Note: Descriptions are shown in the official language in which they were submitted.
~vo 94/00095~13 ~12 ~ PCI`/US93/06143
USE OF CALPAIN INHIBITORS IN THE INHIBlTION
AND TREATMENT OF MEDICAL CONDITIONS ASSOCIATED WITH
INCREASED CALPAIN ACTIVITY
Background of the Invention
The present invention relates generally to medical treatments involving the
inhihitirJn of calcium-activated proteases, such as Calpain. More specifically, the
present invention relates to the treatment of neurodegenerative conditions, coronary
10disease, circulatory pathology, cataract formation, and other medical conditions
~ccori~ted with calcium-activated protease activity using inhibitors of these proteases.
Neural tissues, inr~ iing brain, are known to possess a large variety of
proteases, inrhl~ling at least two calcium-stim~ ted proteases, termed calpain I and
calpain II, which are activated by micromolar and millimolar Ca2+ concentrations,
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respectively. C~lr~inc are a family of calcium activated thiol proteases that are present
in many tissues and use a cysteine residue in their catalytic mech~nicm Calpain II is
the predomin~nt form, but calpain I is found at synapses and is thought to be the form
involved in long term potentiation, synaptic plasticity and cell death.
Thiol proteases are flictinguiched from serine proteases, metalloproteases and
other proteases by their me~h~nicm of action and by the amino acid residue (cysteine)
that partiriratçs in substrate attack. Although several thiol proteases are produced by
plants, these proteases are not common in m~mm~lc, with cathepsin B (a lysosomalenzyme), other ~thçpcinc and the calpains being among the few representatives of this
family that have been described in m~mm~lc Calpain I and calpain II are the bestdescribed of these, but several other members of the calpain family have been reported.
Other Ca2+-activated thiol proteases may exist, such as those reported by
Yoshihara et al., in J. Bio~ Che~, 265:5809-5815 (1990). The term "Calpain" is used
hereinafter to refer to any Ca2+-activated thiol proteases including the Yoshihara
enzyme and calpains I and II.
Although C~lr~inc degrade a wide variety of protein substrates, cytoskeletal
proteins seem to be particularly susceptible to attack. In at least some cases, the
products of the proteolytic digestion of these proteins by Calpain are distinctive and
persistent over time. Since cytoskeletal proteins are major components of certain types
of cells, this provides a simple method of detecting Calpain activity in cells and tissues.
Specifically, the accumulation of the breakdown products (nBDP's") of spectrin, a
cytoskeletal protein, has been associated with the activation of Calpain. Thus, calpain
activation can be measured indirectly by assaying the proteolysis of the cytoskeletal
protein spectrin, which produces a large, distinctive and biologically persistent
breakdown product when attacked by calpain (Siman, Baudry, and Lynch, Proc. NatLAcad Sci USA 81:3572-3576 (1984); incorporated herein by reference). In neural
tissues, activation of C~lp~inc, as evidenced by accumulation of these BDP's, has been
observed in many neurodegenerative conditions. For example, these phenomena havebeen observed after denervation resulting from focal electrolytic lesions, in genetic
abnormalities, after excitotoxicity, following ischemia in gerbils and rats, following
aflminictration of the toxins kainate and colr~ ne in rats, an in human Alzheimer's
disease. l~lr~inc have also been shown to degrade the lens proteins alpha-crystallin,
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~13~121 . ~
vimentin, and actin in vitro, and have been implicated in the degradation of cardiac
muscle proteins and other tissues.
Commercially available in vitro inhibitors of Calpain include peptide aldehydes
such as lc:upep~in (Ac-Leu-Leu-Arg-H) and Ac-Leu-Leu-Nle-H, as well as
epoxysucrin~t~s such as E-64. These compounds are not useful in inhihiting Calpain in
Central Nervous System ("CNSn) tissue in vivo because they are poorly membrane
p-,~...eal1t and, accordingly, do not cross the blood brain barrier very well. Some of
these compounds have also been found to have other adverse side effects. For
example, lelJ~,eptin has been found to be harmful to heart cells and to adversely affect
blood clotting (Toyo-Oka, et al., Jpn. Heart J., 23(5):829 (1982)). Also, many of these
inhihitors are poorly specific and will inhibit a wide variety of proteases in addition to
C~lrqin Thus, no effective therapy has yet been developed for most
neurodegenerative diseases and concliticnc Mill.ons of individuals suffer from
neurodegene~ative diseases and thus, there is a need for therapies effective in treating
and preventing these diseases and conditions.
Cathepsin B is involved in muscular dystrophy, myocardial tissue damage, tumor
met~ct~ciC~ and bone resorption. In addition, a number of viral processing enzymes,
which are essential for viral infection, are cysteine proteases. Inhibitors of cysteine
proteases would thus have multiple therapeutic uses.
These commercially available compounds are based upon peptide structures that
are believed to interact with the substrate binding site of Calpain. Active groups
~cco-i~ted with the Calpain inhibitors then either block or attack the catalytic moiety of
Calpain in order to inhibit the enzyme.
In addition, other types of compounds that are not commercially available
which inhibit cysteine proteases and are thought to possess i/t vitro Calpain inhibitory
activity have been reported. Examples of such compounds include the peptide
diazomethanes and peptide diazomethyl ketones. See Rich, D.H., Inhibitors of ~stei~le
proteinases, in Protease Inhibitors. ppl53-178 (A.J. Barrett and G. Salversen, Eds.,
Elsevier, New York, 1986), the disclosure of which is hereby incorporated by reference.
Peptide diazomethyl ketones are potentially carcinogenic and along with peptide
dia~omethanes are thought to be poorly membr,,ne permeant and to have low
specificity.
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a~
There is some evidence that certain particular inhibitors of Calpain have certain
therapeutic utilities. For example, leupeptin can f~rilit~te nerve repair in primates.
nY~ct~tin (also known as EST, Ep-460 or E-64d), a derivative of E-64, is believed to
have utility in the treatment of mllscul~r dystrophy. E-64d, while not having signifi~nt
protease inhibitory activity itself, is believed to be converted to more potent forms,
such as to E-64c, inside a m:~mm~ n body.
Evidence from electrophysiological studies suggests that one of the earliest
factors in the chain of reactions leading to cell death is an increase in intracellular-free
calcium as a consequence of Ca2+ channel opening and/or energy depletion.
Intracf~llul~r calcium is likely to produce a large number of consequences, including the
activation of a large number of enzymes, including proteases, such as Calpain, lipases
and kinases. An increase in intrac~ r calcium is also thought to induce changes in
gene expression.
Ischemia, head trauma and stroke have all been associated with the release of
glllt~m~te in amounts large enough to lead to excitotoxicity, the toxicity resulting from
the actions of certain amino acids on neurons of the CNS. The excess glutamate and
other factors, such as free radical damage of membranes or energy depletion, cause an
increase in intracellular Ca2+. It is known that an excess of intracellular Ca2+ leads to
several effects believed to be associated with neuronal cell damage, including
destruction of cell structures through activation of phospholipase and Calpain, as well
as free radical production resulting from activation of phospholipase and xanthine
oxidase. Many other factors have been associated with neurotoxicity. For example,
reductions in action potentials and changes in a wide variety of chemical markers are
known to be associated with neurons exposed to ischemic conditions.
The excitotoxic death of nerve cells following ischemia is the result of a cascade
of events which begins with energy depletion, followed by release of glutamate,
stimulation of glutamate receptors, and an elevation of intracellular calcium. See, e.g.,
Meldrum, "Excitotoxicity in Ischemia: An Overview," in C~lel,lov~scular Diseases,
Ginsberg et al. (eds.), Raven Press, New York, pp. 47-60 (1989). Since many
researchers believe that excitotoxicity plays a large role in the pathology of stroke and
ischemia, much recent research has focused upon developing drugs which reduce
excitotoxicity by acting at specific stages of the excitotoxic cascade.
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Elevations in intr~c~ r calcium have been proposed to play a central role in
the induction of excitotoxic cell death. See, e.g. Meldrum et al., Trends Plla~naco~ Sci,
11:379-387 (1990). Many attempts to prevent excitotoxicity have focused upon blocking
the NMDA subtype of glutamate receptor, which functions as a calcium channel.
Although E~ut~m~te toxicity is calcium dependent, it is clear that calcium influx through
the NMDA receptor is not the sole culprit in excitotoxicity. The correlation between
NMDA antagonist mediated reduction in glutamate-induced intracellular Ca2+ and cell
rescue is poor. Further, agents acting at non-NMDA type calcium channels are
effective inhibitors of ehlS~m~te toxicity and excitotoxicity appears to involve not only
calcium influx through both NMDA and non-NMDA calcium channels but also the
release of Ca2+ from intrac~ r stores. Thus, the mechanism by which Ca2+
becG~ s elevated is still unknown.
It is dear that elevated Ca2+ is a prime intract~llul~r mediator of excitotoxicity.
Elevations of intrac~llul~r calcium modulate many effects, including the activation of
the calcium-dependent thiol proteases calpain I and II. Calpain has been shown to be
activated during excitotoxicity, and calpain activation can be detected early following
ischemia.
Calpain action results in the irreversible cleavage of cellular proteins and
alterations in their function, and this degradative function fits in well with a possible
role in cell death. Further, leupeptin, a calpain inhibitor, has been shown to reduce
ischemic damage in gerbils and to reduce hypoxic damage in rat hippocampal slices.
Much of what is known about excitotoxicity derives from studies of neurons in
vi~o. Primary cultures of cerebral cortex, hippocampal and cerebellar neurons are killed
by exposure to glutamate or glutamate analogs. Recently, glutamate has been reported
to kill pheochromocytoma PC12 cells in a calcium-dependent manner.
Increases in intrac~ r calcium and subsequent calpain activity have also been
linked to other pathological conditions. It has been found, for example, that inexperimental cataracts induced in mice, increased calcium levels have been recorded
just before the onset of cataract formation. The size of infarcted heart tissue in
ischemic myocardium can also be reduced by the administration of calpain inhibitors
(Toda, et al., lpn. Heart J., 30:375-86 (1989); Toyo-Oka, Drug Res., 36(1):671-75 (1986)).
Notwithstanding the foregoing understanding of certain aspects of neurotoxicity,
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~381~-4
no effective therapy has yet been developed for most neurodegenerative diseases and
conditions of the CNS. Millions of individuals suffer from these diseases and
con-1itionc Thus, there is a need for therapies effective in treating and preventing
these tiice~ces and conditions.
In addition to being involved in cytotoxicity, proteases such as calpain have also
been linked to the regulation of cellular growth. However, the mech~nicmc of such
regulation have not been well studied. Some protease inhibitors inhibit cellularproliferation, for example, while others tonh~nre it. Because calpains are ubiquitousJy
distributed in m~mm~ n cells but apparently do not contribute to normal protein
catabolism or general protein turnover, they appear to serve a regulatory role in such
cells. However, the me~ h~nicmc of such regulation have not been well studied. While
some calpain inhibitors have been shown to inhibit cellular proliferation and thus cell
cycling, the specific point in the reproductive cycle at which such inhibition occurs is
not yet known.
An underst~nrling of the regulation of cell cycling is relevant to the development
of treatments for cancer, because cancer cells grow without regulation of such cell
cycling. Chemotherapy treatments for cancer sometime take the form of administering
chemicals which will kill cells that are passing through the cell cycle and actively
dividing while sparing those cells which are not dividing. In one such form of
chemotherapy, drugs which interfere with the replication of the DNA of cells during
the "S" (synthesis) phase of the cell cycle are a-iminictered to a patient. This treatment,
however, will only be effective in killing cells in the S phase. Thus, a drug must be
present in a patient's body for long enough so that all of the cancer cells in the patient
progress through the S phase. Since chemotherapeutic agents kill non-cancerous cells
which are dividing as well as cancerous cells, the timing and duration of
chemotherapeutic drug ~minictration is critical to successful therapy.
There exists, therefore, a need for compounds which are capable of
manipulating the cell cycle, resulting in a shortened duration of chemotherapy and
greater efficacy of the chemotherapeutic agent.
The processes of angiogenesis and vascular repair both depend upon smooth
muscle cell proliferation, since smooth muscle cells play an essential part in the
functioning of blood vessels as well as other organs. Smooth muscle cells are
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stim~ ted to proliferate following vascular injury by a number of different factors,
in~lnrling PDGF (platelet derived growth factor). This is normally a desirable process
which is necess.ll~ for healing. However, following therapeutic angioplasty for the
- opening of obstructed arteries, the proliferation of the smooth muscle cells can result in
rect~nocic~ the blockage of the previously opened artery. Austin G.E., et al., J. Am.
ColL Cardiology, 6:369-377 (1985). This is a .c~ fi~nt problem in the clinical use of
~ngi~pl~cty, and a need therefore eYists for a drug which can inhibit the proliferation of
the smooth muscle cells.
~d~1itil n~lly, proteases such as calpain have also been linked to the regulation
of smooth muscle contraction. However, the mech~nicrn by which contractility and the
m~int~n~nce of the tonically contracted state is regulated in smooth muscle is not well
understood. Many agents which act to decrease contractility of smooth muscle have
little or no efficacy at inhihiting the establishmer.t of the tonic state or reversing the
tonic contractile state once çst~hli~hed.
The tonic contraction of smooth muscle is a normal process. In some cases,
however, such tonic contraction can lead to serious pathological conditions. For~Y~mpl~, contraction of the bronchial smooth muscle leads to shortness of breath and
other symptoms of asthma. Contraction of the coronary arteries can lead to angina,
partial coronary hypoxia and subsequent loss of coronary function. Contraction of the
smooth muscle in cerebral arteries can lead to cerebral vasospasm and hypoxia of the
brain tissue, a serious condition that can leave patients mentally disabled and
permanently brain damaged.
Summary of the Invention
One aspect of the present invention is a method of synchronizing the
reproductive cycle of actively dividing cells. In this method, an amount of a Calpain
Inhibitor which is pharmacologically effective to block the progression of cells from G
phase into S phase is administered to the cells. The Calpain Inhibitor can be one of
the Peptide Keto-Compounds, the Halo-Ketone Peptides, or the Substituted
Heterocyclic Compounds. In one embodiment, the cells to be treated in this method
are located in v~vo in a m~mm:~l, so that the administering step of the method
co~ .ises anminict~ring a Calpain Inhibitor to cells in a mammal. Alternatively, the
a-lminictPring step can comprise a-lminictering a Calpain Inhibitor to cells in v~tro. In
WO 94/00095 PCI`/US93/06143
21~8124
one preferred embodiment, the adminictering step of this method comprises
a-1minict~ring a Peptide Keto-Compound. Calpain Inhibitors can be administered in
this method either intravenously, intramuscularly, intraperitoneally, topically, orally, or
by direct appli~ti- n to cells.
S In another aspect, the present invention co,.",lises a method of blocking the
pl~,~es~ion of the cell cycle from Gl phase to S phase in actively dividing cells in a
m~mm~l In this method, a m~mmal is ac~mini~t~red an amount of a Calpain Inhibitor
which is pharmacologically effective to block the plo~"ession of the cell cycles of
actively dividing cells in the m~mm~l from Gl phase into S phase. The Calpain
Inhibitor can be one of the Peptide Keto-Compounds, the Halo-Ketone Peptides, orthe Suhstinlted Heterocyclic Compounds. In one preferred embodiment, the CalpainTnhihitor is a Peptide Keto-Compound. Calpain Inhibitors can be a-lminict~red
according to this method either intravenously, intramuscularly, intraperitoneally,
topically, orally, or by direct application to living cells. In one embodiment, the Calpain
Inhibitor is a~mini~tered by direct application, where such direct application can
comprise either applying a gel to an area of living cells, driving microspheres loaded
with the Calpain Inhibitor into tissue colll~ ing the living cells, or injecting a solution
cont~ining the Calpain Inhibitor directly into tissue colllL,lisil~g such living cells.
In yet another aspect, the present invention comprises a method of enhancing
the efficacy of chemotherapy in the treatment of cancer in a human patient. Thismethod co"" lises a~minict~ring to the cancerous cells of the patient an amount of a
Calpain Inhibitor which is pharmacologically effective to block the progression of the
cell cycles of such cancerous cells from Gl phase to S phase, and thereafter
a-lminictçring to the cells a chemotherapeutic agent. The Calpain Inhibitor in this
method is selected from the group consisting of Peptide Keto-Compounds, Halo-
Ketone Peptides, and Substituted Heterocyclic Compounds. In one preferred
embodirnent, the Calpain Inhibitor is a Peptide Keto-Compound. The Calpain
Inhibitor in this method can be a~lminictered intravenously, intramuscularly,
intraperitoneally, topically, orally, or by direct application to the cancerous cells. The
chemotherapeutic agent can be aciminict~red beginning 24-48 hours after the
~,~lmini~tration of the Calpain Inhibitor, at which time the cell cycles of the patient's
cancerous cells which were treatable with the Calpain Inhibitor will be synchronized.
WO 94/00095 PCI/US93/06143
A further aspect of the present invention includes a method of determining the
effectiveness of a chemothelapculic agent, collll.lisillg growing cancerous cells in vitro,
a~lmini~t~ring to such cancerous cells an amount of a Calpain Inhibitor which is- effective to block the ylo~ ion of the cells from Gl phase into S phase,
a-l nini~t.oring to the cells the chemotherapeutic agent in an amount sufficient to kill the
cells, and thereafter determining the amount of cell death that occurs. The amount of
cell death that occurs in this method is indicative of the effectiveness the
chemotherapeutic agent tested.
Another aspect of the present invention is a method of increasing the efficiencyof cell transforrnation and thus increasing the efficiency of integration of foreign DNA
into living cells. This method CG",plises a.l nini~t~ring to a population of cells
COlll~lisil-g actively dividing cells an amount of a Calpain Inhibitor which is
pharm~cn'ogir~11y effective to block the progression of the cell cycles of the cells from
Gl phase into S phase, di~continuing the ~imini~tration of the Calpain Inhibitor, and
thereafter introducing foreign DNA into the population of cells. The Calpain Inhibitor
in this method is selected from the group consi:,ling of Peptide Keto-Compounds, Halo-
Ketone Peptides, and Substituted Heterocyclic Compounds. In one embodiment, the
Calpain Inhibitor is a Peptide Keto-Compound. The administration of the Calpain
Inhibitor in this method can continue for the length of one cell cycle in the population
of living cells. The target of the Calpain Inhibitor can be a population of cells located
in a m~mm:ll, which can be a~1mini~tered a Calpain Inhibitor intravenously,
intramuscularly, intraperitoneally, topically, orally, or by direct application to the
population of cells in the m~mm~1 In another embodiment, the Calpain Inhibitor is
a~lmini~t~red instead to a population of cells in vitro.
The present invention provides methods of treating a variety of medical
conditions associated with calcium-activated protease activity in a m~mm~1 by
aclminictering the Calpain inhibitors of the present invention to that mammal. These
Calpain inhibitors are Peptide Keto-Compounds, Halo-Ketone Peptides, and
Substituted Heterocyclic Compounds. Particularly preferred compounds for this use
include the Peptide Ketoamides, such as Z-Leu-Abu-CONH-Et, Z-Leu-Phe-CONH-Et
and Z-Leu-Phe-CONH(CH2)2C6H5. Administration of the inhibitors can be through
any of a variety of routes. These routes include all of the following types of
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a-lminictration: intravenous, intraperitoneal, intramuscular, oral, topical treatment such
as through ointments (int~ ing ophth~lmi~ ointments), eye drops, contact lenses,catheter, directly onto tissues such as blood vessels or cardiac tissue during surgery, or
injection into the pericardial space.
Specific medical conditions which can be treated with these Calpain Inhibitors
include cardiac muscle tissue damage. After a m~mm~l with cardiac muscle tissue
damage has been il1~ntifi~d, that m~mm~l can be treated with a Calpain Inhibitor.
~l~mm~lc at risk for developing cardiac muscle tissue damage can also be treated with
the present Calpain TnhihitQrs. A~lminict~ring these Inhibitors to such m~mm~lc
protects them from the cardiac tissue damage experienced by m~mm~lc which are not
so protected.
In another embodiment of the present invention, cataracts are treated by the
a-lmini.ctration of a Calpain Inhibitor. If a m~mm~l has already developed cataracts,
the development of the cataracts can be slowed or arrested through the administration
of a Calpain Inhibitor. On the other hand, if a m~mm~l has been identified as being at
risk for developing cataracts in the future, the development of cataracts in such a
m~mm~l can be prevented or slowed through the administration of a Calpain Inhibitor.
A variety of other tissues and conditions can also be treated with the novel
Calpain Inhibitors of the present invention. Skeletal and smooth muscle damage, for
example, can be treated by identifying a mammal with such tissue damage and
adminictçring a Calpain Inhibitor to that m~mm~l. Vasospasm, a condition of a
particular 'Kind of smooth muscle, the vascular tissue, can also be reversed in a m~mm~l
identified as having this condition by the administration of Calpain Inhibitors.Erythrocytes damaged by the proteolytic activity of Calpain in hypertensive mammals
can also be treated with the Calpain Inhibitors of the present invention.
In one aspect, the present invention provides methods of halting or inhibiting
the proliferation of smooth muscle cells both in vivo and in vitro by administering a
Calpain Inhibitor. These Calpain Inhibitors are Peptide Keto-Compounds, Halo-
Ketone Peptides, and Substitllted Heterocyclic Compounds. Particularly preferredcompounds for this use include the Peptide Ketoamides, such as Z-Leu-Phe-CONH-Et.
A-lminictration of Ihe inhibitors can be through any of a variety of routes. These
routes include all of the following types of administration: intravenous, intramuscular,
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intraperitoneal, topical, oral, or by direct application. Preferred Peptide Keto-
Compounds useful in the present invention include (Ph)2CHCO-Leu-Phe-CONH-CH2-
2-Py; Z-Leu-Nva-CONH-CH2-2-Py; Z-Leu-Phe-CONH-CH2CH(OH)Ph; (Ph)2CHCO-
Leu-Abu-CONH-CH2CH(OH)Ph; Z-Leu-Phe-CONH2; Z-Leu-Abu-CONH-
CH2CH(OH)Ph; and Z-Leu-Phe-CONHEt.
Direct application of the Calpain Inhibitors can be through various means.
Such means include using a gel or ointment containing the inhibitor to coat the surface
of the balloon of a balloon catheter or onto another surgical instrument that is inserted
into the blood vessel during angioplasty. Alternatively, the gel may be applied directly
to an area of vascular tissue which has been treated by angioplasty during the surgical
procedure. Another route of a-lminictration comprises driving microspheres whichhave been loaded with a Calpain Inhibitor directly into the mammal's blood vessel.
This can be accomrliched by applying the microsphcres to the surface of the balloon or
other surgical instrument used during the angioplasty procedure. The microspheres are
driven into the arterial wall, where they lodge and release the Calpain Inhibitor over
time.
Specific medical conditions which can be treated with these Calpain Inhibitors
include the treatment of a m~mm~l to prevent restenosis of a blood vessel following
angioplasty. After a m~mm~l which has undergone angioplasty has been identified, that
m~mm~l can be treated with a Calpain Inhibitor. Mammals at risk for developing
restenosis can also be treated with the present Calpain Inhibitors. Administering these
Inhibitors to such m~mm~lc protects them from the smooth muscle cell proliferation
experienced by m~mm~lc which are not so protected.
In another aspect, the present invention provides a method of inhibiting tonic
smooth muscle contraction in a m~mm~l susceptible to inapplopliate contraction in a
smooth muscle thereof. The method includes administering to the smooth muscle anamount of a Calpain Inhibitor that is pharmacologically effective to suppress the
contraction thereo The Calpain Inhibitor is one of the Peptide Keto-Compounds,
Halo-Ketone Peptides or Substituted Heterocylic Compounds. Preferably, the inhibitor
is ~c~minict.ored intravenously, intramuscularly, intraperitoneally, topically, orally, by
injection into cclebrospinal fluid, by inhalation, or by direct application to the smooth
muscle, such as by applying directly to an area of smooth muscle. Direct application
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can also be by driving mi,_lu~k~"es loaded with the Calpain Inhibitor into the smooth
muscle. Rel~Y~tinn of the smooth muscle is preferably induced.
In an additional aspect, the present invention provides a method of treating
coronary v~o~ ... in a m~mm~1 In this aspect, the method includes administering to
the m~mm~1 an amount of a Calpain Inhibitor which is pharmacologically effective to
stop v~osr~m of coronary tissue in the m~mm~1 The Calpain Inhibitor is one of the
Peptide Keto-Compounds, Halo-Ketone Peptides or Substituted Heterocylic
Compounds. In a prefe,~ed embodiment, the coronary tissue is surgically exposed and
a solution of Calpain Inhibitor is applied directly to the tissue. Preferably, the coronary
tissue con~p~;ses a coronary artery. In a preferred embodiment of this aspect, the
m:lmm:~1 is suffering from angina and the method comprises a treatment for the angina.
In still another aspect of the invention, there is provided a method of treatingbronchial v~co ,l~a~... in a m~mm~1 This method includes administering to the m~mm~1
an amount of a Calpain Inhibitor which is pharmacologically effective to stop
v~so~p~m of bronchial tissue in the m~mm~1 The Calpain Inhibitor is one of the
Peptide Keto-Compounds, Halo-Ketone Peptides or Substituted Heterocylic
Compounds. The bronchial tissue can be surgically exposed and a solution of Calpain
Inhibitor applied directly to the tissue. In a preferred embodiment of the method, the
m~mm~1 is suffering from asthma and the method cOIll~liscs a treatment for the
asthma.
Yet another aspect of the invention relates to a method of treating cerebral
v;.~os~ ... in a m~mm~1 This method includes administering to the mammal an
amount of a Calpain Inhibitor which is pharmacologically effective to stop vasospasm of
cerebral tissue in the m~mm~1 The Calpain Inhibitor is one of the Peptide Keto-
Compounds, Halo-Ketone Peptides and Substituted Heterocylic Compounds. The
cerebral tissue can be surgically exposed and a solution of Calpain Inhibitor applied
directly to the tissue. In one embodiment of this aspect of the invention, the Calpain
Inhibitor can be injected into the m~mm~1's ce~eb-o~l)inal fluid.
One aspect of the present invention provides a method of medical treatment for
a medical condition in a m~mm~1 In this method, a pharmaceutical composition
containing a morpholine Peptide Keto-Compound is administered to the mammal. Thecomposition is ~clminictered in an amount that is pharmacologically effective to treat
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the condition. The condition is one which is associated with increased proteolytic
activity of Calpain. The morpholine Peptide Keto-Compound can be either a
C-terminal or N-terminal morpholine Peptide Keto-Compound, such as cardiac muscle
- tissue damage, cataracts, skeletal muscle damage, vq-cocpacm or restenosis following
cardiac angioplasty.
Another aspect of the present invention also provides a method of medical
treatment for a medical condition in a mqmmql In this method, a pharmaceutical
composition contqining a Peptide ~toqmidç, Subclass C is administered to the
mqmmq-l This co.~pG~ iOn is arlminictçred in an amount that is pharmacologicallyeffective to treat the condition. The condition that can be treated with this method is
also one qcsociq-ted with increased proteolytic activity of Calpain, such as cardiac
muscle tissue damage, cataracts, skeletal muscle damage, vasospasm or restenosisfollowing cardiac qngjrrlqcty.
One of skill in the art will reco~ e that the present Calpain Inhibitors can be
used to counteract the harmful effects acsoriqted with calpain activity which arise in a
number of medical conditions and ~icçq-c~s Therefore, the treatment of such
conditions with the present Calpain Inhibitors is within the scope of the present
invention.
These and other features and advantages of the present invention will become
apparent from the detailed description which follows, considered together with the
attached drawings and claims.
Brief Description of the Fi~ures
Figure 1 shows the percentage of inhibition of glutamate-induced cell death
through the addition of glutamate and various Calpain Inhibitors relative to control
where no ~utqmqte was added.
Figure 2 shows that Calpain inhibitor reduces cell death following glutamate
exposure. PC12 cells were exposed to 7.5mM glutamate with the indicated
concentration of inhibitor, as described in the text, for 24 hours. Cell viability was
assayed using the MTI assay. Values are expressed as % of naive control ~ sem.
Figure 3 shows the dependence of the ability of Calpain inhibitors to reduce cell
death on gll-tqmqte concentration. PC12 cells were incubated with the indicated
concentration of glutamate and no inhibitor (circles), 20uM Z-Leu-Nva-CONH(CH2)3
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morpholine (triangles), or 30uM Z-Leu-Phe-CONHCH2CH3 (squares) for 24 hours and
cell viability was assayed by M~IT. Values cA~7ressed as % of naive control + sem.
Figure 4. Delayed addition of calpain inhibitor. Glut~m~te (7.5mM) was added
at 0 time and Z-Leu-Phe-CONHCH2CH3 (squares) or Z-Leu-Nva-CONH(CH2)3
morpholine (triangles) added at the indicated times to final concentrations of 100uM
each. Cell viability was measured 24 hours after the addition of glutamate by the MTT
assay. Values e,~r,le.,sed as % of naive control ~ sem.
Figure S graphically depicts the effects of Z-Leu-Phe-CONH-Et and Z-Leu-
Abu-CONH-Et on the size of infarction produced upon MCA occlusion in male rats.
Figure 6 shows the effects of Z-Leu-Abu-CO2Et, a Peptide Keto-Compound,
and CI1 (Ac-Leu-Leu-Me-H) relative to control slices on survival of hippocampal slices
exposed to 10 minutes exposure of anoxic atmosphere where both of these compounds
were added at their optimal inhibitory concentration at both l hour and 2 hour
incubation times.
Figure 7 shows the evoked potential amplitude for control, CI1 treated and Z-
Leu-Abu-CO2Et treated hippocampal slices over a time course during which the slices
are exposed to anoxic atmosphere.
Figure 8 shows the percent recovery of EPSP from severe hypoxia over the
course of one hour incubation for Z-Leu-Phe-CONH-Et and Z-Leu-Phe-C02Et.
Figure 9 shows a comparison of the effect of the presence of CI1 or Z-Leu-Phe-
CO2Et on survival of hippocampal slices expressed as the duration of anoxia (in
minutes) before fiber volley disappearance.
Figure 10 shows the effects of CI1 compared with control on the behavioral and
convulsive effects of kainic acid.
25 - Figure 11 shows the amount of spectrin BDP's in rat brains exposed to kainate
for control and CIl treated rats.
Figure 12 graphically depicts the effect of several different Calpain Inhibitors on
contraction of isolated arteries induced by endothelin (ET-1). Drug A is Z-Leu-Abu-
CONHEt, Drug B is Z-Leu-Phe-CONHEt, Drug C is 1,10-Phenanthroline and Drug D
is TLCK (Tosyl-Lysine-chloromethylketone).
Figure 13 graphically depicts the effect of several other Calpain Inhibitors on
contraction of isolated arteries induced by endothelin (ET-1). Drug E is Z-Leu-Phe,
WO 94/00095 PCI`/US93/06143
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~i3~12~
-15-
Drug F is Z-Leu-Phe-CONHEt (the same as drug B), Drug G is Z-Leu-Phe-
CONH(CH2~,2Ph, Drug H is Ac-Leu-Leu-Me-H (Calpain Inhibitor I), Drug I is Gly-
Gly-Gly and Drug J is (Ph)2CHCO-Leu-Abu-CONH-CH2CH(OH)Ph.
- Figure 14 shows the effect of Calpain Inhibitors on contraction of isolated
arteries induced by phorbol dibutyrate (PDB). Drugs E through J are as in Figure 15.
Figure 15 graphically depicts the effect of Calpain Inhibitors on smooth muscle
resting tension. Drugs E through J are as in Figure 13.
Figure 16 shows the dose-dependent inhibition of oxyhemoglobin-induced
constriction by a Calpain Inhibitor, Z-Leu-Phe-CONH(CH2)3, of the present invention.
Figure 17 shows an example of the time course of artery constriction in an
artery constricted by subarachnoid hemorrhage (SAH) and treated with a Calpain
Inhibitor, Z-Leu-Phe-CONH(CH2)3, of the present invention.
Figure 18 shows the summary of data from three animals in which a Calpain
Inhibitor, Z-Leu-Phe-CONH(CH2)3, of the present invention reversed constrictionslS caused by SAH.
Figure 19 graphically depicts the effects of 7-Leu-Phe-CONHEt and
Ph2CHCO-Leu-Abu-CONH-CH2CH(OH)Ph on the proliferation of cultured bovine
smooth muscle cells.
Figure 20 shows the continued viability of smooth muscle cells after treatment
with a Calpain Inhibitor, despite a complete inhibition of cell proliferation.
Figure 21 graphically depicts the blocking of the progression into S phase of
bovine aortic smooth muscle cells (BASMC) after treatment with the Calpain Inhibitor
Ph2-CHCO-Leu-Abu-CONH-CH2CH(OH)Ph. In this graph, "Drug C" is Ph2-CHCO-
Leu-Abu-CONH-CH2CH(OH)Ph ("Drug C" elsewhere may be a different compound).
2S Figure 22 graphically depicts the synchronous progression into S phase of HeLa
and AT-2 cells after the Calpain Inhibitor Ph2-CHCO-Leu-Abu-CONH-CH2CH(OH)Ph
was washed out of the medium in which such cells were maintained. In this graph,"Drug C" is also Ph2-CHCO-Leu-Abu-CONH-CH2CH(OH)Ph (though "Drug C"
elsewhere may be a different compound).
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Detailed Description of the Invention
A. INTRODU~-l ION
We have discu~/ered that Calpain activation is an event central to many cases ofbrain atrophy and degeneration and that inhibition of Calpain alone is sufficient to
S inhibit or prevent cell deterioration and loss. Thus, we have further discovered that
inhihitit n of Calpain provides protection from neurotoxicity associated with many
neurodegenerative conditions and dic~cloc
In accordance with the furey,oi~g discoveries, we believe that the elevation of
intracPlhll~r calcium associated with neuropathological conditions in neuronal cells
activates Calpain and sets in motion the digestion of neuronal cells from within. We
believe there may be other mech~nicrnc of activation of Calpains associated with these
conditions. Accordingly, one aspect of the present invention is directed to inhibition
and treatment of the neurodegeneration and other diseases associated with this
digestion through the inhibition of Calpain activity. Thus, part of this aspect of the
present invention is to prevent the neurodegeneration and other pathology caused by
this digestion through the iZl vivo administration of Calpain inhibitors. By way of
~Y~mp1e, and not of limitation, diseases and conditions which can be treated using this
aspect of the present invention include neurodegeneration following excitotoxicity, HIV-
induced neuropathy, ischemia, denervation following ischemia or injury, subarachnoid
hemorrhage, stroke, multiple infarction dementia, Akheimer's Disease (AD),
Parkinson's Disease, Huntington's Disease, surgery-related brain damage and other
neuropatholo~ir~l conditions.
As stated above, spectrin BDP's have been found to be associated with Calpain
activation in vivo. We have observed that in each instance of neurodegeneration in
2~ which BDP's characteristic of Calpain activation are detected, Calpain activation is
lor~li7ed to the brain areas most vulnerable to the particular pathogenic manipulation.
In addition, as judged by histological methods, Calpain activation precedes overt
evidence of neurodegeneration. Accordingly, Calpain activation is spatially and
temporally linked to impending or ongoing cell death in the brain. Thus, we believe
that Calpain activation is an important mech:~nicm of cell damage and death in many
pathological conditions, including neuropathological conditions. Moreover, there is
evidence that the activation of Calpains is an early event in the death of cells including
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neural cells. This is in contrast to other known proteases which are activated at later
stages of cell death. Thus, we believe that, advantageously, inhibition of Calpain
activity provides intervention at an early stage of cell death, prior to significant
deterioration of cellular machinery.
Another aspect of the involvement of C~lp~in~ in neurodegeneration is the
involvement of these proteins in regenerating systems. It is known that developing or
regenerating axons are somehow inhibited from further development in a stabilization
process called the "stop pathway." This stabilization can occur when axons have
reached their targets; however, in some systems stabilization can also occur at
inap~ iate places. One researcher has developed evidence that this stop pathway
operates at least in part by the activation of intracellular Calpain and that inhibition of
Calpain can interfere with stabilization (Luizzi, 1990). We believe that Calpaininhihitnrs, when used in accordance with the present invention, can advantageously aid
regeneration and recovery of neural tissue after injury, in addition to inhibiting
neurodegeneration.
Another aspect of the present invention is our discovery that at least three
classes of compounds, the substituted isocoumarins, the peptide keto-compounds and
the Halo-Ketone Peptides have Calpain inhibitory activity. We have further discovered,
as will be described hereinbelow, that these three classes of compounds exhibit
additional properties that render them especially useful as therapeutically effective
compounds in the treatment of neurodegenerative conditions and diseases.
Calpain has also been implicated in the pathogenesis of a number of other
medical conditions. The inhibition of Calpain is capable of slowing the progress of
these diseases and of preventing certain conditions altogether.
The formation of cataracts, for exar~ple, has been linked to Calpain activity inm~mm~ n lenses. In in vivo models of cataract formation, increased Calpain activity
has been documented just before the onset of detectable cataract formation. Calpain
activity has also been observed to decrease after a cataract has formed in a lens,
leading to the inference that calpain activity is involved in the formation of cataracts.
Moreover, we have shown that there are increased levels of spectrin breakdown
products found in in vitro models of cataract formation. The presence of such spectrin
breakdown products is known to be reflective of increased Calpain activity. Thus, we
WO 94/00095 PCI /US93/06143
~:~38124
believe that by a~minictering the Calpain Inhibitors of the present invention, the
forrnation of cataracts can be prevented or slowed.
Calpain activity has also been implicated in producing myocardial infarctions.
Calpain activity is regul~ted by intrac~ r calcium concentrations, and increasedintr~1hl1~r calcium in myocardial tissues has been observed when the myocardium is
cut off from its supply of oYygen due to ischemia. Cell damage and ultimately cell
death results from such ic-h.omi~ The increased proteolytic activity of Calpain due to
increased levels of intraçe11n1~r calcium during ischemia is therefore a contributor to or
direct cause of cell death during cardiac ischemia. Cardiac tissue damage can thus be
prevented or minimi7ed with the present Calpain Inhibitors.
Calpain is also believed to be an important regulator of cell growth. Several
Calpain Inhibitors have been found, for eY~mp1e, to inhibit smooth muscle cell
proliferation. Such proliferation is in fact necessary to repair injured smooth muscle
tissue. Following therapeutic angioplasty, however, smooth muscle cell proliferation
may result in restenosis of the opened blood vessel. Calpain Inhibitors may thus be
used to prevent the smooth muscle cell proliferation which results in the restenosis of
blood vessels.
Other disease conditions can be treated with Calpain Inhibitors as well. Calpainhas been shown to degrade the constituents of skeletal and smooth muscle cells, and
has been imp1i~ated in causing vasospasm. Increased Calpain activity has also been
shown in the blood cells of hypertensive patients, and has been shown to be five times
as active in degrading proteins in such cells as in the cells of non-hypertensive patients.
Calpain Inhibitors therefore can reduce or eliminate the harmful effects of Calpain
activity in these tissues.
We have also found that Calpain Inhibitors inhibit tonic smooth muscle
contraction. These compounds are useful in the treatment of animals or humans for
the purpose of preventing or reducing the smooth muscle contraction associated with
vaso~as,l, and bronchospasm.
The present invention includes the use of a variety of Calpain Inhibitors and
methods for using these inhibitors to treat disease conditions. Specifically, Substituted
Heterocyclic Compounds, Peptide Keto-Compounds, and Halo-Ketone Peptides have
been found to be effective in treating the foregoing conditions as well as other diseases.
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Unless otherwise stated, the Calpain Inhibitors of the present invention refers to the
novel Substituted Heterocyclic Compounds, Peptide Keto-Compounds, and Halo-
Ketone Peptides described herein.
Several Calpain Inhibitors have also been found to play a role in the regulationof the reproductive cycle of the cell. These compounds can be used in the treatment of
cancer in animals or humans along with other chemotherapeutic agents in order to~nh~nce the effe~Li~,ness of such agents. By synchronizing the growth of rapidlydividing cells, these compounds can increase the effectiveness of chemotherapeutics
that act at a specific stage in the cell cycle, such as at DNA replication.
By synchronizing the cell cycles of cells, Calpain Inhibitors are also useful inincreasing the efficiency of cell transformation. Transformation results from the
incorporation of foreign DNA into a cell. Such incorporation is increased when cells
are synthesizing DNA. Thus, by synchronizing cells to the DNA synthetic portion of
the cell cycle, the cells will be more efficiently transformed by foreign DNA introduced
into the cells.
B. SUBST~TUTED HETEROCYCLIC COMPOUNDS
One particular class of compounds exhibiting Calpain inhibitory activity, when
used in accordance with the present invention, are the substituted heterocyclic
compounds. These compounds include the substituted isocoumarins. The substitutedheterocyclic compounds are known to be excellent inhibitors of serine proteases. As
cced hereinbelow, we have now discovered that these compounds are also
inhibitors of calpain I and calpain II, and also of other Calpains. Additionally, as also
~liccl-csed below, we have found that, unlike most known inhibitors of Calpains, these
substituted heterocyclic compounds are not effective as inhibitors of papain or
cathepsin B. Thus, we believe that the substituted heterocyclic compounds provide a
relatively specific means of inhibiting C~lp~inc while not affecting other thiol proteases.
One particular class of substituted heterocyclic compounds with Calpain
inhibitory activity are the isocoumarins having cationic substituents. These substituted
heterocyclic compounds are referred to herein as the "Class I Substituted
Isccoumarins." The Class I Substituted Isocoumarins are known to be excellent
inhibitors of several serine proteases, including bovine thrombin, human thrombin,
WO 94/00095 PCI/US93/06143
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human factor Xa, human factor XIa, human factor XIIa, bovine trypsin, human plasma
plqcmin, human tissue plasminogen activator, human lung tryptase, rat skin tryptase,
human leukocyte ~l~st~ce, porcine pancreatic ~ ct ~C~, bovine chymotrypsin and human
leukocyte cathepsin G. The Class I Substituted Isocoumarins inhibit the serine
proteases by reaction with the active site serine to form an acyl enzyme, which in some
cases may further react with another active site nucleophile to form an additional
covalent bond. We have disc()~,e,ed that the Class I Substituted Isocoumarins also
react with C~lr~in We believe that the merh~nicm of action of Calpain inhibition is
sirnilar to that of the inhibition of serine proteases since the reaction mech~nicm of
C~lp~inc is similar to that of the serine proteases.
The Class I Substituted Isocoumarins having Calpain inhibitory activity have thefollowing structural formula:
O
(I)
or a pharrn~ceuti~lly acceptable salt, wherein
Z is selected from the group consisting of C16 alkoxy with an amino group
attached to the alkoxy group, Cl 6 alkoxy with an isothiureido group attached to the
alkoxy group, C1 6 alkoxy with a guanidino group attached to the alkoxy group, C1 6
alkoxy with an amidino group attached to the alkoxy group, Cl 6 alkyl with an amino
group attached to the alkyl group, C1 6 alkyl with an isothiureido group attached to the
alkyl group, C1 6 alkyl with an guanidino group attached to the alkyl group, C1 6 alkyl
with an amidino group attached to the alkyl group,
R is selected from the group consisting of O=C=N-, S=C=N-, AA-NH-, AA-
AA-NH-, AA-O, AA-AA-O-, M-NH-, M-AA-NH, M-AA-AA-NH-, M-O-, M-AA-O-,
M-AA-AA-O-,
WO 94/00095 PCr/VS93/06143
- ~3812 i
wherein AA r~,~r~sents alanine, valine, leucine, isoleucine, proline, methionine,
phenyl~l~nin.o, tryptophan, glycine, serine, threonine, cysteine, tyrosine, beta-alanine,
norleucine, norvaline, alpha-aminobutyric acid, epsilon-aminocaproic acid, citrulline,
rd-uAy~ -oline, ornithine or sarcosine,
wherein M reple,ents NH2-CO-, NH2-CS-, NH2-SO2-, X-NH-CO-, X-NH-CS,
X-NH-SO2, X-CO-, X-CS-, X-SO2-, X-O-CO-, or X-O-CS-,
wherein X r~resents Cl 6 alkyl, Cl 6 fluoroalkyl, Cl 6 alkyl substituted with K,Cl 6 fluoroalkyl ~bs~ ed with K, phenyl, phenyl substituted with J, phenyl
~licubStituted with J, phenyl tricubstituted with J, naphthyl, naphthyl substituted with J,
naphthyl disubstituted with J, naphthyl trisubstituted with J, C1 6 alkyl with an attached
phenyl group, C1 6 alkyl with two attached phenyl groups, C1 6 alkyl with an attached
phenyl group substituted with J, or C1 6 alkyl with two attached phenyl groups
substituted with J,
wherein J rep~esents halogen, COOH, OH, CN, NO2, Cl 6 alkyl, Cl 6 alkoxy,
C1 6 alkylamine, C1 6 dialkylamine, or C1 6 alkyl-O-CO-,
wherein K represents halogen, COOH, OH, CN, NO2, NH2, C16 alkylamine,
C1 6 dialkylamine, or C16 alkyl-O-CO-,
Y is selected from the group consisting of H, halogen, trifluoromethyl, methyl,
OH and methoxy.
The compounds of Formula (I) can also contain one or more substituents at
position B as shown in the following structure:
8 O
R~
wherein electronegative substituents such as NO2, CN, CI, COOR, and COOH
will increase the reactivity of the isocoumarin, and electropositive substituents such as
WO 94/00095 PCI/US93/06143
1 2 ~
NH2, OH, alkoxy, thioalkyl, alkyl, alkylamino, and dialkylamino will increase its
stability. Neutral substituents could also increase the stability of acyl enzyme and
iu~pruve the effe~ ness of the inhibitors.
The following compounds are representative of the Class I Substituted
Isocou.. arins of the present invention:
4-chloro-3-(3-isothiureidopropoxy)isocoumarin (CiTPrOIC)
7-(benzylcarbamoylamino)-4-chloro-3-(3-
isothiureidopl opu~)i~ocoum~rin (PhCH2NHCONH-CiTPrOIC)
7-(phenylcarbamoylamino)-4-chloro-3-(3-
isothiureidopropoxy)isocoumarin (PhNHCONH-CiTPrOIC)
7-(acetylamino)-4-chloro-3-(3-
isothiureidopropoxy)isocoumarin (CH3CONH-CiTPrOIC)
7-(3-phe..~l~,opionylamino)-4-chloro-3-(3-
isothiureidopropoxy)isocoumarin (PhCH2CH2CONH-CiTPrOIC)
7-(phenylacetylamino)-4-chloro-3-(3-
isothiureidopropoxy)isocoumarin (PhCH2CONH-CiTPrOIC)
7-(L-phenylalanylamino)-4-chloro-3-(3-
isothiureidopropoxy)isocoumarin (L-Phe-NH-CiTPrOIC)
7-(N-t-butylu~.;albo..yl-L-phenylalanylamino)-4-chloro-3-(3-
isothiureidopropû~)isocoumarin (Boc-L-Phe-NH-CiTPrOIC)
7-(D-phenylalanylamino)-4-chloro-3-(3-
isothiureidûpropoxy)isocoumarin (D-Phe-NH-CiTPrOIC)
7-(N-t-butyloxycarbonyl-D-phenylalanylamino)-4-chloro-3-
(3-isothiureidopropoxy)isocoumarin (Boc-D-Phe-NH-CiTPrOIC)
7-(benzylcarbamoylamino)-4-chloro-3-(2-
isothiureidoethoxy)isocoumarin (PhCH2NHCONH-CiTEtOIC)
7-(phenylcarbamoylamino)-4-chlûro-3-(2-
isothiureidoethûxy)isocoumarin (PhNHCONH-CiTEtOIC)
7-(isopropylcarbamoylamino)-4-chloro-3-(2-
isothiureidoethoxy)isocoumarin ((CH3)2CHNHCONH-CiTEtOIC)
7-(phenylacetylamino)-4-chloro-3-(2-
isothiureidoethoxy)isocoumarin (PhCH2CONH-CiTEtOIC)
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7-(L-phenylalanylamino)-4-chloro-3-(2-
isothiureidoethoxy)isocoumarin (L-Phe-NH-CiTEtOIC)
7-(N-t-butyloxy~ l,o-l~l-L-phenylalanylamino)-4-chloro-3-(2-
isothiureidoethoxy)isocoumarin (Boc-L-Phe-NH-CiTEtOIC)
7-(D-phenylalanylamino)-4-chloro-3-(2-
isothiureidoethoxy)isocoumarin (D-Phe-NH-CiTEtOIC)
7-(N-t-butyloxyc~.- 1,G.. yl-D-phenylalanylamino)-4-chloro-3-(2--
isothiureidoethoxy)isocoumarin (Boc-D-Phe-NH-CiTEtOIC)
7-(N-t-butyloxycarbonyl-L-alanyl-L-alanylamino)-4-chloro-3-(2-
isothiureidoethoxy)isocoumarin (Boc-Ala-Ala-NH-CiTEtOIC)
7-(L-alanyl-L-alanylamino)-4-chloro-3-(2-
isothiureidoethoxy)isocoumarin (Ala-Ala-NH-CiTEtOIC)
7-( 1-naphthylcarbamoylamino)-4-chloro-3-(2-
isothiureidoethoxy)isocoumarin (NaphthylNH-CiTEtOIC)
7-((S)-a -methylbenzylc~l~l,a...oylamino)-4-chloro-3-(2-
isothiureidoethoxy)isocoumarin (S-C6Hs(CH3)CHNHCONH-CiTEtOIC)
7-((R)-a-methylbenzylcarbamoylamino)-4-chloro-3-(2-
isothiureidoethoxy)isocoumarin (R-C6H5(CH3)CHNHCONH-CiTEtOIC)
7-dansylamino-4-chloro-3-(2-isothiureidoethoxy)isocoumarin (DansylNH-CiTEtOIC)
7-phenylthiocarbamoylamino-4-chloro-3-(2-
isothiureidoethoxy)isocoumarin (PhNHCSNH-CiTEtOIC)
7-(m-carboxyphenylthiocarbamoyl)amino-4-chloro-3-(2-
isothiureidoethoxy)isocoumarin (m-COOH-PhNHCSNH-CiTEtOIC)
7-(p-carboxyphenylthiocarbamoyl)amino-4-chloro-3-(2-
isothiureidoethoxy)isocoumarin (p-COOH-PhNHCSNH-CiTEtOIC)
7-amino-4-chloro-3-(3-isothiureidopropoxy)isocoumarin
(AClIIC)
Isocoumarins with basic substituents are also known to be effective inhibitors of
serine proteases. See Powers et al, U.S. Patent No. 4,845,242, the disclosure of which is
hereby incorporated by reference; This class of compounds, referred to herein as the
"Class II Substituted Isocoumarins," along with thc other substituted heterocyclic
compounds, is believed to be effective in the use of the present invention.
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The Class II Substituted Isocoumarins have the following structural formula:
~Z
or a pharm~eutir~lly acceptable salt, wherein:
R is selected from the group consisting of -N-H-C(=NH)-NH2, -C(=NH)NH2,
Cl 6 alkyl with an attached amino, and C1 6 alkyl with an attached isothiureido of the
formula -S-C( + NH2~ )NH2~
Z is s~1~eted from the group consi~ ,g of H, halogen, Cl 6 alkyl, C1 6 alkyl with
an attached phenyl, Cl 6 fluorinated alkyl, Cl 6 alkyl with an attached hydroxyl, Cl 6
alkyl with an attached Cl 6 alkoxy, Cl 6 alkoxy, C16 fluorinated alkoxy, Cl 6 alkoxy with
an attached phenyl, benzyloxy, 4-fluolube.~yloxy, -OCH2C6H 4R' (2-substituent), -
OCH2C6H4R' (3-substituent), -OCH2C6H4R' (4-substituent), -OCH2C6H3R2' (2,3-
substitu~nts), -OCH2C6H3R2' (2,4-substituents), -OCH2C6H3R2' (2,5-substituents), -
OCH2C6H3R2' (2,6-substituents), -OCH2C6H3R2' (3,4-substituents), and OCH2C6H3R2'(3,5-substituents) .
R' is selected from the group cor.si~i"g of H, halogen, trifluoromethyl, NO2,
cyano, methyl, methoxy, acetyl, carboxyl, OH, and amino.
Y is selected from the group consisting of H, halogen, trifluoromethyl, methyl,
OH, and methoxy.
Alternately, the Class II Substituted Isocoumarins are represented by structure
(II) where,
Z is selected from the group consisting of C1 6 alkoxy with an attached
isothiureido, Cl 6 alkoxy with an attached guanidino, C16 alkoxy with an attached
amidino, Cl 6 alkyl with an attached amino, C16 alkyl with an attached isothiureido,
Cl 6 alkyl with an attached guanidino, Cl 6 alkyl with an attached amidino,
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R is s~1~cted from the group collsi~Lil~g of H, OH, NH2, NO2 halogen, C1 6
alkoxy, Cl 6 fluorinated alkoxy, Cl 6 alkyl, Cl 6 alkyl with an attached amino, M-AA-
NH-, M-AA-O-,
wherein AA represents alanine, valine, leucine, isoleucine, proline, methionine,phenylalanine, tryptophan, glycine stèrine, threonine, cysteine, tyrosine, asparagine,
~11t~mine, aspartic acid, elut~mir acid, lysine, arginine, histidine, beta-alanine,
norleucine, norvaline, alpha-aminobutyric and epsilon-aminocaponic acid, citrulline,
h~ u~y~loline, ornithine and sarcosine,
wherein M represents H, lower alkanoyl having 1 to 6 carbons, carboxyalkanoyl,
Lyd~u~lkanoyl, amin-alkanoyl, benzene sulfonyl, tosyl, benzoyl, and lower alkyl
sulfonyl having 1 to 6 carbons,
Y is selected from the group consisting of H, halogen, trifluoromethyl, methyl,
OH and methoxy.
As a further alternative, the Class II Substituted Isocoumarins are represented
by structure (II) where
R is selected from the group consisting of -N-H-C(=NH)-NH2, -C(=NH)NH2,
Cl 6 alkyl with an attached amino, Cl 6 alkyl with an attached isothiureido,
Z is selected from the group consisting of Cl 6 alkoxy with an attached amino,
Cl 6 alkoxy with an attached isothiureido, Cl 6 alkoxy with an attached guanidino, Cl 6
alkoxy with an attached ~mitlino, Cl 6 alkyl with an attached amino, Cl 6 alkyl with an
attached guanidino, Cl 6 alkyl with an attached amidino,
Y is selected from the group consis~ing of H, halogen, trifluoromethyl, methyl,
OH and methoxy.
The following compounds are representative of the Class II Substituted
Isocoum~rinc
3-(3-aminopropoxy)isocoumarin,
3-(3-aminopropoxy)-4-chloroisocoumarin,
3-(2-isothiureidoethoxy)-4-chloroisocoumarin,
3 -(3-isothiureidopropoxy)-4-chloroisocoumarin,
7-amino-3-(3-isothiureidopropoxy)-4-chloroisocoumarin,
7-guanidino-3-methoxyisocoumarin,
7-guanidino-3-methoxy-4-chloroisocoumarin,
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~1~81Z~
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7-guanidino-3-ethoxyisocoumarin,
7-gu~ni~lino-3-ethoxy-4-chloroisocoumarin,
7 gu~ni~ino-3-(2-phenylethoxy)isocoumarin,
7 gll~nirlino-3-(2-phenylethoxy)-4-chloroisocoumarin.
Still another class of susbstituted heterocyclic compounds useful in the presentinvention is referred to herein as the "Class III Heterocyclic Compounds" and have the
following ~lu~lu-~ll formula:
~ z~
(III) l~9~ X
wherein
Z is selected from the group consisting of CO, SO, SO2, CCI and CF,
Y is selected from the group consisting of O, S and NH,
X is selected from the group consi~.li"g of N and CH, and
R is selected from the group col~sis~ g of Cl 6 alkyl (such as methyl, ethyl andpropyl), C14 alkyl containing a phenyl (such as benzyl), and Cl 6 fluoroalkyl (such as
trifluoromethyl, pentafluoroethyl, and heptafluoroplo~,yl).
The Z group must be electrophilic since it interacts with the active site serineOH group of the serine protease. The R group must be uncharged and hydrophobic.
One or more of the carbons in the R group could be replaced by O, S, NH and other
such atomic groups as long as the R group maintains its hydrophobic character.
The following compounds are representative of the Class III Heterocyclic
Compounds:
2-trifluoromethyl-4H-3,1-be,-7~ .7il-e-4-one,
2-pentafluGroethyl-4H-3, l-ben7nY~7ine-4-one,
2-heptafluo~ opro~yl-4H-3, 1-benzoxazine-4-one,
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2-methyl-4H-3, 1-bel-7G ~ e-4-one,
2-propyl-4H-3,l-ben70~Y~7ine-4-one,
2-benzyl-4H-3,1-b~ n;.~ e4-one,
2-heptafluorol), o~yl-4-quinazolinone,
2-propyl-4-ql-in~7nlinone,
2-benzyl-4-q--in~7c-1inone,
2-(C6HsCCl2)-4-chloroquin~701ine, and
2-propyl-4-chloroquin~7- 1ine.
The Class III Heterocyclic Compounds are ~ic-~lnsed in Powers et al., U.S. Patent No.
4,847,202, the ~lic-losllre of which is hereby incorporated by reference.
Other s~bstinlted heterocyclic compounds have been prepared earlier for other
)OSeS, such as 3-chloroisocoumarin, Davies and Poole, J. Chem. Soc., pp. 1616-1629
(1928); 3-chloro and 3,4-dichloroisocoumarin, Mi!evskaya, et al., Zltur. Org. K~lim.
9:2145-2149 (1973); 3-methyl and 4-carboxy-3-methylisocoumarin, Tirodkar and
Usgaonkar, Ind J. Chem 7:1114-1116 (1969); 7-nitro and 7-aminoisocoumarin,
Choksey and Usgaonkar, I)l~ J. C~tem. 14B:596-598 (1976). The disclosures of all of
the preceding articles are hereby incorporated by reference. These other substituted
isocoumarins are also believed to exhibit Calpain inhibitory activity when used in
accordance with the present invention.
Still other substituted isocoumarins which have been prepared recently for
inhibition of serine proteases are 3-chloroisocoumarin, Harper, et al., J. A. Chem. Soc.
105:6518-6520 (1983); 3,4-dichloroisocoumarin, Harper, et al., Biochemistry 24:1831-
1841 (1985); 3-alkoxy-7-amino-4-chloroisocoumarin, Harper and Powers, J. Am. Clte~7l.
Soc 106:7618-7619 (1984), Harper and Powers, Bioc/temistry 24:7200-7213 (1983);
additional substituted isocoumarins with basic groups (aminoalkoxy, guanidino orisothiureidoalkoxy), Kam, et al., Biochemistry 27:2547-2557 (1988); 7-substituted 3-
alkoxy-4-chloroisocoumarins, Powers, et al., J. Cell Biochem. 39:33-46 (1989) and
Powers, et al.. Biochemistry 29:3108-3118 (1990). The disclosures of all of the
preceding articles are hereby incorporated by reference. We believe that the foregoing
compounds. which exhibit serine protease inhibitory activity, also exhibit Calpain
inhibitory activity when used in accordance with the present invention. All of the
foregoing isocoumarin compounds, including the Class I and II Substituted
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Isocoumarins, the Class III Substituted Heterocyclic Compounds and the other
s--hstituted heterocyclic compounds useful in the practice of the present invention shall
be referred to collectively hereinafter as the "Substituted Heterocyclic Compounds."
The term "Substituted Heterocyclic Compound" shall be used to refer to any particular
species of these compounds.
The preparation of the various Substituted Heterocyclic Compounds is
illustrated by Examples SHC1-SHC9.
EXAMPLE SHCl
Preparation of 7-(phenylcarbamoylamino)-4-chloroisocoumarin was synthesized
as previously described in Powers, et al., Biochemistry, 29:3108-3118 (1990). This
compound (0.32 g, 1 mmole) was mixed with phenyl isocyanate (0.12g, 1 mmole) in 5
ml of THF and the reaction mixture was stirred at r.t. overnight. The product
7-(phenylcalba.,.oylamino)-4-chloro-3-(2-bromoethoxy)isocoumarin precipitated out,
yield 40%, m.p. 215-217C, mass ~.e~ ... m/e = 437.9 (M+)> Anal. Calc. for
C18Hl4N2O4ClBr: C, 49.40; H, 3.22; N, 6.40; Cl, 8.10. Found: C,49.48; H, 3.25; N,6.34;
Cl, 8.12. The phenylcarbamoylamino compound (0.1 g, 0.23 mmole) was heated with
0.02 g of thiourea (0.26 mmole) in 10 ml of THF at 70C overnight. The final product
precipitated out, yield 0.04 g, 36%, m.p. 161-163C (dec.), mass spectrum (FAB+) m/e
= 433 (M-Br). Anal. Calc. for C19H18N4O4ClBrS:0.25 THF: C, 45.12; H, 3.86; N,
10.53; Cl, 6.67. Found: C, 44.83; H, 3.92; N, 10.12; Cl, 6.41.
7-(Ethylcarbamoylamino)-4-chloro-3-(2-isothiureidoethoxy)isocoumarin,
7-(t-butylcarbamoylamino)-4-chloro-3-(2-isothiureidoethoxy)isocoumarin,
7-(benzylthiocarbamoylamino)-4-chloro-3-(2-isothiureidoethoxy)isocoumarin, 7-
(ethylthiocarbamoylamino)-4-chloro-3-(2-isothiureidoethoxy)isocoumarin, 7-(4-
fluorobenzyl) thiocarbamoylamino-4-chloro-3-(2-isothiureidoethoxy) isocoumarin, and 7-
(2,5-dimethylbenzyl) thiocarbamoylamino-4-chloro-3-(2-isothiureidoethoxy) isocoumarin
can be prepared by the same procedure.
EXAMPLE SHC2
Preparation of 7-(acetylamino)-4-chloro-3-(3-isothiureidopropoxy) isocoumarin:
7-Amino-3(3-bromopropoxy)-4-chloroisocoumarin was synthesized as previously
described (Kam, et al., supra). This compound (0.33 g, 1 mmole) was heated with 0.15
g of acetic anhydride (1.5 mmole) in 20 ml of dry THF. After a few minutes, a yellow
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solid lule~;luit~ted out. After 3 hrs, the solution was concentrated to 5 ml, and the solid
was filtered to give 0.37 g of 7-(acetylamino)-4-chloro-3-(3-bromopropoxy) isocoumarin,
m.p. 170-172C; mass spectrum: m/e = 375 (M+). The acetylated isocoumarin (0.15
g, 0.4 mmole) was treated with thiourea (0.036 g, 0.47 mmole) to give 0.9 g of the final
product, (yield 50%), m.p. 180-181C, mass spectrum m/e = 370 (M+-Br). Anal. Calc.
for C15H17N3O4ClBrS: C, 39.97; H, 3.80; N, 9.32; Cl 7.87. Found: C, 39.86; H 3.83; N,
9.29; a, 7.85.
7-trifluoroacetylamino-4-chloro-3-(3-isothiureidopropoxy) isocoumarin, 7-
heptafluoroblJlylu~lamino-4-chloro-3-(3-isothiureidopropoxy) isocoumarin, 7-
succinylaminû-4-chloro-3-(3-isothiureidopropoxy) isocoumarin, and 7-(o-phthalyl)amino-
4-chloro-3-(3-isothiureidopropoxy) isocoumarin can be prepared by the same procedure.
EXAMPLE SHC3
Preparation of 7-(benzylcarbamoylamino)-4-chloro-3-(3-isothiureidopropoxy)
isocoumarin:
7-(benzylcarbamoylamino)-4-chloro-3(3-bromopropoxy) isocoumarin was
prepared from the reaction of benzyl isocyanate with 7-amino-4-chloro-3-(3-
bromoplopuAy) isocoumarin as described above, m.p. 188-189C, mass spectrum: m/e= 359 (M+ -benzyl). The final product was obtained from the reaction of 7-
(benzylcarbamoylamino)-4-chloro-3-(3-bromopropoxy) isocoumarin with thiourea as
described above (yield 74%), m.p. 165-166C; mass spectrum (FAB+) m/e = 461
(M+-Br). Anal. Calc. for C21H22N4O4ClBrS:0.75 THF: C, 48.36; H, 4.70; N, 9.40; Cl,
6.56. Found: C, 48.13; H, 4.87; N, 9.65; Cl, 6.15.
EXAMPLE SHC4
Preparation of 7-(phenylacetylamino)-4-chloro-3-(2-isothiureidoethûxy)
isocoumarin:
7-Amino-4-chloro-3-(2-bromoethoxy) isocoumarin (0.15 g, 0.47 mmole) was first
mixed with phenylacetyl chloride (0.09 g, 0.55 mmole) in 10 ml of THF, triethylamine
(0.05 g, 0.47 mmole) was then added and the reaction mixture was stirred at r.t.overnight. After Et3N HCl salt was removed by filtration, the product 7-
(phenylacetylamino)-4-chlorû-3-(2-bromoethoxy) isocoumarin was crystallized fromTHF and Pet. ether (yield, 73%), m.p. 165-169C; mass spectrum; m/e = 436.7 (M+).
The phenylacetyamino derivative (0.1 g) was heated with thiourea (0.02 g) to give the
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product 0.05 g (yield, 40%), m.p. 115-120C; mass spectrum (FAB+) m/e = 432 (M+
-Br). Anal. Calc. for C20H1gN3O4ClBrS 0.5 H2O: C 45.99; H, 3.83; N, 8.05; Cl, 6.80.
Found: C, 46.09; H, 4.17; N, 8.02; Cl, 6.79.
EXAMPLE SHC5
Preparation of 7-(R-a-methylbenzylcarbamoylamino)-4-chloro-3-(2-
isothiureidoethoxy) isocoumarin:
7-(R-a-methylbenzylcdlba,.,oylamino)-4-chloro-3-(2-bromoethoxy) isocoumarin
was synthesized in the same manner as described above, m.p. 183-185C; mass
spectrum m/e = 464 (M+). This compound (0.1 g) reacted with thiourea (0.02 g)
under the same condition described above to form the final product 7-(R-a-
methylbenzylcarbamoylamino)-4-chloro-3-(2-isothiureidoethoxy) isocoumarin (0.078 g),
m.p. 143-150C; mass spe~,llu,., (FAB+) m/e = 461 (M+ -Br). Anal. Calc. for
C21H22N4O4ClBrS 0.5H2O: C, 45.75; H, 4.35; N, 10.17; Cl, 6.44. Found: C, 44.95; H,
4.31; N, 10.02; Cl, 6.36.
EXAMPLE SHC6
Preparation of 7-(D-phenylalanylamino)-4-chloro-3(2-isothiureidoethoxy)
icocou~ rin:
Boc-D-Phe (0.33 g, 1.2 mmole) reacted with 1,3-dicyclohexylcarbodiimide (0.13
g, 0.6 mmole) in 10 ml THF at 0C for 1 hour to form the symmetric anhydride, and
then 7-amino-4-chloro-3(2-bromoethoxy) isocoumarin (0.2g, 0.6 mmole) was added.
The reaction was stirred at r.t. overnight and the precipitate 7-(Boc-D-Phe-amino)-4-
chloro-3-(2-bromoethoxy) isocoumarin was formed (0.29 g, 71%). TLC one spot, m.p.
180-182C; mass spectrum m/3 = 566(M+). Anal. Calc. for C25H26N2O6ClBr: C,
53.07; H, 4.63; N, 4.95; Cl 6.27. Found: C, 53.25: H, 4.66; N, 4.87; Cl, 6.24. Boc-D-Phe
compound (0.2 g, 0.35 mmole) was reacted with thiourea (0.027 g, 0.35 mmole) in the
same manner to give 7-(Boc-D-phenylalanylamino)-4-chloro-3-(2-isothiureidoethoxy)
isocoumarin (0.14 g), yield 62%, mass spectrum (FAB+) m/e = 561 (M+ -Br). This
compound (0.1 g) was dissolved in 3 ml of THF at 0C and then the solvent was
evaporated to dryness. The final product precipitated out after addition of ether, one
spot on TLC (CH3CN:H20:AcOH = 8:1:1); mass spectrum (FAB+) m/e = 462 (M+ -
Br -CF3COO).
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7-Boc-alanylamino-4-chloro-3-(2-isothiureidoethoxy) isocoumarin, 7-
benzoylamino-Ala-4-chloro-3(2-isothiureidoethoxy) isocoumarin, 7-benzoylamino-Phe-4-
chloro-3-(2-isothiureidoethoxy) isocoumarin and 7-Boc-valylamino-4-chloro-3-(2-
isothiur~ oe~hm~y) isocoumarin can be prepared by the same procedure.
S EXAMPLE SHC7
Preparation of 7-(Boc-alanylalanylamino)-4-chloro-3-(2-isothiureidoethoxy)
isocoumarin:
7-(Boc-alanylalanylamino)-4-chloro-3-(2-bromoethoxy) isocoumarin was
syntheci7ed in the same manner, m.p. 147-151C; mass spectrum m/e = 561 (M+).
Anal. Calc: C, 47.12: H, 4.85. Found: C, 47.18; H, 4.87. This compound (0.2 g) was
reacted with thiourea (0.03 g) by the same procedure to form 7-(Boc-
alanylalanylamino)-4-chloro-3-(2-isothiureidoethoxy) isocoumarin (0.04 g), mass
spectrum m/e = 556 (M+ -Br).
7-(Alanylalanylamino)-4-chloro-3(2-isothiureidoethoxy) isocoumarin was
prepared by de~ ing of Boc-Ala-Ala-NH-CiTEtOlC with trifluoroacetic acid, mass
spectrum (FAB+) m/e = 456 (M+ -Br -CF3COO).
EXAMPLE SHC8
Preparation of 7-(phenylthiocarbamoylamino)-4-chloro-3-(2-isothiureidoethoxy)
isocoumarin:
7-(Phenylthiocarbamoylamino)-4-chloro-3-(2-bromoethoxy) isocoumarin was
prepared from the reaction of phenyl isothiocyanate with 7-amino-4-chloro-3-(2-
bromoethoxy) isocoumarin, yield 59%, m.p. 157-158C; mass spectrum m/e = 361 (M+-PhNH+1). Anal. Calc.: C, 48.36; H, 3.39. Found: C, 48.26; H, 3.40. The bromoethoxy
compound was then reacted with thiourea by the same procedure to give the final
product, yield 32%; mass spectrum (FAB+) m/e 449 (M+ -Br).
EXAMPLE SHC9
Preparation of 7-(m-carboxyphenylthiocarbamoylamino)-4-chloro-3-(2-
bromoethoxy) isocoumarin was prepared from the reaction of m-carboxyphenyl
isothiocyanate with 7-amino-4-chloro-3-(2-bromoethoxy) isocoumarin, yield 64%, m.p.
157-158C; mass spectrum m/e 361 (M+ -(COOH)PhNH+-Br).
7-(3-Fluorobenzoyl)amino-4-chloro-3-(2-isothiureidoethoxy) isocoumarin, 7-(3-
nitrobenzoyl) amino-4-chloro-3-(2-isothiureidoethoxy) isocoumarin, 7-
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diphenylacetylamino-4-chloro-3-(2-isothiureidoethoxy) isocoumarin, 7-
Ji~hl~ylp~opionylamino-4-chloro-3-(2-isothiureidoethoxy) isocoumarin, 7-(p-
tnlllen~sulfonyl) amino-4-chloro-3-(2-isothiureidoethoxy) isocoumarin, and 7-(a-toluenesulfonyl) amino-4-chloro-3-(2-isothiureidoethoxy) isocoumarin can be prepared
from the reaction of co~ onding 7-substit~lted-4-chloro-3-(2-bromoethoxy)
iCocoum~rin with thiourea as described above. 7-substituted-4-chloro-3-(2-
bromoethoxy) isocoumarin can be synthesized by reacting 7-amino-4-chloro-3-(2-
bromoethoxy) isocol-lllarin with appropliate acid chloride or sulfonyl chloride in the
presence of Et3N.
7-Ethoxycarbonylamino-4-chloro-3-(2-isothiureidoethoxy) isocoumarin, 7-
benzyl~ dll,ol.~lamino-4-chloro-3-(2-isothiureidoethoxy) isocoumarin, and 7-
phenoxycarbonylamino-4-chloro-3-(2-isothiureidoethoxy) isocoumarin can be prepared
from the reaction of 7-substituted-4-chloro-3-(2-bromoethoxy) isocoumarin with
thiourea. 7-Ethoxycarbonylamino-4-chloro-3-(2-bromoethoxy) isocoumarin, 7-
benzyloxycarbonylamino-4-chloro-3-(2-bromoethoxy) isocoumarin and 7-
phenoxycarbonylamino-4-chloro-3-(2-bromoethoxy) isocoumarin can be synthesized by
reacting 7-amino-4-chloro-3-(2-bromoethoxy) isocoumarin with the corresponding
chloroformate.
C. ~kl' l lL~E KETO-COMPOUNDS
Peptide a-ketoesters, peptide a-ketoacids, and peptide a-ketoamides are
transition state analog inhibitors for serine proteases and cysteine proteases. While
these ~ cses of compounds are chemically distinguishable, for simplicity, all of these
compounds will be referred to collectively herein as the "Peptide Keto-Compounds".
The interactions of peptides with serine and cysteine proteases are designated
herein using the nomenclature of Schechter and Berger, Bioclten~ Biopllys. Res.
Commun., 27:157-162 (1967), incorporated herein by reference. The individual amino
acid residues of a substrate or inhibitor are designated P1, P2, etc. and the
corresponding subsites of the enzyme are designated S1, S2, etc. The scissile bond of
the substrate is P1-P1'. The primary recognition site of serine proteases is S1. The
most important recognition subsites of cysteine proteases are S1 and S2. There are
additional recognition sites at the prime subsites such as S1' and S2'.
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Amino acid residues and hlnr~ing groups are designated using standard
abbreviations using nomenrl~tllre rules presented in J. BioL Chem., 260:14-42 (1985),
incorporated herein by reference. An amino acid residue (AA) in a peptide or
inhihitor structure refers to the part structure -NH-CHR1-CO-, where R1 is the side
S chain of the amino acid AA. A peptide a-ketoPcter residue would be deci~n~ed
-AA-CO-OR which rc~.le~.e..ts the part structure -NH-CHRl-CO-CO-OR. Thus, the
ethyl ketoester derived from benzoyl alanine would be designated Bz-Ala-CO-OEt
which represents C6HsCO-NH-CHMe-CO-CO-OEt. Likewise, peptide ketoacid
residues would be clPci~n~ted -AA-CO-OH. Further, peptide ketoamide residues aredecigr ~ted -AA-CO-NH-R. Thus, the ethyl keto amide derived from Z-Leu-Phe-OH
would be designated Z-Leu-Phe-CO-NH-Et which represents C6H5CH2OCO-NH-
CH(CH2CHMe2)-CO-NH-CH(CH2Ph)-CO-CO-NH-Et.
Peptide a-kPtoestPrs containing amino acid residues with hydrophobic side
chain at the P1 site have also been found to be excellent inhibitors of several cysteine
proteases inrlurline papain, cathepsin B and calpain. Calpains can be inhibited by
peptide inhibitors having several different active groups. Structure-activity relationships
with the commercially available i~t vitro inhibitors of Calpain, such as peptide aldehydes,
have revealed that C~lp~inc strongly prefer Leu or Val in the P2 position. Theseenzymes are inhihited by inhibitors having a wide variety of amino acids in the P1
position, but are generally more effectively inhibited by inhibitors having amino acids
with nonpolar or hydrophobic side chains in the P1 position. Thus, we have discovered
that another particular class of compounds exhibiting Calpain inhibitory activity, when
used in accordance with the present invention, are the Peptide Keto-Compounds.
These are compounds of the general structure:
o
M-(aa)n-C-Q-R
or a pharm~reutir~lly acceptable salt, wherein:
M represents NHi-CO-, NH2-CS-, NH2-SO2-, X-NH-CO-, X-NH-CS-,
X-NH-SO2-, X-CO-, X-CS-, X-SO2-, X-O-CO-, or X-O-CS-, H, acetyl,
carbobenzoxy, succinyl, methyloxysuccinyl, butyloxycarbonyl;
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X is selected from the group consi~ling of C1b alkyl, C1b fluoroalkyl,
Clb alkyl ~ul,~l il uted with J, Clb fluoroalkyl ~ub~liluled with J, 1-admantyl,9-fluorenyl, phenyl, phenyl substituted with K, phenyl ~1icubstituted with K,
phenyl l~;~ub~ uled with K, naphthyl, naphthyl substituted with K, naphthyl
~ b~ ed with K, naphthyl tricubstituted with K, Clb alkyl with an attached
phenyl group, Clb alkyl with two attached phenyl groups, Clb alkyl with an
attached phenyl group substituted with K, and C1b alkyl with two attached
phenyl groups ~ul,~ led with K;
J is selected from the group consi~ling of halogen, COOH, OH, CN
NO2, NH2, C1b alkoxy, C1 6 alkylamine, C16 dialkylamine, C1b alkyl-O-CO-,
Clb alkyl-O-CO-NH, and C1 6 alkyl-S-;
K is selected from the group consi~ling of halogen, C1 6 alkyl, C1-6
perfluoroalkyl, Cl 6 alkoxy, NO2, CN, OH, CO2H, amino, C16 alkylamino, C2 l2
dialkylamino, Clb acyl, and Clb alkoxy-CO-, and C16 alkyl-S-;
aa Ic~Jle~,c.lts a blocked or unblocked amino acid of the L or D
configuration, preferably selected from the group consisting of: alanine, valine,
leucine, isoleucine, proline, methionine, methionine sulfoxide, phenylalanine,
tryptophan, glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine,
aspartic acid, glutamic acid, lysine, arginine, histidine, phenylglycine, beta-
alanine, norleucine (nle), norvaline (nva), alpha-aminobutyric acid (abu),
epsilon-aminocaproic acid, citrulline, hydroxyproline, homoarginine, ornithine or
sarcosine;
n is a number from 1 to 20;
Q is O or NH,
R represents H, C1b alkyl, C1 6 fluoroalkyl, Cl 6 chloroalkyl, benzyl, C1b
alkyl s~lbsliluled with phenyl, Cl 6 alkyl with an attached phenyl group
substituted with K.
Thus, the Peptide Keto-Compounds can be divided into the Peptide Ketoesters,
Peptide Ketoacids and Peptide Ketoamides. Each of the compounds can also be
classified based on the number of amino acids contained within the compound, such as
an amino acid peptide, dipeptide, tripeptide, tetrapeptide, pentapeptide and so on.
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We have found certain subrl~ccloc of Peptide a-Ketoester compounds to be
particularly useful as Calpain Inhibitors when used in accordance with the present
invention. These sub~l~cces are referred to herein as the Dipeptide a-Ketoesters(Subclass A), the Dipeptide a-Ketoesters (Subclass B), the Tripeptide a-Ketoesters
(Sub~l~cc A), the Tripeptide a-Ketoesters (Subclass B), the Tetrapeptide a-Ketoesters
and the Arnino Acid Peptide a-Ketoesters. All of these subclasses are considered to be
to be within the class of Peptide Keto-Compounds.
The Dipeptide a-K~toest~rs (Subclass A) are compounds of the formula:
Ml-AA2-AAl-cO-O-Rl
or a pharm~ceutir~lly acceptable salt, wherein
Ml rc~rese.lts H, NH2-CO-, NH2-CS-, NH2-S02-, X-NH-CO-, X2N-CO-,
X-NH-CS-, X2N-CS-, X-NH-SO2-, X2N-SO2-, X-CO-, X-CS-, X-SO2-, X-O-CO-, or X-
O-CS-;
X is selected from the group consisting of C1 10 alkyl, C1 10 fluoroalkyl, C1 10alkyl ~ub~ uled with J, Cl 10 fiuoroalkyl substituted with J, 1-admantyl, 9-fluorenyl,
phenyl, phenyl substituted with K, phenyl disubstituted with K, phenyl trisubstituted
with K, naphthyl, naphthyl substituted with K, naphthyl disubstituted with K, naphthyl
tricubstituted with K, C1 10 alkyl with an attached phenyl group, C1 l0 alkyl with two
attached phenyl groups, C1 10 alkyl with an attached phenyl group substituted with K,
and C1 10 alkyl with two attached phenyl groups substituted with K, C1 10 alkyl with an
attached phenoxy group, and C1 10 alkyl with an attached phenoxy group substituted
with K on the phenoxy group;
J is selected from the group consisting of halogen, COOH, OH, CN, NO2, NH2,
C1 10 alkoxy, C1 10 alkylamine, C2 12 dialkylamine, C1 10 alkyl-O-CO-? C1 10 alky
NH-, and C1 10 alkyl-S-;
K is selected from the group consisting of halogen, C1 10 alkyl, C1 10
perfluoroalkyl, Cl 10 alkoxy, NO2, CN, OH, C02H, amino, Cl 10 alkylamino, C2 12
dialkylamino~ C1-C10 acyl, and C1 10 alkoxy-CO, and C1 10 alkyl-S-;
AAl is a side chain blocked or unblocked amino acid with the L configuration,
D configuration, or no chirality at the a-carbon selected from the group consisting of
alanine, valine, leucine, isoleucine, proline, methionine, methionine sulfoxide,phenylalanine, tryptophan, serine, threonine, cysteine, tyrosine, asparagine, glutamine,
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aspartic acid, ~ut~mi~ acid, lysine, arginine, histidine, phenylglycine, beta-alanine,
norleucine, norvaline, alpha-aminobutyric acid, epsilon-aminocaproic acid, citrulline,
l,~d.u~,ûline, ornithine, homoarginine, sarcosine, indoline 2-carboxylic acid, 2-
azetidine~d,l,u~Lc acid, pi~ecoli~ic acid (2-piperidine carboxylic acid), O-methylserine,
O-~thylse.il~e, S-methylcysteine, S-ethylcysteine, S-benzylcysteine, NH2-
CH(CH2CHEt2)-COOH, alpha-aminoheptanoic acid, NH2-CH(CH2-1-napthyl)-COOH,
NH2-CH(CH2-2-napthyl)-COOH, NH2-CH(CH2-cyclohexyl)-COOH, NH2-CH(CH2-
cydopentyl)-COOH, NH2-CH(CH2-cyclobutyl)-COOH, NH2-CH(CH2-cyclopropyl)-
COOH, trifluoroleucine, and hexafluoroleucine;
AA2 is a side chain blocked or unblocked amino acid with the L configuration,
D configuration, or no chirality at the a-carbon selected from the group consisting of
leucine, isoleucine, proline, methionine, methionine sulfoxide, phenylalanine,
tryptophan, glycine, serine, threonine, cysteine, tyrosine, asparagine, glut~mine, aspartic
acid, glut~mi~ acid, lysine, arginine, histidine, phenylglycine, beta-alanine, norleucine,
norvaline, alpha-aminobutyric acid, epsilon-aminocaproic acid, citrulline,
hydroxyproline, ornithine, homoarginine, sarcosine, indoline 2-carboxylic acid, 2-
azetidinecarboxylic acid, pipecolinic acid (2-piperidine carboxylic acid), O-methylserine,
O-ethylserine, S-methylcysteine, S-ethylcysteine, S-benzylcysteine, NH2-
CH(CH2CHEt2)-COOH, alpha-aminoheptanoic acid, NH2-CH(CH2-1-napthyl)-COOH,
NH2-CH(CH2-2-napthyl)-COOH, NH2-CH(CH2-cyclohexyl)-COOH, NH2-CH(CH2-
cyclopentyl)-COOH, NH2-CH(CH2-cyclobutyl)-COOH, NH2-CH(CH2-cyclopropyl)-
COOH, trifluoroleucine, and hexafluoroleucine;
R1 is selected from the group consisting of H, C1 20 alkyl, Cl 20 alkyl with a
phenyl group attached to the C1 20 alkyl, and C1 20 alkyl with an attached phenyl group
substituted with K.
The Dipeptide a-Ketoesters (Subclass B) are compounds of the structure:
Ml-AA-NH-CHR2-CO-CO-O-R
or a pharmaceutically acceptable salt, wherein
M1 represents H, NH2-CO-, NH2-CS-, NH2-SO2-, X-NH-CO-, X2N-CO-,
X-NH-CS-, X2N-CS-, X-NH-SO2-, X2N-SO2-, X-CO-, X-CS-, X-SO2-, X-O-CO-, or X-
O-CS-;
WO 94/00095 ~13 ~12 ~ `
X is selected from the group consisting of C1 10 alkyl, C1 10 fluoroalkyl, C1 10alkyl s.~ u~ed with J, Cl 10 fluoroalkyl substituted with J, 1-admantyl, 9-fluorenyl,
phenyL phenyl ~ ed with K, phenyl ~iicubstitllted with K, phenyl trisubstituted
with K, naphthyl, naphthyl s~lb~ ted with K, naphthyl r~icubstituted with K, naphthyl
S ~ b ,~ .ted with K, Cl 10 alkyl with an attached phenyl group, Cl 10 alkyl with two
attached phenyl groups, C1 10 alkyl with an attached phenyl group substituted with K,
and C1 10 alkyl with two attached phenyl groups substituted with K, C1 10 alkyl with an
attached phenoxy group, and C1 10 alkyl with an attached phenoxy group substituted
with K on the phenoxy group;
J is selected from the group concictine of halogen, COOH, OH, CN, NO2, NH2~
C1 10 alkoxy, C1 10 alkylamine, C2 12 dialkylamine, C1 10 alkyl-O-CO-, C1 10 alkyl-O-CO-
NH-, and C1 10 alkyl-S-;
K is selected from the group consisting of halogen, C1 10 alkyl, C1 10
perfluoroalkyl, C1 10 alkoxy, NO2, CN, OH, CO2H, amino, C1 10 alkylamino, C2 12
dialkylamino, C1-C10 acyl, and C1 l0 alkoxy-CO-, and C1 l0 alkyl-S-;
AA is a side chain blocked or unblocked amino acid with the L configuration, D
configuration, or no chirality at the a-carbon selected from the group consisting of
alanine, valine, leucine, isoleucine, proline, methionine, methionine sulfoxide,phenylalanine, tryptophan, glycine, serine, threonine, cysteine, tyrosine, asparagine,
~ut~mine, aspartic acid, glllt~mi~ acid, lysine, arginine, histidine, phenylglycine, beta-
alanine, norleucine, norvaline, alpha-aminobutyric acid, epsilon-aminocaproic acid,
citrulline, hydroxyproline, omithine, homoarginine, sarcosine, indoline 2-carboxylic acid,
2-azetidinecarboxylic acid, pipecolinic acid (2-piperidine carboxylic acid), O-
methylserine, O-ethylserine, S-methylcysteine, S-ethylcysteine, S-benzylcysteine, NH2-
CH(CH2CHEt2)-COOH, alpha-aminoheptanoic acid, NH2-CH(CH2-1-napthyl)-COOH,
NH2-CH(CH2-2-napthyl)-COOH, NH2-CH(CH2-cyclohexyl)-COOH, NH2-CH(CH2-
cyclopentyl)-COOH, NH2-CH(CH2-cyclobutyl)-COOH, NH2-CH(CH2-cyclopropyl)-
COOH, trifluoroleucine, and hexafluoroleucine;
R2 r~lesents C1 8 branched and unbranched alkyl, C1 8 branched and
unbranched cyclized alkyl, or C18 branched and unbranched fluoroalkyl;
~1381æ~
WO 94/00095 PCI /US93/06143
R is selected from the group consisting of H, Cl 20 alkyl, C1 20 alkyl with a
phenyl group atta~ hed to the Cl 20 alkyl, and Cl 20 alkyl with an attached phenyl group
substituted with K
The Tripeptide ~-K~toesters (Su~l~cc A) are compounds of the structure:
SM3-AA-AA-AA-CO-O-R
or a pharm~reuti~lly acceptable salt, wherein
M3 represe.~ts H, NH2-CO-, NH2-CS-, NH2-S02-, X-NH-CO-, X2N-CO-,
X-NH-CS-, X2N-CS-, X-NH-SO2-, X2N-SO2-, X-CO-, X-CS-, X-SO2-, T-O-CO-, or X-
O-CS-;
X is selected from the group consi~ling of C1 10 alkyl, C1 10 fluoroalkyl, C1 10alkyl ~lb~ ed with J, C1 10 fluoroalkyl substituted with J, 1-admantyl, 9-fluorenyl,
phenyL phenyl s~ ;n~ted with K, phenyl ~ u~ ed with K, phenyl tric~ stitl-ted
with K, naphthyl, naphthyl suhstitl-ted with K, naphthyl disubstituted with K, naphthyl
b~l;luled with K, Cl 10 alkyl with an attached phenyl group, C1 l0 alkyl with two
attached phenyl groups, C1 10 alkyl with an attached phenyl group substituted with K,
and C1 10 alkyl with two attached phenyl groups substisuted with K, C1 l0 alkyl with an
attached phenoxy group, and C1 l0 alkyl with an attached phenoxy group substituted
with K on the phenoxy group;
T is selected from the group consisting of C1 l0 alkyl, Cl 10 fluoroalkyl, C1 l0alkyl sul,~ ed with J, Cl 10 fluoroalkyl substituted with J, 1-admantyl, 9-fluorenyl,
phenyl, phenyl substituted with K, phenyl disubstituted with K, phenyl trisubstituted
with K, naphthyl, naphthyl substituted with K, naphthyl ~ lb~ ted with K, naphthyl
ub~ uled with K, C2 10 alkyl with an attached phenyl group, C1 10 alkyl with twoattached phenyl groups, C1 10 alkyl with an attached phenyl group substituted with K,
and C1 10 alkyl with two attached phenyl groups substituted with K;
J is selected from the group consi~ g of halogen, COOH, OH, CN, NO2, NH2,
Cl 10 alkoxy, Cl 10 allylamine, C2 12 dialkylamine, Cl 10 alkyl-O-CO-, C110 alkyl-O-CO
NH-, and C1 10 alkyl-S-;
K is selected from the group consisting of halogen, C1 l0 alkyl, C1 10
perfluoroalkyl, C1 10 alkoxy, NO2, CN, OH, CO2H, amino, C1 10 alkylamino, C2 12
dialkylamino, C1-Cl0 acyl, and C1 10 alkoxy-CO-, and C1 10 alkyl-S-;
WO 94/00095 PCI/US93/06143
hl38 12~
-39-
AA is a side chain blocked or unblocked amino acid with the L configuration, D
configuration, or no chirality at the -carbon selected from the group consisting of
alanine, valine, leucine, isoleucine, proline, methionine, methionine sulfoxide,phenylalanine, tryptophan, glycine, serine, threonine, cysteine, tyrosine, asparagine,
S ~t~mine, aspartic acid, ghlt~mir acid, lysine, arginine, histidine, phenylglycine, beta-
alanine, n-lrleurine, norvaline, alpha-aminobutyric acid, epsilon-aminocaproic acid,
citrulline, hydluA~loline, ornithine, homoarginine, sarcosine, indoline 2-carboxylic acid,
2-azetidinecarboxylic acid, pipecolinic acid (2-piperidine carboxylic acid), O-
mclh~ls~ e~ O-ethylserine, S-methylcysteine, S-ethylcysteine, S-benzylcysteine, NH2-
CH(CH2CHEt2)-COOH, alpha-aminoheptanoic acid, NH2-CH(CH2-l-napthyl)-COOH,
NH2-CH(CH2-2-napthyl)-COOH, NH2-CH(CH2-cyclohexyl)-COOH, NH2-CH(CH2-
cyclopentyl)-COOH, NH2-CH(CH2-cyclobutyl)-COOH, NH2-CH(CH2-cyclopropyl)-
COOH, tri_uoroleucine, and h~Y~fl~loroleucine;
R is selected from the group consisting of H, C2 20 alkyl, C1 20 alkyl with a
phenyl group att~r~lrd to the Cl 20 alkyl, and Cl 20 alkyl with an attached phenyl group
"~b,l;tu~ed with K.
The Tripeptide a-Ketoesters (Subclass B) are compounds of the structure:
M3-AA-AA-NH-CHR2-CO-CO-O-R
or a pharm~reutir~lly acceptable salt, wherein
M3 represents H, NH2-CO-, NH2-CS-, NH2-SO2-, X-NH-CO-, X2N-CO-,
X-NH-CS-, X2N-CS-, X-NH-SO2-, X2N-SO2-, X-CO-, X-CS-, X-SO2-, T-O-CO-, or X
O-CS-;
X is selected from the group consi~ling of C1 10 alkyl, Cl 10 fluoroalkyl, C1 10alkyl sukstituted with J, C1 10 _uoroalkyl substin-ted with J, 1-admantyl, 9-fluorenyl,
phenyL phenyl substituted with K, phenyl ~iicubstituted with K, phenyl trisubstituted
with K, naphthyl, naphthyl substituted with K, naphthyl disubstituted with K, naphthyl
tricllkstituted with K, C1 10 alkyl with an attached phenyl group, C1 10 alkyl with two
attached phenyl groups, C1 10 alkyl with an attached phenyl group substituted with K,
and C110 alkyl with two attached phenyl groups substituted with K, C1 10 alkyl with an
attached phenoxy group, and C1 l0 alkyl with an attached phenoxy group substituted
with K on the phenoxy group;
WO94/00095 Z1~ 8~ PCI/US93/06143
10-
T is s.o1ected from the group consi~ g of Cl 1o alkyl, Cl 1o fluoroalkyl, C1 10
aL~cyl substit~1ted with J, Cl 10 fluoroal'kyl substituted with J, 1-admantyl, 9-fluorenyl,
phenyl, phenyl substitl1ted with K, phenyl tlicubstituted with K, phenyl trisubstituted
with K, naphthyl, naphthyl substituted with K, naphthyl disubstituted with K, naphthyl
S l~ ;L~ d with K, C2 l0 alkyl with an attached phenyl group, Cl 10 alkyl with two
attached phenyl groups, C1 10 alkyl with an attached phenyl group substituted with K,
and Cl 10 alkyl with two ~tt~ ed phenyl groups ~ul,~ ted with K;
J is selected from the group consi~ling of halogen, COOH, OH, CN, NO2, NH2,
Cl 10 alkoxy, Cl 10 alkylamine, Cl 10 dialkylamine, Cl 10 alkyl-O-CO-, Cl 1o al'kyl-O-CO-
NH-, and C1 10 alkyl-S-;
K is selected from the group consi~ling of halogen, C1 10 alkyl, C1 10
perfluoroalkyl, Cl 1o allcoxy, NO2, CN, OH, CO2H, amino, Cl 1o alkylamino, C2 l2dialkylamino, C1-Cl0 acyl, and C1 l0 alkoxy-CO-, and C1 l0 alkyl-S-;
AA is a side chain blocked or unblocked amino acid with the L configuration, D
configuration, or no chirality at the a-carbon selected from the group consisting of
alanine, valine, leucine, isoleucine, proline, methionine, methionine sulfoxide,phenylalanine, tryptophan, glycine, serine, threonine, cysteine, tyrosine, asparagine,
~mine' aspartic acid, glutamic acid, Iysine, arginine, histidine, phenylglycine, beta-
alanine, norleucine, norvaline, alpha-aminobutyric acid, epsilon-aminocaproic acid,
citrulline, hydroxyproline, ornithine, homoarginine, sarcosine, indoline 2-carboxylic acid,
2-azetidinecarboxylic acid, pipecolinic acid (2-piperidine carboxylic acid), O-
methylserine, O-ethylserine, S-methylcysteine, S-ethylcysteine, S-benzylcysteine, NH2-
CH(CH2CHEt2)-COOH, alpha-aminoheptanoic acid7 NH2-CH(CH2-1-napthyl)-COOH,
NH2-CH~CH2-2-napthyl)-COOH, NH2-CH(CH2-cyclohexyl)-COOH, NH2-CH(CH2-
cydopentyl)-COOH, NH2-CH(CH2-cyclobutyl)-COOH, NH2-CH(CH2-cyclopropyl)-
COOH, trifluoroleucine, and hexafluoroleucine;
R2 represcnts C1 8 branched and unbranched alkyl, C1 8 branched and
unbranched cyclized alkyl, or Cl 8 branched and unbranched fluoroal'kyl;
R is selected from the group consi~ling of H, C1 20 alkyl, C1 20 alkyl with a
phenyl group attached to the C1,20 alkyl, and C120 alkyl with an attached phenyl group
substituted with K.
~'O 94/00095 ` PCI`/US93/06143
- ~13812`4
-41-
The Tetrapeptide -Ketoesters are compounds of the structure:
M3-AA4-AA-AA-AA-CO-O-R
or a pharm~reuti~ ~lly acceptable salt, wherein
M3 rc~rese~)ts H, NH2-CO-, NH2-CS-, NH2-SO2-, X-NH-CO-, X2N-CO-,
X-NH-CS-, X2N-CS-, X-NH-SO2-, X2N-SO2-, X-CO-, X-CS-,~X-SO2-, T-O-CO-, or X-
O-CS-;
X is selected from the group corlcicting of C1 10 alkyl, C1 10 fluoroalkyl, C1 10
alkyl SU~ ed with J, Cl 10 fluoroalkyl substituted with J, 1-admantyl, 9-fluorenyl,
phenyl, phenyl ~ul.~ ted with K, phenyl disubstituted with K, ~henyl trisubstituted
with K, naphthyl, naphthyl substituted with K, naphthyl disubstituted with K, naphthyl
ubslit~-ted with K, Cl 10 alkyl with an attached phenyl group, Cl 10 alkyl with two
attached phenyl groups, Cl 10 alkyl with an attached phenyl group substituted with K,
and C1 10 alkyl with two attached phenyl groups substituted with K, C1 10 alkyl with an
attached phenoxy group, and C1 10 alkyl with an attached phenoxy group substituted
with K on the phenoxy group;
T is selected from the group consisting of C1 10 alkyl, C1 10 fluoroalkyl, C1 10alkyl substituted with J, C1 10 fluoroalkyl substituted with J, 1-admantyl, 9-fluorenyl,
phenyl, phenyl substituted with K, phenyl disubstituted with K, phenyl trisubstituted
with K, naphthyl, naphthyl substituted with K, naphthyl disubstituted with K, naphthyl
~ ul.~ lted with K, C2 l0 alkyl with an attached phenyl group, Cl 10 alkyl with two
attached phenyl groups. C1 10 alkyl with an attached phenyl group substituted with K,
and C1 10 alkyl with two attached phenyl groups substituted with K;
J is selected from the group consisting of halogen, COOH, OH, CN, NO2, NH2,
C1 10 alkoxy, C1 10 alkylamine, C2 12 dialkylamine~ C1 10 alkyl-O-CO-~ C1-10 alkyl O
NH-, and Cl 10 alkyl-S-;
K is selected from the group consisting of halogen, C1 10 alkyl, C1 10
perfluoroalkyl, C1 10 alkoxy, NO2, CN, OH, CO2H, amino, C1 10 alkylamino, C2 12
dialkylamino, C1-C10 acyl, and C1 10 alkoxy-CO-, and C1 10 alkyl-S-;
AA is a side chain blocked or unblocked amino acid with the L configuration, D
configuration, or no chirality at the a-carbon selected from the group consisting of
alanine, valine, leucine, isoleucine, proline, methionine, methionine sulfoxide,phenylalanine, tryptophan, glycine, serine, threonine, cysteine, tyrosine, asparagine,
WO 94/00095 PCI`/US93/06143
^~138~
-42-
glut~mine, aspartic acid, ~llt~mir acid, Iysine, arginine, histidine, phenylglycine, beta-
alanine, norleucine, norvaline, alpha-aminobutyric acid, epsilon-aminocaproic acid,
citrulline, hydroxyproline, ornithine, homoarginine, sarcosine, indoline 2-carboxylic acid,
2-azetidinecalbu,.ylic acid, pipecolinic acid (2-piperidine carboxylic acid), O-S mGlLyl~G~ e~ O-ethylserine, S-methylcysteine, S-ethylcysteine, S-benzylcysteine, NH2-
CH(CH2CHEt2)-COOH, alpha-aminoheptanoic acid, NH2-CH(CH2-1-napthyl)-COOH,
NH2-CH(CH2-2-napthyl)-COOH, NH2-CH(CH2-cyclohexyl)-COOH, NH2-CH(CH2-
cydopentyl)-COOH, NH2-CH(CH2-cydobutyl)-COOH, NH2-CH(CH2-cyclopropyl)-
COOH, trifluorr,l~urine, and hexafluoroleucine;;
AA4 is a side chain blocked or unblocked amino acid with the L configuration,
D configuration, or no chirality at the a-carbon selected from the group consisting of
leucine, isoleucine, methionine, methionine sulfoxide, phenylalanine, tryptophan,
glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid,
~llt~mic acid, Iysine, arginine, histidine, phenylglycine, beta-alanine, norleucine,
norvaline, alpha-aminobutyric acid, epsilon-aminocaproic acid, citrulline,
I,ydlu~ioline, ornithine, homoarginine, sarcosine, indoline 2-carboxylic acid, 2-
azetidinecarboxylic acid, pipecolinic acid (2-piperidine carboxylic acid), O-methylserine,
O-ethylserine, S-methylcysteine, S-ethylcysteine, S-benzylcysteine, NH2-
CH(CH2CHEt2)-COOH, alpha-aminoheptanoic acid, NH2-CH(CH2-l-napthyl)-COOH,
NH2-CH(CH2-2-napthyl)-COOH, NH2-CH(CH2-cyclohexyl)-COOH, NH2-CH(CH2-
cyclopentyl)-COOH, NH2-CH(CH2-cydobutyl)-COOH, NH2-CH(CH2-cyclopropyl)-
COOH, trifluoroleucine, and hexafluoroleucine;
R is selected from the group consisting of H, C1 20 alkyl, C1 20 alkyl with a
phenyl group attached to the C1 20 alkyl, and C120 alkyl with an attached phenyl group
s~ll,s~ ed with K.
The Amino Acid Peptide a-Ketoesters are compounds of the structure:
Ml-AA-CO-O-R
or a pharmaceutically acceptable salt, wherein
M1 represents H, NH2-CO-, NH2-CS-, NH2-SO2-, X-NH-CO-, X2N-CO-,
X-NH-CS-, X2N-CS-, X-NH-SO2-, X2N-SO2-, Y-CO-, X-CS-, X-SO2-, X-O-CO-, or X-
O-CS-;
WO 94/00095 PCI`/US93/06143
3 8 1 2 ,~ ! `
X is selected from the group conC;~ g of C1 10 alkyl, C1 10 fluoroalkyl, C1 10
alkyl ~ub~l;luled with J, Cl 10 fluoroalkyl substituted with J, 1-admantyl, 9-fluorenyl,
phenyl, phenyl s~ led with K, phenyl tlicubstituted with K, phenyl trisubstituted
with K, naphthyl, naphthyl substituted with K, naphthyl disubstituted with K, naphthyl
~,;~.,I,~Iil-l~ed with K, Cl 10 alkyl with an attached phenyl group, Cl 10 alkyl with two
attached phenyl groups, C1 10 alkyl with an attached phenyl group substituted with K,
and C1 10 alkyl with two attached phenyl groups substituted with K, C1 10 alkyl with an
attached phenoxy group, and C1 10 alkyl with an attached phenoxy group substituted
with K on the phenoxy group;
Y is selected from the group consi~Lillg of C6 l0 alkyl, Cl 10 fluoroalkyl, Cl 1o
alkyl ~lhs~ .led with J, Cl 10 fluoroalkyl suhctituted with J, 1-admantyl, 9-fluorenyl,
phenyl substituted with K, phenyl rlicuhstituted with K, phenyl trisubstituted with K,
naphthyl, naphthyl substituted with K, naphthyl disubstituted with K, naphthyl
ub~liluled with K, Cl 10 alkyl with an attached phenyl group, Cl 10 alkyl with two
attached phenyl groups, C1 10 alkyl with an attached phenyl group substituted with K,
and C1 10 alkyl with two attached phenyl groups substituted with K;
J is selected from the group consisting of halogen, COOH, OH, CN, NO2, NH2,
C1 10 alkoxy, C1 10 alkylamine, C2 12 dialkylamine, C1 10 alkyl-O-CO-, C1 10 alky
NH-, and C1 10 alkyl-S-;
K is selected from the group consis~ g of halogen, C1 1n alkyl, C1 10
perfluoroalkyl, C1 10 alkoxy, NO2, CN, OH, CO2H, amino, C1 10 alkylamino, C2 12
dialkylamino, C1-C10 acyl, and C1 10 alkoxy-CO-, and C1 10 alkyl-S-;
AA is a side chain blocked or unblocked amino acid with the L configuration, D
configuration, or no chirality at the a-carbon selected from the group consisting of
alanine, valine, leucine, isoleucine, proline, methionine, methionine sulfoxide,phenylalanine, tryptophan, glycine, serine, threonine, cysteine, tyrosine, asparagine,
t~mine~ aspartic acid, glutamic acid, lysine, arginine, histidine, phenylglycine, beta-
alanine, norleucine, norvaline, alpha-aminobutyric acid, epsilon-aminocaproic acid,
citrulline, I.yd~ y~loline, ornithine, homoarginine, sarcosine, indoline 2-carboxylic acid,
2-azetidinecarboxylic acid, pipecolinic acid (2-piperidine carboxylic acid), O-
methylserine, O-ethylserine, S-methylcysteine, S-ethylcysteine, S-benzylcysteine, NH2-
CH(CH2CHEt2)-COOH, alpha-aminoheptanoic acid, NH2-CH(CH2-1-napthyl)-COOH,
WO 94/00095 PCI`/VS93/06143
~138l2~
-44-
NH2-CH(CH2-2-napthyl)-COOH, NH2-CH(CH2-cyclohexyl)-COOH, NH2-CH(CH2-
cyclopentyl)-COOH, NH2-CH(CH2-cyclobutyl)-COOH, NH2-CH(CH2-cyclopropyl)-
COOH, trifluoroleucine, and hexafluoroleucine;
R is s~l~orted from the group consi~.ting of H, Cl 20 alkyl, Cl 20 alkyl with a
phenyl group att~rhed to the C1 20 alkyl, and C1 20 alkyl with an attached phenyl group
s~ ed with K.
A few amino acid and peptide ketoesters and ketoacids have been previously
reported. Colnfo,lh and Cornforth inJ. Chen~ Soc, 93-96 (1953), incorporated herei
by reference, report the synthesis of the ketoari.1c PhCH2CO-Gly-CO-OH and
Ac-Gly-CO-OH upon hydrolysis of heterocyclic molecules. Charles et al. in J. Chem.
Soc. Perki~ 1139-1146 (1980), incorporated herein by reference, use ketoesters for the
synthesis of bicyclic heterocydes. They report the synthesis of n-Bu-CO-Ala-CO-OEt,
Pr-CO-Ala-CO-OEt, cyclopentyl-CO-Ala-CO-OEt, Pr-CO-Phg-CO-OEt, and
Bz-Ala-CO-OEt. Hori et al. in Peptia~'es: Struct~re and Function-Proceedings of tlZe Ninth
Amencan Peptide Symposium (Deber, Hruby, and Kopple, Eds., Pierce Chemical Co.),pp 819-822 (1985), inco",o,dted herein by reference, report Bz-Ala-CO-OEt,
Bz-Ala-CO-OH, Z-Ala-AIa-Abu-CO-OEt, Z-Ala-Ala-Abu-CO-OBzl, and
Z-Ala-Ala-Ala-Ala-CO-OEt (Abu = 2-aminobutanoic acid or a-aminobutyric acid) andreport that these compounds inhibit ~l~C~ce Trainer in Tre~lds Pltaml. Sci. 8:303-307
(1987), incorporated herein by reference, comments on one of these compounds.
Burkhart, J. et a~ in Tetrahedron Lett. 29:3433-3436 (1988), incorporated herein by
reference, report the synthesis of Z-Val-Phe-CO-OMe and Bz-Phe-CO-OMe.
Angelastro et a~ in J. Me~ Cllem 33:13-16 (1990), incorporated herein by
reference, report some a-ketoesters which are inhibitors of calpain and chymotrypsin.
Hu and Abeles inArc~ Bioche~r~ Biophys. 281:271-274 (1990), incorporated herein by
reference, report some peptidyl a-ketoamides and a-ketoacids which are inhibitors of
cathepsin B and papain. Peet et al. in J. Me~ Che,~n 33:394-407 (1990), incorporated
herein by reference, report some peptidyl a-ketoesters which are inhibitors of porcine
pancreatic e!~ct~c~, human neutrophil elastase, and rat & human neutrophil
cathepsin G.
WO 94/0009~ PCI/US93/06143
~1~512'~
-45 -
The following Peptide Ketoester compounds are representative of the Peptide
Keto-Compounds found to be useful as Calpain inhibitors within the context of the
present invention:
Bz-DL-Ala-COOEt
Bz-DL-Ala-COOBzl
Bz-DL-Ala-COOnBu
Bz-DL-Phe-COOEt
Bz-DL-Ala-COOCH2-C6H4-CF3 (para)
Bz-DL-Arg-COOEt
Bz-DL-Lys-COOEt
Z-Ala-DL-Ala-COOEt
Z-Ala-DL-Ala-COOBzl
Z-Ala-DL-Ala-COOnBu
MeO-Suc-Ala-DL-Ala-COOMe
Z-Leu-Nva-COOEt
Z-Leu-Nle-COOEt
Z-Leu-Phe-COOEt
Z-Leu-Abu-COOEt
Z-Leu-Met-COOEt
Z-Phe-DL-Phe-COOEt
H-Gly-DL-Lys-COOEt
H-Ala-DL-Lys-COOEt
H-Pro-DL-Lys-COOEt
H-Phe-DL-Lys-COOEt
25 - Z-Ala-Ala-DL-Ala-COOEt
Z-Ala-Pro-DL-Ala-COOEt
Z-Ala-Ala-DL-Abu-COOEt
Z-Ala-Ala-DL-Abu-COOBzl
Z-Ala-Ala-DL-Abu-COOCH2-C6H4-CF3 (para)
MeO-Suc-Val-Pro-DL-Phe-COOMe
H-Leu-Ala-DL-Lys-COOEt
Z-Ala-Ala-Ala-DL-Ala-COOEt
WO 94/00095 PCI/US93/06143
1 3 8 1 ~ ~
46-
MeO-Suc-Ala-Ala-Pro-DL-Abu-COOMe.
Z-Leu-Phe-COOEt
PhCO-Abu-COOEt
(cH3)2cH(cH2)2co-Abu-cooLt
S CH3CH2CH)2CHCO-Abu-COOEt
Ph(CH2)6CO-Abu-COOEt
Z-Leu-4-a-Phe-COOEt
Z-Leu-Leu-Abu-COOEt
Z-Leu-Leu-Phe-COOEt
2-NapS02-Leu-Abu-COOEt
2-NapSO2-Leu-Leu-Abu-COOEt
Z-Leu-NLeu-CO2Et
Z-Leu-Phe-CO2Bu
Z-Leu-Abu-CO2Bu
Z-Leu-Phe-CO2Bzl
Z-Leu-Abu-CO2Bzl.
We have found certain subclasses of Peptide Ketoacid Compounds to be
particularly useful when used in accordance with the present invention. These are
s~brl~cses are the Dipeptide a-Ketoacids (Subclass A), the Dipeptide a-Ketoacids(Subclass B), the Tripeptide a-Ketoacids, the Tetrapeptide a-Ketoacids and the Amino
Acid peptide a-Keto~ritlc All of these are considered to be within the class of Peptide
Keto-Compounds.
The Dipeptide a-Krto~ci~ic (Subclass A) are compounds of the structure:
Ml-AA-NH-CHR2-CO-CO-OH
or a pharm~ceutir~lly acceptable salt, wherein
Ml represents H, NH2-CO-, NH2-CS-, NH2-SO2-, X-NH-CO-, X2N-CO,
X-NH-CS-, X2N-CS-, X-NH-SO2-, X2N-SO2-, X-CO-, X-CS-, X-SO2-, X-O-CO-, or X-
O-CS-;
X is selected from the group concicting of C1 10 alkyl, Cl 10 fluoroalkyl, C1 10alkyl substituted with J, Cl 10 fluoroalkyl substituted with J, 1-admantyl, 9-fluorenyl,
phenyl, phenyl substituted with K, phenyl disubstituted with K, phenyl trisubstituted
with K, naphthyl, naphthyl substituted with K, naphthyl disubstituted with K, naphthyl
WO 94/00095 PCI/US93/06143
213~12i
. .
b~.l ;l uled with K, C1 l0 alkyl with an attached phenyl group, Cl 10 alkyl with two
attached phenyl groups, C1 ,0 alkyl with an attached phenyl group substituted with K,
Cl 10 alkyl with two attached phenyl groups substituted with K, C1 ~0 alkyl with an
attached phenoxy group, and C1 ,0 alkyl with an attached phenoxy group substituted
with K on the phenoxy group;
J is selected from the group consi..ling of halogen, COOH, OH, CN, NO2, NH2,
Cl 10 alko~y, Cl 1O alkylamine, C2 12 dialkylamine, Cl 10 alkyl-O-CO-, Cl 10 alkyl-O-CO-
NH-, and C1 10 alkyl-S-;
K is selected from the group consisting of halogen, C1 10 alkyl, Cl 10
perfluoroalkyl, C1 ,0 alkoxy, NO2, CN, OH, CO2H, amino, C1 10 alkylamino, C2 12
dialkylamino, C1-C1o acyl, and C1 ~0 alkoxy-CO-, and C1 10 alkyl-S-;
AA is a side chain blocked or unblocked amino acid with the L configuration, D
configuration, or no chirality at the a-carbon selected from the group consisting of
alanine, valine, leucine, isoleucine, proline, methionine, methionine sulfoxide,phenylalanine, tryptophan, glycine, serine, threonine, cysteine, tyrosine, asparagine,
elllt~mine, aspartic acid, eJut~mir acid, lysine, arginine, histidine, phenylglycine, beta-
alanine, nnrleurine, norvaline, alpha-aminobutyric acid, epsilon-aminocaproic acid,
citrulline, hydroxyproline, ornithine, homoarginine, sarcosine, indoline 2-carboxylic acid,
2-azetidinecarboxylic acid, pipecolinic acid (2-piperidine carboxylic acid), O-
methylserine, O-ethylserine, S-methylcysteine, S-ethylcysteine, S-benzylcysteine, NH2-
CH(CH2CHEt2)-COOH, alpha-aminoheptanoic acid, NH2-CH(CH2-1-napthyl)-COOH,
NH2-CH(CH2-2-napthyl)-COOH, NH2-CH(CH2-cyclohexyl)-COOH, NH2-CH(CH2-
cyclopentyl)-COOH, NH2-CH(CH2-cyclobutyl)-COOH, NH2-CH(CH2-cyclopropyl)-
COOH, trifluoroleucine, and hexafluoroleucine;
R2 le~lesents C1 8 branched and unbranched alkyl, C1 8 branched and
unbranched cyclized alkyl, or Cl 8 branched and unbranched fluoroalkyl.
The Dipeptide a-Ketoacids (Subclass B) are compounds of the structure:
Ml-AA2-AA,-CO-OH
or a pharm~reutir~lly acceptable salt, wherein
M1 represents H, NH2-CO-, NH2-CS-, NH2-SO2-, X-NH-CO-, X2N-CO-,
X-NH-CS-, X2N-CS-, X-NH-SO2-, X2N-SO2-, X-CO-, X-CS-, X-S02-, X-O-CO-, or X-
O-CS-;
WO 94/0009~ PCI/US93/06143
~ 1 3 ~ ~ 2 Ll
-48-
X is selected from the group col,si~ling of C1 l0 alkyl, C1 10 fluoroalkyl, C1 10
alkyl s.lba,l;luled with J, Cl 1o fluoroalkyl substituted with J, 1-admantyl, 9-fluorenyl,
phenyl, phenyl ~ub~ uled with K, phenyl r~icubst~ ted with K, phenyl trisubstituted
with K, naphthyl, naphtl.~l substit~lted with K, naphthyl disubstituted with K, naphthyl
S ~ ub~l;lu~ed with K, Cl 1o alkyl with an attached phenyl group, Cl 1o alkyl with two
attached phenyl groups, Cl l0 alkyl with an attached phenyl group substituted with K,
and Cl l0 alkyl with two attached phenyl groups sul.~ u(ed with K, Cl l0 alkyl with an
attached phenoxy group, and C1 10 alkyl with an attached phenoxy group substituted
with K on the phenoxy group;
J is selected from the group consisting of halogen, COOH, OH, CN, NO2, NH2,
C1 ,0 alkoxy, Cl l0 alkylamine, C2 12 dialkylamine, C1 10 alkyl-O-CO-, C1 ln alkyl-O-CO-
NH-, and Cl l0 alkyl-S-;
K is s~ çted from the group coll~ia~ing of halogen, Cl 1o alkyl, Cl 10
perfluoroalkyl, Cl l0 alkoxy, NO2, CN, OH, CO2H, amino, Cl l0 alkylamino, C2 12
dialkylamino, Cl-Cl0 acyl, and Cl l0 alkoxy-CO-, and C1 10 alkyl-S-;
AAl is a side chain blocked or unblocked amino acid with the L configuration,
D cnnfi~-ation, or no chirality at the a-carbon selected from the group consisting of
alanine, valine, leucine, isoleucine, proline, methionine, methionine sulfoxide,phenylalanine, tryptophan, serine, threonine, cysteine, tyrosine, asparagine, glutamine,
aspartic acid, glutamic acid, lysine, arginine, histidine, phenylglycine, beta-alanine,
norleucine, norvaline, alpha-aminobutyric acid, epsilon-aminocaproic acid, citrulline,
I,~d~ ,loline, ornithine, homcarginine, sarcosine, indoline 2-carboxylic acid, 2-
azetidinecarboxylic acid, pipecolinic acid (2-piperidine carboxylic acid), O-methylserine,
O-ethylserine, S-methylcysteine, S-ethylcysteine, S-benzylcysteine, NH2-
CH(CH2CHEt2)-COOH, alpha-aminoheptanoic acid, NH2-CH(CH2-1-napthyl)-COOH,
NH2-CH(CH2-2-napthyl)-COOH, NH2-CH(CH2-cyclohexyl)-COOH, NH2-CH(CH2-
cyclopentyl)-COOH, NH2-CH(CH2-cyclobutyl)-COOH, NH2-CH(CH2-cyclopropyl)-
COOH, trifluoroleucine, and hexafluoroleucine;
AA2 is a side chain blocked or unblocked amino acid with the L configuration,
D configuration, or no chirality at the a-carbon selected from the group consisting of
alanine, valine, leucine, isoleucine, proline, methionine, methionine sulfoxide,phenylalanine, tryptophan, glycine, serine, threonine, cysteine, tyrosine, asparagine,
WO 94/0009~ ~ 1 3 ~ ~ 2 i PCI`/US93/06143
-49-
glllt~mine, aspartic acid, ghlt~mir acid, lysine, arginine, histidine, phenylglycine, beta-
alanine, norleucine, norvaline, alpha-aminobutyric acid, epsilon-aminocaproic acid,
citrulline, hydroxyproline, ornithine, homoarginine, sarcosine, indoline 2-carboxylic acid,
2-azetidinecarboxylic acid, pipecolinic acid (2-piperidine carboxylic acid), O-
methylserine, O-ethylserine, S-methylcysteine, S-ethylcysteine, S-benzylcysteine, NH2-
CH(CH2CHEt2)-COOH, alpha-aminoheptanoic acid, NH2-CH(CH2-1-napthyl)-COOH,
NH2-CH(CH2-2-napthyl)-COOH, NH2-CH(CH2-cyclohexyl)-COOH, NH2-CH(CH2-
cyclopentyl)-COOH, NH2-CH(CH2-cyclobutyl)-COOH, NH2-CH(CH2-cyclopropyl)-
COOH, trifluoroleucine, and hexafluoroleucine.
The Tripeptide a-~toar;~C are compounds of the structure:
Ml-AA-AA-AA-CO-OH
or a pharm~ceutir~lly acceptable salt, wherein
M1 represents H, NH2-CO-, NH2-CS-, NH2-SO2-, X-NH-CO-, X2N-CO-,
X-NH-CS-, X2N-CS-, X-NH-SO2-, X2N-SO2-, X-CO-, X-CS-, X-SO2-, X-O-CO-, or X-
O-CS-;
X is selected from the group consisting of C1 10 alkyl, Cl l0 fluoroalkyl, C1 10alkyl substituted with J, C1 10 fluoroalkyl substituted with J, 1-admantyl, 9-fluorenyl,
phenyl, phenyl substituted with K, phenyl disubstituted with K, phenyl trisubstituted
with K, naphthyl, naphthyl substituted with K, naphthyl disubstituted with K, naphthyl
trisubstituted with K, C1 10 alkyl with an attached phenyl group, C1 10 alkyl with two
attached phenyl groups, C1 10 alkyl with an attached phenyl group substituted with K,
and C1 10 alkyl with two attached phenyl groups substituted with K, C1 10 alkyl with an
attached phenoxy group, and C1 10 alkyl with an attached phenoxy group substituted
with K on the phenoxy group;
J is selected from the group consisting of halogen, COOH, OH, CN, NO2, NH2,
Cl 10 alkoxy, Cl 10 alkylamine, C2 12 dialkylamine, Cl 10 alkyl-O-CO-, Cl 10 alkyl-O-CO-
NH-, and C1 10 alkyl-S-;
K is selected from the group consisting of halogen, C1 10 alkyl, C1 10
per~luoroalkyl, Cl 10 alkoxy, NO2, CN, OH, C02H, amino, Cl 10 alkylamino, C2 l2
dialkylamino, C1-C10 acyl, and C1 10 alkoxy-CO-, and C1 10 alkyl-S-;
AA is a side chain blocked or unblocked amino acid with the L configuration, D
configuration, or no chirality at the a-carbon selected from the group consisting of
WO 94/0009~ PCI'/US93/06143
~13~
-50-
alanine, valine, leucine, isoleucine, proline, methionine, methionine sulfoxide,phenylalanine, ~ ophan, glycine, serine, threonine, cysteine, tyrosine, asparagine,
glllt~mine, aspartic acid, glut~mi~ acid, lysine, arginine, histidine, phenylglycine, beta-
alanine, norleucine, norvaiine, alpha-aminobutyric acid, epsilon-aminocaproic acid,
citrulline, I-yd~u~oline, ornithine, homoarginine, sarcosine, indoline 2-carboxylic acid,
2-~7eti-~ineC~ ?~o~ylic acid, pipecolinic acid (2-piperidine carboxylic acid), O-
methylserine, O-elhyl;~;.ille, S-methylcysteine, S-ethylcysteine, S-benzylcysteine, NH2-
CH(CH2CHEt2)-COOH, alpha-aminoheptanoic acid, NH2-CH(CH2-1-napthyl)-COOH,
NH2-CH(CH2-2-napthyl)-COOH, NH2-CH(CH2-cyclohexyl)-COOH, NH2-CH(CH2-
cyclopentyl)-COOH, NH2-CH(CH2-cyclobutyl)-COOH, NH2-CH(CH2-cyclopropyl)-
COOH, trifluoroleucine, and hexafluoroleucine
The Tetrapeptide -Kçtoa~ 1c are compounds of the structure
Ml-AA-AA-AA-AA-CO-OH
or a phar?m~eutir~lly acceptable salt, wherein
M1 lep~ejents H, NH2-CO-, NH2-CS-, NH2-SO2-, X-NH-CO-, X2N-CO-,
X-NH-CS-, X2N-CS-, X-NH-SO2-, X2N-SO2-, Yl-CO-, X-CS-, X-SO2-, X-O-CO-, or X-
O-CS-;
X is selected from the group consisting of Cl l0 alkyl, Cl 10 fluoroalkyl, C1 10alkyl substituted with J, Cl 10 fluoroalkyl ~ubsli~uled with J, l-admantyl, 9-fluorenyl,
phenyl, phenyl substituted with K, phenyl ~licubstitllted with K, phenyl trisubstituted
with K, naphthyl, naphthyl substituted with K, naphthyl disubstituted with K, naphthyl
ub~ uted with K, Cl 10 alkyl with an attached phenyl group, Cl 10 alkyl with twoattached phenyl groups, C1 10 alkyl with an attached phenyl group substituted with K,
and C1 10 alkyl with two attached phenyl groups substituted with K? C1 10 alkyl with an
attached phenoxy group, and Cl 10 alkyl with an attached phenoxy group substituted
with K on the phenoxy group;
Y1 is selected from the group consisting of C2 10 alkyl, C1 10 fluoroalkyl, C1 10
alkyl substituted with J, C1 10 fluoroalkyl substituted with J, 1-admantyl, 9-fluorenyl,
phenyl, phenyl ~ub~liluled with K, phenyl di~ul~ uled with K, phenyl trisubstituted
with K, naphthyl, naphthyl substituted with K, naphthyl disubstituted with K, naphthyl
I,isu?ù~liluted with K, Cl 10 alkyl with an attached phenyl group, Cl 10 alkyl with two
WO 94/00095 ,2 1 3 8 1 2 4 PCI/US93/06143
att~^hed phenyl groups, C1 10 alkyl with an attached phenyl group substituted with K
and C1 l0 alkyl with two attached phenyl groups substituted with K;
J is selected from the group consisting of halogen, COOH, OH, CN, NO2, NH2,
Cl ,o alkoxy, Cl 10 alkylamine, C2 l2 dialkylamine, Cl 10 alkyl-O-CO-, Cl 10 alkyl-O-CO-
NH-, and C1 10 alkyl-S-;
K is selected from the group consisting of halogen, C1 l0 alkyl, C1 10
perfluoroalkyl, C1 l0 alkoxy, NO2, CN, OH, CO2H, amino, C1 10 alkylamino, C2 12
dialkylamino, C1-C10 acyl, and C1 10 alkoxy-CO-, and C1 10 alkyl-S-;
AA is a side chain blocked or unblocked amino acid with the L configuration, D
configuration, or no chirality at the a-carbon selected from the group consisting of
alanine, valine, leucine, isoleucine, proline, methionine, methionine sulfoxide,phenylalanine, tryptophan, glycine, serine, threonine, cysteine, tyrosine, asparagine,
~mine, aspartic acid, gl~t~mir acid, lysine, arginine, histidine, phenylglycine, beta-
alanine, norleucine, norvaline, alpha-aminobutyric acid, epsilon-aminocaproic acid,
citrulline, llyd~ oline, ornithine, homoarginine, sarcosine, indoline 2-carboxylic acid,
2-azetidinecarboxylic acid, pipecolinic acid (2-piperidine carhoxylic acid), O-
methylserine, O-ethylserine, S-methylcysteine, S-ethylcysteine, S-benzylcysteine, NH2-
CH(CH2CHEt2)-COOH, alpha-aminoheptanoic acid, NH2-CH(CH2-1-napthyl)-COOH,
NH2-CH(CH2-2-napthyl)-COOH, NH2-CH(CH2-cyclohexyl)-COOH, NH2-CH(CH2-
cyclopentyl)-COOH, NH2-CH(CH2-cyclobutyl)-COOH, NH2-CH(CH2-cyclopropyl)-
COOH, trifluoroleucine, and hexafluoroleucine.
The Amino Acid Peptide o~-Ketoacids are compounds of the structure:
Ml-AA-CO-OH
or a pharm~euti~lly acceptable salt, wherein
Ml reple~ ts H, NH2-CO-, NH2-CS-, NH2-S02-, X-NH-CO-, X2N-CO-,
X-NH-CS-, X2N-CS-, X-NH-SO2-, X2N-SO2-, Y2-CO-, X-CS-, X-SO2-, X-O-CO-, or X-
O-CS-;
X is selected from the group consisting of C1 10 alkyl, C1 10 fluoroalkyl, C1 l0alkyl substituted with J, C1 10 fluoroalkyl substituted with J, 1-admantyl, 9-fluorenyl,
phenyl, phenyl substituted with K, phenyl disubstituted with K, phenyl trisubstituted
with K, naphthyl, naphthyl substituted with K, naphthyl disubstituted with K, naphthyl
tricubstituted with K, C1 l0 alkyl with an attached phenyl group, C1 10 alkyl with two
WO 94/00095 PCI`/US93/06143
~3~
-52-
attached phenyl groups, C1 10 alkyl with an attached phenyl group substituted with K,
and C1 10 alkyl with two attached phenyl groups substituted with K, C1 10 alkyl with an
att~rhed phenoxy group, and C1 10 alkyl with an attached phenoxy group substituted
with K on the phenoxy group;
Y2 is selected from the group COI.si~ )g of C1 10 alkyl, C1 10 fluoroalkyl, C1 10
alkyl substituted with J, Cl 1o fluoroalkyl sul 5tituted with J, 1-admantyl, 9-fluorenyl,
phenyl substituted with K, phenyl ~ lb~ uted with K, phenyl trisubstituted wieh K,
naphthyl, naphthyl ~b~l;luled with K, naphthyl ~ ub5tituted with K, naphthyl
l. ;~.lb~ ted with K, Cl 10 alkyl with an attached phenyl group, Cl 10 alkyl with two
attached phenyl groups, C1 10 alkyl with an attached phenyl group substituted with K,
and C1 10 alkyl with two attached phenyl groups substituted with K;
J is selected from the group consi~li,.g of halogen, COOH, OH, CN, NO2, NH2,
C1 10 alkoxy, C1 10 alkylamine, C2 12 dialkylamine, C1 10 alkyl-O-CO-, C1 10 alkyl-O-CO-
NH-, and C1 10 alkyl-S-;
K is s~lected from the group consi~ling of halogen, C1 l0 alkyl, C1 l0
perfluoroalkyl, C1 10 alkoxy, NO2, CN, OH, CO2H, amino, C1 l0 alkylamino, C2 12
dialkylamino, C1-C10 acyl, and C1 10 alkoxy-CO-, and C1 10 alkyl-S-;
AA is a side chain blocked or unblocked amino acid with the L configuration, D
configuration, or no chirality at the a-carbon selected from the group consisting of
alanine, valine, leucine, isoleucine, proline, methionine, methionine sulfoxide,phenylalanine, tryptophan, glycine, serine, threonine, cysteine, tyrosine, asparagine,
glut~mine, aspartic acid, giut~mir acid, lysine, arginine, histidine, phenylglycine, beta-
alanine, norleucine, norvaline, alpha-aminobutyric acid, epsilon-aminocaproic acid,
citrulline, hyd,u~loline, ornithine, homoarginine, sarcosine, indoline 2-carboxylic acid,
2-azetidinecarboxylic acid, pipecolinic acid (2-piperidine carboxylic acid), O-
methylserine, O-ethylserine, S-methylcysteine, S-ethylcysteine, S-benzylcysteine, NH2-
CH(CH2CHEt2)-COOH, alpha-aminoheptanoic acid, NH2-CH(CH2-1-napthyl)-COOH,
NH2-CH(CH2-2-napthyl)-COOH, NH2-CH(CH2-cyclohexyl)-COOH, NH2-CH(CH2-
cyclopentyl)-COOH, NH2-CH(CH2-cyclobutyl)-COOH, NH2-CH(CH2-cyclopropyl)-
COOH, trifluoroleucine, and hexafluoroleucine.
~VO 94/00095 ~ 1 3 ~ ~ 2 i PCI`/US93/06143
. ", . ~ .
The following Peptide K.otoarid compounds are representative of the Peptide
Keto-Compounds found to be useful as Calpain inhibitors within the context of the
present invention:
Bz-DL-Lys-COOH
Bz-DL-Ala-COOH
Z-Leu-Phe-COOH
Z-Leu-Abu-COOH.
The peptide a-keto~mides are transition state analogue inhibitors for cysteine
proteases, such as Calpain. We have found that Peptide a-ketoamides containing
amino acid residues with hydrophobic side chains at the Pl site are excellent inhibitors
of several cysteine proteases including calpain I and calpain II.
We have found six ~ubul-c~ps of the peptide ketoamides to be particularly
effective in inhibiting Calpain. These subrl~cc~oc are referred to herein as Dipeptide
a-K~to~mides (Subclass A), Dipeptide a-Ketoamides (Subclass B), Dipeptide
a-Kçto~Tnides (Subclass C, Types 1 through 6), Tripeptide a-Ketoamides, Tetrapeptide
a-K~to~mides and Amino Acid a-Ketoamides. All of these subclasses are consideredherein to be within the class of Peptide Keto-Compounds.
The Dipeptide a-K~to~mides (Subclass A) have the following structural
formula:
M1-AA-NH-CHR2-CO-CO-NR3R4
or a pharm~reutir~lly acceptable salt, wherein
M1 lc~resents H, NH2-CO-, NH2-CS-, NH2-SO2-, X-NH-CO-, X2N-CO-,
X-NH-CS-, X2N-CS-, X-NH-SO2-, X2N-SO2-, X-CO-, X-CS-, X-SO2-, X-O-CO-, or X-
O-CS-;
X is selected from the group consisting of Cl 10 alkyl, Cl 10 fluoroalkyl, Cl 10alkyl substituted with J, C1 10 fluoroalkyl substituted with J, 1-admantyl, 9-fluorenyl,
phenyl, phenyl substitut~o~l with K, phenyl disubstituted with K, phenyl trisubstituted
with K, naphthyl, naphthyl substituted with K, naphthyl disubstituted with K, naphthyl
Llisul,~liLuLed with K, Cl 10 alkyl with an attached phenyl group, Cl 10 alkyl with two
attached phenyl groups, C1 10 alkyl with an attached phenyl group substituted with K,
Cl 10 alkyl with two attached phenyl groups substituted with K, C1 10 alkyl with an
WO 94/0009~ PCr/US93/06143
~2~3~:~X~
attached phenoxy group, and Cl 10 alkyl with an attached phenoxy group substituted
with K on the phenoxy group;
J is selected from the group consi~ling of halogen, COOH, OH, CN, NO2, NH2,
C1 10 alkoxy, C1 10 alkylamine, C2 12 dialkylamine, C1 10 alkyl-O-CO-, Cl 10 alkyl-O-CO-
S NH-, and Cl l0 alkyl-S-;
K is selected from the group consi;,ling of halogen, Cl 10 alkyl, Cl 1o
perfluoroalkyl, Cl 10 alkoxy, NO2, CN, OH, CO2H, amino, Cl 10 alkylamino, C2 l2
dialkylamino, C1-C10 acyl, C1 10 alkoxy-CO-, and C1 l0 alkyl-S-;
AA is a side chain blocked or unblocked amino acid with the L configuration, D
configuration, or no chirality at the a-carbon selected from the group consisting of
alanine, valine, leucine, isoleucine, proline, methionine, methionine sulfoxide,phenylalanine, tryptophan, glycine, serine, threonine, cysteine, tyrosine, asparagine,
El,.l~-..;.~e, aspartic acid, g]l~t~mi~ acid, lysine, arginine, histidine, phenylglycine, beta-
alanine, norleucine, norvaline, a-aminobutyric acid, epsilon-aminocaproic acid,
citrulline, hydroxyproline, ornithine, homoarginine, sarcosine, indoline 2-carboxylic acid,
2-azetidinecarboxylic acid, pipecolinic acid (2-piperidine carboxylic acid), O-
methylserine, O-ethylserine, S-methylcysteine, S-ethylcysteine, S-benzylcysteine, NH2-
CH(CH2CHEt2)-COOH, a-aminoheptanoic acid, NH2-CH(CH2-1-napthyl)-COOH,
NH2-CH(CH2-2-napthyl)-COOH, NH2-CH(CH2-cyclohexyl)-COOH, NH2-CH(CH2-
cyclopentyl)-COOH, NH2-CH(CH2-cyclobutyl)-COOH, NH2-CH(CH2-cyclopropyl)-
COOH, trifluoroleucine, and hexafluoroleucine;
R2 is selected from the group consi~ling of Cl 8 branched and unbranched alkyl,
Cl 8 branched and unbranched cyclized alkyl, and Cl 8 branched and unbranched
fluoroalkyl;
R3 and R4 are selected independently from the group consisting of H, C1 20
alkyl, C1 20 cyclized alkyl, C120 alkyl with a phenyl group attached to the C1 20 alkyl,
C120 cyclized alkyl with an attached phenyl group, C1 20 alkyl with an attached phenyl
group sub~ uled with K, C1 20 alkyl with an attached phenyl group disubstituted with
K, C120 alkyl with an attached phenyl group trisubstituted with K, C120 cyclized alkyl
with an attached phenyl group substituted with K, C1 10 alkyl with a morpholine [-
N(CH2CH2)O] ring attached through nitrogen to the alkyl, C1 ,0 alkyl with a piperidine
ring attached through nitrogen to the alkyl, C1 10 alkyl with a pyrrolidine ring attached
94/00095 ~ 1 3 ~ PCI /VS93/06143
through nitrogen to the alkyl, C1 20 alkyl with an OH group attached to the alkyl, -
CH2CH2OCH2CH2OH, C1 10 with an attached 4-pyridyl group, C1 10 with an attached
3-pyridyl group, C1 10 with an attached 2-pyridyl group, C1 10 with an attached
cyclohexyl group, -NH-CH2CH2-(4-hyd,uAyluhenyl), and -NH-CH2CH2-(3-indolyl).
S The Dipeptide a-K~to~mides (Subclass B) have the following structural formula:
Ml-AA2-AAl-CO-NR3R4
or a pharm~eutir~lly acceptable salt, wherein
M1 rc~resents H, NH2-CO-, NH2-CS-, NH2-SO2-, X-NH-CO-, X2N-CO-,
X-NH-CS-, X2N-CS-, X-NH-SO2-, X2N-SO2-, X-CO-, X-CS-, X-SO2-, X-O-CO-, or X-
O-CS-;
X is selected from the group consisting of C1 10 alkyl, Cl 10 fluoroalkyl, C1 l0alkyl substituted with J, Cl 10 fluoroalkyl substituted with J, 1-admantyl, 9-fluorenyl,
phenyl, phenyl s.~b~ u~ed with K, phenyl disubstituted with K, phenyl trisubstituted
with K, naphthyl, naphthyl s~ (ed with K, naphthyl disubstituted with K, naphthyl
tri~ubstituted with K, C1 10 alkyl with an attached phenyl group, C1 10 alkyl with two
attached phenyl groups, C1 10 alkyl with an attached phenyl group substituted with K,
C1 10 alkyl with two attached phenyl groups substituted with K, C1 10 alkyl with an
attached phenoxy group, and Cl l0 alkyl with an attached phenoAy group substituted
with K on the phenoxy group;
J is selected from the group consisting ûf halogen, COOH, OH, CN, NO2, NH2,
C1 10 alkoxy, C1 10 alkylamine, C2 12 dialkylamine, C1 10 alkyl-O-CO-. C1 10 y
NH-, and C1 10 alkyl-S-;
K is selected from the group consisting of halogen, C1 10 alkyl, C1 10
perfluoroalkyl, C1 10 alkoxy, NO2, CN, OH, CO2H, amino, C1 10 alkylamino, C2 12
dialkylamino, C1-C10 acyl, and C1 10 alkoxy-CO-, and C1 10 alkyl-S-;
AAl is a side chain blocked or unblocked amino acid with the L configuration,
D configuration, or no chirality at the a-carbon selected from the group consisting of
alanine, valine, leucine, isoleucine, proline, methionine, methionine sulfoxide,phenylalanine, tryptophan, serine, threonine, cysteine, tyrosine, asparagine, glutamine,
aspartic acid, glutamic acid, lysine, arginine, histidine, phenylglycine, beta-alanine,
norleucine, norvaline, a-aminobutyric acid, epsilon-aminocaproic acid, citrulline,
hydluAy~loline, ornithine, homoarginine, sarcosine, indoline 2-carboxylic acid, 2-
WO 94/0009~ PCI/US93/06143
~13~i2~
-56-
azetidinecarboxylic acid, pipecolinic acid (2-piperidine carboxylic acid), O-methylserine,
O-~lhylse~ e, S-methylcysteine, S-ethylcysteine, S-benzylcysteine, NH2-
CH(CH2CHEt2)-COOH, a-aminoheptanoic acid, NH2-CH(CH2-1-napthyl)-COOH,
NH2-CH(CH2-2-napthyl)-COOH, NH2-CH(CH2-cyclohexyl)-COOH, NH2-CH(CH2-
cyclopentyl)-COOH, NH2-CH(CH2-cyclobutyl)-COOH, NH2-CH(CH2-cyclopropyl)-
COOH, trifluoroleucine, and hexafluoroleucine;
AA2 is a side chain blocked or unblocked amino acid with the L configuration,
D configuration, or no chirality at the a-carbon selected from the group consisting of
alanine, valine, leucine, isoleucine, proline, methionine, methionine sulfoxide,phenylalanine, tryptophan, glycine, serine, threonine, cysteine, tyrosine, asparagine,
;ne, aspartic acid, glutamic acid, lysine, arginine, histidine, phenylglycine, beta-
alanine, norleucine, nor~aline, a-aminobutyric acid, epsilon-aminocaproic acid,
citrulline, hydlu~y,u~oline, ornithine, homoarginine, sarcosine, indoline 2-carboxylic acid,
2-azetidinecarboxylic acid, pipecolinic acid (2-piperidine carboxylic acid), O-
methylserine, O-ethylserine, S-methylcysteine, S-ethylcysteine, S-benzylcysteine, NH2-
CH(CH2CHEt2)-COOH, a-aminoheptanoic acid, NH2-CH(CH2-1-napthyl)-COOH,
NH2-CH(CH2-2-napthyl)-COOH, NH2-CH(CH2-cyclohexyl)-COOH, NH2-CH(CH2-
cyclopentyl)-COOH, NH2-CH(CH2-cyclobutyl)-COOH, NH2-CH(CH2-cyclopropyl)-
COOH, trifluoroleucine, and hexafluoroleucine;
R3 and R4 are selected independently from the group consisting of H, Cl 20
alkyl, Cl 20 cyclized alkyl, C1 20 alkyl with a phenyl group attached to the C1 20 alkyl,
Cl 20 cyclized alkyl with an attached phenyl group, Cl 20 alkyl with an attached phenyl
group substituted with K, Cl 20 alkyl with an attached phenyl group disubstituted with
K, Cl 20 alkyl with an attached phenyl group trisubstituted with K, Cl 20 cyclized alkyl
with an attached phenyl group substi~uted with K, Cl 10 alkyl with a morpholine
[-N(CH2CH2)0] ring attached through nitrogen to the alkyl, Cl 10 alkyl with a
piperidine ring attached through nitrogen to the alkyl, Cl 10 alkyl with a pyrrolidine ring
attached through nitrogen to the alkyl, Cl 20 alkyl with an OH group attached to the
alkyl, -CH2CH2OCH2CH2OH, Cl 10 with an attached 4-pyridyl group, C1 l0 with an
attached 3-pyridyl group, Cl 10 with an attached 2-pyridyl group, Cl 10 with ;3n attached
cyclohexyl group, -NH-CH2CH2-(4-hydroxyphenyl), and -NH-CH2CH2-(3-indolyl).
WO 94/00095 21~ 812 4 PCr/US93/06143
~ . ,
The Dipeptide a-Keto~mides (Subclass C, Type 1) have the following structural
formula:
MlCO-AA2-AAl-CO-NH-CH2CH(OH)-R
or a pharrn~reuti~lly acceptable salt, wherein
M1 is selected from the group consi~li"g of C1 1 alkyl monosubstituted with
phenyl, Cl 4 alkyl .~ u~ed with phenyl, Cl4 alkyl monosubstituted with 1-naphthyl,
C1~l alkyl monosubstituted with 2-naphthyL Cl ,~ alkoxy monosubstituted with phenyl,
Cl ~ alkoxy ~I;c~b~ ed with phenyl, ArCH20-, ~rO-, ArCH2NH-, and ArNH-;
wherein Ar is selected from the group consi:,ling of phenyl, phenyl
mono~ub~ ed with J, phenyl llicubstituted with J, 1-naphthyl, 1-naphthyl
mono~ ted with J, 2-naphthyl, and 2-naphthyl monosubstituted with J;
J is selected from the group concicting of halogen, OH, CN, NO2, NH2, COOH,
CO2Me, CO2Et, CF3, Cl 4 alkoxy, Cl4 alkylamine, C2 8 dialkylamine, Cl4
perfluoroalkyl, and -N(CH2CH2)2O;
AA2 is an amino acid with the L configuration, D configuration, or DL
configuration at the a-carbon selected from the group co~ ling of alanine, valine,
leucine, isoleucine, proline, histidine, methionine, methionine sulfoxide, phenylalanine,
serine, threonine, phenylglycine, norleucine, norvaline, alpha-aminobutyric acid,
O-methylserine, O-ethylserine, S-methylcysteine, S-ethylcysteine, S-benzylcysteine,
NH2-CH(CH2CHEt2)-COOH, alpha-aminoheptanoic acid,
NH2-CH(CH2-cyclohexyl)-COOH, NH2-CH(CH2-cyclopentyl)-COOH,
NH2-CH(CH2-cyclobutyl)-COOH, NH2-CH(CH2-cyclopropyl)-COOH,
5,5,5-trifluoroleucine, and hexafluoroleucine;
AA1 is an amino acid with the L configuration, D configuration, or DL
configuration at the a-carbon selected from the group consi~lh~g of alanine, valine,
leucine, ic~ u~ine, proline, histidine, methionine, methionine sulfoxide, phenylalanine,
arginine, lysine, tryptophan, glycine, serine, threonine, cysteine, tyrosine, asparagine,
glut~mine, aspartic acid, glutamic acid, phenylglycine, norleucine, nolvaline,
alpha-aminobutyric acid, O-methylserine, O-ethylserine, S-methylcysteine,
S-ethylcysteine, S-benzylcysteine, NH2-CH(CH2CHEt2)-COOH, alpha-aminoheptanoic
acid, NH2-CH(CH2-1-napthyl)-COOH, NH2-CH(CH2-2-napthyl)-COOH,
NH2-CH(CH2-cyclohexyl)-COOH, NH2-CH(CH2-cyclopentyl)-COOH,
WO 94/0009~ PCI/US93/06143
~13~12~
NH2-CH(CH2-cyclobutyl)-COOH, NH2-CH(CH2-cyclopropyl)-COOH,
5,5,5-trifluoroleucine, and hexafluoroleucine;
Rl is selected from the group coU~ ng of phenyl, phenyl monosubstituted with
J, phenyl ~ ubstituted with J, phenyl tl;c~ ;tu~ed with J, pentafluorophenyl,
~ R2 oR2 R20 oR2
l-naphthyl, 1-naphthyl monosubstituted with J, 1-naphthyl ~ ul~l ;lu(ed with J,
2-naphthyl, 2-naphthyl monosubstituted with J, 2-naphthyl disubstituted with J,
2-pyridyl, 2-quinolinyl, and 1-isoquinolinyl;
R2 represe..ts Cl4 alkyl substituted with phenyl, phenyl and phenyl substituted
with J.
Dipeptide a-Keto~mides (Subclass C, Type 2) have the following structural
formula:
MlCO-AA2~AAl~CO~NH~(CH2)n~R3
or a pharm~reutir~lly acceptable salt, wherein
M1 is selected from the group consisting of Cl4 alkyl monosubstituted with
phenyl, Cl4 alkyl ~ b~ uled with phenyl, Cl4 alkyl monosubstituted with 1-naphthyl,
C14 alkyl monosubssitllted with 2-naphthyl, Cl4 alkoxy monosubstituted with phenyl,
Cl4 alkoxy ~ b~ u~ed with phenyl, ArCH20-, ArO-, ArCH2NH-, and ArNH-;
wherein Ar is selected from the group consisting of phenyl, phenyl
monosubstituted with J, phenyl ~ ubstituted with J, 1-naphthyl, 1-naphthyl
monosubstituted with J, 2-naphthyl, and 2-naphthyl monosubstituted with J;
J is selected from the group consisting of halogen, OH, CN, NO2, NH2, COOH,
CO2Me, CO2Et, CF3, Cl4 alkoxy, Cl4 alkylamine, C2 8 dialkylamine, C
perfluoroalkyl, and -N(CH2CH2)2O;
AA2 is an amino acid with the L configuration, D configuration, or DL
configuration at the a-carbon selected from the group consisting of alanine, valine,
leucine, isoleucine, proline, histidine, methionine, methionine sulfoxide, phenylalanine,
serine, threonine, phenylglycine, norleucine, norvaline, alpha-aminobutyric acid,
WO 94/0009~ ~13 8 12 ~ PCr/US93/06143
.
, .
59
O-methylserine, O-ethylserine, S-methylcysteine, S-ethylcysteine, S-benzylcysteine,
NH2-CH(CH2CHEt2)-COOH, alpha-aminoheptanoic acid,
NH2-CH(CH2-cydohexyl)-COOH, NH2-CH(CH2-cyclopentyl)-COOH,
N,H2-CH(CH2-cyclobutyl)-COOH, NH2-CH(CH2-cyclopropyl)-COOH,
5,5,5-trifluoroleucine, and h~Y~fluoroleucine;
AAl is an amino acid with the L configuration, D configuration, or DL
configuration at the a-carbon selected from the group consi:,ling of alanine, valine,
leucine, isoleucine, proline, histidine, methionine, methionine sulfoxide, phenylalanine,
arginine, lysine, tryptophan, glycine, serine, threonine, cysteine, tyrosine, asparagine,
~ ;ne~ aspartic acid, glut~mi-~ acid, phenylglycine, norleucine, norvaline,
alpha-aminobutyric acid, O-methylserine, O-ethylserine, S-methylcysteine,
S-ethylcysteine, S-benzylcysteine, NH2-CH(CH2CHEt2)-COOH, alpha-aminoheptanoic
acid, NH2-CH(CH2-1-napthyl)-COOH, NH2-CH(CH2-2-napthyl)-COOH,
NH2-CH(CH2-cyclohexyl)-COOH, NH2-CH(CH2-cyclopentyl)-COOH,
NH2-CH(CH2-cyclobutyl)-COOH, NH2-CH(CH2-cyclopropyl)-COOH,
5,5,5-trifluoroleucine, and hexafluoroleucine;
n= 1-3;
R3 is selected from the group consi~ g of 2-furyl, 2-furyl monosubstituted with
J, 2-pyridyl, 2-pyridyl monosubstituted with J, 3-pyridyl, 3-pyridyl monosubstituted with
J, 4-pyridyl, 4-pyridyl monosu~stituted with J, 2-quinolinyl, 2-quinolinyl monosubstituted
with J, 1-isoquinolinyl, 1-isoquinolinyl monosubstituted with J,
~ ~N~r ~ ~N~ ~O HNJ~3
.. Me ~ ' O~N
O Me
Me NJ~N,~ J~ ~Me O
O~N N O~N N N andHN NH
Me Me ~_~
~(CH2)4CONH(CH2)2NH
WO 94/00095 PCI`/US93/06143
~ ~ 3
-60-
Dipeptide a-~C~toamides (Subclass C, Type 3) have the following structural
formula:
M3~(CH2)q~CO~AA2~AAl~CO~NH~CH2CH(OH)~R
or a pharm~reutir~lly acceptable salt, wherein
M3 is selected from the group CQh` `I ;--g of 2-furyl, 2-tetrahydrofuryl, 2-pyridyl,
3-pyridyl, 4-pyridyl, 2-pyrazinyl, 2-quinolinyl, 1-tetrahydroquinolinyl, 1-isoquinolinyl,
2-tetral,ydl~isoquinolinyl, and -N(CH2CH2)20;
q = 0-2;
AA2 is an amino acid with the L configuration, D configuration, or DL
configuration at the a-carbon selected from the group con~i~ling of alanine, valine,
leucine, isoleucine, proline, histidine, methionine, methionine sulfoxide, phenylalanine,
serine, threonine, phenylglycine, norleucine, norvaline, alpha-aminobutyric acid,
O-methylserine, O-ethylserine, S-methylcysteine, S-ethylcysteine, S-benylcysteine,
NH2-CH(CH2CHEt2)-COOH, alpha-aminoheptanoic acid,
NH2-CH(CH2-cyclohexyl)-COOH, NH2-CH(CH2-cyclopentyl)-COOH,
NH2-CH(CH2-~;ydobulyl)-COOH, NH2-CH(CH2-cyclopropyl)-COOH,
5,5,5-trifluoroleucine, and hexafluoroleucine;
AAl is an amino acid with the L configuration, D configuration, or DL
configuration at the a-carbon selected from the group consisting of alanine, valine,
leucine, isoleucine, proline, histidine, methionine, methionine sulfoxide, phenylalanine,
arginine, lysine, tryptophan, glycine, serine, threonine, cysteine, tyrosine, asparagine,
."i.~e, aspartic acid, ~ lt~mir acid, phenylglycine, norleucine, norvaline,
alpha-aminobutyric acid, O-methylserine, O-ethylserine, S-methylcysteine,
S-ethylcysteine, S-benylcysteine, NH2-CH(CH2CHEt2)-COOH, alpha-aminoheptanoic
acid, NH2-CH(CH2-1-napthyl)-COOH, NH2-CH(CH2-2-napthyl)-COOH,
NH2-CH(CH2-cyclohexyl)-COOH, NH2-CH(CH2-cyclopentyl)-COOH,
NH2-CH(CH2-cydobutyl)-COOH, NH2-CH(CH2-cyclopropyl)-COOH,
5,5,5-trifluoroleucine, and hexafluoroleucine;
94/00095 ~ 1 3 8 1 2 4 PCr/US93/06143
-61-
Rl is selected from the group cor.~:~l;..g of phenyl, phenyl monosubstituted with
J, phenyl rl;~u1"~ lted with J, phenyl ~ Ib~litu~ed with J, pentafluorophenyl,
oR2 oR2 R20
~ ~oR2 ~oR2 ~OR2
1-naphthyl, 1-naphthyl mono~-ll,,l;l~led with J, 1-naphthyl ~icubstituted with J,
2-naphthyl 2-naphthyl monosubstituted with J, 2-naphthyl disubstituted with J,
2-pyridyl, 2-quinolinyl, and 1-isoquinolinyl;
R2 represents Cl4 alkyl ~ub~ uled with phenyl, phenyl and phenyl substituted
with J.
J is selected from the group consi~ lg of halogen, OH, CN, NO2, NH2, COOH,
C02Me, CO2Et, CF3, Cl 1 alkoxy, Cl ~ alkylamine, C2 8 dialkylamine, C
perfluoroalkyl, and N(CH2CH2)2O;
Dipeptide a-Ketoamides (Subclass C, Type 4) have the following structural
formula:
M3~(CH2)q~CO~AA2~AAl~CO~NH~(CH2)n~R3
or a pharm~euti~lly acceptable salt, wherein
M3 is selected from the group consi~ling of 2-furyl, 2-tetrahydrofuryl, 2-pyridyl,
3-pyridyl, 4-pyridyl, 2-pyrazinyl, 2-quinolinyl, 1-tetrahydroquinolinyl, 1-isoquinolinyl,
2-tetrahydroisoquinolinyl, and-N(CH2CH2)2O;
q= 0-2;
AA2 is an amino acid with the L configuration, D configuration, or DL
configuration at the a-carbon selected from the group consisting of alanine, valine,
leucine, isoleucine, proline, histidine, methionine, methionine sulfoxide, phenylalanine,
serine, threonine, phenylglycine, norleucine, norvaline, alpha-aminobutyric acid,
O-methylserine, O-ethylserine, S-methylcysteine, S-ethylcysteine, S-benzylcysteine,
NH2-CH(CH2CHEt2)-COOH, alpha-aminoheptanoic acid,
NH2-CH(CH2-cyclohexyl)-COOH, NH2-CH(CH2-cyclopentyl)-COOH,
NH2-CH(CH2-cyclobutyl)-COOH, NH2-CH(CH2-cyclopropyl)-COOH,
5,5,5-trifluoroleucine, and hexafluoroleucine;
WO 94/00095 ~, ~ PCr/US93/06143
AAl is an amino acid with the L configuration, D configuration, or DL
configuration at the a-carbon selected from the group consisting of alanine, valine,
leucine, icol-u- ine, proline, histidine, methionine, methionine sulfoxide, phenylalanine,
arginine, lysine, tryptophan, glycine, serine, threonine, cysteine, tyrosine, asparagine,
S ~ e, aspartic acid, g]Ut~mir acid, phenylglycine, norleucine, norvaline,
alpha-aminobutyric acid, O-methylserine, O-ethylserine, S-methylcysteine,
S-ethylcysteine, S-benzylcysteine, NH2-CH(CH2CHEt2)-COOH, alpha-aminoheptanoic
acid, NH2-CH(CH2-1-napthyl)-COOH, NH2-CH(CH2-2-napthyl)-COOH,
NH2-CH(CH2-cyclohexyl)-COOH, NH2-CH(CH2-cyclopentyl)-COOH,
NH2-CH(CH2-cyclobutyl)-COOH, NH2-CH(CH2-cyclopropyl)-COOH,
5,5,5-trifluoro~AuAin~ and hexafluoroleucine;
n = 1-3;
R3 is selected from the group consisting of 2-furyl, 2-furyl monosubstituted with
J, 2-pyridyl, 2-pyridyl monosubstituted with J, 3-pyridyl, 3-pyridyl monosubstituted with
J, 4-pyridyl, 4-pyridyl monos-lbslilu~ed with J, 2-quinolinyl, 2-quinolinyl monosubstituted
with J, 1-isoquinolinyl, 1-isoquinolinyl monosubstituted with J,
Q-- ¢~ ~N , ¢~
~ ~N~r N N ~;~ , O~3~,
~[ ,~ Me J~ NMe ~Me
o N N ,O N N N O
Me Me ' and HN NH
~(cH2)4coNH(cH2)2N
J is selected from the group consi~ g of halogen, OH, CN, N02, NH2, COOH,
CO2Me, CO2Et, CF3, Cl 4 alkoxy, Cl4 alkylamine, C2 ~ dialkylamine, Cl 4
perfluoroalkyl, and N(CH2CH2)2O;
WO 94/00095 PCI'/US93/06143
-- ~13812,^4,,,
-63 -
Dipeptide a-K~to~mides (Subclass C, Type 5) have the following structural
formula:
M4-(CH2) -O-CO-AA2-AAl-CO-NH-CH2CH(OH)-R
or a pharm~reutir~lly acceptable salt, wherein
M4 is selected from the group consisting of 2-furyl, 2-tetrahydrofuryl, 2-pyridyl,
2-pyrazinyl, 2-quinolinyl, 2-tetrahydroquinolinyl, 1-isoquinolinyl, and
1 -tetrahydroisoquinolinyl;
q = 0-2;
AA2 is an amino acid with the L configuration, D configuration, or DL
configuration at the a-carbon selected from the group consisting of alanine, valine,
leucine, isoleucine, proline, histidine, methionine, methionine sulfoxide, phenylalanine,
serine, threonine, phenylglycine, norleucine, norvaline, alpha-aminobutyric acid,
O-methylserine, O-ethylserine, S-methylcysteine, S-etnylcysteine, S-benzylcysteine,
NH2-CH(CH2CHEt2)-COOH, alpha-aminoheptanoic acid,
NH2-CH(CH2-cyclohexyl)-COOH, NH2-CH(CH2-cyclopentyl)-COOH,
NH2-CH(CH2-cyclobutyl)-COOH, NH2-CH(CH2-cyclopropyl)-COOH,
5,5,5-trifluoroleucine, and hexafluoroleucine;
AAl is an amino acid with the L configuration, D configuration, or DL
configuration at the a-carbon selected from the group consisting of alanine, valine,
leucine, isoleucine, proline, histidine, methionine, methionine sulfoxide, phenylalanine,
arginine, Iysine, tryptophan, glycine, serine, threonine, cysteine, tyrosine, asparagine,
gl~It~mine, aspartic acid, glutamic acid, phenylglycine, norleucine, norvaline,
alpha-aminobutyric acid, O-methylserine, O-ethylserine, S-methylcysteine,
S-ethylcysteine, S-benzylcysteine, NH2-CH(CH2CHEt2)-COOH, alpha-aminoheptanoic
acid, NH2-CH(CH2-1-napthyl)-COOH, NH2-CH(CH2-2-napthyl)-COOH,
NH2-CH(CH2-cyclohexyl)-COOH, NH2-CH(CH2-cyclopentyl)-COOH,
NH2-CH(CH2-cyclobutyl)-COOH, NH2-CH(CH2-cyclopropyl)-COOH,
5,5,5-trifluoroleucine, and hexafluoroleucine;
WO 94/00095 PCI`/US93/06143
~13~124 ` - -
-64-
R1 is selected from the group concicting of phenyl, phenyl monosubstituted with
J, phenyl f~ bi~l;luled with J, phenyl ~ itu~ed with J, pentafluorophenyl,
oR2 oR2 R20
5 ~ ~oR2 ~oR2 ~_oR2
1-naphthyl, 1-naphthyl mono~lb~l;tuled with J, 1-naphthyl disubstituted with J,
2-naphthyl 2-naphthyl monos~ ..(ed with J, 2-naphthyl disubstituted with J,
2-pyridyl, 2-quinolinyl, and 1-isoquinolinyl;
R2 ~ e~e.lts C14 alkyl substituted with phenyl, phenyl and phenyl substituted
with J.
J is selected from the group con~i~ling of halogen, OH, CN, NO2, NH2, COOH,
CO2Me, CO2Et, CF3, C14 alkoxy, C14 alkylamine, C2 8 dialkylamine, C14
pe~uoroalkyl, and N(CH2CH2)20;
Dipeptide a-Ketoamides (Subclass C, Type 6) have the following structural
formula:
M4~(CH2)q~0~CO~AA2~AAl~CO~NH~(CH2)n~R3
or a pharm~reutir~lly acceptable salt, wherein
M4 is selected from the group consisting of 2-furyl, 2-tetrahydrofuryl, 2-pyridyl,
2-pyrazinyl, 2-quinolinyl, 2-tetrahydroquinolinyl, 1-isoquinolinyl, and
1-tetral..ydl oisoquinolinyl;
q = 0-2;
AA2 is an amino acid with the L configuration, D configuration, or DL
configuration at the a-carbon selected from the group consisling of alanine, valine,
leucine, isoleucine, proline, histidine, methionine, methionine sulfoxide, phenylalanine,
serine, threonine, phenylglycine, norleucine, norvaline, alpha-aminobutyric acid,
O-methylserine, O-ethylserine, S-methylcysteine, S-ethylcysteine, S-benzylcysteine,
NH2-CH(CH2CHEt2)-COOH, alpha-aminoheptanoic acid,
NH2-CH(CH2-cyclohexyl)-COOH, NH2-CH(CH2-cyclopentyl)-COOH,
NH2-CH(CH2-cyclobutyl)-COOH, NH2-CH(CH2-cyclopropyl)-COOH,
S,S,S-trifluoroleucine, and hexafluoroleucine;
WO 94/00095 PCI/US93/06143
~ 1 3 8 1 2 ~
-65 -
AAl is an amino acid with the L configuration, D configuration, or DL
configuration at the a-carbon selected from the group consis~ g of alanine, valine,
leucine, isoleucine, proline, histidine, methionine, methionine sulfoxide, phenylalanine,
- arginine, lysine, lly~lophan, glycine, serine, threonine, cysteine, tyrosine, asparagine,
S ell~t~mine, aspartic acid, ~hlt~mir- acid, phenylglycine, norleucine, norvaline,
alpha-aminobutyric acid, O-methylserine, O-ethylserine, S-methylcysteine,
S-ethylcysteine, S-benzylcysteine, NH2-CH(CH2CHEt2)-COOH, alpha-aminoheptanoic
acid, NH2-CH(CH2-1-napthyl)-COOH, NH2-CH(CH2-2-napthyl)-COOH,
NH2-CH(CH2-cyclohexyl)-COOH, NH2-CH(CH2-cyclopentyl)-COOH,
NH2-CH(CH2-cyclobutyl)-COOH, NH2-CH(CH2-c~lo~l olJyl)-COOH,
5,5,5-trifluornlçurinç~ and hexafluoroleucine;
n= 1-3;
R3 is selected from the group cc~ l;..g of 2-furyl, 2-furyl monosubstituted withJ, 2-pyridyl, 2-pyridyl monosubstituted with J, 3-pyridyl, 3-pyridyl monosubstituted with
J, 4-pyridyL 4-pyridyl mono~ ;t~led with J, 2-quinolinyl, 2-quinolinyl monosubstituted
with J, 1-isoquinolinyl, 1-isoquinolinyl monosubstituted with J,
t , , M~ I ' I ' ~ '
o O Me
~ . ~ `N~Me
and HN NH
~(CH2)4CONH(CH2)2NI
J is selected from the group consisting of halogen, OH, CN, NO2, NH2, COOH,
C02Me, CO2Et, CF3, Cl4 alkoxy, C1 ~ alkylamine, C2 8 dialkylamine, Cl ,~
perfluoroalkyl, and N(CH2CH2)2O.
WO 94/00095 PCr/US93/06143
~38~
The Tripeptide a-K~to~milles have the following structural formula:
Ml-AA-AA-AA-CO-NR3R4
or a pharm~eutit ~lly acceptable salt, wherein
M1 r~lesents H, NH2-CO-, NH2-CS-, NH2-SO2-, X-NH-CO-, X2N-CO-,
X-NH-CS-, X2N-CS-, X-NH-SO2-, X2N-SO2-, X-CO-, X-CS-, X-SO2-, X-O-CO-, or X-
O-CS-;
X is sel~cted from the group consi~ .g of Cl 10 alkyl, Cl 10 fluoroalkyl, Cl 10
alkyl substituted with J, Cl 10 fluoroalkyl substituted with J, 1-admantyl, 9-fluorenyl,
phenyl, phenyl ~ub~ u(ed with K, phenyl f~i~ubstituted with K, phenyl trisubstituted
with K, naphthyl, naphthyl substituted with K, naphthyl disubstituted with K, naphthyl
t~ ed with K, C1 l0 alkyl with an attached phenyl group, Cl 10 alkyl with two
attached phenyl groups, Cl 10 alkyl with an att~hed phenyl group substituted with K,
C1 10 alkyl with two attached phenyl groups substituted with K, C1 10 alkyl with an
attached phenoxy group, and C1 10 alkyl with an attached phenoxy group substituted
uith K on the phenoxy group;
J is selected from the group consi~ling of halogen, COOH, OH, CN, NO2, NH2,
C1 10 alkoxy, C1 10 alkylamine, C2 12 dialkylamine. C1 10 alkyl-O-CO-~ C1-10 alkyl O CO
NH-, and C1 l0 alkyl-S-;
K is selected from the group consisting of halogen, C1 10 alkyl, C1 10
perfluoroalkyl, Cl l0 alkoxy, NO2, CN, OH, CO2H, amino, Cl l0 alkylamino, C2 12
dialkylamino, C1-C10 acyl, and C1 l0 alkoxy-CO-, and Cl 10 alkyl-S-;
AA is a side chain blocked or unblocked amino acid with the L configuration, D
configuration, or no chirality at the a-carbon selected from the group consisting of
alanine, valine, leucine, isoleucine, proline, methionine, methionine sulfoxide,phenylalanine, tryptophan, glycine, serine, threonine, cysteine, tyrosine, asparagine,
t~mine, aspartic acid, glut~mir acid, lysine, arginine, histidine, phenylglycine, beta-
alanine, norleucine, norvaline, a-aminobutyric acid, epsilon-aminocaproic acid,
citrulline, hyd~uAyl~loline, ornithine, homoarginine, sarcosine, indoline 2-carboxylic acid,
2-æetidinecarboxylic acid, pipecolinic acid (2-piperidine carboxylic acid), O-
methylserine, O-ethylserine, S-methylcysteine, S-ethylcysteine, S-benzylcysteine, NH2-
CH(CH2CHEt2)-COOH, a-aminoheptanoic acid, NH2-CH(CH2-1-napthyl)-COOH,
NH2-CH(CH2-2-napthyl)-COOH, NH2-CH(CH2-cyclohexyl)-COOH, NH2-CH(CH2-
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cyclopentyl)-COOH, NH2-CH(CH2-cyclobutyl)-COOH, NH2-CH(CH2-cyclopropyl)-
COOH, erifluoroleucine, and hexafluoroleucine;
R3 and R4 are selected independently from the group consisting of H, C1 20
alkyl, Cl 20 cyclized alkyl, Cl 20 alkyl with a phenyl group attached to the Cl 20 alkyl,
S Cl 20 cyclized alkyl with an attached phenyl group, Cl 20 alkyl with an attached phenyl
group substituted with K, Cl 20 alkyl with an attached phenyl group disubstituted with
K, Cl 20 alkvl with an attached phenyl group ~ ub~ uled with K, Cl 20 cyclized alkyl
with an attached phenyl group ~ub~l;tu(ed with K, Cl 10 alkyl with a morpholine
[-N(CH2CH2)O] ring attached through nitrogen to the alkyl, C1 10 alkyl with a
piperidine ring attached through nitrogen to the alkyl, C1 10 alkyl with a pyrrolidine ring
attached through nitrogen to the alkyl, C1 20 alkyl with an OH group attached to the
alkyl, -CH2CH2OCH2CH2OH, C1 l0 with an attached 4-pyridyl group, C1 l0 with an
attached 3-pyridyl group, C1 10 with an attached 2-pyridyl group, Cl 10 with an attached
cyclohexyl group, -NH-CH2CH2-(4-hydroxyphenyl), and -NH-CH2CH2-(3-indolyl).
The Tetrapeptide a-Ketoamides have the following structural formula:
Ml-AA-AA-AA-AA-CO-NR3R4
or a pharm~reuti~lly accept~ble salt, wherein
Ml leplesents H, NH2-CO-, NH2-CS-, NH2-S02-, X-NH-CO-, X2N-CO-,
X-NH-CS-, X2N-CS-, X-NH-SO2-, X2N-SO2-, X-CO-, X-CS-, X-SO2-, X-O-CO-, or X-
O-CS-;
X is selected from the group consisting of Cl l0 alkyl, Cl l0 fluoroalkyl, Cl l0alkyl substituted with J, Cl l0 fluoroalkyl substituted with J, l-admantyl, 9-fluorenyl,
phenyl, phenyl substituted with K, phenyl ~lin~bstituted with K, phenyl trisubstituted
with K, naphthyl, naphthyl substituted with K, naphthyl disubstituted with K, naphthyl
tri~ubstituted with K, C1 10 alkyl with an attached phenyl group, C1 l0 alkyl with two
attached phenyl groups, Cl 10 alkyl with an attached phenyl group substituted with K,
C1 10 alkyl with two attached phenyl groups substituted with K, C1 10 alkyl with an
attached phenoxy group, and C1 10 alkyl with an attached phenoxy group substituted
with K on the phenoxy group;
J is selected from the group consisting of halogen, COOH, OH, CN, NO2, NH2,
C] 10 aLI~oxy, Cl 10 alkylamine, C2 l2 dialkylamine, C1 10 alkyl-O-CO-, C1 10 alkyl O
NH-, and C1 10 alkyl-S-;
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K is selected from the group consi~ ,g of halogen, C1 10 alkyl, C1 10
perfluoroalkyl, C1 10 alkoxy, NO2, CN, OH, CO2H, amino, C1 10 alkylamino, C2 12
dialkylamino, C1-C10 acyl, and C1 10 alkoxy-CO-, and C1 10 alkyl-S-;
AA is a side chain blocked or unblocked amino acid with the L configuration, D
configuration, or no chirality at the a-carbon selected from the group consisting of
alanine, valine, leucine, icolell~ine, proline, methionine, methionine sulfoxide,
phenylalanine, tryptophan, glycine, serine, threonine, cysteine, tyrosine, asparagine,
glllt~mine, aspartic acid, glut~mic acid, lysine, arginine, histidine, phenylglycine, beta-
alanine, norleucine, norvaline, a-aminobutyric acid, epsilon-aminocaproic acid,
citrulline, hydtu~y,u,oline, ornithine, homoarginine, sarcosine, indoline 2-carboxylic acid,
2-azetidinecarboxylic acid, pipecolinic acid (2-piperidine carboxylic acid), O-
methylserine, O-ethylserine, S-methylcysteine, S-ethylcysteine, S-benzylcysteine, NH2-
CH(CH2CHEt2)-COOH, c~-aminoheptanoic acid, NH2-CH(CH2-l-napthyl)-COOH,
NH2-CH(CH2-2-napthyl)-COOH, NH2-CH(CH2-cyclohexyl)-COOH, NH2-CH(CH2-
cyclopentyl)-COOH, NH2-CH(CH2-cyclobutyl)-COOH, NH2-CH(CH2-cyclopropyl)-
COOH, trifluoroleucine, and hexafluoroleucine;
R3 and R4 are selected independently from the group consisting of H, C1 20
alkyl, C1 20 cyclized alkyl, C1 20 alkyl with a phenyl group attached to the C1 20 alkyl,
C1 20 cyclized alkyl with an attached phenyl group, C1 20 alkyl with an attached phenyl
group substituted with K, Cl 20 alkyl with an attached phenyl group ~licub5tituted with
K, Cl 20 alkyl with an attached phenyl group trisubstituted with K, Cl 20 cyclized alkyl
with an attached phenyl group substituted with K, C1 l0 alkyl with a morpholine
[-N(CH2CH2)O] ring attached through nitrogen to the alkyl, C1 10 alkyl with a
piperidine ring attached through nitrogen to the alkyl, C1 10 alkyl with a pyrrolidine ring
attached through nitrogen to the alkyl, C1 20 alkyl with an OH group attached to the
alkyl, -CH2CH2OCH2CH2OH, C1 10 with an attached 4-pyridyl group, C1 10 with an
attached 3-pyridyl group, C1 10 with an attached 2-pyridyl group, C1 10 with an attached
cydohexyl group, -NH-CH2CH2-(4-hydroxyphenyl), and -NH-CH2CH2-(3-indolyl).
The Amino Acid ~-Ketoamides have the following structural formula:
M1-AA-CO-NR3R4
or a pharmaceutically acceptable salt, wherein
~vo 94/00095 PCI/US93/06143
1 2 ~ ~ `
-69-
Ml le~"esents H, NH2-CO-, NH2-CS-, NH2-SO2-, X-NH-CO-, X2N-CO-,
- X-NH-CS-, X2N-CS-, X-NH-SO2-, X2N-SO2-, X-CO-, X-CS-, X-SO2-, X-O-CO-, or X-
O-CS-;
X is selected from the group collsi~ling of C1 10 alkyl, C1 10 fluoroalkyl, C1 10
alkyl sul.~ u(ed with J, Cl 10 fluoroalkyl substituted with J, 1-admantyl, 9-fluorenyl,
phenyl, phenyl ~ l;luled with K, phenyl lic-lbstituted with K, phenyl trisubstituted
with K, naphthyl, naphthyl substituted with K, naphthyl disubstituted with K, naphthyl
ted with K, Cl 10 alkyl with an attached phenyl group, Cl 10 alkyl with two
attached phenyl groups, C1 10 alkyl with an attached phenyl group subs-ituted with K,
C1 l0 alkyl with two att~rhed phenyl groups substituted with K, C1 10 alkyl with an
attached phenoxy group, and C1 10 alkyl with an attached phenoxy group substituted
with K on the phenoxy group;
J is selected from the group co..si~ ,g of halogen, COOH, OH, CN, NO2, NH2,
C1 10 alkoxy, C1 10 alkylamine, C2 12 dialkylamine, C1 10 alkyl-O-CO-, C1 l0 alkyl-O-CO-
NH-, and C1 10 alkyl-S-;
K is selected from the group consisting of halogen, Cl l0 alkyl, C1 10
perfluoroalkyl, C1 10 alkoxy, NO2, CN, OH, CO2H, amino, C1 10 alkylamino, C2 12
dialkylamino, C1-C10 acyl, and C1 10 alkoxy-CO-, and C1 10 alkyl-S-;
AA is a side chain blocked or unblocked amino acid with the L configuration, D
configuration, or no chirality at the a-carbon selected from the group consisting of
alanine, valine, leucine, i~ol~u~ine, proline, methionine, methionine sulfoxide,phenylalanine, tryptophan, glycine, serine, threonine, cysteine, tyrosine, asparagine,
~lut~mine, aspartic acid, ~ut~mic acid, lysine, arginine, histidine, phenylglycine, beta-
alanine, norleucine, norvaline, a-aminobutyric acid, epsilon-aminocaproic acid,
citrulline, hydroxyproline, ornithine, homoarginine, sarcosine, indoline 2-carboxylic acid,
2-azetidinecarboxylic acid, pipecolinic acid (2-piperidine carboxylic acid), O-
methylserine, O-ethylserine, S-methylcysteine, S-ethylcysteine, S-benzylcysteine, NH2-
CH(CH2CHEt2)-COOH, a-aminoheptanoic acid, NH2-CH(CH2-1-napthyl)-COOH,
NH2-CH(CH2-2-napthyl)-COOH, NH2-CH(CH2-cyclohexyl)-COOH, NH2-CH(CH2-
cyclopentyl)-COOH, NH2-CH(CH2-cyclobutyl)-COOH, NH2-CH(CH2-cyclopropyl)-
COOH, trifluoroleucine, and hexafluoroleucine;
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R3 and R4 are selected independently from the group consisting of H, C1 20
alkyL C1 20 cyclized alkyl, C1 20 alkyl with a phenyl group attached to the Cl 20 alkyl,
Cl 20 cyclized allcyl with an attached phenyl group, Cl 20 alkyl with an attached phenyl
group ,~ ;luled with K, Cl 20 alkyl with an attached phenyl group disubstituted with
K, Cl 20 alkyl with an ~tt:-^hed phenyl group tr;~ ed with K, Cl 20 cyclized alkyl
with an attached phenyl group ~ b~ ed with K, Cl 10 alkyl with a morpholine
[-N(CH2CH2)O] ring attached through nitrogen to the alkyl, C1 10 alkyl with a
pi~ ;dil~e ring attached through nitrogen to the alkyl, Cl 10 alkyl with a pyrrolidine ring
att:~hed through nitrogen to the alkyl, Cl 20 alKyl with an OH group attached to the
alkyl, -CH2CH20CH2CH20H, Cl 10 with an attached 4-pyridyl group, Cl 10 with an
attqched 3-pyridyl group, C1 10 with an attached 2-pyridyl group, C1 10 with an attached
cyclohexyl group, -NH-CH2CH2-(4-h~dlu~h/_~yl), and -NH-CH2CH2-(3-indolyl).
The Applicants are aware of only a single peptide ketoamide reported in the
literature. This compound is Z-Phe-NHCH2CO-CO-NH-Et (Z-Phe-Gly-CO-NH-Et).
The compound is reported by Hu and Abeles (supra) to be an inhibitor of papain (Ki =
1.5 mM) and cathepsin B (Ki = 4 mM).
The following Peptide K~to~mide compounds are representative of the Peptide
Keto-Compounds found to be useful as Calpain inhibitors within the context of the
present invention:
Z-Leu-Phe-CONH-Et
Z-Leu-Phe-CONH-nPr
Z-Leu-Phe-CONH-nBu
Z-Leu-Phe-CONH-iBu
Z-Leu-Phe-CONH-Bzl
Z-Leu-Phe-CONH-(CH2)2Ph
Z-Leu-Abu-CONH-Et
Z-Leu-Abu-CONH-nPr
Z-Leu-Abu-CONH-nBu
Z-Leu-Abu-CONH-iBu
Z-Leu-Abu-CON~I-Bzl
Z-Leu-Abu-CONH-(CH2)2Ph
Z-Leu-Abu-coNH-(cH2)3-N(cH2cH2)2o
WO 94/00095 PCI /US93/06143
'- ~13~12li
Z-Leu-Abu-CONH-(CH2)7CH3
Z-Leu-Abu-CONH-(CH2)20H
Z-Leu-Abu-coNH-(cH2)2o(cH2)2oH
Z-Leu-Abu-CONH-(CH2)l7CH3
S Z-Leu-Abu-CONH-CH2-C6H3[3,5-(OCH3)2]
Z-Leu-Abu-CONH-CH2-C4H4N
Z-Leu-Abu-CONH-(CH2)sOH
Z-Leu-Abu-CONH-CH2CH(OCH3)2
Z-Leu-Abu-CONH-CH2CH(OC2Hs)2
Z-Leu-Abu-CONH-CH2-C6H8[1,3,3-(CH3)3-5-OH]
Z-Leu-Abu-CONH-(CH2)2C6H4(4-OH)
Z-Leu-Abu-CONH-(CH2)2C6H4(2-OCH3)
Z-Leu-Abu-CONH-(CH2)2C6H4(3-OCH3)
Z-Leu-Abu-CONH -(CH2)2C6H4(4-OCH3)
Z-Leu-Abu-CONH-CH2CH(OH)Ph
Z-Leu-Abu-CONH-CH2CH(OH)C6H4(4-OCH3)
Z-Leu-Abu-CONH-CH2CH(OH)C6H2[2,4,6-(OCH3)3]
Z-Leu-Abu-CONH-CH2CH(OH)C6H4[4-N(CH3)2]
Z-Leu-Abu-CONH-CH2CH(OH)C6Fs
Z-Leu-Abu-CONH-CH2CH(OH)C6H4(3-CF3)
Z-Leu-Abu-CONH-CH2CH(OH)C6H4(3-OPh)
Z-Leu-Abu-CONH-CH2CH(OH)C6H4(4-OPh)
Z-Leu-Abu-CONH-CH2CH(OH)C6H4(4-OCH2Ph)
Z-Leu-Abu-CONH-CH2CH(OH)C6H4-3-OC6H4(3-CF3)
Z-Leu-Abu-CONH-CH2CH(OH)C6H4-3-OC6H3(3,4-Cl2)
Z-Leu-Abu-CONH-CH2CH(OH)C6H3[3,4-(OCH2Ph)2]
Z-Leu-Abu-.CONH-CH2CH(OH)- l-CloH7
Z-Leu-Abu-CONH-CH2CH(OH)-2-ClOH7
Z-Leu -Phe-CONH-CH2CH(OH)Ph
Z-Leu-Phe-CONH-CH2CH(OH)C6H4[4-N(CH3)2]
Z-Leu-Phe-CONH-CH2CH(OH)C6Fs
Z-Leu-Phe-CONH-CH2CH(OH)C6H4(3-CF3)
WO 94/00095 PCr/US93/06143
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Z-Leu-Phe-CONH-CH2CH(OH)C6H4(3-OPh)
Z-Leu-Phe-CONH-CH2CH(OH)C6H4(4-OPh)
Z-Leu-Phe-CONH-CH2CH(OH)C6H4(4-OCH2Ph)
Z-Leu-Phe-CONH-CH2CH(OH)C6H4-3-OC6H4(3-CF3)
S Z-Leu-Phe-CONH-CH2CH(OH)C6H4-3-OC6H3(3,4-C12)
Z-Leu-Phe-CONH-CH2CH(OH)C6H3(3,4-(OCH2Ph)2)
Z-Leu-Abu -CONH-CH2-2-furyl
Z-Leu-Abu-CONH-CH2-2-tetrahydrofuryl
Z-Leu-Abu-CONH-CH2-2-pyridyl
Z-Leu-Abu-CONH-CH2-3-pyridyl
Z-Leu-Abu-CONH-CH2-4-pyridyl
Z-Leu-Abu-CONH-(CH2)2-2-pyridyl
Z-Leu-Abu-CONH-CH2-2-pyridyl(3-COOCH3)
Z-Leu-Abu-CONH-CH2-2-pyridyl(S-COOCH3)
Z-Leu-Abu-CONH-(CH2)2-2-(N-methylpyrrolyl)
Z-Leu-Abu-CONH-(CH2)3- 1-imidazolyl
Z-Leu-Abu-CONH-(CH2)2-4-morpholinyl
Z-Leu-Abu-CONH-(CH2)3-4-morpholinyl
Z-Leu-Abu-CONH-(CH2)3- 1 -pyrrolidinyl-2-one
Z-Leu-Abu-CONH-CH2)2-3-indolyl
Z-Leu-Abu-CONH-CH2-2-quinolinyl
Z-Leu-Abu-CONH-CH2- 1-isoquinoline
Z-Leu-Abu-CONH-(CH2)3- 1-tetrahydroquinolinyl
Z-Leu-Abu-CONH-(CH2)3-2-tetrahydroisoquinolinyl
Z-Leu-Abu-CONH-CH2-8-caffeinyl
Z-Leu-Abu-CONH-CH2-2-(4-methyl-2-thiazolyl)
Z-Leu-Abu-CONH-CONH-(CH2)2NH-biotinyl
Z-Leu-Abu-CONH-CH2-3-pyridyl-N-oxide
Z-Leu-Abu-CONH-CH2-6-uracil
Z-Leu-Phe-CONH-CH2-2-pyridyl
Z-Leu-Phe-CONH-(CH2)3-4-morpholinyl
Z-Leu-Phe-CONH-CH2-2-quinolinyl
WO 94/00095 ~ l 3 8 1 2 ~ PCI/US93/06143
. .
Z-Leu-Phe-CONH-CH2- 1-isoquinolinyl
Z-Leu-Phe-CONH-(CH2)3- 1-tetrahydroquinolinyl
Z-Leu-Phe-CONH-(CH2)3-2-tetrahydroisoquinolinyl
Z-Leu-Phe-CONH-(CH2)2-NH-biotinyl
S Z-Leu-Nva-CONH-CH2CH(OH)Ph
Z-Leu-Nva -CONH-CH2-2-pyridyl
Z-Leu-Nva-CONH-(CH2)3-4-morpholinyl
CH30CO(CH2)2CO-Leu-Abu-CONHEt
2-furyl-CO-Leu-Abu-CONHEt
2-tetrahydrofuryl-CO-Leu-Abu-CONHEt
3-pyridyl-CO-Leu -Abu -CONHEt
2-pyrazyl-CO-Leu-Abu-CONHEt
2-quinolinyl-CO-Leu-Abu-CONHEt
1 -isoquinolinyl-CO-Leu-Abu -CONHEt
4-morpholinyl-CO-Leu-Abu-CONHEt
Ph(CH2)2CO-Leu-Abu-CONHEt
1 -ClOH7CH2CO-Leu-Abu-CONHEt
Ph2CHCO-Leu-Abu -CONHEt
Ph2CHCO-Leu-Abu-CONH-CH2CH(OH)Ph
Ph2CHCO-Leu-Abu-CONH-CH2-2-pyridyl
Ph2CHCO-Leu-Abu-CONH-(CH2)3-4-morpholinyl
Ph2CHCO-Leu-Phe-CONH-CH2CH(OH)Ph
Ph2CHCO-Leu-Phe-CONH-CH2-2-pyridyl
Ph2CHCO-Leu-Phe-CONH-(CH2)3-4-morpholinyl
We studied the inhibition mech~nicm of the Peptide Keto-Compounds in both
serine and thiol proteases. A crystal structure of one a-ketoester bound into the active
site of the serine protease, porcine pancreatic ~ t:~ce, has been completed. The active
site Ser-195 oxygen of the enzyme adds to the carbonyl group of the ketoester to form
a tetrahedral intermediate which is stabilized by interactions with the oxyanion hole.
This structure resembles the tetrahedral intermediate involved in peptide bond
hydrolysis and proves that a-ketoesters are transition-state analogs. His-57 is hydrogen
bonded to the carbonyl group of the ester functional group, the peptide backbone on a
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section of the PPE polypeptide backbone hydrogen bonds to the inhibitor to form a
B-sheet, and the benzyl ester is directed toward the S' subsites. The side chain of the
P1 amino acid residue is located in the S1 pocket of the enzyme. Interactions with
k~to~micles would be similar except that there is the possibility of forming an additional
l,~dloge,l bond with the NH group of the ketoamide functional group. If R is a longer
substitu.ont, then it would make favorable interactions with the S' subsites of the
enzyme. s
_ Ser-1g5
0~`lr N ~ ~1~ o l~ 3 S' subsites
0 o H ,0; ~H
H ,~ ~hole ~ Hjs-57
Val-216 Ser-214
Phe-214
In the case of keto~ c~ there would be no R group to interact with the S'
sub~it~s Therefore, these inhibitors would be expected to be slightly less potent than
the ketoesters and ketoamirlec However, unexpectedly, certain ketoacid compoundshave been found to have surprisingly high activity when used in the context of the
present invention. In particular, Z-Leu-Phe-COOH and Z-Leu-Abu-COOH have been
found to be extremely potent inhibitors of C~lp~in~
The active site of cysteine proteases shares several features in common with
serine proteases in~lurling an active site histidine residue. In place of the Ser-19~,
cysteine proteases have an active site cysteine residue which would add to the ketonic
carbonyl group of the peptide ketoa~ c/ ketoesters, or ketoamides to form an adduct
very similar to the structure described above except with a cysteine residue replacing
the serine-195 residue. Additional interactions would occur between the extendedsubstrate binding site of the cysteine protease and the inhibitor that would increase the
binding affinity and specificity of the inhibitors.
The Peptide Keto-Compounds bind to the proteases inhibited thereby using
many of the interactions that are found in complexes of a particular individual enzyme
with its substrates. In order to design an inhibitor for a particular cysteine protease, it
is necessa,y to: 1) find the amino acid sequences of good peptide substrates for that
enzyme, and 2) place those or similar amino acid sequences into a Peptide Keto-
WO 94/00095 2 ~ 3 8 1 ~ ~ PCI /US93/06143
Compound. This design strategy will also work when other classes of peptide inhibitors
are used in place of the peptide substrate to gain information on the appropliate
sequen~e to place in the Peptide Keto-Compound inhibitor. Thus, we are able to
predict the structure of new inhibitors for other proteases based on knowledge of their
substrate specificities. Once a good inhibitor structure for a particular enzyme is
found, it is then possible to change other characteristics such as solubility orhydrophobicity by adding subs-itu~nts to the M or R groups.
Ad-litionql interactions with the enzyme can be obtained by tailoring the R
group of the inhihitor to imitate the amino acid residues which are preferred by an
individual protease at the S1' and S2' subsites. For example, ketoamides with R =
alkyl substituted with phenyl would interact effectively with serine and cysteine
proteases which prefer Phe, Tyr, Trp residues at P1' and/or P2'. Likewise, the M1
group can be tailored to interact with the S subsites of the enzyme. This designstrategy will also work when other classes of peptide inhibitors are used in place of the
peptide substrate to gain information on the app~ liate sequence to place in thek~toqmide inhibitor. Thus, we are able to predict the structure of new inhibitors for
other serine and cysteine proteases based on knowledge of their substrate specificities.
Once a good inhibitor structure for a particular enzyme is found, it is then possible to
change other characteristics such as solubility or hydrophobicity by adding substituents
to the M1 or R groups.
In the case of Calpain, a cysteine protease, a known inhibitor sequence is the
peptide aldehyde, Ac-Leu-Leu-Me-H (also known as Calpain Inhibitor 1 and
hereinafter designated as "CI1"). This inhibitor, in addition to a related peptide
aldehyde inhibitor Ac-Leu-Leu-Nme-H (also known as Calpain Inhibitor II) are
commercially available from Calbiochem of La Jolla, California. We have discovered
that peptide -ketoesters with aromatic amino acid residues in P1 are good inhibitors
of the thiol proteases, cathepsin B, papain and Calpain. Additionally, we have
discovered that peptide a-ketoester and peptide -ketoamides with either aromatic
amino acid residues or small hydrophobic alkyl amino acid residues at P1 are good
inhibitors of Calpain.
Our discovery of Peptide Keto-Compounds effective as Calpain Inhibitors was
made through assay of the Peptide Keto-Compounds as reversible inhibitors. Various
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concentrations of inhibitors in dimethylsulfoxide (DMSO) were added to the assaymixture, which contained buffer and substrate. The reaction was started by the
addition of the enzyme and the hydrolysis rates were followed spectrophotometrically
or fluorimetrically. 88 mM KH2PO4, 12 mM Na2HPO4, 1.33 mM EDTA, 2.7 mM
cysteine, pH 6.0 was used as a buffer for cathepsin B; and 20 mM Hepes, 10 mM
CaCl2, 10 mM B-mercaptoethanoL pH 7.2 buffer was utilized for calpain I and calpain
LI.
All peptide thioester hydrolysis rates were measured with assay mixtures
cont~ining 4,4'-dithiodipyridine (e324 = 19800 M~lcm~1; Grasetti & Murray, Arc~t.
Biochel?~ Biophys., 119:41-49 (1967)). Papain was assayed with Bz-Arg-AMC or
Bz-Arg-NA (Kanaoka et al., Cllem. Phan~ BulL, 25:3126-3128 (1977)), and the AMC
(7-amino-4-methylcoumarin) release was followed fluorimetrically (excitation at 380 nm,
and f~mi~inn at 460 nm). Cathepsin B was assayed with Z-Arg-Arg-AFC (Barrett andKirschke, Methods Enymo~, 80:535-561 (1981)), and the AFC (7-amino-4-
trifluoromethylcoumarin) release was followed fluorimetrically (~Yrit~tion at 400 nm,
and emission at 505 nm). Calpain I from human erythrocytes and calpain II from
rabbit were assayed using Suc-Leu-Tyr-AMC (Sasaki et al., J. Biol. C~lem.
259:12489-12494 (1984), hereby incorporated by reference), and the AMC (7-amino-4-
methylcoumarin) release was followed fluorimetrically (excitation at 380 nm, andemission at 460 nm). Enzymatic hydrolysis rates were measured at various substrate
and inhihitnr concentrations, and Ki values were determined by Dixon plot.
Table PKC1 shows the inhibition constants (Ki) for papain, cathepsin B, calpain
I, and calpain II.
The inhibition constants for papain shown in Table PKC1 were measured in
0.05 M Tris-HCl, pH 7.5 buffer, containing 2mM EDTA, SmM cysteine (freshly
prepared), 1% DMSO, at 25C, using NQ-Benzoyl-Arg-AMC as a substrate, except that
those values of inhihitinn constants for papain marked with an "e" in Table PKC1 were
measured in 50 mM Tris-HCl, pH 7.5 buffer, containing 20 mM EDTA, 5 mM cysteine,9% DMSO, at 25C, using N-Benzoyl-Arg-NA as a substrate.
w~ 94/00095 PCl`/US93/06143
21'~12~
TABLE PKC1
Inhibition of Cysteine Proteases by
Peptide Ketoesters and Ketoacids
S Kj(l~M)
Compounds
pa cgb cIc CIId
Z-Leu-Abu-COOEt 0.04 0.4
Z-Leu-Phe-COOEt 0.23 0.4
Z-Leu-Me-COOEt 0.12 0.18
Z-Leu-Nva-COOEt 30 1.2
Bz-DL-Phe-COOEt 500e 64
Z-Phe-DL-Phe-COOEt 1.8 0.1
Z-Phe-DL-Ala-COOEt 3.6 3.2
Z-Ala-Ala-DL-Ala-COOEt 1.5 2.2 200
Z-Ala-Ala-DL-Abu-COOEt 0.9 10 50 200
Z-Ala-Ala-DL-Abu-COOBzl 30 60
Z-Ala-Ala-DL-Nva-COOEt 30 0.1
Z-Ala-Pro-DL-Ala-COOEt 26 66
MeO-Suc-Val-Pro-DL-Phe-COOMe 1.1 0.1
2.9e
Z-Ala-Ala-Ala-DL-Ala-COOEt 2.1 10.0
MeO-Suc-Ala-Ala-Pro-Abu-COOMe 0.7 6.0 100
ap = Papain CCI = Calpain I
- bCB = Cathepsin B dCII = Calpain II
WO 94~00095 PCI/US93/0614
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It can be seen from the data in Table PKC1 that the dipeptide ketoesters with
Abu, Phe, or Nle in the P1 site and Leu in the P2 site are potent inhibitors of calpain I
and calpain II. Tripeptides with Abu or Ala in the P1 site and Ala in the P2 site are
also seen to be inhibitors of Calpain, albeit somewhat weaker inhibitors than the
dipeptides. Thus, in accordance with the foregoing description of the design of Peptide
Keto-Compound inhibitors, we believe that Peptide Keto-Compounds based on these
and similar structures will exhibit Calpain inhibitory activity.
Assay of Inhibitory Potency of Pephde a-ketoamides HEPES, heparin, and
A23187 were obtained from Calbiochem. Suc-Leu-Tyr-AMC and chromogenic
substrates were obtained from Sigma. Calpain I was purified from human erythrocytes
according to the method of Kitahara (Kitahara, et a~, J. Biochem. 95:1759-1766 (1984))
omitting the Blue-Sepharose step. Calpain II from rabbit muscle and cathepsin B were
purchased from Sigma. Papain was pur~l,ased from Calbiochem.
Peptide a-ketoqmid~s were assayed as lcv~sible enzyme inhibitors. Various
cunrel-t.dtions of inhihitors in Me2SO were added to the assay mixture which contained
buffer and substrate. The reaction was started by the addition of the enzyme and the
hydrolysis rates were followed spectrophotometrically or fluorimetrically.
Calpain I from human erythrocytes and calpain II from rabbit were assayed
using Suc-Leu-Tyr-AMC (Sasaki et aL, J. Biol. ale~7t~ 259:12489-12494 (1984);
incorporated herein by reference), and the AMC (7-amino-4-methylcoumarin) release
was followed fluorimetrically (excitation at 380 nm, and emmision at 460 nm). Calpains
were assayed in 25 mM Tris pH = 8.0, 10 mM CaC12. Fluorescence was followed
using a Gilson FL-lA fluorometer or a Perkin-Elmer 203 Fluorescence spectrometer.
Cathepsin B was assayed in 20 mM sodium acetate pH = 5.2, 0.5 m~ dithiothreitol
using Bz-Phe-Val-Arg-p-nitroanilide as substrate. Alternately, cathepsin B was assayed
with Z-Arg-Arg-AFC (Barrett and Kirschke, Metl~od~ Enymo~ 80:535-561 (1981);
incol~olated herein by reference), and the AFC (7-amino-4-trifluoromethylcoumarin)
release was followed fluorimetrically (excitation at 400 nm and emmision at 505 nm).
Papain was assayed in 100 mM KPO4, 1 mM EDTA, 2.5 mM cysteine pH = 6.0 using
Bz-Arg-AMC or Bz-Arg-NA (Kanaoka et aL, Clzent. Phamt. BulL 25:3126-3128 (1977);incorporated herein by reference) as a substrate. The AMC
(7-amino-4-methylcoumarin) release was followed fluorimetrically (excitation at 380 nm,
~"') 94/00095 2 1 3 ~ 12 ~ PCI/US93/06143
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and .ornmic;nn at 460 nm). Enzymatic hydrolysis rates were measured at various
substrate and inhibitor concentrdtions, and Ki values were determined by either
Li..e~-e~vef-Burk plots or Dixon plots.
A 0.1 M Hepes, 0.5 M NaCl, pH 7.5 buffer was utilized for human leukocyte
elastase (HLE), porcine pancreatic elastase tPPE), chymotrypsin and cathepsin G. A
0.1 Hepes, 0.01 M CaC12, pH 7.5 buffer was utilized for trypsin, plasmin, and
co~g~ ti~n c,~,..es. A 50 mM Tris.HCl, 2 mM EDTA, S mM cysteine, pH 7.5 was
used as a buffer for papain. A 88 mM KH2PO4, 12 mM Na2HPO4, 1.33 mM EDTA,
2.7 mM cysteine, pH 6.0 solution was used as a buffer for cathepsin B. A 20 mM
Hepes, 10 mM CaC12, 10 mM mercatoethanol, pH 7.2 buffer was utilized for calpain I
and calpain II.
HLE and PPE were assayed with MeO-Suc-Ala-Ala-Pro-Val-NA and
Suc-Ala-Ala-Ala-NA, re~l,ce~ively (Nakajima et al., J. Biol. Chen~ 254:4027-4032 (1979);
incorporated herein by reference). Human leukocyte cathepsin G and chymotrypsin Aa
were assayed with Suc-Val-Pro-Phe-NA (Tanaka et aL, Bioche~nistry 24:2040-2047
(1985); inco.~ol~ted herein by reference). The hydrolysis of peptide 4-nitro~nilid~ was
measured at 410 nm (e410 = 8800 M~1cm~l; Erlanger et al., Arch. Biocller7t. Biopllys.
95:271-278 (1961); incorporated herein by reference). Trypsin, thrombin, human
plasma kallikrein, porcine pancreatic kallikrein, human factor XIa, and human plasmin
were assayed with Z-Arg-SBzl or Z-Gly-Arg-SBu-i (McRae et aL, Biochemistry
20:7196-7206 (1981); incorporated herein by reference). All peptide thioester
hydrolysis rates were measured with assay mixtures containing 4,4'-dithiodipyridine
(e324 = 19800 M~1cm~1; Grasetti & Murray,Arch. Biocllem. Biopllys. 119:41-49 (1967);
incorporated herein by reference).
Structure-Activity Relationships. Table PKC2 shows the inhibition constants (Ki)for calpain I, calpain II and cathepsin B. Changing the R group on the amide
~i~ifir~ntly i~ v~s the inhibitory potency toward calpains. Dipeptide a-ketoamides
with Abu, Phe, and Nva in the P1 site and Leu in the P2 site are potent inhibitors of
these cysteine proteases. The presence of a hydrogen bond donor in the S1' subsite of
the cysteine proteases which may be interacting with the N-H on the ketoamide
functional group is indicated since ~licubstituted amides were much less effective
inhibitors. Derivatives of Z-Leu-AA-CONHR where the R group contained a hydroxy
WO 94/0009~ PCI /US93/06143
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or allcoxy ~,roup, such as (CH2)sOH and CH2CH(OC2Hs)2, are very good inhibitors of
the ~lp~in~ The prescence of an aromatic ~roup in P1' position of the peptide
ketoami~e inhibitor resulted in improved inhibitory potency for calpains which indicates
the prescence of hydrophobic residues in the S' subsites of both c~lp~in~ The
derivatives Z-Leu-AA-CONH(CH2)nR where R was phenyl, phenyl substituted with
hydroxy or alkoxy ~,roups and naphthyl, are also very good inhibitors of calpains and
cathep~in B. Derivatives of Z-Leu-Abu-CONH(CH2)nR where the R ~roup contained
a heterocylic group which has both a hydrophobic moiety with an electronnegativeatom, are among the best inhibitors for calpains and cathepsin B. For example
Z-Leu-Nva-CONHCH2-2-pyridyl is the best inhibitor of calpain I.
Z-Leu-Abu-CONHCH2-2-pyridyl is the best inhibitor of calpain II respectively in this
series, but its isomers, Z-Leu-Abu-CONH-CH2-3-pyridyl and
Z-Leu-Abu-CONH-CH2-4-pyridyl, are substantially poorer inhibitors.
TABLE PKC2. Tr~!' of Cysteine Proteases by Peptide a-}~toq ;~ with the
Slr~ es Z-Leu~ CONHR.
R Kj (llM)
Cal I Cal II Cat B
AA = a-aminobutyric acid
(CH2)2oH 0.8 0.078
4.5
(CH2)5OH 0 5 0.051
0.28
(CH2)2O(CH2)2OH 0.65 0.16
2.0
CH2CH(OCH3)2
CH2CH(Oc2Hs)2 0.2
CH2-C6H8( 1,3,3-(CH3)3-5-OH) 0.42 0.069
0.89
(CH2)2C6H4(4-OH) 0.38 0.06
(CH2)2C6H4(2-OcH3) 0.13 0.16
0.63
- ~13 ~12 4 PCl/US93/06143
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(CH2)2C6H4(3-OcH3) 0.11 0.086
0.31
(CH2)2C6H4(4-OCH3) 0.12 0.046
O.M
CH2C6H3(3,5-(OcH3)2) 2.3 0.022
1.8
CH2-2-furyl 0.80 0.033
6.0
CH2-2-tetral,y-J- orulyl 0.33 0.066
10 4.5
CH2-2-pyridyl 0.64 0.017
3.0
CH2-3-pyridyl 0.12
1.2
CH2-4-pyridyl 1.1 0.11
6.4
(CH2)2-2-pyridyl 0.41 0.47
0.20
CH2-2-pyridyl(3-COOCH3) ca. l lO
CH2-2-pyridyl(5-COOCH3) ca.28
(CH2)2-2-(N-methylpyrrole) 0.16 0.076
1.2
(CH2)3-l-imi~7~-1yl 0.29 0.068
9.9
(CH2)24-morpholinyl 1.0 0.16
2.5
(CH2)3-4-morpholinyl 0.14 0.041
6.9
- (CH2)3-l-pyrrolidine-2-one 1.2 0.27
30 2.0
(CH2)2-3-indolyl 0.3 0.05
CH2-2-quinolinyl 0.13
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CH2-1-isoquinolinyl 0.25
0.3
(CH2)3-1-tetrahydroquinolinyl 0.37
(CH2)3-2-tetrahydroisoquinolinyl 0.3 1
CH2-8-caffeine 32.0
CH2-2-(4-methylthiazole) 34.0
(CH2)2NH-biotinyl 0.65
CH2-3-pyridyl-N-oxide 9.5
CH2-6-uracil 9.0
AA = phenylalanine
CH2-2-pyridyl 0.65
0.27
(CH2)3-4-morpholinyl 0.22
CH2-2-quinolinyl 0.11 0.023
0.34
CH2- 1-isoquinolinyl 2.4
9.6
(CH2)3-1-tetrahydroquinolinyl 0.38
(CH2)3-2-tetrahydroisoquinolinyl 0.22
(CH2)2NH-biotinyl 0.22
AA = Norvaline
CH2-2-pyridyl 0.019 0.12
(CH2)3-4-morpholinyl 0.25 0.10
4.2
Table PKC3 shows the inhibition constants (Kj) of Z-Leu-AA-
CONH-CH2CH(OH)R. The hydrophobic moiety substituted with CH2CH-X (X =
electronegative atoms such as O, N) resulted in good inhibitor structures.
Z-Leu-Abu-CONH-CH2CH(OH)C6Fs is the best inhibitor for calpain I, and
Z-Leu-Abu- CONH-CH2CH(OH)Ph is the best inhibitor for calpain II respectively inthis series.
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TABLE PKC3. Tr~ ;ti~ of Cysteine Proleases by Peptide a-Ketoq ides with the
Slru~lur~s Z-Leu-AA-CONH-CH2CH(OH)-R.
R Ki
Cal I Cal IICat B
AA = a-aminobutyric acid
Ph 1.1 0.0150.37
C6H4(4-OCH3) 0.24
C6H2(2,4,6-(OCH3)3) 0.38
C6H4(4-N(CH3)2)
C6Fs 0.05
C6H4(3-CF3) 0.35
C6H4(3-OPh) 0.90
C6H4(4-OPh) 0.10
C6H4(4-OcH2ph) 0.08
C6H4-3-OC6H4(3-CF3) 0 07
C6H~ -3-oC6H3(3.1-Cl2) 0.27
C6H3(3,4-(OcH2ph)2) 0.23
1-C1oH7 0.12
2-CloH7 0.35
AA = phenylalanine
Ph 1.3 0.052.1
C6H4(4-N(CH3)2) 0.62
C6Fs 0.70
C6H4(3-CF3) 0.46
C6H4(3-OPh) 0.60
C6H4(4-Oph) 0.20
C6H4(4-OcH2ph) 0.20
C6H4-3-OC6H4(3-CF3) . 0.18
- C6H4-3-oc6H3(3~4-cl2)
C6H3(3~4-(ocH2ph)2)
AA = Norvaline
Ph 7.8 11
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In general, replacement of the Z group (PhCH2OCO-) by related aromatic
groups also resulted in good inhibitor structures (Table PKC4).
Table PKC4. Inhibition of Cysteine Proteases by Peptide a-lcPtnq i~es with the
S St... .s MlCO-Leu-AA-CONH-R.
Ml R KI (~lM)
Cal I Cal II
Cat B
AA = a-aminobutyric acid
Z Et 0.5 0.232.4
CH3OcO(cH2)2 Et 3.8
2-furyl Et 0.85
2-tetrahydrofuryl Et 18.5
3-pyridyl Et 1.30
2-pyr~zinyl Et 0.30
2-quinolinyl Et 0.5
1-isoquinolinyl Et 0.35
4-morpholinyl Et 7.9
Ph(CH2)z Et
1-CloH7cH2 Et
Ph2CH Et 5.0
Ph2CH CH2CH(OH)Ph 0.75 0.20
Ph2CH CH2-2-pyridyl 0.5 0.092.8
Ph2CH (CH2)3-4-morpholinyl 0.8 0.112.3
AA = phenylalanine
Ph2CH CH2CH(OH)Ph 10 0.73
Ph2CH CH2-2-pyridyl 1.1 0.362.2
Ph2CH (CH2)3-4-morpholinyl 0.76 0.0743.8
2 1 3 8 1 2 lJ PCI`/US93/06143
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Preparation of pep~ide ~-ketoesters. The peptide a-ketoesters are prepared by a
two step Dakin-West procedure. This procedure can be utilized with either amino acid
derivatives, dipeptide derivatives, tripeptide derivatives, or tetrapeptide derivatives as
shown in the following scheme:
O
M-(AA)n-OH -- > Enol Ester -- > M-(AA)n-CO-R.
The precursor peptide ((AA)n) can be prepared using standard peptide
chemistry procedures, including those that are well described in publications such as
The Peptides~ Analysis. Synthesis, Biology. 1-9 (1979-1987), published by Academic
Press ('~he Peptides") and Syntl~ese von Peptiden in Houben-Wevl Methoden der
Organischen Chemie, 15, Parts 1 an-i 2, (1974) published by Georg Thieme Verlag
("Houben-Wevl"); both references hereby incorporated herein by reference.
The M group can be introduced using a number of different reaction schemes.
For example, it could be introduced directly on an amino acid as shown in the following
scheme:
H-(AA)n-OH -- > M-(AA)n-OH.
Alternatively, the M group can be introduced by reaction with an amino acid ester,
followed by removal of the ester group to give the same product, as shown in thefollowing scheme:
H-(AA)n-OR'--> M-(AA)n-OR'--> M-(AA)"-OH.
These and other techniques for introduction of the M group are well
documented in the The Peptides, Houhen-Weyl, and many other textbooks on organicsynthesis. For example reaction with cyanate or p-nitrophenyl cyanate would introduce
a carbamyl group (M = NH2CO-). Reaction with p-nitrophenyl thiocarbamate would
introduce a thio carbamyl group (M = NH2CS-). Reaction with NH2S4O2Cl would
introduce the NH2SO2- group. Reaction with a substituted alkyl or aryl isocyanate
would introduce the X-NH-CO- group where X is a substituted alkyl or aryl group.Reaction with a substituted alkyl or aryl isothiocyanate would introduce the X-NH-CS-
group where X is a substituted alkyl or aryl group. Reaction with X-SO2-Cl wouldintroduce the X-SO2- group. Reaction with a substituted alkyl or arvl acid chloride
would introduce an acyl group (M = Y-CO-). For example, reaction with
MeO-CO-CH2CH2-CO-Cl would give the Y-CO- group when Y is a C2 alkyl
WO 94/0009~ PCr/US93/06143
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substituted with a C1 alkyl-OCO- group. Reaction with a substituted alkyl or aryl
thioacid chloride would introduce a thioacyl group (M = Y-CS-). Reaction with an a
substituted alkyl or aryl sulfonyl chloride would introduce an X-SO2- group. Forexample reaction with dansyl chloride would give the X-SO2- derivative where X was a
napthyl group monosubstituted with a dimethylamino group. Reaction with a
substituted alkyl or aryl chloroformate would introduce a X-O-CO- group. Reaction
with a subsSituted alkyl or aryl chlorothioformate would introduce a X-O-CS-. There
are many alternate reaction schemes which could be used to introduce all of the above
M groups to give either M-AA-OH or M-AA-OR'.
The M-AA-OH derivatives could then be used directly in the Dakin-West
reaction or could be converted into the dipeptides, tripeptides, and tetrapeptides
M-AA-AA-OH, M-AA-AA-AA-OH, or M-AA-AA-AA-AA-OH which could be be used
in the Dakin-West reaction. The substituted peptides M-AA-AA-OH,
M-AA-AA-AA-OH, or M-AA-AA-AA-AA-OH could also be prepared directly from
H-AA-AA-OH, H-AA-AA-AA-OH, or H-AA-AA-AA-AA-OH using the reactions
described above for introduction of the M group. Alternately, the M group could be
introduced by reaction with carboxyl blocked peptides M-AA-AA-OR',
M-AA-AA-AA-OR', or M-AA-AA-AA-AA-OR', followed by the removal of the
blocking group R'.
The R group in the ketoester structures is introduced during the Dakin-West
reaction by reaction with an oxalyl chloride Cl-CO-CO-O-R. For example, reaction of
M-AA-AA-OH with ethyl oxaiyl chloride Cl-CO-CO-O-Et gives the keto ester
M-AA-AA-CO-O-Et. Reaction of M-AA-AA-AA-AA-OH with Cl-CO-CO-O-Bzl
would give the ketoester M-AA-AA-AA-AA-CO-O-Bzl. Clearly a wide variety of R
groups can be introduced into the ketoester structure by reaction with various alkyl or
arylalkyl oxalyl chlorides (Cl-CO-CO-O-R).
The oxalyl chlorides are easily prepared by reaction of an alkyl or arylalkyl
alcohol with oxalyl chloride Cl-CO-CO-Cl. For example, Bzl-O-CO-CO-CI and
n-Bu-O-CO-CO-Cl are prepared by reaction of benzyl alcohol and butanol, respectively,
with oxalyl chloride in yields of 50% and 80% (Warren and Malee, J. C/lro~nat~,
64:219-222 (1972); incorporated herein by reference).
~) 94/00095 2 ~ 3 8 1 2 ~ `~ PCI /US93/06143
K~oto~tirlc M-AA-CO-OH, M-AA-AA-CO-OH, M-AA-AA-AA-CO-OH,
M-AA-AA-AA-AA-CO-OH, are generally prepared from the corresponding ketoesters
M-AA-CO-OR, M-AA-AA-CO-OR, M-AA-AA-AA-CO-OR,
M-AA-AA-AA-AA-CO-OR by alkaline hydrolysis. In some cases, it may be necessary
to use other methods such as hydrogenolysis of a benzyl group (R = Bzl) or acid
cleavage (R = t-butyl) to obtain the keto~ l The alternate methods would be usedwhen the M group was labile to alkaline hydrolysis.
The various peptide ketoamide sub~l~cc.os including M-AA-NH-CHR2-CO-CO-
NR3R4 (Dipeptide Ketoamides, Subclass A), M-AA-AA-CO-NR3R4 (Dipeptide
K~to~mides, Subclass B), M1CO-AA2-AA1-CO-NH-CH2CH(OH)-R1 and five others
presented above (Dipeptide a-Ketoamides, Subclass C, Types 1 through 6), M-AA-AA-
AA-CO-NR3R4 (Tripeptide Ketoamides), M-AA-AA-AA-AA-CO-NR3R4 (Tetrapeptide
Ketoamides) and Ml-AA-CO-NR3R4 (Amino Acid Ketoamides), were prepared
indirectly from the co"~ onding ketoesters. The ketone carbonyl group was first
protected as shown in the following scheme and then the ketoamide was prepared by
reaction with an amine H-NR3R4. The illustrated procedure should also work with
other protecting groups.
20 M1-AA2~ ~ `R ' ~ M2~N$~0~R
R, O R1
H-NR3R4
H ~R3
M,-M2~ ~N~R4
'~R, ~)
In addition to the scheme outlined above, a ketoacid could be used as a
precursor to produce a corresponding ketoamide. Blocking the ketone carbonyl group
of the ketoacid and then coupling with an amine H-NR3R4 using standard peptide
WO 94/0009~ PCI /US93/0614t
~ 1 3 ~
coupling reagents would yield an intermediate which could then be deblocked to form
the keto~mide.
K~to~mides MlCO-AA-AA-CONHR were prepared indirectly from the
ketoesters. The ketone carbonyl group is first protected as shown in the following
S scheme and then the k~to~mide is prepared by reaction with an amine RNH2. The
product is easily isolated from the reaction mixture when using this procedure. This
procedure will also work with other ketone protecting groups. In addition, the
corresponding ketoacid can be used as a precursor to the a-ketoamide via coupling with
an amine RNH2 using standard peptide coupling reagents would result in formation of
the peptide a-ketoamide. O ~
MlCO-Leu ~ ~ -- MlCO-Leu
R~ R~
/ RNH2
M~CO-Leu ~ ~NHR
R~
General Sy~tlletic Metltods for Peptide Keto-Con~pou~lds
The techniques for synthesis of a wide variety of amines are described in many
publications. For example, Evans et al. in J.Org.Cllem 39:914 (1974) reported the
syntheses of phenylethanol derivatives with alkylamino, alkoxyamino and phenyloxyamino
groups. Katrizky et aL in J. C~lenl. Soc:2404-2408 (1956), Fife et aL in Heterocycles
22(1):93-96 (1984), and Heterocycles 22(5):1121-1124 (1984), and Isoda et aL in Chemical
and P1lan77aceutic~1 Bulleti~l 28:1408-1414 (1980) reported the syntheses of pyridine
derivatives with alkylamino and COOR groups. Nagata et aL in Yakugaku Zasshi
83:679-682 (1963) reported the syntheses of quinoline derivatives with alkylamino groups.
Zimmer et al. in Tetralledro~l Letters 2~:2805-2807 (1968) reported the syntheses of
isoquinoline derivatives with alkylamino groups. Aroyan et aL in Iz~. Aka~ Nauk Ann.
SSR, I~tinl. Nauki 18(1):76-82 (1965) reported the syntheses of tetrahydroquinoline
derivatives with alkylamino groups. Yonan, (U.S. 3,245,997 (Cl. 260-288), April 12, 1966.
2 pp) reported the syntheses of tetrahydroisoquinoline derivatives with alkylamino groups
Rybar et al. in Cllem. Comnlun. 35:1415-1433 (1970), Golovchinskaya et aL in J. Ge~leral
Chen~ 22:599-603 (1952), and Nantka-Namirski et aL in Acta. Polo~z. Phan~l. l:S- 12 (1974)
reported the syntheses of caffeine derivatives with alkylamino groups. Goldberg et aL in
~'~ 94/00095 PCI/US93/06143
-
2~12~ `
-89-
J.Chem.Soc:1372 (1947) reported the syntheses of methylthiazole derivatives withalkylamino groups. Mizuno et al. inJ.Org.Che~t. 39:1250 (1974) reported ~he syntheses of
pyridine-N-oxide derivatives with alkylamino groups. Wade inJ.Heterog~clic ateln. 23:981
(1986) reported the syntheses of uracil derivatives with alkylamino groups. All of the
above citations are incorporated herein by reference.
Unless otherwise noted, materials were obtained from commercial suppliers and
used without further purification. Melting points were taken with a Bachi capillary
apparatus and are uncorrected. IH NMR spectra were determined on a Varian Gemini300. Chemical shifts are expressed in ppm (ô) relative to internal tetramethylsilane. Flash
column chromatography was performed with Universal Scientific Inc. silica gel 0-63.
Electron-impact mass spectra (MS) of ncvel compounds were determined with a Varian
MAT 112S spectrometer. The purity of all compounds was checked by thin- layer
chromatography on Baker Si250F silica gel plates using the following solvent system: A,
CHCl3:MeOH = 20:1 v/v; B, CHCl3:MeOH = 100:1 v/v; C, AcOEt; D, CHCl3:MeOH
= 10:1 v/v; E, n-BuOH:AcOH;py:H2O = 4:1:1:2 v/v; F, CHCl3:MeOH = 5:1 v/v; G,
AcOEt:MeOH = 10:1 v/v; H, (i-Pr)2O; I, CHCI3:MeOH:AcOH = 80:10:5 v/v; J,
CHCl3:MeOH:AcOH = 95:5:3 v/v; K, AcOEt:AcOH = 200: 1 v/v; L, CHCI3; M,
CHCl3:MeOH = 50:1 v/v.
Amino acid methyl ester hydrochlorides were prepared according to M. Brenner
etal.,Helv. Che/7l.Acta33:5G8(1950);36:1109(1953)inascaleover 10mmoloraccording
to Rachele, J. Org. C/lel7z. 28:2898 (1963) in a scale of 0.1-1.0 mmol.
Yield (%) mp (C) m.p. (literature)
DL-Nva-OCH3HCl, 100 113-116 116-117
L-Ile-OCH3 HCl, 98 90-91 98-100
L-Phe-OCH3 HCl, 98 159-161 158-160
DL-Abu-OCH3 HCl, 100 148-150 150-151
L-Leu-OCH3 HCI 100 145.5-146.5 147
DL-Nle-OCH3 HCl 93 120-121 122-123
4-Cl-Phe-OCH3HCl 98 184-185 (decomp.) 185-186
N-Acylamino acids was synthesized via Schotten-Baumann reaction as in Bergmann
and Zervas, Chel7t. Ber., 65:1192 (1932) in t'ne case when the aql group was
phenylsulphonyl, 2- naphthylsulphonyl or benzoyl.
WO 94/0009~ PCr/US93/06143
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Yield (%) mp (C) TLC (Rf, eluent)
2-NapSO2-L-Leu-OH 49 115-116 0.58I
2-NapSO2-DL-Abu-OH 51 150-151 0.50I
2-NapSO2-L-Phe-OH 57 148-148.5 0.48K
PheSO2-DL-Abu-OH 44 142-143 0.51K
PhCO-DL-Abu-OH 64 141-142 0.64K
N-Acylamino acids with 4-methylpentanoic, 2-(1- propyl)pentanoic and
7-phenylheptanoic group was synthesized in a two step synthesis. The N-acylamino acid
methyl ester was obtained first and then was hydrolysed to the free N-acylamino acid.
N-Acylamillo Acid Metltyl Esters (General Procedure). To a chilled (10 C) slurry
of the app~u~liate amino acid methyl ester hydrochloride (20 mmol) in 100 ml benzene
was added slowly (temp. 10-15 C) 40 mmol triethylamine or N- methylmorpholine and
then the reaction mixture was stirred for 30 minutes at this temperature. Then 18 mmol
of applc,yliate acid chloride (temp. 10-15 C) was added slowly to the reaction mixture
and the reaction mixture was stirred overnight at room temperature. The precipiatated
hydrochloride was filtered, washed on a funnel with 2 x 20 ml benzene, and the collected
filtrate was washed successively with 2 x 50 ml 1 M HCI, 2 x 50 ml 5% NaHCO3, 1 x 100
ml H20, 2 x 50 ml satd. NaCl and dried over MgSO1. After evaporation of the solvent in
vacuo (rotavaporator), the residue was checked for purity (TLC) and used for the next
step (hydrolysis).
Yield (%) mp (C)
(cH3)2cH(cH2)2co-DL-Abu-ocH3 80 oil
(CH3CH2CH2)2CHCO-DL-Abu-OCH3 96 117-118
Ph(CH2)6CO-DL-Abu-OCH3 72 oil
Hydrolysis (Ge~teral Procedure). To a solution of 10 mmole of the appropriate
N-acylamino acid methyl ester in 100 ml of methanol was added in one portion 11.25 ml
of 1 M NaOH (11.25 mmol) and the reaction mixture was stirred three hours at room
temperature. Then the reaction mixture was cooled to 0 C (ice- salt bath) and acidified
to pH = 2 with 1 M HCI aq. To this reaction mixture was added 100 ml ethyl acetate,
transferred to a separatory funnel and organic layer separated. The water layer was
saturated with solid NaCl or (NH4)2SO4 and reextracted with 2 x 50 ml AcOEt. Thecollected organic layer was washed with 2 x 50 ml H20, decolorized with carbon, and dried
~ ') 94/00095 PCI/US93/06143
21~124
-91-
over MgS04. After evaporation of the solvent in vacuo (rotavaporator), the residue was
checked for purity (TLC) and in the case of contamination was crystallized from an
a~ opliate solvent.
Yield (%) mp (C)
(CH3)2CH(CH2)2CO-DL-Abu-OH 92 110.5-112
(CH3CH2CH2)2CHCO-DL-Abu-OH 99 126-127 (n-octane)
Ph(CH2)6CO-DL-Abu-OH 89 110-112 (n-octane)
N-Acyldipeptide methyl esters were synthesized via the HOBt-DCC method in a
DMF solution as in Konig and Geiger, C/lem. Ber., 103:788 (1970).
Yield (%) mp (C) TLC (Rf, eluent)
Z-Leu-DL-NVa-OCH3 80 112-113 0.37 B
Z-Leu-L-Phe-OCH3 83 86-87 0.85 A
0.39 B
Z-Leu-L-Ile-OCH3 97 oil 0.79 A
0.43 B
Z-Leu-DL-Abu-OCH3 99 86-88 0.33 B
0.26 H
Z-Leu-L-Leu-OCH3 80 91-92 0.79 G
Z-Leu-DL-NLeu-OCH3 97 111- 111.5
Z-Leu-4-CI-Phe-OCH3 65 112-132 0.77 J
(liquid crystal?) 0.68 K
2-NapS02-Leu-DL-Abu-OCH3
99 oil 0.59 A
2-NapS02-Leu-L-Leu-OCH3
97-98.5 0.63 A
N-Acyldipeptides were obtained by hydrolysis of the appropriate methyl esters via
a general hydrolysis procedure. In the case of N-sulphonyldipeptide methyl esters, 1
equivalent of the methyl ester was hydrolyzed with 2.25 equivalent of 1 molar NaOH
because of form a sulfonamide sodium salt.
WO 94/00095 PCI/US93/06143
~i33i24
-92-
Yield (%) mp (C) TLC (Rf, eluent)
Z-Leu-DL-NVa-OH 100 117-118.5 0.11 A
Z-Leu-L-Phe-OH 92 105-106.5 0.28 C
0.55 G
Z-Leu-L-ILe-OH 79 77-79 0.22 A
0.52 C
Z-Leu-DL-Abu-OH 99 glass 0.61 G
Z-Leu-L-Leu-OH 97 glass 0.56 I
Z-Leu-DL-NLeu-OH 98 95-96
Z-Leu-4-Cl-Phe-OH 87 104-114 0.48 K
(liquid crystal?)
2-NapSO2-Leu-DL-Abu-OH
97.4 180-195 (decomp) 0.58 I
2-NapSO2-Leu-L-Leu-OH
94.0 68-70 0.52 I
N-Acytripeptide methyl esters were synthesized via HOBt- DCC method in DMF
solution as in Konig and Geiger, supra.
Yield (%) mp (C) TLC (Rf, eluent)
Z-Leu-Leu-Abu-OCH3 87 140-141.5 0.50 A
Z-Leu-Leu-Phe-OCH3 76 158-159 0.83 J
2-NapSO2-Leu-Leu-Abu-OCH3
97 >200 0.52 A
N-Acyltripeptide were obtained through hydrolysis of the appropriate methyl esters
via general hydrolysis procedure. In the case of N-sulphonyltripeptide methyl ester, 1
equivalent of methyl ester was hydrolyzed with 2.25 equivalent of 1 molar NaOH to form
the sulfonamide sodium salt.
Yield mp (C) TLC (Rf, eluent)
Z-Leu-Leu-Abu-OH 97 glass 0.69 I
Z-Leu-Leu-Phe-OH 98 glass 0.44 K
2-NapSO2-Leu-Leu-Abu-OH
193-195 0.53 I
(decomp.) 0.32 J
~'" 94/00095 PCI/US93/06143
~13812~
-93 -
We have also discovered a process for the synehesis of a-ketoamides with the
structures
M-CO-AA2-AA1-CO-NH-R and
M-CO-AA3-AA2-AA1-CO-NH-R,
wherein
M is selected from the group consisting of C1 4 alkyl monosubstituted with phenyl,
Cl4 alkyl tlicubstituted with phenyl, Cl 4 alkyl monosubstituted with 1-naphthyl, Cl 4 alkyl
monosubstituted with 2-naphthyl, C14 alkoxy monosubstituted with phenyl, Cl4 alkoxy
disubstituted with phenyl, ArlCH2O-, Ar1O-, Ar1CH2NH-, ArlNH- and
Heterocyclel(CH2)q-;
Arl is selected from the group consisting of phenyl, phenyl monosubstituted withJ, phenyl disubstituted with J, 1-naphthyl, 1-naphthyl rnonosubstituted with J, 2-naphthyl,
and 2-naphthyl monosubstituted with J;
J is selected from the group consisting of halogen, OH, CN, NO2, NH2, COOH,
CO2Me, C02Et, CF3, C1 4 alkoxy, C1 4 alkylamine, C2 ~ dialkylamine. C1 4 perfluoroalkyl,
and -N(cH2cH2)2o;
Heterocyclel is selected from the group consisting of 2-furyl, 2-tetrahydrofuryl,
2-pyrazinyl, 3-pyridyl, 4-pyridyl, 2-quinolinyl, I-tetrahydroquinolinyl, 1-isoquinolinyl,
2-tetrahydroisoquinolinyl, and -N(CH2CH2)2O;
q = 0-2;
AA1, AA2 and AA3 are side chain blocked or unblocked a-amino acids with the L
configuration, D configuration, or DL configuration at the a-carbon selected independently
from the group consisting of alanine, valine, leucine, isoleucine, histidine, proline,
methionine, methionine sulfoxide, phenylalanine, serine, threonine, phenylglycine,
norleucine, norvaline, arginine, Iysine, tryptophan, glycine, cysteine, tyrosine, asparagine,
glutamine, aspartic acid, glutamic acid, alpha-aminobutyric acid, O-methylserine,
O-ethylserine, S-methylcysteine, S-ethylcysteine, S-benzylcysteine,
NH2-CH(CH2CHEt2)-COOH, alpha-aminoheptanoic acid,
NH2-CH(CH2-cyclohexyl)-COOH, NH2-CH(CH2-cyclopentyl)-COOH,
NH2-CH(CH2-cyclobutyl)-COOH, NH2-CH(CH2-cyclopropyl)-COOH,
WO 94/0009C, PCI/US93/0614~
'~13812l~
-94-
NH2-CH(CH2- 1-naphthyl)-COOH, NH2-CH(CH2-2-naphthyl)-COOH,
5,5,5-trifluoroleucine, and hexafluoroleucine;
R is selected from the group consisting of H, C1 20 alkyl, C1 20 cyclized alkyl, C1 20
alkyl with a phenyl group attached to the Cl 20 alkyl, Cl 20 cyclized alkyl with an attached
phenyl group, C1 20 alkyl with an attached phenyl group substituted with K, C1 20 alkyl with
an attached phenyl group tli~ubstituted with K, Cl 20 alkyl with an attached phenyl group
tri~ubstituted with K, C1 20 cyclized alkyl with an attached phenyl group substituted with
K, C1 10 alkyl with a morpholine [-N(CH2CH2)O] ring attached through nitrogen to the
alkyl, C1 10 alkyl with a piperidine ring attached through nitrogen to the alkyl, C1 l0 alkyl
with a pyrrolidine ring attached through nitrogen to the alkyl, C1 20 alkyl with an OH
group attached to the alkyl, -CH2CH2OCH2CH2OH, C1 10 with an attached 4-pyridyl
group, Cl 10 with an attached 3-pyridyl group, C1 10 with an attached 2-pyridyl group, C1 10
with an attached cyclohexyl group, -NH-CH2CH2-(4-hydroxyphenyl),
-NH-CH2CH2-(3-indolyl), CH2CH(OH)-Ar2 and (CH2)n-Heterocycle2;
K is selected from the group consisting of halogen, C1 10 alkyl, C1 10 perfluoroalkyl,
Cl 10 alkoxy, N02, CN, OH, C02H, amino, Cl 10 allcylamino, C2 l2 dialkylamino, Cl 10 acyl,
and C1 10 alkoxy-CO, and C1 10 alkyl-S-;
Ar2 is selected from the group consisting of phenyl, phenyl monosubstituted withJ, phenyl disubstituted with J, phenyl trisubstituted with J, pentafluorophenyl,CGH4(3-R2) C6H4(4-R2). C6H3(3-4-(R2)2. C6H2(2,4,6-(OR2)3, I-naphthyl, 1-naphthyl
monosubstituted with J, 1-naphthyl disubstituted with J, 2-naphthyl, 2-naphthyl
monosubstituted with J, 2-naphthyl disubstituted with J, 2-pyridyl, 2-quinolinyl, and
1 -isoquinolinyl;
R2 represents C1 4 alkyl substituted with phenyl, phenyl and phenyl substituted with
J.
Heterocycle2 is selected from the group consisting of 2-furyl, 2-furyl
monosubstituted with J, 2-tetrahydrofuryl, 2-pyridyl, 2-pyridyl monosubstituted with J,
3-pyridyl, 3-pyridyl monosubstituted with J, 4-pyridyl, 4-pyridyl monosubstituted with J,
2-pyrazinyl, 2-quinolinyl, 2-quinolinyl monosubstituted with J, 1-isoquinolinyl,1-isoquinolinyl monosubstituted with J, 1-tetrahydroquinolinyl, 2-tetrahydroisoquinolinyl,
12 ~ PCI/US93/06143
3-indolyl, 2-pyridyl-N-oxide, 3-pyridyl-N-oxide, 4-pyridyl-N-oxide, 2-(N-methyl-2-pyrrolyl),
1-imidazolyl, 1-pyrrolidinyl-2-one, 2-(5-methyl-3-thiazolyl), (CH2)2-NH-biotin;
HNJ~ O Me
O~N O~N N O~N N
H Me Me
co~ isillg the steps:
(a) Protecting the a-ketone carbonyl of a peptidyl a-ketoester with the
10structures
M-CO-AA2-AAl-COOR6 and
M-co-AA3-AA2-AA l-CR6
wherein
R6 is selected from the group consisting of Cl 6 alkyls and Cl 6 alkyls
monosubstituted with phenyl,
by treatment with a blocking reagent in the presence of a Lewis acid in an organic
solvent at 0-100 C for 1-48 hours, wherein
the preferred blocking reagent is 1,2-ethanedithiol;
the preferred Lewis acids are selected from the group consisting of BF3.Et2O,
4-toluene sulfonic acid, AlCl3 and ZnC12;
the preferred organic solvents are selected from the group consisting of CH2CI2,CHCl3, Et2O and THF;
(b) Treating the product with a primary amine RNH2 in an organic solvent at
0-100 C for 1-72 hours, wherein
the preferred organic solvents are selected from the group consisting of EtOH,
THF, CH2Cl2 and DMF;
(c) Removing the blocking group from the a-carbonyl to give the desired
peptidyl a-ketoamide.
We have also discovered another process for the synthesis of peptidyl a-ketoamides
with the structures
M-CO-AA2-AAl-CO-NH-R and
M-CO-AA3-AA2-AAl-CO-NH-R,
WO 94/00095 PCI/US93/0614~
2 ~ S~ 2 4
-96-
wherein
M is selected from the group consi~til,g of Cl 4 alkyl monosubstituted with phenyl,
Cl ~ alkyl disubstituted with phenyl, C1~ alkyl monosubstituted with l-naphthyl, Cl 4 alkyl
monosubstitllted with 2-naphthyl, Cl4 alkoxy monosubstituted with phenyl, C14 alkoxy
disubstituted with phenyl, Ar1CH2O-, Ar1O-, Ar1CH2NH-, ArlNH- and
Heterocyclel(CH2)q-;
Ar1 is selected from the group consisting of phenyl, phenyl monosubstituted withJ, phenyl disubstituted with J, 1-naphthyl, l-naphthyl monosubstituted with J, 2-naphthyl,
and 2-naphthyl monosubstituted with J;
J is selected from the group consisting of halogen, OH, CN, NO2, NH2, COOH,
C02Me, C02Et, CF3, Cl4 alkoxy, Cl~ alkylamine, C2 ~ dialkylamine, Cl ,~ perfluoroalkyl,
and -N(cH2cH2)2o;
Heterocycle1 is selected from the group consisting of 2-furyl, 2-tetrahydrofuryl,
2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrazinyl, 2-quinolinyl, l-tetrahydroquinolinyl,
1-isoquinolinyl, 2-tetrahydroisoquinolinyl, and -N(CH2CH2)2O;
q = 0-2;
AAl, AA2 and AA3 are side chain blocked or unblocked a-amino acids with the L
configuration, D configuration, or DL configuration at the a-carbon selected independently
from the group consisting of alanine, valine, leucine, isoleucine, histidine, proline,
methionine, methionine sulfoxide, phenylalanine, serine, threonine, phenylglycine,
norleucine, norvaline, arginine, Iysine, ttyptophan, glycine, cysteine, tyrosine, asparagine,
glutamine, aspartic acid, glutamic acid, alpha-aminobutyric acid, O-methylserine,
O-ethylserine, S-methylcysteine, S-ethylcysteine, S-benzylcysteine,
NH2-CH(CH2CHEt2)-COOH, alpha-aminoheptanoic acid,
NH2-CH(CH2-cyclohexyl)-COOH, NH2-CH(CH2-cyclopentyl)-COOH,
NH2-CH(CH2-cyclobutyl)-COOH, NH2-CH(CH2-cyclopropyl)-COOH,
NH2-CH(CH2-l-naphthyl)-COOH, NH2-CH(CH2-2-naphthyl)-COOH,
5,5,5-trifluoroleucine, and hexafluoroleucine;
R is selected from the group consisting of H, C1 20 alkyl, C1 20 cyclized alkyl, C1 20
alkyl with a phenyl group attached to the Cl 20 alkyl, Cl 20 cyclized alkyl with an attached
phenyl group, C1 20 alkyl with an attached phenyl group substituted with K, C1 20 alkyl with
an attached phenyl group disubstituted with K, C1 20 alkyl with an attached phenyl group
94/00095 ~ 1 3 8 ;L 2 ~ PCr/US93/06143
-97 -
trisubstituted with K, Cl 20 cyclized alkyl with an attached phenyl group substituted with
K, C1 10 alkyl with a morpholine [-N(CH2CH2)O] ring attached through nitrogen to the
alkyl, C1 10 alkyl with a piperidine ring attached through nitrogen to the alkyl, Cl 10 alkyl
- with a pyrrolidine ring attached through nitrogen to the alkyl, C120 alkyl with an OH
group attached to the alkyl, -CH2CH2OCH2CH2OH, C1 10 with an attached 4-pyridyl
group, C1 10 with an attached 3-pyridyl group, C1 l0 with an attached 2-pyridyl group, C1 l0
with an attached cyclohexyl group, -NH-CH2CH2-(4-hydroxyphenyl),
-NH-CH2CH2-(3-indolyl), CH2CH(OH)-Ar2 and (CH2)n-Heterocycle2;
K is selected from the group consisting of halogen, C1 l0 alkyl, C1 l0 perfluoroalkyl,
C1 10 alkoxy, NO2, CN, OH, CO2H, amino, Cl ln alkylamino, C2 12 dialkylamino, Cl l0 acyl,
and C1 10 alkoxy-CO-, and C1 l0 alkyl-S-;
Ar2 is selected from the group consisting of phenyl, phenyl monosubstituted withJ, phenyl disubstituted with J, phenyl trisuhsti~uted with J, pentafluorophenyl,
6H4(3 ORz), C6H4(4-R2)- C6H3(3.4-(R2)2. C6H2(2,4,6-(OR2)3, 1-naphthyl, 1-naphthyl
monosubstituted with J, 1-naphthyl disubstituted with J, 2-naphthyl, 2-naphthyl
monosubstituted with J, 2-naphthyl disubstituted with J, 2-pyridyl, 2-quinolinyl, and
1 -isoquinolinyl;
R2 represents C14 alkyl substituted with phenyl, phenyl and phenyl substituted with
J.
Heterocycle2 is selected from the group consisting of 2-furyl, 2-furyl
monosubstituted with J, 2-tetrahydrofuryl, 2-pyridyl, 2-pyridyl monosubstituted wit'n J,
3-pyridyl, 3-pyridyl monosubstituted with J, 4-pyridyl, 4-pyridyl monosubstituted with J,
2-pyrazinyl, 2-quinolinyl, 2-quinolinyl monosubstituted with J, 1-isoquinolinyl,1-isoquinolinyl monosubstituted with J, 1-tetrahydroquinolinyl, 2-tetrahydroisoquinolinyl,
3-indolyl, 2-pyridyl-N-oxide, 3-pyridyl-N-oxide, 4-pyridyl-N-oxide, 2-(N-methyl-2-pyrrolyl),
1-irnidazolyl, 1-pyrrolidinyl-2-one, 2-(5-methyl-3-thiazolyl), (CH2)2-NH-biotin;
HNJ~ ,~ Me
O~N O~N N O~N N
H Me Me
comprised of the steps: .
(a) Hydrolyzing a peptidyl a-ketoester with the structures
M-CO-AA2-AA,-COOR6 and
WO 94/00095 PCI /US93/0614
-98-
M-co-AA3-AA2-AAl-cooR6
wherein
R6 is selected from the group consisting of Cl 6 alkyls and Cl 6 alkyls
monosubstituted with phenyl;
by treating the peptidyl a-ketoester with a hydrolysis reagent in an applopliate solvent at
0-100 C for 1-24 hours to give the corresponding peptidyl a-ketoacid, wherein
the preferred hydrolysis reagents are selected from the group consisting of NaOH,
KOH, EtONa and EtOK;
the preferred solvent are selected from the group consisting of water, MeOH,
EtOH, THF and DMF;
(b) Coupling the product peptidyl a-ketoacid with a primary amine RNH2 in
an organic solvent at 0-100 C for 1-72 hours to give the desired peptidyl a-ketoamide,
wherein
the preferred coupling conditions are selected from the group consisting of
treatment with 1,1-carbonyldiimidazole, treatment with dicyclohexylcarbodiimide, and
treatment with dicyclohexylcarbodiimide-1-hydroxybenzotriazole;
the preferred organic solvents are selected from the group consisting of CH2Cl2,CHCl3, DMF and THF.
We have also discovered a process for the synthesis of peptidyl a-ketoamides with
the structures
M-CO-AA2-AAl-CO-NH-R and
M-CO AA3-AA2-AA1-CO-NH-R,
wherein
M is selected from the group consisting of C1 4 alkyl monosubstituted with phenyl,
C14 alkyl disubstituted with phenyl, C1 4 alkyl monosubstituted with 1-naphthyl, Cl4 alkyl
monosubstituted with 2-naphthyl, C1 4 alkoxy monosubstituted with phenyl, C14 alkoxy
disubstituted with phenyl, Ar1CH2O-, ArlO-, Ar1CH2NH-, Ar1NH- and
Heterocyclel(CH2)q-;
Arl is selected from the group consisting of phenyl, phenyl monosubstituted withJ, phenyl disubstituted with J, 1-naphthyl, l-naphthyl monosubstituted with J, 2-naphthyl,
and 2-naphthyl monosubstituted with J;
V~'' 94/00095 ~ 3~ ` PCr/US93/06143
99
J is selected from the group consisting of halogen, OH, CN, NO2, NH2, COOH,
C02Me, C02Et, CF3, Cl 4 alkoxy, C1 4 alkylamine, C2 8 dialkylamine, Cl ~ perfluoroalkyl,
and -N(cH2cH2)2o;
Heterocycle1 is selected from the group consisting of 2-furyl, 2-tetrahydrofuryl,
2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrazinyl, 2-quinolinyl, 2-tetrahydroquinolinyl,
1-isoquinolinyl, 1-tetrahydroisoquinolinyl, and -N(CH2CH2)2O;
q = 0-2;
AAl, AA2 and AA3 are side chain blocked or unblocked a-amino acids with the L
configuration, D configuration, or DL configuration at the a-carbon selected independently
from the group consisting of alanine, valine, leucine, isoleucine, histidine, proline,
methionine, methionine sulfoxide, phenylalanine, serine, threonine, phenylglycine,
norleucine, nor~aline, arginine, Iysine, tryptophan, glycine, cysteine, tyrosine, asparagine,
e~ut~mine, aspartic acid, glutamic acid, alpha-aminobutyric acid, O-methylserine,
O-ethylserine, S-methylcysteine, S-ethylcysteine, S-benzylcysteine,
NH2-CH(CH2CHEt2)-COOH, alpha-aminoheptanoic acid,
NH2-CH(CH2-cyclohexyl)-COOH, NH2-CH(CH2-cyclopentyl)-COOH,
NH2-CH(CH2-cyclobutyl)-COOH, NH2-CH(CH2-cyclopropyl)-COOH,
NH2-CH(CH2-l-naphthyl)-COOH, NH2-CH(CH2-2-naphthyl)-COOH,
5,5,5-trifluoroleucine, and hexafluoroleucine;
R is selected from the group consisting of H, Cl 20 alkyl, Cl 20 cyclized alkyl, C1 20
alkyl with a phenyl group attached to the Cl 20 alkyl, Cl 20 cyclized alkyl with an attached
phenyl group, Cl 20 alkyl with an attached phenyl group substituted with K, Cl 20 alkyl with
an attached phenyl group disubstituted with K, Cl 20 alkyl with an attached phenyl group
trisubstituted with K, Cl 20 cyclized alkyl with an attached phenyl group substituted with
K, Cl 10 alkyl with a morpholine [-N(CH2CH2)O] ring attached through nitrogen to the
alkyl, Cl lq alkyl with a piperidine ring attached through nitrogen to the alkyl, Cl 10 alkyl
with a pyrrolidine ring attached through nitrogen to the alkyl, Cl 20 alkyl with an OH
group attached to the alkyl, -CH2CH2OCH2CH2OH, C1-10 with an attached 4-pyridyl
group, C1 10 with an attached 3-pyridyl group, Cl l0 with an attached 2-pyridyl group, Cl 10
with an attached cyclohexyl group, -NH-CH2CH2-(4-hydroxyphenyl),
-NH-CH2CH2-(3-indolyl), CH2CH(OH)-Ar2 and (CH2)n-Heterocycle2;
WO 94/00095 PCI /US93/0614~
~i3~,14~
-100-
K is selected from the group consisting of halogen, C1 l0 alkyl, C1 10 perfluoroalkyl,
Cl 10 alkoxy, NO2, CN, OH, CO2H, amino, Cl 10 alkylamino, C2 12 dialkylamino, Cl 10 acyl,
and C1 l0 alkoxy-CO-, and C1 10 alkyl-S-;
Ar2 is selected from the group consisting of phenyl, phenyl monosubstituted withJ, phenyl licubstituted with J, phenyl trisubstituted with J, pentafluorophenyl,
C6H4(3-R2)~ C6H4(4-R2). C6H3(3-4-(R2)2. C6H2(2,4,6-(OR2)3, 1-naphthyl, 1-naphthyl
monosubstituted with J, 1-naphthyl disubstituted with J, 2-naphthyl, 2-naphthyl
monosubstituted with J, 2-naphthyl c~icubstituted with J, 2-pyridyl, 2-quinolinyl, and
1-isoquinolinyl;
R2 represents C14 alkyl substituted with phenyl, phenyl and phenyl substituted with
J
Heterocycle2 is selected from the group consisting of 2-furyl, 2-furyl
monosubstituted with J, 2-tetrahydrofuryl, 2-pyridyl, 2-pyridyl monosubstituted with J,
3-pyridyl, 3-pyridyl monosubstituted with J, 4-pyridyl, 4-pyridyl monosubstituted with J,
2-pyrazinyl, 2-quinolinyl, 2-quinolinyl monosubstituted with J, 1-isoquinolinyl,1-isoquinolinyl monosubstituted with J, 1-tetrahydroquinolinyl, 2-tetrahydroisoquinolinyl,
3-indolyl, 2-pyridyl-N-oxide, 3-pyridyl-N-oxide, 4-pyridyl-N-oxide, 2-(N-methyl-2-pyrrolyl),
1-imidazolyl, 1-pyrrolidinyl-2-one, 2-(5-methyl-3-thiazolyl), (CH2)2-NH-biotin;
HNJ~ ,~ O Me
O~N O~N N O~N N
H Me Me
consisting of treating a peptidyl a-enolester derived from a peptidyl a-ketoester with the
structures
M-CO-AA2-AAl-COOR6 and
M-CO-AA3-AA2-AAl-COOR6
wherein
R6 is selected from the group consisting of Cl 6 alkyls and C1 6 alkyls
monosubstituted with phenyl;
with a primary amine RNH2 in an organic solvent at 0-100 C for 1-72 hours to give the
desired peptidyl a-ketoamide, wherein
the preferred organic solvents are selected from the group consisting of CH2Cl2,EtOH, DMF and THF.
~ ~ 3 8 1 ~ ~ PCI/US93/06143
-101-
The following examples, Examples PKC1-PKC6S, are given to illustrate the
synthesis of Peptide Keto-Compounds:
EXAMPLE PKC1
- Z-Ala-DL,Ala-COOEt. This compound was synthesi7ed by a modified Dakin-West
procedure as in Charles et al., J. ChenL Soc. Perkin I:1139-1146 (1980). To a stirred
solution of Z-Ala-Ala-OH (880 mg, 3 mmole), 4-dimethylaminopyridine (15 mg, 0.31mmole), and pyridine (0.8 mL, 10 mmole) in tetrahydrofuran (3 mL) was added ethyl
oxalyl chloride (0.7 mL,6 mmole) at a rate sufficient to initiate refluxing. The mixture was
gently refluxed for 3.5 h. The mixture was treated with water (3 mL) and stirredvigorously at room temperature for 30 min. The mixture was extracted with ethyl acetate.
The organic extracts were dried and evaporated to obtain the residue (1.45 g). The
residue was chromatographed on silica gel and eluted with CH2Cl2 to give the enol ester
product, oil (500 mg,37%); single spot on tlc, Rf2 = 0.67 (CHCl3:MeOH = 9:1); MS, m/e
= 451 (M++1). To a stirred suspension of the enol ester (210 mg, 0.47 mmol) in
anhydrous ethanol (1 mL) at room temperature was added dropwise a solution of sodium
ethoxide in ethanol until a clear yellow solution re,u!-~d. The ethanol was then removed
and the residue was treated with ether. The ether solution was washed with water, dried,
and evaporated to give a residue. This residue was chromatographed on a silica gel and
the product was eluted with methylene chloride. The solvent was removed, and thepeptide ketoester Z-Ala-DL-Ala-CO2Et was obtained as an semi-solid (150 mg, 92 %);
single spot on tlc, Rf1 0.58 (CHCI3:MeOH = 5:1); MS, m/e = 351 (M+ + l). Anal. Calcd.
for C17H22O6N2 1/3 H2O: C~ 57.29; H, 6.22; N~ 7.86. Found: C~ 57.23; H, 6.36; N~ 8.17.
EXAMPLE PKC2
Z-Ala-Ala-DL,Ala-CO2Et. This compound was prepared from Z-Ala-Ala-Ala-
OH using the same procedure as described in Example PKC1. The product was
c;yst~lli7ed from ethyl ether in 23% yield; single spot on tlc, Rf2 = 0.31 (CHCl3:MeOH
= 9:1); mp 143-144 C; MS, m/e = 421 (M+). Anal. Calcd. for C20H27O7N3: C, 56.99;
H, 6.46; N, 9.97. Found: C, 56.96; H, 6.49; N, 9.92.
EXAMPLE PKC3
Z-Ala-Ala-DL-Abu-CO2Et. This compound was prepared from Z-Ala-Ala-DL-
Abu-OH in 11% yield by the procedure described in Example PKC1; sin~le spot on tlc,
Rf2 = 0.60 (CHCl3:MeOH = 9: 1); mp 111-113 C; MS, m/e = 436 (M+ + 1). Anal.
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Calcd. for C21H29O7N3 1/3 H2O: C, 57.13; H, 6.75; N, 9.51. Found: C, 57.38; H, 6.82;
N, 9.62.
EXAMPLE PKC4
Z-Ala-Ala-DL~Nva-C02Et. This compound was prepared from Z-Ala-Nva-OH
S in 20% yield by the procedure described in Example PKCl; single spot on tlc, Rf1 =
0.64 (CHCl3:MeOH = 5:1); MS, m/e = 450 (M+ + 1). Anal. Calcd. for
C22H3lO7N3H2O: C, 56.51; H, 7.11; N, 8.99. Found: C, 56.42; H, 7.08; N, 9.06.
EXAMPLE PKC5
ZAla-Pro-DL,Ala-CO2Et. This compound was prepared from Z-Ala-Pro-Ala-
OH dicyclohexylamine in 19% yield by the procedure described in Example PKC1;
single spot on tlc, Rf2 = 0.55 (CHC13:MeOH = 9:1); MS, m/e = 447 (M+). Anal.
Calcd. for C22H29O7N3 1/2 H2O: C, 57.88; H, 6.62; N, 9.21. Found: C, 57.65; H, 6.68;
N, 9.17.
EXAMPLE PKC6
Z-Ala-Ala-Ala-DL,Ala-CO2Et. The compound was prepared from Z-Ala-Ala-
Ala-Ala-OH in 7% yield by the procedure described in Example PKC1; single spot on
tlc, Rf2 =0.40 (CHCl3:MeOH = 9:1); mp. 163-165 C; MS, m/e = 493 (M++1). Anal.
Calcd. for C23H32O8N4 1/2 H20: C, 55.08; H, 6.63; N, 11.17. Found: C, 54.85; H, 6.53;
N, 11.14.
EXAMPLE PKC7
Bz-DL~Phe-CO2Et. This compound was prepared from Bz-Phe-OH in 36%
yield by the procedure described in Example PKC1, oil, single spot on tlc, Rf2 = 0.61
(CHCl3:MeOH = 9:1); MS, m/e = 325 (M+). Anal. Calcd. for Cl9H19O4N 1/3 H2O:
C, 68.86; H, 5.98; N, 4.22. Found: C, 69.10; H, 6.09; N, 4.38.
EXAMPLE PKC8
MeO-Suc-Ala-DL,Ala-CO2Me. This compound was prepared from MeO-Suc-
Ala-Ala-OH in 22% yield by the same procedure as described in Example PKC1,
except that sodium methoxide in methanol was used for enol ester hydrolysis, single
spot on tlc, Rf2 = 0.43 (CHCl3:MeOH = 9:1); MS, m/e = 317 (M+ + 1). Anal. Calcd.for C13H20O7N4 1/3 H2O: C, 48.44; H, 6.46; N, 8.69. Found: C, 4856; H, 6.39; N, 8.69.
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EXAMPLE PKC9
MeO-Suc-Ala-Ala-Pro-DL-Abu-CO2Me. This compound was prepared from
MeO-Suc-Ala-Ala-Pro-DL-Abu-OH in 22% yield by the procedure described in
FY~mple PKC8; foam, single spot on tlc, Rfl = 0.66 (CHCl3:MeOH = 5:1). Anal.
Calcd. for C22H34OgN4H2O C, 51.53; H, 7.02; N, 10.85. Found: C. 51.11; H, 7.03; N,
10.88.
EXAMPLE PKC10
MeO-Suc-Val-Pro-DL~Phe-C02Me. This compound was prepared from MeO-
Suc-Val-Pro-Phe-OH in 42% yield by the same procedure as described in Example
PKC8; foam, single spot on tlc, Rf2 0.57 (CHCI3:MeOH = 9:1); MS, m/e = 517 (M+).
Anal- Calcd- for C26H358N3 2/3 H2O: C, 58.96; H, 6.90; N, 7.93. Found C 58 92; H
6.96; N, 7.89.
EXAMPLE PKCll
Bz-DL~Ala-CO2-n-Bu. This compound was prepared from Bz-Ala-OH in 45%
yield by the procedure described in Example PKC1, except that n-butyl oxalylchloride
was used for the Dakin-West reaction and sodium n-butoxide in n-butanol was used for
enol ester hydrolysis; colorless oil, single spot on tlc, Rr2 = 0.72 (CHCI3:MeOH = 9:1);
MS, m/e = 277 (M+).
EXAMPLE PKC12
Bz-DL,Ala-CO2Bzl. This compound was prepared from Bz-Ala-OH in 2G~c
yield by the procedure described in Example PKC1, except that benzyl oxalyl chloride
was used in place of ethyl oxayl chloride and sodium benzyloxide in benzyl alcohol was
used for enol ester hydrolysis; single spot on tlc, Rf2 =0.69 (CHCI3:MeOH = 9:1); mp
95-97 C; MS, m/e = 312 (M+ + 1). Anal. Calcd. for C18H17O4N.1/2 H20: C, 67.48;
H, 5.66; N, 4.37. Found: C, 67.78; H, 5.55; N, 4.66.
EXAMPLE PKCl3
Z-Ala-DI~Ala-C02-n-Bu. This compound was prepared from Z-Ala-Ala-OH in
14% yield by the procedure described in Example PKC1, except that n-butyl oxalylchloride was used in the Dakin-West reaction and sodium rt-butoxide was used for enol
ester hydrolysis; oil, single spot on tlc, Rf2 = 0.45 (CHC13:MeOH = 9:1); MS, m/e =
378 (M+). Anal. Calcd. for C1gH26O6N2 l/3 H2O: C, 59.35; H, 7.00; N, 7.29. Found:
C, 59.41; H, 7.03; N, 7.10.
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EXAMPLE PKC14
Z-Ala-DL,Ala-C02Bzl. This compound was prepared from Z-Ala-Ala-OH in
36% yield by the procedure described in Example PKC1, except that benzyl oxalyl
chloride was used in the Dakin-West reaction and sodium benzyloxide in benzyl alcohol
was used for enol ester hydrolysis; single spot on tlc, Rf2 = o 55 (CHC13:MeOH = 9:1);
MS, m/e = 413 (M+ + 1). Anal. Calcd. for CæH24O6N2: C, 64.06; H, 5.87; N, 6.79.
Found: C, 63.79; H, 5.95; N, 6.72.
EXAMPLE PKC15
Z-Ala-Ala-DL,Abu-CO2Bzl. This compound was prepared from Z-Ala-Ala-Abu-
OH in 31% yield by the procedure described in Example PKC1, except that benzyl
oxalyl chloride was used in the Dakin-West reaction and sodium benzyloxide in benzyl
alcohol was used for enol ester hydrolysis; single spot on tlc, Rf2 = 0.40 (CHCl3:MeOH
= 9:1); mp 124-125 C; MS, m/e = 498 (M++1). Anal. Calcd. for C26H3lO7N32/3
H2O: C, 61.28; H, 6.39; N, 8.24. Found: C, 61.14; H, 6.65; N, 7.94.
EXAMPLE PKC16
Bz-DL-Ala-COOH. The hydrolysis procedure of Tsushima et al., J. Orp. Chem.,
49:1163-1169 (1984) was used. Bz-DL-Ala-CO2Et (540 mg, 2.2 mmol) was added to a
solution of 650 mg of sodium bicarbonate in an aqueous 50% 2-propanol solution (7.5
mL of H2O and 2-propanol) and stirred at 40 C under nitrogen. After adding ethyl
acetate and a saline solution to the reaction mixture, the aqueous layer was separated
and acidified with 2N HCl and extracted with ethyl acetate. The organic layer was
dried over magnesium sulfate and the solvent was removed under reduced pressure.The crude hydrolysis product was chromatographed on silica gel and eluted with
methylene chloride and methanol to obtain an oil (150 mg, 31%); single spot on tlc, Rf4
= 0.68 (n-butannl ~-~etic acid:pyridine:H20 = 4:1:1:2). Anal. Calcd. for CllHll04N.3/4
H2O: C, 56.28; H, 5.37; N, 5.97. Found: C, 56.21; H, 5.46; 5.66.
EXAMPLE PKC17
Z-Leu-DI,Nva-COOEt. This compound was prepared from Z-Leu-Nva-OH in
60 % yield by the procedure described in Example PKC1; oil, one spot on tlc, Rf =
0.49 (CHCI3:MeOH = 20:1). NMR (CDCI3) ~: 0.91 (t, 9H), CH3; 1.25 (t, 3H), CH3;
1.38 (q, 2H), OCH2CH3; 1.64 (m, 6H), CH2; 1.85 (m, lH), CH(CH3)2; 4.34 (m, lH)
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CH2CH(NHCOOCH2Ph)CONH; 5.12 (d, 3H) NHCH(CO)CH2 and OCH2Ph; 5.32 (d,
lH) NH; 6.71 (d, lH) NH; 7.36 (s, SH) Ph.
Z-Leu-DL-Nva-enol ester, the precursor of Z-Leu-DL-Nva-COOEt was
synthesized by the same procedure as described in Example PKCl and purified by
column chromatography, oil, one spot on tlc. NMR (CDCl3) ~: 0.96 (t, 9H); 1.25 (t,
3H); 1.41 (t, 2H); 1.54 (m, 4H); 1.72 (m, 3H); 2.80 (t, 2H); 4.20 (q, 2H); 4.43 (q, 2H);
5.16 (q, 2H); 5.23 (s, lH); 7.37 (m, 5H); 11.33 (s, lH).
EXAMPLE PKC18
Z-Leu-DI~Phe-COOEt. This compound was prepared from Z-Leu-Phe-OH in
30 % yield by the procedure described in Example PKCl; oil, one spot on tlc, Rf =
0.47 (CHC13:MeOH = 50:1). NMR (CDCl3) ô: 0.88 (d, 9H), OCH2CH3 and
(CH3)2CH; 1.35 (q, 2H), OCH2CH3; 1.56 (q, 2H), (CH3)2CHCH2CH; 3.03 (m, lH),
(CH3)2CH; 4.32 (m, 2H), NHCH(CO)CH2; 5.08 (s, 4H) CH2Ph; 5.40 (m, lH) NH; 6.61
(d, lH) NH; 7.31 (s, SH) Ph; 7.35.(s, SH) Ph.
lS Z-Leu-DL-Phe-enol ester, the precursor of Z-Leu-DL-Phe-COOEt was
synthesized by the same procedure as described in Example PKCl and purified by
column chromatography, oil, one spot on tlc. NMR (CDCl3) ~: 0.86 (t, 3H); 0.99 (t,
3H); 1.24 (t, 3H); 1.40 (t, 3H); 1.52 (m, 2H); 1.83 (m, 2H); 4.23 (m, 4H); 4.39 (q, 2H);
5.10 (t, 2H); 5.18 (s, lH); 7.26 (m, SH); 7.34 (m, 5H); 8.89 (s, lH).
EXAMPLE PKCl9
Z-Leu-D~Abu-COOEt. This compound was prepared from Z-Leu-Abu-OH in
33 % yield by the procedure described in Example PKCl; oil, one spot on tlc, Rf =
0.66 (CHCl3:MeOH = 20:1). NMR (CDCl3) ~: 0.96 (t, 9H), OCH2CH3 and
(CH3)2CH; 1.26 (t, 3H), CH2CH2CH3; 1.37 (q, 2H), OCH2CH3; 1.66 (q, 2H),
(CH3)2CHCH2CH; 2.00 (m, IH), CH(CH3)2; 4.12 (q, 2H) CHCH2CH3; 4.34 (m, lH)
NHCH(CONH)CH2CH(CH3)2; 5.12 (q, 3H) CH2Ph and CONH(Et)CHCOCOO; 5.29
(t, lH) NH; 6.79 (d, lH) NH; 7.35 (s, SH~ Ph.
Z-Leu-DL-Abu-enol ester, the precursor of Z-Leu-DL-Abu-COOEt was
synthesized by the same procedure as described in Example PKCl and purified by
column chromatography, oil, one spot on tlc. NMR (CDC13) ~: 0.98 (t, 6H); 1.12 (t,
3H`; 1.24 (t, 3H); 1.41 (t, 3H); 1.73 (m, 4H); 2.85 (q, 2H); 4.20 (q, 2H); 4.31 (m, lH);
4.42 (q, 2H); S.lS (q, 2H); 5.21 (s, lH); 7.34 (m, SH); 11.29 (s, lH).
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EXAMPLE PKC20
Ala-DL-Lys-COOEt HCI. To a solution of N-carbobenzyloxyalanyl-Ne-
carbobenzyloxylysine (1.88 g, 3.9 mmol), 4-dimethylaminopyridine (21 mg, 0.17 mmol),
and pyridine (1.0 mL, 12.4 mmol) irl THF (7 mL) was added ethyl oxalyl chloride (0.9
mL, 8.0 mmol) at a rate suffi- ient to start refluxing. The mixture was refluxed gently
for 3 hr, treated with water (4 mL), and stirred vigorously at room temperature for 30
min. The mixture was extracted with ethyl acetate, the organic extracts were washed
with water, dried over MgSO4 and evaporated to give an oily residue (1.56 g). To a
solution of the enol ester (1.56 g, 2.7 mmol) in anhydrous ethanol was added dropwise
a solution of sodium ethoxide in ethanol at room temperature until the solution turned
clear yellow. Ethanol was removed and the residue was dissolved in ethyl acetate. The
organic solution was washed with water, dried over MgSO4, and evaporated to give a
residue. This residue was then purified by column chromatography and the productwas eluted with chloroform-methanol. The solvent was removed and Z-Ala-DL-Lys(Z)-
CO2Et was obtained as a h~-oscopic powder (328 mg, 16 ~G), single spot on tlc, Rf2 =
0.53 (CHCI3:MeOH = 9:1); MS, m/e = 542 (M++l).
N-Carbobenzoxyalanyl-DL-Necarbobenzoxylysine keto ethyl ester, Z-Ala-DL-
Lys(Z)-CO2Et (328 mg, 0.61 mmol) was deprotected with liquid HF containing anisole
at 0 C for 30 min. The HF was removed under reduced pressure. The residual oil
was dissolved in absolute ethanol. HCI/ethanol was added to the solution, and ethanol
was removed in vacuo. The residue was washed by decantation with ether to give asemi solid (216 mg, 100 %); single spot on tlc (n-butanol:acetic acid:pyridine:H2O =
4:1:1:2).
EXAMPLE PKC21
Bz-DL-Lys-COOEtHCI. This compound was prepared from Bz-DL-Lys(Z)-
COOEt in 62% yield by the procedure described in Example PKC20; one spot on tlc,Rf4 = 0.57 (n-butanol:acetic acid:pyridine:H2O = 4:1:1:2). The precursor, Bz-DL-Lys(Z)-COOEt was prepared from Bz-Lys(Z)-OH in 100% yield by the procedure
described in Example PKC1; powder, one spot on tlc, Rr2 = o 75 (CHCI3:MeOH =
9:1); MS m/e = 440 (M+). Anal. Calcd. for C24H28O6N22/3 H2O: C, 63.70; H,
N, 6.19. Found: C, 63.49; H, 6.51; N, 5.92.
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EXAMPLE PKC22
Bz-DL,Arg-COOEt HCI. This compound was prepared from Bz-DL-Arg(Z)-
COOEt in 99% yield by the procedure described in Example PKC20; one spot on tlc,Rf4 = 0.71 (n-but~nol-aceti~ acid:pyridine:H2O = 4:1:1:2), Sakaguchi reagent positive.
Bz-DL-Arg(Z)-COOEt was prepared from Bz-DL-Arg(Z)-OH in 19% yield by the
procedure described in Example PKC20, Rf2 = 0.38 (CHCl3:MeOH = 9:1); mp 140-
142 C; MS, m/e = 468 (M+). Anal. Calcd for C24H286N4 C, 61.53; H~ 6-02; N~
11.96. Found: C, 61.96; H, 6.48; N, 12.34.
EX~MPLE PKC23
H-Gly-DL-Lys-COOEt2HCI. This compound was prepared from Z-Gly-DL-
Lys(Z)-COOEt in 92% yield by the procedure described in Example PKC20; Rf4 =
0.21 (n-butanol-acetir acid:pyridine:H2O = 4: 1: 1:2). Z-Gly-DL-Lys(Z)-COOEt wasprepared from Z-Gly-Lys(Z)-OH in 9% yield by the procedure described in Example
PKC20, one spot on tlc, Rfl = 0.68 (CHCI3:MeOH = 5:1); MS, m/e = 528 (M++1).
EXAMPLE PKC24
H-Pro-DL-Lys-COOEt 2HCI. This compound was prepared from Z-Pro-DL-
Lys(Z)-COOEt in 100% yield by the procedure described in Example PKC20; one spoton tlc (n-butannl ~ceti~ acid:pyridine:H20 = 4:1:1:2). Z-Pro-DL-Lys(Z)-COOEt wasprepared from Z-Pro-Lys(Z)-OH in 15% yield by the procedure described in ExamplePKC20; Rf2 = 0.73 (CHCI3:MeOH = 9:1); MS, m/e 568 (M+ + 1).
EXAMPLE PKC25
H-Phe-DL-Lys-COOEt 2HCI. This compound was prepared from Z-Phe-DL-
Lys(Z)-COOEt in 39% yield by the procedure described in Example PKC20; one spot
on tlc (n-butannl-~cetic acid:pyridine:H2O = 4:1:1:2). Z-Phe-DL-Lys(Z)-COOEt wasprepared from Z-Phe-Lys(Z)-OH as previously described in 9% yield, Rf2 = 0.68
(CHCI3:MeOH = 9:1); MS, m/e = 482 (M+).
EXAMPLE PKC26
H-Leu-Ala-DL-Lys-COOEt2HCI. This compound was prepared from Z-Leu-
Ala-DL-Lys(Z)-COOLt in 52% yield by the procedure described in Example PKC20;
one spot on tlc (M-butanol:acetic acid:pyridine:H2O = 4:1:1:2).
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Z-Leu-Ala-DL,Lys(Z)-COOEt was prepared from Z-Leu-Ala-DL-Lys(Z)-OH in
5% yield by the previously described Dakin West reaction, Rf3 = 0.34 (CHCl3:MeOH= 19:1); MS, m/e = 609 (M+-OCH2CH3).
EXAMPLE PKC27
S Simple Amino Acid, Di- and ~npeptide Enol Esters (General Procedure). A
modified Dakin-West procedure was used (Charles et al., supra) and is illustrated with
the synthesis of Z-Leu-DL-Phe-EE. To a stirred solution of Z-Leu-Phe-OH (6.19 g,15.0 mmol), 4- dimethylaminopyridine (0.183 g; 1,5 mmol) and pyridine (4.75 g, 4,85 ml,
60 mmol) in tetrahydrofuran (45 ml) warmed 50 C was added ethyl oxalyl chloride(4.30 g, 3.52 ml, 31.5 mmol) at a rate sufficient to initiate refluxing. The mixture was
then heated at a gentle reflux for 4 h. After cooling to room temperature the mixture
was treated with water (25 ml) and stirred vigorously at room temperature for 30 min.
The mixture was extracted with ethyl acetate (l50 ml) and after separation of the
organic layer, the water layer was saturated with solid (NH4)2SO4 and re-extracted
2-times with 25 ml ethyl acetate. The combined organic phases were washed 2-times
with 75 ml water, 2-times with 50 ml of satd. NaCI, decolorized with carbon and dried
over MgSO4. After evaporation of the solvent, the crude enol ester (8,36 g, 98%) was
flash-chromatographed on silica gel and the product was eluted with a AcOEt. Thesolvent was evaporated in vacuo (rotavaporator) and the pure enol ester was obtained
as a oil (7.22 g, 85%); single spot on TLC, Rf = 0.84, A; 0.68, C.
Z-Leu-Nva-EE This compound was prepared from Z-Leu-Nva- OH using the
general procedure and purified by flash chromatography on silica gel using
CHCl3:MeOH = 50:1 v/v as eluent. Yield 95%, single spot on TLC, Rf = 0.92, C;
0.28, L.
Z-Leu-Abu-EE. This compound was prepared from Z-Leu-Abu- OH in 78%
yield the general procedure described above. Purification by flash obramatography on
silica gel. Eluent, CHCl3:MeOH = 50:1 v/v, single spot on TLC, Rf = 0.86, A.
PfiCO-Abu-EE. This compound was prepared from PhCO-Abu-OH in 26%
yield by the general procedure as described above. Purification by flash
chromatography on silica gel. Eluent CHCl3, single spot on TLC, Rf = 0.60, M.
(CH3)2CH(CH2)2CO-Abu-EE. This compound was prepared from
(CH3)2CH(CH2)2CO-Abu-OH in 82% yield by the general procedure as described
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above. Purification by flash chromatography on silica gel. Eluent AcOEt, single spot on
TLC, Rf = 0.72, C.
(CH3CH2CH2)2 CH CO-Abu-EE. This compound was prepared from
(CH3CH2CH2)2CH CO-Abu-OH in 100% yield by the general procedure described
above. Purification by flash chromatography on silica gel. Eluent AcOEt, single spot
on TLC, Rf = 0.78, C; 0.81, K.
Ph(CH2)6CO-Abu-EE. This compound was prepared from
Ph(CH2)6CO-Abu-OH in 86% yield by the general procedure described above.
Purification by flash chromatography on silica gel. Eluent AcOEt. Single spot on TLC,
Rf =0.74, C.
Z-Leu-4-CI-Phe-EE This compound was prepared from Z-Leu-4-Cl-Phe-OH in
69% yield by the general procedure described above. Purification by flash
chromatography on silica gel. Eluent AcOEt, sing!e spot on TLC, R~ = 0.77, C; 0.78,
K.
Z-Leu-Leu-Abu-EE. This compound was prepared from Z2-Leu-Leu-Abu-OH
in 62% yield by the general procedure described abo-e. Purification by flash
chromatography on silica gel. Element CHCI3:MeOH = 50:1 v/v. Single spot on TLC,Rf = 0.89, A; 0.75, M.
Z-Leu-Leu-P~e-EE. This compound was prepared from Z-Leu-Leu-Phe-OH in
60% yield by the general procedure described above. Purification by flash
chromatography on silica gel. Eluent CHC13:MeOH = 50:1 v/v. Single spot on TLC,
Rf = 0.80, K; 0.70, M.
2-NapSO2-Leu-Leu-Abu-EE. This compound was prepared from
2-NapSO2-Leu-Abu-OH in 73% yield by the general procedure described above.
Purification by flash chromatography on silica gel. Eluent AcOEt, single spot on TLC,
Rf = 0.71, K; 0.54, C.
2-NapS02-Leu-Leu-Abu-EE. This compound was prepared from
2-NapSO2-Leu-Leu-Abu-QH in 74% yield by the general procedure described above.
Purification by flash chromatography on silica gel. Eluent AcOEt: AcOH = 200:1 v/v.
Sirlgle spot on TLC, Rf = 0.69. K.
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EXAMPLE PKC28
Z-Leu-Phe-COOEt. Single Aminoacid, Di-a~ld 'rnpeptide- ketoesters (General
Procedure). To a stirred solution of 8.53 g (15.0 mmol) of Z-Leu-Phe-EE in 40 mlanhydrous ethanol at room temperature was added dropwise a solution of sodium
ethoxide (0.204 g; 3.0 mmol) in 20.0 ml anhydrous ethanol. The color of the reaction
mixture change from colorless or pall yellow to deep yellow or orange dependent on
enol-ester. Then the reaction mixture was stirred at room temperature for 4-5 hours,
the ethanol was then evaporated in vacuo (rotavaporator) and the residue treated with
200 ml ethyl ether (or 200 ml ethyl acetate in the case of the tripeptide). The ether
(ethyl acetate) solution was washed with 2 x 75 ml H2O, 2 x 75 ml satd. NaCl,
decolorized with carbon and dried over MgSO4. After evaporation of solvent, the
crude product 6.09 g (89.7%) was flash chromatographed on silica gel using CHCl3:
MeOH = 50:1 v/v. Evaporation of solvent give pure Z-Leu-Phe-COOEt (4.08 g;
58.0%) as a thick oil. Single spot on TLC, Rf = 0.60, A; 0.47, M. Mass spectrum,FB-MS [(M + 1)/Z] = 469.
EXAMPLE PKC29
Z-Leu-Nva-COOEt. This was prepared by the preceding general procedure.
Purification by flash chromatography on silica gel, eluent CHC13: MeOH = 100:1 v/v,
yield 86.6%, thick, colorless oil, single spot on TLC, ~f = 0.49, A; 0.37, M. Mass
spectrum FB-MS [(M + 1)tZ~ = 421.
EXAMPLE PKC30
Z-Len-Abu-COOEt. This was prepared by the preceding general procedure.
Purification by flash chromatography on silica gel, eluent CHCI3, yield 82%, thick, pale
yellow oil, single spot on TLC, Rf = 0.66, A. Mass spectrum, CI-MS [(M+ 1)/Z] =
407.
EXAMPLE PKC31
PhCO-Abu-COOEt. This was prepared by the preceding general procedure.
Purification by flash chromatography on silica gel, eluent CHCl3:MeOH = 50:1 v/v,
yield 83%, oil, single spot on TLC, Rf = 0.44, M. Mass spectrum, M/Z 263 (M+);
CI-MS, 264 ((M+1)/Z).
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EXAMPLE PKC32
(CH3)2CH(CH2)2CO-Abu-COOEt. This was prepared by the preceding general
procedure. Purification by flash chromatography on silica gel, eluent AcOEt, yield
43%, oil, single spot on TLC, Rf = 0.56, C. Mass spectrum EI-MS M/Z 257 (M+);
FB-MS, [(M + 1)/Z] = 258.
EXAMPLE PKC33
CH3CH2CH)2CHCO-Abu-COOEt. This was prepared by the preceding general
p,ocedu,e. Purification by flash chromatography on silica gel, eluent CHCI3:MeOH =
50: 1 v/v, thick, yellowish oil, yield 66%, single spot on TLC, Rf = 0.80, C; 0.66, M.
Mass spectrum EI-MS M/Z = 285 (M + ); CI-MS, [(M + 1)/Z] = 286.
EXAMPLE PKC34
Ph(CH2)6CO-Abu-COOEt. This was prepared by the preceding general
procedure. Purification by flash chromatography on silica gel, eluent CHCl3:MeOH =
50:1 v/v, yield 64%, pale yellow oil, single spot on TLC, Rf = 0.29, M. Mass spectrum
EI-MS M/Z = 347 (M + ), FB-MS, [(M + 1)/Z] = 348.
EXAMPLE PKC3~
Z-Leu-4-Cl-Phe-COOEt. This was prepared by the preceding general
procedure. Purification by flash chromatography on silica gel, eluent AcOEt, yield
100%, colorless oil, single spot on TLC, Rf = 0.71, C. Mass spectrum FB-MS M/Z =503(M + ).
EXAMPLE PKC36
Z-Leu-Leu-Abu-COOEt. This was prepared by the preceding general
procedure. Purification by flash chromatography on silica gel, eluent CHCl3:MeOH =
50:1 v/v, yield 79.2%, very thick, colorless oil, single spot on TLC, Rf = 0.28. M. Mass
s~e~ .l. FB-MS, [(M+1)/Z] = 520.
EXAMPLE PKC37
Z-Leu-Leu-Phe-COOEt. This was prepared by the preceding general procedure.
Purification by flash chromatography on silica gel, eluent CHC13:MeOH = 50:1 v/v,
yield 33%, oil, single spot on TLC, Rf = 0.56, M. Mass spectrum, FB-MS, [(M+ 1)/Z]
= 582.
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EXAMPLE PKC38
2-NapSO2-Leu-Abu-COOEt. This was prepared by the preceding general
procedure. Purification by flash chromatography on silica gel, eluent CHCI3:MeOH =
50:1 v/v, yield 38%, thick oil, single spot on TLC, Rf = 0.71, K; 0.54, A. Mass
spectrum FB-MS, [(M + 1)/Z] = 463.
EXAMPLE PKC39
2-NapS02-Leu-Leu-Abu-COOEt. This was prepared by the preceding general
procedure. Purification by flash chromatography on silica gel, eluent AcOEt:AcOH =
200:1 v/v, yield 61%, semi-solid, single spot on TLC, Rf = 0.67, K. Mass spectrum
FB-MS, [(M + 1 )/Z] = 576.
EXAMPLI; PKC40
Z-Leu-Met-CO2Et. This compound was prepared by the above procedure.
Yellow oil, single spot on TLC, Rf = 0.52 (CHCI3:CH30H=50:1), yield 46% (from
dipeptide), MS (FAB) 454 (m+ 1).
EXAMPLE PKC41
Z-Leu-NLeu-CO2Et. This compound was prepared by the above procedure.
Pale yellow oil, single spot on TLC, Rf = 0.57 (CHCI3:CH30H = 50:1), yield 53%
(from dipeptide), MS (FAB) 434 (m+ 1).
EXAMPLE PKC42
Synt~tesis of n-But~l Oxal~l C/tloride. This was prepared by a literature procedure
(Warren and Malee, supra). N-Butanol (0.1 mol. 7.41 g) was added dropwise to oxalyl
chloride (0.5 mol. 63.5 g) at -10 C. After the addition was completed, the reaction
mixture was stirred for 20 min. at r.t. and distilled, giving 15.0 g (91.18 mol. 91%) of
the product n-butyl oxalyl chloride, bp 58-60 C (0.6 mm Hg).
Z-Leu-Phe-CO2Bu. This compound was prepared from Z-Leu- Phe-OH and
butyl oxalyl chloride in 43% yield by the procedure described for the synthesis of
Z-Leu-Phe-CO2Et, except that butyl oxalyl chloride was used in place of ethyl oxalyl
chloride and sodium butyloxide in butanol was used for enol ester hydrolysis. Single
spot on TLC, Rf = 0.54 (CHCI3:CH3OH = 50:1) MS(FAB) m/e = 497 (m+ 1), lH
NMR (CDCI3) ok.
~ ~ ~ 81 2 ~ PCr/US93/06143
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EXAMPLE PKC43
Z-Leu-Abu-CO2Bu. This compound was prepared by the above procedure.
Single spot on TLC, Rf = 0.53 (CHCI3:CH30H = 50:1), yield = 36%, pale yellow oil,
MS (FAB) m/e = 435 (M+ 1), 1H NMR (CDCI3) ok.
S EXAMPLE PKC44
Synthes~s of Benyl Oxalyl CIZIOride. Benzyl alcohol (0.15 mol. 16 g) was added
dropwise to oxalyl chloride (0.75 mol. 95 g) at 5-10 C. After the addition was
complete, the reaction was stirred for 20 min. at r.t. The excess oxalyl chloride was
distilled and recycled. Then the mixture was distilled under vacuo, giving 26 g (0.12
mol. 86%) of benzyl oxalyl chloride, bp. 110-112 C (0.6 mm-Hg). HlNMR (CDCI3)
7.39 (s, SH), 5,33 (s.2H).
Z-Leu-Phe-CO2Bzl. This compound was prepared from Z-Leu- Phe-OH and
benzyl oxalyl chloride in 17% yield by the procedure described in the synthesis of
Z-Leu-Phe-CO2Et, except that benzyl oxalyl chloride was used in place of ethyl oxalyl
chloride and sodium benzyloxide in benzyl alcohol was used for enol ester hydrolysis.
Single spot on TLC, Rr = 0.63 (CHCI3:CH30H = 50:!). Pale yellow solid, mp 117-119
C. MS(FAB) m/e = 532 (m+ 1). HlNMR ok.
EXAMPLE PKC45
Z-Leu-Abu-CO2Bzl. This compound was prepared by the above procedure.
Single spot on TLC. Rf = 0.51 (CHCl3:CH30H = 50:1), pale yellow oil, MS(FAB) m/e= 469 (m+1), yield = 26%.
EXAMPLE PKC46
Z-Leu-Phe-COOH. Dipeptide Ketoacids (General Procedure). To a stirred
solution of 0.53g (1,13 mmol) Z-Leu-Phe-COOEt in 6.0 ml methanol was added 1.27 ml
(1.27 mmol) lM NaOH. The color of the reaction mixture turned dark yellow and a
small amount of solid was deposited. The reaction was run at room temperature and
progress of the hydrolysis was checked on TLC. After 24 h. no more substrate wasdetected. The reaction mixture was chilled in one ice bath at 5 C, acidified with lM
HCl to pH = 3 and extracted with AcOEt (2 x 50 mL). The organic extract were
washed with 2 x 50 ml H2O and if necessary, decolorized with carbon and dried over
MgSO4. After evaporation of the solvent (rotavaporator), the residue (thick oil) were
titura.ed with 2 x 25 ml n-hexane and dried in vacuo. Yield 0.39 g (78%) of colorless,
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very thick oil. TLC, main spot at Rf = 0.24, trace of impurity at Rf = 0.78, I. Mass
spectrum, FB-MS [(M+ 1)/Z] = 441.
EXAMPLE PKC47
Z-Leu-Abu-COOH. This compound was prepared from Z-L-Leu- Abu-COOEt
in 83% yield by the general procedure as described above; TLC, main spot at Rf =0.14, trace of impurity at Rf = 0.73, I. Mass spectrum, FB-MS [(M+ 1)/Z] = 379.
EXAMPLE PKC48
ZLeu-Phe-CONH-Et. To a stirred solution of Z-Leu-Phe-OH (20 g, 48.5
mmole), 4-dimethylaminopyridine (0.587 g, 4.8 mmole),and pyridine (15.7 ml, 194
mmole) in anhydrous THF (100 ml) was added ethyl oxalyl chloride (11.4 ml, 101.8mmole) at a rate sufficient to initiate refluxing. The mixture was gently refluxed for 4
hours, cooled to room temperature, and water (80 ml) was added. The reaction
mixture was stirred vigorously for 30 min, and extracted with ethyl acetate (3 x 100 ml).
The combined organic layers were washed with water (2 x 100 ml), saturated sodium
chloride (2 x 100 ml), decolorized with decolorizing carbon, dried over magnesium
sulfate, and concentrated, leaving a dark orange Oil. Chromatography on a silica gel
column with CHCl3/CH30H (50:1 v/v) afforded 14.63 g (y = 53 %) of Z-Leu-Phe-
enolester. The product was a yellow Oil. Single spot on TLC, Rf = 0.77
(CHCL3/CH30H 50:1). NMR (CDCl3) ok.
To a stirred pale yellow solution of the Z-Leu-Phe-enolester (14.63 g, 25.73
mmole) in anhydrous ethanol (50 ml) was added a solution of sodium ethoxide (0.177
g, 2.6 mmole) in ethanol (5 ml). The orange solution was stirred for 3 hours at room
temperature, then the ethanol was evaporated and the residue was treated with ethyl
ether (300 ml). The ether layer was washed with water (2 x 100 ml), saturated sodium
chloride (2 x 100 ml), dried over magnesium sulfate, and concentrated, leaving a orange
oil. Chromatography on a silica gel column with CHCl3/CH30H (50:1 v/v) afforded
7.76 g (y = 64 %) of the a-ketoester Z-Leu-Phe-COOEt. The product was a yellow oil.
Single spot on TLC, Rf = 0.44 (CHCl3/CH30H 50:1). NMR (CDCl3) ok. MS (FAB,
calcd. for C26H32N2O6: 468.6), m/e = 469 (M+ 1).
The a-carbonyl group of Z-Leu-Phe-COOEt was protected by following
procedure. A solution of Z-Leu-Phe-COOEt ( 1 g, 2.13 mmole) in 5 ml of CH2Cl2 was
added 1,2-ethanedithiol (0.214 ml, 2.55 mmole), followed by 0.5 ml of boron trifluoride
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etherate. The solution was stirred overnight at room temperature. Water (20 ml) and
ethyl ether (20 ml) were added. The organic layer was separated, washed with water (2
x 10 ml), saturated sodium chloride (2 x 10 ml), dried over magnesium sulfate, and
evaporated to afford 0.98 g (y = 84 %) yellow semisolid.
S The protected a-keto~ster (0.98 g, 1.8 mmole) was dissolved in ethanol (5 ml),
cooled to 0-5 C in a ice bath, and ethylamine was bubbled through the solution until
2.43 g (54 mmole) had been added. The reaction mLxture was allowed to warm to
room temperature slowly, and stirred overnight. The mixture was filtered, a white
p.~ipilate was removed, leaving a yellow semisolid. Chromatography on a silica gel
column with CHC13/CH30H (30:1 v/v) afford 0.63 g (y = 75 %) of Z-Leu-Phe-CONH-
Et. The product was a pale yellow solid. Single spot on TLC, Rf = 0.60
(CHCI3/CH30H 20:1); mp 145-147 C. Anal. calcd. for C26H33N3O5 467.56; C. 66.79;H, 7.11; N,8.99; found: C, 66.59; H, 7.09; N, 8.95. NMR (CDC13) ok. MS (FAB) m/e= 468 (M + 1)
EXAMPLE PKC49
Z-Leu-Phe-CONH-nPr. This compound was synthesized from the protected a-
ketoester and propylamine in 92 % yield by the procedure described in Example
PKC48. Single spot on TLC, Rf = 0.50 (CHCI3/CH30H 50:1); mp 152-153 C. Anal.
calcd. for C27H3sN3O5: 481.57; C, 67.33; H, 7.33; N, 8.72. Found: C, 67.21; H, 7.38; N,
8.64. NMR (CDC13) ok. MS (FAB) m/e = 482 (M+ 1).
EXAMPLE PKC50
~Leu-Phe-CONH-nBu. This compound was synthesized from the protected a-
k~toester and butylamine in 67 % yield by the procedure described in Example PKC48.
Single spot on TLC, Rf = 0.50.(CHC13/CH30H 50:1); mp 152-153 C. Anal. calcd. for
4H37N305: 495.59; C, 67.85; H, 7.52; N, 8.48. Found: C, 67.70; H, 7.57; N, 8.43.NMR (CDCI3) ok. MS (FAB) m/e = 496 (M+ 1).
EXAMPLE PKC51
Z-Leu-Phe-CONH-iBu. This compound was synthesized from the protected a-
ketoester and isobutylamine in 53 % yield by the procedure described in Example
PKC48. Single spot on TLC, Rf = 0.54 (CHCI3/CH30H 50:1); mp 152 C. Anal.
calcd. for C28H37N305: 495.59; C, 67.85; H, 7.52; N, 8.48. Found: C, 67.77; H, 7.56; N,
8.40. NMR (CDCI3) ok. MS (FAB) m/e = 496 (M+ 1).
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EXAMPLE PKC52
Z-Leu-Phe-CONH-Bzl. This compound was synthesized from the protected a-
ketoester and benzylamine in 40 % yield by the procedure described in Example
PKC48. After reacting overnight, ethyl acetate (60 ml) was added. The mixture was
filtered to remove a white precipitate. The solution was washed with cooled 1 N HCl
(3 x 25 ml), water (1 x 20 ml), saturated sodium chloride (2 x 20 ml), and dried over
magnesium sulfate. The solution was evaporated leaving a yellow solid.
Chromatography on a silica gel column with CHCl3/CH30H 30:1 v/v) afforded a
yellow solid. Single spot on TLC, Rf = 0.45 (CHCl3/CH30H 30:1); mp 160-162 C.
Anal. calcd. for C3lH3sN3O5: 529.61; C, 70.30; H, 6.66; N, 7.93. Found: C, 70.18; H,
6.67; N, 7.99. NMR (CDCl3) ok. MS (FAB) m/e = 530 (M+ 1).
EXAMPLE PKC53
Z-Leu-Phe-CONH-(CH2)2Ph. This compound was synthesized from the
protected a-ketoester and phenethylamine in 50 % yield by the procedure described in
E~ample PKC52. Single spot on TLC, Rf = 0.50 (CHCl3/CH30H 30:1); mp 151-153
C. Anal. calcd. for C32H37N3O5: 543.66; C, 70.70; H, 6.86; N, 7.73. Found: C, 70.54;
H, 6.88; N, 7.74. NMR (CDCl3) ok. MS (FAB) m/e = 544 (M+ 1).
EXAMPLE PKC51
Z-Leu-Abu-CONH-Et. This compound was synthesized from protected a-
ketoester derived from Z-Leu-Abu-CO2Et and ethylamine in 64 % yield by the
procedure described in Example PKC48. Single spot on TLC, Rf = 0.3G
(CHCl3/CH30H 50;1); mp 130-132 C. Anal. calcd. for C2lH3lN3Os: 405.45; C, G2.20;
H, 7.71; N, 10.36. Found: C, 61.92; H, 7.62; N, 10.31. NMR (CDCl3) ok. MS (FAB)
m/e = 406 (M + 1).
EXAMPLE PKCSS
Z-Leu-Abu-CONH-nPr. This compound was synthesized from the
corresponding protected a-ketoester and propylamine in 47 % yield by the procedure
described in Example PKC48. Single spot on TLC, Rf = 0.28 (CHCI3/CH30H 50:1);
mp 134-135 C. Anal. calcd. for C22H33N3Os: 419.50; C, 62.98; H, 7.93; N, 10.02.Found: C, 62.84; H, 7.97; N, 9.94. NMR (CDCl3) ok. MS (FAB) m/e = 420 (M+ 1).
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EXAMPLE PKC56
Z-Leu-Abu-CONH-nBu. This compound was synthesized from the
col~e~onding protected a-ketoester and butylamine in 42 % yield by the proceduredescribed in Example PKC48. Single spot on TLC, Rr = 0 54 (CHCI3/CH30H 50:1);
mp 135-136 C. Anal. calcd. for C23H3sN3Os: 433.53; C, 63.71; H, 8.13; N, 9.69.
Found: C, 63.48; H, 8.07; N, 9.67. NMR (CDCl3) ok. MS (FAB) m/e = 434 (M+ 1).
EXAMPLE PKC57
Z-Leu-Abu-CONH-iBu. This compound was synthesized from the
corresponding protected a-ketoester and isobutylamine in 65 % yield by the procedure
described in ExamplePKC48. Single spot on TLC, Rf = 0.25 (CHCl3/CH30H 50:1);
mp 133-135 C. Anal. calcd. for C23H3sN3O5: 433.52; C, 63.72; H, 8.14; N, 9.69.
Found: C, 63.46; H, 8.10; N, 9.60. NMR (CDCl3) ok. MS (FAB) m/e = 434 (M+1).
EXAMPLE PKC58
Z-Leu-Abu-CONH-Bzl. This compound was synthesized from the corresponding
protected a-ketoester and benzylamine in 29 ~o yield by the procedure described in
Example PKC52. Single spot on TLC, Rf = 0.56 (CHf'13/CH3OH 30:1); mp 140-141
C. Anal. calcd. for C26H33N30s: 467.54; C, 66.79; H, 7.11; N, 8.99. Found: C, 66.65;
H, 7.07; N, 8.93. NMR (CDCI3) ok. MS (FAB) m/e = 468 (M+ 1).
EXAMPLE PKC59
Z-Leu-Abu-CONH-(CH2)2Ph. This compound was synthesized from the
co,lei,pondingprotected a-ketoester and phenethylamine in 51 % yield by the
procedure described in Example PKC52. Single spot on TLC, Rf = 0.44
(CHCl3/CH30H 30:1); mp 156-157 C. Anal. calcd. for C2~H3sN3Os: 481.59; C, 67.34;
H, 7.33; N, 8.72. Found: C, 67.38; H, 7.33; N, 8.78. NMR (CDCI3) ok. MS (FAB)
m/e = 482 (M+ 1).
EXAMPLE PKC60
Z-Leu-Abu-CONH-(CH2)3-N(CH2CH2)20. This compound was synthesized
from protected a-ketoester and 4(3-aminopropyl)morpholine in 33 % yield by the
procedure described in Example PKC48. After reacting overnight, ethyl acetate (80 ml)
was added. The mixture was filtered to remove a white precipitate. The solution was
washed with water (3 x 20 ml), saturated sodium chloride (2 x 20 ml), and dried over
magnesium sulfate. The solution was evaporated leaving a yellow oil. Chromatography
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on a silica gel column with CHCl3/CH30H (10:1 v/v) afforded a yellow semisolid,
which was recryst~lli7ed from ethyl acetate/hexane to obtain a pale yellow solid. Single
spot on TLC, Rf = 0.42 (CHCI3/CH30H 10:1); mp 125-126 C. Anal. calcd. for
C26H40N4O6: 504.63; C, 61.88; H, 7.99; N, 11.10. Found: C, 61,69; H, 7.95; N, 11.07.
NMR (CDCl3) ok. MS (FAB) m/e = 505 (M+ 1).
EXAMPLE PKC61
Z-Leu-Abu-CONH-(CH2)7CH3 This compound was synthesized from the
corresponding protected a-ketoester and octylamine in 67 % yield by the procedure
described in Example PKC52. It was white solid. Single spot on TLC, Rf = 0.55
(CHCl3/CH30H 30:1); mp 134-135 C. Anal. calcd. for C27H43N3O5: 489.66; C, 66.23;
H, 8.85; N, 8.58. Found: C, 66.19; H, 8.81; N, 8.61. NMR (CDCI3) ok. MS (FAB)
m/e = 490 (M+1).
EXAMPLE PKC62
Z-Leu-Abu-CONH-(CH2)20H. This compound was synthesized from the
corresponding protected a-ketoester and ethanolamine in 29 % yield by the procedure
described in Example PKC60. The product was a white sticky solid. Single spot onTLC, Rf = 0.42 (CHCl3/CH30H 10:1); mp 151-153 C. Anal: calcd. for C21H31N3O6:
421.49; C, 59.84; H, 7.41; N, 9.97. Found: C, 59.11; H, 7.44; N, 9.81. NMR (CDCI3)
ok. MS (FAB) m/e = 422 (M+ 1).
EXAMPLE PKC63
Z-Leu-Abu-CONH-(CH2)20(CH2)20H. This compound was synthesized from
the corresponding protected a-ketoester and 2-(2-aminoethoxy)ethanol in 34 % yield by
the procedure described in Example PKC60. The product was white sticky solid.
Single spot on TLC, Rf = 0.42 (CHCl3/CH30H 10:1); mp 103-105 C. Anal.: calcd.
for C23H3sN3O~: 465.55; C, 59.34; H, 7.58; N, 9.03. Found: C, 59.23; H, 7.58; N, 9.01.
NMR (CDCl3) ok. MS (FAB) m/e = 466 (M+ 1).
EXAMPLE PKC64
Z-Leu-Abu-CONH-(CH2)l7CH3. This compound was synthesized from the
corresponding protected a-ketoester and octadecylamine in 12 % yield by the
procedure described in Example PKC52. The product was a pale yellow solid. Single
spot on TLC, Rf = 0.54 (CHCl3/CH30H 30:1); mp 134-136 C. Anal: calcd. for
v~) 94/00095 PCI/US93/06143
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C37H63N3O5: 629.92; C, 70.55; H, 10.08; N, 6.67. Found: C, 70.71; H, 10.14; N, 6.75.
NMR (CDCl3) ok. MS (FAB) m/e = 630.2 (M+ 1).
EXAMPLE PKC65
ZLeu-Abu-CONH-CH2-C6H3(OCH3)2. This compound was synthesized from
the col,esponding protected a-ketoester and 3,5-dimethoxybenzylamine in 4S ~O yield
by the procedure described in Example PKC52. The product was yellow sticky solid.
Single spot on TLC, Rf = 0.44 (CHC13/CH30H 30:1); mp 153-155 C. Anal.: calcd.
for C28H37N307: 527.62; C, 63.74; H, 7.07; N, 7.96. Found: C, 63.66; H, 7.09; N, 7.92.
NMR (CDC13) ok. MS (FAB) m/e = S28.8 (M+ 1).
EXAMPLE PKC66
Z-Leu-Abu-CONH-CH2-C4H4N. This compound was synthesized from the
cull~s~ul,ding protected a-ketoester and 4-(aminomethyl)pyridine in 45 % yield by the
procedure described in Example PKC60. The product was greenish yellow solid.
Single spot on TLC, Rf = 0.55 (CHCI3/CH30H 10:1); mp 124-126 C. Anal: calcd. for
C2sH32N40s: 468.55; C, 64.08; H, 6.88; N, 11.96. Found: C, 63.88; H, 6.87; N, 11.96.
NMR (CDCI3) ok. MS (FAB) m/e =469 (M+ 1).
EXAMPLE PKC67
Z-Leu-Abu-CONH-(CH2)5OH. This compound was synthesized from
1,3-dithiolane derivative of Z-Leu-Abu-COOEt and S-amino-1-pentanol. To a solution
of protected a-ketoester (1 mmol) in ethanol (3 mL) was added 5-amino-1-pentanol (3
mmol) and stirred overnight at r.t. To the mixture was added AcOEt (25 mL) and
white precipitate was filtered. The filtrate was washed with cold lN HCI (2 x 10 mL),
water (1 x 10 mL), saturated NaCI (2 x 10 mL) and dried over MgSO4. After
evaporation of the solvent, chromatography on a silica gel column using solvent
2S CHCl3/CH30H 10:1 followed by precipitation from AcOEt/hexane afforded a white
solid (42% yield). Single spot on TLC, Rf = 0.54 (CHCI3/CH30H 10:1), mp 122-123
C. lH NMR (CDC13) ok, MS (FAB) m/e = 464 (M+ 1). Anal: calcd. for
C24H37N30G, 463; C, 62.18; H, 8.04; N, 9.06. Found: C, 61.S2; H, 7.96; N, 8.98.
EXAMPLE PKC68
Z-Leu-Abu-CONH-(CH2)2OH. This is an alternative synthesis for the
compound designated in Example PKC 62. This compound was synthesized from
1,3-dithiolane derivative of Z-Leu-Abu-COOEt and ethanolamine by the procedure
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described in Example PKC67, and purified by column chromatography using solvent
CHCl3/CH30H 10:1 (40% yield). White solid, single spot on TLC, Rf = 0.42
(CHCl3/CH30H 10:1), mp 151-154 -C. 1H NMR (CDCl3) ok, MS (FAB) m/e = 422
(M+ 1). Anal: calcd. for C21H31N3O6, 421; C, 59.84; H, 7.41; N, 9.97. Found: C, 59.11;
H, 7.44; N, 9.81.
EXAMPLE PKC69
Z-Leu-Abu-CONH-(CH2)20(CH2)20H. This is an alternative synthesis for the
compound de~ign~ted in FY~mple PKC 63. This compound was synthesized from
1,3-~lithiol~ne derivative of Z-Leu-Abu-COOEt and 2-(2-aminoethoxy)ethanol by the
procedure described in Example PKC67, and purified by column chromatography using
solvent CHC13/CH30H 10:1 (34% yield). White solid, single spot on TLC, Rf = 0.42(CHCl3/CH30H 10:1), mp 103-105 -C. 1H NMR (CDCl3) ok, MS (FAB) m/e = 466
(M+ 1). Anal: calcd. for C23H3sN3O7, 465; C, 59.30; H, 7.58; N, 9.02. Found: C, 59.23;
H, 7.58; N, 9.01.
EXAMPLE PKC70
Z-Leu-Abu-CONH-CH2CH(OCH3)2. This compound was synthesized from
1,3--lithiol~ne derivative of Z-Leu-Abu-COOEt and aminoacetaldehyde dimethylacetal
by the procedure described in Example PKC67, and purified by column
chromatography using solvent CHCl3/CH30H 20: 1 (25% yield). White solid, single
spot on TLC, Rf = 0.47 (CHCl3/CH30H 20:1), mp 99-102 ~C. 1H NMR (CDCl3) ok,
MS (FAB) m/e = 466 (M+ 1). Anal: calcd. for C23H3sN3O7, 465; C, 59.30; H, 7.58; N,
9.02. Found: C, 58.95; H, 7.71; N, 9.00.
EXAMPLE PKC71
Z-Leu-Abu-CONH-CH2CH(OC2H5)2. This compound was synthesized from
1,3-~lithi- l~ne derivative of Z-Leu-Abu-COOEt and aminoacetaldehyde diethylacetal,
and purified by column chromatography using solvent CHCl3/CH30H 20:1 (36%
yield). White solid, single spot on TLC, Rf = 0.37 (CHCl3/CH30H 20:1), mp 100-103
C. lH NMR (CDCI3) ok, MS (FAB) m/e =494 (12%, M+ 1), 448 (100%, M+ 1-45).
Anal: calcd. for C2sH39N3O7, 493; C, 60.83; H, 7.96; N, 8.51. Found: C, 60.73; H, 7.98;
N, 8.42.
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EXAMPLE PKC72
~Leu-Abu-CONH-CH2-C6H8(1,3,3-(CH3)3-S-OH). This compound was
~rnthesized from 1,3-~1ithi~ ne derivative of Z-Leu-Abu-COOEt and
3-aminomethyl-3,5,5-trimethyl-cyclohexanol, and purified by column chromatography
using solvent CHCl3/CH30H 30:1 (51% yield). White solid, single spot on TLC, Rf =
0.55 (CHCl3/CH30H 30:1), mp 59-61 C. 1H NMR (CDCl3) ok, MS (FAB) m/e =
532 (M+ 1). Anal; calcd. for C29H4sN3O6,531; C, 65.51; H, 8.53; N, 7.90. Found, C,
65.21; H, 8.55, N, 7.81.
EXAMPLE PKC73
Z-Leu-Abu-CONH-(CH2)2C6H,~(4-OH). This compound was synthesized from
1,3-t1ithiol~ne derivative of Z-Leu-Abu-COOEt and 4-(2-aminoethyl)phenol, and
purified by column chromatography using solvent CHCl3/CH30H 30:1 (60~o yield).
White solid, single spot on TLC, Rf = 0.56 (CHC13/CH30H 30:1), mp 151-153 ~C. 1HNMR (CDCI3) ok, MS (FAB) m/e = 498 (M+ 1). Anal: calcd. for C27H3sN3O6, 497;
C, 65.17; H, 7.09; N, 8.45. Found, C, 65.16; H, 7.13, N, 8.52.
EXAMPLE PKC74
Z-Leu-Abu-CONH-(CH2)2C6H4(2-OCH3). This compound was synthesized from
1,3-~lithinl~ne derivative of Z-Leu-Abu-COOEt and 2-methoxyphenethylamine, and
purified by column chromatography using solvent CHCI3/CH30H 50:1 (71% yield).
Yellow solid, single spot on TLC, Rf = 0.47 (CHCl3/CH30H 50:1), mp 101-103 C.
lH NMR (CDCl3) ok, MS (FAB) m/e = 512 (M+ 1). Anal; calcd. for C2~,H37N306,
511; C, 65.73; H, 7.29; N, 8.'1. Found, C, 65.50; H, 7.31; N, 8.19.
EXAMPLE P~iC75
Z-Leu-Abu-CONH-(CH2)2C6H4(3-OCH3). This compound was synthesized from
1,3-dithiolane derivative of Z-Leu-Abu-COOEt and 3-methoxyphenethylamine, and
purified by column chromatography using solvent CHCI3/CH30H 50:1 (56% yield).
Yellow solid, single spot ~m TLC, Rf = 0.46 (CHCI3/CH30H 50:1), mp 99-100 ~ ~C. lH
NMR (CDCl3) ok, MS (FAB) m/e =512 (M+ 1). Anal: calcd. for C2~,,H37N306, 511; C,65.73; H, 7.29; N, 8.21. Found, C, 65.62; H, 7.34; N, 8.16.
EXAMPLE PKC76
ZLeu-Abu-CONH-(CH2)2C6H4(4-OCH3). This compound was synthesized from
1,3-di;hiolane derivative of Z-Leu-Abu-COOEt and 4-methoxyphenethylamine, and
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purified by column chromatography using solvent CHC13/CH30H 50:1 (50% yield).
White solid, single spot on TLC, Rf = 0.46 (CHCI3/CH30H 50:1), mp 152-155 --~C. lH
NMR (CDCl3) ok, MS (FAB) m/e = 512 (M+l). Anal: calcd. for C2,3H37N306, 511;
C, 65.73; H, 7.29; N, 8.21. Found, C, 65.64; H, 7.30; N, 8.19.
5EXAMPLE PKC77
Z-Leu-Abu-CONH-CH2C6H3(3,5-(OCH3)2. This compound was synthesized
from 1,3-dithiolane derivative of Z-Leu-Abu-COOEt and 3,5-dimethoxyphenethylamine,
and purified by column chromatography using solvent CHCI3/CH30H 50:1 (50~0
yield). Wllite solid, single spot on TLC, Rf = 0.46 (CHCl3/CH30H 50:1), mp 153-155
C. lH NMR (CDCI3) ok, MS (FAB) m/e = 528 (M+ 1). Anal: calcd. for
C28H37N307, 527; C, 63.74; H, 7.07; N, 7.96. Found, C, 63.66; H, 7.09; N, 7.92.
EXAMPLE PKC78
Z-Leu-Abu-CONH-CH2CH(OH)Ph. This compound was-synthesized from
1,3-dithiolane derivative of Z-Leu-Abu-COOEt and 2-amino-1-phenylethanol by the
procedure described in Example PKC67, and purified by column chromatography using
solvent CHCl3/CH30H 10:1(50% yield). White solid, single spot on TLC, Rf = 0.48
(CHC13/CH30H 10:1), mp 152-154 -C. lH NMR (CDCI3) ok, MS (FAB) m/e = 498
(M+ 1). Anal: calcd. for C27H3sN3O6, 497; C, 65.17; H, 7.09; N, 8.44. Found, C, 65.06;
H, 7.05; N, 8.50.
EXAMPLE PKC79
Z-Leu-Abu-CONH-CH2CH(OH)C6H,~(4-OCH3). This compound was
synthesized using 2-amino-1(4-methoxy)phenylethanol and purified by column
chromatography using solvent AcOEt/hexane 3:2 (26% yield). Yellow solid, single spot
on TLC, Rf = 0.56 (AcOEtlhexane 1:1), mp 128-129 C. lH NMR (CDCI3) ok, MS
(FAB) m/e = 528 (M+ 1). Anal: calcd. for C28H37N307, 527; C, 63.74; H, 7.07; N,
7.96. Found, C, 63.44; H, 7.08; N, 7.82.
EXAMPLE PKC80
Z-Leu-Abu-CONH-CH2CH(OH)C6H2(2,4,6-(OCH3)3). This compound was
synthesized using 2-amino-1(2,4,6-trimethoxy)phenylethanol and purified by column
chromatography using solvent CHCI3/CH30H 20:1 followed by CHCI3/CH30H 10:1
(29% yield). Yellow solid, single spot on TLC, Rf = 0.54 (CHCI3/CH30H 10:1), mp
170-172 ---C. lH NMR (CDC13) ok, MS (FAB) m/e = 588 (90%, M+ 1), 570 (100~G,
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M+ 1-18). Anal: calcd. for C30H41N3Og, 587; C, 61.31; H, 7.03; N, 7.15. Found, C,
60.86; H, 7.29; N, 6.95.
EXAMPL~ PKC81
Z-Leu-Abu-CONH-CH2CH(OH)C6H4(4-N(CH3)2). This compound was
synthesized using 2-amino-1(4-dimethylamino)phenylethanol and purified by columnchromatography using solvent AcOEt/hexane 6:1 (23% yield). Yellow solid, single spot
on TLC, Rf = 0.41 (AcOEt/hexane 6:1), mp 104 - -C (dec.). lH NMR (CDCl3) ok, MS
(FAB) m/e = 523 (M+ 1-18). Anal: calca. for C29H40N4O6, 540; C, 64.42; H, 7.45; N,
10.36. Found, C, 64.27, H, 7.42; N, 10.34.
EXAMPLE PKC82
Z-Leu-Abu-CONH-CH2CH(OH)C6F5. This compound was synthesized using
2-amino-1-pentafluorophenylethanol and purified by column chromatography using
solvent CHCL3/CH30H 10:1 (66% yield). White solid, single spot on TLC, Rf = 0.28(CHCl3/CH30H 10:1), mp 167-171 -~C. 1H NMR (DMSO-d6) ok, MS (FAB) m/e =
570 (M+ 1-18). Anal: calcd. for C27H30N3O6Fs, 587; C, 55.19; H, 5.14; N, 7.15. Found,
C, 56.13; H, 5.58; N, 7.20.
EXAMPLE PKC83
Z-Leu-Abu-CONH-CH2CH(OH)C6H4(3-CF3). This compound was synthesized
using 2-amino-1(3-trifluoromethyl)phenylethanol and purified by column
chromatography using solvent CHC13/CH30H 10:1 (72% yield). Dark yellow
s~micolid, single spot on TLC, Rf = 0.48 (CHCl3/CH30H 10:1). 1H NMR (CDCl3)
ok, MS (FAB) m/e = 566 (M+ 1). Anal: calcd. for C28H34N306F3, 565; C, 59.46; H,
6.06; N, 7.42. Found, C, 59.12; H, 6.18; N, 7.14.
EXAMPLE PKC84
Z-Leu-Abu-CONH-CH2CH(OH)C6H4(3-OPh). This compound was synthesized
using 2-amino-1(3-phenoxy)phenylethanol and purified by column chromatography
using solvent CHCl3/CH30H 10:1 (67% yield). Yellow oil, single spot on TLC, R~ =0.54 (CHCl3/CH30H 10:1). 1H NMR (CDCl3) ok, MS (FAB) m/e = 590 (53%,
M+ 1), 572 (100%, M+ 1-18). Anal: Calcd. for C33H39N307, 589; C, 67.21; H, 6.66; N,
7.12. Found, C, 66.76; H, 6.25; N, 7.06.
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EXAMPLE PKC8~
Z-Leu-Abu-CONH-CH2CH(OH)C6H4(4-OPh~. This compound was synthesized
using 2-amino-1(4-phenoxy)phenylethanol and purified by column chromatography
using solvent CHC13/CH30H 20:1 (48% yield). Yellow semisolid, single spot on TLC,
Rf = 0.22 (CHCl3/CH30H 20:1), mp 55-60: C. 1H NMR (CDCl3) ok, MS (FAB) m/e
= 590 (47%, M+ 1),572 (100%, M+ 1-18). Anal: calcd. for C33H39N307, 589; C, 67.21;
H, 6.66; N, 7.12. Found, C, 67.30; H, 6.67; N, 7.10.
EXAMPLE PKC86
Z-leu-Abu-CONH-CH2CH(OH)C6H4(4-OCH2Ph). This compound was
synthesized using 2-amino-1(4-benyloxy)phenylethanol and purified by column
chromatography using solvent CHC13/CH30H 20:1 (39~o yield). Yellow solid, singlespot on TLC, Rf = 0.40 (CHCl3/CH30H 20:1), mp 59-62 ~C. lH NMR (CDCI3) ok,
MS (FAB) m/e = 604 (M+ 1). Anal: calcd. for C34H4lN307, 603; C, 67.64; H, 6.84; N,
6.96. Found, C, 67.50; H, 6.87; N, 6.90.
EXAMPLE PKC87
Z-Leu-Abu-CONH-CH2CH(OH)C6H~-3-OC6H1(3-CF3). This compound was
synthesized using 2-amino-1(3-(3'-trifluoromethyl)phenoxy)phenylethanol and purified
by column chromatography using solvent CHCI3/CH30H 30:1 (57~O yield). Yellow
solid, single spot on TLC, Rf = 0.40 (CHCI3/CH30H 30:1), mp 97-101 ~ C. IH NMR
(CDCI3) ok, MS (FAB) m/e = 658 (M+ 1). Anal: calcd. for C34H38N3O7F3, 657; C,
62.09; H, 5.82; N, 6.39. Found, C, 62.05; H, 5.84; N, 6.42.
EXAMPLE PKC88
Z-Leu-Abu-CONH-CH2CH(OH)C6H4-3-OC6H3(3,4-Clt). This compound was
synthesized using 2-amino-1(3-(3',4'-dichloro)phenoxy)phenylethanol and purified by
column chromatography using solvent CHCl3/CH30H 20:1 (55% yield). Yellow solid,
single spot on TLC, Rf = 0.28 (CHCI3/CH30H 20:1), mp 63-67 -~C. lH NMR
(CDCl3) ok, MS (FAB) m/e = 659 (M+ 1). Anal: calcd. for C33H37N3O7CI2, 658; C,
60.18; H, 5.66; N, 6.38. Found, C, 59.37; H, 5.12; N, 6.16.
EXAMPLE PKC89
Z-Leu-Abu-CONH-CH2CH(OH)C6H3(3,4-(OCH2Ph)2). This compound was
synthesized using 2-amino-1(3,4-dibenzyloxy)phenylethanol and purified by columnchromatography using solvent CHCI3/CH30H 10:1 (60~o yield). White solid, single
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spot on TLC, Rf = 0.48 (CHCI3/CH30H 10:1), mp 101-104 C. lH NMR (CDCI3) ok,
MS (FAB) m/e = 710 (M+1). Anal: calcd. for C4,H47N308, 709; C, 69.37; H, 6.67; N,
5.92. Found, C, 68.23; H, 6.70; N, 6.08.
EX~MPLE PKC90
ZLeu-Abu-CONH-CH2CH(OH)-1-Cl0H7. This compound was synthesized using
2-amino-1(1-naphthyl)phenylethanol and purified by column chromatography using
solvent AcOEt/hexane 1:1 (15% yield). Pale orange solid, single spot on TLC, Rf =
0.48 (AcOEt/hexane 1:1), mp 63-71: ~C. 1H NMR (CDCI3) ok, MS (FAB) m/e = 548
(M+ 1). Anal: calcd. for C31H37N3O6, 547; C, 67.99; H, 6.81; N, 7.67. Found, C, 67.73;
H, 7.03; N, 7.40.
EXAMPLE PKC91
Z-Leu-Abu-CONH-CH2CH(OH)-2-ClOH7. This compound was synthesized using
2-amino-1(2-naphthyl)phenylethanol and purified by column chromatography using
solvent AcOEt/hexane 3:2 (17% yield). Orange solid, single spot on TLC, Rf = 0.39
(AcOEt/hexane 3:1), mp 137-140 ~C. 1H NMR (CDCI3) ok, MS (FAB) m/e = 548
(M+ 1). Anal: calcd. for C31H37N3O6, 547; C, 67.99; H, 6.81; N, 7.67. Found, C, 68.15;
H, 6.83; N, 7.43.
EXAMPLE PKC92
Z-Leu-Phe-CONH-CH2CH(OH)Ph. This compound was synthesized using
2-amino-1-phenylethanol and purified by column chromatography using solvent
CHCl3/CH30H 10:1 (46% yield). White solid, single spot on TLC, Rf = 0.72
(CHCl3/CH30H 10:1), mp 164-166 C. 1H NMR (CDCl3) ok, MS (FAB) m/e = 560
(M+ 1). Anal: calcd. for C32H37N3O6, 559; C,68.67; H, 6.66; N, 7.51. Found, C, 68.46,
H, 6.68, N, 7.50.
EXAMPLE PKC93
Z-Leu-Phe-CONH-CH2CH(OH)C6H4(4-N(CH3)2). This compound was
prepared using 2-amino-1(4-dimethylamino)phenylethanol and purified by column
chromatography with solvent CHCl3/CH30H 10:1 (22% yield). Yellow solid, single
spot on TLC, Rf = 0.68 (CHCl3/CH30H 10:1), mp 130 C (dec.). lH NMR (CDCI3)
ok, MS (FAB) m/e = 603 (35%, M+ 1), 585 (100%, M+ 1-18). Anal: calcd. for
C34H42N4O6, 602; C, 67.75, H, 7.02, N, 9.29. Found, C, 66.43; H, 7.06; N, 9.22.
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EXAMPLE PKC94
Z-Leu-Phe-CONH-CH2CH(OH)C6Fs. This compound was prepared using
- 2-amino-1-pentafluorophenylethanol and purified by column chromatography using
solvent CHCl3/CH30H 20:1 (47% yield). White solid, single spot on TLC, Rf = 0.45(CHCl3/CH30H 20:1), mp 191-192 - C. lH NMR (DMSO-d6) ok, MS (FAB) m/e =
632 (100%, M+ 1-18). Anal: calcd. for C32H32N3O6F5, 649; C, 59.16; H, 4.96; N, 6.46.
Found, C, 61.18; H, 5.37; N, 6.68.
EXAMPLE PKC95
Z-Leu-Phe-CONH-CH2CH(OH)C6H4(3-CF3). This compound was prepared
using 2-amino-1(3-trifluoromethyl)phenylethanol and purified by column
chromatography using solvent CHC13/CH30H 20:1 (42% yield). Dark yellow
se~ olid single spot on TLC, Rf = 0.48 (CHCI3/CH30H 10~ H NMR (CDCI3)
ok, MS (FAB) m/e = 628 (M+ 1). Anal: calcd for C33H36N36F3~ 627; C~ 63-15; H~
5.78; N, 6.69. Found, C, 63.24; H, 5.82; N, 6.65.
EXAMPLE PKC96
Z-Leu-Phe-CONH-CH2CH(OH)C6H4(3-OPh). This compound was prepared
using 2-amino-1(3-phenoxy)phenylethanol and purified by column chromatography
using solvent CHCl3/CH30H 20:1 (50% yield). Yellow semisolid, single spot on TLC,
Rf = 0.25 (CHCl3/CH30H 20:1). lH NMR (CDC13) ok, MS (FAB) m/e = 652
(M+ 1). Anal: Calcd. for C38H41N3O7, 651; C, 70.02; H, 6.34; N, 6.44. Found, 69.67;
H, 6.60; N, 6.23.
EXAMPLE PKC97
Z-Leu-Phe-CONH-CH2CH(OH)C6H4(4-OPh). This compound was prepared
using 2-amino-1(4-phenoxy)phenylethanol and purified by column chromatography
using solvent CHCl3/CH30H 30:1 (30% yield). Yellow semisolid, single spot on TLC,
Rf = 0.20 (CHCl3/CH30H 30:1), mp 146-149 ---C. 1H NMR (CDCl3) ok, MS (FAB)
m/e = 652 (25%, M+ 1), 634 (100 %, M+ 1-18). Anal: calcd. for C38H41N3O7, 651; C,
70.02; H, 6.34; N, 6.44. Found, 70.14; H, 6.36; N, 6.38.
EXAMPLE PKC98
Z-Leu-Phe-CONH-CH2CH(OH)C6H4(4-OCH2Ph). This compound was
prepared using 2-amino-1(4-benzyloxy)phenylethanol and purified by column
chromatography using solvent CHC13/CH30H 20:1 (49% yield). Yellow solid, single
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spot on TLC, Rf = 0.45 (CHCl3/CH30H 20:1), mp 133-134 - C. 1H NMR (CDCl3) ok,
- MS (FAB) m/e = 666 (M+ 1). Anal: calcd. for C39H43N307, 665; C, 70.35; H, 6.51; N,
6.31. Found, 69.55; H, 6.46; N, 6.25.
EXAMPLE PKC99
S Z-Leu-Phe-CONH-CH2CH(OH)C6H4-3-OC6H4(3-CF3). This compound wasprepared using 2-amino-1(3-tri~uoromethyl)phenoxy)phenylethanol and purified by
column chromatography using solvent CHCl3/CH30H 20:1 (52% yield). Yellow solid,
single spot on TLC, Rf = 0.23 (CHCl3/CH30H 20:1), mp 142-143 -C. lH NMR
(CDCl3) ok, MS (FAB) m/e = 720 (M+ 1). Anal: calcd. for C39H40N3O7F3, 719; C,
65.08; H, 5.60; N, 5.72. Found, C, 64.66; H, 5.58; N, 5.72.
EXAMPLE PKC100
Z-Leu-Phe-CONH-CH2CH(OH)C6H4-3-OC6H3(3,4-C12). This compound was
prepared using 2-amino-1(3-(3',4'-dichloro)phenoxy)phenylethanol and purified bycolumn chromatography using solvent CHCl3/CH30H 20:1 (41% yield). Yellow solid,
single spot on TLC, Rf = 0.40 (CHCl3/CH30H 20:1), mp 136-137 ~ ~C. lH NMR
(CI)Cl3) ok, MS (FAB) m/e = 721 (M+ 1). Anal: calcd. for C38H39N307Cl2, 720; C,
63.33; H, 5.45; N, 5.83. Found, C, 62.78; H, 5.09; N, 5.42.
EXAMPLE PKC101
Z-Leu-Phe-CONH-CH2CH(OH)C6H3(3,4-(OCH2Ph)2). This compound was
prepared using 2-amino-1(3,4-dibenzyloxy)phenylethanol and purified by column
chromatography using solvent CHCl3/CH30H 20:1 (45% yield). Yellow solid, single
spot on TLC, Rf = 0.42 (CHC13/CH30H 20:1), mp 149-152 ~ C. 1H NMR (CDCl3) ok,
MS (FAB) m/e = 772 (M+1). Anal: calcd. for C46H49N308, 771; C, 71.57; H, 6.39; N,
5.44. Found, C, 71.33; H, 6.45; N, 5.41.
EXAMPLE PKC102
ZLeu-Abu-CONH-CH2-2-Furgl. This compound was synthesized from
1,3-dithiolane derivative of Z-Leu-Abu-COOEt and 2-furfurylamine by the procedure
described in Example PKC67, and purified by column chromatography using solvent
CHCl3/CH30H 20:1 (43% yield). White solid, single spot on TLC, Rf = 0.68
(CHCl3/CH30H 10:1), mp 138-139 C. lH NMR (CDCl3) ok, MS (FAB) m/e = 458
(M+ 1). Anal: calcd. for C24H3lN306, 457; C, 63.00; H, 6.83; N, 9.18. Found, C, 62.22;
H, 6.72; N, 9.00.
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EXAMPLE PKC103
Z-Leu-Abu-CONH-CH2-2-Tetral.~.lr~ru. ~l. This compound was synthesized
using 2-tetral,yd,orulru,.ylamine and purified by column chromatography using solvent
CHC13/CH30H 20:1 (35% yield). Yellow solid, single spot on TLC, Rf = 0.59
(CHCl3/CH30H 20:1), mp 126-128 C. 1H NMR (CDCl3) ok, MS (FAB) m/e = 462
(M+ 1). Anal: calcd. for C24H3sN3O6, 461; C, 62.45; H, 7.64; N, 9.10. Found, C, 62.37;
H, 7.63; N, 9.19.
EXAMPLE PKC104
Z-Leu-Abu-CONH-CH2-2-Pyridyl. This compound was synthesized from
1,3-dithiolane derivative of Z-Leu-Abu-COOEt and 2-aminomethylpyridine. After
reacting overnight at r.t., to the mixture was added AcOEt (25 mL) and white
prec;yi~te was filtered. The filtrate was washed with water (3 x 10 mL), saturated
NaCl (2 x 10 mL) and dried over MgSO4. After evaporation of the solvent,
chromatography on a silica gel column using solvent CHC13/CH30H 10:1 followed byprecipitation from AcOEt/hexane afforded a yellow solid (50% yield).
Single spot on TLC, Rf = 0.50 (CHCl3/CH30H 10:1), mp 117-119 C. lH NMR
(CDC13) ok, MS (FAB) m/e = 469 (M+ 1). Anal: calcd. for C25H32N4Os, 468; C,
64.08; H, 6.88; N, 11.96. Found, C, 63.93; H, 6.86; N, 11.85.
EXAMPLE PKC105
Z-Leu-Abu-CONH-CH2-3-Pyridyl. This compound was synthesized from
1,3-c1ithinl~ne derivative of Z-Leu-Abu-COOEt and 3-aminomethylpyridine by the
procedure described in Example PKC104, and purified by column chromatography
using solvent CHCl3/CH30H 10:1 (35% yield). Yellow solid, single spot on TLC, Rf =
0.54 (CHCl3/CH30H 10:1), mp 122-123: C. lH NMR (CDCl3) ok, MS (FAB) m/e =
469 (M+ 1). Anal: calcd. for C25H32N4O5, 468; C, 64.08; H, 6.88; N, 11.96. Found, C,
63.98; H, 6.91; N, 11.97.
EXAMPLE PKC106
Z-Leu-Abu-CONH-CH2-4-Pyridyl. This compound was synthesized using
4-aminomethyl-pyridine and purified by column chromatography using solvent
CHCl3/CH30H 10:1 (45% yield). Yellow solid, single spot on TLC, Rf = 0.55
(CHCl3/CH30H lG:l), mp 124-126 ---C. lH NMR (CDCl3) ok, MS (FAB) m/e = 469
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_,
-129-
(M+ 1). Anal: calcd. for C2sH32N4O5, 468; C, 64.08; H, 6.88; N, 11.96. Found, C,63.88; H, 6.87; N, 11.96.
EXAMPLE PKC107
Z-Leu-Abu-CONH-(CH2)2-2-Pyridyl. This compound was synthesized using
2-(2-aminoethyl)pyridine and purified by column chromatography using solvent
CHCl3/CH30H 10:1(53% yield). Yellow solid, single spot on TLC, Rf = 0.60
(CHCl3/CH30H 10:1), mp 128-130 C. 1H NMR (CDCI3) ok, MS (FAB) m/e = 483
(M+1). Anal: calcd. for C26H34N4O5, 482; C, 64.71; H, 7.10; N, 11.61. Found, C,
64.04; H, 7.05; N, 11.49.
EXAMPLE PKC108
Z-Leu-Abu-CONH-(CH2)2-2-(N-Melh~ .."le). Th. ompound was
synthesized from protected Z-Leu-Abu-COOEt and 2(2-aminoethyl)-1-methylpyrrole by
the procedure described in Example PKC104, and purified by column chromatographyusing solvent CHCl3/CH30H 30:1 (16% yield). Orange semisolid, single spot on TLC,
Rf = 0.34 (CHCl3/CH30H 30:1), mp 120-123 ~C. lH NMR (CDCl3) ok, MS (FAB)
m/e = 485 (M+1). Anal: calcd. for C26H36N4O5, 484; C. 64.44; H, 7.48; N, 11.56.
Found, C, 64.02; H, 7.26; N, 11.21.
EXAMPLE PKC109
ZLeu-Abu-CONH-(CH2)3-~ q7S~Iyl. his compound was synthesized using
1(3-aminopropyl)imidazole by the procedure described in Example PKC104, and
purified by column chromatography using solvent CHC13/CH30H 10:1 (27% yield).
Yellow semisolid, single spot on TLC, Rf = 0.33 (CHCI3/CH30H 10:1), mp 52-55 ~C.1H NMR (CDCl3) ok, MS (FAB) m/e = 486 (M+ 1). Anal: calcd. for C25H3sNsO
485; C, 61.83; H, 7.26; N, 14.42. Found, C, 60.90; H, 7.21; N, 13.87.
EXAMPLE PKC110
ZLeu-Abu-CONH-(CH2)2-4-M~ he'inyl. This compound was synthesized
using 4-(2-aminoethyl)morpholine and purified by column chromatography using
solvent CHCl3/CH30H 10:1 (SS~o yield). Yellow semisolid, single spot on TLC, Rf =
0.49 (CHCI3/CH30H 10:1), mp 124-126 - ~C. 1H NMR (CDC13) ok, MS (FAB) m/e =
491 (M+ 1). Anal: calcd. for C2sH38N4O6, 490; C, 61.15; H, 7.81; N, 11.42. Found, C,
61,08; H, 7.85; N, 11.34.
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EXAMPLE PKClll
Z-Leu-Abu-CONH-(CH2)3 4-Mo.~ho!;nyl. This compound was synthesized
using 4-(3-aminopropyl)morpholine and purified by column chromatography using
solvent CHCl3/CH30H 10:1 (42% yield). Yellow semisolid, single spot on TLC, Rf =0.50 (CHCl3/CH30H 10:1), mp 125-126 - C. 1H NMR (CDCI3) ok, MS (FAB) m/e =
505 (M+ 1). Anal: calcd. for C26H40N406, 504; C, 61.88; H, 7.99; N, 11.10. Found, C,
61,69; H, 7.95; N, 11.07.
EXAMPLE PKC112
Z-Leu-Abu-CONH-(CH2)3-l-Pyrrolidinyl-2-one. This compound was prepared
from Z-Leu-Abu-COOH and 1-(3-aminopropyl)2-pyrrolidinone, and purified by columnchromatography using solvent CHCI3/CH30H 10:1 (33% yield). White semisolid,
single spot on TLC, Rf = 0.51 (CHCI3/CH30H 10:1). lH NMR (CDCI3) ok, MS
(FAB), m/e = 503 (M+ 1). Anal: calcd. for C26H38N406, 502; C, 62.13; H, 7.62; N,11.14. Found, C, 62.02; H, 7.71; N, 10.52.
EXAMPLE PKC113
Z-Leu-Abu-CONH-(CH2)2-3-Indolyl. This compound was prepared from
Z-Leu-Abu-COOH and 3-(2-aminoethyl)indole and purified by column chromatography
using solvent CHCI3/CH30H 30:1 (18% yield). White semisolid, single spot on TLC,Rf = 0.47 (CHCl3/CH30H 30:1). 1H NMR (CDCI3) ok, MS (exact FAB), m/e = 521
2745.
EXAMPLE PKC114
Z-Leu-Abu-CONH-CH2-2-Quinolinyl. This compound was prepared from
1,3-dithiolane derivative of Z-Leu-Abu-COOEt and 2-aminomethylquinoline by the
procedure described in Example PKC104, and purified by column chromatography
using solvent AcOEt/hexane 2:1 (16% yield). Yellow solid, single spot on TLC, Rf =
0.27 (AcOEt/hexane 2:1), mp 135-138 ~ C. 1H NMR (CDC13) ok, MS (FAB) m/e =
519 (M+ 1). Anal: calcd. for C29H34N405, 518; C, 67.16; H, 6.60; N, 10.80. Found, C,
66.89; H, 6.68; N, 10.61.
EXAMPLE PKCllS
Z-Leu-Abu-CONH-CH2-l-lsoquinolinyl. This compound was prepared using
1-aminomethylisoquinoline and purified by column chromatography using solvent
AcOEt/hexane 2:1 (12~o yield). Yellow solid, single spot on TLC, Rf = 0.34
213~12~
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(AcOEt/hexanel:1), mp 121-125 ~-~C. 1H NMR (CDCl3) ok, MS (FAB) m/e = 519
(M+1). Anal: calcd. for C29H34N4Os, 518; C, 67.16; H, 6.60; N, 10.80. Found, C,
67.11; H, 6.61; N, 10.83.
EXAMPLE PKC116
Z-Leu-Abu-CONH-(CH2)3-1-Tetrahydroquinolinyl. This compound was
synthesized using N-aminopru~Jylletraquinoline and purified by column
chromatography using solvent CHCl3/CH30H 30:1 (40% yield). Oil, single spot on
TLC, Rf = 0.26 (CHCl3/CH30H 20:1). lH NMR (CDCl3) ok, MS (FAB) m/e = 551
(M+ 1). Anal: calcd. for C3lH42N405, 550; C, 67.61; H, 7.69; N, 10.17. Found, C,67.15; H, 7.42; N, 10.02.
EXAMPLE PKC117
Z-Leu-Abu-CONH-(CH2)3-2-Tetral,~l~o;co~ l. This compound was
synthesized using N-amino~lu~ylisotetraquinoline and purified by column
chromatography using solvent CHC13/CH30H 20:1 (20% yield). Yellow semisolid,
single spot on TLC, Rf = 0.51 (CHC13/CH30H 20:1). lH NMR (CDCI3) ok, MS
(FAB) m/e = 551 (M+ 1). Anal: calcd. for C3lH42N,10s, 550; C, 67.61; H, 7.69; N,10.17. Found, C, 67.23; H, 7.32; N, 9.98.
EXAMPLE PKC118
Z-Leu-Abu-CONH-CH2-8-Caffeine. This compound was synthesized using
8-aminomethylcaffeine and purified by column chromatography using solvent
CHCl3/CH30H 20:1 (30% yield). YelJow solid, single spot on TLC, Rf = 0.35
(CHCi3/CH30H 10:1), mp 171-177 -~C (dec.). 1H NMR (CDCl3) ok, MS (FAB) m/e
= 556 (16%, M+ 1-28), 471 (100%, M+ 1-113). Anal: calcd. for C2~H37N7O7, 583; C,57.62; H, 6.39; N, 16.79. Found, C, 57.70; H, 6.48; N, 16.69.
EXAMPLE PKCll9
Z-Leu-Abu-CONH-CH2-2-(4-Methyl-2-thiazolyl). This compound was prepared
using synthesized 2-aminomethyl-4-methylthiazole and purified by column
chromatography using solvent AcOEt/hexane 6:1 (26% yield). Orange semisolid, single
spot on TLC, Rf = 0.4Q (AcOEt/hexane 6:1). lH NMR (CDCl3) ok, MS (FAB, calcd.
for C24H32N4O5S, 488) m/e = 489 (3%, M+ 1), 376 (100%, M+ 1-113).
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EXAMPLE PKC120
Z-Leu-Abu-CONH-(CH2)2NH-Biotinyl. This compound was prepared from
Z-Leu-Abu-COOH and biotinylethylenediamine hydrochloride. Biotin (1 g, 4.1 mmole)
was dissolved in 20 mL of DMF at 70 C and cooled to 40 C, CDI (0.97 g, 6 mmole)
in 3 mL of DMF was then added and white precipitate were appeared. After stirring
at r.t. for two hours, ethylenediamine (1.34 mL, 20 mmole) in 10 mL of DMF was
added and stirred for another 3 hours. After evaporating DMF, the semisolid residue
was dissolved in 50 mL of refluxed methanol and the unreacted biotin was removed by
filtration. The solution was evaporated to dryness, the residue was washed with CHCI3
to remove the imidazole, dissolved in 6 mL of water, acidified to pH 3.0 with lN HCI,
and evaporated to dryness. The crude product was crystallized from methanol to give
1.04 g of biotinylethylenediamine hydrochloride (79% yield). Long spot on TLC, Rf =
0.21 (butanol:AcOH:H2O = 4:1:1), mp 241-242 _C. lH NMR is consistent with the
structure.
To a stirred solution of Z-Leu-Abu-COOH (0.6 g, 1.58 mmol) in DMF (15 mL)
was added HOBt (0.22 g, 1.58 mmol), DCC (0.49 g, 2.38 mmol), and stirring continued
for 2 hours at r.t.(mixture A). To a stirred solution of biotinylethylenediaminehydrochloride (0.6 g, 1.85 mmol) in DMF (10 mL) was added TEA (0.28 mL, 2.03
mmol) at 0-5 ---C and stirred for 2 hours at r.t.(mixture B). To the stirred mixture A
was added mixture B and stirred 3 days. After filtering, the filtrate was evaporated to
get a semisolid which was washed with H2O (30 mL), lM HCl (30 mL), H2O (30 mL)
and dried under vacuum. Chromatography on a silica gel column using solvent
CHCl3/CH30H 5:1 afforded a yellow solid (42 % yield). Long spot on TLC, Rf = 0.41
(CHCl3tCH3OH 5:1), mp 188-192 C (dec.). lH NMR (DMSO-d6) ok, MS (FAB)
m/e = 647 (M+ 1). Anal: calcd. for C3lH46N6O7S, 646; C, 57.56; H, 7.17; N, 12.99.
Found, C, 57.04; H, 7.21; N, 13.29.
EXAMPLE PKCl21
Z-Leu-Abu-CONH-CH2-3-Pyridyl-N-oxide. This compound was prepared from
Z-Leu-Abu-COOH and 3-aminomethylpyridine-N-oxide, and purified by column
chromatography using solvent CHCl3/CH30H 20:1 (15% yield). Yellow oil, long spoton TLC, Rf = 0.40 (CHCl3/CH30H 5:1). lH NMR (CDCl3) ok, MS (FAB, calcd. for
C25H32N4O6, 484) m/e = 485 ( 2%, M+ 1), 372 (lOO~o, M+ 1-113).
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EXAMPLE PKC122
Z-Leu-Abu-CONH-CH2-6-Uracil. This compound was prepared from
Z-Leu-Abu-COOH and 6-aminomethyluracil and purified by column chromatography
using solvent CHCl3/CH30H 10:1 (1.5% yield). Brown oil, long spot on TLC, Rf =
0.28 (CHCl3/CH30H 10:1). 1H NMR (CDCl3) ok, MS (FAB, calcd. for C24H31N5O7,
501) m/e = 389 (100%, M+ 1-113).
EXAMPLE PKC123
Z-Leu-Phe-CONH-CH2-2-Pyridyl. This compound was prepared from
1,3-~ithinl~ne derivative of Z-Leu-Phe-COOEt and 2-aminomethylpyridine by the
procedure described in Example PKC104, and purified by column chromatography
using solvent CHCl3/CH30H 20:1(41% yield) . Yellow solid, long spot on TLC, Rf =0.40 (CHCl3/CH30H 20:1), mp 144-146 -C. lH NMR (CDCl3) ok, MS (FAB) m/e =
531 (M+ 1). Anal: calcd. for C30H34N4O5, 530; C, G7.91; H, 6.46; N, 10.56. Found, C,
67.64; H, 6.50; N, 10.64.
EXAMPLE PKC124
Z-Leu-Phe-CONH-(CH2)3-4-Morpholinyl. This compound was prepared from
1,3-dithiolane derivative of Z-Leu-Phe-COOEt and 4-(3-aminopropyl)morpholine, and
purified by column chromatography using solvent CHCl3/CH30H 10:1(40% yield) .
Yellow solid, long spot on TLC, Rf = 0.55 (CHCl3/CH30H 10:1), mp 155-156 C. IH
NMR (CDCl3) ok, MS (FAB) m/e = 581 (M+ 1). Anal: calcd. for C31H42N4O6, 566.
C, 65.70; H, 7.47; N, 9.89. Found, C, 65.64; H, 7.49; N, 9.84.
EXAMPLE PKC125
Z-Leu-Phe-CONH-CH2-2-Quinolinyl. This compound was prepared using
2-aminomethylquinoline and purified by column chromatography using solvent
AcOEt/hexane 1:1 (33% yield) . Yellow solid, long spot on TLC, Rf = 0.30
(AcOEt/hexane 1:1), mp 131-135 ~ C. 1H NMR (CDCl3) ok, MS (FAB) m/e = 581
(M+ 1). Anal: calcd. for (~34H36N405, 580; C, 70.32; H, 6.25; N, 9.65. Found, C,70.31; H, 6.27; N, 9.63.
EXAMPLE PKC126
Z-Leu-Phe-CONH-CH2-1-Isoquinolinyl. This compound was prepared using
1-aminomethylisoquinoline and purified by column chromatography using solvent
AcOEt/hexane 1:1 (7% yield). Yellow solid, single spot on TLC, Rf = 0.45
WO 94/0009~ PCr/US93/0614~
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(AcOEt/hexane 1:1), mp 169-173 C. 1H NMR (CDCI3) ok, MS (FAB) m/e = 581
(M+ 1). Anal: calcd. for C34H36N4Os, 580; C, 70.32; H, 6.25; N, 9.65. Found, C, 70.05;
H, 6.29; N, 9.47.
EXAMPLE PKC127
ZLeu-Phe-CONH-(CH2)3-l-Tetrah~d.v~uin~linyl. This compound was
prepared usirlg N-aminopropyltetraquinoline and purified by column chromatography
using solvent CHCl3/CH30H 30:1 (40% yield). Yellow solid, single spot on TLC, Rf =
0.58 (CHCl3/CH30H 20:1), mp 115-120 -C. 1H NMR (CDCl3) ok, MS (FAB) m/e =
613 (M+ 1). Anal: calcd. for C36H44N4Os, 612; C, 70.56; H, 7.24; N, 9.14. Found, C,
70.46; H, 7.26; N, 9.19.
EXAMPLE PKC128
Z-Leu-Phe-CONH-(CH2)3-2-Tetlahydroiso4l :)!;nyl. This compound was
prepared using N-aminopropylisotetraquin~line and purified by column
chromatography using solvent CHCl3/CH30H 20:1 (51% yield). Yellow solid, single
spot on TLC, Rf = 0.62 (CHCl3/CH30H 10:1), mp 107-111 -C. lH NMR (CDCl3) ok,
MS (FAB) m/e = 613 (M+ 1). Anal: calcd. for C36H44N4Os, 612; C, 70.56; H, 7.24; N,
9.14. Found, C, 69.61; H, 7.25; N, 9.05.
EXAMPLE PKC129
Z-Leu-Phe-CONH-(CH2)2NH-biotinyl. This compound was prepared from
Z-Leu-Phe-COOH and synthesized biotinylethylenediamine hydrochloride by the
procedure described for Example PKC120, and purified by column chromatography
using solvent CHCl3/CH30H 5:1 (35% yield). White solid, long spot on TLC, Rf =
0.42 (CHCl3/CH30H 5:1), mp 204-206 C (dec.). 1H NMR (DMSO-d6) ok, MS
(FAB) m/e = 709 (M+ 1). Anal: calcd. for C36H48N6O7S, 708; C, 60.99; H, 6.82; N,11.85. Found, C, 61.03; H, 6.83; N, 11.77.
EXAMPLE PKC130
Z-Leu-Nva-CONH-CH2CH(OH)Ph. This compound was synthesized from
1,3-dithiolane derivative of Z-Leu-Nva-COOEt and 2-amino-1-phenylethanol by the
procedure described in Example PKC67, and purified by column chromatography using
solvent CHCl3/CH30H 10:1 (54% yield). White solid, single spot on TLC, Rf = 0.56(CHCl3/CH30H 10:1), mp 75-77 - ~C. 1H NMR (CDCl3) ok, MS (FAB, calcd. for
C~8H37N3O6, 511) m/e = 512 (M+ 1).
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EX~MPLE PKC131
ZLeu-Nva-CONH-CH2-2-Pyridyl. This compound was prepared from
1,3-.lithi~ ne derivative of Z-Leu-Nva-COOEt and 2-aminomethylpyridine by the
procedure described in Example PKC104, and purified by column chromatography
using solvent CHC13/CH30H 10:1(50% yield) . Yellow solid, long spot on TLC, Rf =0.55 (CHCl3/CH30H 10:1), mp 65-70 C. 1H NMR (CDCl3) ok, MS (FAB, calcd. for
C26H34N4OS,482) m/e = 483 (M+ 1)
EXAMPLE PKC132
~Leu-Nva-CONH-(CH2)3 1 MV.,~-';nyl. This compound was prepared from
1,3-dithiolane derivative of Z-Leu-Nva-COOEt and 4-(3-aminopropyl)morpholine, and
purified by column chromatography using solvent CHCl3/CH30H 10:1(37% yield) .
Yellow solid, long spot on TLC, Rf = 0.23 (CHCl3/CH30H 10:1), mp 108-110 ~C. lH
NMR (CDC13) ok, MS (FAB, calcd. for C27H42N4O6, 518) m/e 519 (M+1).
EXAMPLE PKC133
CH30CO(CH2)2CO-Leu-Abu-CONHEt. To a solid Z-Leu-Abu-CONHEt (1 g,
2.47 mmol) was added a solution of hydrogen bromide in acetic acid (30 wt%, 1.52 mL,
7.40 mmol) at r.t. The mixture was vigorously stirred for 1 hour during this time all of
the ketoamide dissolved in acetic acid. The reaction was quenched with Et2O (30 mL)
then separated. The semisolid was triturated and washed successively with Et20 (5 x
30 mL). After removing solvent, the residue was dried under vacuum, leaving a very
I,~dloscopic solid. lH NMR (CDCl3) showed loss of Z group. The yield was 70-80%.To a stirred solution of mono-methylsuccinate (0.28 g, 2.13 mmol) in DMF (10
mL) was added DCC (0.44 g, 2.13 mmol) and HOBt (0.29 g, 2.13 mmol). The mixture
was stirred for 2 hours at r.t.(mixture A). To a stirred solution of
Leu-Abu-CONHEt.HBr (0.5 g, 1.42 mmol) in DMF (5 mL) was added TEA (0.2 mL,
1.42 mmol) at 0-5 -- C and stirred for 30 min (mixture B). To the stirred mixture B was
added mixture A at 0-5 - C and the reaction was stirred overnight at r.t. After
evaporation of the solvent, AcOEt (40 mL) was added, the precipitate was filtered, the
filtrate was washed with 0.25 N HCl (10 mL), H2O (20 mL), 10% Na2CO3 (3 x 20 mL),
H2O (20 mL), satd. NaCl (2 x 20 mL), dried over MgSO4, and concentrated.
Chromatography on a silica gel column with solvent CHCl3/CH30H 10:1 afforded a
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yellow semisolid (42% yield). Single spot on TLC, R~ = 0.43 (CHCl3/CH30H 10:1).
H NMR (CDCl3) ok, MS (FAB, calcd for C18H31N3O6~ 385) m/e = 386 (M+ 1).
EXAMPLE PKC134
2-Furyl-CO-Leu-Abu-CONHEt. This compound was synthesized using 2-furoic
acid by the procedure described for compound 67 and purified by column
chromatography using solvent CHCI3/CH30H 30:1 (39% yield). Yellow solid, single
spot on TLC, Rf = 0.51 (CHCl3/CH30H 10:1), mp 58-59 C. 1H NMR (CDC13) ok,
MS (FAB) m/e = 366 (M+ 1). Anal: calcd. for Cl8H27N30s, 365; C, 59.16; H, 7.44; N,
11.50. Found, C, 58.12; H, 7.53; N, 11.64.
EXAMPLE PKC13~
2-T~lnlh~dl~)rul~l-CO-Leu-Abu-CONHEt. This compound was synthesized
using 2-tetrahydrofuroic acid and purified by column chromatography using solvent
CHCl3/CH30H 30:1 (41% yield). Yellow oil, single spot on TLC, Rf = 0.54
(CHCl3/CH30H 10:1). 1H NMR (CDCl3) ok, MS (FAB, calcd. for C18H31N3Os, 369)
m/e = 370 (M+1).
EXAMPLE PKC136
3-Pyridyl-CO-Leu-Abu-CONHEt. This compound was synthesized using
nicotinic acid and purified by column chromatography using solvent CHCI3/CH30H
10:1 (49% yield). Yellow solid, single spot on TLC, Rf = 0.56 (CHCl3/CH30H 10:1),
mp 57-61 - C. lH NMR (CDa3) ok, MS (FAB) m/e = 377 (M+ 1). Anal: calcd. for
C1gH28N4O4, 376; C, 60.58; H, 7.49; N, 14.92. Found, C, 60.05; H, 7.51; N, 14.58.
EXAMPLE PKC137
2-F~,.,~i..~l-CO-Leu-Abu-CONHEt. This compound was synthesized using
2-pyrazinecarboxylic acid and purified by column chromatography using solvent
CHCl3/CH30H 10:1 (18% yield). Yellow solid, single spot on TLC, Rf = 0.33
(CHCl3/CH30H 10:1), mp 51-56 ~ -C. 1H NMR (CDC13) ok, MS (FAB) m/e = 378
(M+1). Anal: calcd. for C18H27N5O4, 377; C, 57.29; H, 7.16; N, 18.56. Found, C,
56.74; H, 7.28; N, 18.32.
EXAMPLE PKC138
2-Quinolinyl-CO-Leu-Abu-CONHEt. This compound was synthesized using
quinaldic acid and purified by column chromatography using solvent AcOEt/hexane 1:1
(45% yield). Orange solid, single spot on TLC, Rf = 0.48 (AcOEt/hexane 1:1), mp
~'')94/00095 ~13~t~ PCI/US93/06143
-
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56-59 . ~C. 1H NMR (CDCl3) ok, MS (FAB) m/e = 427 (M+ 1). Anal: calcd. for
C23H30N404, 426; C, 64.79; H, 7.09; N, 13.13. Found, C, 64.98; H, 7.45; N, 12.48.
EXAMPLE PKC139
l-T~4 ^';nyl-CO-Leu-Abu-CONHEt. This compound was synthesized using
1-isoquinoline carboxylic acid and purified by column chromatography with solvent
AcOEt/hexane 1:1 (46% yield). Red solid, single spot on TLC, Rf = 0.47
(AcOEt/hexane 1:1), mp 104-106 C. lH NMR (CDC13) ok, MS (FAB) m/e = 427
(M+ 1). Anal: calcd. for C23H30N404, 426; C, 64.79; H, 7.09; N, 13.13. Found, C,65.00; H, 7.31; N, 12.96.
EXAMPLE PKC110
4-M~."b-~l;nyl-CO-Leu-Abu-CONHEt. This compound was synthesized from
4-morpholinecarbonyl chloride (1 mmol), Leu-AbuCONH-EtHBr ( 1 mmol) and TEA
(2.5 mmol), and purified by column chromatography using solvent CHCl3/CH30H 10:1(33% yield). Yellow oil, single spot on TLC, Rf = 0.45 (CHCl3/CH30H 10~ H
NMR (CDCl3) ok, MS (FAB, calcd- for C18H32N45~ 384) m/e = 385 (M+ 1)-
EXAMPLE PKCt41
Ph(CH2)2CO-Leu-Abu-CONHEt. This compound was synthesized from
1,3-rlithit l~ne derivative of Ph(CH2)2CO-Leu-Abu-COOEt and EtNH2, and purified by
column chromatography using solvent CHC13/CH30H 30:1 (72% yield). Yellow solid,
single spot on TLC, Rf = 0.23 (CHCl3/CH30H 30:1), mp 134-136 ~ C. IH NMR
(CDCl3) ok, MS (FAB) m/e = 404 (M+ 1). Anal: calcd. for C22H33N304, 403; C,
65.48; H, 8.24; N, 9.60. Found, C, 65.52; H, 8.30; N, 9.42.
EXAMPLE PKC142
1-ClOH7CH2CO-Leu-Abu-CONHEt. This compound was synthesized from
1,3-dithinl~ne derivative of 1-C1OH7CO-Leu-Abu-COOEt and EtNH2, and purified by
column chromatography using solvent CHC13/CH30H 30:1 (67% yield). Yellow solid,
single spot on TLC, Rf = 0.47 (CHC13/CH30H 30:1), mp 201-203 ~C. lH NMR
(CDCl3) ok, MS (FAB) m/e = 440 (M+ 1). Anal: calcd. for C25H33N304, 439; C,
68.31; H, 7.57; N, 9.56. Found, C, 68.19; H, 7.52; N, 9.49.
EXAMPLE PKC143
Ph2CHCO-Leu-Abu-CO~HEt. This compound was synthesized from
1,3-dithiolane derivative of Ph2CHCO-Leu-Abu-COOEt and EtNH2, and purified by
WO 94/00095 PCI`/US93/06143
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column chromatography using solvent CHCl3/CH30H 10:1 (24% yield). Yellow solid,
single spot on TLC, Rf = 0.40 (CHCl3/CH30H 10:1), mp 78-83 -~C. lH NMR
(CDCl3) ok, MS (FAB) m/e = 467 (M+ 1). Anal: calcd. for C27H3sN3O4, 466; C,
69.65; H, 7.58; N, 9.02. Found, C, 70.04; H, 7.72; N, 8.72.
S EXAMPLE PKC144
Ph2CHCO-Leu-Abu-CONH-CH2CH(OH)Ph. This compound was synthesized
from 1,3-dithiolane derivative of Ph2CHCO-Leu-Abu-COOEt and
2-amino-1-phenylethanol, and purified by column chromatography using CHCl3
followed by solvent CHC13/CH30H 30:1(30% yield). Yellow solid, single spot on
TLC, Rf =0.40 (AcOEt/hexane 1:1), mp 178-180 -~C. 1H NMR (CDCl3) ok, MS
(FAB) m/e = 558 (M+ 1). Anal: calcd. for C33H39N30s, 557; C, 71.07; H, 7.05; N,
7.53. Found, C, 70.93; H, 7.10; N, 7.46.
EXAMPLE PKC145
Ph2CHCO-Leu-Abu-CONH-2-CH2-Pyridyl. This compound was prepared from
1,3--~ithiol~ne derivative of Ph2CHCO-Leu-Abu-COOEt and 2-aminomethylpyridine,
and purified by column chromatography using CHC13 following by solvent
CHCl3/AcOEt 7:3 (9% yield), mp 161-163. Yellow solid, single spot on TLC, Rf =
0.30 (CHCl3/CH30H 10:1). 1H NMR (CDCl3) ok, MS (FAB) m/e = 529 (M+ 1).
Anal: calcd. for C31H36N4O4, 528; C, 70.43; H, 6.8G; N, 10.60. Found, C, 70.42; H, 6.91;
N, 10.47.
EXAMPLE PKC146
Ph2CHCO-Leu-Abu-CONH-N-(CH2)3-M~,",~^Iinyl. This compound was
prepared from 1,3-dithiolane derivative of Ph2CHCO-Leu-Abu-COOEt and
N-aminopropylmorpholine, and purified by column chromatography using CHCl3
followed by solvent CHC13/AcOEt 7:3 (25 % yield), mp 170-174. Yellow solid, single
spot on TLC, Rf = 0.25 (CHC13/CH30H 10:1). lH NMR (CDC13) ok, MS (FAB) m/e
= 565 (M+ 1). Anal: calcd. for C32H44N4Os, 564; C, 68.06; H, 7.85; N, 9.92. Found, C,
67.22; H, 7.77; N, 9.75.
EXAMPLE PKC147
Ph2CHCO-Leu-Phe-CONH-CH2CH(OH)Ph. This compound was prepared
from 1,3-dithiolane derivative of Ph2CHCO-Leu-Phe-COOEt and
2-amino-1-phenylethanol, and purified by crystallization from CHC13/ether (16% yield).
213 ~ ~ 2 4
-139-
Yellow solid, single spot on TLC, Rf = 0.41 (AcOET/CH30H 9:1), mp 192-196 -C.
1H NMR (CDCl3) ok, MS (FAB) m/e = 620 (M+ 1). Anal: calcd. for C38H41N3O5,
619; C, 73.64; H, 6.67; N, 6.78. Found, C, 72.00; H, 6.62; N, 6.41.
EXAMPLE PKC148
,~
Ph2CHCO-Leu-Phe-CONH-CH2-2-Pyridyl. This compound was syn~hesized
from 1,3-~1ithinl~ne derivative of Ph2CHCO-Leu-Phe-COOEt and
2-aminomc:thyl~,idine, and purified by column chromatography using CHCl3 following
by solvent CHCl3/AcOEt 9:1 (9% yield). Yellow solid, single spot on TLC, Rf = 0.33
(AcOET/CH30H 9:1), mp 160-162 C. lH NMR (CDCl3) ok, MS (FAB) m/e = S91
(M+1). Anal: calcd. for C36H38N4O4, 590; C, 73.20; H, 6.48; N, 9.48. Found, C, 69.91;
H, 6.29; N, 8.98.
EXAMPLE PKC149
Ph2CHCO-Leu-Phe-CONH-(CH2)3-4-M~ 1. This compound was
srth~ci7ed from 1,3--lithiQI~ne derivative of Ph2CHCO-Leu-Phe-COOEt and
N-aminopropylmorpholine, and purified by column chromatography using AcOEt
following by crystallization from AcOEt/ether (20 % yield). Yellow solid, single spot
on TLC, Rf = 0.45 (AcOET/CH30H 9:1), mp 158-160 C. 1H NMR (CDCI3) ok, MS
(FAB) m/e = 627 (M+ 1). Anal: calcd. for C37H46N4Os, 626; C, 70.90; H, 7.40; N,
8.94. Found, C, 70.05; H, 7.43; N, 8.68.
A variety of techniques for certain synthetic steps in the synthesis of the
Peptide Keto-Compounds can be used. Additional synthetic procedures are providedin the following two FY~mpl,~s
EXAMPLE PKC150
D- ~'' ,~' ea (L)-Leu-(L)-Abu-CONH-Et. The structure of Dimethylurea-(L)-Leu-(L)-
Abu-CONH-Et is shown below:
CH~ <c ~C ~ ~CH3
O O
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This compound was produced through synthesis of the reactant Dimethylurea-(L)-Leu-
(L)-Abu hydroxy ethyl amide.
(L)-Leucine (1.31 g (10 mmoles)) was placed in a 3 neck round-bottomed flask,
equipped with two pre.".lle eq~li7ing dropping funnels. 12.S mL of 1.0 N NaOH (12.5
mmoles) was added to the flask and then the mixture was cooled on ice, 12.5 mL of 1.0
N NaOH was placed in one dropping funnel and 1.15 mL (12.5 mmoles) of
dimethylcarbamoyl chloride was placed in the other. The contents of the additionfunnels were added to the flask cimult~neously over ten minlltes The mixture wasallowed to react for an ad~1itinn~l fifteen minutes. The reaction was then washed twice
with 15 mL of ethyl acetate. The aqueous layer was cooled on ice and acidified to a
pH of 2 with 1.0 N HCl. The aqueous layer was extracted three times with 15 mL of
ethyl acetate. The combined organics were dried over magnesium sulfate, filtered and
con-~çnt-ated in vacuo. There remained 0.10 g of a white solid (5%) which possessed
an Rf value of 0.31 using 91:8:1 chloroform:methanol:acetic acid as the eluent.
Boc-Abu hydroxy ethyl amide (0.233 g, 0.894 mmoles) was dissolved in S mL of
dioxane followed by the addition of 20 mL of 4N HCl/dioxane. The reaction mixture
was allowed to react for two hours. After this time, the reaction mixture was
concentrated in vacuo and used immediately in the next step. The HCl Abu hydroxyethyl amide was dissolved in 30 mL of anhydrous DMF and cooled on an ice bath for
ten minutes. To this solution was added 0.217 g (1.07 mmoles) of morpholineleucine
urea, 0.46 mL (2.68 mmoles) of diisopropylethylamine and 0.133 g (0.984 mmoles) of 1-
hydroxybenzot~iazole (HOBt) and allowed to equilibrate for thirty minutes. After this
time, 0.188 g (0.984 mmoles) of EDC suspended in 10 mL of anhydrous DMF was
added and the reaction mixture was allowed to react overnight. The reaction mixture
was concentrated in vacuo and the resulting residue was purified by silica gel column
chromatography employing 90:10 chloroform:methanol as the eluent. There remained0.2044 g (66.56% yield) of a white solid with Rf value of 0.38 in the solvent system
detailed above.
0.100 g (0.291 mmoles) of Dimethylurea-Leu-Abu hydroxy ethyl amide was
dissolved in 10 mL of methylene chloride and cooled in an ice bath. To this mixture
was added 0.487 mg (.003 mmoles) of 2,2,6,6-tetramethyl-1-piperidinyloxy, free radical
(TEMPO) and .014 mL (0.291 mmoles) of an aqueous KE~,r solution (S.9S g of KBr
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dissolved in 25 mL of water). The reaction mix was stirred vigorously while four 80
microliter portions of a lM aqueous sodium hypochlorite (pH 9.5) were added at 15
minute intenals. After this time the reaction mixture was analyzed by TLC employing
90:10 chloroform:methanol to check for completeness of the reaction. If the reaction
s.,
was not complete another portion of TEMPO and another regimen of the sodium
hypochlorite solution should be added. This reaction required three additional
regimens of TEMPO and sodium hypochlorite.
When the reaction was deemed complete by TLC, the layers were separated.
The aqueous layer was extracted with methylene chloride (3 x 10 mL). The combined
organic layer was washed with 10% HCl (1 x 10 mL), 30 mL of a 100 mL stock solution
of 10% HCl cont:~inin~ 1.6 g of KI, 10% sodium thiosulfate (2 x 30 mL) and brine ~1 x
30 mL). The organic layer was then dried over magnesium sulfate, filtered, and
con-~entrated in vacuo. The crude material was triturated with petroleum ether to give
an off-white solid which was recryst~lli7ed from ethyl acetate:hexane. There remained
0.048 g (48.5% yield) of a white solid with an Rf value of 0.43 in the solvent system
detailed above.
TLC analysis of the product on silica gel gave an Rf value of 0.43 in the solvent
system detailed above. HPLC analysis was performed on a Vydac C4 column (4.6 x
250 mm) at 60C using a gradient of 15-25% B/30 minutes (A=0.1% TFA in water,
B=0.1% TFA in acetonitrile). The product had a retention time of 14.49 minutes and
a purity of 97%.
Analyses of the final product provided the following results: Mass spectrum
analysis found (M+H)+ at m/z 343. Elemental analysis for ClGH30N4O4 found 55.80
C, 8.70 H and 15.97N while calculated values were 56.12 C, 8.83 H and 16.36N. For
1HNMR (600 MHz, d6-DMSO) analysis, the shifts observed were 8.65(t,1H),
8.10(d,1H), 6.07(d,1H), 4.85(m,1H), 4.20(m,1H), 3.12(m,2H), 2.77(s,6H), 1.77(m,1H),
1.63(m,1H), 1.48(m,2H), 1.40(m, lH), 1.02(t,1H), 0.85(m,9H).
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EXAMPLE PKCl51
Boc-(L)-Leu-(L)-Abu-CONH-Et. The structure of Boc-(L)-Leu-(L)-Abu-CONH-Et is
shown below:
S ~<
Boc~ c~ ~c~3
This compound was produced by synthesis of the reactant Boc-(L)-Leu-(L)-Abu
hydroxy ethyl amide.
Boc-Abu hydroxy ethyl amide (0.233 g, 894 mmoles) was dissolved in 5 mL of
dioxane followed by the addition of 20 mL of 4N HCl/dioxane. The reaction mixture
was allowed to react for two hours. After this time, the reaction mixture was
conr~ntrated in vacuo and used immediately in the next step. The HCl Abu hydroxyethyl amide prepared above, was dissolved in 25 mL of anhydrous DMF and cooled on
an ice bath for ten minutes. To this solution was added 0.267 g (1.07 mmoles) ofmorpholineleucine urea, 0.46 mL (2.68 mmoles) of diisoplo~,ylethylamine and 0.133 g
(0.984 mmoles) of 1-hydroxybenzotriazole (HOBt) and allowed to equilibrate for thirty
minutes After this time 0.188 g (0.984 mmoles) of EDC suspended in 10 mL of
anhydrous DMF was added and the reaction mixture was allowed to react overnight.The reaction mixture was concentrated in vacuo and the resulting residue redissolved in
100 mL of chloroform. The solution was washed twice with 50 mL of both saturatedsodium bicarbonate and brine. The organic layer was dried over magnesium sulfate,
filtered and concentrated in vacuo. The crude material was purified by silica gel
column chromatography employing 90:10 chloroform:methanol as the eluent. There
remained 0.1841 g (55.12% yield) of a white solid with and Rf value of 0.42 in the
solvent system detailed above.
Boc-Leu-Abu hydroxy ethyl amide (.0823 g, 0.22 mmoles) was dissolved in 10
mL of methylene chloride and cooled in an ice bath. To this mixture was added 0.325
mg (.002 mmoles) of 2,2,6,6-tetramethyl-1-piperidinyloxy, free radical (TEMPO) and
.011 mL of an aqueous KBr solution (5.95 g of KBr dissolved in 25 mL of water). The
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reaction mix was stirred vigorously while four 60 microliter portions of an lM aqueous
sodium hypochlorite (pH 9.5) were added at 15 minute intervals. After this time the
reaction mixture was analyzed by TLC employing 90:10 chloroform:methanol to check
for cr~mrl~teness of the reaction. If the reaction was not complete another portion of
S TEMPO and another regimen of the sodium hypochlorite solution should be added.
This reaction required one additional regiment of TEMPO and sodium hypochlorite.When the reaction was deemed complete by TLC, the layers were separated.
The aqueous layer was extracted with methylene chloride (3 x 10 mL). The combined
organic layer was washed with 10% HCl (1 x 10 mL), 30 mL of a 100 mL stock solution
of 10% HCl containing 1.6 g of KI, 10% sodium thiosulfate (2 x 30 mL) and brine (1 x
30 mL). The organic layer was then dried over magnesium sulfate, filtered and
concentrated in vacuo. The crude material was triturated with petroleum ether to give
an off-white solid which was recryst~lli7ed from ethyl acetate:hexane. There remained
0.067 g (82.3% yield) of a white solid with an Rf value of 0.52 in the solvent system
detailed above.
TLC analysis of the product on silica gel gave an Rf value of 0.52 in the solvent
system detailed above. HPLC analysis was performed on a Vydac C4 column (4.6 x
250 mm) at 60C using a gradient of 25-35% B/30 minutes (A=O.l~o TFA in water,
B=0.1% TFA in acetonitrile). The product had a retention time of 21.05 minutes and
a purity of 99.14%.
Analyses of the final product provided the following results: Mass spectrum
analysis found (M+H)+ at m/z 372. Elemental analysis for C1~H33N3O~ found 57.84
C, 8.84 H and 11.05 N while calculated values were 58.20 C, 8.95 H and 11.05 N. For
1HNMR (600 MHz, d6-DMSO) analysis, the shifts observed were 8.66(t,1H),
8.06(d,1H), 6.85(d,1H), 4.88(m,1H), 3.99(m,1H), 3.12(m,2H), 1.77(m,1H), 1.77(m,1H),
1.60(m,1H), 1.51(m,1H), 1.35(br s,11H), 1.02(t,3H), 0.86(m,9H).
Morpholine Peptide Keto-Compounds
As is clear from the foregoing description of the Peptide Keto-Compounds, the
term Peptide Keto-Compound as used herein also includes the morpholine Peptide
Keto-Compounds. These morpholine compounds can be classified in any of the various
classes or subrl~cses and types of Peptide Keto-Compounds referred to hereinabove.
Thus, for example these compounds include the morpholine Peptide Ketoacids, the
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morpholine Peptide K~to~mid~c and the morpholine Peptide Ketoesters. The
morpholine Peptide Keto-Compounds can include either N-terminal or C-terminal
morpholine groups. In the N-terminal morpholine Peptide Keto-Compounds, the M
group (or M1, M2, M3, M4 group) includes a morpholine ring that can, in some
ci~ nres, include the nitrogen of the N-terminal amino acid. The C-terminal
morpholine Peptide Keto-Compounds include a morpholine ring that is part of the
C-te~rnin:~l R (or R1 etc.) group of the compound. In certain examples of these
compounds, the R-group in~ d~ an alkyl morpholine, as in the compound described
above in Example PKC140.
The morpholine Peptide Keto-Compounds can be produced using synthesis
te~hniqu.os generally similar to those used for synthesis of other Peptide Keto-Compounds.
The C-terminal morpholine Peptide Keto-Compounds can be produced using
the general method of production of Peptide Ketoamides, which are derived from the
corresponding Peptide Ketoesters. In the case of C-terminal Peptide Keto-Compounds,
the Peptide Ketoesters can be reacted with N-amino alkyl morpholine to produce the
C-terminal N-alkyl morpholine derivative of the Peptide Ketoester. One such
procedure is shown hereinabove as Example PKC140.
The N-terminal morpholine Peptide Keto-Compounds can be produced using
the general scheme outlined above, wherein a morpholine compound is substitued for
other N-terminal hlocl~ing groups. However, other methods of synthesis can also be
used. The following Example shows one exemplary procedure for production of
N-terminal morpholine compounds.
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Example PKC152
M~.p}-l- ez (L)-Leu-(L)-Abu-CONH-Et. The structure of Morpholineurea-(L)-
Leu-(L)-Abu-CONH-Et is shown below:
.
O ~ O
31 ~ (L) ¦l S~-- 11 X
Ten grams of N-t-butyl~,A~callJ..A.y-(L)-a-aminobutric acid (N-Boc-(L)-Abu) was
dissolved in 100 ml of anhydrous tetrahydrofuran (THF). To this solution was added
9.4 mL of diisopropylethylamine and 25.61 g (49.2 mmoles) of PyBOP. The solutionwas allowed to equilibrate for 10 minutes. Following equilibration, a solution of 5.28 g
(54.1 mmoles) of N,O-dimethylhydroxylamine hydrochloride dissolved in 5 mL of
acetonitrile and cont~ining 25.6 mL of N,N-diisop,opylethylamine (54.1 mmoles) was
added. The reaction was stirred overnight at room temperature.
The reaction mixture was then concentrated in vacuo and redissolved in 200 mL
of ethyl acetate. The ethyl acetate layer was washed three times with 1.0 N HCI (100
mL), three times with saturated sodium bicarbonate (100 mL) and three times withbrine (100 mL). The reaction mixture was dried over magnesium sulfate, filtered and
concenl~ated in vacuo giving a yellow oil. The crude product was purified by silica gel
chromatography using 2:1 ethyl acetate:hexane as the eluent. The product was isolated
as a white solid (77% yield) with an Rf of 0.77 on silica employing the same solvent
system used above.
Anhydrous ethyl ether (75 mL) and 0.9 g (23.7 mmoles) of lithium aluminum
hydride were placed in a 500 mL round-bottomed flask. The suspension was cooled in
an ice bath for ten minutes. A pres~7~te equalizing dropping funnel, containing 4.5 g
(18.4 mmoles) of Boc-Abu hydroxamate dissolved in 75 mL of anhydrous ethyl ether,
was attached to the round bottom flask and the contents were added dropwise over one
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hour, with continued cooling. The reaction mixture was allowed to react for an
adrlitinn~l two hours at room temperature.
The reaction mixture was then cooled in an ice bath and a cold solution of
potassium hydrogen sulfate (5.4 g in 230 mL of water) was slowly added to the reaction
flask and allowed to react for an additional 10 minutes. The aqueous and organiclayers were separated and the aqueous layer was extracted with anhydrous ethyl ether
(3 x 100 mLs). The combined organic layer was washed 3 x 100 mLs each with 1.0 NHCL saturated sodium bicarbonate and brine and then dried over magnesium sulfate,
filtered and
concentrated in vacuo. The product was isolate as a white solid (63% yield), with an
Rf of 0.90 on silica, using 2:1 ethyl acetate:hexane as the eluent.
N-Boc abuinal (4.00 g (21.39 mmoles)) was dissolved in 26 mL of methanol and
cooled on ice. To this was added a cold solution of 2.67 g of sodium bisulfite dissolved
in 54 mL of water. This reaction was stirred overnight at 4C. 265 mL of ethyl acetate
was then added to the above reaction mix followed by a solution of 1.08 g (22 mmoles)
of sodium cyanide dissolved in 80 mL of water, and then stirred overnight at 4C. The
aqueous and organic layers were separated and the aqueous layer was extracted twice
with 50 mL of ethyl acetate. The combined organics were dried over magnesium
sulfate, filtered and evaporated in vacuo leaving a clear colorless oil (70% yield). TLC
analysis on silica using 1:1 ethyl acetate:hexane as the eluent showed the product to
have an Rf of 0.69. The Boc-Abu cyanohydrin was used without further purification.
The Boc-Abu cyanohydrin isolated was dissolved in 120 mL of 4N HCI/dioxane.
60 mL of water was then added to the reaction mixture and it was refluxed overnight.
The reaction mixture was IOt~vapped to dryness leaving a brown solid. The solid was
dissolved in water and extracted three times with 100 mL of ethyl acetate. The
aqueous layer was then concen~rated in vacuo and rotdvdpped from ethyl ether three
times. This material was used without further purification.
HClAbu hydroxy acid (2.9 g (17.16 mmoles)) was dissolved in 51 mL of 2:1
dioxane:water and placed in an ice bath. To this was added 42.5 mL (42.5 mmoles) of
lN sodium hydroxide. The reaction was allowed to cool and 6.12 g (28.04 mmoles) of
di-tert-butyl dicarbonate was then added. The pH of the reaction was m~int~ined
between 9.5 and 10 by the addition of base. Following an overnight reaction time it
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was worked up as follows. The dioxane was rotavapped off and an additional 15 mL of
water was added to the reaction mixture. The water was covered with a layer of ethyl
acetate and cooled on ice. The pH of the aqueous layer was adjusted to 2.5 with 3N
HCl. The organic and aqueous layers were separated and the aqueous layer was
extracted twice with 50 mL of ethyl acetate. The combined organic layers were dried
over magnesium sulfate, filtered, and evaporated in vacuo leaving a brown viscous oil.
The crude material was purified by silica gel chromatography using 91:8:1
chloroform:methanol:acetic acid as the eluent. There remained 1.140 g Boc-Abu
hydroxy acid (26.3% yield from Boc-Abuinal). TLC analysis on silica using the
same system detailed above showed the product to be one spot with an Rf value of0.22.
Boc-Abu hydroxy acid (0.96 g (4.13 mmoles)) was dissolved in 35 mL of
dimt:thylroll,.amide (DMF) and cooled in an ice hath. 0.78 mL (12.4 mmoles) of 70%
triethylamine and 0.84 g (6.2 mmoles) of 1-hydroxybenzotriazole (HOBT) were added
and allowed to equilibrate for thirty minutes. After this time 1.0 g (5.22 mmoles) of 1-
(3-dimethylaminopropy)-3-ethylcarbodiimide hydrcchloride (EDC) suspended in 10 mL
of DMF was added. The reaction was allowed react at room temperature overnight.
The reaction was then rotavapped to dryness and redissolved in 100 mL of
chloroform and washed three times with 35 ml of saturated sodium bicarbonate andthen brine. The mixture was dried over magnesium sulfate, filtered and concentrated
in vacuo. The crude material was purified by silica gel column chromatography
employing 9:1 ethyl acetate:hexane. 0.938 g (85% yield) of product was isolated which
possessed and Rf value of 0.55 in the above solvent system.
Boc-Abu hydroxy ethylamide (0.233 g, 0.894 mmoles) was dissolved in 5mL of
dioxane followed by the addition of 20 mL of 4N HCI/dioxane. The reaction mixture
was allowed to react for two hours. After this time, the reaction mixture was
concentrated in vacuo and used immediately in the next step.
(L)-Leucine (1.31 g (10 mmoles)) was placed in a 3-neck round-bottom flask,
equipped with two pressure equalizing dropping funnels. 12.5 mL of 1.0N NaOH (12.5
mmoles) was added to the flask and then the mixture was cooled on ice. 12.5 mL of
1.0 N NaOH was placed in one dropping funnel and 1.46 mL (12.5 mmoles) of
morpholinecarbonyl chloride was placed in the other. The contents of the addition
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funnels were added to the flask ~irnult~neously over ten minutes. The mixture was
allowed to react for an adrlition~l twenty minutes. The reaction mixture was then
washed twice with 15 mL of ethyl acetate. The aqueous layer was cooled on ice and
acidified to a pH of 2 with 1.0 N HCl. The aqueous layer was extracted three times
with 15 mL of ethyl acetate. The combined organics were dried over magnesium
sulfate, filtered and concentrated in vacuo. There remained 0.48 g of a white solid
(20% yield) which pos~essed an Rf value of 0.45 using 91:8:1
chloroform:methannl acetir acid as the eluent.
Boc-Abu hydroxy ethyl amide (0.266g) was dissolved in 5 mL of dioxane
followed by the addition of 20 mL of 4N HCl/dioxane. The reaction mixture was
allowed to react for two hours. After this time, the reaction mixture was concentrated
in vacuo and used immediately in the next step. The HCl Abu hydroxy ethyl amide
was dissolved in 30 mL of anhydrous DMF and cooled on an ice bath for ten minutes.
To this solution was added 0.30 g (1.23 mmoles) of morpholineleucine urea, 0.5~ mL
(3.07 mmoles) of diisop~o~Jylethylamine and 0.152 g (1.13 mmoles) of 1-
hydroxybenzotriazole and allowed to equilibrate for thirty minutes. After this time,
0.218 g (21.13 mmoles) of EDC suspended in 10 mL anhydrous DMF was added and
the reaction mixture was allowed to react overnight. The reaction mixture was
concentrated in vacuo and the resulting residue was purified by silica gel column
chromatography employing 90:10 chloroform:methanol as the eluent. There remained0.2414 g (61.04% yield) of a white solid with an Rf value of 0.36 in the solvent system
detailed above.
Boc-(L)-Leu-(L)-Abu hydroxy ethyl amide (0.1225 g (0.317 mmoles)) was
dissolved in 10 mL of methylene chloride and cooled in an ice bath. To this mixture
was added 0.5 mg (.00317 mmoles) of 2,2,6,6-tetramethyl-1-piperidinyloxy, free radical
(TEMPO) and .0159 mL (.0317 mmoles) of an aqueous KBr solution (5.95 g of KBr
dissolved in 25 mL of water). The reaction mix was stirred vigorously while four 87
microliter portions of a lM aqueous sodium hypochlorite (pH 9.5) were added at 15
minute intervals. After this time the reaction mixture was analyzed by TLC employing
90:10 chloroform:methanol to check for completeness of the reaction. If the reaction
was not complete another portion of TEMPO and another regimen of the sodium
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hypochlorite solution should be added. This reaction required three additional
regimens of TEMPO and sodium hypochlorite.
When the reaction was deemed complete by TLC, the layers were separated.
The aqueous layer was extracted with methylene chloride (3 x 10 mL). The combined
organic layer was washed with 10% HCl (1 x 10 mL), 30 mL of a 100 mL stock solution
of 10% HCl cont~ining 1.6 g of KI, 10% sodium thinslllf~te (2 x 30 mL) and brine (1 x
30 mL). The organic layer was then dried over magnesium sulfate, filtered and
con-~ntrated in vacuo. The crude material was triturated with petroleum ether to give
an off-white solid which was recryst~lli7ed from ethyl acetate hexane. There remained
0.048 g (39.6% yield) of a white solid with an Rf value of 0.32 in the solvent system
detailed above.
TLC analysis of the product on silica gel gave an Rf value of 0.32 in the solvent
system detailed above. HPLC analysis was performed on a Vydac C4 column (4.6 x
250 mm) at 60C using a gradient of 15-25% B/30 minutes (A=O.l~o TFA in water,
B=0.1% TFA in acetonitrile). The product had a retention time of 14 minutes and a
purity of 97.8%. Analyses of the final product provided the following results: Mass
spectrum analysis (FABMS) found (M+H)+ at m/z 385. Elemental analysis for
C18H32N4Os found 56.14 C, 8.24 H and 14.36 N while calculated values were 56.23 C,
8.39 H and 14.57 N. For 1HNMR (600 MHz, d6-DMSO) analysis, the shifts observed
were 8.65(t,1H), 8.10(d,1H), 6.41(d,1H), 4.85(m,1H), 4.20(m,1H), 3.51(m,4H),
3.26(m,4H), 3.12(m,2H), 1.75(m,1H), 1.62(m,1H), 1.48(m,2H), 1.40~m,1H), 1.02(t,3H),
0.85(m,~H).
D. HALO-KETONE ~k~ ES
Halomethyl ketone peptides are irreversible inhibitors for serine proteases and
cysteine proteases. This class of compounds includes peptides having a variety of
halomethyl groups at their C-terminus. These halomethyl groups include -CH2X, -
CHX2 and CX3, where X ~ es~nts any halogen. A number of analogous compounds
have been synthesized, including the amino-halo ketones and the diazo-ketone peptides.
Although these analogous compounds are chemically distinguishable, all of these
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haloketone compounds are believed to have a similar mech~nicm of action.
Accordingly, for cimpli~ity, all of the foregoing compounds will be referred to
collectively herein as the "Halo-Ketone Peptides."
The reactivity of haloketones has generally been found to be in the order I >
Br > Cl > F. However, in.;,tasil-g the reactivity of the haloketone in this way can lead
to acceleration of competing side effects. Thus, it is preferable to increase the
reactivity of the halomethyl ketone peptides by altering the peptide structure.
In s~lecting a proper inhibitor for Calpain, the same basic peptide structure
selection techniques as used for the Peptide Keto-Compounds can be used. Once a
peptide structure has been identified, the most effective C-terminus grouping can be
empirically determined through kinetic inhibition studies of each of the compounds
with Calpain.
Many of the Halo-Ketone Peptides are available commercially. For example,
Leu-CH2Cl, Phe-CH2Cl, Z-lys-CH2Cl, Tosyl-LysCH2Cl (TLCK), Tosyl-PheCH2Cl
(TPCX), Z-Gly-Leu-Phe-CH2CI, Z-Phe-Ala-CH2Cl, z-Phe-Phe-CH2Cl, D-Phe-Pro-Arg-
CH2Cl, MeoSuc-Phe-Gly-Gly-Ala-CH2CI, MeoSuc-Ala-Ala-Pro-Ala-CH2Cl, MeoSuc-
Ala-Ala-Pro-Val-CH2Cl, Ala-Ala-Pro-Val-CH2Cl, Ala-Ala-Phe-CH2Cl, Suc-Ala-Ala-Pro-
Phe-CH2Cl and D-Val-Leu-Lys-CH2Cl are all available from suppliers such as Enzyme
Systems Products of Livermore, California. From the same suppliers, the following
diazomethyl ketone peptides are available: Leu-CHN2, Z-Phe-Phe-CHN2, Z-Phe-Ala-
CHN2, Z-Phe-Pro-CHN2, Z-Lys-CHN2 and Gly-Phe-CHN2. In addition, the production
of a-amino fluoro ketone peptides has been described in United States Patent
No. 4,518,528 to David W. Rasnick, the disclosure of which is hereby incorporated by
this reference.
The preparation of various Halo-Ketone Peptides is reviewed in Metho~s in
Enymology, 46:197-208 (1977), the ~ sllre of which is hereby incorporated by
reference. Briefly, halomethyl ketone derivatives of blocked amino acids are readily
prepared by the reaction of mineral acids (hydrohalic) with the corresponding
diazomethyl ketone. Iodomethyl ketones are prepared by reaction of a halo-ketonewith NaI, since reaction with HI with a diazomethyl ketone yields the methyl ketone.
A number of different blocking groups can be used, including benzyloxycarbonyl (Z)
and t-butyloxycarbonyl (Boc). The diazomethyl ketone is prepared by reaction of
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diazo",cthane with the appropliate acid activated by means of dicyclohexylcarbodiimide
(DCCI), by the mixed anhydride method.
Unblocked amino acid chloromethyl ketones can be prepared by reaction of
benzyluAy~.,l,ollyl blocked derivatives with HBr or HOAc, trifluoroacetic acid, or by
5 },.~d~(,ge"ation.
Synthesis of peptide chloromethyl ketones can be accomplished simply by
coupling an dpp~opliate peptide or amino acid with an unblocked amino acid
chloromethyl ketone. A few dipeptides can be converted directly to the chloromethyl
ketone using the mixed anhydride and CH2N2 followed by HCI.
Various synthetic problems are encountered in the preparation of chloromethyl
ketone derivatives of basic amino acids. The side chain usually must be blocked during
synthesis, and cliffirllltie5 are often encountered during removal of the blocking group.
Use of trifluoroacetic acid or HF was eventually fourld to give a good conversion to
product.
A number of examples of the preparation of Halo-Ketone Peptides have been
reported in the literature, inrlu-ling a co"")rehensive review of over 100 amino acid
derivatives and approxi",ately 60 peptide derivatives listed in J.C. Powers, in
"Chemistry and Biochemistry of Amino Acids, Peptides and Proteins," Vol. 4, Dekker,
New Yor~ 1977), the tli~rlnsure of which is hereby incorporated by reference. Those
20 of skill in ~ne art will recognize how to locate a multitude of examples of the
production of the Halo-Ketone Peptides. Accordingly, no additional examples are
provided herein.
E. TN VTTRO USES
In addition to the foregoing classes of compounds now d;scovered to possess
25 Calpain inhibitory activity, we believe that a large number of other such compounds
exist. In view of the largè number of inhibitors of Calpain of different classes we
disclose herein, all of the known, newly discovered and yet undiscovered inhibitors of
Calpain shall be referred to hereinafter collectively, using the term "Calpain Inhibitor.r
The Calpain Inhibitors may be used in vitro for a variety of purposes to inhibit30 unwanted Calpain activity. For example, the Calpain Inhibitors may be used in vitro to
prevent proteolysis that occurs in the process of production, isolation, purification,
storage or transport of peptides and proteins.
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The Calpain Inhibitors described herein can also be used in vitro to prevent
further degradation of tissue samples from occurring after preparation of the samples.
This in vitro prevention of degradation can be especially useful in the preparation of
assays for neurodegeneration wherein the assay comprises a test for the products of
Calpain activity in the tissues, such as assays for breakdown products (BDP's) of
cyto~ P~I co",pol-ents such as spectrin, MAP2, actin binding protein and tau. P.Seubert et aL, Neuroscience, 31:195 (1989), the licrl~sure of which is hereby
incorporated by reference, disclose an exemplary method of quantitating the amount of
spectrin BDP's as an inrlir~inn of Calpain activity.
The Calpain Inhibitors of this invention are also useful in a variety of other
expe,i,l,e"~al procedures where proteolysis due to Calpains is a significant problem.
For eY~mple, indusion of the Calpain Inhibitors in radioimmunoassay experiments can
result in higher sensitivity. The use of the Calpain Inhibitors in plasma fractionation
procedures can result in higher yields of valuable plasma proteins and can make
purification of the proteins easier. The Calpain Inhibitors disclosed here can be used
in cloning experiments utilizing recombinant or transfected bacterial or eukaryotic cell
cultures in order to increase yield of purified recombinant product.
To use the Calpain Inhibitors in vitro, the Calpain Inhibitors are dissolved in an
organic acid, such as dimethylsulfoxide (DMSO) or ethanol, and are added to an
aqueous solution containing the protease which is to be inhibited, such that the final
concentration of organic solvent is 25% or less. The Calpain Inhibitors may also be
added as solids or in suspension.
F. TREATMENT OF NEURODEGENERATION
We have discovered that the Calpain Inhibitors are useful ill vivo to treat
pathologies in which excess proteolysis by Calpains is involved. Such pathologies are
believed to include neuropathologies such as neurodegeneration resulting from
excitotoxicity, HIV-induced neuropathy, ischemia, denervation, injury, subarachnoid
hemorrhage, stroke, multiple infarction dementia, Alzheimer's Disease (AD),
Huntington's Disease, surgery-related brain damage, Parkinson's Disease, and other
pathologicàl conditions.
In additional itl vivo uses, peptide a-ketoamide can be used to control protein
turnover, muscular dystrophy, myocardial tissue damage, and bone resorption as shown
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in Tables PKC2, PKC3, and PKC4 by effective inhibition of Iysosomal cathepsin B.Peptide a-ketoqmit1es can also be used as neuroprotectants or for the treatment of
ischemia, stroke, restenosis or Alzheimer's disease as shown in Tables PKC2, PKC3,
and PKC4 by effective inhibiton of calpain I and calpain II.
51. T~ of Ir~h;~ rs
In order to identify Calpain Tnhihitors that are useful in the practice of the
present invention for treatment or inhibition of neurodegenerative conditions and
e~cf C, it is ill"~ ant to identify those inhibitors posessing significant Calpain
inhibitory activity. It is also important to identify those Calpain Inhibitors having a
high degree of specificity for inhibition of Calpain, in order to avoid interference with
other biological processes when the Calpain Inhibitor is introduced into a mammal
requiring treatment for neurodegeneration. Because all thiol proteases are believed to
exert their effect through a similar mechanism of action, our primary concern was to
identify those Calpain Inhibitors having substantial inhibitory activity against Calpain,
but relatively weak or no activity against other thiol proteases. Accordingly, in order to
identify such Calpain Inhibitors, we tested a variety of Calpain Inhibitors for their
ability to inhibit calpains I and Il, and compared this data with the ability of the same
Calpain Inhibitors to inhibit Cathepsin B, another thiol protease. Those CalpainInhibitors with high i)t vitro inhibitory activity against Calpain and a relatively lower
activity against Cathepsin B are believed to be most useful for i~t vivo therapy.
Examples lA through lC show the results of these studies for a variety of Calpain
Inhibitors.
EXAMPLE lA
Inhibition by Substituted Heterocvclic Compounds
The isocoumarins are irreversible inhibitors of Calpain. We obtained ICso
values for a variety of these Calpain Inhibitors as a kinetic analysis of these
compounds. Purified Calpains can be assayed using the fluorogenic substrate succinyl-
leucine-tyrosine-methylaminocoumarin (available commercially) or by measuring the
release of acid-soluble peptides from casein because we have found that the
isocoumarins inhibit casein proteolysis by Calpain.
Calpains I and II were purified by the method of (Yoshimura, et al. 1983).
(Kitahara, supra) provides an alternative purification scheme. Calpain II may
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alternatively be purchased from Sigma Chemical Co. as "Calcium Activated NeutralProtease." In this assay, purified Calpain was incubated with 14C-methylated casein in
the presence of various Heterocyclic Compounds and the amount of acid-soluble
radioactivity released by the action of Calpain was measured. The ICso values were
determined as the concentration of Heterocyclic Compound compound at which 50% of
the Calpain activity was inhihited Table lA shows ICso values for various Isocoumarin
Compounds.
TABLE lA
INHIBITION OF CALPAINS BY SUB~il ll U I ~:l) ISOCOUMARINS
ICso (uM)
Calpain I Calpain II
CiTPrOIC 100 70
NH2-CiTPrOIC (ACITIC) 10 120
PhCH2NHCONH-CiTPrOIC80 30
CH3CONH-CiTPrOIC 700 80
L-Phe-NH-CiTPrOIC 30
BOC-L-Phe-NH-CiTPrOICno inhibition >200
PhCH2NHCONH-CiTEtOIC90
PhCH2CONH-CiTEtOIC 30
D-Phe-NH-CiTEtOIC 200
Thus, it can be seen from Table lA that a variety of the Isocoumarin
Compounds have ci~nifir~nt Calpain inhibitory activity at low concentrations.
EXAMPLE lB(i)
Protease Inhibition by Peptide Keto-Compounds
The Peptide Keto-Compounds are reversible inhibitors of Calpains and other
thiol proteases. The Ki values for the inhibition of calpain 1, calpain II and Cathepsin
B were determined for several Peptide Keto-Compounds. Inhibition of calpain I from
human erythrocytes and calpain II from rabbit muscle were assayed using
Suc-Leu-Tyr-amidomcthylcoumarin as substrate in an assay buffer of 20mM HEPES
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pH=7.2, 10mM CaC12, 10mM B-mercaptoethanol. Cathepsin B from bovine spleen was
assayed using Z-Lys~-nitrophenylphosphate as substrate.
Table lB(i) shows the results of the studies of Example lB(i). The Ki value for
the inhibition of C-~lp~in~ and cathepsin B by several Peptide Keto-Compounds are
S shown in ~M (micromolar). The values for leupeptin, which is commercially available
from Calbiochem of La Jolla, California, are shown for comparison.
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Table lB(i)
K; VALUES FOR ~ E KETO-COMPOUNDS
Inhibitor Calpain I Calpain II Cathepsin B
Leupeptin 0.32 0.43 6
Z-Ala-Ala-Ala-CO2Me 200 ---- 1.5
Z-Ala-Ala-Abu-CO2Et 50 200 0.9
Z-Leu-Phe-CO2Et 0.23 0.4 > 50
Z-Leu-Me-CO2Et 0.12 0.18 18
Z-Leu-Abu-CO2Et 0.04 0.4 14
Z-Leu-Nva-COOEt 1.2 30
It can be seen from the results in Table lB(i) that the Peptide Keto-Compounds
inhibit Calpain with Ki values similar or superior to leupeptin. In particular, Z-Leu-
Phe-CO2Et, Z-Leu-Nle-CO2Et and Z-Leu-Abu-CO2Et were found to possess greater
Calpain inhibitory activity than leupeptin. In addition, these particular compounds
were highly specific to Calpain, with lower inhibitory activity toward Cathepsin B than
leupeptin.
EXAMPLE lB(ii)
Protease Inhibition of Peptide Keto-Compounds
We tested the ability of an additional group of Peptide Keto-Compounds to
inhibit several proteases in order to evaluate their specificity for Calpain. The results
of these studies are shown in Table lB(ii).
Table lB(iu). Inhibition of Calpain I, Calpain II, Cathepsin B (CathB), Chymotrypsin (Chym), PP
Fl~ct~ce and Papain
Inhibitor Kj(~M)
Calpain I Calpain II CathB Chym elasta papain
se
Z-Leu-Abu-COOEt 4.5 0.4 30 > 100 > 100 220
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T h!................................................. Kj(~M)
Calpain I Calpain II CathB Chym elasta papain
se
ZLeu-Abu-COOnBu 1.8 4 ~100 25 10
Z-Leu-Abu-COOBz 9.5 0.47 4 40 >100 40
Z-Leu-Leu-Abu-COOEt 1.8 2.6 22 >100 25
2-NapSO2-Leu-Leu-Abu-CO OEt 16 1.4 25 35 47
2-NapCO-Leu-Leu-Abu-COOEt 0.09 > 300 28
Tos-Leu-Leu-Abu-COOEt 33 69 25 28
Ph-(C H3)2-co-Leu-Abu-co OEt 1.2
Z-Leu-Abu-COOH 0.075 0.022 1.5 >150 >150
Z-Leu-Abu-CONHEt 0.5 0.23 2.4 >150 65
Z-Leu-Abu-CONHnPr 0.25 8 >300 2
Z-Leu-Abu-CONHnBu 0.2 13 >300 5
Z-Leu-Abu-CONHiBu 0.14 4 >300 40
ZLeu-Abu-CONHBz 0.35 2 >300
Z-Leu-Abu-CO N H-(CH2)2-Ph 0.022
Z-Leu-Abu-CO N H-(C H2)3-Mpl 0.041
Z-Leu-Abu-CONH-(CH2)7CH3 0.0 19
Z-Leu-Abu-CO N H-(C H2)2O H . 0.078
Z-Leu-Abu-CONH- 0. 16
(CH2)2o(cH2)2oH
Z-Leu-Phe-COOEt 1.8 0.4 340 0.025 >100 75
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or K~ M)
Calpain I Calpain II CathB Chym elasta papain
se
Z-Leu-Phe-COOnBu 5.0 1.1 15 0.15> 100 15
Z-Leu-Phe-COOBz 3.4 1.6 45 1.6> 100 45
Z-Leu-Leu-Phe-COOEt 1.4 1.9 42 0.26> 100 15
Z-Leu-Phe-COOH 0.0085 0.0057 4.5 76 > 150
Z-Leu-Phe-CONHEt 7.0 0.32 6 73>150
Z-Leu-Phe-CONHnPr 15.0 0.05 3 18> 300
Z-Leu-Phe-CONHnBu 0.028 3 8> 100
Z-Leu-Phe-CONHiBu 0.065 4 24
Z-Leu-Phe-CONHBz 0.046
Z-Leu-Phe-CONH(CH2)2Ph 0.024 (2)
Z-Leu-Me-COOEt 0.18 20 2.2 190
Z-Leu-Nva-COOEt 1.4 1.2 25 1602.3 150
Z-Leu-Met-COOEt 1.0 1.5 55 1.75> 100 140
Z-Leu-4-Cl-Phe-COOEt <4.0 0.4 50 0.9> 100 150
Table lB(ii) shows the inhibition constants (KI) for cathepsin B, calpain I, andcalpain II with peptide ketoamides. Dipeptide Ketoamides with Abu and Phe in the P
site and Leu in the P2 site are potent inhibitors of calpain I and calpain II. Z-Leu-
Abu-CONH-Et is a better inhibitor of calpain I than Z-Leu-Phe-CONH-Et by 14 fold.
Repl~cement of the Z group (PhCH2OCO-) by similar groups such as PhCH2CH2CO-,
PhCH2CH2SO2-, PhCH2NHCO-, and PhCH2NHCS- would also result in good inhibitor
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structures. One good inhibitor of calpain II is Z-Leu-Abu-CONH-(CH2)2-Ph.
Ch~nging the R3 and R4 groups s gnificantly improves the inhibitory potency toward
calpain II. The best Dipeptide Ketoamide inhibitors are those which have long alkyl
side chains (e.g. Z-Leu-Abu-CONH-(CH2)7CH3), alkyl side chains with phenyl
~vb~ ed on the alkyl group (e.g. Z-Leu-Abu-CONH-(CH2)2-Ph), or alkyl groups witha morpholine ring substituted on the alkyl group (e.g. Z-Leu-Abu-CONH-(CH2)3-Mpl,
Mpl = -N(CH2CH2)20). Dipeptide a-keto~mides with a small aliphatic amino acid
residue or a Phe in the P1 site are also good inhibitors for cathepsin B. The best
inhihitor is Z-Leu-Abu-CONHEt and replacement of the Z (PhCH2OCO-) by
PhCH2CH2CO-, PhCH2CH2SO2-, PhCH2NHCO-, and PhCH2NHCS- would also result
in good inhibitor structures.
EXAMPLE lB(iii)
Stability of Peptide Keto-Compounds
We determined the half-life in minutes of several Peptide Keto-Compounds in
both plasma and liver homogenates. The results of the determinations of stability of
the compounds in plasma and liver homogenates are shown in Table lB(iii).
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Table lB(iii). Stability in Plasma and in Liver of Peptide Keto-Ccmp L_ d~;.
Tr~;~ to, t1/2
plasma liver
Z-Leu-Abu-COOEt 2.8
2-NapSO2-Leu-Leu-Abu-COOEt ~ 60
2-NapCO-Leu-Leu-Abu-COOEt 25
Tos-Leu-Leu-Abu-COOEt 30
Z-Leu-Abu-COOH > 60 > 60
Z-Leu-Abu-CONHEt > 60 > 60
Z-Leu-Abu-CONHnPr > 60 > 60
Z-Leu-Abu-CONHnBu > 60 > 60
Z-Leu-Abu-CONHiBu > fi0
Z-Leu-Abu-CONHBz > 60 > 60
1~ Z-Leu-Phe-COOEt 7.8
ZLeu-Phe-COOnBu 7.7
Z-Leu-Phe-COOBz 1.9
Z-Leu-Phe-COOH > 60 > 60
Z-Leu-Phe-CONHEt > 60 > 60
Z-Leu-Phe-CONHnPr > 60 > 60
Z-Leu-Phe-CONHnBu ~ 60 ~ 60
Z-Leu-Phe-CONHiBu ~ 60
Z-Leu-Phe-CONH(CH2)2Ph > 60
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~rh;h-~ ~ tl/2
plasma liver
Z-Leu-Nle-COOEt 3.7
Z-Leu-Nva-COOEt 2.8
Z-Leu-Met-COOEt 8
It can be seen from the data in Table lB(iii) that the Peptide Keto-Compounds
are generally quite stable in plasma and liver homogenates. However, it is also shown
that the Peptide a-keto~mides were substantially more stable in both plasma and liver
than the corresponding peptide a-ketoesters
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EXAMPLE lC
Protease Inhibition by Halo-Ketone Peptides
The Halo-Ketone Peptides, like the substituted isocoumarins, are irreversible
inhibitors of Calpain. We determined the Kapp/[I] values for various members of this
class of compounds against Calp~in~ I and II. For comparison, we also determinedthese values against the additional thiol proteases Papain and Cathepsin B for at least
one Halo-Ketone Peptide. These Kapp values are not directly comparable to the Ki or
ICso values deterrnined above for other classes of inhibitors.
We assayed Calpain I and II using Suc-leu-tyr-amidomethylcoumarin. Papain
was assayed using benzoyl-arg-4-nitroanilide, and Cathepsin B (bovine) was assayed
using CBZ-lys-4-nitrophenyl ester. We followed the progress curve method of Tian and
Tsou, Biochemist~y, 21:1028-1032 (1982), the disclosure of which is hereby incorporated
by reference, to derive kinetic data. Briefly, this method makes use of the equation:
[P0] = V[S]/K
(1 + [S]/K)A[y
where [PO] represents the concentration of product formed at a time approaching
infinity, A is the Kapp in the presence of substrate (S), K is the Michaelis constant and
[y is the concentration of the inhibitor. Since [S] and [Y] are known and V and K can
be determined, Kapp can be readily determined.
The Kapp/[I] for various Halo-Ketone Peptides are shown in Table lC.
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TABLE lC
KINETIC PARAMETERS OF HALO-KETONE ~ L)ES
Inhibitor CI CII3 P CB
S Z-Gly-Leu-Phe-CH2Cl 2840001 946000
Boc-Gly-Leu-Phe-CH2Cl 902000 540000 290000
Z-Leu-Phe-CH2Cl 2250002 585000
Z-Gly-Leu-Ala-CH2Cl 210000
Ac-Leu-Phe-CH2Cl 259001 33400
Z-Val-Phe-CH2Cl 27200
Z-Ala-Phe-CH2Cl 2400
Ac-Ala-Ala-Ala-Ala-CH2Cl 1300
CI = Calpain I 1 - Rat
CII = Calpain II 2 Human
P = Papain 3 - Rabbit
CB = Cathepsin B
It can be seen from the results in Table lC that the Halo-Ketone Peptides
inhibit Calpain with relatively high Kapp/[I] values. In particular, Z-gly-leu-phe-CH2Cl,
Boc-gly-leu-phe-CH2Cl, Z-leu-phe-CH2Cl and Z-gly-leu-ala-CH2Cl were found to
possess ~igr if ir~nt Calpain inhibitory activity. In addition, Boc-gly-leu-phe-CH2CI was
shown to be somewhat specific to Calpain, with lower inhibitory activity toward
Cathepsin B or Papain than toward Calpain. The results shown in the table reveal that
Z-gly-leu-phe-CH2Cl and Boc-gly-leu-phe-CH2Cl produce similar inhibitory effects.
Thus, the blocking group is not shown to have a great effect on Calpain inhibitory
activity.
The kinetic constants of other irreversible Calpain Inhibitors include the
following with Kapp/[I] in parentheses: E-64 (7500), E64-d (23000) and Z-leu-leu-tyr-
CHN2 (230000). E-64 is commercially available from Sigma Chemical Co.. and is
shown here to be a poor inhibitor of Calpain. 7.-leu-leu-tyr-CHN2 is a diazomethyl
peptide compound, here shown to possess significant Calpain inhibitory activity.
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2. Tr~ 'in-~ of Calpain in Neural Tissues
In order to evaluate the inhibition of Calpain by the various Calpain Inhibitorsin neural tissues, we assayed the Calpain Inhibitors using the known ability of Calpain
to cleave spectrin, a protein component of neuronal and other tissue, into BDP's. In
this assay, more effective Calpain Inhibitors will prevent the conversion of spectrin into
BDP's. Example 2 is one example of such an assay.
EXAMPLE 2
Inhibition of Calpain in
Crude Brain Extracts hv Calpain Inhibitors
The activity of Calpain in crude brain extracts was measured by ~Y~mining the
Ca2+-stimulated proteolysis of the endogenous substrate spectrin. Brain tissue was
homogenized in 10mM Tris pH=7.4, 0.32M sucrose, lmM EGTA, lmM dithiothreitol a
nd the nuclei and debris removed by low speed centrifugation. Various Calpain
Inhibitors were added to the supernatant in a DMSO vehicle and a calcium salt (final
effective concentration about 1.2mM) added to start the reaction. Proteolysis ofspectrin was evaluated by western blot as described by Seubert, et al., Brair~ Res.,
459:226-232 (1988), the disclosure of which is hereby incorporated by reference.Briefly, a known quantity of a spectrin-containing sample treated with Calpain is
separated by SDS-PAGE and immunoblotted with anti-spectrin antibody. The amount
of spectrin immunoreactivity found corresponding to the characteristic BDP's is
indicative of the amount of spectrin activity present in the sample. An examplary
method for quantitating BDP's is to assay Spectrin BDP's by homogenizing brain parts
in 20mM Tris pH=7.2, .32M sucrose, 50~M Ac-Leu-Leu-nLeu-H on ice. Homogenates
are then mixed 1:1 with 10% SDS, 5% B-mercaptoethanol, lO~c glycerol, 10mM Tris
pH=8.0, 0.5% bromophenolblue, heated to 95C, and subjected to elec;rophoresis in
4-1/2 % polyacrylamide gels. The proteins in the gels are transferred to nitrocellulose
and the spectrin and BDP's detected using a rabbit polyclonal anti-spectrin antibody
and established immunodetection methods. The amount of spectrin and BDP's in each
sample can be quantitated by densitrometric scanning of the developed nitrocellulose.
Due to Calpain's requirement for Ca2+, in the absence of Ca2+ little or no
spectrin proteolysis occurred, regardless of the presence of inhibitor, while in the
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presence of Ca2+ the spectrin was >95% cleaved to BDP's within 40 min. if no Calpain
Inhibitor is added.
Both leupeptin and CI1 showed inhibition in the system of Example 2. In
addition, the following compounds of the Substituted Heterocydic Compounds were
found to produce sienifir~nt inhibition at 100 I-M:
3-chloroisocoumarin
3,4-dichloroisocoumarin
3-benzyloxy-4-chloroisocoumarin
7-(acetylamino)-4-chloro-3-(propoAy)-isocoumarin
4-chloro-3-(3-isothiureidopropoxy)isocoumarin
7-amino-4-chloro-3-
(3-isothiureidopropoxy)isocoumarin
7-(benzylcarbamoylamino)-4-chloro-3-
(3-isothiureidopropoxy)isocoumarin
7-(phenylcarbamoylamino)-4-chloro-3-
(3-isothiureidopl opuAy)isocoumarin
7-(acetylamino)-4-chloro-3-
(3-isothiureidopropoxy)isocoumarin
7-(3-phenylpropionylamino)-4-chloro-3-
(3-isothiureidoptu~oAy)isocoumarin
7-(phenylacetylamino)-4-chloro-3-
(3-isothiureidopropoxy)isocoumarin
7-(L-phenylalanylamino)-4-chloro-3-
(3-isothiureidopropoxy)isocoumarin
7-(benzylcarbamoylamino)-4-chloro-3-
(3-isothiureidoethoxy)isocoumarin
7-(phenylcarbamoylamino)-4-chloro-3-
(3-isothiureidoethoxy)isocoumarin
7-(D-phenylalanylamino)-4-chloro-3-
(3-isothiureidoethoxy)isocoumarin.
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The following compounds of the Halo-Ketone Peptides were also found to
produce si~nifir~nt inhibition at 100 I~M:
Z-Leu-Phe-CH2Cl
Ac-Leu-Phe-CH2Cl
S Z-Gly-Leu-Phe-CH2CI
Boc-Gly-Leu-Phe-CH Cl
Ac-Val-Phe-CH2Cl
Z-Gly-Leu-Ala-CH2CI.
In addition, the following compounds of the Peptide Keto-Compounds were
found to produce cignifi--~nt inhibition at 100 ~M:
Bz-DL-Phe-COOEt
Z-Leu-Nva-COOEt
Z-Leu-Me-COOEt
Z-Leu-Phe-COOEt
Z-Leu-Abu-COOEt
Z-Leu-Met-COOEt
Z-Ala-Ala-DL-Abu-COOEt
MeO-Suc-Val-Pro-DL-Phe-COOMe
Z-Ala-Ala-Ala-DL-Ala-COOEt
MeO-Suc-Ala-Ala-Pro-DL-Abu-COOMe.
Z-Leu-Phe-COOEt
Thus, the Substituted Heterocyclic Compounds, Peptide Keto-Compounds and
Halo-Ketone Peptides, in addition to leupeptin and CI1, provide inhibition in brain
homogenates.
3. In vivo Inhibition of N~,.o~ r~.tion through Infusion Techniques
In order to demonstrate that the inhibition of Calpain activity alone is sufficient
to inhibit neurodegeneration in vivo, we tested the ability of the Calpain Inhibitor,
leupeptin, to inhibit neurodegeneration in gerbils subjected to transient ischemia.
As stated above, leupeptin is poorly membrane permeant. Therefore, leupeptin
is not expected to cross the blood-brain barrier ("BBB") very well. Accordingly, in
order to provide the brain with sufficient leupeptin to adequately inhibit Calpain
activation, we used brain infusion techniques. Through the use of these techniques we
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were able to subject brain tissues to intimate contact with leupeptin for sustained
periods of time. Example 3A is provided to show the in vivo protection from
neurodegeneration found during one such study.
EXAMPLE 3A
In Vivo Protection Against Neurodegeneration
A small cannula was implanted in the right lateral ventricle of adult gerbils, and
secured to the skull with dental cement. An Alzet micro-osmotic pump was attached to
the cannula for intracerebroventricular perfusion. The pump was filled with either
saline alone (control) or lt:uyey~ (20 mg/ml in saline). After three days perfusion
with either the control solution or with the leupeptin solution, transient ischemia was
induced by bilaterally clamping the carotid arteries for a period of ten minutes. Core
temperatures were taken during and following ischemia, with no differences notedbetween control and leupeptin treated animals. Fourteen days later, the animals were
carrifired by Nembutal overdose and transcardial perfusion of a 10% solution of
paraformaldehyde in PBS. Coronal sections of the brain were stained with cresyl violet
and were examined for the extent of neuronal loss. The control gerbils exhibited the
typical damage found in the CAl field following ischemia, with a 72% loss of neurons.
However, the leupeptin treated gerbils showed far less neurodegeneration, with only a
I5 % loss of neurons.
The results of Example 3A cannot be explained by changes in thermoregulation,
since core temperatures did not differ between the groups. Accordingly, we believe
that the Calpain inhibitory activity of leupeptin is responsible for the observed
differences in neuronal cell loss. In order to further quantitate the differences, and
verify that leupeptin produced a Calpain inhibitory effect within the observed regions of
the brain, we performed a related series of experiments. In this series of experiments,
spectrin BDP's were measured in the leupeptin treated and control animals. As
csed above, these BDP's are indicative of the amount of Calpain activity occurring
within the tissue. Example 3B is provided to demonstrate the results of these
experiments.
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EXAMPLE 3B
In Vivo Inhibition of Calpain Activity
Impl~ntation surgeries and clamping of the carotid arteries were performed as
above with a control-ischemia group (n=4) and a leupeptin-ischemia group (n=5). A
third group of animals (n=4) received implantation with pumping of saline, but was not
subjected to ischemia. Animals were sacrificed by decapitation 30 minutes after
clamping of the arteries. The brains were rapidly removed and placed in cold
homogenization buffer (0.32 M sucrose, 10 mM Tris-HCl, 2 mM EDTA, 1 mM EGTA,
100 IIM leupeptin and 1 llgtml of the Halo-Ketone Compound, tos-phe-CH2CI
(TPCK)). The CA1 region of the hippocampus was then dissected. The samples from
both control and leupeptin treated animals were then prepared for SDS-PAGE and
immunoblotting with labeled anti-spectrin antibody, as described above in connection
with in vitro uses of the Calpain Inhibitors. The control animals exhibited a marked
increase in the levels of BDP's relative to the gerbils not subjected to ischemia. These
BDP's co-migrated with BDP's observed after in vitro proteolysis of spectrin with
C~lp~in The brain tissue from the leupeptin treated gerbils exhibited apploxil..ately
25% of the BDP's observed in the control ischemia treated gerbils.
Another group of gerbils (n=3) were sacrificed immediately after ischemia
without leupeptin treatment in order to observe the effects of ischemia without
reoxygenation. These gerbils exhibited a similar amount of increase of BDP's as the
control-ischemic gerbils observed after a 30 minute reperfusion period.
Thus, the results of Example 3B indicate that leupeptin exerts its
n~ oprotective effect through the inhibition of Calpain activation. The results also
indicate that the observed proteolysis of spectrin was an effect of ischemia, and not
secondary to the reoxygenation. Accordingly, the results indicate that inhibition of
Calpain activity in vivo produces a neuroprotective effect.
Although the foregoing studies demonstrate that leupeptin can inhibit
neurodegeneration in vivo, leupeptin is not the therapeutic drug of choice because of
the need to infuse the drug directly into the brain for an extended period of time to
exert its neuroprotective effect. This is due to the relatively poor ability of this
compound to cross the BBB. Accordingly, it is believed that a more therapeutically
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practical way to inhibit neurodegeneration would be to use more membrane permeant
Inhibitor of Calpain.
4. Platelet P~. --hility
In accordance with our disc~elies demonstrated in Examples 3 and 3A, we
believe that having a compound cross the BBB and enter CNS tissue is a key
characteristic of a therapeutically useful approach to treat or inhibit neurodegeneration
within the CNS. Use of Calpain inhibitors that have P~lh~nred membrane permeability
is one such approach. Thus, we measured the ability of various Calpain inhibitors to
penetrate the platelet membrane and inhibit Calpain that is normally contained in
platelets. As shown below in the following examples, our results indicate that particular
compounds of the Heterocyclic Compounds, Peptide Keto-Compounds and Halo-
Ketone Peptides, in addition to the Peptide Aldehyde, CI1, exhibit good membranepermeability.
As an indication of the membrane permeability of the various Calpain
Inhibitors, we measured the ability of various Calpain Inhibitors to penetrate platelet
membranes and inhibit the Calpain normally found within platelets. The membrane of
pl~t~letc is believed to have many similarities to the BBB and accordingly, suchexperiments are believed to provide a good indication of the ability of the various
Calpain Inhibitors to cross the BBB. Example 4 shows the results of some of these
platelet experiments using the Calpain Inhibitors of the present invention.
EXAMPLE 4A
Membrane Permeation of Calpain Inhihitors
Platelets were isolated by a modification of the method of Ferrell and Martin, J.
Bio~ ChenL, 264:20723-20729 (1989), the disclosure of which is hereby incorporated by
reference. Blood (15-20 ml) was drawn from male Sprague-Dawley rats into lOOmM
EDTA-citrate containing 10 units heparin, and centrifuged 30 minutes at 1600 rpm at
room temperature. The plasma was resuspended in 15ml buffer 1 (136mM NaCI,
2.7mM KCI, 0.42mM NaH2PO4, 12mM NaHCO3, 2mM MgC12, 2 mg/ml BSA (Sigma),
5.6mM glucose, 22mM Na3Citrate pH 6.5) and platelets were isolated at 2200 rpm at
room temperature of 25 minutes. Platelets were resuspended to 107 cells/ml in buffer
2 (136mM NaCI, 2.7mM KCI, 0.42 NaH2PO4, 12mM NaHCO3, 2mM MgCI, 1 mg/ml
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BSA, 5.6mM glucose, 20mM HEPES pH 7.4) and allowed to "rest" for a minimum of
10 minutes at room temperature before use.
Platelets were incub~ted for 5 minutes in the presence of inhibitor. In order toprovide sllffiri~ont intr~c~ r calcium to activate Calpain, the calcium ionophore
S A23187 was added to a final coTIr~ntration of 11~M. After a further S minute
inrub~tion, the pl~tf~let~ were harvested by centrifugation (1 min 10,000 x g) and
resuspended in 10% sodium dodecyl sulfate, 10mM Tris pH=8.0, 5%
B-mercaptoethanol, 0.02% bromophenol blue, and heated to 95 C for 5 min. Samples
were subjected to SDS-PAGE on 6% mini gels and transferred to nitrocellulose
(Schleicher and Schuell BA83) for 2 hours at lOOmA/gel in an LKB Novablot. Filters
were blocked for 10 minutes in 0.25% gelatin, t% BSA, 0.25% Triton X100, 0.9%
NaCl, 10mM Tris-Cl pH 7.5, incubated overnight in the same solution containing
antibody to rat spectrin, washed 3 X 10 minutes with lOmM Tris-Cl pH 7.5, 0.5~G
Triton X100, incubated 4 hours in wash buffer plus alkaline phosphatase conjugated
goat anti-rabbit antibody (Biorad), and washed as above. Filters were developed using
the Biorad AP conjugate substrate kit. Spectrin immunoreactivity on the filters was
qu~ntitated by densitometry.
The inhibition of Calpain within platelets as measured by the proteolysis of theendogenous Calpain substrate spectrin in the presence of inhibitors was assayed for a
variety of Calpain Inhibitors. The poorly permeant inhibitors leupeptin and E-64 had
little effect on intrac~ollul:lr Calpain. In contrast, the highly membrane permeant
Heterocyclic Compounds, Peptide Keto-Compounds, and Halo-Ketone Peptides
effectively inhibited platelet Calpain.
The following Heterocyclic Compounds were found to produce significant
inhibition at 100 I~M in the system of Example 4:
3-chloroisocoumarin
4-chloro-3-(3-isothiureidopropoxy)isocoumarin
7-amino-4-chloro-3-
(3-isothiureidopropoxy)isocoumarin
7-(benzylcarbamoylamino)-4-chloro-3-
(3-isothiureidopropoxy)isocoumarin
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7-(phenylcarbamoylamino)-4-chloro-3-
(3-isothiureidopropoxy)isocoumarin
7-(acetylamino) ~1 -chloro-3-
(3-isothiureidopl o~uAy)isocoumarin
7-(3-phe.,yl~,ru~ionylamino)-4-chloro-3-
(3-isothiureidopropoxy)isocoumarin
7-(phenylacetylamino)-4-chloro-3-
(3-isothiureidopropoxy)isocoumarin
7-(L-phenylalanylamino)-4-chloro-3-
(3-isothiureidopropoxy)isocoumarin
7-(benzylcarbamoylamino)-4-chloro-3-
(3-isothiureidoethoxy)isocoumarin
7-(phenylcarbamoylamino)-4-chloro-3-
(3-isothiureidoethoxy)isocoumarin
7-(D-phenylalanylamino)-4-chloro-3-
(3-isothiureidoethoxy)isocoumarin .
The following Halo-Ketone Peptides were found to produce ~ignifi~nt
inhibition at 100 ~M in the system of Example 4: -
Z-Leu-Phe-CH2Cl
Ac-Leu-Phe-CH2CI
Z-Gly-Leu-Phe-CH2Cl
Boc-Gly-Leu-Phe-CH2Cl.
The following Peptide Keto-Compounds were found to produce significant
inhibition at 100 I.M in the system of Example 4:
Z-Ala-Ala-D,L-Abu-COOEt
Z-Ala-Ala-Ala-D,L-Ala-COOEt
MeO-Suc-Ala-Ala-Pro-D,L-Abu-COOMe
Z-Leu-Phe-COOEt
Z-Leu-Nle-COOEt
Z-Leu-Nva-COOEt
Z-Leu-Abu-COOEt
Z-Leu-4-Cl-Phe-COOEt
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Z-Leu-Leu-Abu -COOEt
Z-Leu-Leu-Phe-COOEt
2-NapSO2-Leu-Abu-COOEt
2-NapSO2-Leu -Leu -Abu-COOEt
S Z-Leu-Met-CO2Et
Z-Leu-NLeu-CO2Et
Z-Leu-Phe-CO2Bu
Z-Leu-Abu-C02Bu
Z-Leu-Phe-CO2Bzl
Z-Leu-Abu-CO2Bzl
Z-Ala-Ala-D,L-Abu-COOBzl
Z-Leu-Phe-COOH
Z-Leu-Abu-COOH.
Among those compounds found to exhibit Calpain inhibitory activity in the
homogenate system of FY~mple 2, we found at least three compounds which failed to
exhibit Calpain inhibitory activity in the platelet system of Example 4. These
compounds are leupeptin, MeO-Suc-Val-Pro-D,L-Phe-COOMe and Bz-D,L-Phe-
COOEt. Leupeptin is known to be poorly membrane permeant, thus confirming that
the platelet assay will exclude known poorly membrane permeant compounds.
Accordingly, the two Peptide Ketocompounds found not to provide Calpain inhibitory
activity within platelets are also believed to be poorly membrane permeant, and would
not be expected to cross the BBB.
EXAMPLE 4B
Quantitative Studies of Platelet Membrane Permeability
We performed additional quantitative or semi-quantitative studies on several
Peptide Keto-Compounds using the assay of Example 4A, except that ICso values were
determined as the concentration at which 50% of the Calpain activation present in
controls occurred. Results are shown in Table 4B. For the semi-quantitative assays,
indicated with +'s in Table 4B, "+" indicates detectable inhibition at 100 ~M, "+ +"
indicates cignifi~ntly more inhibition than "+", and "+ + +" indicates no detectable
activation of Calpain detectecl
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TABLE 4B
Platelet Assay of Peptide l~eto ides, Ketoesters and Ket~P ~ .
Tr~ ;ts~r IC50
s
Z-Leu-Abu-COOEt 42
ZLeu-Abu-COOnBu 28
Z-Leu-Abu-COOBz + +
Z-Leu-Leu-Abu-COOEt 40
2-NapSO2-Leu-Leu-Abu-COOEt 100
Tos-Leu-Leu-Abu-COOEt 30
Z-Leu-Abu-COOH 8
Z-Leu-Abu-CONHEt 1.5
Z-Leu-Abu-CONHnPr 70
Z-Leu-Abu-CONHnBu 2.0
Z-Leu-Abu-CONHiBu 28
Z-Leu-Abu-CONHBz 1.5
Z-Leu-Phe-COOEt 42
Z-Leu-Phe-COOnBu + + +
Z-Leu-Phe-COOBz + +
Z-Leu-Leu-Phe-COOEt + +
Z-Leu-Phe-COOH 6.5
Z-Leu-Phe-CONHEt 1.7
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ICso
Z-Leu-Phe-CONHnPr 24
Z-Leu-Phe-CONHnBu 38
ZLeu-Phe-CONHiBu 22
Z-Leu-Phe-CONH(CH2)2Ph 3.0
Z-Leu-Nle-COOEt 20
Z-Leu-Nva-COOEt 40
Z-Leu-Met-COOEt +
Z-Leu-4-Cl-Phe-COOEt +
Table 4B shows that peptide a-ketoamides and ketoacids were much more
effective than col.~:~onding peptide ketoesters in this platelet assay. E~xtending the
R3 group to an alkyl group or an alkyl group substituted with a phenyl group increased
the membrane permeability of the inhibitors as indicated by increased potency in the
platelet assay. In view of these results, Applicants believe that extending the R group
to include longer alkyl groups or alkyl groups substituted with phenyl groups would
increase the membrane permeability of a given inhibitor.
In view of the foregoing, the results of Examples 4A and 4B support our belief
that CI1 and the Substituted Heterocyclic Compounds, Peptide Keto-Compounds and
Halo-Ketone Peptides are believed to be membrane permeant and therefore, are
expected to be effective in crossing the BBB subsequent to i)l vivo administration of the
compounds.
5 I2rd~ of Gl~ t~ Toxicity
To further identify those Calpain Inhibitors likely to possess pharmacologicallyactive n~uloprotective ability, we tested the ability of the Calpain Inhibitors to protect
against glutamate excitotoxicity. Excess extrac.oll~ r glutamate is thought to play a key
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role in the induction of neuropathology in ischemia, which is accompanied by Calpain
activation. In support of this role for excess glutamate, cultured N18-RE-105 (aneuroblastoma-retinal hybrid) cells can be killed by the addition of glutamate into the
culture medium. This glut~m~te-mediated cytotoxicity is calcium dependent and can be
reduced through a number of merh~ni~mc including free radical scavengers, blockers of
the N-type voltage-sensitive calcium channel, and quisqualate-subtype glutamate
~nt~gonict~ Thus, glnt~m~te-mediated 'Killing of N18-RE-105 cells is an in vitro model
for neuropathology.
Accordingly, we tested the ability of the Calpain Inhibitors to inhibit glutamate-
induced cell death in these cells in order to establish that the Calpain Inhibitors can
decrease or prevent glutamate-induced death Of N18-RE-105 cells. Some of these tests
are shown in Example SA.
EXAMPLE SA
Inhibition of Glutamate-lnduced Cell Death
Stock cultures of N18-RE-105 cells were maintained in Dulbecco's modified
Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS) and supplemented
with hypoxanthine, aminopterin and thymidine (HAT). Subconfluent cultures were split
and plated into 96-well plates. Twenty-four hours after plating the cells were exposed
to fresh media containing glutamate and various concentrations of Calpain inhibitors.
Control cells were not treated with glutamate. The treated cells received SmM
~h1t~m~ee and leupeptin (51lg/ml) or the other Calpain Inhibitors listed in Figure 1 at
31~g/ml. Conversion of MTT was measured 19 hours later as described. Nineteen
hours after the onset of exposure, cell viability was quantitated by measuring the extent
to which the cells convert 3(4,5-dimethylthiazol-2-yl)-2-5-diphenyltetrazolium bromide
(MTr) to a blue formazan product, which occurs in the mitochondria of living but not
dead cells (Pauwels et al.; 1988). A higher absorbance is indicative of greater cell
viability.
Figure 1 shows the percent of blue formazan product remaining after treatment
with gl11t~m~t~, relative to control where no glutamate was added. Thus, it can be seen
that with vehicle plus glutamate but no inhibitor, less than 70% of the mitochondrial
act~;vity remains. However, Figure 1 shows that several Calpain inhibitors, including
leupeptin, CI1 and representatives of the Heterocyclic Compounds, Peptide Keto-
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Compounds and Halo-Ketone Peptides protect N18-RE-105 cells against glutamate
toxicity. The Peptide Keto-Compound Calpain inhibitor, Z-Ala-Ala-Abu-C02Et, the
Substinlted Heterocyclic Compounds, CITPrOIC and ACITIC, and the Halo-Ketone
Peptide, TPCK comrlet~ly blocked the toxic effects of glutamate, resulting in 100% or
S greater of the formazan product as seen with cells not treated with glutamate. Thus,
Example 5 shows that these Calpain Inhibitors effectively block cell death in an in vitro
model for neuropathology. Accordingly, this data further supports our discovery that
Calpain Inhibitors are neuroprotective in vivo.
We have discuve,ed that glutamate-induced cell death in pheochromocytoma
PC12 cells can be prevented by the membrane permeant calpain inhibitors
Z-Leu-Phe-CONHCH2CH3 and Z-Leu-Nva-CONH(CH2)3 morpholine. These
inhibitors rescue a greater proportion of the PC12 cells than calpain inhibitor 1 (Ac-
Leu-Leu-Norleucinal) although higher concentrations can be required in certain
in~t~nres Z-Leu-Nva-CONH(CH2)3 morpholine also induces short processes in both
the presence and absence of glutamate. We observed reduction in glutamate-induced
cell death is observed even when calpain inhibitors are added several hours after
glut~m~te These observations are the first demonstration that Calpain inhibitors can
rescue cells in vitro from glutamate toxicity and support the critical role of calpain
activation in excitotoxicity. These experiments provide still further support that
inhibition of calpain is useful in the treatment of neurodegeneration, such as stroke and
ischemia.
The rat pheochromocytoma PC 12 is described by Greene et al, Proc. NatL
Acad Sci USA, 73:2424-2427 (1976), the disclosure of which is hereby incorporated by
reference. This tissue expresses the NMDA subtype of glutamate receptors.
Treatment of PC12 cells with glutamate for 24 hours produces death of 80% of thecells as measured by conversion of Glutamic acid, 3-[4,5-dimethylthazol-2-yl]-2,5-
diphenyltetrazolium bromide (MTT) into its blue formazan product. When PC12 cells
are exposed simultaneously to glutamate and calpain inhibitor, cell death is reduced.
Thus, these cells can be used as an effective model to determine the effectiveness of
the various calpain inhibitors to alleviate cell death. The experimental procedures we
used to evaluate glutamate toxicity are described below in Example 5B.
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F-Y~ SB
Glutamate Toxicity Assay in Pheochromocytoma Cells
Cells of the RC72 subclone of the rat pheochromocytoma PC12 were grown in high
- glucose Dulbecco's modified Eagle's medium (DME) without glutamine with 10% fetal
S bovine serum and 5% horse serum and gentamycin. These cells are available from Dr.
David Schubert of the Salk Institute. Media, horse serum, dialyzed fetal calf serun ~nd
gentamycin were from Irvine Scientifir Fetal calf serum was from BioCell.
Prior to plating, cells were cultured for 2 passages (4 days) in the same media with
~I~t~mine. Cells were plated at 10,000 cells/well into 96 well plates coated with collagen
and grown for 24 hours prior to experiments.
Exposure to glutamate was performed in DME without glutamine with 10~o
dialyzed fetal calf serum and 50 ug/ml gentamycin. Calpain inhibitors were added to
cultures from DMSO stocks. Final DMSO concentrations did not exceed 0.1%.
After 24 hours exposure to glutamate and inhibitors, 20ul of 7.5 mg/ml Glutamic
acid,3-[4.5-dimethylthazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) in PBS was added
to each well. MTT is available from Sigma. The cultures were incubated for 60 minutes
and the media carefully removed. Detergent (10% Triton X-100, 0.4% concentrated HC1
in ,soplopanol) was added and incubated for 10 minutes on a shaking table before the
plates were read using a microplate reader. The difference between the absorbance at 655
and 595 nm was used as a measure of viability. All experiments were normalized to
untreated cells in the same plate.
We tested a number of Calpain inhibitors for their ability to rescue PC12 cells from
glut~m~te toxicity using the experimental protocol described in the foregoing example.
The results are shown in Table 5B.
TABLE SB
Inhibitor Concentration l.M % of control
Ac-Leu-Leu-Arg-H (leupeptin) 5000 6.9
Ac-Leu-Leu-Nle-H (calpain inhibitor 1) 3 61
34
E64 100 17
TPCK 100 12
Cystatin C 0.1IU/ml 23
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Z-Leu-Phe-CONHCH2CH3 100 77
Z-Leu-Abu-CONHCH2CH3 100 26
Z-Leu-Abu-CONH(CH2)3 morpholine 100 6.7
Z-Leu-Abu-CONH(CH2)2 phenyl 100 28
Z-Leu-Abu-CONH2 100 11
Z-Leu-Nva-CONHCH2CH3 50 69
Z-Leu-Phe-CONH(CH2)3 morpholine 100 50
Z-Leu-Nva-CONH(CH2)3 morpholine 100 89
Reduction of glutamate-mediated cell death is produced by several different calpain
inhibitors. Z-Leu-Phe-CONHCH2CH3 and Z-Leu-Nva-CONH(CH2)3 morpholine
appeared to provide the best results in rescuing cells from glutamate toxicity. Inhibitors
related to Z-Leu-Phe-CONHCH2CH3 also rescue PC12 cells from glutamate toxicity,
although with varying efficacy. Substitution of Abu for Phe or Nva in the P1 position
decreases the efficacy of the compounds. Several calpain inhibitors, including leu~c~til-
and E64, did not rescue the cells. Leupeptin and E64 are known to be poorly cell-
penetrating, providing further support for membrane-permeance as an important factor
in the pharmacological effectiveness of the calpain inhibitors as used in the present
invention. The poor calpain inhibitors cystatin C and TPCK also did not rescue the cells.
This result is consistent with our conclusion that celi death is specifically the result of
calpain activation and does not involve another protease with related specificity.
We further studied the more effective compounds, Z-Leu-Phe-CONHCH2CH3 and
Z-Leu-Nva-CONH(CH2)3 morpholine. Figure 2 shows the results obtained using the
procedures of the foregoing Example using different concentration of these two
compounds along with calpain inhibitor 1 (CI1), a peptide aldehyde. The concentrations
we used are as indicated. CI1 prevents glutamate-induced cell death at concentrations as
low as 3uM but its efficacy does not increase but rather decreases with increasing
concentration. We have observed toxicity by Cl1 at higher concentrations; however, Cl1
is not toxic by itself at concentrations below 10uM. Thus, we believe that this toxicity
explains the observed increase in cell death at higher concentrations of CI1. The calpain
inhibitors Z-Leu-Phe-CONHCH2CH3 and Z-Leu-Nva-CONH(CH2)3 morpholine exhibit
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typical sigmoidal dose-response curves for cell rescue and produce nearly complete rescue
at high concentrations.
We observed no toxicity of Z-Leu-Phe-CONHCH2CH3 or
Z-Leu-Nva-CONH(CH2)3 morpholine at any concentration tested. This reduction of cell
death by these compounds is dose-dependent with ICso values of 20-50uM. The IC50values of 20-50uM for Z-Leu-Phe-CONHCH2CH3 and Z-Leu-Nva-CONH(CH2)3
morpholine are ci~nifir~ntly above the Ki's for calpain I or calpain II for these compounds.
The Ki values for Z-Leu-Phe-CONHCH2CH3 and Z-Leu-Nva-CONH(CH2)3 morpholine
are 200 nM and 250nM, respectively, using human erythrocyte calpain I, and 22nM and
100nM, respectively, using rabbit muscle calpain II. There are two possible explanations
for the difference between the ICso values we measured and the Ki's for these compounds
using purified C~lp~in less than complete penetration of the cell or the metabolism of
the inhibitors by the cells. As dicc-lcsed above, ~he poorly permeant inhibitor leupeptin
is ineffective at preventing cell death. Thus, we believe that the difference between the
Ki and IC50 values is due to membrane permeance effects.
We also evaluated the effect of glutamate concentration on the ability of
Z-Leu-Phe-CONHCH2CH3 or Z-Leu-Nva-CONH(CH2)3 morpholine to alleviate cell
death. Figure 3 shows the results obtained when PC12 cells were incubated with the
in~ tçd concentration of glutamate and no inhibitor (circles), 20uM
Z-Leu-Nva-CONH(CH2)3 morpholine (triangles). or 30uM Z-Leu-Phe-CONHCH2CH3
(squares) for 24 hours and cell viability was assayed by Ml-r, as described in the Example.
Values are e,~lessed as % of naive control + sem. At submaximal concentrations of these
compounds, the rescuing effect can be overcome by high concentrations of glutamate.
Thus, it is clear that the rescue of PC12 cells from glutamate toxicity is related to both the
concentration of glut:3m~te and to the concentration of inhibitor. We believe that the
dependence on glutamate concentration is the result of the activation of multiple pathways
of cell damage by the high concentration of glutamate. There is ample evidence for
calpain-independent meçh~nicmc of excitotoxic cell death. Observations by others that
~ntinYi(l~nts such as vitamin E also rescue these cells from glutamate toxicity provide
support for the idea that such calpain-independent mechanisms are operative. However,
we have shown that inhibition of calpain alone is sufficient to alleviate cell death. Thus,
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the use of Calpain inhibitors in accordance with the present invention is a signfir~nt
unexpected finding.
We also studied the ability of Calpain inhibitors to alleviate cell death after the
cells have been exposed to eJut~m~e The results of this analysis are shown in Figure 4.
Glnt~mate at 7.5mM was added at 0 time and Z-Leu-Phe-CONHCH2CH3 (squares) or
Z-Leu-Nva-CONH(CH2)3 morpholine (triangles) added at the indi~ated times to final
concentrations of 1001.M each. Cell viability was measured 24 hours after the addition of
e~l-t~m~te. by the MTT assay. Values are expressed in the Figure as % of naive control
~ sem. It can be seen that addition of calpain inhibitors after the cells have been exposed
to glllt~m~te is only partially effective. Advantageously, some rescue of cell death is still
observed if inhibitor is added as long as 2 hours after glutamate. However, by 8 hours
after the ghlt~m~te addition, inhibitors no longer have an effect. Accordingly, it is
advantageous to a~i~nini~ter the Calpain inhibitors of the present invention within two
hours of ell-t~m~te activation. Interestingly, at 8 hours after the addition of glutamate, the
cells are still largely normal in appearance and are 100% viable by MTT assay regardless
of the presence of inhibitor. This suggests that Calpain cleaves one or more cellular
proteins which are e~nti~l for cellular functioning, and this perturbation results in cell
death some hours later. Thus, it is still desirable to administer Calpain inhibitors within
two hours of glutamate release, even where cell or tissue morphology remains normal.
We also evaluated morphological changes produced by the Calpain inhibitors. We
exposed cells to 100 ~M Z-Leu-Nva-CONH(CH2)3 morpholine alone or to 100 I~M
Z-Leu-Nva-CONH(CH2)3 morpholine and 7.5 IlM glutamate. Cells exposed to both
treatment~ show an altered morphology with short processes extending from the cell body
which are rarely seen in the absence of inhibitor but are occasionally seen with glutamate
alone in surviving cells. Inhibitor at high concentrations does not prevent some of the
glutamate-induced decrease in the number of adherent cells, but these non-adherent cells
remain viable as measured by Ml'r conversion. Incubation of PC12 cells with
Z-Leu-Nva-CONH(CH2)3 for longer times (up to 72 hours) does not cause the expansion
of these short processes into longer neurites, nor does it cause cytotoxicity. This
morphological effect is not seen consistently with different calpain inhibitors, and is not
caused by Cl1;
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Excitotoxicity in vivo, as well as other forms of neurodegeration, are accompanied
by the breakdown of the cytoskeletal protein spectrin, which we believe is mediated by
calpain. The breakdown of spectrin in vivo, as well as the digestion of spectrin by calpain
in vivo, produces not only the re~luction in the amount of intact spectrin but also a
characteristic doublet of spectrin breakdown products (BDP's) of molecular weight 150 and
155kDa. These BDP's appear to be unusually persistent in vivo. The detection of spectrin
BDP's can be used as an assay for cellular degeneration, especially neurodegeneration.
See U.S. Patent No. 5,118,606 to Lynch et al., the disclosure of which is herebyincorporated by reference.
We exposed PC12 cells to glutamate for 24 hours. Cells were extracted with
CHAPS and analyzed for spectrin breakdown by western assay, as described in U.S. Patent
No. 5,118,606. Analysis of the PC12 cells after glutamate toxicity reveals a decrease in the
amount of intact spectrin but no striking increase in the 150 and 155kDa BDP's. The
decrease in the amount of spectrin immunoreactivity cannot be accounted for by loss of
protein from the samples as equal amounts of protein were loaded in each lane. Thus, in
this assay the BDP's that are usually seen upon proteolysis appear to be degraded into
small fragments not recognized in the western assay either directly or through the SBDP's
more rapidly than is observed in vivo. We also added either Z-Leu-Phe-CONHCH2CH3or Z-Leu-Nva-CONH(CH2)3 morpholine to the samples exposed to glutamate, and
in~lnded in the western assay. The loss of spectrin immunoreactivity was prevented by the
addition of either calpain inhibitor.
Our results show that calpain inhibitors can rescue PC12 cells from glutamate
toxicity. Thus, inhibition of calpain represents an exciting new approach to theamelioration of ischemic and excitotoxic damage in stroke and other neurodegenerative
proc~ s.
6. PPdu~ of Infarction upon MCA O~ ;o~
Stroke is a significant health problem in the human population. Strokes are
occlusions of cerebral arteries producing a decreased blood flow to brain regions, which
cause cell death through oxygen and nutrient deprivation. This type of lesion can be
modeled in rats by surgical occlusion of the middle cerebral artery (MCA). Several models
for MCA occlusion have been developed, and all give substantially similar results.
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MCA occlusion produces a large volume of infarcted brain tissue 24 hours after
oc~1u;,ion. Previous studies have shown that the size of the infarct as judged by TTC
staining does not increase after the first 24 hours post-occlusion. Thus, we used an MCA
occlusion model in order to test the ability of Calpain inhibitors to prevent
neurodegeneration. This model is described in FY~mple 6.
EXAMPLE 6
MCA Occlusion Model for Neurodegeneration
Male Sprague-Dawley albino rats weighing apploxi--,ately 250-300 grams were
anesthetized with pentobarbital (70 mg/kg, i.p.). The neck region was shaven and a 2 cm
incision was made. The superficial fascia was teased away with tissue forceps and blunt
tip tissue scissors using a spread method. The right common carotid artery was isolated
away from the vagus nerve and tied off with a single suture (3.0 silk). The external carotid
was permanently ocrl--ded by suturing. The bifurcation of the internal carotid and
pterygopalatine arteries was exposed and a single microaneurysm clip was placed on the
pterygopalatine. Another microaneurysm clip was placed on the common carotid just
proximal to the external/internal bifurcation. A suture was placed loosely around the
common carotid and a lumen was made in the vessel with the tip of a 25g needle. A 40
mm nylon suture was prepared by melting the tip to smooth the pointed end and marked
with a dot exactly 17.5 mm from the melted end. The suture was inserted into the lumen
of the artery as far as the vessel clip, the clip is removed and the suture advanced until the
marking was at the bifurcation of the internal and external carotid arteries. This places
the end of the suture in the circle of Willis just beyond the source of the middle cerebral
artery and occludes this artery. The loose suture around the carotid is tied lightly to keep
the nylon suture in place. The microaneurysm clip on the pterygopalatine artery was
removed, the incision is closed and the animals are allowed to recover in heated recovery
cages.
Twenty-four hours after occlusion, the brains of these animals were removed and
sliced into 2mm sections. The sections were stained using 2,3,5-triphenyltetrazolium
chloride as in Lundy, et al., J. PhannacoL Meth., 16:201-214 (1986). Absence of red color
development indicated tissue damage or death. The sizes of the infarcted tissue zone
(area with red stain) and impaired zone (area with partial development of red color) were
evaluated using quantitative morphometry.
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Drugs or vehicle were administered by infusion into the femoral vein. All animals
received the same volume of drug or vehicle (20% dimethyl sulfoxide/80% propylene
glycol) via a catheter attached to an Alzet osmotic minipump (24 hr pump, 8 ~l/hr, 90 ul
total volume).
The model of Example 6 was used to determine the size of infarcted area for
control (vehicle, i.v.) and with a~lmini~tration of each of two Calpain inhibitors: Z-Leu-
Phe-CONH-Et and Z-Leu-Abu-CONH-Et. These results are depicted graphically in
Figure 5. It can be seen that aclminictration of either of the Calpain inhibitors Z-Leu-Phe-
CONH-Et or Z-Leu-Abu-CONH-Et produces a reduction in the size of the infarcted area.
7. Ir~ib;tin~ of Anoxic and Hypoxic Damage
The CA1 region of hippocampus is a brain area particula~ly vulnerable to ischemic
damage and other insults involving excitatory amino acids. The hippocampus is also a
major focus of cell degeneration in Alzheimer's disease. Neural cells in slices in vi~o
degenerate following hypoxia through the same chain of events (including reperfusion
effects) observed in vivo during and after ischemia. We believe that studies of
degeneration of neural slices in the presence of the various Calpain Inhibitors is an
effective indicator of the membrane permeance of the Calpain Inhibitors. Accordingly, we
believe that these studies provide a model for the treatment and inhibition of
neurodegeneration in vivo. Similar studies for determining the efficacy of compounds
useful in the treatment of neurodegeneration in accordance with the present invention can
be performed using other models, such as protection against degeneration in platele~s or
cells in culture.
It is believed that hypoxia is a major cause of neurotoxicity in a variety of
neurodegenerative diseases and conditions, such as stroke and head injury. Thus, we
con-luctecl further studies using hippocampal slices to show that the various Calpain
inhibitors, advantageously, can increase survival of hippocampal nerve cells during
exposure to hypoxic or anoxic conditions. An initial screening procedure was first used to
q~ tatively determine whether the various Calpain Inhibitors can provide neuroprotection
from anoxia in hippocampal slices. An example of these initial screening procedures is
shown by Example 7A.
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EXAMPLE 7A
~nitial Screen for Inhibition of Anoxic Damage
Hippor~mp~l slices (400 um) were prepared from Sprague Dawley rats (6 to 7
weeks) and m~int~ined in an interface chamber at 35 C using conventional techniques, i.e.,
the lower surface of the slice received a constant perfusion (0.5 ml/min) of ACSF, while
the upper surface was exposed to a moist atmosphere of O2:CO2 (95%:5%) exchanged at
a rate of 2 L/min. The ACSF medium contains (in mM): NaCl (124), KCl (3), KHPO4
(2.5), CaCl2 (3.4), NaHCO3 (26) and D-Glucose (10). Field excitatory post-synaptic
rei~yonses were recorded from stratum radiatum of CAlb in response to stimulation of
Schaffer-commi~ ral fibers in CAla or CAlc. The depth of the recording electrode was
optimized and evoked responses were collected at a rate of one evoked response every 30
seconds.
For the initial screening procedure, 14 to 16 slices are harvested from the
hippocampus of a single rat and placed in a common ACSF bath. Each slice is tested in
sequPnre to determine the m~gnitude of its pre-anoxic evoked le~ponse. Five s~imul~tion
pulses (each 0.1 ms (milli~econd) in duration) were presented over a 15 second interval.
The largest evoked resL~onse was noted and recorded for each slice.
Following this, the slices were incubated for one hour, with either drug or vehicle
alone added to the ACSF. After the one hour drug incubation period, the oxygen-enriched
atmosphere of the chamber was made anoxic by substituting nitrogen for oxygen (N2 =
95%; C2 = 5%). The slices were retained in this anoxic environment for 10 minutes,
following which the oxygen-enriched atmosphere (2 = 95%; C2 = 5%) was
reest~hli~hed.
The slices were given the opportunity to recover for 30 minutes following
reoxygenation whereupon each was stimulated and the maximum evoked potential
determined, as described above during the pre-anoxia period. Those slices which, after
anoxia, produced a maximum evoked potential of greater than 50% of that observed prior
to anoxia were defined as surviving slices.
Results of the studies of Example 7A are shown in Figure 6. Figure 6 shows the
effects of Z-Leu-Abu-CO2Et, a Peptide Keto-Compound, and CI l relative to control slices
on survival of hippocampal slices exposed to 10 minutes exposure of anoxic atmosphere.
As seen in this figure, when the control slices are deprived of oxygen for 10 minutes in the
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absence of drug, virtually all fail to survive, as measured by their ability to elicit 50% of
their pre-anoxia evoked response. In accordance with this finding, few if any recover upon
reu~ ation. Figure 6 also shows that when CI1 or Z-Leu-Abu-C02Et are added to the
ACSF, the slices are protected from the effects of anoxia, evidenced by a substantial
~ropo~ lion of slices eliciting evoked potentials.
Finally, it can be seen that Z-Leu-Abu-CO2Et is cigrlifir~ntly more effective inprotecting against anoxia and preventing degradation of slices at the minim~l 1 hour
inf ub~tinn time, and at lower corl~Pntrations than CI1. This effect is believed to be due
to the superior membrane permeance of the Peptide Keto-Compounds.
Table 7A shows further data from the studies of Example 7A.
TABLE 7A
PERCENT OF SLICES SURVlVING TEN MINUTES ANOXIA
Compound Dose (uM) Incutation Time Survival
Control ---- 1 hour <1%
Leupeptin 1000 3 hours 50%
CI1 20n 2 hours 53%
SHC 20 1 hour 50%
HKP 50 1 hour 50%
PKC 100 1 hour 70%
It can be seen from the data in Table 7A that all of the Calpain Inhibitors tested
provide increased survival. SHC, a Substi~uted Heterocyclic Compound is ACITIC; HKP,
a Halo-Ketone Peptide, is Boc-Gly-Leu-Phe-CH2Cl; and PKC, a Peptide Keto-Compound,
is Z-Leu-Abu-C02Et. All are shown to be highly effective in influencing survival times.
Leu~ is seen to be the least effective neuroprotectant. Thus, we believe that ACITIC,
Boc-Gly-Leu-Phe-CH2Cl and Z-Leu-Abu-CO2Et are more effective in influencing survival
because of their membrane permeability. Accordingly, the results shown in Table 7A
support our belief that Calpain Inhibitors with membrane permeability are effective
neuroprotectants.
To further elucidate the ability of Calpain Inhibitors to provide neuroprotection
to hippocampal slices, and to provide a more quantitative indication of the membrane
permeability of these Calpain Inhibitors, we measured the effect of various Calpain
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Inhibitors on the evoked response on a single neuronal slice before during and after
anoxia. These studies are shown in Example 7B.
EXAMPLE 7B
Inhibition of Anoxic Damage
As in Example 7A, hippoc~mpal slices (400 ~m) were prepared from Sprague
Dawley rats (6-7 weeks) and m~int~ined in an interface chamber at 35C using
conventi- n~l techniques, i.e. the lower surface of the slice received a constant perfusion
(0.5 ml/min) of an artificial ce,c:blos~inal fluid (ACSF), while the upper surface was
exposed to a moist atmosphere of O2:CO2 (95%:5%) exchanged at a rate of 2 L/min. The
ACSF medium contains (in mM): NaCl (124), KCl (3) KHPO4 (1.25), MgSO4 (2.5), CaCl2
(3.4), NaHCO3 (26) and D-Glucose (10). Field excitatory post-synaptic responses were
recorded from stratum ra~ tllm of CAlb in response to stimulation of Schaffer-
cornmicsural fibers in CAla or CA1c. The depth of the recording electrode was
optimized and evoked responses were collected at a rate of one evoked response every 30
seconds.
After establishing a stable baseline of evoked responses (appr~ xi",ately 10
minutes), ACSF containing Calpain Inhibitor was washed into the chamber and slices were
incubated for a period of one hour. After incubation evoked responses were againrecorded and the change in the amplitude of the responses from baseline levels was noted.
No effect of the inhibitors tested on baseline evoked responses was observed.
For anoxia experiments, incubation in the drug-containing medium was followed
by replacement of the O2:CO2 (95%:5%) atmosphere with N2:CO2 (95%:5%). Slices were
exposed to this anoxic e"~/~ o",~,ent until disappearance of the pre-synaptic fiber volley and
for two minutes (severe anoxia) longer (total time in anoxic environment app~ i".~t~ly
7-8 minutes in control case). Effects of Calpain Inhibitors on the functional recovery of
the slices after the anoxic episode were then measured. Recovery of the evoked potential
(EPSP) slope and amplitude by the drug treated slices can be compared to control slices
to determine the relative efficacy of various Calpain Inhibitors.
Figure 7 shows the EPSP amplitude in millivolts for control CI1 treated and Z-
Leu-Abu-CO2Et (a Peptide Keto-Compound) treated hippocampal slices in the studies of
Example 7B. The periods of anoxia are represented by the black bars under the graph.
It can be seen in Figure 7 that the control slices deprived of oxygen in the absence of drug
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display a gradual reduction of EPSP and abruptly lose fiber volley activity about 5-6
minutes after the beginning of anoxia. Reoxygenation at or before this point leads to
co.,.plete functional recovery after about 20 minutes of reoxygenation, but reoxygenation
after this point does not. In the latter case the lecuvered EPSP slope and amrlittlde
become pro~.eisi~,ly reduced as the duration of anoxia post-fiber volley disappearance
(post-FVD) increases. After severe anoxia (2 minutes post-FVD), slices recover only 15%
of the EPSP slope.
In contrast to the control slices, recovery begins to occur shortly after the end of
anoxia for the treated slices. Figure 7 shows a comparison of the effects on EPSP
~mplitude produced in the presence of no inhibitor; the Peptide Keto-Compound, Z-Leu-
Abu-CO2Et and CI1. Z-Leu-Abu-CO2Et produces a recovery from severe anoxia superior
to that seen with CI1.
Figure 8 shows the percent recovery of EPSP from severe hypoxia using the
peptide ketoester Z-Leu-Phe-CO2Et and its corresponding peptide ketoamide Z-Leu-Phe-
CONH-Et. These studies were performed in a manner similar to that of Example 7B,except using a hypoxic environment in place of the anoxia of Example 7B. It can be seen
that use of the peptide ketoamide results in essentially complete (near 100%) recovery
from hypoxia while the peptide ketoester produces a partial recovery. The control slices
experienced little or no recovery.
An interesting characteristic that we have discovered for certain Calpain Inhibitors
is their ability to lengthen the period of exposure to anoxia required to produce fiber
volley disappearance (FVD). Typically, under control anoxia conditions, fiber volley
disappearance occurs in less than six minutes (Figure 9). The Peptide Keto-Compound,
Z-Leu-Phe-CO2Et, substantially lengthens the period of exposure to anoxia required to
produce FVD. This is an important advantage of the use of this Peptide Keto-Compound
for neuroprotection because slices can be expected to recover completely if reoxygenated
before fiber volley disappearance. Thus, treatment with this Peptide Keto-Compound is
expected to produce a greater percentage of recovery of cells from incipient
neurodegenerative conditions. It is believed that other representatives of the Peptide
Keto-Compounds as well as of other classes of Calpain Inhibitors also provide this effect.
Table 7B shows the perecentage of recovery of pre-anoxia synaptic tr~ncmic~ion
(evoked potential amplitude) of slices treated with various Calpain Inhibitors or of control
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slices. All of these slices were exposed to ten minutes of anoxia according to the protocol
of Example 7B.
TABLE 7B
PERCENT RECOVERY OF SYNAPTIC TRANSMISSION AF~ER ANOXL~
S Compound Concentration ~0 Recovery
Control --- 15
CI1 200 35
SHC 20 60
HKP 50 30
PKC-1 100 38
PKC-2 100 55
The results shown in Table 7B provide further evidence that the peptide aldehyde,
CI1, as well as the Substituted Heterocyclic Compounds (SHC) represented by ACITIC,
Halo-Ketone Peptides (HKP) rc~re;,ented by Boc-Gly-Leu-Phe-CH2Cl, and Peptide Keto-
Compounds (PKC) represented by Z-Leu-Phe-CO2Et (PKC-1) and Z-Leu-Abu-CO2Et
(PKC-2) are sufficiently membrane permeant to provide neuroprotection through Calpain
inhibition.
CI1, which is at least partially membrane permeant, produces some effect, however,
does not signifir~ntly lengthen the period of anoxia required to suppress electrical activity.
For example, see Figure 9. Thus, compared to control, or even compared to leupeptin and
CI1, the Substituted Heterocyclic Compounds, Peptide Keto-Compounds and Halo-Ketone
Peptides can increase the degree of recovery after anoxic episodes while producing the
additional advantage of extending the amount of time slices can tolerate anoxia and
thereby recover completely.
An important effect of the Peptide Keto-Compounds and other membrane
permeant Calpain Inhibitors is that they are ~i~nifi~ntly more effective in lower doses
than less permeable Calpain Inhibitors such as CI1. Although CIl is shown to be at least
somewhat membrane permeant due to its ability to affect slice survival, the moremembrane-permeant inhibitors provide signifir~ntly increased protection. Thus, the more
highly membrane-permeant Calpain Inhibitors are believed to be especially effective in
treating and inhibiting neurodegeneration.
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The results of the studies of Examples 7A and 7B show that the Substituted
Heterocyclic Compounds, Peptide Keto-Compounds and Halo-Ketone Peptides are
membrane-permeant Calpain Inhibitors which are believed to be especially effective in
treating and inhihiting neurodegeneration. The results also show that Peptide Keto-
Compounds, and perhaps representatives of other classes, can extend the duration of
anoxia required to suppress electrical activity in hippoc~mp~l slices. As discussed above,
these effects are important advantages of these compounds.
8. in vivo Neuroprotection by Calpain Inhibitors
As ~ cced above, therapeutics useful for influencing the function of cells within
the CNS must cross the BBB to reach their targets within the CNS. Non-BBB permeant
compounds might, in addition to the brain infusion techniques described above, be
a~lminictered via intraventricular administration, but this also severely limits their
usefulness in practice. In order to test the in vivo effectiveness of the Calpain Inhibitors
to cross the BBB and become therapeutically useful, we tested the ability of intraperitoneal
injection of the Calpain Inhibitors to protect against excitotoxic damage in vivo. Protection
was measured by evaluating changes in behavior of rats after injection with kainate. These
studies are shown in Example 8A.
EXAMPLE 8A
Protection A~ainst Behavioral Changes from Excitotoxic Damage hy Peripherally
Administered Calpain Inhihitors
~ats (male Sprague-Dawley, 200i5 gms) were injected intraperitoneally with
12mg/kg kainic acid in sallne vehicle and either 200~l DMSO (dimethylsulfoxide) or
4.6mg calpain inhibitor dissolved in the same volume of DMSO. The rats were
observed for six hours following the injections and the kainate-induced behavioral
symptoms and convulsions scored on a scale of 0-6 (0=no symptoms; 1=wet dog
shakes; 2=salivation and chewing; 3=at least one convulsive episode; 4=repeated or
sustained convulsions; 5=convulsions, including rearing and falling; 6=convulsions
followed by death within the 6 hrs post injection).
Figure 10 shows the effects of CIl on the behavioral and convulsive effects of
- 30 kainic acid. In the control group, over half the animals showed symptoms greater than
mild behavioral symptoms, and many exhibited overt convulsions, presumably reflecting
seizure activity within the brain. Unexpectedly. in the inhibitor treated group, the
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in~ n~e and severity of convulsions was reduced. Thus, this data suggests that
Calpain Tnhihjtors have an anti-convulsive effect. This effect is a distinct advantage in
the use of Calpain Inhibitors in epilepsy-related neurodegenerative conditions and in
stroke, which is often ac~ompanied by seizures.
In order to more clearly demonstrate that the behavioral and anti-convulsive
effects seen with the Calpain Inhibitors result from inhibition of Calpain we tested the
brain tissues of the rats from Example 8A for accumulation of spectrin BDP's. Ascu~sed above, these BDP's are associated with Calpain activity and with the
neurodegeneration ~ccori~ted therewith.
EXAMPLE 8B
Protection Against Sr)ectrin Breakdown from Excitotoxic
Damage by Peripherally Administered Calpain Inhibitors
Four days following the injection of kainate in the rats from Example 8A, the
brains of the rats were removed and assayed for spectrin BDP's. Spectrin BDP's were
assayed by homogenizing brain parts in 20mM Tris pH = 7.2, .32M sucrose, 50~M Ac-
Leu-Leu-nLeu-H on ice. Homogenates were mixed l:l with lO~o SDS, 5%
B-mercaptoethanol, 10% glycerol, 10mM Tris pH=8.0, 0.5% bromophenolblue, heated
to 95C, and subjected to electrophoresis in 4-l/2% polyacrylamide gels. The proteins
in the gels were transferred to nitrocellulose and the spectrin and BDP's detected using
a rabbit polyclonal anti-spectrin antibody and established immunodetection methods.
The amount of spectrin and BDP's in each sample was quantitated by densitometricsc~nning of the developed nitrocellulose.
Figure 11 shows the results of Example 8B. It can be seen that kainate
stimulated the breakdown of spectrin in both Calpain Inhibitor treated and control rats.
However, treated rats exhibited significantly less BDP's. These results verify that
Calpain activity in the brains of the treated rats was reduced. An unexpected
observation was that even those treated animals that exhibited severe seizures had
~ignifi~ntly less spectrin breakdown than untreated animals subjected to kainate. Thus
Calpain Inhibitor treatment reduced both the behavioral/convulsive effects of kainate
and the activation of calpain in the most severely affected ~nim~l.c
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9 C~
All of the foregoing studies support our discovery that Calpain Inhibitors
provide in vivo protection against neurodegeneration associated with anoxia,
excitotoxicity and other causes. Thus, these Calpain inhibitors possess neuroprotective
activity against a variety of in vivo neurodegenerative diseases and conditions, including
excitotoxicity, HIV-induced neuropathy, ischemia following denervation or injury,
subarachnoid hemorrhage, stroke, multiple infarction dementia, Alzheimer's Disease
(AD), Huntington's Disease, Parkinson's Disease, surgery-related brain damage and
other pathological conditions.
Those Calpain Inhibitors which possess significant Calpain Inhibitory activity in
vitro and also meet at least one of the foregoing or different tests for membrane
permeability are excellent c~n~ tes for treatment of neurodegeneration.
G. TREATMENT OF NON-NEUROLOGlCAL CONDITIONS
A number of medical conditions associated with increased Calpain activity can
be treated with the Calpain Inhibitors of the present invention. These Inhibitors are
a~lminictered to a m~mm~l having a medical condition which is caused at least in part
by the proteolytic activity of Calpain. Specific medical conditions are described below
which benefit from the administration of Calpain Inhibitors.
1. Treatment of Cardiac Muscle Tissue Damage
Damage to the cardiac muscle tissue of a mammal can be slowed or prevented
by the administration of Calpain Inhibitors. To treat a mammal, such as a human
patient, who has cardiac muscle tissue damage with Calpain Inhibitors, that patient is
first identified by screening patients for those with symptoms of having cardiac tissue
damage. Examples of such patients include those who have experienced heart attacks.
2~ Other groups likely to have experienced cardiac tissue damage include victims of
violent assault whose thoracic cavities have received a physical insult, as well as those
who have suffered from viruses or other pathogenic agents known to attack the heart
muscle. After identifying such people, routine tests, such as ultrasound and magnetic
resonance im~ging, are then used to determine whether or not they actually have
suffered cardiac muscle tissue damage.
Calpain Inhibitors can be administered to people with damaged myocardial
tissue in a number of ways. The most direct method of administration is the injection
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of up to a liter of a Calpain Inhibitor solution directly into the damaged tissue, where
the Calpain Inhibitor is at a concentration in the range of between 0.001 mg/ml and 10
mg/ml, and preferably 0.01 mg/ml, of a Calpain Inhibitor. Any pharm~ceuti~lly
acceptable carrier vehicle may be used to carry the Calpain Inhibitor. This method of
aflmini~tration is normally used only when the myocardium is exposed and undergoing
treatmçnt, as during surgery. The direct infusion of a Calpain Inhibitor into the heart
in the foregoing concentrations with a catheter, or the injection of such a Calpain
Inhibitor into the pericardial space, in order to achieve the local administration of the
Inhibitor is another way of treating the heart muscle when tissue damage is occurring.
The intravenous or intramuscular administration of a Calpain Inhibitor is
preferred, however, when a patient is not undergoing surgery or other invasive
treatment. A Calpain Inhibitor in a pharm~reutirllly acceptable solution is injected
once daily in a dosage of between app,uAil.,ately O.OOl mg/kg of bûdyweight and 100
mg/kg, and preferably between O.Ol mg/kg and 10 mg/kg, to treat cardiac muscle tissue
damage. More preferably, a Calpain Inhibitor is administered several times per day at
approprlately reduced dosages. More preferably still, a Calpain Inhibitor is infused
slowly into a patient by drip infusion, to ensure that the Calpain Inhibitor is present in
the bloodstream in a relatively constant concentration. Of course, oral formulations of
a Calpain Inhibitor can also be administered, and other methods of administration
known to the art can be used as well.
Calpain Inhibitors can also be used to treat a mammal at risk for suffering
damage to that m~mm~l's cardiac tissue. Thus, myocardial infarctions can be
prevented or decreased in size through the administration of a Calpain Inhibitor to
such a m~mm~l A mammal, such as a person, at risk for suffering damage to its
myocardial tissue is identified by screening a population for people with symptoms that
indicate a higher-than-average risk for suffering a heart attack, including shortness of
breath, obesity, high blood pressure, and high levels of cholesterol in the blood.
Preferably, people who have been diagnosed as having cardiac ischemia, who have
experienced a mild heart attack, or who have other symptoms which indicate that they
are at risk for suffering a serious heart attack in the near future are identified. Those
at risk for suffering cardiac tissue damage can be at least partially protected from such
damage by taking Calpain Inhibitors prophylactically. The screening of a population
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for people who would benefit from Calpain Inhibitor therapy is normally done by a
physician in the course of eY~mining that physician's patients, but might also be done
through a health screening program sponsored by an employer or a school.
- Once an individual who would benefit from the administration of a Calpain
S Inhibitor has been identified, an Inhibitor can be administered in a number of ways.
One method of administration is to inject an Inhibitor only once at a relatively large
dosage in order to treat a person suffering from acute myocardial ischemia (a heart
attack). In this case, between appluxil.,ately 0.001 mg/kg and 100 mg/kg, and
preferably between 0.01 mg/kg and 1 mg/kg, of an Inhibitor suspended or dissolved in
an appio~liate pharm~ceutir ll carrier can be injected intravenously into such an
individual. In this way, the acute damage to the myocardium suffered by heart attack
victims can be avoided or reduced.
Inhibitors can also be injected several times over the course of a period of time,
or they can be infused intravenously at a steady rate for a period of time in order to
protect an individual at risk for suffering cardiac tissue damage. Individuals are
aflmini~t~red between a~pluAi"lately 0.001 mg/kg and 100 mg/kg, and preferably
between 0.01 mg/kg and 10 mg/kg, of a Calpain Inhihitor daily. This route of
a~lminictration is preferred for individuals who are at high risk for suffering a
myocardial infarction in the near future, and such administration may be continued as
long as such a risk of having a heart attack remains.
In addition, other methods of administering a Calpain Inhibitor are possible to
protect an individual from myocardial tissue damage. The oral administration of a
Calpain Inhibitor in tablet or liquid form is preferable for long term Calpain Inhibitor
therapy, because such routes of administration are easier for an individual to
a-lminictçr to him or herself. Between ~ppluxil-,ately 0.001 mg/kg and 100 mg/kg, and
preferably between 0.01 mg/kg and 10 mg/kg, of a Calpain Inhibitor is administered
daily in this form of Calpain Inhibitor therapy.
EXAMPLE 9
The effect of a Calpain Inhibitor is tested on rabbits by inducing a region of
ischemic myocardium and atlmini~tering a Calpain Inhibitor. Cardiac ischemia is
induced in rabbit myocardium by ligating the coronary artery followed by ligation of the
branches of the left circumflex artery adjacent to the ischemic (cyanotic) area when the
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epicardial cyanotic area reaches 0.75 to 0.80 of the length of the long axis of the left
ventricle (measured between the atrioventricular groove and the cardiac apex). This
produces a coronary infarct of regular size. A Calpain Inhibitor is administeredintravenously at 10 mg/kg/hour for two hours beginning either 5 minutes before or just
after the ligations. In both cases, groups treated with a Calpain Inhibitor exhibit
smaller infarcts than those treated with a pharmaceutical carrier alone without a
Calpain Inhibitor.
EXAMPLE 10
A human diagnosed as having suffered a mild heart attack is prescribed an oral
formulation of a Dipeptide a-Ketoamide (Subclass A) Calpain Inhibitor providing 2
mg/kg of the inhibitor. The formulation is taken once per day to protect the individual
in the event he or she suffers a further heart attack.
EXAMPLE 11
A human who has been picked up by an emergency team within several minutes
of suffering a heart attack is a~mini~tered an injectable composition providing 10
mg/kg of a Dipeptide a-Ketoamide (Subclass B) Calpain Inhibitor. In this way
permanent tissue damage to the myocardial tissue is avoided.
2. Tre? : t of Skeletal Muscle Tissue Damage
Damage to muscle tissues can also be prevented or reduced through the
administration of Calpain Inhibitors. Research has shown that Calpain is involved in
the degeneration of muscle tissues. During the autolysis of muscle fibers, for example,
Calpain degrades the Z-disc of skeletal muscle myofibrils. Calpain also degradesmyosin, a muscle protein, over a wide range of protease concentrations. Since Calpain
is activated by elevated intracellular levels of calcium, any rise in such levels can lead
to damage to a muscle cell. Any condition in which intracellular calcium levels become
elevated, therefore, can be treated with a Calpain Inhibitor in order to prevent or limit
the damage to skeletal muscle tissue due to the activity of Calpain.
Calpain Inhibitors can be used to treat skeletal muscle tissue damage caused by
a variety of factors. A physical insult to skeletal muscle tissue that damages the muscle
cell membrane and results in increased intracellular levels of calcium, for example, can
be treated with a Calpain Inhibitor, which blocks the proteolysis of muscle cell
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constituents. Muscular dystrophy, a condition characterized by elevated intracellular
calcium levels, can also be treated with Calpain Inhibitors.
Identifying an individual with skeletal muscle tissue damage is normally done bya ~l.y~ic;an. A physician, for example, can identify an individual with the clinical
symptoms of muscular dystrophy. Other damage to skeletal muscle tissue can likewise
be ~ gnosetl
As with the treatment of myocardial tissue, the route of administration of
Calpain Inhibitors to skeletal muscle tissue will vary, depending on the site of the
damaged tissue. An injury to the bicep is most directly treated through an
intramuscular injection of a Calpain Inhibitor into the bicep muscle of the arm, the site
of injury. A more systemic condition, such as muscular dystrophy, however, is better
treated through a systemic injection of a Calpain Inhibitor. Such an injection can be
inlramuscular, intraperitoneal, or intravenous. Alternatively, the oral administration of
Calpain Inhibitors can effect systemic administration of such Inhibitors.
EXAMPLE 12
An individual is diagnosed with Duchenne's muscular dystrophy by a physician.
To treat that condition, between applu~i",ately 0.1 m ,/kg and 10 mg/kg of a Calpain
Inhibitor in a phosphate-buffered saline solution is injected intravenously into the
individual once per day for the course of the treatment.
3. T~ of Smooth Muscle Injuly
Calpain is involved as well in the breakdown of smooth muscle cells. It has
been reported that certain smooth muscle cell proteins, such as calponin, are degraded
by Calpain. As indicated by studies of the proteolytic proclivity of Calpain in other
tissues, Calpain poses a threat to smooth muscle cells whenever those cells experience
elevated intrac~ r calcium levels. In particular, mammals which have experiencedphysical damage to their smooth muscle tissue or whose blood circulation to their
smooth muscle tissue has been cut off would benefit from the administration of Calpain
Inhibitors. Prophylactic administration of such Inhibitors reduces the damage done by
such conditions. Administration of Calpain Inhibitors after smooth muscle tissue has
been damaged, however, is also beneficial since such Inhibitors can slow or stop the
plo~;~ess of the proteolytic activity of Calpain in such tissues.
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The identification of an individual with smooth muscle tissue damage, such as
an intestinal ulcer, is normally done by a physician. After diagnosing an individual as
having damage to her smooth muscle tissue, the physician can give a Calpain Inhibitor
to that individual to slow further damage to such smooth muscle tissue
The administration of a Calpain Inhibitor to smooth muscle tissue can be
accomplished by any method known to the art. While intramuscular injection is a
possible route of delivery, in most cases intravenous, intraperitoneal, or oral delivery
will be preferred, depending on the location of the damaged tissue. For example,someone with an ulcer is more effectively treated with an oral delivery of a Calpain
Inhibitor, since the Inhibitor can then coat the affected area directly in the course of
passing through the digestive system. lntravenous delivery can also be used, however,
and would be preferable if the Inhibitor is itself upsetting to the digestive system of the
individual or if the low pH of the stomach interferes with the effectiveness of the
Inhibitor. Between a~p,uxi",ately 0.001 mg/kg and 100 mg/kg, and preferably between
0.01 mg/kg and 10 mg/kg is used to treat smooth muscle tissue damage.
EXAMPLE 13
An individual is diagnosed as having an ulcer by her physician, and is prescribed
a Calpain Inhibitor along with other ulcer medications. App, oxh"ately 2 mg/kg of the
Calpain Inhibitor is administered orally in tablet form to the individual with every meal
as long as the individual is experiencing the ulcer.
4. Tr.~- I of Smooth Muscle Contraction
The tonic contraction of smooth muscle in appropriate circumstances is a
normal process. However, in inappropriate circ~-mct~ncec tonic contraction can lead to
serious pathological conditions. For example, contraction of the bronchial smooth
muscle leads to the shortness of breath and other symptoms of asthma. Contraction of
the coronary artéries can lead to angina, partial coronary hypoxia and subsequent loss
of coronary function. Contraction of the smooth muscle in cerebral arteries can lead to
cerebral v~co~p~m and hypoxia of the brain tissue, a serious condition that can leave
patients mentally disabled and permanently brain damaged.
Angina generally results from both an occlusive and a spastic component. In
some patients, angina is largely a result of either the occlusive or spastic component.
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Advantageously, the Calpain Inhibitors of the present invention can be used to treat
both co",yone.lts of angina.
V~cosp~cm is a condition which affects smooth muscles, particularly blood
vessels. V~cocp~cm is a sllct~ined spastic or tonic contraction of the vascular tissue.
Such contractions are associated with a rise in intracellular calcium levels. Calpain
activity has been linked to these contractions, and contracted blood vessels can be
dilated with Calpain Inhibitors.
An individual experiencing vacocracm is normally identified by a physician. A
physician might, for example, detect v~cospacrn during surgery, or might detect
vasospasm indirectly through the observation of external symptoms. Vasospasm is also
often detected by angiogram.
Vacos~ frequently occur as a result of subarachnoid hemorrhage, which
causes blood dots. Such blood clots are believed to provide factors that promotevacosr~cm. Such hemorrhaging is therefore an indication that an individual is at risk
for v~cosp~cm
It is one of the surprising discoveries of the present invention that Calpain
Inhibitors can be used to block the establishment of the tonic state, or to relax tonically
contracted smooth muscle. We have found Calpain Inhibitors of several classes to be
particularly useful in this regard, including the Substituted Heterocyclic Compounds,
Peptide Keto-Compounds, Peptide Aldehydes, Halo-Ketone Peptides. Various
sub~l~cces of compounds within these broad classes that we have found to be useful in
the inhibition of tonic smooth muscle contraction include the peptide ketoamides,
chloromethyl ketone peptides, epoxysuccinates, diazomethane peptides and peptidealdehydes.
The Calpain Inhibitors of the present invention provide a number of advantages
in the treatment of smooth muscle contraction. These inhibitors provide unexpectedly
high efficacy in the treatment of smooth muscle contraction, such as in the treatment of
v~cosracm disorders of many types. Furthermore, we have shown that Calpain
Tnhihitors of the present invention are additionally beneficial because they have
relatively little effect on resting tension in smooth muscle. Thus, we consider these
Calpain Inhibitors to be particularly useful in the treatment of human subjects without
adverse side effects.
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The ~dmini~tration of Calpain Inhibitors to prevent smooth muscle tonic
contraction or to relax tonically contracted smooth muscle can be by any of a number
of methods known to those of skill in the art. Such methods include the systemicdelivery of Calpain Inhibitors through intravenous, intraperitoneal, or intramuscular
injection, or through oral delivery, as described hereinbelow. Administration of the
Calpain Inhibitor for this purpose can also be through use of a catheter system such as
will be readily known to those having skill in the art. A solution of Calpain Inhibitor
can be injected directly into the ce-elJlu~pinal fluid. Aerosolization of solutions
cont~ining a Calpain Inhibitor is a preferred mode of administration. Finely dispersed
dry powders can also be used successfully. Other known methods of delivery are also
acceptable. Thus, formulations including Calpain Inhibitors can be of many forms,
in~ ine tablets, troches, solutions, powders and the like, as described herein.
In order to induce dilation of a spastic or tonically contracted blood vessel and
thereby reverse v~o~p~cm, a Calpain Inhibitor can be administered by direct topical
application to the blood vessel. This method, of course, necessitates the exposure of
the blood vessel so that it can be physically manipulated, and thus requires surgery. A
Calpain Inhibitor at a concentration of approximately 1 - 500 ~lM can be topically
applied to achieve vasodilation.
However, when it is desired to treat vasospasm without surgery, a Calpain
Inhibitor can be ar~mini~tered intravenously. In this event, between appluxilllately
0.001 mg/kg and 100 mg/kg, and preferably between 0.01 mg/kg and 10 rrig/kg, of a
Calpain Inhibitor suspended or dissolved in a pharm~ceutic~lly acceptable carrier is
adminictered once to an individual. The oral administration of a Calpain Inhibitor in
like amounts is also possible, although this route of administration would not be as fast-
acting as intravenous administration.
For inhibition of other tonic smooth muscle contractions, between
al~L,luAi,nately 0.001 mg/kg body weight and 100 mg/kg body weight of a Calpain
Inhibitor can be a-lminictered daily, divided into one to eight doses, or via continuous
infusion intravenously. More preferably, the daily dosage to an individual to prevent or
relax tonic smooth muscle contraction would provide between 0.01 mg/kg to 10 mg/kg
body weight. Optimum dosages can be determined for each particular Calpain
Inhibitor using techniques known to those having ordinary skill in the art.
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Higher concentrations of Calpain Inhibitors can be administered through the
direct application of such Inhibitors to the smooth muscle tissue. For example, the
Calpain Inhibitor can be loaded into a microsphere and the microsphere driven into
the smooth muscle to effect direct application. For the relaxation of spastic arteries,
the artery can be surgically exposed and a solution of Calpain Inhibitor applied directly
to the artery. Calpain Inhibitors can also be delivered through the use of a slow-
release compound, such as a gel or ointment, applied directly to the smooth muscle
tissue during surgery. Local a~ nictration strategies include inhalation formulations
for use in treating bronchospasm.
In the following examples, certain specific Calpain Inhibitor compounds were
tested in order to verify the results of the present invention. These drugs are
specifically iclentified hereinabove in the Brief Description of the Figures, upon
reference to the figures referred to in the Examples. Drugs A, B, F (the same drug as
B), G, H, J and CX are all Peptide Keto-Compounds that are inhibitors of Calpain.
Drug H is another inhibitor of Calpain. Drugs C, E and I are not inhibitors. Drug D
is a relatively poor inhibitor that is a compound of the Halo-Ketone Peptide class.
EXAMPLE 14
Isolated arteries in vitro were treated with 10-~M endothelin (ET) to induce
contraction of the smooth muscle. The arteries were then treated with Calpain
Inhibitor at a concentration of between 10-7M and 104M. The results of this procedure
are shown in Figures 12 and 13 for Calpain Inhibitors A-J, as described hereinabove in
the Brief Description of the Figures. These Figures show that administration of a
Calpain Inhibitor can effectively reduce endothelin-induced contraction of isolated
arteries in vitro.
EXAMPLE 15
Isolated arteries in vitro were treated with 10-7M phorbol dibutyrate (PDB) to
induce contraction of the smooth muscle. The arteries were then treated with
compounds E-J (as described hereinabove in the Brief Description of the Figures) at a
. concentration of between 10~M and 104M. The results of this procedure are shown in
Figure 14. This Example demonstrates that administration of a Calpain Inhibitor in
accordance with the present invention can effectively reduce PDB-induced contraction
of isolated arteries ill vitro.
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EXAMPLE 16
Isolated arteries in vi~ro were treated with Calpain Inhibitors E-J (as described
hereinabove in the Brief Description of the Figures) at concentrations of 3x10~M,
10-sM, and 3x10-sM. The tension in the arteries, in mg, was then measured. The
results are shown in Figure 15. This example verifies that Calpain Inhibitors of the
present invention have relatively little effect on resting tension in smooth muscle.
EXAMPLE 17
The protective effect of Z-Leu-Phe-CONH(CH2)3 on the acute phase of
v~cosp~.~m was evaluated in live rabbits. Arterial contraction measured for
ay~luAilllately 10 minutes in order to establish a baseline. Z-Leu-Phe-CONH(CH2)3 at
concentrations ranging from 100 IlM to 300 ~M, or vehicle was then administered to
the ~nim~ At a time appluAilllately 60 minutes after establishment of a baseline,
oxyhemoglobin was administered in order to induce constriction. Figure 16 shows that
higher concentrations of Calpain Inhibitor resulted in less constriction, with virtually no
cons~ ion occurring in the animal receiving the Calpain Inhibitor at 300 ~-M.
Constriction was reversed by a~lminic~ration with aCSF.
EXAMPLE 18
Figure 17 provides an example of the effect of a Calpain Inhibitor of the
present invention on an artery in a live animal that was constricted by subarachnoid
hemorrhage (SAH). The "normal" resting size of the vessel, as estimated from an age-
matched control animal, was apptoAilllately 750 ~m. The vessel was constricted to
a~pl~Y;,..~tPIy 400 ~m following hemorrhage. Perfusion of aCSF alone for 90 minutes
had no effect. Z-Leu-Phe-CONH(CH2)3 at 100 ~LM reversed the SAH-induced
constriction by app~ ;,.,m~tely 60% to about 600 ~m.
EXAMPLE 19
Three rabbits were subjected to SAH resulting in constriction of cerebral
arteries. Z-Leu-Phe-CONH(CH2)3 was then administered to produce a concentration
of 100~M and the amount of relaxation measured. Figure 18 shows the summary of
data from all three ~nim~l~ In all three, Z-Leu-Phe-CONH(CH2)3 reversed
constrictions induced by SAH. The constricted diameter is taken as the "100% value"
in this graph (unlike in Figure 16) and the relaxation is expressed as a percentage of
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the 100 % value. The data were analyzed in this fashion because the absolute amount
of consstriction following SAH varies from animal to animal.
EXAMPLE 20
A human diagnosed as suffering from tonic smooth muscle contraction is
S intramuscularly adn inictered ap,u,uAi.. ately 10 mg of a Calpain Inhibitor, such as Z-
Leu-Phe-CONH(CH2)2Ph, in a phosphate buffered saline solution by intravenous
injection at least once per day for appru~il..ately one week or until it is determined that
the risk of tonic contraction has subsided.
EXAMPLE 21
A human diagnosed as suffering from angina associated with coronary artely
~co~ cln is orally ar~rninicttored appro"ir..ately 100 mg of Z-Leu-Phe-CONHEt. The
Calpain Inhibitor is delivered by surgically exposing the artery and applying a solution
of Calpain Inhibitor in a phosphate buffered saline solution directly to the artery.
EXAMPLE 22
A human diagnosed as suffering from asthma associated with bronchospasm is
a~iminictered between appr..~;."~t~ly 100 mg of (Ph)2CHCO-Leu-Abu-CO~H-
CH2CH(OH)Ph by inhalation. The Calpain Inhibitor is delivered by inhalation of aformulation cont~ining the Calpain Inhibitor directly into the patient's lungs.
EXAMPLE 23
A human diagnosed a suffering from cerebral vasospasm is administered
~ppru~i...ately 100 mg of Z-Leu-Phe-CONHEt into the CSF. The Calpain Inhibitor is
delivered by injecting said Calpain Inhibitor in a phosphate buffered saline solution
directly into the patient's ce[el),o~,inal fluid.
EXAMPLE 24
During surgery, a blood vessel is discovered to be experiencing tonic smooth
muscle contraction. A solution containing a 100~.M solution of a Calpain Inhibitor is
topically applied to the contracted blood vessel. If one application fails to produce full
dilation of the blood vessel, further topical applications are performed.
EXAMPLE 25
Cerebral vacosp~crn in an individual is detected, and a solution containing
approximately 2 mg/kg of a Calpain Inhibitor is dissolved in phosphate buffered saline
and then injected intravenously into the individual.
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EXAMPLE 26
We tested the ability of several Calpain Inhibitors to dilate ex vivo blood vessels
treated to induce vaco.~l.?c.... We found that Calpain Inhibitor-1 provided low activity
in indu~ing v~codil~tion, while at least three Peptide K~.to~mide Calpain Inhibitors
within the scope of the present invention provided appreciably greater activity. We
found that highly lipophilic compounds were particularly effective. Thus, we found that
each of Z-Leu-Phe-CONH(CH2)2C6Hs, Z-Leu-Phe-CONHEt, and Z-Leu-Abu-
CONHEt were more effective than Calpain Inhibitor-I; however these compounds arelisted in decreasing order of effectiveness.
5. Tre~ ~ of Hypertenci-- Related Injury
The activity of ~alr~cta~in, a natural inhibitor of Calpain, is significantly reduced
in the erythrocytes of individuals who have hypertension. The activity of Calpain in
such cells concomitantly increased, and both functional and structural lesions have been
observed in the erythrocytes of hypertensive mammals. Calpain Inhibitors can
therefore be adminictered to a hypertensive m~mmal in order to counteract the
harmful effects of hypertension on such a mammal's erythrocytes caused by the
increased proteolytic activity of Calpain in such cells.
An individual with hypertension is diagnosed by a physician, and in conjunction
with other therapies to lower that individual's blood pressure a Calpain Inhibitor is
adminictered to that individual. Approximately I - 10 mg/kg of a Calpain Inhibitor is
administered to the individual daily, preferably in an oral formulation for ease of
adrninictration.
EXAMPLE 27
An individual is diagnosed as having hypertension. Approximately 2 mg/kg of a
Calpain Inhibitor is adminictered daily in tablet form to that individual until the
individual's blood pre~ e returns to a normal range.
6. Tre~ of Cataracts
Calpain has been implicated in the causation of cataracts. In a murine model of
cataractogenesis, Calpain was found to have a very high activity in the lenses of mice
just before visible cataracts appeared. The activity of Calpain then decreased as
cataracts formed on the lenses of such mice. Other indicators of Calpain activity
include the fact that the concentration of calcium increases markedly in the lenses of
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such mice as cataracts begin to develop. Proteins similar to the il2 vitro reaction
products of the degradation of the lens protein crystallin by Calpain are also found in
the lenses of such mice.
The formation of cataracts is linked to a number of external and internal
factors, int~lurling age, diabetes, hereditary t~ aceC UV radiation, drugs such as
steroids, and toxic cherni~ Calpain Inhibitors can protect people exposed to agents
known to cause cataracts, or who show signs of developing cataracts, through the~rlrnini~tration of a Calpain Inhibitor to such people before the development of a
cataract.
Calpain Inhibitors can also be administered to people who have not developed
fully mature cataracts, in order to halt or slow the progress of cataract development.
Such treatments can potentially save millions of dollars in medical care costs. In one
study, it was estimated that if cataract development in individuals could be slowed for
10 years or more, over $600 million (in 1973 dollars) could be saved in annual medical
care costs in the U.S. alone.
A person having a cataract who would benefit from treatment with a Calpain
Inhibitor is easily identified by a physician trained in diagnosing cataracts. Such a
person is then treated through the administration of Calpain Inhibitors to slow or halt
the progress of cataract formation.
Individuals who would benefit from protection from cataract formation using
Calpain Inhibitors can also be identified by a physician. For example, patients whose
families have a history of developing cataracts are candidates for treatment with
Calpain Inhibitors. Preventative therapy with Calpain Inhibitors is also indicated for
people with diabetes or who are regularly exposed to agents. such as steroids, which can
cause cataracts.
The administration of Calpain Inhibitors for the treatment or prevention of
cataracts can be by any of a number of methods. Cataracts can be treated by the
systemic delivery of Calpain Inhibitors through intravenous, intraperitoneal, orintramuscular injection ur through oral delivery. Between ap,uluAi~ately 0.001 mg/kg
and 100 mg/kg of a Calpain Inhibitor is administered daily to an individual to prevent
or slow cataract development.
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Higher concentrations of Calpain Inhibitors can be delivered, however, through
the direct application of such Inhibitors to the eyes. Direct application can be done
either through the injection of Calpain Inhibitors into the eye, or through the topical
~pplir~tion of Calpain Inhibitors which have been suspended in or miAed with eyedrops, ointments, or solutions which are then placed on the eye. Calpain Inhibitor
solutions can also be soaked into contact lenses which then deliver a Calpain Inhibitor
to the eye slowly over time. Between appluAil"ately 0.00001 mg/kg and 1 mg/kg, and
preferably between 0.0001 mg/kg and 0.1 mg/kg, are administered daily in such direct
applications.
EXAMPLE 28
An elderly individual with partially developed cataracts is treated with eye drops
containing Calpain Inhibitors. One drop in each eye is administered twice daily. The
eye drop solution is formulated so that each drop contains between 0.001 mg/ml and 1
mg/ ml of a Calpain Inhibitor.
EXAMPLE 29
An individual whose family has a history of cataract development is
~lminictered a Calpain Inhibitor in tablet form to be taken once daily. Each tablet
contains approxi."ately 2 mg of a Calpain Inhibitor ~pproxi",ately per kilogram of
bodyweight.
7. Tl 2nt ~ ~ of Restenosis
When blood vessels become blocked by arterial plaques and fatty deposits,
therapeutic angioplasty can be used to open such stenotic regions. One of the most
commonly used angioplasty procedures, coronary balloon angioplasty, makes use of a
catheter which has an inflatable balloon at its distal end. In this procedure, the
catheter is inserted into the arterial lumen, and the distal end of the catheter is guided
to the stenotic region. Once positioned within the stenotic area, the balloon iseApanded in order to flatten the arterial plaque against the wall of the vessel.Other types of mecll~nic~l procedures for opening stenoses within the
v~cr~ lre have also been used. These include the use of lasers or atherectomy
devices to remove occlusions. Similar procedures for mechanically opening stenoses
are also performed in heart valves (valvuloplasty) and peripheral vessels (peripheral
angioplasty).
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Smooth muscle cell proliferation of the vascular wall is a normal response to
various physiological stimuli, inr~ ling those ~cso~ ted with procedures for
me~h~ni-~lly opening stenoses. Such stimuli can trigger the proliferation of smooth
- muscle cells and the migration of these cells to the vascular subintima, where they
S continue to proliferate. Smooth muscle cell proliferation is normally a desirable
process, one in fact that is often nec.osc~ry for healing, but which is not necessary for
recovel y from angioplasty.
However, following therapeutic angioplasty for the opening of obstructed
arteries, the proliferation of smooth muscle cells can result in restenosis and blockage
of the opened artery. As many as 50% of the patients who undergo successful coronary
angioplasty can develop recurrent coronary artery obstructions following the procedure
due to such rest~no~i~ It would therefore be of great medical value to be able to
inhibit smooth muscle cell proliferation in order to prevent restenosis following
angioplasty.
It is one of the surprising discoveries of the present invention that Calpain
InhibitGrs can be used to inhibit smooth muscle cell proliferation and thereby prevent
restenosis following angioplasty. A class of Calpain Inhibitors which are particularly
useful in this application are the Peptide Keto-Compounds. These inhibitors are potent
inhibitors of calpain. For example, Z-Leu-Phe-CONHEt inhibits the proliferation of
cultured bovine smooth muscle cells with an IC50 of around l50 IlM. Ph2CHCO-Leu-Abu-CONH-CH2CH(OH)Ph has been found to inhibit the proliferation of cultured
bovine smooth muscle cells with an IC50 of around 60 ~lM. Other Calpain Inhibitors
which are effective in preventing restenosis can be determined by routine
experimentation, such as through thymidine incorporation into cultured bovine aortic
smooth muscle cells. Additional Calpain Inhibitors believed to be particularly effective
in this regard are the Halo-Ketone Peptides, and the Substituted heterocyclic
Compounds.
The administration of Calpain Inhibitors to inhibit smooth muscle cell
proliferation and to prevent restenosis can be by any of a number of methods known to
those of skill in the art. Such methods include the systemic delivery of CalpainInhibitors through intravenous, intraperitoneal, or intramuscular injection, or through
oral delivery. Formulations used for injection may also contain elements such as
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ethanol, ethoxylated castor oil, and N,N'-dimethyl acetamide, which serve to solubilize
the hydrophobic Calpain Inhibitors. Other art-known methods of delivery are also, of
course, possible. Between approximately 0.001 mg/kg and 100 mg/kg of a Calpain
Inhibitor can be a~lmini~tered daily to an individual to inhibit smooth muscle cell
S proliferation and to prevent restenosis.
Higher concentrations of Calpain Inhibitors can also be administered through
the direct application of such Inhibitors to the vascular tissue. For example, during a
coronary balloon angioplasty procedure, a balloon catheter can be inserted into the
desired blood vessel and Calpain Inhibitors can be delivered through the catheter. One
such method of application involves passing a balloon through the lumen of a vessel to
the site of a vascular lesion or stenosis, after which the balloon is inflated in order to
flatten plaque against the wall of the vessel. In this method, a Calpain Inhibitor is
directly applied to the blood vessel through the angioplasty balloon. In anothermethod, the angioplasty balloon or another tool used during the angioplasty procedure
is coated with the Calpain Inhibitor so that the Calpain Inhibitor is applied directly to
the site of stenosis. Alternatively, the Calpain Inhibitor can be loaded into a
microsphere and the microsphere can be driven into the injured tissue to effect direct
application. A Calpain Inhibitor can also be delivered through the use of a slow-
release compound, such as a gel or ointment, applied directly to the injured tissue
during surgery.
EXAMPLE 30
Two cultures of bovine aortic smooth muscle cells at low density were serum
starved. One cell culture was treated with about 10-l00 ~M Z-Leu-Phe-CONHEt, andthe other culture was treated with about 10-100 IlM Ph2CHCO-Leu-Abu-CONH-
CH2CH(OH)Ph. Treatment with Calpain Inhibitor began 6 hours prior to stimulationwith serum. The cells were then stimulated to divide by changing the old, serum free
media to the same media containing 10% fetal bovine serum. The results of this
procedure for two Calpain Inhibitor compounds are shown in Figure 19. This Figure
shows that the peptide keto compounds Z-Leu-Phe-CONHEt and Ph2CHCO-Leu-Abu-
CONH-CH2CH(OH)Ph effectively inhibit the proliferation of cultured bQvine smoothmuscle cells with an IC50 of around 150 ~M and 60 ~M, respectively. Results of this
same testing using several different Calpain Inhibitors are shown in Table 30.
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TABLE 30
Tr~ iof BASMC Prolif~
Stru~Activity R~l~ti~nChir
structure IC
(Ph)2CHCO-Leu-Phe-CONH-CH2-2-Py 30
Z-Leu-Nva-CONH-CH2-2-Py 30
Z-Leu-Phe-CONH-CH2CH(OH)Ph 40
(Ph)2CHCO-Leu-Abu-CONH-CH2CH(OH)Ph 60
Z-Leu-Phe-CONH2 75
Z-Leu-Abu-CONH-CH2CH(OH)Ph 130
Z-Leu-Phe-CONHEt 150
Z-Leu-Abu-CONHEt > 200
(Ph)2CHCO-Leu-Abu-COOEt no inhib
EXAMPLE 31
To ensure that the inhibition of cell proliferation in Example 30 was not due toa toxic effect on the cells, the bovine aortic smooth muscle cells treated according to
the procedure of Example 30 were further treated for one week with about 10-100 ~M
Ph2CHCO-Leu-Abu-CONH-CH2CH(OH)Ph. Figure 20 shows that treatment with
Calpain Inhibitor did not cause significant cell death despite a complete inhibition of
proliferation. In addition, no increase in trypan blue permeability was seen after
treatment for 1 week with the aforementioned Calpain Inhibitor, thus indicating the
continued viability of the cells. Furthermore, the antiproliferative effect of the Calpain
Inhibitor (Ph)2CHCO-Leu-Abu-CONH-CH2CH(OH)Ph was found to be rapidly
reversed upon washout of the drug, another indication of the continued viability of the
smooth muscle cells.
EXAMPLE 32
A human diagnosed as undergoing restenosis is administered between
app.oxil,lately between 0.01 mg/kg and 100 mg/kg of a Calpain Inhibitor in a
phosphate buffered saline solution by intravenous injection at least once per day for
a~luAill,ately one week or until it is determined that the risk of restenosis has
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EXAMPLE 33
A human who is determined to be at risk for developing restenosis, such as
someone who has undergone angioplasty, is adminictered between about 0.01 mg/kg
and 100 mg/kg of a Calpain Inhibitor in phosphate buffered saline by intravenousinjection once per day for appro~ .ately one week or until it is determined that the
risk of restenosis has passed.
EXAMPLE 34
A human undergoing coronary balloon angioplasty is administered a Calpain
Inhibitor to prevent restenosis. The Calpain Inhibitor is delivered by means of an
ointment containing between about 0.2 - 10~ (2 g/kg to 100 g/kg) of a Calpain
Inhibitor. The ointment is coated on the surface of the balloon used in the angioplasty
procedure. The Calpain Inhibitor is thus delivered directly to the injured tissue when
the balloon is inflated during the procedure.
8. Synchroni7qtinn of the Cell Cycle
The ubiquitous distribution of calpain in mammalian cells, taken together with
the fact that the cleavage of substrate proteins by calpain does not appear to be part of
protein catabolism or general protein turnover, points to a regulatory role for calpain in
cells. While not wishing to be bound by any particular theory, we believe that a cell's
progression through the cell cycle is dependent upon the calpain mediated cleavage of
regulatory proteins. This theory has been deduced from our discovery that the
inhibition of calpain in a cell with the Calpain Inhibitors of the present invention
prevents the progression of the cell's reproductive cycle *om the Gl phase to the S
phase. Thus, we have found that Calpain Inhibitors can be used to synchronize the
reproductive cycles of cells.
An experiment which was conducted to shou that Calpain Inhibitors block the
passage of cells from Gl to S phase is shown in Figure 21. By serum starving the cells
used in this experiment, these cells were all blocked at the Gl phase due to lack of
sufficient nutrients to allow passage into the S phase. In this experiment, serum was
added to the cells in order to allow them to pass into S phase, and the DNA content of
the cells was analyzed with a flow cytometer. The cells were divided into several
groups, and the Calpain Inhibitor Ph2-CHCO-Leu-Abu-CONH-CH2CH(OH)Ph was
added to each of these groups at the various points in time following serum addition
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shown above the top row of squares (at 0, 12, 18, 21, 24" 30, or 48 hours after the
~rlllitir~n of serum to the cells). The peaks shown in each of the individual squares
depict the amount of DNA in the cells of the analyzed group at the designated point in
time, where the leftmost peak in each of these squares represents the amount of DNA
in cells which are in the Gl phase. The peak to the right of this G1 peak replesellts
the amount of DNA which has accumulated in cells which have passed through the Sphase.
The numbers shown along the diagonal axis of the graph represent the time
after the adllition of serum that the Calpain Inhibitor Ph2-CHCO-Leu-Abu-CONH-
CH2CH(OH)Ph was added to each of the samples of the cells. Thus, the leftmost
square in each row represents the amount of DNA in cells which have not been
exposed to a Calpain Inhibitor. As can be seen from the first row of squares, when
Calpain Inhibitor was added at the time of serum addition, the cells thus treated
remained in G1 phase and did not progress through to the S phase (as shown by the
absence of a second, right-shifted peak). However, if Calpain Inhibitor is added after
some cells have already begun passing into S phase, such as at 18 or 21 hours after
serum addition, then these cells will continue on in the cell cycle. When such cells
again reach Gl phase, however, they will be prevented from progressing, as shown in
the squares repr~senting cells sampled at 48 hours, in which only a very small right-
shifted peak is visible in all of the cell cultures.
The discovery that Calpain Inhibitors can block the cell cycle has been utilizedto devise a treatment for cancer. This treatment involves the synchronization of the
cell cydes of cancer cells, followed by a course of chemotherapy. According to this
embodiment of the present invention, a patient is first treated with a Calpain Inhibitor,
which blocks the patient's actively dividing cells, including cancer cells, from passing
from the G1 phase (the "gap" between mitosis and the beginning of DNA synthesis) to
the S phase. After the patient has been treated with the Calpain Inhibitor for the
length of one cell cycle, all of the patient's cancerous cells will be in the Gl phase.
Treatment with the Calpain Inhibitor is then stopped, thereby allowing the actively
dividing cells to enter the S phase. All of the cancer cells which have been exposed to
the Calpain Inhibitor will then progress synchronously into the S phase. At this point,
a chemotherapeutic agent which interferes with proper DNA replication is
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a~minict.ored to the patient. Since all of the cancerous cells exposed to the calpain
inhibitor will be progressing synchronously into the S phase when the chemotherapeutic
agent is a~lmini~tered, all of these cells will be affected by the agent. This course of
treatment can be repeated in order to treat cells which did not get previously exposed
to the calpain inhibitor.
Figure 22 shows that cancer cells in particular are susceptible to having their
cell cycles blocked by Calpain Inhibitors, and that such blockage can be reversed by
removing the Calpain Inhibitor. In the experiment illustrated by Figure 22, the cell
cycles of cells from AT-2 and HeLa cell lines were synchronized through the use of a
the Calpain Inhibitor Ph2-CHCO-Leu-Abu-CONH-CH2CH(OH)Ph. However, we have
also found that P388 leukemia cells, L1210 leukemia cells, and human mveloma cells
can also be similarly synchronized through the use of a Calpain Inhibitor. Thus, it is
believed that all cancer cells can be treated according to the methods of the present
invention
As in Figure 21, the squares in Figure 22 depict the results of subjecting cells to
flow cytometry analysis, in which the amount of DNA in cells is quantitated. The cells
used in this experiment were first exposed to the Calpain Inhibitor Ph2-CHCO-Leu-
Abu-CONH-CH2CH(OH)Ph for 48 hours, after which the cell medium (which
contained this Calpain Inhibitor) in which the cells were suspended was changed. At
time 0 in this figure, when the cell medium was changed, most of the AT-2 (top row)
and HeLa (bottom row) cells were in Gl phase, as shown by the presence of a large
left-shifted peak and only a very small right-shifted peak. After washing out the
medium containing the Calpain Inhibitor, the cells began to progress through S phase,
until after 30 hours quite a number of cells had progressed through the cell cycle (as
shown by the significantly larger right-shifted peak). Thus, Calpain Inhibitors can be
used to synchronize the cell cycles of cancer cells and allow them to synchronou sly pass
into S phase so that they can be effectively treated with a chemotherapeutic agent.
One of the benefits of using Calpain Inhibitors in conjunction with a
chemotherapeutic agent is that the use of the chemotherapeutic agent can be
~;~ron~inued after the length of the S phase of a patient's cancer cells, rather than
requiring the agent to be administered for a full cell cycle in order to affect all
treatable cancer cells. This results in a shorter duration of treatment and therefore a
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l~ssçning of the discomfort and side effects of the chemotherapy. The efficacy of the
chemotherapeutic agent is also increased.
In order to determine the length of a cancerous cell's cell cycle or the length of
such a cell's S phase, the type of cancer cell should first be determined. This can be
done, for example, by taking a biopsy of the tissue which is or is believed to be
cancerous. Once the type of cancer cell is determined, the length of that cell's cell
cycle and of the S phase of that cell's cycle can be appruxi..,ately determined by
reference to information known to those of skill in the art. Alternatively, cancerous
tissue extracted during the biopsy can be observed in order to determine the length of
the cell cycle of the cells in such cancerous tissue and the length of the S phase of the
cell cycle of such cells. Since the S phase of a cell's reproductive cycle typically makes
up a relatively short period in the cell's reproductive cycle, it is anticipated that
st~ndard chemotherapy regimens which target the DNA synthesis of actively dividing
cancer cells can be considerably shortened through the use of Calpain Inhibitorsaccording to this aspect of the present invention.
The a~ ninictration of Calpain Inhibitors to synchronize the cell cycle can be by
any of a number of methods known to those of skill in the art. Such methods include
the systemic delivery of a Calpain Inhibitor through intravenous, intraperitoneal, or
intramuscular injection, or throur,h oral delivery. Administration of a Calpain Inhibitor
can also be accomplished through the use of a catheter. Other methods of delivery
known to those of skill in the art are as well possible.
A pharmacologically effective does of a calpain inhibitor for blocking the cell
cycle of cells from p~ ,l esail)g from G phase to S phase of between approximately
0.001 mg/kg and 100 mg/kg Calpain Inhibitor can be administered daily to an
individual to cause synchronization of the cell cycle. Preferably, between 1 and 50
mg/kg of a Calpain Inhibitor are administered to such an individual. In one possible
course of treatment, a patient can be administered 1 mg/kg of a Calpain Inhibitor.
Appru~il..ately 24-48 hours after the patient has received this dose of the Calpain
- Inhibitor, the patient is administered between about 60-75 mg/m2 of adriamycin, a
chemotherapeutic drug sold by Adria Laboratories. Dublin, Ohio. This course of
treatment can then be repeated approximately every 21 days or otherwise as needed
until the cancer is eradicated or in remission.
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Higher concentrations of Calpain Inhibitors than those ~liccll~sed above can also
be ~lmini~t~red through the direct application of such Inhibitors to living cells. For
eY~mple, the Calpain Inhibitor can be loaded into a microsphere, and the microsphere
can then be driven into tissue to effect direct application to cancer cells. A Calpain
Inhibitor can also be delivered through the use of a slow-release compound, such as a
gel or ointment, which is applied directly to cells. Other strategies for the local
a~lrnini~tration of a ~alpain Inhibitor include the injection of a solution containing a
Calpain Inhibitor directly into a malignant tumor to effect synchronization of the cell
cycles of the cancerous cells of the tumor.
The Calpain Inhibitors of the present invention can also be used in assessing
the effectiveness of a chemotherapeutic agent. By synchronizing the cell cycles of cells
grown in vitro, chemotherapeutic agents that interfere with DNA synthesis can beassayed most effectively. Thus, to growing cancerous cells, an amount of a Calpain
Inhibitor is arlrninictered to synchronize the cells at the end of Gl phase. The Calpain
Inhibitor can then be rinsed or washed out in a manner well known to those having
ordinary skill in the art. Thereafter, the cells are allowed to enter S phase, and a
potential chemotherapeutic agent is allrninict~red to the cells in an amount believed to
kill cancerous cells. The number of killed cells can be determined using any of a
number of techniques known in the art, such as by measuring the cells' ability to
convert MTT into its blue product. The more effective chemotherapeutic agents will be
more effective at killing cells at low dosages.
EXAMPLE 35
Determinin~ the Len ths of the Phases of a Cell Cvcle
A biopsy is performed on a patient who has been determined Io have cancer,
and cancerous cells are thereby removed from the patient. These cells are given a brief
pulse of 3H-thymidine and are then washed. Samples of these cells are taken at various
times over the course of apploxi",ately 24 hours. Autoradiographs are then prepared
from these samples. Initially, the cells that are in the S phase are radiolabeled, while
cells in the G2, M and Gl phases are not labeled. After a length of time equal to the
length of the G2 phase, the labeled cells will enter the M phase. By monitoring when
labeled cells pass into the M phase, and then eventually re-enter the M phase, one can
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determine the average durations of the G2, M, S and Gl phases of the cell cycle, as
well as the length of the entire cell cycle.
EX~MPLE 36
Inhibition of the Passage of Cells into S Phase with a Calpain Inhibitor
Bovine aortic smooth muscle cells (BASMC) were grown in tissue culture medium
for at least 48 hours. These cells were then serum starved for an additional 48 hours,
resulting in a population of cells arrested in Gl. The cells were then exposed to media
cont~ining about 10% fetal bovine serum to stimulate cell cycle progression and division.
At various times following the addition of the serum, the Calpain Inhibitor Ph2-CHCO-
Leu-Abu-CONH-CH2CH(OH)Ph was added to the culture medium to a final
con~ entration of 70mM. Cells from these cultures were removed and stained with DNA
dye and analyzed for DNA content using fluorescence activated cell counting.
The results of this experiment are shown in Figure 21. This experiment
demonstrates that cells exposed to a Calpain Inhibitor at the time of serum addition do
not increase their DNA content over the course of the experiment, which means that they
do not progress into the S phase of the cell cycle. The addition of serum to serum-starved
cells normally allows cells to p~u~ ss synchronously into the S phase. The addition of a
Calpain Inhibitor 18 or more hours after the addition of serum, however, does not inhibit
the increase in DNA content and the subsequent division of the cells. Thus, Calpain
Inhibitors act to block the progression of the cell cycle into the S phase.
EXAMPLE 37
Removal of a Calpain Inhihitor from a Cell Culture Arrested in Gl Phase Will
Allowthe Culture to Pro~ress to S Phase
Two cell cultures, one of HeLa cells and one of AT-2 cells, were each grown in
the presence of serum and the Calpain Inhibitor Ph2-CHCO-Leu-Abu-CONH-
CH2CH(OH)Ph at a final concentration of 70mM for 48 hours. The culture media was then replaced with media lacking this Calpain Inhibitor. This allowed the cells to progress
through the cell cycle. At various times after the removal of the Calpain Inhibitor the cells
were stained with DNA dye and analyzed using fluorescence activated cell counting.
The results of this experiment are illustrated in Figure 21. Both cell types were
predominantly in the Gl phase after 48 hours of treatment with Calpain Inhibitor, as
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shown by their normal DNA content. After washout of the Calpain Inhibitor, both cell
types progressed into the S phase, as shown by the increase in their DNA content. Thus,
it was shown that cells can be made to synchronously progress into the S phase of the cell
cycle after being treated with a Calpain Inhibitor after removal of the Calpain Inhibitor.
s
EXAMPLE 38
Use of Substituted Isocoumarins In Chemotherapy
A human diagnosed as having a cancerous tumor is administered a Substituted
Isocoumarin. Such administration is performed by injecting directly into the tumor a
solution containing applu~i.. ately 1 mg/kg of a Substituted Isocoumarin in phosphate
buffered saline. Reginning 24-48 hours after administration of the Substituted
Isocoumarin, 70 mg/m2 of adriamycin (Adria Laboratories, Dublin, OH) is administered
to the patient. This treatment is repeated at 21 day intervals until the tumor is eradicated
or in remission.
EXAMPLE 39
Use of Peptide Ketoamides in Chemotherapy
A human diagnosed as having a cancerous tumor is administered a Peptide
Keto~mi~1e. Such administration is performed by injecting directly into the tumor a
solution containing app~oxin~ately 1 mg/kg of a Peptide Ketoamide in phosphate buffered
saline. Beginning 24-48 hours after administration of the Peptide Ketoamide, 70 mg/m2
of adriamycin (Adria Laboratories, Dublin, OH) is administered to the patient. This
treatment is repeated at 21 day intervals until the tumor is eradicated or in remission.
EXAMPLE 40
Use of Peptide Ketoacids in Chemotherapy
A human diagnosed as having a cancerous tumor is administered a Peptide
Ketoacid. Such administration is performed by injecting directly into the tumor a solution
containing app-o~i...ately 1 mg/kg of a Peptide Ketoacid in phosphate buffered saline.
Bçginning 24-48 hours after ~minictration of the Peptide Ketoacid. 70 mg/m2 of
adriamycin (Adria Laboratories, Dublin, OH) is administered to the patient. Thistreatment is repeated at 21 day intervals until the tumor is eradicated or in remission.
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EXAMPLE 41
Use of Peptide Ketoesters in Chemotherar)v
A human diagnosed as having a cancerous tumor is administered a Peptide
Ketoester. Such adminictration is performed by injecting directly into the tumor a solution
containing ap~l~ u",ately 1 mg/kg of a Peptide Ketoester in phosphate buffered saline.
BeginninE 2448 hours after adminictration of the Peptide Ketoester, 70 mg/m2 of
adriamycin (Adria Laboratories~ Dublin, OH) is administered to the patient. Thistreatment is repeated at 21 day intervals until the tumor is eradicated or in remission.
*. In.. ~a~ g the Efficiency of Cell Transt~. -tjon
With the discovery that Calpain Inhibitors can prevent a cell from entering the S
phase of the cell cycle, we have found that such Inhibitors can be used to increase the
efficiency with which cells are transformed with DN~.. When foreign DNA is introduced
(transformed) into a cell, such DNA can be incorporated into the genome of that cell.
Whether such incorporation takes place depends upon the presence of DNA splicing and
r~plir~rion enzymes which are most active during the S phase of the cell cycle. Thus, the
efficiency of the incorporation of foreign DNA can be increased by introducing the foreign
DNA into a population of cells which have been synchronized in the S phase using a
Calpain Inhibitor.
In this aspect of the present invention, cells in vitro~ such as mammalian cells in
culture, can first be synchronized as described above by administering a Calpain Inhibitor
to such cells. A dose of a Calpain Inhibitor which is pharmacologically effective to block
the cell cycle of cells form progressing from Gl phase to S phase is between ap~"o~i,.,ately
10 I-M and 500 mM. Such a dose can be administered to the cells by adding the Calpain
Inhibitor in solution to the media in which the cells are suspended. Following the addition
of the Calpain Inhibitor, the cells will pass into the Gl phase and remain in that state until
the Calpain Inhibitor is washed out of the cell media or until it is used up by the cells.
Once the cells are thus synchronized in the Gl phase, the Calpain Inhibitor can be
removed from the cell media by removing the cell media and adding fresh medium. After
allowing 5llffirjrnt time to allow the cells to pass into the S phase, the cells can be
transformed by methods known to those of skill in the art. For example, the methods
dicrltsed in Molecular Cloni/lg (Sambrook~ Jr., Fritsch, E.F., and Maniatis, T., Molecular
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Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY
(1989)) or in Short Protocols in Molecular Biolo~y, (Ausbel, et al. eds., Sllon Protocols in
MolecularBiology, John Wiley & Sons (1989)), which are hereby incorporated by reference,
can be used to transform the cells. In a preferred embodiment, exogenous oligonucleotides
are included in vector sequences for transformation of the cells. In this embodiment, the
exogenous oligonucleotides preferably code for protein are operatively linked to a
promoter sequence for transcription and later translation. Following transformation, the
cells can be used in a variety of ways known to those having skill in the art.
In one embodiment, this method can be used to treat a mammal which has a
disease caused by a genetic mutation that results in a protein deficiency in a particular
tissue. In this embodiment, cells of the affected tissue can be removed from a patient
synchronized in the G, phase as described above. After allowing the cells to pass into the
S phase, these cells are transformed by methods known to the art, for example byintroduction of a viral vector carrying exogenous nucleotide sequences. The cells
transformed with oligonucleotides coding for a normal gene can be retransplanted into the
patient from whom the cells were taken, where they will then be able to function normally
due to the incorporation of the normal gene.
EXAMPLE 42
Use of Calpain Tnhibitors to Increase Efficiencv of Transformation in Gene Therapy
A human is diagnosed with sickle cell anemia, which is caused by a genetic
mutation that results in a deficiency of normal hemoglobin in red blood cells.
Hematopoietic bone marrow cells are removed from the patient and put into culture in
vi~ro in the presence of 100 mM of a Calpain Inhibitor, which causes the cells to
synchronize at the G1 phase. After synchronization, the Calpain Inhibitor is washed out,
allowing the cells to proceed to the S phase. The cells are then transformed with foreign
DNA which includes the normal gene coding for hemoglobin. After transformation with
such foreign DNA, the cells are reintroduced into the patient, where they will repopulate
the bone marrow and produce normal hemoglobin protein in red blood cells.
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10. Other Non-N~ I Uses
It is known that a large number of medical conditions and diseases are associated
with an increase in the activity of Calpain and other calcium-activated proteases. We
therefore believe that the compositions of the present invention are beneficial in treating
a large number of these other conditions, and the treatment of these other conditions can
properly be considered within the scope of the present invention.
H. DRUG DELIVERY
The ability of the various Calpain Inhibitors to penetrate plasma membranes is acignifir~nt advantage of these compounds from a pharmaceutical perspective. We believe
that this ability, advantageously, allows the Calpain Inhibitors to provide excellent
permeation of the blood-brain barrier. This is in contrast to many pharmaceuticals,
especially peptides, which often exhibit poor permeation of the blood-brain barrier. Thus,
we believe that the Calpain Inhibitors will exhibit excellent results as pharmaceutically
neuroprotective agents.
For treatment of neurodegeneration and other medical conditions, the Calpain
Inhibitors can be a(lminictered orally, topically, intraperitoneally or parenterally. The term
"parenteral" as used herein includes all non-oral delivery techniques including transdermal
aAminic~ration~ subcut~neous injection, intravenous, intramuscular or intrasternal injection,
intrathecal injection (directly into the CNS) or infusion techniques.
The dosage depends primarily on the specific formulation and on the object of the
therapy or prophylaxis. The amount of the individual doses as well as the administration
is best determined by individually ~c.c~scing the particular case. However, in preferred
compositions, the dosages of Calpain Inhibitors per day are preferably in the range of
1 ,ug/kg total body mass to 100 mgtkg total body mass, more preferably in the range of
10 llg/kg total body mass to 10 mg/kg total body mass.
The pharmaceutical compositions containing the active ingredient may be in a form
suitable for oral use, for example as tablets, troches, lozenges, aqueous or oily suspensions,
dispersible powders or granules, emulsions, hard or soft capsules or syrups or elixirs.
- Dosage levels of the order to 0.2 mg to 140 mg per kilogram of body weight per day are
useful in the treatment of above-indicated conditions ( 10 mg to 7 gms per patient per day).
The amount of active ingredient that may be combined with carrier materials to produce
a single dosage form will vary depending upon the host treated and the particular mode
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of a~lminictration. However, typically, a single dose will contain sufficient Calpain Inhibitor
to provide a complete day's dosage in a single orally acceptable form.
For injection, the therapeutic amount of the Calpain Inhibitors or their
pharm~reutir~lly acceptable salts will normally be made by subcutaneous injection,
S intravenous, intr~ml~scul~r~ intraperitoneal or intrasternal injection, or by intrathecal
injection (directly into the brain). For injection, the therapeutic amount of the peptide
a-keto~mides or their pharmaceutically acceptable salts will normally be in the dosage
range from 0.2 to 140 mg/kg of body weight. In order to provide a single day's dose with
a single injection, the pharmaceutical compositions for parenteral administration will
contain, in a single dosage form, from about 70 llg to about 7 g of Calpain Inhibitor per
dose of from about 0.5 ml to about 1 liter of carrier solution. In addition to the active
ingredient, these pharm~re~ltir~l compositions will usually contain a buffer, e.g. a
phosphate buffer that keeps the pH in the range from 3.5 to 7 and also sodium chloride,
and can also contain m~nnitol or sorbitol for adjusting the isotonic pressure. In a
preferred form of these compositions, DMSO or other organic solvent is added in order
to assist the introduction of the Calpain Inhibitor across membranes.
Additionally, lipids can be introduced into the pharmaceutical compositions in
order to facilitate entry of the Calpain inhibitor compounds into tissue of the CNS. These
compositions are prepared in accordance with methods known to those of skill in the art.
Briefly, a lipid such as, phosphatidyl choline, cholesterol, other well-known lipid carrier or
mixtures thereof, is mixed with the active compound along with a solvent, the solvent is
dried off and the material reconsLilllted in saline. The compositions can also indude other
ingredients known to those of ordinary skill in the art, such as detergents, surfactants or
emulsifying agents.
A composition for topical application or infusion can be formulated as an aqueous
solution, lotion, jelly or an oily solution or suspension. A composition in the form of an
aqueous solution is obtained by dissolving the Calpain Inhibitor in aqueous buffer solution
of pH 4 to 6.5 and, if desired, adding a polymeric binder. An oily formulation for topical
~pplir~tirJn is obtained by suspending the Calpain Inhihitor in an oil, optionally with the
addition of a swelling agent such as aluminium stearate and/or a surfactant. The addition
of DMSO to these topical compositions is believed to allow at least partial penetration of
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the active Calpain Inhibitor into the blood stream after application of the composition to
the skin of a patient to allow for transdermal administration.
For treatment of neurodegeneration resulting from excitotoxicity, HIV-induced
- neuropathy, ischemia following denervation or injury, subarachnoid hemorrhage, stroke,
mnltiple infarction dementia, Al7heimer's Disease (AD), Huntington's Disease, surgery-
related brain damage, Parkinson's Disease, and other pathological conditions, the Calpain
Inhibitors or pharmaceutically acceptable salts thereof may be administered orally or
parenterally. The dosage depends primarily on the specific formulation and on the object
of the therapy or prophylaxis. The amount of the individual doses as well as theadministration is best determined by individually assessing the particular case.In many acute neurodegenerative conditions and events, such as stroke and head
injury, it is important to deliver the Calpain Inhibitor as soon after injury as is practicable.
Thus, it is preferable to identify those individuals who have suffered stroke, head injury
or other injury in which neurodegeneration is associated or is likely to occur, and to begin
a~lminictration of a Calapin Inhibitor within 1 minute to 2 hours after the event, in order
to prevent as much neurodegeneration as possible.
A particular application of the Calpain Inhibitors within the scope of the present
invention is the application of these compounds during surgery to prevent
neurodegeneration associated therewith. For example, for surgeries performed under
general anesthesia, hypoxic conditions can occur throu~h inadequate perfusion of the CNS
while under anesthesia. Additionally, many major surgeries of the cardiovascular system
require that a patient's heart be stopped and that perfusion be maintained through
artificial means. In such surgeries, there is an increased danger of hypoxia occurring
within the CNS, which can also result in neurodegeneration. Moreover, during
neurosurgeries, there is an inherent risk of neurodegeneration resulting from
infl~mm~tion, bleeding, hemorrhaging and the like. Such neurodegeneration can beinhibited by infusion with a solution containing Calpain Inhibitor. However,
neurodegeneration resulting from neurosurgery can also be reduced prophylactically by
atlminictration of a Calpain Inhibitor through any of the foregoing administration
techniques. Such administration is also believed to inhibit or prevent neurodegeneration
associated with the use of anesthesia or with the use of artifical means of perfusion during
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major surgeries. A surgical patient can also have Calpain Inhibitor administered
throughout surgery through intravenous drip.
The following examples are intended to illustrate certain neuroprotective uses of
the Calpain Inhibitors within the scope of the ~resent invention. As such, they are not
meant to limit the invention in any way.
EXAMPLE 35
A Neuroprotective Composition for Intravenous Injection
500 ~g CH3CONH-CiTPrOIC from Example SHC2
4 ml Propylene GJycol
1 ml DMSO
EXAMPLE 36
A N~u-or)~-)tective Composition for Intravenous Drip
250 mg Z-Leu-Phe-CONH-Et from Example PKC 48
1000 ml Phosphate Buffered Saline (pH 6.0)
10 ml DMSO
EXAMPLE 37
A Neuroprotective Composition fnr Transdermal Applicatinn
25 mg Z-Leu-DL-Abu-COOEt from Example PKC19
3 ml Phosphate Buffered Saline (pH 6.0)
2 ml DMSO
EXAMPLE 38
Neuroprotection after Head Injury
A first group of patients who are victims of head trauma is given 2 ml of the
injectable composition of Example 30 intravenously within ten minutes of the time of
injury. A second group of similarly matched patients does not receive the composition.
The first group of patients exhibits markedly fewer and less severe outward symptoms of
neurodegeneration, such as dementia, memory loss and paralysis.
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EXAMPLE 39
- Neuroprotection Durin~ Sur~ery
A patient about to undergo a triple bypass heart surgery is a-1mini~tered 500 ml of
the co."posilion of FY~mrle 31 per hour using an intravenous drip system. Duringsurgery, the patient's heart is stopped and perfusion continued through artificial means.
Although complications develop while restarting the heart and disconnecting the patient
from the artificial means of perfusion, the patient becomes conscious within several hours
of surgery. Within a few days, the patient's mental status is normal with no indications of
neurodegeneration.
For the inhibition of smooth muscle cell proliferation in the treatment or
prevention of restenosis, Calpain Inhibitor can also be administered directly to the site of
injured smooth muscle tissue. Such a~mini~tration can be accomplished, for example, by
means of an ointment or gel applied to the surface of a balloon or other surgical tool used
in an angioplasty procedure. In this way, if damage is done during angioplasty that would
otherwise result in r~ct~nocic~ restenosis can be prevented.
The direct administration of a Calpain Inhibitor to the site of injured tissue can
also be accomplished by loading the Calpain Inhibitor into microspheres and imbedding
the microspheres into the injured tissue. This can be accomplished by applying the
microspheres to the surface of the balloon used in the angioplasty procedure. When the
balloon is inflated inside thi artery, the force of the expansion drives the microspheres into
the arterial wall, where they become lodged. The microspheres then release the Calpain
Inhibitor slowly over time and provide local application to the injured tissue.
For the treatment of numerous medical conditions, Calpain Inhibitors can be
injected in solutions either intravenously, intraocularly, intramuscularly, intraperitoneaUy,
or intrasternally. These solutions will contain a Calpain Inhibitor in the range of from
about 70 llg to about 7 g per dose in about 0.5 ml to 1 liter of a pharmaceutically
acceptable carrier solution. The solution preferably contains a buffer, such as a phosphate
buffer, that keeps the pH in the range of about 3.5 to 7. The solution also preferably
contains applu~ -.ately 9000 mg/l of sodium chloride (0.9% saline), as well as mannitol
or sorbitol for adjusting the isotonic pressure. DMSO at 0.01 to 10 ml/liter can also be
used in injectable solutions of the present invention in order to potentiate the Calpain
WO 94/00095 PCI/US93/06143
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Inhibitor, or help it to penetrate membranes. Other additives such as ethanol or ethoxylated oils can also be used.
When using Calpain Inhibitors to protect cardiac, skeletal, or smooth muscle tissue
from damage, intravenous drip infusion is preferred to the periodic injection of such
S Inhibitors. A solution suitable for intravenous infusion can be prepared by suspending
app~ tely 250 mg of a Calpain Inhibitor in 1000 ml of an aqueous solution of
phosphate buffered saline containing 10 ml DMSO. However, in the treatment of
muscular Jy~trophy, or when treating a condition for a long period of time with Calpain
Inhibitors, an oral formulation of a Calpain Inhibitor is preferred. Such an oral
formulation can be in the form of a tablet, in which a powdered form of a Calpain
Inhibitor is mixed with a pharm~ceuti~lly acceptable filler material capable of being
formed into a tablet.
In the treatment of cataracts, the injectable solutions referred to above can bea-lminictered by soaking them into a contact lens, which is then worn for a period of time
long enough to allow the solution to diffuse into the eye from the lens. Other methods of
delivering Calpain Inhibitors to an eye with the injectable solutions described above
include the ~c~mini~tration of eye drops co,.,p,;sing the solution.
Calpain Inhibitors can also be formulated in an ointment for administration to the
eye. This can be accomplished by dissolving a Calpain Inhibitor in an aqueous solution
and then adding a pharm~ceutir~lly acceptable polymeric binder. A Calpain Inhibitor can
also be directly dissolved or suspended in such a polymeric binder.
For the treatment or prevention of tonic smooth muscle contraction, Calpain
Inhibitor can also be administered directly to the smooth muscle, including application to
coronary tissue. Such administration can be accomplished by means of an ointment, gel
or solution applied directly to the smooth muscle during surgery. Direct administration
can also be accomplished by loading the Calpain Inhibitor into a microsphere andimbedding the microsphere into the smooth muscle tissue. The microsphere then releases
the Calpain Inhibitor slowly over time and provides local appliction to the tissue.
For the treatment of cerebral v~cospasm, a solution of a Calpain Inhibitor can be
injected directly into the cerebrospinal fluid of the patient. For the treatment of
bronchospasm, such as that which occurs in asthmatic patients, a solution containing a
Calpain Inhibitor can be inhaled directly into the patient's lungs.
94/ooo9~ PCI /US93/06143
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The following additional examples are provided to further illustrate certain
embodiments of pharm~ceuti~l colllposilions within the scope of the present invention.
EXAMPLE 40
An Injectable Composition for Non-Neuroloeical Uses
1 mg Z-Leu-Abu-CONH-Bzl from Example PKC58
4 ml Propylene Glycol
1 ml DMSO
EXAMPLE 41
An Ophthalmic Solution for Treatin~ Cataracts
500 ~lg Z-Leu-Abu-CONH-iBu from Example PKC57
S ml sterile phosphate buffered saline
EXAMPLE 42
A Solution for Topical Application to a Tonic.
Contracted Blood Vessel
2 mg Z-Leu-Abu-CONH-(CH2)3-N(CHCH2)2O from Example PKC60
l ml DMSO
10 ml sterile phosphate buffered saline
It will be appreciated that certain variations may suggest themselves to those skilled
in the art. The foregoing detailed description is to be clearly understood as given by way
of illustration, the spirit and scope of this invention being interpreted through reference
to the appended claims.
