Note: Descriptions are shown in the official language in which they were submitted.
WO 92/118~0 2 ~ 9 8 ~ û 9 P~/U!~i91/097~6
1-
USE OF CALP~IN INHlBl'rORS IN THE ~IIBI'lION
A~D TREATMENT OF NEURODEGENERATII:)N
Baclc~round of the Invention
The present invention reiat~s generally to the field of neuroprotectants and more
5specifically to the use of inhibitors of calciuln-sti}nulated proteases, such as ~alpain, as
therapeutics for neurodegeneration.
Neural tissues, including brain, are knowT~ to possess a large variety OI proteases,
including at least two calcium-stirnulated proteases, termed calpain l and calpain II, v~hich
are activated by micromolar and millirnolar Ca2+ concentrations, respectively. Calpains are
10a family of calci~n activated thiol proteases that are present in many tissues. Calpain 11 is
the predominant forrn, but calpain I is found at synapses and is thought to be the form
involved in long terrn potentiation and synaptic plasticity.
Thiol proteases are distinguished from serine proteases, metalloproteases and other
proteases by their mechanism of action and by the amino acid residue (cysteine) that
15participates in substrate attack. Although several thiol proteases are produced by plants,
these proteases are not cornrnon in marnmals, ~vith cathepsin B (a lysosomal er~ne), other
cathepsins and the calpains being among the few representa~ives of this ~arnily that have
been described in mammals. Calpain I and calpain II are the best described of these, but
several other members of the calpain ~amily have been reported.
20Other Ca2+-act~vated thiol proteases may e~st, such as those reported by Yoshihara
et aL in J. Biol. Chem. 265:5809-5815 (1990). The term "Calpain" is used hereinafter to
.. refer to any Ca2+-activated thiol proteases induding the Yoshihara enzyTne and calpa~ns I
and II.
While Calpains degrade a wide ~ariety of protein substrates, cytoskeletal prote~ns
25 -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 ~ime.
Since cytoskeletal proteins are major components of certain types of cells, this provides a
sirnple method of detecting Calpain activity in cells and tissues. Specifically, the
accumulation of the breakdown products ("BDP'sn) of spectrin, a cytoskeletal protein, has
30been associated with the aclivation of Calpain. In neural tissues, act~vation of Calpains, as
evidenced by accurnulation of these BDP's, has been obsened in many neurodegenerative
conditions, including denervation resulting from focal electrolytic lesions, genetic
abnormalities, excitotoxicity, Alzheimer's disease, following ischemia in gerbils and following
E:U~S7-1TUTE~ SHEET
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,.. ~ .. .` , , ~ ... .- . . . .
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.. .. . ~.... . . . .
W092/11850 PCI/US91/097~ ~
2~986~9
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adn~inistration of the to~ins kainate and colchicine in rats, when adininistered peripherally
or centra~ly.
Comrnercially availabie ~ vitro inhibitors of Calpa~n include peptide aldehydes such
as leupeptin (Ac-l~u~Leu-Arg-H), as well as epo~ysuccinates such as E~64. These
S compounds are not useful in inhibiting Calpain in Central Nervous System (nCNSn) tissue
in vivo because they are poorly membrane perrneant and, acs~ordingly, do not cross the
blood brain barTier veIy well. Also, many of these in~u~itors are poorly specific and will
inhibit a wide var~e~ of protea~ses in addition to Calpain. 'Ihese commercially available
compounds are based upon peptide structures that are believed to interact ~nth the substrate
binding site of Calpain. Active groups associated with the Calpa~n inhibitors then either
block or attacl~ Ihe catalytic moiety of Calpain in order to inhibit the enzyïne.
In addition, other types of compounds thought to possess in vitro Calpain inhibitory
activity that are not commercially available have been reported. Examples of such
compounds indude the peptide diazomethanes. See Rich, D~., Inhibitors of ~steine1~ proteinases, in Protease Inhibitors, AJ Barrett and G ~alversen, Eds., Elsevier, New York,
1986, ppl53-178, the disclosure of which is hereby incorporated by reference. These peptide
diazomethanes are similarly thought to be poorly membrane penneant and non-specific.
There is some evidence that certain particular inhibitors of Calpain have certain
therapeutic utilities. For example, leupeptin can facilitate nerve repair in prirnates.
Loxastatin (also known as EST, Ep460 or E-64d), a derivative of E-64, ~s believed to have
utiliy in the treatment of muscular dystrophy. E-64d, while not having significant protease
inhibitory activity itself, is believed to be converted to more potent fonns, such as to E-64c,
inside a marnmalian body.
Evidence from electrophysiological studies suggests that one of the ear~iest 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. Intracellular calcium is
lilcely to produce a large nwnber of consequences, including the activation of a large number
of en~yTnes, including p~oteases, such as Calpain, lipases and kinases. An increase in
intracellular calcium is also thoudlt to induce changes in gene expression.
Ischenua, head traurna and stroke have all been associated with the rele~ase of
glutamate in amounts large enough to lead to excitotoxicity, the to~icity resulting from the
actions of certain amino acids on neurons of the CNS. The e~cess glutamate and other
factors, such as free radical damaBe of membranes or energy depletion, cause an increase
in intracellular Ca2+. It is known that an excess of intraceliular Ca2+ leads to several effects
SUBSTITUTI~ S~E~T
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believed to be associated with neuronal cell damage, including destruction of cell structures
through activation of phospholi~ase and Calpain, as well as free radical production resulting
from ac~ivation of phospholipase and xanthine oxidase. Many other factors have been
- associated with neuroto~city. For example, reductions in action potentials and changes in
a wide variety of chemic~l markers are known to be associated wi~h neurons exposed to
ischemic conditions.
Notwithstanding the foregoing understanding of certain aspects of neuroto~city, no
effecdve therapy has yet been developed for most neurodegenera~ive diseases and conditions
of the CNS. Millions of individuals suffer from these diseases and conditions. Thus, there
is a need for therapies effective in treadng and preventing these diseases and eonditions.
Summarv of the Tnvention
The present invention provide the use of a Calpain inhibitor compound or a
pharmaceutically acceptable salt or derivativf~ thereof for the manufacture of a medicament
for inhibiting or treating neurodegeneration in a mammal hanng or likely to experience a
neuropathology associated with neurodegeneration. In certain embodiments of this use, the
neurodegeneration is occurring due to e~citotoxicity, HIV-induced neuropathy, ischemia
following denervation or injury, subarachnoid hemorrhage, stroke, mul~iple infarction
dementia, Alzheimer's Disease, Huntington's Disease, or Parkinson's Disease. Themediument can comprise a pharmaceutically acceptable carrier and is for parenteral
2û administration, such as for transdermal administration, subcutaneous injection, intravenous,
intramuscular or intrasternal injection, intrathecal injection directly into the CNS or
infusion. The mediument can also be in a forrn suitable for oral use. In some
embodirnents, the medicarnent is for substantially preventing neurodegeneration in a patient
undergoing surgery during and subsequent to the surgery, such as for a patient undergoing
neurosurgery, cardiovascular surgery or a surgery using general anesthesia. The Calpain
inhibitor compound preferably enters t~ssue of the C~S of the mammaL such as through use
of a membrane-permeant Calpin inhl~itor.
The present invention also provides an in vitro method of selecting Calpain
Inhibitors for use as Calpain ~hibitor protectants in the in vivo treatment or inhibition of
degeneration. This method includes identifying compounds having Calpain inhibitory
activity vitro~ and identi~ing those compounds with Calpain inhibito~y activity that are
membrane permeant through an vjtro assay for membrane permeance. The irL~i~o assay
for membrane permeance can include providing a plurality of tissue portions from a
mammal; treating a~ least one, but not a~, of the tissue portions with a Calpain Inhibitor;
WO 92/11850 2 ~ 9 8 ~ 0 9 PCI'/US91/097~
subjecting the tissue portions to an eYent that can cause degeneraeion in untreated eissue;
measuring the amount of degeneration that occurs in ehe tissue poreions; and comparing the
arnount of degeneration that occurs in the treated tissue portions with ths amount of
degeneration occurring in the untreated tissue portions. The amount of degeneration in the
treated tissue portions being less than the amount of degeneration ir~ the untreated tissue
portions indicates that the Calpain Inhibitor is membrane-permeant. The tissue portions
in some embodiments are brain sli~es, platelets or ceUs in cultllre. The measuring step in
some embodirnen~s involves analyzing the tissue portions for the presence of the BDP's of
a cytoskeletal eomponent such as spectrin, MAP2, actin binding protein or tau. The
measuring step can also include measuring the electncal actiYity of the tissue portions. The
in vitro assay for membrane per~neance can b~ performed by measuring the ability of the
Calpain Inhibitor to penetrate platelet membranes and inhibit endogenous Calpain of the
platelets.
Ihe present inYention also proYides the use of a Substituted Hetero~yclic Compound
or a pharmaceuticaUy acceptable salt or derIvatiYe ~hereof for the manufacture of a
medicament for inhbiting or treating neurodegeneration in a mammal haYing or likely to
experience a neuropathology associated with ne~lrodegeneration. The medicarnent is
preferabk~r for inhibiting or treating neurodegeneration of the CNS. The Substituted
Heterocyclic Compound preferably is a member of the Class I Substituted ~socoumarins, the
Class II Substituted Isocournarins or the Class m Heterocyclic Compounds. More
preferably, the Substituted Heterocyclic Compound is 3-chloroisocournarin; a 3,4-
dichloroisocoumarin; a 3-alkoxy-7-arnino 1-chloroisocoumarin; or a 7-substituted 3-alkoxy~-
chloroisocoumarin. The use of Clairn 17, wherein said Substituted Heterocyclic Compound
isCl~PrOIC,NH2-Cl~PrOIC,PhCH2NHCONH-C~PrOIC, CH3CONH~CiTPrOIC,L-Phe-
NH-ClTPrOIC, PhG~I2NHCONH~Cl~EtOIC, PhCH2CONH-Cs~EtOIC, or D-Phe-NH-
' CiTEtOIC.
Another aspect of the present invention provides the use of a Peptide Keto-
Compound hav~ng Calpain inhl~itory activity or a pharmaceutically acceptable salt or
derivative thereof for the manufacture of a medicament for in}~iting or treatingneurodegeneration in a marnmal having or likely to e~perience a neuropathology associated
with neurodegeneration. The medicament is preferably for inhibiting or treating
neurodegeneration of the GNS. The Peptide Keto-Compound preferably is a peptide a-
ketoester, a peptide a-ketoacid or a peptide a-ketoamide. More preferably, the Peptide
Keto-Compound is a member of one of the following su~classes: Dipeptide a-Ketoesters
SUBS~tTUTE SHEET
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;: WO 92/1185û 2 ~ 9 ~ ~ ~ 9 PCI/lUS91/09786
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(Subclass A), Dipeptide ~-Ketoesters (Subclass B), Tripeptide a-Ketoesters (Subclass A),
Tripeptide -Ketoesters (Subclass B), Tetrapeptide ~-Ketoesters, Amino Acid Peptide
-Ketoesters, Dipeptide a-Ket~acids (Subclass A), Dipeptide -Ketoacids (Subclass B),
Tn~eptide -Keeoacids, Tetrapeptide a-Ketoacids ~nd Arnino A~id Peptide ~-Ketoacids,
Dipeptide ~-Ketoa~nides (Subclass A), Dipeptide a-Ketoarnides (Subclass B), T~ipeptide
c~-Ketoarnides, Tetrapeptide a-Xetoarnides or Amino Acid a-Ketoarnides.
Still another aspect of the present invention provides the use of a Halo-Ketone
Peptide having Calpain in~ubitory ac~vity or a pharrnaceutically acceptable salt or derivative
thereof Ior the manufacture of a medicament for inh~biting or treating neurodegeneration
in a mammal having or ]ikely to experience a neuropathology associated with
neurodegeneration. The medicament is preferably for inhibiting or treating
neurodegeneration of the CNS. The Halo-Ketone Peptide can be an arnino-halo ketone
peptide or a diazo-ketone peptide.
The uses of the present invention of Substituted Heterocrclic Compounds, PeptideKeto-Compounds or Halo-Ketone Peptides can be used in connection with
neurodegeneration associated with e~citotoxicity, HIV-induced neuropathy, ischernia,
subarachnoid hemorrhage, stroke, bra~ seizure, major heart attack, multiple in~arction
dementia, Alzheirner's Disease, Huntington's Disease or Parkinson's Disease. Themedicament of these uses can indude a pharmaceutically acceptable carrier, and be for
parenteral administration, such as transdermal administration, subcutaneous injection,
intravenous, intramuscular or intrasternal injection, intrathecal injection directly into the
CNS or an infusion technique. The medicament can also be in a form suitable for oral use.
These uses can also be in coMection with neurodegeneration is occurring from ischemia-
inducing events, stroke, head injury, major heart attaclc, brain seizure, near drowning, carbon
monoxide poison~ng, surgery-related brain damage or another event known to causeneurodegeneration.
Another aspect of the present invention provides a method of minim~ing proteolysis
in an in ~o sample containing peptides or proteins during or following the prccessing,
production, preparation, isolation, purification, storage or transport of the samples,
comprising ehe addition eo the sasnple of a Substituted Hetero~clic Compound or a Peptide
Keto-Compound that is a member of one of the following subclasses: Dipeptide
a-Ketoesters (Subclass A), Dipeptide a-Ketoesters (Subclass B), Tripeptide a-Ketoesters
(Subclass A), Tripeptide a-Ketoesters (Subclass 8), Tetrapeptide a-Ketoesters, Arnino Acid
Peptide -Ketoesters, Dipeptide a-Ketoacids (Subclass A), Dipeptide a-Ketoacids (Subclass
~VO 92/11850 2 ~ 9 8 6 ~ 9 -6- PCI/US91/~97~ ~
B), Tripeptide ~-Ketoacids, Tetrapeptide a-Ketoacids, Amino Acid Peptide a-Ketoacids,
Dipeptide ~-Ketoarnides (Su~class A), Dipeptide a-Ke20arnides (Subclass B), Tripeptide
a-Ketoamides, Tetrapeptide a-Ketoarnides or Amino Acid a-Ketoam~des. The presentinvention also provides a method of n~nim~g degradation resul~ting from Calpain act~vity
S in a tissue sample during or following preparation of the sample, comprising the addition
to the sample of a Substituted Heterocyclic Compound, Peptide Keto-Compound or a Halo-
Ketone Peptide. The sample can be a whole organ and the addition of compound
comprises perfusion of the organ with the compound disso~ved in fluid. Preferably, following
the addition step, the tissue sample is used in an assay for neurodegeneratiQn wherein the
assay comprises a test for the products of Calpain activity in the tissue samples. The
addition of compound in either of these methods can include the addition of a Peptide
a-Ketoacid to the sample. Preferably, this Peptide a-Ketoacid comprises a compound that
is a member of one of the fo110wing subclasses: Dipeptide a-Ketoacids (Subclass A),
Dipeptide a-Ketoacids (Subclass B), Tripeptide -Ketoacids, Tetrapeptide a-Ketoacids or
Arnino Acid Peptide a-Ketoacids.
Yet another aspect of the present invention provides pharmaceutical compositionsfor the treatment or inhibition of neurodegeneration. These compositions include a
pharmacologically effective neuroprotective amount of a Substituted Hetero~yclicCompound, Peptide Keto-Compound or Halo-Ketone Peptide, or pharmaceutically
acceptable salts or derivatives thereof in a pharmaceutically acceptable forrnulation
containing a carrier material. In one preferred embodirnent, a Peptide Keto-Compound is
included in the composition where~n said Peptide Keto-Compound comprises a compound
from one of the following subclasses: Dipeptide a-Ketoesters (Subclass A), Dipeptide
a-Ketoesters (Subclass B), Tripeptide -Ketoesters (Subclass A), Tripeptide a-Ketoesters
(SubclassB), Tetrapeptide a-Ketoesters, orAminoAcid Peptide a-Ketoesters. This Pepdde
Keto-Compound is preferabb one of the following compounds: Bz-DL-Ala-COOEt,
Bz-DL-Ala-COOCH2-C6H4-CF3 (para), Bz-DL-Lys-COOEt, PhCO-Abu-COOEt,
(CH3)2CH(CH2)2CO-Abu-COOEt, CH3CH2CH)2CHCO-Abu-COOEt or
Ph(CH2)6CO-Abu-COOEt. Another preferred Peptide Keto-Compound is one of the
following compounds: Z-Ala-DL-Ala-COOEt, Z-Ala-DL-Ala-COOBzl,
Z-Ala-DL-Ala-COOnBu, Z-Leu-Nva-COOEt, Z-Leu-Me-COOEt, Z-Leu-Abu-COOEt,
~-Leu-Met-COOEt, Z-Leu-Phe-COOEt, Z-Leu-4-Cl-Phe-COOEt,
~-NapSO2-Leu-Abu-COOEt, Z-Leu-NLeu-CO2Et, Z-Leu-Phe-C02Bu, Z-Leu-Abu-CO2Bu,
Z-Leu-Phe-CO2Bzl, MeO-Suc-Ala~DL-Ala-COOMe, or Z-Leu-Abu-CO2Bzl. Still other
SU6~ I tTUl ~ SHFET
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WO 92/11850 2 ~ ~ g ~ Q 9 P~/US91/09786
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Peptide Keto-Compounds ~r use in ~he preferred compositions are one of ~he followin~
compounds: 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-AbuCOC)CH2-C6H4-CF3 (para), H-Leu-Ala-DL-Lys-COO~t,
ZLeu-Leu-Abu-COOEt, ZL~u-Leu-Phe-COOEt, MeO-Suc-Val-Pro-D~Phe-COOMe or
2-NapSO2-IRu-Ieu-Abu-COOEt. A preferred Peptide Keto-Compound could also be
MeO-SucAla-Ala-Pro-DI~Abu-COOMe or Z-Ala-Ala-Ala-DI,Ala-COOEt. Still other
preferred Peptide Keto-Compouulds would be a compound ~om one of the following
subclasses: Dipeptide a-Ketoacids (Subclass A), Dipeptide ~-Ketoacids (Subclass B),
Tripeptide a-Ketoacids, Tetrapeptide a-Ketoacids or the Amino Acid Peptide a-Ketoacids.
Thus, preferred Peptide Keto-Compounds could be one of the following compounds:
Bz-DL-Lys-COOH, BznDI~Ala-COOH, ZLeu-Phe-COOH or ZLeu-Abu-COOH. A
compound ~om one of the following subclasses: Dipeptide a-Ketoamides (Subclass A),
Dipeptide a-Ketoamides (Subclass B), Tripeptide a-Xetoamides, Tetrapeptide
a-Ketoamides or Amino Acid a-Ketoamides, could also be used in the preferred
compositions. Thus, another preferred Peptide Ke~o-CompouIld would be one of the~ollow~g compounds: ~Leu-Phe-CC)NH-Et, ~Leu-Phe-CONH-nPr, ~Leu-Phe-CONH-
nBu, Z-Leu-Phe-CONH-iBu, Z-Leu-Phe-CONH-:Bzl, ~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-CONH-Bzl, ~Leu-Abu-CONH-(CH2)2Ph, Z-Leu-Abu-CONH-(CH2)3-
N(CH2CH~2O, ZLeu-Abu-CONH-(C~H2)7CH3, ZLeu-Abu-CONH-(C~I2)20H, ZLeu-Abu-
CONH-(CH2)20(CH2)20H, Z-Leu-Abu-CONH (CH2)l7CH3, ZLeu Abu CONH-CH2-
C6H3(0CH3)2 or Z-Leu-Abu-CONH-CH2~C4H4N. The composition can also include a
Substituted Heterocyclic Compound such as one of the Class I Substi~u~ed Isocoumarins,
Class lI Substituted Isocoumarins or Class m Heterocyclic Compounds. Preferred
Substituted Hetero~yclic Compounds are 3-chloroisocoumar~n, a 3,4-dichloroisocoumarin,
a 3-allco~y-7-amino 1~1oroisocoumarin, a 7-substitu~ed 3-alkoxy-4~chloroisocournarin;
CiTPrOIC, NH2-Cl~TPrOIC, PhCH2NHCONH-ClTPrOIC, CH3CONH-ClTPrOIC, L-Phe-
NH-GTPrOIC, PhCH2NIICONH-ClTEtOIC, PhCH2CONH-ClTEtOIC, or D-Phe-NH-
CiTEtOIC In these compositions, the composition is preferably in dosage ~orm comprising
from 70 llg to 7 g of active ingredient in each dose, and the carrier material includes a
liquid, wherein ~he composition is ~n dosage form and wherein each dose comprises ~om
05 ml to 1 liter of said carrier material. The compositions can additionally include at least
one of the follo~ing: DMSO or other organic solvent, a lipid carrier, a detergent, a
.~
:, ~ ~ ,, '- :
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WO 9Z/11850 2 0 9 8 ~ ~ ~ PCr/US91/o97~ ;-
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surfactant, or an emulsifying agent. These compositions can be su~table for parenteral
administration or in a forrn suitable for topical application. The compositions can be in a
variety of forms, such as an aqueous solution, a lotion, a jelly, an oily solution, or an oily
suspension.
S The present invention also provides the use of a Substituted Heterocyclic Compound
as a medicament, the use of a Halo-Ketone Peptide as a medicament, and the use of a
Peptide Keto-Compound as a medi~ament, wherein said Peptide Xeto-Compound is a
compound from one of the following subclasses: Dipeptide ~-Ketoesters (Subclass A),
~ipeptide a-Ketoesters (Subclass B), Tripeptide a-Ketoesters (Subclass A), Tripeptide
~-Ketoesters (Subclass :E3), Tetrapeptide a-~etoesters, or Amino Acid Peptide a -Ketoesters.
Preferred Peptide Keto-Compounds in this use ~nclude any one of the Peptide Keto-
Compounds desaibed above in connection with the pharmaceutical compositions.
Brief Summarv of the Fi@lres
Figure 1 shows the percentage of i~u~ition of glut~nate-induced cell death through
the addition of ~lutamate and various Calpain Inhibitors rela~ve to control where no
glu~amate was added.
Figure 2 graphically depic~s the e~ects of ZLeu-Phe-C:ONH-Et (CX269) and ZLeu-
A~u-CONH-Et (CX275) on the size of iT~rction produced upon MCA occlusion in malerats.
~igure 3 shows the effects of CX216 (ZLeu-Phe-CO2Et, a Peptide Keto-
Compound), and C:I1 (Ac-Leu-Leu-Me-H) rela~ve to ~ontrol slices on survival of
hippocampal slices exposed to 10 minutes exposure of anoxic atrnosphere where both of
these compounds were added at their optimal inhibitory concentradon at both 1 hour and
2 hour incubation times.
Figure 4 shows the evoked potential amplitude for control, CI1 treated and CX218treated hippocampal slices over a time course during which the slices sre exposed to anoxic
atmosphere.
Figure 5 shows the percent recovery of EPSP from severe hypoxia over the course
of one hour incubation for ~Leu-Phe-CONH-Et (CX269) and ~Leu-Phe-CO2Et (CX216).
Figure 6 shows a comparison of the ef~ect of the presence of CI1 or CX216 on
survlval of hippocampal slices expressed as the duration of anoxia (in minutes) before fiber
volley disappearance.
Figure 7 shows the effects of CI1 compared with control on the behavioral and
convulsive effects of kainic acid.
~;UBSTITUTE SHEEJ
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W(:~ 92/11850 2 0 ~ PC11US91/097~6
"~., ~
Figure 8 shows the amount of spectrin BDP's in rat brains e~posed to kainate forcontrol and CI1 treated rats.
Detailed Description of the Invention
A. INTRODUCI ION
We haYe discovered that Calpain activation is an event central to many cases of
- bra~n atrophy and degeneration and that inhibition of Calpain alon~ is su~icient to inhibit
or prevent cell deterioration and loss. Thus, we have further discovered that inhibition of
C~ain provides protertion from neuroto~ncity associated with many neurodegenerative
conditions and diseases.
In accordance with the foregoing di~coveries, we believe that the elevation of
intracellular ~alcium a~sociated with neuropathological condition~ in neuronal cells activates
Calpain and sets in motion the digestion OI neuronal ce11s from within. We believe there
may be other mechanisms of activation of Calpains associated with these conditions.
Accordirlgly, o~e aspect of ~he present invention is di~ected to inhibidon and treatment of
1~ the neurodegeneration and o~her diseases associated with this dic,estion through the
inhibition of Calpain actiYity. Thus, part of this aspect of the present invention is to prevent
the neurodegeneradon and other pathology caused by this diges~ion through the vivo
adrninistration of Calpain inhll~itors. By way of example, and not of lirnitation, diseases and
conditions which can be treated using this aspect of the present invendon include
20 neurodegeneradon following e~citotoxicity, HIV-induced neuropashy, ischenua, denervation
foJlowingischemia orinjury, subarachnoid hemorrhage, stroke, multipleinfarction dementia,
Alzheimer's Disease (AD), Parkinson's Disease, Huntington's Disease, surgery-related brain
damage and other neuropathological conditions.
As stated above, spectrin BDP's have been found to be associated with Calpa~n
25 activation in vivo, We have obsened that in each instance of neurodegeneration in which
BDP's characteristic of Cal~ain activation are detected, Calpain activation is localized to the
brain areas most vulnerabIe to the particular pathogenic manipulation. Isl addition, as
judged by histological methods, C:alpain activation precedes overt evidence of
neurodegenerat;on. Accordingly, CalpaM activation is spatially and temporally lir~ced to
30 impending or ongoing ce~l death in the brain. Thus, we believe that Calpain activation is
an important mechanism of cell damage and death in many pathological conditions,induding neuropathological conditions. Moreover, there is eYidence that the activation
of Calpains is an early event in the death of cells including neural cells. This is in contrast
to other known proteases which are activated at later stages of cell death. Thus, we believe
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wo 92/11850 2 0 9 8 6 a 9 pcr/us91~o978 ~
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that, advantageously, inhibition of Calpain activity provides intervention at an early stage
of cell death, pnor to significant deterioration of ee1lular machinery.
Another aspect of the involvement of Calpains in neurodegeneration is the
involvement of these proteins in regenerating systems. It is Icnown that developing or
regenerating axons are somehov 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 stab;lization can also occur at inappropriate places.
One researcher has de~eloped evidence that this stop pathway operates at least in part by
the activation of intracellular Calpain and that in11ibition of Calpain can interfere vnth
stabilization (Luizi, 1990). We believe that Calpain inhibitors, when used in accordance
with the present invPntion, can advantageously aid regeneration and recovery of neural
tissue after injury, in addition to inhl~iting neurodegeneration.
Another aspect of the present invention is our diseovery that at least three classes
of compounds, the substituted isocournarins, 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 e~pecially useful as therapeutically effective compounds in the ereatment
of neurodegenerative conditions and diseases.
B. SUBSTl~TED HEleROCYCIIC COMPOU~DS
One particular class of compounds exhibiting Calpain inhibitory activity, ~hen used
in accordance with the present invention, are the substituted heterocyclic compounds. These
compounds include the substituted isocoumarins. The substituted heterocyclic compounds
are known to be excellent inhl~itors of serine proteases. As discussed hereinbelow, we have
now discovered that these compounds are also inhibitors of calpain I and calpa~n I~, and also
2~ of other Calpains. Additionally, as also diuussed below, we have found that, unlLke most
~ known inhibitors of Calpains, these substituted heterocyclic compounds are not effective as
ir~ibitors of papain or cathepsin B. Thus, we believe that the sl~bstituted heteros~yclic
compounds provide a relatively specific means of inhibiting Calpams while not affecting
other thiol proteases.
One particular dass of substituted heterocyclic compounds with Calpain inhibitory
' aceivity are the isocoumarins having cationic substituents. These substituted hetero~yclic
compounds are referred to herein as the "Class I Substituted Isocoumanns." The aass I
Substituted Isocoumarins are known to be e~cellent inhibitors of several serine proteases,
induding bovine thrombin, human thrombin, hu nan factor Xa, human factor X~a, lluman
!E~IUE~STIITU~E SH~T
~ WO92/11~,50 ~ 36~ PC~/US91/0~7f~6
factor X[Ia, bovine trypsin, human plas,ma plasmin, hurnan tissue plasrninogen activator,
hurnan lung tryptase, rat skin tryptase, human leukocyte elastase, porcine pancreatic
elastase, bovine ch,vmotrypsin and human leuko~yte cathepsin G. The Class I Substituted
Isocoumarins inhibit the serine proteases by reaction with the active site serme to form an
acyl er~ne, which in some cases may further react with another active site nucleophile to
form an additional covalent bond. We have discovered that the Class I Substituted
Isocoumarins also react with Calpain. We believe that the mechanism of action of Calpain
inllibition is similar to that of the iinh~bition of serine proteases since the reaction
mechanism of Calpains is similar to that of the serine proteases.
The Class I Subsdtuted Isocoumarins having Calpain in~u~itory activity have the
follo~ing structural forrnula:
~ O
(I) ~2
or a phannaceutically acceptable salt, wherein
Z is selected from the group consisdng of C1-6 aLlco~y with an amino group attached
to the alkoxy group, C1-6 alkoxy with an isothiureido group attached to the aL~coxy group,
Cl-6 aL~coxy with a guanidino group attached to the aL~coxy group, C1-6 allcoxy with an
a nidino group attached to the allco:y group, C1-6 alkylwith an arnino group attached to the
alkyl group, C1-6 allyl with an isothiureido group attached to the allyl group, C1-6 aLlcyl
with an guanidino group attached to the allyl group, C1-6 aLcyl with an an~idino group
attached to the alkyl group,
R is selected from the g;oup consist~ng of O=C=N-, S= C=N-, AA-NH-, AA-AA-
NH~ O,AA-AA-O-, M-NH-, M-A~A-NH, M-AA-AA-NH-, M-O-, M-AA-O-, M-AA-AA-
O-,
wherein AA repre~,erlts alanine, valine, leucine, isoleucine, proline, methionine,
phenylalanine, tryptophan, glycine, serine, tnreonine, cysteine, tyrosine, beta-alanine,
~BSTITUT SHE.ET
.: , ' : -
WO 92/11850 2 a 9 8 6 0 9 PCr/lJS91/0978
-12-
norleucine, nonaline, alpha-aminobutyric acid, epsilon-aminocaproic acid, citrulline,
hydroxyproline, ornithine or sarcosine,
wherein M represents NH2-CO-, NH2~ , NH2-S02-, X~ CO, X-NH-CS, X-
NH-S02, X-CO-, X-CS-, X-S02-, X-O-CO-, or X-O-CS-,
S wherein X represents C1-6 allyl, C1-6 fluoroalkyL C1-6 aLtcyl substituted with K,
C1-6 fluoroalkyl substituted wilh K, phenyl, phenyl substituted with J, phenyl disubstituted
with J, phenyl trisubstituted with J, naphthyl, naphthyl subsdtuted ~ith J, naphthyl
disubstituted with J, naphthyl trisubstituted with J, C1-6 aLkyl with an attached phenyl group,
C1-6 allyl with two attached phenyl groups, C1-6 allyl with an attached phenyl group
substituted with J, or C1-6 aL~cyl with t~o attached phenyl groups substituted with J,
wherein J represents halogen, COOH, OH, CN, N02, C1-6 allyl, C1-6 aL~coxy, C1-6
all~lamine, C1-6 dialkylarnine, or C1-6 alkyl-O-CO-,
wherein K represents halogen, COOH, OH, CN, N02, NH2, C1-6 aLkylamine, C1-6
diaL'cylan~ine, or C1-6 alkyl-O-CO-,
Y is selected from the group consisting of H, halogen, trifluoromethyL methyl, OH
and metho~y.
The compounds of Formula (I) can also contain on or more substituents at position
B as shown in the following structure:
8 0
R~ o
E3~2
y ` -
wherein electronegative substituents such as N02, CN, CI, COOR, and COOH will
increase the reactivity of the isocou narin, and electropositive substituents such as NH2, OH,
aLcoxy, thioallyL allyl, allylamino, and diaLcylamino will increase its stability. Neutral
substituents could also increase the stability of acyl enzgme and improve the effectiveness
of she inhibitors.
The following compounds are representative of the Class I Substituted Isocoumarins
of ehe present invention:
S~ STITUTE SHEE~ T
`-!W092/ll850 2098~a9 PCr/US91/09786
.
4-chloro 3-(3-isothiureidopropoxy)isocoumarin (CiTPrOIC)
7-(benzylcarbamoylamino)4-chloro-3-(3-
isothiureidopropoxy)isocoumarin (PhCH2NHCONH-CiTPrOIC)
7-(phenyl~arbamoylamino)~-chloro-3-(3-
isothiureidopropoxy)isocoumarin (PhNHCONH-CiTPrOIC)
7-(acetylarnino)~-chloro-3-(3-
10 ~sothiureidopropoxy)isocoumann (CH3CONH-CiTPrOIC)
7-(3-phenylpropionylarnino) 4~hloro-3-(3-
isothiureidopropoxy)isocoumarin (PhCH2OE12CONH-CiTPrOIC)
7-(phenylacetylamino)4-chloro-3-(3-
isothiureidopropoxy)isocoumarin (PhCH2CONH-CiTRrOIC)
7-(L-phenylalanylamino)~-chloro-3-(3-
isothiureidopropoxy)isocoumarin (L-Phe-NH-CiTPrOIC)
7-(N-t-butyloxycarbonyl-I~phenylalanylamino) 1-chloro-3-(3-isothiureidopropoxy)isocoumarin
(Boc-L-Phe-NH-CiTRrOIC)
7~ phenylalanylamino) 4-chloro-3-(3-
isothiureidoprapoxy)isocoumarin (D-Phe~ CiTPrOIC)
7-(N-t-butyloxycarbonyl-D-phenylalanylam~no)4-chloro-3-
(3-isothiureidopropoxy)isocoumarin (Boc-D-Phe~ ClTPrOIC)
7-(benzylcarbamoylamino)4-chloro-3-(2-
isothiureidoethoxy)isocoumarin (PhCH2NHCONH-ClTEtOIC)
7-(phenylcarbamoylamino)4-chloro-3-(2-
isothiureidoethoxy)isocou~narin (PhNHCONH-CiTEtOIC)
3~ .
7-(isopropylcarbamoylamino)4-chloro-3-(2-
isothiureidoethoxy)isocoumarin ((CH3)2CHNHCONH-CiTEtOIC)
7-(phenylacetylamino)-4-chloro-3-(2-
isothiureidoethoxy)isocoumarin OEhCH2CONH-CiTEtOIC)
7-(L-phenylalanylamino)-4-chloro-3-(2-
isothiureidoethoxy)isocoumarin (L-Phe-NH-Cl~ tOIC)
7-(N-t-butylo~ycarbonyl-L,phenylalanylamino)-4-chloro-3-(2-isothiureidoetho~y)isocoumarin
(Boc-L-Phe-NH-Cl~EtOIC)
7-(D-phenylalanylamino)-4-chloro-3-(2-
isothiureidoethoxy)isocoumarin (D-Phe-NH-CiTEtOIC~
SlJBSrlllJTE SHE~T
WO 92/11850 2 ~ 9 8 6 0 9 Pcr/usgl/097~! ~
-14-
7-(N-t-butylo~ycarbonyl-D-phenylalanyl~nino)4 chlo}o-3-(2-isothiureidoetho~y)isocoumar~n
(Boc-D-Phe~ Cl~EtOIC)
7-(N-t-butyloxycarbonyl-L-alanyl-L-alanylamino)-4-chloro-3-(2-
isothiureidoethoy~isocoumarin (13Oc-Ala-Ala-NH-CiTEtOIC)
7-(L-alanyl-L-alanylamino)~-chloro-3-(2-
isothiureidoethoxy)iso~oumar~n (ala-Ala-NH-ClTEtOIC3
7 (1-naphthylcarbamoylamino)~-chloro-3-(2-
isothiureidoethoy)iss)coumarin (:NaphthylNH-CiTEtOIC)
7-((S) a methylben~ylcarbamoylamino)~-chloro-3-(2-
isothiureidoetho~y)isocoumann (S-C6H5(CH3)CHNHCONH-C~ tOIC3
15 7~((R)-a-methylbenzylcarbamoylarnino)-4 chloro-3-(2-
isothiureidoetho~y)isocoumarin (R-C6H5(CH3~CHNHCONH-Cl~EtOIC)
7-das~sylan~ino~-chloro-3-(2-isothiureidoetho~y)isocoumarin (DansylNH-CiTEtOIC)
7-phenylthiocarbamoylamino~-chloro-3-(2-
isothiureidoeth~xy)isocournarin (PhNHCSNH-Cl~EtOIC)
7-(m~arboxyphenylthiocarbamoyl)amino 1-chloro-3-(2-
isothiureidoetho~y)isocoumarin (m-COOH-PhNHCSNH-CiTEtOIC)
7-(p~arboxyphenylthiocarbamoyl)amino 4-chloro-3-(2-
isothiureidoethoxy)isocoumarin (p-COOH-PhNHCSNH-CiTEtOIC)
7-amino~-chloro-3-(3-isothiureidopropoxy)isocoumarin
(AClTIC)
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 tbe other substituted heterocyclic compounds, is
believed to be effective in the use of the present invention.
SI~E'r
WO 92/11850 2 ~ ~ ~ 6 a ~ PCI`/US91/09786
", .
-15
The Class II Substituted Isoc~umarins have the following structural formula:
O
S p~ O
~Z
Y
or a pharmaceutically acceptable salt, wherein
R is selected from the group consisting of -N-H-C(=NH)-NH2, -C(=NH)NH2, Cl 6
aL~cyl with an attached amino, and Cl~ alkyl with an attached isothiureido of the formula -
s-c(+NH2+)N~2~
Z is selected from the group consisung of H, halogen, Clb allyl, Cl 6 allyl with an
attached phenyL Cl 6 ~uorina~ed aLlyL Cl 6 allyl with an attached hydroxyl, Cl 6 allyl with
an attached Cl~ alkoxy, Cl.6 aLtco~y, Cl 6 fluorinated alko~y, Cl 6 aL~co~y with an attached
phenyL benzyloxy, 4-fluorobenylo~y, -OCH2C~I 4R' (2-substituent), -OCH2C6H4R' (3-
substituent), OCH2C6H4R' (4-substituent), -OCH2C6H3R2' (2,3-substituents), -
OCH2C6H3R2' (2,4-substi~uents), -OCH2C6H3R2' (2,5-substituents), -OCH2C6H3R2' (2,6-
substituents), -OCH2C6H3R2' (3,4-substituents), and OCH2C6H3R2' (3,5-substituents).
R' is selected from the ~oup consisting of H, halogen, trifluoromethyL N02, 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 Substityted Isocoumarins are represented by structure pI)
where,
Z is selected from the group consisting of C~ coxy with an attached isothiureido,
Cl 6 alkoxy with an a~tached guanidino, Cl 6 aL~coxy with an attached amidino, Cl.6 aLlcyl with
an anached amino, Cl 6 allcyl with an attached isothiureido, Cl 6 allyl with an attached
guanidino, Cl.6 aLlcyl with an attached asnidino,
R is selected from the group consisting of H, OH, NH2, NO2 halogen, C~ coxy,
C1.6 fluorinated aL~coxy, Cl 6 alkyl, C1 6 allyl with an attached amino, ~ , M-AA-O-,
~. ~n~ n~rt 1T~ C~FFT
WO 92/11850 2 0 9 8 6 0 9 Pt~r/US91/t)97~i .
-16-
wherein AA represents alanine, valine, leucine, ~soleuc~ne, proline, methior~ne,phenylalanine, tryptophan; glycine, ser~ne, threonine, cyste~ne, tyrosine, asparagine,
glutamine, aspartic acid, glutan~ic acid, lysine, ar~nine, histidine, beta-alanine, norleucine,
norvaline, alpha-aminobutyric and epsilon-aminocaponic acid, ci~rulline, hydroxyproline,
on~ithine and sarcosine,
wherein M represents H, lower alkanoyl having 1 to 6 cazbons, carbo~ canoyl,
hydroxyaLkanoy1 amin-alkanoyL benzene sulfonyl tosyL benzoyl, and lower aLlcyl sulfonyl
hav~ng 1 to 6 carbons,
Y is selected from the group consist~ng of H, halogen, trifluorome~hyL methyl OHand 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,
allyl with an attached an~ino, Cl 6 allyl with an attached isothiureido,
Z is selected ~om the group consisting of Cl 6 alkoxy with an attached amino, Cl 6
alkoxy with an attached isothiureido, Cl 6 alkoxy with an auached guanidino, Cl 6 aL~;ox~
with an attached amidino, C1 6 alkyl with an attached arnino, Cl 6 alkyl with an attacheo
guanidino, Cl 6 alkyl with an attached amidino,
Y is selected from the group consisting of H, halogen, trifluoromethyL methyL OHand methoxy.
The following compounds are representative of the Class II Substituted
Isocoumarins:
3-(3-ammopropoxy)isocoumarin,
3-(3-ammopropoxy)~-chloroisocoumar~n,
3-(2-isothiureidoethoxy)4-chloro~socoumarin,
3-(3-isothiureidopropoxy)4 chloroisocoumarin,
7-amino-3-(3-isothiureidopropoxy)~hloroisocoumarin,
7-g,uanidino-3-methoxyisocoumarin,
7-guanidino-3-methoxy 4-chloroisocoumarin,
7-guar~idino-3-etho~yisocournarin,
7-guanidino-3-ethoxy~hloroisocournarin,
7-guanidino-3 -(2-phenylethoxy)isocoumarin,
7-guanidino-3-(2-phenyletho~y)~-chloroisocoumarin.
S~J71~E SWEET
WO 92/11850 2 ~ 9 ~ PCT/US91/09786
17~
Still another class of susbstituted heterocyclic compounds useful in the presentin~ention ~s referred to herein as the "Class m Heterocyclic Compounds" ~nd have the
follow~ng structural formula:
.
~Y
(m) ~ x 1~
wherein
Z is selected from the group consisting of CO, SO, SO2, CCl and CF,
Y is selected from the group consisting of O, S and NH,
X is selected from the group consisting of N and CH, and
R is selected from the group consisting of Cl 6 aLtcyl (such as methyl, ethyl and
propyl), Cl ~ allyl containing a phenyl (such as benzyl), and Clb fluoroaL~cyl (such a~
trifluoromethyL pentafluoroethyL and hepta~uoropropyl).
The Z group must be electrophilic since it interacts with ~he active site serine OH
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 ~oup maintains its hydrophobic character.
The following compounds are representatire of the Class m Heterocyclic
Compounds:
2-trifluorome~hyl 1H-3,1-benzo~azine4-one,
2-pentafluoroethyl-4H-3,1-benzoxazine-4-one,
2-heptafluoropropyl-4H-3,1-benzoxazine-4-one,
2-methyl4H-3,1-benzoa~ine~one,
2-propyl-4H-3,1-benzoa~azine-4-one,
2-benzyl-4H-3,1-be~L~oaxa~ine-4-one,
2-hepta~uoropr3pyl4-quinazolinone,
2-propyW-quina~olinone,
2-benzyl-4-quinazolinone,
SU3S~lTU~E SHEET
,
'`.. . ` ~ . `
.` ` `
` . ~`. ` ~. ~ . ` .
WO 92/11850 2 ~ 9 g ~ 9 PCI/US91tO978 ;~`
-18-
2-(C6HsCCl2)4~hloroql~inazoline, and
2-propyl-4-chloroql~ina~oline.
The Class m Heterocyclic Compounds are disclosed in Powers et al., U.S. Patent No.
4,847,202, the disclssure of which is hereby incorporated by reference.
Other substituted heterocyelic ~ompounds have been prepared earlier for other
purposes, such as 3-chloroisocoun~arin, Davies and Poole, J. Chem. Soc., pp. 1616-1629
(1928); 3~hloro and 3,4-dichloroisocoumarin, Milevslcaya, Belins3caya, and Yagupol'sku,
Zhur. Org. Xhim. 9, pp. 2145-2149 (1973); 3-methyl and 4 carbo~y-3-methylisocoumarin,
Tirodkar and Usgaonkar, Ind. J. Chem. 7, pp. 1114-1116 (1969); 7-nitro and 7-
aminoisocoumarin, Cholcsey and Usgaonlcar, Ind. J. Chem. 14B, pp. 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-chloroisocournarin, Harper, Hemrr~i, and Powers, J. A. Chem. Soc.
105, pp. 6518-6S20 (1983); 3,4-dichloroisocournarin, Harper, Hemmi, and Powers,
Biochemistly 24, pp. 1831-1841 (1985); 3-alkoxy-7-arnino~chloroisocoumarin, Harper and
Powers, J. Am. Chem. Soc. 106, pp. 7618-7619 (1984), ~Iarper and Powers, Biochemistry 24,
7200-7213 (1983); additional substituted isocoumarins with basic groups (aminoalkoxy,
guanidino or isothiureidoallcoxy), Kam, Fujikawa and Power~, Biochemistry 27, pp.
2S47-25S7 (1988); 7-substituted 3-~lko~y~^chloroisocoumarins, Powers, K~m, Narasimhan,
Olelcsys~yn, Hernandez and Ueda, J. Cell Biochem. 39, pp. 33-46 (1989) and Powers,
Oleksyszyn, Narasimhan, Kam, Radhakrishnan and Meyer, Jr. Biochemistry 29, 3108-3118
(1990). The disclosures of all of the preceding articles are herebyincorporated by reference.
We believe that the foregoing compounds, which exhibit serine pro~ease inhibitory activity,
also e~hibit Calpa~n inhbitory acti~ity when used in accordance with the pres.ent invention.
All of the ~oregolng isocoumarin compounds, including the Class I and II Substitu~ed
Isocoumarins, the Class m Substituted Heterocyclic Compounds and the other substituted
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 par~icular species of these compounds.
The preparation of the various Substituted Heterocyclic Compounds is illustrated by
Examples SHC1-SHC9.
~:U8~:~TUTE SHEET
. .
.
W0 92/11850 2 0 9 ~ 6 ~ ~ PCl'/US91/09786
-19-
EXAMPLE SHCl
Preparation of 7-(phenylcarbamoylam~no)~4-chloroisocoumar~n was synthes~zed as previously
described (Powers, e~ aL, Biochem~stn~ 29, 3108-3118 (1990)). This compousld (0.32 g 1
mtnole) was mLYed with phenyl isocyanate (0.12g, 1 mmole) in 5 ml of THF and the reaction
S rni~ture was st~Ted at r.t. overnight. The product 7-(phenylcarbamoy~am~no)~-chloro-3-(2-
bromoethoxy)isocoumarm precipi~ated out, yield 40~o, m.p. 215-217 C, mass spectrum m/e
= 437.9 (M~ Anal. Calc. for Cl8Hl4N204ClBr: C, 49.4~; H, 322; N, 6.40; Cl, 8.10.Found: C,49.48; H, 3 25; N,634; Cl, 8.12. The phenylcarbarnoylan~no compound (0.1 g,
0.23 ~slole) was heated with 0.02 g of thiourea (0~6 mmole) in 10 ml of THF at 70C
overmght. The final product prec~pitated out, yield 0.04 g, 36%, m.p. 161-163C (dec.),
mass spectrum (FAB+) m/e = 433 (M-Br). Anal. Calc. for ClgHl8N404ClBrS: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-isothiureidoetho~y)isocoumarin,
7-(benzylthiocarbamoylamino)-4-chloro-3-(2-isothiureidoethoxy)isocoumarin, 7-
(ethylthiocarbamoylamino)~-chloro-3-(2-isothiureidoethoxy)isocoumarir~-(4-fluorobenzyl)
thiocarbamoylamino4-chloro-3-(2-isothiureidoethoxy) isocoumarin, and 7-(2,5-
dimethylbenzyl) thiocarbamoylamino4-chloro-3-(2-isothiureidoethoxy) isocoumarin can be
prepared by the same procedure.
EXAMPLE SHC~2
Preparation of 7-(acetylamino)~-chloro-3-(3-isothiureidopropoxy) isocoumarin:
7-Amino-3(3-bromopropoxy)~chloroisocoumarin was synthesi2ed as previously
described (Kam, et al., 1988). This compound (033 g, 1 mmole) was heated with 0.15 g of
acetic anhydride (1.5 mmole) in 20 rnl of dry Th~. After a few n~inutes, a yellow solid
precipitated out. After 3 hrs, the solution was concentrated to 5 ml, and the solidr was
filtered to give 037 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, 0.4mmole) was treated with thiourea (0.036 g, 0.47 mmole) to ~ive 0.9 E~ o~ the final product,
(yield 50%), m.p. 180-181C, maæ spectrum m/e = 370 (M+-Br). Anal. Calc. for
Cl5Hl7N304ClBrS: C, 39.97; H, 3.80; N, 932; Cl 7.87. Found: C, 39.86; H 3.83; N, 9.29;
CL 7.~5.
7-trifluoroacetylamino-4-chloro-3-(3-isothiureidopropoxy) isocoumarin, 7-
hepta~uorobutyroylamino 1-chloro-3-(3-isothiureidopropoxy)isocoumarin,7-succinylamino-
~n ~ 8~
.. ,, . ~ .-
.
. ~ ... . .
., ~ , -
.. . . . ..
WO 92/118~0 2 ~ 9 8 6 0 9 PCI/lJS91/0978~
-20 -
4-chloro-3-(3-isothiureidopropoxy) isocoumarin, and 7-(o-phthalyl)amino~-chloro-3 (3
isothiureidopropoxy) isocoumarisl can be prepared by the sarne procedur~.
E:XA~'LE SlH~
Preparation of 7 (benzylcarbamoylarnino)~-chloro-3-(3-isothiureidopropO~y)
isocouunarin:
7-(benzylcarbamoylamino)-4~chloro-3(3-bromopropo~y) isocoumarin was prepared
from the reaction of benzyl isoqanate with 7-amino4-chloro-3-(3-bromopropoxy)
isocoumarin as descn~ed above, m.p. 188-189C, mass spectrum. m/e = 359 (M+ -ben~yl).
The final product was obtained ~om the reaction of 7-(ben~ylcarbamoylamino)4-chloro-3-
(3-bromopropo~y) isocoumarin with thiourea as described aboYe (yield 74%), m.p.
165-166C; mass spectrum (FAB+) m/e - 461 (M+-Br). AnaL Calc. for
C2lH22N404Cl~3rS:0.75 1~: C, 48.36; H, 4.70; N, 9.40; CL 6.56. Found: C, 48.13; H, 4.87;
N, 9.65j Cl, 6.15.
EXAMPLE SHC4
Preparationof7-(phenylacetylamino)~-chloro-3-(2 isothiureidoethoxy)isocoumarin:
7~Amina-4-chloro-3 (2-bromoethoxy) isocoumarin (0.15 g, 0.47 mmole) was first
n~Lxed 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 mi~ture was stirred at r.t. overnight. After
Et3NHCl salt was removed by filtration, the product 7-(phenylacetylamino)~-chloro-3-(~-
bromoethoxy) isocoumarin was crystallized from l~ 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 product 0.05 g (yield, 40%), m.p. 115-120 C;
mass spectrum (FAB+) m/e - 432 (M+ -Br). Anal. Calc. for C20HlgN304ClBrS 0.5 H20:
C 45.9~; 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-~-methylbenzylcarbamoylamino)-4-chloro-3-(2-
isothillreidoethoxy) isocoumann:
7-(R a-methylbenzylcarbamoylamino) 1 -chloro-3-(2-bromoelhoxy) isocoumann was
synthes~zed 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 de~cribed above to form the finalproduct 7-(R-a-methylben~ylcarbamoylamino) 1 -
chloro-3-(2-isothiureidoetho~y) isocoumarin (0.078 g), m.p. 143-150C; mass spectrum
(FAB+) m/e 5 461 (M+ -Br). Anal. Calc. for C2lH2;~N404ClBrS0.5H20: C, 45.75; H,
4.35; N, 10.17; Cl, 6.M. Found: C, 44.95; H, 4.31; N, 10.02; CL 6.36.
Sl3BSm~UTE SHE~ET
.
: . :. -
. WO 92/118~iO 2 8 ~ ~ ~ 0 9 PC~/US91/09786
EX~MPLE SHC6
P~eparatio!l of 7-(D-phenylalanylamino)-4-chloro-3(2-isothiureidoethoxy)
isocousnarin:
- soc-D-phe (0.33 g, 1.2 mrnole) reacted with 1,3-dicyclohexylcarbodiimide (0.13 g, 0.6
- S mmole) in 10 ml THF at 0C for 1 hour ~o forrn the symmetric anhydride, and then
7-amino-4~hloro-3(2-bromoetho~y) isocoumarin (0.2g,0.6 mmole) was added. The reaction
was stirred at r.t. overnight and the precipitate 7-(Boc-D-Phe-ammo)~-chloro-3-
(2-bromoethoxy) isocoumarin was formed (0.29 g, 71%). TLC one spot, m.p. 180-182 C;
mass spectrum m/3 = 566(M~). Anal. Calc. for C25H2~jN206ClBr: 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. E}oc-D-Phe compound (0.2 g, 0.35
rnmole) was reacted with thiourea (0.027 g, 035 mrnole) in the same maMer 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). Thi5 compound (Q.1 g) was dissolved in 3 rnl
of l~ 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
spectnlm (FAB+) m/e = 462 (M+ -Br -CF3C00).
7-Boc-alanylamino-4-chloro-3-(2-isothiureidoethoxy) isocoumarin, 7-benzoylamino-Ala-4-chloro-3(2-i~.othiureidoetho~y) isocoumarin, 7-benzoylamino-Phe-4-chloro-3-(2-
isothiureidoethoxy) isocoumarin and 7-Boc-valylamino4-chloro-3-(2~isothiureidoethoxy)
isocoumarin can be prepared by the same procedure.
EXAMPLE SHC7
Preparation of 7-(Boc-alanylalanylamino)-4-chloro-3-(2-isothiureidoethoxy)
isocoumarin:
7-(Boc alanylalanylamino)-4-chloro-3-(2-bromoethoxy) isocoumarin was synthesized2S 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 ~orm 7-(Boc-alanylalanylamino)-4-chloro-3-(2-
isothiureidoetho~y) isocoumann (0.04 g), mass spectrwTI m/e = 556 (M+ -Br).
7-(Alanylalanylamino)-4-chloro-3(2-~sothiureidoethoxy) isocoumarin was prepared
hy deblocking of 130c-Ala-Ala-NH-CiTEtOIC with trifluoroacetic acid, mass spectrum
` (FAB~ m/e - ~56 (M~ -Br-CF3COO).
EXAMPLE SHC8
Preparation of 7-(phenylthiocarbamoylamino) 1-chloro-3-(2-isothiureidoethoxy)
isocoumarin:
~IQ~,~TttTF ~LJI~=T
wo 92/11850 2 ~ ~ 8 6 0 3 PCI/US91/0978~
-2~-
7-(Phenylthiocarbamoyl~mino)~chlorG3-(2-bromoethoxy) isocournal~n was prepared
from the re~ction of phenyl isothiocyanate with 7-amino~-chloro-3-(2-bromoe~hoxy)
isocournarin,yield59%,m.p. 157-158C;massspectrwnm/e = 361 (M~ -PhNH+1). Anal.
Calc.: C, 48.36; H, 3.39. Found: C, 48.26; H, 3.40. The bromoetho~y compound was then
reacted with thiourea by the same procedure to give the final product, yield 32%; mass
spectrum (FAB+) m/s 449 (M+ -Br~.
EXAMPLE SHC9
Preparationof7-(m-carboxyphenylthiocarbamoylamino)~-chlo~o-3-(2-bromoethoxy)
isocoumarin was prepared from the reaction OI m~arboxyphenyl isothiocyanate with 7-
amino-4-chloro-3-(2-bromoetho~zy) isocoumarin, yield 64%, m.p. 157-158 C; mass spectrum
m/e 361 (M+ -(COOH)PhNH+-Br).
7-(3-Fluorobenzoyl)a~uno 1-chloro-3-(2-isothiureidoethoxy) isocoumarin, 7-(3-
nitrobenzoyl)amino-4-chloro-3-(2-isothiureidoethoxyysocoumarin,7-diphenylacetylarnino-4-
chloro-3-(2-isothiureidoeshoxy) isocoumarin, 7-diph~nylpropionylanuno~-chloro-3-(2
isothiureidoethoxy) isocoumarin, 7-(p-toluenesulfonyl) amino-4-chlo~o-3-(2-
isothiureidoethoxy) isocoumarin, and 7-(~-toluenesulfonyl) amino~-chloro-3-(2-
isothiureidoethoxy) isocoumarin can be prepared from the reaction of corresponding 7-
substituted-4-chloro-3-(2~bromoethoxy) isocoumarin with thiourea as descnbed above. 7-
substituted-4-chloro-3-(2-bromoethoy) isocoumarin can be synthesized by reacting 7-amino-
4-chloro-3-(2-bromoethoxy) isocoumarin with appropriate acid chloride or sulfonyl chloride
in the presence of Et3N.
7-Ethoxycarbonylamino4-chloro-3~(2-isothiureidoethoxy) isocoumarin, 7-
benzyloxycarbonylamino-4-chloro-3-(2-isothiureidoethoxy) isocoumarin, and 7-
phenoxycarbonylan~ino-4-chloro-3-(2-isothiureidoethoxy) isocoumarin c~n be prepared from
T~ 25 the reaction of 7 substituted-4-chloro-3-(2-bromoethoxy) isocoumarin wi~h ~h-iourea. 7-
Ethoxycarbonylamino4-chloro-3-(2-bromoethoxy~socownarin,7-benzylo;~ycarbonylan~ino~-
chloro-3-(2-bromoethoxy) isocouunarin and 7-phenoxycarbonylamino-4~hloro-3-(~-
bromoethoxy) isocoumarin c~n be synthes~zed by reac~ing 7amino-4-chloro-3-(2-
bromoethoxy) isocoumarin with the corresponding chloroforrrlate.
30 C. PE~IlPE KETO-CO.MPOUNDS
Peptide a-ketoesters, peptide a-ketoacids, and peptide a-ketoamides are transition
state analog inhibitors for serine proteases and cysteine proteases. While these subclasses
of compounds are chemically distinguishable, for simplicity, all of these compounds will be
referred to collectively herein as the "Peptide Keto-Compol~ndsn.
SU~3~TITVTE S~E~
. . . . .
WO 92/1~850 2 ~ 9 ~ ~ Q 9 Pcr/us91~o9786
-23-
The ~nserac~ions of peptides with serine and cysteine proteases are designated herein
us~ng the nomenclature of Schechter, I., and Berger, A., 1967, Biochem. Biophys. Res.
Corr~nun. 27: 157-162 (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 band of the substrate is
P1-P1'. The pIimary recognition site of serine proteases is S1. The most important
recognition subsites of cysteine proteases are S1 and S2.
Amino acid residues and ~locking groups are designated using standard abbreviations
[see J. Biol. Chem. 260, 14-42 (1985) for nomenclature rules; incosporated herein by
reference]. An ar~ino acid residue (AA) in a peptide or inhibitor structure refers to the
part structure -NH-C~IR1-CO-, where R1 is the side chain of the ar~uno acid AA. A
peptide a-ketoester residue would be designated -AA-CO-OR which represents the part
structure -NH-CHR1-CO-CO-OR. Thus, the ethyl ketoester derived f~om benzoyl alar~ine
would be designated Bz-Ala-CO-OEt which represents C6H5CO-NH-CHMe-CO-CO-O~t.
Likewise, peptide ketoacid residues residues would be designated -AA-CO-OH. Further,
peptide ketoamide residues are designated -AA-CO-NH-R l~hus, the ethyl keto amide
derived from ZLeu-Phe-OH would be designated ZLeu-Phe-CO~NH-Et which represents
C6H5CH20CO NH-CH(CH2CHMe2)-CO-NH-CH(CH2Ph)-C:O-CO-NH Et.
Peptide a-ketoesters containing amino acid residues with hydrophobic side chain at
the P1 site have also been found to be excellent inhîbitors of several cysteine proteases
including papain, cathepsin B and calpain.
Calpains can be inhibited by peptide inhibitors having several different active groups.
Structure-activity relationships with the commercially available in vitro inhibitors of Calpain,
such as peptide aldehydes, have revealed that Calpains strongly prefer Leu or Val in the P2
position. These enymes are inhibited by inhibitors having a wide variety of amino acids
~n the P1 position, but are generally more effectively inhibi`ted by inhibitors ha~ing amino
acids with nonpolar or hydrophobic side chains in the P1 position. Thus, we have discovered
that another particular class of compounds exhibiting Calpa~n inhibitory activity, vhen used
in accordance ~vith the present invention, are the Peptide Keto-Compounds. These are
compounds of the general structure:
., O
Il
M-~aa)n-C-Q-R
:
~'
8lJB~J'rE S~EE~
': " ' - ` ' ` `
WO92/11850 2~9~603 PCI/US91/097~
-24-
or a pharmaceutically acseptable salt, wherein:
M represents NH2-CO-, NH2-CS-, NH~-S02-, X-NH-CO-, X-NH~
X-NH-S02-, X-CO-, X-CS-, X-S02-, X-O-CO-, or X-O-CS-, H, acetyl, carbobenzo~v,
succinyL methylo~ysuccinyl, butyloxycarbonyl;
X is selected from the group consisting of C1-6 aL~cyL C1-6 fluoroallyL C1-6
aLtcyl substituted with J, C1-6 fluoroaLlcyl substituted with J, 1-admantyL 9-fluorenyL
phenyl, phenyl substitu~ed with K, phenyl disubstituted with K, phenyl trisubstituted
with K, naphthyl, naphthyl substituted with K, naphthyl disubstituted with K,
naphthyl trisubstituted with K, C1-6 alkyl with an attached phenyl ~oup, C1-6 allyl
with two attached phenyl groups, C1-6 aLkyl with an attached phenyl group
substituted with K, and C1-6 allyl with t~o attached phenyl groups substituted with
K; .
J is selected from the group consisting of halogen, C(:)OH, OH, CN, N02,
NH2, C1-6 aL~coxy, C1-6 alkylamine, Cl-6 diallylarnine, C1-6 alkyl-O-CO-, C1-6
aL'cyl-O-CO-NH, and C1-6 aL~cyl-S-;
K is selected from the group consisting of halogen, C1-6 all~yL C1-6
perfluoroalkyL C:1-6 alko~y, N02, CN, OH, C02H, arnino, C1-6 allylamino, C2-12
diallylarnino, C1-C6 a~yl, and C1-6 allcoxy-CO-, and C1-6 aL~cyl-S-;
aa represents a blocked or unbloclced amino acid of the L or D
configuration, preferably selected from the group consisting of: alanine, valine,
leucine, isoleucine, proline, methior~ine, methionine sulfoxide, phenylalanine,
tryptophan, glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine,
aspartic acid, glutamic acid, lysine, arginine, histidine, phenylglycine, beta-alanine,
norleucine (nle), no~aline (nva), alpha-asl~inobutyric wid (abu), epsilon-
aminocaproic acid, citrulline, hydro~yproline, homoarginine, ornithine or sarcosine;
n is a number ~om 1 to 20;
Q is O or NH,
R represents H, C1-6 allyL C1-6 fluoroallyL C1-6 chloroal}yl bei~yl, Cl-6
aLlcyl substituted with phenyl, C1-6 al}yl 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 un also be class ied
based on the number of an~ino acids contained within the compound, such as an an~ino acid
peptide, dipeptide, tripeptide, tetrapeptide, pentapeptide and so on.
SUB~3~1TUTE SHE~T
WO 92t11850 ~ ~ 9 ~ ~ ~ 3 PCr/US91/09786
We have ~ound certa~n subelasses of Peptide -Ketoester compounds to be
particularl~ useful as Calpain Inhibitors when used in accordance with the present invention
These sub~lasses are ~eferred to herein as the Dipeptide ~-Ketoesters (Subclass A), the
Dipeptide ~-~etoesters (Subclass B), the Tripeptide a-Ketoesters (Subclass A), the
Tripeptide a-Ketoesters (Subc:lass B), the Tetrapeptide a-Ketoesters and the Amino Acid
Peptide a-Ketoes~ers. All of these subclasses are considered to be to be within the class of
Peptide Keto-Compounds.
The Dipeptide a-Ketoe~ters (Subclass A) are compounds of the formula:
Ml-AA2-AAl-cO'O-Rl
or a phannaceutically acceptable s lt, wherein
Ml represeDts H, NH2-CO-, NH2-CS-, NH !-S02-, X-NH-CO-, X2N-CO-, X-NH-CS-,
X2N-CS-, X-NH-SO2-, X2N-S02-, X-CO-, X-CS-, X-S02-, X-O-CO-, or X-O-CS-;
X is selected from the group COnSistiDg of Cl 10 alkyL C1 10 fluoroalkyl, Cl 10 alkyl
substituted with J, Cl 10 fluoroallyl substituted with J, 1-admantyL 9-~uorenyL phenyL phenyl
1~ substituted with K, phenyl disubstituted with K~ phenyl trisubstituted with K, naphthyl,
naphthyl substituted with K, naphthyl disubstituted with K, naphthyl trisubstituted with K,
Cl 10 allyl with an attached phenyl group, Cl 10 allyl with two attached phenyl groups, Cl 10
allyl with an attached phenyl group substituted with K, and Cl 10 aL~cyl with two attached
phenyl groups substituted ~nth K, Cl 10 allcyl with an attached phenoxy group, and Cl 10 aL~cyl
with an attached pheno~r group substituted with K on the pheno~y group;
J is selected from the group consisting of halogen, COOH, OH, CN, N02, NH2, C
10 allcoxy, Cl 10 alkylamille, C2 12 dial~ylamine, Cl-10 allyl-O-~O-- Cl-10 a~
and Cl 10 alkyl-S-;
K is selected from the group consisting of halogen, Cl 10 aLtcyL Cl 10 perfluoroalkyl,
Cl 10 alkoxy, NO2, CN, OH, CO2H, amino, Cl 10 alkylamino, C2 l2 diallylamino, Cl-C10 acyl,
and Cl 10 alko~y-CO-, and C1 10 aDyl-S-;
AA~ is a side chain blocked or unblocked amino acid with the L configuration, D
configuration, or no chirality at the ~-carbon selected from ~he group consisting of alanine,
valine, leucine, isoleucine, proline, methionine, methionine sulfoxide, phenylalar~ine,
tryptophan, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid,
glutamic acid, lysine, arginine, histidine, phenylglycine, beta-alanine, norleucine, norvaline,
alpha-arninobutyric acid, epsilon-aminocaproic acid, citrulline, hydroxyproline, ornithine,
homoarginine, sarcosine, indoline 2~rboxylic acid, 2-azetidinecarboxylic acid, pipecolinic
acid (2-piperidine carbo~ylic acid), O-methylserine, O-ethylserine, S-methylcysteine, S-
~ I~h'TITl ITF .~lEFr
,
.
: . .
WO 92/11850 2 ~ 9 ~ ~ 9 PCI/U591/097~
-26-
ethyl~ysteine, S-benzylcysteine, NH2-CH(CH2CHEt2)-COOH, alpha-amilloheptanoic acid,
NH2-CH(CH2-l-napthyl)-COOH, NH2-C~I(CH2-2-napthyl)-COOH, NH2-CH(CH2-
cyclohe~yl)-COOH, NH2-CH(CH2-cyclopentyl)-COOH, NH2-CH(CH2-cyclobutyl)-COOH,
NH2-CH(CH2-cyclopropyl)-COOH, tri~uoroleuc~ne, and hexafluoroleucine;
AA2 iS a side chain blocked or unblocked arnino acid with the L configuration, Dconfiguration, or no ch~rality at the a-carbon selected from the group consisting of leucine,
isoleucine, proline, rnethionine, methionine sulfoxide, phenylalamne, tryptophan, glycLne,
serine, threor~ne, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid,
Iysine, arg~nine, histidme, phenylglycine, beta-alanine, norleucine, norvalisie, alpha-
arninobutyric acid, epsilon-arninocaproic acid, citrulline, hydroxyproline, ornithine,
homoarg~nine, sarcosine, indoline 2-carboxylic acid, 2- 7etidislecarboxylic acid, pipecolinic
acid (2-piperidine carboxylic acid), O-methylserine, O-e~hylserine, S-methylcysteine, S-
ethylcysteine, S-benzyl~ysteine, NH2-CH(CH2C~t2)-COOH, alpha-arninoheptanoic acid,
NH2-CH(CH2-l-napthyl)-COOH, NH2-CH(CH2-2-napthyl)-COOH, NH2-CH(CH,-
cyclohexyl)~COOH, NH2-CH(CH2-cyclopentyl)-COOH, NH2-CH(CH2-cyclobutyl)-COOH,
NH2-CH(CH2-cyclopropyl)-COOH, trifluoroleucine, and hexafluoroleucine;
Rl is selected from the group consisting of H, Cl 20 allyl, Cl 20 alkyl with a phenyl
group attached to the Cl 20 alkyL and Cl 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 pharrnaceutically acceptable salt, wherein
Ml represents H, NH2-CO-, NH2-CS-, NH2-S02-, X-NH-CO-, X2N-CO-, X-NH-CS-,
X2N-CS-, X~ SO2-, X2N-S02-, X-CO-, X-CS-, X-S02-, X-O-CO-, or X-O-CS-;
X is selected from the group consisting of Cl 10 aLIyL Cl 10 nuoroal~yL C1 iO allyl
substituted with J, Cl 10 1uoroalkyl substituted with J, 1-adrnantyl, 9-fluorenyL phenyL phenyl
substituted with K, phenyl disubstituted with K, phenyl trisubstituted with K, naphthyl,
naphthyl substituted with K, naphthyl disubstitutet with Kl naphthyl trisubstituted with K.
Cl 10 allyl with an attached phenyl ~oup, Cl 10 alkyl with two attached phenyl ~roups, Cl 10
aLkyl with an attached phenyl group substituted with K, and Cl 10 al}yl with two attached
phenyl groups substituted with K, Cl 10 alkyl with an attached phenoxy group, and Cl 10 aL~cyl
with an attached pheno~y group substituted with K on the phenoxy group;
SUBSTIT9JTE SHEET
. . .
2~9~9
s WO 92/t1850 PCI/US91/09786
J is selected f~om the group corlsisting of halogen, COOH, OH, CN, N02, NH2, C
10 aL~07cy, Cl 10 aL~cylamine~ 2 diallylar~e, Cl 10 allyl-O-CO-, C1 lo a~ky
and Cl 10 allyl-S-;
K is selected from the ~roup consisting of halogen, Cl 10 alky~ Cl 10 perfluoroalkyl,
Cl 10 alko~y, N02, CN, OH, C02H, amino, Cl 10 allylamino, C2-12 diallylamino, Cl-C10 acyl,
and Cl 10 aL~coxy-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 ~om the group consisting of alanine,
valine, leucine, isoleucine, proline, methionine, methionine sulfoqcide, phenylalanine,
tryptophan, glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid,
glutam~c acid, Iysine, arginine, histidine, phenylglycine, beta-alanine, norleucine, no~valine,
alpha-a~unobu~yric acid, epsilon-aminocaproic acid, citrulline, hydro~yproline, ornithine,
homoargi}~ine, sarcosine, indoline 2~arboxylic acid, 2-a~etidinecarboxylic acid, pipecolinic
acid (2-piperidine carboxyiic acid), O-methylserine, O-ethylserine, S-methylcysteine, S-
e~hylcysteine, S-benzylcysteine, NH2-CH(CH2~;~e2)-COOH, alpha-ars~inoheptanoic acid,
NH2-CH(CH2 l-napthyl)-COOH, NH2-CH(CH2-2-napthyl)-COOH, NH2-CH(CH2-
~yclohexyl)-COOH, ~I2-CH(CH2~yclopentyl)-COO~, NH2-CH(CH2-cyclobutyl)-COOH,
NH2-CH(CH2~yclopropyl)-COOH, trifluoroleucine, and hexafluoroleucine;
R2 represents Cl 8 branched and unbranched allyL Cl 8 branched and uulbranched
cyclized alkyl; or Cl ~ branched and unbranched ~uoroalkyl;
R is selected from the group consisting of X Cl 20 alkyl, Cl 20 alkyl with a phenyl
group attached to the Cl 20 alkyL and Cl 20 alkyl with an attached phenyl group substituted
with K
The Tripeptide a-Ketoesters (Subclass A) are compounds of the structure:
M3-AA-AA-AA-CO-O-R
or a phannaceutically acceptable salt, wherein
M3 represents H, NH2-CO-, NH2-CS-, NH2-SO2-, X-NH-CO-, X2N-CO-, X~ 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 consisting of Cl 10 alkyl, Cl 10 ~uoroalkyl, C1 10 a~cyl
30 substituted with J, Cl 10 fluoroallcyl substituted with J, 1-adman~yl, 9-fluorenyl, phenyL phenyl
substituted with K, phenyl disubstituted with K, phenyl trisubsti~uted with K, naphthyl,
naphthyl substituted with K, naphthyl disubsùtutPd with K, naphthyl trisubstituted with K,
Cl 10 alkyl with an attached phenyl group, Cl 1o alkyl with two attached phenyl ~roups, Cl 10
aLlyl with an attached phenyl group substituted with K~ and Cl 10 aL~cyl with two attached
SUBSTIT~
-
.
WO 92/11850 PCI/US91/097~
2~98~09
-28-
phenyl groups substituted with K~ Cl 10 allyl with an attached phenoxy group, and Cl 10 alkyl
with an attached pheno~;y group substituted with K on the phenoxy ~oup;
T is selected ~om the group consisting of Cl 10 alkyL Cl 10 ~uoroalkyi Cl 10 alkyl
substituted with J, Cl 10 fluoroalkyl substituted with J, 1-admantyL 9-~luoreny1 phenyL phenyl
S substituted ~ith K, phenyl disubstituted with K, phenyl trisubstituted with K, naphthyl,
naphthyl substituted with K, naphthyl disubstituted with K, naphthyl trlsubstituted with K
C2 1o allyl with an atta~hed phenyl group, Cl 10 alkyl with two attached phenyl groups, Cl 10
allyl with an attached phenyl group substituted with K, and Cl 10 allcyl with two attached
phenyl groups substituted with K;
J is selected from the group cons~ting of halogen, COOH, OH, CN, NO2, NH~, Cl
10 aL1coxy, Cl 10 allyla~e, C~l2 dialkylamLne, Cl 10 alkyl-O-CO-, Cl 10 alkyl-O-CO-NH-,
and Cl 10 allyl-S-;
K is selected from the group consisting of halogen, Cl 10 allyl, Cl 10 perfluoroallyl,
Cl 10 aLko~y, N02, CN, OH, C02H, at~.uno, Cl 10 allylamino, C2 l2 dialkylals~no, Cl-C10 acyl,
and Cl 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, glutamine, aspar~ic acid,
glutamic acid, lysine, arguune, histidine, phenyl~ycine, beta-alanine, norleucine, norvaline,
alpha-aminobutyric acid, epsilon-an~inocaproic acid, citrulline, hydroxyproline, ornithine,
hornoarginine, sarcosine, indoline 2-carboxylic acid, 2-azetidinecarboylic acid, pipecolinic
acid (2-piperidine carboxylic acid), O-methylserine, O-ethylserine, S-methylcysteine, S-
ethylcysteine, S-benzylcysteine, NH2-C~I(CH2CH~t2)-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-CEl(CH2-cyclopropyl)-COOH, trifluoroleucine~ and hexafluoroleucine;
R is selected frosn the group consisting of H, C2 20 allyl, Cl 20 allyl with a phenyl
group attached to the Cl 20 alkyL and Cl 20 a1~cyl with an attached phenyl ~oup substituted
with K
The Tripeptide a-Ketoesters (Su~xlass B) are compounds of the structure:
M3-AA-AA-NH-~IR2-CO-CO-O-R
or a pharmaceutically acceptable salt, wherein
SUBSTITIJTE SHEE~
.,
, :
.
, ' ` .~';~.,.WO 9~/11850 2 ~ 9 ~ ~ O ~ PCI`/US9~/09786
-29-
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-(: O-, or X-O-CS-;
X is selected from the g~oup consisting of Cl 10 aL~cyl, Cl 10 fluoroaLlcyL Cl 10 alkyl
substituted with J, Cl 10 fluoroaLtcyl substituted with J, 1-admantyL 9-fluorenyL phenyl, phenyl
substituted with X, phenyl disubstituted with K, phenyl trisubstituted with K, naphthyl,
naphthyl substituted with K, naphthyl disubstituted with K, naphthyl trisubstituted with K,
Cl 10 aLtcyl with an attached phenyl group, Cl 10 allyl with two attached phenyl groups, Cl 10
aL~cyl with an attached phenyl group substituted with K, and Cl 10 aLIcyl with two attached
phenyl roups substituted wi~h K, Cl 10 aLkyl with an attached phenoxy group, and Cl 10 aLlcyl
with an attached phenoxy ~roup substituted with K on the phenoxy group;
T is selected from ~he ~roup consisting of Cl 10 alkyL Cl 10 1uoroalkyL Cl 10 aLtcyl
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 trisubstituted with K,
S:~2 10 aLtcyl with an attached phenyl group, Cl 10 aL~cyl with two attached phenyl g~oups, Cl 10
~ yl with an attached phenyl group substituted with K, and Cl l0 aLkyl with two attached
phenyl groups substituted ~ith K;
J is selected from the group consisting of halogen, COOH, OH, CN~ NO2, NH2, C
10 alkoxy, Cl 10 allylaro~e, Cl lo diallylamme, C1 10 alkyl-O-CO, C1 10 Y
~!0 and Cl lo allyl-S-;
K is selected from the group consisting of halogen, Cl 10 allyl, Cl 10 periluoroaLcyl,
1-10 alkoxy, N02, C'N, OH, C02H, am~no, Cl 10 alkylarnino, C2 l2 dialkylamino, Cl-C10 a~l,
and Cl 10 alkoxy-CO-. and Cl.10 a~l-S-;
AA is a side chain blocked or unblocked alTuno acid with the L configuratiorl, Dconfiguration~ or no chirality at the a-carbon selected from the group consisting of alanine,
valine, leucine, isoleucine, prol;ne, methionine, methionine sulfoxide, phenylalanine,
tryptophan, glyc~ne, serine, threonine, cysteine, tyrosine, asparagine, glutals~ine, aspartic acid,
glutan~ic acid, bsine, argir~ine, histidine, phenyl~ycine, beta-alanine, norleucine, non~aline,
alpha-arninobutyric acid, epsilon-aminocaproic acid, citrulline, hydro~proline, ornithine,
homoarginine, sarcosine, indoline 2-carboxylic acid, 2-azetidinecarbo~ylic acid, pipecolinic
acid (2-piperidine carbc~ylic acid), CJ-methy~senne, O-ethylserine, S-methylcysteine, S-
ethylcysteine, S-benzylcysteine, NH2-CH(CH2~t2)-COOH, alpha-aminoheptanoic acid,
NH2-CH(CH2-1-napthyl)-COOH, NH2-CH(CH2-2-napthyl)-COOH, NH2-CH(CH2-
WO 92/11850 8 6 0 9 P~US91/097
~30-
~yclohexyl)-COOH, NH2-CH(ÇH2~yclopentyl)-COOH, NH2-C:H(CH2 cyclobutyl)-COOH,
NE2-CH(CH2 cyclopropyl)-COOH, trifluoroleucine, and hexa~luoroleucine;
R2 represents Cl 8 branched and unbranched alkyl, Cl 8 branched and unbranched
cyclized allyL or Cl 8 branched and unbranched fluoroallcyl;
S R is selected from the group consisting of H, Cl 20 alkyL Cl 20 alkyl with a phenyl
group attached to the Cl 20 al~yL and Cl 20 alkyl with an attached phenyl group substituted
with K
The Tetrapeptide a-Ketoesters are compounds of the structure:
M3-AA4-A~ AA-CaO-R
or a phannaceutically acceptable salt, wherein
M3 represents H, NH2-CO-, NH2-CS-, NH2-SOr, 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 consisting of Cl 10 alkyL Cl 10 fluoroalkyL Cl 10 aL~cyl
substituted with J, Cl 10 fluoroalkyl substituted with J, l-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,
Cl 10 alkyl with an attached phenyl group, Cl 10 aLlcyl with two attached phenyl groups, Cl 10
alkyl ~ith an attached phenyl group substituted with K, and Cl 10 aLcyl with two attached
phenyl groups substi~uted with K, Cl 10 alkyl with an attached phenoxy group, and C~ 10 allcyl
with an attached phenoxy group substituted with K on the phenoxy group;
T is selected from the group consisting of Cl 10 allyl, Cl 10 ~uoroaLkyl, Cl 10 a~l
substitutedwithJ, Cl 10fluoroalkylsubstitutedwithJ, 1-admantyL 9-fluorenyl, phenyl, phenyl
substituted with K, phenyl disubstituted ~rith X, phenyl trisubstituted with K, naphthyl,
naphthyl substituted with K, naphthyl disubstituted with K, naphthyl trisubstituted with K,
~5 C 10 alkyl~nth an attached phenyl group, Cl 10 aL~cylwith two attached phenyl groups, Cl 10
aL~cyl with an attached phenyl group substituted with K, and Cl l0 allyl with two attached
phenyl groups substituted with K;
J is selected from the group consisting of halogen, COOH, OH, CN, NO2, NH2, Cl
10 alkoxy, Cl 10 aL~ylam~ne, C2 12 dialkylamine, Cl 10 allyl-O-CO-, Cl 10 allyl-O-CO-NH-,
and Cl 10 allyl-S-;
K is selected ~om the group consisting of halogen, Cl 10 allyL Cl 10 perfluoroallyL
Cl 10 alko%y, NO2, CN, OH, CO~I, amino, Cl 1o alkylamino, C2 l2 diaL~cylan~ino, Cl-C10 ac~l,
and Cl 10 alkoxy-C-, and C1 10 alkyl-S-;
~ttR~TtTlil^ E SHE~ET
,
.'
. -
WO 92/1~850 2 ~ 3 PCl/lJS91/09786
-31-
AA is a side chain blocked or unblocked amino acid with the L configuration, D
confi~uration, or no chirality a~ the a~rbon selected from the group consisting of alanine,
valine, leucine, isoleucine, pro~ine, methionine, methionine sulfoxid~, phenylalanine,
t~ptophan, glycine, serine, threonine, cysteine, fyrosine, asparagine, ~lutars~ine, aspartic acid,
S glutamic acid, Iysine, arginine, histidine, phenylglycine, beta-alanine, norleucine, nonaline,
alpha-arninobutyric acid, epsilon-aminocaproic acid, cit~ulline, hydro~yproline, ornithine,
homoarginine, sarcosine, indoline 2 car~y1ic acid, 2-azetidinecarbo~ylic acid, pipecolinic
acid (2-piperidine carba~lic acid), O-methylserine, O~ethy3serine, S-methylcysteine, S-
ethyl~ysteine, S-benzyl~ysteine, N~I2 CH(CH2C~HEt2)-COOH, alpha-aminoheptanoic acid,
NH2-CH(CH2-1-napthyl)-COOH, NH2-CH(CH2-2-napthyl)-COOH, NH2-CH(CH2-
cyclohe~yl)-COOH, NH2~ H2~yclopentyl)-COOH, NH2-CH(CH2~yclobutyl)-COOH,
NH2-CH(CH2-cyclopropyl).COOH, tri~uoroleucine, and hexafluoroleucine;;
AA4 iS a s;de chain blocked or unblocked amino acid ~ith the L corfiguration, D
conf;guration, or no chirality at the a-carbon selected from the group consisting of leucine,
isoleucine, methionine, methion~ne sulfoxide, phenylalanine, tryptophan, glycine, serine,
threonine, cysteine, tyrosine, aspa~agine, glutamine, aspartic acid, glutamic acid, lysine,
arg~nine, histidine, phenylglycine, beta-alanine, norleucine, nor~aline, alpha-aminobutyric
acid, epsilon-aminocaproic acid, citrulline, hydroxyproline, orni~ne, 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-GEI(CH2C~t2)-COOH, alpha-aminoheptanoic acid, NH2-CH(CH2- 1
napthyl)-COOH,NH2-CH(CH2-~-napthyl)-COOH,NH2-CH(CH2~yclohexyl)-COOH,NH2-
CH(CH2-cyclopentyl).COOH, NH2-CH(CH2-cyclobutyl)-COOH, NH2-CH(CH2-
cyclopropyl).COOH, tri~uoroleucine, and hexa~uoroleucine;
2S R is selected from the group consisting of H, Cl 20 aLIcyL Cl 20 alkyl with a phenyl
group attached to the Cl 20 alkyL and Cl-20 allcyl with an attached phenyl group substituted
with K
The Amino Acid Peptide a-Ketoessers are compounds of the structure:
Ml-AA-CO-O-R
or a phasmaceutically acceptable salt, wherein
Ml represents H, NH2-CO-, NH2-CS-, NH2-SO2-, X-~IH-CO-, X2N CO, X
X2N-CS-, X-NH-SO2-, X2N-S02-, Y-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 fluoroallyL Cl 10 a~lyl
substituted with J, Cl 10 fluoroallyl substituted with J, 1-admantyL 9-fluorenyL phenyl, phenyl
Sl.18S~ITUTE SHEET
` `
WO 92/118~0 2 0 9 g 6 0 9 -32- PCT/U~91/0978 `
substituted with K, phenyl disubstituted with K, phenyl trisubstituted with K, naphthyL
naphthyl substituted with K, naphthyl disubstituted with K, naphthyl tr~substituted with X,
Cl 10 aLIcyl with an attached phenyl group, Cl 10 allyl with two attached phenyl groups, Cl 10
aL1cyl with an attached phenyl group substituted with K, and Cl 10 alkyl with two attached
S phenyl ~oups substituted with K, Cl 10 al~yl with an attached phenoy group, and Cl 10 alkyl
with an attached pheno~y group substituted ~ith X on the phenoxy group;
Y is selected from the group consisting of C~10 a~yL Cl 10 ~uoroaL~cyL Cl 10 alkyl
substituted ~nth J, Cl-10 fluoroaL~cyl substituted with J, l-admantyL 9-fluorenyL phenyl
substituted with ~, phenyl disubstituted with K, phenyl trisubstituted w~th K, naphthyL
naphthyl substituted with K, naphthyl disubstituted with B, naphthyl trisubstituted with K,
Cl 10 alkyl with an attached phenyl group, Cl 10 aLkyl with two attached phenyl groups, Cl 10
aLlcyl with an attached phenyl group substituted with K, and Cl 10 alkyl with two attached
phenyl groups substituted with K;
J is selected from the group consisting of halogen, COOH, OH, CN, N02, NH2, Cl
1~ lo aLkoxy, Cl 10 aL~cylamme, C2 12 diaLkylamine, Cl 10 allyl-O-CO-, Cl 10 allyl-O-CO-NH-,
and Cl 10 alkyl-S-;
K is selected from the group consisting of halogen, Cl 10 allyl, Cl-10 perfluoroa~l
C~ lo alko~y, N02, CN, OH, ~2~ amino, C1 10 alkylamino, C~ 12 diaLkylan~no, Cl-C10 acyl,
and Cl 10 aLcoy-CO-, and Cl l0 allyl-S-;
AA is a side chain ~olocked or unblocked amino acid with ~he L configuration, D
configuration, or no chirali~y at the a-carbon selected from the group consisting of alanine,
valinej leucine, isoleucine, proline, methionine, methionine sulfo dde, phenylalanine,
tryptophan, glycine, serine, threonine, cysteine, tyrosine, asparagine, glutam~ne, aspartic acid,
glutamic acid, lysine, arginine, histidine, phenylglycine~ beta-alanine, norleucine, norvaline,
2~ alpha-an~inobutyric acid, epsilon-arninocaproic acid, citrulline, hydroxyproline, ornithine,
homoarg~ni~le, sarcosine, indoline 2-carboxylic acid, 2-azetidinecarbo4ylic acid, pipecolinic
acid (2-piperidme carboylic acid), O-methylserine, O-ethylserine, S-methylcysteine, S-
ethylcysteine, S-benzylcysteine, NH2-CH(CH2C~IEt2)-COO~ 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, trinuoroleucine, and he~ uoroleucine;
R is selected ~om the group consisting of H, Cl 20 alkyL Cl-20 alkyl with a phenyl
group attached to the Cl 20 alkyL and Cl 20 aL'cyl with an attached phenyl group substituted
with K
C~ ~ITI ~TF .S}~F~T
-- ,
WO 92/11850 2 0 9 8 6 0 3 PCI'/US91/09786
,-- t
....
33
The follow~ng Peptide Ketoester compounds are representative of the Peptide Keto-
Compounds found to be useful as Calpa~n ;nhibitors Withi~l the conte~t of the present
~nvention:
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
ZAla-DL,Ala-COOEt
ZAla-DL,Ala-COOBzl
~AIa-DL-Ala-COOnBu
MeO-Suc-Ala-DL,Ala-COOMe
~Leu-N~ra-COOEt
Z-Leu-Me-COOEt
~Leu-Phe-COOEt
ZLeu-Abu-COOEt
ZLeu-Met-COOEt
Z-Phe-DL,Phe-COOEt
H-Gly-DI~Lys-COOEt
H-Ala-DL-Lys-COOEt
H-Pro-DL-Lys-COOEt
H-Phe-DL-Lys-COOEt
Z-Ala-Ala-DL-Ala-COOEt
~AIa-Pro DL Ala COOEt
ZAla-Ala-DL,Abu-COOEt
ZAla-Ala-DL-Abu-COOBzl
Z Ala-Ala-DI,Abu-COOCH2-C6H4-CP3 (para)
MeO Suc-Val-Pro-DL Phe COOMe
H-Leu-Ala-DL~Lys-COOEt
ZAla-Ala-Ala-DL-~la-COOEt
MeO-Suc-Ala-Ala-Pro-DL-Abu COOMe.
ZLeu-Phe-COOEt
SUlE~;~lT~alTE S~EEr
~;i,
:
~"
..
WO92/11850 0986a9 PCI/US~1/097
-34-
PhCQ-Abu-COOEt
(c~3)2cH(cH2)2co-Abu~coc)Et
C$I3CH2CE)2CHCO.Abu-COOEt
Ph~CH2)6CO-Abu-COOEt
- ZLeu4~ Phe-~OOEt
Z-Leu-Leu-Abu-COOEt
~Leu-Leu-Phe-COOEt
2-NapS02-Leu-Abu-COOEt
2-NapS02-Leu-Leu-Abu-COOEt
ZLeu-NLeu-C02Et
ZLeu-Phe-C02Bu
Z-Leu-Abu-C02Bu
Z-Leu-Phe-CO2Bzl
ZLeu-Abu-C02BzL
1~ We have found certain subclasses of Peptide Ketoacid Compounds to be partieularly
useful when used in accordance with the present inVentiQn. These are sul~classes are the
Dipeptide a-Ke~oacids (SubclassA), theDipeptide a-Ketoaeids (SubclassB), the Tripeptide
-Ketoacids, the Tetrapeptide a-Ketoacids and the Amilio Acid peptide ~-Ketoacids. All
of these are considered to be within the c lass of Peptide Xeto-Compounds.
The Dipeptide a-Ketoacids (Subclass A) are compounds of the structure:
Ml-AA-NH-CHR2-CO-CO-OH
or a phannaceutically acceptable salt, wherein
Ml represents H, NH2-CO-, NH2 CS-, NH2 S02-, X-NH-CO~, X2N-CO-, X-NH-CS-,
X2N-CS-, X-NH~S02-, X2N-S02-, X-CO-, X-CS-, X-S02-, X-O-CO-, or 2{-O-CS-;
X is selected from the group consisting of Cl 1o allyl, Cl 10 fluoroaL~cyL Cl 10 allyl
substituted with J, Cl 10 fluoroalkyl substituted with J, 1~admantyl, 9-iluorenyL phenyl, phenyl
substituted with K, phenyl disubstituted with K, phenyl trisubstituted with K, naphthyl,
naphthyl substituted with K, naphthyl disubstituted with K, naphthyl trisubstituted ~ith K,
Cl ~0 a;lyl with an attached phenyl group, C1 iO alkyl with two attached phenyl groups, Cl 10
30 alkyl with an attacl~ed phenyl group substituted with K, Cl 10 aLlcyl with ~wo attached phenyl
groups substituted with K, Cl 10 allyl with an attached pheno~y group, and Cl 10 alkyl with
an attached pheno~y group substituted with K on the phenoxy group;
SUBSTITUTE SHEE~
.: :
.. ~,.. ,, . . .,, ~.......... , . , ; , .
WO92/11850 2~98~a~ Pcr/US91/09786
-35-
J is selected ~om the group consisting of halogen, COOH, OH, CN, NO2, NH2, Cl
10 aL~cwy, C1 10 allylamille, C2 ~ aLlylamme, Cl 10 allyl~ CO-, Cl 10 allyl-O-CO-NH.,
and Cl 10 alkyl-S-;
K is selected ~om the group consisting of halogen, Cl 10 allyl Cl 10 pe~uoroalkyl
S Cl 10 alko~y, NO2, CN, OH, CO2H, amino, Cl 10 allylamino, C2-l2 diallyla~ o, Cl-C10 acyL
and Cl 10 alkoxy-CO-, and C1 10 allcyl-S-;
AA is a side chain blocked or unblocked an~ino acid with the L con~iguration, D
configuration, or no chirality at the a-carbon selected from the group consisting of alanine,
valine, leucine, isoleucine, proline, methionine, methionine sulfo~ide, phenylalanine,
t yptophan, glyc~ne, serine, threonine, cysteine, tyrosine, asparagine, glutan~ne, aspar~ic acid,
glutan~c acid, lysine, ar~e, histidine, phenylglycine, beta-alanine, norleucine, norvaline,
alpha-a~.ûnobutyric acid, epsilon-aminocaproic acid, citrul~ine, hydro~yproline, ornithine,
homoargi~une, sarcosine, indoline 2-carboxylic acid, 2-azetidinecarboxylic acid, pipecolinic
acid (2-piperidine carbo~ylic acid), O-methylserine, O-etllylserine, S-methylcysteine, ~-
ethylcysteine, S-benzylcysteine, NH2-~l(CH2~Et2)-COOH, alpha-anunoheptanoic acid,
NH2-CH(CHrl-napthyl)-COOH, NH2 CH(CH2-2-napthyl)-COOH, NH2-C~(CH2-
cyclohexyl)-COOH, NH2-CH(CH2~yclopentyl)-COOH, NH2-GH(CH2~yclobutyl~-COOH,
NH2-C~I(CH2-cyclopropyl)-COOH, trifluoroleucine, and hexafluoroleucine;
R2 represents Cl 8 branched and unbranched allyL Cl ~ branched and unbranched
cyclized alkyl, or Cl 8 branched and unbranched fluoroalkyl.
The Dipepdde ~-Ketoacids (Subclass B) are compounds of the structure:
Ml-AA2-AAl-CO-OH
or a pharmaceudcally acceptable salt, Yvherein
Ml represents H, NH2-CO-, NH2-CS-, NH2-S02-, X-NH-CO-, X2N-CO-, X-NH-CS-,
X2N-CS-, X-NH-S02-, X2N-S02-, X-CO-, X-CS-, X-SO2-, X-O-CO-, or X-O-~S-;
X is selected from the group consisdng of Cl 10 allyL Cl 10 fluoroalkyL Cl 10 alkyl
substi~uted with J, Cl 10 fluoroaDcyl substituted with J, 1-adman~qL 9-nuorenyL phenyL phenyl
substituted with K, phenyl disubstituted with K, phenyl trisubstituted with K, naphthyl,
naphthyl substituted with K, naphthyl disubstituted with K, naphthyl tnsubstituted with K,
Cl 10 aLcyl with an attached phenyl group, Cl 10 aLcyl with two attached phenyl groups, Cl 10
allyl ~nth an attached phenyl ~oup substituted with K, and Cl 10 allyl with rwo attached
phenyl groups substituted with K, Cl 10 alkyl with an attached pheno~y ~oup, and Cl 10 alkyl
with an attached pheno~y group substituted with K on the phenoxy group;
S~3BS~7'Ur~ SHEE~
.
. .
,
:
wO 92/11850 2 0 9 8 6 0 9 PCI/US91/097B~
-36-
J is selected from the group consisting of halogen, COOH, OH, CN, N02, NH2, C1
10 aL~co~y, C1 10 aLlyla~e, C~12 dia~lam~e, Cl 10 allyl-O-CO-, Cl 10 al}yl-O-CO-NH,
and C1 l0 allcyl-S-;
K is selected from the group consisting of halogen, Cl 10 a~yL cl lo perfluoroallyL
110 aLkoxy, N02, CN, OH, C02H, amino, Cl 10 allylamino, C2 ~2 diallylan~ino, Cl-C10 acyL
and Cl 10 alkoxy-CO~, and C1 10 alXyl-S-;
AAl is a side chain blocked or unblocked amino acid wi~h the L configuration, D
configlaration, or no chirality at the ~-carbon selected f~om ~he group consistirlg of alanine,
valine, leuune, isoleucLqe, proline, methionine, methionine sulfo~de, phenylalanine,
tryptophan, serine, threonine, cysteine, tyrosine, asparag~ne, glutamine, aspartic acid,
glutamic acid, lysine, arginine, histidine, phenylg}ycine, beta-alanine, norleucine, norvaline,
alpha-aminobutyric acid, epsilon-aminouproic acid, citrulline, hydroxyproline, ornithine,
homoarginine, sarcosine, ~ndoline 2 carbo~y1ic acid, 2-a~etidinecarbo~ylic acid, pipecolinic
acid (2-piperidine carbo~y~c acid), O-methylserine, O-ethylserine, S-methylcysteine, S-
ethylcysteine, S-ben2ylc~s~eine,NH2-CH(CH2CHE~ COOH, alpha-aminoheptanoic acid,
NH2-CH(CH2-l-napthyl)-COOH, NH2~CH(CH2-2-napthyl)-COOH, NH2-CEI(CH2-
cydohe~yl).COOH, NH2 CH(CH2~clopentyl)-COO~ I2-CH(~H2~yclobutyl)-COOH,
NH2-CH(CH2-cyclopropyl)-COOH, trifluoroleucine, and he1~afluoroleucine;
M2 is a side chain blacked or unblocked ~o acid with the L configuration, D
configuration, or no chirality at the a carbon selected from the group consLr.ting of alarline,
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-alaninet norleucine, norvaline,
alpha-aminobutyric acid, epsilon-aminocaproic acid, citrulline, hydro~proline, ornithine,
homoarginine, sarcosine, indoline 2 carbo7y1ic acid, 2-azetidinecarbo~ylic acid, pipecolinic
acid (2-piperidine carba~ylic acid), O-methylserine, O-ethylserine, S-methylcysteine, S-
ethylcysteine, S-ber2ylcysteine, NH2-C~(GH2CHEt2)-COOH, alpha-am~noheptanoic acid,
NH2-CH(CH2-l-napthyl)-COOH, NH2-CH(CH2-2-napthyl)-COOH, NH2-CH(CH2-
cyclohexyl)-COOH, NHyC~ H2~yclopentyl)-COOH, NH2-GH(CH2~yclobutyl)-COOH,
NH2-CH(CH2~yclopropyl)-COOH, tri~uoroleucine, and hexa~uoroleuc~ne.
The Tripeptide a-Ketoacids are compounds of the s~ucture:
Ml-AA-AA-AA-CO-OH
or a pharmaceutically acceptable salt, wherein
' .
SU~3ST~TU~ ~ffEEt
,
WO 92/118~0 P~/US91/09786
i 209~
-37-
Ml represents H, NH2-CO-, N~I2-CS-, NH2-SO2-, X-NH-CO-, X2N-CO-, X-NH-CS-,
X2N-CS-, X-NH-S02-, X2N~02-, X-CO-, X-CS-, X-S02-, X-O-CO-, or X-O-CS-;
X is selected fcom the ~oup consisting of Cl 10 alkyL Cl 10 fluoroalkyL Cl 10 alkyl
subsdtuted with J, Cl 10 fluo~oaL~cyl 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 L;subsdtuted with K, naphthyl trisubstituted with K,
Cl 10 allyl with an attached pheslyl ~oup, Cl 10 allyl with two attached phenyl groups, Cl 10
al}yl with an attached phenyl group substituted ~nth K, and Cl~10 aLlcyl with two attached
phenyl groups substituted with K, Cl 10 alkylwith an attached pheno~y group, and Cl 10 alkyl
with an attached pheno~y group substituted with K on the pheno~y group;
J is selected ~om the group consisting of halogen, COOH, OH, CN, N02, NH2, Cl
10 lko~y, Cl-10 a~ylamme, C2 12 diaLlylamme, Cl 10 alkyl-O-CO, Cl 10 allyl-O-CO-NH,
and C1 10 allyl-S-;
K is selected from the group consisting of halogen, Cl 10 al}yL Cl lo pD-rfluoroalkyL
1-10 xy, N2~ CN, OH, C2H- armno, Cl 10 allylamillo, C2 12 dialkylamino, Cl-C10 acyL
and Cl 10 alko~y-CO-, and Cl 10 aLk~l-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 sulfo~de, phenylalanine,
tryptophan, glycine, serine, threonine, cysteine, tyrosine, asparagine, ~lutamine, aspar~ic acid,
glutamic acid, bsine, arginine, histidine, phenylglycine, beta-alanine, norleucine, norvaline,
alpha-aminobutyric acid, epsilon-aminocaproic acid, citrulline, hydrw~yproline, ornitbine,
homoarginine, sarcosine, indoline 2 car~o~ylic acid, 2-azetidinecarbo~ylic acid, pipeco~;nic
acid (2-piperidine carbo~ylic acid), O-methylserine, O-ethylserine, S-methylcysteine, S-
etbylcysteine, S-benzylcysteine, NH2-CH(CH2C~t2)-COOH, alpha-aminoheptanoic acid,
NH2-CH(CH2-l-napthyl)-COOH, NHrCH(CH2-2-napthyl)-COOH, NH2-CH(CH2-
cycloheyl) COOH, NH2-C~I(CH2-cydopentyl)-COOH, NH2-CH(CH2~:yclobutyl)-COOH,
NH2-CH(GEI2 cydopropyl)-COOH, tri~luoroleucine, and he~a~uoroleucine.
The Tetrapeptide a-Ketoacids are compounds of the structure:
Ml-AA-~A-AA-AA-CO-OH
or a pharmaceu~ically acceptable salt, wherein
Ml represents H, N~2-CO-, NH2-CS-, NH2-S02-, X-NH-CO-, X2N-CO-, X-NH-CS-
X2N-CS-, X-NH-S02-, X2N-SO2-, Yl-CO-, X-CS-, X-SO2-, X-O-CO-, or X-O-CS-;
SU138TITUTE SHFET
WO 92/11850 2 ~ 9 8 6 ~ 9 PCI/US91/0978~
-38-
X is selected ~om the group consisting of Cl 10 aLkyl Cl 10 ~uoroalkyL Cl 10 aLI~yl
substituted w~th J, Cl 10 fluoroallyl substituted with J, 1-admantyL 9-fluorenyL phenyL phenyl
substituted w~th K, phenyl disubstituted with K, phenyl trisubstituted with K, naphthyL
naphthyl substituted with K, ~phthyl disubstituted with K, naphthyl trisubstituted with K,
S Cl 10 alkyl w~th an attached phenyl ~oup, Cl 10 all~l with two attached phenyl groups, Cl 10
aLkyl with an attached phenyl group substi~uted with K, and Cl 10 aLkyl ~ith two attached
phenyl groups substituted with K, C1 l0 aL~cyl with an attached pheno~y group, and Cl 10 alkyl
with an attached pheno.y group substitut d w~th K on the phenoy group;
'Yl is selected f~om the group conslsting of C2 lo aLlcyL Cl lO fluoroalkyL C1 l0 aLlyl
substitutedw~thJ, C1 1OfluoroallcylsubstitutedwithJ, 1-a~mantyL 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,
Cl 10 allyl wi~h an attached phenyl group, Cl 10 alkyl with two attached phenyl ~oups, Cl 10
allyl with an attached phenyl group substituted with K, and Cl 10 alkyl with two attached
phenyl groups subsdtuted vrith K;
J is selected from the group consisting of halogen, COOH, OH, CN, NO2, NH2, Cl
10 alko~y, Cl 10 alkylamine, C 12 dialkylamine, Cl 10 alkyl-O-CO-, Cl 10 alkyl-O-CO-NH-,
and Cl 1o alk~l-S-;
K is selected from the group consisting of halogen, Cl 1o alkyL Cl 1o per~uoroalkyl,
Cl 10 alkoxy, N02, CN, OH, C02H, amino, Cl 1O allylamino, C2.12 dialkylarnino, Cl-C10 acyl,
and Cl 10 alko~y-CO-, and Cl 10 aLtcyl~;
AA is a side chain blocked or unblocked amino acid with the L configuration, D
configuration, or no chirality a~ the a~on selected from the group consisting of alanine,
valine, leuc~ne, ~soleucine, proline, methionine, methionine sulfo~ide, phenylalanine,
tryptophan, glycine, senne, threonine, ~ysteine, tyrosine, asparagine, glutamine, aspartic acid,
gluhn~ic acid, }ys3ne, arginine, histidine, phenylglycine, beta-alanine, norleucine, norvaline,
alpha-aminobutyric acid, epsilon~ nocaproic aciL citrulline, hydro~yproline, ornithine,
homoargmine, sarcosine, ~ndoline 2-carbo~ylic acid, 2-azetidinecarbo~ylic acid, pipecolinic
acid (2-piperidine carbo~ylic acid), O-methylsenne, O-ethylserine, S-methylcysteine, S-
ethylcysteine, S-ben~ylcysteine, NH2-C~I(CH2C:HEt2)-COOH, alpha-an~inoheptanoic acid,
NH2-CH(CH2-l napthyl)-COOH, NH2-CH(CH2-2-napthyl)-COOH, NH2-CH(CH2-
cyclohexyl)-COOH, NH2-CH(CH2~yclopentyl)-COOH, NH2-CH(CH2~dobutyl)-COOH,
NH2-CH(CH2-cydopropyl)-COOH, trifluoroleucine, and hexafluoroleucine.
The Amino Acid Peptide a-Ketoacids are compounds of the structure:
SU~STITI ITF .~L~r:~T
- .
`,' ~`' '''' ' ~ :,
.....
WO 92/11850 ~ ~ ~ 3 ~ o 9 PCI'/US91/09786
-39-
M~ CO-OH
or a pharmaceutically acceptable salt, wherein
Ml represents H, NH2-CO-, NH2-CS-, NH2-S02-, X N~ CO, 2
2 , X N~-S02-, X2N~02-, Y2-C0-7 X-CS-, X-~02-~ X-O-CO-, or X-O-~S-;
S X is selected from the group consisting of Cl 10 allyl, Cl 10 ~uoroalkyl, Cl 10 alkyl
substituted with J, C 1 10 fluoroalkyl substituted with J, 1-admantyl, 9-~uurenyl, phenyl, phenyl
substituted with X, phenyl disubstituted with iK, phenyl trisubstituted with K, naphthyl,
naphthyl substituted with K, naphthyl disu~sdtuted ~nth K, naphthyl trisubstituted with K,
Cl 10 allyl with an attached phenyl group, C1 l0 allyl ~nth two attached phenyl groups, C1 l0
al}yl with an attached phenyl group sllbstituted with K, and Cl 10 allyl with two attached
phenyl groups substituted with K, Cl 10 allyl with an at~ached pheno~y group, and Cl~10 allyl
with an attached phenoxy group subseituted with K on ~he pheno~y group;
Y2 is selec~ed ~om the group conslsting of Cl 10 aL~cyL C1 10 ~uoroa~yl, C1 10 alkyl
subs~ituted ~vith J, Cl 10 fluoroallyl substituted ~nth J, 1-adman~L 9^fluorenyL phenyl
substituted wi~h K, phenyl disubstituted with K, phenyl trisubstituted with K, naphthyl,
naphthyl substituted with K, naphthyl disubstituted ~th K, naphthyl trisllbs~cituted 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 substitu~ed wlth K, and Cl 10 allyl ~nth two attached
phenyl groups substituted with K;
J is selected f~om the group consisting of halogen, COOH, OH, CN, NO2, NH2, C
10 allco~y, C1 10 allcylamine, C~12 dialkylamine, C1 10 allykO-CO-, C1 10 aLkyl O
and Cl 1o allyl~-;
K is selected from the group consisting of halogen, Cl-10 alkyl Cl 10 per~uoroalkyl,
C1 10 aL~coxy, NO2, CN, O~I, CO2H, amino, Cl 10 allylamino, C2 l2 diaLIcylalruno, Cl-C10 acyl,
and Cl 10 alkoxy-CO-, and C1 10 aLlyl~
AA is a side chain blocked or unblocked amino acid with ~he L con~iguration, D
configuration, or no chirality at the a-carbon selected from the group consisting of alanine,
valine, leucine, isoleucine, proline, methionine, methionine sulfo~ude, phenylalanine,
tryptophan, glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid,
g~utamic acid, lysine, arginine, histidine, phenylglycine, beta-alunine, norleucine, norvaline,
alpha.an~inobutyric acid, epsilon-aminocaproic acid, citrulline, hydro7yproline, ornithine,
homoar~pnine, sarcosine, indoline 2-carboxylic acid, 2-azetidinecarbo~lic acid, pipecolinic
acid (2-piperidine car~oxylic acid), O-methylserine, O-ethylserine, S-methylcysteine, S-
ethylcysteine, S-berzylcysteine, NH2-CH(CH2CHEt2)-COOX alpha-aminoheptanoic acid,
SW3STlTl3TE SH@ET
. ~ .
WO 92/11850 2 ~ ~ 2 6 ~ ~ PCI`/US91~0978
10-
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 h~afluoroleucine.
The following Peptide ~etoacid compounds are represen~ative of the Peptide Keto-S Compounds found to be useful as Calpain inhibitors withirl the context of the present
invention:
:E3z DL,Lys-COOH
Bz-DI,Ala-COOH
Z-Leu-Phe-COOH
i~T eu Abu-COOH.
The peptide -Icetoamides are transition state analogue inhibitors for cysteine
proteases, such as Calpain. We have found that Peptide a-ketoarnides containing amino
acid residues with hydrophobic side chains at the Pl site are excellent inhibitors of several
cysteine proteases including calpain I and calpain IL
We have found fiYe subclasses of the peptide ketoamides to be particularly efEective
in inhl~iting Calpain. These subclasses are referred to herein as Di~eptide a-Ketoarnides
(SubclassA),Dipeptide a~Ketoamides (SubclassB), Tripeptide a-Ketoan~ides, Tetrapeptide
a-Ketoamides and Amino Acid a-Ketoamides. All of these subclasse~ are considered herein
to be withn the class of Peptide Keto-Compounds
The Dipeptide a-Ketoamides (Subclass A) haYe the following structural formula:
Ml AA NH-CHR2 CO-CO-NR3R4
or a phannaceutically acceptable salt, wherein
Ml represents H, NH2-CO-, NH2-CS-, N~I2-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 3s selected from the group consisting of Cl 10 alkyL Cl 10 fluoroaLIcyL ~ 0 alkyl
substituted with J, Cl 10 fluoroaLl~yl substituted with J, 1-admantyL 9-fluorenyL phenyl, phenyl
substituted wi~h K, phenyl disubstituted with K, phenyl tri~ubstituted with K, naphthyL
naphthyl substituted with K, naphthyl disubstituted with K, naphthyl tnsubstituted with E
Cl 10 aLkyl with an attached phenyl group, Cl 10 alkyl with two attached phenyl groups, Cl 10
aLlcyl with an attached phenyl group substituted with K, Cl 10 alkyl with two attached phenyl
groups substituted with K, Cl 10 alkyl with an attached phenoxy group, and Cl 10 allyl with
an attached pheno~y group subsdtuted with K on the pheno~y group;
5UI~STITUTE SHEET
. .
WO 92/11850 2 ~ P~/US91/09786
J is selected from the group consistirlg of halogen, COOH, OH, CM, NO2, NH2, Cl
10 alkoxy, C1 10 ~ylamine, Cz 12 diallyla~une, C1 10 alkyl-O-CO-, C1 l0 alkyl O CO NH,
and Cl 10 alkyl-S-;
K is selected from the group consisti~g of h~logen, Cl 10 a~L cl lo per~UoroalksL
Cl 10 alkoxy, N02, CN, OH, C02H, amino, Cl 10 allylan~ino, C2 l2 diallylamirlo, Cl-C10 acyl,
Cl 10 aL~o~y-CO-, and Cl 10 alkyl-S-;
AA is a side chain blwked or unblocked amino acid with the L coIIfiguration, D
configuration, or no chirality at the a-carbon selested from the group consisting of alanine,
valine, leucine, isoleucine, proline, methionine, methionine sulfo~ide, phenylalanine,
tryptophan, glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspar~ic acid,
glutamic acid, lysine, arginine, histidine, phenylglycine, beta-alanine, norleucine, norvaline,
a-arninoblltyric acid, epsilon-a~T~nocaproic acid, citrulline, hydroxyproline, ornithine,
homoarginine, sarcosin, indoline 2-carboylic acid, 2-azetidinecarbo~ylic acid, pipecolinic
acid (2-piperidine carboxylic acid), O-methylserine, O-ethylserine, S-methylcysteine, S-
ethylcysteine, S-beDzylcysteine, NH2-CH(CH2CHEt2)-COOH, -aminoheptanoic acid, NH2-
CH(CH2-1-napthyl)-COOH, NH2-C~H(CH2-2-napthyl)-COOH, NH2-~H(CH2~yclohexyl)-
C9X NH2-CH(~E12-cyclopen.yl)-COOH, NH2-CH(CH2 cyclobu~l)-C9OH, NH2-
CH(CH2-cyclopropyl)-COOH, tri~uoroleucine, and he~a~uoroleucine;
R2 is selected from the group consisting of Cl 8 branched and unbranched allyl, Cl..8
branched and unbranched cyclized alkyl, and Cl 8 branched and unbranched fluoroa~kyl,
R3 and R4 are selected independently from the group consisting of H, Cl 20 aL~cyl,
Cl 20 cyclized alkyL Cl 20 allyl with a phenyl group attached to the Cl 20 alkyl, Cl 20 cyclized
alkyl with an attached phenyl group, Cl 20 a1kyl with an attached phenyl ~oup substitu~ed
with K, Cl 20 alkyl with an attached phenyl group disubstituted with K, Cl 20 alkyl with an
attached pbenyl group trisubstituted with K, Cl 20 cyclized alkyl with an attached phenyl
group substituted with K, Cl 10 allyl with a morpholine-[-N(CH2CH2)O] ring attached
through nitrogen to the all~yL Cl 10 allyl with a piperidine ring attached through nitrogen
to the alkyL Cl 10 aLlcyl with a pyrrolidine ring attached through nitrogen to the allyL Cl 20
aLlcyl with an OH group attached to the aLlcyL -CH2CH2C)CH2C~2OH, Cl 10 with an
attached 4-pyridyl group, Cl 1o with an attached 3-pyridyl group, Cl 10 with an attached 2-
pyridyl group, Cl 10 with an attached cyclohe1yl group, -NH-C~H2CH2-(4-hydroyphenyl), and
NH-C~2C~2.(3-indolyl).
The Dipeptide a-Ketoamides (Subclass B) have the following structural forrnula:
Ml-AA2-AAl-CO-NR3R4
~:LI~IM ITF c ~l~
`
:' :
WO 92/11850 2 ~ 9 ~ 6 0 9 P~/US91/097B~. r
~2-
or a pharrnaceutically acceptable salt, wherein
Ml represents H, NHz-CO-, NH2-C:~S-, NH2-S02-, X-NH-CO-, X2N-CO-, X-NH-CS-,
X2N-CS-, X-NH-S02-, X2N^S02-, X-CO-, X-CS-, X-S02-, X-O-CO-, or X-O-CS-;
X is selected from the group consisting of Cl 10 allyL Cl 10 fluoroalkyL C1 10 alkyl
Ssubstituted with J, Cl 10 fluoroaL~cyl substituted with J, 1-admantyL 9-~uorenyL phenyL phenyl
substituted with K, phenyl disubstituted with ~, phenyl trisubstituted with K, naphthyL
naphthyl substituted with ~, naphthyl disubstit~ted with K, naphthyl trisubstituted with K,
Cl 10 aL~cyl with an attached phenyl group, C~ 10 allyl with two attached phenyl groups, Cl 10
allyl with an attached phenyl group substituted ~nth K, Cl 10 allyl ~vith two attached phenyl
10groups substituted with K, Cl 10 allyl with an attached pheno~ roup, and Cl 10 alkyl with
an attached pheno~y group substituted with lE~ on the pheno~y group;
J is selected from the group consisting of halogen, COOH, OH, CN, N02, NH2, Cl
10 al~co~y, Cl 10 a~ylamLne, C2 12 diallylamme, Cl 10 a~l-O-CO-, Cl 10 allyl-O-CO-NH-,
and Cl 10 allyl~-;
15K is selected from the group consisting of halogen, Cl 10 alkyL Cl 10 pP.~uoroalX~L
0 alko~, N02, CN, OH, CO2H, amino, Cl 10 allylamino, C2 12 diallylamino, Cl-C10 acyl,
and C1 10 alko~y-CO-, and Cl 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 f~om the group consisting of alanine,
20valine, leuane, isoleucine, proline, methionine, methionine sulfo~ide, phenylalanine,
tryptophan, serine, threonine, g~steine, tyrosine, asparagine, glu~nine, aspartic acid,
glutaïnic acid, lysine, arginine, histidin4 phenylglycin4 beta-alanine, norleucine, norvaline
a-an~inobu~yric acid, epsilon-aminocaproic acid, citrulline, hydroxyproline, orTIitnine,
homoarginine, sarcosine, indoline 2-carboxylic acid, 2-azetidinecarboxylic acid, pipecolinic
25acid (2-piperidine carboxylic acid), O-methylserine, O-ethylserine, S-methylcysteine, S-
- ethylcysteine, S-benzyl~ystein4 NH2 CH(CH2t;~t2)-COOH, ~-aminoheptanoicacid, NHz-
CH(CH2 1 napthyl) COOH, N~2-CH(CH2~2 napthyl) COOH, NH2~CH(CH2-cydohexyl)-
COOH, NH2-~I(CH2~clopentyl)-COC)H, NHrC~H(CH2-cyclobutyl)-COOH, NH2-
CH(CH2~ydopropyl)-COOH, tri~luoroleucin4 and hexafluoroleucine;
30AA2 is a side chain blocked or unblocked amino acid with the L configuration, D
con~iguration, or no chirality at the a-carbon selected ~om the group consisting of alanine,
valine, leucine~ isoleucine, proline, methionine, methionine sulfoxide, phenylalanine,
tryptophan, glyc~ne, serin4 threonine, cysteine, ~qrosine, asp~ragine, glutamine, aspartic acid,
glutamic acid, Iysine, arginine, histidine, phenylglycine, beta-alanine, norleucine, norvali, e,
SlJBSTiTUT'- S~.EET
.
.
., - ` :
..: wo 92~11850 2 ~ Q ~, pcr/vs91/o9786
~3-
a-aminobutyric acid, epsilon-aminocaproic acid, citrulline, hydro~yproline, ornithine,
homoarginine, sarcosine, indoline 2~carboxylic acid, 2-azetidinecarboxylic acid, pipecolinic
acid (2-pi~eridine earbo~ylic acid), O-methylser~ne, O-ethylserine, S-methylcysteine, S-
ethylcysteine, S-benzylcysteine, NH2-CH(CH2~ )-COOH, ~ -aminoheptanoic acid, NH2-
S CH(CH2-1-napthyl)-COOH, NH2-CH(CH2-2-napthyl)-COOH, NH2~CH(C~H2-cyclohexyl)-
COOH NH2-CH(CH2~yclopentyl)-C::OOH, NX2-CH(OEI2~yclobutyl) COOH, 2
C~(CH2~yclopropyl)-COC)H, tri1uoroleuane, and he~a~uoroleucine;
R3 and R4 are selected independentl~ f~om the group co~sisting of H, C1 20 alkyLC1 20 cyclized allyL cl 20 aL~yi w~th a phenyl group attached to the C~ kyL C~ 20 cy~zed
alkyl with an attached phenyl group, Cl 20 allyl ~ith an attached phenyl group subs~ituted
with K, Cl 20 allyl with an attached phenyl group disubstituted with K, C1 20 aL~cyl with an
attached phenyl group trisubstituted with K, C~ cycli~ed aIkyl with an attached phenyl
~oup substituted with K, Cl 10 aLlcyl with a morpholine ~-N(C~2C~2)0] ring attached
through nitrogen to the aL~cyL C~ 0 allyl with a piperidine ring attached through nitrogen
tO the aL~cyL Cl 1o allyl with a pyrrolidine ring attached through r~itrogen to the aLIcyL Cl 20
alkyl with an OH group attached to the a~yL -CH2CH20CH2~H20H, Cl 10 with an
attached 4-py~dyl group, Cl 10 with an attached 3-pyndyl group, Ci 10 q ith an attached 2-
pyridyl group, Cl-10 with an attached cyclohe1yl group, -NH-CH2~I2 (4-hydro~yphenyl), and
-NH-CH2CH2-(3-irldobl~.
The Tripeptide a-Ketoarnides have the follo~ing structural formula:
Ml-AA AA-AA-CO NR3R4
or a pharmaceutically acceptable salt, wherein
Ml represents ~ NH2-CO-, NH2-CS-, NH2~O2-, X-NH-CO-, X2N-CO-, X-NH-CS-,
X2N C5-, X-NH SO2-, X2N~02-, X CO-, X-t:~S-, X-SO2-, X-O-CO-, or X-O-CS-;
2~ X ~s selected ~om the group consisting of Cl 10 aLIyL Cl 10 fluoroalkyl~ C1-10 aLIcyl
substituted with J, Cl 10 fluoroalkyl substisu~ed with J, 1-admantyl, 9-fluorenyL phenyL phenyl
subsdtuted vvith ~, phenyl disubstituted with K, phenyl trisubstituted ~/ith K, naphthyL
naphthyl substituted with K, naphthyl disubstituted with K, naphthyl ~risubstituted with K,
Cl 10 alkyl with an attached phenyl group, Cl 10 allcyl with two attached phenyl groups, Cl 10
aLlcyl with an attached phenyl group substituted with K, Cl 10 allyl with ~o attached phenyl
~oups substituted with K, Cl 10 allyl ~nth an attached pheno~y group, and C 1-10 alkyl with
an attached pheno~y group substituted with K on the pheno~y group;
SV8S~ HFF-r
. . , ~ ~ ,
, ......................................................................... .
WO 92/11850~ ~) 9 8 6 0 9 PCr/US91/09786 ~..
J is selected from the g~oup cons~sting of halogen, COOH, C)H, CN, NO2, NH2, Cl
10 aL~co~y, C1 10 allylam~ne, C2 12 dia31ylam~ne, Cl 10 allyl-O-CO-, Cl 10 allyl-O-CO-NH-,
and Cl 10 aUyl-S-;
K is selected from the group consisting of halogen, Cl 10 allyL Cl 10 per~uoroalkyL
S Cl 10 alkoxy, N02, CN, OH, C02H, all~ino, 1-10 allylamino, C2 12 dialkylarl~ino, Cl-C10 acyL
and Cl 10 aLIco~y-CO-~ and Cl 10 ~l~-;
AA is a side chain blocked or unblocked amino acid with the L configuration, D
config~ation, or no c~ira~ a~ the a carbon selected from the ~roup consisting nf alanine,
valine, leucine"soleucine, proline, methionine, methionine sulfoxide, phenyl~lanine,
t~yptophan, glyeine, 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, hydro~yproline, ornithine,
homoarginine, sarcosine, indoline 2~arboxylic acid, 2-azetidinecarboxy~ic acid, pipecolinic
acid (2-piperidine carb~ylic acid~, O-methylsemle, O-e~hylserine, S-methylcys~eine, S-
eshylcysteine, S-benzylcysteine, NEI2-CH(GH2CHEt2)-COOH, a -an~inoheptanoic acid, NH2-
CH(CH2-1-napthyl)-COOH, NH2-CH(CH~2-napthyl)-COOH, NH2-CH(CH2 çyclohe~
COOH, NH2-CH(CH2~clopentyl~COOH, NH2-~I(CH2-cyclobutyl)-COOH, NH2-
CH(CH2~yclopropyl)-COOH, ~rifluoroleucine, and he~afluordeucine;
R3 and R4 are ~elected ~ndependently from the group consisting of H, Cl 20 aL~cyl,
C1 20 cyclized alkyl, C1 ~0 alkyl ~nth a phenyl group attached to the Cl 20 aLIyL cl 20 cyclized
allyl ~nth an attached phenyl group, Cl 20 allyl ~ith an attached phenyl group substituted
with K, Cl 20 aLlcyl with an attached phenyl group disubstituted with X, C120 allyl with an
attached phenyl group trisubstituted with K, ~-20 cyclized alkyl with an attached phenyl
group substituted with ~, Cl 10 alkyl with a morpholine [-N(CH2CH2)0] ring attached
~5 through l.utrogen to the allyl, Cl 10 aL~cyl ~nth a piperidine ring attached through nitrogen
to the alkyl, Cl 10 allc,vl with a py~olidine ring attached through nitrogen to the allyl, Cl 20
alkyl with an OH group attached to the alkyl, -CH2CH20CH2CH20H, Cl 10 wlth an
attached 4-pyridyl group, Cl-10 with an a~tached 3-pyridyl group, Cl 10 with an attached 2-
pyridyl group, Cl 10 ~qth an athched cydohe~yl group, -NH-CH2CH2-(4-hydro~yphenyl), and
-NH-CH2C~z(3-indolyl).
The Tetrapeptide a-Ketoarnides have the following structural formula:
Ml-AA-AA-AA-AA-CO-NR3R4
or a pharmaceutically acceptable salt, wherein
SU~STITUT SHE3ET
. . ~ .~ , . .~ . . .
wo 92/11850 2 ~ 0 9 Pc~/us9l/o97s6
~5-
Ml represents H, NH2-CO-, NH2-CS-, NH2-SO2-, X-NH-CO, X~N CO, X NH CS,
X2N-CS-, X-NH-SO2-, X2N-S02-, X-CO-, X-~S-, X-S02-, X-O~CO-, or X-O-CS-;
X is selected from the group consisting of Cl,10 alkyL Cl 10 fluoroaL~cyL Cl,10 aLkyl
substituted with J, Cl 1(~ fluoroaLlcyl substituted with J, l-admantyL 9 fluorenyL phenyl, phenyl
S substitu~ed with X, phenyl disubstituted with K, phenyl trisubstituted with K, naphthyL
naphthyl substituted with K, naphthyl disubstituted with K, naphthyl trisubstituted with X,
C 1-10 allyl v~,ith an attached phenyl group, Cl 10 aLkyl with two attached phen~l groups, Cl 10
allyl with an attached phenyl group substituted ~rith K, Cl,1o allyl with two attached phenyl
groups substituted with K, Cl,10 allyl with an attached pheno~y group, and Cl,10 aLkyl with
an athched pheno~y group substituted with K on the pheno~y group;
J is selected from ~he ~oup consisting of halogen, COOH, OH, CN, NO2, NH2,
10 alkoxy, Cl 10 allylamine, C~l2 diallylamine, C~ 0 all~yl-O-CO-, C1 10 alky
and Cl 10 allyl-S-;
K is selected from the group consisting of halogen, Cl 10 alkyL Cl 10 pe~uoroaL~yl,
Cl 10 alkoxy, NO2, CN, OH, CO2H, amino, Cl 10 aL~cylamino, C2-12 dialkylan~ino, Cl-C10 acyl,
and Cl 10 alko~y-CO-, and C1 l0 alkyl-S-;
AA is a side ~hain blocked or unbloclced asT~no ac~d w~th the L configuration, Dconfiguration, or no chirality at the a-car~on selected from the group consisting of alal~ine,
valine, leucine, isoleucine, proline, methionine, methionine sulfo~de, phenyialanine,
tryptophan, glycine, serine, threon~ne, cysteine, tyrosine, asparagine, glutamine, aspartic acid,
~latamic acid, lysine, arginine, histidine, phenylglycine, beta-alanine, norleucine, norvaline,
-aminobutyric acid, epsilon-aminocaproic acid, citrulline, hydro~yproline, ornithine,
homoarginine, sarcosine, indoline 2-carboxylic acid, 2-azetidinecarbo~ylic acid, pipecolinic
acid (2-piperidine carba~ylic acid), O methylserine, O-e~hylserine, S-methylcysteine, S-
ethylcysteine, S-benzyl~ysteine, NH2-CH(CH2CHEt2)-COOH, c~-am~noheptanoic acid, NH2-
CH(C~I2-l-napthyl)-COOH, NH2-CH(C~H2-2-napthyl)-t:OOH, NH2 CH(CH2 cyclohe~yl)-
COOH, NHrCH(CH2-cyclopentyl)-COOH, NH2-CH(CH2-cyclobutyl)-COOH, NH2-
CH~CH2~yclopropyl)-COOH, trifluoroleucine, and he sa~uoroleucine;
R3 and R4 are selected independently ~om the group consisting of H, Cl 20 allyl,Cl 20 ~yclized alkyl, Cl ~0 alkyl vrith a phenyl group attached to the Cl 20 allyL Cl 20 cyclized
aL~cyl with an attached phenyl group, Cl 20 allyl with an attached phenyl group substi~uted
with lK, Cl 20 allyl with an attached phenyl group disubstituted with K, Cl 20 allyl 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 mo~pholine [-N(GH2C:EI2)O] ring attached
SUBSTITUTE SH~ET
2~9g6~
WO 92/118~0 P~/VS91/09786-: -
~6-
through nitrogen to the alkyL cl lo aLtcyl with a piperidine ring attaohed through nitrogen
to the all~L cl lo allyl with a pyrrolidine r~ng attached through nitrogen to the allyL Cl 20
allyl with an OH group a~tached to the allyL -~H2CH20C~2CH20H, Cl 10 with an
at~ched 4-pyndyl group, Cl 10 w~th an attached 3-pyridyl ~oup, Cl 10 with an attached 2-
S pyridyl group, Cl 10 with an attached ~yclohe~yl group, -NH-C~H[2~2-(4-hydro~yphenyl), and
-NH-CH2CH2-(3-indolyl).
I~e Amino Acid ~-Ketoamides have the f~llowing s~ructural fosmula:
Ml-AA-CO-NR3R4
or a pharmaceutically acceptable salt, where~n
Ml represents H, NH2-CO-, NH2-C:S-, NH2-SO2-, X-NH-CO-, X2N CO, X NH
X2N~CS-, X-NH~02-, X2N~02-, X-CO-, X-CS-, X-S02-, X-O~ O-, or X-O-C~;-;
X is selected ~om the group consistmg of Cl 10 allyL Cl 10 fluoroallyL C1 10 alkyl
substitutedwithJ, Cl 1O~uoroallcylsubstitutedwithJ, l-admantyL 9-fluorenyL phenyL phenyl
substituted with K, phenyl disubstituted with K, phenyl trisubstieuted with K, naphthyl,
naphthyl subseituted with K, naphthyl disubstituted with K, naphthyl trisubstituted wiéh K,
Cl 10 aLtcyl with an attached phenyl group, Cl 10 allyl ~nth two attached phenyl groups, Cl 10
allyl with an attached phenyl group subs~itu~ed ~ith K, Cl 10 aL~cyl wi~h two attached phenyl
groups substituted with K, Cl 10 alkyl with an attached pheno~sy group, and Cl 10 aLkyl with
an attached pheno~y group substituted with K on the pheno~y ~oup;
J is selected from the group consist~ng of halogen, COOH, OH, CN, NO2, NH2, C
10 aL~o~y, Cl-10 allylamine, C2 12 diaLlcylamine, Cl-10 alkyl-O-CO-, Cl 10 aL~cyl-O-CO~
and Cl 10 allyl~-;
K is selected from the group consisting of halogen, Cl 10 allyL Cl 10 perfluoroallyl,
C1 10 alka~y, NO~, CN, OH, CO2H, arnino, Cl 10 allylamino, C2 l2 diallylan~ino, Cl-C10 acyl,
and Cl 10 alko~y-CO-, and C1 10 alkyl~-;
AA is a side chain blocked or unblocked am~no acid ~th the L configuration, D
configuration, or no chirality at the at-car~on selected from the group consisting of alan~ne,
valine, leucine, isoleucine, proline, methionine, methionine sulfo~ide, phenylalanirle,
tryptophan, glycine, serine, threonine, cysteine, tyrosine, asparagine, glutan~ine, aspartic acid,
glutamic acid, lysine, arginine, histidine, phenylglycine, beta-alanine, norleucine, norvaline,
~-arninobutyric acid, epsilon-aminocaproic acid, ci~rulline, hydro~yproline, ornithine,
homoarginine, sarcosine, indoline 2~arbo~y]ic acid, 2-azetidinecarboxylic acid, pipecolinic
acid (2-piperidine carbo~ylic acid), O-methylserine, O-ethylserine, S-methylcysteine, S-
ethylcysteine, S-ben~ylcysteine, NH2 CH(CH2CHEt2)-COOH, a-aminoheptanoic acid, NlH2-
S~JBSTITUTE SHEET
--
'~ WO 92/1 1850 2 ~ 9 ~ ~ ~ 9 PCI/US91/09786
~7-
CH(CH2-1-napthyl)~COOH, NH2~CH(~I2-2-napthyl)-COOH, NH2 CH(CH2-cyclohe~yl)-
COOH, NH2-CH(OEI2~yclopentyl)-COOH, NH2-CH(CH2~clobutyl)~COOH, NH2-
CH(CH2 cycloprupyl)-COOH, tri~uoroleucine, and he~luordeucine;
R3 and R4 ~re selected independentl~ f~om the ~oup consisting of H, Cl 20 allyl,S C1 20 cyclized allyL C1 20 allyl with a phenyl group a2tached to the C1 20 alkyll C1 20 cyclized
al~yl ~ith an attached phenyl group, Cl 20 allyl with an attached phenyl ~oup substituted
with K, Cl 20 alkyl ~vith an at2ached phenyl group disubstituted with K, Cl 20 allyl with an
attached phenyl ~oup trisubstituted with K, Cl 20 cyclized allyl ~ith an a~tached phenyl
group ~ubstituted with K, Cl 10 alkyl ~vith a morpholine [-N(CH2C~2~O] nng attached
through nitrogen to the ~yL Cl 1o aLlcyl with a piperidine nng attached through ni~rogen
to ~he allyL Cl 10 alkyl with a pq~rolidine nng attached through nitrogen to the aLlcyl, Cl 20
allcyl with an OH group attached to ~he aL~cyL -C~2CH2OCH2CH2OH, Cl 10 with an
attached 4-pyridyl group, Cl 10 with an attached 3-pyridyl ~oup, Cl 10 with an attached 2-
pyridyl group, Cl 10 with an attached cyclohe;cyl group, NH CH2CH2 (4-hydro~yphenyl), and
NH-CH2CH2-(3-indolyl).
The Applic~nts are aware of only a single peptide ketoamide reported in the
literature. This compound is Z-Phe-NHOEI2C0-C0-NH-Et (Z-Phe-Gly C0-NH-Et). The
compound is reported in Hu and Abeles ~4rch Bioche~L BiopJ~s.. 281, 271-274 (1990)] to
be an inhibitor of papain and cathepsin B.
The follo~nng Pep~ide Ketoan~ide compounds are representa~ve of the Peptide
Keto-Compounds found to be useful as Calpain inhibitors ~ithin the conte~t of the present
invention:
Z-Leu-Phe-CONH-Et
ZLeu-Phe-CONH-nPr
ZLeu-Phe CONH-nBu
Z-Leu-Phe-CONH-iBu
ZLeu-Phe-CON~I-13zl
Z-Leu-Phe-CONH-(CH~2Ph
ZLeu-Abu-CONH-Et
ZLeu-Abu-CONH-nPr
ZLeu-Abu-CONH-nBu
Z-Leu-Abu-CONH-iBu
Z-Leu-Abu-CONH-Bzl
Z Leu-Abu-CONH-(CH2)2Ph
SlJaSTlTUTE SHEET
.
.
~v~u~
WO 92/11B50 P~/US91/09786 -
-48-
Z Leu-AbU-coNH-(cH2)3 N(cH2c~2)2o
ZLeu-Abu-CONH-(CH2~7CH3
Z-Leu-Abu-CONH-(CH~)20H
ZLeu-Abu-CONH~ I2)2o(cH2)2oH
Z-Leu-Abu-CONH~ CH3
~Leu-Abu-CONH-CH2-c6H3(0cH3)2
Z-I,eu Abu-CONH-CH2-C4~I4N
We studied the inhibition mechanism of the Peptide Keto-Compouulds in both senneand thiol proteases. A cryshl structure of one a-ketcester bound into the active site of the
senne protease, porcine pancreatic elastase, has been completed. Ille active site Ser-l9~
o~ygen of the enzyme adds to the carbonyl group of the ketoester to ~onn a te~rahedral
~ntermediate which is stabili~ed by mteractions with the oyanion hole. This structure
resembles the tetrahedral intermediate involved in peptide bond hydrolys~s and proves that
a-ketoesters are transition-state analogs. His-57 3s hydrogen bonded to the carbonyl group
of the ester functional group, the peptide backbone on a section of the polypeptide
backbone hydrogen bonds to the inhibitor to forrn a B-sheet, and the ben;cyl 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. Intsractions with ketoarnides would be similar ~cept ~hat there is
the possibility of forming an additional hydrogen bond w~th the NH group of the ketoamide
functional group.
~ the case of ketoacids, there would be no R group to interact with the S' subsites~
Therefore, these inhibitors would be e1~pected to be slightly less potent than the ketoesters
and ketoamides. However, une~pectedly, certain ketoacid compounds have been found to
have surpnsingly high activity when used in the conte~t of the present invention. In
particular, Z-Leu-Phe-COOH and ~Leu-Abu COOH have been found to be e~tremely
potent inhbiton of Calpains.
The active site of ~ysteine proteases shares ~everal features in common with serir~e
proteases including an active site histidine residue. In place of the Ser-19~, ~ysteine
proteases have an active site cysteine residue which would add to the ketonic carbonyl group
of the peptide ketoacids, ketoesters, or ketoamides to fonn an adduct very similar to the
structure described above egcept ~ith a cysteine residue replacing the serine-195 residue.
Additional interactions would occur between the e~ended substrate binding site of ~he
~ysteine protease and the inhl~itor that would increase the binding affini~q and specificity
of the inhibitors.
SUBSTITUTE~ SHEET
. . - . . . ... ~ ...... .. .. ..
; . - ~
. . . . - . : ............. .
~, . ~ ` ~ . ...
! ,''.2~ O 92/ll8s0 2 ~ 9 ~ ~ O ~ P~/US91/0978S
~9.
The Peptide Keto-Compounds bind to the proteases inhl~ited thereby using many
of the interactions that are found in comple~es of a particular ind~Yidual en~yme wi~h its
substrates. In order to design an inhl~itor ~or a particular cysteirle protease, it L'' necessary
to: 1) find the amino acid sequences of good peptide substrates for that enzyme, and 2)
S place those or si~nilar amino acid sequences into a Peptide Keto-Compound. This design
strate~y will also work when other classes of peptide inhibitors are used in place of the
peptide substrate to gain infonnation on the appropriate sequence to place in the Peptide
Keto-Compound inhibitor. Thu~., we are able to predict the structure of new inhibitors for
other proteases based on h~owledge of ~heir substrate specificities. C)nce a good inhibitor
structure for a particular enzyme is found, it is then possl~le to change other characteristics
such as solubility or hydrophobicity by adding substituents to the M or R groups.
Ln the case of Calpain, a known inhibitor sequence is the peptide aldehyde,
Ac-Leu-Leu-Nle-H (also hlown as Calpain Inhl~itor 1 and hereinafter designated as HCIl").
This inhibitor, in addition to a related peptide aldehyde inhl~itor Ac-Leu-Leu-Nme-H (also
hlown as Calpan Inhibitor II) are commercially available from Calbiochem of La Jolla,
California. We have discovered that peptide ~-ketoesters with aromatic an~ino acid residues
in P1 are good inhibitors of the thiol proteases, ca~hepsin B, papain and Calpain.
Additionally, we have disco~vered that peptide a-ketoe~ter and peptide a-ketoamides with
either aromatic amino acid residues or small hydrophobic al~yl amino acid residues at P1
are good inhibitors of Calpain.
Our discovery of Peptide Keto-Compounds effective as Calpain Inhibitors was madethrough assay of the Peptide Keto-Compounds as reversible inhibitors. Various
concentrations of inhibitors in dimethylsulfoxide (DMSO~ 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.
88 mM K~I~Pt:)4, 12 mM Na2HPO4, 133 mM EDTA, 2.7 mM cystelne, pH 6.0 was used asa buffer for cathepsin B; and 20 mM Hepes, 10 mM CaC12, 10 mM B-mercaptoethanoL pH
7.2 buf~er was utilized for calpain I and calpain II.
All peptide thioester hydrolysis rates were measured with assay n~i~tures conta~ing
4,4'-dithiodipyridine [e324 = 19800 M-lcm-1; ~asetti & Murray, Arch. Biochem. Biophys.
119, pp 41-49 (1967)]. Papain was assayed with Bz-Arg-AMC or Bz-Arg-NA ~rKanaoka et
al., Chem. Pharm. Bull. 25, 3126-3128 (1977)], and the AMC (7-an~ino-4-methylcoumarin)
release was followed fluorimetrically (e~citation at 380 nm, and emission at 460 nm).
Cathepsin B was assayed with Z-Arg-Arg-AFC [Barrett and ~irschke, Methods Enzymol.
SUE~5Tm~ JF~T
~'
WO 92/11850 2 ~ 9 ~ 6 0 9 PC~r/US91/0978f-.,
-50-
80, 535-561 (1981)j, and theAFC (7~amino-4-trifluoromethylcoumarin) releasewas followed
~luorimetrically (e~citation at 400 ~n, and en~ission at 505 nm). Calpain I ~om human
erythrocyte~. and calpain II ~om rabbit were assayed using Suc-Leu-~r-AMC [Sasalci et al.,
J. Bio~ ChenL 259, 12489-12494 (1984), hereby incorporat~ by reference], and the AMC
5 (7-amino~-methylcoumarin) rele~e was followed fluorimetrically (e~citation at 380 nm, and
emi~sion at ~60 nm). ~atic hydrolysis rates were measured at various substrate and
inh~oitor eoncentrations, and Ki value3 were detern~ned by Di~on plot.
Table PKC1 shows the inhibidon constants (Ki) ~or papaint 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 75 buffer, containing 2mM EDTA9 ~mM ~ysteine (freshly prepared), 1%
DMSO, at 25C, using N~-Benzoyl-Arg-AMC as a substrate, ~cept that those values of
inhibition constants for papain marked with an ~e~ in Table PKC1 were measured in 50 mM
Tris-HCL pH 7.5 buf~er, containing 2û mM EDTA, S mM qsteine, 9% DMSO, at 25C,
using N~-Berlzoyl-Arg-NA as a substrate.
S'Ji3S~lTUTE SHE~T
..
: WOg2/11850 2~609 PCl~l~S9l/09786
-51-
TABLE PKC1
l~bition Qf C~ysteine Pro~eases bv
Peptide Ketoesters and Ketoacids
.
S Ki(mM)
Compounds
pa GBb ac CIId
ZLeU-AbU-COOFt 0.04 0.4
ZLeu-Phe-COOE~t 023 0.4
ZLeu-Me-COOE~t 0.12 0.18
Z-Leu-Nva-COOEt 30 1.2
Bz-DL-Phe-COOEt 500e ~4
Z-Phe-DL,Phe-COOEt 1.8 0.1
~Phe-DL,Ala-COOEt 3.6 3~
Z-Ala-A.la-DL-Ala-COOEt 15 22 200
:ZAla-Ala-DL,Abu-COOEt 0.9 10 50 200
Z-Ala-Ala-DI~Abu~OOBzl 39 60
ZAla-Ala-DL Nva-COOEt 30 0.1
~AIa-Pro-D~Ala-COOEt 26 66
MeO Suc Val-Pro-DL~Phe-COOMe 1.1 0.1
2,~e
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 Cc~ pain I
bCB=Cathepsin B dC~=Cal~ain 11
~ O~!TITl IT~ U~Ç:T
. ~ ~ . ... . ~
wo 92/11850 2 0 ~ 8 G 0 3 Pcr/usgl/o97~
-52-
It can be seen from the data in Table PKCl that the dipeptide ketoesters with Abu,
Phe, or Nle in the Pl site and Leu in the P2 site are potent inhibitors of calpain I and
Gllpain ~. Tripeptides with Abu or Ala in the Pl site and Ala in the :P2 site are also seen
to be inhibitors of Calpain, albeit somewhat weaker inhibitors than the dipeptides. Thus,
S in accordance ~ith the foregoing descri~tion of the design of Peptid~ Keto-Compound
inhibitors, we believe that Peptide Keto-Compounds b~sed on these and similar structu~es
will ex~ibit Calpain inhibitoly act~ity.
The peptide a-ketoesters are prepared by a two step Dakin-West procedure. This
pro~edure can be utilized with either amino acid derivatiYes~ dipeptide derivatives, tripeptide
derivatives, or tetrapeptide derivatives as shown in the follo~ing scheme:
o
Il
M-(AA)n-OH -- ~ Enol Ester ~ > M-(AA)n-CO-R.
l~e precursor peptide ((AA)n) can be prepared using s~ndard peptide chernistry
procedures, including those that are well described in publications such as The Peptides,
Analysis, Synthesis, Biology, Vol. 1-9, published in 19791987 by Academic Press ("The
Peptidesn) and Houben-Weyl Methoden der Organischen Chemie, VoL 15, Parts 1 and 2,
Synthese von Peptiden, published by Georg Thieme Verlag, Stuttgart in 1974 (nHouben-
Weyln) (both r~ferences hereby incorporated herein by reference).
The M group can be introduced usmg a number OI different reaction schemes. For
example, it could be introduced directly on an anuno acid as shown in the following scheme:
H-(AA)n-OH > M-(AA)n-OH.
Alternatively, the M group caD be introduced by reaction ~nth an arnino acid ester, followed
by removal of the ester group to g~ve the same product, as shown n the followmg scheme:
~(AA)n-OR'--~ M,-(AA)n-OR'--~ M-(AA)n-OH.
These and other techniques for introduction of the M group are well docurnented
in the The Peptides, Houben WeyL and many other tectl?ooks on orgamc synthesis. For
example reaction v/ith cyanate or p-nitrophenyl cyanate would introduce a carbamyl group
(M = NH;!CO-). Reaction with p-nitrophenyl thiocarbamate ~ould introduce a thio
carbamyl group (M = NH2CS-). Reaction with N~I2S402a would introduce the NH2SC)2-
group. Reaction with a substituted aLtcyl or aryl isocyanate would introduce ~he X-NH-CO-
group where X is a substituted allyl or aryl group. Reaction with a substituted allyl or aryl
isothiocyanate would introduce the X~ group where X is a substituted alkyl or aryl
group. Reaction with X-S02-Cl would introduce the X-SO2- group. Reaction with a
S~JBSTIT~JTE SHEET
. ` : : . . ~ - :
~ .
,A WO 92/11850 2 ~ 9 ~ ~ ~ 9 PCI/US91/09786
-53-
subslituted allyl or aryl acid chloride would introduce an a~yl group (M = Y-C0-). For
e~ample, reaction with MeO-C0-GH~CH2-C0 Cl would gIve ~he Y C0- ~oup when Y is
a C2 allyl substituted w~th a C~ yl OC0- group. Reaction with a substituted aL~cyl or aryl
thioacid chloride would introduce a thioacyl group (M = Y~ ). Reaction with an asubstituted allyl or aryl sulfonyl chloride would introduce an X-S02- group. For e~ample
reaction with dansyl chloride would g~ve the X S02- derIvative where X was a napthyl group
monosubstituted with a dimethylamino group. Reaction with a substituted aLl~yl or aryl
chlorofonna~e would introduce a X-0-C0- group. Re~ion with a substitu~ed allyl or aryl
chloro~iofonnate 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~ OR'. The M-AA-OH dernratives 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. Alternately1 the M group could be introduced by reaction with
carbo~ylblockedpeptides 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 o~alyl chloride Cl-CO-CO-O-R. For example, reaction of
M-AA AA-OH w~th ethyl o~alyl chloride Cl-CO-CO-O-Et gives the keto ester
M-AA-AA-CO-O-Et. Reaction of M-AA-AA-AA-AA-OH~ith Cl-CO-CO-O-Bzlwould ~ve
the ketoes~er 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 ,rarious allyl or arylalkyl oxalyl
chlorides (Cl-CO-CO-O-R).
The oxalyl chlorides are easily prepared by reaction of an aLlcyl or arylalkyl alcohol
with oxalyl chloride Cl CO-CO-CL For e~Eunple, Bzl-O-CO-CO-Cl and n-Bu-O-CO-CO-Cl
are prepared by reaction of benzyl alcohol and butanoL respec~vely, ~vith oxalyl chloride in
yields of 50% and 80% [Warren, C. B., and Malee, E. J., J. Chmmatograpty 64, 219-222
(1972); incorporated herein by reference].
Ketoacids M-AA-CO-OH, M-AA-AA-CO-OH, M-AA-AA-AA-CO-OH,
M-AA-AA-~A.AA-CO-OH, are generally prepared ~om the corresponding ketoesters
M-AA-CO-OR, M-AA-AA-CO-OR, M-AA-~A-AA-CO-OR, M-AA-AA-AA-AA-Co-OR
SlJBSrlTlJ~E SHEET
:.
W O 9 2 / 1 1 8 5 0~ p C r / U S 9 1 t 0 9 7 8 f
by aL~caline hydrolysis. In some cases, it may be necessary to use other methods such as
hydrogenolysis of a berlzyl ~oup (R = Bzl) or acid cleavage (R = t-butyl) to obtaill the
ketoacid. The alternate methods ~vould be used when the M group was labile to aL~caline
hydrolysis.
S The various peptide ketoamide subclasses (M-AA-NH-CHR2-CO-CO-NR3R4
(Di~eptide Ketoarnides (Subclass A)), M-AA-AA-CO-NR3R4 (Dipeptide Ketoamides
(SubclassB~), M-AA-AA-~A-CO-NR3R4 (InpeptideKetoamides~, M- AA-AA-AA-AA-CO-
NR3R4 (tetrapeptide Icetoarnides) and M~ CO-NR3R4 (Amino Acid Ketoamides)) were
prepared indirectly f~om the corresponding ketoesters. Ihe ketone carbonyl group was first
protected as shown in the following scheme and then the !cetoamide was prepared by
reaction with an amine H-NR3R4. The illustrated procedure should also work wi~h other
protecting groups.
Ml-AA~ ~ `R ~ -AA2~N~o~R
~ H-NR3R4
Ml-M2~ ~ ~R4
R1 o
In addition to the scheme outlined above, a ketoacid could be used as a precursor
to produce a corresponding ketoamide. Blocldng the ketone carbonyl group of the ketoacid
and then couplingwith an amine H-NR3R4 using standard peptide coupling reagents would
yield an intenned~ate ~rhich could then be deblocl~ed to form the ketoamide.
Generol Syn~etfc Method~ for Peptide Reto-Compounds
Unless otherwise noted9 materials were obtained from commercial suppliers and
used ~nthout further purification. Melting points were taken ~1vith a B~chi capillaly
apparatus and are uncorrected. lH NMR spectra were detenT~Lned on a Varian Gemini 300.
Chemical shifts are e~ressed in ppm (~) relative to internal ~etramethylsilane. Flash
column chromatography was per~ormed with Universal Scientific Inc. silica gel 0-63.
~:llBSTlTUTE S~IEET
,
~- .
: ; ~ :
WO 92/11850 2 ~ ~ 8 6 ~ 9 P~US9l/09786
Electron-impact mass spectra (MS) of novel compounds were detern~ined with a Varian
MAT 112S pe~rometer. The purity of all compounds was checked by thill- laye~
chromatography orl Baker Si250F silica gel plates Usillg th~ following solvent s,vstem: A,
CHC13:MeOH = 20:1 v/v; B, CHG13: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, CHC13:MeOH = 5:1 v/v; G,
AcOEt:MeOH = 10:1 v/v; H, (i-Pr)20; I, CHC13:MeOH:AcOH = 80:10:5 ~/v; J,
CHCl3:MeOH:AcOH = 95:5:3 v/v; K, AcOEt:AcOH = 200:1 v/v; L, C~C13; M,
C~IC13:MeOE = 50:1 v/v
Amino acid methyl ester hydrochlorides were prepared aecording to M. Brenner e~
al.[~le~. Chen~ Acta 33,568 (1950);36,1109 (1953)] Ln a s~e over 10 rnunol or according
to ~achele ~. Ch~. Chen~ 28, 2898 (1963)]in a s~e ofO.1-1.0 n~nol.
Yield (%) mp (~) m.p. (literature)
DL-Nva-OCH3HCL 100 113-116 116-117
Ir~e-OC~3 HCL 98 9C-91 98-100
L-Phe-OCH3 HCl, 98 159-161 158-160
DI,Abu-OGH3HCI, 100 148-150 150~151
L-Leu-OCH3 HC1 100 145.S~146.5 147
DL~Me-OCH3 Ha 93 120-121 122-123
4-Cl-Phe-C)C~I3Ha 98 18`4-185 (decomp.) 185-186
N-Acylamino acids was synthesized via Schotten-Baumann reaction lM. Ber~nann,
L. Zenas, Chem Ber. 65, 1192 (1932)] in the case when the acyl group was phenylsulphonyl,
2- naphthylsulphonyl or benzoyl.
STITUTE S HE~T
.
~ .
.
.
.
2~9~0~
WO 92/11850 PCr/lJS91/09786
~5~
Yield (%) mp (C) TLC (R~, eluent)
2-NapSO2-L-Leu-OH 49 115-116 058I
2-NapSO2-D~Abu-OH 51 150-151 050I
2-NapS02-L-Phe-OH 57 148-1485 0.48K
PheSO2-DL-Abu-OH 44 142-143 0.51K
PhCO-DL,Abu-OH 64 141-142 0.64K
N-A~ylamino 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-acylan~ino acid.
N-Acyl~ no Acid Met*yl Este~s (Gener~ Pr~cedl~re). To a chi~led (10 C) slurry of
the appropriate amino acid methyl ester hydrochloride (20 mmol) in 100 ml benzene was
added slowly (temp. 10-15 C) 40 rnmol triethylamine or N- methylmo~pholine and then the
reaction mixture ~as stirred for 30 n~inutes at this temperature. Then 18 rnmol of
appropriate acid chloride (temp. 10-15 C) was added slowly to the reaction mi~ure and
the reaction n~ture was stirred overnight at room temperature. The p~ecipiatatedhydrochloride was filtered, washed on a funnel with 2 ~ 20 ml benzene, and the collected
filtrate was washed successivdy with 2 ~ 50 ml 1 M HCI, 2 x 50 rnl 5% NaHC03, 1 x 100 rnl
H20, 2 x 50 rnl satd. NaCl and dried over MgSO4. After evaporation of the solvent in vacuo
(rotavaporator), the residue was checked for purity (ILC~ and used for the next step
(hydrolysis).
Yield (%) mp (C)
(cH3)2cH(cH2)2co-DLAbu ocH3 80 oil
(CH3CH2CH2)2CHCO-DL-Abu-OCH3 96 117-118
Ph(CH2)6CO-DL,Abu-OCH3 72 oil
Hyd~o~is (Get~eral Proced~e). To a solu~ion of 10 mmole of the appropriate
N-as~yiamino acid methyl ester in 100 rnl of methanol was added in one portion 11.25 ml of
1 M NaOH (11~ mmol? and the reaction mi~ture was stirred three hours at room
temperature. Then the reaction n~Ll~ture was cooled to 0 C (ice- salt bath) and acidified
to ~1 = 2 with 1 M Ha aq. To this reaction m~ture was added 100 ml ethyl acetate,
transferred to a separatory funnel and organic layer separated. ~he water layer was
saturated with solid Naa or (NH4)2SO4 and ree3~tracted with 2 ~ 50 ml AcOEt. Tlse
collected organic layer was washed with 2 ~ 50 rnl H20, decolorized with carbon, and dried
over MgS04. After evaporation of the solvent in ~vacuo (rotavaporator), the residue was
~ SUBSTITUTE SHlEEt
-
.. . . . ... ; .
`- ~ .
. ' : . .
~ ` wo 92/11g50 2 ~ 9 3 6 ~ 9 PCr~lJS91/09786
cheeked for puri~ (TLC) and in the case of contan~ination was crystallized ~om an
appropriate solvent.
Yield (%) mp (C)
(CH3)2CH(CH2)2CO DL-Abu-OH 92 1105-112
(CH3CH2CH2)2 C~HCO-DL-Abu OH 99 126-127 (n-octane)
Ph(CH2)5CO-DI~Abu-OH 89 110-112 (n-octane)
N~Ac3rldipeptide methyl esters were syllthesized via ~he HOBt-DCC method in a
DMF solution [K8 ug and Beiger, C)~em Ber. 103, 788 (1970)].
Yield (%) mp (C) TLC (Rf, eluent)
Z-Leu-DL-NVa-OCH3 80 112-113 0.37 B
ZLeu-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-OCH~ 99 86-88 033 B
0.26 H
Z-Leu-L-Leu-OCH3 80 91-92 0.79 G
Z-Leu-DL-NLeu-OCH3 97 111-1115
Z Leu-4 Cl-Phe-OCH3 65 112-132 0.77 J
(liquid c~ystal?) 0.68 K
2-NapSO2-Leu-DL-Abu-OCH3
99 oil 0~9 A
2-NapSO2 Leu L-Leu-OCH3
97-98.5 0.63 A
N-Acyldipeptides were obtained by hydrolysis of the appropriate methyl esters via
a generàl hydrolys~s procedure. In the case of N-sulphonyldipeptide methyl esters, 1
equivalent of the methyi.ester was hydrolyzed with 2.25 equivalent of 1 molar NaOH
because of forrn a sulfonamide sodium salt.
SUlBSTlTlJTE SHEIET
WO 92~11850 2 ~ 9 8 ~ ~ 3 I'CTtUS91/09786 r::
Yield (~o) mp (C) TLC (R~, eluent)
Z-I.eu DL-~a-OH 100 117-118.5 0.11 A
ZLeu-L-Phe OH 92 105-106.5 0.28 C
055 G
Z Leu-L~ OH 79 77-79 0.22A
05~C
~leu-DL-Abu OX 99 gl~ss 0.61 G
~Leu-L~.Leu-OH 97 glass 056I
ZDLeu-DI,NLeu-O~I 98 95~96
ZLeu~-Cl-Phe-OH 87 104-114 0.48 K
(liquid crystal?)
2-NapS02-Leu-DL-Abu-OH
97.4 180-195 (decomp) 0.58 I
2-NapSC)2-Leu-L-Leu~OH
94.0 68-70 052 I
N-Acytripeptide methyl esters were synthesized via HOBt- DCC me~hod in DMF
solution LrKonig and Geiger, Chem. Ber. 103, 78B (1970)].
Yield (%) mp (C) TLC (Rf, eluent)
~Leu-~eu-Abu-0C~I3 87 140-1415 050 A
20~Leu-Leu-Phe-OGH3 76 158-159 0.83 J
2-NapS02-Leu Leu-Abu-OC~I3
97 ~200 0~2 A
N-Acyltripeptide were obtained through hydrolysis of the appropriate methyl esters
via general hydrolysis procedure. Ln the case of N-sulphonyltripeptide me~hyl ester, 1
25 equivalen~ ~f methyl ester was hydrolyzed with 2~5 equivalent of 1 molar NaOH to form
the sulfonamide ssdium salt.
Y.ield mp (C) TLC (R~, eluent)
~Leu~Leu-Abu-OII 97 glass 0.69 I
ZLeu-Leu-Phe OH 98 glass 0.44K
302-NapS02-Leu Leu-Abu-OH
- 85 193-195 0.~3 I
(decomp.) 032 J
The following examples, Examples PKC1-PKC65, are given to illus~rate the synthesis
35 of Peptide Keto-Compounds:
SUBSTITVTE SHEET
, . .
, : . -
WO 92/11850 2 ~ ~ 8 6 ~ 9 PCTtUS9l/09786
759~
EXAMPI,E PKCl
-DL~ COOEt. This compound was s~mthesized by a modi~ied Daki~i-West
procedure [Charles et al., J. Chem. Soc. Perlcin L 1139-1146, (1980)~. To a stirred solution
of ZAla-Ala-OH (880 mg, 3 mmole), 4 dimethylaminopyridine (15 mg, 0.31 mmole), and
pyridine (0.8 mL 10 mmole) in tetrahydrofuran (3 mL) was added ethyl o~alyl chloride (0.7
mL, S rnmole~ at a rate sufficient to ~n~tiate reflu~g. The n~ture was gently reflu~ed for
35 h. The mi~ture was treated ~ith water (3 mL) and stirred vigorously at room
~emperature for 30 n~in. The m~ture was e~tracted w~th ethyl acetate. The organic e~tracts
were dried and evaporated to~ ol~tah ~e- residue (1.45 g). The residue was
chromatographed on silica gel and eluted with CH2Cl2 to g~ve the enol ster product, oil
(500 mg, 37~O); single spot on tlc, R~2 = 0.67 (CHCl3:MeOH = 9:1); MS, m/e = 451(M++1). To a stirred suspension of the enol ester (~10 mg, 0.47 mrnol) ~n anhydrous
ethanol (1 mL) at room temperature was added dropwise a solution of sodium ethoxide in
ethanol until a dear yello~ soiution resulted. Ihe ethanol was then removed and the
residue was trea~ed 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 duted ~nth methylene chloride. The solvent was removed, and the peptide
ketoester ZAla-DL,Ala-C02Et was obtained as an sen~i-solid (1~0 mg, 92 %); single spot
on tlc, Rf1 058 (C~Ia3:MeOH ~ 5:1); MS, m/e = 351 (M~1). Anal. Calcd. for
C17H2206N2-1/3 H20: C7 57.29; H, 622; N, 7.86. Found: C, 57.23; H, 6.36; N, 8.17.
EXAMPI,E PKC2
Z~Ala-Ala-DL~ CO2Et. This compound was prepared ~om ZAla-Ala-Ala-OH
using the same procedure a~ described in E~ample PKC1. The product was crystalli~ed
from ethyl ether in 23% yield; sDgle spot on tlc, Rf2 ~ 031 (CHC13:MeOH = 9:1); mp
143-144 ~C; MS, m/e ~ 421 (M+).~AIial. C~llcd. fbr C2~H27O7N3: C, S6.99; X 6-46; N,
9.97. Found: C, 56.96; H, 6.49; N, 9.92.
EXAMPI.E PKC3
Z-Ala-Ala-DL,-Abu-C02Et. This compound was prepared from ZAla-Ala-DL-
Abu-OH in 11% yield by the procedure descnbed in Example PKC1; single spot on tlc,
Rf2 = 0.60 (CHCl3:MeOH = 9:1); mp 111-113 C; MS, m/e = 436 (M+~1). Anal.
Calcd. for C21H2907N3-1/3 H20: C, 57.13; H, 6.75; N, 9.51. Found: C, 57.38; H, 6.82;
N, 9.62.
~ n~lTl ITr CUE~T
. . .
WO 92/11850 2 ~ 9 8 5 9 ~ PCI/lJS91/09786
-60-
E~AMPLE PKC4
~AIa~Ala-l)L~Nva-CS:)2Et. This compound was prepared from ~AIa-Nva-OH in
20% yield by l:he procedure descri~ed in E~ample PKCl; single spot on tlc, R~l = 0.64
(CHCl3:MeOH = 5:1); MS, m/e = 450 (M~+l). AnaL C~lcd. for C~H3lO7N3H2O: C,
56.51; lH, 7.11; N, 8.99. Found: C, 56.42; H, 7.08; N, 9.06.
EXAMPLE PKC~
Z-Ala-Pro.I)~Ala-CO2Et. ~his compound was prepared ~rom ~AIa-Pro-Ala-
OH dicycloh~ylamine ~n 19% yield b~ the proeedLIre descnbed in ~ample PKCl; sing~e
spot on tlc, ~2 = 055 (G~CI3:MeOH 8 9:1); MS, m/e = 447 (M~). An~l. Calcd. ~or-
C~2H2907N3 1/2 H20: C, 57.88; H, 6.62; N, 9.21. Foumd: C, 57.65; H, 6.68; N, 9.17.
EX~IPLE PKC6
Z-AIa-A~la-~-DL-~la CO2Et~ The compound was prepared from ~AIa-Ala-Ala-
Ala-OH in 7% yield by the procedure described in ~ample PKC1; single SpOt on tlc, Rf2
-0.40 (C~Ht:13:MeOH = 9:1); mp. 163-165 C; MS, m/e = 493 (M++1). Anal. Calcd.
for C23H3208N4-1/2 X20: C, 55.08; H, 6.63; N, 11.17. Found: C, 54.85; H, 6.53; N,
11.14.
EXAMPLE PKC7
Bz-DI,Phe-CO2Et. ~ compound was prepared ~om Bz-Phe-OH in 36% yield
by the procedure descnbed in E~cample PKC1, oil, single spot on tlc, Rf2 = 0.61
(CHCl3:MeOH = 9:1); MS, m/e ~ 325 (M~). AnaL Calcd. for C~ 904N l/3 H20: C,
68.86; H, 5.98; N, 4 22. Found: C, 69.10; H, 6.09; N, 438.
EX~MPIE PKC8
M~O-Suc~Ala.DL Ala-CO2M~ This compound was prepared from MeO-Suc Ala-
Ala OH n 22% yield by the same procedure as descrlbed in E~ample PKC1, e~cept that
sodium metho~ide in methanol was used for enol ester bydrolysis, single Spot~orl tlc,
- 0.43 (CHC13:MeOH = 9:1); MS, m/e ~ 317 (M~ + 1). AnaL Calcd. for
Cl3H2~07N4 1/3 H20: C, 48.44; H, 6.46; N, 8.69. Found: C, 4856; H, 6.39; N, 8.69.
SUBSTITUTE S~5EET
.
wO 92/118S0 2 ~ ~ ~ 6 ~ 9 P~/US91/097~6
~1-
E~MPLE PKC9
MeO Suc~ P~DI~Abu~CO2M[g. This compolmd was prepared ~om
MeO-Suc-Ala-Ala-Pro-DL,Abu~OH in 22% yi~ld b~ the proeedure described in ExamylePKC8; foam, single spot on tlc, Rfl = 0.66 (CHCl3:MeOH = 5:1). Anal. Calcd. for
S C22H3409N4H20: C, 51.53; H, 7.02; N, 10.85. Found: C. 51.11; H, 7.03; N, 10.88.
EXAMPLE PKC10
MeO-Suc-Val.P~DL-Ph~Ct)2~Me. I~is compound was prepared from MeO-
Suc Val-Pro-Phe OH in 42% yield by the same procedure as descnbed in E7~ample
PKCB; foam, single spot on ~ 2 057 (~ICl3:MeOH = ~:1); MS, m/e - 517 (M+).
10 Anal. Calcd. ~or C~6H350~N3 2/3 H20: C, 58.96; H, 6.90; N, 7.93. Found: C, 58.92; H,
6.96; N, 7.89.
EXAMPLE PKC11
Bz-DL,Ala-CO2-n-Bu~ This compound was prepared from Bz-Ala-OH in 45%
yield by the procedure descnbed in E~ample PKC1, e~c pt that n-butyl oxalylchloride
15 was used for the Dakin-West reaction and sodi~n n-butoxide in n-butanol was used for
enol ester hydrolysis; coloriess oil, single spot on tlc, R~2 = 0.72 (CHCl3:MeOH = 9:1);
MS, m/e = 277 (M+).
EXAMPLE PKC12
Bz DI,Als C02BzL Ihis compound was prepared ~om Bz-Ala-OH in 26% yield
20 by the procedure described in Example PKC1, el~cept that benzyl oxalyl chloride was
used in place of ethyl oxayl chloride and sodiurn benzylo~ide in benzyl alcohol was used
for enol ester hydrolysis; single spot on tlc, ~2 e0.69 (CHCI3:MeOH = 9:1); mp 95-97
C; MS, m/e - 312 (M-~ +1). AnaL Calcd. for Cl8Hl704N.l/2 H20: C, 67.48; H, 5.66;
N, 437. Found: C, 67.78; H, 5.SS; N, 4.66.
EX~MPLE PKC13
ZAla-DL~Ala-CO2 ~-Bu. This compound was prepared from Z-Ala-Ala-OE in
14% yield by the procedure described in ~ample PKC1, except that n-but~yl oxalylchloride was used in the Daldn-West reaction and s~dium n-butoxide was used for enol
ester hydrolysis; oil, single spot on tlc, Rf2 = û.45 (CEIC:13:MeOH = 9:1); MS, m/e =
30 378 (M~). Anal. Calcd. for Cl~H;606Nil/3 H20: C, 5935; H, 7.00; N, 729. Found: C,
59.41; H, 7.03; N, 7.10.
EXAMPLE PKC14
:~Ala-DL,Ala-CO~BzL This compound was prepared ~om ZAla-Ala-OH In
36% yield by the procedure described in Example PKC1, except that benzyl oxalyl
SUBSTITUT SHI1ET
~ . ; . ~. . . .
209~ t
WO 92/11850 Pcr/us9l/o9786:
-62-
chloride was used in the Dakisl-West reaction and sodiwn be~lo~ide in benzyl alcohol
was used for enol ester hydro~sis; single spot on tlc, R~2 = 0.55 (CHCl3:MeOH ~ 9:1);
MS, m/e = 413 (M+ ~1). AnaL Calcd. ~r C22H24O6N2~ C, 64.06; H, 5.87; N, 6.79.
Found: C, 63.79; H, 5.95; N, 6.72.
EXAI~LE PKC15
~AIa-Ala-DL,Abll-CO2BzL l~is compound was prepared ~om Z~Ala-Ala-Abu-
OH in 31~o yield by the proc~dure descnbed in E%ample PXC1, ~s~ept that ben~yl o2a~l
chlonde ~as osed in ~he Dal~n-West reaction and sodium benz~ ide in benzyl alcohol
~as used for enol ester hydrolysis; sin~e spot on tk; R~2 = 0 40 (CHa3:MeOH = 9:1);
mp 124-125 C; MS, m/e = 498 (M I + 1). Anal. Calcd. for C2~H3lQ7N3 2/3 H20: C,61.28; H, 639; N, 8.24. Found: C, 61.14; H, 6.65; N, 7.94.
E~AMPLE PKC16
Bz-DI,.ALq-COO}I. The hydrolysis procedure of Tsushima et al. F Org. Chem.
49, 1163-1169 (1984)] was used. Bz-DL,Ala-C02Et (540 mg, 2.2 ;s3~nol) was added to a
solution of 650 mg of sodium bicarbonate in an aqueous 50% 2-propanol solution (7
mL of H20 and 2-propanol) and stirred at 40 C under n~trogen. After adding ethyl
acetate and a saline solu~ion to the reaction mi~ture, the aqueous layer was separated
and acidified with 2N HCl and e1~tracted with ethyl acetate. The organic layer was dried
over magnesiurn sulfate and the solvent was removed under reduced pressure. The
cmde hydrol~sis product was chromatographed on silica gel and eluted with methylene
chloride and methanol to obtain an oil (150 mg, 31%); single spot on tlc, R~4 = 0.68 (n-
butanol:acetic acid.~yridlne:H2O - 4:1:1:2). Anal. Calcd. for CllH1104N.3/4 H~O: C,
5628; H, 537; N, 5.97. Found: C, 5621; H, ~.46; 5.66.
Eg~MPLE PKC17
Z-Leu-I~L-Nva-COOEt. This compound ~vas prepared ~om Z-Leu-Nva-OH in 60
% yield by the procedure descn~d in E~ample P}~C1; oil, one spot on tlc, Rf = 0.49
(CHGI3:MeOH Y 20:1). NMR ~CDCI3) ~: 0.91 (t, 9H), C~I3; 1.25 (t, 3H), CH3; 1.38 (q,
2H), OCH2C~3; 1.64 (m, 6H), C$I2; 1.85 (m, 1H), CH(CH3)2; 434 (m, 1H)
CH2CEI(NHCOOC~I2Ph)CONH; 5.12 (d, 3H) NHGH(CO)CH2 and OCH2Ph; 532 (d,
lH) NH; 5.71 (d, 1H) NH; 736 (s, ~H) Ph.
Z-Leu-DI,Nva-end ester, the preeorsor OI Z-I eu-DL-Nva-COOEt was
synthesized by the same procedure as descn~ed in Example PKC1 and purified by
column chromatography, oiL one spot on tlc. N~IR (CDCl3) ~: 0.96 (t, 9H); 1.25 (t,
SVB~;TITUTE~ SH~ET
,
. .
2 ~ 3
WO 92~118~;0 PCI`/US91~09786
-63 -
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);
.16 (q, 2~I); 5.23 ts, lH); 737 (m, SH); 1133 ~s, lH).
EXAMPIE PKC18
Z L6u DI,Phe-COOEt. This compound was prepared from ~Leu-Phe-OH ~n 30
% yield by the procedure descn~d in E~ample P~Cl; oil~ one spot on tlc, Rf = 0.47
(CHC13:MeOH - 50:13. NMR (CDGI3) ~: 0.88 (d, 9H), OCH2CH3 aIId (CH3)2CH; 1.35
(q, 2H), OCH2CH3; 156 ~q, 2~), (CH3)2CEIGH2 ::H; 3.03 (m, lH), (CH3)2CH; 4.32 (m,
2~I), NHCH(CO)C~H2; 5.08 (s, 4~) GH2Ph; ~.40 (m, lH) NH; 6.61 (d, l~I) NH; 7.31 (s,
5H) Ph; 73S.(s, ~H) Ph.
Z-Leu-DL~Phe enol ester, the precur~or of Z-Leu-DL-Phe~COOEt was
synthesized by the same procedure as descnbed in E~ample PKC1 and puri~ied by
column chromatography, oil, one spot on tlc. NMR (CDCl3) B: 0.86 (t, 3H); 0.99 (t,
3H); 1.24 (t, 3H); 1.40 (t, 3H); 152 (m, 2H); 1.83 (m, 2H); 4 23 (m, 4H); 4.39 (q, 2H);
5.10 (t, 2H); ~.18 (s, 1H); 7.26 (m, SH); 734 (m, ~H); 8.89 (s, 1~I).
EXAMPLE PKC:l9
2-Leu-DL Abu COOEt. This oompound was prepared from Z-Leu-Abu-OH in
33 % yield by the procedure de~bed in Example P~C1; oiL one spot on tlc, Rf = 0.66
(CHC13 McOH a 20:1). NMR (CDCl3) ~: 0.96 (t, 9H), OG~2CH3 uld (CH3)2CH; 1.26
(t, 3H), CH2CH2CH3; 137 (q, 2~), OGH2S~H3; 1.66 (q, 2H), (C~3)2~CH2CH; 2.00
(m, lH), CH(CH3)2; 4.12 ~q, 2H) GHGH2CH3; 4-34 (m, 1H)
NHCH(CONH)CH2CH(C~H3)2; 3.12 (q, 3H) CE[2Ph and CONH~Et)CHCOCOO; ~.29 (t,
lH) NH; 6.79 (d, lH) NH; 735 (s~ SH) Ph.
Z Leu-DL-Abu-enol ester, the precursor of Z Leu-DL-Abu-COOEt was
~gnthes~zed ~y the same procedure as descnbed in E%iample PKC1 and puri~ed by
column chromatography, o~, one spot on tlc. NMR (CDCI3) ~: a.ss (t~ 6H); 1.12 (t,
3H); 1.24 (t, 3H); 1.41 (t, 3H); 1.73 (m, 4H); 2.86 (q, 2H~; 4.20 (q, 2H); 431 (m, lH);
4.42 (q, 2H); 5.15 (q, 2H); 5.21 (s; 1H); 734 (m, ~H~; 11.29 (s, lH).
EXAMPI,E PKC20
Ala-DI,I,ys-COOEtHCL To a solution OI N-carbobenylo%yalanyl-Ne-
,i 30 carbobenzylo~ylysine (1.88 g, 3.9 mmol~, 4-dime~hylan~inopyridine (21 mg, 0.17 mmol),
and pyndine (1.0 rnL 12.4 mmol) in l~ (7 mL) was atded ethyl oxalyl chloride (0.9
mL 8.0 mmol) at a rate sufEicien~ to start refluxing. The mi~ture was refllLxed gently for
3 hr, treated with water (4 mL), and stirred vigorously at room temperature for 30 min.
The ~ ture was extracted with ethyl acetate, the organic extracts were washed with
SV~TITLIT~ S~FFT
., ~, .
..
. .
.
u u ~J ~
WO 92/~1850 ~CI/US91/09786 ~^
water, dried over MgSO4 and evaporated to give an oily residue (156 g). To a solution
of the enol ester (1.56 g, 2.7 snmol) in anhydrous ethanol was added dropwise a solution
of sodium etho~de in ethanol at room temperature until the solution turned ~learyellow. Eth~nol w~c remo~ed and the residue was dissolved in ethyl acetate. The
S organic soluaion was washed with water, riried over MgSO4, and evaporated to g~ve a
residue. This residue was then purifi d b~ colurnn chromatography and the product was
eluted with chlorofonn-methanol. The solvent was remo~ed and Z~ DL-Lys(Z)-
C02Et was obt~uned as a hygroscopi~ powder (328 mg, 16 %), single spot on tlc, R~2 =
053 (CEIC13:MeOH = 9:1); MS, m/e = 542 (M~ + 1).
N-Carbobenzoxyalanyl-DL,N~carbobenzo~ylysine keto ethyl ester, ZAla-DL-
Lys(Z)-CO2Et (328 mg, 0.61 mmol) ~as deprotected with liquid HF contai~ing anisole at
O C for 30 min. The HF was removed under reduced pressure. I~e residual oil wasdissolved in absolute ethanol. HCI/ethanol was added to the solution, and ethanol was
removed h vacuo. The residue was washed by decantation with ether to give a semisolid (216 mg, 1~0 %); single spot on tlc (n-butanol:acetic acid:pyridine:H20 = 4:1:1:2).
EXAMPLE PKC21
Bz-DL.-Lys-COOES~ICL This compound wasprepared ~rom Bz-DL-Lys(Z)-
COOEt in 62% yield by the procedure descnbed in E1~ample PKC20; one spot on tlc, Rf4
= 057 (n-bu~nol:acetic acid pyridine:H20 = 4:1:1:2). The precursor, Bz-DL-Lys(Z)-
COOEt w~s prepared ~om Bz-Lys(Z)-OH in 100% yield by the procedure described in
Example PKCl; powder, one spot on tlc, E~f2 = 0.75 (CHC13:MeOH = 9:1); MS, m/e =440 (M ~ ). Anal- Calcd. for C24H2806N2~/3 H20: C, 63.70; H, 653; N, 6.19. Found: C,
63.49; H, 651; N, 5.92.
EXAMPLE PKC~2
3~-DL Arg COOEt X~L This compound was prepared from Bz-DL-Arg(Z)-
COOEt in 99% yield by the procedure descn~ed in E;~mple PKC20; one spot on tlc, Rf4
= 0.71
(n-butanol:acetic acid:pyridine:H20 = 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 E~ample PKC20, R~2 = 038 (C:HG13:MeOH = 9:1); mp 140-142 C; MS,
m/e = 468 (M+). AnaL Calcd. for C24H2~O6N4: C, 6153; H, 6.02; N, 11.96. Found: C,
61.96; H, 6.48; N, 1234.
8U13ST~UTE SHE~ET
.~ - . WO 92/1 18~0 2 ~ ~ 8 ~ ~ 9 Pcr/US91/09786
~5-
EXAM[PLE PK~3
H-GI~-DL,Lys-COOEt 2HCL l'his compo~nd was prepared from ZC;ly-DL-
Lys(Z)-COO:E~ in 92% yield b~ the pro~edure descn~ed in E~ample PKC20; Rf4 = 0.21
(n-butanol:acetic acid:pyridine:H2O = 4:1:1:2). ZC;ly-DL-Lys(Z~-COOEt was prepared
from ZGly-Lys(Z)-OH in 9% yield by the procedure described in E~ample PKC20, orle
spot on tlc, Rf1 = 0.68 (CHC13:MeOH = 5:1); MS, m/e = 528 (M+ + 1).
~LE PKa4
H PFo-DL~Lys-COOEt21HCL Thi~ compound was prepared from Z-Pro-DL-
Lys(Z)-COOEt in 100% yield by the procedure descn~ed in E~ample PKC20; one spot
on tlc (n-butanol:acetic acid:~dine:H2O = 4:1:1:2). ZPro-DL-Lys(Z)-COOEt was
prepared from
ZPro Lys(Z) OH in 15% yield by the procedure desoribed in E;~ample PKC20; Rf2 =
0.73 (CHC13:MeOH = 9:1); MS, m/e 568 (M~ + 1).
EXAMPLE PKC25
H-Ph~DL,Lys-COOEt 2HCI. This compound was prepared f~om ~Phe-DL-
Lys(Z)-COOEt in 39% yield by the procedure descnbed in E~ample PKC~0; one spot on
tlc (n-butanol:acetic acid:pyridine:H2O = 4:1:1:2). Z-Phe-DL,Lys(Z)-COOEt was
prepared ~om ZPhe-Lys(Z)-OH as previously de~ ed in 9% yield, Rf2 = 0.68
(C~ICl3:MeOH = 9:1); MS, m/e ~ 482 (M~).
EXAMPLE PKC26
H Leu Ala D~L~ COOEt~HCL Thi~ compound was prepared from Z-Leu-Ala-
DL Lys(Z)-COOEt in 52% yield by the procedure described in E~ample PKC20; one
spot on tlc (n butand:acetic acid:pyridine:H20 2 4:1:1:2).
~Leu Ala DL,Ly~(Z)~COOEt was prepared from Z Leu-Ala DL-Lys(Z)-OH in
S~o yield by the pre~iously described Dakin West reaction, R~3 ~ 0.34 (CHCl3:MeOH =
19:1); MS, m/e = 609 (M~-C)CH2~H3)-
EXAMPLE PKC27
S~le Am no Acid, Df- and T~fpeptide Enol Esters (Çeneral Proce~re). A
modi~ied Dakir~-West procedure was used [Charles et aL, J. Che~ Soc Per~ , 1139
- 30 (1980)~ and is illustrated ~nth the sgnthesis of Z-IAu-DL~Phe-EE. To a stirred solution
of ~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 rnmol) in ~etrahydrofuran (45 ml) warmed 50 C was added
ethyl oxalyl chloride (430 g, 352 ml, 31.5 rnmol) at a ra~e su~icient to initiate re~uxing.
I~e rnD~ture was then heated at a gentle refl~ for 4 h. After cooling to room
~1 IR~tTI ITF ~ FFT
. . . .
- -
: ,
WO 92/118~0 PCr/US91/0978t
-66-
temperature the rni~ture vas treated v.rith water (25 rnl) and stirred v;gorously at room
temperature for 30 ~in. rhe rn~xture was ~xtracted with ethyl acetate (150 ml) and after
separation of the organic layer, the ~vater layer was saturated with solid (NH4)2S04 and
re-e~tracted 2-times v~ith 2~ ml ethyl acetate. The combined organic phases were washed
2-tirnes ~ith 75 rnl water, 2-times with 50 rnl of satd. NaCL decolori~ed with ca~bon and
dried over MgSO4. After evaporation of the solvent, the crude enol ester (8,36 g, 98%)
was Elash-chromatographed on silica gel and the product wa~ eluted with a AcOEt. The
solvent was evaporated in vacuo ~rotavaporator~ and the pure enol ester was obtained as
a oil (7.22 g, 85%); single spot on TLC, R~ = 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
CHC13: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 p~epared from ZLeu-Abu- OH in 78%
yield the general procedure described above. Purifioation by flash obramatography on
silica geL $1uent, CHC:13:MeOH = ~0:1 v/Y, sillgle spot on IrLC, Rf = 0,86, A.
PhCO-Abu-EE. This compound was prepared ~om PhCO-Abu-OH in 26% yield
by the general procedure as descnbed above. Purification by flash chromatography on
silica gel. Eluent CHC13, single spot on TLC, R~ = 0.60, M.
(CH3)2CH(CH2)2CO-Abu~ This compoundwas prepared from
(CH3)2CH(CH2)2CO-Abu-OH in 82% yield by the general procedure as described
above. Purification by flash chromatography on silica gel. Eluent AcOEt, sin~le spot on
TLC, Rf = 0.72, C.
(CH3CH2CH~)2 CH CO-Abu-EE. 'rhis compound was prepared from
(CH3GH2CH2)2CH CO-Abu OH in 100% yield by the general praeedure described
above. Purification by ~ash chromatography on silica geL Eluent AcOEt, sin~le spot on
TLC, Rf = 0.78, C; 0.81, K
Ph(CH~)6CO-Abu-EE. This compound was prepared from
Ph(CH2)6CO-Abu-OH in 86% yield by the general procedure descnbed above.
Purification by flash chromatography on silica gel. Eluent AcOEt. Single spot on Tl C,
R~ =0.74, C.
Z-Leu4-CI-Phe-EE. T~s compound was prepared from ZLeu- 4-Cl-Phe-OH in
69% yield by the general procedure described above. Purification by flash
chromatography on silica geL Eluent AcOEt, single spot on TLC, Rf = 0.77, C; 0.78, K.
!~:U~TlTlJTF ~ IF1~T
` WO92 2~8~9
/11850 PCI'/US~t/09786
-67-
Z-Leu-Leu-~bu-EE This compound was prepared from Z2-Leu- Leu-Abu-OH
in 62% yield by the general procedure descIibed above. Puri~cation by flash
chroma~ography on silica geL :~lement CHC:13:MeOH = 50:1 v/v. Single spot on TLC,
Rf = 0.89, A; 0.75, M.
Z-Leu-Leu-F'he-EE. ~s compound was prepared from ZLeu- Leu-Phe-OH in
60% yield by the general procedure descnbed above. ~ication by fl~sh
chromatography on silica geL ~luent CHa3:MeOH = 50:1 v/v. Sin~e spot on TLC, Rf
= 0.80, K; 0.70, M.
2-NapSO~ Leu-Abu-E;E;. This compoun~ was prepared from
lQ 2-NapSO~-Leu-Abu-OH in 73% yield by the ~eneral procedure descn~ed above.
Purification by flash chromatography on silica gel. Eluent AcOEt, single spot on TLC,
Rf =-0.71, K; 05~, C.
2-Nap50~Leu-Leu-Abu-E;E;. ~is compound was prepared 30m
2-NapSO2-Leu-Leu-Abu-OH in 74% yield by the general procedure descnbed above.
Purification by ~ash chromatography Oll silica gel. Eluen~ AcOEt: AcOH = 200: 1 v/v.
Single spot on II~ = 0.69. K
Z-Leu~he-COOEt. Sulgk Arr~ D~nd Tnpeptide- k~tocste~ (Gerleral
Procedure). To a stirred solution of B.53 g (15.0 mmol) of ZLeu-Phe-EE in 40 ml
anhydrous ethanol at room temperature was added dropwi3e a solution of sodium
ethoxide (0.204 g; 3.0 mmol) in 20.0 ml anhydrous ethanol. The c~lor of the reaction
n~i~nlre change from colodess or pall yellow to deep yellow or orange dependent on
enol-ester. Then the reaction rni~ure was stirred at room temperature for 4-5 hours, the
ethanol was then evaporated in va~uo (rohvaporator) 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 ~ 75 r~ H~O, 2 ~ 75 JTII satd. NaCl, decolorized with
carbon and dried over MgS04. After ~vaporation of solvent, the crude product 6.09 g
(89.7%) was flash chromatographed on silica gel using CHC13: MeOH - 50:1 v/v.
Evapora~ion of ~olvent give pure Z-Leu-Phe-COOEt (4.08 g, 58.0%) as a thick oil.Single spot on ILC, R~ = 0.60, A; 0.47, M. Mass spectrum, FB-MS [~M+ 1~/Z3 = 469.
EXA~LE PKC28
2.Leu.Nva.COOEt. ~is was prepared by the preceding general procedure.
Purification by flash chromatography on silica gel, eluent CEICI3: MeOH = 100:1 v/v,
yield 86.6%, thick colorless oil, single spot on TLC, Rf = 0.49, A; 037, M. Massspe~rusn F~3-MS ~(M~1)/Z] = 421.
~Tl ITF .
. . .. - ~ .
:
.
~, ',.
wO92~11850 2~6a~ PCI/US91/097~6^-
-68-
EXAMPLE PKC29
-Abu-COOE~ ~s was prepared by the preceding general pr~edure.
Purification by :~ch chromatography on silica gel, eluent CHCl3, yield 82%, thick pale
yellow oil, single spot on TLC, R~ = 0.66, A. Mass spectrum, CI-MS [(M+ 1)/Z] = 407.
S EXAMPLE PKC3D
PhCO-Abu-COOEt. I~is was prepared by the preceding general procedure.
Puri~cation by flilsh chromatography on silica gel, eluent CHM3:MeOH = 50:1 v/v,yield 83%, oiL sin~e spot on TLC, Rf = 0.44, M. Mass spectn~m, M/Z 263 (M~
CI-MS, 264 ((M+ 1~/Z).
EXAMPLE PKC31
(CH3)2CH(~Ia)2CO Abu-CVOEt. ~s was prepared by the preceding general
proeedure. Purification by ~ash chromatography on silica geL eluent AcOEt, yield 43%,
oil, sin~le spot on TLC, Rf = 056, C. Mass spectmm EI-MS M/7 257 (M+); FB-MS,
[(M+1)tZ3 = 258.
13;XAMPLE PKC32
CH3CH2CH)2CHCO-Abu-COOEt. Ihis was prçpared by the preceding general
procedure. Purification b~ flash chromatography on silica geL eluent CHC13:MeOH =
50: 1 v/v, thick, yello~h oiL yield 66%, single spot on TLC, R~ = 0.80, C; 0.66, M.
Mass spectrwn EI-MS M/Z = 285 (M+); CI-MS, [(M+ 1)/Z] = 286.
EXAMPLE PKC33
Ph(CH2)6CO-Abu COOE~ This was prepared by the preceding general
procedure. Purification by flash chromatography on silica geL eluent CHC13:MeOH =
50:1 v/v, yield 64~o, pale yellow oiL single spot on TLC, Rf = 0.29, M. Mass spectrum
EI-MS M/Z - 347 (M~), FB-MS, [(M~1)/Z3 ~ 348-
EXM~LE PKC~4
Z~Leu~CI~Phe~COOEt. Ihis was prepared by the preceding gener 1 procedure.
Purification by ~ash chromatography on silica geL eluent AcOEt~ yield 100%, colorless
oil, s~ngle spot on TLC, Rf - 0.71, C. Mass spectrum FB-MS M/Z = 503(M ' ).
EX~MPLl: PKC35
Z-Leu-Leu-Abu-COOE~. This was prepared by the preceding general procedure.
Purification by flash chromatography on silica geL eluent C~ICl3:MeOH - 50:1 v/v, yield
79.2%, very thiclc, colorless oiL sDngle spot on TLC, R~ z 0.28. M. Mass spectrum
~3-MS, l(M~ 1)/Z] = 520.
SllBS~TU~E S~E~E~'
.
. . . ..
-~ WO 92/11~50 2 ~ 9 PCr/U~;91/09786
.~9
EXAMPLE P~C36
u-Phe-COOEt. Ihis was prepared by the preceding general procedure.
Purificaeion by flash chromatography on silica geL eluent CHC13:MeOH = 50:1 v/v, yield
33%, oiL single spot on TLC, Rf = 056, M. Mass spectrurn, FB-MS, [(M+ 1)/Z] = 582.
S EXAMtPLE PXC:37
2-NapSO2-Leu-Abu-COOEt. This was prepared by the preceding general
procedure. Puri~cation by flash chromato~raphy on silica gel, eluent ~C13:MeOH =50:1 v/v, yield 38%, thick oil, sin~ le spot on TLC, Rf = 0.71, K; 054, A. M~LSS spectrum
- F~3-MS, [(M+1)/Z] = 463.
EXAMPLE PK~,8
2-NapS02-Leu-Leu-Abu-COOEt. lllis was prepared by the preceding general
procedure. Purification by flash chromatography on silica geL eluent AcOEt:AcOH =
200:1 v/v, yield 61%, serni-solid, sMgle spot on rLC, Rf = 0.67, K Mass spec~rumFB-MS, [(M+1)/Z] = 576.
EXAMPLE PKC39
Z-Leu-Met-C02Et. This compound was prepared by the above procedure.
Yellow oil, sin~e spot on TLC, Rf = O.S2 (C~C13:CH30H=50:1), yield 46% (from
dipeptide), MS (FAB) 454 (m+ 1).
E~AMPLE PKC40
Z-Leu-NLeu-C02Et. This compound was prepared by the above procedure. Pale
yellow oil, single spot on TLC, Rf = 057 (CHCl3:CH3C)H = 50:1), yield 53% (~om
dipeptide), MS (FAB) 434 (m+ 1).
EXAMPLE P~41
Syn~hesis of n-Butyl (~ l Chloride. This was prepared by a liserature procedure
[Warren and Malee, J. Chromat. 64, 219-æ2 (197~)]. N Butanol (o.i mol. 7.41 g) was ~
added dropwise to o~alyl cblodde (05 mol. 635 g) at -10 C. After the addition was
completed, the reaction mi~ure was stirred for 20 Trun. at r.t. and distilled, giving 15.0 g
(91.18 moL 91%) of the product n-bu~l o%alyl chloride, bp 58-60 ~C (0.6 mITI Hg).
Z-Leu-Pbe CO2Bu. This compound was prepared f~om ZLeu- Phe-OH and
butyl oxalyl chloride in 43% yield by the procedure described for the synthesis of
ZLeu-Phe-CO2Et, e~cept that butyl o~alyl 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 (CHCl3:CH30H = 50:1) MS(FAB) m/e = 497 (mt 1), lH NMR
(CDC13? ok.
SUBSTITUTE SHEET
,
WO 92/11850 2 ~ 9 8 5 ~ 9 Pcr/u5g1/09786
-70-
E:~PIE Pl~C42
Z-Leu-Abu-CO2Bu. This compound was prepared by the above procedure.
Single spo~ on TLC, R~ = 0.53 (CHCl3:CH3C)E = 50:1), yield = 36%, pale yellow oiL
MS (F~B) m/e = 435 (M~ H NMR (CDa3) ok.
EX~MPLE PK~3
Synthesfs of Ben~yl O~yl atlorid~ Ben~yl alcohol (0.15 mol. 16 g) was added
dropwise to oxalyl chloride (0.75 mol. 95 g) at S-10 C. After the addition was complete,
the reaction was st rred for 20 min. at r.t. Ille es:cess ~alyl chloride was distilled and
re~,rcled. Then the mi~ture was distilled under vacuo, gIving 26 g (0.12 mol. 86%) of
ben~yl o~alyl chloride, bp. 110-112 C (0.6 mm-Hg3. HlNMR (CDCl3) 7.39 (s, SH), 5,33
(s.2H3.
Z-Leu-Phe-CO2BzL This compound was prepared from ZLeu- Phe OH and
benzyl oxalyl chloride in 17~o yield by the procedure described in the synthesis of
Z Leu-Phe-CO2Et, e~cept that benzyl oxalyl chloride was used in place of ethyl oxalyl
lS chloride and sodium benzyloxide in ?oenzyl alcohd was used for enol ester hydrolysis.
Single spot on TLC, Rf = 0.63 (CHCI3:CH3~:)H = 50:1). Pale yellow solid, mp 117-119
C. MS(FAB) m/e = 532 (m~ 1). H1NMR ok
EXAMPLE PKC44
ZLeu-Abu-CO2BzL This compound was prepared by the above procedure.
Single spot on TLC. R~ = 051 (CHC:13:CH30H = SO:1), pale yellow oil, MS(FAB) m/e- 469 (m+ 1), yield = 26%.
EXAMPLE PKC45
~Leu-Phe~COOH. D.~peptide Ketoacids (General Procedure). To a stirred
solution of 053g (1,13 rnmol) ZLeu-Phe-COOEt in o.O ml methanol was added 1.27 ml
~25 (1.27 mrnol) 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 checlced on TLC. After 24 h. no more substrate was
detectet. The reaction mi~ re was chilled in one ice bath at 5 C, acitified with lM
HCl to pH = 3 and e~tracted with AcOEt (2 ~c 50 mL). The organic extract were
washed with 2 x 50 ml H20 and if nec~sary, decolorized with carbon and dried over
MgSO4. After evaporation of ~he solvent (rotavaporator3, the residue (thick oll) were
titurate~ with 2 ~ ml n-he~ane and dried in vacuo. Yield 0.39 g (78%) of colorless,
very thick oil. TLC, main spot at Rf = 024, tra e of impurity at Rf = 0.78, I. Mass
spectrusn, FB-MS [(M+ l)/Z] = 441.
SU8STITUTE SHEET
.
`~- , . - .
WO 92/11~50 2 a 9 ~ 6 0 9 PCI'/US91/09786
-71-
E~MPLE PKC46
2-Leu-Abu-COOH. This compound w~c pr~pared from ZL-Leu- Abll-COOEt in
83% yield by the general procedure as descn~d above; TLC, main spot at ~ = 0.14,trace of impur~ty at Rf = 0.73, I. Mass spe~, F~3-MS [(M~ l)/y = 379-
S - . EXAMPLE PKC47
~L~u-Pb~CONH-Et. To a stirred solution of ZLeu-Phe-OH (20 g, 48.5
rnmole), ~dimethylaminopyridine (0587 6 4.8 mmole),and pyridine ~15.7 ml, 194
rnmole) in anhydrous TElF (100 rsll) was added et}lyl o~alyl chloride tll.4 mL 101.8
mmole3 at a rate sufEicient to ~itiate refllmng. The mi~ture was gentb reflu~ced for 4
hours, cooled to room temperature, and water (80 rnl) was added. The reaction mi~ture
was stirred vigorously for 30 min, and e~racted with ethyl acetate (3 x 100 ml). The
combined or~an~c layers were washed with water (2 ~ 100 ml), sa~urated sodium chloride
(2 ~ lOO ml), decolorized with decolorizing car~on, drie~ over magnesium sulfate, and
concentrated, leaving a dark orange oil. Chromatography on a silica gel rolumn with
CHCl3/CH30H (50:1 v/v) af~orded 14.63 g (y = 53 %) of ~Leu-Phe-enolester. The
product was a yellow oil. S~ngle spot on TLC, R~ = 0.77 (CHCL3/CH30H 50:1). NMR
(CDC13) olc.
To a stirred pale yello~v soludon of the ZLcu-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 rnmole) in ethanol (5 rnl). 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 rnl). The ether layer was washed with water (2 ~ 100 ml), saturated sodium
chloride (2 x lOO ml), dried over magnesium sulfa~e, and concentrated, leaving a orange
oil. Chromatography on a silica gel column with CHC 13/CH30H (SO:l v/v) af~orded7.76 g (y - 64 %) of the a-l~etoester Z-Leu-Phe-COOEt. The product was a yellow oil.
Single spot on TI,C, R~ - 0.44 (CHa3/CH30H 50:1). NMR (CDC13) ok. MS (FAB,
calcd. for C2ÇH32N206: 468.6), m/e = 469 (M+ 1).
The a-carbonyl group of ZLeu~Phe-COOEt was protected by following
procedure. A solution of Z-Leu-Phe-COOEt (1 g, 2.13 rnmole) in 5 ml of CH2Ck wasadded 1,2-ethanedithid (0.214 ml, 2.5~ mmole), followed by 05 ml of boron trifluoride
etherate. The solution was stirred o~ernight at room tempera~ure. 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 ~c 10 ml), dried over magnesium sulfate, and
evaporated to a~ord 0.98 g (y = 84 %) yellow semisolid.
.` :
:. :
v v t~ ~J
wo 92/ll~5~ P~r/ussl/097s6 -- -
The protec:ted a ketoester (0.98 g, 1.8 mmole) was dissolved in ethanol (5 rnl),cooled to 0-5 C in a ice bath, and ethylalsline was bubbled through the solution Illltil
2.43 g (54 mmole) had been added. The reaction mi~ture was allowed to warm to room
temperature slowly, and stirred overnight. The m~ure was filtered, a white precipitate
S was removed, leaving a yellow semisolid. Chromato~aphy on a silica gel column with
C~ICl3/CH30H (30:1 v/v) afEord 0.63 g (y = 75 %) of ZLeu-Phe-C:ONH-Et. The
produc~ was a pale yellow solid. Single spot on TLC, Rf - 0.60 (CHCl3/C~I30H 20~mp 145-147 C. AnaL cal~. for C26H33N30s 46756; ~, 66.79; H, 7.11; N,839; found:C, 6659; H, 7.09; N, 8.95. NMR (CDCl3) ok. MS (~;AB) m/e- - 468-(M+ 1).
EXAMPLE PKC48
Z-Leu-Phe-CONH-~Pr. This compound was synthes~zed ~om the protected a-
ke~oester and propylamine in 92 % yield by the prou~dure desc~ibed in Example PKC47.
Single spot on TLC, Rf = O.S0 (CHCl3/CH30H 50:1); mp 152-153 C. Anal. calcd. for
C27H3sN30S: 48157; C, 6733; H, 733; M, 8.72. Found: C, 67.21; H, 738; N, 8.64. NMR
(CDCl3) ok. MS (FAB) m/e = 482 (M+l).
EXAMPLE PKC49
~Leu Phe-CONH-nBu. This compound was ~ynthesized f~om the protected a-
ketoester and butylan~ine in 67 ~o y~eld by the proeedure descn~d in E~ample PKC47.
Single spot on TLC, R~ = 050.(GHC13/GH30H 50:1); mp 152-153 C. Anal. calcd. forC28H37N30s: 49S59; C, 67.8S; H, 752; N, 8.48. Found: C, 67.70; H, 7~7; N, 8.43. NMR
(GDCl3) ok. MS (P`AB) m/e = 496 (M+ 1).
EX~MPLli~ PKC50
Z-Leu-Phe-CONH-lBlL This compound was synthesi~ed from the protected a
ketoester and Isobutylamine in 53 % yield by the procedure descrl~ed in Example
PKC47. S~ngle spot on TLC, Rf = 054 (CHC13/CH30H S0:1); mp lg2 ~G ~n~L
calcd. for C28H3~N305: 49S59; C, 67.85; H, 7~2; N, 8.48. Found: C, 67.77; H, 7~6; N,
8.40. NM~ (CDCl3) ok. MS (~ m/e = 496 (M+ 1).
EXAMPLE PKCSl
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 PKC47.
After reacting overnight, ethyl acetate (60 ml) was added. The mD~ture 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 sodiurn chloride (2 ~ 20 rnl), and dried over ma~esium
sulfate. The solution was evaporated leaving a yellow solid. Chromatcgraphy on a silica
SUE~STITUTE SHEET
,
~,. WO92/11850 2~ 9 PCr/US91/097~6
gel column with CHC13/CH30H 30:1 v/v) afforded a yellow solid. Single spot on TLC,
Rf = 0.45 (CHCl3/CH30H 30:1); mp 16~162 ~C. AnaL calcd. for C3l~I3sN305: 529.61;C, 7030; H, 6.66; N, 7.93. Found: C, 70.18; H, 6.67; N, 7.99. NMR (CDC13) ok. MS(FAB) m/e = 530 (M+ 1).
S EXAMPLE PKC52
~IRu-Phe-CONH-(CH2)2Ph ~is compound was synthes~ed from the
protected a-ketoester and phenethylamine in S0 % ~neld by the procedure descn~oed ~n
~ample PKC51. Sin~e spot on TLC, Rf = 050 ~GEIC~3/CE30H 30:1); mp 151-153 ~C.
AnaL calcd. for C32H37N305: 543.66; C, 70.70; H, 6.86; N, 7.73. Found: C, 70.54; H,
6.88; N, 7.74. NMR (CDCI3) o~ MS (FAB) m/e = 544 (M+ 1).
I5XAMPLE PKC53
~L~u-Abu-CONH-Et. This compound was sgnth~ized ~am protected a-
ketoester derived ~om Z-Leu-Abu-C02Et and ethylamine in 64 % y~eld by the
procedure descn~ed In E:cample PKC47~ Single spot on TLC, Rf = 0.36
lS (CHCl3/CH30H 50jl); mp 130-132 C. AnaL calcd. for C21H31N305: 405.4~; C, 62.20;
H, 7.71; N, 1036. Found: C, 61.92; X 7.62; N, 1031. NMR (CDC13) ok. MS (FAB)
m/e ~ 406 (M+1).
EXAMPL~: PXCS4
Z-Leu-Abu-CONH-IlPr. This compound ~vas sgnthe3ized ~om lhe corresponding
protected a-ketoester and propyl~e in 47 % yield by the procedure descnbed in
Example PKC47. S~gle spot on TLC, E~f = 0.28 (CEIC13/CH3OH 50:1); mp 134-135 C.
Anal. calcd. for C22H33N30s: 41950; C, 62.98; H, 7.93; N, 10.02. Found: C, 62.84; H,
7.97; N, 9.94. NMR (CDC:13) ok. MS (FAB) m/e ~ 420 (M+ 1).
EXAMPLE PKC55
Z Leu Abu CONH n~lL This co~npound was ~9nthesized ~om the corresponding
prote ted a-ketoester and butylamine in 42 ~o yidd by the procedure desc~bed in
E~ample PKC47. Single spot on TLC, Rf = 054 (GHC~3/~I3OH 50:1); mp 135-136 C.
Anal. calcd. ~or C23H3sN30s: 433~3; C, 63.71; H, 8.13; N, 9.69. Found: C, 63.48; H,
8.07; N, 9.67. NMR (cDa3) ok. MS (FAB) m/e = 434 (M+ 1).
EXAMPI,E PKC~6
Z-Leu.Abu-CONH.iBu. This compound was synthes~7ed :~om the corresponding
protected a-ketoester and isobutylam~ne in 6~ % yield by the pro~edure described in
Example PKC47. Single spot on TLÇ, Rf z 0.:;~ (CHC13/CH30H 50:1); mp 133-135 ~C.
~3uBsTlT~lTE S~EET
.: .
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W0 92/1 1850 2 ~ 9 8 6 ~ 9 PCT/U~9t/09780~ '`
-74-
Anal. calcd. for C23~I3~N30s: 43352; C, 63.72; H, 8.14; N, 9.69. Found: C, 63.46; H,
8.10; N, 9.60. NMR (CDCl3) o~ MS (FA13) m/e = 434 (M+ 1).
E~MPLE PKC57
~Leu-Abu-CONH-BzL This compound was synthesized from the corresponding
protected a-ket~ester and bes~ylamine in 29 % yield by the procedure described in
E~ample PKC51. Single spot on TLC, Rf = 056 (CHCl3/CH30H 30:1); mp 140-141 C.
Anal. calcd. for C26H33N305: 46754; C, 66.79; H, 7.11; N, 8.99. Found C, 66.65; H,
7.07; N, 8.93. NMR (CnCl3) ok. MS (FAB) m/e = 468 (M+ 1).
EXAMPLE PKC58
~Leu-Abu-CONH-(CH2)2Ph. This compound was synthesized from the
corresponding protected ~-ketoester and phenethyla~ne ~n 51 ~o yield by the procedure
descri~ed in Example PXC51. Single spot on TLC, R~ = 0.44 (CHC13/CH30H 30:1); mp156-157 C. AnaL calcd. for ~7H35N30s: 48159; C, 6734; H, 733; N, 8.72. Found: C,
67.38; H, 733; N, 8.78. NMR (C~DC13j ok MS ~A13) m/e = 482 (M+ 1).
EXAMPLE PKC59
Z-Leu-Abu-CONH-(CH2)3-N(CH2CH2)20. This compound was synthesized from
protected a-ke~oester and 4(3-aminopropyl)morpholine in 33 % yield by the procedure
described in E~ample PKC47. After re:acting oven~ight, ethyl acetate (80 ml) was added.
The m~ture wæ filtered so remove a w~ite precipitate. Ihe solusion was washed with
water (3 ~ 20 ml), saturated sodium c~loride (2 x 20 ml), and dried over magnesiurn
sulfate. The solusion was evaporated leaving a yellow oil. Chromatography on a silica
gel column with ~Ha3/CH30H (10:1 v/v) afforded a yellow sen~solid, which was
recrystallized from ethyl acetate/h~ane to obtain a pale yellow solid. Sin~le spot on
TLC, Rf - 0.42 (CHa3/CH30H 10:1); mp 12S-126 C. Anal, calcd. for C~6H40N406:
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 - SOS (M+1).
EX~MPLE PKC60
Z Leu Abu-CO~H-(CH2)7CH3 l~is compound was synthesized from the
correspondjng protected a-ketoester and ocqlamine in 67 % yield by the proceduredescribed in E.sample PKCS1. It was white solid. S ngle spot on TLC, R~ = 0.55
(CHC13/C~I30H 30:1); mp 134-135 C. AnaL calcd. for C2~H43N305: 489.66; C, 66.23;
H, 8.8S; N, 8.58. Found: C, 66.19; H, 8.81; N, 8.61. NMR (CDCl3) ok. MS (FAB) m/e
= 490 (M+ 1)-
SUBSTITUTE SffE~
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WO 92/11~S0 2 ~ 3 8 ~ ~ 3 PCI/IJS91/0978~
E~LE PKC61
Z-Leu-~bll-CONH-(CH2)20H. Ihis wmpound w~ synthesized ~om the
corresponding protected a-ketoes~er and ethaIlolamine in 29 % yield by l:he procedure
descnbed in Example PKC59. The product was a whi~e sticky sclid. Single spot on
S TLC, Rf = 0.42 (CHC13/CH30H 10:1); mp 151-153 C. Anal: calcd. for C21H31N306:
421.49; C, 59.84; H, 7.41; N, 9.97. Found: C, 59.11; H, 7.M; N, 9.81. NMR (CDCl3) ok.
MS (FAB) m/e o 4æ (M+ 1).
EXAMPLE PKC62
Leu-~bu-CONH~ I2)20(C~I2)20~. This compound was synthes~ed i~om the
corresponding protected a-ketoesser and 2-(2-aminoetho~y)e~hanol in 34 % yield by the
procedure descrl~ed in E~ample PKC59. The product was white sticly solid. Single spot
on TLC, Rf = 0.42 (CHC13/CH30H 10:1); mp 103-105 C. Anal.: calcd. for
C23H35N307: 465.35; C~, 5934; H, 758; N, 9.03. Fow~d: S~, ~9.23; H, 758; N, 9.01. NMR
(CDC13) ok. MS (FAB) m/e = 466 (M+1).
EXAMPLE PKC63
L4u Abu-CONH-(CH2)17C~3. ~is compound was synthesized ~om the
corresponding protected a-ketoester and oc~adecylamine in 12 % yield by the procedure
described in E~nple ~KC~ e product was a pale yellow solid. Single spot on TLC,
Rf = 054 (CHCl3/CH30H 30:1); mp 134-136 C. Anal: calcd. for C37~I63N30s: 629.92;
C, 7055; H, 10.08; N, 6.67. Found: C, 70.71; H, 10.14; M, 6.75. NMR (CDa3) ok. MS
(FAB) m/e = 630.2 (M-t 1).
EXAMPLE PKC64
Z L.eu-Abu CONH CH2 C6H3(0CH3)2. Thi~ compound was synthes~zed ~om the
corresponding protected e ketoester and 3,5 dime~ho ~ybenzylarsline in 45 % yield by the
procedure descnbed in l~ample PKC~1. Ihe product w~ yellow sticky soIid. Sin~le
spot on TLC, R~ = 0.44 (CHC~3/CH30H 30:1); mp 153-1~5 C. Anal.: calcd. for
C28H37N307: 527.62; C, 63.74; ~, 7.07; N, 7.96. Found: C, 63.66; H, 7.09; N, 7.92. NMR
~CDC13) ok. MS (FAB) m/e = 528.8 (M+ 1).
EXAMPLE PKC6~
2~Leu-Abu-CONH CH~ C4H4N. ~is compound was s5nthesized from the
corresponding protected a-ketocster and 4-(aminomethyl)pyridine in 45 % yield by the
procedure described in E~ample PKCS9. The product was ~eenish yellow solid. Single
spot on TLC, Rf = 05S (CHC13/GH30H 10:1); mp 124-126 C. A~ calcd. for
. ,.. ,: . , . ~ . .
wo 9~ 850 2 0 9 ~ ~ Q 9 PCI/US9l/0978~
C25H3~N40s: 468.55; C, 64.08; H, 6.88; N, 11.96. Found: C, 63.88; H, 6.87; N, 11.96.
NMR (CDCl3) ok. MS (FAB) m/e =469 (M[+1).
D. HALO-~TONE P~ES
Halomethyl ketone peptides are irreversl~le inhl~itors for serine proteases and
S cysteine proteases. This elass of compounds includes peptides having a variety of
halomethyl groups at their C-tern~nus. These halomethyl groups include CH2X CHX2
and CX3, where X represents any halogen. A number of analogous compounds have
been synthesized, induding the amino-halo ketones and the diazo-ketone peptides.Although these analogous oompoi~nds are chemically distinguishable, all of thesehaloketone compounds are believed to have a si~slilar mechanism OI aetion. Accordingly,
for simplicity, all of the foregoing compounds will be referred to collectively herein as
the "Hal~Ketone Peptides."
The reactivity of haloketones has generally been found to be in the order I ~ Br> Cl > F. However, incre~cing the react~ity of the haloketone in this way can lead to
acceleration of competing side effects. Thus, it is preferable to incre~se the reactivity of
the halomethyl ketone peptides by altering the peptide structure.
In selecting 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 effecti~e C-tern~inus grouping can be
empirically detennined through kinetic inhl~ition studies of each of the compounds with
Calpain.
Many of the Halo-Ketone Peptides are available commercially For e~ample,
Leu-CH2CL Phe-CH2CL Z-lys-CH2CL Tosyl-LysCH2Cl (TLCK), Tosyl-PheCH2Cl
(TPC~), ZGly-Leu-Phe-CH2CL 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-GH2CI are all available from suppliers such as En~ne
Systems Products of I~vennore, California. From the same suppliers, thz following
diazomethyl ketone peptides are available: Leu-CHN2, Z-Phe-Phe-CHN2, Z-Phe-Ala-
C~IN2, ZPhe-Pro-CHN2, ZLys-CHN2 and Giy-Phe-CHN2. In addidon, the production
of a-amino fluoro ketone pepddes 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.
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l~e preparation of various ~lo-Ketone Pep~ides is reviewed in kIethods in
E~, 46:197-208 (1977), the disclosure of whieh is hereby incorporated by
reference, Brie~y, halomethyl ke~one derr~a~ves of blocked ~o acids are read~y
prepared by the reaction of n~neral acid~ (hydrohalic) with the corresponding
5 diazomethyl ketone. Iodomethyl ketones are prepared by re~ctisn of a halo-ketone with
NaI, since reaction with HI with a di~omethyl ketone yields ~he rnethyl ketone. A
3umber of different bl~ng ~oups can be used, including benzylo~ycarbonyl (Z) arld t-
butylo~ycarbonyl (BOC). The ~iazomethyl ketone is prepared by reactis~ of
diazomethane with the appropriate acid ~a~ed by means of dicyclohe1ylcarbodiiTnide
10 (~ -CI), by the mixed anhydride method.
Unbloc3ced asl~ino acid chloromethyl ketones can be prepared by reaction of
benzyloxycarbonyl blocked derrvatives wi~h Br or HOAc, trifluoroacetic acid, or by
hydrogenation.
Synthesis of peptide chloromethyl Icetones can be accomplished simply by
coupling an appropriate peptide or amino acid with an unblocked an~ino acid
chloromethyl ketone. A few dipeptides can be converted directly to the chloromethyl
ketone using the n~Lsed anhydride and CH2N2 foll~wed by HC:L
Vanous synthetic problems are encountered in the preparation of chloromethyl
ketone derrvat;sves of basic amino acids. I~e side chain usually must be blocked during
20 ~ynthesis, and di~iculties are often encountered dunng removal of the blocking group.
Use of trifluoroacetic acid or HF was eYentually found to give a good conversion ~o
product.
A number of exarnples of the preparation of Halo-Ketone Pepddes have been
reported in the literature, including a comprehensive re~e v of over 100 amino acid
25 derivatives and appro~natley 60 peptide derivatrves Listed in J.C. Powers, in aChen~istry
and Biochernistry of Anuno Acids, Peptide~ and Prote~ns,~ Vol. 4, Dekker, New York
(1977), the disclosure of which is hereby incorporated by reference. Those of skill in the
art will recognize how to locate a multitude of e~amplæ of the produstion of the Halo-
Ketone Peptides. Acoordingly, no addidonal G~nples are pro~rided herein.
30 ~. V~USES
In addition to the foregoing classes of compounds now discovered to possess
Calpain inhl~itory activity, we ~elieve that a large number of other such compounds
exist. In vie~v of the large number of inhibitors of Calpain of different classes we
SuBsr~ u~rr
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W092/11850 2~9~a9 PCr/us91/o97~li
disclose herein, all of the h~own, newly discovered and yet undiseovered ~nhibitors of
Calpain shall be referred to hereinafter collectively, using the term "Calpain Lnhibitor.~
The Calpain Lnhibitors may be used vi~o for a variety of purposes to inhibit
unwanted Calpain ac~vity. For e~ample, the Calpain Inhibitors may be used in vitro to
S prevent proteolysis that occurs in the process of production, isolation, purification,
storage or transport of peptides and proteins.
The Calpain Inhibitors descnbed herein ca~ also be used vitro to prevent
further degradation of ~issue sarnple3 ~om oecurring a~er preparation of the samples.
This vitro prevention of degrad~tion can ~e esp cially useful in the preparation of
assays for neurodegenera~ion where~n the ass~y comprises a test for the products of
Calpain activity in the tissues, such as assays for breakdown products (BDP's) of
cytoskeletal eomponents such as spectrin, MAP2, actin binding protein and tau. P.
Seubert et aL, in Neur~science~ 31:195 (1989), the di~closure of which is herebyincorporated by reference, disclose an exemplary method of quantitating the amount of
1~ spectrin BDP's as an indication of Calpain activity.
The ~alpa~n Is~ubitors of this mvention are also useful in a variety of other
experirnental pro~edures where proteolys~s due to Ca4ains is a signifiunt problem. For
example, inclusion of the Calpain Inhibitors in radioin~nunoassay exp~riments can resul~
in higher sensiti~nty. The use of the Calpain In}~ibitors in plasma fractionation
procedures can result m higher yields of valuable plasma proteins and un make
purification of the proteins easier. The C~alpain Inl~ibitors disclosed here can be used ~n
clomng experiments utilizing recombinant or transfected bacterial or eukaryo~ic cell
cultures in order to increase yield of purified recombinant product.
To use the Calpain ~ibitors in vitro. the Calpain Inhibitors are dissolved in an25 ~ organic acid, such as d~methylsulfo~ide (DMSO) or ethanol, and are added to an
aqueous soludon containing the protease which is to be inhl~ited, such that the final
concentration of organic so}vent is 25% or less. The Calpain Inhibitors may also be
added as solids or in suspension.
F. TREAl'MENT OF ~EURODEGENE~ATIOI~
We have discovered that the Calpain Inh~bitors are use~l in vivo to treat
pathologies in which e~cess proteo~sis by Calpains is involved. Such pathologies are
believed to include neuropathologies such as neurodegeneration resulting from
excitotoxicity, H~V-induced neuropathy, ischernia, denenation, injury, subarachnoid
hemorrhage, stroke, multiple infarction dementia, Alzheimer's Disease (AD),
SU135T~UTE SHEEr
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wo 92/11850 2 ~ 9 ~ PCl/U591/09786
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Hun~ington's Dise~se, surgery-related brain damage, Parkinson's Disease, and other
pathological conditions.
1. Identification of Inhibi~ors
In order to identify Calpain Inhl~itors that are useful in the practice of the
present invention for treatment or inhl~ition of neurodegenera~ve conditions anddiseases, it is important to identi~ ~hose inhl~itors posessing sigrlificant Calpain
inhibitory a~vity. It is also import~nt to identi~ those Calpain Inhibitors having a high
degree of specificity for inhib n of C~ain, in order to aYoid interference with other
biological processes when the Calpain Inhl~itor is introdu~d into a mammal requiring
treatment for neurodegeneration. Because all thiol proteases are believed to e~er$ their
effect through a similar mechanism ~ action, our prirnary concern ~as to identify those
Calpai~ itors having substantial inhl~itory act~ity against Calpain, but relatiYely
weak or no ac~vity against other thiol protease~. Accordingly"n order to identify such
Calpain Inhibitors, we tested a variety of Calpain Inhl~itors for their ability to inhibit
calpains I and II, and compared thls data with the ability of the same Calpa~n Lnhibitors
to irlhibit Cathepsin B, another thiol pro~ease. Ihose Calpain Inhibitors with high
vitro inh~itory acsivi~r against Calpain and a relative~y lower activiq against S ~athepsin B
are believed to ~e most useful for ~vno therapy. E~arnples lA through lC show the
results of these studies for a variety of Calpain Inhl~itor~.
EXAMPLE 1~
InhibitiQn b~ Substitused ~terocyclic C-ompounds
The isocownarins are irreve~sible inhl~itors of Calpain. We obtained ICso valuesfor a variety of these Calpa~n Inhibitors as a kinetic analysis of these compounds.
Purified Calpains can be assayed using the fluorogenic substrate succinyl-leucine-
2S ~ros~ne-methylarninocownarin (available commercially) or by measuring the release of
acid-soluble peptides from casein bec~use we have found that the isocoumarins inhibit
casein proteolysis by Calpain.
Calpains I and ~ were purified by the method of (Yoshisnura, et aL 1983).
(Kitahara 1984) provides an altemative puri~cation scheme. Calpain II may alternatively
be purchased from Sigma Chemical Co. as "Calcium Activated Neutral Prote se.~ Ln this
assay, purified Calpain was incubated with l4C-methylated casein in the presence of
various H[eterocyclic Compounds and the amount of acid-soluble radioactivity released by
the action of Calpain was measured. The ICso values were determined as the
~ ~o~!~rlT~ S~
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concentration of Heterocyclic ~mpound compound at which SO~o of the Calpa~n activity
was inhibited. Table ~A shows I~ values for various Isocoumar~n Compounds.
5UB~m~ SH~E~
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~ABLE L~
INH~llION OF CALPAINS BY SUlBSTl~D ISOCOUMARINS
~LCs~ (UM! -
CalpainI Calpa~n tI
ClTPrOIC 100 70 ~`
NH2-CiTPrOIC (ACl~IC) 10 120
~12NHCONH-CiTPrOIC 80 30
CH3CONH.Cl~PrOIC 700 80
L-Phe-NH-Cl~PrOIC - 30
BOC-L~Phe-NH-CilPrOICno inhl~ition ~200
PhCH2NHCONH-Cl~EtOIC 90
PhC~I2CONH-CiTEtOIC . 30
D-phe~NH-CiTEtOIC 200
I?lus, it can be seen from Table ~ that a variety of the Isocoumarin
C:ompounds have significant ~alpain inhibitory activity at low concentrations.
EX~MP~ lB(I)
Prote~se~nhibition by Peptide Keto-Compounds
The Peptide Keto-Compounds are reYersl~le inhibitors of Calpa~ns and other
20 thiol proteases. The }4 values for the inhibition of calpain I, calpain Il and Cathepsin B
were detern~ned for several Peptide Keto-Compounds, Inhibition of calpain I ~om
human erythro~es and calpain II from rabbit musde were assayed using
Suc.Leu.Tyr.amidomethylcoumarin as substrate in an assay buffer of 20mM HEPES
pH=7.2, lOmM CaC12, lOmM ~mercaptoethanol. Cathepsin B from bovine spleen was
assayed us~ng Z-Lys-4-nitrophenylphosphate as substrate.
Table lB(i) shows the results of the studies of E:sample lB(i). The Ki value forthe inhibition of Calpains and cathepsin B by several Peptide Keto-Compounds areshown in ~lM (micromolar). The values for leupeptin, which is cornmercially available
from Calbiochem of La Jolla, Califonua, are shown for comparison.
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Tabl~ lB(i)
Kj YALUES FOR PEPTIDE KETO-COMPOUNDS
Inhibitor Calpain I Calpain II Cathepsin B
Leupeptin 032 0.43 6
~Ala-Ala-Ala-C02Me 200 ~ 1.5
ZAla-Ala-Abu-CO2Et 50 200 0.9
~Leu-Phe-C02Et 0.23 0.4 ~ 50
Z-Leu-Me-CO2Et 0.12 0.18 i8
ZLeu-Abu-C02Et 0.04 0-4 ld,
ZLeu-Nva-COO~t 1.2 30
It can be seen ~om the results in Table lB(i) that the Peptide Keto-Compo mds
inhibit C:alpain with Ki values similar or superior to leupeptin. In particular, Z-Leu-Phe-
C02Et, ZLeu-Nle-C02Et and Z-Leu-Abu-C02Et were found ~o possess ~eater Calpa~n
inhibitory actiYity than leupeptin. In addition, these partiallar compounds were highly
specific to Calpain, with lo ver inl~ibitory act~vity toward Cathepsin B than leupeptin.
EX~LE lB(~)
otease Ir~ibition o~ Peptide Keto-ComFour~ds
We tested the ability of an additional group of Peptide Keto-Compounds to
inhibit several proteases in order to e~aluate their specificity for Calpain. The results of
these studies are shown ln Table lB(ii).
S~STITUTE SHIE~
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Table lB(i~). Inhibition of Calpa~n I, Ca~ain II, Cathepsin B, PP Elastase ~nd Papain
Inhibitor Kl( ~M)
. __ . .
Calpain I Calpai~ II CathB Ch~m elasta papa~n
~e
,..... ... .. _ . ~ ..
Z-L~u-Abu-COOEt 45 0.4 30 ~100 ~100 220
. _ . _ . . . . , _ __ .. ___
Z Leu Abu COOnBu 1~8 4 ~ lû0 25 10
_ . ~
Z-Leu-Abu-COOBz . 95 0.47 4 40 > 100 40~ _ _ ............................................. Y . . _
Z-Leu-Lek .~bu-COOEt 1~8 2~6 ~2 > 100 25
.
2-NapSO2-Leu-Leu-Abu-COOEt 16 1.4 25 35 47
_ _ , , . . . . . .
2-N~CO-Le- - _eu-Abu-COOEt 0.09 > 300 28
I _ . _ . .
T~s-Leu-Leu-A~u-COOEt 33 69 25 28
I_ _ _ . _ .
Ph-(CH3)2-CO-Leu-Abu-COOEt 1.2
. _ .- , ..
Z-Leu-Abu-COOH 0.075 0.022 1.5 > 150 > 150
I _ _ _
Z-Leu-Abu-CON~t 05 0.23 2.4 > 150 65
_ ~ __ __
Z I~u~Abu CON~:Pr 0~5 8 >300 2
I... _ . _ __
Z-Leu-Abu-CONHnBu . 0~ 13 ~ 300 5
I .. _ _ _ . ,__
Z-Leu~Abu~ ONHiBu 0.14 4 >300 40 . -
I . _ _ _ .
Z-Leu-Abu-CON~ 035 2 > 300
1 . . . .,
Z-Leu-Abu-CONH-tC~ 2-Ph 0.022
I - _ _ _ _ _ __
Z-I~-Abu-CONH-(C~I2)3-Mpl 0.041
~ . . _ _
Z-Leu-Abu-CONH-(CH2)~I3 . 0.019
, ._ _ _ ,, , _ . .
Z-Leu-Abu-CONH-(CH2)20H 0.078
_______ _._ _
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WO 92/11850 PCl/US91/09786- -
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, ~ =
¦ Inhibitor ~(I-M)
I . . _ . . . . ..
Calpa;n I Ca~ain II Ca~hB Chym elasta papa~n
~ se
I . _... . _ _ _ . __ _ _ ___
¦ Z-Leu-Abu-CONH- 0.16
¦ (CH2)2t)(~H2)2oH
_ _ __ _ . ~
Z-Leu-Phe-COOEt 1.8 0.4 340 0.025 > 100 75
_~ =.__~ . . ~ . - __ _ _ _.
Z-Leu-Phe-COOnBu 5.0 1.1 15 0.15 > 100 15
Z-L~-Phe-COOBz 3.4 1.6 45 1.6 > 100 4~
: , .,, __ .
Z-Leu-Leu-Phe-COOEt 1.4 _ 42 0.26 > 100 15
Z-Leu-Phe COOH 0.0085 0.0057 45 76 > 150
~ ....................... . _
Z-Leu-Phe-CON~t 7.0 032 6 73 ~150
___ . . . _ __ ,,
Z~Leu Phe-CON~ 15.0 0.05 3 18 ~300
I _ . _ _
Z-L~-Phe-CON~Bu 0.028 3 8 ~100
I _ . .. _ _ . . ... . .. I
. Z-Leu-Phe-CONHiBu 0.065 4 24
_ . _ _
Z Leu-Phe-CONHBz 0.046
I ......... _ _ . _ _
~: Z-Leu-Phe CONH(CH2)2Ph 0.024 (2)
I _ _ . __
Z-Leu-Me-COOEt 0.18 20 2.2 190
I . ....... _ _ ..
Z.T ~--~va-COOEt 1.4-- - -1.2 25 160 23 150
. I . . .
Z-Leu-Met-COOEt 1.0 15 55 1.75> 100 140
I _ , _ . . -
Z-Leu-4-Cl-Phe-COOEt <4.0 0.4 ~0 0.9> 100 150
. !~ ~ _ _. _ _ . _ ., =
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Table lB(ii) shows the inhibition Qnstants (KI) for cathepsin B, calpain L and
calpain II with peptide ketoamides. Dipeptide Ketoamides with Abu and Phe in the P
site and Leu in the P2 site are pot~nt inhibitors of calpain I and calpain Ir. ZLeu-
Abu-CONH-Et is a better inhibitor of calpain I than ZLeu-Phe-CONH-Et by 14 fold.Replacement of the Z group (PhC~I20CO-) by similar groups such as PhCH;2CH2CO-,
PhCH2CH2S02-, PhCH2NHCO-, and PhCH2NHCS- would also result in good inhibitor
structures. The best irlllibitor of calpain II is ZLeu-Abu-CONH-(CH2)2-Ph. Changing
the R3 and R4 groups significantly improv~s the inllibito~y potency toward calpain II.
I~e ~ Dipeptide Ketoamide inhibitors are those which have long allyl side chains(e.g. ~,Leu-Abu-CONH-(CH2)7CH3), al}yl side chains with phenyl substituted on the
alk~ Proup (e.g. Z-Leu-Abu-CONH-(CH~)2-Ph), or a aL~yl groups with a morpholine
rir.& 9s,l~stituted on the alkyl group [e.g. Z-Leu-Abu-CONH~ I2)3-Mpl, Mpl = -
N(CH2CH2)20]. Dipeptide a-ketoamides with a small aliphatic amino acid residue or
a Phe in the Pl site are also good inhi~itors for ca~hepsin B. The best inhibitor is z
Leu-Abu-CONH-Et and replacement of the ~ (PhCH20CO-) by PhCH2CH2CO-,
PhCH2CH2S02-, PhCH2NHCO-, and PhCH2NHCS- would also result in good inhibitor
structures.
EXAMPLE lB(lil)
Stabilitv of Peptide Keto~ompQunds
We determined tbe half-life of several Peptide Keto-Compounds in both plasma
an~ liver homogenates. The results of the determinations of stabiliq of the compounds
in plasma and liver homogenates are sho~n in Table lB(iii).
SUE3STITUTE SI~EET
.: , - . .
.. .
:
~v~ u~
WO 92J11850 pcr/us9l/n9786
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Table 1B(~ii). S~bility 3~ Plasma and in I,i~er o~ Pep~ide Keto-Compolmds.
Inhibitor tl/2
. li~er
~ __ _
S ~Leu-Abu-COOEt 2
. . . ..... _ . . , .. ..... .
2-NaEISO2 Leu Leu-Abu COOEt ~ 60
I - . .
2-NapCO-I,eu-1 eu-Abu~C(:30Et 25
~__ __
Tos-Leu-Leu-Abu-COOEt 30
Z-Leu-Abu-COOH ~ 60 ~60
~ __
Z-Leu-Abu-CONHEt ~ 60 ~60
I _
Z-Leu-Abu CONHnPr ~ 60 ~60 l
~Leu-Abu-CONHnBu ~ 60 ~60 ¦
_ __
Z-Leu-Abu-CONHlBu ~ 60
_
ZLeu-Abu-CON}~z ~ 60 ~li0 l
I .......... . . . . , .
Z-Leu-Phe-COOEt 7.8
I __ I
Z-I~!u.Phe.COOnBu 7.7
. ............ . , . _
u-Phe-COOBz 1.9
ZLeu-Phg-COOH :- 60 ~60
, _
Z-Leu-Phe-CONHRt ~ 60 ~60
. 11
~Leu-Phe-CONHnPr ~ CO ~60 ¦
I _ .Il
ZLeu-Phe-CONHnBu ~ 60 ~60
. . 11
. LeuRhe-CONHlBu 60 _¦
Z-Leu-Ph~-CONH(CH2)~Ph > 60
I . _
Z-L~u-Nle~COOEt 3.7
5l~13Sl~TE SHEET
... . .
: ~ '
- ~ WO 92/11850 2 ~ 0 9 PCr/US91/09786
Illhibitor tll
plasma li~er l
. _ . . I
Z-Leu-N~a-COOEt 2.8
__ ..
Z~Leu-Met-COOE;t 8
_= ..
It can be seen from the data in Table 1B(i-i) that ~he Pep~ide Keto-Compounds
S are generally quite stable in plasma and l~ver homogenates. However, it is also shown
that the Peptide a-ketoamides v ere substantially more stable in both plasma and liver
than the corresponding peptide a-ketoesters
EXAMPIE 1C
otease Inhibition bv Halo-Xetone Peptides
The Halo-Ketone Peptides, ~ike the s~stituted isocoumarins, are irreversible
inhibitors of Calpa n. We detern~ined the X8pp/[I~ ~alues for various members of ~his
class of compounds aga~nst Calpains I and IL ~or comparison, we also determined
these values against the addi~ional thiol proteases Papain and Cathepsin B for at least
one Halo-Ketone Peptide. These Ka~p values are not directly comparable to the Ki or
ICSo values determined abo~e for other classes of inhibitors.
We assayed Calpain I and II using Suc-leu~ amidomethylcoumarin. Papain
was assayed using benz~yl-arg-4-nitroanilide, and Cathepsin B (bovine) ~as assayed
using CBZ-lys 4-nitrophenyl ester. We followed the progress cun~e method of Tian and
Tsou, Biochemi~trY~ 21:1028-1032 (1982), the disclosure of which ~s hereby incorporated
by reference, to derrve l~netic data. Briefly, this method makes use of the equation:
Vls]/K
(1 + [S]/K)A[~
where [PJ represents the concentration of product formed at a tilTle approachinginf;ni~, A is the ~,p in the presence of substrate (S), K is the Michaelis constant and
2~ ~Y3 is the concentra~ion of the inhibitor. Since lS~ and [Yl are known and V and K can
be deterrr~ined, ~,p can be readily deterrnined.
The Kapp/~ for ~arious Halo-Ketone Peptides are shown in Table lC.
c ~ IR.~::TITI ITI~ C!LI''E:T
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WO 92/11850 2 0 9 8 ~ 0 9 PCT/US91/09786 . :^
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TABLE lC
KINETIC P~RAh~RS OF Halo-Keto~ Peptides
Inhibitor CI C:~13 P CB
ZGly-Leu-Phe-CH2C1 2840001946000
Boc-Gly-Leu-Phe C~I2C1 902000 ~40000 290000
ZLeu Phe-~I2Cl ~oo02 585000
Z-Gly-Leu-Ala-CH2Cl 210000
Ac Leu-Phe-CH2C1 259001 33400
lQ Z-Val-Phe-CH2Cl 27200
ZAla-Phe-CH2C1 2400
Ac-Ala-Ala-Ala-Ala-CH2C1 1300
CI = Calpain I l - Rat
CII = Calpain II 2 Human
P - Papain 3 - Rabbi~
CB = Cathepsin B
It can be seen from the results in Table lC that the Hal~Ketone Peptides
inhibit Calpain with relatively high Kapp/[I values. In particular, Z-gly-leu-phe-CH2Cl,
Boc-gly~leu-phe.C~I2C:I, Z-leu-phe-CH2Cl and ~gly-leu-ala-CEI2a were found to
possess significant Calpain inhl~itory ac~vity. In addition, Boc-gly-leu-phe-CH2Cl was
shown to be somewhat specific to Calpain, with lower inhibitory acti~.~ity toward
Cathepsin B or Papaln than toward Calpain. The results shown in the table reveal that
Z gly-leu-phe.CH2Cl and Boc-gly leu-phe-CH2a produce similar inhibitory effec~s. - -
Thus, the blocking group is not shown to have a great effect on Calpain inhibitory
ac~vity.
The kinetic constants of other irreversl~le Cal~ain Inhl~itors include the
following with Kapp/W in parentheses: E 64 (7500), E64-d (23000~ and Zleu-leu-~yr-
CHN2 (230000). E-64 is cornrnercially ava~able from Sigma Chemical Co., and is
shown here to be a poor inhibitor of Calpain. ~leu-leu-tyr-CH~2 is a diazomethylpeptide compound, here shown to possess significant Calpain inhibitory activity. 2. Inhibition oî C~lpai~ ln Neural Tissues
SUBST~TVT S)~EEJ
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In order to evaluate the inhibition of Calpa~n by the various C`alpain Inhibitors
in neural tissues, we assayed the Cal~ain Inhibitors using the known ability of Calpain
to cleave spe~rin, a proeein component of neuronal and other tissue, into Bl)P's. In
this assay, more effective Calpain Inhl~itors will prevent the con~ersion of spectrin into
S BDP's. Example 2 is one e~ample of such an assay.
EXA~LE 2
~bf~
Crude Brais~ l~tracts ~ C~a~pain Inkibitors
~he actIvity of Cal~ain in crude brain e~tracts vas measured by e~amining the
Ca 5-su~;ated proteo~sis of the endogenous substrate spectrin. Brain tissue was
homogenized in 10mM Tris pH=7.4, 032M sucrose, lmM EGTA, lmM dithiothreitol
and the nuclei and debris . - ~ved by low speed centrifugation. Various Calpain
Ir, ~itors were added so the supernatant in a DMSO vehicle and a calcium salt (final
e~ective concentration about 1.2mM) ad~ed ~o start the reaction. Proteolysis of
spectrin was e~aluated by western blot as described by Seubert, et al. (Brain Research,
459:226-232, 1988), the disclosure of which is hereby incorporated by reference.~efly, a known quantity of a spectrin~ontaining sample treated with Calpain is
separated by SDS-PAG~ and immunoblotted with anti-spectrin antibody. The amount
of spectrin immunoreactiviq found corresponding to the characteristic BDP's is
indicative of the amount of spectrin activity present in the sample. An e~amplary
methnd for quantitating BDP's is to assay Spectrin BDP's by homogenizin'g brain parts
in _umM Tris '?~ =72, 32M sucrose, 50,uM Ac-Leu-Leu-nLeu-H on ice. Homogenates
are then mi~ea 1:1 with 10% SDS, S% B-rnercaptoethanoL 10% glyceroL lOmM Tris
pH=8.0, 0,5% bromophenolblue, heated to 95C, and subjected to electrophoresis in
4 1/2% polyac~rlamide gels. ~e proteins in the gels are transferred to nitrocellulose
and the spectrin and BDP's detected using a rabbit polyclonal anti-spectrin antibody
and es~ablished immunodetection methods. The amount of spec~rin and BDP's in each
sample can be quantitated b~ densitrometric scanning of the developed nitrocellulose.
Due to Calpain's requirement for Ca2~, in the absence of Ca2+ little or no
- 30 spectrin proteolysis occurred, regardless of the pr~sence of inhibitor, while in the
presence of Ca2+ the spe~rin ~as ~ 95% cleaved to BDP's within 40 min. if no Calpain
Lnhibitor ~s added.
a~TsTl ITF ~ Ec~
~. .. . . . . " .. -
WO 92/ll8s0 2 ~ 9 ~ ~ ~ 3 PCr/US91/0978;; -
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Both leupep~n and CIl showed inhl~ition in the system of E~ample 2. In
addition, the follow~ng compounds of ~he Substituted Heterocyclic Compounds werefound to produce signi~icant inhibition at 100 IlM:
3-chloroisocoumar~n
3,4-dichloroisocoumann .
3-benzyloxy~-chloroi~oco unarin
7-(ace~qlamino)~-chloro-3-(propoy)-isocoumarin
4-chloro-3-(3-isothiureidopropo~y)isocoumarin
7-amino~-chloro-3-
(3-isothiureidopropo~y)isocouma~n
7-(benzylcarbamoylam~no) 1~hloro-3-
(3-Lsothiureidopropo~y)isocoumarin
7-(phenylcarbamoylan~ino) 1~hloro-3-
(3-isothiureidopropo~y)isocoumarin
7-(acetylamino) 1~hloro-3-
(3-isothiureidopropo~y)isocoumar~n
7-(3-phenyJpropionylar~uno) 1-chlor~3-
(3-isothiureidopropo~y)is~oumarin
7-(phenylacetylamino)~-chloro-3-
ZO (3-isothiureidopropoxy)isocoumarin
7-~-phenylalanylamino)-4 chloro-3-
(3-isothiureidopropo~y)isocoumar~n
7-(benzylcarbamoylarnino)~-chloro-3-
(3-iso~hiureidoetho~y)isocoumarin
7-(phenylcarbamoyl~nino)~ chlor~3-
(3-isothiureidoetho~;y)isocoumarin
7-(D-phenylalanylamino)-4~hloro-3-
- (3-~sothiureidoetho~y)isocoumarin.
The following compounds of the ~Ialo-Ketone Peptides were also found to
produce signifîGant iTl}ùbition at 100 I~M:
ZLeu-Phe-OE12CI
Ac-Leu-Phe-CH2Cl
Z-Gly-Leu-Phe-CH2Cl
Boc-Gly-Leu-Phe-C~I2Cl
SUBST~TUTE S~IFFT
:
. ~ :. WO 92/11850 2 ~ 9 g 6 ~ 9 PClr/US91/09786
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Ac-Val-Phe-CH2CI
~Gly-Leu-~la-CH2C 1.
In addition, the ~ollowin~ compDunds of the Peptide Keto-Compounds were
found to produce ~i~3nifiGmt inhibition at 100 ~M:
5Bz-I)L-Phe-COOEt
~Leu-Nva-COOEt
ZLeu-Nle-COOEt
~Leu-Phe~OC)Et
~-~eu-Abu-COOEt
10_eu-Met-COOEt
Z-Ala-Ala-DI~Abu-COOEt
MeO-Suc-Val-Pro-DL-Phe-COOMe
~AIa-Ala-Ala-DL-Ala-COOEt
MeO-Suc-Ala-Ala-Pro-DL-Abu-COOMe.
15ZLeu-Phe-COOEt
Thus, the Substituted Heterocyclic Compounds, Peptide Keto-Compounds and
Halo-Ketone Peptides, in addition to leupeptin and ~1, provide in~ubition in brain
homogenates.
3. V~Q Inhib}tioD o~ N~urodegener~tion ~hrough Infusion Techniques
In order to demonstrate that the inhibi~ion of Calpain activ~ty alone is sufficient
to inhibit neurodegeneration vivo, we tested the ability of the Calpain In}~ibitor,
. ~upeptin, to inhl~it neurodegeneration in gerbils subjected to transient ischemia.
As stated abo~e, leupeptin is poorh~r mcmbrane permeant. Therefore, leupeptin
is not e~pected to cross the blood-brain barrier ("BBB") very well. Accordingly, in
order to provide the braill with sufficient leupeptin to adequately inl~oit Calpain
activation, w~ used brain illfusion techniques. Through thc use of these techniques we
were able to subject brain tissues to intimate contact with leupeptin for sustained
periods of time. E~ample 3A is provided to show the in vivo protection ~om
neurodegeneration found during one such study.
EXA~LE 3A
~Vivo Protectio~ nst~eurode~eneration
A small cannula was implanted in the right lateral ventricle of adult gerbils, and
secured to the skull with dental cement. An Al~et n~icro-osmotic pwalp was attached to
the cannula for intracerebroventri~ular perfusion. The pump was filled with either
~T~ F ~
WO 92/11850 2 0 9 8 G 0 9 r PCltU591/0978~-
-g2-
saline alone (control) or leupeptin (20 mg/ml in saline). After three days perf~sion
with ei~her the control solution or with the leupeptin solution, transient ischem~a was
induced by b;laterally clamping the c~otid artenes for a period of ten mmutes. Core
temperatures were taken dur~ng and follo~rlg ischen~a, with no di~erences noted
S between control and leupeptin treated animals. Fourteen days l~ter, the animals were
sacriiced by Nembutal overdose and transcardial per~usion of a 105'o solution of
paraformaldehyde in PBS. Coronal sections of the brain were stained ~ith cresyl violet
and were e~amined for the e~tent of neuronal loss. The control gerbils e~bited the
typical damage found in the CA1 field following ischern~a, ~th a 72% loss of neurons.
However, the leupeptin treated ger~ils showed f~r less neurodegeneration, with only a
15% loss of neurons.
The results of E~ample 3A cannot be e~plained by changes in thermoregulation,
since core temperatures did not differ between the groups. Accordingly, we believe
that the Calpain inhibitors~ act~vity of leupeptin is responsl31e for the observed
difEerences in neuronal cell loss. In order to further ~uantitate the differences, and
veri~ that leupeptin produced a Calpain inhibitory effect within the observed re~ons of
the brain, we performed a related series sf ~per~nents. In this series of e~periments,
spectrin BDP's were measured in the leupeptin treated and control animals. As
discussed above, these BDP's are indieative of the amount of CalE)ain activity occurring
within the tissue. E~ample 3B is provided to demonstrate the results of these
experirnents.
EXAMPLE 3B
In Vivo In}likitior~of C~ ain ~ctivitv
Implantation surgeries and clamping of the carotid arteries were performed as
2~ above with a control-ischen~a group (n-4) and a leupeptin-ischernia group (n=5). A
third group of animals (n=4) recesved imp~antation with pumping of s line, but was not
subjected to ischen~ia Animals were sacTificed by decapitation 30 n~inutes afterclamping of the arteries. The brains were rapidl~ removed and placed in oold
homogenization buffer (0.32 M sucrose, 10 mM Tris-HCL 2 mM ED~A, 1 mM EGTA,
100 ~M leupeptin and 1 ~g/ml of the Halo-Ketone Compound, tos-phe-C~I2Cl
(TPCK)). The CA1 region of the hippocampus was then dissected. I~e samples from
both control and leupeptin treated anirnal~ were then prepared for SDS-PAGE and
~mmunoblotting with labeled anti-spectrin antibo~r, as described above in connec~ion
with in vi~ro uses of the Calpain Inhl~ito~s. lhe wn~rol animals exhl~ited a marked
SUBST~ rE~ T
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WO 92/11850 210 9 g ~ 0 3 PCr/lJS91/09786
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increase in the levels of BDP's reLatne to the gerbils not subjected to ischemia. These
BDP's co-m~grated with BDP's observed after ~ proteolysis of spectr~n with
Calpain. The brain t~ssue from the leupeptin treated gerbils e~hl~ited appro~mately
25% of the BDP's observed in the ~ontrol ischenua treated gerbils.
S Another group of gerbils (n=3) were sacrificed ilT~nediately after lschernia
without leupeptin treatment in order to obserYe the effects of ischemia wi~hout
reo~ygenation. These gerbils e~ ited a similar amoun~ of increase of 13DP's as the
control-ischernic gerbils obsened after a 30 minute reperfusion penod.
~;.e -~ results of E~ample 3B indicate that leupeptin ~erts its
neuroprotec~ive effect through the inhl~ition of Cal~ain activation. The results also
indicate that the observed proteo~sis of spectrin was an efEect of ischemia, and not
secondary to the reo~ygenation. Acoordingly, the results indicate that inhl~ition of
Calpain activity in VIVO produces a neuroprotective efEect.
Although the foregoing studies demons~ate ~hat leupeptin can inhibit
neurodegeneration in vivo, leupeptin is noe ~he therapeutic drug of choice because of
.he need to infuse the drug direc~ into the brain for an e stended period of time to
exert its neuroprotective ef~ect. This is due to the relative~ poor abili~ of ~his
compound to cross the BBB. Accordingly, it is beli~ved that a more therapeutically
practical way to inhibit neurodegeneration would be to use more membrane permeant
Inhibitor of Calpain.
4. Platelet Pe~eablllty
In accordance ~nth our discoveries demonstrated in Examples 3 and 3A, we
believe that ha~ing a compound cross the BBB and enter ~::NS tissue is a key
characteristic of a therapeutiul~y u~eful approach to treat or inhibit neurodegeneration
within the CNS. Use of Calpain inhibitors that have enhanced membrane permeability
is one such approach. Thus, wc measured the ability of various Calpain inhl~itors to
penetrate the platelet membrane and inhibit Calpain that is normally contained in
platelets. As shown beloYv in the following e~amples, our results ind;.cate that particlalar
compounds of the Hetero~yclic Compounds, Peptide Keto-Compounds and Halo-
3a Ketone Peptide~" in addition to ~he Peptide Aldehyde, CI1, ead~ibis good membrane
permeability.
As an indication of the membrane penneability of the various Calpain
Inhibitors, we measured the abili~ of various Calpa~n Inhibitors to penetrate platelet
. memb~aDes and inhibit the Calpain nonnally found within platelets. I~e membrane of
8VEISTiTUTE SHI~
.
wo 92tll850 2 0 9 ~ G 0 9 PCI/US91/0978t~
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platelets is believed to have many simila~ities to the BBB and accordin~ly, such~perirnents are believed to provide a good ~ndication of the ability of the various
Calpa~n Inhibitors to cross the BBB. E~ample 4 shows the results of some of these
platelet e~periments using the Cal~ain Inhl~itors of the present invention.
- 5 EX~MPLE 4A
Membrane Penneation of Calpain Inhibitors
Platelets were isolated b~,r a modification of the method of Ferrell and Mar~in, 1
Biol. Chem.. 264:20723-20729 (1989), ~he disclosure of which is hereby incorporated by
refer~nce. Blood (15-20 rnl) was drawn ~om male Sprague-Dawley rats into 100mM
EDTA-citrate contaiII~ng 10 units hepar~ns and centrifuged 30 sn~nutes at 1600 rpm at
room temperature. The plasma was resuspended in 15ml bu~er 1 (136mM NaCL
2.7mM KCL 0.42mM NaH2PO4, 12mM NaHCO3, 2mM MgCI2, 2 mg/ml BSA (Sigma),
5.6mM glucose, 22mM Na3Citrate pH 65) and platelets were isolated at 2200 rpm atFoom temperature of 25 minutes. Platelets were resuspended to 107 cells/ml in buffer
2 (136mM NaCL 2.7rnM KCI, 0.42 NaH2PO4, 12mM NaHCO3, 2mM MgCL 1 mg/rnl
BSA, 5.6mM glucose, 20mM HEPES pH 7.4) and allowed to "rest~ for a rn~r~murn of
10 minutes at room temperature before use.
Platelets were incubated for ~ r unutes in the presence of in}~ibitor. In order to
provide sufficient intracellular calcium to ac~ate Calpain, the calciwn ionophore
A23187 was added to a final concentration of 1~ M. After a further 5 minute
incubation, the platelets were harvested by centrifugation (1 min 10,000xg) and
resuspended in 10% sodium dodecyl sulfa~e, 10mM Tris pH-8.0, 5%
B-mercaptoethanoL 0.02% bromophenol blue, and heated to 95 C for ~ min.
Samples were subjected to SD~PAGE on 6% nuni gels and ~ansferred to
nitrocellulose (Schleicher and Schue~l BA83) for 2 hours at 100rnA/gel in an I KR
Novablot. Filters were blocked for 10 rninutes in 0.25% gelatin, 1% BSA, 0.25% Triton
X100, 0.9% NaCL 10mM Tns-C:l pH 7.5, incubated overnight in the same solution
contairung antibody to rat spe~rin, washed 3 X 10 minutes with 10mM Tris-Cl pH 7.5,
05% TAton X100, incubated 4 hours in wash buffer plus al3c~line phosphatase
conjugated goat anti-rabbit antl~o~y (Biorad), and washed as above. Filters weredeveloped using the Biorad AP conjugate substrate Icit. Spectrin ~nunoreact~ity on
she filters was quantitated ~y densitometry.
The inhbition of Calpain within platelets as measured by the proteolysis of the
endogenous Calpain substrate spectrin in the presence of in}u~itors was assayed for a
C!l 1~2CTITI lT_ ~LI ~T
: : :
:. :
... . .
`, .
~ ` ' . ' ` '
WO 92~11850 PCT/US91/09786
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Yarie~ of Calpa~n Inhl~itors. Ihe poorly permeant inhibitors leupeptin and E-64 had
lit~le effect on intracellular Cal~ain. In contrast, the highly membrane permeant
Heterocyclic Compounds, Peptide Ke~Compounds, and Halo-Ketone Peptides
effec~ively inhl~ited platelet Calpain.
The following Heteros~yclic Compounds were found to produce significant
ition at 100 IlM in the ~stem of ~ample 4:
3~hloroisoeoumarin
4~hlor~3 -(3-~;othiureidopropoy)~umarin
7-an~no~hloro-3-
(3-isothiureidopropo~y)isoooumarin
7-(benzylcarbamoylam~no) 1~hloro-3-
(3-~othiureidopropo~y)~socoumar~n
7-(phenylcarbamoyl~o)~hloro-3-
(3-isothiureidopropo~y)isocoumann
7-(acetylan~ino)-4-chloro-3
(3-isothiureidopropo~sy)~soco~
7-(3-phenylpropionylan~ino)-4-chloro-3-
(3 -isothiureidopropo~y)isocournarin
7-(phenylacetylan~ino)-4-chloro-3-
(3-isothiureidopropo~y)isocoumarin
7-(L-phenylalanyl~nino)-4~hloro-3-
(3-isothiureidopropoxy)isocoumarin
7-(benzylcarbamoylam~no)- 1-chloro-3-
(3-1sothiureidoetho~cy)~socoumar~n
2~ 7-(phenylcarbamoylamino)~chloro-3-
(3-isothiureidoethoxy)i~ocoumar3n
7-(D-phenylalanylamino) 4-chloro-3-
(3-isothilreidoethoxy)isocournar~n.
The fo~lowing Halo-Ketone Pep~ides were fo1md to produce significant
inhl~ition at 100 ~M in the system of E~ample 4:
ZLeu-Phe-CH2CI
Ac-Leu-Phe-CH2C:l
ZGly-Leu-Phe-CH2Cl
Boc-Gly-Leu-Phe-CH2GI.
SUBS~ITU~E SHEEll'
WO 92/11X50 2 ~ 9 ~ 6 ~ 9 PCr/US91/09786.~
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The following Peptide Ket~Co~npounds were f~u~d to produce s~gn~ficant
inhibition at 100 I-M in ~he system of ~ample 4:
~AIa-Ala-D,~Abu-COC)Et
ZAla-Ala-Ala-D,~Ala-COOEt
MeO-Su~-Ala-Ala-Pro-D,L,Abu-COOMe
ZLeu-Phe-COOEt
ZLeu-Nle-COOEt
~Leu-Nva-COOEt
~I~u-Abu-COOEt
ZLeu-Abu
ZLeu 1-CI-Phe-CC)OEt
~IAu-Leu-Abu-COOEs
Z-Leu-Leu-Phe-COOEt
2-NapS02-Leu-Abu~COOEt
2-NapSO2-Leu-Leu-Abu-COOEt
Z-Leu-Met-C02Et
Z-Leu-NLeu-C02Et
Z-Leu-Phe-C02Bu
Z-Leu-Abu-C02Bu
Z-Leu-Phe-C02Bzl
Z-Leu-Abu-CO2B~I
Z-Ala-Ala-D,l,A~u-COOBzl
2; Leu-Phe-COOH
ZLeu-Abu-COO~I.
Among those compounds found to exhibit Calpain in}libito~r act~vity in the
homogenate system of E~ample 2, we found at least shree compounds which failed to
exhibit Calpain in}~ibitory activity in the platdet ~;ystem of E~ample 4. These
compounds are leupeptin, MeO-Suc-~al-Pro-D,L,Phe-COOMe and Bz-D,L-Phe-
COO~t. Leupeptin is hlown to ~e poorly membrane permeant, thus confi~ning that
the platelet assay will e~cclude hlo~n poorl~r membrane permeant compounds.
Aocordingly, ~he two Pep~de Ke~ocompounds found not to provide Calpain inhibitory
activity within platelets are also believed to be poor~ membrane permeant, and would
not be expected to cross the BBB.
EXAMPLE 4B
SllE3S~lTUTE SHEf~T
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~- :
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"~. WO 92/11850 2 ~ 3 ~ ~ 0 9 PCI`/US91/097g6
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C2uantitative Studies o~latelet Membrane Permeabilitv
We performes~ addi~ional gL~ntita~ve or semi-quantita~ive studies on several
Peptide Keto-Compolmds using the assay of E~ample 4A, e~cept that IC50 values were
deterrnined as the concentration at ~vhich 50% of the Cal~ain act~vation present in
S controls occurred. Results are shown in Table 4B. For the semi-quantitative assays,
-~ed with +'s in Table 4B, ~+" indicates detectable inhibition at 100 ~M, "+ +"
inaicates significantly more ir~.ubition than "+~, and "+ + ~" indicates no detectable
activation of Calpain detected.
S11~31STITUTE SHE~ET
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wo 92~1~8~0 2 0 9 ~ p~/us9l/n978~ `
-98-
TABLE 4B
Platelet Assa~ of Peptide Ketoamides9 Ket~sters and Ke~oacids
- ~ .
Inhibitor IC
S l
_ ~ . I
~Leu-Abu-COOEt 42
. ............ . __ I
Z-Leu-Abu-COOnBu
Z-IAu-Abu-COOBz + + l
. _ I
Z-Leu-Leu-~bu COOEt 4û l
~ . I
2-NapSO2-Leu-Leu-Abu-COOEt 100
I . . __
¦ Tos-Leu-Leu-Abu-COO~t 30
~I~.UrCOON 8
Z-Leu-Abu-CON~t 1.5
. . , , ,, . _ .
ZLeu-Abu-CON~Pr 70
.
Z-Leu-Abu-CON~Bu 2.0
Z-Leu-Abu-CONH~u 28
Z-Leu-Abu-CONBz 15
.
Z-Leu-Phe-COOEt 47
. _
Z-Leu-Phe-COOnBu + + +
_ _ _ _
Z-Leu-~he-COOBz + +
: _
Z-Leu-Leu-Phe-COOEt + +
_~ . .
Z-Leu-Phe-COOH 65
Z-Leu-Phe-CONHEt 1.7
I . _
¦ Z-Leu-Phe-CONHrlPr
SU13~iTUTE Sl JEE t
.. . . .
.
I.`Wo92/11850 2a~0~ PCr/US91/0~786
99
__ _ = .
Inhibitor ICso
Z-Leu-Phe-CONHnBu 38
Z-Leu-Phe-CONHiBu 2~!
~ ~r .
Z l t ~-Phe-CONH(CEI2)2Ph 3.0
,, _ __ _ ~ .
ZLeu-Me-COOEt
5ZLeu-Nva-COOEt 40
... .. . , _
~L~u-Met-COOEt +
I .
Z-Leu~ Phe-COOEt +
1, , ~ ,
Table 4B shows that peptide a-ketoamides and ketoauds were much more
ef~ective than corresponding peptide ketoesters in thi~ platelet assay. E~tending the
R3 group to an alkyl group or an allyl group substituted with a phenyl group increased
the membrane permeability of the inh~itors as indicated by increased potency in the
platelet assay. In view of these results, Applicants believe that ectending the 1~ group
to indude longer allyl groups or alkyl groups substituted with phenyl groups would
increase the membrane permeability of a ~ven inhibitor.
In view of the foregoin& the results of E~amples 4A and 4B support our belief
that CI1 and the Substituted Heterocydic Compounds, Peptide Keto Compounds and
Halo~Ketone Peptides are believed to be membrane permeant and therefore, are
expected to be effective in crossing Ihe BBB subsequent to in vivo administration of the
eompounds.
5. Glutamate To~clt~
To further identii y thase Calpain Inhibitors lilcely to possess pharmacologically
active neuroprotective ability, we tested the abiliq of the Calpain ~hll~itors to protec~
against glu~nate e~citoto~naty. Excess e~racellular glutamate is ~hought to play a key
role in the induction of neuropathology in ischemia, which is acoompanied by Wpain
activation. In support of this role for e1~cess glutamate, cultured Nl8~ 105 (a
neuroblastoma.retinal hybrid) cells can be killed by the addition of glutamate into the
culture medium. This glutamate.mediated cytoto~ici~ is calciurn dependent and can be
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reduced ~hrough a number of mechanisms, including free radical scavengers, blockers of
the N-type volta~e-sensitive calcium channel, and quisqu~late-subtype glutamate
antagonists. Thus, glutamate-mediated killing of N18-~lOS eells is an ~B model
for neuropathology.
S According~, we tested She ability of the Calpain Inhibitors to inhibit glutamate-
induced cell death in these cells in order to establish that the Calpain ~hibitors can
decrease or prevent glutama~e-induced d~ath Of N18-~105 cells. Some of these tests
are shown in ~ample 5.
~LE
S~ock cultures of N18-RE 105 cells were maintained in Dulbecco's modified
~agle's medium (DMEM) containing 10% fetal bonne serusn (FBS) and supplemented
with hypo~anthine, ~inopterin and thymidine (HA~. Subcos~fluent cultures were split
and plated into 96-well plates. Iwenty four hours a~er plating the cells were exposed
15 ~o ~esh media containing glutamate and various concentrations of Calpain inhibitors.
Control cells wer~ not treated with glutamate. The treated cell~ recei~red SmM
glu~nate and leupeptin (5,ug/ml) or the other ~pa~n Inhibitors listed in Figure 1 at
3~g/ml. Conversion of ~IT was measured 19 hours later as descn~ed. Nineteen
hours after the onset of e~osure, cell viabiliq was quandtated by measunng the e~ent
20 to which the cells convert 3(4,5-dimethyltJuawl-2 yl)-2-~diphenyltetrazolium bromide
(~) to a blue formazan product, which sowrs in the r utochondria of If ving but not
dead cells (Pauwels et al., 1988). A higher absorbance is indicatlve of greater cell
viability.
Figure 1 sho~. the percent of blue formazan product remail~ng after treatment
25 with glutamate, relative to control where no ~utamate was added. Thus, it can be seen
that with vehicle plus glutama~e but no inhl~itor, less than 70% of the mitochondrial
activity remains. However, Figure 1 shows that u~eral Calpain i~bitors, including
leupeptin, Gll and representa~ves of the Heteroc yclic Compounds, Peptide Keto-
Compounds and Halo-Ketone Peptides protect N18-R~105 cells agaDst glutamate
30 toxicity. Tile Peptide Keto-Compound Calpain inhlbitor, ZAla-Ala-Abu-CO2Et, the
Substituted Heterocyclic Compounds, ClTPrOIC and A~IC, and the Halo-Xetone
Peptide, TPCK complete~ blocked the toxic effects of glutamate, resulting in 100% or
~eater of the form n product as seen with cells not treated with glutamate. Thus,
E~ample ~ shows that these Calpain Inhibitors efEec~ve~r block cell death in an vitro
SU8STlTllTE SHEE~
WO ~2/11850 PCT/US91/09786
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model for neuropatholo~r. Accordingly, this data further supports our discoYery that
Calpain Ir~bitors are neuroprotect~ve ~ n~o.
6. Redu~iosl of I~a~ion o~on MCA O~ ioll
Stroke is a significant health problem in the hurnan population. Strokes are
S occlusions of cerebr~l arteries produc~ng a decreased blood flow to bra~n re~ons, which
cause cell death through oy~en and nutnent dep~vation. I~is type of lesion can be
modeled in rats by sur~cal ooclusion of the middle cerebral artery (MCA). Several
models for MCA occlusion have beerl developed, and all ~ive substantially similar
results.
MCA occlusion produces a large volume of infarcted brain tissue 24 hours after
occlusion. Previous studies ha~e shown that the size of the infarct as judged by ITC
sta~ing does not increase after the first 24 hours post-oeclusion. ~us, we used an
MCA occlusion model in order to test the ability of Calpain inhibitors to prevent
neurodegeneration. lhis model is descn~ed in E~ample 6.
EXAMPLE 6
~IYsion Model for ~eurode~eneration
Male Sprague-Da~vley albino rat~ weighing appro~mately 250-300 grams were
anesthetized with pentobarbital (70 mg/kg, i.p.). The neck region was shaven and a 2
cm incision was rnade. The superficial f~u ia ~vas teased away with tissue forceps and
blunt tip tissue scissors using a spread method. The right comrnon carotid artery was
isolated away ~om ~he vagus nerve and tied off with a single suture (3.0 siL~;). The
external carotid was permanently occluded by sut~ing. Ihe bifurcation of the internal
carotid and pterygopalatine arteries was e~osed and a single microaneulysm c lip was
placed on the ptenygopalatine. Another microaneutysm dip was placed on the common
urotid just pro~mal to the e~ternal/internal bifurution. A suture ~ras placed loosely
around the co~Tunon carotid and a lumen was made in the vessel with the tip of a 25g
needle. A 40 mm nylon suture was prepared l~y melting the tip ~o smooth the pointed
end and marked with a dot ~actb 175 mm from the melted end. The suture was
inserted into the lumen of the artery as far a~ the vessel clip, the clip is removed and
the suture advanced until the marldng was at the bifurcation of the internal andex~ernal carotid arteries. I~ia places the end of the suture in the circle of Willis just
beyond the source of the middle cerebral ar~ery and oecludes this arsery. The loose
suture around ~he caro~id is tied lightly to keep the nylon suture in plau. The
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n~lcroaneurysm clip on the ptPrygopalatine artery was removed, the incision is closed
and the animals are allowed to re~over in heated recovery cages.
Twenty-four hours after oeclusion, the brains of the~e animals were removed
and sliced into 2rnm sections. The sections were stained using 2,3,5-
S triphenyltetrazolium chloride (see Lundy, EF, Solik BS, Frank, RS, Laq, PS, Combs,
DJ, Zelenock, GB, and D'Ale~y, LG, Mo~hometric ev luation of brain infarcts in rats
and gerbils, J. Pharmaco~ ~eth 16,201-214, 1986). Absence of red color development
indicated tissue damage or death. The s~ of the infarcted tissue zone (area with red
stain) and impaired zone (area with partial development of red color) were evaluated
using quantitative morphome~y.
Drugs or vehicle were administered by infusion into the femoral vein. All
animals received the same volume of drug or vehide (20% dimethyl su~fo~ide/80%
propylene ~ycol) via a catheter attached to an Alzet osmo~ic milLipusnp (24 hr pump, 8
ul/hr, 90 ul total volume).
The model of E~a nple 6 was used to determine the size of infarcted area for
control (vehicle, i.Y.) and with adrminissration of each of ~wo Calpain inhibitors: ZLeu-
Phe-CONH-Et (CX269) and Z Leu-Abu-CONH-Et (CX275). These results are
depicsed graphically in Figure 2. It can be ~een that administration of either of the
Calpain inhl~itors Z-Leu Phe CONH-Et (CX269) or Z-Leu-Abu-CO~H ~t(CX~7~)
produees a reduction in the size of the infarcted area.
7. Inhlbltioo o~ Anoxic and H~ lc Da~nage
The CA1 region of hippocampus ~s a brain area par,sicular~ ~ulnerable to
ischemic damage and other insults imolving cxcitatory amino acids. The hippoc mpus
is also a major focus of cell degeneration in Al~he~mer's disease. Neural cells in slices
degenerate following hypoxia throuBI the se chain of events (including reperfusion
effects) observed vo dur~ng and after ischemia. We believe that snldies of
degeneration of neural slices in the pre~ence of the various Calpain ~h~itors is an
effec~ve indicator of the membrane permeance of the Calpain Inhl~itors. Accordingly,
we believe that these studies provide a model for the trea~nent and inhl~ition OI
neurodegeneration vo. Similar studies for de~ennining the e~icacy of compounds
useful in the treatment of neurodegeneration in accordance ~ith the present invention
can be performed using other models, such as protection against degeneration in
platelets or cells in culture.
-
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-103-
It ~s be~ieved that hypo~a is a major eause of neuroto~icity ~n a variety of
neurodegenerat~ve diseases and conditions, such a~ stroke and head injur~1. Thus, we
eonducted further studies u~ing hippocampal slices to show that the vanuus Calpain
itors, advantageou~b, can ~ncre~se sunmal of hippocampal ne~ve cells during
e~posure to hypo~ic or anox~c conditions. An initial screening procedure was first used
to qualitatively detennine whether the various Calpain In}h~itors Wl provide
neuroprotection from ano~a in hippocampal slices A~ ample of these initial
screen~ng procedures is sh~vn by E~ample 7~
E~MPLE 7A
itial Screenfor Inhll?ition of ~o~ic Dama~
Hippocampal slices (400 um) were prepared frorn Sprague Dawley rats ~6 to 7
We~l~) and maintained in an interface chamber at 35C using conventional techniques,
i.t~ ~e lower ~u~ce of the slice received a constant peffusion (05 ml/m~zl) of ACSF,
while the upper surf~ce was e1~posed to a moist atmosphere f 2:CO2 (gS%:5%)
e~changed at a rate of 2 L/min. The AC~;F medium contains (in mM): NaCl ~124),
KCl (3), KHPO4 (25), CaCl2 (3.4), NaHCO3 (26~ and D-Glucose (10). Field ~citatory
post-~naptic responsss were recorded from stratum radiatu n of CAlb in response to
stimulation of Schaffer-cornmissural fi~ers ~n 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 com non ACSF bath. Each slice is tested
in sequence to deterrnine the magnitude of its pre-ano~c evoked response. Five
stimulation pulses (each 0.1 ms (millisecond) in duration) were presented over a 15
second ~ntenal. The largest evokcd response was noted and recorded for each slice.
Following this, the slices were incubated for one hour, with either drog or
vehicle alone added to the ACSF. After the one hour drug incubation period, the
oxygen-enriched atmosphere of the chamber was rnade anoxic by substituting nitrogen
for oxygen (N2 = 95%; Co2 = 5%). The slices were retained in this ano~ic
environment for 10 n~inutes, following which the a~ygen-enriched a~mosphere (o2 =
95%; C2 = 5%) was reestablished.
Ihe slice~ were given the opportunity to recover for 30 rninutes following
reoxygenation whereupon each was stimulated and the ma~nurn evoked po~ential
determined, as descn~ed above durin~the pre-ano~a period. Those slices which, after
!~1 IR.`t:T~ IT~ ~
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WO 92/11850 2 ~ 9 ~ ~ O ~ pCI/lJS91/0~7~6'" "
ano~iaa, produced a ma~imLun evolces~ potential of greater than 50% of that observed
prior to ano~ia were dei;ned as sun~ing slices.
Results of the studies of E~ample 7A are show~ in Figure 3. Figure 3 shows
the effects of CX218 (Z-Leu-Abu-CO2E~t, a Peptide Ket~Compound), and CI1 relative
to control slices on sumYal OI hippocampal slices e~osed to 10 minutes esposure of
ano~c atmosphere. As ~een ~rl this figure, when the control slices are depr~ed of
oxygen for 10 Iminutes ill the absence of drug, virtually all f~il to sur~e, as measured
by their ability to elicit SO~o of their pre-anoxia eYoked response. In accordance with
this finding, few if any recover upon reoxygenation. Figure 3 also shows that when CI
or CX218 are added to the ACSF, the slices are protected ~om the efEects of ano~ia,
evidenced by a substantial proportion of slices eliciting eYoked potentials.
Finally, i~ e seen that CX218 is significantly more effective in protecting
against anox~a and preventing degrada~ion of slices at the minimal 1 hour incubation
time, and at lower concentrations than CI1. This efEect is believed to be due ~o the
superior membrane permeance of the Peptide Keto-Compounds.
Table 7A shows further data from the studies of Example 7A.
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! WO 92/11850 2 ~1 9 8 6 ~ 9 PCl[/VS91/097~6
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TABLE 7A
PEROEMT OF SLIOES SUBVIVINÇ: TEN M[INImS ~NOXld.
Compound Dose (u ~rubation T~ne Sunnval
Control ~ 1 hour <l~o
S Leupeptin lOOO . 3 hours 50%
~Il 200 2 hours 53~0
CX13 (SHC) 20 l hour ~O~o
CX89 (CKP) SO 1 hour ~O~b
CX2l8 ~ C) 1~ l hour 70%
i~, It can be seen from the d~ta in Table 2 that all of the Calpain ~h~ ors tested
provide ~ncreased scl~viYaL CX~3 (AClTlC, a Substituted Hetero~yclic Compound
(S~ . CX89 (Boe -Cly-Leu-Phe-cH2~1~ a Halo-Ketone Peptide (HKP)) and CX~18
((Z1~Abu-C02Et, a Peptide Keto-Compound (PKG)), are each shown to ~e highly
ef~ective in influencing sun~Ival times. Leupeptin ~s seen to be the least ef~ ve
neuroprotective. Thus, we believe that CX13, CX89 and CX218 (ZLeu-Abu-C02Et)
are more effec~ve in influencing sumsal be~use of their membrane permeability.
Accordingly, the results shown in Table 7A support our belief that Calpain Inhibitors
with membrane permeability are ef~ective neuroprotectants.
To ~rther eluu~idate the ability of Calpain Inhibitors to provide neuroproeection
to hippocampal slices, u~d to provide a more quantita~e indica~ion of the membrane
perrneability of these Calpain Inhibitors, we measured the effeet of vanous Calpain
Inhibitors on the c~oked response on a single neuronsl slice before, during and after
anoxia. These srldies are shown in E~ample 7B.
EXAMPLE 7B
llhiki~iQn of AnQ~UC r)a,Illa8e
As ~n Esample 7A, hippocampal sL;ces (400 ym) were prepared from Sprague
Davley rats (6-7 veeks3 and main~ained in ~n interf~ce chamber at 3S~C using
conventional techniques, ie. the lower eur~ce of the slice recerved a constant perfusion
(05 ml/m~n) of an arti~al cerebrospinal fluid (A~SF), while the upper surface was
exposed to a moist atmosphcre of 02:C02 (95%:5%) e~changed at a rate of 2 L/min.The ACSF medium con~ains (in m~: NaCI (124), KC:I (3), K~04 (1.25), MgS04
(2.5), CaC12 (3.4), NaHC03 (26) and D-Glucose (10). Field sxcitatory post-synaptic
responses ~rere recorded from stratum radiatum of CAlb in response to stimulation of
Schaffer co~sural fibers ~ CA1a or CA1c. The depth of the recording ele~rode
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was optimized and evoked responses were collected at a rate of one evoked response
every 30 seconds.
Af~er establishing a stable baseline of evoked responses (appro~mately 10
rninutes~, ACSF contail~ing ~pain In}~l~itor was washed into the chamber and slices
were iT~cubated for a period of one hour. After incubation, evoked responses were
again recorded and the change in the amplitude of the responses from base~ine levels
was noted. No effect of the inhibitors tes~ed on ba~eline evoked responses was
obsened.
For ano7~a ~eriments, incubation in the drug-containing medium was fo~lowed
by replacement of the 02:C02 (95%:5%) atmosphere ~nth N2:C02 (95%:5%). Slices
were e~posed to this anoac environment until disappearance of the pre-synaptic fiber
volley and for two n~inutes (se~ere ano~ia) longer (total time in ano~ic erlviromnent
appro~imately 7-8 minutes in control case). Effects of Calpain ~hibitors on the
functional recovery of the slices after the anwnc episode were then measured.
Recovery of the evoked po~en~ial (~PSP) slope and arDplitude by the drug treatedslices can be compared to con~rol slices to d~tennine the relative e~icacy of various
Calpain Inhibitors.
Figure 4 shows the E~PSP amp~itude in rnill~volts for con~ol, C:[1 treated and
CX218 (a Peptide Ket~Compound) ~eated hippocampal slices in the studies of
E~ample 7B. It can ~e seen in Figure 4 that the control slices deprived of o~ygen in
the absence of drug display a gradual reduction of EPSP and abruptly lose ~ber volley
activity about S-6 minute3 after the begin~ing of ano~ia. Reo~ygenation at or before
th~s point leads to ~omplete functional recovery after about 20 minuteg of
reo~ygenation, but reo~ygenation ~ this point does not. In the latter case the
recovered EPSP slope and amplitude become progresslvely reduced as the duration of
ano~ post fiber valley disappearance (post-FVD) increases. After severe anoxia (2
minutes post-FVD), slices re~over only 15% of the EPSP ~ope.
In contrast to the control slices, recove~y begins to oceur shortly after the end of
anoxia for the treated slices. Figure 4 shows a comparison of the e~ects on EPSPamplitude produced irl the presence of no inhl~itor; the Peptide Keto-Compo~md,
CX2lK, (~Leu-Abu-COæt) and CI1. CX218 produces a recovery ~om severe anoxia
superior to that seen with CI1.
Figure S sh~ the percent recovery of EPSP ~om seYere hypo~a using the
peptide ketoester CX 216 (~Leu-Phe-CO2Et) and its corresponding peptide
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-' WO 92~11850 2 ~ 9 8 G O ~ PCI'/US91/09786
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ketoan~ide CX269 (Z-Leu-Ph~CONH-Es). These studies were performed ill a manner
similar to that of Example 7B, el~cept USiDg a hypo~ic environrnent in place of the
a~o~a of ~ample 7B. It can be seen that use of the peptide ketoamide results in
essentially complete (near 100%) reeovery ~om hypo~a while the peptide ketoesterS produces a par~ial recovery. I~e control ~lices ~peAenced little or no recovery.
An interesting charactedstic that we have discovered for certain Calpain
bitors Is their abili~y to lengthen the period of e1~posure to ano~ia required to
produce ~r volley disappearance (FVD). 15pi~, under control ano~a conditions,
fiber volley disappearance o:urs in less than s~ minutes (~igure 6). The Peptide
10 Keto-Compound, CX-216, su~stantialb lengthens the period of e~posure to ano;~a
required to produce FVD. This is an important advantage of the use of this Peptide
Keto Compound for neuroprote~ion be~use slices can be e~pected to recover
completely if reo~ygenated before fiber voiley disappearanc ~. Thus, treatment wi~h this
Peptide Keto-Compound is e~pected to produce a greater percen~ge of recove~ of
cells from incipient neurodegenerative conditions. It is believed that other
representatives of the Peptide Keto-Compounds as well as of other classes of ~Ipain
itors also provide this effect.
Table 7B shows the perecenhge of recovery of pre-anoxia synaptic transmission
(evoked potential amplitude) of slices treated with various Calpain Lnhibitors or of
20 control slices. All of these slices were acposed to ten minutes of ano~a according to
the protocol of E~ample 7B.
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T~BI~S 7B
Pl~RCE;NT RECOVE;~ OF SYNA~C TRANSMISSION A~R ANOXL9
ÇQm~!lD~ al~ % Recover~
Control -- lS
S CI1 200 35
CX13 (SHC) 20 60
CX89 (a~) 50 30
CX216 (PKC) 100 38
CX218(PKC) 10~ 5~
The results shown in Table 7B provide fu~her eYidence tha~ the peptide
aldehyde, C:I1, as well as the Substituted Heteroeyclic Compounds, Peptide Keto-Compounds and Halo-Ketone Peptides are sufficiently membrane permeant to provideneuroprotection through Calpain inhl~itiolL
CIl, which is at least p~y membrane permeant~ produces some effect,
1~ howeYer, does not significantly lengthen the period of ano~ia required to suppress
electrical act~ . Thus, compared to controL or even compared to leupeptin and CI1,
the Substituted Hetero~yclic Compounds, Pep~ide Keto-Gompounds and Halo-Ketone
Peptides can increase the degree of recovery after ano~ic episodes while producing the
additional advanhge of ~tending the amount of tirne slices carl tolerate ano~a (and
thereby recover completd~
An important efEect of the Peptide Keto-Compounds and other membrane
permeant Calpa~n Inhibitors ~s that they are ~ignificantly more effec~ve in lower doses
than less permeable Calpain In~bitors such as C:I1. Although C~I1 is sho~m to be at
least ~omewhat membrane penneant due to its abili~r to ~ect slice su~iYal, the more
2~ membrane-permeant inkibitors provide significantly increased protection. Thus, the
more highly membrane-permeant Calpa~n Inhibitor~ are believed to be especially
ef~ective in ~eating and inhlUting neurodegeneration.
The results of the studies of Esamples 7A and 7B show tha~ the Substituted
Hetero~yclic Compounds, Peptide Keto-Compounds and Hal~Ke~one Peptides are
membrane-permeant CalpaLn ~h~ ors which are believed to ~e especially effective in
treating and inhl~iting neuro~egeneration. The results also show that Peptide Keto-
Compounds, and perhaps representatives of other classes, can ~end the duration of
anoxia required to suppress electlical activity in hippooampal islices.. As discussed
above, these effects are ~mportant advantages of these compo~mds.
~1 IR~TIT~ ITF .~F~T
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8. ~ NeuroprotectioD by Calpa~ I~ibitors
As discussed above, therapeutics useful for influencing the function of cells
within the CNS must cross the BBB to reach their targets within the CNS. Non-BBBpermeant compounds might, in addition to the brain infusion techniques de~cribedabove~ be administered v~a intraventricular administration, but this also se~erely limits
their usefulness in practice. In order to test the vivo effectiveness of ~he Calpain
klhl~itors to cross the BBB and become therapeuticalh~r useful, we tested the ability of
mtraperitoneal injection of ~he Calp~ Inhl~itors to pro~ec~ a~st ~citoto~ic damage
~YiVQ, Protection w~ measured ~ aluating dlanges in beha~ of rats after
inie~ion ~ith Icainate. These studies are sho~m in Example 8A.
EXAM[PLE 8A
Admiristered Calpain Inhibltors
Ra~s (male Sprague-Da~ley, 200~5 gms) were injected intraperitone311y with
12mg/kg kainic acid in saline vehicle and ether 200~l DMSO (dirnethylsulfo~ide) or
4.6mg calpain inhibitor dissolYed in the same volwne of DMSO. The ra~s were
obserYed for six hours following the injections and the kaina~e-induced behavioral
symptoms and convulsions soored 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; 53convul3ions, including rearing and falling; 63convulsions
followed by death ~nthin the 6 hr~ post injection).
Figure 7 shows the effects of CI1 on the beha~ioral and con~rulsive effects of
l~ainic acid. In the control group, over half the anin~ls showed symptoms greater ~han
mild behavi~ral symptoms, and many e1~hibited overt convulsions, presumably reflecting
seizure acti~ity within the brain. Une~pectedly, in the inhibitor t~ated group, the
incidence and severity of convulsions was reduced. Thu~, this data suggests thatCalpain Inhibitors have an anti-conmlsive effect. Ihis effect is a distinct advantage in
the use of Calpain Inh~itors in epilep~-related neurodegenerative conditions and in
stroke, which is often accompanied by seizures.
Ln order to rnore clearly demonstrate that the behavioral and anti~on~rulsive
effects seen with the Calpain Inhibitors result from inhbition of Calpain we tested the
brain tissues of the rats from E~ample gA for accumulation of spectrin BDP's. Asdiscussed above, these BDP's are associated with Calpain activity and with the
neurodegeneration a~sociated therewith.
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EXA~LE 8B
Protect n ~inst Speçtrin Breakdown ~om E~citoto~ic
Damage bv Peri~?herallv ~dministered Calpain Inhibitors
Four days follo~ing the inje~ion of kain~te in the rats from 3~ample 8A, the
S brains of the rats were removed and assayed for spectrin BDP's. Spectrin BDP's ~ere
assayed by homogenizing brain parts in 20mM Tris pH=7.2, 32M sucrose, 50~M Ac-
Leu-Leu-nLeu-H on ice. Homogenate~ were mixed lol with 10% SDS, ~%
B-mercaptoethanol, 10% glyceroL lOmM Tris pH=8.0, 05% bromophenolblue, heated
to 9~C, and subjeaed to electrophoresLs in 4-1/2% poiyacryl~rnide gels. 'rhe prote~ns
in the gels were transferred to l~itrocellulose and the spectrin and BDP's detected using
a rabbit polyclonal anti-spectrin antlbody arld established Dnmunodetection methods.
The amount of spe~trin and BDP's in each sample was quantitated by densitometricscanning of the developed nitrocellulose.
Figure 8 shows the results of ~ample 8B. It can be seen that kainate
stimulated the breakdo~n of spectrin in both Cal~ain Inhl~itor treated and con~rol rats.
However, treated rats e~u'oited significant~ less BDP's. These results veri~y that
Calpain ac~vity in the brains of the treated rats was reduced. An une~pected
observation was that wen those treated animals that ~hibited severe seizures hadsigruficantb less spectrin brea3cdo~n than untreated animals subjected to kainate. Thus
Calpain Lnhibitor treatment reduced b~oth the behavioral/convulsive effects of kainate
and the activation of calpain in the most severely affected animals.
9. CoDtluslon
All of the foregoing studies support our discovery that Calpain Inhibitors
provide ~2 protection against neurodegeneration asso~ated with ano~ia,
e~citoto~ci~r and other cause-s. Thu5, these Calpain inhibitors possess neuroprotective
activiq against a varie~r of v~vo neurodegenerative di~es and condidons, including
ex~itotoxici~ induced neuropathy, ischemia following denenation or injury,
subarachnoid hemorrhage, stroke, multiple i~rction dementia, Al~heimer's Disease(AD), Huntington's Disease, Parkinson's D~ease, surgery-related brain damage andother pathological conditions.
Those Calpain Inhibitors which possess significant Calpain Inhibitory ac~vi~ in
vitro and also meet at least one of the foregoing or different tests for membrane
permeability are e~cellent candidates ~or treatment of neurodegeneration.
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The ability of ~he various Calpain I~ahibitors to penetrate pl~sma membranes is
a si~icant advantage of these compounds from a pharmaceutical perspective. We
believe that ~his ability, advantageously, allows the ~ain Inhibitors to provideexcellent permeation of the blood-brain barrier. This is in contrast ~o many
phannaceuticals, especially pept;des, which often e~it poor penneatioII of the blood-
bra~n banier. Thus, we believe that the Cal~ain In~bitors w~l e~bit e1~cellent results
as pha~maceutically neuroprot~e agents.
For tr~tment of neurodegeneration, ~he Calpain Inhibitors can b~ adrninistered
orally, topically or parenterally. The term ~parenteral" a3 wed herein includes all non-
oral delivery techniques includ~ng transdermal admini~tration, subcutaneous injection,
intravenous, intrarnuscular or intrasternal irijection, intrathecal injection (directly into
the CNS) or infusion techniques.
The dosage depends primari~y on the specific formulation and on the object of
the therapy or prophyla~s. '~e amount of the individual doses a~, well as the
administration is best de~ermined by inclividually assessing the particular case.
However, in preferrsd compositions, the dosages of Calpain Inhibitors per day are
preferably in the range of 1 ~g/kg ~otal body mass to 100 mg/lcg total body mass, more
preferab~r in the range of 10 I g/kg total body mass to 10 mg/kg total body mass.
The pharmaceutical compositions contain~ng the active ingredient may be in a
form s~utable for oral use, for ex~nple as tablets, troches, lozen~es, aqueous or oily
suspensions, dispersible powders or granules, emulsions, hard or soft capsules or syrups
or eli~drs. The amount of active ingredient that may be combined with carrier
materials to produce a sin~le dosage ~orm will vary depending upon the host treated
and the pardcular mode of adn~inistration. However, typically, a single dose will
2~ contain sufficient Calpain Inhl~itor to provide a complete dars dosage in a single orally
ac eptable form.
For injecdon, the therapeutic amount of the Calpain Inhibitors or their
pha~naceutically acceptable salts will normally be made by subcutaneous injection,
intravenous, intrarnuscular or intrastemal injection, or by intrathecal injection (directly
into th brain). In order to provide a ~ingle dars dose with a single injection, the
pharmaceutical compositions for parenteral administration will contain"n a single
dosage fonn, from about 70 l~g to about 7 g of ~ain ~ibitor per dose of from
about 05 ml to about 1 liter of carrier solution. In addition to the active ingredient,
these pharmaceudcal compositions ~vill usuaLly contain a buffer, e.g. a phosphate buffer
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that keeps the pH in the range from 35 to 7 and also sodiuun ehloride, and c~n also
contain mannitol or sorbitol for adjusting the isotonic pressure. In a preferred form of
these compositions, DMSO or other organie so}vent is added in order to assist the
introduction of the Calpain Inhibitor across membranes.
Additionally, lipids c~n be introduced in~o the pharmaceutical compositions in
order to $acilitate entry of the Calpain inhibi~or compounds into tissue of the CNS.
These compositions are prepared in accorda~ce Yvith methods lalown to those of skill in
the art. Briefly, a lipid sueh a~, phosphatidyl ~oline, ch~lesteroL other well-hlown
lipid carrier or ~tures thereo~ nth the ac~ve compound along with a
solvent, the solvent is d ied off and the material reeonstituted in saline. The
compositions can also include other ingredients ~own to those of ordirlary skill ~ the
art, such as detergents, surfactants or emulsif~ing 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 ~y dissolving the Calpain Inhl~itor in aqueous
buf~er solution of pH 4 to 65 and, if desired, adding a polymeric binder. An oily
fonnulation for topical application is obtained by suspending the Calpain Inh~itor in
an oil, optionally with the addition of a s~elling 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 the ac~ve Calpain Inhl~itor into the blood
stream after application of the composition to the skin of a patient to aUow fortransdermal adn~nistration.
Por treatment of neurodegeneration resulting from e~citotoxici~t HIV-induced
neuropathy, ischen~a following dener~ation or injwy, subarachnoid hemorrhage, stroke,
multiple infarction dementi~, Alzheimer's Disease (A13),~Huntington's Disease, surgery-
related bra~n damage, Parkinson's Disease, and other pathological conditions, the
Calpain Inhibitors or pharmaceudcally acceptable salts thereof may be admin~tered
orally or parenterally. The dosage depends primarily on the specific fonnulation and
on the objec~ of the therapy or prophyla~s. I~e amount of the individual doses as well
as the adn~il~istration is best detemuned by individually assessing the particular case.
In many acute neurodegenerative conditions and events, such as stroke and
head isljury, it is Lmportant to del~ver the C~ain ~hl~itor as soon after injury as is
practicable. Thus, it is preferable to identify those individuals who have suf~ered
stroke, head injury or other inju~y in which neurodegen4ration is associated or is likely
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to occur, and to begin administration of a C~apin ~hibl~or within 1 minute to 2 hours
after the event, in order to preven~ as much neurodegeneration as possible.
A particular application of ~he Calpain Inhl~itors within the scope of ~he
present invention is the application of these compounds dunng surgery to preventS rl~urodegeneration associated therewith. For example, for surgeries perforTned under
general anesthesia, hypo~ic conditions can occur through inadequate perfusion of the
CNS while under anesthesia. Additionally~ many major surgerie~ of the cardiovascular
system require tnat a patient's heart be stopped and that perfusion be maintained
through ~icial means In such surgeries, ~here ~s an increased danger of hypo~ia
10 occurring within the C NS, which can also result in r1eurodegeneration, Moreover,
during neurosurgeries, there is an inheren~ risk of neurodegeneration resulting from
inflammation, bleeding, hemorrhaging and the like. Such neurodegenera~ion can be~nk e~lby infusion with a solution conta~ning Calpain Inhibitor. However,
Il . ...generation resulting from neurosurgery can also be reduced prophylactically by
adr~inistration of a Calpain Inhl~itor through any of the foregoing adrninistration
techniques. Such administration is also believed to inhl~it or prevent
neurodegeneration associated with the u~e of anesthesia or with the use of artifical
means of perfusion during major surgeries. A surgical pa~ient can also have Calpa~n
Inhibitor administered throughout surgery throu~ intravenous drip.
l~e foJlowing e~amples are intended to illustrate certain neuroprotective uses of
the Calpain ~}ubitors within the scope of the present invention. As such, they are not
.oi lo limit the invention in any way.
EXAMPLE 9
~ Neuroprotective Composition fo~Intravenous Inje~tion
500 ~g CH3CONH-ClTPrOIC from E~ample SHC2
4 ml Propylene Glycol
1 rnl DMS--
EXAMPLE lO
A Neuroprotective Composition fQr Intrave~ous Drip
250 mg ZLeu-Phe-CONH-Et ~om E~ample PKC 47
1000 rnl Phosphate BufEered Saline (pH 6.0)
10 ml D MSO
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EX~MPLE 11
A l~europrotectiv~o~ositio~or Transdermal Application
25 mg~Leu-DL,Abu-COOEt ~rom E~ample PKC19
3 mlPhosphate BufEered Saline (pH 6.0)
2 rnlDMSO
EXAMPLE: 12
eu~o~rote~o~L after ~ead ~ury
A first group of p~tient3 who are victims of head trauma is given 2 ml of the
injectable composition of E~ample 9 intravenous~ hin ten minutes of the time of
injury. A second group of similar~ matche~ patients does not recer~re the composition.
Ille first group of patients e~its marked~ fewer and les5 severe outward symptoms
of neurodegeneration, such as dementia, memory loss and parahJsis.
EXAMPLE 13
~europrote~n Durin~ Sureerv
A patient about to undereo a triple bypass heart surge~y is administered 500 rnlof the composition of E~ample 10 per hour using an intravenous drip system. During
surgery, the patient's heart is stopped and perfusion continued through ar~ifiçial means.
Although compLications devdop while re~tarting the heart and disconne~ing the patient
from the arti~al means of perfusion, the patient becomes consuous within severalhours of surgery. Within a few days, the patient's mental sta~us is nonnal with no
indications of neurodegeneration.
It wDl b~ appreuated that certain variations may ~uggest themselves to those
skilled in the art. The foregoing detailed description is to be clearly understood as
8 iven by way of illustration, the spirit and scope of this imention being interpreted
7C throuBh refercnc~ to the appended rJaims.
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