Note: Descriptions are shown in the official language in which they were submitted.
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STATINE-DERIVED TETRAPEPTIDE INHIBTTORS OF BETA-SECRETASE
Field of the Invention
This invention is directed to compounds useful in treatment of Alzheimer's
disease and
more specifically to compounds that are capable of ~i-secretase enzyme
inhibition, thus
interfering with cleavage of amyloid precursor protein to produce beta-
amyloid, a major
component of the amyloid plaques found in the brains of Alzheimer's sufferers.
to Background of the Invention
Alzheimer's disease (AD) is a progressive degenerative disease of the brain
primarily
associated with aging which results in loss of memory and orientation. As the
disease
progresses, motor, sensory and linguistic abilities are also affected until
there is global
1 ~ impairment of multiple cognitive functions of the brain. These cognitive
losses occur
gradually, but typically lead to severe impairment and eventual death in the
range of four
to twelve years.
Alzheimer's disease is characterized by two major pathologic observations in
the brain:
2o neurofibrillary tangles and amyloid (or neuritic) plaques. Neurofibrillary
tangles occur
not only in Alzheimer's disease but also in other dementia-inducing disorders,
while
amyloid plaques are peculiar to AD. Smaller numbers of these lesions in a more
restricted
anatomical distribution are found in the brains of most aged humans who do not
have
clinical AD. Amyloidogenic plaques and vascular amyloid angiopathy also
characterize
25 the brains of individuals with Trisomy 21 (Down's Syndrome) and Hereditary
Cerebral
Hemorrhage with Amyloidosis of the Dutch-Type (HCHWA-D). At present, a
definitive
diagnosis of AD usually requires observing the aforementioned lesions in the
brain tissue
of patients who have died with the disease or, rarely, in small biopsied
samples of brain
tissue taken during an invasive neurosurgical procedure.
Neurofibrillary tangles are characterized as networks of microtubules and
microfilaments
which were once structural supports running symmetrically through the nerve
cells that
transported nutrients but have degenerated into dysfunctional tangled masses.
They can
be described histologically as non-membrane bound bundles containing paired,
helically
3~ wound filaments (PHF) that are approximately 10 nm in length and located in
the
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perinuclear cytoplasm of certain neurons. Major components of paired helical
filaments
are highly phosphorylated tau proteins (PHF-tau) of 60 kDa, 64 kDa and 68 kDa.
A~3
peptide is also a component of these tangles. Tau belongs to the family of
microtubule-
associated proteins and plays a role in the microtubule assembly and
stabilization. In
certain other neurodegenerative disorders, including corticobasal degeneration
(CBD),
progressive supranuclear palsy (PSP) and Pick's disease, hyperphosphorylated
tau
proteins also accumulate in brain tissue in association with abnormal
filaments. Recent
research indicates that the pattern of hyperphosphorylation and the resulting
ultrastructure
of the helical filaments are somewhat different in each type of disease.
Amyloid plaques, on the other hand, are peculiar to and a defining feature of
AD.
Amyloid plaques are predominantly composed of amyloid beta peptide (A~3, also
sometimes designated as (3A4). A/3 is derived by proteolysis of the amvloid
precursor
protein (APP) and is comprised of 39-43 amino acids. Several proteases called
secretases
1 ~ are involved in the processing of APP. It appears that the abnormal
processing and
deposition of A(3 in areas of the brain responsible for cognitive activities
is a major factor
in the development of D. Cleavage of APP at the N-terminus of the A(3 peptide
by (3-
secretase and the C-terminus by one or more y-secretases constitutes the
amyloidogenic
pathway, i.e., the pathway by which A(3 peptide is formed. Cleavage of APP by
a-
2o secretase and the same or a different gamma secretase produces a-sAPP, a
secreted form
of APP that does not result in amyloid plaque formation. This alternate
pathway precludes
the formation of AB. It has been proposed that A(3 peptide accumulates as a
result of the
processing of APP by [3-secretase and that therefore inhibition of the
activity of this
enzyme is desirable for treatment of AD. See for example, ,Q-Amyloid and
Treatment
2~ Opportunities for Alzheimer's Disease, Sabbagh, M., et al., Alz. Dis. Rev.
3, 1-19,
(1997).
Several lines of evidence indicate that progressive cerebral deposition of
particular
amyloidogenic proteins, (3-amyloid proteins, (A~), play a seminal role in the
pathogenesis
3G of AD and can precede cognitive symptoms by years or decades. See, Selkoe,
Neuron
6:487 (1991). A~3 is released from neuronal cells grown in culture and is
present in
cerebrospinal fluid (CSF) of both normal individuals and AD patients. See.
Seubert et al.,
Nature 39:325-327 (1992).
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Although diffuse deposition of A(3 peptide occurs in most all humans with
aging, the
formation of amyloid plaques occur only in AD patients. Formation of these
plaques is
believed to occur over a period of years or even decades. The A(3 peptide in
amyloid
plaques is always folded in a particular three-dimensional pattern called a
beta-pleated
sheet and appears to be chemically modified as well, which could explain the
association
of the A(3 peptides into the larger, denser plaques, rather than the diffuse
deposits
normally seen. Associated with this central core of A(3 peptide in the amyloid
plaque are
surrounding abnormal neurites and several types of altered glial cells. Glial
cells normally
1o associate with neurons and perform support and protective functions. On the
outside of
the plaque are reactive astrocytes, which are a type of glial cell typically
found in injured
brain areas. Additionally many other biochemical components, including
enzymes,
proteoglycans and apolipoproteins are present in the plaques. For a discussion
of the
formation of these plaques see for example: Sabbagh, M., et al., cited supra.
1~
The neurons touching the amyloid plaques are progressively debilitated and
ultimately
die. At present it is not known whether the A(3 peptide is neurotoxic in
itself or if the
secondary features of the amyloid plaques, e.g., the abnormal glial cells,
cause the nerve
cells to die. Researchers have demonstrated that the A(3 peptide has
neurotoxic effects in
2o vitro. Still other researchers have demonstrated that the 25-35 amino acid
sequence of A(3
peptide is similar to that of substance P, an endogenous neuropeptide compound
present
in certain brain tissues and having neuroexcitatory effects. Co-administration
of substance
P in the study blocked the neurotoxic effect of A~3 peptide in rats. See: An
in vivo model
for the neurodegenerative effects of beta amvloid and protection by substance
P. Kowall
2~ NW, et al., Proc Natl Acad Sci USA 88 (16) p7247-51 (1991). Another study
reports that
A~3 peptide is neurotoxic through its interference with Ca++ homeostasis.
Korotzer A.R.,
et al., Differential regulation by beta-amvloid peptides of intracellular free
Ca2+
concentration in cultured rat microglia. Eur. J. Pharmacol., 288 (2):125-30
1995..
Further, some studies have proposed that A(3 peptide is responsible for the
hyperphosphorylation of tau, a microtubule associated protein, which results
in formation
of PHFs and neurofibrillary tangles as described above. Thus, with A~3 peptide
clearly
linked to the formation of amyloid plaques and implicated in the formation of
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neurofibrillary tangles in AD, there is a need for agents and methods for
reduction of A(3
peptide in vivo.
At present there are no published means for specifically inhibiting the (3-
secretase
enzyme, or even structural identification of the (3-secretase enzyme is or a
peptide
sequence of its active site. Hov~~ever, a commonly assigned application naming
John
Anderson, Guriqbal Basi, et al. as inventors and entitled: (3-Secretase Enzyme
Compositions and Methods, identifies the enzyme and methods of use thereof.
Additionally, a commonly assigned application naming Varghese John, Jay Tung.
Roy
to Hom and Larry Fang as inventors and entitled: Dipeptide Inhibitors of ~3-
Secretase,
describes and claims dipeptide inhibitors of the (3-secretase enzyme. The two
above-
identified applications are being filed on the same day as the present
application. The
contents of these co-pending applications are hereby incorporated by reference
in their
entirety for all purposes. Additionally, US 4 636 491 to Bock, et al.
discloses certain
1~ tetrapeptides having renin inhibitory activity. Some of the compounds
disclosed in the
broadest Markush description by Bock encompass some of the compounds of the
present
invention. However, all the specific examples of Bock are directed to the
sequence: Phe-
His-Sta-Leu or to the same sequence containing derivatives of statine . The
sequence is
neither claimed, nor operative in the (3-secretase inhibition disclosed
herein. It is believed
2o that the compounds claimed herein are patentable as a selected subgenus of
the broad
Markush disclosure of Bock.
Detailed Description of the Invention
This invention is directed to the discovery of a class of compounds that
inhibit ~3-amyloid
peptide production by preferentially binding to and inhibiting the proteolytic
function of
the (3-secretase enzyme. Inhibition of (3-secretase enzyme stops or reduces
the production
of A(3 from APP and thus reduces or eliminates the formation of amyloid
plaques and
other types of A(3 deposition in the brain. Therefore, the compounds are
useful in the
o prevention of Alzheimer's Disease in patients susceptible to AD andior in
the treatment
of patients with AD in order to inhibit further deterioration in their
condition. No~~ it has
been discovered that the compounds of the present invention provide inhibitors
of the (3-
secretase enzyme.
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The invention relates to compounds of formula I
O R3 OH O R'
N N N B
\H ~ ~ 'H
O Ra O Rz O )
Formula 1
wherein
A is a straight or branched chain alkanoxy or alkenoxy of 1 to ~ carbon atoms,
aryl,
arylalkyl, the aryl being optionally substituted with 1 to 2 carbon atoms or
halogen,
adamantyloxy, or 4-aminobutanoic acid;
1o B is selected from the group consisting of hydroxy,
C02
\Q Z CO2R Z
-~~N-~--CR'~L-Q-C02R ~ -~~~N~CR'~-C02R
E (i) E (ii)
( C02R)
Z
T
--~ M O-COzR -~N--~CR'~L~ T
(iii) ' E (iv) T
O
and -.~N z O
I
E x
(v) D
wherein a dithered line represents a point of attachment at B of formula 1;
D is H or an oxo group;
L is a 5 or 6 membered saturated, unsaturated or aromatic heterocycle having
from I to 3
1 ~ heteroatoms chosen from nitrogen, oxygen or sulfur. or a saturated,
unsaturated or
aromatic carbocycle of 3 to 6 carbon atoms, any group represented by L having
optional
substitution with R', OR', or halogen;
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Q is a bond, or is a straight chain linking group of 1 to 3 non-hydrogen atoms
chosen
from the group consisting of -CHI-, -O-, and -NH- wherein O and N may not be
adj acent;
ring M is a stable 5 to 7-membered saturated, unsaturated or aromatic
heterocycle having
up to 2 additional N atoms and optionally having 1 to 2 atoms of O and S,
T is independently selected from the group consisting of H, OH, NO~,
C(O)N(R)z, F,
C~-C~ alkoxy, hydroxymethyl and CF3, wherein at least one T is other than H;
x is an integer of 1 to 3,
y is an integer of 1 to 6,
to z is 0 (zero), 1 or 2,
R' is independently H, -OH, C,-C ~ alkyl or phenyl,
R is independently H, C,- C 4 alkyl, or phenyl,
and E is H, or C~-C ~ alkyl;
R, is C,-C; alkyl;
1 ~ RZ is 2-propyl, 2- methylpropyl- or phenyl optionally substituted with R',
OR' or
halogen;
R3 is phenyl, C,-C; alkyl, or 1-(?-methylthio-)ethyl-;
R4 is 2-propyl, 2-butyl or 2-methylpropyl;
and stereoisomers, hydrates or pharmaceutically acceptable salts or esters
thereof to
2o reduce the formation of A[3 peptide.
Still further, it is an object of the present invention to provide
pharmaceutical
compositions containing such (3-secretase inhibitors in a pharmaceutically
acceptable
earner. Other objects of the invention will become apparent from reading the
'. specification and appended claims.
Detailed Description of the Invention
Natural amino acids are available in abundance, and a great array of non-
naturally
o occuring amino acids have been prepared by techniques well known to those
skilled in the
art of organic synthesis. Roberts and Vellaccio provide a comprehensive
listing of non-
natural amino acids, and techniques for the synthesis of many variations
thereof in The
Peptides, Vol. 5: Analysis, Synthesis, Biology; Academic Press, I~~' 1983. A
more recent
description of additional routes to chirally pure non-natural amino acids is
in: Asvmntetric
6
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WO 00/77030 PCT/US00/16643
synthesis of a amino acids from carbolzvdrates as chiral templates; Cintas, P.
Tetrahedron, 47 (32), 6079-111 (1991). Thus one skilled in the art can
synthesize the
amino acid precursors used in the preparation of the compounds of the
invention by a
judicious selection of one or more of the methods outlined above, which
articles are
hereby incorporated by reference.
Statine is a non-standard amino acid residue present in pepstatin that
provides this peptide
its inhibitory activity (Rich, D.H., J. Med. Chenz. 28, p. 262 (1985).
Interestingly,
pepstatin has no inhibitory activity in assays with ~3-secretase. Statine has
the chemical
1o name (3S, 4S)-4-amino-3-hydroxy-6-methylheptanoic acid, and is further
identified in the
Merck Index (11'h ed.) at monograph no. 8759, and is available commercially,
such as
through the Sigma-Aldrich catalog. The three letter abbreviation given to
statine in the
peptide art is Sta. The (3S, 4S) stereoisomer is designated Sta(s) or
statine(s).Statine
derivatives are also well known in the literature and can be prepared by
methods
is disclosed in United States Patent 4,397,786. Other methods are described in
the series
cited above (The Peptides, Vol. 5: Analysis, Synthesis, Biology; Academic
Press, NY
1983] and by Bringmann et al. in Synlett (5), pp. 253-255 (1990); by Kessler
and
Schudok in Synthesis (6) pp. 457-8 (1990); and by Nishi and Morisawa in
Heterocycles
29(9), 1835-42 (1989).
~S
Unless defined otherwise, all scientific and technical terms used herein have
the same
meaning as commonly understood by one of skill in the art to which this
invention
belongs. All patents and publications referred to herein are hereby
incorporated by
reference in their entirety for all purposes.
Optical Isomers-Diastereomers-Geometric Isomers
Some of the compounds described herein contain one of more asymmetric centers
and
may thus give rise to enantiomers, diastereomers, and other stereoisometric
forms which
3o may be defined in terms of absolute stereochemistry as (R)- or (S)- . or as
(D)- or (L)- for
amino acids. The present invention is meant to include all such possible
diastereomers
and enantiomers as well as their racemic and optically pure forms. Optically
active (R)-
and (S), or (D)- and (L)- isomers may be prepared using chiral synthons or
chiral
reagents, or resolved using conventional techniques. When the compounds
described
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WO 00/77030 PCT/US00/16643
herein contain olefinic double bonds or other centers of geometric asymmetry,
and unless
specified otherwise, it is intended that the compounds include both E and Z
geometric
isomers. Likewise, all tautomeric forms are intended to be included.
Stereoisomers refer to molecules wherein the same atoms attach to one other in
the same
order, but the positioning of the attachment varies so that two molecules may
not be
spatially identical; they are classified according to the number and symmetry
of the chiral
centers in each molecule. Chiral centers are atoms to which the same kinds of
atoms are
attached but have more than one possibility for arrangement around the chiral
center
1n~ atom. There are two types of stereoisomers: diastereomers and enantiomers.
Diastereomers are two molecules which are stereoisomers that have the same
connectivity
but are not mirror images of each other. Chiral centers in diastereomers are
arranged so
that an internal plane of symmetry exists in the molecule. The chemical and
physical
1 ~ properties of diastereomers tend to differ because different spatial shape
changes the ways
in which the molecules interact.
Enantiomers are are two molecules which are exact mirror images of one other,
because
each chiral center is a reflection of the chiral center of the other
enantiomer. Enantiomers
2o have identical chemical and physical properties, which make separations
based on their
physical properties extremely difficult. Enantiomers are usually labeled R and
S (for
right-handed and left-handed) to distinguish them.
Racemic mixtures are defined as mixtures of two mirror image forms of the same
molecule (enantiomers) in equal amounts.
Although stereoisomers may not vary greatly on a chemical level, on the
biological level,
different stereoisomers isomers "fit" differently into the various protein
receptors that
drive biochemical processes and thus stereoisomers, and frequently even
enantiomers, do
?o not bind equally. Therefore, enantiomers of the same compound can have
different effects
within the human body.
Many of the embodiments of the present invention embrace the residue -or side
chain- of
a naturally occurring a-amino acid, it is to be noted that each a-amino acid
has a
_ characteristic "R-group". the R-group being the residue -or side chain-
attached to the a-
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carbon atom of the amino acid. For example, the residue of glycine is H, for
alanine it is
methyl, for valine it is 2-propyl, for methionine it is methylthioethyl. The
specific
residues of the naturally occurring a-amino acids are well known to those of
skill in the
art. See, e.g, A. L. Lehninger, Biochemistry: The Molecular Basis of Cell
Structure and
Function, 1975 (or any edition thereafter), Worth Publishers, NY, see,
particularly
Chapter 4). As used herein, the residues of naturally occurring a-amino acids
are the
residues of those about twenty a-amino acids found in nature which are
incorporated into
a protein by the specific recognition of the tRNA molecule with its cognate
mRNA codon
in humans.
to
Those non-naturally occurnng a-amino acid residues embraced by the present
invention
are known to those of skill in the art. Statine, as discussed above in the
background of the
art is a commercially available y-amino acid. In the compounds herein the
preferred
stereoisomer is the S configuration and the absolute stereochemistry is (3S,
4S).
Other modified or non-usual amino acids are 2-aminobutyric acid (Abu) and
phenylglycine (Phg), In amino acids of this type a change is made in the side
chain of the
amino acid, usually by varying the length or substitution thereon. For
instance, 2-
aminobutyric acid is an a-amino acid that varies from valine by the removal of
one of the
2o methyl groups from the side chain. Phenylglycine is a homolog of the
naturally-occurnng
amino acid phenylalanine, which lacks the methylene linkage between the
peptide
backbone and the phenyl group found in phenylalanine. Norleucine (Nle) is a
slightly
different example where the branching methyl group of leucine is shifted
rather than
deleted to make a non-branching (normal) chain having the same number of
carbon atoms
?~ as leucine. These unusual amino acids can be incorporated into peptide
chains using the
standard peptide linkage synthetic procedures described below for the
naturally-occurring
amino acids.
As used herein, the term "alkyl" includes the straight, branched-chain and
hydrocarbons,
0 the number of carbons atoms being generally specified. Where not specified
the alkyl
groups preferably contain from about I up to about 12, more preferably 1 to 6,
and most
preferably 1 to 5 carbons. Exemplary of such moieties are methyl, ethyl, n-
propyl,
isopropyl, n-butyl, t-butyl, sec-butyl, pentyl, n-hexyl, n-nonyl, n-decyl, and
the like. The
9
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WO 00/77030 PCT/US00/16643
term "lower alkyl " includes C, -CS alkyl groups such as methyl, ethyl, n-
propyl,
isopropyl, n-butyl, s-butyl, t-butyl, n-pentyl, and the like.
As used herein, "heteroatom(s)" is/are selected from O, N or S, unless
otherwise
specified.
Alkenes are alkyl groups containing at least one C-C double bond (-C=C-).
Exemplary of
alkenyl moieties are 2-methyl-2-propenyl, 2-methyl-1-propenyl, propenyl, I-
butenyl, 2-
butenyl, 3-butenyl, 2,2-difluoroethenyl, as well as those straight and
branched chained
to moieties having more than one double bond.
The term "lower alkyl " includes C~ -C; alkyl groups such as methyl, ethyl, n-
propyl,
isopropyl, n-butyl, s-butyl, t-butyl, n-pentyl, and the like.
1 ~ The term "halo" and "halogen" refer to chloro, bromo, fluoro, and iodo.
"Lower alkenyl" refers to those C~ -C~ unsaturated groups such as vinyl, 1-
propene-2-yl,
I-butene-4-yl, I-pentene-5-yl, 2-methyl-2-butene-4-yl and the like.
2o The term "alkanoxy" refers to those groups having an alkyl moiety from 1 to
6 carbon
atoms linked to an oxygen atom. This oxygen is linked to the carbon atom of
another
group. Examples of alkanoxy groups are: methoxy, ethoxy, propoxy, butoxy, iso-
butoxy,
and the like.
25 The term "alkenoxy" includes C~ -C6 groups having a C-C double bond and an
oxygen
atom, such as ethenyloxy, propenyloxy, iso-butoxyethenyl and the like.
The term "amine" includes pirmary, secondary and tertiary amine which may be
in
straight or branched chains or, in the case of secondary and tertiary amines
within rings,
3o and are optionally substituted with, C1 -C~ acyloxy, C~ -C6 alkyl, C~ -C~
alkoxy, nitro,
carboxy, carbamoyl, carbanoyloxy, cyano halogen, amino and the like.
The terms "carboxyl", "carboxylate" and "carbamoyl" are all terms referring to
functional
groups containing the a carbon atom double bonded to an oxygen as well as
single
3, bonded to another oygen, in the case of carbamoyl the carbon is addionally
bonded to a
nitrogen atom. These terms all include the corresponding pharmaceutically
acceptable C;
-C~ alkyl and aryl esters.
to
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WO 00/77030 PCT/US00/16643
The term "aryl" includes 3 to 8 membered stable saturated or unsaturated
organic
monocyclic rings having 0 to 4 hetero atoms selected from S, O, and N; and 7
to 10
membered organic stable, saturated or unsaturated, bicyclic rings having 0 to
5 hetero
atoms selected from S, O, N; both of which may be substituted by halo, C, -C~
alkyl, C~ -
C6 alkoxy, C~ -C6 alkenyl, C~ -C~ alkynyl, substituted C, -C~ alkyl, C~ -C~
substituted
alkoxy, substituted CZ -C6 alkenyl, or substituted C~ -C6 alkynyl, hydroxy,
amino, nitro,
cyano, carboxy, hydroxymethyl, aminomethyl, carboxymethyl, C~ -C4 alkylthio,
C~ -Ca
alkanoyloxy, carbamoyl, or halo-substituted C~ -C~, alkyl. The term "aryl"
also includes
fused ring carbocyclic and heterocyclic moieties having at least one aromatic
nucleus.
The term "arylalkyl" refers to any of the above aryl groups also having an
alkyl radical
though which the group connects to the larger structure. Preferred aryl and
aralkyl
moieties are phenyl, benzyl, phenethyl, 1- and 2-naphthyl, naphthylmethyl, 5-,
6-, 7-, and
8- quinolinyl, benzofuryl, indenyl, or indanyl, benzimidazolyl, indolyl,
benzothiophenyl,
1 ~ indole-3-ethyl and 5-, 6-, 7-, and 8-tetrahydroisoquinoline. Other
examples of such ring
systems may be found in J. Fletcher, O. Dermer, R. Fox, Nomenclature of
Organic
Compounds, pp. 20-63 ( 1974), and in the Examples herein.
The terms substituted alkyl, substituted alkenyl, substituted alkynyl and
substituted
2o alkoxy are these radicals substituted with halogen, hydroxy, amino, C~ -C~
acyloxy, nitro,
carboxy, carbamoyl, carbamoyloxy, cyano, or C~ -C6 alkoxy, and may be
substituted one
or more times with the same or a different group, preferably substituted 1 to
3 times.
A linking group is defined as a divalent linear chain facilitating the reach
of a binding
2s group, for example a carboxyl group to binding sites within the target
enzyme. The
facilitation occurs as a result of either or both the extension of the group
at a particularly
advantageous distance from another binding site on the molecule or in
providing an
advantageous flexibility to the group in being able to adopt a conformation
that allows for
optimal binding. Particular examples of the group Q which may be present in
compounds
30 of the invention include groups of formula: -CHz-(CHZ)n-; -O-(CH~)n-; NH-
(CH~)n-; -O-
CHI-O-; -O-CHI-NH-; NH-CHZ-NH- wherein n=0, 1 or 2.
A pharmaceutically acceptable salt is any salt which substantially retains the
activity of
the parent compound and does not impart any deleterious or undesirable effect
on the
1i
WO 00/77030 CA 02376420 2001-12-14 pCT~S00/16643
subject to whom it is administered and in the context in which it is
administered. Such
salts are those formed with pharmaceutically acceptable canons, e.g., alkali
metals, alkali
earth metals, etc. A pharmaceutically acceptable ester is any ester formed on
a parent
carboxylic acid which substiantially retains the activity of the parent
compound and does
not impart any deleterious or undesirable effect on the subject to whom it is
administered
and in the context in which it is administered. A pharmaceutically acceptable
excipient or
diluent is any excipient which retains the activity of the therapeutic
compound with which
it is admixed and does not impart any deleterious or undesirable effect on the
subject to
whom it is administered and in the context in which it is administered.
Abbreviations used in this specification represent the following:
Ac= acetyl (methylcarbonyl)
bd = broad doublet
BOC=tert-butoxycarbonyl
bs = broad singlet
CBZ=benzyloxycarbonyl
d = doublet
DMF = dimthylformaide
EDC = ethyl-1-(3-dimethylaminopropyl)carbodiimide
2o EtOAc = ethyl acetate
eq. = equivalents
HOBT = 1-hydroxybenzotriazole
ICso = inhibitory concentration of a compound where the enzyme activity is
reduced by
half.
m = multiplet
mass. spec. or MS= mass spectrum
nmr = nuclear magnetic resonance spectrum
Rr = ratio of movement of a substance on a thin layer chromatogram in
comparison to the
movement of the solvent front.
3o s = singlet
t = triplet
TEA = triethylacetate
TFA = trifluoroacetic acid
THF = tetrahvdrofuran
3 ~ TLC = thin layer chromatography
8 = units of measurement for nuclear magnetic resonance spectroscopy which are
relative
to a standard, e.g. trimethyl silane.
~L = microliter
pM = micromolar (an expression of concentration in micromoles! liter)
The terms N-terminal or N-terminus refer to that terminal or end group of a
peptide
bearing the free or derivatized amino group of an amino acid residue.
Likewise, C-
terminal or C-terminus refers to that terminal or end Group of a peptide
bearing the free or
12
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derivatized carboxy group of an amino acid residue. The term "capping group"
refers to a
non-amino acid moiety bonded to the C- or N-terminal of the peptide chain.
Examples of
N-terminal capping groups used in peptide synthesis are BOC (t-
butoxycarbonyl,) and
CBZ (benzyloxycarbonyl) and glutamic acid. Other capping groups are acetyl and
adamantyloxycarbonyl. Non-limiting examples of are shown in Table 1.
Methods of Synthesis
Reaction scheme 1 illustrates the construction of some of the peptides
provided herein
and the variety of reactions that may be used to prepare intermediates from
which
to compounds for formula 1 may be prepared, and provides a generic method for
the
synthesis of the tetrapeptides. Although statine and valine are employed as
the N-terminal
amino acids. It is to be understood that other peptides may be also be
substituted in place
of valine and that derivatives of statine may be employed as alternatives in
the steps
below to provide further tetrapeptides as provided herein.
Scheme 1
NHS-val-OBzI . HCl + Boc-statine(s) A ~ Boc-Sta(s)-val-OBzI
B
T
1) Boc-AA1-OH
Bo~-AAl-sta(s)-vat-oB~t C NH3-Sta(s)-vat-oB~t
B
t) R-AA2-OH
D
NH;-AA 1-Sta(sl-V al-OBzI R-A pr, -AAl-Sta(s)-v al-OBzt
E
F
R-Aft,-AAl-Sta(s)-val-NH-R ~--- R-AA2-AAl-Sta(s)-vat-OH
H~r~-R
KOH
R-AA2-AA1-Sta(s)-vat-NH-R-CO 2CH~ ~ R-AAa-AA,-Sta(s)-val-NH-R-CO ~H
G
13
W~ 00/77030 CA 02376420 2001-12-14 pCT/US00/16643
This scheme may be varied as desired by selecting a different carboxy
protected amino
acid residue, for example, a.-amino-butyric acid (Abu) or phenylglycine (Phg)
in Step A
to couple with statine. Or alternatively, selecting a different statine
derivative, such as
Phe-Sta, wherein the 2-methyl-propyl group is replaced by benzyl, to be
coupled with the
starting amino acid in Step A. Derivatives of statine are known in the art and
described in
United States Patent 4,397,786.
Experimental Methods
1U
Step A: Coupling of the C-terminal amino acid with statine.
Boc-Sta(s) (1.0 equiv.) was dissolved in 30 mL of dry dichloromethane, then
HOBT (2.0
equiv.), H~N-Val-OBzI .HCI (1.0 equiv.) and TEA (~ equiv.) were added and the
mixture
was stirred for 20 minutes. EDC (1.2 equiv.) was then added and allowed to
stir
1 ~ overnight under an atmosphere of nitrogen. The reaction was diluted with
water and
extracted with EtOAc (3x). The organic layers were washed with aqueous citric
acid
(2x), sat. NaHC03 (2x), brine, then dried with MgSO.~.
Step B: Removal of the Boc-protecting group from the resulting dipeptide.
2o The Boc-protecting group of the dipeptide was dissolved in a
trifluoroacetic
acid/methylene chloride (1/1) solution. The reaction was monitored by TLC to
confirm
the consumption of starting material at which time the solvents were removed
under
vacuum to yield the free amine which was used without further purification.
Step C: Coupling deprotected amine with a selected amino acid residue (AAA).
Boc-AAA-OH (1.0 equiv.) is dissolved in 30 mL of dry dichloromethane, then
HOBT
(2.0 equiv.), HzN-Stat-Val-OBzI .HC1 (1.0 equiv.) and TEA (5 equiv.) were
added and all
was stirred for 20 minutes. EDC (1.2 equiv.) is added and the mixture is
stirred overnight
under an atmosphere of nitrogen. The reaction is then diluted with water and
extracted
o with EtOAc (3x). The organic layers are washed with aqueous citric acid
(2x), sat.
NaHC03 (2x), brine, then dried over MgSO:~.
Removal of the Boc-protecting group from the resulting tripeptide is
accomplished as in
Step B above.
;;
Step D: Coupling of the tripeptide with a selected amino acid residue (AAA).
1-~
CA 02376420 2001-12-14
WO 00/77030 PCT/US00/16643
R-AAA-OH (1.0 equiv.) is dissolved in 30 mL of dry dichloromethane, then HOBT
(2.0
equiv.), HEN-AAA-Stat-Val-OBzI.HCI (1.0 equiv.) and TEA (~ equiv.) are added
and the
reaction mixture is stirred for 20 minutes. EDC (1.2 equiv.) is then added to
the mixture
and is allowed to stir overnight under an atmosphere of nitrogen. The reaction
is diluted
with water and extracted with EtOAc (3x). The organic layers are washed with
aqueous
citric acid (2x), sat. NaHC03 (2x), brine, then dried over MgSO,~.
Step E: Removal of the Carboxybenzyl (Cbz) protecting group from the C-
terminus
to R-AAA-AAA-Stat-Val-OBz ( 1.2 g) is dissolved in 100 ml of MeOH and Pd/C
(1g, 10%)
is added. The reaction is subjected to a hydrogen gas atmosphere of SOpsi for
2 hours.
The resulting slurry is filtered through a pad of celite, and rotoevaporated
to yield the
desired material.
1 ~ Step F: Coupling of the C-terminal end of the tetrapeptide with a
functionalized
amine
R-AAA-AA,-Stat-Val-OH (1.0 equiv.) is dissolved in 30 mL of dry
dichloromethane,
then HOBT (2.0 equiv.), HZN-R (1.0 equiv.) and TEA (5 equiv.) were added and
the
reaction mixture is stirred for 20 minutes. EDC ( 1.2 equiv.) is added and
allowed to stir
20 overnight under an atmosphere of nitrogen. The reaction is diluted with
water and
extracted with EtOAc (3x). The organic layers are washed with aqueous citric
acid (2x),
sat. NaHC03 (2x), brine, then dried over MgSOa.
The amine groups (R-NH2) of the examples shown below are commercially
available
unless otherwise indicated by reference to a citation in a journal. Some of
the compounds
are supplied as the methyl ester, if a carboxylic acid function is present. If
only the free
carboxylic acid group is available commercially, the methyl ester can be
prepared as
indicated in step H below.
.o Step G: Cleavage of the C-terminal capping group methyl ester to provide
free
carboxylic acid or carboxvlate salt
The methyl ester ( 1 equiv.)Yis dissolved in a suitable solvent (MeOH/water,
dioxane~water, or THF/water). Hydroxide (2-20 equiv., KOH, NaOH, or LiOH) is
added
and the reaction mixture is allowed to stir until the all of the ester is
converted to acid as
evidenced by TLC. Volatile solvents are removed and the reaction is acidified
with citric
CA 02376420 2001-12-14
WO 00/77030 PCT/US00/16643
acid. The resulting precipitate is collected and characterized to insure the
desired material
is obtained.
Step H: Preparation of the methyl ester for the C-terminal capping reaction
The selected acid is dissolved in dry methanol. HCL (gas) is bubbled through
the mixture
for ~ minutes. The reaction is then stirred overnight and rotoevaporated to
yield the
desired methyl ester.
Step J: Aminoarvl diesters to Aminocyclohexyl diesters
to
An example of this ring hydrogenation is with dimethyl -aminophthalate. The
amino-
phthalate in acetic acid (12%v/v) was added 1.2~ g of 5% rhodium on alumina
(50%
wiw), the mixed slurry was saturated with hydrogen at 55 PSI and shaken for a
total of 72
hrs. Upon completion of the hydrogenation the reaction was filtered through
Celite and
1 ~ dried over anhydrous sodium sulfate. Filtration and subsequent
rotoevaporation yielded
crude product, which was then subjected to silica gel chromatographic
purification to
provide dimethyl aminocyclohexyldicarboxylate as a pale white solid. See also:
Fieser &
Fieser, Reagents for Org. Syn. 4, 418 and Freifelder, M.; Ng, Y. H.; Helgren,
P. F.; J.
Org. Chem., 30, 2485-6.
Step K: Formation of azide
The Halide (5.5 mmole) was dissolved in dry DMF and sodium azide ( 6.88 mmole)
was
added. The reaction was stirred overnight under nitrogen at 40° C.
Workup: the reaction
was concentrated under vacuum and partitioned between ethyl acetate and water.
The
organic layer was dried by MgS04. The azide product was obtained in 89 %
yield.
Step L: Reduction of Azide
3o The azide (2.44 mmole) was dissolved in THF, and Pt02 (catalytic amount).
The reaction
was shaken on a Parr shaker in the presence of hydrogen (20-30 psi) for one
hour.
Workup: Filtered through celite and was rinsed with methanol. The filtrate was
concentrated down to obtain the desired amine product in92% yield.
Step M: Proctection of amine
3;
The amine (9.9 mmole) and triethylamine (9.9mole) was dissolved in dry
dichloromethane and (Boc)~O was added.The reaction was stirred overnight under
16
CA 02376420 2001-12-14
WO 00/77030 PCT/US00/16643
nitrogen. Workup: the reaction was concentrated on a rotary evaporator and the
residue
was taken up with ethyl acetate and then washed with water, citric acid,
sodium
bicarbonate, and brine. The organic solvent was dried over MgSO.~ (86%).
Step N: N-methylation
The amine (4.1 mmole) was dissolved in THF and methyl iodide was added,
followed by
sodium hydride. The reaction was stirred overnight under nitrogen. Workup: the
reaction
was concentrated on a rotary evaporator and the residue was taken up with
ethyl acetate
1o and then washed with water, citric acid, sodium bicarbonate, and brine. The
organic
solvent was dried over MgS04 .
Step O: Bromination
1~ The methyl ester (26.9 mmole), benzoyl peroxide (0.455 mmole), and NBS
(26.9 mmole)
were dissolved in benzene and kept at reflux overnight under N~, after which
the solution
was concentrated to dryness. The remaining solid was filtered and washed with
hot water,
then dried on high vacuum overnight (81 %).
20 Steps K, L and O are employed in the order set forth below:
Br
method O / ~ ~ method K
benzoyl peroxide \ / NaN
NBS '
3 O~ OCH3
NH2
Method L / \ incorporation into
H~/Pd/C ~ \ ~ / ~ dipeptide
O OCH3
Mixtures of final products or intermediates obtained can be separated on the
basis of the
physico-chemical differences of the constituents, in known manner, into the
pure final
products or intermediates, for example by chromatography, distillation,
fractional
crystallization, or by formation of a salt if appropriate or possible under
the
circumstances.
1~
CA 02376420 2001-12-14
WO 00/77030 PCT/US00/16643
For the purpose of classification of the inhibitor activities of the compounds
of the present
invention. the Examples given below have been grouped by their IC;o
concentrations.
Those compounds of Group IV have an IC;oof greater than 200 ~M. The compounds
of
Group III have an IC;oconcentration of from between 100 ~M and 200 ~M and are
the
preferred compounds of the invention. The compounds of Group II have an
IC;oconcentration of from between 10 ~M and 99 ~M and are the more preferred
compounds of the invention. The compounds of Group I have an IC;concentration
of <
~M and are the most preferred compounds of the invention.
Table 1
Enzyme inhibition assay results for structures having the peptide backbone:
O R3 OH O
H H
A-N N N N B
H H
O R2 O
In Examples 1-26: R~ is 2-methylpropyl, R3 is methylthioethyl and A is tert-
1 ~ butyloxycarbonyl. -
Example IC5o Group B
1 IV OMe '!
i
III ' OH
3 IV ~H \ /
I
I
Example ICSO Group B
I
4 i IV N
I
' I
H
j I
5 ~~i III i
,nM- N ~
1S
CA 02376420 2001-12-14
WO 00/77030 PCT/US00/16643
O-
6 II ~n~" N
H
O
7 III ,M.-N O-
O
OH
g ~N
II
O
9 II ,~",.-N ~ O H
O
~ C02H
II
I
11 I OH
~H
0
12 III ,nr- N
H ~ ~N
13 I o
~OH
~
~
/ OO
H
19
CA 02376420 2001-12-14
WO 00/77030 PCT/US00/16643
Example IC5o Group B
0
14 I , ,M,_ N
H ~ ~ ~OH
I I ~''N OH
O
0
16 IV NHz
NH2
~' N i
H
O
I H O
17 II ~ ,r~-N
OH
18 II ~N O
O
19 III
OH
II ~-N ~ co,H
'i
I
21 II
O
22 III N~ o~
0
CA 02376420 2001-12-14
WO 00/77030 PCT/US00/16643
R ICso Group B
O
23 III ,M.-N
~N
H
24 III
25 II
~H ~ / NHZ
26 I C02H
H
f ~-N
COzH
27 II CozH
H
f ~-N
C02H
28 I ,~..-N Co2H
H
29 II f""_ N ~
CO2H
30 I
~~~-N~C02H
H
H
31 II ~-N
co2H
21
CA 02376420 2001-12-14
WO 00/77030 PCT/US00/16643
R ICSO Group B
32 I
~H/~COzH
CO2H
i 33 no data HO
II / \
~''. H i
34 I ~ ~N
H
COZH
I
3 5 II ,N".. N II
/ I
HOZC
36 1 ~ off
~~--N NO2
H
Table 1 continued for variations of amino acid substituents
and/or N-terminal capping groups.
i
R ICSO Group Structure
R~ is benzyl, R3
is phenyl,
A is acetyl and B
is 4-
37 I
aminomethyl-benzoic
acid
R~ is 2-methylpropyl,
R3 is I
i
phenyl, A is benzyloxy-
3g II
carbonyl and B is
4-amino- I
methyl-benzoic acid
i
CA 02376420 2001-12-14
WO 00/77030 PCT/US00/16643
R ICSO Group Structure
R~ is ?-methvlpropyl. R: is
39 II ' phenyl. A is butvloxv-
carbonvl and B is 4-amino-
methvlbenzoic acid
The followinU non-limitinw examples describe a method by which each of the
compounds
of Table 1 and as numbered therein were prepared following the procedure
outlined
generally in Scheme 1.
Example 1
Steps .=~ throuUh F were employed. substitutin~l H?'~-Val-OMe Ithe methyl
ester) for the
m benzvloxvcarbonvl protected startin; material. and selecting L-Met as A~~
and L-~ al as
AA, .
Molecular Formula~ C~~H;.~N.~O~S
Molecular ~~~ 618.87
eight
H-nmr (solvent) (CDCIz ) a 7.43(d. 1H); 7.20(d, 1H); ~.99(d.
1H); ~.20(d.
1H); 4.50(m. 1H); 3.90-3.80(m, 1H); 3.66(s,
3H); 2.50-
?.30(m, 6H); 2.1-1.90(m. 6H); 1.~6(m. ?H);
1.44(s, 9H);
0.99(m. 18H)
C-nmr (solvent) (CDCI~ ) a 190.067, 17?.??9. 168.61. 117.913.
70.817.
70.574. 60.181. X7.383. x'_.124. 40.916.
40.?34. 30.1?3.
j 1?8.187. X4.778.'3.0??. '_'1.706. 19.119.
18.978. 17.802.
1J.169
I Ma~~ ~nec i ~ 619.?
~TH-)
Example 2
The product of Example I was further subjected to Step G to remove the methyl
ester.
leaving the free C-terminal acid.
Molecular Formula C~RH:~N.~O~S I
I Molecular ~~ eight 604.80 i
H-nmr (solvent) (CD~OD) a 8.33(d. 1H): 7.00(d. 1H): 4.>j(m. 1H); 4.33(d.
?H): 4.1-3.81m. 4H); ~'.501m. ?H): 2.42(d. 2H): ~.101m. ?H): j
?.081 s, 2H1: ~.OOIs. 3H): 1.~~(m. 3H): 1.44(s. 9H): 1.33(m,
j 1H): 0.99(m. 18H1
SUBSTITUTE SHEET (RULE 26)
CA 02376420 2001-12-14
WO 00/77030 PCT/US00/16643
C-nmr (solvent) (CD;OD) c~ 194.624.
~ 174.332. 173.972.
100.475. 71.73
7.
71.612. X9.118. ?.699. 41.7?7. 41.?~6.
~-1.14?. 31.741.
31.498. 31.130. 26.01 ~. ~'~.824, ?3.7~~.
2S.716. 22.563.
~ 22.164. _'0.667. 19.640. 18.74. 18.261
19.781.
Mass spec (MH-) ~ 60~
Example 3
Steps .A through F were employed, selecting L-Met as AAi and L-Val as A:~~ and
using_
benzvlamine as R'.v;H~ in step F.
Molecular Formula Cz;H;~N;O~S
Molecular V%ei~ht~ 693.91
H-nmr (solvent) ' (DMSO-d) 6 7.88(d, 1H); 7.77(d. 1H); 7.41(d.
1H): 7.34(m,
~H): 6.88(d. 1H): 4.88(d. 1H); 4.45(m. IH):
4.36(d. 2H):
3.88(m. 2H); 3.45(6. 2H); 2.44(m. 2H): 2.221m.
2H): 2.101 s.
3H); 1.9-1.80(m. 2H); 1.~0(m. 3H); 1.44(s.
9H); 1.38(m.
1H). 0.98(m. 18H)
C-nmrosolventl (DMSO-dl d 172.190. 171.846. 171.66. 1-10.?36.
I~S.864.
1?7.91~. 127.39. 78.734. X8.474. ~l.?24.
42.642. 32.61 7.
30.760. 30.070. 28.714. 24.745, 23.816,
22.287. 19.763.
18.674. 18.447. 1 x.194
Mass s ec (MH-1 694
Example 4
Steps .A through F were employed, selecting L-Met as AAA and L-Val as AAA and
using
I-pentanamine as RNH~ in step F.
Molecular Formula~ C~;H~,~N;O~S
Molecular % eightI 673.9>
H-nmr (solvent) (DMSO-d) c~ 7.89(d. 1H); 7.66(d. 1H); 7.44(d.
1H): 6.88(d.
1H):4.88(d, lH):.~.4~(m. lH):3.78(m.2H):
3.1-3.OIm.2H l: '
2.50(m, 2H); 2.22(d, 1H); 2.04(6. 3H); 2.0-1.80(m,
2H);
1.~~(m. 3H1: 1.44(6. 9H): 1.32(t, 3H1; 0.98(m.
18H)
C-nmr (solvent) (DMSO-d) a 187.944, 183.453, 171.67, 164.00.
16.194.
117.413. 114.615, 117.413, 114.61 ~. 100.429.
61.405,
58.43, X1.169, 32.66. 30.830, 30.04, 29.216.
29.106.
28.714. 24.748. 23.831. 22.303. 19.209,
18.494. 1 x.178.
14.371
Mass s ec (MH-) I 674
1u Example 5
Steps A through F were employed, selecting L-Met as AA, and L-t% al as AAA and
using
ethvlamine as RNH, in step F.
~ Molecular Formula ~ C;«H;-Nr,O-S
-t
SU8ST1TUTE SHEET (RULE 26)
CA 02376420 2001-12-14
WO 00/77030 PCT/US00/16643
' Molecular Weight ~ 631.87 !
H-nmr (solyentl (CDCI: ) 6 7.66(b6.1 ): %.44(bs.lH): 7.20(s. 1H); 7.10(b6.
1H): ~.20(bs. 1H); 4.~~(bs. 1H); 4.1-3.9(m. ~H); 3.66(6. 1H);
', 3.4-3.20(m. 2H): 2.6-2.40(m. ~H); 2.20-1.99(m. 5H);
1.66(bs, 1H): 1.44(d. 9H1; 1.401 s, 1H): 1.20(m. 3H); 0.99(m.
18H)
C-nmr (solvent) (CDC1, ) a 200.280; 190.067: 172.589: 172.001; 1~?.799;
149.272: 59.758: 34.246: 30.233; ?9.67: 28.219; 24.739;
?x.967: ? 1.909: 19.292: 19.022: 17.826: 15.18: 14.628 I
!, Mass spec (MH+) 632.2
Example 6
Steps A through F were employed. selecting L-Met as a.A, and L-~'al as A.A~
and using
methyl 4-aminobutanoate as RI~'H~ in step F.
Molecular Formula Cz~H~,~N;OoS I
', Molecular Vv 703.93
eight
I H-nmr (solvent) (DMSO-d) d 4.88(d. 1H): 4.441m. 1H); 4.11(m.
2H1: 3.88(m. ';
3H); 3.66(s, 3H); 3.44(d. 2H): 3.101m. 2H):
2.43(m, 1H1:
?.2~(t. 2H); 2.14(d. 2H); 2.04(6. 3H): 1.88(m.
2H): 1.60(t,
j 2H): 1.44(6, 9H): 1.401 m. 1H): 0.98(m.
18H)
C-rnnr (solvent) (DMSO-d) a 173.821. 172.089. 171.67. 78.750.
60.417.
58.46. X2.367. 51.77, X1.091, 32.617. 31.277,
30.728.
30.619, 30.078, 28.714. 24.983. 24.76. 23.784,
22.303,
19.724. 19.693, 18.674, 18.494. 1 x.478
~ Mass spec (MH+) ~ 704 j
Example 7
Steps A through F were employed, selecting L-Met as AA, and L-Val as ~~~ and
using
methyl 3-aminopropanoate as RNH~ in step F.
Molecular Formula C;~H;~N;OoS ~I
Molecular Weight 689.91 j
H-nmr (solvent) (DMSO-d) c~ 8.11(d, 1H); 7.98(d, 1H): 7.66(d,
1H); 7.~~(d.
1H); 6.88(d. 1H); 4.88(d. 1H); 4.43(m. 1H);
4.14(m, 1H):
i
I 3.77(m, 3H): 3.66(6. 3H): 2.(m. 4Hj; ?.~4(m,
2H); 2.21(d.
2H); 2.01(6, 3H); 1.89(m. 2H); 1.4~Im, 1H);
1.44(6, 9H);
1.33(m. 1 H): 0.98(m. 18H)
j C-nmr (solvent) (DMSO-d) b 205.602. 177.67. 172.747. 171.634.
114.616.
100.421, 78.70, X9.790. X8.348. ~ 1.867.
~ 1.108, 3.28?.
34.099. 30.799. 30.070. 28.714. 23.8~~.
22.721. 19.732.
19.622. 18.721. 18.462. 15.313. I x.186
I Mass spec (MH+)j 689.9
Examele 8
,;
SUBSTITUTE SHEET (RULE 26)
CA 02376420 2001-12-14
WO 00/77030 PCT/US00/16643
The product of Example 6 was further subjected to Step G to remove the methyl
ester.
leavin; the free C-terminal acid.
I Molecular Formula Cz~Hr,~~;0~>S
I Molecular ~~% ~ 689.91
eieht
H-nmr (solvent) (CD,OD) c~ 4.~6(m. 1H), 4.20(((1,.01-3.981m.
1H); 4 2H);
3.32(m. 2H): 2.50(m. 2H); 2.10(m. 3H);
2.24(m, 2H):
2.00(m, 4H); 1.88(t, 2H); .44(s. 9H);
1.J~(m, 1H); 1 1.44(s.
9H): 1.34(m. 1 H): 0.99(m.
18H)
C-nmr (solvent) (CD~OD) b 173.682. 74.206. .463. 43.527,
71.408. X4 41.444.
39.72. 32.188. 31.397, 31.18,?x.730. X3.598,
28.716.
' ''~.30~. ?0.63. 19.66. 18.60.l~.? 3~
18.206.
~I Mass spec 690.1
( MH+)
Example 9
_ The product of Example 7 was further subjected to Step G to remove the
methyl ester
leaving the free C-terminal acid.
Wlolecular Formula ~ C"H;-N:OuS
j Molecular V~ 675.89 i
eight
H-nmr (solvent) (CD~OD) d 4.45(m, 1H); 4.1-4.0(m. 2H); 4.0-3.98(m.
3H):
3.54(m, 2H); 2.56(m, 3H); 2.40(m, 2H); 2.1-2.0(m.
4H);
1.66(m, 2H): 1.44(m. llHl; 1.22(t. 2H);
0.981m. 18H)
C-nrrtr (solvent) (CDzOD) a 174.231, 173.666, 100.478, 74.167.
-;'1.392,
43.804, 41.62, 36.413, 34.47, 32.627, 31.444.
31.185,
30.856. 28.724, 2~.8~5, 23.622. 22.266.
19.758. 1.259
Mass s ec (MH+) 676
Example 10
Steps A through F were employed. selecting L-Met as A:A, and L-~ al as .-~.~=
and usin~T
1o methyl ?-aminomethvlthiazole-~-carboxvlate as RI~'H~ in step F and then
removal of the
methyl ester according to step G. The methyl 2-aminomethvlthizaole-~-
carboxylate was
prepared according to Synthesis 1986, page 992.
Molecular Formula C~~H;~N~,OgS i
r Molecular 'VeiQht 744.98
H-nmr (solvent) (CDzOD) b 9.10(m. 1H); 8.881m. 1H); 8.26(s.
1H); 7.>j(m.
1H); 4.78(m. 2H); 4.6~(m. 1H); 4.33(m. 2H):
4.1-3.9(m.
~
I 'i
3H): 3.33(2H); 2.50-2.10(m, 4H): 2.10-1.901
m, 4H); 1.60(m.
1H): 1.44(m. (OH): 1.30(m. (H); 0.991 m.
18H) I
C-nmr (solvent) (CD;OD) c~ 168.31, 123.778. 6~.?78. X4.203.
26.316.
25.603, 25.164. 24.82 7, 22.810. 22.672.
21.810. 19.780.
17.664. 16.229. 16.12 7. 13.9 3. 1'.499.
12.224. 11.981. 9.99 ~I
I Mass spec (MH+1 ~ 745.'' I
SUBSTITUTE SHEET (RULE 26)
CA 02376420 2001-12-14
WO 00/77030 PCT/US00/16643
Example 11
Steps .a throu~~h F were employed, selectiny~ L-Met as A:~i and L-~'al as .-
~.~- and usin~~
methyl 4-aminomethvlbenzoate as R1~1H= in step F and then removal of the
methyl ester
according to step G.
Molecular Formula Cz,,H;~,N;O~S i
I
_ ~ 737.96
Molecular Weight
H-nmr (solvent) ~ (MeOD) 6 8.88(m. 1H); 7.889d. ?H): 7.44(d.
~H): -1.~~(m.
2H): 4.22(m, 1H); 4.0-3.9(m. 2H): ?.J-2.3(m.
3H): ?.14(m.
1H); ?.0-1.9(m. 4H): 1.~~(m, 1H); 1.44(s.
9H1: 1.33(m. 1H):
0.991 m_ 18H)
C-nrrlr Isolventl (MeOD) d 171.99. 171.1 7. 171.027. 166.761.
14?.4.
127.964. 1?7.759, 1?J.447, 77.899. 68.4J?.
X9.114, ~7.7J0.
~1.~ 73. 49.16. 40.87. 38.71?, 38.401. ?9.
3 34, 28.611.
28.41 ~. 28.266. 25.803. ~'?.9~3. 20.71
~. I 9. 345. 16.86?.
16.797. 1 x.7:17. 15.23 1?.314
Mass spec IMH-) I 738.3
Example 1?
Steps .~ tlu-ough F were employed, selecting L-Met as .SA, and L-~'al as .-
~.~~ and using
methyl 4-aminomethvlpyridine as RNH~ in step F.
Molecular Formula C,.~H;aN~,O~S
Molecular Weight 694.94
Mass spec (MH+) 695.?
1~u Example 13
Steps A through F were employed. selecting_= L-Met as A.A, and L-Val as .~.~~
and using
methyl 4-aminomethvlbenzosvacetate as RNH= in step F and then removal of the
methyl
ester according to step G. Methyl 4-aminomethvlbenzoxvacetate was prepared
according
to the procedure provided in J. Med. Chem. 1998. Vol. 31. No. 10
I Molecular FormulaC;;H~,~N;O~nS i
Molecular V-ei~ht 767.99
H-nmr (solvent) (MeOD): 6 7.?~ (t, 1H), 6.98 Im. ?H). 6.77
(s. ?H). 4.77 Is.
~H), 4.44 (m, 1h). 4.3? (m. 1h), 3.8 Im.
3H). 3. 33 (s. ?h1. ?.4 '!
(m. ~ H). ?.1 (m. 1H), 1.66 (m. 1h). 1.44
(s. 9h), 0.99 (m.
18H)
SUBSTITUTE SHEET (RULE 26)
CA 02376420 2001-12-14
WO 00/77030 PCT/US00/16643
i C-ntnr ~ solvent) ( MeOD l: c~ 169.033. 165.1 7 8. 16 7.998. 167.573 .
166.7.x-1.
l 63.686. 1 3.801. I?-1.6 7 1. 12-1.61-. 1?0.?'_'?. 1 1 ~.6~0.
108.690. 108.463. 7.1.839. 6.41 1. X9.81 ~. ».93~. X3.80?.
)4.J48. J-1. 3-1-l, -18.403. ~IS.O 7-1, -16.067, 3J.6~ 1. 3).463.
3J.3?~'. '6.489. 36.14-1. ~'~.6-1?, '_').)O1. ~~.3-1J. 3).078,
X4.819. 22.711. 19.741. 17.661. 16.206. 13.729. 13.88.
12.593. 1 ~.? 16. 9.246
Mass spec (MH-) ~ 768 '
Example 14
Steps _=~ through F were employed. selecting L-Met as Aa, and L-V'al as Aa~
and using
methyl 3-aminomethvlbenzoate as RNH_ in step F and then removal of the methyl
ester
according to step G.
Molecular Formula Cn,H;~\; OoS
~ Molecular WeiUht 737.96
I H-nmrlsolventl (CDzOD;'CDC1:)o8.0(s.lH):7.891d.IH1:7.89(s.lH):
7.66(6. 1H); ;.~~(d. 1H): -.~O(d. 1H): 7.=1-1(t.
1H): 6.88(d.
! 1H): 6.7 7(d, 1H); 4.6-4.-1(m. 3H): -1.33(m.
1H): 4.0-3.81m.
3H): ?.~-2.3(m. 4H); 2.22(m. 1H): 2.1-1.9(m.
~H): l.~-
1.4((m. 11H): 1.40(m. 1H); 0.99(m. 18H)
C-nmr (solvent) (CD~ODiCDCI~) d 169.088, 168.123, 168.04,
139.924, i
134.44. 126.984. 124.217, 123.684. 7~.2~~,
~6.0~3. X4.603. ;
48.685. 4.94?, 3.698. 3.494. 29.444, 25.932.
2~.6~0,
25.292, ?x.227, 25.078, 24.106, 23.032,
19.866. 17.899,
16.527. 14.019. 12.859. 12.449. 12.271,
9.583
Mass s ec (MH~-)
Example 1~
Steps .-~ through F were employed. selecting L-Met as A.~, and L-Val as ~..~=
and using)
m methyl -1-aminocvclohexvlcarboxvlate as RNH~ in step F and then removal of
the methyl
ester according to step G.
Molecular Formula~
C~;H~
~N;
O~S
Molecular ~% ~ 729.97
eight
(solvent) (CD~OD) a 8.34(d, 1H): 8.11(d. 1H); 7.98(d.
H-nm 1H); 7.~~(d.
r
1H): 6.88(d, 1H): 4.~~Im. 1H); -1.?3(m.
1H); 3.9-3.8(m. ~H):
3.34(m. 1H); 2.6-2.~(m. 3H1; 2.32(D. 2H);
2.0(S. 3H):
1.98(M. 2H); 1.67(m, 8H); 1.44(6. 9H): 1.33(m.
1H);
0.99(m. 18H)
C-nmr (solvent) (CDzOD) d 172.991, 168.2.11. 168.163, 167.849.
167.?22.
74.761. 6.419. x4.383. 48.191. -16.663,
3~.46J. 3~.?36.
34.726. 26.387. 26.87o. ~s.os~. 2-1.091.
22.688. ~o.~ss.
19.79. 17.671. 16.23 7 . I 3.73. 12.616.
12.428. 9.183
~ Mass spec (MH-I '~ 730.
~S
SUBSTITUTE SHEET (RULE 26)
WO 00/77030 CA 02376420 2001-12-14 pCT/US00/16643
Example 1 f,
Steps _-~ through F were employed. selecting_ L-'Vlet as .~..-~, and L-Val as
.-~.~- and using
?-amino~lutaramide as RNH, in step F.
Molecular Formula I C:;H~, \-OoS
Molecular V-ei~ht I 731.90
H-nmr solvent) (CD;OD) > 5.:~=1(d, 1H): S.?''fd. 1H): 7.S61d. 1H): 6.58(m.
1H). -1.~6fm. 1H): -1.331m. 1H): -1.0-3.91m. 3H): 3.33(m.
?H): ~'.~~(m. ?H); 2.-131m. ?Hl: ?.l-1.9(m. ~H1: 1.~~(m.
~ IH): l.-l~fs.9Hl: 1.33(m. 1H):0.991m. 18H1
C-nrrtr ~soivent) I fCD;OD) ~~ 17-1.129. 96.~0~. 80.711. 71.~~9. b1.971.
~9.~40.
>9.~s_. s-l.ls9. s~.7s6. s~.~so..~l.-X37. ~~.~,~. ;1.6Ø
?8.700. ?~.78~. ?3.786. '_'~.?11. ~'~.1~0. 19.7S1. 19.47.
18.6? 1. 18.149. 1 s.?3~
l~lass spec (V1H-1 I 7 31
E~am~le 1'"
Steps .~ throu~Th F were employed. selectin'r L-:Vlet as A_a, and L-Val as .-
~.~~ and using=
methyl I-aminocyclopropane-1-carbolvlate as RNH~ in step F and then removal of
the
methyl ester according to step G.
I Molecular Formula ~ Cz~H:-~';04S
I Molecular ~~% eight ~ 687.90
i, '.Mass spec I:VIH~) I 688
Example 18
Steps _-~ through F were employed. selecting L-1%Iet as .-~.~~ and L-~'al as _-
~_-~= and using?
3-amino-dihydro-~(3H)-furanone us RNH, in step F.
I Molecular Fornmla I Cz~H;-\;O:,S
I Molecular Weight ~ 687.90
Mass spec f MH-) ~ 688.s
Example 19
Steps .-~ through F were employed. selecting L-Wet as .~.~, and L-Val as .~_-~-
and usin~T
methyl -1-aminobutan-1-of as R'viH- in step F.
~loiecular Fonmuia . C;~H,. \;O~S
Molecular ~VeiUht ' 6 7~.9~
SU8ST1TUTE SHEET (RULE 26)
CA 02376420 2001-12-14
WO 00/77030 PCT/US00/16643
j H-nmrlsolventl ~ (CD;ODIc~S.33(m. 1H):8.11(m. 1H):4.SSIm. 1H):=l.ll(m.
1H): -1.0_;.91I11. ?H): ~.S~Im. ?H): ~. ~31m. ?H): ~.30(m. ',
2H): 2.SSIm. 2H1: 2.1-1.91m. =1H): 1.661 m. ?H): 1.-14(x. 9H);
1.331m. 1H1:0.991m. 18H)
I Mass spec ( MH-) ~ 676 I
Example 20
Steps A through F were employed. selectin'T L-Met as A:A, and L-V'al as AAA
and using
methyl 3-aminobenzoate as RI~TI-~~ in step F and then removal of the methyl
ester
according to step G.
I Molecular Formula C3SHS7NS09S
I Molecular V% eight 723.94 _ I
I Mass snec 1 MH~-1 724 I
Example 21
Steps A throu~_=h F were empioved. selecting L-Met as AA, and L-~~al as A.~=
and using_
methyl 4-piperidine-acetate as RNH, in step F and then removal of the methyl
ester
according to step G.
Molecular Formula C,;H~"N;O~S
Molecular Weight 729.98
H-nmr (solvent) (CD~OD) d 8.22(m, 1H); 7.66(m. 1H): 6.88(d.
1H); 4.87(m,
1H); 4.SS(m. 1H); 4.0-3.8(m. 3H): 3.22(m.
2H1; 2.6-2.3(m,
2H); 2.22fm. 2H); 2.15(m. 1H); 2.0-1.91m.
SH): 1.77(m.
1H): 1.SS(m. 1H): 1.44fm, 9H): 1.331m. 1H):
0.99(18H) I
I
C-rtmr (solvent) (CD~OD) x173.96=1, 166.095. 1?6.??~. 80.680.
',"1.447, ',
I 61.854. 61.854, JS.427, ~-1.165. 4 3.401-1.
-12.'_'S 3. -11.444. '
11.045. 3-1.320. 33.6:16. 3_'.S 10. 31.890.
31.06 i. 30.824.
! 28.685. 2S.78S. 23.739. 2'.179. 20.0-10.
19.765. 18.57=1.
18.425. 18.221. 15.227 I
Mass spec IMH--I ~ 730
Example 23
i ~ Steps A through F were employed. selectin'1 L-Met as AA. and L-~-al as AA=
and using
ethyl piperazine-N'-carboxvlate as RI~TH= in step F and then removal of the
etiyl ester
according to step G.
Molecular Formula ~ C;;H~,~Nr,O~,S '
Molecular V eight ~ 744.98
SUBSTITUTE SHEET (RULE 26)
CA 02376420 2001-12-14
WO 00/77030 PCT/US00/16643
H-nmr (solvent) (CD;OD) c~ 7.55(d. 1H): -i.66(d. 2H): 4.551m. 1H): 4.12(m,
2H): 4.1-3.8(m.4Hl: 3.~-3.2(m. 4H1; 2.551m. 2H); 2.20(m,
~H); 2.1-1.90(m. 5H): 1.66(m. 1H); 1.411(x, 9H); 1.33(m.
4H): 0.99(m. 18H)
C-nmr (solvent) (CD;OD) a 174.873, I 7.1.01 I. 173.925, 173.847. 1','2.592, '
157.231, 80.688, 71.455, 62.967, 61.791. 55.631. 54.126.
52.942. 49. 878. 49.306. 49.024, 46.837. 43.020. 41.382.
41.154, 32.619. 31.726. 31.107, 28.755, 23.802. 22.250.
19.820. 18.668. 18.558. 1 x.290. 14.883
Mass spec (MH+) ~ 745.4
Example 23
Steps A through F were employed, selecting L-Met as AA, and L-Val as AAA and
using
1-acetamido-2-aminoethane as RNH, in step F.
Molecular Formula C~~H~,nN~,O~S
Molecular Weight 688.93
H-nmr Isolventl (MeOD): a 8.33 (m, 2H). 8.01 (d. 1H), 7.66
Id. 1h1. 6.88 (d.
1h), 4.56 Im. 1H), 4.22 Im, 1H). 3.8 (m.
3h). 3.44 (m. 2H),
2.56 (m, 2H). 2.33 (d. 2H). 2.2 (m. 8H),
1.66 (m. 1H1. 1.44
(s, 9H1. 1.33 (m. (H). 0.99 (m, 18H) '
C-nmr (solvent) (MeOD): d 194.616. 174.466, 155.044, 96.512,
76.275,
60.764, 54.400, 52.182. 41.562, 41.280,
39.916, 32.345,
31.579, 31.248, 31.154. 28.693. 25.816,
23.637, 22.650,
22.281. 19.750, 19.632. 18.613. 18.143,
15.204
Mass spec (MH+) ~ 689
E~am~le 24
Steps A through F were employed. selecting L-Met as AA, and L-Val as AAA and
using
1-amino 4-fluorobenzene as RNH~ in step F.
1U
Molecular Formula C;aH;~:N;O-S
Molecular Weight 697.91
H-nmr (solvent) (MeOD): d 7.66 (m, 1H). 7.00 (m. 1H), 4.55 (m, 1H). 4.34
(m, 1H), 4.22 (d, 2H), 4.0 (m, 4H), 3.22 (s, 9H), 2.66 (m.
2H). 2.45 (m. 2H), 1.9 (m, 5H), 1.66 (m. 1H). 1.44 Is. 9H).
1.33 (m. 1 H). 0.99 ( 18H1
~~lass spec ( MH+) 699
Example 2~
Steps A throw=h F were employed. selecting L-Met as AA, and L-Val as AAA and
using
-t-aminomethvlbenzocarboxamide as RNH- in step F.
SUBSTtTUTE SHEET (RULE 26)
CA 02376420 2001-12-14
WO 00/77030 PCT/US00/16643
Molecular Forn~ula ! C;~,H~,~~',.O~S
Molecular ~~'ei~ht ! 736.95
j H-nmrlsoiventt j (CD:OD)~>8.33(d. IH): 7.67(d.3H): 7.44(d.'_'H):.~.~~Im.
1H): 4.0-3.90(m, 3H): 3.331m. 3H): 3.101m.4H1: 3'.~-3.4(m.
3H): 3.01(d.3Hl: l.bblm. 1H1: 1.44(s.9H1:0.991m. 1SH)
i,
~ C-nmr Isolventl (CD:OD) ci 169.174: 168.~s9: 16 5.45: 165.31'_': 16S.139:
168.0~~: 166.21 I : 1 ?.479: 135.481: 138.31 7 : 1 ~7.66~:
133.010: I'_'?.971: 133.603: 1~''_'.~~~: 74.8~~: 6.419:
X6.054: X4.673: ~=1.446: 45.33s: 48.335: 46.640: 4~.9~8:
I 37.45: 3~.~41: 3~.~41: 3~.?67: 3~.1~7: 36.644: ~~.456:
3~.''S3: 3 x.109: 33.6~'_: 19.755: 17.601: 16.0 7 3: 13.69:
1'' 616 I'' X40 9 14-1 3 054
~ :Mass spec ( ~~ZH-1 I 737.4
Example 26
Steps A through F were employed. selecting L-stet as AA, and L-Val as AA= and
usin~~
.~-dicarbowcvclohewlamine as R~TI-I= in step F. The amine was prepared as
follows:
To ~.~ ~ of dimethvl ~-aminoisophthalate in 40 ml of methanol!4.8m1 acetic
acid ( I ~°,Wwn .vas
added 1.3~ U of ~°~ rhodium on alumina(50°,r ww. the mired slum'
was saturated with
hydrogen at ~~ PSI and shaken for a total of 73 hrs. L;pon completion of they
hydrogenation the
reaction was filtered through Celite and dried over anhydrous sodium sulfate.
Filtration and
to subsequent rotoeyaporation yielded 3.8Ja of crude product. which was then
subjected to silica
gel chromatographic purification to afford 0.768 of Di-Methyl 1-
Aminocvclohexvl-3, ~-Di-
Carboxvlate as a pale white solid. See also: Fieser 8: Fieser. Reagents for
Org. Sy. .1~. 415 and
Freifelder. ~I.: N_. 1'. H.: Helgren. P. F.: J. Org. Chem.. 30. 2485-G.
I 'Molecular Formula . C36H63\~O11S
Molecular V eight
I tlc Rf (solvent)~
Rf--0.0~
(
10%MeOH,-'DCMI
Purification: I
I
.
Acid,~Base
Washes.
3.
Trituration
w
Ether.~Filtration
Mass s ec IMH-1 t 773.3(ESI Nee.)
Synthetic Route Method A. B, E. J, F. G
Description Method J: Amino-Arv_~1 Diesters to Amino-Cyclohesyl
Diesters
Example 2?
Prepared in a manner similar to Example 36. except that dimethyl 6-
aminoterephthalate
was utilized in the preparation of '_'.~-dicarboxvcvclohesviamine.
I Molecular Formula ! C36H6s~~Ol IS
I 'Molecular V'eiUht I 77,
I tlc Rf 1 solvent t l Rf=0.3~ ( 10°,oiVIeOH. DCM 1
Purification: ' 1. .-~cid.~Base V ashes. 3. Trituration w Ether Filtration
;,
SUBSTITUTE SHEET (RULE 26)
CA 02376420 2001-12-14
WO 00/77030 PCT/US00/16643
I Mass spec ('_VIH~I~ %%-1.4(ESI -Positivel i
I Elemental Analysis-Calc
I"~,)
Found (r)
I Synthetic Route Method A, B. E. J. F. G
Descnption
Method J: Amino-Arvl Diesters to Amino-Cvclohexvl
' '
! Diesters
Example 28
Prepared using methyl .~-aminoocvclhexylacetate.
~ Molecular Formula~ Cz~,H~;N;OoS
I Molecular 't'ei~ht~ 744
H-nmr (solvent) (MeOD) a 8.32(d. 1H); 8.00(d. 1H); 7.66(d.
1H); 4.66(m,
1H); 4.20(d, 2H); 4.0-3.88(m, 3H); 3.46(m.
2H); ?.66(m.
1H); 2.40(d, 2H); ?.30(d. 2H); ?.1-1.90(m,
4H); 1.77(m,
4H): 1.44(s, 9H): 1.33(m. 1H); 0.99(m. 18H)
I C-nmr ( solvent (MeODI d 177.003, 17s.060, 174. I 9 7. 173.91
1 s. 173.194. !
80.78s, 71.427. 61.9?7. 60.:~?~', s4.?93.
s?.631. 41.486. j
41.204. 4O.2OO, 33.26. 3?.64~. ~?.~98. 3?.347,
31.84. !
31.70. 31.s94. 31.108. ~9.so9. ?8.788. X8.710,
~s.841.
?3.678, ??.?83. 19.77s. 19.66s. 18.61 s.
18.s3. 1 s.197
Mass s ec (MH+) 744
Synthetic Route Method F, J. G
Description
Example 29
Prepared according to the initial preparation used to prepare Example 11 but
Steps M and
N are inserted prior to the removal of the ester alcohol. Arrtinomethylbenzoic
acid was
first protected with 1 equivalent of BOC and then the '~'-protected amine was
methylated
with methyl iodide. The BOC group was subsequently removed and the '~-methvl-
aminomethvlbenzoic acid was coupled with the tetrapeptide.
~ Molecular Formula~ Cz~H~,,N;OQS
Molecular V~ 7s 1.97
eight
H-nmr (solvent) (MeOD) 8 8.10(m, 2H); 7.4s(m. 2H); 4.66(m.
sH); 4.0-
3.80(m, 4H); 3.20(m. 2H): ?.90(m. 3H): ?.60(m.
1H);
2.40(m, 2H); ?.?-2.0(m. =1H); 1.66(m, 1H):
1.44(s. 9H):
1.33(m. 1H): 0.99(m, 18H)
C-nmr lsolvent) (MeOD) a 174.934, 17-1.448, 174.24-1. 17-1.041.
173.947.
169.71-1, 143.86s, 131.418. 131.136. 1?8.816,
80.738.
71.s83, 61.896. 61.770. s6.190, ss.939.
s-l.ls'?, X3.039, Ii
s2.098, 49.8s 7. -11.470. 36.078. 36.046.
3?.s I 9. 32.018.
31.093. ?8.694, ?s.810. X3.709. ~~.I 73.
19.77s. 19.63.1. I
18.61 ~. 18.4?7. 1 s.??9 I
~ Mass s>'ec I 7s?
1'vIH-~)
SUBSTITUTE SHEET (RULE 26)
CA 02376420 2001-12-14
WO 00/77030 PCT/US00/16643
~ Synthetic Route Descrynon ~ Method F. M.N.G
Example 30
The amine utilized was dimethvl I-amino-1. ~-propane-dicarboxv_ late and then
the
dicarboxvlate was saponified to provide the dicarbo~vlic acid.
I Molecular Formula I, C;,Hs~>N;O"S I
~r Molecular ~~'eiaht I 733.91 I
~ H-nmr (solvent) (MeOD) d 8.4~(d. 1H); 8.?~(d. 1H); 7.60(d. 1H): 4.>j(m.
I 1H): 4.2~(d. ?H): 4.01-3.80(m, 3H); 3.401 m. ?H): ~.60(m.
I IH); ?.60(m. 1H): '_'.40(m. 2H); ?.2-1.9(m. 4H); 1.66(m.
I 1H1: 1.44(s. 9H); 1.33(m. 1H); 0.991 m. 18H)
~ C-nmr (solvent) (MeOD) d 176.67, 175.012, 174.809. 174.244. 174.197.
173.978, 80.769. ~I.~O~, 61.880. 60.?18. ~4.?30. ~~.913,
?.678. 41.~ 17. 41.? 19. 3?.409. 31.688. 3 I .?34. 31.077.
?8.694. ''7.644. '_'~.8?6. ?3.741. ??.?~?. 19.79. 19.66.
I
18.61 ~. 18.474. 1 ~.? 1 3
I Mass spec IMH-1 a~ 734
Synthetic Route Method F, G
I Description
Example 31
The tetrapeptide was coupled to the N-terminal capping group. The cappin~,~
groups was
prepared by reacting 1-bromo-I-(4-benzoic acid) ethane with sodium azide,
reducing the
to resulting azide to the amine and coupling the product with the
tetrapeptide.
Molecular Formula C;;H,,,N;OaS
I Molecular ~Veiaht 71.97 'I
~ H-nmr (solvent)I (MeOD .' CCIzD) ~~ 7.98(d. ?H); 7.361 m.
?H): ~.031m. 1H):
4.661 m. 1H); 4.3'_'~m. ?H): 4.0-3.9(m.
~H): '_'.~6(m. 1H): _'.1- '
1.9(m. 7H): 1.661 m. 1H): 1.44(s. 9H1; 1..
3~m. 1H): 0.99(m.
18H)
I C-nmr (solvent) (MeOD / CCI~D) a 174.244, 173.931. 13?.~6?,
130.88.
130.039, 126.997. ''1.36. 69.18. 41.17.
41.?98. 40.106.
31.563. 30.089. 25.83. ?x.888. Z4.88~. ?4.007.
'_'3.709.
?2.424. '_'?.346. 19.S??. 18.677. 15.339.
14.461, 11.43
~ Mass s ec ('MH+) 752
Synthetic Route Method F, K, L. G
I Descri tion
Example 3?
Prepared in a manner similar to Example ?6, except that dimethvl ~-
aminophthalate was
1 ~ utilized in the preparation of 3.4-dicarboxvcvclohewlamine.
I Molecular Formula I C36H6 3N~01 I S
SUBSTITUTE SHEET (RULE 26)
CA 02376420 2001-12-14
WO 00/77030 PCT/US00/16643
I Molecular ~~ eight I 7 7 3
I tlc Rf f solvent ) I Rf=0.19 ( 10° ~MeOH,'DCM ) I
Purification: I 1. :acid,%Base Washes. ~. Trituration w
Ether Filtration j
Mass spec 1=VIHT)~ 774.~(ESI -Positive) I
Synthetic Route Method A, B, E, J, F. G
Description Method J: Amino-Arv_ 1 Diesters to Amino-Cyclohexyl
Diesters
Example 33
The tetrapeptide was coupled with 1-amino-indan-2-of using step F.
I Molecular Formula~ C;,H,,,N;OsS
Molecular ~~ 736
eight
H-nmr (solvent) (MeOD) c~ 7.99(m, 1H); 7.66(m. 1H); 7.4-7.2(b,
4H);
6.88(m. 1H): ~.33(b, 2H); 4.66(b, 1H); 4.~0(m.
2H): 4.1-
3.91m, 3H): 3.33(b, 2H1; 3.10(m. 1H); 3.0-2.9(m.
1H); 2.6-
2.4(m. 2H1: '_'.22(m. 1H): 2.0-1.91m. 4H):
1.44(s. 10H);
0.99 m. 18H1
I C-nmr Isoivent) (MeOD) t~ 168.484. 133.887. 1?3.0~7. 1'_'1.97
~. 120.274.
119.77. 68.060, 6.317. ~~.747. ~4.6~0. ~~.7~3.
48.466.
46.906. 3.45 7 . 3~.1 ~0. 34.844. 26. 340.
~~.4~4. ?x.031.
22.688. 19.788, 17.66. 16.229. 13.808, 1?.~
; 7. 12.412,
9.207
Mass s ec (MH+) 736
Synthetic Route Method F,
Descri tion
EYam~le 34
The methyl ester of 2-carbow-cvclohexvlamine was prepared accordinU to setp H.
then
coupled with the tetrapeptide and the free carboxlvic acid was generated
according to step
G.
Molecular Formula C~~,H~,:N~,O~S
~
Molecular V'eiaht 730 I
~
H-nmr (solvent) (MeOD) 8 8.3~(m. 1H); 8.2~(m. 1H); 7.6~Im,
1H); 6.80(m. I
1H): 4.6~(m. 1H): 4.20(m. 2H); 4.0-3.901
m. 3H); 3.3~(m,
1H): 2.6-2.40(m. 3H); 2.20(b,s. 2H); 1.77(m.
2H): 1.661m.
2H): 1.44(s. (Hl: 1.33(m. 1H): 0.99(m. 18H1
C-nmr Isolventj (MeODI a 172.991. 172.91'_'. 168.163, 167.07
3. 74.78>.
6.434. ~4.~09. 45.348. 46.381. 3~.3~3. ?9.9~
. ~6.9~9,
X6.849. 26.41 S. ~'~.109. 23.424. ?3.34.
22.7 3~. 19.286.
I 17.71 1. 16.433, 16.3. 13.808. 13.666. 1
x.66 3. 1'_'.38. ;
I ~ 9.238
I
Synthetic Route ~ Method H. F. G
Description ~
SUBSTITUTE SHEET (RULE 26)
CA 02376420 2001-12-14
WO 00/77030 PCT/US00/16643
Example 3~
The tetrapeptide was coupled with the methyl ester of phenvlUlycine and the
ester ~=roup
was subsequently removed.
Molecular Formula C~~,H_;<>N;O<,S
Molecular Weight 737
H-nmr Isoiventl (MeOD) e> 7.44-7.30(m. 5H); 5.40(s, IH);
4.551m. 1H);
4.01(m. 2H): 3.98(m. 1H); 3.22(s. 2H): 2.44(m.
2H); 2.1-
2.0(m. 5H); 1.66(m. (HI: 1.33(m. (H): 0.99(m.
18H)
C-nmr ( solvent) (MeOD) d 177.058. 123.794. 123.331. 122.924.
19.764.
~
17.679. 16.159. 13.729. 13.627. 12.553 t
Mass s ec (MH~-> 738
Synthetic Route Method F, G
Description
Example 36
The tetrapeptide was coupled with ?-(5-(?-hvdromnitrobenzene))-I-ethanamine.
Molecular Formula C~~,H~,nN~,O~nS
_ 768.98 I
Molecular Weiuht
H-nmr (solvent) (MeOD) 8 8.00(s, 1H); 7.66(d, 1H); 7.10(d,
1H); 4.55(m,
1H); 4.0-3.90(m, 3H); 3.55(M, 2H); 3.40(S.
2H); 2.80(m.
2H); 2.6-2.50(m, 2H); 2.40(m, 1H); 2.1-1.90(m,
4H);
1.60(m. 1H): 1.44(s, 9H): 1.30(m. 1H); 0.99(m,
18H)
C-nmr (solvent) (MeOD) 8 169.174, 168.139, 168.037. 167.912,
152.479.
133.066, 129.562. 126.741. 120.079. 120.00,
115.133,74.863. 65.897. 56.076.54.634, 54.289.
48.513.
48.262.46.663.46.052.35.283.29.052.29.005.26.254.
25.486. 25.298. 25.486. 25.298. 25.172.
25.109.
22.680.19.83 5. 17.711. 1 7 .640. 16.2 53.
16.088. 13.596.
12.546. 12.075.9.199
Mass s ec 1 MH+ 769
~
Synthetic Route Method F
Description
1~~~ Example 37
The compound was prepared in a manner similar to Example 1 1 with the
following
variations: Phenyl-statine (Phe-Sta) was employed in place of statine and
phenylalycine
(Pha) was selected as AA, and N-Ac-L-Val was used as AA_
Molecular Formula ( C39H49N508 l
Molecular Weight ~ 715
Mass spec (MH-~ ~ 716.3(APCI)
c>
SUBSTITUTE SHEET (RULE 26)
CA 02376420 2001-12-14
WO 00/77030 PCT/US00/16643
Example 38
The compound was prepared in a manner similar to Example I 1 with the
following
variations: Phenylglvcine (Phg) was selected as AA, and carbobenzyloxy (Cbz)-L-
Val
was selected as AA~.
Molecular FormulaI C.~~H;~N;Oa
I Molecular V%ei~ht 772.92
H-nmr(solvent) (DMSO): b 8.63 (m. 1H). 8.461(m, 1H), 7.8991'd.
2H).
7.399 (m, )OH), x.571 (m. 1H), 4.982 (m.
2H1. 4.335 (m.
2H), 4.157 (m, 1H). 3.833 (m, 1H), 2.317
(d. 1H), 1.999 (m.
3H), 1.335 (m. 1H), 1.141 (m. 1H), 0.808
(m. 1~H), 0.64
(d. 1H), 0.0 (d. 1H)
Mass s ec (MH+) 774
Example 39
The compound was prepared in a manner similar to Example 11 with the following
variation:
~ a Phenvl~lvcine IPha ) was selected as AA, .
I Molecular Formula CmH;,N;On I
I Molecular V% 739.90
eight
i H-nmr (solvent) (MeOD): d 8.88 (d. 1H), 8.66 (d, 1h). 8.44
(d. 1H), 7.99 (d,
2H), 7.66 (d, 1H), 7.~ (m, 6H), ~.5~ (d,
IH). 4. (m, 1H),
4.34 (m, 2H), 3.8 (m. 3H), 3.5~ (m, 1h),
3.44 (m. 2H), 2.~~
(m, 2H), 2.2 (m. 2H). 1.X5 (m, 1H), 1.44
(s, 9H), 1.33 (m.
1 H), 0.99 (m, 18H)
C-nmr (solvent) (MeOD): 8 168.210, 166.470. 139.696, 125.079,
124.734.
( 123.927 123.721, 123.574, 123.433, 123.229.
122.884.
122.743, 122.594, 1??.~24, 74.628. 69.724,
~~.~74. X3.035.
46.036, 37.846, 3 7.744. 3.543. 35.369,
26.136. 2~.~17.
i
24.843. 3.793. 22.719. ??.62~. 19.803. 19.631.
17.622.
17.46. 16.190. 1 ~.9~~. 13.698. 13.729.
1?.~46. 1?.342
j Mass spec lMH+)~ 740
Enzyme Inhibition Assay
t~
Purpose/Rationale:
The MBP-Cl'_'~ assay, determines relative inhibition of (3-secretase cleavage
of an MPB-
C12~ substrate by the compounds assayed. Human brain ~3-Secretase from the
concentrated HiQ pool prepared 7;16!97 in 0.20°,o Triton is used in the
assay.
Inhibition data was obtained from an ELISA which uses an anti-MBP capture
antibody
)on precoated and blocked 96-well high binding plates) followed by incubation
with
diluted enzyme reaction supernatant, incubation with an anti-SV'192 specific
biotinylated
reporter antibody and incubation with streptavidilvalkaline phosphatase.
Detection was
SUBSTITUTE SHEET (RULE 26)
CA 02376420 2001-12-14
WO 00/77030 PCT/US00/16643
effected by a fluorescent substrate signal on cleavage by the phosphatase. The
ELISA
only detected cleavage following Leu 596 at the substrate's Swedish APP 751
mutation
site.
Compounds were diluted in a 1:1 dilution series to a six-point concentration
curve (two
wells per concentration) which took up one 96-plate row per compound tested.
Relative
compound inhibition potency was determined by calculating the concentration of
compound that showed a fifty- percent reduction in detected signal compared to
the
enzyme reaction signal in the control wells with no added compound.
Procedure:
to
Each of the test compounds was weighed out into a vial and DMSO was added to
make
up a l OmM solution. To obtain a final compound concentration of 200 gM at the
high
point of a 6-point dilution curve, 100 uL of the 10 mM solution was added to
well C 1 of a
96-well V-bottom plate. Fifty ~L of DMSO was added to odd wells of row C
across the
1 ~ plate and 1:1 serial dilutions were made. 10 ~L of each dilution was added
to each of two
wells on row C of a corresponding V-bottom plate to which 190 ~L of 52 mM
NaOAc/7.9% DMSO, pH 4.5 were pre-added. The NaOAc diluted compound plate was
spun down to pellet precipitant and 20 ~L/well was transferred to a
corresponding flat-
bottom plate to which 30 ~L of ice-cold enzyme-substrate mixture (2.5 ~L MBP-
C125
2o substrate, 0.03 ~L enzyme and 24.5 ice cold 0.09% TX100 per 30 ~1) was
added. The
compound concentration in the final enzyme reaction was thus 50 times less
than the
starting concentration. The final reaction mixture of 200 ~M compound for the
highest
curve point was in 5% DMSO, 20 mM NaAc, 0.06° o TX100, at pH 4.5. The
enzyme
reaction was started by warming the plates to 37° C. After 90 minutes
at 37° C, 200
2~ gL/well cold specimen diluent was added to stop the reaction and 20 ~L/well
was
transferred to a corresponding a-MBP coated ELISA plate, containing 80 ~L/well
specimen diluent. This reaction was incubated overnight at 4oC and the ELISA
was
developed the next day using a 2 hr. incubation with a-192SW followed by
Streptavidin-
AP conjugate and flourescent substrate. The signal was read on a fluorescent
plate
3o reader.
Methods for treating Alzheimer's disease and other diseases characterized by
deposition
of A 3Lpeptide.
3R
CA 02376420 2001-12-14
WO 00/77030 PCT/US00/16643
This invention also relates to a method of treatment for patients suffering
from disorders
or diseases which can be attributed to A(3 plaque formation as previously
described and,
more specifically, a method of treatment involving the administration of the
(3-secretase
inhibitors of formula 1 as the active constituents.
Accordingly, the compounds of formula lcan be used among other things in the
treatment
Alzheimer's disease, and in diseases and indications resulting from the over-
expression of
A(3-peptide such as found in certain genetic defect diseases such as plaque
formation
associated with Trisomy 21 (Down's Syndrome) and Hereditary Cerebral
Hemorrhage
with Amyloidosis of the Dutch-Type (HCHWA-D).
As mentioned above, compounds of formula 1 are useful in medicine since they
are active
as inhibitors of (3-secretase. Accordingly another aspect, this invention
concerns:
1~ a method of management (by which is meant treatment or prophylaxis) of
disease or
conditions mediated by (3-secretase in mammals, in particular in humans, which
method
comprises administering to the mammal an effective, amount of a compound of
formula 1
above, or a pharmaceutically acceptable salt or ester thereof; and
2o a compound of formula (I) for use in human or veterinary medicine,
particularly in the
management (by which is meant treatment or prophylaxis) of diseases or
conditions
mediated by ~3-secretase; and
the use of a compound of formula (I) in the preparation of a composition for
the
2~ management (by which is meant treatment or prophylaxis) of diseases or
conditions
mediated by ~3-secretase.
The disease or conditions referred to above include Alzheimer's disease,
plaque
formation associated with Trisomy 21 (Down's Syndrome) and Hereditary Cerebral
3o Hemorrhage with Amyloidosis of the Dutch-Type (HCHWA-D).
For the treatment of diseases characterized by the overproduction and
deposition of A/3-
ppeptide, the compunds of formula (I) may be administered orally, topically,
parenterally,
by inhalation spray or rectally in dosage unit formulations containing non-
toxic
pharmaceutically acceptable carriers, adjuvants and vehicles. The term
parenteral as used
39
CA 02376420 2001-12-14
WO 00/77030 PCT/LTS00/16643
herein includes subcutaneous injections, intravenous, intramuscular,
intrasternal injection
or infusion techniques. The compounds may be administered in an amount from
about 0.1
mg/kg/day to about S00 mg/kg/day. Preferred amounts for daily administration
are from
about 1 mg/ kg to about 50 mg/kg. It will be understood however, that the
amount of the
compound actually administered will be determined by a physician in the light
of the
relevant circumstances, including the condition to be treated, the chosen
route of
administration, the actual compound administered, the age, weight and response
of the
individual patient, the severity of the patient's symptoms, and the like.
Formulation of pharmaceutical compositions
Compositions are provided that contain therapeutically effective amounts of
the
compounds of formula 1. The compounds are preferably formulated into suitable
pharmaceutical preparations such as tablets, capsules or elixirs, for oral
administration or
in sterile solutions or suspensions for parenteral administration. Typically
the compounds
described above are formulated into pharmaceutical compositions using
techniques and
procedures well known in the art.
About 1 to 500 mg of a compound or mixture of compounds for Formula 1 or a
2o physiologically acceptable salt or ester is compounded with a
physiologically acceptable
vehicle, carrier, excipient, binder, preservative, stabilizer, flavor, etc.,
in a unit dosage
form as called for by accepted pharmaceutical practice. The amount of active
substance in
those compositions or preparations is such that a suitable dosage in the range
indicated is
obtained. The compositions are preferably formulated in a unit dosage form,
each dosage
containing from about 2 to about 100 mg, more preferably about 10 to about 30
mg of the
active ingredient. The term "unit dosage from" refers to physically discrete
units suitable
as unitary dosages for human subj ects and other mammals, each unit containing
a
predetermined quantity of active material calculated to produce the desired
therapeutic
effect, in association with a suitable pharmaceutical excipient. Preferably,
the compound
of formula I above is employed at no more than about 20 weight percent of the
pharmaceutical composition, more preferably no more than about 15 weight
percent, with
the balance being pharmaceutically inert carrier(s).
CA 02376420 2001-12-14
WO 00/77030 PCT/US00/16643
To prepare compositions, one or more compounds of formula 1 are mixed with a
suitable
pharmaceutically acceptable carrier. Upon mixing or addition of the
compound(s), the
resulting mixture may be a solution, suspension, emulsion or the like.
Liposomal
suspensions may also be suitable as pharmaceutically acceptable earners. These
may be
prepared according to methods known to those skilled in the art. The form of
the resulting
mixture depends upon a number of factors, including the intended mode of
administration
and the solubility of the compound in the selected carrier or vehicle. The
effective
concentration is sufficient for ameliorating the symptoms of the disease,
disorder or
condition treated and may be empirically determined.
Pharmaceutical earners or vehicles suitable for administration of the
compounds provided
herein include any such carriers known to those skilled in the art to be
suitable for the
particular mode of administration. In addition, the active materials can also
be mixed with
other active materials that do not impair the desired action, or with
materials that
supplement the desired action or have other action. The compounds may be
formulated as
the sole pharmaceutically active ingredient in the composition or may be
combined with
other active ingredients.
In instances in which the compounds exhibit insufficient solubility, methods
for
2o solubilizing compounds may be used. Such methods are known to those of
skill in this
art, and include, but are not limited to, using cosolvents, such as
dimethylsulfoxide
(DMSO), using surfactants, such as Tween~, or dissolution in aqueous sodium
bicarbonate. Derivatives of the compounds, such as salts of the compounds or
prodrugs of
the compounds may also be used in formulating effective pharmaceutical
compositions.
2~
The concentrations of the compounds are effective for delivery of an amount,
upon
administration, that ameliorates the symptoms of the disorder for which the
compounds
are administered. Typically, the compositions are formulated for single dosage
administration.
The compounds of formula 1 may be prepared with carriers that protect them
against
rapid elimination from the body, such as time release formulations or
coatings. Such
earners include controlled release formulations, such as, but not limited to,
41
CA 02376420 2001-12-14
WO 00/77030 PCT/US00/16643
microencapsulated delivery systems, The active compound is included in the
pharmaceutically acceptable carrier in an amount sufficient to exert a
therapeutically
useful effect in the absence of undesirable side effects on the patient
treated. The
therapeutically effective concentration may be determined empirically by
testing the
compounds in known in vitro and in vivo model systems for the treated
disorder.
The compositions can be enclosed in ampoules, disposable syringes or multiple
or single
dose vials made of glass, plastic or other suitable material. Such enclosed
compositions
can be provided in kits.
The concentration of active compound in the drug composition will depend on
absorption, inactivation and excretion rates of the active compound, the
dosage schedule,
and amount administered as well as other factors known to those of skill in
the art.
1s The active ingredient may be administered at once, or may be divided into a
number of
smaller doses to be administered at intervals of time. It is understood that
the precise
dosage and duration of treatment is a function of the disease being treated
and may be
determined empirically using known testing protocols or by extrapolation from
in vivo or
in vitro test data. It is to be noted that concentrations and dosage values
may also vary
2o with the severity of the condition to be alleviated. It is to be further
understood that for
any particular subject, specific dosage regimens should be adjusted over time
according
to the individual need and the professional judgment of the person
administering or
supervising the administration of the compositions, and that the concentration
ranges set
forth herein are exemplary only and are not intended to limit the scope or
practice of the
2> claimed compositions.
If oral administration is desired, the compound should be provided in a
composition that
protects it from the acidic environment of the stomach. For example, the
composition can
be formulated in an enteric coating that maintains its integrity in the
stomach and releases
,0 the active compound in the intestine. The composition may also be
formulated in
combination with an antacid or other such ingredient.
42
CA 02376420 2001-12-14
WO 00/77030 PCT/US00/16643
Oral compositions will generally include an inert diluent or an edible carrier
and may be
compressed into tablets or enclosed in gelatin capsules. For the purpose of
oral
therapeutic administration, the active compound or compounds can be
incorporated with
excipients and used in the form of tablets, capsules or troches.
Pharmaceutically
compatible binding agents and adjuvant materials can be included as part of
the
composition.
The tablets, pills, capsules, troches and the like can contain any of the
following
ingredients, or compounds of a similar nature: a binder, such as, but not
limited to, gum
l0 tragacanth, acacia, corn starch or gelatin; an excipient such as
microcrystalline cellulose,
starch and lactose, a disintegrating agent such as, but not limited to,
alginic acid and corn
starch; a lubricant such as, but not limited to, magnesium stearate; a
gildant, such as, but
not limited to, colloidal silicon dioxide; a sweetening agent such as sucrose
or saccharin;
and a flavoring agent such as peppermint, methyl salicylate, and fruit
flavoring.
When the dosage unit form is a capsule, it can contain, in addition to
material of the
above type, a liquid carrier such as a fatty oil. In addition, dosage unit
forms can contain
various other materials which modify the physical form of the dosage unit, for
example,
coatings of sugar and other enteric agents. The compounds can also be
administered as a
2o component of an elixir, suspension, syrup, wafer, chewing gum or the like.
A syrup may
contain, in addition to the active compounds, sucrose as a sweetening agent
and certain
preservatives, dyes and colorings and flavors.
The active materials can also be mixed with other active materials which do
not impair
2~ the desired action, or with materials that supplement the desired action.
Solutions or suspensions used for parenteral, intradermal, subcutaneous, or
topical
application can include any of the following components: a sterile diluent,
such as water
for injection, saline solution, fixed oil, a naturally occurring vegetable oil
like sesame oil,
3o coconut oil, peanut oil, cottonseed oil, etc. or a synthetic fatty vehicle
like ethyl oleate or
the like, polyethylene glycol, glycerine, propylene glycol or other synthetic
solvent;
antimicrobial agents, such as benzyl alcohol and methyl parabens;
antioxidants, such as
ascorbic acid and sodium bisulfate; chelating agents, such as
ethylenediaminetetraacetic
4.
CA 02376420 2001-12-14
WO 00/77030 PCT/US00/16643
acid (EDTA); buffers, such as acetates, citrates and phosphates; and agents
for the
adjustment of tonicity such as sodium chloride or dextrose. Parenteral
preparations can be
enclosed in ampoules, disposable syringes or multiple dose vials made of
glass, plastic or
other suitable material. Buffers, preservatives, antioxidants and the like can
be
incorporated as required.
If administered intravenously, suitable carriers include physiological saline
or phosphate
buffered saline (PBS), and solutions containing thickening and solubilizing
agents, such
as glucose, polyethylene glycol, and polypropyleneglycol and mixtures thereof.
Liposomal suspensions, including tissue-targeted liposomes, may also be
suitable as
pharmaceutically acceptable carriers. These may be prepared according to
methods
known to those skilled in the art. For example, liposome formulations may be
prepared as
described in U.S. Pat. No. 4,522.811.
The active compounds may be prepared with earners that protect the compound
against
rapid elimination from the body, such as time release formulations or
coatings. Such
carriers include controlled release formulations, such as, but not limited to,
implants and
microencapsulated delivery systems, and biodegradable, biocompatible polymers,
such as
collagen, ethylene vinyl acetate, polyanhydrides, polyglycolic acid,
polyorthoesters,
polylactic acid and others. Methods for preparation of such formulations are
known to
those skilled in the art.
While this invention has been described with respect to various specific
examples and
embodiments, it is to be understood that the invention is not limited thereby
and should
only be construed by interpretation of the scope of the appended claims.
44
