Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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CYSTEINE PROTEASE INHIBITORS
Bac ,ground of the Invention
Numerous cysteine pretenses have been identified in biological systems. A
"protease" is
an enzyme which degrades proteins or peptides into smaller components. The
term "cysteine
protease" refers to pretenses which are distinguished by the presence of a
cysteine residue which
plays a critical rale in the catalytic process. Mammalian systems, including
humans, normally
degrade and process proteins via a variety of mechanisms including the actions
of cysteine
pretenses. However, when present at elevated levels or when abnormally
activated, or where
introduced into a biological system in the context of a viral, bacterial or
parasitic infection,
cysteine pretenses are thought to be involved in numerous pathophysiological
processes and
disease states.
For example, calcium-activated neutral pretenses ("calpains") comprise a
family of
intracellular cysteine pretenses which are ubiquitously expressed in mammalian
tissues. Three
major calpains have been identified: calpain I and II, and p94. The calpain
family of cysteine
pretenses has been implicated in many diseases and disorders, including
stroke,
neurodegeneration, such as Alzheimer's disease, amyotrophy and motor neuron
damage; acute
central nervous system injury, muscular dystrophy, bone resorption, platelet
aggregation,
cataracts and inflammation. Calpain I has been implicated in excitatory amino-
acid induced
neurotoxicity disorders including ischemia, hypoglycemia and epilepsy. The
cysteine protease
p94, a muscle-specific member of the calpain family, has been identified as a
gene product
responsible.for limb girdle muscular dystrophy (Barrett A.J., et al. ICOP
Newsletter, 1-2 (1996)).
Lysosomxl cysteine pretenses or cathepsins (including cathepsins B, C, H, L,
S, O and
02/K) belong to the papain superfamily of cysteine pretenses. They are widely
distributed and
differentially expressed among tissues. Intracellularly, they serve a variety
of digestive and
processing functions. Extracellularly, they may be involved in tissue
remodeling and in
pathologies such as arthritis, inflammation, myocardial infarction,
Alzheimer's disease, cancer
and muscular dystrophy (Elliott E., et al., Per. in Drug Disc. and Des., 6: 12-
32 (1996)).
Interleukin-1 (3 converting enzyme ("ICE'S is a member of the caspase family
of cysteine
pretenses which catalyzes the formation of interleukin-1 p (IL-1 (i), as well
as the formation of
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interferon-y inducing factor (IGIF) from their inactive precursors, proIL-1 ~3
and pro-IGIF,
respectively. Interleukin-lei is an immunoregulatory protein implicated in
inflammation,
diabetes, septic shock, rheumatoid arthritis and Alzheimer's disease. ICE
and/or other caspases
have also been linked to the apoptotic cell death of neurons which is
implicated in a variety of
neurodegenerative disorders including Parkinson's disease, ischemia and
amyotrophic lateral
sclerosis (ALS)(Dinarello C., et al., New Eng. J. Med, 328: 106-113 (1993)).
Cysteine proteases are also produced by various viral pathogens and appear to
be
involved in every stage of reproduction including DNA and RNA translation and
synthesis, and
capsid formation (Gorbalenya A., et al., Per. In Drug Disc., 6:64-86 (1996);
Krausslich et al.,
Ann. Rev. Biochem., 57:701-54 (1988)). Examples of viral pathogens include
Picornaviridae,
which includes the genera Enterovirus, Rhinovirus, Cardiovirus, and
Aphthovirus, which cause
numerous human disease syndromes, ranging from fatal paralysis, encephalitis,
meningitis,
hepatitis and myocarditis to the common cold (Krausslich et al., Ann. Rev.
Biochem., 57:701-54
(1988)). The picornaviral 3C proteinases, which are produced by all
picornaviruses, are
responsible for processing viral polyproteins, an essential stage in viral
growth (Malcolm B., et
al. Biochemistry, 34:8172-8179 (1995)).
In addition, parasitic cysteine proteinases play significant roles in host-
parasite
interactions and pathogenesis (Robertson C., et al., Pers. in Drug Disc. and
Des., 6:99-118
(1996)). For example, most of the proteinase activity detected in trypanosomes
and various
Leishmania species has been characterized as belonging to the cysteine
protease class. Other
proteases are produced by Clostridium histolyticum and malaria parasites, such
as Plasmodium
falciparum and Plasmodium vinckei strains, and Schistosoma.
Cancer procoagulant, CP, a cysteine proteinase from malignant cells, has
emerged as a
probable activator of the coagulation system in cancer (Alessio M.G., et al.,
Eur. J. Haematol,
45: 78-81 (1990); Gordon S., Methods in Enz., 244:568-581 (1994); Gordon S.,
Sem. in Thromb.
and Hemo., 18, 4:424-433 (1992)).
Existing cysteine protease inhibitors are primarily irreversible in nature;
only weakly
inhibit the enzymatic activity of the targeted protease and/or are toxic.
Thus, there is a need for
effective inhibitors of cysteine proteases as therapeutic and as prophylactic
agents for the
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treatment and/or prevention of cysteine protease mediated pathologies.
Summary ref the Invention
The present invention relates to cysteine protease inhibitors of the general
formula (I):
N-Y
Z~X~Ri
(I)
wherein Z is a cysteine protease binding moiety, Z being a carbonyl containing
group, preferably
an amino carbonyl containing group, wherein the carbon of the heterocycle is
attached directly to
the carbonyl group of Z.
In the above formula (I), R, is alkyl or alkenyl optionally substituted with 1-
3 halo or
hydroxy; alkylamino, dialkylamino, alkyldialkylamino; or cycloalkyl,
alkylcycloalkyl,
alkenylcycloalkyl, (Cs-C,~aryl, (Cs-C,~arylalkyl or (Cs-C,Z)arylalkenyl
optionally comprising 1-
4 heteroatoms selected from N, O and S, and optionally substituted with halo,
cyano, vitro,
haloalkyl, amino, aminoalkyl, dialkylamino, alkyl, alkenyl, alkynyl, alkoxy,
haloalkoxy,
carboxyl, carboalkoxy, alkylcarboxamide, (Cs-C6)aryl, -O-(Cs-C6)aryl,
arylcarboxamide,
alkylthio or haloalkylthio; and
X and Y are independently O, S or N, where N is optionally substituted with
alkyl or
alkenyl optionally substituted with 1-3 halo atoms; (Cs-C6)aryl, arylalkyl or
arylalkenyl
optionally comprising 1-3 heteroatoms selected from N, O and S, and optionally
substituted with
halo, cyano, vitro, haloallcyl, amino, aminoalkyl, diallcylamino, alkyl,
alkenyl, alkynyl, alkoxy,
haloalkoxy, carboxyl, carboalkoxy, alkylcarboxamide, arylcarboxamide,
alkylthio or
haloalkylthio; provided that at least one of Y or X is N. It will be
understood that where Y or X
is substituted nitrogen, both Y and X must be nitrogen.
In one embodiment, R, is methyl, dimethylamino, phenyl or benzyl optionally
substituted
with methyl, halo, methylenedioxy, methoxy, dimethoxy, trimethoxy,
trifluoromethyl and
dimethylamino.
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According to severai preferred embodiments, X is O and Y is N; X is N and Y is
O; or
both X and Y are N.
Typically Z comprises 1 to 5 amino acid residues or mimetics thereof. Thus, Z
may, for
example, comprise a pentapeptidyl, tetrapeptidyl, tripeptidyl or dipeptidyl
binding moiety.
According to a preferred embodiment, Z is of the formula (II):
R'
4 \
~5-'~4'~3-~2-~' I'
R4
(II)
wherein AA,, AAA, AA3, AA, and AAs are independently an amino acid residue or
amino acid
residue mimetic; a direct bond or absent; and
R, and R,' are independently -C(O)Rs, -C(O)NHRs, -S(O)iRs, -C(O)OR,, -CR, or
Rs,
where Rs is H, alkyl, alkenyl or alkynyl optionally substituted with halo,
cyano, vitro, haloalkyl,
amino, aminoalkyl, dialkylamino, haloalkoxy, carboxyl, carboalkoxy or
alkylcarboxamide;
cycloalkyl, alkyicycloalkyl, (Cs-C,Z) aryl or (Cs-C,2}arylalkyl optionally
comprising 1-4
heteroatoms selected from N, O and S, and optionally substituted with halo,
cyano, vitro,
haloalkyl, amino, aminoalkyl, dialkylamino, haloalkoxy, carboxyl, carboalkoxy,
alkylcarboxamide, alkyl, alkenyl; alkynyl or (Cs-C,Z)aryl; or absent; or
together R, and R4' form a
ring comprising 5-7 atoms selected from C, N, S and O. Typical terminal R,
groups include Cbz,
succinic acid derivatives of the formulas -C(O)CH(-CHZCH(CH3)~CHZCOOH, -
C(O)CHiCHiCOOH, and -C(O)CHZCHZC(O)OC(CH~)3; toluenesulfonyl, methane
sulfonyl,
FMOC, (t)-menthyloxy-CO- aad acetyl.
Preferably, the amino acids are selected from arginine or an arginine mimetic,
proline;
aspartic and glutamic acid and the aryl and alkyl esters thereof; alanine and
glycine optionally
substituted at the a-carbon or a-nitrogen with alkyl, cycloalkyl or aryl;
leucine, isoleucine;
cysteine optionally substituted at the sulfur atom with alkyl, alkenyl or
phenyl optionally
substituted with halo, cyano, vitro, haloallcyl, amino, aminoalkyl,
dialkylamino, alkyl, alkoxy,
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haloalkoxy, carboxyl, carboalkoxy, alkylcarboxamide, arylcarboxamide,
alkylthio or
haloalkylthio; phenylalanine, homo-phenylalanine, dehydro-phenylalanine,
indoline-2-carboxylic
acid; tetrahydroisoquinoline-2-carboxylic acid optionally substituted with
halo, cyano, vitro,
haloalkyl, amino, aminoalkyl, dialkylamino, alkyl, alkoxy, haloalkoxy,
carboxyl, carboalkoxy,
alkylcarboxamide, arylcarboxamide, alkylthio or haloallcylthio; tyrosine,
serine or threonine
optionally substituted with alkyl or aryl; tryptophan, histidine, methionine,
valine, norvaline,
norleucine, octahydroindole-2-carboxylic acid; asparagine, glutamine and
lysine optionally
substituted at the nitrogen atom with alkyl, alkenyl, alkynyl, alkoxyalkyl,
alkylthioalkyl,
alkylaminoalkyl, dialkylaminoalkyl, carboxyalkyl, alkoxycarbonyl alkyl or
cycloalkyl,
I O bicycloalkyl, cycloalkyl alkyl, bicycloalkyl alkyl or fused aryl-
cycloalkyl alkyl optionally
comprising 1 or more heteroatoms selected from N, O and S.
Alternatively, AA, is of the formula (IIIa):
N
O
2
(IIIa)
wherein X' is CRZ' or N; and
R2, Rz' and R~" are independently H; alkyl or alkenyl optionally substituted
with 1-3
halo, hydroxy, thin, allcylthio, amino, alkylamino, dialkylamino,
alkylguanidinyl,
dialkylguanidinyl, guanidinyl; -RCOR', -RCOOR', -RNR'R"R° or -
RC(O)NR'R" where R is
alkyl or alkenyl, and R', R" and R° are independently H, alkyl,
alkenyl, cycloalkyl or (Cs-
C6)aryl; or cycloalkyl, alkylcycloalkyl, alkenylcycloalkyl, alkyl-oxyaryl,
alkyl-thioaryl, (Cs-C,2)
aryl, (Cs-C,Z)arylalkyl or (Cs-C,~arylalkenyl optionally comprising 1-4
heteroatoms selected
from N, O and S, and optionally substituted with hydroxy, halo, cyano, vitro,
haloalkyl, amino,
aminoalkyl, dialkylamino, amidine, alkylamidine, dialkylamidine, alkyl,
alkenyl, alkynyl,
alkoxy, haloalkoxy, carboxyl, carboalkoxy, alkylcarboxamide, (Cs-Cs)aryl, -O-
(Cs-C6)aryl,
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arylcarboxamide, alkylthio or haloalkylthio; or
RZ and R2' together with X' form a ring comprising 4-7 atoms selected from C,
N, S and
O, said ring optionally subsitituted with hydroxy, halo, cyano, vitro,
haloalkyl, amino,
aminoalkyl, dialkylamino, amidine, alkylamidine, dialkyl amidine, alkyl,
alkenyl, alkynyl,
alkoxy, haloalkoxy, carboxyl, carboalkoxy, alkylcarboxamide, (Cs-C6)aryl, -O-
(Cs-C6)aryl,
arylcarboxamide, alkylthio or haloalkylthio.
AA2 may be a residue of the formula (IIIb):
R3
N~
R.,3 O
(IIIb)
IS or selected from a residue mimetic of formulas IV to XXIV:
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i 6 R6
I ..W N~.
( " ''~ n ,Y )m w' 0
N )m N I N.X»
Q O
p O ~ R3
(IV) (~ (vl)
R6
O G- R6 O G- R6
v I O i I N~ O N~ O
~. N.X,.X
p ~ I
O ~ R3 O R
(VIII)
(1X)
(VII) 3
R6
R6 O R6. R7 0 R,6
R
( "W~ O 7wN~ O R,7 O
N'X»~N~X» G~N~N~X»
!II
O R O I O I
R
(7C) 3 (7u) R3 (XII) 3
O R6 O R6 O
R'7 ~~ O V1~ y~ O V )" \N 1 /
I X» U N
G~N N,X,~ ~ O
0 R 0 0 X
O I 3 Ol
(7CIII) R3 (XIV) (XV) (7CVI)
_ R6 ~ / ~
\ / ~ \ I R6 O N
N R3 R3. ~ /
N ~'N~X/ N~X/ N O
'~ O X
(XVII) O (XViI1) 0
R6 0
\ / \ I O / ~
N R N O
N,X/R3 ~N~X~ 3 I N'X»N~NwX»
'IO
O 0 O R O
(X7q)0 (X)QI) (X?QII) 3 (7CXIV) ~3
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wherein X" is CR'3 or N;
R3, R', and R"3 are independently H; alkyl or alkenyl optionally substituted
with 1-3
halo, hydroxy, thio, alkylthio, amino, alkylamino, dialkylamino,
alkylguanidinyl,
dialkylguanidinyl, guanidinyl; -RCOR', -RCOOR' or -RC(O)NR'R" where R is alkyl
or alke~nyl,
and R' and R" are independently H, alkyl, alkenyl, cycloalkyl or {Cs-C6)aryl;
or cycloalkyl,
alkylcycloalkyl, alkenylcycloalkyl, alkyl-oxyaryl, alkyl-thioaryl, (Cs-C,2)
aryl, (CS-C,i)arylalkyl
or (Cs-C,Z)arylalkenyl optionally comprising 1-4 heteroatoms selected from N,
O and S, and
optionally substituted with hydroxy, halo, cyano, vitro, haloalkyl, anuno,
aminoalkyl,
dialkylamino, amidine, alkylamidine, dialkylamidine, alkyl, alkenyl, alkynyl,
alkoxy,
haloalkoxy, carboxyl, carboalkoxy, alkylcarboxamide, (CS-C6)aryl, -O-(Cs-
C6)aryl,
arylcarboxamide, alkylthio or halaalkylthio;
m is 0, 1 or 2;
n is 0, 1 or 2;
G is -C(O)-, -NHC(O)-, -S{O)2-, -OC{O)-, -C- or a direct bond;
R6, R,, R'6, R'~ are independently H, alkyl, alkenyl, halo, alkoxy, carboxyl,
carboallcoxy,
amino, aminoalkyl, dialkylamino; cycloalkyl, (Cs-C6) aryl or (Cs-C6) arylalkyl
optionally
comprising 1-3 heteroatoms selected from N, O and S, and optionally
substituted with alkyl,
alkenyl, alkynyl, halo, cyano, vitro, haloalkyl, haloalkoxy, amino,
alkylamino, dialkylamino,
alkoxy, haloalkoxy, carboxyl, carboalkoxy, alkylcarboxamide, alkyithio,
guanidine,
alkylguanidine, dialkylguanidine, amidine, alkylamidine or dialkylamidine; and
U, V, W and Y' are independently or together N, C, C(O), N(Rg) where R.g is H,
alkyl,
halo, alkoxy, carboalkoxy, cycloalkoxy, carboxyl, alkylthio, amino,
alkylamino, dialkylamino, or
aryl, fused aryl or cycloalkyl optionally comprising 1 or more heteroatoms
selected from O, S
and N, and optionally subsituted with halo or alkyl; N(R,o) where R,o is H,
alkyl, alkenyl or
cycloallcyl, aryl, arylalkyl or fused aryl-cycloallcyl optionally comprising 1-
4 heteroatom~
selected from N, O and S, and optionally substituted with alkyl, alkenyl,
alkynyl, halo, cyano,
vitro, haloalkyl, haloalkoxy, amino, alkylamino, diatkylamino, alkoxy,
haloalkoxy, carboxyl,
carboalkoxy, alkylcarboxamide, alkylthio, guanidine, alkylguanidine,
dialkylguanidine, amidine,
alkylamidine or dialkylamidine; or C(R")(R,~ where R" and R,2 are
independently or together
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H, alkyl, alkythio, alkythioalkyl or cycloalkyl, alkylcycloalkyl, phenyl or
phenyl alkyl optionally
subsituted with guanidine, carboalkoxy, hydroxy, haloalkyl, alkylthio,
aikylguanidine,
dialkylguanidine, amidine, alkylamidine or dialkylamidine.
In a preferred embodiment, X' and X" are C, and R'Z and R'3 are H.
In another embodiment, X' and/or X" are N.
Where Z is a calpain binding moiety, preferably R~ is benzyl optionally
substituted with
alkoxy; HzNC(--'NH2)NHCHzCH2CHZ-; -R'-C(=+NHZ)NHZ; -R'-NHC(--'NR")NR°;
or -R'-
NR"R° where R' is cycloalkyl, aryl or arylalkyl optionally substituted
with one or more
heteroatoms selected from N, S or O; and R" and R° are alkyl or
cycloalkyi; or CH3SCHzCH2-,
HOOC(CHz),CH2-, cyclohexyl-CHZ-, imidazolyl-CHZ, benzyl optionally substituted
with OH or
-O-benzyl, _(CH,)iCHCHz-, (CH,)zCH-, CH3CHZCHZ- or CH3(CHZ)~CHz-; and R, is -
CH,-benzyl,
benzyl, (CH,),C-, (CH3),CCHZ-, (CH3)ZCH-, CH~(CHZ)ZCHZ-, CH~CHzCH(CH3)- or
(CH3)zCHCH,-. Preferably, Rs is benzyl, isoquinolinyl, quinolinyl, naphthyl or
HOOCCH~C(CH~CH(CH3)Z)-; or R, is Cbz wherein the phenyl is optionally
substituted with
vitro. Additionally, R, may be toluenesulfonyl, methanesulfonyl, FMOC or (+)-
menthyl-
oxy-CO-.
In one embodiment, AA3 is leucine, AA4 and AA3 are direct bonds or absent, and
Rs is
alkyl.
Several particular embodiments include those where Z is
R~-Leu-Leu-Leu-
R4-Leu-Leu-;
R,~Leu-Leu-Phe-;
R,-Leu-Abu-;
R4-Val-Phe-;
R,-Leu-Leu-Nle-;
R,-Ala-t-BuGly-Val-
R~-t-BuGly-Val-
R~-Leu-Leu-Met-; or
R,-Leu-Nle-.
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Preferably, Z is Cbz-Leu-Nle-; or Cbz-Leu-Val-.
Z may also be a cysteine cathepsin binding moiety, where preferably RZ is CHI-
,
(CH3)ZCH-, (CH,)zCHCHz-, CH,(CHz)ZCHz-, HZNC(--'NHZ)NHCH~CHZCHz-; -R'-
C(--'NHZ)NHz; -R'-NHC(='NR")NR°; or -R'-NR"R° where R' is
cycloalkyl, aryl or arylallcyl
S optionally substituted with one or more heteroatoms selected from N, S or O;
and R" and R° are
alkyl or cycloalkyl; benzyl or -CHZ-benzyl optionally substituted with OH or -
OR' where R' is
alkyl or aryl; CH,CH(-O-benzyl)- or benzyl-S-CHi-; and R, is H, (CH3)iCH-,
(CH3)iCHCHZ-,
CH,(CHZ)zCH2-, HZN{CH~)3CH~-, H,N(CH2)zCH~-, HZNC(='NHZ}NHCH2CHZCH2-; -R'-
C(='NH,)NH2; -R'-NHC(='NR")NR°; or -R'-NR"R° where R' is
cycloalkyl, aryl or arylalkyl
optionally substituted with one or more heteroatoms selected from N, S or O;
and R" and R° are
alkyl or cycloalkyl; benzyl, benzyl substituted with hydroxy and halo; or
(naphthyl)-CHz-.
In one embodiment, Z is a cathepsin B binding moiety, where preferably, RZ and
R3 are
independently benzyl, -CHZ-benzyl, HzNC(='NHZ)NHCHZCHzCHz-; -R'-C{='NH~NHZ; -
R'-
NHC(='NR")NR°; or -R'-NR"R° where R' is cycloalkyl, aryl or
arylalkyl optionally substituted
with one or more heteroatoms selected from N, S or O; and R" and R° are
alkyl or cycloalkyl;
H~N(CHZ)3CH~- or HzN(CH~)2CHz-; and preferably AA3 is Ile, Leu, absent or a
direct bond.
According to a particular embodiment, -AAZ-AA,- are selected from:
-Phe-hPhe-;
-Arg-hPhe-;
-Arg mimetic-hPhe-;
-Leu-hPhe-; and
-Onn-hPhe.
Z may be a cathepsin L binding moiety, where R3 is preferably benzyl or
(CH3~CHCHi-;
and R2 is -CHZ-bcnzyl.
Where Z is a cathepsin S binding moiety; preferably RZ and R3 are alkyl; more
preferably
(CH,)ZCH-, (CH3~CHCHZ- or CH,(CH~ZCHZ-.
In another embodiment, R, is benzyl, (CH3)zCHCHZ- or (CH3~CH-; and R2 is -CHZ-
benzyl. According to one particular embodiment, AA3, AA, and AAf are direct
bonds or absent;
R, is benzyl, isoquinolinyl, quinolinyl, naphthyl or HOOCCHZC(CHiCH(CH3)2)-;
or R, is Cbz.
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Where Z is a cathepsin H binding moiety; Z is preferably
R,-hPhe-; or
HCl~hPhe-.
Z may also be a cathepsin K binding moiety; where preferably R3 is benzyl,
(CH3)zCHCHz- or (CH~~CH-; and preferably AA3 is Gly; and AA, is Val or D-Val.
In another embodiment,
AA, is Arg, Arg mimetic or hPhe;
AAZ is Pro;
AA, is Gly; and
AA4 is VaI or D-Val; or
preferably Z is
R,-Pro-AA,-;
R4-Gly-Pro-AA,-;
R4-Val-Gly-Pro-AA,-;
1 S D-Val-Gly-Pro-AA,-; or
R,-D-Val-Gly-Pro-AA,; where
AA, is Apa, Arg or Arg mimetic, or hPhe.
Other embodiments include compounds where Z is
R,,-AA3-Leu-hPhe-;
R,-AAA-Phe-hPhe-; or
R,-AA3-Val-hPhe-;
where AA3 is Gly, Val, D-Val, a direct bond or absent.
Where Z is a caspase binding moiety; preferably RZ is -RCOOR'; where
preferably R is
-CHi- and preferably R' is H; where preferably AAA and AAA are amino acid
residues and AAs is
a direct bond.
Where Z is an interleulcin-1 ~i converting enzyme binding moiety; AA4 may be
optionally
substituted tyrosine or leucine; AA, may be valine, glutamate or an ester of
glutamate; and R3
may be -CH3 or (CH3)ZCH-.
In another embodiment, R3 is -CH3 or imidazolyl-CH2-; AA3 is valine or
glutamate; and
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R, is -CHI.
Z may also be Ry-AAS-AA4 AAA-Pro-AA,; where AA, is Asp or Asp ester; where -
AAs-
AA,-AA,- may be -Ala-; -Glu-; -Val-; -Tyr-Ala-; -Tyr-Glu-; -Tyr-Val-; -Leu-Ala-
; -Leu-GIu-; or
-Leu-Val-.
In yet a further embodiment where Z is an interleukin-1 p converting enzyme
binding
moiety, AA, is of the formula (VI);
wherein X" is CR'3 where preferably R'3 is H; and
R, is -RCOOR' where R is alkyl or alkenyl, and R' is H, alkyl, alkenyl,
cycloalkyl or (Cs-
C6) aryl. In another, AA, and AAs are direct bonds or absent, AA3 is Tyr or
Tyr(O-R') or a direct
bond or absent; RZ is -RCOOR' where R is alkyl or alkenyl, and R' is H, alkyl,
alkenyl,
cycloalkyl or (Cs-C6) aryl; R6 is phenyl or benryl substituted with halo; and
Rs may be benzyl,
isoquinolinyi, quinolinyl, naphthyl or HOOCCHiC(CH,CH(CH3)z)-.
Where Z is a YAMA binding moiety, preferably Rz is -RCOOR' where preferably R
is
-CHI- and AA4 is Asp or an ester thereof. In another embodiment, AA3 is
optionally substituted
glutamine or glutamic acid or an ester thereof. In yet another embodiment RZ
is (CH3)2CH- or
CH,SCHzCH2-.
Where Z is a FLICE binding moiety, preferably Ri is -RCOOR', where preferably
R is
-CHz-; AAQ is optionally substituted lysine; and preferably AA3 is glutamic
acid; and R3 is
(CH,)ZCH-.
Z may also be a viral or microbial cysteine protease binding moiety. In one
embodiment,
Z is a gingipain binding moiety. Where Z is a gingipain K binding moiety; RZ
is preferably
-RNR'R"R° where preferably R is (C,-C,)alkyl; R' is H; and preferably
R" and R° are H or (C,-
C~)alkyl. In one embodiment RZ is 'H3N(CHZ)3CH2-. Where Z is a gingipain R
binding moiety,
preferably Ri is HzNC(='NH~NHCHZCHZCHz-; -R'-C(=iNH~NHZ; -R'-
NHC(=*NR")NR°; or
-R'-NR"R° where R' is cycloalkyl, aryl or arylalkyl optionally
substituted with one or more
heteroatoms selected from N, S or O; and R" and R° are alkyl or
cycloalkyl.
According to one embodiment, AAz is proline, where Z is R,-Leu-Pro-AA,-, where
AA,
is arginine or an arginine mimetic.
Z may also be a human coronavirus protease binding moiety, where preferably Ri
is
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HzNC(='NH~)NHCH~CHzCH2-; -R'-C(='NHZ)NH2; -R'-NHC(='NR")NR°; or -
R'-NR"R°
where R' is cycloalkyl, aryl or arylalkyl optionally substituted with one or
more heteroatoms
selected from N, S or O; and R" and R° are alkyl or cycloalkyl; and
preferably R3 is (CH3)zCH-,
{CH,)ZCHCH~- or CH3(CHZ)iCHz-; AA3 is Asp or ester thereof, Leu, Arg or Arg
mimetic, or
direct bond; AA, and AAs are direct bonds or absent; and Rs is alkyl.
Where Z is a hepatitis A virus 3C proteinase binding moiety, RZ is preferably
-RC(O)NR'R" where R' and R" are H or -CH,; or RCOOR' where R' is CH,; and AAA
and AA4
are amino acid residues. Preferably, AA4 is Leu; R~ is -CH3 and AA3 is Ala.
Z may also be a hepatitis A virus 3C proteinase binding moiety, where Z is
R4-Leu-AA3-Thr-Gln-;
R,-Trp-AA3-Thr-Gln-;
R4-Val-AA3-Thr-Gln-;
R,-Ile-AA,-Thr-Gln-; or
R~-D-Leu-AA,-Thr-Gln-;
where AA3 is Arg or Arg mimetic.
Where Z is an Ad2 23K protease binding moiety, RZ and R3 are preferably H; AA3
is
alanine; AA, is leucine; AAs is a direct bond; and R, is absent.
Where Z is a human rhinovirus 3C protease binding moiety, preferably RZ is
RCOOR'
where R is -CHi-; R3 is benzyl; and AA3 is leucine, isoleucine or a direct
bond.
In yet a further embodiment, RZ is -RC(O)NR'R" where R' and R" are H, -CH3 or -
CHZCH3; or RCOOR' where R' is -CH; or -CHzCH3.
Z may also be a human picornain 2A protease; where R3 is -CH(OR')CH3 where R'
is H,
alkyl or aryl; and preferably Ri is a hydrophobic side chain. Alternatively,
AA, is Val or
dehydro-Phe; AAz is Pro; and AA3 is Val. Examples include compounds CE-2072,
CE-2060 and
CE-2061, the structures of which are shown below.
In another embodiment, Z is
R4-Ala-Ala-Pro-Val-; or
R,-Ala-Ala-Pro-Ala-.
Additionally, Z may be a protozoan protease binding moiety, such as a
Trypanosoma,
13
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WO 99/54317 PCT/US99/08501
Leishmania or Schistosoma protease binding moiety. The protease may be a
cathepsin L- or
cathepsin B-like protease. In one embodiment, Rz is HZN(CHZ)3CHi-,
HZNC(=TNH,)NHCHZCH2CHz-; -R'-C{--'NHZ)NH2; -R'-NHC(--+NR")NR°; or -
R'-NR"R°
where R' is cycloalkyl, aryl or arylalkyl optionally substituted with one or
more heteroatoms
selected from N, S or O; and R" and R° are alkyl or cycloalkyl; -CH,-
benzyl or benzyl
optionally substituted with OH; and preferably R, is benzyl, (CH3)ZCHCHZ- or
(CH3)ZCH-; and
AA3 is Phe, Leu, Pro or a direct bond. In one example, R, is Boc or Suc.
Z may be also selected from -Pro-Phe-Arg-; -Phe-Arg-;-Val-Leu-Lys-; -Leu-Val-
Tyr-;
Suc-Leu-Tyr- or -Phe-Ala-.
Where Z is a Plasmodium protease binding moiety, preferably R2 is (CH~)iCH-, -
CHZ-
benzyl, benzyl or phenyl optionally substituted with hydroxyl; HZNC(--
'NHZ)NHCHzCHzCH2-;
-R'-C(--'NHz)NH=; -R'-NHC(--'NR")NR°; or -R'-NR"R° where R' is
cycloalkyl, aryl or
arylalkyl optionally substituted with one or more heteroatoms selected from N,
S or O; and R"
and R° are alkyl or cycloalkyl; or -R'-N(R")(R°) where R' is
alkyl, and R" and R° are alkyl or
cycloalkyl; or alkylimidazoyi; and R3 is benzyl, (CH3)ZCHCHz-, (CH,)ZCH-,
HOCHz- or
-CH20R'.
In one embodiment, Z is
R,-Phe-Arg-;
R,-Phe-(arginine mimetic)-;
R4-Phe-Lys-;
R,-Leu-hPhe-;
R,,-Val-Leu-Arg-;
R4-Phe(e-Z)-Lys-;
R,-Val-Leu-(Arg mimetic)-
R,-Phe-Val-; or
R,-Phe-Ser(OBzI)-.
In another embodiment, Z is
R,-Phe-AA,-; or
R,-Leu-AA,-;
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WO 99/54317 PCT/US99/08501
where AA, is optionally substituted lysine; and where R, may be morpholino. In
a further
embodiment, AA3, AA4 and AAs are direct bonds or absent, and R, is Cbz.
The present invention also provides methods of inhibiting the enzymatic
activity of one
or more cysteine proteases comprising contacting a protease with an inhibitory
amount of a
compound described herein.
Preferably the compound is selected from
[2-[5-(3-methylbenzyl)-1,3,4-oxadiazolyl]carbonyl]-2-(S)-methylpropyl]-L-
phenylalanamide-(3R)-(isobutyl)succinic acid;
Acetyl-L-leucyl-N-[1-[2-[5-phenyl]-1,3,4-oxadiazolyl]carbonyl]-4-(guanidino)-
butyl-L-
leucyl amide;
Acetyl-L-leucyl-N-[1-[3-[5-methyl]-1,2,4-oxadiazolyl]carbonyl]-ethyl-L-leucyl
amide;
Acetyl-L-leucyl-N-[1-[3-[5-methyl]-1,2,4-oxadiazolyl]carbonyl]-4-(guanidino)-
butyl-L-
leucyl amide;
Acetyl-L-tyrosinyl-L-valyl-N-[1-[2-[(5-phenyl)-1,3,4-oxadiazolyl]carbonyl]-2-
carboxy-
ethyl]-L-alanine amide;
Acetyl-L-Aspartyl-Valyl-N-[1-[2-[(5-phenyl)-1,3,4-oxadiazolyI] carbonyl]-2-
(carboxy)-
ethyl]-L-glutamyl amide;
(Benzyloxycarbonyl)-L-valyl-N-[I-(2-[5-(3-methylbenzyl)_1,3,4-
oxadiazolyl]carbonyl)-
2-(S)-methylpropyl]-L-prolinamide;
(t-butoxysuccinyl)-L-valyl N-[1-[3-[5-(3-trilluoromethylbenzyl)-1,2,4-
oxadiazolyl]carbonyl]-2-benzylidone]-L-prolinamide; and
Carboxysuccinyl-L-valyl-N-[1-[3-[5-(3-trifluoromethylbenzyl)-1,2,4-
oxadiazolyl]carbonyl]-2-benzylidone]-L-prolinamide.
The present invention also provides a method of inhibiting cancer cell growth
or tumor
progression or tumor metastasis or invasion, by inhibiting the enzymatic
activity of cysteine
proteases associated with such growth or progression, such as cathepsin B or
cathepsin L.
Further provided is a method of inhibiting microbial cell or viral growth or
reproduction
by inhibiting the enzymatic activity of cysteine proteases associated with
such growth or
reproduction. Suitable pathogenic targets include, by example only, hepatitis
A virus 3C
CA 02329712 2000-10-20
WO 99/54317 PCTNS99/08501
proteinase, hepatitis C virus endopeptidase 2, piconnain 3C rhinovirus
protease,
encephalomyelitis virus endopeptidase 2 and picornain 2A protease.
The present invention also provides a method of treating the symptoms
associated with
allergic responses by inhibiting the enrymatic activity of cysteine proteases
associated with
certain allergens, such as, for example Der p I.
The invention provides a method of treating the symptoms associated with
neurodegenerative disorders, such as Alzheimer's.disease, Parkinson's disease,
multiple
sclerosis. The invention further provides a method of treating the symptoms
associated with
stroke.
Further provided is a method of treating the symptoms associated with
inflammatory and
degenerative diseases, such as arthridities, including rheumatoid arthritis or
osteoarthritis, or
periodontal disease.
As used herein, the term "cysteine protease binding moiety" means a chemical
group
capable of binding to the substrate binding site of a cysteine protease,
typically defined in the
literature as the S,-S" site. The term includes both peptides and peptide
mimetics. Preferably,
the binding moiety is selected such that when linked to the keto-heterocycle,
the moiety provides
the resulting compound with inhibitory activity against the target cysteine
protease of less than
100 wM (K; value); and more preferably of less than 10 ~M.
As used herein, the term "optionally substituted" means, when substituted,
mono to fully
substituted.
As used herein, the term "independently" means that the substituents may be
the same ar
different.
As used herein, the term "alkyl" means C,-C,s, however, preferably C,-C,.
As used herein, the term "alkenyl" means C,-C,s, however, preferably C,-C,.
As used herein, the term "alkynyl" means C,-C,s, however, preferably C,-C.,.
It will be understood that alkyl, alkenyl and alkynyl groups, whether
substituted or
unsubstituted, may be linear or branched.
As used herein, the term "aryl," unless otherwise stated, means aryl groups
preferably
comprising 5 to 12 carbons, and more preferably 5 to 6 carbons. Unless
otherwise indicated, the
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WO 99/54317 PCT/US99/08501
term aryl includes mono-and bi-cyclic, as well as fused ring systems. As used
herein, the term
"arylalkyl" includes mono-substituted alkyl groups (e.g., benzyl), as well as
di-substituted alkyl
groups such as -alkyl(phenyl)z (e.g., -CH(phenyl)Z). As used herein, where the
term "arylalkyl"
or "arylalkenyl" is defined by the general formula (Cx Cy)arylalkyl or (Cx
CY)arylalkenyl, x and y
refer to the number of carbons making up the aryl group. The alkyl group is as
defined above.
As used here, the term "arylalkenyl" includes aryl compounds having an alkenyl
chain
comprising 1-3 or more double bonds. Exemplary arylalekenyl groups include =CH-
CHZ-aryl
and -CH=CH-aryl, where aryl is preferably phenyl.
As used herein, the term "arginine mimetic" means an amino acid residue with a
side
chain substituent of the formula -R'-C(--'NH,)NHz; -R'-NHC(--'NR")NR°;
or -R'-NR"R° where
R' is cycloalkyl, aryl or arylalkyl optionally substituted with one or more
heteroatoms selected
from N, S or O; and R" and R° are alkyl or cycloalkyl.
As used herein, the term "Cbz" means benzyloxycarbonyl; and the term "Mu "
means
morpholino.
Pharmaceutically acceptable salts of the compounds described above are within
the scope
of the invention.
Figure 1 shows the inhibition of the production of mature IL-1 p in THP-1 cell
line by
certain compounds of the present invention.
Figures 2A and 2B show the inhibition of the production of mature IL-1 ~i in
whole blood
by certain compounds of the present invention.
Figure 3 is a schcmadc representation of the synthesis of a compound according
to the
invention (CM-0019).
The present invention provides compounds which are useful as cysteine protease
inhibitors. These compounds are characterized by their relatively low
molecular weight,
reversible inhibition, high potency and selectivity with respect to various
types of cysteine
17
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WO 99/54317 PCT/US99/08501
proteases. The compounds can be implemented to prevent, alleviate and/or
otherwise treat
diseases which are mediated by the effects associated with the presence of
cysteine proteases.
Their usage is of particluar importance as they relate to various human
treatments in vivo and as
well as diagnostic tools in vitro.
Peptidyl inhibitors of serine proteases comprising serine protease binding
moieties and
certain keto-heterocycles have been previously described (see WO 96/16080). It
has been
surprisingly found that compounds comprising cysteine protease binding
moieties and these
keto-heterocycles are highly potent and specific inhibitors of a wide variety
of cysteine proteases
as well. The inhibiting activity can be directed against any cysteine protease
by identifying the
binding moiety specific for that protease. The characteristics for the P, . .
. P~ residues (using
substrate nomenclature by Schechter and Berger (Biochem. Biophys. Res. Commun.
27: 157
{1967); Biochem. Biophys. Res. Commun. 32: 898 (1968) ), which define the
minimum
recognition sequence of enzymes for small synthetic peptide substrates or
inhibitors are known
for many enzymes or can be determined by measuring rates of hydrolysis of
various substrates.
Some examples are listed in Table 1.
Table 1. Cvsteine proteases and exemplary recognition elements.
Cysteine P 1 P2 Other Reference
Protease
Calpain I large hydrophobicLeu, bulky 18
and II
e.g. Nva, aliphatic,
Phe, Abu hPhe
Calpain I Arg or Arg- t-butyl-Gly, 38
Leu,
mimetic, Vat, hPhe
Lys, Tyr,
Val, NIe,
Tyr(O-
Bzl), Leu,
Abu, Phe
Papain hPhe, Arg bulky, non-polar, 22
or its
mimetics, Phe
Agly,
Aala
Cathepsins bulky hydrophobicVal, Leu 18, 21, 22,
25
Cysteine residues
like hphe,
(in general)Phe, Met,
Abu,
Nva, or Arg
and its
m imetics
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WO 99/54317 PCT/US99/08501
Cathepsin hPhe, Phe, Phe, Arg P3-large 42
B Tyr, or its
Ser(OBzI), mimetics, hydrophobic
Leu, Tyr,
Thr(OBzI), Np2, Lys, aromatic,
Ornithine Ile
Cys(SBz1),
Arg or
its mimetics,
Gly
Cathepsin Val, Nle, Leu, Phe, 42, 43
S hPhe, Phe Val
Cathepsin hPhe, Lys Phe, Leu 42
L
Cathepsin Arg or its Pro, Leu, P3 - Leu 44
K mimetics, Phe, Val
hPhe, Leu
Cathepsin Arg or its
H mimetics,
hPhe
Caspases Asp P4 - determines22, 40
the
specificity
within
the caspase
family
Interleukin-lAsp P3 - Val, 22, 36, 41,
p Glu or 45, 48
converting ester thereof
enzyme
P4 - Tyr;
Leu
Caspase 3 Asp P4 - Lys 40, 41
(YAMA)
Caspase 8 Asp Val P3 - Glu 40, 41
(FLICE)
P4 - Asp
Picomain Gln or its Phe, Gly P3 - Ile 9
3C
derivatives P4 - small
(e.g.
dimethylGln, hydrophobic
Azogln), residues
Glu and
its derivatives
Human RhinovirusGln or its Phe P3 - Leu 8
3C protease derivatives
(e.g.
dimethylGln,
Azogln),
Glu and
its derivatives
Hepatitis Gln or its Ala, Val, P3 - Arg 14
A Virus Leu, Nle, or iu
3C proteinasederivativesPhe mimetics,
(e.g. Ala
dimethylGln, P4 - hydrophobic
Azogln); residues
Glu and
its derivatives
Human CoronaArg or its Val, Leu, P3 - Asp 1
mimetics Nle or its
Virus protease esters
Hepatitis Leu Leu P3 - Arg 8
C Virus or its
endopeptidase mimetics
2
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WO 99/54317 PCT/US99/08501
Ad2 23K proteaseGly Gly P3 - Ala, 8
P4 - Leu
Trypanosome,Arg or its Phe, Leu, P3 - Pro 23
mimetics, Vat ,Val, Leu
Leishmania Lys, Tyr,
Aia
protease
Picornain Gln or its Thr, Gly, P3 - Ala 9, 46
2A Pro
derivatives
(e.g.
dimethylGln,
Azogln),
Glu and
its derivatives,
Tyr,
Val, Ala
Gingipain Lys, Om 30, 47
K
Gingipain Arg or its Pro, Leu P3 - Leu 30
R mimetics
Malaria! hPhe, Arg large hydrophobicP3 - Val 23a
or its
hemoglobinasemimetics, residues,
Lys, Val, e.g. Phe,
Ser(OBzI), Leu
InnNva,
Tyr
Nva=norvaline; Abu=a-aminobutyric acid; Agly=azaglycyl; Aala=azaalanyl; Np2=2-
naphthylalanine; Nle=norleucine; Eps=epoxysuccinyl
In addition to altering the binding moiety Z, the substituent on the
heterocycle {i.e., R,)
can be varied to further increase the specificity of these compounds toward
the desired cysteine
protease.
By way of example, the compound CM-0019 comprises the binding moiety specific
for
papain and a substituted 1,3,4-oxadiazole:
w
I
0 ~ o
H NH N-N
0
O CH3 O ~CH3 i
H3C I
CH3 w
H3C
CM-0019
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WO 99/54317 PCT/US99/08501
By way of further example, the compounds CQ-0010 and CQ-0011 inhibit caspases.
Compounds CQ-0002 and CQ-0008 are analogs of leupeptin, the structure of which
is provided
below for comparison.
CH3 CH3
O / 1CH3 O O ! 'CH O
_ 3
H3 NH NH ~ ~ H3C NH ~ 'NH
NH~ / ~ NH~ H
p CH3 'Of O ~ ~ O CH3 ''O
H3 H3
CQ-0002 NH H Leupeptin NH~
NHx
Compounds CQ-0004, 0008, 0010 and 0011 are represented below:
CH3
O "CH3 O N_O , OH
H3C NH NH NH y ~ CH
N CH3 O v p 3 O
O CH3 O NH ~ N-N
H3C~NH NH ~ ' /
CH3
O CH O O
H3C~ 3 ~O
CQ-0010 OH
p CH3 O
N-O
H3C\'NH ~~NH / ~ HO H O
~'N CH3
O CH3 O O
O O O _
CH3 ~ NH H3 NH NH NH NH
C~.ooo8 ~ O J~ O
H3~ CH3
OH
CQ-0OI I
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WO 99/54317 PCTNS99/08501
Other specific inhibitors include compounds CE-2072, CE-2060 and CE-2061,
which
have shown inhibitory activity against picornain 2A protease (100% inhibition
at 100 pM):
' o3C CH H3C CH3 -
N
O~NH
O NH O
O
O /
CE-2072
CH3
/
H3C CH3
O
H3 O ~~ N I N-O
NH /
H3C~ O NH N
CH3 O O o
/
1$ CE-2060
CFy
H3C CH3
O
HO ~ N
i
0 0 ~ N
0 0
/
CE-2061
CFy
The compounds of the present invention, salts thereof, and their intermediates
can be
prepared or manufactured as described herein or by various methods known in
the chemical art,
as well as by extension and modification of methods previously described (see
WO 96/16080,
incorporated herein by reference).
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WO 99/54317 PCT/US99/08501
An alternative method has been used where suitably protected peptides are
converted by
the action of an activating coupling reagent such as BOP-CI or HBTU to a
Weinreb amide. The
Weinreb amide is then reacted with a 5-substituted 2-lithio-1,3,4-oxadiazole
at appropriate
temperatures ranging from -78 °C to ~25 °C in a suitable solvent
such as THF or ether to provide
the desired keto-oxadiazoles in a single step. Protecting groups, if present,
are then removed to
provide the enzyme inhibitors in an efficient and convergent manner. A number
of effcient
methods to synthesize 5-substituted I,3,4-oxadiazoles are known in the art.
Conveniently, these
compounds can be synthesized in a single step by refluxing hydrazides of
common carboxylic
acids with excess ethyl orthoformate at high temperature. The excess
orthoformate is hydrolyzed
in the workup and the 5-substituted 1,3,4-oxadiazoles are often obtained in
essentially pure form
without further purification necessary. This entire method of synthesis is
illustrated in general
form in scheme 1 below. Instances where Rz correlates to the amino acid side
chains of aspartic
acid, arginine, and alanine are provided in the Examples.
IS
Scheme 1.
o R~ He~ru o R, cH,
~OH 01EA ~ N
AAn N ~'I( ~ AAn N ~OCH~
1
R~z O H.~.1V+ R~z O
H ~ 1 OCH~
~N~ t. Ethyl orthofarmate o ~R
I Ri
Li
O 2. n-Bu-Li -78 C
N~--R
AAn N~O
R~z 1fO
23
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WO 99/54317 PCTNS99/08501
where AA" means AAZ . . . AAs.
The compounds described herein are useful in inhibiting the activity of
cysteine
proteases, by contacting the compound with the targeted protease, either in an
in vivo or an in
vitro environment. As used herein, the term "contacting" means directly or
indirectly causing the
inhibitor and the protease to come into physical association with each other.
Contacting thus
includes physical acts such as placing the inhibitor and protease together in
a container, or
administering the inhibitors to a patient. Thus, for example, administering a
compound of the
invention to a human patient evidencing a disease or disorder associated with
abnormal and/or
aberrant activity of such proteases in a method for inhibiting the enzymatic
activity of such
proteases which are associated with disease or disorder, falls within the
scope of the definition of
the term "contacting."
Pharmaceutically acceptable salts of the cysteine protease inhibitors also
fall within the
scope of the compounds as disclosed herein. The teen "pharmaceutically
acceptable salts" as
used herein includes organic and inorganic acid addition salts such as
chloride, acetate, maleate,
fumarate, tartrate and citrate. Examples of pharmaceutically acceptable metal
salts are alkali
metal salts such as sodium salt or potassium salt, alkaline earth metal salts
such as magnesium
salt and calcium salt, aluminum salt and zinc salt. Examples of
pharmaceutically acceptable
ammonium salts are ammonium salt, trishydroxymethylaminomethane and
tetramethylammonium salt. Examples of pharmaceutically acceptable amino acid
addition salts
are salts with lysine, glycine and phenylalanine.
Cysteine proteases which may be inhibited by the compounds described herein
include
mammalian, bacterial, parasite, viral, fungal, insect and plant cysteine
proteases. Cysteine
proteases include papain, actinidain, aleurain (barley), allergen
(Dermatophagoides), allergen
(Euroglyphus), ananain (Arranas comosus), asclepain (Asepias syriaca),
bleomycin hydrolase,
calotropin (Calotropis), caricain, clostripain, cathepsin B, cathepsin H,
cathepsin L, cathepsin S,
cathepsin O, cathepsin K, cathepsin T, chymopain, cysteine aminopeptidase
(Lactococcus),
cysteine endopeptidases 2 and 3 (barley), cysteine endopeptidases (Brassica
napus), cysteine
endopeptidase (Caenorhabditis), cysteine endopeptidases 1 and 2
(Dictyostelium), cysteine
endopeptidase (Entamoeba), cysteine endopeptidases 1 and 2 (Haemonchus),
cysteine
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WO 99/54317 PCT/US99/08501
endopeptidase (Hemerocallis), cysteine endopeptidases 1, 2 and 3 (Homarus),
cysteine
endopeptidase (Leishmania), cysteine endopeptidase (mung bean), cysteine
endopeptidase
(Ostertagia), cysteine endopeptidase (pea), cysteine endopeptidase
(Plasmodium), cysteine
protease tpr (Porphyromonas), cysteine endopeptidase {Tetrahymena), cysteine
endopeptidase
(Theileria), cysteine endopeptidase (tobacco), cysteine endopeptidase
(Trypanosoma), dipeptidyl
peptidase I, endopeptidase (baculovirus ofAutographa), endopeptidase EP-C1
(Phaseolus
vulgaris), glycyl endopeptidase, oryzain (includes a, (i and y) (rice),
bromelain (including stem-
and fruit bromelain), ficin, thaumatopain (Thaumatococcus); gingipain R and
gingipain K;
calpains, including calpain (Schistosoma), calpain I, calpain II, calpain p94,
calcium-binding
protein PMP41, sol gene product (Drosophila); streptopain and cysteine
endopeptidase
(Porphyromonas); picarnain 2A, picornain 3C, apothovirus endopepiidase,
cardiovirus
endopeptidase, comovirus endopeptidase, nepovirus endopeptidase; tobacco etch
virus NIa
endopeptidase, hepatitis C virus endopeptidase 2, adenovirus endopeptidase;
tobacco etch virus
HC-proteinase; chestnut blight virus p29 endopeptidase; chestnut blight virus
p48 endopeptidase;
sindbis virus nsP2 endopeptidase; mouse hepatitis virus endopeptidase, avian
infectious
bronchitis virus endopeptidase; a-clostripain; ubiquitin carboxyl-terminal
hydrolase;
deubiquinating enzyme (DOA4 protein), ubiquitin-specific processing peptidase
1, ubiquitin-
specific processing peptidase 2, ubiquitin-specific processing peptidase 3,
tre oncogene protein
{human), unp protein (mouse); hemoglobinase (Schistosoma), legumain (jack
bean); interleultin
converting enzyme and caspases, such as caspase 2 (ICH-1), caspase 3 (CPP32,
YAMA), caspase
4 (ICEreI-II), caspase S (ICEreI-III), caspase 6 (Mch2), caspase 7 (Mch3),
caspase 8 (FLICE,
MchS), caspase 9 (Mch6, ICE-LAP6), caspase 10 (Mch4); pyroglutamyl-peptidase
I; microsomal
ER60 protein endopeptidase; prepiliii leader peptidase; PRRS arteritis virus
PCP a-
endopeptidase, equine arteritis virus Nsp2 endopeptidase; foot and mouth
disease virus L
proteinase; hepatitis A viral protease; human corona virus protease;
encephalomyelitis virus
endopeptidase; malarial hemoglobinase; drosophila hedgehog virus gene product;
dipeptidyl
peptidase I (cathepsin C); Der pl (dust mite); y-glutamyl hydrolase; Actinide
(Actinidia); yeast
cysteine proteinase E, yeast proteinase D, yeast proteinase F; cancer
procoagulant; and
histolysin. Enzyme inhibitors for cysteine proteases may be useful as
potential therapeutic
CA 02329712 2000-10-20
WO 99154317 PCT/US99/08501
drugs for humans or animals, as diagnostic or research tools, as antibacterial
agents, herbicides,
fungicides or pesticides. Potential indications for cysteine protease
inhibitors described herein,
used in prophylaxis, cure or therapy, include:
Cardiovascular disorders--ischemia reperfusion injury from transplantation
andlor
vascular surgery, angiogenesis, neovascularization, acute cardiac allograft
dysfunction, ischemic
cardiac damage, chemotherapy-induced myocardial suppression;
Inflammatory disorders - rheumatoid arthritis and other inflammatory
arthritidies,
inflammatory bowel disease, inflammatory peritonitis, sepsis, systemic
inflammatory response
syndrome, multiple organ failure;
Musculo-skeletal disorders--osteoarthritis, osteoporosis, muscular dystrophy,
myositis;
Neurological disorders-- multiple sclerosis, stroke, Alzheimer's disease,
prion-associated
disorders, ataxia telangiectasia, central nervous system injury;
Pulmonary disorders-- asthma, COPD, adult respiratory distress syndrome,
Wegener's
granulomatosis, emphysema;
Allergic, immunologic and autoimmune disorders--house dust mite allergy,
transplant
rejection, graft verses host disease, Type 1 diabetes mellitus, autoirnmune
thyroiditis, psoriasis,
antibody-mediated autoimrnune diseases, lupus erythematosus, Sjogren's
syndrome,
autoimmune encephalomyelitis;
Solid tumors, lymphomas, leukemias and other malignancies and related
disorders--acute
and chronic myelogenaus leukemia, neuronal cancer, cancer invasion and
metastasis, tumor
angiogenesis, B and T cell lymphomas, acute and chronic lymphocytic leukemia,
resistance to
chemotherapy, cancer associated coagulopathies (including deep venous
thrombosis, coronary
artery disorder, pulmonary embolism, disseminated intravascular coagulation),
Hodgkins disease,
carcinomas of the colon, liver, lung, breast, kidney, stomach, pancreas,
esophagus, oral pharynx,
intestine, thyroid, prostate, bladder, brain; osteo-sarcoma, chondro-sarcoma
and liposarcoma;
neuroblastoma; melanoma; and carcinomas derived from amnion and/or chorion);
Infectious diseases and associated syndromes--septic shock (including Gram-
negative
sepsis), HIVinfection and AIDS, genital herpes, zoster, chickenpox, EBV
infections and
encephalitis, CMV-choreoretinitis or encephalitis, cytomegalovirus infections
in neonates
26
CA 02329712 2000-10-20
WO 99!54317 PCT/US99/08501
(including related pneumonitis), opportunistic infections in immunocompromised
individuals
(including AIDS and transplant patients), dysentery, hepatitis C, hepatitis A,
keratoconjuctovitis,
bronchopneumonia (including pneumonia in immunocompromised individuals),
gastroenteritis,
malaria, rhinovirus, polio, enterovirus infections, common cold, aseptic
meningitis, foot and
mouth disease, Klebsiella pneumonia infection, escherichia coli or
staphylococcus epidermidis,
leprosy bacteremia, otitis media, lambiiasis, non-atopic sinusitis, fulminant
hepatitis;
Kidney disorders--polycystic kidney disease, glomerulonephritis;
Other miscellaneous disorders--periodontal disease, alcohol hepatitis,
prostate
hypertrophy, trauma, cutaneous mastocytosis, radiation- and HIV-induced
diarrhea, cachexia
(including acompanying cancer and malnutrition).
Examples of cysteine proteases and associated disease are described in Table
2.
Table 2.
Cysteine Protease Disease State References
Interleukin 1 (3 Stroke, traumatic Patel, et al., FASEB,
convening brain 10:587-
enzyme (ICE, Caspaseinjury, organ transplant597 (1996);.
1)
rejection and septicBarr et al., BiofTechnology,
shock.
Inflammatory disorders12:487-493 (1994);
Epstein,
including the arthritides,New Eng. J. Med.,
such 328:106-
as rheumatoid arthritis113 (1993).
and
osteoarthritis, inflammatory
bowel disease, ulcerative
colitis, pancreatitis,
and
inflammatory peritonitis,
asthma.
27
CA 02329712 2000-10-20
WO 99/54317 PGT/US99/08501
MAMA (Apopain, CPP32,Diseases in which Barr et al., BiolTechnology,
Caspase 3), FLICE disregulated apoptosis12:487-493 (1994)
(MchS, plays a
Caspase 8), and role in pathology:
other
caspases Solid tumors, B cell
lymphoma, chronic
lymphocytic leukemia,
prostate hypertrophy,
preneoplastic liver
foci,
resistance to chemotherapy,
stroke, Alzheimer's
disease,
Parkinson's disease,
multiple
sclerosis, prion-associated
disorders, ataxia
telangiectasia, ischemic
cardiac damage,
chemotherapy-induced
myocardial suppression,
AIDS, type I diabetes,
lupus
erythematosus, Sjogren's
syndrome,
glomeruionephritis,
dysentery, inflammatory
bowel disease, radiation-
and
HN induced diarrhea,
polycystic kidney
disease,
anemia or erythropoiesis.
Malarial HemoglobinaseMalaria Rockett, et al.,
FEB, 259:257-
259 (1990)
28
CA 02329712 2000-10-20
WO 99/54317 PCT/US99/08501
Der p 1 Asthma, house dust Kalsheker, et al.,
mite Biochem.
allergy Biophys. Research
Comm.,
221:59-61 (1996)
Gingipain K and Adult PeriodontitisWingrove, et al.,
J. Biol.
S Gingipain R Chem., 267:18902-18907
(1992); DiScipio
et al.,
Immun., 87:660-667
(1996)
Cathepsin B, CathepsinOsteoarthritis, Velasco, et al.,
L, osteoporosis, J. Biol.
Cathepsin S, Cathepsinrheumatoid arthritis,Chem., 269:27136-27142
O and
Cathepsin K Alzheimer's disease,(1994); Takeda et
cancer al., FEBS
invasion and Metastasis,Letters, 359:78-80
(1995);
Parkinson's disease,Elliott et al., Persp.
in Drug
leukemia, lymphoma,Disc. and Des., 6:12-32
hodgkin's disease, (1996)
tumors,
including those
of the
bladder, brain,
lung,
pancreas, prostate,
stomach
and thyroid
Cancer Procoagulant Cancer (including Alessio et al., Eur.
carcinomas J.
of the liver, lung,Haematology, 45:78-8I
breast,
kidney, colon, kidney;(1990); Gordon, Seminars
osteo-, in
chondro-, and liposarcoma;Thrombosis and Hemostasis,
neuroblastoma; melanoma;18:424-433 (1992)
nonlympocytic leukemia;
lymphocytic leukemia)
29
CA 02329712 2000-10-20
WO 99/54317 PCT/US99/08501
Calpain I and II Osteoporosis, stroke,Karlsson, et al.,
CNS Neurobiology
injury, Alzheimer'sofAging, 16:901-906
disease (1995);
Additionally, diseasesSquier; et aL, J.
Cell.
involving dysregulatedPhysiol., 159:229-237
apoptosis as listed(1994).
for caspase
above.
Calpain p94 Muscular dystrophy Calpain p94 and limb-girdle
muscular dystrophy,
/COP
Letters 1996.
Hepatitis C Vitvs Hepatitis C Grakoui, et al.,
Proc. Nat.
Endopeptidase 2 and Acad Sciences, 90:10583-
Hepatitis C Virus 10587 (1993)
NS3
Endopeptidase
Picornain 2A and Rhinovirus, polio, Palmenberg, J. Cell.
Picornain enterovirus
3C Proteases infection, common Biochem. 33:191-198
cold, (1987);
aseptic meningitis,Cordingley, et al.,
polio J.
virology, 5037-5045
(Dec
1989)
Hepatitis. A Viral Hepatitis A Krausslich, et al.,
Protease Annu. Rev.
Biochem., 57:701-754
( 1988).
Foot and Mouth DiseaseFoot and mouth diseaseRoberts et al., Virology
Virus L Protease (Cattle) 213:140-146 (1995)
Although the compounds described herein and/or their salts may be administered
as the
pure chemicals, it is preferable to present the active ingredient as a
pharmaceutical composition.
The invention thus further provides the use of a pharmaceutical composition
comprising one or
CA 02329712 2000-10-20
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more compounds and/or a pharmaceutically acceptable salt thereof, together
with one or more
pharmaceutically acceptable carriers thereof and, optionally, other
therapeutic and or
prophylactic ingredients. The carriers) must be 'acceptable' in the sense of
being compatible
with the other ingredients of the composition and not deleterious to the
recipient thereof.
Pharmaceutical compositions include those suitable for oral, topical or
parenteral
(including intramuscular, subcutaneous and intravenous) administration. The
compositions may,
where appropriate, be conveniently presented in discrete unit dosage forms and
may be prepared
by any of the methods well known in the art of pharmacy. Such methods include
the step of
bringing into association the active compound with liquid carriers, solid
matrices, semi-solid
carriers,.finely divided solid carriers or combination thereof, and then, if
necessary, shaping the
product into the desired delivery system.
Pharmaceutical compositions suitable for oral administration may be presented
as discrete
unit dosage forms such as hard or soft gelatin capsules, capsules, cachets or
tablets each
containing a predetermined amount of the active ingredient; as a powder or as
granules; as a
solution, a suspension or as an emulsion. The active ingredient may also be
presented as a bolus,
electuary or paste. Tablets and capsules for oral administration may contain
conventional
excipients such as binding agents, fillers, lubricants, disintegrants, or
wetting agents. The tablets
may be coated according to methods well known in the art., e.g., with enteric
coatings.
Oral liquid preparations may be in the form of, for example, aqueous or oily
suspension,
solutions, emulsions, syrups or elixirs, or may be presented as a dry product
for constitution with
water or other suitable vehicle before use. Such liquid preparations may
contain conventional
additives such as suspending agents, emulsifying agents, non-aqueous vehicles
(which may
include edible oils), or preservative.
The compounds may also be formulated for parenteral administration (e.g., by
injection,
for example, bolus injection or continuous infusion) and may be presented in
unit dose form in
ampules, pre-filled syringes, small bolus infusion containers or in multi-dose
containers with an
added preservative. The compositions may take such forms as suspensions,
solutions, emulsions
in oily or aqueous vehicles, and may contain formulatory agents such as
suspending, stabilizing
and/or dispersing agents. Alternatively, the active ingredient may be in
powder form, obtained
31
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WO 99/54317 PCT/US99/08501
by aseptic isolation of sterile solid or by lyophilization from solution, for
constitution with a
suitable vehicle, e.g., sterile, pyrogen-free water, before use.
For topical administration to the epidermis, the compounds may be formulated
as
ointments, creams or lotions, or as the active ingredient of a transdermal
patch. Suitable
transdermal delivery systems are disclosed, for example, in Fisher et al.
(U.S. Patent No.
4,788,603) or Bawas et al. (tJ.S. Patent No. 4,931,279, 4668,504 and
4,713,224). Ointments and
creams may, for example, be formulated with an aqueous or oily base with the
addition of
suitable thickening and/or gelling agents. Lotions may be formulated with an
aqueous or oily
base and will in general also contain one or more emulsifying agents,
stabilizing agents,
dispersing agents, suspending agents, thickening agents, or coloring agents.
The active
ingredient can also be delivered via iontophoresis, e.g., as disclosed in U.S.
Patent Nos.
4,140,122, 4,383,529, or4,051,842.
Compositions suitable for topical administration in the mouth include unit
dosage forms
such as lozenges comprising active ingredient in a flavored base, usually
sucrose and acacia or
tragacanth; pastilles comprising the active ingredient in an inert base such
as gelatin and glycerin
or sucrose and acacia; mucoadherent gels, and mouthwashes comprising the
active ingredient in
a suitable liquid carrier.
When desired, the above-described compositions can be adapted to provide
sustained
release of the active ingredient employed, e.g., by combination thereof with
certain hydrophilic
polymer matrices, e.g., comprising natural gels, synthetic polymer gels or
mixtures thereof.
The pharmaceutical compositions according to the invention may also contain
other
adjuvants such as flavorings, coloring, antimicrobial agents, or
preservatives.
The compositions may also be administered via inhalation, using a suitable
delivery
vehicle.
It will be further appreciated that the amount of the compound, or an active
salt or
derivative thereof, required for use in treatment will vary not only with the
particular salt selected
but also with the route of administration, the nature of the condition being
treated and the age and
condition of the patient and will be ultimately at the discretion of the
attendant physician or
clinician.
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In general, however, a suitable dose will be in the range of from about 0.5 to
about 100
mg/kg/day, e.g., from about 10 to about 75 mglkg of body weight per day, such
as 3 to about 50
mg per kilogram body weight of the recipient per day, preferably in the range
of 6 to 90
mg/kg/day, most preferably in the range of 15 to 60 mg/kg/day.
$ The compound is conveniently administered in unit dosage form; for example,
containing
to 1000 mg; conveniently 10 to 750 mg, most conveniently, 50 to 500 mg of
active ingredient
per unit dosage form.
Ideally, the active ingredient should be administered to achieve peak plasma
concentrations of the active compound of from about 0.5 to about 75 pM, most
preferably, about
1 to 50 pM, most preferably, about 2 to about 30 uM. This may be achieved, for
example, by the
intravenous injection of a 0.05 to 5% solution of the active ingredient,
optionally in saline, or
orally administered as a bolus containing about 1-100 mg of the active
ingredient. Desirable
blood levels may be maintained by continuous infusion to provide about 0.01-
5.0 mg/kglhr or by
intermittent infusions containing about 0.4-15 mg/kg of the active
ingredient(s).
The desired dose may conveniently be presented in a single dose or as divided
doses
administered at appropriate intervals, for example, as two, three, four or
more sub-doses per day.
The sub-dose itself may be further divided, e.g., into a number of discrete
loosely spaced
administrations; such as multiple inhalations from an insufflator or by
application of a plurality
of drops into the eye.
The inhibitors described herein may be also used for the detection and
quantification of
the activity of a cysteine protease in a pure sample, mixture or biological
fluid or tissue. The
activity can be measured with a protease substrate in the absence and presence
of a known
concentration of the inhibitor. Specific inhibitors can also be used to
confirm that the observed
activity is due to a particular protease.
The inhibitors described herein may also be used to identify and purify
cysteine
proteases. The inhibitors can be covalently linked to a solid support, such as
an affinity column
or beads used in batch methods, and used to purify a protease or enrich a
mixture containing the
protease. The inhibitor may be linked to the solid support or bead either
directly or via a linker
of variable length, such that linkage does not interfere with the binding
properties (see, e.g.,
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WO 99/54317 PCT/US99/08501
Thornberry, N., Methods in Enz., 244:615-31 ( 1994))
While the invention has been described in connection with specific embodiments
thereof,
it will be understood that it is capable of further modifications and this
application is intended to
cover any variations, uses, or adaptations of the invention following, in
general, the principles of
the invention and including such departures from the present disclosure as
come within known or
customary practice within the art to which the invention pertains and as may
be applied to the
essential features hereinbefore set forth, and as follows in the scope of the
appended claims.
The following examples are given to illustrate the invention and are not
intended to be
inclusive in any manner:
Abbreviations used herein are defined as follows:
DMF - dimethylformamide; HBTU - 2-(1H-benzotriazole-1-yl)-1,1,3,3-
tetramethyluronium
hexafluorophosphate; DIEA - diisopropylethylamine; THF - tetrahydrofiuan;
CH3CN -
acetonitrile; EDTA-Naz - ethylenediaminetetraacetic acid disodium salt; Mtr -
4-methoxy-2,3,6-
trimethylbenzene sulfonyl; Bop-Cl - bis(2-oxo-3-oxazolidinyl)phosphinic
chloride; EtOH -
ethylalcohol; EtOAc - ethyIacetate; LDA - lithium diisopropylamide; EDCI - 1-
(3-
dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; NMM - N-methyl
morpholine;
HOBT - 1-hydroxybenzotriazole; TFA - trifluoroacetic acid.
Example I - Synthesis of (~2-~5-(3-methylberuyl)-1,3,4-oxadiazolylJcarbonylJ-2-
(S)-
methylpropylJ-L phenylalanamide-(3R)-(isobutyl)succinic acid (CM-0019).
The intermediate (benzyloxycarbonyl)-L-valyl-N-[1-(2-[5-(3-methylbenzyl)-1,3,4-
oxadiazolyl]hydroxymethyl)-2-(S)-methylpropyl]-L-prolinamide was prepared as
follows:
a. 3-(S)-Amino-2-(R,S)-hydroxy-4-methyl pentanoic acid
To a solution containing 3-(S)-[(benzyloxycarbonyl)amino]-2-acetoxy-4-
methylpentanenitrile (see example 1 of WO 96/16080) (15.2 g, SO.O mmol) in 183
mL of
dioxane was added 183 mL of concentrated hydrochloric acid and 7.45 mL of
anisole. The
34
CA 02329712 2000-10-20
WO 99/54317 PCT/US99/08501
reaction mixture was heated to reflux overnight. The hydrolysis reaction was
allowed to cool to
room temperature and then concentrated in vacuo. The resulting aqueous
solution was extracted
with ether (2X). The aqueous phase was placed on a Dowex SOXB-100 column (H+
form,
preeluted with deionized water to pH = 7). The column was eluted with 2.0 N
ammonium
hydroxide and the pure fractions concentrated to afford S.S3 g (7S %) of 3-(S)-
amino-2-(R, S)-
hydroxy-4-methylpentanoic acid as a pale yellow solid. FAB MS [M+H] m/z;
Calcd: 148,
Found: 148.
b. 3-(S)-((Benzyloxycarbonyl)amino]-2-(R,S)-hydroxy-4-methyl pentanoic acid.
To a solution under an atmosphere of nitrogen containing 1.0 g (6.8 mmol) of 3-
(S)-
amino-2-(R,S)-hydroxy-4-methylpentanoic acid in 9.S mL of 1 N NaOH and 10 mL
of dioxane
was added i .43 g (8.4 mmol) of benzyl chloroformate. The pH was maintained
above pH 8 with
1 N NaOH as needed. The reaction mixture was allowed to stir at room
temperature overnight.
The reaction was diluted with water and washed with ether. The aqueous layer
was acidified with
1 N HCl to pH = 2 and extracted with ether (2X). The combined organic layers
were dried over
magnesium sulfate, filtered and evaporated in vacuo to afford 1.75 g (92%) of
3-(S)-
[(benzyloxycarbonyl)aminoJ-2-(R,S)-hydroxy-4-methylpentanoic acid as a light
yellow viscous
oil. FAB MS [M+H) m/z; Calcd: 282, Found: 282.
c. 3-(S)-~(Benzyloxylcarbonyl)amino]-2-(R,S)-acetoxy-4-methyl pentanoic acid.
To a solution of 3-(S)-[benzyloxycarbonyl)amino]-2-(R,S)-hydroxy-4-
methylpentanoic
acid (1.70 g, 6.04 mmol) and pyridine (4.9 mL) was added acetic anhydride (S.7
mL, 6.17 g, 60.4
mmol) dropwise at room temperature. The reaction was allowed to stir overnight
and was diluted
with ethyl acetate and washed with water (2X). The organic layer was dried
over magnesium
sulfate, filtered and evaporated in vacuo to give a thick oil. The residue was
purified by column
chromatography on silica gel with 1S% methanol/dichloromethane to afford 1.56
g (80%) of 3-
2S (S)-[(benzyloxycarbonyl)amino)-2-(R, S)-acetoxy-4-mcthyl pentanoic acid as
a light yellow
viscous oil. FAB MS [M+H] m/z; Calcd: 324, Found: 324.
d. 1-~(3-Methylpherrylacetyl)-2-(2-(R,S)-acetoxy)-3-(S)-
~(benzyloxycarbonyl)aminoJ-4-
methylpentanoylJ hydrazine.
To a solution containing 3-(S)-[(benzyloxycarbonyl)amino]-2-(R,S)-acetoxy-4-
CA 02329712 2000-10-20
WO 99!54317 PCT/US99/08501
methylpentanoic acid (2.3 g, 7.11 mmol) in 40 mL of DMF under a nitrogen
atmosphere at O°C
was added 1.31 g (9.69 mmol) of HOBT and 1.36 g (7.09 mmol) of EDCI. After
stirring for 30
minutes, 1.20 g (7.3 I mmoi) of 3-methylphenyl acetic hydrazide [prepared
analogously to the
monoacid hydrazides cited by Rabins et. al. (J. Org. Chem., 30:2486 (1965))]
and 1.0 mL (9.10
mmol) of NMM were added. The reaction was allowed to warm to room temperature
and stir
overnight. The reaction was diluted with ethyl acetate and washed with 5%
potassium hydrogen
sulfate, saturated sodium bicarbonate, brine and water. The organic phase was
dried over
magnesium sulfate, filtered and evaporated under reduced pressure. The residue
was purified by
column chromotography on silica gel with 10% methanol/dichloromethane to
afford 2.31 g
{89.0%) of the title compound as a white solid. FAB MS [M+H] m/z; Calcd: 470,
Found: 470.
e. I - j2- j5-(3-Methyl benzyl)-I , 3, 4-oxodiazolylJ-I -acetoxy-2-(S)-
(berrryloxycarborryl)arninoJ-3-methylbutane.
A solution containing 2.31 g (4.92 mmol) of 1-[(3-methylphenylacetyl)-2-(2-(R,
S)-
acetoxy)-3-(S)-[(benzyloxycarbonyl)amino]-4-methylpentanoyl]hydrazine in 25 mL
of pyridine
and 1.88 g (9.86 mmol) of toluene sulfonyl chloride was heated at reflux under
a nitrogen
atmosphere for 72 hours. The solvent was removed under reduced pressure and
the residue
dissolved in ethyl acetate and washed with water. The organic phase was dried
over magnesium
sulfate, filtered and evaporated under reduced pressure. The residue was
purified by column
chromatography on silica gel with 5% ethyl acetate/hexane to afford 1.41 g
(63.5%) of the title
compound. FAB MS (M+H] m/z; Calcd: 452, Found: 452.
f. I - j2- j5-(3-Methylbenzyl)-1, 3, 4-oxadiazolyl)J-2-(S)-(benzyl
oxycarborryl)am inoJ-3-
methylbutan-I-ol.
A solution containing 1.80 g (3.99 mmol) of 1-[2-[5-(3-methylbenzyl)-1,3,4-
oxadiazolyl)-
1-1-acetoxy-2-(S)-(benzyloxycarbony!)amino]-3-methylbutane and 0.72 g (5.21
mmol) of
potassium carbonate in 30 mL of methanol and 8 mL of water was allowed to stir
at room
temperature for 30 minutes. The solvent was removed under reduced pressure and
the residue
dissolved in ethyl acetate and washed with water. The organic phase was dried
over magnesium
sulfate, filtered and evaporated under reduced pressure. The residue was
purified by column
chromatography on silica gel with 60% ethyl acetate/hexane to afford 1.46
(89.3%) of the title
36
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WO 99/54317 PCT/US99/08501
compound. FAB MS [M+H] m/z; Calcd: 410, Found: 410.
g. l-~2-(5-(3-Methylbenzyl)-1,3,4-oxadiazolylJ-2-(S) Amino-3-methylbutan-I-of
hydrochloride.
To a solution containing 1.31 g (3.20 mmol) of 1-[2-[5-(3-methylbenzyl)-1,3,4-
S oxadiazolyl]-2-(S)-(benzyloxycarbonyl)amino]-3-methylbutan-1-of in 25 mL of
trifluoroacetic
acid under a nitrogen atmosphere at O °C was added 0.43 mL (3.94 mmol)
of thioarusole. The
reaction was allowed to warm to room temperature overnight. The solvent was
removed under
reduced pressure and the residue dissolved in ether and cooled to -78°C
under a nitrogen
atmosphere. To this solution was added 3 mL (3 mmol) of 1 N hydrochloric acid
in ether. The
resulting white solid was allowed to settle and the ether decanted. Additional
ether was added
and decanted (3X). The solid was dried under vacuum to afford 0.92 g (92.2%)
of the title
compound. FAB MS [M+HJ m/z; Calcd: 276, Found: 276.
(4S)-4-Benzyl-3-(4 =(methyl)pentanoylJ-2-oxazolidinone
a. 4-Methylvaleric acid (6.56 g, 55.6 mmol) was dissolved in dry CHZCIZ(40 ml)
under
N, and chilled to 4°C. Oxalyl chloride (5.4 mL, 63.5 mmol) was added;
followed by 4 drops of
dry DMF. Rapid COZ evolution occured. The reaction mixture was allowed to warm
to ambient
temperature over 2 h; no more COZ evolution was apparent. Solvents were
stripped by rotary
evaporation and the acid chloride was distilled in vacuo. 'H-NMR (300 MHz,
CDCl3) 8 0.88-
0.93 (m, 6H), 1.59-1.64 (m, 3H), 2.90 (t, 2H, J= 7.5 Hz).
b. (S)-(-)-4-benzyl-2-oxazolidinone (8.93 g, 50.4 mmol) was dissolved in dry
THF under
Nz and chilled to -78°C. nButyl lithium (1.6 M in hexane, 32 mL, 50.4
mmol) was added
dropwise to maintain temperature < -70°C. The mixture was stirred 25
min. at -78 °C, then a
solution of the acid chloride prepared above in dry THF (30 mL) was added
dropwise to maintain
temperature <-65°C. The reaction mixture was stirred overnight and
allowed to warm to 15°C.
The reaction was quenched by careful addition of saturated NH,CI (70 mL). THF
was removed
under reduced pressure and the resultant aqueous slunry was extracted with
EtOAc (100 mL).
The organic layer was washed with 0.5 N NaOH, HBO, brine. The organic layer
was dried over
MgS04, filtered and evaporated in vacuo to return 12.7 g of crude yellow oil.
The crude material
37
CA 02329712 2000-10-20
WO 99/54317 PC'T/US99/08501
was purified by silica gel chromatography (10% EtOAc/hexane) and dried in
vacuo to return 9.0
g (69% yield) of pale yellow oil. C-18 HPLC RT = 16.5 min., 96% pure at 21 S
nm (10-100%
solv. B/25 min; solvent A= 0.1% (v/v)TFA/H20; solvent B = 0.1%
TFA/acetonitrile; FAB-MS
m!z 276(M+H) ;'H-NMR (300 MHz, CDCI,) x0.94 (d, 6H, J=6.3 Hz, e-[(CH3)Z]),
1.53-1.72
(m, 3H, [i CH2,g CH), 2.76 (dd, 1H, J=13.3, 9.6 Hz, oxazolidinone 5-CHI), 2.90-
2.97 (m, 2H),
3.29 {dd, 1H, J=13.2, 3.3 Hz, oxazolidinone SC~IH), 4.15-4.20 (m, 2H), 4.64-
4.70 (m, 1H,
oxazolidinone 4-CH), 7.17-7.39 (m, SH, Ph-H).
c. (4S)-4-Ben.,ryl-3-(2'R)-2 =~((tert-butoxycarbonyl)methylJ-4
=(methyl)pentanoylJ-2-
oxazolidinone.
Diisopropylamine (5.05 mL, 36 mmol) was diluted with dry THF (20 mL) and
chilled to
-20°C under NZ. n-Butyl lithium (1.6 M in hexane, 23 mL, 36 mmol) was
added dropwise to
maintain the temperature <-10°C. The temperature was increased to
4°C and stir ed 30 minutes
to generate LDA. The flask was chilled to -78°C and (4S)-4-Benzyl-3-[4'-
(methyl)pentanoyl]-
2-oxazolidinone in dry THF {1S mL) was added dropwise to maintain the
temperature < -70°C.
The reaction was stirred 30 minutes at -78°C then t-butylbromoacetate
(4.9 mL, 33 mmol) in dry
THF was added dropwise to maintain the temperature <-65°C. The mixture
was stirred and
allowed to warm to -10°C overnight. After 15 hours, the reaction was
quenched by careful
addition of water followed by evaporation of the THF. Water (100 mL) was added
to the slurry.
and the crude mixtrure was extracted with EtOAc (100 mL). The organic layer
was washed with
water and brine; then dried over MgSO,, filtered and dried in vacuo to leave
13.3 g crude yellow
oil. Silica gel chromatography in 15% EtOAclhexane returned 7.84 g (61% yield
) of a white
solid product.
C-18 HPLC RT = 19.5 min., 99% pure at 215 nm (10-100% solv. B/25 min; solvent
A= 0.1%
(v/v)TFA/H~O; solvent B = 0.1% TFA/acetonitrile; FAB-MS mlz 390 (M+H)', 334 (M-
tBu+H)'.
'H-NMR (300 MHz, CDCh) 80.92 (d, 3H, J=6.0 Hz), 0.94 (d, 3H, J=5.8 Hz), 1.28-
1.40 (m, 1H,
C~IvIe2),1.43 (s, 9H3), 2.49 (dd, 1H, J=16.7, 4.6 Hz), 2.72 (dd, 1H, J=10.3,
1.1 Hz), COzC
(CH3)3), 2.76 (dd, 1H, J=10.3, 2.2 Hz, Ph-CHI, 3.35 (dd, 1H, J=13.5, 3.1 Hz),
4.15-4.18 (m,
2H), 4.21 -4.26 (m, 1H), 4.52-4.61 (m, 1H), 7.27-7.34 (m, SH, Ph-H).
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d. (2R)-2-((tert-Butoxycarborryl)methylJ-4-(methyl)pentanoic acid
(4S)-4-Benzyl-3-(2'R)-2'-[[(tert-butoxycarbonyl)methyl]-4'-(methyl)pentanoyl]-
2-
oxazolidinone (5.89, 15.1 mmol) was dissolved in dry THF(100 mL) and water (25
mL) was
added. The mixture was chilled to 4°C under NZ. HZOZ (7.6 mL) was added
followed by
S dropwise addition of LiOH (0.76 g, 18.2 mmol) in H20 (20 mL) over 20
minutes. The mixture
was stirred for 1 hour and allowed to warm to ambient temperature. The mixture
was again
chilled in an ice bath and quenched by addition of Na2S03 (3.1 g) in water (20
mL). THF was
removed by rotovap, the remaining aqueous layer was washed with EtOAc (4x70
mL), then
acidified to approx. pH 2 with conc. HCl after layering with fresh EtOAc. The
mixture was
immediately extracted with EtOAc (3x80mL). The combined EtOAc extracts were
dried over
MgS04, filtered, and evaporated to return 3.29 g clear oil (95% crude yield)
which showed no
traces of starting material by HPLC. This material was used without further
purification.
'H-NMR (300 MHz, CDC13) 80.90 (d, 3H, J=6.4 Hz, CH3), 0.94 (d, 3H, J=6.5 Hz,
CH3), 1.20-
1.48 (m, 1H, CHMez), 1.44 (s, 9H, C(CH,),), 1.50-1.72 (m, 2H), 2.37(dd, 1H,
J=16.4, 2.4 Hz),
2.59 (dd, 1 H, J=16.4, 2.6 Hz), 2.80-2.95 (m, 1 H).
e. Tert-butyl(3R)-3-(isobutyl)succinyl-L phenylalartyl methyl ester.
To a solution of tent-butyl-(3R)-3-(isobutyl)succinate (10.82 g, 47.0 mmols),
in 90 ml of
dry DMF was added HBTU (17.45 g, 46.0 mmols), followed by DIEA (18.43 g, 142.6
mmols).
After stirring for 10 min, L-phenylalanine methyl ester hydrochloride (10.0 g,
46.36 mmols) was
added. This was allowed to stir at room temperature overnight. The solvent was
removed under
reduced pressure and the residue dissolved in 200 ml ethyl acetate. This
solution was washed
with water, 1 M HCI (2x), saturated NaHC03 (2x), brine, and the organics were
dried with
anhydrous MgSO,,. The mixture was filtered and the solvent removed under
reduced pressure.
The residue crystallized to an off white solid upon sitting overnight, giving
13.3 g (74%) of tert-
butyl(3R)-3-(isobutyl)succinyl-L-phenylalanyl methyl ester. C-18 HPLC RT =16.9
min., 98%
pure at 215 nm (10 to 100% solvent B/25 min; solvent A = 0.1% TFA/HZO; solvent
B = 0.1%
TFA/acetonitrile).
FAB-mass spectrum: m/z (M+H)' = 392; Theory = 392.
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'H NMR (CDCl3) 8 [0.85 (d, J= 7.5 hz); 0.88 (d, J= 7.5 hz); 6H]; [1.10-1.23
(m, 1H)];
[1.44 (s, 9H)]; [1.45-1.65 (m, 2H)]; [2.22-2.32 (m, 1H)]; [2.50-2.66 (m, 2H)];
[3.10 (d, J= 6 Hz}
2H]; [3.69 (s,3H)]; [4.82-4.92 (m, 1H)]; [6.19 (d, J= 9 hz), lH]; [7.13-7.33
(m, 5H)].
f. Tert-butyl(3R)-3-(isobutyl)succinyl-L phenylalanine.
A solution of tert-butyl(3R)-3-(isobutyl)succinyl-phenylalanyl methyl ester
(2.0 g, 5.10
mmols} in 5 ml methanol was cooled to 4°C in an ice bath. To this
solution was added 4 ml of
an aqueous solution of lithium hydroxide (333 mg, 7.94 mmols.), and this
solution was stirred
and allowed to warm to room temperature overnight. The solution was
concentrated to an oil
under reduced pressure. The residue was dissolved in 100 ml ethyl acetate,
washed with 10%
citric acid, water, and dried with anhydrous MgSO,. The mixture was 'filtered
and the solvent
was removed under reduced pressure, vacuum dried overnight to give 1.8 g
(95%}of tert-
butyl(3R)-3-(isobutyl)succinyl-L-phenylalanine as a light yellow oil. C-18
HPLC RT = 14.7
min., 95% pure at 254 nm (10 to 100% solvent B/25 min; solvent A = 0.1%
TFA/HZO; solvent B
1 S = 0.1% TFA/acetonitrile).
FAB mass spectrum: M+H = 378; theory = 378.
'H NMR (CDC13) 8 [0.83 (d,J = 6.0 hz); 0.85 (d, J = 6.0 hz); 6H]; [1.10-1.25
(m, 1H)];
[1.44 (s, 9H)]; [1.45-1.65 (m, 2H)]; [2.24-2.35 (m, 1H)]; [2.48-2.58 (m, 2H)];
[3.07-3.27
(m,2H)]; [4.79-4.93 (m, 1H)]; [6.36 (d, J = 9 Hz), 1H]; [7.20-7.41 (m, 5H)].
zo
g. Tert-butyl(3R)-3-(isobutyl)succirryl-~(2-(5-(3-methylbenryl)-1,3,4-
oxadiazolylJ-(R,S)-
hydroxymethylJ-2-(S)-methylpropylJ-L phenylalaninamide.
To a solution of tert-butyl(3R)-3-(isobutyl)succinyl-phenylalanine (1.8 g,
4.80 mmols) in
40 ml'DMF was added HOST (676 mg, 5.0 mmols). This was cooled in an ice bath
to 4°C.
25 EDCI (921 mg, 4.80 mmols) was then added. After stirring for 30 minutes, a
solution of [2-(5-
(3-methylbenzyi)-1,3,4-oxadiazolyl]-2-(S)-amino-3-methylbutan-1-(R,S)-of
hydrochloride(1.50
g, 4.24 mmols) in 20 ml. DMF was added dropwise, followed by N-methyl
morpholine (0.77 g,
7.66 mmols) and the reaction allowed to stir and warm to room temperature
overnight. Most of
the solvent was removed under reduced pressure and the mixture was diluted
with ethyl acetate.
CA 02329712 2000-10-20
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It was then washed with saturated NaHC03, 5% KHSO,, brine, and the organics
dried with
anhydrous MgSO,. The mixture was filtered and the solvent removed under
reduced pressure.
The residue was purified by column chromatography (silica gel, ethyl
acetate:hexane 50:50 to
65:35) to give 1.30 g, 43% of tert-butyl(3R)-3-(isobutyl)succinyl-[[2-[5-(3-
methylbenzyl)-1,3,4-
oxidiazolyl]-(R,S)-hydroxymethyl]-2-(S)-methylpropylJ-L-phenylalaninamide as
an off white
foamy solid. C-18 HPLC RT = 18.3, 18.7 min. diastereomers, 90% pure at 215 nm
(10 to 100%
solvent BI25 min; solvent A = 0.1 % TFA/HzO; solvent B = 0.1 %
TFA/acetonitrile).
FAB mass spectrum: m/z (M+H)' = 635; theory = 635.
h. tert-Butyl(3R)-3-(isobutyl)succinyl-~~2-(S-(3-methylbenzyl)-1,3,4-
oxadiazolylJcarbonylJ-2-
(S)-methylpropylJ-L phenylalaninamide.
To a stirred mixture ofN-chlorosuccinimide (1.07g, 8.0 mmols) in 25 ml dry
toluene at
4 ° C was added 0.84 ml ( 11.45 mmols) dimethyl sulfide (DMS) under a
nitrogen atmosphere. A
white precipitate formed after the addition of DMS. After 30 minutes, the
resulting suspension
was cooled to -25 °C using a carbon tetrachloride and dry ice bath. A
solution of tert-butyl(3R)-
3-(isobutyl)succinyl-[[2-[5-(3-methylbenzyl)-1,3,4-oxadiazolyl]-(R,S)-
hydroxymethyl]-2-(S)-
methylpropylJ-L-phenylalaninamide (1.25g, 1.97 mmols) in 30 ml dry toluene was
added
dropwise. The resulting mixture was stirred for 1.5 h at -25°C and 1.19
ml (8.5 mmols) of
triethylamine was added. After 15 minutes, the cold bath was removed, and the
reaction
monitored by TLC; silica gel; ethyl acetate:hexane (30:70). After 1 h, the
mixture was diluted
with 500 ml ethyl acetate and washed with saturated NaHC03 brine and the
organics dried with
anhydrous MgSO,,. The mixture was filtered and the solvent removed under
reduced pressure.
The residue was purified by column chromatography (silica gel,
methanol:chloroform, 0.5:99.5
to 2.5:97.5) to give tert-butyl(3R)-3-(isobutyl)succinyl-[[2-[5-(3-
methylbenzyl)-1,3,4-
oxadiazolyl]carbonylJ-2-(S)-methylpropylJ-L-phenylalaninamide as an off white
foamy solid;
1.0 g, (80.2%). C-18 HPLC RT = 20.2, 20.7 min. diastereoisomers, 90% pure at
215 nm (10 to
100% solvent B/25 min; solvent A = 0.1% TFA/H20; solvent B = 0.1%
TFAlacetonitrile).
FAB mass-spectrum: m/z (M+H); = 633; theory = 633.
41
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i. jj2-j5-(3-methylbenzyl)-1,3,4-oxadiazolylJcarbonylJ-2-(S)-methylpropylJ-L-
phenylalaninamide-(3R)-(isobutyl)succinic acid.
To a solution of tert-butyl(3R)-3-(isobutyl)succinyl-[2-[S-(3-methylbenryl)-
1,3,4-
oxadiazolylJcarbonylJ-2-(S)-methylpropylJ-L-phenylalaninamide (1.0 g, 1.58
mmol) in 25 ml
dichloromethane (DCM) cooled to 4°C in an ice bath, was added 25 ml
trifluoroacetic acid
(TFA). This was stirred for 1 h. The solvent and TFA are removed under reduced
pressure,
followed by coevaporation with DCM (3x). The material was purified via
gradient RP-HPLC
CH,CN:H~O (25:75 to 100:0 in 60 minutes) to give 292 mg (32%, 0.51 mmols) of
which 52 mg
was pure as a white solid after lyophilization. C-18 HPLC RT = 15.8 min., 95%
pure at 215 nm
( 10 to 100% solvent B/25 min; solvent A = 0.1 % TFA/HZO; solvent B = 0.1 %
TFA/acetonitrile).
FAB Mass spectrum: m/z (M+H)' = 577; theory = 577. 'H-NMR(400 MHz, CDCI;)8
0.76 (d,
3H, J=6.8 Hz), 0.85 (d, 3H, J=6.4 Hz), 0.87 (d, 3H, J=6.4 Hz), 0.93 (d, 3H,
J=6.8 Hz), 1.25-
1.32 (m, 1H), 1.48-1.61 (m, 2H), 2.28-2.36 (m, 2H), 2.35 (s, 3H), 2.44-2.49
(m, 1H), 2.6I-2.69
(m, 2H), 2.95 (dd, 1H, J=13.6, 8.4 Hz), 3.09 (dd, 1H, J=16.6,6.4 Hz),4.24 (s,
2H), 4.67 (dt, 1H,
J=8.0, 6.8 Hz), 5 .19 (dd, 1 H, J=8.4, 6.0 Hz), 6.52 (br. d, 1 H, J=8.4 Hz),
6.81 (br. d, l H, J=7.6
Hz), 6.94-6.99 (m, 1H), 7.10-7.19 (m, 7H). '3C-NMR(100 MHz, CDC13) a 17.26,
19.54, 21.33,
22.13, 22.71, 25.75, 30.84, 31.82, 36.74, 38.16, 40.54, 41.29, 55.16, 61.55,
126.1, 126.8, 128.6,
128.8, 129.0, 129.1, 129.7, 132.5, 136.2, 139.0, 160.2, 167.9, 171.6, 175.0,
175.7, 184.4.
Example II -Acetyl-L-leucyl-N jl-j2-j(5 phenyl)-1,3,4-oxadiazolyl]carbonyl]-4-
(guanidino)-
butyl]-L-leucylJamidE (CQ-0002).
2-Pherryl-1,3,4-oxadiazole intermediate
Benzoyl hydrazide (200 mg) freshly crystallized from chloroform was suspended
in 5 mL
of triethyl orthoformate and heated at reflux under nitrogen in a 160
°C oil bath for 3 hours. The
mixture was cooled to room temperature, chilled in ice, and treated with 50 mL
water and 10 mL
10% KHS04 solution. The mixture was stirred approximately 2 minutes then 50 mL
of EtOAc
was added and stirring continued for 10 minutes. The organic layer was
separated and the
aqueous layer was extracted three times with ethyl acetate. All ethyl acetate
layers were
combined and were washed with 10% sodium bicarbonate solution and saturated
sodium chloride
42
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WO 99/54317 PCT/ITS99/08501
solution. Drying over sodium sulfate, rotary evaporation and further drying
under high vacuum
provided 204 mg of an analytically pure oil which crystallizes upon standing.
Commercially
available benzoylhydrazide (Aldrich) in this reaction may be used, but the
resulting product
often contains a minor impurity which can be removed following the
cyclization, by flash
chromatography on silica gel eluting with 0 - 10% acetone in hexane.
'H-NMR-CDCI3 7.49-7.62 (m, 3H), 8.12 (d, J=6, 2H), 8.49 (s, 1H).
A. Acetyl-L-leucyl-L-leucyl-arginine(Mtr) (N methyl)-(N Methoxy)-amide: Acetyl-
Leu-
Leu-OH (133 mg) and arginine (Mtr) -N-methyl-N-methoxy amide (200 mg) were
dissolved in
10 mL of DMF and were treated with 243 uL of DIEA and 212 mg of HBTU. The
reaction
stirred at room temperature for 1 S hours and was worked up according to
method A. Drying
over Na,SO,, rotary evaporation of the solvent and flash chromatography on
silica gel (50%
acetone in hexane) provided 270 mg of the title compound as a foam.
B. Acetyl-L-leucyl-N jl-j2-j(S phenyl)-1,3,4-oxadiazolylJcarbonylJ-4-j(4-
methoxy-
2,3,6-trimethyl-benzenesulphonyl)-guanidinoJ-butylJ-L-leucyl amide: 2-phenyl-
1,3,4-oxadiazole
(194 mg) in 2 mL of dry THF was chilled to -78° C. n-Butyllithium (1.46
mmole) was added as
a 2.5 M solution in hexanes. The reaction stirred 20 minutes at -?8°C
and was then placed in a
0°C cooling bath. Acetyl-Leu-Leu-Arg-(Mtr)-N-(CH3)-OCH, was then added
in 2 mL of dry
THF. The reaction was placed in a room temperature water bath and stirred 1
hour, then the
solution was chilled to 0 ° C, and 20 mL of saturated ammonium chloride
solution was added
under nitrogen with rapid stirring. After the several minutes of vigourous
stirring the solution
was extracted with EtOAc. The ethyl acetate solution was washed with saturated
sodium chloride
solution, dried over sodium sulfate and concentrated to a pale brown oil by
rotary evaporation.
C. Acetyl-L-leucyl-N jl-j2-j(S pherryl)-1,3,4-oxadiazolylJcarbo~rylJ-4-
(guanidi»o)-
butylJ-L-leucyl amide: One half of the crude product from step B was dissolved
in a pre-formed
solution of 2 mL of TFA and 100 uL thioanisole. The reaction stirred under
nitrogen for 4 hours.
The solvent was removed in vacuo, and the product was precipitated with dry
ether. The
precipitate was taken up in methanol and concentrated in vacuo, the residue
was triturated with
43
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WO 99/54317 PCT/US99/0$501
dry ether, and dried in vacuo to provide 22 mg of the title compound as a
colorless powder.
Samples for biological testing were obtained by reverse phase C 18
chromatography (5-80%
CH3CN, 0.1 % TFA, over 40 minutes). MS m/z (M+H)' 571 (CQ-0002).
S Example III -Synthesis ofAcetyl-L-leucyl-N jl-j3-j5-methyl)-1,2,4-
oxadiazolylJcarbonylJ-ethyl-
L-leucyl amide (CQ-0004)
A. N'-benryloxycarbonyl-L-alanine (N methyl-N methoxy)amide: Cbz-L-Alanine
(1.0 g)
was dissolved in 10 mL dry DMF with 1.55 mI, of DIEA. HBTU (1.78 g) was added
and the
reaction Stirred 30 minutes. Dimethyl hydroxyl amine hydrochloride (0.87 g)
was added
followed by 1.55 mL additional DIEA. The reaction stirred approximately 15
hours at room
temperature. Work up according to general method A, drying over anhydrous
sodium sulfate,
rotary evaporation, and drying under high vacuum produced 0.96 g of a
colorless solid.
B. N'-benzyloxycarbonyl-L-alaninal: A solution of 6 mL of 1 M lithium aluminum
hydride in THF was chilled under nitrogen to 0°C and a solution of
compound A (0.68 g ) in 4
mL DMF was added dropwise. After stirring 15 minutes at 0°C the
reaction was carefully
quenched with 20 mL of EtOAc and 10 mL of 10% KHSO, solution. The organic
layer was
washed with 1 N HCl and 10% NaHC03 solution. Drying over sodium sulfate,
removal of the
solvent by rotary evaporation, and drying under high vacuum provided 0.38 g of
a colorless oil.
C. 2-(R,S)-3-(S)-j(benzyloxycarbonyl)amino)-1-hydroxy-butanenitrile: Compound
B
(1.2 g) triethylamine (0.532 mL) and acetone cyanohydrin (1.56 mL) were
dissovled in 10 mL
of dry CHZCIz and stirred at room temperature for approximately 1 S hours. The
solvent was
removed in vacuo and the residue was taken up in EtzO and washed with
saturated sodium
chloride solution. Drying over anhydrous sodium sulfate, rotary evaporation,
and pumping under
high vacuum provided 1.3 g of the cyanohydrin.
D. 2-(R,S)-3-(S)-j(benryloxycarborryl)amino)-1-acetoxy-butanenitrile: Compound
C
(1.3 g) was dissolved in 2 mL of dry pyridine and was treated with 3.17 mL of
acetic anhydride.
The reaction stirred at room temperature for 3 hours and was then diluted with
ethyl acetate and
washed with water. Drying of sodium sulfate, rotary evaporation, and pumping
under high
vacuum provided 1.34 g of the title compound as an oil.
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E. I-(R,S)-2-(S)-1-j(N hydroxy)carboximideamidoJ-I-acetoxy-2-
j(benryloxycarbonyl)-
amino) propane: Compound D (1.34 g) was dissolved in 21 mL of EtOH and 4.2 mL
of water
and treated with hydroxylamine hydrochloride (0.422 g) and sodium acetate
(0.991 g) and heated
at 40 C for 3 hours. The solvent was removed in vacuo and the residue was
suspended in EtOAc
S and washed with water. Drying over sodium sulfate and evaporation of the
solvent provided 1.1
g of crude rizaterial which was used without further purification.
F. 1-(R, S)-2-(S)-1- j3- j5-(methyl)-I , 2, 4-oxadiazvlylJ-I -acetoxy-2(benryl
oxycarbonyl)-
amino)) propane: Compound E (0.45 g) was suspended in 5 mL toluene and treated
with 185
uL of acetic anhydride. The reaction was refluxed for approximately 15 hours,
after which the
solvent was removed by rotary evaporation and purified by flash chromatography
on silica gel
eluting with 1:1 hexane:EtOAc to provide 0.36 g of title compound.
G. I -(R,S)-2-(S)-I -j3- j5-(methyl))-l, l, 4-oxadiazolylJ-2-
j(benzyloxycarbonyl)-aminoJ-
propan-I-ol: Acetate F (180 mg) was dissolved in 3 mL of MeOH and treated with
a solution of
90 mg of KZCO~ in 1 mL of water. After approximately 20 minutes the reaction
mixture was
1 S diluted with EtOAc and washed with water. Drying over MgSO~, rotary
evaporation and drying
under high vacuum provided 0.160 g of the title compound.
H. 1-(R,S)-2-(S)-I-j3-j5-(methyl)-1,2,4-oxadiazolylJ-2-amino) propan-l-of TFA
salt:
Compound G was taken in 2 mL of trifluoroacetic acid and chilled to
0°C. Thioanisole (100 uL)
was added and the reaction was allowed to warm to room temperature and stirred
approximately
15 additional hours. The solvent was removed in vacuo and traces of remaining
TFA were
removed by rotary evaporation from dichloromethane and methanol. The crude
product was
partially purified by elution through a pre-packed C18 mini-column (Waters Sep-
Pak) with
acetonitrile in water. Lyophilization of appropriate fractions provided the
title compound 0.14 g,
which was used without further purification.
1. 1-(R,S)-2-(S)-(Benzyloxycarbonyl)-L-leucyl-N jl-j(3-j5-(methyl)-1,2,4-
oxadiazolylJ-
hydroxymethylJ-ethyl J-L-leucine amide: Compound H (0.14 g) and Acetyl-Leu-Leu-
OH were
dissolved in DMF (3 mL) and were treated with DIEA (90 uL) and HBTU (234 mg).
The
reaction was allowed to stir approximately 1 S hours at room temperature. The
reaction mixture
was diluted with EtOAc and washed with water. The water wash was extracted
with
CA 02329712 2000-10-20
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dichloromethane. All organic layers were combined and concentrated in vacuo.
The residue was
purified by preparative C 18 reverse phase chromatography (5-60% CH3CN, 0.1 %
TFA) to
provide 0.110 g of the title compound upon lyophilization.
J. Acetyl-L-leucyl-N ~1-~3-~5-methyl-1,2,4-oxadiazolylJcarbonylJ-ethylJ-L-
leucyl amide:
N-chlorosuccinimide (75.4 mg was suspended in dry toluene and chilled to
0°C. Dimethyl
sulfide (60 uL) was added and the suspension stirred 30 minutes at 0°C
and was then chilled to -
25°C. Compound I ( 60 mg) was added in 2 mL dichloromethane and the
reaction stirred 2.5
hours at -25°C. Triethyl amine (84 uL) was added and the reaction
warmed to room
temperature. After stirring 1 hour the reaction mixture was diluted with EtOAc
and was washed
with water. Drying over anhydrous sodium sulfate and removal of the solvent by
rotary
evaporation provided 60 mg of crude product. Flash chromatography on silica
gel provided 30
mg of the title compound as a colorless solid. MS 424. (M+H).
1 H-NMR a 0.89 -0.94 (m, 12 H), 1.50 (d, J=9.6, 3H), 1.53-1.69 (m, 6H), 2.01
{s, 3H), 2.70 (s,
3H), 4.50-4.52 (m, 2H), 5.34-5.39 (m, 1H) 6.23 (d, J=11, 1H), 6.81 (d, J=10.9,
lI~, 7.07 (d,
J=8.8, 1 H). 13C-NMR a 12.4 , 17.6, 22.1, 22.2, 22.8 (2 carbons), 23.1, 24.7,
24.8, 40.8, 41.1,
52.7, 164.1, 170.3, 171.3, 172.3, 178.3, 190.
Example IV -Acetyl-L-leucyl-N ~l-~3-~S-methyl-1,2,4-oxadiazolylJcarbonylJ-4-
(guanidino)-
butylJ-L-leucyl amide (CQ-0007)
A. N'-t-butoxycarbonyl-L Arg(Mtr)-(N methyl-N methoxy)amide: Boc-L-Arg(Mtr)-OH
5.00 g (10.3 mmole) was suspended in dry DMF (10 mL),followed by N,O dimethyl
hydroxylamine hydrochloride (1.25 g) and DIEA (5.4 mL). HBTU (4.28 g) was
added and the
reaction stirred approximately 15 hours at room temperature. The reaction was
worked up
according to general method A, and the EtOAc solution was dried over Na2S0,
and concentrated
to 5.23 g of a colorless foam.
B. N'-t-butoxycarborryl-L-(Mtr)-argininal: Compound A (2 .00 g) was dissolved
in 20
mL of dry THF and chilled to 0°C. To this solution was added 4.72 mL of
a 1 M solution of
LiAIH, in THF, dropwise over 30 minutes at 0°C. The reaction was
quenched at 0°C by the
slow addition of 50 mL EtOAc, followed~by 15 mL of 10% KHS04 solution. The
mixture was
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WO 99/54317 PCT/US99/08501
partitioned between 100 mL EtOAc and 50 mh 1 N HCl solution. The organic layer
was washed
with 1 N HCl solution and saturated sodium chloride solution. The mixture was
dried over
anhydrous sodium sulfate and concentrated by rotary evaporation . Drying under
high vacuum
provided 1.74 g of a white solid.
C. 2-(R,S)-3-(S)-~(t-butoxycarbonyl)amino)-6-((4-methoxy-2, 3, 6-trimethyl-
benze»esulphonyl)-guanidinoJ-2-hydroxy-hexanenitrile: Compound B (1.70 g) was
dissolved in
25 mL of methanol and was treated with 0.941 g of potassium cyanide. The
reaction was allowed
to stir at room temperature for approximately 15 hours. The reaction mixture
was then
partitioned between 150 mL EtOAc and 25 mL 1 N HCI. The organic layer was
washed with 1 N
HCl and dried over anhydrous sodium sulfate solution. Rotary evaporation and
further drying
under high vacuum provided 1.62 g of the title compound.
D. 2-(R,S)-3-(S)-((t-butoxycarbonyl)amino)-6-~(4=methoxy-2, 3, 6- trimethyl-
benzenesulphonyl)-guanidinoJ-2-acetoxy-hexanenitrile: Compound C (1.62 g) was
dissolved in
10 mL of dry pyridine and treated dropwise with 0.62 mL of acetic anhydride.
The reaction was
allowed to stir at room temperature for 3 hours. The solution was diluted with
100 mL EtOAc
and washed three times with equal volumes of I N HCl after drying over
anhydrous sodium
sulfate the solution was concentrated by rotary evaporation to an oil, and
purified by flash
chromatography on silica gel (50-75% EtOAc in hexanes, step gradient) to
provide 0.79 g of the
title compound and 0.64 g of mixed fractions containing traces of compound B.
E. 1-(R,S)-2-(S)-1-((N hydroxy)carboximideamidoJ-1-acetoxy-2-~(t-
butoxycarbonyl)-
aminoJ-S-((4-methoxy-2,3,6-trimethyl-benzenesulphonyl)-guanidinoJ pentane:
Compound D
(0.79 g ) was dissolved in 45 cnL of EtOAc and 3.9 mL of water and was treated
with 0.174 g of
sodium acetate and 0.129 g of hydroxylamine hydrochloride. In an analogous
fashion the mixed
fractions containing compound D (0.64 g) were dissolved in 36.5 mL ethanol and
3.2 mL of
water and were treated with 0.141 g of sodium acetate and 0.105 g of
hydroxylamine
hydrochloride. The reactions were heated at 45 °C for 4 hours with
stirring. HPLC analysis
showed very similar profiles for both reactions. The reactions were diluted
with EtOAc, washed
with water and saturated sodium chloride solution and were then dried over
anhydrous sodium
sulfate and concentrated by rotary evaporation. The combined products were
purified by flash
47
CA 02329712 2000-10-20
WO 99/54317 PCT/US99/08501
chromatography (1-4 % MeOH in EtOAc, step gradient) to provide 0.77 g of the
title compound.
F. I -(R, S)-2-(S)-1- j3- j5-(methyl)-I , 2, 4-oxadiazolylJ-2-amino-S- j(4-
methoxy-2, 3, 6-
trimethyl-benzenesulphonyl)-guanidinoJ pentan-I-of triJluoroacetate salt:
Compound E (0.74 g)
was dissolved in 6.5 mL of dry chloroform and treated with 0.27 mL of triethyl
amine and 0.153
mL of acetic anhydride and allowed to stir 4 hours at room temperature. The
reaction was diluted
with 50 mL toluene and refluxed for approximately 15 hours in a 120°C
oil bath. The volatile
solvents were removed by rotary evaporation and the residue was worked up
according to
method A. Drying over sodium sulfate, concentration by rotary evaporation, and
flash
chromatography on silica gel eluting with EtOAc provided 0.34 g of a colorless
oil. A portion of
this material 0.17 g was dissolved in 4 mL of MeOH and chilled to 0°C.
To this solution was
added 90 uL of a 4 N solution of KzC03. The reaction stirred two hours and was
then partitioned
between 40 mL of EtOAc and 5 mL water. The organic layer was washed with
saturated sodium
chloride solution and dried over sodium sulfate. The ethyl acetate was removed
by rotary
evaporation and traces of ethyl acetate were removed by rotary evaporation
from
dichloromethane. The resulting residue was diluted in 1.33 mL of
dichloromethane and chilled
to 0°C. Trifluoroacetic acid (0.57 mL) was added and the reaction
stirred 1.5 hour at 0°C. The
solvent was rapidly removed in vacuo and the product was dissolved in
dichloromethane and
concentrated to dryness by rotary evaporation.
G. I-(R,S)-2-(S)-L-leucyl-N jl-j(3-j5-(methyl)-1,2,4-oxadiazolylJ-
hydroxymethylJ-4-j(4-
methoxy-2,3,6-trimethyl-benzenesulphonyl)-guanidino)J-butyl)-L-leucine amide:
Compound F
(146 mg) and Acetyl-Leu-Leu-OH (82 mg) were dissolved in 5 mL of dry DMF and
treated with
200 uL of DIEA, followed by 30 mg of HBTU. After 5 minutes an additional 100
uL of DIEA
was added and the reaction stirred approximately 15 hours at room temperature.
The reaction
was diluted with EtOAc and washed with saturated NaHC03 solution and saturated
sodium
chloride solution. After removal of the solvent by rotary evaporation the
product was purified
by preparative C18 reverse phase chromatography (5-60% CH3CN, 0.1% TFA) to
provide 122
mg of the title compound.
H. Acetyl-L-leucyl-N jl-j3-j(5-methyl)-1,2,4-oxadiazolyl)carbonyl)-4-j(4-
methoxy-
2,3,6-trimethyl-benzenesulphonyl)-guanidinoJ-butyl)-L-leucyl amide: N-
chlorosuccinimide (45
48
CA 02329712 2000-10-20
WO 99/54317 PCT/US99/0$501
mg) and dimethyl sulfide (61 uL) in 2.5 mL of toluene were chilled to
0°C with stirring. Stirred
at 0°C for 30 minutes. The mixture was then chilled to ~-25°C in
a dry ice/carbon tetrachloride
bath, then compound G (100 mg) was added by dropwise addition in a mixture of
2.5 mL of
dichloromethane and 1.5 mL of toluene. The reaction stirred at -25 °C
for 3 hours then 100 uL of
triethyl amine was added. After 5 minutes the cooling bath was removed, and
the reaction stirred
1 hour. The reaction mixture was diluted with EtOAc, and washed with saturated
sodium
bicarbonate solution and saturated sodium chloride solution. The solution was
dried over
anhydrous sodium sulfate solution and concentrated to an oil.
I. Acetyl-L-leucyl-N('I-j3-j(5-methyl)-1,2,4-oxadiazolylJcarbonylJ-4-
(guanidino)-butyl-
Z-leucyl amide: Compound H was taken up in 1.75 mL of TFA and chilled to
0°C. Thioanisole
(90 uL) was added and the reaction stirred 1 hour at 0°C, and 4 hours
at room temperature. The
volatile solvents were removed by rotary evaporation, and residual TFA was
removed by adding
dichloromethane and concentrating to dryness on the rotovap. Reversed phase C
18 preparative
chromatography provided the title compound. FAB MS m/z (M+H)r 509 (CQ-0008).
Example V - Synthesis of Acetyl-L-tyrosinyl-L-valyl-N jl - j2- j(S phenyl)-l,
3, 4-
oxadiazolylJcarbonylJ-2-carboxy-ethylJ-L-alanine amide (CQ-0010)
A. N'-Benryloxycarbonyl-L Aspartyl(O-t-butyl) N methyl-N methoxy amide: Cbz-L-
Aspartic Acid (0-t-butyl) (1.0 g, 2.93 mmole), N,O-dimethyl hydroxyl amine
hydrochloride
(0.357 g, 3.66 mmole, were suspended in 15 mL of DMF and treated with 1.53 mL
(8.79
mmoles) of DIEA under N~ atmosphere. HBTU (1.22 g, 3.22 mmoles) was added and
the
reaction stirred approximately 15 hours at room temperature. The reaction
mixture was worked
up according to general extractive work up method A. Drying over NazS04,
rotary evaporation
of the solvent and further drying under high vacuum provided 1.1 g as a
colorless glassy solid.
B. L-Aspartyl-(O-t-butyl) N methyl-N methoxy amide: Compound A (1 g) was
dissolved
in 20 mL methanol containing 5% (v/v) formic acid. The solution was
deoxygenated with
nitrogen bubbling then treated with approximately 200 mg of palladium black.
The reaction
stirred under nitrogen for 3 hours, and was then filtered through celite. The
celite was washed
well with methanol and the filtrates were combined and concentrated by rotary
evaporation.
49
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WO 99/54317 PCT/US99/08501
Residual methanol and formic acid were chased off by the addition and rotary
evaporation of
CHZCIz and 50:50 CHZCIz:hexane. Drying under high vacuum provided 790 mg of an
oil.
C. Acetyl-L-tyrosinyl-L-Valyl-L-Alanyl-L-Aspartyl-(O-t-butyl) N methyl-N
methoxy
amide: Compound B (150 mg) and Acetyl-Tyr-Val-Ala-OH (230 mg , prepared using
S conventional peptide synthesis) were combined and suspended in 10 mL of DMF.
DIEA (305
uL) was added followed by HBTU. Product from normal extractive work up, and
the 1 N HCl
washes were combined after evaporation and purified by preparative HPLC
chromatography (S -
60% CH3CN 0.1 % TFA, over 30 minutes) to provide a lyophilized fraction of 85
mg of 94%
pure material, which was carried on to the anion coupling reaction.
D. Acetyl-L-tyrosinyl-L-valyl-N jl-j2-j(5 phenyl)-1,3,4-oxadiazolylJcarbonylJ
2-
(carboxy-t-butyl)-ethylJ-L-alanine amide: 2-Phenyl-1,3,4,-oxadizaole (i69 mg,
1.16 mmole)
was dissolved in 2 mL dry THF, and chilled to -78 C. n-Butyl lithium (510 uL,
2.5 M solution
in hexane) was added via syrringe, after 20 minutes compound C (88 mg, 0.145
mmole) was
added via syrringe in 3 mL dry THF and the reaction was allowed to warm to
room temperature.
After 15 minutes 20 mL of saturated NH4C1 solution was carefully added under
nitrogen, and the
solution was rapidly stirred for several minutes. The resulting solution was
extracted with
EtOAc, dried over Na2S0,, and concentrated. The resulting product was
dissolved in HZO/
CH3CN and concentrated by freeze-drying. Reverse phase preparative HPLC
chromatography
(5-60% CH3CN, 0.1 % TFA, 30 minute gradient) provided 25.8 mg of a colorless
powder upon
lyophilization.
E. Acetyl-L-tyrosirryl-L-valyl-N jl-j2-j(S phenyl)-1,3,4-oxadiazolylJcarbonylJ-
2-
carboxy-ethylJ-L-alanine amide: Compound D (25 mg) was treated with 2 mL of
TFA and
stirred at room temperature for 2 hours. The TFA was removed on the rotovap,
and remaining
entrained solvent was removed by adding CH2Clz and CH~CN and evaporating. The
crude
product was purified by reverse phase HPLC chromatography (5- 60% CH3CN,
0.1%.TFA, 30
minute gradient). Lyophilization of appropriate fractions provided 15.7 mg of
a colorless
lyophilate. Maldi MS M+Na 659 observed. MS FAB (M+H)' 637.
1 H-NMR: a 0.7? (m, 6H), 1.1-1.2 (m, 3H), 1.74 (s , 3H) 1.9 ( m, 1 H), 2.57 -
2.8 (m, 2H), 2.75-
3.34 (m, 2H) 4.14 (m, 1 H) 4.3 (m, 1 H), 4.44 (m, 1 H) 5.3 (m, 1 H), 6.61 (m,
2H), 7.01 (m, 2H),
CA 02329712 2000-10-20
WO 99/54317 PCT/US99/08501
7.70 (m, 3H) 7.73 (m, 2H) 7.74 - 8.00 (m, 2H) 8.1 (m, 2H) 8.78 (m, 1H), 9.13
(bs, 1H) 12.65 (bs,
1H). "C-NMR - d 17.8, 17.9, 19.0, 22.3, 30.6, 34.9, 36.3, 47.5, 52.7, 54.0,
57.0, 114.6, 122.3,
127.2, 129.5, 129.9, 132.9, 155.6, 159.6, 164.9, 169.0, 170.2, 171.1, 171.3,
172.2, 172.3, 183.8.
Example VI -Acetyl-L Aspartyl-Valyl-N (1-~2-((S phenyl)-1,3,4-oxadiazolylJ
carbonyl)-2-
(carboxy)-ethyl)-L=glutamyl amide (CQ-0011)
A. Acetyl-L-Aspartyl(Ot-Bu)-L-Valyl-L-Glutamyl (O-t-Bu)-L-Aspartyl-(O-t-butyl)
N
methyl-N-methoxy amide: Acetyl-Asp(O-t-Bu)-Val-Glu-(O-t-Bu)-OH (0.302 g, 0.586
mmoles,
prepared by conventional peptide synthesis) and H-Asp-(O-t-Bu)-N-(CH3)-OCH3
(0.150 g, 0.645
mmole, prepared as in example VIII) were combined in 5 mL of DMF and DIEA (305
uL) was
added. HBTU (277 mg) was added. After 2 hours an additional 200 uL of DIEA was
added and
the reaction was allowed to stir approximately 15 hours at room temperature.
The reaction was
worked up according to method A, dried over Na=SO., and concentrated to an
oil. Preparative
reverse phase chromatography (C18, 5 - 60% CH3CN, 0.1% TFA, 30 minute
gradient), and
lyophilization of appropriate fractions provided 0.231 g of a colorless
lyophilate.
B. Acetyl-L-Aspartyl (O-t-Bu)-Valyl-N ~1-~2-((S phenyl)-1, 3, 4-
oxadiazolylJcarborrylJ 2-
(carboxy-O-t-butyl)-ethyl)-L-glutamyl(O-t -Bu) amide: 2-Phenyl-1,3,4;
oxadiazole (161 mg, 1.1
mmole) was dissolved in 2 mL dry THF, and chilled to -78 °C. n-Butyl
lithium (485 uL, 2.5 M
solution in hexane) was added via syringe, after 20 minutes compound A (100
mg, 0.138
mmole) was added via syringe in 3 mL dry THF and the reaction was allowed to
warm to room
temperature. After 60 minutes 20 mL of saturated NH4Cl solution was carefully
added under
nitrogen, and the solution was rapidly stirred for several minutes. The
resulting solution was
extracted with EtOAc, dried over NazS04 and concentrated. The resulting
product was dissolved
in HZO/ CH3CN and concentrated by freeze-drying. Reverse phase preparative
HPLC
chromatography (5-60% CH,CN, 0.1% TFA, 30 minute gradient) provided 34 mg of a
colorless
powder upon lyophilization.
C. Acetyl-L Aspartyl-Valyl-N (1-(2-((S pherryl)-1,3,4-oxadiazolylJ carborrylJ-
2
(carboxy)-ethyl)-L-glutamyl amide: Compound B (25 mg) was treated with 2 mL
tnL of TFA
and stirred at room temperature for 5.75 hours. The TFA was removed on the
rotovap, and
51
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WO 99/54317 PCT/US99/08501
remaining entrained solvent was removed by adding CHZCIZ and CH~CN and
evaporating. The
crude product was purified by reverse phase HPLC chromatography (5-60% CH3CN,
0.1 % TFA,
30 minute gradient). Lyophilization of appropriate fractions provided 15.1 mg
of a colorless
lyophilate. Maldi MS M+Na 669 observed. MS FAB (M+H)' 647.
'H-NMR 6 0.73 (m, 6H), 1.75 - 1.95 (m, 2H), 1.82 (s, 3H), 1.88 (m, 2H), 2.40 -
2.70 (m, 2H)
2.75 -3.05 (m, 2H), 4.14 (m, 1 H), 4.30 (m, 1 H), 4.58 (m, 1 H), 5.33 (m, 1 H)
7.55 (m, I H) 7.61-
7.73 (m, 3H), 8.02 (m, 1H), 8.10 (m, 2H), 8.25 (m, 1H) 8.72 - 8.82 {m, 1H),
12.3 (bs, 3H). "C-
NMR a 17.5, 18.9, 22.3, 27.2, 29.8, 30.6, 34.8, 35.5, 49.3, 51.2, 52.7, 57.0,
122.3, 127.2, 129.5,
132.9, 159.6, 164.9, 169.4, 170.4, 170.5, 171.2, 171.3, 173.7, 183.8.
Example VII - General extractive work up Method A
The reaction mixture was diluted with 5-10 volumes of EtOAc and washed three
times
each with equivalent volumes of 1 N HCl solution, then saturated NaHC03
solution, and finally
with saturated NaCI solution.
Example VIII - Inhibitory Activity Against Cathepsin B and L, Papain and
Gingipain
The enzyme cathepsin B (E.C. 3.4.22.01) was obtained from Calbiochem (San
Diego,
CA); cathepsin L (E.C. 3.4.22.15) from Athens Research and Technology Inc.
(Athens,GA); and
papain (E.C. 3.4.22.02) from Sigma (St. Louis, MO).
Cbz-Phe-Arg-NHMec (-NHMec: 7-(4-methyl)coumarylamide) was obtained from
BachemCalifonzia, inc. (Torrance, CA). All other reagents were obtained from
Sigma.
The enzymes used in enzyme assays with methylcoumarylamides were activated as
described elsewhere (Barnet, et al., Methods Enrymol. 80:535-56I (1981);
Briimme, et al.,
Biochem. J., 264: 475-481 (1989). Cathepsin L was assayed in 0.34 M sodium
acetate buffer, pH
5.5, containing 0.1 % (v/v) Brij 35, 2.5 mM dithiothreitol (DTT) and 5 mM Na2-
EDTA.
Cathepsin B was assayed under the same conditions, except that the buffer was
adjusted to pH 6.
Papain was assayed in 50 mM sodium phosphate buffer, pH 6.8, containing 0.2 M
sodium
chloride, 2 mM DTT, 1 mM Na2-EDTA and 0.025 % Brij 35 (v/v).
Initial velocities of enzymatic reactions were measured
spectrofluorometrically (.1~ = 370
52
CA 02329712 2000-10-20
WO 99/54317 PCT/US99/08501
nm, ~.~m 460 nm) with a Quanta Master QM1 (Photon Technologies International,
South
Brunswick, N.l). Stock solutions of the enzymes were diluted into the buffer,
equilibrated at
room temperature, and preincubated without or with increasing concentrations
of inhibitors. The
reactions were started by addition of substrate. A total of 4 to 8 inhibitor
concentrations were
used to determine IC5° values. In all cases the substrate
concentrations were much smaller than
the IC", value, and the ICso values measured approximated the K; directly
(Cheng, et al.,
Biochemical Pharmacology, 22:3099-3108 (1973)).
Gingipain assay - All assays were carried out in a 96 well microtiter plate
reader and
cleavage of BAPNA (Na-benzoyl-DL-arginine p-nitroanilide hydrochloride) was
detected at 405
nm.
All assays were performed as follows: 180 pl assay buffer (50 mM Tris, 5 nM
CaClz and
10 nM cysteine, at pH 7.6) was mixed with 10 pl gingipain R (RGP). The mixture
was
incubated for 5 min. at room temperature to reduce and activate RGP. 10 pl of
each inhibitor
were added at various concentrations. These mixtures were incubated for 10
min. at room
temperature to allow the inhibitors to complex with RGP. SO pl of 10 mM BAPNA
substrate
was added. A two minute assay was performed with a final volume of 250 pl, and
a final
BAPNA concentration of 2 mM.
2 mM BAPNA was sufficient excess of substrate such that substrate depletion
did not
occur within a 10 minute assay time.. For this reason, two minute assays were
performed
whereby the Vm,~ in mOD/min change in absorbance at 405 nm was used as the
initial velocity
reading. In order to titrate RGP against leupeptin and to determine %
activity, these velocity
readings were transformed on a percent scale where the 100% control contained
no inhibitor.
The initial velocity values were also entered into Graphpad Prism regression
program along with
the various inhibitor concentrations to obtain the IC,° values. All
data represents the minimum
of duplicates, and at times triplicate scts.
Assay results are presented in Table 3. As shown, CM-0019B is an inhibitor of
papain
and cathepsin L and more selective against cathepsin B than is leupeptin.
Compound CQ-0002,
which shares the same recognition sequence {Leu-Leu-Arg) with the broad
spectrum inhibitor
leupeptin, is nearly as potent as leupeptin versus cathepsin B, but
surpisingly has.,a much higher
53
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WO 99/54317 PCT/US99/08501
degree of specificity. In addition, compound CQ-0002 inhibits gingipain R with
a potency
equivalent to that of leupeptin. Compounds CQ-0004 and CQ-0008 are also potent
and selective
cathepsin L inhibitors.
Table 3.
K; (nM] Values
for Cysteine
Protease
Inhibitors
COMPOUND PAPAW CATHEPSIN.B'CATHEPSIN GINGIPAIN
L' R'
Leupeptin 1.0 t 0.06 6.1 t 1.2 0.62 t 0.10 20.8 (IC50)
CM-0019Bb 85 3,000 100
CQ-0002 1,2001280 324 t 46 6.0 f 0.98 28 (IC50)
CQ-0004 25600 t 27200 t 190061 t 14
5340
CQ-0008 8590 t 18601240 t 182 7.13 t 0.32
' Human enzyme.
b "B"denotes remake of larger quantities of corresponding CQ number.
Example IX - Inhibitory Activity Against Caspases
Assay for ICE inhibition
To examine the ability of the caspase family inhibitors, CQ-0010 and CQ-0011,
to inhibit
human IL-1 (3 production, two different assays were employed. In the first,
the human monocytic
cell line, THP-1, was stimulated with E. colt lipopolysaccharide (LPS serotype
0127-88; Sigma
Chemical Co., St. Louis, MO) in the presence and absence of the inhibitors.
This cell line
synthesizes and secretes IL-1 ~i and TNFa as well as other cytokines upon LPS
stimulation. The
second assay used freshly-isolated human whole blood similarly stimulated with
LPS.
THP-1 assay: Two x 106 THP-1 cells were added to 24 well plates in 1 ml RPMI
supplemented with 1% FCS, glutamine and 5 x 10'5 M mercaptoethanol. Two-fold
serial
dilutions of the inhibitors, CQ-0010, CQ-0011 and the commmercially available
Ac-YVAD-
CHO (Biomol Research Laboratories Inc., Plymouth Meeting, PA), were
preincubated with the
cells for 15 min at 37°C. LPS was then added to a final concentration
of 1 ug/ml and the plates
54
CA 02329712 2000-10-20
WO 99/54317 PCT/US99/08501
incubated for 4 hr at 37°C. All incubations were carried out in a
humidified incubator with 5%
COZ in air.
Supernatants were harvested after 4 hr and assayed by ELISA for the presence
of TNFa
and IL-1 (3 using commercially available kits (PerSeptive Biosystems,
Framingham, MA and
R&D Systems, Minneapolis, MN, respectively).
Human whole blood assay: Heparinized whole blood (19.7 U heparin per ml) from
healthy volunteers was collected and dispensed into 12 x 75 mm polystyrene
tubes (0.25 ml per
tube). The inhibitors, CQ-0010, CQ-0011 and Ac-YVAD-CHO were dissolved in
DMSO, then
diluted and added to the tubes in 0.25 ml and preincubated with the blood for
15 min at 37°C.
LPS was then added to a final concentration of 10 or 100 ug/ml.
The tubes were loosely-capped and incubated in a water bath for 4 hr at
37°C after which
they were immersed briefly in an ice-water bath. Supernatants were harvested
by centrifugation
and stored at -70°C. The presence of TNFa and IL-1 (i was detected by
commercially-available
ELISA kits.
Assay for other caspase and granryme B inhibition
Inhibition constants were measured photometrically for YAMA (caspase 3), Lap3
(caspase 7), FLICE (caspase 8), Mch2 (caspase 6) and granzyme B. The buffer
used for all
enzymes consisted of 50 mM Hepes, 100 mM sodium chloride, 10% (v/v) sucrose,
0.1% (v/v)
CHAPS and 10 mM dithiothreitol (DTT). In the case of granzyme B, only 1 mM DTT
was used.
Enzymes were incubated at 37°C for 10 minutes in 100 pL well plates and
synthetic
substrate and inhibitor were added simultaneously. Final substrate
concentration was 20 ~M in
all cases. The synthetic substrate Ac-DEVD-pNA was used for all caspases and
Succ-AAPD-
pNA was used for graazyme B. The appearance of product was monitored over 10
minutes at
410 nm using a Spectromax 340 and ICS curves were calculated from the initial
slopes at
varying inhibitor concentrations and inhibition constants were calculated.
The results are shown in Table 4.
CA 02329712 2000-10-20
WO 99/54317 PCT/US99/08501
Table 4.
Inhibition of
Caspases -
Comparison
with Ac-YVAD-CHO
Caspases Ac-YVAD-CHO Ac-YVAD-het Ac-DVED-het
(CQ-0010) (CQ-0011)
yAMA (CPP32, 20 3.3 s0.1
Caspase 3)
Mch2 (Caspase 100 33 6.7
6)
Lap3 Not Active Not Active 50.03 (slow)
(Mch3, Caspase
7)
FLICE 3.7 s0.02 50.03 (slow)
(MchS, Caspase
8)
ICE 0.3b 0.3b 3b
(Caspase 1) 0.3 - 0.5' 0.3 - 0.5'
Granzyme Not Active Not Active Not Active
' Values given are lt; Iri ilM, unless Vmarwmo utumamu.
IC50 (i1M) values of reduction of IL-1 p release from THP-1 cell line.
' IC50 (lIM) values of reduction of IL-1 ~i release in whole blood assay.
The results indicate that CQ-0010 is an extremely potent and specific
inhibitor of IL-1 ~i
production, capable of almost completely inhibiting the production of this
cytolcine at 5 pM
(Figure 1) while having no dose-dependent effect on levels of TNFa produced
(results not
shown). The IC~o of CQ-0010 was estimated from these dose curves to be 0.3
IIM. CQ-0011 also
inhibited IL 1 p production but with approximately 10-fold less potency
(Figure 1; Table 4).
In the whole blood assay, CQ-0010 was again equipotent to Ac-YVAD-CHO with an
ICso
of 0.3-0.5 pM (Figures 2a and b).
It should be noted that CQ-0010 was equipotent to the aldehyde equivalent (Ac-
YVAD-
CHO) in inhibiting ICE, but showed improved inhibition against FLICE with a K;
of s 20 nM.
56
CA 02329712 2000-10-20
WO 99/54317 PCT/US99/0850i
Compound CQ-0011 is a potent inhibitor of Lap3 and FLICE. The compounds are
selective and
potent caspase inhibitors as shown by their inactivity with respect to
granryme B.
10
20
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CA 02329712 2000-10-20
WO 99/54317 PCT/US99/08501
The following references are incorporated herein:
1. Appleyard G., et al., J. Virol., 66:363-366
(1985).
2. Alessio M., et al., Eur J. Haematol., 45:78-81
(1990).
3. Barr P., et al., BiolTech., 12:487-493 (May
1994).
3a. Barrett A., et al. ICOP Newsletter, 1-2 (Dec.
1996).
4. Dinarello C., et al., New Eng. J. of Med.,
328:106-I 13 (1993).
5. Discipio R., et al. Immunolo,~, 87:660-667
(1996).
6. Dolle R., et al., J. Med Chem., 39:2438-2440
(1996).
7. Elliott E., et al., Per. in Drug Disc. and
Des:, 6:12-32 (1996).
8. Gorbalenya A., et al., Per. in Drug Disc. and
Des., 6:64-86 (1996).
9. Gordon S., Meth. in Enz., 244:568-581 (1994).
10. Gordon S., Sem. in Thromb. and Hemo., 18,4:424-433
(1992).
11. Grakoui A., et al., Proc. Natl. Acad Sci. LISA,
90:10583-10587 {1993).
12. Hewitt, C., J. Exp. Med, 182:1537-1544 (1995).
14. Jewell D., et al., Biochem., 31,34:7862-7869
(1992).
15. Kalsheker N., et al., Biochem. and Biophys.
Res.~ Comm., 221:59-61 (1996).
16. Karlsson,1., et al., Neurobio. ofAging, 16,6:901-906
(i995).
17. Muller-Ladner U, et al., Pers. in Drug Disc.
and Des., 6:87-98 (1996).
18. Li Z., et al., J. Med Chem., 39:4089-4098 (1996).
19. Malcolm B., et al., Biochem., 34:8172-8179
(1995).
20. Tushar P., et al., FASEB, 10:587-597 (1996).
21. Figueiredo-Pereira M., et al., J. oJNeurochem.,
62:1989-1994 (1994).
22. Rasnick D., Pers. in Drug Disc. and Des., 6:47-63
(1996).
23. Robertson C., et al., Pers. in Drug Disc. and
Des., 6:99-118 (1996).
23a. Rockett, K., et al., FEBS, 259,2:257-259 (1990).
24. Jean D., et al., Biochem. J., 312:961 (1995).
25. Storer, A., Pers. in Drug Disc. and Des., 6:33-46
(1996).
26. Squier M., et al., J. oJCell. Phys.,159:229-237
(1994).
58
CA 02329712 2000-10-20
WO 99/54317 PCT/US99/08501
27. Takeda A., et al., FEBS, 359:78-80 (1995).
28. Tchoupe J., et al., BBA, 1076:149-151 (1991).
29. Yoshida K., et al., Jap. Cir. J., 59:40 (1995).
30. Wingrove,1., J. Bio. Chem., 267,26:18902-18907 (1992).
31. WO 96/16080
32. U.S.5,498,616
33. WO 95/26958
34. WO 96/30396
3~5. Krausslich et aL, Ann. Rev. Biochem., 57:701-54 (1988).
36. Livingston, J. Cell. Biochem., 64:19-26 (1997).
37. Matsumura et al., J. Cardio. Pharm. 22:135-142 (1993).
38. Iqbal M., Bioorg. & Med. Chem. Lett., 7:539-544 (1997).
39. Tao, M., et al., Bioorg. & Med Chem. Lett., 5:3009-3012 (1996).
40. Miller, J. Cell. Biochem., 64:2-10 (1997).
41. Alnemri,J. Cell. Biochem., 64:33-42 (1997).
42. Broemme et al., JBC, 269:30238-30242 (1994).
43. Broernme et al., Biochem. J., 264:475-481 (1989).
44. Bossard et al., J. Biol. Chem., 271:12517-12524 (1996).
45. Dolle et al., J. Med Chem., 39:2438-2440 (1996).
46. Molla et al., J. Virology, (Aug. 1993).
47. Scott et al., JBC 268, 7935-7942 (1993).
48. Talanian et al., J.B.C., 272:9677-9682 (1997).
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