U.S. patent application number 10/127324 was filed with the patent office on 2003-05-22 for caspase inhibitors and uses thereof.
Invention is credited to Diu-Hercend, Anita, Golec, Julian, Hercend, Thierry, Knegtel, Ronald, Lang, Paul, Miller, Andrew, Miller, Karen, Mortimore, Michael, Weber, Peter.
Application Number | 20030096737 10/127324 |
Document ID | / |
Family ID | 23092531 |
Filed Date | 2003-05-22 |
United States Patent
Application |
20030096737 |
Kind Code |
A1 |
Diu-Hercend, Anita ; et
al. |
May 22, 2003 |
Caspase inhibitors and uses thereof
Abstract
The present invention relates to novel classes of compounds of
formula I which are caspase and TNF-alpha inhibitors. This
invention also relates to pharmaceutical compositions comprising
these compounds. The compounds and pharmaceutical compositions of
this invention are particularly well suited for inhibiting caspase
and TNF-alpha activity and consequently, can be advantageously used
as agents against caspase-, interleukin-1-("IL-1"), apoptosis-,
interferon-.gamma. inducing factor- (IGIF), interferon-.gamma.-
("IFN-.gamma."), or TNF-alpha mediated diseases, including
inflammatory diseases, autoimmune diseases, destructive bone
disorders, proliferative disorders, infectious diseases, and
degenerative diseases. This invention also relates to processes for
preparing the compounds of this invention. This invention also
relates to methods for inhibiting caspase and TNF-alpha activity
and decreasing IGIF production and IFN-.gamma. production and
methods for treating caspase-, interleukin-1, apoptosis-, and
interferon-.gamma.-, and TNF-alpha mediated diseases using the
compounds and compositions of this invention.
Inventors: |
Diu-Hercend, Anita;
(Charenton le Pont, FR) ; Golec, Julian; (Ashbury,
GB) ; Hercend, Thierry; (Chareton le Pont, FR)
; Knegtel, Ronald; (Abingdon, GB) ; Lang,
Paul; (Viuz-en-Sallaz, FR) ; Miller, Andrew;
(Upton, GB) ; Miller, Karen; (Newbury, GB)
; Mortimore, Michael; (Burford, GB) ; Weber,
Peter; (Abingdon, GB) |
Correspondence
Address: |
FISH & NEAVE
1251 AVENUE OF THE AMERICAS
50TH FLOOR
NEW YORK
NY
10020-1105
US
|
Family ID: |
23092531 |
Appl. No.: |
10/127324 |
Filed: |
April 19, 2002 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
60285051 |
Apr 19, 2001 |
|
|
|
Current U.S.
Class: |
514/1.5 ;
514/1.9; 514/12.2; 514/16.4; 514/16.8; 514/16.9; 514/17.8;
514/18.6; 514/18.9; 514/19.6; 514/19.8; 514/3.7; 514/3.8; 514/4.3;
514/6.9; 544/157; 544/162; 544/337; 544/386; 544/59; 546/22;
546/226 |
Current CPC
Class: |
A61P 17/04 20180101;
A61P 19/00 20180101; A61P 37/00 20180101; A61P 37/02 20180101; A61P
11/00 20180101; C07D 405/06 20130101; A61P 9/00 20180101; A61P
37/08 20180101; A61P 19/02 20180101; A61P 29/00 20180101; A61P
37/06 20180101; A61P 31/00 20180101; A61P 19/10 20180101; A61P 1/16
20180101; A61P 1/00 20180101; A61P 17/06 20180101; A61P 31/06
20180101; C07D 417/06 20130101; C07D 401/06 20130101; A61P 31/14
20180101; A61P 11/02 20180101; A61P 31/12 20180101; A61P 21/04
20180101; A61P 25/14 20180101; A61P 13/12 20180101; A61P 25/16
20180101; A61P 31/18 20180101; A61P 11/06 20180101; A61P 1/02
20180101; A61P 27/02 20180101; A61P 1/04 20180101; A61P 35/02
20180101; A61P 43/00 20180101; A61P 25/08 20180101; A61P 21/00
20180101; A61P 7/06 20180101; A61P 3/04 20180101; A61P 25/00
20180101; A61P 9/04 20180101; A61P 35/00 20180101; C07D 409/06
20130101; A61P 25/28 20180101; A61P 17/00 20180101; A61P 39/02
20180101; A61P 3/10 20180101; A61P 17/14 20180101; A61P 17/02
20180101; C07D 211/60 20130101; A61P 7/00 20180101; A61P 27/14
20180101; A61P 31/04 20180101; A61P 31/20 20180101; A61P 35/04
20180101; A61P 41/00 20180101; A61P 25/32 20180101; A61P 5/14
20180101; A61P 1/18 20180101; A61P 9/10 20180101; A61P 19/06
20180101 |
Class at
Publication: |
514/7 ; 514/19;
544/59; 544/157; 544/162; 544/386; 544/337; 546/22; 546/226 |
International
Class: |
A61K 038/05; C07K
005/04; C07D 279/12; C07D 211/06; C07D 241/04; C07D 265/30 |
Claims
We claim:
1. A compound of the formula I: 70wherein: R.sup.1 is hydrogen, CN,
CHN.sub.2, R, or --CH.sub.2Y; R is an aliphatic group, a
substituted aliphatic group, an aryl group, a substituted aryl
group, an aralkyl group, a substituted aralkyl group, a
non-aromatic heterocyclic group, or a substituted non-aromatic
heterocyclic group; Y is an electronegative leaving group, --OR,
--SR, --OC.dbd.O(R), or --OPO(R.sup.3) (R.sup.4); R.sup.3 and
R.sup.4 are independently R or OR; R.sup.2 is CO.sub.2H,
CH.sub.2CO.sub.2H, or optionally substituted esters, amides or
isosteres thereof; A is C.dbd.O or SO.sub.2; X.sup.1 is oxygen,
sulfur, --NH, or --CH.sub.2, wherein --NH is optionally substituted
by an alkyl group, a cycloalkyl group, a (cycloalkyl)alkyl group,
an amino acid N-terminal protecting group, or COR and --CH.sub.2 is
optionally substituted by fluorine, an alkyl group, a cycloalkyl
group, a (cycloalkyl)alkyl group, an aralkyl group, an aryl group,
an alkyloxy group, an alkylthioxy group, an aryloxy group, an
arylthioxy group, an oxo group (i.e., .dbd.O), or a NHCOR group;
X.sup.2 is oxygen, sulfur, --NH, or --CH.sub.2, wherein --NH is
optionally substituted by an alkyl group, or an amino acid
N-terminal protecting group and --CH.sub.2 is optionally
substituted by an alkyl group, an aryl group, an alkyloxy group, an
alkylthioxy group, an aryloxy group, an arylthioxy group, or an oxo
(i.e., .dbd.O) group, a NHCOR group; X.sup.1 and X.sup.2 optionally
form part of a phenyl ring that is fused to the adjoining ring Q;
X.sup.3 is CH.sub.2 or X.sup.2 and X.sup.3 optionally form part of
a phenyl ring that is fused to the adjoining ring Q, provided that
when X.sup.2 forms a ring with X.sup.3 then X.sup.2 does not form a
ring with X.sup.1; any two hydrogens attached to adjacent positions
in ring Q are optionally replaced by a double bond; and Z is an
optionally substituted ring selected from the group consisting of a
carbocyclic, an aryl, a saturated heterocycle, a partially
saturated heterocycle, and a heteroaryl wherein the ring is
connected to A at a ring carbon; or a pharmaceutically acceptable
derivative thereof.
2. The compound of claim 1 wherein R.sup.1 is CH.sub.2Y and Y is F,
OR, SR, or --OC(.dbd.O) (R).
3. The compound of claim 2 wherein Y is F.
4. The compound of claim 2 wherein R.sup.2 is CO.sub.2H, an ester,
amide, or carboxylic acid isostere.
5. The compound of claim 4 wherein R.sup.2 is CO.sub.2H.
6. The compound of claim 4 wherein X.sup.1 and X.sup.2 are each
CH.sub.2, or X.sup.1 and X.sup.2 combine to form part of an
optionally substituted phenyl ring fused to ring Q.
7. The compound of claim 6 wherein X.sup.1 and X.sup.2 are each
CH.sub.2.
8. The compound of claim 7 wherein A is CO.
9. The compound of claim 8 wherein Z is an optionally substituted
aryl which is connected to A at a ring carbon.
10. The compound of claim 1 selected from Table 1 below:
2TABLE 1 Representative Compounds 71 No. Z 1 72 2 73 3 74 4 75 5 76
6 77 7 78 8 79 9 80 10 81 11 82 12 83 13 84 14 85 15 86 16 87 17 88
18 89 19 90 20 91 21 92 22 93 23 94 24 95 25 96
11. A pharmaceutical composition comprising: a) a compound or a
pharmaceutically acceptable derivative thereof according to any one
of claims 1-10; and b) a pharmaceutically acceptable carrier,
adjuvant or vehicle.
12. A method for treating or preventing a disease selected from a
group consisting of an IL-1 mediated disease, an apoptosis mediated
disease, a TNF-alpha mediated disease, an inflammatory disease, an
autoimmune disease, a destructive bone disorder, a proliferative
disorder, an infectious disease, a degenerative disease, a skin
disease, a disease associated with cell death, an excess dietary
alcohol intake disease, a viral mediated disease, retinal disorder,
uveitis, inflammatory peritonitis, osteoarthritis, pancreatitis,
asthma, adult respiratory distress syndrome, glomerulonephritis,
rheumatoid arthritis, systemic lupus erythematosus, scleroderma,
chronic thyroiditis, Grave's disease, autoimmune gastritis,
diabetes, autoimmune hemolytic anemia, autoimmune neutropenia,
thrombocytopenia, chronic active hepatitis, myasthenia gravis,
inflammatory bowel disease, Crohn's disease, psoriasis, atopic
dermatitis, contact dermatitis, scarring, graft vs host disease,
organ transplant rejection, organ apoptosis after burn injury,
osteoporosis, leukemias and related disorders, myelodysplastic
syndrome, multiple myeloma-related bone disorder, acute myelogenous
leukemia, chronic myelogenous leukemia, metastatic melanoma,
Kaposi's sarcoma, multiple myeloma, haemorrhagic shock, sepsis,
septic shock, burns, trauma, systemic inflammatory response
syndrome, multiple organ dysfunction syndrome, Shigellosis,
Alzheimer's disease, Parkinson's disease, Huntington's disease,
Kennedy's disease, prion disease, cerebral ischemia, epilepsy,
myocardial ischemia, acute and chronic heart disease, myocardial
infarction, congestive heart failure, atherosclerosis, coronary
artery bypass graft, spinal muscular atrophy, amyotrophic lateral
sclerosis, multiple sclerosis, HIV-related encephalitis, aging,
alopecia, neurological damage due to stroke, ulcerative colitis,
traumatic brain injury, spinal chord injury, hepatitis-B,
hepatitis-C, hepatitis-G, yellow fever, dengue fever, Japanese
encephalitis, various forms of liver disease, renal disease,
polycystic kidney disease, H. pylori-associated gastric and
duodenal ulcer disease, HIV infection, tuberculosis, and meningitis
in a subject comprising the step of administering to said subject a
compound or a pharmaceutically acceptable derivative thereof
according to any one of claims 1-10 or a pharmaceutical composition
according to claim 11.
13. The method according to claim 12, wherein the disease is an
apoptosis mediated disease, an inflammatory disease, an autoimmune
disease, a destructive bone disorder, a proliferative disorder, an
infectious disease, a degenerative disease, a disease associated
with cell death, an excess dietary alcohol intake disease, a viral
mediated disease, inflammatory peritonitis, glomerulonephritis,
diabetes, autoimmune hemolytic anemia, autoimmune neutropenia,
thrombocytopenia, chronic active hepatitis, scarring, graft vs host
disease, organ transplant rejection, osteoporosis, leukemias and
related disorders, myelodysplastic syndrome, metastatic melanoma,
haemorrhagic shock, sepsis, septic shock, burns, trauma, systemic
inflammatory response syndrome, multiple organ dysfunction
syndrome, Shigellosis, Alzheimer's disease, Parkinson's disease,
Huntington's disease, Kennedy's disease, prion disease, cerebral
ischemia, epilepsy, myocardial ischemia, acute and chronic heart
disease, myocardial infarction, congestive heart failure,
atherosclerosis, coronary artery bypass graft, spinal muscular
atrophy, amyotrophic lateral sclerosis, multiple sclerosis,
HIV-related encephalitis, aging, alopecia, neurological damage due
to stroke, traumatic brain injury, spinal chord injury,
hepatitis-B, hepatitis-C, hepatitis-G, various forms of liver
disease, renal disease, polycystic kidney disease, H.
pylori-associated gastric and duodenal ulcer disease, HIV
infection, tuberculosis, or meningitis.
14. A method for inhibiting a caspase-mediated function in a
subject comprising the step of administering to said subject a
compound or a pharmaceutically acceptable derivative thereof
according to any one of claims 1-10 or a pharmaceutical composition
according to claim 11.
15. A method for decreasing TNF-alpha levels or activity in a
subject comprising the step of administering to said subject a
compound or a pharmaceutically acceptable derivative thereof
according to any one of claims 1-10 or a pharmaceutical composition
according to claim 11.
16. A method for decreasing IGIF- or IFN-.gamma. production in a
subject comprising the step of administering to said subject a
compound or a pharmaceutically acceptable derivative thereof
according to any one of claims 1-10 or a pharmaceutical composition
according to claim 11.
17. A method for treating complications associated with coronary
artery bypass grafts comprising the step of administering to said
subject a compound or a pharmaceutically acceptable derivative
thereof according to any one of claims 1-10 or a pharmaceutical
composition according to claim 11.
18. A method for preserving cells comprising the step of bathing
the cells in a solution of a compound or a pharmaceutically
acceptable derivative thereof according to any one of claims
1-10.
19. A method according to claim 18, wherein said compound or
pharmaceutically acceptable derivative thereof is used for an organ
transplant or for preserving blood products.
20. A method of treating cancer comprising the step of
administering a compound or a pharmaceutically acceptable
derivative thereof according to any one of claims 1-10 or a
pharmaceutical composition according to claim 11 wherein said
compound or composition is used as a component of
immunotherapy.
21. The method according to any one of claims 12-19 wherein said
compound, derivative or composition is administered with an
additional therapeutic agent.
22. The method of claim 21 wherein said additional therapeutic
agent is selected from a group consisting of a thrombolytic agent,
an anti-inflammatory agent, a matrix metalloprotease inhibitor, a
lipoxygenase inhibitor, a cytokine antagonist, an
immunosuppressant, an anti-cancer agent, an anti-viral agent, a
cytokine, a growth factor, an immunomodulator, a prostaglandin, and
an anti-vascular hyper-proliferation compound.
23. A method for inhibiting TNF-mediated conditions in a subject
comprising the step of administering to said subject a compound or
a pharmaceutically acceptable derivative thereof according to any
one of claims 1-10 or a pharmaceutical composition according to
claim 11.
24. The method according to claim 23, wherein said TNF-mediated
conditions are selected from a group consisting of restinosis,
inflammatory diseases of the central nervous system, demyelinating
diseases of the nervous system, multiple sclerosis, septic
arthritis, aneurysmal aortic disease, traumatic joint injury,
peridontal disease, macular degeneration, diabetic retinopathy,
occular inflammation, keratoconus, Sjogren's syndrome, corneal
graft rejection, cachexia, and anorexia.
25. A method for identifying a compound that decreases TNF-alpha
levels in a cell culture comprising the steps of administering a
compound or a pharmaceutically acceptable derivative thereof
according to any one of claims 1-10 or a pharmaceutical composition
according to claim 11 to the cell culture and comparing the amount
of TNF-alpha present to the amount of TNF-alpha present in a cell
culture that has not been treated with the compound.
26. A method for identifying a compound that decreases TNF-alpha
activity in a cell culture comprising the steps of administering a
compound or a pharmaceutically acceptable derivative thereof
according to any one of claims 1-10 or a pharmaceutical composition
according to claim 11 to the cell culture and comparing the amount
of TNF-alpha present to the amount of TNF-alpha present in a cell
culture that has not been treated with the compound.
27. A method for decreasing TNF-alpha levels or activity in a cell
culture comprising the step of administering to the cell culture a
compound or a pharmaceutically acceptable derivative thereof
according to any one of claims 1-10 or a pharmaceutical composition
according to claim 11.
28. A kit comprising a caspase inhibitor and a tool for measuring
TNF-alpha levels or activity.
29. A method for identifying a compound for decreasing TNF-alpha
levels in a subject comprising administering a compound or a
pharmaceutically acceptable derivative thereof according to any one
of claims 1 to 10 or a pharmaceutical composition comprising the
compound and comparing the TNF-alpha levels present in the subject
before and after treatment with the compound.
30. A method for identifying a compound for decreasing TNF-alpha
activity in a subject comprising administering a compound or a
pharmaceutically acceptable derivative thereof according to any one
of claims 1 to 10 or a pharmaceutical composition comprising the
compound and comparing the TNF-alpha activity present in the
subject before and after treatment with the compound.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional Patent
Application No. 60/285,051, filed Apr. 19, 2001, the content of
which is incorporated by reference.
FIELD OF THE INVENTION
[0002] This invention is in the field of medicinal chemistry and
relates to novel compounds, and pharmaceutical compositions
thereof, that inhibit caspases and/or TNF-alpha that mediate cell
apoptosis and inflammation and inhibit pathophysiologic effects of
excessive amounts of TNF-alpha in a mammal. The invention also
relates to processes for preparing and methods of using the
compounds and pharmaceutical compositions of this invention to
treat diseases where caspase and/or TNF-alpha activity is
implicated.
BACKGROUND OF THE INVENTION
[0003] Apoptosis, or programmed cell death, is a principal
mechanism by which organisms eliminate unwanted cells. The
deregulation of apoptosis, either excessive apoptosis or the
failure to undergo it, has been implicated in a number of diseases
such as cancer, acute inflammatory and autoimmune disorders,
ischemic diseases and certain neurodegenerative disorders (see
generally Science, 1998, 281, 1283-1312; and Ellis et al., Ann.
Rev. Cell. Biol., 1991, 7, 663).
[0004] Caspases are a family of cysteine protease enzymes that are
key mediators in the signaling pathways for apoptosis and cell
disassembly (Thornberry, Chem. Biol., 1998, 5, R97-R103). These
signaling pathways vary depending on cell type and stimulus, but
all apoptosis pathways appear to converge at a common effector
pathway leading to proteolysis of key proteins. Caspases are
involved in both the effector phase of the signaling pathway and
further upstream at its initiation. The upstream caspases involved
in initiation events become activated and in turn activate other
caspases that are involved in the later phases of apoptosis.
[0005] Caspase-1, the first identified caspase, is also known as
interleukin converting enzyme or "ICE."Caspase-1 converts precursor
interleukin-1.beta. ("pIL-1.beta.") to the pro-inflammatory active
form by specific cleavage of pIL-1.beta. between Asp-116 and
Ala-117. Besides caspase-1 there are also eleven other known human
caspases, all of which cleave specifically at aspartyl residues.
They are also observed to have stringent requirements for at least
four amino acid residues on the N-terminal side of the cleavage
site.
[0006] The caspases have been classified into three groups
depending on the amino acid sequence that is preferred or primarily
recognized. The group of caspases, which includes caspases 1, 4,
and 5, has been shown to prefer hydrophobic aromatic amino acids at
position 4 on the N-terminal side of the cleavage site. Another
group which includes caspases 2, 3 and 7, recognizes aspartyl
residues at both positions 1 and 4 on the N-terminal side of the
cleavage site, and preferably a sequence of Asp-Glu-X-Asp. A third
group, which includes caspases 6, 8, 9 and 10, tolerates many amino
acids in the primary recognition sequence, but seems to prefer
residues with branched, aliphatic side chains such as valine and
leucine at position 4.
[0007] The caspases have also been grouped according to their
perceived function. The first subfamily consists of caspases-1
(ICE), 4, and 5. These caspases have been shown to be involved in
pro-inflammatory cytokine processing and therefore play an
important role in inflammation. Caspase-1, the most studied enzyme
of this class, activates the IL-1.beta. precursor by proteolytic
cleavage. This enzyme therefore plays a key role in the
inflammatory response. Caspase-1 is also involved in the processing
of interferon gamma inducing factor (IGIF or IL-18) which
stimulates the production of interferon gamma, a key
immunoregulator that modulates antigen presentation, T-cell
activation and cell adhesion.
[0008] The remaining caspases make up the second and third
subfamilies. These enzymes are of central importance in the
intracellular signaling pathways leading to apoptosis. One
subfamily consists of the enzymes involved in initiating events in
the apoptotic pathway, including transduction of signals from the
plasma membrane. Members of this subfamily include caspases-2, 8, 9
and 10. The other subfamily, consisting of the effector capsases 3,
6 and 7, is involved in the final downstream cleavage events that
result in the systematic breakdown and death of the cell by
apoptosis. Caspases involved in the upstream signal transduction
activate the downstream caspases, which then disable DNA repair
mechanisms, fragment DNA, dismantle the cell cytoskeleton and
finally fragment the cell.
[0009] Knowledge of the four amino acid sequence primarily
recognized by the caspases has been used to design caspase
inhibitors. Reversible tetrapeptide inhibitors have been prepared
having the structure
CH.sub.3CO--[P4]-[P3]-[P2]-CH(R)CH.sub.2CO.sub.2H where P2 to P4
represent an optimal amino acid recognition sequence and R is an
aldehyde, nitrile or ketone capable of binding to the caspase
cysteine sulfhydryl. Rano and Thornberry, Chem. Biol. 4, 149-155
(1997); Mjalli, et al., Bioorg. Med. Chem. Lett. 3, 2689-2692
(1993); and Nicholson et al., Nature 376, 37-43 (1995).
Irreversible inhibitors based on the analogous tetrapeptide
recognition sequence have been prepared where R is an
acyloxymethylketone (--COCH.sub.2OCOR"), wherein R' is exemplified
by an optionally substituted phenyl such as 2,6-dichlorobenzoyloxy,
and where R is COCH.sub.2X wherein X is a leaving group such as F
and Cl. Thornberry et al., Biochemistry 33, 3934 (1994); and Dolle
et al., J. Med. Chem. 37, 563-564 (1994).
[0010] The utility of caspase inhibitors to treat a variety of
mammalian disease states associated with an increase in cellular
apoptosis has been demonstrated using peptidic caspase inhibitors.
For example, in rodent models, caspase inhibitors have been shown
to reduce infarct size and inhibit cardiomyocyte apoptosis after
myocardial infarction, to reduce lesion volume and neurological
deficit resulting from stroke, to reduce post-traumatic apoptosis
and neurological deficit in traumatic brain injury, to effectively
treat fulminant liver destruction, and to improve survival after
endotoxic shock. Yaoita et al., Circulation, 97, 276 (1998); Endres
et al., J Cerebral Blood Flow and Metabolism, 18, 238, (1998);
Cheng et al., J. Clin. Invest., 101, 1992 (1998); Yakovlev et al.,
J Neuroscience, 17, 7415 (1997); Rodriquez et al., J. Exp. Med.,
184, 2067 (1996); and Grobmyer et al., Mol. Med., 5, 585
(1999).
[0011] In general, the peptidic inhibitors described above are very
potent against some of the caspase enzymes. However, this potency
has not always been reflected in cellular models of apoptosis. In
addition peptide inhibitors are typically characterized by
undesirable pharmacological properties such as poor oral
absorption, poor stability and rapid metabolism. Plattner and
Norbeck, in Drug Discovery Technologies, Clark and Moos, Eds.
(Ellis Horwood, Chichester, England, 1990).
[0012] Recognizing the need to improve the pharmacological
properties of the peptidic caspase inhibitors, peptidomimetic and
non-natural amino acid peptide inhibitors have been reported.
[0013] WO 96/40647 discloses ICE inhibitors of the formula: 1
[0014] wherein B is H or an N-terminal blocking group; R.sub.1 is
the amino acid side chain of the P.sub.1 amino acid residue wherein
the P.sub.1 amino acid is Asp; P.sub.n is an amino acid residue or
a heterocyclic replacement of the amino acid wherein the
heterocycle is defined in the application; R.sub.4 is hydroxyl,
alkoxyl, acyl, hydrogen, alkyl or phenyl; m is 0 or a positive
integer; and X is N, S, O, or CH.sub.2.
[0015] WO 97/22619 discloses inhibitors of interleukin-1.beta.
converting enzyme of the formula: 2
[0016] wherein X.sup.1 is --CH; g is 0 or 1; each J is
independently selected from the group consisting of --H, --OH, and
--F, provided that when a first and second J are bound to a C and
said first J is --OH, said second J is --H; m is 0, 1, or 2; T is,
inter alia, --CO.sub.2H; R.sub.1 is 3
[0017] where each Z is independently CO or SO.sub.2; R.sub.3 is as
defined in the application; each X is independently selected from
the group consisting of .dbd.N-- and .dbd.CH--; and R.sub.20 is
chosen from a group containing 4
[0018] WO 98/16502 discloses aspartate ester inhibitors of
interleukin-1.beta. converting enzyme of the formula: 5
[0019] wherein R.sup.1 is, inter alia,
R.sup.5N(R.sup.a)CHR.sup.6CO--; R.sup.2 is certain groups; R.sup.6
is H, C.sub.1-6 alkyl, --(CH.sub.2).sub.naryl,
--(CH.sub.2).sub.nCO.sub.2R.sup.a, hydroxyl substituted C.sub.1-6
alkyl, or imidazole substituted C.sub.1-6 alkyl; each R.sup.a is
independently hydrogen, C.sub.1-6 alkyl or (CH.sub.2).sub.naryl;
and R.sup.5 is, inter alia, CONR.sup.aR.sup.a.
[0020] WO 99/18781 discloses dipeptide apoptosis inhibitors having
the formula: 6
[0021] where R.sub.1 is an N-terminal protecting group; AA is a
residue of any natural .alpha.-amino acid, or .beta.-amino acid;
R.sub.2 is H or CH.sub.2R.sub.4 where R.sub.4 is an electronegative
LG such as F, Cl, TsO--, MeO--, ArO--, ArCOO--, ArN-- and ArS--;
and R.sub.3 is alkyl or H.
[0022] WO 99/047154 discloses dipeptide apoptosis inhibitors having
the formula: 7
[0023] where R.sub.1 is an N-terminal protecting group; AA is any
non-natural amino acid or amino acid residue; and R.sub.2 is an
optionally substituted alkyl or H as defined in the
application.
[0024] WO 00/023421 discloses (substituted) acyl dipeptide
apoptosis inhibitors having the formula: 8
[0025] where n is 0, 1, or 2; q is 1 or 2; A is a residue of any
natural or non-natural amino acid; B is a hydrogen atom, a
deuterium atom, C1-10 straight chain or branched alkyl, cycloalkyl,
phenyl, substituted phenyl, naphthyl, substituted naphthyl,
2-benzoxazolyl, substituted 2-oxazolyl, (CH.sub.2).sub.m
cycloalkyl, (CH.sub.2).sub.mphenyl, (CH.sub.2).sub.m(substituted
phenyl), (CH.sub.2).sub.m(1- or 2-naphthyl),
(CH.sub.2).sub.mheteroaryl, halomethyl, CO.sub.2R.sub.13,
CONR.sub.14R.sub.15, CH.sub.2ZR.sub.16, CH.sub.2OCOaryl,
CH.sub.2OCO(substituted aryl), CH.sub.2OCO(heteroaryl),
CH.sub.2OCO(substituted heteroaryl), or
CH.sub.2OPO(R.sub.17)R.sub.18, where R.sub.13, R.sub.14, R.sub.15,
R.sub.16, R.sub.17, R.sub.18 and m are as defined in the
application; R.sub.2 is selected from a group consisting of
hydrogen, alkyl, cycloalkyl, phenyl, substituted phenyl, and
(CH.sub.2).sub.mNH.sub.2; R.sub.3 is hydrogen, alkyl, cycloalkyl,
(cycloalkyl)alkyl, phenylalkyl, or substituted phenylalkyl; X is
CH.sub.2, C.dbd.O, O, S, NH, C(.dbd.O)NH or CH.sub.2OCONH; and Z is
an oxygen or a sulfur atom.
[0026] WO 00/061542 discloses dipeptide apoptosis inhibitors having
the formula: 9
[0027] where R.sub.1 is an optionally substituted alkyl or hydrogen
group; R.sub.2 is hydrogen or optionally substituted alkyl; Y is a
residue of a natural or non-natural amino acid; R.sub.3 is an
alkyl, saturated carbocyclic, partially saturated carbocyclic,
aryl, saturated heterocyclic, partially saturated heterocyclic or
heteroaryl group, wherein said group is optionally substituted; and
X is O, S, NR.sub.4, or (CR.sub.4R.sub.5).sub.n where R.sub.4 and
R.sub.5 are, at each occurrence, independently selected from the
group consisting of hydrogen, alkyl and cycloalkyl, and n is 0, 1,
2, or 3; or X is NR.sub.4, and R.sub.3 and R.sub.4 are taken
together with the nitrogen atom to which they are attached to form
a saturated heterocyclic, partially saturated heterocyclic or
heteroaryl group, wherein said group is optionally substituted; or
X is CR.sub.4R.sub.5, and R.sub.3 and R.sub.4 are taken together
with the carbon atom to which they are attached to form a saturated
carbocyclic, partially saturated carbocyclic, aryl, saturated
heterocyclic, partially saturated heterocyclic or oxygen-containing
heteroaryl group, wherein said group is optionally substituted; and
provided that when X is O, then R.sub.3 is not unsubstituted benzyl
or t-butyl; and when X is CH.sub.2, then R.sub.3 is not H.
[0028] Generally, the term tumor necrosis factor (TNF) refers to
two closely related cytokines (encoded by separate genes) known as
tumor necrosis factor-alpha (TNF, cachectin) and tumor necrosis
factor-beta (lymphotoxin, TNF-beta). Both cytokines interact with
the same cell membrane receptors, and both have been implicated as
pathogenic mediators of human illness.
[0029] TNF-alpha participates in the signaling pathways that
regulate cell apoptosis and inflammation. TNF-alpha is also known
as TNFSF2, TNFA and DIF. TNF-alpha is a pro-inflammatory mammalian
protein capable of inducing cellular effects by virtue of its
interaction with specific cellular receptors. It is produced
primarily by activated monocytes and macrophages.
Lipopoly-sacccharide (LPS, also called endotoxin), derived from the
cell wall of gram negative bacteria, is a potent stimulator of
TNF-alpha synthesis.
[0030] Due to the deleterious effects which can result from an
over-production or an unregulated production of TNF-alpha,
considerable efforts have been made to regulate the serum level of
TNF-alpha. The pathology of a number of diseases are affected by
TNF-alpha, including, restinosis, inflammatory diseases of the
central nervous system, demyelinating diseases of the nervous
system, multiple sclerosis, septic arthritis, aneurysmal aortic
disease, traumatic joint injury, peridontal disease, macular
degeneration, diabetic retinopathy, occular inflammation,
keratoconus, Sjogren's syndrome, corneal graft rejection, cachexia,
and anorexia.
[0031] While a number of caspase and TNF-alpha inhibitors have been
reported, it is not clear whether they possess the appropriate
pharmacological properties to be therapeutically useful. Therefore,
there is a continued need for small molecule caspase and TNF-alpha
inhibitors that are potent, stable, and have good penetration
through membranes to provide effective inhibition of apoptosis in
vivo. Such compounds would be extremely useful in treating the
aforementioned disease states where caspase enzymes and/or
TNF-alpha cytokines play a role.
SUMMARY OF THE INVENTION
[0032] It has now been found that compounds of this invention and
pharmaceutical compositions thereof are particularly effective as
inhibitors of caspases, regulators of TNF-alpha levels or activity
and inhibitors of cellular apoptosis and inflammatory responses.
These compounds have the general formula I: 10
[0033] wherein:
[0034] R.sup.1 is hydrogen, CN, CHN.sub.2, R, or --CH.sub.2Y;
[0035] R is an aliphatic group, a substituted aliphatic group, an
aryl group, a substituted aryl group, an aralkyl group, a
substituted aralkyl group, a non-aromatic heterocyclic group, or a
substituted non-aromatic heterocyclic group;
[0036] Y is an electronegative leaving group, --OR, --SR,
--OC.dbd.O(R), or --OPO(R.sup.3) (R.sup.4);
[0037] R.sup.3 and R.sup.4 are independently R or OR;
[0038] R.sup.2 is CO.sub.2H, CH.sub.2CO.sub.2H, or optionally
substituted esters, amides or isosteres thereof;
[0039] A is C.dbd.O or SO.sub.2;
[0040] X.sup.1 is oxygen, sulfur, --NH, or --CH.sub.2, wherein --NH
is optionally substituted by an alkyl group, a cycloalkyl group, a
(cycloalkyl)alkyl group, an amino acid N-terminal protecting group,
or COR and --CH.sub.2 is optionally substituted by fluorine, an
alkyl group, a cycloalkyl group, a (cycloalkyl)alkyl group, an
aralkyl group, an aryl group, an alkyloxy group, an alkylthioxy
group, an aryloxy group, an arylthioxy group, an oxo group (i.e.,
.dbd.O), or a NHCOR group;
[0041] X.sup.2 is oxygen, sulfur, --NH, or --CH.sub.2, wherein --NH
is optionally substituted by an alkyl group, or an amino acid
N-terminal protecting group and --CH2 is optionally substituted by
an alkyl group, an aryl group, an alkyloxy group, an alkylthioxy
group, an aryloxy group, an arylthioxy group, or an oxo (i.e.,
.dbd.O) group, a NHCOR group; X.sup.1 and X.sup.2 optionally form
part of a phenyl ring that is fused to the adjoining ring Q;
[0042] X.sup.3 is CH.sub.2 or X.sup.2 and X.sup.3 optionally form
part of a phenyl ring that is fused to the adjoining ring Q,
provided that when X.sup.2 forms a ring with X.sup.3, then X.sup.2
does not form a ring with X.sup.1;
[0043] any two hydrogens attached to adjacent positions in ring Q
are optionally replaced by a double bond; and
[0044] Z is an optionally substituted ring selected from the group
consisting of a carbocyclic, an aryl, a saturated heterocycle, a
partially saturated heterocycle, and a heteroaryl wherein the ring
is connected to A at a ring carbon.
[0045] The compounds of this invention are potent inhibitors of
caspase and TNF activity. They have inhibiting activity across a
range of caspase targets with good efficacy in cellular models of
apoptosis and inflammation. In addition, these compounds are
expected to have improved cell penetration and pharmacokinetic
properties and, as a consequence of their potency, have improved
efficacy against diseases where caspases and/or TNF-alpha are
implicated.
[0046] The invention also relates to methods for inhibiting the
release of TNF-alpha from various cells or decreasing TNF-alpha
levels or activity using the compounds and compositions of this
invention. The invention also relates to methods for identifying
agents useful for decreasing TNF-alpha levels or activity and
treating TNF-alpha mediated diseases. The invention additionally
relates to kits comprising a compound or composition of this
invention and a tool for measuring TNF-alpha levels or
activity.
DETAILED DESCRIPTION OF THE INVENTION
[0047] This invention provides novel compounds and pharmaceutically
acceptable derivatives thereof that are particularly effective as
caspase inhibitors and/or regulators of TNF-alpha levels or
activity. The invention also provides methods for using the
compounds to treat caspase and/or TNF-alpha mediated disease states
in mammals. The compounds have the general formula I: 11
[0048] wherein:
[0049] R.sup.1 is hydrogen, CN, CHN.sub.2, R, or --CH.sub.2Y;
[0050] R is an aliphatic group, a substituted aliphatic group, an
aryl group, a substituted aryl group, an aralkyl group, a
substituted aralkyl group, a non-aromatic heterocyclic group, or a
substituted non-aromatic heterocyclic group;
[0051] Y is an electronegative leaving group, --OR, --SR,
--OC.dbd.O(R), or --OPO(R.sup.3) (R.sup.4);
[0052] R.sup.3 and R.sup.4 are independently R or OR;
[0053] R.sup.2 is CO.sub.2H, CH.sub.2CO.sub.2H, or optionally
substituted esters, amides or isosteres thereof;
[0054] A is C.dbd.O or SO.sub.2;
[0055] X.sup.1 is oxygen, sulfur, --NH, or --CH.sub.2, wherein --NH
is optionally substituted by an alkyl group, a cycloalkyl group, a
(cycloalkyl)alkyl group, an amino acid N-terminal protecting group,
or COR and --CH.sub.2 is optionally substituted by fluorine, an
alkyl group, a cycloalkyl group, a (cycloalkyl)alkyl group, an
aralkyl group, an aryl group, an alkyloxy group, an alkylthioxy
group, an aryloxy group, an arylthioxy group, an oxo group (i.e.,
.dbd.O), or a NHCOR group;
[0056] X.sup.2 is oxygen, sulfur, --NH, or --CH.sub.2, wherein --NH
is optionally substituted by an alkyl group or an amino acid
N-terminal protecting group and --CH.sub.2 is optionally
substituted by an alkyl group, an aryl group, an alkyloxy group, an
alkylthioxy group, an aryloxy group, an arylthioxy group, or an oxo
(i.e., .dbd.O) group, a NHCOR group; X.sup.1 and X.sup.2 optionally
form part of a phenyl ring that is fused to the adjoining ring
Q;
[0057] X.sup.3 is CH.sub.2 or X.sup.2 and X.sup.3 optionally form
part of a phenyl ring that is fused to the adjoining ring Q,
provided that when X.sup.2 forms a ring with X.sup.3, then X.sup.2
does not form a ring with X.sup.1;
[0058] any two hydrogens attached to adjacent positions in ring Q
are optionally replaced by a double bond; and
[0059] Z is an optionally substituted ring selected from the group
consisting of a carbocyclic, an aryl, a saturated heterocycle, a
partially saturated heterocycle, and a heteroaryl wherein the ring
is connected to A at a ring carbon.
[0060] As used herein, the following definitions shall apply unless
otherwise indicated. The term "condition" or "state" refers to any
disease, disorder or effect that produces deleterious biological
consequences in a subject.
[0061] According to this invention, "TNF" or "TNF alpha" refers to
TNF-alpha.
[0062] The term "subject" refers to an animal, or to one or more
cells derived from an animal. Preferably, the animal is a mammal,
most preferably a human. The cells can be in any form, including
but not limited to cells retained in tissue, cell clusters,
immortalized cells, transfected or transformed cells, and cells
derived from an animal that have been physically or phenotypically
altered.
[0063] The term "patient" as used in this application refers to any
mammal, preferably humans.
[0064] The term "interferon gamma inducing factor" or "IGIF" refers
to a factor which is capable of stimulating the endogenous
production of IFN-.gamma..
[0065] The term "aliphatic" means straight chain, branched or
cyclic C.sub.1-C.sub.12 hydrocarbons which are completely saturated
or contain one or more units of unsaturation. For example, suitable
aliphatic groups include substituted or unsubstituted linear,
branched or cyclic alkyl, alkenyl, or alkynyl groups and hybrids
thereof such as (cycloalkyl)alkyl, (cycloalkenyl)alkyl and
(cycloalkyl)alkenyl. The term "alkyl" used alone or as part of a
group or larger moiety refers to both straight and branched chains
containing one to twelve carbon atoms.
[0066] The term "halogen" means F, Cl, Br, or I. The term
"heteroatom" means nitrogen, oxygen or sulfur and shall include any
oxidized form of nitrogen and sulfur, such as N(O), S(O), and
S(O).sub.2 and the quaternized form of nitrogen. It is stood that
the compounds of the present invention are limited to those
existing in nature or chemically stable.
[0067] A combination of substituents or variables is permissible
only if such a combination results in a stable or chemically
feasible compound. A stable compound or chemically feasible
compound is one that is not substantially altered when kept at a
temperature of 40.degree. C. or less, in the absence of moisture or
other chemically reactive conditions, for at least a week.
[0068] The term "aryl", used alone or as part of a group or larger
moiety, refers to monocyclic or polycyclic aromatic carbon ring
systems, and monocyclic or polycyclic heteroaromatic ring systems
containing one or more heteroatoms, having five to fourteen atoms.
Such groups include, but are not limited to, phenyl, naphthyl,
anthryl, furanyl, thienyl, pyrrolyl, oxazolyl, thiazolyl,
imidazolyl, pyrazolyl, isoxazolyl, isothiazolyl, oxadiazolyl,
triazolyl, thiadiazolyl, pyridinyl, pyridazinyl, pyrimidinyl,
pyrazinyl, triazinyl, indolizinyl, indolyl, isoindolyl, indolinyl,
benzofuranyl, benzothiophenyl, indazolyl, benzimidazolyl,
benzthiazolyl, purinyl, quinolizinyl, quinolinyl, isoquinolinyl,
cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl,
1,8-naphthyridinyl, pteridinyl, carbazolyl, acridinyl, phenazinyl,
phenothiazinyl, phenoxazinyl, tetrahydrofuranyl, phthalimidinyl,
tetrazolyl, and chromanyl.
[0069] The term "aralkyl" refers to an alkyl substituted by an aryl
group. The term "heteroaryl" refers to an aryl group containing one
or more heteroatoms. The term "heteroaralkyl" refers to an aralkyl
group containing one or more heteroatoms.
[0070] The term "heterocyclic group" or "heterocycle" refers to
saturated and unsaturated monocyclic or polycyclic ring systems
containing one or more heteroatoms and with a ring size of three to
eight. Such groups include, but are not restricted to, aziranyl,
oxiranyl, azetidinyl, tetrahydrofuranyl, pyrrolinyl, pyrrolidinyl,
dioxolanyl, imidazolinyl, imidazolidinyl, pyrazolinyl,
pyrazolidinyl, pyranyl, piperidinyl, dioxanyl, morpholinyl,
dithianyl, thiomorpholinyl, piperazinyl, trithianyl, quinuclidinyl,
oxepanyl, and thiepanyl. The term "heterocyclic ring", whether
saturated or unsaturated, also refers to rings that are optionally
substituted. The term "heterocyclylalkyl" refers to an alkyl group
substituted by a heterocyclic ring.
[0071] The term "carbocyclic group" or "carbocyclyl" refers to
saturated or unsaturated non-aromatic monocyclic or polycyclic
carbon ring systems which can be fused to aryl or heterocyclic
groups. Examples could include cyclohexyl, cyclopentyl, cyclobutyl,
cyclopropyl, indanyl, tetrahydronaphthyl and the like. The term
"carbocyclylalkyl" refers to an alkyl substituted by a carbocyclic
group.
[0072] An aryl group (including a heteroaryl) or an aralkyl
(including a heteroaralkyl) group, such as benzyl or phenethyl, can
contain one or more substituents. Examples of suitable substituents
of an aryl or aralkyl group include halogen, CF.sub.3, --R.sup.5,
--OR.sup.5, --OH, --SH, --SR.sup.5, protected OH such as acyloxy
and those described in Wuts and Greene, Protective Groups in
Organic Synthesis, 3.sup.rd Edition John Wiley & Sons, 1999),
--NO.sub.2, --CN, --NH.sub.2, --NHR.sup.5, --N(R.sup.5).sub.2,
--NHCOR.sup.5, --NHCONHR.sup.5, --NHCON(R.sup.5).sub.2,
--NR.sup.5COR.sup.5, --NHCO.sub.2R.sup.5, --CO.sub.2R.sup.5,
--CO.sub.2H, --COR.sup.5, --CONHR.sup.5, --CON(R.sup.5).sub.2,
--S(O).sub.2R.sup.5, --SONH.sub.2, --S(O)R.sup.5,
--SO.sub.2NHR.sup.5, or --NHS(O).sub.2R.sup.5, where R.sup.5 is an
aliphatic or a substituted aliphatic group, preferably having one
to three carbons, or an aryl or a substituted aryl group, with the
proviso that when R.sup.5 is an substituted aryl group, said aryl
can not be substituted by an substituted aryl.
[0073] An aliphatic group or a non-aromatic heterocyclic ring can
contain one or more substituents. Examples of suitable substituents
of an aliphatic group or a non-aromatic heterocyclic ring include
those listed above for an aryl or aralkyl group as well as the
following: .dbd.O, .dbd.S, .dbd.NNHR.sup.6, .dbd.NN(R.sub.6).sub.2,
.dbd.N--OR.sup.6, .dbd.NNHCOR.sup.6, .dbd.NNHCO.sub.2R.sup.6,
.dbd.NNHSO.sub.2R.sup.6, and .dbd.NR.sup.6, wherein R.sub.6 is an
aliphatic group or a substituted aliphatic group.
[0074] A substitutable nitrogen on an aromatic or non-aromatic
heterocyclic ring can be optionally substituted. Suitable
substituents on the nitrogen include R.sup.6, COR.sup.6,
S(O).sub.2R.sup.6, and CO.sub.2R.sup.6.
[0075] The term "electronegative leaving group" has the definition
known to those skilled in the art (see March, Advanced Organic
Chemistry, 4.sup.th Edition, John Wiley & Sons, 1992). Examples
of electronegative leaving groups include halogens such as F, Cl,
Br, and I, aryl- and alkyl-sulfonyloxy groups,
trifluoro-methanesulfonyloxy, OR.sup.7, SR.sup.7,
--OC.dbd.O(R.sup.7), --OPO(R.sup.8)(R.sup.9), where R.sup.7 is an
aliphatic group, an aryl group, an aralkyl group, a carbocyclic
group, a carbocyclylalkyl group, a heterocyclic group, or an
heterocyclylalkyl group; and R.sup.8 and R.sup.9 are independently
R.sup.7 or OR.sup.7.
[0076] The term "amino acid N-terminal protecting group" has the
definition known to those skilled in the art. Examples of amino
acid N-terminal protecting groups include those described in Wuts
and Greene, Protective Groups in Organic Synthesis, 3.sup.rd
Edition John Wiley & Sons, 1999).
[0077] Isosteres or bioisosteres of carboxylic acids and esters
result from the exchange of an atom or a group of atoms to create a
new compound with similar biological properties to the parent
carboxylic acid or ester. The bioisosteric replacement can be
physicochemically or topologically based. An example of an
isosteric replacement for a carboxylic acid is CONHSO.sub.2J where
J is an alkyl group such as methyl, ethyl, propyl, butyl, and the
like.
[0078] Compounds of this invention where R.sup.2 is CO.sub.2H or
CH.sub.2CO.sub.2H, .gamma.-ketoacids or .delta.-ketoacids
respectively, can exist in solution as either the open form (a) or
the cyclized hemiketal form (b) (y=1 for .gamma.-ketoacids, y=2 for
.delta.-ketoacids). The representation herein of either isomeric
form is meant to include the other. 12
[0079] Likewise it will be apparent to one skilled in the art that
certain compounds of this invention can exist in tautomeric forms
or hydrated forms, all such forms being within the scope of the
invention. Unless otherwise stated, structures depicted herein are
also meant to include all stereochemical forms of the structures;
i.e., the R and S configurations for each asymmetric center.
Therefore, single stereochemical isomers as well as enantiomeric
and diastereomeric mixtures of the present compounds are within the
scope of the invention. Unless otherwise stated, structures
depicted herein are also meant to include compounds that differ
only in the presence of one or more isotopically enriched atoms.
For example, compounds having the present structures except for the
replacement of a hydrogen by a deuterium or tritium, or the
replacement of a carbon by a .sup.13 C- or .sup.14C-enriched carbon
are within the scope of the application.
[0080] A preferred R.sup.1 group is CH.sub.2Y where Y is an
electronegative leaving group, OR, SR, or --OC(.dbd.O)(R) and most
preferably Y is F.
[0081] Preferably R.sup.2 is CO.sub.2H, esters, amides or isosteres
thereof.
[0082] X.sup.1 is preferably CH.sub.2; X.sup.2 is preferably
CH.sub.2; or X.sup.1 and X.sup.2 combine to form part of an
optionally substituted phenyl ring fused to ring Q. More preferably
X.sup.1 and X.sup.2 are both CH.sub.2.
[0083] A is preferably CO.
[0084] Z is preferably an optionally substituted aryl which is
connected to A at a ring carbon.
[0085] Representative examples of compounds of the present
invention are shown below in Table 1.
1TABLE 1 Representative Compounds 13 No. Z 1 14 2 15 3 16 4 17 5 18
6 19 7 20 8 21 9 22 10 23 11 24 12 25 13 26 14 27 15 28 16 29 17 30
18 31 19 32 20 33 21 34 22 35 23 36 24 37 25 38
[0086] The compounds of this invention can be prepared in general
by methods known to those skilled in the art for analogous
compounds, as illustrated by the general Schemes I and II below and
by the preparative examples that follow. 39
[0087] Reagents: (a) CbzOSuc/THF/TEA; (b) LiOH/THF/H.sub.2O; (c)
EDC/DMAP/HOBt; (d) H.sub.2/10% Pd on C/EtOAc; (e) TBTU/DIPEA/DMF;
(f) Dess-Martin periodinane; (g) TiCl.sub.4/DCM; and (k)
TFA/DCM.
[0088] Scheme I shows a general approach for making the present
compounds. The starting ester hydrochloride 1 is first protected as
a carbamate using a known amino acid N-protecting protocol, for
example Cbz-OSuc (benzyloxycarbonyl-O-succinimidyl) in THF
(tetrahydrofuran) in the presence of base, such as TEA
(triethylamine, step a). The ester 2 is then hydrolyzed using base
or, when the ester is a t-butyl group, using trifluoroacetic acid
(TFA). The acid 3 is then coupled with the amino alcohol 4, using
for example, EDC (1-(3-dimethyl-aminopropyl)-3-ethylcarb- odiimide
hydrochloride), DMAP (4-dimethylaminopyridine) and HOBt
(1-hydroxybenzo-triazole), to provide 5. Depending on the nature of
R.sup.1 and R.sup.2, an amino ketone can be used in place of 4,
which avoids the subsequent oxidation step. In the case of
fluoromethyl ketones where R.sup.1 is CH.sub.2F, the amino alcohol
4 can be obtained according to the method of Revesz et al.,
Tetrahedron Lett., 1994, 35, 9693. The carbamate 5 is then
deprotected using catalytic hydrogenation, for example, H.sub.2
with Pd on C in EtOAc (ethyl acetate), or acidolysis. The amine 6
is then N-substituted with the desired acylating or sulfonylating
agent using standard methods as shown, for example, by step e, TBTU
(2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium
tetrafluoroborate) in DMF (dimethylformamide) in the presence of
DIPEA (diisopropylethylamine). The hydroxyl group in compound 7 is
then oxidized by standard methods as shown, for example, by step f.
Finally, compound 8 is treated appropriately according to the
nature of R.sup.2 to generate I, using TiCl.sub.4 in DCM
(dichloromethane) or TFA in DCM. For example, if I requires R.sup.2
to be a carboxylic acid, then R.sup.2 in 4 is preferably an ester
and the final step in the scheme is hydrolysis. 40
[0089] Reagents: (h) DIPEA/DMF/TBTU/.sup.{circle over
(Z)}--CO.sub.2H; (l)
[0090] DIPEA/DCM/.sup.{circle over (Z)}--COCl; (i)
LiOH/THF/H.sub.2O; (m) KOH/MeOH/H.sub.2O; (j) EDC/DMAP/HOBt; (f)
Dess-Martin periodinane; (g) TiCl.sub.4/DCM; and (k) TFA/DCM.
[0091] Scheme II represents an alternative approach for making the
present compounds. The starting ester hydrochloride 1 is first
reacted with either a carboxylic acid or an acid chloride using
known amide bond forming reactions. The amide 9 is then hydrolyzed
using base. The acid 10 is then coupled with the amino alcohol 4 to
provide 11. Depending on the nature of R.sup.1 and R.sup.2, an
amino ketone can be used in place of 4, which avoids the subsequent
oxidation step. In the case of fluoromethyl ketones where R.sup.1
is CH.sub.2F, the amino alcohol 4 can be obtained according to the
method of Revesz et al., Tetrahedron Lett., 1994, 35, 9693. The
hydroxyl group in compound 11 is then oxidized by standard methods
as shown, for example, by step f. Finally compound 12 is treated
appropriately according to the nature of R.sup.2 to generate I. For
example, if I requires R.sup.2 to be a carboxylic acid, then
R.sup.2 in 4 is preferably an ester and the final step in the
scheme is hydrolysis.
[0092] Certain compounds of this invention can be obtained as
follows. The parent heterocyclic esters 1 or their acids or
derivatives used in scheme I are either commercially available or
can be prepared using standard methods. For example, the parent
heterocyclic ester 1 where X.sup.1 are each CH.sub.2 is
commercially available (H-homoproline-OMe). The parent heterocyclic
ester 1, where X.sup.1 is CH.sub.2; and X.sup.2 is oxygen, can be
prepared by standard methods (Wolfe et al., Tetrahedron Lett.,
1979, 3913). The parent heterocyclic ester 1, where X.sup.1 is
CH.sub.2 and X.sup.2 is sulfur, can be prepared by standard methods
(Miyazaki et al., Bull. Chem. Soc. Jpn., 1993, 66, 536). The parent
heterocyclic ester 1, where X.sup.2 is CH.sub.2; and X.sup.1 is
oxygen, can be prepared by standard methods (Kogami et al., Bull.
Chem. Soc. Jpn., 1987, 60, 2963; Asher et al., Tetrahedron Lett.,
1981, 141; and Brown et al., J. Chem. Soc. Perkin Trans. 1,1985,
2577). The Fmoc N-protected amino acid is also commercially
available. The parent heterocyclic ester 1, where X.sup.2 is
CH.sub.2; and X.sup.1 is sulfur, can be prepared by standard
methods (Kogami et al., Bull. Chem. Soc. Jpn., 1987, 60, 2963; and
Sakai et al., Chem. Pharm. Bull., 1981, 29, 1554). The
corresponding free amino acid is also commercially available. The
parent heterocyclic esters 1, where X.sup.1-2 is CH.sub.2 and have
various substituents can be prepared by standard methods (Shuman et
al., J. Org. Chem., 1990, 55, 738; Agami et al., Synlett, 1997,
799; and Nazih et al., Synlett, 1998, 1337).
[0093] The compounds of this invention are designed, inter alia, to
inhibit caspase activity and/or decrease TNF-alpha levels or
activity. These compounds can be assayed, for example, for their
ability to inhibit apoptosis, inhibit the release of IL-1.beta.,
inhibit caspase activity, and/or decrease TNF-alpha levels or
activity. Assays for each of the activities are known in the art
and are described below in detail in the Examples. Accordingly,
these compounds are capable of targeting and inhibiting events in
caspase- (for example, IL-1-), apoptosis-, IGIF-, IFN-.gamma.-, and
TNF-.alpha.-mediated diseases, and the ultimate activity of the
relevant protein in inflammatory diseases, autoimmune diseases,
destructive bone, proliferative disorders, infectious diseases, and
degenerative diseases.
[0094] Compounds of this invention also inhibit conversion of
pro-IGIF into active, mature IGIF by inhibiting ICE. The term
"interferon gamma inducing factor" or "IGIF" refers to a factor
which is capable of stimulating the endogenous production of
IFN-.gamma..
[0095] Because ICE is essential for the production of mature IGIF
(IL-18), inhibition of ICE effectively blocks initiation of
IGIF-mediated physiological effects and symptoms, by inhibiting
production of mature IGIF. IGIF is in turn essential for the
production of IFN-.gamma.. ICE therefore effectively blocks
initiation of IFN-.gamma.- mediated physiological effects and
symptoms, by inhibiting production of mature IGIF and thus
production of IFN-.gamma..
[0096] Compounds of this invention also inhibit the release of
TNF-alpha from activated cells.
[0097] The pharmaceutical compositions and methods of this
invention, therefore, will be useful for controlling caspase and
TNF-alpha activity in vivo. The compositions and methods of this
invention will thus be useful for controlling caspase, IL-1, IGIF,
IFN-.gamma., or TNF-alpha levels in vivo and for treating or
reducing the advancement, severity or effects of caspase, IL-1-,
apoptosis-, IGIF-, IFN-.gamma.-, or TNF-alpha mediated conditions,
including diseases, disorders or effects.
[0098] According to another embodiment, the invention provides a
composition comprising a compound of this invention or a
pharmaceutically acceptable derivative thereof, as described above,
and a pharmaceutically acceptable carrier.
[0099] According to another embodiment, the compositions of this
invention can further comprise another therapeutic agent. Such
agents include, but are not limited to, a thrombolytic agent such
as tissue plasminogen activator and streptokinase, an
anti-inflammatory agent, a matrix metalloprotease inhibitor, a
lipoxygenase inhibitor, a cytokine antagonist, an
immunosuppressant, an anti-cancer agent, an anti-viral agent, a
cytokine, a growth factor, an immunomodulator (e.g., bropirimine,
anti-human alpha interferon antibody, IL-2, GM-CSF, methionine
enkephalin, interferon alpha, diethyldithiocarbamate, tumor
necrosis factor, naltrexone and rEPO), a prostaglandin, or an
anti-vascular hyperproliferation compound.
[0100] The term "pharmaceutically acceptable carrier" refers to a
non-toxic carrier that can be administered to a patient, together
with a compound of this invention, and which does not destroy the
pharmacological activity thereof.
[0101] Pharmaceutically acceptable carriers that can be used in
these compositions include, but are not limited to, ion exchangers,
alumina, aluminum stearate, lecithin, serum proteins, such as human
serum albumin, buffer substances such as phosphates, glycine,
sorbic acid, potassium sorbate, partial glyceride mixtures of
saturated vegetable fatty acids, water, salts or electrolytes, such
as protamine sulfate, disodium hydrogen phosphate, potassium
hydrogen phosphate, sodium chloride, zinc salts, colloidal silica,
magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based
substances, polyethylene glycol, sodium carboxymethylcellulose,
polyacrylates, waxes, polyethylene-polyoxypropyle- ne-block
polymers, polyethylene glycol and wool fat.
[0102] A "pharmaceutically acceptable derivative" means any
pharmaceutically acceptable salt, ester, salt of an ester or other
derivative of a compound of this invention which, upon
administration to a recipient, is capable of providing, either
directly or indirectly, a compound of this invention or an
inhibitorily active metabolite or residue thereof. The methods for
preparing salts or esters of a compound of this invention are known
to one of skill in the art.
[0103] Pharmaceutically acceptable derivatives of the compounds of
this invention include, without limitation, esters, amino acid
esters, phosphate esters, metal salts and sulfonate esters.
[0104] In pharmaceutical compositions comprising only a compound of
formula I as the active component, methods for administering these
compositions can additionally comprise the step of administering to
the subject an additional agent. Such agents include, but are not
limited to, a thrombolytic agent such as tissue plasminogen
activator and streptokinase, an anti-inflammatory agent, a matrix
metalloprotease inhibitor, a lipoxygenase inhibitor, a cytokine
antagonist, an immunosuppressant, an anti-cancer agent, an
anti-viral agent, a cytokine, a growth factor, an immunomodulator
(e.g., bropirimine, anti-human alpha interferon antibody, IL-2,
GM-CSF, methionine enkephalin, interferon alpha,
diethyldithiocarbamate, tumor necrosis factor, naltrexone and
rEPO), a prostaglandin, or an anti-vascular hyperproliferation
compound. When a second agent is used, the second agent can be
administered either as a separate dosage form or as part of a
single dosage form with the compounds or compositions of this
invention.
[0105] The amount of compound present in the above-described
compositions should be sufficient to cause a detectable decrease in
the severity of the disease, in caspase activity and/or cell
apoptosis, or in TNF-alpha activity and/or cell apoptosis as
measured by any of the assays described in the Examples.
[0106] The compounds of this invention can be employed in a
conventional manner for controlling IGIF and IFN-.gamma. levels in
vivo and for treating diseases or reducing the advancement or
severity of effects which are mediated by a caspase, IL-1,
apoptosis, IGIF, IFN-.gamma. or TNF-alpha. Such methods of
treatment, their dosage levels and requirements can be selected by
those of ordinary skill in the art from available methods and
techniques.
[0107] If pharmaceutically acceptable salts of the compounds of
this invention are utilized in these compositions, those salts are
preferably derived from inorganic or organic acids and bases.
Included among such acid salts are the following: acetate, adipate,
alginate, aspartate, benzoate, benzene sulfonate, bisulfate,
butyrate, citrate, camphorate, camphor sulfonate,
cyclopentanepropionate, digluconate, dodecylsulfate,
ethanesulfonate, fumarate, glucoheptanoate, glycerophosphate,
hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide,
hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate,
methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate,
pamoate, pectinate, persulfate, 3-phenyl-propionate, picrate,
pivalate, propionate, succinate, tartrate, thiocyanate, tosylate
and undecanoate. Base salts include ammonium salts, alkali metal
salts, such as sodium and potassium salts, alkaline earth metal
salts, such as calcium and magnesium salts, salts with organic
bases, such as dicyclohexylamine salts, N-methyl-D-glucamine, and
salts with amino acids such as arginine, lysine, and so forth.
[0108] Also, the basic nitrogen-containing groups can be
quaternized with such agents as lower alkyl halides, such as
methyl, ethyl, propyl, and butyl chloride, bromides and iodides;
dialkyl sulfates, such as dimethyl, diethyl, dibutyl and diamyl
sulfates, long chain halides such as decyl, lauryl, myristyl and
stearyl chlorides, bromides and iodides, aralkyl halides, such as
benzyl and phenethyl bromides and others. Water or oil-soluble or
dispersible products are thereby obtained.
[0109] The compounds utilized in the compositions and methods of
this invention can also be modified by appending appropriate
functionalities to enhance selective biological properties. Such
modifications are known in the art and include those which increase
biological penetration into a given biological system (e.g., blood,
lymphatic system, central nervous system), increase oral
availability, increase solubility to allow administration by
injection, alter metabolism and alter rate of excretion.
[0110] According to a preferred embodiment, the compositions of
this invention are formulated for pharmaceutical administration to
a mammal, preferably a human being.
[0111] Such pharmaceutical compositions of the present invention
can be administered orally, parenterally, by inhalation spray,
topically, rectally, nasally, buccally, vaginally or via an
implanted reservoir. The term "parenteral" as used herein includes
subcutaneous, intravenous, intramuscular, intra-articular,
intra-synovial, intrasternal, intrathecal, intrahepatic,
intralesional and intracranial injection and infusion techniques.
Preferably, the compositions are administered orally or
intravenously.
[0112] Sterile injectable forms of the compositions of this
invention can be aqueous or oleaginous suspension. These
suspensions can be formulated according to techniques known in the
art using suitable dispersing or wetting agents and suspending
agents. The sterile injectable preparation can also be a sterile
injectable solution or suspension in a non-toxic parenterally
acceptable diluent or solvent, for example as a solution in
1,3-butanediol. Among the acceptable vehicles and solvents that can
be employed are water, Ringer's solution and isotonic sodium
chloride solution. In addition, sterile, fixed oils are
conventionally employed as a solvent or suspending medium. For this
purpose, any bland fixed oil can be employed including synthetic
mono-or di-glycerides. Fatty acids, such as oleic acid and its
glyceride derivatives are useful in the preparation of injectables,
as are natural pharmaceutically-acceptable oils, such as olive oil
and castor oil, especially in their polyoxyethylated versions.
These oil solutions or suspensions can also contain a long-chain
alcohol diluent or dispersant, such as carboxymethyl cellulose or
similar dispersing agents which are commonly used in the
formulation of pharmaceutically acceptable dosage forms including
emulsions and suspensions. Other commonly used surfactants, such as
Tweens, Spans and other emulsifying agents or bioavailability
enhancers which are commonly used in the manufacture of
pharmaceutically acceptable solid, liquid, or other dosage forms
can also be used for the purposes of formulation.
[0113] If a solid carrier is used, the preparation can be tableted,
placed in a hard gelating capsule in powder or pellet form, or in
the form of a troche or lozenge. The amount of solid carrier will
vary, e.g., from about 25 mg to 400 mg. When a liquid carrier is
used, the preparation can be, e.g., in the form of a syrup,
emulsion, soft gelatin capsule, sterile injectable liquid such as
an ampule or nonaqueous liquid suspension. Where the composition is
in the form of a capsule, any routine encapsulation is suitable,
for example, using the aforementioned carriers in a hard gelatin
capsule shell.
[0114] A syrup formulation can consist of a suspension or solution
of the compound in a liquid carrier for example, ethanol,
glycerine, or water with a flavoring or coloring agent. An aerosol
preparation can consist of a solution or suspension of the compound
in a liquid carrier such as water, ethanol or glycerine; whereas in
a powder dry aerosol, the preparation can include e.g., a wetting
agent.
[0115] Formulations of the present invention comprise an active
ingredient together with one or more acceptable carrier(s) thereof
and optionally any other therapeutic ingredient(s). The carrier(s)
should be "acceptable" in the sense of being compatible with the
other ingredients of the formulation and not deleterious to the
recipient thereof.
[0116] The pharmaceutical compositions of this invention can be
orally administered in any orally acceptable dosage form including,
but not limited to, capsules, tablets, and aqueous suspensions or
solutions. In the case of tablets for oral use, carriers that are
commonly used include lactose and corn starch. Lubricating agents,
such as magnesium stearate, are also typically added. For oral
administration in a capsule form, useful diluents include lactose
and dried cornstarch. When aqueous suspensions are required for
oral use, the active ingredient is combined with emulsifying and
suspending agents. If desired, certain sweetening, flavoring or
coloring agents can also be added.
[0117] Alternatively, the pharmaceutical compositions of this
invention can be administered in the form of suppositories for
rectal administration. These can be prepared by mixing the agent
with a suitable non-irritating excipient which is solid at room
temperature but liquid at rectal temperature and therefore will
melt in the rectum to release the drug. Such materials include
cocoa butter, beeswax and polyethylene glycols.
[0118] The pharmaceutical compositions of this invention can also
be administered topically, especially when the target of treatment
includes areas or organs readily accessible by topical application,
including diseases of the eye, the skin, or the lower intestinal
tract. Suitable topical formulations are readily prepared for each
of these areas or organs.
[0119] Topical application for the lower intestinal tract can be
effected in a rectal suppository formulation (see above) or in a
suitable enema formulation. Topically-transdermal patches can also
be used.
[0120] For topical applications, the pharmaceutical compositions
can be formulated in a suitable ointment containing the active
component suspended or dissolved in one or more carriers. Carriers
for topical administration of the compounds of this invention
include, but are not limited to, mineral oil, liquid petrolatum,
white petrolatum, propylene glycol, polyoxyethylene,
polyoxypropylene compound, emulsifying wax and water.
Alternatively, the pharmaceutical compositions can be formulated in
a suitable lotion or cream containing the active components
suspended or dissolved in one or more pharmaceutically acceptable
carriers. Suitable carriers include, but are not limited to,
mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters
wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and
water.
[0121] For ophthalmic use, the pharmaceutical compositions can be
formulated as micronized suspensions in isotonic, pH adjusted
sterile saline, or, preferably, as solutions in isotonic, pH
adjusted sterile saline, either with or without a preservative such
as benzylalkonium chloride. Alternatively, for ophthalmic uses, the
pharmaceutical compositions can be formulated in an ointment such
as petrolatum.
[0122] The pharmaceutical compositions of this invention can also
be administered by nasal aerosol or inhalation. Such compositions
are prepared according to techniques well known in the art of
pharmaceutical formulation and can be prepared as solutions in
saline, employing benzyl alcohol or other suitable preservatives,
absorption promoters to enhance bioavailability, fluorocarbons,
and/or other conventional solubilizing or dispersing agents known
in the art.
[0123] It will be recognized by one of skill in the art that the
form and character of the pharmaceutically acceptable carrier or
diluent is dictated by the amount of active ingredient with which
it is to be combined, the route of administration, and other
well-known variables.
[0124] The above-described compounds and compositions are
particularly useful in therapeutic applications relating to an IL-1
mediated disease, an apoptosis mediated disease, an inflammatory
disease, an autoimmune disease, a destructive bone disorder, a
proliferative disorder, an infectious disease, a degenerative
disease, a skin disease, a disease associated with cell death, an
excess dietary alcohol intake disease, a viral mediated disease,
retinal disorders, uveitis, inflammatory peritonitis,
osteoarthritis, pancreatitis, asthma, adult respiratory distress
syndrome, glomerulonephritis, rheumatoid arthritis, systemic lupus
erythematosus, scleroderma, chronic thyroiditis, Grave's disease,
autoimmune gastritis, diabetes, autoimmune hemolytic anemia,
autoimmune neutropenia, thrombocytopenia, chronic active hepatitis,
myasthenia gravis, inflammatory bowel disease, Crohn's disease,
psoriasis, atopic dermatitis, contact dermatitis, scarring, graft
vs host disease, organ transplant rejection, organ apoptosis after
burn injury, osteoporosis, leukemias and related disorders,
myelodysplastic syndrome, multiple myeloma-related bone disorder,
acute myelogenous leukemia, chronic myelogenous leukemia,
metastatic melanoma, Kaposi's sarcoma, multiple myeloma,
haemorrhagic shock, sepsis, septic shock, burns, trauma, systemic
inflammatory response syndrome, multiple organ dysfunction
syndrome, Shigellosis, Alzheimer's disease, Parkinson's disease,
Huntington's disease, Kennedy's disease, prion disease, cerebral
ischemia, epilepsy, myocardial ischemia, acute and chronic heart
disease, myocardial infarction, congestive heart failure,
atherosclerosis, coronary artery bypass graft, spinal muscular
atrophy, amyotrophic lateral sclerosis, multiple sclerosis,
HIV-related encephalitis, aging, alopecia, neurological damage due
to stroke, ulcerative colitis, traumatic brain injury, spinal chord
injury, hepatitis-B, hepatitis-C, hepatitis-G, yellow fever, dengue
fever, Japanese encephalitis, various forms of liver disease, renal
disease, polycystic kidney disease, H. pylori-associated gastric
and duodenal ulcer disease, HIV infection, tuberculosis, and
meningitis.
[0125] The above-described compounds and compositions are also
useful in therapeutic applications relating to a TNF mediated
disease. The phrase "TNF-alpha mediated disease" means, all
diseases states in which TNF-alpha plays a role, either by
excessive production or release of TNF-alpha, itself, or by
TNF-alpha causing an event that triggers or exacerbates the
disease, such as production or release of another
pathophysiological biochemical agent, or cytokine. In one preferred
embodiment, TNF-alpha plays a direct role.
[0126] Such TNF-alpha mediated diseases can include, e.g.,
restinosis, inflammatory diseases such as inflammatory diseases of
the central nervous system, demyelinating diseases of the nervous
system, multiple sclerosis, septic arthritis, aneurysmal aortic
disease, traumatic joint injury, peridontal disease, macular
degeneration, diabetic retinopathy, occular inflammation,
keratoconus, Sjogren's syndrome, corneal graft rejection, cachexia,
and anorexia.
[0127] Excessive TNF-alpha tissue levels have been implicated in
mediating or exacerbating a number of diseases including:
rheumatoid arthritis, rheumatoid spondylitis, osteoarthritis, gouty
arthritis and other arthritic conditions; also general sepsis,
gram-negative sepsis, septic shock, endotoxic shock, toxic shock
syndrome, adult respiratory distress syndrome (ARDS), cerebral
malaria, chronic pulmonary inflammatory disease, silicosis,
asbestosis, pulmonary sarcoidosis, bone resorption diseases, graft
vs. host reactions, allograft rejections; also fever and myalgias
due to bacterial or viral infections, such as influenza; cachexia
secondary to acquired immune deficiency syndrome (AIDS), keloid
formation, scar tissue formation, Crohns disease, ulcerative
colitis, or pyresis; a number of "autoimmune diseases" such as
multiple sclerosis, autoimmune diabetes, and systemic lupus
erythematosus.
[0128] TNF-alpha inhibitors are useful in the treatment of a
variety of allergic, traumatic and other injurious disorders
including: asthma, chronic bronchitis, atopic dermatitis,
urticaria, allergic rhinitis, allergic conjunctivitis,
eosiniophilic granuloma, ulcerative colitis, Crohn's disease,
reperfusion injury of the myocardium and brain, chronic
glomerulonephritis, and adult respiratory distress syndrome
(ARDS).
[0129] The compounds of this invention can inhibit the release of
TNF-alpha and thus can be useful for inhibiting or blocking several
pathophysiological effects of TNF-alpha at injury or surgery sites
and thus also inhibit the release of other pathophysiological
biochemical products from cells such as histamines, prostaglandins,
bradykinins, and peroxidases.
[0130] As discussed above, TNF-alpha inhibitors can be very
effective in the treatment of disorders which follow cellular,
tissue or organ injury or surgery, and can be as effective, or even
more potent, than corticosteroids or immunosuppressants without
producing the side effects common to these agents.
[0131] This invention also relates to a therapeutic method of (1)
inhibiting TNF-alpha release from cells and (2) preventing the
untoward, toxic or lethal effects of excessively high tissue levels
of TNF-alpha in a mammal, including a human. This method comprises
administering to a mammal an effective TNF-alpha inhibiting
quantity of one or more of the above compounds. This method also
can be used for the prophylactic treatment or prevention of certain
TNF-alpha mediated or exacerbated diseases amenable thereto. The
invention provides a method for the treatment of allergic,
traumatic, radiation, chemical, microbial and other injurious
disorders by administering to a mammal, including a human, in need
thereof an effective amount of such compounds.
[0132] The compounds, by inhibiting or blocking the release of
TNF-alpha or decreasing TNF-alpha levels and activity, as well as
the pathophysiologic actions of excessive levels of TNF-alpha in
each of these circumstances, directly facilitate the arrest or
resolution of the tissue or organ damage, and facilitates the
restoration of normal function. Together, these actions relate
their novel use in treating tissue trauma, or other injury
disorders caused by infection, allergy, immunologic phenomena,
burns, radiation exposure, neoplastic disease, toxic chemicals and
expressed as cardiovascular damage, neurologic injury, renal
damage, liver damage, pancreatic damage, as well as ascites,
localized edema, dermal damage and dermal blister.
[0133] The term "inhibiting the release of TNF-a" means:
[0134] a) decrease of in vivo TNF-alpha levels in a mammal such as
a human; or
[0135] b) a down regulation of TNF-alpha levels in vitro or in
vivo; or
[0136] c) a down regulation of TNF-alpha activity, by inhibition of
the direct synthesis of TNF-alpha or a post-translation event.
[0137] The compounds can be useful in inhibiting the release of
TNF-alpha by monocytes, macrophages, neuronal cells, endothelial
cells, epidermal cells, mesenchymal cells (for example:
fibroblasts, skeletal myocytes, smooth muscle myocytes, cardiac
myocytes) and many other types of cells.
[0138] The term "condition" or "state" refers to any disease,
disorder or effect that produces deleterious biological
consequences in a subject.
[0139] The level of TNF-alpha protein in the blood or cell of a
patient or a cell culture (i.e., in the cells and/or in the culture
media) can be determined by assaying for immunospecific binding to
TNF-alpha or to proteins that are known to be produced as a result
of the presence of active TNF-alpha. Such assays are known in the
art. For example, the immunoassays which can be used include, but
are not limited, to competitive and non-competitive assay systems
using techniques such as western blots, radioimmunoassays, ELISA
(enzyme linked immunosorbent assay), "sandwich" immunoassays,
immunoprecipitation assays, precipitin reactions, gel diffusion
precipitin reactions, immunodiffusion assays, agglutination assays,
complement-fixation assays, immunoradiometric assays, fluorescent
immunoassays, FACS analysis and protein A immunoassays. Such assays
are well known in the art (see, e.g., Ausubel et al, eds, 1994,
Current Protocols in Molecular Biology, Vol. 1, John Wiley &
Sons, Inc., New York, which is incorporated by reference herein in
its entirety).
[0140] Competitive binding assays can also be used to determine the
level of TNF-alpha. One example of a competitive binding assay is a
radioimmunoassay comprising the incubation of labeled proteins from
cells expressing TNF-alpha(e.g., .sup.3H or .sup.125I) with a
TNF-alpha antibody in the presence of increasing amounts of
unlabeled TNF-alpha, and the detection of the TNF-alpha antibody
bound to the labeled TNF-alpha.
[0141] TNF-alpha levels can also be assayed by activity assays
known in the art. For example, samples of treated cell cultures or
from blood from patients can be used in TNF-alpha activity assays
known in the art, e.g., J. Immunol. Methods, 1995, 178, 71-76;
Burns, 1994, 20(1), 40-44.
[0142] Dosage levels of between about 0.01 and about 100 mg/kg body
weight per day, preferably between 0.5 and about 75 mg/kg body
weight per day and most preferably between about 1 and 50 mg/kg
body weight per day of the active ingredient compound are useful in
a monotherapy.
[0143] Typically, the pharmaceutical compositions of this invention
will be administered from about 1 to 5 times per day or
alternatively, as a continuous infusion. Such administration can be
used as a chronic or acute therapy. The amount of active ingredient
that can be combined with the carrier materials to produce a single
dosage form will vary depending upon the host treated and the
particular mode of administration. A typical preparation will
contain from about 5% to about 95% active compound (w/w).
Preferably, such preparations contain from about 20% to about 80%
active compound.
[0144] When the compositions of this invention comprise a
combination of a compound of formula I and one or more additional
therapeutic agents, both the compound and the additional agent
should be present at dosage levels of between about 10% to 80% of
the dosage normally administered in a monotherapy regime.
[0145] Upon improvement of a patient's condition, a maintenance
dose of a compound, composition or combination of this invention
can be administered, if necessary. Subsequently, the dosage or
frequency of administration, or both, can be reduced, as a function
of the symptoms, to a level at which the improved condition is
retained when the symptoms have been alleviated to the desired
level, treatment should cease. Patients can, however, require
intermittent treatment on a long-term basis upon any recurrence or
disease symptoms.
[0146] It should also be understood that a specific dosage and
treatment regimen for any particular patient will depend upon a
variety of factors, including the activity of the specific compound
employed, the age, body weight, general health, sex, diet, time of
administration, rate of excretion, drug combination, and the
judgment of the treating physician and the severity of the
particular disease being treated. The amount of active ingredients
will also depend upon the particular compound and other therapeutic
agent, if present, in the composition.
[0147] One embodiment of this invention provides a method for
treating or preventing an IL-1- mediated disease or an
apoptosis-mediated disease in a subject comprising the step of
administering to the subject any compound, pharmaceutical
composition, or combination described herein.
[0148] Another embodiment of this invention provides a method for
inhibiting a caspase-mediated function in a subject comprising the
step of administering to the subject any compound, pharmaceutical
composition, or combination described.
[0149] Another embodiment of this invention provides a method for
decreasing IGIF or IFN-.gamma. production in a subject comprising
the step of administering to the subject any compound,
pharmaceutical composition, or combination described.
[0150] Another embodiment of this invention provides a method for
treating complications associated with coronary artery bypass
grafts in a subject comprising the step of administering to the
subject any compound, pharmaceutical composition, or combination
described herein.
[0151] Another embodiment of this invention provides a method for
preserving cells comprising the step of bathing the cells in a
solution of any compound described herein. Such method using
caspase inhibitors has been reported [Schierle et al., Nature
Medicine, 5, p. 97 (1999); and Natori et al., Transplantation, 68,
pp. 89-96 (1999)]. The amount of caspase inhibitor needed will
depend on the effectiveness of the inhibitor for a given cell type
and the length of time required to preserve cells from apoptotic
cell death.
[0152] Another embodiment of this invention provides a method for
preserving cells needed for an organ transplant or for preserving
blood products, using any compound, pharmaceutical composition, or
combination described herein. Li et al., Transfusion, 40, pp.
1320-1329 (2000).
[0153] Another embodiment of this invention provides a method for
treating various forms of cancer in a subject comprising the step
of administering to the subject any compound, pharmaceutical
composition, or combination described herein as a component of
immunotherapy. Droin et al., Oncogene, 16, pp. 2885-2894 (1998);
Boudard et al., Leukemia, 14, pp. 2045-2051 (2000); Faderl et al.,
Clinical Cancer Research, 5, pp. 4041-4047 (1999); Ozoren et al.,
Cancer Research, 60, pp. 6259-6265 (2000); Sasaki et al., British
Journal of Urology, 81, pp. 852-855 (1998); and Hedlund et al.,
Prostate, 36, pp. 92-101 (1998).
[0154] In a preferred embodiment, the invention provides a method
of treating a mammal, having one of the aforementioned diseases,
comprising the step of administering to said mammal a
pharmaceutically acceptable composition described above. In this
embodiment, if the patient is also administered another therapeutic
agent or caspase inhibitor, it can be delivered together with the
compound of this invention in a single dosage form, or, as a
separate dosage form. When administered as a separate dosage form,
the other caspase inhibitor or agent can be administered prior to,
at the same time as, or following administration of a
pharmaceutically acceptable composition comprising a compound of
this invention.
[0155] A kit according to this invention comprises a compound or a
pharmaceutically acceptable derivative thereof or pharmaceutical
composition of this invention and a tool for measuring TNF alpha
levels and/or activity in vitro or in vivo. The kit can further
comprise instructions for using the contents of the kit. A tool for
measuring TNF-alpha levels of this invention refer to materials
that can be used to measure the TNF gene product (i.e., RNA or
protein) or activity. Such methods are described for example above.
Thus, a tool according to this invention can include e.g., an
anti-TNF antibody, a TNF-alpha DNA probe or a genetically
engineered cell line responsive to TNF alpha levels described
above.
[0156] The methods for identifying a compound or composition that
decreases TNF-alpha activity and/or levels according to this
invention include methods for screening of a plurality of compounds
or compositions for their ability to decrease TNF-alpha activity
and/or levels. For example, high-throughput screening is a desired
embodiment of this invention. According to one embodiment of this
invention, high-throughput screening can be achieved by having
cells in culture in a plurality of wells in a microtiter plate,
adding a different compound or composition to each well and
comparing the TNF-alpha levels and/or activity in each cell culture
to the TNF-alpha levels or activity present in a cell culture in a
control well. Controls that are useful for the comparison step
according to this invention include cells or subjects that have not
been treated with a compound or composition and cells or subjects
have been treated with a compound or composition that is known to
have no effect on TNF-alpha levels or activity. According to one
embodiment of this invention, the high throughput screening is
automated so that the steps including the addition of the cells to
the plate up to the data collection and analysis after addition of
the compound or composition are done by machine. Instruments that
are useful in the comparison step of this invention, e.g.,
instruments that can detect labeled objects (e.g., radiolabelled,
fluorescent or colored objects) or objects that are themselves
detectable, are commercially available and/or known in the art.
Accordingly, compounds and compositions according to this invention
that are useful for decreasing TNF-alpha levels and/or activity can
be quickly and efficiently screened.
[0157] In order that this invention be more fully understood, the
following preparative and testing examples are set forth. These
examples are for the purpose of illustration only and are not to be
construed as limiting the scope of the invention in any way.
EXAMPLES
Example 1
[3S/R
(2S)]-5-Fluoro-4-oxo-3-[1-(2-phenyl-thiazole-4-carbonyl)-2-piperidin-
ecarboxamido]-pentanoic Acid (Compound 1)
[0158] 41
[0159] Method A:(S)-1-(Benzyloxycarbonyl) 2-piperidine-carboxylic
Acid Methyl Ester 42
[0160] A stirred suspension of (S) -Piperidine-carboxylic acid
methyl ester hydrochloride (2.00 g, 11.13 mmol) in anhydrous THF
(40 ml) at room temperature was treated with triethylamine (3.41
ml, 24.50 mmol). The reaction mixture was stirred at room
temperature for 30 min before the addition of
N-(benzyloxycarbonyloxy)succinimide (3.05 g, 12.23 mmol). The
resulting mixture was stirred for 2 hr, before being diluted with
ethyl acetate (20 ml), washed with 2 N HCl, saturated aq.
NaHCO.sub.3, saturated aq. NaCl, dried (Na.sub.2SO.sub.4), filtered
and concentrated. The residue was purified by flash chromatography
(20% ethyl acetate in hexane) to afford the sub-title compound as a
colourless oil (2.0085 g, 65%): .sup.1H NMR (400 MHz, CDCl.sub.3)
.delta.1.19-1.1.54 (2H, m), 1.58-1.80 (4H, m), 2.18-2.33 (1H, m),
2.90-3.15 (1H, m), 3.66-3.81 (3H, m), 4.03-4.21 (1H, m), 4.81-5.25
3H, m), 7.28-7.45 (5H, m).
[0161] Method B: (S)-1-(Benzyloxycarbonyl)-2-piperidine-carboxylic
Acid 43
[0162] A stirred solution of
(S)-1-(benzyloxy-carbonyl)-2-piperidinecarbox- ylic acid methyl
ester (2.00 g, 7.21 mmol) in THF (20 ml) at room temperature was
treated with water (10 ml). Lithium hydroxide (190 mg, 7.93 mmol)
was added and the resulting mixture stirred at room temperature for
3 hr. An additional quantity of lithium hydroxide (40 mg, 1.67
mmol) was added and the resulting mixture was stirred for 2 hr
prior to the removal of the organic solvent. The resulting solution
was washed with diethyl ether and the remaining aqueous layer was
made acidic with 2 N HCl prior to a second extraction step with
ethyl acetate. The organic layer was then recovered, dried
(Na.sub.2SO.sub.4), filtered and concentrated to reveal a colorless
oil (1.9927 g, 105%) which crystallized upon standing: .sup.1H NMR
(400 MHz, CDCl.sub.3) .delta. 1.30-1.88 (5H, m), 2.22-2.41 (1H, m),
3.00-3.21 (1H, m), 4.08-4.25 (1H, m), 4.91-5.30 (3H, m), 7.27-7.48
(5H, m).
[0163] Method C: [3S/R, 4S/R,
(2S)]-5-Fluoro-4-hydroxy-3-[1-(benzyloxycarb-
onyl)-2-piperidinecarboxamido]-pentanoic Acid Tert-Butyl Ester
44
[0164] A stirred mixture of (S)-1-(benzyloxy
carbonyl)-2-piperidinecarboxy- lic acid (4.82 g, 18.31 mmol),
3-amino-5-fluoro-4-hydroxy-pentanoic acid tert-butyl ester (3.99 g,
19.25 mmol), HOBt (2.72 g, 20.13 mmol), DMAP (2.57 g, 21.04 mmol)
and anhydrous THF (60 ml) was cooled to 0.degree. C. before EDC
(3.86 g, 20.13 mmol) was added. The mixture was allowed to warm to
room temperature over 16 hrs before being concentrated under
reduced pressure. The residue was purified by flash chromatography
(60% ethyl acetate in hexane) to afford the sub-title compound as a
white foam (7.3754 g, 72%): .sup.1H NMR (400 MHz, CDCl.sub.3)
.delta. 1.31-1.80 (14H, m), 2.20-2.38 (1H, m), 2.49-3.07 (3H, m),
3.11-3.70 (1H, m), 3.80-4.58 (4H, m), 4.70-5.28 (1H, m), 6.58-7.05
(1H, m), 7.23-7.48 (5H, m)
[0165] Method D: [3S/R, 4S/R,
(2S)]-5-Fluoro-4-hydroxy-3-[2-piperidinecarb- oxamido]-pentanoic
Acid Tert-Butyl Ester 45
[0166] A stirred solution of [3S/R, 4S/R,
(2S)]-5-fluoro-4-hydroxy-3-[1-(b-
enzyloxycarbonyl)-2-piperidinecarboxamido]-pentanoic acid
tert-butyl ester (7.37 g, 16.29 mmol) in ethyl acetate (150 ml) was
treated with 10% Pd/C (830 mg). The reaction mixture was then
thoroughly degassed and placed under a hydrogen balloon. The
resulting mixture was stirred at room temperature for 3 hrs after
which it was filtered through celite and concentrated to the
sub-title compound as a colorless gum (5.17 g, 100%): .sup.1H NMR
(400 MHz, CDCl.sub.3) .delta. 1.16-2.00 (15H, m), 2.51-2.78 (3H,
m), 2.99-3.09 (1H, m), 3.18-3.28 (1H, m), 3.93-4.56 (4H, m),
7.39-7.58 (1H, m); .sup.19F (376 MHz, CDCl.sub.3) .delta. -229.34
(t), -229.42 (t), -229.87 (t), 230.02 (t).
[0167] Method E: [3S/R, 4S/R,
(2S)]-5-Fluoro-4-hydroxy-3-[1-(2-phenyl-thia-
zole-4-carbonyl)-2-piperidine-carboxamido]-pentanoic Acid
Tert-Butyl Ester 46
[0168] A stirred solution of [3S/R, 4S/R,
(2S)]-5-fluoro-4-hydroxy-3-[2-pi- peridinecarboxamido]-pentanoic
acid tert-butyl ester (520 mg, 1.63 mmol), in DMF (9.7 ml) at room
temperature was treated with DIPEA (311 .mu.l, 1.80 mmol). The
resulting mixture was allowed to stir for 30 min before being
treated with 2-phenyl-thiazole-4-carboxylic acid (335 mg, 1.63
mmol) and TBTU (524 mg, 1.63 mmol). The mixture was stirred at room
temperature for 16 hr and then diluted with ethyl acetate. The
resulting solution was washed with 2 N HCl, saturated aq.
NaHCO.sub.3, saturated aq. NaCl, dried (Na.sub.2SO.sub.4), filtered
and concentrated to reveal an oil. The residue was purified by
flash chromatography (60% ethyl acetate in hexane) to afford the
sub-title compound as a colorless oil (463 mg, 56%): .sup.1H NMR
(400 MHz, CDCl.sub.3) .delta. 1.10-1.85 (15H, m), 2.22-2.89 (3H,
m), 3.09-4.78 (6H, m), 5.20-5.43 (1H, m), 7.40-7.56 (3H, m),
7.81-8.11 (3H, m).
[0169] Method F: [3S/R,
(2S)]-5-fluoro-4-oxo-3-[1-(2-phenyl-thiazole-4-car-
bonyl)-2-piperidinecarboxamido]-pentanoic Acid Tert-Butyl Ester
47
[0170] A stirred solution of [3S/R, 4S/R,
(2S)]-5-Fluoro-4-hydroxy-3-[1-(2-
-phenyl-thiazole-4-carbonyl)-2-piperidinecarboxamido]-pentanoic
acid tert-butyl ester (462 mg, 0.91 mmol) in anhydrous DCM (25 ml)
was treated with 1,1,1-triacetoxy-1,1-dihydro-1,2-benziodoxol-3
(1H)-one (426 mg, 1.00 mmol) at 0.degree. C. The resulting mixture
was kept at 0.degree. C. for 2 hr, diluted with DCM, and washed
with saturated aq. Na.sub.2S.sub.2O.sub.3.5H.sub.2O, saturated aq.
NaHCO.sub.3, saturated aq. NaCl. dried (Na.sub.2SO.sub.4) and
concentrated. The residue was purified by flash chromatography (33%
ethyl acetate in hexane) to afford the sub-title compound as a
white solid (376 mg, 82%): IR (solid) 1731, 1619, 1506, 1460, 1363,
1260, 1158 cm.sup.-1; .sup.1H NMR (400 MHz, CDCl.sub.3) .delta.
1.16-1.81 (14H, m), 2.25-2.42 (1H, m), 2.69-3.25 (3H, m), 4.48-5.46
(5H, m), 7.36-8.32 (7H, m); .sup.13C NMR (100 MHz, CDCl.sub.3)
.delta. 21.08, 21.29, 21.33, 25.24, 25.72, 27.23, 28.14, 36.60,
41.33, 41.52, 46.10, 46.27, 52.06, 52.77, 52.84, 82.25, 82.40,
82.66, 83.81, 85.68, 125.09, 125.49, 126.50, 126.69, 127.02,
127.10, 129.46, 129.55, 131.00, 131.18, 132.85, 133.04, 133.29,
150.75, 150.92, 163.50, 163.65, 165.17, 168.07, 168.14, 170.07,
170.23, 171.37, 202.87, 203.02; .sup.19F (376 MHz, CDCl.sub.3)
.delta. -231.36, -231.69, -231.86, -232.28; MS (LR, ES) Calculated
for C.sub.25H.sub.30FN.sub.3O.sub.5S: 503.5974.
[0171] Method G: [3S/R,
(2S)]-5-Fluoro-4-oxo-3-[1-(2-phenyl-thiazole-4-car-
bonyl)-2-piperidinecarboxamido]-pentanoic Acid 48
[0172] A stirred solution of [3S/R,
(2S)]-5-fluoro-4-oxo-3-[1-(2-phenyl-th-
iazole-4-carbonyl)-2-piperidinecarboxamido]-pentanoic acid
tert-butyl ester (370 mg, 0.73 mmol) in anhydrous DCM (20 ml) was
treated with a 1 M solution of titanium tetrachloride in DCM (3.67
ml, 3.67 mmol) at -10.degree. C. The resulting mixture was warmed
to 0.degree. C. and kept at this temperature for 1 hr. The reaction
mixture was then diluted with DCM, and washed with 2 N HCl,
saturated aq. NaCl, dried (Na.sub.2SO.sub.4) and concentrated. The
residue was purified by reverse phase HPLC (acetonitrile/water) to
afford the title compound as a white foam (102 mg, 31%): IR (solid)
1798, 1736, 1674, 1617, 1517, 1479, 1470, 1265; .sup.1H NMR (400
MHz, d.sub.6-DMSO+TFA) .delta. 1.18-1.79 (4H, m), 2.08-2.28 (1H,
m), 2.42-3.50 (5H, m), 4.08-5.40 (4H, m), 7.45-8.28 (6H, m),
8.41-8.67 (1H, m); .sup.13C NMR (100 MHz, d.sub.6-DMSO+TFA) .delta.
19.07, 19.24, 23.29, 23.69, 25.63, 25.72, 26.42, 26.49, 31.54,
33.23, 43.65, 46.14, 50.81, 50.91, 51.56, 51.64, 56.26, 56.34,
80.07, 81.87, 81.97, 83.66, 83.75, 102.43, 102.47, 102.63, 102.66,
125.17, 125.24, 128.19, 129.52, 131.40, 149.51, 162.07, 162.61,
162.67, 165.34, 165.55, 169.71, 169.86, 170.64, 170.72, 171.75,
201.84, 201.20, 201.34; .sup.19F (376 MHz, d.sub.6-DMSO) .delta.
-226.74, -226.82, -226.84, -227.00, -230.37, -230.60, -230.83,
-232.41, -232.55, -232.62, -232.7; MS (LR, ES) calculated for
C.sub.21H.sub.22FN.sub.3O.sub.5S: 447.4890, ES-446.408,
ES+448.184.
Example 2
[3S/R
(2S)]-5-Fluoro-4-oxo-3-[1-(isoquinoline-1-carbonyl)-2-piperidinecarb-
oxamido]-pentanoic Acid, Trifluoroacetic Acid Salt (Compound 2)
[0173] 49
[0174] Method H:
(S)-1-(Isoquinoline-1-carbonyl)-2-piperidinecarboxylic Acid Methyl
Ester 50
[0175] A stirred solution of (S)-1-Piperidine-carboxylic acid
methyl ester hydrochloride (1.00 g, 5.57 mmol), in DMF (20 ml) at
room temperature was treated with DIPEA (2.12 ml, 12.25 mmol). The
resulting mixture was allowed to stir for 30 min before being
treated with 1-isoquinolinecarboxylic acid (964 mg, 5.57 mmol) and
TBTU (1.79 g, 5.57 mmol). The mixture stirred at room temperature
for 4 hr, diluted with ethyl acetate, washed with saturated
aq.NaHCO.sub.3, saturated aq. NaCl, dried (Na.sub.2SO.sub.4),
filtered and concentrated. The residue was purified by flash
chromatography (67% ethyl acetate in hexane) to afford the
sub-title compound as a colourless gum (1.05 g, 63%): .sup.1H NMR
(400 MHz, CDCl.sub.3) .delta. 1.00-2.51 (6H, m), 3.05-3.38 (2H, m),
3.60-3.95 (3H, m), 4.35-4.95 (1H, m), 5.70-5.80 (1H, m), 7.55-7.95
(3H, m), 8.13-8.29 (1H, m), 8.48-8.61 (1H, m)
[0176] Method I:
(S)-1-(Isoquinoline-1-carbonyl)-2-piperidinecarboxylic Acid 51
[0177] A stirred solution of
(S)-1-(isoquinoline-1-carbonyl)-2-piperidinec- arboxylic acid
methyl ester (1.05 g, 3.52 mmol) in THF (20 ml) at room temperature
was treated with water (10 ml). Lithium hydroxide (84 mg, 3.51
mmol) was then added and the resulting mixture was stirred at room
temperature for 16 hrs. The resulting mixture was concentrated to
remove the organic solvent. The resulting solution was then washed
with diethyl ether and the remaining aqueous layer was made acid
with 2 N HCl. The resulting solution was extracted with ethyl
acetate and the organic layer was separated, dried
(Na.sub.2SO.sub.4), filtered and concentrated to reveal a white
solid (902 mg, 90%): .sup.1H NMR (400 MHz, d.sub.4-MeOH) .delta.
1.20-2.52 (6H, m), 3.10-3.39 (2H, m), 4.10-4.90 (1H, m), 7.68-8.55
(5H, m).
[0178] Method J: [3S/R, 4S/R,
(2S)]-5-Fluoro-4-hydroxy-3-[1-(isoquinoline--
1-carbonyl)-2-piperidinecarboxamido]-pentanoic Acid Tert-Butyl
Ester 52
[0179] A stirred mixture of
(S)-1-(isoquinoline-1-carbonyl)-2-piperidineca- rboxylic acid (278
mg, 0.98 mmol), 3-amino-5-fluoro-4-hydroxy-pentanoic acid
tert-butyl ester (213 mg, 1.03 mmol), HOBt (145 mg, 1.07 mmol),
DMAP (137 mg, 1.12 mmol) and anhydrous THF (25 ml) was cooled to
0.degree. C. then EDC (206 mg, 1.07 mmol) was added. The mixture
was allowed to warm to room temperature during 16 hrs then
concentrated under reduced pressure. The residue was purified by
flash chromatography (5% methanol in DCM) to afford the title
compound as a white foam (425 mg, 92%): .sup.1H NMR (400 MHz,
CDCl.sub.3) .delta. 1.20-3.30 (17H, m), 3.95-4.18 (2H, m),
4.29-4.64 (4H, m), 4.86-5.01 (1H, m), 7.65-8.00 (4H, m), 8.10-9.00
(3H, m); .sup.19F (376 MHz, CDCl.sub.3) .delta. -229.39, -229.41,
-229.57, -229.60, -229.64, -229.69, -230.41, -231.08.
[0180] [3S/R,
(2S)]-5-Fluoro-4-oxo-3-[1-(isoquinoline-1-carbonyl)-2-piperi-
dinecarboxamido]-pentanoic Acid Tert-Butyl Ester 53
[0181] This sub-title compound was prepared using procedures
similar to those described in method F as a white foam (268 mg,
63%): .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 1.20-2.60 (15H, m),
2.69-4.30 (4H, m), 4.83-5.78 (4H, m), 7.23-7.35 (1H, m), 7.57-8.00
(1H, m), 8.13-8.30 (1H, m), 8.45-8.72 (1H, m), 9.08-9.73 (1H, m);
.sup.19F (376 MHz, CDCl.sub.3) .delta. -231.53, -231.69, -231.70,
-232.13; MS (LR, ES) calculated for C.sub.25H.sub.30FN.sub.3Os:
471.5334, ES-470.327, ES+472.270.
[0182] Method K: [3S/R,
(2S)]-5-Fluoro-4-hydroxy-3-[1-(isoquinoline-1-carb-
onyl)-2-piperidinecarboxamido]-pentanoic Acid Trifluoroacetic Acid
Salt 54
[0183] An ice cooled solution of trifluoroacetic acid (5 ml) in
anhydrous DCM (5 ml) was added to a stirred ice cold solution of
[3S/R,
(2S)]-5-Fluoro-4-oxo-3-[1-(isoquinoline-1-carbonyl)-2-piperidine-carboxam-
ido]-pentanoic acid tert-butyl ester (240 mg, 0.51 mmol) in
anhydrous DCM (15 ml). The mixture was stirred at 0.degree. C. for
2 hr and 4.degree. C. for 40 hr. The mixture was concentrated under
reduced pressure and the residue was dissolved in dry DCM. This
process was repeated four times in order to remove excess
trifluoroacetic acid. The gum was triturated with diethyl ether to
afford the title compound as an off white solid (126 mg, 53%): IR
(solid) 1794, 1736, 1646, 1441, 1250, 1198, 1150, 1055 cm.sup.-1;
.sup.1H NMR (400 MHz, d.sub.6-DMSO+TFA) .delta. 1.18-2.36 (6H, m),
2.59-3.45 (4H, m), 4.10-5.51 (4H, m), 7.60-8.78 (7H, m); .sup.13C
NMR (100 MHz, d.sub.6-DMSO +TFA); .sup.19F (376 MHz, d.sub.6-DMSO)
.delta. -226.75, -226.81, -227.00, -232.62, -232.66, -233.09; MS
(LR, ES) calculated for C.sub.21H.sub.22FN.sub.3O.sub.5: 415.42,
ES-414.269, ES+416.198.
Example 3
[3S/R,
(2S)]-5-Fluoro-4-oxo-3-[1-benzoyl-2-piperidinecarboxamido]-pentanoi-
c Acid (Compound 3)
[0184] 55
[0185] Method L: (S)-1-Benzoyl-2-piperidinecarboxylic Acid Methyl
Ester 56
[0186] A stirred suspension of (S)-Piperidine-carboxylic acid
methyl ester hydrochloride (1.009 g, 5.65 mmol) in anhydrous DCM (7
ml) at 0.degree. C. was treated with diisopropylamine (3 ml, 17.34
mmol) and then benzoyl chloride (0.72 ml, 6.21 mmol). The resulting
mixture was then stirred at 0.degree. C. for 4 hr, before being
diluted with DCM. The resulting solution was washed with 1 N HCl,
saturated aq. NaHCO.sub.3, saturated aq. NaCl, dried (MgSO.sub.4),
filtered and concentrated to reveal an oil. The residue was
purified by flash chromatography (20% ethyl acetate in hexane) to
afford the title compound as a colourless oil (1.221 g, 87%):
.sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 1.25-1.80 (5H, m),
2.10-2.39 (1H, m), 2.75-3.27 (1H, m), 3.55-3.68 (0.66H, m),
3.70-3.79 (3H, m), 5.41-5.54 (0.66H, m), 5.41-5.53 (0.66H, m),
7.26-7.46 (5H, m).
[0187] Method M: (S)-1-Benzoyl-2-piperidinecarboxylic Acid 57
[0188] A stirred solution of (S)-1-benzoyl-2-piperidinecarboxylic
acid methyl ester (1.221 g, 4.94 mmol) in a solution of methanol (5
ml) in water (5 ml) at 0.degree. C. was treated with potassium
hydroxide (305 mg, 5.43 mmol). The resulting mixture was stirred at
0.degree. C. for 2 hr. A further quantity of potassium hydroxide
(111 mg, 1.97 mmol) was then added and the resulting mixture was
stirred for 1.5 hr, then concentrated. The resulting aqueous
solution was then washed with DCM and the remaining aqueous layer
was made acid with 1 N HCl. The resulting solution was extracted
with ethyl acetate and the organics were seperated, dried
(MgSO.sub.4), filtered and concentrated to reveal a crystalline
solid (870 mg, 76%): .sup.1H NMR (400 MHz, CDCl.sub.3) 1.21-1.78
(5H, m), 1.98-2.27 (1H, m), 2.71-3.20 (1H, m), 3.28-3.38 (3H, m),
3.41-3.53 (0.5H, m), 4.21-4.47 (1H, m), 5.11-5.25 (0.5H, m),
7.27-7.39 (2H, m), 7.40-7.50 (3H, m).
[0189] The title compound was then prepared by subjecting
(S)-1-Benzoyl-2-piperidinecarboxylic acid to procedures similar to
those described in methods J, F and K. The product was isolated
after RP-HPLC (MeCN/H.sub.2O) as a white foam (25 mg, 10% last
step): IR (solid) 3318, 2944, 1787, 1736, 1675, 1611 cm.sup.-1;
.sup.1H NMR (400 MHz, d.sub.6-DMSO) .delta. 1.37-1.63 (5H, m),
2.05-2.18 (1H, m), 2.60-2.94 (2H, m), 3.25-3.46 (2H, m), 4.34-4.77
(2H, m), 5.12-5.29 (2H, m), 7.34-7.90 (5H, m), 8.12-8.58 (1H, m);
.sup.13C NMR (100 MHz, d.sub.6-DMSO) .delta. 20.54, 24.64, 25.11,
26.87, 27.80, 34.65, 45.82, 52.42, 58.46, 83.42, 126.37, 127.20,
128.74, 129.75, 129.86, 136.37, 171.38, 172.22, 173.33, 202.70,
202.82; .sup.19F (376 MHz, d.sub.6-DMSO) .delta. -226.52, -226.71,
-226.84, -226.91, -230.13, -232.28, -232.39, -232.62, -232.66; MS
(FAB+ve, HR) calculated for C.sub.18H.sub.21FN.sub.2- O.sub.5 (MH+)
365.1513, found 365.1519.
Example 4
[3S/R,
(2S)]-5-Fluoro-4-oxo-3-[1-(2-methyl-4-trifluoromethyl-thiazole-5-ca-
rbonyl)-2-piperidinecarboxamido]-pentanoic Acid (Compound 4)
[0190] 58
[0191] The title compound was prepared using procedures similar to
those described in methods A-G. The product was isolated as a white
foam (27.1 mg, 8% last step): IR (solid) 1794, 1736, 1632, 1436,
1355, 1203, 1165, 1126 cm.sup.-1; .sup.1H NMR (400 MHz,
d.sub.6-DMSO+TFA) .delta. 1.14-1.74 (6H, m), 2.01-2.23 (1H, m),
2.42-3.55 (7H, m), 4.06-4.80 (2H, m), 5.00-5.39 (2H, m), 8.02-8.71
(1H, m); .sup.13C NMR (100 MHz, d.sub.6-DMSO+TFA) .delta. 18.87,
19.07, 23.25, 23.85, 25.88, 31.47, 33.03, 33.20, 33.40, 44.18,
46.05, 46.16, 50.78, 50.82, 50.94, 51.02, 51.14, 51.26, 51.42,
56.58, 56.94, 80.08, 81.84, 81.93, 82.05, 83.73, 83.82, 102.44,
102.63, 116.52, 116.68, 116.90, 120.79, 122.31, 124.80, 125.55,
125.65, 129.02, 129.24, 129.32, 133.75, 135.65, 160.84, 161.00,
168.93, 169.18, 169.39, 170.64, 171.78, 172.64, 200.93, 201.26,
201.40; .sup.19F (376 MHz, d.sub.6-DMSO) .delta.-61.54, -226.61,
-226.76, -226.86, -227.02, -228.01, -229.32, -229.86, -230.48,
-231.39, -232.37, -232.55, -232.59, -232.69; MS (LR, ES) calculated
for C.sub.17H.sub.19F.sub.4N.sub.3O.sub.5S: 453.4157, ES-452.327,
ES+454.141.
Example 5
[3S/R,
(2S)]-5-Fluoro-4-oxo-3-[1-(7-methoxy-benzofuran-2-carbonyl)-2-piper-
idine-carboxamido]-pentanoic Acid (Compound 5)
[0192] 59
[0193] The title compound was prepared using procedures similar to
those described in methods A-G. The product was isolated as a white
foam (24.0 mg, 12% last step): IR (solid) 1794, 1736, 1627, 1589,
1427, 1269, 1203 cm.sup.-1; .sup.1H NMR (400 MHz, d.sub.6-DMSO+TFA)
.delta. 1.20-1.80 (4H, m), 2.10-2.29 (1H, m), 2.52-3.66 (5H, m),
4.05-5.42 (4H, m), 6.92-7.49 (4H, m), 8.29-8.90 (1H, m); .sup.13C
NMR (100 MHz, d.sub.6-DMSO+TFA) .delta. 20.52, 20.7524.64, 25.21,
27.00, 32.91, 34.61, 44.97 (CH.sub.2), 47.73, 52.41, 52.66, 52.91,
53.25, 57.77 (CH, CH.sub.3), 83.44, 85.21 (CH.sub.2), 103.94,
104.13 (C), 108.71, 113.92, 124.78 (CH), 128.60, 143.56, 145.57,
148.54, 158.17, 158.55, 158.93, 159.32, 160.59, 160.78, 170.91,
172.21, 173.28, 202.66, 202.80; .sup.19F (376 MHz, d.sub.6-DMSO)
.delta. -75.65, -226.85, -226.94, -228.09, -230.52, -230.83,
-232.64, -232.74, -232.96.; MS (LR, ES) calculated for
C.sub.21H.sub.23FN.sub.2O.s- ub.7: 434.4251, ES-433.386,
ES+435.171.
Example 6
[3S/R,
(2S)]-5-Fluoro-4-oxo-3-[1-(3-chloro-benzo[b]thiophene-2-carbonyl)-2-
-piperidinecarboxamido]-pentanoic Acid (Compound 6)
[0194] 60
[0195] The title compound was prepared using procedures similar to
those described in methods A-G. The product was isolated as a white
foam (163 mg, 38% last step): IR (solid) 1803, 1736, 1674, 1622,
1527, 1417, 1269 cm.sup.-1; .sup.1H NMR (400 MHz, d.sub.6-DMSO+TFA)
.delta. 1.20-1.80 (6H, m), 2.00-2.30 (1H, m), 2.39-3.60 (3H, m),
4.20-4.82 (2H, m), 4.98-5.40 (2H, m), 7.42-7.69 (2H, m), 7.72-7.94
(1H, m), 7.99-8.20 (1H, m), 8.23-8.70 (1H m); .sup.13C NMR (100
MHz, d.sub.6-DMSO+TFA) .delta. 18.87, 19.07, 23.25, 23.85, 25.88,
31.47, 33.03, 33.20, 33.40, 44.18 (CH.sub.2), 46.05, 46.16, 50.78,
50.82, 50.94, 51.02, 51.14, 51.26, 51.42, 56.58, 56.94 (CH), 80.08,
81.84, 81.93, 82.05, 83.73, 83.82 (CH.sub.2), 102.44, 102.63,
116.52, 116.68, 116.90 (C), 120.79, 122.31, 124.80, 125.55, 125.65
(CH), 129.02, 129.24, 129.32, 133.75, 135.65, 160.84, 161.00,
168.93, 169.18, 169.39, 170.64, 171.78, 172.64, 200.93, 201.26,
201.40; .sup.19F (376 MHz, d.sub.6-DMSO +TFA) .delta. -226.55,
-226.79, -226.87, -226.97, -229.76, -229.88, -230.67, -231.15,
-232.35, -232.50, -232.56, -232.61; MS (LR, ES) calculated for
C.sub.20H.sub.20ClFN.sub.2O.sub.5S: 454.9082, ES-453.296,
ES+455.12.
Example 7
[3S/R,
(2S)]-5-Fluoro-4-oxo-3-[1-(3-chloro-thiophene-2-carbonyl)-2-piperid-
inecarboxamido]-pentanoic Acid (Compound 7)
[0196] 61
[0197] The title compound was prepared using procedures similar to
those described in methods H-J, F and K. The product was isolated
as a white foam: IR (solid) 1784, 1736, 1670, 1612, 1522, 1450,
1269 cm.sup.-1; .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 1.13-1.80
(5H, m), 2.01-3.60 (5H, m), 4.11-5.36 (4H, m), 7.02-7.17 (1H, m),
7.70-7.90 (1H, m), 8.40-8.61 (1H, m); .sup.19F (376 MHz,
CDCl.sub.3) .delta. -226.90 (t), -232.65 (m); Low Res. MS (ES)
calculated for C.sub.16H.sub.18ClFN.sub.2O.- sub.5S: 404.8477,
ES-403.23, ES+405.062.
Example 8
[3S/R,
(2S)]-5-Fluoro-4-oxo-3-[1-(benzofuran-2-carbonyl)-2-piperidinecarbo-
xamido]pentanoic Acid (Compound 8)
[0198] 62
[0199] The title compound was prepared using procedures similar to
those described in methods A-G. The product was isolated as a white
foam (3.7 mg, 6% last step): .sup.1H NMR (400 MHz, DMSO+TFA)
.delta. 0.70-1.80 (5H, m), 2.10-3.60 (5H, m), 4.03-5.40 (4H, m),
7.20-7.83 (5H, m), 8.35-8.80 (1H, m); .sup.19F (376 MHz,
CDCl.sub.3) .delta. -226.75, -226.89, -232.71; Low Res. MS (ES)
calculated for C.sub.20H.sub.21CFN.sub.2O.sub.6- : 404.3986,
ES-403.359, ES+405.165.
Example 9
[3S/R,
(2S)]-5-Fluoro-4-oxo-3-[1-[2-(3-trifluoromethyl-phenyl)-thiazole-4--
carbonyl]-2-piperidinecarboxamido]-pentanoic Acid (Compound 9)
[0200] 63
[0201] The title compound was prepared using procedures similar to
those described in methods A-G. The product was isolated as a white
foam (31 mg, 11% last step): IR (solid) 1789, 1741, 1617, 1512,
1441, 1417, 1327, 1231, 1169, 1122 cm.sup.-1; .sup.1H NMR (400 MHz,
d.sub.6-DMSO+TFA) .delta. 1.19-1.81 (5H, m), 2.04-3.55 (5H, m,
Asp), 4.04-5.39 (4H, m), 7.64-8.36 (5H, m), 8.42-8.62 (1H, m);
.sup.13C NMR (100 MHz, d.sub.6-DMSO+TFA) .delta. 20.54, 24.71,
25.13, 27.12, 27.88, 33.06, 34.54, 34.67, 45.29, 47.62, 52.28,
52.42, 53.09, 57.78, 83.22, 83.44, 85.22, 103.92, 104.11, 122.90,
125.50, 125.85, 127.35, 128.21, 129.95, 130.26, 130.58, 130.78,
131.07, 133.77, 151.09, 163.56, 163.97, 165.03, 171.12, 171.28,
172.10, 172.17, 202.81; .sup.19F (376 MHz, d.sub.6-DMSO) .delta.
-61.76, -226.75, -226.85, -226.96, -227.04, -230.23, -230.35,
-230.85, -232.49, -232.6, -232.64, -232.83; MS (LR, ES) calculated
for C.sub.22H.sub.21F.sub.4N.sub.3O.sub.5S: 515.4874, ES-514.361,
ES+516.167.
Example 10
[3S/R,
(2S)]-5-Fluoro-4-oxo-3-[1-[2-(3-trifluoromethyl-phenyl)-furan-4-car-
bonyl]-2-piperidinecarboxamido]-pentanoic Acid (Compound 10)
[0202] 64
[0203] The title compound was prepared using procedures similar to
those described in methods A-G. The product was isolated as a white
foam (82 mg, 18% last step): IR (solid) 1794, 1736, 1670, 1603,
1522, 1431, 1331, 1255, 1165 cm.sup.-1; .sup.1H NMR (400 MHz,
d.sub.6-DMSO +TFA) .delta. 1.25-3.58 (10H, m), 4.18-5.34 (4H, m),
6.98-7.41 (2H, m), 7.62-7.78 (2H, m), 7.91-8.13 (2H, m), 8.10-8.80
(1H, m); .sup.13 C NMR (100 MHz, d.sub.6-DMSO +TFA) .delta. 19.18,
19.42, 31.48, 33.13, 33.32, 50.87, 50.96, 81.95, 81.98, 83.73,
83.76, 102.49, 102.54, 102.69, 107.73, 116.84, 118.78, 119.28,
121.49, 123.66, 124.20, 126.62, 126.91, 128.68, 129.00, 129.16,
129.21, 129.31, 145.80, 145.87, 151.28, 151.39, 157.83, 158.40,
169.27, 169.64, 170.60, 170.70, 171.80, 201.16, 201.30, 201.43;
.sup.19F (376 MHz, d.sub.6-DMSO +TFA) .delta. -61.63, -226.79,
-232.56; MS (LR, ES) calculated for
C.sub.23H.sub.22F.sub.4N.sub.2O.sub.6: 498.4352, ES-497.313,
ES+499.233.
Example 11
[3S/R,
(2S)]-5-Fluoro-3-{[1-(3-methyl-5-phenyl-thiophene-2-carbonyl)
piperidine-2-carbonyl]-amino}-4-oxo-pentanoic Acid (Compound
11)
[0204] 65
[0205] Method N: 5-Bromo-3-methyl-thiophene-2-carbaldehyde
[0206] A solution of 3-methylthiophene-2-carbaldehyde (10 g, 0.079
mol) in dichloromethane (10 ml) was added dropwise to a stirred
solution of bromine (4.08 ml, 0.079 mol) in dichloromethane (15 ml)
at room temperature. The resulting mixture was heated to reflux
temperature for 3 hours before cooling to room temperature, washed
with water (3.times.50 ml), saturated NaHCO.sub.3 solution
(2.times.25 ml), dried (MgSO.sub.4) and the solvent removed at
reduced pressure to give the sub-title compound as a brown solid
(14.7 g, 66% yield): .sup.1H NMR (400 MHz, CDCl.sub.3)
.delta..sub.H 2.60 (3H, s), 6.97 (1H, s) 9.20 (1H, s).
[0207] Method O: 3-Methyl-5-phenyl-thiophene-2-carbaldehyde
[0208] To a solution of 5-bromo-3-methyl-thiophene-2-carbaldehyde
(1.00 g, 4.88 mmol) in ethylene glycol dimethyl ether (9 ml) was
added phenylboronic acid (0.773 g, 6.34 mmol), 2M Na.sub.2CO.sub.3
solution (6.3 ml) and Pd(PPh.sub.3).sub.4 [0.282 g, 0.24 mmol). The
mixture was heated for 18 hours, cooled and the solvent removed at
reduced pressure to leave a brown residue which was partitioned
between water (15 ml) and dichloromethane (20 ml). The organic was
separated, washed with water (2.times.5 ml), brine (10 ml), dried
(MgSO.sub.4) and the solvent removed at reduced pressure to give a
brown oil. Purification by flash column chromatography (6:1 petrol
40-60.degree. C./ethyl acetate) gave the sub-title compound as a
yellow oil (0.90 g, 91% yield): .sup.1H NMR (400 MHz, CDCl.sub.3)
.delta..sub.H 2.75 (3H, s), 7.25 (1H, s) 7.35-7.75 (5H, s), 10.05
(1H, s).
[0209] Method P: 3-Methyl-5-phenyl-thiophene-2-carboxylic Acid
[0210] To a stirred solution of
3-methyl-5-phenyl-thiophene-2-carbaldehyde (0.200 g, 0.99 mmol) and
2-methyl-2-butene (2.77 g, 0.040 mol) in dimethylformamide (4 ml)
at 0.degree. C. was added NaClO.sub.2 (0.894 g, 9.89 mmol) and
NaH.sub.2PO.sub.4 (1.09 g, 7.91 mmol) in water (5 ml). The solution
was allowed to warm to room temperature and stirred for 18 hours.
The solvent was removed at reduced pressure and the residue
partitioned between dichloromethane (10 ml) and 1N HCl solution (10
ml). The organic was separated, the aqueous layer extracted with
dichloromethane (2.times.5 ml). The combined organic layers were
dried (MgSO.sub.4) and the solvent removed at reduced pressure to
give a yellow oil. Purification by flash column chromatography 50%
ethyl acetate/petrol 40-60.degree. C.) gave the sub-title compound
as a white solid (0.14 g, 69% yield): .sup.1H NMR (400 MHz,
CDCl.sub.3) .delta..sub.H 2.65 (3H, s), 7.25 (1H, s) 7.35-7.80 (5H,
s).
[0211] Method Q: [3S/R,
(2S)]-5-Fluoro-3-{[1-(3-methyl-5-phenyl-thiophene-- 2-carbonyl)
piperidine-2-carbonyl]-amino}-4-oxo-pentanoic Acid
[0212] The title compound was prepared from
3-methyl-5-phenyl-thiophene-2-- carboxylic acid using procedures
similar to those described in Methods F, H-K. The product was
isolated as a white foam (0.066 g, 81% yield): IR (film) 1781.5,
1715.3, 1668.0, 1597.1, 1441.0, 1190.3 cm-1; .sup.1H NMR (400 MHz,
DMSO) .delta..sub.H 1.00-1.80 (6H, m), 1.90-2.30 (4H, m), 2.70-3.90
(4H, m), 4.10-5.50 (4H, 2.times.m), 7.30-8.60 (6H, 4.times.m);
.sup.19F NMR (376 MHz, DMSO) -61.7, -224.3, -226.7, -226.8, -227.5,
-232.7, -233.4; MS (FAB+ve, HR) Calculated for
C.sub.23H.sub.24FN.sub.2O.- sub.5S (MH-) 459.52, found 459.40.
Example 12
[3S/R,
(2S)]-5-Fluoro-3-({1-[3-methyl-5-(3-trifluoromethyl-phenyl)-thiophe-
ne-2-carbonyl]-piperidine-2-carbonyl}-amino)-4-oxo-pentanoic Acid
(Compound 12)
[0213] 66
[0214] The title compound was prepared from
3-methylthiophene-2-carbaldehy- de using procedures similar to
those described in Methods F, H-K, N-P. The product was isolated as
a pale pink solid (0.16 g, 94%): IR (film) 1784.0, 1726.9, 1664.9,
1588.7, 1436.2, 1326.6, 1164.6 cm-1; .sup.1H NMR (400 MHz, DMSO)
.delta..sub.H 1.00-1.80 (6H, m), 1.90-2.30 (4H, m), 2.70-4.05 (4H,
m), 4.10-5.40 (4H, m), 7.00-9.00 (6H, m); .sup.19F NMR (376 MHz,
DMSO) -62.0, -224.3, -226.7, -226.9, -227.5, -232.6, -232.7,
-233.4; MS (FAB+ve, HR) Calculated for
C.sub.24H.sub.23F.sub.4N.sub.2O.su- b.5S (MH-) 527.41, found
527.52.
Example 13
[3S/R,
(2S)]-5-Fluoro-4-oxo-3-({1-[5-(3-trifluoromethyl-phenyl)-thiophene--
2-carbonyl]-piperidine-2S-carbonyl}-amino)-pentanoic Acid (Compound
13)
[0215] 67
[0216] The title compound was prepared from
thiophene-2-carbaldehyde using procedures similar to those
described in Methods F, H-K, N-P. The product was isolated as a
pale yellow solid (0.23 g, 93%): IR (film) 1784.0, 1722.1, 1664.9,
1588.7, 1531.5, 1321.8, 1164.6 cm-1; .sup.1H NMR (400 MHz, DMSO)
.delta..sub.H 0.90-1.85 (6H, m), 2.00-2.40 (1H, m), 2.45-3.50 (3H,
m), 3.90-5.55 (4H, m), 7.00-9.05 (7H, m); .sup.19F NMR (376 MHz,
DMSO) -61.7, -224.3, -226.7, -226.8, -227.5, -227.6, -232.7,
-233.4; MS (FAB+ve, HR) Calculated for
C.sub.23H.sub.23F.sub.4N.sub.2O.sub.5S (MH+) 515.51, found
515.35.
Example 14
[3S/R,
(2S)]-5-Fluoro-4-oxo-3-{[1-(pyridine-2-carbonyl)-piperidine-2-carbo-
nyl]-amino}-pentanoic Acid (Compound 14)
[0217] 68
[0218] The title compound was prepared from pyridine-2-carboxylic
acid using procedures similar to those described in Methods F, H-K.
The product was isolated as a white solid (0.10 g, 93%):IR (film)
2945.4, 1650.5, 1446.6, 1186.5, 1139.9 cm.sup.-1; .sup.1H NMR (400
MHZ, CDCl.sub.3) .delta..sub.H 1.40-1.80 (6H, m), 2.20-2.50 (1H,
m), 2.69-3.12 (2H, m), 3.29-3.35 (1H, m), 3.48-3.51 (1H, m),
4.47-5.29 (3H, m), 7.37-9.11 (6H, m); .sup.19F NMR (376 MHz,
CDCl.sub.3) -231.69, -231.56, -231.44; MS (FAB+ve, HR) Calculated
for C.sub.17H.sub.21FN.sub.3O.sub.5 (MH+) 366.36, found 366.4.
Example 15
[3S/R,
(2S)]-3-{[1-(Biphenyl-3-carbonyl)-piperidine-2-carbonyl]-amino}-5-f-
luoro-4-oxo-pentanoic Acid (Compound 15)
[0219] 69
[0220] The title compound was prepared from 3-biphenylcarboxylic
acid using procedures similar to those described in Methods F, H-K.
The product was isolated as a white solid (0.13 g, 97%): IR (film)
2930.9, 1782.2, 1723.7, 1668.6, 1596.1, 1444.2, 1174.7 cm-1;
.sup.1H NMR (400 MHz, CDCl.sub.3) .delta..sub.H 1.40-1.90 (6H, m),
2.19-2.41 (1H, m), 2.70-3.30 (3H, m), 3.70-3.85 (1H, m), 4.30-5.50
(3H, m), 7.35-7.70 (11H, m); .sup.19F NMR (376 MHz, CDCl.sub.3)
-229.60, -229.88; MS (FAB+ve, HR) Calculated for
C.sub.24H.sub.26FN.sub.2O.sub.5 (MH+) 441.46, found 441.4.
Biological Methods
Example 16
Enzyme Assays
[0221] The assays for caspase inhibition are based on the cleavage
of a fluorogenic substrate by recombinant, purified human
Caspases-1, -3, -7 or -8. The assays are run in essentially the
same way as those reported in WO01/42216.
[0222] The compounds of examples 1-15 possess
K.sub.inactvalues>5,000 M.sup.-1s.sup.-1 against caspases-1, -3
and -8.
Example 17
Inhibition of IL-1.beta. secretion from Mixed Population of
Peripheral Blood Mononuclear Cells (PBMC)
[0223] Processing of pre-IL-1.beta. by caspase-1 can be measured in
cell culture using a variety of cell sources. Human PBMC obtained
from healthy donors provides a mixed population of lymphocyte and
mononuclear cells that produce a spectrum of interleukins and
cytokines in response to many classes of physiological stimulators.
The assay conditions used for inhibition of IL-1.beta. secretion
from mixed population of peripheral blood mononuclear cells can be
found in WO01/42216.
[0224] The inhibitory potency of the compounds can be represented
by an IC.sub.50 value, which is the concentration of inhibitor at
which 50% of the mature IL-1.beta. is detected in the supernatant
as compared to the positive controls. Compound 10 of this invention
showed an IC.sub.50 of less than 0.5 .mu.M in inhibition of
IL-1.beta. secretion from peripheral blood mononuclear cells as
determined by the above methods.
Example 18
Anti-Fas Induced Apoptosis Assay
[0225] Cellular apoptosis can be induced by the binding of Fas
ligand (FasL) to its receptor, CD95 (Fas). Conditions for an assay
to measure the effect of compounds on the inhibition of the
caspase-8-mediated apoptotic pathway can be found in
WO01/42216.
[0226] Compound 3 of this invention showed an IC.sub.50 of less
than 0.05 .mu.M in the FAS induced apoptosis assay.
Example 19
Inhibition of TNF Release from Whole Blood
[0227] Human blood was freshly drawn from healthy donors and
collected in vacutainers. Blood was diluted 1:2 in PBS (tissue
culture, pyrogen free) in a sterile bottle and inverted to mix
well. Aliquots of 0.5 ml of blood mixture were dispensed into
cluster tubes in 96 well format.
[0228] Dilutions of the test compounds were prepared in RPMI by
taking 100 mM DMSO stocks of the compounds and diluting 1:10 in
RPMI medium in eppendorfs, to give a 10 mM stock. 1:5 serial
dilutions were prepared from the stock solutions.
[0229] LPS was kept at a frozen stock (-20 degrees C.) at 1 mg/ml
in PBS and then diluted to 1:10 with RPMI medium and finally
diluted in the medium again 1:350. 501l of each test compound
(first concentration was 100uM) were added to the blood samples and
then stimulated with 10 .mu.l LPS (final concentration in the well
is 5 ng/ml). The contents were gently mixed using an 8 well
multi-channel pipette and incubated at 37.degree. C. over night. At
the end of the incubation time, contents were gently mixed, then
spun down at 1000.times. g for 5 mins at 20.degree. C. The serum
supernatants were transferred to a fresh plate without disturbing
the RBCs and diluted 1:2 with the diluent RD6C.
[0230] TNF-alpha levels of supernatants were assayed using the R+D
systems ELISA kit, using R+D systems protocol. Samples were read at
450 nm. Most preferred compounds of this invention showed IC.sub.50
of less than 6 .mu.M in the LPS-induced TNF-alpha assay in whole
blood.
[0231] Compound 10 of this invention showed an IC.sub.50 of less
than 6 .mu.M (5044 nM) in the LPS induced TNF-alpha assay in whole
blood.
[0232] While we have described a number of embodiments of this
invention, it is apparent that our basic examples can be altered to
provide other embodiments, which utilize the compounds and methods
of this invention. Therefore, it will be appreciated that the scope
of this invention is to be defined by the appended claims rather
than by the specific embodiments, which have been represented by
way of example.
* * * * *