U.S. patent application number 09/943103 was filed with the patent office on 2002-08-01 for acetylenic sulfonamide thiol tace inhibitors.
This patent application is currently assigned to American Cyanamid Company. Invention is credited to Chen, James M., Levin, Jeremy I..
Application Number | 20020103163 09/943103 |
Document ID | / |
Family ID | 26852116 |
Filed Date | 2002-08-01 |
United States Patent
Application |
20020103163 |
Kind Code |
A1 |
Levin, Jeremy I. ; et
al. |
August 1, 2002 |
Acetylenic sulfonamide thiol TACE inhibitors
Abstract
The present invention relates to acetylenic aryl sulfonamide
thiols which act as inhibitors of TNF-.alpha. converting enzyme
(TACE). The compounds of the present invention are useful in
disease conditions mediated by TNF-.alpha., such as rheumatoid
arthritis, osteoarthritis, sepsis, AIDS, ulcerative colitis,
multiple sclerosis, Crohn's disease and degenerative cartilage
loss.
Inventors: |
Levin, Jeremy I.; (New City,
NY) ; Chen, James M.; (Stoddard Court, NJ) |
Correspondence
Address: |
WYETH
FIVE GIRALDA FARMS
MADISON
NJ
07940
US
|
Assignee: |
American Cyanamid Company
Madison
NJ
|
Family ID: |
26852116 |
Appl. No.: |
09/943103 |
Filed: |
August 30, 2001 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
09943103 |
Aug 30, 2001 |
|
|
|
09492974 |
Jan 27, 2000 |
|
|
|
60155218 |
Jan 27, 1999 |
|
|
|
Current U.S.
Class: |
514/79 ; 514/126;
514/127; 514/601; 546/22; 548/413; 558/199; 564/80 |
Current CPC
Class: |
C07D 295/13 20130101;
A61P 19/02 20180101; C07C 323/49 20130101; A61K 31/5375
20130101 |
Class at
Publication: |
514/79 ; 514/126;
514/127; 514/601; 546/22; 548/413; 558/199; 564/80 |
International
Class: |
A61K 031/675; C07F
009/28; C07F 009/547; C07F 009/02 |
Claims
What is claimed:
1. The invention provides TACE and MMP inhibitors having the
formula: B wherein B is 33wherein: W is oxygen or sulfur; X is
SO.sub.2 or --P(O)--R.sub.10; Y is aryl or heteroaryl as defined
below, with the proviso that X and Z may not be bonded to adjacent
atoms of Y; Z is O, NH, CH.sub.2 or S; R.sub.1 is hydrogen, aryl,
alkyl of 1-6 carbon atoms, alkenyl of 2-6 carbon atoms, alkynyl of
2-6 carbon atoms; R.sub.2 is hydrogen, aryl or heteroaryl as
defined below, cycloalkyl of 3-6 carbon atoms,
-C.sub.5-C.sub.7-cycloheteroalkyl, alkyl of 1-6 carbon atoms,
alkenyl of 2-6 carbon atoms, alkynyl of 2-6 carbon atoms; or
R.sub.1 and R.sub.2, together with the atom to which they are
attached, may form a ring wherein R.sub.1 and R.sub.2 represent a
divalent moiety of the formula: 34wherein Q=a carbon-carbon single
or double bond, O, S, SO, --N--R.sub.11, or --CONR.sub.15; m=1-3;
r=1 or 2, with the proviso that when Q is a bond, r is equal to 2;
Aryl is phenyl or naphthyl optionally substituted by one to two
substituents selected from R.sub.7, where R.sub.7 is as defined
below; Heteroaryl is defined as 35optionally mono- or di-
substituted by R.sub.7, wherein K is defined as O, S or
--NR.sub.15; R.sub.3 is hydrogen or alkyl of 1-6 carbon atoms; or
R.sub.1 and R.sub.3, together with the atoms to which they are
attached, may form a 5 to 8 membered ring wherein R.sub.1 and
R.sub.3 represent divalent moieties of the formulae: 36wherein Q
and m are as defined above; A is aryl or heteroaryl; s is 0-3; u is
1-4; R.sub.4 and R.sub.5 are each, independently, hydrogen, alkyl
of 1-6 carbon atoms, --CN, --CCH; R.sub.6 is hydrogen, aryl,
heteroaryl, alkyl of 1-6 carbon atoms, alkenyl of 2-6 carbon atoms,
alkynyl of 2-6 carbon atoms, cycloalkyl of 3-6 carbon atoms or
-C.sub.5-C.sub.7-cycloheteroalkyl as defined below; R.sub.7 is
hydrogen, halogen, alkyl of 1-6 carbon atoms; alkenyl of 2-6 carbon
atoms; alkynyl of 2-6 carbon atoms, cycloalkyl of 3-6 carbon atoms,
--OR.sub.8, --CN, --COR.sub.8, perfluoroalkyl of 1-4 carbon atoms,
--O-- perfluoroalkyl of 1-4 carbon atoms, --CONR.sub.8R.sub.9,
--S(O).sub.nR.sub.8, --OPO(OR.sub.8)OR.sub.9,
--PO(OR.sub.8)R.sub.9, --OC(O)NR.sub.8R.sub.9,
--C(O)NR.sub.8OR.sub.9, --COOR.sub.8, --SO.sub.3H,
--NR.sub.8R.sub.9, --N[(CH.sub.2).sub.2].sub.2NR.sub.8,
--NR.sub.8COR.sub.9, --NR.sub.8COOR.sub.9,
--SO.sub.2NR.sub.8R.sub.9, --NO.sub.2, --N(R.sub.8)SO.sub.2R.sub.9,
--NR.sub.8CONR.sub.8R.sub.9,
--NR.sub.8C(.dbd.NR.sub.9)NR.sub.8R.sub.9, -tetrazol-5-yl,
--SO.sub.2NHCN, --SO.sub.2NHCONR.sub.8R.sub.9, phenyl, heteroaryl
as defined above, or -C.sub.5-C.sub.7-cycloheteroalkyl as defined
below; wherein --NR.sub.8R.sub.9 may form a pyrrolidine,
piperidine, morpholine, thiomorpholine, oxazolidine, thiazolidine,
pyrazolidine, piperazine, or azetidine ring; wherein
-C.sub.5-C.sub.7-cycloheteroalkyl is defined as 37wherein K is
defined as above; R.sub.8 and R.sub.9 are each, independently,
hydrogen, alkyl of 1-6 carbon atoms, cycloalkyl of 3-6 carbon
atoms, aryl, heteroaryl or -C.sub.5-C.sub.7-cycloheteroalkyl;
R.sub.10 is alkyl of 1-6 carbon atoms, cycloalkyl of 3-6 carbon
atoms, aryl or heteroaryl as defined above; R.sub.11 is hydrogen,
alkyl of 1-6 carbon atoms, cycloalkyl of 3-6 carbon atoms, aryl,
heteroaryl, --S(O).sub.nR.sub.8, --COOR.sub.8, --CONR.sub.8R.sub.9,
--SO.sub.2NR.sub.8R.sub.9 or --COR.sub.8; R.sub.12 and R.sub.13 are
independently selected from H, --OR.sub.8, --NR.sub.8R.sub.9, alkyl
of 1-6 carbon atoms, alkenyl of 2-6 carbon atoms, alkynyl of 2-6
carbon atoms, cycloalkyl of 3-6 carbon atoms, aryl, heteroaryl,
--COOR.sub.8; --CONR.sub.8R.sub.9; or R.sub.12 and R.sub.13
together form a -C.sub.3-C.sub.6-cycloalkyl of 3-6 carbon atoms or
a -C.sub.5-C.sub.7-cycloheteroalkyl ring; or R.sub.12 and R.sub.13
together with the carbon to which they are attached, form a
carbonyl group; with the proviso that R.sub.10 and R.sub.12 or
R.sub.11 and R.sub.12 may form a cycloheteroalkyl ring, wherein
cycloheteroalkyl is as defined above, when they are attached to
adjacent atoms; R.sub.14 is --OR.sub.8, --NR.sub.8R.sub.9, alkyl of
1-6 carbon atoms, cycloalkyl of 3-6 carbon atoms, aryl or
heteroaryl; R.sub.15 is hydrogen, aryl, heteroaryl, alkyl of 1-6
carbon atoms or cycloalkyl of 3-6 carbon atoms; and n is 0-2; or a
pharmaceutically acceptable salt thereof.
2. A compound according to claim 1 wherein B is 38or a
pharmaceutically acceptable salt thereof.
3. A compound according to claim 2 wherein X is SO.sub.2; or a
pharmaceutically acceptable salt thereof.
4. A compound according to claim 2 wherein X is SO.sub.2, and Z is
oxygen; or a pharmaceutically acceptable salt thereof.
5. A compound according to claim 2 wherein X is SO.sub.2, Z is
oxygen and R.sub.4 and R.sub.5 are hydrogen; or a pharmaceutically
acceptable salt thereof.
6. A compound according to claim 2 wherein X is SO.sub.2, Z is
oxygen, R.sub.4 and R.sub.5 are hydrogen, and R.sub.6 is
--CH.sub.2OH or methyl; or a pharmaceutically acceptable salt
thereof.
7. A compound according to claim 1 wherein R.sub.1 is hydrogen,
such that this compound has the absolute stereochemistry as shown
in structure 1a below. 39
8. A compound of formula II, with the proviso that R.sub.6 is not
hydrogen and R.sub.4, R.sub.5 and R.sub.6 are as defined in claim
1. 40
9. A compound of formula III, with the proviso that R.sub.6 is not
hydrogen wherein J is fluorine, bromine, chlorine, 1,2,4-triazolyl,
benzotriazolyl or imidazol-yl, and R.sub.4, R.sub.5 and R.sub.6 are
as defined in claim 1. 41
10. A method of inhibiting pathological changes mediated by
TNF-.alpha. converting enzyme (TACE) in a mammal in need thereof
which comprises administering to said mammal a therapeutically
effective amount of a compound having the formula according to
claim 1.
11. The method according to claim 8 wherein the condition treated
is rheumatoid arthritis, graft rejection, cachexia, inflammation,
fever, insulin resistance, septic shock, congestive heart failure,
inflammatory disease of the central nervous system, inflammatory
bowel disease or HIV infection.
12. A pharmaceutical composition comprising a compound having the
formula according to claim 1.
13. A compound according to claim 1 which is selected from the
group consisting of
4-But-2-ynyloxy-N-((1R)-2-mercapto-1-methyl-ethyl)-N-methyl-
benzene-sulfonamide.
Description
FIELD OF INVENTION
[0001] This invention relates to acetylenic aryl sulfonamide thiols
which act as inhibitors of TNF-.alpha. converting enzyme (TACE).
The compounds of the present invention are useful in disease
conditions mediated by TNF-.alpha., such as rheumatoid arthritis,
osteoarthritis, sepsis, AIDS, ulcerative colitis, multiple
sclerosis, Crohn's disease and degenerative cartilage loss.
BACKGROUND OF THE INVENTION
[0002] TNF-.alpha. converting enzyme (TACE) catalyzes the formation
of TNF-.alpha. from membrane bound TNF-.alpha.; precursor protein.
TNF-.alpha. is a pro-inflammatory cytokine that is believed to have
a role in rheumatoid arthritis [Shire, M. G.; Muller, G. W. Exp.
Opin. Ther. Patents 1998, 8(5), 531; Grossman, J. M.; Brahn, E. J.
Women's Health 1997, 6(6), 627; Isomaki, P.; Punnonen, J. Ann. Med.
1997, 29, 499: Camussi, G.; Lupia, E. Drugs, 1998, 55(5), 613.]
septic shock [Mathison, et. al. J. Clin. Invest. 1988, 81, 1925;
Miethke, et. al. J. Exp. Med. 1992, 175, 91.], graft rejection
[Piguet, P. F.; Grau, G. E.; et. al. J. Exp. Med. 1987, 166,
1280.], cachexia [Beutler, B.; Cerami, A. Ann. Rev. Biochem. 1988,
57, 505.], anorexia, inflammation [Ksontini, R,; MacKay, S. L. D.;
Moldawer, L. L. Arch. Surg. 1998, 133, 558.], congestive heart
failure [Packer, M. Circulation, 1995, 92(6), 1379; Ferrari, R.;
Bachetti, T.; et. al. Circulation, 1995, 92(6), 1479.],
post-ischaemic reperfusion injury, inflammatory disease of the
central nervous system, inflammatory bowel disease, insulin
resistance [Hotamisligil, G. S.; Shargill, N. S.; Spiegelman, B.
M.; et. al. Science, 1993, 259, 87.] and HIV infection [Peterson,
P. K.; Gekker, G.; et. al. J. Clin. Invest. 1992, 89, 574;
Pallares-Trujillo, J.; Lopez-Soriano, F. J. Argiles, J. M. Med.
Res. Reviews, 1995, 15(6), 533.]], in addition to its
well-documented antitumor properties [Old, L. Science, 1985, 230,
630.]. For example, research with anti-TNF-.alpha. antibodies and
transgenic animals has demonstrated that blocking the formation of
TNF-.alpha. inhibits the progression of arthritis [Rankin, E. C.;
Choy, E. H.; Kassimos, D.; Kingsley, G. H.; Sopwith, A. M.;
Isenberg, D. A.; Panayi, G. S. Br. J. Rheumatol. 1995, 34, 334;
Pharmaprojects, 1996, Therapeutic Updates 17 (October),
au197-M2Z.]. This observation has recently been extended to humans
as well as described in "TNF-.alpha. in Human Diseases", Current
Pharmaceutical Design, 1996, 2, 662.
[0003] It is expected that small molecule inhibitors of TACE would
have the potential for treating a variety of disease states.
Although a variety of TACE inhibitors are known, many of these
molecules are peptidic and peptide-like which suffer from
bioavailability and pharmacokinetic problems. In addition, many of
these molecules are non-selective, being potent inhibitors of
matrix metalloproteinases and, in particular, MMP-1. Inhibition of
MMP-1 (collagenase 1) has been postulated to cause joint pain in
clinical trials of MMP inhibitors [Scrip. 1998, 2349, 20] Long
acting, selective, orally bioavailable non-peptide inhibitors of
TACE would thus be highly desirable for the treatment of the
disease states discussed above.
[0004] U.S. Pat. Nos. 5,455,258, 5,506,242, 5,552,419, 5,770,624,
and 5,817,822 as well as European patent application EP606,046A1
and WIPO international publications WO9600214 and WO9722587
disclose non-peptide inhibitors of matrix metalloproteinases and/or
TACE of which the aryl sulfonamnide hydroxamic acid shown below is
representative. Additional publications disclosing sulfonamide
based NIP inhibitors which are variants of the
sulfonamide-hydroxamate shown below, or the analogous
sulfonamide-carboxylates, are European patent applications
EP-757037-A1 and EP-757984-A1 and WIPO international publications
WO9535275, WO9535276, WO9627583, WO9719068, WO9727174, WO9745402,
WO9807697, WO9831664, WO9833768, WO9839313, WO9839329, WO9842659
and WO9843963. The discovery of this type of MMP inhibitor is
further detailed by MacPherson, et. al. in J. Med. Chem., (1997),
40, 2525 and Tamura, et. al. in J. Med. Chem. (1998), 41, 640.
1
[0005] Publications disclosing .beta.-sulfonamide-hydroxamate
inhibitors of MMPs and/or TACE in which the carbon alpha to the
hydroxamic acid has been joined in a ring to the sulfonamide
nitrogen, as shown below, include U.S. Pat. No. 5,753,653, WIPO
international publications WO9633172, WO9720824, WO9827069,
WO9808815, WO9808822, WO9808823, WO9808825, WO9834918, WO9808827,
Levin, et. al. Bioorg. & Med. Chem. Letters 1998, 8, 2657 and
Pikul, et. al. J. Med. Chem. 1998, 41, 3568. 2
[0006] The patent applications DE19,542,189-A1, WO9718194, and
EP803505 disclose additional examples of cyclic sulfonamides as MMP
and/or TACE inhibitors. In this case the sulfonamide-containing
ring is fused to a aromatic or heteroaromatic ring. 3
[0007] Examples of sulfonamide hydroxamic acid MMP/TACE inhibitors
in which a 2 carbon chain separates the hydroxamic acid and the
sulfonamide nitrogen, as shown below, are disclosed in WIPO
international publications WO9816503, WO9816506, WO9816514 and
WO9816520 and U.S. Pat. No. 5,776,961. 4
[0008] Analogous to the sulfonamides are the phosphinic acid amide
hydroxamic acid MMP/TACE inhibitors, exemplified by the structure
below, which have been disclosed in WIPO international publication
WO9808853. 5
[0009] Sulfonamide MMP/TACE inhibitors in which a thiol is the zinc
chelating group, as shown below, have been disclosed in WIPO
international application 9803166. 6
[0010] It is an object of this invention to disclose aryl
sulfonamide hydroxamic acid MMP/TACE inhibitors in which the
sulfonyl aryl group is para-substituted with a substituted butynyl
moiety or a propargylic ether, amine or sulfide.
SUMMARY OF THE INVENTION
[0011] The invention provides TACE and MMP inhibitors having the
formula:
[0012] B
[0013] wherein B is 7
[0014] wherein:
[0015] W is oxygen or sulfur;
[0016] is SO.sub.2 or --P(O)--R.sub.10;
[0017] Y is aryl or heteroaryl as defined below, with the proviso
that X and Z may not be bonded to adjacent atoms of Y;
[0018] Z is O, NH, CH.sub.2 or S;
[0019] R.sub.1 is hydrogen, aryl, alkyl of 1-6 carbon atoms,
alkenyl of 2-6 carbon atoms, alkynyl of 2-6 carbon atoms;
[0020] R.sub.2 is hydrogen, aryl or heteroaryl as defined below,
cycloalkyl of 3-6 carbon atoms, -C.sub.5-C.sub.7-cycloheteroalkyl,
alkyl of 1-6 carbon atoms, alkenyl of 2-6 carbon atoms, alkynyl of
2-6 carbon atoms;
[0021] or R.sub.1 and R.sub.2, together with the atom to which they
are attached, may form a ring wherein R.sub.1 and R.sub.2 represent
a divalent moiety of the formula: 8
[0022] wherein
[0023] Q=a carbon-carbon single or double bond, O, S, SO,
--N--R.sub.11, or --CONR.sub.15;
[0024] m=1-3;
[0025] r=1 or 2, with the proviso that when Q is a bond, r is equal
to 2;
[0026] Aryl is phenyl or naphthyl optionally substituted by one to
two substituents selected from R.sub.7, where R.sub.7 is as defined
below;
[0027] Heteroaryl is defined as 9
[0028] optionally mono- or di- substituted by R.sub.7, wherein K is
defined as O, S or --NR.sub.15;
[0029] R.sub.3 is hydrogen or alkyl of 1-6 carbon atoms;
[0030] or R.sub.1 and R.sub.3, together with the atoms to which
they are attached, may form a 5 to 8 membered ring wherein R.sub.1
and R.sub.3 represent divalent moieties of the formulae: 10
[0031] wherein Q and m are as defined above;
[0032] A is aryl or heteroaryl;
[0033] s is 0-3;
[0034] u is 1-4;
[0035] R.sub.4 and R.sub.5 are each, independently, hydrogen, alkyl
of 1-6 carbon atoms, --CN, --CCH;
[0036] R.sub.6 is hydrogen, aryl, heteroaryl, alkyl of 1-6 carbon
atoms, alkenyl of 2-6 carbon atoms, alkynyl of 2-6 carbon atoms,
cycloalkyl of 3-6 carbon atoms or -C.sub.5-C.sub.7-cycloheteroalkyl
as defined below;
[0037] R.sub.7 is hydrogen, halogen, alkyl of 1-6 carbon atoms;
alkenyl of 2-6 carbon atoms; alkynyl of 2-6 carbon atoms,
cycloalkyl of 3-6 carbon atoms, --OR.sub.8, --CN, --COR.sub.8,
perfluoroalkyl of 1-4 carbon atoms, --O-- perfluoroalkyl of 1-4
carbon atoms, --CONR.sub.8R.sub.9, --S(O).sub.nR.sub.8,
--OPO(OR.sub.8)OR.sub.9, --PO(OR.sub.8)R.sub.9,
--OC(O)NR.sub.8R.sub.9, --C(O)NR.sub.8OR.sub.9, --COOR.sub.8,
--SO.sub.3H, --NR.sub.8R.sub.9,
--N[(CH.sub.2).sub.2].sub.2NR.sub.8, --NR.sub.8COR.sub.9,
--NR.sub.8COOR.sub.9, --SO.sub.2NR.sub.8R.sub.9, --NO.sub.2,
--N(R.sub.8)SO.sub.2R.sub.9, --NR.sub.8CONR.sub.8R.sub.9,
--NR.sub.8C(.dbd.NR.sub.9)NR.sub.8R.sub.9,
--NR.sub.8C(.dbd.NR.sub.9)N(C.- dbd.OR.sub.8)R.sub.9,
--NR.sub.8C(.dbd.NR.sub.9)N(SO.sub.2R.sub.8)R.sub.9, tetrazol-5-yl,
--SO.sub.2NHCN, --SO.sub.2NHCONR.sub.8R.sub.9, phenyl, heteroaryl
as defined above, or -C.sub.5-C.sub.7-cycloheteroalkyl as defined
below;
[0038] wherein --NR.sub.8R.sub.9 may form a pyrrolidine,
piperidine, morpholine, thiomorpholine, oxazolidine, thiazolidine,
pyrazolidine, piperazine, or azetidine ring;
[0039] wherein -C.sub.5-C.sub.7-cycloheteroalkyl is defined as
11
[0040] wherein K is defined as above;
[0041] R.sub.8 and R.sub.9 are each, independently, hydrogen, alkyl
of 1-6 carbon atoms, cycloalkyl of 3-6 carbon atoms, aryl,
heteroaryl or -C.sub.5-C.sub.7-cyclohetero-alkyl;
[0042] R.sub.10 is alkyl of 1-6 carbon atoms, cycloalkyl of 3-6
carbon atoms, aryl or heteroaryl as defined above;
[0043] R.sub.11 is hydrogen, alkyl of 1-6 carbon atoms, cycloalkyl
of 3-6 carbon atoms, aryl, heteroaryl, --S(O).sub.nR.sub.8,
--COOR.sub.8, --CONR.sub.8R.sub.9, --SO.sub.2NR.sub.8R.sub.9 or
--COR.sub.8;
[0044] R.sub.12 and R.sub.13 are independently selected from H,
--OR.sub.8, --NR.sub.8R.sub.9, alkyl of 1-6 carbon atoms, alkenyl
of 2-6 carbon atoms, alkynyl of 2-6 carbon atoms, cycloalkyl of 3-6
carbon atoms, aryl, heteroaryl, --COOR.sub.8; --CONR.sub.8R.sub.9;
or R.sub.12 and R.sub.13 together form a
-C.sub.3-C.sub.6-cycloalkyl of 3-6 carbon atoms or a
-C.sub.5-C.sub.7-cycloheteroalkyl ring; or R.sub.12 and R.sub.13,
together with the carbon to which they are attached, form a
carbonyl group;
[0045] with the proviso that R.sub.10 and R.sub.12 or R.sub.11 and
R.sub.12 may form a cycloheteroalkyl ring, wherein cycloheteroalkyl
is as defined above, when they are attached to adjacent atoms;
[0046] R.sub.14 is --OR.sub.8, --NR.sub.8R.sub.9, alkyl of 1-6
carbon atoms, cycloalkyl of 3-6 carbon atoms, aryl or
heteroaryl;
[0047] R.sub.15 is hydrogen, aryl, heteroaryl, alkyl of 1-6 carbon
atoms or cycloalkyl of 3-6 carbon atoms;
[0048] and n is 0-2;
[0049] or a pharmaceutically acceptable salt thereof.
[0050] The invention is further directed to a process for making
compounds of structure B involving one or more reactions as
follows:
[0051] 1) converting a compound of formula I, or a salt or solvate
thereof, 12
[0052] into a compound of formula II 13
[0053] 2) converting a compound of formula II above, or a salt or
solvate thereof, to a compound of formula III: 14
[0054] wherein J is fluorine, bromine, chlorine, 1,2,4-triazolyl,
benzotriazolyl or imidazol-yl, and R.sub.4, R.sub.5 and R.sub.6 are
as defined above;
[0055] The invention is still further directed to a process for
making compounds of structure B involving one or more reactions as
follows:
[0056] 1) converting phenol, or a salt or solvate thereof, into a
compound of formula IV: 15
[0057] 2) converting a compound of formula IV above, or a salt or
solvate thereof, to a compound of formula II above.
[0058] Alkyl, alkenyl, alkynyl, and perfluoroalkyl include both
straight chain as well as branched moieties. The definitions of
alkyl, alkenyl, alkynyl, cycloalkyl and phenyl include alkyl,
alkenyl, alkynyl, cycloalkyl and phenyl moieties which are
unsubstituted (carbons bonded to hydrogen, or other carbons in the
chain or ring) or may be mono- or poly-substituted with R.sub.7.
When a moiety contains more than substituent with the same
designation (i.e., alkyl tri-substituted with R.sub.7) each of
those substituents (R.sub.7 in this case) may be the same or
different. Halogen means bromine, chlorine, fluorine, and
iodine.
[0059] The compounds of this invention may contain an asymmetric
carbon atom and some of the compounds of this invention may contain
one or more asymmetric centers and may thus give rise to optical
isomers and diastereomers. While shown without respect to
stereochemistry, the present invention includes such optical
isomers and diastereomers; as well as the racemic and resolved,
enantiomerically pure R and S stereoisomers; as well as other
mixtures of the R and S stereoisomers and pharmaceutically
acceptable salts thereof. It is recognized that one optical isomer,
including diastereomer and enantiomer, or stereoisomer may have
favorable properties over the other. Thus when disclosing and
claiming the invention, when one racemic mixture is disclosed, it
is clearly contemplated that both optical isomers, including
diastereomers and enantiomers, or stereoisomers substantially free
of the other are disclosed and claimed as well.
[0060] Pharmaceutically acceptable salts can be formed from organic
and inorganic acids, for example, acetic, propionic, lactic,
citric, tartaric, succinic, fumaric, maleic, malonic, mandelic,
malic, phthalic, hydrochloric, hydrobromic, phosphoric, nitric,
sulfuric, methanesulfonic, naphthalenesulfonic, benzenesulfonic,
toluenesulfonic, camphorsulfonic, and similarly known acceptable
acids when a compound of this invention contains a basic moiety.
Salts may also be formed from organic and inorganic bases,
preferably alkali metal salts, for example, sodium, lithium, or
potassium, when a compound of this invention contains an acidic
moiety.
[0061] Preferred compounds of the invention are those having the
formula:
[0062] B
[0063] wherein B is: 16
[0064] or a pharmaceutically acceptable salt thereof.
[0065] More preferred compounds of the invention are those in which
B is: 17
[0066] and X is SO.sub.2; or a pharmaceutically acceptable salt
thereof.
[0067] More preferred compounds of the invention are those in which
B is: 18
[0068] and X is SO.sub.2, and Z is oxygen; or a pharmaceutically
acceptable salt thereof.
[0069] More preferred compounds of the invention are those in which
B is: 19
[0070] and X is SO.sub.2, Z is oxygen and R.sub.4 and R.sub.5 are
hydrogen; or a pharmaceutically acceptable salt thereof.
[0071] Still more preferred compounds of the invention are those in
which B is: 20
[0072] and X is SO.sub.2, Z is oxygen, R.sub.4 and R.sub.5 are
hydrogen, and R6 is --CH.sub.2OH or methyl; or a pharmaceutically
acceptable salt thereof.
[0073] Still more preferred compounds are those in which B is:
21
[0074] wherein R.sub.1 is hydrogen, such that this compound has the
absolute stereochemistry as shown in structure 1a above.
DETAILED DESCRIPTION OF THE INVENTION
[0075] The invention compounds are prepared using conventional
techniques known to those skilled in the art of organic synthesis.
The starting materials used in preparing the compounds of the
invention are known, made by known methods or are commercially
available.
[0076] Those skilled in the art will recognize that certain
reactions are best carried out when other potentially reactive
functionality on the molecule is masked or protected, thus avoiding
undesirable side reactions and/or increasing the yield of the
reaction. To this end, those skilled in the art may use protecting
groups. Examples of these protecting group moieties may be found in
T. W-. Greene, P. G. M. Wuts "Protective Groups in Organic
Synthesis", 2.sup.nd Edition, 1991, Wiley & Sons, New York.
Reactive side chain functionalities on amino acid starting
materials are preferably protected. The need and choice of
protecting groups for a particular reaction is known to those
skilled in the art and depends on the nature of the functional
group to be protected (hydroxy, amino, carboxy, etc.), the
structure and stability of the molecule of which the substituent is
part and the reaction conditions. Those skilled in the art will
recognize that the nature and order of the synthetic steps
presented may be varied for the purpose of optimizing the formation
of the compounds of the invention.
[0077] When preparing or elaborating compounds of the invention
containing aryl, heteroaryl or heterocyclic rings, those skilled in
the art recognize that substituents on that ring may be prepared
before, after or concomitant with construction of the ring. For
clarity, substituents on such rings have been omitted from the
schemes herein below.
[0078] The thiol compounds of the invention, 1a-1c, are prepared
according to Scheme 1 by converting an alcohol, 2, into the
corresponding thioester or dithioester, 1b, via a Mitsunobu
procedure using reagents such as triphenylphosphine, diethyl
azodicarboxylate and thiolacetic acid. Conversion of 1b into thiol
1a is accomplished through hydrolysis or reductive cleavage of the
ester using sodium methoxide, sodium borohydride or similar
reagents. Thiol 1a may be converted into the corresponding
disulfide using methods compatible with acetylenic substituents,
such as oxidation with air or oxygen.
[0079] Alternatively, the alcohol moiety of 2 can be converted into
a leaving group J, where J is a halide, tosylate, mesylate,
triflate or similar functionality to give compound 3. Reaction of 3
with nucleophiles such as sodium sulfide, thiolacetic acid,
dithiolacetic acid, or similar agents or their salts, then provides
1a or 1b. 22
[0080] Alcohols 2 may be prepared as shown in Scheme 2.
Amnino-alcohol 4 (R.sub.30.dbd.H), or its hydroxyl protected analog
wherein R.sub.30 is a suitable masking group such as trialkylsilyl
or tetrahydropyran can be sulfonylated or phosphorylated with 7,
wherein J is as described in Scheme 1, in the presence of a tertar
amine base, or pyridine, to provide 8. Alkylation of N--H compound
8 with R.sub.3J and a base such as potassium carbonate or sodium
hydride in a polar aprotic solvent such as acetone,
N,N-dimethylformamide (DMF), or tetrahydrofuran (THF) provides
sulfonamide 9. Compound 9 is also available through direct reaction
of 7 with an N-substituted amino-alcohol derivative, 5. Compound 5
is available via alkylation or reductive alkylation of amine 4, or
by amination of 6 or the corresponding epoxide. Conversion of 9
into the alcohol is then performed in a manner consistent with the
choice of protecting group R.sub.30 and the presence of a
carbon-carbon triple bond. 23
[0081] Another route to compounds 9 is shown in Scheme 3. Compound
7, for example the sulfonyl chloride, can react with a primary
amine or ammonia to give compounds 10 or 11, respectively.
Alkylation of 10 with 6 or an analogous epoxide provides 9
directly, whereas 11 can be alkylated with R.sub.3J followed by 6
(or vice versa) or an epoxide to give 9. 24
[0082] Methods of preparation of sulfonylating agents 7 are shown
in Scheme 4. Thus, sulfonic acid salts 12, where ZR.sub.50 is a
hydroxy, thiol or substituted amino moiety may be alkylated with
acetylenes 13, where J is a suitable leaving group such as halogen
mesylate, tosylate, or triflate to give 14. Acetylenes 13 are
commercially available or known compounds, or they may be
synthesized by known methods by those skilled in the art. The
sulfonic acid salts 14 may be converted into the corresponding
sulfonyl chloride or other sulfonylating agent 7 by known methods,
such as reaction with oxalyl chloride or other reagent compatible
with substituents R.sub.4, R.sub.5 and R.sub.6 and the acetylene.
Alternatively, the disulfide 15 may be converted into di-acetylene
16 by reaction with compounds 13, followed by reduction of the
disulfide bond to provide the analogous thiols which may be
converted into 7 by known methods. Alkylation of the phenol,
thiophenol, aniline or protected aniline 17 with 13 to give 18,
followed by reaction with chlorosulfonic acid provide sulfonic
acids 19 which are readily converted into 7 with oxalyl chloride or
similar reagents. Thiophenols 20 are also precursors to 7 via
protection of the thiol, alkylation of ZH, where Z is O, N or S,
and deprotection of the sulfur followed by oxidation to the
sulfonic acid 19. 25
[0083] The phosphorus containing analogs of 7 may be prepared using
similar methodology, as shown in Scheme 5. 26
[0084] The acetylemic side chain may also be appended after
sulfonylation or phosphorylation of the amino acid derivative, as
shown in Scheme 6. Thus, the amino-alcohol derivatives 4 and 5 can
be sulfonylated or phosphorylated with compounds 23, where
ZR.sub.50 is hydroxy or protected hydroxy, thiol or amine, and, if
necessary, alkylated as in Scheme 2, to give 24. Removal of the
R.sub.50 masking group to gove 25 and subsequent alkylation of the
resulting phenol, thiol or amine with 13 provides 9. In the case
where ZR.sub.50 is equal to OH, no deprotection step is required to
give 25. Alternatively, the OR.sub.30 moiety of 24 may be converted
into the analogous thioester, thiol or disulfide, as shown in
Schemes 1 and 2, prior to deprotection of the ZR.sub.50 moiety of
compound 24. Subsequent alkylation of the unmasked --ZH group would
then provide the compounds of the invention. 27
[0085] The propargylic amine analogs of 9 can be synthesized as
shown in Scheme 7 starting from the amino-alcohol derivatives 4
and/or 5. Sulfonylation or phosphorylation with para-nitro aryl
compound 26, for example 4-nitrobenzenesulfonyl chloride, followed
by alkylation with R.sub.3J (for 4) using a base such as potassium
carbonate or sodium hydride in DME provides 27. Reduction of the
nitro moiety with hydrogen and palladium on carbon, tin chloride or
other known method to give aniline 28 and subsequent alkylation
with 13 then provides 9. Aniline 28 may also be derivatized (29)
prior to alkylation with 13 and then deprotected after the
alkylation step. As in Scheme 6, compound 29 can first be converted
into its thioester, disulfide or protected thiol analog, followed
by appending the propargyl group, via alkylation with 13, and
subsequent deprotection of the aniline to provide compounds 1a-1c
of the invention. 28
[0086] Acetylenic derivatives 9 are also accessible via the fluoro
compounds 31, readily prepared from the amino-alcohol derivatives 4
and/or 5 by reaction with fluoroaryl 30, as shown in Scheme 8.
Displacement of the fluorine of 31 in the presence of a base such
as sodium hydride with a masked hydroxy, thiol, or amino group
(HZR.sub.70, where R.sub.70 is a suitable protecting group) in a
polar aprotic solvent such as DMF, followed by deprotection gives
32, which can then be aylated with 13 to provide 9. Conversion of
31 to 32 where Z is sulfur, might also be accomplished with
Na.sub.2S, K.sub.2S, NaSH or KS(C.dbd.S)OEt. The fluorine of 31 can
also be displaced in a polar aprotic solvent with the propargylic
derivative 33, where Z is O, S or NH, in the presence of a base
such as sodium hydride, to give 9 directly. As described for
Schemes 6 and 7 the order of synthetic operations may be changed
such that the acetylenic moiety is appended after conversion of
OR.sub.30 into the corresponding thioester, disulfide or protected
thiol. 29
[0087] Compound 9, wherein Z is a methylene group, is available via
34, as shown in Scheme 9. Benzylic bromination of 34 with
N-bromosuccinimide in a chlorinated hydrocarbon solvent provides
bromide 35. This is followed by displacement of the bromide with
the appropriate propynyl cuprate to provide sulfonamide 9. 30
[0088] Some of the methods available for the derivatization of
compounds of structure 9 (for the case wherein R.sub.6 is hydrogen)
are shown in Scheme 10. Metallation of the terminal acetylene 9
followed by addition of an aldehyde or alkl halide, sulfonate or
triflate provides derivatives 36 and 37. Reaction of 9 with
formaldehyde and an amine provides the Mannich addition product 38.
Cyanogen bromide addition to 38 gives the propargylic bromide 39
which may be displaced with a variety of nucleophiles to give, for
example, ethers, thioethers and amines, 40. Palladium catalyzed
coupling, reactions of 9 provide the aryl or heteroaryl acetylenes
41. It is recognized by those skilled in the art of organic
synthesis that the successful use of these methods is dependent
upon the compatibility of substituents on other parts of the
molecule. Protecting, groups and/or changes in the order of steps
described herein may be required. 31
[0089] The following specific examples illustrate the preparation
of representative compounds of this invention. The starting
materials, intermediates, and reagents are either commercially
available or can be readily prepared following standard literature
procedures by one skilled in the art of organic synthesis.
EXAMPLE 1
4-But-2-ynyloxy-benzenesulfonic acid sodium salt
[0090] To a solution of 52.35 g (0.225 mol) of
4-hydroxybenzenesulfonate sodium salt in 1L of isopropanol and 225
mL of a 1.0N solution of sodium hydroxide was added 59.96 g (0.45
mol) of 1-bromo-2-butyne. The resulting mixture was heated to
70.degree. for 15 h and then the isopropanol was removed by
evaporation in vacuo. The resulting white precipitate was collected
by filtration, washed with isopropanol and ether and dried in vacuo
to give 56.0 g (100%) of the butynyl ether as a white solid.
EXAMPLE 2
4-But-2-ynyloxy-benzenesulfonyl chloride
[0091] To a 0.degree. solution of 43.8 mL (0.087 mol) of 2M oxalyl
chloride/dichloro-methane solution in 29 mL of dichloromethane was
dropwise added 6.77 mL (0.087 mol) of DMF followed by 7.24 g (0.029
mol) of the product of Example 1. The reaction mixture was stirred
for 10 minutes at 0.degree. then let warm to room temperature and
stirred for 2 days. The reaction was then poured into ice and
extracted with 150 mL of hexanes. The organics were washed with
water and brine, dried over Na.sub.2SO.sub.4, filtered and
concentrated in vacuo to provide 6.23 g (88%) of the sulfonyl
chloride as a yellow solid; m.p. 63-65.degree. C. EI Mass Spec:
243.9 (M.sup.+).
EXAMPLE 3
But-2-ynyloxy-benzene
[0092] To a solution of 6.14 g (0.023 mol) of triphenylphosphine
dissolved in 100 mL of benzene and 40 mL of THF was added 1.75 mL
(0.023 mol) of 2-butyn-1-ol. After five minutes 2.00 (0.023 mol)
phenol, dissolved in 10 mL of THF, was added to the reaction
followed by 3.69 mL (0.023 mol) of diethyl azodicarboxylate. The
resulting reaction mixture was stirred for 18 h at room temperature
and then concentrated in vacuo. The residue was chromatographed on
silica oel eluting with ethyl acetate/hexanes (1:10) to provide
2.18 g (70%) of the butynyl ether as a clear liquid. EI Mass Spec:
146.0 MH+
EXAMPLE 4
4-But-2-ynyloxy-benzenesulfonyl chloride
[0093] To a solution of 0.146 g (1.0 mmol) of the product of
Example 3 in 0.3 mL of dichloromethane in an acetone/ice bath under
N.sub.2 was dropwise added a solution of 0.073 mL (1.1 mmol) of
chlorosulfonic acid in 0.3 mL of dichloromethane. After the
addition was complete, the ice bath was removed and the reaction
was stirred at room temperature for 2 h. To the reaction was then
dropwise added 0.113 mL (1.3 mmol) of oxalyl chloride, followed by
0.015 mL DMF. The reaction was heated to reflux for 2 h and then
diluted with hexane and poured into ice water. The organic layer
was washed with brine, dried over sodium sulfate, and concentrated
in vacuo to provide 0.130 mg (53%) of the desired product as a
light brown solid.
EXAMPLE 5
4-But-2-ynyloxy-N-(2-hydroxy-1-methyl-ethyl)-benzenesulfonamide
[0094] To a solution of 0.279 g (3.718 mmol) of (R)
-(-)-2-amino-1-propanol in 2.6 mL of THF and 0.9 mL of water was
added 0.62 mL of triethylamine followed by 1.00 g (4.09 mmol) of
4-but-2-ynyloxy-benzenesulfonyl chloride and the resulting mixture
was stirred at room temperature for 15 h. The reaction was then
diluted with ethyl acetate and washed with 5% HCl solution and
water, dried over MgSO.sub.4, filtered and concentrated in vacuo.
The resulting solid was washed with ether and dried in vacuo to
provide 0.873 g (83%) of the sulfonamide as a white solid.
Electrospray Mass Spec: 283.8 (M+H).sup.+
EXAMPLE 6
4-But-2-ynyloxy-N-(2-hydroxy-1-methyl-ethyl)-N-methyl-benzenesulfonamide
[0095] To a solution of 0.400 g (1.413 mmol) of the product of
Example 5 in 3.0 mL of DMF was added 0.585 g (4.240 mmol) of
potassium carbonate followed by 0.132 mL (2.12 mmol) of iodomethane
and the resulting mixture was stirred at room temperature for 12 h.
The reaction was then diluted with ether and washed with water,
dried over MgSO.sub.4, filtered and concentrated in vacuo to
provide 0.354 g (84%) of the N-methyl sulfonamide as a white solid.
Electrospray Mass Spec: 297.9 (M+H).sup.+
EXAMPLE 7
Thioacetic acid
S-{2-[(4-but-2-ynyloxy-benzenesulfonyl)-methyl-amino]-prop-
yl}ester
[0096] To a 0.degree. C. solution of 0.302 g (1.017 mmol) of the
product of Example 6 and 0.293 g (1.118 mmol) of triphenylphosphine
in 4.0 mL of THF was added 0.176 mL (1.118 mmol) of diethyl
azodicarboxylate. The resulting mixture was stirred for 0.5 h at
0.degree. C. and then concentrated in vacuo. The residue was
chromatographed on silica gel eluting with ethyl acetateihexanes to
provide 0.228 g (63%) of the thioacetate as a colorless oil.
Electrospray Mass Spec: 355.9 (M+H).sup.+
EXAMPLE 8
4-But-2-ynyloxy-N-((1R)-2-mercapto-1-methyl-ethyl)-N-methyl-benzenesulfona-
mide
[0097] To a solution of 0.168 g (0.473 mmol) of the product of
Example 7 in 2.1 mL of methanol was added 0.092 g (1.704 mmol) of
sodium methoxide. After stirring at room temperature for 2 h the
reaction was quenched with 5% HCl solution and extracted with
ether. The organics were dried over Na2SO4, filtered and
concentrated in vacuo. The residue was chromatographed on silica
gel eluting with ethyl acetate/hexanes in a gradient from (1:10) to
(1:3) to provide 0.148 g (100%) of the thiol as a colorless oil.
Electrospray Mass Spec: 313.9 (M+H).sup.+
EXAMPLE 9
4-But-2-ynyloxy-N-(2-hydroxy-1-methyl-ethyl)-N-(2-morpholin-4-yl-ethyl)-be-
nzenesulfonamide
[0098] According to the procedure of Example 6, 0.400 g (1.413
mmol) of the product of Example 5 and 0.289 g (1.555 mmol) of
4-(2-chloroethyl)morpholine hydrochloride provided 0.334 g (60%) of
the N-morpholinoethyl sulfonamide as a colorless oil. Electrospray
Mass Spec: 397.0 (M+H).sup.+
Pharmacology
[0099] The ability of the compounds of the invention, or their
pharmaceutically acceptable salts, to inhibit matrix
metalloproteinases or TACE and, consequently, demonstrate their
effectiveness for treating diseases modulated by matrix
metalloproteinases or TACE is shown by the following in vitro
assays.
Test Procedures for Measuring MMP-1, MMP-9, and MMP- 13
Inhibition
[0100] These standard pharmacological test procedures are based on
the cleavage of a thiopeptide substrates such as
Ac-Pro-Leu-Gly(2-mercapto-4-- methyl-pentanoyl)-Leu-Gly-OEt by the
matrix metalloproteinases MMP-1, MMP-13 (collagenases) or MMP-9
(gelatinase), which results in the release of a substrate product
that reacts calorimetrically with DTNB
(5,5'-dithiobis(2-nitro-benzoic acid)). The enzyme activity is
measured by the rate of the color increase. The thiopeptide
substrate is made up fresh as a 20 mM stock in 100% DMSO and the
DTNB is dissolved in 100% DMSO as a 100 mM stock and stored in the
dark at room temperature. Both the substrate and DTNB are diluted
together to 1 mM with substrate buffer (50 mM HEPES pH 7.5, 5 mM
CaCl.sub.2) before use. The stock of enzyme is diluted with buffer
(50 mM HEPES, pH 7.5, 5 mM CaCl.sub.2, 0.02% Brij) to the desired
final concentration. The buffer enzyme, vehicle or inhibitor, and
DTNB/substrate are added in this order to a 96 well plate (total
reaction volume of 200 .mu.l) and the increase in color is
monitored spectrophotometrically for 5 minutes at 405 nm on a plate
reader and the increase in color over time is plotted as a linear
line.
[0101] Alternatively, a fluorescent peptide substrate is used. In
this test procedure, the peptide substrate contains a fluorescent
group and a quenching group. Upon cleavage of the substrate by an
MMP, the fluorescence that is generated is quantitated on the
fluorescence plate reader. The assay is run in HCBC assay buffer
(50 mM HEPES, pH 7.0, 5 mM Ca.sup.+2, 0.02% Brij, 0.5% Cysteine),
with human recombinant MMP-1, MMP-9, or MMP-13. The substrate is
dissolved in methanol and stored frozen in 1 mM aliquots. For the
assay, substrate and enzymes are diluted in HCBC buffer to the
desired concentrations. Compounds are added to the 96 well plate
containing enzyme and the reaction is started by the addition of
substrate. The reaction is read (excitation 340 nm, emission 444
nm) for 10 min. and the increase in fluorescence over time is
plotted as a linear line.
[0102] For either the thiopeptide or fluorescent peptide test
procedures, the slope of the line is calculated and represents the
reaction rate. The linearity of the reaction rate is confirmed
(r.sup.2>0.85). The mean (x.+-.sem) of the control rate is
calculated and compared for statistical significance (p<0.05)
with drug-treated rates using Dunnett's multiple comparison test.
Dose-response relationships can be generated using multiple doses
of drug and IC.sub.50 values with 95% CI are estimated using linear
regression.
Test Procedure for Measuring TACE Inhibition
[0103] Using 96-well black microtiter plates, each well receives a
solution composed of 10 .mu.L TACE (final concentration 1
.mu.g/mL), 70 .mu.L Tris buffer, pH 7.4 containing 10% glycerol
(final concentration 10 mM), and 10 .mu.L of test compound solution
in DMSO (final concentration 1 .mu.M, DMSO concentration <1%)
and incubated for 10 minutes at room temperature. The reaction is
initiated by addition of a fluorescent peptidyl substrate (final
concentration 100 .mu.M) to each well and then shaking on a shaker
for 5 sec.
[0104] The reaction is read (excitation 340 nm, emission 420 nm)
for 10 min. and the increase in fluorescence over time is plotted
as a linear line. The slope of the line is calculated and
represents the reaction rate.
[0105] The linearity of the reaction rate is confirmed
(r.sup.2>0.85). The mean (x.+-.sem) of the control rate is
calculated and compared for statistical significance (p<0.05)
with drug-treated rates using Dunnett's multiple comparison test.
Dose-response relationships can be generate using multiple doses of
drug and IC.sub.50 values with 95% CI are estimated using linear
regression.
Human Monocytic THP-1 Cell Differentiation Assay For Soluble
Proteins (THP-1 Soluble Protein Assay)
[0106] Mitogenic stimulation of THP-1 cells cause differentiation
into macrophage like cells with concomitant secretion of tumor
necrosis factor (TNF-_) and TNF receptor (TNF-R p75/80 and TNF-R
p55/60) and Interleukin-8 (IL-8), among other proteins. In
addition, non-stimulated THP-1 cells shed both the p75/80 and the
p55/60 receptors over time. The release of membrane bound TNF-_ and
possibly TNF-R p75/80 and TNF-R p55/60, but not IL-8, is mediated
by an enzyme called TNF-_ converting enzyme or TACE. This assay can
be used to demonstrate either an inhibitory or a stimulatory
compound effect on this TACE enzyme and any cytotoxic consequence
of such a compound.
[0107] THP-1 cells (from ATCC) are a human monocytic cell line
which were obtained from the peripheral blood of a one year old
male with acute monocytic leukemia. They can be grown in culture
and differentiated into macrophage like cells by stimulation with
mitogens.
[0108] For the assay, THP-1 cells are seeded from an ATCC stock
which was previously grown and frozen back at 5.times.106/ml/vial.
One vial is seeded into a T25-flask with 16 mls of RPMI-1640 with
glutamax (Gibco) media containing 10% fetal bovine serum, 100
units/ml penicillin, 100 .mu.g/ml streptomycin, and
5.times.10.sup.-5 M 2-mercapto-ethanol (THP-1 media). Each vial of
cells are cultured for about two weeks prior to being used for an
assay and then are used for only 4 to 6 weeks to screen compounds.
Cells are subcultured on Mondays and Thursdays to a concentration
of 1.times.105/ml.
[0109] To perform an assay, the THP-1 cells are co-incubated in a
24 well plate with 50 ml/well of a 24 mg/ml stock of
Lipopolysacharide (LPS) (Calbiochem Lot#B13189) at 37iC in 5%
CO.sub.2 at a concentration of 1.091.times.10.sup.6 cells/ml (1.1
ml/well) for a total of 24 hours. At the same time, 50 ml/well of
drug, vehicle or THP-1 media is plated in appropriate wells to give
a final volume of 1.2 ml/well. Standard and test compounds are
dissolved in DMSO at a concentration of 36 mM and diluted from here
to the appropriate concentrations in THP-1 media and added to the
wells at the beginning of the incubation period to give final
concentrations of 100 mM, 30 mM, 10 mM, 3 mM, 1 mM, 300 nM, and 100
nM. Cell exposure to DMSO was limited to 0.1% final concentration.
Positive control wells were included in the experiment which had
mitogen added but no drug. Vehicle control wells were included as
well, which were identical to the positive control wells, except
that DMSO was added to give a final concentration of 0.083%.
Negative control wells were included in the experiment which had
vehicle but no mitogen or drug added to the cells. Compounds can be
evaluated for their effect on basal (non-stimulated) shedding of
the receptors by replacing the LPS with 50 ml/well of THP-1 media.
Plates are placed into an incubator set at 5% CO2 and at 37o C.
After 4 hours of incubation, 300 ml/well of tissue culture
supernatant (TCS) is removed for use in an TNF-_ ELISA. Following
24 hours of incubation, 700 ml/well of TCS is removed and used for
analysis in TNF-R p75/80, TNF-R p55/60 and IL-8 ELISAs.
[0110] In addition, at the 24 hours timepoint, and the cells for
each treatment group are collected by resuspension in 500
.mu.l/well of THP-1 media and transferred into a FACS tube. Two
ml/tube of a 0.5 mg/ml stock of propidium iodide (PI) (Boerhinger
Mannheim cat. # 1348639) is added. The samples are run on a Becton
Dickinson FaxCaliber FLOW cytometry machine and the amount of dye
taken up by each cell is measured in the high red wavelength (FL3).
Only cells with compromised membranes (dead or dying) can take up
PI. The percent of live cells is calculated by the number of cells
not stained with PI, divided by the total number of cells in the
sample. The viability values calculated for the drug treated groups
were compared to the viability value calculated for the vehicle
treated mitogen stimulated group ("vehicle positive control") to
determine the "percent change from control". This "percent change
from control" value is an indicator of drug toxicity.
[0111] The quantity of soluble TNF-_, TNF-R p75/80 and TNF-R p55/60
and L-8 in the TCS of the THP-1 cell cultures are obtained with
commercially available ELISAs from R&D Systems, by
extrapolation from a standard curve generated with kit standards.
The number of cells that either take up or exclude PI are measured
by the FLOW cytometry machine and visualized by histograms using
commercially available Cytologic software for each treatment group
including all controls.
[0112] Biological variability in the magnitude of the response of
THP-1 cell cultures requires that experiments be compared on the
basis of percent change from "vehicle positive control" for each
drug concentration. Percent change in each soluble protein
evaluated from the "vehicle positive control" was calculated for
each compound concentration with the following formula: 1 % Change
= pg / ml ( compound ) - pg / ml ( veh pos control ) pg / ml ( veh
pos control ) - pg / ml ( veh neg control ) .times. 100
[0113] For the soluble protein (TNF-_, p75/80, p55/60, IL-8)
studies under stimulated conditions, the mean pg/ml of duplicate
wells were determined and the results expressed as percent change
from "vehicle positive control". For the soluble protein (p75/80
and p55/60 receptors) studies under non-stimulated conditions, the
mean pg/ml of duplicate wells were determined and the results
expressed as percent change from "vehicle positive control"
utilizing the following formula: 2 % Change = pg / ml ( compound
neg control ) - pg / ml ( veh neg control pg / ml ( veh neg control
) .times. 100
[0114] IC.sub.50 values for each compound are calculated by
non-linear regression analysis using customized software utilizing
the JUMP statistical package.
[0115] For the cell viability studies, the viabilities (PI
exclusion) of pooled duplicate wells were determined and the
results expressed as % change from "vehicle positive control". The
viability values calculated for the compound treated groups were
compared to the viability value calculated for the "vehicle
positive control" to determine "percent change from control" as
below. This value "percent change from control" is an indicator of
drug toxicity. 3 % Change = % live cells ( compound ) % live cells
( veh pos control ) - 1 .times. 100
[0116] References:
[0117] Bjornberg, F., Lantz, M., Olsson, I., and Gullberg, U.
Mechanisms involved in the processing of the p55 and the p75 tumor
necrosis factor (TNF) receptors to soluble receptor forms.
Lymphokine Cytokine Res. 13:203-211, 1994.
[0118] Gatanaga, T., Hwang, C., Gatanaga, M., Cappuccini, F.,
Yamamoto, R., and Granger, G. The regulation of TNF mRNA synthesis,
membrane expression, and release by PMA- and LPS-stimulated human
monocytic THP-1 cells in vitro. Cellular Immun. 138:1-10, 1991.
[0119] Tsuchiya, S., Yamabe, M., Yamagughi, Y., Kobayashi, Y.,
Konno, T., and Tada, K. Establishment and characterization of a
human acute monocytic leukemia cell line (THP-1). Int. J. Cancer.
26:1711-176, 1980.
[0120] Results of the above in vitro and in vivo matrix
metalloproteinase inhibition, TACE inhibition and THP standard
pharmacological test procedures are given in Table 1 below.
1TABLE 1 32 Example # MMP-1.sup.a MMP-9.sup.a MMP-13.sup.a
TACE.sup.a THP 8 6,300 2,700 679 273 3 .sup.aIC.sub.50 (nM) .sup.b%
Inhibition @ 3 .mu.M
[0121] Based on the standard pharmacological test procedures
described above, the compounds of this invention are useful in the
treatment of disorders such as arthritis, tumor metastasis, tissue
ulceration, abnormal wound healing, periodontal disease, graft
rejection, insulin resistance, bone disease and HIV infection.
[0122] The compounds of this invention are also useful in treating
or inhibiting pathological changes mediated by matrix
metalloproteinases such as atherosclerosis, atherosclerotic plaque
formation, reduction of coronary thrombosis from atherosclerotic
plaque rupture, restenosis, MMP-mediated osteopenias, inflammatory
diseases of the central nervous system, skin aging, angiogenesis,
tumor metastasis, tumor growth, osteoarthritis, rheumatoid
arthritis, septic arthritis, corneal ulceration, proteinuria,
aneurysmal aortic disease, degenerative cartilage loss following
traumatic joint injury, demyelinating diseases of the nervous
system, cirrhosis of the liver, glomerular disease of the kidney,
premature rupture of fetal membranes, inflammatory bowel disease,
age related macular degeneration, diabetic retinopathy,
proliferative vitreoretinopathy, retinopathy of prematurity, ocular
inflammation, keratoconus, Sjogren's syndrome, myopia, ocular
tumors, ocular angiogenesis/neovascularization and corneal graft
rejection.
[0123] Compounds of this invention may be administered neat or with
a pharmaceutical carrier to a patient in need thereof. The
pharmaceutical carrier may be solid or liquid.
[0124] Applicable solid carriers can include one or more substances
which may also act as flavoring agents, lubricants, solubilizers,
suspending agents, fillers, glidants, compression aids, binders or
tablet-disintegrating agents or an encapsulating material. In
powders, the carrier is a finely divided solid which is in
admixture with the finely divided active ingredient. In tablets,
the active ingredient is mixed with a carrier having the necessary
compression properties in suitable proportions and compacted in the
shape and size desired. The powders and tablets preferably contain
up to 99% of the active ingredient. Suitable solid carriers
include, for example, calcium phosphate, magnesium stearate, talc,
sugars, lactose, dextrin, starch, gelatin, cellulose, methyl
cellulose, sodium carboxymethyl cellulose, polyvinylpyrrolidine,
low melting waxes and ion exchange resins.
[0125] Liquid carriers may be used in preparing solutions,
suspensions, emulsions, syrups and elixirs. The active ingredient
of this invention can be dissolved or suspended in a
pharmaceutically acceptable liquid carrier such as water, an
organic solvent, a mixture of both or pharmaceutically acceptable
oils or fat. The liquid carrier can contain other suitable
pharmaceutical additives such a solubilizers, emulsifiers, buffers,
preservatives, sweeteners, flavoring agents, suspending agents,
thickening agents, colors, viscosity regulators, stabilizers or
osmo-regulators. Suitable examples of liquid carriers for oral and
parenteral administration include water (particularly containing
additives as above, e.g., cellulose derivatives, preferable sodium
carboxymethyl cellulose solution), alcohols (including monohydric
alcohols and polyhydric alcohols, e.g., glycols) and their
derivatives, and oils (e.g., fractionated coconut oil and arachis
oil). For parenteral administration the carrier can also be an oily
ester such as ethyl oleate and isopropyl myristate. Sterile liquid
carriers are used in sterile liquid form compositions for
parenteral administration.
[0126] Liquid pharmaceutical compositions which are sterile
solutions or suspensions can be utilized by, for example,
intramuscular, intraperitoneal or subcutaneous injection. Sterile
solutions can also be administered intravenously. Oral
administration may be either liquid or solid composition form.
[0127] The compounds of this invention may be administered rectally
in the form of a conventional suppository. For administration by
intranasal or intrabronchial inhalation or insufflation, the
compounds of this invention may be formulated into an aqueous or
partially aqueous solution, which can then be utilized in the form
of an aerosol. The compounds of this invention may also be
administered transdermally through the use of a transdernal patch
containing the active compound and a carrier that is inert to the
active compound, is non-toxic to the skin, and allows delivery of
the agent for systemic absorption into the blood stream via the
skin. The carrier may take any number of forms such as creams and
ointments, pastes, gels, and occlusive devices. The creams and
ointments may be viscous liquid or semi-solid emulsions of either
the oil in water or water in oil type. Pastes comprised of
absorptive powders dispersed in petroleum or hydrophilic petroleum
containing the active ingredient may also be suitable. A variety of
occlusive devices may be used to release the active ingredient into
the blood stream such as a semipermeable membrane covering a
reservoir containing the active ingredient with or without a
carrier, or a matrix containing the active ingredient. Other
occlusive devices are known in the literature.
[0128] The dosage to be used in the treatment of a specific patient
suffering a MMP or TACE dependent condition must be subjectively
determined by the attending physician. The variables involved
include the severity of the dysfunction, and the size, age, and
response pattern of the patient. Treatment will generally be
initiated with small dosages less than the optimum dose of the
compound. Thereafter the dosage is increased until the optimum
effect under the circumstances is reached. Precise dosages for
oral, parenteral, nasal, or intrabronchial administration will be
determined by the administering physician based on experience with
the individual subject treated and standard medical principles.
[0129] Preferably the pharmaceutical composition is in unit dosage
form, e.g., as tablets or capsules. In such form, the composition
is sub-divided in unit dose containing appropriate quantities of
the active ingredient; the unit dosage form can be packaged
compositions, for example packed powders, vials, ampoules,
prefilled syringes or sachets containing liquids. The unit dosage
form can be, for example, a capsule or tablet itself, or it can be
the appropriate number of any such compositions in package
form.
* * * * *