CA2358955A1 - N-carboxymethyl substituted benzolactams as inhibitors of matrix metalloproteinase - Google Patents

Abstract

The present invention provides a method of inhibiting matrix metallo-proteinases (MMPs) in a patient in need thereof comprising administering to the patient an effective matrix metalloproteinase inhibiting amount of the N-carboxymethyl substituted benzolactams of formula (1) wherein A is -OH or -NRR'. Such inhibitors are useful in treating neoplasms, atherosclorosis, and chronic inflammatory diseases. The present invention also provides novel N-carboxymethyl substituted benzolactams of formula (1a) wherein A is -NRR'.

Description

t11V1tC LVLy-t1 CA 02358955 2001-06-28 N-CARBOXYMETHYL SUBSTITUTED BENZOLACTAMS AS
INHIBITORS OF MATRIX METALLOPROTEINASE
i o BACKGROUND OF THE INVENTION
The matrix metalloproteinases (MMPs) are a family of zinc containing endopeptidases which are capable of cleaving large biomolecules such as the collagens, proteoglycans and gelatins. Expression is upregulated by pro-inflammatory cytokines and/or growth factors. The MMP's are secreted as inactive zymogens which, upon activation, are subject to control by endogenous inhibitors, for example, tissue inhibitor of metalloproteinases (TINIP) and a2-macroglobulin. Chapman, K.T. et al., J. Med.
Chem.
36, 4293-4301 (1993); Beckett, R.P. et al., DDT 1, 16-26 (1996). The characterizing feature of diseases involving the enzymes appears to be a stoichiometric imbalance between active enzymes and endogenous inhibitors, leading to excessive tissue disruption, 2o and often degradation. McCachren, S.S., Arthritis Rheum. 34, 1085-1093 (1991).
The discovery of different families of matrix metalloproteinase, their relationships, and their individual characteristics have been categorized in several reports.
Emonard, H.
et aL, Cell Molec. Biol. 36, 131-153 (1990); Birkedal-Hansen, H., J. Oral Pathol. 17, 445-451 (1988); Matrisian, L.M., Trends Genet. 6, 121-125 (1990); Murphy, G.J.P.
et al., FEBS Lett. 289, 4-7 (1991); Matrisian, L.M., Bioessavs 14, 455-463 (1992);
Flynn et al., WO-A 95/21854, WO-A 95/21839, US Patent 5,424,425, and US Patent 5,491,143; De et a.l., WO-A 96/29313; Karanewsky et al., EP-A 0599444; and Tourw6 et al., Bioorg. &
Med. Chem. Lett. 2, No. 10, 1305-1308 (1992). Three groups of MMPs have been 3o delineated: the collagenases which have triple helical interstitial collagen as a substrate, the gelatinases which are proteinases of denatured collagen and Type IV
collagen, and the stromelysins which were originally AMENDED SHE~T

characterized as proteoglycanases but have now been identified to have a broader proteolytic spectrum. Examples of specific collagenases include fibroblast collagenase (MMP-1), neutrophil collagenase (MMP-8), and collagenase 3 (MMP-13). Examples of gelatinises include 72 kDa gelatinise (gelatinise A; MMP-2) and 92 kDa gelatinise (gelatinise B; MMP-9). Examples of stromelysins include stromelysin 1 (MMP-3), stromelysin 2 (MMP-10) and matrilysin (MMP-7). Other MMPs which do not fit neatly into the above groups include metalloelastase (MMP-12), membrane-type MMP (MT-MMP or MMP-14) and stromelysin 3 (MMP-11). Beckett, R.P. et al., supra.
Over-expression and activation of MMPs have been linked with a wide range of diseases such as cancer; rheumatoid arthritis; osteoarthritis; chronic inflammatory disorders, such as emphysema and smoking-induced emphysema; cardiovascular disorders, such as atherosclerosis; corneal ulceration; dental diseases such as gingivitis and periodontal disease; and neurological disorders, such as multiple sclerosis. For example, in adenocarcinoma, invasive proximal gastric cells express the 72 kDa form of collagenase Type IV, whereas the noninvasive cells do not. Schwartz, G.K. et al., Cancer 73, 22-27 (1994). Rat embryo cells transformed by the Ha-ras and v-myc oncogenes or by Ha-ras alone are metastatic in nude mice and release the 92 kDa gelatinase/collagenase (MMP-9). Bernhard, E.J. et al., Proc. Natl. Acid. Sci.
91, 4293-4597 (1994). The plasma concentration of MMP-9 was significantly increased (P
<
0.01) in 122 patients with gastrointestinal tract cancer and breast cancer.
Zucker, S. et al., Cancer Res. 53, 140-146 (1993). Moreover, intraperitoneal administration of batimastat, a synthetic MMP inhibitor, gave significant inhibition in the growth and metastatic spread and number of lung colonies which were produced by intravenous injection of the B16-BL6 murine melanoma in C57BL/6N mice. Chirivi, R.G.S. et al., Int. J. Cancer 58, 460-464 (1994). Over-expression of TIMP-2, the endogenous tissue inhibitor of MMP-2, markedly reduced melanoma growth in the skin of immunodeficient mice. Montgomery, A.M.P. et al., Cancer Res. 54, 5467-5473 (1994).
Accelerated breakdown of the extracellular matrix of articular cartilage is a key feature in the pathology of both rheumatoid arthritis and osteoarthritis.
Current evidence suggests that the inappropriate synthesis of MMPs is the key event.
Beeley, N.R.A. et al., Curr. Opin. Ther. Patents, 4(1), 7-16 (1994). The advent of reliable diagnostic tools have allowed a number of research groups to recognize that stromelysin SUBSTITUTE SHEET (RULE 26) is a key enzyme in both arthritis and joint trauma. Beeley, N.R.A. et al., Id.; Hasty, K.A. et al., Arthr. Rheum. 33, 388-397 (1990). It has also been shown that stromelysin is important for the conversion of procollagenase to active collagenase.
Murphy, G. et al., Biochem. J. 248, 265-268 (1987).
Furthermore, a range of MMPs can hydrolyse the membrane-bound precursor of the pro-inflammatory cytokine tumor necrosis factor a (TNF-a). Gearing, A.J.H.
et al., Nature 370, 555-557 (1994). This cleavage yields mature soluble TNF-a and the inhibitors of MMPs can block production of TNF-a both in vitro and in vivo.
Gearing, A.J.H. et al., Id.; Mohler, K.M. et al., Nature 370, 218-220 (1994); McGeehan, G.M. et al., Nature 370, 558-561 (1994). This pharmacological action is a probable contributor to the antiarthritic action of this class of compounds seen in animal models.
Beckett, R.P. et al., supra.
Stromelysin has been observed to degrade the a~-proteinase inhibitor which regulates the activity of enzymes such as elastase, excesses of which have been linked to chronic inflammatory disorders such as emphysema and chronic bronchitis.
Beeley, N.R.A. et al., supra.; Wahl, R.C. et al., Annual Reports in Medicinal Chemistry 25, 177-184 (1990). In addition, a recent study indicates that MMP-12 is required for the development of smoking-induced emphysema in mice. Science, 277, 2002 (1997).
Inhibition of the appropriate MMP may thus potentiate the inhibitory activity of endogenous inhibitors of this type.
High levels of mRNA corresponding to stromelysin have been observed in atherosclerotic plaques removed from heart transplant patients. Henney, A.M., et al., Proc. Natl. Acad. Sci. 88, 8154-8158 (1991). It is submitted that the role of stromelysin in such plaques is to encourage rupture of the connective tissue matrix which encloses the plaque. This rupture is in turn thought to be a key event in the cascade which leads to clot formation of the type seen in coronary thrombosis. MMP inhibition is thus a preventive measure for such thromboses.
Collagenase, stromelysin and gelatinase have been implicated in the destruction of the extracellular matrix of the cornea. This is thought to be an important mechanism SUBSTTTUTE SHEET (RULE 26) of morbidity and visual loss in a number of ulcerative ocular diseases, particularly those following infection or chemical damage. Burns, F.R. et al., Invest. O~thalmol.
and Visual Sci. 32, 1569-1575 (1989). The MMPs present in the eye during ulceration are derived either endogenously from infiltrating leucocytes or fibroblasts, or exogenously S from microbes.
Collagenase and stromelysin activities have been identified in fibroblasts isolated from inflamed gingiva and the levels of enzyme have been correlated with the severity of the gingivitis observed. Beeley, N.R.A. et al., supra.; Overall, C.M. et al., J.
Periodontal Res. 22, 81-88 (1987).
Excessive levels of gelatinise-B in cerebrospinal fluid has been linked with incidence of multiple sclerosis and other neurological disorders. Beeley, N.R.A. et al., supra.; Miyazaki, K. et al., Nature 362, 839-841 (1993). The enzyme may play a key role in the demyelination of neurones and the breakdown of the blood brain barrier which occurs in such disorders.
SUMMARY OF THE INVENTION
The present invention provides a method of inhibiting matrix metallo-proteinases (MMPs) in a patient in need thereof comprising administering to the patient an effective matrix metalloproteinase inhibiting amount of the N-carboxymethyl substituted benzolactams of formula (1):
Rz O ~ O
N

formula ( 1 ) wherein A is selected from the group consisting of -OH and -NRR';
wherein R and R' are independently selected from the group consisting of hydrogen and C~-C~ alkyl or R and R' taken together with the nitrogen atom to which they are attached form a N-morpholino, N-piperidino, N-pyrrolidino, or N-isoindolyl;
SUBSTTTUTE SHEET (RULE 26) R~ is selected from the group consisting of hydrogen, C,-C6 alkyl, -(CHz)a-COZRS, -(CHZ)a-C(O)NHz, -(CHZ)4NHz, -(CHz)3-NH-C(NH)NH2, -(CHz)2-S(O)b-CH3, -CHZ-OH, -CH(OH)CH3, -CHI-SH, -(CHZ)d-Are, and -CH?-Arz;
wherein a is 1 or 2;
b is 0, 1, or 2;
d is an integer from 0 to 4;
R; is selected from the group consisting of hydrogen, C1_Ca alkyl, and benzyl;
Are is a radical selected from the group consisting of and i i R6 R~
wherein R~ is from 1 to 2 substituents independently selected from the group consisting of hydrogen, halogen, C~_C4 alkyl, hydroxy, and Cl_Ca alkoxy;
R~ is selected from the group consisting of hydrogen, halogen, C~_C4 alkyl, and C ~ _C4 alkoxy;
Ar2 is a radical selected from the group consisting of / N
~~ and I(NH
NH
RZ is from 1 to 2 substituents independently selected from the group consisting of hydrogen, halogen, hydroxy C~_C4 alkyl, and C~_C4 alkoxy;
R3 is selected from the group consisting of C~-C~ alkyl, -(CH?)",-W, -(CHz)p-Ar3, -(CHZ)k-COZR9, -(CHZ)n,-NR8~S0?-Y~, and -(CHZ)n,-Z-Q
wherein m is an integer from 2 to 8;
p is an integer from 0-10;
k is an integer from 1 to 9;
W is phthalimido;
SUBSTTTUTE SHEET (RULE 26) Ar3 is selected from the group consisting of N
' R
z3 ~ J ~NH
N
and ~
i S O N
wherein R23 is from 1 to 2 substituents independently selected from the group consisting of hydrogen, halogen, C~-C4 alkyl, and C,-Ca alkoxy;
R8' is hydrogen or C~-C~ alkyl;
R9 is hydrogen or Cl-C~ alkyl;
YI is selected from the group consisting of hydrogen, -(CHZ)~-Ar4, and -N(RZa)z wherein jis0orl;
R24 each time selected is independently hydrogen or Cl-C~ alkyl or are taken together with the nitrogen to which they are attached to form N-morpholino, N-piperidino, N-pyrrolidino, or N-isoindolyl;
Ar4 is Rzs wherein RZ; is from 1 to 3 substituents independently selected from the group consisting of hydrogen, halogen, C~-C4 alkyl, and C~-C4 alkoxy;
Z is selected from the group consisting of -O-, -NR$-, -C(O)NRg-, -NRBC(O)-, -NRgC(O)NH-, -NRBC(O)O -, and -OC(O)NH-;
wherein Rg is hydrogen or C~-C~ alkyl;
Q is selected from the group consisting of hydrogen, -(CHZ)"-Y2, and -(CHZ)XY3;
wherein n is an integer from 0 to 4;
SUBSTTTUTE SHEET (RULE 26) YZ is selected from the group consisting of hydrogen, -(CH~)n-Ar; and -(CHZ),-C(O)OR2~
wherein Ar5 is selected from the group consisting of ( Rz6 and i w N
wherein R2~ is from 1 to 3 substituents independently selected from the group consisting of hydrogen, halogen, C,-C4 alkyl, and C ~ -Ca alkoxy;
h is an integer from 0 to 6;
t is an integer from 1 to 6;
Rz~ is hydrogen or C~-C~ alkyl;
x is an integer from 2 to 4;
Y3 is selected from the group consisting of -N(R28)2, N-morpholino, N-piperidino, N-pyrrolidino, and N-isoindolyl;
wherein RZg each time taken is independently selected from the group consisting of hydrogen and C,-C~ alkyl;
R4 is selected from the group consisting of hydrogen, -C(O)Rlo, -C(O)-(CHz)q-K
and -S-G
wherein Rio is selected from the group consisting of hydrogen, C~_C4 alkyl, phenyl, and benzyl;
q is 0, l, or 2;
K is selected from the group consisting of SUBSTITUTE SHEET (RULE 26) _g_ / ~N
-N V I -N
N
/ Ril '~ and -N

N-Rii Rii wherein V is selected from the group consisting of a bond, -CHZ-, -O-, -S(O),--, -NRZ~-, and -NC(O)RZ2;
wherein r is 0, 1, or 2;
R2~ is selected from the group consisting of hydrogen, Cl_C4 alkyl, and benzyl;
R22 is selected from the group consisting of hydrogen, -CF3, C,-C~o alkyl, phenyl, and benzyl;
Rl ~ each time taken is independently selected from the group consisting of hydrogen, C~_Ca alkyl, and benzyl;
G is selected from the group consisting of / (CHz) W _"irRlz V, (CHZ)~
y Rl3 Vz (CHz) w \1 / (CHz)' ( CHz ) w \
Ri4 / ( CHz )~NHRls COzRl6 and N /
wherein SUBSTITUTE SHEET (RULE 26) _g_ w is an integer from 1 to 3;

R~Z is selected from the group consisting of hydrogen, C~-C~ alkyl, -CHZCHzS(O)uCH3, and benzyl;
wherein a is 0,1, or 2;
R~3 is selected from the group consisting of hydrogen, hydroxy, amino, Cl-C6 alkyl, N-methylamino, N,N-dimethylamino, -CO~R», and -OC(O)R, ~;
wherein R» is hydrogen, -CH20-C(O)C(CH3)3, C~_Ca alkyl, benzyl, or diphenylmethyl;
R~8 is hydrogen, C~-C~ alkyl or phenyl;
R~4 is 1 or 2 substituents independently selected from the group consisting of hydrogen, C~_C4 alkyl, CI_C4 alkoxy, or halogen;
V ~ is selected from the group consisting of -O-, -S-, and -NH-;
VZ is selected from the group consisting of -N- and -CH-;
V3 is selected from the group consisting of a bond and -C(O)-;
V4 is selected from the group consisting of -O-, -S-, -NR~ ~-, and -NC(O)RZO-;
wherein R» is hydrogen, CI_C.~ alkyl, or benzyl;
RZO is hydrogen, -CF3, C~-Coo alkyl, or benzyl;
R,; is selected from the group consisting of hydrogen, C,-C~, alkyl and benzyl;
R» is selected from the group consisting of hydrogen and C,_Ca alkyl;
and stereoisomers, pharmaceutically acceptable salt, and hydrate thereof.
Novel N-carboxymethyl substituted benzolactams are encompassed by formula ( 1 ). Some of these novel compounds are described by of formula ( 1 a), below, which is encompassed by formula ( 1 ). The present invention provides novel N-carboxymethyl substituted benzolactams of formula (1a):
SUBSTITUTE SHEET (RULE 26) Rz p ~ O
S N
R4 NH Aa formula ( 1 a) wherein Aa is -NRR';
wherein R and R' are independently selected from the group consisting of hydrogen and Cl-C6 alkyl or R and R' taken together with the nitrogen atom to which they are attached form a N-morpholino, N-piperidino, N-pyrrolidino, or N-isoindolyl;
R~ is selected from the group consisting of hydrogen, C~-C~ alkyl, -(CHz)a-COzR;, -(CHz)a-C(O)NHz, -(CHz)aNHz, -(CHz)3-NH-C(NH)NHz, -(CHz)z-S(O)n-CH3, -CHz_ OH, -CH(OH)CH3, -CHz-SH, -(CHz)d-Arl, and -CHz-Arz;
wherein a is 1 or 2;
bis0,l,or2;
d is an integer from 0 to 4;
R; is selected from the group consisting of hydrogen, C,_C4 alkyl, and benzyl;
Ar, is a radical selected from the group consisting of and ~
i R6 R~
wherein R~ is from 1 to 2 substituents independently selected from the group consisting of hydrogen, halogen, C ~ _C4 alkyl, hydroxy, and C, _C4 alkoxy;
R~ is selected from the group consisting of hydrogen, halogen, C~_Ca alkyl, and C,_Ca alkoxy;
Arz is a radical selected from the group consisting of SUBSTITUTE SHEET (RULE 26) / N
Ifi and NH ~
NH
wherein m is an integer from 2 to 8;
p is an integer from 0-10;
k is an integer from 1 to 9;
W is phthalimido;
Are is selected from the group consisting of N
NH
N
x and i S O N
wherein R23 is from 1 to 2 substituents independently selected from the group consisting of hydrogen, halogen, C~-Ca alkyl, and C,-Ca alkoxy;
Rg~ is hydrogen or C~-C~ alkyl;
R~ is hydrogen or C,-C6 alkyl;
Y~ is selected from the group consisting of hydrogen, -(CHZ)~-Ar4, and -N(Rz.~) wherein jis0orl;
Rz4 each time selected is independently hydrogen or C~-C~ alkyl or are taken together with the nitrogen to which they are attached to form N-morpholino, N-piperidino, N-pyrrolidino, or N-isoindolyl;
Ara is RZ is from 1 to 2 substituents independently selected from the group consisting of hydrogen, halogen, hydroxy C~_C4 alkyl, and C,_C4 alkoxy;
R3 is selected from the group consisting of C~-C~ alkyl, -(CHZ)",-W, -(CHZ)p-Are, -(CHZ)k-COzR~, -(CH?)m-NRg~SO2-1'~, and -(CH~)~,-z-Q
SUBSTITUTE SHEET (RULE 26) ~ R25 wherein Rzs is from 1 to 3 substituents independently selected from the group consisting of hydrogen, halogen, C,-C4 alkyl, and C~-C4 alkoxy;
Z is selected from the group consisting of -O-, -NR8-, -C(O)NR8-, -NR~C(O)-, -NR~C(O)NH-, -NRgC(O)O -, and -OC(O)NH-;
wherein R$ is hydrogen or Cl-C6 alkyl;
Q is selected from the group consisting of hydrogen, -(CHZ)n-Y2, and -(CHz),~Y~;
wherein n is an integer from 0 to 4;
Y~ is selected from the group consisting of hydrogen, -(CHZ),,-Ar> and -(CHz)t-C(O)ORz~
wherein Ar; is selected from the group consisting of i R26 and ~
N
wherein Rz~ is from 1 to 3 substituents independently selected from the group consisting of hydrogen, halogen, C,-C4 alkyl, and C~-C4 alkoxy;
h is an integer from 0 to 6;
t is an integer from 1 to 6;
Rz~ is hydrogen or C,-C~, alkyl;
x is an integer from 2 to 4;
Y3 is selected from the group consisting of -N(R2~)2, N-morpholino, N-piperidino, N-pyrrolidino, and N-isoindolyl;
wherein SUBSTITUTE SHEET (RULE 26) R2~ each time taken is independently selected from the group consisting of hydrogen and C~-C~ alkyl;
R4 is selected from the group consisting of hydrogen, -C(O)R,o, -C(O)-(CHZ)q-K
and -S-G
wherein Rlo is selected from the group consisting of hydrogen, C,_C4 alkyl, phenyl, and benzyl;
q is 0, 1, or 2;
K is selected from the group consisting of ~N
N V I -N
~--J ~N
Rli and -N
Rll N-Rli Rii wherein V is selected from the group consisting of a bond, -CHZ-, -O-, -S(O)~-, -NRZ~-, and -NC(O)RZZ;
wherein ris0, l,or2;
R2~ is selected from the group consisting of hydrogen, C,_Ca alkyl, and benzyl;
R2z is selected from the group consisting of hydrogen, -CF3, C~-Coo alkyl, phenyl, and benzyl;
R> > each time taken is independently selected from the group consisting of hydrogen, C~_C4 alkyl, and benzyl;
G is selected from the group consisting of SUBSTTTUTE SHEET (RULE 26) (CH )~R
z ~~(~''' iz / (CHz)~V~
R13 Vz (CHz)w ~~ / (CHz)\
N v3~N'~
( CHz ) w / Ri4 / (CHz) W ",,rNHRls ~~(C''~OZR16 and N /
wherein w is an integer from 1 to 3;
R~2 is selected from the group consisting of hydrogen, C~-C~, alkyl, -CHzCH?S(O)~,CH3, and benzyl;
wherein a is 0,1, or 2;
R13 is selected from the group consisting of hydrogen, hydroxy, amino, C~-C~ alkyl, N-methylamino, N,N-dimethylamino, -COZRI~, and -OC(O)R~ ~;
wherein R,~ is hydrogen, -CHzO-C(O)C(CH3)3, C~_C~ alkyl, benzyl, or diphenylmethyl;
R» is hydrogen, C~-C~ alkyl or phenyl;
R~4 is 1 or 2 substituents independently selected from the group consisting of hydrogen, C~_C4 alkyl, C~_Ca alkoxy, or halogen;
V~ is selected from the group consisting of -O-, -S-, and -NH-;
V~ is selected from the group consisting of -N- and -CH-;
V3 is selected from the group consisting of a bond and -C(O)-;
V4 is selected from the group consisting of -O-, -S-, -NR,~-, and -NC(O)Rzo-;
wherein R» is hydrogen, C,_C4 alkyl, or benzyl;
SUBSTITUTE SHEET (RULE 26) R~~ is hydrogen, -CFA, C,-C» alkyl, or benzyl;
R,; is selected from the group consisting of hydrogen, C~-C~, alkyl and benzyl;
R,~ is selected from the group consisting of hydrogen and C~_C4 alkyl;
and stereoisomers, pharmaceutically acceptable salt, and hydrate thereof.
In addition, the present invention provides a composition comprising an assayable amount of a compound of formula ( 1 a) in admixture or otherwise in association with an inert carrier. The present invention also provides a pharmaceutical composition comprising an effective matrix metallo-proteinases inhibitory amount of a compound of formula (la) in admixture or otherwise in association with one or more pharmaceutically acceptable carriers or excipients.
As is appreciated by one of ordinary skill in the art the compounds of formula (1) exist as stereoisomers. Specifically, it is recognized that they exist as stereoisomers at the point of attachment of the substituents R,, R~, and -SR4, R,2, and -NHRI; and at the point of attachment of the group -NHC(O)CH(R3)(SR4) to the benzolactam.
Where indicated the compounds, whether of formula (1), starting materials, or intermediates, follow either the (+)- and (-)- designation for optical rotation, the (D)- and (L)-designation of relative stereochemistry, or the Cahn-Ingold-Prelog designation of (R)-and (S)- for the stereochemistry of at specific positions in the compounds represented by formula (1) and intermediates thereof. Any reference in this application to one of the compounds of the formula ( 1 ) is meant to encompass either specific stereoisomers or a mixture of stereoisomers.
The specific stereoisomers can be prepared by stereospecific synthesis using enantiomerically pure or enantiomerically enriched starting materials which are well known in the art. The specific stereoisomers of amino acid starting materials are commercially available or can be prepared by stereospecific synthesis as is well known in the art or analogously known in the art, such as D. A. Evans, et al. J. Am.
Chem.
Soc., 112, 4011-4030 (1990); S. Ikegami et al. Tetrahedron, 44, 5333-5342 (1988);
W.Oppolzer et al. Tet. Lets. 30, 6009-6010 (1989); Synthesis of Optically Active a-Amino-Acids, R. M. Williams (Pergamon Press, Oxford 1989); M. J. O'Donnell ed.: a-SUBSTITUTE SHEET (RULE 26) Amino-Acid Synthesis, Tetrahedron Symposia in print, No. 33, Tetrahedron 44, No. 17 (1988); U. Schollkopf, PureAppl. Chem. 55, 1799 (1983); U. Hengartner et al.
J. Orb.
Chem., 44, 3748-3752 (1979); M. J. O'Donnell et al. Tet. Lets., 2641-2644 (1978); M.
J. O'Donnell et al. Tet. Lets. 23, 4255-4258 (1982); M. J. O'Donnell et al. J.
Am. Chem.
Soc., 110, 8520-8525 (1988).
The specific stereoisomers of either starting materials or products can be resolved and recovered by techniques known in the art, such as chromatography on chiral stationary phases, enzymatic resolution, or fractional recrystallization of addition salts formed by reagents used for that purpose. Useful methods of resolving and recovering specific stereoisomers are known in the art and described in Stereochemistry of Organic Compounds, E. L. Eliel and S. H. Wilen, Wiley (1994) and Enantiomers, Racemates, and Resolutions, J. Jacques, A. Collet, and S. H. Wilen, Wiley (1981).
As used in this application:
a) the term "halogen" refers to a fluorine atom, chlorine atom, bromine atom, or iodine atom;
b) the term "C,-C6 alkyl" refers to a branched or straight chained alkyl radical containing from 1 to 6 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, pentyl, hexyl, etc.;
c) the term "C~-C4 alkyl" refers to a saturated straight or branched chain alkyl group containing from 1-4 carbon atoms and includes methyl, ethyl, propyl, isopropyl, n-butyl, s-butyl, isobutyl, and t-butyl;
d) the term "C~-Coo alkyl" refers to a branched or straight chained alkyl radical containing from 1 to 6 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, etc.;
e) the term "C~-C4 alkoxy" refers to a straight or branched alkoxy group containing from 1 to 4 carbon atoms, such as methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, t-butoxy, etc.;
SUBSTIT>(JTE SHEET (RULE 26) f) the designation " '~~~~" " refers to a bond for which the stereochemistry is not designated;
g) the designation " " refers to a bond that protrudes forward out of the plane of the page.
h) the designation " °°""" " refers to a bond that protrudes backward out of the plane of the page.
i) as used in the examples and preparations, the following terms have the meanings indicated: "g" refers to grams, "mg" refers to milligrams, "qg" refers to micrograms.
"mol" refers to moles, "mmol" refers to millimoles, "nmole" refers to nanomoles, "L"
refers to liters, "mL" or "ml" refers to milliliters, "pL" refers to microliters, "°C" refers to degrees Celsius, "Rf' refers to retention factor, "mp" refers to melting point, "dec"
refers to decomposition, "bp" refers to boiling point, "mm of Hg" refers to pressure in millimeters of mercury, "cm" refers to centimeters, "nm" refers to nanometers, "brine"
refers to a saturated aqueous sodium chloride solution, "M" refers to molar, "mM" refers to millimolar, "qM" refers to micromolar, "nM" refers to nanomolar, "HPLC"
refers to high performance liquid chromatography, "HRMS" refers to high resolution mass spectrum, "DMF" refers to dimethylformamide, "qCi" refers to microcuries, "i.p." refers to intraperitoneally, "i.v." refers to intravenously, and "DPM" refers to disintegrations per minute;
j) for substituent Z, the designations -C(O)NR8-, -NRgC(O)-, -NR$C(O)NH-, -NRgC(O)O-, and -OC(O)NH- refer to the functionalities represented, respectively, by the following formulae showing the attachment of the group (Q):
SUBSTITUTE SHEET (RULE 26) W) \N . \N/\Ni O2) I (Q) , I H
I
Ra Ra Ra O O
\NJ"~o~ (Q> and \o~N~ (Q>
I ~ ' R H
a these designations are referred to hereinafter as amido, amide, urea, N-carbamoyl, and O-carbamoyl, respectively;
k) the term "pharmaceutically acceptable salts thereof refers to either an acid addition salt or a basic addition salt.
The expression "pharmaceutically acceptable acid addition salts" is intended to apply to any non-toxic organic or inorganic acid addition salt of the base compounds represented by formula (1) or any of its intermediates. Illustrative inorganic acids which form suitable salts include hydrochloric, hydrobromic, sulphuric, and phosphoric acid and acid metal salts such as sodium monohydrogen orthophosphate, and potassium hydrogen sulfate. Illustrative organic acids which form suitable salts include the mono-, di-, and tricarboxylic acids. Illustrative of such acids are for example, acetic, glycolic, lactic, pyruvic, malonic, succinic, glutaric, fumaric, malic, tartaric, citric, ascorbic, malefic, hydroxymaleic, benzoic, hydroxybenzoic, phenylacetic, cinnamic, salicyclic, 2-phenoxybenzoic, p-toluenesulfonic acid, and sulfonic acids such as methane sulfonic acid and 2-hydroxyethane sulfonic acid. Such salts can exist in either a hydrated or substantially anhydrous form. In general, the acid addition salts of these compounds are soluble in water and various hydrophilic organic solvents, and which in comparison to their free base forms, generally demonstrate higher melting points.
The expression "pharmaceutically acceptable basic addition salts" is intended to apply to any non-toxic organic or inorganic basic addition salts of the compounds represented by formula ( 1 ) or any of its intermediates. Illustrative bases which form suitable salts include alkali metal or alkaline-earth metal hydroxides such as sodium, potassium, calcium, magnesium, or barium hydroxides; ammonia, and aliphatic, SUBSTITUTE SHEET (RULE 26) alicyclic, or aromatic organic amines such as methylamine, dimethylamine, trimethylamine, and picoline.
As with any group of structurally related compounds which possess a particular utility, certain groups and configurations of substituents are preferred for the compounds of formula (1). Preferred embodiments are given below:
The compounds in which R~ is selected from the group consisting of Cl-C~, alkyl and -(CHZ)dArl are preferred;
The compounds in which R~ is -(CHZ)dAr~ and Are is phenyl or substituted phenyl are more preferred;
Compounds in which R4 is selected from the group consisting of hydrogen, -C(O)R,o and -S-G are preferred;
Compounds in which R4 is hydrogen, are more preferred;
Compounds in which R4 is -C(O)R,o and Rio is Cl_Ca alkyl more preferred;
Compounds in which A is -OH are preferred; and Compounds in which A is -NRR' wherein R is hydrogen and R' is methyl are preferred.
Examples of compounds encompassed by formula ( 1 ) and ( 1 a) of the present invention include the following. It is understood that the examples encompass all of the isomers of the compound and mixtures thereof. This list is meant to be representative only and is not intended to limit the scope of the invention in any way:
2-(4-(2-Thio-3-phenyl-propionyl-amino)-3-oxo-1,3,4,5- tetrahydro-benzo[c]azepin-2-yl)-4-methyl-valeric acid, (S)-N- 1-(2-methylpropyl)-2-(thio)-ethylamine, disulfide; 2-(4-(2-Thio-3-phenyl-propionyl-amino)-3-oxo-1,3,4,5- tetrahydro-benzo[c]azepin-2-yl)-4-methyl-valeric acid, (R)-1-( 2-methylpropyl)-2-(thio)-ethylamine, disulfide;
2-(4-(2-Thio-3-phenyl-propionyl-amino)-3-oxo-1,3,4,5- tetrahydro-benzo[c]azepin-2-yl)-methyl-valeric acid, L-cysteine ethyl ester, disulfide; 2-(4-(2-Thio-3-phenyl-propionyl-SUBSTTTUTE SHEET (RULE 26) amino)-3-oxo-1,3,4,5- tetrahydro-benzo[c]azepin-2-yl)-4-methyl-valeric acid, N-acetyl-L-cysteine ethyl ester, disulfide; 2-(4-(2-Thio-3-phenyl-propionyl-amino)-3-oxo-1,3,4,5- tetrahydro-benzo[c]azepin-2-yl)-4-methyl-valeric acid, L-cysteine, disulfide; 2-(4-(2-Thio-3-phenyl-propionyl-amino)-3-oxo-1,3,4,5- tetrahydro-benzo[c]azepin-2-yl)-4-methyl-valeric acid, benzylthio, disulfide; 2-(4-(2-Thio-3-phenyl-propionyl-amino)-3-oxo-1,3,4,5- tetrahydro-benzo[c]azepin-2-yl)-4-methyl-valeric acid, ethylthio, disulfide; 2-(4-(2-Thio-3-phenyl-propionyl-amino)-3-oxo-1,3,4,5- tetrahydro-benzo[c]azepin-2-yl)-4-methyl-valeric acid, disulfide, 2-hydroxyethylthio, disulfide; 2-(4-(2-Thio-3-phenyl-propionyl-amino)-3-oxo-1,3,4,5- tetrahydro-benzo[c]azepin-2-yl)-4-methyl-valeric acid, t-butyl ester, 2-pyridylmethylthio, disulfide; 2-(4-(2-Thio-3-phenyl-propionyl-amino)-3-oxo-1,3,4,5- tetrahydro-benzo[c]azepin-2-yl)-4-methyl-valeric acid, 2- thioacetic acid morpholine carboxamide, disulfide; 2-(4-(2-Thio-3-phenyl-propionyl-amino)-3-oxo-1,3,4,5- tetrahydro-benzo[c]azepin-2-yl)-4-methyl-valeric acid, t-butyl ester, benzylthio, disulfide; 2-(4-(2-Thio-3-phenyl-propionyl-amino)-3-oxo-1,3,4,5- tetrahydro-benzo[c]azepin-2-yl)-4-methyl-valeric acid, t-butyl ester, ethylthio, disulfide; 2-(4-(2-Thio-3-phenyl-propionyl-amino)-3-oxo-1,3,4,5-tetrahydro-benzo[c]azepin-2-yl)-4-methyl-valeric acid, t-butyl ester, disulfide, 2-hydroxyethylthio, disulfide; 2-(4-(2-Thio-3-phenyl-propionyl-amino)-3-oxo-1,3,4,5-tetrahydro-benzo[c]azepin-2-yl)-4-methyl-valeric acid, t-butyl ester, 2-pyridylmethylthio, disulfide; 2-(4-(2-Thio-3-phenyl-propionyl-amino)-3-oxo-1,3,4,5-tetrahydro-benzo[c]azepin-2-yl)-4-methyl-valeric acid, t-butyl ester, 2-thioacetic acid morpholine carboxamide, disulfide.
As used herein the term "amino acid" refers to naturally occurring amino acids as well as non-naturally occurring amino acids having substituents encompassed by R~
and Rz as described above. The naturally occurring amino acids included are glycine, alanine, valine, leucine, isoleucine, serine, methionine, threonine, phenylalanine, tyrosine, tryptophan, cysteine, histidine, aspartic acid, asparagine, glutamic acid, glutamine, arginine, ornithine, and lysine. Non-naturally occurring amino acids within the term "amino acid," include without limitation, the D-isomers of the naturally occurring amino acids, norleucine, norvaline, alloisoleucine, t-butylglycine, methionine sulfoxide, and methionine sulfone. Other non-naturally occurring amino acids within the term "amino acid," include without limitation phenylalanines, phenylglycines, SUBSTITUTE SHEET (RULE 26) homophenylalanines, 3-phenylpropylglycines, 4-phenylbutylglycines; each including those substituted by R~ and R6' as described above; and 1-naphthylalanines and naphthylalanines; including those substituted by R~ and R~~ as described above.
The compounds of formula (1) can be prepared by utilizing techniques and procedures well known and appreciated by one of ordinary skill in the art. To illustrate, general synthetic schemes for preparing starting material, intermediates, and the compounds of formula (1) are set forth below. In the reaction schemes below, the reagents and starting materials are readily available to one of ordinary skill in the art and all substituents are as previously defined unless otherwise indicated.
SUBSTITUTE SHEET (RULE 26) Reaction Scheme A
O ~ Rz Y
OH + ~ O
R3~ N
NHz A ' (2) step 1 o R
(3) Rz O ~ O
Y N
NH A' R3' (4) O Rl step 2 step 6 step 4 / Rz / Rz O ~ O O ~ O
Y N
NH A' R4 S NH N A' (5) R ( step 3 ~ z step 5 o ~ o HS N
NH A' R3 O Rl (6) optional deprotection step/
modification Rz O ~ O
,S N

formula ( 1 ) SUBSTITiJTE SHEET (RITLE 26) In Reaction Scheme A, step l, a compound of formula (3) is coupled with a with an appropriate acid derivative of formula (2) to give a compound of formula (4). Such coupling reactions are well known in the art.
An appropriate compound of structure (3) in one in which RZ is as desired in the final compound of formula ( 1 ), R~ as desired in the final product of formula ( 1 ) or gives rise after deprotection to R~ as desired in the final product of formula (1), and A' is A or gives rise to A upon deprotection and modification, if desired, to A as desired in the final compound of formula (1). Appropriate compounds of formula (3) can be prepared by methods described herein and by methods well known and appreciated in the art as described in PCT International Publication Number WO 95/21854, published 17 August 1995 and European Patent Application Publication Number 0 599 444 Al, published 1 June 1994.
An appropriate compound of formula (2) is one in which R;- is R3 as desired in the final product of formula (1) or gives rise after deprotection to R3 as desired in the final product of formula (1) and Y is a protected thio substituent or Y may be a protected hydroxy substituent or bromo which gives rise upon selective deprotection and displacement or displacement and further deprotection and/or elaboration, if required, to -SR4 as desired in the final product of formula (1). Alternately, an appropriate compound of formula (2) may also be one in which R3~ gives rise to R3~~ which, upon further reaction, gives rise R3 as desired in the final product of formula ( 1 ), as described in Reaction Scheme B, and Y is a protected thio substituent. In addition, an appropriate compound of formula (2) may also be one in which the stereochemistry at the R;~ and Y
bearing carbon is as desired in the final product of formula (1) or gives rise after displacement to the stereochemistry as desired at that carbon in the final product of formula (1).
The use and selection of appropriate protecting groups is within the ability of those skilled in the art and will depend upon compound of formula (2) to be protected, the presence of other protected amino acid residues, other protecting groups, and the nature of the particular R3 and/or R4 groups) ultimately being introduced.
Compounds SUBSTTTUTE SHEET (RULE 26) of formula (2) in which Y is bromo and protected thio are commercially available or can be prepared utilizing materials, techniques, and procedures well known and appreciated by one of ordinary skill in the art or described herein. See PCT Application WO
96/11209, published 18 April 1996. Examples commercially available compounds of formula (2) in which Y is bromo include 2-bromopropionic acid, 2-bromobutyric acid, 2-bromovaleric acid, 2-bromohexanoic acid, 6-(benzoylamino)-2-bromohexanoic acid, 2-bromoheptanoic acid, 2-bromooctanoic acid, 2-bromo-3-methylbutyric acid, 2-bromoisocaproic acid, 2-bromo-3-(5-imidazoyl)proionic acid, (R)-(+)-2-bromopropionic acid, (S)-(-)-2-bromopropionic acid.
For example, a compound of formula (3) is contacted in a coupling reaction with a compound of formula (2). The compound of formula (2) may be converted to an activated intermediate; such as and acid chloride, an anhydride; a mixed anhydride of aliphatic carboxylic acid, such as formic acid, acetic acid, propionic acid, butyric acid, isobutyric acid, pivalic acid, 2-ethylbutyric acid, trichloroacetic acid, trifluoroacetic acid, and the like; of aromatic carboxylic acids, such as benzoic acid and the like; of an activated ester, such as phenol ester, p-nitrophenol ester, 2,4-dinitrophenol ester, pentafluorophenol ester, pentachlorophenol ester, N-hydroxysuccinimide ester, N-hydroxyphthalimide ester, 1-hydroxy-1H-benztriazole ester, O-azabenztriazoyl-N,N,N',N'-tetramethyluronium hexafluoro-phosphate and the like; activated amide, such as imidazole, dimethylpyrazole, triazole, or tetrazole; or an intermediate formed in the presence of coupling agents, such as dicyclohexylcarbodiimide or 1-(3-dimethyaminopropyl)-3- ethylcarbodiimide.
The reaction is carried out in a suitable solvent, such as dichloromethane, chloroform, tetrahydrofuran, or dimethylformafide. The reaction generally is carried out at temperaures of from about -20°C to the refluxing temperature of the solvent and generally requires 1 to 48 hours. The product can be isolated and purified by techniques well known in the art, such as filtration, extraction, evaporation, chromatography, and recrystallization.
In Reaction Scheme A, step 2, a compound of formula (4) in which Y is hydroxy or bromo gives rise to a compound of formula (5) in which Y is protected thio.
SUBSTTTUTE SHEET (RULE 26) A compound of formula (4) in which Y is hydroxy (obtained from protected hydroxy compounds of formula (2)) undergoes a displacement reaction with an appropriate thio introducing reagent by the method of Mitsunobu to give a compound of formula (5) in which Y is a protected thio substituent. For example, a compound of formula (4) in which Y is hydroxy reacts with thioacetic acid or thiobenzoic acid, triphenylphosphine, and diethylazodicarboxylate in a suitable aprotic solvent, such as tetrahydrofuran to give a compound of formula (5) in which Y is thioacetyl or thiobenzoyl. The product can be isolated and purified by techniques well known in the art, such as extraction, evaporation, trituration, lyophilization, chromatography, and recrystallization.
A compound of formula (4) in which Y is bromo undergo a displacement reaction with an appropriate thio introducing reagent to give a compound of formula (5) in which Y is protected thio substituent which gives rise upon deprotection and subsequent elaboration, if desired, the -SR4 as desired in the final compound of formula (1).
For example, a solution of p-methoxybenzylmercaptan in a suitable organic solvent such as dimethylformamide is degassed and treated with a suitable base such as sodium hydride. After about 1 to 2 hours, a solution of a compound of formula (4) in which Y is bromo is added. The reaction may benefit from the addition of a suitable catalyst, such as tetra-n-butylammonium iodide. The reaction mixture is carried out for 1 to 25 hours at temperatures ranging from 0°C to about 100°C.
The product can be isolated and purified by techniques well known in the art, such as extraction, evaporation, trituration, lyophilization, chromatography, and recrystallization.
In Reaction Scheme A, step 3, a compound of formula (5) in which Y is protected thio undergoes selective deprotection to give a compound of formula (6).
Protected thio substituents include thioesters, such as thioacetyl or thiobenzoyl, thioethers, such as thiobenzyl, thio-4-methoxybenzyl, thiotriphenylmethyl, or thio-t-butyl, or unsymmetrical sulfides, such as dithioethyl or dithio-t-butyl. The use and selective removal of thio protecting groups is well known and appreciated in the art and described in Protective Groups in Or anic Synthesis, Theodora W. Greene (Wiley-Interscience, 2nd Edition, 1991).
SUBSTTTUTE SHEET (RULE 26) Alternately, in Reaction Scheme A, step 4, a compound of formula (4) in which Y is protected thio is selectively deprotected to give a compound of formula (6), as described above, in Reaction Scheme A, step 3.
In Reaction Scheme A, step 5, a compound of formula (6) undergoes a undergoes modification reaction to give a compound of formula (7). Such modification reactions include, thiol esterification and disulfide formation.
Compounds of formula (7) in which R4 is -C(O)R,o or -C(O)-(CHZ)q-X group can be synthesized by thiol esterifications according to techniques well known and appreciated by one of ordinary skill in the art, such as those disclosed in U.S. Pat. Nos.
5,424,425, issued Jun. 13, 1995.
For example, in a thiol esterification a compound of formula (6) is contacted with about an equimolar amount of an appropriate acid, such as HO-C(O)R~o or HO-C(O)-(CHZ)q-X in the presence of a suitable coupling agent to give a compound of formula (6) in which R4 is -C(O)R~o or -C(O)-(CHZ)q-X. The reaction is carried out in the presence of a coupling agent such as 2-fluoro-1-methylpyridinium p-toluenesulfate, (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride), carbonyldiimidazole, (1-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline, or diethylcyanophosphonate in a suitable aprotic solvent such as methylene chloride. The reaction is generally carried out at temperature of between -20°C and the boiling point of the solvent. Generally, the reaction requires 1 to 24 hours. The product can be isolated and purified by techniques well known in the art, such as extraction, evaporation, trituration, lyophilization, chromatography, and recrystallization.
Compounds of formula (7) in which R4 is -S-G group can be synthesized according to techniques well known and appreciated by one of ordinary skill in the art, as disclosed in PCT Application No. WO 95/21839, published 17 August 1995 and U.S.
Patent Nos. 5,491,143, issued February 13, 1996, and 5,731,306, issued March 24, 1998, and Rogues, B.P. et al., J. Med. Chem. 33, 2473-2481 (1992).
SUBSTITUTE SHEET (RULE 26) For example, in a disulfide formation a compound of formula (6) is contacted with an appropriate compound of formula (8).
~s~ ~
G S N
(g) An appropriate compound of formula (8) is one which gives G as desired in the final product of formula ( 1 ) or gives rise upon deprotection to G as is desired in the final product of formula (1). In addition, the compound of formula (8) may have stereochemistry as desired in the final product of formula (1). The reaction is carried out in a suitable solvent, such as ethanol, methanol, dichloromethane, or mixtures of ethanol or methanol and dichloromethane. The solvent is degassed by passing a stream of nitrogen gas through it for 15 minutes before the reaction is carried out.
The reaction is carried out using from 1.0 to 4.0 molar equivalents of an appropriate compound of formula (8). The reaction is carried out at temperatures of from 0°C to the refluxing temperature of the solvent, with a temperature of 10°C to 30°C
being preferred. The reaction generally requires from 1 to 48 hours. The product can be isolated by techniques well known in the art, such as extraction, evaporation, and precipitation and can be purified by chromatography and recrystallization.
In Reaction Scheme A, step 6, a compound of formula (4) in which Y is hydroxy or bromo is displaced by an appropriate thiol, HSR4, by the methods described in Reaction Scheme A, step 2, to give a compound of formula (7). In Reaction Scheme A, step 6, an appropriate thiol HSR4 is one which gives R4 as desired in the final product of formula ( 1 ) or gives rise to R4 as desired in the final product of formula ( 1 ). Also in Reaction Scheme A, step 6, a compound of formula (4) in which Y is bromo can be displaced by an appropriate thio ester, Ph3S-C(O)-(CHz)q-X by techniques well known and appreciated in the art, as disclosed in U.S. Pat. No. 5,424,425, issued Jun. 13, 1995.
In Reaction Scheme A, a compound of formula (5), (6), or (7) is optionally deprotected to give a compound of formula (1). Such deprotection reactions are well known appreciated in the art and may include selective deprotections of protecting groups on A', R,, R2, R3, and R4, as required to give the desired compound of formula (1).
SUBSTTTUTE SHEET (RULE 26) Reaction Scheme B
o ~ o 0 0 N i, l g ; ' g R3' ($) p ~ ~ p (12), step 1 step 4 '.
/~~~~ / '~ optional / ' step 5 o i. o o ~ o i ;i ,, / ~; i y ' N i~~, ~ ~ ~ N ~ , ~N~ ~ ~ A. ~NJ~/ ~/ A. I.
g II ~ ~ g .I
o Rl ~ ~ o ~ optional (9) ( 11 ) step 5 ' optional step 2 step 3 I
/ice ~ ;
optional ~ ~~
step 5 o ~ o p / o _- __ ~ li ~ ;I
Y,~ C~ N~I~A~ R4 S~N'~1~N~A
H ~ ~ ~ g i, p ~ ~ p (10) formula (1) In Reaction Scheme B, step 1, an appropriate compound of formula (5) is deprotected, hydrolyzed, or reduced to give a compound of formula (9). In Reaction Scheme B, step l, an appropriate compound of formula (5) is one in which A' is A or gives rise to A upon deprotection and modification, if desired, in the final product of formula (1) and R~, and RZ are as desired in the final product of formula (1) or give rise upon deprotection to R~ and/or RZ as desired in the final product of formula (1). In Reaction Scheme B, step 1, an appropriate compound of formula (S) is one in which R3' gives rise to a compound of formula (9) in which R3» is R3 as desired in the final product of formula (1) or undergoes further derivitizatior~ (step 2) to give a compound of SUBSTITUTE SHEET (RULE 26) formula ( 10) in which R3 is as desired in the final product of formula ( 1 ).
In Reaction Scheme B, step 1, an appropriate compound of formula (S) is one in which Y is -SR4 as desired in the final compound of formula ( 1 ) or Y gives rise upon deprotection (step 3) and further functionalization (step 4) or deprotection (step S) to give -SR4 as desired in S the final product of formula ( 1 ). In Reaction Scheme B, the use of compounds of formula (S) in which Y is a protected thio group, such as thioacetyl, thiobenzoyl, 4-methoxybenzylthio or t-butylthio is preferred.
For example, in a deprotection a compound of formula (S) in which R3~ is -(CHZ)m-W (phthalimido group) is contacted with a molar excess of hydrazine monohydrate to give a compound of formula (9) in which R3-~ is -(CHZ)m-NHRB in which R8 is hydrogen. The reaction is typically carried out in a protic organic solvent, such as methanol or ethanol. The reaction is generally carried out at room temperature for a period of time ranging from S-24 hours. The product can be isolated by techniques 1 S well known in the art, such as extraction, evaporation, and precipitation and can be purified by chromatography and recrystallization.
Alternately, for example, in a deprotection of a compound of formula (S) in which R;~ is -(CHZ)m-NR8-t-Boc is contacted with a molar excess of a suitable acid to give a compound of formula (9) in which R;» is -(CHZ)m-NHRx. The reaction is typically carried out in a organic solvent, such as methanol, ethanol, ethyl acetate, diethyl ether, or dioxane. Suitable acids for this reaction are well known in the art, including hydrochloric acid, hydrobromic acid, trifluoroacetic acid, and methanesulfonic acid. The reaction is generally carried out at temperatures from about 0°C to about 2S room temperature for a period of time ranging from 1-10 hours. The product can be isolated by techniques well known in the art, such as extraction, evaporation, and precipitation and can be purified by chromatography and recrystallization.
For example, in a hydrolysis a compound of formula (S) in which R3~ is -(CHZ)m-C(O)OPg3 and Pg3 is methyl or ethyl is contacted with about 1 to 2 molar equivalents of lithium hydroxide, sodium hydroxide, or potassium hydroxide to give a compound of formula (9) in which R3w is -(CHZ)m-COZH. The reaction is carried out in a suitable solvent, such as methanol, ethanol methanol/water mixtures, ethanol/water mixtures, or tetrahydrofuran/water mixtures and generally requires 1 to 24 hours. The SUBSTITUTE SHEET (RULE 26) reaction is carned out at temperatures of from about 0°C to the refluxing temperature of the solvent. The resulting acid is isolated and purified by techniques well known in the art, such as acidification, extraction, evaporation, and precipitation and can be purified by trituration, precipitation, chromatography, and recrystallization.
For example, in a reduction a compound of formula (5) in which R;~ is -(CHZ)m_~-COZPg3 in which Pg3 is methyl or ethyl is contacted with a suitable reducing agent, such as lithium borohydride, lithium aluminum hydride, diisobutylaluminum hydride, 9-borabicyclo(3.3.1)nonane, preferably lithium borohydride to provide a compound of formula (9) in which R;w is -(CHZ)n,-1-CHZOH. The reaction is carried out in a suitable solvent, such as dichloromethane, tetrahydrofuran, or toluene, with tetrahydrofuran being preferred. The reaction is carried out at a temperature of from about -30°C to about 50°C and generally requires from 2 to 12 hours. The product can be isolated by quenching, extraction, evaporation, and precipitation and can be purified by trituration, chromatography, and recrystallization.
In Reaction Scheme B, step 2, a compound of formula (9) undergoes a derivitization reaction to give a compound of formula (10) in which R3 is as desired in the final product of formula (I). Such derivitization reactions include hydrolysis of esters and ester formations as are well known in the art, ether formation, amine alkylation, formation of amides, urea formation, carbamate formation, and formation of sulfonamide.
For example, in an ether formation a compound of formula (9) in which R3» is -(CHZ)m_,-CHZOH is contacted with 1 to 10 molar equivalents of a suitable akylating agent to give a compound of formula (IO) in which R3 is -(CHZ)m-Z-Q in which Z
is -O-.
A suitable alkylating agent is one which transfers Q or protected Q as desired in the final product of formula (1), such as benzyl bromide, benzyl chloride, substituted benzyl bromide, substituted benzyl chloride, ethyl bromoacetate, t-butyl bromoaceate, ethyl 3-chloropropionate, ethyl 3-bromopropionate, ethyl 5-bromovalerate, ethyl 4-bromobutyrate, 3-chloropropionamide, 2-bromoethylbenzene, substituted 2-bromoethylbenzene, 1-chloro-3-phenylpropane, 1-bromo-4-phenylbutane, and the like, or nitrogen mustards, including 2-dimethylaminoethyl chloride, 2-diethylaminoethyl SUBSTITUTE SHEET (RULE 26) chloride, and 3-dimethylaminopropyl chloride. The reaction is carried out in a suitable solvent, such as diethyl ether, tetrahydrofuran, dimethylformamide, dimethyl sulfoxide, or acetonitrile and using a suitable base, such as sodium hydride, potassium hydride, potassium t-butoxide, and lithium diisopropylamide. The reaction is generally carried out at temperatures of -70°C and room temperature and require from about 1-24 hours.
The product can be isolated by techniques well known in the art, such as extraction, evaporation, and precipitation and can be purified by chromatography and recrystallization.
Alternately, as appreciated by those skilled in the art, an ether formation can also be carried out by a procedure similar to the one above using a compound of formula (9) in which R3w is -(CHZ)",_~-CHZOH in which the hydroxy group is first converted to a leaving group, such as chloro, bromo, or mesylate and a suitable alcohol which transfers Q or protected Q as desired in the final product of formula (1), such as benzyl alcohol, substituted benzyl alcohol, phenol, substituted phenol, and the like. The conversion of hydroxy to leaving groups, such as chloro, bromo, and mesylate are well known and appreciated in the art.
For example, in an amine alkylation a compound of formula (9) in which R;-~ is -(CHZ)n,-NHRB is contacted with 1 to 10 molar equivalents of a suitable akylating agent to give a compound of formula ( 10) in which R3 is -(CHz)m-Z-Q in which Z is -NR~-.
The reaction may be carried out after protection of the amine function of R;~-in which R8 is hydrogen by a suitable protecting group, such as benzyl or t-Boc. For an amine alkylation a suitable alkylating agent is one as described above for the ether formation and also includes alkylhalides, such as methyl iodide, methyl bromide, ethyl bromide, propyl bromide, propyl chloride, butyl bromide, butyl chloride, and the like.
The reaction is carried out in a suitable solvent, such as methanol, ethanol, dimethylformamide, or pyridine and using a suitable base, such as sodium carbonate, triethylamine, N,N-diisopropylethylamine or pyridine. The reaction is generally carried out at temperatures of room temperature to the refluxing temperature of the solvent and require from about 1-24 hours. The product can be isolated by techniques well known in the art, such as extraction, evaporation, and precipitation and can be purified by chromatography and recrystallization.
SUBSTITUTE SHEET (RULE 26) WO 00/40564 PCT/iJS99/28339 Alternately, for example, in an amine alkylation a compound of formula (9) in which R;w is -(CHZ)m-NHz is contacted in a reductive alkylation with a suitable aldehyde to give a compound of formula (10) useful as an intermediate for preparing compounds in which R3 is -(CHZ)m-Z-Q in which Z is -NR8-. A suitable aldehyde is one which transfers Q or protected Q as desired in the final product of formula (1 ), such as benzaldehyde and substituted benzaldehydes. The reaction is carried out in a suitable solvent, such as methanol, ethanol, tetrahydrofuran, or mixtures of methanol or ethanol and tetrahydrofuran. The reaction may be carried out in the presence of a drying agent, such as sodium sulfate or molecular sieves. The reaction is carried out in the presence of from 1.0 to 6.0 molar equivalents of a suitable reducing agent, such as, sodium borohydride or sodium cyanoborohydride with sodium cyanoborohydride being preferred. It may be advantageous to maintain the pH in the range of about 4 to 6. The reaction is generally carried out at temperatures of from 0° C to the refluxing temperature of the solvent. Generally, the reactions require 1 to 72 hours. The product can be isolated by techniques well known in the art, such as extraction, evaporation, and precipitation and can be purified by chromatography and recrystallization.
For example, in an amido formation a compound of formula (9) in which R;» is is -(CHZ)m-COZH is contacted with a suitable amine in an amide formation to give a compound of formula (10) in which R3 is -(CHZ)m-Z-Q in which Z is amido. Such amide formation reactions using carboxy activation or suitable coupling agents are well known in the art and described above. A suitable amine, HNRBQ, gives rise to R8 and Q
as desired in the final product of formula (1), such as methylamine, ethylamine, propylamine, butylamine, N-methyl benzylamine, benzyl ~3-alanine, 4-(3-aminopropyl)morpholine, and the like.
For example, in an amide formation a compound of formula (9) in which R;w is is -(CHZ)m-NHRB is contacted with a suitable carboxylic acid in an amide formation to give a compound of formula (10) in which R3 is -(CHz)m-Z-Q in which Z is amide.
Such amide formation reactions using carboxy activation or suitable coupling agents are well known in the art and described above. Suitable carboxylic acids, QC(O)-OH, are SUBSTTTUTE SHEET (RULE 26) ones give rise to Q as desired in the final product of formula (1), such as benzoic acid, substituted benzoic acids, phenyl acetic acids, substituted phenylacetic acids, mono-t-butyl malonate, and the like.
For example, in a urea formation a compound of formula (9) in which R3» is is -(CHZ)n,-NHRB is contacted with an appropriate isocyanate, O=C=N-Q, to give a compound of formula (10) in which R3 is -(CHZ)m-Z-Q in which Z is urea. An appropriate isocyanate is one which gives rise to Q as desired in the final product, such as phenyl isocyanate, substituted phenyl isocyanate, napthyl isocyanate, ethyl isocyanatoacetate, and the like. The reaction is carried out by adding an equivalent of, or a slight molar excess of, an appropriate isocyanate is added to a solution of a compound of formula (9) in which R;w is -(CHZ)m-NHRg in a suitable solvent, such as diethyl ether, benzene, or toluene. The reaction is carried out at temperature of from about 0°C to the refluxing temperature of the solvent and require about 1-24 hours. The product can be isolated and purified by techniques well known in the art, such as filtration, extraction, evaporation, trituration, chromatography, and recrystallization.
For example, in an N-carbamoyl formation a compound of formula (9) in which R3» is -(CHZ)m-NHRB is contacted with an appropriate chloroformate to give a compound of formula (10) in which R3 is -(CHZ)m-Z-Q in which Z is N-carbamoyl. An appropriate chloroformate is one which gives rise to Q as desired in the final product of formula ( 1 ).
Examples of chloroformates include benzyl chloroformate, naphthyl chloroformate, phenyl chloroformate, and substituted phenyl chloroformates, such as 4-chlorophenyl chloroformate, 4-methylphenyl chloroformate, 4-bromophenyl chloroformate, 4-fluorophenyl chloroformate, 4-methoxyphenyl chloroformate and the like. The reaction is carried out by adding an equivalent of, or a slight molar excess of, an appropriate chloro formate to a solution of a compound of formula (9) in which R3w is -(CHZ)",-NHRB in a suitable solvent, such as toluene, tetrahydrofuran, dimethylformamide, dichloromethane, pyridine, or chloroform. The reaction is carried out in the presence of an excess of a suitable base, such as triethylamine, sodium carbonate, potassium bicarbonate, pyridine or N,N-diisopropylethylamine. The reaction is carried out at a temperature of from -70°C to the refluxing temperature of the solvent and generally SUBSTITUTE SHEET (RULE 26) requires from 30 minutes to 24 hours. The product can be isolated and purified by techniques well known in the art, such as extraction, evaporation, chromatography, and recrystallization.
For example, in an O-carbamoyl formation a compound of formula (9) in which R3» is -(CHZ)",_~-CHZOH is contacted with an appropriate isocyanate, as defined above for urea formation, to give a compound of formula (10) in which R3 is -(CHZ)m-Z-Q in which Z
is O-carbamoyl. The reaction is carried out in a suitable solvent, such as diethyl ether, tetrahydrofuran, dimethylformamide, or acetonitrile. The reaction may be facilitated by the use of catalytic amount of a suitable base, such as sodium hydride, potassium hydride, or potassium t-butoxide. The reaction is generally carried out at temperatures of from -20°C to room temperature and require from about 1-24 hours. The product can be isolated by techniques well known in the art, such as extraction, evaporation, and precipitation and can be purified by chromatography and recrystallization.
For example, in a sulfonamide formation to prepare a compound in which R3 is -(CHZ)m-NR8~S02-Y~, a compound of formula (9) in which R3» is -(CHZ)m-NHRg is contacted with an appropriate sulfonamide forming reagent. An appropriate sulfonamide forming reagent, such as a sulfonyl chloride, Y~ S(O)ZC1, or sulfonyl anhydride, Y~(O)zS-O-S(O)2 Y~, is one which gives rise to Y~ as desired in the final product. Examples of appropriate sulfonamide forming reagents are, benzenesulfonyl chloride, dansyl chloride, N-morpholinylsulfonyl chloride, N-piperidinylsulfonyl chloride, 2,4,5-trichlorobenzenesulfonyl chloride, 2,5-dichlorobenzenesulfonyl chloride, 2,4,6-triisopropylbenzenesulfonyl chloride, 2-mesitylenesulfonyl chloride, 4-bromobenzenesulfonyl chloride, 4-fluorobenzenesulfonyl chloride, 4-chlorobenzenesulfonyl chloride, 4-methoxybenzenesulfonyl chloride, 4-t-butylbenzenesulfonyl chloride, p-toluenesulfonyl chloride, 2,3,4-trichlorobenzenesulfonyl chloride, 2,5-dimethoxybenzenesulfonyl chloride, 4-ethylbenzenesulfonyl chloride, 3,4-dimethoxybenzenesulfonyl chloride, 2,6-dichlorobenzenesulfonyl chloride, 3-bromobenzenesulfonyl chloride, 4-n-butylbenzenesulfonyl chloride, benzenesulfonic anhydride, 4-toluenesulfonic anhydride, and 2-mesitylenesulfonic anhydride. The reaction is carried out in a suitable solvent, SUBSTITUTE SHEET (RULE 26) such as tetrahydrofuran, dichloromethane, pyridine, or chloroform and in the presence of an excess of a suitable base, such as triethylamine, sodium carbonate, pyridine, or N,N-diisopropylethylamine. The reaction is carried out at a temperature of from -50°C to the refluxing temperature of the solvent. The reaction generally requires from 30 minutes to 24 hours. The product can be isolated and purified by techniques well known in the art, such as extraction, evaporation, chromatography, and recrystallization.
In Reaction Scheme B, optional step 3, a compound of formula (10) in which R3 is as desired in the final product of formula (1) undergoes a selective thiol deprotection to give a compound of formula (11). Such selective thiol deprotections using suitable protecting groups are well known and appreciated in the art as discussed in Reaction Scheme A, step 4, above.
In Reaction Scheme B, step 4, a compound of formula (11) undergoes a modification reaction as described in Reaction Scheme A, step 5, above, to give a compound of formula (7).
In Reaction Scheme B, optional step 5, a compound of formula (10), (11), or (12) is deprotected to give a compound of formula (1) as discussed in Reaction Scheme A, above.
Alternate routes for preparing the compounds of formula (2) in which Y is bromo are presented in Reaction Schemes C.1 and C.2.
Reaction Scheme C.l OH ~ R3 OH
NHz Br (20) (2 in which Y is bromo) In Reaction Scheme C.l, an appropriate cx-amino carboxylic acid of formula (20) is deaminobrominated to give a compound of formula (2) in which Y is bromo. An appropriate a-amino carboxylic acid of formula (20), and protected forms thereof, is one SUBSTTTUTE SHEET (RULE 26) which is one in which R3~ is as described above in Reaction Scheme A, step 8, above.
In addition, a,-amino carboxylic acid of formula (20) may also be one in which the stereochemistry at the R;~ bearing carbon gives rise after displacement to the stereochemistry as desired at that carbon in the final product of formula (1).
Such appropriate a-amino carboxylic acid of formula (20), are commercially available or may be readily prepared by techniques and procedures well known and appreciated by one of ordinary skill in the art. For example, L-alanine, D-alanine, L-valine, D-valine, D-norvaline, L-leucine, D-leucine, D-isoleucine, D-tert-leucine, glycine, L-glutamic acid, D-glutamic acid, L-glutamine, D-glutamine, L-lysine, D-lysine, L-ornithine, D-ornithine, (D)-(-)-2-aminobutyric acid, D-threonine, D-homoserine, D-allothreonine, D-serine, D-2-aminoadipic acid, D-aspartic acid, D-glutamic acid, D-lysine hydrate, 2,3-diaminopropionic acid monohydrobromide, D-ornithine hydrochloride, D,L-2,4-diaminobutyric acid dihydrochloride, L-meta-tyrosine, D-4-hydroxyphenylglycine, D-tyrosine, L-phenylalanine, D-phenylalanine, D,L-2-fluorophenylalanine, beta-methyl-D,L-phenylalanine hydrochloride, D,L-3-fluorophenylalanine, 4-bromo-D,L-phenylalanine, L-phenylalanine, L-phenylglycine, D-phenylglycine, D,L-4-fluorophenylalanine, 4-iodo-D-phenylalanine, D-homophenylalanine, D,L-2-fluorophenylglycine, D,L-4-chlorophenylalanine, and the like, are all commercially available and the methods in D. A. Evans, et al. J. Am. Chem. Soc., 112, 4011-(1990); S. Ikegami et al. Tetrahedron, 44, 5333-5342 (1988); W.Oppolzer et al.
Tet.
Lets. 30, 6009-6010 (1989); Synthesis of Optically Active a,-Amino-Acids, R.
M.
Williams (Pergamon Press, Oxford 1989); M. J. O'Donnell ed.: a.-Amino-Acid Synthesis, Tetrahedron Symposia in print, No. 33, Tetrahedron 44, No. 17 (1988); U.
Schollkopf, PureAppl. Chem. 55, 1799 (1983); U. Hengartner et al. J. Org.
Chem., 44, 3748-3752 (1979); M. J. O'Donnell et al. Tet. Lets., 2641-2644 (1978); M. J.
O'Donnell et al. Tet. Lets. 23, 4255-4258 (1982); M. J. O'Donnell et al. J. Am. Chem.
Soc., 110, 8520-8525 (1988).
The deaminobromination described in Reaction Scheme C.1 can be performed utilizing conditions described in Compagnone, R.S. and Rapoport, H., J. Ors.
Chem., 51, 1713-1719 (1986); U.S. Pat. No. 5,322,942, issued June 21, 1994;
Overberger, C.G.
and Cho, L, J. Org. Chem., 33, 3321-3322 (1968); or Pfister, K. et al., J. Am.
Chem.
Soc., 71, 1096-1100 (1949).
SUBSTTTUTE SKEET (RULE 26) For example, an a,-amino carboxylic acid of formula (20) and a suitable bromide, such as hydrogen bromide or potassium bromide in acidic solution, such as sulfuric acid, is treated with sodium nitrite. The reaction temperature is carried out a temperatures of from about -25°C to about ambient temperature and require about 1 to 5 hours. The product can be isolated and purified by techniques well known in the art, such as acidification, extraction, evaporation, chromatography, and recrystallization to give the compound of formula (11)in which Y is bromo. The product can be isolated and purified by techniques well known and appreciated in the art, such as acidification, basification, filtration, extraction, evaporation, trituration, chromatography, and recrystallization.
Reaction Scheme C.2 OH -~ ~ OH
(21) sr (2 in which Y is bromo) In Reaction Scheme C.2, an appropriate carboxylic acid of formula (21 ) is brominated to give compound of formula (2) in which Y is bromo. An appropriate carboxylic acid of formula (21), and protected forms thereof, is one which is one in which R;~ is as defined in Reaction Scheme A, step 8, above. In addition, carboxylic acid of formula (21) may also be one in which the stereochemistry at the R3~
bearing carbon gives rise after displacement to the stereochemistry as desired at that carbon in the final product of formula ( 1 ).
For example, a mixture of a carboxylic acid of formula (21 ) and dry red phosphorous are treated dropwise with bromine at temperature ranging from about -20°
to about 10°C. The reaction mixture is then warmed to room temperature and then heated to about 80°C for about 2-5 hours. The reaction mixture is then cooled to room temperature, poured into water containing sodium bisulfate, and neutralized using solid sodium carbonate. The aqueous layer is extracted and acidified with a suitable acid, such as concentrated hydrochloric acid. The precipitate is collected by filtration and SUBSTITUTE SHEET (RULE 26) dried to give the compound of formula (2) in which Y is bromo. The product can be isolated and purified by techniques well known and appreciated in the art, such as acidification, basification, filtration, extraction, evaporation, trituration, chromatography, and recrystallization.
Compounds of formula (20) and (21 ) in which R3~ is a -(CHz)m-W for use in Reaction Schemes C.1 and C.2 are prepared according to Reaction Scheme D.1 and D.2.
Racoon Scheme D. l HzN- ( CHz )~ ~ ~ N-- ( CHG ) m OH OH
O
(22) (21 ) in which R3. is W-(CH2)m In Reaction Scheme D.1 an appropriate w-amino carboxylic acid of formula (22) is converted to an compound of formula (21 ) in which R3> is W-(CHZ)m-. An appropriate cu-amino carboxylic acid of formula (2) is one in which m is as desired in the final product of formula (1) and are readily available in the art. For example, the reaction is carried out in a suitable polar solvent, such as water, ethanol, diethyl ether, tetrahydrofuran, or a water/ethanol solvent mixture using a suitable base, such as sodium carbonate and N-carbethoxyphthalimide. The reaction mixture is typically stirred at about ambient temperature for 1-5 hours. The product can be isolated and purified by techniques well known in the art, such as acidification, extraction, evaporation, chromatography, and recrystallization to give the desired compound of formula (21) in which R3~ is W-(CHz)m-.
SUBSTTTUTE SHEET (RITLE 26) Reaction Scheme D.2 HzN- ( CHZ ) m HzN- ( CHZ ) m (23) off step 1 off NHz (24) NHPg4 step 2 \ N- ( CHz ) m OH
(25) NHPg~
step 3 ° 1 \N- (CH2) m OH

(20) in which R3. is W_(CH2)m_ Reaction Scheme D.2, step l, an appropriate a,c~-diamino acid of formula (23) undergoes a selective N-a-protection to give an N-a-protected-c~-diamino acid of formula (24). An appropriate a,c~-diamino acid of formula (23) is one in which m is as desired in the final product of formula ( 1 ).
For example, a selective N-a-protection of a suitable a,c~-diamino acid, such as L-lysine (formula (23) in which m is 4), is accomplished by masking the c~-amino group by formation of a benzylidene imine. The benzylidene imine is formed by dissolving L-lysine monohydrochloride in lithium hydroxide and cooling the solution to a temperature ranging from about 0° to 10°C. Freshly distilled benzaldehyde is then added and the solution is shaken. N-cu-benzylidene-L-lysine is recovered by filtration and evaporation. The a-amino group of the N-c~-benzylidene-L-lysine then undergoes SUBSTITUTE SHEET (RULE 26) protection, such as the introduction of a Cbz or t-Boc group, followed by hydrolytic cleavage of the imine in situ to give N-a-benzyloxy-carbonyl-L-lysine.
Accordingly, N-c~-benzylidene-L-lysine is added to a mixture of sodium hydroxide and ethanol, cooled to a temperature of from about -5° to about -25°C. Then, precooled solutions of benzyloxycarbonyl chloride in a solvent, such as ethanol, is added to the reaction mixture. The temperature is maintained in a range of from about -10° to about -25°C during the course of addition, and may allowed to rise afterwards. The reaction mixture is then acidified using a suitable acid, such as precooled hydrochloric acid, and N-a-benzyloxycarbonyl-L-lysine, which corresponds to formula (24) where m is 4, is recovered by filtration evaporate and recrystallization.
In Reaction Scheme D.2, step 2, N-a-benzyloxycarbonyl-L-lysine or other compounds of formula (24) is converted to c~-phthalimido-a-benzyloxycarbonyl-L-lysine or other cu-phthalimido-a-aminoprotected carboxylic acid of formula (25) by the method described in Reaction Scheme D.l, above.
In Reaction Scheme D.2, step 3, the ~-phthalimido-a-aminoprotected carboxylic acid of formula (25) is deprotected to give compound of formula (20) in which R3' is W-(CI IZ)n,-.
For example, c~-phthalimido-a-benzyloxycarbonyl-L-lysine is contacted with hydrogen in the presence of a hydrogenation catalyst, such as 10%
palladium/carbon.
The reactants are typically contacted in a suitable solvent mixture such as ethanol, methanol, water, ethanol/water mixtures, or methanol/water mixtures. The reactants are typically shaken under a hydrogen atmosphere of 35-45 psi at room temperature for a period of time ranging from 5-24 hours. The product is typically recovered by filtration and evaporation of the solvent.
A route for preparing the compounds of formula (2) in which Y is protected thio is presented in Reaction Scheme F. The reagents and starting materials are readily available to one of ordinary skill in the art. In Reaction Scheme H
all substituents, unless otherwise indicated, are as previously defined.
SUBSTITUTE SHEET (RULE 26) Reaction Scheme F

Br~
~o-Pgs step 1 (26) (27) step 2 o i o step 3 OH ~ _ O Pgs R3 R,' s (2 in which Y is (28) protected thio) In Reaction Scheme F, step l, a bromoacetate of formula (26) is contacted with an appropriate thiol to give a protected acetic acid ester of formula (27). In a bromoacetate of formula (26) Pg5 is a protecting group, such as methyl, ethyl, t-butyl, and benzyl. An appropriate thiol is one which gives rise to a protected thio group, Y, in the product of formula (11). The use of 4-methoxybenzylmercaptan is preferred.
For example, a bromoacetate of formula (26) is contacted with an appropriate thiol in a suitable organic solvent, such as dimethylformamide.
Advantageously, the solvent is degassed. The reaction is carried out using a suitable base, such as sodium hydroxide, triethylamine, or N,N-diisopropylethylamine. The reaction is carried out at temperatures of from about -50° to about ambient temperature and requires about 1 to 72 hours. The protected acetic acid ester of formula (27) can be isolated and purified by methods well known and appreciated in the art, such as extraction, evaporation, chromatography, and distillation, and recrystallization.
In Reaction Scheme F, step 2, the protected acetic acid ester of formula (27) is alkylated with an appropriate akylating agent to give a compound of formula (28). In Reaction Scheme F, step 2, an appropriate alkylating agent is one which transfers R3~
which is R3 as desired in the final product of formula (1) or gives rise after deprotection to R3 as desired in the final product of formula ( 1 ) or gives rise to R3w as defined in SUBSTITUTE SHEET (RULE 26) Reaction Scheme B, step 1. Appropriate alkylating agents include alkylhalides, such as methyl iodide, methyl bromide, ethyl bromide, propyl bromide, propyl chloride, butyl bromide, butyl chloride, and the like; benzyl bromide, benzyl chloride, substituted benzyl bromide, substituted benzyl chloride, ethyl bromoacetate, t-butyl bromoaceate, ethyl 3-chloropropionate, ethyl 3-bromopropionate, ethyl 5-bromovalerate, ethyl 4-bromobutyrate, 3-chloropropionamide, 2-bromoethylbenzene, substituted 2-bromoethylbenzene, 1-chloro-3-phenylpropane, 1-bromo-4-phenylbutane, and the like, N-(2-bromoethyl)phthalimide, , N-(3-bromopropyl)phthalimide, N-(4-bromobutyl)phthalimide, and the like; 1-bromo-2-phenylethane, 1-bromo-3-phenylpropane, 1-bromo-4-phenylbutane, and the like.
For example, a protected acetic acid ester of formula (27) is alkylated with an appropriate alkylating agent. The reaction is carried out in a suitable solvent, such as diethyl ether, tetrahydrofuran, dimethylformamide, and toluene using a suitable base, such as sodium hydride, potassium hydride, potassium t-butoxide, lithium bis(trimethylsilyl)amide, sodium bis(trimethylsilyl)amide, potassium bis(trimethylsilyl)amide, or lithium diisopropylamide. The reaction is generally carried out at temperatures of about -70°C to about room temperature and require from about 1-24 hours. The product can be isolated by techniques well known in the art, such as extraction, evaporation, and precipitation and can be purified by chromatography and recrystallization.
In Reaction Scheme F, step 3, the compound of formula (28) the carboxy protecting group Pg~ is selectively removed to give a compound of formula (3b) in which Y is protected thio. Such deprotection of esters to acids in the presence of suitable thio protecting groups are well known and appreciated in the art.
A general preparation of compounds of formula (3) is set forth in Reaction Scheme G.
SUBSTITUTE SHEET (RULE 26) Reaction Scheme G
/ Rz / Rz step 1 OH ~ Cl (30) OH OSOzCH3 (31) step 2 iH3 iH3 S\ /S
III( / RZ step 3 / Rz N
O I
N
s =o (33) (32) step 4 / R2 step 5 ~ ~ O / Rz NHz /
N
// o off (34) off (35) / Rz step 6 NHZ

~Sl (CH3) 3 (36) step 7 SUBSTITUTE SHEET (RULE 26) Reaction Scheme G, Cont.
step 7 / O ~ Rz step 8 / O ~ R
/ N
N O
o ~ O
O HN \O-t-Bu O O
O
O~ R1 ~
sl ~CH3~ 3 (3751 (CH3) 3 (38) step 9 Rz w O ~ Rz O O
o step 10 N
0-t-Bu O
O HN O-t-Bu O Rl O
off (39) R1 (40) step 11 Rz O
N
NHz O-t-Bu O R, (3 in which A' is O-t-Bu and as the 4-(S)-isomer) In Reaction Scheme G, step 1, an appropriate compound of structure (30) is functionalized to give the corresponding compound of structure (31 ). An appropriate compound of structure (30) in one in which RZ is as desired in the final product of formula ( 1 ).
For example, an appropriate compound of formula (30) is treated initially with lithium chloride and a suitable non-nucleophilic base, such as collidine SUBSTITUTE SHEET (RULE 26) in a suitable solvent such as dimethylformamide. This is followed by treatment with a suitable mesylating agent, such as mesyl chloride. The reaction is typically carried out at a temperature range of from -30°C to room temperature, preferably 0°C and for a period of time ranging from 2-10 hours. The product is recovered from the reaction mixture by extractive methods as are known in the art and may be purified by chromatography.
In Reaction Scheme G, step 2, the methanesulfonate functionality of the compound of formula (31) is eliminated and the chloro substituted with iodo to a compound of formula (32).
For example, a compound of formula (31 ) is treated with a suitable non-nucleophilic base, such as potassium tert-butoxide in a suitable aprotic organic solvent, such as diethyl ether. The reaction is typically carried out at a temperature range of from -30°C to room temperature, preferably 0°C and for a period of time ranging from 15 minutes to hours to give the corresponding 1-chloromethyl-2-vinyl-benzene derivative which is recovered from the reaction mixture by extractive methods as are known in the art. The 1-chloromethyl-2-vinyl-benzene derivative is then treated with a suitable iodinating agent, such as sodium iodide, in a suitable solvent, such as acetone. The reaction is carried out at a temperature range of from room temperature to reflux temperature of the solvent and for a period of time ranging from 15 minutes to hours. The product is recovered from the reaction mixture by extractive methods as are known in the art.
In Reaction Scheme G, step 3, a compound of formula (32) is subjected to an addition, elimination reaction with 2-(bis-methylsulfonyl-methyleneamino)- 1-(10,10-dimethyl-3,3-dioxo-3-thia-4-aza-tricyclo( 5.2.1.0 1,5)dec-4-yl)-ethanone to give the corresponding compound of formula (33).
For example, the anion of 2-(bis-methylsulfonyl-methyleneamino)- 1-(10,10-dimethyl-3,3-dioxo-3-thia-4-aza-tricyclo( 5.2.1.0 1,5)dec-4-yl)-ethanone is formed by treating 2-(bis-methylsulfonyl-methyleneamino)-1-(10,10- dimethyl-3,3-dioxo-thia-4-aza-tricyclo(5.2.1.0 1,5)dec-4-yl)-ethanone with a suitable non-nucleophilic base, such as n-butyllithium in a suitable aprotic organic solvent, such as SUBSTITUTE SHEET (RULE 26) WO 00/40564 PCT/iJS99/28339 tetrahydrofuran. The reaction is carried out at a temperature range of from -78°C to -30°C, preferable -78°C and for a period of time ranging from 30 minutes to 2 hours.
A compound of formula (32) is then added and the reaction is carried out at a temperature range of from -78°C to room temperature for a period of time ranging from 1-24 hours. The product is recovered from the reaction mixture by extractive methods as are known in the art and may be purified by chromatography.
In Reaction Scheme G, step 4, a compound of formula (33) is hydrolyzed to give a compound of formula (34).
For example, a compound of formula (33) is treated with a suitable acid such as aqueous hydrochloric acid in a suitable organic solvent such as tetrahydrofuran.
The reaction is carried out at a temperature range of from -10°C to room temperature and for a period of time ranging from 30 minutes to 20 hours. Evaporation of the solvent followed by treatment with inorganic base such as aqueous lithium hydroxide in a suitable organic solvent, such as tetrahydrofuran. The reaction is carned out at a temperature range of from -10°C to room temperature and for a period of time ranging from 30 minutes to 10 hours. After acidification, the corresponding 2-amino-3-(2-vinyl-phenyl)-propionic acid derivative of formula (34) was isolated by evaporation of solvents.
In Reaction Scheme G, step 5, the amino functionality of a compound of formula (34) is protected to give the corresponding compound of formula (35).
For example, a compound of formula (34) is treated with an appropriate phthalimide protecting agent, such as N-carbethoxyphthalimide in the presence of a suitable non-nucleophilic base, such as aqueous sodium carbonate. The reaction is carried out at a temperature range of from -10°C to room temperature and for a period of time ranging from 1 - 10 hours. The product is recovered from the reaction zone by extractive methods as are known in the art and may be purified by chromatography.
In Reaction Scheme G, step 6, the carboxylic acid functionality of a compound of formula (35) is esterified to give a compound of formula (36).
SUBSTITUTE SHEET (RULE 26) For example, a compound of formula (35) is treated with 2-(trimethylsilyl)ethanol in the presence of a suitable non-nucleophilic base, such as pyridine, in a suitable organic solvent, such as tetrahydrofuran. The reaction is carried out at a temperature range of from -30°C to room temperature and for a period of time ranging from 30 minutes to 2 hours. A coupling agent, such as 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) is then added and the reaction carried out at a temperature range of from -30°C to room temperature for a period of time ranging from 10 - 48 hours. The product is recovered from the reaction zone by extractive methods as are known in the art and may be purified by chromatography.
In Reaction Scheme G, step 7, the vinyl functionality of a compound of formula (36) is oxidized to give a compound of formula (37).
For example, a compound of formula (36) is treated with ozone in a suitable organic solvent such as methylene chloride and methanol. The reaction is carried out at a temperature range of from -78°C to -50°C and for a period of time necessary for a blue color to persist. After purging the reaction with nitrogen and quenching by methods known in the art, such as addition of dimethylsulfide and pyridine, the product is recovered from the reaction zone by extractive methods as are known in the art and may be purified by chromatography.
In Reaction Scheme G, step 8, a compound of formula (37) is subjected to reductive amination with an appropriate amino acid, tert-butyl ester derivative to give a compound of formula (39). In Reaction Scheme G, step 8, an appropriate amino acid, tert-butyl ester derivative is one in which R~ is as desired in the final product of formula (1) or gives rise to R~ as is desired in the final product of formula (1). In addition, an appropriate amino acid, tert-butyl ester derivative is one in which the stereochemistry is as desired in the final product of formula (1). As is appreciated by those skilled in the art, the use of amino acid, tert-butyl ester derivatives give rise to compounds of formula (3) in which A' is -O-tert-butyl. It is also understood that appropriate amino acid derivatives can have a variety of carboxy substituents give rise to compounds of formula (3) in which A' is other than -O-tert-butyl.
SUBSTITUTE SHEET (RULE 26) For example, a compound of formula (37) is treated with an appropriate amino acid, tert-butyl ester derivative in an appropriate polar organic solvent, such as methanol under dehydrating conditions, such as molecular sieves. The reaction is carried out at a temperature ranged of from -10°C to reflux temperature of the solvent, preferably room temperature, and for a period of time ranging from 30 minutes to 10 hours. A suitable reducing agent, such as sodium cyanoborohydride, is then added and the reaction is carried out at a temperature range of from -10° to reflux temperature of the solvent, preferably room temperature, and for a period of time ranging from 30 minutes to 24 hours. The product is recovered from the reaction zone by extractive methods as are known in art and may be purified by chromatography. As one skill in the art would realize, those amino acid, tert-butyl ester derivatives wherein R~ has a reactive functionality, the reactive functionality may be protected prior to the reductive amination reaction of step 8. The selection and utilization of suitable protecting groups is well known by one of ordinary skill in the art and is described in Protective Groups in Or anic Synthesis, Theodora W.
Greene, (Whey 1981 ).
In Reaction Scheme G, step 9, the ester functionality of a compound of formula (39) is hydrolyzed to give a compound of formula (40).
For example, a compound of formula (39) is treated with an appropriate fluoride reagent, such as tetrabutylammonium fluoride in a suitable organic solvent, such as tetrahydrofuran. The reaction is carried out at a temperature range of from -10°C to room temperature and for a period of time ranging from 30 minutes to hours.
The product is recovered from the reaction zone by extractive methods as are known in art and may be purified by chromatography.
In Reaction Scheme G, step 10, a compound of formula (39) is subjected to a ring closure amination reaction to give a compound of formula (40).
For example, a compound of formula (40) is treated with a suitable activating agent, such as isobutylchloroformate, in the presence of a suitable non-nucleophilic base, such as N-methylmorpholine in a suitable organic solvent, such as tetrahydrofuran. The reaction is carried out at a temperature range of from -10°C to SUBSTITUTE SHEET (RULE 26) reflux temperature of the solvent and for a period of time ranging from 30 minutes to hours. The product is recovered from the reaction zone by evaporation and may be purified by chromatography.
5 In Reaction Scheme G, step 1 l, the protecting group of a compound of formula (40) is removed to give a compound of formula (3) in which A' is -O-tert-butyl as the 4-position (S)-isomer.
For example, the phthalimide protecting groups of a compound of formula 10 (40) can be removed using hydrazine monohydrate in a suitable protic solvent such as methanol. The reaction is carried out at a temperature range of from -10°C to room temperature and for a period of time ranging from 2 hours to 4 days. The product is recovered from the reaction zone by filtration and evaporation.
The following examples present typical syntheses as described in the Reaction Schemes above. These examples and preparations are understood to be illustrative only and are not intended to limit the scope of the invention in any way.

Synthesis of 2-(4-amino-3-oxo-1,3,4,5-tetrahydro-benzofc~azepin-2-yl)-4-methyl-valeric acid, tert-butyl ester Dissolve homophthalic acid (22.2 g, 0.123 mmol) in tetrahydrofuran (250 mL) and add dropwise at room temperature to a slurry of lithium aluminum hydride (15.5 g, 0.407 mol) in tetrahydrofuran (500 mL). Heat at reflux for 18 hours, cool in an ice bath and carefully add, by dropwise addition, water (16 mL), followed by 50% sodium hydroxide (16 mL). Remove the ice bath, add water slowly with stirring and stir until the gray precipitate turns white and evolution of gas ceases.
Filter, wash solids with methylene chloride, dry (MgS04 ) and evaporate the solvent in vacuo to give 2-(2- hydroxymethyl-phenyl)-ethanol as a viscous oil ( 18.4 g, 98%).
Mix 2-(2-hydroxymethyl-phenyl)-ethanol ( 12.0 g, 78.8 mmol) and collidine (23 mL, 0.17mo1) and treat with lithium chloride (7.35 g, 0.173 mmol) in dimethylformamide (125 mL). Cool in an ice bath and treat, by dropwise addition, with mesyl chloride ( 13.4 mL). Stir at 0°C for 4 hours, partition between ice water (300 mL) and a 1:1 mixture of ether:pentane (2X400 mL). Wash the organic layer SUBSTITUTE SHEET (RULE 26) with a saturated solution of CuS04 (2X200 mL), dry (MgS04 ) and purify by silica gel chromatography (2.5:1 hexane/ethyl acetate followed by 2:1 hexane/ethyl acetate followed by 3:2 hexane/ethyl acetate) to give methanesulfonic acid 2-(2-chloromethyl-phenyl)-ethyl ester as a pale yellow oil (8.8 g, 45%).
Dissolve methanesulfonic acid 2-(2-chloromethyl-phenyl)-ethyl ester (8.8 g, 35.4 mmol) in ether (80 mL) and cool to -35° C. Add potassium t-butoxide (10 g, 89 mmol) and stir for 30 minutes. Add water (50 mL) and ether (150 mL), extract, dry (NaZS04) and purify by silica gel chromatography (2:3 methylene chloride/pentane) to give 1-chloromethyl-2-vinyl-benzene as a colorless oil (4.43 g, 82%).
Dissolve 1-chloromethyl-2-vinyl-benzene (4.0 g, 26 mmol) in acetone (100 mL) and add sodium iodide (4.5 g, 30 mmol). Heat at gentle reflux for 30 minutes.
Cool, add water (150 mL) and extract with pentane (200 mL). Dry (MgS04 ) and evaporate the solvent in vacuo to give 1-iodomethyl-2-vinyl-benzene (95%).
Dissolve 2-(bis-methylsulfanyl-methyleneamino)-1-(10,10- dimethyl-3,3-dioxo-3-thia-4-aza-tricyclo(5.2.1.0 1,5)dec-4-yl)-ethanone (7.91 g, 21.0 mmol) in tetrahydrofuran (100 mL) and cool to -78°C. Treat, by dropwise addition, with 1.6 M n-butyllithium in hexane (13.1 mL, 21 mmol). Stir for 1.5 hours, then add hexamethylphosphotriamide (HMPA) (4.25 mL, 24.4 mmol). Stir for 15 minutes and add, via cannula, a solution of 1-iodomethyl-2-vinyl-benzene (6.1 g, 25 mmol) in tetrahydrofuran (100 mL). Stir overnight at room temperature, partition between saturated ammonium chloride (2X75 mL) and ethyl acetate (400 mL). Dry (Na2S04) and purify by silica gel chromatography (2.5:1 hexane/ethyl acetate) to give 2-(bis-methylsulfanyl-methyleneamino)-1-(10,10-dimethyl- 3,3-dioxo-3-thia-4-aza-tricyclo(5.2.1.0 1,5)dec-4-yl)- 3-(2-vinyl-phenyl)-propan-1-one as a white solid (4.Sg).
Dissolve 2-(bis-methylsulfanyl-methyleneamino)-1-(10,10- dimethyl-3,3-dioxo-3-thia-4-aza-tricyclo(5.2.1.0 1,5)dec-4-yl)-3-(2-vinyl-phenyl)-propan-1-one (5.21 g, 10.6 mmol) in tetrahydrofuran (100 mL) and ) and 0.75 M hydrochloric acid (100 mL). Stir at room temperature for 24 hours, evaporate the solvent in vacuo to give the hydrochloride salt as a white solid. Dissolve in tetrahydrofuran (200 mL) and water (SOm), add lithium hydroxide monohydrate (1.9 g, 4.5 mmol) and stir at room temperature under a nitrogen atmosphere for 4 hours. Extract into methylene chloride (200 mL) and wash with 2N sodium hydroxide (50 mL). Acidify to pH 2-3 SUBSTITUTE SHEET (RULE 26) while cooling in an ice bath and concentrate in vacuo to give 2-Amino-3-(2-vinyl-phenyl)-propionic acid as an off white solid (3.40 g, 100%).
Dissolve 2-amino-3-(2-vinyl-phenyl)-propionic acid (3.40g) in water (75 mL) and add sodium carbonate (1.97 g, 18.6 mmol) and N-carbethoxyphthalimide (2.81 g, 12.8 mmol). Stir for 2.5 hours, wash with methylene chloride (200 mL), acidify to pH 1 with cold concentrated hydrochloric acid and extract with ethyl acetate (3X200 mL), dry (Na~S04), evaporate the solvent in vacuo and purify by silica gel chromatography (1:1:0.02 hexane/ethyl acetate/acetic acid) followed by recrystallization (isopropanol) to give 2-(1,3-dioxo-1,3-dihydro-isoindol-2-yl)-3-(2-vinyl-phenyl)- propionic acid as a pale yellow solid (2.47g).
Dissolve 2-(1,3-dioxo-1,3-dihydro-isoindol-2-yl)-3-(2-vinyl-phenyl)-propionic acid (2.47 g, 7.69 mmol) in tetrahydrofuran (35 mL) and cool in an ice bath. Treat with pyridine (1.6 mL, 20 mmol) and 2-(trimethylsilyl)ethanol (2.3 mL, 16 mmol). Stir for 30 minutes and add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) (2.21 g, 11.5 mmol). Stir for 22 hours at 5°C, then at room temperature for 1.5 hours. Cool to 0°C, add 0.6 times all reagents and stir at room temperature overnight. Dilute with ethyl acetate (150 mL), wash with 5% sulfuric acid (40 mL) and saturated sodium hydrogen carbonate (40 mL).
Back extract with methylene chloride (100 mL), wash with brine (30 mL) and dry (Na2S04). Evaporate the solvent in vacuo and purify by silica gel chromatography (2:1 hexane/ethyl acetate) to give 2-(1,3-dioxo-1,3-dihydro-isoindol-2-yl)-3-(2-vinyl-phenyl)- propionic acid, 2-trimethylsilanyl-ethyl ester (2.61 g, 81 %).
Dissolve 2-(1,3-dioxo-1,3-dihydro-isoindol-2-yl)-3-(2-vinyl-phenyl)-propionic acid, 2-trimethylsilanyl-ethyl ester (2.61 g, 6.19 mmol) in methylene chloride (70 mL) and methanol (75 mL). Cool to -78°C and treat with ozone until a blue color persists. Purge with nitrogen and add dimethylsulfide (7 mL) and pyridine (0.35 mL). Allow to warm to room temperature gradually overnight. Partition between methylene chloride ( 100 mL) and water (40 mL). Extract the aqueous with methylene chloride (50 mL), dry (Na2S04) and purify by silica gel chromatography (2.5:1 hexane/ethyl acetate) to give the title compound as a colorless viscous oil (2.65 g, 100%). Step h: 2-(2-(2-(1,3-Dioxo-1,3,dihydro-isoindol-2-yl)-2-(2-trimethylsilanyl- ethoxycarbonyl)-ethyl)-benzylamino)-4-methyl- valeric acid, tert-butyl ester SUBSTITUTE SHEET (RULE 26) Dissolve 2-(1,3-dioxo-1,3-dihydro-isoindol-2-yl)-3-(2- formyl-phenyl)-propionic acid, 2-trimethylsilanyl-ethyl ester (250 mg, 0.590 mmol) in methanol (15 mL) and treat with L-leucine tert-butyl ester hydrochloride (0.66 g, 3.0 mmol). Stir at room temperature for 2 hours with 3A molecular sieves, add sodium cyanoborohydride (0.6 mL of a 1.OM solution in tetrahydrofuran, 0.6 mmol), stir for 0.5 hours, add additional sodium cyanoborohydride (0.3 mL) and stir for 5 hours.
Filter through filter aid, evaporate the solvent in vacuo and partition the residue between methylene chloride ( 100 mL) and saturated sodium hydrogen carbonate (40 mL). Dry (Na2S04), evaporate the solvent in vacuo and purify by silica gel chromatography (5:1 hexane/ethyl acetate followed by 3:1 hexane/ethyl acetate) to give 2-(1,3-dioxo-1,3-dihydro-isoindol-2-yl)-3-(2-formyl- phenyl)-propionic acid, 2-trimethylsilanyl-ethyl ester (221 mg, 63%).
Dissolve 2-(2-(2-(1,3-dioxo-1,3,dihydro-isoindol-2-yl)-2-(2- trimethylsilanyl-ethoxycarbonyl)-ethyl)-benzylamino)-4- methyl-valeric acid, tert-butyl ester (221 mg, 0.372 mmol) in tetrahydrofuran (5 mL) and treat with tetrabutylammonium fluoride (0.43 mL of a 1.OM solution in tetrahydrofuran, 0.43 mmol). Stir for 1.5 hours, evaporate the solvent in vacuo and dissolve the residue in ethyl acetate (75 mL). Wash with 1N hydrochloric acid (25 mL) and brine (25 mL).
Dry (Na2S04) and evaporate the solvent in vacuo to 2-(2-(2-carboxy-2-(1,3-dioxo-1,3,dihydro-isoindol-2-yl)-ethyl)-benzylamino)-4-methyl-valeric acid, tert-butyl ester as a white solid (188mg).
Dissolve 2-(2-(2-carboxy-2-(1,3-dioxo-1,3,dihydro-isoindol-2-yl)-ethyl)-benzylamino)-4-methyl-valeric acid, tert-butyl ester (188mg) in tetrahydrofuran (10 mL) and cool in an ice bath. Add sequentially, N-methylmorpholine (86pL, 0.78 mmol), and isobutylchloroformate (55pL, 0.43 mmol). Stir for 2 hours, filter, wash salts with dry tetrahydrofuran, evaporate the solvent in vacuo and purify by silica gel chromatography (l :l hexane/ethyl acetate) to give 2-(4-((1,3-dioxo-1,3,dihydro-isoindol-2-yl)-3-oxo-1,3,4,5- tetrahydro-benzo[c]azepin-2-yl)-4-methyl-valeric acid, tert-butyl ester as a white solid (64 mg, 93%).
Dissolve 2-(4-((1,3-dioxo-1,3,dihydro-isoindol-2-yl)-3-oxo-1,3,4,5- tetrahydro-benzo[c]azepin-2-yl)-4-methyl-valeric acid, tert-butyl ester (160 mg, 0.336 mmol) in methanol (3 mL) and treat with a solution of hydrazine monohydrate (0.40 mL, 0.40 mmol) in methanol. Stir at room temperature for 65 hours, filter through filter aid, wash SUBSTITUTE SHEET (RULE 26) with methylene chloride, filter through filter aid and dry (MgS04 ). Evaporate the solvent in vacuo to give the title compound (93 mg, 80.2% for this step).

2-(4-(2-Benzoylsulfanyl-3-phenyl-propionyl-amino)- 3-oxo-1,3,4,5-tetrah, benzo[clazepin-2-yl)-4-methyl- valeric acid / \
O ~ O
/ S~ N
NH OH
O O
1.1 Synthesis of 2-(4-(2-benzoylsulfanyl-3-phenyl-propionyl-amino)-3-oxo-1,3,4,5-tetrahydro-benzo[c]azepin-2-yl)-4-methyl-valeric acid, tert-butyl ester Dissolve 2-(4-amino-3-oxo-1,3,4,5-tetrahydro-benzo[c]azepin-2-yl)-4-methyl-valeric acid, tent-butyl ester (93 mg, 0.27 mmol) in methylene chloride (3 mL) and treat with (S)-3-phenyl-2-benzoylthiopropionic acid (115 mg, 0.40 mmol) and EEDQ (100 mg, 0.40 mmol). Stir at room temperature for 18 hours, evaporate the solvent in vacuo, dissolve the residue in ethyl acetate (40 mL) and wash with 5%
sulfuric acid (15 mL) when with saturated sodium hydrogen carbonate (15 mL).
Dry (NazS04), evaporate the solvent in vacuo and purify by silica gel chromatography (6:1 hexane/ethyl acetate followed by 2.5:1 hexane/ethyl acetate) to give the title compound as a colorless oil (141 mg, 85%).
1.2 Synthesis of 2-(4-(2-benzoylsulfanyl-3-phenyl-propionyl-amino)- 3-oxo-1 3 tetrahydro-benzo[clazepin-2-yl)-4-methyl- valeric acid Dissolve 2-(4-(2-benzoylsulfanyl-3-phenyl-propionyl-amino)-3-oxo-1,3,4,5-tetrahydro-benzo[c]azepin-2-yl)-4-methyl-valeric acid, tert-butyl ester (141 mg, 0.229 mmol) in methylene chloride (5 mL) and treat with anisole (0.12 mL, 1.15 mmol) then with trifluoroacetic acid (1.5M). Stir at room temperature for 15 hours, partition between ethyl acetate (25 mL) and brine ( 15 mL). Wash the organic layer with brine (15 mL), dry (NazS04) and purify by silica gel chromatography (1:1 SUBSTTTiJT'E SHEET (RULE 26) hexane/ethyl acetate followed by 1:1:0.01 hexane/ethyl acetate/acetic acid) to give the title compound as a white solid (157mg).
SUBSTITUTE SHEET (RULE 26) 2-(4-(2-Mercapto-3-phenyl-propionyl-amino)-3-oxo- 1 3 4,5-tetrahydro-benzof clazepin-2-yl)-4-methyl-valeric acid / \
o ~ o HS~ N
NH OH
O
2.1 Synthesis of 2-(4-(2-mercapto-3-phenyl-propionyl-amino)-3-oxo- 1 3 4 5-tetrahydro-benzo[c]azepin-2-yl)-4-methyl-valeric acid Dissolve 2-(4-(2-benzoylsulfanyl-3-phenyl-propionyl-amino)-3-oxo-1,3,4,5-tetrahydro-benzo[c]azepin-2-yl)-4-methyl-valeric acid (0.229 mmol) in degassed methanol (3 mL) and cool in an ice bath. Treat with degassed 1 M aqueous lithium hydroxide (1.0 mL) and stir, allowing the ice bath to warm gradually over 3 hours. With the reaction at 0°C, acidify with 5% hydrochloric acid.
Partition between methylene chloride (75 mL) and water (25 mL), dry (Na2SOa) and purify by silica gel chromatography (3:1:0.01 hexane/ethyl acetate/acetic acid followed by 1:1:0.01 hexane/ethyl acetate/acetic acid to give the title compound as a white solid (84 mg, 80.8%).

Synthesis of 2-bromo-6-phthalimidohexanoic acid Combine 6-aminohexanoic acid (6-aminocaproic acid) (8.0 g, 60 mmol) and water (100 mL). Add sodium carbonate (6.84 g, 64 mmol) and N-carbethoxyphthalimide (14.0 g, 64 mmol). After 1.5 hours, extract the reaction mixture with ethyl acetate (100 mL). Cool the aqueous layer in an ice bath and acidify using concentrated hydrochloric acid to give a solid. Collect the solid by filtration, rinse with water, and dry to give 6-phthalimidohexanoic acid (12.7 g, 80% yield).
Combine 6-phthalimidohexanoic acid (12.7 g, 48 mmol) and dry red phosphorous ( 1.95 g, 63 mmol). Cool in an ice bath and add dropwise bromine ( 12.7 mL, 246 mmol). Warm to room temperature and then heat to 80°C. After 3 hours, cool the reaction mixture to ambient temperature, pour into water (300 mL) containing SUBSTTTUTE SHEET (RULE 26) sodium bisulfate, and neutralize using solid sodium bicarbonate and extract with diethyl ether (about 150 mL). Acidify the aqueous layer with concentrated hydrochloric acid give a solid. Collect the solid by filtration and dry to give the title compound ( 15 g, 91.5% yield, 73.2% for both steps).

Synthesis of (R)-2-bromo-6-phthalimidohexanoic acid Combine (R)-2-N-carbobenzyloxy-6-aminohexanoic acid ((R)-Na-Cbz-lysine) (14.0 g, 50 mmol) and water (500 mL). Add sodium carbonate (5.65 g, 53 mmol) and N-carbethoxyphthalimide (13.15 g, 60 mmol). After 1.5 hours, acidify using concentrated hydrochloric acid to give a solid. Collect the solid by filtration, rinse with water, and dry to give (R)-2-N-carbobenzyloxy-6-phthalamidohexanoic acid.
Combine (R)-2-N-carbobenzyloxy-6-phthalamidohexanoic acid obtained above, methanol (200 mL), 10% palladium-on-carbon ( 1 g) and treat with hydrogen at atmospheric pressure. After 18 hours, filter, add to the filtrate a solution of hydrochloric acid in methanol (50 mL, 1 M, 50 mmol), and evaporate in vacuo to give (R)-2-amino-6-phthalamidohexanoic acid hydrochloric acid salt.
Combine (R)-2-amino-6-phthalamidohexanoic acid hydrochloric acid salt (12.5 g, 40 mmol) and a 2.5 M aqueous sulfuric acid solution (40 mL). Cool in a salt-ice bath.
Add 49% aqueous hydrobromic acid solution (13.2 g). Add dropwise over about 20 minutes, an aqueous solution of sodium nitrite (2.8 g, 40 mmol, in 20 mL of water).
After 3 hours, warm to ambient temperature. After 18 hours, collect the resultant solid by filtration, rinse with water and dry in vacuo to give a residue.
Chromatograph the residue on silica gel eluting with 1/1 ethyl acetate/dichloromethane containing 5%
acetic acid to give the title compound.
FXAMPT F '~
2-(4-(2-Mercapto-6-phthalimidohexanoyl-amino)-3-oxo-1,3,4, 5-tetrahydro-benzo(c)azepin-2-~)-4-methyl-valeric acid SUBSTTTUTE SHEET (RULE 26) / \

i /
N N
NH OH
O SH O
3.1 Synthesis of2-(4-(2-bromo-6-phthalimidohexanoyl-amino)-3-oxo-1,3 4 5-tetrahydro-benzo[c]azepin-2-yl)-4-methyl-valeric acid t-butyl ester Combine 2-(4-amino-3-oxo-1,3,4,5-tetrahydro-benzo[c]azepin-2-yl)-4-methyl-valeric acid, tent-butyl ester (0.34 mmol), 2-bromo-6-phthalimidohexanoic acid (0.189 g, 0.56 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (0.106 g, 0.55 mmol), and 1-hydroxybenztriazole hydrate ((75 mg, 0.56 mmol) in dicloromethane (8 mL). After 17 hours, evapoarte in vacuo to give a residue, partition the residue between ethyl aceate and an aqueous 5% sulfuric acid solution (about 20 mL). Separate the layers, extract the organic layer with a saturate aqueiosu sodium bicarbonate solution and then brine, dry over NazSOa, filter, and evaporate in vacuo to give the title compound.
3.2 Synthesis of 2-(4-(2-(p-methoxvbenzylthio)-6-bhthalimidohexanovl-aminol-3-oxo-1,3,4,5-tetrahydro-benzo[c]azepin-2-yl)-4-methyl-valeric acid t-butyl ester Combine p-methoxybenzylmercaptan (0.08 mL, 0.57 mmol) and sodium hydride (17 mg, 60% oil dispersion, 0.42 mmol) in degassed dimethylformamide (3 mL).
After 1 hour, tetra-n-butylammonium iodide (about 5 mg) and 2-(4-(2-bromo-6-phthalimidohexanoyl-amino)-3-oxo-1,3,4,5-tetrahydro-benzo[c]azepin-2-yl)-4-methyl-valeric acid t-butyl ester (0.27 mmol). After 20 hours, quench be the addition of a saturated aqueous ammonium chloride solution and dilute with water (about 5 mL).
Extract with ethyl acetate (about 75 mL). Separate the layers and extract the orgainc layer with brine, dry over Na2S0~, filter, and evaporate in vacuo to give the title compound.
3.3 Synthesis of 2-~4-(2-mercapto-6-phthalimidohexanoyl-amino 3-oxo-1,3,4,5-tetrahydro-benzoL~azepin-2-yl)-4-methyl-valeric acid SUBSTITUTE SHEET (RULE 26) Combine 2-(4-(2-(p-methoxybenzylthio)-6-phthalimidohexanoyl-amino)-3-oxo-1,3,4,5-tetrahydro-benzo[c]azepin-2-yl)-4-methyl-valeric acid t-butyl ester (0.20 mmol), mercury (II) acetate (0.084 g, 0.26 mmol), and anisole (0.23 mL, 2.1 mmol) in dichloromethane (6.6 mL). Cool in an ice bath and degas by repeatedly cycles of vacuum and filling the vessel with nitrogen gas. Add trifluoroacetic acid (2.5 mL).
After 1 hour, warm to ambient temperature. After 3 hours, purge with hydrogen sulfide (gas) for about 10 minutes. Filter and evaporate in vacuo to give a residue.
Repeatedly, combine the residue and carbon tetrachloride and evaporate in vacuo to give the title compound.

Synthesis of (L)-phenylalanine-N-methyl amide trifluoroacetic acid salt Combine t-Boc-(L)-phenylalanine (8.00 g, 30.2 mmol) and tetrahydrofuran (20 mL). Cool to about -30°C and add sequentially N-methylmorpholine (3.5 mL, 32 mmol) and then isobutyl chloroformate (4.5 mL, 35 mmol). After 10 minutes, add 40%
aqueous methylamine (13 mL, 380 mmol). After 2 hours, concentrate the reaction mixture in vacuo, combine the evaporated reaction mixture and dichloromethane (125 mL) and extract with an aqueous 1M hydrochloric acid solution and then a saturated aqueous sodium bicarbonate solution. Dry the organic layer over NazS04, filter, and evaporate in vacuo to give t-Boc-(L)-phenylalanine-N-methyl amide, which is used without further purification.
Combine t-Boc-(L)-phenylalanine-N-methyl amide (8.4 g, 30 mmol), methylene chloride (100 mL), and trifluoroacetic acid (20 mL). After 3 hours, evaporate in vacuo to give a residue. Repeatedly, combine the residue and carbon tetrachloride and toluene remove residual trifluoroacetic acid by coevaporation and evaporate in vacuo to give a residue. Triturate the residue with diethyl ether to yield the title compound as a solid (9.12 g, 100%).

Synthesis of 2-(4-amino-3-oxo-1,3,4,5-tetrahydro-benzo[c]azepin-2- 1~)-3-phenyl-pro~ionic acid, N-methyl amide Prepare by the method of Preparation 1 using (L)-phenylalanine-N-methyl amide trifluoroacetic acid salt to give the title compound.
SUBST>rTUTE SHEET (RULE 26) 2-(4-(2-Mercapto-6-phthalimidohexanoyl-amino)-3-oxo-1,3,4,5-tetrah, benzo(c]azepin-2-yl)- 3-phen ~~1-propionic acid, N-methyl amide / \

i /
N N

O SH O
4.1 Synthesis of 2-(4-(2-bromo-6-phthalimidohexanoyl-amino)-3-oxo-1,3,4,5-tetrahydro-benzo[c]azepin-2-yl)- 3-phenyl-propionic acid, N-methyl amide Prepare by the method of Example 3.1 using 2-(4-amino-3-oxo-1,3,4,5-tetrahydro-benzo[c]azepin-2-yl)-3-phenyl-propionic acid, N-methyl amide and 2-bromo-6-phthalimidohexanoic acid to give the title compound.
4.2 Synthesis of 2-(4-(2-(p-methoxybenzylthio)-6-phthalimidohexanoyl-amino)-3-oxo-1,3,4,5-tetrahydro-benzo[c]azepin-2-yl)- 3-phen ~~1-propionic acid, N-methyl amide Prepare by the method of Example 3.2 using 2-(4-(2-bromo-6-phthalimidohexanoyl-amino)-3-oxo-1,3,4,5-tetrahydro-benzo [c] azepin-2-yl)-3-phenyl-propionic acid, N-methyl amide and p-methoxybenzylmercaptan to give the title compound.
4.3 Synthesis of 2-(4-(2-mercapto-6-phthalimidohexanoyl-amino)-3-oxo-1,3,4,5-tetrahydro-benzoic]azepin-2-yl)- 3-phen ~~l-propionic acid, N-methyl amide Prepare by the method of Example 3.3 using 2-(4-(2-(p-methoxybenzylthio)-6-phthalimidohexanoyl-amino)-3-oxo-1,3,4,5-tetrahydro-benzo[c]azepin-2-yl)-3-phenyl-propionic acid, N-methyl amide, mercury (II) acetate, anisole, and trifluoroacetic acid to give the title compound.
The present invention provides a method of inhibiting matrix metalloproteinase (MMP) to a patient in need thereof comprising administering to the patient an effective matrix metalloproteinase inhibiting amount of a compound of formula ( 1 ).
SUBSTITUTE SHEET (RULE 26) As used herein, the term "patient" refers to warm-blooded animals or mammals, including guinea pigs, dogs, cats, rats, mice, hamsters, rabbits and primates, including humans. A patient is in need of treatment to inhibit MMP when it would be beneficial to the patient to reduce the physiological effect of active MMP. For example, a patient is in need of treatment to inhibit MMP when a patient is suffering from a disease state characterized by excessive tissue disruption or tissue degradation, such as, but not limited to, a neoplastic disease state or cancer; rheumatoid arthritis;
osteoarthritis;
cardiovascular disorders, such as atherosclerosis; corneal ulceration; dental diseases, such as gingivitis or periodontal disease; and neurological disorders, such as multiple sclerosis; chronic inflammatory disorders, such as emphysema and especially smoking-induced emphysema.
The identification of those patients who are in need of treatment to inhibit MMP
is well within the ability and knowledge of one skilled in the art. A
clinician skilled in the art can readily identify, by the use of clinical tests, physical examination and medical/family history, those patients who are suffering from disease states characterized by excessive tissue disruption or tissue degradation.
An "effective matrix metalloproteinase inhibiting amount" of a compound of formula (1) is an amount which is effective, upon single or multiple dose administration to the patient, in providing relief of symptoms associated with MMP and is thus effective in inhibiting MMP-induced tissue disruption and/or MMP-induced tissue degradation. As used herein, "relief of symptoms" of MMP-mediated conditions refers to decrease in severity over that expected in the absence of treatment and does not necessarily indicate a total elimination or cure of the disease. Relief of symptoms is also intended to include prophylaxis.
An effective matrix metalloproteinase inhibiting dose can be readily determined by the use of conventional techniques and by observing results obtained under analogous circumstances. In determining the effective dose, a number of factors are considered including, but not limited to: the species of the patient; its size, age, and general health; the specific disease involved; the degree of involvement or the severity of the disease; the response of the individual patient; the particular compound administered; the mode of administration; the bioavailability characteristics of the SUBSTITUTE SHEET (RULE 26) WO 00/40564 PCT/iJS99/28339 preparation administered; the dose regimen selected; and the use of concomitant medication.
An effective matrix metalloproteinase inhibiting amount of a compound of formula (1) will generally vary from about 0.1 milligram per kilogram of body weight per day (mg/kg/day) to about 300 milligrams per kilogram of body weight per day (mg/kg/day). A daily dose of from about 1 mg/kg to about 100 mg/kg is preferred.
A neoplastic disease state refers to an abnormal state or condition characterized by rapidly proliferating cell growth or neoplasm. Neoplastic disease states for which treatment with a compound of formula (1) will be particularly useful include:
Leukemias, such as, but not limited to, acute lymphoblastic, chronic lymphocytic, acute myeloblastic and chronic myelocytic; Carcinomas and adenocarcinomas, such as, but not limited to, those of the cervix, oesophagus, stomach, small intestines, colon, lungs (both small and large cell), breast and prostate; Sarcomas, such as, but not limited to, oesteroma, osteosarcoma, lipoma, liposarcoma, hemangioma and hemangiosarcoma;
Melanomas, including amelanotic and melanotic; and mixed types of neoplasias such as, but not limited to carcinosarcoma, lymphoid tissue type, follicullar reticulum, cell sarcoma and Hodgkin's Disease. Neoplastic disease states for which treatment with a compound of formula ( 1 ) will be particularly preferred include carcinomas and adenocarcinomas, particularly of the breast, prostate and lung.
Atherosclerosis is a disease state characterized by the development and growth of atherosclerotic lesions or plaque. The identification of those patients who are in need of treatment for atherosclerosis is well within the ability and knowledge of one of ordinary skill in the art. For example, individuals who are either suffering from clinically significant atherosclerosis or who are at risk of developing clinically significant atherosclerosis are patients in need of treatment for atherosclerosis. A
clinician of ordinary skill in the art can readily determine, by the use of clinical tests, physical examination and medical/family history, if an individual is a patient in need of treatment for atherosclerosis.
The term "chronic inflammatory disease" refers to diseases or conditions characterized by persistent inflammation in the absence of an identifiable irritant or SUBSTITUTE SHEET (RULE 26) microbial pathogen. Inflammatory diseases for which treatment with a compound of formula (1) will be particularly useful include: emphysema, chronic bronchitis, asthma, and chronic inflammation, and especially smoking-induced emphysema.
In effecting treatment of a patient, a compound of formula ( 1 ) can be administered in any form or mode which makes the compound bioavailable in effective amounts, including oral and parenteral routes. For example, the compound can be administered orally, subcutaneously, intramuscularly, intravenously, transdermally, topically, intranasally, rectally, inhalation, and the like. Oral and inhalation administration is generally preferred. One skilled in the art of preparing formulations can readily select the proper form and mode of administration depending upon the disease state to be treated, the stage of the disease, and other relevant circumstances.
Remington's Pharmaceutical Sciences, 18th Edition, Mack Publishing Co. (1990).
A compound of formula ( 1 ) can be administered in the form of pharmaceutical compositions or medicaments which are made by combining a compound of formula ( 1 ) with pharmaceutically acceptable earners or excipients, the proportion and nature of which are determined by the chosen route of administration, and standard pharmaceutical practice.
The pharmaceutical compositions or medicaments are prepared in a manner well known in the pharmaceutical art. The carrier or excipient may be a solid, semi-solid, or liquid material which can serve as a vehicle or medium for the active ingredient.
Suitable carriers or excipients are well known in the art. The pharmaceutical composition may be adapted for oral or parenteral use and may be administered to the patient in the form of tablets, capsules, suppositories, solution, suspensions, gels, ointments, aerosol or the like.
The pharmaceutical compositions may be administered orally, for example, with an inert diluent or with an edible carrier. They may be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, a compound of formula ( 1 ) may be incorporated with excipients and used in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, chewing gums and the like.
These preparations should contain at least 4% of a compound of formula ( 1 ), the active SUBSTTTUTE SHEET (RULE 26) ingredient, but may be varied depending upon the particular form and may conveniently be between 4% to about 70% of the weight of the unit. The amount of the active ingredient present in compositions is such that a unit dosage form suitable for administration will be obtained.
The tablets, pills, capsules, troches and the like may also contain one or more of the following adjuvants: binders such as microcrystalline cellulose, gum tragacanth or gelatin; excipients such as starch or lactose, disintegrating agents such as alginic acid, Primogel, corn starch and the like; lubricants such as magnesium stearate or Sterotex;
glidants such as colloidal silicon dioxide; and sweetening agents such as sucrose or saccharin may be added or a flavoring agent such as peppermint, methyl salicylate or orange flavoring. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier such as polyethylene glycol or a fatty oil.
Other dosage unit forms may contain other various materials which modify the physical form of the dosage unit, for example, as coatings. Thus, tablets or pills may be coated with sugar, shellac, or other enteric coating agents. A syrup may contain, in addition to the present compounds, sucrose as a sweetening agent and certain preservatives, dyes and colorings and flavors. Materials used in preparing these various compositions should be pharmaceutically pure and non-toxic in the amounts used.
For the purpose of parenteral therapeutic administration, the compounds of the present invention may be incorporated into a solution or suspension. These preparations should contain at least 0.1 % of a compound of the invention, but may be varied to be between 0.1% and about 50% of the weight thereof. The amount of the active ingredient present in such compositions is such that a suitable dosage will be obtained.
Preferred compositions and preparations are able to be determined by one skilled in the art.
The solutions or suspensions may also include one or more of the following adjuvants: sterile diluents such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents;
antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylene diaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of SUBSTITUTE SHEET (RULE 26) toxicity such as sodium chloride or dextrose. The parenteral preparation can be enclosed in ampules, disposable syringes or multiple dose vials made of glass or plastic.
The compounds of the present invention may also be administered by inhalation, such as by aerosol or dry powder. Delivery may be by a liquef ed or compressed gas or a suitable pump system which dispenses the compounds of the present invention or a formulation thereof. Formulations for administration by inhalation of compounds of formula (1) may be delivered in single phase, bi-phasic, or tri-phasic systems. A variety of systems are available for the administration by aerosol of the compounds of formula (1). Dry powder formulations are prepared by either pelletizing or milling the compound of formula (1 ) to a suitable particle size or by admixing the pelletized or milled compound of formula (1) with a suitable Garner material, such as lactose and the like. Delivery by inhalation includes the necessary container, activators, valves, subcontainers, and the like. Preferred aerosol and dry powder formulations for 1 S administration by inhalation can be determined by one skilled in the art.
The selective MMP-12 inhibitors of the present invention can be evaluated by the procedures that follow.
EXAMPLE A
Source and Activation of proMMP-1 ProMMP-1 (EC 3.4.24.7; interstitial collagenase) was purified from culture medium of human rheumatoid synovial fibroblasts stimulated with macrophage-conditioned medium according to Okada, Y. et al., J. Biol. Chem. 261, 14245-(1986). The active MMP-1 was obtained by treatment of proMMP-1 with trypsin (5 pg/mL) at 37°C for 30 minutes, followed by addition of soybean trypsin inhibitor (50 ~g/mL).
Determination of Inhibition Constant (K;) for MMP-1 The activated MMP-1 is assayed using a fluorogenic substrate, Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NHz, Knight, C.G. et al., FEBS Lett. 296, 263-266 (1992), at 37°C in 2.0 mL of assay buffer containing 50 mM Tris, pH 7.6, 0.2 M
sodium chloride, SO mM calcium chloride, and 0.02% Brij-35. The increase in fluorescence due to cleavage of Gly-Leu peptide bond by MMP-3 was monitored with Perkin-Elmer LSSOB
SUBSTITUTE SHEET (RULE 26) Fluorimeter (~.e~ 328 nm, 7~e", 393 nm, excitation slit 2.5, emission slit 10). Substrate and inhibitor stock solutions were made in DMF. For determination of K; values for MMP-1 inhibitors, a series of intermediate inhibitor solutions were prepared in DMF
and 1 or 2 ~L of the diluted inhibitor solution was mixed with 1 ~L of 2 mM
substrate solution in DMF in a quartz cuvette containing 2 mL of assay buffer. The enzyme (10 ~L of 0.2 ~M MMP-3 dilution in assay buffer) was added at the last to start the reaction.
For routine measurement of a K; value for a reversible, competitive inhibitor, the initial rates in the presence of at least four inhibitor concentrations (two concentrations above K; and two concentrations below K;) were measured using [S] = 1 pM (« Km) and [MMP-1 ] = 0.8 nM. Under these conditions, the measured K;, app is close to true K;.
Calculation of K; Values The K; for a competitive inhibitor is calculated using:
vo/v; _ (1 + [I]/K;, app) and K; = K;, app/(1 + [S]/Km), where vo is the initial rate in the absence of inhibitor, v; is the initial rate in the presence of inhibitor at the concentration of [I], [S] is the substrate concentration, and Km is the Michaelis constant.
If slow binding is observed (i.e. if the approach to the binding equilibrium is slow), the final steady-state rate rather than the initial rate is taken as v;.
FXAMPT F R
Source and Activation of nroMMP-2 Recombinant MMP-2 was purified from the fermentation broth of yeast Pichia pastoris that carries the integrated MMP-2 gene into its chromosome. In brief, the full-length cDNA for MMP-2 was obtained by reverse transcription of RNA from human melanoma A375M cell line by the reverse transcriptase polymerise chain reaction (RT-PCR) using sequence specific oligonucleotides. The nucleotide sequence was confirmed by Taq cycle sequencing. The cDNA was ligated into the Pichia pastoris expression vector pHIL-D2 in such a way that the expression of pro-MMP-2 is under the control of the methanol inducible alcohol oxidise promoter. The expression construct was digested with either SaII or NsiI and used to transform the Pichia pastoris strains KM71 and SMD1168. A large-scale culture of a selected clone designated was performed in a high cell density fermentor and the recombinant MMP-2 was purified from the culture supernatant by gelatin-sepharose 4B (Pharmacia). The enzyme SUBSTITUTE SHEET (RULE 26) WO 00/40564 PCT/iJS99/28339 is sufficiently pure at this stage for routine measurement of inhibition. If desired, however, the enzyme may be further purified by AcA 44 gel filtration (Spectra).
Determination of Inhibition Constant (K;) for MMP-2 The active MMP-2 was obtained by activation of proMMP-2 at 37°C
for 1 h with 4-aminophenylmercuric acetate which was then removed by a Sephadex G-50 spin column. The enzyme is assayed using a fluorogenic substrate, Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NHZ, at 37°C in 2.0 mL of assay buffer containing 50 mM
Tris, pH 7.6, 0.2 M sodium chloride, 50 mM calcium chloride, 0.02% Brij-35, and 50 pM (3-mercaptoethanol. The increase in fluorescence is monitored (~,eX 328 nm, 7~,en, 393 nm).
Substrate and inhibitor stock solutions are made in DMF. The enzyme is added at the last to start the reaction. For routine measurement of a K; value for a reversible, competitive inhibitor, the initial rates in the presence of at least four inhibitor concentrations (two inhibitor concentrations above K; and two below K;) are measured using [S] = 1 ~M («Km) and [MMP-2] = 0.4 nM. Under these conditions, the measured K;, app is close to true K;.
EXAMPLE C
Source and Activation of proMMP-3 ProMMP-3 (EC 3.4.24.17; Stromelysin-1 ) was purified from culture medium of human rheumatoid synovial fibroblasts stimulated with macrophage-conditioned medium according to Okada, Y. et al., J. Biol. Chem. 261, 14245-14255 (1986).
The active MMP-3 was obtained by treatment of proMMP-3 with trypsin (5 pg/mL) at 37°C
for 30 minutes, followed by addition of soybean trypsin inhibitor (50 ~g/mL).
Aliquots of the activated MMP-3 were stored at -20°C.
Determination of Inhibition Constant (K;) for MMP-3 The activated MMP-3 is assayed using a fluorogenic substrate, Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH~, Knight, C.G. et al., FEBS Lett. 296, 263-266 (1992), at 37°C in an assay buffer containing 50 mM Tris, pH 7.6, 0.2 M sodium chloride, 50 mM
calcium chloride, and 0.02% Brij-35. The increase in fluorescence due to cleavage of Gly-Leu peptide bond by MMP-3 was monitored with Perkin-Elmer LS50B
Fluorimeter (~eX 328 nm, a,em 393 nm, excitation slit 2.5, emission slit 10). Substrate and inhibitor stock solutions were made in DMF and 0.1 % HCl-DMF, respectively. For SUBSTITUTE SHEET (RULE 26) determination of K; values for MMP-3 inhibitors, a series of intermediate inhibitor solutions were prepared in 0.1 % HC1-DMF and 1 or 2 p,L of the diluted inhibitor solution was mixed with 1 ~L of 2 mM substrate solution in DMF in a quartz cuvette containing 2 mL of assay buffer. The enzyme (10 pL of 0.2 ~,M MMP-3 dilution in assay buffer) was added at the last to start the reaction. For routine measurement of a K;
value for a reversible, competitive inhibitor, the initial rates in the presence of at least four inhibitor concentrations (two concentrations above K; and two concentrations below K;) were measured using [S] = 1 ~M (« Km) and [MMP-3] = 1 nM. Under these conditions, the measured K;, app is close to true K;.
Calculation of K; Values The K; for a competitive inhibitor is calculated using:
vo/v; _ (1 + [I]/K;, app) and K; = K;, app/(1 + [S]/K",), where vo is the initial rate in the absence of inhibitor, v; is the initial rate in the presence of inhibitor at the concentration of [I], [S] is the substrate concentration, and K", is the Michaelis constant.
If slow binding is observed (i.e. if the approach to the binding equilibrium is slow), the final steady-state rate rather than the initial rate is taken as v;.
EXAMPLE D
Source of MMP-12 (macrophage metalloelastase) MMP-12 (EC 3.4.24.65) was cloned, expressed and purified according to Shapiro, S.D. et al., J Biol. Chem. 268, 23824-23829 (1993). Autoactivation resulted in the fully processed active form of the enzyme. Aliquots of MMP-12 were stored at -70C.
Determination of the inhibition constant (Ki) for MMP-12.
The potency of inhibitors of MMP-12 was measured using either quartz cuvettes or microtiter plates. The activity of MMP-12 was measured using a fluorogenic substrate, Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2, Knight, C.G. et al., FEBS
Lett.
296,263-266 (1992), at 25C in an assay buffer containing 50 mM Tris, pH 7.6, 0.2 M
sodium chloride, 50 mM calcium chloride, and 0.02% Brij-35. The increase in fluorescence due to cleavage of Gly-Leu peptide bond by MMP-12 was monitored with a Perkin-Elmer LSSOB Fluorimeter (7~ex 328 nm, 7~em 393 nm, excitation slit 2.5, emission slit 10) for the cuvette assay and with a Molecular Devices Fmax fluorescence plate reader (7~ex 320 nm, ~,7~em 405 nm) for the microtiter plate assay.
Substrate and SUBSTITUTE SHEET (RULE 26) inhibitor stock solutions were made in N,N,dimethylformamide (DMF) and 0.1%

DMF, respectively.
Ki values were determined using the cuvette method by preparing a series of intermediate inhibitors solutions in 0.1 % HC1-DMF and mixing the inhibitor with substrate (final concentration 2 qM) in a quartz cuvette containing 2 rnl of assay buffer.
MMP-12 was added to start the reaction at a concentration of 2 nM and progress curves were generated. For routine measurement of a Ki value for a reversible competitive inhibitor, the initial rates in the presence of at least four inhibitor concentrations ( two concentrations above and two concentrations below the Ki) were measured [S] =
2 qM
(«~) and [MMP-12] = 2 nM. Under these conditions, the measured Ki,app is close to the true Ki.
Ki values were determined using the microtiter plate method in a manner similar to that described for the cuvette method with some modifications. Four different inhibitor concentrations (50 ql in assay buffer)of each compound were added to separate wells of a microtiter plate and substrate was added (100 ql) to get a final concentration of 4 mM. MMP-12 was added to a final concentration of 2 nM (50 ~l) to start the reaction. Cleavage of substrate was recorded every 30 seconds for 30 minutes and progress curves were generated.
Calculation of Ki values The Ki for a competitive inhibitor was calculated using: Vo/Vi =
(1+[I]/Ki,app) and Ki = Ki,app/(1+[S]/Km), where Vo is the initial rate in the absence of inhibitor, Vi is the initial rate in the presence of inhibitor at the concentration of [I], [S] is the substrate concentration, and Km is the Michaelis constant. If slow binding is observed (i.e. if the approach to the binding equilibium is slow), the final steady-state rate rather than the initial rate is taken as Vi.
SUBSTITUTE SHEET (RULE 26)

Claims (32)

WHAT IS CLAIMED IS:

1. Use of an effective amount of matrix metalloproteinase inhibiting compound of the formula or a pharmaceutically acceptable salt thereof for the preparation of a pharmaceutical composition useful in the treatment of a patient in need thereof, wherein A is selected from the group consisting of -OH and -NRR';
wherein R and R' are independently selected from the group consisting of hydrogen and C6 alkyl or R and R' taken together with the nitrogen atom to which they are attached form a N-morpholino, N-piperidino, N-pyrrolidino, or N-isoindolyl;
R1 is selected from the group consisting of hydrogen, C1-C6 alkyl, -(CH2)n-CO2R5, -(CH2)a-C(O)NH2, -(CH2)4NH2, -(CH2)3-NH-C(NH)NH2, -(CH2)2-S(O)b-CH3, -CH2-OH, -CH(OH)CH3, -CH2-SH, -(CH2)d-Ar1, and -CH2-Ar2;
wherein a is 1 or 2;
b is 0, 1, or 2;
d is an integer from 0 to 4;
R5 is selected from the group consisting of hydrogen, C1-C4 alkyl, and benzyl;
Ar1 is a radical selected from the group consisting of <IMGs>
wherein R6 is from 1 to 2 substituents independently selected from the group consisting of hydrogen, halogen, C1-C4 alkyl, hydroxy, and C1-C4 alkoxy;

R7 is selected from the group consisting of hydrogen, halogen, C1-C4 alkyl, and C1-C4 alkoxy;
Ar2 is a radical selected from the group consisting of R2 is from 1 to 2 substituents independently selected from the group consisting of hydrogen, halogen, hydroxy C1-C4 alkyl, and C1-C4 alkoxy;
R3 is selected from the group consisting of C1-C6 alkyl, -(CH2)m-W, -(CH2)p-Ar3, -(CH2)k-CO2R9, -(CH2)m-NR8'SO2-Y1, and -(CH2)m-Z-Q
wherein m is an integer from 2 to 8;
p is an integer from 0-10;
k is an integer from 1 to 9;
W is phthalimido;
Ar3 is selected from the group consisting of wherein R23 is from 1 to 2 substituents independently selected from the group consisting of hydrogen, halogen, C1-C4 alkyl, and C1-C4 alkoxy;
R8' is hydrogen or C1-C6 alkyl;
R9 is hydrogen or C1-C6 alkyl;
Y1 is selected from the group consisting of hydrogen, -(CH2)j-Ar4, and -N(R24)2 wherein j is 0 or 1;

R24 each time selected is independently hydrogen or C1-C6 alkyl or are taken together with the nitrogen to which they are attached to form N-morpholino, N-piperidino, N-pyrrolidino, or N-isoindolyl;
Ar4 is wherein R25 is from 1 to 3 substituents independently selected from the group consisting of hydrogen, halogen, C1-C4 alkyl, and C1-C4 alkoxy;
Z is selected from the group consisting of -O-, -NR8-, -C(O)NR8-, -NR8C(O)-, -NR8C(O)NH-, -NR8C(O)O -, and -OC(O)NH-;
wherein R8 is hydrogen or C1-C6 alkyl;
Q is selected from the group consisting of hydrogen, -(CH2)n-Y2, and -(CH2)x Y3;
wherein n is an integer from 0 to 4;
Y2 is selected from the group consisting of hydrogen, -(CH2)h-Ar5 and -(CH2)r-C(O)OR27 wherein Ar5 is selected from the group consisting of wherein R26 is from 1 to 3 substituents independently selected from the group consisting of hydrogen, halogen, C1-C4 alkyl, and C1-C4 alkoxy;
h is an integer from 0 to 6;
t is an integer from 1 to 6;
R27 is hydrogen or C1-C6 alkyl;
x is an integer from 2 to 4;

Y3 is selected from the group consisting of -N(R28)2, N-morpholino, N-piperidino, N-pyrrolidino, and N-isoindolyl;
wherein R28 each time taken is independently selected from the group consisting of hydrogen and C1-C6 alkyl;
R4 is selected from the group consisting of hydrogen, -C(O)R10, -C(O)-(CH2)q-K
and -S-G
wherein R10 is selected from the group consisting of hydrogen, C1-C4 alkyl, phenyl, and benzyl;
q is 0, 1, or 2;
K is selected from the group consisting of wherein V is selected from the group consisting of a bond, -CH2-, -O-, -S(O)r-, NR21-, and -NC(O)R22;
wherein r is 0, 1, or 2;
R21 is selected from the group consisting of hydrogen, C1-C4 alkyl, and benzyl;
R22 is selected from the group consisting of hydrogen, -CF3, C1-C10 alkyl, phenyl, and benzyl;
R11 each time taken is independently selected from the group consisting of hydrogen, C1-C4 alkyl, and benzyl;
G is selected from the group consisting of wherein w is an integer from 1 to 3;
R12 is selected from the group consisting of hydrogen, C1-C6 alkyl, -CH2CH2S(O)u CH3, and benzyl;
wherein a is 0,1, or 2;
R13 is selected from the group consisting of hydrogen, hydroxy, amino, C1-C6 alkyl, N-methylamino, N,N-dimethylamino, -CO2R17, and -OC(O)R18;
wherein R17 is hydrogen, -CH2O-C(O)C(CH3)3, C1-4 alkyl, benzyl, or diphenylmethyl;
R17 is hydrogen, C1-C6 alkyl or phenyl;
R14 is 1 or 2 substituents independently selected from the group consisting of hydrogen, C1-C4 alkyl, C1-C4 alkoxy, or halogen;
V1 is selected from the group consisting of -O-, -S-, and -NH-;
V2 is selected from the group consisting of -N- and -CH-;
V3 is selected from the group consisting of a bond and -C(O)-;
V4 is selected from the group consisting of -O-, -S-, -NR19-, and -NC(O)R20-;
wherein R19 is hydrogen, C1-C4 alkyl, or benzyl;
R20 is hydrogen, -CF3, C1-C10 alkyl, or benzyl;

R15 is selected from the group consisting of hydrogen, C1-C6 alkyl and benzyl;
R16 is selected from the group consisting of hydrogen and C1-C4 alkyl;
and stereoisomers, pharmaceutically acceptable salt, and hydrate thereof.

2. Use of an effective amount of matrix metalloproteinase inhibiting compound of the formula or a pharmaceutically acceptable salt thereof for the preparation of a pharmaceutical composition useful in the treatment of a neoplastic disease state, wherein A is selected from the group consisting of -OH and -NRR';
wherein R and R' are independently selected from the group consisting of hydrogen and C6 alkyl or R and R' taken together with the nitrogen atom to which they are attached form a N-morpholino, N-piperidino, N-pyrrolidino, or N-isoindolyl;

R1 is selected from the group consisting of hydrogen, C1-C6 alkyl, -(CH2)a-CO2R5, -(CH2)a-C(O)NH2, -(CH2)4NH2, -(CH2)3-NH-C(NH)NH2, -(CH2)2-S(O)b-CH3, -CH2-OH, -CH(OH)CH3, -CH2-SH, -(CH2)d-Ar1, and -CH2-Ar2;
wherein a is 1 or 2;
b is 0, 1, or 2;
d is an integer from 0 to 4;
R5 is selected from the group consisting of hydrogen, C1-C4 alkyl, and benzyl;
Ar1 is a radical selected from the group consisting of wherein R6 is from 1 to 2 substituents independently selected from the group consisting of hydrogen, halogen, C1-C4 alkyl, hydroxy, and C1-C4 alkoxy;
R7 is selected from the group consisting of hydrogen, halogen, C1-C4 alkyl, and C1-C4 alkoxy;
Ar2 is a radical selected from the group consisting of R2 is from 1 to 2 substituents independently selected from the group consisting of hydrogen, halogen, hydroxy C1-C4 alkyl, and C1-C4 alkoxy;

R3 is selected from the group consisting of C1-C6 alkyl, -(CH2)m-W, -(CH2)p-Ar3, -(CH2)k-CO2R9, -(CH2)m-NR8'SO2-Y1, and -(CH2)m-Z-Q
wherein m is an integer from 2 to 8;
p is an integer from 0-10;
k is an integer from 1 to 9;
W is phthalimido;
Ar3 is selected from the group consisting of wherein R23 is from 1 to 2 substituents independently selected from the group consisting of hydrogen, halogen, C1-C4 alkyl, and C1-C4 alkoxy;
R8' is hydrogen or C1-C6 alkyl;
R9 is hydrogen or C1-C6 alkyl;
Y1 is selected from the group consisting of hydrogen, -(CH2)j-Ar4, and -N(R24)2 wherein j is 0 or 1;
R24 each time selected is independently hydrogen or C1-C6 alkyl or are taken together with the nitrogen to which they are attached to form N-morpholino, N-piperidino, N-pyrrolidino, or N-isoindolyl;
Ar4 is wherein R25 is from 1 to 3 substituents independently selected from the group consisting of hydrogen, halogen, C1-C4 alkyl, and C1-C4 alkoxy;
Z is selected from the group consisting of -O-, -NR8-, -C(O)NR8-, -NR8C(O)-, -NR8C(O)NH-, -NR8C(O)O -, and -OC(O)NH-;
wherein R8 is hydrogen or C1-C6 alkyl;
Q is selected from the group consisting of hydrogen, -(CH2)n-Y2, and -(CH2)x Y3;
wherein n is an integer from 0 to 4;
Y2 is selected from the group consisting of hydrogen, -(CH2)h-Ar5 and -(CH2)t-C(O)OR27 wherein Ar5 is selected from the group consisting of wherein R26 is from 1 to 3 substituents independently selected from the group consisting of hydrogen, halogen, C1-C4 alkyl, and C1-C4 alkoxy;
h is an integer from 0 to 6;
t is an integer from 1 to 6;
R27 is hydrogen or C1-C6 alkyl;

x is an integer from 2 to 4;
Y3 is selected from the group consisting of -N(R28)2, N-morpholino, N-piperidino, N-pyrrolidino, and N-isoindolyl;
wherein R28 each time taken is independently selected from the group consisting of hydrogen and C1-C6 alkyl;
R4 is selected from the group consisting of hydrogen, -C(O)R10, -C(O)-(CH2)q-K
and -S-G
wherein R10 is selected from the group consisting of hydrogen, C1-C4 alkyl, phenyl, and benzyl;
q is 0, 1, or 2;
K is selected from the group consisting of wherein V is selected from the group consisting of a bond, -CH2-, -O-, -S(O)r-, -NR21-, and -NC(O)R22;
wherein r is 0, 1, or 2;
R21 is selected from the group consisting of hydrogen, C1-C4 alkyl, and benzyl;
R22 is selected from the group consisting of hydrogen, -CF3, C1-C10 alkyl, phenyl, and benzyl;
R11 each time taken is independently selected from the group consisting of hydrogen, C1-C4 alkyl, and benzyl;
G is selected from the group consisting of wherein w is an integer from 1 to 3;
R12 is selected from the group consisting of hydrogen, C1-C6 alkyl, -CH2CH2S(O)u CH3, and benzyl;
wherein a is 0,1, or 2;
R13 is selected from the group consisting of hydrogen, hydroxy, amino, C1-C6 alkyl, N-methylamino, N,N-dimethylamino, -CO2R17, and -OC(O)R18;
wherein R17 is hydrogen, -CH2O-C(O)C(CH3)3, C1-C4 alkyl, benzyl, or diphenylmethyl;
R18 is hydrogen, C1-C6 alkyl or phenyl;
R14 is 1 or 2 substituents independently selected from the group consisting of hydrogen, C1-C4 alkyl, C1-C4 alkoxy, or halogen;
V1 is selected from the group consisting of -O-, -S-, and -NH-;
V2 is selected from the group consisting of -N- and -CH-;
V3 is selected from the group consisting of a bond and -C(O)-;
V4 is selected from the group consisting of -O-, -S-, -NR19-, and -NC(O)R20-;
wherein R19 is hydrogen, C1-C4 alkyl, or benzyl;
R20 is hydrogen, -CF3, C1-C10 alkyl, or benzyl;

R15 is selected from the group consisting of hydrogen, C1-C6 alkyl and benzyl;
R16 is selected from the group consisting of hydrogen and C1-C4 alkyl;
and stereoisomers, pharmaceutically acceptable salt, and hydrate thereof.

3. Use of an effective amount of matrix metalloproteinase inhibiting compound of the formula or a pharmaceutically acceptable salt thereof for the preparation of a pharmaceutical composition useful in the treatment of atherosclerosis, wherein A is selected from the group consisting of -OH and -NRR';
wherein R and R' are independently selected from the group consisting of hydrogen and C6 alkyl or R and R' taken together with the nitrogen atom to which they are attached form a N-morpholino, N-piperidino, N-pyrrolidino, or N-isoindolyl;

R1 is selected from the group consisting of hydrogen, C1-C6 alkyl, -(CH2)a-CO2R5, -(CH2)8-C(O)NH2, -(CH2)4NH2, -(CH2)3-NH-C(NH)NH2, -(CH2)2-S(O)b-CH3, -CH2-OH, -CH(OH)CH3, -CH2-SH, -(CH2)d-Ar1, and -CH2-Ar2;
wherein a is 1 or 2;
b is 0, 1, or 2;
d is an integer from 0 to 4;
R5 is selected from the group consisting of hydrogen, C1-C4 alkyl, and benzyl;
Ar1 is a radical selected from the group consisting of wherein R6 is from 1 to 2 substituents independently selected from the group consisting of hydrogen, halogen, C1-C4 alkyl, hydroxy, and C1-C4 alkoxy;
R7 is selected from the group consisting of hydrogen, halogen, C1-C4 alkyl, and C1-C4 alkoxy;
Ar2 is a radical selected from the group consisting of R2 is from 1 to 2 substituents independently selected from the group consisting of hydrogen, halogen, hydroxy C1-C4 alkyl, and C1-C4 alkoxy;
R3 is selected from the group consisting of C1-C6 alkyl, -(CH2)m-W, -(CH2)p-Ar3, -(CH2)k-CO2R9, -(CH2)m-NR8'SO2-Y1, and -(CH2)m-Z-Q
wherein m is an integer from 2 to 8;
p is an integer from 0-10;
k is an integer from 1 to 9;
W is phthalimido;
Ar3 is selected from the group consisting of wherein R23 is from 1 to 2 substituents independently selected from the group consisting of hydrogen, halogen, C1-C4 alkyl, and C1-C4 alkoxy;
R8' is hydrogen or C1-C6 alkyl;

R9 is hydrogen or C1-C6 alkyl;
Y is selected from the group consisting of hydrogen, -(CH2)j-Ar4, and -N(R24)2 wherein j is 0 or 1;
R24 each time selected is independently hydrogen or C1-C6 alkyl or are taken together with the nitrogen to which they are attached to form N-morpholino, N-piperidino, N-pyrrolidino, or N-isoindolyl;
Ar4 is wherein R25 is from 1 to 3 substituents independently selected from the group consisting of hydrogen, halogen, C1-C4 alkyl, and C1-C4 alkoxy;
Z is selected from the group consisting of -O-, -NR8-, -C(O)NR8-, -NR8C(O)-, -NR8C(O)NH-, -NR8C(O)O -, and -OC(O)NH-;
wherein R8 is hydrogen or C1-C6 alkyl;
Q is selected from the group consisting of hydrogen, -(CH2)n-Y2, and -(CH2)x Y3;
wherein n is an integer from 0 to 4;
Y2 is selected from the group consisting of hydrogen, -(CH2)h-Ar5 and -(CH2)t-C(O)OR27 wherein Ar5 is selected from the group consisting of wherein R26 is from 1 to 3 substituents independently selected from the group consisting of hydrogen, halogen, C1-C4 alkyl, and C1-C4 alkoxy;

h is an integer from 0 to 6;
t is an integer from 1 to 6;
R27 is hydrogen or C1-C6 alkyl;
x is an integer from 2 to 4;
Y3 is selected from the group consisting of -N(R28)2, N-morpholino, N-piperidino, N-pyrrolidino, and N-isoindolyl;
wherein R28 each time taken is independently selected from the group consisting of hydrogen and C1-C6 alkyl;
R4 is selected from the group consisting of hydrogen, -C(O)R10, -C(O)-(CH2)q-K
and -S-G
wherein R10 is selected from the group consisting of hydrogen, C1-C4 alkyl, phenyl, and benzyl;
q is 0, 1, or 2;
K is selected from the group consisting of wherein V is selected from the group consisting of a bond, -CH2-, -O-, -S(O)r-, -NR21-, and -NC(O)R22;
wherein r is 0, 1, or 2;
R21 is selected from the group consisting of hydrogen, C1-C4 alkyl, and benzyl;
R22 is selected from the group consisting of hydrogen, -CF3, C1-C10 alkyl, phenyl, and benzyl;

R11 each time taken is independently selected from the group consisting of hydrogen, C1-C4 alkyl, and benzyl;
G is selected from the group consisting of wherein w is an integer from 1 to 3;
R12 is selected from the group consisting of hydrogen, C1-C6 alkyl, -CH2CH2S(O)u CH3, and benzyl;
wherein u is 0,1, or 2;
R13 is selected from the group consisting of hydrogen, hydroxy, amino, C1-C6 alkyl, N-methylamino, N,N-dimethylamino, -CO2R17, and -OC(O)R18;
wherein R17 is hydrogen, -CH2O-C(O)C(CH3)3, C1-C4 alkyl, benzyl, or diphenylmethyl;
R18 is hydrogen, C1-C6 alkyl or phenyl;
R14 is 1 or 2 substituents independently selected from the group consisting of hydrogen, C1-C4 alkyl, C1-Ca alkoxy, or halogen;
V1 is selected from the group consisting of -O-, -S-, and -NH-;
V2 is selected from the group consisting of -N- and -CH-;
V3 is selected from the group consisting of a bond and -C(O)-;

V4 is selected from the group consisting of -O-, -S-, -NR19-, and -NC(O)R2O-;
wherein R19 is hydrogen, C1-C4 alkyl, or benzyl;
R20 is hydrogen, -CF3, C1-C10 alkyl, or benzyl;
R15 is selected from the group consisting of hydrogen, Cl-C6 alkyl and benzyl;
R16 is selected from the group consisting of hydrogen and C1-C4 alkyl;
and stereoisomers, pharmaceutically acceptable salt, and hydrate thereof.

4. Use of an effective amount of matrix metalloproteinase inhibiting compound compound of the formula or a pharmaceutically acceptable salt thereof for the preparation of a pharmaceutical composition useful in the treatment of chronic inflammatory disease, wherein A is selected from the group consisting of -OH and -NRR';
wherein R and R' are independently selected from the group consisting of hydrogen and C6 alkyl or R and R' taken together with the nitrogen atom to which they are attached form a N-morpholino, N-piperidino, N-pyrrolidino, or N-isoindolyl;
R1 is selected from the group consisting of hydrogen, C1-C6 alkyl, -(CH2)a-CO2R5, -(CH2)a-C(O)NH2, -(CH2)4NH2, -(CH2)3-NH-C(NH2, -(CH2)2-S(O)b-CH3, -CH2-OH, -CH(OH)CH3, -CH2-SH, -(CH2)d-Ar1, and -CH2-Ar2;
wherein a is 1 or 2;
b is 0, 1, or 2;
d is an integer from 0 to 4;

R5 is selected from the group consisting of hydrogen, C1-C4 alkyl, and benzyl;
Ar1 is a radical selected from the group consisting of wherein R6 is from 1 to 2 substituents independently selected from the group consisting of hydrogen, halogen, C1-C4 alkyl, hydroxy, and C1-C4 alkoxy;
R7 is selected from the group consisting of hydrogen, halogen, C1-C4 alkyl, and C1-C4 alkoxy;
Ar2 is a radical selected from the group consisting of R2 is from 1 to 2 substituents independently selected from the group consisting of hydrogen, halogen, hydroxy C1-C4 alkyl, and C1-C4 alkoxy;
R3 is selected from the group consisting of C1-C6 alkyl, -(CH2)m-W, -(CH2)p,-Ar3, -(CH2)k-CO2R9, -(CH2)m-NR8-SO2-Y1, and -(CH2)m-Z-Q
wherein m is an integer from 2 to 8;
p is an integer from 0-10;
k is an integer from 1 to 9;
W is phthalimido;
Ar3 is selected from the group consisting of wherein R23 is from 1 to 2 substituents independently selected from the group consisting of hydrogen, halogen, C1-C4 alkyl, and C1-C4 alkoxy;
R8 is hydrogen or C1-C6 alkyl;
R9 is hydrogen or C1-C6 alkyl;
Y1 is selected from the group consisting of hydrogen, -(CH2)j-Ar4, and -N(R24)2 wherein j is 0 or 1;
R24 each time selected is independently hydrogen or C1-C6 alkyl or are taken together with the nitrogen to which they are attached to form N-morpholino, N-piperidino, N-pyrrolidino, or N-isoindolyl;
Ar4 is wherein R25 is from 1 to 3 substituents independently selected from the group consisting of hydrogen, halogen, C1-C4 alkyl, and C1-C4 alkoxy;
Z is selected from the group consisting of -O-, -NR8-, -C(O)NR8-, -NR8C(O)-, -NR8C(O)NH-, -NR8C(O)O -, and -OC(O)NH-;
wherein R8 is hydrogen or C1-C6 alkyl;
Q is selected from the group consisting of hydrogen, -(CH2)n-Y2, and -(CH2)x Y3;
wherein n is an integer from 0 to 4;
Y2 is selected from the group consisting of hydrogen, -(CH2)h-Ar5 and -(CH2)t-C(O)OR27 wherein Ax5 is selected from the group consisting of wherein R26 is from 1 to 3 substituents independently selected from the group consisting of hydrogen, halogen, C1-C4 alkyl, and C1-C4 alkoxy;
h is an integer from 0 to 6;
t is an integer from 1 to 6;
R27 is hydrogen or C1-C6 alkyl;
x is an integer from 2 to 4;
Y3 is selected from the group consisting of -N(R28)2, N-morpholino, N
piperidino, N-pyrrolidino, and N-isoindolyl;
wherein R28 each time taken is independently selected from the group consisting of hydrogen and C1-C6 alkyl;
R4 is selected from the group consisting of hydrogen, -C(O)R10, -C(O)-(CH2)q-K
and -S-G
wherein R10 is selected from the group consisting of hydrogen, C1-C4 alkyl, phenyl, and benzyl;
q is 0, l, or 2;
K is selected from the group consisting of wherein V is selected from the group consisting of a bond, -CH2-, -O-, -S(O)r-, -NR21-, and -NC(O)R22;
wherein r is 0, 1, or 2;
R21 is selected from the group consisting of hydrogen, C1-C4 alkyl, and benzyl;

R22 is selected from the group consisting of hydrogen, -CF3, C1-C10 alkyl, phenyl, and benzyl;
R11 each time taken is independently selected from the group consisting of hydrogen, C1-C4 alkyl, and benzyl;
G is selected from the group consisting of wherein w is an integer from 1 to 3;
R12 is selected from the group consisting of hydrogen, C1-C6 alkyl, -CH2CH2S(O)u CH3, and benzyl;
wherein u is 0,1, or 2;
R13 is selected from the group consisting of hydrogen, hydroxy, amino, C1-C6 alkyl, N-methylamino, N,N-dimethylamino, -CO2R17, and -OC(O)R18;
wherein R17 is hydrogen, -CH2O-C(O)C(CH3)3, C1-C4 alkyl, benzyl, or diphenylmethyl;
R18 is hydrogen, C1-C6 alkyl or phenyl;
R14 is 1 or 2 substituents independently selected from the group consisting of hydrogen, C1-C4 alkyl, C1-C4 alkoxy, or halogen;
V1 is selected from the group consisting of -O-, -S-, and -NH-;
V2 is selected from the group consisting of -N- and -CH-;

V3 is selected from the group consisting of a bond and -C(O)-;
V4 is selected from the group consisting of -O-, -S-, -NR19-, and -NC(O)R20-;
wherein R19 is hydrogen, C1-C4 alkyl, or benzyl;
R20 is hydrogen, -CF3, C1-C10 alkyl, or benzyl;
R15 is selected from the group consisting of hydrogen, C1-C6 alkyl and benzyl;
R16 is selected from the group consisting of hydrogen and C1-C4 alkyl;
and stereoisomers, pharmaceutically acceptable salt, and hydrate thereof.

5. Use of an effective amount of matrix metalloproteinase inhibiting compound of the formula or a pharmaceutically acceptable salt thereof for the preparation of a pharmaceutical composition useful in the treatment of smoking-induced emphysema, wherein A is selected from the group consisting of -OH and -NRR';
wherein R and R' are independently selected from the group consisting of hydrogen and C6 alkyl or R and R' taken together with the nitrogen atom to which they are attached form a N-morpholino, N-piperidino, N-pyrrolidino, or N-isoindolyl;
R1 is selected from the group consisting of hydrogen, C1-C6 alkyl, -(CH2)a -CO2R5, -(CH2)a -C(O)NH2, -(CH2)4NH2, -(CH2)3-NH-C(NH)NH2, -(CH2)2-S(O)b -CH3, -CH2-OH, -CH(OH)CH3, -CH2-SH, -(CH2)d -Ar1, and -CH2-Ar2;
wherein a is 1 or 2;
b is 0, 1, or 2;

d is an integer from 0 to 4;
R5 is selected from the group consisting of hydrogen, C1-C4 alkyl, and benzyl;
Ar1 is a radical selected from the group consisting of wherein R6 is from 1 to 2 substituents independently selected from the group consisting of hydrogen, halogen, C1-C4 alkyl, hydroxy, and C1-C4 alkoxy;
R7 is selected from the group consisting of hydrogen, halogen, C1-C4 alkyl, and C1-C4 alkoxy;
Ar2 is a radical selected from the group consisting of R2 is from 1 to 2 substituents independently selected from the group consisting of hydrogen, halogen, hydroxy C1-C4 alkyl, and C1-C4 alkoxy;
R3 is selected from the group consisting of C1-C6 alkyl, -(CH2)m-W, -(CH2)p-Ar3, -(CH2)k-CO2R9, -(CH2)m-NR8'SO2-Y1, and -(CH2)m-Z-Q
wherein m is an integer from 2 to 8;
p is an integer from 0-10;
k is an integer from 1 to 9;
W is phthalimido;
Ar3 is selected from the group consisting of wherein R23 is from 1 to 2 substituents independently selected from the group consisting of hydrogen, halogen, C1-C4 alkyl, and C1-C4 alkoxy;
R8' is hydrogen or C1-C6 alkyl;
R9 is hydrogen or C1-C6 alkyl;
Y1 is selected from the group consisting of hydrogen, -(CH2)j-Ar4, and -N(R24)2 wherein j is 0 or 1;
R24 each time selected is independently hydrogen or C1-C6 alkyl or are taken together with the nitrogen to which they are attached to form N-morpholino, N-piperidino, N-pyrrolidino, or N-isoindolyl;
Ar4 is wherein R25 is from 1 to 3 substituents independently selected from the group consisting of hydrogen, halogen, C1-C4 alkyl, and C1-C4 alkoxy;
Z is selected from the group consisting of -O-, -NR8-, -C(O)NR8-, -NR8C(O)-, -NR8C(O)NH-, -NR8C(O)O -, and -OC(O)NH-;
wherein R8 is hydrogen or C1-C6 alkyl;
Q is selected from the group consisting of hydrogen, -(CH2)n-Y2, and -(CH2)x Y3;
wherein n is an integer from 0 to 4;
Y2 is selected from the group consisting of hydrogen, -(CH2)h-Ar5 and -(CH2)t-C(O)OR27 wherein Ar5 is selected from the group consisting of wherein R26 is from 1 to 3 substituents independently selected from the group consisting of hydrogen, halogen, C1-C4 alkyl, and C1-C4 alkoxy;
h is an integer from 0 to 6;
t is an integer from 1 to 6;
R27 is hydrogen or C1-C6 alkyl;
x is an integer from 2 to 4;
Y3 is selected from the group consisting of -N(R28)2, N-morpholino, N-piperidino, N-pyrrolidino, and N-isoindolyl;
wherein R28 each time taken is independently selected from the group consisting of hydrogen and C1-C6 alkyl;
R4 is selected from the group consisting of hydrogen, -C(O)R10, -C(O)-(CH2)q-K
and -S-G
wherein R10 is selected from the group consisting of hydrogen, C1-C4 alkyl, phenyl, and benzyl;
q is 0, 1, or 2;
K is selected from the group consisting of wherein V is selected from the group consisting of a bond, -CH2-, -O-, -S(O)t-, -NR21-, and -NC(O)R22;
wherein r is 0, 1, or 2;
R21 is selected from the group consisting of hydrogen, C1-C4 alkyl, and benzyl;

R22 is selected from the group consisting of hydrogen, -CF3, C1-C10 alkyl, phenyl, and benzyl;
R11 each time taken is independently. selected from the group consisting of hydrogen, C1-C4 alkyl, and benzyl;
G is selected from the group consisting of wherein w is an integer from 1 to 3;
R12 is selected from the group consisting of hydrogen, C1-C6 alkyl, -CH2CH2S(O)u CH3, and benzyl;
wherein u is 0,1, or 2;
R13 is selected from the group consisting of hydrogen, hydroxy, amino, C1-C6 alkyl, N-methylamino, N,N-dimethylamino, -CO2R17, and -OC(O)R18;
wherein R17 is hydrogen, -CH2O-C(O)C(CH3)3, C1-C4 alkyl, benzyl, or diphenylmethyl;
R18 is hydrogen, C1-C6 alkyl or phenyl;
R14 is 1 or 2 substituents independently selected from the group consisting of hydrogen, C1-C4 alkyl, C1-C4 alkoxy, or halogen;
V1 is selected from the group consisting of -O-, -S-, and -NH-;
V2 is selected from the group consisting of -N- and -CH-;
V3 is selected from the group consisting of a bond and -C(O)-;

V4 is selected from the group consisting of -O-, -S-, -NR19-, and -NC(O)R20-;
wherein R19 is hydrogen, C1-C4 alkyl, or benzyl;
R20 is hydrogen, -CF3, C1-C10 alkyl, or benzyl;
R15 is selected from the group consisting of hydrogen, C1-C6 alkyl and benzyl;
R16 is selected from the group consisting of hydrogen and C1-C4 alkyl;
and stereoisomers, pharmaceutically acceptable salt, and hydrate thereof.

6. Use according to any one of Claims 1-5 wherein R1 is selected from the group of C1-C6 alkyl and -(CH2)d Ar1-

7. Use according to any one of Claims 1-5 wherein R4 is -(CH2)d Ar1 and Ar1 is phenyl or substituted phenyl.

8. Use according to any one of Claims 1-5 wherein R4 is selected from the group consisting of hydrogen, -C(O)R10 and -S-G.

9. Use according to any one of Claims 1-5 wherein R4 is hydrogen.

10. Use according to any one of Claims 1-5 wherein R4 is -C(O)R10 and R10 is C4 alkyl.

11. Use according to any one of Claims 1-5 wherein A is -OH.

12. Use according to any one of Claims 1-5 wherein A is -NRR' wherein R is hydrogen and R' is methyl.

13. A compound of the formula wherein Aa is -NRR';
wherein R and R' are independently selected from the group consisting of hydrogen and C6 alkyl or R and R' taken together with the nitrogen atom to which they are attached form a N-morpholino, N-piperidino, N-pyrrolidino, or N-isoindolyl;
R1 is selected from the group consisting of hydrogen, C1-C6 alkyl, -(CH2)a-CO2R5, -(CH2)a-C(O)NH2, -(CH2)4NH2, -(CH2)3-NH-C(NH)NH2, -(CH2)2-S(O)b-CH3, -CH2-OH, -CH(OH)CH3, -CH2-SH, -(CH2)d-Ar1, and -CH2-Ar2;
wherein a is 1 or 2;
b is 0, 1, or 2;
d is an integer from 0 to 4;
R5 is selected from the group consisting of hydrogen, C1-C4 alkyl, and benzyl;
Ar1 is a radical selected from the group consisting of wherein R6 is from 1 to 2 substituents independently selected from the group consisting of hydrogen, halogen, C1-C4 alkyl, hydroxy, and C1-C4 alkoxy;
R7 is selected from the group consisting of hydrogen, halogen, C1-C4 alkyl, and C1-C4 alkoxy;
Ar2 is a radical selected from the group consisting of R2 is from 1 to 2 substituents independently selected from the group consisting of hydrogen, halogen, hydroxy C1-C4 alkyl, and C1-C4 alkoxy;
R3 is selected from the group consisting of C1-C6 alkyl, -(CH2)m-W, -(CH2)p-Ar3, -(CH2)k-CO2R9, -(CH2)m-NR8'SO2-Y1, and -(CH2)m-Z-Q
wherein m is an integer from 2 to 8;
p is an integer from 0-10;
k is an integer from 1 to 9;
W is phthalimido;
Ar3 is selected from the group consisting of wherein R23 is from 1 to 2 substituents independently selected from the group consisting of hydrogen, halogen, C1-C4 alkyl, and C1-C4 alkoxy;
R8' is hydrogen or C1-C6 alkyl;
R9 is hydrogen or C1-C6 alkyl;
Y1 is selected from the group consisting of hydrogen, -(CH2)j-Ar4, and -N(R24)2 wherein j is 0 or 1;
R24 each time selected is independently hydrogen or C1-C6 alkyl or are taken together with the nitrogen to which they are attached to form N-morpholino, N-piperidino, N-pyrrolidino, or N-isoindolyl;
Ar4 is wherein R25 is from 1 to 3 substituents independently selected from the group consisting of hydrogen, halogen, C1-C4 alkyl, and C1-C4 alkoxy;
Z is selected from the group consisting of -O-, -NR8-, -C(O)NR8-, -NR8C(O)-, -NR8C(O)NH-, -NR8C(O)O -, and -OC(O)NH-;
wherein R8 is hydrogen or C1-C6 alkyl;
Q is selected from the group consisting of hydrogen, -(CH2)n-Y2, and -(CH2)x Y3;
wherein n is an integer from 0 to 4;
Y2 is selected from the group consisting of hydrogen, -(CH2)h-Ar5 and -(CH2)t-C(O)OR27 wherein Ar5 is selected from the group consisting of wherein R26 is from 1 to 3 substituents independently selected from the group consisting of hydrogen, halogen, C1-C4 alkyl, and C1-C4 alkoxy;
h is an integer from 0 to 6;
t is an integer from 1 to 6;
R27 is hydrogen or C1-C6 alkyl;
x is an integer from 2 to 4;
Y3 is selected from the group consisting of -N(R28)2, N-morpholino, N-piperidino, N-pyrrolidino, and N-isoindolyl;
wherein R28 each time taken is independently selected from the group consisting of hydrogen and C1-C6 alkyl;
R4 is selected from the group consisting of hydrogen, -C(O)R10, -C(O)-(CH2)q-K
and -S-G
wherein R10 is selected from the group consisting of hydrogen, C1-C4 alkyl, phenyl, and benzyl;
q is 0, 1, or 2;
K is selected from the group consisting of wherein V is selected from the group consisting of a bond, -CH2-, -O-, -S(O)r-, -NR21-, and -NC(O)R22;
wherein r is 0, 1, or 2;
R21 is selected from the group consisting of hydrogen, C1-C4 alkyl, and benzyl;
R22 is selected from the group consisting of hydrogen, -CF3, C1-C10 alkyl, phenyl, and benzyl;
R11 each time taken is independently selected from the group consisting of hydrogen, C1-C4 alkyl, and benzyl;
G is selected from the group consisting of wherein w is an integer from 1 to 3;
R12 is selected from the group consisting of hydrogen, C1-C6 alkyl, -CH2CH2S(O)u CH3, and benzyl;
wherein u is 0,1, or 2;
R13 is selected from the group consisting of hydrogen, hydroxy, amino, C1-C6 alkyl, N-methylamino, N,N-dimethylamino, -CO2R17, and -OC(O)R18;
wherein R17 is hydrogen, -CH2O-C(O)C(CH3)3, C1-C4 alkyl, benzyl, or diphenylmethyl;
R18 is hydrogen, C1-C6 alkyl or phenyl;
R14 is 1 or 2 substituents independently selected from the group consisting of hydrogen, C1-C4 alkyl, C1-C4 alkoxy, or halogen;
V1 is selected from the group consisting of -O-, -S-, and NH-;
V2 is selected from the group consisting of -N- and -CH-;
V3 is selected from the group consisting of a bond and -C(O)-;
V4 is selected from the group consisting of -O-, -S-, -NR19-, and -NC(O)R20-;
wherein R19 is hydrogen, C1-C4 alkyl, or benzyl;
R20 is hydrogen, -CF3, C1-C10 alkyl, or benzyl;

R15 is selected from the group consisting of hydrogen, C1-C6 alkyl and benzyl;
R16 is selected from the group consisting of hydrogen and C1-C4 alkyl;
and stereoisomers, pharmaceutically acceptable salt, and hydrate thereof.

14. A compound according to Claim 13 wherein R1 is selected from the group consisting of C1-C6 alkyl and -(CH2)d Ar1.

15. A compound according to Claim 13 wherein R1 is -(CH2)d Ar1 and Ar1 is phenyl or substituted phenyl.

16. A compound according to Claim 13 wherein R4 is selected from the group consisting of hydrogen, -C(O)R10 and -S-G.

17. A compound according to Claim 13 wherein R4 is hydrogen.

18. A compound according to Claim 13 wherein R4 is -C(O)R10 and R10 is C1-C4 alkyl.

19. A compound according to Claim 13 wherein Aa is -NRR' wherein R is hydrogen and R' is methyl.

20. A pharmaceutical composition comprising an effective matrix metallo-proteinases inhibitory amount of a compound of any one of Claims 13-19 in admixture or otherwise in association with one or more pharmaceutically acceptable carriers or excipients.

21. A pharmaceutical composition comprising the compound as set forth in claim useful in the treatment of a patient in need thereof.

22. A pharmaceutical composition comprising the compound as set forth claim 1 useful in the treatment of a neoplastic disease state.

23. A pharmaceutical composition comprising the compound as set forth claim 1 useful in the treatment of atherosclerosis.

24. A pharmaceutical composition comprising the compound as set forth claim 1 useful in the treatment of chronic inflammatory disease.

25. A pharmaceutical composition comprising the compound as set forth claim 1 useful in the treatment of smoking-induced emphysema.

26. The pharmaceutical composition according to any one of Claims 21-25 wherein R1 is selected from the group of C1-C6 alkyl and -(CH2)d Ar1.

27. The pharmaceutical composition according to any one of Claims 21-25 wherein R1 is -(CH2)d Ar1 and Ar1 is phenyl or substituted phenyl.

28. The pharmaceutical composition according to any one of Claims 21-25 wherein R4 is selected from the group consisting of hydrogen, -C(O)R10 and -S-G.

29. The pharmaceutical composition according to any one of Claims 21-25 wherein R4 is hydrogen.

30. The pharmaceutical composition according to any one of Claims 21-25 wherein R4 is -C(O)R10 and R10 is C1-C4 alkyl.

31. The pharmaceutical composition according to any one of Claims 21-25 wherein A
is -OH.

32. The pharmaceutical composition according to any one of Claims 21-25 wherein A
is NRR' wherein R is hydrogen and R' is methyl.