CA2253796C - Inhibition of matrix metalloproteases by acetylene containing compounds - Google Patents

Abstract

Matrix metalloprotease inhibiting compounds, pharmaceutical compositions thereof and a method of disease treatment using such compounds are presented. The compounds of the invention have the generalized formula:
(see formula I) where R15 is selected from the group comprising: HOCH2, (n-Pr)2NCH2, CH3CO2CH2, EtOCO2CH2, HO(CH2)2, CH3CO2(CH2)2, HO2C(CH2)2, OHC(CH2)3, HO(CH2)4, 3-HO-Ph, and PhCH2OCH2; and R16 is (see formula II or III) These compounds are useful for inhibiting matrix metalloproteases and, therefore, combating conditions to which MMP's contribute, such asosteoarthritis, rheumatoid arthritis, septic arthritis, periodontal disease, corneal ulceration, proteinuria, aneurysmal aortic disease, dystrophobic epidermolysis, bullosa conditions leading to inflammatory responses, osteopenias mediated by MMP activity, tempera mandibular joint disease, demyelating diseases of the nervous system, tumor metastasis or degenerative cartilage loss following traumatic joint injury, and coronary thrombosis from athrosclerotic plaque rupture. The present invention also provides pharmaceutical compositions and methods for treating such conditions.

Description

Inhibition of l~rlatrix Metalt~oproteases by acetylene containing compounds BACKGR1~C''.rD 4F TFIE IIYV'ENTIOH
Field of the Invention This invention relates to enzyne inhibitors. and more particularly, to novel biaryl acetylene containing compounds or derivatives thereof useful for inhibiting matrix metalloproteases.
Description of the Related Art The matrix metalloproteases (a.k.a. matrix metalloendo-proteinases or VL~tPs) are a familu of zinc endoproteinases which include, but are not limited to, interstitial collagenase !a.k.a..
VIVtP-I ), stromelysin (a.k.a.. proteoglycanase, crattsin, or H~-3), gelatinase .4 (a.k.a..
7~kDa-gelatinase or ~-2) and gelati.nase B (a.k.a.. 95kDa-gelatinase or :vLl~iP-9). These VLVIPs are secreted by a variety of cells including fibroblasts and chondrocytes, along with natural proteinaceous inhibitors known as TIlviPs (issue Inhibitor of MGtallo,~roteinase).
All of these'.vffVIPs are capable: of destroying a variety of connective tissue components of 1 ~ articular cartilage or basement membranes. 'Each MMP is secreted as an inactive proenzyme which must be cleaved in a subsequent step before it is able to exert its own proteolytic activity. In addition to the matrix destroying el:fect,' certain of these MMPs such as MIV)P-3 have been implicated as the in vivo activator for other MMPs such as MMP-1 and MMP-9 (Ito, et al., Arch Biochem Biophys. ~, 211, 1988; Ogata., et a1.,1. Biol. Chem. ~7, 3581, 1992).
Thus, a cascade -'0 of proteolytic activity can be initiated by an excess of MIvIP-3. It foliows that specific IvllVlP-3 inhibitors should iimit the activity of other MMPs that are not directly inhibited by such inhibitors.
It has also bees reported that M~Q'-3 can crave and thereby inactivate the endogenous inhibitors of other proteinases such as elastase (Winyard, et al., FENS Lens.
~Q, 1, 91, 1991).

Inhibitors of 1~~P-3 could thus influence the activity of other destructive proteinases by modifying the level of their endogenous inhibitors.
.~ number of diseases are thought to be mediated by excess or undesired matrix-destrovng metalloprotease activity or by an imbalance in the ratio of the l~~iPs to the TL~fPs. These include:
a~ osteoarthritis (Woessner, et al., J. Hiol.Chem. ?59(6). 3633, 1984; Phadke, et al., J. Rheumatol.
t~. 8~'_'. 1983), bi rheumatoid arthritis (Mullins, et al.. Biochim. Biophys.
acts 69~, 117. 1983:
V'oolley, et al., arthritis Rheum. ?0, 1231, 1977; Gravallese, et al., Arthritis Rheum. 3~, 1076.
1991 ), c) septic arthritis (Williams, et al., arthritis Rheum. 3 533, 1990), d) tumor metastasis l Reich. et al.. Cancer Res. 48, 3307. 1988, and ;~fatrisian, et- al., Proc.
Nat'1. Acad. Sci.. USA 3 IO 9-11 3. 1986), e) periodontal diseases (Overall, et al., J. Periodontal Res. 3, 81, 1987), f) corneal ulceration Bums, et al.. Invest. Opthalmol. Vis. Sci. ~, 1569, 1989), g) proteinuria (Baricos, et al., Biochem. J. 254, 609, 1988), h) coronary thrombosis from atherosclerotic plaque rupture (Henney, et al.. Proc. Nat'1. Acad. Sci., USA 8~, 8154-8158, 1991 ), l) aneurysmal aortic disease (Vine, et aJ., Clin. Sci. 81, 333, 1991), j) birth control (Woessner, et al.. Steroids 54 491, 1989), k) dystrophobic 15 epidetmolysis bullosa (Kronberger, et al., J. Invest. Dermatol. 79 208, 1982), and 1) degenerative cartilaee loss following traumatic joint injury, m) conditions leading to inflammatory responses, osteopenias mediated by MMP activity, n) tempero mandibular joint disease, o) demyelating diseases of the nervous system (Chantry, et al., J. Neurochem. 50 688, 1988).
The need for new therapies is especially important in the case of arthritic diseases. The '0 primary disabling effect of osteoarthritis (OA), rheumatoid arthritis (RA) and septic arthritis is the progressive loss of articular cartilage and thereby normal joint function. No marketed pharmaceutical agent is able to prevent or slow this cartilage loss, although nonsteroidal anti-SUBSTITUTE SHEET (RULE 26) inflammatory drugs (NSA)Ds) have been given to control pain and swelling. The end result of these diseases is total loss of joint function which is only treatable by joint replacement surgery. VtViP
inhibitors are expected to halt or reverse the progression of cartilage loss and obviate or delay surgical intewention.
Proteases are critical elements at several stages in the progression of metastatic cancer. In :his process. the proteoUtic degradation of structural protein in the basal membrane allows for :xpansion of a tumor in the primary site, evasion from this site as well as homing and invasion in distant, secondary sites. Also, tumor induced angiogenesis is required for tumor growth and is dependent on proteolytic tissue remodeling. Transfection experiments with various apes of prote3ses have shown that the matrix metalloproteases play a dominant role in these processes in particular gelatinases A and B (LLI~iP-2 and ~tMP-9, respectively). For an overview of this field see 'tullins. et al.. Biochim. Biophys. ~cta 69~, 177, 1983; Ray, et al., Eur.
Respir. J. 7 2062, 1994;
Birkedal-Hansen, et al., Crit. Rev. Oral Biol. ~ted. 4, 197, 1993.
Furthermore, it was demonstrated that inhibition of degradation of extracellular matrix by 1 ~ the native matrix metalloprotease inhibitor TIMP-2 (a protein) arrests cancer growth (DeClerck, et al.. Cancer Res. ~?. 701, 1992) and that TIMP-2 inhibits tumor-induced angiogenesis in experimental systems (Moses, et al. Science 2~8, 1408. 1990). For a review, see DeCIerck, et al., .inn. V. Y. Acad. Sci. ~, 222, 1994. It was further demonstrated that the synthetic matrix metalloprotease inhibitor batimastat when given intraperitoneally inhibits human colon tumor growth and spread is an orthotopic model in nude mice (Wang, et al. Cancer Res. ~, 4726, 1994) and prolongs the survival of mice bearing human ovarian carcinoma xenografts (Davies, et. al., SUBSTITUTE SHEET (RULE 26) Cancer Res. ~~, 2087, 1993). The use of this and related compounds has been described in Brown.
et al.. ~'~'O-9321942 A2.
There are several patents and patent applications claiming the use of metalloproteinase inhibitors ;or the retardation of metastatic cancer, promoting tumor recession. inhibiting cancer cell proliferation, slowing or preventing cartilage loss associated with osteoarthritis or for treatment of ether diseases as noted above (e.g. Lew, et al., WO-9519965 A l : Beckett. et al.. WO-9519956 :~ l Beckett. et al., WO-9519957 A1: Beckett, et al.. WO-9519961 A1; Brown. et al..
WO-9321942 A?:
Cnrnrttin. et al., WO-9421625 Al; Dickens. et al., U.S. Pat. Vo. 4,599.361:
Hughes, et al., LT.S. Pat.
~o. 5.190.937; Broadhurst, et al.. EP 574758 Al; Broadhurn, et al., EP 276436;
and Vlyers, et al., EP ~~0~73 Al. The preferred compounds of these patents have peptide backbones with a zinc complexing group (hydroxamic acid, thiol, carboxylic acid or phosphinic acid) at one end and a variety of sidechains. both those found in the natural amino acids as well as those with more novel functional groups. Such small peptides are often poorly absorbed, exhibiting low oral bioavailability. They are also subject to rapid proteolvtic metabolism, thus having short half lives.
1 ~ As an example, batimastat, the compound described in Brown, et al., WO-9321942 A2, can only be given intra peritoneally.
Certain 3-biphenoylpropanoic and 4-biaryloylbutanoic acids are described in the literature as anti-inflammatory, anti-platelet aggregation, anti-phlogistic, anti-proliferative, hypolipidemic, antirheumatic, analgesic, and hypochoiesterolemic agents. In none of these examples is a reference made to MIvIF' inhibition as a mechanism for the claimed therapeutic effect.
Certain related compounds are also used as intermediates in the preparation of liquid crystals.
SUBSTITUTE SHEET (RULE 26~

Specifically. Tomcufcik, et al., US patent 3.784,701 claims certain substituted bettzovlpropionic acids to treat inflammation and pain. These compounds include -biphenoylpropanoic acid (a.ic.a. fenbufen) shown below.
' 0 / off - o Feabufen Child. et a1..1. Phann. Sci. 66, 466, 1977 describes structure-activity relationships of several analoes of fenbuien. These include several compounds in which the biphenyl ring system is substituted or the propanoic acid portion is substituted with phenyl, halogen, hydroxyl or methyl, or the carboxylic acid or carbonyl functions are converted to a variety of derivatives. Rio compounds are described which contain a 4'-substituted biphenyl and a substituted propanoic acid portion combined in one molecule. The phenyl (compounds XLIX and LXXVIn and methyl (compound XL,VII) substituted compounds shown below were described as inactive.
1 ~ _ _ O ~ 0 CHI
/ ~ / OH ~ / ~ / OH
O O
~.IX 70.VII
O
/ OH
O
LXXVB
Kameo, et al., Chew. Phann. Bull. ~ø, 2050, 1988 and Totnizawa, et al.. JP
patent 62132825 A2 describe certain substituted 3-biphenoylpropionic acid derivatives aad analogs thereof including S
SUBSTfTUTE SHEET (RULE 26) the followine. Various compounds with other substituents on the propionic acid portion are described, but they do not contain biphenyl residues.

X : / \ / off s wherein X = H. ~'-Br. ~'-Cl. 4'-CHI, or 2'-Br.
Cousse, et al., Eur. J. Vied. Chem. ~, .~~, 1987 describe the following methyl and methylene substituted 3-biphenoyl-propanoic and -propenoic acids. The corresponding compounds in which the carbonyl is replaced with either CH,OH or CH, are also described.
_ O O
/ \ / OH X v / \ / OH
p v I O
0 ~ 0 / \ / ~ OH X v / \ / OH

1 ~ whezetn X = H. C1. Hr. CHsO, F, or NHi.
Nichl, et al. DE patent 1957750 also describes certain of the above methylene substituted biphenovlpropanoic acids.
El-Hashash, et al., Revue Roum. Chim. ~3. 1581, 1978 describe produce derived from (3-aroyl-acrylic acid epoxides including the following biphenyl compound. No compounds '0 substituted on the biphenyl portion are described.
SUBSTITUTE SHEET (RULE 26) cH,1 \1 CH, OH
Kitamura. et al.. JP patent 60209539 describes certain biphenyl compounds used as intermediates for the production of liquid crystals including the following.
The biphenyl is not substituted in these intermediates.
/ ~ o off R' p wherein R' is an alkvi of l-l0 carbons.
Thyes. et al.. DE patent 2854475 uses the following compound as an intermediate. The biphenyl group is not substituted.
O
off l~ O
Sammour, et al., Egypt J. Chem. ]"~, 311. 1972 and Couquelet, et al., Hull.
Soc. Chim. Fr.
9, 3196. 1971 describe certain dialkylamino substituted biphenoylpropanoic acids including the following. In no case is the biphenyl group substituted.
R' R=

O ~N

OH

~ / ~ /

O

wherein R', RZ = alkyl, benzyl, H, or, together with the nitrogen, morpholinyl.

SUBSTITUTE SHEET (RULE 26~

Others have disclosed a series of biphenyl-containing carboxylic acids, illusu~ated by the compound shown below, which inhibit neural endopeptidase hiEP 24.11 ), a membrane-bound unc metalloprotease (Stanton. et al., Bioorg. :fed. Chem. Lett. .~, X39, 1994;
Lombaett. et al.. Bioore.
~ted. Chem. Lett. -t. .71 ~. 1994: Lombaert, et al.. Bioorg. Med. Chem. Lett.
=. 1.~5. 199: Lombaert.
et al.. Bioorg. Lied. Chem. Lett. ~, 1 ~ 1. 1995).

Ph0-P~ ~~ ~.~COZH
Ph0 1-I
It has been reported that N-carboxyalkyl derivatives containing a biphenvlethylgiycine.
illustrated by the compound shown below, are inhibitors of stromelysin-t (VC~iP-3), 72 kDA
gelatinise (:~-2) and collagenase (burette, et al.. WO-929689).
F
1~ I \
0 ~H~
P6. ~ ~~ ~~C~H
IiO
CHI CHI
CHI
.0 It would be desirable to have effective MMP inhibitors which possess improved bioavailability and biological stability relative to the peptide-based compounds of the prior art, and SUBSTITUTE SHEET (RULE 26) which can be optimized for use against particular target ViMPs. Such compounds are the subject of t5e present application.
The development of efficacious l~~iP inhibitors would afford new therapies for diseases mediated by the presence of, or an excess of VL'uP activity, including osteoarthritis. rheumatoid arthritis. septic arthritis. tumor metastasis, periodontal diseases, corneal ulcerations, and proteinuria.
Several inhibitors of VtMPs have been described in the literature, including thiols (Beszant, et al..
J. ~ted. Chem. ~, 4030, 1993), hydroxamic acids (Wahl, et al. Bioorg. died.
Chem. Lets. ~. 349.
199: Conway. et al. J. Exp. Vied. 1~2_, 449. 1995; Porter, et al., Bioorg.
Vied. Chem. Lett. 4,'_7.1, 1994: Tomczuk. et al., Bioorg. Vted. Chem. Lett. ~, 343, 1995; Castelhano, et al.. Bioorg. Vied.
Chem: Lztt. ~, 141, 1995), phosphorous-based acids (Bird, et al. 3. Med. Chem.
37, 1~8, 1994;
l~torphy. et al.. Bioorg. Med. Chem. Left. .1, 2747, 1994; Komlewicz, et al..
J. Med. Chem. 3~. 263.
1990), and carboxylic acids (Chapman, et al. J. Vied. Chem. ,3~, -X293, 1993:
Brown, et al.. J. Vted.
Chem. 37, 674. 1994; Morphy, et al., Bioorg. Vied. Chem. Lett. ~ 2747, 1994;
Stack, et al.. :arch.
Biochem. Biophys. ?~7, 240, 1991: Ye. et al., J. Vied. Chem. 7 206, 1994;
Grobelny, et al., ! ~ Biochemistry 2~, 6145, 1985; Mookhtiar, et al., Biochemistry 27, 4299.
1988). However, these inhibitors eenerally contain peptidic backbones, and thus usually exhibit low oral bioacuvity due to poor absorption and short half lives due to rapid proteolysis. Therefore, there remains a need for improved MMP inhibitors.
=0 SUMMARY OF THE INVENTION
This invention provides compounds having matrix metalloprotease inhibitory activity. These compounds are useful for inhibiting matrix metalloproteases and, therefore, combating conditions SUBSTITUTE SHEET (RULE 26~

to which VfVtP's contribute. Accordingly, the present invention also provides pharmaceutical compositions and methods for treating such conditions.
The compounds described relate to a method of treating a mammal comprising administering to the mammal a matrix metalloprotease inhibiting amount of a compound according to the invention sufficient to:
~a) alleviate the effects of osteoarthritis, rheumatoid arthritis, septic arthritis. periodontal disease, corneal ulceration, proteinuria, aneurysmal aortic disease, dvstrophobic epidermolysis. bullosa, conditions leading to inflammatory responses, osteopenias mediated by ~tl~tP activity, tempero mandibular joint disease, demyelating diseases of the nervous system;
(b) retard tumor metastasis or degenerative cartilage loss following traumatic joint injury;
(c) reduce coronary thrombosis from athrosclerotic plaque rupture; or (d) effect birth control The compounds of the present invention are also useful scientific research tools for studying l ~ functions and mechanisms of action of matrix metalloproteases in both in vtvo and in virro systems.
Because of their N(lv>P-inhibiting activity, the present compounds can be used to modulate MMl' action, thereby allowing the researcher to observe the effects of reduced MMP
activity in the experimental biological system under study.
This invention relates to compounds having matrix metalloprotease inhibitory activity and ?0 the generalized formula:
TxA-B-D-E-G . (L) io SUBSTITUTE SHEET {RUtE 26) In the above generalized formula IL). T~A represents a substituted or unsubstituted aromatic 6-membered ring or heteroaromatic ~ - 6 membered ring containing 1 - '_ atoms independently selected from the group of N, O, or S. T represents a substituted acerylenic moiety.
In the generalized formula (L), B represents an aromatic 6-membered ring or a heteroaromatic ~ - 6 membered ring containing 1 - 2 atoms independently selected from the group of ~. O. or S. It is referred to as the B ring or B unit. When N is employed in conjunction with either S or O in the B ring, these heteroatoms are separated by at least one carbon atom.
In the generalized formula (L), D represents \ \ \ \.H \,H
~C=O ~C='dOH GCS~ j 'OH ~ ~H
l~
In the generalized formula (L), E represents a chain of n carbon atoms bearing m substituents R° in which the R6 groups are independent substituents, or constitute spiro or nonspiro rings. Rings may be formed in two ways: a) two groups R6 are joined, and taken together with the chain atoms) to which the two R6 ~oup(s) are attached, and any intervening chain atoms, constitute a 3 - 7 l . membered ring, or b) one group R° is joined to the chain on which this one group R6 resides, and taken toeether with the chain atoms) to which the Rb group is attached, and any intervening chain atoms, constitutes a 3 - 7 membered ring. The number n of carbon atoms in the chain is 2 or 3, and the number m of R6 substituents is an integer of 1 - 3. The number of carbons in the totality of R°
groups is at least two.
30 Each group R° is alkyl, alkenyl, alkynyl, heteroaryl, non-aromatic cyclic, and combinations thereof optionally substituted with one or more heteroatoms as dexribed more fully below. In the //
SUBSTITUTE SHEET (RULE 26) generalized formula (L), E is a substituted mono- or bicyclic moiety optionally substituted ~zth one or more heteroatoms.
In the eeneralized formula (L). G represents -PO,H, . -of O ~ 0 R ~ 3 ~-'.~1 C N H ~t ' ~ H H ~t . or in which Vi represents -CO,H. -COi'1(R"l, or -CO,R'=, and R" represents any of the side chains of the ! 9 noncyclic naturally occurring amino acids.
"lie most preferred compounds of the invention are:
O R~6 !0 R~s-~=0 ~ / ~ / OH

where R's is selected tom the group comprising: HOCH2, VteOCH:, (n-Pr):NCH,.
CH,CO:CH,, EtOCO,CH,. HO(CH,),, CH,CO:(CH,),, HO:C(CH2)~, OHC(CH),, HO(CH~, Ph, 3-HO-Ph, and PhCH,OCH,; and R'b is Ph 1 ~ ~ or /~ N
O
Pharmaceutically acceptable salts of these compounds are also within the scope of the tnventton.
In most related reference compounds of the prior art, the biphenyl portion of the molecule ~4 is unsubstituted, and the propanoic or butanoic acid portion is either unsubstituted or has a single methyl or phenyl group. Presence of the larger phenyl group has beea reported to cause prior art compounds to be inactive as anti-inflammatory analgesic agents. See, for example, Child, et a1.,1.
IL
SUBSTITUTE SHEET (RULE 26) Pharm. Sci. ~~, X66 (19??). By contrast, it has now beta fotmd drat compounds which c~thibit Fotent Vf~tP inhibitory activity contain a substituent of significant size on the propanoic or butanoic portion of the molecule. The biphenyl portions of the best 1~1V~ inhibitors also preferably contain a substituent on the ~' position, althou~ah when the propanoic or butanoic portions ate optimally substituted, the unsubstituted biphenyl compourats of the invention have sufficient activity to be considercd realistic drug candidates.
The fot~egoing merely summarizes certain aspects of the peesmt invention and is not t0 inc~d. nor should it be construed, to limit the invcnaon in any way.
DESCRIPTION OF THE PREFERRED EMBODIME~y'fS
More particularly, the compounds of the present invention are materials having matrix metalloprotease inhibitory activity and the generalized formula:
T~.~,-&D-tr-G (L) 1~ in which T,.~ represents a substituted or unsubst'ttuted aromatic or heteroaromatic moiety selected from the group consisting of:
/ /
T ~~ T ~~ T ~~.1 T=
T /N Ni T /O Tx Ni Tx S
R
T ~ T. . ~ T; ~N J T.
" N. J NON N N
N
!3 T
W I
in which R' represents H or alkyl of 1 - 3 carbons.
ThrouQttout this application. in the displayed chemical structures, an open bond indicates the point at ~chich the structure joins to another soup. For example, Rso /I
where R'' is l0 is the structure I
In these structures, the aromatic ring is referred to as the A ring or A unit, and T represents 1 ~ a substituent group, referred to as a T group or T unit. T is a substituted acetylenic moiety and x is The B ring of generalized formula (L) is a substituted or unsubstitutcd aromatic or heteroaromatic ring, in which any substituents are groups which do not cause the molecule to fail to fit the active site of the target enzyme, or disrupt the relative conformations of the A and B rings, '0 such that they would be detrimental. Such substituents may be moieties such as lower alkyl, tower alkoxy, C'~I, NOi, halogen, etc., but are not to be limited to such groups.
In the generalized formula (L), H represents an aromatic or heteroaromatic ring selected from the group consisting of:
/' SUQSTlTUTE SHEET (RULE 26) / / ~~ / ~ ~ /

R l y ~ ~ / ~ '~1 R' -' ~~ N ~ / N
i~
R
--~ N~-R, R R N
j R~
N~ l=N / /
N~ '' , I
~I , ~N~ ~N~ ~ N
--y---N N
~'J~ N' I N II / I /
W N /~ ~N ~ .N
N N
in which R' is defined as above. These rings are referred to as the B ring or B unit.
In the generalized formula (L), D represents the moieties:
H H
~C=O ~ =NOH ~ =S % OOH ~ ~H
Ln the generalized formula (L), E represents a chain of n carbon atoms bearing m substituents R°, referred to as R6 groups or R °units. The R °groups are independent substituents, or constitute spiro or nonspiro rings. Rings may be formed in two ways: a) two groups R° are joined, and taken together with the chaia atoms) to which the two R6 groups) are attached, and any intervening chain atoms, constitute a 3 - 7 membered ring, or b) one group R6 is joined to the chain on which this one group Rb resides, and taken together with the chain atoms) to which the R6 group is attached, and /S
SUBSTITUTE SHEET (RULE 26) any intervening chain atoms, constitutes a 3 - 7 membered ring. The number n of carbon atoms in the chain is ? or 3, and the number m of R6 substituents is an integer of 1 -3. 'Ihe number of carbons in the totality of R° stoups is at least two.
Each soup R6 is independently selected from the group consisting of the substituents listed s below as items 1 ) - 14).
1 ) alkyl of 1 - 10 carbons, provided that if the A unit is phenyl, the B unit is phcnylene, m is 1. n is ?. and the alkyl group is located on the alpha carbon relative to the D unit. then x is 1 or 3:
.) aryl of 6 - 10 carbons, provided that if the A unit is phenyl, the B unit is pheny lene, the aryl group is phenyl, n is 2, and m is 1 or ?, then x is l or 2;

3) hcteroaryl comprising 4 - 9 carbons and at least one N, O, or S heteroatom;
~) arylalkyl in which the aryl portion contains 6 - 10 carbons and the alkyl portion contains 1 - 8 carbons;
~) heteroaryl-alkyl in which the heteroaryi portion comprises 4 - 9 carbons and at 1 ~ least one N, O, or S heteroatom, and the alkyl portion contains 1 - 8 carbons;
6) alkenyl of 2 - 10 carbons;
7) aryl-alkenyl in which the aryl portion contains 6 - 10 carbons and the alkenyl portion contains 2 - S carbons;
8) heteroaryl-alkenyl in which the heteroaryl portion comprises 4 - 9 carbons and 30 at least one N, 0, or S heteroatom and the alkenyl portion contains 2 -5 carbons;
9) alkynyl of 2 - 10 carbons;

SUBSTITUTE SHEET (RULE 26) 10) aryl-alfcrnyi in which the aryl portion contains 6 - 10 carbons and the allc~rvl portion contains ? - ~ carbons;
1 1 ) heteroaryl-alkynyl in which the heteroaryt portion comprises 4 - 9 carbons and at feast one ~. O, or S heteroatom and the alkynyi portion contains ? - ~
carbons:
1?)-(CH,),R' in which t is 0 or an integer of 1 - ~ and R' is selected from the group consisting of:

w ~ l -N I ) -N I _N
r r O
r ~ w O\\ / O
w r _N N Rt II Rt _N
0 ~~ N'~O 0 I
O O O
NRt _N _'N I NRt O O O
t t 1 1 R O R t R O -N_O-Rt -N_C-N_Fi .-N_C_ORt -Nt Rt r ~ ~~~)u N
as well as corresponding hevtcroaryl moieties in which the aryl portion of as aryl-containing R' group comprises 4 - 9 carbons and at least one N, 0, or S heteroatom. In such R' groups, Y represents O or S; F;' is as defined above, and a is 0, 1, or 2 provided that when R' is t /-'/. fR )~ w'.~1, R1 Y
or and the A unit is phenyl, the 13 unit is phenylene, m is l, n is 2, and t is 0, rhea x is 1 or 2.
1. )-(CH:),ZR' in which v is an interger of 1 to 4, Z represents -S-. -S(0)-. -S0,-. or -O-, and R' is selected from the group consisting of alkyl of 1 to 12 carbons, aryl of 6 to 10 carbons, heteroaryl comprising .~ to 9 carbons and at least one ~. 0. or S
heteroatom:
awlalkyl in which the aryl portion contains 6 to 12 carbons and the alkyl portion contains 1 to -f carbons: heteroarytalkyl in which the aryl portion contains 6 to 12 carbons and at least one N. O, or S heteroarom and the alkyl portion contains ! to 4 carbons; -C(O)R9 in which the R9 represents alkyl of 2 to 5 carbons, aryl of 6 to 10 carbons, heteroaryl comprising 4 to 9 carbons and at least one N, 0, or S heteroatom, and arylalkyl in which the aryl portion contains 6 to 10 carbons or is a heteroaryl comprising 4 to 9 carbons and at least one ~I, O, or S heteroatom, and the alkyl portion contains 1 to 4 carbons with the provisos that when R8 is -C(O)R9, Z is -S- or-O-; when Z is -O--, Rg may also be -(C,,HZ,,O)tR9 in which v, t and R9 are as defined above: and when the A unit is phenyl, the B unit is phenylene, m is 1, n is 2. and v is 0, then x is 1 or ~; and 1.~~{CH:)",SiR'°~ in which w is an integer of 1 to 3, aad R'°
represeau alkyl of 1 to carbons.
In addition, aryl or hcteroaryl portions of any of the T or Rb groups optionally may bear up 30 to two substitueats selected from the group consisting of -(CH~,C(R"xR'i)OH, -(CH~~,OR", -(CH,)ySR", -(CH~~,S(O)R", -(CH~)yS(O~R". -(CH=~,SO~N(R"}r, -{CHI)a,N(R"~, -(CH,)"N(R")COR'i, -OC(R"~O- in which both oxygen atoms arc connected to the aryl ring, ~8 -~CH,)~COR". -(CH,hCON(R"),, -ICH,)~CO,R". -(CH,)~OCOR".-halogen. -CHO. -CF;. -NO:.
-C~. and -R'Z. in which v is 0 - 4: R" represents H or alley! of I - .t carbons: and R'= represents alkyl of f --I carbons.
In the eeneralized formula (L). G represents -PO,H, -Vh 0 ~ 0 R" N-N
-C-N-H-~t . -C-~-~-M , or ~~ .N
N
in which VI represents -CO,H. -CON(R" ),, or -CO,R'2, and R" represents anv of the side chains of the 19 noncyclic naturally occurring amino acids.
Pharmaceutically acceptable salts of the compounds falling within the generalized formula I 0 ( L ) are also within the invention.
The G unit is most preferably attached to the E unit at the carbon ~i to the D
unit and is preferably a carboxylic acid group.
It is to be understood that as used herein, the term "alkyl" means straight, branched, cyclic, and polycyclic materials. The term "haloalkyl" means partially or fully halogenated alkyl groups 1 ~ such as -1 CH,):C1. -CF, and -C6F,3, for example.
In one of its embodiments, the invention relates to compounds of generalized formula (L) in which at least one of the units A, B, and R° comprises a heteroaromatic ring. Preferred heteroaromatic ring-containing compounds are those in which the heteroaryl groups are heteroaryl of ~ - 9 carbons comprising a 5 - 6 membered heteroaromatic ring containing O, S, or NR' when the ?0 ring is 5-membered, and N when said ring is 6-membered. Particularly preferred heteroaromatic ring-containing compounds are those in which at least one of the A and B units comprises a thiophene ring. When A unit is thiophene, it is preferably connected to B unit at position 2 and SUBSTITUTE SHEET (RULE 26~

WO 97!43245 PCT/L1S97107921 caries one substituent croup T on position ~. When B unit is thiophene, it is preferably connected throueh positions 2 and 5 to D and A units respectively.
In the generalized formula (L), the A and B rings are preferably phenyl and phenvlene.
respectively, the .W ing preferably bears at least one substituent group T
preferably located on the position furthest from the position of the A ring which is connected to the B
ring, the D unit is preferably a carbonyl group, and the G unit is preferably a carboxyl group.
In another embodiment. the invention relates to compounds of generalized formula (L), in the E unit of which n is 2 and m is 1. These compounds thus possess two carbon atoms between the D unit and the G unit, and carry one substituent on this two-carbon chain.
In another of iu embodiments, the invention relates to compounds of generalized formula (L) in which the A ring is a substituted or unsubstituted phenyl group, the H
ring is p-phenylene, and aryl portions of any aryl-containing Rb moieties contain only carbon in the rings. These compounds thus contain no heteroaromatic rings.
In another of its embodiments, the invention relates to compounds of generalized formula I ~ ~ L ) in which m is l and Rb is an independent substituent. These compounds are materials which contain only a single substituent R° on the E unit, and this substituent in not involved in a ring.
Preferred compounds within this subset have the formula / ~ / C CHiCHR6COOH
T
?0 in which x is 1 aad the substituent group T is located on the 4- position of the A ring, relative to the point of attachment between the A and B rings. The para substituent group T of this subset is more preferably acetylene containing moities selected from the following group:
MeOCH2C=C-, (»-i~
SUBSTITUTE SHEET (RULE 26) Pr),VCH,C=C-, CH~CO:CH:C=C-, EtOCO,CH,C=C-. HOCH:C=C-, HO(CH,~,C=C-.
CH..CO=(CHIC=C-. HO=C(CH~C=C-. OHC(CH~~C=C-. HO(CH,),C=C-. PhC=C-. 3-HO-PhC=C
and PhCH,OCH,C=C-.
Other compounds of general formula (L) in which Rb is -(CH=),R' have t as an integer of 1-~.
Preferred compounds of general formula (L) in which R6 is -(CH:)~ZR' have v as an integer of 1-~l and Z as -S- or -O-. additional compounds of general formula (L) in which R6 is alkyl contain ~
or more carbons in said alkyl and those in which R6 is an~lalkyl contain 2-3 carbons in the alkyl portion of said arylalkyl.
In another of its embodiments. the invention relates to compounds of generalized formula IO (L) in which the number of substituents m on the E unit is 2 or 3; and when m is 2, both groups R6 are independent substituents, or together constitute a spiro ring, or one group R6 is an independent substituent and the other constitutes a spiro ring; and when m is 3, two groups R6 are independent substituents and one group R6 constitutes a ring, or two groups R6 constitute a ring and one group R° is an independent substituent, or three groups Rb are independent substitucnts. This subset l ~ therefore contains compounds in which the E unit is di- or tri-substituted, and in the disubstituted case any rings formed by one or both R6 groups are spiro rings, and in the trisubstituted case, the R°
groups may form either spiro or nonspiro rings.
in another of its embodiments, the invention relates to compounds of generalized formula (L) in which the number of substituents m on the E unit is 1 or 2; and when m is 1, the group R6 30 constitutes a nonspiro ring; and when m is 2, both groups R6 together constitute a nonspiro ring or one group R6 is an independent substituent and the other constitutes a nonspiro ring. 'Ibis subset =1 SUBSTITUTE SHEET (RULE 26~

therefore contains compounds in Which the E unit carries one or two substituents R6, and at least one of these substituents is involved in a nonspiro ring.
More particularly, representative compounds of generalized formula (L) in which one or more of the substituent groups 1t6 are involved in formation of nonspiro rings have E units of the following structures:
- (fh.aR°~) (CR°.~t.~a-"
a (Ht.eR°~)---(CR°~t.~?
~ (R,~Jr , ~'(lit.eR°eC~~R°~t.~-(C.~..~ \ttR~°)~C
- (HZaR6aC) (CRsoH~.Ja""
tHt.bR°dC~'t~ Rae~'tt.~1 ~lRt~
- (HZaR°,C) (CR°Wale"'~
(R°~. ~a (Ht-eR°dC)----.--I °~t.e) tR~Jt -(Ht-aR°~~~(CR°eHt.~-(G~-r~ \ ~ tRt4h~
_. (~
- (HZ.,R°,C) (CRa,,HZ.~dw' ....
( \ _..
t~
(R )k - (H2.aR°aC) (CR°,H2-a~a""' (H~.eR°dC?w'- I~ et'rt~l ( -"-r"'_'~'(Ru~c , 8ttd m inwhichais0, l,or2;bis0or i;cis0or l;dis0orl;c+dis0or leis 1-S;fis 1-4;gis3 - S; h is 2 - 4; i is 0 - 4; j is 4 - 3; k is 0 - 2; the total number of groups R6 is 0, 1, or 2; U represents O, S, or NR'; and z is 1 or 2; each group R'4 is independently selected from the group consisting of alkyl of 1 - 9 carbons; arylalkyl in which the alkyl portion contains 1 - 7 carbons and the aryl portion contains 6 - 10 carbons; alkenyl of 2 - 9 carbons; aryl-substituted alkenyl in which the alkenyl portion contains 2 - 4 carbons and the aryl portion contains 6 -10 carbons;
alkynyl of 2 - 9 carbons;
aryl-substituted alkynyl in which the alkynyl portion contains 2 - 4 carbons and the aryl portion contains 6 - 10 carbons; aryl of 6 - 10 carbons; -CORi; -COZR3; -CON(Ri~; -(CH~rR' in which t is 0 or an integer of 1 - 4; and -(CHZ),,ZR' in which v is 0 or an integer of 1 to 3, and Z represents -S-or -O-. R', R', and R' have been defined above.
Preferred compounds of generalized formula (L) in which one or more of the substituent groups R6 are involved in the formation of nonspim rings have E units of the following structures:
- (HziR°,C) ~ R°~2-~Id'-' - (H2.aR°sC1 (CR°aH2-a)d' (H~.bR° C)---(CR°eH~.~ (H~.bR°eCy--'(CR°n~'~~.~
l~~Jr , -~ -(R"h (C,H~ (CoH~.y~
_" (H2.vR°sC) (CR°~2.Jd-' ~R°
(H~.eR ~( et'i~.~
lx"),, (C;Hx.~t in which a, b, c, d, (c + d), e, g, i, k, the total number of groups R6, U, and R" are as defined above.
'?2a The substituent group 'T is preferably sn acetylene containing moiety with the general formula:
R'°(CH~aC=C-where n is 1-4 and R~° is selected from the group consisting of HO-, Me0-, (n-Pr~N-, CH3C02-, CH3CHiOC02-, HOzC-, OHC-, Ph-, 3-HO-Ph- and PhCHZO-, provided that when R~° is Ph or 3-HO-Ph,n=0.

WO 97!43245 PCT/US97/07921 Vtost preferably, T is: ~teOCH,C=-C-. (n-Pr),NCH:C=C-. CH,CO,CH,C=C-.
EtOCO:CH,C=C-. HOCH,C=C-. HO(CH,),C=C-. CH,CO,(CH:),C=C-. HO,C(CH,)_C=C-.
OHCiCH:),C=C-, HO(CH,),C=C-, PhC=C-, 3-HO-PhC=C- and PhCH,OCH,C=C-.
The subscript x. which defines the number of T substituents, is preferably 1 or '_', most preferably 1. and when x is 1 the T is preferably on the 4- position of ring A.
The A ring is preferably a phenyl or thiophene ring, most preferably phenyl.
The B ring is preferably a 1,-1-phenylene or ?.~-thiophene ring, most preferably 1.-~-phenylene.
The D unit is most preferably a carbonyl group.
In the E soup. R6 is preferably:
1 ) arylalkyl wherein the aryl portion contains 6 - 10 carbons and the alkyl portion contains 1 - 8 carbons;
'_' ) -(CH=),R' wherein t is 0 or an integer of 1 - ~ and R' is an imidoyl group containing an aromatic residue: or l ~ 3 ) -(CH=)~ZRa wherein v is 0 or an integer of 1 - :~ , Z is S or 0, and R' is aryl of 6 - 10 carbons or arylalkyl wherein the aryl portion contains 6 to 12 carbons and the alkyl portion contains 1 to 4 carbons.
The group R° is most preferably the following, wherein, any aromatic moiety is preferably substituted:
'_0 1 ) arylalkyl wherein the aryl portion is phenyl and the alkyl portion contains 1 - 4 carbons;
~ZS~
SUBSTITUTE SHEET (RULE 26) WO 97/43245 PCTlUS97/07921 2) -(CH:)~R' wherein t is an integer 1 - 3, and R' is ~1-(1.?-naphthalene-dicarbox-imidoyl), N-(2.3-naphthalene-dicarboximidoyl), or ~-(1.8-naphthalene-dicarboximidovl);
or'.~I-phthalimidoyl.
-! CH:),ZRB wherein v is an integer of 1 - 3. Z is S, and R~ is phenyl.
The more preferred compounds of generalized formula (L) have R° units of the following structures:

Ph or /~ N I I

l0 Those skilled in the art will appreciate that many of the compounds of the invention exist in enantiomeric or diastereomeric forms, and that it is understood by the art that such stereoisomers generally exhibit different activities in biological systems. This invention encompasses all possible stereoisomers which possess inhibitory activity against an wimp, regardless of their stereoisomeric designations, as well as mixtures of stereoisomers in which at least one member possesses inhibitory 1~ activity.
The most prefered compounds of the present invention are as indicated and named in the Iist below:
I) RIS 4'-(3-hydroxy-1-propynyl~y-oxo-a-(3-phenylpropyl)-[i,l'-biphenyl)-~-butanoic acid, tI) S-4'-(3-hydroxy-1-propynyl)-y-oxo-a-{3-phenylpropyl)-{1,1'-biphenyl)-butanoic acid, ?0 III) R-4'-(3-hydroxy~1-propynyl)-y-oxo-a-(3-phenyipropyl)-[1,1'-biphenyl]-4-butanoic acid, N) 4'-(3-methoxy-1-propynyl)-y-oxo-a-{3-phenylpropyl)-[1,1'-biphenyl]-4-butanoic acid, V) y-oxo-a-(3-phenytpropyir4'-{3-propyl-1-hexynyl~[1,1'-biphenyl)-4-butanoic acid, ZS
SUBSTITUTE SHEET (RULE 26~

VI) ~'-[3-(acetyloxy)-1-propvnyl]-y-o!co-a-(3-phenyipropyl)-[1,1'-biphenyl]-.~-butanoic acid.
~'1T1 ~'-[3-[(ethoxvcarbonvl)oxy]-1-propvnyl]-y-oxo-a-(3-phenylpropyli-[l.l'-biphenyl]--t.
butanoic acid.
~'iII~ ~'-~-~-hydroxy-1-butv~nyi)-y-oxo-a-(3-phenylpropyl)-[l.l'-biphenyl]--~-butanoic acid.
3 L~C) -1'-[3-(acetyloxy)-1-propy~yl]-y-oxo-a-(3-phenylpropyl)-[1.1'-biphenyl]-~-butanoic acid, Y) .~'-(~-carboxy-1-butvnyl)-y-oxo-a-(3-phenylpropyl)-[1,1'-biphenyl]-i-butanoic acid, YI) y-oxo--t'-(3-oxo-1-penrynyl)-a-(3-phenylpropyl)-[1.1'biphenyl]-4-butanoic acid.
~CII) ~'-(6-hydroxy-1-henynyl)-y-oxo-a-(3-phenylpropyl)-[1,1'-biphenyl]-i-butanoic acid, lIII) y-oxo-~'-(phenylethynyl)-a-(3-phenyipropyl)-[1.1'-biphenyl]-~-butanoic acid and ~CIV) .i'-[3-hydroxyphenyl)ethvnyl]-y-oxo-a-(3-phenylpropyl)-[I.1'-biphenyl]-.~-butanoic acid, yCV) 1,3-dihydro-1.3-dioxo-a-[2-oxo-2-[4'-[3(phenylmethoxy)-1-propvnvl][1,1'-biphenyl]-.~-yl]ethyl]-2H-isoindole-2-butanoic acid, and XVn 1.3-dihydro-a-[2-[4'-(hydroxyethynyl)[1,1'-biphenyl]-4-yl]-2-oxoethyl]-I.3-dioxo-?N
isoindole-2-butanoic acid.
1~
General Preparative Methods:
The compounds of the invention may be prepared readily by use of known chemical reactions and procedures. Nevertheless, the following general preparative methods are presented to aid the reader in synthesizing the inhibitors, with more detailed particular examples being '_0 presented below in the experimental section describing the working examples. :a,.ll variable groups of these methods are as described in the generic description if they are not specifically defined below. The variable subscript n is independently defined for each method. When a variable group SUBSTITUTE SHEET {RULE 26) with a given syTrtbol (i.e R9) is used more than once in a given structure, it is to be understood that zach of these groups may be independently varied within the range of definitions for that svtnbol.
Geaera! Method A - '/he compounds of this invention in which the rings A and B
are substituted phem~l and phenylene respectively are conveniently prepared by use of a Friedel-Crafrs reaction of a substituted biphenyl VIII with an activated acyl-containing intermediate such as the succinic or glutaric anhydride derivative ViIII or acid chloride MIV in the presence of a Lewis acid catalyst such as aluminum trichloride in an aprotic solvent such as i,l.'_.2-tetrachloroethane. 'Ihe well known Friedel-Crafrs reaction can be accomplished with use of many alternative solvents and acid catalysts as described by Berliner. Org. React., ~, '_'?9, 1949 and Heaney, Comp. Org. Synth.
~ ?3.1991.
If the anhydride :vIIII is monosubstituted or multiply-substituted in an unsymmetrical way, the raw product VtI-A often exists as a mixture of isomers via attack of the anhydride from either of the two carbonyls. The resultant isomers can be separated into pure forms by crystallization or chromatography using standard methods known to those skilled in the art.
1 ~ When they are not commercially available, the succinic anhydrides ;~tIII
can be prepared via a Stobbe Condensation of a dialkyl succinate with an aldehyde or ketone (resulting in side chain R°), followed by catalytic hydrogenation, hydrolysis of a hemiester intermediate to a diacid. and then com~etsion to the anhydride IvtIIII by reaction with acetyl chloride or acetic anhydride. Alternatively, the hemiester intermediate is convened by treatment with thionyl chloride or oxalyl chloride to the acid chloride NllV. For a review of the Stobbe condensation, including lists of suitable solvents and bases see Johnson and Daub, Org. React. 6_, 1, 1951.
2~
SUBSTITUTE SHEET (RULE 26) This method, as applied to the preparation of 1~LQT (R6 = H, isobuyt and H. n-pentvt), has been described Wolanin- et al.. C; S Patent 4,77 t .038.
~Iet6od a O a ut~
Lewis acid - - ' iT)~% / \ / + O O Soi~~;% / \ / ~ Re Rb 0 ~n R~R6 ~I n !vQII
n=2or3 or O OR~z ~ OR's C1 R6 R6 0 ~~ % / \ / ~Rb Rs 0 /n ' - ' /n ~V :vt1-A-2 n=2or3 - - + O p O 0 / \ / Lewis acid - - CO H
(T)x~ (CHI" ~~ Solvent ~~% / \ /
R (CH~~ R ~, ~I IvQII-A ~-p-33 1~ nsa-3 Base O
CO H
('>~x / \ /
(~z)~ Ru Ivll-A-4 Method A is especially useful for the preparation of cyclic compounds such as MI-A-3, in which two R6 groups are connected in a methylene chain to form a 3-7 member ring. Small ring (3-5 ZS
SUBSTITUTE SHEET (RULE 26~

member) anhydrides are readily available only as cis isomers which yield cis invention compounds ELI-.~-~. The traps compounds ~-A--t are then prepared by treatment of W-A-3 with a base such as DBL' in THF. The substituted four member ring starting material anhydrides such as ~-A-1 are formed in a photochemical ?-? reaction as shown below. This method is especially useful for the preparation of compounds in which R" is acetoxy or acetoxymethylene.
:after the subsequent Friedel-Crafts reaction the acetate can be removed by basic hydrolysis and the carboxyl protected by conversion to 2-(trimethylsilyl)ethyl ester. The resultant intermediate with R" = CH,OH can be converted to invention compounds with other R'j groups by using procedures described in General Method G.
p\~p 0 ~ 0 UV tight acaouiuzle -1 Ri4 The Friedel-Crafts method is also useful when double bonds are found either between C-2 and C-3 of a succinoyl chain (from malefic anhydride or l-cyclopentene-1,2-dicarboxylic anhydride, 1 ~ for e~cample) or when a double bond is found in a side chain, such as in the use of itaconic anhydride as starting material to yield products in which two Rb groups are found on one chain carbon together to form an exo-methylene (=CHI) group. Subsequent uses of these compounds are described in Methods D.
General Method B - Altemativeiy the compounds MI can be prepared via a reaction sequence involving mono-alkylation of a dialkyl malonate MVI with an alkyl halide to form intermediate MVB, followed by alkylation with a haiomethyl biphenyl ketone MVIZI to yield intermediate NBC. Compounds of s~uucture IvBX are then hydrolyzed with aqueous base and heated SUBSTITUTE SHEET (RULE 26) to decarboxyiate the malonic acid intermediate and yield VII-B-2 (Method B-1 ). By using one equivalent of aqueous base the esters V1I~B-2 with R'= as alkyl are obtained, and using more than m~o equivalents of base the acid compounds (R'= = H) are obtained. Optionally, heat is not used and the diacid or acid-ester III-B-1 is obtained.
Alternatively, the diester intermediate ~~(I~C can be heated with a strong acids such as concentrated hydrochloric acid in acetic acid in a sealed tube at about 110 'C
for about 2:~ hr to yield ViI-B-1 (R'= = H). Alternatively, the reaction of MVI with MVIII can be conducted before that with the alkyl halide to yield the same MIX (Method 8-2).
Alternatively, a diester intermediate .W~IX, which contains R'= = allyl, can be exposed to Pd catalysts in the presence of pvrrolidine to yield MI-B-? (R'~ = H) (Dezeil, Tetrahedron Lett. ~, X371. 1990.
Intermediates MVII are formed from biphenyls MB in a Friedel-Craft reaction with haloacetyl halides such as bromoacetyl bromide or chioroacetyl chloride.
Alternatively, the biphenyl can be reacted with acetyl chloride or acetic anhydride and the resultant product 1 ~ halogenated with, for example. bromine to yield intermediates MVIII (X =
Br).
vlethod B has the advantage of yielding single regio isomers when Method A
yields mixtures. Method B is especially useful when the side chains R6 contain aromatic or hetemaromatic rings that may participate in intramolecular acylation reactions to give side products if Method A
were to be used. This method is also very useful when the R° group adjacent to the carboxyl of the ?0 final compound contains hetematoms such as oxygen, sulfur, or nitrogen, or more complex functions such as imide rings.
SUBSTITUTE SHEET (RULE 26) METHOD B

X~R6 X~BrotCl ~x ewis acid 0 O~ H OR~~
I) ' I'R6 OR°i Lewis acid 0 0 ~tVI
X
21 Halogenation ' /
. R6 R6 ~ Aah. base c-r)~~
vtvt~ B- I ) o B-2) s O R ORis X R 0 R6 0 ORiZ ~. H ORti ,"QX - - 0 base 0 'vtVfI
I 0 m ~ / ~ / R6 Rs Saong acid 1 ) Aqueous base Heat or whoa 2) Acid Rli , ~yl, Pd(Ph3P)a~
0 pyrrolidine 0 R6 OH a 0 R6 H a _ OR _ OR
/ 6 R6 O ~ / R6 R6 O
~x R
I ~ ~-B.I ~-B-2 When R6 contains selected functional groups Z, malonate MVII can be prepared by alkylating a commercially available unsubstituted malonate with prenyl or allyl halide, subject this product to ozonalysis with reductive work-up, and the desired z group can be coupled via a '_0 ~Iitsunobu reaction (Mitsunobu, Synthesis 1, 1981 ). Alternatively, the intermediate alcohol can be subjected to alkylation conditions to provide malonate MVII containing the desired Z group.
3~
SUBSTITUTE SHEET (RULE 26) 1 ) NaH, TFff . they isoprenyl bromide i ~' 2) 0~, CH:CI: then Rs R O~R ;~a;gH,, VieOH R~iO~OR~' ~0 ~O 3) Mitsuaobu ~O ~0 3) CBr,, or Ph~P , 4) Alkylanoa R6 - (CH:)=Z
General Method C - Especially useful is the use of chiral I~PLC to separate the enantiomers of racemic product mixtures (see, for example, Arit, et al., Chem. Int. Ed.
Engl. ,~, 30, i 991 ). The compounds of this invention can be prepared as pure enantiomers by use of a chiral auxiliary route.
See. for example, Evans. Aldtzchimica Acts. 1 ~(2), ~3, 1982 and other similar references known to one skilled in the art.
General rlethod D - Compounds in which R6 are alkyl- or aryl- or heteroaryl-or acyl- or heteroarylcarbonyl-thiomethylene are prepared by methods analogous to those described in the patent WO 90/05719. Thus substituted itaconic anhydride MXVI (n = 1) is reacted under Friedel-Crafts conditions to yield acid MI-D-1 which can be separated by chromatography or 1 ~ crystallization from small amounts of isomeric MI-D-5. :alternatively, MI-D-Ss are obtained by reaction of invention compounds MI-D-4 (from any of Methods A through C) with formaldehyde in the presence of base.
Compounds MI-D~1 or MI-D-5 are then reacted with a mercapto derivative MXVII
or VIXVIII in the presence of catalyst such as potassium carbonate, ethyldiisobutylamine, ?0 tetrabutylamtnonium fluoride or free radical initiators such as azobisisobutyronitrile (AIBI~ in a solvent such as diethylfotinamide or tetrahydrofuran to yield invention compounds MI-D-2, MI-D-3, MI-D-6, or MI-D-7.
3y SUBSTITUTE SMEET (RULE 26) Viethod D
o a - ~R6 O Lev~ns~c~d / ' ~R6 ~ O SOIV~ / / ~ ~ ~ 6 6 'R R Jo 0 NOM
o - ! or 2 - ~Q~D-5 0 R~ R~ 0 R6 R~
~ OH Fo_ rm~dehyd ~ - - o OH
/ O Bite mij, / ~

'.vQ-D-4 NQ-D-S
O SRS
OH
Vii, / ~ / RR61 H . S . R, ~I NQ-D-2 'D"! or O
OII ;vOMII 0 S-~-R9 HS~ R9 OH
~R R~, O

Re R6 OH

/n \\
H.S.Rs NaCVII ('17,r~ ~ ~ ~ ~ O
M-I-D-5 S' R~
0 ~ :vt-I-D-6 ~~ R9 or Rs R6 OH

In \~
O
'_'0 '.vi-I-D-7 O

;3 SUBSTITUTE SHEET (RULE 26) General Method E - Biaryi compounds such as those of this application may also be prepared by Suzuki or Stille cross-coupling reactions of aryl or heteroaryl metallic compounds in which the metal is zinc. tin. magnesium. lithium, boron, silicon, copper, cadmium or the like with an aryl or heteroay 1 halide or triflate (trifluoromethane-sulfonate) or the like. In the equation below either Vlet or X is the metal and the other is the halide or triflate (OTf). Pd(com) is a soluble complex of palladium such as tetrakis(triphenylphosphine)-palladium(O) or bis-Itriphenylphosphine)-palladium(I)1) chloride. These methods are well known to chose skilled in the art. See, for example.
Suzuki. Pure Appl. Chem. 63, Z13, 1994; Suzuki, Pure Appl. Chem. ø~3, 419.
1991; and Farina and Roth. "Metal-Organic Chemistry" Volume 5 (Chapter 1 ), 1994.
The starting materials :~ixXBII (B = 1..~-phenylene) are readily formed using methods analogous to chose of methods A, B, C, or D but using a halobenzene rather than a biphenyl as starting material. When desired, the materials in which X is halo can be converted to those in which X is metal by reactions well known to those skilled in the art, such as treatment of a bromo intermediate with hexamethylditin and palladium tetrakistriphenylphosphine in toluene at reflux to 1 ~ yield the trimethyltin intetTrtediate. The starting materials 1v00~ (B =
heteroaryl) are most conveniently prepared by method C but using readily available heteroaryl rather than biphenyl starting materials. The intermediates MXXII are either commercial or easily prepared from commercial materials by methods well known to those skilled in the art.
Method E
?0 (T)tA-Met + X-B-E-G -i ('I~;A-B-D~E-G
VIXXII MXXIII Pd(com) MI-E
T, x. A, B, E and G as in Structure (L) Met = Metal and X = Halide or Triflate SUBSTITUTE SHEET (RULE 26) or stet = Halide or Triflate arid X = Metal These general methods are useful for the preparation of compounds for which Friedel-Crafts s reactions such as those of Methods A, B, C, or D would lead to mixtures with various biaryf acylation patterns. Lfethod E is also especially useful for the preparation of products in which the aryl soups. ~ or B, contain one or more heteroatoms (heteroaryls) such as those compounds that contain thiophene, furan, pyridine, pyrrole, oxazoie, thiazole, pyrimidine or pyrazine rings or the like instead of phenyls.
General ytethod F - When the Rb groups of method F form together a 4 - 7 member carbocyclic ring as in Intermediate VQ~CV below, the double bond can be moved out of conjugation with the ketone soup by treatment with two equivalents of a strong base such as lithium diisopropylamide or lithium hexamethylsilylamide or the like followed by acid quench to yield compounds with the structure MXXVI. Reaction of :vQ~XVI with mercapto derivatives using methods analogous to 1 ~ those of General Method D then leads to cyclic compounds MI-F-I or MI-F-2.
?0 SUBSTITUTE SHEET (RULE 26) Method F

\ - _ - t~W
/ \ o off iT)x - \ ~ fCHJ" Solvent (T)=
HII \ ~ ( CH=)"-W~QN
n - I-3 / \ O OH
H.S. Ri (T~~ - \ ~ (CHI" \
O
O II
/ \ 0 OH SH~ R9 LXVIII
(T)X ~~ ~ (CHI" ~ S O

-F-1 R~ / \ OH
\ / (CHJn Ci7~ -S~O
:vQ-F-2 General Method G - The compounds of this invention in which two Rb groups are joined to form a substituted 5-member ring are most conveniently prepared by method G. In this method l~ acid CII (R = H) is prepared using the protocols described in Tetrahedron ~7, Suppl., 411, 1981.
The acid is protected as an ester [eg. R = benryl (8n) or 2-(trimethyisilyl)ethyl (TMSE)J by use of coupling agents such as 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and procedures well known to those skilled in the art. Substituted bromobiphenyl CIa is converted to its Grignard reagent by treatment with magnesium and reacted with CI to yield alcohol CVI.
?0 alcohol CVI is eliminated via base treatment of its mesylate by using conditions well known to those skilled in the art to yield olefin CVII. Alternatively CITI is converted to a uimethyltin intermediate via initial metallation of the bromide with n-butyllithium at low temperature (-78 °C) SUBSTITUTE SHEET (RULE 26) followed by treatment with chloronimethyltin and CI is converted to an enoltriflate (C~ by reaction wsth ?- (~1 ~l-bis(ttifluoromethylsulfonyl)amino)-~-chloropvridine in the presence of a strong aprotic base. The tin and enoltriflate intermediates are then coupled in the presence of a Pd° catalyst. CuI
and AsPh, ~o yield directly intermediate CVII. Ozonoiysis of CVB (workup with methylsufide) ~~ields aldehyde CVm. Alternatively treatment with OsO, followed by HIO, converts CVII to cVm.
Viethod G ~ ~1 w , ROC RFC I ~ _ BrMg / ~ \ \ ~ ~ CVI
(T~ OH
Cl CV 1) MsC!
C>PyN.I.~ 2) elimination Mg ROiC _ ROiC
'~ OTf X
/ + ~ ~\~ Pd(0~ /
1) HuLi ~ CILI: X = Br C~ 1) Ozone 2) CISnMe~ CN: X = SnMe~
2) MtSMt 1~
O CO=R O COiR
J~ ,,,cHO
,,vCHiOH
m~~ ~ ~~ m=%J cviB
or ~u~bu.
TsC1 acytatioa or alkylation o ~o=~ o co=R
?0 ,,.CHzOTs ~ ~ ~~ ,,.CHZZ
alkytanon ~ ~ W
mi J cx m~ ~ c~a 3~
SUBSTITUTE SHEET (RULE 26) Conversion of key intermediate CVI>I to the targeted patent compounds is accomplished in several ways depending on the identity of side chain function Z. Reaction of CVIII with Vl%ittig reagents followed by hydrogenation yields products in which Z is alkyl and or arylalkyl. Selective reduction of aldehyde CVTII with a reducing agent such as lithium tris [(3-ethyl-3pentyl)oxy~altnninurtt hydride (LTEPA) yields alcohol C13C. The alcohoi is converted to phenyl ethers or a variety of heteroatom substituted derivatives used to generate sidechain Z via the Llitsunobu conditions well known to those skilled in the art (see Mitsunobu, Synthesis. l, 1981).
Alternatively the alcohol of CL~C is convened to a leaving group such as tosylate (C3~ or bromide by conditions well known to those skilled in the art and then the leaving group is displaced by an appropriate nucleophile. Several examples of this type of reaction can be found in Norman, et al., J. Died. Chem. 37, 252. 1994. Direct acylation of the alcohol CIX yields invention compounds in which Z = OAcyt and reaction of the alcohol with various alkyl halides in the presence of base yields alkyl ethers. In each case a final step is removal of acid blocking group R to yield acids (R
= H) by using conditions which depend on the stability of R and Z, but in all cases well known to 1 ~ those skilled in the art such as removal of benzyl by base hydrolysis or of 2-ltrimethylsilyl)ethyl by treatment with tetrabutylammonium fluoride.
General il~tet6od H - Amides of the acids of the invention compounds can be prepared from the acids by treat;rtent in as appropriate solvent such as dichloromethane or dimethylfotmamide with a primary or secondary amine and a coupling agent such as dicyclohexylcarbodiimide. These ?0 reactions are well known to those skilled in the art. The amine component can be simple alkyl or arylalkyi substituted or can be amino acid derivatives in which the carboxyl is blocked and the amino group is free.

SUBSTITUTE SHEET (RULE 2~

General Method I - The compounds of this invention in which (T); is an alky~nyl or substituted alkynyl are prepared according to general method I (Austin. J.
Org. Chem. -~6, =X80.
1981 ). Inurmediate VfX is prepared according to methods A. B, C. D or G by starting with commercial VfItI IR' = 9r). Reaction of W~C with substituted acetylene 1~LYI
in the presence of Cu(i) % palladate reagent gives invention compound VtI-I-1. In certain cases.
R' may be an alcohol blocked as trialkylsilyl. In such cases the silyl group can be removed by treatment with acids such as tritluoroacetic acid or HF - pyridine reagent.
Method I
/Rs ~ /Rs C (CH:), 1l7 Q fCHi). R3(~:~-C=CH / \ OH
,/ \ " ~ OH
/ (EthN Rs(CHih-C=Cue- \ /
~'t1. C Cul nao~~ W-I-1 dichlorob~~atp6myl pbosphme pailadate Suitable pharmaceutically acceptable salts of the compounds of the present invention include addition salts formed with organic or inorganic bases. The salt forming ion derived from such bases t ~ can be metal ions, e.g., aluminum, alkali metal ions, such as sodium or potassium, alkaline earth metal ions such as calcium or magnesium, or an amine salt ion, of which a number are known for this purpose. Examples include ammonium salts, arylalkylamines such as dibenzylamine and .V..~'-dibenrylethylenediamine, lower alkylamines such as methylamine, r-butylamine, procaine, lower alkylpiperidines such as N ethylpiperidine, cycloalkylamines such as cyclohexylamine or ?0 dicyclohexylamine, 1-adarnantyiamine, benTathine, or salts derived from amino acids like. arginine, lysine or the like. The physiologically acceptable salts such as the sodium or potassium salts and the amino acid salts can be used medicinally as described below and are preferred.

SUBSTITUTE SHEET (RULE 26~

These and other sale which are not necessarily physiologically acceptable are useful in isolating or purifying a product acceptable for the purposes described below.
For example, the use of commercially available enantiomerically pure amines such as (+)-cinchonine in suitable solvents can yield salt crystals of a single enatiomer of the invention compounds, leaving the opposite enantiomer in solution in a process often referred to as "classical resolution." As one enantiomer of a given invention compound is usually substantially greater in physiological effect than its antipode, this active isomer can thus be found purified in either the crystals or the liquid phase. The salts are produced by reacting the acid fotin of the invention compound with an equivalent of the base supplying the desired basic ion in a medium in which the salt precipitates or in aqueous medium and then lyophilizing. The free acid form can be obtained from the salt by conventional neutralization techniques, e.g.. with potassium bisulfate, hydrochloric acid, etc.
The compounds of the present invention have been found to inhibit the matrix metalloproteases l~tNiP-3. GIMP-9 and MMP-2, and to a lesser extent MMP-1, and are therefore useful for treating or preventing the conditions referred to in the background section. As other 1 ~ Vil~lPs not listed above share a high degree of homology with those listed above, especially in the catalytic site, it is deemed that compounds of the invention should also inhibit such other MMPs to varying degrees. Varying the substituenu on the biaryl portions of the molecules, as well as those of the propanoic or butanoic acid chains of the claimed compounds, has been demonstrated to affect the relative inhibition of the listed MMPs. Thus compounds of this general class can be "tuned" by selecting specific substituenu such that inhibition of specific MIvIF'(s) associated with specific pathological conditions can be enhanced while leaving non-involved MMPs less affected.
SUBSTITUTE SHEET (RULE 26~

The method of treating matrix metalloprotease-mediated conditions may be practiced in mammals. including humans, that exhibit such conditions.
The inhibitors of the present invention are contemplated for use in veterinary and human applications. For such purposes, they will be employed in pharmaceutical compositions containing active in~edient(s) plus one or more pharmaceutically acceptable carriers, diluents, fillers, binders.
and other excipients. depending on the administration mode and dosage form contemplated.
Administration of the inhibitors may be by any suitable mode known to those skilled in the art. Examples of suitable parenteral administration include intravenous, intraarcicular, subcutaneous and intramuscular routes. Intravenous administration can be used to obtain acute regulation of peak t 0 plasma concentrations of the drug. Improved half life and targeting of the drug to the joint cavities may be aided by entrapment of the drug in liposomes. It may be possible to improve the selectivity of tiposomal targeting to the joint cavities by incorporation of ligands into the outside of the liposomes that bind to svnovial-specific macromolecules. Alternatively intramuscular, intraarticular or subcutaneous depot injection with or without encapsulation of the drug into degradable 1 ~ microspheres e.g., comprising poly(DL-lactide-co-glycolide) may be used to obtain prolonged sustained drug release. For improved convenience of the dosage form it may be possible to use an i.p. implanted reservoir and septum such as the Percuseal system available from Pharmacia.
Improved convenience and patient compliance may also be achieved by the use of either injector pens (e.g. the Novo Pin or Q-pen) or needle-free jet injectors (e.g. from Bioject, Mediject or Becton ?0 Dickinson). Prolonged zero-order or other precisely controlled release such as pulsatile release can also be achieved as needed using implantable pumps with delivery of the drug through a cannula SUBSTITUTE SHEET (RULE 26~

into tile svnovial spaces. Examples include the subcutaneously implanted osmotic pumps available from ALZA, such as the ALZET osmotic pump.
~iasat delivery may be achieved by incorporation of the drug into bioadhesive particulate carriers 1_'00 um) such as those comprising cellulose, polyacrylate or polycarbophil, in conjunction with suitable absorption enhancers such as phospholipids or acylcarnitines.
Available systems include those developed by DanBiosys and Scios Nova.
.~ noteworthy attribute of the compounds of the present invention in contrast to those of various peptidic compounds referenced in the background section of this application is the demonstrated oral activity of the present compounds. Certain compounds have shown oral bioavailabiliry in various animal models of up to 90 - 98 %. Oral delivery may be achieved by incorporation of the drug into tablets, coated tablets, dragees, hard and soft gelatine capsules, solutions, emulsions or suspensions. Oral delivery may also be achieved by incorporation of the drug into enteric coated capsules designed to release the drug into the colon where digestive protease activity is low. Examples include the OROS-CT/OsmetTM and PULSINCAPT'" systems from ALZA
1 ~ and Scherer Drug Delivery Systems respectively. Other systems use azo-crosslinked polymers that are degraded by colon specific bacterial azoreductases, or pH sensitive polyacrylate polymers that are activated by the rise in pH at the colon. The above systems may be used in conjunction with a wide range of available absorption enhancers.
Rectal delivery may be achieved by incorporation of the drug into suppositories.
''0 The compounds of this invention can be manufactured into the above listed formulations by the addition of various therapeutically inert, inorganic or organic carriers well known to those skilled in the art. Examples of these include, but are not limited to, lactose, corn starch or derivatives !f t SUBSTITUTE SHEET (RULE 2b~

thereof, talc, vegetable oils, waxes, fats, polyols such as polyethylene glycol, water, saccharose, alcohols, glycerin and the like. Various preservatives, emulsifiers, dispersants, flavorants, wetting aeents. antioxidants, sweeteners, colorants, stabilizers, salts, buffers and the like are also added, as required to assist in the stabilization of the formulation or to assist in increasing bioavailability of the active ingredients) or to yield a formulation of acceptable flavor or odor in the case of oral dosing.
The amount of the pharmaceutical composition to be employed will depend on the recipient and the condition being treated. The requisite amount may be determined without undue experimentation by protocols known to those skilled in the art. Alternatively, the requisite amount l 0 may be calculated, based on a determination of the amount of target enzyme which must be inhibited in order to treat the condition.
The matrix metalloprotease inhibitors of the invention are useful not only for treatment of the physiological conditions discussed above, but are also useful in such activities as purification of metalloproteases and testing for matrix metalloprotease activity. Such activity testing can be both 1 s in vitro using natural or synthetic enzyme preparations or in vivo using, for example, animal models in which abnormal destructive enzyme levels are found spontaneously (use of genetically mutated or transgenic animals) or are induced by administration of exogenous agents or by surgery which disrupts joint stability.
,p E~,4MPLES
The following examples are offered for illustrative purposes only and are not intended, nor should they be construed, to limit the invention in any way.
9~3 SUE3STITUTE SHEET (RULE 26) General Procedt:res:
All reactions were performed in flame-dried or oven-dried glassware under a positive pressure of argon and were stirred magnetically unless otherwise indicated.
Sensitive liquids and solutions were transferred via syringe or cannula and were introduced into reaction vessels through rubber septa. Reaction product solutions were concentrated using a Buchi evaporator unless otherwise indicated.
Viaterials:
Commercial grade reagents and solvents were used without further purification except that diethyl ether and tetrahydrofuran were usually distilled under argon from benzophenone keryl, and methylene chloride was distilled under argon from calcium hydride. Many of the specialty organic or organometallic starting materials and reagents were obtained from Aldrich.
1001 West Saint Paul Avenue. Milwaukee, WI X3233. Solvents are often obtained from EM Science as distributed by V WR Scientific.
1 ~ Chromatography:
Analytical thin-layer chromatography (TLC) was performed on Whatmans pre-coated glass-backed silica gel 60 A F-254 250 ~cm plates. Visualization of spots was effected by one of the following techniques: (a) ultraviolet illumination, (b) expostue to iodine vapor, (c) immersion of the plate in a 10% solution of phosphomolybdic acid in ethanol followed by heating, and (d) ?0 immersion of the plate in a 3% solution of p-anisaldehyde in ethanol containing 0.5% concentrated suifiu~ic acid followed by heating.
Column chromatography was performed using 230-400 mesh EM Sciences silica gel.
SUBSTITUTE SHEET (RULE 26) Analytical high perfotmancc liquid chromatography (I-~LC) was performed at t mL min'' on a 4.6 x 250 mm Microsorb~ cohunn monitored at 288 nm. and semi-preparative HPLC was performed at 24 mL min' on a 21.4 x 250 mm Microsorb~ column monitored at 288 nm.
Instrumentation:
Melting points (mp) were determined with a Thomas-Hoover melting point apparatus and are uncorrected.
Proton ('H) nuclear magnetic resonance (NMR) spectra wire measured with a General Electric GN-OMEGA 300 (300 MHz) spectrometer, and carbon thirteen ("C) NMR
spectra were measured with a General Electric GN-OMEGA 300 (75 MHz) spectrometer. 'Most of the compounds synthesized in the experiments below were analyzed by nmr, and the spectra were consistent with the proposed strucnues in each case.
Mass spectral (MS) data were obtained on a Kratos Concept 1-H spectrometer by liquid-cesium secondary ion (LCIMS), an updated version of fast atom bombardment (FAB). Most 1 ~ of the compounds synthesized in the experiments below were analyzed by mass spectroscopy, and the spectra were consistent with the proposed structures in each case.
General Comments:
For mufti-step procedures, sequential steps are noted by numbers.
~5 SUBSTITUTE SHEET (RULE 26~

Examyle 1- Preyaration of Com~ionnd I

v ~ ~OEt 0 OEt Step 1 A dry 2-L, three-necked, round-bottomed flask was equipped with a stir bar. a pressure equalizing addition funnel, an argon inlet and a thermometer. The flask was charged with a suspension of sodium hydride (8.4 g of 95% NaH; 0.33 moi) in dry THF (700 mL) and was cooled with an ice water bath. Diethyl malonate (48.54 g, 0.30 mol) was added dropwise from the addition funnel over 25 min. Stirring was continued for 1.5 h before adding 1-bromo-3-phenylpropane (47 I O mL. -61 g, -0.30 mol) over 10 min via the addition funnel. Rinses of the addition funnel (THF. 3 x 10 mL) were added to the reaction mixture and stirring was continued for 30 min. The addition funnel and thermometer were replaced with a reflex condenser and stopper, and the reaction was heated at reflex for 19 h. The mixture was cooled to room temperature and then with an ice water bath. Distilled water (400 mL) was slowly added with stirring. The layers were separated and the 1 ~ aqueous phase was extracted with chloroform ( 100 mL). The combined organics were washed with 10% HCl (250 mL) and the separated aqueous phase was back-extracted with chloroform ( 100 mL).
The combined organics were washed with saturated NaHCO, (250 mL) and the separated aqueous phase was back-eacu~acted with chloroform ( 100 mL). The organics were dried (Na=SO,) and concentrated to yield a yellow oil which was purified by distillation through a Vigreux column at 20 reduced pressure (0.4 torr). The fiaction boiling at 124-138 'C was cleaa desired product (57.21 g, 0.206 mol; 68% yield). TLC (50% hexanes-dichloromethane): Rt= 0.32.
SUBSTITUTE SHEET (RULE 26~

O
Br Br Step 2 A 1-L, one-necked, round bottom flask was equipped with a rubber septum and an argon inlet. The flask was charged with a solution of commercially available 4-bromobiphenyl (~O.OOg, 0.'_ 1 ~ mol) in dichloromethane (100mL). Hromoacetyl bromide (2l.OmL. -t8.7g, 0.230 mot) was added via syringe and the solution was cooled with an ice water bath to 0 JC, while A1C13 (34.3g, 0.'_' ~ 8 mol) was added portionwise. Gas evolved >iom the opaque olive green reaction mixture.
l0 After 2~h at room temperature, the reaction mixture was cautiously poured into a cold saturated aqueous NaHCO~ solution. The resulting mixture was extracted with three 200mL
portions of ethyl acetate, and the combined organic layers were dried over Na,.SO, and concentrated to afford the desired pmduct as a yellow solid in quantitative yield. TLC (30%
dichloromethane-hexanes), Ri = 0.30.
l~
20 Step 3 A dry 2-L, three-necked, round-bottomed flask was equipped with a magnetic stir bar, as argon inlet, and a pressure equalizing addition funnel. The flask was charged with a solution of the product of step 1 {63.0 g, 0.227 mol) in THF (500 mL). "Ihe reaction vessel was cooled with an ice y~
SUBSTITUTE SHEET (RULE 26) water bath while soditun hydride (5.40 g of 9S% NaH, 0.214 mol) was added slowly in portions.
The reaction mvetvre was stirred for 1 h at 0 °C, and a solution of the product of step 2 (80.0 g, 0.31 ~
mol) in dt~.~ THF (300 mL) was added via addition funnel over ca. 20 min. The deep orange reaction mi~cture was stirred at room temperature under argon for 3 h. The reaction vessel was cooled in an ice water bath while distilled water ( 1 SO mL) was added cautiously. The aqueous phase was exnacted with three 300 mL portions of ethyl acetate, the combined organic phases were dried over VtgSO" and concentrated to afford 124 g of a dark orange oil. This material was used in the following operation without purification.
The orange oil was dissolved in 400 mL of 1:1 THF:methanol, and added to an aqueous ~aOH solution (4 N, S00 mL. '_'.00 mot). The reaction mixture was stirred for 24 h at room temperature, 48 h at SO °C, and 24 hotus at room temperature. The majority of MeOH was removed in vacuo and the residue extracted with a 200 mL portion of 1:1 ethyl acetate:hexanes and a 200 mL
portion of hexanes. The aqueous phase was acidified with HCI, extracted with two 200 mL portions, and three 100 mL portions of ethyl acetate. The combined organic phases were dried over MgSO, 1 ~ and concenuated to afford a quantitative yield of diacid. TLC ( 10%
methanol-chloroform with 1 acetic acid): R.r=0.45.
P~
Step 4 The unpurified diacid from step 3 was dissolved in 1,4-dioxane (500 mL) aad heated to reflex for 24 h. The solvent was removed in vacuo, and a 10 g portion of the residue SUBSTITUTE SHEET (RULE 26) chromatographed on silica gel (gradient elution with 10-50% ethyi acetate-hexanes containing 1 acetic acid) to afford 0.840 g ( 10%) of the desired product as a yellow solid. MP 174 °C.
0 Pb HOCHtC=C ~ ~ ~ ~ OH
I

Step 5 - .a one-necked, 15-mL, round-bottomed flask equipped with a rubber septum and an argon needle inlet was charged with '_'.6 mL of diethylamine, the product of step 4 (0.300 g, 0.667 mmol).
propargyl alcohol ( 1.0 mL, 0.96 g, 17 mmol), copper (I) iodide (0.0220 g, 0.11 ~ mmol), and craps-dichlorobis(triphenylphosphine)palladate (0.110 g, 0.157 mmol). The resulting mixture was stirred for .~ d at room temperature. The reaction mixture was concentrated (290 mg residue) and part of the residue (90 mg) was purified via column chromatography on 50 g of silica get {20% ethyl acetate-hexanes with 0.5% acetic acid) afforded the coupling product as a white solid (0.035 g, 40%) of coupling product as a white solid. MP 130 °C.
~xampte 2 and Example 3 - Preparation of Compounds II and I~
Example 2 and Example 3 were prepared by chiral separation of Example 1 on a Chiralcel ~.D r column (2 cm x 25 em) using 5% EtOH. 4.75% H,O and 0.095% HOAc in CH,CN, flow rate ?0 mLmin.
Example 2: First off Chiralcel AD ~ column; ' H NMR (300 N~iz, CDCI,) b 8.02 (d, J = 8.4 Hz, ?0 2H),7.67(d,J=8.7Hz,2H),7.58(d,J=8.7Hz,2H),7.53(d,J=8.4Hz,2H),7.17-7.33(m,5 H), 4.54 {s, 2 H), 3.46 (dd, J = 8.1, 16.8 Hz, 1 H), 3.14-3.02 (m, 2 H), 2.65 (t, J = 7.2 Hz, 2 H), 1.64-1.84 (m, 4 H).
g9 SUBSTITUTE SHEET (RULE 26~

Esampie 3: Second off Chiralcel AD ~ column; 'H NMR (300 MHz, CDCI,) E 8.02 (d. J = 8.4 Hz ' H).7.67(d,J=8.7Hz,2 H).7.58(d.J=8.7Hz,2H).7.53(d,J=8.4Hz2H),7.17-7.33 (m.~
H).-~.p4(s,2H),3..~6(dd,J=8.I, 16.8 Hz, 1 H),3.14-3.02(m,2H),2.65(t,J=7.2Hz,2H), 1.64-1. 84 ( m. 4 H ).
Example 6 - PreQaration of Compound VI
.~ one-necked, 10-mL, round-bottomed flask equipped with a rubber septum and an argon needle inlet was charged with 0.~ mL of pyridine, Example 1 (0.0070 g, 0.014 mmol), and acetic anhydride (0.020 mL. 22 mg, 0.21 mmol). The reaction mixture was stirred for 2 h at room temperature, and then added to 30 mL of 1N HC1. The resulting mixture was extracted with three 30 mL portions of ethyl acetate, the combined organic phases were dried over ~gSO,, and concentrated. Purification via HPLC (2.5% ethyl acetate-dichloromethane with 0.01%
trifluoroacetic acid) afforded 3 mg (38%) of Example 6. MP 137 °C.
Example 7 - Preparation of Compound VII
1 ~ ~ one-necked, 15-mL. round-bottomed flask equipped with a rubber septum and an argon needle inlet was charged with 2 mL of triethylamine, 2 mi of THF, compound I
(0.0570 g, 0.134 mmol), and ethyl chlorofotmate (0.032 mL. 36 mg, 0.34 mmol). The reaction mixture was stirred for 16 h at room temperature and then added to 50 mL of 1N HCI. The resulting mixture was extracted with three 50 mL portions of ethyl acetate, the combined organic phases were dried over ?0 VigSO,, and concentrated. Column chromatography on 10 g silica gel (40%
ethyl acetate-hexanes with 0.~% HOAc) followed by purification via HPLC (1.5% ethyl acetate-dichloromethane with 0.01% trifluoroacetic acid) afforded 1 mg (1.5%) of Example 7. MS (FAB-LSIMS) 499 [M+H)-.
So SUBSTITUTE SHEET (RULE 26) ExamRie 11- Preyaration of Compo~pd XI
:~ one-necked, 2~-mL. round-bottomed flask equipped with a rubber septum and an argon needle inlet was charged with 1 mL of CH:CI=, Example 12 (0.012 g, 0.026 mmol), and the Dess-Martin reagent (16 mg, 0.038 mmol) prepared according to Dess, et al.. J.
Org. Chem. 48.
4166. 1983. The resulting mixture was stirred for 30 min at 0 °C, diluted with 30 mL of ethyl acetate, and washed with two 20 mL portions of 1N HCI. The organic layer was dried over ~IgSO"
and concentrated. Purification via HPLC ( 1.6% ethyl acetate-dichloromethane with 0.01 trifluoroacetic acid) afforded 1 mg (9%) of Example 11. 'H NMR (300 MHz.
CDCI,) b 9.70 (t. J
= 1.3 Hz. 1 H), 8.05 (d, J = 8..~ Hz. 2 H), 7.70 (d, J = 8.4 Hz, 2 H), 7.65 (d. J = 8.4 Hz, 2 H). 7..~4 (d. J= 8.4 Hz. 2 H). 7.16-7.33 (m. ~ H), 3.46 (dd. J= 8.1, 16.8 Hz, 1 H), 3.14-3.02 (m. 2 H), 2.67 (t. J = 7.'_ Hz. 2 H), 2.48 (t, J = 7.5 Hz. 2 H). 2.41 (dt. J = 1.3 Hz and 6.3 Hz, 2 H), 1.96 (m, 2 H), 1.64-1.84 (m. 4 H).
The above methods for the preparation of Example 1, Example 2, Example 6, Example 7, and lrxample 11 were used to prepare the following series of biphenyl containing products.

SUBSTITUTE SHEET RULE 26~

WO 97!43245 PCT/LJS97/07921 rABLE I

0 Ph I ~ I ~ OH

comp R isomer m.p.(C)lother characterization I HOCH,C=C R.S 130 II HOCH,C=C S 'H NVtR (300 IvBiz. CDCI,) b 8.02 (d, J =

8.4 Hz, 2 H), 7.67 (d, l = 8.7 Hz.
2 H), 7.58 !d. J = 8.7 Hz, 2 H), 7.53 {d, J
= 8.4 Hz.
2 H), 7.17-7.33 (m, ~ H), 4.54 (s, 2 H), 3.46 (dd, J

= 8.1.16.8 Hz, 1 H), 3.02-3.14 (m, 2 H), 2.65 (t, J = 7.2 Hz. 2 H), 1.64-1.84 (m, 4 H).

III HOCH,_C=C R 'H NMR (300 IvB-Iz, CDCI,) b 8.02 (d, J =

8.4 Hz. 2 H), 7.67 (d, J
= 8.7 Hz, 2 H), 7.58 (d. J = 8.7 Hz, 2 H), 7.53 (d, J
= 8.4 Hz.
2 H), 7.17-7.33 (m, ~ H), 4.54 (s, 2 H), 3.46 {dd.
J

= 8.1,16.8 Hz, 1 H), 3.02-3.14 (m, 2 H), 2.65 (t, J = 7.2 Hz, 2 H), 1.64-1.84 (m, 4 H).

N MeOCH,C=C RS 136 V (-Pr),NCH,C=C R.S ' MS (FAB-LSIMS) 510 [M+H)' VI CH,CO,CH~C=C RS 137 VII EtOCO:CH:C=C R,S leis (FAB-LSIMS) 499 [M+H]' VIII HO(CH=)zC=C R,S 124 IX CH,CO:(CH~)~C=CR,S MS (FAB-LSIMS) 483 [M+H)-.

X HOzC(CH~C=C R,S 184 XI OHC(CHZ),C=C R,S 'H NMR
(300 MHz, CDCh) b 9.70 (t, J=

1.3 Hz, 1 H), 8.05 (d, J
= 8.4 Hz, 2 H), 7.70 (d, J = 8.4 Hz, 2 H), 7.65 (d, J
= 8.4 Hz, 2 H), 7.44 (d, J=
8.4 Hz, 2 H), 7.15-7.35 (m, 5 H), 3.46 (dd, J
= 8.1, 16.8 Hz, 1 H), 3 .14-3.02 (m, 2 H), 2.67 (t, J a 7.2 Hz, 2 H), 2.48 (t, J

= 7.5 Hz, 2 H), 2.41 (dt, J = 1.3 Hz and 6.3 Hz, 2 H), 1.96 (m, 2 H), 1.64-1.84 (m, 4 H).

fZ
SUBSTITUTE SHEET (RULE 26~

XII HO(CH,),C=C R.S 123 PhC=C R,S 154 XIV 3-HO-PhC=C RS 237 Eramgle 15 - Prenaratioe of Compound XV
0' ~ N' ~ 0 J
~0 0 Step 1 A solution of sodium hydride (4.35 g, 181 mmol) in freshly distilled THF (100 mL) was cooled to 0 °C and treated with commercially available diallyl malonate (35.0 g, 190 mmol) over :~0 min via a dropping funnel. After stirring at room temperature for 30 min.
:W(2-bromoethyl)phthalimide {43.9 g, 247 mmol) was added to the solution in one portion and the 1 ~ mixture was heated to reflux. After 48 h the solution was cooled to 0 °C, quenched with 2N HCl and concentrated to about 20% of its original volume. The concentrate was diluted with ethyl acetate (300 mL) and washed successively with saturated aqueous solutions of K:CO~ and NaCI.
The organic layer was dried over MgSO" filtered and concentrated under reduced pressure.
Purification by flash column chromatography (gradient elution with 5-25% ethyl acetate-hexanes) afforded diallyl 2-phthalimidoethylmalonate (41.2 g, 64%) as a colorless oil.
'H NMR (300 MHz, CDCI,) b 7.82 (m, 2H), 7.72 (m, 2H), 5.85 (m, 2H), 5.30 (m. 2H), 5.22 (m, 2H), 4.60 (m, 4H), 3.80 (t, J = 6.6 Hz, 2H), 3.46 (t, J = 7.2 Hz, 1 H), 2.30 (dd, J = 13.8, 6.9 Hz, 2H).

SUBSTITUTE SHEET (RULE 26) 0 CO=Allyl 0 CO,Allyl \ /

Br Step 2 A solution of the product of step 1 (x.20 g, 1.x.6 mmol) in treshly distilled THF (100 mL~
was cooled to 0 °C, while NaH (385 mg, 16.1 mmol) was slowly added.
After 40 minutes. the reaction mixture was warmed to room temperature, the product of Example l, step 2 (.~.>j g, 1.x.6 mmoi) was added in portions, and the miacture was stirred for 24 h. The reaction mixture was cooled to 0 'C, quenched slowly with 2~I HC1 (300 mL), extracted with one 150 mL
portion of dichloromethane and two 100 mL portions of dichloromethane. The combined organic phases were dried over MgSO" filtered and concentrated to afford 6.~0 g (71%} of the desired product which was used in step 3 without purification. 'TLC (30% ethyl acetate.hexanes): Rf=
0.4.
O
N
1> > \ /
Step 3 A solution of the product of step 2 (6.50 8,10.4 mmol) in 1,4-dioxane ( 100 mL) was cooled to 0 ~C, while tetrakis(triphenylphosphine)paliadium (0.180 g, 146 mmol) and pyrrolidine (2.40 mL, 29.'? mmol) were added sequentially. ABer stirring for 2 h at 0 'C and 4 h at room temperature, the reaction mixttae was poured into 2N HCl ( 100 mL). The resulting mixture was extracted with four 100 mL portions of dichloromethane, the combined organic phases were dried over MgSO,, and concentrated to give the diacid as a yellow solid (9.70 g). A 3.8 g sample of this material was dissolved in 1,4-dioxane (150 mL) and heated at reflex for 1 h. After cooling to room temperature, sy SUBSTITUTE SHEET (RULE 26~

the solution was concentrated and the residue was chromatographed on 300 g silica gel (gradient with ~%-15% methanol-dichloromethane) to give the desired acid (0.300 g) which was further punned ~1a recrystallization to afford 0.170 g (59% overall yield from step 21 of the desired product as a white cn~stalline solid. MP 209-210 °C.
j Ph~ 0 ~--C=CH
Step -t A one-necked, 100-mL. round-bottomed flask equipped with a rubber septum and an argon needle inlet containing 2 ml of THF was charged with NaH (435 mg, 17.2 mmol) and cooled to 0 'C while propargyl alcohol {1.0 mL, 0.963 g, 17.2 mmol) was added via syringe over ca. S min. The resulting mixture was stirred at 0 °C for 10 min and at room temperature for 30 min. Henzylbromide ( 1.8 ml. 2.59 g, 15.1 mmol) was added, the reaction mixture was stirred at room temperature for 36 h, poured into pentane (150 mL), and washed with a 100 mL portion of brine.
'Ihe solvent was removed via distillation and the residue (3.5 g of a yellow oil) was used in step 5 directly. 'H ~iR
(300 \~iHz, CDCI,) 5 7.36-7.31 (m. 5 H), 4.61 (s, 2H), 4.17 (d, J=2.4 Hz, 2 H), 2.47 (t, J= 2.4 Hz, 1 H).
\ /
O .N. ,0 O
Ph~O~C-C / \ / \ OH
O
Step 5 "f he procedure of Example 1, step 5 was used to prepare Example I 5 using the product from step 4 and the product of step 3 as starting materials. MP 151 ~C.
SUBSTITUTE SHEET (RULE 26~

xamp]e 16 - PreQaration of Compound XVI
t-BuM~SiO
~--C=CH
Step 1 ~ one-necked, 100-mL, round-bottomed flask equipped with a rubber septum and an argon needle inlet was charged with propargyl alcohol (1.0 mL. 0.963 g, 17.2 mmol), ether f20 ml). and cooled to 0 °C while NaH (435 mg, 17.2 mmol) was added siowly. The resulting mixture was stirred at room temperature for 1 h, and t-buryldimethylsilyl chloride (2.60 g, 17.2 mmol) was added.
The reaction mixture was stirred at room temperature for 6 h, poured into hexane ( 150 mL), and washed with 1 N HCI. The organic phase was dried over :vIgSO, and concentrated to atTord 2.88 g of a yellow oil which was used in step 2 without purification. 'H NMR (300 biz, CDCh) b 4.29 ~d. J = ~.1 Hz. 2 H), 2.37 (t. J=2.1 Hz, 1 H), 0.89 (s, 9 H), 0.11 (s, 3 H).

~ \ \ /
Step 2 The procedure of Example l, step 2 was used to prepare the desired acetyl biphenyl using commercially available 4-iodobiphenyl and aceryi chloride. TLC ( 10% ethyl acetate-hexanes): R~-1 ~ 0.3.

t-suMe=sio~ / \ / \
c-_-c Step 3 The procedure ofExample 1, step 5 was used to prepare the desired biphenyl acetylene using the product of step 1 and the product of step 2. TLC ( 10% ethyl acetate-hexanes): Rf- 0.4.

-SUBSTITUTE SHEET (RULE 26~

r-HuMeZSiO~ ~ ~ ~ ~ ~ 8r C=C
Step ~i .a one-necked. ~0-mL. round-bottomed flask equipped with a rubber septum and an argon s needle inlet was charged with ~ mL of THF, the product of step 3 (1.06 g, ?.94 mmol), and cooled to -78 'C while potassium hexamethyldisilazide (617 mg. ?.94 mmol) was added dropwise via svrinee. The reaction mixture was stirred at -78 °C for 30 min, trimethylsilyl chloride (0.374 mL.
0.3.0 g, '_.94 mmol) was added dropwise via syringe, and the resulting mixture was stirred at -78 'C for 3 h. The reaction mixture was warmed to 0 °C for 1 h. N
bromosuccinimide (0.540 g, 2.94 I 0 mmoll was added, and the mixture was allowed to warm to room temperature and stirred 16 h. The reaction mixture was poured into a 100 mL portion of aqueous saturated NH,CI, and extracted with three ~0 mL portions of dichloromethane. The combined organic phases were dried over MgSO, and concenuated. Column chromatography on 200 g of silica gel (gradient elution with 0- 5% ethyl acetate-hexanes) afforded 0.264 g (20%) of the bromomethyl ketone. 'TLC ( 10%
ethyl 1 ~ acetate-hexanes): R~ 0.5.
O _N_ ,O

t-BuMezSiO - OH
~-C=C / \ ~ /

ss SUBSTITUTE SHEET (RULE 26~

Step 5 The procedures of Example 13, steps 2-3 were used to prepare the desired biphenyl phthalimide using the product of step 4. TLC (~0% ethyl acetate-hexanes with 1 % acetic acid): R=
0.3.
\ /
0 -N- ' 0 HO - OH
~-C'-__C / \ \ /

Step 6 A one-necked. ~0-mi.. round-bottomed flask equipped with a rubber septum and an argon needle inlet was charged with 10 mL of CH_Cl,, the product from step ~ (0.040 g, 0.067 mmol), and '_ mL of 1-ff'-pyridine. The resulting mixture was stirred for 10 minutes at room temperature, diluted with a 7~ mL portion of water, and extracted with a 75 mL portion of CH=C1:.
The organic phase was dried over MgSO, and concentrated. Column chromatography on 5 g of silica gel (25% ethyl acetate-hexanes with 1 % HOAc) afforded 6 mg ( 19%) of Example 16. MP 145 °C.
1 ~ Example 17 Biological Assays of Invention Compounds P218 Quenched Fluorescence Assay for MMP Inhibition:
The P218 quenched fluorescence assay (Microfiuorometric Profiling Assay) is a modification of that originally described by Knight, et al.. FEBS Lett. ~, 263, 1992 for a related substance and a variety of matrix metalloproteinases (MMPs) in cuvettes. The assay was run with each invention compound and the three MMPs, Mmp-3, MMP-9 and Nlivlp-2, analyzed in parallel, adapted as follows for a 96-well microtiter plate and a Hamilton A'T~
workstation.

SUBSTITUTE SHEET (RULE 26~

P218 Fluoro~~stTate: P218 is a synthetic substrate containing a -~-acetyl-7-methoxycoumarin ( I~iCA) group in the N-terminal position and a 3-(2. -~-dinitrophenvl)-L-2.3-diaminopropionyl (DPA) group internally. This is a modification of a peptide reported by Knight ( 199'_'1 that was used as a substrate for matrix metalloproteinases. Once the P'_18 peptide is cleaved putative clip site at the Ala-Leu bond), the fluorescence of the MCA group can be detected on a tluorometer with excitation at 328 rtm and emission at 393 nm. P218 is currently being produced BACHE~t exclusively for Bayer. P218 has the structure:
H-VIC?~-Pro-Lys-Pro-Leu-Ala-Leu-DPA-Ala-Arg-NH2 (MW 1332.2) Recombinant Hutnan CHO Stromelvsin l:~:HP-31 Recombinant Human CHO Pro-;t-f.'l-fP-3: Human CHO pro-stromelysin-257 (pro-~L\~iP-3) was expressed and purified as described by Housley, et al., J. Biol. Chem.
?~,$, 4481. 1993.
.-lcrivation ojPro-~yfMP-3: Pro-MMP-3 at 1.72 ~M ( 100 ~g/mL) in 5 mM Tris at pH 7.5, 5 mNt CaCI,. ~5 mM NaCI, and 0.005% Brij-35 MMP-3) activation buffer) was activated by incubation with TPCK (N-tosyl-(L)-phenylalanine chloromethyl ketone) trypsin (1:100 wlw to pro-Vi:~tP-3) at 25 'C for 30 min. The reaction was stopped by addition of soybean trypsin inhibitor (SBTI; 5:1 Ww to trypsin concentration). This activation protocol results in the formation of 45 kDa active ~L~LP-3. which still contains the C-terminal portion of the enzyme.
Preparation of Human Rer~mbinant Pro-Gelatinase A IMMP-21:
Recombinant Human Pro-~~fMP-2: Human pro-gelatittase A (pro-MMP-2) was prepared ?0 using a vaccinia expression system according to the method of Fridman, et al., J. Biol. Chem. 2~7, 15398, 1992.
SUBSTITUTE SHEET (RULE 26) activation of Pro-~l~LtfP-2: Pro-MMP-2 at 252 mglmL was diluted 1:5 to a final concentration of SO p.glmL solution in 25 mM Tris at pH 7.5, ~ mM CaCI,. 1 ~0 mM NaCI, and 0.005°'° Brij-35 (1~-2 activation buffer). p-Aminophenylmercuric acetate ( APMA) was prepared in 10 m-~t c3.~ mymL) in 0.05 NaOH. The AP'v1A solution was added at l;'20 the reaction volume for a final AMPA concentration of 0.~ mM, and the enzyme was incubated at 37 °C for 30 min.
:activated MMP-2 (l5 mL) was dialyzed twice vs. 2 L of MMP-2 activation buffer (dialysis membranes were pre-treated with a solution consisting of 0.1 % BSA in bLl~tP-2 activation buffer for 1 min. followed by extensive H,O washing). The enzyme was concentrated on Centricon concentrators (concentrators were also pre-treated a solution consisting of 0.1% BSA in MMP-2 activation buffer for 1 min.. followed by washing with H,O. then MMP-2 activation buffer) with re-dilution followed by re~oncentration repeated twice. The enzyme was diluted to 7.5 mL (0.5 times the original volume) with MNLP-2 activation buffer.
Preparation of Human Recombinant Pro-Gelatinase B (l4iMP-9)~
Recombinant Human Pro-~~fMP-9: Human pro-gelatinase B (pro-I~iMP-9) derived from 1 ~ L;937 cDNA as described by Wilhelm, et al. J. Biol. Chem. ~~,~4, 17213, 1989 was expressed as the full-length form using a baculovirus protein expression system. The pro-enzyme was purified using methods previously described by Hibbs, et a1.1. Biol. Chem. 2~Q, 2493, 1984.
Activation ojPro-MMP-9: Pro-MMP-2 20 pg/mL in 50 mM Tris at pH 7.4, l OmM
CaCI,, 1 ~0 mM NaCI, and 0.005% Btij-35 (I~-9 activation buffer) was activated by incubation with 0.5 30 mMp-aminophetrylmercuric acetate (APMA) for 3.5 h at 37 °C. The enzyme was dialyzed against the same buffer to revmove the A.PMA.
SUBSTITUTE SNEET (RULE 26~

he strumeatatios:
Hamiltion .~ficrolab .~T Plus: The :vL'viP-Profiling Assay is performed robotically on a Hamilton ~LicroLab AT PlusD. The Hamilton is programmed to: ( 1 ) serially dilute up to 11 potential inhibitors automatically from a 2.~ mM stock in 100% DMSO; (2) distribute substrate followed by inhibitor into a 96 well Cytofluor plate; and (3) add a single enzyme to the plate with mixing to start the reaction. Subsequent plates for each additional enzyme are prepared automatically by beginning the program at the substrate addition point, remixing the diluted inhibitors and beginning the reaction by addition of enzyrrte. In this way, all M~ assays were done using the same inhibitor dilutions.
.tfillipore Cvtofluor 1l. Following incubation, the plate was read on a Cy~tofluor II
fluorometric plate reader with excitation at 340 nM and emission at 395 nM
with the gain set at 80.
Buffe 1.licrofluoromerric Reaction Buffer (MRB): Dilution of test compounds, enzymes, and P~ 18 substrate for the microfluorometric assay were made in microfluorometric reaction buffer consisting 1 ~ of ~0 mM 2-(tai-morpholinokthanesulfonic acid (IvIES) at pH 6.5 with 10 mM
CaCI:, 150 mM NaCI, 0.005% Brij-3~ and 1% DMSO.
a h s:
~I~f~NP .'I~ficrofluorometric Prof ling Assay. The assay is done with a final substrate concentration of 6 p.M P218 and approximately .~ to .8 nM'.~LMP with variable drug concentrations.
'_'0 The Hamilton is programmed to serially dilute up to 11 compounds from a 2.5 mM stock (100%
DMSO) to lOx the final compounds concentrations in the assay. Initially, the instrument delivers various amounts of microfluoromentric reaction buffer (MRB) to a 96 tube rack of 1 ral Marsh SUBSTITUTE SHEET (RULE 26) WO 97!43245 PCT/ITS97/07921 dilution tubes. the instrument then picks up 20 ~1 of inhibitor (2.5 mM) from the sample rack and mixes it with a buffer in row A of the Marsh rack, resulting in a ~0 ~tM drug concentration. The inhibitors are then serially diluted to 10, 5, 1. .2, .OS and .01 ~M. Position 1 on the sample rack contains only DViSO for the "enzyme-only" wells in the assay, which results in no inhibitor in column 1, rows A through H. The instrument then distributes 107 u! of P? 18 substrate (8.2 uM in VtRB) to a single 96 well cvtofluor microtiter plate. The instrument re-mixes and loads 14.~ ~1 of diluted compound from rows A to G in the Marsh rack to corresponding rows in the microtiter plate.
( Row H represents the "background" row and 39.5 ~l of MR.B is delivered in placed of drug or enzyme). The reaction is started by adding 25 ~1 of the appropriate enzyme (at 5.86 times the final enzyme concentration) from a BSA treated reagent reservoir to each well, excluding Row H, the "background" row. (The enzyme reservoir is pretreated with 1% HSA in ~0 m:'vf Tris, pH 7.5 containing 150 mM NaC 1 for 1 hour at room temp., followed by extensive H,O
washing and drying at room temp.).
~~frer addition and mixing of the enzyme, the plate is covered and incubated for 25 min. at 1 ~ 37 'C. Additional enzymes are tested in the same manner by beginning the Hamilton program with the distribution of P218 substrate to the microtiter plate, followed by re-mixing and distribution of the drug from the same Marsh rack'to the microtiter plate. The second (or third, etc.) MMP to be tested is then distributed from a reagent rack to the microtiter plate with mixing, prior to covering and incubation. This is repeated for all additional MMP's to be tested.
lCSO Determination in Micro~luorornetric Assay: Data generated on the Cytofluor II is copied from an exported ".CSV" file to a master Excel spreadsheet. Data from several different MMPs (one 96 well plate per MMP) were calculated simultaneously. The percent inhibition is SUSSTIT'UT"E SNEET (RULE 2'6~

determination for each drug concentration by comparing the amount of hydrolysis (fluorescence units generated over '_'~ minutes of hydrolysis) of wells containing compound with the wertzyme only" wells in column 1. Following subtraction of the background the percent inhibition was calculated as:
((Control values - Treated values)/Control values) x 100 Percent inhibitions were determined for inhibitor concentrations of 5, l, 0.~, 0.1, 0.02. 0.00 and.
0.001 ~M of drug. Linear regression analysis of percnet inhibition versus log inhibitor concentration was used to obtain IC,o values.
Tab a COMP.i~ MMP-3 Fluorogenic MMP-9 FluorogenicMMP2 Fluorogenic II 2184 I = 35% 252 VII 67 '460 103 Dt 57 542 37 X 203 I = 27% 108 XI 1730 I = 24% 596 XII 56.6 614 36 SUBSTITUTE SHEET (RULE 26) XIV 125 I = 46% 85 ~f V 11 ~CVt 4 2 1 Other embodiments of the invention will be apparent to those skilled in the art from a consideration of this specification or practice of the invention disclosed herein. It is intended that the specification and examples be considered as exemplary only, with the true scope and spirit of the invention being indicated by the following claims.
d~
SUBSTITUTE SHEET (RUtE 26)

Claims (12)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMS ARE DEFINED AS FOLLOWS:

1. ~A compound having the general formula:
and pharmaceutically acceptable salts thereof, wherein:
R15 is selected from the group of HOCH2, (n-Pr)2NCH2, CH3CO2CH2, EtOCO2CH2, HO(CH2)2, CH3CO2(CH2)2, HO2C(CH2)2, OHC(CH2)3, HO(CH2)4, 3-HO-Ph-, and PhCH2OCH2; and R16 is

2. A pharmaceutical composition comprising the compound according to claim 1 and a pharmaceutically acceptable carrier.

3. Use of a therapeutically effective amount of the compound according to claim 1 to inhibit a matrix metalloprotease in a mammal in need of such therapy.

4. Use of a therapeutically effective amount of the composition according to claim 2 to inhibit a matrix metalloprotease in a mammal in need of such therapy.

5. The use according to claim 3 or 4, wherein said effective amount is sufficient to:

(a) alleviate the effects of osteoarthritis, rheumatoid arthritis, septic arthritis, periodontal disease, corneal ulceration, proteinuria, aneurysmal aortic disease, dystrophobic epidermolysis, bullosa, conditions leading to inflammatory responses, osteopenias mediated by MMP activity. tempero mandibular joint disease, demyelating diseases of the nervous system;
(b) retard tumor metastasis or degenerative cartilage loss following traumatic joint injury;
(c) reduce coronary thrombosis from athrosclerotic plague rupture; or (c) effect birth control.

6. The use according to any one of claims 3, 4 or 5, wherein said mammal is a human.

7. The use according to claim 5, wherein the effect is to alleviate the effects of osteoarthritis.

8. The use according to claim 5, wherein the effect is to retard tumor metastasis.

9. Use of the compound of claim 1 in the preparation of a medicament.

10. The use according to claim 9, wherein the medicament is for inhibition of metalloprotease activity.

11. Use of the composition of claim 2 in the preparation of medicament.

12. A compound selected from the group:
I) R/S 4'-(3-hydroxy-1-propynyl)-.gamma.-oxo-.alpha.-(3-phenylpropyl)-[1,1'-biphenyl)-4-butanoic acid II) S-4'-(3-hydroxy-1-propynyl)-.gamma.-oxo-.alpha.-(3-phenylpropyl)-[1,1'-biphenyl]-4-butanoic acid, III) R-4'-(3-hydroxy-1-propynyl)-.gamma.-oxo-.alpha.-(3-phenylpropyl)-[1,1'-biphenyl]-4-butanoic acid, IV) ~4'-(3-methoxy-1-propynyl).gamma.-oxo-.alpha.-(3-phenylpropyl)-[1,1'-biphenyl]-4-butanoic acid, V) ~.gamma.-oxo-.alpha.-(3-phenylpropyl)-4'-(3-propyl~-1-hexynyl)-[1,1'-biphenyl]-4-butanoic acid, VI) ~4'-[3-(acetyloxy)-1-propynyl]-.gamma.-oxo-.alpha.-(3-phenylpropyl)-[1,1'-biphenyl]-4-butanoic acid.
VII) ~4'-[3-[(ethoxycarbonyl)oxy]-1-propynyl]-.gamma.-oxo-.alpha.-(3-phenylpropyl)-[1,1'-biphenyl]-4-butanoic acid.
VIII) 4'-(4-hydroxy-1-butynyl)-.gamma.-oxo-.alpha.-(3-phenylpropyl)-[1,1'-biphenyl]-4-butanoic acid, IX) ~4'-[3-(acetyloxy)-1-propynyl]-.gamma.-oxo-.alpha.-(3-phenylpropyl)-[1,1'-biphenyl]-4-butanoic acid, X) ~4'-(4-carboxy-1-butynyl)-.gamma.-oxo-.alpha.-(3-phenylpropyl)-[1,1'-biphenyl]-4-butanoic acid, XI) .gamma.-oxo-4'-(5-oxo-1-pentynyl)-.alpha.-(3-phenylpropyl)-[1,1'-biphenyl)-4-butanoic acid, XII) 4'-(6-hydroxy-1-henynyl)-.gamma.-oxo-.alpha.-(3-phenylpropyl)-[1,1'-biphenyl]-4-butanoic acid, XIII) .gamma.-oxo-4'-(phenylethynyl)-.alpha.-(3-phenylpropyl)-[1,1'-biphenyl]-4-butanoic acid, XIV)~ 4'-[3-hydroxyphenyl)ethynyl]-.gamma.-oxo-.alpha.-(3-phenylpropyl)-[1,1'-biphenyl)-4-butanoic acid, XV) ~1,3-dihydro-1,3-dioxo-.alpha.-[2-oxo-2-[4'-[3(phenylmethoxy)-1-propynyl][1,1'-biphenyl]-4-yl]ethyl]-2H-isoindole-2-butanoic acid, and XVI) ~1,3-dihydro-.alpha.-[2-[4'-(hydroxyethynyl)[1,1'-biphenyl]-4-yl]-2-oxoethyl]-1,3-dioxo-2H-isoindole-2-butanoic acid-