AU710759B2 - Inhibition of matrix metalloproteases by acetylene containing compounds - Google Patents

Description

WO 97/43245 PCT/US97/07921 Inhibitors of MPvt-3 could thus influence the activity of other destructive proteinases by modifying the level of their endogenous inhibitors.

A number of diseases are thought to be mediated by excess or undesired matrx-destroying metalloprotease activity or by an imbalance in the ratio of the MMPs to the TLMPs. These include: a) osteoarthntis (Woessner, et al., J. Biol.Chem. 259(6). 3633, 1984; Phadke, et al., J. Rheumatol.

852. 1983). b) rheumatoid arthritis (Mullins, et al.. Biochim. Biophys. Acta 695, 117. 1983: Woolley. et al.. Athritis Rheum. 20, 1231, 1977; Gravallese, et al., Arthritis Rheum. 34, 1076.

1991). c) septic arthritis (Williams, et al., Arthritis Rheum. 3 533, 1990), d) tumor metastasis SReich. et al., Cancer Res. 48, 3307. 1988, and Matrisian, et al., Proc. Nat'l. Acad. Sci., USA 83 9413. 1986), e) periodontal diseases (Overall, et al., J. Periodontal Res. 2, 81, 1987), f) corneal ulceration (Bums. et al., Invest. Opthalmol. Vis. Sci. 30, 1569, 1989), g) proteinuria (Baricos, et al., Biochem. J. 254, 609, 1988), h) coronary thrombosis from atherosclerotic plaque rupture (Henney, et al.. Proc. Nat'l. Acad. Sci., USA 88, 8154-8158, 1991), i) aneurysmal aortic disease (Vine, et al., Clin. Sci. 81, 233, 1991), j) birth control (Woessner, et al.. Steroids 54 491, 1989), k) dystrophobic epidermolysis bullosa (Kronberger, et al., J. Invest. Dermatol. 29 208, 1982), and I) degenerative cartilage 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. 5, 688, 1988).

The need for new therapies is especially important in the case of arthritic diseases. The primary disabling effect of osteoarthritis 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) WO 97/43245 PCT/US97/07921 inflammatory drugs (NSAIDs) 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. MMP inhibitors are expected to halt or reverse the progression of cartilage loss and obviate or delay surgical intervention.

Proteases are critical elements at several stages in the progression of metastatic cancer. In this process. the proteolytic degradation of structural protein in the basal membrane allows for expansion 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 types of proteases have shown that the matrix metalloproteases play a dominant role in these processes in particular gelatinases A and B (MMP-2 and MMP-9. respectively). For an overview of this field see Mullins. et al., Biochim. Biophys. Acta 695, 177, 1983; Ray, et al., Eur. Respir. J. 7, 2062, 1994; Birkedal-Hansen, et al., Crit. Rev. Oral Biol. Med. 4, 197, 1993.

Furthermore, it was demonstrated that inhibition of degradation of extracellular matrix by the native matrix metalloprotease inhibitor TIMP-2 (a protein) arrests cancer growth (DeClerck, et al., Cancer Res. 2, 701, 1992) and that TIMP-2 inhibits tumor-induced angiogenesis in experimental systems (Moses. et al. Science 28, 1408, 1990). For a review, see DeClerck, et al., Ann. N. Y. Acad. Sci. 732, 222, 1994. It was further demonstrated that the synthetic matrix metalloprotease inhibitor batimastat when given intraperitoneally inhibits human colon tumor growth and spread in an orthotopic model in nude mice (Wang, et al. Cancer Res. 54 4726, 1994) and prolongs the survival of mice bearing human ovarian carcinoma xenografts (Davies, et. al., 3 SUBSTITUTE SHEET (RULE 26) SWO 97/43245 PCT/US97/07921 Cancer Res. 53 2087. 1993). The use of this and related compounds has been described in Brown.

et al.. WO-9321942 A2.

There are several patents and patent applications claiming the use of metalloproteinase inhbitors for the retardation of metastatic cancer, promoting tumor regression. inhibiting cancer cell proliferation, slowing or preventing cartilage loss associated with osteoarthritis or for treatment of other diseases as noted above Levy. et al.. WO-9519965 A1; Beckett. et al.. WO-9519956 A Beckert.et al. WO-9519957 Al: Beckett, et al.. WO-9519961 Al; Brown. et al.. WO-9321942 .A2: Crimmin. etal., WO-9421625 Al; Dickens, et al.. U.S. Pat. No. 4,599,361: Hughes, et al., U.S. Pat.

No. 5.190.937; Broadhurst, et al.. EP 574758 Al; Broadhurst, et al., EP 276436; and Myers, et al., EP 520573 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 proteolytic metabolism, thus having short half lives.

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 hypocholesterolemic agents. In none of these examples is a reference made to MMP 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) WO 97/43245 PCT/US97/07921 Specificall. Torncufcik, et al., US patent 3.784,701 claims certain substituted benzovlpropionj'c acids to treat inflamrmation and pain. These compounds include S -biphenovipropanoic acid fenbufen) shown below.

\/OH

0 Fenbufen Child. et al.. J. Pharm. Sci. 66, 466, 1977 describes structure-activity relationships of several analogs of fenbufen. These include several compounds in which the biphenyi ring systemn is substituted or the propanoic acid portion is substituted with phenyl, halogen, hydroxyl or methyl, or the carboxylic acid or carbonyl fuinctions are converted to a variety of derivatives. No compounds are described which contain a 4'-substituted biphenyl and a substituted propanoic acid portion combined in one molecule. The phenyl (compounds XLIX and DMCU) and methyl (compound XLVII) substituted compounds shown below were described as inactive.

0

H

OOH

Kamneo, etal., Chemn. Pharm. Bull. 2050, 198 8 and Tornizawa, et al.. JP patent 62132825 A2 describe certain substituted 3 -biphenoylpropiomic acid derivatives and analogs thereof including SUBSTTUTE SHEET (RULE 26) WO 97/43245 PCT/US97/07921 the following. Various compounds with other substiruents on~ the propionic acid portion are described. but they do not contain biphenyl residues.

0 wherein X H. 4'-Br. 4 t -Cl. 4'-CH 3 or 2 '-Br.

Cousse. et al., Eur. J. Med. Chemi. 1987 describe the following methyl and methylene substituted 3 -biphenoyl-propanoic and -propenoic acids. The corresponding compounds in which the carbonyi is replaced with either CHOH or CH-, are also described.

a 0 0 x 7 OH

OH

0 x ~OH 0 0 wherein X Cl. Br. CHO, F, or NH 2 Nichi, et al. DE patent 1957750 also describes certain of the above methylene substituted biphenovipropanoic acids.

EI-Hashash, et al., Revue Rouni. Chim. 2, 1581, 1978 describe products derived from f3-arovl-acrylic acid epoxides including the following biphenyl compound. No compounds.

.0 substituted on the biphenyl portion are described.

SUBSTITUTE SHEET (RULE 26) WO 97/43245 PCTIUJS97/07921

CH)

0

OH

R 0 KTyes. et al.. J patent 253 sesreslown copoind asphn iopndsemediae The sbien theup isnterbstiute.

0 Sarnmour, et al., Egypt J. Chem. 15, 311. 1972 and Couquelet, et al., Bull. Soc. Chim. Fr.

2, 3196, 1971 describe certain dialkylamnino substituted biphenoylpropanoic acids including the following. In no case is the biphenyl group substituted.

R' R 2 0 wherein R' alkyl, benzyl. H, or, together with the nitrogen, morpholinyl.

I7 SUBSTITUTE SHEET (RULE 26) WO 97/43245 PCT/US97/07921 Others have disclosed a series of biphenyi-contairung carboxylic acids, illustrated by the compound shown below, which inhibit neural endopeptidase (NE? 24.11), a membrane-bound zinc metalloprotease (Stanton. et al., Bioorg. Mied. Chem. Lent. 4, 539, 1994; Lombaert, et al.. Bioorg-.

Med. Chem. Len. 2715. 1994: Lombaert etal.. Bioorg. Med. Chem. Let.t. 145. 1995: Lombaert.

et al., Bioorg. Med. Chem. Lett. .,15 1. 1995).

0 0 PhO- P N,,_C2 PhO It has been reported that N-carboxyalkvl derivatives containing a biphenviethyiglycine.

illustrated by the compound shown below, are inhibitors of stromelysin-1 MP-) 72 kDA gelatinase (MMP-2) and collagenase (Durette, et al.. WO-9529689).

F

Nzt Ph.4

CH

3

CH)

CH

3 It would be desirable to have effective MMND inhibitors which possess improved bioavailability and biological stability relative to the peptide-based compounds of the prior art, and 8 SUBSTITUTE SHEET (RULE 26) WO 97/43245 PCT/US97/07921 which can be optimized for use against particular target NM~s. Such compounds are the subject of the present application.

The development of efficacious M1vfP inhibitors would afford new therapies for diseases mediated by the presence of. or an excess of M'v~f activity, including osteoartu-itis. rheumatoid septic arthritis. tumor metastasis, periodontal diseases. corneal ulcerations, and proteinuria.

Several Inhibitors of MIMPs have been described in the literature, including thiols (Beszant. et al..

J. NMed. Chem. 403)0, 199'3). hydroxamic acids (Wahl. et al. Bioorg. Mied. Chem. Lett. 49.

1995. Conway, et al. J. Exp. Med. jJ, 449, 1995; Porter, et al., Bioorg. Med. Chem. Lett. 4, 274 1, 1994: TomcvzIc. et al.. Bioorg. Med. Chem. Lett. 5, 343, 1995; Castelhano, et al.. Bioorg. Med.

Chem. Lett. i, 1415, 1995), phosphorous-based acids (Bird, et al. J. M~ed. Chem. 37, 158, 1994: Morphv. etal., Bioorg. Med. Chem. Lett. 4, 2747, 1994; Kortvlewicz, et al.. J. Med. Chem. 3.263.

1990). and carboxylic acids (Chapman. et al. J. Med. Chem. 36 4293, 1993; Brown, et al., J. Med.

Chem. 37, 674. 1994; Morphy, et al., Bioorg. Med. Chem. Lett. 4, 2747, 1994; Stack et al.. Arch.

Biocem. iopys. .i7 240, 1991: Ye. et al., J. Med. Chem. 37, 206, 1994, Grobelny, et a.

Biochemistry 24, 6145, 1985; Mookhtiar. et al., Biochemistry 27 4299. 1988). However, these inhibitors aenerally contain peptidic backbones, and thus usually exhibit low oral bioactivity due to poor absorption and short half lives due to rapid proteolysis. Therefore, there remains a need for improved MMP inhibitors.

SUMMARY OF THE rNVENTION 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) WO 97/43245 PCT/US97/07921 to which MIMP'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: Ia) alleviate the effects of osteoarthritis, rheumatoid arthritis, septic arthritis. periodontal disease, coreal 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; retard tumor metastasis or degenerative cartilage loss following traumatic joint injury; reduce coronary thrombosis from athrosclerotic plaque rupture; or effect birth control The compounds of the present invention are also useful scientific research tools for studying functions and mechanisms of action of matrix metalloproteases in both in vivo and in vitro systems.

Because of their MMP-inhibiting activity, the present compounds can be used to modulate

MMP

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 the generalized formula: T,A-B-D-E-G.

(L)

/o SUBSTITUTE SHEET (RULE 26) WO 97/43245 PCT/US97/07921 In the above generalized formula T,A represents a substituted or unsubstituted aromatic 6-membered ring or heteroaromatic 5 6 membered ring containing I 2 atoms independently selected from the group of N, O, or S. T represents a substituted acetylenic moiety.

In the generalized formula B represents an aromatic 6 -membered ring or a heteroaromatic 5 6 membered ring containing I 2 atoms independently selected from the group of N. 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 D represents S N ,H H C=O C=NOH C=S C C S0 'OH /'H In the generalized formula E represents a chain of n carbon atoms bearing m substituents

R

6 in which the R 6 groups are independent substituents, or constitute spiro or nonspiro rings. Rings may be formed in two ways: a) two groups Rare joined, and taken together with the chain atom(s) to which the two R 6 group(s) are attached, and any intervening chain atoms, constitute a 3 7 membered ring, or b) one group R. is joined to the chain on which this one group R 6 resides, and taken together with the chain atom(s) to which the R 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 R' substituents is an integer of I 3. The number of carbons in the totality of R 6 groups is at least two.

Each group R' is alkyl, alkenyl, alkynyl, heteroaryl, non-aromatic cyclic, and combinations thereof optionally substituted with one or more heteroatoms as described more fully below. In the SUBSTITUTE SHEET (RULE 26) WO 97/43245 PCTIUS97/07921 gleneralized formula E is a substituted mono- or bicyc lic moiety optionally substituted wihone or more heteroatorns.

In the generalized formula G represents

-PO

3

-M.

00 R1 3 -C-N-C-M -C-N-C-M or H 14 H'N in which M represents -COH. or -COR' 2 and R" 3 represents any of the side chains of the 19 noncyciic naturally occurring amino acids.

The most preferred compounds of the invention are: 0 R16

OH

R -C C wvhere R" is selected from the group comprising:

HOCH

2 MeOCH., (n-Pr),NCH,,

CH,CO,CH,,

EtOCO,CH,, HO(CH4,,

,CH

3 C0:(CH,,

HOC(CH

2 2

OHC(CH

2 3

HO(CH

2 Ph, 3H0..Ph. and PhCH.OCH,; and R 1 6 is Ph or 0 Pharmaceutically acceptable salts of these compounds are also within the scope of the invention.

In most related reference compounds of the prior art, the biphenyl portion of the molecule 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 been reported to cause prior art compounds to be inactive as anti-inflarnmatory analgesic agents. See, for example, Child, et al., J.

SUBSTITUTE SHEET (RULE 26) WO 97/43245 PCT/US97/07921 Pharm. Sci. 66, 466 (1977). By contrast. it has now been found that compounds which exhibit potent MMP inhibitory activity contain a substituent of significant size on the propanoic or butanoic portion of the molecule. The biphenyl portions of the best MvMP inhibitors also preferably contain a substiruent on the 4' position, although when the propanoic or butanoic portions are optimally substituted, the unsubstituted biphenyl compounds of the invention have sufficient activity to be considered realistic drug candidates.

The foregoing merely summarizes certain aspects of the present invention and is not intended, nor should it be construed, to limit the invention in any way. All of the patents and other publications recited in this specification are hereby incorporated by reference in their entirety.

DESCRIPTION OF THE PREFERRED

EMBODIMENTS

More particularly, the compounds of the present invention are materials having matrix metalloprotease inhibitory activity and the generalized formula: T,A-B-D-E-G

(L)

in which T,A represents a substituted or unsubstituted aromatic or heteroaromatic moiety selected from the group consisting of:

N

RTN S T N

R'

X "Oo N T 13 SUBSTITUTE SHEET (RULE 26) SWO 97/43245 PCT/US97/07921 in which R' represents H or alkyl of I 3 carbons.

Throuhout this application in the displayed chemical structures, an open bond indicates the point at which the structure joins to another group. For example, Rs where R is is the structure 0^X) In these structures, the aromatic ring is referred to as the A ring or A unit, and T represents a substituent group, referred to as a T group or T unit. T is a substituted acetylenic moiety and x is

I.

The B ring of generalized formula is a substituted or unsubstituted 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, such that they would be detrimental. Such substituents may be moieties such as lower alkyl, lower alkoxy, CN, NO,, halogen, etc., but are not to be limited to such groups.

In the generalized formula B represents an aromatic or heteroaromatic ring selected from the group consisting of:

/H

SUBSTITUTE SHEET (RULE 26) WO 97/43245 PCT/US97/07921 R1

N

N

N s

N

R

R

N

r 0

N

oN k 1 0 17

N

/-N

Ri

N

N

N0

N

N

NN

in which R' is defined as above. These rings are referred to as the B ring or B unit.

In the generalized formula D represents the moieties: ,H H C=0 C=NOH C=S H

CCH

'OH

H

In the generalized formula E represents a chain of n carbon atoms bearing m substituents referred to as R'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 chain atom(s) to which the two R 6 group(s) are attached, and any intervening chain atoms, constitute a 3 7 membered ring, or b) one group R' is joined to the chain on which this one group R 6 resides, and taken together with the chain atom(s) to which the R' group is attached, and SUBSTITUTE SHEET (RULE 26) WO 97/43245 PCT/US97/07921 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 R 6 substituents is an integer of I 3. The number of carbons in the totality ofR 6 groups is at least two.

Each group R 6 is independently selected from the group consisting of the substituents listed S below as items 1) 14).

1) alkyl of I 10 carbons, provided that if the A unit is phenyl, the B unit is phenylene. m is 1. n is 2. and the alkyl group is located on the alpha carbon relative to the D unit. then x is I or 2; 2) aryl of 6 10 carbons, provided that if the A unit is phenyl, the B unit is phenylene, the aryl group is phenyl. n is 2. and m is I or 2, then x is I or 2; 3) heteroaryl comprising 4 9 carbons and at least one N, O, or S heteroatom; 4) arylalkyl in which the aryl portion contains 6 10 carbons and the alkyl portion contains 1 8 carbons: heteroaryl-alkyl in which the heteroaryl portion comprises 4 9 carbons and at 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 5 carbons; 8) heteroaryl-alkenyl in which the heteroaryl portion comprises 4 9 carbons and at least one N, O, or S heteroatom and the alkenyl portion contains 2 -5 carbons; 9) alkynyl of 2 10 carbons; /6 SUBSTITUTE SHEET (RULE 26) aryl-alklnyl in which the aryl portion contains 6 10 carbons and the alknvl portion contains 2 5 carbons; I 1) heteroaryl-alkynyl in which the heteroarvl portion comprises 4 9 carbons and at least one N. O. or S hereroatom and the alkvnyl portion contains 2 5 carbons: 12)-(CH),R 7 in which t is 0 or an integer of I 5 and R' is selected from the group consisting of: o 0 N -N-N o 0o

O

0 0 0 N 0 O0 R0 a. a -N -N N o

-N

0 10 R 2 0 Rc RD R 0 to :I II 11I

-N-C-N-R

2

-N-C-OR'

-N Y NY -R as well as corresponding heteroaryl moieties in which the aryl portion of an ayl-co ging

R

7 group comprises 4 9 carbons and at least one N,0O, or S heteroatom. In such R' groups, Y represents O or S; R' is as defined above, and u is 0, 1, or 2 provided that when R' is

N

-N Y N 2 or and the A unit is phenyl, the B unit is phenviene. m is 1. n is 2. and t is 0. then x is I or 2.

I3)-(CH.),ZR in which v is an interger of 1 to 4, Z represents or and R' is selected from the group consisting of alkyl of I to 12 carbons. aryl of 6 to carbons, heteroaryl comprising 4 to 9 carbons and at least one N. O. or S heteroatom: arvlalkyl in which the aryl portion contains 6 to 12 carbons and the alkyl portion contains I to 4 carbons: hetemroarvialkyl in which the aryl portion contains 6 to 12 carbons and at least one N. O. or S hereroatom and the alkyl portion contains I to 4 carbons; in which the R' represents alkyl of 2 to 6 carbons, aryl of 6 to 10 carbons, heteroary comprising 4 to 9 carbons and at least one N. O. 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 N, 0.

or S hereroatom, and the alkvl portion contains 1 to 4 carbons with the provisos that when R'is Z is or when Z is R'may also be 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 2; and 0: 14)-(CH),SiR' 0 3 in which w is an integer of I to 3, and R 0 represents alkvi of 1 to 2 carbons.

In addition, aryl or heteroaryl portions of any of the T or R' groups optionally may bear up to two substituents selected from the group consisting of -(CH 2 -(CH 2 )0R", -(CHSR", (CH2)S(0)R". -(CH2SO2NR", in which both oxygen atoms are connected to the aryl ring, WO 97/43245 PCTIUS97/07921 (CH (CH,),CO,R (CH:),OCOR" .halogen. -CHO, -CF 3

-NO..

-CN. and -R 2 in which y is 0 4: R" represents H or alkvl of I 4 carbons: and R' represents alkvl of 1 4 carbons.

In the generalized formula G represents -PO,H, -M.

0 0 R 3

-N

-C-N-C-M -C-N-C-M or

N

in which M represents -COH. or -COR".

2 and R" represents any of the side chains of the 19 noncyclic naturally occurring amino acids.

Pharmaceutically acceptable salts of the compounds falling within the generalized formula are also within the invention.

The G unit is most preferably attached to the E unit at the carbon P 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 such as -CF, and -C 6 F 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 6 comprises a heteroaromatic ring. Preferred heteroaromatic ring-containing compounds are those in which the heteroaryl groups are heteroaryl of 4 -9 carbons comprising a 5 6 membered heteroaromatic ring containing O, S, or NR' when the 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 /9 SUBSTITUTE SHEET (RULE 26) WO 97/43245 PCT/US97/07921 carries one substituent group T on position 5. When B unit is thiophene, it is preferably connected through positions 2 and 5 to D and A units respectively.

In the generalized formula the A and B rings are preferably phenyl and phenylene.

respectively. the A ring 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 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 its embodiments, the invention relates to compounds of generalized formula in which the A ring is a substituted or unsubstituted phenyl group, the B ring is p-phenylene. and aryl portions of any aryl-containing R' 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 in which m is 1 and R 6 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 s_

C-CHCHR'COOH

in which x is I and 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: MeOCHC-C-, (nto SUBSTITUTE SHEET (RULE 26) WO 97/43245 PCT/US97/07921 Pr):NCH,C=C-, CHCOCHC=C-, EtOCOCH,C=C., HOCH,CC.C,

HO(CH,):CC-.

CHCO:(CH),C- HO,C(CHC--C.

OHC(CH,)

3 C HO(CH),C-C-. PhC-C.. 3-HO.PhC

C

and PhCHOCH,C-C-.

Other compounds of general formula in which R 6 is 7 have t as an integer of Preferred compounds of general formula in which R 6 is have v as an integer of 1-4 and Z as or Additional compounds of general formula in which R 6 is alkvl contain 4 or more carbons in said alkyl and those in which R 6 is arylalkyl contain 2-3 carbons in the alkvl portion of said arylalkyl.

In another of its embodiments, the invention relates to compounds of generalized formula in which the number of substituents m on the E unit is 2 or 3; and when m is 2, both groups R 6 are independent substituents, or together constitute a spiro ring, or one group R 6 is an independent substituent and the other constitutes a spiro ring; and when m is 3, two groups R 6 are independent substituents and one group R 6 constitutes a ring, or two groups R constitute a ring and one group R" is an independent substituent, or three groups R 6 are independent substituents. This subset 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 R 6 groups are spiro rings, and in the trisubstituted case, the R 6 groups may form either spiro or nonspiro rings.

In another of its embodiments, the invention relates to compounds of generalized formula in which the number of substituents m on the E unit is I or 2; and when m is I, the group R 6 constitutes a nonspiro ring; and when m is 2, both groups R 6 together constitute a nonspiro ring or one group R 6 is an independent substituent and the other constitutes a nonspiro ring. This subset

I

SUBSTITUTE SHEET (RULE 26) WO 97/43245 PCTIUS97/07921 therefore contains compounds in which the E Unit carries one or two substituents Re. and at least one of these substituents is involved in a nonspiro ring.

Mfore particularly, representative compounds Of generalized formula in which one or more of the substituent groups R' are involved in formation of nonpr ring have E units of the following structures:

(C,

2 eakJ (H2.R6.C) (CR4H,- (CReH-.J \1 (R2 h 2 -0

(R

1 4 )k 1

(N

24

R

1 C) (CR.H 2 4 and in which ais 0, 1, or 2;b is 0orI; cis 0or 1; dis 0or 1; c+d is 0or 1; eis 1 5; fis 1 gis 3 5; h is 2 i is 0 j isO0- 3; k is0.-2; the total number of groups R is 0 1, or 2; U represents 0. S, or NR'; and z is 1 or 2; each group R" 4 is independently selected from the group consisting of.

ZZ

SUBSTITUTE SHEET (RULE 26) alkvl Of I 9 car-bons; arylalkyl in which the alkyl portion contan I abn n h rip ion contains 6 10 carbons: alkenyl of 2 9 carbons, aryl -substituted alkenyl in which the alkenyi portion contains 2 4 carbons and the aryl portion contains 6 10 carbons; alkynyl of 2 9 carbons: aryl-substiruted alkNmyl in which the alkynyl portion cont~ains 2 4 carbons and the ary portion contains 6 10 carbons; aryl of 6 10 carbons; -COR'; -CO,R 3 -CON(R 2 in which t is o or an integzer of I 4: and -(CH..)VZR 8 in which v is 0 or an integer of I to 3. and Z represents

-S-

or R 7 and RW have been defined above.

Preferred compounds of generalized formula in which one or more of the substiruent groups R' are involved in the formation of nonspiro rings have E units of the following st~ucrures: 2 R'C C O 4 tN6,R',C) /C4/" in whcI.b .d c+de ,i .tettlnubro mp .adR r sdfndaoe

I%~

0 R3 sPHr3-O ,n=0 *WO 97/43245 PCTIUS97/07921 Ntost preferably, T is: MeOCH,Cmc.. (n-Pr)2NCH,CnC-, CH3COCHC=C.

EtOCO-CH,C!=C- HOCH.C-=C-. HO(cH2):C CHCO,(Cl{,)2C=C.

HOC(CH.}C-=C.

OHC&'HI,C=C.

HO(CH,)

4 cCC. PhC=C-, 3 -HO-PhC=C.. and PhCH,OCH,C=C..

The subscript x. which defines the number of T substituents, is preferably I or 2, most preferably 1. and when x is I the T is preferably on the 4- position of ring A.

T'he A ring is preferably a phenvi or thiophene ring, most preferably phenyl.

The B ring is preferably a I 4 -phenylene or 2 .5-thiophene ring, most preferably I .4-phenviene.

The D unit is most preferably a carbonyl group.

In the E group.

R

6 is preferably: 1) arvlalkyl wherein the aryl portion contains 6 10 carbons and the alkyl portion contains I 8 carbons; 2) wherein t is 0 or an integer of I 5 and R' is an iznidoyl group containing an aromatic residue-, or 3) -(CH.,ZR8 wherein v is 0 or an integer of 1 Z is S or 0. and R' is aryl of 6 10 carbons or arvialkyl wherein the aryl portion contains 6 to 12 carbons and the alkyl portion contains 1 to 4 carbons.

The group R 6 is most preferably the following, wherein, any aromatic moiety is preferably substituted: 1) arylalkyl wherein the aryl portion is phenyl and the alkyl portion contains I 4 carbons; SUBSTITUTE SHEET (RULE 26) WO 97/43245 PCT/UJS97/07921 2) wherein iannegr1-3adR 7 isN(.nhtanedcbx 1 Isa nee ,adRi -1.-ahhln-iabx imidov N_23 npthl -dic~uarboxiuovi), or l.8.naphthalene.dicarboximido.i or N-phthalimidovl.

3-(CH4)ZR' wherein v is an integer of I 3. Z is S. and R' is phenyi.

The more preferred compounds of generalized formula have R' units of the following Structures: r" Ph or

N

to 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 MMP, regardless of their stereoisomenic designations, as well as mixtures of stereoisomers in which at least one member possesses inhibitory activity.

The most prefered compounds of the present invention are as indicated and named in the list below: I) R/S 4'-(3-hydroxy- l-propynyl-y-oxo-a.(3-phenylpropyl)( 1,1 '-biphenyl1-4-bumnoic acid.

11) S. 4'-{3-hydroxy- Il-propynyl)yoxo..a-(3.phenylpropy 1) 1,1 '-biphenyll-butanoic acid, 111) R4.{(3-hydroxy-l-propynyl).y-oxo.a(3 -phenyipropyi)[ 1,1 '-biphenyl]-4-butanoic acid, IV) 4 '-(3-methoxy- I -propynyl)-y-oxo-a-(3-phenylpropyl) 1,1 '-biphenyll.4-butanoic acid, V) y-oxoa(3phenyipropyl)-4-{3-pmpy.. 1 -hexynyl)-[ 1,1 I-biphenyl]-4-butanoic acid, SUBSTITUTE SHEET (RULE 26) WO 97/43245 PCTIUS97/07921 VI) 4 '-(3)-(acetyloxv)- l-propynyII-y.oxoa(3-phenipropyl) 1.1 -biphenvi-4buaoic acid.

VII' 4 [-[(ethoxvcarbonvl )oxv I propvnyll) -y.0 phenvi pro pvi 1,1 V-biphenyl 14 butanojc acid, VIII) 4-hvdroxN I -burvnvl)-Y-oxo-a-(3.phenylpropyl) 1.1 '-biphenvll-4-butanojc acid, DO) 4 '-[3-(acetyloxy)- Il.propymyi1.Y-Oxo-a(3phenvlpropy 1.1 F-biphenyll-4-butanoic acid, X) 4 '-(4-carboxy- I-butynvi -oxo-ct-(3 -phenyipropyl 1.1'-biphenyl 1-4-butanoic acid, XI) y-oxo-4'-(5.oxo- Il-penrqnyl)-a-(3 phenylpropyI)-[ 1.1 lbiphenvll..4-butanoic acid.

XII) 4'-(6-hydroxv- I-henynvi -oxo-a-(3 -phenylpropyl)- 1,1'-biphenyl

I-

4 -butanoic acid, XIII) '(-oxo- 4 '-(phenyiethynyl -phenylpropyl)r[ 1.1 '-biphenyl]..4-butanoic acid and XIV) 4 '-[3-hydroxyphenY)ethyy]-y-oxo-a-(3.phenvlpropyl)( 1,1 'biphenyll-4buanoic acid, XV) 1.3 -dihvdro-1.-ixoa[-oo2 I 3phnietoy--propynyl] [1,1 '-biphenyl]-4yl ]ethyl] -2H-isoindole..2 -buaoic acid. and XVI) 1.3 -dihydro-ct.[2.[4'-(hydroxyethynyl)[ 1,1 '-biphenyl)y]..v-.2.oxoethyl]. I,3-dioxo-2Hisoindole-2-butanoic acid.

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 presented below in the experimental section describing the working examples. All 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 2.6 SUBSTITUTE SHEET (RULE 26) WO 97/43245 PCT/US97/07921 with a given symbol (i.e R 9 is used more than once in a given structure, it is to be understood that each of these groups may be independently varied within the range of definitions for that symbol.

General Method A The compounds of this invention in which the rings A and B are substituted phenvl and phenylene respectively are conveniently prepared by use of a Friedel-Crafts reaction of a substituted biphenyl NM with an activated acyl-containing intermediate such as the succinic or glutaric anhydride derivative NMII or acid chloride MIV in the presence of a Lewis acid catalyst such as aluminum trichloride in an aprotic solvent such as 1.1.2.

2 -tetrachloroethane. The well known Friedel-Crafts reaction can be accomplished with use of many alternative solvents and acid catalysts as described by Berliner. Org. React., 5, 229. 1949 and Heaney, Comp. Org. Synth.

733. 1991.

If the anhydride MIII is monosubstituted or multiply-substituted in an unsymmetrical way, the raw product MI-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.

When they are not commercially available, the succinic anhydrides MIIL can be prepared via a Stobbe Condensation ofa dialkyl succinate with an aldehyde or ketone (resulting in side chain R 6 followed by catalytic hydrogenation, hydrolysis of a hemiester intermediate to a diacid. and then conversion to the anhydride MI 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 MIV. For a review of the Stobbe condensation, including lists of suitable solvents and bases see Johnson and Daub, Org. React. 6, 1, 1951.

SUBSTITUTE SHEET (RULE 26) WO 97/43245 PCTIUS97/07921 This method. as applied to the Preparation of NOf (R 6 isobutyl and H. n-penrvl). has been described Wolanin. et al.. US Patent 4,771.-038, Method A (T 0: vUI n-=2 or 3 or U UH- :0 Lewis acid Solvent 6 R 6 0 R1 2 0 OR' 6 0 MIf-A-2 MIffV n =2 or 3 MM MEIl-A n 0-3 Lewis acid Solvent (m 1 NU-A-3 Base

CO

2

H

MI-A-4 Method A is especially useful for the Preparation of cyclic compounds such as MI-A-3, in which two R 6 groups are connected in a methylene chain to form a 3-7 member ring. Small ring 28 SUBSTITUTE SHEET (RULE 26) WO 97/43245 PCT/US97/07921 member) anhydrides are readily available only as cis isomers which yield cis invention compounds NU-A-3. The trans compounds MI-A-4 are then prepared by treatment of MI-A-3 with a base such as DBU in THF. The substituted four member ring starting material anhydrides such as MIM-A

I

are formed in a photochemical 2-2 reaction as shown below. This method is especially useful for the preparation of compounds in which R" 4 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' 4 CH,OH can be converted to invention compounds with other R" 4 groups by using procedures described in General Method G.

0 0 0 0 0 0 1,,R 4 UJV light acetomtnile MV -Y MI-A-I R14 The Friedel-Crafts method is also useful when double bonds are found either between C-2 and C-3 ofa succinoyl chain (from maleic anhydride or l-cyclopentene-1,2-dicarboxylic anhydride, for example) 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 R 6 groups are found on one chain carbon together to form an exo-methylene group. Subsequent uses of these compounds are described in Methods D.

General Method B Alternatively 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 MVII, followed by alkylation with a halomethyl biphenyl ketone MVIII to yield intermediate MIX. Compounds of structure MDI are then hydrolyzed with aqueous base and heated SUBSTITUTE SHEET (RULE 26) WO 97/43245 PCT/US97/07921 to decarboxylate the malonic acid intermediate and yield MI-B-2 (Method By using one equivalent of aqueous base the esters MI-B-2 with R' as alkyl are obtained, and using more than two equivalents of base the acid compounds H) are obtained. Optionally, heat is not used and the diacid or acid-ester NMI-B-I is obtained.

Alternatively, the diester intermediate MIX 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 24 hr to yield MI-B-I (R' 2 Alternatively, the reaction of MVI with MVIII can be conducted before that with the alkyl halide to yield the same MIX (Method B-2).

Alternatively, a diester intermediate MXIX, which contains R' 2 allyl, can be exposed to Pd catalysts in the presence of pyrrolidine to yield MI-B-2 (R 2 H) (Dezeil, Tetrahedron Lett. 28, 4371. 1990.

Intermediates MVII are formed from biphenyls M in a Friedel-Craft reaction with haloacetyl halides such as bromoacetyl bromide or chloroacetyl chloride. Alternatively, the biphenyl can be reacted with acetyl chloride or acetic anhydride and the resultant product halogenated with. for example. bromine to yield intermediates MVI (X Br).

Method B has the advantage of yielding single regio isomers when Method A yields mixtures. Method B is especially useful when the side chains R 6 contain aromatic or heteroaromatic 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 6 group adjacent to the carboxyl of the final compound contains heteroatoms such as oxygen, sulfur, or nitrogen, or more complex functions such as imide rings.

SUBSTITUTE SHEET (RULE 26) WO 97/43245 METHOD B PCTIUS97/07921 -0 x ewis acid 0 1>TR6 Lewis acid 0

X

2) Halogeiinan6 MV MVI

(X-R

6 0 R1 0 [H

OR"

2 H OR 12 O MVl I Anh. base 0 R OR' 2 H OR'1 2 O MWI 0 -0 R 6

OH

\/1 MI-B- I MJ-B-2 When R' contains selected fuinctional groups Z, maionate 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 'itsunobu reaction (Mitsunobu, Synthesis 1, 198 Alternatively, the intermediate alcohol can be subjected to alkylation conditions to provide malonate MVII containing the desired Z group.

31 SUBSTITUTE SHEET (RULE 26) WO 97/43245 PCT/US97/07921 1) NaK TJF, e isoprenyl bronmide R 120 R141 2) 03, CHIC1I then R2 RO Na6BH, NeOH ROOR 2 0 0 3)MiUtunobu 0 y MV! 3) CBr 4 or Ph 3 P 0VI 4) Alkylation M"H± General Method C -Especiallv use fWz is the use of chiral HPLC to separate the enantiomers of racemic product mixtures (see, for example, Auit, et al., Chem. Int. Ed. Engl. j11 199 The compounds of this invention can be prepared as pure enantiomers by use of a chiral auxiliary route.

See. for example, Evans. Aidrichimica Acta, 23, 1982 and other similar references known to one skilled in the art.

General Method D -Compounds in which R' are alkyl- or aryl- or heteroarvl- or acvl- 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 crystallization from small amounts of isomeric MI-D-5. Alternatively, MI-D-5s 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-S are then reacted with a mercapto, derivative MXVII or MXVIII in the presence of catalyst such as potassium carbonate, ethyidiisobutylamine, tetrabutylammoniuxn fluoride or free radical initiators such as azobisisobutyronitrile (AIBN) in a solvent such as diethylforrnamide or tetrahydrofuran to yield invention compounds MI-D-2, MI-D-3, MI-D-6, or MI-D-7.

31- SUBSTITUTE SHEET (RULE 26) WO 97/43245 WO 9743245PCTIUS97/07921 Method D 0 KJ-

(R-

TX0 0 L e vis acid O F Solvent C a I or 2 O 6 R 6R'R 0 R'R' OH Formnaldehyde

~O

(T),4J 0 M. .4-D-4S 0 HS JR 9 0SR'

OH

CTX RR6 0 MI.D-2n or 0 (TX~ \/OH NG-D-3 R6 R 6OH 0 ,M-I-D-6

SR

or M-1-D-S S52 XI 0 Mv HS

,R

9 R 6R

OH

/n 0

SR

SUBSTITUTE SHEET (RULE 26) WO 97/43245 PCT/US97/07921 General Method E Biaryl 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 heteroaryl halide or triflate (trifluoromethane-sulfonate) or the like. In the equation below either Met 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(triphenyiphosphine)-palladium(O) or bis- (triphenylphosphine).

palladium(III) chloride. These methods are well known to those skilled in the art. See, for example.

Suzuki. Pure Appl. Chem. 6, 213, 1994; Suzuki, Pure Appl. Chem. 6, 419. 1991; and Farina and Roth. "Metal-Organic Chemistry" Volume 5 (Chapter 1994.

The starting materials MXXIII (B 1.4-phenylene) are readily formed using methods analogous to those 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 yield the trimethyltin intermediate. The starting materials MXXIII (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 (T),A-Met X-B-E-G

(T),A-B-D-E-G

MXXII MXXII 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) WO 97/43245 PCT/US97/07921 or Met Halide or Triflate arid X Metal These general methods are useful for the preparation of compounds for which Friedel-Crafts reactions such as those of Methods A, B, C, or D would lead to mixtures with various biaryl acylation patterns. Method E is also especially useful for the preparation of products in which the aryl groups. A or B. contain one or more heteroatoms (heteroaryls) such as those compounds that contain thiophene, furan. pyridine, pyrrole. oxazole, thiazole, pyrimidine or pyrazine rings or the like instead of phenyls.

General Method F When the R 6 groups of method F form together a 4 7 member carbocyclic ring as in Intermediate MXXV below, the double bond can be moved out of conjugation with the ketone group 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 MXXVI with mercapto derivatives using methods analogous to those of General Method D then leads to cyclic compounds MI-F-I or MI-F-2.

SUBSTITUTE SHEET (RULE 26) WO 97/43245 PCTIUS97/07921 Method F 0 0 0 0 Lewisacid

OH

Solvent (Ty x(H) n- 1-3 MI--i0

OH

f o t 22 00O tysly~ty (T)EJ y seo ~(Hih,,npoy)..tycbdiiehdoclrd n brombipeny CS iscnendt Geea byho us h opud o hsivnining hcontonrs e noneto The aid i proecte as n estr fe. R benyl Cifio 2-t itysiconeted (ToS~ a y seth otf intrmeiat vi iitil mtalatin f te bomie wthn-butyllithium at low temperature (-78 0

C)

SUBSTITUTE SHEET (RULE 26) WO 97/43245 PCT/US97/07921 followed by treament with Chlorotrmethyitin and CI is converted to an eno trifate (CII by reaction wAith [YY-Nbis(tifluommethvis fonvi.)anmio].5.chlioropy.idine in the presence of a strong aprotic base. The tin and enoltriflate intermediates are then coupled in the presence of a Pd 0 catalyst. CuIi and AsPh, to yield directly intermediate CVII. Ozonolysis of CVII (workup with methylsufide) yiel ds aldehyde CVIII. Alternatively treatment with 0504 followed by H10 4 converts CV11 to

CVIII.

Method G ROC R02C~--K C1CV 1) MsCI Cl~yN Mg2) eiimjnanann R02C OWR0 2 C xr 2H C ui~n e C I X Bu r c IV 1 O zone 2) csn.C3 CV: sme)2) MeSMe 0 COR

,""CH

2 0H red

-AC

(TXx CVIII or TsC1 Mitsunobu, acylaaion or alkylation 2000

R

R A2~ I 1 CH2Z N "lationN N M" 0 (T ACX CXx SUBSTITUTE SHEET (RULE 26) WO 97/43245 PCT/US97/07921 Conversion of key intermediate CVm to the targeted patent compounds is accomplished in several ways depending on the identity of side chain function Z. Reaction of CVIII with Witig reagents followed by hydrogenation yields products in which Z is alkyl and or arylalkyl. Selective reduction of aldehyde CVm with a reducing agent such as lithium trs [(3-ethyl- 3pentyl)oxy]aluminum hydride (LTEPA) yields alcohol CDX. The alcohol is convened to phenyl ethers or a variety of heteroatom substituted derivatives used to generate sidechain Z via the Mitsunobu conditions well known to those skilled in the art (see Mitsunobu, Synthesis. 1, 1981).

Alternatively the alcohol of CLX is converted to a leaving group such as tosylate (CX) 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. Med. Chem. 37, 2552. 1994. Direct acylation of the alcohol CIX yields invention compounds in which Z OAcyl 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 those skilled in the art such as removal of benzyl by base hydrolysis or of 2-(trimethylsilyl)ethyl by treatment with tetrabutylammonium fluoride.

General Method H Amides of the acids of the invention compounds can be prepared from the acids by treatment in an appropriate solvent such as dichloromethane or dimethylformamide with a primary or secondary amine and a coupling agent such as dicyclohexylcarbodiimide. These reactions are well known to those skilled in the art. The amine component can be simple alkyl or arylalkyl substituted or can be amino acid derivatives in which the carboxyl is blocked and the amino group is free.

18 SUBSTITUTE SHEET (RULE 26) WO 97/43245 PCT/US97/07921 General Method I The compounds of this invention in which is an alkynyl or substituted alkynyl are prepared according to general method I (Austin. J. Org. Chem. 46, 2280.

1981). Intermediate MX is prepared according to methods A. B, C, D or G by starting with commercial MIm Br). Reaction of MX with substituted acetylene MXI in the presence of Cu(I) palladate reagent gives invention compound MI-1-1. In certain cases. R 3 may be an alcohol blocked as trialkylsilyl. In such cases the silyl group can be removed by treatment with acids such as trifluoroacetic acid or HF pyridine reagent.

Method I R MXI R 0 (CH2), 0 (M) c) R (CH, -CCH OH

OH

(t)IN RI(CHI-C-c 0 C 0 ul tras.

O

dichlorobisnphenyl phosph-e palladate 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 can be metal ions, 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.V-dibenzylethylenediamine, lower alkylamines such as methylamine. t-butylamine, procaine, lower alkylpiperidines such as N-ethylpiperidine. cycloalkylamines such as cyclohexylamine or dicyclohexylamine, 1-adamantylamine, benzathine, 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.

3, SUBSTITUTE SHEET (RULE 26) WO 97/43245 PCT/US97/07921 These and other salts 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 form 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, with potassium bisulfate, hydrochloric acid, etc.

The compounds of the present invention have been found to inhibit the matrix metalloproteases MMP-3. MMP-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 MMPs 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 substituents 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 substituents such that inhibition of specific MMP(s) associated with specific pathological conditions can be enhanced while leaving non-involved MMPs less affected.

SUBSTITUTE SHEET (RULE 26) WO 97/43245 PCT/US97/07921 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'redient(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, intraarticular, subcutaneous and intramuscular routes. Intravenous administration can be used to obtain acute regulation of peak 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 liposomal targeting to the joint cavities by incorporation of ligands into the outside of the liposomes that bind to synovial-specific macromolecules. Alternatively intramuscular, intraarticular or subcutaneous depot injection with or without encapsulation of the drug into degradable microspheres 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 the Novo Pin or Q-pen) or needle-free jet injectors from Bioject, Mediject or Becton 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

I/

SUBSTITUTE SHEET (RULE 26) WO 97/43245 PCT/US97/07921 into the synovial spaces. Examples include the subcutaneously implanted osmotic pumps available from ALZA, such as the ALZET osmotic pump.

Nasal delivery may be achieved by incorporation of the drug into bioadhesive particulate carriers (<200 such as those comprising cellulose, polyacrylate or polycarbophil. in conjunction with suitable absorption enhancers such as phospholipids or acylcamitines. Available systems include those developed by DanBiosys and Scios Nova.

A 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 bioavailability 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/OsmetT and PULSINCAP T systems from ALZA 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.

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 UBSTITUTE SHEET (RULE 2 SUBSTITUTE SHEET (RULE 26) WO 97/43245 PCT/US97/07921 thereof, talc, vegetable oils, waxes, fats, polyols such as polyethylene glycol, water, saccharose.

alcohols, glycerin and the like. Various preservatives, emulsifiers, dispersants. flavorants. wetting agents. 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 ingredient(s) 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 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 ofmetailoproteases and testing for matrix metalloprotease activity. Such activity testing can be both 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.

EXAMPLES

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.

SUBSTITUTE SHEET (RULE 26) WO 97/43245 PCT/US97/07921 General Procedures: 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.

Materials: Commercial grade reagents and solvents were used without further purification except that diethyl ether and tetrahydrofuran were usually distilled under argon from benzophenone ketyl, 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 53233. Solvents are often obtained from EM Science as distributed by VWR Scientific.

Chromatography: Analytical thin-layer chromatography (TLC) was performed on Whatman" pre-coated glass-backed silica gel 60 A F-254 250 um plates. Visualization of spots was effected by one of the following techniques: ultraviolet illumination, exposure to iodine vapor, immersion of the plate in a 10% solution of phosphomolybdic acid in ethanol followed by heating, and (d) immersion of the plate in a 3% solution of p-anisaldehyde in ethanol containing 0.5% concentrated sulfuric acid followed by heating.

Column chromatography was performed using 230-400 mesh EM Science* silica gel.

SUBSTITUTE SHEET (RULE 26) WO 97/43245 PCT/US97/07921 Analytical high performance liquid chromatography (HPLC) was performed at I mL minon a 4.6 x 250 mm Microsorb' column monitored at 288 nm, and semi-preparative HPLC was performed at 24 mL min' on a 21.4 x 250 mm MicrosorbD column monitored at 288 nm.

Instrumentation: Melting points (mp) were determined with a Thomas-Hoover melting point apparatus and are uncorrected.

Proton nuclear magnetic resonance (NMR) spectra were measured with a General Electric GN-OMEGA 300 (300 MHz) spectrometer, and carbon thirteen 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 structures 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 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 multi-step procedures, sequential steps are noted by numbers.

lir SUBSTITUTE SHEET (RULE 26) WO 97/43245 PCT/US97/07921 Examle 1- PreDaration of ComDound I 0 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 mol) 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 l-bromo-3-phenylpropane (47 mL. -61 g, -0.30 mol) over 10 min via the addition funnel. Rinses of the addition funnel (THF. 2 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 reflux condenser and stopper, and the reaction was heated at reflux 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 aqueous phase was extracted with chloroform (100 mL). The combined organics were washed with HC1 (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-extracted with chloroform (100 mL). The organics were dried (NaSO,) and concentrated to yield a yellow oil which was purified by distillation through a Vigreux column at reduced pressure (0.4 torr). The fraction boiling at 124-138 'C was clean desired product (57.21 g, 0.206 mol; 68% yield). TLC (50% hexanes-dichloromethane): Rf= 0.32.

14 SUBSTITUTE SHEET (RULE 26) WO 97/43245 PCT/US97/07921 Br- Step 2 A I-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 (50.00g, 0.215 mol) in dichloromethane (100mL). Bromoacetyl bromide (21.0mL. 48.7g, 0.230 mol) was added via syringe and the solution was cooled with an ice water bath to 0°C, while AICI 3 34 .3g, 0.258 mol) was added portionwise. Gas evolved from the opaque olive green reaction mixture.

After 24h at room temperature, the reaction mixture was cautiously poured into a cold saturated aqueous NaHCO 3 solution. The resulting mixture was extracted with three 200mL portions of ethyl acetate, and the combined organic layers were dried over NaSO, and concentrated to afford the desired product as a yellow solid in quantitative yield. TLC (30% dichloromethane-hexanes),

R,

=0.30.

OH

OH

0 Step 3 A dry 2-L, three-necked, round-bottomed flask was equipped with a magnetic stir bar, an 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). The reaction vessel was cooled with an ice 47 SUBSTITUTE SHEET (RULE 26) WO 97/43245 PCT/US97/07921 water bath while sodium hydride (5.40 g of 95% NaH, 0.214 mol) was added slowly in portions.

The reaction mixture was stirred for I h at 0 OC, and a solution of the product of step 2 (80.0 g, 0.215 mol) in dry THF (300 mL) was added via addition funnel over ca. 20 min. The deep orange reaction mixture was stirred at room temperature under argon for 3 h. The reaction vessel was cooled in an ice water bath while distilled water (150 mL) was added cautiously. The aqueous phase was extracted with three 300 mL portions of ethyl acetate, the combined organic phases were dried over MgSO,, 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 NaOH solution (4 N, 500 mL. 2.00 mol). The reaction mixture was stirred for 24 h at room temperature, 48 h at 50 and 24 hours 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 ofhexanes. The aqueous phase was acidified with HC1, extracted with two 200 mL portions, and three 100 mL portions of ethyl acetate. The combined organic phases were dried over MgSO, and concentrated to afford a quantitative yield of diacid. TLC (10% methanol-chloroform with 1% acetic acid): Rf=0.45.

O Ph

OH

0 Br Step 4 The unpurified diacid from step 3 was dissolved in 1,4-dioxane (500 mL) and heated to reflux for 24 h. The solvent was removed in vacuo, and a 10 g portion of the residue #8 SUBSTITUTE SHEET (RULE 26) WO 97/43245 PCT/US97/07921 chromarographed on silica gel (gradient elu~tion with 10-50% ethyl acetate-hexanes containing 1% acetic acid) to afford 0.840 g of the desired product as a yellow solid. MP 174 OC.

0 Ph HOCHIC-SC

/\OH

0 Step 5 A one-necked, 1 5-mL. round-bottomed flask equipped with a rubber septum and an argon needle inlet was charged with 2.6 rnL of diethylamine, the product of step 4 (0.300 g, 0.667 mmol).

propargyl alcohol (1 .0 rnL. 0.96 g, 17 rnmol), copper iodide (0.0220 g, 0. 115 mmol), and trans-dichlorobis(trphenvlphosphine)palladate 110 g, 0. 157 rnmol). The resulting mixture was stirred for 4 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 gel (20% ethyl acetate-hexanes with acetic acid) afforded the coupling product as a white solid (0.035 g, of coupling product as a white solid. MP 130 TC.

Exampnle 2 and Examakle- Prearation of Compounds 11 and III Example 2 and Example 3 were prepared by chiral separation of Example 1 on a Chiralcel AD column (2 cm x 25 cm) using 5% EtOR, 4.75% H,O and 0.095% HOAc in CH 3 CN, flow rate mLimin.

Example 2: First off Chiralcel AD column; 'H NMR (300 MHz, CDC1 3 8 8.02 J 8.4 Hz, 2 7.67 J 8.7 Hz, 2 7.58 J 8.7 Hz, 2 7.53 J 8.4 Hz, 2 7.17-7.33 (mn, 4.54 2 3.46 (dd, J 8.1, 16.8 Hz, 1 3.14-3.02 (mn, 2 2.65 J 7.2 Hiz, 2 H), 1.64-1.84 (mn, 4 H).

SUBSTrIUTE SHEET (RULE 26) WO 97/43245 PCTIUS97/07921 Example 3: Second off Chiralcei AD 'column; 'H NMR (300 MfHz, CDCI,) 8 8.02 J 8.4 Hz.

2 7.67 J 8.7 Hz, 2 7.58 J 8.7 Hz, 2 H) 7.53 J 8.4 Hz. 2 7.17-7.33 (mn. 4.54 2 3.46 (dd, J 8.1, 16.8 Hz, I 3.14-3.01 (in, 2 2.65 J 7.2 Hz 2 H), 1.64-1.84 (in. 4 H).

Example 6 Preparation of Cornaound V1 A one-necked, 10-mi. round-bottomed flask equipped with a rubber septum and an argon needle inlet was charged with 0.5 rnL of pyridine, Example 1 (0.0070 g, 0.0 14 inmnol), and acetic anhvdride (0.020 mL. 22 ing, 0.21 inmol). The reaction mixture was stirred for 2 h at room temperature, and then added to 3 0 m.L of IN HCI. The resulting mixture was extracted with three 30 rnL portions of ethyl acetate, the combined organic phases were dried over MgSO,, and concentrated. Purification via HPLC ethyl acetate-dichloromethane with 0.0 1% trifluoroacetic acid) afforded 3 mng of Example 6. LMP 137 TC.

Example 7 Preparation of Compound

VII

A one-necked, 15-rnL. round-bottomed flask equipped with a rubber septum and an argon needle inlet was charged with 2 rnL of triethylamine. 2 ml of THE, compound 1 (0.0570 g, 0.134 rnzol). and ethyl chloroforinate (0.032 inL. 36 mng, 0.34 inmol). The reaction mixture was stirred for 16 h at room temperature and then added to 50 mL of IN HCI. The resulting mixture was extracted with three 50 mL portions of ethyl acetate, the combined organic phases were dried over MgSO 4 and concentrated. Column chromnatography on 10 g silica gel (40% ethyl acetate-hexanes with 0.5% HOAc) followed by purification via HPLC ethyl acetate-dichloromethane with 0.01% trifluoroacetic acid) afforded 1 mng of Example 7. MS (FAB-LSIM4S) 499 (M+I{J.

SUBSTITUTE SHEET (RULE 26) WO 97/43245 PCTIUS97/07921 Example -11 Preparation of Compound X1 A one-necked, 25-mL. round-bottomed flask equipped with a rubber septum and an argon needle inlet was charged with I m.L of CH 1 Example 12 (0.012 g, 0.026 nimol), and the Dess-.Martin reagent (16 mg, 0.038 nimol) prepared according to Dess. et al., J. Org. Chem 48, 4156. 1983. The resulting mixture was stirred for 30 min at 0 0 C, diluted with 30 mL of ethyl acetate. and washed with two 20 mL portions of IN HCI. The organic layer was dried over MgSO 4 and concentrated. Purification via HPLC ethyl acetate-dichloromethane with 0.01% trifluoroacetic acid) afforded I mg of Example 11. 'H INM (300 MHz. CDCI 3 5 9.70 J 1.3' Hz. I 8.05 J= 8.4 Hz. 2 7.70 J 8.4 Hz, 2 7.65 J= 8.4 Hz, 2 7.44 J= 8.4 Hz, 2 7.15-7.3 5 (in. 5 3.46 (dd. J 8.1, 16.8 Hz, 1 3.14-3.02 (in. 2 2.67 J= 7.2 Hz,.2 2.48 J= 7.5 Hz, 2 2.41 (dt, J= 1.3 Hz and 6.3 H7, 2 1.96 (mn,2 H), 1.64-1.84 (in, 4 H).

The above methods for the preparation of Example 1, Example 2. Example 6. Example 7, and Example 11I were used to prepare the following series of biphenyl containing products.

215 SUBS1iTUTE SHEET (RULE 26) WO 97/43245 WO 9743245PCT/US97/07921 TABLE

I

0 Ph R OH 0

COMP

I HOCH.C!!C [I HOCH,CaC III HOCH,C=-C isomer M.p.( 0 C)/other characterization R.S 130 S 'H NMR (300 MHz. CDCI,) 6 8.02 J1 8.4 Hz, 2 7.67 J 8.7 Hz, 2 7.58 J 8.7 Hz, 2 7.53 J3 8.4 Hz. 2 H), 7.17-7.33 (mn, 5 4.54 2 3.46 (dd, J 8.1. 16.8 Hz, 1 3.02-3.14 (in, 2 2.65 J 7.2 Hz. 2 1.64-1.84 (in, 4 H).

R 'H NMR (300 N'f~z. CDCI 3 6 8.02 J1 8.4 Hz. 2 7.67 J 8.7 Hz, 2 7.58 J 8.7 Hz. 2 7.53 J 8.4 Hz. 2 H), 7.17.7.33 (mn, 5 4.54 2 3.46 (dd, J 8.1, 16.8 Hz, I 3.02-3.14 2 2.65 (t,J =7.2HRz, 2H), 1.

6 4 -1.

8 4 4H).

R. S 136 IV lMeOCH,CzC v (n-Pr)2NC{,C-=C IVII

CH

3

CO,CH

2

C-=C

VII EtOCO,CH,CsC VII

HO(CH,)

2 CzC IX CH3CO,(CH 2 2

CE=C

X

HO

2

C(CH-

2 2

C-=C

xi

OHC(CH

2 3

CEC

a' S R- S R's R

S,

MS (FAB-LS EMS) 5 10 [M+H] 137 MS (FAB-LSIMS) 499 fM+H]- 124 IMS (FAB-LSIv1S) 483 R.'S 'H NMR (300 Mflz, CDCI 3 6 9.70 J= 1.3 Hz, 1 8.05 J1= 8.4 Hz. 2 7.70 J= 8.4 Hz, 2 7.65 J= 8.4 Hz, 2 M), 7.44 J= 8.4 Hz, 2 7.15-7.3 5 (mn,5 H), 3.46 (dd, J 8.1, 16.8 Hz, I 3.14-3.02 (mn, 2 2.67 J1 7.2 Hz, 2 2.48 J 7 .5 Hz, 2H), 2.41 (dt, J= 1.3 Hz and 6.3 Hz, 2H), 1.

9 6 2 M, 1.

6 4 -1.84 4H).

SUBSTTUTE SHEET (RULE 26) I WO 97/43245 WO 9743245PCT/US97/07921 Xfl HO(CH,),C-=c R.S 123 XMI PhC=-C R, S 154 XIV 3-HO-PhC-=C KS 237 EX2Mnole_15 Preparation of Compound

XV

0 NO0 0 Step 1 A solution of sodium hydride (4.35 g, 181 nunol) in freshly distilled THF (100 niL) was coo led to 0 'C and treated with commercially available diallyl malonate (3)5.0 g, 190 mmol) over min via a dropping funnel. After stirring at room temperature for 30 min., iV(2-bromoethyl)phthaimide (43.9 g, 247 mniol) was added to the solution in one portion and the mixture was heated to reflux. After 48 h the solution was cooled to 0 quenched with 2N HCI and concentrated to about 20% of its original volume. The concentrate was diluted with ethyl acetate (3 00 rnL) and washed successively with saturated aqueous solutions of K,CO3 and NaCl.

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-phthaimxidoethylmalonate (41.2 g, 64%) as a colorless oil. 'H NNM (300 Mflz, CDC1 3 6 7.82 (mn, 2H), 7.72 (in, 2H), 5.85 (mn, 2H), 5.30 (in, 2H), 5.22 (mn, 2H), 4.60 (mn, 4K, 3.80 J= 6.6 Hz, 2H), 3.46 J =7.2 Hz, I1H), 2.30 (dd, J= 13.8, 6.9 Hz, 2H).

53 SUBSTITUTE SHEET (RULE 26) WO 97/43245 PCT/US97/07921 0 C0AAUyl O CO Allyl Br S Step 2 A solution of the product of step 1 5 .20 g, 14.6 mmol) in freshly distilled THF (100 mL) was cooled to 0 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 1. step 2 (4.55 g, 14.6 mmol) was added in portions, and the mixture was stirred for 24 h. The reaction mixture was cooled to 0 quenched slowly with 2N HCI (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 6.50 g of the desired product which was used in step 3 without purification. TLC (30% ethyl acetate-hexanes): Rf= 0.4.

0 COH 0 Step 3 A solution of the product of step 2 (6.50 g, 10.4 mmol) in 1,4-dioxane (100 mL) was cooled to 0 while tetrakis(triphenylphosphine)palladium (0.180 g, 146 mmol) and pyrrolidine (2.40 mL, 29.2 mmol) were added sequentially. After stirring for 2 h at 0 'C and 4 h at room temperature, the reaction mixture was poured into 2N HCI (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 A 3.8 g sample of this material was dissolved in 1,4-dioxane (150 mL) and heated at reflux for 1 h. After cooling to room temperature, SUBSTITUTE SHEET (RULE 26) WO 97/43245 PCT/US97/07921 the solution was concentrated and the residue was chromatogaphed on 300 g silica gel (gadient with 5%-15% methanol-dichloromethane) to give the desired acid (0.300 g) which was further purfied ia recrystallization to afford 0.170 g (59% overall yield from step 2) of the desired product as a white crystalline solid. MP 209-210 o

C.

Ph O 0

CCH

Step 4 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 Nail (435 mg, 17.2 rmeol) and cooled to 0 C while prpargl alcohol (1.0 L, 0.963 g, 17.2 o) was added via syringe ver a. 5 The resulting mixture was stirred at 0 C for 10 min and at room temperature for 30 min. Benzy. bromide (1.8 mi. 2.59 g, 15.1 rnmol) was added, the reaction mixture was stirred at room tempeture for 36 h, poured into pentane (150 mL), and washed with a 100 mL portion of brine. The solvent was removed via distillation and the residue (3.5 g of a yellow oil) was used in step 5 directly. 'H NMR (300 MHz, CDC 3 6 7.36-7.31 5 4.61 2H), 4.17 (d,J=2.4 Hz, 2 2.47 (t,J=2.4

H

1 H).

0 N

O

Ph 0

OH

Step 5 The procedure of Example 1, step 5 was used to prepare Example 15 using the product from step 4 and the product of step 3 as starting materials. MP 151 'C.

SUBSTITUTE SHEET (RULE 26) WO 97/43245 PCTIUS97/079 2 1 EXZWD~~~e 16 r~rtOnf CowLRH Oud Xvr t-BuMe,Sio

C=CH

Step 1 A one-necked, I O0-mI.,, round-bottomed flask equipped with a rubber septum and an argon needle inlet was charged with propargyl alcohol (1.0 rnL. 0.963 g, 17.2 Mmol). ether (20 ml). and cooled to 0 *C while Na- (435 mg, 17.2 mmol) was added slowly. The resulting mixture was stirred at room temperature for 1 h. and t-butyldimethylsilyj chloride (2.60 g, 17.2 nimol) was added.

The reaction mixture was stirred at room temperature for 6 h, poured into hexane (150 rm.L). and washed with I N HCI. The organic phase was dried over MgSO 4 and concentrated to afford 2.88 g of a yellow oil which was used in step 2 without purification. 'H NMRQ(00,MI-z. CDCl 3 64.29 J= 2. Hz2 H),2.37(t. =2.1 Hz, I 0 8 9 9H)o0n1 3 H).

0 Step 2 The procedure of Example 1, step 2 was used to prepare the desired acetyl biphenyl using comnlerclally available 4 -iodobiphenyl and acetyl chloride. TLC (10% ethyl acetate-hexanes):

R=

0.3.

t-B M e 2 S O OCk Step 3 The procedure of Example 1, step 5 was used to prepare the desired biphenyl acetylene using the product of step I and the product of step 2. TLC (10% ethyl acetate-hexanes): Rx 0.4.

SUBSTITUTE SHEET (RULE 26) WO 97/43245 PCTIUS97/r7921 I- u e C =C 0\ Br Step 4 A one-necked. 50-rnL. round-bottomed flask equipped with a rubber septum and an argon needle inlet was charged with 5 ML of THF. the product of step 3 1 .06 g, 2.94 mmol), and cooled to -78 TC while potassium hexamethyldisilazide (617 Mg. 2.94 mmol) was added dropwise via syringe. The reaction mixture was stirred at -78 TC for 30 min, trimethylsilyl chloride (0.374 mL.

0.3120 g, 2.94 mmol) was added dropwise via syringe, and the resulting mixture was stirred at -78 TC for 3 h. The reaction mixture was warmed to 0 0 C for 1 h. V-bromosucciniride (0.540 g, 2.94 mrnol) 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

4 CI. and extracted with three 50 rnl portions of dichioromethane. The combined organic phases were dried over MgSO, and concentrated. Column chromatography on 200 g of silica gel (gradient elution with 0- 5% ethyl acetate-hexanes) afforded 0.264 g of the bromomethyl ketone. TLC (10% ethyl acetate-hexanes): 0 NO0 0 /E C

O

SUBSTITUTE SHEET (RULE 26) WO 9743245PCT/US97/0792 1 Step 5 The procedures of Example 15, steps 2-3 were used to prepare the desired biphenyl phthalimjde using the product of step 4. TLC (50% ethyl acetate-hexanes with I1% acetic acid):

R=

0.3 0 NO0 0 HO~

OH

Step 6 A one-necked. 50-rnL. round-bottomed flask equipped with a rubber septum and an argon needle hnet was charged with 10 m.L of CH,CI,, the product from step 5 (0.040 g, 0.067 mnmol), and 2 rnL of l-F-pvridine. The resulting mixture was stirred for 10 minutes at room temperature. diluted with a 75 mL portion of water, and extracted with a 75 niL portion of CHWCl,. 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 of Example 16. NIP 145 *C.

Example 17 Biological Assays of Invention Compounds P218 uenched Fluorscenc Assay for MMP Inhibition: The P218 quenched fluorescence assay (Micro fluorometric Profiling Assay) is a modification of that originally described by Knight, et al.. FEBS Lett. 296, 263, 1992 for a related.

210 substance and a variety of matrix metalloproteinss (MM~s) in cuvettes. The assay was run with each invention compound and the three MM~s, Mmp-3, MNvI-9 and MMP-2, analyzed in parallel, adapted as follows for a 96-well microtiter plate and a Hamilton AT workstation.

so SUBSTITUTE SHEET (RULE 26) WO 97/43245 PTU9/72 P218 Fluorovenic Substrate: P218 is a synhetic substrate containing a 4 -acewl.-7.

methoxycounar.m MCA) group in the N-terrnjnal position and a 4 -dinitrophenv]Lj..diaminopropionyt (DPA) group internally. This is a modification of a peptide reported by Knight (1992) that wvas used as a substrate for matrix metalloproteinases. Once the P218 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 ran and emission at 393 nmn. P218 is currently being produced BACHEM exclusively for Bayer. P218 has the structure: H-MCA-Pro..Lys.PTo.LCu-/aLeuDPAAaIrgN (MNW 13-32.2) Recominan Humn CH Stromelvsin (MMP-3) Recombinant Human CHO Pro-.MMP.3: Human CH0 pro-stromelysin.-257 (pro-TNP3) was expressed and purified as described by Housley, et al., J. Biol. Chem. 268, 4481. 1993.

.Activation of Pro.A4MP3: Pro-MiMP-3 at 1. 72 ptM (100 ptg/mL) in 5nM Tris at pH- 7.5, mM CaCI,, 25 mM NaCI, and 0.005% Brij-35 MMP-3) activation buffer) was activated by incubation with TPCK (N-tosyl-(L).phenylaanin chloromethyl ketone) trypsin 100 w/w to prois MMIP-3) at 25 'C for 30 min. The reaction was stopped by addition of soybean trypsin inhibitor (SBTI; 5:1 wfw to trypsin concentation). This activation protocol results in the formation of 45 kDa active MMP-3. which still contains the C-terminal portion of the enzyme.

Prepraton o Hu an ecombinant Pro-Geatinse A (MMP-ZL: Recombinat~n Human Pro-,VMP-2: Human pro-gelatinase A (pro-MNp.2) was prepared using a vaccinia expression system according to the method of Fridrnan, et al., J. Biol. Chem. 267, 15398. 1992.

SIBSTIUT SHEET (RULE 26) W O 97 43245P C T U S 97/07921 A4ctval'on of Pro.-jMj.

2 Pro-Mj"_2. at 252 inmjM was diluted 1:5 to a final concentration of 50 4M/nL solution in 25 M.M Tris at PH 7.5, 5 MM CaCI,, 150 MM NaCI. and 0.005% Brij-35 (NvfM.2 activation buffer). P-Ailophenylmercu.jc acetate (AkPMA) was Prepared n 10 rnM' (3-5 mw.nL) in 0.05 NaOH. Trhe APM ,A solution was added at 1,,20 the reaction volume for a final AMPA concentration of 0.5 MM and the enzymewainuteat3 0 Cor0 Activated MMP.2 (15 n1L) was dialyzed twice vs. 2 L Of MMP-2 activation buffer (dialysis membranes were pretreated with a solution consisting Of 0. 1% BSA in MLMP-2 activation buffer for I mi. followed by extensive HO washing). The enlzyme was concentr.ated on Centricon concentrators (concentrators were also pre-treated a solution consisting Of 0. 1% BSA in NM-~2 activation buffer for 1 mini.. followed by washing with H.O, then MAQf-2 activation buffer) with redilution followed by re-concentration repeated twice. The enzyme was diluted to 7.5 nML (0.5 times the original volume) with MMAP-2 activation buffer.

PreR aration of Huma Rc mb inant ProjtnsGB( M Recombinant Human pr.M.t 9.~q Human pro-gelatns B (PrO-M.p.9) derived from L93 7 cDNA as described by Wilhelm, et al. J. Biol. Chem. 264, 17213, 1989 was expressed as the fi-llength form using a baculovirus protein expression system. The pro-enzyme was Purified using methods previously described by Hibbs, et al. J. Biol. Chem. 260. 2493, 1984.

A4ctlvaiOn of PrO-U-MJ.9: Pro-MMY.2 20 jig/mi in 50 nUM Tris at PH 7.4, 10OmM CaC., 150 m.M NaCI, and 0.005% Brij-35 (NM"-9 activation buffer) was activated by incubation with mM P-amiPheylneruri acetate (ApMA) for 3.5 h at 3 7 0 C. The enzyme was dialyzed against the same buffer to revmove the Aph4A.

SUBSTITUTE SHEET (RULE 26) WO 97/43245 PCT/US97/07921 Instumentation Harniltion Aficrolab AT Plus: The -N~tPT-Pro filing Assay is performed roboticaily on a l-amiton Microlab AT Plus", The Harrilton is programmed to: serially dilute up to I I potential inhibitors automatically from a 2.5 mM stock in 100% DM50O; distribute substrate followed by inhibitor into a 96 well Cytofluor plate; and 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 enzyme. In this way, all \Wvf assays were done using the same inhibitor dilutions.

Millipore Cvtofluor 1I. Following incubation. the plate was read on a Cytofluor 11 fluoromeuic plate reader with excitation at 340 nM and emission at 395 nM with the gain set at BUffers: Nlicrofluorometric Reaction Buffer (MRB): Dilution of test compounds, enzymes, and P2118 substrate for the rnicrofluorometric assay were made in microfluorometrc reaction buffer consisting of 50 m. -Nmrhln~taeufri acid (MES) at pH 6.5 with 10 m.M CaCI,, 150 mM NaCI, 0.005% Brij-35 and 1% DMS0.

3Lethods: AM Vicrofluorometrnc Profiling Assay. The assay is done with a final substrate concentration of 6 gtM P218 and approximately .5 to .8 WM MW? with variable drug concentrations.

The Hamilton is programmed to serially dilute up to 11I compounds from a 2.5 trm stock (100%/ DMSO) to l Ox the final compounds concentrationsj in the assay. Initially, the instrument delivers various amounts of microfluoromentric reaction buffer to a 96 tube rack of 1 ml Marsh SUBSTITUTE SHEET (RULE 26) WO 97/43245 PCT/US97/0792 1 dilution tubes. The instrument then picks up 20 il 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 50 AM drug concentration. The inhibitors are then serially diluted to 10, 5, .05 and .01 pM. Position I on the sample rack contains only DMSO for the "enzyme-only" wells in the assay, which results in no inhibitor in column I. rows A through H. The instrument then distributes 107 1 of P218 substrate (8.2 uM in IMRB) to a single 96 well cytofluor microtiter plate. The instrument re-mixes and loads 14.5 4l 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 Pl of MRB is delivered in placed of drug or enzme). The reaction is started by adding 25 pl 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 "back g r ound" row. (The enzyme reservoir is pretreated with 1% BSA in 50 mM Tris, pH containing 150 mM NaCI for I hour at room temp., followed by extensive HO washing and drying at room temp.).

After addition and mixing of the enzyme, the plate is covered and incubated for 25 min. at 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 rackto 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.

IC50 Determination in Microfluorometric 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 4t.

SUBSTITUTE SHEET (RULE 26) WO 97/43245 PTU9/72 determination for each drug concentration by comparilng the amount of hydrolysis (fluorescence units generated over 25 minutes of hydrolysis) of wells containijng compound with the **en~zzme only" wells in colun 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, 1, 0.5, 0.1. 0.02. 0.005 and.

0.00! P.M of drug. Linear regression analysis of percniet inhibition versus log inhibitor concentration was used to obtain

IC,

0 values.

Tabe SUBSTITUTE SHEET (RULE 26) WO 97/43245 PCT/US97/07921 S 125 I 46% XV 11 40 XVI 4 2 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.

SUBSTITUTE SHEET (RULE 26) 1. A matrix metalloprotease inhibiting compound having the general formula R'"-CC

OH

O 0 wherein R" is selected from the group consisting of HOCH 2 (n-Pr) 2

NCH

2

CH

3

CO

2

CH

2 EtOCO 2

CH

2

HO(CH

2 2

CH

3

CO

2

(CH

2 2

HO

2

C(CH

2 2

OHC(CH

2

HO(CH

2 4 3-HO-Ph, and PhCH20CH 2 and R16 is 0 or and pharmaceutically acceptable salts thereof.

2. A composition having matrix metalloprotease inhibitory activity, comprising a compound of claim 1 and a pharmaceutically acceptable carrier.

3. A method of inhibiting matrix metalloprotease activity in a mammal comprising administration of an effective amount of the matrix metalloprotease inhibitor compound of *claim 1 to said mammal.

4. The method of claim 3 wherein said mammal is a human.

A method of treating a m mmmal comprising administering to the mammal a matrix metalloprotease inhibiting amount ofa compound according to claim 1 sufficient to: metalloprotease inhibiting amount of a compound according to claim 1 sufficient to:

Claims (2)

1. 6. The method of claim 5 wherein the effect is alleviation of osteoarthritis.
2. 7. The method of claim 5 wherein the effect is retardation of tumor metastasis. SUBSTITUTE SHEET (RULE 26) ABSTRACT OF THE DISCLOSURE 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 formulas: 0 R' 1 O where R 15 is selected from the group comprising: HOCH 2 MeOCH 2 (n-Pr) 2 NCH 2 CH 3 CO 2 CH 2 EtOCO 2 CH 2 HO(CH 2 2 CH 3 CO 2 (CH 2 2 HO 2 C(CH 2 2 OHC(CH 2 3 HO(CH 2 4 Ph, 3-HO-Ph, and PhCH20CH 2 and R 16 is 0 Ph or V 0 ft .f S* These compounds are useful for inhibiting matrix metalloproteases and, therefore, combating ":conditions to which MMP's contribute, such as osteoarthritis, rheumatoid arthritis, septic arthritis, periodontal disease, corneal ulceration, proteinuria, aneurysmal aortic disease, dystrophobic epidermolysis, bullosa, conditions leading to inflammatory responses, S"osteopenias mediated by MMP activity, tempero mandibular joint disease, demyelating diseases of the nervous system, tumor metastasis or degenerative cartilage loss flowing S* traumatic joint injury, and coronary thrombosis from athrosclerotic plaque rupture. The present invention also provides pharmaceutical compositions and methods for treating such conditions.