BACKGROUND OF THE INVENTION

Field of the Invention

This invention relates to enzyme inhibitors, and more particularly, to novel substituted biar l oxobu yπc acid compounds or derivatives thereof useful for inhibiting matrix metailoproteases Description of the Related Art The matπx metailoproteases (a.k.a. mamx metalloendo-proteinases or MMPs) are a family of zinc endoprotemases which include, but are not limited to, interstitial coilagenase ia.k.a.. MP- 1 ). stromelysm (a.k.a.. proteogiycanase. transm, or MP-3), gelatinase A (a.k.a.. 72kDa-geiatιnase or .VtMP-2) and gelatinase B (a.k.a.. 95kDa-geiatιnase or MMP-9). These MMPs are secreted by a variety of cells including fibroblasts and chondrocytes, along with natural proteinaceous inhibitors known as TIMPs (Iissue Inhibitor of MetalloErotemase).

All of these MMPs are capable of destroying a variety of connective tissue components of articular cartilage or basement membranes. Each MMP is secreted as an mactive proenzyme which must be cleaved in a subsequent step before it is able to exert its own proteoiytic activity. In addition to the matrix destroying effect, certain of these MMPs such as MMP-3 have been implemented as the in vivo activator for other MMPs such as MMP- 1 and MMP-9 (no, _et .. Arch

Biochem Biophys. 2j&L 211 (1988); Ogata, et al., J. Biol. Chem. 2SL 3581 (1992)). Thus, a cascade of proteoiytic activity can be initiated by an excess of MMP-3. It follows that specific MMP-3 inhibitors should limit the activity of other MMPs that are not directly inhibited by such inhibitors.

It has also been repoπed that MMP-3 can cleave and thereby inactivate the endogenous inhibitors of other proteinases such as elastase ( inyard. et al., ^' FEBS Letts. 229, 1. 91 ( 1991 n Inhibitors of MMP-3 could thus influence the activity of other destrucuve protetnases bv modifying the lev el of their endogenous inhibitors. A number of diseases are thought to be mediated by excess or undesired matrix-destroying metal loprotease activity or by an unbalance in the ratio of the MMPs to the TIMPs. These include: ai osreoarthntis fWoessner. et al.. J. Biol.Chem.. 259(6). 3633 (1984); Phadke. et al., J Rheumatol. 10, 852 1983)), b) rheumatoid arthritis (Mullins. et al.. Biochim. Biophys. Acta 695, 1 17 ( 1983)), Woo Hey. et at.. Arthritis Rheum. 20, 1231 (1977); Gravallese, et al.. Arthritis Rheum. 34. 1076 1 1991 )1. c ) septic arthritis (Williams, et al.. Arthritis Rheum. 33_, 533 (1990)), d) tumor metastasis

(Reich, et al.. Cancer Res., 4&, 3307 (1988). and Matrisian. et al., Proc. Nat'l. Acad. Sci., USA £3, 9413. ( 1986)), e) periodontal diseases (Overall, et al., J. Periodontal Res. 2Z, 81 (1987)), corneal ulceration (Bums, et al.. Invest. Opthalmol. Vis. Sci. 3J), 1569 (1989)), g) proteinuria (Bancos. 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 (1991)), 0 aneurysmal aortic disease (Vine, et al.. Clin. Sci. §1, 233 (1991)), j) birth control (Woessner, et al., Steroids 5_4 _> 491 ( 1989)), k) dystrophobic epidermolysis bullosa (Kronberger, et al., J. Invest. Dermatol. 7_9, 208 (1982)). and 1) 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. 50, 688 (1988)).

The need for new therapies is especially important in the case of arthritic diseases. The primary disabling effect of osteoarthntis (OA), rheumatoid arthritis (RA) and septic arthritis is the

7-

progressiv e loss of articular cartilage and thereoy normal joint function. No marketed

pharmaceutical agent is able to prevent or slow this cartilage loss, although noπsteroidal anti- Lriilarnrnatory drugs ( SAJDs) 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 proteoiytic degradation of structural protein in the basal membrane allows for expansion of a rumor in the primary site, e asion from this site as well as homing and invasion m distant, secondary sites. Also, rumor induced angiogenesis is required for rumor growth and is dependent on proteoiytic tissue remodeling. Transfection experiments with vaπous types of proteases have shown that the matrix metailoproteases play a dominant role in these processes in particular gelat ases 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. _t Eur. Respir. J. 7, 2062 (1994); Birkedal-Hansen. et al., Cπt. Rev. Oral Biol. Med. 4, 197 (1993).

Furthermore, it was demonstrated that inhibition of degradauon of extracellular matrix by the native matrix metalloprotease inhibitor TIMP-2 (a protein) arrests cancer growth ( eClerck, et

al.. Cancer Res. 52, 701 (1992)) and that TIMP-2 inhibits tumor-induced angiogenesis in experimental systems (Moses, et al. Science 245, 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 mtrapeπtoneally inhibits human colon tumor growth and spread in an orthotopic model in nude mice (Wang, et al. Cancer Res. 5^ , 4726 (1994))

and prolongs the survival of mice bearing human ovaπan carcinoma xenografts (Davies. et. al..

Cancer Res. 53, 087 ( 1993)). The use of th s and related compounds has been descπbed in Brown. et l . WO-9321942 . \2 (931 1 1 1).

There are sev eral patents and patent applications claiming the use of metalloproteinase inhibitors for the retardauon of metastatic cancer, promoting rumor regression, inhibiting cancer cell proliferation, slowing or preventing cartilage ioss associated with osteoarthruis or for treatment of other diseases as noted above (e.g. Levy, et al.. WO-9519965 Al; Beckett, et al., WO-9519956 Al .

Beckett, et ai. WO-9519957 Al; Beckett, et al., WO-9519961 Al; Brown, et al.. WO-9321942 A2;

CrjTirrun. et al., 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 A 1 ; 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, carboxyiic acid or phosphinic acid) at one end and a ariety 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 proteoiytic metabolism, thus having short half lives.

As an example, batirnastat, the compound described in Brown, et al., WO-9321942 A2, can only be given intra peπtoneally.

Certain 3-biphenoylpropanoιc 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.

4-

SUBSTΠTΠΈ SHEET (RULE 26)

Specifically, Tomcufcik, et al.. L ^' S patent 3.784,701 claims certain substi _t u _t ed benzoylpropioruc acids to treat inflammation and pain. These compounds include 3-bφhenoylpropanoιc acid (a.k.a. fenbu en) shown below

Fenbufen

Child, et al.. J. Pharm. Sci.. 66_, 466 ( 1977) descπbes structure-activity relationships of se eral analogs of fenbufen. These include several compounds in which the biphenyl πng system is substituted or the propanoic acid portion is substituted with phenyl, halogen, hydroxyl or methyl, or the carboxyiic acid or carbonyl funcuons are converted to a variety of deπvatives. No compounds are descπbed which contain a 4'-substιtuted biphenyl and a substituted propanoic acid portion combined in one molecule. The phenyl (compounds XLLX and LXXVH and methyl (compound XLVIT) substituted compounds shown below were described as inactive.

ameo, et al., Chem. Pharm. Bull., 3 $, 2050 (1988) and Tomizawa, et al., JP patent 62132825 A2 describe certain substituted 3-biphenoylpropionic acid derivatives and analogs thereof

including the following. Vaπous compounds with other substiruents on the propionic acid poπion are descπbed. but they do not contain biphenyl residues

serein X = H. 4'-Br, 4'-Cl. 4'-CH ₃ , or 2'-Br.

Cousse. et al.. Eur. J ed. Chem., 22, 45 (1987) descnbe the following methyl and memv ene subsututed 3-bιphenoyl-propanoιc and -propenoic acids. The coσesponding compounds in which the carbonyl is replaced with either CH _: OH or CH, are also descnbed.

wherein X = H. Cl. Br. CH,O. F. or NH,.

Nichl, et al. DE patent 1957750 also describes certain of the above methylene substituted btphenoylpropanoic acids.

El-Hashash, et al., Revue Roum. Chim. 23, 1581 ( 1978) descnbe products deπved from β-aroyl-acrylic acid epoxides including the following biphenyl compound. No compounds

substituted on the biphenyl portion are descnbed.

itamura. et al.. J? 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.

wherein R ¹ is an alkyl of 1-10 carbons.

Thyes, et al., DE patent 2854475 uses the following compound as an intermediate. The biphenyl group is not substituted.

Sammour, et al., Egypt J. Chem. ϋ, 31 1 (1972) and Couquelet, et al., Bull. Soc. Chim. Fr.

2, 3 196 ( 1971 ) describe certain dialkylamino substituted biphenoylpropanoic acids including the following. In no case is the biphenyl group substituted.

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

Others have disclosed a seπes of biphenyl-contairung carboxyiic acids, illustra _t ed bv _t he compound shown below, which inhibit neural endopeptidase i EP 24 1 1 ), a membrane-bound zinc metalloprotease (Stanton. et al., Bioorg. Med. Chem. Lett. 4, 539 ( 1994); Lombaert, et ai . Bioorε Med Chem Lett 4. 2715 ( 1994); Lombaert. et al., Bioorg Med. Chem. Lett. _. 145 ( 1995). Lombaert. et al.. Bioorg. Med. Chem. Lett. 5, 151 ( 1995)).

It has been reported that N-carboxyalkyl deπvatives containing a biphenylethyiglycme. illustrated by the compound shown below, are inhibitors of stromeiysιn-1 (MMP-3), 72 DA gelatinase (MMP-2) and collagenase (Dure te, et al., WO-9529689).

It would be desirable to have effective MMP inhibitors which possess improved bioavailability and biological stability relative to the peptide-based compounds of the pπor an, and which can be optimized for use against particular target MMPs. Such compounds are the subject of the present application.

The development of efficacious MMP inhibitors would afford new therapies for diseases mediated by the presence of. or an excess of MMP activity, including osteoaπhπtis. rheumatoid arthntis. septic arthπus. rumor metastasis, peπodontal diseases, comeal ulcerations, and proteinuπa. Sev eral inhibitors of MMPs have been descπbed in the literature, including thiols (Beszant. et al.. J Med. Chem. 36, 4030 (1993). hydroxamic acids (Wahl. et al. Bioorg. Med. Chem. Lett. 5, 349

( 1 95 ) Conway. et al. J. Exp. Med. U 2, 49 ( 1995); Porter, et al.. Bioorg. Med. Chem. Lett. 4, 2741 1 1994V. Tomczuk. et al.. Bioorg. Med. Chem. Lett. 5, 343 ( 1995); Castelhano. et al.. Bioorg. Med. Chem. Lett. 5_, 1415 ( 1995)). phosphorous-based acids (Bird, et al. J. Med. Chem. 37, 158 ( 1994); Morohy. et al.. Bioorg. Med. Chem. Lett. 4, 2747 (1994); Kortylewicz, et al.. J. Med. Chem. H, 263 1 1990)). and carboxyiic acids ( Chapman, et ai. J. Med. Chem. 3 , 4293 ( 1993); Brown, et al. J

Med. Chem. 37, 674 (1994); Morohy. et al.. Bioorg. Med. Chem. Lett. 4, 2747 (1994); Stack, et al.. Arch. Biochem. Biophys. 287, 240 (1991); Ye. et al.. J. Med. Chem. 17, 206 (1994); Grobelny, et al.. Biochemistry 24, 6145 ( 1985); Mookhtiar. et al.. Biochemistry 21 4299 (1988)). However. these inhibitors generally 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 INVENTION

This invention provides compounds having mamx metalloprotease inhibitory activity. These compounds are useful for inhibiting matπx metailoproteases and, therefore, combating conditions to which MMP's contribute. Accordingly, the present invention also provides pharmaceuucal compositions and methods for treating such conditions.

The compounds descnbed relate to a method of treating a mammal compπsmg administering to the mammal a matπx metalloprotease inhibiting amount of a compound according to the invention aurfϊciept to t a i allev iate the effects of osteoarthπtis. rheumatoid arthπtis. septic arthπtis. peπodontal disease, comeal ulceration, protemuπa, aneurysmal aortic disease, dystrophobic epidermolysis, bullosa. conditions leading to inflammatory responses, osteopenias mediated by MMP activity, tempero mandibular joint disease, demyeiatiπg diseases of die nervous system;

( b ) retard tumor metastasis or degenerative cartilage loss following traumatic joint in _j ury; (c) reduce coronary thrombosis from athrosclerotic plaque rupture; or

(d) temporaπly reduce fertility (i e , act as effective birth control agents).

The compounds of the present invention are also useful scientific research tools for studying functions and mechanisms of action of matπx metailoproteases in both in vivo and in vuro 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 expeπmental biological system under study.

This invention relates to compounds having matπx metalloprotease inhibitory activity and the generalized formula:

(T) _X A-B-D-E-G (L) In the above generalized formula (L), (T).A represents a substituted or unsubstituted aromatic 6-membered ring or heteroaromatic 5 - 6 membered nng containing 1 - 2 atoms of N, O, or S. T represents one or more substituent groups, the subscnpt x represents the number of such

/€?

In the above generalized formula (L), T,A represents a substituted or unsubstituted aromatic 6-membered ring or heteroaromatic 5 - 6 membered πng containing 1 - 2 atoms lndependenth selected from the group of N. O. or S. T represents a substituted acetylenic moiety

I the generalized formula (L). B represents an aromatic 6-membered nng or a heteroaromatic 5 - 6 membered nng containing 1 - 2 atoms independently selected from the group of N. O. or S It is referred to as the B nng or B unit. When N is employed in conjunction with either S or O in the B nng. these heteroatoms are separated by at least one carbon atom.

In the generalized formula (L). D represents

C=0 C = N0H C =S

In the generalized formula (L). E represents a chain of n carbon atoms beanng m substiruents R ⁶ in which the R ⁶ groups are independent substiruents, or constitute spiro or nonsptro nngs. Rings may be formed in two ways: a) two groups R ⁶ 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 nng, or b) one group R ⁶ 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 nng. The number n of carbon atoms in the chain is 2 or 3, and the number m of R* substituents is an integer of 1 - 3. The number of carbons in d e totality of R* groups is at least two. Each group R' is alkyl, aikenyl, alkynyl, heteroaryl, non-aromatic cyclic, and combinations thereof optionally substituted with one or more heteroatoms as described more fully below. In the

//

In the generalized formula (1). E represents a linear or cyclic alkyl moiety substituted wi _t h a mono- or bi-heterocychc πng structure

Ln the generalized formula (L), G represents -PO-H, -M

in which M represents -CO _: H. -CON(R") _: or -CO _; R'-\ and R' ³ represents any of the side chains of the 19 noncyclic naturaily occumng ammo acids.

Pharmaceutically acceptable salts of these compounds are also within the scope of the invention In most related reference compounds of the pπor 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 iarger phenyl group has been reported to cause pπor art compounds to be inactive as anti-inflammatory analgesic agents. See, for example. Child, et al.. J Pharm. Sci 6§, 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 MMP inhibitors also preferably contain a substituent on the 4'-posιuon, 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 mereiy summaπzes 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.

1%

DESCRIPTION OF THE PREFERRED EMBODIMENTS

More particularly, the compounds of the present invention are matenals having matnx metalloDrotease inhibttorv activity and the generalized formula

(T\A-B-D-E-G ( L) in which (TVA. represents a substituted or unsubstituted aromatic or heteroaromatic moiety selected rrom the group consisting of

T -Λ-

Throughout this application, in the displayed chemical structures, an open bond indicates the point at hich the structure joins to another group. For example.

where R ⁵⁰ is

is the structure

/)

in w mch R ¹ represents H or alkyl of 1 - 3 carbons

Throughout this app cauon. in the displayed chemical structures, an open bond indicates the point at which the structure joins to another group For example,

w here R' ^ϋ is

is the structure

In these structures, the aromatic πng is referred to as the A ring or A unit, and T represents a substituent group, referred to as a T group or T un t. T is a substituted acetylemc moiety and x is

1

The B πng of generalized formula (L) is a substituted or unsubstituted aromatic or heteroaromatic πng, in which any substituents are groups which do not cause the molecule to fail to fit the acuve site of the target enzyme, or disrupt the relative conformations of die A and B πngs, such that they would be detrimental. Such substituents may be moieties such as lower alkyl, lower alkoxy, CN, NO ₂ , halogen, etc., but are not to be limited to such groups.

In the generalized formula (L), B represents an aromatic or heteroaromauc πng selected from the group consisting of:

fr

portion compπses 4 - 9 carbons and at least one N. O. or S heteroatom and the alkyl portion contains 1 - 4 carbons.

R represents H. alkyl of 1 - 12 carbons; aryl of 6 - 10 carbons; heteroaryl compπsing 4 - 9 carbons and at least one N. O. or S heteroatom; arylalkyl in which the ary l portion contains 6 - 10 carbons and the alkyl portion contains 1 - 4 carbons; heteroaryl-alkyl in which the heteroaryl portion compnses 4 - 9 carbons and at least one N, O, or S heteroatom and the alkyl portion contains 1 - 4 carbons, alkenyl of 2 - 12 carbons; alkynyl of 2 - 12 carbons; -(C^H^O)^' in which q is 1 -3. r is 1 - 3. and R' is H provided q is greater than 1. or R ^s is alkyl of 1 - 4 carbons, or phenyl; -(CH _: VX in which s is 2 - 3 and X is halogen: or -C(0)R ² .Any unsaruration in a moiety which is attached to Q or which is part of Q is separated from any N. 0, or S of Q by at least one carbon atom, and the number of substituents, designated x, is 0, I . or 2.

The substituent group T can also be an acetylene containing moiety with the general formula:

R ^J0 (CH _j ),C=C —

where n is 1 -4 and R ³⁰ is selected from the group consisting of: HO-, MeO-. N(n-Pr),-. CH,CO,-,

CH,CH,OCO _: -, HO,C-, OHC-. Ph-, 3-HO-Ph-. and PhCH _: 0-, provided that when R ^J0 is Ph or 3- HO-Ph. n = 0

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

IS

in which R ¹ is defined as above. These πngs are refeσed to as the B ring or B unit.

Compounds of the general formula (L) include those in which the combination (T) -A-B has the structure:

R N^ -Z- CH - ~ ~ where Z may be (CH,) _e -C ₆ H ₄ -(CH,) _f or (CH,) _r e = 0-8, f - 0-5 and g = 0-14, r is 0-6. R ¹³ may be a straight, or cyclic alkyl group of 6-12 carbons atoms, preferably of 7-1 1 carbon atoms, and optionally may bear one or more pharmaceutically acceptable substituents which are discussed more fully below.

R ¹⁵ may also be a polyether of the formula R"O(C _: H ₄ O) _h in which me subscript "h" is 1 or 2, and the group R" is a straight, branched or cyclic alkyl group of 1-5 carbon atoms, preferably of 1 -3 carbon atoms and straight, or phenyl, or benzyl. R" optionally may bear one or more pharmaceutically-acceptable substiruents which are discussed more fully below.

R' ⁵ may also be a substituted alkynyl group of the formula:

inch the subscπpt "b ^" is 1 - 10 and the group R" is H-. HO- or R ³⁴ 0- and the group is preferably the HO- group. R- ⁴ may be an alkyl group of 1 -3 carbon atoms, or phenyl or benzyl R ³³ optionally may near one or more pharmaceutically-acceptable substituents which are discussed more fully below

R ^; ' may also be H, Cl. MeO or

w herein n is 0-4. R" is C _: H,, allyl. or benzyl. in the generalized formula (L), D represents the moieties:

\ \ \ \ ,H \ ,H

C =0 C = NOH C=S ,C ,C\

/ / / OH H

In the generalized formula (L), E represents a moiety having the following formula:

herem r is 0-2 and R ⁰ is a mono- or bi- heterocvclic structure. When r=0 the above structure takes the form

When r is 1 or 2, a cyclobutyl or cyclopentyl ring is formed, respectively. Each ring of the mono- or bi- heterocylic structures comprise 5-7 membered rings substituted with 1-3 heteroatoms independently selected from N, S, and O; one or two carbons of d e ring are optionally carbonyl carbons; any sulfur of the ring is optionally -S(O)- or -S(O) ₂ -; one or more ring members are optionally substituted with one or two methyl groups; and the heterocyclic structure is attached to

π

and the A unit is phenyl, the B unit is phenylene. m is 1 , n is 2, and t is 0. then x is 1 or 2

13 ⁾ -( CH _; χZR* in which v is an interger of 1 to 4, Z represents -S-, -S(O)-. -SO,- or -0-. and R ⁸ is selected from the group consisting of alkyl of I to 12 carbons, aryl of 6 to 10 carbons, heteroaryl compπsing 4 to 9 carbons and at least one N. O. or S heteroatom. arylalkyl in which the aryl portion contains 6 to 12 carbons and the alkyl portion contains

1 to 4 carbons, heteroarylaikyi in which the aryl portion contains 6 to 12 carbons and at least one N, O. or S heteroatom and the alkyl portion contains 1 to 4 carbons, -C(0)R in which the R represents alkyl of 2 to 6 carbons, aryl of 6 to 10 carbons, heteroaryl compnsing 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 compπsing 4 to 9 carbons and at least one N. 0. or S heteroatom, and the alkyl portion contains 1 to 4 carbons with the provisos that when R ^J ιs -C(O)R ⁹ . Z is -S- or -O-. when Z is -0-. R ⁸ may also be -(C^O)^ ³ in which q, r. and R ⁵ are as defined above, and when me 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

in which w is an integer of 1 to 3, and R ¹⁰ represents alkyl of I to

2 carbons.

In addition, aryl or heteroaryl portions of any of the T or R ⁶ groups optionally may bear up to two substituents selected from the group consisting of -(CH ₂ ) _y C(R")(R ^, )OH, -(CH ₂ ) _y OR",

-(CH,) _V SR", -(CH ₂ ) _v S(O)R", -<CH ₂ ) _y S(O) ₂ R", -(CH ₂ ) _y SO ₂ N(R") ₂ , -(CH^O*"),,

-(CH _: ) _v N(R")COR ¹² , -OC(R") _: O- in which boϋi oxygen atoms are connected to the aryl πng,

/8

The B πng is preferably a 1 ,4-phenylene or 2.5-thiophene ring, most preferably .4-phenyiene.

The D unit is most preferably a carbonyl group.

In the E unit, r is preferably 0 or 2 and R ⁴⁰ is preferably one of the following:

or PhCH _: OCH,OCH ₂ -. The G unit is most preferably a carboxyiic acid group and is attached to the E unit at the 2 position, i.e., d e carbon atom of the E unit beta to die D unit.

ή

It is to be understood that as used herein, the term ^■• alkyl" means straight, branched, cvclic. and polycyclic matenals. The term "'haioaikyl" means partially or fully halogenated alkyl groups such as -(CH _: ) _; CI, -CF _; and - F _π for example.

The B πng of generalized formula ( L) is a substituted or unsubstituted aromatic or heteroaromatic πng, in which any substiruents are groups which do not cause the molecule to fail to tit the active site of the target enzyme, or disrupt the relative conformations of the A and B πngs. such that they would be detnmental Such groups 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 (L). the A and B πngs are preferably phenyl and phenylene. respectively, the A πng preferably bears at least one substituent group T preferably located on the position furthest from the position of the A πng which is connected to the B πng, the D unit is preferably a carbonyl group, and the G unit is preferably a carboxyl group.

Certain embodiments include compounds having matnx metalloproteinase inhibitory activity and the following generalized formula:

where Z = (CH,) _« -C ₆ H ₄ -(CH ₁ ) _f or (CHj) _r e ^» 0-8, f = 0-5, g = 0-14, r is 0-6 and where y is 0, 2. or

R' ⁵ may be H, Cl, MeO or

R ¹ wherein n is 0-4, R" is C ₂ H , allyl or benzyl, and R*° is one of:

and -CH OCH _: OCH,Ph.

The most preferred compounds of generalized formula (L) are

wherein T is selected from a group consisting of:

7-1

r is 0-2. and R ⁴⁰ is selected from the group consisting of:

and -CH,OCH,OCH,Ph. τ*

The invention also relates to certain intermediates useful in the symhesis of some of the claimed inhibitors. These intermediates are compounds having die generalized formula

where Bn is benzyl, TMSE is trimethylsilyl ethyl and R*° is as defined above.

2-2-

SUBSTITLΠΈ SHEET (RULE 26)

Those skilled in the art will appreciate that many of the compounds of the invention exist in enantiomeπc or diastereomeπc 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 hich possess inhibitory activity against an MMP, regardless of their stereotsomeπc 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) 2-[(4'-chloro[ l .r-biphenyl]-4-yl)carbonyl]-5-[(4-oxo-1.2.3-benzotπazin-3(4 H)-yl)methyl]- cyclopentanecarboxylic acid,

II) 2 - ( (4'-chloro[ 1 , 1 '-biphenyl ]-4-yl )carbony I ] -5 -[phenoxymethoxymethyl ] - cyclopentanecarboxylic acid,

III) 2-[4'-chloro[ l , -biphenyl]-4-yl)carbonyl]-5-[[(l-pyπ-olidinyithioxomemyl)th io]methyI]- cyclopentanecarboxyiic acid,

yl)methyl]-cyclopentanecarboxylic acid,

V) 2-[(4'-chloro[ l ,l'-biphenyl]-4-yt)carbonyl]-5-[ l -oxo-2(lrY)-phthalazinyl)methyl]- cyclopentanecarboxyiic acid.

VI) 2-[4*-chloro[l ,l'-biphenyl]-4-yl)carbonyl]-5-[(2-oxo-3(2H)-benzoxazolyl)me thyl]- cyclopentanecarboxylic acid,

VU) 2-[(4'-chloro(l, -biphenyl]-4-yl)carbonyl]-5-[5,5-dimethyl-2,4-dioxo-3-oxazol idinyl- methyl]-cyclopentanecarboxylic acid,

Z

Vffl ⁾ 2-{ ⁽ 4 ^' -chloro[l.r-bιphenyl]-4-yl ⁾ carbonyl]-5-[ ⁽ 2.4-dioxo-3-thιazohdιnyl)methyl]- cyclopentanecarboxyhc acid.

LX ⁾ 2-[ ⁽ 4 ^' -chloro[l.r-biphenyl]-4-yl ⁾ carbonyl]-5-[2.4.5-tπoxo-l-ιmιdazolιdinyl)methyl]- yciopentanecarboxyl acid

X ⁾ 2-[ ⁽ 4 ^' -chloro(l ^, t ^' -biphenyl]-4-yl)carbonyl]-5-[ ⁽ 3,6-dihydro-2.6-dioxo-l(2H)- pyπmidιnyl)methyl]-cyclopentanecarboxyiιc acid,

XI ⁾ 2-[ ⁽ 4 ^' -chloro[l.r-bιphenyl]-4-yl ⁾ carbonyl]-5-[3,4-dihydro-2.4-dιoxo-l(2H)- py-πmιdinyπmethyl]-cyciopentanecarboxyiic acid,

XII ⁾ 2-[ι4 ^' -chloro(l, -biphenyl]-4-yl)carbonyl]-5-[(l,4-dihydro-2.4-dιoxo-3(2H- quιnazolinyi)methyl]-cyclopentanecarboxyiic acid,

XIIΪ ⁾ 2-[(4'-chloro[l.r-biphenyl]-4-yl)carbonyl]-5-[3,4-dihydro-1. 3-dioxo-2(lH)- ιsoquιnolinyl)methyl]-cyclopentanecarboxyiic acid, XrV) 2-[(4 ^, -chioro[l,r-biphenyl]-4-yl)carbonyl]-5-[(l,4-dihydro-4 -oxo-3(2H)- quιnazolinyl)methyl]-cyclopentanecarboxylic acid, XV) 2-(4 ^, -chloro[l,r-biphenyl]-4-yl)carbonyl]-5-[(1.3-dihydro-3 -oxo-2H-indazol-2-yl)methyl]- cyclopentanecarboxylic acid, XVI) 2-[(4'-chloro[l,r-biphenyl]-4-yl)carbonyl]-5-[2,3-dihydro-lH -benzimidazol-l-yl)methyl]- cyclopentanecarboxylic acid, XVIT) 2-[(4 ^* .chloro(l,l'-biphenyl]-4-yl)carbonyl]-5-[(3,4-dihydro- l,4-dioxo-2(lH)- phthalazinyl)methyl]-cyclopentanecarboxylic acid,

XVLTT) R/S α-[2-(4 ^, -chloro[l,r-biphenyl3-4-yl)-2-oxoethyl]-l-oxo- 2(lH)-phthalazinebutanoic acid,

ZM-

XIX) R-α-[2- ⁽ 4 ^' hloro(l, r-bιphenyl]-4.yt )-2-oxoethyl ]-l -oxo- 2(lH)-phthalazιnebutanoιc acid.

XX ⁾ S- -(2-(4' hloro[ l . r-bιphenyl]-4-y ).2-oxoethy ]-l -oxo- 2( lH)-phthaiazinebutanoιc acid.

XXI ) -[2-(4'-chloro( 1.1 '-bιphenyl]-4-y|)-2-oxoethyl]-4-oxo- 1.2.3 ,-Benzotπazιne-3(4H)-butanoιc acid, and XXII) α-[2-(4'-chloro[ l .r-bιphenyl]-4-yl)-2-oxoethyl]-2.3-dihydro-5-methyI-2-oxo- l H- 1.4- benzodiazepιne-1 -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 die expeπmental section describing the working examples. All variable groups of these methods are as described in the generic description if diey are not specifically defined below. The variable subscπpt n is independently defined for each method. When a variable group with a given symbol (i.e R') is used more than once tn a given structure, it is to be understood that each of these groups may be independently varied within the range of definitions for tiiat symbol.

General Method A - The compounds of this invention in which the rings A and B are substituted phenyl and phenylene respectively are conveniently prepared by use of a Friedel-Crafts reaction of a substituted biphenyl MD with an activated acyl-containing intermediate such as the succinic or glutaric anhydride derivative Mill or acid chloride MTV 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

15

acid catalysts as descπbed by Berliner. Org. React.. 5, 229. 1949 and Heaney, Comp. Orε. Syn _t h _. 2. "33. 1991.

If the anhydride MLTJ is monosubstiruted or multiply-substituted in an unsymmetrical way. the raw product MI-A often exists as a mixture of isomers via attack of the anhydπde from either of the two carbonyls. The resultant isomers can be separated into pure forms by crystallization or chromatography using standard metiiods known to those skilled in the art.

When they are not commercially available, the succinic anhydπdes Mill can be prepared via a Stobbe Condensauon of a 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 conv ersion to the anhydπde MHI by reaction with acetyl chloπde or acetic anhydride. Alternatively, the hemiester intermediate is converted by treatment with thionyl chloride or oxalyl chloride to the acid chlonde MTV. For a review of the Stobbe condensation, including lists of suitable solvents and bases see Johnson and Daub. Org. React., 6_, 1 ,1951.

This method, as applied to the preparation of MITJ (R ⁶ = H, isobutyl and H, n-penryl), has been descπbed Wolanin, et al.. US Patent 4.771.038.

Zέ

n = 0-3

Base

Method A is especially useful for the preparation of cyclic compounds such as MI-A-3, in which two R* groups are connected in a methylene chain to form a 3-7 member ring. Small ring (3-5 member) anhydrides are readily available only as cis isomers which yield cis invention compounds

MI-A-3. The trans compounds MI-A-4 are tiien 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 MU1-A-1

TΠ

are formed in a photochemical 2-2 reaction as shown below. This method is especially useful for the preparation of compounds in which R ¹⁴ is acetoxy or acetoxymethylene. After the subsequent Fπedel-Crafts reaction the acetate can be removed by basic hydrolysis and the carboxyi protected by conversion to 2-( tnmeu ylsilyl)ethyl ester. The resultant intermediate with R ^{| J} = CHOH can be converted to invention compounds with other R ^u groups by using procedures descπbed in

General Method G.

The Fπedel-Crafts method is also useful when double bonds are found either between C-2 and C-3 of a succinoyl chain (from maleic anhydride or l -cyclopentene-l ,2-dicarboxylic anhydride, for example) or when a double bond is found in a side chain, such as in die use of itaconic anhydπde as starting mateπal to yield products in which two R ⁶ groups are found on one chain carbon together to form an exo-mediylene (=CH _: ) 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 MVTJ, followed by alkylation with a halomethyl biphenyl ketone MVUI to yield intermediate MD Compounds of structure MLX are then hydrolyzed with aqueous base and heated to decarboxylate the malonic acid intermediate and yield MI-B-2 (Method B-l). By using one equivalent of aqueous base the esters MI-B-2 with R ^ι: as alkyl are obtained, and using more ύian

2.8

two equivalents of base the acid compounds (R ¹ - = H) are obtamed. Optionally, heat is not used and the diacid or acid-ester MI-B-1 is obtained.

Alternatively, the diester intermediate MLX can be heated with a strong acids such as concentrated hydrochloπc acid in acetic acid in a sealed tube at about 1 10 ^' C for about 24 hr to y ield MI-B-1 ( R' ^: = H) Alternatively, the reaction of MVI with MVITJ can be conducted before that with the alkyl haiide to yield the same MLX (Method B-2).

Alternatively, a diester intermediate MXLX, which contains R ^{ι :} = allyl, can be exposed to Pd catalvsts in the presence of pyτrolidine to yieid MI-B-2 (R ¹² = H) (Dezeil. Tetrahedron Lett. 2J5, 4371 1990

Intermediates MVII are formed from biphenyis MLT in a Fπedel-Craft reaction with haloacetyl haiides such as bromoacetyl bromide or chloroacetyl chloπde. Alternatively, the biphenyl can be reacted with acetyl chloπde or acetic anhydπde and the resultant product halogenated with, for example, bromine to yield intermediates MVTH (X = Br).

Method B has the advantage of yielding single regio isomers when Memod A yields mixtures. Method B is especially useful when the side chains R* 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 die final compound contains heteroatoms such as oxygen, sulfur, or nitrogen, or more complex functions such as imide rings.

l°\

MI-B-1 MI-B-2

When R ⁶ contains selected functional groups Z, malonate MVII can be prepared by aikylating 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 Mitsunobu reaction (Mitsunobu, Synthesis 1 , 1981 ). .Alternatively, d e intermediate alcohol can be subjected to alkylation conditions to provide malonate MVII containing die desired Z group.

3>o

General Method C - Especially useful is the use of chiral HPLC to separate the enantiomers of racemic product mixtures (see. for example, .Ant. et al.. Chem. Int. Ed. Engl. 1 , 30 (1991 )). The compounds of this invennon can be prepared as pure enantiomers by use of a chiral auxiliary route. See. for example. Evans. Aldrichimica Acta. !5_(2), 23, 1982 and otiier similar references known to one skilled in the art.

General Method D - Compounds in which R* 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 itacomc 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 tiirough C) with formaldehyde in the presence of base.

Compounds MI-D-1 or MI-D-5 are then reacted with a mercapto derivative MXVπ or MX VIII in the presence of catalyst such as potassium carbonate, ethyldiisoburylarrune, tetrabutylammonium fluoride or free radical initiators such as azobisisobutyronitrile (AJBN) in a

solvent such as diediylformamide or tetrahydrofuran to yield invention compounds MI-D-2. MI-D-3. \fl-D-6. or MI-D-7

Method D

^

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 heteroaryi metallic compounds in which the metal is zinc, tin. magnesium, lithium, boron, silicon, copper, cadmium or the like with an aryl or heteroar l halide or tπflate (tπfluoromeuiane-sulfonate) or the like. In the equation below either Met or X is the metal and the other is the halide or tπflate (OTf)- Pd(com) is a soluble complex of palladium such as tetrakιsi tπphenyiphosphine)-palladium(O) or bis- (triphenylphosphinei- palladium(uT) chloride. These methods are well known to tose skilied in the art. See. for example. Suzuki. Pure Appl. Chem. 63., 213 (1994): Suzuki. Pure Appl. Chem. £1 419 ( 1991 ); and Faπna and Roth. ^• "Metal-Organic Chemistry" Volume 5 (Chapter 1 ), 1994. The starting matenals MXXm (B = 1 ,4-phenylene) are readily formed using methods analogous to those of methods A. B, C, or D but using a halobenzene rather tiian a biphenyl as starting material. When desired, die 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 tπmethyltin intermediate. The starting materials MXXHI (B ~ heteroaryl) are most conveniently prepared by method C but using readily available heteroaryl rather than biphenyl starting matenals. The intermediates MXXD. are either commercial or easily prepared from commercial materials by methods well known to those skilled in die art.

Method E (T)-A-Met + X-B-E-G ► (T)„A-B-D-E-G

MXXD MXXπi Pd(com) MI-E

T, x, A, B, E and G as in Structure (L)

Met = Metal and X = Halide or Triflate

or

Met = Halide or Triflate arid X = Metai

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 vaπous biary acylation patterns. Metiiod 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, pyπmidine or pyrazine nngs or the like instead of phenyls. General Method F - When the R" groups of metiiod F form togedier 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 hexamethylsilvlamide or the like followed by acid quench to yield compounds with the structure MXXV I. 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.

2

Method F

General Method G - The compounds of this invention in which two R* groups are joined to form a substituted 5-member πng are most conveniently prepared by method G. In this method acid CLLT (R = H) is prepared using die protocols descπbed in Tetrahedron 37, Suppl., 1 1 ( 1981 ).

The acid is protected as an ester [eg. R = benzyl (Bn) or 2-(trimethylsilyl)ethyl (TMSE)] by use of coupling agents such as l-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and procedures well known to those skilled in the art. Substituted bromobiphenyl Cm is converted to its Grignard reagent by treatment with magnesium and reacted with CLII to yield alcohol CVI. Alcohol CVI is eliminated via base treatment of its mesyiate by using conditions well known to those skilled in the art to yield oiefin CVϋ.. Alternatively CHI is converted to a trimethyltin intermediate via initial metallation of the bromide with n-butyllithium at low temperature (-78 °C)

followed by treatment with chlorotπmethyltin and CI is converted to an enoltπflate (CUD bv reaction

with 2- rN\N ^' -bis< fluoromemy1sulfonyi)amino]-5-chloropyndine in the presence of a strong aprotic base. The tm and enoltπflate intermediates are then coupled in die presence of a Pd ^ύ catalyst. Cul and AsPh to y ield directly intermediate CVU. Ozonolysis of CVU (workup with methvlsutlde) y ields aldehyde CVEH. Alternatively treatment with OsO, followed by HIO, converts CVϋ to

CVIII Method G

⁽ _*

Conversion of key intermediate CVTH to the targeted patent compounds is accomplished in several ways depending on the identity of side chain function Z. Reaction of CVTH with Wittie reagents followed by hydrogenation yields products in which Z is alkyl and or arylalky l. Selectiv e reduction of aldehyde CVITJ with a reducing agent such as lithium tπs [(3 -ethyl - penrynoxy]aluminum hydπde (LTEPA) yields alcohol CLX. The alcohol is converted to phenvl ethers or a vaπety of heteroatom substituted denvatives used to generate sidechain Z via the Mitsunobu reaction using conditions well known to those skilled in the art (see Mitsunobu. Sy nthesis. 1 ( 1 981 )). 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 men the leaving group is displaced by an appropπate 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 CLX yields in ention compounds in which Z = OAcyl and reaction of die alcohol with various alkyl haiides in the presence of base yields alkyl etiiers. 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 ail cases well known to those skilled in the art such as removal of benzyl by base hydrolysis or of 2-(tπmethylsιlyl)ethyl by treatment with tetrabutylammonium fluoride.

General Method H - Amides of die acids of the invention compounds can be prepared from the acids by treatment in an appropriate solvent such as dichloromediane 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 deπvatives in which the carboxvl is blocked and _t he ammo group is free.

General Method I - The compounds of this invention in which (T), is an alkynyl or suDstituted alkyny l are prepared according to general method 1 (Austin. J Org. Chem. 46, 2280 ( 1981 ) ). Intermediate MX is prepared according to methods A. B, C, D or G by starting wi _t h commercial MIΗ (R ¹ = Br). Reaction of MX with substituted acetylene MXI in the presence of Cud) . palladate reagent gives invention compound MI-I-1. In certain cases. R ³ may be an alcohol blocked as tπalkyisilyl. In such cases the silyl group can be removed by treatment with acids such as tπfluoroacetic acid or HF - pyπdine reagent. Method I

Suitable pharmaceutically acceptable salts of the compounds of die present invention include addition salts formed with organic or inorganic bases. The salt forming ion derived from such bases can be metal ions, e.g., aluminum, alkali metal ions, such as sodium of 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 dibe zylamine and .V.N-dibenzylethylenediamine, lower alkylamines such as methylamine, r-butyiamine, procame, lower alkylpiperidines such as N-ethylpiperidine, cycloalkylamines such as cyclohexyiamine or dicyclohexylamine, 1 -adamantylamine, benzathine, or salts derived from amino acids like arginine.

3&

lysine or the like. The physiologically acceptable sans such as the sodium or potassium salts and the ammo acid salts can be used medicinally as descπbed below and are preferred.

These and other salts which are not necessaπly physiologically acceptable are useful in isolating or puπfymg a product acceptable for the purposes descπbed below For example, die use of commercially available enantiomeπcally pure amines such as (^)-cιnchonιne in suitable solvents can y ield 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 dian its antiDode. this acuve isomer can thus be found puπfied 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, e.g.. with potassium bisulfate. hydrochloπc acid, etc.

The compounds of the present invention have been found to inhibit the matπx metailoproteases 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 die background section. As other MMPs not listed above share a high degree of homology with those listed above, especially in die 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 relaαve inhibition of the listed MMPs. Thus compounds of tiϋs general class can be "tuned" by

W

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

The method of treating matnx metalloprotease-mediated conditions may be practiced in mammals, including humans, which exhibit such conditions The inhibitors of die present invention are contemplated for use in veteπnary and human applications For such purposes, they will be employed in pharmaceutical compositions containinε active ingredient! s) plus one or more pharmaceutically acceptable earners, 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 an. Examples of suitable parenteral administration include intravenous, intraarticular, subcutaneous and intramuscular routes. Intravenous administration can be used to obtain acute regulation of peak piasma concentrations of the drug. Improved half-life and targeting of the drug to the joint cavities may be aided by entrapment of die drug in hposomes. It may be possible to improve die selectivity of liposomal targeting to the joint cavities by incorporation of ligands into d e outside of the hposomes diat bind to synovial-specific macromolecules. Alternatively intramuscular, intraarticular or subcutaneous depot injection with or without encapsulation of the drug into degradable microspheres e.g., compπstng poly(DL-lactιde-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 l p. implanted reservoir and septum such as the Percuseai 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 Dickinson). Prolonged zero-order or other precisely controlled release such as pulsatile release can

4o

aiso oe achieved as needed using impiantable pumps w th delivery of the drug through a cannula 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 panicula _t e earners ( 200 _*m) such as uiose compπsing cellulose, polyacrylate or polycarbophil. in conjunction with suitable absorption enhancers such as phospho pids 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 iose of anous 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 vaπous a mal 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 enteπc coated capsules designed to release the drag into die colon where digestive protease activity is low. Examples include the OROS-CT/Osmet™ and PULSINCAP™ systems from ALZA and Scherεr 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 nse in pH at the colon. The above systems may be used in conjunction with a ide range of available absorption enhancers. Rectal delivery may be achieved by incorporation of die drug into suppositories.

The compounds of this invention can be manufactured into die above listed formulations by the addition of various dierapeutically inert, inorganic or organic carriers well known to diose skilled

in the art. Examples of these include, but are not limited to. lactose, co starch or de aπves thereof, talc, vegetable oils, waxes, fats, pol ols such as polyethylene glycol. water, saccharose. alcohols, glyceπn and the tike Vaπous preservatives, emulsifiers. dispersants. flavorants. wertine 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 ιngredιent(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, d e requisite amount may be calculated, based on a determinanon 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 puπfication of metailoproteases and testing for matπx metalloprotease activity. Such activity testing can be both in vitro using natural or syndietic enzyme preparations or in vivo using, for example, ammal 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.

41

Experimental: General Procedures:

All reactions were performed in flame-dned or oven-dned glassware under a positive pressure of argon and were stirred magnetically unless otherwise indicated. Sensitive liquids and solutions were transferred via synnge or cannuia 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 puπfϊcation except diat diethyl ether and tetrahydrofuran were usually distilled under argon from benzophenone ketyl. and mediylene chioπde was distilled under argon from calcium hydride. Many of die specialty organic or organometallic starting matenals and reagents were obtained from Aldrich, 1001 West Saint Paul Avenue. Milwaukee, WT 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 am plates. Visualization of spots was effected by one of the following techniques: (a) ultraviolet illumination, (b) exposure to iodine vapor, (c) immersion of the plate tn 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 sulfuπc acid followed by heating, and e) immersion of the plate in a 5% solution of potassium permanganate in water containing 5% sodium carbonate followed by heating.

Column chromatography was performed using 230-400 mesh EM Science ¹ silica eel

Analytical h gh performance liquid chromatography ( HPLC) was performed at 1 L mm on a 4 6 x 250 mm Microsorb ⁸ column monitored at 288 nm. and semi-preparative HPLC was Dertbrmed at 24 mL min on a 21.4 x 250 mm Microsorb* column mo tored at 288 nm. Instrumentation:

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

Proton ( ' H) nuclear magnetic resonance fNMR) spectra were measured with a General

Electπc GN'-OMEGA 300 (300 MHz) spectrometer, and carbon thirteen ( ^{| J} C) NMR spectra were measured with a General Electπc GN-OMEGA 300 (75 MHz) spectrometer. Most of die compounds synthesized in the expenments 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 ratos Concept 1-H spectrometer by liquid-cesium secondary ion (LCLMS), an updated version of fast atom bombardment (FAB). Most of the compounds systhesized in the expenments 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. Vaπations within steps are noted by letters. Dashed lines in tabular data indicates point of attachment. Example 1 - Preparation of Compound I

Step 1. A solution of exo-oxobicyclo [2.2.1] heptane-7-carboxylic acid [prepared usine the protocols descπbed in Author. Tetrahedron. 37, suppl.. 41 1. 1981 (3.04 g, 19 7 mmol) in CH-CK (45 mL) was cooled to 0°C and treated with 2-(tπmethylsιlyl) ethanol (2.7 mL. 18.6 mmol). EDC (3 94 g. 20 55 mmol) and DMAP (0.1 1 g, 0.9 mmoi). After warming to room temperature and stimn for 2 hrs.. d e reaction mixture was quenched with water and diluted with CH,C1 . After separating the layers, the organic phase was washed with satd. aq. N ^' aCl. dried over MgS0 ₄ and concentrated. Puπfication by MPLC (0-25% EtOAc-hexanes) provided the target compound (3.9 g, 78%) as a colorless oil. H NMR (CDCI,) 6 4.18 (m. 2H), 2.88 (m. 2H). 2.76 (m, 1 H . 2.05 (m, 4H), 1.50 (m. 2H). 0 99 (t. J=8.4Hz. 2H). 0 99 (s, 9H).

Step 2. A solution of the ketone from step 1 (3.18 g, 12.50 mmol) and 2-[N.N- bιs(tπfluoromethylsulfonyl) amιno]-5-chloropyridine (6.6 g, 16.30 mmoi) in THF was cooled to - "8 "C and carefully treated with a 0.5M solution of HMDS in toluene (24 mL, 12 mmol). After the addition was complete and the solution stirred for 2 h, the reaction mixture was quenched widi water (30 mL), warmed to room temperature and diluted with EtOAc. The two phases were die separated. The organic layer was washed with satd. ^' aq. NaCl, dried over MgSO, and concentrated. Puπfication by MPLC (0-15% EtOAc-hexanes) provided the target compound (4.2 g, 91%) as a colorless oil. 'H NMR (CDCI ₃ ) 0 5.75 (d, J=4.8Hz, I H), 4.13 (t, J=9.0Hz, 2H), 3.18 (m, 2H), 2.62

(m, IH), 2.62 (m, 2H), 1.41 (t, J=9.3Hz, IH), 1.23 (t, J-9.1Hz, IH), 0.96 (t, J=8.4Hz, 2H), 0.04 (s, 9H).

Step 3. A solution of 4-chlorobiphenyl (3 0 g, 15 9 mmol) in acetic acid ( 50 mL) was carefully treated with broπune ( 1 1 mL. 20.7 mmol) at room temperature. The reaction mixture was heated to reflux for 4 h, cooled to room temperature and treated with excess propene until the mixrure became clear. The solution was concentrated to a thick siurry. diluted with CH.CK ( 50 mL) and washed successively with water and 2N NaOH. The organic extract was dπed over MgSG^, filtered and concentrated. Punfication by re-crystallization form EtOAc gave the aryl bromide , 3 57g. 84%) as a white crystalline solid. Η NMR (CDCI,) δ 7.57 (m, 2H), 7 48 (m. 2H), 7 41 < m. 4H)

Step 4. A solution of 4-bromo-4'-chlorobiphenyl (8.0 g, 30.0 mmol) in THF (120 mL) was cooled to -78 °C and carefully treated with Λ-BuLi (19.7 mL, 1.6 M solution in hexanes, 31.5 mmol). After stimng for 1 h, the mixture was treated with chlorotπmethyltin (33 mL, 1.0 M soln., 33.0 mmol). After an additional 30 min., the solution was warmed to room temperature and concnetrated. The off-white solid was diluted with CH _: C1 _: (300 mL) and washed successively with water and satd. aq. NaCl. The organic layer was dried over MgSO , filtered and concentrated. Purification by MPLC (hexanes) gave the desired aryltin compound (9.38 g, 89%) as a white crystalline solid. Η NMR (CDCI,) δ 7.62 (m, 6H), 7.54 (m, 2H), 0.39 (s, 9H).

Η

Step 5. A solution of the inflate from step 2 (4.2 g, 10 89 mmol). Cul (0.215 g, 1.1 mmol).

ΛsPh- ( 0 339 g, 1 1 mmol). CUPdfMeCN (0 215 g, 0 56 mmol) and a few crystals of BHT m 1 - methyl-2-pyrrolidinone < 1 1 5mL) was lowered into an oil bath preheated to 85 ⁵ C. After stimng 4 mm . the bipheny ltin denvanve from step 4 (7 3 g, 20 7 mmol) was added in one portion. The mixture was stirred for 30 mm., cooled to room temperature and diluted with EtOAc. After separating die phases, me aq. layer was back extracted with EtOAc and the combined organic layers dned over MgSO , filtered and concentrated. The resulting residue was adsorbed on silica gel and purified by MPLC (0-15% EtOAc-hexanes) to give the coupled product (4.0 g, 86%) as a white cry stalline solid. Η NMR (CDCI,) 6 7 52 (m, 6H), 7.42 (m, 2H), 6.40 (d. J=3.3Hz, IH), 4.19 (t, J= 10 2Hz. 2H), 3.58 (m. I H). 3.23 (m. I H). 2.60 (m. IH). 1.95 (m. 2H), 1.20 (m. 2H). 1.02 (d. J=7 5Hz. 2H). 0.08 (s, 9H).

Step 6. A solution of die olefin from step 5 (3.60 g, 8.47 mmol) in 10% MeOH-CH,CU (200 mL) was cooled to -78 °C and treated with ozone as a gas added directly into die reaction mixture ( 10 min., I L/min.). After TLC indicated the absence of starting material the solution was purged with argon (15 min.), treated with methylsulfide (13 mL) and warmed to room temperature. After stirring overnight, the solution was concentrated to a residue which was purified by MPLC (0-15%

M

EtOAc-hexanes ⁾ to give a mixture of the desired aldehyde and corresponding dimethyl acetal. The product mixture was dissolved in acetone (45 mL) and treated with CSA (0 192 g, 0 83 mmol ) and water ⁽ 0 1 mL. 16 5 mmol ⁾ After stimng overnight, the solution was concentrated and punned bv MPLC 0- ! 5° o EtOAc-hexanes) to give the desired aldehyde ( 3 45 g, 89%) as a colorless oil NMR (CDCI,) 6 9 78 ⁽ d. J=9 0Hz, I H), 8 05 (d, J=6.6Hz. 2H), 7 65 (d. J=6 6Hz, 2H). 7 55 (d.

J=9 0Hz. 2H). 7 44 (d. J=9 0Hz. 2H). 4 15 (m. 3H). 3 87 (t. J=7 2Hz , I H). 3 15 (m. l H). 2 20 ι m. I H). 2 03 (m. I H). 1 86 (m. I H). 1 58 (s. I H). 1 25 (t. J=6 9Hz, I H), 0 93 (m. 2H). 0 00 (s. 9H)

Step 7. A solution of lithium aluminum hydride ( 1.9 mL, 1.0 M THF) in THF (6 mL) was

treated with 3-ethyl-3-pentanol (0.83 g, 5 77 mmol) and heated to a gentle reflux for 1 h. The mixture was then cooled to room temperature.

A solution of the aldehyde intermediate from step 6 (0.85 g, 1.86 mmol) in THF (15 mL) was cooled to -78 ^ύ C and treated with the previously prepared solution of LTEPA in THF via cannula in a dropwise manner. After the addition was complete, the solution was stirred at -78 °C for 4 h and subsequently quenched with 2N HC1 (4 6 mL). The reaction mixture was diluted with EtOAc and washed with water. The organic layer was dπed over MgSO , filtered and concnetrated. Purification by MPLC (5-40% EtOAc-hexanes) afforded die desired aldehyde (0.640 g, 75%) as a white crystalline solid. Η NMR (CDC1 ₃ ) δ 8.05 (d, J=8.7Hz, 2H), 7.65 (d, J=8.5Hz, 2H), 7.55 (d,

J=8 4Hz, 2H), 7 44 (d, J=8.4Hz, 2H), 4.15 (m, 2H), 3.76 (t, J=6.3Hz , 2H), 3.28 (t, J=6.3Hz, 2H ,

2.48 ιm. lH ⁾ . 2.35 (L J=6 0Hz. I H ⁾ . 2.18 (m. I H), 1 91 (m. 2H). 1.57 (s. I H). 1.35 (t. J=6 9Hz. I H) 0 91 i m. 2HI- 0 01 (s, 9H).

Step 8. A solution of the aicohol from step 7 (0 050 g, 0.109 mmol). tnphenylphosphine ι 0 057 g. 0 217 mmol) and benzo-1.2.3-tnazιn-4(3H)-one (0 034 g, 0.231 mmol) in THF (2.5 mL) was treated with diethyl azodicarboxylate (0 035mL. 0.222 mmol). The mixture was stirred at room temperature for 16 hrs.. concentrated under reduced pressure and puπfied by MPLC (0-20% EtOAc- nexanes) to give the target compound (0 034g, 53%). TLC. R _f 0.16 (silica. 20% EtOAc-hexanes)

Step 9. A solution of the ester from step 8 (0.031 g, 0.052 mmol) in CH,C1 _: (2 mL) was cooled to 0°C and treated with TFA (0.25 mL). After stimng for 5 h, the solution was concentrated under reduced pressure and purified via flash column chromatography (0-5% MeOH-CH _: CL) to give the desired acid (0.023 g, 90%) as a white crystalline solid. MP 198-199°C.

Example 2 - Preparation of Compound II

4β

Step 1. The benzyl ester was prepared in a manner analogous to the one descπbed for _t he corresponding 2-tnmemylsilyl ester intermediate < example 1. steps 1 -7). Ln this case, benzyl alcohol was used instead of 2-trimethy!sily edιanol in step 1.

Step 2. A solution of the intermediate from step I (0.020 g, 0.045 mmol) and diisopropylethylamine (0.025 mL. 0.144 mmol) in CH.CI, (1.5 mL) was treated with benzyl chioromethyledier (0.016 mL. 0.099 mmol) and stirred at room temperature for 6 h. Purification of the concentrated reaction mixture, by flash column chromatography (5-20% EtOAc-hexanes) provided the desired ether (0.022g, 86%). TLC: R 0.25 (silica, 20% EtOAc-hexanes).

Step 3. A solution of d e intermediate benzyi ester from step 2 (0.020 g, 0.035 mmol) in THF

(0.4mL) and edianol (0.4mL) was treated with NaOH solution (0.14 mL, 0.5 g 10 mL water). After stimng for 45 min. At room temperature, the mixture was diluted with EtOAc and quenched with

2N HC1 (0.2 ml). The organic layer was washed with satd. aq. NaCl, dried over MgS0 ₄ and concnetrated to give the desired acid (0.012g, 72%). MP 112- 1 13°C.

Example 3 - Preparation of Compound III

β

Step 1. A solution of the alcohol from example 2. step 1 ( 0.100 g, 0.223 mmol) and α;:sopropyϊedιylamιne ι0 05 mL. 0 287 mmol) in CH _: CI, (3 0 mL) was treated with p- toiuenesulfonyl chloπde (0.048 g, 0.249 mmol) and a crystal of DMAP. The mixture was stirred t room temperature for lόhrs.. concentrated under reduced pressure and punned by MPLC (0-20% EtOAc-hexanes) to give the desired tosylate (0 1 18 g, 88%). TLC: R.0.23 (silica, 0-20% EtOAc- hexanes ).

Step 2. A solution of the tosylate from step 1 (0.039 g, 0.066 mmol) and 18-crown-6 (0 044 . 0 166 mmol) in DMF (0.7 mL) was treated with sodium pyrrolidine dithiocarbamate ι.0.035g, 0 165 mmol) and stirred at room temperature for 16 h. The reaction mixture was diluted with EtOAc and water. .After separatmg die phases, the organic layer was washed with satd. aq. NaCI. dπed over MgSO ₄ . filtered and concentrated. Purification by MPLC (3-15% EtOAc-hexanes) provided the desired product (0.038 g, 99%). TLC. P _y O.34 (silica, 0-20% EtOAc-hexanes).

Step 3. The deprotecπon of die benzyl ester intermediate from step 2 was accomplished using the same protocol as described for example 2 in step 3. MP 177-178°C. The above methods for preparation of Examples 1-3 were, or could be used to prepare the series of biphenyl containing products found in Table 1.

Table 1

51-

SUBSTmJTE SHEET (RULE 26)

53

SUBSTrrUTE SHEET (RULE 26)

Example 18 - Preparation of Compound XVIII

Step I. A solution of pthlaiazinone ( 1.00 g, 6.84 mmol), triphenylphosphme ( 1.79 g. 6.84 mmol ) in THF ( 25 mL) was cooled to 0 °C and treated with 2- bromo ethanol (0.480 mL. 6.84 mmol) and diediyl azocarboxylate ( 1.07 mL. 6.84 mmol). After stimng I h at 0 °C, the solution was warmed to room temperature and striπed for an additional 12 h. The resulting mixture was concentrated and purified by flash column chromatography (35% ethyl acetate-hexanes ) to afford 1 .40 g (81%) of bromo ethyl phthalazinone as a white solid. TLC: Py 0.65 (40%ethyl acetate- hexane).

Step 2. A solution of sodium hydride (0.040 g, 1.54 mmol) in THF (5 mL) was cooled to 0

'C and carefully treated with diallyl malonate (0.260 g, 1.41 mmol). After warming to room temperature and stirring for 20 min., bromo etiiyl phtiialazinoπe from step 1 (0.325 g, 1.28 mmol) was added in one poπion and the mixture was heated to reflux for 18 h. The reaction mixture was

diluted with saturated aq. NH ₄ CI (20 mL) and EtOAc (20 mL). The resulting organic phase was washed with water, dπed over MgSO ₄ , filtered, and concentrated to afford 0 240 g (52%) of a yello w oil TLC. PL- 0.60 (40% ethvl acetate-hexane).

Step 3. A 2 . three-necked, round bottom flask was equipped with a mechanical stirrer. a diermometer and an argon inlet. The flask was charged with a solution of 4-chlorobιphenyl (48 30 g. 0 256 mol) in dichloromethane (500 mL). Bromoaceryl bromide (23 mL. 0.26 mol) was added via syringe and the solution was cooled with an ice bath to an tntemal temperature of 3 ^J C The thermometer was temporaπly removed and AlCI, was added poπionwise over 5 min. The internal temperature rose to 10 °C and white gas evolved from the opaque olive green reaction mixture. After 24 hrs. of stirring, the reaction was quenched by cautiously pouring into cold 10% HC1 ( ID. The organic layer became cloudy yellow green. Chloroform was added to help dissolve the solids. but the organic layer never became transparent. The organics were concentrated on a rotary ev aporator and dπed further under vacuum. The crude product was a pale green solid ( - 82g) which was recrystallized from hot ethyl acetate to give l-(2-bromoethanone)-4-(4-chlorophenyl)-benzene as brown needles (58.16 g). Concentration of the mother liquor followed by addition of hexanes delivered a second crop of crystals (1 1.06 g) which gave an NMR spectrum identical to that of the first crop. The total yield of the product was 87%. TLC: Py 0.30 (silica, 70% hexanes- dichlormethane).

Step 4. A solunon of sodium hydride ⁽ 0.020 g, 0.775 mmol) in THF (2.0 mL) was cooled to

0 C and carefully treated with the diester from step 2. The ice bath was removed and the resul _t ing mixture was stiπed for 20 min. The reaction mixture was re-cooied to 0°C and treated widi 1 -(2- bromoethanone)-4-(4-chlorophenyl)-benzene (0.200 g, 0.646 mmol) in one poπion. The mix _t ure w as warmed to room temperature over 30 min and subsequently heated to reflux for 12 hrs. The reaction mixture was added to satd. aq. NH,C1 (10 mL) and diluted with EtOAc (10 mL). The resulting organic phase was washed wnth water ( 10 mL), dried over MgS0 ₄ , filtered and concentrated to afford 0.327 g (78%) of a yellow oil. TLC: Pγ O.40 (silica, 40% ethyl acetate- he ane).

Step 5. A solution of the diester product from step 4 (0.327 g, 0.558 mmol) in 1,4 dioxane ( mL) was treated witii tetrakis(triphenylphosphine)pailadium (0.006 g, 0.005 mmol) in one portion and pyτrolidone (0.102 mL, 1.22 mmol) added dropwise over 15 min. After stirring for 30 min. at room temperature, the reaction mixture was diluted wid IN HCl (20 mL) and EtOAc (20 mL). The resulting organic phase was washed with satd. aq. NaCl, dried over MgSO ₄ , filtered, and concentrated to provide the diacid as a crude brown oil which was immediately carried on to step 6. TLC: PγO.29 (silica, 5% methanol-methylene chloride).

Step 6. A solution of the diactd product from step 5 in 1.4 dioxane (25 mL) was heated to reflux for 24 h. After cooling to room temperature, the resulting mixture was concentrated to a gray solid Recry stalhzation from ethyl acetate afforded 0 044g ( 18%, two steps) of compound XV HI as a white solid. MP 232°C. TLC. R,0 5 (silica. 10% methanol-methylene chloπde).

Example 19 - Preparation of Compound XIX _/ COjtBu

CO _: ιBu

Step 1. A solution of sodium hydnde (0 040 g, 1 54 mmol) in THF (lOOmL) was cooled to

0°C and treated with di-tert-butyl malonate (20.7 3 mL, 92.47 mmol) dropwise via dropping runnel, over 20 min. After stimng at room temperature for 30 mm., 3,3-dιmethylallyl bromide (9 7 mL. 83 22 mmol) was added. After stimng an additional 19 h. the reaction mixture was diluted widi 10% HCl solution (100 mL) and EtOAc ( 100 mL) The resulting organic phase was washed with satd aq NaCl, dπed over MgSO ₄ , filtered, and concentrated to afford 25 74 g (94%)of a crude yellow oil. TLC. P _γ 0.60 (silica. 10% etiiyl acetate-hexane).

TOitflu ^H0 — CO,tBu

Step 2. A solution of the crude olefin from step I (25.74 g, 90.50 mmol) in CH ₂ C1 _: (350 mL) and methanol (90.mL) was cooled to -78 °C and purged with O, for 20 mm. O ₃ was bubbled through the soiuuon until a blue color remained (2h). The solution was purged with 0 for 20 mm.; unul the

5 ^* 7

solution became colorless. After warming to 0 C. NaBH ₄ (3.42 g, 90.50 mmol) was added in one portion. .After several minutes the ice bath was removed and the mixture was stirred overnight. The mixture was concentrated, re-diluted in CH _; CL , washed with water ( 100 mL), 10% HCl ( 100 mL). bnne ι50 mL >. dπed over MgSO ₄ , filtered and concentrated into a coloriess oil. Puπfication of 15.0 g of crude matenal by flash chromatography (30% ethyl acetate-hexanes) afforded 6.86g (50%) as a colorless oil. TLC. R^O.30 (silica, 35% ethyl acetate-hexane).

Step 3. The malonate intermediate was prepared in a manner analogous to the one described for the preparation of example 18. step I . For this example, benzo- 1 ,2,3-triazin-4(3H)-one was used in place of phuialazinone and the alcohol form step 2 was used in place of 2-bromo edianol. TLC:

R,0.40 (silica, 40% etiiyl acetate-hexane).

Step 4. The dial ylated malonate intermediate was prepared in a manner analogous to the one described for the preparation of example 18, step 2. In this example, the monoalkylated malonate from step 3 was allcylated with die 1 -<2-bromoethone)-4-(4-chlorophenyl benzene. TLC: R 0.50 (silica, 40% ethyl acetate-hexane).

5β

Step 5. A solution of the diester from step 4 (4 61 g. 0.746 mmol) in 1.4 dioxane ₁ 10 mL) was treated with 4N HCl and heated to reflux for 10 h. After concnetratmg to an oil. die residue was purified by flash chromatography (0-10% methanoi-dichlorometiiane to give a yellow solid. MP ! 95 C Example 20 and Example 21 - Preparation of compunds XX and XXI

Example 19 was separated by chromatography on a chiral HPLC column (CH L EtOAc- hexanes ) Example 20 was the first to come off the column. Example 21 eluted second. Example 20. MS (F.AB-LSMIS) 462 [M+H] ^'

Example 21. .Anal. Calculated for C _: ,H _:c C!N - C, 65 01 ; H. 4.36, N, 9.10. Found C, 64.70: H. 4 06. N. 8.72.

Example 22 - Preparation of Compound XXII

Br _N _ ^ -C0 ₂ tBu CO,tBu

Step 1. A solution of di-rerr-butyl (2-hydroxyethyl)malonate (0.500 g, 1.92 mmoi), PPh,

( 0 555 g. 2.12 mmol) and CBr ₄ (0.704 g, 2.12 mmol) in CH _: C1 _: (4.0 mL) was stiπed at 0 °C for 5 min.. then warmed to room temperature. After stimng for an additional 16 h, die reaction mixture was concentrated in vacuo and purified via column chromatography (5-10% ethyl acetate-hexanes) to give 0.615 g (99%) of the desired product. TLC: PyO.7 (silica, 10% EtOAc-hexanes).

S°l

Step 2. A flask containing 1.3-dιhydro-5-methyl-2H- 1.4-benzodιazepιn-2-one (0 324 g. 1 03 mmol ⁾ and Cs CO ₃ (0 900 g. 2.76 mmol) was dπed under vacuum, flushed with Ar and charεed with a solution of di-t -butyl ⁽ 2-bromoethyl) malonate (0 300 g, 0 929 mmol) in DMF (3 0 mL) at 0 ³ C The mixture was stirred at 0 °C for 1 min.. room temperature for 15 mm., and 120 'C for 1 h. The reaction mixture was diluted with EtOAc (250 mL) and washed with water (2 x 50 mL).

The organic layer was separated, dπed over MgSO ₄ and concentrated. Puπfication by column chromatography (50-100% ethyi acetate-hexanes) afforded 0 017g of the desired product TLC R. 0 5 i sihca. 100% EtOAc).

Step 3. A flask containing the mono alkylated malonate from step 2 (0.37 g) and sodium butoxide (.009 g, 0.089 mmol) was vacuum dπed. flushed with Ar and diluted with THF ( 1 0 mL) at 0°C. After stirring at 0 °C for 30 min.. die reaction mixture was charged with 4-bromoacetyl-4'- chlorobiphenyl (0.027 g, 0.089 mmol) and subsequently stirred at room temperature for an additional 5 h. The reaction mixture was diluted with CH _: CU (75 mL) and washed with water (25 mL). The organic layer was separated, dried over MgSO ₄ and concentrated. Crude puπfication by column chromatography (50-100% ethyl acetate-hexanes) afforded die desired product (0.100 g, 0.154 mmol) which was used directly in step 4.

&0

Step 4. A solution of the malonate from step 3 (0.100 g, 0.154 mmol) in formic acid ( 1 0 mL ι was stirred at room temperature for 6 hrs. The resulting solution was concentrated in vacuo and used directly in step 5

Step 5. A solution of the product from step 4 in 1,4 -dioxane (2.0 mL) was heated to 100 ^} C for 1 6 h. After cooling to room temperature, the solvent was removed vacuo. Purification bv column chromatography (etiiyl acetate-hexanes-AcOH. 60:40: 1 ) afforded 0.020 g of a mixture which contained the desired product. The mixture was purified via HPLC on a C18 column

( acetonitrile-water) to furnish 2 mg of the target compound. HRMS 489.15720 (m- ), (calc. 488.15029).

Example 23 Biological Assays of Invention Compounds P218 Quenched Fluorescence Assay for MMP Inhibition:

The P218 quenched fluorescence assay (Microfluorometric Profiling Assay) is a modification of that originally described by Knight, et al., FEBS Lett. 296, 263 (1992) for a related substance and a vaπery of matπx metal loproteinases (MMPs) in cuvettes. The assay was run with each invention compound and the three MMPs, MMP-3, MMP-9 and MMP-2, analyzed in parallel, adapted as follows for a 96-well microtiter plate and a Hamilton AT* workstation.

P2I8 Fluorogenic Substrate: P218 is a synthetic substrate containing a 4-acetyi-7- methoxycoumarin ( MCA) group in the N-terminal position and a 3-[2, 4-dinitrophenyl]-L-2,3-

diammopropionyl (DPA) group internally This is a modification of a peptide reported by Knigh _t ( 1992) that was used as a substrate for mamx metalloproteinases. Once die P 18 peptide is cleaved ( putative clip site at the Ala-Leu bond), the fluorescence of he MCA group can be detected on a fluorometer with excitation at 328 nm and emission at 393 nm P218 is currently being produced B ACHEM exclusively for Bayer. P218 has the structure:

H-MCA-Pro-Lys-Pro-Leu-Λ/α-Ieu-DPA-Ala-Arg-NH2 (MW 1332.2) Recombinant Human CHO Stromelvsin (MMP-3)

Recomb ant Human CHO Pro-MMP-3: Human CHO pro-stromelysιn-257 (pro-MMP-3) was expressed and punfied as descπbed by Housley, et al.. J Biol. Chem. 268. 4481 (1993) Activation of Pro-MMP-3 Pro-MMP-3 at 1.72 μ.M ( 100 μg mL) in 5 mM Tπs at pH 7.5, 5 m.M CaCl,, 25 mM NaCl, and 0.005% Brij-35 (MMP-3 activation buffer) was activated by incubation wvdi TPCK (N-tosyl-(L)-phenylalanιne chlorometiiyl ketone) trypsm ( 1 100 w/w to pro- MMP-3) at 25 °C for 30 mm. The reaction was stopped by addition of soybean trypsin inhibitor (SBTI; 5.1 w/w to trypsin concentrauon). This acnvation protocol results in the formation of 45 kDa active MMP-3, which still contains the C-terminal portion of the enzyme.

Preparation of Human Recombinant Pro-Gelatinase A fMMP-2

Recombinant Human Pro-MMP-2: Human pro-gelatinase A (pro-MMP-2) was prepared using a vaccinia expression system according to die method of Fridman, et al., J. Biol. Chem. 267. 15398 (1992). Activation of Pro-MMP-2: Pro-MMP-2 at 252 mg mL was diluted 1 :5 to a final concentration of 50 μg mL solution in 25 mM Tris at pH 7.5, 5 mM CaCl ₂ , 150 mM NaCl, and 0.005% Brij-35 (MMP-2 activanon buffer). p-Aminophenylmercuric acetate (APMA) was prepared

in 10 mM (3.5 mg/mL) in 0.05 NaOH. The APMA solution was added at 1/20 the reaction volume for a final AMPA concentration of 0.5 mM. and the enzyme was incubated at 37 °C for 30 mm _. Activated MMP-2 ( 15 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 MMP-2 activation buffer for 1 min. followed by extensive H,0 washing). The enzyme was concentrated on Centπcon 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 _: 0. then MMP-2 activation buffer) with re-dilution followed by re-concentration repeated twice. The enzyme was diluted to 7.5 mL (0.5 times the onginal volume) with MMP-2 activation buffer. Preparation of Human Recombinant Pro-Gelatinase B (MMP-9):

Recombinant Human Pro-MMP-9: Human pro-geiatinase B (pro-MMP-9) deπved from U937 cDNA as described by Wilhelm, et al. J. Biol. Chem. 2JJ4, 17213 (1989) was expressed as the full-lengτh form using a baculovirus protein expression system. The pro-enzyme was punned using methods previously described by Hibbs, et al. J. Biol. Chem. 26j 2493 (1984). Activation of Pro-MMP-9: Pro-MMP-2 20 μg/mL in 50 mM Tris at pH 7.4, lOmM CaCl _: ,

1 0 mM NaCl, and 0.005% Brij-35 (MMP-9 activation buffer) was activated by incubation with 0.5 mM /?-aminophenylmercuric acetate (APMA) for 3.5 h at 37 °C. The enzyme was dialyzed against the same buffer to revmove the APMA.

Instrumentation.

Hamiltion Microlab AT Plus: The MMP-Profiling Assay is performed robotically on a Hamilton MicroLab AT Plus*. The Hamilton is programmed to: (1) serially dilute up to 11 potential

inhibitors automatically from a 2.5 mM stock in 100% DMSO: (2) distribute substrate followed by inhibitor into a 96 well CytoΩuor plate: and (3) add a single enzyme to die plate with mixing _t o s _t aπ the reacuon. Subsequent plates for each additional enzyme are prepared automatically by beginning the program at the substrate addition point, remixing the diluted inhibitors and beginning die reaction by addition of enzyme. In dus way, all MMP assays were done using the same inhibitor dilutions. fillφore 11 Following incubation, the plate was read on a Cytofluor Q fluoromemc plate reader widi excitation at 340 nM and emission at 395 nM with the ga set at 80 Buffers: icrσfluorometric Reaction Buffer (MRB): Dilution of test compounds, enzymes, and P218 substrate for die microfluorometnc assay were made m microfiuorometnc reaction buffer consisting of 50 mM 2-(N-mo hoIino)cthanesulfonιc acid (MES) at pH 6 5 with 10 mM CaCI ₂ , 150 mM NaCl. 0 005% Bπj-35 and 1% DMSO

Methods:

MMP Microfluorometnc Profiling Assay The assay is done with a final substrate concentrauon of 6 μM P218 and approximately 5 to 8 nM MMP with vaπable drug concentrations. The Hamilton is programmed to seπally dilute up to 1 1 compounds from a 2.5 mM stock (100% DMSO) to 1 Ox the final compounds concentrations in die assay. Initially, the instrument delivers vaπous amounts of microfiuoromentπc reaction buffer (MRB) to a 96 tube rack of 1 ml Marsh dilunon tubes. The instrument then picks up 20 μl of inhibitor (2.5 mM) from the sample rack and mixes it with a buffer m row A of the Marsh rack, resulting in a 50 μM drug concentrauon. The

⁽ A

SUBSTΓTUTE SHEET (RULE 26)

inhibitors are then seπally diluted to 10, 5, 1 , .2, 05 and .01 μM. Position I on the sample rack contains only DMSO for the ^' 'enzyme-only" wells m the assay, which results in no inhibitor in column I, rows A through H. The instrument then distributes 107 μl of P218 substrate (8 2 u.M in MRB) to a single 96 well cytofluor microtiter plate. The instrument re-mixes and loads 14 5 ul of diluted compound from rows A to G in the Marsh rack to coπesponding rows in the microtiter plate

( Row H represents the "background" row and 39 5 μl of MRB is delivered in placed of drug or enzyme) The reaction is started by adding 25 μl of die appropπate enzyme (at 5 86 times the final enzyme concentration) from a BSA treated reagent reservoir to each weil. excluding Row H. die 'background" row (The enzyme reservoir is pretreated with 1% BSA in 50 mM Tns, pH 7 5 containing 150 mM NaCl for 1 hour at room temp., followed by extensive H _: O washing and drying at room temp.).

After addition and mixing of the enzyme, the plate is covered and incubated for 25 mm. at 37°C Additional enzymes are tested in die same manner by beginning the Hamilton program with the distπbution of P218 substrate to the microtiter plate, followed by re-mixing and distπbution of the drug from the same Marsh rack to die microtiter plate. The second (or third, etc.) MMP to be tested is then distributed from a reagent rack to the microtiter plate with mixing, pπor to coveπng and incubation. This is repeated for all additional MMP's to be tested.

IC50 Determination in Microfluorometnc 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 determination for each drug concentration by comparing the amount of hydrolysis (fluorescence units generated over 25 minutes of hydrolysis) of wells containing compound with the "enzyme

only ^" wells in column 1. Following subtraction of the background the percent inhibi _t ion was calculated as:

((Control values - Treated values VControl values) x 100 Percent inhibitions were determined for inhibitor concentrations of 5. 1. 0.5. 0.1 , 0.02. 0.005 and. 0.001 μM of drug. Linear regression analysis of percent inhibition versus log inhibi _t or concentration was used to obtain IC« ₀ values.

Table 1

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Other embodiments of the invention will be apparent to those skilled m the an from a consideration of this specification or practice of the invention disclosed herein. It is intended tiiat the specification and examples be considered as exemplary only, with the true scope and spiπt of the invention being indicated by the following claims.

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