WO1997043238A9 - Substituted oxobutyric acids as matrix metalloprotease inhibitors - Google Patents

Abstract

The present invention provides pharmaceutical compositions and methods for treating certain conditions comprising administering an amount of a compound or composition of the invention which is effective to inhibit the activity of at least one matrix metalloprotease, resulting in achievement of the desired effect. The compounds of the present invention are either of generalized formula (I) wherein y is 0, 2, or 3, r is 0-6, Z is (CH2)7 or (CH2)e-C6H4-(CH2)f, wherein e is 0-1 and f is 1-6, and R15 is -H, -Cl, -OMe or (a), (b) wherein n is 0-4, R17 is C2H5, allyl, benzyl, and R16 is (c) wherein t is 0-1, x is 0-4, and R4 is one of the following: halide, alkyl of 1-6 carbons, OR, NR¿2?, NO2 (R = H or alkyl of 1-6 carbons). 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, osteopenias mediated by MMP activity, tempero mandibular joint disease, demyelating diseases of the nervous system, tumor metastasis or degenerative cartilage loss following traumatic joint injury, and coronary thrombosis from atherosclerotic plaque rupture. The present invention also provides pharmaceutical compositions and methods for treating such conditions.

Description

Substituted Oxobutyric Acids as Matrix Metalloproteinase Inhibitors

BACKGROUND OF THE INVENTION Field of the Invention

This invention relates to enzyme inhibitors, and more particularly, to novel oxoburyπc acids compounds or derivatives thereof useful for inhibiting matnx metalloproteases. Description of the Related Art

The matnx metalloproteases (a.k.a. matnx metalloendo-protemases or MMPs i are a famrn of zinc endoproteinases which include, but are not limned to. interstitial collagena≤e (a.k.a.

MMP- 1 ). stromelysin (a.k.a.. proteoglycanase. transin. or MMP-3), gelatinase A (a.k.a.. 72kDa-geiaunase or MMP-2) and gelatinase B (a.k.a.. 95kDa-gelatιnase or MMP-9). These MMPs are secreted by a variety of cells including fibroblasts and chondrocytes. along with natural proteinaceous inhibitors known as TTMPs (Tissue Inhibitor of MetalloProteinase). All of these MMPs are capable of destroying a vanery of connective tissue components of articular cartilage or basement membranes. Each MMP is secreted as an inactive proenzyme which must be cleaved in a subsequent step before it is able to exert its own proteolytic activity In addition to the matnx 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 (Ito. et al.. Arch Biochem Biophys. 67, 21 1 ( 1988); Ogata, et al.. J. Biol. Chem., 267, 3581 (1992)). Thus, a cascade of proteolytic 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 reported that MMP-3 can cleave and thereby inactivate the endogenous inhibitors of other protemases such as elastase (Wmyard. et al . FEBS Lens 229, 1 , 91 (1991 )) Inhibitors of MMP-3 could thus influence the activity of other destructive protemases by modifying the level of their endogenous inhibitors A number of diseases are thought to be mediated by excess or undesired matπx-destroying metalloprotease activity or by an imbalance in the ratio of the MMPs to the TIMPs These include a) osteoarthπus (Woessner, et al , J. Biol Chem , 259(6). 3633 (1984), Phadke. et al . J Rheumatol 10, 852 (1983)), b) rheumatoid arthπtis (Mullins, et al.. Biochim, Biophys Acta 695, 1 17 (1983). Woo Hey, et al , Arthπtis Rheum 20, 1231 (1977), Gravallese, et al., Arthπtis Rheum 34, 1076 ( 1991 )) c) septic arthπtis (Williams, et al . . rthπtis Rheum H, 533 ( 1990)), d) tumor metastasis

(Reich, et al . Cancer Res , 48, 3307 (1988). and Matπsian. et al., Proc Nat'l Acad Sci . USA £3, 9413 (1986)), e) peπodontal diseases (Overall, et al., J Peπodontal Res 22, 81 (1987)), cornea! uiceration (Bums, et al., Invest. Opthalmol Vis Sci 2 _, 1569 (1989)), g) protetnuna (Baπcos. 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 )), I) aneurysmal aortic disease (Vine, et al . Can Sci & 233 (1991)), j) birth control (Woessner, et al., Steroids 54, 491 (1989)). k) dystrophobic epidermolysis bullosa (Kronberger. et al . J Invest Dermatol 79, 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 £0, 688 (1988))

The need for new therapies is especially important in the case of arthπtic diseases The pπmary disabling effect of osteoarthπtis (OA). rheumatoid arthπtis (RA) and septic arthπtis 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- inflammatory drugs (NS AIDs) 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 dela> surgical intervention.

Proteases are cπtical elements at several stages in the progression of metastatic cancer. In this process, the proteolytic degradation of structural protem in the basal membrane allows for expansion of a tumor in the pπmary site, evasion from this site as well as homing and invasion in distant, secondary sues. Also, tumor induced angiogenesis is required for tumor growth and is dependent on proteolytic tissue remodeling Transfection experiments with vanous types of proteases have shown that the matnx metalloproteases play a dominant role in these processes in particular gelaunases 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. 1, 2062 (1994); Birkedal-Hansen, et al., Crit. Rev Oral Biol. Med. 4, 197 (1993). Furthermore, it was demonstrated that inhibition of degradation of extracellular matnx by the native matnx metal loprotease inhibitor TIMP-2 (a protein) arrests cancer growth (DeClerck, et al.. Cancer Res. 52, ^"701 (1992)) and that TTMP-2 inhibits tumor-induced angiogenesis in expeπmental systems (Moses, et al. Science 248, 1408 (1990)). For a review, see DeClerck. et al., Ann. N Y Acad. Sci. 732, 222 ( 1994). It was further demonstrated that the synthetic matnx metal loprotease inhibitor batimastat when given lntraperitoneally 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., Cancer Res. £3, 2087 (1993)). The use of this and related compounds has been described in Brown, e al.. WO-9321942 A2 (931 1 1 1 ). There are several patents and patent applications claiming the use of metalloprotemase inhibitors for the rctardauon of metastatic cancer, promoting rumor regression, inhibiting cancer cell proliferation, slowing or preventing cartilage loss associated with osteoarthπtis or for treatment of other diseases as noted above (e.g. Levy, et al., WO-9519965 A 1. Beckett, et al . WO-9519956 A ! . Beckett et al., WO-9519957 Al . Beckett et al., WO-9519961 A l , Brown, et al., WO-9321942 A2.

Cnmmin. et al . WO-9421625 A 1. 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 complexmg group (hydroxamic acid, thiol. carboxylic acid or phosphinic acid) at one end and a vaπety of sidechains. both those found in the natural ammo acids as well as those with more novel functional groups Such small peptides are often poorly absorbed, exhibiting low oral bioavailabi ty. They are also subject to rapid proteolytic metabolism, thus having short half lives. As an example, baumastat. the compound descπbed in Brown, et al., WO-9321942 A2. can only be given intrapeπtoneally. Others have disclosed a seπes of biphenyl-containing carboxylic acids, illustrated by the compound shown below, which inhibit neural endopeptidase (NEP 24 11 ), a membrane-bound zinc metalloprotease (Stanton. et al., Bioorg. Med. Chem. Lett 4, 539, 1994. Lombaert. et al.. Bioorg Med. Chem. Lett. 4, 2715 (1994), Lombaert. et al., Bioorg. Med. Chem Lett 5, 145 ( 1995), Lombaert. et al., Bioorg. Med. Chem. Lett. 5, 151 ( 1995))

It has been reported that N-carboxyalkyl derivatives containing a biphenylethylglycine. illustrated by the compound shown below, are inhibitors of stromelysin- 1 (MMP-3). 72 kDA gelatinase (MMP-2) and collagenase (Durette, et al., WO-9529689).

It would be desirable to have effective MMP inhibitors which possess improved bioavailabihty and biological stability relative to the peptide-based compounds of the prior art, 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 osteoarthπtis. rheumatoid arthritis, sepuc arthritis, tumor metastasis, peπodontal diseases, cornea! uicerations. and proteinuria. Several inhibitors of MMPs have been described 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 ( 1995); Conway, et al. J. Exp. Med. L82, 449 ( 1995); Porter, et al., Bioorg. Med. Chem. Lett. 4, 2741 (1994); Tomczuk, et al.. Bioorg. Med. Chem. Lett. 5, 343 (1995); Casteihano, et al., Bioorg. Med. Chem. Lett. 5, 1415 (1995)), phosphorous-based acids (Bird, et al. J. Med. Chem. H, 158 (1994), Morphy, et al.. Bioorg Med Chem Lett 4, 2747 (1994), Kortyiewicz. et al , J Med Chem

21, 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 Biochem Biophys 287, 240 (1991 ), Ye. et al , J Med Chem 37, 206 (1994). Grobeiny, et al . Biochemistry 24, 6145 (1985), Mookhtiar. et al , Biochemistry 2, 4299 (1988))

However, these inhibitors generally contain peptidic backbones, and thus usually exhibit low oral bioacmity due to poor absorption and short half lives due to rapid proteolvsis Therefore, there remains a need for improved MMP inhibitors

SUMMARY OF THE INVENTION

This invention provides compounds having matnx metal loprotease inhibitory activity These compounds are useful for inhibiting matnx metalloproteases and, therefore, combating conditions to which MMPs contribute Accordingly, the present invention also provides pharmaceutical compositions and methods for treating such conditions The compounds descπbed relate to a method of treating a mammal compnsmg admirusteπng to the mammal a matnx metalloprotease inhibiting amount of a compound according to the invention sufficient to

(a) alleviate the effects of osteoarthπtis, rheumatoid arthntis, septic arthntis, peπodontal disease, comeal ulceration, proteinuπa, aneurysmal aortic disease, dystrophobic epidermolysis, bullosa, conditions leading to inflammatory responses, osteoperuas mediated by MMP activity, tempero mandibular joint disease, demyelatmg diseases of the nervous system,

(b) retard tumor metastasis or degenerative cartilage loss following traumatic joint injury;

(c) reduce coronary thrombosis from athrosclerotic plaque rupture; or (d) effect birth control

The compounds of the present invention are also useful scientific research tools for studying funcuons and mechanisms of action of matnx metalloproteases in both in vivo and in vitro systems

Because of their MMP-inhibmng 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 matnx metal loprotease inhibitory activity and the generalized formula

A-D-E-G (L) A represents alkyl. allyl-, benzyioxy-, or 3-propynyl alkyl groups as well as the structure

R -^ _^Z-CH₂ where Z = (CH_:)_t-QH₄-(CH_:)_f or (CH_:)_g. e = 0-8, f = 0-5, g = 0-14 and wherer is 0-6 R^{l 5} may be -H, -Cl. -OMe or

wherein n is 0-4. R'⁷ is -C_:H_}. -allyl. or -benzyl.

In the generalized formula (L), D represents

\ \ \ \ ,H \ ,H

C=0 C = NOH C =S C_x C.

/ / / OH / H

In the generalized formula (L), E represents a chain of n carbon atoms beanng m substituents R⁶ in which the R⁶ groups are independent substituents. or constitute spiro or nonspiro nngs. Rings may be formed in two ways: a) two groups R⁶ are joined, and taken together with the chain atom(s) to which the two R^β group(s) are attached, and any intervening chain atoms, constitute a 3 - 7 embered πng, or b) one group R⁶ is joined to the chain on which this one group R⁶ resides, and taken together with the chain atom(s) to which the R⁶ group is attached, and any intervening chain atoms, constitutes a 3 - 7 membered ring. The number n of carbon atoms in the chain is 2 or 3, and the number m of R⁶ substituents is an integer of 1 - 3. The number of carbons in the totality of R⁶ groups is at least two.

Each group R* is alkyl. alkenyl. alkynyl, aryl, heteroaryl. non-aromatic cyclic, and combmauons thereof optionally substituted with one or more hetero-atoms as described more fully below

In the generalized formula (L). E is a substituted mono- or bicyclic moiety optionally substituted with one or more heteroatoms.

In the generalized formula (L), G represents -CO H, -PO₃H_;, -M,

m which M represents -C0₂H, -CONfR"),, or -CO,R¹². where R" is H or alkyl of 1 - 4 carbons. R'- IS alkyl of 1 - 4 carbons, and R^u represents any of the side chains of the ! 9 noncyclic naturally occurπng amino acids. Certain embodiments include compounds having matnx metal loproteinase inhibitory activity and the following generalized formula:

where Z = (CH₂)_e-C₆H₄-(CH₂)_f or (CH₂)_P e = 0-8, f = 0-5, g = 0-14 and where y is 0, 2, or 3.

R" may be H, Cl, MeO or

wherein n is 0-4, R¹⁷ is C_:H„ allyl, or benzyl, and R¹⁶ is one of

where t is 0-2, x is 0-4 and R" is one of the following: halide, alkyl of 1-6 carbons, OR. NR,. NO_: (R = H or alkyl of 1 -6 carbons).

The foregoing merely summanzes 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 incoφorated by reference in their entirety.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

More particularly, the compounds of the present invention are mateπals having matnx metalloprotease inhibitory activity and the generalized formula:

A-D-E-G (L) in which A represents alkyl of 9-14 carbons or alkyloxv of 9-18 carbons, or allyloxy-, benzyloxy-, or propynyl- alkyl of 9-18 carbons. A is also represented by the structure:

R ^Z-CH₂ where Z = (CH_:)_e- H₄-(CH₂)_f or (CH₂)_g, e = 0-8, f = 0-5, g = 0-14 and where r is 0-6. R^{l 5} may be -H. -Cl. -OMe or

wherein n is 0-4, R¹⁷ is C,H₅. allyl, or benzyl.

In the generalized formula (L), D represents the moieties

\ \ \ \ .H \ ,H

C=0 C = NOH C =S C_s C,

/ / / OH / H

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

where R is

is the structure

In the generalized formula (L), E represents a chain of n carbon atoms beaπng m substituents R⁶. referred to as R⁶ groups or R⁶ units. The R⁶ groups are independent substituents, or constitute spiro or nonspiro πngs. Rings may be formed in two ways: a) two groups R^β are joined, and taken together with the cham atom(s) to which the two R⁶ group(s) are attached, and any intervening chain atoms, constitute a 3 - 7 membered πng. or b) one group R⁶ is joined to the chain on which this one group R⁶ resides, and taken together with the chain atom(s) to which the R ⁶group is attached, and any intervening chain atoms, constitutes a 3 - 7 membered πng. The number n of carbon atoms in the chain is 2 or 3, and the number m of R⁶ substituents is an integer of 1 - 3. The number of carbons in the totality of R* groups is at least two.

Each group R⁶ is independently selected from the group consisting of the substituents listed below as items 1) - 14): 1) alkyl of 1 - 10 carbons;

2) aryl of 6 - 10 carbons;

3) heteroaryl compπsing 4 - 9 carbons and at least one N, O. or S heteroatom;

4) arylalkyl in which the aryl portion contains 6 - 10 carbons and d e alkyl portion contains 1 - 8 carbons; 5) heteroaryl-alkyl in which the heteroaryl portion compπses 4 - 9 carbons and at least one N, O, or S heteroatom. and the alkyl portion contains 1 - 8 carbons;

6) alkenyi of 2 - 10 carbons;

7) aryl-alkenyl in which the aryl portion contains 6 - 10 carbons and the alkenyi portion contains 2 - 5 carbons;

8) heteroaryl-alkenyl in which the heteroaryl portion compπses 4 - 9 carbons and at least one N, O. or S heteroatom and the alkenyi portion contains 2 -5 carbons.

9) alkynyl of 2 - 10 carbons;

10) aryl-alkynyl in which the aryl portion contains 6 - 10 carbons and the alkynyl portion contains 2 - 5 carbons.

1 1 ) heteroaryl -alkynyl in which the heteroaryl portion compπses 4 - 9 carbons and at least one N. O, or S heteroatom and the alkynyl portion contains 2 - 5 carbons;

12)-(CH;,),R⁷ in which t is 0 or an integer of 1 - 5 and R⁷ιs selected from the group consisting of:

as well as corresponding heteroaryl moieαes in which the aryl portion of an arvl-containing R⁷ group compπses 4 - 9 carbons and at least one N. 0. or S heteroatom In such R⁷ groups, Y represents O or S. and u is 0, 1. or 2 provided that when R⁷ is

—

and the A un t is phenyl. the B un t is phenyiene. m is 1. n is 2. and t is 0, and x is 1 or 2

R¹ represents H or alkyl of 1 - 3 carbons R^: represents H, alkyl of 1 - 6 carbons, aryl of 6 - 10 carbons, heteroaryl compπsing 4 - 9 carbons and at least one N. O, or S heteroatom. arylalkyi in which the aryl portion contains 6 - 10 carbons and the alkyl portion contains 1 - 4 carbons, or heteroaryl-alkyl in which the heteroaryl portion compπses 4 - 9 carbons and at least one N, O, or

S heteroatom and the alkyl portion contains 1 - 4 carbons

R⁵ represents alkyl of 1 - 4 carbons, aryl of 6 - 10 carbons, heteroaryl compπsing 4 - 9 carbons and at least one N, O, or S heteroatom, arylalkyi in which the aryl portion contains 6 - 10 carbons and the alkyl portion contains 1 - 4 carbons, or 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

13) -{CHjXPR'in which v is an interger of 1 to 4, P represents -S-, -S(O)-, -SO_:-, or -O- and R¹ is selected from the group consisting of alkyl of 1 to 12 carbons, aryl of 6 to

10 carbons, heteroaryl compπsing 4 to 9 carbons and at least one N, O, or S heteroatom, arylalkyi in which the aryl portion contains 6 to 12 carbons and the alkyl portion contains 1 to 4 carbons; heteroaiylalkyl 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(O)R⁹ in which the R' represents alkyl of 2 to 6 carbons, aryl of 6 to 10 carbons, heteroaryl compπsing 4 to

9 carbons and at least one N, O, or S heteroatom; and arylalkyi in which the aryl portion contains 6 to 10 carbons or is a heteroaryl compπsing 4 to 9 carbons and at least one N, O. or S heteroatom, and the alkyl portion contains 1 to 4 carbons, with the provisos that when R⁸ is -C(O)R . Z is -S- or -O-, when Z is -0-, R⁸ may also be -(C„H_2qO)-Rⁱ in which q. r. and

R⁵ are as defined above; and when the A un t is phenyl, the B unit is phenylene. m is 1. n is 2. and v is 0, then x is 1 or 2.

14)

in which w is an integer of 1 to 3. and R'° represents alkyl of 1 to

2 carbons. In addiuon. aryl or heteroaryl portions of any of the R⁶ groups optionally may bear up to two substituents selected from the group consisting of -(CH₂\C(R")(R'²)OH, -(CH₂),OR". -( CH₂\SR".

-(CH₂),S(O)R", -(CH₂)_vS(O),R , ' kCH₂χSO,N(R )₂, -(CH₂χN(R )₂. -(CH₂)_VN( )COR .

-OC(R" )₂O- in which both oxygen atoms are connected to the aryl πng, -(CH,).COR".

-(CH^CONfR''^, -(CH₂χC0₂R". -(CH₂\OCOR" -halogen. -CHO, -CF₃, -NO₂, -CN, and -R^ι:, in which y is 0 - 4: R" represents H or alkyl of 1 - 4 carbons; and R'² represents alkyl of 1 - 4 carbons.

In the generalized formula (L). G represents -CO₂H. -PO,H₂, -M,

O ll \ / O ι ι R l ¹³ I N-N >'

— C-N-C-M , —C-N-C-M , or — ^ -^N

H H N in which M represents -CO_:H, -CON(R")_:, or -CO_:R¹². and R'³ represents any of the side chains of the 19 noncyclic naturally occumng ammo acids.

Pharmaceutically acceptable salts of the compounds falling within the generalized formula (L) are also within the invention. It is to be understood that as used herein, the term "alkyl" means straight, branched, cyclic. and polycyclic materials. The term "haloaikyl" means partially or fully halogenated alkyl groups such as -(CH_;)₂C1. -CF₃ and -C₆F₁₃ for example.

In one embodiment the invention relates to compounds of generalized formula CL). wherein n is 2 and m is 1 in the E unit. 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 CL) in which the number of substituents m on the E unit is 2 or 3; and when m is 2, both groups R⁶ are independent substituents, or together constitute a spiro πng. or one group R* is an independent substituent and the other constitutes a spiro πng; and when m is 3. rwo groups R⁶ are independent substituents and one group R⁶ constitutes a πng, or two groups R 'constitute a ring and one group R6 is an independent substituent. or three groups R⁶ are independent substituents. This subset therefore contains compounds in which the E unit is di- or tπ- substituted, and in the disubstituted case any rings formed by one or both R⁶ groups are spiro ππgs, and in the tπsubstituted case, the R⁶ groups may form either spiro or nonspiro πngs.

In another of its embodiments, the invention relates to compounds of generalized formula (L) in which the number of substituents m on the E unit is 1 or 2; and when m is 1 , the group R⁶ constitutes a nonspiro ring; and when m is 2. both groups R⁶ together constitute a nonspiro πng or one group R⁶ is an independent substituent and the other constitutes a nonspiro πng. This subset therefore contains compounds in which the E unit cames one or two substituents R⁶, and at least one of these substituents is involved in a nonspiro πng.

More particularly, representative compounds of generalized formula (L) in which one or more of the substituent groups R⁶ are involved in formation of nonspiro πngs have E units of the following structures:

in which a is 0, 1 , or 2, b is 0 or 1 , c is 0 or 1 , d is 0 or 1 , c + d is 0 or 1 , e is 1 - 5, f is 1 - 4, g is 3

- 5, h is 2 - 4. l is 0 - 4, j is 0 - 3. k is 0 - 2, the total number of groups R⁶ is 0. 1 , or 2, U represents O. S. or NR' , and z is 1 or 2, Each group R¹⁴ is independently selected from the group consisting of alkvl of 1 - 9 carbons; arylalkyi in which the alkyl portion contains 1 - 7 carbons and the aryl portion contains 6 - 10 carbons, alkenyi of 2 - 9 carbons, aryl-substituted alkenyi in which the alkenyi portion contains 2 - 4 carbons and the aryl portion contains 6 - 10 carbons; alkynyl of 2 -

9 carbons, aryl -substituted alkynyl in which the alkynyl portion contains 2 - 4 carbons and the aryl portion contains 6 - 10 carbons; aryl of 6 - 10 carbons; -COR²; -CO,R³; -CONCR²)^ -(CH^p n which t is 0 or an integer of 1 - 4, and -(CH₂).ZR^β in which v is 0 or an integer of 1 to 3, and Z represents -S- or -O- R', R², R\ R⁶, R⁷, and R' have been defined above Preferred compounds of generalized formula (L) in which one or more of the substituent groups R⁶ are involved in formation of nonspiro πngs have E units of the following structures:

— >

in which a. b. c. d, ic ->- d), e, g. 1. k. the total number of groups R⁶, U, and R'⁴ are as defined above.

Other compounds of generalized formula (L) have R⁶ units of the following structures:

where n is 0-1.

Most preferred compounds of the general formula (L) include those of the following general formula

wherein y is 0 (i.e., there is no πng structure), 2 (cyclobutyl), or 3 (cyclopentyl), r is 0-6. Z is (CH_:)₇ or (CH₂).-C₆H₄-(CH₂)_f, wherein e is 0-1 and f is 1 -6. and R¹⁵ is -H, -Cl. -OMe or

R¹⁷'° *_. . HO^≡≡^ wherein n is 0-4, R¹⁷ is -C₂H,, -allyl, -benzyl, and R¹⁶ is

where x is 0-4. t is 0-2, and R⁴ is one of the following: halide. alkyl of 1 -6 carbons, OR, NR . NO_:

(R = H or alkyl of 1 -6 carbons).

Those skilled in the art will appreciate that many of the compounds of the invention exist

in enantiomeπc or diastereomenc 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 stereoisomeπc designauons. as well as mixtures of stereoisomers m 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) 1 ,3-dihydro- 1 ,3-dioxo-α-(2-oxododecyl)-2H-isoιndole-2-butanoic acid,

II) l ,3-dihydro-l ,3-dioxo-α-(2-oxoundecyl)-2H-isoindole-2-butanoic acid,

III) 1 ,3-dihydro- 1 ,3-dioxo-α-(2-oxotridecyl)-2H-isoindole-2-butanoic acid,

IV) 1 ,3-dihydro-l ,3-dioxo-α-(2-oxotetradecyl)-2H-isoindole-2-butanoic acid, V) 1.3-dihydro-1.3-dioxo-α-(2-oxopentadecyl)-2H-isomdole-2-butanoιc acid,

VI) 1 ,3-dihydro- 1.3-dioxo-α-(2-oxohexadecyl)-2H-isomdole-2-butanoιc acid,

VII) γ-oxo-α-(2-phenylethyl)-benzeneheptanoιc acid.

VIII) γ-oxo-α-(2-phenylethyl)-benzenehexanoιc acid, and EX) γ-oxo-α-(2-phenylethyl)-benzenepentanoιc acid The compounds of the invention may be prepared by use of known chemical reactions and procedures. Nevertheless, the following general preparative methods are presented to aide the reader in synthesizing the inhibitors. More detailed procedures for particular examples are presented below in die experimental section. In the general methods the following generic descriptions apply. The group designated P represents a protecting group. It may be appreciated by one skilled in the act that a variety of different protecting groups may be used to protect a potentially reactive functional group (e.g. carboxylic acid, alcohol) and that the particular choice will depend upon the reaction conditions required to prepare a given target compound. A description of such protecting groups may be found in: Green and Wuts, Protective Groups in Organic Synthesis, 2nd Ed.. John Wiley and Sons. New York. 1991.

The group designated X represents a leaving group, It is well known to those skilled in the an that several different functional groups such as halides, mesylates, tosylates and triflates may serve as a leaving groups. It is also known that the choice of a particular leaving group typically depends on such factors as the reactivity of the nucleophile, stability of the compound and ease of synthesis.

General Method A - The compounds of the invention where E does not contain a ring are conveniently prepared using an (α-halomethyl ketone and a substituted malonate derivative. The α-halomethyl ketone intermediate CIO (X = Cl, Br) can be conveniently prepared from a carboxylic acid or a methyl ketone. From carboxylic acid Cl, treatment with oxalyl chloride and a catalytic amount of DMF in a solvent like trimethylsilylchloride provides the corresponding acid chloride. Subsequent treatment with excess diazomethane followed by anhydrous HCI or HBr provides intermediate CIE. Alternatively, intermediate CIII can be prepared from methyl ketone CII via the corresponding silyl enol ether by treatment with N-bromosuccinimide (NBS). The silylenol ether is conveniently prepared from the methyl ketone by treatment with triniethylsilylchlotide (TMSCI) and a base like lithium hexamethyldisilazide (LHMDS). General procedures for the preparation of α-halomethyl ketones are well-known to those skilled in the art. For additional references see Corey, et al., Tetrahedron Lett. 25, 495 (1984) and Reuss, et al., J. Org. Chem. 39, 1785 (1974).

Methods for the preparation of substituted malonate derivatives (CV) are also well-established in the literature. Typically, an unsubstimted malonate derivative is treated with a base like NaH or KOt-Bu in a polar aprotic solvent, and then alkylated with a substituted halide. Similarly, alkylation of mono alkylated intermediate CV with α-halomethyl ketone CHI provides dialkylated intermediate, CVI. It should be appreciated by those skilled in the art that sidechain Y may be the sidechain desired in the final target or a simple handle to further elaborate that portion of the molecule at a latter stage of the synthesis. If sidechain Y is die desired sidechain, then intermediate CVI can simply be deprotected and decarboxylated using well-known procedures to give target compound CVHI. The conditions used to deprotect intermediate CVI will depend on the type of protecting group used. Some convenient protecting groups used to synthesize the compounds of the invention include methyl, allyl, benzyl and tert-butyl. Methods to incorporate and remove these groups are well-known to those skilled in the art (see above reference). The choice of protecting group used in the synthesis will depend on such factors as functional group compatibility, ease of synthesis and availability of starting materials.

If the target compound CXI contains a moiety Q which is sensitive to the reaction conditions used in the alkylation steps then an intermediate sidechain Y can be used. In this case, a protected ethanol group such as CH₂CH₂OTBS can be conveniently incoφorated as this handle. Intermediate CV with Y = -CH_jCH,OTBS can be prepared by using TBSOCH₂CH₂Br as Y-X in the first alkylation step. TBSOCH₂CH₂Br can be prepared from HOH₂CH₂Br by methods well-known to those skilled in the art. The protecting group can be removed to provide the corresponding alcohol which may be converted to phenyl ethers or a variety of heteroatom substituted derivatives used to generate sidechain Q via the Mitsunobu reaction. The Mitsunobu reaction is well known to those skilled in the art; see Mitsunobu, Synthesis 1 (1981 ), and Hughes, Organic Reactions 42, 335 ( 1992). Alternatively, the alcohol intermdiate is converted to a leaving group such as tosviate or bromide and 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). After the desired sidechain Q is incorporated to form CX, the malonate moiety can be deprotected and decarboxylated to provide the target compound CXI. In some cases the ketone moiety of intermediate CHI may need to be protected to avoid undesired side reactions. If required, protection as an acetal using the protocols like those described in Hwu. et al.. J. Org. Chem. 50, 3946 (1 85), is generally preferred,

o M 1 . . COCl,_{2 t} o M , ^{1 ) T} L^MHM^SCD¹ S o II

_R ^-₀H 2) CH,N, R ^^{/ X} 2) NBS R ^CH, ci ^{3) rø} cm en

0 0

Y-X

NaH. THF PO OP Nafi THF PO AA OP

CV crv

General Method B - The compounds of this invention in which two R⁶gr°ιιps are joined to form a substituted 5-member ring E are most conveniently prepared by method B. In this method acid MI (R=H) is prepared using the protocols described in Beeley, et al., Tetrahedron 37 Suppl., 411 (1981 ). The acid is protected as an ester [e.g., R= benzyl (Bn) or 2-(trimethylsiiyl)ethyl (TMSE)] by use of coupling agents such as l-(3-dimnethylaminopropyl-3-ethylcarbodiimide) hydrochloride and procedures well known to those skilled in the art. The Grignard reagent Mil [prepared from the corresponding bromide by treatment with magnesium] is reacted with MI (R = Bn, TMSE) to yield alcohol MOI. Alcohol MIH is eliminated via base treatment of its mesylate using conditions well known to those skilled in the art to yield olefin MTV Ozonolysis of MTV (workup with methvsulfide) yields aldehyde MV Alternatively, treatment with OsO₄ followed by H<10₆ converts MIV to MV

Conversion of key intermediate MV to the targeted patent compounds is accomplished in several ways depending on the identity of side chain function J Reaction of MV with Wittig reagents followed by hydrogenation yields products in which J is alkyl, aryl or arylalky 1 Selective reduction of aldehyde MV with a reducing agent such as lithium tπs[(3-ethyl-3-penryl)oxy]alumιnum hydπde (LTEPA) yields alcohol MVI The alcohol may be converted to phenyl ethers or a vaπety of heteroatom substituted deπvatives used to generate sidechain R¹⁶ via the Mitsunobu reaction The Mitsunobu reaction is well known to those skilled in the art, see Mitsunobu. Synthesis 1 (1981), and Hughes, Organic Reactions-, 42, 335 (1992) Alternatively alcohol MVI is converted to a leaving group such as tosylate MVII or bromide by conditions well known to those skilled in the art and then the leaving group is displaced by the appropπate nucleophile Several examples of this type of reaction can be found in Norman, et al , Med Chem 37, 2552 (1994) Direct acylation of the alcohol MVI yields compounds in which J = OAcyl and reaction of the alcohol with vanous 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 J, but in all cases well known to those skilled in the art Removal of the benzyl group, for example, may be accomplished by base hydrolysis or hydrogenolysis, whereas deprotection of the 2-(tnmethylsιlyl)ethyl ester is typically earned out by simple treatment with tetrabutylammonium fluoride

Suitable pharmaceutical K acceptable salts of the compounds of the present

include addition salts formed with organic or inorganic bases The salt forming ion deπved from such bases can be metal ions, e g , aluminum, alkali metal ions, such as sodium or potassium, alkaline earth metal ions such as calcium or magnesium, or an amine salt ion. of which a number are known for this purpose Examples include ammonium salts, arylalkvlamines such as dibenzylamine and \ \-dibenzylethyienediamine. lower alkylamines such as methylamine, .-butylamine, procaine. lower aikylpipeπdines such as Λ-ethylpipeπdine cycloalkvlamines such as cyclohexylamine or dιc\ciohexylarrune. 1 -adamantylamιne. benzathme. or salts deπved from amino acids like arginine. lvsine or the like The physiologically acceptable salts such as the sodium or potassium salts and the ammo acid salts can be used medicinally as descπbed below and are preferred These and oU er salts which are not necessaπly physiologically acceptable are useful in isolating or puπfying a product acceptable for the purposes descπbed below For example, the use of commercially available enantiomeπcally pure amines such as (-t-)-cιnchonme 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 mvention compound is usually substantially greater in physiological effect than its antipode. this active 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. hydrochioπc acid. etc.

The compounds of the present invention have been found to inhibit the matnx 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.

The method of treating matnx metal loprotease-mediated conditions may be practiced in mammals, including humans, which exhibit such conditions.

The inhibitors of the present invention are contemplated for use in veteπnary and human applications. For such purposes, they will be employed in pharmaceutical compositions containing active mgredιent(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 posomal 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 e g . compπsing poly(DL-lactιde-co-glycolιde) 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 Percuseal system available from Pharmacia Improv ed convenience and patient compliance may also be achieved by the use ot 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 also be achieved as needed using implantable pumps with 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 paniculate earners ( 200 um) such as those compnsing cellulose, polvacrvlate 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 attπbute of the compounds of the present invention in contrast to those of various peptidic compounds referenced m the background section of this application is the demonstrated oral activity of the present compounds Certain compounds have shown oral bioavailabihty in vanous 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 capsuies designed to release the drug into the colon where digestive protease activity is low Examples include the OROS-CT'Osmet™ and PULSINCAP™ 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 com unction with a wide range of available absorption enhancers.

Rectal delivery may be achieved by incorporation of the drug into suppositoπes.

The compounds of this invention can be manufactured into the above listed formulations by the addition of vanous therapeutically inert, inorganic or organic earners well known to those skilled in the art. Examples of these include, but are not limited to, lactose, com starch or derivatives thereof, talc, vegetable oils, waxes, fats, polyols such as polyethylene glycol. water, saccharose, alcohois. glyceπn and the like. Vanous 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 bioavailabihty 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, 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 of metalloproteases and testing for matnx metalloprotease activity Such activity testing can be both m 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 ammals) or are induced by admimstration of exogenous agents or by surgery which disrupts joint stability

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

EXAMPLES General Procedures:

All reactions were performed in flame-dπed 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 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 puπfication except that diethyl ether and tetrahydrofuran were usually distilled under argon from benzophenone ketyl. and methylene chloride was distilled under argon from calcium hydπde. Many of the specialty organic or organometallic starting mateπals and reagents were obtained from Aldπch, 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 sihca gel 60 A F-254 250 um plates. Visualization of spots was effected by one of the following techmques: (a) ultraviolet illumination, (b) exposure to iodine vapor, (c) 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-arusaidehyde in ethanol containing 0.5% concentrated suifuπc acid followed by heating.

Column chromatography was performed using 230-400 mesh EM Science* silica gel .Analytical h gh performance liquid chromatography (HPLC) was performed at 1 mL min ^' on a 4 6 \ 250 mm Microsorb* column monitored at 288 nm. and semi-preparative HPLC was performed at 24 mL mm^'1 on a 21 4 x 250 mm Microsorb* column monitored at 288 nm. Instrumentation:

Melting points (mp) were determined with a Thomas-Hoover melting point apparatus and are uncorrected. Proton (Η) nuclear magnetic resonance (NMR) 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 the compounds synthesized m the expeπments 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 jatos Concept 1 -H spectrometer by hquid-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

EXAMPLES 1 - 6 - Preparation of Compounds I- VI Step 1 A solution of sodium hydnde (4.35 g, 1 81 mmol) in freshly distilled THF (100 mL) was cooled to 0 °C and treated with commercially available dially malonate (35 0 g, 190 mmol ) over 40 min via a dropping funnel After stirnng at room temperature for 30 min. Λ^'-(2- bromoethyOphthahmide (43 9 g, 247 mmol) was added to the solution in one portion and the mixture was heated at reflux After 48 h the solution was cooled to 0°C, quenched with 2N HCI and concentrated to about 20% of its oπginal volume The concentrate was diluted with ethyl acetate (300 mL) and washed successively with saturated aqueous solutions of K CO-, and NaC l The organic layer was dned over MgSO₄, filtered and concentrated under reduced pressure Puπfication by flash column chromatography (gradient eiution with 5-25% ethyl acetate-hexanes) provided diallyi 2-phthalιmιdoethylmalonate (451.2 g. 64%) as a colorless oil 'H NMR (300 MHZ, CDC1 ,) δ 7 82 (m. 2H). 7 72 (m. 2H). 5 85 (m. 2H). 5 30 (m. 2H). 5 22 (m, 2H). 4 60 (m. 4H). 3 80 (t. J = 6 6 Hz. 2H). 346 (t. = 7 2 Hz. 1H). 2 30 (dd. J = 13 8. 6 9 Hz. 2H) The product of the above- descπbed reaction is illustrated below

Step 2 A one-necked, 50-mL, round-bottomed flask equipped with a rubber septum and an argon needle inlet was charged with 12 mL THF. tπmethylsilyl chloπde (0 83 ml. 0 710 g, 6.54 mmol). lithium hexamethyldisilazide (6.50 ml. 1.0 M in THF, 6.50 mmol), and cooled to -78 °C while a solution of 2-dodecanone (1.19 g. 6.46 mmol) in 8.0 ml THF was added dropwise over a period of 30 mm via cannula. The resulting mixture was stirred at -78 °C for 30 min. Λ- bromosuccinimide (1.27 g. 7.13 mmol) was added, and the reaction mixture was stirred at -78 °C for 30 min. diluted with 200 ml of pentane. and washed with three 50 mL portions of bπne. The organic phase was dried over Na_}SO₄ and concentrated to provide 2.5 g of a yellow solid Column chromatography on 100 g of silica gel (gradient elution with 3-5% ethyl acetate-hexanes) afforded 0.680 g (40%) of the bromomethyl ketone as a white solid. TLC (5% ethyl acetate-hexanes) R₇=0 4 The product of the above-descπbed reaction is illustrated below:

O

Step 3. A one-necked. 25 -mi. round-bottomed flask equipped with a rubber septum and an argon needle inlet was charged with 3 ml of THF and the product of step 1 (314 mg. 0.978 mmol). The resulting mixture was cooled to 0 °C and sodium t-butoxide (88.0 mg, 97% pure. 0.888 mmol) was added. .After 30 mm. a solution of the product of step 2 (250 mg, 0.950 mmol) in 3 ml of THF was added dropwise via synnge. The resulting mixture was allowed to warm to room temperature and stirred for 16 h. The reaction mixture was diluted with 100 ml of CH₂C1_: and washed with three 30 ml portions of brine. The organic layer was dned over MgSO₄ and concentrated. Column chromatography on 40 g silica gel (gradient elution with 10-30% ethyl acetate-hexanes) afforded 0.300 g (63%) of the desired product as a white solid. TLC (30% ethyl acetate-hexanes) Rf=0.5.

The product of the above-described reaction is illustrated below:

Step 4 Preparation of Example 1. A one-necked. 154-mL. round-bottomed flask equipped with a rubber septum and an argon needle inlet was charged with 2 mL of dioxane. the product of step 3 ( 300 mg. 0.556 mmol). pyrrolidine (0.12 ml, 0.102 g, 1 44 mmol). and tetrakιs(mphenylphospιne)palladιum ( 10.0 mg. 0.0086 mmol). The resulting mixture was exposed to a slight vacuum to degas the solution and argon was reintroduced. The reaction mixture was stirred at room temperature for 12 h. the dioxane and pyrrolidine were removed in vacuo. and the residue was redissolved in 2 ml dioxane. The resulting mixture was exposed to a slight vacuum to degas the solution and argon was reintroduced. The reaction mixture was heated at 1 15 ^CC for 4 h. 85 °C for 12 h. and concentrated. Column chromatography on 10 g of silica gel (30% ethyl acetate- hexanes with 0.5% acetic acid) afforded 0.137 g (59%) of Example 1 as a white solid (MP 89- 90°C). The product of d e above-descπbed reaction is illustrated below:

The above methods for the preparation of Example 1 were used to prepare the following examples (TABLE I) using the appropriate bromoketones in step 3. TABLE I

'Preparation of 1 -bromo-2-tetradecanone A one-necked. 100-mL. round-bottomed flask equipped with a rubber septum and an argon needle inlet was charged with 16 mL CC 1₄. 1.2-epoxιtetradecane (2 0 mL. 1 66 g, 7 82 mmol), polyvinyl pyndine (1 00 g), and bromine (0.20 ml, 0.62 g, 3 88 mmol) The resulting mixture was stirred at room temperature under irradiation of a desk lamp (soft white, 60 W) for 30 mm. The reacuon mixture was diluted with a 1 1 mixture of hexane:ethyl acetate (150 ml), washed with a 50 mL portion of saturated NaHCOv and washed with a 50 mL portion of bπne The organic layer was dned over MgSO„ and concentrated Column chromatography on 100 g of silica gel (gradient eluϋon with 5-10% ethyl acetate-hexanes) afforded 0 440 g (39%) of 1 -bromo-2- tetradecanone as a white solid TLC (5% ethyl acetate-hexanes) R, = 0.4

EXAMPLES 7 ■ 9 - Preparation of Compounds VTI-IX

Step 1. A solution of 4-(4-methoxyphenyl)-butyπc acid (3 04 g, 15 4 mmol) in CH,C1 , (45 mL) was treated with oxalyl chlonde (1 1 6 mL, 2.0 M soln. in dichloromethane) and DMF ( 1 drop) The solution was heated to reflux for 2 h. cooled to 0°C and treated with an excess of diazomethane (ether soln.). After stiirmg an additional 30 mm. excess 4 M HCI (soln. in 1.4-dιoxane) was added and the mixture was warmed to room temperature and stirred ovemite The solution was concentrated under reduced pressure, diluted with ethyl acetate and successively washed with water, satd. aq NaHCO₃ and satd. aq. NaCl The organic phase was dned over MgSO filtered and concentrated. Purification by MPLC (5-25% EtOAc-hexanes) provided the target compound (2 45 g. 70%) as a colorless oil. TLC R/ 0 45 (silica, 15% ethyl actate-hexane). The resulting compound is illustrated below:

Step 2. A solunon of sodium hydnde (6 35 g. 264 mmol) in THF (500 mL) was treated with diethyl malonate (47.35 mL, 312 mmol) After stirnng for 2 h. (2-bromoethyl)benzene (32.8 mL, 240 mmol) was carefully added to the reaction mixture. Following the addition, the solution was heated to a gentle reflux for 16 h. cooled to 0°C and quenched with 2 N HCI . The resulting solution was concentrated under reduced pressure, diluted widi EtOAc and washed with satd. aq. NaCl . The organic layer was dried over N!.:SO₄ and concentrated. Vacuum distillation ( 1 5 mm Hg) provided the substituted malonate (444 g. 71%) as a colorless oil TLC. R/ 0 52 (silica, 20% ethyl actate- hexane). The resulting compound is illustrated below

Step 3. A solution of malonate from step 2 (3.50 g, 13.2 mmol) in DME (5 mL) was treated with NaOEt (0.67 g, 9.9 mmol) and stirred for 30 mm. While the solution was stimng, a separate flask containing a solution of the α-chloro ketone from step 1 (0.95 g, 4.2 mmol) in DME (5 mL) was treated witii Lil (0.62 g, 4.6 mmol), stirred for 15 min. and cannulated into the first solution. After stimng ovemite, the reaction mixture was diluted with EtOAc and washed with water and satd aq NaCl The orgamc layer was dned over MgSO₄, filtered and concentrated Puπfication by MPLC (5-20% EtOAc- hexanes) provided the desired malonate (0 41 g. 21 %) as a colorless oil TLC R/ 0 30 (silica, 20% ethyl actate-hexane) The resulting compound is illustrated below

Step 4 - Preparation of Example 7. A solution of the diester from step 3 (0 19 g, 0 42 mmol) in ethanol (3 mL) was treated with 2 N NaOH (0.5 mL) and stirred at room temperature After stimng for 16 h. the soln was concentrated under reduced pressure, diluted with ethyl acetate, and washed with aq K._:CO The aqueous layer was acidified to pH 1 with 2 N HCI . and extracted with ethyl acetate The orgamc layer was dned over MgSO₄. filtered and concentrated The resulting diacid was dissolved in 1 ,4-dιoxane (3 mL) and heated to 65 °C After stimng for 24 h, the soln was concentrated and puπfied by flash column chromatography (2-4% MeOH-CH₂Cl ₂) to give the targeted compound (72 1 mg. 49%) MP 70-71 °C The resulting compound (Example 7) is illustrated below

The above methods for the preparation of Example VU were used to prepare the following examples (Table U) TABLE π

EXAMPLE 10 Biological Assays of Invention Compounds P218 Quenched Fluorescence Assav for MMP Inhibition:

The P218 quenched fluorescence assay ( icrofluorometπc Profiling Assay) is a modification of that oπgmally descπbed by Kmght, et al . FEBS Lett. 296. 263, 1992 for a related substance and a vaπety of matnx metalloproteinases (MMPs) in cuvettes The assay was run with each invenuon 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.

P218 Fluorogenic Substrate:

P218 is a synthetic substrate containing a 4-acetyl-7-methoxycoumaπn ( MCA) group in the N-terminal position and a 3-[2, 4-dιnιtrophenyl]-L-2,3-dιamιnopropionyl (DPA) group internally. This is a modification of a peptide reported by Knight ( 1992) that was used as a substrate for matnx 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 fluorometer with excitation at 328 nm and emission at 393 nm. P218 is currently being produced BACHEM exclusively for Bayer. P218 has the structure: H-MCA-Pro-Lys-Pro-Leu- _/α-Zeu-DPA-Ala-Arg-NH2 (MW 1332.2)

Recombinant Human CHO Stromelvsin (MMP-3)

Recombinant Human CHO Pro-MMP-3: Human CHO pro-stromelysιn-257 (pro-MMP-3) was expressed and purified as descπbed by Housley, et al., J. Biol. Chem. 268. 4481 (1993)

Activation of Pro-MMP-3 : Pro-MMP-3 at 1.72 μM (100 μg/mL) in 5 mM Tris at pH 7.5. 5 M CaCl_:. 25 mM NaCl. and 0 005% Brij-35 MMP-3) activation buffer) was activated by incubation with TPCK (N-tosyl-(L)-phenylalanine chloromethyl ketone) trypsin (1 : 100 w/w to pro- MMP-3) at 25 °C for 30 min. The reaction was stopped by addition of soybean trypsin inhibitor (SBTI; 5 : 1 w/w to trypsin concentration). This activation 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 the method of Fridman. et ai.. 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,, 150 mM NaCl, and

0.005%o Brij-35 (MMP-2 activation buffer). /7-Ammophenylmercuric acetate (APMA) was prepared in 10 mM (3.5 mg/mL) in 0.05 NaOH. The APMA solution was added at 1/20 die reaction volume for a final AMPA concentration of 0.5 mM, and die enzyme was incubated at 37 °C for 30 min. 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_:O washing). The enzyme was concentrated on Centricon concentrators (concentrators were also pre-treated with 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 oπginal volume) with MMP-2 activation buffer. Preparation of Human Recombinant Pro-Gelatinase B (MMP-9):

Recombinant Human Pro-MMP-9: Human pro-gelatinase B (pro-MMP-9) derived from U937 cDNA as described by Wilhelm, et al. J. Biol. Chem. 264, 17213 (1989) was expressed as the full-length form using a baculovirus protein expression system. The pro-enzyme was purified using methods previously described by Hibbs. et al. J. Biol. Chem. 260, 2493 (1984).

Activation of Pro- MMP -9: Pro-MMP-2 20 μg/mL in 50 mM Tris at pH 7.4, lOmM CaCl₂. 150 mM NaCl, and 0.005% Brij-35 ( MP-9 activation buffer) was activated by incubation with 0.5 mM

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 1 1 potential inhibitors automatically from a 2.5 mM stock in 100% DMSO; (2) distribute substrate followed by inhibitor into a 96 well Cytofluor plate; and (3) add a single enzyme to the plate with mixing to start the reaction. Subsequent plates for each additional enzyme are prepared automatically by beginning the program at the substrate addition point, remixing the diluted inhibitors and beginning the reaction by addition of enzyme. In this way, all MMP assays were done using die same inhibitor dilutions. Milhpore Cytofluor II Following incubation, the plate was read on a Cytofluor II fluorometnc plate reader wi excitation at 340 nM and emission at 395 nM with the ga set at 80 Buffers:

Microfluoromemc Reaction Buffer (MRB): Dilution of test compounds, enzymes, and P218 substrate for the microfluorometπc assay were made in microfluorometπc reaction buffer consisting of 50 mM 2-(N-morpholιno)ethanesulfonιc acid (MES) at pH 6.5 widi 10 mM CaCl₂, 150 mM NaCl. 0.005% Brij-35 and 1 % DMSO Methods:

MMP Microfluoromemc Profiling Assay The assay is done with a final substrate concentration of 6 uM P218 and approximately 5 to 8 nM MMP with vanable drug concentrations.

The Hamilton is programmed to senally dilute up to 1 1 compounds from a 2.5 mM stock (100% DMSO) to l Ox the final compounds concentrations in the assay Initially, the instrument delivers various amounts of microfluoromentnc reaction buffer (MRB) to a 96 tube rack of 1 ml Marsh dilution tubes. The instrument then picks up 20 μl of inhibitor (2.5 mM) from the sample rack and mixes it with a buffer in row A of the Marsh rack, resulting m a 50 μM drug concentration. The inhibitors are then serially diluted to 10. 5, 1 , 2. .05 and 01 μM. Position 1 on die sample rack contains only DMSO for the "enzyme-only" wells in the assay, which results in no inhibitor in column 1, rows A through H. The instrument then distπbutes 107 μl of P218 substrate (8.2 μM in MRB) to a single 96 well cytofluor microtiter plate. The instrument re-mixes and loads 14.5 μl of diluted compound from rows A to G in the Marsh rack to corresponding rows in die 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 well, excluding Row H, d e "background" row. (The enzyme reservoir is pretreated with 1% BSA in 50 mM Tris, pH 7.5 containmg 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. Add onal enzymes are tested in the same manner by beginning the Hamilton program with the distnbution of P218 substrate to the microtiter plate, followed by re-mixing and distπbution of the drug from the same Marsh rack to the microtiter plate. The second (or third, etc.) MMP to be tested is then distributed from a reagent rack to the microtiter plate with mixing, pπor to coveπng and incubation This is repeated for all additional MMP's to be tested.

IC50 Determination in Microfluoromemc 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 compaπng the amount of hydrolysis (fluorescence units generated over 25 minutes of hydrolysis) of wells containing compound widi the "enzyme only" wells in column 1 Following subtraction of the background the percent inhibition was calculated as:

((Control values - Treated values)/Control values) x 100 Percent inhibitions were determined for inhibitor concentrations of 5, 1, 0 5. 0.1. 0.02,0 .005 and, 0.001 μM of drug. Linear regression analysis of percnet inhibition versus log inhibitor concentration was used to obtain IC₅₀ values. Profiling Assay Data for Invention Compounds.

All IC₅₀ values are expressed as nM. When "I = x %" is shown, x represents the % inhibition at 5 μM.

Table III

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 spiπt of the invention being indicated by the following claims

Claims

We claim:

1 A matrix metalloproteinase-inhibiting compound having die generalized formula.

wherem y is 0, 2, or, 3, r is 0-6. Z is (CH₂)₇ or (CH₂)_e-C₆H₄-(CH₂)_f, wherem e is 0-1 and f is 1 -6; R¹⁵

wherein n is 0-4, R" is C₂H₅, allyl. benzyl, and R¹⁶ is

wherem t is 0-1, x is 0-4, and R⁴ is one of the following: halide. alkyl of 1-6 carbons, OR, NR₂, NO, (R = H or alkyl of 1-6 carbons).

2. A method of inhibiting matnx metalloprotease activity compπsing providing an effective matnx metalloprotease-inhibiting amount of a compound according to ciaim 1.

3 A matnx metalloprotease inhibiting composiuon compπsing a compound according to claim 1 and a pharmaceutically acceptable carrier.

4. A method of treating a mammal comprising administenng to the mammal a matnx metalloprotease inhibiting amount of a compound according to claim 1 sufficient to:

(a) alleviate the effects of osteoarthritis, rheumatoid arthritis, septic arthritis, periodontal disease, comeal ulceration, proteinuria, aneurysmal aortic disease, dystrophobic epidermolysis, bullosa. conditions leading to inflammatory responses, osteopenias mediated by MMP activity, tempero mandibular joint disease, demyelating diseases of the nervous system;

(b) retard tumor metastasis or degenerative cartilage loss following traumatic joint injury;

(c) reduce coronary thrombosis from athrosclerotic plaque rupture; or (d) effect birth control.

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

6. The method of claim 4 wherein the effect is alleviation of osteoarthritis.

7. The method of claim 4 wherein the effect is retardation of tumor metastasis.