CN1163466C - Substituted oxobutanoic acid derivatives as matrix metalloproteinase inhibitors - Google Patents

Abstract

The present invention provides pharmaceutical compositions and methods for treating certain diseases associated with matrix metalloproteases, comprising administering an amount of a compound or composition of the present invention effective to inhibit at least one matrix metalloprotease, to achieve the desired effect. The compound of the present invention is any one of the general formula : wherein y is 0, 2 or 3, r is 0-6, and Z is (CH)₂)₇Or (CH)₂)_e-C₆H₄-(CH₂)_fWherein e is 0-1, f is 0-5, and R¹⁵is-H, -Cl, -OMe or (a), (b), wherein n is 0-4, R¹⁷Is C₂H₅Allyl, benzyl, and R¹⁶Is (c) wherein t is 0-2, x is 0-4, and R⁴Is one of the following: halide, C1-C6 alkyl, OR, NR₂、NO₂(R ═ H or alkyl of 1 to 6 carbons).

Description

Substituted oxobutyric acid derivatives as matrix metalloproteinase inhibitors

field

The present invention relates to enzyme inhibitors, and more particularly to novel oxobutyric acid compounds or derivatives thereof useful as inhibitors of matrix metalloproteinases.

background

Matrix metalloproteinases (also known as matrix metalloendoproteases or MMPs) are a class of zinc endoproteases that include, but are not limited to, interstitial collagenase (also known as MMP-1), stromelysin (also known as mucin protease, transin, or MMP-3), gelatinase A (also known as 72 kDa-gelatinase or MMP-2) and gelatinase B (also known as 95 kDa-gelatinase or MMP-9). These MMPs are secreted by a variety of cells including fibroblasts and chondrocytes, together with natural protease inhibitors known as TIMPs (tissue inhibitors of metalloproteinases).

All of these MMPs can damage various connective tissue components of articular cartilage or basement membrane. Each MMP is secreted as an inactive zymogen that must be cleaved in a subsequent step before it can exhibit its proteolytic activity. Some of these MMPs, such as MMP-3, have been implicated as in vivo activators of other MMPs, such as MMP-1 and MMP-9, in addition to their matrix-disruptive effects (Ito, A.; Nagase, H. Biochemistry and Biophysics Libraries (Arch. Biochem. Biophys.) 267 , 211-6 (1988); Ogata, Y.; Enghild, JJ; Nagase, H., J. Biochem., 267 , 3581-4 (1992)). Thus, a range of proteolytic activities can be triggered by excess MMP-3. This means that a particular MMP-3 inhibitor will 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 thus inactivate endogenous inhibitors of other proteases such as elastase (Winyard, P.G.; Zhang, Z.; Chidwick, K.; Blake, D.R.; Carrell, R.W.; Murphy, G. FEBS Lett. 279, 91-4 (1991)). Thus, MMP-3 inhibitors can affect the activity of other destructive proteases by altering their endogenous inhibitor levels.

Many diseases are thought to be mediated by excess or inappropriate matrix-destructive metalloprotease activity, or by an imbalance in the ratio of MMPs to TIMPs. These diseases include: a) osteoarthritis (Woessner, J.F., Jr.; Selzer, M.G., J. Biochem. 259, 3633-8 (1984) and Phadke, K.J. Rheumatol. 10, 852-60 (1983)), b ) rheumatoid arthritis (Mullins, D.E.; Rohrlich, S.T.Biochim.biophys.Acta695, 117-214 (1983), Woolley, D.E.; Crossley, M.J.; Evanson, M.J.ArthritisRheum.20, 1231-9 (1977), and Gravallese, E.M.; Darling, J.M.; Ladd, A.L.; Katz, J.N.; Glimcher, L.H. Arthritis Rheum. 34, 1076-84 (1991), c) septic arthritis (Williams, R.J., III; Smith, R.L.; Schurman, D.J.Arthritis Rheum.33, 533-41(1990)), d) tumor metastasis (Reich, R.; Thompson, E.W.; Iwamoto, Y; Martin, G.R.; Deason, J.R; Fuller, G.C.; Miskin, R. Cancer Res .48, 3307-12 (1988) and Matrisian, L.M.; et al Proc. (PeridontalRes.) 22, 81-8 (1987)), f) corneal ulcer (Burns, F.R. et al., Ophthalmology Research (Invest. Ophthalmol. Vis. Sci.) 30, 1569-75 (1989)), g) protein Urine (Baricos, W.H. et al., Biochem. J. 254, 609-12 (1988)), h) Coronary thrombosis due to rupture of atherosclerotic plaques (Davies, M.J. et al., Proceedings of the National Academy of Sciences USA (Proc. Natl. Acad. Sci. U.S.A.) 88, 8154-8 (1991)), i) Aneurysmal aortic disease (Vine, N. et al., Clin. Sci. 81, 233-9 (1991)), j ) birth control (Woessner, J.F., Jr. et al., Steroids 54, 491-9 (1989)), k) dystrophic epidermolysis bullosa (Kronberger, A. et al., J. Invest. Dermatol. 79, 208-11 (1982)), and 1) degenerative cartilage loss due to traumatic joint injury, conditions causing inflammatory reactions, osteopenia mediated by MMP activity, temporomandibular joint diseases, demyelinating diseases of the nervous system, etc. (Chantry, A. et al., J. Neurochem. 50, 688-94 (1988)).

The need for new treatments is especially important in the case of joint diseases. The major disabling effect of osteoarthritis (OA), rheumatoid arthritis (AR) and septic arthritis is the progressive loss of articular cartilage and adjacent normal joint function. There are no medications on the market that can prevent or slow this loss of cartilage, although nonsteroidal anti-inflammatory drugs (NSAIDs) can control pain and swelling. The end result of these diseases is a complete loss of joint function, which can only be treated by joint replacement surgery. MMP inhibitors are expected to halt or reverse the process of cartilage loss avoiding or delaying surgical intervention.

Proteases are key elements at several stages in the process of metastatic cancer. In this approach, proteolytic degradation of structural proteins in the basement membrane causes the spread of the tumor at the first site, exits from and returns from the site, and develops at a distant second site. Furthermore, tumor-induced angiogenesis is required for tumor growth and relies on proteolytic tissue modification. Infection experiments with various types of proteases have shown that matrix metalloproteinases, in particular, gelatinases A and B (MMP-2 and MMP-9, respectively) play important roles in these processes. For an overview of these fields see Mullins, D.E et al, Biochim. Biophys. Acta 695, 177-214 (1983), Ray. J. M. et al, Eur. Respir. J. 7, 2062-72 (1994) and Birkedal-Hansen, H et al , Crit. Rev. Oral. Biol. Med. 4, 197-250 (1993).

Moreover, it can be shown that inhibition of degradation of the extracellular matrix by the natural matrix metalloproteinase inhibitor TIMP-2 (a protein) prevents cancer growth (De Clerck, Y.A et al., Cancer Research, 52, 701-8 (1992)), Also, TIMP-2 inhibits carcinoma-induced angiogenesis in experimental systems (Moses, M.A et al., Science, 248, 1408-10 (1990)). For a review see DeClerck, Y. et al., Ann. N. Y. Acad. Sci. 732-222-32 (1994). The synthetic matrix metalloproteinase inhibitor batimastat has also been shown to inhibit human colon tumor growth when administered intraperitoneally and spread in nude mice in an orthotopic model (Wang, X et al., Cancer Research, 54, 4726-8 (1994)) and prolong the survival of rats bearing human ovarian carcinoma exografts (Davies, B et al., Cancer Research, 53, 2087-91 (1993)). The use of this and related compounds has been described in Brown et al. WO-A-9321942A2 (931111).

There are several patents and patent applications disclosing metalloproteases useful for arresting metastatic cancer, promoting tumor regression, inhibiting cancer cell proliferation, delaying or preventing cartilage loss associated with osteoarthritis, or for the treatment of other diseases noted above Inhibitors (eg WO-A-9519965, WO-A-9519956, WO-A-9519957, WO-A-9519961, WO-A-9321942, WO-A-9421625, USP-4599361; USP-5190937; EP- 0574758A1, published Dec. 22, 1993; EP-026436A1, published Aug. 3, 1988; and EP-0520573A1, published Dec. 30, 1992). The preferred compounds of these patents have a peptide backbone with a zinc ligand (hydroxamic acid, thiol, carboxylic acid or phosphinic acid) at one end and many side chains, both found in natural amino acids and with newer of functional groups. Such small peptides are often poorly absorbed and exhibit low oral bioavailability. They also undergo rapid proteolytic metabolism and have a short half-life. As an example, batimastat, the compound described in Brown et al. WO-A-9321942A2, can only be administered intraperitoneally.

Others have disclosed a series of biphenyl-containing carboxylic acids, examples of which are shown below, which inhibit neural endopeptidase (NEP24.11), a membrane-bound zinc metalloprotease (Stanton, et al., Bioor .Med.Chem.Lett.4, 539,1994; Lombaert, et al., Bioor.Med.Chem.Lett.4, 2715, 1994; Lombaert, et al., Bioor.Med.Chem.Lett.5, 145 , 1995; Lombaert, et al., Bioor. Med. Chem. Lett. 5, 151, 1995).

N-carboxyalkyl derivatives containing biphenylethylglycine, exemplified by the compounds shown below, have been reported to be stromelysin-1 (MMP-3), 72 kDa gelatinase (MMP-2) and collagenase (Durette, et al., WO-9529689).

Potent MMP inhibitors that have improved bioavailability and biological stability relative to prior art peptide-based compounds and that can be optimally used against specific target MMPs would be needed. Such compounds are the subject of the present application.

The development of effective MMP inhibitors will provide therapeutic potential for diseases caused by the presence of MMP activity, or mediated by excess MMP activity, including osteoarthritis, rheumatoid arthritis, septic arthritis, tumor metastasis, periodontal disease, corneal ulcer , The treatment of proteinuria. Several MMP inhibitors have been described in the literature, including thiols (beszant, et al., J. Med. Chem. 36, 4030, 1993), hydroxamic acids (Wahl, et al., Bioor. Med. Chem. .Lett.5,349,1995; Conway, et al., J.Exp.Med.182,449,1995; Porter, et al., Bioor.Med.Chem.Lett.4,2741,1994; Tomczuk, et al ., Bioor.Med.Chem.Lett.5,343,1995; Castelhano, et al., Bioor.Med.Chem.Lett.5,1415,1995), containing phosphoric acid (Bird, et al., J.Med.Chem .37,158,1994; Morphy, et al., Bioor.Med.Chem.Lett.4,2747,1994; Kortylewicz, et al., J.Med.Chem.33,263,1990) and carboxylic acid (Chapman , et al., J.Med.Chem.36,4293; Brown, et al., J.Med.Chem.37,674,1994; Morphy, et al., Bioor.Med.Chem.Lett.4,2747,1994 ; Stack, et al., Arch.Biochem.Biophys.287,240,1991; Ye, et al., J.Med.Chem.37,206,1994; Grobelny, et al., Biochemistry 24,6145,1985; Mookhtiar, et al., Biochemistry 27, 4299, 1988). However, these inhibitors generally contain a peptide backbone, exhibit low oral bioactivity due to their low absorption, and exhibit a short half-life due to rapid proteolysis. Therefore, improved MMP inhibitors are needed.

Invention Summary

The present invention provides compounds having matrix metalloproteinase inhibitory activity. These compounds are useful for inhibiting matrix metalloproteinases and, therefore, against diseases caused by MMPs. Accordingly, the present invention also provides pharmaceutical compositions and methods of treating these conditions.

The compound relates to a method of treating a mammal, comprising: administering to the mammal a matrix metalloproteinase inhibitory amount of a compound of the invention sufficient to:

(a) Relief of osteoarthritis, rheumatoid arthritis, septic arthritis, periodontal disease, corneal ulcer, proteinuria, aortic disease of aneurysm, dystrophic epidermolysis blister, conditions leading to inflammatory response, by Osteopenia mediated by MMP activity, temporomandibular joint disease, or demyelinating disease of the nervous system;

(b) Block of tumor metastasis or degenerative cartilage loss caused by traumatic joint injury;

(c) reducing coronary thrombosis caused by atherosclerotic plaque rupture;

or (d) achieve birth control.

The compounds of the invention are also useful as scientific tools for studying the function and mechanism of matrix metalloproteinases in in vitro and in vivo systems. Due to their MMP-inhibitory activity, the compounds of the present invention are useful for modulating MMP action, thereby enabling researchers to observe the effect of reduced MMP activity in experimental biological systems under study.

The present invention relates to compounds having matrix metalloproteinase inhibitory activity and the following general formula:

A-D-E-G (L)

A stands for alkyl; allyl; benzyloxy; or 3-propynylalkyl and the structure:

where Z=(CH ₂ ) _e -C ₆ H ₄ -(CH ₂ ) _f or (CH ₂ ) _g , e=0-8, f=0-5, g=0-14, and r is 0-6 ,

R ¹⁵ can be -H, -Cl, -OMe or

Wherein: n=0-4, R ¹⁷ is -C ₂ H ₅ , allyl, or benzyl.

In the general formula (L), D represents:

In the general formula (L), E represents a chain of n' carbon atoms with m substituents R ⁶ , wherein R ⁶ is an independent substituent, or constitutes a helical or non-helical ring. Rings can be formed in two ways: a) two ^R6 groups are combined and together with the chain atoms to which the two ^R6 groups are attached, and any intermediate chain atoms form a 3-7 membered ring, or b) one ^R6 group is bonded to It rests on the chain, and together with the chain atoms to which the ^R group is attached, and any intermediate chain atoms form a 3-7 membered ring. The number n' of carbon atoms in the chain is 2 or 3, and the number m of R ^substituents is an integer of 1-3. The number of carbon atoms in all R ^groups is at least 2.

Each ^R6 is alkyl, alkenyl, alkynyl, aryl, heteroaryl, non-aromatic ring, and combinations thereof optionally substituted with one or more heteroatoms as described in more detail below.

In the general formula (L), G represents -CO ₃ H, -PO ₃ H ₂ , -M,

或

or

Wherein, M represents -CO ₃ H, -CON(R ¹¹ ) ₂ , or -CO ₂ R ¹² , wherein R ¹¹ is H or an alkyl group with 1-4 carbon atoms. R ¹² represents an alkyl group with 1-4 carbons, and R ¹³ represents any side chain of 19 kinds of acyclic natural amino acids.

Certain embodiments include compounds having matrix metalloprotease activity and having the formula:

Wherein, Z=(CH ₂ ) _e -C ₆ H ₄ -(CH ₂ ) _f or (CH ₂ ) _g , e=0-8, f=0-5, g=0-14, and y=0, 2 or 3.

R ¹⁵ can be H, Cl, MeO or

Wherein, n is 0-4, R ¹⁷ is C ₂ H ₅ , allyl, or benzyl, and R 16 is one of the following:

Wherein t is 0-2, X is 0-4, and R ⁴ is: halide, 1-6 carbon alkyl, OR, NR ² , NO ₂ (R=H or 1-6 carbon alkyl).

The foregoing merely outlines certain aspects of the invention without limiting the invention in any sense. All patents and other publications cited in this specification are hereby incorporated by reference in their entirety.

Description of preferred options

More specifically, the compounds of the present invention are substances having matrix metalloproteinase inhibitory activity and having the following formula:

A-D-E-G (L)

A represents an alkyl group of 9-14 carbon atoms; allyloxy-; benzyloxy-; The alkyl group and its structure:

where Z=(CH ₂ ) _e -C ₆ H ₄ -(CH ₂ ) _f or (CH ₂ ) _g , e=0-8, f=0-5, g=0-14, and r is 0-6 .

R ¹⁵ can be -H, -Cl, -OMe or

Wherein: n=0-4, R ¹⁷ is -C ₂ H ₅ , allyl, or benzyl.

In the general formula (L), D represents:

Throughout this application, in the chemical structures shown, an open bond refers to the point at which the structure bonds to another group. like:

where ^R50 is

Refers to the structure

In the general formula (L), E represents a chain of n carbon atoms with m substituents R ⁶ called R ⁶ groups or R ⁶ units). R ⁶ is an independent substituent, or constitutes a helical or non-helical ring. Rings can be formed in two ways: a) two ^R6 groups are combined and together with the chain atoms to which the two ^R6 groups are attached, and any intermediate chain atoms form a 3-7 membered ring, or b) one ^R6 group is bonded to It rests on the chain, and together with the chain atoms to which the ^R group is attached, and any intermediate chain atoms form a 3-7 membered ring. The number n of carbon atoms in the chain is 2 or 3, and the number m of R ^substituents is an integer of 1-3. The number of carbon atoms in all R ^groups is at least 2.

Each R ⁶ is independently selected from the substituents listed in the following 1)-14):

1) 1-10 carbon alkyl group;

2) 6-10 carbon aryl;

3) Heteroaryl containing 4-9 carbons and at least one N, O or S heteroatom;

4) Aralkyl, wherein the aryl part contains 6-10 carbons and the alkyl part contains 1-8 carbons;

5) Heteroaryl-alkyl, wherein the heteroaryl group contains 4-9 carbons and at least one N, O, or S heteroatom, and the alkyl portion contains 1-8 carbons;

6) 2-10 carbon alkenyl;

7) Aryl-alkenyl, wherein the aryl part contains 6-10 carbons and the alkenyl part contains 2-5 carbons;

8) Heteroaryl-alkenyl, wherein the heteroaryl part contains 4-9 carbons and at least one N, O, or S heteroatom, and the alkene part contains 2-5 carbons;

9) 2-10 carbon alkynyl;

10) Aryl-alkynyl, wherein the aryl part contains 6-10 carbons and the alkynyl part contains 2-5 carbons;

11) Heteroaryl-alkynyl, wherein the heteroaryl part contains 4-9 carbons and at least one N, O, or S heteroatom, and the alkynyl part contains 2-5 carbons;

(1)-(CH ₂ ) _t R ⁷ , wherein t is an integer of 0 or 1-5, and R ⁷ is selected from

and corresponding heteroaryl moieties, wherein the aryl moiety of aryl-containing ^R7 contains 4-9 carbons and at least one N, O, or S heteroatom. In this ^R7 group, Y represents O or S, and μ = 0, 1 or 2, provided that when ^R7 is

或

or

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

R ¹ represents H or an alkyl group of 1-3 carbons, R ² represents H; an alkyl group of 1-6 carbons; an aryl group of 6-10 carbons; containing 4-9 carbons and at least one N, O or Heteroaryl with S heteroatoms; aralkyl in which the aryl portion contains 6 to 10 carbons and the alkyl portion contains 1 to 4 carbons; or in which the heteroaryl portion contains 4 to 9 carbons and at least one Heteroaryl-alkyl with N, O, or S heteroatoms and the alkyl moiety contains 1 to 4 carbons.

R ³ represents an alkyl group of 1-4 carbons; an aryl group of 6-10 carbons; a heteroaryl group containing 4-9 carbons and at least one N, O or S heteroatom; wherein the aryl part contains 6- Aralkyl of 10 carbons with 1-4 carbons in the alkyl portion; or 4-9 carbons in the heteroaryl portion and at least one N, O or S heteroatom with 1-4 carbons in the alkyl portion carbon heteroaryl-alkyl.

1.13)-(CH ₂ ) _v P′R ⁸ , wherein v is an integer of 1-4, P′ represents -S-, -S(O)-, -SO ₂ -, or -O-, and R ⁸ is selected Alkyl of 1-12 carbons; Aryl of 6-10 carbons; Heteroaryl of 4-9 carbons and at least one N, O or S heteroatom; wherein the aryl moiety contains 6-12 Aralkyl containing 1-4 carbons in the alkyl portion; heteroaryl in which the aryl portion contains 6-12 carbons and at least one N, O or S heteroatom and the alkyl portion contains 1-4 carbons -C(O)R ⁹ , wherein R ⁹ represents an alkyl group of 2-6 carbons, an aryl group of 6-10 carbons, containing 4-9 carbons and at least one N, O or S heteroatom and aralkyl groups in which the aryl portion contains 6 to 10 carbons or 4 to 9 carbons and at least one N, O, or S heteroatom and the alkyl portion contains 1 to 4 carbons base, with the proviso that when R ⁸ is -C(O)R ⁹ , Z is -S- or -O-; when Z is -O-, R ⁸ is also -(C _q H _2q O) _r R ⁵ , and When the A unit is phenyl, the B unit is phenylene, m is 1, n is 2, and v is 0, then x is 1 or 2.

14) -(CH ₂ ) _w SiR ¹⁰ 3, wherein W is an integer of 1-3, and R ¹⁰ represents an alkyl group of 1-2 carbons.

In addition, the aryl or heteroaryl portion of any R ⁶ group may optionally bear up to two substituents selected from: -(CH ₂ ) _y' C(R ¹¹ )(R ¹² )OH, -( CH ₂ ) _y' R ¹¹ , -(CH ₂ ) _y' SR ¹¹ , -(CH ₂ ) _y' S(O)R ¹¹ , -(CH ₂ ) _y' S(O) ₂ R, -(CH ₂ ) _y′ SO ₂ N(R″) ₂ , -(CH ₂ ) _y′ N(R ¹¹ ) ₂ , -(CH ₂ ) _y′ N(R ¹¹ )COR, -OC(R ¹¹ ) ₂ O-, Two of the oxygen atoms are linked to the aromatic ring, -(CH ₂ ) _y′ COR ¹¹ , -(CH ₂ ) _y′ CON(R ¹¹ ) ₂ , -(CH ₂ ) _y′ CO ₂ R ¹¹ , -( CH ₂ ) _y′ OCOR ¹¹ , -halogen, -CHO, -CF ₃ , -NO ₂ , -CN and -R ¹² , wherein y' is 0-4; R ¹¹ represents H or an alkyl group with 1-4 carbons ; and R ¹² represents an alkyl group of 1-4 carbons.

In the general formula (L), G represents -CO ₂ H ₁ (O ₃ H ₂ ), -M,

或

or

Where M represents -CO ₂ H, -CON(R ¹¹ ) ₂ , or -CO ₂ R ¹² , R ¹² represents an alkyl group with 1-4 carbons, and R ¹³ represents any side chain of 19 kinds of acyclic natural amino acids .

Pharmaceutically acceptable salts of these compounds are also within the scope of the general formula (L) of the present invention.

It should be understood that the term "alkyl" as used herein refers to straight chain, branched chain, cyclic, and polycyclic species. The term "haloalkyl" refers to partially or perhalogenated alkyl groups such as -(CH ₂ ) ₂ Cl, -CF ₃ and C ₆ H ₁₃ .

In one embodiment, the invention relates to compounds of general formula (L), wherein in the E unit n is 2 and m is 1 . Thus, these compounds have two carbon atoms between the D and G units and carry a substituent on the two-carbon chain.

In another of its embodiments, the invention relates to compounds of general formula (L), wherein the number of substituents m on the E unit is 2 or 3; and when m is 2, the ^two R are independently substituted group, or together form a spiral ring, or one ^R6 group is an independent substituent and the other forms a spiral ring; and when m is 3, two ^R6 groups are independent substituents and one ^R6 group constitutes A ring, or two R ⁶ groups form a ring and one R ⁶ group is an independent substituent, or three R ⁶ groups are independent substituents. Thus, this substructure includes compounds in which the E unit is di- or trisubstituted, and in the case of disubstituted any ring formed by one or two ^R groups is a helical ring, and in the case of trisubstituted In the case of , the R ⁶ group can form a helical or non-helical ring.

In another of its embodiments, the present invention relates to compounds of general formula (L), wherein the number of substituents m on the E unit is 1 or 2; and when m is 1, the ^R group constitutes a non-helical ring; and When m is 2, two R ⁶ together form a non-helical ring or one R ⁶ is an independent substituent and the other forms a non-helical ring. Thus, this substructure includes compounds in which the E unit bears one or two ^R6 substituents and at least one substituent is included in a non-helical ring.

More specifically, representative compounds of general formula (L), wherein one or more ^R substituents are involved in the formation of a non-helical ring, have an E unit of the following structural formula.

where 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; i is 0-4; j is 0-3; k is 0-2; the total number of ^R6 is 0, 1, or 2; U represents O, S or ^NR1 ; and z is 1 or 2; any R is independently selected from: an ^alkyl group of 1-9 carbons; an aralkyl group wherein the alkyl portion contains 1-7 carbons and the aryl portion contains 6-10 carbons; Alkenyl groups of 2 to 9 carbons; aryl-substituted alkenyl groups in which the alkenyl moiety contains 2 to 4 carbons and the aryl moiety contains 6 to 10 carbons; alkynyl groups of 2 to 9 carbons; Wherein the alkynyl group contains 2-4 carbons and the aryl part contains 6-10 carbon aryl substituted alkynyl groups; 6-10 carbon aryl groups; -COR ² ; -CO ₂ R ³ ; -CON( R ⁶ ) ₂ ; -(CH ₂ ) _t R ⁷ , wherein t is an integer of 0 or 1-4; and -(CH ₂ ) _v Z′R ₈ , wherein v is an integer of 0 or 1-3, and Z' represents -S- or -O-. R ¹ , R ² , R ³ , R ⁶ , R ⁷ and R ⁸ are as defined above.

Preferred compounds of general formula (L), wherein one or more ^R substituents are involved in the formation of non-helical rings, have E units of the following structure:

and

wherein a, b, c, d, (c+d), e, g, i, k, the total number of R ⁶ , U, and R ¹⁴ are as defined above. Other compounds of general formula (L) have ^R units of the formula:

or

where n" is 0-1.

The most preferred compounds of general formula (L) include compounds of the following general formula

Where y is 0 (i.e. acyclic structure), 2 (cyclobutyl), or 3 (cyclopentyl), r is 0-6, Z is (CH ₂ ) ₇ or (CH ₂ ) _e -C ₆ H ₄ -(CH ₂ ) _f , where e is 0-1, and f is 0-5, R ¹⁵ is -H, -Cl, -OMe or

where n is 0-4, R ¹⁷ is -C ₂ H ₅ , allyl, benzyl, and R ¹⁶ is

Wherein x is 0-4, t is 0-2, and R ⁴ is any of the following: halogen, alkyl of 1-6 carbons, OR, NR ₂ , NO ₂ (R=H or alkane of 1-6 carbons base).

Those skilled in the art will recognize that many of the compounds of the present invention exist in enantiomeric or diastereomeric forms, and it is known in the art that such stereoisomeric forms often exhibit different activities in biological systems. The present invention includes all possible stereoisomers having inhibitory activity against MMPs, regardless of their stereoisomeric design, as well as mixtures of stereoisomers in which at least one has inhibitory activity.

The most preferred compounds of the invention are indicated and named below:

I) 1,3-dihydro-1,3-dioxo-α-(2-oxododecyl)-2H-isoindole-2-butyric acid,

II) 1,3-dihydro-1,3-dioxo-α-(2-oxoundecyl)-2H-isoindole-2-butyric acid,

III) 1,3-Dihydro-1,3-dioxo-α-(2-oxotridecyl)-2H-isoindole-2-butyric acid,

IV) 1,3-Dihydro-1,3-dioxo-α-(2-oxotetradecyl)-2H-isoindole-2-butyric acid,

V) 1,3-dihydro-1,3-dioxo-α-(2-oxopentadecyl)-2H-isoindole-2-butyric acid,

VI) 1,3-dihydro-1,3-dioxo-α-(2-oxohexadecyl)-2H-isoindole-2-butyric acid,

VII) γ-oxo-α-(2-phenylethyl)-phenylheptanoic acid,

VIII) γ-oxo-α-(2-phenylethyl)-phenylhexanoic acid,

IX) γ-Oxo-α-(2-phenylethyl)-valeric acid.

The compounds of the present invention can be prepared by using known chemical reactions and techniques. However, the following general preparations are provided to assist the reader in synthesizing the inhibitors, with more detailed specific examples in the experimental section describing the working examples below.

The general method is illustrated by the following general description. A group defined as P represents a protecting group. Those skilled in the art will appreciate that a variety of protecting groups can be used to protect potentially reactive functional groups (eg, carboxylic acids, alcohols) and the specific choice is based on the reaction conditions required to prepare the target compound. A description of such protecting groups can be found in: Green and Wuts, Protecting Groups in Organic Synthesis, 2nd Edition, John Wiley and Sons, New York, 1991.

The group designated X represents a leaving group. It is well known to those skilled in the art that several different functional groups such as halide, mesylate, tosylate and triflate can be used as leaving groups. It is also known that the choice of a particular leaving group is generally based on such factors as nucleophilic reactivity, stability of the compound, and ease of synthesis.

General Procedure A - Compounds of the invention wherein E does not contain a ring can be conveniently prepared using (α-halomethyl ketones and substituted malonate derivatives. The α-halomethyl ketone intermediate CIII (X=Cl , Br) can be prepared conveniently from carboxylic acids or ketones. From the carboxylic acid CI, treatment with oxalyl chloride in a solvent such as trimethylsilyl chloride and a catalytic amount of DMF provides the corresponding acid chloride. Subsequent treatment with an excess of Diazomethane followed by treatment with anhydrous HCl or HBr affords intermediates CIII.Alternatively, intermediates CIII can also be prepared from ketones CII by treatment of silyl enol ethers with N-bromosuccinimide (NBS). Silyl enol ethers are conveniently prepared from methyl ketones by treatment with trimethylsilyl chloride (TMSCI) and a base such as hexamethyldisilazide (LHMDS). Preparation of α-halomethyl ketones General methods are well known to those skilled in the art. For further references see Corey et al., Tetrahedron Lett. 25 , 495 (1984) and Reuss et al., J. Org. Chem., 39 1785 (1974).

Methods for preparing substituted malonate derivatives (CVs) are also well established in the literature. Typically, unsubstituted malonate derivatives (CV) are treated with bases such as NaH or Kot-Bu in polar aprotic solvents, followed by alkylation with substituted halides. Similarly, alkylation of the monoalkylated intermediate with the alpha-halomethyl ketone CIII gives the dialkylated intermediate, CVI. It will be appreciated by those skilled in the art that the side chain of Y may be the side chain that is desired in the final object or that part of the molecule that can be easily manipulated later in the synthesis to further process the molecule. If the side chain Y is the desired side chain, the intermediate CVI can be easily deprotected and decarboxylated by known methods to give the desired compound CVIII. Conditions for deprotecting intermediate CVI depend on the type of deprotecting group used. Some convenient protecting groups useful in the synthesis of compounds of this invention include methyl, allyl, benzyl and tert-butyl. Methods for insertion and removal of these groups are well known to those skilled in the art (see above). The choice of protecting groups for this synthesis is based on factors such as compatibility of functional groups, ease of synthesis, and availability of starting materials.

The intermediate side chain Y can be used if the target compound CXI contains a moiety Q which is sensitive to the reaction conditions used in the alkylation step. In this case, this manipulation conveniently inserts _a protected ethanol group such as _CH2CH2OTBS . The intermediate CV _of Y= _-CH2CH2OTBS can be prepared _{using TBSOCH2CH2Br} as YX in the first alkylation step. _TBSOCH2CH2Br can be prepared _{from HOH2CH2Br} by methods well known to those skilled in the art _. Removal of the protecting group provides the corresponding alcohol, which can be converted to the phenyl ether or various heteroatom substituted derivatives for generation of side chain Q by the Mitsunobu reaction. The Mitsunobu reaction is well known to those skilled in the art; see Mitsunobu , Synthesis I (1981), and Hughes, Organic Reactions 42 , 335 (1992). Alternatively, the alcohol intermediate is converted to a leaving group such as tosylate or bromide and displaced by a suitable nucleophile. Several examples of such reactions are found in Norman et al., J. Med. Chem., 37 , 2552 (1994). After insertion of the desired side chain Q to form CX, the malonate moiety was deprotected and decarboxylated to give the target compound CXI. In some cases, the ketone moiety of intermediate CIII may need to be protected to avoid undesired side reactions. Protection as acetals is generally preferred, if desired, by methods such as those described in Hwu et al., J. Org. Chem., 50 , 3946 (1985).

General Method B - Compounds of the invention wherein two ^R6 groups are joined to form a substituted 5-membered ring E are most preferably prepared by Method B. In this method, the acid MI (R=H) is prepared as described in Beeley et al., Tetrahedron 37 Suppl., 411 (1981). The acid is protected as an ester with a linker such as 1-(3-dimethylaminopropyl-3-ethylcarbodiimide) hydrochloride and methods well known in the art [e.g., R = benzyl (Bn) or 2-(trimethylsilyl)ethyl (TMSE)]. Reaction of Grignard reagent MII (prepared from the corresponding bromide by magnesium treatment) with MI (R=Bn, TMSE) affords alcohol MIII. Alcohol MIII is eliminated to afford alkene MIV by base treatment of its mesylate using conditions well known to those skilled in the art. Ozonation of MIV (done with dimethylsulfide) affords the aldehyde MV. Alternatively _, MIV was converted to MV by treatment with _OsO4 followed by _H5IO6 .

The conversion of the key intermediate MV to the desired proprietary compound was carried out in several ways based on the identity of the side chain function J. Reaction of MV with a Wittig reagent followed by hydrogenation affords products in which J is alkyl, aryl or aralkyl. Selective reduction of aldehyde MV with a reducing agent such as lithium tris[(3-ethyl-3-phenyl)oxy]aluminum hydrochloride (LTEPA) yields alcohol MVI. This alcohol can be converted to phenyl ether or various heteroatom substituted derivatives to generate side chain ^R16 by 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 intermediates are converted by conditions well known in the art, such as tosylate The leaving group of MVII or bromide is replaced by a suitable nucleophile. Several examples of this type of reaction are found in Nerman et al., Journal of Medical Chemistry, 37 , 2552 (1994). The direct acylation of the alcohol MVI produces and reacting the alcohol with various alkyl halides in the presence of a base to give alkyl ethers. In each case, the final step is removal of the acid protecting group R to give the acid (R=H)), which The conditions used depend on the stability of R and J, but each is well known to those skilled in the art. For example, removal of the benzyl group can be accomplished by base hydrolysis or hydrogenolysis, while the 2-(trimethylsilyl)ethyl ester is generally deprotected by a facile treatment with tetrabutylammonium fluoride.

Suitable pharmaceutically acceptable salts of the compounds of the present invention include addition salts formed with organic or inorganic bases. Salt-forming ions derived from such bases may be metal ions, for example, aluminum, alkali metal ions such as sodium or potassium, alkaline earth metal ions such as calcium or magnesium, or amine salt ions, many of which are known for this purpose. . Examples include ammonium salts, arylalkylamines such as dibenzylamine and N,N-dibenzylethylenediamine, lower alkylamines such as methylamine, tert-butylamine, procaine, lower alkylpiperamines Pyridines such as N-ethylpiperidine, cycloalkylamines such as cyclohexylamine or dicyclohexylamine, 1-adamantylamine, benzathine, or salts derived from amino acids such as arginine, lysine, etc. Physiologically acceptable salts such as sodium or potassium salts and amino acid salts can be used pharmaceutically as described below, and are preferred.

These and other salts which do not need to be physiologically acceptable are useful in isolating or purifying products which are acceptable for the purposes described below. For example, commercially available enantiomerically pure amines such as (+)-cinchonine in a suitable solvent can yield crystals of the individual enantiomer salts of the compounds of the invention in a process commonly referred to as "classical resolution", at The opposite enantiomer remains in solution. Because the enantiomer of a given compound of the present invention is physiologically much larger than its enantiomer, the active isomer can be purified in crystalline or liquid phase. The salt is produced by reacting the acid form of the compound with an equivalent of a base providing a basic ion in a medium in which the salt is precipitated or in an aqueous medium followed by lyophilization. The free acid form can be obtained from salts by common neutralization techniques, for example, with potassium bisulfate, hydrochloric acid, and the like.

The compounds of the present invention have been found to inhibit the matrix metalloproteinases MMP-3, MMP-9, and MMP-2, and to a lesser extent MMP-1, and are therefore useful in the treatment or prevention of the disorders listed in the Background section. Since other MMPs not listed above share a high degree of homology with those listed above, especially at the catalytic site, it can be considered that the compounds of the present invention should also inhibit such other MMPs to varying degrees . Varying the substituents on the biaryl moiety of the molecule, as well as the substituents on the propionic or butyric acid chains of the claimed compounds, has been shown to affect the relative inhibition of the listed MMPs. Thus, compounds of this general class can be "tuned" by selection of specific substituents so that inhibition of specific MMPs associated with specific pathological conditions is enhanced, while MMPs not included are less affected.

Methods of treating matrix metalloproteinase-mediated disorders can be practiced in mammals, including humans, exhibiting such disorders.

The inhibitors of the present invention are contemplated for veterinary and human use. Accordingly, they will be used in pharmaceutical compositions containing the active ingredient plus one or more pharmaceutically acceptable carriers, diluents, fillers, binders, and other excipients, depending on Mode of administration and desired dosage form.

Administration of the inhibitor can be by any suitable means 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 achieve acute modulation of peak plasma concentrations of the drug. Improved half-life and drug targeting to the joint cavity can be enhanced by entrapping the drug within liposomes. The selectivity of liposome targeting to the joint cavity can be improved by incorporating ligands into the periphery of liposomes bound to synovial fluid-specific macromolecules. Alternatively, intramuscular, intraarticular, or subcutaneous depot injection into degradable microspheres, for example, microspheres comprising poly(DL-lactide-co-glycolide), with or without drug encapsulation, Can be used to obtain sustained drug release. For dosage form improved convenience, i.p. implanted reservoirs and spacers such as the Percuseal system available from Pharmacia are available. Improved convenience and patient compliance can also be achieved with injection pens (such as Novo Pin or Q-pen) or needle-free jet injectors (such as those available from Bioject or Becton Dickinson). Delayed zero-order or other precisely controlled release, such as pulsatile release, can also be achieved by implantable pumps that deliver the drug through the cannula to the synovial fluid space as needed. Examples include subcutaneous implantation of osmotic pumps obtained from ALZA, such as ALZET osmotic pumps.

Nasal delivery can be achieved by incorporating the drug into bioadhesive particulate carriers (<200 μm), such as those comprising cellulose, polyacrylate or polycarbophil, in combination with suitable absorption enhancers such as phospholipids or acylcarnitines. Commercially available systems include those developed by DanBiosys and Scios Nova.

Contrary to the various peptide compounds listed in the background section of this application, the outstanding contribution of the compounds of the present invention is the oral activity exhibited by the compounds of the present invention. Certain compounds have shown bioavailability as high as 90-98% in various animal models. Oral delivery can be achieved by incorporating the drug into tablets, coated tablets, dragees, hard or soft capsules, solutions, emulsions or suspensions. Oral delivery can also be achieved by incorporating the drug into enteric-coated capsules designed to release the drug into the colon, where digestive protease activity is low. Examples include the OROS-CT/Osmet ^(TM) and PULSINCAP( ^TM) systems available from ALZA and Scherer Drug Delivery Systems, respectively. Other systems use azo-crosslinked polymers that are degraded by colon-specific bacterial azoreductase, or pH-sensitive polyacrylate polymers that are activated by raising the pH in the colon. The above systems can be used in combination with a wide range of absorption enhancers.

Rectal delivery can be achieved by incorporating the drug into a suppository.

The compounds of the present invention can be prepared into the above-mentioned preparations by adding various therapeutically inert inorganic or organic carriers known to those skilled in the art. Examples of these include, but are not limited to, lactose, corn starch or derivatives thereof, talc, vegetable oils, waxes, fats, polyols such as polyethylene glycol, water, sucrose, alcohols, glycerol, and the like. Various preservatives, emulsifiers, dispersants, flavoring agents, wetting agents, antioxidants, sweeteners, coloring agents, stabilizers, salts, buffers, etc. are also added as needed to help stabilize the preparation or help increase the activity The bioavailability of ingredients, resulting in a preparation with an acceptable taste or smell in the case of an oral dosage form.

The amount of pharmaceutical composition employed will depend on the recipient and the condition being treated. The required amount can be determined by one skilled in the art without undue experimentation. Alternatively, the required amount can be calculated based on determining the amount of the target enzyme that must be inhibited to treat the condition.

The matrix metalloproteinase inhibitor of the present invention can not only be used for treating the above-discussed diseases, but also can be used for the purification of metalloproteinase and the test of matrix metalloproteinase activity. Such activity tests can be performed in vitro with natural or synthetic enzyme preparations or, for example, where abnormal destructive enzyme levels are found naturally (with genetically mutated or transgenic animals) or by administration of exogenous agents or by destruction of joints. Stability surgery was induced in vivo in animal models.

The following examples are for illustration only and do not limit the present invention in any sense.

Example

General process:

Unless otherwise stated, all reactions were performed in flame-dried or oven-dried glassware under a positive pressure of argon with magnetic stirring. Sensitive liquids and solutions are transferred by syringe or catheter and introduced into the reactor through a rubber septum. Unless otherwise stated, the reaction product solutions were concentrated using a Buchi evaporator.

raw material:

Commercial grade reagents and solvents were used without further purification, only diethyl ether and tetrahydrofuran were distilled under argon with benzophenone ketyl and methylene chloride was distilled with calcium hydride under argon. A number of specific organic or metalorganic starting materials and reagents were obtained from Aldrich, 1001 West Saint Paul Avenue, Milwaukee, WI 53233. Solvents are generally obtained from EM Science as classified by VWR Scientific.

Chromatography:

Analytical thin layer chromatography (TLC) was performed on ^Whatman® pre-coated glass-supported silica gel GHLF 250mm plates. The coloration of the spots was performed by one of the following techniques: (a) UV light irradiation, (b) exposure to iodine vapor, (c) immersion of the plate in a 10% ethanol solution of phosphomolybdic acid followed by heating, and (d) immersion of the plate Immerse in a 3% ethanol solution of methoxybenzaldehyde containing 0.5% concentrated sulfuric acid and heat.

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

instrument:

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

The proton (1h) nuclear magnetic resonance (NMR) spectrum was measured with a General Electric GN-OMEGA300 (300MHz) spectrometer, and the carbon 13 ( ¹³ C) NMR spectrum was measured with a General Electric GN-OMEGA300 (75MHz) spectrometer. Most of the compounds synthesized in the experiments below were analyzed by NMR, and in each case the spectra were consistent with the hypothesized structures.

Mass spectral data were acquired with a kratos Conceβt 1-H mass spectrometer by liquid-cesium secondary ionization (LCIMS), the latest version of fast atom bombardment (FAB). Most of the compounds synthesized in the following experiments were analyzed by mass spectrometry, and in each case the spectra were consistent with the hypothesized structures.

General explanation:

For multi-step processes, successive steps are numbered.

Embodiment 1-6 prepares compound I-VI

Step 1 A solution of sodium hydride (4.35 g, 181 mmol) in freshly distilled THF (100 ml) was cooled to 0°C and replaced with commercially available diallyl malonate (35.0 g, 190mmol) treatment. After stirring at room temperature for 30 minutes, N-(2-bromoethyl)phthalimide (43.9 g, 247 mmol) was added to the solution in one portion and the mixture was heated at reflux. After 48 hours, the solution was cooled to 0°C, quenched with 2N HCl and concentrated to approximately 20% of its original volume. The concentrate was diluted with ethyl acetate ₍ 300 ml) and washed successively with saturated aqueous solutions of _K2CO3 and NaCl. The organic layer was dried over _MgSO4 , filtered and concentrated under reduced pressure. Purification by flash column chromatography (5-25% ethyl acetate-hexanes) gave diallyl 2-phthalimidoethylmalonate (451.2 g, 64%) as a colorless oil.

¹ H NMR (300MHZ, CDCl ₃ ) δ7.82(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=6.6Hz, 2H), 346(t, J=7.2Hz, 1H), 2.30(dd, J=13.8, 6.9Hz, 2H).

The product of above-mentioned reaction is as follows:

Step 2 A one-neck 50ml round bottom flask equipped with a rubber septum and an argon needle was charged with 12ml THF, trimethylsilane chloride (0.83ml, 0.710g, 6.54mmol), lithium hexamethyldisilazide (6.50ml, 1.0M solution in THF, 6.50mmol) and cooled to -78°C while adding 2-dodecanone (1.19g, 6.46mmol) dropwise in 8.0ml via cannula over a period of 30 minutes of THF solution. The resulting mixture was stirred at -78°C for 30 minutes. N-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 pentane and washed three times with 50 ml brine respectively. The organic phase was dried over _Na2SO4 and concentrated to give 2.5 _g of a yellow solid. Column chromatography on 100 g of silica gel (3-5% ethyl acetate-hexane gradient) gave 0.680 g (40%) of bromomethyl ketone as a white solid. TLC (5% ethyl acetate-hexane), _Rf = 0.4. The product of the above reaction is as follows:

Step 3 A one neck 25ml round bottom flask fitted with a rubber septum and an argon needle guide was charged with 3ml THF and the product of Step 1 (314mg, 0.978mmol). The resulting mixture was cooled to 0 °C and sodium tert-butyrate (88.0 mg, 97% purity, 0.888 mmol) was added. After 30 minutes, a solution of the product from step 2 (250 mg, 0.950 mmol) in 3 ml THF was added dropwise via syringe. The resulting mixture was warmed to room temperature and stirred for 16 hours. The reaction mixture was diluted with 100 mL _CH2Cl2 and washed three _times with 30 mL brine each. The organic layer was dried over MgSO ₄ and concentrated. Column chromatography on 40 g of silica gel (eluting with a gradient of 10-30% ethyl acetate-hexanes) gave 0.300 g (63%) of the resulting product as a white solid. TLC (30% ethyl acetate/hexanes) _Rf = 0.5. Above-mentioned reaction product is as follows:

Step 4 Preparation of Example 1. To a one neck 154ml round bottom flask equipped with a rubber septum and an argon needle cannula was added 2ml of dioxane, the product of step 3 (300mg, 0.556mmol), pyrrolidine (0.12ml, 0.102g, 1.44mmol), and tetra( Triphenylphosphine) palladium (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 hours, the dioxane and pyrrolidine were removed in vacuo, and the residue was redissolved in 2 mL of dioxane. The resulting mixture was exposed to a slight vacuum to degas the solution and argon was reintroduced. The reaction mixture was heated at 115°C for 4 hours. 85°C, 12 hours, and concentrated. Column chromatography on 10 g of silica gel (30% ethyl acetate-hexane with 0.5% acetic acid) yielded 0.137 g (59%) of the white solid of Example 1 (MP 89-90°C). Above-mentioned reaction product is as follows:

The following examples (Table 1) were prepared using the method described above for the preparation of Example 1 via the appropriate bromoketone in Step 3

Table I

^a Preparation of 1-bromo-2-tetradecanone: 16ml CCl ₄ , 1,2-epoxytetradecane (2.0ml, 1.66 g, 7.82mmol), benzylpyrrolidine (1.00g), and bromine (0.20ml, 0.62g, 3.88mmol). The reaction mixture was stirred for 30 min at room temperature under desk light illumination (soft white, 60 W). The reaction mixture was diluted with a 1:1 hexane:ethyl acetate mixture (150 mL), washed with 50 mL portions of saturated _NaHCO3 , and washed with 50 mL of brine. The organic layer was dried over _MgSO4 and concentrated. 100 g of silica gel column chromatography (5-10% ethyl acetate-hexane gradient elution) gave 0.440 g (39%) of 1-bromo-2-tetradecanone as a white solid. TLC (5% ethyl acetate-hexane), _Rf = 0.4.

      Example 7-9-Preparation of Compound VII-IX

Step 1 Treat 4-(4-methoxyphenyl)-butyric acid (3.04 g, 15.4 mmol) in _CH2 with oxalyl chloride (11.6 ml, 2.0 M solution in dichloromethane) and DMF (1 drop) _Cl2 (45ml) solution. The solution was heated to reflux for 2 hours, cooled to 0°C and treated with excess diazomethane (in ether). After stirring for another 30 minutes, excess 4M HCl (in 1,4-dioxane) was added and the mixture was warmed to room temperature and stirred overnight. The solution was concentrated under reduced pressure, diluted with ethyl acetate and washed successively with water, saturated aqueous NaHCO ₃ and saturated aqueous NaCl. The organic phase was dried over MgSO ₄ , filtered and concentrated. Purification by MPLC (5-25% ethyl acetate-hexane) gave the title compound (2.45 g, 70%) as a colorless oil. TLC: Rf _0.45 (silica gel, 15% ethyl acetate-hexane). The resulting compound has the following formula:

Step 2 A solution of NaH (6.35 g, 264 mmol) in THT- (500 ml) was treated with diethyl malonate (47.35 ml, 312 mmol). After stirring for 2 hours, (2-bromoethyl)benzene (32.8ml, 240mmol) was carefully added to the reaction mixture. The solution was then heated to slight reflux for 16 hours, cooled to 0°C and quenched with 2N HCl. The resulting solution was concentrated under reduced pressure, diluted with EtOAc and washed with saturated aqueous NaCl. The organic layer was dried over MgSO ₄ and concentrated. Vacuum distillation (1.5 mmHg) gave the substituted malonate (44.4 g, 71%) as a colorless oil. TLC: Rf _0.52 (silica gel, 20% ethyl acetate-hexane). The resulting compound has the following formula:

Step 3: A solution of the malonate (3.50 g, 13.2 mmol) from Step 2 in DME (5 ml) was treated with NaOEt (0.67 g, 9.9 mmol) and stirred for 30 minutes. While stirring the solution, the separatory flask containing the α-chloroketone (0.95 g, 4.2 mmol) from step 1 in DMF (5 ml) was treated with LiI (0.62 g, 4.6 mmol), stirred for 15 min, and socketed Tube to first solution. After stirring overnight, the reaction mixture was diluted with EtOAc and washed with water and saturated aqueous NaCl. The organic layer was dried over _MgSO4 , filtered and concentrated. Purification by MPLC (5-20% EtOAc-hexanes) gave the resulting malonate (0.41 g, 21%) as a colorless oil. TLC: Rf _0.30 (silica gel, 20% ethyl acetate-hexane). The resulting compound has the following formula:

Step 4 - Preparation of Example 7 A solution of the diester from Step 3 (0.19g, 0.42mmol) in ethanol (3ml) was treated with 2N NaOH (0.5ml) and stirred at room temperature. After stirring for 16 hours, the solution was concentrated under reduced pressure, diluted with ethyl acetate, and washed with _{aqueous K2CO3} . The aqueous layer was acidified to pH 1 with 2N HCl and extracted with ethyl acetate. The organic layer was dried over _MgSO4 , filtered and concentrated. The resulting diacid was dissolved in 1,4-dioxane (3ml) and heated to 65°C. After stirring for 24 hours, _the solution was concentrated and purified by flash column chromatography (2-4%, MeOH- _CH2Cl2 ) to give the title compound (72.1 mg, 49%). MP 70-71°C.

Gained compound (embodiment 7) is as follows

The procedure described above for the preparation of Example VII was used to prepare the following examples (Table II).

Table II

Example 10

Biological tests of compounds of the present invention

P218 Quenching Fluorescence Test of MMP Inhibitors

The P218 quenched fluorescece assay (Profiling assay) was originally described by CGKnight et al., FEBS Letters, 296, 163-266 (1992), in cuvettes for relevant bottom substances and modifications of many matrix metalloproteinase (MMP) assays. The assay was performed with each example compound of the invention and three MMPs, MMP-3, MMP-9 and MMP-2, suitable for analysis in parallel in 96-well microtiter plates and Hamilton AT ^- workstations as follows.

P218 Fluorogenic Substrates

P218 contains a 4-acetyl-7-methoxycoumarin (MCA) group at the N-terminal position and a 3-(2,4-dinitrophenyl)-(L)-2 , a synthetic substrate for the 3-diaminopropionyl (DPA) group. This is a modification of a peptide reported by Knight (1992) as a substrate for matrix metalloproteases. Upon cleavage of the P218 peptide (putative cleavage site on the Ala-Leu bond), the fluorescence of the MCA group can be detected on a fluorometer with excitation at 328nm and emission at 393nm. P218 is currently manufactured exclusively for Bayer Corp by BACHEMBioscience, Inc. P218 has the following structure: H-MCA-Pro-Lys-Pro-Leu-Ala-Leu-DPA-Ala-Arg- _NH2 (MW 1332.2)

Recombinant human CHO stromelysin (MMP-3):

Recombinant human CHO Pro-MMP-3: Human CHO Pro-stromelysin 257 (pro-MMP-3) was expressed and purified as described by T.J. Housley et al., J. Biol. Chem., 268, 4481-4487 (1993).

Activation of Pro-MMP-3: Pro-MMP-3 at 1.72 μM (100 μg/mL) in MMP- 3 Activate by incubating with TPCK (N-methylsulfonyl-(L)-phenylalanine chloromethyl ketone) trypsin (1:100 w/w to pro-MMP) in the activation buffer for 30 minutes at 25°C. The reaction was stopped by adding soybean trypsin inhibitor (SBTI; 5:1 w/w to trypsin concentration). This method of activation results in the formation of a 45 kDa active MMP-3, which also contains the C-terminal portion of the enzyme.

Preparation of Human Recombinant Pro-Gelatinase A (MMP-2):

According to the method of R. Fridman et al., Journal of Biochemistry, 267, 15398-405, (1992), human recombinant Pro-MMP-2: human pro-gelatinase A (Pro-MMP-2) was prepared using the vaccinia expression system.

Activation of Pro-MMP-2: 252 mg/mL of Pro-MMP-2 in MMP-2 activation buffer consisting of 25 mM Tris at pH 7.5, 5 mM CaCl, 150 mM CaCl, and 0.005% Brij-35 diluted 1:5 to a final concentration of 50 mg/mL. p-Aminophenylmercury acetate (APMA) was prepared at 10 mM (3.5 mg/mL) in 0.05 N sodium hydroxide. The APMA solution was added at 1/20 of the reaction volume to make a final APMA concentration of 0.5 mM, and the enzyme was incubated at 37°C for 30 minutes. Activated MMP-2 (15 mL) was added to 2L of MMP-2 Activation Buffer (permeable membrane was pretreated with a solution consisting of 0.1% BSA in MMP-2 Activation Buffer for 1 min, followed by extensive water washing). The enzyme was re-diluted in a Centricon concentrator (the concentrator was also pretreated with a solution consisting of 0.1% BSA in MMP-2 activation buffer for 1 min, followed by a water wash followed by a wash with MMP-2 activation buffer), followed by repeated re-treatment. Concentrated twice. The enzyme was diluted to 7.5 mL (0.5 times the original volume) with MMP-2 activation buffer.

Preparation of Human Recombinant Pro-Gelatinase B (MMP-9)

The human recombinant Pro-MMP-9 derived from U937 cDNA as described in S.M.Wilhelm et al., Biochemical Journal, 264, 17213-17221 (1989): human recombinant Pro-gelatinase B (Pro -MMP-9) expressed as a full-length form. The pro-enzyme was purified by the method described by M.S. Hibbs et al., J. Biol. Chem., 260, 2493-500 (1984).

Activation of Pro-MMP-9: Pro-MMP-9 (20 μg /mL) was activated by incubation with 0.5 mM p-aminophenylmercury acetate (APMA) for 3.5 hours at 37°C. The enzyme was dialyzed against the same buffer to remove APMA.

instrument:

Hamilton Microlab AT Plus ^_ : MMP-Profiling assays were automated with HamiltonMicrolab AT Plus ^_ . Hamilton was programmed to: (1) automate serial dilutions to 11 potential inhibitors with 2.5 mM stock solutions of inhibitors in 100% DMSO; (2) distribute substrates in 96-well Cytofluor plates, inoculate to distribute inhibitors; and (3) adding individual enzymes to the plate, mixing to initiate the reaction. Subsequent plates of each additional enzyme are automatically prepared by starting the program at the moment of substrate addition, mixed with diluted inhibitor, and the reaction initiated by addition of enzyme. In this way, all MMP assays were performed with the same inhibitor dilutions.

Millipore Cytofluor II; after incubation, plates were read on a Cytofluor II fluorescence reader with excitation at 340nm, emission at 395nm, and magnification set at 80.

Buffer:

Microfluorometer Reaction Buffer (MRB): For microfluorometer assays, dilutions of test compounds, enzymes, and P218 substrates were prepared in 50 mM 2-(N-morpholino)ethanesulfonic acid (MES) at pH 6.5 with Prepare in Microfluorometer Reaction Buffer (MRB) consisting of 10 mM calcium chloride, 150 mM sodium chloride, and 0.005% Brij-35 and 1% DMSO.

method:

MMP Microfluorometer Profile Analysis Test. The assay was performed with a final P218 concentration of 6 [mu]M, approximately 0.5 to 0.8 nM activated MMP, and variable inhibitor concentrations. Hamilton was programmed in the assay to serially dilute 11 compounds from a 2.5 mM stock (100% DMSO) to 10-fold the final compound concentration. Initially, the instrument delivered various volumes of Microfluorometer Reaction Buffer (MRB) into 1 mL Marsh dilution tubes in a 96-tube rack. The instrument takes 20 μL of inhibitor (2.5 mM) and mixes it with the buffer in row A of the Marsh rack, resulting in an inhibitor concentration of 50 μM. The inhibitor was then serially diluted to 10, 5, 1, 0.2, 0.05 and 0.01 [mu]M. Position 1 of the sample holder contained only DMSO for the "enzyme only" wells used in the assay, which resulted in no inhibitor in column 1, rows A through H. The instrument then dispenses 107 μL of P218 into individual 96-well Cytofluor microtiter plates. Remix the instrument and load 14.5 μL of the diluted compound from row A to G on the Marsh rack into the corresponding row of the microtiter plate. (Row H represents the "background" row. To this was added 39.5 μL of microfluorometer reaction buffer instead of drug or enzyme). Row H, the "background" row, was excluded by adding 25 [mu]L of the appropriate enzyme (5.86 times the final enzyme concentration) from the BSA-treated reagent reservoir to each well. (Enzyme tanks were pretreated with 1% BSA in 50 mM Tris pH 7.5 containing 150 mM NaCl for 1 hour at room temperature, followed by extensive washing with water and drying at room temperature).

After enzyme addition and mixing, the plate was covered and incubated at 37°C for 25 minutes. Additional enzymes were tested in the same manner by initiating the Hamilton procedure, dispensing the P218 substrate in the microtiter plate, followed by remixing, and dispensing the drug onto the microtiter plate from the same Marsh rack. The second (or third, etc.) MMP to be tested is then dispensed from the reagent rack onto the microtiter plate, mixed then covered and incubated.

_IC50 Determination in Microfluorometer Assay: Data generated on the Cytofluor II was copied from the exported "CSV" file to the Excel spreadsheet of the householder. Data from various MMPs (one 96-well plate per MMP) were calculated simultaneously. The percent inhibition for each drug concentration was determined by comparing the amount of hydrolysis (fluorescence units produced at 25 minutes of hydrolysis) for wells containing compound to "enzyme only" wells in column 1. The background was subtracted and percent inhibition was calculated as follows:

((control value - treated value)/control value) x 100 Percent inhibition was determined for inhibitor concentrations of 5, 1, 0.5, 0.1, 0.02, 0.005 and 0.001 [mu]M. Linear regression analysis of percent inhibition versus logarithm of inhibitor concentration was used to obtain _IC50 values.

The profile analysis test data of compound of the present invention

table 5

MMP-profile analysis data. All _IC50 values used are expressed in nM. When "I=x%" is displayed, x represents % inhibition at 5 μM.

table 5

compound MMP-3 fluorophore _IC50 MMP-9 Fluorophore _IC50 MMP-2 fluorophore _IC50 I 4800 120 1300 II 5000 240 1850 III 2950 91 750 IV 2380 155 515 V 3384 150 1100 VI I = 45% 235 1020 VII I=3% I = 18% VIII I = 13% I = 15% IX I=9% I = 18%

Other aspects of the invention will be apparent to those skilled in the art from consideration of the specification or practice of the invention disclosed herein. The specification and examples are to be considered as illustrative only, with the true scope and spirit of the invention set forth in the appended claims.

Here Ph is phenyl, Me is methyl, THF is tetrahydrofuran, Bu is butyl, tBu

is tert-butyl and Et is ethyl.

Claims (7)

1. A compound inhibiting matrix metalloproteinases having the general formula: where y is 0, 2 or 3, r is 0-6, Z is (CH ₂ ) ₇ or (CH ₂ ) _e -C ₆ H ₄ -(CH ₂ ) _f , where e is 0-1 and f is 0-5; R ¹⁵ is -H, -Cl, -OMe or

where n is 0-4, R ¹⁷ is C ₂ H ₅ , allyl, benzyl, and R ¹⁶ is

Wherein t is 0-2, x is 0-4, and R ⁴ is any one selected from: halide, 1-6 carbon alkyl, OR, NR ₂ , NO ₂ , where R=H or 1- Alkyl group of 6 carbons. 2. A method of inhibiting the activity of a matrix metalloprotease in vitro comprising providing a compound of claim 1 in an effective matrix metalloprotease inhibitory amount. 3. A composition for inhibiting matrix metalloproteinases, comprising the compound of claim 1 and a pharmaceutically acceptable carrier. 4. The application of the compound of claim 1 in the preparation of the pharmaceutical composition for treating mammals, comprising giving the compound of claim 1 of matrix metalloproteinase inhibitory amount to mammals, it is enough: (a) have the effect of alleviating osteoarthritis, rheumatoid arthritis, septic arthritis, periodontal disease, corneal ulcer, proteinuria, aortic disease of aneurysm, dystrophic epidermolysis blister, conditions leading to inflammatory response, Effects of osteopenia mediated by MMP activity, temporomandibular joint disease, or demyelinating disease of the nervous system; (b) to block cartilage loss following tumor metastasis or traumatic joint injury; (c) reduce coronary thrombosis caused by rupture of atherosclerotic plaque; or (d) Achieve birth control. 5. The use of claim 4, wherein said mammal is a human. 6. Use according to claim 4, wherein said effect is reduction of osteoarthritis. 7. The use according to claim 4, wherein said effect is to block tumor metastasis.