JP3417951B2 - Substituted oxybutyric acids as matrix metalloprotease inhibitors - Google Patents

Description

BACKGROUND OF THE INVENTION Field of the Invention The present invention relates to novel oxybutyric acid compounds or derivatives thereof useful for inhibiting enzyme inhibitors, particularly matrix metalloproteases.

Related Art Matrix metalloproteases (also known as matrix metalloendo-proteinases or MMPs) are
Interstitial collagenase (also known as MMP-1), stromelysin (also known as proteoglycanase, transin or MMP-3), gelatinase A (72k
It belongs to the family of zinc endoproteinases, including but not limited to Da-gelatinase, also known as MMP-2) and gelatinase B (also known as 95kDa-gelatinase or MMP-9). These MMPs are expressed by TI cells by a variety of cells including fibroblasts and chondrocytes.
It is secreted together with a natural protein inhibitor known as MP (Tissue Inhibitor of MetalloProteinase).

All these MMPs are capable of destroying various connective tissue components of articular cartilage or basement membrane. Each MMP is
It is secreted as an inactive proenzyme to be degraded in subsequent steps before it can exert its own proteolytic activity. In addition to this matrix-destroying effect, these particular MMPs, such as MMP-3, are
Satisfies the conditions as an in vivo activator for MP-1 and MMP-9 (Ito et al., Arch Biophys. 267, 211 (1
988); Ogata. Et al., J. Biol. Chem., 267, 3581 (1992)).
Therefore, the cascade of proteolytic activity is associated with excess MMP-
It can be started by 3. Therefore, a specific MMP
-3 inhibitors should limit the activity of other MMPs that are not directly inhibited by such inhibitors.

MMP-3 can be destroyed, with other proteinases
For example, it has been reported that an endogenous inhibitor of elastase can be inactivated (Winyard. Et al., FEBES L.
etts, 279, 1, 91 (1991)). Therefore, inhibitors of MMP-3 could have been able to influence the activity of other destructive proteinases by modifying the levels of their endogenous inhibitors.

Many diseases are believed to be mediated by excessive or unwanted matrix-disrupting metalloprotease activity or by an imbalance in the ratio of MMPs and TIMPs. This includes a) osteoarthritis (Woessner, et al., J. Biol. Chem., 2
59 (6), 3633 (1984); Phadke, et al., J. Rheumatol. 10,85.
2 (1983)), b) rheumatoid arthritis (Mullins, et al., Bio
chim.Biophys.Acta 695,117 (1983); Woolley, et al., Art
hritis Rheum. 20, 1231 (1977); Gravallese, et al., Arth
ritis Rheum.34,1076 (1991)), c) septic arthritis (williams et al., Arthritis Rheum.33,533 (1990)),
d) Tumor metastasis (Reich et al., Cancer Res., 48, 3307 (198
8); and Matrisian et al., Proc. Nat'l. Acad. Sci. USA 8
3,9413 (1986)), e) Periodontal disease (Overall. Et al., J. Period)
ontal Res.22,81 (1987), f) Corneal ulcer (Burns
, Invest.Ophthalmol.Vis.Sci.30,1569 (1989)),
g) Proteinuria (Baricos et al., Biochem. J. 254,609 (198
8)), h) Coronary thrombosis from rupture of atherosclerotic plaque (Hen
ney et al., Proc. Nat'l, Acad. Sci. USA88, 8154 (1991)),
i) Aortic aneurysm disease (Vine, et al., Clin. Sci. 81,233 (199
1)), j) Birth control (Woessner et al., Steroids 54,491)
(1989)), k) dystrophic epidermolysis bullosa (kronbergr
, J. Invest. Dermatol. 79,208 (1982)) and l) traumatic joint damage following loss of degenerated cartilage, m) inflammatory response mediated by MMP activity, symptoms leading to osteopenia, n) temporal Mandibular joint disease, o) Demyelination of the nervous system (Chantry et al., J. Neurochem. 50,688 (1988)).

The need for new treatments is especially important in the case of joint inflammation. The primary damaging effect of osteoarthritis (OA), rheumatoid arthritis (RA) and septic arthritis is the progressive loss of articular cartilage and its normal joint function. Although nonsteroidal anti-inflammatory drugs (NSAIDs) have been given to control pain and swelling, there are no over-the-counter drugs that can prevent or delay this articular cartilage loss. The end result of these diseases is a total loss of joint function that can only be treated by joint replacement surgery. MMP inhibitors are expected to stop or slow the progression of cartilage loss and obviate or delay surgical procedures.

Proteases are important factors in several stages of metastatic cancer progression. In this process, the proteolytic disintegration of structural proteins in the basement membrane allows for tumor expansion in the primary site, escape from this site and regression and invasion at a distant second site. Tumor-induced angiogenesis is also required for tumor growth and depends on proteolytic tissue remodeling. Transfection experiments with different types of proteases show that matrix metalloproteases play a predominant role in these processes in specific gelatinases A and B (MMP-2 and MMP-9, respectively). Is shown. For an overview of this area, see Mullin
Biochem.Biophys.Acta.695,177 (1983); Eur.Respir.J.7,2062 (1994) such as Ray; Crit.Rev.Oral such as Birkedal-Hansen. .Biol.M
ed. 4,197 (1993).

Furthermore, the original matrix metalloproteinase inhibitor
Blocking extracellular matrix breakdown by TIMP-2 (protein) arrests cancer growth (DeClerck, et al., Cancer Res. 5).
2,701 (1992)), TIMP-2 blocks tumor-induced angiogenesis in an experimental system (Moses et al., Science 248, 1407 (19).
90)) is shown. See Ann.NYAcad.Sci.732,22 (1994) by DeClerck et al. For a summary. Furthermore, the synthetic matrix metalloprotease inhibitor batimastart inhibits human rectal tumor growth and spread in fresh specimens in nude mice when it is applied intraperitoneally (Wang et al., Cancer Rev. 54. , 4726 (1994)),
And prolong the survival of mice bearing human ovarian cancer xenografts (Davies, et al., Cancer Res. 53, 2087 (199
3)). The use of this and related compounds is
n) etc. in WO-9321942A2 (931111).

Use of metalloproteinase inhibitors for delaying metastatic cancer, promoting tumor regression, inhibiting cancer cell growth, delaying or preventing cartilage loss associated with osteoarthritis, or for treating other diseases mentioned above. There are many patents and patent applications claiming that (eg Levy et al. WO-9519965A1;
Beckett et al WO-9519956A1; Beckett et al WO-9519957A1;
Beckett et al. WO-9519961A1; Brown, et al., WO-9321942; Cri.
mmin et al. WO-9421625A1; Dickens et al. U.S. Patent No. 459936
1; Hughes et al. U.S. Patent No. 5190937; Broadhurst et al. EP 574
758A1; EP276436 by Broadhurst et al .; and EP520573 by Myers et al.
A1). The advantageous compounds of these patents have a peptide backbone with a zinc complexing group (hydroxamic acid, thiol, carboxylic acid or phosphinic acid) at one end and various side chains, both of which are natural amino acids. Found in the same manner and likewise has a plurality of novel functional groups. Such small peptides are often poorly absorbed and exhibit poor oral bioavailability. They have a short half-life because they are subject to a rapid proteolytic mechanism. As an example, only the compounds described in WO-9321942A2 by Batimastart or Brown et al. Can be given intraperitoneally.

Others describe a series of biphenyl-containing carboxylic acids and describe membrane-bound zinc metalloproteases with compounds such as those shown below that inhibit neutral endopeptidase (NEP24.11) (Stanton et al. Bioorg Ned.Chem.L
ett.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)).

The N-carboxyalkyl derivative containing biphenylethylglycine represented by the following formula is stromelysin-
1 (MMP-3), an inhibitor of 72kDA gelatinase (MMP-2) and collagenase (WO-952968 of Durette et al.
9).

Effective MMPs with improved bioavailability and biostability to peptide-base compounds in the known literature, which can be optimized for use against specific target MMPs
It was desired to have an inhibitor. Such compounds are the object of the present invention.

Development of effective MMP inhibitors provides a novel treatment for diseases mediated by the presence or excess of MMP-activity including osteoarthritis, rheumatoid arthritis, septic arthritis, tumor metastasis, corneal ulcer and proteinuria. Should be. Several inhibitors of MMPs have been described in the literature, including thiols (Beszant. Et al., J. Med. Chem. 36, 4030 (1993)), hydroxamic acid ((Wahl et al., Bioorg. , Med.Chem.Lett.5,349
(1995); Conway et al., J. Exp. Med. 182,449 (1995); Porte
R et al., Bioorg, Med. Lett. 4, 2741 (1994); Tomczuk et al., Bio
org.Med.Chem.Lett.5,343 (1994); Castelhano, et al., Bio
org.Med.Chem.Lett.5,1415 (1995)), phosphorus-based acids (Bird, et al., J.Med.Chem.37,158 (1994); Morphy et al., Bio.
org.Med.Lett.4,2747 (1994); Kortylewicz et al., J.Med.C.
hem.33,263 (1990)) and carboxylic acid (Chapman et al., JM.
ed.Chem.36,4293 (1993); Brown et al., J. Ned.Chem.37,674.
(1994); Morphy et al., Bioorg. Med. Chem. Lett. 4, 2747 (19.
94); Stack et al., Arch. Biochem. Biophys. 287,240 (199
1); Ye et al., J. Med. Chem. 37, 206 (1994); Grobelny et al., Bi
ochemistry 24,6145 (1985); Mookhtiar et al., Biochemist
ry 27,4299 (1988). However, these inhibitors generally have a peptide backbone and therefore usually exhibit low oral bioactivity due to low absorbency and short half-life due to rapid proteolysis. Therefore, there remains a need for improved MMP inhibitors.

SUMMARY OF THE INVENTION The present invention provides compounds having matrix metalloprotease inhibitory activity. These compounds are useful for inhibiting matrix metalloproteases and therefore for combating conditions involving MMPs. Accordingly, the invention also provides pharmaceutical compositions and methods for the treatment of such conditions.

The compound described in mammals comprises a compound of the present invention in an amount that inhibits matrix metalloprotease, (a) osteoarthritis, rheumatoid arthritis, septic arthritis,
Of periodontal disease, corneal ulcer, proteinuria, aortic aneurysm disorder, vegetative epidermolysis bullosa, symptoms causing inflammatory response mediated by MMP activity, osteopenia, temporomandibular joint disease, demyelination of nervous system To reduce the effects, (b) delay subsequent traumatic joint damage of tumor metastasis or degenerated cartilage loss, (c) reduce coronary thrombosis from atherosclerotic plaque rupture, or (d) enable birth control The present invention relates to a method for treating a mammal, which comprises applying a sufficient amount to the mammal.

The compounds of the present invention are also useful scientific tools for studying the function and mechanism of action of matrix metalloproteases in vivo or in vitro. Those MMP-
On the basis of their inhibitory activity, the compounds according to the invention can also be used to modulate the action of MMPs, whereby the investigators are able to reduce the effect of the reduced MMP activity in the experimental biological system under study. It is possible to observe.

The present invention relates to compounds having metalloprotease inhibitory activity and having the general formula: ADGE (L) where A is alkyl; allyl-, benzyloxy- or 3-propynylalkyl. Group, as well as structure: The expressed, wherein, Z is _{(CH 2) e -C 6 H} 4 - is (CH _{2) f} or _{(CH 2) g, e =} 0~8, f = 0~5, g = 0~1
4, r is 0 to 6, R ¹⁵ is —H, —Cl, —OMe or Where n is 0-4 and R ¹⁷ is -C ₂ H ₅ , -allyl or -benzyl].

D in the general formula (L) is Represents

In the general formula (L), E represents a chain of n'carbon atoms having m substituents R ⁶ , wherein the R ⁶ groups are independent substituents or constitute a spiro or non-spiro ring. is doing. The ring may be formed in two ways: a) together with a chain atom to which two groups R ⁶ are bound and to which two R ⁶ groups are bound, and any intervening chain atoms. A 3 to 7 membered ring, or b) one group R ⁶ with one group thereon
R ⁶ is attached to the remaining chain, together with the chain atom to which R ⁶ is attached and any intervening chain atom
It forms a seven-member ring. The number n 'of carbon atoms of this in the chain is 2 or 3, the number m of R ⁶ substituents is an integer of 1-3. The total number of carbons in the R ⁶ group is at least 2.

Each group R ⁶ is alkyl, alkenyl, alkynyl, aryl, heteroaryl, non-aromatic rings and combinations thereof optionally substituted by one or more of the following heteroatoms.

The G in the general formula _{(L), -CO 2 H,} -PO 3 H 2, -M, Where M is --CO ₂ H, --CON (R ¹¹ ) ₂ or --CO
₂ R ¹² wherein R ¹¹ is H or alkyl of 1 to 4 carbon atoms, R ¹² is alkyl of 1 to 4 carbon atoms and R ¹³ is
Represents any side chain of 19 acyclic naturally occurring amino acids.

Certain embodiments include compounds having matrix metalloproteinase inhibitory activity and having the general formula: Wherein Z is _{(CH 2) e -C 6 H} 4 - (CH 2) f or (CH _{2) g}
And e = 0 to 8, f = 0 to 5, g = 0 to 14, y is 0, 2 or 3, and R ¹⁵ is H, Cl, MeO or Where n is 0-4 and R ¹⁷ is —C ₂ H.
₅ , allyl or benzyl, and R ¹⁶ is one of the following: Where t is 0-2, x is 0-4, and R ⁴ is one of the following: halide, alkyl of 1-6 carbon atoms, OR, NR ₂ , NO ₂ (R = H or alkyl of 1 to 6 carbons)].

The foregoing merely summarizes particular aspects of the invention and is not intended to limit the invention in any way. The patents or other references cited in this specification are hereby incorporated by reference in their entirety.

Advantageous Embodiments More particularly, the compounds of the invention are substances having a matrix metalloprotease inhibitory activity and the following general formula: A-D-E-G (L) where A is a carbon number of 9-14. , Alkyloxy of 9 to 18 carbons or allyloxy-, benzyloxy or propynyl-alkyl of 9 to 18 carbons].

A has the structure: (Wherein Z is _{(CH 2) e -C 6 H} 4 - (CH 2) f or (CH _{2) g}
And e = 0 to 8, f = 0 to 5, g = 0 to 14, and r is 0.
Is to 6, R ¹⁵ is -H, -Cl, -MeO or Where n is 0-4 and R ¹⁷ is —C ₂ H.
₅ , which is allyl or benzyl).

D in the general formula (L) is a group: Represents

Throughout this specification, open bonds in the depicted structures indicate the point at which this structure is attached to other groups. For example Where R ⁵⁰ is The structure is Is.

In the general formula (L), E is m in relation to the R ⁶ group or R ⁶ unit.
Represents a chain of carbon atoms n having R ⁶ substituents. The R ⁶ group is an independent substituent or constitutes a spiro or non-spiro ring. Ring may be formed in two ways: a) two groups R ⁶ are combined, it 3-7 membered together with the two chain atoms R ⁶ are attached to and any intermediary chain atoms whether or b) one group R ⁶ constitutes a ring, united with the chain there are one radical R ⁶ thereon, and it in linkage atom and any intermediary chain R ⁶ groups are attached 3-7 together with the atom
Form a ring of members. The number n of carbon atoms in this chain is 2
Or 3, and the number m of R ⁶ substituents is an integer of 1 to 3.
The total number of carbon atoms in the radical R ⁶ is at least 2.

Each group R ⁶ is independently selected from the group consisting of the substituents listed under 1-14) below: 1) alkyl of 1-10 carbons 2) aryl of 6-10 carbons 3) carbon 4-9 and at least one N, O or S
Heteroaryl having heteroatoms 4) the aryl portion of which has carbon atoms 6-10, the alkyl portion has aryl carbon atoms 1-8, and the heteroaryl portion of which has carbon atoms 4-9, and at least one A heteroaryl having an N, O or S heteroatom, the alkyl moiety having 1-8 carbons; an alkyl; 6) an alkenyl having 2-10 carbons; 7) the aryl moiety having 6-10 carbons, Aryl-alkenyl where the alkenyl moiety has 2-5 carbons; 8) the heteroaryl moiety has 4-9 carbons, has at least one N, O or S heteroatom, and the alkenyl moiety has 2-5 carbons. 9) an alkynyl having 2-10 carbons; 10) an aryl- having an aryl moiety having 6-10 carbons and an alkynyl moiety having 2-5 carbons; 11) Heteroaryl-alkynyl, the heteroaryl moiety of which has carbons 4-9 and at least one N, O or S heteroatom, the alkynyl moiety of which has carbons 2-5; (CH ₂ ) _t R ⁷ (where t is 0 or an integer from 1 to 5 and R ⁷ is selected from the following group: And the aryl portion of the aryl-containing R ⁷ group is carbon 4
9 and corresponding heteroaryl groups having at least one N, O or S heteroatom). In such R ⁷ group, Y represents O or S, u is 0, 1 or 2, provided that R ⁷ is Where the A unit is phenyl, the B unit is phenylene, m is 1, n is 2, and t is 0.
And x is 1 or 2.

R ¹ represents H or an alkyl group having 1 to 3 carbon atoms. R ² is H; alkyl having 1 to 6 carbons; aryl having 6 to 10 carbons;
9, a heteroaryl having at least one N, O or S heteroatom; an aryl moiety thereof having 6 to 10 carbon atoms and an aryl moiety having 1 to 4 carbon atoms; or a heteroaryl portion thereof having 4 carbon atoms. Have ~ 9,
Represents a heteroaryl-alkyl having at least one N, O or S heteroatom and the alkyl moiety having 1-4 carbons.

R ³ is alkyl having 1 to 4 carbons; aryl having 6 to 10 carbons;
A heteroaryl having 4 to 9 carbon atoms and at least one N,
A heteroaryl having an O or S heteroatom; the aryl part of which has 6 to 10 carbons and the alkyl part of which has 1 to 1 carbon
4 represents an arylalkyl having 4; or a heteroaryl part thereof having 4 to 9 carbons, having at least one N, O or S heteroatom, wherein the alkyl part has 1 to 4 carbons. .

_{13) - (CH 2) v} P'R 8 ( here, v is an integer from 1 to 4, P 'is -S -, - S (O) -, - SO 2 - or -O-
R ⁸ is alkyl having 1 to 12 carbons; aryl having 6 to 10 carbons; heteroaryl having 4 to 9 carbons and at least one N, O or S heteroatom; and the aryl portion thereof having 6 to 12 carbons. An arylalkyl having an alkyl moiety having 1 to 4 carbons; the aryl moiety having 6 to 12 carbons, having at least one N, O or S heteroatom,
The alkyl moiety is selected from heteroarylalkyl having 1 to 4 carbons); -C (O) R ⁹ (wherein R ⁹
Is an alkyl having 2 to 6 carbons, an aryl having 6 to 10 carbons, a heteroaryl having 4 to 9 carbons and at least one N, O or S heteroatom;
~ 10 or heteroaryl is a heteroaryl having 4-9 carbons and at least one N, O or S heteroatom, wherein the alkyl moiety represents arylalkyl having 1-4 carbons), when R ⁸ is -C (O) R ^9, Z is a -S- or -O-; Z is -O-
R ⁸ may be — (C _q H _2q O) _r R ⁵ , where the A unit is phenyl, the B unit is phenylene, m is 1 and n is 2 And when v is 0, x is 1 or 2), 14)-(CH ₂ ) _w SiR ¹⁰ ₃ (where w is an integer from 1 to 3 and R ¹⁰ is carbon 1 ~ 2 alkyl).

In addition, any R ⁶ group aryl or heteroaryl may have up to _two substituents selected from the following groups: — (CH ₂ ) _{y ′} C (R ¹¹ ) (R ¹² ). _{OH, - (CH 2) y} '
_{^{OR 11, - (CH 2)}} y 'SR 11, - (CH 2) y' S (O)
_{^{R 11, - (CH 2)}} y 'S (O) 2 R 11, - (CH 2) y' SO 2 N
(R) ₂ ,-(CH ₂ ) _y'N (R) ₂ ,-(CH ₂ ) _y'N
(R) COR, -OC (R 11) 2 O- ( wherein both oxygen atoms are connected to the aryl _{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 to 4 and R ¹¹ is H
Or alkyl having 1 to 4 carbons; R ¹² represents alkyl having 1 to 4 carbons.

The G in the general formula _{(L), -CO 2 H,} -PO 3 H 2, -M, (Wherein M represents —CO ₂ H, —CON (R ¹¹ ) ₂ or —CO ₂ R ¹² , and R ¹¹ represents any of the side chains of naturally occurring 19 acyclic amino acids). Represent

Also included within the scope of the invention are pharmaceutically acceptable salts of the compounds within formula (L).

The term “alkyl” as used herein means straight chain, branched chain, cyclic and polycyclic materials. The term "haloalkyl", partially or fully halogenated alkyl groups such as - (CH _{2) 2} Cl, means a -CF ₃ and -C ₆ F _13.

In one embodiment, the invention relates to compounds of general formula (L), wherein n in the E unit is 2 and m is 1. Therefore, these compounds have two carbon atoms between the D and G units, and one on each of these two carbon chains.
Has 4 substituents.

In another aspect, the invention provides a compound of general formula (L) wherein the number m of substituents on the E unit is 2 or 3,
When m is 2, both groups R ⁶ are independent substituents or together form a spiro ring or one group R ⁶ is an independent substituent and the other is When forming a spiro ring, or when m is 3, two groups R ⁶ are independent substituents, and one group R ⁶ forms a ring or two groups R ⁶ form a ring. And one group R ⁶ is an independent substituent or three groups R ⁶ are independent substituents. Thus, this subset is such that the E units in the formula are di- or tri-substituted, and when di-substituted 1 or 2 R ⁶
Any ring formed by a group is a spiro ring, and when tri-substituted, the R ⁶ group contains a compound capable of forming a spiro or non-spiro ring.

In another embodiment, the invention relates to compounds of the general formula (L) in which the number m of substituents on the E unit is 1 or 2 and when m 1 the group R ⁶ is non- A spiro ring; when m is 2, two groups R ⁶ together form a non-spiro ring, or one group R ⁶ is an independent substituent and the other is non- Constituting a spiro ring). This assembly unit thus comprises compounds in which the E unit in the formula has 1 or 2 substituents R ⁶ , at least one of these substituents being contained in the non-spiro ring.

In particular, representative compounds of general formula (L) in which one or more substituents R ^{6 in the} formula are involved in the formation of a non-spiro ring have an E unit of the structure: [Wherein 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 to 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 radicals R ⁶ is 0, 1 or 2; U represents O, S or NR ¹ and z is 1 or 2;
Each group R ¹⁴ is independently an alkyl having 1 to 9 carbons, an arylalkyl having an alkyl moiety having 1 to 7 carbons and an aryl moiety having 6 to 10 carbons, an alkenyl having 2 to 9 carbons, An aryl-substituted alkenyl group in which the alkenyl moiety has 2-4 carbons and the aryl moiety has 6-10 carbons, an alkenyl of 2-9 carbons; the alkynyl moiety has 2-4 carbons and the aryl moiety has 6-carbons Aryl-substituted alkynyl having 10; aryl having 6 to 10 carbons; -CO
R ² ; -CO ₂ R ³ ; -CON (R ² ) ₂ ;-( CH ₂ ) _t R ⁷ (where t is 0 or an integer of 1 to 4); and-(CH ₂ ) _v Z ′ R
⁸ (where v is 0 or an integer of 1 to 3 and Z ′ is —S
-Or-O-), R ¹ ,
^{R 2, R 3, R 6} , R 7 and R ⁸ have the above meanings.

Preferred compounds of general formula (L) in which one or more of the substituents R ^{6 in} the formula are involved in the formation of the non-spiro ring are the compounds of the structure E
Have units: (In the formula, a, b, c, d, (c + d), e, g, i,
k, the total number of radicals R ⁶ , U and R ¹⁴ are as defined above).

Other compounds of general formula (L) have R ⁶ units of the structure: (In the formula, n ″ is 0 to 1).

Most preferred compounds of general formula (L) include those of the following general formula: [In the formula, y is 0 (that is, there is no ring structure), 2 (cyclobutyl) or 3 (cyclopentyl), r is 0 to 6, and Z is (CH ₂ ) ₇ or (CH ₂ ) _e- C. ₆ H ₄ — (CH ₂ ) _f , where e is 0 to 1, f is 0 to 5, R
¹⁵ is -H, -Cl, -OMe or Where n is 0-4 and R ¹⁷ is —C ₂ H ₅ , —
Allyl, -benzyl, R ¹⁶ is (In the formula, x is 0 to 4, t is 0 to 2, and R ⁴ is one of the following: halide, alkyl having 1 to 6 carbons, OR,
NR ₂ , NO ₂ (R = H or alkyl having 1 to 6 carbons)
Represents].

It will be apparent to those skilled in the art that many of the compounds of this invention exist in diastereomeric forms between enantiomers and such stereoisomers generally exhibit different activities in biological systems. The present invention includes all possible stereoisomers having inhibitory activity against MMPs, regardless of their stereoisomeric name, as well as mixtures of stereoisomers of which at least one has inhibitory activity.

The most advantageous compounds of the invention are described and named as follows: 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) -benzenehepta Acid, VIII) .gamma.-oxo-.alpha.-(2-phenylethyl) - benzene hexanoic acid and IX) .gamma.-oxo-.alpha.-(2-phenylethyl) - benzene pentanoic acid.

The compounds of the present invention can be prepared using known chemical reactions and methods. Nevertheless, to assist the reader in synthesizing this inhibitor, the following general method of preparation is described.
More detailed methods for specific examples will be found in the experimental part below.

In a general manner, the following general description is used. The group described by P means a protecting group. Those skilled in the art will appreciate that in practice a wide variety of protecting groups can be used to protect potentially reactive functional groups (eg carboxylic acids, alcohols), the particular choice being made to prepare a given target compound. It can be seen that it depends on the reaction conditions required. A description of such protecting groups exists in the following literature: Green and Wut
s, Protective Groups in Organic Synthesis, 2nd Ed., J
ohn Wiley and sons, New York, 1991.

The groups described by X represent releasable groups, which are well known to the person skilled in the art, that several different functional groups such as halides, mesylates, tosylates and triflates can act as releasable groups. The choice of a particular leaving group typically depends on such factors as the reactivity of the nucleophile, the stability of the compound and the ease of synthesis.

General Method A-Compounds of the invention in which E in the formula is ring-free are advantageously prepared using α-halomethylketones and substituted malonate derivatives. This α-halomethylketone intermediate CIII (X = Cl, Br) can be advantageously prepared from carboxylic acids or methylketones. Treatment of the carboxylic acid CI with oxalyl chloride and a catalytic amount of DMF in a solvent such as trimethylsilyl chloride provides the corresponding acid chloride. Subsequent treatment with excess diazomethane followed by treatment with anhydrous HCl or HBr provides intermediate CIII
Cause Intermediate CIII is methyl ketone CII to N-
It can also be prepared via the corresponding silyl enol ether by treatment with bromsuccinimide (NBS).
Silyl enol ethers are advantageously prepared from methyl ketones by treatment with bases such as trimethylsilyl chloride (TMSCI) and lithium hexamethyldisilazide (LHMDS). General methods for the production of α-halomethyl ketones are well known to those skilled in the art. For additional reference, see Corey et al., Tetrahedrion Lett. 25,495 (1
984) and Reuss, et al., J. Org. Chem. 39, 1785 (1974).

Methods for the production of substituted malonate derivatives (CV) have also been established in the literature. Typically, the unsubstituted malonate derivative is treated with a base such as NaH or KOt-Bu in a polar aprotic solvent and then alkylated with a substituted halide. Similarly, alkylation of a monoalkylated intermediate CV with α-halomethylketone CIII is accomplished by dialkylated intermediate CV
Serve I. It should be appreciated by those skilled in the art that the side chain Y may be the desired side chain in the final target or that further achievement of that part of the molecule in later steps of the synthesis is a straightforward process. is there. When side chain Y is the desired side chain, intermediate CVI can be prepared using well known methods,
Easily deprotected and decarboxylated to target compound C
It can be VIII. The conditions used to deprotect the intermediate CVI depend on the type of protecting group used. Some advantageous protecting groups used to synthesize the compounds of the present invention include methyl, allyl, benzyl and t-butyl. Methods of introducing and removing these groups are well known to those of skill in the art (see references above). The choice of protecting groups used in this synthesis depends on several factors such as functional group compatibility, ease of synthesis of starting materials and availability.

Intermediate side chains Y can be used if the target compound CXI has a group Q which is sensitive to the reaction conditions used in the alkylation step. In this case, CH ₂ CH ₂ OT
Introducing a protected ethanol group such as B can be advantageous as the treatment. Y = Intermediate CV with -CH ₂ CH ₂ OTB can be produced by using a TBSOCH ₂ CH ₂ Br as Y-X in the first alkylation step. TBSOCH ₂ CH ₂ Br can be produced from HOH ₂ CH ₂ Br by methods well known to those skilled in the art. The protecting group can be removed to provide the corresponding alcohol, which is converted to a phenyl ether or to various heteroatom-substituted derivatives used to generate the side chain Q via a Mitsunobu reaction. can do. This Mitsunobu reaction is well known to those skilled in the art (Mitsunobu, Synthesis I (198
1) and Hughes, Organic Reactions 42, 335 (1992)). The alcohol intermediate can also be converted to a releasable group such as tosylate or bromide and replaced with a suitable nucleophile. Some examples of this type of reaction are given in Norman et al., J. Med. Chem. 37,2552 (199
4). After introducing the desired side chain Q to form CX, the malonate group can be deprotected and decarboxylated to provide the target compound CXI. In some cases it is necessary to protect the ketone group of intermediate CIII to avoid undesired side reactions. If necessary, J.Org.Chem.5 by Hwu et al.
Protections such as acetals using protocols such as those described in 0,3946 (1985) are generally advantageous.

General Method B-Compounds of the invention in which two R ^{6 s} in the formula are taken together to form a substituted 5-membered ring E are described in Method B
Is most advantageously manufactured by In this method, Beetley et al., Tetrahedron 37, Supplement., 411 (198
The acid MI (R = H) is prepared using the protocol described in 1). This acid is a coupling agent such as 1- (3
-Dimethylaminopropyl-3-ethylcarbodiimide) hydrochloride and protected as an ester [ie R = benzyl (Bn) or 2- (trimethylsilyl) ethyl (TMSE)] using methods well known to those skilled in the art. Grignard reagent MII [prepared from the corresponding bromide by treatment with magnesium] reacts with MI (R = Bn, TMSE) to give alcohol MIII. MIII is removed via base treatment of the mesylate using conditions well known to those skilled in the art to yield the olefin MIV. Ozonation of MIV (post-treatment with methyl sulfide) yields an aldehyde. MV is also converted to MV by OsO ₄ and subsequent treatment with H ₆ IO ₆ .

Conversion of the key intermediate MV to the target patented compound is accomplished in several ways, depending on the uniqueness of the side chain functional group J.
The hydrogenation following the reaction of MV with Wittig reagent
This results in a product where J is alkyl, aryl or arylalkyl. Selective reduction of the aldehyde MV with a reducing agent such as lithium tris [(3-ethyl-3-pentyl) oxy] aluminium hydride (LITEPA) yields the alcohol MVI. This alcohol can be converted to a phenyl ether or to various heteroatom-substituted derivatives used to generate the side chain R ¹⁶ that undergoes a Mitsunobu reaction. This Mitsunobu reaction is well known to those skilled in the art (Mitsunobu, Synthesis I (1981) and Hughes,
Organic Reactions, 42, 335 (1992)). Alcohol MVI can be converted to a leaving group such as tosylate MVII or bromide under conditions well known to those skilled in the art and then the leaving group can be replaced with a suitable nucleophile.
Some examples of this type of reaction are Norman (Norma
n) et al., Med. Chem. 37, 2552 (1994). Direct acylation of the alcohol MVI yields a compound where J in the formula is O-acyl and reaction of this alcohol with various alkyl halides in the presence of a base yields an alkyl ether. In each case, in the final step, R
And depending on the stability of J, in all cases the acid blocking group R is removed to yield the acid (R = H) using conditions well known to those skilled in the art. Removal of the benzyl group can be achieved by, for example, basic hydrolysis or hydrogenolysis,
Deprotection of (trimethylsilyl) ethyl ester is typically carried out by a brief treatment with tetrabutylammonium fluoride.

Suitable pharmaceutically acceptable salts of the compounds of this invention include addition salts formed with organic or inorganic bases. Salt-forming ions derived from such bases are metal ions,
It can be, for example, aluminium, 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. For example, ammonium salts, arylalkylamines such as dibenzylamine and N, N-dibenzylethylenediamine, lower alkylamines such as methylamine, t-butylamine, procaine, lower alkylpiperidines such as N-ethylpiperidine, cycloalkylamines. For example, salts derived from cyclohexylamine or dicyclohexylamine, 1-adamantylamine, benzathine, or amino acids such as arginine, lysine or the like. Conservatively tolerated salts, such as sodium or potassium salts and amino acid salts, can be advantageously used in medicine as described below.

These and other salts that are not necessarily systematically acceptable are useful in isolating or purifying acceptable products for the purposes described below. For example, a commercially available enantiomerically pure amine in a suitable solvent,
By using, for example, (+)-cinchonine, it is possible to obtain salt crystals of a single enantiomer of a compound of the invention, leaving the opposite enantiomer in solution, in a manner often referred to as "classical resolution". it can. Since the one enantiomer of the compound of the invention obtained is, in physiological effect, usually essentially larger than its enantiomer, this active isomer was found to be purified crystallinely or in the liquid phase. obtain. The salts are prepared by reacting the acid form of a compound of the invention with an equivalent amount of a base that provides the desired basic ion in a medium in which the salt precipitates or an aqueous medium, followed by lyophilization. Manufactured. The free acid form can be obtained from the salt by conventional neutralization methods, eg using potassium bisulfate, hydrochloric acid and the like.

The compound of the present invention is a matrix metalloprotease.
It has been found to suppress MMP-3, MMP-9 and MMP-2 and small amounts of MMP-1 and is therefore useful for treating or preventing related conditions in the background. Since other MMPs not mentioned above share a high degree of homology with those mentioned above, especially at the catalytic site, the compounds of the invention likewise inhibit such other MMPs to a lesser extent. It is speculated that it should change. Altering the substituents on the biaryl portion of the molecule has been shown to affect the relative inhibition of MMP's listed in those of the propanoic acid or butyric acid chains of the claimed compounds as well. Thus, this general group of compounds can be "tuned" by selecting special substituents, thus increasing the inhibition of special MMP (s) associated with special pathological conditions. However, the remaining non-involved MMPs are less affected.

The method of treating a matrix metalloprotease-mediated condition can be practiced in mammals, including humans, who exhibit such conditions.

The inhibitors of the present invention are intended for use in veterinary and human administration. The inhibitors of the present invention, for such purposes, depending on the intended mode of administration and dosage form, in addition to one or several active ingredients in particular, are one or several pharmaceutically acceptable. Used in pharmaceutical compositions containing excipients, diluents, fillers, binders and other excipients.

Administration of this inhibitor can be by any suitable method known to those of skill in the art. Examples of suitable parenteral administration include intravenous, intraarterial, subcutaneous and intramuscular. Intravenous administration can be used to obtain an acute adjustment of peak plasma concentration of the drug. Improved half-life and targeting of the drug to the joint space can be aided by introducing the drug into liposomes. By incorporating the ligand outside the liposomes that bind to the synovial fluid-specific macromolecule, it is possible to improve the selectivity of liposome targeting to the joint space. For example, selective intramuscular, intravenous or subcutaneous depot injection of the drug with or without encapsulation of the drug in degradable microspheres composed of poly (DL-lactide-co-glycolide) was prolonged. It can also be used to obtain sustained drug release. Due to the improved convenience of the dosage form, it is possible to use ip implant reservoirs and septa such as the Percuseal system available from Pharmacia. Improved convenience and patient compliance is achieved with injector pens (eg Novo Pin or Q-pen) or needleless jet injectors (eg Bioject.Mediject).
Or Becton Dickinson). Prolonged zero-dimensional or other precisely coordinated release, e.g. pulsatile release, is achieved by administration of the drug through the cannula into the synovial space, optionally using an implantable pump. You can also Examples include subcutaneous implant osmotic pumps available from ALZA, such as ALZET osmotic pumps.

Nasal administration may be carried out with bioadhesive particle excipients (<200 mg) in combination with a suitable absorption enhancer, such as phospholipids or acylcarnitines.
μm), for example by placing the drug in what consists of cellulose, polyacrylate or polycarbophil. Acquisition systems include Dan Biosys and Scios Nova
Includes those developed by.

In comparison to the properties of the various peptide compounds referred to in the background part of this application, a noteworthy property of the compounds of the invention is the above mentioned oral efficacy of the compounds of the invention. Certain compounds have been tested in various animal models at 90%
It showed oral bioavailability up to ~ 98%. Oral administration is
It can be achieved by formulating the drug in tablets, coated tablets, dragees, hard and soft gelatin capsules, solutions, emulsions or suspensions. Oral administration can also be achieved by formulation of the drug in enteric-coated capsules designed to release the drug in the colon, which has low digestive protease activity. Examples include ALZA and Sc respectively
OROS-CT / Osmet ^TM from herer Drug Delivery Systems
And the PULSINCAP ^™ system. Other systems use azo-crosslinked polymers that are degraded by colon-specific bacterial azoreductases, or pH-sensitive polyacrylate polymers that are activated by raising pH in the colon. The system described above can be used with a wide range of effective absorption enhancers.

Rectal administration can be accomplished by incorporating the drug into a suppository.

The compounds of the present invention can be made into the above formulations by the addition of various therapeutically inert, inorganic or organic excipients known to those of ordinary skill in the art. Examples are lactose, corn starch or derivatives thereof, talc, vegetable oils, waxes, fats,
It includes, but is not limited to, polyols such as polyethylene glycol, water, sucrose, alcohols, glycerin and the like. In addition, various preservatives, emulsifiers,
A dispersant, a flavoring agent, a wetting agent, an antioxidant, a sweetening agent, a coloring agent, a stabilizer, a salt, a buffering agent, etc. may be added, if necessary, to help stabilize the formulation, or one or several kinds of activities. It is added to aid in enhancing the bioavailability of the ingredient or, in the case of oral administration, to obtain an acceptable flavor or scent formulation.

The amount of pharmaceutical composition to be used depends on the patient to be treated and the condition. The required amount can be determined without undue experimentation with protocols known to those skilled in the art. The required amount can also be calculated based on a measure of the amount of target enzyme that must be suppressed to treat the condition.

The matrix metalloprotease inhibitor of the present invention is
Not only are they useful for the treatment of the above mentioned physiological conditions, but also in activities such as purification of metalloproteases, and in testing matrix metalloprotease activity. Such activity tests are carried out in vitro using natural or synthetic enzyme preparations or, for example, in animal models in which abnormally disrupted enzyme concentrations are naturally found (use of genetically abrupt displacement or transgenic animals). ), Or in vivo using an animal model in which the aberrantly disrupted enzyme levels are caused by the administration of external agents or by surgery that disrupts binding stability.

The present invention will be described in detail with reference to the following examples, but the present invention is not limited thereto.

EXAMPLES General Procedures: All reactions were carried out under positive pressure of argon in flame-dried or oven-dried glass vessels and magnetically stirred unless otherwise stated. Sensitive liquids and solutions were transferred via syringe or cannula and introduced into the reaction vessel through a rubber septum. Reaction product solutions were concentrated using a Buchi evaporator unless otherwise stated.

Raw materials: Commercial grade reagents and solvents were used without further purification, but always diethyl ether and tetrahydrofuran were distilled from benzophenone ketyl under argon,
Methylene chloride was removed by distillation from calcium hydride under argon. Many special organic or organometallic starting materials and reagents are available from Aldrich, 1001 West Saint Paul Aven.
Obtained from ue, Milwaukee, WI53233. Solvents are often VMR Sc
Obtained from EM Science, supplied by ientific.

Chromatography:   Analytical Thin Layer Chromatography (TLC), Whatman
Pre-coated glass backed silica gel plates (pre-coa
ted glass−backed silica gel 60AF−254 250 μm plate
s) done above. Spot visualization is one of the following methods
In (a) UV illumination, (B) iodine vapor
Exposure, (c) 10% solution of phosphomolybdic acid in ethanol
Soaking the plate in, then heating, and (d) 0.5%
P-anisalde in ethanol containing sulfuric acid at a concentration
Immersing the rate in a 3% solution of hydrid, then heating.

  Perform column chromatography on a 230-400 mesh EM.
  Science Performed using silica gel.

  28 high performance liquid chromatography (HPLC) for analysis
4.6 × 250 mm Microsorb monitoring at 8 nm 1m on the column
Semi-preparative HPLC monitored at 288 nm, performed at l / min 2
1.4 × 250 mm Microsorb Run at 24 ml / min on the column.
It was.

Measurement operation: Melting point (mp) is measured by Thomas-Hoover melting point device (Thomas
-Hoover melting point apparatus) and was not corrected.

The proton ( ¹ H) nuclear magnetic resonance (NMR) spectrum is
Carbon 13 ( ¹³ C) NMR spectrum was measured using an Electric GN-OMEGA 300 (300 MHz) spectrometer
It measured using the Electric GN-OMEGA 300 (75MHz) spectrometer. Most of the compounds analyzed in the
And its spectrum was in each case in agreement with the proposed structure.

Mass spectrum (MS) data can be obtained from liquid-cesium secondary ion (LCIMS),
Obtained on the Kratos Concept 1-H spectrometer with the latest version of fast atom bombardment (FAB). Most of the compounds analyzed in the experiments below were analyzed by mass spectroscopy, the spectra of which were in each case in agreement with the proposed structure.

General notes:   For the multi-step method, the sequential steps are numbered.

Examples 1-6 Preparation of compounds I-VI First stage. A solution of sodium hydride (4.35 g, 181 mmol) in freshly distilled THF (100 mL) was cooled to 0 ° C. and marketed with diallyl malonate (35.0 g, 190 mmol) over 40 min. Processed via dropping funnel. After stirring for 30 minutes at room temperature, N- (2-bromoethyl) phthalimide (43.9 g, 247 mmol) was added in portions to this solution and the mixture was heated under reflux. After 48 hours, the solution was cooled to 0 ° C., quenched with 2N HCl and concentrated to about 20% of its initial volume. Concentrate the ethyl acetate (300m
It was diluted with l) and subsequently washed with a saturated aqueous solution of K ₂ CO ₃ and NaCl. The organic phase was dried over MgSO _4, filtered, and concentrated under reduced pressure. Purification by flash column chromatography (gradient elution with ethyl acetate-hexane 5-25%) gave diallyl-2-phthalimidoethyl malonate (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, 2
H), 346 (t, J = 7.2Hz, 1H), 2.30 (dd, J = 13.
8, 6.9Hz, 2H). The products of the above reaction are shown below: Second stage. In a 50 ml 1 neck round bottom flask equipped with rubber septum and needle for argon, THF 12 ml, trimethylsilyl chloride (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., at the same time, TH
A solution of 2-dodecanone (1.19 g, 6.46 mmol) in F8.0 ml was added dropwise via cannula over 30 minutes. 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 minutes, diluted with 200 ml pentane and washed 3 times with 50 ml brine each. The organic phase was dried over Na ₂ SO ₄ and concentrated to give a yellow solid 2.5 g. Column chromatography on 100 g of silica gel (gradient elution with ethyl acetate-hexane 3-5%) gave 0.680 g (40%) of bromomethyl ketone as a white solid. TLC (ethyl acetate-hexane 5%) _Rf = 0.4.

  The products of the above reaction are shown below.

Third stage. A 25 ml 1 neck round bottom flask equipped with a rubber septum and a needle inlet for argon was charged with 3 ml THF and the first stage product (314 mg, 0.978 mmol). The resulting mixture was cooled to 0 ° C. and sodium t-butoxide (8
8.0 mg, purity 97%, 0.888 mmol) was added. After 30 minutes, a solution of the second stage product (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. CH the reaction mixture
_It was diluted with 100 ml of ₂ Cl _{2 and} washed 3 times with 30 ml of saline each. The organic phase was dried over MgSO _4, and concentrated. 40 g of silica gel
0.300 g of the desired product by column chromatography on (gradient elution with ethyl acetate-hexane 10-30%).
(63%) was obtained as a white solid. TLC (ethyl acetate-hexane 30%) _Rf = 0.5.

  The products of the above reaction are shown below.

Fourth stage. Production of Example 1. In a 154 ml 1 neck round bottom flask equipped with a rubber septum and needle for argon, dioxane 2 ml, third stage product (300 mg, 0.556 mmol), pyridine (0.12 ml, 0.102 g, 1.44 mmol) and tetrakis. (Triphenylphosphine) palladium (1
0.0 mg, 0.0086 mmol). The resulting mixture was exposed to a weak vacuum for degassing the solution and reintroduced with argon. The reaction mixture was stirred at room temperature for 12 hours, dioxane and pyrrolidine were removed in vacuo and the residue was dioxane.
Redissolved in 2 ml. The resulting mixture was exposed to a weak vacuum for degassing the solution and reintroduced with argon. The reaction mixture was heated at 115 ° C. for 4 hours, 85 ° C. for 12 hours and concentrated. Column chromatography on 10 g of silica gel (ethyl acetate-hexane 30% with 0.5% acetic acid) gave 0.137 g (59%) of the product of Example 1 as a white solid (MP89-90).
° C). The products of the above reaction are shown below: The above procedure for the preparation of Example 1 was used to prepare the following Example (Table 1) with the appropriate bromoketone in the third step.

a) Production of 1-bromo-2-tetradecanone: CCl ₄ 16ml, 1,2-epoxytetradecane (2.0ml, 1.66g, in a 100ml 1 neck round bottom flask equipped with a rubber septum and a needle inlet for argon. 7.82 mmol), polyvinylpyridine (1.00 g) and bromine (0.20 ml, 0.62 g, 3.88 mmol) were charged. The resulting mixture was stirred at room temperature for 30 minutes under irradiation of a table lamp (soft white, 60W). The reaction mixture was diluted with a 1: 1 mixture of hexane: ethyl acetate (150 ml), washed with 50 ml of saturated NaHCO _{3 and} 50 ml of brine. The organic phase was dried over MgSO _4, and concentrated. Column chromatography on 100 g of silica gel (gradient elution with ethyl acetate-hexane 5-10%) afforded 0.440 g (39%) of 1-bromo-2-tetradecanone as a white solid. TLC (ethyl acetate-hexane 5%) R _f =
0.4.

Examples 7-9 Preparation of compounds VII-IX First stage. A solution of 4- (4-methoxyphenyl) -butyric acid (3.04 g, 15.4 mmol) in CH ₂ Cl ₂ (45 ml) was treated with oxalyl chloride (11.6 ml, 2.0 M solution in dichloromethane) and DMF (1 drop). did. The solution was heated at reflux for 2 hours, cooled to 0 ° C. and treated with excess diazomethane (ether solution). After stirring for an additional 30 minutes, excess 4M HCl (solution in 1,4-dioxane) was added, the mixture was warmed to room temperature and stirred overnight. The solution was concentrated under reduced pressure, diluted with ethyl acetate, followed by water, saturated aqueous Na.
Wash with HCO ₃ and saturated aqueous NaCl. The organic phase was dried over MgSO _4, filtered, and concentrated. Purification by MPLC (EtOAc
-Hexane 5-25%), target compound (2.45g, 70%)
Was obtained as a colorless oil. TLC: _Rf 0.45 (silica, ethyl acetate-hexane 15%). The compound obtained is shown below: Second stage. Sodium hydride (6.35 in THF (500 ml)
g, 264 mmol) in diethyl malonate (47.3
5 ml, 312 mmol). After stirring for 2 hours, (2
-Bromoethyl) benzene (32.8 ml, 240 mmol)
Was carefully added to the reaction mixture. Following this addition, the solution was heated gently under reflux for 16 hours, cooled to 0 ° C.,
Quenched with 2N HCl. The resulting solution was concentrated under reduced pressure, diluted with EtOAc and washed with saturated aqueous NaCl. The organic phase was dried over MgSO _4, and concentrated. Vacuum distillation (1.5mmH
g) gave the substituted malonate (44.4 g, 71%) as a colorless oil. TLC: _Rf 0.52 (silica, ethyl acetate-hexane 20%). The compound obtained is shown below: Third stage. A solution of malonate (3.50 g, 13.2 mmol) from the second step in DME (5 ml) was added with NaOEt (0.67 g, 9.9
Mmol) and stirred for 30 minutes. While stirring the solution, another flask containing a solution of the α-chloroketone from the first step (0.95 g, 4.2 mmol) in DME (5 mL) was treated with LiI (0.62 g, 6 mmol), Stir for 15 minutes and cannulate the first solution. After stirring overnight, the reaction mixture was diluted with EtOAc, washed with water and saturated aqueous NaCl. The organic phase was dried over MgSO _4, filtered, and concentrated. Purification by MPLC (EtOAc-hexane 5-20%)
At this time the desired malonate (0.41 g, 21%) was obtained as a colorless oil. TLC: _Rf 0.30 (silica, ethyl acetate-hexane 20%). The compound obtained is shown below: Fourth Stage-Production of Example 7. Third in ethanol (3 ml)
A solution of the diester from step (0.19 g, 042 mmol) was treated with 2N NaOH (0.5 ml) and stirred at room temperature. After stirring for 16 h, the solution was concentrated under reduced pressure, diluted with ethyl acetate, washed with K ₂ CO ₃ water. The aqueous phase was acidified to pH 1 with 2N HCl and extracted with ethyl acetate. The organic phase was dried over MgSO _4, filtered, and concentrated. The resulting diacid was dissolved in 1,4-dioxane (3 ml) and heated to 65 ° C. After stirring for 24 h, the solution was concentrated and _{(MeOH-CH 2 Cl 2 2~4} %) is purified by flash column chromatography, the target compound (72.1mg, 49%) was obtained. MP70
~ 71 ° C. The resulting compound (Example 7) is shown below.

The procedure of Example VII above was used for the preparation of the next example (Table II).

Example 10 Biological assay of compounds of the invention Fluorescence assay of P218 quenching for MMP inhibition:   P218 Quenching fluorescence assay (trace fluorescence profiling assay
(Mikrofluorometric Profiling Assay))
Material and various matrix metallopro
Knight, et al., FEBS Let on Thease (MMPs)
A modification of it first described by t.296, 263, 1992.
Is. This assay was run for each compound of the invention and three species.
Using MMPs MMP-3, MMP-9 and MMP-2,
Analyze in parallel and perform the 96-well microtitration assay as follows:
(96-well microtiter plate) and Hamilton AT
At a workstation (Hamilton AT workstation)
Applied

P218 Fluorogenic substrate: P218 is a 4-acetyl-7-methoxycoumarin (MCA) group at the N-terminal position and 3- [2,4-dinitrophenyl] -L-2,3-diaminopropionyl (DPA) inside. It is a synthetic substrate having a group. This is a variant of the peptide reported by Knight (1992), which was used as a substrate for matrix metalloproteases. Cleavage of the P218 peptide (putative cleavage site is Ala-Leu bond),
The fluorescence of the MCA group can be detected with a fluorimeter, excitation at 328 nm and emission at 393 nm. P218 is generally exclusively
Manufactured by Bayer as BACHEM. P218 has the following structure: H-MCA-Pro-Lys-Pro-Leu-Ala-Leu-DPA-Ala.
-Arg-NH2 (MW1332.2) Recombinant human CHO stromelysin (MMP-3) Recombinant human CHO pro-MMP-3: Human CHO pro-Stromelysin-257 (pro
-MMP-3) and expressed by Housley, et al., J. Biol. Che.
Purified as described by M. 268,4481 (1993).

Activation of pro-MMP-3: Tris 5 mM, pH 7.5, CaCl ₂
1.72 μm (100 μg / mL) of pro-MMP-3 in 5 mM, NaCl 25 mM and Brij-350.005% (MMP-3 activation buffer)
, TPCK (N-tosyl- (L) -phenylalanine chloromethyl ketone) trypsin (1: 100 w / w vs. pro-MMP-
In addition to 3), it was activated by keeping it at 25 ° C. for 30 minutes. Soybean trypsin inhibitor (SBTI;
It was stopped by the addition of 5: 1 w / w to trypsin concentration).
The result of this activation protocol is the formation of active MMP-3 (45 kDa) which still contains the C-terminal part of the enzyme.

Production of human recombinant pro-gelatinase A (MMP-2) Recombinant human pro-MMP-2: human pro-gelatinase A (pro-MMP-2) was prepared by Fridman, et al., J. Biol. Che.
m.267, 15398 (1992) using the vaccinia expression system.

Activation of pro-MMP-2: pro-MMP-2 (252 mg / ml
) Is diluted 1: 5 and Tris 25 mM, pH 7.5, CaCl ₂ 5 m
M, NaCl 1 50 mM and Brij-35 0.005% (MMP-2 activation buffer) to a final concentration of 50 μg / ml and p-aminophenyl meliclic acid acetate (APMA) at 10 mM (3.5 mM) in NaOH 0.05. (mg / ml). The APMA solution was added in a 1/20 reaction volume to a final APMA concentration of 0.5 mM and the enzyme was incubated at 37 ° C for 30 min.
The temperature was kept constant for a minute. Activated MMP-2 (15 ml), MMP-2
It was dialyzed twice against 2 liters of activation buffer (the dialysis membrane was pretreated with a solution of 0.1% BSA in MMP-2 activation buffer for 1 minute, followed by a large amount of H ₂ O). Washed).
Enzymes were concentrated on Centricon concentrators (concentrators also used BSA in MMP-2 activation buffer).
Pre-treatment with a solution consisting of 0.1% for 1 minute, followed by washing with H ₂ O, then MMP-2 activation buffer), rediluting,
Subsequently, reconcentration was repeated twice. The enzyme was diluted with MMP-2 activation buffer to 7.5 ml (0.5 times the initial volume).

Production of Human Recombinant Pro-Gelatinase B (MMP-9) Recombinant Human Pro-MMP-9: Wilheim, et al. J. Biol. Che
U937c D as described by m.264, 17213 (1989).
Human pro-gelatinase B (pro-MM derived from NA
P-9) as a baculovirus protein expression system (baculovirus
Protein expression system) was used to express the full-length form. Hibbs, et al. J. Biol. Chem. 260, 2493
This pro-enzyme was purified using the method previously described by (1984).

Activation of pro-MMP-9: Tris 50 mM, pH 7.4, CaCl ₂ 1
Pro-MMP-2 (20 μg / ml) in 0 mM, NaCl 150 mM and Brij-35 0.005% (MMP-9 activation buffer) together with 0.5 mM p-aminophenyl meliclic acid acetate (APMA) at 37 ° C. It was activated by keeping it at a constant temperature for a period of time. To remove APMA, the enzyme was dialyzed against the same buffer.

Measurement operation:   Hamiltion Microlab AT Plus: MMP-Profiling
The Hamilton Microlab assay (MMP-Profiling Assay)
AT Plus It is done by the autopilot above. This Hamilton is
It is programmed as follows: (1) DMSO 100% in
Stock solution of 2.5mM automatically suppresses 11 potentials
(2) Substrate, then inhibitor
Are distributed in 96-Wellcht Fluor plates;
(3) While mixing the single enzymes to start the reaction
Add to plate. Next plate for each added enzyme
Start the program at the point of substrate addition and dilute suppression
Remix the agents and start the reaction by adding the enzyme
By automatically manufacturing. In this way, all MMP
The determination was performed using the same inhibitor dilution.

Millipore Cytofluor II. Following incubation, plates were placed on a Cytofluor II fluorescence plate reader for 340 n.
Read at excitation at M and emission at 395 nM, gain at 80.

Buffer: Microfluorescence Reaction Buffer (MRB): Dilutions of test compound, enzyme and P218 substrate for microfluorescence assay at pH 6.5, C
aCl ₂ 10 mM, NaCl 150 mM, and Brij-35 0.005% and DM
Prepared in a trace fluorescence reaction buffer consisting of 50 mM 2- (N-morpholino) ethanesulfonic acid (MES) with 1% SO.

Method: MMP microfluorescence profiling assay: This assay has various concentrations of drug, P218 (6 μM) and MMP about 0.5.
Performed at a final substrate concentration of ˜0.8 nM. Hamilton is programmed to serially dilute 11 compounds at a final compound concentration of 10x from 2.5 mM stock solution (DMSO 100%) during the assay. First, the instrument is a marsh (M
arsh) up to 96 dilution tubes, trace fluorescence reaction buffer (MR
Different amounts of B) are shown. Then, the instrument is
20μl of inhibitor (2.5mM) was picked up and mixed with the buffer of the A-row of Marsh rack, resulting in a drug concentration of 50μM
Occurs. The inhibitor is then continuously added at 10, 5, 1, 0.
Dilute to 2, 0.05 and 0.01 μM. Position on the material rack 1
Contains only DMSO for the "enzyme only" wells in the assay, which results in column 1, rows A through H without inhibitor. The instrument is then P218 substrate
Dispense 107 μl (8.2 μM in MRB) into a single 96-well cytofluor microtiter plate. The machine is remixed and 14.5 μl of diluted compound are placed in the corresponding rows in the microtiter plate, rows A through G on the Marsh rack. (Column H represents the "background" column, 39.5 μl MRB is given instead of drug or enzyme). The reaction is initiated by the addition of 25 μl of the appropriate enzyme (at a final enzyme concentration of 5.86 fold) from the SBA-treated reagent reserve to each well, except for row H, the “background” row. (The enzyme reserve is BSA1 in Tris 50 mM, pH 7.5 containing NaCl 150 mM.
% At room temperature for 1 hour, then washed with excess H ₂ O and dried at room temperature).

After adding and mixing the enzyme, cover the plate and incubate at 37 ° C.
Hold at constant temperature for 25 minutes. Additive enzymes are tested in a similar manner by initiating the Hamiltonian program for partitioning P218 substrate into microassay concentration plates, followed by remixing and partitioning of drug into microtiter plates from the same Marsh rack. Then, the second (or third, etc.) MMP to be tested,
Dispense from reagent rack to microtiter plate, mix, then cover and incubate. All additional MMs to test this
Repeat for P.

IC50 measurement by micro-fluorescence assay: Export of data obtained by Cytofluor II (exported “CSV” file, master Excel spreadsheet)
Copy to. Data from several different MMPs (96 well plates per MMP 1) were calculated simultaneously. Suppression%
Is a measurement for each drug concentration by comparing the amount of hydrolysis in the wells containing the compound (fluorescent units that occur over 25 minutes of hydrolysis) to the "enzyme only" wells in column 1. After background subtraction,% inhibition was calculated as follows: ((control value-treated value) / control value) x 100% inhibition was 5, 1, 0.5, 0.1, 0.02, 0.005 and 0.001 of drug. It was measured for an inhibitor concentration of μM. Linear regression analysis of percent inhibition against inhibitor concentration (log), was used to obtain IC _50.

Profiling Assay Data for Compounds of the Invention All IC ₅₀ values are expressed as nM. When showing "I = x%", x represents% inhibition at 5 μM.

Other embodiments of this invention will be apparent to those skilled in the art from consideration of this specification or practice of the invention provided herein.
The specification and examples are considered exemplary only, and the true scope and spirit of the invention is set forth in the following claims.

─────────────────────────────────────────────────── ─── Continued Front Page (51) Int.Cl. ⁷ Identification Code FI A61P 13/02 A61P 13/02 15/00 15/00 19/02 19/02 21/00 21/00 27/02 27/02 29/00 101 29/00 101 35/04 35/04 43/00 111 43/00 111 C07D 209/48 C07D 209/48 Z (72) Inventor Michael Van Zand USA Connecticut Gilford Barker Hill Drive 56 (72) Inventor David Earl Briterley United States Connecticut Branford Stoney Creek Road 240 (56) References Table 10-509146 (JP, A) Table 11-506097 (JP, A) Table 11-510517 (JP) , A) Special table 11-510518 (JP, A) Special table 11-510821 (JP, A) Special table 11-511179 (JP, A) Special table 2000 -502343 (JP, A) Special Table 2001-509783 (JP, A) (58) Fields surveyed (Int.Cl. ⁷ , DB name) CA (STN) CAOLD (STN) REGISTRY (STN)

Claims (6)

(57) [Claims] 1. A general formula: [In the formula, y is 0, 2 or 3, r is 0 to 6,
Z is (CH _{2) 7} or _{(CH 2) e -C 6 H} 4 - represents a (CH _{2) f,} where, e is 0 to 1, f is an 0 to 5: R ¹⁵
Is -H, -Cl, -OMe or Wherein n is 0-4, R ¹⁷ is C ₂ H ₅ , allyl, benzyl and R ¹⁶ is Where t is 0-2, x is 0-4, R ⁴ is one of the following: halide, alkyl of 1-6 carbon atoms, OR, NR ₂ , NO ₂ , (R = H or alkyl having 1 to 6 carbons]. 2. A matrix metalloprotease inhibitor composition containing the compound according to claim 1 and a pharmaceutically acceptable carrier. 3. A medicament for treating a condition caused by matrix metalloprotease, which comprises the compound according to claim 1. 4. A medicine for treating a condition caused by matrix metalloprotease, comprising: (a) osteoarthritis, rheumatoid arthritis, septic arthritis,
Of periodontal disease, corneal ulcer, proteinuria, aortic aneurysm disease, dystrophic epidermolysis bullosa, symptoms leading to inflammatory response mediated by MMP activity, osteopenia, temporomandibular joint disease, demyelination of nervous system Reduce the effects, (b) delay traumatic joint damage following tumor metastasis or degenerative cartilage loss, (c) reduce coronary thrombosis from atherosclerotic plaque rupture, or (d) enable birth control The medicine according to claim 3, which is intended for the purpose. 5. The purpose is to reduce osteoarthritis,
The medicine according to claim 4. 6. The purpose is to delay tumor metastasis,
The medicine according to claim 4.