MXPA01006763A - Use of substituted 4-biarylbutyric and 5-biarylpentanoic acid derivatives as matrix metalloprotease inhibitors for the treatment of respiratory diseases - Google Patents

Abstract

Novel 4-Biarylbutyric and 5-Biarylpentanoic Acid Derivatives, use of substituted 4-Biarylbutyric and 5-Biarylpentanoic Acid Derivatives as Matrix Metalloprotease Inhibitors for the Treatment of Respiratory Diseases, pharmaceutical compositions containing them, and a process for using them. The compounds of the invention have the generalized formula (I) (T)xA-B-D-E-CO2H wherein A is an aryl or heteroaryl rings;B is an aryl or heteroaryl ring or a bond;each T is a substituent group, x is 0, 1, or 2;the group D represents (a), (b), (c), or (d);the group E represents a two or three carbon chain bearing one to three substituent groups which are independent or are involved in ring formation, possible structures being shown in the text and claims;and each of the substituents on E is an independent substituent;and include pharmaceutically acceptable salts thereof.

Description

Use of 4-barylbutyric and substituted 5-biaryl pentanoic acid derivatives as inhibitors of matrix metalloprotases for the treatment of respiratory diseases FIELD OF THE INVENTION This invention relates to the use of enzyme inhibitors, and more particularly, to new and known 4-biarylbutyric acids and 5-biarylpentanoic acid inhibitors of matrix metalloproteases and their derivatives, for the prevention and treatment of respiratory diseases.

BACKGROUND Derivatives of substituted 4-barylbutyric and 5-biarylpentanoic acids are described as inhibitors of matrix metalloproteases in WO 96/15096, WO 97/43237, WO 97/43238, WO 97/43239, WO 97/43240, WO 97. / 43245, WO 97/43247 and WO 98/22436.

Matrix metalloproteases (matrix metalloendoproteinases or MMPs) are a family of zinc endoproteinases that include, but are not limited to ace, interstitial collagenase (MMP-1), stromelysin (proteoglycanase, transin or MMP-3), gelatinase A (72 kDa gelatinase or MMP-2), "neutrophil collagenase (MMP-8), gelatinase B Ref: 130479 (95 kDa gelatinase or MMP-9), macrophage elastase (MMP-12) and human collagenase 3 (MMP-13). These MMPs are secreted by a series of cells including fibroblasts, chondrocytes, granulocytes and macrophages, together with natural inhibitors of proteinases known as TIMP (metalloproteinase tissue inhibitors).

All these MMPs are capable of destroying a series of connective tissue components of articular cartilage or basal membranes and a wide variety of extracellular matrix proteins. Each MMP is secreted in the form of an inactive proenzyme that must be cleaved at a later stage before being able to exert its own proteolytic activity. In addition to the killing effect of the matrix, certain of these MMPs, such as MMP-3, have been applied as activators in vivo of other MMPs, such as MMP-1 and MMP-9 (A. Ho, H. Nagase , Arch. Biochem. Biophys., 267, 211-216 (1988), Y. Ogata, JJ Enghild, H. Nagase, J. Biol. Chem., 267, 3581-3584 (1992)). Thus, a cascade of proteolytic activity can be initiated by an excess of MMP-3. It follows that specific inhibitors of MMP-3 should limit the activity of other MMPs that are not directly inhibited by such inhibitors.

In addition to its ability to degrade extracellular matrix proteins, it has further been shown that MMP-12 hydrolyzes elastin (P.P. Mecham, TJ Broekelmann, CJ Fliszar, SD Shapiro, HG Welgus, Senior MRI, J. Biol. Chem. , 272, 18071-18076 (1997)). This activity is shared by other MMP enzymes, specifically MMP-2 and MMP-9.

It has also been reported that MMP-3, MMP-9 and MMP-12 can cleave and thus inactivate the endogenous inhibitors of other proteinases, such as elastase (PG Winyard, Z. Zhang, K. Chidwick, DR Blake, RW Carrell, G. Murphy, FEBS Letts., 279, 1, 91-94 (1991); TJ Gronski, RL Martin, DK Kobayashi, BC Walsh, MC Holman, M. Huber, HE Van Wart, SD Shapiro, J. Biol. Chem ., 272, 12189-12194 (1997)). The inhibitors of these MMP enzymes could thus influence the activity of other destructive proteinases by modifying the level of their endogenous inhibitors.

The macrophages of MMP-12 knockout mice have decreased ability to degrade extracellular matrix components and penetrate into reconstituted basal membranes, both in vitro and in vivo (JM Shipley, RL Wesselschmidt, DK Kobayashi, TJ Ley, SD Shapiro, PNAS, 93., 3942-3946 (1996)). These results support the hypothesis that MMP-12 is necessary for extracellular proteolysis mediated by macrophages and tissue invasion. In addition, MMP-12 knockout mice do not develop emphysema nor show elevated levels of macrophage in response to tobacco smoke, whereas wild-type mice do (RD Hautamaki, DK Kobayashi, Senior RM, SD Shapiro, Science, 277, 2002-2004 (1997)). Therefore, there is strong evidence supporting the role of MMP-12, secreted by activated alveolar macrophages, in the development of pulmonary emphysema. In patients with emphysema and smoking subjects both MMP-1, -8, -9 and -12 released from alveolar macrophages and neutrophils are implicated in the pathogenesis of COPD.

Through its proteolytic activity, the matrix metalloproteases are involved in a series of respiratory diseases, for example the following: asthma; chronic obstructive pulmonary disease, including chronic bronchitis and emphysema; cystic fibrosis; bronchiectasis, respiratory distress syndrome in adults (ARDS); allergic respiratory disease, including allergic rhinitis; diseases linked to the production of TNF, "including acute pulmonary fibrotic diseases, pulmonary sarcoidosis, silicosis, miner's pneumoconiosis, alveolar damage.

Evidence of the involvement of matrix metalloproteases in various respiratory diseases is provided by the following references: a) COPD, -Finlay et al. Thorax, 1997, 52, chronic bronchitis 502 and emphysema Am. J. Resp. Crit .. Care Med. 1997, 156, 240 - Cateldo et al. Am. J. Resp. Crit. Care Med. 1998, 157, A502 - Sedura et al. Am. J. Resp. Crit. Care Med. 1998, 157, A568 - Shapiro et al. J. Biol. Chem. 1993, 268, 23824 - Kostan et al. Am. J. Resp. Crit. Care Med. 1998, 157, A143 - Riccobono Eur. Resp. J. 1997, 10, 26S b) bronchiectasis - Sepper et al. Chest 1995, 107, 1641 - Sepper et al. Eur. Resp. J. 1997, '10, 278S c) fibrosis - Delacourt et Am. J. Resp. Cystica al. Crit. Care Med. 1995, 152, A 764 - Power et al. Am. J. Resp. Crit. Care Med. 1994, 150, 818 d) asthma - Shute et al. Int. Arch. Allergy Immunol. 1997, 111 • 10 - Ohno et al. Am. J. Resp. Cell. Mol. Biol. 1997, 16, 212 - Mantino et al. Am. J. Resp. Cell. Mol. Biol. 0. 5 • 1997 ,. 17, 583 - Okada et al. Am. J. Resp. Cell. Mol. Biol. 1997, 17, 519 .20 e) SDRA - Delclaux et Am. J. Physiol. to the. 1997, 272, L442.

In addition to chronic lung diseases, it is believed that a number of other conditions are mediated by activity ^ 25 excess or undesired matrix destroying metalloprotease or by an imbalance of the ratio of MMP to TIMP or by the action of TNF release.

MMP inhibitors may also be useful in the inhibition of other mammalian metalloproteases such as the adamalysin family (or ADAM), whose members include the TNFa converting enzyme (TACE) and ADAM-10, which can produce the release of TNF from the cells.

SUMMARY This invention relates to the use for the prevention and treatment of respiratory diseases of new and known compounds with matrix metalloprotease inhibitory activity of generalized formula (I): (T) xA-B-D-E-C02H (i; In the above generalized formula (I), (T) XA represents an aromatic or heteroaromatic 6-membered 6-membered ring containing from 1 to 2 N, O or S atoms, substituted or unsubstituted. T represents one or more substituent groups, the subscript x represents the number of said substituent groups and A represents the aromatic or heteroaromatic ring, designated as ring A or unit A. When N is used together with S or O in ring A, these heteroatoms are separated by at least one carbon atom.

The substituent group (s) T is (independently) selected from the group constituted. by halogen; I rent; haloalkyl; haloalkoxy; alkenyl; alkynyl; - (CH -.) PQ, where p equals 0 or an integer from 1 to 4; -alkenyl-Q, the alkenyl moiety comprising 2 to 4 carbons; and alkynyl-Q, the alkynyl moiety comprising 2 to 7 carbons. In the last three groups Q is selected from the group consisting of aryl, heteroaryl, -CN, -CHO, -N02, -C02R2, -0C0R2, -SOR3, -S02R3, -CON (R4) 2, -SON (R) 2 , -? OR2, -N (R4) 2 / -N (R2) COR2, -N (R2) C02R3, -N (R2) C0N (R) 2, -CHN4, -OR4 and -SR4.

In these formulas, R 2 represents H, alkyl, aryl, heteroaxyl, arylalkyl or heteroarylalkyl. R3 represents alkyl, aryl, heteroaryl, arylalkyl or heteroarylalkyl. R4 represents H; I rent; aril; heteroaryl; Arylalkyl; heteroarylalkyl; alkenyl; alkynyl; alkylenoxy; polyalkylenoxy; alkylenethio or alkyleneamino terminated with H, alkyl or phenyl; haloalkyl; lower alkoxycarbonyl; or acyl When two R4 groups are located on a nitrogen, they can be linked via a bond to form a heterocycle, such as, for example, a morpholine ring, thiomorpholine, pyrrolidine or piperidine.

The unsaturation in a moiety that is attached to Q or that is part of Q is separated from any N, O or S of Q by at least one carbon atom. Ring A may be unsubstituted or may carry up to 2 substituents T. Accordingly, the subscript x is 0, 1 or 2.

In the generalized formula (I), B represents a bond or an aromatic 6-membered ring or a 5- to 6-membered heteroaromatic ring containing from 1 to 2 N, 0 or S atoms, optionally substituted. When B is a ring, it is designated as ring B or unit B. When N is used together with S or 0 in ring B, these heteroatoms are separated by at least one carbon atom. There may be 0 to 2 T substituents in ring B.

In the generalized formula (I), D represents H: c--: 0 > : C --------- NN (RZ) 2: NOR2 < OH wherein R2 is defined as above and each R2 may be the same or different.

In the generalized formula (I), E represents a chain of n carbon atoms bearing m substituents R6, the groups R > independent substituents, or constituting spiro or non-spiro rings. The rings can be formed in two ways: a) two R6 groups are joined together and taken together with the atom (s) of the chain to which the two R6 groups are attached, and with any intermediate atom of the chain, constitute a ring of 3 to 7 members, or b) a group R is attached? to the chain in which this group R ° resides, and taken together with the atom (s) of the chain to which the R6 group is attached, and with any intermediate atom of the chain, constitutes a ring of 3 to 7 members. The number n of carbon atoms in the chain is from 2 to 4, and the number m of substituents R6 is an integer from 1 to 3.

Each R group is independently selected from the group consisting of: * fluorine; * hydroxyl, with the proviso that an individual carbon atom can not carry more than one hydroxyl group; *I rent; * aryl; * heteroaryl; * arylalkyl; * heteroarylalkyl; * alkenyl; * alkenyl substituted with aryl; * alkenyl substituted with heteroaryl; * alkynyl, - * alkynyl substituted with aryl; * alkynyl substituted with heteroaryl; * - (CH), R7, where t equals 0 or an integer from 1 to 5 and R7 is selected from the group consisting of: * N- phthalimidoyl; * N- (1,2-naphthalenedicarboxyimidoyl); * N- (2,3-naphthalenedicarboxyimidoyl), - *? - (1,8-naphthalenedicarboxyimidoyl); *? - indoloil; *? - (2-pyrrolidinonyl); * N-succinimidoyl; *? - maleimidoílo; * 3-hydantoinyl; * 1, 2,4-urazolyl; * amido; * urethane; *urea; * substituted or unsubstituted non-aromatic heterocycles containing and connected by an? atom, comprising one or two?, O, S, SO or S02 additional and containing zero, one or two carbonyls, and optionally bearing a ring of benzene or condensed pyridine; *Not me; * the corresponding heteroaryl radicals in which the aryl portion of an aryl-containing group R7 comprises from 4 to 9 carbons and at least one heteroatom of N, O or S; * - (CH2), ZR8 where v is 0 or an integer from 1 to 4, Z represents: and Rs is selected from the group consisting of: * alkyl; * aryl; * heteroaryl; * to ilalkyl; * heteroarylalkyl; and * -C (0) R9, wherein R9 represents alkyl of at least two carbons, aryl, heteroaryl, arylalkyl or heteroarylalkyl; and with the additional conditions that -when R8 is -C (0) R9, Z is S or O; when Z is O, R8 can be alkyleneoxy or polyalkyleneoxy terminated with H, alkyl or phenyl; Y * alkyl substituted with trialkylsilyl.

In addition, the aryl or heteroaryl portions of any of the groups T or R6 can optionally carry up to two substituents selected from the group consisting of - (CH2KC (R4) (R3) 0H, - (CH2) yOR4, - (CH2) and SR4, - (CH2), S (O) R4, - (CH:) _. S (0) 2R4 - (CH2) and S02N (R4) 2, - (CH2) and N (R4) 2, - (CH2) _ ,? (R4) COR12, -OC (R4) 20- wherein both oxygen atoms are connected to the aryl ring, (CH2) and C0R4, - (CH2), CO? (R4) 2, - (CH2) and C02R4, - ( CH2) and OCOR4, -halogen, -CHO, -CF3, -? 02, -C? And R3, wherein y is from 0 to 4. R3 and R4 are defined as above, in addition, any two R4 that are attached to a nitrogen can be linked to form a heterocycle such as a morpholine ring, thiomorpholine, pyrrolidine or piperidine.

The pharmaceutically acceptable salts of these compounds, as well as the commonly used prodrugs of these compounds, such as the O-acyl derivatives of the compounds of the invention containing hydroxyl groups, are also within the scope of the invention.

In the more related reference compounds of the prior art, the biphenyl portion of the molecule is unsubstituted, and the propanoic or butanoic acid portion is unsubstituted or has a single methyl or phenyl group. The presence of the major phenyl group has been reported to cause the prior art compounds to be inactive as anti-inflammatory analgesic agents. See, for example, R.G. Child, et al., J. Pharm. Sci., 66, 466-476 (1977). In contrast, it has now been discovered that compounds which exhibit potent MMP inhibitory activity contain a substituent of significant size in the propanoic or butanoic portion of the molecule. The biphenyl portions of the best MMP inhibitors also preferably contain a substituent at the 4 'position, although when the butanoic or propanoic portions are optimally substituted, the unsubstituted biphenyl compounds of the invention have sufficient activity to be considered realistic drug candidates.

The compounds of the present invention exhibit good activity for MMP-2, MMP-3, MMP-8, MMP-9, MMP-12 and MMP-13. and a good selectivity for these MMPs against other MMPs such as MMP-1 and MMP-7.

In addition to the compounds described above, the invention is. it also relates to pharmaceutical compositions with matrix metalloprotease inhibitory activity, said compositions comprising a compound of the invention as described above, and in greater detail in the detailed description below, and a pharmaceutically acceptable carrier.

As a result of the above-mentioned selectivity, the compounds of the present invention are especially suitable for the treatment of respiratory diseases.

Therefore, the invention relates. also to a method of treating a mammal such as a human, a farm animal or a domestic animal, to achieve an effect, the effect being the prevention or treatment of asthma; chronic obstructive pulmonary disease including chronic bronchitis and emphysema; cystic fibrosis; bronchiectasis, respiratory distress syndrome in adults (ARDS); allergic respiratory disease, including allergic rhinitis; diseases linked to the production of TNF (?), including acute lung fibrotic diseases, pulmonary sarcoidosis, silicosis, miner pneumoconiosis, alveolar damage, the method comprising administering an amount of a compound of the invention as described above, and in more detail in the detailed description below, which is effective to inhibit the activity of at least one matrix metalloprotease, resulting in the achievement of the desired effect.

DETAILED DESCRIPTION The most particularly preferred for use for the prevention and treatment of respiratory diseases are compounds with matrix metalloprotease inhibitory activity of generalized formula: (T) xA-B-D-E-C02H (I) wherein (T)? A represents a substituted or unsubstituted aromatic or heteroaromatic moiety selected from the group consisting of: wherein R 1 represents H or alkyl of 1 to 3 carbons In these structures, the aromatic ring is designated as ring A or unit A, and each T represents a substituent group, designated as a group T or unit T. The substituent groups T are independently selected from the group consisting of: halogens -F, -Cl, -Br and -I; alkyl of 1 to 10 carbons; haloalkyl of 1 to 10 carbons; haloalkoxy of 1 to 10 carbons; alkenyl of 2 to 10 carbons; alkynyl of 2 to 10 carbons; - (CH2) pQ, where p is 0 or an integer from 1 to 4; -alkenyl-Q, in which the alkenyl residue comprises from 2 to 4 carbons; and -alkynyl-Q, wherein the alkenyl moiety comprises from 2 to 7 carbons. In each of the last three groups Q is selected from the group consisting of aryl of 6 to 10 carbons; heteroaryl comprising from 4 to 9 carbons and at least one heteroatom of N, O or S; -CN; -CHO; -NO ,; "-C02R2; -OCOR2; -SOR3; -S02R3; -CON (R4) 2; -S02N (R) 2; -C (O) R2; -N (R4) 2; -N (R2) COR2; -N (R) C02R3; -N (R2) CON (R4) 2; -CHN,; OR4 and -SR4 The groups R2, R3 and R4 are defined as above.

R- represents H; alkyl of 1 to 6 carbons; aryl of 6 to 10 carbons, heteroaryl comprising from 4 to 9 carbons and at least one heteroatom of α, O or S; arylalkyl in which the aryl portion contains from 6 to 10 carbons and the alkyl portion contains from 1 to 4 carbons; or heteroarylalkyl in which the heteroaryl portion comprises from 4 to 9 carbons and at least one heteroatom of α, 0 or S and the alkyl portion contains from 1 to 4 carbons.

R3 represents alkyl of 1 to 4 carbons; aryl of 6 to 10 carbons; heteroaryl comprising from 4 to 9 carbons and at least one heteroatom of N, O or S; arylalkyl in which the aryl portion contains from 6 to 10 carbons and the alkyl portion contains from 1 to 4 carbons; or heteroaryl alkyl wherein the heteroaryl portion comprises from 4 to 9 carbons and at least one heteroatom N, O or S and the alkyl portion contains from 1 to 4 carbons.

R4 represents H, alkyl of 1 to 12 carbons; aryl of 6 to 10 carbons; heteroaryl comprising from 4 to 9 carbons and at least one heteroatom of N, O or S; arylalkyl in which the aryl portion contains from 6 to 10 carbons and the alkyl portion contains from 1 to 4 carbons; heteroarylalkyl in which the heteroaryl portion comprises from 4 to 9 carbons and at least one heteroatom of N, S or O and the alkyl portion contains from 1 to 4 carbons; alkenyl of 2 to 12 carbons; alkynyl of 2 to 12 carbons; - (CqH2qO) rR5 where q is from 1 to 3, r is from 1 to 3 and R5 is H, with the proviso that q is greater than 1, or R5 is alkyl of 1 to 4 carbons; or phenyl; alkyl-terminated with H, alkyl of 1 to 4 carbons, or phenyl; alkylene-terminated with H, alkyl of 1 to 4 carbons or phenyl; - (CH2) SX where s is from 1 to 3 and X is halogen; -C (0) OR2; Ó -C (0) R2.

When two R4 groups are located on a nitrogen, they can be linked via a bond to form a heterocycle, such as, for example, a morpholine ring, thiomorpholine, pyrrolidine or piperidine.

Any unsaturation in a residue that is attached to Q or that is part of Q is separated from any N, 0 or S by at least one carbon atom, and the number of substituents, designated x, is 0, 1 or 2.

In the generalized formula (I), B represents an optionally substituted aromatic or heteroaromatic ring or ring selected from the group consisting of: wherein R1 is defined as above. These rings are designated as ring B or unit B. There may be from 0 to 2 substituents T on ring B, where T is defined as above.

In the generalized formula (I), D represents the residues Ai .0 = 0 '^ c' ^ .C = NN (R-), O C = N? R ~ ^. OH - ^ wherein R2 is defined as above and each R2 may be the same or different.

In the generalized formula (I), E represents a chain of n carbon atoms carrying m substituents R6, designated as Rn groups or R6 units. The R6 groups are independent substituents, or constitute spiro or non-spiro rings. The rings can be formed in two ways: a) two Rf 'groups are joined and taken together with the atom (s) of the chain to which the two R6 groups are attached, and with any intermediate atom of the chain, they constitute a ring of 3 to 7 members, or b) a group R6 is attached to the chain in which this group R6 resides, and taken together with the atom (s) of the chain to which the group is attached R6, and with any intermediate atom of the chain, constitutes a ring of 3 to 7 members. The number n of carbon atoms in the chain is from 2 to 3, and the number m of substituents Rú is an integer from 1 to 3.

Each Rfi group is independently selected from the group consisting of the substituents listed below as points 1) -16). 1) fluorine: 2) hydroxyl, with the proviso that an individual carbon atom can not carry more than one hydroxyl group; 3) alkyl of 1 to 10 carbons; 4) aryl of 6 to 10 carbons; 5) heteroaryl comprising from 4 to 9 carbons and at least one hetero-iron of N, O or S; 6) arylalkyl, in which the aryl portion contains from 6 to 10 carbons and the alkyl portion contains from 1 to 8 carbons; 7) heteroarylalkyl, wherein the heteroaryl portion comprises from 4 to 9 carbons and at least one heteroatom of N, O or S, and the alkyl portion contains from 1 to 8 carbons; 8) alkenyl of 2 to 10 carbons; 9) arylalkenyl, wherein the aryl portion contains from 6 to 10 carbons and the alkenyl portion contains from 2 to 5 carbons; 10) heteroarylalkenyl, wherein the heteroaryl portion contains from 4 to 9 carbons and at least one heteroatom of N, O or S and the alkenyl portion contains 2 to 5 carbons; 11) alkynyl of 2 to 10 carbons; 12) arylalkynyl, wherein the aryl portion contains from 6 to 10 carbons and the alkynyl portion contains from 2 to 5 carbons; heteroarylalkyl, wherein the heteroaryl portion comprises from 4 to 9 carbons and at least one heteroatom of N, O or S, and the alkynyl portion contains from 2 to 5 carbons; ) where t is 0 or an integer from 1 to 5 and R7 is selected from the group consisting of: .25 as well as the corresponding heteroaryl moieties, wherein the aryl portion of an aryl-containing group R7 comprises from 4 to 9 carbons and at least one heteroatom of N, 0 or S. In said groups R7, Y represents O or S; R1, R2 and R3 are as defined above, and u is 0, 1 or 2; . ) - (CH2) VZR8, where v is 0 or an integer from 1 to 4; Z represents -S-, -S (O) -, -S02-, -O-, carbonyl or -CH (OH) -; and R8 is selected from the group consisting of: alkyl of 1 to 12 carbons; aryl of 6 to 10 carbons; heteroaryl comprising from 4 to 9 carbons and at least one heteroatom of N, 0 or S; arylalkyl, wherein the aryl portion contains from 6 to 12 carbons and the alkyl portion contains from 1 to 4 carbons; heteroarylalkyl, wherein the aryl portion comprises from 4 to 9 carbons and at least one heteroatom of N, 0 or S and the alkyl portion contains from 1 to 4 carbons; -C (0) R9, wherein R9 represents an alkyl of 2 to 6 carbons, aryl of 6 to 10 carbons, heteroaryl comprising from 4 to 9 carbons and at least one heteroatom of N, O or S, or arylalkyl, wherein the aryl portion contains from 6 to 10 carbons or is heteroaryl comprising from 4 to 9 carbons and at least one heteroatom of N, O or S, and the alkyl portion contains from 1 to 4 carbons, under the conditions of which - when R8 is -C (0) R9, Z is -S- or -O-; - when Z is -0-, R8 can also be - (CqH2q0) rR5, where q, r and R5 are as defined above, - 16) - (CH2) ttSi (R10) 3, where w is an integer from 1 to 3 and Ru? represents an alkyl of 1 to 4 carbons.

In addition, the aryl or heteroaryl portions of any of the groups T or R6 can optionally carry up to two substituents selected from the group consisting of - (CH2) _C (R4) (R3) OH, - (CH2) yOR4), - (CH2) and SR4), - (CH2) and S (O) R4), - (CH2)} S (0) 2R4), - (CH2) and S02N (R4)) 2, - (CH2) .N (R4)) 2, - (CH,) .N (R4)) COR3, -OC (R4)) - in which both oxygen atoms are connected to the aryl ring, - (CH2) and COR4, - (CH2), CON (R4)) 2, - (CH2) and C02R4); - (CH2) and OCOR4, -halogen, -CHO, -CF ,, -N02, -CN and -R3, wherein y is from 0 to 4; R3 is defined as above, R4 is defined as above and in addition any two R4 which are attached to a nitrogen may be linked to form a heterocyl, such as a morpholine ring, thiomorpholine, pyridine or piperidine.

The pharmaceutically acceptable salts of these compounds, as well as the commonly used prodrugs of these compounds, such as the O-acyl derivatives of these compounds, are also within the scope of the invention.

In the compounds of the invention, the following is preferred.

Substituent group T, when on ring A, is preferably halogen, 1-alkynyl-Q or an ether OR 4, where R 4 is preferably alkyl of 1 to 12 carbons or arylalkyl in which the aryl portion is 6 to 10 carbons and the alkyl portion contains 1 to 4 carbons. It is most preferred that T is halogen, -C = C- (CH2) t0H, where t is an integer from 1 to 5, and when T is OR4, R4 is alkyl of 1 to 6 carbons or benzyl.

The subscript x, which defines the number of substituents T, is preferably 1 or 2, most preferred 1, and this substituent T is preferably in position 4 of ring A.

Ring A is preferably a phenyl or thiophene ring, most preferably phenyl. Ring A preferably carries at least one substituent group T, preferably located at the position farthest from the position of ring A which is connected to ring B.

Remainder B of the generalized formula (I) is a substituted or unsubstituted aromatic or heteroaromatic ring or bond, wherein any of the substituents are groups that do not cause the molecule to fail to conform to the activp site of the target enzyme, or that they alter the relative conformations of rings A and B, so that they are harmful. Said groups can be, or are not limited to, p.sub.2), residues such as lower alkyl, lower alkoxy, CN,? 0, halogen, etc. The residue B is preferably a 1,4-phenylene or 2,5-thiophene ring, most preferably 1-phenylene.

Unit D is most preferably a carbonyl or a -CHOH- group.

The Rfl group is preferably: 1) arylalkyl, the aryl portion containing 6 to 10 carbons and containing the alkyl portion of 1 to 8 carbons; 2) - (CH2) tR7 where t equals 0 or an integer from 1 to 5 and where R7 is an imidoyl group condensed with an aromatic residue, or the group 1, 2, 3-benzotriazin-4 (3H) -one-3 - ilo; or 3) - (CH2) ZR8, where v equals 0 or an integer from 1 to 4, Z being equal to S or 0, and R8 being aryl of 6 to 10 carbons or arylalkyl wherein the aryl portion contains 6 to 12 carbons and the alkyl portion contains 1 to 4 carbons.

The group R6 is most preferably one of the following, and in these, any aromatic moiety is preferably substituted: 1) arylalkyl, the aryl portion being equal to phenyl and the alkyl portion containing 1 to 4 carbons; 2) - (CH ^ R7, where t is an integer from 1 to 3, and where R7 is N-phthalimidoyl, 1, 2, 3-benzotriazin-4 (3H) -one-3-yl, N- (1, 2) naft al.enodi carboximidoyl), N- (2, 3-naphthalenedicarboximidoyl) or N- (1,8-naphthalenodicarboximidoyl); 3) - (CH2) ZR8, where v is an integer from 1 to 3, where Z S and R8 are phenyl.

It is understood that as used herein, the term "alkyl" means linear, branched, cyclic and polycyclic materials. The term "haloalkyl" means partially or fully halogenated alkyl groups such as - (CH2) 2C1, -CF3 and -C6FI3 / for example.

In one of its embodiments, the invention relates to compounds of generalized formula (I) in which at least one of the units A, B, T and R6 comprises a heteroaromatic ring. Preferred heteroaromatic ring-containing compounds are those in which the heteroaryl groups are heteroaryl of 4 to 9 carbons comprising a 5-6 membered heteroaromatic ring containing O, S or NR1, when the ring is 5 members, and N when the aforementioned ring is 6 members. Particularly preferred heteroaromatic ring-containing compounds are those in which at least one of the units A and B comprises a thiophene ring. When unit A is thiophene, it is preferably connected to unit B in position 2 and carries a substituent group T in position 5. When unit B is thiophene, it is preferably connected through positions 2 and 5 to the units D and A respectively.

In another embodiment, the invention relates to compounds of generalized formula (I), in unit E of which, n is 2 and ra is 1. These compounds thus possess two carbon atoms in unit D and carboxyl group , and they carry a substituent in this two carbon chain.

In another of its embodiments, the invention relates to compounds of generalized formula (I) in which ring A is a substituted or unsubstituted phenyl group, ring B is p-phenylene, and aryl portions of residues T and R? containing aryl contain only carbon in the rings. These compounds thus do not contain heteroaromatic rings.

In another of its embodiments, the invention relates to compounds of generalized formula (I) wherein m is 1 and R6 is an independent substituent. These compounds are materials that contain only a single R6 substituent in unit E, and this substituent is not involved in a ring.

Preferred compounds of general formula (I) wherein R1 'is ~ (CH2), R7, have t as an integer of 1 to 5. Preferred compounds of general formula (I) wherein R6 is - (CH2) , ZRS have v as an integer from 1 to 4 and Z as -S- or -O-. Preferred compounds of general formula (I) wherein R6 is alkyl contain 4 or more carbons in said alkyl, and those in which R6 is arylalkyl contain from 2 to 3 carbons in the alkyl portion of said arylalkyl.

In another of its embodiments, the invention relates to compounds of generalized formula (I) in which the number of substituents m in unit E is 2 or 3; and when m is 2, both R6 groups are independent substituents; or together they constitute a spiro ring, or a group R6 is an independent substituent and the other constitutes a spiro ring; and when m is 3, two R R groups are independent substituents and one R6 group constitutes a ring, or two R6 groups constitute a ring and one R6 group is an independent substituent, or three R6 groups are independent substituents. This subset therefore contains compounds in which the unit E is di- or trisubstituted, and in the disubstituted case, any of the rings formed by one or both of the groups R6 are spiro rings, and in the tri-substituted case, the R6 groups can be form spiro or non-spiro rings.

In another of its embodiments, the invention relates to compounds of generalized formula (I) in which the number of substituents m in unit E is 1 or 2; and when m is 1, the group Rf > it constitutes a non-spiro ring; and - when m is 2, both groups R6 together constitute a non-spiro ring or a R group? it is an independent substituent and the others constitute a non-spiro ring. This subset therefore contains compounds in which the unit E carries one or two substituents R6, and at least one of these substituents is involved in a non-spiro ring.

More particularly, representative compounds of generalized formula (I) in which one or more of the substituent groups R? are involved in the formation of non-spiro rings, have units E of the following structures: 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 to 5; f is 1 to 4; . g is 3 to 5; h is 2 to 4; i is 0 to 4; j is 0 to 3; k is 0 to 2; the total number of groups R6 is 0, 1 or 2; U represents O, S or NR1; and z is 1 or 2. Each R14 group is independently selected from the group consisting of: alkyl of 1 to 9 carbons; arylalkyl wherein the alkyl portion contains from 1 to 7 carbons and the aryl portion contains from 6 to 10 carbons; alkenyl of 2 to 9 carbons; alkenyl substituted with aryl wherein the alkenyl portion contains from 2 to 4 carbons and the aryl portion contains from 6 to 10 carbons; alkynyl of 2 to 9 carbons; alkynyl substituted with aryl wherein the alkynyl portion contains from 2 to 4 carbons and the aryl portion contains from 6 to 10 carbons; aryl of 6 to 10 carbons; -COR2; -CH (OH) R2; -C02R3; -CON (R2) 2; - (CH2) tR7 where t is 0 or an integer from L to 4; and - (CH2) VZR8 where v is 0 or an integer from 1 to 3, and Z represents -S-, S (O), S02 or -O-. R1, R7 and R8 are defined above.

Other preferred compounds of generalized formula (I) in which one or more of the substituent groups R6 is involved in the formation of non-spiro rings have units E of the following structures: where a, b, c, d, (c + d), e, g, i, k, the total number of groups Rf ', U and R14 are as defined above. -10 Other preferred compounds for use for the prevention and treatment of respiratory diseases of generalized formula (I), in which one or more of the substituent groups R6 is involved in the formation of non-spiro rings, have the formula wherein the subscript x is 1 or 2; a substituent T is • 20 located in position 4 of ring A, with respect to the point of union between rings A and B; e is 2 or 3 and R14 is as defined above.

The most preferred compounds are those shown in table I: .25 -25 Table 1 In the above structures, the term "racemate" in the case of cyclopentane derivatives, represents the trans, trans diastereomer, that is, for examples C-VII, C-XI, C-XVI to C-XVIII and C -XXIX to C-XXXV, a 1S / R, 2S / R, 5R / S ratio.

The use of the following compound is especially preferred: (+) -4- (4'-Chloro- [1,1'-biphenyl] -4-yl) -2- [2- (1,3-dioxo-1,3-dihydro-2H-isoindol-2) acid -yl) ethyl] -4-oxobutanoic In another aspect of the invention the following new compounds of general formula (! ') Are provided.

CO-E-C02H representing a residue 3 -carboxyl-5-R7-pentan-1-on-l-yl and substituents T and R7 having the meaning indicated in the following table: Table 2 The following compound is especially preferred: Acid (+) -2- [2- (1, 3-dioxo-l, 3-dihydro-2H-isomodol-2-? L) ethyl-4- (4'-ethoxy- [1,1'-biphenyl] -4-il) -4-oxobutanoic In another aspect of the invention, the use of compounds of general formula (I1) is a preferred embodiment. in which T is (C, -C4) alkoxy, chloride, bromide, fluoride, acetoxy, hydroxy, cyano, trifluoromethyl or trifluoromethoxy, CO-E-C02H represents a residue 3-carboxyl-5-R7-pentan-l-on-l-yl- or 2-carboxyl-3 - (R7-methyl) cyclopentan-1-yl) carbonyl-, and R7 represents a group of formula and their salts-The use of the following compound is especially preferred: Acid (+) -2-12- (1, 3-dioxo-l, 3-dihydro-2H-isoindol-2-yl) ethyl] 4- (4'-ethoxy [1,1'-biphenyl] -4- il) -4-oxobutanoic, General Preparative Methods: The compounds of the invention can be prepared by the use of known chemical reactions and procedures as described in detail in WO 96/15096, WO 97/43237, WO 97/43238, WO 97/43239, WO 97/43240, WO 97/43245, WO 97/43247 and WO 98/22436. However, the following general preparatory procedures are presented as an aid to the reader to synthesize the inhibitors. The general procedures A to K can be used to prepare 4-baryl-4-oxobutanoic acids, 4-aryl-4-oxobutanoic acids, 5-biaryl-5-oxopentanoic acids or 5-aryl-5-oxopentanoic acids appropriately substituted. These general procedures are also found in the document WO 9615096 (May 23, 1996), together with illustrative preparations of the keto acids. The choice of a specific synthetic process is dictated by the condition that the conditions used do not give rise to undesired changes in the T or R6 residues of the prepared compounds.

All the variable groups of these procedures are as described in the general description if they are not specifically defined below. The variable subscript n is defined independently for each procedure. When a variable group with a given symbol (ie R6 or T) is used more than once in a given structure, it is understood that each of these groups can be varied independently within the scope of definitions of that symbol. As defined above, the compounds of the invention contain as unit E a chain of 2 to 3 carbon atoms carrying 1 to 3 substituents R6 which are not defined as H. In contrast, it is observed that in the schemes of the general process then, the groups Rf 'are used as if their definition included H, to show when said R6 groups can exist in the structures, and for ease of drawing. However, no change is intended in the. definition of R6 for this non-standard use. Thus, only for purposes of the schemes of the general procedure below, Rd can be H in addition to the remains indicated in the definition of R6. The latest compounds contain 1 to 3 non-hydrogen R6 groups, General Procedure A - The key intermediates in which rings A and B are phenyl and substituted phenylene, respectively, are conveniently prepared by the use of a Friedel-Crafts reaction of a biphenyl II substituted with an intermediate containing activated acyl such as the derivative III of glutaric succinic anhydride or the acid chloride IV in the presence of a Lewis acid catalyst, such as aluminum trichloride, in an aprotic solvent such as 1,1,1,2-tetrachloroethane. The well known Friedel-Crafts reaction can be carried out with many solvents and alternative acid catalysts as described in E. Berliner, Org. React, 5, 229 (1949) and H. Heany, Comp. Org. Synth , 2, 733 (1991).

Process A If the anhydride III is monosubstituted or asymmetrically multisubstituted, the crude product I-A often exists in the form of a mixture of isomers by attacking the anhydride by either of the two carbonyls. The resulting isomers can be separated into the pure forms by crystallization or chromatography, using conventional procedures known to those skilled in the art.

When they are not commercially available, the anhydrides I -? - 4 Succinic III can be prepared by a Stobbe condensation of a dialkyl succinate with an aldehyde or ketone (which results in the R6 chain), followed by catalytic hydrogenation, hydrolysis of a half ester intermediate to a diacid and then conversion to the anhydride III by reaction with acetyl chloride or acetic anhydride. Alternatively, the half ester intermediate is converted by treatment with thionyl chloride or oxalyl chloride to the acid chloride IV wherein R 12 is lower alkyl. For a review of Stobbe condensation, including lists of suitable solvents and bases, see W.S. Johnson and G.H. Daub, Org. React. , 6, 1 (1951). This procedure, applied to the preparation of III (Rc '= H, isobutyl and H, n-pentyl) has been described in.D. Wolanin, et al., U.S. Patent 4,771,038, September 13, 1988.

Process A is especially useful for the preparation of key cyclic intermediates such as I-A-3 in which two R6 groups are connected in a methylene chain to form a 4 to 7 membered ring. Small ring anhydrides (3 to 5 membered) are readily available only as cis isomers, which provide cis I-A-3 compounds of the invention. The trans I-A-4 compounds are then prepared by treatment of I-A-3 with a base such as DBU in THF.

The anhydrides starting material substituted four-membered rings, such as III-A-1, are formed in a 2 + 2 photochemical reaction as shown below. This process is especially useful for the preparation of compounds in which R14 is acetoxy or acetoxymethylene. After the subsequent reaction of Fri del-Crafts, the acetate can be removed by basic hydrolysis and the carboxyl protected by conversion to (2-tritymethylsilyl) ethyl ester. The resulting intermediate with R14 = CH2OH can be converted into key intermediates with other R14 groups by using procedures described in general procedure K.

The Friedel-Crafts procedure is also useful when there are double bonds between C-2 and C-3 of a succinoyl chain (of maleic anhydride or 1-cyclopentane-1,2-dicarboxylic anhydride, for example) or when it is found a double bond in a side chain, such as in the use of itaconic anhydride as starting material, providing products in which two R6 groups found on a carbon chain together form an exomethylene group (= CH2). Subsequent uses of these compounds are described in procedures D and E.

General Procedure B - Alternatively, the key intermediates can be prepared by a reaction sequence involving monoalkylation of a dialkyl VI malonate with an alkyl halide, forming intermediate VII, followed by alkylation with a halomethylbiphenyl ketone VIII, providing intermediate IX . The compounds of structure IX are then hydrolysed on an aqueous base and then heated to decarboxylate the malonic acid intermediate and provide I-B-2 (process B-1). When using an aqueous base equivalent, esters I-B-2 are obtained with R '~ as alkyl, and using more than two equivalents of base, the acidic compounds are obtained (RI2 = H). Optionally, no heat is used and the diacid or ester of acid I-B-1 is obtained. Alternatively, the diester intermediate IX can be heated with a strong acid such as concentrated hydrochloric acid in acetic acid in a sealed tube at about 110 ° C temperature for about 24 hours, providing I-B-2 (R12 = H).

Alternatively, the reaction of VI with VIII can be carried out before that of the alkyl halide, providing the same IX (method B-2).

Intermediates VIII are formed from biphenyls II in a Friedel-Crafts reaction with haloacetyl halides such as bromoacetyl bromide or chloroacetyl chloride. Alternatively, the biphenyl can be reacted with acetyl chloride or acetic anhydride and the resulting product be halogenated, for example with bromine, to provide intermediates VIII (X = Br).

Process B has the advantage of providing individual regioisomers, while Process A provides mixtures. Process B is especially useful when the R6 side chains contain aromatic or heteroaromatic rings which can participate in intramolecular acylation reactions by providing side products if the process A is used. This procedure is also very useful when the group R6 adjacent to the carboxyl of the The final compound contains heteroatoms such as oxygen, sulfur or, nitrogen, or more complex functions such as imide rings.

Procedure B I-U-I-B-2 General Procedure C - The use of chiral HPLC to separate the enantiomers from mixtures of racemic key intermediates is especially useful (see, for example, D. Arlt, B. Boemer, R. Grosser and W. Lange, Angew.Chem. In Ed. Engl 30 (1991), n ° 12). The key intermediates are prepared in the form of pure enantiomers by the use of an auxiliary chiral route, see for example: D.A. Evans, Aldrichimica Acta, .15 (2), 23 (1982) or other similar references known to one skilled in the art.

C-1 The acid halide X is reacted with the lithium salt of the chiral auxiliary XI (R is often isopropyl or benzyl), providing intermediate XII, which in turn is rented at low temperatures (typically lower than -50 b C) with the haloterc-butyl acetyl compound XIII, yielding the pure isomer XIV. The use of XI of opposite chirality provides XIV of opposite chirality. The conversion of XIV to the enantiomerically pure diacid XV is accompanied by treatment with lithium hydroxide / hydrogen peroxide in THF / water, followed by acids such as trifluoroacetic acid. Compound XV is then converted to the enantiomerically pure anhydride III-A by treatment with acetyl chloride. The use of a Friedel-Crafts reaction as in procedure A, then converts III-A to I-C-1.

C-2 The biphenyl II starting material can also be reacted first in a Friedel-Crafts reaction, as described above, with succinic anhydride, followed by Fisher esterification with a lower alcohol, such as methanol, in the presence of a strong acid. , such as sulfuric acid, forming the acyl derivative IC-2. The carbonyl group of this material is then blocked as a ketal such as that formed by treatment with 1,2-bistrimethylsilyl-oxyethane in the presence of a catalyst such as trimethylsilyl triflate in a suitable solvent. Many other ketal derivatives and reaction conditions familiar to those skilled in the art can be used at this stage. Basic hydrolysis of the ester, followed by the reaction of the resulting I -C-3 with XI in the presence of an amide coupling agent, such as l- (3-dimethylaminopropyl) -3-ethylcarbodiimide, provides the amide I-C-4. Reaction of this chiral amide with an alkylating agent such as alkyl or arylalkyl triflate or halide provides the enantiomerically enriched product IC-5, which can be converted to I-C-6 cephalic acid by treatment with a weak base such as hydroxide. of lithium / hydrogen peroxide and then in ketoacid IC-7 by treatment with an acid. These unlocking steps can be performed in any order.

Procedure C-1 £ 0 > 25 Procedure C-2 General Procedure D - The key intermediates are prepared in which Rft are alkyl- or aryl- or heteroaryl- or acyl- or heceroarylcarbonylthiomethylenes by methods analogous to those described in patent publication WO 90/05719. A) Yes, substituted itaconic anhydride XVI (n = l) is reacted under Friedel-Crafts conditions to provide I-D-1 acid, which can be separated by chromatography or crystallization of small amounts of isomeric I-D-5. Alternatively, I-D-5 is obtained by reaction of the key intermediates I-D-4 (from any of procedures A to C) with formaldehyde in the presence of a base.

The compounds l-D-1 or I-D-5 are then reacted with a mercapto derivative XVII or XVIII in the presence of a catalyst. such as potassium carbonate, ethyldiisobutylamine, tetrabutylammonium fluoride or free radical initiators such as azobisisobutyronitrile (AIBN) in a solvent such as dimethylformamide or tetrahydrofuran, providing the key intermediates lD-2, ID-3, -D-6 or ID-7.

Procedure D General Procedure E - The reaction of maleic anhydride XIX optionally substituted under Friedel-Craf s conditions with II provides the key intermediate I-E-1, which in turn is reacted with any of the Mercapto XVII derivatives -25 or XVIII, providing the key intermediates I-E-2 or I-E-3, or with the substituted amine XX providing the key intermediate I-E-4. The esterification of I-E-1 (R 6 = H) with CH 3 I / DBU, followed by reagent XXI and AgF and then basic hydrolysis, provides the key intermediate of pyrrolidine I-E-5 R 1 can be various alkyl or arylalkyl groups, including benzyl. Reaction of the ester intermediate (from step 2) with benzyloxycarbonyl chloride in THF at reflux followed by hydrolysis provides the key intermediates where Ru is benzyloxycarbonyl.

Procedure E General procedure F - Biaryl key intermediates such as those of this application can also be prepared by cross-coupling reactions of Suzuki or Stille of metallic aryl or heteroaryl compounds in which the metal is zinc, tin, magnesium, lithium, boron, silicon, copper, cadmium or the like with a halide or aryl triflate or heteroaryl (trifluoromethanesulfonate) or the like. In the following equation, Met or X is the metal and the other is the halide or triflate. Pd (com) is a soluble complex of palladium such as tetrakis (triphenylphosphine) -palladium (0) or bis (triphenylphosphine) palladium (II) chloride. These methods are well known to those skilled in the art. See, for example, A. Suzuki, Pure Appl. Chem., 66., 213-222 (1994); A. Suzuki, Pure Appl. Chem., 63, 419-422 (1991) and V. Fariña and G. Roth, "Metal-Organic Chemistry", volume 5 (Chapter 1), 1994.

Starting materials XXIII (B = 1, 4-phenylene) are easily formed using procedures analogous to processes A, B or C, but using a halobenzene instead of a biphenyl as the starting material. When desired, the materials in which X is halo can be converted to those in which X is metal by reactions well known to those skilled in the art, such as the treatment of a bromine intermediate with hexamethyldistane and tetrakistriphenylphosphine of palladium in toluene at reflux, yielding the trimethyltin intermediate. "The starting materials XXIII (B = heteoaryl) are most conveniently prepared by process C, but using readily available heteoaryl starting materials instead of biphenyl. Intermediates XXII are commercial or are prepared easily from commercial materials by methods well known to those skilled in the art.

These general procedures are useful for the preparation of key intermediates for which Friedel-Crafts reactions, such as those of procedures A, B, C, D or E, would lead to mixtures with various biaryl acylation patterns. Process F is also especially useful for the preparation of key intermediates where aryl groups A or B contain one or more heteroatoms (heteroaryl), such as those compounds containing thiophene, furan, pyridine, pyrrole, oxazole, thiazole rings, pyrimidine or pyrazine or the like in place of phenyls.

Procedure F (T), A-Met + X-B-D-E-C02H * - (T) xA-B-D-E-CO, H XXII XXIII (Pd) com I-F T, x, A, B, E and D as in the structure I Met = metal and X = halide or triflate Met = halide or triflate and X = metal General procedure G - When the R groups of procedure F together form a carbocyclic ring of 4 to 7 members as in intermediate XXV below, the double bond can be displaced from the conjugation with the ketone group by treatment with two equivalents of a strong base, such as lithium diisopropylamide or lithium hexamethylsilylamide or the like, followed by acid inactivation, providing compounds of structure XXVI. The reaction of XXVI with mercapto derivatives using procedures analogous to those of general procedure D then leads to the key intermediate I-G-1 or I-G-2.

Procedure G -L5 General Procedure H - The key intermediates in which two R6 groups form a carbocyclic ring of 4 to 7 members, as in I-H below, and R14 is alkyl or arylalkyl, are prepared according to the H process. The starting material XXVII with two equivalents of a strong base such as lithium diisopropylamide (LDA) followed by an alkyl or arylalkyl halide (R14X), providing the intermediate XXVIII. This material is then reduced to alcohol with a reducing agent capable of "selective" reduction of the ketone, such as sodium borohydride, followed by > Dehydration with triphenylphosphine / diethyl azodicarboxylate (DEAD) in a suitable solvent such as THF under reflux afforded XXIX. The hydrolysis of the ester with aqueous base followed by the formation of amide with RI2ONHR?;! (R is (C 1 -C 4) alkyl, but usually CH 3) in the presence of a coupling agent such as dicyclohexyldiimide (DCC) provides XXX. Other acyl activating groups well known to those skilled in the art, such as acyl chlorides or mixed anhydrides, could be used in place of XXX. The substituted biphenyl halide XXXI is reacted with an alkyl lithium, such as two equivalents of tert-butyllithium, yielding the lithiated biphenyl XXXII, which is then reacted with the activated acyl compound XXX. The resulting intermediate XXXIII is then reacted with diethylaluminum cyanide, providing intermediate XXXIV which is then hydrolyzed with aqueous acid, yielding the key intermediate I-H which is purified by chromatography on silica gel, yielding the pure isomers.

Procedure H n = 1 - 4 XXVIII XXVII]) reduction. 2) dehydration I-H General Procedure I - Key intermediates are prepared in which two R6 groups together form a pyrrolidine ring according to procedure I. The starting material XXXV (L-pyroglutaminol) is reacted with acid catalysis with benzaldehyde XXXVI (may be substituted), providing the bicyclic derivative XXXVII. A double bond is then introduced using the phenylosinyl methodology, well known to those skilled in the art, providing XXXVIII, which in turn is reacted with a vinyl copper complex (I), providing a conjugate addition product XXXIX. Said reactions, in which Lig may be, for example, another vinyl or halide equivalent, are well known to those skilled in the art. Reduction with hydride (lithium aluminum hydride or the like) of XXXIX followed by conventional blocking with, for example, tert-butyldylmethylsilyl chloride, provides XXXX which in turn is reacted with a benzyl chloroformate XXXXI, optionally substituted, providing XXXXII. The ozonolysis of this intermediate followed by reducing processing (dimethisulfide, zinc / acetic acid or the like) leads to aldehyde XXXXIII. Reaction of this aldehyde with a biphenyl organometalic such as XXXII provides alcohol XXXIV. Unblocking the silyl group with, for example, tetrabutylammonium fluoride, followed by oxidation with, for example, pyridinium dichromate or the like, provides the key intermediate 1-1-1 wherein R14 is a carbobenzyloxy group.

Alternatively, the carbobenzyloxy group is removed by reaction with hydrogen and a catalyst such as palladium on carbon, yielding the unsubstituted key intermediate 1-1-2, optionally followed by 'N-alkylation, providing key intermediate 1-1-3 . These final stages are well known to those skilled in the art. Alternatively, intermediate XXX can be directly treated with ozone followed by the other steps of this procedure, providing 1-1-3, wherein R14 is optionally substituted benzyl instead of as in 1-1-1.

This method is especially useful for preparing single enantiomers because the starting material XXXV is available in the form of either of the isomers or in the form of D-pyroglutaminol, providing enantiomeric products.

Procedure I General Procedure J - The key intermediates in which E represents a substituted chain of 3 carbons are prepared by process J. Intermediates XXXXVII, if not available from commercial sources, are prepared by reaction of an activated biphenylcarboxylic acid derivative XXXXV with substituted acetic acid XXXXVI, which has been converted to its bis-anion with two equivalents of a strong base, such as LDA followed by heating to decarboxylate the intermediate ketoacid. The product XXXXVII is then treated with the methylenemalonate derivative XXXXVIII in the presence of a strong base such as sodium hydride, by providing the substituted malonate XXXXIX.This malonate can be further alkylated, under conditions familiar to those skilled in the art, by providing L which in turn is treated with acid and then heated to provide the key medium I. As an alternative, the final alkylation can be omitted, providing products in which the Rf 'adjacent to the carboxyl is H. Alternatively, XXXXVII can be alkylated with ester L-3-halopropionate in the presence of a base such as LDA, providing the lJ-2 ester which can then be hydrolyzed with aqueous base to provide the key intermediate lJ-3 with acid treatment.This procedure is especially useful if any of the groups R6 contains aromatic residues.

Procedure J 1-J-l Process K - The key intermediates in which two R6 groups are joined to form a substituted 5-membered ring, are most conveniently prepared by method K. In this procedure, LIT acid (R = H) is prepared using the described protocols in Tetrahedron, vol. 37, suppl. 1981, 411. The acid is protected in the form of an ester (R = benzyl or 2- (trimethylsilyl) ethyl) by the use of coupling agents such as 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and well processes. known to those skilled in the art. The substituted bromobiphenyl LIII is converted to its Grignard reagent by treatment with magnesium, which is then reacted with LII, providing the alcohol LIV. LIV alcohol is removed by basic treatment of its mesylate using conditions well known to those skilled in the art, providing the LV olefin. Alternatively, Lili is converted to a trimethyltin intermediate by an initial metalation of the bromide with n-butyllithium at a low temperature (-78 ° C) followed by treatment with chlorotrimethyldin and LII is converted to an enol triflate by reaction with 2- [N, N-bis (rifluoromethyl-sulfonyl) amino] -5-chloropyridine in the presence of a strong aprotic base. The tin and enol triflate intermediates are then coupled in the presence of a catalyst of Pd °, Cul and AsPh3,. directly providing the intermediate LV. Ozonolysis of LV (processing with methyl sulfide) provides the aldehyde LVI. The alternative treatment with Os04 followed by HI04 converts LV into LVI.

The conversion of intermediate LVI into the key intermediate I-K is achieved in various ways, depending on the identity of the X-function of the side chain. The reaction of LVI with Wittig reagents followed by hydrogenation provides products where X is alkyl, aryl or arkalkyl. The reduction of the aldehyde LVI with LAH provides the alcohol I-K (X = OH). The alcohol is converted to phenylethers or N-phthalimidoyl compounds by the use of the appropriate starting materials and under Mitsunobu conditions well known to those skilled in the art; see 0. Mitsunobu, Synthesis', 1 (1981). Alternatively, the IK alcohol (X = 0H) is converted to a leaving group such as tosylate (X = OT) or bromide (X = Br) under conditions well known to those skilled in the art and then the leaving group is displaced with nucleophiles of sulfur or azide, providing products with "X = thioether or azide, which in turn are reduced and acylated by providing amides (X = NHacyl). Direct acylation of the alcohol I-K (X = OH) provides key intermediates where X = Oacyl and the reaction of the alcohol with various alkyl halides in the presence of base provides alkyl ethers (X = OR 2). In each case, the final step is the removal of the acid blocking group R, by providing acids (R = fl) using conditions that depend on the stability of R and X, but in all cases well known to the person skilled in the art, such as removal of benzyl by basic or 2- (trimethylsilyl) ethyl hydrolysis by treatment with tetrabutylammonium fluoride.

Procedure K LV ozone, then methyl sulfide 10 The pharmaceutically acceptable salts of the compounds of The present invention containing an acidic moiety includes the addition salts formed with organic or inorganic bases. The salt-forming ions derived from said bases can be metal ions, for example aluminum, alkali metal ions, such as sodium or potassium, metal ions. alkaline earth metal such as calcium or magnesium, or an amine salt ion, of which a number of them are known for this purpose. Examples include ammonium salts, arylalkylamines such as dibenzylamine and N, N-dibenzylethylenediamine, lower alkylamines such as methylamine, t-butylamma, procaine, '25 lower alkylpiperidines such as N-ethylpiperidine, cycloalkylamines such as cyclohexylamine or dicyclohexylamine, 1-adamantylamine, benzathine or salts derived from amino acids such as arginine, lysine or the like. Physiologically acceptable salts such as the sodium or potassium salts and the amino acid salts can be used medicinally as described below and are preferred.

The pharmaceutically acceptable salts of the compounds of the present invention which contain a basic moiety include the addition salts formed with organic or inorganic acids. The salt-forming ions derived from said acids can be halide ions or natural or non-natural carboxylic or sulfonic acid ions, of which a number of them are known for this purpose. Examples include chlorides, acetates, tartrates or salts derived from amino acids such as glycine or the like. Physiologically acceptable salts, such as chloride salts and amino acid salts, can be used medicinally as described below and are preferred.

These and other salts, which are not necessarily physiologically acceptable, are useful in the isolation and purification of an acceptable product for the purposes described below.

The salts are produced by reacting the acid form of the compound of the invention with one equivalent of the base that supplies the desired basic ion, or the basic form of the compound of the invention with one equivalent of the acid that supplies the desired acid ion , in a medium in which the salt precipitates either in an aqueous medium and then with lyophilization. The free acid or base form of the compounds of the invention can be obtained from the salt by conventional neutralization techniques, for example with potassium bisulfate, hydrochloric acid, sodium hydroxide, sodium bicarbonate, etc.

The compounds of the present invention are expected to inhibit matrix metalloproteases MMP-2, MMP-3, MMP-8, MMP-9, MMP-12, MMP-13 and the related protease TACE, as well as the release of TNFc- in vivo, and it is therefore expected that they will be useful for the treatment or prevention of the conditions cited in the background section. Since other MMPs not listed above share a high degree of homology with those enumerated above, especially at the catalytic site, it is considered that the compounds of the invention should also be capable of inhibiting said other MMPs to varying degrees.

By varying the substituents on the biaryl portions of the molecules, as well as those of the R6 groups of the claimed compounds, it is expected that the relative inhibition of the listed MMPs will be affected. Thus, compounds of this general class can be "adapted" by the selection of specific substituents, so that the inhibition of specific MMPs associated with specific pathological conditions can be enhanced, leaving without implicating the less affected MMPs.

The compounds of the present invention exhibit good activity for MMP-2, MMP-3, MMP-8, MMP-9, MMP-12 and MMP-13, and a good selectivity for these MMPs against other MMPs such as MMP-1 and MMP-7.

As a result of the above-mentioned selectivity profile, the compounds of the present invention. invention are especially suitable for the treatment of respiratory diseases.

The method of treatment of conditions mediated by matrix metalloproteases or mediated by the release of TNFc. it can be practiced in mammals, including humans, that exhibit these conditions.

The inhibitors of the present invention are contemplated for use in veterinary and human applications. For these purposes, more than one or more pharmaceutically acceptable vehicles, diluents, fillers, binders or other excipients will be used in pharmaceutical compositions containing active ingredient (s), depending on the mode of administration and the dosage form contemplated.

The administration of the inhibitors can be by any suitable mode known to those skilled in the art. Examples of suitable parenteral administration include intravenous, intraarticular, subcutaneous, and intramuscular routes. Intravenous administration can be used to obtain an accurate regulation of the plasma peak concentrations of the drug. The half-life and targeting of the drug to the articular cavities can be improved by entrapping the drug in liposomes. It may be possible to improve the selectivity of the liposomal targeting to the articular cavities by incorporating ligands outside of the liposomes that bind to specific synovial macromolecules. Alternatively, injection of intramuscular, intraarticular or subcutaneous stock with or without encapsulation of the drug in degradable microspheres, comprising for example poly (DL-lactide-co-glycolide) can be used to obtain sustained sustained drug release. To improve the convenience of the dosage form, it may be possible to use an ip and septum implanted reservoir, such as the Percuseal system available from Pharmacia. An improvement in the convenience and compliance of the patient can also be achieved by the use of injection pens (for example Novo Pin or Q-pen) or needle-free jet injectors (for example from Bioj ect, Mediject or Becton Dickinson). A prolonged zero distribution or other precise control release such as pulsed release can also be achieved as needed, using implantable pumps with drug delivery through a cannula in the synovial spaces. Examples include the subcutaneously implanted osmotic pumps available in ALZA, such as the ALZET osmotic pump.

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

Oral delivery can be achieved by incorporating the drug into tablets, coated tablets, dragees, hard and soft gelatin capsule, solutions, emulsions or suspensions. Oral delivery can also be achieved by incorporation of the drug in enteric coated capsules designed to release the drug in the colon when the digestive protease activity is low. Examples include the OROS-CT / OSmet ™ and PULSINCAP ™ systems from ALZA and Scherer Drug Deliver Systems, respectively. Other systems use azor-crosslinked polymers that are degraded by colonic-specific bacterial azorreductases or pH-sensitive polyacrylate polymers that are activated by increasing the pH in the colon. The above systems can be used together with a wide range of available absorption enhancers.

Rectal delivery can be achieved by incorporating the drug in suppositories.

The compounds of this invention can be manufactured in the formulations listed above by the addition of various therapeutically inert, inorganic or organic carriers well known to those skilled in the art. Examples of these include, but are not limited to them, lactose, corn starch or derivatives thereof, talc, vegetable oils, waxes, fats, polyols such as polyethylene glycol, water, sucrose, alcohols, glycerin and the like. Various preservatives, emulsifiers, dispersants, flavorings, wetting agents, antioxidants, sweeteners, colorants, stabilizers, salts, buffers and the like are also added, as required to assist in the stabilization of the formulation or to help increase the bioavailability of the the active ingredient (s) or to provide an acceptable taste or aroma formulation in the case of oral dosage.

The amount of pharmaceutical composition to be employed will depend on the recipient and the condition to be treated. The necessary amount can be determined without undue experimentation by protocols known to those skilled in the art. Alternatively, the amount needed can be calculated based on a determination of the amount of target enzyme that must be inhibited to treat the condition. It is expected that the compounds of the invention will generally be administered in doses in the range of 0.01-100 mg per kg of body weight per day.

The matrix metalloprotease inhibitors of the invention are useful not only for the treatment of. the physiological conditions discussed above, but are also useful in activities such as metalloprotease purification and matrix metalloprotease activity assay. Said activity assay can be either in vitro, using natural or synthetic enzyme preparations, or in vivo, using for example, animal models in which abnormal destructive enzymatic levels have been spontaneously found (use of genetically mutated or transgenic animals) or have induced by the administration of exogenous agents or by surgery that alters the stability of the joints.

Biological protocols The inhibitory activities of the compounds of the invention can be determined against matrix metalloproteases and the production of TNFa as described below.

MMP inhibition assay by inactivated fluorescence of P218: This assay is adapted from that described by Knight et al., FEBS. Letters, '296, 263-266 (1992) for MMP-3 and a related substrate.- The hydrolysis rate of the synthetic substrate H-MCA-Pro-Lys-Pro-Leu-Ala-Leu-DPA-Al'a- Arg-NH2 (P218) by the respective MMP is fluorimetrically controlled, using an excitation wavelength of 340 nm and an emission wavelength of 395 nm, in the presence or absence of test compounds. The substrate is initially prepared with 100% DMSO to a concentration of 1 x 10 ~ 2 M, then diluted in assay buffer to a final concentration of 20 μM. The test compounds (10 mM in DMSO) are diluted in assay buffer to an initial concentration of 0.3-1000 nM. These are diluted to a final concentration in the assay of 0.03 nM to 100 nM. The reaction is initiated by the addition of substrate to a final concentration of 20 μM. The total assay volume in a 96-well microtiter plate is 150 μl. The cutting of the substrate between the Leu-Ala residues allows the fluorescence of the MCA group to be detected by a fluorimeter (Cytofluor II) according to an excitation of 340 nm and an emission at 395 nm. The change in fluorescence is continuously monitored for a period of 40 minutes.

The K's are calculated, using the procedure described by Williams and Morrison, Methods in Enzymology, 63., 437-467 (1979) to measure K, apareme for strong binding inhibitors, and is summarized as follows: River/ . l -v.-v ,,) - * K? Apparent x v, / v0 + [E] " [I] and [E] 0 are the concentrations of inhibitor and enzyme, and ^ v ,, are the reaction rates with / without • inhibitor. [I] u is equal to IC50 when v, is half of v0, so that: IG?, = 0, 5 x [E] 0 + K. Apparent The IC 50 are determined at each enzyme concentration using Xlfit software, the K, aparant is then determined graphically. from the graph of IC50 versus concentration of MMP, using the intersections to estimate the K? aparemc. Thus, the intersection values in IC50 = 0 'and [E] 0 = 0 are equal to -2 x K, aparcnle and K, apareme, respectively. The IC5U values are calculated using the% inhibition values at each enzyme concentration, making sure that the data is taken from the linear part of the reaction velocity curves. The K can then be calculated with the equation: ? -? ~ K? jp.?ru?tc / (^ - + S) / Km where S = substrate concentration and K ". = dissociation constant.

The test conditions are modified as follows for each of the MMPs used: MMP-1 (interstitial collagenase of human gingival fibroblast) Pro-MMP-1 was supplied by Jack Windsor [Windsor et al, J. Bi ol, Chem. 269 (42), 26201-26207 (1994)]. The proenzyme was activated by incubation in trypsin / 1 mM AEBSF 1:20 for 10 minutes at 25 ° C temperature. Km value: 30 μM Test buffer: The assay is carried out in buffer containing 50 mM HEPES, 10 mM CaCl 2, pH 7.0. Enzyme concentrations for IC5Ü determinations: 0.1 μg / ml Enzyme concentrations for K determinations,: 0.1-0.8 μg / ml.

MMP-2 (gelatinase A) Gelatinase A (MMP-2) is prepared using a vaccine expression system according to the procedure of R. Fridman et al., J. Biol. Chem. , 267, 15398 (1992). Assay Buffer: The assay is carried out in buffer containing 50 mM Tris, 150 mM NaCl, 10 mM CaCl 2, 0.005% Brij-35 at pH 7.0.

Enzyme concentrations for IC5U determinations: 0.078 μg / ml MMP-3 (stromelysin) Preparation of Recombinant Truncated Stromelysin (MMP-3): Truncated pro-stromelysin-257 is expressed in a soluble form ~~ of E. coli as described in Marcy et al., Biochemistry, 30., 6476-6483, 1991 (see also Cancer Treat, Res. 61, 1-41 (1992)). The soluble truncated proestromelysin is purified by a modification of the monoclonal antibody affinity chromatography method described in Housley et al., J. Biol. Chem., 268, 4481-4487, 1993. Assay buffer: The assay is carried out in buffer containing 50 mM HEPES, 10 M CaCl 2, pH 7.0. 9d Enzyme concentrations for IC50 determinations: 0.8 μg / ml.

MMP-7 (matrilysin) Catalytic domain expressed in E. coli, using the vector pET14, provided by Dr. Steve Saphiro, Jewish Hospital at the Washington University Medical Center, St. Louis, Missouri, USA Assay buffer: the assay is carried out in buffer containing 50 mM HEPES, 10 mM CaCl 2, pH 7.0.

Enzyme concentrations for the IC5U determinations: 0.3 μg / ml.

MMP-8 (neutrophil collagenase) The recombinant truncated form (met80-gly242) was obtained according to the protocol of Knauper, Eur. J. Biochem. 189, 296-300 (1990).: * Assay Buffer: The assay is carried out in buffer containing 50 mM HEPES, 10 mM CaCl 2, pH 7.0.

Enzyme concentrations for the IC50 determinations: "9.4 μg / ml.

MMP-9 (gelatinase B) MMP-9 is isolated by modifying the previously described procedures of Hibbs et al. (J. Biol. Chem., 260, 2493-2500, 1984) and Wilhelm et al. (J. Biol. Chem., 264., 17213-17221, 198_9). Briefly, preparations of polymorphonuclear leukocytes (PMN) are isolated as described above from 3 or more units of freshly extracted whole blood. The cells are resuspended in phosphate-buffered saline (PBS) containing phorbol myristate acetate 100 ng / ml (PMA) in the presence of diisopropyl fluorophosphate (DFP) 50 M, leupeptin and aprotinin 1 μg / ml and catalase 1 mg / ml for 1 hour at 37 ° C temperature. The supernatants are collected by centrifugation (300 x g) and the mixtures are frozen at -70 ° C. All chromatographic procedures are performed at 4 ° C temperature. The thawed samples are concentrated five times using an Amicon chamber equipped with a YM-10 membrane. The concentrate is dialysed under pressure, against 0.02 M Tris-HCl, 0.1 M NaCl, 1 mM CaCl, 1 μM ZnCl 2, 0.001% Brij-35, 0.02% sodium azide (NaN 3). , pH 7.5 and applied to DEAE ion exchange chromatography resin which is pre-equilibrated with the same buffer at a flow rate of 0.4 ml / min. The column is washed extensively with the same buffer and the gelatinase is eluted as 4 ml fractions from the column with 0.02 M Tris-HCl, 0.5 M NaCl, 21 mM CaCl, 1 μM ZnCl 2, Brij-9? 35 to 0.001%, 0.02% sodium azide (NaN3), pH 7.5. Fractions containing gelatinase are observed by gelatin zymography (see below), they are loaded onto an agarose gelatin affinity resin and washed with the same buffer. The gelatinase activity is eluted at a flow rate of 1 ml / min from the column in the form of 1 ml fractions with 0.02 M Tris-HCl, 1 M NaCl, 1 mM CaCl, ZnCl, 1 μM, Brij -35 to 0.001%, 0.02% NaN3, pH 7.5 containing 10% dimethylsulfoxide (DMSO). Fractions containing gelatinase activity are pooled and dialyzed against 0.005 M Tris-HCl, 5 M NaCl, 0.5 mM CaCl 2, 0.1 μM ZnCl 2, 0.001% Brij-35, pH 7.4. The protein content associated with the material is determined with a micro-BCA assay (Pierce, Rockford, IL), lyophilized and reconstituted to the desired working concentration (100 μg / ml). Recombinant human pro-MMP-9 is also expressed in baculovirus.

K value, ": 22 μM Assay buffer: The assay is carried out in buffer containing 50 mM HEPES, 150 mM NaCl, 10 mM CaCl 2, Brij-35 al 0.005%, pH 7.0. Enzyme concentrations for IC50 determinations: 0.38 μg / ml Enzyme concentrations for K determinations,: 0.38-1.00 μg / ml. 9Í MMP-12 (macrophage elastase) MMP-9 was isolated according to the following procedure: Human GenePool cDNA libraries of lung, spleen and human brain were obtained from Invitrogen (Groningen, The Netherlands). Purified oligonucleotides were purchased by HPLC in BioTez (Berlin, Germany), strain DH5c. of E. coli in Life Technologies (Heidelberg, Germany), strain BL21 (DE3) in Novagen (Heidelberg, Germany). The pfu DNA polymerase and pBlueScriptII-KS (+) were from Stratagene. (Heildelberg, Germany). The ADÑ plasmid and the DANN gel isolation kits were purchased from Qiagen (Hilden, Germany). All restriction enzymes and DNA modifying enzymes were purchased from New England Biolabs (Schwalbach, Germany). The HiTrapQ (5 ml) was from Pharmacia Biotech (Freiburg, Germany). The Vydac C4-RP HPLC columns (214TP54: 300A, 5μm, 4.6x250mm; 214TP1022; 300A, 10μM, 22x250mm) were from Promochem (Wesel, Germany). The fluorogenic substrate P218 was purchased from Polypeptide Laboratories. (Wolfenbüttel, Germany) Ultrapure urea was purchased from Schwarz-Mann (Cleveland, OH). Ready-to-use PAGE-SDS gels to use NuPAGE of 10% bis-tris polyacrylamide and MOPS flow buffer NuPAGE 20x were obtained at Novex (Frankfurt am Main, Germany) . The 10% AMPA and all other chemicals were from Sigma (Deisenhofen, Germany).

Molecular cloning of MMP-12: The CDS of human MMP-12 was cloned (accession number in GenBank: L23808; accession number SwissProt: P39900) by PCR of the human normal spleen cDNA library with the Oligo-428 specific primers (front primer with the BamHl site underlined: 5 '-aaa ttt aaa crga tcc gcc acc atg aag ttt ctt cta ata ctg ctc ctg-3') and Oligo-431 (reverse primer with the underlined EcoRl site: 5 '-aaa ttt aaa gaa ttc att aac aac caa acc age tat tgc ttt tca-3 '). The removal of an internal EcoRl site was carried out by PCR mutagenesis with Oligo 434 (underlined mutated base: 5'-gcc tcc tg atg tgt agt cca gaa ctc gtc ctc ate gaa atg tgc atc-3 ') The reaction by PCR (100 μl volume) was carried out with Pfu DNA polymerase in a GeneAMp 2400 PCR system from Perkin Elmer with 25 cycles of denaturation (94 ° C, 1 minute), fixation (60 ° C, 1 minute) and extension (72 ° C, 2 minutes). The PCR product was purified with gel by electrophoresis on 0.8% agarose gels / EtBr, separated from the agarose gel with the isolation kit of QiaEx2 DNA was excised with the restriction endonucleases Bamñl / EcoRl and purified again. This material was ligated to the cloning vector pBlueScriptII-KS (+), which had previously been digested with BamHl and EcoRl cuts and dephosphorylated with calf intestine alkaline phosphatase. The recombinant plasmid (pMYZ180) was transformed into the E. coli strain DH5a. Plasmid DNA was isolated from various clones with the Qiagen plasmid preparation kit and analyzed by restriction digestion. The complete CDS DNA sequence of MMP-12 was determined by dideoxy sequencing with terminating dye chemistry in an ABl 377 sequencer. Sequence analysis and all subsequent bioinformatic work was performed with the Lasergene software package from DNAStar ( Madison, WT). Analysis of the sequence of the pMYZ 180 construct (4369 bp) identified silent mutations in the MMP-12 CDS at positions 802 (G-> A), 1402 (T-> C), 1429 (C-> 4). T) and 2002 (T-> C), which did not affect the amino acid sequence of the protein.

Construction of expression vectors: To test the best strategy for both protein expression and protein purification, several different expression constructs were generated by PCR with full-length or partial sequences of the MMP-12 gene plus various affinity tags for purification. The following constructs were prepared: pMYZ187: The coding sequence for full length human MMP-12 was cut by digestion with BamHl / EcoR1 from pMYZ180, ligated to pET-28a cut with BamHI / EcoRI "and the complete coding sequence of the final construct was verified by Double-stranded DNA sequencing.

PMYZ188: The coding sequence for full-length human MMP-12 was cut by digestion with BamHl / EcoR1 from pMYZ180, ligated to pET-32a cut with BamHI / EcoRI, and the complete coding sequence of the final construct was verified by Double-stranded DNA sequencing.

PMYZ139: The coding sequence for full-length human MMP-12 was cut by digestion with BamHl / EcoR1 from pMYZ180, ligated to pMYZ173 (a derivative of pET28a with an amino terminal mark of the GB1 domain) cut with BamHI / EcoRI and verified the complete coding sequence of the final construct by double-stranded DNA sequencing.

PMYZ19: The coding sequence for amino acids 1-2-79 of human MMP-12 was amplified by PCR with primers Oligo-428 (forward primer with the BamHl site underlined: 5 '-aaa ttt aaa gga tcc gcc acc atg aag ttt ctt cta ata ctg ctc ctg-31) and Oligo-486 (reverse primer with the underlined EcoRl site: 5 '-ttt aaa ttt gaa ttc att atg gtt ctg aat tgt cag gat ttg gca-3') of pMYZldO, was cut with BamHI / EcoRI, was ligated to pET-28a cut with BamHI / EcoRI, and the complete coding sequence of the final construct was verified by double-stranded DNA sequencing.

PMYZ195: The coding sequence for amino acids 1-264 of human MMP-12 was amplified by PCR with Oligo-428 primers (forward primer with the BamHl site underlined: 5 '-aaa ttt aaa gga tcc gcc acc atg aag ttt ctt cta ata ctg ctc ctg-3 ') and Oligo-487 (reverse primer with the underlined EcoRl site: 5' -ttt aaa ttt gaa ttc att agt ctc cat here ggg act gaa tgc cac-3 ') of pMYZ180, was cut with BamHI / EcoRI was ligated to pET-28a cut with BamHI / EcoRI, and the complete coding sequence of the final construct was verified by double-stranded DNA sequencing. pMYZ198: The coding sequence for amino acids "1-279 of human MMP-12 was amplified by PCR with primers Oligo-428 (forward primer with the BamHl site underlined: 5 '-aaa ttt aaa gga tcc gcc acc atg aag ttt ctt cta ata ctg ctc ctg-3 ') and Oligo-486 (reverse primer with the underlined EcoRl site: 5' -ttt aaa ttt gaa ttc att atg gtt ctg aat tgt cag; gat ttg gca-31) of pMYZ180, was cut with BamHl / EcoR1, pET-32a cut with BamHI / EcoRI was ligated, and the complete coding sequence of the final construct was verified by double-stranded DNA sequencing.

PMYZ199: The coding sequence for amino acids 1-264 of human MMP-12 was amplified by PCR with Oligo-428 primers (forward primer with the BamHl site underlined: 5 '-aaa ttt aaa gga tcc gcc acc atg aag ttt ctt cta ata ctg ctc ctg-3 ') and Oligo-487 (reverse primer with the underlined EcoRl site: 5' -ttt aaa ttt gaa ttc att agt ctc cat here ggg act gaa tgc cac-3 ') of pMYZ180, was cut with BamHI / EcoRI was ligated to pET-32a cut with BamHI / EcoRI, and the complete coding sequence of the final construct was verified by double-stranded DNA sequencing.

PMYZ200: The coding sequence for amino acids 1-279 of human MMP-12 was amplified by PCR with primers Oligo-428 (forward primer with the BamHl site underlined: 5 '-aaa ttt aaa gga tcc gcc acc atg aag ttt ctt cta ata ctg ctc ctg-31) and Oligo-486 (reverse primer with the underlined EcoRl site: 5 '-ttt aaa ttt gaa ttc att atg gtt ctg aat tgt cag gat ttg gca-3') of pMYZ180, cut with BamHl / EcoRI, was ligated to pMYZ173 cut with BamHI / EcoRI, and the complete coding sequence of the final construct was verified by double-stranded DNA sequencing.

PMYZ201: The coding sequence for amino acids 1-264 of human MMP-12 was amplified by PCR with primers Ol'igo-428 (forward primer with the BamHl site underlined: 5 '-aaa ttt aaa gga tcc gcc acc atg aag ttt ctt cta ata ctg ctc ctg-3 ') and Oligo-487 (reverse primer with the underlined EcoRl site: 5' -ttt aaa ttt gaa ttc att agt ctc cat here ggg act gaa tgc cac-3 ') of pMYZ180, was cut with BamHI / EcoRI, ligated to pMYZ173 cut with BamHI / EcoRI, and the complete coding sequence of the final construct was verified by double-stranded DNA sequencing. pMYZ215 ': The coding sequence for amino acids 17-279 of human MMP-12 was amplified by PCR with primers Oligo-513 (forward primer with the N-site underlined: 5' -aaa ttt aaa cat atg ctt ecc ctg aac age tet here age ctg-3 ') and Oligo-486 (reverse primer with the underlined EcoRl site: 5' -ttt aaa ttt gaa ttc att atg gtt ctg aat tgt cag gat ttg gca-3 ') of pMYZ180, was cut with Ndel / EcoRI was ligated to pET-28a cut with Ndel / EcoRI, and the complete coding sequence of the final construct was verified by double-stranded DNA sequencing.

PMYZ216: The coding sequence for amino acids 17-264 of human MMP-12 was amplified by PCR with Oligo-513 primers (forward primer with Ndel underlined site: 5 '-aaa ttt aaa cat atg ctt ecc ctg aac age tet here age ctg-3 ') and Oligo-487 (reverse primer with the underlined EcoRl site: 5' -ttt aaa ttt gaa ttc att agt ctc cat here ggg act gaa tgc cac-3 ') from pMYZ180, cut with Ndel / EcoRI was ligated to pET-28a cut with Ndel / EcoRI, and the complete coding sequence of the final construct was verified by double-stranded DNA sequencing.

PMYZ217: The coding sequence for amino acids 17-279 of human MMP-12 was amplified by PCR with primers Oligo-514 - (forward primer with the Ncol site underlined: 5 '-aaa ttt aaa gcc atg gct ctt ecc ctg aac age tet here age ctg-3 ') and Oligo-486 (reverse primer with the underlined EcoRl site: 5' -ttt aaa ttt gaa ttc att atg gtt ctg aat tgt cag gat ttg gca-3 ') of pMYZ180, was cut with Ncol / EcoRI was ligated to pET-32a cut with Ncol / EcoRI, and the complete coding sequence of the final construct was verified by double-stranded DNA sequencing.

PMYZ218: The coding sequence for amino acids 17-264 of human MMP-12 was amplified by PCR with primers Oligo-514 (forward primer with the Ncol site underlined: 5 '-aaa ttt aaa gcc atg gct ctt ecc ctg aac age tet here age ctg-3 ') and Oligo-487 (reverse primer with the EcoRl site underlined: 5' -ttt aaa ttt gaa ttc att agt ctc cat here ggg act gaa tgc cac-3 ') from pMYZ180, cut with Ncol / EcoRI, was ligated to pET-32a cut with Ncol / EcoRI, and the complete coding sequence of the final construct was verified by double-stranded DNA sequencing.

PMYZ219: The coding sequence for amino acids 17-279 of human MMP-12 was amplified by PCR with primers Oligo-515 (forward primer with the BamHl site underlined: 5 '-aaa ttt aaa gga tcc ctt ecc ctg aac age tet here age ctg-3 ') and Oligo-486 (reverse primer with the underlined EcoRl site: 5' -ttt aaa ttt gaa ttc att atg gtt ctg aat tgt cag gat ttg gca-3 ') of pMYZ180, cut with BamHl / EcoRI was ligated to pMYZ173 cut with BamHI / EcoRI, and the complete coding sequence of the final construct was verified by double-stranded DNA sequencing. pMYZ220: The coding sequence for amino acids 17-264 of human MMP-12 was amplified by PCR with primers Oligo-515 (forward primer with the BamHl site underlined: 5 '-aaa ttt aaa gqa tcc ctt ecc ctg aac age tet here age ctg-3 ') and Oligo-487 (reverse primer with the underlined EcoRl site: 5' -ttt aaa ttt gaa ttc att agt ctc cat here ggg act gaa tgc cac-3 ') from pMYZ180, cut with BamHl / EcoRI was ligated to pMYZ173 cut with BamHI / EcoRI, and the complete coding sequence of the final construct was verified by sequencing of AD? double chain.

PMYZ227: The coding sequence for amino acids "1.00-279 of human MMP-12 was amplified by PCR with primers Oligo-533 (forward primer with the site? Underlined: 5 '-aaa ttt aaa cat atg ttc agg gaa atg cca ggg ggg ecc gta tgg-3 ') and Oligo-486 (reverse primer with the underlined EcoRl site: 5' -ttt aaa ttt qaa ttc att atg gtt ctg aat tgt cag gat, ttg gca-3 ') of pMYZldO, cut with? del / EcoRl, ligated to pET-29a cut with? del / EcoRl, and the complete coding sequence of the final construct was verified by double-stranded DNA sequencing.

PMYZ228: The coding sequence for amino acids 100-264 of human MMP-12 was amplified by PCR with primers Oligo-533 (forward primer with Ndel underlining site: 5 '-aaa ttt aaa cat atg ttc agg gaa atg cca ggg ggg ecc gta tgg-3 ') and Oligo-487 (reverse primer with the underlined EcoRl site: 5' -ttt aaa ttt gaa ttc att agt ctc cat here ggg act gaa tgc cac-3 ') of pMYZ180, cut with Ndel / EcoRI, was ligated to pET-28a cut with Nde / EcoRI, and the complete coding sequence of the final construct was verified by double-stranded DNA sequencing.

Bacterial expression: Charges of one liter of LB medium (10 g of tryptone, 5 g of yeast extract, 10 g of NaCl) containing ampicillin (200 μg / ml) or wedge (10Q μg / ml) were inoculated with 20 ml of each overnight culture of E. coli BL21 (DE3) cells with the appropriate expression vector. The cells were cultured at 37 ° C temperature to an OD600 of about 0.8 before induction with IPTG (final concentration 1 mM). Incubation was continued at 37 ° C temperature for 4 hours before collecting by centrifugation. The sediments were frozen. Cells immediately and were kept at -20 ° C until use. Aliquots (100 μl) of each cell culture were taken and protein expression was analyzed in 10% PAGE-SDS gels.

Protein purification: Cell pellets, frozen from 1 liter of bacterial cells were thawed, dissolved in 50 mM Tris-HCl, pH 8.0, with 15% glycerol, sonicated with 4 pulses of 10 seconds and centrifuged for 30 minutes. minutes at 20,000 rpm with a JA-20 rotor. After removing the supernatant, the inclusion bodies were dissolved in the pellet overnight at room temperature with 50 ml of 8 M urea, 50 mM Tris-HCl, pH 8.0 with gentle agitation. The next day, the solution was centrifuged for 30 minutes at 20,000 rpm with a JA-20 rotor and the supernatant was used for further purification. Charges of 5-10 ml were applied to a 5 ml HiTrapQ ion exchange column, eluting with 8 M urea, 50 mM Tris-HCl, pH 8.0 (buffer A); After loading the sample and washing the column, the protein was eluted by increasing the salt concentration with a solution of urea d M, 50 mM Tris-HCl, pH 8.0 + 1.0 M NaCl (buffer B) in a linear gradient of 0-50% buffer B in 40 column volumes. Purity was assayed in the fractions with protein elution at about 30% buffer B by denaturing SDS-PAGE and mass spectrometry, and fractions containing MMP-12 were pooled together. Charges of 5-10 ml of this solution were taken, acidified to a final concentration of 10% acetic acid and injected onto a preparative C4-RP-HPLC column (Vydac 214RP1022; 300 A, 10 μm, 22x250 mm) with a flow rate of 10 ml / min at a Waters HPLC chromatography work station. The starting buffer was H20 / 0.1% TFA (buffer A) and the elution buffer was 90% CH3CN / 10% H2O / 0.1% TFA (buffer B). The pure protein eluted as a single peak at approximately 35% B buffer and the protein-containing fractions were analyzed again by denaturing SDS-PAGE and mass spectrometry. The pooled fractions of MMP-12 protein were frozen in liquid N2 and lyophilized for 3-5 days. "The lyophilized protein was stored at -20 ° C temperature.

Value of K ". : 5.4 μM Assay buffer: The assay is carried out in buffer containing 50 mM HEPES and 10 mM CaCl 2, pH 7.0. Enzyme concentrations for IC5U determinations: 0.3 μg / ml Enzyme concentrations for K determinations: 0.044-0.98 μg / ml.

MMP-13 (human collagenase 3) Rat pro-MMP-13 was obtained according to the protocol of Roswit, Arch. Biochem. Biophys., 225, 285-295 (1983) and activated by incubation in trypsin 1:10.

Assay Buffer: The assay is carried out in buffer containing 20 mM Tris, pH 7.5, 250 mM NaCl, 5 mM CaCl 2, 0.05% NaN 3, 0.005% Brij. Enzyme concentrations for IC5U determinations: 0.3 μg / ml.

The IC50 values of the selected compounds are given in the following table 3. The compound numbers represent the compounds described in table 1: ta a 3 The following data illustrates the selectivity of the examples of the invention to MMP-2, MMP-3, MMP-8, MMP-9, MMP-12 and MMP-13. The sample numbers represent the examples described in the experimental part: * IC 50 table 4 In vitro functional assays 1. Human alveolar macrophages Human alveolar macrophages were obtained by bronchoscopy of healthy smoking volunteers. The cells were centrifuged and resuspended at 2 x lOVml. Traffic of alveolar macrophages (+ or - lipopolysaccharide 2.5 μg / ml) was induced through an artificial basement membrane (Matrigel) by human MCP-1 (5 ng / mlj for a period of 48-98 hours). (la, 2/3, 5/3) -2- { [4 '-chloro- (1, 1' -biphenyl) -4-yl] carbonyl.}. -5- [(1,3-dihydro) - 1, 3-dioxo-2H-isoindol-2-yl) methyl] cyclopentanecarboxylic acid inhibited the trafficking induced by MCP-1 of human alveolar macrophages to t-raves of this artificial basement membrane (IC50 <1 μM).

Murine peritoneal macrophages Mouse murine peritoneal macrophages were obtained 5 days after an intraperitoneal injection of ti-oglicolate. The cells were centrifuged and resuspended at 2 x 106 / ml. Traffic of peritoneal macrophages was induced through an artificial basement membrane (Matrigel) by murine MCP-1 (5 ng / ml) for a period of 48-98 hours. The acid (lo., 2/3, 5/3) - 2-. { [4'-chloro- (1,1'-biphenyl) -4-yl] carbonyl} -5- [(1,3- dihydro-l, 3-di oxo-2 H -isoi ndo l-2-yl) methyl] cyclopentanecarboxylic acid inhibited the trafficking of murine peritoneal macrophages through this artificial basement membrane (IC50 = 1 μM).

Production of TNFo? induced by LPS in mice The inhibitory properties in vivo of the selected compounds can be determined using a model of in vivo production of TNFa induced by murine LPS. BALB / c mice (Charles River Breeding Laboratories, Kingston, NY) are treated in groups of ten with vehicle or compound. After one hour, endotoxin (lipopolysaccharide (LPS) of E. coli 100 mg) is administered intraperitoneally (ip). After 90 minutes, the animals are sacrificed by asphyxiation with carbon dioxide and the plasma of the individual animals is obtained by cardiac puncture in heparinized tubes. The samples are clarified by centrifugation at 12,500 g for 5 minutes at 4 ° C temperature. The supernatants are decanted into new tubes, which are stored as needed at -20 ° C. Serum TNFa levels are measured using a commercial murine TNF ELISA kit (Genzyme).

Other embodiments of the invention will become apparent to one skilled in the art upon consideration of this specification or the practice of the invention described herein. It is understood that the specification and examples are considered illustrative only and the true scope and spirit of the invention is indicated by the following claims.

Abbreviations: DMF: N, N-dimethylformamide RT: Room temperature THF: Tetrahydrofuran Preparation examples EXAMPLE 1 Acid 2 - '[2 - (1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl) ethyl] -4- (4'-ethoxy - [1-, 1'-biphenyl] -4-yl) -4-oxobutanoic Intermediate ÍA 4 -Etoxi -1, 1 '-biphenyl Iodoethane (68.7 g, 35.57 ml, 440.6 mmol) was added to a suspension of 50 g (170.2 mmol) of 4-hydroxy-l, 1'-biphenyl and 40.6 g (293, 75 mmol) of K2CO3 in 600 ml of acetone. The resulting reaction mixture was stirred at reflux for 16 hours. After cooling to room temperature, the acetone was removed under reduced pressure, the residue was dissolved in ethyl acetate and extracted with water. The aqueous layers were extracted 3 times with ethyl acetate, the combined organic phases were dried (Na2SO4) and evaporated to yield 56 g of the desired compound as a colorless solid.

Yield: 56 g (96%) NMR-H (d (1-DMSO): 7.55-7.65 (m, 4H), 7.42 (t, J = 8 Hz, 2H), 7.3 (t, J = 8 Hz, 1H), 6.95-7.05 (m, 2H), 4.07 (q, J = 7 Hz, 2H), 1.35 (t, J = 7 Hz, 3H).

Intermediate IB 2 - . 2-Bromo-l - (4'-ethoxy - [1,1'-biphenyl] -4-yl) -1-ethanone A solution of 56 g (282 mmol) of intermediate IA in 1.5 1 of CH 2 C 12 was cooled to 0 ° C, and placed under an argon atmosphere. Bromoacetyl bromide (85.5 g, 36, d ml, 423 mmol) was added and then A1C13 (37.41 g, 2d0.53 mmol) was added in portions over 60 minutes. After the addition was complete, the mixture was stirred for 20 hours and warmed to room temperature. The mixture was then poured onto a stirred mixture of 2 kg of ice / 500 ml of concentrated HCl. The organic layer was separated, washed with 2 N HCl and water, dried (Na 2 SO 4) and evaporated. The crude product was purified by recrystallization (acetonitrile) to afford 49.3 g (54%) of a white solid.

NMR-'H (d (1-DMS0): d, Od (d, J = d Hz, 2H), 7.81 (d, J = 8 Hz, 2H), 7.73 (d, J = 8 Hz , 2 Hz), 7.06 (d, J = 8 Hz, 2H), 4.95 (s, 2H), 4.1 (q, J = 7 Hz, 2H), 1.36 (t, J = 7 Hz, 3H).

Intermediate 1C 2- [2- (1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl) ethyl] -2 - [2 - (4'-ethoxy - [1,1 '-biphenyl] Di (tert-butyl) -4-yl) -2-oxoethyl] malonate A solution of intermediate 5F (3.2 g, 10 mmol) in 50 ml of DMF was added dropwise to a suspension of NaH (500 mg, 12.5 mmol) in 20 ml of DMF and stirred for 30 minutes at room temperature. room temperature. Intermediate IB (3.9 g, 10 mmol) in 30 ml of DMF was added slowly and the resulting mixture was stirred for 4 hours at room temperature. The reaction was quenched with a saturated solution of NH 4 Cl, extracted twice with diethyl ether, washed with saturated NaHCO 3, water and brine, dried (Na 2 SO 4) and evaporated. The crude product was purified using flash chromatography (hexane / ethyl acetate: l / 1).

Yield: 4.96 g (71%) NMR-'H (d? -DMSO): 8.02 (d, J = 8 Hz, 2H), 7.68-7.61 (m, 8H), 7.05 (d, J = d Hz, 2H ), 4.1 (q, J = 7 Hz, 2H), 3.72 (s, 2H), 3.55-3.65 (m, 2H), 2.26-2, d7 (m, 2H) , 1.33-1.4 (m, 18H).

EXAMPLE 1 2- [2- (1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl) ethyl] -4- (4'-ethoxy- [1,1 '-biphenyl] -4 acid. -il) -4-oxobutanoic 2.2 g (3.51 mmol) of intermediate 1C was added in one portion to a cooled (0 ° C) mixture of CH2C12 and trifluor acetic acid 1: 1. The reaction mixture was stirred overnight at room temperature, evaporated and dried in vacuo. The residue was dissolved in 5 ml of dioxane and heated at reflux for 5 hours. The reaction mixture was evaporated, the residue was triturated with diethyl ether, stirred for 15 minutes and filtered. The remaining solid was dried in vacuo. Yield: 1.21 g (73%) 1H-NMR (d6-DMSO): 12.28 (s, 1H), 8.02 (d, J = 8 Hz, 2H), 7.67-7.9 (m, 8H), - 7.05 (d, J = d Hz, 2H), 4.1 (q, J = 7 Hz, 2H), 3.72 (t, J = 7.5 Hz, 2H) , 3.49 (dd, J = 17.5 Hz, J = d Hz, 2H), 3.28 (dd, J = 17.5 Hz, J = 5 Hz, 2H), 2.78-2.95 (m, 1H), 1.76-2.13 (m, 2H), 1.38 (t, J = 7 Hz, 3H).

The racemate of Example 1 was separated into its pure enantiomers by chiral HPLC using a commercially available 5 μm Kromasil KR 100-5-CHI-DMB phase. A solvent mixture consisting of 50% isohexane and a 50% mixture of tert-butyl methyl ether / dichloromethane / glacial acetic acid (480: 40: 1) at a constant flow rate of 25 ml / min was used.

Example 2: Acid (+) -2- [2- (1,3-dioxo-l, 3-dihydro-2H-isoindol-2-yl) ethyl] 4- (4'-ethoxy - [1,1'-biphenyl] -4-yl) -4-oxobutanoic acid Fastest Elution Enantiomer: Yield: 374 mg (42%) [c.] 23n = + 6.33 ° (c = 0.47 in THF) Example 3 Acid (-) -2- [2- (1, 3-dioxo-l, 3-dihydro-2H-isoindol-2-yl) ethyl] 4- (4'-ethoxy - [1,1 'bif nil] - 4-yl) -4-oxobutanoic Slower elution enantiomer: Yield: 321 mg (36%) Example 4 Acid (+) -4- (4'-chloro- [1,1'-biphenyl] -4-yl) -2- [2- (1,3-dioxo-1,3-dihydro-2H-isoindol -2 -yl) ethyl] -4-oxobutanoic The compound of Example 4 was prepared according to the procedure given for Example 268 in WO 96/15096. [c. D = + 5.5 ° (c = 0.525 in THF).

Example 5 Acid (rae) -4- [4 '- (acetyloxy) - [1,1'-biphenyl] -4-yl] -2- [2- (1,3-dioxo-1 (3-dihydro-2H-) indole-2-yl) ethyl] -4-oxobutanoic acid Intermediate 5A Acetate 4 '- (2-bromoacetyl) - [l, l' -biphenyl] -4-yl A solution of 50 g (236 mmol) of [1, 1 '-biphenyl] -4-yl acetate in 500 ml of dichloromethane was placed under argon and cooled to 0 ° C. Bromacetyl bromide (31.6 ml, 363 mmol) was added, followed by aluminum chloride (94.3 g, 707 mmol.), Which was added in portions with vigorous stirring for 30 minutes. 0 ° C temperature for another 30 minutes and at room temperature overnight, then the mixture was slowly poured into 500 ml of ice-cold 10% HCl and extracted three times with dichloromethane, the organic layer was dried over Na2SO4. filtered and evaporated The residue was triturated with diisopropyl ether / isopropanol 1: 1, filtered and the remaining solid was dried in vacuo.

Yield: 73.3 g (93.4%) NMR-'H (CDC13): d = 2.34 (s, 3H), 4.48 (s, 2H), 7.21 (m, 2H), 7.66 (m, 4H), d, 0d ( m, 2H).

Intermediate 5B 2 - . 2 - (Benzyloxy) -1-ethanol Ethylene glycol (742.5 g, 11.96 mmol) was added to a solution of sodium hydroxide lentils (475.2 g, 11, dd mmol) in 4-50 ml of water maintained at dO ° C of temperature. Then benzyl chloride (302.8 g, 2.39 mmol) was added at 65 ° C temperature, and the resulting suspension was vigorously stirred at 120 ° C overnight. After cooling to room temperature, the mixture was poured into ice water and extracted five times with diethyl ether. The combined organic layers were washed with brine, dried over Na 2 SO 4, filtered and evaporated. The remaining residue was then distilled in vacuo and the relevant fractions were collected (e.g. 95-125 ° C at 0.1-1 mbar (10-100 Pa)).

Yield: 175 q (48.1%) of a colorless liquid NMR-? (CDC13): d = 2.09 (tr, 1H), 3.60 (m, 2H), 3.77 (m, 2H), 4.56 (s, 2H), 7.35 (m, 5H) .

Intermediate 5C Benzyl -2-chloroethylether Thionyl chloride (41.2 mL, 567.6 mmol) was slowly added to a mixture of intermediate 5B (90 g, 80% pure, 473.1 mmol) and N, N-dimethylaniline (76.5 mL, 597 , 5 mmol) maintaining the reaction temperature at 50 ° C by cooling with ice-water. After stirring at 50 ° C temperature for 1 hour, additional portions of N, N-dimethylaniline (15.3 ml, 119.5 mmol) and thionyl chloride were added. (8.2 rrl, 113.5 mmol), and the mixture was stirred at 50 ° C for another 2 hours and at room temperature overnight. The solution was then poured into an ice-water mixture (200 ml) and conc. HCl. (100 ml) and extracted three times with dichloromethane. The combined organic layers were washed twice with 10% HCl and twice with water, dried over NaSO 4, filtered and evaporated. The remaining residue was then distilled in vacuo (water pump) and the relevant fractions were collected.

Yield: 69.1 g (85.5%) of a colorless liquid.

NMR-'H (CDC13): d = 3.69 (m, 4H), 4.59 (s, 2H), 7.35 (m, 5H) Intermediate 5D 2- [di (tert-butyl) 2- (benzyloxy) ethyl] malonate] Di (tert-butyl) malonate (151.4 g, 686 mmol) at 50 ° C was added dropwise to a suspension of potassium tert-bucylate (77 g, 686 mmol) in 500 ml of tert. -butanol. Sodium iodide (10.33 g) was then added, followed by the dropwise addition of intermediate 5C (117.1 g, 686 mmol) at 40-50 ° C temperature. The resulting slurry was stirred at 70 ° C for two days. During this time, two additional portions of potassium tert-butylate (15.4 g each, 70 mmol) were added. The mixture was then poured onto ice-water and extracted three times with diethyl ether. The organic layers were dried over NaSO4, filtered and evaporated. The crude product was finally purified by column chromatography using a gradient of cyclohexane / ethyl acetate (70: 1-> 15: 1).

Performance; 134 g (55.8%) of a colorless oil.

NMR-'H (DMSO-d6): d = 1.38 (s, 18H), 1.96 (q, 2H), 3.31 (tr, 1H), 3.41 (tr, 2H), 4, 43 (s, 2H), 7.31 (m, 5H).

Intermediate 5E Di (tert-butyl) 2- (2-hydroxyethyl) malonate A solution of intermediate 5D (46.5d g, 132.9 mmol) in 300 ml of ethanol was hydrogenated at atmospheric pressure in the presence of 10% palladium on carbon (2.0 g). After stirring for 3 hours at room temperature, another 1.0 g portion of palladium catalyst was added and stirring was continued at room temperature overnight. The mixture was then filtered through Celite, evaporated and the crude product was purified by column chromatography using a gradient of dichloromethane / methanol (70: 1-> 30: 1).

Yield: 23.3 g (67.2%) of a pale yellow oil.

NMR-1 (CDCl 3): d = 1.47 (s, ldH), 1.96 (tr, 1H), 2.0d (q, 2H), 3.36 (tr, 1H), 3.72 ( q, 1H).

Intermediate 5F 2- (1, 3-Dioxo-1,3-dihydro-2H-isoindol-2-yl) ethyl] malonate di (tert-bu tyl) They were added successively to a stirred solution of intermediate 5E (30.0 g, 115.2 mmol) in 255 ml of dry THF, phthalimide (21.4 g, 144.1 mmol), triphenylphosphine (35.1 g, 132, 5 mmol) and, at 0 ° C temperature, diethyl azodicarboxylate 122, 1 g, 126, d mmol). The resulting solution was stirred overnight by warming to room temperature, then diluted with ethyl acetate and washed twice with water and with brine. The organic phase was dried over Na 2 SO, filtered and evaporated. The crude product was finally purified by column chromatography using a gradient of cyclohexane / dichloromethane / ethyl acetate (7: 1: 1-> 5: 1: 1).

Yield: 10.02 g (22.3%) of a white solid.

NMR-'H (DMSO-d: d = 1.37 (s, IdH), 2.03 (q, 2H), 3.30 (tr, 1H), 3.63 (tr, 2H), 7.85 (m, 4H).

Intermediate 5G 2- . { 2 - [4 '- (Acetyloxy) - [1,1'-biphenyl] -4-yl] -2-oxoethyl} -2 - (1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl) ethyl] malona to di (tert-butyl) A solution of intermediate 5F (6.5 g, 16.69 mmol) in 60 ml of dry THF was added slowly to 0 ° C of temperature under argon atmosphere to a suspension of sodium hydride (0.51 g, suspension 80% in mineral oil, 16.86 mmol) in 30 ml of dry THF. After stirring at 30-40 ° C temperature for 30 minutes, the mixture was again cooled to 0 ° C temperature and a solution of intermediate 5A (5.6 g, 16, d6 mmol) was added dropwise at 60 ml of dry THF. The mixture was then stirred overnight, heating to room temperature. Additional portions of sodium hydride (0.1 g, 3.4 mmol) and intermediate 5A (1.12 g, 3.4 mmol) were added at 0 ° C temperature, and stirring was continued at room temperature for another 3 hours The reaction mixture was quenched by the addition of a saturated solution of ammonium chloride (100 ml) and brine (200 ml) and extracted twice with ethyl acetate. The organic phase was dried over Na 2 SO 4, filtered and evaporated. The crude product was finally purified by column chromatography using a gradient of dichloromethane / ethyl acetate (50: 1-> 30: 1).

Yield: .4, 41 g (41.2%) of an off-white solid.

NMR-'K (DMSO-d0): d = 1.36 (s, 18H), 2.32 (m, 5H), 3.61 (tr, 2H), 3.73 (s, 2H), 7, 28 (d, 2H), 7.81 (m, 8H), 6.04 (d, 2H).

Example 5 Acid (rae) -4 - [4 '- (acetyloxy) - [1,1'-biphenyl] -4-yl] -2 - [2- (1,3-dioxo-1,3-dihydro-2H-isoindole -2 -yl) ethyl] -4 -oxobu tanoic Intermediate 5G (400 mg, 0.62 mmol) was dissolved at 0 ° C in a mixture of dichloromethane (5 ml) and trifluoroacetic acid (5 ml). After stirring at room temperature for 1.5 hours, 10 ml of toluene was added and the reaction mixture was evaporated. The residue was dried in vacuo, then redissolved in 20 ml of dioxane, and the solution was heated to reflux for 6 hours. The mixture was evaporated to dryness, the residue was triturated with diethyl ether, filtered and the remaining solid was dried in vacuo to provide the final product.

Yield: 261 mg (86.2%) of an off-white solid.

NMR-H (DMSO-d6): d = 1.95 (m, 2H), 2.31 (s, 3H), 2, d9 (m, 1H), 3.38 (m, 2H), 3, 72 (tr, 2H), 7.28 (d, 2H), 7.84 (m, 8H), 8.07 (d, 2H), 12.33 (br, 1H).

Example 6 Acid (rae) -2- [2- (1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl) ethyl] -4- (4'-hydroxy - [1,1'-biphenyl] -4-il) -4-oxobutanoic Intermediate 6A 2- [2- (1, 3-Dioxo-l, 3-dihydro-2H-isoindol-2-yl) ethyl] -2- [2- (4'-hydroxy- [1,1'-biphenyl] -4 di- (tert-butyl) -2- oxoethyl] malonate Anhydrous potassium carbonate in fine powder (2.15 g, 15.58 mmol) was added to a solution of intermediate 5G (2.0 g, 3.12 mmol) in 90 ml of a THF / methanol / ethanol mixture (30 g. : 50: 10) The resulting suspension was vigorously stirred at room temperature for 45 minutes, then diluted with ethyl acetate and filtered, the filtrate was concentrated in vacuo to half its original volume, diluted again with acetate ethyl acetate and then poured into an ice-cold pH 4 buffer solution, the aqueous phase was extracted twice with ethyl acetate, and the organic layers were dried over Na2SO4, filtered and evaporated. , 700 mg was dissolved (approximately 1.1 mmol) of the residue (containing some material with the ring open) in 20 ml of dichloromethane and 1-hydroxy-1H-benzotriazole hydrate (189 mg, 1.23 g) was added at 0 ° C. mmol) and N '- (3-dimethylaminopropyl) -N-ethylcarbodiimide hydrochloride (229 mg, 1.18 mmol). The reaction mixture was stirred at room temperature for 3 days, then diluted with dichloromethane and washed twice with a buffer solution at pH 4 and with a saturated solution of sodium hydrogen carbonate. The organic phase was dried over Na2SO4, filtered and evaporated. The crude product was finally purified by column chromatography using a gradient of dichloromethane / methanol (100: 1-> 80: 1).

Yield: 494 mg (71%) of a white solid.

NMR-'H (DMSO-d6): d = 1.38 (s, 18H), 2.31 (m, 2H), 3.60 (m, 2H), 3.70 (s, 2H), 6, 90 (d, 2H), 7.61 (d, 2H), 7.78 (m, 6H), 8, 00 (d, 2H), 9, 76 (s, 1H).

EXAMPLE 6 Acid (rae) -2 - [2 - (1,3-dioxo-1,3-dihydro-2H-i or indole-2-yl) ezyl] -4 - (4'-hydroxy - [1 , 1 '-biphenyl] -4 -yl) -4-oxobutanoic acid Intermediate 6A (490 mg, 0.82 mmol) was dissolved at 0 ° C in a mixture of dichloromethane (7.5 ml) and trifluoroacetic acid (7.5 ml). After stirring at room temperature for 30 minutes, 7 ml of toluene was added and the reaction mixture was evaporated. The residue was dried under vacuum, then redissolved in 15 ml of dioxane and the solution was heated to reflux for 4.5 hours. After cooling to room temperature, diethyl ether (15 ml) was added to the reaction mixture, and the precipitated product was collected by filtration. The filtrate was evaporated to dryness, the residue triturated with diethyl ether, which contained a few drops of methane, and filtered again, providing a second crop of final product.

Yield: 299 mg (82.6%) of a white solid.

NMR-H (DMSO-d6): d = 1.94 (m, 2H), 2.87 (m, 1H), 3.38 (m, 2H), 3.70 (tr, 2H), 6, 89 (d, 2H), 7.61 (d, 2H), 7.75 (d, 2H), 7.85 (m, 4H), 8.01 (d, 2H), 9.76 (sa, 1H) ), 12.30 (sa, 1H).

Example 7 Acid (rae) -4- (4'-chloro- [1,1 'bif' enyl] -4-yl) -2- [2- (4,6-dimethoxy -1,3-dioxo-1,3-dihydro) -2H- isoindol-2-yl) ethyl] -4-oxob tanoi co.

Intermediate 7A 2-Bromo-l- (4'-chloro- [1,1'-biphenyl] -4-yl) -1-ethanone The intermediate was prepared as described in the indicated reference WO 96/15096.

Intermediate 7B 2 - . 2 - [2 - (4, 6-Di-toxi-1,3-di-oxo-1, 3-dihydro-2H-i-soindol-2-yl) -ethyl] -malone to di (tert-butyl) They were added successively to a stirred solution of intermediate 5E (8.4 g, 32.2 mmol) in 100 ml of dry THF, 3,5-dimethoxyphthalimide (10.0 g, 48.3 mmol), triphenylphosphine (11.1 mmol). g, 41.8 mmol) and, at 0 ° C temperature, diethyl azodicarboxylate (6.7 g, 38.6 mmol). The resulting solution was stirred overnight, heating to room temperature. After filtration, the filtrate was diluted with ethyl acetate and washed twice with water and brine. The organic phase was dried over Na2SO4, filtered and evaporated. The crude product was finally purified by column chromatography using a gradient of dichloromethane / ethyl acetate (70: 1-> 30: 1).

Yield: 2.69 g (18.6%) of a white solid, 1H-NMR (DMSO-d6): d = 1.38 (s, 18H), 1.97 (q, 2H), 3.23 (tr, 1H), 3.54 (tr, 2H), 3.92 (s, 6H), 6, d9 (d, 1H), 6.97 (d, 1H).

Intermediate 7C 2- [2- (4'-Chloro- [1,1'-biphenyl] -4-yl) -2-oxoethyl] -2- [2- (4,6-dimethoxy-1,3-dioxo-1, 3-dihydro-2H-isoindol-2-yl) ethyl] -di-tert-butylmalonate A solution of intermediate 7B (2.69 g, 5.9 d mmol) in 30 ml of dry THF at 0 ° C temperature was added dropwise to a suspension of sodium hydride (0.18 g) under an argon atmosphere. g, 80% suspension in mineral oil, 6.04 mmol) in 20 ml of dry THF. After stirring at 30-40 ° C temperature for 3Q minutes, the mixture was again cooled to 0 ° C temperature and a solution of intermediate 3A (1.87 g, 6.04 mmol) was added dropwise to the solution. 20 ml of dry THF. The mixture was then stirred overnight, heating to room temperature. Additional portions of sodium hydride (36 mg, 1.2 mmol) and intermediate 7A (374 mg, 1.2 mmol) were added at 0 ° C temperature, and stirring was continued at room temperature for three days. The reaction mixture was quenched by the addition of a saturated solution of ammonium chloride (40 ml) and brine (80 ml) and extracted twice with ethyl acetate. The organic phase was dried over Na, S04, filtered and evaporated. The crude product was finally purified by column chromatography using a gradient of dichloromethane to dichloromethane / ethyl acetate £ 20: 1-5.

Yield: 1.51 g (37.2%) of a white solid.

R, = 0.51 (dichloromethane / ethyl acetate 20: 1) = 0.59 (cyclohexane / ethyl acetate 1: 1).

ESI-MS: m / z = 678 [M + H] +, 622 ([M + H] + -C 4 H 8), 566 ([M + H] + - 2 x C 4 H b), 548 ([M + H] + -2 x C 4 H 8 / -H20).

Example 7 4- (4'-chloro- [1,1'-biphenyl] -4-yl) -2- [2- (4,6-dimethoxy -1,3-dioxo-1,3-dihydro-2H-isoindole) -2-il) ethyl] -4-oxobutanoic Intermediate 7C (1.5 g, 2.2 mmol) was dissolved at 0 ° C in a mixture of dichloromethane (10 ml) and trifluoroacetic acid (10 ml). After stirring at room temperature for 45 minutes, 10 ml of toluene was added and the reaction mixture was evaporated.

The residue was dried under vacuum, then redissolved in 20 ml of dioxane and the solution was heated to reflux for 6 hours. The mixture was evaporated to dryness, the residue was triturated with diethyl ether, filtered and the remaining solid was dried in vacuo to provide the final product.

Yield: 1.07 g (92.1%) of an off-white solid.

NMR-'H (DMSO-d0): d = 1.89 (m, 2H), 2, d4 (m, 1H), 3.39 (m, 2H), 3.63 (tr, 2H), 3 , 92 (s, 6H), 6, 88 (d, 2H), 6.97 (d, 1H), 7.5d (d, 2H), 7.83 (m, 4H), 8.07 (d, 2H), 12.30 (ss, 1H). Examples 8 and "9 (+) and (-) -4- (4 '-Bromo- [1,1'-biphenyl] -4-yl) -2- [2- (1,3-dioxo-1,3-dihydro-2H- isoindol -2-yl) ethyl] -4-oxobutanoic acid 4- (4'-Bromo- [1,1'-biphenyl] -4-yl) -2- [-2- (1,3-dioxo-1,3-dihydro-2H-isoindol -2- acid was prepared. il) ethyl] -4-oxobutanoic racemic essentially as described in the indicated reference WO 96/15096.

NMR-H (DMSO-d6): d = 1.95 (m, 2H), 2.8d (ra, 1H), 3.38 (m, 2H), 3.72 (tr, 2H), 7, 72 (m, 4H), 7.85 (m, 6H), 8.0d (d, 2H), 12, 33 (br, 1H). 1.0 g (1.97 mmol) of this material was separated into the pure enantiomers by chiral HPLC using a phase Kromasíl KR 100-5-CHI-DMB of 5 μm commercially available.

A solvent mixture consisting of 40% isohexane was used. and a mixture of tert-butylmethylether / dichloromethane / glacial acetic acid (480: 40: 1) at 60% at a constant flow rate of 25 ml / min.

Example 8 Enantiomer A eluting the first: Yield: 309 mg (31%). [a] D- ° = + 3.87 ° (c = 0.458 g / 100 ml THF) Example 9 Enan íomer B eluting the second: Yield: 240 mg (24%) [a] -5.98 ° (c = 0.462 g / 100 ml THF). Examples 10 and 11 Acid (+) and (-) -4- (4'-chloro- [1,1'-biphenyl] -4-yl) -2- [2- (5,7-dioxo-5, 7 -dihydro- 6H- [1, 3] dioxolo [4,5-f] isoindol-6-yl) -ethyl] -4-oxobu anoi co 4- (4'-Chloro- [1,1'-biphenyl] -4-yl) -2- [2- (5,7-dioxo-5,7-dihydro-6H- [1,3] was prepared. -radioxolo- [4, 5-f] isoindol-6-yl) ethyl] -4-oxobutanoic racemic essentially as described in the indicated reference WO 96/15096.

NMR-? (DMSO-dc): d = 1.91 (m, 2H), 2.85 (m, 1H), 3.38 (m, 2H), 3.66 (tr, 2H), 6.26 (s, 2H), 7.39 (s, 2H), 7.58 (d, 2H), 7.62 (m, 4H), 8.0fe (d, 2H), 12.31 (sa, 1H). 0.70 g (1.38 mmol) of this material was separated into its pure enantiomers by chiral HPLC using a commercially available Kromasil KR 100 -5-CHI-MDB 5 μm phase. A solvent mixture consisting of 40% isohexane and a mixture of tert-butylmethylether / dichloromethane / glacial acetic acid (480: 40: 1) at 60% was used at a constant flow of 25 ml / min.

Example 10 Enantiomer A eluting the first: Yield: 261 mg (37.3%) [a] DI = + 13.99 ° (c = 0.955 g / 100 ml of THF) Example 11 Enantiomer B eluting the second: Yield : 228 mg (32.6%) [a. 2d_ D - 15.15 ° (c = 0.991 g / 100 ml of THF) Example 12 4- (4'-cyano- [1,1'-biphenyl] -4-yl) -4-oxo-2- (2- [4 -oxo-1,2,3-benzotriazin-3 (4H) - il] ethyljbutanoic Intermediate 12A (2-Bromoacetyl) - [1,1 '-biphenyl] -4-carbonyl trile 4.68 g of aluminum chloride (35.15 mmol) are dissolved in 45 ml of dichloromethane and treated dropwise with 3.38 g (16.74 mmol) of bromacetyl bromide at 0 ° C temperature. After 30 minutes, 3 g (16.74 mmol) of 4-cyanobiphenyl dissolved in 15 ml of dichloromethane are added dropwise. The reaction mixture is stirred overnight at room temperature, added to ice water and extracted twice with dichloromethane. The organic phase is washed with water and brine, dried and evaporated. The residue is triturated with petroleum ether, filtered and dried. Yield: 4.24 g (83%). 200 MHz, NMR-? (CDC13): 4.49 (s, 2H), 7.73 (m, 6H), 8.11 (d, 2H).

Intermediate 12B 2 - . 2 - [2 - [4'-Cyano- [1,1'-biphenyl] -4-yl) -2-oxoethyl] -2-. { 2- [4-oxo-1,2,3-benzotriazin-3 (4H) -yl] ethyl-malonate di (tert-butyl) A solution of 2.59 g (6.67 mmol) of intermediate 13C in 20 ml of DMF is added drop by drop to a suspension of 0.333 g of NaH (60% in mineral oil) in 20 ml of DMF. The mixture is stirred for 30 minutes and a 2 g solution is added. (6.67 mmol) of intermediate 12A in 20 ml of DMF The mixture is stirred for 2.5 hours at room temperature, poured into 150 ml of NH 4 Cl solution and extracted with ethyl acetate. Organic phase with water and brine, dried over MgSO and evaporated, The residue was purified by chromatography to give 0.62 g (15%). 200 MHz, NMR-'H (CDC13): 2.71 (s, 2H), 3.81 (s, 2H), 4.53 (m, 2H), 7.75 (m, 7H), 7.91 (m, 1H), 8.10 (m, 3H), 8.30 (m, 1H).

Example 12 Acid 4 - (4'-cyano- [1,1'-biphenyl] -4-yl) -4 -sxo-2-. { 2 - [4-oxo-1, 2, 3-benzotriazin-3 (4H) -yl] ethyl} butanoic Dissolve 0.61 g (1 mmol) of intermediate 12B in 10 ml of dichloromethane / trifluoroacetic acid (1: 1) at 0 ° C temperature and stir the mixture for 2 hours at room temperature. After adding toluene, the reaction mixture is evaporated to dryness and the residue is suspended in 10 ml of dioxane. The solution is stirred under reflux for 6 hours and at room temperature overnight. The solvent is removed in vacuo and the residue is purified by HPLC to afford 96 mg (21%). 200 MHz, NMR-'H (CDCl 3): 2.28 (m, 2H), 2.49 (m, 2H), 3.26 (m, 1H), 4.58 (m, 2H), 7.72. (m, 10H), 8.16 (m, 1H), 8.38 (m, 1H).

Example 13 4-OXO-2- acid. { 2- [4-oxo-l, 2, 3-benzotriazin-3 (4H) -yl] tyl} -4 - [4 '- (trifluoromethoxy) - [1, 1' -biphenyl] -4 -yl] bu tanoic Intermediate 13A 1- [4 '- (Trifluoromethoxy) - [1,1' -biphenyl] -4 -yl] -1-ethanone To a mixture of 30 g (126 mmol) of 4- (txifluoromethoxy) -1,1-biphenyl and 21 g of aluminum t-chloride (157 mmol) in 120 ml of nitrobenzene at a temperature below 20 ° C were added.9.87 g (126 mmol) of acetic acid chloride. The reaction mixture is stirred for 2 hours at 0 ° C temperature, added to 240 ml of ice-water and 42 ml of concentrated HCl and extracted with ethyl acetate. ethyl. The organic phase is washed with water and brine and the solvents are removed in vacuo. The solvent is triturated with petroleum ether, filtered and dried. Another batch can be obtained from the filtrate after crystallization at 4 ° C of temperature, giving a total of 23.5 g (66%). 200 MHz, NMR-'H (CDCl 3): 2.65 (s, 3H), 7.33 (d, 2H), 7.58 (m, 4H), 8, 06 (d, 2H).

Intermediate 13B 2 -Bromo-1 - [4 '- (trifl uoromethoxy) - [1, 1' -biphenyl] - 4 -i l] -1 ethanone 12.27 g (43.8 mmol) of intermediate 13A are dissolved in a mixture of 150 ml of methanol, 150 ml of ethanol and 50 ml of ether with gentle stirring. 5.914 g (56.9 mmol) of trimethyl ester of boronic acid are added at room temperature and 7.35 g of bromine (45.9 mmol) are added dropwise. The mixture is stirred, until the disappearance of the rs-o-brown color. The solvents are removed in vacuo and the residue is purified by chromatography (cyclohexane / ethyl acetate; affording 6.2 g (39%). 200 MHz, NMR-'H (CDCl 3): 4.49 (s, 2H), 7.33 (d, 2H), 7.68 (dd, 4H), 8.10 (d, 2H).

Intermediate 13C 2- . { 2- [4-Oxo-l, 2, 3-benzotriazin-3 (4H) -yl] ethyl} di (tert-butyl) malonate 46.2 g (177 mmol) of intermediate 5E are dissolved in 600 ml of THF. 69.8 g (266 mmol) of triphenylphosphine and 39.2 g (266 mmsl) of 1, 2, 3-benzotriazin-4 (3H) -one are added. 46.4 g (266 mmol) of DEAD are added dropwise. The reaction mixture is stirred overnight at room temperature. The solvent is removed in vacuo and the product is obtained by chromatography (cyclohexane / ethyl acetate 6: 1).

Yield: 51.8 g (63%). 200 MHz, NMR-'H (CDC13): 1.43 (s, 18H), 2.43 (quadr., 2H), 3.30 (t, 1H), 4.57 (t, 2H), 7, 80 (m, 1H), 7.94 (m, 1H), 8.16 (dd, 1H), 8.36 (dd, 1H).

Intermediate 13D 2- . { 2- [4-Oxo-1, 2, 3-benzotriazin-3 (4H) -yl] ethyl} -2- . { 2-oxo-2- [4 '- (trifluoromethoxy) - [1,1'-biphenyl] -4-yl] ethyl} diTterc-butyl malonate) It is added dropwise to a suspension of 0.52 g (12.9 mmol) of sodium hydride (60% suspension in mineral oil) in 20 ml of DMF, a "solution of 4.04 g (10.37 g). mmol) of intermediate 13C in 30 ml of DMF After stirring for 30 minutes at room temperature a solution of 3.73 g (10.37 mmol) of intermediate 13B in 30 ml of DMF is added dropwise and stirred The reaction mixture was poured for 2 hours at room temperature, the reaction mixture was poured into a solution of NH4C1, extracted with ethyl acetate and the organic phase was washed with water and brine, after drying and evaporation of the solvents, the purify the product by chromatography to give 2.09 g (30%). 200 MHz, NMR-? (CDC13): 2.70 (m, 2H), 3.82 (s, 2H), 4.54 (m, 2H), 7.32 (d, 2H), 7.62 (m, 5H), 8 , 07 (m, 4H), 8.30 (m, 1H).

Example 13 Acid 4 -oxo -2-. { 2- [4 -oxo-l, 2, 3-benzotriazin-3 (4H) -yl] ethyl} -4 - [4 '- (trifluoromethoxy) - [1, 1' -biphenyl] -4-yl] butanoic A solution of 2.09 g (3.13 mmol) of intermediate 13C in 15 ml of dichloromethane and 15 ml of trifluoroacetic acid is stirred at room temperature for 2 hours. After adding toluene, the solvents are removed in vacuo. The residue is dissolved in 30 ml of dioxane, the solution is heated at reflux for 6 hours and stirred at room temperature overnight. The solvents were removed in vacuo, the residue was triturated with ether, the precipitate was collected by filtration and dried, yielding 0.51 g (32%). 200 MHz, NMR-'H (DMSO-d6): 2.18 (m, 2H), 2.98 (m, 1H), 3.51 (m, 2H), 4.52 (m, 2H), 7 , 52 (d, 2H), 7.89 (m, 5H), 8.10 (m, 3H), 3.24 (m? 2H), 12.39 (s, 1H).

Example 14 and 15 Acid (+) and (-) - (1S *, 2S *, 5R *) -2- [(4'-chloro- [1,1'-biphenyl] -4-yl) caxbonyl] - 5 - [(1,3-di-oxo-1,3-dihydro-2H-y soindol-2-yl) methyl] cyclopentanecarboxylic acid The racemic compound was prepared essentially following the procedure for Example 360 of WO 96/15096. 4.20 g (8.61 mmol) of this material were separated into the pure enantiomers by chiral HPLC using a commercially available Kromasil KR 100-5-CHI-MDB phase of 5 μl. A solvent mixture consisting of 30% isohexane and a mixture of tert-butylmethylether / dichloromethane / glacial acetic acid (480: 40: 1) at 70% was used at a constant flow rate of 25 ml / min.

Example 14 Enantiomer A eluting first: Yield: 1.72 g (41.0%) [a] + 44.26 ° (c = 0.464 g / 100 ml THF) Example 15 Enantiomer B eluting second: Yield: 1.59 g (37.9%) [c D2 '= - 43.70 ° (c = 0.575 g / 100 ml THF) It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Claims (1)

1. CLAIMS Having described the invention as above, the content of the following claims is claimed as property: r 1. The use of compounds with matrix metalloprotease inhibitory activity of generalized formula: (T) xA-B-D-E-C02H wherein (a) (T) XA represents a substituted or unsubstituted aromatic or heteroaromatic moiety selected from the group consisting of: wherein R1 represents H or alkyl of 1 to 3 carbons; and each T represents a substituent group, independently selected from the group consisting of: * the halogens -F, -Cl, -Br and -I; * alkyl of 1 to 10 carbons; *. haloalkyl of 1 to 10 carbons; * haloalkoxy of 1 to 10 carbons; * alkenyl of 2 to 10 carbons; * alkynyl of 2 to 10 carbons; * - (CH2) pQ, wherein p is 0 or an integer from 1 to 4. * -alkenyl-Q, said alkenyl moiety comprising 2 to 4 carbons, and * -alkynyl-Q, said alkynyl moiety comprising 2 to 7 carbons, and Q is selected from the group consisting of aryl of 6 to 10 carbons, heteroaryl comprising from 4 to 9 carbons and at least one heteroatom of N, 0 or S, -CN, -CHO, -N02, -C02R2, -OCOR2, - SOR3, -S02R3, -CON (R4) 2, -S02N (R4) a, -C (0) R2, -N (R4) 2, -N (R2) COR2, -N (R2) C02R3, -N ( R2) CON (R4) 2, -CHN4, -OR4 and -SR4; wherein R2 represents H; alkyl of 1 to 6 carbons; aryl of 6 to 10 carbons, heteroaryl comprising from 4 to 9 carbons and at least one heteroatom of N, O or S; or arylalkyl wherein the aryl portion contains from 6 to 10 carbons and the alkyl portion contains from 1 to 4 carbons; or heteroarylalkyl wherein the portion The heteroaryl comprises from 4 to 9 carbons and at least one heteroatom of N, 0 or S and the alkyl portion contains from 1 to 4 carbons; R3 represents alkyl of 1 to 4 carbons; 15 aryl of 6 to 10 carbons; heteroaryl comprising from 4 to 9 carbons and at least one heteroatom of N, O or S; arylalkyl in which the aryl portion contains from 6 to 10 carbons and the portion The alkyl contains from 1 to 4 carbons; or heteroaryl alkyl wherein the heteroaryl portion comprises from 4 to 9 carbons and at least one heteroatom N, O or S and the alkyl portion contains from 1 to 4 carbons; 25 R 14 represents H; alkyl of 1 to 12 carbons; aryl of 6 to 10 carbons; heteroaryl comprising from 4 to 9 carbons and at least one heteroatom of N, 0 or S; arylalkyl in which the aryl portion contains from 6 to 10 carbons and the alkyl portion contains from 1 to 4 carbons; heteroarylalkyl wherein the heteroaryl portion comprises from 4 to 9 carbons and at 1G minus one hetero atom of N, S or O and the alkyl portion contains 1 to 4 carbons; alkenyl of 2 to 12 carbons; alkynyl of 2 to 12 carbons; - (CqH2qO) rR5 where q is l to 3, r is l to 3 and R5 is H, with the proviso that q is greater than 1, or alkyl of 1 to 4 carbons, or phenyl; alkylenethium terminated with H, alkyl of 1 to 4 carbons, or phenyl; alkyleneamine terminated with H, alkyl of 1 to 20 4 carbons or phenyl; - (CH2) SX where s is 1 to 3 and X is halogen; -C (0) OR2; or -C (0) R2; and with the conditions that a) when two groups R4 are located on a nitrogen, can be linked by a bond forming a heterocycle, and b) an unsaturation in a residue that is attached to Q or that is part of Q is separated from any N, O or S of Q by at least a carbon atom, and x is 0, 1 or 2; (b) B represents a bond or an optionally substituted aromatic or heteroaromatic ring containing 0 to 2 substituents T, said substituents having independently the meaning specified in (a), wherein the B rings are selected from the group consisting of: wherein R1 is as defined above; (c) D represents wherein R2 is defined as above and each R2 may be the same or different; (d) E represents a chain of n carbon atoms carrying m substituents R6, said R groups being independent substituents, or constituting spiro or non-spiro rings in which a) two R6 groups are joined and taken together with the atom (s) of the chain to which the two R6 groups are attached, and with any intermediate atom of the chain, they constitute a ring of 3-7 members, or b) a group R6 is attached to the chain in which this group R6 resides, and taken together with the atom (s) of the chain to which the group is attached. group R ?, and with any intermediate atom of the chain, constitutes a ring of 3-7 members; wherein n is 2 or 3; m is an integer from 1 to 3; each R6 group is independently selected from the group consisting of: * fluorine; * • hydroxyl, with the proviso that an individual carbon atom can not carry more than one hydroxyl group; * alkyl of 1 to 10 carbons; aryl of 6 to 10 carbons; heteroaryl comprising from 4 to 9 carbons and at least one heteroatom of N, O or S; arylalkyl in which the aryl portion contains from 6 to 10 carbons and the alkyl portion contains from 1 to 8 carbons; heteroarylalkyl in which the heteroaryl portion comprises from 4 to 9 carbons and at least one heteroatom of N, O or S and the alkyl portion contains from 1 to 8 carbons; alkenyl of 2 to 10 carbons; arylalkenyl, the aplo portion containing from 6 to 10 carbons and containing the alkenyl portion from 2 to 5 carbons; Heteroarylalkyl, containing the heteroaryl portion of 4 to 9 carbons and at least one heteroatom of N, O or S and containing the alkenyl portion of 2 to 5 carbons; alkynyl of 2 to 10 carbons; Arylalkynyl, the aryl portion containing from 6 to 10 carbons and containing the alkynyl portion from 2 to 5 carbons; heteroarylalkyl, the heteroaryl portion comprising 4 to 9 carbons and at least one 25 heteroatom of N, 0 or S, and containing the alkynyl portion of 2 to 5 carbons; - (CH) tR7, where t is 0 or an integer from 1 to 5; and R7 is selected from the group consisting of: 10 fifteen twenty 25 • 25 and the corresponding heteroaryl moieties in which the aryl portion of an aryl-containing group R7 comprises from 4 to 9 carbons and at least one heteroatom of N, O or S; where Y represents O or S; R1, R2 and R3 are as defined above, and u is 0, 1 or 2; Y - (CH2) ZR8, wherein v is 0 or an integer from 1 to 4; and Z represents Rs is selected from the group consisting of: alkyl of 1 to 12 carbons; aryl of 6 to 10 carbons; Heteroaryl comprising from 4 to 9 carbons and Four . at least one heteroatom of N, O or S; arylalkyl, the aryl portion containing 6 to 12 carbons and containing the alkyl portion of 1 to 4 carbons; Heteroarylalkyl, the aryl portion comprising 4 to 9 carbons and at least one heteroatom of N, O or S and containing the alkyl portion of 1 to 4 carbons; -C (0) R9, wherein R9 represents an alkyl of 2 to 6 carbons, aryl of 6 to 10 carbons, heteroaryl comprising from 4 to 9 carbons and at least one heteroatom of N, O or S, or arylalkyl in that the aryl portion contains 6 to 10 carbons or is heteroaryl comprising from 4 to 9 carbons and at least one heteroatom of N, 0 or S, and the alkyl portion contains from 1 to 4 carbons; with the conditions that when R8 is -C (0) R9, Z is -S- or -0-; when Z is -O-, R8 can also be - (CqH2qO) rR3 in which q, r and R5 are as defined above; * - '(CH2)? If (R, 0) 3, where it is an integer from 1 to 3; and R, n represents an alkyl of 1 to 2 carbons. and with the proviso that the aryl or heteroaryl portions of any of the groups T or R6 can optionally carry two substituents selected from the group consisting of - (CH2hC (R4) (R3) 0H, - (CH2) yOR4, - (CH2) and SR4, - (CH2) > S. (0) R4, - (CH2) and S (0) 2R4, - (CH2).}. S02N (R4) 2, - (CH2) N (R4), , - (CH2) and N (R4) COR3, -OC (R4) 20- in which both oxygen atoms are connected to the aryl ring, - (CH ^ COR4, - (CH2) and CON (R) 2, - (CH2 ) > C02R4, - (CH2) _V0C0R4, -halogen, -CHO, -CF3, -N02, -CN, and R3, wherein y is from 0 to 4, and R3 and R4 are defined as above, and any two R4 that are attached to a nitrogen may be linked to form a heterocycle; and pharmaceutically acceptable salts and prodrugs thereof for the manufacture of drugs for the treatment and prevention of respiratory diseases. The use of compounds according to claim 1, with matrix metalloprotease inhibitory activity of generalized formula: (T) xA-B-D-E-C02H wherein (a) (T) XA represents a substituted or unsubstituted aromatic or heteroaromatic moiety selected from the group consisting of: represents a substituent group,. independently selected from the group consisting of: * the halogens -F, -Cl, -Br and -I; * alkyl of 1 to 10 carbons; * haloalkyl of 1 to 10 carbons; * alkenyl of 2 to 10 carbons; * alkyl of 2 to 10 carbons; * - (CH2) pQ, wherein p is 0 or an integer from 1 to 4. * -alkenyl-Q, wherein said alkenyl residue comprises from 2 to 4 carbons, and * -alkyl-Q, in the that the said alkyl residue comprises from 2 to 7 carbons, and it is selected from the group consisting of -OR4 and -SR4. in which R4 represents H; alkyl of 1 to 12 carbons; aryl of 6 to 10 carbons; heteroaryl comprising from 4 to 9 carbons and at least one heteroatom of N, O or S; arylalkyl in which the aryl portion contains from 6 to 10 carbons and the alkyl portion contains from 1 to 4 carbons; heteroarylalkyl in which the heteroaryl portion comprises from 4 to 9 carbons and at least one heteroatom of N, S or O and the alkyl portion contains from 1 to 4 carbons; -C (0) OR2; Ó -C (0) R2; and * with the proviso that an unsaturation in a moiety that is linked to Q or that is part of Q is separated from any N, O or S of Q by at least one carbon atom, and x is 0, 1 or 2; (b) B represents an optionally substituted phenyl or thienyl ring containing from 0 to 2 T substituents, said substituents having independently the meaning specified in (a). (c) D represents (d) E represents a chain of n carbon atoms carrying m substituents R6, wherein said R ° groups are independent substituents, or constitute non-spiro rings in which two R6 groups are joined and taken together with the ) , atom (s). of the chain to which the two R6 groups are linked, and with any intermediate atom of the . chain constitute a ring of 5 to 6 members; and in which • n is 2 or 3; m. . is an integer 1 or 2; _: .- each group Rd is selected independently of -;; 'group consisting of: * arylalkyl in which the aryl portion -. contains 6 to 10 carbons and the portion; alkyl contains from 1 to 8 carbons; * - (CH2) tR7, in which. E is 0 or an integer from 1 to 5; and R7 is selected from the group consisting of: in ", those" R "is independently selected from the group consisting of: H, aryl of 6 to 10 carbons - (CH2hZR8, where v is 0 or an integer from 1 to 4, and Z represents Rs is selected from the group consisting of: alkyl of 1 to 12 carbons; aryl of 6 to 10 carbons; heteroaryl comprising from 4 to 9 carbons and at least one heteroatom of N, O or S; arylalkyl, the aplo portion containing 6 to 12 carbons and containing the alkyl portion of 1 to 4 carbons; heteroarylalkyl, the aryl portion comprising 4 to 9 carbons and at least one heteroatom of N, O or S and containing the alkyl portion of 1 to 4 carbons; -C (0) R9, R9 represents alkyl of 2 to 6 carbons, aryl of 6 to 10 carbons, heteroaryl comprising from 4 to 9 carbons and at least one heteroatom of N, O or S, or arylalkyl, containing the aryl portion of 6 to 10 carbons or being heteroaryl comprising from 4 to 9 carbons and at least one heteroatom of N, O or S, and containing the alkyl portion of 1 to 4 carbons; with the conditions that - when R8 is -C (0) R9, Z is S or O; - when Z is O, R8 can also be - (CqH2q0) rR5, where q, r and R5 are as defined above; Y * - (CH2) ttSi (R '°) 3, w being equal to an integer from 1 to 3; and R10 representing an alkyl of 1 to 2 carbons. -with the condition of the aryl or heteroaryl portions of any of the groups T or R6 can optionally carry up to two substituents selected from the group consisting of OR4, N (R4) 2, -OC (R4) 20-, wherein both oxygen atoms are connected to the aryl ring, CON (R4) 2, OCOR4, halogen, -N02, and alkyl of up to 6 carbon atoms being R4 as defined above, - and the pharmaceutically acceptable salts and prodrugs thereof for the manufacture of drugs for the treatment and prevention of respiratory diseases. The use of a compound of claim 1 or 2, wherein at least one of the units A, B, T and Rñ comprises a heteroaromatic ring for the manufacture of drugs for the treatment and prevention of respiratory diseases. The use of a compound of claim 1 or 2, wherein in said unit E, n is 2 and m is 1, for the manufacture of drugs for the treatment and prevention of respiratory diseases. The use of a compound of claim 1 or 2, wherein A is B is p-phenylene and D is a carbonyl group, for the manufacture of drugs for the treatment and prevention of respiratory diseases. The use of a compound of claim 1 or 2, wherein the compound is selected from the following group: 25 25 25 25 25 25 for the manufacture of drugs for the treatment and prevention of respiratory diseases. Compounds of general formula (I ') CO-E-C02H representing a residue 3-carboxyl-5-R7-pentan-1-on-l-il -characterized because the substituents T and R -have the meanings indicated in the following table: An acid compound (+) -2 - [2 - (1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl) ethyl] -4- (4'-ethoxy- [1,1'- biphenyl] -4-yl) -4-oxobutanoic acid. The use of compounds of general formula (I1) in which T is alkoxy (-C, chloride, bromide, fluoride, acetoxy, hydroxy, cyano, trifluoromethyl or trifluoromethoxy, CO-E-C02H represents a residue 3 -carboxyl-5-R'-pent-an-1-on-1-yl- or 2-carboxy 1 - 3 - (R 1 -methyl) cyclopentan-1-yl) carbonyl-, Y R7 represents a group of formula and its salts, for the manufacture of drugs for the treatment and prevention of respiratory diseases. The use of the compound (+) -2- [2- (1, 3-dioxo-l, 3-dihydro-2H-isoindol-2-yl) ethyl-4- (4'-ethoxy- [1,1'-biphenyl] - 4-yl) -4-oxobutanoic for the manufacture of drugs for the treatment and prevention of respiratory diseases. The use of the compound (+) -4- (4'-chloro- [1,1'-biphenyl] -4-yl) -2- [2- (l, 3-dioxo-1,3-dihydro-2H-isoindole -2 -yl) ethyl] -4-oxobutanoic for the manufacture of drugs for the treatment and prevention of respiratory diseases. The use of a compound according to any of claims 1 to 6 or 9 to 11 in the manufacture of a medicament for the treatment of a condition mediated by MMP-2, MMP-3, MMP-9, MMP-12 and / or MMP - 13 A method for the prevention of a condition mediated by MMP-2, MMP-3, MMP-9, MMP-12 and / or NMP-13, characterizes ±) perqué caiprerd- e the administration? of an effective amount of a substance according to any of claims 1 to 6 or 9 to 11. The use of a compound according to any of claims 1 to 6 or 9 to 11 for the treatment or prevention of asthma; chronic obstructive pulmonary disease including chronic bronchitis and emphysema; cystic fibrosis; bronchiectasis, respiratory distress syndrome in adults (ARDS); allergic respiratory disease, including allergic rhinitis; diseases linked to the production of TNF, "including acute pulmonary JTibrotic diseases, pulmonary sarcoidosis, silicosis, miner's pneumoconiosis, alveolar damage in mammals, such as humans, farm animals or domestic animals. The use of a composition with rretakprobsase inhibitory activity of the matrix, characterizes the caiprenfe park with scpp of any of claims 1 to 12 and a pharmaceutically acceptable vehicle for the manufacture of drugs for the treatment and prevention of respiratory diseases. . A composition containing the compounds according to claims 7 or 8. A composition according to claim 16 for the treatment and prevention of acute and chronic inflammatory processes.