Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
  • A61K31/19—Carboxylic acids, e.g. valproic acid
  • A61K31/192—Carboxylic acids, e.g. valproic acid having aromatic groups, e.g. sulindac, 2-aryl-propionic acids, ethacrynic acid 
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

• This invention relates to the use of enzyme inhibitors, and more particularly, to new and known matrix metalloprotease-inhibiting 4-Biarylbutyric Acids and 5-Biaryl- penta-noic Acids and derivatives thereof, for the prevention and treatment of Multiple Sclerosis.
• MS Multiple Sclerosis
• MMPs Matrix metalloproteinases
• MMP-2 Matrix metalloproteinases
• MMP-9 92 kDa gelatinase
• collagenases MMP-1
• MMP-7 macrophage metalloelastase
• MMP-12 membrane-bound MMPs 1 -4.
• Hydroxamic acid derivatives with non-selective MMP-inhibitory activities were shown to be herapeutically efficacous in different animal models of Multiple Sclerosis (J. Neuroimmunol. 1997, 74, 85-94; Ann. Neurol. 1998, 44, 35-46).
• This invention relates to the use for the prevention and treatment of Multiple Sclerosis of compounds having matrix metallprotease inhibitory activity of the generalized formula (I) :
• (T) X A represents a substituted or unsubstituted aromatic 6-membered ring or heteroaromatic 5 - 6 membered ring containing 1 - 2 atoms of N, O, or S.
• T represents one or more substituent groups, the subscript x represents the number of such substituent groups, and A represents the aromatic or heteroaromatic ring, designated as the A ring or A unit.
• N is employed in conjunction with either S or O in the A ring, these heteroatoms are separated by at least one carbon atom.
• the substituent group(s) T are independently selected from the group consisting of halogen; alkyl; haloalkyl; haloalkoxy; alkenyl; alkynyl; -(CH2)pQ in which p is 0 or an integer of 1 - 4; -alkenyl-Q in which the alkenyl moiety comprises 2 - 4 carbons; and alkynyl-Q in which the alkynyl moiety comprises 2 - 7 carbons.
• Q in the latter three groups is selected from the group consisting of aryl, heteroaryl, -CN, -CHO, -N ⁇ 2, -CO2 2 , -OCOR 2 , -SOR 3 , -SO2R 3 , -CON(R4) 2 , -S ⁇ 2N(R 4 )2, -COR , -N(R 4 )2, -N(R 2 )COR 2 , -N(R 2 )CO2R 3 , -N(R 2 )CON(R 4 )2, -CHN4, -OR 4 , and -SR 4 .
• R 2 represents H, alkyl, aryl, heteroaryl, arylalkyl, or heteroaryl- alkyl.
• R 3 represents alkyl, aryl, heteroaryl, arylalkyl, or heteroaryl-alkyl.
• R 4 represents H; alkyl; aryl; heteroaryl; arylalkyl; heteroaryl-alkyl; alkenyl; alkynyl; alkyleneoxy, polyalkyleneoxy, alkylenethio or alkyleneamino terminated with H, alkyl, or phenyl; haloalkyl; lower alkoxycarbonyl; or acyl.
• a heterocycle such as, for example, a morpholine, thiomorpholine, pyrrolidine, or piperidine ring.
• Unsaturation in a moiety which is attached to Q or which is part of Q is separated from any N, O, or S of Q by at least one carbon atom.
• the A ring may be unsubstituted or may carry up to 2 substituents T. Accordingly, the subscript x is 0, l, or 2.
• B represents a bond or an optionally substituted aromatic 6-membered ring or a heteroaromatic 5 - 6 membered ring containing 1 - 2 atoms of N, O, or S.
• B is a ring, it is referred to as the B ring or B unit.
• N is employed in conjunction with either S or O in the B ring, these heteroatoms are separated by at least one carbon atom.
• D represents
• R.2 is defined as above and each R ⁇ may be the same or different.
• E represents a chain of n carbon atoms bearing m substituents R6> in which the R" groups are independent substituents, or constitute spiro or nonspiro rings. Rings may be formed in two ways: a) two groups R ⁇ are joined, and taken together with the chain atom(s) to which the two R6 group(s) are attached, and any intervening chain atoms, constitute a 3 - 7 membered ring, or b) one group R ⁇ is joined to the chain on which this one group R6 resides, and taken together with the chain atom(s) to which the R ⁇ group is attached, and any intervening chain atoms, constitutes a 3 - 7 membered ring.
• the number n of carbon atoms in the chain is 2 to 4, and the number m of R° substituents is an integer of 1 - 3.
• Each group R6 is independently selected from the group consisting of:
• R-7 is selected from the group consisting of:
• R° is selected from the group consisting of:
• R ⁇ represents alkyl of at least two carbons, aryl, heteroaryl, arylalkyl, or heteroaryl-alkyl;
• R 8 when Z is O, R 8 may also be alkyleneoxy or polyalkyleneoxy terminated with H, alkyl, or phenyl;
• aryl or heteroaryl portions of any of the T or R6 groups optionally may bear up to two substituents selected from the group consisting of -(CH 2 ) y C(R 4 )(R 3 )OH, -(CH 2 ) y OR 4 , -(CH 2 ) y SR 4 , -(CH 2 ) y S(O)R 4 -(CH 2 ) y S(O) 2 R 4 -(CH 2 ) y SO 2 N(R )2, -(CH 2 ) y N(R 4 ) 2 , -(CH 2 ) y N(R 4 )COR 12 ,
• the invention also relates to pharmaceutical compositions having matrix metalloprotease inhibitory activity, which compositions comprise a compound of the invention as described above and in more detail in the detailed description below, and a pharmaceutically acceptable carrier.
• the invention also relates to a method of treating a mammal such as a human, a farm animal, or a domestic pet, to achieve an effect, in which the effect is treatment and prevention of Multiple Sclerosis.
• (T) X A represents a substituted or unsubstituted aromatic or heteroaromatic moiety selected from the group consisting of:
• Rl represents H or alkyl of 1 - 3 carbons.
• the aromatic ring is referred to as the A ring or A unit
• each T represents a substituent group, referred to as a T group or T unit.
• Substituent groups T are independently selected from the group consisting of: the halogens -F, -Cl, -Br, and -I; alkyl of 1 - 10 carbons; haloalkyl of 1 - 10 carbons; haloalkoxy of 1 - 10 carbons; alkenyl of 2 - 10 carbons; alkynyl of 2 - 10 carbons; -(CH2)pQ in which p is 0 or an integer 1 - 4; -alkenyl-Q in which the alkenyl moiety comprises 2 - 4 carbons; and -alkynyl-Q in which the alkenyl moiety comprises 2 - 7 carbons.
• Q in each of the latter three groups is selected from the group consisting of aryl of 6 - 10 carbons; heteroaryl comprising 4 - 9 carbons and at least one N, O, or S heteroatom; -CN; -CHO; -NO2; -CO2R 2 ; -OCOR 2 ; -SOR 3 ; -SO2R 3 ; -CON(R )2 ; -SO2N(R 4 )2 ;
• R 2 , R 3 , and R 4 are defined as follows.
• R 2 represents H; alkyl of 1 - 6 carbons; aryl of 6 - 10 carbons; heteroaryl comprising 4 - 9 carbons and at least one N, O, or S heteroatom; arylalkyl in which the aryl portion contains 6 - 10 carbons and the alkyl portion contains 1
• heteroaryl-alkyl in which the heteroaryl portion comprises 4 - 9 carbons and at least one N, O, or S heteroatom and the alkyl portion contains 1 - 4 carbons.
• R 3 represents alkyl of 1 - 4 carbons; aryl of 6 - 10 carbons; heteroaryl comprising 4 - 9 carbons and at least one N, O, or S heteroatom; arylalkyl in which the aryl portion contains 6 - 10 carbons and the alkyl portion contains 1 - 4 carbons; or heteroaryl-alkyl in which the heteroaryl portion comprises 4 - 9 carbons and at least one N, O, or S heteroatom and the alkyl portion contains
• R 4 represents H; alkyl of 1 - 12 carbons; aryl of 6 - 10 carbons; heteroaryl comprising 4 - 9 carbons and at least one N, O, or S heteroatom; arylalkyl in which the aryl portion contains 6 - 10 carbons and the alkyl portion contains 1
• heteroaryl-alkyl in which the heteroaryl portion comprises 4 - 9 carbons and at least one N, O, or S heteroatom and the alkyl portion contains 1 - 4 carbons; alkenyl of 2 - 12 carbons; alkynyl of 2 - 12 carbons; -(CqH2qO) r R ⁇ in which q is 1-3, r is 1 - 3, and R ⁇ is H provided q is greater than 1, or R ⁇ is alkyl of 1 - 4 carbons, or phenyl; alkylenethio terminated with H, alkyl of 1-4 carbons, or phenyl; alkyleneamino terminated with H, alkyl of
• R 4 groups When two R 4 groups are situated on a nitrogen, they may be joined by a bond to form a heterocycle, such as, for example, a morpholine, thiomorpholine, pyrrolidine, or piperidine ring.
• a heterocycle such as, for example, a morpholine, thiomorpholine, pyrrolidine, or piperidine ring.
• Any unsaturation in a moiety which is attached to Q or which is part of Q is separated from any N, O, or S of Q by at least one carbon atom, and the number of sub- stituents, designated x, is 0, 1, or 2.
• B represents a bond or an optionally substituted aromatic or heteroaromatic ring selected from the group consisting of:
• Rl is defined as above and each R 1 may be the same or different.
• These rings are refened to as the B ring or B unit.
• D represents the moieties
• E represents a chain of n carbon atoms bearing m substituents R ⁇ , refened to as R° groups or R" units.
• the R6 groups are indepen- dent substituents, or constitute spiro or nonspiro rings.
• Rings may be formed in two ways: a) two groups R ⁇ are joined, and taken together with the chain atom(s) to which the two R6 group(s) are attached, and any intervening chain atoms, constitute a 3 - 7 membered ring, or b) one group R" is joined to the chain on which this one group R6 resides, and taken together with the chain atom(s) to which the R6 group is attached, and any intervening chain atoms, constitutes a 3 - 7 membered ring.
• the number n of carbon atoms in the chain is 2 or 3
• the number m of R ⁇ substituents is an integer of 1 - 3.
• Each group R ⁇ is independently selected from the group consisting of the substitu- ents listed below as items 1) - 16).
• heteroaryl comprising 4 - 9 carbons and at least one N, O, or S heteroatom
• arylalkyl in which the aryl portion contains 6 - 10 carbons and the alkyl portion contains 1 - 8 carbons; 7) heteroaryl-alkyl in which the heteroaryl portion comprises 4 - 9 carbons and at least one N, O, or S heteroatom, and the alkyl portion contains 1 - 8 carbons;
• aryl-alkenyl in which the aryl portion contains 6 - 10 carbons and the alkenyl portion contains 2 - 5 carbons;
• heteroaryl-alkenyl in which the heteroaryl portion comprises 4 - 9 carbons and at least one N, O, or S heteroatom and the alkenyl portion contains 2 -5 carbons;
• aryl-alkynyl in which the aryl portion contains 6 - 10 carbons and the alkynyl portion contains 2 - 5 carbons;
• heteroaryl-alkynyl in which the heteroaryl portion comprises 4 - 9 carbons and at least one N, O, or S heteroatom and the alkynyl portion contains 2 - 5 carbons;
• R 7 groups Y represents O or S;
• Rl> R2> and R 3 are as defined above, and each R 1 , R 2 andR 3 may be the same or different; and u is 0, 1, or 2;
• R 8 is selected from the group consisting of: alkyl of 1 to 12 carbons; aryl of 6 to 10 carbons; heteroaryl comprising 4 - 9 carbons and at least one N, O, or S heteroatom; arylalkyl in which the aryl portion contains 6 to 10 carbons and the alkyl portion contains 1 to 4 carbons; heteroaryl-alkyl in which the aryl portion comprises 4 - 9 carbons and at least one N, O, or S heteroatom and the alkyl portion contains 1 - 4 carbons; -C(O)R 9 in which R 9 represents alkyl of 2 - 6 carbons, aryl of 6 - 10 carbons, heteroaryl comprising 4 - 9 carbons and at least one N, O, or S
• aryl or heteroaryl portions of any of the T or R" groups optionally may bear up to two substituents selected from the group consisting of -(CH 2 )yC(R )(R 3 )OH, -(CH 2 )yOR 4 ), -(CH 2 ) y SR 4 ), -(CH 2 ) y S(O)R 4 ),
• the substituent group T when it is on the ring A, is preferably halogen, 1-alkynyl-Q, or an ether OR 4 wherein R 4 is preferably alkyl of 1 - 12 carbons or arylalkyl in which the aryl portion is 6 - 10 carbons and the alkyl portion contains 1 - 4 carbons. Most preferably, T is halogen, or ⁇ C ⁇ C— (CH ) t OH in which t is an integer of 1 -
• R 4 is alkyl of 1 - 6 carbons, or benzyl.
• the subscript x which defines the number of T substituents, is preferably 1 or 2, most preferabl y 1, and this substituent T is preferabl y on the 4- position of ring A.
• the A ring is preferably a phen y l or thiophene ring, most preferabl y phen y l.
• the A ring preferabby bears at least one substituent group T, preferabl y located on the position furthest from the position of the A ring which is connected to the B ring.
• the B moiety of generalized formula (I) is a bond or a substituted or unsubstituted aromatic or heteroaromatic ring, in which an y substituents are groups which do not cause the molecule to fail to fit the active site of the target enz y me, or disrupt the relative conformations of the A and B rings, such that the would be detrimental.
• groups may be, but are not limited to, moieties such as lower alkyl, lower alkox y , CN, NO2, halogen, etc.
• the B moiety is preferabty a 1 ,4-phen lene or 2,5- thiophene ring, most preferabl y l ,4-phen y lene.
• the compounds according to the invention can exist in stereoisomeric forms which either behave as image and minor image (enantiomers), or which do not behave as image and minor image (diastereomers).
• the invention relates both to the enantiomers or diastereomers and their respective mixtures. These mixtures of the enantiomers and diastereomers can be separated into the stereoisomerically uniform constituents in a known manner.
• the D unit is most preferably a carbonyl or a -CHOH- group.
• the group R 6 is preferably:
• arylalkyl wherein the aryl portion contains 6 - 10 carbons and the alkyl portion contains 1 - 8 carbons;
• v is 0 or an integer of 1 - 4
• Z is S or O
• R 8 is aryl of 6 - 10 carbons or arylalkyl wherein the aryl portion contains 6 to 12 carbons and the alkyl portion contains 1 to 4 carbons.
• the group R 6 is most preferably one of the following, and in these, any aromatic moiety is preferably substituted:
• arylalkyl wherein the aryl portion is phenyl and the alkyl portion contains 1 - 4 carbons; 2) -(CH2)tR ⁇ wherein t is an integer of 1 - 3, and R? is N-phthalimidoyl, 1,2,3- benzotriazin-4(3H)-one-3-yl, N-( 1 ,2-naphthalenedicarboximidoyl), N-(2,3- naphthalenedicarboximidoyl), or N-(l,8-naphthalenedicarboximidoyl); or
• alkyl means straight, branched, cyclic, and polycyclic materials.
• haloalkyl means partially or fully halo- genated alkyl groups such as -(CH2)2C1, -CF3 and -C6F13, for example.
• the invention relates to compounds of generalized formula (I) in which at least one of the units A, B, T, and R" comprises a heteroaromatic ring.
• Prefened heteroaromatic ring-containing compounds are those in which the heteroaryl groups are heteroaryl of 4 - 9 carbons comprising a 5 - 6 membered heteroaromatic ring containing O, S, or NR* when the ring is 5- membered, and N when said ring is 6-membered.
• Particularly prefened heteroaromatic ring-containing compounds are those in which at least one of the A and B units comprises a thiophene ring.
• a unit is thiophene, it is preferably connected to B unit at position 2 and canies one substituent group T on position 5.
• B Unit is thiophene, it is preferably connected through positions 2 and 5 to D and A units respectively.
• the invention relates to compounds of generalized formula (I), in the E unit of which n is 2 and m is 1. These compounds thus possess two carbon atoms between the D unit and carboxyl group, and carry one substituent on this two-carbon chain.
• the invention relates to compounds of generalized formula (I) in which the A ring is a substituted or unsubstituted phenyl group, the B ring is p-phenylene, and aryl portions of any aryl-containing T and R ⁇ moieties contain only carbon in the rings. These compounds thus contain no heteroaromatic rings.
• the invention relates to compounds of generalized formula (I) in which m is 1 and R ⁇ is an independent substituent. These compounds are materials which contain only a single substituent R ⁇ on the E unit, and this substituent in not involved in a ring.
• -(CH2)vZR 8 have v as an integer of 1-4 and Z as -S- or -O-.
• the invention relates to compounds of generalized formula (I) in which the number of substituents m on the E unit is 2 or 3; and when m is 2, both groups R ⁇ are independent substituents, or together constitute a spiro ring, or one group R ⁇ is an independent substituent and the other constitutes a spiro ring; and when m is 3, two groups R ⁇ are independent substituents and one group R6 constitutes a ring, or two groups R 6 constitute a ring and one group R 6 is an independent substituent, or three groups R6 are independent substituents.
• This subset therefore contains compounds in which the E unit is di- or trisubstituted, and in the disubstituted case any rings formed by one or both R° groups are spiro rings, and in the trisubstituted case, the R° groups may form either spiro or nonspiro rings.
• the invention relates to compounds of generalized formula (I) in which the number of substituents m on the E unit is 1 or 2; and when m is 1, the group R ⁇ constitutes a nonspiro ring; and when m is 2, both groups R ⁇ together constitute a nonspiro ring or one group R 6 is an independent substituent and the other constitutes a nonspiro ring.
• This subset therefore contains compounds in which the E unit carries one or two substituents R ⁇ and at least one of these substituents is involved in a nonspiro ring.
• each group R ⁇ 4 is independently selected from the group consisting of: alkyl of 1 - 9 carbons; arylalkyl in which the alkyl portion contains 1 - 7 carbons and the aryl portion contains 6 - 10 carbons; alkenyl of 2 - 9 carbons; aryl-substituted alkenyl in which the alkenyl portion contains 2 - 4 carbons and the aryl portion contains 6 - 10 carbons; alkyl of 1 - 9 carbons; arylalkyl in which the alkyl portion contains 1 - 7 carbons and the aryl portion contains 6 - 10 carbons; alkenyl of 2 - 9 carbons; aryl-substituted alkenyl in which the alkenyl portion contains 2 - 4 carbons and the aryl portion contains 6 - 10 carbons; alkyl of 1 - 9 carbons; aryl-substituted alkenyl in which the alkenyl portion contains 2 - 4 carbons and the
• T is (C ⁇ -C )-alkyl, (C ⁇ -C 4 )-alkoxy, chloride, bromide, fluoride, acetoxy, hydroxy, cyano, trifluoromethyl or trifluoromethoxy,
• CO-E-CO 2 H represents a 3-carboxyl-5-R 7 -pentan-l-on-l-yl- or a 2-carboxyl-3-(R 7 - methyl)-cyclopentan- 1 -yl)carbonyl-residue, wherein
• R represents a group of the formula
• the compounds of the invention may be prepared by use of known chemical reactions and procedures as described in details in WO 96/15096, WO 97/43237, WO 97/43238, WO 97/43239, WO 97/43240, WO 97/43245, WO97/43247 and WO
• variable groups of these methods are as described in the generic description if they are not specifically defined below.
• the variable subscript n is independently defined for each method.
• R ⁇ or T a variable group with a given symbol
• each of these groups may be independently varied within the range of definitions for that symbol.
• the compounds of the invention contain as the E unit a chain of 2 or 3 carbon atoms bearing 1 to 3 substituents R" which are not defined as H.
• R ⁇ groups are used as if their definition includes H, to show where such R" groups may exist in the structures, and for ease in drawing.
• R ⁇ may be H in addition to the moieties set forth in the definition of R6.
• the ultimate compounds contain 1 to 3 non-hydrogen groups R ⁇ .
• the raw product I-A often exists as a mixture of isomers via attack of the anhydride from either of the two carbonyls
• the resultant isomers can be separated into pure forms by crystallization or chromatography using standard methods known to those skilled in the art.
• the succinic anhydrides III can be prepared via a Stobbe Condensation of a dialkyl succinate with an aldehyde or ketone
• Method A is especially useful for the preparation of cyclic key intermediates such as I-A-3 in which two R ⁇ groups are connected in a methylene chain to form a 4-7 membered ring.
• Small ring (3-5 member) anhydrides are readily available only as cis isomers which yield cis invention compounds I-A-3.
• the trans compounds I-A-4 are then prepared by treatment of I-A-3 with a base such as DBU in THF.
• key intermediates can be prepared via a reaction sequence involving mono-alkylation of a dialkyl malonate VI with an alkyl halide to form intermediate VII, followed by alkylation with a halomethyl biphenyl ketone VIII to yield intermediate IX.
• Compounds of structure IX are then hydrolyzed with aqueous base and then heated to decarboxylate the malonic acid intermediate and yield I-B-2 (Method B-l).
• reaction of VI with VIII can be conducted before that with the alkyl halide to yield the same IX (Method B-2).
• Intermediates VIII are formed from biphenyls II in a Friedel-Craft reaction with haloacetyl halides such as bromoacetyl bromide or chloroacetyl chloride.
• Method B has the advantage of yielding single regio isomers whereas Method A yields mixtures.
• Method B is especially useful when the side chains R ⁇ contain aromatic or heteroaromatic rings that may participate in intramolecular acylation reactions to give side products if Method A were to be used.
• This method is also very useful when the R ⁇ group adjacent to the carboxyl of the final compound contains heteroatoms such as oxygen, sulfur, or nitrogen, or more complex functions such as imide rings.
• Acid halide X is reacted with the lithium salt of chiral auxiliary XI (R is often isopropyl or benzyl) to yield intermediate XII, which in turn is alkylated at low temperatures (typically under -50°C) with halo-tert-butylacetyl compound XIII to yield pure isomer XIV.
• R is often isopropyl or benzyl
• XII alkylated at low temperatures (typically under -50°C)
• halo-tert-butylacetyl compound XIII to yield pure isomer XIV.
• the use of opposite chirality XI yields opposite chirality XIV.
• Conversion of XIV to the enantiomerically pure diacid XV is accomplished by treatment with lithium hydroxide/hydrogen peroxide in THF/water, followed by acids such as trifluoroacetic acid.
• the compound XV is then converted to enantio
• Biphenyl starting material II may also first be reacted in a Friedel-Crafts reaction as earlier described 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 to fonn acyl derivative I-C-2.
• the carbonyl group of this material is then blocked as a ketal such as that formed by treatment with 1,2-bistrimethyl- silyloxyethane in the presence of a catalyst such as trimethyl-silyltriflate in a suitable solvent.
• a catalyst such as trimethyl-silyltriflate
• Pd(com) is a soluble complex of palladium such as tetrakis(triphenylphosphine)-palladium(0) or bis-(triphenylphos-phine)-palla- dium(II) chloride.
• palladium such as tetrakis(triphenylphosphine)-palladium(0) or bis-(triphenylphos-phine)-palla- dium(II) chloride.
• 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 treatment of a bromo intermediate with hexamethylditin and palladium tetrakistriphenylphosphine in toluene at reflux to yield the trimethyltin intermediate.
• the intermediates XXII are either commercial or easily prepared from commercial materials by methods well known to those skilled in the art.
• Method F is also especially useful for the preparation of key intermediates in which the aryl groups A or B contain one or more heteroatoms (heteroaryls) such as those compounds that contain thiophene, furan, pyridine, pynole, oxazole, thiazole, pyrimidine or pyrazine rings or the like instead of phenyls.
• heteroaryls such as those compounds that contain thiophene, furan, pyridine, pynole, oxazole, thiazole, pyrimidine or pyrazine rings or the like instead of phenyls.
• This material is then reduced to the alcohol with a reducing agent capable of selective reduction of the ketone such as sodium borohydride, followed by dehydration with triphenylphosphine / diethyl azo- dicarboxylate (DEAD) in a suitable solvent such as THF at reflux to yield XXIX.
• a reducing agent capable of selective reduction of the ketone such as sodium borohydride
• DEAD triphenylphosphine / diethyl azo- dicarboxylate
• a suitable solvent such as THF at reflux
• Lig can be, for example, another equivalent of vinyl group or halide
• Hydride reduction lithium aluminum hydride or the like
• XXXIX followed by standard blocking with, for example, t-butyldimethylsilylchloride yields XXXX which in turn is reacted with an optionally substituted benzylchloroformate XXXXI to yield XXXXII.
• Ozonolysis of this intermediate followed by reductive workup leads to aldehyde XXXXIII.
• the carbobenzyloxy group is removed by reaction with hydrogen and a catalyst such as palladium on carbon to yield the unsubstituted key intermediate 1-1-2 optionally followed by N- alkylation to yield key intermediate 1-1-3.
• a catalyst such as palladium on carbon
• the intermediate XXXX can be directly treated with ozone followed by the other steps of this method to yield 1-1-3, in which R* 4 is optionally substituted benzyl rather than as in 1-1-1.
• This malonate can be further alkylated under conditions familiar to those skilled in the art to yield L which in turn is treated with acid and then heated to yield key intermediate 1-J-l.
• the final alkylation can be omitted to yield products in which the R6 adjacent to the carboxyl is H.
• XXXXVII can be alkylated with 3- halopropionate ester LI in the presence of base such as LDA to yield ester l-J-2 which can then be hydrolyzed with aqueous base to yield key intermediate l-J-3 upon treatment with acid. This method is especially useful if any of the groups R6 contain aromatic residues.
• Method K The key intermediates in which two R ⁇ groups are joined to form a substituted 5-member ring are most conveniently prepared by method K.
• Sub- stituted bromobiphenyl LIII is converted to its Grignard reagent by treatment with magnesium which is then reacted with LII to yield alcohol LIV.
• Alcohol LIV is eliminated via base treatment of its mesylate by using conditions well known to those skilled in the art to yield olefin LV.
• LIII is converted to a trimethyltin intermediate via initial metallation of the bromide with n-butyllithium at low temperature (-78°C) followed by treatment with chlorotrimethyltin and LII is converted to an enoltriflate by reaction with 2-[N,N-bis(trifluoromethylsulfonyl)- amino]-5-chloropyridine in the presence of a strong aprotic base.
• Suitable pharmaceutically acceptable salts of the compounds of the present invention that contain an acidic moiety include addition salts formed with organic or inorganic bases.
• the salt forming ion derived from such bases can be metal ions, e.g., aluminum, alkali metal ions, such as sodium of potassium, alkaline earth metal ions such as calcium or magnesium, or an amine salt ion, of which a number are known for this purpose.
• Examples include ammonium salts, arylalkylamines such as dibenzylamine and N,N-dibenzylethylenediamine, lower alkylamines such as methylamine, t- butylamine, procaine, lower alkylpiperidines such as N-ethylpiperidine, cyclo- alkylamines such as cyclohexylamine or dicyclohexylamine, 1 -adamantylamine, benzathine, or salts derived from amino acids like arginine, lysine or the like.
• the physiologically acceptable salts such as the sodium or potassium salts and the amino acid salts can be used medicinally as described below and are prefened.
• Suitable pharmaceutically acceptable salts of the compounds of the present invention that contain a basic moiety include addition salts formed with organic or inorganic acids.
• the salt forming ion derived from such acids can be halide ions or ions of natural or unnatural carboxylic or sulfonic acids, of which a number are known for this purpose. Examples include chlorides, acetates, tartrates, or salts derived from amino acids like glycine or the like.
• the physiologically acceptable salts such as the chloride salts and the amino acid salts can be used medicinally as described below and are prefened.
• the salts are produced by reacting the acid form of the invention compound with an equivalent of the base supplying the desired basic ion or the basic form of the invention compound with an equivalent of the acid supplying the desired acid ion in a me- dium in which the salt precipitates or in aqueous medium and then lyophilizing.
• the free acid or basic form of the invention compounds can be obtained from the salt by conventional neutralization techniques, e.g., with potassium bisulfate, hydrochloric acid, sodium hydroxide, sodium bicarbonate, etc.
• the compounds of the present invention are expected to inhibit the matrix metallo- proteases MMP-1, MMP-2, MMP-3, MMP-8, MMP-9, MMP-12, MMP-13, and the related protease TACE, as well as the release of TNF ⁇ in vivo, and are therefore expected to be useful for treating or preventing the conditions refened to in the background section.
• MMPs not listed above share a high degree of homology with those listed above, especially in the catalytic site, it is deemed that compounds of the invention should also inhibit such other MMPs to varying degrees. Varying the substituents on the biaryl portions of the molecules, as well as those of the R ⁇ groups of the claimed compounds, is expected to affect the relative inhibition of the listed MMPs.
• compounds of this general class can be "tuned" by selecting specific substituents such that inhibition of specific MMP(s) associated with specific pathological conditions can be enhanced while leaving non-involved MMPs less affected.
• the method of treating matrix metalloprotease-mediated or TNF ⁇ release-mediated conditions may be practiced in mammals, including humans, which exhibit such conditions.
• inhibitors of the present invention are contemplated for use in veterinary and human applications. For such purposes, they will be employed in pharmaceutical compositions containing active ingredient(s) plus one or more pharmaceutically ac- ceptable carriers, diluents, fillers, binders, and other excipients, depending on the administration mode and dosage form contemplated.
• Administration of the inhibitors may be by any suitable mode known to those skilled in the art.
• suitable parenteral administration include intravenous, intra- articular, subcutaneous and intramuscular routes.
• Intravenous administration can be used to obtain acute regulation of peak plasma concentrations of the drug.
• Improved half-life and targeting of the drug to the joint cavities may be aided by entrapment of the drug in liposomes. It may be possible to improve the selectivity of liposomal targeting to the joint cavities by incorporation of ligands into the outside of the lipo- somes that bind to synovial-specific macromolecules.
• intramuscular, intraarticular or subcutaneous depot injection with or without encapsulation of the drug into degradable microspheres e.g., comprising poly(DL-lactide-co-glycolide) may be used to obtain prolonged sustained drug release.
• degradable microspheres e.g., comprising poly(DL-lactide-co-glycolide)
• an i.p. implanted reservoir and septum such as the Percuseal system available from Pharmacia.
• Improved convenience and patient compliance may also be achieved by the use of either injector pens (e.g. the Novo Pin or Q-pen) or needle-free jet injectors (e.g. from Bioject, Mediject or Becton Dickinson).
• Prolonged zero-order or other precisely controlled release such as pulsatile release can also be achieved as needed using implantable pumps with delivery of the drug through a cannula into the synovial spaces.
• implantable pumps with delivery of the drug through a cannula into the synovial spaces.
• Examples include the subcutaneously implanted osmotic pumps available from ALZA, such as the
• Nasal delivery may be achieved by incorporation of the drug into bioadhesive particulate carriers ( ⁇ 200 ⁇ m) such as those comprising cellulose, polyacrylate or polycarbophil, in conjunction with suitable absorption enhancers such as phospholipids or acylcarnitines.
• bioadhesive particulate carriers ⁇ 200 ⁇ m
• suitable absorption enhancers such as phospholipids or acylcarnitines.
• Available systems include those developed by DanBiosys and Scios Nova.
• Oral delivery may be achieved by incorporation of the drug into tablets, coated tablets, dragees, hard and soft gelatine capsules, solutions, emulsions or suspensions.
• Oral delivery may also be achieved by incorporation of the drug into enteric coated capsules designed to release the drug into the colon where digestive protease activity is low.
• enteric coated capsules designed to release the drug into the colon where digestive protease activity is low.
• examples include the OROS-CT/OsmetTM and PULSINCAPTM systems from ALZA and Scherer Drug Delivery Systems respectively.
• Other systems use azo- crosslinked polymers that are degraded by colon specific bacterial azoreductases, or pH sensitive polyacrylate polymers that are activated by the rise in pH at the colon. The above systems may be used in conjunction with a wide range of available absorption enhancers.
• Rectal delivery may be achieved by incorporation of the drug into suppositories.
• the compounds of this invention can be manufactured into the above listed formulations by the addition of various therapeutically inert, inorganic or organic caniers well known to those skilled in the art.
• examples of these include, but are not limited to, lactose, corn starch or derivatives thereof, talc, vegetable oils, waxes, fats, polyols such as polyethylene glycol, water, saccharose, alcohols, glycerin and the like.
• the amount of the pharmaceutical composition to be employed will depend on the recipient and the condition being treated. The requisite amount may be determined without undue experimentation by protocols known to those skilled in the art. Alter- natively, the requisite amount may be calculated, based on a determination of the amount of target enzyme which must be inhibited in order to treat the condition. It is expected that the compounds of the invention generally will 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 treatment of the physiological conditions discussed above, but are also useful in such activities as purification of metalloproteases and testing for matrix metalloprotease activity.
• activity testing can be both in vitro using natural or synthetic enzyme preparations or in vivo using, for example, animal models in which abnormal destructive enzyme levels are found spontaneously (use of genetically mutated or transgenic animals) or are induced by administration of exogenous agents or by surgery which disrupts joint stability.
• Inhibitory activities of the compounds of the invention against matrix metalloprote- ases and production of TNF ⁇ may be determined as described below.
• MMP-9 is isolated 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, 1989). Briefly, polymorphonuclear leukocytes (PMN) preparations are isolated as described above from 3 or more units of freshly drawn whole blood.
• PMN polymorphonuclear leukocytes
• Cells are resuspended in phosphate buffered saline (PBS) containing 100 ng/ml phorbol myristate acetate (PMA) in the presence of 50 mM di-isopropylfluo- rophospate (DFP), 1 ⁇ g/ml leupeptin and aprotinin, and 1 mg/ml catalase for 1 hr at 37°C.
• DFP di-isopropylfluo- rophospate
• DFP di-isopropylfluo- rophospate
• 1 ⁇ g/ml leupeptin and aprotinin 1 mg/ml catalase for 1 hr at 37°C.
• Supernatants are collected by centrifugation (300 x g) and the samples are frozen at -70°C. All chromatographic methods are performed at 4°C. Thawed samples are concentrated 5-fold using an Amicon chamber equipped with a YM-10 membrane
• the concentrate is pressure dialyzed against 0.02M Tris-HCl, 0.1 M NaCI, 1 mM CaCl2 , 1 ⁇ M ZnCl2, 0.001% Brij-35, 0.02% sodium azide (NaN3) s pH 7.5 and applied to DEAE ion exchange chromatography resin which is previously equilibrated with the same buffer at a flow rate of 0.4 ml/min.
• the column is extensively washed with the same buffer and gelatinase is eluted as 4 ml fractions from the column with 0.02M Tris-HCl, 0.5 M NaCI, 1 mM CaCl2, 1 ⁇ M ZnCl2, 0.001% Brij-35, 0.02% NaN3 ; pH 7.5.
• Gelatinase containing fractions are observed by gelatin zymography (see below), loaded onto a gelatin agarose affinity resin and washed with the same buffer.
• Gelatinase activity is eluted at a flow rate of 1 ml/min from the column as 1 ml fractions with 0.02M Tris-HCl, 1 M NaCI, 1 mM CaCl2, 1 ⁇ M ZnCl2, 0.001% Brij-35, 0.02% NaN3 ; pH 7.5 containing 10% dimethyl sulfoxide (DMSO).
• the fractions containing gelatinase activity are pooled and dialyzed against 0.005M Tris-HCl, 5mM NaCI, 0.5 mM CaCl2 , 0.1 ⁇ M ZnCb,
• the protein content associated with material is determined with a micro-BCA assay (Pierce, Rockford, IL), lyophilized and reconstituted to a desired working concentration (100 ⁇ g/ml).
• MMP-2 Gelatinase A
• Truncated Prostromelysin-257 is expressed in a soluble form in E.coli as described by Marcy et al., Biochemistry, 30, 6476-6483, 1991. Soluble truncated prostro- melysin is purified by a modification of the monoclonal antibody affinity chromatography method described by Housley et al., j. Biol. Chem., 268, 4481-87, 1993.
• This assay was originally described by Knight et al., FEBS Letters, 296, 263-266, 1992, for a related substrate. The assay is run continuously in a 3.0ml cuvette using a Perkin-Elmer LS 50 B Spectrofluorimeter at 25 ⁇ C in a final volume of 2.0 mis.
• P218 substrate (lOmM) in 100% DMSO is diluted to a final concentration of 2.0 micromolar ( ⁇ M) into assay buffer: 50mM MES, pH 6.5 containing 150mM NaCI, lOmM CaC12, 0.005% Brij-35, and l%(v/v) DMSO.
• Test compounds(lOmM) in DMSO are diluted in assay buffer at an initial concentration of 10 to 100 micro- molar. These are diluted to a final concentration in the assay from 10 nM to 1 ⁇ M depending upon their potency previously determined in primary thiopeptilide assay described above.
• the reaction is initiated by the addition of recombinant stromelysin (MMP-3) at a final concentration of 1.0 nM.
• MMP-3 recombinant stromelysin
• the fluorescent MCA group is detected using an excitation wavelength of 328 nanometers and an emission wavelength of 393 nanometers.
• the assay is linear from 0.2 to 5nM MMP-
• this substrate has a K of 16 ⁇ M at pH 6.5 and a kcat/K w value of 56,000M-lsec-l.
• This assay is adapted from the one described by Knight et al., FEBS Letters, 296, 263-266 (1992) for MMP-3 and a related substrate.
• the rate of hydrolysis of the synthetic substrate H-MCA-Pro-Lys-Pro-Leu-Ala-Leu-DPA-Ala-Arg-NH 2 (P218) by human recombinant MMP-12 is monitored fluorometrically, using an excitation wavelength of 340 nm and an emission wavelength of 395 nm, in the presence or absence of the test compounds.
• the assay is canied out in buffer containing 50 mM HEPES and 10 mM CaCl 2 at pH 7.0.
• the substrate is made up initially in 100%> DMSO to a concentration of 1 x 10 " M, then diluted in assay buffer to a final con- centration of 20 ⁇ M.
• Test compounds (10 mM in DMSO) are diluted in assay buffer at an initial concentration of 0.3-1000 nM. These are diluted to a final concentration in the assay from 0.03 nM to 100 nM.
• the reaction is initiated by the addition of substrate at a final concentration of 20 ⁇ M.
• the total assay volume in a 96 well micro- titre plate is 150 ⁇ l.
• Cleavage of the substrate between the Leu- Ala residues allows the fluorescence of the MCA group to be detected on a fluorometer (Cytofluor II) following excitation at 340 nm and emission at 395 nm. Change in fluorescence is continually monitored for a 40 min period.
• [I]o and [E] 0 are inhibitor and enzyme concentrations, and v, / v 0 are reaction veloci- ties with / without inhibitor.
• [I]o is equal to IC 50 when v, is half v 0 , so that:
• IC 50 's are determined at each enzyme concentration (0.044-0.98 ⁇ g/ml) using Xlfit software. K, appa r ent is then determined graphically from the plot of IC 50 versus MMP-
• IC 50 values are calculated using % inhibition values at each enzyme concentration, ensuring data is taken from the linear part of the reaction rate curves. The K, can then be calculated from the equation:
• K, K, appar e nt / (1 +S)/K m
• This assay is run with a protocol analogous to that reported for MMP-3 inhibition using the synthetic peptide P218 and each of the three enzymes and measuring quenched fluorescence.
• This assay can be run with each invention compound with the three enzymes in parallel as adapted for a 96-well microtitre plate using a Hamilton AT ® workstation.
• the following examples illustrate the selectivity of compounds of the invention for specific human MMPs:
• the in vivo inhibitory properties of selected compounds can be determined using a murine LPS induced TNF ⁇ production in vivo model.
• BALB/c mice Charles River Breeding Laboratories; Springfield, NY
• endotoxin E. coli lipopolysaccharide (LPS) 100 mg
• LPS lipopolysaccharide
• animals are euthanized by carbon dioxide asphyxiation and plasma is obtained from individual animals by cardiac puncture into heparinized tubes.
• the samples are clarified by centrifugation at 12,500 x g for 5 min at 4 °C.
• the supernatants are decanted to new tubes, which are stored as needed at -20 °C.
• TNF ⁇ levels in sera are measured using a commercial murine TNF ELISA kit (Genzyme).
• Acute EAE Experimental Autoimmune Encephalomyelitis
• PRP ⁇ 39- ⁇ 5 ⁇ myelin constituent proteolipid protein
• Bordetella pertussis Bordetella pertussis.
• EAE-diseased mice develop clinical signs around 10 days after immunization which are accompanied by rapid loss of body weight and which resolve between day 16 and 20. Histopathologically this acute disease is characterized by CNS perivascular and parenchymal inflammation and, if at all, minimal demyelination. Infiltrates are mostly composed of T-cells and macrophages.
• Acute EAE is induced in female Lewis rats by inoculation of a spinal cord-adjuvant emulsion into each hind paw of the animals. Animals show signs of disease (body weight loss, neurological deficits) starting around day 10 after immunization and resolving between days 17-23.
• Animals are treated by per os (p.o.) administration of the test compound. Treatment starts on day 6 post inoculation and stops on day 17.
• the compound is administered at 3 different doses (e.g. 1, 10, 30 mg/kg) once daily. Body weight and appearance of neurological signs are checked on a daily basis.
• Chronic relapsing EAE is induced by adoptive transfer of PLP 139 _ ⁇ 51 -responsive lymph node cells to naive female SJL/J recipient mice by injection into the tail vein. 6-8 days later, the mice develop paralysis and incontinence, acute symptoms which conespond to a period of intense inflammation, demyelination and some Wallerian degeneration in the central nervous system. The acute phase peaks from 14-20 days post transfer (dpt) of lymph node cells is followed by remission. The chronic phase of EAE, characterized by spontaneous relapses with severe clinical symptoms, occurs after 21 dpt.
• test compound is administered daily by p.o. application at 3 different doses (e.g.
• Treatment starts on day 21 post transfer and stops on day 78.
• Neurological impairment in EAE mice is evaluated every 1-4 days after disease induction.
• the clinical scores of each individual animal are used to score relapses and to calculate relapse frequency and mean clinical score.
• lodoethane (68.7 g, 35,57 mL, 440.6 mmol) was added to a suspension of 50 g (170.2 mmol) of 4-hydroxy- 1,1 '-biphenyl and 40.6 g (293.75 mmol) K 2 CO 3 in 60 mL acetone.
• the resulting reaction mixture was stined under reflux for 16 hours.
• the acetone was removed under reduced pressure, the residue was dissolved in ethyl acetate and extracted with water.
• the aqueous layers where extracted 3 times with ethyl acetate, the combined organic phases dried (Na 2 SO ) and evaporated to yield 56 g of the desiered compound as a colorless solid.
• Example 2 The racemate of Example 1 was separated into its pure enantiomers via chiral HPLC using a commercially available 5 ⁇ m Kromasil KR 100-5-CHI-DMB phase. A solvent mixture consisting of 50%o iso-hexane and 50% of a tert-butylmethyl ether / dichloromethane / glacial acetic acid mixture (480:40: 1 ) was employed at a constant flow rate of 25 ml/min.
• Example 2 A solvent mixture consisting of 50%o iso-hexane and 50% of a tert-butylmethyl ether / dichloromethane / glacial acetic acid mixture (480:40: 1 ) was employed at a constant flow rate of 25 ml/min.
• Example 2 A solvent mixture consisting of 50%o iso-hexane and 50% of a tert-butylmethyl ether / dichloromethane / glacial acetic acid mixture (480:40: 1 ) was employed at
• This intermediate was prepared as described in the indicated reference WO 96/15096.
• Racemic 4-(4'-Bromo[ 1,1 '-biphenyl]-4-yl)-2-[2-(l, 3-dioxo-l, 3-dihydro-2H-isoindol- 2-yl)- ethyl]-4-oxobutanoic acid was prepared essentially as described in the indicated reference WO 96/15096.
• Racemic 4-(4'-Chloro[l,l'-biphenyl]-4-yl)-2-[2-(5,7-dioxo-5,7-dihydro-6H-[l,3]-di- oxolo-[4,5-fJisoindol-6-yl)ethyl]-4-oxobutanoic acid was prepared essentially as described in the indicated reference WO 96/15096.
• the racemic compound was prepared essentially following the procedure for Example 360 of WO 96/15096.
• Example 16 The compound of Example 16 was separated into its pure enantiomers by HPLC on a Kromasil 100-5-CHI-DMB (5 ⁇ m) column using a mixture consisting of n-heptane :
• Example 20 700 mg of the compound of Example 19 were separated into the pure enantiomers by HPLC on a Kromasil 100-5-CHI-DMB (5 ⁇ m) column using a mixture consisting of n-heptane : (0.2%> acetic acid/TBME) : dichloromethane (40 : 54 : 6 by volume) as eluant.
• Example 20
• ⁇ 2-[4-oxo-l,2,3-benzotriazin-3(4H)-yl]ethyl ⁇ malonate is dissolved in a mixture of 4ml dichloromethane and 4ml trifluoroaceticacid and the resulting solution is stined for 2 hrs at room temperature. 10ml toluene are added and the solvents are removed in vacuo. The residue is taken up in 8ml 1,4-dioxane and the resulting solution re- fluxed for 6 hrs and stined at room temperature for 12hrs. The solvent is removed in vacuo and the product obtained by trituration with ethylacetate.
• Example 22 The compound of Example 22 was separated into its pure enantiomers by HPLC on a Kromasil 100-5-CHI-DMB (5 ⁇ m) column using a mixture consisting of i-heptane : dichloromethane : acetic acid (480 : 40 : 1 by volume) as eluant.
• Example 25 The compound of Example 25 was separated into its pure enantiomers by HPLC in analogy to Example 21.

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