WO1993014096A1

WO1993014096A1 - Phosphono derivatives of amino acids as metalloproteinase inhibitors

Info

Publication number: WO1993014096A1
Application number: PCT/GB1993/000086
Authority: WO
Inventors: Nigel Robert Arnold Beeley; Thomas Andrew Millican; John Richard Morphy
Original assignee: Celltech Limited
Priority date: 1992-01-15
Filing date: 1993-01-15
Publication date: 1993-07-22
Also published as: AU3358593A; GB9200826D0; EP0581923A1; JPH06506483A; CA2105309A1

Abstract

Compounds of formula (I) are described wherein R represents a -P(O)(X?1R6)X2R7¿ group, where X?1 and X2¿, which may be the same of different, is each an oxygen or a sulphur atom, and R?6 and R7¿, which may be the same or different each represents a hydrogen atom or an optionally substituted alkyl, aryl, or aralkyl group; R1 represents a hydrogen atom or an optionally substituted alkyl, alkenyl, aryl, aralkyl, heteroaralkyl or heteroarylthioalkyl group; R2 represents an optionally substituted alkyl, alkenyl, cycloalkyl, cycloalkylalkyl, aryl, aralkyl, aralkoxy, or aralkylthio group, or an amino (-NH¿2?), substituted amino, carboxyl (-CO2H) or esterified carboxyl group; R?3¿ represents a hydrogen atom or an alkyl group; R4 represents a hydrogen atom or an alkyl group; R5 represents a group -[Alk]¿nR?8 where Alk is an alkyl or alkenyl group optionally interrupted by one or more -O- or -S- atoms or -N(R9)- groups [where R9 is a hydrogen atom or a C¿1-6?alkyl group], n is zero or an integer 1, and R?8¿ is an optionally substituted cycloalkyl or cycloalkenyl group; X represents an amino (-NH¿2?), or substituted amino, hydroxyl or substituted hydroxyl group; and the salts, solvates and hydrates thereof. The compounds are metalloproteinase inhibitors and in particular have a selective gelatinase action, and may be of use in the treatment of cancer to control the development of tumour metastasises.

Description

PHOSPHONO DEtHVATIVES OF AMINO ACIDS AS METALLOPROTEINASE

INHIBITORS

FIELD OF THE INVENTION

This invention relates to a novel class of phosphonopeptidyl derivatives, to processes for their preparation and to their use in medicine.

BACKGROUND TO THE INVENTION

In normal tissues, cellular connective tissue synthesis is offset by extracellular matrix degradation, the two opposing effects existing in dynamic equilibrium. Degradation of the matrix is brought about by the action of proteinases released from resident connective tissue cells and invading inflammatory cells, and is due, in part, to the activity of at least three groups of metalloproteinases. These are the collagenases, the gelatinases (or type-IV collagenases) and the stromelysins. Normally these catabolic enzymes are tightly regulated at the level of their synthesis and secretion and also at the level of their extracellular activity, the latter through the action of specific inhibitors, such as α₂-macroglobulins and TIMP (tissue inhibitor of metalloproteinase), which form inactive complexes with metalloproteinases.

The accelerated, uncontrolled breakdown of connective tissues by metalloproteinase catalysed resorption of the extracellular matrix is a feature of many pathological conditions, such as rheumatoid arthritis, corneal, epidermal or gastric ulceration; tumour metastasis or invasion; periodontal disease and bone disease. It can be expected that the pathogenesis of such diseases is likely to be modified in a beneficial manner by the administration of metalloproteinase inhibitors and numerous compounds have been suggested for this purpose [for a general review see Wahl, R.C. ≤l al Ann. Rep. Med. Chem. 25, 175-184, Academic Press inc., San Diego (1990)]. Certain phosphonopeptides have been described as collagenase inhibitors in European Patent Specification No. 320118 and International Patent Specifications Nos. WO 91/15516 and WO 91/15507.

SUMMARY OF THE INVENTION

We have a now found a new class of phosphonopeptidyl derivatives, members of which are metalloproteinase inhibitors and which, in^' particular, advantageously possess a potent and selective inhibitory action against gelatinase.

There is now much evidence that metalloproteinases are important in tumour invasion and metastasis. Tumour cell gelatinase, in particular, has been associated with the potential of tumour cells to invade and metastasise. Tumour invasion and metastasis is the major cause of treatment failure for cancer patients, and the use of a selective gelatinase inhibitor such as a compound of the present invention which is capable of inhibiting tumour cell invasion can be expected to improve the treatment of this disease.

Thus according to one aspect of the invention we provide a compound of formula (1)

R

wherein R represents a -P(0)(X¹ R⁶)X²R⁷ group, where X¹ and X², which may be the same or different, is each an oxygen or a sulphur atom, and R⁶ and R⁷, which may be the same or different each represents a hydrogen atom or an optionally substituted alkyl, aryl, or aralkyl group;

R¹ represents a hydrogen atom or an optionally substituted alkyl, alkenyl, aryl, aralkyl, heteroaralkyl or heteroarylthioalkyl group;

R² represents an optionally substituted alkyl, alkenyl, cycloalkyl, cycloalkylalkyl, aryl, aralkyl, aralkoxy, or aralkylthio group, or an amino (- NH₂), substituted amino, carboxyl (-C0₂H) or esterified carboxyl group;

R³ represents a hydrogen atom or an alkyl group;

R⁴ represents a hydrogen atom or an alkyl group;

R⁵ represents a group -[Alk^R⁸ where Alk is an alkyl or alkenyl group optionally interrupted by one or more -O- or -S- atoms or -N(R⁹)- groups [where R⁹ is a hydrogen atom or a C₁_₆alkyl group], n is zero or an integer 1 , and R⁸ is an optionally substituted cycloalkyl or cycloalkenyl group;

X represents an amino (-NH₂), or substituted amino, hydroxyl or substituted hydroxyl group;

and the salts, solvates and hydrates thereof.

It will be appreciated that the compounds according to the invention can contain one or more asymmetrically substituted carbon atoms, for example those marked with an asterisk in formula (I). The presence of one or more of these aysmmetric centres in a compound of formula (I) can give rise to stereoisomers, and in each case the invention is to be understood to extend to all such stereoisomers, including enantiomers and diastereoisomers, and mixtures, including racemic mixtures, thereof.

In the formulae herein, the -line is used at a potential asymmetric centre to represent the possibility of R- and S- configurations, the — « line and the — — line to represent an unique configuration at an asymmetric centre.

When the groups R¹ and/or R² in compounds of formula (I) each represents an optionally substituted alkyl or alkenyl group, it may be, for example, a straight or branched C,_₆ alkyl or C₂.₆alkenyl group, such as a methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, n-pentyl, i-pentyl, n-hexyl, ethenyl, 1 -propenyl, 1 -butenyl or 2-butenyi group optionally substituted by one or more C_|_₆alkoxy, e.g. methoxy, ethoxy, propoxy, C,_₆alkylthio, e.g. methylthio, ethylthio, propylthio, C₆_₁₂arylC.,_galkoxy, e.g. phenylC,_₆ alkoxy such as benzyloxy, aralkylthio, e.g phenylC^galkylthio such as benzylthio, amino (-NH₂), substituted amino, [such as -NHR^{1 0}, where R¹⁰ is a C^g alkyl e.g. methyl or ethyl, C₆_₁₂arylC_|_₆alkyl, e.g. phenylC,_₆alkyl, such as benzyl,

C₆.₁₂aryl, e.g. phenyl, C₃.₈cycloaIkyl, e.g. cyclohexyl, or C₃.₈cycloalkylC . ₆alkyl, e.g. cyclohexylmethyl group], carboxyl (-C0₂H) or -C0₂R^{1 2} [where R¹² is as defined below] groups.

Aryl groups represented by R¹ and/or R² in compounds of formula (I) include C₆.₁₂ aryl groups such as phenyl or 1-.or 2-naphthyl groups.

Aralkyl groups represented by R¹ and/or R² include C₆_₁₂arylC.,_₆alkyl groups such as phenylC^galkyl, or 1- or 2-naphthylC^galkyl, for example benzyl, phenylethyl, phenylpropyl, phenylbutyl, phenylpentyl, 1- or 2- naphthylmethyl, naphthylethyl, naphthylpropyl, naphthylbutyl or naphthylpentyl groups.

When the group R¹ in compounds of formula (I) is a heteroaralkyl group, it may be for example a Cg.gheteroarylC^galkyl group, such as an optionally substituted pyrrolylmethyl, furanylmethyl, thienyimethyl, imidazolylmethyl, oxazolylmethyl, thiazolylmethyl, pyrazolylmethyl, pyrrolidinylmethyl, pyridinylmethyl, pyrimidinylmethyl, morpholinylmethyl, or piperazinylmethyl group.

Heteroarylthioalkyi groups represented by R¹ include C₃.₆heteroarylthioC₁. ₆alkyl groups such as optionally substituted pyrrolylthiomethyl, furanylthio methyl , oxazoiylthiomethyl , thiazolylthiomethyl , py razo ly It hio methyl, py rrolidiny It hio methyl, py ridiny It hi o methyl, pyrimidinylthiomethyl, morpholinylthiomethyl, or piperazinylthiomethyi groups.

Optional substituents which may be present on heteroaralkyl or heteroarylthioalkyi groups represented by R¹ include those discussed below in relation to R¹ and/or R² when these groups are for example aralkyl or aralkylthioalkyl groups.

Cycloalkyl groups represented by the group R² in compounds according to the invention include C₃.₈cycloalkyl groups such as cyclopentyi or cyclohexyl groups.

When R² is a cycloalkylalkyl group it may be for example a C3.3CycloalkylC._j_ 6^a'^kv' 9^rouP ^{sucn as a} cyclopentylC,_₆alkyl or cyclohexylC^galkyl group, for example a cyclopentyimethyl, cyclopentylethyl, cyclopentylpropyl, cyclopentylbutyl, cyclohexylmethyl, cyclohexylethyl, cyciohexylpropyl, or cyclohexylbutyl group.

When R² is an aralkoxy or an aralkylthio group it may be for example a C₆_ ₁₂arylC,_₆alkoxy or C₆_₁₂arylC₁_₆alkylthio group such as a phenylC₁.

₆alkoxy or phenylC_|.₆alkyti.io group, e.g. a benzyloxy, phenylethoxy, phenylpropoxy, phenylbutoxy, benzylthio, phenylethylthio, phenylpropylthio or phenylbutylthio group.

The cycloalkyl, cycloalkylalkyl, aryl, aralkyl, aralkoxy or aralkylthio groups represented by R¹ and/or R² in compounds of formula (I) may each optionally be substituted in the cyclic part of the group by one, two or more substituents [R¹¹] selected from halogen atoms, e.g. fluorine, chlorine, bromine or iodine atoms, or Chalky!, e.g. methyl or ethyl, C.,_₆alkoxy e.g. methoxy or ethoxy, C₂.₆alkylenedioxy, e.g. ethylenedioxy, haloCμgalkyl, e.g. tri-fluoromethyl, C^galkylamino, e.g. methylamino or ethylamino, C-μ gdialkylamino, e.g. dimethylamino or diethylamino, amino (-NH₂), nitro, cyano, hydroxyl (-OH), carboxyl (-C0₂H), -C0₂R⁹, where R⁹ is as defined above, C,_₆alkylcarbonyl, e.g. acetyl, sulphonyl (-S0₃H), C,_ ₆alkyfsulphonyl, e.g. methylsulphonyl, aminosulphonyl (-S0₂NH₂), C^g alkylaminosulphonyl, e.g. methylamiπosulphonyl or ethylaminosulphonyl, C^gdialkylaminosulphonyl e.g. dimethylaminosulphonyl or diethylaminosulphonyl, carboxamido (-CONH₂), Cμgalkylaminocarbonyl, e.g. methylaminocarbonyl or ethylaminocarbonyl, C,_₆dialkylaminocarbonyl, e.g. dimethylaminocarbonyl or diethylaminocarbonyl, sulphonylamino (- NHS0₂H), C-_{| .}galkylsulphonylamino, e.g. methylsulphonylamino or ethylsu lpho nylami no , o r C^ dialkylsulphonylamino, e.g. dimethylsulphonylamino or diethylsulphonylamino groups. It will be appreciated that where two or more R¹¹ substituents are present, these need not necessarily be the same atoms and/or groups. The R¹¹ substituents may be present at any ring carbon atom away from that attached to the rest of the molecule of formula (I). Thus, for example, in phenyl groups any substituents may be present at the 2-, 3-, 4-, 5- or 6- positions relative to the ring carbon atom attached to the remainder of the molecule.

When the group R² in compounds of formula (I) is a substituted amino group, this may be for example a group -NHR¹ ° where R¹ ° is as defined above. Esterified carboxyl groups represented by R² include groups of formula - C0₂R^{1 2} where R^{1 2} is a straight or branched, optionally substituted C₁.₈alkyl group such as a methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl or t- butyl group; a C₆.₁₂arylC₁.₈alkyl group such as an optionally substituted benzyl, phenylethyl, phenylpropyl, 1-naphthylmethyl or 2-naphthylmethy! group; a C_6.12aryl group such as an optionally substituted phenyl, 1- naphthyl or 2-naphthyl group; a Cg.^aryloxyC^galkyi group such as an optionally substituted phenyloxymethyl, phenyloxyethyl , 1 - naphthyloxymethyl or 2-naphthyloxymethyl group; an optionally substituted C,_₈alkanoyloxyC_|_₈alkyl group, such as a pivaloyloxymethyl, propionyloxyethyl or propionyloxypropyl group; or a C₆_₁₂aroyloxyC₁..₈alkyl group such as an optionally substituted benzoyloxyethyl or benzoyloxypropyl group. Optional substituents present on the groups R¹² include for example one or more halogen atoms such as fluorine, chlorine, bromine or iodine atoms, or C₁.₄alkyl, e.g. methyl or ethyl, or C₁.₄alkoxy, e.g. methoxy or ethoxy, groups.

When the groups R³ and R⁴ in compounds of formula (I) are alkyl groups, they may be for example C,_₆alkyl groups such as methyl or ethyl groups.

The groups R⁶ and/or R⁷ in compounds of formula (I) may each be a hydrogen atom or an optionally substituted straight or blanched C _j__₆a.kyl_t e.g. methyl, ethyl, n-propyl, i-propyl, n-butyl or i-butyl, C₆.₁₂aryl, e.g. phenyl, or Cg.₁₂arylC₁.₆alkyl_l e.g. benzyl, phenylethyl or phenylpropyl group. Optional substituents present on alkyl groups of this type include one or more C_|_₆alkoxy, e.g. methoxy, ethoxy, or C₁.₆alkylthio, e.g. methylthio, or ethylthio groups or an optionally substituted C₆.₁₂aryloxy, e.g. phenyloxy,

C₆.₁₂arylthio e.g. phenylthio, Cg.^aryiCμgalkoxy e.g. benzyloxy or C₆_ ₁₂arylC_|_₆alkylthio e.g. benzylthio. Optional substituents present on the group R⁶ or R⁷ when it is an aryl or aralkyl group or an alkyl group substituted by an aryloxy or arylthio group include R¹¹ groups present on the cyclic part of R⁶ or R⁷ as defined above.

When the group Alk is present in compounds of formula (I) it may be a straight or branched C,_₆alkyl, e.g. methyl, ethyl, n-propyl i-propyl, n-butyl, i- butyl, n-pentyl or n-hexyl or C₂.₆alkenyl e.g. ethenyl or 1-propenyl group optionally interrupted by one or more -O- or -S- atoms or -N(R⁹)- groups where R⁹ is a hydrogen atom or a C-μgalkyl group such as a methyl group.

The group R⁸ in compounds of formula (I) may represent a C₃.₈cycloalkyl, e.g. cyclopentyi or cyclohexyl, or C₃.₈cycioalkenyl e.g. cyclopentenyl or cyclohexenyl, group optionally substituted by one, two or more Cμgalkyl, e.g. methyl or ethyl, C^galkoxy, e.g. methoxy or ethoxy, C,_galkylthio, e.g. methylthio, or hydroxyl groups.

When X in the compounds of formula (I) represents a substituted amino group it may be for example a group of formula -NR¹³R¹⁴, where R¹³ and

R¹ , which may be the same or different, is each a hydrogen atom (with the proviso that when one of R¹³ or R¹⁴ is a hydrogen atom, the other is not) or an optionally substituted straight ot branched alkyl group, optionally interrupted by one or more -O- or -S- atoms or -N(R⁹)- or aminocarbonyloxy

[-NHC(0)0-] groups or R^{1 3} and R¹⁴, together with the nitrogen atom to which they are attached, may form an optionally substituted C₃.₆cyclic amino group optionally possessing one or more other heteroatoms selected from -O- or -

S-, or -N(R⁹)- groups.

When R¹³ and/or R¹⁴ is an alkyl group it may be for example a C,_galkyl group such as a methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, ort- butyl group, optionally interrupted by one or more -0- or -S- atoms, or - N(R⁹)- or aminocarbonyloxy groups and may be for example a methoxymethyl, ethoxymethyl, methoxyethyl, ethoxyethyl or ethylaminocarbonyloxymethyl group. The optional substituents which may be present on such groups include hydroxyl (-OH), carboxyl (-C0₂H), esterified carboxyl (-C0₂R¹²), carboxamido (-CONH₂), substituted carboxamido, e.g. a group -CONR¹³R¹⁴ where NR¹³R¹⁴ is as defined herein, amino (-NH₂), substituted amino, for example a group of formula -

NR^{1 2}R¹³, or aryl, e.g. C₆_ι₂ aryl such as phenyl, optionally substituted by one, two or more R¹¹ substituents selected from those listed above in relation to the group R².

Particular examples of cyclic amino groups represented by -NR¹³R¹⁴ include morpholinyl, imidazolyl, piperazinyl, pyrrolyl, oxazolyl, thiazolyl, pyrazolyl, pyrrolidinyl, pyridinyl and pyrimidinyl groups.

When the group X is a substituted hydroxyl group it may be for example a group -OR¹³ where R¹³ is as defined above, other than a hydrogen atom.

Salts of compounds of formula (1 ) include pharmaceutically acceptable salts, for example acid addition salts derived from inorganic or organic acids, such as hydrochlorides, hydrobromides, hydroiodides, p-toluene sulphonates, phosphates, sulphates, acetates, trifluoroacetates propionates, citrates, malonates, succinates, lactates, oxalates, tartarates and benzoates.

Salts may also be formed with bases. Such salts include salts derived from inorganic or organic bases,, for example alkali metal salts such as sodium or potassium salts, alkaline earth metal salts such as magnesium or calcium salts, and organic amine salts such as morpholine, piperidine, dimethylamine or diethylamine salts.

The group R in compounds of formula (I) may in particular be a - P(0)(OR⁶)OR⁷, e.g. a -P(0)(OH)OR⁷ group, or a -P(0)(SH)OR^? or -

P(0)(OH)SR⁷ group. Examples of such groups include -P(0)(OCH₃)OCH₃, - P(0)(OCH₂CH₃)OCH₂CH₃, -P(0)(OH)OH, -P(0)(OH)SH, -P(0)(SH)OH, - P(0)(OH)OCH₃, -P(0)(OH)SCH₃, -P(0)(OH)OCH₂CH₃, -P(0)(OH)OPh, - P(0)(OH)SPh, -P(0)(OH)OCH₂Ph or -P(0)(OH)SCH₂Ph, where Ph is a phenyl group optionally substituted by one or more substituents R¹¹.

In the compounds of formula (I) the group R¹ may in particular be a C_j _₆alkyl group such as a methyl group, an aralkyl group such as benzyl group, an arylthioalkyl group such as a phenythiomethyl group or a heteroarylthioalkyi group such as thienylthiomethyl, pyridinylthiomethyl or pyrimidinylthiomethyl group or is especially a hydrogen atom.

The group R² may be in particular an optionally substituted Chalky!, C₃_ gcycloalkyl, C₃.₈cycloalkylC₁.₆alkyl, C₆.₁₂aryl, Cg.^arylC^galkoxy or C₆_ _{1 2}aralkylthio group and, especially, a Cg_₁₂arylC.,_galkyl group. Particular types of these groups are optionally substituted C₃.₆ alkyl, such as n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, n-pentyl or i-pentyl; cyclopentyi; cyclohexyl; cyclopentylC,_galkyl, such as cyclopentylC₃.₆alkyl, e.g. cyclopentylpropyl, cyclopentylbutyl, or cyclopentylpentyl; phenyl; α- or β- naphthyl; phenyiCμgalkoxy, e.g. phenylethoxy, phenylpropoxy or phenylbutoxy; phenylC^_g alkylthio, e.g. phenylethylthio, phenylpropyfthio or phenylbutylthio; and, especially, phenyiC^galkyl such as phenylC₃.₆alkyl e.g. phenylpropyl, phenylbutyl or phenylpentyl; or 1 - or 2-naphthylC₁_₆alkyl such as 1- or 2-naphthylC₃.₆alkyl, e.g.. 1- or 2-naphthylpropyl, naphthylbutyl or naphthylpentyl. Each of these cycloalkyl or aryl groups may be substituted, by one two or more substituents R¹¹ described above.

The groups R³ and R⁴ in compounds of formula (I) may each in particular be a methyl group, or, especially, a hydrogen atom.

The group R⁵ in compounds of formula (I) may be in particular a group - AlkR⁸, where R⁸ is an optionally substituted cycloalkyl or cycloalkenyl group. Thus, the group R⁵ in compounds of formula (I) may be an optionally substituted C₃_₈cycloalkylC,_₆alkyl [e.g. cyclopentylC^galkyl such as cyciopentylmethyl or cyclopentylethyl, or cyclohexyC_|_₆alkyl such as cyclohexylmethyl or cyclohexylethyl], C₃_₈cycloalkenylC._|_galky. [e.g. cyclopentenylC_|_₆alkyl such as cyclopentenylmethyl or cyclohexβnylC

₆alkyl such as cyclohexenylmethyl], cycloalkylC,_₃alkoxyCι_₃alkyl [e.g. cyclopentylmethoxymethyl, cyclohexylmethoxymethyl] Cg.gcycloalkenylC^ ₃alkoxyC, _.3al ky l [e . g . cyc lo pe n te n y l m et h o xy m et h yl o r cyclohexenylmethoxymethyl] C₃.₈cycloalkylC₁.₃alkylthioC₁.₃alkyii {e.g. cyclopentyimethylthiomethyl or cyclohexylmethylthiomethyl] or C₃_

₈cycloalkenylC₁_3alkylthioC₁.₃alkyl [e.g. cyclopentenylmethylthiomethyl or cyclohexenylmethylthiomethyl], C₃.₈cycloalkyC₁.₃alkylaminoC₁.₃alkyl [e.g. cyclopentylrπethylaminomethyl, or cyclohexylmethylaminomethylj or C₃_ gCycloalkenylC^galkyaminoC^galkyl [e.g. cyclopentenylmethylaminomethyi or cyclohexenylmethylaminomethyl] group.

The group X in compounds of formula (I) may be in particular an amino (- NH₂) or -NR ^{1 3}R^{1 4} group. Particular -NR ^{1 3}R^{1 4} groups are -NHR^{1 4} groups. Groups of this type include those where R ^{1 4} is a C,_₆alkyl group, for example a methyl, ethyl, or n-propyl group, optionally interrupted by one or more -O- or -S- atoms or -N(R⁹) [e.g. -NH- or -N(CH₃)-] or aminocarbonyloxy groups, and optionally substituted by a hydroxyl, carboxyl, carboxyalkyl, e.g. carboxymethyl, carboxamido, amino, -NR^{1 3}R¹⁴, [for example di-C₁. ₆alkylamino such as dimethylamino, C,_₆alkylamino such as methylamino, or C₃.₆ cyclic amino such as morpholinyl, pyrrolidinyl or pyridinyl] or phenyl optionally substituted by one, two or more R¹¹ substituents.

A particularly useful group of compounds according to the invention is that of formula (I) wherein R⁵ is an AlkR⁸, group, where Alk is a C^g alkyl and R⁸ is a C₃.₈ cycloalkyl or C₃.₈ cycloalkenyl group. Another particularly useful group of compounds according to the invention is that of formula (I) where R² is an optionally substituted alkyl, cycloalkyl, cycloalkylalkyl, aryl, aralkoxy or aralkylthio group.

A further particularly useful group of compounds of formula (I) are those wherein X is an amino or substituted amino group.

In general, in compounds of formula (I) the groups R¹ , R³ and R⁴ is each preferably a hydrogen atom.

An especially useful group of compounds according to the invention has the formula (ia)

wherein R, R², R⁵ and X are as defined for formula (I); and the salts, solvates and hydrates thereof.

A particularly useful group of compounds of formula (la) are those wherein R represents a P(0)(OH)OR⁷ group; R² represents an optionally substituted alkyl, alkenyl, cycloalkyl, cycloalkylalkyl, aryl, aralkoxy or aralkylthio group;

R⁵ represents a group -AlkR⁸, where Alk is a C^_₆ alkyl group and R⁸ is a cycloalkyl or cycloalkenyl group;

X is an amino (-NH₂) or substituted amino group; and the salts, solvates and hydrates thereof. Particuiarly useful compounds of formula (la) are those wherein R⁵ is a group -AlkR⁸, and R⁸ is an optionally substituted cyclohexyl group.

Compounds of this type in which R⁵ is a cyclohexylC_{1 -6}alkyl group, particularly a cyclohexylmethyl group, are especially useful.

Other useful compounds of formula (la) include those wherein R² represents a C₃.₆alkyl group, particularly an iso-butyl or n-pentyl group, or a cycloalkylC₃.₆alkyl group, particularly a cyclohexylpropyl, cyclohexylbutyl or cyclohexylpentyl group, or especially an optionally substituted phenylC₂. ₆alkyl group particularly an optionally substituted phenylethyl phenylpropyl, phenylbutyl or phenylpentyl group. Optional substituents on the phenyl group may be one, two or more R¹¹ groups as defined for compounds of formula (I).

In the compounds of formula (la) X may be a -NH₂ group or a group - NR^{1 3}R^{1 4} as defined for compounds of formula (I).

An especially useful group of compounds according to the invention has the formula (la) wherein R² is an optionally substituted phenylC_3.6alkyl group, especially an optionally substituted phenylpropyl or phenylbutyl group, R⁵ is a cyclohexylmethyl group; and X is a amino (-NH₂) or NR^{1 3}R^{1 4} group. Compounds of this type wherein X is -NH₂ or -NHR^{1 3} are particularly useful.

In general, compounds of formula (I) wherein R is a -P(0)(OH)OH group are particularly preferred.

In the compounds of formulae (I) and (1 a), when the group R⁵ is a cycloalkylC.,_₆alkyl group then the chiral centre to which this group is attached preferably has a S-configuration. The compounds according to the invention may be prepared by the following processes. In the description and formulae below the groups R,

R¹ , R², R³, R⁴, R⁵ and X are as defined above, except where otherwise indicated. It will be appreciated that functional groups, such as amino, hydroxyl or carboxyl groups, present in the various compounds described below, and which it is desired to retain, may need to be in protected form before any reaction is initiated. In such instances, removal of the protecting group may be the final step in a particular reaction. Suitable amino or hydroxyl protecting groups include benzyl, benzyloxycarbonyl or t- butyloxycarbonyl groups. These may be removed from a protected derivative by oatalytic hydrogenation using for example hydrogen in the presence of a metal catalyst, for example palladium on a support such as carbon in a solvent such as an alcohol e.g. methanol, or by treatment with trimethylsiiyi iodide or trifluoroacetic acid in an aqueous solvent. Suitable carboxyl protecting groups include benzyl groups, which may be removed from a protected derivative by the methods just discussed, or alkyl groups, such as a t-butyl group which may be removed from a protected derivative by treatment with trifluoroacetic acid in an aqueous solvent. Other suitable protecting groups and methods for their use will be readily apparent. The formation of the protected amino, hydroxyl or carboxyl group may be achieved using standard alkylation or esterification procedures, for example as described below.

Thus according to a further aspect of the invention a compound of formula (I) may be prepared by coupling an acid of formula (II)

(II) or an active derivative thereof, with an amine of formula (III)

followed by removal of any protecting groups.

Active derivatives of acids for formula (II) include for example acid anhydrides, or acid halides, such as acid chlorides.

The coupling reaction may be performed using standard conditions for amination reactions of this type. Thus, for example the reaction may be achieved in a solvent, for example an inert organic solvent such as an ether, e.g. a cyclic ether such as tetrahydrofuran, an amide e.g. a substituted amide such as dimethylformamide, or a halogenated hydrocarbon such as dichloromethane at a low temperature, e.g. -30°C to amibient temperature, such as -20°C to 0°C, optionally in the presence of a base, e.g. an organic base such as an amine, e.g. triethylamine or a cyclic amine such as N- methylmorpholine. Where an acid of formula (II) is used, the reaction may additionally be performed in the presence of a condensing agent, for example a diimide such as N.N'-dicyclohexylcarbodiimide, advantageously in the presence of a triazoie such as l-hydroxybenzotriazoie. Alternatively, the acid may be reacted with a chloroformate for example ethylchloroformate, prior to reaction with the amine of formula (III).

Free hydroxyl or carboxyl groups in the starting materials of formulae (II) and (III) may need to be protected during the coupling reaction. Suitable protecting groups and methods for their removal may be those mentioned above. Compounds of formula (II) for use in this reaction are preferably those wherein at least one of R⁶ or R⁷ in the group R is other than a hydrogen atom. Conveniently, each of R⁶ and R⁷ is an optionally substituted alkyl, aryl or aralkyl group. Such groups, when present in compounds of the invention may be cleaved as described below to yield other compounds of the invention whe_.rein R⁶ and/or R⁷ is each a hydrogen atom.

It will be appreciated that where a particular steroisomer of formula (I) is required, this may be obtained by resolution of a mixture of isomers following the coupling reaction of an acid of formula (II) and an amine of formula (III). Conventional resolution techniques may be used, for example separation of isomers by Chromatography e.g. by use of high performance liquid chrormatography. Where desired, however, appropriate homochiral starting materials may be used in the coupling reaction to yield a particular stereo isomer of formula (I). Thus, in particular process a compound of formula (la) may be prepared by reaction of a compound of formula (lla)

with an amine of formula (Ilia)

(Ilia)

as described above. Compounds of formula (I) wherein R is a group -P(0)(X¹ R⁶)X²R⁷ and R⁶ and/or R⁷ is a hydrogen atom may be prepared from a corresponding compound of formula (I) wherein R⁶ and/or R⁷ is an optionally substituted alkyl, aryl or aralkyl group by a cleavage reaction, usir.g for example a reagent such as trialkylsilyl halide, e.g. a trialkylsilyl bromide such as bromotrimethylsilane, in an inert solvent such as a halogenated hydrocarbon e.g. dichloromethane, or an aqueous acid or alkali; or, when R⁶ and/or R⁷ is an aralkyl group by hydrogenolysis using reagents and conditions as described below for the preparation of intermediates of formula (II).

In the following description of the preparation of intermediate compounds the groups R⁶ and/or R⁷ in the group R are preferably other than a hydrogen atom.

Intermediate acids of formula (II) may be prepared from a corresponding ester of formula (IV)

where R¹⁵ is an aralkyl group, such as a benzyl group, by hydrogenolysis, for example by reaction with hydrogen in the presence of a metal catalyst, e.g. palladium, on a support such as carbon. The reaction may be performed in a solvent such as an alcohol, e.g. methanol optionally at an elevated pressure and temperature. The intermediates of formula (IV) may be prepared by reaction of an acrylate of formula (V)

with a phosphϊte:P(OR¹⁶)(X¹ R⁶)X²R⁷ where R^{1 6} is a leaving group, for example a silyl group such as a trialkylsilyl group e.g. a trimethylsiiyi group, at an elevated temperature.

Acrylates of formula (V) may be prepared by reaction of an mono-ester of formula (VI)

with an aldehyde R¹CHO or polymer thereof e.g. paraformaldehyde or paraldehyde in the presence of a base, for example an organic base such as piperidine. The reaction may be performed in a solvent, such as pyridine, optionally at an elevated .temperature.

Mono-esters of formula (VI) may be prepared by hydrolysis of the corresponding di-ester of formula (VII)

using a base, for example an alkali hydroxide such as potassium hydroxide, in an inert solvent such as dioxan at a low temperature e.g. around 0°C.

Diesters of formula (VII) may be prepared by alkylation of the corresponding malonates of formula R¹⁵OCOCH₂C0₂R¹⁵ with a halide R²Hal, where Hal is a halogen atom such as a chlorine or bromine atom in the presence of a base, e.g. a hydride such as sodium hydride in a solvent such as tetrahydrofuran at ambient temperature.

Malonates of formula R¹⁵OCOCH₂C0₂R¹⁵ are either known compounds or may be prepared by methods analogous to those used for the preparation of the known compounds.

Intermediate phosphites of formula :P(OR^{1 6})(X¹ R⁶)X²R⁷ for use in the preparation of intermediates of formula (IV) may be prepated by reaction of a phosphite HP(0)(X¹ R⁶)X²R⁷ with an appropriate amine (R¹⁵)₂NH e.g. a silazane, at an elevated temperature, e.g. the reflux temperature.

Phosphites of formula HP(0)(X¹R⁶)X²R⁷ are either known compounds or may be prepared by methods analogous to those used for the preparation of the known compounds.

In an alternative process, intermediate acids of formula (II) may be prepared by reaction of an acid R²CH₂C0₂H with a phosphonate P(0)(X¹R⁶)(X²R⁷)CH₂OR¹⁶ where R¹⁶ is a leaving group, for example a trifluoromethylsulphonyloxy group in the presence of a base such as n- butyllithium in a solvent such as tetrahydrofuran. Phosphonates for use in this reaction may be prepared from the corresponding compound P(0)(X¹R⁶)(X²R⁷)CH₂OH by reaction with paraformaldehyde in the presence of a base such as triethylamine at an elevated temperature followed by reaction with a halide R¹⁶Hal in the presence of a base such as sodium hydride in a solvent such as an ether. Phosphonates P(0)(X¹R⁶)(X²R⁷)CH₂OH are either known compounds or may be prepared by methods analogous to those used for the preparation of the known compounds.

Intermediates )f formula (III) and acids of formula R CH₂C0₂H are either known compounds, or may be prepared from known starting materials by methods analogous to those used for the preparation of the known compounds.

The homochiral acids of formula (lla) may be prepared according to another feature of the invention by oxidation of an oxazolidinone of formula (VIII)

(where Ph is a phenyl group) using an oxidising agent such as peroxide, e.g. hydrogen peroxide in a solvent such as an ether e.g: a cyclic ether such as tetrahydrofuran, at a low temperature, e.g. around 0°C followed by treatment with a base, such as lithium hydroxide, at an elevated temperature. The compounds of formula (VIII) are novel, particularly useful, intermediates for the preparation of stereoisomers of formula (la) and form a further aspect of the invention.

The compounds of formula (VIII) may be prepared by reaction of an acyl halide RCH₂CH(R²)COHal (where Hal is a halogen atom such as chloride, bromine or iodine atom) with a solution of (S)-4-(phenylmethyl)-2- oxazolidinone in the presence of a base such as n-butyl lithium in a solvent such as tetrahydrofuran at a low temperature, e.g. around -78°C.

Acyl halides RCH₂ CH(R²)COHal may be prepared by treatment of the corresponding known acids RCH₂CH(R²)C0₂H with conventional halogenating agents for example thionyl halides under standard reaction conditions.

The compounds according to the invention are potent and selective inhibitors of gelatinase. The activity and selectivity of the compounds may be determined by the use of appropriate enzyme inhibition test for example as described in Example A hereinafter. In our tests using this approach, compounds according to the invention have been shown to inhibit gelatinase with Ki values in the picomolar-nanomolar range and to have particularly useful selectivity for gelatinase over stromelysin and collagenase.

The ability of compounds of the invention to prevent tumour cell invasion may be demonstrated in a standard mouse model. Thus, briefly, nude mice may be inoculated with a tumour cell line showing gelatinase - dependent invasion and the ability of compounds according to the invention to reduce subsequent lung tumour colonisation may be evaluated in accordance with standard procedures. In out tests, compounds according to the invention, when administered intravenously at 1 mg/kg to mice in the above model have reduced lung tumour colonisation to negligable levels, and do not cause any adverse effects at this dose.

The compounds according to the invention can be expected to be of use to prevent tumour cell metastasis and invasion. The compounds may therefore be of use in the treatment of cancer, particularly in conjunction with radiotherapy, chemotherapy or surgery, or in patients presenting with primary tumours, to control the development of tumour metastasises. Thus, according to a further aspect of the invention we provide a compound of formula (I) for use in the treatment of cancer to control the^" development of tumour metastasises. Particular cancers may include breast, melanoma, lung, head, neck or bladder cancers.

For use according to this aspect of the invention, the compounds of formula (I) may be formulated in a conventional manner, optionally with one or more physiologically acceptable carriers, diluents or excipients.

Thus according to a further aspect of the invention we provide a pharmaceutical composition comprising a compound of formula (I) and a pharmaceutically acceptable diluent, carrier or excipient.

in a still further aspect the invention provides a process for the production of a pharmaceutical composition comprising bringing a compound of formula (I) into association with a pharmaceutically acceptable diluent, carrier or excipient.

Compounds for use according to the present invention may be formulated for oral, buccal, parental or rectal administration or in a form suitable for nasal administration or administration by inhalation or insufflation.

For oral administration, the pharmaceutical compositions may take the form of, for example, tablets or capsules prepared by conventional means with pharmaceuticaily acceptable excipients such as binding agents (e.g. pregeiatinised maize starch, polyvinylpyrrolidone or hydroxypropl methylcellulose); fillers (e.g. lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g. magnesium stearate, talc or silica); disintegrants (e.g. potato starch or sodium glycollate); or wetting agents (e.g. sodium lauryl sulphate). The tablets may be coated by methods well known in the art. Liquid preparations for oral administration may take the form of, for example, solutions, syrups or suspensions, or they may be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents, emulsifying agents, non-aqueous vehicles; and preservatives. The preparations may also co'ntain buffer salts, flavouring, colouring and sweetening agents as appropriate.

Preparations for oral administration may be suitably formulated to give controlled release of the active compound.

For buccal administration the compositions may take the form of tablets or lozenges formulated in conventional manner.

The compounds of formula (I) may be formulated for parental administration by injection e.g. by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form. The compositions for injection may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilising and/or dispersing agents. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g. sterile pyrogen-free water, before use.

The compounds of formula (I) may also be formulated in rectal compositions such as suppositories or retention enemas, e.g. containing conventional suppository bases such as cocoa butter or other glycerides.

In addition to the formulations described above the compounds of formula (I) may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation or by intramuscular injection.

For nasal administration or administration by inhalation the compounds for use according to the present invention are conventiently delivered in the form of an aerosol spray presentation for pressurised packs or a nebuliser, with the use of suitable propellant, e.g. dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.

The compositions may, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the active ingredient. The pack or dispenser device may be accompanied by instructions for admininstration.

The doses of compounds of formula (I) used to control the development of tumour metastasises will vary depending on the condition of the patient to be treated but in general may be in the range around 0.5mg to 50mg/kg body weight, particularly from about 1 mg to 40mg/kg body weight. Dosage units may be varied according to the route of administration of the compound in accordance with conventional practice.

DescriDtion of Specific Embodiments

The invention is further illustrated in the following non-limiting Examples.

In the Examples, the following abbreviations are used:

RT - room temperature DCCI - N,N'-dicyclohexylcarbodiimide DMF - dimethylformamide THF - tetrahydrofuran TFA - trifluoroacetic acid RPHPLC reverse phase high performance liquid chromatography HOBT- N-hydroxybenzotriazole Et₂0 - diethylether EDC - 1 -(3-dimenthyiami nopropyl)-3-ethylcarbodiimide, hydrochloride

All temperatures are in °C

Intermediate 1

Benzvl .2-benzvloxvcarboxvl-5-phenvl. pentanoate

To a suspension of NaH (5.04g) in dry THF (200ml) was added dropwise over a period of 1 hour, dibenzyl malonate (56.86g, 50ml) and the temperature allowed to rise to 43°. 1-Bromo-3-phenylpropane (39.8g,

30.4ml) was added and the reaction kept at RT for 3 hours. The mixture was refluxed for 4 hours, cooled, poured into H₂0/NaCI/NH₄CI (400ml) and extracted with CH₂CI₂ (4 x 100ml). The organic layer was dried (MgS0₄) and concentrated to give a yellow oil. The ϋϋg. compound (63.6g) was purified on stlica gel (Merck 9385) using Et₂0/hexaπe (5:95) —

Et₂0/hexane (25:75).

¹HNMR (CDCI₃) δ 7.0-7.40 (15H, m), 5.15 (4H, s), 3.45 (1 H, d, d), 2.60 (2H, t), 1.85-2.05 (2H, m), 1.55-1.65 (2H, m)

Intermediate 2

2-Benzvloxvcarbonvl-5-phenvlpentanoic acid

To Intermediate 1 (63.6g) dissolved in 1,4-dioxan (400m!) was added KOH (8.87g) in H₂0 (110ml) at 0°. The mixture was allowed to rise to RT and stirred overnight. The solvent was removed in vacuo and H₂0 (100ml) added. Extraction with Et₂0 removed any undesired diester and the aqueous phase was acidified (pH~1 ) with 1.0M HCI. The product was extracted into Et₂0 (3 x 100ml), dried (MgS0₄) and concentrated to give the title compound (29.0α..

¹HNMR (CDCI₃) δ 10 (1 H, bs) 7.10-7.60 (10H, m), 5.20 (2H, s) 3.48 (1H, t), 2.52 (2H, t), 1.86-2.10 (2H, m), 1.60-1.80 (2H, m) IntφrmQ te 3

0-Benzvl-2-.3-phenvl.propvlDroD-2-enoate

Paraformaldehyde (380mg) was added to a stirred solution of the

Intermediate 2 (2.6g) dissolved in anhydrous pyridine (25ml) containing piperidine (118mg, 137μl). The reaction was heated at 60° for 2.1/2 hours, cooled, poured into 1.0 MHCl and extracted with Et₂0 (3 x 50mL). The Et₂0 layer was dried (MgS0₄), and concentrated to give the tills, compound (1 -3g).

¹HNMR (CDCI₃) δ 7.12-7.36 (10H, m), 6.21 (1 H, d), 5.55 (1 H, d), 5.20 (2H, s), 2.62 (2H, t), 2.37 (2H, t), 1.76-1.88 (2H, m).

Intermediate

Pimet yltrimethy|gi|yiphQ-Phite

A mixture of dimethylphosphite (9.18ml) and hexamethyldisilazane (25.32ml) was heated under reflux (bath temperature 140°) for 3h. The crude mixture was distilled in vacuo (approximately 5mm Hg) through a

12cm vigueux column and the fraction distilling at bath temperature 95°- 110° collected to give the Hue. compound (7.20g) ¹ HNMR (CDCI3) δ/ppm 3.50 (s, 3H), 3.46 (s, 3H), 0.26 (s, 9H).

Intermediate 5

Benzvl .2-dimethoxvphosphinvlmethvl-5-Dhenvl.pentanoate

A mixture of Intermediate 4 (1.877g) and Intermediate 3 (1.044g) were stirred at 70° (N2 atom) for 18h. Water (10ml) and chloroform (10ml) were added and the mixture stirred vigrously for 0.5h. The mixture was partitioned between water (10ml) and chlorofom (15ml) and the aqueous layer further extracted with chloroform (20ml). The organic layers were dried (MgS0₄) and concentrated in vacuo to a crude oil 1.55g. This was chromatographed on silica 60 ('Merck' 9385) eluting with ethyl acetate-methanol (2%) to give the title compound as an oil (0.3423g)

¹HNMR (CDCI₃) δ 7.35 (5, 5H), 7.77 (t, 2H), 7.17 (t, 1 H), 7.08 (d, 2H), 5.115 (s, 2H), 2.83 (m, ¹ H), 2.57 (t, 2H), 2.29 dd and 2.23 dd (-together 1 H), 1.87 dd and 1.83 dd (together ¹ H) and 1.76-1.50 ppm (br in 4H). -

Intermediate 6

2-Dimethoxyphosphinylmethyl-5-phenylpentanoic acid A mixture of Intermediate 5 (0.3397g) and 10% palladium on carbon catalyst (50mg) in methanol (20ml) was stirred under an atmosphere of hydrogen for 18h. tic silicagel 60 (chloroform-methanoi 10% eluent) showed conversion to one product with Rf0.23. The mixture was filtered and concentrated in vacuo to give the title compound as a colourless oil (0.2498g)

¹ HNMR (CDCI₃) δ 7.30-7.19 (m, 3H), 7.16 (d, 3H) 3.76 (s, 3H)_r 3.72 (s, 3H),

2.79 (m, 1 H), 2.63 (t, 2H), 2.30 (dd) and 2.24 (dd)- together 1 H, 1.88 (dd) and 1.82 (dd) - together 1 H and 1.76-1.66 (m, 4H) ppm.

Intermediate 7

L-β-cyclQhg>.ylalaπiπ _?-N-(2-phgpylgt yl) amid? tBoc-β-cyclohexyl-L-alanine (1.35g, 5mmoL) was dissolved in dry CH2CI2-

4-Nitrophenol (695mg, 5mmoL) was added followed by DCCI (1.03g,

5mmoL). After 1 hour at room temperature the reaction was concentrated in vacuo. ether was added and the solution filtered. The residue was concentrated in vacuo . dissolved in CH₂Cl2 (10ml) and phenethylamine (690μl, 5.5mmoL) was added. The reaction was poured into NaHC0₃ and extracted with CH2CI2 (3 x 20ml), was dried (l^SO^ and concentrated [n vacuo. Purification on silica gel (Merck 9385) using CH2Cl2→CH₂Cl2/MeOH 85:15) gave a clean oil (900mg) which was dissolved in CH2CI2 T A (9:1 ) and left a RT for 30 min. The reaction was concentrated in vacuo. dissolved in CH2CI2 (50ml) and poured into Na2C0₃ (aq). The organic layer was separated, dried (Na2Sθ4) and concentrated in vacuo to give an oil which was purified on silica gel (Merck 9385) using C^C^/methanol/triethylamine 96:3:1 to give the title compound as an oil (500mg).

¹ H NMR (CDCL3) δ 0.95 (m, 2H), 1.25 (m, 6H), 1.55 (bs, 2H), 1.65 (m, 5H), 2.8 (t, 2H, J=6HZ), 3.4 (dd, 1 H, J=3 and 10HZ), 3.5 (dd, 2H, J=6 and 12HZ), 7.2 (m, 5H)

Intermediate 8

O-.Diethyl.hvdroxymethylphosphonate

A mixture of diethyl phosphite (20g) paraformaldehyde (4.44g) and triethylamine (2ml) was heated to 60°C for 1 hour. The reaction was then cooled and partitioned between chloroform and aqueous citric acid. The organic layer was separated, washed with brine, dried (MgS0₄) and evaporated to give the tili__. compound as a clear colourless oil. (20.73g).

1HNMR (CDCI3) δ 4.2(4H, m); 3.9 (2H, d); 1.35 (6H, t). Iπtermediate 9

0-,Diethvl.trifluoromethvlsulDhonvlnxvmethvlDhosphonate

To a suspension of sodium hydride (3.85g) in dry ether (200ml) at -30°C was added by syringe trifluoromethanesulphonyi chloride (25g). A solution of Intermediate 8 (20.73g) in dry ether (20ml) was added dropwϊse over 20 minutes maintaining the internal temperature at -20°C. After stirring the suspension for 2 hours at -20°C, the excess hydride was removed by filtering through Celite. The filtrate was diluted with dichloromethane, washed twice with 10% aqueous sodium bicarbonate, separated, dried (MgS0₄) and evaporated to give the tills com^pound as a pale yellow oil.

The tills compound was purified on silica, eluting with 35% ethyl acetate in hexane (10.5g).

¹H NMR (CDCI₃) δ 4.60 (2H, d); 4.25 (4H, m); 1.40 (6H, t)

Intermediate 10

2-Diethoxvphos^phinvimethvl-5.4-methylphenvl.pentanoic acid

A solution of n-butyllithium in hexane (13ml) was added to a solution of di- isopropylamine (2.94ml) in dry THF (30ml) at 0°C. After stirring for 30 minutes, a solution of p-toluylvaleric acid (2.0g) in dry THF (15ml) was added and the reaction mixture was warmed to 35°C for 30 minutes. After cooling to room temperature, hexamethylphosphorus triamide (1.89ml) was added, followed by Intermediate 9 (3.12g) as a solution in THF (15ml). The reaction was stirred for 3 hours at room temperature then quenched with aqueous citric acid, extracted into Et₂0, dried (MgS0₄) and evaporated. The residue was chromatographed on silica, eluting with 3 - 10% CH₃OH in CH₂CI₂, to give the title compound as a clear gum (1.26g).

¹HNMR (CDCI₃) δ 7.05 (4H, m); 4.05 (4H, m); 2.5-2.85 (3H, m); 2.3 (3H, s); 2.25 (1 H, m); 1.6- 1.9 (5H, m); 1.25 (3H, t).

Example 1

[3-Phosphono-2R.S-phenvlpropvl-1 -oxoproDvn-L-β-cvclohexvlalanine-N-.2- phenylethyl. amide, dimethylester

A solution of Intermediate 6 (151.7mg) in dry tetrahydrofuran (3.0ml) was cooled to -30° and treated with N-methylmorpholine (59.1μl) and ethyl chloroformate (49.0μl). The mixture was stirred at -30° for 1 h and a solution of Intermediate 7 (240.8mg) and N-methylmorphorpholine (59.1 ml) in dry DMF (2.0ml) added dropwise at -30°C. The mixture was allowed to warm up to RT overnight and the solvent removed in vacuo. The residue was partitioned between water (15mi) and ethyl acetate (20ml) and the aqueous layer further extracted with ethyl acetate (2 x 20ml). The combined organic layers were dried (MgS0₄) and concentrated in vacuo to give an oil (304.6mg) which was chromatographed on silicagel 60 ('Merck' 9385) eluting with ethyl acetatemethanol (2%) to give the tills compound as a mixture of diastereoisomers and as an oil (189.4mg)

1 HNMR (CDCI₃) δ 7.64 (br, t, 0.5H), 7.40-7.10 (br, m, 10H), 6.31 (br, t, 0.5H)

5.17 (d, 0.5H), 5.78 (d, 0.5H), 4.5 (br, dt) and 4.14 (dd) -together ¹ H, 3.72 (s, 3H), 3.69 (s, 3H), 3.45 (m, 2H), 2.83 (t) and 2.69 (t) - together 2H, 2.60 (br, q,

2H), 2.45 (br, m, ¹ H) 2.35-2.05 (m, 1 H) and 2.0-0.8 (br, m, 18H) ppm. Examole 2

[3-Phosphono-2R-phenylproDyl-1 -oxopropyl]-L-β-cvclohexylalanine-N-.2- phenylethyl) amide

A solution of the compound of Example 1 (189.4mg) in dry dichloromethane (5.0ml) was treated with bromotrimethylsiiane (1.0ml) and the mixture stirred overnight at room temperature. Methanol (15.0ml) was added and the mixture stirred at room temperature for 0.5h. The solvent was removed in vacuo, the residue azeotroped with ethanol (40ml) and then dissolved in ethanol water (1 :1) (30ml). The solution was stirred at room temperature for 0.5h and the solvent removed in vacuo to give a mixture of RS and SS diastereoisomers as an oily solid. This was separated by preparative HPLC on a Dynamax C18RP column (21.4mm dia) using 0.1 % TFA-H₂0 (A) and 0.1% TFA-CH₃CN(C) and gradient elution between (70:30) and 5:95) respectively. Peak 1 (elution time 14.71 mins) yielded the RS isomer (20.0mg) as a white solid.

¹ HNMR (CDCI₃) δ 7.27-6.95 (br, m, 10H, 4.25 (t, ¹ H), 3.27 (m, 2H)2.75-2.40 (m, 5H), 2.03 (dd) and 1.95 (dd) -together 1 H and 1.80-0.68 (br, m, 18H) ppm

Peak 2 (elution time 15.98 mins) yielded the SS isomer (58.4mg) as a white solid.

¹ HNMR (CDCI₃) δ 7.35-7.0 (br, m, 10H), 4.25 (dd, 1 H), 3.30 (br, t, 2H), 2.80-

2.40 (br, m, 5H), 2.15-1.94 (m, 1 H) and 1.85-0.6 ppm (br, m, 18H)

The following compounds were prepared in a similar manner to the compound of Example 2 by reaction of Intermediate 6 with an appropriate amide, and subsequent hydrolysis of the resulting dimethyl ester followed by separation of diastereoisomers, as described above. Example 3

[3-Phosphono-2S-phenvlpropyl-1 -oxopropyl]-L-β-cyclohexvlalani ne-β- alanine

¹ HNMR (CDCIg) δ 7.15-6.90 (m, 5H), 4.19 (dd, 1 H), 3.14 (m, 2H), 2.65-2.40 (m, 3H), 2.34 (t, 2H), 1.99 (dd) and 1.96 (dd) -together 1 H and 1.70-0.52 (br, m, 18H)

Example 4

[3-Phosphono-2R-phenvlpropvl-1 -oxoprθDvll-L-β-cvclohexvlalanine ¹ HNMR (CDCI₃) δ 7.30-7.10 (m, 5H), 4.26 (br, t, 1 H), 3.28 (m, 2h), 2.74-2.46 (m, 3h), 2.35 (t, 2H), 2.03 (dd) and 1.97 (dd) together 1 H and 1.90-0.54 ( r, m, 18H

Example 5

alanine. methyl ester

¹ HNMR (CDCI₃) δ 7.22-7.0 (m, 5H), 4.25 (dd, 1 H), 3.56 (s, 3H), 3.32 (d, tr,

2H), 2.70-2.46 (m, 3H), 2.44 (t, 2H), 2.14 (br, h, 1 H) and 1.80-0.65 ppm (br, m, 18H) Examole 6

[3-Phosphono-2R.S-phenylpropyl-1 -oxopropyll-L-β-cvclohexylalanine-N- [4.3-aminopropvl.morpholinel amide, bromine salt

ISOMER 1¹H_,δ (DMSO db) 8.48 (br, t, ~ 0.5H), 8.30 (d, - 0.5H), 7.34-7.20 (m, 2H), 7.20-7.10 (br, d, 3H), 4.16 (m, 1 H), 3.95 (m, 2H) 3.80-3.0 (br, m) overlapped with H₂0, 2.60 (m, 1 H) 2.0 (br, q, 1 H) and 1.80-0.60 (br, m, 18H).

ISOMER 2 ¹ HNMR δ (DMSO db) 8.57 (br, t, ~ 0.5H) 8.27 (d, ~ 0.5H), 7.30- 7.20 (m, 2H), 7.20-7.10 (br, d, 3H) 4.14 (m, 1 H), 3.92 (m, 2H), 3.80-3.0 (br, m) overlapped with H₂0, 2.65 (m, 1 h), 1.95 (br, q, 1 H) and 1.75-0.60 (br, m, o 18H) ppm

Example 7

I2ιElιasβiιana--2B,a;H; iiι i&£ cyclohexylalanine-N-(2-phenylethyl. amide, diethylester

A solution in dry DMF (5ml) of Intermediate 10 (250mg), triethylamine s (101.4μl); N-hydroxybenzotriazole (98.6mg), Intermediate 7 (200mg) and

EDC (140mg) was stirred at room temperature for 5 hours under nitrogen. The reaction mixture was partitioned between Et₂0 and aqueous citric acid, the organic layer separated, washed with aqueous NaHC0₃, dried (MgS0₄) and evaporated to give the tills compound (320mg) as a ^'50:50 mixture of o diastereomers .

¹H NMR (CDC1₃) δ 7.9(0.5H,t); 7.0-7.3 (9H, m); 4.5 (0.5H, m); 4.5 (0.5H, m); 4.35 (0.5H, m); 4.0

(4H, m); 3.40 (2H, m); 2.70-2.90 (2H, m); 2.40-2.10 (8H, m); 0.80-2.0 (23H, m). Example 8

_vciohexvlalanine-N-.2-phenvlethvl.-amide

A solution of the compound of Example 7 (320mg) in dry CH₂CI₂ (20ml) was treated with trimethylsiiylbromide (2ml) at room temperature for 2.5 days.

The reaction was quenched with methanol (5ml) and volatile material was removed. The two diastereoisomers were separated by reverse phase

HPLC (Dynamax 60A). Eluent: 50 - 100% acetonitrile in 0.1% TFA/water, to yield the R-isomer (49mg). ¹H NMR (CD₃0D) δ 7.15-7.30 (5H, m); 7.0 (4H, m); 4.35 (1 H, t); 3.35 (2H, m); 2.0-2.75 (10H, m);

0.80-1.85 (17H, m).

EXAMPLE A

The activity and selectivity of the compounds of the invention may be determined as described below.

All enzyme assays to determine Ki values were performed using the peptide substrate Dnp-Pro-Leu-Gly-Leu-Trp-Ala-D-Arg-NH₂. [M. Sharon Stock and Robert D. Gray. JBC 264. 4277-81 , 1989). The enzymes cleave at the Gly- Leu bond which can be followed fluorimetrically by measuring the increase in Trp fluorescence emission associated with the removal of the quenching dinitrophenol (Dnp) group.

Essentially, enzyme (e.g. gelatinase, stromelysin, collagenase) at 0.08-2nM; a range of inhibitor concentrations (0.1 -50 x Ki) and substrate (approx.

20μm) are incubated overnight in 0.1 M Tris/HCI buffer, pH 7.5, containing 0.1 M NaCI, 10mM CaCl2 and 0.05%. Brij 35 at either room temperature or

37°C depending on the enzyme. The reaction is stopped by adjusting the pH to 4 using 0.1 M sodium acetate buffer and the fluorescence read at an excitation wavelength of 280nm and emission wavelength of 346nm.

K: values can be established using the equation for tight-being inhibition:-

where V₀ is the initial rate of reaction in the absence of inhibitor, V_j is the initial rate in the presence of inhibitor, [E] is the total enzyme concentration and [j] the total inhibitor concentration in the reaction mixture.

For stromelysin and collagenase, Kj (app) was assumed to approximate to the true K_j as [S] « K_m for the substrate hydrolysis. For gelatinase the K_j was determined by performing the analyses at several substrate concentrations. A plot of K_j(app) vs. [S] then gave the true K_j as the value of the y-axis intercept.

The following results were obtained with compounds according to the invention:

Ki.nM.

Claims

1. A compound of formula (1 )

R¹ represents a hydrogen atom or an optionally substituted alkyl, alkenyl, aryl, aralkyl, heteroaralkyl or heteroarylthioalkyi group;

R² represents an optionally substituted alkyl, alkenyl, cycloalkyl, cycloalkylalkyl, aryl, aralkyl, aralkoxy, or aralkylthio group, or an amino

(-NH₂), substituted amino, carboxyl (-C0₂H) or esterified carboxyl group;

R³ represents a hydrogen atom or an alkyl group;

R⁴ represents a hydrogen atom or an alkyl group;

R⁵ represents a group -[Alkj_pR⁸ where Alk is an alkyl or alkenyl group optionally interrupted by one or more -O- or -S- atoms or -N(R⁹)- groups [where R⁹ is a hydrogen atom or a C_|_₆aikyl group], n is zero or an integer 1 , and R⁸ is an optionally substituted cycloalkyl or cycloalkenyl group;

X represents an amino (-NH₂), or substituted amino, hydroxyl or substituted hydroxyl group; and the salts, solvates and hydrates thereof.

2. A compound according to Claim 1 wherein R represents a -P(0)(OR⁶)OR⁷ group where R⁶ and R⁷, which may be the same or different each represents a hydrogen atom or an optionally substituted alkyl, aryl or arlkyl group.

3. A compound according to Claim 2 wherein R is a -P(0)(OH)OH group.

4. A compound according to any one of the preceding claims wherein R¹ is a hydrogen atom.

• _

5. A compound according to any one of the preceding claims wherein R³ is a hydrogen atom.

6. A compound according to any one of the preceding claims wherein R⁴ is a hydrogen atom.

7. A compound according to any one of the preceding claims wherein R² is an optionally substituted alkyl, cycloalkyl, cycloalkylalkyl, aryl, aralkoxy or aralkylthio group.

8. A compound according to Claim 7 wherein R² is an optionally substituted alkyl group.

9. A compound according to any one of the preceding claims wherein R⁵ is an AlkR⁸ group where Alk is a C_1-6 alkyl group optionally interrupted by one or more -O- or -S- atoms or -N(R⁹)- groups, where R⁹ is a hydrogen atom or a Cι_₆ alkyl group, and R⁸ is an optionally substituted C_3-8 cycloalkyl or C₃.₈ cycloalkenyl group.

10. A compound according to any one of the preceding claims wherein X is an amino or substitued amino group.

11. A compound of formula (1a)

(-NH₂), substituted amino, carboxyl (-C0₂H) or esterified carboxyl group;

R³ represents a hydrogen atom or an alkyl group;

R⁴ represents a hydrogen atom or an alkyl group;

R⁵ represents a group -[Alk^R⁸ where Alk is an alkyl or alkenyl group optionally interrupted by one or more -O- or -S- atoms or -N(R⁹)- groups

[where R⁹ is a hydrogen atom or a C,_₆alkyl group], n is zero or an integer 1 , and R⁸ is an optionally substituted cycloalkyl or cycloalkenyl group;

12. A compound according to Claim 11 wherein R represents a P(0)(OH)OR⁷ group;

R² represents an optionally substituted alkyl, alkenyl, cycloalkyl, cycloalkylalkyl, aryl, aralkoxy or aralkylthio group;

R5 represents a group -AlkR⁸, where Alk is a C_1-6 alkyl group and R⁸ is a cycloalkyl or cycloalkenyl group;

X is an amino (-NH₂) or substituted amino group; and the salts, solvates and hydrates thereof.

13. A compound according to Claim 12 wherein R is a -P(0)(OH)OH group.

14. A compound according to Claim 12 or Claim 13 wherein R⁵ represents a cyclohexylC-₍.₆alkyl group;

15. A compound according to Claim 14 wherein R⁵ represents a cyclohexylmethyl group.

16. A compound according to Claims 12 to 14 wherein R² is an optionally substituted phenylC₂.₆alkyl group.

17. A compound according to Claim 16 wherein R² represents an optionally substituted phenylpropyl group.

18. A pharmaceutical composition comprising a compound according to any one of Claims 1 to 17 and a pharmaceutically acceptable diluent, carrier or excipient.

19. A process for preparing a compound of formula (1 ) as defined in Claim 1 , said process comprising coupling an acid of formula (II)

or an active derivative thereof, with an amine of formula (III)

followed by removal of any protecting groups.