CA2105309A1 - Phosphono derivatives of amino acids as metalloproteinase inhibitors - Google Patents

Abstract

Compounds of formula (I) are described wherein R represents a -P(O)(X1R6)X2R7 group, where X1 and X2, which may be the same of 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 (-CO2H) 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]nR8 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. 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

WO 93/14096 PCr/~B93/00086 ~ i u ~ ~ ~ 9 PHOSP~ONO DERIVATIVES OF AMINO AC~:)S AS MEIALU)PRal~EINASE
INHIBIIORS

FIELD QF 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-lV 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 S of specific inhibitors, such as ~2-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 pathoiogical 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. .~I ~1 Ann.
Rep. Med. Chem. 25,175-184, Academic Press Inc., San Diego (1990)].

WO 93/14096 ~ 1 ~t ~J ~i i3 9 PCr/GB93/00086 Certain phosphonopeptides have been described as collagenase inhibitors in European Patent Specification No. 320118 and International Patent Specifications Nos. WO 91/15~16 and WO 91/15507.

SUMMARY OF THE INVENTION

We have a now found a new class of phosphonopeptidyl d~rivatives, 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 (I) ~O R~

wherein R represents a-P(O)(X1R6)X2R7 group, where X1 and X2, which may be the same or different, is each an oxygen or a sulphur atom, and R6 ..

.

wo 93/t4096 pcr/GBs3/ooo86 2 i ~ U ~'1 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 (-CO2H) or esterified carboxyl group; :

R3 represents a hydrogen atom or an alkyl group:

R4 represents a hydrogen atom or an alkyl group;

R~ represents a group -lAlk]nR8 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), 0! 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 .
, .. . .

wo 9~/14096 pcr/GBs3~ooog6 t ~ ~ 4 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 C1 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-butenyl group optionally substituted by one or more C~ 6alkoxy, e.g. methoxy, ethoxy, propoxy, C1 6alkylthio, e.g.
methylthio, ethylthio, propylthio, C6 ~2arylC~ 6alkoxy, e.g. phenylC1 6 alkoxy such as benzyloxy, aralkylthio, e.g phenylC~ 6alkylthio such as benzylthio, amino (-NH2), substituted amino, [such as -NHR1 0, where R1 0 is a C1 6 alkyl e,g. methyl or ethyl, C6 12arylC1 6alkyl, e.g. phenylC1 6alkyl, such as benzyl, C6 12aryl, e.g. phenyl, C3 8cycloalkyl, e.g. cyclohexyl, or C3 8cycloalkylC1 6alkyl, e.g. cyclohexylmethyl group], carboxyl (-CO2H) or -CO2R12 [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 12arylC1 6alkyl groups such as phenylC1 6alkyl, or 1- or 2-naphthylC1 6alkyl, 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 C3 6heteroarylC1 6alkyl group, such as an optionally substituted pyrrolylmethyl, furanylmethyl, thienylmethyl, imidazolylmethyl, oxazolylmethyl, thiazolylmethyl, pyrazolylmethyl, pyrrolidinylmethyl, pyridinylmethyl, pyrimidinylmethyl, morpholinylmethyl, or piperazinylmethyl group.

Heteroarylthioalkyl groups represented by R1 include C3 6heteroarylthioC1 6alkyl groups such as optionally substituted pyrrolylthiomethyl, furanylthiomethyl, oxazolylthiomethyl, thiazolylthiomethyl, pyrazolylthiomethyl, pyrrolidinylthiomethyl, pyridinylthiomethyl, pyrimidinylthiomethyl, morpholinylthiomethyl, or piperazinylthiomethyl groups.

Optional substituents which may be present on heteroaralkyl or heteroarylthioalkyl 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 cyclopentyl or cyclohexyl groups.

When R2 is a cycloalkylalkyl group it may be for example a C3 8cycloalkylC1 6alkyl group such as a cyclopentylC1 6alkyl or cyclohexylC1 6alkyl group, for example a cyclopentylmethyl, cyclopentylethyl, cyclopentylpropyl, cyclopentylbutyl, cyclohexylmethyl, cyclohexylethyl, cyclohexylpropyl, or cyc!ohexylbutyl group.

When R2 is an aralkoxy or an aralkylthio group it may be for example a C6 12arylC1 6alkoxy or C6 12arylC1 6alkylthio group such as a phenylC1 6alkoxy or phenylC1 6alkythio group. e.g. a benzyloxy, phenylethoxy, WO 93/ l 4096 PCT/G B93/00086 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 (1) 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 C1 6alkyl, e.g. methyl or ethyl, C1 6alkoxy e.g.
methoxy or ethoxy, C2 6alkylenedioxy, e.g. ethylenedioxy, haloC1 6alkyl, e.g.
tri-fluoromethyl, C~ 6alkylamino, e.g. methylamino or ethylamino, C1 dialkylamino, e.g. dimethylamino or diethylamino, amino (-NH2), nitro, cyano, hydroxyl (-OH), carboxyl (-CO2H), -CO2R9, where R9 is as defined above, C1 6alkylcarbonyl, e.g. acetyl, sulphonyl (-SO3H), C1 6alkylsulphonyl, e.g. methylsulphonyl, aminosulphonyl (-SO2NH2), C1 6 alkylaminosulphonyl, e.g. methylaminosulphonyl or ethylaminosulphonyl, C1 6dialkylaminosulphonyl e.g. dimethylaminosulphonyl or diethylaminosulphonyl, carboxamido (-CONH2), C1 6alkylaminocarbonyl, e.g. methylaminocarbonyl or ethylaminocarbonyl, C1 6dialkylaminocarbonyl, e.g. dimethylaminocarbonyl or diethylaminocarbonyl, sulphonylamino (-NHSO2H), C1 6alkylsulphonylamino, e.g. methylsulphonylamino or ethylsulphonylamino, or. C1 6dialkylsulphonylamino, 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 (1). 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 (1) is a substituted amino group, this may be for example a group -NHR1 0 where R1 0 is as defined above.

. . . . . . . . . . ... . .

Wo 93/14096 ~, PCr/Gs93/~oox6 ;~, 1 il ~ .i U

Esterified carboxyl groups represented by R2 include groups of formula -CO2R1 2 where R1 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-naphthyimethyl group; a C6 12aryl group such as an optionally substituted phenyl, 1-naphthyl or 2-naphthyl group; a C6 12aryloxyC1 8alkyl group such as an optionally substituted phenyloxymethyl~ phenyloxyethyl, 1 naphthyloxymethyl or 2-naphthyloxymethyl group; an optionally substituted C1.8alkanoyloxyC1.8alkyl 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 ~alkoxy, 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 C1 6alkyl groups such as methyl or ethyl groups.

The groups R6 and/or R7 in compounds of formula (I) may eac.h be a hydrogen atom or an optionally substituted straight or blanched C1 6alkyl, e.g. methyl, ethyl, n-propyl, i-propyl, n-butyl or i-butyl, C6 12aryl, e.g. phenyl, or C6 12arylC1 6alkyl, e.g. benzyl, phenylethyl or phenylpropyl group.
Optional substituents present on alkyl groups of this type include one or more C1 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, C6.~2arylC1.6alkoxy e.g. benzyloxy or C6 12arylC1 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 .
.

Wo 93/1~096 pcr/GBs3/ooo86 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 C1 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 C1 6alkyl group such as a methyl group.

- The group R8 in compounds of formula (I) may represent a C3 8cycloalkyl, e.g. cyclopentyl or cyclohexyl, or C3 8cycloalkenyl e.g. cyclopentenyl or cyclohexenyl, group optionally substituted by one, two or more C1 6alkyl, e.g. methyl or ethyl, C1 6alkoxy, e.g. methoxy or ethoxy, C1 6alkylthio, 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 R14, 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(O)O-] groups or R13 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 C1 6alkyl group such as a methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, or t-butyl group, optionally interrupted by one or more -O- or -S- atoms, or -N(R9)- or aminocarbonyloxy groups and may be for example a methoxymethyl, ethoxymethyl, methoxyethyl, ethoxyethyl or . . . . , . ~ . . ~

. : .

wo 93/14096 ~ ) U ~3 pcr/GB93/ooo86 ethylaminocarbonyloxymethyl group. The optional substituents which may be present on such groups include hydroxyl (-OH), carboxyl (-CO2H), esterified carboxyl (-CO2R12), 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 -NR12R13, or aryl, e.g. C6 12 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 oR1 3 where R1 3 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(O)(OR6)0R7, e.g. a -P(O)(OH)OR7 group, or a -P(O)(SH)OR7 or -P(O)(OH)SR7 group. Examples of such groups include -P(O)(OCH3)0CH3,-wo 93/14096 pcr/GBs3/ooo86 ,t ~ U ~ , P(O)(OCH2CH3)OCH2CH3, -P(O)(OH)OH, -P(O)(OH)SH, -P(O)(SH)OH, P(O)(OH)OCH3, -P(O)(OH)SCH3, -P(O)(OH)OCH2CH3, -P(O)(OH)OPh, P(O)(OH)SPh,-P(O)(OH)OCH2Ph or-P((:))(OH)SCH2Ph, where Ph is a phenyl group optionally substituted by one or more substituents R~

In the compounds of formula (I) the group R1 may in particular be a C1 6alkyl group such as a methyl group, an aralkyl group such as benzyl group, an arylthioalkyl group such as a phenythiomethyl group or a heteroarylthioalkyl group such as thienylthiomethyl, pyridinylthiomethyl or pyri.midinylthiomethyl group or is especially a hydrogen atom.

The group R2 may be in particular an optionally substituted C1 6alkyl, C3 8cycloalkyl, C3 8cycloalkylC1 6alkyl, C6 12aryl, C6 12arylC1 6alkoxy or C6 12aralkylthio group and, especially, a C6 12arylC1 6alkyl 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; cyclopentyl;cyclohexyl; cyclopentylC1 6alkyl, such as cyclopentylC3 6alkyl, e.g.
cyclopentylpropyl, cyclopentylbutyl, or cyclopentylpentyl; phenyl; ~- or ~-naphthyl; phenylC1 6alkoxy, e.g. phenylethoxy, phenylpropoxy or phenylbutoxy; phenylC1 6 alkylthio, e.g. phenylethylthio, phenylpropylthio or phenylbutylthio; and, especially, phenylC1 6alkyl 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 R~1 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) rnay be in particular a group -AlkR8, where R3 is an optionally substituted cycloalkyl or cycloalkenyl group.

.
: . : . , . . ... . .

.

WO 93/14096 PCT/G1~93/OOn86 .i ,J V 9 Thus, the group R5 in compounds of formula (1) may be an optionally substituted C3 8cycloalkylC1 6alkyl [e.g. cyclopentylC1 6alkyl such as cyclopentylmethyl or cyclopentylethyl. or cyclohexyC1 6alkyl such as cyclohexylmethyl or cyclohexylethyl], C3 8cycloalkenylC1 6alkyl [e.g.
cyclopentenylC1 6alkyl such as cyclopentenylmethyl or cyclohexenylC1 6alkyl such as cyclohexenylmethyl], cycloalkylC1 3alkoxyC1 3alkyl [e.g.
cyclopentylmethoxymethyl, cyclohexylmethoxymethyl] C3 8cycloalkenylC1 3alkoxyC1 3alkyl [e.g. cyclopentenylmethoxymethyl or cyclohexenylmethoxymethyl] C3 8cycloalkylC1 3alkylthioC1 3alkyl [e.g.
cyclopentylmethylthiomethyl or cyclohexylmethylthiomethyl] or C3 8cycloalkenylC1 3alkylthioC1 3alkyl [e.g. cyclopentenylmethylthiomethyl or cyclohexenylmethylthiomethyl], C3 8cycloalkyC~ 3alkylaminoC1 3alkyl [e.g.
cyclopentylmethylaminomethyl, or cyclohexylmethylaminomethyl3 or C3 8cycloalkenylC1 3alkyaminoC1 3alkyl [e.g. cyclopentenylmethylaminomethyl or cyclohexenylmethylaminomethyl] group.

The group X in compounds of tormula (I) may be in particular an amino (-NH2) or-NR13R14 group. Particular -NR13R14 groups are -NHR14 groups.
Groups of this type include those where R14 is a C1 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, -NR13R14, [for example di-C
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 grol~p of compounds according to the invention is that of formula (I) wherein R5 is an AlkR8, group, where Alk is a C1 6 alkyl and R8 is a C3 8 cycloalkyl or C3 acycloalkenyl 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 (la) R ~ .J~ X ' O R5 (la) 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(o)(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 C1 6 alkyl group and R8 is a cycloalkyl or cyc!oalkenyl group;

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

: .

, WO 93/14096 PCr/GB93/OOOX6 Particularly useful compounds of formula (la) are those wherein R5 is a group -AlkR3, and R8 is an optionally substituted cyclohexyl group.
Compounds of this type in which R5 is a cyclohexylC1 6alkyl 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, o 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 -NR1 3R14 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 phenylC3 6alkyl group, especially an optionally substituted phenylpropyl or phenylbutyl group, R5 is a cyclohexylmethyl group; and X is a amino (-NH2) or NR13R14 group.
Compounds of this type wherein X is -NH2 or -NHR13 are particularly useful.

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

In the compounds of formulae (I) and (1 a), when the group R5 is a cycloalkylC1 6alkyl group then the chiral centre to which this group is attached preferably has a S-configuration.

- : - , - . .
, ' ~ ' ' .

wo 93/14096 pcr/GBs3/ooo86 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 c'atalytic 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 trimethylsilyl 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 (Il) R ~,OH
R1 o (Il) ., . , . . ~ -' ., ' - .

- . . .. ~ . .~ .
.
.

WO 93/14096 PC r/GB93/ooos6 J~V3 or an active derivative thereof. with an amine of formula (111) HN
~ X
R4 R5 (111) followed by removal of any protecting groups~

Active derivatives of acids for formula (Il) include for example a~id anhydrides, or acid halides, such as acid chlorides~

The coupling reaction may be performed using standard conditions ~or 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 et~er, e.g. a cyclic ether such as tetrahydrofuran, an amide e.g. a substituted arnide such as dimethylformamide, or a halogenated hydrocarbon such as dichloromethane at a low temperaturs, e.g. -30C to amibient temperature, such as -20C to 0C, 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 (Il) is used, the reaction r~3ay additionally be performed in the presence of a condensing agent, ~tor example a diimide such as N,N'-dicyclohexylcarbodiimide, advantageousiy in the presence of a triazole such as l-hydroxybenzotriazole Alternatively, the acid may be reacted with a chloroformate for example ethylchlorotormate, prior to reaction with the amine of formula (111)~

Free hydroxyl or carboxyl groups in the starting materials of formulae (Il) and (1ll) may need to be protected during the coupling reaction. Suita~le protecting groups and methods for their removal may be those men~ion~d above~

. ~ . : . ~ : . . . .: .
, . , . ~ . . ,,, -w ' ;~ ~ ~ V V~ -16-Compounds of formula (Il) 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 wherein 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 (Il) and an amine of formula (111). 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 (1). Thus, in particular process a compound of formula (la) may be prepared by reaction of a compound of formula (lla) R ~OH
o (lla) with an amine of formula (Illa) HN~X

R5 (Illa) as described above.

' : .. ,-,, . ~ ,~

.
~. ~ , , .

wo 93/14096 Pcr/GB93/ooo86 Compounds of formula (I) wherein R is a group -P(o)(X1R6)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, using 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 (Il).

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 (Il) may be prepared from a corresponding ester of formula (IV) R~,OR1C
R1 (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 20elevated pressure and temperature.

i .. . . . -., . .

. . , ~ i u .~ ~ ~ 9 -1 8-The intermediates of formula (IV) may be prepared by reaction of an acrylate of formula (V) ~,R15 R1 o (V) with a phosphite:P(OR16)(X1R6)X2R7 where R16 is a leaving group, for example a silyl group such as a trialkylsilyl group e.g. a trimethylsilyl group,at an elevated temperature.

Acrylates of formula (V) may be prepared by reaction of an mono-ester of formula (Vl) O O
HOJ~oR15 R2 (Vl) 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 (Vl) may be prepared by hydrolysis of the corresponding di-ester of formula (Vll) ,, . ., . . . ~ .
, . . - - -. . . . - , .
, . -wo 93/14096 ~- PCT/GB93/00086 ~ i iJ ~ù 9 , g o o R15OJ~oR1s R2 (Vll) 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 OC.

Diesters of formula (Vll) may be prepared by alkylation of the corresponding malonates of formula R15OCoCH2CO2R15 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 R15OCoCH2CO2R15 are either known compounds or may be prepared by methods analogous to those used for the preparation of the known compounds.

Intermediate phosphites of ~ormula :P(oR16)(X1R6)X2R7 for use in the preparation of intermediates of formula (IV) may be prepated by react!on of a phosphite HP(o)(X1R6)X2R7 with an appropriate amine (R15)2NH e.g. a silazane, at an elevated temperature, e.g. the reflux temperature.
.
Phosphites of formula HP(o)(X1R6)X2R7 are either known compounds or rnay be prepared by methods analogous to those used for the preparation of the known compounds.

In an alternative process, intermediate acids of formula (Il) may be prepared by reaction of an acid R2CH2CO2H with a phosphonate P(O)(X1R6)(X2R7)CH2OR16 where R16 is a leaving group, for example a - . . ..

- : . ~: ,, . . . .,~ . .
. .. . - . . . . . .

WO 93~140~6 PCr/GB93/00086 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(O)(X1R6)(X2R7)CH20H 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(O)(X1R6)(X2R7)CH20H are either known compounds or may be prepared by methods analogous to those used for the preparation of the known compounds.

Intermediates-~f formula (111) and acids of formula R2CH2CO2H 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 (Vlll) ,1~
R 1~ N O

O
~ Ph (Vlll) (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 0C followed by treatment with a base. such as lithium hydroxide, at an elevated temperature.

-` ' ' . '. ' ' W093/t4096 `. . ~ I PCr/GB93/00086 The compounds of formula (Vlll) 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 tVIII) 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 -78C.

Acyl halides RCH2 CH(R2)COHal may be prepared by treatment of the corresponding known acids RCH2CH(R2)CO2H 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 1mg/kg to mice in the above model - . . . . .

.

.

wo 93/14096 pcr/GB93/ooo86 ; 9 -22-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 tl) 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 ofa pharmaceutical composition comprising bringing a compound of formula (1) 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 pharrnaceutical compositions may take the form of, for example, tablets or capsules prepared by conventional means with wo 93/1~096 pcr/GB93/ooo86 ~it3~iJ9 pharmaceutically acceptable excipients such as binding agents (e.g.
pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropl methylcellulose); fillers (e.g. Iactose, 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 vehi¢les; and preservatives. The preparations may also contain 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 -. . ~
- . .
.. - .. . . . ..

: - ~
. . . . . . .
. . . . . . .

Wo 93/14096 pcr/GBs3looo86 ù 3 -24-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 ~Omg/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.

. - . ~ -- - ~ ......

- ~2~ il 9 DescriDtion of S~eCi~ic Embo~imen~

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-dimenthylaminopropyl)-3-ethylcarbodiimide, hydrochloride All temperatures are in C

.. . . -- . . - . : .:

. - , . ~ . . . . ~ .

- . ~ ' , .

WO 93/14096 PCI`/GB93/00086 ù 9 -26-lntermediate 1 E~nzyl (2-benzvloxvcarboxvl-5-phenyl! eentanoate To a suspension of NaH (5.049) in dry THF (200ml) was added dropwise over a period of 1 hour, dibenzyl malonate (~6.869, 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 H2O/NaCI/NH4CI (400ml) and extracted with CH2CI2 (4 x 100ml). The organic layer was dried (MgSO
and concentrated to give a yellow oil. The ~!~ cQmpound (63.6g) was purified on s~lica gel (Merck 938~) using Et2O/hexane (5:95) Et2O/hexane (25:75).

1 HNMR (CDCI3) ~ 7.0-7.40 (15H, m), 5.15 (4H, s), 3.45 (1 H, d, d), 2.60 (2H, t),1.85-2.0~ (2H, m),1.55-1.65 (2H, m) Intermediate 2 2-Benzyloxycar~ -ehenvleentanoic acid To Intermediate 1 (63.69) dissolved in 1,4-dioxan (400ml) was added KOH
(8.879) in H2O (110ml) at 0. The mixture was allowed to rise to RT and stirred overnight. The solvent was removed in vacuo and H2O (lOOml) added. Extraction with Et2O removed any undesired diester and the aqueous phase was acidified (pH~1) with 1.0M HCI. The product was extracted into Et2O (3 x 100ml), dried (MgSO4) and concentrated to give the title comDound (29.0g).

1HNMR (CDCI3) ~ 10 (1H, bs) 7.10-7.60 (10H, m), 5.20 (2H, s) 3.48 (1H, t), 2.~2 (2H, t),1.86-2.10 (2H, m),1.60-1.80 (2H, m) WO 93/1~096 i` PCl`/GB93/0~086 j~ 2 V ~ ~ ~) 9 lntermediate 3 O-Benzvl-2-(3-Dhenvl~proeylproD-2-enoate Paraformaldehyde (380mg) was added to a stirred solution of the Intermediate 2 (2.69) dissolved in anhydrous pyridine (25ml) containing s piperidine (118mg, 137111). The reaction was heated at 60 for 2.1/2 hours, cooled, poured into 1.0 MHCI and extracted with Et2O (3 x 50mL). The Et2O - ^
layer was dried (MgSO4), and concentrated to give the title compound (1.3g).
1HNMR (CDCI3) â 7.12-7.36 (10H, m), 6.21 (1H, d), 5.55 (1H, d), 5.20 (2H, s), 2.62 (2H, t),2.37 (2H, t),1.76-1.88 (2H, m).

Intermedi~ 4 Dimethvltrimethvlsilvl~hosDhite A mixture of dimethylphosphite (9.18ml) and hexamethyldisilazane (25.32ml) was heated under reflux (bath temperature 140) for 3h. The ` 15 crude mixture was distilled in vacuo lapproximately 5mm Hg) through a 12cm vigueux column and the traction distilling at bath temperature 95-11 0 collected to give the ~!~ compound (7.20g) HNMR (CDCI3) O/ppm 3.50 (s,3H),3.46 (s, 3H),0.26 (s,9H).

Intermediate 5 BenaLl (~-~imethoxvQh~hinylmethvl-5-Qhenvl)r~entanoate 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 .. . . . - ;

. . . . . .
-WO 93/14096 ~ PCT/GB93/00086 w ~ 3 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 (MgSO4) and concentrated in vacuo to a crude oil 1.559. This was chromatographed on silica 60 ('Merck' 9385) eluting with ethyl acetate-methanol (2%) to give the title comDound as an oil (0.34239) HNMR (CDCI3) ~ 7.35 (5, 5H), 7.77 (t, 2H), 7.17 (t, 1H), 7.08 (d, 2H), 5.115 (s, 2H), 2.83 (m,1H), 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).

o Intermediate 6 2-DimethoxvehosDhinvlmethvl-5-Dhenylpentanoic acid A mixture of Intermediate 5 (0.33979) and 10% palladium on carbon catalyst (50mg) in methanol (20ml) was stirred under an atmosphere of hydrogen for 18h. Tlc silicagel 60 (chloroform-methanol 10% eluent) showed conversion to one product with RfO,23. The mixture was filtered and concentrated in vacuQ to give the ~ com~ound as a colourless oil (0.24989) 1HNMR (CDCI3) ~ 7.30-7.19 (m, 3H), 7.16 (d, 3H) 3.76 (s, 3H), 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) and1.82 (dd) - together 1 H and 1.76-1.66 (m, 4H) ppm.

Intermediate 7 L ~-cyclohexylalanine-N-(2-phenvlethvl~ amide tBoc-,B-cyclohexyl-L-alanine (1.359, 5mmoL) was dissolved in dry CH2C12.

4-Nitrophenol (695mg, 5mmoL) was added followed by DCCI (1.03g, 5mmoL). After 1 hour at room temperature the reaction was concentrated m vaçuo, ether was added and the solution filtered. The residue was Wo 93/14096 pcr/Gs93/ooo86 ù 9 concentrated in vacuo, dissolved in CH2CI2 (10ml) and phenethylamine :
(690111, 5.5mmoL) was added. The reaction was poured into NaHC03 and extracted with CH2CI2 (3 x 20ml), was dried (Na2S04) and concentrated in ~Q. Purification on silica gel (Merck 93~5) using CH2CI2~CH2CI2/MeOH 85:15) gave a clean oil (9OOmg) which was dissolved in CH2C12/TFA (9:1) and left a RT for 30 min. The reaction was concentrated in~L~o~ dissolved in CH2C12 (~Oml) and poured into Na2C03 (aq). The organic layer was separated, dried (Na2S04) and concentrated in vacuo to give an oil which was purified on silica gel (Merck 9385) using CH2CI2/methanol/triethylamine 96:3:1 to give the title comDound as an oil (500mg).

1 H NMR (CDCL3) ~ 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 1 OHZ), 3.5 (dd, 2H, J=6 and 12HZ), 7.2 (m,5H) Intermediate 8 O-(Diethyl)hvdroxvmethvlohosphonate A mixture of diethyl phosphite (209) paraformaldehyde (4.44g) and triethylamine (2ml) was heated to 60C 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 (MgSO4) and evaporated to give the title compound as a clear colourless oil. (20.73g).

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

~ .
.
. , ~ . ' ' : :
.

.
.

wo 93/14096 pcr/GB93/ooo86 , 3 -30-lntermediate 9 O-(Diethvl)trifluoromethvlsuleh~nyloxvmethylehosDhonate To a suspension of sodium hydride (3.85g) in dry ether (200ml) at -30C
was added by syringe trifluoromethanesulphonyl chloride (25g). A solution of Intermediate 8 (20.739) in dry ether (20ml) was added dropwise over 20 minutes maintaining the internal temperature at -20C. After stirring the `
suspension for 2 hours at -20C, the excess hydride was removed by filtering through Celite. The filtrate was diluted with dichloromethane, washed twice with 10% aqueous sodium bicarbonate, separated, dried (MgSO4) and evaporated to give the title compound as a pale yellow oil.
The Ii~L~. comeound was purified on silica, eluting with 35% ethyl acetate in hexane (10.59).

H NMR (CDCI3) ~ 4,60 (2H, d); 4.25 (4H, m); 1.40 (6H, t) Intermediate 1 0 2-DiethoxvDhosDhinylmethyl-5L~-methylehenyl~Dentanoic acid A solution of n-butyllithium in hexane (13ml) was added to a solution of di-isopropylamine (2.94ml) in dry THF (30ml) at 0C. 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 35C 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 ~t2O, dried (MgSO4) and evaporated. The residue was chromatographed on silica, eluting with 3 - 10% CH30H in wo 93/ 14096 Pcr/ G s93/00086 ~ i ù ~ , 9 CH2CI2~ to give the title compound as a clear gum (1.26g).

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

Exam~ 1 . . .

3-PhosDhono-2R S-~henvlproDvl-1 -oxoeroovl]-L-,B-cyclohexvlalanine-N-~2-~henylethvl) amide. dimethvlester A solution of Intermediate 6 (151.7mg) in dry tetrahydrofuran (3.0ml) was cooled to -30 and treated with N-methylmorpholine (59.1~L1) and ethyl chloroformate (49.0111). The mixture was stirred at -30 for 1 h and a solution of Intermediate 7 (240.8mg) and N-methylmorphorpholine (59.1ml) in dry DMF (2.0ml) added dropwise at -30C. The mixture was allowed to warm up to RT overnight and the solvent removed in vacuo. The residue was partitioned between water (15ml) and ethyl acetate (20ml) and the aqueous layer further extracted with ethyl acetate (2 x 20ml). The combined organic layers were dried (MgSO4) and concentrated in ~uo to give an oil (304.6mg) which was chromatographed on silicagel 60 ('Merck' 9385) eluting with ethyl acetatemethanol (2%) to give the title comeound as a mixture of diastereoisomers and as an oil (189.4mg) 1 HNMR (CDCI3) ~ 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 1H, 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.

.. . .

6 PCT/G~93/00086 Example 2 [3-Phos~hono-2R-~henyleropvl-1 -oxo~ro~yl]-L-~-cvclohexvlalaninç-N-(2-Dhenvlethyl) amide A solution of the compound of Example 1 (189.4mg) in dry dichloromethane (5.0ml) was treated with bromotrimethylsilane (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
diastereoisome`~s as an oily solid. This was separated by preparative HPLC
on a Dynamax C18RP column (21.4mm dia) using 0.1% TFA-H2O (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) ~ 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.

1HNMR (CDC13) ~ 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,1H) and 1.85-0.6 ppm (br, m,18H) The following compounds w-ere 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.

WO 93/14096 PCI`/GB93/00086 ù 9 Example 3 ~-[3-PhosDhono-2S-Dhenvlpro,~1-1-oxoeroDy~l-L-~~clohe~y!~lanine-~-alanine 1HNMR (CDCI3) ~7.15-6.90 (m, 5H), 4.19 (dd,1H), 3.14 (m~ 2H), 2.65-2.40 s (m, 3H), 2.34 (t, 2H),1.99 (dd) and 1.96 (dd) -together 1 H and 1.70-0.52 ~r, m,18H) Examl~le 4 ~-Pho~hono-2R-phenvlDroDvl-1 -oxoDropyl]-L-,B-cvclohexylalanine HNMR (CDCI3) ~ 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 (br, m,18H

Example 5 ~,-Pho ~ ho~Q ~ Qhenv~QQyl-1-oxQQ ~ yl ~ ~ ~ Lohe ~l~l~ine-~-alanine. methvl ester 1HNMR (CDCI3) ~ 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, 1H) and 1.80-0.65 ppm (br, m,18H) wo 93/1~096 pcr/Gss3/ooo86 J t v Example Ç

[3 -Pho spho no-2 R.S- phQ~Qropv 1-1 -oxop ropy 11- L-~-cycl ohexvlal anin e-N_ ~4(3-a~noeroDvl)morpholine] amide. bromine salt ISOMER i 1H ~ (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, 1H), 3.95 (m, 2H) 3.80-3.0 (br, m) overlapped with H2O,2.60 (m,1H) 2.0 (br, q, lH) and 1.80-0.60 (br, m,18H).

ISOMER 2 1HNMR ~ (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,1H), 3.92 (m,2H), 3.80-3.0 (br, m) overlapped with H2O, 2.65 (m, lh), 1.95 (br, q, 1H) and 1.75-0:60 (br, m, 18H) ppm Example 7 ~3:phos~hQnQ:2B~ 4 mQ~hvl~hQn~ QcQ~ QXQ~Q~Q~
cyclohexylalanine-N-(2-phenylethyl!amide. diethylester A solution in dry DMF (5ml) of Intermediate 10 (250mg), triethylamine (101.4~L1); N-hydroxybenzotriazole (98.6mg), Intermediate 7 (200mg) and EDC (140mg) was stirred at room temperature for 5 hours under nitrogen.
The reaction mixtur0 was partitioned between Et2O and aqueous citric acid, the organic layer separated, washed with aqueous NaHCO3, dried (MgSO4) and evaporated to give the ~L~!Q compound (320mg) as a 50:50 mixture of diastereomers.
H NMR (CDCI3) 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).

,, WO 93/1~096 ~ PC'r/GB93/00086 Example 8 ~3 PhQs,QhQno 2B,S ~4 meIh~!~hen~lLQ o~
cvclohexvlalanine-N-(2-phenylethvl!-amide A solution of ~he compound of Example 7 (320mg) in dry CH2CI2 (20ml) was treated with trimethylsilylbromide (2ml) at room temperature for 2.~ 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) 7.15-7.30 (5H, m); 7.0 (4H, m); 4.35 (1 H, t); 3.35 (2H, m); 2.0-2.75 (1 OH, m);
0.80-1.85 (1 7H, 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.
20~1m) are incubated overnight in G.1M Tris/HCI buffer, pH 7.~, containing 0.1M NaCI, 10mM CaCI2 and 0.05%. Brij 35 at either room temperature or 37C depending on the enzyme. The reaction is stopped by adjusting the - . ., . ~ ~
... . .

wo 93/14096 pc~r/GBs3/ooo86 pH to 4 using 0.1M sodium acetate buffer and the fluorescence read at an excitation wavelength of 280nm and emission wavelength of 346nm.

Kj values can be established using the equation for tight-being inhibition:---~/(Kj(app) + [1]2 + 2 (Kj(app) - [I])[E] + [E]2 (Kj(app)+ [I] - [
2[q ~ J

where V0 is the initial rate of reaction in the absence of inhibitor, Vj is the initial rate in the presence of inhibitor, [E] is the total enzyme concentrationand [~] the total inhibitor concentration in the reaction mixture.

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

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

. Ki(nM) Compound of Collagenase Stromelysin-1 Gelatinase-72KD
Examele No.
2 25~1M 701 18.9 4 22.1 ~M 1 .7311M46.5 8 1 7~1M 277 2.5 ., : . , . . . . : .

. ~. ..
,. . . .. . . .

Claims (19)

1. A compound of formula (1) (I) wherein R represents a -P(O)(X1R6)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 (-CO2H) 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]nR8 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.

2. A compound according to Claim 1 wherein R represents a -P(O)(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(O)(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 C1-6 alkyl group optionally interrupted by one or more -O- or -S- atoms or -N(R9)- groups, where R9 is a hydrogen atom or a C1-6 alkyl group, and R8 is an optionally substituted C3-8 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) (1a) wherein R represents a -P(O)(X1R6)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 (-CO2H) 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]nR8 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.

12. A compound according to Claim 11 wherein R represents a P(O)(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 C1-6 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(O)(OH)OH group.

14. A compound according to Claim 12 or Claim 13 wherein R5 represents a cyclohexyiC1-6alkyl 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) (II) or an active derivative thereof, with an amine of formula (III) (III) followed by removal of any protecting groups.