EP0612324A1 - Phosphonopeptides with collagenase inhibiting activity - Google Patents

Abstract

L'invention concerne des dérivés de phosphore possédant la structure (I), les procédés de préparation de ceux-ci et leur utilisation en tant qu'inhibiteurs de la collagénase.The invention relates to phosphorus derivatives having the structure (I), processes for their preparation and their use as collagenase inhibitors.

Description

Phosphonopeptides with collagenase inhibiting activity.

The present invention relates to novel phosphorus

derivatives, processes for their preparation and their use in medicine. In particular, the present invention relates to their use as inhibitors of enzymes of the collagenase family of neutral metalloproteases, for treating arthritic and other diseases. The mammalian collagenase family of enzymes comprises a number of proteases, exemplified by interstitial (type I) collagenase itself, the stromelysins (also known as

proteoglycanases or transins), fibroblast and

polymorphonuclear leucocyte gelatinases (also known as collagen-IV-ases), and 'pump-1' (putative metalloprotease 1, uterine metalloprotease) [Goldberg et al, J. Biol. Chem. 2610, 6600, 1986; Whitham et al, Biochem. J. 240, 913, 1986; Breathnach et al, Nucleic Acids Res., 15, 1139, 1987; Muller et al, Biochem. J., 253, 187, 1988; Collier et al, J. Biol. Chem., 263, 6579, 1988; Murphy et al, Biochem. J., 258, 463, 1989; Quantin et al, Biochem. (N.Y.), 28, 5327, 1989;

Birkedal-Hansen, J. Oral Pathol., 17, 445, 1988; P. Basset et al, Nature 348, 699, 1990]. Membership of the mammalian collagenase family of proteases is evident by possession of a number of highly characteristic and experimentally

verifiable properties as described in EPA 401963 (Beecham Group), which can be adopted as criteria for allocation to this family of enzymes. As a particular example of the therapeutic value of

inhibitors of the collagenase family of enzymes, such as are disclosed in the present invention, chronic arthritic diseases leading to extensive loss of the collagen,

proteoglycan and elastin components of the cartilage, bone and tendons within the joints, should be amenable to

treatment with inhibitors of the collagenases,

proteoglycanases (stromelysins) and gelatinases currently thought to be the major enzymes involved. These enzymes have been detected in extracts of synovial and cartilage tissue, and have also been extensively studied in tissue cultures of a wide range of connective tissues.

Apart from control of the biosynthesis, secretion and activation of the enzymes, the most important natural regulation of these enzymes in normal and diseased states, is considered to be the endogenous production of inhibitors such as the Tissue Inhibitor of Metalloproteinases and alpha-2 macroglobulin. An imbalance between the local levels of the proteolytic enzymes and of their natural inhibitors will allow destruction of connective tissue components to occur. The compounds described in the present invention, being synthetic and low molecular weight inhibitors of this family of enzymes, offer a therapeutically useful way in which a more normal or non-pathological balance between inhibition and enzymic activity can be restored: they thus act to complement and supplement the endogenous enzyme inhibitors. Indeed, because these enzymes usually act only within restricted pericellular environments, before being

inactivated by inhibitors circulating in the blood and present in most inflammatory exudates, the low molecular weight inhibitors disclosed here may be more effective than endogenous proteinaceous inhibitors that are excluded by their size from the localized regions of connective tissue destruction. EPA-320118 (Beecham Group) discloses a class of phosphorus derivatives having activity as inhibitors of collagenase and utility in the treatment of rheumatoid arthritis and related diseases in which collagenolytic activity is a contributing factor.

Novel phosphorous derivatives have now been discovered, which are collagenase inhibitors and thus of potential utility in the treatment of diseases in which collagenolytic activity and tissue remodelling is implicated.

According to the present invention there is provided a compound of general formula (I), or a salt thereof:

in which,

R₁ is -(CH₂)_n-W where n is 0-6 and W is amino,

optionally-substituted phenyl, -CONR₅R₆, -NR₅COR₆,

NR₅CO₂CH₂R₆ or NR₅CONR₅R₆ where R₅ is hydrogen or C_1-6 alkyl and R₆ is hydrogen, C_1-6 alkyl, optionally-substituted phenyl or heteroaryl, or R₅ and R₆ together with the

nitrogen atom to which they are bonded form a 5-, 6- or 7-membered ring with an optional oxygen or sulphur atom or an optionally substituted second nitrogen atom in the ring; or W is -S(O)_p-R₇ where p is 0, 1 or 2 and R₇ is C_1-6alkyl; or W is a group of sub-formula (a), (b), (c) or (d):

where in sub-formula (a), (b) and (c) A represents an optionally-substituted mono or bicyclic aryl or heteroaryl ring, and in sub-formula (c) and (d) q is an integer from 1 to 3;

R₂ is C_3-6 alkyl;

R₃ is hydrogen, C_1-6alkyl, -CH₂-Z where Z is optionally substituted phenyl or heteroaryl, -(CH₂)_rNR₈R₉,

-(CH₂)_rNHCOR₁₀, -(CH₂)_rNR₁₁c(=NR₁₂)NR₈R₉,

-(CH₂)_rCONH(CH₂)_sNR₈R₉ or -(CH₂)_r-R₁₃ where r is 1 to 6, s is 2 to 4, each of R₈ and R₉ is independently hydrogen or C_1-6alkyl or R₈ and R₉ together with the nitrogen atom to which they are bonded form a 5-, 6- or 7-membered ring with an optional oxygen or sulphur atom or an optionally

substituted second nitrogen atom in the ring, R₁₀ is

C_1-6alkyl or -(CH₂)_tNR₈R₉ where t is 1 or 2 and R₈ and R₉ are as defined above, R₁₁ is hydrogen or C_1-6alkyl or R₁₁ and R₈, together with the nitrogen atoms to which they are bonded, form an optionally substituted 5-, 6- or 7-membered ring, R₁₂ is hydrogen or C_1-6alkyl and R₁₃ is an optionally substituted piperidyl ring or R₃ is a group:

where R₁₄ is hydrogen, C_1-6alkyl or optionally substituted benzyl and R₁₅ is hydrogen or C_1-6alkyl; and R₄ is hydrogen, C_1-6alkyl or -(CH₂)_rNR₈R₉ in which r, R₈ and R₉ are as defined for R₃; or R₃ and R₄ are joined together as -(CH₂)_m- where m is an integer from 4 to 12, or R₃ and R₄ are joined as - (CH₂ ) x-NR_{1 6}- (CH₂ ) _y- where x is an integer from 1 to 9, y is an integer from 2 to 10, and the moiety - (CH₂ ) _x- is adjacent to the carbon atom bearing R₃ marked with an asterisk in formula (I), and R₁₆ is selected from hydrogen, C_1-6alkyl, C_2-6alkanoyl, C_1-6alkoxycarbonyl, aroyl, aralkyl or aralkyloxycarbonyl in each of which the aryl moiety is optionally substituted. C_1-6alkyl groups (either alone or as part of another group), may be a straight chain or branched.

Optional substituents for aryl and heteroaryl groups may be selected from OH, C_1-6 alkyl, C_1-6 alkoxy, halogen,

-NHCO(C_1-6) alkyl, -NHCOPh and -CONR₅R₆, where Ph is

optionally substituted phenyl and R5 and Rg are as defined above.

The term aryl includes phenyl and naphthyl. It will be understood that the term heteroaryl includes aromatic heterocyclic groups containing one or more

heteroatoms and includes 5- or 6-membered monocyclic and 9-or 10-membered bicyclic heteroaryl groups which preferably contain one or two heteroatoms selected from nitrogen, oxygen and sulphur. When Z is 9- or 10-membered bicyclic heteroaryl the two rings are fused with one 5- or 6-membered ring preferably containing a single heteroatom, for example indolyl. When R₅ and R₆ or R₈ and R₉ groups combined with their appended nitrogen atom to form a heterocyclic ring, typical examples of a suitable ring structure are piperidine, or pyrrollidine, piperazine and morpholine. When such groups contain a second nitrogen heteroatom this may be optionally substituted, for example, by a C_1-6alkyl group.

Suitably R₁ is -(CH₂)_n-W where n is 0, 1, 2 or 3. Suitably W is amino, phenyl, N-phthalimido or 1,8- naphthalenedicarboxamido each of which may be optionally substituted, NHCO₂CH₂R₆ where R₆ is optionally substituted phenyl or S(O)_pCH₃ where p is 0, 1 or 2.

Preferably R₁ is 2-hydroxyphenyl, -(CH₂)_n-w where n is 2 and W is amino, phenyl, 2-hydroxyphenyl, NHCO₂CH₂Ph,

N-phthalimido, 4-bromo-1, 8-naphthalenedicarboxamido, 7,9- dioxo-8-azaspiro [4,5]decyl, methylmercapto, methylsulphinyl or methylsulphonyl, or R₁ is -(CH₂)_n-w where n is 1, 2 or 3 and W is a 1,8-naphthalenedicarboxamido group.

Suitably R₂ is a C4 alkyl group, such as n-butyl, iso-butyl or sec-butyl. Preferably R2 is iso-butyl.

Suitably R₃ is benzyl, C_1-6alkylamino, 4-hydroxybenzyl, C_1-6alkoxybenzyl such as 4-methoxybenzyl, or 9- or 10-membered fused bicyclic heteroarylmethyl such as

3-indolylmethyl. Preferably R₃ is benzyl, 4-methoxybenzyl, -(CH₂)₄NH₂ or 3-indolylmethyl.

Suitably R₄ is methyl, ethyl or -(CH₂)_rNR⁸R⁹. Preferably R₄ is methyl or -(CH₂)₂NR⁸R₉ where R⁸ and R⁹ are both hydrogen or R⁸ and R⁹ together with the nitrogen atom to which they are bonded form a pyrrolidine or N-methylpiperazine group.

Suitably, when groups R₃ and R₄ are combined as -(CH₂)_m-then m = 10, resulting in a lactam structure based on a 13-membered ring.

Suitably, when groups R₃ and R₄ are combined as

-(CH₂)_x-NR₁₆-(CH₂)_y- then x and y have values such that R₃ and R₄ form part of an 11- to 16-membered azalactam

structure and R₁₆ is hydrogen, methyl, benzyl, t-butoxycarbonyl or benzyloxycarbonyl. Preferably, when groups R₃ and R₄ are combined they form a group -(CH₂)₁₀-.

Particular compounds of this invention include:

N-[N-(1-phosphono-1-(2-hydroxyphenyl)methyl)-leucyl]

-N,O-dimethyl-(S)-tyrosinamide,

N-[N-(1-phosphono-3-(1,8-naphthalenedicarboximide)propyl)- (S)-leucyl]-N,O-dimethyl-(S)-tyrosinamide,

N-[N-(1-phosphono-3-phthalimidopropyl)-(S)-leucyl]-(S)-phenylalanine-N-methylamide,

N-[N-(1-phosphono-4-(1,8-naphthalenedicarboximido)butyl)-(S)-leucyl]-(S)-phenylalanine-N-methylamide,

N-[N-(1-phosphono-2-(1,8-naphthalenedicarboximido)ethyl-(S)- leucyl]-(S)-phenylalanine-N-methylamide,

N-[N-(1-phosphono-3-(1,8-naphthalenedicarboximido)propyl)- (S)-leucyl]-(S)-phenylalanine-N-methylamide,

N-[N-(1-phosphono-3-phenylpropyl)-(S)-leucyl]-(S)-phenylalanine-N-methylamide,

N-[N-(1-phosphono-3-(4-brom-o-1,8-naphthalenedicarboximido)propyl)-(S)-leucyl]-(S)-phenylalanine

methylamide,

N-[N-(1-phosphono-3-(benzyloxycarbonylamino)propyl)-(S)-leucyl]-(S)-phenylalanine methylamide,

N-[N-(1-phosphono-3-(2-hydroxyphenyl)propyl)-(S)-leucyl]-(S)-phenylalanine methylami-de,

N-[N-(1-phosphono-3-(methylmercapto)propyl)-(S)-leucyl]-(S)-phenylalanine-N-methylamide,

N-[N-(1-phosphono-3-(methylsulphinyl)propyl)-(S)-leucyl]-(S)-phenylalanine-N-methylamide,

N-[N-(1-phosphono-3-(methylsulphonyl)propyl-(S)-leucyl]-(S)-phenylalanine-N-methylamide,

N-[N-(1-phosphono-3-(1,8-naphthalenedicarboximido)propyl)- (S)-leucyl]-(S) tryptophan-N-methylamide,

N-[N-(1-phosphono-3-(1,8-naphthalenedicarboximido)propyl)-(S)-leucyl-(S)-lysine-N-methylamide,

N-[N-(1-phosphono-3-(1,8-naphthalenedicarboximido)propyl)- (S)-leucyl]-(-)-aminoazacyclotridecan-2-one, N-[N-(1-phosphono-3-(1,8-naphthalenedicarboximido)propyl)- (S)-leucyl]-(S)-lysine-N-(aminoethyl) amide,

N-[N-(1-phosphono-3-(1,8-naphthalenedicarboximido)propyl)- (S)-leucyl]-(S)-lysine-N-(ethylpyrrolidine) amide,

N-[N-(1-phosphono-3-(1,8-naphthalenedicarboximido)propyl)- (S)-leucyl]-(S)-lysine-N-(ethyl-N-methylpiperazine) amide, N-[N-(1-phosphono-3-[8-(7,9-dioxo-8-azaspiro[4,5]decyl)]- propyl)-(S)-leucyl]-(S)-phenylalanine-N-methylamide, or N-[N-(1-phosphono-3-[8-(7,9-dioxo-8-azaspiro[4,5]decyl)]- propyl)-(S)-leucyl]-(S)-lysine-N-methylamide,

and pharmaceutically acceptable salts thereof.

The compounds of formula (I) may form salts with bases e.g. sodium hydroxide. The compounds of formula (I) have a basic nitrogen atom and may form acid addition salts e.g.

hydrochloride, hydrobromide, sulphate, phosphate, acetate, fumarate, maleate, citrate, lactate, tartrate, oxalate and similar acid addition salts. Such compounds form part of the present invention.

The compounds of formula (I) have at least two, and may have three or more asymmetric centres and therefore exist in more than one stereoisomeric form. The invention extends to all such forms and to mixtures thereof, including racemates, and diastereoisomeric mixtures.

Preferred isomers are those having the (S)- or (-) -configuration at the chiral centre bearing R₃ when R₃ is other than hydrogen, and those having the (S) -configuration at the chiral centre bearing R₂, both marked with an

asterisk in formula (I).

The present invention also provides a process for the preparation of a compound of formula (I) which comprises cleaving a group R₂₀ from a compound of formula (II): wherein R₂₀ is C_1-6alkyl, optionally substituted phenyl or optionally substituted benzyl and R₂₁ is hydrogen,

C_1-6alkyl or optionally substituted benzyl and R_1' R₂, R₃ and R₄ are as defined in formula (I), and where necessary, converting R₂₀ to hydrogen by a further cleavage reaction.

Cleavage of R₂₀ and, where necessary, R_21, may be carried out in aqueous acid or alkali or using a trimethylsilyl halide, preferably bromotrimethylsilane, in an inert

solvent, for example dichloromethane. Benzyl esters may alternatively be removed by hydrogenolysis or other standard debenzylation procedures.

When both R₂₀ and R₂₁ are C_1-6alkyl, cleavage of R₂₀ only - to give to a compound of formula (II) in which R₂₀ is hydrogen and R₂₁ is C_1-6alkyl, may be carried out by

treatment with excess alkali under mild conditions, for example with aqueous sodium hydroxide in an alcoholic solvent at room temperature.

Similarly, where R₂₀ is optionally substituted benzyl and R₂₁ is C_1-6alkyl, the benzyl group only may be cleaved by hydrogenolysis to give a compound of formula (II) in which R₂₀ is hydrogen and R₂₁ is C_1-6alkyl.

Cleavage of an R₂₁ C_1-6alkyl group may thereafter be carried out as described above to give the compound of formula (I).

When R₂₁ is not hydrogen, then cleavage of both R₂₁ and R₂₀ is conveniently effected in a single reaction. Preferably R₂₀ and R₂₁ are both C_1-6alkyl, such as methyl or ethyl, or R₂₀ and R₂₁ are both benzyl. Compounds of formula (II) are believed to be novel and form a further aspect of the invention.

Compounds of formula (II) may be prepared by treating a compound of formula (III):

in which R₁, R₂, R₂₀ and R₂₁ are as defined in formula (II) (except that R₂₁ is not H), with a compound of formula (IV)

in which R₃ and R₄ are as defined in formula (I).

The reaction is preferably carried out in the presence of a coupling agent, such as dicyclohexylcarbodiimide or

1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide

hydrochloride in the presence of 1-hydroxybenzotriazole, or using 1,1'-carbonyldiimidazole, in an inert solvent such as dichloromethane or acetonitrile.

Selective cleavage of the group R₂₁ may then be carried out using the procedures described above for the preparation of compounds of formula (I) to give compounds of formula (II) in which R₂₁ is hydrogen.

Alternatively, compounds of formula (II) in which R₂₀ and R₂₁ are C_1-6alkyl or optionally substituted benzyl may be prepared by the reaction of a compound of formula (V): in which R₂, R₃ and R₄ are as defined in formula (I), with a compound of formula (VI):

in which R₁ is as defined in formula (I), R₂₀ and R₂₁ are C_1-6alkyl or optionally substituted benzyl and R₁₇ is a leaving group such as halogen, methanesulphonyloxy or trifluoromethanesulphonyloxy, in the presence of a base such as triethylamine or Proton Sponge (1,8-bis(dimethylamino)naphthalene), or using anhydrous potassium carbonate in an alcoholic solvent.

Where R₁₇ is an oxygen-based leaving group, for example trifluoromethanesulphonyloxy, which is preferred,

displacement of the leaving group is conveniently carried out in the presence of Proton Sponge in an inert solvent such as acetonitrile or dichloromethane, over a period of several days in the absence of light.

As a further alternative, compounds of formula (II) in which R₂₀ and R₂₁ are C_1-6alkyl, optionally substituted aryl or optionally substituted benzyl may be prepared by reaction of a compound of formula VII:

in which R₁, R₂, R₃ and R₄ are as defined for formula (I) with a compound of formula (VIII):

in which R₁₈ is C_1-6alkyl and R₂₀ and R₂₁ are as defined for formula (II) provided that R₂₁ is not hydrogen and

thereafter removing the phosphonic acid protecting group.

The reaction is suitably carried out in an organic solvent such as dichloromethane at reduced temperature, e.g. -10 to 5°C. The intermediate compounds of formula (III) may be prepared by treating a compound of formula (IX) or a salt thereof:

in which R₁, R₂₀ and R₂₁ ^{are as} defined in formula (III), with a compound of formula (XA) or (XB) or a salt thereof:

in which R₂ is as defined in formula (I), R₁₇ is as defined in formula (VI) and R₁₉ is hydrogen or a carboxyl protecting group, and thereafter removing the R₁₉ carboxyl protecting group. When a compound of formula (XB) is used, the reductive amination may be carried out by hydrogenation over a noble metal catalyst such as palladium on carbon or by reaction with sodium cyanoborohydride at pH 6 to 7. Lower alkyl alcohol solvents such as methanol and ethanol are suitable for both reactions. These reactions may be carried out in the presence of molecular sieves.

A hydrogenation reaction is preferred but this process precludes the use of compounds of formulae (IX) and (XB) in which any of R₂₀, R₂₁ or R₁₉ is benzyl. Preferably a carboxyl protecting group is a methyl or ethyl ester. Ester protecting groups may be removed under standard basic hydrolysis conditions using dilute base such as 1 Normal aqueous sodium hydroxide in methanol.

When the compound of formula (IX) is in the form of the free base, the compound of formula (XB) is suitably an α-keto ester (R₁₉ = C_1-6alkyl).

When the compound of formula (IX) is a salt, such as the hydrochloride salt, the compound of formula (XB) is suitably a salt of an α-keto acid (R₁₉ = H), for example the sodium salt.

The preparation of compounds of formula (III) using a compound of formula (XA) may be carried out under standard alkylation conditions. A halogen leaving group is

preferably bromine and an oxygen-based leaving group is preferably trifluoromethanesulphonyloxy.

Compounds of formula (III) may alternatively be prepared by condensing a compound of formula (XI) or a salt thereof:

in which R₂ is as defined in formula (I) and R₁₉ is a carboxyl protecting group with an aldehyde, R₁-CHO in which R₁ is as defined in formula (I) and treating the

condensation product with an appropriate phosphite, for example dimethyl phosphite, diphenyl phosphite or dibenzyl phosphite and thereafter removing the carboxyl protecting group. The carboxyl group is conveniently protected as an alkyl or benzyl ester which may be removed using standard hydrolysis or hydrogenation conditions.

As described above in connection with reductive amination of compounds of formula (XB), where a benzyl protecting group R₁₉ is removed by hydrogenation then R₂₀ and R₂₁ are

restricted to C_1-6alkyl.

As a- further alternative, compounds of formula (III) may be prepared by treating a compound of formula (XII):

in which R₁ and R₂ are as defined in formula (I) and R₁₉ is a carboxyl protecting group as defined for formula (XI) with a compound of formula (VIII) as hereinbefore defined using conditions as described for the reaction of compounds of formulae (VII) and (VIII).

A further alternative preparation of compounds of formula (III) may be carried out by reacting a compound of formula (VI) as hereinbefore defined with a compound of formula (XI) as hereinbefore defined, using conditions as described for the reaction of compounds of formula (V) with compounds of formula (VI), and thereafter removing the protecting group R₁₉. Suitable carboxyl protecting groups include alkyl, benzyl and trialkylsilyl groups. A trialkylsilyl protecting group. for example trimethylsilyl, is especially useful in that it may be readily incorporated, in situ, for example by

addition of hexamethyldisilazane to the reactants in

acetonitrile in the presence of triethylamine, and

selectively removed in aqueous methanol, without imposing any limitations on the value of R₂₀ and R₂₁. Other

silylating agents include trimethylsilyl chloride and

N, N-diethyltrimethylsilylamine. An R₁₉ alkyl carboxyl protecting group may be removed by base hydrolysis, for example using sodium hydroxide in aqueous methanol.

It will be appreciated that where the carboxyl protecting group R₁₉ is alkyl, R₂₀ and R₂₁ may0 be alkyl or benzyl derivatives, but where R₁₉ is a benzyl group, R₂₀ and R₂₁ are limited to alkyl.

When compounds of formula (III) are prepared by this route, it is preferred that R₂₀ and R₂₁ are benzyl and R₁₇ is trifluoromethanesulphonyloxy in the compound of formula (VI) and R₁₉ is trimethylsilyl or methyl in the compound of formula (XI). Compounds of formula (V) may be prepared by treating a compound of formula (XIII):

in which R₂ is as defined in formula (I) and wherein the amino group is optionally protected, with a compound of formula (IV) as hereinbefore defined, in the presence of a coupling agent as hereinbefore described for the preparation of compounds of formula (II) from compounds of formulae (III) and (IV). Compounds of formula (VII) may be prepared by reaction of an aldehyde R₁-CHO in which R₁ is as defined in formula (I) with an amine of formula (V) as hereinbefore defined in an organic solvent such as dichloromethane at reduced

temperature (e.g. -10 to 5°C) in the presence of magnesium sulphate.

Compounds of formula (VIII) may be prepared by reaction of the corresponding phosphite, for example dimethylphosphite or dibenzylphosphite with a trialkylsilylhalide, for example trimethylsilyl chloride, in an inert solvent such as

dichloromethane at reduced temperature (e.g. -10 to 5°C) in the presence of a base such as a trialkylamine. Compounds of formula (VI) may be prepared from

hydroxyalkylphosphonate derivatives by conversion of the hydroxyl group to the leaving group R₁₇ by conventional methods. For example, where R₁₇ is trifluoro- methanesulphonyloxy, trifluoromethanesulphonic anhydride may be added to a solution of the hydroxyalkylphosphonate in an inert solvent such as dichloromethane, the reaction being carried out at reduced temperature under an inert

atmosphere, according to the general method of E. Vedejs et al., Journal of Organic Chemistry 50, 2165, (1985).

Hydroxyalkylphosphonate compounds may in turn be prepared by reaction of the corresponding phosphite, for example

dibenzylphosphite, with an aldehyde R₁-CHO in which R₁ is as defined in formula (I) according to the general method of F. Texier-Boullet and A. Foucaud, Synthesis, 916 (1982).

Benzyl and alkyl phosphites are either commercially

available compounds or can be prepared from commercially available starting materials by standard methods. Intermediate compounds of formula (IX) are either known compounds or may be prepared from known aminoalkyl

phosphonic acid derivatives using standard procedures to introduce the R₂₀ and R₂₁ as required. Protection of the amine function during these reactions may be necessary.

Introduction of an R₂₀ or R₂₁ methyl group may be effected by reaction with diazomethane in a suitable inert solvent. Compounds of formula (IX) of fixed configuration may be prepared by the general method of R. Jacquier et al.,

Phosphorus and Sulfur 36, 73, 1983.

The compounds of formulae (IV) and (XIII) are either known amino acid derivatives or can be made from these derivatives by known methods. Compounds of formula (XA) and (XB) are either known compounds or may be prepared from known

compounds by known methods. Compounds of formula (XII) may be prepared from an aldehyde R₁-CHO as hereinbefore defined and a compound of formula (XI) as hereinbefore defined by a procedure analogous to that described for the preparation of compounds of formula (VII).

The intermediates of formulae (II), (III), and certain intermediates of formula (IX) disclosed herein are novel compounds and form an aspect of the present invention as do the described processes for their preparation.

Where obtainable, pharmaceutically acceptable salts of the compounds of formula (I) may be formed conventionally by reaction with the appropriate acid or base. As mentioned previously, the compounds of formula (I) exist in more than one diastereoisomeric form. Where the

processes of the invention produces mixtures thereof, the individual isomers may be separated one from another by chromatography e.g. column chromatography or HPLC.

Alternatively, separate diastereoisomeric compounds of formula (I) can be obtained by using stereoisomerically pure starting materials or by separating desired isomers of intermediates at any stage in the overall synthetic process, and converting these intermediates to compounds of formula (I). It will be appreciated that where a single diastereoisomer of a compound of formula (I) is prepared by more than one process variant as hereinbefore described, each of which allows a different chiral centre to be defined, it may be possible to deduce the configuration at a chiral centre which is not pre-determined using a particular process variant.

Furthermore, it will be appreciated that although the absolute configuration at a particular chiral centre may not be known, it is possible to characterise a given

diastereoisomer relative to its epimer or to another

diastereoisomer using MNR spectroscopy or optical rotation.

In a further aspect, the present invention provides

compounds of formula (I) or pharmaceutically acceptable salts thereof for use as active therapeutic agents,

particularly as agents for treatment of conditions in which degradation of connective tissue and other proteinaceous components of the body occurs, such as musculo-skeletal disorders resulting from collagenolytic activity,

particularly rheumatism and/or arthritic conditions, and tissue remodelling.

Compounds of formula (I) also have potential utility in the treatment of cancer; for preventing myelin degradation in the central and peripheral nervous system; for the treatment or prevention of colonic damage such as irritable bowel disease; and in other conditions in which members of the collagenase family of neutral metalloproteases have

pathological or other roles.

The present invention further provides a pharmaceutical composition, which comprises a compound of formula (I), or a pharmaceutically acceptable salt thereof, and a

pharmaceutically acceptable carrier.

A composition of this invention is useful in the treatment of musculo-skeletal disorders, particularly arthritic diseases and for modulation of tissue remodelling as well as having potential utility in the treatment of the diseases described above. A composition of the invention, which may be prepared by admixture, may contain a diluent, binder, filler,

disintegrant, flavouring agent, colouring agent, lubricant or preservative in conventional manner. These conventional excipients may be employed in conventional manner, for example as in the preparation of compositions of related peptide enzyme inhibitors, such as the ACE inhibitor

enalapril.

A composition of the invention may be adapted for oral, topical, rectal or parenteral administration but oral administration is preferred. Parenteral compositions may be administered intravenously, intramuscularly, intraarticularly, intradermally, sub-cutaneously or into the cerebro-spinal fluid.

Preferably, a pharmaceutical composition of the invention is in unit dosage form and in a form adapted for use in the medical or veterinarial fields. For example, such

preparations may be in a pack form accompanied by written or printed instructions for use as an agent in the treatment or prophylaxis of any of the disorders mentioned above.

The suitable dosage range for the compounds of the invention may vary from compound to compound and may depend on the condition to be treated. It will also depend, inter alia, upon the relation of potency to absorbability and the mode of administration chosen. The compound or composition of the invention may be

formulated for administration by any route, the preferred route depending upon the disorder for which treatment is required, and is preferably in unit dosage form or in a form that a human patient may administer to himself in a single dosage.

Compositions may, for example, be in the form of tablets, capsules, sachets, vials, powders, granules, lozenges, reconstitutable powders, or liquid preparations, for example solutions or suspensions, or suppositories.

The compositions, for example those suitable for oral administration, may contain conventional excipients such as binding agents, for example syrup, acacia, gelatin,

sorbitol, tragacanth, or polyvinylpyrrolidone; fillers, for example lactose, sugar, maize-starch, calcium phosphate, sorbitol or glycine; tableting lubricants, for example magnesium stearate; disintegrants, for example starch, polyvinylpyrrolidone, sodium starch glycollate or

microcrystalline cellulose; or pharmaceutically acceptable wetting agents such as sodium lauryl sulphate.

Solid compositions may be obtained by conventional methods of blending, filling, tableting or the like. Repeated blending operations may be used to distribute the active agent throughout those compositions employing large

quantities of fillers. When the composition is in the form of a tablet, powder, or lozenge, any carrier suitable for formulating solid pharmaceutical compositions may be used, examples being magnesium stearate, starch, glucose, lactose, sucrose, rice flour and chalk. Tablets may be coated according to methods well known in normal pharmaceutical practice, in particular with an enteric coating. The composition may also be in the form of an ingestible

capsule, for example of gelatin containing the compound, if desired with a carrier or other excipients. For example, in a hard gelatin capsule containing the required amount of a compound of the invention in the form of a powder or

granulate in intimate mixture with a lubricant, such as magnesium stearate, a filler, such as macrocrystalline cellulose, and a disintegrant, such as sodium starch

glycollate.

Compositions for oral administration as liquids may be in the form of, for example, emulsions, syrups, or elixirs, or may be presented as a dry product for reconstitution with water or other suitable vehicle before use. Such liquid compositions may contain conventional additives such as suspending agents, for example sorbitol, syrup, methyl cellulose, gelatin, hydroxyethylcellulose,

carboxymethylcellulose, aluminium stearate gel, hydrogenated edible fats; emulsifying agents, for example lecithin, sorbitan monooleate, or acacia; aqueous or non-aqueous vehicles, which include edible oils, for example almond oil, fractionated coconut oil, oily esters, for example esters of glycerine, or propylene glycol, or ethyl alcohol, glycerine, water or normal saline; preservatives, for example methyl or propyl p-hydroxybenzoate or sorbic acid and, if desired, conventional flavouring or colouring agents.

The compounds of this invention may also be administered by a non-oral route. In accordance with routine pharmaceutical procedure, the compositions may be formulated, for example for rectal administration as a suppository or for parenteral administration in an injectable form. For injection, for example by intra-articular injection or by injection into the cerebro-spinal fluid or via other routes which will gain access to sites of demyelination, such as by intramuscular, intradermal or subcutaneous injection, as freely soluble solutions or as poorly dispersed depot stores, the compounds of the invention may be presented in an aqueous or

non-aqueous solution, suspension or emulsion in a

pharmaceutically acceptable liquid, e.g. sterile

pyrogen-free water or a parenterally acceptable oil or a mixture of liquids, which may contain bacteriostatic agents, anti-oxidants or other preservatives, buffers or solutes to render the solution isotonic with the blood, thickening agents, suspending agents or other pharmaceutically

acceptable additives. Such forms will be presented in sterile unit dose form such as ampoules or disposable injection devices or in multi-dose forms such as a bottle from which the appropriate dose may be withdrawn or a solid form or concentrate which can be used to prepare an

injectable formulation.

For topical and percutaneous administration, the

preparations may also be presented as an ointment, cream, lotion, gel, spray, aerosol, wash, skin paint or patch. A unit dose for treating diseases in which enzymes of the collagenase family are involved will generally contain from 10 to 1000 mg and preferably will contain from 10 to 500 mg, in particular 10, 50, 100, 150, 200, 250, 300, 350, 400, 450 or 500 mg. The composition may be administered one or more times a day, for example 2, 3 or 4 times daily, so that the total daily dose for a 70 kg adult will normally be in the range 10 to 3000 mg. Such a dosage corresponds to

approximately 0.15 to 50 mg/kg per day. Alternatively, in particular for injection, the unit dose will contain from 2 to 200 mg of a compound of the invention and be administered in multiples, if desired, to give the desired daily dose.

The present invention additionally provides a method of treating conditions in which degradation of connective tissue and other proteinaceous components of the body occurs which comprises administering to the mammal in need of such treatment an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof. The present invention also provides the use of a compound of formula (I) or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for use in the treatment of conditions in which degradation of connective tissue and other proteinaceous components of the body occurs.

The following Descriptions and Examples illustrate the preparation of compounds of the invention. All temperatures are expressed in °C.

Description 1

N- [1- (Dxe-thoxyphosph±nyl) -1- (2-hydroxyphenyl)methyl] - leucine, benzyl ester (D1)

Salicylaldehyde (1.83g), leucine benzylester p-toluene sulfonate salt (5.9g) and triethylamine (2.1 ml) in toluene (100 ml) were heated at reflux for 2 h in a Dean-Stark apparatus. The solution was cooled and solvent removed in vacuo. Diethyl phosphite (3.2g) was added and the solution heated at 120°C for 24 h. The reaction mixture was cooled and column chromatography on silica gel, eluting with ethyl acetate, gave the title compound as a yellow oil (2.8g). Description 2

N-[1-(Diethoxyphosphinyl)-1-(2-hydroacyphenyl)methyl]-leucine (D2) The title compound (1.0g) was prepared from (Dl) (1.4g) by hydrogenation at atmospheric pressure over 10% palladium on charcoal in ethanol.

Description 3

N-[N-(1-Diethoxyphosphinyl-1-(2-hydroxyphenyl)methyl)-leucyl]-N,O-dimethyl-L-tyrosinamide (D3)

A solution of N-[1-(diethoxyphosphinyl)-1-(2-hydroxy-phenyl)methyl-leucine (D2) (0.5g) in dry acetonitrile was cooled to 0°C in an ice-bath and 1,1'-carbonyldiimidazole added. The mixture was kept at 0°C for 1 h and then

O-methyl-(S)-tyrosine N-methylamide in the minimum volume of acetonitrile was added dropwise with stirring. The mixture was allowed to warm to room temperature and stirred for a further 2 days and then evaporated to dryness. The residue was taken up in chloroform and washed with dilute citric acid and water. The organic layer was dried with anhydrous sodium sulphate, filtered and evaporated to dryness to give the crude product. Purification by column chromatography on silica gel with initially chloroform rising to 10%

methanol/chloroform as eluant gave the title compound

(0.43g).

Observed EI M+563.

Description 4 3-(1,8-Naphthalenedicarboximide)propanol (D4)

3-Amino-1-propanol (3.75g) was dissolved in dichloromethane (500 ml) and dimethylformamide (50 ml) and naphthalic anhydride (9.9g) added, followed by triethylamine (6.9 ml). The solution was stirred at room temperature for 2h and 1-hydroxybenzotriazole (8.75g) and 1-ethyl-3(3-dimethylaminopropyl)carbodiimide hydrochloride (13.4g) added and the solution stirred at room temperature for 24h. The solution was filtered, washed with water, 1N hydrochloric acid, saturated sodium bicarbonate solution and dried with

anhydrous sodium sulphate. The solution was filtered and the solvent evaporated in vacuo to give the title compound as a pale yellow solid (7.5g). Description 5

3-(1,8-Naphthalenedicarboximido)propanal (D5)

A solution of oxalyl chloride (3.5 ml) in dichloromethane (100 ml) was cooled to -60°C and dimethylsulphoxide (5.95 ml) in dichloromethane (10 ml) added slowly over 15 min. The alcohol (9g) (D4) in dichloromethane (100 ml) was added dropwise over 30 min, and the solution stirred at -60°C for 15 min. The reaction mixture was allowed to warm to -15°C, cooled to -60°C and triethylamine (24.5 ml) added. The solution was allowed to warm to room temperature, washed with water, dilute hydrochloric acid, 10% sodium carbonate solution and dried with anhydrous sodium sulphate. The solution was filtered and the solvent evaporated in vacuo to give the title compound as a white solid (8.3g). δ (CDCI₃) : 2.86 (2H, dt), 4.53 (2H, t), 7.75 (2H, t), 8.22 (2H, d), 8.56 (2H, d), 9.90 (1H, s).

Description 6

N-[N-(1-(Dibenzyloxyphosphinyl)-3-(1,8-naphthalene- dicarboximido)propyl)-(S)-leucyl]-N,O-dimethyl-(S)- tyrosinamide (D6)

3-(1,8-Naphthalenedicarboximido)propanal (3.2g) (D5) in dichloromethane (50 ml) was addedto a solution of N-((S)- leucyl)-N,O-dimethyl-(S)-tyrosinamide (4.04g) in

dichloromethane (50 ml) at 0°C. Magnesium sulphate was added and the reaction mixture stirred at 0°C for 15 min, then at room temperature for 15 min. Dibenzyl trimethylsilylphosphite (4.25g), prepared by the method of J.I.G. Cadogan et al. [Tetrahedron 1990, 46,

7175], was added via syringe to the above reaction mixture under nitrogen at 0°C. The solution was allowed to warm to room temperature and stirred for 18h, washed with water, 10% citric acid solution and saturated sodium chloride solution. The solution was dried with anhydrous sodium sulphate, filtered and the solvent evaporated in vacuo to give an orange oil. Purification by column chromatography on silica gel, eluting with ethyl acetate, gave a single diastereoisomer.

Isomer D6A (0.9g) δ (CDCI₃) : 0.81 (6H, dd), 0.90-2.35 (6H, m), 2.77 (3H, d),

2.87 (2H, m), 3.22 (2H, m), 3.67 (3H, s), 4.12 (2H, t), 4.74 (1H, m), 4.95 (4H, m), 6.78 (2H, d), 7.10 (2H, d), 7.31 ( 10H, s ) , 7 . 60 ( 1H, d) , 7 . 74 ( 3H, t ) , 8 . 22 (2H, d) , 8 .5 ! (2H, d) .

Observed FAB (M+H)⁺ 819. C₄₆H₅₁N₄O₈P requires M 818.

Further elution gave a slower running single

diastereoisomer.

Isomer D6B (1.8g) δ (CDCI₃) : 0.82 (6H, dd), 1.12-2.24 (6H, m), 2.69 (3H, d), 2.83 (1H, m), 3.13 (2H, m), 3.62 (1H, m), 3.68 (3H, s), 4.15 (1H, m), 4.27 (1H, m), 4.60 (1H, t), 4.95 (4H, m), 6.59 (1H, d), 6.76 (2H, d), 7.15 (2H, d), 7.30 (10H, m), 7.62 (1H, d), 7.76 (2H, t), 8.23 (2H, d), 8.60 (2H, d).

Observed FAB (M+H) ⁺ 819. C₄₆H₅₁N₄O₈P requires M. 818.

Description 7

3-(Phthalimido)propanol (D7)

3-Amino-1-propanol (7.5g) was dissolved in ethanol (200 ml), N-carbethoxyphthalimide (21.9g) was added and the solution stirred at room temperature for 24h. The solvent was evaporated in vacuo and the residue dissolved in chloroform and washed with water (2x). The chloroform solution was dried with sodium sulphate, filtered and the solvent

evaporated in vacuo to give the title compound (12.7g).

Description 8

3-(Phthalimido)propanal (D8) The title compound (11.5g) was prepared from 3- (phthalimido)propanol (D7) (12.7g) by the procedure

described in Description 5. δ (CDC1₃) : 2.89 (2H, dt), 4.05 (2H, t), 7.74 (2H, m), 7.86 (2H, m), 9.83 (1H, s).

Description 9

N-[N-(1-(Dimethoxyphosphinyl)-3-phthalimidopropyl)-(S)- leucyl]-(S)-phenylalanine-N-methylamide (D9)

3-(Phthalimido)propanal (4.06g) (D8) in dichloromethane (50ml) was added to a solution of (S)-leucyl-(S)-phenylalanine-N-methylamide (5.82g) in dichloromethane (50ml) at 0°C. Magnesium sulphate was added and the

reaction mixture stirred at 0°C for 15 min, then at room temperature for 15 min.

Dimethyl trimethylsilylphosphite (3.3g), prepared by the method of J.I.G. Cadogan et al. [Tetrahedron 1990, 46,

7175] was added via syringe to the above reaction mixture under nitrogen at 0°C. The solution was allowed to warm to room temperature and stirred for 18h, washed with water, 10% citric acid solution and saturated sodium chloride solution. The solution was dried with anhydrous sodium sulphate, filtered and the solvent evaporated in vacuo to give an orange oil.

Purification by column chromatography on silica gel, eluting with ethyl acetate, followed by 10% methanol/ethyl acetate gave the title compound as a mixture of 2 diastereoisomers (8g) (D9). δ (CDCI₃) : 0.85 (6H, m), 0.95-2.18 (5H, m) , 2.67 (1-4H, d) , 2.75 (H4H, d), 2.80-3.32 (3H, m), 3.55 (½H, t), 3.72 (8H, m), 3.84 (½H, m) , 4.60 (V6H, q), 4.75 (½H, m), 6.22 (½H,d) 7.22 (5-4H, m), 7.53 (1H, t), 7.74 (3H, m) , 7.85 (2H, m) .

Observed FAB (M+H) ⁺ 587 C₂₉H₃₉N4O₇P requires M. 586

The title compound was also prepared as a single

diastereoisomer (D9A) (0.09g) from the acid (D24A) (0.1g) and (S)-phenylalanine-N-methylamide (0.05g) by the procedure described in Description 19. δ (CDCI₃) : 0.88 (6H, d), 1.28 (2H, m), 1.60 (2H, m), 1.84 (1H, m), 2.08 (1H, m), 2.67 (3H, d), 3.14 (2H, m), 3.55 (1H, t), 3.75 (8H, m), 3.84 (1H, m), 4.60 (1H, q), 6.26 (1H, d), 7.23 (5H, m),7.51 (1H, d), 7.73 (2H, m), 7.85 (2H, m)

Description 10 4-(1,8-Naphthalenedicarboximido)butanol (D10)

The title compound (4.5g) was prepared from 4-amino-1-butanol (4.45g) by the procedure described in Description 4. Description 11

4-(1,8-Naphthalenedicarboximido)butanal (Dll)

The title compound (3.96g) was prepared from 4-(1,8-naphthalenedicarboximido) butanol (D10) (4.38g) by the procedure described in Description 5. δ (CDCI₃) : 2.10 (2H, 2 overlapping t), 2.60 (2H, t), 4.20 (2H, t), 7.70 (2H, t), 8.17 (2H, d), 8.58 (2H, d, 9.83

(1H, s).

Description 12 N-[N-(1-(Dibenzyloxyphosphinyl)-4-(1,8-naphthalenedicarboximido)butyl)-(S)-leucyl]-(S)-phenylalanine-N- methylamide (D12)

The title compound (3.33g) was prepared as a mixture of 2 diastereoisomers from 4-(1,8-naphthalenedicarboximido)butanal D11) (3.96g), (S)-leucyl-(S)-phenylalanine-N- methylamide (4.32g) and dibenzyl trimethylsilylphosphite (4.99g) by the procedure described in Description 6. δ (CDCl₃) : 0.67 (3H, dd), 0.79 (3H, dd), 0.99-2.03 (8H, m), 2.56 (1½H, d), 2.74 (1½H, d), 2.70-3.69 (4H, m), 4.12 (2½H, m), 4.50 (½H, q), 4.93 (4H, m), 6.23 (½H, d), 7.23 (15H, m), 7.74 (3H, m), 8.22 (2H, m), 8.61 (2H, d).

Observed FAB (M+H)⁺ 803. C₄₆H₅₁N₄O₇P requires M 802.

Description 13 2-(1,8-Naphthalenedicarboximido)ethanol (D13)

The title compound (7g) was prepared from ethanolamine

(3.05g) by the procedure described in Description 4. Description 14

2-(1,8-Naphthalenedicarboximido)ethanal (D14)

The title compound (3.15g) was prepared from 2-(1,8-naphthalenedicarboximido) ethanol (D13) (3.21g) by the procedure described in Description 5. δ (CDCI₃) : 5.05 (2H, s), 7.73 (2H, t), 8.24 (2H, d), 8.60 (2H, d), 9.74 (1H, s).

Description 15 N-[N-(1-(Dibenzyloxyphosphinyl)-2-(1,8-naphthalene-dicarboximido)ethyl)-(S)-leucyl]-(S)-phenylalanine-N-methylamide (D15)

The title compound (3.0g) was prepared as a mixture of 2 diastereoisomers from 2-(1,8-naphthalenedicarboximido)-ethanal (D14) (3.13g), (S)-leucyl-(S)-phenylalanine-N-methylamide (3.81g) and dibenzyl trimethylsilylphosphite (4.4g) by the procedure described in Description 6. δ (CDCl₃) : 0.19 (1½H, d), 0.39 (1½H, d), 0.70 (3H, dd), 0.70-1.48 (3H, m), 2.22 (½H, dd), 2.68 (1½H, d), 2.74 (1½H, d), 2.74-3.65 (3½H, m), 4.30 (1H, m), 4.49 (1½H, m), 4.74 (½H, m), 4.98 (4H, m), 6.20 (½H, m), 6.78 (1H, dd), 6.86 (½H, d), 7.22 (15H, m), 7.76 (2H, t), 8.22 (2H, d), 8.57 (2H, d).

Observed FAB (M+H)⁺ 775. C₄₄H₄₇N₄O₇P requires M 774.

Description 16

N-[N-(1-(Dibenzyloxyphosphinyl)-3-(1,8-naphthalene-dicarboximido)propyl)-(S)-leucyl]-(S)-phenylanine-N-methylamide (D16) The title compound (2.5g) was prepared from 3-(1,8-naphthalenedicarboximido) propanal (D5) (3.5g), (S)-leucyl- (S)-phenylalanine-N-methylamide (4.03g) and dibenzyl

trimethylsilylphosphite (4.65g) by the procedure described in Description 6.

Purification by column chromatography on silica gel, eluting with a gradient of 0-5% methanol/ethyl acetate gave a single diastereoisomer. Isomer D16A

δ (CDCI₃) : 0.77 (6H, dd), 0.83-2.03 (5H, m), 2.17 (1H, m), 2.77 (3H, d), 2.77-3.37 (3H, m), 4.21 (2H, t), 4.78 (1H, m), 4 . 96 (4H, m) , 7 .24 (15H, m) , 7 .62 (1H, d) , 7 .76 (2H, t) ,

8.23 (2H, d) , 8.57 (2H, d) .

Observed FAB (M+H) ⁺ 789. C₄₅H₄₉N₄O₇P requires M 788. Analysis: C₄₅H₄₉N₄O₇P requires C, 68.51; H, 6.26; N, 7.10%. Found C, 68.44; H, 6.17; N, 7.55%.

Further elution gave a slower running diastereoisomer. Isomer D16B

δ (CDCl₃) : 0.79 (6H, dd), 1.05-2.28 (5H, m), 2.66 (3H, d), 2.78 (1H, m), 3.17 (2H, m), 3.58 (1H, t), 4.20 (2H, m), 4.63 (1H, m), 4.93 (4H, m), 6.63 (1H, d), 7.21 (15H, m), 7.65 (1H, d), 7.75 (2H, t), 8.21 (2H, d), 8.58 (2H, d).

Observed FAB (M+H) ⁺ 789. C₄₅H₄₉N₄O₇P requires M 788.

Analysis: C₄₅H₄₉N₄O₇P requires C, 68.51; H, 6.26; N, 7.10%, Found C, 68.83; H, 6.18; N, 7.40%.

Description 17

N-[1-(Dibenzyloxyphosphinyl)-3-phenylpropyl]-(S)-leucine, methyl ester (D17)

Hydrocinnamaldehyde (4.62g) in dichloromethane (50 ml) was added to a solution of (S)-l-ucine methyl ester (5.0g) in dichloromethane (50 ml) at 0°C. Magnesium sulphate was added and the reaction mixture stirred at 0°C for 15 min, then at room temperature for 15 min.

Dibenzyl trimethylsilylphosphite (11.6g), prepared by the method of J.I.G. Cadogan et al. [Tetrahedron 1990, 46,

7175], was added via syringe to the above reaction mixture under nitrogen at 0°C. The solution was allowed to warm to room temperature and stirred for 18h, washed with water, 10% citric acid solution and saturated sodium chloride solution. The solution was dried with anhydrous sodium sulphate, filtered and the solvent evaporated in vacuo to give a yellow oil.

Purification by column chromatography on silica gel, eluting with a mixture of pentane:ether:acetone (10:9:1), gave a single diastereoisomer.

Isomer D17A (1.47g) δ (CDCl₃) : 0.88 (6H, dd), 1.40 (1H, m), 1.61-1.90 (2H, m),

2.10 (1H, m), 2.56-2.92 (5H, m), 3.63 (3H, s), 3.75 (1H, t),4.97 (4H, m), 7.25 (15H, m).

Observed EI M⁺ 524.

Further elution gave a slower running single diastereoisomer

Isomer D17B (0.61g)

δ (CDCI₃) : 0.87 (6H, dd), 1.40 (2H, m), 1.75 (2H, m), 2.05 (1H, m), 2.66 (1H, m), 2.84 (2H, m), 3.42 (1H, t), 3.58 (3H, s), 4.93 (4H, m), 7.26 (15H, m).

Observed EI M⁺ 524. Analysis: C₃₀H₃₈NO₅P requires C, 68.82; H, 7.73; N, 2.68% Found C, 68.87; H, 7.23; N, 2.37%.

Description 18 N-[1-(Dibenzyloxyphosphinyl)-3-phenylpropyl]-(S)-leucine (D18)

The methyl ester (D17A) (1.35g) was dissolved in dioxan (30 ml) and water (15 ml). Sodium hydroxide (0.12g) was added and the solution was stirred at room temperature for 24h. The dioxan was evaporated in vacuo and the aqueous solution washed with ether, acidified with 2N hydrochloric acid and extracted with ethyl acetate (2x). The organic extracts were dried with sodium sulphate and the solvent evaporated in vacuo to give the title compound, as a white solid (0.65g), as a single diastereoisomer (D18A). δ (CDCI₃) : 0.87 (6H, dd), 1.48 (2H, m), 1.77 (2H, m), 2.13 (1H, m), 2.86 (2H, m), 3.76 (1H, t), 5.02 (4H, m), 7.16 (5H, m), 7.33 (10H, s).

Observed EI M⁺ 510

[α]_D ²² = -12.08° (c=l% in methanol)

Analysis: C₂₉H₃₆NO₅P .1/2H₂O requires C, 67.17; H, 7.19; N, 2.70%.

Found C, 67.13; H, 6.93; N, 2.79%.

Similarly, the title compound was prepared, as a pale yellow oil (0.22g), as a single diastereoisomer (D18B) from the methyl ester (D17B) (0.52g). δ (CDCI₃) : 0.88 (6H, t), 1.38 (1H, m), 1.62 (2H, m), 1.85 (1H, m), 2.10 (1H, m), 2.75 (3H, m), 3.37 (1H, t), 5.03 (4H, m), 7.16 (5H, m), 7.33 (10H, s). Observed EI M⁺ 510

Description 19

N-[N-(1-(Dibenzyloxyphosphinyl)-3-phenylpropyl)-(S)-leucyl]- (S)-phenylalanine-N-methylamide (D19)

The acid (D18A) (0.6g) was dissolved in dichloromethane (20 ml), cooled to 0°C and 1-hydroxybenzotriazole (0.23g) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (0.33g) were added. The solution was stirred at 0°C for 15 min, then at room temperature for 15 min, cooled to 0°C and (S)-phenylalanine-N-methylamide (0.24g) in dichloromethane (20 ml) added. The solution was stirred at room temperature for 24h, washed with water, 10% citric acid solution and saturated sodium chloride solution. The organic solution was dried with anhydrous sodium sulphate and the solvent evaporated in vacuo to give a beige solid (0.69g).

Purification by column chromatography on silica gel, eluting with ethyl acetate gave the title compound, as a white solid (0.52g), as a single diastereoisomer (D19A). δ (CDCl₃) : 0.80 (6H, d), 1.20 (2H, m), 1.50 (1H, m), 1.73 (2H, m), 2.00 (1H, m), 2.54 (1H, m), 2.65 (3H, d), 2.73 (2H, m), 3.02 (2H, m), 3.57 (1H, t), 4.54 (1H, q), 4.97 (4H, m), 6.05 (1H, broad s), 7.19 (10H, m), 7.33 (10H, s). Observed FAB (M+H)⁺ 670. C₃₉H₄₈N₃O₅P requires M 669.

[α]_D ²² = -30.85° (c=0.98% in methanol)

Analysis: C₃₉H₄₈N₃O₅P requires C, 69.94; H, 7.22; N, 6.27%. Found C, 70.05; H, 7.28; N, 6.38%.

Similarly, the title compound was prepared from the acid (D18B) (0.20g), as a pale yellow oil (0.23g), as a single diastereoisomer (D19B). δ (CDCI₃) : 0.78 (6H, dd), 1.02-1.32 (3H, m), 1.65-2.14 (3H, m), 2.60 (3H, m), 2.76 (3H, d), 2.92 (1H, dd), 3.14 (1H, dd), 3.32 (1H, dd), 4.80 (1H, m), 5.00 (4H, m), 7.00 (1H, d), 7.20 (10H, m), 7.33 (10H, m), 7.63 (1H, broad d).

Observed FAB (M+H)⁺ 670. C₃₉H₄₈N₃O₅P requires M 669. Description 20

N-[1-(Dimethoxyphosphinyl)-3-(1,8-naphthalene

dicarboximido)propyl]-(S)-leucine, benzyl ester (D20)

3-(1,8-Naphthalenedicarboximido)propanal (D5) (9.5g) was added to a solution of (S)-leucine benzyl ester, p-toluene sulphonate salt (14.75g) and triethylamine (5.8ml) in dichloromethane (100ml). Molecular sieves and magnesium sulphate were added and the mixture stirred under nitrogen for 18h. The solution was filtered and added dropwise to dimethyl trimethylsilylphosphite (7.5g), prepared by the method of J.I.G.Cadogan et al [Tetrahedron 1990, 46, 7175], under nitrogen at 0°C. The solution was allowed to warm to room temperature and stirred for 18h, washed with water, 10% citric acid solution, saturated sodium bicarbonate solution and saturated sodium chloride solution. The solution was dried with anhydrous sodium sulphate, filtered and the solvent evaporated in vacuo to give a yellow oil.

Purification by column chromatography on silica gel, eluting with a mixture of ether:ethyl acetate (1:1) gave a single diastereoisomer (D20A) (5.51g). δ (CDCl₃) : 0.95 (6H, dd), 1.53 (2H, t), 1.75 - 2.00 (3H, m), 2.27 (1H, m), 2.98 (1H, m), 3.72 (3H, d), 3.80 (3H, d), 3.90 (1H, t), 4.20 (1H, m), 4.46 (1H, m), 5.65(2H,m), 7.35 (5H, m), 7.77 (2H, t), 8.23 (2H, d), 8.62 (2H, d). ³¹P: δ (CDCI₃) : 28.73

Observed CI (M+H)⁺ 567, C₃₀H₃₅N₂O₇P requires M.566

[α]_D ²² = -14.94 (c=0.38, MeOH)

Analysis: C₃₀H₃₅N₂O₇P requires C, 63.60; H, 6.23; N,4.94% Found C, 63.28; H, 6.20; N, 4.83% Further elution gave a slower running single

diastereoisomer.

Isomer D20B (5.23g) δ (CDCI₃) : 0.93 (6H, dd), 1.53 (2H, t), 1.80 (2H, m), 2.17 (1H, m), 3.10 (1H, m), 3.63 (1H, t), 3.71 (3H, d), 3.77 (3H, d), 4.29 (1H, m), 4.44 (1H, m), 5.15 (2H, m), 7.36 (5H, m), 7.75 (2H, t), 8.21 (2H, d), 8.59 (2H, d).

³¹P: δ (CDCI₃) : 29.65

Observed CI (M+H) ⁺ 567 C30H35N2O7P requires M 566 [α]_D ²² = -13.20 (c=0.44, MeOH)

Analysis: C₃₀H₃₅N₂O₇P.H₂O requires C, 61.64; H, 6.38;

N, 4.79%

Found C, 61.56; H, 6.35; N, 4.62%.

Description 21 N-[1-(Dimethoxyphosphinyl)-3-(1,8-naphthalene

dicarboximido)propyl]-(S)-leucine (D21)

The benzyl ester (D20A) (3.7g) was dissolved in methanol (100ml) and hydrogenated over 10% palladium on charcoal at atmospheric pressures for 3h. The solution was filtered and the solvent evaporated in vacuo to give the title compound as a single diastereoisomer (2.7g) (D21). δ (CDCI₃) : 0.96 (6H, t), 1.58 (2H, m), 1.89 (2H, m), 2.26 (1H, m), 3.12 (1H, m), 3.76 (3H, d), 3.83 (3H, d), 4.29 (1H, m), 4.47 (1H, m), 7.67 (2H, t), 8.14 (2H, d), 8.49 (2H, d)

³¹P: δ (CDCI₃) : 29.22

Observed FAB (M+H) ⁺ 477 C₂₃H₂₉N₂O₇P requires M 476 [α] _D ²² = -1 .50 (c = 1 .00, MeOH)

Analysis: C₂₃H₂₉N₂O₇P. H₂O requires C, 55.87; H, 6.31; N, 5.66%

Found C, 55.67; H, 5.84; N, 5.66%

Description 22 N-[N-(1-(Dimethoxyphosphinyl)-3-(1,8-naphthalene

dicarboximido)propyl)-(S)-leucyl]-(S)-phenylalanine-N- methylamide (D22)

The title compound (1.2g) was prepared from the acid (D21) (0.95g) and (S)-phenylalanine-N-methylamide (0.24g) by the procedure described in Description 19. δ (CDCI₃) : 0.89 (6H, dd), 1.26 (2H, m), 1.62 (1H, m), 1.84 (2H, m), 2.11 (1H, brond s), 2.60 (3H, d), 2.85 (1H, m), 3.20 (2H, m), 3.55 (1H, t), 3.71 (3H, d), 3.78 (3H, d), 4.19 (1H, m), 4.33 (1H, m), 4.65 (1H, q), 6.56 (1H, d), 7.23 (5H, m), 7.71 (1H, d), 7.76 (2H, t), 8.23 (2H, d), 8.60 (2H, d).

Description 23

N-[1-(Dimethoxyphosphinyl)-3-phthalimidopropyl]-(S)-leucine, benzyl ester (D23)

The title compound was prepared from 3-(phthalimido-propyl)propanal (3.36g) and (S)-leucine benzyl ester, p-toluene sulphonate salt (6.51g) by the procedure described in Description 20.

Purification by column chromatography on silica gel, eluting with a mixture of ether:ethyl acetate (1:1) gave a single diastereoisomer.

Isomer D23A (1.62g) δ (CDCI₃) : 0.92 (6H, m), 1.38 - 1.92 (5H, m), 2.13 (1H, m), 2.93 (1H, m), 3.47 - 4.06 (3H, m), 3.73 (3H, d), 3.79 (3H, d), 5.13 (2H, m), 7.33 (5H, m), 7.70 (2H, m), 7.83 (2H, m).

Further elution gave a slower running single

diastereoisomer.

Isomer D23B (1.52g) δ (CDCI₃) : 0.90 (6H, dd), 1.50 (2H, t), 1.70 - 2.20 (4H, m) 2.95 (1H, m), 3.53 (1H, t), 3.70 (3H, d), 3.75 (3H, d), 3.75 - 4.03 (3H, m), 5.14 (2H, m), 7.35 (5H, m), 7.70

(2H, m), 7.83 (2H, m)

Description 24

N-[1-(Dimethoxyphosphinyl)-3-phthalimidopropyl]-(S)-leucine (D24)

The title compound was prepared as a single diastereoisomer (D24A) (0.97g) from the benzyl ester (D23A) (1.52g) by the procedure described in Description 21. δ (CDCI₃) : 0.96 (6H, dd), 1.55 (2H, m), 1.85 (2H, m), 2.20 (1H, m), 3.03 (1H, dt), 3.78 (8H, m), 3.98 (1H, m), 7.70 (2H, m), 7.83 (2H, m)

Similarly, the title compound was prepared as a single diastereoisomer (D24B) (0.83g) from the benzyl ester (D23B) (1.4g) δ (CDCI₃) : 0.96 (6H, d), 1.55 (2H, m), 1.85 (2H, m), 2.15 (1H, m), 2.98 (1H, m), 3.44 (1H, m), 3.78 (8H, m), 7.73 (2H, m), 7.85 (2H, m)

³¹ P: δ (CDCI₃) : 29.01 Description 25

3-(4-Bromo-1,8-naphthalenedicarboximido)propanol (D25) A mixture of 4-bromophthalic anhydride and 3-amino-1- propanol (12g) in ethanol (200ml) was heated under reflux for 30 min. The solution was allowed to cool to room temperature and the solvent evaporated in vacuo to give a solid which was washed with ester to give the title compound (12.2g) (D25). δ (CDCl₃) : 2.00 (2H, dt), 3.60 (2H, t), 4.35 (2H, t), 7.88 (1H, t), 8.07 (1H, d), 8.44 (1H, d), 8.60 (1H, d), 8.70 (1H, d)

Description 26

3-(4-Bromo-1,8-naphthalenedicarboximido)propanal (D26) The title compound (10.3g) (D26) was prepared from the alcohol (12.2g) (D25) by the procedure described in

Description 5. δ (CDCI₃) : 2.90 (2H, dt), 4.54 (2H, t), 7.85 (1H, t), 8.04 (1H, d), 8.40 (1H, d), 8.59 (1H, dd), 8.66 (1H, dd), 9.90 (1H, s)

Description 27 N-[1-(Dimethoxyphosphinyl)-3-(4-bromo-1,8-naphthalene dicarboximido)propyl]-(S)-leucine, trimethylsilylethyl ester (D27)

The title compound (D27) was prepared from 3-(4-bromo-1,8-naphthalenedicarboxamido)propanal (D26) (3.0g) and (S)-leucine, trimethylsilylethyl ester (2.17g) by the procedure described in Description 20. Purification by column chromatography on silica gel, eluting with 40 - 60° petrol, increasing to 20% ethyl acetate/40 -60° petrol gave a single diastereoisomer Isomer D27A (1.0g) δ (CDCl₃) : 0.96 (8H, m), 1.48 (2H, t), 1.82 (2H, m), 2.25 (1H, m), 2.94 (1H, m), 3.75 (8H, m), 4.15 (2H, m), 4.24 (1H, m), 4.52 (1H, dt), 7.81 (1H, t), 8.02 (1H, d), 8.39 (1H, d), 8.56 (1H, d), 8.63 (1H, d)

Further elution gave a slower running single diasterisomer.

Isomer D27B (0.95g) δ (CDCI₃) : 0.91 (8H, m), 1.47 (2H, t), 1.8 (2H, m), 2.13 (1H, m), 3.06 (1H, m), 3.51 (1H, t), 3.73 (3H, d), 3.79 (3H, d), 4.12 (3H, m), 4.26 (1H, m), 4.41 (1H, m), 7.80 (1H ,t), 8.00 (1H, d), 8.38 (1H, d), 8.54 (1H, d), 8.62 (1H, d)

Description 28

N-[1-(Dimethoxyphosphinyl)-3-(4-bromo-1,8-naphthalene dicarboximido)propyl]-(S)-leucine (D28)

The trimethylsilylethyl ester (1.0g) (D27A) was dissolved in tetrahydrofuran (50 ml) and the solution cooled to 0°C. A solution of IM tetrabutylammonium fluoride (1.5ml) in tetrahydrofuran (10ml) was added dropwise under nitrogen and the solution stirred at room temperature for 24h. Water (20ml) was added and the solution cooled to 0°C and

acidified with 2N hydrochloric acid. The solution was extracted with ethyl acetate (3x) and the combined organic extracts washed with water, dried with anhydrous sodium sulphate and the solvent evaporated in vacuo to give the title compound as a single diastereoisomer (0.8g) (D28A) δ (CDCI₃) : 1.04 (6H, m) , 1.73 (2H, m) , 2 . 00 - 2 . 60 (3H, m) , 3 .88 (7H, m) , 4.21 (2H, m) , 4.40 (2H, m) , 7.55 (1H, t) , 7. 82 (1H, t) , 8 .05 (1H, d) , 8 .32 (2H, t) Similarly the title compound was prepared as a single diastereoisomer (0.28g) (D28B) from the trimethylsilylethyl ester (0.33g) (D27B). δ (CDCI₃) : 0.98 (6H, d), 1.20 - 2.20 (5H, m), 3.07 (1H, m), 3.34 (1H, m), 3.50 (1H, t), 3.82 (6H, t), 4.34 (2H, t), 7.87 (1H, t), 8.06 (1H, t), 8.40 (1H, d), 8.60 (1H, d), 8.66 (1H, d)

Description 29

N-{N-(1-(Dimethoxyphosphinyl)-3-(4-bromo-1,8- naphthalenedicarboxjmido)propyl]-(S)-leucyl]-(S)-phenylalanine-N-methylamide (D29) The title compound was prepared from the acid (1.0g) (D28A) and (S)-phenylalanine-N-methylamide (0.65g) by the method described in Description 19.

Purification by column chromatography on silica gel, eluting with 5% methanol/ethyl acetate gave the title compound as a single diastereoisomer (0.3g) (D29A) δ (CDCI₃) : 0.89 (6H, d), 1.27 (2H, m), 1.62 (1H, m), 1.69 (2H, m), 2.26 (1H, m), 2.70 (3H, d), 2.84 (1H, m), 2.93 -3.25 (1H, m), 3.56 (1H, t), 3.72 (3H, t), 3.79 (3H, d), 4.17 (1H, m), 4.31 (1H, m), 4.58 (1H, m), 6.48 (1H, d), 7.23 (5H, m), 7.68 (1H, d), 7.87 (1H, t), 8.06 (1H, d), 8.42 (1H, d), 8.60 (1H, d), 8.68 (1H, d) Similarly, the title compound was prepared as a single diastereoisomer (0.1g) (D29B) from the acid (0.28g) (D28B) and (S)-phenylalanine-N-methylamide (0.18g). δ (CDCI₃) : 0.85 (6H, dd), 1.13 (2H, m) , 1.57 (2H, m), 1.90 - 2.24 (2H, m), 2.80 (3H, d), 3.00 (2H, m) , 3.28 (2H, m), 3.70 (3H, d), 3.75 (3H, d), 4.27 (2H, t) , 4.77 (1H, m) , 7.23 (5H, m), 7.50 (1H, d), 7.62 (1H, d), 7.87 (1H, t), 8.05 (1H, d), 8.40 (1H, d), 8.60 (1H, d), 8.65 (1H, d)

Description 30

N-[N-(1-(Dibenzyloxyphosphinyl)-3-(2-benzyloxy-phenyl)propyl)-(S)-leucyl]-(S)-phenylalanine-N-methylamide (D30)

The title compound (4.0g) was prepared from 3-(2-benzyloxy-phenyl) propanol (4.8g), (S)-leucyl-(S)-phenylalanine-N-methylamide (5.82g) and dibenzyltrimethylsilyl phosphite (6.7g) by the procedure described in Description 6.

Observed FAB (M+H)⁺ 776 C₄₆H₅₄N₃O₆P requires M. 775

Description 31

N-[N-(1-(Dimethoxyphosphinyl)-3-(methylmercapto)propyl)-(S)- leucyl]-(S)-phenylalanine-N-methylamide (D31)

The title compound (4.1g) (D31) was prepared from 3-(methyl- mercapto)propanal (2.08g), (S)-leucyl-(S)-phenylalanine-N- methylamide (5.82g) and dimethyltrimethylsilyl phosphite (3.3g) by the procedure described in Description 9.

Purification by column chromatography on silica gel, eluting with 3% methanol/ethyl acetate gave the title compound as a mixture of 2 diastereoisomers (4.1g) (D31) δ (CDCl₃) : 0.90 (6H, m), 0.90-1.95 (6H, m), 2.08 (1½H, s), 2.10 (1½H, s), 2.60 (2H, t), 2.73 (1½H, d), 2.76 (1½H, d), 2.82-3.12 (2H, m), 3.26 (1½H, m), 3.57 (½H, m), 3.74 (3H, d), 3.78 (3H, d), 4.55 (½H, q), 4.76 (½R, m), 6.14 (½H, broad s), 7.22 (5H, m), 7.38 (½H, broad s), 7.53 (½H, d), 7.64 OSH, d).

Observed FAB (M+H)⁺ 488 C₂₂H₃₈N₃O₅PS requires M 487

Description 32

N-[N-(1-(Dimethoxyphosphinyl)-3-(methylsulphinyl)propyl)- (S)-leucyl]-(S)-phenylalanine-N-methylamide (D32)

Sodium periodate (0.86g) in water (5ml) was added to a solution of N-[N-(1-(dimethoxyphosphinyl)-3-(methyl-mercapto)propyl)-(S)-leucyl]-(S)-phenylalanine-N-methylamide (0.98g) (D31) in methanol (50ml). The solution was stirred at room temperature for 2h and the solvent evaporated in vacuo. The residue was dissolved in

chloroform, washed with water, saturated sodium chloride solution and dried with anhydrous sodium sulphate. The solution was filtered and the solvent evaporated in vacuo to give the title compound. Purification by column chromatography on silica gel, eluting with 5% methanol/chloroform gave the title compound as a mixture of 2 diastereoisomers (0.63g) (D32) δ (CDCl₃) : 0.87 (6H, m), 1.03-2.35 (6H, m), 2.60 (1½6H, d), 2.66 (1½H,d), 2.70 (1½6H, d), 2.73 (1½H, d), 2.77-3.10 (4½4H, m), 3.22 (1H, dd), 3.60 (½H, m), 3.77 (3H, d), 3.80 (3H, d), 4.53-4.77(1H, m), 6.14 (½H, broad s), 7.10 (½H, broad s), 7.22(5H, m), 7.50(½H, d), 7.57 (½H, d).

Observed FAB (M+H)⁺ 504 C₂₂H₃₈N₃O₆PS requires M 503

Description 33

N-[N-(1-(Dimethoxyphosphinyl)-3-(methylsulphonyl)propyl)- (S)-leucyl]-(S)-phenylalanine-N-methylamide (D33)

A solution of sodium periodate (4.26g) in water (20ml) was added to N-[N-(1-(dimethoxyphosphinyl)-3-(methylmercapto)propyl)-(S)-leucyl]-(S)-phenylalanine-N- methylamide (0.98g) (D31) in methanol (50ml). The solution was stirred at room temperature for 72h and the solvent evaporated in vacuo. The residue was dissolved in

chloroform, washed with water, saturated sodium chloride solution and dried with anhydrous sodium sulphate. The solution was filtered and the solvent evaporated in vacuo to give the title compound. Purification by column chromatography on silica gel, eluting with 10% methanol/ethylacetate gave the title compound as a mixture of 2 diastereoisomers (0.52g) (D33) δ(CDCl₃) : 0.84 (6H, m), 1.02-2.40 (7H, m), 2.70 (1½H, d), 2.74 (1½H, d), 2.93 (1½H, s), 3.01 (1½H, s), 3.03 (2H, m),

3.20 (2H, m), 3.40 (½H, m), 3.58 (½H, m), 3.78 (6H, m), 4.56 (1/2H, q), 4.66 (½H, m), 5.73 (½H, d), 6.80 (½H, d), 7.23 (5H, m), 7.44 (1H, t) Observed FAB (M+H) ⁺ 520 C₂₂H₃₈N₃O₇PS requires M 519 Description 34

N-[N-(1-(Dimethoxyphosphinyl)-3-(1,8-naphthalene

dicarboximido) propyl)-(S)-leucyl]-(S)-phenylalanine-N- methylamide (D34) The title compound (0.53g) was prepared as a single

diastereoisomer (D34) from the acid (0.6g) (D21) and (S)- tryptophan-N-methylamide (0.31g) by the procedure described in Description 19. δ(CDCl₃) : 0.89 (6H, dd), 1.35 (2H, m), 1.72 (3H, m), 1.96 (1H, m), 2.71 (3H, d), 2.78 (1H, m), 3.27 (1H, dd), 3.44 (1H, dd), 3.55 (1H, m), 3.62 (3H, d), 3.75 (3H, d), 3.94 (1H, m), 4.17 (1H, m), 4.74 (1H, q), 6.42 (1H, d), 6.97 (2H, m), 7.14 (1H, d), 7.27 (1H, s), 7.62 (2H, dd), 7.77 (2H, t), 8.24 (2H, d), 8.43 (1H, broad s), 8.59 (2H, d)

Description 35

N-[N-(1-(Dimethoxyphosphinyl)-3-(1,8-naphthalene

dicarboximido)propyl)-(S)-N_ε-benzyloxycarbonyllysine-N-methylamide (D35)

The title compound (1.4g) was prepared as a single

diastereoisomer (D35) from the acid (0.95g) (D21) and (S)-N_ε-benzyloxycarbonyllysine-N-methylamide (0.42g) by the

procedure described in Description 19. δ(CDCl₃): 0.99 (6H, t), 1.32-2.04 (11H, m), 2.23 (1H, m),

2.74 (3H, d), 3.00 (1H, m), 3.73 (3H, d), 4.22-4.55 (3H, m), 5.09 (2H, s), 5.18 (1H, m), 6.50 (1H, d), 7.33 (5H, m), 7.57 (1H, d), 7.75 (2H, t), 8.23 (2H, d), 8.60 (2H,d) ³¹P δ(CDCl₃) : 29.96 m.p. 128-132°C Observed FAB (M+H)+ 742 C₃₈H₅₀N₅O₉P requires M 751

[α]_D ²² = -22.49 (c=0.98%,MeOH)

Analysis: C₃₈H₅₀N₅O₉P requires C, 60.71; H, 6.70; N, 9.32% Found C, 60.37; H, 6.47; N, 9.12%

Description 36

N-[N-(1-(Dimethoxyphosphinyl)-3-(1,8-naphthalene

dicarboximido) propyl)-(S)-leucyl]-(-)-aminoazacyclo

tridecan-2-one (D36)

The title compound (0.75g) was prepared as a single

diastereoisomer (D36) from the acid (0.6g) (D21) and (-)-3-aminoazacyclotridecan-2-one (0.3g) ([α]_D ²⁰ = -63.6 (c=l% in methanol) by the procedure described in Description 19. δ(CDCl₃): 0.95 (6H, dd), 1.28 (16H, m), 1.52 (2H, m), 1.65-2.11 (5H, m), 2.25 (1H, m), 2.85 (1H, m), 3.04 (1H, broad s), 3.62 (2H, m), 3.71 (3H, d), 3.79 (3H, d), 4.36 (2H, m), 4.54 (1H, m), 6.43 (1H, m), 7.63 (1H, d), 7.76 (2H, t), 8.22 (2H, d), 8.60 (2H, d)

Observed FAB (M+H) ⁺ 671 C₃₅H₅₁N₄O₇P requires M 670 Description 37

(S)-N_α-t-Butyloxycarbonyl-N_ε-benzyloxycarbonyllysine-N- (benzyloxycarbonyl aminoethyl) amide (D37)

The title compound (7.2g) was prepared from N_α-t-butyloxy- carbonyl-N_ε-benzyloxycarbonyllysine (5.3g) and 2- (benzyloxycarbonylamino) ethylamine(2.72g) by the procedure described in Description 19. δ(CDCl₃) : 0.90 (15H, m), 1.75 (1H, m), 3.17 (2H, q), 3.32 (4H, broad s), 4.00 (1H, q), 5.10 (4H, s), 5.39 (1H, d), 5.56 (1H, t), 6.76 (1H, broad s), 7.33 (10H, S) Description 38

N-[N-(1-(Dimethoxyphosphinyl)-3-(1,8-naphthalene

dicarboximido) propyl)-(S)-leucyl]-(S)-(N_ε-benzyloxy

carbony1)lysine-N-(benzyloxycarbonylaminoethyl)amide (D38)

N_α-t.-Butyloxycarbonyl-N_ε-benzyloxycarbonyllysine-N- (benzyloxycarbonyl aminoethyl) amide (1.22g) (D37) was dissolved in dichloromethane (20ml) and trifluoroacetic acid (15ml) added. The solution was stirred at room temperature for 2h and the solvent evaporated in vacuo. The residue was dissolved in dichloromethane (25ml) and triethylamine added to pH10 to give N_ε-benzyloxycarbonyllysine-N- (benzyloxycarbonylaminoethyl) amide. The title compound was prepared from the above amine (1.0g) and the acid (0.95g) (D21) by the procedure described in Description 19.

Purification by column chromatography on silica gel, eluting with 5% methanol/ethyl acetate gave the title compound as a single diastereoisomer (1.0g) (D38). δ(CDCl₃) : 0.93 (3H, d), 0.97 (3H, d), 1.30-2.10 (11H, m), 2.20 (1H, m), 3.15 (2H, m), 3.30 (4H, broad s), 3.64 (3H, d), 3.74 (3H, d), 4.32 (4H, m), 5.04 (4H, s), 5.15 (1H, broad s), 5.78 (1H, broad s), 7.08 (1H, broad s), 7.28 (10H, m), 7.65 (1H, d), 7.74 (2H, t), 8.22 (2H, d), 8.59 (2H, d).

Observed FAB (M+H) + 915 C_{4 6}H₅₉N₆O₁₁P requires M 914

Description 39

N-[N-(1-(Dimethoxyphosphinyl)-3-(1,8-naphthalene

dicarboximido) propyl)-(S)-leucyl]-(S)-(N_ε-benzyloxycarbonyl) lysine-N-(ethylpyrrolidine) amide (D39) The title compound was prepared from (S)-(N_ε-benzyloxy-carbonyl) lysine-N-(ethylpyrrolidine) amide (0.73g) and the acid (0.83g.) (D21) by the procedure described in

Description 19. Purification by column chromatography on silica gel, eluting with 10% methanol/chloroform gave the title compound as a single diastereoisomer (1.2g) (D39) δ(CDCl₃) : 0.97 (6H, t), 1.34-1.66 (6H, m), 1.66-2.08 (9H, m), 2.25 (1H, m), 2.58 (6H, m), 3.03 (1H, m), 3.20 (2H, q), 3.30 (2H, q), 3.69 (1H, m), 3.72 (3H, d), 3.80 (3H, d),

4.21-4.44 (2H, m), 4.50 (1H, m), 5.09 (2H, s), 5.31 (1H, t), 6.78 (1H, broad s), 7.32 (5H, m), 7.54 (1H, d), 7.74 (2H, t), 8.22 (2H, d), 8.60 (2H, d).

³¹P δ(CDCl₃) : 30.05

Observed FAB (M+H) + 835 C₄₃H₅₉N₆O₉P requires M 834 [α]_D ²² = -17.21 (c=1.00, MeOH) Description 40

(S)-N_α-t-Butyloxycarbonyl-N_ε-benzyloxycarbonyllysine-N- (ethyl-N-methylpiperazine) amide (D40)

The title compound (4.2g) was prepared from N_α-t- butyloxycarbonyl-N_ε-benzyloxycarbonyl lysine (3.8g) and 1- aminoethyl-4-methyl piperazine (1.43g) by the procedure described in Description 19. δ(CDCl₃): 1.42 (9H, s), 1.50 (7H, m), 1.80 (1H, m), 2.30 (3H, s), 2.50 (10H, m), 3.19 (2H, q), 3.23 (2H, q), 4.05 (1H, m), 4.95 (1H, m), 5.08 (2H, s), 5.17 (1H, m), 6.55 (1H, m), 7.36 (5H, s).

Observed FAB (M+H)⁺ 506 C₂₆H₄₃N₅O₅ requires M 505

Description 41 N-[N-(1-(Dimethoxyphosphinyl)-3-(1,8-naphthalene

dicarboximido) propyl)-(S)-leucyl]-(S)-(N_ε-benzyloxycarbonyl)lysine-N-(ethyl-N-methylpiperazine) amide (D41)

The title compound was prepared from (D40) (1.1g) and the acid (0.92g) (D21) by the procedure described in Description (38).

Purification by colum chromatography on silica gel, eluting with 10% methanol/ethyl acetate gave the title compound as a single diastereoisomer (D41) (1.2g). δ(CDCl₃) : 0.93 (6H, t), 1.30-2.05 (12H, m), 2.26 (3H, s), 2.40 (10H, m), 3.00 (1H, m), 3.22 (4H, m), 3.67 (1H, m), 3.73 (3H, d), 3.80 (3H, d), 4.20-4.42 (2H, m), 4.50 (1H, m), 5.06 (2H, s), 5.25 (1H, m), 6.52 (1H, broadt), 7.30 (5H, s), 7.50 (1H, d), 7.70 (2H, t), 8.20 (2H, d), 8.58 (2H, d).

³¹P δ(CDCl₃) : 29.99 Observed FAB (M+H) ⁺ 864 C₄₄H₆₂N₇O₉P requires M 863 [α]_D ²² = -18.80 (c= 0.74, MeOH) Description 42 3-[8-(7,9-dioxo-8-azaspiro[4,5]decyl)]propanol (D42) 3,3-Tetramethylene glutaric anhydride (8.4g) was dissolved in toluene (200ml) and 3-aminopropan-1-ol (4.5g) in toluene (50ml) added over 5 minutes. The solution was stirred at room temperature for 2h then heated at reflux for 3h. The solution was allowed to cool and solvent evaporated in vacuo to yield the title compound as a pale yellow oil (13.8g) (D42). δ(CDCl₃) : 1.53 (4H, m), 1.72 (6H, m), 2.65 (4H, s), 3.51 (2H, t), 3.92 (2H, t)

Description 43

3-[8-(7,9-dioxo-8-azaspiro[4,5]decyl)]propanal (D43) The title compound (3.3g) (D43) was prepared from the alcohol (3.4g) (D42) by the procedure described in Description 5. δ(CDCl₃) : 1.50 (4H, m), 1.71 (4H, m), 2.60 (4H, s), 2.64 (2H, dt), 4.12 (2H, t), 9.75 (1H, s)

Observed CI M+H 224 Description 44 N-[1-(Dimethoxyphosphinyl)-3-[8-(7,9-dioxo-8- azaspiro[4,5]decyl)] propyl]-(S)-leucine, benzyl ester (D44)

The title compound was prepared from the aldehyde (3.2g) (D43), (S)-leucine, benzyl ester p-toluene sulphonate salt (5.7g) and dimethyl trimethylsilyl phosphite (2.6g) by the procedure described in Description 20.

Purification by colum chromatography on silica gel, eluting with a mixture of 2:1 ether: ethyl acetate, increasing to 1:1 ether:ethyl acetate, gave a single diastereoisomer. Isomer D44A (2.14g) δ(CDCl₃) : 0.92 (6H, t), 1.37-1.87 (13H, m), 1.96 (1H, m), 2.57 (4H, s), 2.82 (1H, m), 3.71 (3H, d), 3.78 (3H, d), 3.83 (2H, m), 4.00 (1H, m), 5.13 (2H, m), 7.34 (5H, m)

³¹P δ(CDCl₃) : 28.74

Observed EI MH⁺ 537 [α]_D ²² = -13.33 (c=1.00, MeOH)

Analysis: C₂₇H_4ON₂O₇P.1/2H₂O requires C, 59.44; H, 7.76; N, 5.13%

Found: C, 59.42; H, 7.65; N, 4.99%

Further elution gave a mixture of 2 diastereoisomers (3.l2g) Further elution gave a slower running single diastereoisomer Isomer D44B (0.70g) δ(CDCl₃) : 0.91 (6H, dd), 1.50 (6H, broad t), 1.70 (7H, m), 1.94 (1H, m), 2.59 (4H, s), 2.93 (1H, m), 3.53 (1H, t), 3.74 (6H, d), 3.84 (1H, m), 4.00 (1H, m), 5.16 (2H, s), 7.34 (5H, m). ³¹P δ(CDCl₃) : 29.55 Observed El MH⁺ 537

[α]_D ²² = -11.47 (c=0.78, MeOH)

Analysis: C₂₇H₄₁N₂O7P.1/2H₂O requires C, 59.44; H, 7.76; N, 5.13%

Found C, 59.27; H, 7.58; N, 4.87%

Description 45

N-[1-(Dimethoxyphosphinyl)-3-[8-(7,9-dioxo-8-azaspiro[4,5]decyl)] propyl]-(S)-leucine (D45)

The--title compound was prepared as a single diastereoisomer (D45A) (1.7g) from the benzyl ester (D44A) (2.1g) by the procedure described in Description 21. δ(CDCl₃) : 0.96 (6H, dd), 1.50 (6H, m), 1.70 (6H, m), 1.74 (1H, m), 2.06 (1H, broad s), 2.60 (4H, s), 2.98 (1H, m), 3.76 (3H, d), 3.81 (3H, d), 3.90 (2H, m), 4.03 (1H, m).

³¹P δ(CDCl₃) : 29.13

Observed FAB (M+H)⁺ 447 C20H35N2O7P requires M 446

[α]_D ²² = -9.11 (c=0.48, MeOH) Similarly, the title compound was prepared as a single diastereoisomer (D45B) (0.5g) from the benzyl ester (D44B) (0.7g) δ(CDCl₃) : 0.96 (6H, dd), 1.50 (6H, broad s), 1.60-1.90 (7H, m), 2.00 (1H, m), 2.59 (4H, s), 2.92 (1H, m), 3.41 (1H, t), 3.73 (1H, m), 3.80 (3H, d), 3.84 (3H, d), 3.92 (1H, m)

Description 46 N-[1-(Dimethoxyphosphinyl)-3-[8-(7,9-dioxo-8- azaspiro[4,5]decyl)] propyl)-(S)-leucyl]-(S)-phenylalanine- N-methylamide (D46)

The title compound was prepared as a single diasteroisomer (0.43g) (D46A) from the acid (D45A) and (S)-phenylalanine-N- methylamide (0.2g) by the procedure described in Description 19. δ (CDCl₃) : 0.87 (6H, dd), 1.20 (2H, m), 1.51 (5H, m), 1.73 (6H, broad s), 1.90 (1H, m), 2.61 (4H, s), 2.73 (3H, d),

3.12 (2H, m), 3.49 (1H, m), 3.72 (3H, d), 3.77 (3H, d), 3.74 (2H, m), 3.90 (1H, m), 4.57 (1H, q), 6.32 (1H, broad d), 7.25 (5H, m), 7.48 (1H, d).

³¹P δ(CDCl₃) : 30.10

Observed FAB (M+H) ⁺ 607 C₃₀H₄₇N₄O₇P requires M 606

[α]_D ²² = -25.38 (c=0.76,MeOH)

Analysis: C₃₀H₄₇N₄O₇P1/2H₂O requires C, 58.52; H, 7.86; N, 9.10%

Found C, 58.67; H, 7.50; N; 9.17%

Similarly, the title compound was prepared as a single diastereoisomer (D46B) (0.46g) from the acid (0.5g) (D45B) and (S)-phenylalanine-N-methylamide (0.22g) δ(CDCl₃) : 0.82 (6H, t), 1.00 (1,Hm), 1.16 (1H, m), 1.50 (5H, m), 1.70 (6H, m), 1.95 (1H, m), 2.60 (4H, s), 2.70 (3H, d), 2.98 (1H, dd), 3.10 (1H, m), 3.30 (1H, dd), 3.73 (3H, d), 3.78 (3H, d), 3.85 (3H, m), 4.77 (1H, m), 7.22 (5H, m), 7.34 (1H, broad d), 7.47 (1H, d)

Description 47 N-[N-(1-(Dimethoxyphosphinyl)-3-[8-(7,9-dioxo-8-azaspiro[4,5]decyl)] propyl)-(S)-leucyl]-(S)-benzyloxycarbonyllysine-N-methylamide (D47) The title compound was prepared as a single diastereoisomer (0.5g) (D47) from the acid (0.45g) (D45A) and (S)-N_ε-benzyloxycarbonyllysine-N-methylamide (0.29g) by the

procedure described in Description 19. δ (CDCl₃) : 0.96 (6H, dd), 1.47 (8H, m), 1.75 (11H, m), 1.98 (1H, m), 2.59 (4H, s), 2.75 (3H, d), 2.80 (1H, m), 3.20 (2H, m), 3.60 (1H, t), 3.72 (3H, d), 3.79 (3H, d), 3.85 (1H, m), 4.00 (1H, m), 4.30 (1H, q), 5.09 (2H, s), 5.17 (1H, broad s), 6.40 (1H, broad d), 7.34 (5H, s), 7.44 (1H, d).

³¹P δ(CDCl₃) : 30.01

Observed FAB (M+H) ⁺ 722 C₃₅H₅₆N₅O₉P requires M 721 [α]_D ²² = -28.87 (c=0.95,MeOH)

Analysis : C₃₅H₅₆N₅O₉P1 /2H₂O requires C, 57 .72 ; H, 7 . 86 ;

N, 9.58%

Found C, 57.22; H.7.37; N, 9.54

Description 48

N-[N-(1-(Dimethoxyphosphinyl)-3-aminopropyl)-(S)-leucyl]- (S)-phenylalanine-N-methylamide (D48)

N-[N-(1-Dimethoxyphosphinyl)-3-phthalimidopropyl)-(S)- leucyl]-(S)-phenylalanine-N-methylamide (lg) (D9) was dissolved in a 0.2M methanol solution of hydrazine (50ml) and the solution stirred at room temperature for 24h. The solvent was evaporated in vacuo and the mixture treated with methanol, filtered and the solvent evaporated in vacuo to give the title compound as a mixture of 2 diastereoisomers (0.7g) (D48) which was used in the next stage without further purification.

Description 49

N-[N-(1-(Dimethoxyphosphinyl)-3-(benzyloxycarbonylamino) propyl)-(S)-leucyl]-(S)-phenylalanine-N-methylamide (D49)

N-[N-(1-(Dimethoxyphosphinyl)-3-aminoρropyl)-(S)-leucyl]- (S)-phenylalanine-N-methylamide (4.5g) (D48) was dissolved in water (20ml) and dioxan (20ml). Benzyl chloroformate (1.8ml) was added and the pH maintained at 9 - 9.5 by the addition of 10% sodium hydroxide solution. After stirring at room temperature overnight, the solution was extracted with chloroform. The organic extracts were washed with water, dried with anhydrous sodium sulphate, filtered and the solvent evaporated in vacuo to give a colourless oil.

Purification by column chromatography on silica gel, eluting with 10% methanol/ethyl acetate gave the title compound as a mixture of 2 diastereoisomers (4.7g) (D49) δ (CDCl₃) : 0.87 (6H, m), 0.96 - 2.17 (6H,m), 2.59 (1½H, d), 2.62 (1½H , d), 2.62 (1½H, d), 2.80 (1H, m), 3.00 (2H, m), 3.24 (2H, m), 3.44 (1H, m), 3.70 (6H, t), 4.66 (1H, m), 5.07 (2H, m), 5.36 (½H, broad t), 6.12 (½H, d), 6.41 (½H, broad t), 7.24 (11H, m), 7.60 (½H,d)

Observed FAB (M+H⁺) 591 C₂₉H₄₃N₄O₇P requires M 590

Example 1

N-[N-(1-Phosphono-l-(2-hydroxyphenyl)methyl)leucyl]-N,O-dimethyl-(S)-tyrosinamide (El)

The diethyl ester (D3) (0.2g) was dissolved in

dichloromethane (10ml) and treated with bromotrimethyl-silane (0.5ml). The solution was stirred at room

temperature for 4 days, methanol (20ml) was added and the solvent evaporated in vacuo to give the crude product.

Column chromatography on reverse phase silica, eluting with a gradient of 5% to 30% methanol in water gave the title compound (0.16g) as a mixture of diastereoisomers. Observed FAB (M+H) ⁺ 508. C₂₄H₃₄N₃O₇P requires M 507.

Example 2

N-[N-(1-Phosphono-3-(1,8-naphthalenedicarboximide)propyl)-(S)-leucyl]-N,O-dimethyl-(S)-tyrosinamide (E2)

The dibenzyl ester (D6A) (0.5g) was dissolved in ethanol (100 ml) and hydrogenated over 10% palladium on charcoal at atmospheric pressure for 24h. The solution was filtered and the solvent evaporated in vacuo to give the title compound as a single diastereoisomer (E2A) (0.38g).

Observed FAB (M+H)⁺ 639. C₃₂H₃₉N₄O₈P requires M 638. Similarly, the dibenzyl ester (D6B) gave the title compound as a single diastereoisomer (E2B).

Observed FAB (M+H)⁺ 639. C₃₂H₃₉N₄O₈P requires M 638. Example 3

N-[N-(1-Phosρhono-3-phthalimidopropyl)-(S)-leucyl]-(S)- phenylalanine-N-methylamide

The dimethyl ester (D9) (1g) was dissolved in

dichloromethane (10ml) and bromotrimethylsilane (1.56g) added under nitrogen at room temperature. The solution was stirred at room temperature for 3h, then refluxed for 1.5h. The solution was allowed to cool and solvent evaporated in vacuo to give a colourless oil which was dissolved in dioxan (5ml) and water (0.5ml) and refluxed for lh. The solvent was evaporated in vacuo to give the title compound as a white crystalline compound (0.7g) as a mixture of 2- diastereoisomers. δ (CD₃OD) : 0.98 (6H, m), 1.55-2.40 (5H, m), 2.65 (3H, s), 2.80-3.25 (3H, m), 3.68 (1½H, m), 3.84 (½H, m), 4.21 (½H, m), 4.43 (½H, t), 4.61 (1H, m), 7.27 (5H, m), 7.84 (4H, m)

Observed FAB (M+H)⁺ 559 C27H35N4O7P requires M.558

The dimethyl ester (D9A) (0.06g) was dissolved in

dichloromethane (10ml) and the solution cooled to -20°C.

Iodotrimethylsilane (0.1ml) was added under nitrogen at -20°C and the solution stirred for 30 min. The solution was allowed to warm to room temperature and stirred for 3h. The solvent was evaporated in vacuo and the residue dissolved in methanol (20ml) and stirred for 2h. Solvent was evaporated in vacuo to give the title compound (0.05g) as a single diastereoisomer.

δ(CD₃OD): 1.00 (6H, t), 1.70 (3H, m), 2.06 (1H, m), 2.42 (1H, m), 2.69 (3H, s), 2.83-3.20 (3H, m), 3.70 (1H, m), 3.86 (1H, m), 4.42 (1H, t), 4.68 (1H, dd), 7.30 (5H, m), 7.85 (4H, m)

Observed FAB (M+H)⁺ 559 C₂₇H₃₅N₄O₇P requires M 558 Example 4

N-[N-(1-Phosphono-4-(1,8-naphthalenedicarboximido)butyl)- (S)-leucyl]-(S)-phenylalanine-N-methylamide (E4)

The title compound (2.7g) was prepared from the dibenzyl ester (D12) (3.23g) by the procedure described in Example 2 δ (CD₃OD): 0.68 (3H, dd), 0.83 (3H, dd), 1.06-1.90 (7H, m), 2.25-3.15 (4H, m), 2.53 (1½H, s), 2.60 (1½H, s), 3.94 (1½H, m), 4.24 (½H, t), 4.50 (1H, m), 7.06 (5H, m), 7.51 (2H, t), 8.14 (2H, d), 8.22 (2H, d). Observed FAB (M+Na)⁺ 645. C₃₂H₃₉N₄O₇P requires M. 622.

Example 5

N-[N-(1-Phosphono-2-(1,8-naphthalenedicarboximido)ethyl-(S)-leucyl]-(S)-phenylalanine-N-methylamide (E5)

The title compound (0.69g) was prepared from the dibenzyl ester (D15) (1.14g) by the procedure described in Example 2, δ (CD₃OD) : 0.27 (H4H, d), 0.42 (1½H, d), 0.96 (3H, d), 0.96-1.80 (3H, m), 2.46 (1½H, s), 2.74 (1½H, s), 2.50-3.30 (3H, m), 4.14 (½H, t), 4.50 (2H, m), 4.66 (1H, m), 4.81 (½H, m), 7.17 (5H, m), 7.76 (2H, t), 8.32 (2H, t), 8.50 (2H, d). Observed FAB (M+H)⁺ 595. C₃₀H₃₅N₄O₇P requires M 594.

Example 6

N-[N-(1-Phosphono-3-(1,8-naphthalenedicarboximido)propyl)-(S)-leucyl]-(S)-phenylalanine-N-methylamide (E6) The title compound (0.63g) was prepared as a single

diastereoisomer (E6A) from the dibenzyl ester (D16A) by the procedure described in Example 2. The title compound (0.4g) was also prepared as a single diastereoisomer (E6A) from the dimethyl ester (D22) by treatment with bromotrimethylsilane (1ml) in dichloromethane (50ml) at room temperature under nitrogen for 48h. The solvent was evaporated in vacuo and the residue dissolved in methanol (25ml) and water (5ml) and stirred for 2 h. The solvent was evaporated in vacuo to give the title compound (E6A) (0.4g) as a white solid. δ (CD₃OD) : 1.02 (6H, dd), 1.78 (4H, m), 2.14 (1H, m), 2.70 (3H, s), 2.96 (1H, dd), 3.12 (1H, dd), 3.99 (1H, m), 4.10

(1H, m), 4.34 (1H, m), 4.78 (1H, dd), 7.22 (5H, m), 7.78

(2H, t), 8.34 (2H, d), 8.53 (2H, d).

³¹P: δ(CD₃OD) : 18.47

Observed FAB (M+H)⁺ 609 C₃₁H₃₇N₄O₇P requires M 608

[α]_D ²² = -25.39 (c=0.81, MeOH) Similarly, the dibenzyl ester (D16B) gave the title compound as a single diastereoisomer (E6B). δ (CD₃OD) : 1.00 (6H, m), 1.76 (3H, m), 2.03 (1H, m), 2.41

(1H, m), 2.68 (3H, s), 2.95 (2H, m), 3.14 (1H, dd), 4.16 (2H, m), 4.46 (1H, t), 4.69 (1H, dd), 7.30 (5H, m), 7.75

(2H, t), 8.30 (2H, d), 8.46 (2H, d)

Observed FAB (M+H) ⁺ 609 C31H37N4O7P requires M 608 Example 7

N- [N- (1-Phosphono-3-phβnylpropyl) - (S) -leucyl] - (S) -phenylalanine-N-methylamide (E7) The dibenzyl ester (D19A) (0.46g) was dissolved in ethanol (100 ml) and hydrogenated over 10% palladium on charcoal at atmospheric pressure for 24h. The solution was filtered and the solvent evaporated in vacuo to give the title compound, as a white solid, as a single diastereoisomer (E7A) (0.35g). δ(CD₃OD) : 0.95 (6H, dd), 1.66 (3H, m), 1.85 (1H, m), 2.14 (1H, m), 2.36 (1H, m), 2.50-2.84 (2H, m), 2.70 (3H, s), 2.92 (1H, dd), 3.07 (1H, dd), 4.40 (1H, t), 4.63 (1H, dd), 7.23 (10H, m).

[α] _D ²² = -26 .24° (c=l% in methanol )

m.p . = 147-150°C .

Observed FAB (M+H) ⁺ 490. C₂₅H₃₆N₃O₅P requires M. 489

Analysis: C₂₅H₃₆N₃O₅P.1/2H₂O requires C, 60.23; H, 7.48; N, 8.43%.

Found C, 59.84; H, 7.29; N, 8.43%.

Similarly, the title compound was prepared from the dibenzyl ester (D19B) (0.14g),as a white solid, as a single

diastereoisomer (E7B) (0.10g). δ (CD₃OD) : 0.81 (6H, dd), 1.52 (3H, m), 1.78 (1H, m), 2.07 (1H, m), 2.52 (3H, s), 2.60 (1H, m), 2.65-3.04 (4H, m), 3.91 (1H, broad s), 4.49 (1H, t), 7.13 (10H, m). [α]_D ²² = -13.71° (c=1% in methanol).

Observed FAB (M+H) ⁺ 490. C₂₅H₃₆N₃O₅P requires M 489.

Example 8

N-[N-(1-Phosphono-3-(4-bromo-1,8-naphthalenedicarboximido)-propyl)-(S)-leucyl]-(S)-phenylalanine-N-methylamide (E8) The title compound was prepared as a single diastereoisomer (0.08g) (E8A) from the dimethyl ester (0.2g) (D29A) by the procedure described in Example 6. δ (CD₃OD) : 1.02 (6H, dd), 1.76 (3H, broad d), 2.02 (1H, broad s), 2.40 (1H, m), 2.68 (3H, s), 2.84 (1H, m), 2.94 (1H, dd), 3.11 (1H, dd), 4.04 (1H, m), 4.16 (1H, m), A . A1 (1H, t), 4.67 (1H, dd), 7.30 (5H, m), 7.79 (1H, t), 7.98 (1H, d), 8.20 (1H, d), 8.45 (2H, t).

Similarly, the title compound was prepared as a single diastereoisomer (E8B) from the dimethyl ester (D29B). δ (CD₃OD) : 0.97 (6H, dd), 1.78 (4H, m), 2.16 (1H, m), 2.94 (1H, dd), 3.08 (1H, dd), 3.20 (1H, m), 3.93 (1H, broad s), 4.05 (1H, broad s), 4.47 (1H, m), 4.70 (1H, t), 7.77 (1H, t), 7.95 (1H, d), 8.20 (1H, d), 8.42 (2H, t)

Example 9

N-[N-(1-Phosphono-3-(benzyloxycarbonylamino)propyl)-(S)-leucyl]-(S)-phenylalanine-N-methylamide (E9)

The title compound was prepared from the dimethyl ester (D49) by treatment with bromotrimethylsilane for 3h as described in Example 6. δ(CD₃OD) : 0.96 (6H, m), 1.55-2.30 (5H, m), 2.70 (3H, m), 2.88-3.21 (5H, m), 3.65 (1H, m), 4.05-4.45 (1H, m), 5.10 (2H, m), 7.30 (10H, m)

Observed FAB (M+H)⁺ 563 C₂₇H₃₉N₄O₇P requires M. 562 Example 10

N-[N-(1-Phosphono-3-(2-hydroxyphenyl)propyl)-(S)-leucyl]- (S)-phenylalanine-N-methylamide (E10)

The title compound was prepared as a mixture of 2

diastereoisomers (2.5g) (E10) from the dibenzyl ester (4g) (D30) by the procedure described in Example 2. Observed FAB (M+H)⁺ 506 C₂₅H₃₆N₃O₆P requires M 505

Example 11

N-[N-(1-Phosphono-3-(methylmercapto)propyl)-(S)-leucyl]-(S)-phenylalanine-N-methylamide (E11)

The title compound was prepared as a mixture of 2

diastereoisomers (0.15g) (E11) from the dimethyl ester

(0.19g) (D31) by treatment with bromotrimethylsilane for 24h as described in Example 6.

Observed FAB (M+H) ⁺ 460 C₂₀H₃₄N₃O₅P requires M 4 Example 12

N-[N-(1-Phosphono-3-(methylsulphinyl)propyl)-(S)-leucyl]- (S)-phenylalanine-N-methylamide (E12)

The title compound was prepared as a mixture of 2

diastereoisomers (0.42g) (E12) from the dimethyl ester

(0.52g) (D32) by treatment with bromotrimethylsilane for 24h as described in Example 6. δ(CD₃OD) : 0.99 (6H, m), 1.60-2.47 (5H, m), 2.70 (3H, m), 2.77-3.53 (8H, m), 4.36 (½H, m), 4.45 (½H, m), 4.70 (1H, m), 7.31 (5H, m) Example 13

N-[N-(1-Phosphono-3-(methylsulphonyl)propyl)-(S)-leucyl]- (S)-phenylalanine-N-methylamide (E13) The title compound was prepared as a mixture of 2

diastereoisomers (0.4g) (E13) from the dimethyl ester (0.5g) (D33) by treatment with bromotrimethylsilane for 48h as described in Example 6. δ(DMSO) : 0.80 (6H, m), 1.22-1.54 (5H, m), 2.43 (1H, m), 2.50

(1½H, d), 2.57 (1½H, d), 2.84 (1H, m), 2.90 (1½H, s), 2.94

(1½H, s), 2.94-3.40 (4H, m), 4.50 (1H, m), 7.22 (5H, m),

8.06 (½H, d), 8.14 (½H, d), 8.33 (½H, d), 8.49 (½H, d) Observed FAB (M+H) ⁺ 492 C₂₀H₃₄N₃O₇PS requires M 491

Example 14

N-[N-(1-Phosphono-3-(1,8-naphthalenedicarboximido)propyl)-(S)-leucyl]-(S) tryptophan-N-methylamide (E14)

The title compound was prepared as a single diastereoisomer (0.4g) (E14) from the dimethyl ester (0.52g) (D34) by treatment with iodotrimethylsilane as described in Example 3. δ(CD₃OD) : 0.65 (3H, d), 0.73 (3H, d), 1.20 (3H, m), 1.52 (1H, m), 1.72 (1H, m), 2.00 (1H, m), 2.45 (1H, m), 2.50 (3H, s), 2.58 (1H, m), 3.45 (1H, t), 4.06 (2H, m), 4.50 (1H, dd), 6.77 (2H, m), 7.07 (1H, s), 7.10 (1H, t), 7.45 (1H, d), 7.70 (2H, t), 8.23 (2H, d), 8.42 (2H, d) ³¹P : δ (CD₃OD) : 24.58

Observed FAB (M+Na)⁺ 670 C₃₃H₃₈N₅O₇P requires M. 647 [α]_D ²² = -20.41 (c=0.8%, MeOH)

Example 15

N-[N-(1-Phosphono-3-(1,8-naphthalenedicarboximido)propyl)- (S)-leucyl-(S)-lysine-N-methylamide hydrocholoride (E15)

The title compound was prepared as a single diastereoisomer (0.62g) (E15) from the dimethyl ester (1.0g) (D35) by

treatment with bromotrimethylsilane for 48h as described in Example 6. The product was dissolved in methanol and

treated with ether/HCl to give the hydrochloride salt. δ(CD₃OD) : 0.90 (6H, t), 1.44 (2H, m), 1.54-1.80 (7H, m), 2.08 (1H, m), 2.40 (1H, m), 2.60 (3H, s), 2.92 (2H, m), 3.26 (1H, m), 4.24 (2H, m), 4.34 (1H, m), 4.45 (1H, t), 7.73 (2H, t), 8.27 (2H, d), 8.45 (2H, d)

³¹P δ(CD₃OD) : 17.00

[α]_D ²² = -9.03 (c=0.89, MeOH)

Observed FAB (M+H)⁺ 590 C₂₈H₄₀N₅O₇P requires M 589

Example 16 N-[N-(1-Phosphono-3-(1,8-naphthalenedicarboximido)propyl)- (S)-leucyl]-(-)-aminoazacyclotridecan-2-one, di-sodium salt (El6)

The title compound was prepared as a single diastereoisomer (0.6g) (E16) from the dimethyl ester (0.7g) (D36) by treatment with bromotrimethylsilane for 48h as described in Example 6. The product was dissolved in methanol/water and converted to the di-sodium salt by the addition of two equivalents of sodium hydroxide. δ(CD₃OD) : 0.98 (6H, m), 1.10-1.98 (22H, m), 2.18 (1H, broad S), 2.68 (2H, m), 3.63 (1H, q), 4.30 (3H, m), 7.75 (2H, q), 8.24 (2H, t), 8.46 (2H, t)

Observed FAB (M+H)⁺ 687 C₃₃H₄₇N₄O₇PNa₂ requires M 686

Example 17

N-[N-(1-Phosphono-3-(1,8-naphthalenedicarboximido)propyl)- (S)-leucyl]-(S)-lysine-N-(aminoethyl) amide dihydrochloride (E17) The dimethyl ester (0.4g) (D38) was dissolved in

dichloromethane (40ml) and a 1.0M ether solution of hydrogen chloride (1.2ml) added. Bromotrimethylsilane (4ml) was added, the solution stirred at room temperature for 48h and the solvent evaporated in vacuo. The residue was dissolved in methanol (20ml) and the solution stirred for 2h. The addition of acetone gave the title compound (0.75g) as a single diastereoisomer (E17). δ (CD₃OD) : 1.02 (3H, d), 1.05 (3H, d), 1.47-2.03 (10H, m), 2.20 (1H, m), 2.54 (1H, m), 3.08 (4H, m), 3.37-3.63 (3H, m), 4.40 (2H, m), 4.58 (1H, t), 7.88 (2H, t), 8.41 (2H, d), 8.62(2H, d) Observed FAB (M+H)⁺ 619 C₂₉H₄₃N_gO₇P requires M 618

Example 18 N-[N-(1-Phosphono-3-(1,8-naphthalenedicarboximido)propyl)- (S)-leucyl]-(S)-lysine-N-(ethylpyrrolidine) amide

dihydrochloride (E18)

The title compound was prepared as a single diastereoisomer (0.4g) (E18) from the dimethyl ester (0.6g) (D39) by the procedure described in Example 17. δ(CD₃OD) : 1.03 (3H, d), 1.06 (3H, d), 1.50-2.22 (13H, m), 2.55 (1H, m), 3.03 (2H, q), 3.12 (2H, m), 3.32 (4H, m), 3.54 (1H, m), 3.66 (1H, m), 3.75 (2H, m), 4.34 (2H, m), 4.62 (1H, t), 7.87 (2H, t), 8.40 (2H, d), 8.50 (2H, d)

³¹P δ(CD₃OD) : 15.71 Observed FAB (M+H)⁺ 673 C₃₃H₄₉N₆O₉P requires M. 672

Example 19

N-[N-(1-Phosphono-3-(1,8-naphthalenedicarboximido)propyl)-(S)-leucyl]-(S)-lysine-N-(ethyl-N-methylpiperazine) amide trihydrochloride (E19)

The title compound was prepared as a single diastereoisomer (0.9g) (E19) from the dimethyl ester (1.1g) (D41) by the procedure described in Example 17. δ(CD₃OD) : 1.03 (6H, dd), 1.55 (2H, m), 1.69-2.03 (7H, m), 2.25 (1H, m), 2.53 (1H, m), 3.01 (1H, t), 3.05 (3H, s), 3.50 (4H, m), 3.65 (1H, m), 3.74 (1H, m), 3.81 (8H, broad s), 4.31-4.47 (4H, m), 4.55 (1H, t), 7.82 (2H, t), 8.35 (1H, d), 8.54 (1H, d)

³¹p δ(CD₃OD) : 17.45 Observed FAB (M+H) ⁺ 702 C₃₄H₅₂N₇O₇P requires M 701 [α]_D ²² = +1.79 (c=0.78, MeOH)

Example 20

N-[N-(1-Phosρhono-3-[8-(7,9-dioxo-8-azaspiro[4,5]decyl)]- propyl)-(S)-leucyl]-(S)-phenylalanine-N-methylamide (E20)

The title compound was prepared as a single diastereoisomer (0.37g) (E20A) from the dimethyl ester (0.4g) (D46A) by treatment witn bromotrimethylsilane for 48h as described in Example 6. δ(CD₃OD) : 0.97 (6H, d), 1.50 (4H, m), 1.72 (8H, m), 2.17 (1H, m), 2.65 (4H, s) , 2.69 (3H, s), 2.75 (1H, m) 2.98 (1H, m), 3.12 (1H, m), 3.77 (2H, m), 4.39 (1H, q), 4.68 (1H, q), 7.28 (5H, m).

³¹P δ(CD₃OD) : 18.54

Observed FAB (M+H)⁺ 579 C₂₈H₄₃N₄O₇P requires M 578 [α]_D ²² = -23.45 (c=0.64, MeOH)

Similarly, the title compound was prepared as a single diastereoisomer (0.41g) (E20B) from the dimethyl ester (0.46g) (D46B)

Example 21

N-[N-(1-Phosphono-3-[8-(7,9-dioxo-8-azaspiro[4,5]decyl)]-propyl)-(S)-leucyl]-(S)-lysine-N-methylamide hydrochloride (E21) The title compound was prepared as a single diastereoisomer (0.3g) (E21) from the dimethyl ester (0.5g) (D47) by the procedure described in Example 17. δ (CD₃OD) : 1.02 (6H, t), 1.55 (6H, m), 1.66-1.87 (11H, m), 1.95 (1H, m), 2.28 (1H, m), 2.72 (4H, s), 2.74 (3H, s), 3.00 (2H, t), 3.23 (1H, m), 3.90 (1H, m), 4.09 (1H, m), 4.42 (1H, q), 4.49 (1H, t) ³¹P δ(CD₃OD) : 18.04

Observed FAB (M+H)⁺ 560 C₂₅H₄₆N₅O₇P requires M.559

[α]_D ²² = -16.72- (c=0.66, MeOH)

Example 22

Pharmaceutical compositions for oral administration may be prepared by combining the following:

1) Solid Dosage Formulation

% w/w

Compound of formula 1 10%

Magnesium stearate 0.5%

Starch 2.0%

HPM cellulose 1.0%

Microcrystalline cellulose 86.5%

The mixture may be compressed to tablets, or filled into hard gelatin capsules.

The tablet may be coated by applying a suspension of film former (e.g. HPM cellulose), pigment (e.g. titanium dioxide) and plasticiser (e.g. diethyl phthalate) and drying the film by evaporation of the solvent. The film coat can comprise 2.0% to 6.0% of the tablet weight, preferably about 3.0%.

2) Capsule

%w/w

Compound of formula 1 20%

Polyethylene glycol 80%

The medicinal compound is dispersed or dissolved in the liquid carrier, with a thickening agent added, if required. The formulation is then enclosed in a soft gelatin capsule by suitable technology. Example 23

A pharmaceutical composition for parenteral administration may be prepared by combining the following:

Preferred Level

Compound of formula 1 1.0%

Saline 99.0% The solution is sterilised and sealed in sterile containers

COLLAGENASE INHIBITOR ASSAY

The test is performed essentially as in Cawston and Barrett, Anal. Biochem. 99, 340-345 (1979). Compounds for testing are dissolved in methanol by sonication and added to

collagenase (purified from culture supernatants from the human lung fibroblast cell line, WI-38) in buffer. After a 5 min pre-incubation at 37°C, the assay tubes are cooled to 4°C and ³H-acetylated rat skin type I collagen is added.

The assay tubes are incubated at 37°C overnight. The ³H-collagen forms insoluble fibrils, which are the substrate for the enzyme.

To terminate the assay, the assay tubes are spun at 12000 rpm for 15 minutes. Undigested ³H-collagen is pelleted, while digested ³H-collagen is found as soluble peptides in the supernatant. A sample of the supernatant is taken for liquid scintillation counting. The activity of collagenase inhibitors (IC₅₀: 50% inhibitory concentration) is expressed as that concentration of

compound that inhibits a known (standard) concentration of enzyme by 50%. The compounds of Examples 2-12 had IC₅₀ values in the range 2.3 × 10^-6 - 1.3 × 10^-8 M.

Claims

Claims:

A compound of the formula (I) or a salt thereof

in which,

R₁ is -(CH₂)_n-W where n is 0-6 and W is amino,

optionally-substituted phenyl, -CONR₅R₆, -NR₅COR₆,

NR₅CO₂CH₂R₆ or NR₅CONR₅R₆ where R₅ is hydrogen or C_1-6 alkyl and R₆ is hydrogen, C_1-6 alkyl, optionally-substituted phenyl or heteroaryl, or R5 and Rg together with the

nitrogen atom to which they are bonded form a 5-, 6- or 7-membered ring with an optional oxygen or sulphur atom or an optionally substituted second nitrogen atom in the ring; or W is -S(O)p-R₇ where p is 0, 1 or 2 and R₇ is C_1-6alkyl; or W is a group of sub-formula (a), (b), (c) or (d):

—

where in sub-formula (a), (b) and (c) A represents an optionally-substituted mono or bicyclic aryl or heteroaryl ring, and in sub-formula (c) and (d) q is an integer from 1 to 3;

R₂ is C_3-6 alkyl;

R₃ is hydrogen, C_1-6alkyl, -CH₂-Z where Z is optionally substituted phenyl or heteroaryl, -(CH₂)_rNR₈R₉,

-(CH₂)_rNHCOR₁₀, -(CH₂)_rNR₁₁C (=NR₁₂)NR₈R₉,

-(CH₂)_rCONH(CH₂)_sNR₈R₉ or -(CH₂)_r-R₁₃ where r is 1 to 6, s is 2 to 4, each of R₈ and R₉ is independently hydrogen or C_1-6alkyl or R8 and R₉ together with the nitrogen atom to which they are bonded form a 5-, 6- or 7-membered ring with an optional oxygen or sulphur atom or an optionally

substituted second nitrogen atom in the ring, R₁₀ is

C_1-6alkyl or -(CH₂)_tNR₈R₉ where t is 1 or 2 and R₈ and R₉ are as defined above, R₁₁ is hydrogen or C_1-6alkyl or R₁₁ and R₈, together with the nitrogen atoms to which they are bonded, form an optionally substituted 5-, 6- or 7-membered ring, R₁₂ is hydrogen or C_1-6alkyl and R₁₃ is an optionally substituted piperidyl ring or R₃ is a group:

where R₁₄ is hydrogen, C_1-6alkyl or optionally substituted benzyl and R₁₅ is hydrogen or C_1-6alkyl; and R₄ is hydrogen, C_1-6alkyl or -(CH₂)_rNR₈R₉ in which r, R₉ and R₉ are as defined for R₃; or R₃ and R₄ are joined together as -(CH₂)_m- where m is an integer from 4 to 12, or R₃ and R₄ are joined as - (CH₂) _x-NR₁₆- (CH₂) _y- where x is an integer from 1 to 9, y is an integer from 2 to 10, and the moiety -(CH₂)_x- is adjacent to the carbon atom bearing R₃ marked with an asterisk in formula (I), and R₁₆ is selected from hydrogen, C_1-6alkyl, C_2-6alkanoyl, C_1-6alkoxycarbonyl, aroyl, aralkyl or aralkyloxycarbonyl in each of which the aryl moiety is optionally substituted.

2. A compound according to claim 1 in which R₁ is 2- hydroxyphenyl, -(CH₂)_n-w where n is 2 and W is amino, phenyl, 2-hydroxyphenyl, NHCO₂CH₂Ph, N-phthalimido, 4-bromo-1,8-naphthalenedicarboxamido, 7,9-dioxo-8-azaspiro[4,5]decyl, methylmercapto, methylsulphinyl or methylsulphonyl, or R₁ is -(CH₂)_n-W where n is 1, 2 or 3 and W is a 1, 8-naphthalenedicarboxamido group.

3. A compound according to claim 1 or 2 in which R₂ is n- butyl, iso-butyl or sec-butyl.

4. A compound according to any one of claims 1 to 3 in which R₃ is benzyl, C_1-6alkylamino, 4-methoxybenzyl,

-(CH₂)₄NH₂ or 3-indolylmethyl and R₄ is methyl or

-(CH₂)₂NR₈R₉ where R₈ and R₉ are both hydrogen or together with the nitrogen atom to which they are bonded, form a pyrrolidine or N-methylpiperazine group, or R₃ and R₄ are combined to form a group -(CH₂)₁₀-.

5. A compound according to any one of claims 1 to 5 in which the chiral centres marked with an asterisk in formula (I) have the (S)-configuration when R₃ is other than

hydrogen.

6. A compound according to claim 1 which is:

N-[N-(1-phosphono-1-(2-hydroxyphenyl)methyl)-leucyl]

-N,O-dimethyl-(S)-tyrosinamide,

N-[N-(1-phosphono-3-(1,8-naphthalenedicarboximide)propyl)- (S)-leucyl]-N,O-dimethyl-(S)-tyrosinamide,

N-[N-(1-phosphono-3-phthalimidopropyl)-(S)-leucyl]-(S)-phenylalanine-N-methylamide,

N-[N-(1-phosphono-4-(1,8-naphthalenedicarboximido)butyl)- (S)-leucyl]-(S)-phenylalanine-N-methylamide,

N-[N-(1-phosphono-2-(1,8-naphthalenedicarboximido)ethyl-(S)-leucyl]-(S)-phenylalanine-N-methylamide,

N-[N-(1-phosphono-3-(1,8-naphthalenedicarboximido)propyl)- (S)-leucyl]-(S)-phenylalanine-N-methylamide, N-[N-(1-phosphono-3-phenylpropyl)-(S)-leucyl]-(S)- phenylalanine-N-methylamide,

N-[N-(1-phosphono-3-(4-bromo-1,8-naphthalene- dicarboximido)propyl)-(S)-leucyl]-(S)-phenylalanine

methylamide,

N-[N-(1-phosphono-3-(benzyloxycarbonylamino)propyl)-(S)- leucyl]-(S)-phenylalaninemethylamide,

N-[N-(1-phosρhono-3-(2-hydroxyphenyl)propyl)-(S)-leucyl]-(S)- phenylalaninemethylamide,

N-[N-(1-phosphono-3-(methylmercapto)propyl)-(S)-leucyl]-(S)- phenylalanine-N-methylamide,

N-[N-(1-phosphono-3-(methylsulphinyl)propyl)-(S)-leucyl]-(S)- phenylalanine-N-methylamide,

N-[N-(1-phosphono-3-(methylsulphonyl)propyl-(S)-leucyl]-(S)- phenylalanine-N-methylamide,

N-[N-(1-phosphono-3-(1,8-naphthalenedicarboximido)propyl)- (S)-leucyl]-(S) tryptophan-N-methylamide,

N-[N-(1-phosphono-3-(1,8-naphthalenedicarboximido)propyl)- (S)-leucyl-(S)-lysine-N-methylamide,

N-[N-(1-phosphono-3-(1,8-naphthalenedicarboximido)propyl)- (S)-leucyl]-(-)-aminoazacyclotridecan-2-one,

N-[N-(1-phosphono-3-(1,8-naphthalenedicarboximido)propyl)- (S)-leucyl]-(S)-lysine-N-(aminoethyl) amide,

N-[N-(1-phosphono-3-(1,8-naphthalenedicarboximido)propyl)- (S)-leucyl]-(S)-lysine-N-(ethylpyrrolidine) amide,

N- [N- (1-phosphono-3- (1, 8-naphthalenedicarboximido) propyl) - (S)-leucyl]-(S)-lysine-N-(ethyl-N-methylpiperazine) amide, N-[N-(1-phosphono-3-[8-(7,9-dioxo-8-azaspiro[4,5]decyl)]-propyl)-(S)-leucyl]-(S)-phenylalanine-N-methylamide, or

N- [N- (1-phosphono-3- [8- (7, 9-dioxo-8-azaspiro [4, 5] decyl) ] -propyl)-(S)-leucyl]-(S)-lysine-N-methylamide,

and pharmaceutically acceptable salts thereof.

7. A process for the preparation of a compound according to claim 1 which process comprises cleaving a group R20 from a compound of formula (II):