AU637903B2 - Phosphonopeptides with collagenase inhibiting activity - Google Patents

Description

OPI DATE 30/10/91 APPLN. ID 76502 91 AOJP DATE 05/12/91

PCT

PCT NUMBER PCT/GB91/00529 INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (51) International Patent Classification 5 (11) International Publication Number: WO 91/15506 C07K 5/06, A61K 37/64 Al (43) International Publication Date: 17 October 1991 (17.10.91) (21) International Application Number: PCT/GB91/00529 (74)Agents: RUSSELL, Brian, John et al.; SmithKline Beecham, Corporate Patents, Great Burgh, Yew Tree Bot- (22) International Filing Date: 5 April 1991 (05.04.91) tom Road, Epsom, Surrey KT18 5XQ (GB).

Priority data: (81) Designated States: AT (European patent), AU, BE (Euro- 9008065.6 10 April 1990 (10.04.90) GB pean patent), CA, CH (European patent), DE (European patent), DK (European patent), ES (European patent), FR (European patent), GB (European patent), GR (71) Applicant (for all designated States except US): BEECHAM (European patent), IT (European patent), JP, KR, LU GROUP P.L.C. [GB/GB]; SB House, Great West Rcad, (European patent), NL (European patent), SE (Euro- Br,ntford, Middl.esex T8. 9DD pean patent), US.

Fowr" A'e.w l4ik^s A Ca$t Nel flOLX4tll.n/" (72) Inventors; and re ./tj d /Add4/e.s e TW bPi s /d k'a 1t Inventors/Applicants (for US only) MARKWELL, Roger, Published Edward [GB/GB]; RAHMAN, Shahzad, Sharooq [BD/ Wi;h international search report.

GB]; WARD, Robert, William [GB/GB]; SmithKline Before the expiration of the time limit for amending the Beecham Pharmaceuticals, Coldharbour Road, The Pin- claims and to be republished in the event of the receipt of nacles, Harlow, Essex CM19 SAD amendments.

'i lo (54) Title: PHOSPHONOPEPTIDES WITH COLLAGENASE INHIBITING ACTIVITY R R 2 1 2 o

O

1 R 4 0-P N N (I) OR H H OR 0 R3 (57) Abstract Azalactam derivatives processes for their preparation and their use as collagenase inhibitors having structure WO 9115506PCT/GB9 1/00529 PHOSPHONOPEPTIDES WITH COLLAGENASE INHIBITING ACTIVITY The poresent 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 neutr~l metalloproteases, for treating arthritic and other diseases.

The mammalian collagenase family of enzymes comprises a number of proteases, exemplified by interstitial (type t collagenase itself, the stromelysins (also known as proteoglycanases or transins), fibroblast and polymorphonuclear l~~eucocyte gelatinases (also known as collagen-IV-ases), and 'pump-i' (putative metalloprotease 1, uterine metalloprotease). Membership of the mammalian collagenase family of proteases is evident by possession of a number of b~ighly characteristic and experimcntally verifiable properties. [Goldberg et al., J. Biol. Chem.

2610, 6600, 1986; Whitham et al., Biochem. 3. 240, 913, 1986; Breathnach et al., Nucleic Acids Res., 139, 1987; Muller et al., Biochem. 253, 187, 1986; Collie= etal., 3. Biol. Chem., 263, 6579, 1988; Murphy et al., Biochem. 258, 463, 1989; Quantin et al., Biochem.

28, 5327, 1989; Birkedal-Hansen, 3. Oral Pathol., 7,445, 1988).

The range of therapeutic applications of the inventic;:.

described hereinafter reflects the fundamental role ,F collagen and other proteinaceous substrates of th~e czllagenase family of e-nzvme.s in the connective tissue matrix throughouat the body. Appli1cations extend to clin-ical interventions in many diseases and Phenomena invol-ving the destruction of collagen and other conneczive tissue components, and also normal or di1-soraerec tissue remodelling.

WO 91/15506 PCIT/GB91/00529 -2- Inhibitors of the collagenase family of enzymes are considered to provide useful treatments for: arthritic diseases, such as rheumatoid and osteoarthritis, soft tissue rheumatism, polychondritis and tendonitis; bone resorption diseases, such as osteoporosis, Paget's disease, hyperparathyroidism and cholesteatoma; the enhanced collagen destruction that occurs in association with diabetes; the recessive classes of dystrophic epidermolysis bullosa; periodontal disease and related consequences of gingival production of collagenase, or of PMNL collagenase release following cellular infiltration to inflamed gingiva, including by combating the greater susceptibility of diabetes patients to periodontal disease; corneal ulceration, e.g. that induced by alkali or other burns, by radiation, by vitamin E or retinoid deficiency; ulceration of the skin and gastro-intestinal tract, and abnormal wound healing; postoperative conditions, including colonic anastomosis, in which collagenase levels are raised; cancer, where members of the collagenase family of enzymes have been implicated in the neovascularization required to support tumour growth and survival Basset et al. Nature, 348, 699, 1990] in the tissue remodelling required to accommodate the growing primary and secondary tumours, and in the penetration of tumour cells through the basement membrane of the vascular walls during metastasis; and demyelinating diseases of the central and peripheral nervous systems, including syndromes in which myelin loss is the primary pathological event and those in which demyelination follows axonal atrophy. The degradation of myelin in these diseases, exemplified by multiple sclerosis, is mediated by members of the collagenase family of enzymes.

WO 91/15506 PCT/GB91/00529 3 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 family of Tissue Inhibitor of Metalloproteases (TIMPS), and alpha-2 macroglobulin. An imbalance between the local levels of the proteolytic enzymes and 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 WO 91/15506 PCT/GB91/00529 4 effective than endogenous proteinaceous inhibitors that are excluded by their size from the localized regions of connective tissue destruction.

European Patent Application 88310492.9 (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 structurally related compounds 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 or a salt, solvate or hydrate thereof:

R

1 R OH 0 OR H H

R

3

(I)

in which, R is hydrogen, C1-6 alkyl or optionally substituted benzyl; R, is hydrogen or C.i_ alkyl; WO 91/15506 PCT/GB91/00529 5 R2 is C3- 6 alxyl; and R3 and R4 are joined together as -(CH2)p-X-(CH2)q- where p is an integer from 1 to 9, q is an integer from 2 to and the moiety -(CH 2 p is adjacent to the carbon atom bearing R 3 marked with an asterisk in formula and X is -NR 5 where R5 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.

Unless otherwise specified, each alkyl group is preferably a C 1 -8 group, more preferably C 1 and may be a straight chain or branched. An aryl moiety is preferably phenyl.

Optional substituents for an aryl moiety may be selected from OH, C 1 -6 alkyl, C1-6 alkoxy and halogen.

R is preferably hydrogen, methyl, ethyl or benzyl, especially hydrogen.

Values for R1 include hydrogen, methyl, ethyl, isopropyl and n-butyl. As an alkyl group, R 1 is pre'Lrably methyl or ethyl.

R

2 is preferably a C 4 alkyl group, such as n-butyl, iso-butyl or sec-butyl, especially iso-butyl.

Values for R 3 and R 4 together include -(CH2)p-X-(CH2) c where p and q have values such that R3 and R 4 form part of an 11- or 13 to 16-membered azalactam structure, and X is

-NR

5 where R 5 is hydrogen, methyl, benzyl, t-butoxycarbonyl or benzyloxycarbonyl.

WO 91/15506 PCT/GB91/00529 6 Most preferred are compounds where R 3 and R4 are joined together as -(CH 2 )p-X-(CH 2 where p is 4 and q is 5 or p is 3 and q is 6 or p is 4 and q is 6 or p is 4 and q is 3 and X is -NR 5 where R5 is hydrogen or methyl.

The compounds of formula may form salts with bases e.g. sodium hydroxide. The compounds of formula have a basic nitrogen atom and may form acid addition salts e.g. with hydrochloric acid. Such compounds form part of the present invention.

Where compounds of formula or salts thereof, form solvates or hydrates, these also form an aspect of the invention.

The compounds of formula 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-configuration at the chiral centre bearing R 2 and the S-configuration at the chiral centre bearing R 3 marked with an asterisk in formula The compounds of formula or their salts, solvates or hydrates are preferably in pharmaceutically acceptable form. By pharmaceutically acceptable form is meant, inter alia, of a pharmaceutically acceptable level of purity excluding normal pharmaceutical additives such as diluents and carriers, and including no material considered toxic at normal dosage levels.

WO 91/15506 PCT/GB91/00529 7 The compounds of formula or their salts, solvates or hydrates are preferably in substantially pure form.

A substantially pure form will generally contain at least by weight, preferably 75%, more preferably 90% and still more preferably 95% or 99% or more of the compound of formula I or its salt or solvate.

Compounds of formula or their salts, solvates or hydrates may be isolated as crystalline solids or in the form of foams or gums.

A preferred pharmaceutically acceptable form is the crystalline form.

The present invention provides the compounds of formula or pharmaceutically acceptable salts, solvates or hydrates 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 also have potential utility in the treatment of cancer; for preventing myelin degradation in the central and peripheral nervous system; and in other conditions in which members of the collagenase family of neutral metalloproteases have pathological or other roles.

The present invention also provides a process for the preparation of a compound of formula which comprises converting a group R 20 to hydrogen by cleaving a group R 20 from a compound of formula (II): WO 91/15506 PCT/GB91/00529 8-

R

1

R

O 202 N R4

OR

2 1

H

R

3

(II)

wherein R 2 0 is alkyl, optionally substituted phenyl, or optionally substituted benzyl and R 21 is hydrogen, alkyl, optionally substituted phenyl, or optionally substituted benzyl and R 1

R

2

R

3 and R 4 are as defined in formula and where necessary, converting R 21 to hydrogen.

Cleavage of R 20 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 or acetonitrile.

Benzyl esters may alternatively be removed by hydrogenolysis or other standard debenzylation procedures.

Phenyl residues may be removed by hydrogenation over platinum oxide.

When both R20 and R21 are alkyl, cleavage of R 2 0 only, to give to a compound of formula (II) in which R20 is hydrogen and R 21 alkyl, which is a compound of formula (I) in which R is alkyl, 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 alkyl, the benzyl group only may be cleaved by WO 91/155T06 PC/GB91/00529 9 hydrogenation to give a compound of formula (II) in which

R

20 is hydrogen and R 2 1 is alkyl.

Cleavage of an R 21 alkyl group may thereafter be carried out as described above to give a compound of formula (I) in which R is hydrogen.

When R in a compound of formula is hydrogen and R 21 in a compound of formula (II) is not hydrogen, then cleavage of both R 21 and R20 is conveniently effected in a single reaction. Preferably R 20 and R21 are both alkyl, such as methyl or ethyl, or benzyl.

It will be appreciated that compounds of formula (II) in which R 21 is hydrogen are themselves compounds of the invention of formula Compounds of formula (II) may be prepared by treating a compound of formula (III):

R

1

R

2 OR21 0 P H O

OR

21 (III) in which R 1

R

2

R

20 and R21 are as defined in formula (II) (except that R21 is not with a compound of formula (IV):

O

2

N

HN NHR 4

R

.3

(IV)

WO 91/15506 PCIGB91/00529 10 in which R 3 and R 4 together are as defined in formula The reaction is preferably carried out in the presence of a coupling agent,, such as dicyclohexylcarbodiimide or (dimethylamino) propyl] carbodiimide hydi';hloride in the presence of 1-hydroxybenzotriazole, or using 1,l'-carbonyldiimidazole, in an inert solvent such as dichloromethane or acetonitrile.

Selective cleavage of the group R 21 may then be carried out using the procedures described above for the preparation of compounds of formula to give compounds of formula (II) in which R2 1 is hydrogen.

The intermediate compounds of formula (III) may be prepared by treating a compound of formula or a salt thereof: 0 P NH 2 2

OR

2 1

(V)

in which R 1

R

2 0 and R21 are as defined in formula with a compound of formula (VIA) or (VIB) or a salt thereof:

R

2

R

2 R11 C0 2

R

12 0 C0 2

R

12 (VIA) (VIB) WO 91/15506 PCT/GB91/00529 11 in which R2 is as defined in formula R 11 is a leaving group such as halogen, methanesulphonyloxy or trifluoromethanesulphonyloxy and R12 is hydrogen or a carboxyl protecting group, and thereafter removing an R12 carboxyl protecting group. Preferred method is the reaction of with When a compound of formula (VIB) 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 and (VIB) in which any of R 20

R

21 or R12 '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 or aqueous potassium hydroxide in 1,4-dioxane.

When the compound of formula is in the form of the free base, the compound of formula (VIB) is suitably an a-keto ester (R 12 alkyl).

When the compound of formula is a salt, such as the hycrochloride salt, the compound of formula (VIB) is suitably a salt of an a-keto acid (R 12 for example the sodium salt.

WO 91/15506 PCT/GB9l/00529 12 The preparation of compounds of formula (III) using a compound of formula (VIA) 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 (VII) or a salt thereof: R2

H

2 N C02 1 2

(VII)

in which R2 is as defined in formula and R12 is a carboxyl protecting group with an aldehyde, R 1 -CHO in which R1 is as defined in formula and treating the condensation product with an appropriate dialkyl or trialkyl phosphite, for example dimethyl 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 (VIB), where a benzyl protecting group R 12 is removed by hydrogenation then R20 and R21 are restricted to alkyl.

Alternatively, compounds of formula (II) in which R20 and R21 are alkyl or optionally substituted benzyl may be prepared by the reaction of a compound of formula (VIII): WO 91/15506 PCT/GB91/00529 13 R2 o

H

2 N NH R 4

R

3

(VIII)

in which R 2

R

3 and R 4 are as defined in formula with a compound of formula (IX):

R

1

OR

2 0 O^ 11

OR

(IX)

in which R 1 is as defined in formula R20 and R21 are alkyl, optionally substituted phenyl, or optionally substituted benzyl and R 11 is a leaving group as defined for formula (VIA), 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 R11 is an oxygen-based leaving group, for example trifluoromethanesulphonyloxy, which is preferred, displacement of the .eaving group is conveniently carriec 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.

WO 91/15506 PCT/GB91/00529 14 A further alternative preparation of compounds of formula (III) may be carried out by reacting a compound of formula (IX) as hereinbefore defined with a compound of formula (VII) in which RL2 is a carboxyl protecting group, using conditions as described for the reaction of compounds of formula (VIII) with compounds of -formula and thereafter removing the protecting group R 12 Suitable carboxyl protecting groups include alkyl, benzyl, trialkylsilyl and trialkylsilylethyl 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 R 12 alkyl carboxyl protecting group may be removed by base hydrolysis, for example using sodium hydroxide in aqueous methanol or potassium hydroxide in aqueous 1,4dioxane.

It will be appreciated that where the carboxyl protecting group R 12 is alkyl, R 20 and R 21 may be alkyl, phenyl or benzyl derivatives, but where R12 is a benzyl group, and P 21 are limited to alkyl and phenyl.

When compounds of formula (III) are prepared by this route, it is preferred that R20 and R21 are benzyl and R,.

is trifluoromethanesulphonyloxy in the compound of formula (IX) and R 12 is trimethylsily) or methyl in the compound of formula (VII).

WOP 91/15506 PCT/GB91/00529 15 Compounds of formula (VIII) may be prepared by treating a compound of formula (VII): R2 1

H

2 N C0 2

R

12

(VII)

in which R2 is as defined in formula R 12 is hydrogen 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 (IX) may be prepared from hydroxyalkylphosphonate derivatives by conversion of the hydroxyl group to the leaving group R 11 by conventional methods.

For example, where R11 is trifluoromethanesulphonyloxy, 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 R 1 -CHO in which R, is as defined in formula 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.

WO 91/15506 PCr/GB9/1/00529 16 Intermediate compounds of formula are either known compounds or may be prepared from known aminoalkyl phosphonic acid derivatives using standard procedures to introduce R20 and R 21 as required.

Protection of the amine function during these reactions may be necessary.

Introduction of an R20 or R 21 methyl group may be effected by reaction with diazomethane in a suitable inert solvent.

Compounds of formula of fixed configuration may be prepared by the general method of R. Jacquier et al., Phosphorus and Sulfur 36, 73, (1988).

Compounds of formula (IV) may be prepared by oxidising the primary alcohol function in a compound of formula

Y

H NH H Z-N- (CH 2 )p N-(CH 2 )q-OH 0

(X)

wherein p and q are as defined in formula Y is a nitrogen protection group, and Z is R5 to give the corresponding aldehyde, followed by removal of Z where Rg is an acyl group; cyclisation and reduction; and thereafter, as necessary, removing the nitrogen protection group Y and interconverting R 5 WO 91/15506 PCT/GB91/00529 17 Suitable nitrogen protection groups include t-butoxycarbonyl (BOC) and benzyloxycarbonyl groups.

The oxidation may be carried out using pyridinium chlorochromate, or under Swern oxidising conditions, for example by treatment with dimethylsulphoxide and an acyl halide followed by triethylamine, as described by D. Swern et al., J. Org. Chem., 43, 2480 (1978). The cyclisation and reductive amination step may be effected by catalytic hydrogenation over a suitable noble metal catalyst, for example palladium on carbon, or by reaction with sodium cyanoborohydride or sodium borohydride. In some cases the yield of azalactams may be increased by carrying out the reductive amination step under acidic conditions.

Nitrogen protection groups may be removed by standard methods. A t-butoxycarbonyl group may be removed by treatment with trifluoroacetic acid at reduced temperature. Where Z is a nitrogen protection group, it may be selected to undergo concomitant cleavage during the cyclisation reaction to give a compound in which R5 is hydrogen. For example, when Z is a benzyloxycarbonyl group, it will be readily removed by catalytic hydrogenation.

An R 5 hydrogen may be interconverted to an R 5 Cl..

6 alkyl, aralkyl or aryl group. The secondary amine group in the azalactam ring may be alkylated to form an R 5 alkyl group.

For example, the amine group may be methylated to form an

R

5 methyl group. The methylation step may be effected by catalytic hydrogenation over a suitable noble metal catalyst, for example palladium on carbon, in the presence of aqueous formaldehyde. Other suitable methylation procedures are described by E. Askitoglu et al., Helv.

WO 91/15506 PCT/GB91/00529 18 Chim. Acta., 68, 750, (1985); E. Engler et al., Helv.

Chim. Acta., 68, 789, (1985); and M. Lennon et al., J.

Chem. Soc. (Perkin 622, (1975).

Compounds of formula may be prepared by reacting a compound of formula (XI):

Y

L

H NH

Z-N-(CH

2 )p OH

(XI)

wherein p, Y and Z are as defined for formula with a compound of formula (XII): H2N-(CH 2 )q-OH (XII) wherein q is as defined for formula The reaction may be carried out using standard procedures for forming an amide from a carboxylic acid and an amine, for example using a coupling agent such as 1,1'carbonyldiimidazole, 1,3-dicyclohexylcarbodiimide or 1-(3dimethylaminopropyl)-3-ethylcarbodiimide.

Compounds of formula (XI) are di-aminoalkanoic acid derivatives. These are known compounds or may be prepared from known starting materials by standard methods.

For example the compound of formula (IV) in which R3 and

R

4 together are -(CH2)p-X-(CH 2 q where p is 3, q is 6 and WO 91/15506 PCT/GB91/00529 19 X is -NH- is prepared frm,. a compound of formula (XI) derived from ornithine which is commercially available.

The compounds of 'formula (IV) in which R 3 and R 4 together are -(CH 2 )p-X-(CH 2 where p is 4, q is 3, 5 or 6 and X is -NH- are prepared from a compound of formula (XI) derived from the amino acid lysine. The compound of formula derived from S-lysine, in which Y is t-butoxycarbonyl and Z is benzyloxycarbonyl, is commercially available.

Similarly, the compound of formula (IV) in which R 3 and R 4 together are -(CH 2 )p-X-(CH 2 where p is 1, q is 8 and X is -NH- may be prepared from 2,3-diaminopropionic acid.

The compounds of formula (VII) are either known amino acid derivatives or can be made from these derivatives by known methods. Compounds of formula (VIA) and (VIB) are either known compounds or may be prepared from known compounds by known methods.

The intermediates of formulae (III), and certain intermediates of formula 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 may be formed conventionally by reaction with the appropriate acid or base. Solvates may be formed by crystallization from the appropriate solvent.

WO 91/15506 PCT/GB9i/00529 20 As mentioned previously, the compounds of formula (I) exist in more than one diastereoisomeric form. Where the processes of the invention produce mixtures thereof, the individual isomers may be separated one from another by chromatography e.g. HPLC.

Alternatively, separate diastereoisomeric compounds of formula 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 It will be appreciated that where a single diastereoisomer of a compound of formula 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 chira-l centre may not be known, it is possible to characterise a given diastereoisomer relative to its epimer by reference to the direction in which the plane of polarised light is rotated.

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

*;VO 91/15506 PCT/GB91/00529 21 A composition of this invention is useful in the treatment of musculo-skeletal disorders, particularly arthritic diseases and for modulation of tissue remodelling.

A composition of the invention also has potential utility in the treatment of cancer; for preventing myelin degradation in the central and peripheral nervous system; and in other conditions in which members of the collagenase family of neutral metalloproteases have pathological or other roles.

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, subcutaneously or into the cerebro-spinal fluid.

Preferably, a pharmaceutical composition of the invention is in unit dosage fortr 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.

WO 91/15506 PC~T/GB91/00529 22 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 em'nying large quantities of fillers. When the composition is in the WO 91/15506 PCT/GB91/00529 23 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 accordinr 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 microcrystalline 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 nonaqueous 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.

WO 01/15506 PCT/GB91/00529 24 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 injrctable form. For injection, for example by intraarticular injection or by injection into the cerebrospinal 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 pyrogenfree water or a parenterally acceptable oil or a mixture of liquids, which may contain bacteriostatic agents, antioxidants 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 WO 91/15506 PCT/GB91/00529 25 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 mulSi.ples, 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, such as rheumatism and/or arthritic conditions in mammals, such as humans, which comprises administering to the mammal in need of such treatment an effective amount of a compound of formula or a pharmaceutically acceptable salt thereof.

The present invention also provides the use of a compound of formula '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 such as rheumatism and/or arthritic conditions.

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

WO 91/15506 WO 9115506PCT'/GB91/00529 -26 Description 1 Dibenzyl (1-hydroxypropyl) phosphonate (DOJ.

0 (PhCH 2

O)

2 P IT OH

C

2

H

The general method of F. Texier-Boullet and A. Foucaud [Synthesis, 1982, 916] was employed. A mixture of dibenzyl phosphite (31.13 ml, 0.14 mole) and propionaldehyde (10.21 ml, 1 equiv.) was stirred at room temperature and basic alumina (70g) added in one portion.

After standing overnight at room temperature chloroform was added and the alumina collected and washed with chloroform. The filtrate was evaporated to dryness and the resulting clear oil chromatographed on silica gel (600g) with gradi-.4cr elution (ether 5% methanol/ether).

The title compound was obtained as a clear oil which solidified on standing (27.82g, A sample was recrystallized from ether/pentane to give a white crystalline solid, m.p. 81-82'C.

Found: C, 64.09; H,6.71. C 17

H

21 0 4

P

1 requirez C, 63.74; H, 6.61%.

5(CDCl 3 1.04(3H,t,J=7Hz), 1.6-1.95(2H,m), 2.21(lH, brs), 3.8(1H, 2 overlapping triplets,J=5 and 4. 97-5.18 (4H, m) 7.34 (10H,s) WO 91/15506 PCT/GB91/00529 27 Description 2 Dibenzyl ((l-trifluoromethanesulphonvloxy)propyl)phosphonate (D2) 0 (PhCHzO) 2 P 0 CF 3

C

2

H

5 0 The title compound was prepared by the general method of E. Vedejs et al. Org. Chem. 1985, 50(12), 2165]. A solution of dibenzyl (l-hydroxypropyl)phos-phonate (Dl) (24.97g, 0.078 mole) in methylene chloride (180 ml) was cooled to -50 0 C under N 2 2,6-Lutidine (11.12 ml, 0.0?5 mole) was added followed by trifluoro- methanesulphonic anhydride (15.1 ml, 0.0898 mole) keeping the temperature -50 0 C. The mixture was allowed to warm slowly to 0°C and then taken into cold ether. The solution was subjected to a rapid aqueous work-up by washing the organic layer with ice-cold water, dilute hydrochloric acid (x2) and finally brine. The organic layer was dried (anhydrous MgSO 4 and evaporated to dryness to give the title compound as a pinkish orange oil (33.77g, 96%) which was used without further purification.

(CDC1 3 1.08(3H,t,J=7Hz), 1.88(2H,m), 4.94(1H,2 overlapping triplets,J=5.5 and 7Hz), 4.88-5.22(4H,m) and 7.35(10H,m).

WO 91/15506 PCT/GB91/00529 28 Description 3 N-(1-(R)-Dibenzyloxyphosphinylpropyl)-(S)-leucine (D3A) and N-(1-(S)-Dibenzyloxyphosphinylpropyl)-(S) -leucine (D3B)

CH

3

CH

3 (PhCH 2

O)

2 P N C0 2

H

H

Method A Following the general method of US 4808741 for the preparation of leucine trimethylsilyl ester a mixture of (S)-leucine 1.15g, 0.0088 mole), hexamethyldisilazane (1.75 ml), and triethylamine (1.38 ml) in acetonitrile (13.5 ml.) was heated at reflux for a total of 4h.

Dibenzyl ((l-trifluoromethanesulphonyloxy)propyl)phosphonate (D2) (4.5g, 0.01 mole) was then added and the mixture maintained at 40-42 0 C for 48h. The reaction can also be carried out at ambient temperature. After cooling the mixture was filtered, washed with methanol and the filtrate evaporated to dryness. The residue was taken up in chloroform and washed with dilute HC1 (x2) and finally water. The chloroform layer was dried (anhydrous Na 2

SO

4 filtered and evaporated to dryness to give an orange gummy solid (3.67g). The crude product was trituratied with the minimum volume of ether/pentane to give a white crystalline solid which after collection, washing with a little cold ether/pentane and drying gave the title compound, R,S isomer (D3A) (0.47g, m.p. 112-115 0

C.

WO 91/15506 WO 9115506PCTrkB91/00529 29 Observed Desorption CI (NH 3 MH 434. C 23

H

32

NO

5 P requires M 433.

EaD0= -23.09' (c=0.97 MeOH) Found: C,63.73; H,7.42; N,3.23. C 3 3 N0 5 P requires C,63.73; H,-7.44; N,3.23%.

S(CDC1 3 0.89 (6H,t) 1.03 (3E,t) 1.25-2.0 2.74 3.28 br 3.73 (1H, br 4.9-5.15 (4H, mn), 7.35 (10H, s).

The other isomer, (S)-dibenzyloxyphosphinyJlpropyl)- (S)-leucine (D3B), can be obtained by preparative HPLC using a Hamilton PRP-1 column, 300 x 7.0mm, 264R with a 40:60 acetonitrile:water eJluent mixture and a flow rate of 4.0 mi/min. Under these conditions the R,S isomer (D3A) elutes first with a retention time of 34.6 min and the S,S isomer (D3B) is well separated at 42.7 min.

For the isomer (D3B): Observed FAB 434. C 23 11 32 N0 5 P requires M 433.

8 (CDC,'l] 3 0.88 (GH,dd), 0.98 1.4 1.52-1.9 (4H,in), 2.72 3.38 4.9-5.15 7.32 (1OH,s).

The S,S isomer (D3B) on coupling with (S)-ainino acid derivatives leads to the S,S,S, series.

Method B A mixture of (S)-leucine methyl ester hydrochloride (0.543g; 0.003 mole), dibenzyl (1-trifluoromethanesulphonyloxy)propyl)phosphonate (D2) (1.35g; 0.003 mole) and anhydrous potassium carbonate (1.0g) in methanol (2 ml) was heated at 50 0 C, with stirring, for 4 hours and then left at room temperature overnight.

WO 91/15506 PCT/GB91/00529 30 The reaction mixture was evaporated to dryness in vacuo, and dissolved in chloroform (5 ml) and filtered. The filtrate, and washings, were combined and chromatographed on silica gel 60 ,(50g) using ethyl acetate-pentane (1:1) as the eluent, to afford a mixture of N-(l-(R)-dibenzyloxyphosphinylpropyl)-(S)-leucine methyl ester and N-(1-(S)-dibenzyloxyphosphinylpropyl)-(S)-leucine methyl ester as an oil (0.55g). The esters can be separated into individual diastereomers by column chromatography on silica gel with initially 50% diethyl ether/pentane as eluent, rising to 100% diethyl ether.

The above mixture of esters (l.lg, 0.0025 mole) in methanol (4.0 ml) was treated with a solution of sodium hydroxide (0.llg; 0.00275 mole) in water (1.5 ml), and the solution was stirred at room temperature overnight. It was evaporated to one third volume, in vacuo,-taken in water and extracted with ether. The aqueous fraction was acidified with citric acid to pH 3-4 and then extracted (5x) with chloroform. The chloroform fraction was dried (Na 2

SO

4 and evaporated to dryness in vacuo to give a mixture of the title compounds (D3A) and (D3B) as an oil that slowly solidified.

Trituration of the product with ether gave N-(l-(R)-dibenzyl-oxyphosphinylpropyl)-(S)-leucine (D3A) (0.34g) as a white crystalline solid, identical to the product obtained by Method A.

Alternatively, the single isomer can be hydrolysed separately. For example N-(1-(S)-dibenzyloxyphosphinylpropyl)-(S)-leucine methyl ester on hydrolysis by the above method gave N-(1-(S)-dibenzyloxyphosphinylpropyl)-(S)-leucine (D3B), m.p. 71-73 0

C.

WO091/15506PC/B9/52 PCr/GB91/00529 31 Descrixption 4 N(X-te rt -But oxvcarbonyl -ben z y oxycarbonyl -lysine- hydroxy) Pentylam' de (D4) H3C

H

H3 N .N CH 2 Ph 100 To a solution of Natr-uoyabnlN-ezl oxycarbonyl-(S)-lysine (7.8g, 21 mmol) in anhydrous dichioromethane (150 ml) maintained at 0 0 C was added 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (4.3g, 22.5 mmol) and 1-hydroxybenzotriazole (3.6g, 26.5 mmole). Tw mix~ture was stirred for 0.5h at 0 0 C, 5-aminopentan--o. (A"K3g, 22.5 mmol) added and stirring continued at room temperature. After 3h the mixture was washed with saturated aqueous NaHCO 3 (60 ml) dried over anhydrous magnesium sulphate and evaporated in vacuo to afford a viscous oil. Purification by flash chromatography [(CHC1 3 :MeOH) (20:1) v/v) gave the titl~a compound (D4) as a clear oil (8.O1g).

Observed 466. C 24

H

39 0 6

N

3 requires M 465.

WO 91/15506 PC'/GB91/00529 32 Description N -tert-Butoxycarbonyl-N-benzyloxycarbonyl- lysine-(4-formyl)butylamide H O H

H

3 C ON O C CH 3 0 N OCH 2 Ph 0 To a stirred solution of oxalyl chloride (1.47g, 12 mmol) in anhydrous dichloromethane (40 ml) maintained under an atmosphere of nitrogen at -60 0 C was added dimethylsulphoxide (1.21g, 15 mmol) dropwise, such that the temperature remained below -50aC. The mixture was left stirring at -60 0 C for 15 minutes, alcohol (D4) (3.6g, 7.7 mmol) diluted in anhydrous dichloromethane (10 ml) was added, and allowed to warm up to -25 0 C over lh. The mixture was then cooled down to -60 0 C, triethylamine (4.7g, 46 mmol) added slowly such that the internal temperature remained below -50 0 C. On completion of addition, the mixture was gradually warmed up to room temperature, washed with water (30 ml) and sat., aq. NaC1 (30 ml). The aqueous washes were back extracted with dichloromethane (2x30 ml) and the combined organic fractions were dried over anhydrous magnesium sulphate and evaporated in vacuo to yield a viscous clear oil.

Purification by flash chromatography [(EtOAc:MeOH) (20:l)v/v] afforded the title compound as an oil (2.8g).

Observed (M+H) 464. C 24

H

37 0 6

N

3 requires M 463.

WO 91/15506 PCFT/GB91/0529 33 Description 6 (S)-3-(N-tert-Butoxycarbonyl)amino-8-(N-benzyloxycarbonyl)-1,8-diazacvclotridecan-2-one (D6) H O H3c°

H

0 OCHzPh Method A The aldeL, e (D5) (1.8g, 3.88 mmol) was dissolved in ethanol (180 ml) and hydrogenated over 5% palladium on charcoal (200 mg) at atmospheric pressure and 35 0 C for 72h. The suspension was filtered through Kieselguhr and evaporated in vacuo to give crude 3-[N-tert-butoxycarbonyl]amino-(S)-1,8-diazacyclotridecan-2-one. The crude amine was dissolved in a mixed solvent system of tetrahydrofuran/water, (6:20 ml) v/v cooled to o0C and treated with benzylchloroformate (0.66g, 3.88 mmol) and excess sodium carbonate to maintain a pH between 10 and 11. The mixture was left stirring at room temperature overnight, washed with ethyl acetate (3x25 ml), and the combined organic fractions dried over anhydrous magnesium sulphate and evaporated in vacuo to afford a clear oil.

Purification by flash chromatography [(EtOAc:MeOH) (20:1)v/v] yielded the title compound (D6) as a white solid (0.2g).

Observed M 447. C 24

H

37 0 5

N

3 requires M 447.

WO 91/15506 P~/GB91/00529 34 Method B The aldehyde (D5) was hydrogenated at about 100 psi over palladium on charcoal in methanol, and then in acidic methanol to afford crude (S)-3-[N-tertbutoxycarbonyl)amino-1,8-diazacyclotridecan-2-one.

The amine was treated with benzylchloroformate and purified as descried in Method A to yield the identical title compound (D6).

Description 7 (S)-3-Amino-8-[N-benzyloxycarbonvl -1,8-diazacyclotridecan-2-one, trifluoroacetate salt (D7) H 0 TFA. H N O OCH2Ph A cooled solution of the lactam (D6) (0.19g, 0.42 mmol) in dichloromethane (5 ml) was treated with trifluoroacetic acid (2 ml). After 0,5h the solvent was evaporated under reduced pressure, the residue diluted with dichloromethane (15 ml) and washed with sat. aq. NaCl ml). The organic fraction was dried over anhydrous magnesium sulphate and evaporated in vacuo to give crude title compound (D7) as an oil. This was used as such without further purification.

WO 91/15506 PCT/GB91/00529 35 Description 8 N- N-(R)-(1-Phosphonopropyl)-(S)-leucyll amino- 8-(N-benzvloxvcarbonyl)-1,8-diazacvclotridecan-2-one, dibenzvl ester (D8)

CH

3

C

2

H

5

H

N

o H O OCH2Ph A solution of N-(1-(R)-dibenzyloxyphosphinylpropyl)- (S)-leucine (D3A) (0.194g, 0.45 mmol) in anhydrous dichloromethane (20 ml) maintained at O0C was treated with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (0.085g, 0.45 mmol) and 1-hydroxybenzotriazole (0.079g, 0.58 mmol). After stirring for the reaction mixture was sequentially treated with lactam (D7) (0.15g) and N,N-diisopropyl-ethylamine (0.12g, 0.89 mmol). Stirring was continued for 18h at room temperature, then the mixture was washed with sat. aq.

NaHCO 3 (2x20 ml), and sat. aq. NaC1 (2x20 ml). The aqueous washes were back-extracted with dichloromethane and the combined organic fractions dried over anhydrous magnesium sulphate, and evaporated in vacuo to yield an oil. On purification by flash chromatography methanol in chloroform), the title compound (D8) was obtained as a clear oil (0.110g).

Observed (M+H) 763. C 42

H

59

N

4 0 7 P requires M 762.

WO 91/15506 WO 9115506PCr/GB91 100529 36 8 l.2(3H,t), 1.25-l.93(18H,m), 2.70(lHi,m), 2.90(lH,rn), 3.18(2H,m), 3.39(2H,dt), 3.62(2H,M), 4.23(lH,m), 4.92-5.12(6H,m), 6.0(2.H,brs), 7.28-7.42 Description 9 Natr-uoyabnl-6bnvoyabnl -ornithine- (6-hydroxy) hexv lamide (D9)

H

3 C~ TH 0~

Z~X

CH3 0 H OQH 2 Ph

H

A solution of Natr-uoyabnl-cbnyoyabnl (S)-ornithine (12g, 0.033 mci) in anhydrous dichioromethane (200 ml) maintained at 0 0 C, was treated sequentially with 1- (3-dimethylaminopropyl) -3ethylcarbodiimide hydrochloride (7.5g, 0.039 mol), and 2.hydroxybenzotriazole (5.3g, 0.039 mol). The solution was stirred at 0 0 C for 1 h, treated with 6-aminohexan-l-ol (4.6 g, 0.039 mol) and left stirring overnight at room temperature. The mixture was then washed with saturated aqueous NaHCO 3 dried over anhydrous magnesium sulphate, and evaporated in vacuo to afford a viscous oil.

Purification by flash chromatography [(CHCl 3 :MeQH) gave the title compound (D9) as a clear oil (9.1g).

observed 466. C 24

H

39 0 6

N

3 requires M 465.

WO 91/15506 PTG9/02 PCr/GB91/00529 37 8(CDCl 3 1.25-l.4(5H,m), l.42-l.62(71H,m), l.78(1H,m), 3.l-3.35(4H,m), 3.6 4.12(111,m), 5.08 (2H, br 5.31(lH,t), 5.42(1H,d), 6.73(lH,brs), 7.3.(5H,m).

Description Natr-uo~eroy-cbnyovabnl -ornithine- )pentyvlamide

H

3

C

0

H

0H

H

The alcohol (D9) (6.5g, 13.9 immol) dissolved in dichioromethane (50 ml) was added to a vigorously stirred mixture of pyridinium chlorochromate (9g, 41.8 mmol) and 4A molecular sieves (20g) in dichloromethane (200 ml).

Additional portions of pyridinium chlorochromate (4g) were added after 30 minutes and,45 minutes. After 1h total reaction time, the mixture was poured into ether (200 ml) and the reaction flask rinsed with ether (3x100 ml) The combined organic fractions were filtered through kieselguhr, and concentrated in vacuo to afford a yellow oil. Purification by flash chromatography E(EtOAc:MeOH) (49;1)v/v then (20:1)v/v] yielded the title compound (D1O) as a viscous oil Observed 464. C 24

H

37

N

3 0 6 requires M 463.

WO 91/15506 PCT/GB91/00529 38 Description 11 (S)-3-(N-tert-Butoxycarbonyl)amino-7-(Nbenzyloxycarbonyl)-1,7-diazacyclotridecan-2-one (D11) H O

H

3 C O N N H3C C 0

N

O OCHPh The aldehyde (D10) (2.5g) in methanol (300 ml) was treated with 5% palladium on charcoal The suspension was hydrogenated at 100 psi and ambient temperature for 48h, treated with 2.5M aqueous hydrochloric acid (2 mi) and hydrogenation continued at the said pressure for a further 24h. The suspension was filtered through kieselguhr and evaporated in vacuo to give crude 3-(N-tert-butoxycarbonyl)amino-(S)-1,7-diazacyclotridecan-2-one. The crude amine was dissolved in a mixed solvent system of tetrahydrofuran/water, (10:40 ml) v/v cooled to 0°C and treated with benzylchloroformate (0.92g) and excess sodium carbonate to maintain a pH between 10 and 11. The mixture was left stirring at room temperature for 4h, solvent partly evaporated in vacuo and the residue extracted with dichloromethane (3x50 ml). The combined organic fractions were dried over anhydrous magnesium sulphate and evaporated in vacuo t& afford a clear oil. Purification by flash chromatography [(EtOAc:MeOH) (50:1) v/v] yielded the title compound (D11) as a white solid (0.2'7g).

Observed M 447. C 2 4

H

3 7 0 5

N

3 requires M 447.

WO 91/15506 WO 9115596PCT/GB91/00529 39 Description 12 -3--Axino-'7- Nbnvoyarovl17 diazacyclotridecan-2-one, trifluoroacetate salt (D12) HO0

TFA.

0 OCH 2 Ph A cooled (O 0 C) solution of the lactam (Dll) (0.13g) in dichloromethane (5 ml) was treated with trifluoroacetic acid (3 ml) After lh the solvent was evaporated under reduced pressure, to afford crude title compound (D12) as an oil,, This was used as such without further purification.

Description 13 -3-tN- (-Phosphonopropyl) S) -leucyll Iamino-7- (N-benzyioxvcarbonyl) 7-diazacyclotridecan-2-one, dibenzyl ester (D13)

CH

3

CH

3 CA H 0 (PhC.1H 2

O)

2 P NH 0 'OMH 2 Ph A solution of N-(1-(R)-dibenzylokcyphosphinylpropyl)- leucine (D3A) (0.115g) in anhydrous dichloromethane ml) maintained at 0 0 C was treated with 1- (3- WO 91/15506 WO 9115596PCI'/GB91/00529 40 dirnethylaminopropyl) -3-ethylcarbodiimide hydrochloride (0.056g) and 1-hydroxybenzotriazole (0.036g) After stirring for 0.5h the reaction mixture was sequentially treated with the 'crude lactam (D12), N,N-diisopropylethylamine (0..075g) and stirring continued at room temperature for 4h. 'The mixture was washed with water ml) and sat. aq. NaHCO 3 (2x20 ml). The aqueous washes were back-extracted with dichloromethane and the combined organic fractions dried over anhydrous magnesium sulphate and evaporated in vacuo to yield an oil. On purification by flash chromatography methanol in chloroform), the title compound (D13) was obtained as a white foam (0.125g).

Observed 763. C 42

H

59

N

4 0 7 P requires M 762.

Description 14 Natr-uovabnl-ebnvovabnl -lysine- (6hYdroxy)hexYlamide_(Dl4)

H

3

C

0

H

H

3 C~ 0p H

CH

3 0 N ><rOCH 2 Ph 0 A solution of Na-tert-butoxycarbonyl-NF--benzyloxycarbonyl-- (S)-lysine (15.8g, 0.042 mol) in anhydrous dichioromethane (200 ml) maintained at 0 0 C, was treated sequentially with 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (9.96g, 0.051 mol) and 1-hydroxybenzotriazole (7.0g, 0.051 mol) The solution was stirred WO 91/15506 WO91/5506PCT/GB9I /00529 41 at 000 for 1h, treated with 6-aminohexan-l-o. (4.7g, 0.046 mol), and left stirring overnight at room temperature.

The mixture was then washed with saturated aqueous NaHCO 3 dried over anhydrous magnesium szalphate, and evaporated in vacuo to afford a viscous oil. Purification by flash chromatography ICHCl 3 :MeOH) (20:1) v/v gave the title compound (D14) as a clear oil (16'g).

Observed 480. C 25

H

41

N

3 0 6 requires M 479.

Descrintion Nater ;u xcroy1-6bnz oyabn -lysine- formyl)pentylamide

H

3 C H 01 CH3,.- N OCH 2 Ph 0 A stirred solution of dimethyl sulphoxide (3.63g, 0.046 mol) in anhydrous dichloromethane (2.00 ml) maintained at 0 C, was treated with oxalylchloride (2.58g, 0.0198 mol) diluted in dichloromethane (10 ml) at such a rate, so as to ensure temperature remained below -500C. After stirring for 20 minutes, the alcohol (D14) (6.35g, 0.013 mol) dissolved in dichloromethane (50 ml) was added dropwise over 5 mins. The mixture was stirred at -6500C for 15 mins, warmed up to -35 0 C, stirred -41or a further mins then cooled down to -600C. The solution was treated with triethylamine (8g, 0.08 mol), warmed up to room WO 91/15506 PCT/GB91/00529 42 temperature, washed with water (2x100 ml), dried over anhydrous magnesium sulphate and solvent evaporated under reduced pressure to afford a viscous oil. Purification by flash chromatography [(EtOAc:MeOH) (30:1) v/v] gave the title compound (D15) as an oil Observed (M+H) 478. C 25

H

39

N

3 0 6 requires M 477.

Description 16 (S)-3-(N-tert-Butoxycarbonyl)amino-7-(Nbenzyloxvcarbonvy)-1,8-diazacyclotetradecan-2-one (D16) T O

H

3 CXO "WN <KN

H

3 C C"0 3H O OCH 2 Ph The aldehyde (D15) (5.0g) in methanol (450 ml) was treated with 5% palladium on charcoal The suspension was hydrogenated at 140 psi and ambient temperature for 48h, treated with 2.5M aqueous hydrochloric acid (3 ml) and hydrogenation continued at the said pressure for a further 24h. The suspension was filtered through Kieselguhr and evaporated in vacuo to give crude (S)-3-(N-tert-butoxycarbonyl)amino-1,8-diazacyclotetradecan-2-one. The crude amine was dissolved in a mixed solvent system of tetrahydrofuran/water, (10:40 ml) v/v cooled to 0°C and treated with benzylchloroformate (2.85g) and excess sodium carbonate to maintain a pH between 10 and 11. The mixture was left stirring at room temperature for 4h, solvent WO 91/15506 W091/5506PCT/GB9I /00529 43 partially evaporated in vacuo and the residue extracted with dichloromethane (3xlOO ml). The organic fraction was dried over anhydrous magnesium sulphate and evaporated in vacuo to afford a clear oil. Purification by flash chromatography [(EtOAc:MeOH) (50:1) v/vI yielded the title compound.(D16) as a white solid (2.0g) m.p. 131.5-134.0'C.

Observed M+r 461. C 25

H

39

N

3 0 5 requires M 461.

Description 17 -3-Amino-B- rN-benzvloxvcarbonvl3-1, 8diazacyclotetredd.can-2-one, trifluoroacetate salt (D17) H1 C TMA. i4 I'L 'N

H

N

0 OCH 2 Ph A cooled (0 0 C) solution of the lactam (D16) (0.175g) in dichioromethane (5mi) was treated with trifluoroacetic acid (3 ml) After lh the solvent was evaporated under reduced pressure, to afford crude title compound (D17) as an oil. This was used as such without further purification.

WO 91/15506 PCT/GB91/00529 44 Description 18 (1-Phosphonopropyl) -leucylli amino-8- (N-benzvloxycarbonyl)-1, 8-diazacvclotetradecan-2-one, dibenzyl ester (D18) 0M3

C

2

H

5

H

(PhCH 2 O)P N 1 HN H

N

O OCH 2 Ph A solution of N-(l-(R)-dibenzyloxyphosphinylpropyl)-(S)leucine (D3A) (0.133g) in anhydrous dichloromethane (10 ml) maintained at 0 C was treated with 1-(3dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (0.065g) and l-hydroxybenzotriazole (0.046g). After stirring for 0.5h the reaction mixture was sequentially treated with the crude lactam (D17) and N,Ndiisopropylethylamine (0.1g) and stirring continued at room temperature for 4h. The mixture was washed with water (10 ml) and sat.aq. NaHCO 3 (2x10 ml). The aqueous washes were back-extracted with dichloromethane and the combined organic fractions dried over anhydrous magnesium sulphate and evaporated in vacuo to yield an oil. On purification by flash chromatography methanol in chloroform), the title compound (D18) was obtained as a white foam (170 mg).

Observed (M+H) 777. C 43

H

61

N

4 0 7 P requires M 776.

WO 91/15506 PCT/GB91/00529 45 Description 19 (S)-3-(N-tert-Butoxycarbonyl)amino-8-(N-methyl)-1,8diazacyclotridecan-2-one (D19) H O

H

3 C 0

H

CH3 N 1o CC) A solution of diazalactam (D6) (0.35g) in methanol (20 ml) was treated with 5% palladium on charcoal (0.2g) and hydrogenated for 24h at atmospheric pressure and room temperature. The suspension was filtered through kieselguhr, and the filtrate diluted with methanol to a -total volume of 50 ml. The solution was treated with palladium on charcoal (0.3g) followed by 40% aqueous formaldehyde (1 ml), and the resulting suspension hydrogenated for 48h at a pressure of 100 psi. The suspension was filtered through Kieselguhr, solvent evaporated in vacuo, to yield an oil which on exposure to diethyl ether solidified. Purification by flash chromatography [(CHCl 3 :MeOH:NH 3 (90:9:0.5) v/v] afforded the title compound (D19) as a pale yellow solid (0.14g).

m.p. 135-137 0

C.

Observed M 327. C 17

H

33

N

3 0 3 requires M 327.

WO 91/15506 PCT/GB91/00529 46 Description (S)-3-Amino-8-fN-methyll-1,8-diazacyclotridecan-2-one, trifluoroacetate salt

H

0

TFA.H

H N N

CH

3 A cooled solution of the diazalactam (D19) (0.175g) in dichioromethane (5 ml) was treated dropwise with trifluoroacetic acid (3 ml). After lh the solvent was evaporated under reduced pressure to afford crude title compound (D20) as an oil. This was used as such without further purification.

Description 21 rN- (I-Phosphonopropyl) -leucyl 11amino- (N-methyl)-1,8-diazacyclotridecan-2-one, dibenzyl ester (D21) a-i 3 C Hc H, H O (PhCH2.0)2 N f" mH(h~l0

N

0 C VH3 A solution of the N-(l-(R)-dibenzyloxyphosphinylpropyl)- (S)-leucine (D3A) (0.133g) in anhydrous dichloromethane ml) maintained at OOC was treated with 1-(3- WO 91/15506 WO 9115506PCT/GR01i/00529 4~7 dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (0.065g) and 1-hydroxybenzotriazole (0.046g). The mixture was allowed to stir for 0.5h at room temperature, then treated with amine (D20) dissolved in dichioromethane (210 ml), followed by N,N-diisopropylethylamine (0.1g).

After stirring for 18h, the mixture was washed with sat.

aqueous NaCl (2x10 ml), dried over anhydrous magnesium sulphate and solvent evaporated in vacuc to afford an oil.

Purification by flash chromatography methanol in chloroform) gave the title compound (D21) as a white foam (0.17g).

Observed 643. C 35

H

55

N

4 0 5 P requires M 642.

Descition 22 Natr-uov bnlN-bnvovabnl -lysine- (3hydroxy),propylamide (D22) H~ 0

H

3 C CH N ORP 0 A stirred solution of Na-tert-butoxycarbonyl-NEbenzyloxycarbonyl- -lysine (17.2g) maintained at 0 0 C in anhydrous dichloromethane (250 ml) was treated with l-(3dimethylaminopropyl) -3-ethylcarbodiimide hydroch.Loride (7.5g) and l-hydroxybenzotriazole The soluition was stirred at 0 0 C for lh, treated with 3-aminopropan-1-ol (4.08g) dissolved in dichloromethane (50 ml) and left WO 91/15506 W091/5506PCT/GB91 /00529 48 stirring for 18h at room temperature. The solution was then washed with sat. aqueous NaHCO 3 dried over anhydrous magnesium sulphate, and evaporated in vacuo to afford. a viscous oil. Purification by flash chromatography E(EtOAc:MeOH) (49:1) v/vJ gave the title compound (D22) as a clear oil (15..2g).

Observed 438. C 22

H

35

N

3 0 6 requires t4 437.

Description 23 Natr-uovabnlN-'bnyovabnl -lysine- (2formvl) ethylamide (D23)

H

3 C 0 H H EC'H N -,(OCH 2 Ph 0 The t.itle compound (D23) was prepared following the pro'cedure as described for the synthesis of N(X-ter-tbutoxycarbonyl-NE-benzyloxycarbonyl- -lysi4ne- (4formyl)butylamide (DS) (yield: 59%).

Observed 436. C 22

H

33

N

3 0 6 requires M 435.

WO 91/15506 PCr/GB91/00529 49 Description 24 (N-tert-Butoxvcarbonvl) amino-8- (Nbenzvloxvcarbonvl) 8-diazaccloundecan-2-one (D24)- C HH3 0 H 0 0

N

CH3 CJJ3 H N C2P 0 The title compound (D24) was prepared following the procedure described for the synthesis of (S)-3-(N-tertbutoxycarbonyl)amino-7-(N-benzyloxy-carbonyl)-1,7diLz acyclotridecan-2-one (D11) (Yield m.p. 172- 174 0

C.

Description -3-Amino-B- rN-benzvloxycarbonvll 8-diazacycloundecan- 2-one, trifluoroacetate salt

HO

TFA H' I N OCH2Ph The title compound (D25) was prepared following the procedure described for the synthesis of (S)-3~-amino-7-[Nbenzyloxycarbonyl-1,7-diazacyclo-tridecan-2-one, trifluoroacetate salt D12), and used without purification.

WO 91/15506 W091/5506PCI/GB9I /00529 Description 26 (-Phosphonopropyl) (S)-leucyll Iamino-8- (N-benzvloxycarbonyl) -diazacycloundecan-2-one, dibenzyl ester (D6

CH

3

C

2 11 5 H 0 (PhCH 2

Q)

2 P NN 00 OGH 2 Ph The title compound (D26) was prepared following the procedure described for the synthesis of (1-phosphonopropyl)-(S)-leucyl) ]amino-7- (Nbenzyloxycarbonyl) 7-diazacyclotridecan-2-one, dibenzyl ester (D13) (Yield: 44%).

Observed FAB (MvI((hC 2 0) 2 pO3+ 473. C 40

H

55

N

4 0 7 P requires M 734.

Description 27 CS) (-Phosphonopropyl) -leucyll Iamino-B- (N-benzvloxycarbonvl) 8-diazacyclotridecan-2-one, dibenzyl ester (D27)

CH

3

CH

3

C

2

H

5 H 0 2 P' NI II0

N-

0 0 CH 2 Ph WO 91/15506 PCT/GB91/00529 51 A solution of N-(1-(S)-dibenzyloxyphosphinylpropyl)-(S)leucine (D3B) (0.27g) in anhydrous dichloromethane (10 ml) maintained at o0C was treated with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (0.125g) and 1hydroxybenzotriazole (0.088g). After stirring for the reaction mixture was sequentially treated with the lactam trifluoroacetate salt (D7) (0.75 mmol) and N,Ndiisopropylethylamine (0.16g). Stirring was continued for 18h at room temperature, then the mixture was washed wji:h sat.aq. NaHCO 3 (2x15 ml), and sat. aq. NaC1 (2x15 ml).

The organic fraction was dried over anhydrous magnesium sulphate, and evaporated in vacuo to yield an oil. On purification by flash chromatography methanol in chloroform), the title compound (D27) was obtained as a clear oil (0.31g).

Observed (M+H) 763. C 42

H

59 0 7

N

4 P requires M 762.

Description 28 -(1-Phosphonopropyl) -(S)-leucyl] amino-8- (N-benzyloxycarbonyl)-1,8-diazacvclotetradecan-2-one, dibenzyl ester (D28)

CH

3

CH

3 2 5 .C2H H 0 A N

N

2 P N 0 H 0 H 0 OCH 2 Ph A solution of N-(1-(S)-dibenzyloxyphosphinylpropyl)-(S)leucine (D3B) (0.091g) in anhydrous dichloromethane ml) maintained at 0 C was treated with 1-(3- WO 91/15506 WO 9115506PCT/GB91/00529 52 dimethylarninopropyl) -3-ethylcarbodiimide hydrochloride (0.04g) and 1-hydroxybenzotriazole (0.028g) After stirring for 0.5h the reaction mixture was, -equentially treated !-ith lact~am trifluoroacetate salt (D17) (0.23 minol) and N,N-diisopropylethylamine (0.054g) Stirring was continued for 18h at room temperature, then the mixture was washed with sat. aq. NaCi (2x10 ml). The organic fraction was dried over anhydrous magnesium siilphate, and evaporated in vac-up to yield an oil.

Purification by flash chromatography methanol in chloroformn) afforded the title compound as an oil (0.105g).

Observed 777. C 43

E{

61

N

4 0 7 P requires M 776.

Description 29 .S--N-[N-R)-(l-Phosphonoethyl) -leucvlllIamino-8- (Nbenzvloxvcarbon\'l)-l.8-diazacvclotridecan 2-one, dibenzyl ester (D29A) and ,-3-[N-.rN-(S)-(-phosphonoethlS) leucvll Iamino-8- (N-benzvloxycarbonyl) -1,8diazacyclotridecan-2-one, dibenz vi ester (D29BI_

CH

3

CH

3

CH

3 H 0 (PhCH 2

O)

2 P N 4 1* N 0 10 H 0

OC

2 Ph A solution of the isomeric mixture of acids (D29A) and (D29B) (0.28g) in anhydrous dichloromethane (20 ml) maintained at 0 0 C was treated with 1- (3dimethylaminopropyl) -3-ethylcarbodiimidle hydrochloride (0.137g) and 1-hydroxybenzotriazole (0.097g) After WO 91/15506 W091/5506PCT/GB91/00S29 53 stirring for 0.5h the reaction mixture was sequentially treated with lactam (D7) (0.072 mmol) and N,Ndiisopropylethylamine (0.185g) Stirring was continued for 18h at room t 'emperature, then the mixture was washed with sat. ag. NaCl (20 ml). The organic fra:tion was dried over anhydrous magnesium sulphate, and evaporated in vacuc to 'ield an oil, On purification by flash chromatography methanol in chloroform), the title compound (D29) was obtained as) oil (0.32g).

Observed FAB 749. C 4 1

H

5 7

N

4 0 7 P requires M 748.

Description (R)-Dibenzyloxvphosphinvlethvl)-(S)-leucine (D30A) and N-C (S)-i.-dibeit-yloxvphosphinylethyl)-(S)-leucine

CH-

3

CH

3

CH

3 2

O)

2 P N -,C0 2

H

01f1 The title mixture of diastereoisomers (D30) was prepared analogously to the method in Description 3, Method B, as a white solid.I Observed FAB 420. C 22

H

30 N0 5 P requires M 419.

WO 91/15506 WO 91/15506PC'I/GB9I /00529 54 Example 1 rN- rN- -1-PhospRhonopropyl) -leucyl I I amino- 1. 8-diazacycordea- n (El) O HO0 NC N 0 H

H

H

A solution of phosphonic diester (D8) (0.11g) in ethanol ml) was hydrogenated over 10% palladium on charcoal at atmospheric pressure for 24h. The solution was filtered through Kieselguhr and solvent evaporated in vacua. The residue was triturated with diethyl. ether (5 ml) to give the title compound as a white solid, 0.057g,. m.p.

178.5-180..5 0

C.

Observed FAB 449. C 20

H

41 0 5

N

4 2 requires M 448.

2.66(lH,rn), 3.05(5H,m), 3.3 (1H,m overlapping with MeOD), 3.58(1E,m), 4.38(lH,m).

WO091/15506 PCr/GB91/00529 Example 2 (S)-3-[N-fN-((R)-l-Phosphonopropvl)-(S)--leucvll ]arino-1,7diazacy clotridecan-2-ne(E2)

CH

3

C

2

H

5

C'

HO N

N

I, I f 0 H 100 A solution of phosphonic diester (D13) (0.09g) in methanol ml) was hydrogenated over 5% palladium on charcoal at atmospheric pressure for 24h. The solution was filtered through kieselguhr and solvent evaporated in vacuo. The residue was triturated with diethyl ether (5 ml) to give the title compound (E2) as a white solid (0.038g).

Observed 449. C 20

H

4 ,0 5

N

4 P requires M 448.

8 (CDCl 3 0.96(EH,d), l.2(3H,t), l.35-l.9(17H,m), 2.49(lH,m), 2.9(1H,m), 2.98(3H,m),3.l2(lH,m), 3.42(lH,m), 4.33(1H,t).

WO 91/15506 W091/5506PCT/GB91/00529 -56- Example 3 N- -1-Phosphonopropyl) -leucylllamino-1,8diazacyclottradecan-2-one C(q3I GHi 3

C

2

H

5 3 HO H5 H 0 HO PK 14 0 i.H

N

A solution of phosphonic diester (D18) (0.09g) in methanol ml) was hydrogenated over 5% palladium on charcoal (0.08g) at atmospheric pressure for 24h. The solution was filtered through kieselguhr and solvent evaporated in vacuo. The residue was triturated with diethyl ether ml) to give the title compound (E3) as a white.solid (0.05g).

Observed FAB 463. C 21

H

43

N

4 0 5 P requires M 462.

3(CD 3 OD): 0.98(6H,t), l.12(3H,t), l.3-2.l(19H,m), 2.7-3.25(EH,m), 3.72(lH,m), 4.45(lH,m), 4.55(lH,m).

WO 91/15506 W091/5506PCr/GB91/00529 57 Examvle 4 -1-Phosphonopropyvl) -leucyll Iamino-8- (N-methyl) -1,8-diazacyclotridecan-2-one (E4).

CH

3

CFI

3

N

CH

3 A solution of phosphonic diester (D21) (0.09g) in methanol ml) was hydrogenated over 5% palladium on charcoal at atmospheric pressure for 24h. The solution was filtered through kieselguhr and the solvent evaporated in vacuo.

The residue was triturated with diethyl ether (5 ml) to give the title compound (E4) as a white solid (0.051g).

Observed 463. C 21

H

43

N

4 0 5 P requires M 462.

(CD

3 0.98(6H,t), l.1(3H,t), 1.4-2.l(17H,m), 2.7(lH,m), 2.88(311,s), 3.05-3.4(6H,m), 3.65(lHi,m), 4.4 (lH,m).

WO 91/15506 WO 9115506PCI/GB9I /90529 -58 Example -l-Phosphonopropyl) -leucyll 1 amino-i. 8diazacycloundecan-2-one, hydrochloride salt c 3

CH

3 HOH 0 0-HH A solution of phosphonic diester (D26) (0.07g) in ethanol ml) was treated with 5% palladium on charcoal (0.06g) .Id hydrogenated for 18h at atmospheric pressure. The suspension was .filtered through kieselguhr, and the solvent evaporated in vacuo to give a pale brown foam.

The foam was dissolved in ethanol, and treated with 1M ethereal HCl to afford the title compound (E5) as a white solid.

1.7-2.1(llH,m), 2.9-3.2(611,m), 3.6(18,m), 4.45(iH,rn), 4.55 (lH,m).

Example 6 diazacyclotridecan-2-one (E6),

CH

3 HO j 54 HO P N' N it H 0 H N 0

H

WO 91/15506 PCT/GB91/00529 59 A solution of phosphonic diester (D27) (0.29g) in methanol ml) was hydrogenated over 5% palladium on charcoal (0.25g) at atmospheric pressure for 24h. The solution was filtered through ,kieselguhr and solvent evaporated in vacuo. The residue was triturated with diethyl ether ml) to give the title compound (E6) as a white solid (0.16g).

Observed FAB (M+H) 449. C 20 H41N 4 0 5 P requires M 448.

8 (CD30D): 1.0(6H,dd), l.l(3H,t), 1.4-2.0(17H,m), 2.45(lH,m), 2.85-3.2(5H,m), 3.38alH,m), 3.58(lH,m), 4.49(lH,t).

Example 7 N-[N-((S)-1-Phosphonopropvl)-(S)-leucvl]]amino-l, 8diazacyclotetradecan-2-one (E7)

CR

3

SC

2

H

5 H 0 11 H H H

H

A solution of phosphonic diester (D28) (0.085g) in methanol (20 ml) was hydrogenated over 5% palladium on charcoal (0.08g) at atmospheric pressure for 24h. The solution was filtered through kieselguhr and solvent evaporated in vacuo. The residue was triturated with diethyl ether (5 ml) to give the title compound (E7) as a white solid (0.045g).

Observed FAB (M+H) 463. C 21

H

43

N

4 0 5 P requires M 462.

WO 91/15506 W091/5506PCT/GB9I/00529 60 2.85-3.25 (EH,m) 3.65 (1H,m) 4.12 (1H,m) 4.49(lE,di).

Example 8 rN- N- -1-Phosphonoethyl) -leucylllIamino-i 8diazacyclotridecan-2-one (E8A) and phosphonoethyl) -leucyll 1amino-li 8-diazacyclotritlecan- 2-one (E8B)

CH

3 HO AH 0 H 0 H" 0 II

H

A solution of-phosphoiic diester (D29) (0.26g) in methanol ml) was hydrogenated Pver 5% palladium on charcoal (0.2g) at atmospheric pressure for 24h. The solution was filtered through Kieselguhr and solvent evaporated in vacuo. The residue was triturated with diethyl ether ml) to give the title compound (EB) as a white solid as a mixture of diastereoisomers.

Observed FAB 435. C 19

H

39

N

4 0 5 P requires M 434.

WO 91/15506 PC T/GB91/00529 61 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 0 C, the assay tubes are cooled to 4 0 C and 3 H-acetylated rat skin type I collagen is added. The assay tubes are incubated at 37 0

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 3 H-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 (IC 50 inhibitory concentration) is expressed as that concentration of compound that inhibits a known (standard) concentration of enzyme by The compounds of Examples E1-E7 had IC 50 values inthe range 6 x 10- 8 3 x 10-6.

Claims (14)

1. A compound of the formula or a salt, solvate or hydrate thereof OH Ol PN 4 OR H R 3 (I) in which, R is hydrogen, C 1 -6 alkyl or optionally substituted benzyl; K 1 is hydrogen or Ci-6 alkyl; R2 is C 3 -6 alkyl; and R3 and R 4 are joined together as -(CH 2 )p-X-(CH 2 q where p is an integer from 1 to 9, q is an integer from 2 to and the moiety -(CH 2 is adjacent to the carbon ,tom bearing R 3 marked with an asterisk in formula and X is -NRg- where R 5 is selected from hydrogen, C 1 6 alkyl, C 2 6 alkanoyl, Cl 1 6 alkoxycarbonyl, aroyl, aralkyl or aralkyloxycarbonyl in each of which the aryl moiety is optionally substituted.

2. A compound according to claim 1 in which R is hydrogen, methyl, ethyl or benzyl. WO 91/15506 PCT/GB91/00529 63

3. A compound according to claim 1 or 2 in which R 1 is hydrogen, methyl, ethyl, isopropyl or n-butyl.

4. A compound according to any one of claims 1 to 3 in which R 2 is n-butyl, iso-butyl or sec-butyl. A.compound according to any one of claims 1 to 4 in which R 3 and R 4 are joined together as -(CH2)p-X-(CH2) q where p and q have values such that R 3 and R 4 form part of an 11- or 13 to 16-membered azalactam structure, and X is -NR 5 where R 5 is hydrogen, methyl, benzyl, t- butoxycarbonyl or benzyloxycarbonyl.

6. A compound according to any one of claims 1 to 5 in which R is hydrogen, R 1 is methyl or ethyl, R 2 is iso- butyl, and R3 and R4 are joined together as -(CH 2 )p-X-(CH 2 where p is 4 and q is 5, p is 3 and q is 6 or p is 4 and q is 6, or p is 4 and q is 3 and X is -NR 5 where R 5 is hydrogen or methyl.

7. A compound according to any one of claims 1 to 6, in which the chiral centres marked with an asterisk in formula have the S-configuration when R3 is other than hydrogen.

8. A compound according to claim 1 which is: (S)-3-(N-[N-((R)-1-phosphonopropyl)-(S)-leucyll]amino- 1,8-diazacyclotridecan-2-one; (S)-3-[N-[N-((R)-1-phosphonopropyl)-(S)-leucyl])amino- 1,7-diazacyclotridecan-2-one; (S)-3-(N-[N-((R)-1-phosphonopropyl)-(S)-leucyl]]amino- 1,8-diazacyclotetradecan-2-one; B2957 64 -1-phosphonopropyl) -leucylJ amino- 8- (N-methyl) 8-diazacyclotridecan-2-one; 1-phoisphonop ropyl1) CS) -leucyl] ]amino- 1, 8-diazacyclound *ecan-2-one, hydrochloride salt; -1-phosphonoprop!) -leucyl] I amino- 1, 8-diazacyclotridecan-2-one; -I -pho sphonop ropy 1) -leucyl] I amino- 1, 8-diazacyclotetradecan-2-one; -1-phosphonoethyl) -leucyl] amino- 8-diazacyclotridecan-2-one and -1-phosphonoethyl) -leucylJ amino- 1, 8-diazacyclotridecan-2-one.

9. A process for the preparation of a compound according to claim 1 which process comprises converting a group R 20 to hydro(_en by cleaving a group R 2 0 from a compound of formula (II): *R R OR 1 0 R substituted penyl anor s y~gnakl optionally substituted phenyl or optionally substituted benzyl and Rl, R 2 R 3 and R 4 are as defined in formula and where necessary, converting 9 21 to hydrogen. A compound of the formula (11) as defined in claim 9 subject to the proviso that R1is not hydrogen. WO091/15506PC/B9/02 PCr/GB91/00529 65

11. A compound according to claim 10 which is N- (-phosphonopropyl) -letkeyl3 3amino- 8- (N-benzyjloxycarbonyl) 8-diazacyclotridecan-2-one, dibenzyl ester; (S)-3-[N-[N-(R)-(l-phosphonopropyl)-(S)-leucyl3 )amino-7- (N-benzyloxycarbonyl) 7-diazacyclotridecan-2-one, dibenzyl ester; (-phosphonopropyl)- CS) -leucyl) 3amino-8- (N-benzyloxycarbonyl) 8-diazacyclotetradecan-2-one, dibenzyl ester; CR) C-phosphonopropyl)- CS) -leucyl) 3amino-B- (N-methyl) 8-diazacyclotridecan-2-one, dibenzyl ester; (S)-3-'[N-N-(R)-(-phosphonopropyl)-(S)-leucyl] ]amino-8- (N-benzyloxycarbonyl) 8-diazacycloundecan-2-one, dibenzy. ester; -(1-phosphonopropyl) -leucyl3 3amino-B- (N-benzyloxycarbonyl) 8-diazacyclotridecan-2-one, dibenzyl ester; (-phosphonopropyl) -3eucyli 3amino-B- (N-benzyloxycarbonyl) 8-diazacyclotetradecan-2-one, dibenzyl ester; -(l-phosphonoethyl) -leucyl) 3amino-8- (N- benzyloxycarbonyl)-l, 8-diazacyclotridecan-2-one, diberizyl ester and 2 (S)-3-(N-(N-(S)-(l-phosphonoethyl)-(S)-leucylJ ]amino-8-(N- bgnzyloxycarbonyl) -1,8-diazacyclotridecan-2-one, dibenzyl ester.

12. A pharmaceutical composition comprising a compound according to any one of claims 1 to 8 or a pharmaceutically acceptable salt, solvate or hydrate thereof, and a pharmaceutically acceptable carrier. -66-

13. A compound according to any one of claims 1 to 8 or a phaimaceutically acceptable salt, solvate or hydrate thereof, for use as an active therapeutic substance.

14. A compound according to any one of claims 1 to 8 or a pharmaceutically acceptable salt, solvate or hydrate thereof, for use in the treatment of conditions in which degradation of connective tissue and other proteinaceous components of the body occurs. The use of a compound according to any one of claims 1 to 8 or a pharmaceutically acceptable salt, solvate or hydrate thereof, in the manufacture of a medicament for the treatment of conditions in which degradation of connective tissue and other proteinaceous components of the body occurs. ^CC4 C/(AH 40 C liG 1

16. Compounds of the formula A or salts, solvates or hydrates thereof or processes for their preparation, substantially as hereinbefore described with reference to the 20 Descriptions or Examples. DATED this 2nd day of February, 1993 Beecham Group p.l.c. By Its Patent Attorneys DAVIES COLLISON CAVE 930202,papcr\dab.765O2spc,66 INTERNATIONAL SEARCH REPORT International Application No PCT/GB 91/00529 1. CLASSIFICATION OF: SUBJECT MATTER (if severdt classification symoola apply, indicate all) According to International Patent Classification (IPC) or to both National Classification and IPC IPCS C 07 K 5/06, A 61 K 37/64 11. FIELDS SEARCHED Minimum Documentation Searched Classification System Classification Symbols IPC 5 C 07 K, A 61 K Documentation Searched other thin Minimum Documentation to the Extent that such Documents are Included In the Filds SearchedI

111. DOCUMENTS CONSIDERED 10 BE RELEVANT Category Citation of Dm.ument, 11 with Indicaion, where appropriate, of the relevant possages It Relevant to Claim No. 13 Y EP, A, 0320118 (BEECHAM GROUP PLC) 1-15 14 June 1989 see page. 3, line 42 page 8, line 41; description 13; example 5; claims 1-16 cited in the application Py EP, A, 0401963 (BEECHAM GROUP PLC) 1-15 12 December 1990 see page 4, line 37 page 11, line descriptions 7,10,11,16; examples 3,4, 6-8; claims 1-11 A Biochemistry.volume 28, no. 12, June 1989, 1-15 American Chemical Society (Washington, DC, US), D. Grobelny et al.: "Binding energetics of phosphorus-containing inhibitors of thermolysin", pages 4948 -4951 see the whole document *Special categories of cited documonts: 10 later document published alter t.he InterioationaI Wling date A" dcumnt drinnp he gnerl stte l te ar whch I no orpriority date and not In connict with 0i'2 tplcation but "Alcocns deto b e o atcl releae citeatwic snt01ted to understand the princ~iple or theory underlying the consderd t bit4),p& iculr rlevnceInvention earlier document but published on or atter the International document of particular relevance,, the claimed Invention fili ng date cannot be considered novel or cannot be considered to document which rnay throw doubts on priority claim~k) or Involve an Invsntiv2 stop which is cited to rtstablish the publication date of another document of particular relevance;' the claimed Invention citation or other sliecial reason (as specifiad) cannot be considered to Involve an Inventive step wilen the I'D" document referring to an or&l disclosure, use, exhibition or document is combinad with one or more other such J~ocu- other means mstits, such combination being obvious to a person skiled document published prior to the International filing date but In the art. later than the priority date claimed O" document member of the same patent family IV. Date of the Actual Compittion of the lnteirnatlonid Search Data of Malling of this International Search Report 28th June 1991, 27 0. q1- International Searching Authority Signature of Authorized Ofcy EUROPEAN PATENT OFFICE ss.MRTNI Form PCTIISA/210 (second sheet) iavjary 19,15) ANNEX TO THE INTERNATIONA ;L SEARCH REPORT ON INTERNATIONAL PATENT APPLICATION NO. GB 9100529 SA 46300 This anntx lists the patent family members relating to the patent documents cited in the above-mentioned international search report. The members are as contained in the European Patent Office EDP ile on 19108191 The European Patent Office is in no way liable for these particulars which are merely given for the purpose of inform~ation. Patent document Publication Patent family Publication cited in search report I date Imember(s) Idate EP-A- 0320118 14-06-89 AU-A- JP-A- US-A- 2507088 1160992 4935404 25-05-89 23-06-89 19-06-90 EP-A- 0401963 12-12-90 AU-A- 5315890 18-10-90 UP-A- 3063294 19-03-91 A. ;Z For momt details about this annex -see Official Journal of t2he European Patent Offic, No. 12182