This invention relates to a series of cycloalkyl-substituted glutaraitiide derivatives which are diuretic agents having utility in a variety of therapeutic areas including the treatment of various c__trriiovascular disorders such as hypertension, heart failure and renal insufficiency.

According to the specification of our European patent application EP-A-0274234 and our pending European patent application No 89305180.5 we describe and claim certain cyclca-Lyl-sub-stituted glutaramide derivatives having an aπ _nocycloal___nec_arboxylate ring as diuretic agents. The present invention provides further related compounds in which the am__rxxycloaIaneca__boxylate ring is replaced by a beta-alanine group or derivative thereof.

The coπpcunds are Dlhhibitors of the zinc-dependent, neutral endcpeptidase E.C.3.4.24.11. This enzyme is involved in the brea own of several peptide hormones, including atrial natriuretic factor (ANF) , which is secreted by the heart and which has potent vasodilatory, diuretic and natriuretic activity. Thus, the cxaipounds of the invention, by inhibiting the neutral endopeptidase E.C.3.4.24.11, can potentiate the biological effects of ANF, and in particular the compounds are diuretic agents having utility in the treatment of a number of disorders, including hypertension, heart failure, angina, renal insufficiency, premenstrual syndrome, cyclical oedema, Menieres disease, hyperaldosteronism (primary and .secondary) pulmonary oedema, ascites, and hypercalciuria. In addition, because of their ability to potentiate the effects of ANF the compounds have

utility in the treatment of glaucoma. As a further result of their ability to inhibit the neutral endopeptidase E.C.3.4.24.11 the <x_πpounds of the invention may have activity in other therapeutic areas including for exanple the treatment of asthma, inflammation, pain, epilepsy, affective disorders, dementia and geriatric confusion, obesity and gastrointestinal disorders (especially diarrhoea and irritable bowel syndrome) , the modulation of gastric acid secretion and the treatment of hyperren__naemia and leukaemia.

The ccπpcunds of the present invention are of the formula:

wherein A cccpletes a 4 to 7 mesonbered carbocyclic ring which may be saturated or ιror_o-unsaturated and which may optionally be fused to a further saturated or unsaturated 5 or 6 membered carbocyclic ring ;

4 each of R and R is independently H, C.. -C alkyl, benzyl or an alternative biolabile ester-forming group;

R is H or C,-C alkyl;

2 3 18 19

R , R , R and R are independently selected from H,

C.-C alkyl, C,-C ₆ alkoxy, a__yl(C.-C ₆ )alkyl, aryl(C -Cg)alkenyl, aryl(C,-C.)alkoxy(C-.-C )alkyl, and hydrox (C,-C )alkyl,

3 19 2 18 or R and R are as defined above and R and R , together with the carbon atom to which they are attached, form a

2-indanylidene group;

5 and R is C,- alkyl, C -C alkenyl, C -C alkynyl,

C,-C_ cycloalkyl, or C,-C-. cycloalkenyl,

5 . or R is C,-C ₈ alkyl substituted by halo, hydroxy, C.-C alkoxy, C.-C alkox (C.-C )alkoxy, C -CL cycloalkyl,

C -C^ cycloalkenyl, aryl, aryloxy, heterocyclyl, -WTR ,

-NR ⁸ COR ⁹ , -NR ⁸ S0 ₂ R ¹⁰ , -CCNR ⁶ R ⁷ or R ⁶ R ⁷ N-(C ₁ -C ₆ )alkoxy;

5 . or R is ,-Cg alkyl substituted by a group of the formula:

R ¹²

11 ' 13 -NR CO-C-R

R ¹³

11 ' 14 -CCNR -C-R i ¹⁵

fi 7 wherein R and R are each independently H, C,-C alkyl, C - cycloalkyl, aryl, aryl(C,-C.)alkyl, C_-C _^ alkoxyalkyl,

1 __ D

6 7 or heterocyclyl; or the two groups R and R are taken together with the nitrogen to which they are attached to form a pyrxolidinyl, piperidino, morpholino, piperazinyl or N-fC- _^ -C.Ja kyl-piperazinyl group;

R ⁸ is H or C- _j ^-C _j alkyl;

9 , R is _j -C, alkyl, CF , aryl, aryl(C.-C.)alkyl, 7 aryl(C.-C )alkoxy, heterocyclyl, C.-C alkoxy or NR R fi 7 wherein R and R are as previously defined;

R is C.-C alkyl, C -C_ cycloalkyl, aryl or heterocycl l;

R is H, .-C- alkyl, aryl or C -C_ cycloalkyl,

R ¹² is R^CONR ¹¹ -, R ^1:L S0 ₂ NR ^1:L -, R ¹⁶ R ¹⁷ N-(CH ₂ ) -, or

R 0-, wherein each R is as previously defined above;

13 14 R and R are each .independently H or C,-C alkyl; or

13 14 . . . .

R is H and R is C,-C ₈ alkyl which is substituted by

OH, _j -C ₄ alkoxy, SH, SCH.., NH-,, aryl

CCONH-, NBLCO-, CO-H, guanidino, aryl, or heterocyclyl;

13 14 . . or the two groups R and R are joined together to for , with the carbon atom to which they are attached, a

5 or 6 merribered carbocyclic ring which may be saturated or and which may optionally be substituted by C,-C alkyl or fused to a further 5 or 6 membered saturated or unsaturated carbocyclic ring;

13 12 14 or R is H, and R and R are linked to form a

2-(N-COR -4-aminopyrrolidinyl) group; R ¹⁵ is R ¹⁶ R ¹⁷ NCO-, R ¹¹ OCO-, R ^1:L 0CH ₂ - or heterocyclyl, wherein R is as previously defined above;

alkyl, and p is 0 or an integer of from 1 to 6; and pharmaceutically acceptable salts thereof and biop__ecursors therefor.

In the above definitions, unless otherwise indicated, alkyl groups having three or more carbon atoms may be .straight or branched-chain. The term aryl as used herein means an arcxnatic hydrocarbon group such as phenyl, naphthyl or biphenyl which may optionally be substituted with one or more OH, CN, CF , C-.-C. alkyl, _~ - -C, alkoxy groups or halo atoms. Halo means fluoro, chloro, bromo or iodo.

The term heterocyclyl means a 5 or 6 membered nitrogen, oxygen or sulphur <_x>ntaining heter cyclic group which, unless otherwise stated, may be saturated or unsaturated and which may optionally include a further oxygen or one to three nitrogen atoms in the ring and which may optionally be benzofused or substituted with for exanple, one or more halo, C.-C alkyl, hydroxy, carbamoyl, benzyl, oxo, a ino or mono or di-(C,-C. alkyl)amino or (C.-C. alkanoyl)amino groups. Particular examples of heterocycles include pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, furanyl, tjetrahydrofuranyl, tetrahydr pyranyl, dioxanyl, thienyl, oxazolyl, isoxazolyl, thiazolyl, indolyl, isoindolinyl, quinolyl, quinoxalinyl, quinazolinyl and benzimidazolyl, each being optionally substituted as previously defined.

The cxaipounds of formula (I) may contain several asymmetric centres and thus they can exist as enantiomers and diastereoisomers. The invention includes both mixtures and the

2 18 3 separated individual isomers. The substituents R , R and R ,

19 R respectively may be of threo or erythro configuration relative

5 to each other. As the carbon atcm to which R is attached constitutes a chiral centre, the ccarpσunds may exist as R or S forms with respect to this centre. The terms erythro and threo are as defined in the article by D-C Ha, D. J. Hart and T-K Yang.

J. Am. Chem. Soc. 19841064819.

The pharmaceutically acceptable salts of the compounds of formula (I) containing an acidic centre are those formed with bases which form non-toxic salts. Examples include the alkali metal salts such as the sodium, potassium or calcium salts or salts with amines such as diethylamine. Compounds having a basic centre can also form acid addition salts with pharmaceutically acceptable acids. Examples include the hydrochloride hyd__cbromide, sulphate or bisulphate, phosphate or hydrogen phosphate, acetate, citrate, fumarate, gluconate, lactate, maleate, succinate and tartrate salts.

The term bioprecursor in the above definition means a pharmaceutically acceptable biologically degradable derivative of the compound of formula (I) which, upon adm_Lnistration to an animal or human being, is converted in the body to produce a ccπpσund of the formula (I) .

A preferred group of compounds are those in which A is (CH )

2'4 and R is H, A thus completing a cyclcpentane ring.

2 3 18 19 Examples of R , R , R and R are H, styryl, methyl, ethyl,

2 lsqprcpyl and methoxy. In a preferred group of cxsrpounds R is

18 3 19 aryl(C -C ₆ )alkenyl, such as styryl, R is H and either R and R

3 19 are both methyl or one of R and R is hydrogen and the other is

2 18 3 methyl, methoxy or isopropyl. When one each of R , R and R ,

19 . R is H and the other is a substituent other than H, these substituents are preferably in the erythro configuration relative to each other.

Also preferred are those cxxipounds of formula (I) wherein R

4 and R are both H (diacids) as well as biolabile mono and di-ester

4 derivatives thereof wherein one or both of R and R is a biolabile ester-forming group.

The term biolabile ester-forming group is well understood in the art as meaning a group which provides an ester which can be readily cleaved in the body to liberate the corre_sponding diacid

4 of formula (I) wherein R and R are both H. A number of such ester groups are well known, for example in the penicillin area or in the case of the ACE-__nhibitor antiLhypertensive agents.

In the case of the coπpounds of formula (I) .such biolabile pro-drug esters are particularly advantageous in providing ccπpounds suitable for oral administration. The suitability of any particular ester^forming group can be assessed by conventional animal or in vitro enzyme hydrolysis studies. Thus, desirably for optimum effect, the ester should only be hydrolysed after absorption; acxxjrdingly, the ester should be resistant to hydrolysis before absorption by digestive enzymes but should be readily hydrolysed by, for example, liver enzymes. In this way the active diacid is released into the bloodstream following oral absorption.

In addition to lower alkyl esters (particularly ethyl) and benzyl esters, suitable biolabile esters include alkanoyloxyalkyl esters, .Including alkyl, cycloalkyl and aryl substituted derivatives thereof, aryloxyalkyl esters, aroyloxyalkyl esters, aralkyloxyalkyl esters, arylesters, aralkylesters, and haloalkyl esters wherein said alkanoyl or alkyl groups have from 1 to 8 carbon atoms and are branched or straight chain and said aryl groups are phenyl, naphthyl or indanyl optionally substituted with one or more C,-C alkyl or C -C alkoxy groups or halo atoms.

4 Thus examples of R and R when they are biolabile groups other than ethyl and benzyl include:

1-(2,2-diet_hylbutyryloxy)ethyl, 2-ethylp__opionyloxymethyl, l-(2-ethylpropionyloxy)ethyl, l-(2,4-d-L_ιethylbenzcyloxy)eth.yL, 1-

(benzoyloxy)benzyl, l-(benzoyloxy)ethyl, 2-methyl-l-propionyloxy- propyl, 2,4,6-triιtethylberιzoyloxymethyl, l-(2,4,6-trimethyl- benzyloxy)ethyl, pivalcyloxymethyl, phenethyl, phenpropyl, 2,2,2- trifluoroethyl, 1- or 2-r_aphthyl, 2,4-d±rethylphenyl, 4-t-butyl- phenyl, 5-(4-methyl-l,3-dioxalynyl-2-onyl)methyl and 5-indanyl.

4 Compounds of the formula (I) wherein one or both of R and R are C.-C alkyl, particularly ethyl, t-butyl or benzyl, are valuable intermediates for the preparation of the diacids wherein

R and R ⁴ are both H.

5 . In a further preferred group of compounds R is methylene substituted by a group of the formula -^HC0CR ¹² R R ¹⁴ ' particularly where R ¹² is NE^, R ^iL CONH- or R ^i:L S0 ₂ H-, R ¹³ is H and

14 R is -(OI.) NH . Particularly preferred are such groups derived

5 from S-lysine, thus especially preferred R substitutents of this

type include N^-ac^tyl-S-lysylaiiiinoirethyl, N^-roethanesulphonyl- S-lysyl-aιτ_inomethyl, N^-p^enylsul^onyl-S-lysyl-a_i___ncsnethyl N^-cyclctoutylca__bonyl-S-lysyl-aιt___r_πetiyl,N^-t-butoxy carbonyl-

S-lysyl-am__nσ_ιethyl and S-lysyl-aituj nethyl.

5 In a further group of preferred compounds R is C,-C _β alkyl substituted by C,-C ₈ alkoxy, particularly methoxyethyl; or wherein

5 . R is C,-C ₈ alkyl substituted by C.-C alkoxy (C -C ) alkoxy, particularly 2-ιτethoxyethoxymethyl.

Particularly preferred compounds are those in which R, R and

4 2 3 2 3 .

R are H, A is (CϊL) ., R is εtyryl, R is methyl, R and R being

18 19 5 in the erythro relative configuration, R and R are H, and R is S-lysylam__nomethyl, N^-acetyl-S-lysylaiiύ-nomethyl and

N^-nethanesulphonyl-S-lysylaiidno-itethyl and biolabile ester derivatives thereof. These cx__rpounds may exist in the R- or S- fo ms, or a mixture thereof, relative to the chiral centre to which R is attached. In the case of the N^-methanesulj_honyl-S- lysylam__nα_nethyl derivative, the S-form is preferred.

The compounds of formula (I) may be prepared by a number of different processes. The basic procedure involves the synthesis of a partially protected geminally disubstituted glutaric acid derivative which is coupled to an amine to give the desired glutaramide. The carboxylic acid group in the amine, if free, or

5 any reactive groups in R , may require protection during the coupling step and such protecting groups are removed in the final stage of the process.

The synthetic route is illustrated in Scheme 1 wherein A, R '

2 3 18 19 . 5* .

R , R , R and R are as previously defined, R is as defined

5 for R with any reactive group therein protected if necessary and

R and R are as defined for R and R excluding H, or they are conventional carboxylic acid protecting groups:

Scheme 1

R ²⁰ 0 ₂ C

(ID

(III)

(IV) The reaction of the cαrpounds of formula (II) and (III) is achieved using conventional amide coupling techniques. Thus in one process the reaction is achieved with the reactants dissolved in an organic solvent, e.g. dichlo__omethane, using a diimide condensing agent, for exanple l-ethyl-3-(dirrethylaιι -nopropyl)- carbcdiimide, or N,N'-dicyclchexylca-±)θά^imide, advantageously in the presence of 1-hydroxybenzotriazole and an organic base such as 4-metlιy__morpholine. The reaction is generally complete after a period of from 12 to 24 hours at room temperature and the product is then isolated by conventional procedures, i.e. by washing with water or filtration to remove the urea by-product and evaporation of the solvent. The product may be further purified by

crystallisation or iraiatography, if necessary. The coπpounds of

4 formula (IV) include compounds of formula (I) wherein R and R are

C -C alkyl or benzyl.

In some cases the coupled product, in protected form, may be subjected to conventional chemical transformation reactions to allow preparation of further ∞πpounds of formula (IV) . Thus for

5 ¹ exanple coπpounds of formula (IV) wherein R contains an ester group may be hydrolysed or hydrogenated to generate the carboxylic acid which may be further reacted, for exanple with an amine, to give amide derivatives.

. . . 5' .

Similarly cαrpounds wherein R contains a substituted or protected amino group (for exanple a benzylamino, d-Lbenzylamino, benzylco_yca__bcnylaπ_ino or t-butyloxyca__bo_τyla_i-__no group) may be converted to the free amines by, for example, hydrogenation or hydrolysis as appropriate. The amines produced may be further reacted; thus for example reaction with a sulphonyl halide yields the corresponding sulphonamides, acylation with an acid chloride or anhydride yields the cx>n_esponding amides, reaction with an isocyanate yields urea derivatives and reaction with a ciiloroformate yields the carbamate respectively. All these transformations are entirely conventional and appropriate cαnditiαns and reagents for their performance will be well known to those skilled in the art as will other variations and possibilities.

The diesters of formula (IV) may be further reacted to give the monoester of diacid derivatives of formula (I) wherein one or

4 both of R and R are H. The conditions used will depend on the

20 21 precise nature of the groups R and R present in the compound

of formula (IV) and a number of variations are possible. Thus for

20 21 example when both of R and R are benzyl, hydrogenation of the

4 product will yield the diacid of formula (I) wherein R and R are

20 21 both H. Alternatively if one of R and R is benzyl and the other is alkyl, hydrogenation will yield a monoester product.

This can be hydrolysed, if desired, to again yield the diacid

20 21 product. When one of R and R is t-butyl, treatment of the compound of formula (IV) with trifluoroacetic acid yields the

20 21 ∞rresporeiing πonoacid. The diester product wherein R and R are benzyl .or lower alkyl can also be treated with trimethylsilyl iodide to produce the dicarboxylic acid product. If some other

20 21 carboxylic acid protecting group is used for R or R then clearly appropriate conditions for its removal must be enployed in the final step to give the ester or diacid product of formula (I) .

2 5 . In the case where the ring A or the substituent R or R is unsaturated, the deprotection must be effected by non-reductive

4 , methods, thus for exanple if either of R and R is benzyl, they may be removed by treatment with trimethylsilyl iodide.

As well as __emσving any protecting group which may be present

5» in R , a number of chemical transformation reactions are possible on the final mono-ester or diacid products as previously described. In each case the product may be obtained as the free carboxylic acid or it may be neutralised with an appropriate base and isolated in salt form.

In a variant of the above p__ocedure, compounds of the formula

5 R Q ft 1 Ω

(I) wherein R is C.-C ₆ alkyl substituted by -NR COR , -NR SO R , -NR ¹¹ COCR ¹² R ¹³ R ¹⁴ or -NR ¹¹ S0 ₂ CR ¹² R ¹³ R ¹⁴ are prepared by a process which involves acylating or sulphonylating a ∞npound of the formula:

(V)

22 8 11 20 21 wherein R is as defined for R or R , R and R are as previously defined and Y is a .-C alkyl group; by reaction with

9 10 12__13 14 an acid of the formula R CX> ₂ H, R SO.H, ^R R CC0 ₂ H, or

RT. R CS0_H, or an activated derivative thereof. The resulting amide or εulphonamide product is then deprotected if required and the diester product hydrolysed to yield the carboxylic acids of

4 formula (I) wherein R and R are each H as previously described.

The coπpounds of foπrnila (V) are prepared following the procedures shown in Scheme 1 but using a coπpound of formula (II)

5» . . . 5* having R as a protected amine derivative. Thus, for example R can contain a bis-[(lS)-phenylethyl]aπ__j_omethyl εubstituent.

Hydrogenation of the coupled product gives the corresponding free

22 . amine of formula (IV) wherein R is H and Y is C3_ . This route is of particular value for the preparation of cxxrpounds having

2(S) stereochemistry in the glutaramide backbone.

The starting spiro-substituted glutaric acid mono esters of formula (II) may be prepared as described in our European patent applications EP-A-0274234 and 89305180.5.

The amines of formula (III) are generally novel cαπpounds (particularly when the substituents have defined stereochemistry) and they are prepared by appropriate synthetic procedures in accordance with literature precedents. In one procedure, following the method of D.J. Hart, K. Kanai, D.G. Thomas and T-K. Yang, J. Qrg. Chem, 1983, 48, 289, the appropriate substituted ethyl propanoate is treated with n- _utyl lithium in the presence of diisopropylamine and the product treated with the appropriate N-(trimethylsilyl)imine, followed by acid hydrolysis to yield the desired amine. The N-(trimethylsilyl)imine may be obtained by reaction of the appropriate aldehyde with lithium bis(trimethyl¬ silyl)amide. These procedures are illustrated in the Preparations given hereafter.

Appropriate coupling and protecting methods for all of the above steps and alternative variations and p__ocedures will be well known to those skilled in the art by reference to standard text books and to the examples provided hereafter.

As previously mentioned, the cαtpounds of the invention are potent inhibitors of the neutral endopeptidase (E.C.3.4.24.11). This enzyme is involved in the breakdown of a number of peptide hormones and, in particular, it is involved in the breakdown of atrial natriuretic factor (ANF) . This hormone consists of a family of related natriuretic peptides, secreted by the heart, of which the major cjlrculating form in humans is kncwn to be the 28 amino-acid peptide referred to as -hANP. Thus, by preventing the

degradation of ANF, fcy endcpeptidase E.C.3.4.24.11, the coπpounds of the invention can potentiate its biological effects and the conpounds are thus diuretic and natriuretic agents of utility in a number of disorders as previously described.

Activity against neutral endopeptidase E.C.3.4.24.11 is assessed using a procedure based on the assay described by J.T. Gafford, R.A. Skidgel, E.G. Erdos and L.B. Hersh, Biochemistry, 1983, 32, 3265-3271. The method involves detern ning the concentration of compound required to reduce by 50% the rate of release of radiolabelled hippuric acid from hippuryl- - phenylalanyl-_ a__ginine by a neutral endopeptidase preparation from rat kidney.

The activity of the conpounds as diuretic agents is determined by measuring their .ability to .Increase urine output and sodium ion excretion in saline loaded conscious mice. In this test, male mice (Charles River CD1, 22-28g) are acclimatised and starved overnight in metabowls. The mice are dosed intravenously via the tail vein, with the test coπpound dissolved in a volume of saline solution equivalent to 2.5% of body weight. Urine samples are collected each hour for two hours in pre-weighed tubes and analysed for electrolyte concentration. Urine volume and sodium ion aicentration frαp the test animals are compared to a control group which received only saline.

For administration to man in the curative or prophylactic ^■ treatment of hypertension, congestive heart failure or renal insufficiency, oral dosages of the coπpounds will generally be in the range of from 4-800mg daily for an average adult patient (70kg) . Thus for a typical adult patient, individual tablets or

capsules contain from 2 to 400ng of active coπpσund, in a suitable pharmaceutically acceptable vehicle or carrier for adn nistration singly, or in multiple doses, once or several times a day. Dosages for intravenous administration would typically be within the range 1 to 400mg per single dose as required. In practice the physician will determine the actual dosage which will be most suitable for an individual patient and it will vary with the age, weight and response of the particular patient. The above dosages are exemplary of the average case but there can, of course, be individual instances where higher or lower dosage ranges are merited, and such are within the scope of this invention.

For human use, the coπpounds of the formula (I) can be administered alone, but will generally be adrrdnistered in admixture with a pharmaceutical carrier selected with regard to the intended route of administration and standard pha__maceutical practice. For exanple, they may be administered orally in the form of tablets containing such excipients as starch or lactose, or in capsules or ovules either alone or in admixture with excipients, or in the form of elixirs or suspensions containing flavouring or colouring agents. They may be injected parenterally, for exanple, intravenously, intramuscularly or subcutaneσusly. For parenteral adndnistration, they are best used in the form of a sterile aqueous solution which may contain other substances, for exanple, enough salts or glucose to make the solution isotonic with blood.

The ccβtrpounds may be adrrdnistered alone but may also be administered together with such other agents as the physician

shall direct to optimise control of blood pressure or to treat congestive heart failure, renal insufficiency or other disorders in airy particular patient in accordance with established medical practice. Thus the coπpounds can be cx>-adnir-istered with a variety of cardiovascular agents, for exanple with an ACE -Inhibitor such as captopril or enalapril to facilitate the control of blood pressure in treatment of hypertension; or with digitalis, or another cardiac stimulant, or with an ACE inhibitor, for the treatment of congestive heart failure. Other possibilities include co-adm__nistration with a calcium antagonist (e.g. nifedipine, amlodopine or diltiazem) a beta-blocker (e.g. atenolol) or an alpha-blocker (e.g. prazosin or doxazosin) as shall be determined by the physician as appropriate for the treatment of the particular patient or condition involved.

In addition to the above, the coπpounds may also be administered in conjunction with exogenous ANF, or a derivative thereof or related peptide or peptide fragment having diuretic/natriuretic activity or with other ANF-gene related peptides (e.g. as described by D. . Vesely et al, Biochem. Biophys. Res. Cccπm., 1987, 143, 186).

Thus in a further aspect the invention provides a pharmaceutical coπposition coπprising a compound of the formula (I) , or a pharmaceutically acceptable salt thereof or biqprecursor therefor, together with a pharmaceutically acceptable diluent or carrier.

The invention also includes a cx∑rrpσund of the formula (I) , or a pharmaceutically acceptable salt thereof or bioprecursor therefor, for use in medicine, particularly for use as a diuretic

agent for the treatment of hypertension, congestive heart failure or renal insufficiency in a human being.

The invention further includes the use of a coirpσund of the formula (I) for the manufacture of a medicament for the treatment of hypertension, heart failure, angina, renal insufficiency, premenstrual syndrome, cyclical oedema, Menieres disease, hyperaldostereonism, pulmonary oedema, ascites, hypercalciuria, glaucoma, asthma, inflammation, pain, epilepsy, affective disorders, dementia and geriatric confusion, obesity, gastrointestinal disorders (iιx;luding diarrhoea) , hyperrenfLraemia, leukaemia, and the modulation of gastric acid secretion.

The preparation of the ∞mpσunds of the invention will now be more particularly illustrated by reference to the following experimental examples, in which Examples 1-30 describe preparations of compounds of the formula (I) and Preparations 1-12 describe preparations of starting amines of formula III. The purity of ∞ pαunds was routinely monitored by thin layer c_h_x_matography using Merck Kieselgel 60 F ₂₅₄ plates. ^" Ti-Nuclear magnetic reasonance spectra were recorded using a Nicolet QE-300 -spectrometer and were in all cases consistent with the proposed structures.

Example 1 2(R,S)-(N -t-Butoxyca-±onyl-N^-_r thane-5ul-honyl-S-lv_5yl- an__-r_cmethyl)-3-ri-(l-carix)xycyclopentyl)1prop-m acid t-butyl ester

(a. 4-Methy__morpholine salt of 2(R,S.amir_omethyl-3-(l- ca_ xιxycyclcpent-yl)propanoic acid t-butyl ester

A stirred mixture of 2 (R,S)-D__benzylaminc ethyl-3-(l- ca_±>oxycyclopentyl)propanoic acid t-butyl ester hydrochloride (1.46g, 3mmol) and 4-πethy__morpholine (3ml) in ethanol (30ml) was hydrogenated over palladium (from 20% Bd OHJ 'C; 2.0g) at 60 psi (4.1 bar).

After 18 hours the π xture was filtered through Arbicel, the solvent evaporated and the residue dried azeotropically with toluene. The required primary amino acid 4-nethy__morpholine salt ∞ntei_r_i_og one mole equivalent of 4-metoylmorphol__ne hydrochloride was obtained.

(b. 2(R,S.-N -t-Butoxyc_arbonyl-N^-methanesulphonyl-S-lysyl- an___nc_πethyl)-3-fl-(l-ca_±oxycyclcpentyl)1p-n- e^ acid t-butyl ester l-(3-Diπethylami_nop__opyl)-3-ethylcaι±)CX3_iimide hyd__ochloride (0.57g, 3mmol) was added to a stirred mixture of N -t-buto-^c^_±oι l-N^-nethanesulphonyl-S-lysine (0.97g, 3mmol) , l-hyd__oxybenzotriazole (0.4g, 3mmol) and 4-methylmorpholine (0.66ml, 6mmol) in dry di hlo__oniethane (20ml). After 0.5 hours, the resulting solution was added to the crude salt described above and the reaction mixture stirred for 16 hours at room temperature, then evaporated under vacuum. The residue was dissolved in ethyl acetate (25ml) and the solution washed successively with ^~ LM

hyd__ochloric acid (2 x 20ml) , saturated aqueous sodium bicarbonate solution (2 x 10ml) , water (20ml) and saturated brine (20ml) , then dried (MgSO ) and evaporated under vacuum. The crude material (2.0g) was purified by cJircmatography on silica gel using an ethyl acetet_a-nιet_ri_nol-^ethyla_r_ine elution gradient (99:1:1 - 90:10:1) to afford the required product (0.68g). Rf 0.35 (silica; ethyl acetete-n thanol-i_scp__opyl-amine/80:20:l) .

Examples 2-4 The cxJirpσunds of Table 1 were obtained by the general method of Example I, using the appropriate S-lysine derivative. Coπpound 4, the S,S-enantio_ner of the (xsnpound of Example 1, was synthesised in analgous fashion from the sodium salt of 2(S)- acid t-butyl ester, described in EP 89308740.3.

Table 1

C0 _? H

Example 5

N-( 1-r2(R.S)-Benzyloxycarbonylpentyl1cyclopentylcarbonyl>- ervth-_o-2--Tethyl-3-styryl-j3-alanine. methyl ester l-(3-D_bτιethylan___nc)prDpyl)-3-et_hylca_± di___rLide hyd__ochloride (1.73g, 9.2πtmol) was added to a stirred mixture of 1-[2(R,S)- benzyloxyc_arbonylpentyl]cyclopentane carboxylic acid (EP 0274234, Exanple 13) (1.46g, 4.6mmol), 1-hydroxybenzotriazole (0.62g, 4.6mmol), erythro-2-π_s1±yl-3-sty_yl-i9-alanir_e, methyl ester (l.Og, 4.6mmol) and 4-metoylrr_3rphol__ne (1.0ml, 9.2mmol) in dry dichLo_x!methane (50ml) . After 16 hours at room tenperature, the reaction solution was evaporated under vacuum and the residue dissolved in ethyl acetate (50ml) . This solution was washed successively with 1M hydrochloric acid (2 x 25ml) , saturated aqueous sodium bicarbonate solution (2 x 25ml) , water (25ml) and saturated brine (25ml) , then dried (MgSO.) and evaporated under vacuum. The crude material was purifie d by chro_ratography on silica gel using a dichloranethane-hexane elution gradient (1:2 - 2:1) to provide the required product as a colourless oil (1.7 g) . Found: C,73.92; H,8.14; N,2.86. C ₃ 2 ^H 4 ₁ ^N0 5 requires C,73.96; H,7.85; N,2.70%.

Examples 6-15

The cc-ipounds of Table 2 were obtained by the general method of Exanple 5 from the appropriate 1-substituted cyclcpentane carboxylic acids and /.-alanine ester derivatives.

Table 2

Ph

Table 2 continued

Table 2 continued

(a) 1/2 H ₂ 0 (b) 1 1/2 H ₂ 0

BOC = t-Butoxycarbonyl

Example 16

N-(1-f2(R,S)-Carboxypentyl1cvclopentylc_arbonyl)-eryr_hro -2- πyathyl-3-stγryl-g-alanine

A stirred mixture of iodine (6.6g, 26mmol) and hexamethyl- disilane (4.3g, 29.3mmol) was heated at 70°C under dry nitrogen for 1.5 hours, then diluted with cyclchexene (80ml) . To this mixture was added a solution of the title product of Example 5 (1.7g, 3.3mmol) in cyclchexene (50ml) , then the resulting mixture stirred at 75°C for 4 hours.

The bulk of the cyclchexene was removed under vacuum and the residue partitioned between ether and IM aqueous sodium hydroxide solution. The aqueous phase was separated, acidified with 2M hydrochloric acid and extracted with ether (2x) . Evaporation under vacuum of the cxaribined and dried (MgSO ) ether extracts gave the required product as a beige foam (0.9g) . Found: C,68.56; H,8.17; N,3.26. C^Ii^NO^ 1/4H-.0 requires C,68.63; H,8.04; N,3.34%.

Examples 17-21 The <_x_πpσunds of Table 3 were obtained by the general method of Exanple 16 from the products of Examples 7-11 respectively.

Table 3

Table 3 continued

Example 22

N-(l-r2(R.S.-<_s__ri3oxy-3-(2-methoxyethoxy)propyl1c_v clopentyl- carbonyl>-ervthro-2-methyl-3-st_yryl-g-alan-_ne

Trifluoroacetic acid (4ml) was added to a stirred solution of the product of Exanple 6 (0.30g, 0.56mmol) in dry dic_lϊLorαmethane (4ml) . After 2 hours at room teπperature the reaction mixture was evaporated under vacuum., then most of the residual trifluoroacetic acid removed azeotropically using dic_ orσmethane (3x) . The residue was partitioned between ether and water, then the aqueous phase adjusted to pH4 with IM aqueous ammonium bicarbonate solution and further .shaking together of the ether and aqueous phases effected. The ether phase was separated and extracted with 0.5M aqueous sodium hydroxide solution (8ml) , then the aqueous phase separated and allowed to stand at room teπperaturε for 16 hours.

The sodium hydroxide solution was acidified to pH 1 with _LM hydrochloric acid, then extracted with ether (2x) . Evaporation under vacuum of the dried (MgSO ₄ ) ether extracts furnished the required product as a colourless oil (0.15g) . Found: C,62.91; H,7.64; N,2.90. C J^^K)-, ILO requires C,62.61; H,7.78; N,2.92%.

Example 23

N-(l- 2(S)-T ιtoxyc_a_-bonyl-3-(N^-met-anesulphonyl-S- lysylamino)-propyl1cyclopentylcarbonyl.-eryth-_o-2-met^yl-3- stryryl- ff-alanine

To a stirred solution of the diester from exanple 15 (0.39g, 0.5mmol) in dioxan (5ml) at room teπperature was added aqueous sodium hydroxide (0.06_πg in 1ml) and the reaction mixture was stirred overnight at room temperature. The solvent was removed under reduced pressure, and the resulting gum partitioned between ethyl acetate (50ml) and water (25ml) . The organic phase was washed with water (3 x 10ml) , dried (MgSO ) , filtered, and evaporated to yield a colourless solid (0.33g) . Oiromatography over silica gel (0.5g) eluting with ethyl acetate/hexane (1:1), adding up to 2% methanol, yielded after examination and evaporation of the appropriate fractions, the title ccπpσund as a solid (190πg). Found: C, 58.85; H, 8.02; N, 7.19%. C _3g H ₆₍₎ N ₄ O ₁₀ S requires: C, 58.92; H, 7.95; N, 7.15%.

Example 24

N-(l-f2 S)-T3utoxycarbonyl-3-(N -πret-hanesulphonyl-S- lysylam__no)-propyl1wclopentanecarboπyl)-erythro-2-methyl-3 - styryl-β-alanine 1-benzoyloxyethyl ester

To a solution of the acid obtained from exanple 23 (0.5g, 0.65_πmol) in acetonitrile (5ml) was added aqueous cesium carbonate (0.106g in 2ml) and the solution evaporated to dryness, azeotroped with toluene and taken up in N,N-d__πethylacetamide (5ml) . 1-Benzoyloxy ethyl chloride (0.12g) was added and the

reaction mixture stirred at room temperature overnight. A further 0.05g of the chloride was added and the πdxture sonicated for 2hr (terrperature rise to 40°C) . The reaction mixture was evaporated under reduced pressure, partitioned between ethyl acetate (150ml) and water (20ml) , and acidified with dilute HC1. The organic phase was washed with water, dried (MgSO ) , filtered and evaporated to yield an oil (0.6g) . <_hronatography over silica gel (60g) in hexane/ethyl acetate (2:1) and combination and evaporation of the desired fractions yielded the title coπpound as an oil (0.32g, 54%). Found: C, 61.66; H, 7.59; N, 6.04%. C ₄₇ H _6g N ₄ 0 S __equires: C, 61.82; H, 7.51; N, 6.14%.

Examples 25-28 The c_oπpσunds of Table 4 were prepared by the method of Example 24, replacing the 1-benzσyl ethyl chloride with the appropriate chloride.

Table 4

Ph

Example 29

N-,l-[2(S)- ι ιtoxyc_a_±>onyl-3-(N^-π^thanesult-honyl-S- lysylaroino)-propyl1cyclopen1_ylcarbonyl)-e_-yt___o-2-methyl -3-sty_-Vl- ff-alanine 5-indanyl ester

A π xture of the acid obtained from example 23 (0.5g, 0.65mmol), 5-indanol (0.22g, 1.6mmol), l-hydroxybenztriazole (O.llg, 0.72mmol), N-me1_hy__morpholine (0.08g, 0.85_πmol) and l-(3-di_met ylamino propyl)-3-etoylca_±»odiimide hyd__ochloride (0.16g, 0.85mmol) in methylene chloride (15ml) was stirred at room teπperature for 3 days. The reaction mixture was concentrated by evaporation in vacuo, taken up into ethyl acetate (200ml) , washed with NaHCO aq (3 x 40ml) and brine (40ml) , dried (MgSO ) , filtered and evaporated to yield an oil (0.7g) . This was c±_romatographed over silica gel (14g) in hexane:ethylacetate (2:1), and after combination of the desired fractions, evaporation yielded the title α_aτpσund as an oil (0.3g). Found: C, 63.93; H, 7.70; N, 6.25%. C ₄₇ H ₆₈ N O S requires: C, 64.06; H, 7.78; N, 6.36%.

Example 30

N-fl-f2(S.-Carboxy-3-(N^-methanesulT tonyl-S-lysylamino.- propyl1c_yclc_pentylcarbonyl)-e3_yth-_o-2-nethyl-3-styryl-g- alanine, ethyl ester. hvc_roc_hloride

A stirred, ice-cold solution of the product from Example 15 (0.95 g, 1.2 mrrol) in dry dichlorαπethane (20 ml) was saturated with dry hydrogen chloride, then the cooling bath removed. After a further 1 hour, the reaction mixture was evaporated under vacuum to afford the required product as a beige foam (0.80 g) . Found: C,53.34; H,7.28; N,7.72. C _χ H N OgS; HC1; 11/4 I^O requires C,53.52; H,7.46; N,8.05%.

Examples 31-33 The coanpounds of Table 5 were obtained by the general method of Exanple 30 from the products of Examples 12, 13 and 14 respectively.

Table 5

(a) 1/4 H ₂ 0 (b) H ₂ 0 (c) 11/4 H ₂ 0

Examples 34-39 The α_mpounds of Table 6 were made by the method of Exanple 0 from the appropriate products from Examples 24-29.

Table 6

H ₂ N

Table 6 continued

Example 40

N-(1-j ^" 2 (S.-Carboxy-3-(N^irrethanesulphonyl-S-lysylaπti-oo,- propyl1cyclopentylcarbonyl -eryrlι_X)-2--rethyl-3-sty-yl-g-alaι--ne

A mixture of the product of Example 30 (0.80g, l.lδmmol) and IM aqueous sodium hydroxide .solution (6.9ml, 6.9mmol) was stirred at room teπperature for 1 hour, then the resulting solution washed with ether (2 x 5ml) and loaded onto a column of strongly acidic ion-exchange resin. The column was washed to neutrality using distilled water, then eluted with 5% aqueous pyridine. Evaporation under vacuum of the appropriate fractions gave a glass which was dissolved in distilled water; freeze drying of this aqueous solution provided the required product as a white foam (0.59g). Found: C,53.64; H,7.59; N,8.42. C^H^N _j OgS. _{2 χ/4} - _j .0 requires C,53.65; H,7.53; N,8.63%.

Examples 41-43 The cαrpσunds of Table 7 were obtained by the general method of Example 40 from the products of Examples 31, 32 and 33 respectively.

Table 7

^

Ph

(a) 2 3/4 E^O (b) 1 1/4 H ₂ 0 (c) 2 1^0

Preparation 1 cis-3-Methyl-4-styryl-2-azetiάlinone (Methodology of D.J. Hart et al., J. Orq. Chem. , 1983, 48, 289)

To a stirred solution of όliisopropylamine (1.19g, 11.9mmol) in dry tetrahydrofuran (20ml) at -40°C under dry nitrogen was added a solution of n-butyllithium in hexane (2.5M; 4.76ml, 11.9mmol) . The resulting solution was stirred for 0.5 hours, whilst being allowed to warm to room teπperature, then cooled to -70°C before dropwise addition of a solution of ethyl propanoate (1.19ml, lqmmol) in dry tetxahydrofuran (20ml), at such a rate that the teπperature did not exceed -65°C. After a further 10 minutes, a solution of N-(trimethylsilyl)C-Lrtnamaldimine [prepared by the addition of trans cinnamaldehyde (1.32g, lOmmol) to a solution of lithium bis(trimethylsilyl)amide in tetxahydrofuran (IM; 10ml, lOmmol) under dry nitrogen at room teπperature] was added, again πa taining the internal teπperature at < 65°C, and stirring was continued for 15 minutes at -70°C before removal of the cooling bath.

The reaction mixture was stirred for a further 16 hours at room terrperature, then diluted with ether (20ml) and washed successively with IM hydrochloric acid (3 x 20ml) , saturated aqueous sodium bicarbonate solution (25ml) , and saturated brine (20ml) . Evaporation under vacuum of the dried (MgS0 ₄ ) organic solution gave a brown oil (2.0g) , which was purified by cihro atography on silica gel using an ether-hexane elution gradient (1:8 - 2:1) to give the required product (0.41g). Found: C,76.64; H,7.10; N,6.97. ^H^NO requires C,76.98; H,7.00; N,7.48%.

Preparations 2-6 The coπpounds of Table 8 were obtained by the general method of Preparation 1 using the appropriate ethyl ester. In the case of ethyl methoxyacetate the trans 0-lactam (Preparation 3) was the major product, in contrast to the other preparations where the cis isomer predominates.

Table 8

Table 8 continued

(a) 1/8 It-O

(b) 1/4 H ₂ 0

(c) 1/4 Et ₂ 0

Preparation 7 e;rvt±i--O-2-^fethyl-3-styryl-j3-alani-ne, methyl ester A inixture of the product of Preparation 1 (3.8g, l8.9ιπmol) and hydrogen chloride in methanol (2M; 75ml) was stirred at room teπperature for 16 hours, then evaporated under vacuum. The residue was partitioned between ethyl acetate and saturated aqueous sodium bicarbonate solution, then the organic phase separated, dried (MgSO ) , and evaporated under vacuum to furnish the required product as a brown oil (3.9g). Found: C,71.78; H,7.98; N,5.72. ₁₃ - ⁱ ₁ - ₇ ^m ₂ ^rec 3 ^uires C,71.20; H,7.82; N,6.39%.

Preparations 8-12 The cxsmpαunds of Table 9 were ci tained by the general method of Preparation 7 from the products of Preparations 2-6 respectively.

Table 9

Ph

(a) 1/8 H ₂ 0