IE53315B1 - Substituted dipeptides,processes for their preparation and pharmaceutical compositions containing them and their use in the inhibition of enkephalinase - Google Patents

Description

<1 53315 This invention relates to certain novel substituted dipeptides and processes for their preparation. The invention also relates to pharmaceutical compositions containing substituted dipeptides including novel dipeptides in 5 accordance with this invention.

A natural opiate receptor agonist, known as "enkephalin", which is believed to be a mixture of two peptides H-Tyr-Gly-Gly-Phe-Met-OH (methionine-enkephalin), and H-Tyr-Gly-Gly-Phe-Len-OH (leucine-enkephalin), both being subsumed 10 hereinafter under the generic name "enkephalin", has been reported to produce a profound analgesia in rats, when injected into the brain ventricles thereof, (Beluzzic et al Nature 260, 625 (1976) ). 2 (31) 258485 (32) 28 April 1981 (31) 258486 (32) 28 April 1981 (31) 258487 (32) 28 April 1981 (31) 233548 (32) 12 Feb. 1981 (31) 233549 (32) 12 Feb. 1981 -· ** S 3 31 s It is known that enkephalin, naturally produced in the body of warm-blooded animals is inactivated by enzymes of the group known as enkephalinases which are also naturally produced in such bodies. It is therefore, of interest to 5 find compounds capable of inhibiting or mitigating the above-mentioned inactivating effect of the enkephalinases.

One aspect of the present invention provides substituted dipeptides of the. formula* Sr R, I1 I5 R.— HC— N—X — CONH CH(CH-) , COR, 1 | 2 y O R2 I and the pharmaceutically acceptable salts thereof, in which R^ is hydrogen; alkyl; halogen loweralkyl; hydroxy lower-alkyl; loweralkoxy loweralkyl; aryloxy loweralkvl; amino loweralkyl; loweralkylamino loweralkyl; di-loweralkyl aminoloweralkyl; acylamino loweralkyl; 15 diarylaminoloweralkyl; arylamino loweralkyl; guanidino loweralkyl; heteroaryl; aryl; aralkyl; mercapto loweralkyl; arylthio loweralkyl; loweralkylaralkyl; alkvlthio loweralkyl; aralkyloxyalkyl; aralkylthio-alkyl; or heteroaryloxyalkyl; wherein the latter ' 20 3 radicals may be substituted with halogen, loweralkyl, loweralkoxy, hydroxy, araino/aminomethyl, carboxyl, cyano and/or. sulfamoyl; or - 53315 alkenyl (preferably loweralkenyl)substituted by a heterocyclic (e.'g. heteroaryl) group or alkyl(e.g. loweralkyl)substituted by a heterocyclic (e.g. heteroaryl) group, the 5 latter two groups optionally being Substi tuted by one or more groups chosen from loweralkyl, hydroxy, loweralkoxy, amino, loweralkylamino, di-loweralkylaraino, acy-lamino, halogeno, halogenoloweralkyl, cyano ^ and/or sulfonamide; is -COOH, COO-(loweralkyl) , -COO- (aryl lower alkyJ), -COO aryl, or the group 0=P—OR^ Kl wherein ‘ is hydrogen, lower alkyl or benzyl; and 15 R^ is hydroxy, alkoxy, benzyloxy, lower alkyl, aryl or benzyl/ R3 is hydrogen or lower alkyl; R. (CH,) R. ,4 , 4 X is -CH- or -N-wherein q is 0 or 1; 20 2.Ω& are chosen from.the groups defined for R^, indolyl (e.g. 3-indolyl), indolylalkyl, 4 53315 (e.g. 3-indolylalkyl), adamantyl (e.g. 1-adamantyl), adamadtylmethyl (e.g. 1-adamantylraethyl) amino methylphenylloweralkyl; y is zero or an integer from 1 to 3; and 5 Rg is chosen from hydroxy,lowera 1 koxy and' the group -NRgRg in .which Rg and Rg are independently hydrogen, aralkyl, or loweralkyl which together with the nitrogen atom to which they are attached may form a 4 to 6 raembered heterocyclic group, one of whose 10 members may be oxygen or sulphur; and wherein, when X is R. r -CH-, and y is zero, the following provisions apply: (1) if r2 is -COOH, -COO(loweralkyl).

-COO(arylloweralkyl) or -COO(aryl), and 15 Rg is -OH, loweralkoxy, alkylaraino, dialkylamino, aralkylamino or diaralkylamino, then R^ is chosen from G"A- in which G is 2- or 3-thienyl or 1- or 2-naphthyl and A is -CHg-- or -CH^-loweralkyl-, (@,H(CH2)n- 20 in which n is Q or an integer from 1 to.8 and m is an integer from 2 to 8, adamantyl-Diethyl,unsubstituted or substituted phenyl, or substituted phenylloweralkyl, in which the substituent(s) for the latter two groups is or are chosen from halogen, trifluororaethyl, 25 nitro, loweralkyl and loweralkoxy; further provided that 5 53315 (ii) if, when R2 and Rg are as set forth in proviso (i), and R^ is hydrogen or methyl, Rg is hydrogen and R5 is hydrogen, lower alkyl, unsubstituted phenyl, 5 benzyl, benzyl substituted by halogen, hydroxy- ' loweralkyl, loweralkoxyloweralkyl, phenoxy lower- alkyl, mercapto loweralkyl, alkylthioloweralkyl, phenylthioloweralkyl or benzyl lower alkyl» then R4 is chosen from G-A as defined above, 6 53315 ^^Vm/H^CH2V as defined above, (but excluding cyclohexyl and cyclohexyl- roetbyl) adamantyl methyl, phenyl, or phenyl lower-alkyl which latter two groups are substituted by 5 nitro, loweralkyl or lower alkoxy; (iii) if, when R^.R^.R^ and Rg are as set forth in proviso (ii) and Rg is amino then R4 cannot be the group-(C232)t-Rx in which t is 0 or 1 and R^ is hydrogen, alkyl, eyelohexyl, phenyl or phenyl sub-10 stituted by halogen, trifluoromethyl or hydroxy; (iv) when Rg is amino, R^ is hydrogen, alkyl C^-C^q, hydroxy-lower-alkyl, lower-aikoxy-lower alkyl, aryloxy-lower-alkyl, heteroaryloxy-loweralkyl, amino-lower-alkyl, loweralkyl-15 aminoloweralkyl, diloweralkylamino-lower alkyl, acylaminoloweralkyl, arylaminolower-alkyl, guanidino-lower-alkyl, alkyl substituted by a heterocyclic group, alkylthioloweralkyl, arylthioloweralkyl. arylloweralkyl, aralkyl-20 oxyloweralkyl, aralkylthioloweralkyl, in which the aryl-group of any foregoing aryl containing substituent may be substituted by halogen, loweralkyl, hydroxy, lower-alkoxy-amino,cyano or sulfon-amido; R2 is -COOH, -COO alkyl, COO aryl, 25 -COO (arylloweralkyl)), Rg is hydrogen, and R^ is hydrogen, lower—alkyl, phenyllower-alkyl, amino-methylphenyllower-alkyl, hydroxy-phenylloweralkyl, hydroxyloweralkyl, acylaminoloweralkyl, aminoloweralkyl, dimethyl 30 aminoloweralkyl, guanidinoloweralkyl, indo- lylalkyl, lower-alkylthio-lower- alkyl; then Rg cannot be a group having the structure 7 - 53313 R15 /l6 \ / (CHj I2 p R15 R16 \ / " ch2 s tCf2>P *15 \ CH Q 'V' !7Λ wherein p is 0,1 or 2 5 R15 iS 1,*βΓ°σ·Β or lower alkyl Rj_g Is lower alkyl, aryl, aralkyl or heteroaryl and Q is C3-Cg cycloalkyl; or the group v, / ‘"i’p wherein p is as defined above and r is 2 or 3. 8 One sub-class of compounds which is of Interest is that . in which : _ I4 53315 X is -CH- , y is 1,2 or 3, and R2 is -COOH, -C00-(loweralkyl), -COQ(arylloweralky]), or -COO^ryl), R3 is hydrogen; 5 R^ is chosen from hydrogen, the group Th-CH2-B- , as defined above, the group Naph-CHj-B- as defined above, 1-adamantyl methyl, cycloloweralkyl, hydroxyloweralkyl, acylaminolower-alkyl.Ce.g. acetylaminoloweralkyl), aminoloweralkyl, di-loweralkylamino loweralkyl, halogeno loweralkyl, guanidino 10 loweralkyl, imadazolyl loweralkyl, indolylloweralkvl, mercaptoloweralkyl, loweralkylthioloweralkyl, unsubstituted and subsituted phenyl, and unsubstitdted and substituted phenylloweralkyl in which the substituent(s) of the latter two groups is/are chosen from halogeno, loweralkyl, lower 15 alkoxy, trifluoromethyl and/or nitro, the group -(CH^L '"'cH-(CH,) " as defined above, and aminomethylphenyl 1oweralkyl.

Another sub-class of compounds which is of interest is that 20 in which: R, I4 X is -(CH)- and R2 is -COOH, -COO(loweralkyl), -COO(aryllower alkyl), or -COQ(aryl) and y is 0, Rj^ is H, alkyl, aralkyl, phenyl, aralky-. loxyalkyl, aralkylthioalkyl or hetero- 25 aryloxyalkyl, the latter three radicals optionally being substituted as defined » above; R3 is hydrogen; 9 53315 R4 is chosen front G— A~ as defined above (caJ~ "(ch) (ch9) — as defined above substituted or unsubstituted phenyl or substituted phenyllowerslkyl in which 5 the substituent(s) for the latter two groups is or are chosen from halogen, trifluoromethyl, nitro, loweralkyl, and loweralkoxy, with the proviso for R4 only that when Rg is the group -N (CH_) or -N Q in which V___/ 2 p \_/ 10 p is 3,4 or 5, and Q is oxygen or sulfur, R4 may also be hydrogen, loweralkyl, indolylloweralkyl, mercaptoloweralkyl, loweralkylthio-loweralkyl, guanidinoloweralkyl, imidazolyl-loweralkyl, hydroxy substituted phenyl and 15 Re is chosen from the groups defined for R, above, J 4 including those set forth in the proviso and R, is chosen from hydroxy, loweralkoxy or o the group -NR’gR’g . wherein R*g and R‘g independently are chosen from hydrogen, 20 loweralkyl, or together with the nitrogen atom to which they are attached are /-N -N^JCH2)p or -N^_Q in which p and Q are as defined above. 53315 Another sub-class of compounds of interest is that in f2 which X is -N- , y is preferably zero, R^ is as defined above with reference to formula I, with the possible exception of alkenyl substituted 5 by a heterocyclic group and alkyl substituted by a substituted heterocyclic group; =» R2 is -COO(aryl), -COO(aralkyl) or preferably -COOH or -COO(loweralkyl)i is hydrogen or loweralkyl; 10 R^ is preferably one of the groups defined for R^ in this group of compounds or CH(CH„) m 2 n as defined above, adamantyl or indolyl, Rg is preferably one of the groups defined for 15 in'this group of compound^, and Rg is preferably -OH.

Yet another sub-class of compounds of interest is that in which R2 is 0=P—OR10 , y is preferably zero, R11 R^ is as defined above with reference to formula I with the possible exception of alkenyl substituted 20 by a heterocyclic group and alkyl substituted by a „ substituted heterocyclic group; Ϊ4 f4 X is -CH- or -N- 11 - 53315 R,, R,, R_, R,n and R,. are as defined above with reference to formula I; Rj is -COOH or -EOO(loweralkyl); R, is -OH;and wherein, when R. is hydrogen, aryl or o' 4 R. |4 heteroaryl X is preferably -CH- · 5 In this Specification the following definitions apply: 1) The terms alkyl, alkoxy and alkenyl signify such groups having from 1 to 20 carbon atoms and include such groups which are straight chained, or branched chained and/or comprise one or more 10 alieyclic rings; 2) The terras lower alkyl, lower alkoxy and lower alkenyl signify such groups having 1 to 8 carbon atoms; 3) The. term aralkyl signifies such groups having 7 to 15 20 carbon atoms in which the alkyl moiety may be straight chained or branched chained and/or comprise one or more alicyclic rings; 4) Heterocyclic - embraces groups having a 4,5 or 6 member ring in which two of the members may be 20 hetero atoms, one of which is nitrogen and the other is oxygen or sulfur; 5) Heteroaryl - embraces 5 or 6 member aromatic rings 25 having 1 or 2 hetero atoms chosen from S, N and 0. 6) Heteroaralkyl - signifies alkyl groups as defined above substituted by heteroaryl as defined above; 12 53315 7) Aryl - denotes a radical derived from an aromatic hydrocarbon by the removal of one hydrogen atom and embraces phenyl, tolyl and· naphthyl, and substituted phenyl wherein the substituents are preferably chosen 5 from halogen, hydroxy, trifluoromethyl, nitro, loweralkoxyaraino, loweralkylamino, di-loweralkylamino, acylamino, loweralkyl, loweralkoxy, amino, cyano and/or sulfanamide.

Among those novel’ compounds defined by formula I; in which 10 R4 I » X is -CH- and R., is other than 0=P— OR1Q , ^11 and y is preferably zero, some which are of interest are, for example, those in which R. is chosen from 1-adamantyl, 1- or 2-naphthyl 15 methyl, 2- or 3-thienyl methyl, halogeno (e.g. fluoro) substituted benzyl, and cyclopropyl-methyl; also those in which R^ is phenyl lower-alkyl e.g. benzyl or phenyl—ethyl, or lower-alkyl, e.g. methyl or ethyl, particularly when R^ is one of the groups mentioned immediately above; and also 20 those in which Rj. is hydrogen or methyl, particularly when R^ and R^ are members of the groups defined immediately above; and also those in which R2 is -COOH or -COO(lower alkyl), e.g. -COOCHg or -COOCjHg, particularly when R^, R4 and R,. are members of the groups set forth immediately 25 above; and also those in which Rg is hydroxy or lower alkoxy, particularly when R^, R2, R^ and Rg are members of the group set forth immediately above. 13 53315 Representative of such novel compounds are the following: N-(L-l^ethoxycarbonylethyl)-L-2-thlenyl-alanylglycine, N-(Ιι-1-ethoxycarbonylethyl)-L-2-thienyl-alanyl-L-alanine, 'N- (L-l-ethoxycarbonylethyl) -Ιι-3-thienyl-alanyl-Ii-alanine, 5 N-(L-l-ethoxycarbonylethyl)-L-3-thienyl-alanylglycine, N- (L-l-carboxyethyl) -L-2- thianylalanyl-L-alanine, N-(L-l-tert-butoxycarbonylethyl)-L-2-thienylalanyl-L-alanine, N-(L-l-carboxy-3-phenylpropyl)-L-3-(3-thienyl)alanyl-L-10 alanine m.p. 192.5°C.-193°Ci N-(D,L-1-carboxy-3-phfenyIpropy1)-L-3-(2?-naphthyl)alanyl-L-alanine m.p. 149°C.-155°C; N-(L-l-ethoxycarbonyl-3-phenylpropyl)-L-3-(2'-thienyl) alanyl-L-alanine, or the maleate salt thereof 15 m.p. 124°C.-124.5°Cf N- (D-l-carboxy- 3 -phenyl propyl) -L-{4-f luqr opheny 1) alanyl-L-alanine m.p. 147°C.-152°C; N-(L-l-carboxy-3-phenylpropyl)-L-3-(1-naphthyl)alanyl-0-alanine, 20 N-(D-l-carboxy-3-phehylpropyl)-L-3-(3-thienyl)alanyl- L-alanine m.p. 163°C.-164°C.

N- (L -l-carboxy-2-(benzylthio)-ethyl)-L-phenylalanyl-β-alanine, H-(L-l-carboxy-2-phenethyl)-L-phenylalanyl-j3-alanine 25 m.p. 218°C.-219°C.

N-(L-l-carboxy-2-phenethyl)-L-phenylalanyl- -aminobutyrie- acid m.p. 209°C.-211°C. - N-(L-l_carboxy-3-phenylpropyl)-L-phenylalanyl-0-alanine hemihydrate m.p. 176°C.-190°C.

N-(L-l-carboxy-3-phenylpropyl)-D,L-3-(1-naphthyl)alanyl- : L-alanine m.p. 186°C.-194°C. and the pharmaceutically acceptable salts thereof. · 14 53315 Among the compounds of formula I in which X is R.

I4 CH2 -N- and 1*2 is carboxy or -COO (loweralkyl), is OH and y is preferably zero, which are of interest are e.g. those in which g R^ and R3 are both hydrogen and Rj is -COO(lowera1kyl)e.g. -COOCjH,.; also those compounds in which -> R^ is hydrogen, alkyl or aralkyl, R4 is 3-indolyl, aryl or heteroaryl, 10 R5 is hydrogen or loweralkyl, and R3 is hydrogen or loweralkyl; also those compounds in which R^ is aralkyl e.g. c6, R2 is carboxy, 15 R^ is aryl e.g. phenyl, and R3 and Rg are both hydrogen; also those compounds in which R^ is alkyl e.g. methyl, R2 is carboxy, 2o R3 is hydrogen, R4 is heteroaryl e.g. 4-imidazolyl, and R5 is loweralkyl e.g. methyl, and those in which R^ is methyl, phenethyl or aralkthioalkyl, 25 R2 is _C00H' R^ is phenyl and R,. is hydrogen, methyl or isobutyl; and those compounds in which 3 R^ is phenethyl or methyl, 30 R2 is -COO (loweralkyl} R3 is hydrogen, R^ is 3-indolyl, heteroaryl or aryl, and 15 5 3 315 Rg is hydrogen or lower alkyl ; Representative examples o£ such compounds are those having the following combinations of substituents: (i) Rj= hydrogen, 5 R2= -COOCjHj., r3= H, R^= 3-indolyl, Rg= H; and (ii) Rj= methyl, IQ Ry= rCOOH, R3= H R^= 4-imidazolyl, Rg= methyl. i Among the compounds of formula X in which R2 is 0=P—or^q y R11 ^ Rg is -OH and y is preferably zero, R.

I4 which are generally of interest when X is -CH- are those in which R^ is hydrogen, alkyl, aryl or aralkyl and R^ is lower alkyl,aryl, aralkyl(e.g. benzyl), heteroalkyl or heteroaryl, for instance such compounds in which R^ is 20 methyl or phenethyl, R3 is hydrogen, Rg is hydrogen or lower alkyl(e.g. methyl), R^Q is hydrogen and R^^ is hydroxy or methoxy; and those which are generally of interest when i4 X is -N- are those in which R^ is hydrogen, alkyl, aryl : and aralkyl and R^ is lower alkyl, aralkyl e.g. benzyl or 25 heteroaralkyl, for instance such compounds in which R^ is methyl or phenethyl, R3 is hydrogen and Rg is hydrogen or 16 lower alkyl e.g. methyl and R^q is hydrogen and R^ is hydroxy or methoxy. 53315 Another group of compounds of particular interest comprises compounds in which R^^ is methyl or phenethyl, R^ is benzyl, 5 Rg is hydrogen or methyl is hydrogen and R^ is methyl R. R,.

I I and X is -CH- or -N- .

We have found that the compounds as defined above with reference to Formula I including those excluded by the proviso (i) but excluding those compounds excluded by provisos 10 (ii), (iii) and (iv) are useful in inhibiting enkephalinase, as described with reference to the in vivo and in vitro tests below.

Representative examples of such compounds» in addition to those given above are as follows: N-(L-l-carboxy-3-phenylpropyl)-L-phenylalanyl-L-alanine, 15 N-(D,L-l-carboxy-ethyl)-L-phenylalanyl-L-alanine, N-(D,L-carboxy-3-phenylpropyl)-L-phenyl-alanyl glycine, N-(L-l-carboxy-3-phenylpropyl)-L-phenyl-alanyl-L-alanine, and N-(L-carboxy-2-methyl propyl)-D,L-phenyl-alanyl-L-alanine, 20 h-(L-l-ethoxycarbonyl-3-phenylpropyl)-D-phenylalanyl-L- alanine and pharmaceutically acceptable salts thereof.

The compounds of the present invention can be prepared by a number of methods as described below. 17 53315 a) One method, broadly speaking, (for producing compounds in which R2 is other than 0=P—OR^Q)comprises reducing a R11 compound having the formula: Rl_ R5 C=N—X — CONHCH(CK-) COR, R 2 y 6 A 2 5 in which R^, and Rg may include suitable protection of any reactive groups, followed by removal of any protecting groups, if necessary, so as to provide a dipeptide of the formula: Rl\ *5 CH NH — X CONHCH (CH2) CORg R2 R, I4 and, if desired, preferably when X is -CH- , when R, is -OH 6 in the resulting compound of formula B, converting R, to an 6 alkoxy group or to the group -NRgRg« The above method may be carried out by reducing a Schiff's base as it is formed in a reaction mixture from a ketone acid or ester and an amino acid or ester according to the overall reaction. 18 ' 53315 R. Η N— X — CONHCH(CH_) COR, + R.~ C=0 ^ 2 y 6 1 | £ R2 D *5 * R,— c = NH~ X — CONHCH (CH_) , COR A | 2 y 6 R2 * E In the above reductive condensation reaction X is preferably R.

I 4 -CH-.

For example, the reaction between the keto acid or ester and the primary amine C can be carried out in solution using for example water or acetonitrile as solvent under 10 substantially neutral conditions in the presence of a .suitable reducing agent, for example sodium cyanoboro-hydride.

Alternatively a Schiff’s base obtained by the reaction of the keto acid or ester C and the amino acid or ester D may 15 be catalytically reduced in the presence of hydrogen at 1-4 atmospheres; suitable catalysts are for example Raney nickel and palladium on carbon e.g. lOXPd on carbon.

Usually the group CORg will be a protected carboxyl group e.g. benzyloxycarbonyl or loweralkoxycarbonyl e.g. t-butyl-20 oxycarbonyl. In such instances the protecting group can be reduced giving a compound of formula I in which Rg is hydroxy, using conventional hydrolysis or hydrogenolysis 19 procedures, for example hydrolysis under basic conditions, for instance by reaction with sodium hydroxide in a suitable solvent. 53315 When Rg is -OH it may be converted to the group -NRgRg or 5 to an alkoxy group by conventional procedures. For example, a compound of formula I may be reacted with SOCl^ or oxalyl chloride to convert the Rg group into an activated ester group, and thereafter reacted with an amine HNRgRg or alcohol to obtain the desired compounds.

Compound C can be prepared by following the reaction sequence given below (the compounds in which X is I4 ' -CH- are exemplified.) R. R= I4 l5 - Ρ—NH — CH — C02H + H2NCH(eH2ry-CORg- £ £ ' 11 R. R_ R, Rc I4 l5 I4 I5 H2N—CHCONHCH(CH2)yCOR6 -(- p — NH— CH—OONHCH(CH2)yC9R6 C H In the above reaction sequence the amino group of the amino acid F may be protected e.g. by any conventional amino protecting group P, for e.g; benzyloxycarbonyl or t-butyloxy carbonyl. 53315 The amino acid F can be condensed with an amino ester derivative G for example using a condensing agent such as dicyclohexylcarbodiimide (DCC) or diphenylphosphoryl azide.

An activating agent, for example 1-hydrodybenzothiazole, 5 may be employed in the condensation reaction.

The amino protecting group P attached to the resulting dipeptide H is then removed by conventional treatment, for example, by treatment with an acid or by hydrogenation, for example, using hydrogen in the presence of a metal catalyst. f4 CH In the case of those compounds in which X is -tithe reduction of the compound A may’for example be-carried out after isolation of the base of formula: R. CH_R, R, lv. I 2 4 15 C=N— N- CONH-CHtCH-) COR, T u--- 2 y 6 R2 15 using a reducing agent e.g. sodium cyanoborohydride under weakly acidic conditions. The resulting compound of the formula CH,R, R, | 2 4 | 5 R.- CH-NH-N - CONH-CH(CB„) CORt 1 | 2 y t> R2 can then be treated, as required to convert Rfi/if it is a 20 carboxy protecting group, by well-known methods such as - hydrolysis or hydrogenation to yield a terminal carboxy group, and likewise Rj/if it is carboxy . protected can be converted, if desired, to carboxyl.

The starting compound T can be formed by the following 25 reaction sequence: 21 53315 0 0 ο II II II PgOC— ΝΗΝΗ_ + HC R.-* PgOC—NHN=CHR, Z 4 9 4 J K h O * ci PgOC— NHNH—CH2R4 M 0 * 5 cod Rg-C (CH2) -CH-NH2.HC1-0=C=N- CH(ffl2) —CDRg N g * o aui. r.

II I ^4 15 PgOC —NH - N —CONHCH (CH2) ^pOORg P 1 fH2R4l R5 · • H_N—N— CONH—CH{CHJ-OOR, Λ Δ Y ' Ό O II R r1c-r2 s ▼ R, CH_R, R.

K , 2 4 (5 C=N -N- CONH—CH{CH.) — .COR, / i γ o R2 t 22 53315 In the foregoing reaction sequence, an N-protected hydrazine M is prepared by condensation of a protected-carboxy substituted hydrazine with an appropriately substituted aldehyde, isolating the resulting compound L and then 5 reducing it e.g. by hydrogenation, with hydrogen using a palladium-on-carbon catalyst, or by treating L with a reducing agent such as sodium borohydride.

The resulting hydrazine M is then reacted with an appro- ' priately substituted isocyanate O to form the N-protected 10 amino peptide £ . Isocyanate 0 can be obtained by treating an oC-amino acid ester hydrochloride N with phosgene in a hydrocarbon solvent, as illustrated in the above reaction sequence, removing the solvent and distilling the residue.

The protected carbaxy.'l group on the terminal amino group of corrpound 15 £ can then be removed by any standard method such as by acid hydrolysis using acetic acid or trifluoroacetic .acid, to yield the free amine R (see for example J. Chem. Soc.

Perkin I, 246 (1976) ), which is then condensed with an appropriately substituted acid ketone or aldehyde S, under 20 basic conditions e.g.’ in the presence of sodium acetate, to yield the hydrazone T. For example, using pyruvic acid ethyl ester as ketone S yields a hydrazone T in which R^ is -CHj and R2 is -COOC^H^. in the above sequence may for example, be methoxycarbonyl. The resulting compound T can then 25 be reduced e.g. by treatment under weakly acidic' conditions with sodium cyanoborohydride,to produce compound E.

Alternatively the primary amine R can be Condensed with ketone or aldehyde S in the presence of a reducing agent such as sodium cyanoborohydride under weakly acidic conditions e.g. in the presence of acetic acid, whereby the hydrazone T will be reduced, as it is formed, to yield the compound E directly. 23 53315 b) The compounds of the present invention can also be prepared by Coupling an amino acid ester having the formula: R5 I5 H„NCH(CH0)- COR, A' l 2 y 6 — 5 with a compound of the formula: R, R,— CH—Ν' — X—COOH 1 I R2 B! in which R^, R2, Rg and R& may include suitable protection of any reactive groups, followed by removal of any protecting groups, so as to produce a dipeptide of the formula R3 rs I I R,-CH- N: — X -CONHCH(CH-) COR, X | 2 y 6 R2 C‘ and thereafter, if desired , preferably when X is R, I4 I -CH- and is other than 0=P— OR^^hen is OH, R11 converting Rg into an alkoxy group or the group -NRgR^ e.g. by the procedure described above. 24 In the case of the preparation of compounds in which X is R, |4 5 3315 -CH- and is other than 0=P—OR^q a compound B’ can R11 be produced by reductive condensation of an appropriately substituted amino acid with keto acid or ester according to 5 the reaction scheme.

R, R, l4 I4 R,~ C=0 + H NCH- C0oH -± R,— CHNHCH—C0oH > - | 2 * ' J. | 2 R2 R2 Preferably, however; the compound B’ is prepared by reductive condensation of an appropriately substituted keto acid with an amino acid ester according to the reaction I4 K.— C=0 + R.— CHNH. -k R.CHNHCHCO-H 4 | 1 | 2 1| i COOH R2 R2 These condensations may be carried out under the conditions described above in connection with the reaction between compounds D and C in method a) . The resulting compound B,’ is then coupled with an amino acid A* in which Rg is e.g. alkoxy or dialkylamino; or other non-reactive group within the above definition of Rg.

Route b) is suitable for those compounds in which R„ is i * 0=P—OR1q . A starting intermediate B1 for such compounds is, R11 25 \ 53315 e.g. the compound R.

I4 R.— CH— NH-CH — CO.H 1 I 2 0=P-ORio R11 D1 in which is alkyl or benzyl. Compound D1 can be - prepared for example by reaction of an amine R.— CH—NH9 B ' 0=P-°RlO R11 E' V with a substituted pyruvic acid of the formula 0=C—CC^H to form a Schiff's base which is reduced, preferably with sodium cvanoborohydride to form compound D'. The compound A' which is reacted in this particular procedure with 10 compound D' is for example an appropriately substituted methyl glycinate (i.e. Rg is -OMe), the conditions being e.g. as outlined above in part a) for reactions between primary amines and keto acids or esters, for instance using a condensing agent e.g. DCC.

Appropriate selection of protecting groups as Rg and R^ aiaW.es the resulting compound selectively to be de-protected at the group Rg, by conventional procedures, to provide a terminal carboxyl group in which Rg is -OH, whilst leaving the group 1*10 position if desired; if desired however, both Rg and ; 20 —OR^q can for example, be hydrolysed to hydroxy under alkaline conditions. 26 53315 Compound B‘ can be coupled with an substituted amino acid A' using standard techniques e.g. carrying out the reaction by first forming an activated ester group by reaction of B' with an activating agent such as 1-hydroxy-5 benzotriazole, followed by reaction with compound A' and treatment with a condensing agent such as 1-13- 'dimethyl-aminopropyl)-3-ethylcarbodi iraide hydrochloride· c) A further method for preparing compounds in which R. f4 X is -CH- comprises reducing a compound of the formula R. rr I4 \5 10 R,— C— N=C — CONH — CH—(CH)—COR, 1 | y o R2 X in which R^, R2, R^, R,. and Rg may include suitable protection of any reactive groups, so as to produce a compound of the formula H R, R- | 14 (5 · 15 R.— CH— N—CH-CONHCH (CH,) —COR, 1 | 2 y o R2 G· followed by removal of any protecting groups. This method is conveniently carried out by condensation of a ketone of the formula R Rr ,4 | 5 0=C— CONH— CH— (CH,)—COR, * · 4 y O Λ 20 with an amino acid or ester of the formula 27 — 53315 Η R,- C- NH~ 1 I *2 £ in the presence of a reducing agent e.g. sodium cyanoboro-hydride.

If desired, the resulting compounds G' having a terminal carfaoxy group (Rg= -OH), and preferably those in which R2 is other than 0=P—OR, _ can be further treated by procedures " I ' R11 described above to form corresponding compounds of formula I in which Rg is alkoxy or -NRgRg.

The intermediate E1 in which R^ is -OH can be prepared by following the reaction sequence given below: R,4CH NaOAC / \ R' CHO + CH-CO-H --i N O 4 |2 2 acoh \y J.

NHCOCH3 H- CH3 Rr i5 H2NCH—COORg „ n+ * 0 R ' η I5 E* f --- R’ ,CH=C— C —NHCH(CH_) COR, h2o 4 I * y 6 NHCOCH3 K’ 28 53315 in which is a group which together with a methyl group by whigh it, Is attached to the peptide chain of the resulting coii^>ound E* constitutes an R4 group.

N-Acgtyl glycine H' is condensed with an appropriately R^ 5 substituted aldehyde (preferably an aryl or heteroaryl or heteroaralkyl aldehyde) in the presence of either acetic anhydride, or of acgtic acid/sodiuru acetate, to yield the substituted azlactgne J1 which is in turn reacted with an R,j substituted glyeinate to yield the R,. substituted N-acetyl dehydro dipeptide which in the presence of aqueous acid is converted t© the corresponding R^, Rg substituted pyruvoylamino acid B'.

Alternatively, the starting compound E1 may be prepared by condensing an R^- substituted pyruvic acid with an R,.-15 substituted glyeinste ester under the influence of dicyclohexyl c§rbodiimide (DCC) followed by alkaline hydrolysis of the resulting ester according to the reaction sequences I4 j5 DCC |4 |5 0=C— C02H + H?NCH(CH2)yaas^-► 0=C— CONHCH(CH2)y CO.Rg L1 M> N1 20 Rg in compound. M1 may for example be methoxy.

Hydrolysis of an ester N1 gives the free keto acid R. Rc " ,4 ,5 0=C— CQNHCH(CH2)y COjH 29 53315 In the case of those compounds in which R2 is 0=P—OR^q *11 method c) can be practised using compounds in which R^q is other than hydrogen i.e. benzyl or lower alkyl. The corresponding compounds in which R^q is hydrogen can readily 5 be obtained by reducing when it is benzyl, and when it is alkyl by basic hydrolysis, using conventional techniques.

Intermediates which . can-be used to.produce compounds in accordance with the invention by this procedure have the formula: R-,--CH-NH„ in which R. n io 1 I 2 10 0=P-ORio R„ P is not hydrogen, and will generally be condensed with a compound of formula E' in the presence of a reducing agent such as sodium cyanoborohydride. The aforesaid intermediates can be prepared for example, by reacting 15 together an R^ substituted aldehyde, ammonia and an R^ substituted alkoxy or benzyloxy phosphonic or phosphinie acid. d) Yet a further method for the preparation of compounds of the invention, comprises reacting an amino acid or ester 20 having the formula 30 53315 H *5 R n_X_CONHCH(ch) COR, el <> Ay o S in which X, .R^ and Rg are as defined above, with a compound of the 'formula I1 Hal— CH Τ' R2 * 5 in which R^, R^, R^„ Rg-and Rg may include suitable protec tion, of any reactive groups, and Hal represents halogen e.g. chlorine or bromine, I4 If desired, preferably when X is -CH- , when Rg is (-0H) the R6 group may be converted to an alkoxy group or the 10 group -NRgRg by the procedures mentioned above.

The reaction of the alkylating agent Τ' with intermediate S' can be carried out under conventional alkylating conditions, preferably in the presence of a base, e.g. a tertiary amine or an inorganic hydroxide, carbonate or bicarbonate. The '15 reaction is usually carried out in water or an organic solvent such as dimethyl formamide or acetonitrile.

V q In the case of the compounds in which X is -N- compounds in which R3 is hydrogen may be prepared by following the ; reaction sequence given below by way of example: 31 53315 S S CH3— OC — CH—NHC N —NHj + HalCK2C02CH3 U I V * F< .¾ b ‘FA ch3o-c-ck— hhc— n—NH - CH2 - co2ch3 5 Hydrolysis of the methylated carboxyl groups yields the compounds in which Rfi is OH and R2 is -COOH. The halide is preferably chloride or bromide. e) Yet a further method is available for preparing those compounds of the invention in which Rj is Ο— P—OR^q/ *11 10 which comprises reacting a phosphite ester or phosphonate ester with an R^, disubstituted compound. as shown below: 0 *5 ii 1 Η— P —OR. . + R— CH=N-X-CONHCH(CH„) COR, 1 •‘'W * 2 y o *11 Au · I5 15 R. CH— NH— X—CONHCHCOR, 1 j 6 °=*-°*10 *11 B" 32 53315 in which R1Q is not hydrogen, and ^ORg is a protected carboxy group e.g. carbomethoxy, and any other reactive groups in R^, and Rg may be suitably protected, followed by removal of the protecting groups as required.

The resulting compound B" may be treated to deprotect the group CORg so as to provide a terminal carboxylic group (Rg=OH). Also the group R^q may if desired be replaced by hydrogen. For example, treatment of compound B" in which R& is lower alkyloxy e.g. methoxy, by hydrolysis, ^ 10 under basic conditions yields compounds of the invention in which R^q. hydrogen and Rg is -OH.

The immediately preceding method is particularly suitable for those compounds in which R, R. i4 i4 X is -N- and also those in which X is -CH- and R. 4 15 is hydrogen, aryl, heteroaryl, adamantyl or indolyl directly attached to the carbon of the peptide chain.

The present invention comprehends the -salts of those compounds of formula I containing one or more free acid or basic · groups, with inorganic or organic acids or bases. Such 20 salts include, for example, alkali metal salts e.g. sodium and potassium salts and salts of organic and inorganic acids, for example, HCl and maleic acid.

The salts may be formed by known methods, for example, by » reacting the free acid or free base forms of the product 25 with at least one equivalent of the appropriate base or acid in a solvent or reaction medium e.g. ethylacetate, in which * the salt is insoluble, or in a solvent which can be removed in vacuo or by freeze-drying. 33 53315 When the reactions described above, either for the preparation of intermediates or the final desired compounds of the invention, involve the generation of water, e.g. condensation reactions/ these reactions may be carried out 5 under azeotropic distillation with a suitable high boiling solvent e.g. toluene or xylene, or in the presence of a dehydrating agent e.g. a molecular sieve.

Various intermediates for the preparation of the compounds » of the invention by the methods described above are 10 commercially available e.g. from Chemical Dynamics Corporation or their preparation is also described in the peptide or general chemical literature e.g. J.H. Jones Comprehensive Organic Chemistry, Vol. 2. p. 819 - 823 1979; references 2 and 29-31, and Tetrahedron Letters No. 4, 1978/ p.375-378. 34 The compounds defined above may possess centres of chirality at their asymmetric -carbon atoms. 53315 Generally, the compounds produced by the processes described herein will be diastereomeric mixtures of compounds 5- which are respectively 'D' and 'L' at least at one of the said centres of chirality.

Separation of such diastereomeric mixtures may be carried out by techniques well known in the art. For example, the respective compounds can be separated by physical means 10 such as crystallization or chromatography, or by the form ation of diastereomeric salts followed by fractional crystallisation.

The invention is concerned with all stereomeric forms of the dipeptide compounds set forth herein.

Those compounds having a configuration similar to that of natural L-amino acids are preferred. Usually natural amino acids are assigned the S configuration according to the cahn-ingold Prelog system. A notable exception is the natural amino acid Cysteine which has the R configuration.

-I* S33t 5 Dipeptide compounds as defined herein inhibit the activity of enkephalinases. Some of these compounds have been found useful in inhibiting Enkephalinase A derived from the striata of rats. In in vitro tests, such compounds, g .A . when used in a concentration range of 10 to 10 M, have reduced the activity of the aforesaid enzyme by 50* or more.

The pnkephalinase used in the aforesaid tests was obtained by separation from the brains of young male Sprague-10 Dowley rats using the procedure described by C. Gorenstein et, al. in Life Sciences, vol., 25, Pages 2065-2070, pergamog Press (1979).

In this procedure the various enkephalin degrading enzyme activities in brain are resolved from one another following 15 the procedure in the "Life Sciences" publication.

The brain (minus cerebellum) from one young Sprague-Dawley rat was first homogenised in 30 volumes of 50 raM Tris buffer, pH 7.4, The resulting homogenate was centrifuged at 50,000xg for 15 mins and the pellet, constituting the membrane-bound 20 enzyme material, resuspended in Tris and washed and centrifuged as described above four times.

The membrane pellet was solubilized by incubating it for 45 mins at 37°c. in the presence of 15 volumes (based on initial brain weight) of 50mM Tris - 1* TRITON X-100 buffer 25 pH 7.4. After centrifugation at 100,000xg for 60 mins to remove non-solubilized material, the Triton-soluble supernatant was layered on a 1.5 x 30 era DEAE Sephacel column previously equilibrated with 50mM Tris - 0.1* Triton, pH 7.4. Material was eluted from the 'column using a 1 litre linear Nad gradient running from 0.0 to 0.4M. Eluant was collected 36 5331S in 7ml fractions, each of which was assayed for enkephalinase acitivity. Under these conditions enkephalinase "A" activity (dipeptidyl carboxypeptidase) is found to elute between 120 and 220ml, followed by aminopeptidase activity 5 (260 to 400ml) and finally enkephalinase "B" activity (dipeptidyl aminopeptidase) between 420 and 450ml.

In the array enkephalinase activity was monitored by radio-metry. The substrate was 3H-met-enkephalin (50.1 Ci/mmol, NEN) diluted in 0.05M Tris buffer, pH 7.4, such that the 10 final reaction mixture concentration was 40nM. Total reaction mixture volume including enzyme and substrate was 250ml.

Incubation was carried out for 90min at 37°C. To kill the reaction» tubes were transferred to a boiling water bath for 15 15min. A 4ml aliquot of the reaction mixture was then spotted on a Baker-Flex Silica Gel IB plate (20 x 20cm) along with unlabelled-standards of met-enkephalin, tyrosine, tyrosyl-glyeine, tyrosyl—glycyl-glycine) and the components co-chromatographed in an isopropanol: ethyl acetate: 20 5¾ wt/vol acetic acid solvent system (2:2:1) which is capable' of resolving met-enkephalin from its breakdown products.

Total running time was approximately 17 hours. TLC tanks were gassed with nitrogen prior to starting the run.

Following the run, the markers were visualized with ninhy-25 drin spray. These spots along with remaining plate regions were cut from the plate and the radioactivity corresponding , to each spot monitored through scintillation counting. 37 53315 Thus the amount of radioactivity in the spots containing respectively Tyr, TyrGly, TyrGlyGly, and undigested raet-enkephalin, was determined.

For each plate, the radio-activity of each spot produced 5 therefrom can be expressed as a percentage of the total radioactivity recovered from the plate.

To eliminate the effect of natural decomposition of met-enkephalin in the absence of enzymes, the percentages of the radio-activity of the Tyr, TyrGly, TyrGlyGly spots of a 10 non-enzyme blank are subtracted from the corresponding values obtained for the experimental samples from each of the runs using enzymes; the resulting values are summed to give net product percentages for each run.

In order to obtain figures for making comparative evalua-15 tions of different dipeptide species, for each species the net product percentage (P) formed in a run in the presence of a respective species and the net product percentage (A) formed in a test run in the absence of the species can be substituted into the formula (A-P)/P. The resulting 20 yalues for different species are used to provide an indication of the comparative inhibiting activity of the various dipeptides. . 38 53315 Also, in in vitro tests, such compounds, when parenterally administered in the dose range of 5 to 100 mg/kg to mice, have been observed to potentiate the analgesic effects of intracerebrally administered 'D-Ala-met-enkephalinamide. pharmaceutical compositions may be in any of the forms known in the art, for example,tablets, capsules or elixirs for oral administration, and sterile solutions or suspensions for parenteral administration.

The dosage forms are advantageously prepared using, in 10 addition to the active dipeptide, a pharmaceutically acceptable and compatible carrier or excipient, binders, preservatives, stabilizers and flavouring agents.

The dosage forms will usually be prepared such as to faci->litate the administration of the active compounds in a 15 dosage in the range 6f from 5 to 100 mg/kg, such doses being administered at intervals of 3 to 8 hours.

Typical acceptable pharmaceutical carriers for use in the formulations described above are exemplified by: sugars such as lactose, sucrose, mannitol and sorbitol; starches 20 such as corn starch, tapioca starch and potato starch; cellulose and derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and methyl cellulose; calcium phosphates such asi di-calcium phosphate and tri-calcium phosphate; sodium sulfate, calcium sulfate, polyvinyl-25 pyrrolidone, polyvinyl alcohol, stearic acid; alkaline earth metal stearates such as magnesium stearate and calcium stearate, stearic acid;vegetable oils such as peanut oil, cotton seed oil, sesame oil, olive oil and corn oil; cationic and anionic surfactants; ethylene glycol polymers; 30 beta-cyclodextrin; fatty alcohols and hydrolysed cereal solids. 39 53315 The following Examples illustrate the invention EXAMPLE 1 Preparation of L-3-(31-thienvl)alanvl-L-alanine trifluoro-acetate salt. a) ti-tert butVloxvcarbonvl-L-3-(31 thienvl) alanine. 2.0g(11.68inM) of L-3-(3-thienyl)alanine (Chemical Dynamics. Inc.) were suspended in 1.6ml triethylamine, and 2.6g of di-tertbutyl· dicarbonate were dissolved in a mixture of 22ml of diraethylformamide (DMF) and 22ml of methanol which had 10 previously been dried over a molecular sieve.

The DMF/methanol solution was added to the suspension with stirring and stirring was continued over a further period of 24 hours at ambient temperature. The resulting, homogenous solution was diluted with 330ml of water and then acidified with citric acid. The resulting solution was extracted 4 times with 80ml of ethyl acetate, the extract was washed with water, then with brine, and dried over magnesium sulphate. The solvent was evaporated to give 3.2g of the title product·. b) N-ter.tbutvl oxvcarbonyl — 1 -3- (31 -thienvl) alanvl-L-alanine ethvl ester.

The 3.2g (11.68mM) of title product from step a) were suspended, together with 1.8g of L-alanine ethyl ester, hydrochloride (Sigma Chemicals) and 3.3g hydroxybenzyltriazole in 25 a mixture of 20ml dimethylformamide and 2.0ml of triethylamine with stirring at ambient temperature. 2.5g of N-(N,N-dimethylaminopropyl)-N1-ethylcarbodiimide hydrochloride and 2ml of triethylamine were then added to the suspension and theresulting mixture was stirred overnight. 300 ml of water was then added to the reaction mixture which was thereafter extracted 4 times with 60ml ethyl acetate. 40 5331 S The combined extracts were washed twice with 70ml of IN sodium bicarbonate, twice with 30ml of IN citric acid, twice with 70ml of water, and once with 70ml brine. The resulting extract was dried over magnesium sulphate and 5 the solvent evaporated to give 5.1g of the title product. c) N-tertbutvloxvcarbonvl-L-S-(31-thienyl)alanvl-L-alanine 4.4g (11.8mM) of the title product from step b) was dissolved in 30ml of methanol. 30ml water was added to the solution followed by l.lg (14.2mM) of NaOH in the form of a 50¾ wt 10 by volume aqueous solution. The reaction mixture was stirred at ambient temperature and after one hour a sample was checked for starting material using thin layer chromatography (tic) on silica gel using a mixture of methanol, chloroform and acetic acid in a volumetric ratio of 5:95:0.5 as the 15 mobile phase. Stirring was continued and the reaction mixture monitored in this way until completion of the reaction was indicated by the absence of starting material.

The reaction mixture was then concentrated under reduced pressure and the residue was mixed with 30ml water; the 20 resulting aqueous solution was washed twice with 30ml of methylene chloride and then acidified to yield a gummy precipitate. This precipitate was extracted with ethyl acetate and the extract dried over magnesium sulfate after which the ethyl acetate was evaporated to give 5.8g of the 25 title product. d) L-3-(31-thienyl)alanvl-L-alanine trifluoroacetate.

The 5.8g of the title product from step c) was dissolved in 15ml methylene chloride with stirring. 15ml trifluoro-acetic acid was added to the solution and the mixture was ; 30 stirred for two hours at room temperature. The reaction mixture was monitored by tic on silica gel using a mixture of methylene chloride, methanol and acetic acid in a volumetric ratio of 95:5:0.5 as the mobile phase. 41 — 53315 When tic indicated the absence o£ starting material the reaction mixture was concentrated to a gummy residue which was dissolved in benzene and distilled to a residue, dissolved again in benzene and distilled yet again to give 5 7g of the title product. e) N-(l-carboxv-3-nhenvlproovl)-L-3- (3‘-thienyl)alanvl- l·-alanine A solution of L-3-(3-thienyl)alanyl-L-alanine trifluoro- : acetate (4.75g, 13.4 m.mol) and 2-oxo-4-jshenylbutyric acid 10 (11,0g, 56.1 m.mols) in water was adjusted to pH 6.8 by addition of IN NaOH, Sodium cyanoborohydride (2.2g, 34.8 m.mol) was added in a single portion to the solution and the resulting reaction mixture was stirred at room temperature for 64 hours. The pH of the resulting ]5 solution was then adjusted to 2.9 by the addition of IN HS, The gummy product which separated from the solution was washed several times with water. The gummy product was then dissolved in 20ml of acetonitrile and the solution diluted with 10ml of ether. Upon Sooling a solid separated 20 and this was collected by filtration. The mother liquors were stood in a refrigerator for two days by which time a white solid had deposited: this solid was filtered off, washed with acetonitrile and dried to yield the N-(L-l) diastereoisomer of the title compound having a m.p. of 25 192.5°C. - 193°C.

The first mentioned solid was recrystallised from ethanol to give the N-(D-l) diastereoisomer having a m.p. of 163°C.-164°C. 42 53315 EXAMPLES 2 ΤΟ 7 By following procedures similar to that of Example 1, using appropriate starting materials, the following compounds and diastereomeric mixtures were prepared: 5 2) N~(L-l-carboxy-3-phenylpropyl)-L-phenylalanyl- L-alanine, m..p. 176°C. - 179°C; 3) N-(D,L-l-carboxy-3-phenylpropyl)-L-3-(21-naphthyl) alanyl-L-alanine, m.p. 149°C.-155°C. 4) H-(L-l-ethoxycarbonyl-3-phenylpropyl)-L-3-(2’- 10 thienyl)alanyl-L-alanine maleate, m.p. 124°C—125 C.

) N- (D,L-l-carboxyl-ethyl)-L-phenylaianyl-L-alanine, m.p, 125°C.-135°C. 6) N- (D-l-carboxy-3-phenylpropyl)-L-(4—fluorophenyl) alanyl-L-alanine, m.p. 147 C.-152 C. ^ 7) N-(D,L -l-carboxy-3-phenylpropyl)-L-phenyl-alanyl- glycine, m.p. 140°C.-145°C.

' EXAMPLE 8 15g (0.07m) of phenylalanine methyl ester hydrochloride were dissolved in 400 ml methanol 2.4g (0.105m) of so-20 diura phenyl pyruvate, followed by 15g powdered 3A molecular seive (Aldrich Chemical) were then added. The slurry was stirred at ambient temperature while 6.6g (0.105m) of sodium cyanoborohydride in 75ml methanol 43 - 53315 were added over a period of 6 hours. The slurry was thereafter stirred for 2 days, then filtered and the filtrate concentrated to a syrup. 300ml of 2 1/2 per. cent, hydrochloric acid were added to the syrup over 3 . hours and the resulting white crystals filtered and dried under reduced pressure to constant weight. A representative .'sample of the dried product was chromatographed on silica gel' thin layer plates using as solvent system a chloroform: methanol: concentrated hydrochloric acid 10 mixture (50:15:1 volumetric ratio) to ascertain the approximate ratio of the diastereoisomers. The chromatogram showed approximately a 50/50 mixture of diastereoisomers with some '3-phenyl-2-hydroxy propionic acid present.

Yield: 24g; m.p. 119 -130 C.

NMR consistent with the desired product.

Upon crystallisation from 400ml ethanol 14.7g of desired compound (again in the' form of a aiastereomeric mixture) was obtained enriched with the faster moving isomer (as O determined by thin layer chromatography (tic) ) , M.P. 135-150* C. 200mg of the' enriched diastereomeric mixture was recrystallized from 3ml of methanol to yield lOOmg .crystals which 44 53315 were nearly pure faster moving isomer (as determined by tic) M.P. 169*— 172°C. 14.5g of the enriched diastereomex'ic mixture was subjected to a recrystallisation procedure using 400ml methanol but S no crystals formed. The solution was concentrated to 200ml, seeded and allowed to stand, λ gelatinous, precepitate formed which was shown by tic to be a mixture of isomers with the slower moving isomer predominant: yield 7.0g.3.0g of this product were subjected to high performance liquid 10 chromatography (hplc) on Waters Prep. 500 Equipment using two silica gel' columns in series, with a solvent system consisting of chloroform: methanol: ammonium hydroxide (500: 150:10 volumetric ratio) at a flow rate of 0.2 litres per minute.' 'Fractions 8 and 9 yielded 450mg of isomer X (see below),:, 15 Fraction 10 yielded 400mg of material which upon recrystallisation' from methanol yielded 150mg of isomer I. Fractions 12 to 15 yielded 1.2g pure isomer IX · (see below). Fraction 11 yielded a mixture of isomer I and isomer XX.

HPLC was carried out on another 3.5g of the same product 20 as in the preceding paragraph using the same solvent ratios and absorbents. Fraction 4 yielded 400mg of pure isomer I, Fraction 5 yielded l.Og of material which when recrystallised from methanol yielded 550mg of substantially pure isomer X, fraction 6 yielded 0.4g of mixture of isomer 25 X and isomer II(fractions 7 to 10 yielded 1.6g of pure isomer II Isomer I M.P. 171°-· 1730 C.

Mass Spectrum M+l 328 282 (M-COOHj 268 Formula % JH2 HC- NH — CH.COOH COOCHg Isomer II M.P.' 149°-151°C 5 Mass Spectrum M+l 328 282 (M-COOH) 268 (M-C00CH3) The filtrate obtained upon filtering the gelatinous precipitate was concentrated to 50 ml and 100 ml of acetonitrile were added 10 thereto with cooling, resulting in precipitation of 1.0 g of white crystals M.P. 167° - 170°C. Thesecrystals were recrystallised from methanol; 0.7 g of white crystals, M.P. 170°-173°C were obtained. 46 53315 L-N-[(1-carboxy-2 phenyl)ethy]3-phenylalanyT-p-alanine methyl ester l.Og (3.05idM) of isomer I were dissolved in 2Oral of dimethyl-form amide (dmf) then were added 520mg (3.4mM) of 1-hydroxy-5 benzotriazolehydrate (HOBT H,0) followed by 472mg (3.4mM) of £-alanine methyl ester hydrochloride, 1.3ml (equivalents) of N-ethylmorpholine (NEM) and 649mg (3.4iriM) of N-(3-di-methylaminopropyl) -W-ethylcarbodiimide hydrochloride (EDCL). The resulting solution' was stirred at room temperature for 48 hours and then poured into '150ml of ice water with stirring and the. aqueous solution extracted twice with 150ml of ethylether. The ethylether layer was washed three times " with 100ml of water, then with water to which a few drops of HCL had been added, and then finally with water. The 15 ether layer was dried over magnesium carbonate, filtered and the filtrate concentrated to a gum; Yield l.lg of title product.

L-N-[(1-carboxy-2-phenyl)ethy1]-phenylalany1-6-a1ani ne The gum,from the previous step' was dissolved :ui 20ml metha-20 nol and'6.61ml of IN sodium hydroxide added dropwise over a period of 10 'minutes with stirring ana external cooling.

The solution was allowed to stand overnight (15-20 hours) at room temperature. The 5 per. bent, hydrochloric acid solution was added dropwise. until the pH of the solution 25 reached 2.5 (the solution being stirred during this addition) whereupon a white solid slowly precipitated, lhe solution was filtered and the precipitate dried under reduced, pressure, at 40*C., to constant weight. 47 - 53315 • · Yield 900mgf M.P. 218-219 C.

Mass Spectrum M/e ’ 385 (M+l) ' 367 (M+l-18) 340 (M+l-COOH) 5 339 (M-45-COOH) 293 (M-91).

EXAMPLES 9 TO 15 By following procedures similar to that of Example 8 using ' appropriate starting materials, the following compounds and diastereomeric mixtures were prepared. 9) S-(L-l-carboxy-3-phenylpropyl) -D,L-3-(l- naphthyl) alanyl-L-alanine m.p. 186°C. - 194°c.

) N- (L-l-carboxy-3-phenylpropyl) -L-3- (1-naphthyl) 15 alanyl-B-alanine, 48 53315 11) N-(L-l-carboxy-2-methylpropyl)-D,L-phenyl-alanyl- L-alanine m.p. 160°C. - 172°C. 12) N-(L-l-carboxy-2-(benzylthio)-ethyl)-L-phenylalanyl- 5 β-alanine m.p. 152°C. - 154°C. 13) N-(L-l-carboxy-'5-phenylpropyl)-L-phenylalanyl-L- alanine 14) N-(L-l-carboxy-2-phenylethyl)-L-phenylalanyl-y - 10 amino butyric acid m.p. 209°Cv - 211°C.

) N-(L-l-carboxy-3-phenylpropyl)-L-phenyl-alanyl-0- alanine hemihydrate m.p. 176°C. - 190°C. 49 53315 EXAMPLE 16 N-(D,L-l-carboxvethvl)-L-ohenvlalanvlnlvcine To a solution of 0.6g {2.5m.mol) of L-phenvlalanylglycine in 5ml of IN NAOH and 20ml water was added dropwise over 5 a period of 1 hour a solution of 0.25g (2.5m.mol) 2- chloropropionic acid in 5ml of IN NaOH; during the addition the reaction solution was maintained in the temperature range of from 80°c. - 90°C, and the pH thereof in the range of 8-9. On completion of the addition the reaction 10 mixture was maintained, by heating, for 15.'.minutes at 80°C. - 90°C. and then evaporated to dryness under reduced pressure and neutralised with 5% wt/vol. aqeous hydrochloric acid. Upon re-evaporation to dryness a grey solid was obtained.

The grey solid was stirred with ether which was then decanted; and the solids were then stirred with cold ethanol. Undissolved solids were filtered from the ethanol solution which was evaporated to dryness to give the title mixture of diastereomers. m.p. 90°C. - 120°C. 50 EXAMPLE 17 53315 a) Preparation of t-butyloxycarbonyl hydrazine To a stirred solution of 0.1ml hydrazine hydrate in 15ml 5 isopropanol was added dropwise a solution of di-t-butyl dicarbonate in isopropanol (the solution having a concentration of 0.45ml of dicarbonate in 7ml of the propanol).

The addition was at the rate of one drop every 7 seconds.

The reaction was stopped after approximately 6/7ths of 10 the t-butyldicarbonate had been added.

The mother liquor was evaporated, and the resulting solid was chromatographed on silica gel using a mixture of chloroform/methanol in a volume ratio of 97:3 as the mobile phase.

Fractions 20 to 33 were collected which after working up in conventional manner yielded 0.105g of the title product as a solid m.p. 34°C. - 35°C.

Re-crystallisation from ether/petroleum ether yielded a solid with m.p. 36.5°C. - 37°C. b) Preparation of Benzaldehvde ΙινάΓΒζιηο^-^ιίνΐ.-Ητ-Ηπν^ι^-ίρ 3.47g of the title product from a) were dissolved in 30ml of ethanol with stirring and 2.79g benzaldehyde were added.

The reaction mixture was heated in a steam bath with stirring for 10 minutes then chilled in an ice water 25 bath. The resulting reaction mixture was filtered to yield the title product (3.50g). 51 The mother liquor was evaporated to a solid which after washing with hexane and filtering yielded 1.56g solids. 53313 The two fractions, were subjected to thin layer chromatography on silica gel plates using chloroform/methanol 5 in a volume ratio of 97:3 as the mobile phase.

Subsequent tests revealed that the two fractions were substantially identical products. The total yield was 5.06g. m.p. 190°C. - 194.5°C. c) Preparation of feenzvlhvdrazino-t-butvlcarboxvlate. lg of the title product from b) was dissolved in 30ml tetrahydrofuran. 0.23g. of a 5% palladium-on-carbon catalyst was added and hydrogenation was carried out for approximately 15 minutes. The reaction mixture was filtered and the mother liquor evaporated yielding 0.96g 15 of the title product. d) Preparation of jnethvl-l-isocvanato-3-methvlpropi-onate. 7g. of L-leucine methyl ester was suspended in 200ml toluene which had previously been dried over calcium 20 hydride. The suspension was stirred and an excess of phosgene gas bubbled -therethrough . After the solids had dissolved the temperature of the reaction mixture was raised to, and maintained at, reflux for a period of half an hour.

After cessation of heating nitrogen gas was bubbled through the resulting solution to remove the excess phosgene and hydrogen chloride reaction product dissolved inthe solution. After this operation the solution was evaporated and the resulting liquid product distilled. 52 53315 [α]^6 = -25.55° (C=5.7% in toluene) (literature -22.4°C.

Annalen 1952/ 575, 217) b-p. 71°C. - 72°C. (45mm Hg) e) N-t-butyloxvcarbonvl-L-a-azaphenvlalanvl-L-leucine 5 methyl ester 2.47g of benzylhydrazino-t-butylcarboxylate (product of -> step c) was dissolved in 15ml toluene with stirring.

Then 1.90g of methyl-l-isocyanato-3-methylpropionate (product of step d) was added, followed by the addition. of 1.12g of triethylamine, to the reaction solution. The resulting reaction mixture was stirred for 16 hours, after which it was diluted with 200ml of ethyl acetate, washed once with 5% wt./vol*aqueous solution of citric acid, then once with brine, and finally once with water.

The solution was dried over sodium sulphate, and, after filtering off the sodium sulphate, was concentrated to a residue which was chromatographed on 75g of silica gel using a 7/3 by volume mixture of hexane and chloroform as the eluant.

After working up in conventional manner 3.’72gi of the title product was obtained. [a]p® = "2.1° (c=3.29% in chloroform). 53 53315 f) α-azaphenvlalanvl-L-leucine methyl ester 8.0 ml of an anhydrous mixture of HCl/CHjCOOH (volumetric ratio 1/80) was cooled to 4°C. under stirring and the product of step e) was added thereto. Stirring was con-5 tinned at 4 °C. for 20 minutes after which the solvent was removed under reduced pressure. The residue was subjected to thin layer chromatography on silica gel using a chloro-form/methanol mixture (98/2 v/v) as the eluant. Appropriate fractions with corresponding rf values were combined yielding 10 0.163g of the title product [a]^ = +1.9° Mass spectrum M+293 . (-HC1) 262 (-m-OCH,) 234 (-m-COjCHg) 15 g) N- (D, L-l-ethoxvcarbonvl-2-phenvl-ethyl) -g-azaphenvl alanvl-L-leucine methvl ester 0.151g (0.458m.mol) of the title product from step f) was dissolved in 3ml methanol and 83.4mg of ethyl-4~phenyl-2-oxobutanoate was added thereto with stirring at 60°C. The 20 reaction was monitored by tic using silica gel plates and a chloroform/acetone mixture (9/1 by volume) as the eluant.

Upon substantial .completion of the reaction 70mg of sodium cyanoborohydride and 4 drops of a 10% wt/vol aqueous solution of acetic acid.were added.

The reaction was monitored by tic using silica gel plates and a mixture of chloroform and methanol (98/2, by volume) as the eluant. A further 70mg of sodium cyanoborohydride and 4 drops of acetic acid solution mentioned above were then added. 54 53315 The reaction solution was allowed to stand for 16 hours, after which the solvent was removed under reduced pressure. 40ml ethylacetate was added to the residue, the resulting solution washed with 20ml of aqueous sodium chloride and 5 then twice with 20ml water. The organic solution was dried over magnesium sulphate and filtered and the solvent removed under reduced pressure.

The residue (0.277g) was chromatographed on silica gel using a mixture of hexane/chloroform ( 7/3 by volume) as 10 the eluant. Appropriate fractions were combined according to their thin layer migration to give the title product. h) N-(D.L-l-carboxv-2-phenvl-ethvl)-a-azaphenvlalanvl- L-leucine 0.428g (0.265m.mol) of the title compound of step g) was 15 dissolved in 10ml aqueous acetone (acetone/water, 3/1 by volume) 1.06ml of aqueous sodium hydroxide solution (pontain- ing 1.06m.mol NaOH) was added to the reaction solution whilst stirring. The reaction was monitored using tic , mixture of with a'chloroform /'methanol/ammonium hydroxide in a 1:1:1 20 volumetric ratio as the eluant. An additional 0.04ml of concentrated sodium hydroxide (containing 0.4m.mol NaOH) solution was added and the monitoring of the reaction continued until the reaction was substantially complete. 10ml of water was added to the reaction mixture which was 25 extracted twice with 30ml portions of ethyl acetate. The extracts were combined, dried and concentrated to a residue.

The residue was chromatographed on silica gel using the lower phase of chloroform : methanol : ammonium hydroxide mixture (2:1:1 volumetric ratio) as the eluant.

Appropriate fractions were combined according to their rf values. The combined fractions were recrystallised from diethyl ether, the mother liquor from the first recrystallisation being further concentrated to provide a second crop of crystals. The total yield of title compound was 43 mg. . 55 53315 EXAMPLE 18 fH2C6H5 |H2CH(CH3)2 C6HsCH-NH-N-COHHCHCOOH 0=P—OH C2H5 A solution of α-azaphenylalanyl-L-leucine methyl ester " (see Example 17) and one equivalent (with respect to the 5 ester) of benzaldehyde' in anhydrous methanol is allowed to stand over a 3A type molecular sieve for 16 hours. To the resulting solution is added one equivalent (with respect to the ester) of ethyl phosphonic acid ethyl ester and a trace of sodium methoxide. The reaction solution is then ^ allowed to stand for 6 to 8 hours. The molecular sieve material is then removed by filtration and the filtrate is concentrated under reduced pressure. To the concentrated solution is added 2.1 equivalents (with respect to the ester) of sodium hydroxide in the form of a IN aqueous 15 solution, and the mixture is stirred for 6 hours at room temperature. The organic solvent is removed under reduced pressure and the residue is diluted with water.

Insoluble material is extracted with ethyl acetate and the aqueous solution is acidified to pH 2.5 by the addition of 20 IN HCl. The resulting precipitate is filtered off and dried to give a mixture of diastereomers of the title compound. 56 EXAMPLE 19 53315 (L'D) CH_- CHNHCH- CO-NHCHCOOH 3 | (L) 0=P—OH I OH (1L)-1-amino ethyl phosphonic acid {see U.S. Patent Specification 4016148) was dissolved in anhydrous methanol 5 and an equimolar quantity of thionyl chloride was added thereto a drop at a time, the solution being kept at 0°C. during the addition. After the addition was completed the resulting solution was allowed to warm to room temperature and stood at this temperature for from 6 to 8 hours. Vola-10 tiles were then removed under reduced pressure at room temperature to yield crude (1-L)-1-aminoethyl phosphonic dimethyl ester hydrochloride.

A solution was made up of the dimethyl ester and an equimolar quantity of sodium phenyl pyruvate in methanol gnd was 15 adjusted to a pH in the range of 6.5 to 7 with IN NaOH solution and treated with a 50% molar excess of sodium cyanoborohydride. The reaction mixture was stirred for 2 days at room temperature, then poured into water. The pH of the resulting aqueous mixture was then adjusted to a 20 value in the range 2 to 3, and the resulting precipitate » extracted with ethyl acetate. The extracts were washed with water and dried. Evaporation of the solvent gave the crude mono-acid of formula: ®2C6H5 CH -— CH- NHCH- CO-H 3 | o=p / N MeO OMe 57 53315 The mono-acid was dissolved in dimethylformamide together with an equivalent (with respect to the mono-acid) of L-alanine methyl ester hydrochloride, and one equivalent (with respect to the mono-acid) of N-ethylmorpholine. The resul-5 ting mixture was cooled to approximately 0°C. and one equivalent· (with respect to the mono-acid) of N-(3-dimethyl-aminopropyl) - N'-ethylcarbodiimide hydrochloride was added thereto.

The reaction mixture was allowed to warm to room temperature 10 and stirred overnight. It was then poured into water and the precipitated product extracted with ethyl acetate.

The extracts were washed with 0.5¾ wt/vol HC1, then with saturated sodium bicarbonate solution, dried and evaporated to yield a compound of the formula: ™2C6H5 CH3 15 CH,- CH-NH- CH-CO -NHCHCOOCH, 0=P OCH, I 3 0CH3 This product was dissolved in methanol, and to the resulting solution was added, drop by drop, 3.1 equivalents with respect thereto of sodium hydroxide in the form of a IN solution. After stirring overnight at room temperature 20 the reaction mixture was concentrated under reduced pressure, diluted with water and adjusted to pH. in the range 3 to 4 with IN HCl solution.

The precipitated products were filtered, washed with water and dried to give a mixture of diastereomers of the title compound. 58

Claims (5)

53315 ELAMS

1. A compound having the formula: i3 I5 R,— BC — Η —X — COKH-CB(CB,)„ COR, i | d γ b *2 I 5. and the pharmaceutically acceptable salts thereof, in which R1 is hydrogen; alkyl; halogen loweralkyl; hydroxy lower-alkyl; loweralkoacy loweralkyl; aryloxy loweralkyl; amino loweralkyl; loweralkylamino loweralkyl; di-loweralkyl aminoloweralkyl; acylamino loweralkyl;-10 diarylarainoloweralkyl; arylaaino loweralkyl; guanidine loweralkyl; heteroaryl; aryl; arielkyl; mercapto loweralkyl; arylthio loweralkyl; loweralkylaralkyl; alkylthio loweralkyl* aralkyloxyalkyl; aralkylthio-alkyl; or heteroaryloxyalkyl; wherein the latter 15 3 radicals may be substituted with halogen, loweralkyl, loweralkoxy, hydroxy, amino/ aminomethyl, carboxyl, cyano and/or sulfamoyl; or alkenyl substituted by a heterocyclic group or alkyl substituted by a heterocyclic group, the latter 20 two groups optionally being substituted by one or more groups chosen from loweralkyl, hydroxy, loweralkoxy, amino, loweralkylamino, di-loweralkylamino, acylamino, halogeno, halogenoloweralkyl, cyano and/or sulfonamide; - 53315 R2 is -COOH, COO-(loweralkyl), -COO-(aryl lower alkyl), -COO aryl, or the group 0=P—OR,A I 10 hi wherein R^q is hydrogen, lower alkyl or benzyl; and i 5 R^ is hydroxy, alkoxy, benzyloxy, lower alkyl, aryl or benzyl; R3 is hydrogen or lower alkyl; R4 (CH_) R, j4 , 2 q 4 X is -CH- or -N- wherein q is 0 or 1; R4 and Rg are chosen from the groups defined for R1, indolyl (e.g. 3-indolyl), indolylalkyl, (e.g. 3-indolylalkyl), adamantyl (e.g. 1-adamantyl), adamantylmethyl (e.g. 1-adamantylmethyl) and amino methylphenylloweralky1; 15. is zero or an integer from 1 to 3; and 53315 Rfi is chosen from hydroxy, loweralkoxy ana the group -KRgR9 in which Rg and Rg are independently hydrogen, aralkyl, or loweralkyl which together with the nitrogen atom to which they are attached may form 5 a 4 to 6 membered heterocyclic group, one of whose members nay be oxygen or sulphur; and wherein, when X is -CE- , and y is zero, the following provisions apply: i (i) if R2 15 -COOE, -COO (loweralkyl) -COO(aryl- 10 loweralkyl) or COO aryl and Rg is -05, lower alkoxy, loweralkylamino, diloweralkylamino aralkylamino or diaralkyl amino, then R^ is chosen from G-A- in which G is 2- or 3- thienyl or 1- or 2- naphthyl^and A is -CHj- or -CHj- 15 lower alkyl -, (CH_) CH(CH-) - in which n is . z ay i n 0 or an integer frSm 1 to 8, and m is an integer from 2 to 8, adamantylmethyl ,ur.sr.bf!ti- tuted or substituted phenyl or substituted phenyl lower alkyl in which the substituent(s) 20 for the latter two groups is or are chosen from halogen, trifluoromethyl, nitro, loweralkyl or loweralkoxy? further provided that (ii) if, when R^ and Rg are as setforth in proviso (i), and R^ is hydrogen or methyl, is hydrogen and 25 Rj is hydrogen, lower alkyl, unsubstituted phenyl, benzyl, benzyl substituted by halogen, hydroxy-loweralkyl, loweralkaxyloweralkyl, phenoxy loweralkyl, mercapto loweralkyl, alkylthioloweralkyl, phenylthioloweralkyl or benzyl lower alkyl, then R. is chosen from G-A as defined above, 4 .. 53315 (cH^CH(CH2)n- as defined above, (but excluding cyclohexyl and cyclohexyl-Vv'—methyl) adamantyl methyl, phenyl, or phenyl lower-alkyl which latter two croups are substituted by 5 nitro, loweralkyl or lower alkoxy; (iii) if, when ,,R^ and Rj are as set forth in proviso (ii) and Rg is amino then R^ cannot be the group-(CHj) ^-R^ in which t is 0 or 1 and R^ is .. hydrogen, alkyl, cyclohexyl, phenyl or phenyl sub-10 stituted by halogen, trifluoromethyl or hydroxy; i (iv) when R& is amino, .R^ is hydrogen, alkyl C^-ClQ, hydroxy-lower-alkyl, lower-alkoxy-lower alkyl, aryloxy-lower-alkyl, heteroaryloxy-loweralkyl, amino-lower-alkyl, loweralkyl-15 aminoloweralkyl, diloweralkylamino-lower alkyl, aeylaminoloweralkyl, arylaminolower-alkyl, guanidino-lower-alkyl, alkyl substituted by a heterocyclic group, alkylthioloweralkyl, arylthioloweralkyl, arylloweralkyl, aralkyl-20 oxyloweralkyl, aralkyithioloweralkyl, in which the aryl-group of any foregoing aryl containing substituent may be substituted by halogen,loweralkyl, hydroxy, lower-alkoxy-amino,cyano or sulfonamide; R2 is -COOH, -COO alkyl, COO aryl, 25 -COO (arylloweralkyl)), R^ is hydrogen, and R^j is hydrogen, lower—alkyl, phenyllower-alkyl, amino-methylphenyllower-alkyl, hydroxy-phenylloweralkyl, hydroxyloweralkyl, acyl-aminoloweralkyl, aminoloweralkyl, dimethyl 3q aminoloweralkyl, guanidinoloweralkyl, indo- lylalkyl,lower-alkylthio-lower-alkyl; then R^ cannot be a group having the structure 5 3 3 i S Ri5 Ae \ / CH 0 X" Ι0?A R15 R16 ... \ / CH2 s V (cf2>p '15 \ CH, Q X" lrf2’'. 5 wherein p is 0,1 or 2 r^5 is hydrogen or lower alkyl is lower alkyl, aryl, aralkyl or heteroaryl and Q is Cj-Cg cycloalkyl; /x or the group \ / (CH ) I P 10 wherein p is as defined above and r is 2 or 3j 53315 and wherein the terms alkyl, alkoxy and alkenyl and above refer to such groups having from 1 to 20 carbon atoms, said groups being straight chained, or branched chained and/or comprising one or more alicyclic rings; loweralkyl and 5 loweralkoxy indicate such groups as aforesaid but having 1 to 8 carbon atoms. 7 ✓ / / / 5331S

2. A compound as defined in Claim 1, wherein X is -CH-, y is 1,2 or

3. , R2 is -COOH, -COO (loweralkyl), -COO (arylloweralkyl) or -COO (aryl), R3 is hydrogen and R4 and R5 are chosen 5 from hydrogen, the group G - A - as defined in Claim 1, 1-adamantylmethyl, cycloloweralkyl, hydroxyloweralkyl, alkyl, acylaminoloweralkyl, aminoloweralkyl, di-lower-alkylaminoloweralkyl, halogeno-loweralkyl, guanidino-loweralkyl, 10 imidazolylloweralkyl, indolylloweralkyl, mer- capto loweralkyl, loweralkylthioloweralkyl, unsubstituted phenyl, unsubstituted or substituted phenylloweralkyl wherein the substituent(s) for the latter two groups is or are chosen from ]5 halogeno, loweralkyl, loweralkoxy, trifluoro- methyl and nitro, (CH_) CH(CH-) - wherein n and m are as defined z m . z n N___/ in Claim 1, and aminomethyl- phenylloweralkyl. 20 3. A compound as defined in Claim 1, in which R, X is -CH- , y is 2ero, and subject to provisions (i), (ii),(iii) and (iv) of „ Claim 1, R1 is H, alkyl, aralkyl, phenyl, aralkyloxy-25 alkyl, or heteroaryloxyalkyl wherein the latter three groups are optionally substi- 5331S tuted as defined in Claim 1, Rj is hydrogen; R^ is chosen from the group G-A- as defined in Claim 1, 5 (CHj ^m ) n an<3 m are as 1 >vs- \/ defined in Claim 1, sub stituted or unsubstituted phenyl or substituted phenylloweralkyl in which the substituent(s) for the latter two 10 groups is or are chosen from halogen, tri- fluoromethyl, nitro, loweralkyl, and lower-alkoxy, with the proviso for only that when Rg is the group -N (CHjIp or -N^_<2 in which 15. is 3,4 or 5, and Q is oxygen or sulfur, R^ may also be hydrogen, loweralkyl, indolyl-loweralxy, mercaptoloweralkyl, loweralkylthio-loweralkyl, guanidinoloweralkyl, imidazolyl-loweralkyl, hydroxy substituted phenyl, 20 R5 is chosen from the groups defined for R^ above, including those set forth in the proviso in this claim, and Rg is chosen from hydroxy, loweralkoxy or the group -NR'gR'g, wherein R'g and R’g indepen-25 dently are chosen from hydrogen, loweralkyl, or together with the nitrogen atom to which they are attached are 53315 or -N^_Q in which p and Q are as defined above.

4. A compound according to Claim 2 or 3, in which is chosen from loweralkyl and phenyl 5 loweralkyl, R2 is chosen from -COOH and -COO-(loweralkyl), R^ is chosen from 2- or 3-thienylmethyl, 1- or 2-naphthylmethy1, benzyl or halogeno-substi-tuted benzyl, and 10 R, is -OH or lower alkoxy, b 5. A compound according to Claim 1, wherein y is 1,2 or 3,' X is -CH- R^ is hydrogen, phenylethyl, benzyl or lower-15 alkylthioloweralkyl, 533.15 R2 is carboxy or loweralkoxy carbonyl, R3 is hydrogen, R^ is benzyl or lower alkyl, Rj is hydrogen and g Rg is hydroxy or loweralkoxy. 6. A compound according to Claim 1, the compound being N-(L-l-ethoxycarbonylethyl)-1,-2-thienyl-alanyl-glycine, 10 N-(L-l-ethoxycarbonylethyl)-L-2-thieny1-alanyl- L-alanine, N-(L-l-ethoxycarbonylethyl)-L-3-thieny1-alanyl-L-alanine, N-(L-l-ethoxycarbonylethyl)-L-3-thienyl-alanyl-15 glycirie, N-(L-l-carboxyethyl)-L-2-thienylalanyl-L-alanine or a pharmaceutically acceptable salt thereof. 68 ? i 53315 7. A compound according to Claim l,the compound being N- (L-l-carboxy-3-pbenylpropyl)-L-3-. (3-thienyl)alanyl-L-alanine N-(D,L-l-carboxy-3-phenylpropyl)-L-3-(Z-naphthyl)alanyl-5 L-aianine N-(L-l-etboxycarbonyl-3-phenylpropyl)-L-3-(2'-thienyl) alanyl-L-alanine, N-(D-l-carboxy-3-phenylpropyl) -L-{4~fluorophenyl)alanyl-L-alanine 10 N-(L-l-carboxy-3-phenyipropyl)-L-3-(1-naphthyl)alanyl- 3-alanine, N- (D-l-carboxy-3-pbehylpropyl) -l-3-(3-thienyl)alanyl-L-alanine N-(L-l-carboxy-2-(benzylthio)-ethyl) -L-phenylalanyl-A -15 alanine, N-(L-l-carboxy-2-phenethyl)-L-phenylalanyl-B-alanine N-(L-l-carboxy-2-phenethyl)-L-phenylalanyl- γ-aminobutyric-acid K- ( L-'l-carboxy-3-pbenylpropyl) -L-phenylalanyl-j3-alanine 20 hemihydrate or N-(L-l-carboxy-3-phenylpropyl)-D,L-3-(1-naphthyl)alanyl-L-alanine ' o.r a pharmaceutically acceptable salt thereof. • S 53315 8- A compound according to Claim 1, in which y is zero, R^ is as defined in Claim 1, R2 is o4_or10 R11 R3' R<' Rt‘ 0 RH an£ x £re es refined in 5 Claim 1, R, is -OH, ^ p ' provided that, when R^ is hydrooen, aryl or hetero- Γ aryl, X is -CH- . 9. A pharmaceutical composition comprising a compound of formula X as defined in Claim 1, or a salt thereof, 10 mixed with a pharmaceutically acceptable carrier or excipient. 10- . A pharmaceutical composition according to claim 9. in which the compound is N- (L-l-carboxy-3-phenyipropyl) -D.L-S- (1-naphthyl)alanyl-15 L-alanine, or N- (L-1-carboxy-3-phenylpropyl) -L-pheny 1-s.lany 1 _ g -alanine hemihydrate. 11. A compound of formula I as defined in Claim 1, including those compounds excluded from Claim 1 by 20 proviso (i) but excluding those compounds excluded s from Claim 1 by provisos (ii), (iii) or liv), for use in the inhibition of enhephalinase. Dated this 17th day of December 1981, TOMKINS & CO., A(pp*jcants' Agents, (signed) . lafc

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