CA1304540C - Substituted dipeptides, processes for their preparation and pharmaceutical compositions containing them and their use in the inhibition of enkephalinase - Google Patents

Abstract

A B S T R A C T

The invention relates to substituted dipeptides having the formula:

and the pharmaceutically acceptable salts thereof, (wherein the substituents R1, R2, R3, R5, R6, R7, R8 and R9 as well as X and y are as defined in the specification).

The invention also relates to processes for preparing such compounds and to pharmaceutical compositions containing them. The compounds particularly useful in the inhibition of enkephalinase.

Description

Sl~O

SUBSTITUTED DIPEPTIDES, PROCESSES FOR THEIR PREPARATION AND
PHARMACEUTICAL COMPOSITIONS CONTAINING THEM
AND THEIR USE IN THE INHIBITION OF
ENKEPHALINASE

This invention relates to certain novel substituted dipeptides and processes for their preparation. The invention also relates to pharmaceutical composi-tions containing substituted dipeptides including novel dipeptides in accordance with this invention.

A natural opiate receptor agonist, known as "enke-phalin", 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-Leu-OH (leucine-enkephalin), both being subsumed 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) ).

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 find compounds capable of inhibiting or mitigating the above-mentioned inactivating effect of the enkephalinases.
X

. .

One aspect of the present invention provides substituted dipeptides of the formula:

13 R~ ¦

Rl--CH--N--CH--CON CH--( C )n Rg wherein Rl is hydrogen, substituted or unsubstituted aryl, heteroaryl where the heteroatom is 0, S or N and substituted or unsubstituted lower-alkyl wherein the substituents on the substituted lower-alkyl are selected from one or more of substituted or unsubstituted aryl, heteroaryl where the hetero-atom is 0, S, or N, substituted or unsubstituted arylthio, amino, loweralkanoyl-amino, mono and di-lower alkylamino, substituted or unsubstituted aryloxy and substituted or unsubstituted aralkylthio; in which the substituents on the substituted aryl group or moiety are selected from one or more of: halogen, loweralkyl, hydroxyl, loweralkoxyl, halo-loweralkyl, nitro, mono-or diloweralkylamino, or cyano:

l3rJ~sco R2 is carbox~ C~U l~weralkyl,--COU-aryl, -CO~-aralkyl hherein the aryl can be substituted as in R , ~R l o -CGO-CH2 -O - C - loheralkyl, -CON wherein Rll Rlo and R~l are the same or differen~ and are selected ~rom hydrogen, loweralkyl, aryl, substituted aryl wherein the substitutents are as in Rl, aralkyl, substituted aralkyl wherein the substituents are on the aryl moiety and are as in Rl; or Rlo ana R
together with the nitrogen ~orm a 5-7 membered saturated ring which can also contain oxygen as part of the ring;

R3 is hydrogen or loheralk~

R4 is hydrogen, ~uranomethyl, thienylmethyl, tetrahydro-naphthylmethyl, 1- or 2-naphthylmethyl, 3-thia-naphthenylmethyl, loweralkyl, cycloloweral~yl, cyclo-loweralkylmethyl, hydroxyloweralkyl, acylamino-lower-alkyl, amino-loweralkyl, mono- or di-loweralkylamino-loweralkyl, loweralkoxyloweralkyl, aral l~oxylower_ alkyl wherein the aryl moiety can be substituted as in Rl, 3-indolyloweralkyl, benzyl, phenyl, substituted aralkyl, e.g. benzyl, aryl, e.g. phenyl, substituted aralkyl, e.g. substituted benzyl and substituted aryl, e.g. substi-tuted phenyl.
R5 is hydrogen or loweralkyl;

R6, R7 and R8 are chosen from the groups defined for R4;

Rg is chosen from lhe groups defined for R2;

n is 0, 1, 2, or 3, 2no ~hen n is 2 or 3, R7 ana ~8 can be on the same or di~erent carbon;

13~D~5.~.:0 possibly with the proviso that when n=O then one or more of the following conditions app1ies:

a) Rl is pyr~dyl, thienyl, furanyl, naphthyl, naphthyl-loweralkyl, or aralkylthio, b) R3 is loweral~yl;

c) R4 is furanomethyl, cycloloweralkyl, aryloxylower-alkyl, thienylmethyl,-tetrahydronaphthyl-methyl;
1- or 2-naphthylmethyl, 3-thianaphthenyl-~ethyl, cyclo-loweralkylmethyl, loweral}ioxylo~er-alkyl, aralkyloxy-loweralkyl, aryl, substitutea aryl, in which the substituents are one cr more of the aryl substituents oelined in Rl, substi~uted arylmethyl substituted on the aryl moiety hy one or more of the aryl substitu-ents defined in Rl ~ith the exception of hyaroxyl;
d~ R6 is acylaminoloweralkyl, mono-or ai-loweralkylamino oweralkyl cr one of the groups mentioned (in(c)) above for R4 excluding phenyl;
or a pharmaceutically acceptable~salt thereof ln this Specification the follo-~ing definitions appl~

1) ~he terms alkyl 2nd alkoxy signify such groups haYing from 1 to 2û car~on atoms ana incluae such groups which are straight chaine~, or branche~ chai~e~ ana/or comprise one or more alicyclic rings;
.

2) ~he terms ~ower alk~l and lower alkoxy signify such groups havlng 1 to 8 carbon atoms;

, 13C~45~0 3) ~rhe t ~ ara~ si gni f ~ cs s~ch oruups ha~i ng 7 tC>
20 car~n ol oms in ~h~c~ the al~ moict~ ra~ be s~ra~gh~ ch2ineo or branche~ chainea an~/~r c~m~r;Se c~ne or more a~ i c~cl i C r:ings;

4) Aryl - denotes a radical derived from an aromatic hydrocarbon by the removal of one hydrogen atom and embraces phenyl, tolyl and naphthyl.

Some preferred compounds in accordance with the invention are those in which n is 1, 2, or 3, Rl is arylloweralkyl, R2 is carboxyl, -COO-loweralkyl, or -COO-arylloweralkyl, R3 is hydrogen, R4 is benzyl, 1- or 2-naphthylmethyl, lower-alkyl, substituted or unsubstituted phenyl-loweralkyl or 2- or 3- thienylmethyl, R6, R7 and R8 are hydrogen, Rg is carboxyl, or /Rlo -CON in which one Or Rlo and R
Rll is hydrogen and the other is loweralkyl, or together with the nitrogen atom to which they are attached Rlo and R
from a 5-7 membered ring containing oxygen as a ring heteroatom, or the pharmaceutically acceptable salts thereof.
Especially preferred are the compounds defined in the preceding paragraph in which n is 1, Rl is l-naphthylmethyl, paratolylmethyl benzyl or phenylethyl, R2 is carboxy, and R4 is paratolylmethyl or benzyl, or the pharmaceutically acceptable salts thereof.

~3U~S4~

Currently moct preferred of ~he novel compounds of this in-~ention are (L)-N-(l-carboxy-3-phenylpropylj-(L)-phenylala-nyl-~-alanine and (L)-N-(l-carboxy-2-phenyl)ethyl-phenylalanyl-~-alanine, (m.pts. 202-204C and 218-219C
respectively).

Other useful novel compounds in accordance with this inven-tion are listed below (where more than one optically active isomeric form exists melting points of other such isomers are sometimes yiven in paranthesis;
N-(L-l-carboxy-3-phenylpropyl)-L-3-(3-thienyl)alanyl-L-alanine m.p. 192.5C - 193C;
N-(D,L-l-carboxy-3-phenylpropyl)-L-3-(2-naphthyl)alanyl-L-alanine m.p. 149C - 155 C;
N-(L-l-ethoxycarbonyl-3-phenylpropyl)-L-3-(2'-thienyl)-alanyl-L-alanine, or the maleate salt thereof m.p- 124C - 124.5C (100-110C);
N-(D-l-carboxy-3-phenylpropyl)-L-(4-fluorophenyl)alanyl-L-alanine m.p. 147C - 152C;
N-(L-l-carboxy-3-phenylpropyl)-L-3-(1-naphthyl)alanyl-~alanine, N-(D-l-carboxy-3-phenylpropyl(-L-3-(3-thienyl)alanyl-L-alanine m.p. 163C - 164C;
N-~L-l-carboxy-3-phenylpropyl)-L-3-(1-naphthyl)alanyl-L-alanine m.p. 155C - 156C;
N-(l-carboxy-3-phenylpropyl)-L-3-(3-thienyl)alanyl-L-alanine and the monoethanolate thereof m.p. 163C-164C;
N-(L-(l-carboxy-2-benzylthio)ethyl)-phenylalanyl-~L~-alanine and the sesequihydrate thereof m.p. 153C - 154C.

~`~ 13~45~

N-tl.-~ carbox~-~-(benz~tllio)-e~hyl)-~_phen~alanyl-~-a~anine~
~-(L-l-carb~xy_2_phenethyl)_L_phenyialan~.l_ ~ -aminobutyric-acid m.p 209 c.-211 C~(179-181C) (L-l-carbo~:y-3-p~en~lprop~ L-pheny~alan~l-B-alanine 189-19iC or the hemihydrate m.p. 176 C - 190 C.

~-(L-l-carb~x~-3_phen~1propyl)_D L-3-~1-naphth~l)alanvl-L-a~anine ~ p 186 C -194 C

(D)-N-(l-ethoxycarbonyl_3_phen lproprl)-phenylalanyl-~-ala-nine (N.P. 101-103 C) or the maleate salt thereof M.P. 94-97 C.

(D)-N-(l-carboxy-3-phenylpropyl)-phenylalanyl-~-alanine ^M.P. 183-185 C (190-192 C) N-L-(l-carboxy-3-phenylpropyl)-L-3-(2-naphthyl)alanyl -L-alanine m.p. 149-155 C.

N-[(L)-l-carboxy-2-(l-naphthyl)ethyl~ -(L)-(~-phenyl-2-amino-butyrcy~-~-alanine m.p. 206-207C.

N-(L)-(l-carboxy-2-phenylethyl)-(L)-hcmophenylalanyl-~-ala-nine m.p. 200-201C.

N-(L)-(l-ethoxycarbDnyl-3-pi~enylp-opyl)-(D)-pheny~alan ~-alanine m.p. 103-105C

N-(L)-(l_carboxy-2-phenylethyl)-(L)-phenylalanyl-~-alanine-2-phenethylamide m.p. 204-205C.

N-(L)-(l-Denzyloxycarbcnyl-2-phenylethyl)-~L)-phenylalan ~-alanlne benzylester l3a4s~0 -- 8 ~

N-(L)~ benz~o~ycarb~n~ 2-phen}~lethy~ L)-phenylalanyl-~-alanine-t2_methyl_3_tri~1uoromethyl)_anilide m.p,ll4-116C, N- [(D,L)-l-car~oxy-2-phenylethyl~ -L-1e~cyl-(L)-phenylalani-ne or the hemihydrate thereof m.p. 178-180 C.

N- [(L)-l-carbGxy-2-phenylethyl3 -(L)-leucyl-~-alanine trifluoroacetate m.p. 70-72C.

N-(L)-(l-carboxy-2-phenylethyl)-(L)-phenylalanyl-~-alanine (2-methyl-3-trifluoromethyl)- anilide m.p. 191-192C.

N-(L)-(l-benzyloxycarb~nyl-2-phenylethyl)-(L)-phenylalanyl-~-alanyl morpholine amide N-[(L)-l-carboxy-2-phenylethyl]-(L)_phen}~lalar.yl-~-alanyl-morpholine amide ~.T~. 1?.7-12~ C.
N-(L-l-ethoxycarbDny~ethyl)-L-2-thienyl-alanylglycine, N-(L-l-ethoxycarbonylethyl)-L-2-thienylalanyl-L-alanine~
N-~L-l-ethoxycarbonyletnyl)-L-3-thienylalanyl-L-alanine~
N-(L-l-ethoxycarbonylethyl)-L-3-thienylalanylglycine~
N-(L-l-ca boxye~hyl)-L-2-thienylalanyl-L-alanine~
(L-l-tert-butoxycarbonyleth~l)-L-2-thienylalanyl-L-alanine , N-(L)-l-carboxy-2-para-methylphenylethyl-(L)-phenylalanyl-~-alanine m.p. 222-224C, N-(l-car~oxy-2-(1-naphthylethyl))-(R)-phenylalanyl-~-alanine m.p. 183-187C, N-((R)-l-benzyloxycarbonyl-2-phenylethyl)-(R)-phenylalanyl-~-alanyl-2,2-dimethylpropionyloxymethyl ester, N-(S)-(l-carboxy-2-phenylethyl)-(s)-paramethylphenylalan alaninet 13~:~4S40 - 8a -and the pharmaceutically acceptable salts thereof~

We have founa that the compounds as defined above with reference to FoYmulal including those excluded by the pI'O-viso are surprisir.gly actiYe and useful in inhibiting en-kephalinase, as described wit~ reference to the in vivo and in ~itro tests below. RepresentatiYe examples of such compGunds, in addition to those given above are as follows:

~3U4S~.O

~- (L-~-carb~ 3-phcn~lprOp}.l )--L-phen~ a~ an~] -L-al anine, m.p. 176-179C.
)~-(D,L_l_car~x~ -eth~l)-L -p hen yl a~ an~l-L-alanin e, N-(D~L-carbo~-3-phen~lpropyl) -L-~h en~l -alan~l alv ci n e, N- tL-l -carbox~-3-phenvlpropyl ) - L-phen~l - a~an~ L -a] a nine, -carb~:y-2_me~hyl propyl ) - D, L-phen~ al an~Tl-L - al a nin e, and the hy~rate (m.p. 163-165 C)and hemihydrate (m.p. 160-172 C) -thereof.
N-(L)-1-carboxy-2-phenylethyl-L-leucyl-L-phenylalanine.

N-(l-carboxy-3-phenylpropyl)-L-phenylalanyl-L-alanyl 1/2 isopropanol m.p. 164-164.5C.
N-(D-l-carboxy-3-phenylpropyl)-L-phenylalanyl-L-alanine m.p. 214-215 C.
N-(L-l-ethoxycar~onyl-3-phenylpropyl)-L-phenylalanyl-L-alanine or the maleate thereof m.p. 128-130 C (115-120C.
N-(l-carboxy-3-phenylpropyl)-L-phenylalanyl-qlycine hemihydrate m.p. 140-145C.
N-(l-carb~xy-l-methyl)- L-phenylalanyl glycine and the hydrate thereof m.p. 125-132C.

and the phar~aceutically acceptable salts thereof.

13~S~.O

The compounds can be prepared by an appropriate one of the Iollo~ing metho~s (in which in the following for-l~ R2~ R3,R4, R5, R6, R7, R8 and Rg and n are as definedabove, including suit2ble protection for any functional group(s)):
a) reducing a compou~d of the formula A at the C-N double bond IR6 17 F N - 1H - CO1 CH - (C) - Rg ---~ I

A
(for producing a compoun~ of formula I in which R3 is H);

b) coupling a compound of formula A' with an amino acid B' H-NfH-tf) - Rg ~ Rl-fH-N- C~- COOH --~

A' B' c) reducing a compound of formula X at the C=N double ~ond H R R R

Rl - C - N=C - CON-CH-(C) - Rg ---t X
~or producing a comp~und of formula I in which R3 is ~ , d) alkylating an amine of formula S' with a compound of formula T' R3 R~4 15 IR7 IRl \ N - CH - CON-FH--(IC)n ~ Rg ~ HallCH ------~I

S' T' ~3(~5.~

Meth~ a) ma~ be car~ied out by reducing the SchifI's ~ase A as it is formed in a r~action mixture from a ketone of formula D and an amine of formula C according to the over-all reaction.

14 . 1~6 17 H2~-C CON CH[~)n 9 Rl 14 .
R - C - ~' C - CO~ CH(C)n -- R~
R2 R5 R~

A

~or e~ample, the reaction between the ketone D
and the primary amine C can be carriea.out in soluti~n using lor example water or acetonitrile as solvent unaer substa~tially neutral conaitions in the ~resence ~F a sUit2ble reaucing agent,for e~ample s~ium c~ranobDro-hydride ~ternatiyely a Schiff~s base obtained by the reaction o~
the keton~ D and the primary amine C may be catalytically reaucea in t~e presence of hyarogen at 1-4 atmDspheres; suitable catalysts are ~or example ~ane~
n~ckel an~ palla~ium on carb~n e ~. lO`Pd . on carb~n ~3~ S~. O

~s~c~ the group Rg wi~1 be a protected carbo~:y~ 9I-OUp e.g. benz~o~:ycarbon~,1 or lowe~a1ko~:ycarb~nyl e.g. t-b~t~
ox)carbon~l In ~uch instances the protecting group can be reàuced a~ing a compound of for~ula I in ~hich Rg is carboxyl ~sing conventional h~aro1~sis or h~àrogeno~sis procedures, fcr e~ample hydrolysis unàer basic conditions, for instance by reaction ~ith sodium hydroxide in a suitable sQlYent .

When Rg is carboxyl it may for example be converted to the ~Rl o group -CON or esterfied by an alkyl, aryl or aralkyl group by conventior.al procedures. For example, a compouna of formula I may be reacted with SOCl2 or oxalyl chloride to convert the -OH function of the Rg group into an activated ester group, and thereafter reacted with an amine HNRlo R
or an appropriate alcohol to obtain the desired compoundc.

Compound C can be prepared by following the reaction ~equence given below l4 R6 p - ~H - CH - C02H ~ H-NCH-(C) - Rg G

R4 R6 R7 l4 R6 77 H2N - C~CINCH (R)n 9 H R5 R8 - 1 3 _ :13(;~4S~ ~

~n thc~ aL~ve reac1 ~o~ se~nucnce 1~1~ ,c~Tlino grcur) oI ~?lc .~mino aci~ r ma~ be protec~e~ e.9 by an~ convcntional amino pr~ec-ting group P, e.g. benzy~o~ycarbonyl or t-b~ty~o~.y carbonyl~

The amino acid r can ~e c~n~ensed wi~h an amin~ ester àerivative G for exa~ple using a condensing agent such a5 dic~clohexylcarbDdiimiae ~DCC) ~r aiphenylph~sPhrYl azide-hn activating agent, ~or example l-hyaroaibenzothiazole~
may be emp~.oye~ in the conaensation reaction.

The amino protecting group P attachea to the resulting dipeptide H is then removea by conventional treatment, ~or example, b~ treatment with an acia or by hyarogenation, for example, using hy2rogen in the presence o~ a ~etal catalyst Method b) can be carried out e.g. by first ~orming compound B' by subjecting to reductive condensatio~ an appropriately substituted amino acid with an..appropriate ketone according to the reacti~n scheme.

- C=O ~ H2NC~ CO2H > Rl- CHNHCH - C02H

2 R2 B' Preferably~ however~the compound B~ is prepared by e~u~ti~e con~ensation o~ an appropriately substituted ketone with a primary amine according to the reaction:
R

R4 IC O Rl ~ 2 ~ RlC~NHCHC~2H

13~s,~,~,o _ 14 -~hese conde~sa~ions ~a~ be carried out under the condi~onS
~escribe~ above in connection with the react~on betwecn compoun~s D and C in metho~ a). ~he resultin~ compDuna B' is then coupled ~ith an amino compound A'`in which Rg is e.g.
alkoxy-carbonyl or dialkylamino carbonyl, or another non-reactive or suitably protected group.
Co~p~r,d ~' c3n be coupled ~ith an amino compound A' using standard techniques e'.g. carrying out the reaction by first f~rming an ac~ivated ester group by reaction of B' wit~ an activating agent such as l-hyaroxy-benzotr~azole~ followed by reaction with comnouna A' ana treatment ~ith a conaensin~ agent such as 1-~3-~~imet~yl-aminoprOpyl)-3-ethylcarbodii~ioe hyàrochl ori de is conveniently carriea out by conaensation of a ketone of Method c) the formula R14 15 l6 17 O=C - CO~ - CH - (C)n - ~9 with a primary amine of the formula Rl C NH2 R2 : F~

in the presence of a reducing agent e.g. sodium cyanoboro-hydri~e.

, - ~5 - 13~45.~1.0 ~he intc-rmc-~la~c- E~ in which .~g i~ c2rl~0~:yl can bc ~,repa-red b~ follo~.~in~ thc r~ac~ior. sequence givcn belo~.:

R' 4 ` ~
~aOAC ~ \
R~4- cH~ ~ 1~2002H A O y J' H' C~

¦6 17 Nc~- (c)-Rg xx ~ ~ O R R
H30 1l ~S 17 E~ ~ R'~CH=C- C - ~HCH(C~n Rg ~COCH3 in ~hich R'4 is a group ~hich together ~ith a ~ethyl group by ~hich it is attachea to the peptiàe chain of the result-ing.compound E' constitutes an R4 gr~up~

h'-~cet~l glycine H' is conoense~ ~ith an appr~priately R~
substituted al~ehyde (pre~erably an aryl or heteroaryl or heteroaralk~l alde~yde) in the presence of àcetic anhydride/sodium acetate, to yield the substituted azlactone J' ~hich is in turD reacte~ ~ith an amino acid XX to yield the N-~cetyl oehydro dipeptide K' ~hich in the presence of aqueous acid is converted to the corresponding pyruvoylamino acid derivative E'~

_ 16-13~45~.~

~erna~i~,ely the s~arting comF~und E~ may b~ prep2rea by condensing an R4- substituted pyruvic acid L~ with anamino acid ester Mt under the influence of dicyclo-hexyl carbodiïmi~e (DCC) followe~ b~7 alkaline h~drolysis of the resulting ester according to the reacti~n sequence:

1 4 6 1 7 DCC R,~ 1 6 1 7 O=C--C02H ~ ~ CH~1 )n 9 1 O=C-- CO~-CH(C) --Rg 5 . 8 R5 8 k' ~ N' Rg in compound M' may for example be methoxycarbonyl.

Hyarol~sis o an ester ~ oives the iree ~eto acio O=C - CONHCH(C~n ~02H

In method d) the reaction of the alkylating agent T' with intermediate S~ can be carriea out ~nder conventional alky-lating conditions, preferably in the presence of a base, e.g.
a tertiary amine or an inorganic hydroxide, carbonate or bicarbonate. The reaction is usually carried out in water or an organic solvent such as dimethyl formamide or acetoni-trile.

l ~ - 13~4S~...O

~he present invention comprehends the sa~ts of those compounoS
of ~ormula I containing one or more free acid or basic:
groups, with inorganic or organic acias or bases. S~ch salts incluae, for example, al~ali metal salts e.g. so~ium an~ pDtassium salts an~ salts of organic and inoraanic acias, f~r example, HCl and maleic acia.

~he salts may be ~orme~ ~y Xnown meth~s, for e~ample, by reacting the 'free'aci~ or ~ree base forms of the proauct ~ith at least one egui~alent of the appropriate base ~r acia in a sol~ent or reaction ~edium e.g. ethylacetate, in which the salt is insoluble, or in a solvent ~hich can be remove~
in vacuo pr by freez`e-'drying.

h~en tne reactions aescribed above, either for the prepara-ti~n of intermeoiates or the final aesired co~pounas of the invention, involve the generation of water, e g. con-densation reactionsj these reactions may be carriea o~t un~er aze~tropic distillation with a suitable high b~iling solvent e g. toluene or xylene, or in the presence of a dehydrating agent e.g. a m~lecular sieve.

, .

~ario~s intermediates for the preparation of the compounds of the invention by the meth~as de's'cribea a~ve are commercially'available e.g.-~rom Chemical ~ynamics CorpDr-ation or their preparation is also describea in the peptiae or general chemical literature e.g. ~.H. Jones ComprehensiVe Organic Chemistry, Vol. 2'. p. 819 - 823 1979; references 2 an~ 29-31, and Tetrahedron Letters N~. 4, lg78, p 375_378.

.

.. ,.~.. ..

13(~45~ 0 C~ ~on,pounns àe'ir,ed ~b~ JU~3Y p~Sr~L'SS CC~J-~J e~s 0~ C))~ t!-~t thclr ~s~ t~ c carbon a~oms.

Genera~ the co~oun~s produce~ bv the ~rocesses descri-beo herein will be diastereomeric ~ixtures ~f compounds ~hich are respecti~ely ~D~ an~ ~L~ at least at one of the sai~ centres of chirality.

Separation of such aiastereomeric ~ixtures ~a~r be carrie~
out by techniques well kno~n in the art ~or example, the respecti~e compounds can be separate~ by physical means such as crystallization or chromatography, or by the form-ation of aiastereomeric salts ~ollowe~ by ~ractional cr~stallisation.

~he inyention is concerned ~ith all stereomeric forms of the ~ipeptiae compDun~s set forth herein.

~hose compounds hauin~ a configuration si~ilar to that of natural L-amino acids are preferred. ~suall~r natural amino acids are assignea the S configuration accorain~ to the Cahn-lngola Prelog s~stem. A notable exception is the natural amino acia cysteine ~hich has the R configuration-- 19 _ 13(~S~ ~

~ )L~ à~- c~ uno~ 5 ~c~ d ~ Li~) ~J~ it ~ Vi-) o~ ~nke~halinases S~me o~ t~ese CO~D~O~naS have ~ecn ~oun~ ~se~u~ in inhibitin9 En};ephalinase ~ deri~ed Iro~
th~ striata ol rats In ~n vitro tests, s~ch compoun~5, ~hen use~ in a concentration range o~ ~0 to lC M~
ha~e re~ucea the acti~ity o~ ~e a~oresaid enzyme b~ 50 or m~re.

~he ~n};ephalinase ~sed in the aforesaia tests ~as obtain-ed by separations from ~he brains o~ young male SpragUe-le~ rats using the proce~ure aescribea by C Gorensteinet~ al. in Life Sclences, vol , 25, Paoes 2065-2~70, pergam~n Press (1979) In this proce~ure the various en~ephalin aegraaing enzyme actiYities in brain are resol~e~ fro~ one another ~ollowin~
a proce~ure describea by Gorenstein et. al. in the aforesaid Life Sciences public~tion. The brain (minus cerebellum) from one young Sprague-Da~ley rat ~-as ~irst hDm~genisea in 30 ~olumes o~ 50 n~5 Tris bu~.er, pH 7.4, The resulting homogenate was centrifugea at 50,000xg for 15 mins ana the pellet, constituting the membrane-bouna enzyme material, resuspenoea in ~ris and washea ana centri-~ge~ as oescribea above ~our ti~es.

The membrane pellet was solubilizea by incubating it ~or 4~ mins at 37-C. in the presence of 15 ~oiumes (basea on initial brain ~eight) of ~Om~5 ~ris - lX T~IT0~ X-100 buffer pH 7 4. After centrifugation at lOO,OOOxg for 60 mins to rem~ye non-solubilizea material, the Triton-soluble super-natant ~2S layerea on a 1 5 Y 30 cm DEhE Sephacel column previously equilibratea with 50~5 Tris -- 0.1~ Triton~ pH 7.4 Material ~as elutea from the co~mn using a 1 litre linear NaCl graaient running from 0.0 to 0.4~s. Eluant ~'2S collectea ;~QJe ~ c~ ~ k , ~.

~ ~o ï3(;'~S~.' 0 ; ] ) 7JIJ ~ ~ r n C ~ C } ~ ; C ? ~ C~ J )1 ~
nasc ~cit i~ity . Unt~r 1 ~esc~ c~ndi ~i ol1S er~};epha~ ir,~sc ''t."
activity (dipep~ l carb~:ypepti~ase) is foun~ to c~u~e between 120 ana 220~1, f~l~owe~ by aminDpeptiaase activit~
(260 t~ 4~ml) an~ Iinally enkephalinase ~B~' activity (dipepti~)~l aminopepti~ase) between 420 and 450m~.

~n the assay enkephalinase activity was m~nitorea by raaio-metry The substrate ~as 3H-met-en~ep~alin (50 1 Ci~mmol, ~E~) ai~utea in 0 05~ Tris bu~fer, pH 7.4, such that the ~inal reaction mixture concentration was 40nM. Total reacti~n mi~-ture ~ol~me incluaing enzyme an~ substrate was 2~0ml.

InCUbatiOn ~2S carrie~ out for s~min at 37C. ~o kill the reaction, tubes were transferrea to a boiling water bath for l~min A 4~1 ali~u~t of the reaction ~i~ture was then spDtte~ on a Baker-~lex Silica Gel lB plate (20 x 2Qcm~
along ~ith unlabelle~_stanaaras of ~et-en~ephalin, tyrosine, tyr~syl-glycine~ tyrosyl _glycyl-clycine) ana the comp~nentS
co-chromatographea in an isopropanol: ethyl acetate:
5~ ~t/yol acetic acia solvent system (2:2:1) which is capable-o~ resolYing met-enkep~alin from its breakao~n proaucts.
~otal running time was approximate~y 17 h~urs. TLC tan~s were gassea ~ith nitrngen prior to startin~ the run.
~ollo~ing the run, the markers were ~isualizea with ninhy-drin spray. ~hese spots along with remaining plate regions were cut from the plate ana the ra~ioacti~ity correspon~ing to each spot ~nitorea through scintillation counting.

S~. 0 Thus the amount of radioactivity in the spots containing respectively Tyr, TyrGly, TyrGlyGly and undigested metenkephalin, was determined.

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

To eliminate the effect of natural decomposition of metenkephalin in the absence of enzymes, the percentages of the radio-activity of the Tyr, TyrGly, TyrGlyGly spots of a 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 evaluations 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 values for different species are used to provide an indication of the comparative inhibiting activity of the various dipeptides and to establish the most active, and, therefore, preferred compounds as identified in the above description.

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 adminis-tered D-Ala-met-enkephalinamide.

13~5~

Pharmaceutical compositions including the active compounds of the invention 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 addltion to the active dipeptide, a pharma-ceutically acceptable and compatible carrier or excipient, binders, preservatives, stabilizers and flavouring agents.

The dosage forms will usually be prepared such as to facilitate the administration of the active compounds in a dosage in the range of from S 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 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 as di-calcium phosphate and tri-calcium phos-phate; sodium sulfate, calcium sulfate, polyvinyl-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 X

~3~145~ ~

-23~

anionic surfactants; ethylene glycol polymers;
beta-cyclodextrin; fatty alcohols and hydrolysed cereal solids.

The following Examples illustrate the invention.

Preparation of N-(l-carboxy-3-phenylpropyl)-L-3-(3 -thienyl)alanyl-L-alanine trifluoro-acetate salt a) N-tritertiarybutyloxycarbonyl-L-3-(3 -thienyl)alanine.

2.0g(11.68mM) 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 dimethylformamide (DMF) and 22ml of methanol (which had previously been dried over a molecular sieve.

The DMF/methanol solution was added to the sus-pension 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.

" X

, . . .

~3U~ O
- 2~-b) N-tritertiarybutyloxycarbonyl-L-3-(3 -thienyl)alanyl-L-alanine ethyl ester.
__ __ The 3.2~ (11.68mM) of title product from s-tep a) were suspended, together with 3.~g of l.-alanine ethyl ester hydro-chloride (Sigma Chemicals) and 3.3g hydroxybenzyltriazole in a mlxture of 20ml dimethylformamide and 2.Oml of triethylamine with stirring at ambient temperature. 2.5g of N-(n,N-dimethylaminopropyl)-N -ethylcarbodiimide hydro-chloride and 2ml of triethylamine were then added to the suspension and the resulting mixture was stirred overnight. 30Oml of water was then added to the reaction mixture which was, thereafter, extracted 4 times with 60ml ethyl acetate.

The combined extracts were washed twice with 70ml of lN sodium bicarbonate, twice with 30ml of lN citric acid, twice with 70ml of water and once with 70ml brine. The resulting extract was dried over magnesium sulphate and the solvent evaporated to give 5.lg of the title product.

c) N-tritertiarybutyloxycarbonyl-L-3(3 -thienyl)alanyl-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 50% wt 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 chromato-:13~45~

-24a-graphy (tlc) on silica gel using a mixture of methanol, chloroform and acetic acid in a volumetric ratio of 5:95:0.5 as the 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 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 title product.

d) L-3-(3 -thienyl)alanyl-L-alanine trifluoroacetate.

The 5.8g of the title product from step c) was dissolved in lSml methylene chloride with stirring.
15ml trifluoroacetic acid was added to the solution and the mixture was stirred for two hours at room temperature. The reaction mixture was monitored by tlc 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.

.

2~ _ 13(~45~0 ~,hc~n ~c in~ica~ed the absence ol star~in~ ma~crial th~
react;on JniXture Was concentra~. ea ~o a 9~v resiaue ~.~hi ch ~'2S dissol~e~ in benzene and distillea to a resi oue, diss~lvea again in benzene ana aistil~e~ yet again tc~ ~7ive 7g of the title pro~uct.

e) ~ carbo~y_3_~hen~.1pro~y~ 3-(3-thienv~alan~
L-a3 anine A solution of L-3-'(3-thi'enyl)alanyl- L-alanine trifluoro-acetate (4 75g, 13.9 m m~l)~'ana 2-oxo-4-phenylbutyric acia (ll.Og, 56.1 m mols) in water ~as ad~usted to p~3 6 . 8 by aadition o~ lN NaOH, Sodium cyanoborohyariae (2.?9, 34.8 m.mol) was a~ed in a single ~Drtion to the solution and the resulting reaction mixture was stirred at roDm temperature for 64 hours. The pH of the resulting s~l~tion was then adjusted to 2 9 by the addition of lN HCl ~he g~mmy product ~hich separated from the solution was washed seYeral times with water. The gummy pr~auct was - then dissolYed in 2~1 of acet'onitrile ana the solution di~te~ with lOml of ether' ~pon co~ling a solia separated -and this was collected b~ fil~ration. ~he mother liquors ~,ere stood in a refrigerator for two da~s by which time a white solid had deposited: this solid was filtered o~f, washed ~ith acetonitrile and arie~ to yield the N-(L-l) diastereoisomer of the title com~ound having a m ~ of 192.5C. - 193C.

~he first mentioned solid was recrystallisea from ethanol, 'to give the ~-(D-l)'diastereoisomer having a m p of 163C.-~64C.
,, ;
~ .
~-~: ' .

.
-13(~4S~.! O

~ ):J~ JTs ~ ~Jr) 7 B~ iol~o~ing pJ~~ce~Llres si~i~ar to that of ~amp~
using appropria~e starting materials, ~l-e ~ol~o~in9 compDun~s an~ aiastere~meric mi~tures were prepar~:

2) N-~L-l-carb~-3-phenyl~ropyl)-L-~henyla~an L-alanine, ~.p. 176 C. - i79 C;

3) ~-tD~L-l-car~oxy-3-phenylpropyl)-L-3-(2~-naohthyl) alanyl-L-alanine,' m.p. 149 C -1~5 C

4) ~-(L-l-ethoxycarbDnyl-3-phenyl~ropyl)-L-3-(2`-thienyl)alanyl-L-alanine maleate, m p 12~C -125 C

D~L-l-carboxyl-eth~Tl)-L-phenyla~-anyl-L-alanine~
m p 125C -13~C

6) N-(D-l-carboxy-3-phenylpropyl) L~(4-fluorophen~
alanyl-L-alanine, ~ p 147 C -152 C.

7) ~ ,L -l-carboxy-3-p~enylpropyl)-L-phenyl-alan glycine, m.p. 140 C.-14~ C

. .
~ E 8 - -. ' . -159 (0.07m) of phenylalanine ~ethyl es'ter hyarochloriae~ere aissolvea ~n 400ml met'ha'nol. 2.4g (~ ~) o~ so- ' dium phenyl pyruvate,'~ollo~ea by 159 powaerea 3A ~ole-culal seive '(Aldrich'Chemical) were then ~odea. ~he slurry W25 stirred at a~ient temperat~re ~hile 6 69 (D.~O~m) of so~ium c~an~bDrohydriae in 75n~ ~et~anDl , ' , .
~ , .

- 27 - 13045~0 ~ere'a~ed Dver a p~rlo~ ~I 6 hours ~he slurry was thereafte~ stirre~ f~r 2 aa~s, then ilterea ana the Ii~trate concentrate~ to a syrup. 3DDml ~ 2 1/~ per_ cent, h~idroc~ ic acia wer~e ~aaae'a to the SylUp oYer 3 h~urs and the' resulting whi'te crystals ~ilterea ana ariea un~er re~ce~ press~re to con~tant wei'ght. A represen-tative sample o~ ~he ariea proauct~~as chromatographea -on silica ~el ~hin layer plates'using as sol~ent syst~m a chl~ro~Im: ~eth'a'nDl: concen'~rate~ hydr~chl~ric acia ~ixture'~ 15-1 v~lumetric ratio) t~ ascertain the ap-pro~:imate Tatio ~f the'aiastere~i~s~mers. The chr~matD--gram sh~we~ appr~:imately a ~0/~0 mixture of'aiastereo-is~er's ~ith 's'ome 3-p~enyl-2-~y'aroxy pr~pionic acia pre-sent.
~ O
'iela: 24g; ~.p. 119 -13D C.
N~ c~nsisten't ~ith'the aesirea pro~uct.

Up~n crystallisation ~rom 400nl ethan~l 14 7y o~ aesirea comp~un~ (a~ain in the ~orm of a ~iastereo~eric n~xture) ;25 obtaine~ en~ichea ~ith the *aster ~Ting isc>~Der (as aetenDinei~ ~y thin layer chromat-~:>grapl y (tlc) ~, ~I P 13~--l~D ~
.

2~0JDg c>f the enri~he~ aiaste~eomeric ~ixture was recrys~al--lizea from 3nil c~ ~ethanc~l to yield l~n~3 crystals ~)~ic~

~ - ~8 - ~3~ 0 e J~L~l?~ ' p~'L- ~tc.l ~r ,~Jr)~ y ~';C~ 5 (3r~ t~ C) ~; . }' ~ C 9 -- 1 7 2 ~ .

14 59 oI the enriche~ ~3ias~ereo~Deric mixture ~as subjected to a ~eclystallisatiOn proce~ure usin~ 4 OOml ~nethanDl but nD crystals for~Ded. ~he solution was cc>ncentratea to 2DDJnl, seeoe~ an~ allc,we~ to stana. A gelatinous p~ecepitate ~O~Dea ~hi ch was shD~n by tlc to ~e a mi~;ture c~f isorDers wi~h the slower ~ ny isD~Der preaominant: yiel.d 7 0g-3.0g f this ~ro~3uct were Su~jectea to high perfor~nance liguia chromatography (h~lc) c~n- ~iaters Prep, ~00 EquipTDent using tw~ silica gel ~ mns in series, with a solYen~: system c~n~iisting of chloro~orm: ~ethanc~ nium hyaro~rlae (50D:
150:10 volumetric i.atio) at a ~low rate c~f 0 2 litres per ~nute. ~ractions -fi ano 9 yieldea 450mg of ~ somer I-FraCtiOn 10 Yie1ae~ 40D~D9 ~ ~Daterial ~hich upon recrystal-lisation fro~ ~ethanDl yielae~ 150mg ~I isome~ I. Fracti~ns 12 to 15 yielaea 1.29 p~re is~er il.(see below). ~raction 11 yielaed a ~ture o~ is~er I ana isomer II

HPLC was carriea out on an~ther 3.5g ~f the same pr~duct as in the pIeceding par~graph ~sing the same s~lvent ra-tios and absorbents Fracti~n 4 yielaea 400mg of pure isDmeI I, Fracti~n 5 yielae~ l ~g o~ material which when recrystalliSed ~r~ methanol- yielaed 55D~g ~ substantially pure isomer 1, fraction 6 yielaed 0_4g o~ xture o~ isomer I a~ isome~ ~ractions ~ t~ iD yielaea 1 6g o~ pure iso~er .

~sc>~Der I ~I P 171 --173 C
ass Spectrum N~l 328 282 ~ ~O~H) 268 (M-COOH3) : 236 (M~
.;................... .
: , ~

13U~5~` 0 Formul a C H2 1~3 HC~ C~.CODH

~s~Der ll M P 14~
- ~ass Spect~um }5~1 328 . .

2 8 2 ~--COD~) 2 6 8 (~--CODCH3) ~e ~iltrate ~ 'La;ne~ up~n ~:filte~ing the yelat~ nDus pIe-cipitate was oc:~ncentrate~ tc, ~ ana lDDrbl ~ acet~nitrile were a~ea there~o uith c~oling~lesul~in~ in precipitat;(~n 0~ l.Og ~ white-c-~sta~s ~ 167 -- -17D ~ ~hese crystals were lecryst-allise~ m ~Dethan~l; 0_7g ~ white crystals, .P 17;-173 C were o~ainea ~3~4S~ O
~ 30-L-N [(l-carboxy-2-phenyl)ethy~ -phenylalanyl-~-alinine methyl ester.

1.0g t3.05mM) of isomer I were dissolved in 20ml of dimethylformamide (dmf) then were added 520mg (3.4mM) of l-hydroxybenzotriazolehydrate (HOBT H2O) followed by 472mg (3.4mM) of ~-alanine methyl ester hydrochloride, 1.3ml (equivalents) of N-ethylmorpho-line (NEM) and 649mg t3.4mM) of N-(3-dimethylamino-propyl)-N-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 ether layer was dried over magnesium carbonate, filtered and the filtrate concentrated to a gum;
Yield l.lg of the title product.

L-N- ~l-carboxy-2-phenyl)ethy~ -phenylalanyl -~-alanine The gum from the previous step was dissolved in 20ml methanol and 6.6ml of IN sodium hydroxide added dropwise over a period of 10 minutes with stirring and external cooling. The solution was allowed to stand overnight (15-20 hours) at room temperature.
The 5 per. cent. hydrochloric acid solution was added dropwise until the pH of the solution reached 2.5 (the solution being stirred during this addition) whereupon a white solid slowly ,, .

13~5~

precipitated. The solution was filtered and the precipitate dried under reduced pressure, at 40C, to constant weight.

Yield 900 mg, m.p. 218 - 219C.

Mass Spectrum M/e 385 (M+l) 367 (M+1-18) 340 (M-~l-COOH) 339 (M-45-COOH) 293 (M-91) By following procedures similar to that of Example 8 using appropriate starting materials, the following compounds and diastereomeric mixtures were prepared.

9) N-(L-l-carboxy-3-phenylpropyl)-D,L-3-(l-naphthyl)alanyl-L-alanine m.p. 186C - 194C.

10) N-(L-l-carboxy-3-phenylpropyl)-L-3-(l-naphthyl)alanyl-~-alanine ~, , _ 32 - ~3~5~

( L - ~ - ca r- bc,.~ 2 ,.~T,~ t ))y ~ ~r oJ~ L- ~ n y ~ - ~ ] a L-a~anine m.p. ~60 C. - 172 C.

12) N-(L-l-carbD~-y-2-(benzy~thio)-ethyl)-L-pheny~a~an ~-a~anine ~ p 152C. - 1~9C

13) N-(L-l-carboxy-~phenylpropyl)-~-phenylalanyl-L
alanine 19) N-(L-l-carboxy_2-phenylethyl)-L-phenylalanyl-~ -amin~-~utyric acid m.p. 209~C. - 211~C.

1~) N-(L-l-carboxy-3-phenylpropyl)-L-phenyl-alanyl-~-alanine hemihyarate or the hemihydrate thereof m.p. 176 C. - 190 C.

,~, .

- ?,3 - 13(~9~5~0 ~ >,~ ']~
h~ IJ~ a_-?}~ yc~ n~ a~ an~ o] vc~ n(~ .
~1~ a So~U~ion of 0.69 (2 5m mc~l) o~ L-phen}~alany~9~ycin in 5ml of 1l~ I~AOH an~ 2ûml uat.er is aaacd drop~iSe over a perio~ o~ 1 hour a solut~on oI 0 25g (2 5m.mol ) 2-ch~oropropionic acid in 5r~l1 o~ aO~; auring the ad~3i t ion the reaction solution is ~l~aintainea in the ~emperature Iange ~>I Irom 80C _ 90C, and t.he pH thereof in the ranoe oI 8-9 C~n co3Dpletion of the aaditior~ the reacti~n roixture ~s maintainea, by heatin~ or l~.minutes at 80C _ 90C ana then evapnratea to arynes~i unaer reau-ce~ pressure ana neutralisea ~.~ith 5X wt/vol aqueous hydrochloric acia ~pon re-eva~ration tc~ aryness a grey solid is obtainea.

~he grey solid is stirrea with ether ~hich is then decantea; and the solias are then stirrea ~.~ith cold ethanol Un~issolvea solias are filterea from the ethanol solution which is evaporatea to ~3ryness to give the title mi~:ture of aiastereomers. ~n p 9D C -- 120 C

~, .

Claims (13)

1. A compound having the formula:

I

and the pharmaceutically acceptable salts thereof, in which R1 is hydrogen; alkyl; halogen-loweralkyl; hydroxy-loweralkyl; loweralkoxy-loweralkyl; aryloxy-loweralkyl; amino-loweralkyl; loweralkylamino-loweralkyl; di-loweralkyl aminoloweralkyl;
acylamino-loweralkyl; di-arylaminoloweralkyl;
arylamino-loweralkyl; guanidino loweralkyl; heteroaryl consisting of aryl where the heteroatom is 0, S or N;
aryl selected from phenyl, tolyl or naphthyl; aralkyl;
mercapto-loweralkyl; arylthio-loweralkyl; loweralkyl-aralkyl; alkylthio loweralkyl; aralkyloxyalkyl;
aralkylthio-alkyl; or heteroaryloxyalkyl;

wherein the latter 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-two heterocyclic groups optionally being substituted by one or more groups chosen from loweralkyl, hydroxy, loweralkoxy, amino, lower-alkylamino, di-loweralkylamino, acylamino, halogeno, halogenoloweralkyl, cyano and/or sulfonamido;

R2 is -COOH, -COO(loweralkyl), -COO-(aryl-loweralkyl), -COO-(aryl)-COO-CH2-O-?-loweralkyl, wherein R20 and R21 are the same or different and are selected from hydrogen, loweralkyl, aryl, aralkyl, or R20 and R21 taken together form a saturated 5-7 membered ring which can also contain oxygen as a ring heteroatom, or the group wherein R10 is hydrogen, loweralkyl or benzyl; and R11 is hydroxy, alkoxy, benzyloxy, loweralkyl, aryl or benzyl;

R3 is hydrogen or loweralkyl;

X is or wherein q is 0 or 1;

R4 is chosen from the groups defined for R1, indolyl, indolylalkyl, aminomethyl-phenylloweralkyl, 3-thianaphthenylmethyl and tetrahydronaphthyl-methyl, R5 is hydrogen or loweralkyl, Y is zero or an integer from 1 to 3;

R6, each R7 and each R8 are independently chosen from the groups defined for R4;

R9 is chosen from the groups defined for R2, and when y is 2 or 3, R7 and R8 can be on the same or different carbon atom, subject to the proviso that when X is -, y=0, and R2 is not 0=?-OR10 , then one or more of the following conditions applies:

a) R1 is pyridyl, thienyl, furanyl, naphthyllower-alkyl, naphthyl or aralkylthio;

b) R3 is loweralkyl;

c) R4 is furanomethyl, cycloloweralkyl, aryllower-alkyl, thienylmethyl, tetrahydronaphthyl-methyl;
1- or 2-naphthylmethyl, 3-thianaphthenyl-methyl, cycloloweralkylmethyl, loweralkoxyloweralkyl, aralkoxy-loweralkyl, aryl, substituted aryl in which the substitutents are one or more of:
halogen, loweralkyl, hydroxyl, loweralkoxyl, haloloweralkyl, nitro, mono- or di-loweralkylamino, or cyano, and substituted aryl methyl substituted on the aryl moiety, by one or more of the aryl substituents defined in this proviso with the exception of hydroxyl;

d) R6 is acylaminoloweralkyl, mono- or di-lower-alkylaminoloweralkyl or one of the groups mentioned above [in (c)] for R4 excluding phenyl.

2. A compound as defined in claim 1, wherein R4 is chosen from 3-indolyl and 3-indolylalkyl.

3. A compound as defined in Claim 1, in which X is --, and R1 is hydrogen, substituted or unsubstituted aryl, heteroaryl where the heteroatom is 0, S or N, and substituted or unsubstituted loweralkyl wherein the substituents on the substituted loweralkyl are selected from halogen, one or more of substituted or unsubstituted aryl, heteroaryl where the heteroatom is 0, S, or N, substituted or unsubstituted arylthio, amino, loweralkanoyl-amino, mono and di-loweralkylamino, substituted or unsubstituted aryloxy, and substituted or unsubstituted aralkylthio; in which the substit-uents on the substituted aryl groups or moieties are selected from one or more of:
halogen, loweralkyl, hydroxyl, loweralkoxyl, haloloweralkyl, nitro, mono- or di-loweralkyl-amino, or cyano:

R2 is , carboxyl, -COO loweralkyl, -COO-aryl, -COO-aralkyl wherein the aryl or aryl moiety of the aralkyl group can be substituted as in R1, -COO- CH2- 0 -- loweralkyl, wherein R20 and R21 are the same or different and are selected from hydrogen, loweralkyl, unsubstituted or substi-tuted aryl wherein the substituents are as defined in R1 in this claim, unsubstituted or substituted aralkyl wherein the substituents are on the aryl moiety and are as defined in R1 in this claim; or R20 and R21 together with the nitrogen form a 5-7 membered saturated ring which can also contain oxygen as part of the ring;

R4 is hydrogen, furanomethyl, thienylmethyl, tetrahydro-naphthylmethyl, 1- or 2-naphthylmethyl, 3-thianaph-thenylmethyl, loweralkyl, cycloloweralkyl, cyclo-loweralkylmethyl, hydroxyloweralkyl, acylamino-lower-alkyl, amino-loweralkyl, mono- or di-loweralkylamino-loweralkyl, loweralkoxyloweralkyl, aralkoxylower-alkyl wherein the aryl moiety can be substituted as defined in R1 in this claim, 3-indolylloweralkyl, aralkyl, aryl, substituted aralkyl and substituted aryl wherein substituents are as defined for aryl substitutents in R1;

R6, R7 and R8 are chosen from the groups defined for R4 in this claim; and R9 is chosen from the groups defined for R2 in this claim;
and a pharmaceutically acceptable salt thereof.

4. A compound as defined in Claim 3, in which y is 1, 2 or 3, R1 is arylloweralkyl;

R2 is carboxyl, -COO-loweralkyl, or -COO-aryllower-alkyl;

R3 is hydrogen, R4 is benzyl, 1- or 2-naphthylmethyl, loweralkyl, phenyl-loweralkyl or 2- or 3-thienylmethyl;

R5,R6,R7 and R8 are hydrogen;

R9 is carboxyl, or in which one of R20 and R21 is hydrogen and the other is loweralkyl, or together with the nitrogen atom to which they are attached R20 and R21 form a 5-7 membered saturated ring containing oxygen as a ring heteroatom; or a pharmaceutically acceptable salt thereof.

5. A compound as defined in Claim 4, wherein y is 1, R1 is 1-naphthylmethyl, paratolylmethyl, benzyl or phenylethyl;

R2 is carboxy; and R4 is paratolylmethyl or benzyl, or a pharmaceutically acceptable salt thereof.

6. The compound (N)-L-(1-carboxy-2-phenylpropyl)-(L)-phenylalanyl-.beta.-alanine, or (N)-L-(1-carboxy-2-phenylethyl)-(L)-phenylalanyl-.beta.-alanine.

7. A compound according to Claim 1, in which y is as defined in Claim 1, R1 is as defined in Claim 1;

R2 is R3,R4,R5,R6.R7,R8,R10 and R11 are as defined in Claim 1;
R9 is carboxyl;

and the pharmaceutically acceptable salts thereof.

8. A method of preparing a compound of formula I
as defined in Claim 1, which is characterised in that the compound is prepared by an approp-riate method selected from the following methods (wherein in the following formulae R1, R2, R3, R4, R5, R6, R7, R8, R9 and y are as defined for formula 1, including suitable pro-tection of any reactive group(s)):

a) reducing a compound of the formula A at the C=N double bond I

for producing a compound of formula I in which R3 is H;
b) coupling a compound of formula A' with an amino acid B' I;

c) reducing a compound of formula X at the C=N double bond I

for producing a compound of formula I in which R3 is H;

d) alkylating an amine of formula S' with a compound of formula T' I

in which Hal represents halogen;
followed by removal of any protecting groups, if necessary and thereafter, if desired, converting the resulting compound of formula I into another com-pound of formula I and/or forming a pharmaceutically acceptable salt thereof, and if desired isolating a preferred isomer.

9. A method as defined in Claim 8, which comprises using as starting compounds A; or A' and-B'; X; or S' and T'; those in which y is 1, 2 or 3, R1 is arylloweralkyl, R2 is carboxyl, -COO-loweralkyl, -coo-arylloweralkyl, R3 is hydrogen, R4 is benzyl, 1- or 2-naphthylmethyl, loweralkyl, phenyl-loweralkyl or 2- or 3-thienylmethyl, R6, R7 and R8 are hydrogen, and R9 iscarboxyl, or in which one of R10 and R11 is hydrogen and the other is loweralkyl, or together with the nitrogen atom to which they are attached R10 and R11 form a 5 to 7 membered..
ring closure containing oxygen as a ring heteroatom, wherein the substituents are as defined for aryl substituents in Claim 1.

10. A method according to Claim 9, which comprises using as starting compounds, A , A ' and B ', X , or S ' and T', those in which R1 is benzyl or phenylethyl, R2 is carboYy, and R4 is benzyl.

11. A method according to claim 10 which comprises either using as starting materials A, A' and B', X, or S' and T', those in which R1 is benzyl or phenylethyl, R2 is carboxy, R3, if present, is hydrogen, R4 is benzyl, R5, R6, R7 and R8 are hydrogen, and R9 is carboxy, n is 1 or any radicals R1, R2, R3, R4, R5, R6, R7, R8, R9 not in accordance with the first part of this claim are converted to be in accordance with the first part of this claim, followed, if desired or necessary, by isolating the S,S stereoisomer to thereby produce the compound (S)-N-(1-carboxy-3-phenylpropyl)-(S)-phenylalanyl-.beta.-alanine or (S)-N-(1-carboxy-2-phenyl-ethyl)-(S)-phenylalanyl-.beta.-alanine.

12. A method according to claim 8, which comprises condensing a ketone of formula D

R1 - with a primary amine of formula C
in a reactive medium containing a reducing agent so as to effect the reduction of the compound of formula A to the compound of formula I as it is formed therein by said condensation, wherein R1, R2, R4, R5, R6, R7, R8, R9 and y are as defined in Claim 1.

13. A method according to Claim 8, which comprises con-densing a keto acid or ester of formula E' with a primary amine of formula F

in a reactive medium containing a reducing agent so as to effect the reduction of the compound of formula X to the compound of formula I in said reactive medium as it is formed by the aforesaid condensation, wherein R1, R2, R4, R5, R6, R7, R8, R9 and y are as defined in Claim 1.