CA1250091A - Substituted dipeptides, methods for their production, pharmaceutical compositions containing them, method for making such pharmaceutical compositions - Google Patents

Abstract

A B S T R A C T

Dipeptides are disclosed having the formula I
I
in which R1,R2,R3,R4,R5 and R6 are various substituents, p is 0,1 or 2 and the asterisks indicate chiral centres.

The compounds inhibit the action of enkephalinase in mammals.

Pharmaceutical compositions containing the above compounds are described.

Description

~ ~s~l~g~ :

'SUB'STITUTED DIPEPTIDE'S, ME~HODa FOR THEIR PRODUCTION, -PHARMACEUTI'CAL COMPOSITIO~a CONTAINING THEM, METHOD FOR MAKING SUCH PHAR~.~EUTICAL COMPOSITION

Enkephalin is a natural opiate receptor agonist and is believed to be a mixture of two pentapeptides:
H-Tyr-Gly-Gly-Phe-Met-OH (methionine-enkephalin), and H-Tyr-Gly-Gly-Phe-Leu-OH (leucine-enkephalin). Herein-`~ after, the name enkephalin is used generically to em-- brace all such compounds.
' It has been reported by Beluzzi et al., Nature, 260, 625 (1976), that when enkephalin is injected into the b-atn ventriclP. of rats. a pro_ound analgesia is ob-tained. It is also known in the art that enkephalin - is acted upon by a group of enz~mes-known generically as enkephalinases, which are 21so~naturally occurr1ng and is inactivated thereby. ~ ~ ;

European Patent Application No~. 79105015.6, publica-tion No. 12401 discloses certain d1peptide derivati- ;
`~ ~ ves which are described as pos~essing antihyperten-~ ~ 20 sive effects.
~:
European Patent Application'No. 81110337.3 publica-tion No. 54862 discloses certain dipeptide compounds ~ ' :: ~ ,. ~ :

:` :

Zs~ g3L

which possess an enkephalinase inhibitory activity.

The present invention comprises compounds having the formula I

R1f*H-NH-f*H-CONH~(CH2)p-C*-COR6 I
:~ - COR2 R3 R5 or a mixture of enantiomers o- diasterioisomers con-taining the s~me, and the pharmaceutically acceptable salts thereof wherein:
Rl is alkyl having .rom l to 6 carbon ato~s, ada-mantylmethyl, cycloalkylmethyl ha~ing from 4 to 8 , carbon atoms or A-Xm-CnH2n- wherein X is oxygen or :~ sulfur, A is phenyl which may be substituted wi~h the group Y, wherein Y is haloaen, hvdroxy, trifluoro-methyl, alkoxy having from 1 to 6 carbon atoms, alkyl havinq from l to 6 carbon atoms, 2- or 3-furanyl, 2 or 3-thienyl or phenyl [whlch may be substituted : . with halogen, hydroxy, trifluoromethyl, alkoxy having from l to 6 carbon atoms or alkyl naving ,rom l to 6 carbon atomsl, benzyl [the phenyl ring of which may :~
be substituted with the group Y, 25 defined~above3, l- or 2-naphthyl, 2- or 3-furanyl or 2- or 3-thienyl;
. :
: . ~

::

; ' lZSU~9~L
.

m is 0 or 1 an~ n is 0, 1, 2, 3, or 4;
R2 and R6 may be the same or different and are hydroxy, alkoxy havinc _om 1 to & carbon z~oms, B-Xm-CnH2n-O- wherein B is phenvl ~hich may be sub-stituted with the srou? ~', as de ined herein] or 1-or 2-naphthyl, X, m, and n a_e as defined herein pro-vided that when n=0 also m=O; ~X~2~X~a ~ l in which the : alkyl group has 1 to 6 carbor. atoms, -OCH2CO~phenyl ~the phenyl ring of which may be substituted with the group Y, as defined above], l-glycer~l, . o R R
x o ; ~- or -OCH -CH-CH

~ wherein R7 is hydrogen, alkyl having from 1 to 6 car-:~: bon ~toms, or p~enyl wrich m~y be substituted w~th the group Y, as defined above, and ~8~is hydrogen or alkyl ~ 15 having from 1 to 6 carbon atoms; : :
~:~ R2 may also be -NR7R~ wherein R7and R8 are as ~; defined above;
R3 is alkyl having from l to 6 carbon atoms, ::~:
~: cycloalkylmethyl having from 4 to ô carbon atoms, 2- ~ : or 3- thienylmethyl, 2- or 3-furanylmethyl, 1- or 2-na~hthy~methyl, or benzyl the phenyl ring of which may be substituted:with the gro:up Y,~as defined abbve;
R is D-C H2 ~ ~ wherein D lS hydrogen, alkyl having from 1 to 4 carbon:atoms or phenyl which: :
may be substituted with the group Z., wherein Z is ~ ~
, : ~ ~ ~ . : ::: , :
~ ' ' :

:~:
:
, ~ , .. :
.
::, :

:: ~

izs~s~

halogen, hydroxy, trifluoromethyl, alkoxy having from l to 6 carbon atoms, or alkyl having from l to 6 carbon atoms;
m and n are as defined above, S R5 is hydrogen or alkyl having from l to 6 carbon atoms; and ~ is 0, l or 2;
with the proviso that when:

R1 is alkyl having from 1 to 6 atoms, cyclo~
alkylmethyl having 4 to 8 carbon atoms, or A-Xm-Cn~2n-in which (i)A is as defined above except that when A is phenyl or benzyl the phenyl group or phenyl moiety of the benzyl group is optionally substituted by Y' which : is chosen from halogen, hydroxy, trifluoromethyl, alkoxy :~ 15 having 1 to 6 carbon atoms or alk~l havinq 1 to 6 carbon ,t-atoms, n is zero and m is zero, or n is l to 4 and m is ~
zero or l and X is oxygen, or (ii)A is phenyl or ~-benzyl optionally substituted by Y' as defined above, :
n is l to 4, m is one and X is sulphur, t ` 20 R3 is alkyl having 1 to 6 carbon atoms, cycloalkyl-~ methyl having from 4 to 8 atoms, 2- or 3-thienyl-: methyl, 2- or 3-furanylmethyl, l- or 2-naphthyl-methyl, or benzyl, the phenyl ring of which may be `
s~ubstituted by the group Ylas defined above; ...
''''~

...~.
, ,, ~,'''','','"
: : ",,~

: ,, ~ , :i : : :

: , .

.

~zs~9~

_ 4a -R4 is D-CnH2n-Om- is which (i)D is hydrogen, alkyl having from 1 to 4 carbon atoms or phenyl which maybe substituted by Y' as defined above, m is zero and n is 1 to 4, or (ii)D is hydrogen or phenyl optionally sub-stituted by Y' and m and n are both zero, :

~.....
, ", :
:`~

':

.. , . . ., . . . ~
:

:
~' ' ~ - .

lZS~G91 R5 is as defined above, and p is as defined above, then at least one of R2 and :R6 must be chosen from:

B-Xm-CnH2n~0- in which ~ and X are as defined abo~e, m is l and n is 1 to 4, or (ii)B is phenyl substituted by 1- or 3-furanyl, 2- or 3-thienyl or phenyl~which maybe substituted by halogen, hydroxy, trifluoromethyl, alkoxy having 1 to 6 carbon atoms or alkyl having 1 to 6 . _ . .
carbon atoms),m ls zero and n is zero or l to 4; pivalo~l-oxymethoxy~-0-CH2-C-~henyl (in which the phenyl group may :~ .
be substituted by Y as defined above), 1-glyceryl, ~ O or -O-CH~C~ ~2 .. O--: wherein R7 and R8 are as defined above, or R2 is chosen : from -NR7R8 in which R7 is phenyl substituted by ;:~ 15 l- or 3-furanyl, 2- or 3-thienyl or phenyl (which :~ may be substituted by halogen, hydroxy~trifluoro-:~- methyl, alkoxy having l to 6 carbon atoms or alkyl ¦:
~ having l to 6 carbon atoms~, and R8 is as defined above.

.`; In ~ormula 1, the asterisks denote thos-e carbon atoms which may be asymmetric (chiral) centres. The~ vention :~ contemplates all isomers at these centres both in pure ~; form and in admixture.

: :

, :~

~256~gl One group of compounds of for~.-lla I of interest are those in which R4 represents 2 group D-Cn H2n-Om-as defined above in which the sum of n and m is at least one, that is R4 does not represent hydrogen.
Examples of such R4 groups are hydrbxy, methoxy, methyl and benzyl with preferred Yalues being methyl and benzyl. Preferred values for the other groups represented in formula I when R4 has such value are as follows, (subject to the proviso as defined a~oYe)::
Rl is benzyl optionally para substituted by ` chlorine, methoxy, methyl or phenyl, 2-phenylethyl or 1- or 2-naphthylmethyl and is most preferably benzyl or p-phenylbenzyl, R2 and R6 are the same or different and are hydroxy, methoxy, ethoxy, benzyloxy, 2-phenoxyethoxy, 1-glyceryl, '~`' C: C~
~ ~ ' ~ ~:
~ O-C~: -C:~-C-.i2 ~ -.

;
,.A, or (2,2-dimethyl-1-~-oxopropoxy) methoXy, and are ::
:~ 2p most preferably chosen from hydroxy, 2-phenoxy- ~ :
~:: ethoxy, l-glyceryl, ~ .
.
~: ~

~: , . . .
~ .

~5~
- 7 _ CH3 r:-~3 o --O-C~-C~l C~'2 . c_ :~ .
and (2,2-dimethyl-1-oxypropoxy) methoxy, R3 is benzyl, ~ -methylbenzyl, ~- phenylbenzyl, l-naphthylmethyl or 3-thienylmethyl and is most preferably benzyl or ~-phenylbenzyl, and ~ R5 is hydrogen.
: , Another group of compounds of lnterest are those in which one of R2 and R6 ls chosen from ; 2-phenoxyethoxy, l-glyceryl, O O ~<J
-O-C~2~CX Crl2 or (2,2-dimethyl-1-oxopropoxy)methoxy and the other is chosen from the foregoing groups or is hydroxy, methoxy, ethoxy or benzyloxy: when R2 and R6 have such values, the preferred values for Rl R3 and R5 are as set forth in the preceding para-graph, and preferred values for Rg are hydrogen, .` methyl and benzylO

p is preerably one.
; .
~3 :
::

12S~9~

.

As ~sed herein, unless stated otherwise, the terms alkyl and alkoxv deno.e s~ch croups having straigh~ or branchec carbon chains of fro~ 1 to 6 carbon atoms. Tne term pivaloyl-oxymethyl is the trivial or common name for the (2,2-dimeth~
1-oxopropoxy)methyl croup Balogen ineluoes l~orine, chlorine, bromine and iodine.
Certain of the compounàs having structurzl formula I
form salts with pharmaceu.iccily acceptable acids. Hydrochloric, 10 sulfuric, acetic, maleic, 'umaric and the like may be ~tilized.
Compounds having structural formula I wherein R2 and/
or R6 are hydroxy form salts with pharmaceutically acceptable bases. Sodium, pota-.sium and calcium hydroxide 2S well as 15 sodium and potassium carbonate are examples of suitable bases for this purpose. In adoi.ion, salts formed with pharmaceu-tic211y acceptable amines such as, lor example, ammonia, ~-methylglucamine, benzylamine and morpholine are als~

~.i .
contemplate~. ~

Specific compounds haying strtlctural form~la I with-in the scope of invention are those having the names:

1. N-~N-IL-1-(2,2-dlmethyl-1-oxopropoxy)methoxy)carbonyl)-

2-phenylethyl~-L-phenylalanyl]-B-alanine, (2,2-dimethyl-1-~` oxopropoxy)methyl ester;

25 2. N-IN-IL-1-[phenylmethoxy]carbonyl~-2-phenylethyl]-, ~
L-~henyl~lanyl)-~-alanine, (2,2-dimethyl-1-oxopropoxy)methyl ester; ~ ~ ~

. 9 .

g ~l25~:t9~

3. N-[N-[L-1-carboxv-2-(4--phenyl)phenylethyl)-L-. phenylalanyl]-~-alanine:

4. N-IN-lL-[1-l(2,~-dimethvl-l-oxopropoxy)methoxy]
carbonyl~-2-~4-phenvl)phenylethvll-L-phenylalanyl~-B-alanine, ~2,2-dimethyl-~-ox.opropoxy)meth~l ester;

5. N-LN-~L-1-carboxy-2-phenylethyl~-L-~4-phenyl) phenylalanyl1-B-2lanine;

6. N-[N-[L-11-[(2,2-dimethyl-1--oxopropoxy)methoxy)-carbonyl~-2-(4-phenyl)phenylethyl~-L-(4 phenyl)pheny~alanyl-1~ B-alanine, (2,2-dimethyl-i-oxopropoxy)methyl ester;

7. ~-¦N-~L~ (2,~-dimethyl-1-oxopropoxy)methoxy~-carbonyl~-2-phenylethyl~-L-(4-phenyl)phenylalanyl~-B-alanine, (2,2-dimethyl-1-oxopropoxy)methyl ester;
7A. N-tL~l~arboxy-2-phenylethyl~-l-phenylalanyl-~ -. alanine, 2,2-dimethyl-1,3-dioxolan-4-yl-methyl ester m.p.
153--8C;
7s . N-[N-[L[l-~2,2-dimethyl-l,3-dioxoIan-4-yl)methoxy]-carbonyl]-2-phenylethyl]-L-phenylalanyl, malea-te salt;

~ ' ~

~ - :
` , ~:

: ~ :

~ .
~.. ~ . .

.

-10 - 12SC~
.......

8. ~-[N-[L-1-carboxy-2-phenylethyl]-L-phenylalanyl]- .
L-(~-methyl)-B-alanine;

9. N-[N-[L-[1-[(2,2-dimethyl-l-oxopropoxy)methoxy]- -carbonyl~-2-phenylethyl)-L-phenylalanyl]-L-(~-methyl)-B-alanine, (2,2~dimethyl-1-oxopropoxy)methyl ester, lO. W-[N-[(L-1-carboxy-2-phenylethyl)]-L-phenylalanyl]-B-alanine, 2-phenoxyethyl ester;

~ r .':;

' i . . j 11. N-lN-[L-[1-¦(2,2-dimethyl-1-oxopropoxy~methoxy]- : ,.
:;~ carbonyl]-2-phenylethyl)-L-phenylalanyl]-B-alanine;
.

10 12.- N~[N-[L-[1-1(2-phenoxy)ethoxy~carbonyl3 2- ::
, : ;
~ phenylethyl]-L-phenylalanyl]-~-alanine, : : .
~ .. :
:~ 13. N-[N-[L-[1-[(1-oxo-3-isobenzofuranyloxy)]carbonyl]- `:. ~
2 phenylethyl)-~-phenylalanyl-B-alaDine; ~ .
14. N-[N-[L~ [(2,3-dihydroxy)-1-p~ropoxy]carbonyl]-2- : ~ .
phenylethyll-L-phenylalanyl]-B-alanine 15. ~-[N-[L-[1-[(2,2-dimethyl-1,3-d~oxolan-4-yl)methoxy]- ~,.
carbonyl3-2-phenylethyl3-L~pheDylaDaDyl]-B-alanine, ; 16. N-[N-[L-~ (2,2-dimethyl-i-oxOpropoxy)methoxy]-carboDyl~-2-(4-phenyl)phenylethyll-;L-pheDylalanyl}-B-alsDiDe;
17. N-[N-lL-[l-[t2-phenoxy)ethoxy~carbonyl3-2-(4-phenylj-phenylethyll-L-phenylalanyll-B-alanlDe:
.:

' ~ ~ :
: ~ :

: , . ....
, . . .
: . :

:: ,. .

2S~91 18. ~-[N-IL-[1-t(1-oxo-3-isobenzofuranyloxy)]carbonyl]-2 (4-phenyl)phenylethyl]-L-Phenylalan~l]-e-alanine;
19. ~-[N-[L-[1-l(2,3-dihydroxy)-1-propoxylcarbonyl]-2-(4-phenyl)phenylethyl]-L-phenylalan~l]-~-2lanine;
20. N-[N-[L-l1-[(2,2-dimethyl-1,3-dioxolan-4-yl)methoxy]-carbonyl]-2-(4-phenyl)phenylethyl]-L-phenylalanyl]-B-alanine;
21. N-lN-[L-[1-[(2,2-dimethyl-1-oxopropoxy)methoxy]-carbonyl]-2-phenylethyl]-L-(4-phenyl)phenylalanyl]-B-alanine;
22. N-~-[L-[1-[(2-phenoxy)ethoxy~carbonyl] 2-phenylethyl]-L-(4-phenyl)phenylalanyl~-B-alanine;
23. N-[N-[L-[1-[(1-oxo-3-isobenzofuranyloxy)]carbonyl]-2-phenylethyl]-L-(4-phenyl)phenylalanyl]-B-alanine;
24. N-[N-¦L-[1-[(2,3-dihydroxy)-1-propoxy]carbonyl)-2-phenylethyl]-L-(4-phenyl)phenylalanyl)-B-alanine; ;~
25. N-[N-[L-[1-[(2,2-dimethyl-1,3-dioxolan-4-yl)me~hoxy~-carbonyl]-2-p~enylethyl]-L-~4-phenyl)phenylalanyl]-B-alanine;
26. N-[N-[L-[1-l 2,2-dimethyl-1~oxopropoxy)methoxy~
carbonyl]-2-(4-phenyl)phenyle~hyl1-L-(4-phenyl)phenylalanyI3-B-alanine; ~ :`
20. 27 N-[N- 1L-[1-[(2-phenoxy)ethoxy]carbonyl]-2-~-phenyl)~
phenylethyl)-L-~4-phenyl)phenylalanyl]-B-alanine;
~ . :
28. N-[N-[L-[1-[~1-oxo-3-isobenzofuranyloxy)~arbonyl]-2-`~; (4-phenyl?phenylethyl]-L-~4-pbenyl)phenylalanyl)-B-alanine;:
: : 29. N-[N-[L-[1-[t2 r 3-dihydroxy)-1-propoxy]carbonyl~-2-:25 (4-phenyl~phenylethyl)-L-~:4-phenyl)phenylalanyl~-B-alanine;
30. ~-[N-[L-[1-~(2,2-dimethyl-1,3-dioxolan-4-yl~methoxy~-carbonyl]-2-~4-phenyl~phenylethyl]-L-~4-phenyl)phenyIalanyl~
B-alanine;

, - 12 _ ~25~9~

31. N-~N-lL-[l-[(~l2-dimethy~ oxopropoxy)methoxy]
carbonyl~-2-Phenylethyl~-L-phenyl2lanvl]-D~L-~-meth L-alanine;
32. N-~N-[L-[1-¦(2-phenoxy)ethoxy]c2rbonyl]-2-phenylethyl]-L-phenylalanyl~-D,L-u-methyl-~-alanine:
33. N-[~-[L-[1-[(1-oxo-3-isobenzofuranvloxy)~carbonyl)-2-phenylethyl~-L-phenylalanyl~-D,L--~-methyl-B-alanine;
34. N-[N-[L-[1-[(2,3-dihydroxy)-1-propoxy]carbonyl~-2-phenylethyl]-L-phenylalanyl]-D,L-~-methyl-B-alanine;
10 35. N-lN-[L-~ (2,2-dimethyl-1,3-dioxolan-4-yl)methoxy]-carbonyl~-2-phenylethyl~-L-phenylalanyl]-D,L-u-methyl-B-alanine;
36. N-[N-[L-[1-[(2,2-dimethyl-1-oxopropoxy)methoxy]-carbonyl]-2-(4-phenyl)phenylethyl]-L-phenylalanyl]~D,L-~-15 methyl-B-alanine;
37. N-[N-~L-[1-[(2-phenoxy)ethoxy]carbonyl3-2-(4-phenylj-phë-nylethyl]-L-phenylalanyl]-D,L-~-methyl ~-a.lanine;
38. N-EN-IL-[1-[(1-oxo-3-isobenzofuranyloxy)]carbonyl]^
~ 2-(4-phenyl)phenyl~thyl]-L-phenylalanyl~-D,L-~-methyl-; 20 g-alanine;
`~ 39. N-[N-[L-[1-[(2,3-dihydroxy)-1-propoxy]carbonyl]-2-(:4-~ phenyl)phenylethyl~-L-phenylalanyl~-D,L-~-methylB-alanine;
:~ ~ 40. N-~N-[L-[l-[~ 2 r 2-dimethyl-1,3-dioxolan-4-yl)methoxy~-:. : carbonyl]-2-~-phenyl)phenylethyl~-L-phenylalanyl]-D,L~
methyl-B-alanine;
41. N-IN-[L-[1-[t2,2-dimethyl-1-oxopropoxy)methoxy]-carbonyl]-2-phenylethyl~-L-(4-phenyl)phenylalanyll-D,L-~-- methyl-B-alanine;
., : :
;' :
- ~- . :

, ., ~ ' ' '' " :

~25Q~

42. N-[N-[L-[1-[(2-phenoxy)ethoxy]carbonyl]-2-phenylethyl]-L-(4-phenyl)phenylalanyl]-D,L-~-methyl-~-alanine;

43. N-[N-[L-[l-[(l-oxo-3-isobenzofuranyloxy)]carbonyl]-2-phenylethyl]-L-(4-phenyl)phenylalanyl]-D,L-~-methyl-~-alanine;

44-. N-[N-[L-[1-[(2,3-dihydroxy)-1-propoxy]carbonyl]-2-phenylethyl]-L-(4-phenyl)phenylalanyl]-D,L-~-methyl-~-alanine;

45. N-[N-[L-[1-[(2,2-dimethyl-1,3-dioxolan-4-yl)-methoxy]-carbonyl]-2-phenylethyl]-L-(4-phenyl)phenyl-alanyl]-D,L-a-methyl-~-alanine;

46. N-[N-[L-[1-](2,2-dimethyl-1-oxopropoxy)methoxy]-carbonyl]-2-(4-phenyl)phenylethyl]-L-~4-phenyl)phenyl-alanyl3-D,L-~-methyl-~-alanine;

47. N-[N-[L-[1-[(2-phenoxy)ethoxy]carbonyl~-2-(4-phenyl)-phenylethyl]-L-(4-phenyl)phenylalanyl~-D,L-a-methyl-~-alanine;

48. N-[N-[L-[(l-oxo-3-isobenzofuranyloxy)]carbonyl]-, 20 2-(4-phenyl)phenylethyl3-L-(4-phenyl)phenylalanyl]-D, ~ :~
: L a-methyl-~-alanine;

49. N-[N-[L-[1-~(2,3-dihydroxy)-1-propoxy]carbonyl]- .
2-~4-phenyl)phenylethyl]-L-(4~-phenyl)phenylalanyl3-D,L-a-:: methyl-~oalanine;
:
; : .

:
:

:

, , . ~ . :: -i25U~

50. N- N-[L-tl-[(2,2-dimethyl-1,3-dioxolan-4-yl)-methoxy]-carbonyl]-2-(4-phenyl)phenylethyl]-L-(4-phenyl)-phenylalanyl]-D,L-a-methyl-~-alanine;

; 51. N-[N-~L-[1-[(2,2-dimethyl-1-oxopropoxy)methoxy]-: 5 carbonyl]-2-phenylethyl]-L-2 thienylalanyl]-~-alanine;

52. N-[N-[L-[1-[(2,2-dimethyl-1-oxopropoxy)methoxy]
carbonyl]-2-phenylethyl]-L-3-thienylalanyl]-~-alanine;

53. N-[N-[L-~1-[(2,2-dimethyl-1-oxopropoxy)methoxy]-carbonyl]-2-phenylethyl~-L-2-furoalanyl]-~-alanine;

54. N-[N-[L-[1-[(2,2-dimethyl-1-oxopropoxy)methoxy]-carbonyl]-2-phenylethyl]-L-phenylalanyl]-a-methylalanine;

55. N-[N-[L-[1-[(2,2-dimethyl-1-oxpropoxy)methoxy]-carbonyl]-2-phenylethyl]-L-phenylalanyl]-L-a-hydroxy--alanine;
, ~
56. N-[N-[L-[1~[(2,2-dimethyl-1-oxopropoxy)methoxy]-_ carbonyl]-2-phenylethyl]-L-phenylalanyl]-D-a-hydroxy--alanine; :

~: . 57. N:-[N-[L-[1-[(2,2-dimethyl-1-oxopropoxy)methoxy3-~ .
carbonyl]-2-phenylethyl]-L-phenylalanyl]-L-a-methoxy-~-alanine;

.. 58. N-[N-[L-~1-[(2,2-dimethyl-1-oxopropoxy~methoxy~-carbonyl]-2-phenylethyl3-L-phenylalanyl]-D-a-methoxy- ::
~ ~-alanine;

:~ ~: 59. N-[N-~L-[~2l2-dimethyl-l-oxopropoxy)methoxy]
~: ~:; 25 carbonyl]-2-phenylethyl]-L-phenylalanyl]-D~L-a-meth : ~-alanine b~nzyl ester; and .
: ~`` : :
, .

.. ~
: . .

:, ~: ~ .. .. . . .
,.

~s~

60. N-[N-[L-[1-[(2,2-dimethyl-1-ox~propoxy)methoxy]-carbonyl]-2-(4-methoxy)phenylethyl]-L-phenylalanyl-~-alanine.

Also within the scope of the invention are the pharma-ceutically acceptable salts of the above listed specific compounds.

The compounds having structural formula I may be utilized to exert their analgesic effect in the many dosage forms known to the art, such as tablets, capsules or elixirs for oral administration or in sterile solutions or suspensions for parenteral administration. The fore-going pharmaceutical dosage forms are adYantageously prepared using, in addition to a compound of this inven-tion, pharmaceutically acceptable~and compatible excipients, binders~preservatives, stabilizers, flavours and the like. In each of the dosage forms the active compound will be administered in a dosage in the~range of from ab~ut 1 to 100 mg/kg. T,ie doses ~re to be administered at intervals of from 3 to 8 hours. However, _ the quantity and frequency of dosage will depend upon such factors as the severity of the pain, the general ~` physical condition of the patient, including the age .
and weight of the patient and other factors which a person skilled in the art will recognize.

The compounds of this invention may be prepared by using ..
reactions and reagents well known in the po?ypeptide art.

Broadly speaking the compounds of formula I are prepared by an appropriate one of the following methods (in which ~ in the following fo~m~ae`p, Rl,R2,R3,R4,R~ and R6 are as :;:: ~ : :

. , - : .
:
.~ .

~ ~ . . , . ` :. .

~25V~

defined above including identities excluded by the provisoset forth above and including suitable protection of any reactive groups);
a) reducing a compound o' foxmula XX at the C-N
double bond .~ R
Rl- C= -CR-CON... (CH2)p CR4 5 6 ~~ I ;

b) condensing a ke~o acid or ester of fo~mula II
with an amino acid of formula III in a reactive medium containing 2 reducing ag~nt ; . l3 Rl-C(O)COR2 + H2NCHCONH(CH2)p CR4R5COR~
II III

c) coupling an amino acid of formula VIII with - an amino acid of fo~mula V

HC~ CO~H H2~tCH2)p CR4R5 COR
COR2 V V :~

d) reducing a compound of the formula XXI at : :
~ 15 the C=N d u~le bond ;~ Rl IC~ N C CONH(CH2)pCR4R5COR
~ 2 XXI
; : :

' ' ' :~

- : . - ~ - :

:: .

..

lZ~

e) condensins a ket~ acid or ester OL formula X with an amino aci-d in 2 reactive medium cont2inins 2 reducina agent 2 P 4 5 6 1 IHNH2 -~ I

in a reactive medium containing a reaucing agent;

f) alkylating an amine of formula III with a compound of formula XXII

- H NCHCONH(CH ~p CR R
2 2 4 5CR6 + XC~

XXII

in which X is a suitable leaving grouD such as a halogen atom, trifluoromethylsulfonate or : the like, ;
~` followed by the removal of any protecting groups, and thereafter, if necessary or desired, converting a so obtained compound into a compound, or into another compound, of formula I as defined above and/or forming a salt thereof, and if desired isolating a preferred lsomer.
.

' -: ; ,, .. ~ , ~25~) [191 -- l 8 The following overall procedures may be used to prepare the compounds of this invention from readily available : or easily prepared starting materials:

Procedure 1 R
~ S G- NH - CH -CO2H + H2N(CH2)p-CR4R5-cOR6 : VI V

: R13 G-NH-CH-CONH(CH2)pCR4R5-C0 I V
: i .~
: IR3 ~H2NcHcoNH(cH2)pcR4R5coG + R1 C~ ~ 2 ~ .
: .
III II
-3 :
- RlCHNHcHcONH~cH2~pcR4R5coR6 : :
- . . C~2 . ~ .
9 : I ~

In the foregoing reaction sequence, the amino~function o~ Compound VI is protected by an amino protecting group commonly used in the art (G) such as benzyloxycar~bonyl,~ t-butyl-oxycarbonyl or the ~like. Compound Yi is condensed with an aminoester derivative:V wherem~R6 is protected, if necessary or desired, for instance such protected group ~:

.
.

.~.,.. , . .:-, . : .: :

:
. -~25~

R6 may be benzyloxy, t-butyloxy, lower- alkoxy or the like. Condensing agents such as dicyclohexyl carbo-diimide, or diphenylphosphoryl azide may be employed.
Also, activating agents such as 1-hydroxybenzotriazole may be employed in the reaction.

The resulting dipeptide IV is deprotected at the amine terminus by treatment with acids or by hydrogenation using for example, hydrogen and a metal catalyst, The resulting product (III), is then condensed ~according to method (b) as defined above) with a ketoacid or keto-ester (II) in a suitable solvent such as water or aceto-nitrile at a substantially neutral pH in the presence of a reducing agent such as sodium cyanoborohydride or ; other equivalently functioning reducing agent. Alter-natively, the Schiff base resulting from the initial ;.
condensation of II and II~I may be reduced-~according to method (a) as defined above) e.g. by catalytic reduction to give I using hydrogen at a pressure of l - 4 atmos-pheres. The catalytic reduction may be effected using Raney nickel catalyst or 10% palladium on carbon or the , . .
like. ,he compounds of formula I~having i termlnal carboxyl group,~may be prepared fr~om~a corresponding ester by hydrolysis or hydrogenolysis.

In the foregoing reaction sequence, substituents Rl, R2,R3,R4,R~ and~R6; and p are as previously defined, G is a suitable protecting group, e.g. benzyloxy carbonyl or t-butyloxy;carbonyl.

, , ~ :

:

: . .

Procedure 2 R R

II + H2NCH-C02H ~ Rl-fHNHCHCO2H

:` YII VIII

V I I I + H2N ( CH2') pCR4R5COR6~ Rl IHNHCHCONH(CH2)pCR4R5COR6 , ~ COR2 I

: 5 Pr~cedure 2 is commenced by condensing an R3 substituted amino acid with a ketoester II by procedures substan tially as described in Procedure l.The resulting inter-mediate (VIII) Is then coupled (according to method (c) as defincd above) with an R4,R5 substituted amino acid in which R6 is protected and may be for example alkoxy, or a dialkylamino group, or an equivalent protected group ~ to form compounds which after remova} o~ : ~
protecting groups which may be present will pro uce ::
products~of this invention wherein~substituents : 15 R1,R2,R3,R4,~5 and R6; and p are as~defined above.

, - : : ~
.

:

.
. .
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~125q;~

Procedure 3 RllHNH2 ~ O=CCONH(cH2)pcR4R5coR6 IX X
RlCHNH~HCONH(OH2)pCR4R5COR6 ~ - -: I
~ .
An am;no acid or ester (IX) wherein Rl and R2 are ;~ 5 defined as above is condensed with a carbonyl compound (X) under conditions described in Procedures 1 and 2 ~, to prepare I laccording to methods (d) or (e) as defined above). Substituents R3,R4,R5 and R6; and p are as previously described.
:
Procedure 4 Ill + X-~HRl RlCHNHCHR3CONH(CH2)pCR4R5COR6 2 CR~2 ` I

Intermediate III, whose preparation is shown in Procedure ~;~ 1, is reacted with a substituted halo ester under ~ ;~
conventional alkylating conditions, preferably in the presence of a base (a tertiary amine or an inorganic hydroxide, carbonate or bicarbonate). The reaction is ; ~ usually carried out~in water or;ln~an organlc solYent, . : : :

:

, ~ . , - . .
. ~ , , .

.

, lZS~ 91 such as N,N-dimethylformamide or acetonitrile. Substi-tuents Rl,R2,R3,R4,R5 and R6; and p are as previously described.

In the foregoing reaction sequences wherein water is generated by the reactants (e.g. the condensation of compounds II and III in Method 1), the reaction may be effected by azeotropic distillation of the formed water ;~ with suitable high boiling solvents such as toluene or xylene. Alternatively, the reactions may be effected in the presence of dehydrating agents such as molecular sieves or the like.
:
Various of the intermediates for use in the methods and procedures described above can be obtained by following directly or analogously procedures~described in European Patent Application publication No. 54862.
: , Further a number of the intermediates for preparing the compounds ~f this invention ~re commercially aYailabl~
or they may be readily prepared by art recogni~ed - methods. Intermediates for preparing a substantial number of the compounds of this invention are described or the preparation théreof is embodied in publications ~i and treatises re1ating to peptide chemistry such as, J.H. Jones, in "Comprehensive Organic Chemistry", Vo1.2, D. Barton and W.D. Ollis, Editors, Pergamon Press, 1979 pp 819-823 and references 2, and 29-31 cited therein.

The following examples illustrate the preparation of the compounds of the invention.

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~, , .
~ - ` . ~ `
. -' - ~Z5 EXAIlPLE 1 N-~N-[L-l-CARBOXY-2-(4-PHENYL)PHENYLETHYL]-L-PHENYL-ALANYL]-~-ALANINE BENZYL ESTER

A. 4-Phenylphenylpyruvic Acid 409 of Potassium t-butoxide l"as added to 150 ml of diethyl oxalate in small p~rtions with stirring. After the initial exothermic reaction subsided, the reaction mixture was heated on a steam bath under nitrogen in order to dissolve solids~ After cooling down to room temperature, 7~9 of 4-biphenyl acetic acid methyl ester was added in one portion. The mixture was stirred at 60-70~C., for 2 hours while low boiling material was removed under vaccum. On cooling to room temperature, the viscous residue was stirred with 200 ml of ether and 350 ml of water with cooling. The ether layer was separated and - extracted once with IOO ml of water. The aqueous layers were combined, extracted once with ether, made acidic with concentrated HCl (cooling) and extracted with 2 x ~` 300 ml of ether. Some sol;ds did not dissolve in the ether layer and were filtered. The ether layer was _ then evaporated to dryness and the semi-solid resi~due was combined with the preceeding solids. A mixture of `: ~ :
160 ml of conc. HCl and 35C ml of acetic acid was added to the ~solids/semi-solid residue:mlxture, and the ~ ~
25 resulting mixture was heated under~reflux for 2 1/2 ~ ;
hours. On cooli;ng down to 50C., a solid precipi~tated, ~ ;
which was filtered and washed with 150 ml of water. The wet solid was stirred with 150 ml of acetonitrile for five minutes, then~filtered and dried under high Yacuum at room temperature for 3 hours~.~ q2.2 9 of the title compound m.p. 215-218C., were obtained.

:

, : ~ ' , . :' `

~ `'.'` ' ~ ' :12 - 2~ -B. N-[N-[N-tert.-butyloxycarbonyl]-L-phenylalanyl]-~ . . . . . _ .. _ _ . ..
~-alanine benzyl ester To a stirred mixture of 0.023 mole each of di-t-butyl-dicarbonate, L-phenylalanine, ~-alanine benzyl ester p-toluenesulfonate, hydroxybenzotriazole, N-dimethyl-aminopropyl-N'-ethylcarbodi ~ de hydrochloride in 75 ml of dry N,N-dimethylformamidein an ice bath was added 5 ml of N-ethyl-morpholine and the mixture stirred at room temperature for 3 hours, then poured into 600 ml of ice water and extracted with 3 x 150 ml of ether.
The ether layers were combined and extracted once with 150 ml of 0.3N HCl, then twice ~ith 300 ml of water, and then dried over Na2S04, filtered and evaporated down to dryness at 28C in vacuo. 9.0 9 of gummy solid residue were obtained.
. .
C. N-~L-Phenylalanyl)-~-alanine benzyl ester hydro-chloride -~: :
-4.0 9 of the material from Example lB in 25 ml cf ~ ethyl acetate at 0C was stirred with gaseous HCl for ; _ 20 10 min. Stir at 0C for 1 1/2 hours then at 10C for 30 min. A stream of N2 was passed into the solvent to expel excess HCl. The solution was poured into 2Q0 ml ~- of ether with vigourous stirring, and the precipitated solid filtered, and then dried at room temperature under high vacuum for 2 hours to give 3.35 9 of product.

D. N-[N-[L-l-Carboxy-2-(4-phenyl)phenylethyl~-L-phenylalanyl]-~-alanine benzyl ester.
~- A mixture of 9.10 g of N-~L-phenylalanyl3-~-alanine benzyl ester hydrochloride and 8.40 9 of the compound of Example lA in 500 ml of a mixture of tetrahydrofuran-~: :
.

.
~ - :

,` ~ ' ~'` ' `

12S~)091 ether (9:1) was treated with triethylamine to pH 6.6 and stirred at room temperature for 1 1/2 hours. A
solution of 2.0 9 of sodium cyanoborohydride in a mixture of 100 ml of tetrahydrofuranethanol was added dropwise over a period of 2 hours with stirring. Stirring was continued at room temperature overnight. The reaction mixture was concentrated to 75 ml at 40C in vacuo. The residue was stirred with 400 ml each of 0.5N HCl and ether for one hour. The ether layer was dried over sodium sulfate, filtered and evaporated to dryness in vacuo.
A yellow viscous syrup was obtained which was dissolved in 25 ml of ethanol. Solids were formed, on refrigeration over-night, and were filtered off, washed with cold ethanol (4.2 9) and chromatographed on a column of 300 9 of silica gel using a solvent mixture consisting of CHC13:CH3ûH:CH3C02H (200:10:2). The effluent was divided into fractions which were evaporated to dryness in vacuo.
In this manner, 600 mg of a solid product consisting of the title compound, m.p. 192-194C., were obtained.
, :
, :
-_ 20 EXAMPLE 2 N-~N-~L-l-CARBOXY-2-(4-PHENYL)PHENYLETHYL]-L-PHENYL-~ ALANYL]-~-ALANINE.
; ~ A suspension of 59~0 mg of the product from Example 1 in l~Q ml of ethanol was shaken ove!night with 200 mg of ~`~ 25 10% Pd/C in a Parr;apparatus.~ The resulting mixture was diluted with 40 ml of ethanol and 10 ml of water`then heated on a steam~bath briefly until all white precipi-tate dissolved. After cooling, the catalyst was filtered off. The filtrate was evaporated~at 50~C under vacuum to 5 ml. The solld was filtered, washed with ethanol, and dried at room temperature under high vacuum for 6 ::: :
~: : : :

,. .... .
~ :

:
~:

~.2S(J~

hours to yield 155 mg of the title compound m.p. 226-228C.

[a]D26 = 16.8 (C=0.5, DllF).
Analysis Calculated: C, 70.40; H, 6.13; N, 6.08.
Found: C, 70.54; H, 6.26; N, 5.97.

N-[N-[L-[l-[PIVALOYLOXYMETHOXY[CARBONYL[-2-PHENYLETHYL)]-.
L-PHENYLALANYL]-~-ALANINE, PIVALOYLOXYMETHYL ESTER

A. N-(tert.-Butyloxycarbonyl)-~-alanine, pivaloyloxy-methyl es~er Triethylamine (32.4 ml) is added to N-tert.-butyloxy-carbonyl ~-alanine (40 9) in N,N'-dimethylformamide at room temperature under a nitrogen atmosphere in a 1 1 round bottom flask. The solution is stirred 15 minutes 15. and chloromethyl pivalate (36.6 9) [M. Rasmussen &
N.J. Leonard, J. Amer. Chem. ~oc., 89, 5439 (1967)]is added dropwise at 0C. The solution is stirred at room ~~ temperature overnight. The mixture is then diluted with ethyl acetate, filtered, washed with water, then brine, and evaporated to give 709 crude~matPrial~ which was ~; ~ ` chromatographed (silica gel) eluting with 15% ethyl ~ acetate-hexane to give 62.5 9 of product.
.;, : ~ ~
B. ~-Alanine, pivaloyloxymethyl ester, trifluoroacetat-e Trifluoroacetic acid (100 ml) is added to a solution of ~; 25 the product from Example 3A (62 g) in methylene chloride (180 ml) at 0C. The mixture is stirred at room temp-erature for two hours,~and solvent removed in vacuum to give 100 9 product as a pale yellow oil.

:

~ , 9:~

C. N-[L-l-(Phenylmethoxy)carbonyl-2-phenylethyl)]-L-phenylalanine L-Phenylalanine benzyl ester hydrochloride, 190.4 9 (0.652 mole) is suspended in 960 mls absolute methanol, 6.7 1 dry (3A sieves) tetrahydrofuran added and the slurry is stirred while adding about 50 ml triethylamine to bring the pH to 6.5-7Ø The pH is checked with EM
Reagents ColorpHast indicator sticks, range pH 5-10, moistened with water before use. To the neutral slurry is added 200 9 (0.98 mole) sodium phenyl pyruvate hydrate (Sigma), followed by 240 9 crushed 3A molecular sieves. (Sieves may be gound in a mortar and need not be finely powdered. If too fine, they are dif~icult to remove by filtration). The slurry is stirred at ambient temperature while adding a solution of 61.6 9 (0.98 mole) sodium cyanoborohydride in 40 ml methanol plus 300 ml dry tetrahydrofuran dropwise over 5 hours. The reactinn is stirred at ambient temperature for 48 to 72 hours ` . while monitoring the disappearance of benzyl ester by thin layer chromatogra~hy. Ihe reaction mixture is filtered to remove sieves, washing~sieves well with hot methanol, as some product precipitates out on them.

The filtrate is concentrated on a rotary evaporator at 50C to a syrup. This syrup is dissolved in 2.4 1 ether in a 12 1 round bottomed flask and~ stirred in an ice ~ bath while adding 2.4 1 2.5% aqueous HCl. The large - volume of HCN which is generated is passed into a sodium hydroxide trap. The mixture is allowed to st;r for 2.5 to 3 hours while a white solid gradually forms at the interface and evolution o~ HCN stops. The 2-phase mixture is filtered (most of the aqueous phase may be ~ ,, drawn off in a separator before filtcring as the product :~
: ~ :
.... , .. .... ~ ~ ~ .
: -, ~ - - ..
,: ~ ~ . ., : :
. . : .

~:

remains at the interface) and the solid is washed well with ether and dried in vacuo below 50; yield 80-90 9, m.p. 175-180~C. This material is 90-95% pure L,L
isomer as estimated by thin layer chromatography. The crude product i5 re-dissolved in about 10 1 boiling, absolute methanol, some fine white inorganic insolubles filtered and the filtrate concentrated to 5 1 at the boil-ing point, when flocculent white crystals appear. The product is allowed to cool slowly to room temperature and then to 0C for 2-3 hours. The solid is collected and dried in vacuo at 50C; yield wt 57-60 9, m.p. 185-186C, [~]D26 5.9 to 6.3 * (DMS0, C~l~. The product is greater than 98% L,L isomer as estimated by thin layer chromatography and high pressure liquid chromatography analysis. *(results of several runs) j, ~ D. N-(L-l-Benzyloxycarbonyl-2-phenylethyl)-L-phenyl-,, ` alanine pivaloyloxymethyl ester.
: A solution of 4.03 9 (10 m mole) of the product from Example 3C and 1.5 ml (llmmole) triethylamine in 20 = 20 ml DMF is treated at ambient temperature with 1.6 ml (11 mmole~ of chloromethyl-pivalate, stirred at 50-60C for 24 hours, and the resultin~ slurry poured into water and extracted with 3 x 100 ml ~ether. Some insolubles are filtered off, and the ether phase washed with water, dried and concentrated to an oil;
yield 4.8 9; NMR consistent with structure of title~
compound.

E. N-(L-Carboxy-2-phenylethyl)-L-phenylalanine, piva-loyloxymethyl ester .... ~ . ... .
The crude diester from Example 3D (4.8 9) is hydrogenated ~ in a Parr apparatus at 60 psig in 50 ml methanol + 5 ml :,~

'~- ' : ~ :
.
~ ~ , ~25¢~C'~l H20 in the presence of 0.4 9 of 10l Pd/C for 3 hours.
The resulting reaction mixture is filtered and concentrated to a damp solid which is re-crystallized from methanol/
H20. White fluffy needles are filtered off and dried in vacuo; yield 3.0 9, m.p. 122-124C. TLC showed essentially one spot, Rf 0.2, in CHC13/MeOH/HOAc 100:
1:0Ø5.

F. N-(L-l-Pivaloyloxymethylcarbonyl-2~phenylethyl)-L-phenyl-alanine-~-alanine, pivaloyloxymethyl ester -A mixture of 1.0 9 (2.3 mmole) of the product from Example 3E and 0.89 9 (2.3 ~mole) of the product from Example 3B in 25 ml DMF is treated with 1.01 ml (8 mmole) N-ethyl-morpholine followed by 352 mg (2.3 mmole) 1-hydroxybenzotriazole hydrate and 439 mg ~2.3 mmole) 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide hydro-chloride, and allowed to:stir at ambient temperature overnight. Thin layer chromatography still shows starting material present. The reaction mixture is heated to 40-50C., for 6 hours and allowed to stand at room temperature overnight. Tne mixture is poured into water and extracted with several portions of ether, then ~ the ether phase is washed well with water, dried and ; concentrated to an oil (1.2 9). The oil is chromato-graphed on Merck thin layer chromatography grade silica gel 60-G to yield 0.9 9 of oil showing a single spot, Rf 0.4 in EtOAc/Hexane 1:2 (same system as used for column chromatography.) Analysis: Cal¢ulated for C33H44N209: C, 64.69; H, 7.24; N, 4.57.
Found: C, 64.47; H, 7.20; N, 4.29.
~D26 -21.7 (DMF, C=l.O).

. .

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EXAMPLE
N-[N-[L-[1-[(2,2-DIMETHYL-l-OXaPROPOXY)METHOXY]CARBONYL~-2-(4-PHENYL)-PHENYLETHYL]-L-PHEN~'LALANYL]-~-ALANINE
A. N-[D,L-l-Carboxy-2-(4-phenyl)phenylethyl]-L-phenyl-alanine, benzyl ester.
A stirred suspension of 4-phenylphenylpyruvic acid (24.0 9) and L-phenylalanine benzyl ester hydrochloride (23.2 9) in 1 1. of THFlethanol (9:1) was brought to pH 6.6 by gradual addition of triethylamine. In the course of this process, all solids dissolved. After stirring the resul-ting solution for 2 hours at room temperature, a solution of sodium cyanoborohydride (3.5 9) in the same solvent was added dropwise with stirring. The reaction mixture ; was allowed to stir overnight at room temperature.

The reaction mixture was then concentrated to 200 ml under reduced pressure. The residue was poured into 600 ml of 0.3N HCl with cooling and stirring. A gummy solid sepArated. The aqueous material was decanted off, and the remaining solid stirred with 120 ml of ethanol. The ~- 20 resulting solids were filtered, and the wet solids stirred - with 100 ml of fresh ethanol. After standing overnight, the solids were filtered and dried to give 22.3 9 of - solid product.

B. N-[D,L-[1-[~2,2-Dimethyl-l-oxopropoxy)methoxy~-.
carbonyl]-2-(4-phenyl)phenylethyl]-L-phenylalanine, ~ benzyl ester.
:
Triethylamine (3.05 ml) was added to a solution of N-[D,L-l-carboxy-2-(4-phenyl)phenylethyl]-L-phenylalanine, .
benzyl ester (9.6 9) in 30 ml of dimethylformamide. The mixture was stirred at room temperature for 20 minc and .
.~

. . -- . . .

:1~5~

chloromethyl pivalate (3;15 ml) was added. The resulting mixture was heated in a bath at 45-55C for 4 hours with stirring, then allowed to stir at room temperature overnight. The resulting mixture was diluted with 300 ml of water and extracted with three 150 ml portions of ether. The combined ether layers were extracted twice with 100 ml portions of water, and the ether solution dried over anhydrous Na2S04. Filtration and evaporation in vacuo gave 7.5 9 of syrupy product.
:
This material was chromatographed on 135 9 of silica gel eluting with a mixture of ethylacetate-cyclohexane (85:15). Fractions containing the desired diastereo-meric mixture of products were identified by thin-layer ; chromatography, combined, and evaporated to dryness in vacuo to give 5.9 9 of prpduct as a syrup.
. .
C. N-[D,L-[1-~(2,2-Dimethyl-l-oxopropoxy)methoxy]-carbonyl]-2-~4-phenyl)phenylethyl]-L-phenylalanine A sol Uti on of the above product (5.9 9) in 175 ml of _ ethanol was hydrogenated at 15-30 psig over 750 mg. of 10% Pd/C for 2 hours. The reaction mixture was diluted with an additional 250-ml of ethanol and warmed to 45C
to dissolve the precipitated product. Catalyst was `~ filtered from the warm solution, and the filtrate evapor-ated to give a total of 4.4 9 product.

` 25 D. N-[N-[L-[1-[(?,2-Dimethyl-l-oxopropoxy)methoxy]-carbonyl]-2-(4-phenyl)phenylethyl~-L-phenylalanyl]-~
~-alanine, benzyl ester.
-~ N-ethylmorpholine (1.2 ml) was added to a stirred mixture of the above product t3~25 9), N-(N,N-dimethylamino-propyl)-N'-ethylcarbodiimide hydrochto~ride (1.7 g), of hydroxybenzotriazole (1.3 g) and ~-alan;ne benzyl : : : :

' .~zs()a~.l ester p-toluenesulfonate (3.0 9) in 25 ml of dimethyl-formamide. The mixture was stirred at room temperature for 3 hours, diluted with 200 ml of ice-water and extrac-ted with two 125 ml portions of ether. The combined extracts were washed with 250 ml of water and dried over anhydrous MgS04. Filtration and evaporation gave 5.25 9 of residue. Thin-layer chromotography (silica gel, ~; CHC13/EtOAc - 10:1) showed two major products, Rf=0.36 and Rf=0.32 (partially overlapping). This material was chromotographed on 350 9 of silica gel (thin-layer chromotography grade), eluting with CHC13-EtOAc ~100:5).
Fractions containing the pure individual components were identified by thin layer chromatography, combined and evaporated. In this manner 650 mg of faster moYing `15 component (L,L-diastereoisomer) was obtained along with 590 mg of L,D-diastereoisomer.

The final product was obtained by hydrogenating a solution of 650 mg of L, L-diastereo;somer above in 50 ml ethanol oYer 50 mg of 10% Pd'C at 15-45 psig for 3 hours.
Catalyst was filtered off, and the filtrate evaporated to dryness in vacuo at 30C. The residue was chroma-tographed on 50 g of t.l.c. grade silica gel, eluting initially with 300 ml of CHC13/EtOAc ~10:1), then wlth CHC13/CH30H/AcOH (600:10:2). Fractions containing pure product were identi~ied by thin;layer chromatography silica gel)CHC13/CH30H/AcOH (600:10:2), Rf = 0.31.
~ These fractions were combined, evaporated, and the~ ;
:~ residue dried at room temperature in high vacuum over-night. The residue was recrystallized from ether and ~` 30 the product -dried at 45C for 3 1i2 hours in high vacuum. Obtain 250 mg. solid, m.p. 101-103C, ]D26 -22 (c = 0.5, DMF).

- ~:

-~, : , iZSV~91 EXAMPLE S
N-[N-[L-[1-(2,2-DIMETHYL-l-O~OPROPOXY)METHOXY]CARBONYL~-.
2-PHENYLETHYL]-L-PHENYLALANYL~-~-ALANINE
A mixture of 0.9 9 (2.1 mmole) of N-(L-l-carboxy-2-S phenylethyl)-L-phenylalanine, pivaloyloxymethyl ester ~Example 3E) and equimolar amounts of ~-alanine benzyl ester p-toluenesulfonate (73) mg , l-hydroxybenzotria-zole hydrate (321 mg) and 1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide HCl (401 mg) were dissolved in 20 ml DMF containing 0.8 ml (6.3 mmole) N-ethylmorpholine and the mixture stirred at ambient temperature overnight.
The yellow solution was poured into water (150 ml) and extracted with 3 x 100 ml ether. The ether phase was ` washed with water, dried and concentrated to an oil, lS 1.1 9 showing one major spot with minor impurity on thin-layer chromatography.

The total product was chromatographed on 150 9 silioa gel : eluting with ethyl acetate/hexane (30:70). The fractions were combined and concentrated to an oil, ~a]D26 - 20.0h _ 20 (DMF, c = 1) The above-described product (1.5 9) was hydrogenated at 40 psig in 100 ml absolute EtOH over 0.2 9 10~ Pd/C
for 3 hours.

The catalyst was filtered and the filtrate concentrated to an oil which crystallized, upon drying in high vacuum, overnight. On standing under hexane fine colourless crystals were obtained, which on filtration and drying gave 1.2 9 of product, m.p. 93-95C, [~D26 - 27.3 ~; (DMF, c = 1), :, ,..... . :
: - .

~;~S~9~

By following the procedures described in the preceding description and examples, using appropriate intermediates, each of the specific compounds 1-63 listed above can be prepared.

~ 5 In the following table, the shorthand notations C6H5CH2 ; and C7H7 indicate the benzyl groups; and C6H5 indicates the phenyl group. By way of example utilizing the above described methods, the following compounds of the invention were prepared. Except where indicated all compounds have the L absolute stereochemistry at the chiral centres, and ; R5 equal to hydrogen.

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A. N-(L~l-(Phenylmethoxycarbonyl)-2-phenylethyl)-L-phenylalanlne, cyanomethyl ester A solution of 4.03 g (10 mmol) of the product of example 3C
and 2.1 ml (15 mmol) of triethylamine in 150 ml of acetone was treated dropwise at room temperature with 0.95 ml (15 mmol) of chloroacetonitrile. The resulting solution was heated at reflux overnight. It was then concentrated to dryness and partitioned between water and ether. The ether phase was washed with se~eral portions of water, dried, and concentrated to an oil. 4.5 g of material were thus obtained, and used directly in the next step.
B. N-(L-l-(Phenylmethoxycarbonyl)-2-phenylethyl)-L-phenylalanine, 2,2-dimethyl-1,3-dioxolan- 4-yl methyl ester.
The product of the preceeding step - (4.4 g, 10 mmol) and 25 ml (20 mmol) of 2,~-dimethyl-1,3-dioxolan-4-yl methanol (Aldrich Chemical Co.) were combined, then triethylamine (1.4 ml, 10 mmol) and N,N-dimethyl-4-aminopyridine (20 mg) were added. The resulting mixture was stirred under a nitrogen atmosphere at 55-60 for 16 hrs. The reaction mixture was then poured into water and extracted with several portions of ether. The combined ether extracts were washed with several portions of water, dried, and concentrated to give 5.7 g of an oil. This product was chromatographed on 400 g of Merck silica gel 60G, eluting with ethyl acetate-hexane mixtures.
Fractions shown to be pure by thin-layer chromatography were combined, and evaporated to dryness giving 4.2 g oil, ~3D =-5.2 (c 1.3, DMF) C. N-(L-1-(2,2-dimethyl-1,3-dioxolan-4-yl methoxycarbonyl)-2-phenylethyl)-L-phenylalanine.
The product of the preceeding steD was dissolved in 50 ml of tetrahydrofuran and hydrogenated in a Parr apparatus over 0.4 g of 10% Palladium on carbon at room temperature and 60 psig for 4 hrs. The catalyst was filtered, and the filtrate evaporated in vacuo to give a waxy residue 13 g) which was crystallized from ethyl acetate to yield 2.6 g of crystals, mp 140-2.
D. N-(N-tL-~1-(2,2-dimethyl-1,3-dioxolan-4-yl)methoxv)-carbonyl)-2-phenylethyl)-L- phenylalanyl)- ~-alanine, benzyl ester.
A mixture of the product of the preceeding step (2.5 g, 5. 8 mmol), 2.2 g (6.4 mmol~ of ~-alanine benzyl ester p-tol-uenesulfonate, 887 mg (5 . 8 mmol) of l-hydroxybenzotriazole hydrate, 1.1 g (5. 8 mmol) of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and ~3 ml (18 n~ol) of N
ethyl morpholine in 20 ml of dimethylformamide was stirred at room temperature overnight.

, : -, ' `~`:

-36B- 1 ztjo ~ 9 ~

The resulting yellow solution was poured into water and extracted with several portions of ether. The combined ether extracts were washed repeatedly with water, dried over anhydrous potassium carbonate, and concentrated to an oil (3.3 g).
This material was chromatographed on 300 g of Merck silica gel 60, eluting with hexane-ethyl acetate (2:1). Fractions shown to be homogenous by thin-layer chromatogra hy (silica gel F2s4, ethyl acetate-hexane (1:1)) were combined, and sol-vent evaporated to give 2.7 g of oily product, which was used directly in the following step.
E N-(N-(L-(l-t2,2-dimethvl-1,3-dioxolan-4-yl)methoxy)-carbonyl)-2-phenylethyl)-L-phenylalanyl)-~ -alanine.
The product of the preceeding s'ep (2.2 g) was dissolved in 50 ml of absolute ethanol and hydrogenated in a Parr apparatus over 0.2 g of 10% palladium/carbon at 50 psig for 4 hrs. at room temperature. The catalyst was filtered, and the filtrate concentrated to an oil (1.9 g) which solidified.
Recrystallization from ether at -80 gave 1.8 g of solid, mp 80-2,~ 6 = -22.7 (c 1, DMF).Thin-layer chromatography showed a single spot, Rf=0.35 (chloroform-methanol-acetic acid 500:25:2.5).
F. N-(N-(L-(1-(2,2-dimethyl-1,3-dioxolan-4~yl)methoxy)-carbonyl)-2-phenylethyl)-L-phenylalanyl)-~ -alanine, maieate (1:1).
The product of the preceeding step (5.0 g) was dissolved in 200 ml of ether and treated with a solution of maleic acid (1.16 g) in 200 ml ether. Crystals slowly formed upon standing overnight at 5-10 in the refrigerator. These were filtered and dried to give 4.7 g of salt, mp 127-9, ~d3D~= -16.3U
(c 1, DMF). Analysis calculated for C31H38N2011: C;60.58, H;6.23, N;4.55. Found: C; 60.33, H; 6.31, N; 4.48.

. ,~

.
, iZS~O91 The compounds having structural formula I inhibit the activity of enz~mes designated enkephalinases. The compounds are particularly useful for the inhibition of enkephalinase A, which is derived form the striata of both rats and humans. In in vitro tests, selected ! compounds having structural formula I in a concentra-tion range from 10 to 10 6M have been found to inhibit the~ activity of the aforementioned enzyme by 50% or ! more.

The following test procedure was utilized to assay the enkephalinase A inhibition of the compounds having - structural formula I.

Enkephalin (ENK) degrading activity was separated into the following three fract;ions according to the method 15 of Gorenstein and Snyder, ~Life Sci., 25, 2065 (1979):
Enk'ase A (Gly3-Phe4), Aminopeptidase, (AP) (Tyr~-~`~ Gly2), and Enk'ase B (Gly2-Gly3).

Enzyme activity was separated by taking the brain tissue (minus cerebellum) from Sprague-Dawley rats and homo-20 genizing it in 30 volumes of 50 mM Tris buffer, pH 7.4, ~, using a Brinkmann Polytron. The resulting ho~ogenate is centrifuged at 50,000xg for 15 min. The pellet, consti-tuting the membrane bound enzyme material, is washed by ~¦ re-suspending it in Tris and re-centrifuging it 4 times.
.1 .
Following washing, solubilization of the membrane pellet is achie~ed by incubat;ng it for 45 min. at 37C in the presence of 15 volumes (based on initial brain weight) of 50mM Tris-1% Triton*X-100 buffer, pH 7.4. After centrifugation at lOO,OOOxg for 60 m;nutes to remove non-solubilized material, the Triton*soluble supernatant ' ` * Trademark ' ` ~S~Q91 is layered on a 1.5 x 30 cm DEAE Sephacel*column previ-ously equilibrated with 50 mM Tris-0.1% Triton* pH 7.4.
Material is eluted from the column using a 1 liter linear NaCl gradient running from 0.0 to 0.4 M. Eluant is collected in 7 ml fractions, each of which is assayed for enkephalin degrading activity. Under these conditions Enk'ase A activity is found to elute between 120 and 200 ml. followed by AP activity (260 to 4bo ml) and finally ~1 by Enk'ase B activity between 420 and 450 ml.
~ '. ' .
-¦ 10 Enkephalin degrading activity is monitored using a ! radiometric assay. The substrate is 3H-Met5-ENK (50.1 Ci/mmol, New England Nuclear) diluted in 0.05 M Tris buffer, pH 7.4, such that the final reaction mixt~re concentration is 40 ~M. Total reaction mixture volume including enzyme and substrate is 250 ~1. Incubation is carried out for 90 min at 37C. To`stop the reaction, ~; tubes are transferred to a boiling water bath for 15 `; min.

Assay products are se?arated from one another using thin _ 20 layer chromatography. A 4 ~1 aliquot of the reaction mixture is spotted on a Baker-flex*Silica Gel lB plate ~; (20 x 20 cm) along with unlabeled standards (Met5-~;~ ENK, tyrosine, tyrosylglycine, tyrosyl-glycyl-glycine) ~` and the components cv-chromatographed in an isopropanol:
ethyl acetate: 5% acetic acid sol~ent system (2:2:1) ~i which is capable of resolving Met5-ENK from its break-down products. Total running time is approximately 17 ~ hours. TLC tanks are gassed with nitrogen prior toI starting the run. Following the run, markers are visual-., ized with ninhydrin spray. These spots, along with rema;ning plate regions, are cut~from the plate and the * Trademark .

` :

~2S~(~9 radioactivity corresponding to each monitored using liquid scintillation counting. IC50,s are determined using linear regression techniques.

: Utilizing this procedure, the following nanomolar (nM) concentrations for the specified compounds were found to inhibit the action of enkephalinase A by 50% (IC50).

TABLE A

Compound Enkephalinase A
Inhibition IC50 nM
.~ .
A N.A.l B N.A.
,. - C N .A . l D N.A.l :
: _ 15 E ~:~ 230 :
: F 580 G : 6.4 ~: H ~ ~ ~N.A.l ' `~

J 0,9 ~ ~ .. A I

,: ~ , ~ ~ : :
, . -~ No activity at <105nM ~ ;~
.:

. . :. , . .: . ~
;~ . - ~ :
~,. ::

~: :

, .;

i~54~91 The following test procedure ~las utilized to assess the noted compounds' potentiatiation of the analgesic effects of (DAla2-Met )-enkephalinamide (DAEAM). Background for the use of this procedure is given in Chipkin, R.E., Iorio, L.C., Barnett, A., Berger, J., and Billard, W., Regulatory Peptides: From Molecular Biology to Function, edited by E. Costa and M. Trabucchi, Raven Press, New York, 1982, pp. 235-242.

Male CFl mice (19-239) from Charles River Breeding Labs, Mass., are used (N=10/dose or dose combination). Tail-flick testing is done similar to that of Dewey and Harris, Methods in Narcotic Research, Eds., S. Ehrenpreis and -A. Neidle, pp. 101-109, Marcel Dekker, Inc., New York, 1975 using a radiant heat noxious stimulus. Following determination of control latencies (typically 2-3 sec), the mice are first injected (sc or po) with either vehicle or drug and after an appropriate interval inject-ed intracerebroventricularly (icv) with either vehicle : (1~!l of saline) or DAEAM according to Haley and 20 McCormic, Br., J. Pharmacol., 12,12 (1957). Tail flick . _ _ _ latencies are re-determined 30 min. later, as this has previously been determined to be the time of peak analgesia for DAEAM, a cut-off of 10 seconds is employed.

Utilizing this procedure, the following ED50 values~(the dose at which half the test animals displayed analgesia) ~, ~ were obtained for selected compounds.

: ~
::
: ~' ~, . ' ' : : :

;; ' :

:
- - . . :
, ~2S~

.~ ... _.
TABLE B
DAEAM Potentatiation Compound ED50 (route) A 155 mg/k (po) ; 5 B 37 mg/k (po) C 100 mg/k (po) D 100 mg/k (po) ; E 30-60 mg/k (po) F 100 mg/k (sc) - G 50~9 (icv) ~: H 100 mg/k (po) ., ' .' '. .
d~ K 50~49 (icv)2 . ` L 100 mg/k ( po ) ~, ~
_. . ... _ _ ..

~": . It should be noted that compounds;A, B, C, F and H are ::.............. ester derivatives; ~ompound H being an ester derivative - of compound G, and compound L is an amide der;vative.
: Such derivatization is found to confer : 20 oral activity to the p:arent entity which show poo:r :.
~ absorpt;on from the gastrointestinil tract~. These : ~:

:: ` ` :

. : : : ` ;:

~2S~)~9~

derivatives, which show no activity in vitro at 105nM
(see Table A), are bioactivated in vivo to deliver the parent (in vivo enkephalinase A inhibitors) to a site ~: of action within the central nervous system (see Table B).

: ` :

' ;' -

Claims

The embodiments of the invention in which an exclusive property or privilege is claimed, are defined as follows:

1. A method of preparing a compound having the formula I

I

or a mixture of enantiomers or diasterioisomers con-taining the same, and the pharmaceutically acceptable salts thereof wherein:
R1 is alkyl having from 1 to 6 carbon atoms, ada-mantylmethyl, cycloalkylmethyl having from 4 to 8 carbon atoms or A-Xm-CnH2n- wherein X is oxygen or sulfur, A is phenyl which may be substituted with the group Y, wherein Y is halogen, hydroxy, trifluoro-methyl, alkoxy having from 1 to 6 carbon atoms, alkyl having from 1 to 6 carbon atoms, 2- or 3-furanyl, 2-or 3-thienyl, or phenyl [which may be substituted with halogen, hydroxy, trifluoromethyl, alkoxy having from 1 to 6 carbon atoms or alkyl having from 1 to 6 carbon atoms], benzyl [the phenyl ring of which may be substituted with the group Y, as defined above], 1- or 2-naphthyl, 2- or 3-furanyl or 2- or 3-thienyl;

m is 0 or 1 and n is 0, 1, 2, 3, or 4;
R2 and R6 may be the same or different and are hydroxy, alkoxy having from 1 to 8 carbon atoms;
B-Xm-CnH2n-O- wherein B is phenyl [which may be sub-stituted with the group Y, as defined herein] or 1-or 2-naphthyl, X, m, and n are as defined herein pro-vided that when n=0 also m=0; -OCH2OCO-alkyl in which the alkyl group has 1 to 6 carbon atoms; -OCH2CO-phenyl [the phenyl ring of which may be substituted with the group Y, 25 defined above) ; 1-glyceryl, wherein R7 is hydrogen, alkyl having from 1 to 6 car-bon atoms, or phenyl which may be substituted with the group Y, as defined above, and R8 is hydrogen or alkyl having from 1 to 6 carbon atoms;
R2 may also be -NR7R8 wherein R7and R8 are as defined above;
R3 is alkyl having from 1 to 6 carbon atoms, cycloalkylmethyl having from 4 to 8 carbon atoms, 2-or 3- thienylmethyl, 2- or 3-furanylmethyl, 1- or 2-napthylmethyl, or benzyl the phenyl ring of which may be substituted with the group Y, as defined above;
R4 is D-CnH2n-Om- wherein D is hydrogen, alkyl having from 1 to 4 carbon atoms or phenyl which may be substituted with the group Z, wherein Z is halogen, hydroxy, trifluoromethyl, alkoxy having from 1 to 6 carbon atoms, or alkyl having from 1 to 6 carbon atoms;
m and n are as defined above, R5 is hydrogen or alkyl having from 1 to 6 carbon atoms; and p is 0, 1 or 2;
with the proviso that when:

R1 is alkyl having from 1 to 6 atoms, cyclo-alkylmethyl having 4 to 8 carbon atoms, or A-Xm-CnH2n-in which (i)A is as defined above except that when A is phenyl or benzyl the phenyl group or phenyl moiety of the benzyl group is optionally substituted by Y' which is chosen from halogen, hydroxy, trifluoromethyl, alkoxy having 1 to 6 carbon atoms or alkyl having 1 to 6 carbon atoms, n is zero or 1 to 4, and m is zero or [m is] 1 and X
is oxygen, or (ii)A is phenyl or benzyl optionally sub-stituted by Y' as defined above, n is 1 to 4, m is one and X is sulphur, R3 is alkyl having 1 to 6 carbon atoms, cycloalkyl-methyl having from 4 to 8 atoms, 2- or 3-thienyl-methyl, 2- or 3-furanylmethyl, 1- or 2-naphthyl-methyl, or benzyl, the phenyl ring of which may be substituted by the group y' as defined above;
R4 is D-CnH2n-Om is which (i)D is hydrogen, alkyl having from 1 to 4 carbon atoms or phenyl which maybe substituted by Y' as defined above, m is zero and n is 1 to 4, or (ii)D hydrogen or phenyl optionally substituted by Y' and m and n are both zero, R5 is as defined above, and p is as defined above, then at least one of R2 and R6 must be chosen from:

B-Xm-CnH2n-O- in which (i)B and X are as defined above, m is a and n is 1 to 4, or (ii)B is phenyl substituted by 1- or 3-furanyl, 2- or 3-thienyl or phenyl which maybe substituted by halogen, hydroxy, trifluoromethyl, alkoxy having 1 to 6 carbon atoms or alkyl having 1 to 6 carbon atoms), m is zero and n is zero or 1 to 4; pivaloyloxy-methoxy, (in which the phenyl group may be substituted by Y as defined above), 1-glyceryl, wherein R7 and R8 are as defined above, or R2 is chosen from -NR7R8 in which [R7 is phenyl substituted by Y' as defined above] and R8 is as defined above, characterized in that the compound is prepared by an appropriate method selected from the following methods (wherein in the following formulae p,R1,R2,R3,R4,R5, and R6 are as defined above including identities excluded by the proviso set forth above and including protection of any reactive groups);

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

b) condensing a keto acid or ester of formula II
with an amino acid of formula III in a reactive medium containing a reducing agent I;

c) coupling an amino acid of formula VIII with an amino acid of formula V

I

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

e) condensing a keto acid or ester of formula X with an amino acid in a reactive medium containing a reducing agent I

f) alkylating an amine of formula III with a compound of formula XXII

1 in which X is a halogen atom;
followed by the removal of any protecting groups, and thereafter, if necessary or desired, converting a so obtained compound into a compound, or into another compound, of formula I as defined above and/or forming a salt thereof, and if desired isolating a preferred isomer.
2. A method as defined in claim 1 wherein in starting materials XX, III, V, XXI, or X, and in the compound produced, R4 is the group D-CnH2n-O-m as defined in claim 1 in which the sum of n and m is at least 1.
3. A method as defined in claim 1 characterized in that in starting materials XX, II, III, VIII, V, XXI, X, the amino acid of process e), or XXII, and in the compound produced, one of R2 and R6 is 2-phenoxyethoxy, 1-glyceryl.

or (2,2-dimethyl-1-oxopropoxy)methoxy, and the other is chosen from the groups defined above or is hydroxy, methoxy, ethoxy or benzyloxy.
4. A method as defined in claim 1 wherein in starting materials, XX, II, III, VIII, V, XXI, X, the amino acids of process e) and XXII, and in the compound produced, R1 is benzyl optionally para substituted by chlorine, methoxy, methyl, or phenyl, 2-phenylethyl or 1- or 2-naphthylmethyl, R2 and R6 are the same or different and are hydroxy, methoxy, ethoxy, benzyloxy, 2-phenoxyethoxy, 1-glyceryl, or (2,2-dimethyl-1- oxypropoxy)methoxy, and R3 is benzyl, p-methylbenzyl, p-phenylbenzyl, 1-naphthylmethyl or 3-thienylmethyl.
5. A method as defined in claim 1 wherein in starting materials XX, III, V, XXI, X and in the compound produced, R4 is methyl or benzyl and R5 is hydrogen.
6. A method as defined in claim 1 wherein in starting materials XX, II, III, VIII, V, XXI, X, the amine acid to process e), XXIII, and in the compound produced, R1 is benzyl optionally para substituted by chlorine, methoxy, methyl, or phenyl; 2-phenylethyl or 1- or 2-naphthylmethyl, R3 is benzyl, p-methylbenzyl, p-phenylbenzyl, 1-naphthylmethyl or 3-thienylmethyl;
R4 is hydrogen, methyl or benzyl, and R5 is hydrogen.
7. A method as defined in claim 6 wherein starting materials XX, III, V, XXI, X, and in the compound produced, R4 is hydrogen.
8. A method as defined in claims 3, 4 or 6 wherein in starting materials XX, III, V, XXI, X and in the compound produced, p is one.
9. A method as defined in claim 1 wherein either in starting materials XX, II, III, VIII, V, XXI, X, the amino acid of process e), and XXII and in the compound produced, R1 is benzyl, R2 is hydroxy, R3 is benzyl, R4 is hydrogen, R5 is methyl, R6 is hydroxy, and p is one and the stereo configuration of the starting materials is said that the compound produced is N- [N- [L-1-carboxy-2-phenylethyl]-L-phenylalanyl]-L-(?-methyl)-.beta.-alanine, or in starting materials XX, II, III; VIII, V, XXI, X, the amino acid of process e), and XXII, and in the compound produced, R1 is benzyl, R2 is R3 is benzyl, R4 and R5 are hydrogen, R6 is hydroxy and p is one, and the stereo configuration of the starting materials is such that the compound produced is N-[N-[L-[1-[(2,2-dimethyl-1-oxopropoxyl)methoxy]-carbonyl]-2-phenyethyl]-L-phenylalanyl]-.beta.-alanine, or in starting materials XX, II, III, VIII, V, XXI, X, the amino acid of process e), and XXII and in the compound produced, R1 is benzyl, R2 is R3 is benzyl, R4 and R5 are hydrogen, R6 is hydroxy and p is 1, and the stereo configuration of the starting materials is such that the compound produced is N-[N-[L-[1-[(2,2-dimethyl-1,3-dioxolan-4-yl) methoxy]-carbonyl]-2-phenylethyl]-L-phenylalanyl]-.beta.-alanine.

(continued on next page) 10.A compound having the formula I
I
or a mixture of enantiomers or diasterioisomers con-taining the same, and the pharmaceutically acceptable salts thereof wherein:
R1 is alkyl having from 1 to 6 carbon atoms, ada-mantylmethyl, cycloalkylmethyl having from 4 to 8 carbon atoms or A-Xm-CnH2n- wherein X is oxygen or sulfur, A is phenyl which may be substituted with the group Y, wherein Y is halogen, hydroxy, trifluoro-methyl, alkoxy having from 1 to 6 carbon atoms, alkyl having from 1 to 6 carbon atoms, 2- or 3-furanyl, 2-or 3-thienyl, or phenyl [which may be substituted with halogen, hydroxy, trifluoromethyl, alkoxy having from 1 to 6 carbon atoms or alkyl having from 1 to 6 carbon atoms], benzyl [the phenyl ring of which may be substituted with the group Y, as defined above], 1- or 2-naphthyl, 2- or 3-furanyl or 2- or 3-thienyl;

m is 0 or 1 and n is 0, 1, 2, 3, or 4;
R2 and R6 may be the same or different and are hydroxy, alkoxy having from 1 to 8 carbon atoms, B-Xm-CnH2n-O- wherein B is phenyl [which may be sub-stituted with the group Y, as defined herein] or 1-or 2-naphthyl, X, m, and n are as defined herein pro-vided that when n=O also m=O; -OCH2OCO-alkyl in which the alkyl group has 1 to 6 carbon atoms, -OCH2CO-phenyl [the phenyl ring of which may be substituted with the group Y, as defined above], 1-glyceryl, wherein R7 is hydrogen, alkyl having from 1 to 6 car-bon atoms, or phenyl which may be substituted with the group Y, as defined above, and R8 is hydrogen or alkyl having from 1 to 6 carbon atoms;
R2 may also be -NR7R8 wherein R7and R8 are as defined above;
R3 is alkyl having from 1 to 6 carbon atoms, cycloalkylmethyl having from 4 to 8 carbon atoms, 2-or 3- thienylmethyl, 2- or 3-furanylmethyl, 1- or 2-naphthylmethyl, or benzyl the phenyl ring of which may be substituted with the group Y, as defined above;
R4 is D-CnH2n-Om- wherein D is hydrogen, alkyl having from 1 to 4 carbon atoms or phenyl which may be substituted with the group Z, wherein Z is halogen, hydroxy, trifluoromethyl, alkoxy having from 1 to 6 carbon atoms, or alkyl having from 1 to 6 carbon atoms;
m and n are as defined above 5 R5 is hydrogen or alkyl having from 1 to 6 carbon atoms; and p is 0, 1 or 2;
with the proviso that when: .

R1 is alkyl having from 1 to 6 atoms, cyclo-alkylmethyl having 4 to 8 carbon atoms, or A-Xm-CnH2n-in which (i)A is as defined above except that when A is phenyl or benzyl the phenyl group or phenyl moiety of the benzyl group is optionally substituted by Y' which is chosen from halogen, hydroxy, trifluoromethyl, alkoxy having 1 to 6 carbon atoms or alkyl having 1 to 6 carbon atoms, n is zero or 1 to 4, and m is zero or 1 and X
is oxygen, or (ii)A is phenyl or benzyl optionally sub-stituted by Y' as defined above, n is 1 to 4, m is one and X is sulphur, R3 is alkyl having 1 to 6 carbon atoms, cycloalkyl-methyl having from 4 to 8 atoms, 2- or 3-thienyl-methyl, 2- or 3-furanylmethyl, 1- or 2-naphthyl-methyl, or benzyl, the phenyl ring of which may be substituted by the group Y' as defined above;
R4 is D-CnH2n-Om is which (i)D is hydrogen, alkyl having from 1 to 4 carbon atoms or phenyl which maybe substituted by Y' as defined above, m is zero and n is 1 to 4, or (ii)D hydrogen or phenyl optionally substituted by Y' and m and n are both zero, R5 is as defined above, and p is as defined above, then at least one of R2 and R6 must be chosen from:
B-Xm-CnH2n-O- is which (i)B and X are as defined above, m is 1 and n is 1 to 4, or (ii)B is phenyl substituted by 1- or 3-furanyl, 2- or 3-thienyl or phenyl (which maybe substituted by halogen, hydroxy, trifluoromethyl, alkoxy having 1 to 6 carbon atoms or alkyl having 1 to 6 carbon atoms), m is zero and n is zero or 1 to 4; pivaloyloxy-methoxy, (in which the phenyl group may be substituted by Y as defined above), 1-glyceryl, wherein R7 and R8 are as defined above, or R2 is chosen from -NR7R8 in which R7 is phenyl substituted by 1- or 3-furanyl, 2- or 3-thienyl or phenyl (which may be substituted by halogen, hydroxy)trifluoro-methyl, alkoxy having 1 to 6 carbon atoms or alkyl having 1 to 6 carbon atoms), and R8 is as defined above.

A compound of formula I as defined in claim 10, charac-terized in that R4 represents a group D-CnH2n-Om- as defined in claim 10 in which the sum of n and m is at least one.

12. A compound of formula I as defined in claim 1, characterized in that one of R2 and R6 is 2-phenoxy-ethoxy, 1-glyceryl, or (2,2-dimethyl-1-oxopropoxy)methoxy, and the other is chosen from the groups defined above or is hydroxy, methoxy, ethoxy or benzyloxy.

13. A compound as defined in claim 11, characterized in that R1 is benzyl optionally para substituted by chlorine, methoxy, methyl, or phenyl, 2-phenylethyl or 1- or 2-naphthylmethyl, R2 and R6 are the same or different and are hydroxy, methoxy, ethoxy, benzyloxy, 2-phenoxyethoxy, 1-glyceryl, or (2,2-dimethyl -1-oxopropoxy)methoxy, and R3 is benzyl, p-methylbenzyl, p-phenylbenzyl, 1-naphthylmethyl or 3-thienylmethyl.

14. A compound as defined in claim 13, characterized in that R4 is methyl or benzyl and R5 is hydrogen.

15. A compound as defined in claim 12, characterized in that R1 and R3 are as defined in claim 4, R4 is hydrogen, methyl or benzyl, and R5 is hydrogen.

16. A compound as defined in claim 15, characterized in that R4 is hydrogen.

17. A compound as defined in claims 10, 11 or 12 in which p is one.

18. N-[N-[L-1-(2,2-dimethyl-1-oxopropoxy)methoxy]carbonyl]-2-phenylethyl]-L-phenylalanyl]-.beta.-alanine, (2,2-dimethyl-1-oxopropoxy)methyl ester;
N-[N-[L-1-[phenylmethoxy]carbonyl]-2-phenylethyl]-L-phenylalanyl]-.beta.-alanine, (2,2-dimethyl-1-oxopropoxy)methyl ester;
N-[N-[L-1-carboxy-2-(4-phenyl)phenylethyl]-L-phenylalanyl]-.beta.-alanine;
N-[N-[L-[1-[(2,2-dimethyl-1-oxopropoxy)methoxy]
carbonyl]-2-(4-phenyl)phenylethyl]-L-phenylalanyl]-.beta.-alanine, (2,2-dimethyl-1-oxopropoxy)methyl ester;
N-[N-[L-1-carboxy-2-phenylethyl]-L-(4-phenyl)-phenylalanyl]-.beta.-alanine;
N-[N-[L-[1-[(2,2-dimethyl-1-oxopropoxy)methoxy]-carbonyl3-2-(4-phenyl)phenylethyl)-L-(4-phenyl)phenylalanyl-.beta.-alanine, (2,2-dimethyl-1-oxopropoxy)methyl ester;
N-[N-[L-[1-[(2,2-dimethyl-1-oxopropoxy)methoxy]-carbonyl]-2-phenylethyl)-L-(4-phenyl)phenylalanyl]-.beta.-alanine, (2,2-dimethyl-1-oxopropoxy)methyl ester;
N-[N-[L-1-carboxy-2-phenylethyl]-L-phenylalanyl)-L-(.alpha.-methyl)-.beta.-alanine;
N-[N-[L-[1-[(2,2-dimethyl-1-oxopropoxy)methoxy]-carbonyl]-2-phenylethyl]-L-phenylalanyl]-L-(.alpha.-methyl)-.beta.-alanine, (2,2-dimethyl-1-oxopropoxy)methyl ester;
N-[N-[(L-1-carboxy-2-phenylethyl)]-L-phenylalanyl]-.beta.-alanine, 2-phenoxyethyl ester;

N-[N-[L-[1-[(2,2-dimethyl-1-oxopropoxy)methoxy]-carbonyl]-2-phenylethyl]-L-phenylalanyl]-.beta.-alanine;
N-[N-[L-[1-[(2-phenoxy)ethoxy]carbonyl)-2-phenylethyl]-L-phenylalanyl]-.beta.-alanine;
N-[N-[L-[1-[(1-oxo-3-isobenzofuranyloxy)]carbonyl]-2-phenylethyl]-L-phenylalanyl-.beta.-alanine;
N-[N-[L-[1-[(2,3-dihydroxy)-1-propoxy]carbonyl]-2-phenylethyl]-L-phenylalanyl]-.beta.-alanine:
N-[N-[L-[1-[(2,2-dimethyl-1,3-dioxolan-4-yl)methoxy]-carbonyl]-2-phenylethyl]-L-phenylananyl]-.beta.-alanine;
N-[N-[L-[1-[(2,2-dimethyl-1-oxopropoxy)methoxy]-carbonyl]-2-(4-phenyl)phenylethyl]-L-phenylalanyl]-.beta.-alanine;
N-[N-[L-[1-[(2-phenoxy)ethoxy]carbonyl]-2-(4-phenyl)-phenylethyl]-L-phenylalanyl]-.beta.-alanine;
N-[N-[L-[1-[(1-oxo-3-isobenzofuranyloxy)]carbonyl]-2-(4-phenyl)phenylethyl]-L-phenylalanyl]-.beta.-alanine;
N-[N-[L-[1-[(2,3-dihydroxy)-1-propoxy]carbonyl]-2-(4-phenyl)phenylethyl]-L-phenylalanyl]-.beta.-alanine;
N-[N-[L-[1-[(2,2-dimethyl-1,3-dioxolan-4-yl)methoxy)-carbonyl]-2-(4-phenyl)phenylethyl]-L-phenylalanyl]-.beta.-alanine;
N-[N-[L-[1-[(2,2-dimethyl-1-oxopropoxy)methoxy]-carbonyl]-2-phenylethyl]-L-(4-phenyl)phenylalanyl]-.beta.-alanine;
N-[N-[L-[1-[(2-phenoxy)ethoxy]carbonyl]-2-phenylethyl]-L-(4-phenyl)phenylalanyl]-.beta.-alanine;
N-[N-[L-[1-[(1-oxo-3-isobenzofuranyloxy)]carbonyl)-2-phenylethyl]-L-(4-phenyl)phenylalanyl]-.beta.-alanine;

N-[N-[L-[1-[(2,3-dihydroxy)-1-propoxylcarbonyl]-2-phenylethyl]-L-(4-phenyl)phenylalanyl]-.beta.-alanine;
N-[N-[L-[1-[(2,2-dimethyl-1,3-dioxolan-4-yl)methoxy]-carbonyl]-2-phenylethyl]-L-(4-phenyl) phenylalanyl]-.beta.-alanine;
N-[N-[L-[1-[(2,2-dimethyl-1-oxopropoxy)methoxy]-carbonyl]-2-(4-phenyl)phenylethyl]-L-(4-phenyl)phenylalanyl]-.beta.-alanine;
N-[N-[L-[1-[(2-phenoxy)ethoxy]carbonyl]-2-(4-phenyl)-phenylethyl]-L-(4-phenyl)phenylalanyl]-.beta.-alanine;
N-[N-[L-[1-[(1-oxo-3-isobenzofuranyloxy)]carbonyl]-2-(4-phenyl)phenylethyl]-L-(4-phenyl)phenylalanyl]-.beta.-alanine;
N-[N-[L-[1-[(2,3-dihydroxy)-1-propoxy]carbonyl]-2-(4-phenyl)phenylethyl]-L-(4-phenyl)phenylalanyl]-.beta.-alanine;
N-[N-[L-[1-[(2,2-dimethyl-1,3-dioxolan-4-yl)methoxy]-carbonyl]-2-(4-phenyl)phenylethyl]-L-(4-phenyl)phenylalanyl]-.beta.-alanine;
N-[N-[L-[1-[(2,2-dimethyl-1-oxopropoxy)methoxy]-carbonyl]-2-phenylethyl]-L-phenylalanyl]-D,L-.alpha.-methyl-.beta.-alanine;
N-[N-[L-[1-[(2-phenoxy)ethoxy]carbonyl]-2-phenylethyl]-L-phenylalanyl]-D,L-.alpha.-methyl-.beta.-alanine;
N-[N-[L-[1-[(1-oxo-3-isobenzofuranyloxy)]carbonyl]-2-phenylethyl]-L-phenylalanyl]-D,L-.alpha.-methyl-.beta.-alanine;
N-[N-[L-[1-[(2,3-dihydroxy)-1-propoxy]carbonyl]-2-phenylethyl]-L-phenylalanyl]-D,L-.alpha.-methyl-.beta.-alanine;
N-[N-[L-[1-[(2,2-dimethyl-1,3-dioxolan-4-yl)methoxy]-carbonyl]-2 phenylethyl]-L-phenylalanyl]-D,L-.alpha.-methyl-.beta.-alanine;

N-[N-[L-[1-[(2,2-dimethyl-1-oxopropoxy)methoxy]-carbonyl]-2-(4-phenyl)phenylethyl]-L-phenylalanyl]-D,L-.alpha.-methyl-.beta.-alanine;
N-[N-[L-[1-[(2-phenoxy)ethoxy]carbonyl]-2-(4-phenyl)-phenylethyl]-L-phenylalanyl]-D,L-.alpha.-methyl.beta.-alanine;
N-[N-[L-[1-[(1-oxo-3-isobenzofuranyloxy)]carbonyl]-2-(4-phenyl)phenylethyl]-L-phenylalanyl]-D,L-.alpha.-methyl-.beta.-alanine;
N-[N-[L-[1-[(2,3-dihydroxy)-1-propoxy]carbonyl]-2-(4-phenyl)phenylethyl]-L-phenylalanyl]-D,L-.alpha.-methyl.beta.-alanine;
N-[N-[L-[1-[(2,2-dimethyl-1,3-dioxolan-4-Yl)methoxy]-carbonyl]-2-(4-phenyl)phenylethyl]-L-phenylalanyl]-D,L-.alpha.-methyl-.beta.-alanine;
N-[N-[L-[1-[(2,2-dimethyl-1-oxopropoxy)methoxy]-carbonyl]-2-phenylethyl]-L-(4-phenyl)phenylalanyl]-D,L-.alpha.-methyl-.beta.-alanine;
N-[N-[L-[1-[(2-phenoxy)ethoxy]carbonyl]-2-phenylethyl]-L-(4-phenyl)phenylalanyl]-D,L-.alpha.-methyl-.beta.-alanine;
N-[N-[L-[1-[(1-oxo-3-isobenzofuranyloxy)]carbonyl]-2-phenylethyll-L-(4-phenyl)phenylalanyl]-D,L-.alpha.-methyl-.beta.-alanine;
N-[N-[L-[1-[(2,3-dihydroxy)-1-propoxy]carbonyl]-2-phenylethyl]-L-(4-phenyl)phenylalanyl]-D,L-.alpha.-methyl-.beta.-alanine;
N-[N-[L-[1-[(2,2-dimethyl-1,3-dioxolan-4-yl)methoxy]-carbonyl]-2-phenylethyl]-L-(4-phenyl)phenylalanyl]-D,L-.alpha.-methyl-.beta.-alanilne;

N-[N-[L-[1-[(2,2-dimethyl-1-oxopropoxy)methoxy]-carbonyl]-2-(4-phenyl)phenylethyl]-L-(4-phenyl)phenylalanyl]-D,L-.alpha.-methyl-.beta.-alanine;
N-[N-[L-[1-[(2-phenoxy)ethoxy]carbonyl]-2-(4-phenyl)-phenylethyl]-L-(4-phenyl)phenylalanyl]-D,L-.alpha.-methyl-.beta.
alanine;
N-[N-[L-[1-[(1-oxo-3-isobenzofuranyloxy)]carbonyl]-2-(4-phenyl)phenylethyl]-L-(4-phenyl)phenylalanyl]-D,L-.alpha.-methyl-.beta.-alanine;
N-[N-[L-[1-[(2,3-dihydroxy)-1-propoxy]carbonyl]-2-(4-phenyl)phenylethyl]-L-(4-phenyl)phenylalanyl]-D,L-.alpha.-methyl-.beta.-alanine;
N-[N-[L-[1-[(2,2-dimethyl-1,3-dioxolan-4-yl)methoxy]-carbonyl]-2-(4-phenyl)phenylethyl]-L-(4-phenyl)phenylalanyl]-D,L-.alpha.-methyl-.beta.-alanine;
N-[N-[L-[1-[(2,2-dimethyl-1-oxopropoxy)methoxy]-carbonyl]-2-phenylethyl)-L-2-thienylalanyl]-.beta.-alanine;
N-[N-[L-[1-[(2,2-dimethyl-1-oxopropoxy)methoxy]carbonyl]-2-phenylethyl]-L-3-thienylalanyl]-.beta.-alanine;
N-[N-[L-[1-[(2,2-dimethyl-1-oxopropoxy)methoxy]carbonyl]-2-phenylethyl]-L-2-furoalanyl]-.beta.-alanine;
N-[N-[L-[1-[(2,2-dimethyl-1-oxopropoxy)methoxy]carbonyl]-2-phenylethyl]-L-phenylalanyl]-.alpha.-methylalanine;
N-[N-[L[1-[(2,2-dimethyl-1-oxopropoxy)methoxy]carbonyl]-2-phenylethyl]-L-phenylalanyl]-L-.alpha.-hydroxy-.beta.-alanine;
N-[N-[L-[1-[(2,2-dimethyl-1-oxopropoxy)methoxy]carbonyl]-2-phenylethyl]-L-phenylalanyl]-D-.alpha.-hydroxy-.beta.-alanine;

N-[N-[L-[1-[(2,2-dimethyl-1-oxopropoxy)methoxy]carbonyl]-2-phenylethyl]-L-phenylalanyl]-L-.alpha.-methoxy-.beta.-alanine;
N-[N-[L[1[(2,2-dimethyl-1-oxopropoxy)methoxy]carbonyl]-2-phenylethyl)-L-phenylalanyl]-D-.alpha.-methoxy-.beta.-alanine;
N-[N-[L-[I-[(2,2-dimethyl-1-oxopropoxy)methoxy]carbonyl]-2-phenylethyl]-L-phenylalanyl]-D,L-.alpha.-methyl-.beta.-alanine benzyl ester;
N-[N-[L-[1-(2,2-dimethyl-1-oxopropoxy)methoxy]carbonyl]-2,(4-methoxy)phenylethyl]-L-phenylalanyl-.beta.-alanine;
N-[(L)-1-carboxy-2 phenylethyl]-L-phenyl alanyl-L-phenylalanine;
N-[(L)-1-carboxy-2-phenylethyl]-L-leucyl-L-phenylalanine;
and/or a salt of one of the foregoing, which contains a free acid function, with a pharmaceutically acceptable base, or a salt of one of the foregoing with a pharmaceutically acceptable acid.
19. A composition which comprises a compound as defined in claims 10, 11 or 12 in combination with a pharmaceutically acceptable carrier.