PROCEDE DE PRODUCTION DE DERIVES DE DIPEPTIDES

PROCESS FOR THE MANUFACTURE OF DIPEPTIDE DERIVATIVES

Abrégé anglais

ABSTRACT OF THE DISCLOSURE
Process for the manufacture of dipeptide derivatives derived from
histidyl-proline of the general formula
<IMG>
wherein each of the groups R and Z have a range of meanings, and in which
the optically active amino acids preferably have the L-configuration. These
compounds, and their pharmaceutically acceptable salts, find use as psycho-
stimulating agents, or anti-depressive agents.

Revendications

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. A process for the preparation of the dipeptide derivatives of
formula (I):
<IMG> (I)
wherein R1 and R2 have the same or a different meaning and each represents an
alkyl or cycloalkyl radical containing up to six carbon atoms or a benzyl
group or both, together with the nitrogen atom to which these radicals are
attached, represent a five to seven membered heterocyclic ring, or wherein R2
represents a hydrogen atom, R3 and R4 have the same or a different meaning
and each represent hydrogen or an alkyl radical containing from one to three
carbon atoms or together represent an additional bond between the carbon atoms
to which they are attached, R5 represents a hydrogen atom or an alkyl radical
containing from one to three carbon atoms, and wherein Z represents a divalent
group, being one of the structures:
- NH -, <IMG>, - NH - CO -
<IMG> or <IMG>,
wherein R6 and R7 have the same or a different meaning and each represent
hydrogen or an alkyl radical containing from 1 to 3 carbon atoms and salts
of the compounds of formula (I) with pharmaceutically acceptable acids, by
reacting:
18

a) a carboxylic acid of the formulae
<IMG> (II) or <IMG> (IIa)
wherein R3, R4, R5 and Z have the same meaning as above and Y represents a
group cleavable by hydrogenolysis, in the presence of an agent, which is able
to split off water or a functional derivative of an acid of formula (II) or
(IIa), respectively, with a compound of formula (III),
<IMG> (III)
wherein R1 and R2 have the same meaning as above and in which the 1-(3)-
position of the imidazole group of the histidyl residue may be protected
against acylation, or a derivative thereof obtained by reacting the compound
of formula (III) in a manner known per se with a silylating agent derived
from a trialkylsilanol or a dialkylsilanediol (each alkyl radical of said
silylating agents containing from one to three carbon atoms) and splitting
off in a manner known per se the group Y, the protecting group from the
imidazole group contained in the histidyl residue and the silyl groups, if
present; or
b) a compound of the formula,
19

<IMG> (IV)
wherein R3, R4, R5 and Z have the same meaning as above and X represents a
hydrogen atom or a group Y as defined before, or a derivative of the compound
of formula (IV) in which the 1-(3)-position of the imidazole group is pro-
tected reversibly (as described previously) in presence of an agent which is
able to split off water, or a functional derivative of a compound of formula
(IV) with a compound of formula (V)
<IMG> (V)
wherein R1 and R2 have the same meaning as above and splitting off any pro-
tecting groups, if present, or
c) a compound of formula (VI)
(VI)
<IMG>
wherein R3, R4, R5, X and Z have the same meaning as above and W represents a
hydroxy, acyloxy, p-nitrophenoxy, tri- or pentachlorophenoxy-, pentafluoro-
phenoxy, pyridyloxy, phenylmercapto, p-nitrophenylmercapto or cyanomethyloxy
group or the residue of N-hydroxysuccinimide, or a derivative of the compound
of formula (VI) in which the 1-(3)-position of the imidazole group is protected
reversibly as described previously, with an amine of formula (VII),
<IMG> (VII)

wherein R1 and R2 have the same meaning as above and splitting off any pro-
tecting groups, if present; or
d) a compound of formula (III), wherein R1 and R2 have the same meaning
as above, provided that neither of these radicals may be a benzyl group in
this instance, and in which the 1-(3)-position of the imidazole group may be
protected against acylation preferably by a group cleavable by hydrogenolysis,
with an acid of the formula:
<IMG> (VIII)
wherein Hal represents a chlorine or a bromine atom in the presence of an
agent capable of splitting off water or with a functional derivative of such
an acid of formula (VIII), and thereafter dehalogenating the intermediate by
hydrogenolysis and effecting removal of the protecting group, if present to
give such compounds of formula (I) in which R3 and R4 form an additional bond
between the carbon atoms to which they are attached, R5 is hydrogen and Z
represents -N=C(OH)- or -NH-CO-, respectively.
2. Dipeptide derivatives of the formula,
<IMG> (I)
in which R1 and R2 are the same or different and each represent an alkyl or
cycloalkyl radical containing up to six carbon atoms or a benzyl group or
both, together with the nitrogen atom to which they are attached, represent
21

a five to seven membered heterocyclic ring, or in which R2 represents a
hydrogen atom, R3 and R4 are the same or different and each represent hydrogen
or an alkyl radical containing from one to three carbon atoms, or where R3
and R4 together can also represent an additional bond between the carbon atom
to which these radicals are attached, R5 represents a hydrogen atom or an
alkyl radical containing from one to three carbon atoms, and Z represents a
divalent structure which completes the ring to give a five- or six-membered
ring, said divalent structure being selected from the structures,
<IMG> , <IMG> , <IMG> ,
<IMG> and <IMG>,
wherein R6 and R7 are the same or different and each represent hydrogen or an
alkyl radical containing from one to three carbon atoms, and pharmaceutically
acceptable acid salts thereof, whenever prepared by the process of claim 1 or
by an obvious chemical equivalent thereof.
3. Process according to claim 1 wherein in formula I the groups R3
and R4 together represent a double bond, and Z represents one of the divalent
structures -NH-, -HN-CO-, or -N=C(OH)-.
4. Dipeptide derivatives according to claim 2 of the formula:
<IMG>
wherein R1, R2 and R5 have the same meaning as in formula (I) and wherein Z1
represents one of the divalent structures -NH-, -HN-CO-, or -N=C(OH)-,
respectively, and pharmaceutically acceptable acid salts thereof, whenever
22

prepared by the process of claim 3, or by an obvious chemical equivalent
thereof.
5. Process according to claim 1 wherein R2 represents hydrogen, and
R1 is a group R1', and R1' is chosen from an alkyl or cycloalkyl group
containing up to six carbon atoms or a benzyl group.
6. Dipeptide derivatives according to claim 2 of the formula,
<IMG>
wherein R3, R4, R5 and Z have the same meaning as in formula (I) and wherein
R1' represents an alkyl or cycloalkyl radical containing up to six carbon
atoms or the benzyl group and pharmaceutically acceptable acid salts thereof,
whenever prepared by the process of claim 5 or by an obvious chemical equival-
ent thereof.
7. Process according to claim 1 wherein R2 represents hydrogen and R1
represents a straight chain alkyl group containing one to four carbon atoms.
8. Dipeptide derivatives according to claim 2 of the formula
<IMG>
and pharmaceutically acceptable salts thereof whenever prepared by the process
of claim 7 or by an obvious chemical equivalent thereof.
23

9. Process for the preparation of orotyl-L-histidyl-L-proline-N-
methylamide which comprises reacting together the hydrobromide of L-histidyl-
L-proline-methylamide, orotic acid, and L-hydroxybenzotriazole in the pre-
sence of triethylamine and N,N'-dicyclohexyl carbodiimide.
10. Orotyl-L-histidyl-L-proline-N-methylamide whenever prepared
by the process of claim 9 or by an obvious chemical equivalent thereof.
11. Process for the preparation of 5-oxo-6-(D,L)-methyl-thiomorpho-
line-3-(L)-carbonyl-L-histidyl-L-proline-N-methylamide which comprises react-
ing together the hydrobromide of L-histidyl-L-proline methylamide, thiomorpho-
line-6-(D,L)-methyl-5-on-3-(L)-carboxylic acid and 1-hydroxy benzotriazole in
the presence of triethylamine and N,N'-dicyclohexyl-carbodiimide.
12. 5-Oxo-6-(D,L)-methyl-thiomorpholine-3-(L)-carbonyl-L-histidyl-
L-proline-N-methylamide whenever prepared by the process of claim 11 or by an
obvious chemical equivalent thereof.
13. Process for the preparation of orotyl-L-histidyl-L-proline-N(n-
butyl)-amide, which comprises reacting together the hydrobromide of L-histidyl-
L-proline-n-butylamide, orotic acid, and 1-hydroxybenzotriazole in the pre-
sence of triethylamine and N,N'-dicyclohexyl-carbodiimide.
14. Orotyl-L-histidyl-L-proline-N-(n-butyl)-amide whenever prepared
by the process of claim 13 or by an obvious chemical equivalent thereof.
15. Process for the preparation of orotyl-L-histidyl-L-proline-N-
(n-hexyl)-amide which comprises reacting together the hydrobromide of L-
histidyl-L-proline-(n-hexyl)-amide, orotic acid and 1-hydroxybenzotriazole in
the presence of triethylamine and N,N'-dicyclohexyl-carbodiimide.
16. Orotyl-L-histidyl-L-proline-N-(n-hexyl)-amide whenever prepared
by the process of claim 15 or by an obvious chemical equivalent thereof.
24

17. Proccss for the preparation of 5-oxo-6(D,L)-methyl-thiomorpholine
3-(L)-carbonyl-L-histidyl-L-proline-N-(n-hexyl)-amide which comprises
reacting together the hydrobromide of L-histidyl-L-proline(n-hexyl) amide
thiomorpholine-6-(D,L)-methyl-5-on-3-(L)-carboxylic acid and 1-hydroxy
benzotriazole in the presence of triethylamine and N,N'-dicylohexyl-
carbodiimide.
18. 5-Oxo-6(D,L)-methyl-thiomorpholine-3-(L)-carbonyl-L-histidyl-L-
proline-N-(n-hexyl)-amide whenever prepared by the process of claim 17 or by
an obvious chemical equivalent thereof.

Description

i520
The present invention relates to a process for the manufacture of
dipeptide derivatives, which are derived from histidyl-proline, one or both
of the amino acids contained therein being optically active or racemic, but
preferably having the L-configuration. More precisely the invention relates
to the manufacture of compounds of formula (I)
~ ~ R
\ ~ J - NN G~ - C0 - ~ C0 - N \
and salts of these compounds with pharmaceuticaIly acceptable acids.
In formula (I) the radicals Rl and R2 are the same or different, -
and each represent an alkyl or a cycloalkyl radical containing up to six
carbon atoms or a benzyl group or represent - together with the nitrogen
atom to which these radicals are attached - a five to seven membered hetero~
cyclic ring, R2 also may be a hydrogen atom. R3 and R4 are the same or
different and each represent hydrogen or an alkyl radical containing from
one to three carbon atoms. R3 and R4 together also can represent an addition-
al bond between the carbon atoms to which these radicals are attached. R5
represents a hydrogen atom or an alkyl radical containing from one to three
carbon atoms. Z represents a divalent structure which completes the ring to
give a five- or six-membered ring, said divalent structure being selected from
the structures:
pH
- NH -, - N = ~ -, - NH - C0 -,
; 20 lR6 16
- C - S -, and - f o
R7 R7
. .
_ 1 - ~

wherein R6 and R7 are the same or different and each represent hydrogen or an
alkyl radical containing from one to three carbon atoms.
The dipeptide derivatives of formula (I) are acyl derivative~ of
histidyl_proline amides which may be derived from a carboxylic acid of
formula (II):
~' " / S
¦ R4
¦ / 3
\ N / b OOH (II)
H
Preferred acids of formula (II) for use in preparing the dipeptides
of the present invention are orotic acid, imidazolidine-(2)-one-(4)-carboxylic
acid and thiomorpholine-(5)-one-(3)-carboxylic acid. Other suitable acids of
formula (II) are for instance, morpholine-(5)-one-(3)-carboxylic acid, (4)-
carboxy-imidazole-(2)-one, thiomorpholine-(6)-methyl-(5)-one-(3)-carboxylic
acid, 5-methyl-, 5-ethyl- and 5-propyl-orotic acid, and thio rpholine-(5)-
one-(2,2)-dimethyl-(3)-carboxylic acid.
Where R3 and R4 do not represent a second bond between the carbon -
atoms to which said members are connected and/or in the case that R6 and R7
are different, the acyl groups derived from the acid of formula (II) can be
present in the compound of formula (I) in racemic or optically active fonm,
and are preferably in the L_configuration.
If R2 represents a hydrogen atom, Rl preferably is a straight chain
alkyl radical containing up to six carbon atoms, especially a radical contain-
ing one to four carbon atoms.
Where Rl and R2, together with the nitrogen atom to which these
radicals are attached, represent a heterocyclic radical, this group may be
the pyrrolidino-, the piperidino- or the hexamethylenimino-group or the

1(~5;~0
residue of a five to seven membered heterocyclic ring containing at least one
further hetero atom, such as the thiazolidino-, morpholino- or thiomorpholino-
eroup .
Due to the basicity of the histidyl radical the compounds of fonmula
(I) can form salts with acids. A further object of the present invention
accordingly is the preparation of salts of the compounds of formula (I) to
provide pharmaceutically acceptable salts of inoreanic or organic acids, such
as hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, formic
acid, acetic acid, propionic acid, benzoic acid, salicylic acid, phenyl acetic --~
acid and benzenesulfonic acid.
Surprisingly the compounds of formula (I) (and their salts) possess
biological properties, which with respect to their effectiveness are nearly
the same as those of pyroglutamyl-histidyl-proline amide, which compound
normally is designated as "tyrotropine releasing hormone" or nTRHn. The
effects of the new compounds, however, last considerably longer than those of
the known product. Of special value in therapy is that the relation of the
central nervous system stimulating effects to the endocronologic effects is
shifted for the new compounds (if compared with those effects of TRH) favorably
to the pharmacologically valuable properties.
The compounds of formula (I) may be administered orally or parenter-
ally and act quickly For instance on parenteral administration the effects
are observed after 10 minutes.
The most impressive effects in pharmacological tests indicate a
central nervous system stimulating action for the new products. On administer-
ing equal doses of TRH and one of the new products, respectively, to test
animals the new compound has a central nervous system stimulating action which
is several times longer than TRH.
The toxicity of the compound of formula (I) is very low. Due to
these valuable properties the new compounds can be used as therapeutics, for
-- 3 --

lO~S~O
instance as psychostimulating agents or anti-depressive agents. The compounds
are of therapeutic value in animals and humans.
Suitable pharmaceutical preparations containing the compounds of
formula (I) or their salts are tablets for oral use, dragees, granules,
capsules, drops, syrups, sprays for intranasal application or administration
via bronchial sprays, and for parenteral application, sterile a~ueous solutions.
These pharmaceutical preparations are prepared as is known in the prior art
with inert carriers, such as is known for other anti-depressants and CNS-
stimulants used in human and warm-blooded animal therapy.
The therapeutically effective dosage of the new compounds depends on
the severity of the depression to be treated and also on the route of a~min-
istration. Preferably the compounds of the invention are used parenterally in
a dosage of 0.005 to 0.5 mg/kg and orally in a dosage of 0.05 to 5 mg/kg. When
the therapy does not call for such dosages as stated, the dosage _ay be in-
creased or decreased as recommended by the circumstances.
The compounds of formula (I) are prepared by reacting a histidyl-
proline amide (having the desired optical configuration) of formula (III),
IN
H N - C - C0 - ~ C0 - ~ (III),
R2
wherein Rl and R2 have the same meaning as above with an acid of formula (II)
in the presence of an agent, which is able to split off water, especially in
presence of a carbodiimide, preferably dicyclohexyl carbodiim;de, or with a
- functional derivative of an acid of formNla (II), as for instance an acid

lO~iS~O
halide, anhydride, mlxed anhydride, azide or an acti~ated ester thereof.
Instead of the acid of formula (II) or a functional derivative
thereof, also a compound of formula (IIa)
, - _ / R5
I ¦ \ R
/ 3 (IIa),
\ / ~COOH
Y
wherein R3, R4, R5 and Z have the same meaning as above and Y represents a
group cleavable by hydrogenolysis, preferably a carbobenzoxy group or a
substituted carbobenzoxy group, or a functional derivative of the acid of
formula (IIa), as for instance an acid halide, anhydride, mixed anhydride,
azide or an activated ester, may be used.
During the reaction of the histidyl-proline amide of formula (III)
with the acid of fonmula (II) or (IIa) or a derivative thereof, the 1-(3)-
position of the ~midazole group of the histidyl residue may be protected
against acylation.- Suitable protective groups are known from peptide synthesis
to those skilled in the art. Such groups are for instance the carbobenzoxy
group, substituted carbobenzoxy groups, the trityl, o-nitrophenoxy acetyl or
the tert.-butyloxycarbonyl group and other groups known E~ se, which after
the reaction is completed can be split off by hydrolysis or hydrogenolysis.
Instead of the histidyl-proline amide of formula (III) with either a
free or protected imidazole group, a derivative thereof, obtained by reacting
it in a manner known per se with a silylating agent, derived from a triaIkyl-
silanol or a dialkylsilanediol (each alkyl radical thereof containing from one
to three carbon atoms) as for instance hexamethyldisilazene, trimethylchloro-
silane, trimethylsilylacetamide or dimethyldichlorosilane and the known
.,
_ 5 _

S'~
equivalents thereof, may be used in the process of the present invention.After the acylation reaction (which in this case has to be perfor~ed in
absence of a proton active solvent) is finished, the silyl groups can easily
be split off by hydrolysis or alcoholysis.
In preparing the compounds of formula (I) it is possible also to
react a compound of fonmula (IV):
, ` / R5
I ¦ \ R
l\ 1 ~ 3
~ \ N / ~ NH - CH - COOH (IV),
wherein R3, R4, R5 and Z have the same meaning as above and X represents a
hydrogen atom or a group of type Y as defined before, or a derivative of the
compound of formula (IV) in which the 1-(3)-position of the imidazole group is
protected reversibly as described above, in the presence of an agent which is
able to split off water, especially in presence of a carbodiimide, preferably
dicyclohexyl carbodiimide or with a functional derivative of a compound of
formula (IV) as for instance an acid halide, anhydride, mixed anhydride or an
activated ester with a compound of formula (V),
~ ON / (V), `
wherein R1 and R2 have the same meaning as above and thereafter splitting off
any protecting groups; if present.
Furthermore compounds of formula (I) can be prepared by reacting a
compound of formula (VI)
-- 6 --

iO~i5;~0
- _ ~ R5
~ R3 ~NH (VI),
O \ N CO - NH - CH - CO - N ~ O - W
wherein R3, R4, R5, ~ and Z have the same meaning as above and W repreQent~ a
hydroxy, acyloxy, p-nitrophenoxy, tri- or pentachlorophenoxy-, pentafluoro-
phenoxy, pyridyloxy, phenylmercapto, p-nitrophenylmercapto or cyanomethyloxy
group or the residue of N-hydroxysuccinimide, or a derivative of the compound
of formula (VI) wherein the 1-(3)_position of the imidazole group is protected
reversibly in the manner described hereinabove, with an ~mine of formula (VII),
Rl -
HN~/ (VII)
wherein Rl and R2 have the same meaning as above and splitting off any pro-
tecting groups, if present.
Compounds of fonmula (I), (one or both of the amino acid groups
contained therein being optically active or racemic but preferably having the ;~
L,configuration) derived from orotic acid, wherein Rl and R2 have the same
meaning as above, provided that neither of these radicals may be a benzyl
:~ group in this instance, are preferably manufactured by reacting the compound
of formula (III) having the desired optical configuration with an acid of the
formNla:
~o~
IN N OCH (VIII)
1- H

lO~S;~O
wherein Hal represents a chlorine or a bromine atom in the presence of an
agent able to split off water, especially in the presence of a carbodiimide,
preferably dicyclohexylcarbodiimide or with a functional derivative of such an
acid of formula (VIII), as for instance an acid halide, anhydride, mixed
anhydride, azide or an activated ester thereof, and thereafter dehalogenating
the intermediate by hydrogenolysis.
During the reaction of the compound of formula (III) with the acid
of formula (VIII) or the derivative thereof, the 1-(3)-position of the imida-
zol group of the histidyl residue may be protected against acylation. Suit-
able protective groups are those mentioned already hereinabove. In this lastdescribed method, it is especially useful to employ such protective groups as
can be split off by hydrogenolysis (as for instance the carbobenzoxy group,
substituted carbobenzoxy group or the o-nitrophenoxyacetyl group). In this
case the removal of the protective group from the intermediate occurs coin-
cidently with the hydrogenolysis of the halogen atom. Naturally, it is also
possible, however, to protect the imidazole group with such groups, which can
be split off by hydrolysis (as for instance trityl or tert. butyloxycarbonyl
groups and others) and to remove such protective groups from the intermediate
product or from the product obtained by hydrogenolysis of the halogen atom
from the intermediate.
The hydrogenolysis of the halogen atom is preferably carried out by
means of catalytically activated hydrogen. Preferably a noble metal hydrogena-
tion catalyst, as for instance palladium or platinum on charcoal, on barium
sulfate, on alumina, on calcium or barium carbonate and other noble metal
catalysts known E~ se, are used inthehydrogenolysis, which can be perfonmed
under nonmal or increased pressure , preferably at room temperature. Solvents
like water or mixtures of water with lower alcohols (methanol, ethanol) or
with tetrahydrofuran and dioxane are preferably used in the hydrogenolysis
step. It is, however, also possible to use glacial acetic acid for example.

lO~S~O
The hydrogen halide formed during the hydrogenolysis is preferably
bound as soon as it is formed. To that end the hydrogenolysis is preferably
performed in the presence of an agent which is able to bind hydrogen halides.
If the abovementioned preferred or other non-acidic solvents are used, suit-
able hydrogen halide binding agents are for instance magnesium oxide, barium
oxide, aIkali hydroxides, ammonia or ammonium hydroxide, triethyl~mine or
salts of such bases with acids which are very much weaker than the hydrogen
halide being formed, as for instance aIkali carbonates or acetates. Suitable
media for the performance of the hydrogenolysis step also include for instance,
solutions of aIkali metal, aIkaline earth metal, ammonium or amine acetates in
acetic acid.
Mixed anhydrides of the acids of formulae (II), (IIa), (n) and
(VIII) are preferably obtained from trimethylacetic acid or from noesters of
carbonic acid especially those in which the carbonic acid is esterified with
aliphatic alcohols containing from one to four carbon atoms. Suitable
activated esters of such acids are those with p-nitrophenol, tri- or penta-
chlorophenol, pentafluorophenyl, N-hydroxysuccinimide, 2- or 4-~ydroxypyridine,
thiophenol, p-nitrothiophenol, hydroxyacetonitrile, l-hydroxybenzotriazole and
other h~vdroxy- or mercapto compounds conventionally used in peptide chemistry
to prepare activated esters from acids.
The compounds of formula (I) and their salts are relatively stable
products. They can therefore be purified for instance, by dissolving and
reprecipitation, by recrystallization~ and also by column chr~matography or
counter-current distribution.
The following non-limiting examples further illustrate the invention.
All temperature references are uncorrected.
E~llMPI~: 1
a) ~ solution of 12.7 g of methylamine in 350 ml of absolute tetrahydro-
; furan is treated, while stirring, at o& with 71 g of the N-hydroxysuccinLmide

l(U~S~O
ester of benzyloxycarbonyl-L-proline.
The mixture is stirred for 15 minutes at o& and thereafter for two
hours at room temperature. After distilling off the sol~ent in ~ac~um the
residue is dissolved in ethyl acetate, which solution is washed with a 5%
aqueous solution of potassium hydrogen sulfate, a saturated aqueous solution
of sodium hydrogen carbonate and finally with water. The solution is dried
with anhydrous sodium sulfate and evaporated. Thus 46 g (85% of the theoret-
ical yield) of benzyloxycarbonyl-L_proline-N-methylamide are obtained in the
form of a colorless, viscous oil which crystallizes on storage at 3 - 5 C,
but melts at room temperature.
b) 102.3 g of benzyloxycarbonyl-L_proline-methylamide dissolved in
methanol are treated in the presence of freshly prepared palladium black and
22.3 ml of glacial acetic acid with hydrogen. The catalyst is filtered off
and the solution is mixed with 100 ml of 4 N hydrochloric acid, then e~aporated
in vacuum, mixed with absolute ethanol and again evaporated. The crystalline
residue is recrystallized from methanol/ether (3:10). The product is dried in
vacuum over phosphorus pentoxide. Thus 49.7 g (77% of the theoretical yield)
of L_proline-methylamide hydrochloride melting at 165 C are obtained. [~]D =
_ 55.3 (c = l; methanol)-
c) To a suspension of 60.8 g of benzyloxycarbonyl-L_histidinhydrazide -
in 400 ml of dimethylformamide which is chilled to - lS to - 20& are added
228 ml of a 4.38 molar solution of hydrogen chloride in absolute tetrahydrofuran.
- While stirring, 24 ml of tert.butyl nitrite are added in such manner than the
temperature remains below -15 & . Thereafter the mixture is stirred for 30
minutes at - 15 & , chilled to - 45 & and then treated dropwise with 139 ml of
triethylamine at a temperature below - 30 C. 32.9 g of L_proline-methyl~mide
; hydrochloride, 27.8 ml of triethyl~ine and, 15 minutes later, 22 ml of N-
methylmorpholine are added. The reaction mixture is stirred for 24 hours,
during which time it is allowed to wanm to room temperature. The precipitate
_ 10 --

~(t~ ~ S ~ O
fonmed is separated and the filtrate is evaporated under reduced pressure.
The residue is dissolved in 300 ml of water containing a small amount of
hydrochloric acid. On bringing the pH value to 9 by adding a concentrated
aqueous solution of ammonia, an oil separates which is isolated by decanting
the aqueous layer and dissolved in tetrahydrofuran. This solution is diluted
with the same volume of ethyl acetate and then extracted several times with
water. The organic layer, after drying over sodium/sulfate is evaporated
under reduced pressure. After drying the residue over phosphorus pentoxide
in vacuum benzyloxycarbonyl-L histidyl-L_proline-methylA~ide is obtained in
form of a solid foam. Yield: 46.5 g - 58% of the theoretical. []D = ~40 3
(c = l; methanol).
d) 30 g of benzyloxycarbonyl-L histidyl-L proline-methylamide are
dissolved in 100 ml of glacial acetic acid and treated with 100 ml of a 40%
solution of hydrogen bromide in glacial acetic acid. After stirring for one
hour at room temperature, 500 - 600 ml of dry ether are added, the precipitate
formed is separated, washed with dry ether and dried in vacuum over phosphorus
pentoxide and potassium hydroxide. The hydrobromide of L,histidyl-L-proline-
methylamide thus obtained, 12.8 g of thiomorpholine 4 -(D,L)-methyl-5-on-3-(L)-
carboxylic acid and 10.5 g ofl-hydroxybenzotriazole are dissolved in 150 ml of
dimethylformamide, chilled to -5 to 0 and treated with the amount of tri-
ethylamine being equivalent to the amount of hydrogen bromide present in the
mixture. Finally 15 g of N,N1-dicyclohexyl-carbodi;m;de dissolved in a few ml
of dimethylformamide are added. After stirring for 10 minutes in the ice bath
the mixture is stirred for 12 hours, during which time it is allowed to reach
room temperature. The precipitate formed is separated, the filtrate is evapo-
rated under reduced pressure and the residue thus obtained is dissolved in 150
ml of water and stored at 3C. After filtration the filtrate is mixed with an
equal volume of methanol and treated with a cationic e~changer in free acidic
; state as, for instance, the product known under the trade name "Dowex 50 W~ 4n

10~5~
The cationic exchanger carrying the product is separated by filtration, wa~hed
with methanol and water and then it is slurried in methanol/water (1:1) and
treated, while stirring, with ammonia until the pH of the mixture is 9.5.
The exchanger resin is separated and the filtrate is evaporated under reduced
pressure. The residue is recrystallized twice from a small volume of water
and then dried over phosphorus pentoxide. Thus 9.4 g of 5-Oxo-6-(D,L)-methyl-
thiomorpholine-3-(L)-carbonyl-L-histidyl-L,proline-N-methylamide melting at
138 - 139 & are obtained. [a]D = - 48.0 (c = l; methanol).
EXAMPLE 2
The procedure is the same as in example ld, there are used however
11.4 g of orotic acid (anhydrous) instead of the thiomorpholine-6-(D,L)-
methyl-5-on-3(L)-carboxylic acid. The residue remaining after evaporating
the eluate of the cationic exchanger is recrystallized twice from ethanol/
ether (4:1) and once from ethanol. The product is dissolved in water and
lyophylized. After drying over phosphorus pentoxide 5.4 g of orotyl-L-
histidyl-L proline-N-methylamide are obtained. The melting point of the
product is not characterizing. [a]D = -46.6 (c = l; methanol).
The combined mother liquors are evaporated and the residue thus
obtained is recrystall;zed several times from isopropanol. After drying of
the product in vacuum, a second batch of 3.6 g of the desired compound is
obtained.
EXAMPLE 3
The procedure is the same as in example ld, there are used however
35 g benzyloxycarbonyl-L,histidyl-L,proline-cyclohexylamide instead of the
benzyloxycarbonyl-L,histidyl-L,proline-methylamide and 11.4 g of orotic acid
(as in example 2). The eluate of the cationic exchanger is evaporated and the
residue is further purified by column chromatography on silicagel (particle
size 0.063 - 0.200 mm), using ethanol~water (5:1) for elution. On evaporation
of the eluate, ~.6 g of orotyl-L_histidyl-L_prolin-cyclohexylamide are obtained.
_ 12 --

S~
[]24= _54 3 (c = 0.3; methanol).
EXAMPLE 4
a) Using the procedure described in example la, 20 ml of n-butylamine
are reacted with 69.2 g of the N-hydroxy-succinimide ester of benzyloxycarbon-
yl-L-proline. The desired product is recrystallized from ethylacetate/ligroin
(1:1). After drying in vacuum 49.8 g (82 % of the theoretic yield) of
benzyloxycarbonyl_L,proline_(n_butyl)_amide melting at 84-85 & are obtained.
[~]D5= -48.9 (c = 1; methanol).
b) 60.8 g benzyloxycarbonyl-L,proline-(n-butyl)-amide are hydrogenated
in presence of palladium black and 11.6 ml of glacial acetic acid as described
in example lb. Thereafter 50 ml of 4 N hydrochloric acid are added, the mix-
ture is evaporated and the residue dissolved in ethanol. The oil remaining
after distilling off the ethanol is crude L,proline-(n-butyl)-amide-hydro-
chloride, which without further purification is used in the next step.
c) The product obtained in example 4b is used in the procedure of
example lc instead of the L_proline-methylamide-hydrochloride. The residue
obtained on evaporating the reaction mixture in vacuum is treated with water,
then with ammonia (until pH 9.5 is reached). This solution is extracted
several times with ethyl acetate. The combined extracts are washed consecu-
tively with water, 10~ aqueous sodium carbonate solution and water. Theorganic layer is dried over sodium sulfate and evaporated. After drying the
residue in vacuum 53 g (60% of the theoretical yield) of benzyloxycarbonyl-L,
histidyl-L,proline-(n-butyl)-amide are obtained in form of a porous mass.
[~]D = ~ 47.6 (c = 1; methanol).
d) The procedure is as described in example 3, there are used however
33.1 g of benzyloxycarbonyl-I-histidyl-L,proline-n-butylamide and 11.4 g of
orotic acid, the elution from the column of silicagel being made with methanol
in this instance. ~hus 10.8 g (33.4 ~ of the theoretical yield) of orotyl-L,
histidyl-I-proline-N-(n-butyl)-amide are obtained. []D = ~ 55.0 (c = O.S;
_ 13 -

SZO
methanol).
E~IE _
The procedure is the same as in example ld, there are used, however,
35.2 g benzyloxycarbonyl-L,histidyl-L,proline-(n-hexyl)_amide and 11.4 g orotic
acid. The residue obtained on distilling off the dimethylfonmamide is dis-
solved in methanol/water tl:l) and treated (as described) with a cationic
exchanger resin. The eluate of the exchanger resin is evaporated. The resi-
due is dissolved by adding 50 ml of water and 4 N hydrochloric acid until pH 3
is reached. A small amount of impurities is filtered off, the filtrate
evaporated in vacuum and the residue recrystallized twice from n-butanol,
saturated with water. The substance thus obtained is dissolved in water,
which solution is extracted several times with n-butanol. The combined ex-
tracts are evaporated in vacuum. Thus 7.9 g (21% of the theoretical yield) of
orotyl-I-histidyl-L-proline-(n-hexyl) amide-hydrochloride melting at 180-185 C
are obtained. ~a]D = -57.8 (c = 1; methanol).
The combined mother liquors are evaporated and the residue is sub-
jected to counter-current distribution in the system n-butanol/water. Thus
further 5.7 g (15% of the theoretical yield) of the desired product are
obtained.
0 EXAMPL~ 6
a) 24.9 g of benzyloxycarbonyl-I-proline are dissolved in 150 ml of
absolute tetrahydrofuran and then 13.9 ml of triethylamine are added while
stirring. After chil~;ng to - 15C, a solution of isobutyl-chlorocarbonate
in 50 ml of absolute tetrahydrofuran and, after 5 minutes, a solution of 10.1
ml of piperidine in 50 ml of absolute tetrahydrofuran are added dropwise at
this temperature. The mixture is stirred for two hours longer, during which
time it is allowed to reach room temperature, and then evaporated in vacuum.
The residue is treated with ethyl acetate, and this solution is consecutively
extracted with water, 5% aqueous potassium hydrogensulfate solution, saturated
- 14 _

S;~O
aqueous sodium carbonate solution and water. After drying the solution over
sodium sulfate, the ethyl acetate is distilled off and the residue is tri-
turated with ether and then recrystallized from ethylacetate/ligroin (2:3).
Thus 21.2 g (67g of the theoretical yield) of benzyl oxycarbonyl-L,proline-
piperidide are obtained. Melting point 92 & . [a]23 = -21.9 (c = l; methanol).
b) 63.2 g of benzyloxycarbonyl-L_proline-piperidide are treated with
hydrogen in presence of palladium black and 11.6 ml of glacial acetic acid as
described in example lb. After the addition of 50 ml of 4 N hydrochloric
acid the mixture is evaporated and the residue is dissolved in ethanol. The
oil remaining after distil~ing off the ethanol is crude L-proline-piperidide-
hydrochloride, which without further purification is used in the next step.
c) The product obtained in example 6b is used in the procedure described
in example lc instead of the L,proline-methylamide-hydrochloride. The residue
obtained on evaporating the filtered reaction mixture in vacuum is purified as
described in example 4c. The oily residue obtained after distilling off the
ethyl acetate is dissolved in a small amount of methanol and reprecipitated
by addition of ether. The solvents are decanted and the product is dried in
vacuum, whereby 46.5 g (51% of the theoretical yield) of benzyloxycarbonyl-L-
histidyl-L-proline-piperidide are obtained in the fonm of a porous mass.
0 [a]D = ~ 51.3 (c = 1; methanol).
d) The procedure is as described in example 3, there are used however
33.6 g of benzyloxycarbonyl-L,histidyl-2-proline-piperidide and 11.4 g of
orotic acid, the elution from the column of silica gel being made with ethanol
in this instance. Thus 10.9 g (32% of the theoretical yield) of orotyl-I-
histidyl-l_proline-piperidide are obtained. ~a]D = -62.5 (c = 0.5; methanol).
EXAMP~E 7
a) The procedure is the same as in example 6a. However 11.2 ml of benzyl
amine are used instead of the piperidine. The produce is purified by recrys-
tallization from ethyl acetate/ligroin (1:1). After drying in vacuum, 24.3 g
_ 15 --

lO~blS20
(72~ of the theoretical yield) of benzyloxycarbonyl-L_proline-benzyla~ide are
obtained. Melting point: 93 - 94 C. [a]D3 = - 43.5 (c = l; methanol).
b) 67.7 g of benzyloxycarbonyl-L,proline-benzylamide are dissol~ed in
300 ml of glacial acetic acid and treated with 240 ml of a 40% solution of
hydrogen bromide in glacial acetic acid After stirring for 90 minutes at
room temperature, the mixture is evaporated in vacuum. The residue is dissol-
ved in a small amount of methanol and reprecipitated by adding of ether. The
solvents are decanted, and the precipitate is triturated with ether and fin-
ally dried in vacuum. The crude-L,proline-benzylamide-hydrobromide thus
0 obtained is used in the next step.
c) The crude product obtained in example 7b is used in the procedure
described in example 4c. Thus 80.7 g (85% of the theoretical yield) of
benzyloxycarbonyl-L,histidyl-L,proline benzylamide are obtained in the form
of a porous mass. [a]D = - 39.1 (c = 1; methanol).
d) The procedure is as described in example 4d, there are used, however,
35.6 g of benzyloxycarbonyl-I,histidyl-I-proline-benzylamide and Il.4 g of
orotic acid. Orotyl-l,histidyl-L,proline-N-benzylamide is obtained in a yield
of 11.7 g (=33.3% of the theoretical yield. [a]D = - 50.~ (c = 1; methanol).
Following the procedures described above, especially those explained
in the examples, the following compounds of formula (I) can also be prepared,
for example. (In cases where no particulars of the configuration are given,
any of the acid components of the compounds of formula (I), i.e. the proline,
the histidine and the acid of formula (II), can be present in the racemic
state, in the L, or in the D-configuration. As stated already above, the L_
configuration is preferred, however, for said components):
Orotyl-I-histidyl-L,proline-morpholide
L,2-Oxo-imidazolidine-4-carbonyl-l-histidyl-L_proline-N-methylamide
5-iso-propylorotyl-histidyl-proline-N-ethylamide
2-Oxo-5,5-dimethyl-imidazolidine 1-carbonyl-histidyl-proline-N-methylamide.

S;~O
2-Oxo-4,5-dimethyl-imidazolidine 1-carbonyl-histidyl-proline-N-methyla ide.
2-Oxo-5-ethyl-imidazolidine-4-carbonyl-histidyl-proline-N-ethylamide
2-Oxo-5-methyl_5-ethyl-imidazolidine 1-carbonyl-histidyl-proline-N-isopropyl-
amide.
5-Oxo-2,2,6-trimethyl-thiomorpholine-3-carbonyl-histidyl-proline-N-methylamide.
5-Oxo-2,3,6-trimethyl-thiomorpholine_3_carbonyl-histidyl-proline-pyrrolidide
5-Oxo-6,6-dimethyl-thiomorpholine-3-carbonyl-histidyl-proline-N_methylamide
5-Oxo-morpholine-3-carbonyl-histidyl-proline-N-benzylamide
5-Oxo-6-methyl-morpholine-3-carbonyl-histidyl-proline-N-methylamide
5-Oxo-2,6-dimethyl-morpholine-3-carbonyl-histidyl-proline-N-methylamide
5-Oxo-6-(DL)-methyl-thiomorpholine-3(L)-carbonyl-L,histidyl-L_proline-N-(n-
hexyl)-amide. -
S-Oxo-thiomorpholine-3-carbonyl-histidyl-proline-N-methylamide
5-Oxo-thiomorpholine-3-carbonyl-histidyl-proline-N-benzylamide
5-Oxo-thiomorpholine-3-carbonyl-histidyl-L-proline-morpholide.
;
- 17 -