IE44485B1 - Dipeptide derivatives - Google Patents

Abstract

The compounds correspond to the formula: in which the symbols R1 to R5 have the meanings given in Claim 1. The preparation is effected by forming the amide bond in accordance with one of four process variants, starting from amino compounds of the formulae: and the carboxylic acid corresponding in each case to the remainder of the molecule. The reaction is also carried out using functional derivatives of the carboxylic acid or with the 1 and 3 positions of the imidazole group in the histidyl residue being temporarily protected. The compounds show a spectrum of activity which is similar to that of thyrotropin-releasing hormone, but with a longer duration of activity and a stronger stimulatory effect on the central nervous system; they can be used therapeutically as psychostimulants and as antidepressants.

Description

The present invention relates to dipeptide derivatives, to a process for the manufacture thereof and to pharmaceutical preparations containing such derivatives.

The dipeptide derivatives according to the present 5 invention are derived from histidyl-proline, the amino acids contained therein being optically active (D or L) or racemic, but preferably having the L-configuration. More precisely the invention provides compounds of the general formula (I) /'ZRs z c (I) CO—N /R1 I wherein R^ and which may be the same or different, each represents hydrogen or an alkyl or cycloalkyl radical containing up to six carbon atoms or a benzyl group, with the proviso that and R2 cannot both be hydrogen, or R^ and Rj together with the nitrogen atom to which they are attached represent a five to seven numbered heterocyclic ring or may be a hydrogen atom.

Rj and R^ which may be the same or different each represents hydrogen or an alkyl radical containing from one to three carbon atoms or R3 and R^ together may 4 4SS - 3 represent an additional direct bond between the carbon atoms to which they are attached; R 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, the divalent structure being:OH Rc R i !6 !6 -NH-, -N=C- φ -NH-C9, -C-S- or -C-0I i wherein Rg and R? may be the same or different and each represents hydrogen or an alkyl radical containing from one to three carbon atoms and wherein the Z-containing acyl group has the L-, D- or PL-configuration where Rg and R^ do not represent an additional direct bond and/or •where R. and R are different, and addition salts of 6 7 these compounds with pharmaceutically acceptable acids.

The dipeptide derivatives of general formula (I) are acyl derivatives of histidyl-proline amides.

Where Rg and R^ do not represent a second direct bond between the carbon atoms to which they are attached and/or in case that Rg and R? are different, the acyl groups can be present in the compounds of formula (I) in racemic or optically active form (D or L) and are preferably in the L-configuration.

If Rg represents a hydrogen atom, R^ is preferably a straight-chain alkyl radical containing up to six carbon atoms and most preferably one to four carbon atoms.

Where R^ and Rg, together with the nitrogen atom to which they are attached, represent a heterocyclic 4 4 ® 5 radical, this group may be the pyrrolidono, the piperidino or the hexamethyleneimino group or the residue of a fiveto seven-membered heterocyclic ring containing at least one further hetero atom, such as the thiazolidino, morpho5 lino or thiomorpholino group.

Due to the basicity of the histidyl radical the compounds of general formula (I) can form salts with acids. Accordingly, the present invention also provides salts of the compounds of formula (I) with pharmaceutic10 ally acceptable (as salts) inorganic or organic acids, such as hydrochloric acid, hydrobromic acid, sulphuric acid, phosphoric acid, formic acid, acetic acid, propionic acid, benzoic acid, salicylic acid, phanylacetie acid and bensenesulphonic 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 L-pyroglutamyl-L-histidyl-L-proline amide, which compound normally is designated as tyrotropine releasing hormone or "TRH. 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 stimulating effects to the endocrinologic effects is shifted for the new compounds (if compared with those effects of TSH) favourably to the pharmacologically valuable properties.

The compounds of general formula (I) and their pharmaceutically acceptable salts may be administered orally or parenterally and act quickly. For instance on parenteral administration the effects are observed after 10 minutes. -: 4 t S 5 The most impressive effects in pharmacological tests indicate a central stimulating action of the new products. On administering equal doses of TRH and one of the new products, respectively, to test animals the new compound has a central stimulating action which is several times longer than TRH.

The toxicity of the compounds of formula (I) is very low. Due to these valuable properties the new compounds can be used as therapeutics, for instance as psychostimulating agents or anti-depressive agents.

The compounds are of therapeutic value in animals and humans.

Accordingly, the present invention also provides a pharmaceutical composition comprising at least one compound of general formula (X) as previously defined or a pharmaceutically acceptable acid addition salt thereof together with a pharmaceutically acceptable carrier or diluent therefor.

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 aqueous solutions. These pharmaceutical preparations can be prepared by known methods using inert carriers such as are knowrt for other antidepressants and CNS-stimulants used in human and warmblooded 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 administration. Preferably the compounds of the invention are used parenterally in a unit 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 may be increased or decreased as recommended by the particular ' circumstances.

Conveniently the compounds of general formula (I) are prepared by reacting a histidyl-proline amide (having the desired optical configuration) of formula (III), (III) CO — N ZR1 I wherein R^ and Rj are as previously defined, with a compound of formula (II) \ZR5 C (II) R, Z 3 ^c c • X XKZ \ I COOH wherein R^, R^, Rj. and Z are as previously defined, and Y represents hydrogen or a group cleavable by hydrogen15 olysis, preferably a carbobenzoxy group or a substituted carbobenzoxy group, in the presence of an agent which promotes the formation of an amide bond, with elimination of a molecule of water from the reactants. 4 4 8 5 especially in the presence of a carbodiimide and most preferably dicyclohexyl carbodiimide, or a functional derivative as herein defined of the compound of formula (II).

By the term functional derivative is meant a derivative of the carboxylic group which renders it capable of reacting with an amino group with formation of an amido group. Examples of such functional derivatives are acid halides, anhydrides, mixed anhydrides, azides and activated esters.

Preferred acids of formula (II) for use in preparing the dipeptides (I) 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)-carboxyimidazole-(2)-one, thiomorpholine-(6)-methyl-(5)-one(3)-carboxylic acid, 5-methyl-, 5-ethyl- and 5-propylorotic acid, and thiomorpholine-(5)-one-(2,2)-dimethyl(3)-carboxylic acid.

During the reaction of the histidyl-proline amide of formula (III) with the acid of formula (II) or a derivative thereof, the 1-(3)-position of the imidazole group of the histidyl residue may be reversibly protected. By the term reversibly protected we mean that the 1-(3)-position of the imidazole group of the histidyl residue is protected against acylation by means of a protective group which, after the reaction is completed, can be split off e.g. by hydrolysis or hydrogenolysis. Suitable protective groups are known from peptide synthesis to those skilled in the art and include the carbobenzoxy group, substituted carboben2oxy 4 4 8-S groups, the trityl, O-nitrophenoxy acetyl or the tert.butyloxy-carbonyl group and other groups known per se, which after the reaction is completed can be split off by hydrolysis or hydrogenolysis.

Instead of the histidylproline amide of formula (III) with either a free or protected imidazole group it is possible to use a derivative thereof, obtained by reacting it in a manner known per se with a silylating agent which is a trialkylsilanol or a dialkylsilanediol derivative containing the moiety (alkyl)^Si- or (alkyl)2 Si<(each alkyl radical thereof containing from one to three carbon atoms) e.g. hexamethyldisilasane, trimethyl chlorosilane, trimethylsilylacetamide or dimethyldichlorosilane, in the process of the present invention.

The acylation reaction must, in this case, be performed in the absence of a proton active solvent which is defined as one which is capable of liberating at least one proton and hence eliminating the protecting silyl group. After the acylation reaction is finished, the silyl groups can easily be split off by hydrolysis or alcoholysis.

The compounds of formula (I) may also be prepared by reacting a compound of formula (IV)· yRs -is NH c c y \ / y Ή I Y CH„ CO— HH — CH _ COOH (IV) 4448 5 wherein R^, R^, ar*d 2 have the same meaning as above and Y represents a hydrogen atom or a group cleavable by hydrogenolysis defined before, or a derivative of the compound of formula (IV) in which the 1-(3)-position of the imidazole group of the histidyl residue is protected reversibly as herein defined, in the presence of an agent which promotes the formation of an amide bond, with elimination of a water molecule from the reactants, .especially in the presence of a carbodiimide, preferably dicyciohexyl carbodiimide or with a functional derivative (as herein defined) of a compound of formula (IV), or a reversibly protected derivative thereof, as for instance an acid halide, anhydride, mixed anhydride or an activated ester with a compound of formula (V), wherein R^ and R2 have the same meaning as above and thereafter splitting off the protecting group, if present.

Furthermore, compounds of formula (I) can be 20 prepared by reacting a compound of formula (VI) NNH i CH.

(VI) Λ C , CO —NH — CH—CO—N CO — w I 4448 5 wherein R^, Y and z have the same meaning as above and W represents a hydroxy, acyloxy, p-nitrophanory, trichlorophenoxy or pentachlorophenoxy, pentafluorophenoxy, pyridyloxy, phenylmercapto, p-nitrophenylmercapto or cyanomethyloxy group or the residue of N-hydroxysuccinimide of formula CO-CH / I 2 — N CO- ch2 or a derivative of the compound of formula (VI) wherein the 1-(3)-position of the imidasole group of the histidyl residue is protected reversibly in the manner described hereinabove, with an amine of formula (VII), HN (VII) wherein R^ and R2 have the same meaning as above and splitting off the protecting groups, if present.

Compounds of formula (I), (one or both of the amino acid groups contained therein being optically active (D or L) or racemic but preferably having the L-configuration) derived from orotic acid, wherein R^ 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 formula (VIII): 444S5 HN Hal K^ ^COOH Η OH Hal COOH (VIII) wherein Hal represents a chlorine or a bromine atom in the presence of an agent which promotes the formation of an amide bond, with elimination of a water molecule from the reactants, especially in the presence of a carbodiimide most preferably dicyclohexylcarbodiimide or with a functional derivative (as herein defined) of such an acid of formula (VIII), e.g. 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 a derivative thereof, the 1-(3)-position of the imidazole group of the histidyl residue may be protected against acylation. Suitable protective groups are those mentioned already hereinabove. In this last-described 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 groups or the o-nitrophenoxyacetyl group). In this case the removal of the protective group from the intermediate occurs coincidently 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 hydrogenation catalyst, e.g. palladium or platinum on charcoal, on barium sulphate, on alumina, on calcium or barium carbonate and other noble metal catalysts known per se, are used in the hydrogenolysis, which can be performed under normal or increased pressure, preferably at room temperature. Solvents such as water or mixtures of water with lower (i.e. C^-C^ alcohols (e.g. 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.

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 above-mentioned preferred or other non-acidic solvents are used, suitable hydrogen halide binding agents are, for instance, magnesium oxide, barium oxide, alkali hydroxides, ammonia or ammonium hydroxide, triethylamine or salts of such bases with acids which are very much weaker than the hydrogen halide being formed, as for instance alkali carbonates or acetates. suitable media for the performance of the hydrogenolysis step also include for instance, solutions of alkali metal, alkaline earth metal, ammonium or amine acetates in acetic acid. 14 8 5 Mixed anhydrides of the acids of the formulae (II), (Ila), (TV) and (VIII) are preferably obtained from trimethylacetic acid or from monoesters 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-nitroohenol, trichlorophenol or pentachlorophenol, pentafluorophenyl, N-hydroxysuccinimide, 2- or 4-hydroxypyridine, thiophenol, jg-nitrothiophenol, hydroxyacetonitrile, 1-hydroxybenzotriazole and other hydroxy 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 recrystallisation, and also by column chromatography or counter-current distribution.

The following Examples further illustrate the invention. Ratios specified are calculated on a volume basis and all temperature references are uncorrected. No importance was attached to obtaining maximum yields in carrying out the experiments on which the Examples are based.

Example 1 a) A solution of 12.7 g of methylamine in 350 ml of absolute tetrahydrofuran was treated, while stirring, at 0°C with 71 g of the N-hydroxysuccinimide ester of benzyloxycarbonyl-L-proline.

The mixture was stirred for 15 minutes at 0°C and thereafter for two hours at room temperature. d 4 8 S - 14 After distilling off the solvent in vacuum the residue was dissolved in ethyl acetate, which solution was washed with a 5% (w/v) aqueous solution of potassium hydrogen sulphate, a saturated aqueous solution of sodium hydrogen carbonate and finally with water. The solution was dried with anhydrous sodium sulphate and evaporated. Thus 46 g (85% of the theoretical yield) of benzyloxycarbonyl-L-proline N-methylamide were obtained in the form of a colourless, viscous oil which crystallised on storage at 3-5°C, but melted at room temperature. b) 102.3 g of benzyloxycarbonyl-L-proline, Kmethylamide dissolved in methanol were treated in the presence of freshly prepared palladium black and 22.3 ml of glacial acid with hydrogen. The catalyst was filtered off and the solution was mixed with 100 ml of 4 N hydrochloric acid, then evaporated in vacuum, mixed with absolute ethanol and again evaporated. The crystalline residue was recrystallised from methanol diethyl ether (3s10). The product was dried in vacuum over phosphorus pentoxide. Thus 49.7 g (77% of the theoretical yield) of L-proline N-methylamide hydrochloride melting at 165°C were obtained. o [ a] D = -55.3 (c = 1; methanol). c) To a suspension of 60.8 g of benzyloxycarbonyl-L-histidinehydrazide in 400 ml of dimethylformamide which is chilled to -15 to -20°C were added 228 ml of a 4.38 molar solution of hydrogen chloride in absolute tetrahydrofuran. While stirring, 24 ml of tert.-butyl nitrite were added in such manner that the temperature remained below -15°C. Thereafter the 4 4 8 5 mixture was stirred for 30 minutes at ~15°C, chilled to -45°C and then treated dropwise with 139 ml of triethylamine at a temperature below -30°C. 32.9 g of L-proline N-methylamide hydrochloride, 27.8 mi of triethylamine and, 15 minutes later, 22 ml of N-methylmorpholine were added. The reaction mixture was stirred for 24 hours, during which time it was allowed to warm to room temperature. The precipitate formed was separated and the filtrate evaporated under reduced pressure. The residue was 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 separated which was isolated by decanting the aqueous layer and dissolved in tetrahydrofuran.

This solution was diluted with the same volume of ethyl acetate and then extracted several times with water.

The organic layer, after drying over sodium sulphate was evaporated tinder reduced pressure. After drying the residue over phosphorus pentoxide in vacuum benzyl20 oxycarbonyl-L-histidyl-L-proline N-methylamide was obtained in the form of a solid foam. Yield: 46.5 g = 58% of the theoretical. [a] 24- _4o.3° (c - 1; methanol). d) 30 g of benzyloxycarbonyl-L-histidyl-L-proline N-methylamide were dissolved in 100 ml of glacial acetic acid and treated with 100 ml of a 40% (w/v) solution of hydrogen bromide in glacial acetic acid. After stirring for one hour at room temperature, 500-600 ml of dry diethyl ether was added, the precipitate formed was separated, washed with dry diethyl ether and dried in vacuum over phosphorus pentoxide and potassium hydroxide. The hydrobromide of L-histidyl-L-proline - 16 N-methylamide thus obtained, 12.8 g of thiomorpholine6-(DL)-methyl-5 -one -3 - (L)-carboxylic acid and 10.5 g of l-hydroxybenzotriazole were dissolved in 150 ml of dimethylformamide, chilled to -5° to 0° and treated with an amount of triethylamine equivalent to the amount of hydrogen bromide present in the mixture. Finally 15 g of N, Ν'-dicyclohexylcarbodiimide dissolved in a few ml of dimethylformamide were added. After stirring for 10 minutes in the ice bath the mixture was stirred for 12 hours, during which time it was allowed to reach room temperature. The precipitate formed was separated, the filtrate was evaporated under reduced pressure and the residue obtained was dissolved in 150 ml of water and stored at 3°C. After filtration the filtrate was mixed with an equal volume of methanol and treated with a cationic exchanger in free acidic state as, for instance, the product known under the trade name Dowex (Trade Mark) 50 VJX 4. The cationic exchanger carrying the product was separated by filtration, washed with methanol and water and was then slurried in methanol/water (1«1) and treated, while stirring, with ammonia until the pH of the mixture was 9.5. The exchanger resin was separated and the filtrate was evaporated under reduced pressure. The residue was recrystallised twice from a small volume of water and then dried over phosphorus pentoxide.

Thus 9.4 g of 5-0xo-6-(DL)-methyl-thiomorpholine-3-(L)carbonyl-L-histidyl-L-proline N-methylamide melting at 138°C were obtained. [a] 2'= -48.0° (c = 1; methanol). 4 4 8 5 - 17 Example 2 The procedure was the same as described in Example Id, there were used however 11.4 g of orotic acid (anhydrous) instead of the thiomorpholine--6-(SL)-methyl5-one-(L5-carboxylic acid. The residue remaining after evaporating the eluate of the cationic exchanger was recrystallised twice from ethanol/diethyl ether (4:1) and once from ethanol. The product was dissolved in water and lyophylized. After drying over phosphorus pentoxide 5.4 g of orotvl-L-histidyl-L-proline N-methyiamide were obtained. The melting point of the product was not characterising. [<-ϊ]24= -46.6° (c = 1; methanol).

The combined mother liquors were evaporated and the residue thus obtained was recrystallised several times from isopropanol. After drying of the product in vacuum, a second batch of 3.6 g of the desired compound was obtained.

Example 3 The procedure was the same as described in Example Id, there are used however 35 g benzyloxycarbonyl-Lhistidyl-L-proline N-cyclohexylamide instead of the benzyloxycarbonyl-L-histidyl-L-proline N-methylamide and 11.4 g of orotic acid (as in Example 2). The eluate of the cationic exchanger was evaporated and the residue was further purified by column chromatography on silica gel (particle size 0.063-0.200 mm), using ethanol/water (5*1) for elution. On evaporation of the eluate, 8.6 g of orotyl-L-histidyl-L-proline N-cyclohexylamide were obtained. [qj^4= -54.3° (c = 0.3; methanol). 443 5 Example 4 a) Using the procedure described in Example la, ml of n-butylamine were reacted with 69.2 g of the N-hydroxysuccinimide ester of benzyloxycarbonyl-Lproline. The desired product was recrystallised from ethyl acetate/ligroin (1:1). After drying in vacuum 49.8 g (82% of the theoretical yield) of benzyloxycarbonyl-L-proline N-(n-butyl)amide melting at 84-85°C were obtained. [a]25= -49.9° (c = 1; methanol). b) 60.8 g benzyloxycarbonyl-L-proline-(n-butyl)amide were hydrogenated in the 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 were added, the mixture was evaporated and the residue was dissolved in ethanol. The oil remaining after distilling off the ethanol was crude L-proline N-(n-butyl)amide hydrochloride, which without further purification was used in the next step. c) The product obtained in Example 4b was used in the procedure of Example 1c instead of the L-proline N-methylamide hydrochloride. The residue obtained on evaporating the reaction mixture in vacuum was treated with water, then with ammonia (until pH 9.5 was reached). This solution was extracted several times with ethyl acetate. The combined extracts were washed consecutively with water, 10% w/v aqueous sodium carbonate solution and water. The organic layer was dried over sodium sulphate and evaporated. After drying the residue in vacuum 53 g (60% of the theoretical yield) of benzyloxycarbonyl-L-histidyl-L-proline N~(n-butyl)amide were obtained in the form of a porous mass. 444S5 f vi 2"= -47.6° (c = 1; methanol), L - D d) The procedure was as described in Example 3, there were used however 33.1 g of benzyloxyearfconyl-Lh.istidyl-L-proline N-(r-butyl)amide and 11.4 g of orotic acid, the elution from the column of silica gel being made with methanol in this instance. Thus 10.8 g (33.4% of the theoretical yield) of crotyl-L-histidylL-prolir.e N-(n-butyl)amide were obtained. f cl 22--= -55.0° (c - 0.5? methanol). u J p Example 5 Tha procedure was the same as in Example Id, there were used, however, 35.2 g of benzyloxycarbonyl-Lhistidyl-L-proline N-(n-hexyl)amide and 11.4 g of orotic acid. The residue obtained on distilling off the dimethylformamide was dissolved in methanol/water (1:1) and treated (as described) with a cationic exchanger resin. The eluate of the exchanger resin was evaporated. The residue was dissolved by adding 50 ml of water and 4 N hydrochloric acid until pH 3 is reached.

A small amount of impurities was filtered off, the filtrate was evaporated in vacuum and the residue was recrystallised twice from n-butanol, saturated with water. The substance thus obtained was dissolved in water, which solution was extracted several times with n-butanol. The combined extracts were evaporated in vacuum. Thus 7.9 g (21% of the theoretical yield) of orotyl-L-histidyl-L-proline N-(n-hexyl)amide hydrochloride melting at 18O-185°C were obtained. -57.8° (c = 1; methanol).

The combined mother liquors were evaporated and the residue was subjected to counter-current distribution 4 4 8 5 - 20 in the system n-butanol/water. A further 5.7 g (15% of the theoretical yield) of the desired product was obtained.

Example 6 a) 24.9 g of benzyloxycarbonyl-L-proline were dissolved in 150 ml of absolute tetrahydrofuran and then 13.9 ml of triethylamine were added while stirring. After chilling to -15°C, a solution of isobutylchlorocarbonate in 50 ml of absolute tetrahydrofuran and, after 5 minutes, a solution of 10.1 ml of piperidine in 50 ml of absolute tetrahydrofuran were added dropwise at this temperature. The mixture was stirred for two hours longer, during which time it was allowed to reach room temperature, and then evaporated in vacuum. The residue was treated with ethyl acetate, and this solution was consecutively extracted with water, 5% (w/v) aqueous potassium hydrogen sulphate solution, saturated aqueous sodium carbonate solution and water. After drying the solution over sodium sulphate, the ethyl acetate was distilled off and the residue was triturated with diethyl ether and then recrystallised from ethyl acetate/ligroin (2s3) . Thus 21.2 g (67% of the theoretical yield) of benzyloxycarbonyl-L-prolinepiperidide were obtained. Melting point 90°C. [a] 23= -21.9° (c = 1? methanol). b) 63.2 g of benzyloxycarbonyl-L-prolinepiperidide were treated with hydrogen in the 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 was evaporated and the residue was dissolved in ethanol. The oil remaining 4 -3 S 5 after distilling off the ethanol was crude 1.-proline piperidide hydrochloride, which without further purification was used in the next step. c) The product obtained in Example 6b was used in the procedure described in Example 1c instead of the Lproline N-methylamidehydrochloride. The residue obtained on evaporating the filtered reaction mixture in vacuum was purified as described in Example 4c. The oily residue obtained after distilling off the ethyl acetate was dissolved in a small amount of methanol and reprecipitated by addition of diethyl ether. The solvents were decanted and the product was dried in vacuum, whereby 46.5 g (51% of the theoretical yield) of benzyloxycarbonyl-L-histidyl-L-prolinepiperidide were obtained in the form of a porous mass. [aj^= 51.3° (c = 1; methanol). d) The procedure was as described in Example 3 there were used however 33.6 g of benzyloxycarbonyl-Lhistidyl-L-prolinepiperidide 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-L-histidyl-L-prolinepiperidide were obtained. [a]24= -62.5° (c = 0.5 methanol).

Example 7 a) The procedure was the same as described in Example 6a. However 11.2 ml of benzylamine were used instead of the piperidine. The product was purified by recrystallisation from ethyl acetate/ligroin (lil). After drying in vacuum, 24.3 g (72% of the theoretical yield) of ben2yloxycarbonyl-L-proline N-bensylamide were obtained. Melting point: 93—94°C. [a]23= -43.5° (c = 1; methanol). b) 67.7 g of benzyloxycarbonyl-L-proline N-benzyl5 amide were dissolved in 300 ml of glacial acetic acid and treated with 240 ml of a 40% (w/v) solution of hydrogen bromide in glacial acetic acid. After stirring for 90 minutes at room temperature, the mixture was evaporated in vacuum. The residue was dissolved in a small amount of methanol and reprecipitated by adding diethyl ether. The solvents were decanted and the precipitate was triturated with diethyl ether and finally dried in vacuum. The crude L-proline N-benzylamide hydrobromide thus obtained was used in the next step. c) The crude product obtained in Example 7b was used in the procedure described in Example 4c. Thus 80.7 g (85% oi; the theoretical yield) of benzyloxycarbonyl -L-histidyl-L-proline N-benzylamide was obtained in the form of a porous mass. [a] 23= -39.1° (c = 1? methanol). d) The procedure was as described in Example 4d, there were used, however, 35.6 g of benzyloxycarbonylL-histidyl-L-proline Ν-benzylamide and 11.4 g of orotic acid. Orotyl-L-histidyl-L-proline N-benzylamide was obtained in a yield of 11.7 g (= 33.3% of the theoretical yield). [a]23= -50.5 (c = 1, methanol).

Following the procedures described above, espec30 ially those explained in the examples, the following *4485 compounds of formula (15 were prepared. (In cases where no particulars of the configuration are given, any cf 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 Lconfiguration is preferred, however, for said components): Orotyl-L-histidyl-L-prolina morpholide; L-2-Oxo-imidazolidine-4-carbonyl-L-histidyl-Lproline N-methylamide; -isopropylorotyl-histidyl-proline N-ethylamide; 2-Oxo-5,5-dimethyl-imidazolidine-4-carbonylhistidyl-proline N-methylamide; 2-Oxo-4,5-dimethyl-imidazolidine-4-carbonylhistidyl-proline N-methylamide; 2-Oxo-5-ethyl-imidazolidine-4-carbonyl-histidylproline N-ethylamide; 2-Oxo-5-methyl-imidazolidine-4-carbonyl-histidylproline N-isopropylamide; -Oxo-2,2,6-trimethyl-thiomorpholine-3-carbonylhistidyl-proline N-methylamide; -Oxo-2,3,6-trimethyl-thiomorpholine-3-carbonylhistidyl-proline pyrrolidide: -Oxo-6,6-dimethyl-thiomorpholine-3-carbonylhistidyl-proline N-methylamide; -Oxo-morpholine-3-carbonyl-histidyl-proline N-benzylamide; -Oxo-6-methyl-morpholine-3-carbonyl-histidylproline N-methylamide; -Oxo-2,6-dimethyl-morpholine-3-carbonyl-hist idylproline N-methylamide; - 24 5-Oxo-6-(DL)-methyl-thiomorpholine-3-(L)-carbonylL-histidyl-L-proline-N- (n-hejtyl) amide; -Oxo-thiomorphol ine-3 -carbonyl -hi pi idyl -pro.l i no N-methylambda; -Oxo-thiomorpholine-3-carbonyl-histidyl-proline N-benzylamide; -Oxo-thiomorpholine-3-carbonyl-histidyl-L-proline morpholide.

Claims (19)

1. A dipeptide derivative, wherein the araino acids contained therein independently have the D-, Lor DL-configuration, of the general formula (I): N CO — NH— CH—CO—N (I) wherein R^ and R^ which nay be the sane or different each represents hydrogen or an alkyl or cycloalkyl radical containing up to six carbon atoms or a benzyl group,with the proviso that R^ and R 2 cannot both be hydrogen, or and R 9 together with the nitrogen atom to which they are attached, represent a five- to seven-membered heterocyclic ring optionally containing at least one other heteroatom. R„ and R„ which may be the same or different each 3 4 15 represents hydrogen or an alkyl radical containing from one to three carbon atoms, or R^ and R 4 together represent an additional direct bond between the carbon atoms to which they are attached: R represents a hydrogen atom or an alkyl radical 5 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, the divalent structure being: OH R, R, , i 6 ? -3SH-, -N=C- -HH-CO-, -C-S- or -c-O- , wherein R g and R? which may be the same or different each represents hydrogen or an. alkyl radical containing from one to three carbon atoms, and wherein the Z5 containing acyl group has the L-, D- or DL-configuration where R 3 and R^ do not represent an additional direct bond and/or where Rg and R? are different, and pharmaceutically acceptable acid addition salts thereof.

2. A dipeptide derivative as claimed in Claim 1 10 which has the formula: z i c / 1 ¾ CO—N wherein R., and R. and R_ are as defined in Claim 1 and 12 5 wherein Z^ represents one of the divalent structures -NH- or -HM-CO-^-N=C(OH)-, respectively, and pharmaceutic· ally acceptable acid addition salts thereof.

3. A dipeptide derivative as claimed in Claim 1 which has the formulas 4. 4 4 8 5 / ? '5 Xx NK / CEL / \/X I I 0 N z s C0 NH—CH—CO—N-C0 _NH _R„ wherein R , R , R and Z are as defined in Claim 1 and □ 4 0 wherein R^ 1 represents an alkyl or cycloalkyl radical containing up to six carbon atoms or the benzyl group and pharmaceutically acceptable acid addition salts thereof.

4. A dipeptide derivative as claimed in Claim 3, wherein 1 represents a straight-chain alkyl group having one to four carbon atoms.

5. Orotyl-L-histidyl-L-proline N-methylamide and pharmaceutically acceptable acid addition salts thereof.

6. 5-Oxo-6-(DL}-methyl-thiomorpholine-3-(L)carbonyl-L-histidyl-L-proline N-methylamide and pharmaceutically acceptable acid addition salts thereof.

7. Orotyl-L-histidyl-L-proline N-(n-butyl)amide and pharmaceutically acceptable acid addition salts thereof.

8. Orotyl-L-histidyl-L-proline N-(n-hexyl)amide and pharmaceutically acceptable acid addition salts thereof.

9. 5-Oxo-6-(DL)-methyl-thiomorpholine-3-(L)carbonyl-L-histidyl-L-proline N-(n-hexyl)amide and pharmacsutically acceptable acid addition salts thereof.

10. A process for the preparation of a dipeptide derivative of the general formula (I) as defined in 444® S Claim 1 or a pharmaceutically acceptable acid addition salt thereof which comprises reacting a carboxylic acid of the general formula (II): (II) KT COOH I y 5 wherein R^, R^, R^ and Z are as defined in Claim 1 and Y represents hydrogen or a group cleavable by hydrogenolysis in the presence of an agent which promotes the formation of an amide bond, with elimination of a water molecule from the reactants, or a functional derivative 10 (as herein defined) of an acid of the general formula (IIwith a compound of formula (III), wherein R^ and R^ are as defined in Claim 1 and in which the 1-(3)-position of the imidazole group of the 15 histidyl residue may b.e protected against acylation, or a derivative thereof obtained by reacting the compound of formula (ill) with a silylating agent which is a 44-38 trialkyl silanol or a dialkylsilanedicl derivative containing the moiety (alkyl),Si- or (alkyl),Sic (each alkyl radical of said silylating agents containing frorr. one to three carbon atoms), in the absence of a proton active 5 solvent as herein defined, and splitting off in the group Y, the protecting group from the imidazole group contained in the histidyl residue and the silyl groups, if present, and, if desired, reacting the resulting compound of general formula (X) with a pharmaceutically 10 acceptable acid to form a pharmaceutically acceptable acid addition salt thereof.

11. A process for the preparation of a dipeptide derivative of the general formula (X) as defined in Claim 1 or a pharmaceutically acceptable acid addition 15 salt thereof which comprises reacting a compound of the formula: '1 / R 5 C N~ R, NH .c c Z \ W Z \ N' I γ Z R 3 CH„ CO — NH — CH . COOH (IV) •wherein Ry R,, Rg and Z are as defined in Claim 1 and Y represents a hydrogen atom or a group cleavable by 20 hydrogenolysis,or a derivative of a compound of formula (IV) in which the 1-(3)-position of the imidazole group of the histidyl residue is protected reversibly (as herein defined),in the presence of an agent which promotes the formation of an amide bond, with elimination 4 8 S of a water molecule from the reactants, or a functional derivative (as herein defined) of a compound of the formula (IV^or a reversibly protected derivative thereof^ with a compound of formula (V) HN .CON X R 1 .1 wherein R^ and R 2 are as defined in Claim 1 and splitting off the protecting groups, if present, and, if desired, reacting the resulting compound of general formula (I) with pharmaceutically acceptable acid to form a pharma10 ceutically acceptable acid addition salt thereof.

12. A process for the preparation of a dipeptide derivative of the general formula (I) as defined in Claim 1 or a pharmaceutically acceptable acid addition salt thereof which comprises reacting a compound of 15 the formula (VI) N' I Y (VI) co—w wherein R 3 , R^, R^ and Z are as defined in Claim 1, Y represents hydrogen or a group cleavable by hydrogenolysis and W represents a hydroxy, acyloxy, £-nitro20 phenoxy, trichlorophenoxy or pentachlorophenoxy, 4 4 -1 pentafluorophenoxy, pyridyloxy, phenylmercapto, o-nitrophenylnercapto or eyanomethyloxy group or the residue of N-hydroxysuccinimide of formula CO-CH / i z 0— N M CO-CH 2 jor a derivative of the compound of formula (VI) in which the l-{3)-position of the imidazole group of the histidyl amide is protected reversibly (as herein defined)^ with an amine of formula (VII), HN X R i (VII) 10 wherein and R. ? are as defined in Claim 1 and splitting off the protecting groups, if present, and, if desired, reacting the resulting compound of general formula (I) with a pharmaceutically acceptable acid to form a pharmaceutically acceptable acid addition salt thereof. 15

13. A process for the preparation of dipeptide derivative of the general formula (I) as defined in Claim 1 or a pharmaceutically acceptable acid addition salt thereof which comprises reacting a compound of formula (III) as defined in Claim 10 except that neither 20 R^ nor ma Y represent a benzyl group, and in which the 1-(3)-position of the imidazole group of the histidyl residue may be protected against acylation, with an acid of the formula (VIII)i 04485 Ο OH Hal H (VIII) COOH wherein Hal represents a chlorine or a bromine atom, in the presence of an agent which promotes the formation of an amide bond, with elimination of a water molecule 5 from the reactants^or with a functional derivative (as herein defined) of 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 10 Rg and R^ form an additional direct bond between the carbon atoms to which they are attached, R_ is hydrogen 5 and Z represents -M e C(0H)- or -NH-CO-, respectively, and, if desired, reacting the resulting compound with a pharmaceutically acceptable acid to form a pharmaceutio15 ally acceptable acid addition salt thereof.

14. A process for the preparation of a dipeptide derivative of the general formula (I) as defined in Claim 1 or a pharmaceutically acceptable acid addition salt thereof substantially as herein described with 20 reference to any of the specific Examples.

15. A dipeptide derivative of the general formula (I) as defined in Claim 1 or a pharmaceutically acceptable acid addition salt thereof when prepared by a process as claimed in any of Claims 10 to 14.

16. A pharmaceutical composition comprising at least one compound of general formula (I) as defined « 4 4 8 S in Claim 1 or a pharmaceutically acceptable acid addition salt thereof as claimed in any of Claims 1 to 9 and 15 together with a pharmaceutically acceptable carrier or diluent therefor. 5

17. A pharmaceutical composition as claimed in Claim lb which comprises orotyl-L-histidyl-L-proline N-methylamide.

18. A pharmaceutical composition as claimed in Claim 15 or 17 in unit dosage form suitable for oral or 10 parenteral application,

19. . A pharmaceutical composition as claimed in Claim 16 substantially as herein described.