CS261344B1 - (6-Purinylthio) acyl dipeptides and a process for their preparation - Google Patents

Abstract

New N- f(6-purinylthio)acylj -L-alanyl- -D-isoglutamines of the general formula I, in which R3 denotes a hydrogen atom or an amino group, R2 denotes an alkyl group with 1 to 4 carbon atoms, a benzyl group or a hydrogen atom and n denotes 1 or 2, their salts and a process for their preparation consisting in the condensation of (6-purinylthio)alkanoic acid of the general formula II, or its reactive derivative, with an ester of a dipeptide of the general formula III and optionally in the subsequent saponification of the ester thus obtained. The compounds of the general formula I exhibit significant immunostimulatory activity.

Description

The authors of the invention have studied in more detail some analogues of muramyl dipeptides and unexpectedly found that new simple compounds of general formula I, which contain as a carrier of the peptide part of MDP instead of muramic acid cytostatically active 6-mercaptopurine, or 6-thioguanine, bound via an acetic or propionic acid residue, show interesting immunostimulatory effects. The compounds of general formula I contain as a dipeptide L-alanyl-D-isoglutamine, which has the same steric arrangement at the asymmetric centers of amino acids as the dipeptide in MDP: it consists of natural L-alanine, which is peptide-bound to D-isoglutamine, belonging to the steric series of D-amino acids not commonly found in nature.

According to the invention, N-[(6-purinylthio)acyl]-L-alanyl-D-isoglutamines of the general formula I are prepared by reacting the known (6-purinylthio)alkanoic acids of the general formula II

S(CHj) _n COOH ,Ά/γ (id,

R ¹ H (II) , in which R ¹ and n have the above meaning or a reactive derivative thereof, is reacted with the L-alanyl-D-isoglutamine ester of general formula III

in which R denotes an alkyl group with 1 to 4 carbon atoms or a benzyl group, and the ester of general formula I obtained, in which R has the same meaning as R, is optionally saponified to an acid of general formula I, in which R denotes a hydrogen atom.

The reaction of the (6-purinylthio)alkanoic acid of the general formula II with the ester of the dipeptide III is carried out using conventional methods of peptide synthesis, for example by condensation of an active derivative of the acid II, such as a chloride, mixed anhydride, reactive ester or imidazolide, with the dipeptide III in the presence of acid-binding agents, such as triethylamine, diisopropylethylamine, 1-methylpiperidine and the like, or by condensation of the acid II with the dipeptide III in the presence of carbodiimides, such as dicyclohexylcarbodiimide or water-soluble carbodiimides, for example N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride. In the preparation of 2 esters of general formula I, in which R denotes a lower alkyl group or a benzyl group, which are poorly soluble in water, it is advantageous to use those methods in which water-soluble reaction products are formed from the condensation reagents used, such as the carbonyldiimidazole or carbodiimide methods using water-soluble carbodiimides.

In the carbonyldiimidazole method, a solution of the imidazolide acid I is prepared from 1 molar equivalent of acid I and 1.1 molar equivalent of Ν,Ν'-carbonyldiimidazole in a suitable medium, such as dimethylformamide or dimethylsulfoxide, at a temperature of 0 to +25 °C, which is reacted in the same medium and at the same temperature with a solution of 1 molar equivalent of dipeptide ester III in the same solvent. The course of the reaction is monitored by thin-layer chromatography and after the reaction is complete (after 10 to 24 h), the desired product is isolated by conventional methods of preparative organic chemistry, for example by diluting the reaction mixture with water, adjusting the pH, filtering off the crude product and crystallization or absorption of the product dissolved in water onto a suitable ion exchanger, for example cation exchange resin, elution thereof and optionally further purification.

•

In the carbodiimide method, 1.0 to 1.1 molar equivalent of the appropriate carbodiimide, such as dicyclohexylcarbodiimide or N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride, is added to a solution of 1 molar equivalent of acid II and 1 molar equivalent of dipeptide ester III in a suitable solvent, such as dimethylformamide, dimethylsulfoxide, phosphoric acid hexamethyltriamide, and the like, at a temperature of 0 to +5 °C, and the mixture is stirred after the addition of 1 molar equivalent of dipeptide ester III for 1 to 72 h at a temperature ranging from 0 to 40 °C; the course of the reaction is monitored by thin-layer chromatography. After the reaction is complete, if dicyclohexylcarbodiimide is used, the precipitated dicyclohexylurea is filtered off and the filtrate is diluted, after optional evaporation of the solvent under reduced pressure, with water, whereby the sparingly soluble ester of the general formula I crystallizes, or, if a water-soluble carbodiimide is used, the reaction mixture is directly diluted with water and after adjusting the pH to a weakly acidic reaction, the precipitated crude ester is filtered off and purified by column chromatography and/or crystallization, or the water-soluble ester is isolated after diluting the reaction mixture with water using ion exchangers.

Saponification of esters of the general formula I, in which R denotes an alkyl group with 1 to 2 carbon atoms or a benzyl group, to acids of the general formula I, in which R denotes a hydrogen atom, is carried out by the action of 2 to 3 molar equivalents of alkali metal hydroxides, such as sodium or potassium hydroxide, per 1 molar equivalent of ester in a medium of water or a mixture with a lower alcohol, such as methanol or ethanol, at a temperature of 0 to 40 °C, for a period of 2 to 10 h. The course of hydrolysis is monitored by thin-layer chromatography and after saponification of all the ester, the reaction mixture, optionally after distillation of the alcohol, is acidified with a strong acid, such as hydrochloric acid or sulfuric acid, to a pH of about 3.5 to 4 and the separated product is purified by crystallization, preferably from water, after filtration.

The starting compounds of general formula II are known substances or can be prepared by analogous methods; the preparation of (6-purynylthio)alkanoic acids (of general formula II, in which R^ represents a hydrogen atom) is described by Semonský and co-workers /Collection Czechoslov. Chem. Comun.

25, 1091 (1960)/, (2-amino-6-purinylthio)alkanoic acids (of general formula II, in which r1 denotes an amino group) Elionová and co-workers /J. Am. Chem. Soc. 81, 1898 (1959)/.

The necessary esters of L-alanyl-D-isoglutamine of the general formula III are also known. Usually, suitable N-protected derivatives of the said esters are used as starting materials, for example N-ethoxycarbonyl, N-benzyloxycarbonyl or N-tert-butyloxycarbonyl derivatives, from which the protecting groups are removed before the actual reaction by known methods, for example by treatment with a solution of hydrogen chloride in glacial acetic acid or hydrogenolytically, and the free ester of the dipeptide of the general formula III or its salt is obtained, which is used without further purification for the reaction with the acid II.

The immunomodulatory activity of the compounds of general formula I was evaluated by a delayed hypersensitivity test on white guinea pigs, which were administered 0.2 ml of a mixture of ovalbumin (2.5 mg) with the test substance, either in incomplete Freund's adjuvant (FIA) or only in physiological solution, into the left hind paw. The effect of the test substances was measured three weeks after administration, when the skin reaction of the guinea pigs after administration of 10 and 20 µg of ovalbumin was monitored. From the results shown in Table I it is clear that N-£(6-purinylthio)acetylJ-L-alanyl-D-isoglutamine of formula la (i.e. a compound of general formula I, in which R ¹ denotes a hydrogen atom) ch ₃ conh ₂ sch ₂ co- nhchco—nhch(ch ₂ ) ₂ cooh

New

Sr(la) has effects on delayed hypersensitivity in guinea pigs both in mineral oil and when administered only in saline, which substances from the muramylpeptide and desmuramylpeptide groups do not have.

Table

Induction of delayed hypersensitivity in guinea pigs

Tested Dose Skin response after : 24 h (mm) substance Cug) dose ovalbumin dose ovalbumin 10 TUq 20 /uq FIA - 3.7 + 0.31 5.5 + 0.45 ILO 100 6.1 + 0.37 9.8 + 0.55 ⁺ the 100 5.2 + 0.32 9.1 + 0.58 Saline solution 0.5 + 0.75 1.0 + 0.97 MDP 100 1.0 + 0.89 2.4 + 1.48 the 100 2.9 + 0.54 5.5 + 0.63 ⁺

Explanations:

FIA - Freund's incomplete adjuvant (Bayol + Arlacel 4:1)

MDP - muramyl dlpeptide

+. - statistically significant difference

The tests performed show that the compounds of general formula I, especially compound Ia, can be used in therapy as immunostimulating, antineoplastic and anti-infective agents.

For the above purposes, water-soluble, non-toxic salts of those compounds of the general formula I in which R represents a hydrogen atom are also suitable. As said salts, salts with pharmaceutically acceptable inorganic or organic bases can be used, such as salts with alkali metals, alkaline earth metals, ammonium salts and salts with organic amines, for example with triethylamine, ethanolamine, dicyclohexylamine and others.

The preparation of the compounds of formula I is illustrated in more detail in the following examples, which do not limit the scope of the invention. The temperatures are given in degrees Celsius and the specific rotation values refer to substances free of crystal solvent; the percentages are weight percent.

Example 1

N-((6-purinylthio)acetyl)-L-alanyl-D-isoglutamine benzyl ester

To a solution of 0.54 g (3.3 mmol) of carbonyldiimidazole in 6 ml of anhydrous dimethylformamide is added 0.63 g (3 mmol) of (6-purinylthio)acetic acid with stirring and cooling to 0 to +5 °C.

After the acid has dissolved (after about 10 min), a solution of 0.88 g (3 mmol) of L-alanyl-D-isoglutamine benzyl ester in 2 ml of dimethylformamide is added to the obtained imidazolide solution at the indicated temperature and the mixture is stirred for 15 h at the same temperature. The reaction mixture is poured into ice water, acidified with acetic acid to pH 6 and the precipitated product is filtered off (1.03 g, yield 68.7%). After recrystallization of the crude product from aqueous ethanol, the title compound is obtained with mp 206-208 °C and optical rotation ( ^{c =} 0.5, pyridine).

Example 2

N-[(6-purinylthio)acetyl]-L-alanyl-D-isoglutamine methyl ester

To a solution of 0.54 g (2 mmol) of L-alanyl-D-isoglutamine methyl ester hydrochloride in 5 ml of anhydrous dimethylformamide, 0.22 g (2.2 mmol) of triethylamine is added with stirring at room temperature, followed by 0.42 g (2 mmol) of (6-purinylthio)acetic acid. The suspension is cooled to 0 to +5°, 0.45 g (2.2 mmol) of dicyclohexylcarbodiimide is added, and the reaction mixture is stirred at the above temperature for 8 h. The precipitated substance, containing a mixture of dicyclohexylurea with the desired product, is filtered off, and the methyl ester is taken up in 50% aqueous methanol under boiling.

After concentration of the methanolic filtrate under reduced pressure and recrystallization of the separated crude product from water, 0.32 g (37.8% yield) of the title compound with mp 212-215° and optical rotation +4.1° (c - 0.1, water) was obtained. A further portion of the methyl ester can be obtained by diluting the dimethylformamide filtrate with water (250 ml) and chromatographing the aqueous solution on Dower 50 W x 2 cation exchange resin in the H form using water to wash the column and diluted ammonia (1:100) to elute the desired compound.

Example 3

N-[(2-amino-6-puririylthio)acetyl]-L-alanyl-D-isoglutamine methyl ester

To a solution of 0.46 g (2 mmol) of L-alanyl-D-isoglutamine methyl ester in 9 ml of anhydrous dimethylformamide is added 0.45 g (2 mmol) of (2-amino-6-purinylthio)acetic acid, the suspension is cooled to 0 to +5 °, 0.45 g (2.2 μmol) of dicyclohexylcarbodiimide is added and the mixture is stirred for 2 h at the indicated temperature and for 44 h at room temperature. The solids are filtered off, the filtrate is diluted with water (55 ml), a small amount of the separated by-product is filtered off and the filtrate, which contains the desired product, is chromatographed on a Dowex 50 W x 2 column, 100/200 mesh, in the H ⁺ form. After washing the column with water, the methyl ester is eluted with dilute ammonia (1:100) and the residue from the eluate (0.54 g) is chromatographed on a silica gel column using a mixture of chloroform, methanol and concentrated ammonia (4:4:1) for elution. The residue from the combined uniform fractions gives, after recrystallization from a mixture of methanol and acetone, the title compound with mp 133-135° and optical rotation {α]ρθ= +18.6° (c = 0.2, water).

Example 4

N-[3-(6-purinylthio)propionyl]-L-alanyl-D-isoglutamine methyl ester

In the same manner as in Example 3, but using 3-(6-purinylthio)propionic acid as the starting material, after a reaction time of 24 h at room temperature, the title compound is obtained with mp 202-206° (from water) and optical rotation Q>Qp ⁰ = -9.5° (c = 0.2, water).

Example 5

N-J-(6-purinylthio)acetylj-L-alanyl-D-isoglutamine

To a solution of 5.0 g (0.01 mol) of N-(6-purinylthio)acetyl-L-alanyl-D-isoglutamine benzyl ester in 130 ml of 80% aqueous methanol at room temperature, 20.0 ml (0.02 mol) of 1M sodium hydroxide solution are added and the mixture is stirred for 4 h at 20 to 25°. The methanol is distilled off under reduced pressure at a temperature of about 40°, the aqueous solution (about 30 ml) is filtered with activated carbon, the filtrate is acidified with 10.0 ml (0.02 mol) of 2M hydrochloric acid (pH about 3.5 to 4), seeded and left to stand overnight in a refrigerator at a temperature of about +5°. The precipitated product is filtered off.

(2.53 g, yield 62%) and recrystallized from water. The title acid is obtained in the form of colorless needle-like crystals with a melting point of 142-145° and optical rotation = -3.5° (c = 0.2, water).

Examples

In the same manner as in Example 5, by saponification of the corresponding esters with 1M sodium hydroxide solution at room temperature, the following are obtained:

N [(2-amino-6-purinylthio)acetyl J-L-alanyl-D-isoglutamine, m.p. 186 to 188° (from water), £«Έ°= (c = 0.1, water);

N -N 3 -(6-purinylthio)propionyl]-L-alanyl-D-isoglutamine, m.p. 209 to 210°, £0Q 2 O = -10.8° (c = 0.1, water).

Claims (7)

SUBJECT OF THE INVENTION 1. N- (6-Purinylthio) acyl] -L-alanyl-D-isoglutamines of Formula I C ^H 2 conh ₂ S (CH ₂ ) n CO-NHCHCO-NHCH (CH ₂ ) ₂ COOR ² in which r 'represents a hydrogen atom or an amino group, R is C1-C4alkyl, benzyl or hydrogen and n is 1 or 2, and salts thereof. 2. A process for the preparation of N- [(6-purinylthio) acyl] -L-alanyl-D-isoglutamines of formula (I) according to claim 1, characterized in that (6-purinylthio) alkanoic acid of formula (II): SfCH ^ n COOH (II) in which R ¹ has the meaning indicated in claim 1, or a reactive derivative thereof, is reacted with an ester of L-alanyl-D-isoglutamine of Formula III CH, CONH, I ³ T ³ 3 H ₂ N-CHCO-NHCH (CH ₂ ) ₂ COOR (III), □ wherein R represents a C 1 -C 4 alkyl group or a benzyl group, 2 and 3 the obtained ester of the general formula 1 in which R has the same meaning as R ₂ is optionally saponified to the acid of formula I wherein R is hydrogen. 3. A process according to claim 2, wherein the condensing agent for the reaction of the acid of formula II with the ester of the dipeptide of formula III is a carbodiimide such as N, N'-dioyclohexylcarbodiimide or N-ethyl-N ' (dimethylaminopropyl) carbodiimide, in a suitable inert medium. 4. A process according to claim 2, wherein the reactive acid derivative of the formula (II) is an imidazolide thereof prepared in situ by the action of N, N ' -carbonyldiimidazole on an acid in an inert medium. 5. Process according to claim 3 or 4, characterized in that dimethylformamide, dimethylsulfoxide or hexamethylphosphoric triamide is used as inert medium. 6. The process of claim 2 wherein the saponification of the ester of formula I wherein R is C1-C4 alkyl or benzyl to the acid 2 of formula I wherein R is hydrogen is treated with up to 3 mol equivalents of alkali metal hydroxides such as sodium or potassium hydroxide in a suitable environment. 7. The process of claim 6 wherein the saponification is carried out in water or in a mixture of water with a lower alcohol such as methanol or ethanol at a temperature of 0 to 40 ° C.