CS261344B1 - (6-purinylthio)acyldipeptides and method of their production - Google Patents

Abstract

New N- (6-purinylthio) acyl-L-alanyl- -D-isoglutamines of formula I, ve wherein R 3 represents a hydrogen atom or an amino group, R2 denotes an alkyl group having 1 to 4 carbon atoms, benzyl group or the hydrogen atom and n denote 1 or 2, respectively salts and the process for their production in (6-purinylthio) alkanoic acid condensation of formula II, or a reactive thereof derivative, with the dipeptide ester of formula III and optionally in the following saponification of the ester thus obtained. Compounds of Formula I show significant immunostimulatory activity efficiency.

Description

The present inventors have studied in more detail some analogues of muramyl dipeptides and have unexpectedly found that the novel simple compounds of formula I which contain cytostatics active 6-mercaptopurine or 6-thioguanine linked via the acetic or propionic acid moiety instead of muramic acid, exhibit interesting immunostimulatory effects. The compounds of formula I contain as dipeptide L-alanyl-D-isoglutamine having the same steric arrangement at the asymmetric amino acid centers as the dipeptide in MDP: it consists of natural L-alanine, which is peptide-bound to D-isoglutamine belonging to the steric series non-naturally occurring D-amino acids in nature.

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

S (CH3) _n COOH, Ά / γ (id,

R ¹ H (II), wherein R ¹ and n are as defined above or a reactive derivative thereof, is reacted with an L-alanyl-D-isoglutamine ester of formula III

wherein R is C1 -C4 alkyl or benzyl, and the ester of formula (I) in which R is the same as R is optionally saponified to an acid of formula (I) in which R is an atom hydrogen.

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

In the carbonyldiimidazole method, from 1 mol equivalent of acid I and 1.1 mol equivalent of Ν, Ν'-carbonyldiimidazole are prepared in a suitable medium such as dimethylformamide or dimethylsulfoxide, at a temperature of 0 to +25 ° C, a solution of imidazole acid I which is medium and reacted at the same temperature with a solution of 1 mole equivalent of the dipeptide III ester in the same solvent. The progress of the reaction is monitored by thin-layer chromatography and after completion of the reaction (after 10-24 h) the desired product is isolated by conventional preparative organic chemistry procedures, for example by diluting the reaction mixture with water, adjusting pH, filtering the crude product and crystallizing it, or absorbing the product dissolved in water to a suitable product. an ion exchanger, for example a cation exchanger, by eluting it and optionally further purifying it.

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In the carbodiimide method, to a solution of 1 mole equivalent of acid II and 1 mole equivalent of dipeptide III ester in a suitable solvent, such as dimethylformamide, dimethylsulfoxide, hexamethylphosphoric triamide and the like, is added 1.0 to 1.1 mol equivalents at 0 to +5 ° C. an appropriate carbodiimide such as dicyclohexylcarbodiimide or N-ethyl-N '- (3-dimethylaminopropyl) carbodiimide hydrochloride, and the mixture is stirred for 1 to 72 hours at 0 to 40 ° C after addition of 1 mol equivalent of dipeptide III ester; the progress of the reaction was monitored by thin layer chromatography. After completion of the reaction, if dicyclohexylcarbodiimide is used, the precipitated dicyclohexylurea is filtered off and the filtrate is diluted, if necessary by evaporation of the solvent under reduced pressure, with water to crystallize the sparingly soluble ester of the formula I or with water-soluble carbodiimides. water and after adjusting the pH to a slightly 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 dilution of the reaction mixture with water by means of ion exchangers.

The saponification of esters of formula I in which R is C1 -C2 alkyl or benzyl to acids of formula I in which R is hydrogen is carried out with 2 to 3 mol equivalents of alkali metal hydroxides, such as sodium or potassium hydroxide. , per 1 mol equivalent of the ester in water or a mixture with a lower alcohol such as methanol or ethanol at 0 to 40 ° C for 2 to 10 h. The course of hydrolysis is monitored by thin layer chromatography and after saponification of the ester the reaction mixture or After distilling off the alcohol, it is acidified with a strong acid, such as hydrochloric acid or sulfuric acid, to a pH of about 3.5 to 4, and the precipitated product is purified by crystallization, preferably from water, after filtration.

The starting compounds of the formula II are known compounds or can be prepared by analogous methods; the preparation of (6-purynilthio) alkanoic acids (of the general formula II in which R @ 6 denotes hydrogen) has been described by Semonský et al. / Collection Czechoslov. Chem. Comun.

25, 1091 (1960)], (2-amino-6-purinylthio) alkanoic acid (of formula II in which R1 is an amino group) Elionic et al. Am. Chem. Soc. 81, 1898 (1959)].

The L-alanyl-D-isoglutamine esters of formula III are also required. Usually, suitable N-protected derivatives of said esters are used as starting materials, for example N-ethoxycarbonyl, N-benzyloxycarbonyl or N-tert-butyloxycarbonylderivatives, from which, prior to the reaction, known methods, for example by treatment with hydrogen chloride in glacial acetic acid or hydrogenolytically , deprotecting to give the free ester of the dipeptide of formula III or a salt thereof, which is used for the reaction with acid II without further purification.

The immunomodulatory activity of the compounds of formula I was evaluated by a delayed hypersensitivity test in white guinea pigs, administered to the left hind paw 0.2 ml of a mixture of ovalbumin (2.5 mg) with the test substance, either in incomplete Freund's adjuvant (FIA) or physiological only. solution. The effect of the test substances was measured at 3 weeks post-dosing, following a guinea pig skin reaction following administration of 10 and 20 µg ovalbumin. From the results listed in Table I it is evident that £ N- (6-purinylthio) acetylJ-L-alanyl-D-isoglutamine of Formula Ia (i.e. a compound of formula I wherein R ¹ is hydrogen), CH ₃ conh ₂ sch ₂ co- nhchco — nhch (ch ₂ ) ₂ cooh

Ný ·

Sr (1a), has effects on delayed hypersensitivity in guinea pigs both in mineral oil and when administered only in physiological saline, which have no effect from the muramylpeptides and desmuramylpeptides.

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 MOP 100 ALIGN! 6.1 + 0.37 9.8 + 0.55 ⁺ la 100 ALIGN! 5.2 + 0.32 9.1 + 0.58 Fysiol. solution 0.5 + 0.75 1.0 + 0.97 MDP 100 ALIGN! 1.0 + 0.89 2.4 + 1.48 la 100 ALIGN! 2.9 + 0.54 5.5 + 0.63 ⁺

Explanatory notes:

FIA - Freund's Incomplete Adjuvant (Bayol + Arlacel 4: 1)

MDP - muramyldpeptide

+. - statistically significant difference

The tests carried out show that the compounds of the formula I, in particular the compound Ia, can be used in therapy as immunostimulatory, antineoplastic and antiinfective agents.

Water-soluble, non-toxic salts of those compounds of formula I in which R is hydrogen are also suitable for this purpose. Salts with pharmaceutically acceptable inorganic or organic bases such as alkali metal, alkaline earth metal, ammonium and organic amine salts such as triethylamine, ethanolamine, dicyclohexylamine and others can be used.

The preparation of the compounds of formula (I) is illustrated by the following non-limiting Examples. Temperature data are given in degrees Celsius, and specific rotation values refer to crystal free materials; percentages denote 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 was added 0.63 g (3 mmol) of (6-purinylthio) acetic acid while stirring and cooling to 0 to +5 ° C.

After dissolution of the acid (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 imidazolide solution obtained at this temperature, and the mixture is stirred at the same temperature for 15 h. The reaction mixture was poured into ice water, acidified to pH 6 with acetic acid, and the precipitated product was filtered off (1.03 g, 68.7% yield). Recrystallization of the crude product from aqueous ethanol gave the title compound, 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 was added, with stirring, at room temperature, 0.22 g (2.2 mmol) of triethylamine and then charged with 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 for 8 h at this temperature. The precipitated product 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.

Concentration of the methanolic filtrate under reduced pressure and recrystallization of the precipitated crude product from water gave 0.32 g (37.8% yield) of the title compound, m.p. 212 DEG-215 DEG and optical rotation + 4.1 DEG (c = 0.1, water). An additional 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 H form using water to wash the column and dilute ammonia (1: 100) to elute the desired compound.

Example 3

N- (2-Amino-6-purirylthio) 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 was added 0.45 g (2 mmol) of (2-amino-6-purinylthio) acetic acid, cooled to 0 to + 5 °, 0.45 g (2.2 mmol) of dicyclohexylcarbodiimide are added and the mixture is stirred for 2 hours at this temperature and 44 hours at room temperature. The solids were filtered off, the filtrate was diluted with water (55 mL), a small amount of by-product precipitated was filtered off and the filtrate containing the desired product was chromatographed on a Dowex 50 W x 2, 100/200 mesh, H ⁺ form. . After washing the column with water, the methyl ester was eluted with dilute ammonia (1: 100) and the residue from the eluate (0.54 g) was chromatographed on a silica gel column using a 4: 4: 1 mixture of chloroform, methanol and concentrated ammonia. Recrystallization from the combined methanol / acetone mixture gave the title compound (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 of m.p. 202-206 ° (from water) and optical rotation was obtained. Q> Q ⁰ = -9.5 ° (c = 0.2, water).

Example 5

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

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

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

Examples

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

N - [(2-amino-6-purinylthio) acetyl] -1-alanyl-D-isoglutamine, m.p. 186-188 ° (from water), «« ° = (c = 0.1, water);

N- [3- (6-purinylthio) propionyl] -L-alanyl-D-isoglutamine, m.p. 209 DEG-210 DEG, [.alpha.] D @ 20 = -10.8 DEG (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.