CZ520687A3 - process for preparing 1-beta-d-arabinofuranosyl-5-azacytosine - Google Patents

Abstract

The process for preparing 1-beta-d-arabinofuranosyl-5-azacytosine is based on the reaction of two compounds - protected D-arabinofuranosylhalogenide and silylated 5-azacytosine either in chloroform or by heating of the reaction compounds in a vacuum. In the case that the produced intermediate product is protected by benzyl groups it undergoes hydrogenolysis of catalysed palladium to carbon in the presence of an excess of dry hydrogen chloride, which is neutralised after the end of the reaction by anhydrous trialkylamine. If the intermediate product is protected by acyl groups, alcoholysis is used to remove them.

Description

The invention relates to a process for the preparation of 1 -? - D-arabinofuranosyl-5-azacytosine of formula X

13-0-4 Rabinofuranosyl-5-azacytosine (1) is an analogue of 2'-deoxycytidine from which it can be formally derived by two structural modifications, replacing the hydrogen at the 2-position, pointing to the same side of the sugar ring as the pyrimidine base with OH, and replacing the CH group at the 5-position of the pyrimidine nucleus with an isoelectronic nitrogen atom. Each of these individual exchanges resulted in substances with strong anti-leukemic activity in human γν model leukemic cell systems in vivo and in vitro. 1- / 3-O-A rabbi nof u ranose l cythosin, in which only hydrogen at position 2 of the 2'-deoxycytidine sugar moiety is replaced by the OH group, is the most effective preparation for the treatment of human acute myelogenous leukemia. 5-Azacytidine and 2'-deoxy-5-azacytidine, which can be formally derived only by replacing the CH group at the 5-position of the pyrimidine nucleus of the cytidine or 2'-deoxycytidine with an isoelectronic nitrogen atom, also show potent anti-leukemic activity. 5-Azacytidine is successfully used in the clinical treatment of leukemic patients and 2-deoxy-5-azacytidine is currently in the second phase of clinical trials. All of these nucleosides are metabolized in cells in a similar way and the mechanism of chemical degradation of 5-aza-anaLogs is identical. However, each of these substances differs from each other by a set of kinetic parameters * which determines their metabolic disposition in the tumor cell. The relative rates of deamination to inactive uracil nucleosides or 5-azauration of lu, phosphorylation to active 5'-triphosphates, incorporation into RNA or DNA, and spontaneous hydrolysis of the 5-aza-analog triazine ring are examples of kinetic parameters that directly affect the cytocidal activity of these nucleosides. . Tumor cells can also be substantially attenuated by the activity of individual enzymes that catalyze activation or. inactivating these analogs. Variability in biochemical pharmacology appears to be the cause of the different clinical utility of these drugs, as well as the wide range of susceptibility to individual types of leukemia observed in both clinical treatment and model systems.

1- # - D-Arabinofuranosyl-5-azacytosine / 17 is potent against tumor activity in cell culture and animal models. In L1210 murine leukemia, said nucleoside shows somewhat less potent activity than 5-azacytidine or 1- [beta] -D-a rabbinoforascytosine. However, in HT-29 human colon solid tumor cells transplanted in athymic mice, its cytocidal effect is much more pronounced than that of 5-azacytidine or 1-3-D-arabinofuranosylcytosine. The results of the study of the antileukemic effects and the mechanism of biological action of 1- [beta] -D-araninophosphorus in the case of ranose 1-5-aza cytosine are summarized in a review by Vesely: Pharmac. Ther. 28, 227-235 (1985).

1-10-DA for biosynthesis of 1-5-aza cytosin for high efficacy against three human tumors transplanted athymic mice, as well as a number of mouse tumor models, selected for clinical trial / Driscoll, Johns, Plowman: Investi ga ti oo.al New Drugs 3.

331/1985/2. The advantages of this nucleoside for use in human medicine are, in addition to its broad spectrum of anti-tumor effects, its relative selective toxicity to tumor cells and its ability to induce cell differentiation fDalal et al.: Cancer Res. 46, 831 (1986).

1- (5-D-A rabbinofuranosyl-5-aza cytosine), by its chemical structure, belongs to the group of 5-azacytosine nucleosides or 1-glycosyte-5-azacytosines. General methods for the preparation of these compounds are the subject of a number of Czechoslovak patents, possibly copyright certificates / Czechoslovak. U.S. Pat. No. 114716, No. 116297, No. 139542, No. 139549, No. 142531, ó. 147607,

AO No 152016, AO No 152017, AO No 220430 and AO No 221103 /.

The use of lewis acids as catalysts in the synthesis of protected 5-azacytosine nucleosides / NSR pat. No. 2012888) and also the patented microbial preparation of 5-azacytidine alone (U.S. Pat. 3,816.619 /.

Synthesis of 5-azacytosides of new nucleosides has also been the subject of a series of publications by Piskala, Sorm: Collect. Czech. Chem. Commun.

29, 2060 (1964); Pliml, Sorm: ibid. 29, 2576 (1964); Winkley, Robinson J. Org. Chem. 35, 491 (1970); Niedballa, Vorbrugen: ibid. 39, 3572 (1974); Pískala et al .: Nucleic Acids Res., Spec. Publ. No T, s. 17 (1975); Pískala et al .: Nutleic Acids Res., Spec. Publ. No 4, s. 109 (1978); Piskala, Sorm: Nueleic Acid Cheraistry, L. B. Towrtsend, R.S. Tipson, et al., Part 1, 435-441 and 443-449 (1978); Pískala et al .: Nucleic Acids Symposium Series 9, 83 (1981). The microbial preparation of 5-azacytidine by Hanka et al .: Antimicrob has also been reported. Ag. Chemother., 619 (1966); Bergy, Herr, ibid. 625/1966/7. Microbial preparation of 5-azacytidine is not advantageous, since this nucleoside, like other 5-azapyrimidine nucleosides, is unstable in aqueous solutions and consequently its isolation from a complex microbial medium is very difficult and uneconomical. In addition, this procedure is limited to 5-azacytidine and does not allow the preparation of other compounds of this type. Synthetic processes which generally allow to work under anhydrous conditions and therefore without substantial losses are more preferred. The synthetic processes described so far include different variants of two substantially different processes. The first approach is based on protected glycosyl isocyanates, which are converted to end products by a multi-step synthesis, and the second approach consists in the direct glycosylation of the silylated 5-azacytosines and subsequent deprotection.

1- (3-D-Arabinofuranosyl-5-azacytosine) was first prepared via 1- (2,3,5-tri-O-benzyl- [3-D-arabinosyl] -4-methylthio-1,3, 5-triazin-2 (1H) -one, which was obtained by the isocyanate method in analogy to the preparation of similar 1-peracylglycosyl-4-alkylthio-1,3,5-1-azin-2 (1H) -one (Czechoslovak). U.S. Pat. No. 116297 /. Replacing the methylthio group of said intermediate with an amino group (analogous to the preparation)

1-glycosyl-5-azacytosines from similar compounds / Czechoslovak. U.S. Pat. No. 139542 // crystalline 1- (2,3,5-tri-O-benzyl-) - 3-O- and rabi [beta] -5-a was obtained with a cytosity of 23% overall yield (calculated on starting 2), 3,5-tri-O-benzyl-1-O-p-nitrobenzoyl-O-arabic). The debenzylation of tribenzylated rabies of 1-5-3 zacytosine by catalytic hydrogenolysis was performed on a 1 molar scale in methanol in the presence of two equivalents of hydrogen chloride and the use of palladium on carbon as a catalyst. After suctioning off the catalyst, the mixture was neutralized in a conventional manner by filtration through a weakly basic ion exchange column. Recrystatisation of the crude _{from a} very non-fainting product yielded 1- (3-O-a-rabinofuranose-5-azacytosine) in 25% yield. Smear, β-configuration at anomeric carbon, and anticonformation around the CN glycosyte bond resulted from the CO-spectra of this substance. The chiroptical properties of 1- (3-O-arabinofuranosyl-5-azacytosine) were presented at an international symposium, which also briefly mentioned the above-mentioned first synthesis of this nucleoside. Liblice- Castle (Czechoslóvakia), September 26-29, 1972 /. In contrast to 5-azacytidine, which inhibits the growth of Escherichia coli to 100% already at a concentration of 1 µg / ml, 1- [3-D-arabinofuranosyl-5-azacytosine showed no inhibition even at a concentration of 100 µg / ml. The preparation of larger amounts of 1-0-1 > and rabinofuranosyl-5-azacytosine _{from the} need to detect anti-leukemia effects / encountered considerable difficulties in the last step of said synthesis. Already the order of magnitude of the batches in the hydrogenation of tribenzylarabinosyl-5-azacytosine led to a mixture of syrupy decomposition products from which no nucleoside could be recovered at all. Later, however, this critical step was able to control and test the anti-leukemic effects of 1- (3-O-a rabbi nofu ranose 1-5-aza cytosi nuMertes et al .: Nucleic Acids Symposium Series, No 14, 237/1984/7). .

1 / 2.3 _{R-5-tri-0-arabinosyl} benzy1-0-0 / -5-azacytosine was also prepared in 47% yield by direct glycosylation silylated

5-azacytosine 2,3,5-tri-O-benzyl-D-arabinosyl chloride in dichloromethane. However, an attempt to debenzylate with palladium-catalysed hydrogenolysis led only to the decomposition of the triazine ring (Winkley, Robins: J. Org. Chem.). 35, 491 (1970). Later, 1- (3-D-arabinofuranosyl-5-azacytosine) was prepared via its stable 5,6-dihydroderivative. This intermediate was obtained either by reduction of said tribenzylarabisyl-5-azacytosine with sodium borohydride followed by debenzylation of 1- (2,3,5-tri-O-benzyl) - 3D-arabisyl) -5,6-dihydro-5-azacytosine with hydrogenolysis in ethanolic hydrogen chloride, catalyzed by palladium on charcoal, or directly by catalytic hydrogenation of tribenzylarabidose 1-5-azacytosine using the same catalyst, but with a considerable prolongation of the reaction time and an increase in the amount of hydrogen chloride. According to these procedures, the arabinose L-5-azacytosine dihydroderivative in the form of the hydrochloride is further dehydrogenated with atmospheric oxygen under silylation conditions and free 1-γ-D-arabinofuranosyl-5-azacytosine (1) is finally obtained by methanolytic cleavage of trimethylsilyl groups. Beisler et al., Biochem. Pharmacol. 26, 2469 (1977) and J. Med. 2 £ 1230/1979/7. The total yield of this synthesis and the rabies of 1-5-azacytosine is about 30% (calculated on the starting 2,3,5-tri-O-benzyl-1-O-p-nitrobenzoyl-Da rabies nose).

The above arabinosyl-5-azacytosine I syntheses are characterized by relatively low overall yields and considerable elongation. Further extensive research into the preparatively more advantageous direct synthesis of nucleoside I resulted in surprisingly high yields in the glycosylation step and also for the first time perfectly solved the difficult problem of hydrogenolytic debenzylation of tribenzylarabinosyl-5-azacytosine. This made it possible to significantly shorten this synthesis by three times / steps, by hydrogenating the triazine ring, by silylating dehydrogenation and by methanolytic cleavage of trimethylsityl groups, and by increasing the overall yield and the rabinosil-5-azacytosine I, calculated on the starting benzylated and rabinofuranose, from 30% to 90%. Another advantage of the process according to the invention is that the use of the expensive import silylating agent fbis (trimethylsilyl) -trifluoroacetamide 7 is completely eliminated here, and the consumption of imported expensive palladium on coal is at least ten times lower. The process is much less demanding in terms of material, energy and foreign exchange than the known processes.

The method for the preparation of 1- £ arabinofuranosy1-5 -D-azacytosine of the formula I according to the invention consists in that _a protected and Rabbi noses lhalogenid formula II

H, X / 11 /

Where R is benzyl or acyl and X is halogen, reacts with an excess of the silylated 5-azacytosine of formula III

O (111) wherein R is alkyl of 1 to 6 carbon atoms, or by heating the reactants under vacuum and the protected arabinosyl-5-azacytosine obtained in either chloroform without solvent of the general formula

IV

/ IV /

- 6 defective known procedures.

•in 2J r > O o <<= · El Z - <H N - <

o <39 CJ I Cí 'oo! Cí —J

The azacytosine of the formula I according to the invention is characterized in that the protected arabinosyl halide of the formula XI

where R is benzyl or acyl and X is halogen, reacts with an excess of the silylated 5-azacytosine of formula (XIX-γγ-111) (111) where it denotes alkyl of 1 to 6 carbon atoms, either in chloroform or by heating of the reactants to a temperature of 60 to 10 ° C, in a vacuum of 0.0013 to 2.67 kPa without solvent, and the protected arabinosyl-5-azacytosine of the general formula IV obtained

AND

-7 .1

33 r > θ O <o <-> PI <r ~ - <m N - <

O

00 «ςο

- CO 'what oo as £ * wherein R' is benzyl _from the hydrogenolysis _of catalyzed by palladium on charcoal in an alcohol in the presence of an excess of a dialkyl ketone with 3-6 carbon atoms _of which alkyl acetal having from 1 to 6 carbon atoms, for activation of the catalyst with excess dry hydrogen chloride _from which, after completion of the reaction is neutralized with anhydrous trialkylominera wherein the alkyl moieties have from 1 to 6 carbon atoms and _from where R is acyl _of alcoholysis treatment with an alkali metal alkoxide in an alcohol with 1-6 carbon atoms.

R indicates one the same as in formula II, _Z

The reaction of the halide of formula II, wherein R ¹ is benzyl _z with an excess of silylated 5-azacytosine of formula III, proceeds well in chloroform over a wide temperature range of from C 1 to C 2 chloroform. However, the highest yields were obtained by carrying out this condensation at room temperature using 2,3,5-tri-O-benzyl-1'-D-arabinosyl chloride and bis (trimethylsilyl) -5-azacytosine as reactants.

Another feature of the invention is that the reaction of the protected arabinose lhalogenidu of formula II _from wherein R stands for acyl, and X is halogen, with a silylated 5- azacytosine of formula III is carried out in vacuo at temperatures of preferably 90 to 12 (ή ° 0th

In the reaction of a compound of formula II with a compound of formula III, 1.5 to 3.0 mol equivalents of the compound of formula III are used.

AND

The hydrogenolysis of compounds of formula IV wherein R is benzyl is carried _out in an alcohol having 1 to 6 carbon atoms, preferably with methanol. In the hydrogenolysis, 10% by weight of the catalyst is preferably used as catalyst and at most 10% by weight of the starting material at ambient temperature at atmospheric pressure using 1.6 to 1 _of 8 mol equivalents of hydrogen chloride to activate the catalyst.

<1

The stereoselectivity of the reaction of a halide of formula II wherein R is acyl _z with a silylated 5-azaeytosine of formula III is somewhat dependent on the nature of the acyl groups. Good results were obtained using compounds of formula II wherein R 1 was benzoyl

7.7 7 10 or chlorobenzoyl. A lower proportion of the β-anomer was obtained using a compound of formula II wherein R was p-methoxybenzoyl or p-nitrobenzoyl. Again, bis (trimethylsilyl) -5-azacytosine was preferably used in these reactions as it is readily available and the condensation formed by condensation of halogen-trimethylsilane has a low boiling point and can be easily removed from the reaction mixture. Optimal results were obtained using 1.5 to 3.0 mol equivalents of silylated 5-azacytosine. The reaction proceeds over a wide temperature range of from 0 ° C to 100 ° C, but best results were obtained at temperatures of 90 ° C to 120 ° C using a water pump vacuum. Good results have also been obtained with the haloalkylalkylsilane formed by codestilation with chloroform. In this case, the reaction is carried out in a distillation apparatus and the chloroform loss is continuously replenished with fresh solvent during the reaction.

The primary condensation product of a halide of formula II with a silylated 5-azacytosine of formula III is protected arabinosyl-5-azacytosine of formula IV silylated at the amino group at the 4-position of the triazine core. However, during the usual working up of the reaction mixture, the trialkylsityl group is hydrolytically cleaved so that the isolated product is a compound of formula IV.

Hydrogenolysis of a compound of formula IV wherein R is benzyl. The reaction is preferably carried out in methanol in the presence of 10% palladium on carbon catalyst as a maximum of 10% by weight of the starting material at room temperature at atmospheric pressure. The reaction further requires the presence of hydrogen chloride to activate the catalyst. Since the compound of formula IV is weakly basic and neutralizes 1 equivalent of hydrogen chloride, more than 1 equivalent of acid must be used to activate the catalyst. However, too much excess of hydrogen chloride accelerates the undesired hydrogenation of the triazine core. Optimal results were obtained using 1.6 to 1.8 mol equivalents of hydrogen chloride. Acetone, well-available 2,2-dimethoxypropane dimethylacetal (used in considerable excess), has proven useful to maintain the anhydrous environment.

For neutralizing hydrogen chloride, anhydrous triethylamine, whose hydrochloride is very soluble in both methanol and chloroform, has been advantageously used, which facilitates the isolation of the product.

The alcohol of the protected and rabyl-5-azacytosine of formula IV _of 1 wherein R is acyl is preferably carried out in methanol at room temperature by treatment with 0.1 to 1 mol equivalent of sodium methoxide.

In the following, the invention is explained in more detail by way of non-limiting examples.

Example 1

Preparation of 1- / 2,3,5-tri-O-benzyl-ZS-D-a-rabidose l--5-a cysteine

A solution of 2,3,5-tri-O-benzyl-1-O-p-nitrobenzoyl-O-arabinose / 26.5 g; 46.5 mmol (in dry 1,2-dichloroethane (260 ml)) were intensively saturated with dry hydrogen chloride for 30 minutes under ice-cooling and exclusion of external humidity, and then left to stand at 0 ° C for 2 hours. The precipitated p-nitrobenzoic acid is suctioned off rapidly, washed with 1,2-dichloroethane / yield: 7.7 g; 99.1%), the filtrate was evaporated in vacuo at 25 [deg.] C. (bath temperature) and the syrupy residue was distilled with dry toluene (3 * 50 ml). A mixture of 2,5,5-tri-O-benzyl-O-arabinosyl chloride, bis (trimethylsilyl) -5-azacytosine (24.0 g) thus obtained; 93.6 mmol (dry chloroform) (140 ml) were stirred magnetically at room temperature for 5 hours to avoid external moisture and the resulting slightly cloudy solution was allowed to stand for 85 hours. The solution is cooled in an ice bath and shaken with ice-cold saturated sodium bicarbonate solution, which is bubbled with carbon dioxide for 15 minutes before use. The resulting emulsion was suction filtered through a pad of celite and the filter material was washed with chloroform. After drying (MgSO4), the organic layer of the filtrate was evaporated in vacuo to a thick syrup, which was diluted with dry ether (100 ml) and the crystalline slurry was allowed to stand at room temperature for 1 hour. After treatment of the mother liquors, a total of 21.35 g (89.2%) of 1- (2,3,5-tri-O-benzyl-4-D-arabisyl) -5-azacytosine is obtained; m.p. 141-143 ° C (dec.). Chromatography of the last mother liquors on a silica gel column (100 g), eluting with chloroform and crystallization of the major portion from ether gave an additional crop (0.43 g) of the same material. Total yield: 21.78 g (91%).

Example 2

Preparation of 1- / 2 _of 3 _of 5-tr-i-O-benzoyl 1- (3-0-β-rabinosyl) -5-azacytosine

Methyl 1-2,3,5-tri-O-benzoyl-X-D-arabinoside solution / 0.95 g>

mmol) in a mixture of dry 1,2-dichloroethane (5 ml), a 40% solution of hydrobromic acid in acetic acid (5 ml) and acetyl bromide (0.5 ml) was allowed to stand at room temperature for 45 minutes, excluding external moisture. The solvents were evaporated at 25 ° C (bath temperature) and the residue codistilled to remove the residue of hydrogen bromide and acetic acid with dry toluene (3 x 10 mL). A mixture of the thus obtained syrupy 2,3,5-tri-O-benzoyl-6-arabisylbromide and bis (trimethyl3ilyl) -5-azacytosine (1.1 g); 4.2 mmol (after homogenization) are heated under vacuum, water pump at 90-95 ° C (bath temperature) for 2 hours. A solution of the reaction mixture in dry 1,2-dichloroethane (10 ml) was diluted with cold chloroform (20 ml) and shaken with cold saturated sodium carbonate solution (10 ml). The mixture was suction filtered through a pad of celite, the filter material was washed with chloroform and the organic layer of the filtrate was dried (MgSO4) and evaporated in vacuo. The syrupy residue is dissolved in dry acetonitrile (5 ml), a small precipitate of 5-azacytosine is filtered off with suction and the clear filtrate is again evaporated in vacuo. Crystallization of the residue from dry acetonitrile (4 ml) gave 0.36 g (32.5%) of 1- (2,3,5-tri-O-benzoyl- (3-O-a-rabinosyl)).

-5-azacytosin, m.p. 219-220C (decomposition).

When kLad 3

Preparation of 1- (2,3,5-tri-O-benzoyl-γ-β-a rabinosyl) -5-azacytosine

A mixture of crude syrupy 2,3,5-tri-O-benzoyl-O-arabinose Ibromide, prepared from methyl-2,3,5-tri-O-benzoyl-X-O-arabinoside (1.9 g, 4 mmol) according to the procedure according to Example 2, bis (trimethylsilyl) -5-azacytosine (2.05 g> 8 mmol) and dry chloroform (10 ml) were heated in a distillation apparatus for 16 hours so that the mixture slowly distilled and the chloroform was replenished from a separatory funnel to the volume of the reaction mixture remained approximately the same throughout the reaction. After dilution with chloroform (30 ml) and cooling in an ice bath, the solution was shaken with an ice-cold saturated solution of sodium methane (10 ml). The mixture was suction filtered through a pad of celite, the filter material was washed with chloroform and the organic layer of the filtrate was dried (MgSO4) and evaporated in vacuo. The syrup residue is stirred with dry toluene (50 ml), the insoluble material is filtered off with suction and the sulphate filtrate is again evaporated in vacuo. Crystallization of the residue from dry acetonitrile yields 0.50 g (22.5%) of 1- (2,3,5-tri-O-benzyl-1- (3-D) -benzoic acid [5-aza-cy] tos i nuj tt 213 DEG -220 DEG C. (decomposition). After recrystallization from ethanol -

The 1,2,2-dichloroethane melted the product at 221-222 [deg.] (Decomposition).

Example 4

Preparation of 1- (2,3,5-1) -i-O-benzoyl 1- (5-O-a) -benzoic acid l--5-a zacy tosin

Mixture of crystalline 2,3,5-tri-O-benzoyl-α-O-arabinosyl bromide / 1.05 g; 2 mmol (a) bis (trimethylsilyl) -5-azacytosine (1.1 g); 4.2 mmol) was heated to 100-105 ° C for 2 hours after homogenization under water pump vacuum. The mixture is then further processed as in Example 2. Crystallization of the crude product from dry acetonitrile yields 0.36 g (32.5%) of 1- (2,3,5-tri-O-benzoyl- [3-d] rabidose). 1 -5-aza cytosine; 1.1. (Decomposition).

Example 5

Preparation of 1- (2,3,5-1) -1-O-benzoyl- [3- (D) -benzoic acid] -5-a-cyano

To a solution of methyl-2,3,5-tri-O-benzoyl-α-O-arabididium (2.383 g); 5 mmol (in acetic acid (12.5 ml) was added 40% hydrogen bromide in acetic acid (12.5 ml) and the mixture was allowed to stand at room temperature for 20 minutes, excluding external moisture. The solution was diluted with 1,2-dichloroethane (50 mL) and poured onto ice / water (300 mL). The organic layer was shaken with ice-cold saturated solution to sodium carbonate until neutral and after drying (MgSO 4) was evaporated in vacuo at 25 ° C (bath temperature). Mixture of the thus obtained syrupy 2,3,5-tri-O-benzoyl-D-arabinosyl bromide and bis (t-methylsilyl) 12

-5-azacytosine / 2.57 g; 0.01 mmol / after homogenisation is heated in a water-jet vacuum 1.5 hours at 90 to 95 [° ^C / bath temperature /. The mixture is then worked up in analogy to Example 2. 0.717 g (25.3%) of 1- (2,3,5-1) -1-O-benzyl-3-Da-bi-axis 1 is obtained. (-5-azacytosine) mp 218-22 ° C (dec.).

Example 6

Preparation of T- (2, J, 5-tri-O- (o-chlorobenzoyl) - (3-3-a rabinosyl) azazytytine

A mixture of methyl-2,3,5-tri-O-benzoyl-dibenzoic acid (0.949 g, 2 mmol), methanol (5 ml) and methanolic IM-NaOCH3 (0.3 ml) was stirred magnetically at room temperature until dissolution. starting materials. The solution was allowed to stand at room temperature for 24 hours and poured onto a column of weakly acidic ion exchanger (5 ml) in H form prepared in methanol. The column was washed with methanol (50 ml) and eluted with methanol ^. The mixture was evaporated in vacuo.

The residue was stirred with water (10 ml) and the methyl benzoate was removed by shaking into chloroform (10 ml). The aqueous layer was evaporated and the residue was codistilled sequentially with ethanol (2 x 5 mL) and dry pyridine (2 x 5 mL). To a solution of the methyl O-arabinofuranoside thus obtained in dry pyridine (5 ml), o-chlorobenzoyl chloride (0.85 ml) was added under magnetic stirring, ice cooling and exclusion of external moisture. The mixture was allowed to stand at room temperature for 24 hours and diluted with ice water (20 mL). The product was shaken into chloroform (50 ml) and the chloroform layer was successively shaken with 10% sulfuric acid (3 x 10 ml) water (10 ml) and a saturated solution. The sodium bicarbonate (2 x 10 ml) and dried (MgSO4) was evaporated in vacuo. The crude product was purified by silica gel column chromatography (10 g), which was prepared in toluene.

The column was washed with toluene-ethyl acetate (9: 1) and the fractions containing the major portion were evaporated in vacuo. A solution of the thus obtained syrupy methyl-2,3,5-1 R-O- (o-chlorobenzyl) -α-D-arabinoside in a mixture of dry dichloroethane (5 ml), a 40% solution of hydrogen bromide in acetic acid (5 ml) and acetyl bromide (0.5 ml) were allowed to stand at room temperature for 45 minutes under exclusion of external humidity. The solvents were evaporated at 25 ° C (bath temperature) and the residue codistilled with dry toluene (3 x 10 ml). A mixture of the thus prepared syrup-like 2,3,5-tri-O- (o-chlorobenzoyl) -D-arabidobromide and bis (trimethylsilyl) -S-azacytosine (1.1 g); 4.2 mmol / was heated in a water pump vacuum at 95-100 ° C for 2 hours. The mixture is then worked up in analogy to Example 2. Crystallization of the crude product from dry acetonitrile yields 0.30 g of 122,17,1 1- (2,3,5-tri-O- (o-chlorobenzoyl) -). —Disubstituted l--5-aza cytosine; m.p. 198-202 ° C (decomposition) »

After recrystallization from acetonitrile, an analytically pure product is obtained. ° C / decomposition / »

Example 7

Preparation of 1- (2,3,5-1) -1-O- (p-chlorobenzoyl) -3-D- and rabiyl-5-azacytosine

Methyl solution of 1-2,3,5-tri-O- (p-chlorobenzoyl) -benzoic acid du (1.16 g> 2 mmol) in dry 1,2-dichloroethane (5 ml),

A 40% solution of hydrogen bromide in acetic acid (5 ml) and acetyl bromide (0.5 ml) was allowed to stand at room temperature to exclude external moisture. The solvents were evaporated at 25 ° C (bath temperature) and the residue was codistilled with dry toluene (3 × 10 ml). A mixture of the crude crystalline 2,3,5-tri-O- (p-chlorobenzoyl) -D-arabidobiphenyl and bis (trimethylsilyl) -5-azacytosine (1.1 g> 4.2 mmol) thus obtained after homogenization The reaction mixture in dry 1,2-dichloroethane (10 ml) was diluted with cold chloroform (20 ml) and shaken with cold saturated sodium bicarbonate solution (10 ml). ml /. The mixture was suction filtered through a pad of celite, the filter material was washed with chloroform and chloroform-methanol (95: 5) until the product was completely washed out of the filter layer. After drying (MgSO4), the organic layer was evaporated in vacuo and the residue was gradually crystallized from chloroform14

methanol (1: 1) and twice from dimethylformamide-methanol (1: 1). There are obtained 0 _of 431 g (32.6%) of 1- (2,3,5-tri-O- p -chlorobenzoyl) -? -? - arabisyl] -5-α-acytosine; tt 246-247 ^ 0 (decomposition).

Example 8

Preparation of 1-ff-D- and rabinofuranosyl-5-azacytosine

Dry hydrogen chloride solution / 1.204 g; 0.033 mol (in absolute methanol) (106 ml) was allowed to stand at room temperature for 2 hours after the addition of 2,2-dimethoxypropane (10 ml) with exclusion of external moisture. After addition of 1- (2,3,5-tri-O-benzyl) - (3-D-arabinosyl) -5-azacytosine (10.23 g, 0.02 mol), 10% palladium on carbon (1 g) and absolute methanol (300 ml), the mixture is hydrogenated with vigorous magnetic stirring at at room temperature at atmospheric pressure until the rate of hydrogen consumption was significantly slowed down, when the silica gel thin layer chromatography (ethyl acetate-acetone-ethanol-0.1 G05M phosphate buffer pH 7.0 / 4: 1: 1: 1) _r detection in UV light? it contains no benzylated intermediate. The catalyst was filtered off with suction and the clear filtrate was neutralized by adding a solution of dry triethylamine (4 ml); 0.033 mol (absolute methanol) (20 ml). Concentration of the solution in vacuo at 25 ° C (bath temperature) to a volume of about 100 mL gave 4.4 g (91.4%) of 1-? -D-arabinofuranosyl-5-azacytosine; tt 229-231pC (decomposition). The mother liquor is concentrated to a small volume, stirred with chloroform (150 ml) and the precipitate is recrystallized from methanol after suction. This gave an additional 0.240 g (5%) of the product; mp 228-230 ° C (dec.).

Example 9

Preparation of 1- / 3-0-a rabi naphtha in early 1-5-a with cysteine

A mixture of 1- (2,3,5-tri-O-benzoyl-γ-3-O-arabinosyl) -5-α-acytosine (4.41 g); 7.92 mmol), absolute methanol (35 ml) and methanolic IM-NaOCH3 (2.5 ml) were stirred magnetically at room temperature for 24 hours. Acetic acid (0.3 ml) was added and the precipitate was filtered off with suction to give 1.57 g (81.1%) of 1- (3-O-a) rabbinol of ranose 1-5-azacytosin. 228-230 ° C (dec.).

Example 10

In the preparation of [beta] -g-O-a rabbinofuranose-5-a zacytosi nu

1- (2,3,5-tri-O- (o-chlorobenzoyl) -? - D - and rabies 1? -5-aza cytosine? 0.163 gj. 0.25 mmol) was treated analogously to. in Example 9, methanol. 0.044 g (72.1%) of 1- (3-D-arabinofuranosyl-5-azacytosine) was obtained; m.p. 229-23 ° C (dec.).

Example 11

The preparation of 1 -? - O - and rabinofuranosyl-5-azacytosine

1- (2,3,5-1 R-O-) p -chlorobenzoyl-2-D-arabinosyl-5-azacytosine (0.33 g, 0.5 mmol) was subjected to methanolysis analogously to Example 9. 0.07 g (57.3%) of 1-γ-O-arabinofuranosyl-5-azacytosine is obtained; m.p. 228-230lpC (decomposition).

Example 12

In the preparation of 1- / 3-0-a rabiofofananosy-5-a z a cy tosi nu

Crude product obtained from methyl 2,3,3,5-tri-O-benzoyl-4-ol-O-arabididium / 0.95 g; 2 mmol) was dissolved in 1,2-dichloroethane (10 ml) according to the procedure of Example 2 and the nucleoside was precipitated with petroleum ether (60 ml). The precipitate formed is subjected to methanolysis without further purification analogously to Example 9.

There was obtained 0.120 g (24.6%) of 1-43-D-α-rabbinin in the case of ranose 1-5-aza cytosu m.p. 227-229 ^ 0 (decomposition).

Example 13

Comparison of potency of various cytidine derivatives

Activity was determined in L 1210 leukemia mouse cell culture. To exponentially growing cells (50,000 / ml on day 0), test substances were added after 24 hours of growth and after a further 42 hours the number of cells was determined. See Table 1.

Table 1

substance ara-C 5-AdC and ra-5-AC @ 1 -ara-5-AC concentration / mol / 1 / 10 ⁶ 10 ⁶ IQ ' ⁵ i '-5 10 ⁵ X controls 5 23 14 92

control »100% (2.8 * 10 cells / ml), no drug ara-C: arabinosylcytosine added

5-AdC; 5-ara-deoxycytidine ara-5-AC: 1- [4-] -rabinofuranosyl-5-azocytosine-a-5-AC laanomer ara-5-AC

The table shows that ara-5-AC shows high anti-leukemic activity. *

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Claims (2)

-P, 4-m and 4-m, t-e, 2 o1. A process for the preparation of 1- (ODa-rabinofuranose 1-5-a) with a cytosine of formula I ## STR2 ## wherein R is benzyl or acyl and X is halogen reacts with an excess of silylated 5-azacytosine of formula III / ^111/2 wherein R represents an alkyl with 1-6 carbon atoms, in either chloroform or by heating the reactants to a temperature from 60 to 160 DEG C., in vacuo, from 0.0013 to 2.67 kPa without solvent, and the obtained protected arabinosyl-5- azacytosine of formula IV ί -18.1 where R denotes the same as in formula II, if benzyl, hydrogenolysis, catalysed by palladium on carbon, is subjected in an alcohol to the excess of the dialkyl tacetyl of a ketone of 3 to 6 carbon atoms, wherein the alkyls of acetal have 1 to 6 atoms The catalyst is activated with an excess of dry hydrogen chloride, which is neutralized upon completion of the reaction with anhydrous triethylamine, wherein the alkyls have 1 to 6 carbon atoms, and when R is acyl, the alcohols are treated with an alkali metal alkoxide in T to 0-atoms. carbon. 2. Process according to claim 3, characterized in that the reaction of the protected arabinose halide of the general formula II, wherein: R is benzyl and X is halogen, with the silylated 5-azacytosine of formula III, carried out in chloroform at 0 DEG C. and chloroform, preferably at room temperature. 3. A process according to claim 1, characterized in that the reaction of a protected arabinosyl halide of the formula II, wherein: R is acyl and X is halogen, with the silylated 5-azacytosine of formula (III ₎ carried out under vacuum, preferably at temperatures of 90-12 ° C. _z . 4. A process according to claim 1, wherein the reaction of a compound of formula II with a compound of formula III uses 1, 5 to 3.0 mol equivalents of the compound of formula III. sjt4'h / & ei 5. The process according to claim 1, wherein the hydrogenolysis of the compounds of the formula IV in which R1 is benzyl is carried out in an alcohol having 1 to 6 carbon atoms, preferably methanol. 6. Process according to claim 1, characterized in that the genolysis of the compounds of the general formula IV in which R is benzyl is carried out at a catalyst addition rate of 10% by weight / palladium and on carbon in an amount of at most 10. at atmospheric pressure and using 1.6 to 1.8 mol equivalents of hydrogen chloride to activate the catalyst. 7. A process according to claim 1, wherein the hydrogenolysis of compounds of formula IV wherein R1 is benzyl is 2,2-dimethoxypropane as the dialkylacetal of the ketone. ř i 19 8. The process according to b-oxta r, characterized by the hydrogenation of the hydrogen chloride after the hydrolysis, is carried out in that the dry neutralitrile is not dried. Process according to Claim 1, characterized in that the alcohols of the compounds of the formula IV in which R is acyl are carried out with sodium methoxide in methanol. Process for the preparation of 1- / 3-D-arabinofuranosyl-5-azacytosine you The method of quantity indicates the purity Dosa yields, tedious and more expensive Lez falls into the field of anti-cancer drug production. The manufacturing process of the invention allows the production of large coke in high yields at maximum for use in clinical practice. blunt production of this substance provided a lower alite. Moreover, these methods have been very much in view of the raw materials used / e.g. agent, because a large amount of expensive (if not in worse medicines and imported forces) 2- / 2? - $ ^ oC? The process according to the invention involves the reaction of two components, the protected D-arabinofuranosyl halide and the silylated 5-azacytosine, in chloroform or by heating the reactants under vacuum. When the resulting intermediate is protected with benzyl groups, it is subjected to palladium-carbon-catalyzed hydrogenolysis in the presence of an excess of ketone dialkyl acetal to activate the catalyst with an excess of dry hydrogen chloride which is neutralized with anhydrous tri-alkylamine upon completion of the reaction. When the intermediate is protected with acyl groups, alcoholysis is used to remove the intermediate.