CS221103B1 - Method of preparation of 5-azacytosine nucleosides - Google Patents

Description

The invention relates to a process for the preparation of 5-azacytosine nucleosiides of the general formula I

X ⁽ ►

where

R @ ¹ denotes free or protected glycosyl, R @ ^{2 is} hydrogen, C1 -C6 alkyl, C1 -C6 haloalkyl, C1 -C12 aralkyl, phenyl, R ^{@ 3} and R ^{@ 4 are the} same or different hydrogen, alkyl of 1 to 6 carbon atoms, or aralkyl of 7 to 12 carbon atoms.

The most important compounds of this type are the pyrlmidine antimetabolites 5-azacytidine [4-amino-1- [3-D-ribofuranosyl] s: -triazin-2 (1H) -ion] and 2'-deoxy-5-azacytidine [ 4-amino-1- (1,2-deoxy-5S-D-erythno-pentofuranosyl) -s-triazin-2 (1H) -one]. Both substances inhibit the growth of bacteria at low concentrations and have significant carcinostatic effects. Viriostatic activity has also been reported with 5-azacitidine. The biological effects and mechanism of action of these compounds, as well as the results of clinical research on 5-azacythidine, are known (Vesely and Cihak: Pharmac. Ttoer 2, A, 813-840 (1978)). in the United States for the treatment of myelogenous forms of leukemia 2'-Pexy-5-azacytidine is also being tested clinically and both are used as tools for biochemical, pharmacological and molecular biology research. Biological activity, although to a lesser extent ^1, also appears to be 6 and N ⁴ -substituted 5-azacytidine and 2'-deoxy-5-azacytidine derivatives.

5-AzacytOsin nucleosides can be prepared according to Czechoslovak patents or author's certificates No. 114 716, No. 116 297, No. 139 542, No. 139 549, No. 142 531, No. 147607, No. 152 016, and No. 152 017, respectively. AO No. 220 430. The synthesis of these compounds has also been the subject of a number of publications (Pískala, Šorm: Cíllect. Czech. Chem. Commun. 29, 2060 (1964); Pliml, Sorm: ibid. 29, 2576 (1964); Winkltey, Robina). : J. Org. Chem., 35, 491 (1970); Ni-edballa, Vorbruggen: J. Org. Chem., 39, 3572 (1974); Pískala et al., Nucleic Acids Res., Spec. 1, pp. 17 (1975);

2110 3

Pískala et al., Nucleic Acids Res., Spec.

Publ. No 4, p. 109 (1978); Pískala, Sorm: Nucleic Acid Chemistry (Part 1, 435-441, 441 and 443-449 (1978)). 5-Azacytidine was also prepared by the microbial route (Hanka et al., Antimicrob. Ag. Chemofcher. 619 and 625 (1966); US Pat. 3,816,619). Microbial preparation of 5-azacytidine is not advantageous as this nucleoside is not stable in aqueous solutions and consequently its isolation from a complex medium is very difficult and uneconomical. Moreover, the process is limited to this antibiotic and does not allow the preparation of other compounds of this type. Synthetic processes which allow to work under anhydrous conditions and therefore without substantial losses and are of a more general nature are more preferred. Different variants of two fundamentally different approaches are used in the synthesis of 5-azacytosine nucleiosides. The first is based on protected glycosyl isocyanates, which are converted to end products by a multi-step synthesis, and the second is based on the application of conventional direct glycosylation of silylated 5-azacytoins.

The shortest variant of the isocyanate method is described in the Czechoslovak patent No. 139,549. The essence of this process is the reaction of protected glycosyl isocyanates with acylguanidines and subsequent cyclization of the resulting protected N-acylamidino-N'-glycosylureas to protected 5-azacytosinium nucleosides. to free nucleosides. A critical point of this otherwise attractive synthesis is the cyclization of protected N-acylamidino-N'-glycosylureas, which in some cases proceed very slowly even at elevated temperatures and in the presence of desiccants, and the water resulting from cyclization additionally hydrolytically cleaves the non-cyclized acylamidineUrea into arnidinium urea salt acid. The formation of the bis-adduct in the reaction of the isocyanate with acylguanidine according to the cited patent also contributes to a lesser extent to the reduction of the overall yields.

For these reasons, low yields were achieved by applying this process.

Further study of this approach to the synthesis of 5-azacytosine nucleosides revealed that the cyclization of protected N-acylamidino-N‘-glycosylureas takes place virtually quantitatively and very rapidly by the action of a larger excess of strongly basic silylating agents in polar inert solvents.

Mixtures of haloalkylalkylsilanes and trialkylamines may also be used as silylating agents. The ratio of the two components must be chosen so that the trialkylamine predominates in the mixture. Preferably bis (trimethylsilyl) acetamide, bis (trimethylsilyl) trifluoroacetamide or a mixture of chlorotrimethylsilane and triethylamine (1: 1.1) were used. Hexamethyldisilazane, which is often used to silylate heterocyclic compounds, is virtually ineffective. The use of 6-equivalents of the silylating agent has been found to work well in ace3 tonitrile. Under these conditions, cyclization is complete to room temperature within a few minutes. In low-polar solvents, such as benzene, cyclization did not take place under otherwise identical conditions even within 24 hours. Finally, it has been found that undesirable bis-adduct formation in the reaction of isocyanate with acylguanidine can also be avoided by slowly dropping a solution of the isocyanate in an aprotic inert solvent to a vigorously stirred suspension of acylguanidine in the same solvent. Acetone has proven to be very useful as a solvent for this reaction.

Under these conditions, in most cases 80 to 90% of the total yields of the protected nucleosides were achieved. After substantially improving cyclization using silylating agents, this procedure represents the most effective variant of the isocyanate method for the preparation of 5-azacytosine nucleosides. Also, compared to direct glycosylation, the yields obtained are in most cases higher. Taking into account that there is no need to prepare silylated 5-azacytosines, this procedure is also shorter than direct glycosylation.

The present invention, i.e. a process for the preparation of the 5-azacytosine nucleosides of formula I, consists in providing a solution of the protected glycosyl isocyanates of formula II

Rt‘ — N = C — O (II), where Rt‘ is a protected glycosyl in an inert aprotic solvent, adds to the stirred mixture of the acylguanidine of formula III

R ³ R ⁴ \ Z

N

AND

R2-CO-NH-C = NH (III) wherein R ^2, R ³ and R ⁴ means the same as »in the formula I, in an inert aprotic solvent, and the obtained protected N-acylamidin» oN'-glykosylmočovlina of »Formula IV

R ³ R ⁴ >

\ Z N

R ² —CO — NH — C = N — CO — NH — R ⁴ '(IV) wherein

R ⁴ 'protected glycosyl and R ² , R ³ and R ⁴ denote the same as in formula I are subjected to cyclization in an inert aprotic solvent by an excess of bis (trialkylsilyl) amides wherein the alkyl has 1 to 6 carbon atoms or mixtures of halo-trialkylsilanes and trialkylamines wherein the alkyls of these components have 1 to 6 carbon atoms in which the trialkylamine predominates to form a compound of formula I wherein R ¹ is a protected glycosyl and R ² , R ³ and R ⁴ are as defined above, optionally converted in known manner to a free 5-azacytoisinium nucleoside of formula I wherein R ¹ is free glycosyl and R ² , R ³ and R ⁴ are as defined above.

The addition of the isocyanate II to the acylguanidine III is carried out in inert aprotic solvents such as acetone, benzene, 1,2-dichloroethane, chloroform, acetonitrile, dimethylformamide, dimethylsulfoxide and the like. The reaction proceeds very quickly already at room temperature and is completed in a few minutes. Best results were obtained using acetone as solvent. In principle, the reaction is carried out in equimolar ratios of arc components, however, for slightly soluble acylguanidines such as formyl or acetylguanidine, it is advisable to use a minor excess of acylguanidine and remove unreacted material by filtration after completion of the reaction. Most of the resulting protected N-acylamidino-N'-glycosylureas do not appear to tend to crystallize and are obtained in the form of thin foams which, after possible chromatographic purification on a silica gel column, are analytically pure and chromatographically pure. However, from the preparative point of view, the isolation of these intermediates is of no importance and therefore the cyclization is carried out without isolation of these compounds, which considerably shortens the process. The cyclization of the acylaminocurea is also carried out in an inert apriottic solvent, which may in each case be identical to the solvent used in the previous stage of the synthesis and thus can be carried out in a single flask. The cyclization proceeds very well in solvents with a high long-term stability such as acetonitrile. The surprisingly beneficial effect of the silylating agents is believed to be the activation of the acyl carbonyl of the acylamidine ureas of formula (III) by the formation of silyloxyalkyl derivatives which appear to be more readily cyclic than the acylamine diureas. Another favorable factor is that under silylating conditions the water (or trialkylsilane) formed is converted very quickly to an inert hexaalkyldisilyl oxane. Cyclization may further be facilitated by prior shaking of the linear intermediate at one of the nitrogen atoms. This possibility is particularly likely with the acylamidinoma Cocivin of formula III, wherein R ³ and R ⁴ are hydrogen. In such a case the obtained protected 5-azacytosine nucleoside as the N ⁴ -silylderivátů which are soluble in alcohol, which occurs relatively quickly sllylových groups and cleavage nesilylovaný resulting protected nucleoside ¹ is excreted generally in pure crystalline form. Very good results were obtained by using 6 equivalents of the silylating agent at 0 to 50 ° C, preferably at room temperature. Greater surplus no longer led to improved results. When using a smaller excess of the silylating agent, cyclization to the loop does not occur. Preferred silylating agents are flsi (trimethylsilyl) acetamide, bisftrimethylsilyltrifluoroacetamide or a 1: 1.1 mixture of chlorotrimethylsilane and triethylamine. Deprotection is carried out by conventional methods. For the preparation of 5-azacytosime nucleosides, it is preferable to use acyl groups to protect the hydroxyls since these can be removed by alcoholysis, preferably methanolysis, without substantially decomposing the triazine moiety. Amonolysis is less desirable because in some cases the triazine ¹ nucleus is partially opened. The benzyl protecting groups are considerably less suitable, as the triazine moiety of the mucosal leukes is also strongly attacked in their removal.

In the following, the invention is illustrated in more detail by way of example without being limited thereto.

Example 1

Preparation of 2 ‘, 3‘, 5‘-tri-O-benzoyl-5-azacytidine.

A solution of crude 2,3,5-tri-O-benzoyl- [5-D-ribosylisiophthyanoate (prepared by known method from 1.01 g of 10-acetyl-2,3,5-tri-O-benzoyl- [N -dibro] -islo) in 5 ml of dry acetone, 0.174 g of formylguaniidine in 10 ml of dry acetone is added dropwise over 30 minutes to a vigorously magnetically stirred mixture, leaving no external substance at room temperature. The mixture was stirred at room temperature for 30 minutes and a small portion of the undissolved formylguanidine was filtered off. The filtrate was evaporated in vacuo at 40 ° C (bath temperature) and the residue was dissolved in 20 mL dry acetonitrile. 1.5 ml of chlorotrimethylsilane and 1.5 ml of triethylamine are then added successively to the solution under external cooling with ice water. The mixture is left at room temperature for 1 h in the absence of external moisture. It is then diluted with 50 ml of benzene, a soluble triethylammonium chloride which is richly precipitated from the mixture immediately after the addition of triethylamine, is suctioned off, washed thoroughly with benzene and the filtrate is evaporated in vacuo. The residue was dried for about an hour under vacuum at a government pump at room temperature. The residue was dissolved in 5 ml of ethanol and the solution was left to crystallize overnight. 0.9 g (81%) of 2 ‘, 3‘, 5‘-tri-O-benzoly-5-azacytidine is obtained; mp 182-184 ° C (dec.).

Example 1 a d 2

The crude intermediate prepared according to Example 1 by addition of isocyanate to formylguanidine was dissolved in 20 ml of acetonitrile and 3 ml of bis (trimethylsilyl) 221103 acetamide was added to the solution. The mixture was kept in a sealed flask at room temperature for 1 hour and then evaporated in vacuo at 40 ° C (bath temperature), dried in a water pump vacuum at room temperature, dissolved in 5 ml of ethanol and allowed to crystallize overnight. 1.0 g (90%) of 2 2, 3‘, 5‘-tri-O-benzoyl-5-azacytidine is obtained; mp 182-184 ° C (dec.). The same yield was obtained in use

3.5 ml of bis (trimethylsilyl) trifluoroacetamide under otherwise identical conditions.

Example 3

Preparation of 2 ‘, 3‘, 5‘-tri-O-benaoyl-6-methyl-5-azacytidine

Reaction of crude 2,3,5-tri-O-benzoyl-5S-D-ribosyl isocyanate with 0.2 g of acetylguanidine affords 0.91 g (80%) of 2 ', 3', 5-trl- O-benzoyl-6-methyl-5-azacytidine; mp 234-237 ° C (dec.).

Example 4

Preparation of 3 ‘, 5‘-di-O-p-toluoyl-2‘-deoxy-5-azacytidine and its α-D-anomer

Reaction of a 2-deoxy-3,5-di-O-toluioyl-D-erythro-pentosylisocyanate anomeric mixture (prepared from 1.94 g of the corresponding halosylate with 1guanidine form (0.435 g) gave 2.14 g according to Example 2) i.e. 92% of a mixture of 3 ', 5'-di-O-p-toluoyl-2'-deoxy-5-az, acytidine and its αD-yes-mer.

Example 5

Preparation of 2 ‘, 3‘, 5‘-trl-O-benzoyl-6-phenyl-5-azacytidine

Reaction of 1 g of crude 2,3,5-tri-O-benzoyl-S-D-ribosylisocyanate with 0.326 g of benzoylguanidine in 10 ml of acetone followed by cyclization in 15 ml of acetonitrile with 3.5 ml of Ν, Ο-bis (trimethylsilyl) trifluoroacetamide at room temperature for 24 h, 1.05 g (80%) of 2 ', 3', 5'-trl-O-benzoyl-6-phenyl-5-azacytidine, m.p. 116-119 ° C, was obtained by crystallization from 10 ml of ethanol. C.

Example 6

Preparation of 2 ‘, 3‘, 5‘-tri-O-benzoyl-6-benzyl-5-azacytidine

Reaction of 1 g of crude 2,3,5-tri-O-benzloyl / SD-ribiosylisocyanate with 0.345 g of phenylacetylguaniline in 10 ml of acetone followed by cyclization in 10 ml of acetonitrile with a mixture of 1.6 ml of chlorotrimethylsilane and 1.6 ml of triethylamine at temperature room for 45 minutes was obtained by crystallization from 4 ml of ethanol 1.06 g (80%) of 2 ', 3', 5'-tri-O-benzoyl-6-benzyl-5-azacytidine, mp 218-222 ° C ( decomposition).

Example 7

Preparation of 2 ‘, 3‘, 5‘-tri-O-benzoyl-6-chloromethyl-5-azacytidine

Reaction of 1 g of crude 2,3,5-trl-O-benzoyl-N-D-rhosylisocyanate with 0.271 g of chloroacetylguanidine in 10 ml of acetone followed by cyclization in 10 ml of acetonitrile with a mixture of 1.6 ml of chlorotrimethylsilane and 1.6 ml triethylamine for 40 minutes at room temperature, 1.01 g (84%) of 2 ', 3', 5'-tri-O-benzioyl-6-chloromethyl-5-azacytidine, mp 206-210 °, were obtained according to the procedure of Example 1 C (decomposition) (ethanol) Recrystallization from methanol-1,2-dichloroethane gave the melting point of 218-220 ° C (decomposition).

Example 8

Preparation of 2 ', 3', 5'-tri-O-benzoyl-N ⁴ , N ⁴ -dimethyl-5-azacytidine

Reaction of 1 g of crude 2,3,5-tri-O-benzoyl- (3-D-ribioeylisocyanate) with crude N-formyl-N ', N'-diinethylguainidine (prepared in a conventional manner from 2 mmol of dimethylguanidine and 2 mmol of ethyl formate). in 10 ml of acetone followed by cyclization in 10 ml of acetonitrile with 3.0 ml of N, O-bis (trimethylsilyl jacetamide) after chromatography on silica gel and crystallization from ethanol, 0.21 g (18%) of 2 ', 3', 5'-tri-O-benzo-yl-N ⁴ , N ⁴ -dimethyl-5-azacytidine, mp 218-220 ° C (dec.).

Example 9

Preparation of 6-methyl-3 ^', 5'di-Op-toluoyl-2'-deoxy-5-azacytidine and its D-anomer

Reaction of 2 g of crude 3,5-di-Op-toluoyl-2-deoxy-D-erythro-pentosyl isocyanate with 0.5 g of acetylguanidine gives 2.1 g (88%) of the crude mixture of 2'-deoxy- 6-methyl-3 ', 5'-di-O-p-toluoyl-5-azacytidine and its α-D-anomer. Chromatography on a silica gel column with chloroform / mathainol gives the pure β-anomer, m.p. 199 DEG-201 DEG C. (decomposition) (acetonitrile) and pure α-anomer, m.p. 228 DEG-231 DEG C. (decomposition) (1,2-dichloroethane-methanol);

Example 10

Preparation of 6-methyl-5-azacytidine

The mixture, 0.571 g of 2 ', 3', 5'-tri-O-benzoyl-6-methyl-5-azacytidine, 5 ml of methanol and 0.2 ml of a 1M-Na-OCH 3 solution in methanol was magnetically stirred in a sealed flask over - night at room temperature. After acidification with glacial acetic acid, the product is filtered off with suction and washed with methanol. 0.240 g (93%) of 6-methyl-5-azacytidine, m.p. 142 DEG-147 DEG C. (decomposition), was obtained.

Example 11

Preparation of 2‘-deoxy-6-methyl-5-azacytidine

Following the procedure of Example 10, 0.21 g (87%) of 2'-deoxy- is obtained from 0.478 g of 2'-deoxy-6-methyl-3 ', 5'-dl-O-p-toluoyl-5-azacyclidine. 6-methyl-5-azacytidine, mp 190-195 ° C (dec.).

Example 12

Preparation of 6-benzyl-5-azacytidine

Following the procedure of Example 10, from 0.647 g of 2 ', 3', 5'-trl-O-thiobenzyl-6-benzyl-5-azacytidine, 0.25 g (75%) of 6-benzyl- was obtained by crystallization from ethanol. 5-azacytidine; mp 206-208 ° C (dec.).

Example 13

Preparation of 6-phenyl-5-azacytidine

Following the procedure of Example 10, 0.63 g of 2 ', 3', 5'-trl-O-benzloyl-6-phenyl-5-azacytidine was obtained after decathionization using a weakly acidic ion exchanger and crystallization from isopropyl alcohol 0.23 g (71%). phenyl-5-azacytidine, mp 200-201 ° C (example).

Example 14

Preparation of 1- (2-deoxy-α-D-erythro-pentofuranosyl) -6-methyl-5-azacytosine

Following the procedure of Example 10, 0.17 g (70%) was obtained from 0.478 g of 1- (3,5-di-O-p-toluolyl-2-deoxy-α-D-erythro-pentofuranosyl) -6-methyl-5-azacytosine. 1- (2-Deoxy-α-D-erythropenta-furanosyl) -6-methyl-5-azacytoline, mp 182-185 ° C (dec.) (methanol).

Claims (4)

SUBJECT OF THE INVENTION 1. A process for the preparation of 5-azacytosine nucleiosides of the formula I in an inert aprotic solvent and the obtained protected N-acylamidino-N‘-glycosylurea of the formula IV R ³ R ⁴ ( ¹ ) wherein R ¹ denotes free or protected glycosyl, R ² hydrogen, alkyl of 1 to 6 carbon atoms, haloalkyl of 1 to 6 carbon atoms, aralkyl of 7 to 12 carbon atoms, phenyl, R ³ and R ^{3 The} same or different denotes hydrogen, C 1 -C 6 alkyl or C 7 -C 12 aralkyl, characterized in that the solution of protected glycosyl isocyanate of the general formula II Ri'_N = C = O (Π), where R ⁴ 'is a protected glycosyl in an inert aprotic solvent, adds to the stirred mixture of the acylguanidine of general formula III R ³ R ^{4 -} R ² - co - NH - C = NH (III), where R ² , R ³ and R ⁴ denote the same as in formula I, N R ² —CO — NH — C = N — CO — NH — R ⁴ '(IV), wherein R ¹ * is a protected glycosyl and R ² , R ³ and R ⁴ denote the same as in Formula I, undergo cyclization in an Inert an aprotic solvent by treating an excess of bis (trialkylslilyl) amides wherein the alkyl has 1-6 carbon atoms, or a mixture of haloalkylalkylsilane and a trialkylamine wherein the alkyls of these components have 1-6 carbon atoms in which trlalkylamine predominates to form a compound of formula I ¹ is a protected glycosyl and R ² , R ³ and R ⁴ are as defined above, optionally converted in a known manner to a fused 5-azacytosine nucleoside of formula I, wherein R ¹ is free glycosyl and R ² , R ³ and R ⁴ have the above meaning. 2. A process according to claim 1, wherein acetone is used as an inert solvent for the addition of protected glycosyl isocyanate with acylguanidines. 3. A process as claimed in claim 1, wherein the cyclization is carried out in acetonitrile by treatment with 6 equivalents of the silylating agent at temperatures of 0 to 50 ° C. 4. A process according to claim 1 wherein the silylating agent is bis (trimethylsilyl) acetamide bis (trimethylailyl) trifluoroacetamide or a 1: 1 mixture of chlorotrimethylsilane and triethylamine: