Partial acylation of cytidine and its 2'-C-methyl analogue as a tool to functionalize the ribonucleosidic 2’,3’-cis -diol system

Precisely controlled conditions of acylation and 2’,3’-O-isomerization allowed to synthesize tripivaloyl derivatives of cytidine with free 2’-or 3’-hydroxyl group in a simple manner. Acylation of 2’-C-β -methylcytidine proceeded in a different way and resulted in the formation of tripivaloyl 2’-hydroxy nucleoside, or dipivaloyl 2’,3’-dihydroxy compound. All these products may be applied as key intermediates in the regioselective modification of 2’,3’-cis -diol system.


Introduction
Hepatitis C virus (HCV) is responsible for serious blood-borne infections, chronic liver diseases and, in many cases, for hepatocellular carcinoma.Since there is no established vaccine for HCV, many research laboratories have developed new therapeutic agents of anti-HCV activity.In this respect, the nucleoside analogs of the structure of 2'-C-alkyl ribofuranosides 1,2 seem to be promising candidate compounds.One of the most in vitro active compounds of this type is an analog of naturally occurring cytidine (1), 2'-C-β-methylcytidine (2).As it has been shown recently, the ribonucleoside 2 is a potent and selective inhibitor of Flaviviridae in cell culture. 3ecause there is an urgent need for new therapeutic compounds of this type, the structure of 2'-C-β-methylcytidine has been modified in different ways, and some new nucleoside analogs have been obtained, e.g.3'-O-valinyl ester of 2'-C-β-methylcytidine, 3 2'-deoxy- 1,4 and 3'-deoxyribosides, 5 2'-C-β-methyl-1-(β-D-xylofuranosyl)-cytosine, 5 2'-fluoro 6 and 3'-fluoro 5 derivatives, and 3'-methoxynucleoside 5 .All these compounds derived from 2'-C-βmethylcytidine (2) are substituted or modified at either 2'-or 3'-hydroxyl group.Furthermore, as one may conclude from the cited literature, the most difficult synthetic steps have been related to the appropriate protection of 2',3'-cis-diol system prior to the final modification.
Preparation of the 3'-hydroxyl synthons is more difficult and requires an appropriate protection of 2'-and 5'-hydroxyl functions.The 2',5'-bis-substituted key intermediates can be obtained in several steps from unprotected nucleosides, either via partial hydrolysis of 2',3'orthoesters and separation of the resulting 2'-O-and 3'-O-acyl derivatives, 8 or with the use of Markiewicz's reagent, followed by the 2'-O-protection, removal of the 3',5'-O-cyclic protection with fluorides, and subsequent 5'-O-protection with trityl group.Both procedures are tedious and time-consuming.Moreover, a suitable protection of exoamine groups is required for heterocyclic bases other than uracil.
However, synthesis of the 2',5'-bis-protected ribonucleosides may be accomplished in just one step by partial acylation of ribonucleosides. 9,10The method was developed in 1980 th , but it has not been used very often since that time.The procedure involves reaction of unprotected ribonucleosides with a precisely controlled amount of acid chlorides in pyridine.The observed regioselectivity of acylation can be attributed to the "unusual acidity of the 2'-hydroxyl group". 9herefore, the 2'-OH group undergoes acylation faster than 3'-hydroxyl.Here we describe how the latter method can be applied for partial protection of 2'-C-β-methylcytidine (2).

Results and Discussion
Prior to the study on partial acylation of 2'-C-β-methyl analog 2, we performed introductory experiments applying unmodified cytidine (1) as a model compound (Scheme 1).We noticed that the best regioselectivity of protection was obtained in the pivaloyl series, and this was in line with the previous report. 9In this manner, the reaction of 1 with 3.5 equivalents of pivaloyl chloride in pyridine gave O 2' ,O 5' ,N 4 -tripivaloylcytidine (3) as the main product, along with O 2' ,O 3' ,O 5' ,N 4 -tetrapivaloylcytidine (4) as a minor product.The reaction proceeded with high selectivity in a temperature range varying from -5 -20 o C. The tripivaloyl cytidine derivative 3 is not a new compound.In fact, O 2' ,O 5' ,N 4 -tripivaloylcytidine (3) was obtained as reported, and used without isolation in a subsequent one-pot reaction. 11In the present work, however, we have succeeded in crystallization of the product 3 from toluene.In this way, the 3'-OH key intermediate 3 can be now obtained as a crystalline material in a one-step procedure from cytidine (79% yield).The tripivaloyl cytidine derivative 3 can be a useful substrate in the 3'modification reactions.The product 3, however, could not be purified by chromatographic means.Although chromatography on silica gel in chloroform -methanol allowed to obtain the tetrasubstituted compound 4 for structure determination, the main product 3 underwent 2',3'-isomerization to form a mixture of 3 and O 3' ,O 5' ,N 4 -tripivaloyl derivative 5. Similar isomerization reactions of partially acylated ribonucleosides during chromatography were reported previously, 9 but in the present work we managed to isolate the pure 2'-OH isomer 5.When O 2' ,O 5' ,N 4 -tripivaloylcytidine (3) was stirred in a suspension of silica gel in methanol-containing solvents (e.g.chloroform -methanol 4:1, v/v), we observed the preferred formation of its isomer 5 (ratio 5/3 ca.5:1; as proofed by 1 H NMR). The main product of isomerization, O 3' ,O 5' ,N 4 -tripivaloyl derivative 5, was more stable than 3 and could be purified by chromatography in aprotic solvent systems.In this manner, we managed to obtain the second isomer of tripivaloyl cytidine (5).This compound can be applied as a 2'-OH component for 2'-modification reactions; however, its synthesis is slightly more laborious than the 3'-OH compound 3, and we have not succeeded in crystallization of the isomer 5.
In the light of presented experiments in the unmodified cytidine series, we could anticipate a similar reactivity of its close analog, 2'-C-β-methylcytidine (2).However, to our surprise, the treatment of 2 with pivaloyl chloride (3.5 eqs.) in pyridine gave O 3' ,O 5' ,N 4 -tripivaloyl-2'-C-βmethylcytidine (6) in quantitative yield, instead of the expected O 2' ,O 5' ,N 4 -tri-substituted product (Scheme 2).This shows that the tertiary 2'-hydroxyl group is very inert in acylation reactions and therefore, in the presence of 2'-C-β-methyl, the third pivaloyl group is being attached in the 3'-position.Unlike in the case of cytidine, any tetrapivaloyl compound has not been formed, despite of an excess of the acylating reagent.In addition, O 3' ,O 5' ,N 4 -tripivaloyl-2'-C-βmethylcytidine (6) does not undergo 2',3'-O-isomerization and therefore, it may be purified by using chromatographic methods.These experimental facts further emphasize the very low reactivity of 2'-hydroxyl group in 2'-C-β-methylcytidine.According to our literature search, this is the first report on the low reactivity of the tertiary 2'-hydroxyl in the series of 2'-C-βalkylnucleosides.3][14] In conclusion, compound 6 does not seem to be a promising substrate for 2'-modification.To solve the problem of regioselective modification of the 2',3'-diol system in 2'-C-βmethyl-D-ribonucleosides, we decided to take advantage of this low reactivity of 2'-hydroxyl group, assuming that this position could remain unprotected during 3'-modification.To test this idea, O 5' ,N 4 -dipivaloyl-2'-C-β-methylcytidine (7), i.e. a compound in which the 2',3'-diol system was not protected, was synthesized in the reaction of 2 with 2.5 molar eqs. of pivaloyl chloride in pyridine at room temperature.The reaction gave a mixture of O 5' ,N 4 -dipivaloyl-2'-C-βmethylcytidine (7; 70% yield after silica gel column separation), and O 3' ,O 5' ,N 4 -tripivaloyl-2'-Cβ-methylcytidine (6) as a minor product.In our preliminary experiments of modifications, the dipivaloyl ribonucleoside 7 could be selectively substituted in the intermolecular reactions with other acylating reagents (e.g.reaction with mesyl chloride), but in some cases it underwent intramolecular reactions (e.g. the formation of 2',3'-isopropylidene derivative). 15n conclusion, the reaction of partial acylation of ribonucleosides offers a simple and inexpensive route towards new nucleoside analogs.We have developed a very simple laboratory method towards 2'(3')-monohydroxyl cytidine derivatives (5 and 3, respectively), which may be useful in 2'-or 3'-modification approaches.We have shown for the first time that partial acylation in the 2'-C-β-methyl ribofuranosyl proceeds quite differently than in unmodified ribofuranosyl series, and the products (6 and especially 7) can be useful synthons for further chemical modifications in order to obtain new biologically active compounds.