The present invention is the method for incorporation of acetal and acetal ester groups for protection of hydroxyl function. The method is applied in particular in the processes of RNA synthesis. The method can be employed in the synthesis of nucleosides with acetal and acetal ester groups for the protection of hydroxyl functions.

There are a number of reactions in organic chemistry which involve compounds containing hydroxyl or amine groups, in which it is desirable to block temporarily the reactivity of these groups and, for this reason, techniques for temporary blocking of these functions are employed.

To this aim, protecting groups are used. The incorporation of protecting groups must be selective and efficient. The linkage between a protecting group and a blocked functional group must be stable in the conditions of successivereactions, and the unblocking must be selective and efficient, e.g. as a result of applying easily accessible and non-toxic reagents, and/or as a result of using physical factors, e.g. heating, irradiation.

The chemical synthesis of RNA consists of the reaction of condensation of nucleotide units. An activated nucleotide unit binds to the free 5 '-OH group of the growing RNA chain. In this manner, an internucleotide linkage is formed between position 3 ' of one nucleotide and position 5 ' of the other nucleotide. The remaining reactive centres of the nucleotide, not taking part in a given reaction, are temporarily blocked by protecting groups. An appropriate selection of protecting groups enables efficient formation of an internucleotide linkage. An appropriate selection of protecting groups, particularly for 2'-hydroxyl functions, and a method for their incorporation and removal, are the main problems associated with the synthesis of RNA chains.

As a rule , the procedure of functional group protection in the synthe sis of oligoribonucleotides proceeds according to the following scheme:

• blocking of the amine functions in pyrimidine and purine bases of the nucleoside,

• blocking of hydroxyl functions, whereby the blocking is generally conducted in the following sequence of steps:

o regioselective blocking of -OH groups in positions 3' and 5',

o blocking of the -OH group in position 2',

o unblocking of -OH groups in positions 3' and 5',

o re-blocking of the -OH group in position 5' - this stage of blocking is performed with 4,4'-dimethoxytntyl (DMTr).

The unit thus prepared is subjected to a reaction with a phosphitylation reagent in order to incorporate phosphite in position 3 ' . Blocking of amine functions of in pyrimidine and purine bases of nucleosides/nucleotides can be performed in optional order depending on a type of amine protection.

The protecting groupof the 2'-OH function must remain stable until the synthesis of the RNA chain is complete, and its unblocking - which constitutes the final stage of RNA synthesis - cannot adversely affect the synthesized RNA chain.

There are a number of ways known toprotecthydroxyl groups in positions 3' and 5', however theseprotecting groupsare useful at intermediate stages, not at the stage of synthesis of RNA chain fragments. A protectionof the hydroxyl function in position 2' is especially important for the process of RNA chain synthesis.

A protecting group in position 2' has a direct impact on the reactivity and efficiency of internucleotide linkage formation during the chemical synthesis of RNA fragments, and:

1. should represent the smallest possible steric hindrance during internucleotide linkage formation. The larger the protecting group in position 2', the lower the efficiency of internucleotide linkage formation. An important role is played by the order of the carbon atom bound to the oxygen atom in position 2' (1), since it has been proved that the efficiency of in internucleotide linkage formation decreases along with the increase in the order of the carbon atom in position a relative to the oxygen atom in position 2' (2).

2. must be stable in the conditions of RNA chain synthesis: premature unblocking leads to a partial or complete degradation of the RNA chain, and creates the risk of isomerization of internucleotide linkages 3'-5'→ 2'-5'.

3. its incorporation must be selective and efficient.

4. The removal of the blocking group from 2 ' should take place in approximately mildconditions, since basic conditions may induce hydrolysis of the internucleotide linkage, and acidic conditions - isomerization and hydrolysis of the internucleotide linkages.

An important factor in the processes of oligoribonucleotide synthesis is the stability of protection of hydroxyl and amine functions throughout the entire duration of the process of synthesis - from the stage of preparation of the nucleotide unit used in synthesis, through the successive stages of synthesis, isolation of the final product, and the processes of removal of protecting groups from the final product.

The protection of hydroxyl groups, particularly in position 2', is achieved with a range of protecting groups, the most common of which are ethers, silyl ethers, acetals and acetal esters.

1. Ether groups

Czernecki (3) applied a benzyl group to protect 2'-hydroxyl function. The group ensures adequate protection during oligoribonucleotide synthesis, however it is removed by direct hydrogenolysis which may be accompanied by partial hydrogenation of double bonds C5=C6in pyrimidine nucleotide bases.

Ohtsuka (4) used a photolabile 2-nitrobenzyl group removable by UV irradiation, which is why the synthesis of the nucleotide unit and the RNA chain must be conducted without the access of light, and furthermore the removal may induce reactions of photodimerization or photodestruction of nucleobases.

2. Protecting groups based on silyl ethers Ogilvie (5) applied a f-butyldimethylsilyl group for protection of 2'-hydroxyl function. The group is selectively removed with fluoride ions. The i-butyldimethylsilyl protecting group, however, presents a considerably large steric hindrance which has an adverse effect on the formation of the internucleotide linkage, reducing the efficiency of condensation of nucleotide units.

3. Acetal groups

Acetal protecting groups of hydroxyl functions have been known for a long time, however not all of them can be used for protecting the 2'-OH function in the chemical synthesis of RNA.

The acid-labile tetrahydropyran-l-yl (thp) and methoxytetrahydropyran-4-yl (mthp) groups have known applications as protecting groups of 2'-hydroxyl function.

The structure of the thp group has a centre of asymmetry at the acetal carbon atom. In natural ribonucleosides, which are pure enantiomers, the incorporation of thp group results in the formation of a mixture of diastereoisomers, which might cause necessity of otherwise difficult their separation operation. The mthp group has no centre of chirahty. Nevertheless, neither thp nor mthp groups can be employed in the chemical synthesis of RNA if the 5 '-hydroxyl function is blocked with the commonly used DMTr group.

Reese et al. (8) proposed the application of l-[(2-chloro-4-methyl)phenyl]-4- methoxypiperidin-4-yl (ctmp) group, stable in the conditions of DMTr group removal.

Beijer et al. (9) used a l-(2-fluorophenyl)-4-methoxypiperidin-4-yl (fpmp) group which is stable in the conditions of DMTr group removal. All the achiral acetal groups, mthp, ctmp and fpmp, introduce a large steric hindrance, and have a quaternary carbon atom in the a position relative to the 2 '-oxygen atom. Thus, this higher order has an adverse effect on the efficiency of formation of the internucleotide linkage (2).

4. Acetal or acetal ester derivatives of formaldehyde

A method for protecting the hydroxyl function with acetal or acetal ester derivatives of formaldehyde has been known. This protecting groups are sufficiently stable in the acidic environment, as opposed to other acetals. These formaldehyde derived acetals exhibit sufficient stability in acidic conditions which are used for DMTr group removal. The application of acetalderivatives of formaldehyde makes it possible to use various unblocking conditions depending on the characteristics of the formaldehyde derivative, e.g. their structure allows their removal in conditions other than acidic.

Acetal or acetal ester derivatives of formaldehyde do not introduce large steric hindrances since they contain a secondary carbon atom in the a-position relative to the 2' oxygen atom. On account of their mixed nature, the groups maintain stability during the chemical synthesis of RNA.

The patent US5986084 discloses the application of a triisopropylsilyloxymethyl (TOM) group in the synthesis of long RNA fragments. The method of incorporating the protecting group in the 2' position in the nucleotide comprises two stages. At the first stage, a substrate with free 3 '- and 2'-hydroxyl groups is subjected to a reaction with dibutyltin(IV) chloride, producing reactive cyclic 5 '-0-(4,4'-dimethoxytrityl)-2',3'-0-dibutylstannateribonucleo side which, at the second stage, is subjected to a reaction with triisopropylsilyloxmethyl chloride. The resulting product is a m i xture o f tw o n u c l e o s i d e i s o m e rs , n am e l y 5 '-0-(4,4'-dimethoxytrityl)-2'-0- (triisopropylsilyloxymethyl)nucleoside (5'-0-DMTr-2'-0-TOM-nucleoside) a n d 5 '-0-(4,4'- dimethoxytrityl)-3'-0-(triisopropyl silyloxymethyl)nucleo side (5 -0-DMTr-3'-0-TOM- nucleoside). Both isomers require chromatographic separation, as only 5 '-0-DMTr-2'-0-TOM- nucleoside is suitable for RNA synthesis. The yield of synthesis of isomer 5'-0-DMTr-2'-0-TOM- nucleoside is 40%-60%. The unblocking procedure requires the use of fluoride ionsforming appropriate salts and their removal is problematic and calls for an additional oligomer purification procedure.

Ohgi et al. (10) used a cyanoethoxymethyl (CEM) group for blocking the 2'-OH function in a ribonucleoside. The application of this group for 2'-hydroxyl protection turned out to be very efficient, as it allowed obtaining a 1 10-mer RNA chain for the first time

There are two known methods for incorporation of the CEM group. Oghi (9) described a method involving the reaction of blocked 5 '-0-(4,4'-dimethoxytrityl)nucleoside with dibutyltin(IV) chloride. The reaction gives rise to reactive cyclic 5'-0-(4,4'-dimethoxytrityl)-2',3'- O-dibutylstannate-ribonucleoside which, at the next stage, is subjected to a reaction with (2- cyanoethoxy)methyl chloride. The reaction between 2-cyanoethoxymethyl chloride and 5'-0-(4,4'- dimethoxytrityl)-2',3'-0-dibutyl-stannate-ribonucleoside leads to the formation of two isomers: 5'- 0-(4,4'-dimethoxytrityl)-2'-0-(2-cyanoethoxymethyl)nucle o side (5 '-0-DMTr-2'-0-CEM- nucleoside) and 5 '-0-(4,4'-dimethoxytrityl)-3'-0-(2-cyanoethoxymethyl)nucleos ide (5 '-ODMTr- 3'-0-CEM-nucleoside). Both isomers require chromatographic separation, as only 5'-0-DMTr-2'- O-CEM-nucleoside is suitable for RNA synthesis. The efficiency of obtaining isomer 5' -O-DMTr- 2'-0-CEM-nucleosideis 29-51%. Furthermore, (2-cyanoethoxy)methyl chloride used in the process is a cancerogenic compound.

Yoshinobu (11) disclosed a method for incorporation of a 2-cyanoethoxymethyl group protecting the 2'-hydroxyl function of nucleoside, based on the reaction of 3 '- and 5'-protected nucleoside with 2-cyanoethyl methylthiomethyl ether at a very low temperature, in the presence of N-iodosuccinimide and trifluoromethanesulphonic acid. The reactions are performed at a temperature of -45 C since higher temperatures may induce the alkylation of pyrimidine bases. The method requires the use of expensive reagents.

The US patent US8536318 discloses the use of a pivaloyloxymethyl group (PivOM) for the protection of hydroxyl function. The incorporation of pivaloyloxymethyl group into the hydroxyl function is a two-stage process. It consists of the reaction between a nucleoside protected in position 5'-0-(4,4'-dimethoxytrityl) group and free 3'- and 2'-hydroxyl groups with dibutyltin(IV) oxide, resulting in the formation of cyclic 2',3 '-dibutylstannate-ribonucleoside which, at the next stage, reacts with pivaloyloxymethyl chloride. The reaction produces two isomers: 5'-0-(4,4'- dimethoxytrityl)-2'-0-pivaloyloxymethylnucleoside (5'-0-DMTr-2'-0-PivOM-nucleoside) and 5'- 0-(4,4'-dimethoxytrityl)-2'-0-p ivaloylo xym ethyl nuc l eo s i de ( 5 '-0-DMTr-3'-0-PivOM- nucleoside). Both isomers require chromatographic separation, as only 5'-0-DMTr-2'-0-PivOM- nucleosideis suitable for RNA synthesis. The efficiency of obtaining isomer 5 -0-DMTr-2 -0- PivOM-nucleoside is 34-49%. The group is compatible with other protecting groups used in the chemical synthesis of oligoribonucleotides, however the disclosed method for its incorporation into the nucleoside is inefficient and involves multiple stages. Lackey (12) applied a levulinyloxymethyl (ALE) group for protecting the 2'-hydroxyl function during RNA synthesis. In the proposed method, the starting component is 5',3'-0- (tetraisopropyldisiloxane-l,3-diyl)nucleoside which, in a reaction with dimethyl sulphoxide, acetic acid and acetic anhydride, is converted into 5',3'-0-(tetraisopropyldisiloxane-l,3-diyl)-2'-0- (methylthiomethyl)nucleoside which, following isolation and purification, is subjected to a reaction with sulphuryl chloride, forming another intermediate product i.e. 5',3'-0- (tetraisopropyldisiloxane-l,3-diyl)-2'-0-(chloromethyl)nucle oside which, after solvent evaporation, is dissolved in dichloromethane and combined with the crown ether 15-crown-5 (15- C-5) and sodium levulinate (NaLv). The product of the reaction is 5',3'-0- (tetraisopropyldisiloxane-l,3-diyl)-2'-0-levulinyloxymethyl nucleoside.

The method comprises several stages and is time consuming, with an overall duration exceeding 30 hours. Furthermore, column chromatography and expensive crown ethers markedly increase the costs of synthesis.

Known methods for incorporation of acetal or acetal ester groups to protect the 2'-hydroxyl function of nucleosides are inefficient, expensive, and involve multiple stages, which is why they markedly extend the duration and increase the costs of synthesis, and in many cases require the application of harmful substrates such as 2-cyanoethoxymethyl chloride which is a cancerogenic compound.

The purpose of the invention was development of a simple and effective method for incorporation of acetal and acetal ester groups protecting the hydroxyl function in order to use in particular thus blocked compounds in further chemical reactions and to protect the hydroxyl function of the 2' group in nucleosides.

It was unexpectedly proved that it was possible to conduct a reaction between a hydroxyl group and compounds such as a thioacetal derivative of alcohol or a thioacetal ester derivative of acid in the presence of tin(IV) chloride (SnCl ₄ ).

The present invention is a method for incorporation of an acetal or acetal ester group to protect hydroxyl function. The method according to the invention consists of the reaction of an organic compound containing at least one hydroxyl group, soluble in an aprotic solvent, with a compound of the general formula 1, Ri-S-CH ₂ -0-R ₂ (1) where

- Ri represents a Ci_ ₆ alkyl; unsubstituted or substituted benzyl or naphthyl, with substituents including a Ci_ ₇ alkyl, halogen, aminoacyl; in particular Ri represents -CH ₃ -Ph, -Ph(4-Cl), - Ph(4-CH ₃ ), -CH ₂ Ph

- R ₂ represents

• a Ci_i ₅ alkyl;

• alkyl-aryl, in which the alkyl chain contains Ci_ ₅ , whereas aryl contains from 1 to 8 unsubstituted or substituted rings, with substituents including a Ci_ ₇ alkyl, halogen, aminoacyl, tertiary amine group, cyano group;

· a group of the general formula 2

where R ₃ represents:

o a Ci-15 alkyl;

o a ketone group;

o unsubstituted or substituted phenyl, with substituents including a Ci_ ₇ alkyl, halogen, aminoacyl, tertiary amine group, cyano group.

• a group of the general formula 3

R ₄

Si R ₅

Re

(3)

where R4, R ₅ and R5 are different or the same, and represent a Ci_ ₂ 8 alkyl or aryl containing from 1 to 8 rings or trimethylsilyl, where the total number of carbon atoms in the group of formula 3 is no less than 6 and no more than 30,

the presence of SnCl ₄ , in an aprotic solvent. The method according to the invention can be applied for compounds containing at least one hydroxyl group, and the compounds must be soluble in aprotic solvents.

Due to the properties of SnCl ₄ , the method is only suitable for modifying organic compounds that are soluble in aprotic solvents. The method can also be applied for compounds that are insoluble in aprotic solvents, on condition that they are first converted into a form which is soluble in these solvents. For example, ribose is insoluble in aprotic solvents, however incorporation of a protecting group, e.g. a silyltert-butyldimethylsilylgroup, into one or two hydroxyl groups will permit ribose dissolution in aprotic solvents.

The solvents that can be used include halogen derivatives of alkanes, particularly carbon tetrachloride, chloroform, dichloromethane or 1,2-dichloroethane; aromatic solvents, particularly benzene, toluene; cyclic ethers, particularly tetrahydrofuran; nitrile compounds, particularly acetonitrile, or a mixture of these solvents. It is particularly advantageous to use 1,2- dichloroethane.

The reaction is conducted in anhydrous environments, whereby it is possible for the reaction to be conducted in an environment containing trace amounts of water, however if this is the case, an appropriate excess of SnCl ₄ must be ensured because a part of it is bound by water.

In the method according to the invention, the organic compound containing at least one hydroxyl group and an appropriate compound of the general formula 1 is dissolved in a solvent, and then SnCl ₄ is added to the reaction mixture. It is advantageous to introduce SnCl ₄ in the form of a solution in the same solvent as that is the reaction medium or in 1,2-dichloroethane.

SnCl ₄ is used in an amount not smaller than 0.01 mole per one mole of hydroxyl groups which are intended to be exchanged. It is advantageous to use SnCl in an amount from 1 to 6 moles per one mole of hydroxyl groups which are intended to be exchanged most advantageously in an amount from 2.5 to 4.5 moles.

The ratio of the compound of formula 1 to hydroxyl groups can be 1 : 1, however it is advantageous for the compound of formula 1 to be used in excess ranging from 1 to 8. An excess of the compound of formula 1 makes it possible to achieve the highest possible process efficiency within the shortest time. The reaction can be conducted over a broad temperature range, from very low temperatures up to temperatures not exceeding the boiling temperature of the reaction mixture. The reaction is conducted in low temperatures, however not lower than the solidification point of the solvent used. The higher the temperature, the lower the efficiency of obtaining the target product and the greater the quantity of by-products. It is beneficial to conduct the reaction in temperatures from the range from -80 C to 0 C, and most advantageously - below 15 C. Due to that fact, it is advantageous to use solvents with low solidification points.

The reaction of hydroxyl group blocking involves preparation of a solution of a compound containing a hydroxyl group, and the compound of formula 1, followed by cooling of the mixture to a low temperature. After the cooling procedure, SnCl ₄ is introduced, whereupon the reaction is advantageously conducted at a low temperature until the completion of the process. In order to determine the optimum duration of the process, it is advantageous to monitor the course of the blocking reaction by thin-layer chromatography on silica gel plates, or by high-performance liquid chromatography (HPLC). The duration of the reaction depends on the types of substrates and temperature, and generally ranges from 4 to 16 hours.

On completion of the reaction, the product is isolated and purified using known methods. Prior to product isolation it is advantageous to neutralize SnCl ₄ with a neutralizing agent in an amount of at least 4 molar equivalents of SnCl ₄ used. Depending on product stability, neutralization can be performed with:

1. solutions of bases, solutions of hydrogen carbonate salts, if the product is stable in basic conditions, it is advantageous to use sodium or potassium hydrogen carbonate;

2. buffers with pH of 7 (± 0.7) .

Following neutralization, it is advantageous to filter the mixture in order to separate solid products, whereupon the product is isolated from the post-reaction mixture through extraction with an aprotic organic solvent and then purified by known methods.

A product of hydroxyl function protectionwith an acetal or acetal ester protecting group is an original compound in which the site of the hydroxyl group is occupied by the group of formula 4

where R ₂ has the meaning defined above.

In the second aspect, the present invention relates to a method for protecting the hydroxyl function, particularly in position 2' in nucleoside derivatives, consisting of the incorporation of an acetal or acetal ester group. The method according to the invention consists of the reaction between the compound of the general formula 5

where

B represents radicals of nucleobases, particularly uracil, or appropriately protected residuesof adenine, guanine, cytosine, uracil and thymine,

Yi and Y ₂ are the same or different, and represent groups protecting hydroxyl functions in positions 3' and 5 '; in particular, they are silyl groups: triethylsilyl, tert-butyl-dimethyl- silyl, isopropyl-dimethyl-silyl, tert-butyl-diphenyl-silyl, methyl-diisopropyl-silyl, triphenylsilyl, triisopropylsilyl, methyl-di-tert-butylsilyl

or 6

where B has the meaning defined above, and A represents group of formulas 7, 8, 9, 10 and 1 1

and the compound of the general formula 1 (hereinafter referred to as the blocking compound),

R S-CH ₂ -0-R ₂ (i)

where

- i represents a C _w alkyl; unsubstituted or substituted benzyl or naphthyl, with substituents including a Ci_ ₇ alkyl, halogen, aminoacyl; in particular Ri represents -CH _3> -Ph,

-Ph(4-Cl), -Ph(4-CH ₃ ), -CH ₂ Ph

- R ₂ represents

· a Ci_i ₅ alkyl;

• alkyl-aryl in which the alkyl chain contains Ci_ ₅ , and aryl contains from 1 to 8 unsubstituted or substituted rings, with substituents including a Ci_ ₇ alkyl, halogen, aminoacyl, tertiary amine group, cyano group;

• group of the general formula 2

where:

R ₃ represents

o a Ci-15 alkyl;

o ketone group o unsubstituted or substituted phenyl, with substituents including a Ci_ ₇ alkyl, halogen, aminoacyl, tertiary amine group, cyano group,

• group of the general formula 3

where R4, R ₅ and R _< ; are different or the same, and represent a Ci_ ₂ 8 alkyl or aryl containing from 1 to 8 rings or trimethylsilyl, with the total number of carbon atoms in that group is no less than 6 and no more than 30,

in the presence of (SnCl ₄ ), in an aprotic solvent.

According to the invention, the method for the protection of the hydroxyl function in nucleosides consists in particular of incorporation of groups of formulas 12 and 13 in position 2'

The solvents that can be used include halogen derivatives of alkanes, in particular carbon tetrachloride, chloroform, dichloromethane or 1,2-dichloroethane; aromatic solvents: particularly benzene, toluene; cyclic ethers, particularly tetrahydrofuran; nitrile compounds, particularly acetonitrile, or a mixture of these solvents. It is particularly advantageous to use 1,2- dichloroethane.

The reaction is conducted in anhydrous media but it is possible for the reaction to be conducted in an environment containing trace amounts of water, however if this is the case, an appropriate excess of SnCl ₄ must be ensured because a part of it is bound by water.

In the method according to the invention, the compound of the general formula 5 or 6, in which substituents have the meaning defined above, and an appropriate blocking compound are dissolved in a solvent, and then SnCl ₄ is added to the reaction mixture. It is advantageous to introduce SnCl ₄ in the form of a solution in the same solvent as that is the reaction medium or in 1 ,2-dichloroethane. SnCl ₄ is used in an amount not smaller than 0.01 mole per one mole of hydroxyl groups which are intended to be protected. It is advantageous to use SnC in an amount from 1 to 6 moles per one mole of hydroxyl groups which are intended to be protected, most advantageously in an amount from 2.5 to 4.5 moles.

The ratio of the compound of formula 1 to hydroxyl groups can be 1 : 1, however it is advantageous for the compound to be used in excess ranging from 1 to 8. An excess of the compound of the formula 1 makes it possible to achieve the highest possible process efficiency withm the shortest time.

The reaction can be conducted over a broad temperature range, from very low temperatures up to temperatures not exceeding the boiling temperature of the reaction mixture, however the temperature of the reaction must not be higher than the temperature of de-protecting the hydroxyl function in positions 3 ' and 5 '. It is advantageous to conduct the reaction in low temperatures, however not lower than the solidification point of the solvent used. The higher the temperature, the lower the efficiency of the process and the greater the quantity of by-products. It is advantageous to conduct the reaction in the temperature range from -80 C to 0 C, and most advantageous - below 15 C. Due to that fact, it is advantageous to use solvents with low solidification points.

The reaction of hydroxyl group protection involves preparation of a solution of a compound containing a hydroxyl group which is to be protected, and a blocking compound, followed by the cooling of the mixture to a low temperature. After the cooling procedure, SnCl ₄ is introduced, and then the reaction is advantageously conducted at a low temperature until the completion of the process. In order to determine the optimum duration of the process, it is advantageous to monitor the course of the reaction by thin-layer chromatography on silica gel plates, or by high- performance liquid chromatography (HPLC). The duration of the reaction depends on the types of substrates and temperature, and generally ranges from 4 to 16 hours.

On completion of the reaction, the product is isolated and purified using known methods, following the same procedure as in the method according to the first aspect of the invention.

The product of the reaction is the compound of the general formula 14,

where B, R ₂ , Yi and Y ₂ have the meanings defmed above,

or of the formula 15

where A, B and R ₂ have the meanings defmed above.

The protecting groups used in the method according to the invention are unblocked in different conditions, on a case-by-case basis, depending on the structure of the group.

A characteristic property of acetal and acetal ester groups protecting the hydroxyl function is high flexibility in unblocking methods. In principle, all known chemical unblocking methods can be employed, however due to the specific properties of acetal and acetal ester groups stemming from the nature of these compounds, it is possible to select appropriate conditions which are advantageous for a particular application of the compounds protected with these groups. The selection of the most advantageous method of unblocking the hydroxyl function protected with the method according to the invention depends not only on the chemical nature of the blocking group but also on the specific properties of the blockage used.

The removal of acetal and acetal ester groups can be performed with solutions of inorganic bases, e.g. NaOH, KOH, and organic bases, e.g. amines in organic or inorganic solvents, also in their mixtures. It is advantageous to use weak bases (e.g. aqueous ammonia solution, methanol/ammonia solution, ethanol/ammonia solution, methylamine in methanol, n-butylamine in methanol), which is why during the unblocking there is practically no hydrolysis of mtemucleotide linkages. Acetal and acetal ester protecting groups can also be unblocked in acidic conditions, and they are stable in conditions required for the removal of the acid-labile dimethoxytrityl group (DMTr) from the 5 '-hydroxyl position. The groups are stable, which is their advantage, towards weak acid solutions used for the unblocking of the 5 '-hydroxyl group blocked with the acid-labile dimethoxytrityl group (DMTr). In these conditions, the acetal or acetal ester group protecting the 2'-hydroxyl function is stable, which is a significant factor for the chemical synthesis of the RNA chain.

Acetal and acetal ester protecting groups can also be removed in reactions that are specific to a particular blocking group, e.g. in a reaction between the carbonyl group of the ieto-ketoester radical (e.g. levulinyl, Lv, H ₃ CC(0)CH ₂ CH ₂ C(0)-,) and the unblocking reagent i.e. hydrazine solution.

In the third aspect, the subjects of the present invention are new monothioacetals of the general formula 1,

R S-CH ₂ -0-R ₂ (i)

where

• i represents (4-chloro)phenyl

· R ₂ represents o-toluyl, benzoyl, pivaloyl.

New monothioacetals are obtained in a reaction between an appropriate chloride of the general formula 16

where R ₂ has the meanings defined above

and an appropriate derivative of phenylthiomethanol of the general formula 17

_/ S^ _/ OH

^{R l} ( 17)

where Ri has the meaning defined above,

in an aprotic solvent, advantageously in diethyl ether in the presence of an amine. As an example, the reaction is conducted in the following manner: 1 eqval of an appropriate compound of formula 17 is dissolved in diethyl ether and combined with 1 egual of an amine. The solution is cooled down to the temperature of 0 C and, on stirring, 1 egualof an appropriate compound of formula 16 is added. On completion of the reaction, the cooling is stopped and a saturated sodium hydrogen carbonate solution is added successively until carbon dioxide no longer evolves from the reaction mixture. The mixture is separated and the organic layer containing the reaction product is dried, following which the solvent is evaporated and the final product is crystallized.

The method according to the invention is universal and can be applied for the protection of hydroxyl functions with acetal and acetal ester groups. The method can be applied for the protection of hydroxyl groups not only in nucleosides and their analogues, but also in alcohols and complex chemical compounds containing a hydroxyl group.

The procedure of incorporating acetal and acetal ester protecting groups into the hydroxyl function is:

• simple - requires the mixing of two reagents and an addition of SnCl ₄ , · economical - uses reagents which are either inexpensive or easy to obtain.

The method makes use of both known and new compounds, developed specifically for the invention, containing a thioacetal or thioacetal ester group.

The method according to the invention has a particularly advantageous application in the chemical synthesis of RNA and its analogues.

The blockage of the hydroxyl function using the method according to the invention is compatible with other protecting groups applied during RNA chain synthesis; for example, it is stable in the conditions of unblocking of the 3'- and 5' -hydroxyl positions with fluoride ions.

The subject of the invention is presented in the following examples which illustrate the invention but do not limit its scope.

Example 1

In a round-bottom flask, 0.2 g (0.41 mmol) of 3 ',5 '-0-(tetraisopropyldisiloxane-l,3-diyl) was dissolved in 3 ml of dry 1 , 2-dichloroethane, and then 0.9 g (3.29 mmol) of anhydrous benzoyloxymethylthio(4-chloro)benzene was added. To the solution 0.2 g of 4A molecular sieves were added to dry the solution. The flask was closed with a septum provided with argon-filled balloons, and was placed in a cooling bath and cooled down to -25°C; the solution was stirred magnetically. Using a syringe, 1.2 ml of 1.2 M tin(IV) chloride solution (1.43 mmol) in 1,2- dichloroethane were then added. The mixture was stirred for 5 hours, maintaining the temperature of -23°C. The course of the reaction was monitored by TLC (hexane-ethyl acetate-methanol 9:4: 1). On completion of the reaction, a saturated aqueous solution of sodium hydrogen carbonate was added to neutralize tin(IV) chloride until bubbles of gas (carbon dioxide) stopped forming, then the cooling was stopped. Following complete neutralization, the reaction mixture was filtered from white precipitate, and then the raw product was extracted three times from the filtrate with 1,2- dichloroethane (3x8ml). The organic layers were collected and dried over anhydrous sodium (VI) sulphate, following which the solvent was evaporated. The raw product was purified in a chromatographic column packed with silica gel 60 (63-200 μιη) from Merck, using methylene dichloride-methanol (99: 1) as eluents. In this manner, 3 ',5'-0-(tetraisopropyldisiloxane-l,3-diyl)- 2'-0-benzoyloxymethyl-uridine was obtained with 89% yield.

Spectroscopic analysis:

lH NMR (400MHz, CDC1 ₃ ) 0,893-1.089 (28H, m), 3.951, 3.985 (1H, H-5", dd, J=2.4Hz, J=2,4Hz), 4.149, 4.173 (1H, H-4', J=1.6Hz, J=1.6Hz), 4.201-4.264 (2H, H-2 ' , H-3 \ m), 4.378 (1H, H-5 ', d,

J=4.4Hz), 5.68 (1H, H-5, d, J=8Hz), 5.758 (1H, H-l ', d, J=6,4Hz), 5.784-5.800 (2H, OCH ₂ 0, m) 7.407-7.470 (2H, H-Ar, m), 7.561 (1H, H-Ar, t, J=5,6), 7.867 (1H, H-6, d, J=8Hz), 8.071-8.107 (2H, H-Ar, m)

¹³ C NMR (400MHz, CDC1 ₃ ) 12.45, 12.81, 12.99, 13.23 (CH), 16.70, 16.84, 16.92, 17.18, 17.24, 17.31, 17.34, 17.41 (CH ₃ ), 59.25 (C-5'), 67.53 (C-3'), 81.72 (C-2'), 82.63 (C-4'), 88.42 (OCH ₂ 0), 89.33

(C-l'), 101.58 (C-5'), 128.27-133.25 (6C-Ar), 139.51 (C-6), 149.79 (C-2), 163.47 (C-4), 165.83 (C=0)

Example 2

Following the procedure defined in Example 1, a reaction was conducted in 6 ml of 1,2- dichloroethane between 0.5 g ( 1 mmol) of 3 ',5'-0-(tetraisopropyldisiloxane-l,3-diyl)uridine and 1.665 g (6 mmol) of benzoyloxymethylthio(4-chloro)benzene and 2.3 ml of 1 M tin(IV) chloride (2.5 mmol) in 1,2-dichloroethane. The reaction was conducted over a 24-hour period. On completion of the reaction, a saturated aqueous solution of hydrogen carbonate was added until bubbles of gas (carbon dioxide) stopped forming. In this manner, 3',5'-0-(tetraisopropyldisiloxane- l,3-diyl)-2'-0-benzoyloxymethyluridine was obtained with 82% yield. NMR analysis confirmed the structure of the product.

Example 3

Following the procedure defined in Example l,a reaction was conducted in 6 ml of 1,2- dichloroethane between 0.5 g ( 1 mmol) of 3',5'-0-(tetraisopropyldisiloxane-l,3-diyl)uridine and 0.57 g (2 mmol) of benzoyloxymethylthio(4-chloro)benzeneand 1.180 ml of 1.3 M tin(IV) chloride solution (1.54 mmol) in 1,2-dichloroethane. The reaction was conducted over a 48-hour period. On completion of the reaction, a saturated solution of sodium hydrogen carbonate was added until carbon dioxide stopped evolving. In this manner, S'^'-O-itetraisopropyldisiloxane-l^-diyl^'-O- benzoyloxymethyl-uridine was obtained in the yield of 15%. NMR analysis confirmed the structure of the product.

Example 4

Following the procedure defined in Example 1, a reaction was conducted in 2 ml of 1,2- dichloroethane between 0.2 g (0.3 mmol) of N ⁶ -phenoxyacetyl-3',5'-0-(tetraisopropyldisiloxane- l,3-diyl)adenosine and 0.69 g (2 mmol) of benzoyloxymethylthio(4-chloro)benzene and 1.120 ml of 0.9 M tin(IV) chloride solution (1.05 mmol) in 1,2-dichloroethane. The reaction was conducted over a 6-hour period. On completion of the reaction, a saturated solution of sodium hydrogen carbonate was added until carbon dioxide stopped evolving. In this manner, N ⁶ -phenoxacetyl-2'- benzoyloxymethyl-3',5'-(tetraisopropyldisiloxane-l,3-diyl)ad enosine was obtained in the yield of 72%.

Spectroscopic analysis:

Ή NMR (400MHz, CDC1 ₃ ) 9.50 (s, 1H, NH); 8.72 (s, 1H, H-2); 8.25 (s, 1H, H-8); 8.071-8.107 (2H, H-Ar, m) 7.44-6.81 (m, 7H, H -Ar,H-Ar-Pac); 6.22 (d, J = 4.7 Hz, 1H, H-l'); 5.51-5.40 (2H, m OCH20); 5.08 (t, 3JH2' H3',H1' = 4.7 Hz; 1H, H-2 ' ) ; 4.88 (s, 2H, NHCOC¾Ph); 4.55 (q, 3jH37H2',OH3 ',H4'= 4.7 Hz, 1H, H-3 '); 4.28 (m, 1H, H-4'); 3.54 (dd„lH, H-5'); 3.43 (dd, 2JH5"/H5' = 10.7 Hz; 3JH5" H4'= 4 Hz, 1H, H5"); 2.75 (1H, m); 0,893-1.089 (28H, m). ¹³ CNMR (400MHz, CDCI ₃ ) 166.7 (NHCO);165.83 (C=0); 158.6 (C); 157.2 (C, Pac); 152.6 (C2); 151.5 (C6); 148.4 (C4); 144.4, 135.5 (C, Car); 142.2 (C8); 130.1, 129.9, 128.1, 127.9122.4, 115, 114.9, 113.2 (12H, Ar); 123.2 (C5); 89.0 (OCH20); 87.3 (CI'); 84.2 (C4'); 81.7 (C2'); 70.5 (C3'); 68.1

Example 5

Following the procedure defined in Examplel, a reaction was conducted in 6 ml of 1,2- dichloroethane between 0.2 g (0.27 mmol) o f N ² -tert-butylphenoxyacetyl-3',5'-0- (tetraisopropyldisiloxane-l,3-diyl)guanosine and 0.62 g (2.2 mmol) of benzoyloxymethylthio(4- chloro)benzene and 1.2 ml of 067 M tin(IV) chloride solution (0.81 mmol) in dichloroethane. The reaction was conducted over a 6-hour period. On completion of the reaction, a saturated solution of sodium hydrogen carbonate was added until carbon dioxide stopped evolving. In this manner, N ² - tert-butylphenoxacetyl-3',5'-0-(tetraisopropyldisiloxane-l,3 -diyl)-2'-0- benzoyloxymethylguanosine was obtained in the yield of 74%.

Spectroscopic analysis:

¾NMR (400MHz, CDC1 ₃ ) 11.78 (s, 1H, NH-1); 9.09 (s, 1H, NHPac); 7.79 (s, 1H, H-8); 7.36-6.73 (m, 9H, H-Ar, H-tbuPac); 5.99 (H, H-l'); 5.46-5.36 (2H, m, OCH ₂ 0); 5.23 (s, 2H, NHCOCH2Ph); 4.65 (t,

1H, H-2'); 4.34 (m, 1H, H-3'); 4.17 (m, 1H, H-4'); 3.38 (m,lH, H-5'); 3.35 (dd, 2JH5"/H5' = 10.7 Hz; 3JH5"/H4'= 2.9 Hz, 1H, H-5"); 2.49 (d, J= 4.3 Ηζ,ΙΗ, OH3'); 1.15 (s, 9H, tbuPac), 0.893-1.089 (28H, m).

^NMR (400MHz, CDC1 ₃ ) 169.9 (NHCO); 165.83 (C=0), 158.6 (C, Car); 155.2 (C6); 154.2 (4C, tbuPac);

147.7 (C2); 146.5 (C4); 145.9 (C, tbuPac); 144.3, 135.5 (C, ); 137.0 (C8); 130.1, 128.1, 127.1

126.9, 126.8, 114.4, 113.3 (CH, Car); 122.3 (C5); 89.3 (OCH20); 86.1 (CI'); 84.0 (C2'); 82.7 (C4'); 70.4 (C3'); 67.1 (NHCOCH2Ph); 63.1 (C5'); 34.3 (C, PhC(CH3)3); 31.4 (PhC(CH3)3), 16.70, 16.84, 16.92, 17.18, 17.24, 17.31, 17.34, 17.41 (GH ₃ ) ₃ ,12.45, 12.81, 12.99, 13.23 (CH). Example 6

Following the procedure defined in Examplel, a reaction was conducted in 1 ml of 1,2- di chloroethane b etwe en 0.05 g ( 0. 1 mmo l) o f 3 ' , 5 '-0- (tetraisopropyldisiloxtetraisopropyldisiloxane-l,3-diyl)urid ine and 0.266 g (1 mmol) of benzoyloxymethylthio(4-methyl)benzene and 0.45 ml of 1 M tin(IV) chloride (0.45 mmol) in dichloroethane. The reaction was conducted over a 4.5-hour period. On completion of the reaction, a saturated solution of sodium hydrogen carbonate was added until carbon dioxide stopped evolving. In this manner, 3 ',5'-(tetraisopropyldisiloxane-l,3-diyl)-2'-0-(benzoyloxymet hyl)undine was obtained in the yield of 85%/

Spectroscopic analysis:

lR NMR (400MHz, CDC1 ₃ ) 0.893-1.089 (28H, m), 3.951, 3.985 (1H, H-5", dd, J=2.4Hz, J=2.4Hz), 4.149, 4.173 (1H, H-4\ J=1.6Hz, J=1.6Hz), 4.201-4.264 (2H, H-2', H-3 ', m), 4.378 (1H, H-5', d, J=4.4Hz),

5.68 (1H, H-5, d, J=8Hz), 5.758 (1H, H-l \ d, J=6.4Hz), 5.784-5.800 (2H, OCH ₂ 0, m) 7.407-7.470 (2H, H-Ar, m), 7.561 (1H, H-Ar, t, J=5.6), 7.867 (1H, H-6, d, J=8Hz), 8.071-8.107 (2H, H-Ar, m) ¹³ C NMR (400MHz, CDC1 ₃ ) 12.45, 12.81, 12.99, 13.23 (CH), 16.70, 16.84, 16.92, 17.18, 17.24, 17.31, 17.34, 17.41 (CH ₃ ) ₃ , 59.25 (C-5 '), 67.53 (C-3 '), 81.72 (C-2'), 82.63 (C-4'), 88.42 (OCH ₂ 0), 89.33 (C-1 ' 101.58 (C-5'), 128.27-133.25 (6C-Ar), 139.51 (C-6), 149.79 (C-2), 163.47 (C-4), 165.83

(C=0)

Example 7

Following the procedure defined in Example 1, a reaction was conducted in 1 ml of 1,2- dichloroethane between 0.05 g (0. 1 mmol) of 3 ',5 '-0-(tetraisopropyldisiloxane-l,3-diyl)uridine and 0.244 g ( 1 mmol) of benzoyloxymethylthiobenzeneand 0.45 ml of 1 M tin(IV) chloride (0.45 mmol) solution in 1,2-dichloroethane. The reaction was conducted over a 8-hour period. On completion of the reaction, a saturated solution of sodium hydrogen carbonate was added until carbon dioxide stopped evolving. In this manner, 3 ',5 '-0-(tetra-isopropylsiloxane-l,3-diyl)-2'-0- (benzoyloxymethyl)uridine was obtained in the yield of 80%.

Spectroscopic analysis:

lR NMR (400MHz, CDC1 ₃ ) 0.893-1.089 (28H, m), 3.951, 3.985 (1H, H-5", dd, J=2,4Hz, J=2.4Hz), 4.149, 4.173 (1H, H-4', J=1.6Hz, J=1.6Hz), 4.201-4.264 (2H, H-2', H-3 ', m), 4.378 (1H, H-5', d, J=4.4Hz), 5.68 (1H, H-5, d, J=8Hz), 5.758 (1H, H-l \ d, J=6.4Hz), 5.784-5.800 (2H, OC¾0, m) 7.407-7.470 (2H, H-Ar, m), 7.561 (1H, H-Ar, t, J=5.6), 7.867 (1H, H-6, d, J=8Hz), 8.071-8.107 (2H, H-Ar, m) ¹³ C NMR (400MHz, CDC1 ₃ ) 12.45, 12.81, 12.99, 13.23 (CH), 16.70, 16.84, 16.92, 17.18, 17.24, 17.31, 17.34, 17.41 (CH ₃ ), 59.25 (C-5'), 67.53 (C-3 '), 81.72 (C-2'), 82.63 (C-4'), 88.42 (OCH ₂ 0), 89.33 (C- 1 '), 101.58 (C-5'), 128.27-133.25 (6C-Ar), 139.51 (C-6), 149.79 (C-2), 163.47 (C-4), 165.83 (CO) Example 8

Following the procedure defined in Example 1, a reaction was conducted in 5 ml of 1,2- dichloroethane between 0.5 g (1 mmol) of 3 ',5 '-0-(tetraisopropyldisiloxane-l,3-diyl)uridine and 2 g (1 mmol) of toluyloxymethylthio(4-chloro)benzeneand 2.40 ml of 1.25 M tin(IV) chloride (3 mmol) solution in 1,2-dichloroethane. The reaction was conducted over a 12-hour period. On completion of the reaction, a saturated solution of sodium hydrogen carbonate was added until carbon dioxide stopped evolving. In this manner, 3 ',5 '-0-(tetraisopropyldisiloxane-l,3-diyl)-2'-0- (o-toluyloxymethyl)uridine was obtained in the yield of 85 %.

Spectroscopic analysis:

lH NMR (400MHz, CDC1 ₃ ) 0.893-1.089 (28H, m), 2.615 (3H, s, CH ₃ ), 3.951, 3.985 (1H, H-5", dd, J=2.4Hz, J=2.4Hz), 4.149, 4.173 (1H, H-4', J=1.6Hz, J=1.6Hz), 4.201-4.264 (2H, H-2', H-3 ', m), 4.378 (1H, H-5', d, J=4.4Hz), 5.68 (1H, H-5, d, J=8Hz), 5.758 (1H, H-l ', d, J=6.4Hz), 5.784-5.800 (2H, OCH ₂ 0, m) 7.407-7.470 (2H, H-Ar, m), 7.561 (1H, H-Ar, t, J=5.6), 7.867 (1H, H-6, d, J=8Hz), 8.071-8.107

(2H, H-Ar, m)

¹³ C NMR (400MHz, CDC1 ₃ ) 12.45, 12.81, 12.99, 13.23 (CH), 16.70, 16.84, 16.92, 17.18, 17.24, 17.3 1, 17.34, 17.41 (CH ₃ ), 21 .72 (CH ₃ ), 59.25 (C-5 '), 67.53 (C-3 '), 81.72 (C-2'), 82.63 (C-4'), 88.42 (OCH ₂ O), 89.33 (C-l '), 101.58 (C-5'), 128.27-133.25 (6C-Ar), 139.51 (C-6), 149.79 (C-2), 163.47 (C-4), 165.83 (C=0)

Example 9

Following the procedure defined in Example 1, a reaction was conducted in 3 ml of 1,2- dichloroethane between 0.5 g ( 1 mmol) of 3 ',5 '-0-(tetraisopropyldisiloxane-l,3-diyl)uridine and 1.58 g (6 mmol) of pivaloyloxymethylthio(4-chloro)benzene and 2.3 ml of 1 M tin(IV) chloride solution (2.55 mmol) in 1,2-dichloroethane. The reaction was conducted over a 6-hour period. On completion of the reaction, a saturated solution of sodium hydrogen carbonate was added until carbon dioxide stopped evolving. In this manner, 3 ',5 '-0-(tetraisopropyldisiloxane-l,3-diyl)-2'-0- (pivaloyloxymethyl)uridine was obtained in the yield of 75%.

Spectroscopic analysis:

Ή NMR (400MHz, CDC1 ₃ ) 0.955-1. 102 (28H, m), 1.230 (9H, (C/¾ ₃ , s), 3.951, 3.985 (1H, H-5", dd, J=2Hz, J=2Hz), 4.113, 4.137 (1H, H-4 ', dd, J= 1.6Hz, J= 1.6Hz), 4.21-4.303 (3H, H-2',3',5', m), 5.496, 5.561 (2H, (X¾0, dd, J=6.4Hz, J=6.8Hz), 5.668 (1H, H-5, d, J=8Hz), 5.743 (1H, H-l ', s), 7.860 (1H, H-6, d, J=8Hz) ¹³ C NMR (400MHz, CDC1 ₃ ) 12.55, 12.84, 13.06, 13.39 (CH), 16.80, 1691, 16.95, 17.10, 17.21, 17.28, 17.38, 17.46 (CH ₃ ), 26.97 ((CH ₃ ) ₃ ), 38.79 (C(CH ₃ ) ₃ ), 59.30 (C-5'), 67.99 (C-3'), 81.27 (C-2'), 81.61 (C-4'),

87.76 (C-1 ' 89.06 (OCH ₂ 0), 101.53 (C-5), 139.39 (C-6), 149.50 (C-2), 193.03 (C-4), 178.03 (CO) Example 10

Following the procedure defined in Example 1, a reaction was conducted in 2 ml of 1,2- dichloroethane between 0.2 g (0.4 mmol) of 3',5'-0-(tetraisopropyldisiloxane-l,3-diyl)undine and 0.97 g (4 mmol) of pivaloyloxymethylthio(4-methyl)benzene and 2 ml of 0.9 M tin(IV) chloride solution (1.8 mmol) in 1,2-dichloroethane. The reaction was conducted over a 6-hour period. On completion of the reaction, a saturated solution of sodium hydrogen carbonate was added until carbon dioxide stopped evolving. In this manner, 3',5'-0-(tetraisopropyldisiloxane-l,3-diyl)-2'-0- (pivaloyloxymethyl)uridine was obtained in the yield of 75%.

Spectroscopic analysis:

lR NMR (400MHz, CDC1 ₃ ) 0.955-1.102 (28H, m), 1.230 (9H, (C¾) ₃ , s), 3.951, 3.985 (1H, H-5", dd, J=2Hz, J=2Hz), 4.113, 4.137 (1H, H-4 ', dd, J= 1.6Hz, J= 1.6Hz), 4.21-4.303 (3H, H-2',3',5', m), 5.496, 5.561 (2H, OCi¾0, dd, J=6.4Hz, J=6.8Hz), 5.668 (1H, H-5, d, J=8Hz), 5.743 (1H, H-l\ s), 7.860 (1H, H-6, d, J=8Hz)

1 ³ C NMR (400MHz, CDC1 ₃ ) 12.55, 12.84, 13.06, 13.39 (CH), 16.80, 1691, 16.95, 17.10, 17.21, 17.28, 17.38, 17.46 (C¾), 26.97 ((CH ₃ ) ₃ ), 38.79 (C(CH ₃ ) ₃ ), 59.30 (C-5'), 67.99 (C-3'), 81.27 (C-2'), 81.61 (C-4'), 87.76 (C-Γ), 89.06 (OCH ₂ 0), 101.53 (C-5), 139.39 (C-6), 149.50 (C-2), 193.03 (C-4), 178.03 (C=0) Example 1 1

Following the procedure defined in Example 1, a reaction was conducted in 3 ml of 1,2- dichloroethane between 0.25 g (0.51 mmol) of 3 ',5'-0-(tetraisopropyldisiloxane-l,3-diyl)uridine and 0.86 g (5.1 mmol) of benzoyloxymethylthiomethyl ether and 2.3 ml of 1 M tin(IV) chloride (2.3 mmol) solution in 1,2-dichloroethane. The reaction was conducted over an 8-hour period. On completion of the reaction, a saturated solution of sodium hydrogen carbonate was added until carbon dioxide stopped evolving. In this manner, 3',5'-0-(tetraisopropyldisiloxane-l,3-diyl)-2'-0- (benzoyloxymethyl)uridine was obtained in the yield of 80%.

Spectroscopic analysis: ^l R NMR (400MHz, CDC1 ₃ ) 0.981-1.110(m, 28H), 3.977, 4.011 (1H, H-5', dd, J=2.4 Hz, J=2.4Hz,), 4.150, 4.173 (1H., H-4 ' , dd, J=2Hz, J=2Hz,), 4.229-4.283 (3H, H-2' ,3 ',5 " , m,) 4.776, 4.702 (2H, OC¾C ₆ H ₅ dd, J=12Hz, J=12Hz), 4.967, 5.057 (2H, OC¾0, dd, J=6.8Hz, J=6.8Hz), 5.668 (1H, H-5, d, J=8Hz), 5.803 (1H, H-l ', s), 7.311-7.374 (5h, H-Ar, m), 7.885 (1H, H-6, d, J=8Hz)

¹³ C NMR (400MHz, CDC1 ₃ ) 12.58, 12.86, 13.09, 13.37 (CH), 16.83, 16.94, 16.98, 17.06, 17.21, 17.31, 17.39, 17.48 (CH ₃ ), 59.36 (C-5'), 68.03 (OCH ₂ Ar), 69.17 (C-3 '), 77.99 (C-2'), 81.83 (C-4'), 89.34 (C-1 ' 93.07 (OCH ₂ O),101.902 (C-5') 127.63-128.32 (5C-Ar), 137.51 (lC-Ar), 139.34 (C-6), 149.64 (C-2), 163.01 (C-4)

Example 12

Following the procedure defined in Example 1, a reaction was conducted in 3 ml of 1,2- dichloroethane between 0.2 g (0.41 mmol) of 3 ',5 '-0-(tetraisopropyldisiloxane-l,3-diyl)undine and 0.728 g (4.1 mmol) of benzoyloxymethylthiomethyl ether and 1.7 ml of 1 M tin(IV) chloride ( 1 8 mmol) solution in 1,2-dichloroethane. The reaction was conducted over a 12-hour period. On completion of the reaction, a saturated solution of sodium hydrogen carbonate was added until carbon dioxide stopped evolving. In this manner, 3 ',5 '-0-(tetraisopropyldisiloxane-l,3-diyl)-2'-0- (benzoyloxymethyl)uridine was obtained in the yield of 85%.

Spectroscopic analysis:

lR NMR (400MHz, CDC1 ₃ ) 0.893-1.089 (28H, m), 3.951, 3.985 (1H, H-5", dd, J=2.4Hz, J=2.4Hz), 4.149, 4.173 (1H, H-4', J=1.6Hz, J=1.6Hz), 4.201-4.264 (2H, H-2', H-3 ', m), 4.378 (1H, H-5', d, J=4.4Hz),

5.68 (1H, H-5, d, J=8Hz), 5.758 (1H, H-l ', d, J=6.4Hz), 5.784-5.800 (2H, OCH ₂ 0, m) 7.407-7.470 (2H, H-Ar, m), 7.561 (1H, H-Ar, t, J=5.6), 7.867 (1H, H-6, d, J=8Hz), 8.071-8.107 (2H, H-Ar, m) ¹³ C NMR (400MHz, CDC1 ₃ ) 12.45, 12.81, 12.99, 13.23 (CH), 16.70, 16.84, 16.92, 17.18, 17.24, 17.31, 17.34, 17.41 (CH ₃ ), 59.25 (C-5'), 67.53 (C-3 '), 81.72 (C-2'), 82.63 (C-4'), 88.42 (OCH ₂ 0), 89.33 (C- 1 '), 101.58 (C-5), 128.27-133.25 (6C-Ar), 139.51 (C-6), 149.79 (C-2), 163.47 (C-4), 165.83 (C=0)

Example 13

Following the procedure defined in Example 1, a reaction was conducted in 3 ml of 1,2- dichloroethane between 0.2 g (0.52 mmol) of 3 ' ,5 '-di-fert-butyl-silylene-uridine and 1 g (4.2 mmol) of anhydrous triisopropylsilyl(ethylthio)methyl ether and 1.7 ml of 1 M anhydrous tin(IV) chloride ( 1.82 mmol) solution in 1,2-dichloroethane. The reaction was conducted over a 6-hour period. On completion of the reaction, a saturated aqueous solution of hydrogen carbonate was added until bubbles of gas stopped forming. In this manner, 3 ',5'-0-(di-tert-butyl-silylene)-2'-0- [[(triisopropylsilyl)oxy]methyl]uridine was obtained in the yield of 75%.

lH NMR (400MHz, CDC1 ₃ ) δ (ppm): 1.12 (m, 39H), 3.965 (lH,m), 4.152 (1H, H-4'), 4.241 (2H, H-2', H-3\ m), 4.364 (1H, H-5'), 5.62 (1H, d, J=8Hz), 5.732 (1H, d, J=6.4Hz), 5.796 (2H, OCH ₂ 0, m) 7.867 (1H, d, J=8Hz)

¹³ C NMR (400MHz, CDC1 ₃ ) δ (ppm): 11.98, 12.02, 12.05, ((CH ₃ ) ₂ CH), 16.89, 17.08, 17.19, 17.24, 17.31, 17.36 (CH ₃ ), 27.04, 27.09, 27.13, 27.28 27.35, 27.62 (CH ₃ , f-butyl), 32.65, 32.76(CH, /-butyl) 58.98 (C-5'), 67.13 (C-3'X 81.56 (C-2'), 82.32 (C-4'), 88.56 (OCH ₂ 0), 89.17 (C-l'), 101.24 (C-5), 138.53 (C-6), 148.79 (C-2), 162.96 (C-4)

Example 14

Following the procedure defined in Example 1, a reaction was conducted in 3 ml of 1,2- dichloroethane between 0.2 g (0.48 mmol) of 3 ',5'-di-ier/-butoxysilylene-uridine and 1.12 g (3.8 mmol) of anhydrous (p-chlorophenylthiometoxy)-ferf-butyldimethylsilane and 1.7 ml of 1 M anhydrous tin(IV) chloride (1.68 mmol) solution in 1,2-dichloroethane. The reaction was conducted over a 6-hour period. On completion of the reaction, a saturated aqueous solution of hydrogen carbonate was added until bubbles of gas stopped forming. In this manner, 3',5'-0-(di-teri-butoxy- silylene)-2'-0-[[(di-methyl-teri-butylsilyl)oxy]methyl]-urid ine was obtained in the yield of 73%. ¾ NMR (400MHz, CDC1 ₃ ) δ (ppm): 0.16 (m, 6H), 1.02 (m, 9H), 1.35 (m, 18H), 3.965 (lH,m), 4.152 (1H, H-4'), 4.241 (2H, H-2', H-3 ', m), 4.364 (1H, H-5'), 5.62 (1H, d, J=8.2Hz), 5.732 (1H, d, J=6.4Hz),

5.698 (2H, OCHjO, m) 7.867 (1H, d, J=8.2Hz),

1 ³ C NMR (400MHz, CDC1 ₃ ) δ (ppm): -4.9 (CH ₃ ), 23.09, 23.29, 23.54 (CH ₃ , f-butyl), 31.04 (C, /-butyl), 32.04, 32.09, 32.13, 32.28 32.35, 32.62 ((CH ₃ ) ₃ COSi, /-butyl), 58. 64 (C-5'), 68.35 (C-3'), 74.68, 74.79 ((CH ₃ ) ₃ COSi, /-butyl), 82.42 (C-2'), 83.06 (C-4'), 88.93 (OCH ₂ 0), 89.91 (C-l'), 101. 43 (C-5), 138.68 (C-6), 147. 59 (C-2), 163.33 (C-4)

Example 15

A volume of 30 μΐ (0.3 mmol) of dry benzyl alcohol was transferred into a round-bottom flask and then dissolved in 1 ml of dry 1,2-dichloroethane, then 0.166 g (0.6 mmol) of benzoyloxymethylthiobenzene was added in the presence of 4 A molecular sieves. The flask was closed with a septum provided with an argon-filled balloon. The mixture was cooled down to the temperature of -25°C and thereafter, on stirring, 0.6 ml of 0.8 M solution of tin(IV) chloride (0.48 mmol) in 1,2-dichloroethane was added. The reaction was conducted in argon atmosphere. The mixture was stirred magnetically at a temperature of -25°C for 5 hours. Thereafter, the reaction was completed by adding an aqueous solution of sodium hydrogen carbonate until carbon dioxide stopped evolving,then the cooling bath was removed. The white precipitatewas filtered off, and the filtrate was extracted three times with 1,2-dichloroethane (3x3 ml). The organic layers were collected and dried over anhydrous sodium (VI) sulphate. The solvent was evaporated. The raw product was purified on a preparative PLC plate covered with silica gel 60 RP-18, F ₂₅ 4, 1 mm, from Merck, using hexane-dichloromethane 2:3 as the mobile phase. The product was extracted with dichloromethane (15 ml). In this manner, benzyloxymethylbenzoyl was obtained in the yield of 56%.

Spectroscopic analysis:

Ή NMR (400MHz, CDC1 ₃ ) 4.8 (2H, s, CH ₂ ), 5.6 (2H, s, OCH ₂ 0), 7.35-7.679 (8H, m), 8.02 (2H, d, J=lHz) ¹³ C NMR (400MHz, CDC1 ₃ ) 68.02 (1C, C¾), 98.58 (1C, OC¾0), 136.21 (lC-Ar), 126.68-133.02 (11C- Ar), 166.57 (C=0)

Example 16

A portion of 0 .25 0 g ( 0 .4 m m o l ) o f 3 ' , 5 '-0-(tetraisopropyldisiloxane-l,3-diyl)-2'-0- benzoyloxymethyluridine obtained according to Example 1 was dissolved in 6 ml of anhydrous tetrahydrofurane (THF), then 0.5 ml of 1 Mtriethylammonium fluoride in THF was added. The mixture was stirred for 18 hours at room temperature. The course of the reaction was monitored by TLC (methylene chloride-methanol 9: 1). On completion of the reaction, a saturated solution of sodium hydrogen carbonate was added until bubbles of gas stopped forming. The product was extracted six times with 5 ml of methylene dichloride . The organic layers were dried over anhydrous sodium sulphate. The solvent was evaporated. The product, 2'-0-benzoyloxymethyl- uridine, was purified in a chromatography column packed with silica gel 60 (63-200 μιη) from Merck, using methylene dichloride-methanol (96:4) as eluents. The yield was 82%.

Spectroscopic analysis:

lR NMR (400MHz, DMSO-de) 3.546-3.596(m, 1H), 3.60-3.678 (m, 1H), 3.877-3.902 (m, 1H), 4.157-4.227 (m, 1H), 4.405(t, 1H), 5.165 (t, lH), 5.323 (d, 1H, J=5.6 Hz), 5.527 (d, 1H, J=6.4Hz), 5.596 (t, 1H, J=8Hz), 5.937 (t, 1H, J=5.2Hz), 7.666 (t, lH-arom, J=7.2Hz), 7.912-8.057 (m, 3H-arom), 7.491-7.583

(2H, m, lH-arom, 1H-6)

¹³ C NMR (400MHz, DMSO-d ₆ ) 59.25 (C-5'), 67.53 (C-3'), 81.72 (C-2'), 82.63 (C-4'), 88.42 (OCH ₂ 0),

89.33 (C-1' 101.58 (C-5), 128.27-133.25 (6C-Ar), 139.51 (C-6), 149.79 (C-2), 163.47 (C-4), 165.83 (C=0).

Example 17

A portion of 50 mg (0.13 mmol) of 2 '-0-benzoyloxym ethyl -uridine obtained in Example 16 was dissolved in 2.5 ml of THF, then2.5 ml of 2 M solution of n-butylamine in methanol was added. On completion of the unblocking reaction (21 hours), the solvent and amine residue were evaporated from the reaction mixture. The raw product was introduced into a chromatographic column packed with silica gel 60 (63-200 μπι) from Merck, using methylene dichloride-methanol (60:40) as eluents. The collected fraction was evaporated. The isolated product of the unblocking reaction was uridine, which was confirmed by NMR analysis.

Spectroscopic analysis:

¾ NMR (400MHz, DMSO-d ₆ ) 3.516-3.567 (lH,m); 3.567-3.644 (lH,m); 3.823-3.849 (lH,q, J=3.6Hz, J=7.2Hz), 3.939-3.974 (1H, q, J=5.2Hz, 9.2Hz), 3.999-4.039 (1H, q, J=5.6Hz, J=10.8Hz), 5.060-5.089

(2H,m), 5.358 (1H, d, J=5.5Hz), 5.629 (1H, d, J=4Hz, H-5), 5.776 (1H, d, J=5.6Hz, H-l'), 7.880 (1H, d, J=4Hz, H-6), 11.297 (lH,s, ΝΉ)

"C NMR (400MHz, DMSO-d ₆ ) 60.85(C-5'); 69.88(C-3 ') ; 73.54(C-2') ; 84.83 (C-4'); 87.68(C-1') ;

101.75(C-5) ; 140.72(C-6); 150.74(C-2); 163.12(C-4)

Example 18

A portion of 0.250 g ( 0.4 16 mmol) of 3 ' , 5 '-0-(tetraisopropyldisiloxane-l,3-diyl)-2'-0- pivaloyloxymethyluridine obtained according to Example 9 was dissolved in 6 ml of dry tetrahydrofurane (THF), whereupon 0.5 ml of 1 M triethylammonium fluoride in THF was added. The mixture was stirred for 18 hours in room temperature . The course of the reaction was monitored by TLC (methylene chloride-methanol 9: 1). On completion of the reaction, a saturated solution of sodium hydrogen carbonate was added until bubbles of gas stopped forming. The product was extracted six times with 5 ml of methylene di chloride. The organic layers were dried over anhydrous sodium sulphate. The solvent was evaporated. The raw product of the reaction was purified in a chromatographic column packed with silica gel 60 (63-200 μιη) from Merck, using methylene dichloride-methanol (95 :5) as eluents. In this manner, 2'-0-(pivaloyloxymethyl)uridine was obtained in the yield of 84%.

Spectroscopic analysis:

Ή NMR (400MHz, DMSO-dg) 3.546-3.596(m, 1H), 3.60-3.678 (m, 1H), 3.877-3.902 (m, 1H), 4.157-4.227 (m, 1H), 4.405(t, 1H), 5.165 (t, lH), 5.323 (d, 1H, J=5.6 Hz), 5.527 (d, 1H, J=6.4Hz), 5.596 (t, 1H, J=8Hz), 5.937 (t, 1H, J=5.2Hz), 7.491-7.583 (1H, d, H-6, J=6.4 Hz)

¹³ C NMR (400MHz, DMSO-d ₆ ) 59.25 (C-5'), 67.53 (C-3 '), 81.72 (C-2'), 82.63 (C-4'), 88.42 (OCH ₂ 0), 89.33 (C-1 ' 101.58 (C-5'), 128.27-133.25 (6C-Ar), 139.51 (C-6), 149.79 (C-2), 163.47 (C-4), 165.83 (C=0).

Example 19

A portion of 50 mg (0.14 mmol) of 2'-(9-(pivaloyloxymethyl)uridine obtained in Example 18 was dissolved in THF (2.5 ml). Thereafter, 2 M n-butylamine solution in methanol (2.5 ml) was added. On completion of the unblocking reaction (34 hours), the solvent and amine residue were evaporated from the reaction mixture. The post-reaction mixture was introduced into a chromatography column packed with silica gel 60 (63-200 μηι) from Merck, using methylene dichloride-methanol (60:40) as eluents. Uridine was isolated as the product of unblocking. NRM analysis confirmed that the compound resulting from the removal of the protecting group is uridine. Example 20

100 mg (0.16 mmol) of 3',5 '-0-(tetraisopropyldisiloxane-l,3-diyl)-2'-0-(benzoyloxymeth yl)- uridine obtained in Example 1 was dissolved in 2.5 ml of THF . Thereafter, 2.5 ml of 2 M n- butylamine solution in methanol was added. On completion of the unblocking reaction (48 hours), the solvent and amine residue were evaporated from the reaction mixture. The raw product was introduced into a chromatography column packed with silica gel 60 (63-200 μιη) from Merck, using methylene dichloride-methanol (99: 1) as eluents. The isolated product of unblocking of the protecting group in position 2' is 3 ',5'-0-(tetraisopropyldisiloxane-l,3-diyl)uridine.

Spectroscopic analysis: ^l R NMR (400MHz, CDC1 ₄ )0.982-1.096 (28H, m), 3.339 (1H, d, J=2Hz), 3.985, 4.019 (1H, dd, J=2.8Hz, J=2.8Hz), 4.115-4.141 (1H, m), 4.187-4.221 (2H, m), 4.327-4.361 (1H, m), 5.695 (1H, d, J=8Hz, H- 5), 5.734 (1H, s, H-l'), 7.720 (1H, d, J=8Hz, H-6)

¹³ C NMR (400MHz, CDC1 ₄ ) 12.46-13.34 (CH, 4C), 16.78-17.42 ( CH _3> 8Q60.23 (C-5'), 68.89 (C-3'), 75.14 (C-2'), 81.90 (C-4'), 90.91 (C-l '), 101.94 (C-5'), 139.95 (C-6), 150.00 (C-2), 163.26 (C-4).

Example 21

A portion of 96 mg ( 0 . 1 6 mm o l) o f 3 ' , 5 '-0-(tetraisopropyldisiloxane-l,3-diyl)-2'-0- (pivaloyloxymethyl)-uridine obtained in Example 10 was dissolved in THF (2.5 ml). Thereafter, 2.5 ml of 2 M n-butylamine solution in methanol was added. On completion of the unblocking reaction (52 hours), the solvent and amine residue were evaporated from the reaction mixture. The raw product was introduced into a chromatography column packed with silica gel 60 (63-200 μπι) from Merck, using methylene dichloride-methanol (98 :2) as eluents. The isolated product of unblocking of the protecting group in position 2 ' is 3 ' ,5 '-0-(tetraisopropyldisiloxane-l,3- diyl)uridine. Spectroscopic analysis confirmed the identification of the compound - cf. the spectrum in Example 20.

Example 22

A portion of 1 02 mg (0 . 1 6 mmo l) of 3 ' , 5 '-0-(tetraisopropyldisiloxane-l,3-diyl)-2'-0- toluyloxymethyluridine obtained in Example 8 was dissolved in 2.5 ml of THF, then 2.5 ml of 2 M solution of n-butylamine in methanol was added. On completion of the unblocking reaction (72 hours), the solvent and amine residue were evaporated from the reaction mixture. The raw product was introduced into a chromatographic column packed with silica gel 60 (63-200 μηι) from Merck, using methylene dichloride-methanol (98:2) as eluents. The isolated product of unblocking of the protecting group in position 2' is 3 ',5'-0-(tetraisopropyldisiloxane-l,3-diyl)uridine. Spectroscopic analysis confirmed the identification of the compound - cf. the spectrum in Example 20.

Example 23

A portion of 42 μΐ ( 1 .6 mmol) of hydrofluoric acid-pyridine complex (HF-Py, Aldrich) was carefully added, while cooling, to 0.2 ml of pyridine. To the solution at a temperature of 0°C, upon magnetic stirring, 0 . 2 3 0 g ( 0 . 4 m m o l ) o f 3 ' , 5 '-0-(di-tert-butyl-silylene)-2'-0- [[(triisopropylsilyl)oxy]methyl]uridine obtained in Example 13 was added in 6 ml of anhydrous methylene dichlonde. The mixture was stirred for 2 hours at a temperature of 0°C. The course of the reaction was monitored by TLC (methylene chloride-methanol 9: 1 v/v). On completion of the reaction, a saturated solution of sodium hydrogen carbonate was added until bubbles of gas stopped forming. The product was extracted with dichloromethane several times (6x5 ml). The organic layers were dried over anhydrous sodium sulphate. The solvent was evaporated. The product, 2'-0- [[(triisopropylsilyl)oxy]methyl]uridine, was purified in a chromatographic column packed with silica gel 60 (63-200 μιη) from Merck, using methylene dichloride-methanol (96:4) as eluent. In this manner, 2'-0-[[(triisopropylsilyl)oxy]methyl]uridine was obtained in the yield of 76%.

Ή NMR (400MHz, CDC1 ₃ ) δ (ppm): 1.19 (21H,m), 3.784 (m, 1H), 4.049 (m, 1H), 4.075 (m, 1H), 4.234(t, 1H, J=4Hz), 4.364 (t,lH, J=4Hz), 5.518(m, 2H, OCH ₂ 0), 5.698 (d, 1H, J=8Hz), 5.713 (d, 1H, J=3.6Hz), 7.736 (1H, d, H-6, J=8Hz)

¹³ C NMR (400MHz, CDC1 ₃ ) δ (ppm): 12.23, 12.29, 12.34 (CH), 17.54, 17.62, 17.69, 17.86, 18.06, 18.12 (CH ₃ ), 57.96 (C-5'), 68.23 (C-3'), 82.56 (C-2'), 83.24 (C-4'), 89.32 (C-l'), 90.56 (OCH ₂ 0), 101.34 (C-5), 139.53 (C-6), 149.92 (C-2), 163.76 (C-4)

Example 24

Following the procedure defined in Example23, a reaction was conducted by carefully adding 42 μΐ (1.6 mmol) of hydrofluoric acid-pyridine complex (HF-Py, Aldrich), on cooling, to 0.2 ml of pyridine. To the solution at a temperature of 0°C, a portion of 0.220 g (0.4 mmol) of 3',5'-0-(di- tert-butoxy-silylene)-2'-0-[[(di-methyl-t-butylsilyl)oxy]met hyl]uridine in 6 ml of anhydrous methylene dichloride was added at a temperature of 0°C. The reaction was conducted for 2 hours at a temperature of 0°C. On completion of the reaction, a saturated solution of sodium hydrogen carbonate was added until carbon dioxide stopped evolving. In this manner, 2'-0-[[(di-methyl-t- butylsilyl)oxy]methyl]uridine was obtained in the yield of 74%.

Ή NMR (400MHz, CDC1 ₃ ) δ (ppm): 0.96 (m, 6H) 1.106 (m, 9H) 3.794 (m, 1H), 4.0 1 (m, 1H), 4.089 (m, 1H), 4.296 (t, 1H, J=4Hz), 4.384 (t,lH, J=4Hz), 5.496 (m, 2H, OCH ₂ 0), 5.726 (d, 1H, J=8Hz), 5.763 (d, 1H, J=3.6Hz), 7.724(1H, d, H-6, J=8Hz)

¹³ C NMR (400MHz, CDC1 ₃ ) δ (ppm): -4.8 (CH ₃ ), 19.78 (C, /-butyl) 23.09, 23.29, 23.54 (CH ₃ , /-butyl), 57.46 (C-5'), 68.29 (C-3'), 82.56 (C-2'), 83.14 (C-4'), 88.97 (OC¾0), 89.94 (C-l'), 102.34 (C-5), 139.68 (C-6), 148.925 (C-2), 164.23 (C-4) Example 25

A portion of 0. 108 g (0.25 mmol) of 2 '-0-[[(triisopropylsilyl)oxy]methyl]uridine obtained in Example 23 was dissolved in 5 ml of THF, then 0.3 ml of 1 Mtetrabutylammonium fluoride in THF was added. On completion of the unblocking reaction (5 hours), a saturated solution of sodium hydrogen carbonate was added until bubbles of gas stopped forming.The product was extracted with dichloromethane several times (6x10 ml). The organic layers were dried over anhydrous sodium sulphate. The solvent was evaporated. The raw product was introduced into a chromatographic column packed with silica gel 60 (63-200 μιη) from Merck, using methylene dichloride-methanol (60:40) as eluents. The collected fraction was evaporated. The isolated product of the unblocking reaction was uridine, which was confirmed by NMR analysis - cf. the spectrum in Example 17.

Example 26

A portion of 0.1 g (0.25 mmol) of 2'-0-[[(di-methyl-t-butylsilyl)oxy]methyl]uridine obtained in Example 24 was dissolved in 5 ml of THF, then 0.3 ml of 1 Mtetrabutylammonium fluoride in THF was added. On completion of the unblocking reaction (3.5 hours), a saturated solution of sodium hydrogen carbonate was added until bubbles of gas stopped forming.The product was extracted with dichloromethane several times (6x10 ml). The organic layers were dried over anhydrous sodium sulphate. The solvent was evaporated. The raw product was introduced into a chromatography column packed with silica gel 60 (63-200 μιη) from Merck, using methylene dichloride-methanol (60:40) as eluents. The collected fraction was evaporated. The isolated product of the unblocking reaction was uridine, which was confirmed by NMR analysis - cf. the spectrum in Example 17.

Example 27

A portion of 35 ml of anhydrous diethyl ether was transferred into a round-bottom flask and used for dissolving 10 g (0.057 mol) of 4-chlorophenylthiomethanol, whereupon 4.6 ml (0.057 mol) of anhydrous pyridine was added. The solution was placed in a cooling bath with a temperature of 0°C, then on stirring, 8.01 g (0.057 mol) of anhydrous benzoyl chloride was added. The reaction was conducted for 2 hours at a temperature of 0°C, whereupon the cooling bath was removed. During the reaction, pyridine hydrochloride precipitate appeared. On completion of the reaction, a saturated aqueous solution of sodium hydrogen carbonate (ca. 60 ml) was added to the reaction mixture until carbon dioxide stopped evolving. The pyridine hydrochloride precipitate wasdissolved. The ether layer with the product of the reaction was isolated and dried over anhydrous sodium (VI) sulphate. The solvent was evaporated. The product was crystallized from diethyl ether. In this manner, benzoyloxymethylthio-(4-chloro)benzene was obtained in the yield of 89%.

Spectroscopic analysis:

Ή NMR (400MHz, CDC1 ₃ ) 5.634 (2H, s), 7.302-7.317 (2H, H-Ar., m), 7.442-7.475 (4H, H-Ar, m), 7.591

(1H, H-Ar t, J=8), 8.035-8.056 (2H, H-Ar, m )

1 ³ C NMR (400MHz, CDC1 ₃ ) 68.66 (-OCH ₂ S-), 127.41, 128.36, 128.43, 128.73, 129.22, 129.40, 129.42,

129.66, 132.04, 133.06, 133.37, 133.67 (C-arom), 165.67 (C=0)

Example 28

A portion of 10 ml of anhydrous diethyl ether was transferred into a round-bottom flask and used for dissolving 3 g (17 mmol) of 4-chlorophenylthiomethanol, whereupon 1.38 ml (17 mmol) of anhydrous pyridine was added. The mixture was placed in a cooling bath with a temperature of 0°C, whereupon, onstirring, 2.247 ml (17 mmol) of anhydrous o-toluyl chloride was added. The reaction was conducted for 2 hours at a temperature of 0°C, then the cooling bath was removed. During the reaction, pyridine hydrochloride precipitate appeared. On completion of the reaction, a saturated aqueous solution of sodium hydrogen carbonate was added to the reaction mixture until carbon dioxide stopped evolving. The pyridine hydrochloride precipitate was dissolved.The ether layer with the product of the reaction was isolated and dried over anhydrous sodium (VI) sulphate. The solvent was evaporated. In this manner, o-toluyloxymethylthio-(4-chloro)benzene was crystallized from diethyl ether in the yield of 95%.

Spectroscopic analysis:

¾ NMR (400 MHz, CDC1 ₃ ) 2.615 (3H, s, C¾), 5.643 (2H, s, OCH ₂ S), 7.272-7.295 (2H, m, H-Ar), 7.319 (2H, m, H-Ar), 7.321-7.338 (2H, m, H-Ar),7.430-7.449 (1H, m), 7.462-7.479 (2H, m), 7.917-7.937 (1H, m)

1 ³ C NMR (400 MHz, CDC1 ₃ ) 21.72 (CH ₃ ), 68.28 (OC¾S), 125.79-140.72 (12C-Ar) 166.45 (C=0) Example 29

A portion of 2 ml of anhydrous diethyl ether was transferred into a round-bottom flask and used for dissolving 0.5 g (2.8 mmol) of 4-chlorophenylthiomethanol (0.5 g; 2.8 mmol; 1 egual), whereupon 0.3 ml (3.65 mmol) of anhydrous pyridine was added. The mixture was placed in a cooling bath with a temperature of 0°C, then on stirring, 0.37 ml (3 mmol) of anhydrous pivaloyl chloride was added. The reaction was conducted for 60 min at a temperature of 0°C, then the cooling bath was removed. During the reaction, pyridine hydrochloride precipitate appeared. On completion of the reaction, a saturated aqueous solution of sodium hydrogen carbonate was added until carbon dioxide stopped evolving. The pyridine hydrochloride precipitate was dissolved. The ether layer with the product of the reaction was isolated and dried over anhydrous sodium (VI) sulphate. The solvent was evaporated. The product was crystallized from diethyl ether. In this manner, pivaloyloxymethylthio-(4-chloro)benzene was obtained in the yield of 78%.

Spectroscopic analysis:

Ή NMR (400 MHz, CDC1 ₃ ) 1.198 (9H, s), 5.373 (2H,s), 7.285-7.302 (2H, m, H-Ar), 7.385-7.402 (2H, m, H- Ar)

¹³ C NMR (400 MHz, CDC1 ₃ ) 26.9 ((CH ₃ ) ₃ ), 38.78 (C(CH ₃ ) ₃ ), 67.96 (OCH ₂ S), 129.15, 129.23, 131.71,

131.89, 133.24, 136.11 (C-arom), 177.52 (C=0)

Example 30

A portion 4 ml of anhydrous diethyl ether was transferred into a round-bottom flask and used for dissolving 2 g (12 mmol) of 4-methylphenylthiomethanol, then 0.96 ml (12 mmol) of anhydrous pyridine was added. The mixture was placed in a cooling bath with a temperature of 0°C, then on stirring, 1.6 ml (12 mmol) of anhydrous pivaloyl chloride was added. The reaction was conducted for 60 min at a temperature of 0°C, then the cooling bath was removed. During the reaction, pyridine hydrochloride precipitate was formed. On completion of the reaction, a saturated aqueous solution of sodium hydrogen carbonate was added until carbon dioxide stopped evolving. The pyridine hydrochloride precipitate was dissolved. The ether layer with the product of the reaction was isolated and dried over anhydrous sodium (VI) sulphate. The solvent was evaporated. In this manner, pivaloyloxymethylthio-(4-methyl)benzene in the form of an oily liquid was obtained in the yield of 63%. Spectroscopic analysis:

Ή NMR (400MHz, CDC1 ₃ ) 1.198 (9H,s), 2.197 (3H, s), 4.629 (2H, s, (-SCH ₂ 0-)), 7.335-7.368 (4H, m, H- arom)

¹³ C NMR (400MHz, CDC1 ₃ ) 21.05 (CH ₃ ), 26.96 ((CH ₃ ) ₃ ), 38.79 (C(CH ₃ ) ₃ ), 69.77 (-SCH ₂ 0-), 129.78 (2C- arom), 131.06 (C-arom), 131.32 (2C-arom), 137.60 (C-arom), 177.68 (C=0)

Example 31

A portion of 1.36 g (35 mmol) of imidazole was added to a solution of 3 g (17 mmol) of 4- chlorophenylthiomethanol in 20 ml of anhydrous methylene chloride, and cooled down in the c o o l i ng b ath w ith a te m p e ratu re o f 0 °C . The re afte r, 2. 72 g ( 1 8 m m o l ) o f t- butyldimethylchlorosilane was added and the mixture was left to heat to room temperature, then the reaction was conducted for 16 hours. 50 ml of methylene chloride and 50 ml of 5% aqueous solution of NaH ₂ P0 ₄ were added to the mixture. The organic layer was dried with anhydrous sodium sulphate and then evaporated under reduced pressure, obtaining an oil of the raw product which was purified by chromatography in a column with silica gel 60 (63-200 μιη) from Merck, using dichloromethane-hexane (85 : 15) as eluent. In this manner, (p-chlorophenylthiomethoxy)-t- butyldimethylsilane in the form of thick oil was obtained in an amount of 3.5 g, in the yield of ca. 70%.

lR NMR (400 MHz, CDC1 ₃ , ppm): 0.12 (H ₃ CSi, 3H), 0,91 (CH ₃ C, 9H), 5.32 (s, SCH ₂ 0, 2H), 7.32 (m, 2H, Ar), 7.46 (m, 2H, Ar).

¹³ C NMR (400 MHz, CDC1 ₃ , ppm): -4.8, 19.32, 25.35,25.72, 25.98, 69.77 (-SCH ₂ 0-), 129.781, 130.14, 131.06, 131.32, 131.57, 137.60

Example 32

Following the procedure defined in Example 1, a reaction was conducted in 17 ml of 1,2- dichl oroethane b etween 1 g ( 1 .9 mmol) of 3 ' , 5 '-0-(tetraisopropyldisiloxane-l,3- diyl) adenosine and 4.58 g (15.7 mmol) of toluiloxymethylthio(4-chloro)benzene and 1.778 ml of 3,75 M tin(IV) chloride solution (1.05 mmol) in 1,2-dichloroethane. The reaction was conducted over a 24 hour period. On completion of the reaction, a saturated solution of sodium hydrogen carbonate was added until carbon dioxide stopped evolving. In this manner, 2'-0-toluiloxymethyl-3 ',5'-0-(tetraisopropyldisiloxane-l,3-diyl)adenosine was obtained in the yield of 67%. Spectroscopic analysis:

'H NMR (400MHz, CDC1 ₃ ) 8.918 (s, IH, H-2), 8.469 (s, IH, H-8), 7.976-7.954 (IH, H-Ar, m), 7.21-7.31 (m, 3H, H-Ar), 6.50 (s, 2H, NH ₂ ); 6.054 (s IH, Η-Γ), 5.704-5.823 (2H, m OCH ₂ 0), 4.760-4.819 (m, 2H, H-4', H-5'), 4.150-4.183 (m, 2H, H-3 ', H-2'), 4.010-4.049 (m, IH, H-5"), 2.75 (s, 3H), 0.986-1.100 (m, 28H).

¹³ C NMR (400MHz, CDC1 ₃ ) 166.54 (C=0), 152.86 (C); 149.01 (C2), 140.5 (C6); 138.4 (C4), 132.43, 132.01, 131.95, 131.72, 130.96, 129.284 (C-Ar), 129.284 (C8), 125.671 (C5), 88.75 (OCH20), 88.54 (CI '), 81.38 (C4'), 69.059 (C2' i C3 '), 59.810 (C5'), 21.776 (CH ₃ ), 16.890-17.417 (CH ₃ ), 12.650- 13.368 (CH).

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