Phosphoramidite derivative and method of producing rna oligo

FIELD: chemistry.

SUBSTANCE: invention relates to phosphoramidite derivatives of general formula where Bx denotes adenine, guanine, cytosine, thymine or uracil, where the amine group of adenine, guanine and cytosine can be optionally protected by a protective group selected from acetyl and phenoxyacetyl; R1 is a substitute of general formula in which R11, R12 and R13 are identical or different, and each denotes hydrogen or alkoxy; R2a and R2b are identical or different, and each denotes alkyl; and WG1, WG2 denote a cyano group. The invention also pertains to a multistep method of producing the said compounds. The invention also relates to intermediate compounds of the said method, namely: an intermediate ether compound of general formula where L is a halogen or a C1-C5alkylthio group; WG1 is a cyano group; an intermediate compound of general formula where Bx denotes adenine, guanine, cytosine, thymine or uracil, where the amine group of adesine, guanine and cytosine can be optionally protected by a protective group selected from an acetyl group and a phenoxyacetyl group; and WG1 denotes a cyano group; an intermediate compound of general formula where Bx is as described above; R1 is a substitute of general formula (2); an intermediate compound of general formula where Bx is as described above; A is a silicon-containing substitute of general formula or where R6 denotes alkyl and WG1 denotes a cyano group. The invention also relates to a method of producing an oligonucleotide of general formula where each B independently denotes adenine, guanine, cytosine, uracil or thymine; each R independently denotes H or hydroxyl and at least one of R denotes hydroxyl; Z denotes H or a phosphate group; and n is an integer between 1 and 100, involving steps A-G, characterised by use of said phosphoramidite derivatives as a monomer compound of nucleic acid at step B.

EFFECT: high yield.

7 cl, 1 dwg, 21 ex

 

The technical field

The present invention relates to a new derivative of phosphoramidite, which introduced a new protective group on the 2'-hydroxyl group, and the reagent for the introduction of this protective group.

The level of technology

Oligoribonucleotide acid (oligo-RNA) are useful as RNA probes for genetic analysis of pharmaceutical substances on the basis of RNA (antisense RNA, ribozymes, RNA for RNA-mediated regulation of gene expression), artificial enzymes, and aptamers (oligonucleotides that exhibit specific binding molecule-target). The method of solid-phase synthesis for the preparation of oligo-RNA created in the late eighties. In the first message related to this method, used derivatives of phosphoramidites withtert-butyldimethylsilyl group (TBDMS) or triisopropylsilyl group (TIPS) as a protective group for the 2'-hydroxyl group (non-patent document 1).

In the chemical synthesis of oligo-RNA is present far more problems than chemical synthesis oligodeoxyribonucleoside acids (oligo-DNA), consisting only of deoxyribonucleotides.

For example, using the TBDMS group as a protective group for the 2'-hydroxyl group can cause a side reaction, in which the group TBDMS protecting the 2'-hydroximino the group, migrates to the 3'-hydroxyl group during fosfaurilirovania 3'-hydroxyl group. In addition, the use of volumetric substituent, such as a TBDMS group, as a protective group for the 2'-hydroxyl group, can reduce the rate of the condensation reaction in the formation mezhnukleotidnyh due spatial difficulties around the phosphorus atom in the 3'-position, possibly leading to rupture or rearrangement mezhnukleotidnyh during removal of the protective group for the 2'-hydroxyl group after oligomerization.

In order to overcome the above problems, currently exploring more effective ways of synthesis of oligo-RNA.

As a protective group for the 2'-hydroxyl group of the known 1-(2-cyanoethoxy)ethyl (CEE) group, which is removed along with the 3'- and 5'-protective bis-silyl group in a neutral environment, ensuring the destruction of bis-silyl protective group (non-patent document 2).

Based on this information, Wada has developed a derivative of phosphoramidite to obtain oligo-RNA, in which the group CEE, which can be removed in a neutral environment, is introduced at the 2'-hydroxy-group (non-patent document 3 and non-patent document 4). However, since the introduction of the group CEE in position 2'-hydroxyl group leads to the formation of a new asymmetric center ol the th-RNA, in which the 2'-hydroxyl group is protected by a group CEE represent diastereoisomer mixture. Therefore, the purification and isolation of the desired oligo-RNA are more complex. In addition, since the oligo-RNA, which was introduced in the group CEE, have a methyl group on the carbon attached to the oxygen atom at the 2'position, we should expect the emergence of a spatial difficulties around the phosphorus atom attached to the 3'-hydroxyl group, which is causing concerns about a decrease in the efficiency of condensation and reaction rate of condensation.

Non-patent document 1: N.A. Usman et al., Journal of the American Chemical Society, Vol. 109, 7845 (1987).

Non-patent document 2: Wolfgang Pfleiderer et al., Helvetica Chimica Acta, Vol. 81, 1545 (1998).

Non-patent document 3: Takeshi Wada, Bioindustry, Vol. 21, No. 1, 17 (2004).

Non-patent document 4: T. Umemoto et al., Tetrahedron Letters, Vol. 45, 9529 (2004).

DISCLOSURE of INVENTIONS

A task that should be solved by the invention of

The main objective of the present invention is to provide a useful and new derived phosphoramidite for simple and high-yielding method for the synthesis of oligo-RNA. Another objective of the present invention is to provide a new essential connection that can be used to attach the protective group to the 2'-hydroxyl is Noah group of ribose, thus, the protecting group can be removed in a neutral environment.

TOOLS TO SOLVE PROBLEMS

After an intense and persistent research, the authors of the present invention have found a connection through which you can achieve the above purposes, and thus to carry out the present invention.

I. Derived phosphoramidite of the present invention

The present invention may include a derivative of phosphoramidite represented by the following General formula (1) (hereafter in this document referred to as derived phosphoramidite of the present invention).

in which

Bxrepresents a nucleic acid base which may have a protective group;

R1is a Deputy, represented by the following General formula (2)

in which

R11, R12and R13are the same or different and each represents hydrogen or alkoxygroup.

R2aand R2bare the same or different, and each represents an alkyl group, or R2aand R2btaken together with the adjacent nitrogen atom, may form a 5-6-membered saturated cyclic amino group, with a cyclic amino group optionally has an oxygen atom or sulfur with the cave cycle in addition to the adjacent nitrogen atom; and WG1and WG2are the same or different, and each represents an electron-withdrawing group.

Examples of nucleic bases Inxnot specifically limited as long as it represents a nucleic acid base used in the synthesis of nucleic acid, and may include, for example, adenine, guanine, cytosine, uracil or modified form.

A modified form of the nucleic base connection means, in which the nucleic base carries one or more arbitrary substituents.

Examples of the substituent for the modified form Inxmay include halogen, acyl group, alkyl group, arylalkyl group, alkoxygroup, alkoxyalkyl group, a hydroxy-group, amino group, monoalkylamines, dialkylamines, carboxypropyl, cyano and nitro-group. A modified form of Inxmay be substituted by one to three such substituents.

Nucleic basexcan be protected. Especially it is preferable that was protected amino group of the nucleic base with the amino group, such as adenine, guanine and cytosine.

The protective group of the amino group is not particularly limited as long as she represents a protective group used as a protective group of the nucleic acid is you, and it may include, for example, benzoyloxy group, 4-methoxybenzoyl group, acetyl group, propionyl group, butyryloxy group, isobutyryloxy group, phenylacetyl group, phenoxyacetyl group, 4-tert-butylphenoxyacetyl group, 4-isopropylthioxanthone group, and (dimethylamino)methylene group.

Examples of the saturated cyclic amino group, R2may include pyrrolidin-1-ilen group, piperidine-1-ilen group, morpholine-1-ilen group and thiomorpholine-1-ilen group.

Electron-withdrawing group WG1and WG2may include a cyano, a nitro-group, alkylsulfonate and halogen. Among them cyano is preferred.

Examples of the halogen derivative of phosphoramidite of the present invention may include fluorine, chlorine, bromine and iodine.

Examples of acyl groups derived phosphoramidite of the present invention can include linear or branched alkanoyloxy group having 1-6 carbon atoms, and arilou group having 7-13 carbon atoms. More specifically, the acyl group may include, for example, formyl group, acetyl group, n-propionyloxy group, isopropylamino group, n-butanoyloxy group, isobutylamino group,tert-butanoyloxy group, virolog group, hexanoyl group, benzoyloxy group, n is foilow group and levelingaion (ß-acetylphenyl) group.

Examples of alkyl groups derived phosphoramidite of the present invention can include linear or branched alkyl group having 1-5 carbon atoms. More specifically, the alkyl group may include, for example, methyl group, ethyl group, n-sawn group, isopropyl group, n-boutelou group, isobutylene group,Deut-boutelou group,tert-boutelou group, n-pentelow group, isopentyl group, neopentyl group andtert-pentelow group. The alkyl group may be substituted, and examples of the substituent may include halogen, alkyl group, alkoxygroup, cyano and nitro-group. The alkyl group may be substituted by one to three such substituents.

Examples of alkyl part arylalkyl group, alkoxyalkyl group, monoalkylamines, dialkylamines and alkylsulfonate in derived phosphoramidite of the present invention may include alkyl group, identical to the above.

Examples of alkoxygroup derived phosphoramidite of the present invention can include linear or branched alkoxygroup having 1-4 carbon atoms. More specifically, alkoxygroup may include, for example, methoxy group, ethoxypropan, n-propoxylate, isopropoxy, n-butoxypropyl, isobutoxy,Deut-Butoh is the system of groups and tert-butoxypropan. Among them alkoxygroup having 1-3 carbon atoms, are preferred, and methoxy group is more preferred.

Examples alkoxides alkoxyalkyl group derived phosphoramidite of the present invention may include alkoxygroup identical to the above.

Examples of aryl part arylalkyl group derived phosphoramidite of the present invention may include aryl groups having 6 to 12 carbon atoms.

Specifically, the aryl group may include, for example, phenyl group, 1-naftalina group, 2-naftalina group and biphenylene group. The aryl group may be substituted, and examples of the substituent may include halogen, alkyl group, alkoxygroup, cyano and nitro-group. Aryl group may be substituted by one to three such substituents. Examples of halogen, alkyl groups and alkoxygroup, which substituents are alkyl or aryl groups derived phosphoramidite of the present invention, may include, respectively, of the group, is identical to the above.

Derived phosphoramidite of the present invention can be used as a reagent to obtain oligo-RNA. Derived phosphoramidite of the present invention is a derivative of phosphoramidite with security is the Republican broadcasting type in position 2'-hydroxyl group, that can be removed in a neutral environment. In addition, derived phosphoramidite of the present invention differs in that the condensation reaction proceeds in a shorter time and leads to better output during the synthesis of the oligo-RNA compared to traditional derivative of phosphoramidite. This is because the protective group of the ether type, introduced at the 2'-hydroxyl group, is a linear protecting group and, therefore, does not create steric space around the phosphorus atom attached to the 3'-hydroxyl group. Derived phosphoramidite the present invention makes it possible to obtain oligo-RNA of high purity by the way, is essentially identical to the method used in the production of oligo-DNA.

In this document the term "oligo-DNA" means oligonucleosomal acid, having only deoxyribonucleotides. Moreover, in this document the term "oligo-RNA" means oligonucleosomal acid containing at least one ribonucleotide, and which may also have one or more deoxyribonucleotides.

Specific examples of the derivative of phosphoramidite of the present invention can include the following compounds 1-5:

1.

N6-acetyl-5'-O-(4,4'-dimethoxytrityl)-2'-O-(2-cyanoethoxy)adenosine 3'-O-(2-cyanoethyl N,N-diisopropyl sporadic)

2.

N2-acetyl-5'-O-(4,4'-dimethoxytrityl)-2'-O-(2-cyanoethoxy)guanosine 3'-O-(2-cyanoethyl N,N-diisopropylphosphoramidite)

3.

N2-phenoxyacetyl-5'-O-(4,4'-dimethoxytrityl)-2'-O-(2-cyanoethoxy)guanosine 3'-O-(2-cyanoethyl N,N-diisopropylphosphoramidite)

4.

N4-acetyl-5'-O-(4,4'-dimethoxytrityl)-2'-O-(2-cyanoethoxy)citizen 3'-O-(2-cyanoethyl N,N-diisopropylphosphoramidite)

5.

5'-O-(4,4'-dimethoxytrityl)-2'-O-(2-cyanoethoxy)uridine 3'-O-(2-cyanoethyl N,N-diisopropylphosphoramidite)

BRIEF DESCRIPTION of DRAWINGS

The drawing shows a chromatogram obtained by high performance liquid chromatography (HPLC) with reversed phase.

In the drawing, the vertical axis indicates time (min) and the horizontal axis indicates the optical absorption.

The BEST OPTION of carrying out the INVENTION

II. The method of deriving phosphoramidite of the present invention

Derived phosphoramidite of the present invention can be obtained as follows.

The following method of obtaining in those cases when the original substance have a Deputy that has an effect on the reaction (for example, hydroxyl group, amino group and carboxypropyl), these source materials traditionally used in the reaction, protecting them suitable protective group in accordance with known JV the way.

After completion of the reaction, the protective group can be removed by a known method such as catalytic reduction, alkali treatment, acid treatment or the like Derived phosphoramidite of the present invention can be obtained from known compounds or intermediate compounds, which can be easily obtained, for example, by the following steps a-h.

The method of deriving phosphoramidite of the present invention are described in detail below.

(1) phase a:

The process of obtaining the derivative of a nucleoside represented by the following General formulas (15) and (15'), where the protective group of the ether type, which can be removed in a neutral environment, type of 2'-hydroxyl group by exposure to alkylating reagent on a derivative of the nucleoside represented by the following General formula (14)

where

Inx, R1and WG1such as defined above.

Examples of the alkylating reagent can include an ether compound represented by the following General formula (13)

in which

L represents a halogen, killigrew, alkylsulfonyl group or alkylthiols; and WG1such as defined above.

Examples of halogen, aryl part of aristocraty, and alkyl parts alcalali kidney group and ancilliary L can respectively include the same halogen, aryl and alkyl groups as halogen, aryl and alkyl groups derived phosphoramidite of the present invention.

Specific examples of the ether compound (13) may include the following compounds 1 and 2:

1. Chloromethyl 2-cyanoethylene ether

2. 2-Cyanoethylation ether

The ether compound (13) is a new alkylating reagent, which can be entered by the Deputy ether type, which is removed in a neutral environment, in position 2'-hydroxyl group in an alkaline medium, and which is useful as a reagent for obtaining a derivative of phosphoramidite of the present invention.

The ether compound (13) can be obtained through the following stages 1-4.

Stage 1:

The process of obtaining the compound represented by the following General formula (24), by alkyltrimethylenedi alcohol compounds represented by the following General formula (20)

in which

WG1the same as defined above; and

R3represents an alkyl or aryl group.

The compound (24) is an essential connection (13), where L is alkylthiophene.

Examples of alkyl groups for R3may include the same alkyl group as alkyl group derived phosphoramidite of the present invention.

In those cases where R3represents a methyl group, examples alkyltrimethylenedi reagent may include a mixed solvent containing dimethyl sulfoxide, acetic anhydride and acetic acid. The amount of dimethyl sulfoxide, which should be used can vary within the range of 10-200 mol per mol of compound (20) and preferably 20 to 100 mol per mol of compound. The amount of acetic acid, which should be used can vary within the range of 10-150 mol per mol of compound (20) and preferably 20 to 100 mol per mol of compound. The amount of acetic anhydride, which is to be used, may be in the range of 10-150 mol per mol of compound (20) and preferably 20 to 100 mol per mol of compound. The reaction temperature preferably ranges range 0-100ºC. The reaction time varies depending on the type of starting compounds and the reaction temperature, and is preferably from 1 to 48 hours.

Stage 2:

The process of obtaining the compound represented by the following General formula (25), by halogenation of the compound (24).

in which

WG1and R3such as defined above; and

X2represents a halogen.

The compound (25) is a compound where L ether compound(13) is a halogen.

Examples of the halogen in the X2may include the same halogen as the halogen derivative of phosphoramidite of the present invention.

This stage can be performed in a known manner (for example, T. Benneche et al., Synthesis 762 (1983)).

Examples of the solvent to be used is not particularly limited, except in those cases when it is involved in the reaction, and may include, for example, halogenated hydrocarbons such as dichloromethane, chloroform, carbon tetrachloride and 1,2-dichloroethane.

Examples of the halogenation agent may include sulfurylchloride and phosphorus oxychloride.

The amount of halogenation agent, which must be used, respectively, may vary within the range of 1-20 mol per mol of compound (24) and preferably 1-10 mol per mol of compound. The reaction temperature preferably ranges range 0-100ºC. The reaction time varies depending on the type of starting compounds and the reaction temperature, and is preferably from 30 min to 24 hours.

Stage 3:

The process of obtaining the compound represented by the following General formula (25A), by Aristolochia connection (25).

in which

WG1and X2such as defined above; and

R3Arepresents an aryl group.

Connection (25A) of atrial is to place a connection class ether compounds (13), where L is arylthioureas. Examples of aryl groups for R3Amay include aryl group, identical aryl group derived phosphoramidite of the present invention. This stage can be performed in a known manner. Examples of the solvent which must be used in a special way limit, except in those cases when it is involved in the reaction, and may include, for example, dichloromethane and acetonitrile. Examples arylthioureas reagent may include thiophenol and 4-methylthiophenol. The number arylthioureas reagent, which should be used can vary within the range of 1-20 mol per mol of compound (25) and preferably 1-5 mol per mol of compound. The reaction temperature is preferably in the range of 0-100ºC. The reaction time varies depending on the type of starting compounds and the reaction temperature, and is preferably from 1 to 48 hours.

Stage 4:

The process of obtaining the compound represented by the following General formula (24A), by oxidation of the compound (24).

in which

WG1and R3such as defined above.

The compound (24A) is a compound of the class of essential compounds (13), where L is alkylsulfonate group. Examples of the alkyl groups of R3may include alkaline the group, identical alkyl group derived phosphoramidite of the present invention.

This stage can be performed in a known manner. Examples of the solvent to be used is not particularly limited, except in those cases when it is involved in the reaction, and may include, for example, dichloromethane, chloroform, and methanol. Examples of the oxidizing agent may include metallocarborane acid, metaperiodate salt and hydrogen peroxide. The amount of oxidizer, which should be used can vary within the range of 1-10 mol per mol of compound (24) and preferably 1-2 mol per mol of compound. The reaction temperature is preferably in the range of 0-100ºC. The reaction time varies depending on the type of starting compounds and the reaction temperature, and is preferably from 1 to 48 hours.

In those cases, when the alkylating reagent is used as a compound (25), this stage can be performed as follows.

Stage can be performed by reacting the alkylating reagent and a base, with compound (14), which is commercially available or is synthesized in accordance with the known method.

Examples of the solvent which must be used in a special way limit, except in those cases when it is involved in the reaction,and may include, for example, halogenated hydrocarbons such as dichloromethane, chloroform, carbon tetrachloride and 1,2-dichloroethane. The amount of alkylating reagent, which should be used can vary within the range of 1-20 mol per mol of compound (14) and preferably 1-10 mol per mol of compound. At this stage optionally alkylating reagent may be subjected to the reaction by means of an intermediate compound produced by the interaction of the metal-containing reagent and a base, with compound (14). Examples of metal-containing reagent may include dibutyltindilaurate. The amount of metal-containing reagent to be used may vary within the range of 1-20 mol per mol of compound (14) and preferably 1-10 mol per mol of this compound. Examples of the base may include an organic base such as pyridine, 2,6-dimethylpyridine, 2,4,6-trimethylpyridine, N-Mei, triethylamine, tributylamine, N,N-diisopropylethylamine and 1,8-diazabicyclo[5.4.0]-7-undecene. The amount of base that should be used can be in the range of 1-20 mol per mol of compound (14) and preferably 1-10 mol per mol of compound. The reaction temperature preferably ranges within the range of 0 to 120 ºC. The reaction time varies depending on the type of starting compounds and the temperature of the reaction and is preferably from 30 min to 24 hours.

In those cases, when the alkylating reagent is used as a compound (24) or (25), this stage can be performed as follows.

Stage can be performed by reacting an alkylating reagent, acid and reagent for halogenation of the sulfur atom with the compound (14), which is commercially available or is synthesized in accordance with a known method (e.g., M. Matteucci, Tetrahedron Letters, Vol. 31, 2385 (1990)). The amount of alkylating reagent, which should be used can vary within the range of 1-5 mol per mol of compound (14) and preferably of 1.05 to 3 mol per mol of compound. Examples of the acid may include triftormetilfullerenov, triftorbyenzola silver and trimethylsilyltrifluoromethane. The amount of acid that must be used can vary within the range from 0.01 to 20 mol per mol of compound (14) and preferably 0.02 to 10 mol per mol of compound. Examples of the solvent to be used is not particularly limited, except in those cases when it is involved in the reaction, and may include, for example, dichloromethane, chloroform, carbon tetrachloride, 1,2-dichloroethane, benzene, toluene, xylene, tetrahydrofuran, acetonitrile and mixtures thereof. Examples of the halogenation reagent to the sulfur atom, which should be used at this stage, can locate N-bromosuccinimide (NBS) and N-jodatime (NIS). The amount of halogenation reagent to the sulfur atom, which should be used can vary within the range of 1-10 mol per mol of compound (14) and preferably of 1.05 to 5 mol per mol of compound. The reaction temperature is preferably within the range of-78-30ºC. The reaction time varies depending on the type of starting compounds and the reaction temperature, and is preferably from 5 minutes to 5 hours.

In those cases, when the alkylating reagent is used as a compound (24A), this stage can be performed as follows.

Stage can be performed by reacting an alkylating reagent, acid anhydride and a base, with compound (14), which is commercially available or is synthesized in accordance with the known method. The amount of alkylating reagent to be used may be in the range of 1-5 mol per mol of compound (14) and preferably of 1.05 to 3 mol per mol of compound. Examples of the acid anhydride may include the anhydride of triftoratsetata and acetic anhydride. The number of acid anhydride, which is to be used may be within the range from 0.01 to 20 mol per mol of compound (14) and preferably 0.02 to 10 mol per mol of compound. Examples of the base may include tetramethylrhodamine and kallidin. Quantity the quantity of base, which should be used can be within the range from 0.01 to 20 mol per mol of compound (14) and preferably 0.02 to 10 mol per mol of compound. Examples of the solvent to be used is not particularly limited, except in those cases when it is involved in the reaction, and may include, for example, dichloromethane, chloroform, carbon tetrachloride, 1,2-dichloroethane, and mixtures thereof. The reaction temperature preferably ranges range-78-30ºC. The reaction time varies depending on the type of starting compounds and the reaction temperature, and is preferably from 5 min to 24 hours.

(2) stage b:

The process of isolation and purification of the derivative of the nucleoside (15), obtained through the stages (a)

At this stage, a derivative of the nucleoside can be isolated and purified from the mixture obtained through stages (a), by using the standard method of isolation and purification, such as thin layer chromatography, column chromatography on silica gel or the like

(3) stage with:

The process, which is carried out separately from stage b, for connection ribonucleic acid represented by the following General formula (17), where the protective group of the ether type, which can be removed in a neutral environment, type of 2'-hydroxyl group by exposure to alkylating reagent on Obedinenie ribonucleic acid, represented by the following General formula (16)

in which

Inxand WG1such as defined above; and

But remasterise Deputy represented by the following General formula (18a) or (18b)

in which

R6represents an alkyl group.

Examples of alkyl groups for R6may include alkyl group, identical alkyl group derived phosphoramidite of the present invention.

Examples of the alkylating reagent can include alkylating reagents, is identical to the above.

In those cases, when the alkylating reagent is used as a compound (25), this stage can be performed as follows.

Stage can be performed by reacting the alkylating reagent and a base, with compound (16), which is commercially available or is synthesized in accordance with the known method.

Examples of the solvent to be used is not particularly limited, except in those cases when it is involved in the reaction, and may include, for example, halogenated hydrocarbons such as dichloromethane, chloroform, carbon tetrachloride and 1,2-dichloroethane. The amount of alkylating reagent, which is to be used, you may find the change within the range of 1-20 mol per mol of compound (14) and preferably 1-10 mol per mol of compound. At this stage optionally alkylating reagent may be subjected to the reaction by means of an intermediate compound produced by the interaction of the metal-containing reagent and a base, with compound (16). Examples of metal-containing reagent may include dibutyltindilaurate andtert-butylaniline. The amount of metal-containing reagent to be used may be in the range of 1-20 mol per mol of compound (16) and preferably 1-10 mol per mol of this compound. Examples of the base may include an organic base such as pyridine, 2,6-dimethylpyridine, 2,4,6-trimethylpyridine, N-Mei, triethylamine, tributylamine, N,N-diisopropylethylamine and 1,8-diazabicyclo[5.4.0]-7-undecene. The amount of base that should be used can be in the range of 1-20 mol per mol of compound (16) and preferably 1-10 mol per mol of compound. The reaction temperature preferably is within the range of 0 to 120 ºC. The reaction time varies depending on the type of starting compounds and the reaction temperature, and is preferably from 30 min to 24 hours.

In those cases, when the alkylating reagent is used as a compound (24) or (25), this stage can be performed as follows.

Stage can be accomplished through collaboration and caleruaga reagent, acid and reagent for halogenation of the sulfur atom with the compound (16), which is commercially available or is synthesized in accordance with a known method (e.g., M. Matteucci, Tetrahedron Letters, Vol. 31, 2385 (1990)). The amount of alkylating reagent, which should be used can vary within the range of 1-5 mol per mol of compound (16) and preferably of 1.05 to 3 mol per mol of compound. Examples of the acid may include triftormetilfullerenov, triftorbyenzola silver and trimethylsilyl-triftorbyenzola. The amount of acid that must be used can vary within the range from 0.01 to 20 mol per mol of compound (16) and preferably 0.02 to 10 mol per mol of compound. Examples of the solvent that should be used is not particularly limited, except in those cases when it is involved in the reaction, and may include, for example, dichloromethane, chloroform, carbon tetrachloride, 1,2-dichloroethane, benzene, toluene, xylene, tetrahydrofuran, acetonitrile and mixtures thereof. Examples of the halogenation reagent to the sulfur atom, which should be used at this stage may include N-bromosuccinimide (NBS) and N-jodatime (NIS). The amount of halogenation reagent to the sulfur atom, which should be used can vary within the range of 1-10 mol per mol of compound (16) and prefer the Ino of 1.05 to 5 mol per mol of compound. The reaction temperature is preferably within the range of-78-30ºC. The reaction time varies depending on the type of starting compounds and the reaction temperature, and is preferably from 5 minutes to 5 hours.

In those cases, when the alkylating reagent is used as a compound (24A), this stage can be performed as follows.

Stage can be performed by reacting an alkylating reagent, acid anhydride and a base, with compound (16), which is commercially available or is synthesized in accordance with the known method. The amount of alkylating reagent, which should be used can vary within the range of 1-5 mol per mol of compound (16) and preferably of 1.05 to 3 mol per mol of compound. Examples of the acid anhydride may include the anhydride of triftoratsetata and acetic anhydride. The number of acid anhydride, which should be used can vary within the range from 0.01 to 20 mol per mol of compound (16) and preferably 0.02 to 10 mol per mol of compound. Examples of the base may include tetramethylrhodamine and kallidin. The amount of base that should be used can vary within the range from 0.01 to 20 mol per mol of compound (16) and preferably 0.02 to 10 mol per mol of compound. Examples of the solvent which dollars the yen to be used, especially not limited, except in those cases when it is involved in the reaction, and may include, for example, dichloromethane, chloroform, carbon tetrachloride, 1,2-dichloroethane, and mixtures thereof. The reaction temperature is preferably within the range of-78-30ºC. The reaction time varies depending on the type of starting compounds and the reaction temperature, and is preferably from 5 min to 24 hours.

(4) stage d:

The process, which is carried out separately from stages a-C, for connection ribonucleic acid represented by the following General formula (19), by exposure to dimethyl sulfoxide, acetic acid and acetic anhydride in connection ribonucleic acid (16)

in which

A and bxsuch as defined above.

This stage can be carried out by reacting dimethyl sulfoxide, acetic acid and acetic anhydride with the compound (14), which is commercially available or is synthesized in accordance with the known method.

This stage can be carried out by reacting dimethyl sulfoxide, acetic acid and acetic anhydride with the compound (14), which is commercially available or is synthesized in accordance with the known method.

The amount of dimethyl sulfoxide, which should be used is Vano, may vary within the range from 10 to 200 mol per mol of compound (16) and preferably from 20 to 100 times the number of moles per mole of the compound.

The amount of acetic acid, which should be used can vary within the range of 10-150 mol per mol of compound (16) and preferably 20 to 100 mol per mol of compound. The amount of acetic anhydride, which should be used can vary within the range of 10-150 mol per mol of compound (16) and preferably 20 to 100 mol per mol of compound. The reaction temperature preferably is within the range of 10 to 50'C. The reaction time varies depending on the type of starting compounds and the reaction temperature, and is preferably from 30 min to 24 hours.

(5) stage e:

The process for obtaining compounds of ribonucleic acid represented by the following General formula (17), where the protective group of the ether type, which can be removed in a neutral environment, type of 2'-hydroxyl group by exposure to alcohol compounds represented by the following General formula (20), acid and reagent for halogenation of the sulfur atom on a derivative of the nucleoside (19), obtained through stage d.

where

A, bxand WG1such as defined above.

Stage can be carried out by mutual the action of an alcohol compound (20), acid and reagent for halogenation of the sulfur atom with the connection ribonucleic acid (19) in accordance with the known method. Examples of the solvent to be used is not particularly limited, except in those cases when it is involved in the reaction, and may include, for example, dichloromethane, chloroform, carbon tetrachloride, 1,2-dichloroethane, benzene, toluene, xylene, tetrahydrofuran, acetonitrile and mixtures thereof. The amount of alcohol compounds (20), which should be used can be in the range of 1-20 mol per mol of compound (19) and preferably 1-10 mol per mol of compound. Examples of the acid may include triftormetilfullerenov, triftorbyenzola silver and trimethylsilyl-triftorbyenzola. Examples of the halogenation reagent to the sulfur atom may include N-bromosuccinimide (NBS) and N-jodatime (NIS). The amount of halogenation reagent to the sulfur atom, which should be used can vary within the range of 0.1 to 20 mol per mol of compound (19) and preferably 0.2 to 10 mol per mol of compound. The reaction temperature is preferably within the range of-100-20ºC. The reaction time varies depending on the type of starting compounds and the reaction temperature, and is preferably from 5 minutes to 12 hours.

(6) stage f:

The process for obtaining compounds of ribonukleinova acid, represented by the following General formula (21), by removing the protective groups for the 3'and 5'-hydroxyl groups of the compounds of ribonucleic acid (17), obtained in stage C or stage that is

in which

A, bxand WG1such as defined above.

This stage can be carried out by dissolving compound (17) in an organic solvent and interaction with the fluorinating agent and the acid in the form of a mixture with an arbitrary mixing ratio. Examples of the fluorinating agent to be used at this stage may include ammonium fluoride, fluoride, Tetra-n-butylamine (TBAF), triethylamine-trihydrochloride, peridiniidae. The amount of the fluorinating agent to be used can vary within the range of 0.1 to 20 mol per mol of compound (17), and preferably 0.2 to 10 mol per mol of compound. The reaction temperature preferably is within the range of 0 to 120 ºC. The reaction time varies depending on the type of starting compounds and the reaction temperature, and is preferably from 30 min to 24 hours.

(7) stage g:

The process for obtaining compounds of ribonucleic acid (15) by introducing a protective group (R1), which can be removed in an acidic medium, in the 5'-hydroxyl group of the compounds of ribonucleic acid (21), polucen the th stage through f.

in which

A, bx, R1and WG1such as defined above; and

X3represents a halogen.

Examples of the halogen for X3may include halogen, identical to the halogen derivative of phosphoramidite of the present invention. Stage can be performed by reacting R1X3with compound (21) in accordance with the known method. The number R1X3that should be used can be in the range of 1-20 mol per mol of compound (21) and preferably 1-10 mol per mol of compound. Examples of the solvent to be used is not particularly limited, except in those cases when it is involved in the reaction, and may include, for example, acetonitrile and tetrahydrofuran. Examples of the base may include an organic base such as pyridine, 2,6-dimethylpyridine, 2,4,6-trimethylpyridine, N-Mei, triethylamine, tributylamine, N,N-diisopropylethylamine and 1,8-diazabicyclo[5.4.0]-7-undecene. The amount of base that should be used can vary within the range of 1-20 mol per mol of compound (21) and preferably 1-10 mol per mol of compound. The reaction temperature preferably is within the range of 0 to 120 ºC. The reaction time varies depending on the type of the original substances, the reaction temperature preferably ranges from 30 min to 24 hours.

(8) stage h:

The process of obtaining the derivative of phosphoramidite of the present invention posttaliban 3'-hydroxyl group by exposure hospitilised reagent and, if necessary, the activating agent on the derivative of the nucleoside (15)obtained in stage b or stage f.

where

Inx, R1, R2a, R2b, WG1and WG2such as defined above.

Examples hospitilised reagent may include compounds represented by the following General formulas (22) and (23)

in which

R2a, R2band WG2such as defined above; and

X1represents a halogen.

Examples of the halogen for X1may include halogen, identical to the halogen derivative of phosphoramidite of the present invention. This stage represents the reaction fosfaurilirovania 3'-hydroxyl group by reacting hospitilised reagent with compound (15) and can be carried out in accordance with the known method. If necessary, can be used activating agent. Examples of the solvent to be used is not particularly limited, except in those cases when it is involved in the reaction, and may include, for example, acetonitrile and tetrahydrofuran.

The number fo vitiligo reagent, which should be used can vary within the range of 1-20 mol per mol of compound (15) and preferably 1-10 mol per mol of compound. Examples of the activating agent may include 1H-tetrazole, 5-ethylthioethyl, 4,5-dichloroimidazole, 4,5-dicyanoimidazole, benzotriazolyl, imidazolium, peridiniella, N,N-diisopropylethylamine and 2,4,6-kallidin/N-Mei. The amount of the activating agent to be used may be in the range of 1-20 mol per mol of compound (15) and preferably 1-10 mol per mol of compound. The reaction temperature preferably is within the range of 0 to 120 ºC. The reaction time varies depending on the type of starting compounds and the reaction temperature, and is preferably from 30 min to 24 hours. Derived phosphoramidite the present invention thus obtained can be isolated and purified by a method known in nature, such as the concentration, the conversion in the liquid phase, separation, extraction with solvent, crystallization, recrystallization, fractional distillation or chromatography.

III. The way to obtain oligo-RNA

The present invention may include a method of obtaining oligo-RNA, represented by the following General formula (3), including the use of derived phosphoramidite of the present invented the I.

The details are described below.

in which

each independently represents adenine, guanine, cytosine, uracil, thymine or modified form;

each R independently represents H or a hydroxyl group, and at least one R is a hydroxyl group;

Z represents H or phosphate group; and

n is an integer from 1 to 100.

Preferably n is an integer from 10 to 50, and more preferably an integer from 15 to 30.

Examples of the substituent for the modified form may include halogen, acyl group, alkyl group, arylalkyl group, alkoxygroup, hydroxyl group, amino group, monoalkylamines, dialkylamines, carboxypropyl, cyano and nitro-group; and a modified form may be substituted by 1-3 of such substituents. Examples of halogen, aryl groups, alkyl groups, arylalkyl group, alkoxygroup, alkoxyalkyl group, amino group, monoalkylamines and dialkylamines for modified forms can include groups that are identical to corresponding groups derived phosphoramidite of the present invention.

The way to obtain oligo-RNA (3) by using the derivative of phosphoramidite of the present invention may be implementing the known method and, for example, can be carried out by condensation of compounds of the monomer of nucleic acid from the 3' to 5' step by step in accordance with the following stages A-G.

Compounds and reagents, which must be used in the next stage, except derived phosphoramidite of the present invention, not particularly limited, as they usually use in the synthesis of oligo-RNA or oligo-DNA. In addition, all stages can be performed using an automatic synthesizer for DNA or manually as in the case of the use of traditional substances for the synthesis of nucleic acids. The use of an automated synthesizer is desirable from the standpoint of convenience and simplicity of the method and the accuracy of the synthesis. Compounds and reagents described in the following stages a to G, except for the connection of the monomer of nucleic acid is not particularly limited, as they usually use in the synthesis of oligo-DNA or oligo-RNA.

(1) phase A:

The process of obtaining the compound represented by the following General formula (5), by deleting the 5'-hydroxyl group of the compounds represented by the following General formula (4), under the action of acid.

in which

n, R1and WG2such as defined above;

each independently represents hell is in, guanine, cytosine, uracil, thymine or modified form; and each R4independently represents H, alloctype or Deputy, represented by the following General formula (6)

in which

WG1the same as defined above; and

E represents an acyl group or a Deputy, represented by the following General formula (7)

-Q-linker-solid media (7),

in which

Q represents a single bond or a Deputy, represented by the following General formula (8)

in which

WG2the same as defined above; and

T represents H, alloctype, Deputy represented by the above General formula (6) or (7), provided that either E or T is Deputy (7).

Stage carried out by the action of the acid on the compound represented by the following General formula (26a), (26b) [compound (4), where n is 1], which is attached to the solid carrier, or oligo-RNA or oligo-DNA, obtained by executing processes stage a, stage D [compound (4), where n is 2-100], which is attached to a solid carrier (hereafter in this document referred to as the compound attached to the solid media).

in which

xand R1such as defined above;

R2Land R4Lrepresent a Deputy (7);

R2is alloctype; and

R4represents H, alloctype or Deputy (6).

Examples of the acyl part of alloctype R2and R4may include acetyl group, propionyl group, butanoyloxy group, isobutylamino group, benzoyloxy group, 4-methoxybenzoyl group, phenylacetyl group, phenoxyacetyl group, 4-tert-butylphenoxyacetyl group and 4-isopropylthioxanthone group. Examples of the solid carrier can include a glass with a specified pore size (CPG), oxacilline glass with a specified pore size (oxalyl-controlled pore glass (see, e.g., Alul, et al., Nucleic Acids Research, Vol.19, 1527 (1991)), the carrier of TentaGel - based media derived aminopenicillins (see, for example, Wright et al., Tetrahedron Letters, Vol.34, 3373 (1993)) and the porous copolymer of polystyrene and divinylbenzene. Examples of the linker may include 3-aminopropyl group, succinimido group, 2,2'-decanesulfonate and alkylamino long chain (LCAA). Compound (26a) and (26b), which are attached to the solid carrier are in accordance with the known method or acquire on sale, and examples of the preferred option implementation is the connection represented with edowski General formula (27) or (28)

in which

Inx, R1, R4and WG2such as defined above.

Connection (27) and (28), where R4Deputy (6), can be obtained from the derivative of phosphoramidite of the present invention in accordance with the known method. Examples of acids that should be used at this stage may include triperoxonane acid, dichloracetic acid, trichloroacetic acid. Acid, which must be used at this stage, may be diluted in a suitable solvent so as to have a concentration of 1-5%. Examples of the solvent is not specifically limited, except in those cases when it is involved in the reaction, and may include, for example, dichloromethane, acetonitrile, water and mixtures thereof. The reaction temperature preferably is within the range of 20-50'C. The reaction time varies depending on the type of acid and the reaction temperature, and is preferably from 1 min to 1 hour.

The amount of reagent that should be used, preferably varies within the range of 1-100 mol per mol of compound attached to the solid carrier, and more preferably 1-10 mol per mol of compound attached to a solid carrier.

(2) stage:

The process of obtaining the compound represented trail is the fact that the General formula (9), by condensation of Monomeric compounds of the nucleic acid with the compound obtained by the stage And, using an activating agent.

where

Inx, E, n, R1, R4, T and WG2such as defined above.

This stage can be carried out by reacting compounds of the monomer of nucleic acid and the activating agent with the compound attached to the solid carrier. Examples of Monomeric compounds of nucleic acids may include derived phosphoramidite of the present invention and the compound represented by the following General formula (29), which is commercially available.

in which

R1, R2A, R2band WG2such as defined above; and

Inyrepresents a nucleic acid base which may have a protective group.

Examples of nucleic bases Inynot particularly limited, as it represents a nucleic acid base, used for synthesis of nucleic acid, and may include, for example, adenine, guanine, cytosine, thymine, and their modified form. The modified form is identical to the modified form defined above for the Inx. Examples of the substituent for the modified form of the Bymay include halogen, al the ilen group, arylalkyl group, alkoxygroup, hydroxyl group, amino group, monoalkylamines, dialkylamines, carboxypropyl, cyano and nitro-group; and a modified form of Inymay be substituted by one to three such substituents.

Examples of halogen, aryl groups, alkyl groups, arylalkyl group, alkoxygroup, alkoxyalkyl group, amino group, monoalkylamines and dialkylamines for a modified form Inycan include groups that are identical to corresponding groups derived phosphoramidite of the present invention.

Nucleic basexcan be protected, and, in particular, nucleic acid base having an amino group (e.g., adenine, guanine, cytosine) preferably can be protected at the amino group. The protective group of the amino group in Inycan include groups, identical protective groups of an amino group in Inx.

Examples of the activating agent may include activating agents, is identical to the above.

Examples of the solvent for this reaction is not particularly restricted, excepting only those cases where he is involved in the reaction, and may include, for example, acetonitrile and tetrahydrofuran. The reaction temperature preferably is within the range of 20-50'C.

The reaction time varies depending the barb on the type of activating agent and the reaction temperature, and is preferably from 1 min to 1 hour. The amount of agent that must be used, preferably varies within the range of 1-100 mol per mol of compound attached to the solid carrier, and more preferably 1-10 mol per mol of compound attached to a solid carrier.

(3) stage With:

The process of kupirovaniya 5'-hydroxyl groups unreacted compound (5) at the stage of the Century

where

BxE, n, R4, T and WG2such as defined above; and

R5represents methyl or phenoxymethyl group.

This stage represents the reaction for the introduction of protection for 5'-hydroxyl groups, non-reacted in stage (C), and can be carried out by reacting kapiruumi agent with the compound attached to the solid carrier. Examples kapiruumi agent may include acetic anhydride and ferociously anhydride. Capituli agent that should be used can be diluted in a suitable solvent so as to have a concentration of 0.05-1 M examples of the solvent are not particularly limited, except in those cases when it is involved in the reaction, and may include, for example, pyridine, dichloromethane, acetonitrile, tetrahydrofuran and mixtures thereof. In addition, at this stage, if necessary, can be used, for example, 4-dimethyl inoperation, N-Mei as a reaction accelerator. The reaction temperature preferably is within the range of 20-50'C. The reaction time varies depending on the type kapiruumi agent and the reaction temperature, and is preferably from 1 to 30 minutes, the Amount of agent that must be used, preferably varies within the range of 1-100 mol per mol of compound attached to the solid carrier, and more preferably 1-10 mol per mol of compound attached to a solid carrier.

(4) stage D:

The process of turning the phosphoryl group in the phosphate group by reacting the oxidizing agent with compound (9)obtained in stage C.

where

BxE, n, R1, R4, T and WG2such as defined above.

This stage represents a reaction for the conversion of the trivalent phosphorus to pentavalent phosphorus through the oxidizer can be carried out by reacting the oxidizing agent with the compound attached to the solid carrier. Examples of the oxidizing agent may include iodine andtert-butylhydroperoxide.

In addition, the oxidizer, which should be used can be diluted in a suitable solvent so as to have a concentration of 0.05-1 M examples of the solvent are not particularly limited, except in those cases when the and he is involved in the reaction, and may include, for example, pyridine, tetrahydrofuran, water and their mixtures. For example, can be used iodine/water/pyridine-tetrahydrofuran, iodine/pyridine-acetic acid and the agent for peroxide oxidation (tert-butylhydroperoxide/methylene chloride and the like). The reaction temperature preferably is within the range of 20-50'C. The reaction time varies depending on the type of oxidizing agent and the reaction temperature, and is preferably from 1 to 30 minutes, the Amount of agent that must be used, preferably varies within the range of 1-100 mol per mol of compound attached to the solid carrier, and more preferably 1-50 mol per mol of compound.

(5) stage E:

The process of detachment of the compound (11)obtained in stage D, from the solid substrate, and then removing the protective groups each nucleic bases and each 2'-hydroxyl group.

where

B, Bx, E, R, R1, R4, n, T, WG2and Z are such as defined above.

Stage of detachment is a reaction of removal of oligo-RNA having the desired chain length, from solid media and the linker via a chip off the agent and which is carried out by adding a chip off the agent to a solid carrier, which contains oligo-RNA having the desired chain length.

N is this stage can be removed protective group of nucleic bases. Examples of chip off the agent can include a concentrated aqueous solution of ammonia and methylamine. Chip off the agent that should be used at this stage, may be diluted, for example, methanol, ethanol, isopropyl alcohol, acetonitrile, tetrahydrofuran and mixtures thereof. Among them, ethanol is preferred. The reaction temperature may be within the range of 15-75°C., preferably 15-30°C., and more preferably 18-25°C. the Time of reaction for removing the protective groups may vary within the range of 1-30 hours, preferably 1-24 hours, and more preferably 1-4 hours. The concentration of ammonium hydroxide in the solution, which should be used for removing the protective groups may be 20-30% by weight, preferably 25-30% by weight, more preferably from 28 to 30 mass%. The amount of agent that must be used can range from 1 to 100 mol per mol of compound attached to the solid carrier, and preferably from 10 to 50 times the number of mol per mol of compound. The stage of removal of the protective group for the 2'-hydroxyl group is carried out through the actions of the agent for the removal of the protective group for the 2'-hydroxyl group, such as tetrabutylammonium fluoride, trihydrated/salt of triethylamine. Examples of the solvent are not particularly limited, except in those cases when it is involved in the reaction, and may include, for example, tetrahydrofuran, N-organic, pyridine, dimethylsulfoxide, and mixtures thereof. If necessary, as a decontamination agent for Acrylonitrile, which is a by-product at this stage, can be added alkylamino, amicin, thiol, thiol derivatives, or mixtures thereof. Examples of the alkylamine may include the unbranched alkylamino having 1-6 carbon atoms. In particular, alkylamine may include, for example, methylamine, ethylamine, n-Propylamine, n-butylamine, n-pentylamine, n-hexylamine. Examples of amidine may include benzamidine and formamidine. Examples of the thiol may include non-branched thiol having 1-6 carbon atoms. In particular, thiol may include, for example, methyl, ethanthiol, 1-propanethiol, 1-butanethiol, 1-pentanethiol and 1-hexanethiol. Examples of the derivative of the thiol may include the same or different alcohol and ether containing unbranched alkylthio having 1-6 carbon atoms. In particular, the derived thiol may include, for example, 2-mercaptoethanol, 4-mercapto-1-butanol, 6-mercapto-1-hexanol, mercaptoethylamine ether, 2-mercaptoethyl ether, 3-mercaptopropionyl ether, 4-mercaptopropyl ether, 5-mercaptoethylamine ether and 6-mercaptohexanol ether. The reaction temperature may preferably be within the range of 20 to 80. The reaction time varies depending on the type of agent for the removal of the protective group, which should be used, and the reaction temperature and preferably varies within the range of 1-100 hours. The amount of agent that must be used, preferably varies within the range of 50-500 mol per mol removable protective group, and more preferably 50 to 100 mol per mol removable protective group. Oligo-RNA-protected 5'-hydroxyl group, can be isolated and purified from the above reaction mixture by standard methods of separation and purification such as extraction, concentration, neutralization, filtration, centrifugation, recrystallization, column chromatography on silica gel, thin layer chromatography, hydrophobic column chromatography, ion-exchange column chromatography, gel-filtration column chromatography, dialysis, ultrafiltration, etc.

(6) stage F:

The process of removing the 5'-hydroxyl group in the compound (12)obtained in stage E.

where

B, n, R, R1and Z such as the substituents defined above.

This stage represents a reaction for the final removal of the protective group for 5'-hydroxyl group in oligoribonucleotide and can be carried out by the action of the acid on oligo-RNA, derived from solid media. Examples of acid, which is should be used at this stage, may include trichloroacetic acid, dichloracetic acid and acetic acid. At this stage, can be used acid, diluted in a suitable solvent. Examples of the solvent are not particularly limited, except in those cases when it is involved in the reaction, and may include, for example, dichloromethane, acetonitrile, water, buffer solution, pH of which varies from 2 to 5, and mixtures thereof. Examples of the buffer solution may include acetate buffer solution. The reaction temperature preferably ranges within the range of 20-50'C. The reaction time varies depending on the type of acid and the reaction temperature, and is preferably from 1 min to 1 hour. The amount of agent that must be used, preferably varies within the range of 1-100 mol per mol of compound attached to the solid carrier, and more preferably 1-10 mol per mol of compound.

(7) stage G:

The process of isolation and purification of compound (3)obtained in stage F.

Stage separation and purification is a stage of extraction and purification of the desired oligo-RNA from the above reaction mixture by known isolation and purification, which may include, for example, extraction, concentration, neutralization, filtration, separation by centrifugation, re is cristallization, column chromatography with reversed phase (C8-C18), the cartridge-column reversed-phase (C8-C18), cation-exchange column chromatography, anion-exchange column chromatography, gel-filtration column chromatography, high performance liquid chromatography, dialysis, ultrafiltration, and combinations thereof. Examples of the solvent may include acetonitrile, methanol, ethanol, isopropyl alcohol, water and a solvent obtained by mixing in an arbitrary ratio. In this case, for example, the pH of the solution, which should be within the pH range 1-9, can be installed by adding sodium phosphate, potassium phosphate, sodium chloride, potassium chloride, ammonium acetate, acetate of triethylamine, sodium acetate, potassium acetate, Tris-hydrochloric acid or ethylenediaminetetraacetic acid as an additive in a concentration of 1 mm-2 M

Oligo-RNA having the desired chain length can be obtained by repeating stages A-D.

In addition, in this method, apply the compound (26a), where R4is a Deputy (6), the compound (26a), where R4represents H or alloctype, or the compound (26b), where R2is alkyloxy etc.

When using the compound (26a), where R4represents H or alloctype, or is soedineniya (26b), where R2is alkyloxy as an initial matter, it is necessary to apply one or more derivatives of phosphoramidite of the present invention as a compound monomer of nucleic acids.

Moreover, in this method, the isolation and purification of oligo-RNA also carried out by process stage F before process stage E, and then processes the stage G.

EXAMPLES

Now the present invention will be described in more detail referring to examples, which, however, is not limited to the present invention.

Example 1

Chloromethyl-2-cyanoethylene ether

Stage 1

Getting methylthiomethyl-2-cyanoethylene ether

3-Hydroxypropionitrile (32 g, 450 mmol) was dissolved in 450 ml of dimethyl sulfoxide, and to this add 324 ml of acetic anhydride and 231 ml of acetic acid, and the reaction solution was stirred at room temperature for 24 hours.

Sodium bicarbonate (990 g) dissolved in 4.5 l of water and the reaction solution is added dropwise to aqueous sodium bicarbonate solution for 1 hour. The reaction solution is stirred for 1 hour and subjected to extraction with ethyl acetate, and the extract is dried over anhydrous magnesium sulfate, and the solvent is distilled off. The obtained oily product was then purified column chromatography on silica gel with getting 41 methylthiomethyl-2-cyanoethylene ester as a colorless oily product (yield 70%).

1H-NMR (CDCl3): to 2.18 (s, 3H); to 2.66 (t, 2H, J=6.3 Hz); of 3.77 (t, 2H, J=6.3 Hz); 4,69 (2N)

Stage 2

Getting chloromethyl-2-cyanoethylene ether

Methylthiomethyl-2-cyanoethylene ester (3.3 g, 25 mmol) is dissolved in 70 ml of methylene chloride and added dropwise 2 ml of sulfurylchloride (25 mmol), and then the reaction was performed at room temperature for 1 hour.

After completion of the reaction the solvent is distilled off under reduced pressure to obtain 2.5 g of the target compound as a colorless oily product (yield 85%).

Boiling point: 84-85 º C at 0.3 Torr)

1H-NMR (CDCl3): of 2.72 (t, 2H, J=6.3 Hz); to 3.92 (t, 2H, J=6.3 Hz); 5,52 (2N)

Example 2

5'-O-(4,4'-dimethoxytrityl)-2'-O-(2-cyanoethoxy)uridine 3'-O-(2-cyanoethyl N,N-diisopropylphosphoramidite)

Stage 1

Obtaining 5'-O-(4,4'-dimethoxytrityl)-2'-O-(2-cyanoethoxy)uridine

5'-O-(4,4'-dimethoxytrityl), uridine (546 mg, 1 mmol) dissolved in 4 ml of 1,2-dichloroethane, and to this add 452 mg diisopropylethylamine (3.5 mmol), and then to this add 365 mg dibutyltindilaurate (1.2 mmol). The reaction is carried out at room temperature for 1 hour.

Then the reaction is performed at 80 ° C and added dropwise 155,4 mg chloromethyl-2-cyanoethylene ester (1.3 mmol), the reaction solution is stirred for 30 minutes

After completion of the reaction, the reaction solution is added to acadeny aqueous solution of sodium bicarbonate, and subjected to extraction with methylene chloride, and the extract is dried over anhydrous magnesium sulfate, and the solvent is distilled off. The resulting mixture was purified column chromatography on 30 g of silica gel with obtaining 5'-O-(4,4'-dimethoxytrityl)-2'-O-(2-cyanoethoxy)uridine (197 mg, yield 34%).

1H-NMR (CDCl3): 2,47 (d, 1H, J=7.8 Hz); 2,69 (t, 2H, J=6.3 Hz); 3,55 (DD, 1H, J=11,3 and 2.2 Hz); 3,62 (DD, 1H, J=11,3 and 2.2 Hz); 3,83 (C, 6N); a 3.87 (t, 2H, J=6.3 Hz); 4,07-4,08 (m, 1H); 4,32 (DD, 1H, J=5,3, 1.9 Hz); 4,54 (square, 1H, J=5.3 Hz); 4,94, 5,11 (2D, 2H, J=6.9 Hz); 5,32 (d, 1H, J=8,2 Hz); 6,00 (d, 1H, J=1.9 Hz); 6,85-to 6.88 (m, 4H); 7,29-7,41 (m, N); 8,02 (d, 1H, J=8,2 Hz); 8,53 (users, 1H)

Mass spectrometry with ionization of the spark or glow discharge (ESI-Mass): 652 [M+Na]+

Stage 2

Obtaining 5'-O-(4,4'-dimethoxytrityl)-2'-O-(2-cyanoethoxy)uridine 3'-O-(2-cyanoethyl N,N-diisopropylphosphoramidite)

5'-O-(4,4'-dimethoxytrityl)-2'-O-(2-cyanoethoxy), uridine (209 g of 0.332 mmol), obtained in stage 1, dissolved in 2 ml of acetonitrile and added dropwise 23 mg tetrazole (of 0.332 mmol) and 150 mg of 2-cyanoethyl N,N,N',N'-tetraisopropylphosphorodiamidite (0,498 mmol)and the reaction is carried out at 45 º C for 1.5 hour.

After completion of the reaction, the reaction solution is mixed with saturated aqueous sodium bicarbonate solution, and subjected to extraction with ethyl acetate, and the extract is dried over anhydrous magnesium sulfate, and the solvent is distilled off. The resulting mixture was purified column of HRO what ecografia on 20 g of silica gel to obtain the desired compound (200 mg; yield 73%).

Mass spectrometry with ionization of the spark or glow discharge (ESI-Mass): 852 [M+Na]+

Example 3

2'-O-(2-cyanoethoxy)uridine

Stage 1

Obtain 3',5'-O-(tetraisopropyldisiloxane-1,3-diyl)-2'-O-(2-cyanoethoxy)uridine

3',5'-O-(tetraisopropyldisiloxane-1,3-diyl), uridine (150 mg, 0.3 mmol) dissolved in 7 ml of tetrahydrofuran in an argon atmosphere, and added 54 mg methylthiomethyl-2-cyanoethylene ester (0.4 mmol) and 100 mg of molecular sieves 4Å, and the reaction solution is stirred for 10 minutes

The reaction is carried out at 0 ° C and add 2 ml of triftoratsetata (10 mg, 0.06 mmol) in tetrahydrofuran. Then add 92 mg of N-jodatime (0.4 mmol) and the reaction solution is stirred for 1 hour.

After completion of the reaction, the reaction solution is filtered through celite and washed with methylene chloride and the organic layer washed with 1 M aqueous solution of hydrocyanate sodium. The organic layer was washed with saturated aqueous sodium bicarbonate, and dried over anhydrous magnesium sulfate, and the solvent is distilled off.

The obtained solid residue purified by thin-layer chromatography to obtain 3',5'-O-(tetraisopropyldisiloxane-1,3-diyl)-2'-O-(2-cyanoethoxy)uridine (150 mg, yield 85%).

1H-NMR (CDCl3): 0,97-1,12 (m, 28N); 2,68-by 2.73 (m, 2H); 3,78-3,86 (m, 1H); 3.96 points-of 4.05 (m, 2H); 4.1 and 4,30 (m, 4H); 5,0-5,04 (m, 2H); 5,70 (d, 1H, J=8,2 Hz); of 5.75 (s, 1H); of 7.90 (d, 1H, J=8,2 Hz); 9,62 (users, 1H)

Mass spectrometry with ionization of the spark or glow discharge (ESI-Mass): 570 [M+H]+

Stage 2

Obtaining 2'-O-(2-cyanoethoxy)uridine

3',5'-O-(tetraisopropyldisiloxane-1,3-diyl)-2'-O-(2-cyanoethoxy), uridine (200 mg, 0.35 mmol)obtained in stage 1, dissolved in 2 ml of methanol, and to this added 65 mg of ammonium fluoride (1,76 mmol)and the reaction solution is stirred under heating at 50 º C for 5 hours.

After cooling air to the reaction solution was added acetonitrile. The solution is stirred, and filtered, and concentrated.

The obtained solid residue purified column chromatography on silica gel to obtain the target compound (108 mg, yield 94%).

1H-NMR (CD3OD): 2,72 was 2.76 (t, 2H, J=6.2 Hz); 3,68-to 3.92 (m, 4H); 4,00-a 4.03 (m, 1H); 4.26 deaths-4,32 (m, 2H); 4,81-of 4.95 (m, 2H); 5,71 (d, 1H, J=8,1 Hz); 6,00 (d, 1H, J=3.3 Hz); 8,10 (d, 1H, J=8,1 Hz)

Mass spectrometry with ionization of the spark or glow discharge (ESI-Mass): 350 [M+Na]+

Example 4

Obtaining 5'-O-(4,4'-dimethoxytrityl)-2'-O-(2-cyanoethoxy)uridine

2'-O-(2-cyanoethoxy), uridine (14 g, 43 mmol) is subjected to azeotropic distillation with pyridine and then dried with a vacuum pump for 30 minutes

The solid residue is dissolved in 300 ml of tetrahydrofuran, and added dropwise in an argon atmosphere 68 g of pyridine (856 mmol)and 20 g of molecular sieves 4Å, and the mixture is stirred for 10 minutes

To the reaction solution was added in three portions every 1 hour of 19.6 g of 4,4'-dimethoxytrityl (of 57.8 mmol) and the mixture is additionally stirred for 1 hour.

After adding 10 ml of methanol and stirring the reaction solution for 2 min, the reaction solution is filtered through celite and washed with ethyl acetate.

After concentrating the filtrate, the solid residue dissolved in ethyl acetate and washed with saturated aqueous sodium bicarbonate.

After washing the organic layer with saturated brine (saturated NaCl solution) and drying over anhydrous magnesium sulfate, the solvent is distilled off.

The obtained solid residue purified by chromatography on silica gel to obtain the target compound (26.5 g, yield 98%).

Example 5

N4-acetyl-5'-O-(4,4'-dimethoxytrityl)-2'-O-(2-cyanoethoxy)citizen 3'-O-(2-cyanoethyl N,N-diisopropylphosphoramidite)

Stage 1

Obtaining N4-acetyl-5'-O-(4,4'-dimethoxytrityl)-2'-O-(2-cyanoethoxy)cytidine

N4-acetyl-5'-O-(4,4'-dimethoxytrityl)citizen (588 mg, 1 mmol) dissolved in 4 ml of 1,2-dichloroethane, and to this add 452 mg diisopropylethylamine (3.5 mmol), and then add another 365 mg dibutyltindilaurate (1.2 mmol). The reaction is carried out at room temperature for 1 hour.

Then the reaction is avodat at 80 ° C, and added dropwise 155,4 mg chloromethyl-2-cyanoethylene ester (1.3 mmol), and the reaction solution is stirred for 60 minutes

After completion of the reaction, the reaction solution is added to a saturated aqueous solution of sodium bicarbonate and extracted with methylene chloride. The extract is dried over anhydrous magnesium sulfate and the solvent is distilled off. The resulting mixture was purified column chromatography on 30 g of silica gel with obtaining N4-acetyl-5'-O-(4,4'-dimethoxytrityl)-2'-O-(2-cyanoethoxy)cytidine (219 mg, yield 35%).

1H-NMR (CDCl3): are 2.19 (s, 3H); 2,56 (d, 1H, J=8,8 Hz); to 2.65 (t, 2H, J=6.2 Hz); 3,55 (DD, 1H, J=10,5, 2,5 Hz); 3,63 (DD, 1H, J=10,5, 2,5 Hz); 3,82 (C, 6N); 3,86 (t, 2H, J=6.2 Hz); 4.09 to to 4.14 (m, 1H); 4,28 (d, 1H, J=5,1 Hz); of 4.44 figure-4.49 (m, 1H); equal to 4.97, 5,24 (2D, 2H, J=6.9 Hz); 5,96 (s, 1H); 6,86-to 6.88 (m, 4H); to 7.09 (d, 1H, J=6.9 Hz); 7,26-7,42 (m, N); 8,48 (d, 1H, J=6.9 Hz); 8,59 (users, 1H)

Mass spectrometry with ionization of the spark or glow discharge (ESI-Mass): 693 [M+Na]+

Stage 2

Obtaining N4-acetyl-5'-O-(4,4'-dimethoxytrityl)-2'-O-(2-cyanoethoxy)citizen 3'-O-(2-cyanoethyl N,N-diisopropylphosphoramidite)

N4-acetyl-5'-O-(4,4'-dimethoxytrityl)-2'-O-(2-cyanoethoxy)citizen (205 mg, 0,306 mmol)obtained in stage 1, dissolved in 2 ml of methylene chloride, and added 105 mg diisopropylethylamine (0,812 mmol), and added dropwise 116 mg of 2-cyanoethyl N,N-diisopropylchlorophosphoramidite (0.49 mmol). The reaction Rast is the PR subjected to reaction at room temperature for 1 hour.

After completion of the reaction the solvent is distilled off and the resulting mixture was purified column chromatography on 20 g of silica gel to obtain the target compound (242 mg, yield 91%).

Mass spectrometry with ionization of the spark or glow discharge (ESI-Mass): 871 [M+H]+

Example 6

N4-acetyl-2'-O-(2-cyanoethoxy)citadin

Stage 1

Obtaining N4-acetyl-3',5'-O-(tetraisopropyldisiloxane-1,3-diyl)-2'-O-(2-cyanoethoxy)cytidine

N4-acetyl-3',5'-O-(tetraisopropyldisiloxane-1,3-diyl)citizen (1,00 g, 1,89 mmol) and methylthiomethyl-2-cyanoethylene ester (500 mg, with 3.79 mmol) are mixed and the mixture is dissolved in a solvent, mixed with 10 ml of toluene and 10 ml of tetrahydrofuran.

Then add 975 mg triftoratsetata silver and dried by adding molecular sieves 4Å.

While cooling with ice added 370 mg of N-bromosuccinimide (of 2.08 mmol) and the solution stirred for 10 min in a reaction vessel protected from light. In addition, add 70 mg of N-bromosuccinimide (0,39 mmol) and stirred for 25 minutes

After completion of the reaction, the reaction solution is diluted with methylene chloride and washed with saturated aqueous sodium bicarbonate. The extract is dried over anhydrous sodium sulfate and the solvent is distilled off. The resulting mixture was purified column chromatography on silica gel with the teachings of N 4-acetyl-3',5'-O-(tetraisopropyldisiloxane-1,3-diyl)-2'-O-(2-cyanoethoxy)cytidine (936 mg, yield 81%).

1H-NMR (CDCl3): 0,90-1,11 (m, 28N); of 2.28 (s, 3H); 2,62-and 2.79 (m, 2H); 3,78-to 3.89 (m, 1H); 3.96 points-Android 4.04 (m, 2H); 4,19-to 4.23 (m, 3H); 4,30 (d, 1H, J=13,6 Hz); 5,00 (d, 1H, J=6,8 Hz); 5,09 (d, 1H, J=6,8 Hz); 5,77 (s, 1H); 7,44 (d, 1H, J=7.5 Hz); 8,30 (d, 1H, J=7.5 Hz); 10,13 (s, 1H)

Mass spectrometry with ionization of the spark or glow discharge (ESI-Mass): 611 [M+H]+

Stage 2

Obtaining N4-acetyl-2'-O-(2-cyanoethoxy)cytidine

N4-acetyl-3',5'-O-(tetraisopropyldisiloxane-1,3-diyl)-2'-O-(2-cyanoethoxy)citizen (500 mg, 0,819 mmol)obtained in stage 1, is dissolved in a solvent consumption of 2.5 ml of tetrahydrofuran and 2.5 ml of methanol, and add 150 mg of ammonium fluoride (4,10 mmol), and then the reaction solution is subjected to reaction at 50 º C for 4 hours.

After completion of the reaction, the reaction solution was diluted with acetonitrile and filtered, and the solvent is distilled off. The resulting mixture was purified column chromatography on silica gel to obtain the target compound (210 mg, yield 70%).

1H-NMR (D2): To 2.13 (s, 3H); 2,66-a 2.71 (m, 2H); 3.72 points-of 3.78 (m, 3H); 3,90 (DD, 1H, J=13,0, and 2.6 Hz); 4,06-4,11 (m, 1H); 4,20 (DD, 1H, J=7,1, 5,2 Hz); the 4.29 (DD, 1H, J=5,1, 2,9 Hz); a 4.83 (d, 1H, J=7.2 Hz); 4,94 (d, 1H, J=7,2 Hz); 5,95 (d, 1H, J=2,9 Hz); of 7.25 (d, 1H, J=7,6 Hz); of 8.25 (d, 1H, J=7,6 Hz)

Mass spectrometry with ionization of the spark or glow discharge (ESI-Mass): 391 [M+Na]+

Example 7

Obtaining N4-acetyl-5'-O-(4,4'-dimethoxytrityl)-2'-O-(2-cyanoethoxy)cytidine

2'-O-(2-cyanoethoxy)citizen (9,9 g, 26.8 mmol) is subjected to azeotropic distillation with pyridine and then dried with a vacuum pump for 30 minutes the Solid residue is dissolved in 190 ml of tetrahydrofuran, and added dropwise in an argon atmosphere 43 g of pyridine (538 mmol) and 20 g of molecular sieves 4Å, and the mixture is stirred for 10 minutes

To the reaction solution was added in three portions every 1 hour of 11.8 g of 4,4'-dimethoxytrityl (34,9 mmol) and the mixture is additionally stirred for 1 hour.

After adding 2 ml of methanol and stirring the reaction solution for 2 min, the reaction solution is filtered through celite and washed with ethyl acetate.

After concentrating the filtrate by evaporation, the solid residue is dissolved in ethyl acetate and washed with saturated aqueous sodium bicarbonate.

After washing the organic layer with saturated brine (saturated NaCl solution) and drying over anhydrous magnesium sulfate, the solvent is distilled off.

The obtained solid residue purified by chromatography on silica gel to obtain the target compound (15 g, yield 83%).

Example 8

N2-acetyl-5'-O-(4,4'-dimethoxytrityl)-2'-O-(2-cyanoethoxy)guanosine 3'-O-(2-cyanoethyl N,N-diisopropylphosphoramidite

Stage 1

Obtaining N2-acetyl-5'-O-(4,4'-dimethoxytrityl)-2'-O-(2-cyanoethoxy)guanosine

N2-acetyl-5'-O-(4,4'-dimethoxytrityl)guanosine (627 mg, 1 mmol) dissolved in 4 ml of 1,2-dichloroethane, and add 452 mg diisopropylethylamine (3.5 mmol), and then add 365 mg dibutyltindilaurate (1.2 mmol). And then the reaction solution is subjected to reaction at room temperature for 1 hour.

Then the reaction solution is heated up to 80 ° C and added dropwise 155,4 mg chloromethyl-2-cyanoethylene ester (1.3 mmol), and the solution is stirred for 60 minutes

After completion of the reaction, the reaction solution is mixed with saturated aqueous sodium bicarbonate and subjected to extraction with methylene chloride. The extract is dried over anhydrous magnesium sulfate and the solvent is distilled off. The resulting mixture was purified column chromatography on 30 g of silica gel with obtaining N2-acetyl-5'-O-(4,4'-dimethoxytrityl)-2'-O-(2-cyanoethoxy)guanosine (450 mg, yield 63%).

1H-NMR (CDCl3): of 1.92 (s, 3H); 2,47 is 2.51 (m, 2H); 2,68 (users, 1H); 3,30 (DD, 1H, J=10,7, 3.8 Hz); 3,47 (DD, 1H, J=10,7, 3.8 Hz); 3,55-of 3.60 (m, 1H); 3,65-3,70 (m, 1H); 3,74, of 3.75 (2s, 6N); 4,22-to 4.23 (m, 1H); 4,55-4,58 (m, 1H); 4,78, a 4.83 (2D, 2H, J=7.0 Hz); free 5.01 (t, 1H, J=5,1 Hz); of 5.99 (d, 1H, J=5,1 Hz); 6,76-6,79 (m, 4H); 7,17-7,44 (m, N); 7,88 (s, 1H); at 8.36 (users, 1H); 12,06 (users, 1H)

Stage 2

Obtaining N2-acetyl-5'-O-(4,4'-dimethoxytrityl)-'-O-(2-cyanoethoxy)guanosine 3'-O-(2-cyanoethyl-N,N-diisopropylphosphoramidite )

N2-acetyl-5'-O-(4,4'-dimethoxytrityl)-2'-O-(2-cyanoethoxy)guanosine (400 mg, 0,563 mmol)obtained in stage 1, dissolved in 2 ml of methylene chloride, and added 181 mg diisopropylethylamine (1.4 mmol), and added dropwise 161 mg 2-cyanoethyl N,N-diisopropylchlorophosphoramidite (of 0.68 mmol). Then the reaction is carried out at room temperature for 1 hour.

After completion of the reaction the solvent is distilled off and the resulting mixture was purified column chromatography on 20 g of silica gel to obtain the target compound (471 mg, yield 92%).

Example 9

N6-acetyl-5'-O-(4,4'-dimethoxytrityl)-2'-O-(2-cyanoethoxy)adenosine 3'-O-(2-cyanoethyl N,N-diisopropylphosphoramidite)

Stage 1

Obtaining N6-acetyl-5'-O-(4,4'-dimethoxytrityl)-2'-O-(2-cyanoethoxy)adenosine

N6-acetyl-5'-O-(4,4'-dimethoxytrityl)adenosine (22,0 g, 36,0 mmol) is dissolved in 170 ml of 1,2-dichloroethane and added 16.3 g of diisopropylethylamine (126 mmol), and then add to 12.1 g dibutyltindilaurate (and 39.7 mmol). Then the reaction is carried out at room temperature for 1 hour.

Then the reaction solution is heated up to 80 ° C and added dropwise 4,30 g of chloromethyl-2-cyanoethylene ether (36,0 mmol)and the solution stirred for 30 minutes

After completion of the reaction, the reaction solution was added to saturated aqueous solution of bicarb the ATA sodium and subjected to extraction with methylene chloride. The extract is dried over anhydrous magnesium sulfate and the solvent is distilled off. The resulting mixture was purified column chromatography on silica gel with obtaining N6-acetyl-5'-O-(4,4'-dimethoxytrityl)-2'-O-(2-cyanoethoxy)adenosine (7.47 g; yield 33%).

1H-NMR (CDCl3): of 2.51 (t, 2H, J=6.2 Hz); 2,58 (d, 1H, J=5.5 Hz); 2,61 (s, 3H); of 3.45 (DD, 1H, J=10,7, 4.0 Hz); of 3.54 (DD, 1H, J=10,7, 3,2 Hz); 3,62-with 3.79 (m, 2H); 3,79 (s, 6N); 4,25 (userc, 1H, J=4,6 Hz); 4,59 (square, 1H, J=5,2 Hz); 4,87-4,94 (m, 3H); 6,23 (d, 1H, J=4.4 Hz); 6,80-6,83 (m, 4H); 7,22-to 7.32 (m, 7H); 7,40-the 7.43 (m, 2H); to 8.20 (s, 1H); 8,61 (users, 1H); to 8.62 (s, 1H)

Mass spectrometry with ionization of the spark or glow discharge (ESI-Mass): 695 [M+H]+

Stage 2

Obtaining N6-acetyl-5'-O-(4,4'-dimethoxytrityl)-2'-O-(2-cyanoethoxy)adenosine 3'-O-(2-cyanoethyl N,N-diisopropylphosphoramidite)

N6-acetyl-5'-O-(4,4'-dimethoxytrityl)-2'-O-(2-cyanoethoxy)adenosine (10.0 g, 14.4 mmol), obtained in stage 1, is dissolved in 75 ml of methylene chloride, and added 4.7 g of diisopropylethylamine (36 mmol), and added dropwise 4,82 g 2-cyanoethyl N,N-diisopropylchlorophosphoramidite (20.3 mmol). Then the reaction is carried out at room temperature for 1 hour.

After completion of the reaction the solvent is distilled off and the resulting mixture, which is approximately 30 ml of the solvent, purified column chromatography on silica gel to obtain the target compound (12.0 g; yield 93%).

<> Mass spectrometry with ionization of the spark or glow discharge (ESI-Mass): 895 [M+H]+

Example 10

N6-acetyl-2'-O-(2-cyanoethoxy)adenosine

Stage 1

Obtaining N6-acetyl-3',5'-O-(tetraisopropyldisiloxane-1,3-diyl)-2'-O-(2-cyanoethoxy)adenosine

In 8 ml of methylene chloride are suspended 245 mg N-jodatime (1,09 mmol) and 280 mg triftoratsetata silver (1,09 mmol) and the solution dried by adding molecular sieves 4Å.

To the reaction solution was added a solution of N6-acetyl-3',5'-O-(tetraisopropyldisiloxane-1,3-diyl)adenosine (400 mg, 0.73 mmol) and 145 mg methylthiomethyl-2-cyanoethylene ester (1.11 mmol) in 4 ml of methylene chloride under ice cooling and the reaction mixture is stirred for 3 hours.

After completion of the reaction, the reaction mixture was diluted with methylene chloride and washed with aqueous sodium thiosulfate solution and saturated aqueous sodium bicarbonate. The extract is dried over anhydrous magnesium sulfate and the solvent is distilled off. The resulting mixture was purified column chromatography on silica gel with obtaining N6-acetyl-3',5'-O-(tetraisopropyldisiloxane-1,3-diyl)-2'-O-(2-cyanoethoxy)adenosine (201 mg, yield 45%).

1H-NMR (CDCl3): 0,98-1,11 (m, 28N); 2,62 (s, 3H); 2,69 (TD, 2H, J=6,5, 1.5 Hz); 3,81-to 3.89 (m, 1H); as 4.02-4.09 to (m, 2H); 4,17 (d, 1H, J=9.4 Hz); 4,28 (d, 1H, J=13,4 Hz); 4,50 (d, 1H, J=4.5 Hz); 4,67 (l is, 1H, J=8,8, and 4.5 Hz); 5,02 (d, 1H, J=7,0 Hz); 5,08 (d, 1H, J=7,0 Hz); 6,10 (s, 1H); 8.34 per (s, 1H); 8,66 (s, 1H); 8,67 (s, 1H)

Mass spectrometry with ionization of the spark or glow discharge (ESI-Mass): 636 [M+H]+

Stage 2

Obtaining N6-acetyl-2'-O-(2-cyanoethoxy)adenosine

N6-acetyl-3',5'-O-(tetraisopropyldisiloxane-1,3-diyl)-2'-O-(2-cyanoethoxy)adenosine (300 mg, 0.47 mmol)obtained in stage 1, is dissolved in a solvent consumption of 0.1 ml of acetic acid and 2 ml of 0.5 M solution of tetrabutylammonium fluoride, and the reaction solution was stirred at room temperature for 2 hours.

After completion of the reaction the reaction mixture is purified column chromatography on silica gel to obtain the target compound (160 mg, yield 86%).

1H-NMR (DMSO-d6): of 2.25 (s, 3H); 2,53 of 2.68 (m, 2H); 3,41-of 3.46 (m, 1H); 3,56-to 3.64 (m, 2H); 3,69-to 3.73 (m, 1H); 4,00-4,01 (m, 1H); 4,36-4,37 (m, 1H); 4.72 in-4,78 (m, 3H); 5,20 (ushort, 2H); 5,41 (d, 1H, J=5,2 Hz); 6,17 (d, 1H, J=5,7 Hz); 8,66 (s, 1H); 8,72 (s, 1H); of 10.72 (s, 1H)

Mass spectrometry with ionization of the spark or glow discharge (ESI-Mass): 415 [M+Na]+

Example 11

Obtaining N6-acetyl-5'-O-(4,4'-dimethoxytrityl)-2'-O-(2-cyanoethoxy)adenosine

N6-acetyl-2'-O-(2-cyanoethoxy)adenosine (9,50 g, and 24.2 mmol) dissolved in 100 ml of anhydrous pyridine and then dried by concentration. Then the solid residue is dissolved in 100 ml dehydr the appropriate pyridine in an argon atmosphere.

While cooling with ice added 10.7 g of 4,4'-dimethoxytrityl (% 31.2 mmol) and the reaction is carried out at room temperature for 1 hour 20 minutes

After completion of the reaction, the reaction solution is diluted with methylene chloride and washed with water. The extract is dried over anhydrous sodium sulfate and the solvent is distilled off. The resulting mixture was purified column chromatography on silica gel to obtain the target compound (13.8 g; yield 82%).

Example 12

N2-Phenoxyacetyl-5'-O-(4,4'-dimethoxytrityl)-2'-O-(2-cyanoethoxy)guanosine 3'-O-(2-cyanoethyl N,N-diisopropylphosphoramidite)

Stage 1

Obtaining N2-phenoxyacetyl-5'-O-(4,4'-dimethoxytrityl)-2'-O-(2-cyanoethoxy)guanosine

N2-phenoxyacetyl-5'-O-(4,4'-dimethoxytrityl)guanosine (720 mg, 1 mmol) dissolved in 4 ml of 1,2-dichloroethane, and add 452 mg diisopropylethylamine (3.5 mmol), and then add 365 mg dibutyltindilaurate (1.2 mmol). Then the reaction is carried out at room temperature for 1 hour.

Then the reaction is performed at 80 ° C and added dropwise 155,4 mg chloromethyl-2-cyanoethylene ester (1.3 mmol), and the solution is stirred for 60 minutes

After completion of the reaction, the reaction solution is mixed with saturated aqueous sodium bicarbonate and subjected to extraction with methylene chloride. The extract is dried over anhydrous sulfa what Ohm magnesium and the solvent is distilled off. The resulting mixture was purified column chromatography on 30 g of silica gel with obtaining N2-phenoxyacetyl-5'-O-(4,4'-dimethoxytrityl)-2'-O-(2-cyanoethoxy)guanosine (384 mg; yield 48%).

1H-NMR (CDCl3): 2,47 is 2.51 (m, 2H); 2,58 (users, 1H); 3,42 (DD, 1H, J=10,1, 3.8 Hz); 3.46 in (DD, 1H, J=10,1, 3.8 Hz); 3,53 is 3.57 (m, 1H); 3,69-to 3.73 (m, 1H); of 3.77 (s, 6N); 4,24-4.26 deaths (m, 1H); 4,48-4,50 (m, 1H); br4.61 with 4.65 (m, 2H); of 4.83, 4,87 (2D, 2H, J=7.0 Hz); 4,88 (t, 1H, J=5.7 Hz); 6,05 (d, 1H, J=5.7 Hz); 6,80-PC 6.82 (m, 4H); 6,92-of 6.96 (m, 3H); 7,07-7,11 (m, 2H); 7,20-7,42 (m, N); to 7.84 (s, 1H); 8,99 (s, 1H); 11,81 (users, 1H)

Mass spectrometry with ionization of the spark or glow discharge (ESI-Mass): 825 [M+Na]+

Stage 2

Obtaining N2-phenoxyacetyl-5'-O-(4,4'-dimethoxytrityl)-2'-O-(2-cyanoethoxy)guanosine 3'-O-(2-cyanoethyl N,N-diisopropylphosphoramidite)

N2-phenoxyacetyl-5'-O-(4,4'-dimethoxytrityl)-2'-O-(2-cyanoethoxy)guanosine (320 mg, 0,399 mmol)obtained in stage 1, is dissolved in 4 ml of methylene chloride and add to 128.8 mg diisopropylethylamine (0,996 mmol), and added dropwise 141,5 mg 2-cyanoethyl N,N-diisopropylchlorophosphoramidite (0,598 mmol). Then the reaction is carried out at room temperature for 1 hour.

After completion of the reaction the solvent is distilled off and the resulting mixture was purified column chromatography on 30 g of silica gel to obtain the target compound (316 mg; yield 79%).

Mass spectrometry with ionization of the spark or glow time is a poison (ESI-Mass): 1003 [M+H] +

Example 13

N2-phenoxyacetyl-2'-O-(2-cyanoethoxy)guanosin

Stage 1

Obtaining N2-phenoxyacetyl-3',5'-O-(tetraisopropyldisiloxane-1,3-diyl)-2'-O-(2-cyanoethoxy)guanosine

N2-phenoxyacetyl-3',5'-O-(tetraisopropyldisiloxane-1,3-diyl)guanosine (2.0 g, 3.0 mmol) dissolved in 16 ml of tetrahydrofuran, and add 0,99 g methylthiomethyl-2-cyanoethylene ether (7.6 mmol) and 1.0 g of molecular sieves 4Å, and the reaction solution is stirred at 45 º C for 10 min in argon atmosphere.

After adding a solution of 0.68 g of triftoratsetata (4.5 mmol) in 5 ml of tetrahydrofuran and stirring the reaction solution type of 1.02 g of N-jodatime (4.5 mmol) and the reaction solution is stirred for 15 minutes

After adding saturated aqueous sodium bicarbonate solution to the reaction solution and then filtering the reaction solution, the organic layer washed with 1 M aqueous solution of hydrocyanate sodium.

Next, the reaction solution was sequentially washed with water and saturated brine (saturated NaCl), and the extract is dried over anhydrous magnesium sulfate, and the solvent is distilled off.

The obtained solid residue purified column chromatography on silica gel with obtaining N2-phenoxyacetyl-3',5'-O-(tetraisopropyldisiloxane-1,3-diyl)-2'-O-(2-cyan is ethoxymethyl)guanosine (2.0 g; yield 89%).

1H-NMR (CDCl3): 0,99-1,11 (m, 28N); 2,59-2,77 (m, 2H); 3,82-of 4.05 (m, 3H); 4,15 (d, 1H, J=9,3 Hz); 4,25 is 4.35 (m, 2H); to 4.52-4,56 (DD, 1H, J=9,3, a 4.3 Hz); 5,00-5,07 (2D, 2H, J=7.2 Hz); 5,95 (s, 1H); 6,99 for 7.12 (m, 3H); 7,35-7,40 (m, 2H); of 8.09 (s, 1H); 9,38 (users, 1H); 11,85 (users, 1H)

Mass spectrometry with ionization of the spark or glow discharge (ESI-Mass): 766 [M+Na]+

Stage 2

Obtaining N2-phenoxyacetyl-2'-O-(2-cyanoethoxy)guanosine

Prepare a solution consisting of about 0.14 ml of acetic acid (0.14 mmol) and and 2.83 ml of 1 M solution of tetrabutylammonium fluoride in tetrahydrofuran and 2.83 mmol).

N2-phenoxyacetyl-3',5'-O-(tetraisopropyldisiloxane-1,3-diyl)-2'-O-(2-cyanoethoxy)guanosine (1.0 g, 1.35 mmol)obtained in stage 1, dissolved 2.83 ml of tetrahydrofuran, and add the solution prepared above and the reaction is carried out at room temperature for 1 hour in argon atmosphere.

The reaction solution is concentrated under reduced pressure, and the residue is dissolved in methylene chloride, and purified column chromatography on silica gel to obtain the target compound (0,67 g; yield 99%).

1H-NMR (DMSO-d6): 2,59-of 2.66 (m, 2H); 3,41-3,63 (m, 4H); 3,98 (m, 1H); 4,32 (m, 1H); 4,58-to 4.62 (t, 1H, J=5.3 Hz); 4,71-4,78 (DD, 2H, J=13,1, 6,8 Hz); to 4.87 (s, 2H); 5,12 (s, 1H); lower than the 5.37 (s, 1H); 5,97 (d, 1H, J=6,1 Hz); of 6.96-6,99 (m, 3H); 7,28-7,34 (m, 2H); 8,30 (s, 1H); 11,78 (users 2N)

Mass spectrometry with ionization of the spark or glow discharge (ESI-Mass): 50 [M-N] -

Example 14

N2-phenoxyacetyl-5'-O-(4,4'-dimethoxytrityl)-2'-O-(2-cyanoethoxy)guanosin

N2-phenoxyacetyl-2'-O-(2-cyanoethoxy)guanosine (660 mg, 1,32 mmol) is subjected to azeotropic distillation with pyridine and then dried with a vacuum pump for 30 minutes

The solid residue is dissolved in 9 ml of tetrahydrofuran, and added dropwise in an argon atmosphere 2.1 g of pyridine (26,4 mmol) and 600 mg of molecular sieves 4Å, and the reaction solution is stirred for 10 minutes

To the reaction solution was added in three portions every 1 hour 540 mg of 4,4'-dimethoxytrityl (1,58 mmol) and the reaction solution was additionally stirred for 1 hour.

After adding 2 ml of methanol and stirring the reaction solution for 2 min, the reaction solution is filtered through celite and washed with ethyl acetate.

After concentrating the filtrate by evaporation, the residue is dissolved in ethyl acetate and separated by saturated aqueous sodium bicarbonate solution.

After washing the organic layer with saturated brine (saturated NaCl solution) and drying over anhydrous magnesium sulfate, the solvent is distilled off.

The obtained solid residue purified column chromatography on silica gel to obtain the target compound (800 mg, yield 75%).

Example 15

N6-acetyl-3',5'-O-(tetraisopropyl ciloxan-1,3-diyl)-2'-O-(2-cyanoethoxy)adenosine

Stage 1

Obtaining N6-acetyl-3',5'-O-(tetraisopropyldisiloxane-1,3-diyl)-2'-O-methylthioadenosine

N6-acetyl-3',5'-O-(tetraisopropyldisiloxane-1,3-diyl)adenosine (2.00 g, 3.62 mmol) was dissolved in 25 ml of dimethylsulfoxide, and add 17.5 ml of acetic anhydride and 12.5 ml of acetic acid, and the reaction solution was stirred at room temperature for 14 hours.

After completion of the reaction, the reaction solution is added to 200 ml of water, extracted with ethyl acetate and washed with saturated aqueous sodium bicarbonate. The extract is dried over anhydrous sodium sulfate and the solvent is distilled off. The resulting mixture was purified column chromatography on silica gel with obtaining N6-acetyl-3',5'-O-(tetraisopropyldisiloxane-1,3-diyl)-2'-O-methylthioadenosine (1,36 g; yield 61%).

1H-NMR (CDCl3): 0,96-1,11 (m, 28N); of 2.20 (s, 3H); 2,61 (s, 3H); is 4.03 (DD, 1H, J=13,4, 2,4 Hz); 4,18 (d, 1H, J=9.1 Hz); 4,27 (d, 1H, J=13,4 Hz); 4.63 to-4,71 (m, 2H); 5,00 (d, 1H, J=11.5 Hz); 5,07 (d, 1H, J=11.5 Hz); 6,09 (s, 1H); 8,31 (s, 1H); 8,65 (s, 1H); 8,69 (s, 1H)

Mass spectrometry with ionization of the spark or glow discharge (ESI-Mass): 635 [M+Na]+

Stage 2

Obtaining N6-acetyl-3',5'-O-(tetraisopropyldisiloxane-1,3-diyl)-2'-O-(2-cyanoethoxy)adenosine

N6-acetyl-3',5'-O-(tetraisopropyldisiloxane-1,3-diyl)-2'-O-methylthioadenosine (1,00 g, and 1.63 mmol), polucen is th at stage 1, dissolved in 25 ml of tetrahydrofuran.

To the reaction solution was added 5,88 g 3-hydroxypropionitrile (82,7 mmol), and the solution is dried by adding molecular sieves 4Å, and cooled to 45 º C.

To the reaction solution was added 440 mg of N-jodatime (a 1.96 mmol) and then 490 mg triftoratsetata (3,26 mmol) and the reaction solution is stirred at 45 º C for 15 minutes

After completion of the reaction, the reaction solution is neutralized by adding triethylamine while cooling and diluted with methylene chloride. The reaction solution was washed with aqueous sodium thiosulfate solution and saturated aqueous sodium bicarbonate, the extract is dried over anhydrous sodium sulfate and the solvent is distilled off. The resulting mixture was purified column chromatography on silica gel to obtain the target compound (722 mg, 71%yield).

Example 16

Uridylic-[3'→5']-uridylic-[3'→5']-uridylic-[3'→5']-uridylic-[3'→5']-uridylic-[3'→5']-uridylic-[3'→5']-uridylic-[3'→5']-uridylic-[3'→5']-uridylic-[3'→5']-uridylic-[3'→5']-uridylic-[3'→5']-uridylic-[3'→5']-uridylic-[3'→5']-uridylic-[3'→5']-uridylic-[3'→5']-uridylic-[3'→5']-uridylic-[3'→5']-uridylic-[3'→5']-uridylic-[3'→5']-uridylic-[3'→5']-the uridine

Oligo-RNA connections specified in the title, are synthesized by entering a commercially available solid media CPG (glass with a specified pore size) (37 mg, 1 mmol), terasawa 2'/3'-O-benzoyl-5'-O-(4,4'-dimethoxytrityl)uridine, in the column with a glass filter and by using an automatic nucleic acid synthesizer (ExpediteTM: Applied Biosystems (Applied Biosystems)).

Using 5'-O-(4,4'-dimethoxytrityl)-2'-O-(2-cyanoethoxy)uridine 3'-O-(2-cyanoethyl N,N-diisopropylphosphoramidite) as a compound monomer of nucleic acids, tetrazole as a condensation catalyst, a solution of iodine as oxidant, a solution of acetic anhydride and N-methylimidazole as kapiruumi solution.

After 20-fold condensation of compounds of the monomer of nucleic acid oligo-RNA otscheplaut by steps of 10 M aqueous-ethanolic solution of methylamine as a chip off the agent at room temperature for 1-2 hours and the protective groups each phosphate parts removed.

After concentrating the reaction mixture under reduced pressure and remove the unwanted peaks using columns with reversed phase (ODS) the reaction solution is distilled eluent (50 mm triethylamine acetate buffer solution in acetonitrile).

After concentration the residue under reduced pressure the residue is subjected to reaction with 1 M solution of tetrabutylammonium fluoride in tetrahydrofuran at room temperature for 1 hour to remove the protective group for the 2'-hydroxyl group.

After desalting the reaction solution for itnow group for 5'-terminal group is removed by 80% solution of acetic acid (processing at room temperature for 10 min).

After concentration under reduced pressure the aqueous layer was washed with ether and obtain the target compound with high purity without purification.

Time-of-flight mass spectrometry matrix assisted laser desorption ionization (Matrix-Assisted Laser Desorption Ionization Time of Flight Mass Spectroscopy (MALDI-TOF-MS):

The calculated value 6367,52 [M+H]+

Value found 6366,50 [M+H]+

From analytic HPLC with reversed phase, shown in the drawing, it is clear that the compound obtained has a high degree of purity.

Measurement conditions are as follows:

Device for high-performance Liquid Chromatography (HPLC)

Device for aspiration: LC-6A (SHIMADZU CORPORATION)

Detector: SPD-6A (SHIMADZU CORPORATION)

Column HPLC with reversed phase: Mightysil RP-18GP <4.6 mm ⌀×15 cm> (KANTO CALLED)

The column temperature: 35

The gradient mobile phase: linear gradient, 20 min (solution: 0-70%)

Solution A: 50 mm triethylamine acetate buffer solution containing 5% acetonitrile

Solution: 50 mm triethylamine acetate buffer solution containing 90% acetonitrile

The flow rate of mobile phase: 1 ml/min

The wavelength for detection by the spectrophotometer in the UV-visible area: 260 nm

Example 17

Citadell-[3'→5']-uridylic-[3'→5']-uridylic-[3'→5']-adenylyl-[3'→5']-citadell-[3'→5']-guanylyl-[3'→5']-Zaidi the Il-[3'→5']-uridylic-[3'→5']-guanylyl-[3'→5']-adenylyl-[3'→5']-guanylyl-[3'→5']-uridylic-[3'→5']-adenylyl-[3'→5']-citadell-[3'→5']-uridylic-[3'→5']-uridylic-[3'→5']-citadell-[3'→5']-guanylyl-[3'→5']-adenylyl-[3'→5']-uridine

Oligo-RNA connections specified in the title, are synthesized by entering a commercially available solid media CPG (glass with a specified pore size) (37 mg, 1 mmol)containing 2'/3'-O-benzoyl-5'-O-(4,4'-dimethoxytrityl)uridine, in a column with a glass filter and by using an automatic nucleic acid synthesizer (ExpediteTM: Applied Biosystems (Applied Biosystems)).

Using 5'-O-(4,4'-dimethoxytrityl)-2'-O-(2-cyanoethoxy)uridine 3'-O-(2-cyanoethyl N,N-diisopropylphosphoramidite), N4-acetyl-5'-O-(4,4'-dimethoxytrityl)-2'-O-(2-cyanoethoxy)citizen 3'-O-(2-cyanoethyl N,N-diisopropylphosphoramidite), N4-acetyl-5'-O-(4,4'-dimethoxytrityl)-2'-O-(2-cyanoethoxy)adenosine 3'-O-(2-cyanoethyl N,N-diisopropylphosphoramidite) and N2-phenoxyacetyl-5'-O-(4,4'-dimethoxytrityl)-2'-O-(2-cyanoethoxy)guanosine 3'-O-(2-cyanoethyl N,N-diisopropylphosphoramidite) as a compound monomer nucleic acid; 5-ethylthioethyl as a condensation catalyst; solution of iodine as oxidant solution proxyusername anhydride and N-methylimidazole as kapiruumi solution.

After a 19-fold condensation of compounds of the monomers of nucleic acid protective group for 5'-terminal hydroxyl group is removed to the solid phase. Then oligo-RNA otscheplaut through the action of the mixture concentrated in the aqueous solution of ammonia and ethanol (3:1) as a chip off the agent at 40 º C for 4 h and protective phosphate group each part and the base are removed.

After concentrating the reaction mixture under reduced pressure, the residue is subjected to reaction with 1 M solution of tetrabutylammonium fluoride in tetrahydrofuran containing 10% n-Propylamine and 0.6% 2-mercaptoethanol ether, at room temperature for 1 hour to remove the protective group for the 2'-hydroxyl group.

After desalting the reaction solution, the reaction solution is purified by DEAE-ion-exchange resin (TOYOPEARLDEAE-650) to obtain the target compound of high purity (112 OD260; yield 58%).

Here the absorption of ultraviolet radiation with a wavelength of 260 nm (OD260shows the yield of the target compound.

Further in this document, the absorption (OD260means the yield of the target compound.

Mass spectrometry matrix assisted laser desorption ionization, time-of-flight Matrix-Assisted Laser Desorption Ionization Time of Flight Mass Spectroscopy (MALDI-TOF-MS):

The calculated value 6305,9 [M+H]+

Value found 6304,8 [M+H]+

Example 18

Adenylyl-[3'→5']-citadell-[3'→5']-adenylyl-[3'→5']-uridylic-[3'→5']-citadell-[3'→5']-adenylyl-[3'→5']-citadell-[3'→5']-uridylic-[3'→5']-uridylic-[3'→5']-adenylyl-[3'→5']-citadell-[3'→5']-guanylyl-[3'→5']-citadell-[3'→5']-uridylic-[3'→5']-guanylyl-[3'→5']-adenylyl-[3'→5']-guanylyl-[3'→5']-uridylic-[3'→5']-adenylyl-[3'→5']-citadell-[3'→5']-uridylic-[3'→5']-uridylic-[3'→']-citadell-[3'→5']-guanylyl-[3'→5']-adenylyl-[3'→5']-adenylyl-[3'→5']-adenylyl-[3'→5']-uridylic-[3'→5']-guanylyl-[3'→5']-uridine

The target compound synthesized in the same way as is done in example 17 (92 OD260; yield 31%).

MALDI-TOF-MS:

The calculated value 9519,8 [M+H]+

Value found 9520,4 [M+H]+

Example 19

Uridylic-[3'→5']-uridylic-[3'→5']-uridylic-[3'→5']-uridylic-[3'→5']-uridylic-[3'→5']-uridylic-[3'→5']-uridylic-[3'→5']-uridylic-[3'→5']-uridylic-[3'→5']-uridylic-[3'→5']-uridylic-[3'→5']-uridylic-[3'→5']-uridylic-[3'→5']-uridylic-[3'→5']-uridylic-[3'→5']-uridylic-[3'→5']-uridylic-[3'→5']-uridylic-[3'→5']-uridylic-[3'→5']-uridylic-[3'→5']-uridylic-[3'→5']-uridylic-[3'→5']-uridylic-[3'→5']-uridylic-[3'→5']-uridylic-[3'→5']-uridylic-[3'→5']-uridylic-[3'→5']-uridylic-[3'→5']-uridylic-[3'→5']-uridylic-[3'→5']-uridylic-[3'→5']-uridylic-[3'→5']-uridylic-[3'→5']-uridylic-[3'→5']-uridylic-[3'→5']-uridylic-[3'→5']-uridylic-[3'→5']-uridylic-[3'→5']-uridylic-[3'→5']-uridine

The target compound synthesized in the same way as is done in example 17 (254 OD260; yield 65%).

MALDI-TOF-MS:

The calculated value 12185,8 [M+H]+

Value found 12183,3 [M+H]+

Example 20

Adenylyl-[3'→5']-adenylyl-[3'→5']-uridylic-[3'→5']-citadell-[3'→5']-adenylyl-[3'→5']-citadell-[3'→5']-adenylyl-[3'→5']-guanylyl-[3'→5']-adenylyl-[3'→5']-adenylyl-[3'→5']-uridylic-[3'→5']-citadell-[3'→5']-guanylyl-[3'→5']-uridylic-[3'→5']-citadell-[3'→5']-guanylyl-[3'→5']-uridylic-[3'→5']-adenylyl-[3'→5']-uridylic-[3'→5']-guanli�-[3'→5']-citadell-[3'→5']-adenylyl-[3'→5']-guanylyl-[3'→5']-uridylic-[3'→5']-guanylyl-[3'→5']-adenylyl-[3'→5']-adenylyl-[3'→5']-adenylyl-[3'→5']-adenylyl-[3'→5']-citadell-[3'→5']-uridylic-[3'→5']-citadell-[3'→5']-uridylic-[3'→5']-citadell-[3'→5']-uridylic-[3'→5']-uridylic-[3'→5']-citadell-[3'→5']-adenylyl-[3'→5']-adenylyl-[3'→5']-thymidine

The target compound synthesized in the same way as is done in example 17 (75 OD260; yield 19%).

MALDI-TOF-MS:

The calculated value 12731,8 [M+H]+

Value found 12731,7 [M+H]+

Example 21

Uridylic-[3'→5']-guanylyl-[3'→→5']-adenylyl-[3'→5']-adenylyl-[3'→5']-uridylic-[3'→5']-adenylyl-[3'→5']-citadell-[3'→5']-adenylyl-[3'→5']-adenylyl-[3'→5']-adenylyl-[3'→5']-uridylic-[3'→5']-citadell-[3'→5']-adenylyl-[3'→5']-citadell-[3'→5']-adenylyl-[3'→5']-guanylyl-[3'→5']-adenylyl-[3'→5']-adenylyl-[3'→5']-uridylic-[3'→5']-citadell-[3'→5']-guanylyl-[3'→5']-uridylic-[3'→5']-citadell-[3'→5']-guanylyl-[3'→5']-uridylic-[3'→5']-adenylyl-[3'→5']-uridylic-[3'→5']-guanylyl-[3'→5']-citadell-[3'→5']-adenylyl-[3'→5']-guanylyl-[3'→5']-uridylic-[3'→5']-guanylyl-[3'→5']-adenylyl-[3'→5']-adenylyl-[3'→5']-adenylyl-[3'→5']-adenylyl-[3'→5']-citadell-[3'→5']-uridylic-[3'→5']-citadell-[3'→5']-uridylic-[3'→5']-citadell-[3'→5']-uridylic-[3'→5']-uridylic-[3'→5']-citadell-[3'→5']-adenylyl-[3'→5']-adenylyl-[3'→5']-uridylic-[3'→5']-uridylic-[3'→5']-citadell-[3'→5']-uridylic-[3'→5']-uridylic-[3'→5']-uridylic-[3'→5']-adenylyl-[3'→5']-thymidine

The target compound synthesized in the same way as is done in example 17 (83 OD260; yield 15%).

MALDI-TOF-MS:

The calculated value 17476,6 [M+H]+

Value found 17474,6 [M+H]+

INDUSTRIAL APPLICABILITY

Derived phosphoramidite this is subramania has a protective group of the ether type, which type of 2'-hydroxyl group. The protective group of the ether type is a linear protective group, and the spatial structure around the phosphorus atom attached to the 3'-hydroxyl group is not hindered, and therefore, the derivative of phosphoramidite the present invention makes possible the course of the condensation reaction in a shorter time and get the best output in the condensation reaction in the process of synthesizing oligo-RNA compared to traditional derivative of phosphoramidite.

The use of derived phosphoramidite the present invention makes it possible to obtain oligo-RNA of high purity by using a method essentially identical to the method used in the production of oligo-DNA.

1. Derived phosphoramidite represented by the following General formula (1)
,
in which
Inxrepresents adenine, guanine, cytosine, thymine or uracil, where the amino group of adenine, guanine and cytosine can be optionally protected by a protective group selected from acetyl group and phenoxyacetyl group;
R1is a Deputy, represented by the following General formula (2)
,
in which
R11, R12and R13are the same or different, and K is jdy represents hydrogen or C1-Calcaterra;
R2aand R2bare the same or different, and each represents a C1-Saltillo group; and
each WG1and WG2represents cyano.

2. The method for obtaining oligonucleotide represented by the following General formula (3), comprising the following steps A-G, featuring a derivative of phosphoramidite according to claim 1 as Monomeric compounds of nucleic acids at the next stage In

in which
each independently represents adenine, guanine, cytosine, uracil or thymine;
each R independently represents H or a hydroxyl group, and at least one R is a hydroxyl group;
Z represents H or phosphate group; and
n is an integer ranging from 1 to 100,
stage A: obtaining compounds represented by the following General formula (5), by deleting the 5'-hydroxyl group by the interaction of the acid with the compound represented by the following General formula (4)

in which
n is as defined above;
eachxindependently represents adenine, guanine, cytosine, thymine or uracil, where the amino group of adenine, guanine and cytosine can be optionally protected by a protective group selected from acetyl group and Fe is oxyacetylene group; and
R1is a Deputy, represented by the following General formula (2)
,
in which
R11, R12and R13are the same or different and each represents hydrogen or C1-Calcaterra;
each WG2represents cyano;
each R4independently represents H, alloctype, where the acyl fragment represents a C1-Selenolo group or C7-Sol group, or Deputy, represented by the following General formula (6)
,
in which
WG1represents cyano; and
E represents a C1-alkanoyloxy group or C7-Sol group or Deputy, represented by the following General formula (7)
,
in which
Q represents a single bond or a Deputy, represented by the following General formula (8)
,
in which
WG2such as WG2defined above; and
T represents H or alloctype, where the acyl fragment represents a C1-Selenolo group or C7-Sol group, provided that either S or T is Deputy (7),
moreover, the acid is triperoxonane acid, dichloracetic acid or trichloroacetic acid
stage: obtain the compounds represented by the following General formula (9), by condensation of Monomeric compounds of the nucleic acid with the compound obtained in stage A, using an activating agent

in which
Inx, E, n, R1, R4T and WG2such as defined above; and
the activating agent is a 1H-tetrazole, 5-ethylthioethyl, 5-benzylmercaptan-1H-tetrazole, 4,5-dichloroimidazole, 4,5-dicyanoimidazole, benzotriazolyl, imidazolium, peridiniella, N,N-diisopropylethylamine or 2,4,6-kallidin/N-Mei;
stage C: capping the 5'-hydroxyl groups unreacted compound (5) at the stage In using kapiruumi agent

wherex, E, n, R4T and WG2such as defined above; and
R5represents methyl or phenoxymethyl group; and capituli agent is an acetic anhydride or ferociously anhydride,
stage D: the transformation of the phosphoryl group in the phosphate group by reacting the oxidizing agent with compound (9), which is obtained at the stage

where BxE, n, R1, R4T and WG2such as defined above; and
the oxidizing agent is an iodine or tert-butylhydroperoxide;
stage E: repeating stages A-D for receiving the oligonucleotide having the desired chain length,
stage F: cleavage of compound (11)obtained in stage D, the solid carrier and then removing the protective groups each nucleic bases and each 2'-hydroxyl group using a chip off the agent, the agent for the removal of the protective group of the 2'-hydroxyl group and a deactivating agent

where a, BxE, n, R, R1, R4, T, WG2and Z are such as defined above,
chip off the agent is a concentrated aqueous solution of ammonia or methylamine;
agent for the removal of the protective group of the 2'-hydroxyl group is a tetrabutylammonium fluoride or trihydrated/salt of triethylamine; and deactivating agent is alkylamino, amicin, thiol, thiol derivatives,or mixtures thereof,
stage G: remove the 5'-hydroxyl group of compound (12)obtained in stage F

where, n, R, R1and Z are such as defined above,
stage H: isolation and purification of the oligonucleotide (3), obtained in stage G.

3. The ether compound represented by the following General formula (13)
,
in which
L represents a halogen or C1-Sukitooru;
WG1represents cyano.

4. The method of deriving phosphoramidite according to claim 1, comprising the following stages (a-h,
stage a: receiving nucleoside represented by the following General formulas (15) and (15'), where the protective group of the ether type, which can be removed in a neutral environment, type of 2'-hydroxyl group by exposure to alkylating reagent to a nucleoside represented by the following General formula (14)
,
wherexrepresents adenine, guanine, cytosine, thymine or uracil, where the amino group of adenine, guanine and cytosine can be optionally protected by a protective group selected from acetyl group and phenoxyacetyl group;
R1and WG1such as defined in claim 1; and
alkylating reagent is an ether compound represented by the following General formula (13)

in which
L represents a halogen or C1-Sukitooru; and WG1represents a cyano,
stage b: isolation and purification of the derivative of the nucleoside (15)obtained in stage (a),
stage: implemented separately from stage b receiving nucleoside represented by the following General formula (17), where the protective group of the ether type, which can be removed in a neutral environment, type of 2'-hydroxyl group by exposure alkiliruushim the reagent on the nucleoside, represented by the following General formula (16)
,
in which
Bxand WG1such as defined above; and
But remasterise Deputy represented by the following General formula (18a) or (18b)
,
in which
R6represents a C1-Saltillo group; and an alkylating reagent such as defined above,
stage d: implemented separately from stages a-C receiving nucleoside represented by the following General formula (19), by exposure to dimethyl sulfoxide, acetic acid and acetic anhydride in connection ribonucleic acid (16)
,
where a and bxsuch as defined above,
stage e: receiving nucleoside represented by the following General formula (17), where the protective group of the ether type, which can be removed in a neutral environment, type of 2'-hydroxyl group by exposure to alcohol compounds represented by the following General formula (20), acid and reagent for halogenation of the sulfur atom on nuke (19)obtained in stage d

where a, bxand WG1such as defined above;
acid is triftormetilfullerenov acid, triftorbyenzola silver or trimate silyl triftorbyenzola; and halogenation reagent for the sulfur atom is a N-bromosuccinimide (NBS) or N-jodatime (NIS),
stage f: receiving nucleoside represented by the following General formula (21), by removing the protective groups for the 3'and 5'-hydroxyl groups of the nucleoside (17), obtained at the stage or at the stage of e
,
where a, bxand WG1such as defined above,
stage g: receiving nucleoside (15) by introducing a protective group (R1), which can be removed in an acidic medium in a 5'-hydroxyl group of the nucleoside (21)obtained in stage f
,
wherex, R1and WG1such as defined above; and
X3represents halogen,
stage h: derivatization of phosphoramidite represented by the following General formula (1), fosfaurilirovaniem 3'-hydroxyl group by exposure hospitilised reagent and, if necessary, the activating agent on the nucleoside (15)obtained in stage b or stage g

where Bx, R1and WG1such as defined above;
each of R2aand R2brepresents a C1-Saltillo group;
WG2represents cyano;
the activating agent is a 1H-tetrazole, 5-ethylthioethyl, 5-benzylmercaptan-1H-tetrazole, 4,5-dichlo the imidazole, 4,5-dicyanoimidazole, benzotriazolyl, imidazolium, peridiniella, N,N-diisopropylethylamine or 2,4,6-kallidin/N-Mei;
hospitalise reagent is a compound represented by the following General formula (22) or (23)
,
in which
each of R2aand R2brepresents a C1-Saltillo group;
WG2represents cyano; and
X1represents a halogen.

5. Nucleoside represented by the following General formula (21)
,
where Bxrepresents adenine, guanine, cytosine, thymine or uracil, where the amino group of adenine, guanine and cytosine can be optionally protected by a protective group selected from acetyl group and phenoxyacetyl group; and
WG1represents cyano.

6. Nucleoside represented by the following General formula (15)
,
wherexrepresents adenine, guanine, cytosine, thymine or uracil, where the amino group of adenine, guanine and cytosine can be optionally protected by a protective group selected from acetyl group and phenoxyacetyl group;
R1is a Deputy, represented by the following General formula (2)
,
where R11, R 12and R13the same or different, each represents hydrogen or C1-Calcaterra; and
WG1represents cyano.

7. Nucleoside represented by the following General formula (17)
,
wherexrepresents adenine, guanine, cytosine, thymine or uracil, where the amino group of adenine, guanine and cytosine can be optionally protected by a protective group selected from acetyl group and phenoxyacetyl group;
But remasterise Deputy represented by the following General formula (18a) or (18b)
,
where R6represents a C1-Saltillo group and
WG1represents cyano.



 

Same patents:

FIELD: chemistry.

SUBSTANCE: in compound of formula (I): , R1 represents C1-4-alkoxy C3-6cycloalkyl optionally substituted with atom of halogen, hydroxyl, trifluoromethyl, optionally substituted with halogen atom 5-6-member heterocyclyl, in which heteroatoms are selected from oxygen, optionally substituted with halogen atoms phenyl or optionally substituted with halogen atoms 5-6-member heteroaryl, in which heteroatoms are selected from nitrogen and/or sulfur; R2 represents hydrogen or trifluoromethyl; R3 represents hydrogen, optionally substituted with atom of halogen, C3-6cycloalkyl, optionally substituted with atom of halogen, trifluoromethyl, C1-4-alkyl phenyl, optionally substituted with atom of halogen, trifluoromethyl, C1-4-alkoxy heterocyclyl, which has in ring 1-2 heteroatoms, selected from nitrogen, oxygen or sulfur, or optionally substituted with C1-4-alkyl 5-6-member heterocyclyl, which has in ring 1-2 heteroatoms, selected from nitrogen or oxygen, R4 and R5 independently represent hydrogen; X represents covalent bond or lower alkylene; X1 represents covalent bond or lower alkylene, Y represents covalent bond or lower alkylene, optionally substituted with hydroxy or cycloalkyl; and Z represents -C=C-, -R6C=CR7- or -CHR6CHR7-, where R6 and R7 in each position represent hydrogen or lower alkyl.

EFFECT: antilipolytic effect of compounds.

30 cl, 7 dwg, 31 ex

FIELD: chemistry.

SUBSTANCE: invention pertains to the method of obtaining 2-amino-6-azido-9-(2,3,5-tri-O-acetyl-β-D-ribofuranosyl)purine and can be used in organic chemistry and pharmaceutical industry. The method lies in that, 2-amino-6-azido-9-(2,3,5-tri-O-acetyl-β-D-ribofuranosyl)purine and sodium azide interact in the presence of the above mentioned tetrametylammonium chloride boiled for 4 hours in absolute acetonitrile. The obtained compound is cleaned by elution of benzol. The residue is dissolved in chloroform and the desired product is separated during precipitation using hexane.

EFFECT: high degree of purity with high output.

1 ex

FIELD: chemistry.

SUBSTANCE: method implies that suspension 2-amino-6-azido-9-(2,3,5-tri-O-acetyl-β-D-ribofuranozile)purine in 60% anhydrous hydrogen fluoride solution of pyridine is diazotizied with tert-butylnitrite during 1 hour at (-18) - (-22)°C. Reaction mixture is decomposed with cut ice. Reaction product is purified by, flash-chromatography on aluminum oxide. Then produced 2-fluorine-6-azido-9-(2,3,5-tri-O-acetyl-β-D-ribofuranozile)purine is hydrogenated at air pressure in 10% acetic acid solution of absolute ethyl acetate with 10% palladium on carbon solution occurrence during 18 hours. Reaction product is purified in acetonitrile solution by flash-chromatography on aluminum oxide at 50-55°C and crystallized from alcohol.

EFFECT: production of compound of high purity with high output.

2 ex

FIELD: chemistry.

SUBSTANCE: invention applied for relates to process of obtaining 2,6- dichlor-9-(2,3,5-tru-O-acetyl-β-D-ribofuranozyl) purine and may be used in organic chemistry and pharmaceutical industry. The process involves conduction of 2,6- dichlor-9-(2,3,5-tru-O-acetyl-β-D-ribofuranozyl) purine with tret-butyl nitrite in the methylene chloride medium at (-18)-(-22)°C during 2 hours in presence of pyridine hydrochloride and phosphorus oxychloride followed by decomposing the reaction mixture with chipped ice, and cleansing of the target product in methylene chloride with flash-chromatography on silica gel.

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1 ex

FIELD: chemistry.

SUBSTANCE: this invention covers method of production of 2-chloroadenosine and may be used in organic chemistry and pharmaceutical industry. The method includes ammonolysis of 2.6-di-chloro-9-(2,3,5-tri-O-acetyl-(β-O-ribofuranozyl)purine in absolute ethyl acetate saturated with ammonia at 0°C during 3 days with further hydrolysis of obtained 5'-0-acetyl-2-chloro-adenosine with 20% ammonia solution in methanol at 20°C during 6 hours, isolation of desired product from the reaction mixture by boiling in mixture of chloroform and methanol, their volumetric ratio 3:1, and purification by crystallization from water.

EFFECT: production of substance with high purity.

1 ex

FIELD: organic chemistry, medicine.

SUBSTANCE: invention relates to compound of the formula (I) wherein each among R represents independently hydrogen atom, (C1-C6)-alkyl, (C3-C7)-cycloalkyl, phenyl or phenyl-(C1-C3)-alkyl; X and X' represent -CH2OH, -CO2R2, -OC(O)R2, -CH2OC(O)R2 or C(O)NR3R4 wherein R2, R3 and R4 represent independently hydrogen atom (H), (C1-C6)-alkyl substituted optionally with one-three (C1-C6)-alkoxy-groups, (C1-C6)-alkylthio-groups, halogen atoms, hydroxy-, amino-, mono-(C1-C6)-alkyl)-amino-, di-(C1-C6)-alkyl)-amino-group; Z and Z' represent independently (C1-C6)-alkyl broken optionally with one-three sulfur atoms (S) or non-peroxide oxygen atom (O), or they absent; n = 1-3; or to its pharmaceutically acceptable salt. Compounds are agonists of adenosine A2A-receptors and can be used for inhibition of inflammatory response or inflammation treatment.

EFFECT: valuable medicinal properties of compounds.

56 cl, 1 tbl, 21 dwg, 37 ex

The invention relates to nucleoside analogs of formula (1) in which R1represents H or a group protecting the hydroxyl, R2represents H, a group protecting the hydroxyl group of phosphoric acid, a protected group, phosphoric acid or a group of the formula P(R3R4in which R3and R4are the same or different and represent a hydroxyl group, a protected hydroxyl group, alkoxygroup, allylthiourea, cyanoacetylurea, amino group, substituted alkyl group; And represents alkylenes group containing from 1 to 4 carbon atoms, and a represents a substituted purine-9-ilen group or substituted 2-oxopyrimidine-1-ilen group containing at least one Deputy, selected from hydroxyl groups, protected hydroxyl groups, amino groups, protected amino groups, alkyl groups

The invention relates to medicine and provides substances that are effective against tumors and viruses, for which conventional anti-tumor agents and antiviral agents exhibit only insufficient effects, and have cancerostatic action and antiviral effects on different tumor immune

The invention relates to certain oxipurinol the nucleosides, compounds related data oxipurinol the nucleosides, acyl derivatives and compositions that contain at least one of these compounds

The invention relates to purine derivative of L-nucleoside of the formula (I), where R1, R2', R3' and R4- N; R2, R3and R5- HE; Z1- N; Z2selected from N and CH; Z3- NR-, -C(R)2, -S-, where R, same or different, selected from H, Br, NH2, alkyl and alkenyl; Z4selected from C=O, -NR-, -C(R)2- where R, same or different, selected from H and Br; Z5Is N; X is selected from H, HE, SH, -SNH2, -S(O)NH2, -S(O)2NH2Y from H and NH2; W is O, and Y represents NH2then Z3is not a-S-

FIELD: chemistry.

SUBSTANCE: disclosed are α- and β-crystalline forms of 5'-desoxy-N4-carbopentyloxy-5-fluorocytidine of formula (III) , their preparation method through crystallisation of the raw product from a suitable solvent and pharmaceutical compositions based on the said compounds, having anti-cancer activity. The solvent used when preparing the α-modification is an ester or a mixture of ester-containing solvents. The solvent used when preparing the β-modification is a mixture of water and alkanol or a mixture of tetrahydrofuran and diethyl ether or carbon tetrachloride.

EFFECT: obtaining compounds and pharmaceutical compositions based on the said compounds, having anticancer activity.

10 cl, 2 dwg, 7 ex

FIELD: chemistry.

SUBSTANCE: invention refers to anhydrous polymorphic salt representing hemisulphate 1-[4(S)-azido-2(S),3(R)-dihydroxy-4-(hydroxymethyl)-1-(R)-cycloamyl]- cytosine of formula (Ia) possessing extra stability and improved physical properties that facilitates production, transportation of compound and preparation of compositions on the basis of compounds of formula I . Invention also refers to various crystal forms of compounds of formula (Ia), characterised by certain values of lattice constant D (interplanar spacing), to methods of their production from compounds of formula (I), to pharmaceutical compositions on the basis of compounds of formula (Ia) and to method of disease treatment caused by C hepatitis virus, including injection if such treatment is required, of therapeutically effective amount of compound of formula (Ia).

EFFECT: production of compound possessing extra stability and improved physical properties that facilitates production, transportation of compound and preparation of compositions on its basis.

14 cl, 5 dwg, 1 tbl, 9 ex

FIELD: chemistry; medicine.

SUBSTANCE: invention pertains to nucleoside derivatives with general formula (I) , where R1 represents hydrogen; COR5, where R5 is chosen from C1-18 alkyl, phenyl, CH2OPh and CH2Ph; C(=O)OR5, where R5 represents C1-18 alkyl; or COCH(R6)NHR7, where R6 represents C1-5 alkyl, and R7 represents R5OCO, where R5 is C1-18 alkyl, R2 represents hydrogen; COR5 , where R5 is chosen from C1-18 alkyl, C1-18 alkenyl, phenyl or CH2OPh; C(=O)OR5, where R5 is chosen from C1-18 alkyl, C1-18 alkenyl, substituted with low alkyl; C(=O)NHR5, where R5 represents C1-18 alkenyl; or COCH(R6)NHR7, where R6 is chosen from side-chains of natural amino acid and C1-5alkyl, and R7 is chosen from hydrogen and R5OCO, where R5 is C1-18 alkyl; R3 and R4 are the same and are chosen from hydrogen; COR5, where R5 is chosen from C1-18 alkyl, or phenyl; C(=O)OR5, where R5 is C1-18 alkyl, or R3 and R4 together represent C(CH3)2; or their pharmaceutical acidic additive salts; under the condition that, at least one of R1, R2, R3 or R4 is not hydrogen. The invention also relates to pharmaceutical compositions, which have antiviral activity to HCV.

EFFECT: obtaining of a range of new biologically active substances.

15 cl, 2 tbl, 9 ex

The invention relates to nucleoside analogs of formula (1) in which R1represents H or a group protecting the hydroxyl, R2represents H, a group protecting the hydroxyl group of phosphoric acid, a protected group, phosphoric acid or a group of the formula P(R3R4in which R3and R4are the same or different and represent a hydroxyl group, a protected hydroxyl group, alkoxygroup, allylthiourea, cyanoacetylurea, amino group, substituted alkyl group; And represents alkylenes group containing from 1 to 4 carbon atoms, and a represents a substituted purine-9-ilen group or substituted 2-oxopyrimidine-1-ilen group containing at least one Deputy, selected from hydroxyl groups, protected hydroxyl groups, amino groups, protected amino groups, alkyl groups

The invention relates to a derivative copernicia and their pharmaceutically acceptable salts of General formula I

< / BR>
where R1represents a methyl group, R2represents a methyl group, R4represents a hydroxy-group and X represents a methylene group; R1represents a methyl group, R2represents a hydrogen atom, R4represents a hydroxy-group and X represents a methylene group; R1represents a methyl group, R2represents a methyl group, R4represents a hydrogen atom and X represents a methylene group; R1represents a hydrogen atom, R2represents a hydrogen atom, R4represents a hydroxy-group and X represents a methylene group; or R1represents a methyl group, R2represents a methyl group, R4represents a hydroxy-group and X represents a sulfur atom

The invention relates to a method for producing 5'-deoxy-5-ptoluidine formula I, including (a) the interaction of 2',3'-O-isopropylidene-5-ptoluidine formula II with a functional derivative of a sulfonic acids R-SO3H, where R is a (C1-C4)alkyl, triptorelin, unsubstituted, mono-, di - or tizamidine phenyl group, (b) interaction of a derivative of formula IV with iodide of alkaline or alkaline earth metal, (C) hydrolysis of a derivative of formula V in an acidic environment, and (d) recovering a derivative of formula VI with hydrogen or a hydrogen donor

The invention relates to new compounds of formula I Nu-O-Fa, where O is oxygen, Nu is a nucleoside or nucleoside analogue, including such nitrogen base, as adenine, Esenin, cytosine, uracil, thymine; Fa - acyl monounsaturated C18YPD C20-9-fatty acids, which fatty acid etherification hydroxyl group in 5-position of the sugar portion of the nucleoside or nucleoside analog, or a hydroxyl group, an acyclic chain of an analogue of the nucleoside

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to group of compounds of general formula where R1, R2, R3, R4, R5 and R6 independently on each other represent C1-4 alkyl, -SO3H, polysulfated β-glycosyl or polysulfated diglycosyl group, on condition that, at least, one of R1-R6 represents polysulfated β-glycosyl or polysulfated diglycosyl group, or their pharmaceutically acceptable salts, where glycosyl group contains pentopyranose or hexopyranose molecule with configuration of choice, and diglycosyl group contains pentopyranose or hexopyranose molecule with configuration of choice, one hydroxyl group of which is glycosylated by other pentopyranose or hexopyranose molecule with configuration of choice. Invention also relates to pharmaceutical composition to be used in treatment of acute or chronic inflammatory diseases of respiratory ways in mammals on the basis of said compounds or their pharmaceutically acceptable salts.

EFFECT: application of said compounds or their pharmaceutically acceptable salts for obtaining medication for treatment of acute or chronic inflammatory diseases of respiratory ways in mammals.

37 cl, 4 tbl, 19 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to oligosaccharide, suitable for vaccine against meningitis A, which includes first mannose unit, which has spacer in alfa-configuration in C-1, where said spacer is able to conjugate with protein, and bound to second mannose unit by 1,6-bond, which binds C-6 of first unit with C-1 of second unit, 1,6-bond including phosphonate. Invention also relates to methods of obtaining oligosaccharide and improved methods of obtaining mannose derivative, suitable for obtaining immunogenic oligosaccharide. Invention also relates to pharmaceutical composition for induction of immune response, immunogenic composition, capable of inducing formation of protective antibodies against meningitis A and vaccine against meningitis A, which include oligosaccharide.

EFFECT: obtained glycoconjugates have C-phosphonate bond, which is much more stable than natural phosphodiester bonds, as well as higher immunologic activity.

51 cl, 4 dwg, 3 tbl, 16 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to oligosaccharide, suitable for vaccine against meningitis A, which includes first mannose unit, which has spacer in alfa-configuration in C-1, where said spacer is able to conjugate with protein, and bound to second mannose unit by 1,6-bond, which binds C-6 of first unit with C-1 of second unit, 1,6-bond including phosphonate. Invention also relates to methods of obtaining oligosaccharide and improved methods of obtaining mannose derivative, suitable for obtaining immunogenic oligosaccharide. Invention also relates to pharmaceutical composition for induction of immune response, immunogenic composition, capable of inducing formation of protective antibodies against meningitis A and vaccine against meningitis A, which include oligosaccharide.

EFFECT: obtained glycoconjugates have C-phosphonate bond, which is much more stable than natural phosphodiester bonds, as well as higher immunologic activity.

51 cl, 4 dwg, 3 tbl, 16 ex

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