Synthesis of oligonucleotides

FIELD: chemistry.

SUBSTANCE: present invention discloses a method of producing oligonucleotides, involving the following steps: a) providing a hydroxyl-containing compound of formula (A) wherein B is a heterocyclic base and i) R2 is H, a protected 2-hydroxyl group, F, protected amino group, O-alkyl group, O-substituted alkyl, substituted alkylamino or C4'-O2' methylene bridge; R3 is OR'3, NHR"3, NR"3R'"3, wherein R'3 is a group, protected hydroxyl, protected nucleotide of protected oligonucleotide, R"3, R'"3 are independently amine-protecting groups, and R5 is OH or ii) R2 is H, protected 2'-hydroxyl group, F, protected amino group, O-alkyl group, O-substituted alkyl, substituted alkylamino, C4'-O2' methylene bridge; R3 is OH, and R5 is OR5 and R'5 is a hydroxyl-protecting group, protected nucleotide or protected oligonucleotide or iii) R2 is OH, R3 is OR'3, NHR"3, NR"3R'"3, wherein R3 is a hydroxyl-protecting group, protected nucleotide or protected oligonucleotide, R"3, R'"3 are independently amine-protecting groups, and R5 is OR'5 and R'5 is a hydroxyl-protecting group, protected nucleotide or protected oligonucleotide; b) reaction of said compound with a phosphitylation agent in the presence of an activator of formula (I) (activator I), wherein R is alkyl, cycloalkyl, aryl, aralkyl, heteroalkyl, heteroaryl; R1, R2 is H or together form a 5-6-member ring; X1, X2 are independently N or CH; Y is H or Si(R4)3, where R4 is alkyl, cycloalkyl, aryl, aralkyl, heteroalkyl, heteroaryl; B is a deprotonated acid, to obtain a phosphitylated compound; c) reaction of said phosphitylated compound without separation thereof from a second compound of formula (A), wherein R5, R3, R2, B are independently selected but have the same values as given above, in the presence of an activator II other than I.

EFFECT: improved method.

9 cl, 14 ex, 1 dwg

 

The technical field to which the invention relates,

This invention relates to a process for the production of oligonucleotides.

The level of technology

Oligonucleotides are key compounds in the natural Sciences play an important role in various fields. For example, they are used as probes in the field of gene expression analysis, as primers in polymerase chain reaction (PCR) or DNA sequencing.

In addition, there are also many potential therapeutic applications, which include, for example, http://www.multitran.ru/c/m.exe?t=tik oligonucleotides.

A growing number of applications require more oligonucleotides, therefore, there is an urgent need to develop improved methods for their synthesis.

For a General overview see, e.g., "Antisense - From Technology to Therapy" Blackwell Science (Oxford, 1997).

One of the known types of building blocks in the synthesis of oligonucleotides are phosphoramidate; see, for example, S.L. Beaucage, M. H. Caruthers, Tetrahedron Letters 1859 (1981) 22. These phosphoramidite nucleosides, deoxyribonucleosides and their derivatives are commercially available. 3'-O-phosphoramidite used in conventional solid-phase synthesis, but other synthetic procedures also use the 5'-Oh and 2'-O-phosphoramidite. One of the stages in obtaining such phosphoramidites NUS is esedov is fosfaurilirovania (protected) nucleosides. After fosfaurilirovania received humidity usually distinguished with the use of expensive methods of separation, for example chromatography. After highlighting sensitive humidity must be stored in special conditions (for example, at low temperature, dehydrated). During storage the quality of amidites may be reduced by some degree of decomposition and hydrolysis. Can happen both of these adverse reactions, and the results become visible. Most often hydroxyl group and amino group, and other functional groups present in the nucleoside, protect before fosfaurilirovaniem the remaining 3'-, 5'- or 2'-O-hydroxyl group.

These phosphoramidite then condense with the hydroxyl groups of the nucleotides or oligonucleotides. The use of dedicated amidite can also lead to partial hydrolysis by condensation of amidite.

Phosphoramidite are expensive compounds. Regular price of desoxidation is in the range of 40.00 euros per gram. The corresponding building blocks of RNA are even more expensive.

The invention

The objective of the invention is to provide a method of obtaining oligonucleotides that overcomes at least some shortcomings of the prior art.

In one embodiment, the implementation is given the second invention is the method for obtaining oligonucleotide, incorporating the following stages:

a) providing a hydroxyl-containing compounds having the formula

in which

B is a heterocyclic base

and

i) R2represents H, a protected 2'-hydroxyl group, F, a protected amino group, alkyl group, O-substituted alkyl, substituted alkylamino or C4'-O2' methylene bridge,

R3represents OR'3That other"3, NR3R'"3where R'3represents a group protecting the hydroxyl-protected nucleotide or a protected oligonucleotide, R3, R'"3independently from each other represents a group protecting the amine,

and R5represents OH

or

ii) R2represents H, a protected 2'-hydroxyl group, F, a protected amino group, an O-alkyl group, O-substituted alkyl, substituted alkylamino or C4'-O2' methylene bridge,

R3represents OH and

R5represents OR'5and R'5represents a group protecting the hydroxyl-protected nucleotide or a protected oligonucleotide

or

iii) R2represents OH,

R3represents OR'3,Other"3, NR3R'"3where R'3represents a group protecting the hydroxyl-protected nucleotide or Conn the config oligonucleotide, R3, R"'3independently from each other represents a group protecting the amine, and

R5represents OR'5and R'5represents a group protecting the hydroxyl-protected nucleotide or a protected oligonucleotide;

b) interaction of the compounds with fosfaurilirovanny agent in the presence of an activator having the formula I (activator I)

in which

R represents alkyl, cycloalkyl, aryl, aralkyl, heteroalkyl, heteroaryl,

R1, R2represent H or form a 5-6-membered ring,

X1X2independently of one another are N or CH,

Y represents H or Si(R4)3where R4represents alkyl, cycloalkyl, aryl, aralkyl, heteroalkyl, heteroaryl,

B represents deprotonirovannoi acid,

obtaining fosfaurilirovannogo connection

c) interaction of the specified fosfaurilirovannogo connection without highlighting it with a second compound having the formula

in which R5, R3, R2, B is selected independently from each other, but have the same definitions as above,

in the presence of activator II selected from the group consisting of tetrazole, derived tetrazole, 4,5-dicyanoimidazole, trifurcata is as pyridinium and mixtures thereof.

In accordance with this invention fosfaurilirovannoe connection is obtained by fosfaurilirovania hydroxyl group of a nucleoside, nucleotide or oligonucleotide using activators having the formula I, which are preferably derivatives of imidazole.

Received sensitive phosphoramidic without purification or selection condense with the hydroxyl groups of nucleosides, nucleotides or oligonucleotides in the presence of activator II, other than the activator I. Any allocation received phosphoramidite or office amidite from the activator I will not be held. Preferably the reaction proceeds in the same reaction vessel. Activator II is used in the presence of activator I.

Activators condensation of amidite known from the prior art, had a high reactivity in the activation functions of amidite. The use of such activator for procedures fosfaurilirovania also leads to some degree of "excessive reaction" (for example, to the formation of a 3'-3' by-product). To overcome this and other problems, the reactivity of the activator is modulated. In this case, the reaction selectively stops at the level of amidite essentially free of by-products, such as 3'-3'-a by-product. Only this result (the formation of amidite in situ) lets continue the curve to this process by the beginning of the condensation of amidite.

Activator II is able to induce phase condensation. After addition of activator II amidic takes amidino condensation.

Can be used as activator II all activators (other than the activator (I), which are able to activate the received amidic to react with the hydroxyl-containing compound at the stage c); i.e. tetrazole and derivatives tetrazole. Preferred derivatives of tetrazole are benzylmercaptan and ethylthioethyl (ETT). Suitable compounds selected from the group consisting of nitrogen-containing heterocycles having deprotonirovannoi form hydrogen acid, pyridine, and salts of pyridine and mixtures thereof. Nitrogen-containing heterocycles have N0-H bond, i.e. N is not protonated. These compounds can be used in the form of salts by combining with acids, such as acid H+B-in which B-has the same meaning as defined in the claims. Other suitable activator II is pyridine, preferably triptorelin pyridinium.

Preferred compounds selected from the group consisting of tetrazole, derived tetrazole, 4,5-dicyanoimidazole, triptoreline pyridinium and mixtures thereof.

After the condensation is usually applied oxidation (education PO) or sulfonation (education PS). For education PO preferred peroxide the approach. This reaction can be performed without any of the stages of extraction (iodine oxidation requires several stages of extraction).

In the case of sulfonation may use any known reagent for sulphonation (i.e. PADS, S-Tetra, beaucage). The preferred reagent for the formation of PS is sulfur. The difference in the cost of production of evidence in favor of the application of sulfur.

In one implementation, the reaction may proceed in the presence of acetone.

Hospitalise agent can be used in more or less equimolar amount relative to the hydroxyl groups of hydroxyl-containing compounds.

In another embodiment, the implement may be used in excess, for example 3-5 mol/mol, hydroxyl groups in the hydroxyl-containing compound.

In another preferred embodiment, the implementation of the polymeric alcohol is added after stage b) under item 1 [claims]. Suitable polymeric alcohols include polyvinyl alcohol (PVA), commercially available as PVA of 145,000 Merck, Darmstadt. Preferred is a porous PVA with a particle size of >120 µm (80%). Suitable also are membrane with hydroxyl groups or other compounds that can form enols.

Activator I can be used in stoichiometric and catalytic (3-50 mol %, predpochtitel what about - 10-30 mol %) of the amount or in excess.

In a preferred embodiment, the implementation of the activator I has a formula selected from the group consisting of

in which

Y represents H or Si(R4)3where R4represents alkyl, cycloalkyl, aryl, aralkyl, heteroalkyl, heteroaryl,

B represents deprotonirovannoi acid,

R represents methyl, phenyl or benzyl.

These activators are described, for example, in Hayakawa et al, J. Am. Chem. Soc. 123 (2001) 8165-8176.

In one embodiment, the implementation of the activator used in combination with an additive. The additive can be selected from deprotonirovannykh forms of the compounds having formula I, and other heterocyclic bases, for example pyridine. Appropriate ratios between the activator and the additive is 1:1-1:10.

In one of the preferred variants of realization of the activator can be obtained by following the procedure "in situ". In this case, the activator does not allocate that leads to improved results. Hydrolysis or decomposition of the target molecules is suppressed.

For higher output fosfaurilirovania 3'- and/or 5'-position of the oligonucleotides (di, tri, Tetra, Penta, hexa, hepta and oktamery) obtaining activator in situ and in combination with the additive is preferred.

As described above, fosfaurilirovania particularly useful p and the synthesis of oligonucleotides and phosphoramidite building blocks. Therefore, in a preferred embodiment, the implementation of the hydroxyl-containing compound is a sugar residue, such as a nucleoside or derived from the oligomer. Such nucleosides are, for example, adenosine, cytosine, guanosine and uracil, deoxyadenosine, deoxyguanosine, deoxythymidine, detoxication and their derivatives may contain a protective group.

Usually, they shall be suitably protected functional group of the heterocycle and hydroxyl groups, with the exception of one, which should be hospitalizovana. Typical groups which are used as protective for 5'OH groups are dimethoxytrityl, monomethacrylates or tert-butyldimethylsilyl (TBDMS), which allows fosfaurilirovania 3'-OH group. Other possible groups are phosphate and H-phosphonates, see, for example,

In the case of pastefire and phosphodiester R may be selected from alkyl, aryl, alkylaryl. Phenyl is preferred.

Other groups protecting the hydroxyl at the 5', 3' and 2' [provisions], well known in the art, such as TBDMS.

In General, hospitalise agent may be the same as in the reaction of fosfaurilirovania using 1-H-tetrazole.

In the preferred embodiment, it has the formula

in which Z represents a leaving group, for example, -CH2CH2CN, -CH2CH=CHCH2CN, para-CH2C6H4CH2CN, -(CH2)2-5N(H)COCF3, -CH2CH2Si(C6H5)2CH3or-CH2CH2N(CH3)COCF3and R1and R2independently from each other represent a secondary amino group, N(R3)2in which R3represents alkyl having from 1 to about 6 carbon atoms; or R3represents geteroseksualnoe or geterotsyklicescoe ring containing from 4 to 7 atoms and having up to 3 heteroatoms selected from nitrogen, sulfur and oxygen.

Typical hospitalise agent is a 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite.

Other preferred hospitalidade reagents are derivatives of oxazaborolidine, as described in N. Ok et al., J. Am. Chem. Soc. 2003, 125, 8307-8317 included here as a reference. This hospitalise agent allows the synthesis of oligonucleotides in which mezhnukleotidnyh communication stereoselective manner is converted into phosphothioate. Such diastereoselective synthesized mezhnukleotidnyh phosphothioate links have a great influence on the use of phosphothioate as antisense drugs or immunostimulatory agents is s.

Fig. 1 shows a reaction scheme in accordance with this invention.

Suitable examples of deprotonated acid B-are triptorelin, triplet, dichloracetate, mesyl, tosyl, o-chlorophenolate. Acids with a pKa less than 4,5 preferred. Preferably they are of low nucleophilicity.

In one embodiment, the implementation of the reaction is carried out in the presence of molecular sieves for the dehydration reaction medium. In General, water should be removed, or connected by dehydration of the environment during the reaction.

It is also possible to combine the activator I according to this invention with hospitaleros agent and add hydroxyl component later. It is also possible to combine the activator I with hydroxyl-containing compound and then add hospitalise agent.

In the case of supplements the activator is mixed with the hydroxyl component before adding hospitilised agent.

To obtain activator "in situ" of the selected acid is preferably addedafteradd the additive at a controlled reaction temperature.

Hospitalise agent may be added before adding the selected acid or after it.

Regarding addition of acid and hospitalised agent nucleoside component can be added at the end or in the beginning.

In a preferred variant of the implementation of the corresponding base and activator hydroxyl-containing compound and hospitalise agent are combined and the acid is added at the beginning of the reaction.

Fosfaurilirovannoe connection (phosphoramidite) then condense with the hydroxyl group of a nucleoside, nucleotide or oligonucleotide in the presence of activator II.

After the reaction of the compounds described above, the received triavir subjected to oxidation. The oxidation can be used, for example, to obtain a stable phosphate and thiophosphate links.

As used herein, the term "oligonucleotide" also includes oligonucleotide, analogs of oligonucleotides, modified oligonucleotides, nucleotide mimetics and the like in the form of RNA and DNA. In General, these compounds form a skeleton linked Monomeric subunits, where each linked Monomeric subunit is directly or indirectly attached to the residue of the heterocyclic base. Linkages between Monomeric subunits, Monomeric subunit and residues of heterocyclic bases can vary in structure, resulting in a plurality of variants of the resulting compounds.

This invention is particularly useful in the synthesis of oligonucleotides having the formula Xnwhere each X is selected from A, dA, C, dC, G, dG, U, dT and n=2 to 30, preferably 2 to 12, more preferably 2 to 8 or 2 to 6, and their derivatives, provided is a protective group. Modifications known in the art, are modified heterocyclic bases, sugars or links connecting the Monomeric subunit. Variations mezhnukleotidnyh ties described, for example, in WO 2004/011474, starting from the bottom of page 11, is included as a reference.

Typical derivatives are phosphothioate, fastdictionary, methyl and alkyl - phosphonates and phosphonoacetate.

Other typical modifications take place in the sugar residues. Either ribose replaced by another sugar, any one or more provisions are replaced by other groups such as F, O-alkyl, S-alkyl, N-alkyl. The preferred embodiment is a 2'-methyl and 2'-methoxyethoxy. All these modifications are known in the art.

As for the rest of heterocyclic bases, there are many other synthetic substrates that are used in the art, such as 5-methylcytosine, 5-hydroxy-methylcytosine, xanthine, gipoksantin, 2-aminoadenine, 6 - or 2-alkyl derivatives of adenine and guanine, 2-thiouracil. Such modifications are also described in WO 2004/011474, starting on page 21.

When used in the synthesis of these grounds usually have a protective group, for example, N-6-benzyladenine, N-4-benzylation or N-2-isobutylamino. In General, all reactive group which is not p is rednaznachena to participate in subsequent reactions, must be protected, especially hydroxyl groups of sugar.

In embodiments of relating to the synthesis of oligonucleotides, useful for carrying out the reaction in the presence of aldehydes or ketones, which can be used either as a reaction medium or as an additional solvent with other solvents.

Suitable such compounds are able to form enols. Typical compounds have the formula R1R2C=O, in which R1and R2independently of one another represent H or consist of 1-20 carbon atoms, which may form a cyclic structure by themselves, or R1and R2form a cyclic system together, in which R1or R2are not H. Especially preferred ketone is acetone. The presence of acetone inhibits the activity of any quantities of amines, such as Diisopropylamine (DIPA), which are released during the process fosfaurilirovania. This can be used to fosfaurilirovania shorter and longer oligonucleotides with similar results (no decomposition). Other ketone compounds having the formula Rx-C(=O)-Ryin which RXand Ryindependently of one another represent C1-C6alkyl or together form cycloalkyl, can also be is used insofar because they are capable of forming enolate in the presence of, for example, amines with CH2group in α-position.

This invention is further illustrated by the following non-limiting examples.

Example 1

Synthesis of 5'-O-DMTr-T-T-3'-O-Lev of cyanomethylphosphonate via in situ 5'-O-DMTr-T-3'-O-phosphoramidite using trifenatate methylimidazole (MIT)

5.0 g of 5'-O-DMTr-T-3'-OH (9.2 mmol, 1.0 EQ.) and of 2.34 g MIT (to 11.9 mmol, 1.3 EQ.) was dissolved in 100 ml dichloromethane was added 3 g of molecular sieves 3E (angstroms), and the mixture was stirred for 10 minutes was Added to 3.8 ml of 2-cyanoethyl N,N,N',N'-Tetra-isopropylacetanilide (11,9 mmol, 1.3 EQ.). The formation of 5'-O-DMTr-T-3'-O-phosphoramidite was completed after 2 h, then was added 3.28 g of 5'-OH-T-3'-O-Lev (for 9.64 mmol, of 1.05 equiv.) and 51 ml of tetrazole (0,45 M, 22, 95mm mmol, 2.5 EQ.) and was stirred overnight. Formed trehzameshchenny postit was oxidized by the addition of 4.57 g I2, 140 ml of tetrahydrofuran (THF), 35 ml of pyridine and 4 ml of H2O. the Reaction was completed after 10 minutes the Reaction mixture was evaporated, dissolved in 300 ml dichloromethane, was extracted with 200 ml of saturated sodium thiosulfate solution and then was extracted with 200 ml of saturated solution of sodium bicarbonate. The combined aqueous layers were extracted with 30 ml of dichloromethane, the combined organic layers were obezvozhivani Sul is an atom of magnesium, and the solvent evaporated. The output amounted to 9.0 g (colorless foam): 98%; purity (determined by high performance liquid chromatography (HPLC)): 84%.

Example 2

Synthesis of 5'-O-DMTr-dCBz-T-3'-O-Lev of cyanomethylphosphonate through obtaining 5'-O-DMTr-dCBz-3'-O-phosphoramidite in situ using trifenatate methylimidazole (MIT)

108 mg MIT high (0.56 mmol, 1.5 EQ.) and 224 mg of 5'-O-DMTr-dCBz-3'-OH (from 0.37 mmol, 1.0 EQ.) was dissolved in 9 ml of dichloromethane and was added 300 mg of molecular sieves 3E. 140 μl of 2-cyanoethyl N,N,N',N'-tetraisopropylphosphorodiamidite (0.44 mmol, 1.2 EQ.) was added to the mixed solution. The formation of 5'-O-DMTr-dCBz-3'-O-phosphoramidite was completed after 30 minutes the Mixture was filtered, added 125 mg of 5'-OH-T-3'-O-Lev (from 0.37 mmol, 1.0 EQ.) and 2 ml of tetrazole (0,45 M, 0.9 mmol, 2.4 EQ.) and was stirred overnight. Formed trehzameshchenny postit was oxidized by adding 10 ml of oxidizing solution (254 mg I2, and 7.8 ml of THF, 1.9 ml of pyridine and 222 μl of H2O). The reaction was terminated after 30 min. Output (determined by HPLC): 66%.

Example 3

Synthesis of 5'-O-DMTr-dCBz-dGiBu-3'-O-Lev cyanomethylphosphonate of Trevira through obtaining 5'-O-DMTr-dCBz-3'-O-phosphoramidite in situ using trifenatate methylimidazole (MIT)

to 2.57 g of 5'-O-DMTr-dCBz-3'-OH (6.0 mmol, 1.0 EQ.) and 1.76 g MIT (9.0 mmol,1.5 EQ.) was dissolved in 6 ml of acetone and 6 ml of acetonitrile and was added 3.0 g of molecular sieves 3E. 2,46 ml of 2-cyanoethyl N,N,N',N'-tetraisopropylphosphorodiamidite (7,74 mmol, 1.3 EQ.) was added to the mixed solution. The formation of 5'-O-DMTr-dCBz-3'-O-phosphoramidite was completed after 30 minutes the Solution was filtered and added to a solution 2,48 g 5'-OH-GiBu-3'-O-Lev (5.7 mmol, of 0.95 EQ.) and 2.3 g of benzylmercaptan (to 12.0 mmol, 2.0 EQ.) in 20 ml of dichloromethane and 20 ml of acetonitrile and stirred 30 minutes, the Solution containing the formed trehzameshchenny postit was filtered and was sulfurously by adding 14 g of polymer-bound tetrathionate of 25.2 mmol, 4.2 EQ.). The reaction was completed after 16 h Output (determined by HPLC): 84%.

Example 4

Synthesis of 5'-O-DMTr-dCBz-dCBz-3'-O-Lev cyanomethylphosphonate of Trevira through obtaining 5'-O-DMTr-dCBz-3'-O-phosphoramidite in situ using trifenatate methylimidazole (MIT)

10 g of 5'-O-DMTr-dCBz-3'-OH (15.8 mmol, 1.0 EQ.) and 7.75 g MIT (to 39.5 mmol, 2.5 EQ.) was dissolved in 30 ml of dichloromethane and 30 ml of acetonitrile, was added 10 g of molecular sieves 3E and the mixture was stirred for 30 minutes at 9.0 ml of 2-cyanoethyl N,N,N',N'-tetraisopropylphosphorodiamidite (28.4 mmol, 1.8 EQ.) was dissolved in 15 ml dichloromethane and 15 ml of acetonitrile. A solution of 5'-O-DMTr-dCBz-3'-OH and MIT was added dropwise to a stirred solution of 2-cyanoethyl N,N,N',N'-tetraisopropylphosphorodiamidite. The formation of 5'-O-DMTr-dCBz-3'-O-phosphoramidite C which were conducted in 30 minutes This solution was filtered and added to a solution 5,43 g 5'-OH-CBz-3'-O-Lev (12.6 mmol, 0.8 EQ.) and 7.6 g of benzylmercaptan (39,5 mmol, 2.5 EQ.) in 90 ml of dimethylformamide and 450 ml of acetonitrile and stirred for 10 minutes, the Solution containing the formed trehzameshchenny postit, was filtered and was sulfurously by adding 50 g of polymer-bound tetrathionate (90 mmol, 5.7 EQ.). The reaction was completed after 16 h Output (determined by HPLC): 80%.

Example 5

Synthesis of 5'-O-DMTr-dABz-dGiBu-3'-O-Lev cyanomethylphosphonate of Trevira through obtaining 5'-O-DMTr-dABz-3'-O-phosphoramidite in situ using trifenatate methylimidazole (MIT)

5.0 g of 5'-ODMTr-dABz-3'-OH (5.8 mmol, 1.0 EQ.) and 1.8 g MIT (9.2 mmol, 1.6 EQ.) was dissolved in 50 ml acetone and 50 ml of acetonitrile, was added 2.5 g of molecular sieves 3E and the mixture was stirred for 15 min to 3.0 ml of 2-cyanoethyl N,N,N',N'-tetraisopropylphosphorodiamidite (9.5 mmol, 1.6 EQ.) was added to the mixed solution. The formation of 5'-O-DMTr-dABz-3'-O-phosphoramidite was completed after 1 h the solution was filtered and added to a solution 2,22 g 5'-OH-GiBu-3'-O-Lev (5.1 mmol, 0,94 EQ.) and 2.9 g of benzyl-mercaptotetrazole (15.1 mmol, 2.6 EQ.) in 25 ml of dichloromethane and 25 ml of acetonitrile and stirred for 40 minutes, the Solution containing the formed trehzameshchenny postit, filtered and Alferov is whether the addition of 2 g of polymer-bound tetrathionate (3.6 mmol, 3.9 EQ.). The reaction was completed after 16 h Output (determined by HPLC): 71%.

Example 6

Synthesis of 5'-O-DMTr-T-dGiBu-3'-O-Lev cyanomethylphosphonate of Trevira through obtaining 5'-O-DMTr-T-3'-O-phosphoramidite in situ using trifenatatemethylimidazole (MIT)

5.0 g of 5'-O-DMTr-T-3'-OH (9.2 mmol, 1.0 EQ.) and 2.7 g MIT (13.5 mmol, 1.5 EQ.) was dissolved in 50 ml acetone and 50 ml of acetonitrile, was added 2.5 g of molecular sieves 3E and the mixture was stirred for 15 min to 3.0 ml of 2-cyanoethyl N,N,N',N'-tetraisopropylphosphorodiamidite (9.5 mmol, 1.03 EQ.) was added to the mixed solution. The formation of 5'-O-DMTr-T-3'-O-phosphoramidite was completed after 1 h the solution was filtered and added to a solution of 4.44 g of 5'-OH-GiBu-3'-O-Lev (10.2 mmol, 1.1 EQ.) and 5.3 g of benzylmercaptan (27.6 mmol, 1.6 EQ.) in 50 ml of dichloromethane and 50 ml of acetonitrile and stirred for 2 hours, the Solution containing the formed trehzameshchenny postit, was filtered and was sulfurously by adding 30 g of polymer-bound tetrathionate (54 mmol, 5.9 EQ.). The reaction was completed after 16 h Output (determined by HPLC): 90%.

Example 7

Synthesis of 5'-O-DMTr-T-dCBz-dCBz-dCBz-3'-O-Lev cyanomethylphosphonate of Trevira through obtaining 5'-O-DMTr-T-P(S)-dCBz-3'-O-phosphoramidite in situ using trifenatate methylimidazole (MIT)

100 mg of 5'-O-DMr-T-P(S)-dC Bz-3'-OH (0.10 mmol, 1.0 EQ.) and 24.4 mg MIT (0.11 mmol, 1.1 equiv.) was dissolved in 10 ml dichloromethane, was added 200 mg of molecular sieves 4E. 32 μl of 2-cyanoethyl N,N,N',N'-tetraisopropylphosphorodiamidite (0.10 mmol, 1.0 EQ.) was added to the mixed solution. The formation of 5'-O-DMTr-T-P(S)-dCBz-3'-O-phosphoramidite was completed after 24 h was Added 82 mg of 5'-OH-dCBz-3'-P(S)-dCBz-3'-O-Lev (0.09 mmol, 0.9 EQ.) and 366 μl of a solution of tetrazole (0,45 M, 0.16 mmol, 1.6 EQ.) and was stirred for 45 h Formed trehzameshchenny postit was sulfurously by adding 400 mg of polymer-bound tetrathionate within 72 hours Output (determined by HPLC): 58%.

Example 8

Synthesis of 5'-O-DMTr-dCBz-dGiBu-dCBz-dCBz-3'-O-Lev cyanomethylphosphonate of Trevira through obtaining 5'-O-DMTr-dCBz-P(S)-dGiBu-3'-O-phosphoramidite in situ using trifenatate methylimidazole (MIT)

100 mg of 5'-O-DMTr-dCBz-P(S)-dGiBu-3'-OH (0.09 mmol, 1.0 EQ.) and 17.8 mg MIT (0.09 mmol, 1.0 EQ.) was dissolved in 10 ml dichloromethane, was added 200 mg of molecular sieves 4E. To mix the solution was added 28 ál 2-cyanoethyl N,N,N',N'-tetraisopropylphosphorodiamidite (0.09 mmol, 1.0 EQ.). The formation of 5'-O-DMTr-dCBz-P(S)-dGiBu-3'-O-phosphoramidite completed in 3 hours was Added 40 mg of 5'-OH-dCBz-3'-P(S)-dCBz-3'-O-Lev (0.04 mmol, 0.5 EQ.) and 0.9 ml of a solution etitioners the La (0.25 M, 0.23 mmol, 2.5 EQ.) and was stirred for 2 h Formed trehzameshchenny postit was sulfurously by adding 200 mg of polymer-bound tetrathionate within 72 hours Yield 30 mg (14.1 mmol, white crystals): 16%; purity (determined by HPLC): 67%.

Example 9

Synthesis of S'-O-DMTr-dCBz-dCBz-dABz-T-3'-O-Lev cyanomethylphosphonate of Trevira through obtaining 5'-O-DMTr-dCBz-P(S)-dCBz-3'-O-phosphoramidite in situ using trifenatate benzylimidazole (BIT)

100 mg of 5'-O-DMTr-dCBz-P(S)-dCBz-3'-OH (0.09 mmol, 1.0 EQ.) and 46 mg BIT (0,17 mmol, 1.9 EQ.) was dissolved in 5 ml of acetone and 5 ml of acetonitrile, was added 500 mg of molecular sieves 3E. 58 ál 2-cyanoethyl N,N,N',N'-tetraisopropylphosphorodiamidite (0.14 mmol, 1.5 EQ.) was added to the mixed solution. The formation of 5'-O-DMTr-dCBz-P(S)-dCBz-3'-O-phosphoramidite was completed after 1 h was Added 41.3 mg 5'-OH-dABz-3'-P(S)-T-3'-O-Lev (0.05 mmol, of 0.55 EQ.) and 43.7 mg benzylmercaptan (0.23 mmol, 2.5 EQ.) and was stirred for 1.5 h Formed trehzameshchenny postit was sulfurously by adding 500 mg of polymer-bound tetrathionate within 72 hours Output (determined by HPLC): 70%.

Example 10

Synthesis of 5'-O-DMTr-dGiBu-dGiBu-dG!Bu-T-dGiBu-dGiBu-3'-O-Lev of cyanomethylphosphonate through getting 5 O-DMTr-dG lBu-P(O)-dGiBu-3'-O-phosphoramidite in situ using trifenatate methylimidazole (MIT)

200 mg of 5'-O-DMTr-dGiBu-P(O)-dGiBu-3'-OH (0.18 mmol, 1.0 EQ.) and 56 mg MIT (0.27 mmol, 1.5 EQ.) was dissolved in 5 ml of acetone was added 300 mg of molecular sieves. In the mixed solution was added 128 μl 2-cyanoethyl N,N,N',N'-tetraisopropylphosphorodiamidite (BisPhos) (0.4 mmol, 2.2 EQ.). The formation of 5'-O-DMTr-dGiBu-3'-P(O)-dGiBu-3'-O-phosphoramidite ended after 15 minutes was Added 156 mg 5'-OH-dGiBu-T-dGiBudGiBu-3'-O-Lev (0.09 mmol, 1.0 EQ.) and 87 mg of benzylmercaptan (0.46 mmol, 5.0 equiv.) and was stirred for 20 minutes Resulting trehzameshchenny postit was oxidized by the addition of 3.7 ml of oxidizing solution (94 mg I2, 2,9 ml of THF, 0.7 ml of pyridine and 82 μl of H2O). The reaction was terminated after 30 min. Output (determined by HPLC): 51%.

Example 11

Synthesis of 5'-O-DMTr-dG!Bu-T-3'-O-Lev cyanomethylphosphonate of Trevira

200 g (312 mmol) DMTr-dGIBu-3'-OH and 80 g (408 mmol) MIT was dissolved in 400 ml of dichloromethane and 400 ml of acetone. Added 200 g of molecular sieves and 89 ml (1.25 mol) NMI (N-methyl-imidazole). At 15°C to stir the solution was added 109 ml (344 mmol) BisPhos. The formation of 5'-O-DMTr-dGIBu-3'-O-phosphoramidite was completed in 10 min, and the solution was stirred for another 30 minutes to 88.4 g (260 mmol) of 5'-OH-T-3'-O-Lev and of 83.4 g (624 mmol) ETT was dissolved in 600 ml Aceto the a and 600 ml of dichloromethane. Added 100 g of molecular sieves and 86 ml (1,08 mol) NMI. To this stirred solution was added 800 ml of phosphoramidite. The reaction was terminated after 10 min, and cooled mixture (ice bath) was added 46 ml of a solution of peroxide of butanone (Curox M400). The reaction was completed in 5 minutes Conversion (determined by HPLC): 100%.

Example 12

Synthesis of 5'-O-DMTr-dGiBu-T-3'-O-Lev cyanomethylphosphonate of Trevira

1.0 g (1.56 mmol) DMTr-dGiBu-3'-OH and 368 mg (1.88 mmol) MIT was dissolved in 3 ml dichloromethane and 3 ml of acetone. Added 1 g of molecular sieves and 154 μl (1.25 mol) NMI. At 15°C to stir the solution was added 594 ál (of 1.87 mmol) BisPhos. The formation of 5'-O-DMTr-dGiBu-3'-O-phosphoramidite was completed in 10 min, and the solution was left to mix for another 30 minutes 438 mg (1,29 mmol) 5'-OH-T-3'-O-Lev and 396 mg (of 3.07 mmol) ETT was dissolved in 5 ml of acetone and 5 ml of dichloromethane. Added 1 g of molecular sieves and 248 ml (3.61 mol) NMI. To this stirred solution was added 5.5 ml of a solution of phosphoramidite. The reaction was terminated after 10 min and

A) was added 25 mg (7.8 mmol) of sulfur (S8) and 2.5 mg of Na2S×9H2O. the Reaction was terminated after 10 minutes Conversion (determined by HPLC): 100%;

C) was added 25 mg (7.8 mmol) of sulfur (S8). The reaction was completed after 3 hours, the Conversion of 99%.

Example 13

Synthesis of 5'-O-DMTr-T-dCBz-dGiBu-T--dG iBu-3'-O-Lev cyanomethylphosphonate of Trevira

5.0 g (4.9 mmol) DMTr-T-dCBz-3'-OH and 2.4 g (12.3 mmol) MIT was dissolved in 10 ml dichloromethane and 10 ml of acetone. Added 8 g of molecular sieves and 980 µl (12.3 mol) NMI. At 15°C to stir the solution was added 3,13 ml (9,85 mmol) BisPhos. The formation of 5'-O-DMTr-T-dCBz-3'-O-phosphoramidite was completed in 10 min, and the solution was allowed to mix for 30 minutes was Added 100 ml of heptane, decantation and the precipitate formed was added 10 ml of dichloromethane and 10 ml of acetone. 4.44 g (2,79 mmol) 5'-OH - dGiBu-T-T-dGiBu-3'-O-Lev and 1.05 g (of 8.06 mmol) ETT was dissolved in 15 ml of acetone and 15 ml of dichloromethane. Added 5 g of molecular sieves and 640 μl (8,06 mol) NMI. To this stirred solution was added 20 ml of phosphoramidite. The reaction was terminated after 10 min, and added 930 mg (3,09 mmol) PADS. The reaction was terminated after 10 minutes Conversion (determined by HPLC) of 92%.

Example 14

Synthesis of 5'-O-DMTr-dCBz-dABz-dCBz-dABz-dCBz-dABz-dCBz-dABz-3'-O-Lev cyanomethylphosphonate of Trevira

860 mg (0.45 mmol) of 5'-O-DMTr-dCBz-dABz-dCBz-dABz-3'-OH and 133 mg (0.67 mmol) MIT was dissolved in 3 ml dichloromethane and 3 ml of acetone. Added 800 mg of molecular sieves and 55 μl (69 mol) NMI. To the stirred solution was added 214 μl (of 0.65 mmol) BisPhos. the education of 5'-O-DMTr-dC Bz-dABz-dCBz-dABz-3'-3'-O-phosphoramidite was completed in 10 min, and the solution was allowed to mix for 20 minutes was Added 30 ml of heptane, decantation and the precipitate formed was added 5 ml dichloromethane and 5 ml of acetone. 545 mg (0.3 mmol) of 5'-OH-dGiBu-T-T-dGiBu-3'-O-Lev and 117 mg (0.9 mmol) ETT was dissolved in 3 ml acetone, 3 ml of dichloromethane and 0.3 ml of dimethylphthalate (DMF). Added 1 g of molecular sieves and 70 μl (0.9 mmol) NMI. To this stirred solution was added 8 ml of phosphoramidite. The reaction was terminated after 30 min, to the mixture was added 70 μl of a solution of peroxide of butanone (Curox M400). The reaction was terminated after 10 minutes Conversion (determined by HPLC): 80%.

1. The method for obtaining oligonucleotide, comprising the steps:
a) providing a hydroxyl-containing compound having the formula

which is a heterocyclic base and
i) R2is a N-protected 2'-hydroxyl group, F, a protected amino group, an O-alkyl group, O-substituted alkyl, substituted alkylamino or C4'-O2' methylene bridge,
R3represents OR'3That other"3, NR3R"'3where R'3represents a group protecting the hydroxyl-protected nucleotide or a protected oligonucleotide, R3, R"'3independently from the other, represents a group protecting the amine,
and R5HE is a
or
ii) R2is a N-protected 2'-hydroxyl group, F, a protected amino group, an O-alkyl group, O-substituted alkyl, substituted alkylamino or C4'-O2' methylene bridge, R3is a HE, and
R5represents OR'5and R'5represents a group protecting the hydroxyl-protected nucleotide or a protected oligonucleotide
or
iii) R2is a HE,
R3represents OR'3That other"3HR"3R"'3where R'3represents a group protecting the hydroxyl-protected nucleotide or a protected oligonucleotide, R3, R"'3independently from each other represents a group protecting the amine, and
R5represents OR'5and R'5represents a group protecting the hydroxyl-protected nucleotide or a protected oligonucleotide;
b) interaction of the compounds with hospitaleros agent in the presence of an activator having the formula I (activator I)

in which
R represents alkyl, cycloalkyl, aryl, aralkyl, heteroalkyl, heteroaryl,
R1, R2represent H or together form a 5-6-membered ring,
X1, X2independent the one from another are N or CH,
Y represents H or Si(R4)3where R4represents alkyl, cycloalkyl, aryl, aralkyl, heteroalkyl, heteroaryl,
Represents deprotonirovannoi acid,
obtaining fosfaurilirovannogo connection
(C) interaction of the specified fosfaurilirovannogo connection without isolation with a second compound having the formula

in which R5, R3, R2In selected independently from each other, but have the same definitions as above,
in the presence of activator II, different from the activator I.

2. The method according to claim 1, wherein the activator of the formula I has a formula selected from the group including

in which Y is as defined in claim 1,
R represents methyl, phenyl or benzyl.

3. The method according to claim 1 or 2, in which hospitalise agent has the formula
II

in which Z represents a leaving group, a R1and R2independently from each other, are secondary amino groups.

4. The method according to claim 3, in which hospitalise agent is a 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite.

5. The method according to claim 1, in which deprotonirovannoi acid is formed from the group comprising triperoxonane acid, dichloracetic acid, methanesulfonate acid, thrift methansulfonate acid, o-chlorophenolate.

6. The method according to claim 1, wherein the reaction is carried out in the presence of acetone.

7. The method according to claim 1, in which hospitalise agent is used in amounts of from 1.0 to 1.2 mol/mol of hydroxyl groups in the hydroxyl-containing compound.

8. The method according to claim 1, in which hospitalise agent is used in an amount of from 3 to 5 mol/mol of hydroxyl groups in the hydroxyl-containing compound.

9. The method according to claim 1, in which deprotonirovannoi acid is formed from the group comprising triperoxonane acid, dichloracetic acid, methanesulfonate acid, triftormetilfullerenov acid (triplet), o-chloro-phenolate and mixtures thereof.



 

Same patents:

FIELD: medicine, pharmaceutics.

SUBSTANCE: group of inventions refers to a composition of a polynucleotide adjuvant (PICKCa) and methods for using it for immune response manifestation. The polynucleotide adjuvant contains polyriboinosine-polycytidylic acid (PIC) wherein the PIC molecules are heterogenous by molecular weight and have molecular weight 66000 to 1200000 Dalton, at least one antibiotic and one positive ion. The invention also refers to an immunogenic composition containing the polynucleotide adjuvant together with the other immunogenic compositions, such as an antigen specified in viral, bacterial, fungal, parasitic and/or cancer antigens; as well as to methods for using such adjuvant compositions for the generation of immune response, immune response of mucosa on the antigenic compound.

EFFECT: group of inventions provides development of immune response specific for the disease in the host body.

22 cl, 38 dwg, 28 tbl, 7 ex

FIELD: chemistry.

SUBSTANCE: method of producing an aminophosphite monomer of an achiral non-nucleotide insert for modifying oligonucleotides involves dimethoxytritylation of the corresponding alcohol, branching the skeleton of the insert by reacting an amine with an activated carboxylic acid and phosphitylation of the precursor of the end compound in standard conditions. The first step involves synthesis of 4-(2-hydroxyethyl)morpholine-2,3-dione (compound I) by adding diethanolamine solution to a solution of diether of oxalic acid in molar ratio of diether of oxalic acid to diethanolamine equal to 1:1.15, while stirring at room temperature for 15-24 hours. Further, the obtained compound I is dimethoxytrirylated with dimethoxytritylchloride in equimolar ratio of reactants. The obtained compound II is then used for acylation of an aliphatic primary or secondary amine bearing the required functional group(s), at concentration of reactants of 0.1-0.4 M at temperature 25-60°C for 8-24 hours, followed by phosphitylation of the obtained compound 2-cyanoethyl-N,N,N',N' with tetraisopropyl diamidophosphite using a standard method to obtain the end amidophosphite monomer of an achiral non-nucleotide insert.

EFFECT: high output.

4 cl, 4 dwg, 4 tbl, 5 ex

FIELD: medicine.

SUBSTANCE: set contains one base pair showing activity of upstream and downstream primers, and a probe having the following structure: 5' CAAGNACTTCTGTTNCCCCGGACYGA 3'; 5' ATNTNTC AATTGTCANCATAAGC AGCC A 3'; F AM -5' CCTYCGGCNCCTGAYTGCGGCTAATCC 3'-BHQ1.

EFFECT: invention enables high-accuracy identification of the genetic material of human enteroviruses A, B, C, D.

4 dwg, 1 tbl, 1 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to medicine and concerns a pharmaceutical composition for inducing an immune response on tumour-associated macrophagues containing a mini-gene DNA construct which codes a polypeptide in a pharmaceutically acceptable carrier wherein the polypeptide contains three immunogenic legumain fragments bonded together with linker peptides, and wherein each linker peptide consists of the AAA or AAY sequence.

EFFECT: invention provides effective control of breast carcinoma, non-small-cell lung cancer and colorectal carcinoma.

8 cl, 2 ex, 26 dwg

FIELD: chemistry.

SUBSTANCE: invention relates to oligonucleotide-oligocationic molecules AiBjH, which are used in molecular biology, diagnosis and therapeutic versions of use. The oligonucleotide-oligocationic molecules AiBjH can be synthesised via automated phosphorus amidite chemistry and have oligonucleotide fragments Ai and oligocationic fragments Bj. The fragment Ai is an i-dimensional oligonucleotide residue with index i from 5 to 50, in which nucleotide A is an oligomer with natural or synthetic nucleotide bases and/or pentafuranosyl groups and/or native phosphodiester bonds, as well as chemical modifications or substitutions thereof. Fragment Bj is a j-dimensional organic oligocationic fragment with index j from 1 to 50, in which B is selected from a group comprising -HPO3-R1-(X-R2n)n1-X-R3-O-, where R1, R2n and R3, identical or different, denote C1-C5 alkylene, X denotes NH or NC(NH2)2, index n1 = 2 to 20; -HPO3-R4-CH(R5X1)-R6-O-, where R4 denotes C1-C5 alkylene, R5 and R6, identical or different, denote C1-C5 alkylene, and X1 denotes a putrescine, spermidine or spermine residue.

EFFECT: high efficiency of the method.

18 cl, 14 dwg, 5 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: inventions relate to field of biotechnology. iRNK-agents contain sense strand, where said sense strand contains in internal area 15-30 successive nucleotides, which differ from nucleotides of SEQ ID NO:427 sequence not more than by one nucleotide, and antisense strand, where said antisense strand contains in internal area 15-30 successive nucleotides, which differ from nucleotide of SEQ ID NO:428 sequence by not more than one nucleotide.

EFFECT: iRNA agents can contain modifications, which increase their stability in biological sample, cholesterol molecule, non-natural nucleotide base, iRNA-agents are applied for inhibition of expression of PB2 gene of influenza A virus.

73 cl, 9 dwg, 6 tbl, 3 ex

FIELD: chemistry.

SUBSTANCE: invention describes novel polynucleotide and amino acid sequences of Brachyspira hyodysenteriae, which can be used to diagnose diseases in animals, caused by B. hyodysenteriae, to treat or prevent diseases associated with infection with B. hyodysenteriae. The invention describes a cell containing a plasmid containing a polynucleotide, for treating and preventing a disease associated with infection of an animal with B. Hyodysenteriae. The invention describes immunogenic and vaccine compositions for generating immune response in an animal, which contain a polypeptide, a polynucleotide, a cell or a plasmid for treating or preventing infection of animals by B. hyodysenteriae, as well as sets of instruments for diagnosis which contain a monoclonal antibody, capable of biding the disclosed polypeptide or a polypeptide or polynucleotide. The invention enables to successfully diagnose diseases caused by B. hyodysenteriae, prevent or treat animals infected with B. hyodysenteriae. The sequences described herein can be used for diagnosis and therapeutic and/or preventive treatment of animals from diseases caused by other types of Brachyspira, including B. intermedia, B. suantatina, B. alvinipulli, B. aalborgi, B. innocens, B. murdochii and B. pilosicoli.

EFFECT: high efficiency of using the composition.

39 cl, 4 tbl

FIELD: chemistry.

SUBSTANCE: method involves reaction of a hydroxyl-containing compound, which contains a sugar fragment, with a phosphitylation agent in the presence of an activator of formula I , where R = alkyl, cycloalkyl, aryl, aralkyl, heteroalkyl, heteroaryl, R1, R2 = either H, or together form a 5-6-member ring, X1, X2 = independently N or CH, Y = H or Si(R4)3, where R4 = alkyl, cycloalkyl, aryl, aralkyl, heteroalkyl, heteroaryl, B = trifluoroacetate, dichloroacetate, mesyl, tosyl, o-chlorophenolate.

EFFECT: novel method of producing phosphoramidites.

17 cl, 21 ex

FIELD: medicine.

SUBSTANCE: invention describes nucleotide sequence (dwg.2), coding immunogenic polypeptide LcrV(G113), serving the base for construction of recombinant plasmid DNA pETV-I-3455, with the size 6538 bp, which codes immunogenic polypeptide LcrV(G113). Plasmid consists of plasmid pBR322 replicon, β-lactamase gene, determining resistance to ampicillin, T7-promoter, 1ac-operator, f1-replicon and DNA fragment, flanked by the sites for restrictases Ndel and Hindlll, coding synthesis of protein LcrV(G113), which starts from initiating codon ATG. Described is recombinant strain of bacteria E. coli BL21 (DE3)/pETV-I-3455 - producer of immunogenic polypeptide LcrV(G113) with amino acid sequence, represented on dwg.3, where tryptophan in position 113 (W113) is substituted with glycin. Described is method of obtaining said polypeptide by cultivation of strain E. coli BL21(DE3)/pETV-I-3455. Cells are destroyed in buffer solution by ultrasound and polypeptide is isolated successively by gel-permeation chromatography with application of carrier TSK HW-40, anion-exchanging and hydrophobic chromatography.

EFFECT: invention makes it possible to obtain product with high immunogenic and protective activity.

5 cl, 10 dwg, 2 tbl, 5 ex

FIELD: medicine.

SUBSTANCE: what is offered is a method for producing high-polymer yeast RNA of used beer yeast. Used beer yeast concentrated by centrifugation is suspended in an aqueous solution of oleic acid titrated by alkali to pH 7-8. The suspension is kept at 98-102°C for 40-60 minutes. The hot lysate is settled for 20-24 h at room temperature. A supernatant is poured out with using a siphon. Then NaCl is added to the poured out liquid to the concentration 2-3 M. The suspension is kept for 20-96 h at room temperature. Then it is centrifuged without cooling in a low-speed centrifuge. The prepared cakes are consistently washed out in the NaCl solution 2-3 M and 92-96% ethanol by resuspension at room temperature and low-speed centrifugation.

EFFECT: invention promotes spread-out of the raw-material base and simplification of the high-polymer RNA technology.

1 ex

FIELD: chemistry.

SUBSTANCE: present invention relates to a method of producing 5-(4-methyl-1H-imidazol-1-yl)-3-(trifluoromethyl)benzamine , involving reaction of 4-methyl-1H-imidazole or salt thereof with a compound of formula , where X denotes a halogen and Y denotes NH2, in the presence of a suitable base or corresponding transition metal as a catalyst or combination thereof in a suitable solvent. The invention also relates to other versions of the method of producing a compound of formula (I) and intermediate compounds used.

EFFECT: new versions of the method of producing 5-(4-methyl-1H-imidazol-1-yl)-3-(trifluoromethyl)benzamine (I), which is an intermediate compound in synthesis of biologically active compounds.

9 cl, 14 ex

FIELD: chemistry.

SUBSTANCE: invention relates to a new improved method of producing onium tetrafluoroborates through reaction of an onium halide with trialkyloxonium tetrafluoroborate, trialkylsulphonium tetrafluoroborate or triphenylcarbonium tetrafluoroborate, characterised by that the halide has formula (1) [XR4]+ Hal-, where X denotes N, P, Hal denotes Cl, Br or I and R in each case independently denotes a linear alkyl having 1-8 C atoms, or the halide has formula (2) [(R1R2N)-C(=SR7)(NR3R4)]+ Hal- (2), where Hal denotes Br or I R1-R7 each independently denotes a linear alkyl having 1-8 C atoms, or the halide has formula (3) [C(NR1R2)(NR3R4)(NR5R6)]+ Hal- (3), where Hal denotes CI, Br or I and R1-R6 each independently denotes a linear alkyl having 1-8 C atoms, or the halide has formula (4) [HetN]+ Hal- , where Hal denotes CI, Br or I and HetN+ denotes a heterocyclic cation selected from a group comprising imidazolium pyrrolidinium pyridinium where each of substitutes R1' - R4' independently denotes hydrogen, CN, linear or branched alkyl having 1-8 C atoms, dialkylamine containing alkyl groups having 1-4 C atoms but which is not attached to he heteroatom of the heterocyclic ring.

EFFECT: method enables to obtain products with low content of halides with high purity and high output.

5 cl, 12 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: there is described a compound of formula I: or its pharmaceutically acceptable salt, where R2 represents (CR3R4)n-NR5R6 and m, p, q, Ar, R1, R3, R4, R5 and R6 are those as specified in the patent claim and defined as selective 5-NT6 and/or 5-NT2A antagonists. There is also described a pharmaceutical composition containing this compound, and application thereof in preparing drugs for treating diseased conditions of central nervous system chosen from psychoses, schizophrenia, manic depressions, neural disorders, memory impairment, attention deficient syndrome, Parkinson's disease, amyotrophic lateral sclerosis, Alzheimer's disease, malnutrition and Huntington's disease.

EFFECT: preparation of the compounds which can find application in treatment of a diseased condition of central nervous system.

27 cl, 1 tbl, 29 ex

FIELD: chemistry.

SUBSTANCE: present invention relates to compounds of formula (I) and their pharmaceutically acceptable salts and esters. The disclosed compounds have LXR-alpha and/or LXR-beta agonist properties. In formula (I) R1 is hydrogen, halogen; R2 is lower alkyl, flouro-lower alkyl; R3 is hydrogen, phenyl; R4 is hydrogen, hydroxy; R5 is hydrogen; phenyl; R6 is phenyl, a 5-6-member heteroaryl with one or two heteroatoms selected from nitrogen and sulphur, a 9-member bicyclic heteroaryl with a sulphur atom as a heteroatom, which can be optionally substituted with a halogen, or R6 is , R7 is a lower alkyl; R8 is phenyl which is optionally substituted with one substitute selected from a group consisting of halogen, fluoro-lower alkyl, R9-O-C(O)-, R10R11NC(O)-, phenyl-lower alkoxy; R9, R10, R11 independently represent hydrogen or lower alkyl; L is a single bond, lower alkylene or lower alkenylene; m assumes values from 0 to 3; n is equal to 0 or 1.

EFFECT: obtaining a new compound and a pharmaceutical composition which contains the disclosed compound as an active ingredient for therapeutic and/or preventive treatment of diseases.

23 cl, 47 ex

FIELD: chemistry.

SUBSTANCE: invention refers to synthesis of [18F]fluororganic compounds ensured by reaction of [18F]fluoride and relevant halogenide or sulphonate with alcoholic vehicle of formula 1 where R1, R2 and R3 represent hydrogen atom or C1-C18 alkyl.

EFFECT: possibility for mild process with low reaction time and high yield.

21 cl, 2 tbl, 27 ex

FIELD: chemistry.

SUBSTANCE: invention relates to new displaced heterocyclic derivatives that can be used in treatment of diabetes and to reduce the content of cholesterol. In formula m is 1; n is 1; Q is C; A is -(CH2)x2-0-(CH2)x3-, where x2 varies from 1 to 3 and x3 is 0; B is a bond or it is (CH2)x4, where x4 varies from 1 to 2; X represents CH or N; X2, X3, X4, X5, X6 represent C, N, O; provided that one from X2 X3 X4 X5 and X6 represents N; and at least one of X2, X3, X4, X5, and X6 represents C; R1 represents H or C1-C6alkyl; R2 is H; R2a, R2b and R2c can be equal or different and selected from H, C1-C6alkyl, C1-C6alkoxy, halogen or thyano; R3 is selected from phenyloxycarbonile, C1-C6alkyloxycarbonile, phenylcarbinol, phenyl, alkoxy; Y represents CO2R4 (where R4 represents H or C1-C6alkyl); (CH2)m can be not necessarily displaced by 1 substitute.

EFFECT: produced are pharmaceutical composition for treatment of diabetes and to reduce the content of cholesterol.

13 cl, 2 tbl, 22 dwg, 88 ex

FIELD: chemistry of organophosphorus compounds, chemical technology.

SUBSTANCE: invention describes a method for synthesis of monohydroperfluoroalkanes, bis-(perfluoroalkyl)phosphinates and perfluoroalkylphosphonates. Method involves treatment of at least one perfluoroalkylphosphorane with at least one base wherein base(s) are chosen from group consisting of alkali-earth metal hydroxides, metalloorganic compound in useful solvent or at least one organic base and an acid in useful reaction medium. Also, invention describes novel perfluoroalkylphosphonates and bis-(perfluoroalkyl)phosphinates, using novel perfluoroalkylphosphonates and bis-(perfluoroalyl)phosphinates as ionic liquids, catalysts of phase transfer or surfactants.

EFFECT: improved method of synthesis.

18 cl, 19 ex

FIELD: organic chemistry, medicine, pharmacy.

SUBSTANCE: invention relates to using phenylethenyl- or phenylethynyl-derivatives as antagonists of glutamates receptors. Invention describes using compound of the general formula (I):

wherein each among R1, R2, R3, R4 and R5 means independently of one another hydrogen atom, (C1-C6)-alkyl, -(CH2)n-halogen, (C1-C6)-alkoxy-group, -(CH2)n-NRR', -(CH2)n-N(R)-C(O)-C1-C6)-alkyl, phenyl or pyrrolyl that can be unsubstituted or substituted with one or more (C1-C6)-alkyl; each among R, R' and R'' means independently of one another hydrogen atom or (C1-C6)-alkyl; A means -CH=CH- or C≡C; B means ,, , , or wherein R6 means hydrogen atom, (C1-C)-alkyl, -(CH2)n-C(O)OR, or halogen atom; R7 means hydrogen atom, (C1-C6)-alkyl, -(CH2)n-C(O)OR', halogen atom, nitro-group or oxodiazolyl group that can be unsubstituted or substituted with (C1-C6)-alkyl or cycloalkyl; R8 means hydrogen atom, (C1-C6)-alkyl, -(CH2)n-OH, -(CH2)n-C(O)OR'' or phenyl; R9 means (C1-C6)-alkyl; R10 and R11 mean hydrogen atom; R12 means -(CH2)n-N(R)-C(O)-(C1-C6)-alkyl; R13 means hydrogen atom; each R14, R15, R16 and R17 independently of one another means hydrogen atom or (C1-C6)-alkoxy-group; each R18, R19 and R20 independently of one another means hydrogen atom; R21 means hydrogen atom or (C1-C6)-alkyl; R22 means hydrogen atom, (C1-C6)-alkyl or (C1-C6)-alkyl comprising one or more substitutes chosen from groups hydroxy- or halogen atom; R23 means hydrogen atom, (C1-C6)-alkanoyl or nitro-group; each among R24, R25 and R26 independently of one another means hydrogen atom or (C1-C6)-alkyl; n = 0, 1, 2, 3, 4, 5 or 6; X means -O- or -S-; Y means -CH= or -N=, and its pharmaceutically acceptable salts used in preparing medicinal agents designates for treatment or prophylaxis of disorders mediated by mGluR5-receptors. Also, invention describes compounds of the formula (I-A), compound of the formula (I-B-1) given in the invention description, and a medicinal agent used in treatment or prophylaxis of disorders mediated by mGluR5-receptors.

EFFECT: valuable medicinal properties of compounds.

44 cl, 1 tbl, 44 ex

FIELD: organic chemistry, chemical technology.

SUBSTANCE: invention relates to derivatives of adamantine, in particular, to a new method for preparing adamant-1-yl-containing azoles of the general formula I-VIII: wherein R1 means ; R2 means ; R3 means ; R4 means ; R5 means ; R6 means ; R7 means , and R8 means . Indicated derivatives of adamantine are semifinished products used in synthesis of biologically active substances. Proposed method for preparing these compounds involves using a new method for synthesis of adamant-1-yl-containing azoles that includes the addition reaction of azoles: 2-methylimidazole, 3(5)-methylpyrazole and 4-methylpyrazole, 3,4-dinitropyrazole, 1,2,4-triazole, 3-methylpyrazole, 3-nitro-1,2,4-triazole and 5-methyltetrazole to 1,3-dehydroadamantane in the mole ratio of 1,3-dehydroadamantane to azole = 1:1 in diethyl ether medium at temperature 100°C for 4-5 h.

EFFECT: improved preparing method.

8 ex

The invention relates to imidazole derivative of General formula I, where n=0 or 1, R1is hydrogen, alkyl, R2is hydrogen or R2and R3form a double bond, R3is hydrogen, alkyl, R4is hydrogen, alkyl, hydroxy-group, alkoxy, R5is hydrogen or alkyl, or R4and R5form a carboxyl group, R6, R7, R8is hydrogen, alkyl, hydroxy-group, alkoxy, hydroxyalkyl, halogen, X-CHR9-(CHR10)m-, m = 0 or 1, R9and R10is hydrogen or alkyl

FIELD: organic chemistry, chemical technology.

SUBSTANCE: invention relates to derivatives of adamantine, in particular, to a new method for preparing adamant-1-yl-containing azoles of the general formula I-VIII: wherein R1 means ; R2 means ; R3 means ; R4 means ; R5 means ; R6 means ; R7 means , and R8 means . Indicated derivatives of adamantine are semifinished products used in synthesis of biologically active substances. Proposed method for preparing these compounds involves using a new method for synthesis of adamant-1-yl-containing azoles that includes the addition reaction of azoles: 2-methylimidazole, 3(5)-methylpyrazole and 4-methylpyrazole, 3,4-dinitropyrazole, 1,2,4-triazole, 3-methylpyrazole, 3-nitro-1,2,4-triazole and 5-methyltetrazole to 1,3-dehydroadamantane in the mole ratio of 1,3-dehydroadamantane to azole = 1:1 in diethyl ether medium at temperature 100°C for 4-5 h.

EFFECT: improved preparing method.

8 ex

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