RU2131436C1 - Nuclease-resistant oligonucleosides, method of preparing thereof, and nuclease-resistant nucleoside dimer - Google Patents

Abstract

FIELD: biochemistry. SUBSTANCE: described is new nuclease-resistant oligonucleoside containing sequence of general formula I:

wherein each Z is independently R¹ or

wherein W is -D-D-D- wherein each D is independently CHR or NR⁶ wherein R is independently hydrogen, OH, and R⁶ is hydrogen provided that only one of D is NR⁶, W′ is independently W or

, R¹ is independently OH, SH or NR²R³ wherein R² and R³ are independently H or C₁-C₆- alkyl, each R⁵ is independently H or C₁-C₁₂ alkyl, each Y is independently H or OH, each B is independently adenine, citosine, guanine, thymine, uracil or modifications thereof, each e and f is integer from 0 to 50 provided that at least one of each f is at least 1, j is integer from 1 to about 200, K is 0 or integer from 1 to about 197, and each of m and n is independently from 1 to 200, each P is independently integer from 2 to 4; Q is either from 1 to about 197 provided that sum j+k+Q is from about 5 to 200. EFFECT: improved properties of the nuclease-resistant oligonucleosides. 9 cl, 42 ex, 9 tbl

Description

 This application is a partial continuation of application US N 07/562180, filed August 3, 1990; US application N 07/582287, filed September 13, 1990; US application N 07/582456, filed September 13, 1990; and US application N 07/582457, filed September 13, 1990

The present invention relates to compounds, compositions and methods for inhibiting gene expression. The proposed compounds contain
(1) oligonucleoside sequences comprising from about 6 to about 200 bases, with a triatomic bridge between nucleosides or
(2) oligonucleoside sequences, comprising from about 9 to 200 bases, with a diol at either or both of their ends.

 An antisense compound refers to any compound that binds or hybridizes to a nucleotide sequence in any nucleic acid, i.e., DNA or RNA, and thereby inhibits the function or synthesis of said nucleic acid. Due to the ability of antisense compounds to hybridize with both RNA and DNA, such compounds may interfere with gene expression at the level of transcription, RNA processing, or translation.

 Antisense molecules can be constructed and synthesized to prevent transcription of specific genes on mRNA by hybridization with genomic DNA and inhibition of the action of RNA polymerase directly or indirectly. The advantage of delivering DNA to the target is that only insignificant amounts of antisense compounds are needed to provide a therapeutic effect. Alternatively, antisense compounds can be constructed and synthesized by hybridization with RNA to inhibit the mechanisms of post-transcriptional modification (RNA processing) or protein synthesis (translation). As examples of RNAse carriers, matrix RNA (mRNA), transport RNA (tRNA), ribosomal RNA (rRNA) and the like can be mentioned. Examples of processing and translation mechanisms include pre-mRNA splicing with the removal of nitrons, 5 'capping at the end of the mRNA, hybridization stop, and nuclease-mediated mRNA hydrolysis.

 Currently, the development of practical applications of antisense technologies for scientific and therapeutic purposes, however, is complicated by a number of technical tasks, Klausner, A, Biotechnology, 8: 303 - 304, 1990. Synthetic antisense molecules are subject to rapid degradation by nuclease, which is present in target cells . Oligonucleoside sequences of antisense DNA or ZNA, for example, are destroyed by exonucleases that act at both the 5 ′ and 3 ′ ends of the nucleic acid. In addition, endonucleases can cleave DNA or RNA via internal phosphodiester bonds between individual nucleosides. As a result of this splitting, the effective half-life of the introduced antisense compounds is very short, which inevitably leads to the use of large, often taken dosage doses.

 Another problem is associated with the extremely high cost of DNA or RNA when using the currently available semi-automatic DNA synthesizers to produce them. Recently, calculated data have been obtained that the production of one gram of antisense DNA is approximately 100,000 am. dollars (Armstrong, L., Business Week, March 5, 1990, p. 89).

 The next problem relates to the delivery of antisense substances to an organ - or target cell. Antisense substances must have access to the cell nucleus (that is, such substances must penetrate the plasma or nuclear membrane). The need to increase membrane permeability (increased hydrophobicity) should, however, be balanced with respect to the need for water solubility (increased hydrophilicity) in the body spaces with liquid contents, for example, as plasma and cell cytosol.

 Another problem is the resistance of antisense substances, whether they act independently in the body or are hybridized with the target nucleic acid. Oligonucleotide sequences, such as antisense DNA, undergo steric recombination around chiral phosphorus centers.

 The delivery of gene products using antisense agents is the inevitable next step in the manufacture of drugs for humans (see Armstrong, p. 88 above). The successful use of antisense technologies for therapeutic purposes, however, requires finding solutions to the above problems.

 One approach to solving the problem of obtaining antisense compounds with stability, resistance to action, nuclease, low cost, and the ability to access and hybridize with nucleic acid targets throughout the body is to synthesize oligonucleoside sequences with modifications of the structure of a normal phosphate-carbohydrate chain.

 Currently, there are essentially two types of modified chain oligonucleosides known. The first type includes modifications of the normal interoligonucleoside phosphodiester bond. The second type involves modification with the replacement of the phosphodiester bond with non-phosphate interoligonucleoside bonds (Uhlmann, E. and Peyman, A. Chemical Reviews, 9 (4), pp. 544-584, 1990.

 As phosphodiester bonds, as is known, phosphotioates, alkylphosphotriethers, methylphosphonates and alkylphosphoramidates are currently used.

 Phosphorothioate-modified phosphodiester bonds mean phosphodiester bonds where one or more bridging oxygen atoms are replaced by sulfur. Such bonds, however, are not suitable for use in antisense compounds. Preservation of the chiral phosphorus center leads to steric modification of monothioates. Moreover, both mono- and dithioates lack a specific hybridization site, and both quickly eliminate from plasma. The high affinity of phosphotioates for glass and plastic also makes the synthesis of these compounds difficult and ineffective.

 Methyl and ethyl phosphotriethers are prepared by reacting phosphodiester-linked oligonucleosides with anhydrous methanol or ethanol. (Miller, P. S. and others, J. Am. Chem. Soc., 93, pp. 6657-6665, 1971).

 The triester bond in oligodeoxyribonucleotide-ethylphosphotriethers is stable under normal physiological pH conditions, although it can be hydrolyzed with a strong acid or base. Methyl phosphotriesters are less stable than ethyl and other alkyl phosphotriethers at neutral pH due to the possibility of nucleophilic substitution of the methyl group of the triester with the introduction of a solvent. Oligodeoxyribonucleotide ethyl phosphotriethers, as it turns out, are completely not susceptible to hydrolysis by nucleases, and are not susceptible to hydrolysis by nucleases or esterases found in fetal calf serum or human serum (see Uhlmann above).

 Methylphosphonates have several significant drawbacks in terms of their therapeutic potential, including their poor solubility in water, chiral phosphorus centers, inability to regulate stereo-selective synthesis with a high yield of the product, fast plasma clearance, and their excretion in urine.

 Oligodeoxyribonucleoside phosphoramidates have internucleoside bonds containing nitrogen-phosphorus bridges. Such nucleic acid analogs can be obtained from phosphoramidate intermediates or by oxidation of intermediate H-phosphonates in the presence of primary or secondary amines. The preparation of H-phosphonate analogues and the oxidation reaction can be easily carried out in an industrial DNA synthesizer.

 Many non-ionic oligonucleotide sequences containing non-phosphate internucleoside bonds, for example, as carbonate, acetate, carbamate or dialkyl or diaryl silyl derivatives, are known and synthesized.

 Although the carbonate bond is resistant to acid hydrolysis, it is fairly easy to split with a base, and therefore special precautions must be taken when removing the protective groups at the end of the synthesis. Although stable duplexes have been found between poly (dA) analogs containing carboxymethyl internucleotide bonds and poly (U) analogs, other bases have not yet been studied. For example, it is not clear whether the carbonate bond, or not, interferes with the exact reproduction of the duplex.

 It is known that carbamate internucleoside bonds have greater water solubility compared to other internucleoside bridges. The use of carbamate bridges is however limited because thymine carbamates do not form hybrids with complementary DNA, while cytosine carbamates do not hybridize with guanine oligomers.

 The carbamate bond, similarly to the carbonate bond, is stable under physiological conditions. Unlike carbonate bonds, the carbamate bridge is stable against base hydrolysis, and this ability simplifies the synthesis of oligomers containing such a bond. The carbamate bridge is resistant to nuclease hydrolysis.

 As well as carbonate and acetate bonds, the carbamate bridge is not similar to the form of phosphodiester internucleotide bonds. Molecular models, however, propose that this bridge ensure that the oligomer accepts conformations, as a result of which it could form hydrogen-linked complexes with complementary nucleic acids.

A carbamate-linked oligomer containing six thymidine units does not form complexes with either A (pA) ₅ or dA (pA) ₅ . On the other hand, a carbamate-linked oligomer containing six deoxycytosine units forms stable complexes with d- (pG) ₆ and poly (dG). The internucleoside bond of the dialkyl or diphenylsilyl derivatives of oligomeric analogs is almost similar to the tetrahedron form of the normal phosphodiester internucleotide bond. These oligomers are obtained in solution by reacting a properly protected nucleoside-3'-O-dialkyl- or diphenylsilyl chloride or trifluoromethanesulfonyl derivative with a 3'-protected nucleoside in anhydrous pyridine. The latter can be obtained by reacting 5'-O-trityl nucleoside with dialkyl or diphenyldichlorosilane or with bis (trifluoromethanesulfonyl) diisopropylsilane.

 Due to the fact that dialkyl and diphenylsilyl groups are sensitive to acid hydrolysis, it is necessary to be especially careful in selecting protective groups for synthesis. Nucleoside dimers and hexamers with siloxane internucleoside bonds and a method for synthesizing such polymers are described by Ogilvie and Cormier, see, for example, Ogilvie K.K. and Cormier J.F., Tetrahedron Letters, 26 (35), pp. 4159-4162 (1985); Ogilvie K.K and Cormier J.F., Nucleic Acid Research, 16 (10), pp. 4583-4594, 1988.

 Although oligonucleoside sequences linked by carbonate, carbamate and silyl bridges have the necessary resistance to nuclease, which makes it attractive to use them as potential antisense reagents, this ability has not yet been reported. Moreover, the ability of these oligomers to be included by cells in culture was not reported anywhere. A potential disadvantage of these oligomers is reported to be their low solubility in an aqueous medium. The possibility of obtaining the titers necessary for their effective use in biological experiments has not yet been clarified, although the solubility can essentially be increased by introducing hydrophilic groups into the molecules.

 The present invention provides oligonucleotide analogs, compositions based on them, intermediates for their preparation and methods for the synthesis of these new oligonucleotide analogues having stability, resistance to nucleases, specificity for target cells and lipid solubility.

Summary of the invention
The present invention provides nucleotide analogs containing oligonucleoside sequences comprising from about 6 to 200 bases and having a triatomic internucleoside bridge. The specified triatomic internucleoside bridge of such oligonucleoside sequences is represented by the formula
-DDD-
in which each D is independently CHR, oxygen or NR ⁶ , where R is independently hydrogen, OH, SH, or NH ₂ , R ⁶ , hydrogen or C ₁ -C ₂ -alkyl, provided that only one of D is oxygen or group NR ⁶ .

 In a preferred embodiment of the present invention, oligonucleoside sequences include bases selected from the group consisting of adenine, cytosine, guanine, uracil, thymine and their derivatives.

где W означает -D-D-D-, в которой каждый радикал D означает независимо CHR, кислород или NR⁶, где R означает независимо водород, OH, SH, или NH₂, R⁶ водород или C₁-C₂-алкил при условии, что только один из D означает кислород или группу NR⁶;
каждый W' независимо означает W или

где каждый R¹ означает OH, SH, группу NR²R³, в которой R² и R³ каждый независимо означает водород или C₁-C₆-алкил, или группу NHR⁴, где R⁴ означает C₁-C₁₂-алкил;
R⁴ означает C₁-C₁₂-ацил;
каждый Y независимо означает H или OH;
каждый B независимо означает аденин, цитозин, гуанин, урацил, тимин и их модификации;
j означает целое число от 1 до примерно 200;
k означает 0 или целое число от 1 до примерно 197; и
q означает 0 или целое число от 1 до примерно 197, при условии, что сумма j + k + q равна примерно 4 - 200.More specifically, the proposed compounds contain oligonucleoside sequences of the formula I

where W is -DDD-, in which each radical D is independently CHR, oxygen or NR ⁶ , where R is independently hydrogen, OH, SH, or NH ₂ , R ^{6 is} hydrogen or C ₁ -C ₂ -alkyl, provided that only one of D is oxygen or an NR ⁶ group;
each W 'independently means W or

where each R ¹ is OH, SH, an NR ² R ³ group in which R ² and R ^{3 are} each independently hydrogen or C ₁ -C ₆ alkyl, or an NHR ⁴ group, where R ⁴ is C ₁ -C ₁₂ - alkyl;
R ⁴ is C ₁ -C ₁₂ acyl;
each Y independently means H or OH;
each B independently means adenine, cytosine, guanine, uracil, thymine and their modifications;
j is an integer from 1 to about 200;
k is 0 or an integer from 1 to about 197; and
q is 0 or an integer from 1 to about 197, provided that the sum j + k + q is about 4-200.

 The proposed compounds contain oligonucleotide or oligonucleoside sequences that may contain a diol at either or both of their ends.

 As diols, it is preferable to use 1,2-diols (glycols).

 As examples of glycols, mention may be made of polyalkylene glycols, preferably polyethylene glycols or polypropylene glycols. Preferred glycols include tetraethylene glycol and hexaethylene glycol. Polyols in which all but two hydroxyls are protected can also be used as diols.

где каждый Z независимо означает R¹ или

где каждый R¹ означает OH, SH, группу MR²R³, в которой R² и R³ каждый независимо означает водород или C₁-C₆-алкил, или группу NHR⁴, где R⁴ означает C₁-C₁₂-ацил;
каждый R⁵ независимо означает водород или C₁-C₁₂-алкил;
каждый из W, W', Y, B, j, k и q имеет вышеуказанные значения;
каждое e и f независимо друг от друга принимает значения от 0 до 50 при условии, что по крайней мере одно из значений e и f равно по крайней мере 1;
каждое m и n независимо друг от друга принимает значения от 1 до 200; каждое p независимо принимает значение от 2 до 4.In the case when the compounds of the invention are oligonucleoside sequences with a diol at either or both ends, these compounds are expressed by formula II

where each Z independently means R ¹ or

where each R ¹ means OH, SH, a group MR ² R ³ in which R ² and R ³ each independently means hydrogen or C ₁ -C ₆ alkyl, or an NHR ⁴ group, where R ⁴ means C ₁ -C ₁₂ - acyl;
each R ⁵ independently means hydrogen or C ₁ -C ₁₂ alkyl;
each of W, W ', Y, B, j, k and q has the above meanings;
each e and f independently, takes values from 0 to 50, provided that at least one of the values of e and f is at least 1;
each m and n independently from each other takes values from 1 to 200; each p independently takes a value from 2 to 4.

где R означает OH, SH, группу NR²R³, в которой R² и R³ каждый независимо означает водород или C₁-C₆-алкил, или группу NHR⁴, где R⁴ означает C₁-C₁₂-ацил;
R¹ означает водород или C₁-C₁₂-алкил;
олиго (N) означает нативную или модифицированную олигонуклеотидную последовательность, содержащую от примерно 9 до 200 оснований;
каждое e и f независимо друг от друга принимает значения от 0 до 50 при условии, что по крайней мере одно из значений e и f равно по крайней мере 1;
каждое m и n независимо друг от друга принимает значение от 1 до 200;
каждое p независимо принимает значение от 2 до 4.In a preferred embodiment, the sum j + k + q is from about 9 to 50 bases, more preferably from about 12 to 50 bases, and most preferably from about 15 to 18 bases. In this embodiment of the invention, the compounds of the invention comprise oligonucleosides of the formula

where R is OH, SH, an NR ² R ³ group in which R ² and R ^{3 are} each independently hydrogen or C ₁ -C ₆ alkyl or an NHR ⁴ group, where R ⁴ is C ₁ -C ₁₂ acyl;
R ¹ is hydrogen or C ₁ -C ₁₂ alkyl;
oligo (N) means a native or modified oligonucleotide sequence containing from about 9 to 200 bases;
each e and f independently, takes values from 0 to 50, provided that at least one of the values of e and f is at least 1;
each m and n independently from each other takes a value from 1 to 200;
each p independently takes a value from 2 to 4.

 In a preferred embodiment, said oligonucleotide comprises, for example, in a homopolymer or heteropolymer form, any of the combinations dA, dC, dG, T.

где R означают OH, SH, группу NR²R³, в которой R² и R³ каждый независимо означает водород или C₁-C₆-алкил, или группу NHR⁴, где R⁴ означает C₁-C₁₂-ацил;
R¹ означает водород или C₁-C₁₂-алкил;
олиго(N) означает олигонуклеотидную последовательность, содержащую от примерно 9 до 50 оснований;
e и f независимо друг от друга принимает значения от 0 до 50 при условии, что по крайней мере одно из значений e и f равно по крайней мере 1;
m и n независимо друг от друга принимает значение от 0 до 200 при условии, что по крайней мере одно из значений m и n составляет от 1 до 200.In the case where polyethylene glycol is used as glycol, in the indicated embodiment, the compounds contain oligonucleosides of the formula

where R is OH, SH, an NR ² R ³ group in which R ² and R ^{3 are} each independently hydrogen or C ₁ -C ₆ alkyl or an NHR ⁴ group, where R ⁴ is C ₁ -C ₁₂ acyl;
R ¹ is hydrogen or C ₁ -C ₁₂ alkyl;
oligo (N) means an oligonucleotide sequence containing from about 9 to 50 bases;
e and f independently, take values from 0 to 50, provided that at least one of the values of e and f is at least 1;
m and n independently from each other takes a value from 0 to 200, provided that at least one of the values of m and n is from 1 to 200.

 The oligonucleotides of the present invention may contain known internucleoside linker groups, for example, phosphodiester, silyl and other well-known communication groups, provided that they contain an effective amount of the proposed linker and / or diol groups completing the synthesis of the D-D-D sequences of the present invention.

где W означает D-D-D-,
в которой каждый радикал D означает независимо CHR, кислород или NR⁶, где R означает независимо водород, OH, SH, или NH₂, R⁶ - водород или C₁-C₂-алкил при условии, что только один из D означает кислород или группу NR⁶;
каждый B независимо означает аденин, цитозин, гуанин, урацил, тимин и их модификации;
R⁷ означает OH, трет-бутилдиметилсилилокси или фосфорамидитную группу, и R⁸ означает OH, защитную группу или трет-бутилдиметилсилилоксигруппу.The present invention also relates to nucleoside dimers of the formula

where W means DDD-,
in which each radical D is independently CHR, oxygen or NR ⁶ , where R is independently hydrogen, OH, SH, or NH ₂ , R ⁶ is hydrogen or C ₁ -C ₂ alkyl, provided that only one of D is oxygen or a group NR ⁶ ;
each B independently means adenine, cytosine, guanine, uracil, thymine and their modifications;
R ⁷ is OH, a tert-butyldimethylsilyloxy or phosphoramidite group, and R ⁸ is OH, a protective group or a tert-butyldimethylsilyloxy group.

 The present invention also provides a method for inhibiting nuclease hydrolysis of compounds containing oligonucleoside sequences. The specified method involves attaching a diol to any 5 '- or 3' - end of the specified connection, or to each of them. Such diols are attached to the 5 ′ and / or 3 ′ end as a result of the interaction of these oligonucleosides with alkoxytrityldiolcyanophosphine, preferably dimethoxytritylglycol cyanophosphine or monomethoxytrityl glycol cyanophosphine.

The present invention also provides a method for inhibiting nuclease hydrolysis of native or modified nucleotide compounds, which comprises preparing oligonucleoside sequences from about 6 to about 200 bases having a triatomic internucleoside bond represented by the above formula
-DDD-.

 The present invention also provides compositions for inhibiting gene expression, comprising compounds containing oligonucleoside sequences of about 6-200 bases, having the aforementioned triatomic internucleoside bond, and a physiologically acceptable carrier. The specified compound may contain diol y of either of or both ends. Polyethylene glycols are preferably used as diols.

 The present invention also provides a method for inhibiting gene expression, which comprises administering to any mammal, if necessary, an effective dose of a compound containing an oligonucleoside sequence of about 6-200 bases having the above triatomic internucleoside linkage. These compounds may contain a diol at either or both of their ends. Polyethylene glycols are preferably used as diols.

 A brief description of the drawings.

 In FIG. 1a shows a synthetic route for the production of aldehyde nucleoside (Compound 1).

 In FIG. 1b shows a synthetic route to the production of a nucleoside complex with phosphonium iodide (Compound II).

 In FIG. 2 shows a synthetic route for producing nucleoside dimers connected by a 3 atomic carbon bridge between nucleosides using an aldehyde nucleoside and a phosphonium iodide nucleoside obtained according to the scheme of FIG. 1a and 1b, respectively.

 In FIG. Figure 3 shows a synthetic route to the preparation of a thymidine dimer using aldehyde-thymidine and phosphono-iodide-thymidine (Compound I and II, respectively).

In FIG. Figure 4 shows a synthetic route for producing a nucleoside dimer connected by a bridge with 2 carbon atoms and 1 nitrogen atom between nucleosides in the form of 3'-CCN-5 '. These dimers are obtained by reacting nucleosides that contain an aldehyde (CHO) with nucleosides containing a functional amino group (NH ₂ ) under reduction conditions.

In FIG. 5 shows a synthetic route for producing a nucleoside dimer connected by a bridge with 2 carbon atoms and 1 nitrogen atom between nucleosides in the form of 3'-CCN-5 '. These dimers are obtained by the interaction of nucleosides that contain an aldehyde (CHO) and a functional amino group (NH ₂ ) under reduction conditions.

In FIG. 6 shows a synthetic route for the preparation of a thymidine dimer connected by a bridge with 2 carbon atoms and 1 nitrogen atom between nucleosides in the form of 3'-CCN-5 '. These dimers are prepared by reacting thymidines which contain an aldehyde (CHO) with thymidines containing a functional amino group (NH ₂ ) under reduction conditions.

In FIG. 7 shows a synthetic route for the preparation of a thymidine dimer connected by a bridge with 2 carbon atoms and 1 nitrogen atom between nucleosides in the form of 3'-CCN-5 '. These dimers are obtained by the reaction of thymidines, which contain an aldehyde (CHO) functional amino group (NH ₂ ) under reduction conditions.

 A detailed description of the invention.

 The proposed compounds essentially relate to oligonucleotide or oligonucleoside sequences that are resistant to nuclease hydrolysis.

 As used herein, the term “nucleoside” means a compound consisting of a purine or pyrimidine base with penta sugar (pentose).

 As used herein, the term “nucleotide” means a phosphoric acid ester of any nucleoside.

 As used herein, the term “nucleotide” means a polynucleotide containing only phosphodiester internucleoside bonds, for example, “native” DNA or RNA.

 Adenosine (A), guanosine (G), cytidine (C), uridine (U), deoxyadenosine (dA), deoxyguanosine (dG), deoxycytidine (dC), and thymidine can be used as nucleosides.

The compounds of the present invention contain oligonucleoside sequences consisting of from about 6 to 200 bases, with a phosphodiester or triatomic internucleoside bond. The triatomic internucleoside bond (-DDD-) contains
1) three carbon atoms,
2) two carbon atoms and one oxygen atom, or
3) two carbon atoms and one nitrogen atom.

 Oligonucleoside sequences include sequences from native or modified nucleosides.

 As used herein, the term “internucleoside bond” means atoms and molecules that form a bridge between the carbon atom at position 3 of the sugar residue of one base of a native or modified nucleoside and the carbon atom at position 5 of the sugar residue of an adjacent base of such nucleoside. Thus, the proposed nucleosides include A, C, G, U, dA, dC, T or their derivatives, for example, 5-bromo or 5-iodouracil, 5-methylcytosine, isocytosine- (2-amino-4-oxopyrimidine ), isoguanine (2-oxo-6-aminopurine), inosine (6-oxopurin), 5-vinylluracil and 5-vinylcytosine.

The triatomic internucleoside bond is represented by the formula
-DDD-
in which each D is independently CHR, oxygen or NR ⁶ , where R is independently hydrogen, OH, SH, or NH ₂ , R ^{6 is} hydrogen or C ₁ -C ₂ alkyl, provided that only one of D is oxygen or an NR group ⁶ .

где W означает -D-D-D, в которой каждый радикал D означает независимо CHR, кислород или NR⁶, где R означает независимо водород, OH, SH, или NH₂, R⁶ - водород или C₁-C₂-алкил, при условии, что только один из D означает кислород или группу NR⁶;
каждый W¹ означает, независимо означает W или

где каждый R¹ означает OH, SH, группу NR²R³, в которой R² и R³ каждый независимо означает водород или C₁-C₆-алкил, или группу NHR⁴, где R⁴ означает C₁-C₁₂-алкил;
R⁴ означает C₁-C₁₂-ацил;
каждый Y независимо означает H или OH;
каждый B независимо означает аденин, цитозин, гуанин, урацил, тимин и их производные;
j означает целое число от 1 до примерно 200;
k означает 0 или целое число от 1 до примерно 197; и
q означает 0 или целое число от 1 до примерно 197 при условии, что сумма j+k+q равна примерно 4 - 200.The proposed compounds contain oligonucleoside sequences of the formula I

where W is —DDD, in which each radical D is independently CHR, oxygen or NR ⁶ , where R is independently hydrogen, OH, SH, or NH ₂ , R ⁶ is hydrogen or C ₁ -C ₂ alkyl, provided that only one of D is oxygen or an NR ⁶ group;
each W ¹ means independently means W or

where each R ¹ is OH, SH, an NR ² R ³ group in which R ² and R ^{3 are} each independently hydrogen or C ₁ -C ₆ alkyl, or an NHR ⁴ group, where R ⁴ is C ₁ -C ₁₂ - alkyl;
R ⁴ is C ₁ -C ₁₂ acyl;
each Y independently means H or OH;
each B independently means adenine, cytosine, guanine, uracil, thymine and their derivatives;
j is an integer from 1 to about 200;
k is 0 or an integer from 1 to about 197; and
q is 0 or an integer from 1 to about 197, provided that the sum j + k + q is about 4-200.

 In a preferred embodiment, the sum j + k + q is from about 9 to 50 bases, more preferably from about 12 to 25 bases, and most preferably from about 15 to 18 bases.

 The compounds of the invention may contain a diol at either or both ends. Preferred diols include glycols, also known as 1,2-diols, containing two hydroxyl groups at adjacent carbon atoms. As preferred glycols, polyalkylene glycols are used. The term "alkylene", as used herein, means open and branched chain radicals containing from 2 to 4 carbon atoms, possibly substituted, as indicated in this description. Examples of such radicals include ethylene, propylene, isobutylene and the like. Preferred polyalkylene glycols are polyethylene glycols, for example, hexaethylene glycol and tetraethylene glycol. Polyols can also be used as diols, each of the hydroxyls of which, in addition to two, has protective groups.

 These diols are attached to any of the 5 ′ or 3 ′ end of such oligonucleosides, or to each of them via phosphodiester bonds. In one embodiment of the present invention, said diols are attached to only one end of the oligonucleoside chain.

The terminal diol is bound to a residue selected from the group consisting of hydroxyl (OH), sulfhydryl (SH), amino group (NH ₂ ), alkylamino group (NH-alkyl substituted group), dialkylamino (NH [alkyl] ₂ ) and NH [acyl] amido group )

где каждый Z независимо означает R¹ или

где каждый R¹ означает OH, SH, группу NR²R³, в которой R² и R³ каждый независимо означает водород или C₁-C₆- алкил, или группу NHR⁴, где R⁴ означает C₁-C₁₂-ацил;
каждый R⁵ независимо означает водород или C₁-C₁₂ алкил;
каждый из W, W', Y, B, j, k и q имеет вышеуказанные значения; каждое e и f независимо друг от друга принимает значения от 0 до 50 при условии, что по крайней мере одно из значений e и f равно по крайней мере 1;
каждое m и n независимо друг от друг принимает значения от 1 до 200;
каждое p независимо принимает значение от 2 до 4.In the case where glycols are present at either or both ends, the compounds of the invention comprise oligonucleoside sequences of formula II

where each Z independently means R ¹ or

where each R ¹ is OH, SH, an NR ² R ³ group in which R ² and R ^{3 are} each independently hydrogen or C ₁ -C ₆ alkyl, or an NHR ⁴ group, where R ⁴ is C ₁ -C ₁₂ - acyl;
each R ⁵ independently means hydrogen or C ₁ -C ₁₂ alkyl;
each of W, W ', Y, B, j, k and q has the above meanings; each e and f independently, takes values from 0 to 50, provided that at least one of the values of e and f is at least 1;
each m and n independently from each other takes values from 1 to 200;
each p independently takes a value from 2 to 4.

 In a preferred embodiment, the sum j + k + q is from about 9 to 50 bases, more preferably from about 12 to 25 bases, and most preferably from about 15 to 18 bases.

 In another embodiment of the present invention, the compounds of the invention comprise oligonucleotide sequences of from about 9 to 200 bases with a diol at either or both of their ends. In yet another embodiment of the present invention, the compounds of the present invention contain oligonucleotide sequences of from about 2 to 200 bases, having the proposed (-D-D-D -) - connection. Preferred diols include glycols, also known as 1,2-diols, containing two hydroxyl groups at adjacent carbon atoms. As preferred glycols, polyalkylene glycols are used. The term "alkylene", as used herein, means open and branched chain radicals containing from 2 to 4 carbon atoms, possibly substituted, as indicated in this description. Examples of such radicals include ethylene, propylene butylene and the like. As preferred polyalkylene glycols, polyethylene glycols are used. Preferred polyalkylene glycols include polyethylene glycols. Hexaethylene glycol and tetraethylene glycol are most preferred.

 These diols are attached to any of the 5'- or 3'-end of such oligonucleosides, or to each of them via phosphodiester bonds. In one embodiment of the present invention, said diols are attached to only one end of the oligonucleoside chain.

The terminal diol is linked to a residue selected from the group consisting of hydroxyl (OH), sulfhydryl (SH), amino group (NH ₂ ), alkylamino group (NH-alkyl substituted group), dialkylamino (NH [alkyl] ₂ ) and amido group (NH [acyl ]). As used herein, the term “alkylene” means straight or branched chain radicals containing from 1 to 12 carbon atoms, optionally substituted as described herein. As examples of alkyl and dialkylamino groups, methyl, ethyl, propyl, butyl, pentyl, hexyl, dimethyl, diethyl, dipropyl, dibutyl, dipentyl and dihexylamines and the like can be mentioned. As used herein, straight chain or branched "NH- (acyl)" or "amino" radicals containing from 1 to 12 carbon atoms with an O = CHNH ₂ terminal group. Examples of amide groups include methanamide, ethanamide, propanamide, butanamide, pentanamide, hexanamide, heptanamide, octanamide, nonanamide, decanamide, undecanamide and dodecanamide.

где R означает OH, SH, группу NR²R³, в которой R² и R³ каждый независимо означает водород или C₁-C₆ -алкил, или группу NHR⁴, где R⁴ означает C₁-C₁₂ -ацил;
R¹ означает водород или C₁-C₁₂-алкил;
олиго (N) означает нативную или модифицированную олигонуклеотидную последовательность, содержащую от примерно 9 до 200 оснований;
каждое e и f независимо друг от друга принимает значения от 0 до 50 при условии, что по крайней мере одно из значений e и f равно по крайней мере 1;
каждое m и n независимо друг от друга принимает значение от 1 до 200;
каждое p независимо принимает значение от 2 до 4.In one embodiment of the invention, the compounds of the invention comprise oligonucleosides of the formula

where R is OH, SH, an NR ² R ³ group in which R ² and R ^{3 are} each independently hydrogen or C ₁ -C ₆ alkyl or an NHR ⁴ group, where R ⁴ is C ₁ -C ₁₂ acyl;
R ¹ is hydrogen or C ₁ -C ₁₂ alkyl;
oligo (N) means a native or modified oligonucleotide sequence containing from about 9 to 200 bases;
each e and f independently, takes values from 0 to 50, provided that at least one of the values of e and f is at least 1;
each m and n independently from each other takes a value from 1 to 200;
each p independently takes a value from 2 to 4.

 Said oligonucleotide sequence is preferably a homopolymer or heteropolymer sequence containing any of the combinations of dA, dC, dG, T or their analogs.

 In a preferred embodiment, m and n are independently 1 to 8, more preferably m and n are 4. In a preferred embodiment, the oligonucleotide sequences contain from about 9 to 50 bases, more preferably from about 12 up to 25 bases and most preferably from about 15 to 18 bases.

где R означает OH, SH, группу NR²R³, в которой R² и R³ каждый независимо означает водород или C₁-C₆ алкил, или группу NHR⁴, где R₄ означает C₁-C₁₂ ацил;
R¹ означает водород или C₁-C₁₂ алкил;
олиго (N) означает нативную или модифицированную олигонуклеотидную последовательность, содержащую от примерно 9 до 200 оснований;
каждое e и f независимо друг от друга принимает значения от 0 до 50 при условии, что по крайней мере одно из значений e и f равно по крайней мере 1;
каждое m и n независимо друг от друга принимает значение от 1 до 200;
каждое p независимо принимает значение от 2 до 4.In a preferred embodiment, the proposed antisense compounds contain polyethylene glycol at both the 5 ′ and 3 ′ ends and are represented by the formula

where R is OH, SH, an NR ² R ³ group in which R ² and R ³ each independently is hydrogen or C ₁ -C ₆ alkyl, or an NHR ⁴ group, where R ₄ is C ₁ -C ₁₂ acyl;
R ¹ means hydrogen or C ₁ -C ₁₂ alkyl;
oligo (N) means a native or modified oligonucleotide sequence containing from about 9 to 200 bases;
each e and f independently, takes values from 0 to 50, provided that at least one of the values of e and f is at least 1;
each m and n independently from each other takes a value from 1 to 200;
each p independently takes a value from 2 to 4.

где R означает OH, SH, группу NR²R³, в которой R² и R³ каждый независимо означает водород или C₁-C₆ алкил, или группу NHR⁴, где R⁴ означает C₁-C₁₂ ацил;
олиго (N) означает олигонуклеотидную последовательность, содержащую от примерно 9 до 50 оснований;
e, f, m и n каждый независимо друг от друга принимает значение от 1 до 50.In the case where polyethylene glycol is used as glycol, in the indicated embodiment, the compounds contain oligonucleosides of the formula

where R is OH, SH, an NR ² R ³ group in which R ² and R ³ each independently is hydrogen or C ₁ -C ₆ alkyl, or an NHR ⁴ group, where R ⁴ is C ₁ -C ₁₂ acyl;
oligo (N) means an oligonucleotide sequence containing from about 9 to 50 bases;
e, f, m and n each independently from each other takes a value from 1 to 50.

 In a preferred embodiment, said oligonucleotide comprises, for example, in a homopolymer or heteropolymer form, any of the combinations dA, dC, dG, T.

 In other preferred embodiments of the present invention, tetraethylene glycol (TEG) is used as the polyethylene glycol, provided that both m and n are 4, or hexaethylene glycol if both m and n are 6.

 The compounds of the invention are suitable for use as antisense agents. Antisense agents hybridize to a complementary nucleoside sequence in the target nucleic acid, and thereby inhibit the translational and transcriptional function of the specified target nucleic acid. As the target nucleic acid, both PHK and DNA can be used.

 The antisense compounds of the invention comprise oligonucleoside sequences from about 6 to 200 bases having homopolymer or heteropolymer sequences that include bases selected from the group: adenine (A), cytosine (C), guanine (G), uracil (U), thymine ( T) and their derivatives. Specific sequences are selected based on their given targets. The selected sequence is hybridized to the indicated target nucleic acid. As targets, the MYC oncogene, RAC oncogen, and viral nucleic acids can be mentioned.

Compounds of the invention can be obtained by the following methods:
A. Compounds with an internucleoside bridge of three carbon atoms.

 Oligonucleosides connected by a three-carbon internucleoside bridge are synthesized by the interaction of nucleosides with functional aldehyde and ylide groups at positions 3 'and 6', respectively, according to the Wittig method.

 The preparation of nucleoside aldehyde and phosphonium iodide nucleoside from commercially available reagents is illustrated in FIG. 1a and 1b, respectively. The specified aldehyde (Compound 1, obtained according to the scheme of Fig. 1a) is synthesized from the known 3'-allyl-3'-deoxy-5'-0-tert-butyldimethylsilyl-3'-thymidine (Compound A, Fig. 1). The resulting allyl derivative is subjected to oxidation by a regioselective method using a catalytically effective amount of osmium tetroxide and N-methylmorpholine oxide as a co-oxidant. The resulting diol (Compound B, Fig. 1a) is hydrolyzed with sodium periodate to yield an aldehyde in almost quantitative yield.

 The synthesis of phosphonium iodide nucleoside (Compound II, fig. 1b) is carried out from a commercially available 5'-triphenylmethyl nucleoside (Compound C, fig. 1b). A silyl group at the 3'-end is introduced into this tritylated nucleoside using tert-butyldimethylsilyl chloride, followed by removal of the trityl group under acidic conditions. The resulting primary hydroxyl (Compound E, FIG. 1b) is oxidized under Swern conditions to yield an aldehyde (Compound F, FIG. 1b). The crude aldehyde is immediately reacted with an ylide derived from methyltriphenylphosphonium bromide to give 4'-vinyl deoxy-3'-tert-butyldimethylsilyl nucleoside in high yield. The resulting vinyl compound is subjected to regioselective interaction with hydroborate to give the primary alcohol (Compound G, FIG. 1b). The primary alcohol, in turn, is converted to the corresponding iodide (Compound H, Fig. 1b) using triphenylphosphine iodide in the presence of imidazole with an excellent iodide yield. Finally, the obtained iodide is converted to the target nucleoside phosphonium iodide using triphenylphosphine in acetonitrile.

 Ylide is obtained from nucleoside phosphonium iodide using tert-butylate as the base, which is immediately reacted with the aldehyde in high yield of the Wittig compound. The resulting Wittig compound is hydrogenated with hydrogen by a regioselective method on 10% palladium black (10% Pd-C) at atmospheric pressure with a quantitative yield to saturate the double bond of this bridge. From the resulting saturated compound (Compound 2, FIG. 2), the silyl group is removed with tetrabutylammonium fluoride, whereby a diol is obtained (Compound 3, FIG. 2). The 5'-terminal primary hydroxyl diol is then protected by the regio-selective method using dimethyloxytrityl chloride, and the obtained 3'-hydroxyl (Compound 4, Fig. 2) is converted to phosphoramidite (Compound 5, Fig. 2) by reacting it with 2-cyanoethyl-N, N-diisopropylchlorophosphoramidite.

 Nucleoside dimers or higher oligomers with trialkylsilyloxy protecting groups are conjugated to form oligonucleotides of any given chain length. Upon completion of the chain growth reaction, the oligomers obtained are deprotected by conventional methods. For further chain extension in a solid-state synthesizer, where oligomers are bound by phosphate bridges, the terminal hydroxyl groups of these oligomers at the 5'- and 3'-end are properly functionalized using tritylating reagents, for example, dimethoxytrityl chloride and phosphoramidite, respectively.

 B. Compounds with an internucleoside bridge with 2 carbon atoms and 1 oxygen.

 Oligonucleoside sequences with an internucleoside bridge containing 2 carbon atoms and 1 nitrogen atom are synthesized by reacting a 3'-silylated, 5'-toluenesulfonyl nucleoside with a 5'-protected nucleoside.

 3'-Acetyl-5'-aldehyde nucleoside is prepared from a commercially available 3'-acetyl nucleoside according to a conventional method well known to those skilled in the art. The resulting 3'-acetyl-5'-aldehyde nucleoside is then converted to 3'-acetyl-5'-carboxymethoxymethylene nucleoside using the modified Wittig reaction.

 The 5'-methylene process is reduced with sodium borohydride in alcohol, preferably isopropanol, followed by deprotection of the 3'-acetyl group with sodium methylate in alcohol, preferably methanol. After that, the 3'-hydroxy group is protected by a silyl group. In a preferred embodiment, tert-butyldimethylsilyl is used as the silyl group.

 Then 3'-Acetyl-5'-carboxymethoxymethylene nucleoside is additionally reduced with diisobutylaluminium hydride (DJBAL) in tetrahydrofuran to 3'-0-silyl-5'-deoxy-3 '- (2' 'ethanol) of the nucleoside derivative. The 5'-ethanol group is converted to a para-toluenesulfonyl group with the introduction of para-toluenesulfonyl chloride in pyridine. The exocyclic amino group of the main residue of 5'-p-toluenesulfonyl nucleoside can be protected if desired by conventional methods. A benzoyl residue is used as the protective group of the exocyclic amino group of adenine and cytosine. A preferred protecting group of the exocyclic amino group of guanine is the isobutyl residue. Guanine can also be protected at position 06.

 The 3'-O-silyl-5'-para-toluenesulfonyl nucleoside is then reacted with a 5'-protected nucleoside to form a 3'-O-silyl-5'-protected nucleoside dimer with a bicarbon-single oxygen bridge between nucleosides. As the 5'-O-protecting group, trityl 6 and more preferably dimethoxytrityl are preferably used. If desired, the 3'-O-silyl-5'-O-protective nucleoside may have a protective group in the exocyclic amino groups of the main nucleoside residue.

 After that, the protections of these nucleoside dimers are removed and modified at the position of the carbon atom at the 3'-end with a cyanophosphine reagent, preferably 2-cyanoethoxy diisopropylaminophosphone for subsequent use in the solid-phase synthesis by the phosphoramidite chain-building mechanism. (see above Gait).

 Nucleoside dimers or higher oligomers with trialkylsilyloxy protecting groups are conjugated to form oligonucleosides of any given chain length. Upon completion of the chain growth reaction, the oligomers obtained are deprotected by conventional methods. For further chain extension in a solid-state synthesizer, where oligomers are bound by phosphate bridges, the terminal hydroxyl groups of these oligomers at the 5'- and 3'-end are properly functionalized using tritylating reagents, for example, dimethoxytrityl chloride and phosphoramidite, respectively.

 C. Compounds with an internucleoside bridge of 2 carbon atoms and 1 nitrogen.

 Oligonucleoside sequences connected by an internucleoside bridge containing 2 carbon atoms and 1 nitrogen, represented as C-C-N, are obtained by the interaction of nucleosides containing aldehydes with nucleosides that include amino groups under reduction conditions, as shown in Fig. 4.

 Both aldehyde derivatives and amino derivatives are prepared from 3'-allyl-3'-deoxy-5'-O-tert-butyldimethylsilyl-3'-thymidine. The obtained allyl derivative is subjected to oxidation by the regioselective method using a catalytically effective amount of osmium tetroxide and N-methylmorpholine, N-oxide as a co-oxidant with a diol yield. The resulting diol is hydrolyzed with sodium periodate to yield an aldehyde in almost quantitative yield.

 Amino derivatives are obtained from commercially available nucleosides. According to standard methods, the primary hydroxyl group of a nucleoside is converted by regio-selective methods into the tosylate group of para-toluenesulfonyl chloride, followed by conversion of the intermediate into iodide. 3'-Hydroxylated intermediate iodide is protected with tert-butyldimethylsilyl chloride and an azide group introduced by reaction with sodium azide. The functional azide group can be effectively converted to the target amine by reduction using 10% palladium black in a hydrogen atmosphere or by reduction with a Raney catalyst.

 The resulting amine and aldehyde are bonded to each other (reductive amination reaction) in the presence of sodium cyanoborohydride in a buffered medium. The resulting oligonucleoside dimer with a C-C-N bridge between nucleosides is obtained in high yield. The resulting oligonucleoside is reacted with a mixture of trifluoroacetic anhydride and triethylamine to protect secondary aliphatic nitrogen. The silyl group with tertrabutylammonium fluoride is removed from the protected oligonucleoside, and the primary hydroxyl group of the obtained diol is selectively protected with dimethoxytrityl chloride. The remaining secondary hydroxyl group is converted to the desired phosphoramidite by reacting it with 2-cyanoethyl-N, N-diisopropylchlorophosphoramidite.

 Oligonucleosides connected by an internucleoside bridge containing 2 carbon atoms and 1 nitrogen, represented as NCC, are obtained by the interaction of nucleosides containing functional aldehyde and amino groups at the 3'- and 5'-ends, respectively, under reduction conditions, as shown in FIG. . 5.

 Amine and aldehyde moieties are synthesized from commercially available compounds. The amine is obtained from 3-azido-3-deoxythymidine (AZT). The primary hydroxyl group of AZT is protected with dimethoxytrityl chloride, and the resulting azide is converted to the desired amine by the regio-selective method using 10% black in an atmosphere of hydrogen or a Raney catalyst.

 Aldehyde is synthesized from commercially available 5'-dimethoxytritylthymidine. A silyl group is introduced into the tritylated thymidine using tert-butyldimethylsilyl chloride, after which the trityl group is removed in an acidic medium. The resulting primary hydroxyl group is oxidized in Swern medium with the release of the aldehyde. The obtained aldehyde is not isolated, but is used immediately for its reaction with (carbethoxymethylene) triphenylphosphorane, which gives an unsaturated ester. Said unsaturated ester is subjected to regio-selective hydrogenation with 10% palladium black to form a saturated ester in quantitative yield. The resulting saturated ester, in turn, is converted to the desired aldehyde using diisobutylaluminium hydride (DIBAL-H) by a highly selective method.

 Amine and aldehyde are bonded to each other in the presence of cyanoborohydride under reductive amination in a buffered environment. An N-C-C formed bridge between nucleosides is obtained in good yield. The secondary aliphatic nitrogen of the oligonucleoside is protected with a mixture of trifluoroacetic acid and triethylamine. A silyl group is removed from the protected dimer or higher oligonucleoside derivative and the resulting hydroxyl is converted to phosphoramidite using 2-cyanoethyl-N, N-diisopropylchlorophosphoramidite.

 Nucleoside dimers or higher oligomers with trialkylsilyloxy protecting groups are conjugated to form oligonucleosides of any given chain length. Upon completion of the chain growth reaction, the oligomers obtained are deprotected by conventional methods. For further chain extension in a solid-state synthesizer, where the oligomers are bound by phosphate bridges, the terminal hydroxyl groups of these oligomers of the -5'- and 3'-ends are properly functionalized using tritylating reagents, for example, dimethoxytrityl chloride and phosphoramidite, respectively.

D. Compounds containing a diol at any and each of their ends
If necessary, diols are attached to any or each of the ends of the nucleic acids by modifying the solid-phase synthesis method according to the phosphoramidite mechanism (Oligonucleotide Synthesis: A Practical Approach, publ. MJ Gait, pp. 35-81, IRL Press, Washington DC, 1984).

 In accordance with the proposed modification of the solid-phase synthesis method, any diol is attached to one or each of the ends of the oligonucleotide according to the method where the diol interacts with an alkoxytrityl reagent to form a tritylated diol. Preferably, glycol is used as the diol, more preferably polyalkylene glycol, preferably monomethoxytrityl chloride or dimethoxytrityl chloride, and most preferably dimethoxytrityl chloride. The resulting tritylated diols are then reacted with a cyanophosphine reagent to form trityldiol cyanophosphine, which is used as the phosphoramidite reagent (hereinafter referred to as the diol phosphoramidite reagent) in the solid phase synthesis of the compounds of the present invention.

 At the initial stage of solid-phase synthesis, the nucleoside is attached to a solid-phase carrier, preferably to a glass-based carrier with controlled porosity (CGP). Said nucleoside is preferably attached to such a glass carrier via a succinate bridge at the 3'-hydroxyl group. Known other means of immobilizing nucleosides on solid phase carriers and any specialist in the field of oligonucleoside synthesis can easily implement them. Alternatively, for the introduction of a diol at the 3'-end, the diol-phosphoramidite substrate can be immobilized on a solid phase carrier prior to the introduction of the first nucleoside. Any specialist will easily understand the means of modifying such synthesis methods using diol-phosphoramidite reagents. Any number of diols can be attached to the solid phase carrier before administration of the first nucleoside. In a preferred embodiment, 1 to about 50 diols are used. If diols are attached only to the 5'-end, then no diol can be immobilized on a solid phase carrier.

 After the attachment of the first nucleoside or diol (s) to the solid phase carrier, the growth of the oligonucleoside chain occurs in successive stages, namely that the protective group of the 5'-hydroxyl (functional trityl group) is removed, then the 5'-hydroxyl is activated in the presence of a phosphoramidite reagent, then there is 5'-trityl nucleoside, 3'-phosphoramidite, they carry out the capping of unreacted nucleosides and oxidize the phosphorus linking group.

 The protective group at 5'-hydroxyl is removed with an acid, preferably trichloroacetic acid.

 Specialists are well aware of activating agents that can be used in accordance with the proposed method. As preferred activators, tetrazole and activating gold (Beckman Instr. Inc., Palo Alto, Ca) are used.

 Activation occurs in the presence of an introduced nucleoside phosphorimidite or diol phosphoramidite reagent, the latter being replaced by a nucleoside phosphoramidite substrate used in traditional methods of solid phase synthesis if the diol is introduced at the end or both ends of the polynucleotide chain. Unreacted polynucleotide chains are cleaved or capped using capping agents, for example, acetic anhydride and N-methylimidazole.

 An unstable trivalent phosphorus bond is oxidized, for example, in a preferred embodiment with iodine to form a stable pentavalent phosphodiester bond of the oligonucleotide.

 After the assembly of the desired oligonucleotide chain is completed, the phosphate protective groups are removed, the obtained oligonucleotide units are removed from the solid phase carrier and the main protective groups are removed by conventional methods (see Gaits, pp. 67–70 above).

 It will be apparent to those skilled in the art that other methods for synthesizing oligonucleotides can be modified in a similar manner to obtain antisense oligonucleotides with terminal diol groups.

 The present invention is suitable for use in the treatment of mammals suffering from hereditary diseases, or diseases caused by the violation of the mechanisms of expression of genetic information. Currently, studies are underway to create therapeutic methods using antisense compounds in the treatment of viral infections caused, for example, by HIV, cytomegaly virus, herpes simplex virus, hepatitis B, papilloma, picornavirus; cancer of the lungs, intestines, cervix, breast and ovary; allergic diseases of the immune system, such as acquired immunodeficiency syndrome (AIDS), hematologic neoplasia and hyperproliferative disorders (see Armstrong et al., p. 89: Klausher et al., pp. 303 - 304, see above).

Compositions of the invention suitable for inhibiting gene expression include physiologically acceptable carriers and
1) compounds containing oligonucleotide sequences consisting of about 9 to 200 bases, with an internucleoside bond of the formula -DDD; as specified herein, possibly containing a diol at either or both of their ends, or
2) compounds comprising oligonucleotide sequences consisting of about 9 to 200 bases with a diol at either or both ends.

 The proposed compositions suitable for inhibiting gene expression include one or more of the proposed compounds together with one or more non-toxic physiologically acceptable carriers, adjuvants or carriers, generally referred to in this description as carriers for parenteral administration, oral administration in solid or liquid form, rectal or local administration and the like.

 These compositions can be administered to humans and animals as orally, rectally, parenterally (intravenously, intramuscularly or subcutaneously), intracisternally, intravaginally, intraperitoneally, topically (powders, emulsions or drops) or buccally or nasally in the form of an aerosol.

 Compositions of the invention suitable for parenteral administration may include physiologically acceptable sterile aqueous or non-aqueous solutions, emulsions, suspensions or dispersions, and sterile powders for the preparation of injectable solutions or dispersions before use. As examples of suitable aqueous or non-aqueous vehicles, diluents, solvents or carriers, water, ethanol, polyols (propylene glycol, polyethylene glycol, glycerin and the like), their respective mixtures, vegetable oils (for example, like olive oil) and injectable complex can be mentioned esters, for example, as ethyl oleate. The necessary flowability can be achieved by using a coating membrane, for example, lecithin, providing the desired particle size distribution in the case of dispersions and using a surfactant.

 These compositions may also contain adjuvants, for example, as preservatives, wetting, emulsifying and releasing agents. Protection against the action of microorganisms can be provided with various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. In addition, if desired, they can include isotonic agents, for example sugars, sodium chloride and the like. Prolonged absorption of the injectable form can be achieved using agents that delay absorption, for example, aluminum monostearate and gel.

 If desired, and for more efficient distribution, the proposed compounds can be incorporated into substrates with prolonged release or targeted delivery to the target organ, for example, polymer matrices, liposomes and microspheres. The proposed compositions can be sterilized, for example, by filtration through a bacteria-retaining filter or by the administration of aseptic agents in the form of sterile solid formulations that can be dissolved both in sterile water and some other sterile injectable solutions immediately before use.

 As solid dosage forms for oral administration, capsules, tablets, pills, powders and granules can be used. In such solid dosage forms, the active compound is mixed together with at least one inert traditional excipient (or carrier), for example, as sodium citrate or dicalcium phosphate, or with (a) excipients, and dry diluents, for example, starches, lactose, sucrose, glucose, mannitol and silicic acid, (b) binders, for example, carboxymethyl cellulose, gelatin, polyvinylpyrrodlidone, sucrose and acacia, (c) humectants, for example glycerin, (d) disintegrants, such as agar-agar, calcium carbonate potato essential or cassava starch, alginic acid, certain combined silicates and sodium carbonate, (e) absorption retarding agents, such as paraffin, (f) absorption accelerators, for example, quaternary ammonium salts, (g) wetting agents, for example, cetyl alcohol and glycerol monostearate, (h) adsorbents, for example, kaolin and bentonite, and (i) lubricants, for example, talc, calcium stearate, magnesium, solid polyethylene glycols, sodium lauryl sulfate, or mixtures thereof. When used in the form of capsules, tablets and pills, such dosage forms may also contain buffering agents.

 Solid dosage forms of a similar type can also be used as fillers in hard and soft gelatin capsules using, for example, lactose or milk sugar, as well as high molecular weight porethylene glycols and the like.

 Solid dosage forms, for example tablets, dragees, capsules, pills and granules, can be prepared with a coating or a coating, for example, as enteric coatings and with other coatings generally known in the art. These forms may include encapsulating substances, and be of such a composition that provides a sustained release of the active ingredient or ingredients in a specific part of the gastrointestinal tract in a prolonged manner. Examples of such encapsulated compositions suitable for use include polymeric substances and waxes.

 Liquid dosage forms for oral administration may be in the form of physiologically acceptable emulsions, solutions, suspensions, syrups and elixirs. In addition to the active compounds, liquid dosage forms may contain inert diluents traditionally used in this field, for example, as water, or other solvents, solubilizing agents and emulsifiers, for example, ethyl, isopropyl alcohols, ethyl acetate and ethyl carbonate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, vegetable oils, in particular cottonseed, peanut, corn, olive and sesame oils, glycerin, tetrahydrofurfuryl alcohol, and polyethylene glycols are complex e sorbitol fatty acid esters or mixtures of these substances, and the like.

 In addition to inert diluents, the compositions of the invention may also include adjuvants, for example, wetting agents, emulsifiers and suspending agents, sweetening agents, perfumes and aromatic substances.

 Suspensions, in addition to the active compounds, may contain suspending agents, for example, ethoxylated isostearyl alcohols, esterified polyoxyethylene sorbitol and sorbitan, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, or mixtures of these substances and the like.

 Compositions for rectal administration are preferably used in the form of suppositories obtained by mixing the compounds of the invention with appropriate inert excipients or carriers, for example, chocolate butter, polyethylene glycol or suppository wax, which at normal temperatures is in the solid state but becomes liquid at body temperature, and thus melting in the rectal or vaginal opening releases the active ingredient.

 Dosage forms for topical administration of a compound of the invention include ointments, powders, aerosols and inhalers. The active ingredient is mixed under sterile conditions with a physiologically acceptable carrier and any necessary preservatives, buffers or propellants, if necessary. Ophthalmic dosage forms, in particular ophthalmic ointments, powders and solutions are also included in the scope of the present invention.

 The compounds of the invention may also be administered in liposome form. It is known that liposomes are usually derived from phospholipids or other lipid substances. Liposomes are formed by mono- or multilayer hydrated liquid crystals that are dispersed in an aqueous medium. Any non-toxic, physiologically acceptable and metabolizable lipid having the ability to form liposomes can be used. The proposed compositions in liposomal form and in addition to the lipoxygenase-inhibiting compounds of the present invention may contain stabilizers, preservatives, excipients and the like. As preferred lipids, phospholipids and phosphatidylcholines (lecithins), both natural and synthetic, are used.

 Known methods for producing liposomes, see, for example, Methods in Cell Biology, publication Prescott, vol. XI, Academic Press, New York State, New York, p. 33 et seq., 1976.

 The actual concentration of the active ingredient in the compositions of the invention for a single administration may be varied so as to obtain an amount of the active ingredient that is effective to create the desired therapeutic effect of the particular composition and method of administration. The choice of a single dose, therefore, depends on the desired therapeutic effect, method of administration, the required course of treatment and other factors.

 The total daily dose of the compounds of the invention administered to the host organism once or fractionally can be, for example, from about 1 mmol to 5 μl per kg body weight. A single dosage form of the preparation of the present invention may contain a single or divided dose thereof, which can be used to administer a daily dosage. It should be borne in mind, however, that the selection of an individual dosage of a drug for any particular patient depends on many factors, including body weight, general health, gender, diet, time and method of administration, rate of absorption and excretion, combination with other drugs and the severity of the disease to prescribe a course of treatment.

 The following examples illustrate the best embodiment of the present invention and in no case should they be construed as limiting the scope of this description and claims.

 Example 1

 Preparation of 5'-O-dimethoxytrityl-3'-O-tert-butyl-dimethylsilylthymidine.

 Dimethoxytritylthymidine (5.0 g, 9.2 mmol) and imidazole (1.2 g, 18.4 mol) were dissolved in 15 ml of anhydrous dimethylformamide (DMF) and the resulting solution was added to tert-butyldimethylsilyl chloride (1.7 g, 11.5 mmol).

 The reaction mixture was stirred for 4 hours at room temperature, diluted with ethyl acetate, washed with water, saturated sodium chloride and dried with sodium sulfate. The title compound is obtained in quantitative yield.

 Example 2

 Preparation of 3'-O'-tert-butyldimethylsilylthymidine.

 5-O'-dimethoxytrityl-3-O'-tert-butylmethylsilylthymidine, (0.7 g, 1.1 mmol) obtained according to the procedure of Example 1, is treated for 13 hours at room temperature with 13 ml of 3% trifluoroacetic acid in a solution of methylene chloride. The resulting mixture was then neutralized with 5% (w / v) sodium bicarbonate solution. The organic layer is dried with sodium sulfate. The resulting title compound was purified by flash chromatography using a 0-30% gradient of ethyl acetate in methylene chloride. The product yield is 85%.

 Example 3

 Preparation of 3'-O'-tert-butyldimethylsilylthymidine-4'-aldehyde.

Oxalyl chloride (2.88 ml, 33.0 mmol) was added to a thoroughly mixed solution of dry methylene chloride at a temperature of 78 ^° C, followed by dropwise addition of dimethyl sulfoxide (DMSO) (3.12 ml, 4.4 mmol). After 10 minutes, add dropwise over 2 minutes. alcohol (5.6 g, 15.7 mmol) obtained according to the procedure of Example 2 in 20 ml of CH ₂ Cl ₂ and the resulting mixture was stirred for 45 minutes. Thereafter, Et ₃ N (58.1 ml, 8.1 mmol) was added to the reaction solution, and the resulting mixture was stirred for another 45 minutes. Then the reaction mixture was brought to room temperature, washed first with water (2x10 ml), then with brine (10 ml) and dried with sodium sulfate. The resulting crude aldehyde is used in the next synthesis step.

 Example 4

 Preparation of 5'-vinyl-5'-deoxy-3'-O'-tert-butyldimethylsilyldeoxythymidine.

A solution containing sodium bis-trimethylsilylamide (0.6 mmol) is added dropwise to a solution of methyltiphenylphosphonium bromide (0.7 mmol) in dry tetrahydrofuran at a temperature of 0 ^° C. After 30 minutes, a solution containing the corresponding 4'-aldehyde in THF was added dropwise to the resulting solution under a nitrogen atmosphere. The reaction mixture was stirred for 2 hours, diluted with ethyl acetate, washed with water, then brine and dried with sodium sulfate. The resulting title compound was purified by flash chromatography using 20% ethyl acetate in hexane. The yield is 55-60%.

 Example 5

 Preparation of 3'-O'-tert-butyldimethylsilyl-5'-deoxy-5'-hydroxymethylthymidine.

To a solution containing 2M 2-methyl-2-butene (1.6 eq., 1.5 ml, 3 mmol) in 3 ml of anhydrous THF at a temperature of 0 ^o C, 1.6 equiv. 1M borane-tetrahydrofuran complex (3 ml, 2 mmol) in a nitrogen atmosphere.

 the resulting reaction solution was stirred for 10 minutes, followed by the addition of vinylthymidine obtained by the method of Example 4 (0.7 g, 1.9 mmol) in 5 ml of anhydrous THF. The reaction mixture was stirred for 45 minutes and refrigerated for 4 days.

The substrate is prepared using an aqueous solution containing 3.1 eq. 2M caustic soda and 3.1 eq. 30% hydrogen peroxide (preferably adding dropwise hydrogen peroxide to an aqueous solution of sodium hydroxide at a temperature of 0 ^o C with stirring for 10 minutes). Then the resulting solution was slowly added through a dropping funnel to the reaction mixture at 0 ^° C, stirred for 1 hour, removed from the ice bath, diluted with ethyl acetate, washed with water, then with saturated sodium chloride solution and dried with sodium sulfate. The title compound was purified by flash chromatography using a 20 - 80% gradient of ethyl acetate in hexane. The product yield is 62%.

 Example 6

 Preparation of 5'-Iodomethyl-5'-deoxy-3'-O'-tert-butyldimethylsilylthymidine.

 To a solution containing 3'-O'-tert-butyldimethylsilyl-5'-deoxy-5'-hydroxymethylthymidine obtained by the procedure of Example 5 (0.3 g, 0.9 mmol) in dry acetonitrile (5 ml) and ether (3.4 ml ) add 3 equivalents of triphenylphosphine (0.7 g, 2.8 mmol), 4 equivalents of imidazole (0.3 g, 3.7 mmol) and 2.2 equivalents of iodine (0.5 g, 2.8 mmol). The resulting reaction mixture was stirred for 45 minutes and the solvent was evaporated. Ethyl acetate was added to the residue, and the precipitate formed was washed with water, a saturated solution of sodium chloride and dried with sodium sulfate. The title compound was purified by flash chromatography using a 30-50% gradient of ethyl acetate in hexane. The product yield is 90%.

 Example 7

 Preparation of 3'-O'-tert-butyldimethylsilyl-5'-deoxy-5'-thymidylmethylphosphonium iodide.

Triphenylphosphine (1.57 g, 6 mmol) was added to the mixed solution containing 5'-iodomethyl-5'-deoxy-3'-O'-tert-butyldimethylsilylthymidine (480 mg, 1 mmol) obtained by the procedure of Example 6, and the resulting the mixture was heated under reflux for 12 hours at a temperature of 90 ^° C. The reaction mixture was cooled, after which the solvent was distilled off. The title compound was purified by flash chromatography using 5% MeOH in CH ₂ Cl ₂ . The resulting product is obtained in 95 - 96% yield.

 Example 8

 Preparation of 5'-tert-butyldimethylsilyl-3'-deoxy-3 '- (1``, 2' '- dihydroxy-3' '- propyl) thymidine.

Osmium tetroxide (OSO ₄ ) (4 drops, 2.5% (w / v) in butanol is added to a mixed mixture consisting of 3'-deoxy- (2``-propenyl) -3'-deoxy-5'-O '-tert-butyldimethylsilylthymidine obtained according to the procedure described in J.Org.Chem. 1989, 54, p. 2 767 - 2 769 (CKChu et al.) (183 mg, 0.5 mmol) and 4-methylmorpholine N-oxide (53 mg, 0.45 mmol) in anhydrous tetrahydrofuran at a temperature of 0 ^o C. The resulting reaction mixture was then quenched with a 10% aqueous solution of sodium methanesulfite (2.0), stirred for 20 minutes, filtered through a layer of silicon dioxide and diluted with ethyl acetate (25.0 ml). rganicheskuyu phase was washed with water (5.0) and brine, then dried over sodium sulfate. The solvent was evaporated, and the above compound obtained was purified by flash chromatography.

 Example 9

 Preparation of 5'-tert-butyldimethylsilyl-3'-deoxythymide-3'-ml-acetaldehyde.

Sodium periodate (214 mg, 1 mmol) is added to a mixed solution of thymidine diol obtained according to the procedure of Example 2 (200 mg, 0.5 mmol) in THF-H ₂ O (4: 1; 5.0 ml). After 1 hour, the reaction mixture was diluted with ethyl acetate (25 ml), washed with water (2x5 ml), and then with brine and dried. The title compound was purified by flash chromatography using 70% ethyl acetate in hexane.

Osmium tetroxide (OSO ₄ ) (4 drops, 2.5% (w / v) in butanol is added to a stirred mixture consisting of 3'-deoxy- (2``-propenyl) -3'-deoxy-5'-O '-t-butyldimethylsilyl-thymidine obtained according to the procedure described in J.Org.Chem., 1989, 54, pp. 2767-2769 (CKChu et al.) (183 mg, 0.5 mmol) and 4-methylmorpholine-N -oxide (53 mg, 0.45 mmol) in anhydrous tetrahydrofuran at a temperature of 0 ^o C.

 The resulting reaction mixture was then quenched with a 10% aqueous solution of sodium metasulfite (2.0), stirred for 20 minutes, filtered through a pad of silica and diluted with ethyl acetate (25.0 ml). The organic phase is washed with water (5.0) and brine, and then dried with sodium sulfate. The solvent was evaporated, and the above compound was purified by flash chromatography.

 Example 10

 Obtaining thymidine dimers with a three-carbon internucleoside bond.

 The synthesis steps of Example 10a-10e are illustrated in FIG. 3.

10a. Sodium tert-butylate (0.62 ml, 1M solution in THF, 0.62 mmol) was added to a mixed suspension of a phosphonium iodide-based compound obtained by the procedure of Example 7 (241 mg, 0.326 mmol) in dry THF (2.0 ml) at a temperature of -78 ^o C in an atmosphere of nitrogen. After 20 minutes, a 3'-acetaldehyde derivative was added to the resulting reaction mixture, prepared as described in Example 9, (80 mg, 0.22 mmol). After 60 minutes, the reaction mixture was diluted with ethyl acetate (30 ml), washed with water (2 x 5 ml) and dried with sodium sulfate. The solvent was evaporated and the olefin product was purified by flash chromatography using 70% ethyl acetate in hexane. The product yield ranges from 55 - 60%.

10b. 10% palladium black (20 mg) is added to a mixed solution of compound 1 (109 mg) in methanol at a temperature of 25 ^° C. under a hydrogen pressure of 1 atm. After 4 hours, the catalyst was filtered through a pad of celite, and the solvent was distilled off. Thereafter, the obtained compound 2 was separated and purified by flash chromatography in an 80% ethyl acetate solution dissolved in hexane.

10c. Example 2.8 equivalents of tetrabutylammonium fluoride at 0 ^° C. are added to a mixed solution containing Compound 2 (350 mg) in 5.0 ml THF. After 3 hours, the solvent was distilled off, and the resulting Compound 3 was purified by flash chromatography using 10% methanol in CH ₂ Cl ₂ .

 10d. About 0.05 equivalents of 4-dimethylaminopyridine, 1.4 equivalents of triethylamine and 1.2 equivalents of 4,4-dimethoxytrityl chloride are added to a mixed solution of Compound 3 (0.6 mmol) in dry pyridine (4.0 ml). After 2 hours, the reaction mixture was quenched with 2 ml of water, and then diluted with 2.0 ml of ethyl acetate. The organic layer was separated, washed with brine and dried. The resulting Compound 4 was purified by flash chromatography using 5% methanol in methylene chloride.

10e. About 2.0 equivalents of diisopropylethylamine and 1.0 ml of dry dichloromethane (CH ₂ Cl) are added to a stirred solution of Compound 4 (0.5 mmol). After 30 minutes, 0.75 equivalent of 2-cyanoethyl-N, N-diisopropylchlorophosphoramidite was added dropwise to the reaction mixture over 20 minutes, and stirring was continued for another 1 hour. The solvent was evaporated and the resulting Compound 5 was purified by flash chromatography using ethyl acetate (containing 1% triethylamine) in a nitrogen atmosphere.

The above steps of ad synthesis are used to produce dimers containing three-carbon internucleoside bonds, where each of the three carbon atoms is represented by the formula —CH ₂ -.

Any or each of the carbon atoms can be hydroxylated by modifying the method shown in FIG. 3, based on the following. A drop of a 2.5% (w / v) solution of osmium tetroxide in tert-butanol at a temperature of 0 ^° C is added to a mixed solution containing Compound 1 and 4-morpholine-N-oxide (9.1 mg) in 0.8 ml of THF. The resulting reaction mixture was kept at 0 ^° C for 24 hours, then quenched with an aqueous solution of sodium metabisulfite, diluted with ethyl acetate and washed first with water and then with brine. The solvent was evaporated and the resulting hydroxylated dimer was purified by thin layer chromatography using ethyl acetate as an eluent. The specified hydroxylated dimer is then protected, and the 5'- and 3'-ends are modified according to the above steps bd.

 Example 11

 Obtaining hydroxylated three-carbon internucleoside bonds.

 The thymine dimer phosphoramidites obtained in steps a-d are used in the solid-phase synthesis method with phosphoramidite modification to form the oligonucleoside sequences shown in Table 1.

 Oligodeoxynucleotides are synthesized from 3 '- in the direction of the 5'-end.

C = -CH₂-CH₂-CH₂-
Y = тетраэтиленгликоль
Синтез затем протекает в соответствии с модифицированным фосфорамидитным методом. 5' - Концевую гидроксильную группу прикрепленного тимидина подвергают взаимодействию с трихлоруксусной кислотой со снятием защиты 5'-гидроксила. После снятия защиты, прикрепленный тимидин подвергают взаимодействию с активатором, в частности, с тетразолом и фосфорамидитным реагентом, содержащим диметокситритилтетраэтиленгликольцианофосфин. После активации осуществляют кэппинг непрореагировавших 5' гидроксилов при использовании уксусного ангидрида и N-метилмидазола. Полученную фосфорную связь затем окисляют йодом в соответствии со стандартными методиками. В последовательностях 1 и 2, содержащих два тетраэтиленгликолевых остатка (ТЭГ) повторяют вышеописанные стадии снятия защиты, активирования, кэппинга и окисления.Table 1
Sequence - Ref. code
5 'Tp Tp Tp Tp Tp [TcT] p Tp Tp Tp Tp yp yp T3' - 1
5 'Tp Tp Tp Tp Tp Tp Tp [TcT] p Tp Tp pyp уT3' - 2
5 'Tp Tp Tp Tp Tp Tp Tp Tp [TcT] p yp T3' - 3
T = thymidine

C = -CH ₂ -CH ₂ -CH ₂ -
Y = tetraethylene glycol
The synthesis then proceeds in accordance with the modified phosphoramidite method. The 5 ′ terminal hydroxyl group of the attached thymidine is reacted with trichloroacetic acid to deprotect the 5′-hydroxyl. After deprotection, the attached thymidine is reacted with an activator, in particular, tetrazole and a phosphoramidite reagent containing dimethoxytrityl tetraethylene glycol cyanophosphine. After activation, unreacted 5 ′ hydroxyls are capped using acetic anhydride and N-methylmidazole. The resulting phosphorus bond is then oxidized with iodine in accordance with standard techniques. In sequences 1 and 2 containing two tetraethylene glycol residues (TEG), the above steps of deprotection, activation, capping and oxidation are repeated.

 The chain growth then proceeds by carrying out the usual sequential deprotection, activation, capping and oxidation operations using the modification consisting in the fact that the thymidine dimer bound by the three-carbon bridge obtained according to the methods of Examples 1 to 9 is introduced into the chain, if necessary, during the activation stage .

At the end of chain assembly, thymidine oligomers are removed from the microporous glass support using a concentrated solution of ammonium hydroxide. The resulting solution is then treated at a temperature of 55 ^o C for about 8 to 15 hours to remove all the protective groups of Y exocyclic amino groups of the bases.

 Example 12

 Preparation of 3'-O-acetyl-5'-carbomethoxymethyl-5'-deoxythymidine.

About 0.39 g of sodium borohydride is added to a chilled (in an ice bath) mixed mixture of 3.17 g of 3'-O-acetyl-5'-carbomethoxymethylene-5'-deoxythymidine in 95 ml of isopropanol. The resulting mixture was stirred at a temperature of 0 ^o C in nitrogen atmosphere for 30 minutes, then at room temperature for another 4.5 hours.

 The quenched mixture is quenched with 20 ml of methanol, then 200 ml of distilled water for 30 minutes, after which it is extracted with several portions of ethyl acetate. The combined organic extracts are treated with brine and dried over anhydrous magnesium sulfate. The specified desiccant was filtered off and the solvent was evaporated. The title compound is obtained as a residue on a glass carrier in an amount of 2.7 g.

 Example 13

Preparation of 5'-carboxymethoxymethyl-5'-deoxythymidine
About 10 drops (25% (w / v) of methanol solution of sodium methylate are added to a cooled mixed solution containing 2.22 g of 3'-O-acetyl-6'-carboxymethyl-5'-deoxythymidine obtained according to the procedure of Example 12, in about 300 mg dry (passed through a layer of neutral aluminum dioxide) methanol The resulting mixture was stirred in a nitrogen atmosphere without cooling in an ice bath for 20 minutes.

 A small amount of cation exchange resin (Biorad AB 50WX8) is added to the reaction mixture, followed by stirring for 30 minutes. The solvent was evaporated under reduced pressure to leave a residue on the glass in an amount of 2.1 g, which was then treated with warm toluene and, after cooling, filtering and washing with cyclohexane, the crude product was obtained as a white solid, 1.64 g.

 The title compound is further purified from trace amounts of starting material by silica gel column chromatography, eluting with ethyl acetate. Recrystallization from ethyl acetate / hexane gave white crystals.

 Example 14

 Preparation of 3'-O-tert-butyldimethylsilyl-5 '- (2' '- hydroxyethyl) thymidine.

About 19 ml of a 1M solution containing diisobutylaluminium hydride in THF is added to a cooled (-40 ^o - 30 ^o C) mixed solution containing 1.88 g of 3'-O-tert-butyldimethylsilyl-5'-carboethoxymethyl-5'-deoxythymidine in 40 ml of anhydrous THF in an atmosphere of nitrogen. The reaction temperature is then gradually increased to -20 ^o C.

The cooled mixture was then quenched by adding about 3.5 ml of methanol, and the reaction temperature was then raised again to -10 ^° C. About 18 ml of water in 36 ml of THF was added to the heated reaction mixture, and the temperature was again raised to 10 ^° C. Most of the THF was evaporated. under reduced pressure, and the residue was diluted with approximately two volumes of water. The aqueous phase is extracted several times with a mixture of ethyl acetate and chloroform. The combined extracts were washed with chilled 2N hydrochloric acid and then with brine, dried with anhydrous sodium sulfate and filtered. The solvent was removed from the filter under reduced pressure to yield the title compound (about 1.6 g).

 Example 15

 Preparation of 3'-O-tert-butyldimethylsilyl-5 '- (2``-iodoethyl) -5'-deoxythymidine.

About 1 g of p-toluenesulfonyl chloride is added to a solution containing 3'-O-tert-butyldimethylsilyl-5 '- (2''- hydroxyethyl) thymidine obtained according to the procedure of Example 14 in 25-30 ml of anhydrous pyridine, and the resulting mixture is kept at a temperature of about 5 ^° C. for about 19 hours.

 Then the reaction mixture is added to about 200 ml of ice water and extracted several times with diethyl ether. The combined organic extracts were washed with chilled 2N hydrochloric acid, then with water and brine. The washed extracts are dried with anhydrous sodium sulfate and filtered. The solvent was removed from the filtrate under reduced pressure to give a residue on a glass support in an amount of 1.24 g as a para-toluenesulfonyl derivative (derivative).

 About 0.54 g of para-toluenesulfonyl derivative and 0.38 g of sodium iodide are dissolved in 55 ml of dry (molecular sieves 4A) acetone for three days, followed by the introduction of another 0.19 g of sodium iodide with stirring on the last day.

 The reaction mixture was purified by silica gel chromatography, 85 g, eluting with a 25% solution of ethyl acetate in hexane. The solvent was evaporated. 0.4 g of the title 3'-O-tert-butyldimethylsilyl-5 '- (2' '- iodoethyl) -5'-deoxythymidine are obtained. The solvent was evaporated. 0.4 g of the title 3'-O-tert-butyldimethylsilyl-5 '- (2' '- iodoethyl) -5'-deoxythymidine are obtained.

 Example 16

 Preparation of 5'-carbomethoxymethylene-5'-deoxythymidine.

 About 10 drops of a 25% (w / v) methanol solution of sodium methylate are added to a mixed solution containing 1.55 g of 3'-O-acetyl-5'-carbomethoxymethylene-5'-deoxythymidine in 150 ml of dry methanol (passed through a neutral layer aluminum dioxide). The resulting mixture was stirred at room temperature under nitrogen for 6 hours.

 A small amount of cation exchange resin (Biorad AB 50WX8) is added to the reaction mixture, followed by stirring for 10 minutes. The solvent was evaporated under reduced pressure to give a white solid in the amount of 1.3 g, which was then powdered twice with warm toluene, treated with hot ethanol, filtered, cooled, and after drying the title compound was obtained as a white crystalline solid, 0.85 g.

 Example 17

Preparation of 3'-O-tert-butyldimethylsilyl-5 '- (2''- hydroxyethylene) -5'-deoxythymidine
A solution containing 296 mg of 5'-carbethoxymethylene-5'-deoxythymidine was added dropwise in a nitrogen atmosphere to a chilled (ice bath) mixed solution containing 205 mg of imidazole and 227 mg of tert-butyldimethylsilichloride in 1 ml of anhydrous dimethylformamide. After completion of the introduction, the resulting mixture was removed from the ice bath, and stirred at ambient temperature for two hours, then at a temperature of 35 ^o C for another two hours, and finally at a temperature of 40 ^o C for 30 minutes.

 The cooled mixture is then quenched by adding about 2 ml of methanol, followed by adding 2 to 3 volumes of water. The aqueous phase is extracted several times with ethyl acetate. The combined extracts were washed with water, saturated sodium bicarbonate and then with brine, dried with anhydrous sodium sulfate and filtered. The solvent was removed from the filtrate under reduced pressure to yield 0.40 g of 3'-O-tert-butyldimethylsilyl-5 '- (2' '- hydroxyethylene) -5'-deoxythymidine.

About 4 ml of a 1M solution containing diisobutylaluminium hydride in tetrahydrofuran is added dropwise to a solution containing 0.37 g cooled to -30 to -35 ^° C.

3'-O-tert-butyldimethylsilyl-5'-carbomethoxymethylene-5'-deoxythymidine is dissolved in 10 ml of anhydrous tetrahydrofuran at a temperature below 30 ^o C. After the complete introduction of the reagent, the resulting reaction mixture is stirred under nitrogen for another two hours while maintaining the internal temperature in the range of from about -30 ^o to about -20 ^o C.

 Approximately 0.8 ml of methanol is added to the reaction mixture, followed by the addition of a solution containing 4 ml of water in 8 ml of tetrahydrofuran. Most of the more volatile components of THF are evaporated under reduced pressure. The aqueous residue was diluted with approximately two volumes of water and extracted several times with ethyl acetate. The combined extracts were washed with chilled 2N hydrochloric acid and then with brine, dried with anhydrous sodium sulfate and filtered. After evaporation of the filtrate, the title compound, 0.267 g, is obtained in residue. A portion of the product is purified to analytical purity by silica gel chromatography using a 50% solution of ethyl acetate in hexane as an eluent.

 Example 18

 Obtaining thymidine dimers containing an internucleoside bond with 2 carbon atoms and 1 oxygen atom (3'-O-C-C-5 ').

 18a. An equimolar amount of each base and 3'-O-tert-butyldimethylsilyl-5 '- (2``-iodoethyl) -5'-deoxythymidine are added to the mixed solution containing 5'-O-tritylthymidine. The progress of the reaction is controlled by thin layer chromatography (TLC). After completion of the reaction, the target dimer is isolated and purified by flash chromatography.

18b. To a mixed solution containing 3'-O-tert-butyldimethylsilyl-5 '- (2''- iodoethyl) -5'-deoxythymidine, maintaining the temperature at about -5 ^° C, 2 equivalents of base are added, after which the solvent is evaporated to dryness. The resulting residue was redissolved in dimethylformamide, and the equivalent of 5′-dimethoxytrityl-2 ′, 3′-cyclothymidine was added. The resulting reaction mixture was heated to a temperature of about 40 ^° C. and dimer synthesis was monitored by TLC. After completion of the synthesis, the target dimer is isolated and purified by flash chromatography.

 Example 19

 Deprotection at the 3'-end of the dimer obtained in Example 18.

The 3'-tert-butyldimethylsilyl protecting group of protected disers is removed by treating with a THF solution containing the dimer of Example 18 using 2.8 equivalents of tetrabutylammonium fluoride at a temperature of 0 ^° C. After hydrolysis is complete (approximately 3 hours), the solvent is evaporated and the target dimer is isolated and purified flash chromatography.

 Example 20

 Obtaining a functional dimeric unit for automated synthesis.

 The dimeric product obtained in Example 19 was dissolved in dichloromethane and 2 equivalents of diisopropylethylamine were added. The mixture was stirred for 30 minutes, followed by the dropwise addition of 0.75 equivalents of 2-cyanoethyl-N, N-diisopropylchlorophosphoramidite over about 20 minutes. Stirring is continued for another 1 hour, and after evaporation of the solvent, the resulting functionalized dimer is isolated and purified by flash chromatography using ethyl acetate in an inert medium.

 Example 21

 Preparation of 5'-tert-butyldimethylsilyl-3'-deoxy-3 '- (1``, 2' '- dihydroxy-3' '- propyl) thymidine.

Osmium tetroxide (OsO ₄ ) (4 drops, 2.5% (w / v) in butanol is added to a mixed mixture consisting of 3 '- (2''- propenyl) -3'-deoxy-5'-O'- tert-butyldimethylsilylthymidine (183 mg, 0.5 mmol) obtained by the method described in the literature and 4-methylmorpholine-N-oxide (53 mg, 0.45 mmol) in 5.0 ml of anhydrous tetrahydrofuran at a temperature of 0 ^o C. The resulting reaction mixture was then quenched 10% aqueous sodium metabisulfite solution (2.0), stirred for 20 minutes, filtered through a pad of silica and diluted with ethyl acetate (25.0 ml). The organic phase is washed with water (5.0) and brine, and then dried with sodium sulfate, the solvent is evaporated and the above compound is purified by flash chromatography.

 Example 22

 Preparation of 3'-deoxythymide-3'yl-acetaldehyde-5'-O-tert-butyldimethylsilylthymidine.

Sodium periodate (214 mg, 1 mmol) is added to a mixed solution of thymidine diol obtained according to the procedure for Example 21 (200 mg, 0.5 mmol) in THF-H ₂ O (4: 1, 5, 0 ml). After 1 hour, the reaction mixture was diluted with ethyl acetate (25 ml), washed with water (2x5 ml), and then with brine and dried. The title compound was purified by flash chromatography using 70% ethyl acetate in hexane.

 Example 23

 Preparation of 5'-O- (para-toluenesulfonyl) thymidine.

To a mixed solution of thymidine (20 g, 82.6 mmol) in dry pyridine (200 ml) was added para-toluenesulfonyl chloride (47.2 g, 247.6 mmol) at 0 ^° C. under nitrogen. After three hours, the reaction mixture was poured onto ice and then extracted with ethyl acetate. After evaporation of the solvent, the resulting product was recrystallized from a mixture of ethyl acetate and methanol. The named compound is obtained in the form of a white crystalline substance with a yield of 70 - 75%.

 Example 24

 Preparation of 5'-iodo-5'-deoxythymidine.

 Sodium iodide (10 g, 66.7 mmol) was added to a mixed solution of thymidine tosylate (10.65 g, 26.9 mmol) obtained in Example 23 in dry acetone (75 ml) and the resulting mixture was heated under reflux for 16 hours. The solvent was evaporated and the residue was diluted with ethyl acetate. The organic phase is washed with water (2x20 ml) and brine (10 ml), and then dried with sodium sulfate. The named compound is crystallized from methanol in the form of a white crystalline product with a yield of 90 to 95%.

 Example 25

 Preparation of 3'-O-tert-butyldimethylsilyl-5'-iodo-5'-deoxythymidine.

 Imidazole (4.2 g, 61.7 mmol) was added to a mixed solution of thymidine iodide (8.0 g, 24.7 mmol) obtained by the procedure of Example 24 in dry DMF. After five minutes, tert-butyldimethylsilyl chloride (4.47 g, 29.64 mmol) was added to the reaction mixture, followed by stirring for 4 hours. The reaction mixture was then diluted with ethyl acetate (250 ml), washed with water (2x100 ml) and brine. After drying with sodium sulfate, the title compound is purified by flash chromatography using 60% ethyl acetate in hexane in 92% yield.

 Example 26

 Preparation of 3'-O-tert-butyldimethylsilyl-5'-azido-5'-deoxythymidine.

To a mixed solution of 3'-O-tert-butyldimethylsilyl-5'-iodo-5'-deoxythymidine (10.8 g, 20 mmol) obtained by the procedure of Example 25 in dry DMF was added sodium azide (3.9 g, 60 mmol), and the resulting mixture was heated at 0 ^° C. for 12 hours. Then the reaction mixture was diluted with ethyl acetate (200), washed with water (2x50 ml) and brine (50 ml), and then dried with sodium sulfate. The title compound was purified by flash chromatography using 50% ethyl acetate in hexane. The product yield is 92%.

 Example 27

 Preparation of 3'-O-tert-butyldimethylsilyl-5'-amino-5'-deoxythymidine.

 To a mixed solution of thymidine azide (5.0 g, 13.1 mmol) obtained by the method of Example 26 in MeOH, 200 mg of palladium black in a nitrogen atmosphere was added. Nitrogen is then released and replaced with hydrogen. The operation of the release and replacement of gases is repeated twice, while stirring is continued at a hydrogen pressure of 1 atm. within 12 hours. Hydrogen is removed and the catalyst is filtered through a pad of celite. The solvent was evaporated in vacuo. After purification of the crude product by flash chromatography using a 5-10% solution of MeOH in dichloromethane, 85 to 87% of the title compound are obtained.

 Example 28

 Preparation of 3'-azido-3'-deoxy-5'-O-dimethoxytritylthymidine.

 To a mixed solution containing 3'-azido-3'-deoxythymidine (2.67 g, 10 mmol) in dry pyridine (50 ml) was added 4-dimethylaminopyridine (61 mg, 0.5 mmol), triethylamine (1.9 ml, 14 mmol) and 4 , 4'-dimethoxytrityl chloride (4.1 g, 12 mmol) in sequential order. After 3 hours, water was added and the resulting solution was extracted with ethyl acetate (250 ml). The organic phase is separated, washed with brine (50 ml) and dried with sodium sulfate. The title compound was purified by flash chromatography using 5% methanol in methylene chloride. The product yield is 80 - 85%.

 Example 29

 Preparation of 3'-amino-3'-deoxy-5'-O-dimethoxytritylthymidine.

 To a mixed solution of thymidine azide (3.99 g, 40 mmol) obtained by the method of Example 2 in MeOH, 200 mg of 10% palladium black in argon atmosphere was added. Argon gas is then released and hydrogen is introduced. This operation is repeated twice, while stirring is continued at a hydrogen pressure of 1 atm. within 12 hours. Hydrogen is then removed and the catalyst is filtered through a pad of celite. The solvent was evaporated in vacuo. After purification of the crude product by flash chromatography using a 5% solution of MeOH in methylene chloride, 90 to 93% of the title compound are obtained.

¹ H NMR (300 MHz, CDCl ₃ ) δ 7.61 (s, 1H), 7.60 - 7.21 (m, 1H), 6.83 - 6.87 (m, 3H), 6.85 (t, J = 5 Hz, 1H), 3.80 ( s, 6H), 3.81 - 3.73 (m, 2H), 3.53 - 3.49 (m, 1H), 3.38 - 3.33 (m, 1H), 2.36 - 2.33 (m, 1H), 2.25 - 2.20 (m, 1H), 1.5 (s, 3H), IR (neat) ν _max 3 020, 1 697, 1 605, 1 512, 1 246, 1 030 cm.

 Example 30

 Preparation of 5'-O-dimethoxytrityl-3'-O-tert-butyl-dimethylsilylthymidine.

 Dimethoxytritylthymidine (5.0 g, 9.2 mmol) and imidazole (1.2 g, 18.4 mol) are dissolved in 15 ml of anhydrous dimethylformamide (DMF) and the resulting solution is added to tert-butyldimethylsilyl chloride (1.7 g, 11.5 mmol).

 The reaction mixture was stirred for 4 hours at room temperature, diluted with ethyl acetate, washed with water, saturated sodium chloride and dried with sodium sulfate. The title compound is obtained in quantitative yield.

 Example 31

 Preparation of 3'-O'-tert-butyldimethylsilylthymidine.

 5-O'-dimethoxytrityl-3-O'-tert-butyldimethylsilylthymidine (0.7 g, 1.1 mmol) obtained by the procedure of Example 30 was treated for 13 hours at room temperature with 13 ml of 3% trifluoroacetic acid in a solution of methylene chloride. The resulting mixture was then neutralized with 5% (w / v) sodium bicarbonate solution. The organic layer is dried with sodium sulfate. The resulting title compound was purified by flash chromatography using a 0-30% gradient of ethyl acetate in methylene chloride. The product yield is 85%.

 Example 32

 Preparation of 3'-O'-tert-butyldimethylsilylthymidine.

Oxalyl chloride (2.88 ml, 33.0 mmol) was added to a thoroughly mixed solution of dry methylene chloride at -78 ^° C, followed by dropwise addition of dimethyl sulfoxide (DMSO) (3.12 ml, 4.4 mmol). After 10 minutes, add dropwise over 2 minutes. alcohol (5.6 g, 15.7 mmol) obtained by the method of Example 31 in 20.0 ml of CH ₂ Cl ₂ and the resulting mixture was stirred for 45 minutes. Thereafter, Et ₃ N (58.1 ml, 8.1 mmol) was added to the reaction solution, and the resulting mixture was stirred for another 30 minutes. Then the reaction mixture was brought to a temperature of -23 ^o C for 30 minutes. Thereafter, carbethoxymethylene triphenylphosphorane (10.94 g, 31.4 mmol) was added and the reaction mixture was stirred for 12 hours at room temperature. The reaction mixture was then diluted with water (2 x 125 ml) and brine (50 ml) and dried (Na ₂ SO ₄ ). The crude product is purified by flash chromatography using initially 20% ethyl acetate in hexane and then 40% ethyl acetate in hexane. As a result, both trans and cis isomers of the title compound are obtained in a ratio of 3: 1, respectively. The total yield of the product is approximately 72 - 76%.

Trans isomer analytical data
IR (neat) ν _max 3 125, 3 180, 2 982, 2 960, 1 698, 1 490, 1 274, cm ^-1 ;
¹ H NMR (300 MHz, CDCl ₃ ) δ 7.04 (s, 1H), 6.87 (dd, J = 15.6 and 5.4 Hz, 1H), 6.23 (t, J = 6.7 Hz, 1H), 6.03 (dd, J = 15.6 and 1.6 Hz, 1H), 4.33 - 4.28 (m, 1H), 4.14 (q, J = 71 Hz, 2H), 4.16 - 4.12 (m, 1H), 2.28 - 2.19 (m, 1H), 2.09 - 1.98 (m, 1H), 1.87 (s, 3H), 1.23 (t, J = 7.1 Hz, 3H), 0.81 (s, 9H), 0.01 (s, 6H); Calculated for C ₂₀ H ₃₂ O ₆ N ₂ Si;
C 56.58; H 7.60; N, 6.60; Found
C 56.36; H 7.30; N, 6.60.

 Example 33

 Preparation of 3'-O-tert-butyldimethylsilyl-5'-carbethoxymethyl-5'-deoxythymidine.

 To a mixed solution of unsaturated thymidine ester (4.24 g, 10 mmol) obtained by the procedure of Example 32 in EtOAc, 200 mg of 10% palladium black in a nitrogen atmosphere was added. Nitrogen gas was removed under vacuum and hydrogen was introduced. This operation is repeated twice, while stirring is continued at a hydrogen pressure of 1 atm. within 12 hours. Hydrogen is then removed and the catalyst is filtered through a pad of celite. The solvent was evaporated in vacuo. The resulting product is crystallized from a mixture of hexane and ethyl acetate. The title compound is obtained in 95% yield.

IR (neat) ν _max 3 180, 2 925, 2 950, 1 696, 1 486, 1 260, 1 240 cm ^-1 ;
¹ H NMR (300 MHz, CDCl ₃ ) δ 7.20 (s, 1H), 6.11 (t, J = 6.6 Hz, 1H), 4.07 (q, J = 7.1 Hz, 2H), 4.03 - 3.98 (m, 1H) , 2.51 - 2.32 (m, 2H), 24 - 2.15 (m, 1H), 1.18 (t, J = 7.1 Hz, 3H), 0.81 (s, 9H), 0.01 (s, 6H);
For C ₂₀ H ₃₄ O ₆ N ₂ Si;
Calculated C 56.31; H 8.03; N 6.57;
Found C, 55.91; H 7.74; N, 6.50.

 Example 34

 Preparation of 3'-tert-butyldimethylsilyl-5'-deoxy-thymide-5'yl-acetaldehyde.

To a stirred solution of thymidine ester (3.41 g, 8 mmol) obtained in Example 33 in 60 ml of dry CH ₂ Cl ₂ at a temperature of -78 ^° C was added dropwise Di-BAL-H (16.4 ml, 1.0 M solution in hexane, 16.4 mmol) for 3 minutes. After 20 minutes, the reaction mixture was diluted with 300 ml of EtOAc and washed twice with 50 ml of a saturated solution of sodium potassium tartrate. The organic phase is washed with brine and dried (sodium sulfate). The title compound was purified by flash chromatography of 50-70% ethyl acetate. The product yield is 85 - 87%.

 Example 35

Obtaining deoxythymidine dimer containing the internucleoside bond 3'-CCN-5 '(Fig. 3)
35a. To a mixed solution containing thymidine amine from Example 27 (1.07 g, 3 mmol) and thymidine aldehyde from Example 22 (1.38 g, 3.6 mmol) in 50 ml of ethanol and 10 ml of an aqueous buffer solution (pH 5.5. NaH ₂ PO ₄ - NaOH), a solution of NaCNBH ₃ in THF (12 ml, a 1.0M solution in THF, 12 mmol) was added dropwise at a temperature of 5 ^° C. over 1 hour. The reaction mixture was stirred for another 4 hours and then diluted using 2.50 ml of ethyl acetate. The reaction mixture was washed with water (2 x 40 ml) and brine (25 ml), and dried with sodium sulfate. Compound 1 (Fig. 6) was purified by flash chromatography, eluting first with ethyl acetate and then with 5-8% MeOH in CH ₂ Cl ₂ in 62-64% yield.

¹ H NMR (300 MHz, CDCL ₃ ) δ 7.60 (s, 1H), 7.19 (s, 1H), 6.18 (t, J = 6.6 Hz, 1H), 6.087 (t, J = 3.9 Hz, 1H), 4.29 - 4.23 (m, 1H), 4.15 - 3.98 (m, 1H), 3.91 - 1.85 (m, 1H), 3.70 - 3.78 (m, 2H), 2.95 - 2.87 (m, 1H), 2.84 - 2.66 (m, 3H), 2.35 - 2.05 (m, 5H), 1.94 (s, 3H), 1.93 (s, 3H), 1.80 - 1.63 (m, 1H), 1.55 - 1.45 (m, 1H), 0.93 (s, 9H) 0.69 (s, 9H), 0.11 (s, 6H), 0.07 (s, 6H).

35b. Compound 1 (FIG. 6) (1.66 mg, 0.23 mmol) was added to a stirred solution containing trifluoroacetic anhydride (0.32 ml, 2.3 mmol) and triethylamine (0.64 ml, 4.6 mmol) in CH ₂ Cl ₂ (5.0 ml). After 2 hours, the reaction mixture was quenched with an aqueous solution of NaHCO ₃ (5.0 ml) and diluted with EtOAc (25 ml). The organic phase is washed with water (2 x 10 ml), brine (5 ml) and dried (Na ₂ SO ₄ ). Compound 2 (FIG. 6) was purified by flash chromatography using 7% MeOH in CH ₂ Cl ₂ in 91 to 93% yield.

35c. To a stirred solution containing Compound 2 (164 mg, 0.2 mmol) in THF (4.0 ml) was added tetrabutylammonium fluoride (68 mmol) at 0 ^° C. After 2 hours, the solvent was evaporated and the resulting Compound 3 (Fig. 6) was purified by flash chromatography. when using an increasing 5 - 8% polarity of a solution of MeOH in CH ₂ Cl ₂ . The product yield is 90%.

35d. To a mixed solution containing Compound 3 (151 mg, 0.26 mmol) in dry pyridine (3.0 ml) was added 4.4-dimethylaminopyridine (1.6 mg, 0.0128 mmol) and triethylamine (0.057, 0.42 mmol). After 5 minutes, dimethoxytrityl chloride (121 mg, 0.358 mmol) was added to the reaction mixture, and stirring was continued. After two hours, the reaction mixture was diluted with ethyl acetate (25 ml) and washed with water (2 x 10 ml), brine (5 ml) and dried (Na ₂ SO ₄ ). The resulting crude product was purified by flash chromatography using 7% MeOH in CH ₂ Cl ₂ with 91 to 93% yield of Compound 4 (FIG. 3).

35th. Dry diisopropyl ethylamine (0.15 ml, 0.67 mmol) was added to Compound 4 (150 mg, 0.168 mmol), and then CH ₂ Cl ₂ (0.5 ml) was added. The flask was then shaken to dissolve the alcohol and 2-cyanoethyl-N, N-diisopropylchlorophosphoramidite (0.056 ml, 0.25 mmol) was added over 20 sec. After 45 minutes, the reaction mixture was quenched with CH ₃ OH (1.0 ml), diluted with EtOAc (50 ml) and Et ₃ N (1.0 ml), washed with 10% aqueous K ₂ CO ₃ solution (2 x 5.0 ml), and then with brine ( 5 ml) and dried (Na ₂ SO ₄ ). The resulting crude product was purified by flash chromatography using EtOAc in 91–93% yield of Compound 5 (FIG. 6).

 Example 36

 Obtaining deoxythymidine dimer containing the internucleoside bond 3'-N-C-C-5 '(Fig. 4).

36a. To a mixed solution containing the amine from Example 29 (2.72 g, 5 mmol) and the aldehyde from Example 34 (2.29 g, 6 mmol) in 50 ml of ethanol and 10 ml of an aqueous buffer solution (pH 5.5, Na ₂ H ₂ PO ₄ - NaOH ) add a solution of NaCNBH ₃ in THF (12 ml, 1.0 M solution in THF, 12 mmol) dropwise at a temperature of 5 ^o C for 1 hour. The reaction mixture was stirred for another 4 hours and then diluted using 250 ml of ethyl acetate. The reaction mixture was washed with water (2 x 60 ml) and brine, and dried with sodium sulfate. Compounds 1 (Fig. 7) were purified by flash chromatography, eluting first with ethyl acetate and then 5-8% MeOH in CH ₂ Cl ₂ . Compound 1 (Fig. 7) was obtained in 72–74% yield.

¹ H NMR (300 MHz, CDCl ₃ ) δ 7.56 (m, 1H), 7.36 - 7.34 (m, 2H), 7.29 - 7.15 (m, 8H), 7.03 (s, 1H), 6.77 (m, 3H), 6.20 (t, J = 6.0 Hz, 1H), 6.08 (t, J = 6.7 Hz, 1H), 4.01 - 3.97 (m, 2H), 3.84 - 3.72 (m, 1H), 3.72 (s, 6H), 3.71 - 3.63 (m, 1H), 3.48 - 3.22 (m, 2H), 3.30 - 3.22 (m, 1H), 3.48 - 3.32 (m, 2H), 3.30 - 3.32 (m, 1H), 7.52 - (m, 2H ), 2.27 (m, 3H), 2.08 - 1.97 (m, 1H), 1.83 (s, 1H), 1.67 - 1.48 (m, 3H), 1.43 (s, 3H), 1.22 - 1.15 (m, 1H), 0.01 (s, 6H).

36b. Compound 1 (Fig. 7) (210 mg, 0.23 mmol) was added to a mixed solution containing trifluoroacetic anhydride (0.32 ml, 2.3 mmol) and triethylamine (0.64 ml, 4.6 mmol) in CH ₂ Cl ₂ (5.0 ml). After 2 hours, the reaction mixture was quenched with an aqueous solution of NaHCO ₃ (5.0 ml) and diluted with EtOAc (25 ml). The organic phase is washed with water (2 x 10 ml), brine (5 ml) and dried (Na ₂ SO ₄ ). Compounds 2 (FIG. 7) were purified by flash chromatography using 7% MeOH in CH ₂ Cl ₂ . The yield of Compound 2 is 89 to 91%.

36c. To a stirred solution containing Compound 2 (180 mg, 0.2 mmol) in THF (4.0 ml) was added tetrabutylammonium fluoride (0.4 mmol, 1.0 M THF solution, 0.4 mmol) at 0 ^° C. After 2 hours, the solvent was evaporated and the resulting product was purified flash chromatography using increasing polarity, i.e. a MeOH solution from 5 to 8% in CH ₂ Cl ₂ . The yield of Compound 3 (FIG. 7) is 89%.

36d. Dry diisopropyl ethylamine (0.15 ml, 0.67 mmol) was added to Compound 3 (150 mg, 0.168 mmol), and then CH ₂ Cl ₂ (0.5 ml) was added. The flask was then shaken to dissolve the alcohol and 2-cyanoethyl-N, N-diisopropylchlorophosphoramidite (0.056 ml, 0.25 mmol) was added over 20 sec. After 45 minutes, the reaction mixture was quenched with CH ₃ OH (1.0 ml), diluted with EtOAc (50 ml) and Et ₃ N (1.0 ml), washed with 10% aqueous K ₂ CO ₃ solution (2 x 5.0 ml), and then with brine ( 5 ml) and dried (Na ₂ SO ₄ ). The resulting crude product was purified by flash chromatography using EtOAc in 70 to 75% yield of Compound 4.

 Example 37

 Synthesis of deoxythymidine oligomers with internucleoside bond 3'-N-C-C-5 '.

 The thymidine dimer phosphoramidites obtained in stages a through d are used in the modified solid-state synthesis method according to the phosphoramidite mechanism to obtain the oligonucleoside sequences shown in Table 2.

m = 3'-N-C-C-5'
Олигодезоксинуклеотиды синтезируют от 3' - в направлении 5'-конца.table 2
Sequence - Ref. code
5 'TpTpTpTpTpTpTpTT [TnT] pT 3' - 4
5 'TpTpTpTpTp [TnT] pTpTpTpT 3' - 5
5 '[TnT] pTpTpTpTpTpTpTpTTTT 3' - 6
T = thymidine

m = 3'-NCC-5 '
Oligodeoxynucleotides are synthesized from 3 '- in the direction of the 5'-end.

 The initial stage of the synthesis consists in the attachment, i.e. via a 3'-succinate bond, of 5'-dimethoxytrityldeoxythymidine to a CPA carrier (glass substrate with controlled porosity). The 5'-O-dimethoxytrityl protecting group of the attached thymidine is reacted with trichloroacetic acid to deprotect the 5'-hydroxyl.

 The chain growth then proceeds by carrying out the usual sequential deprotection, activation, capping and oxidation operations using a modification leading to the (-NCC -) - linked thymidine dimer obtained according to the methods of Examples 30 to 37, introduced into the chain, if necessary activation stage time.

At the end of chain assembly, thymidine oligomers are removed from the microporous glass support using a concentrated solution of ammonium hydroxide. The resulting solution is then treated at a temperature of 55 ^o C for about 8 to 15 hours to remove all protective groups from exocyclic amino groups of the bases.

 Example 38

 Obtaining the tetraethylene glycol terminal DNA sequence of the anti-RAS oncogen.

38a. Obtaining dimethoxytrityl tetraethylene glycol (DMTTEG). Excess tetraethylene glycol (TEG) (approximately 100 ml) was mixed with approximately 7 ml (5.1 g; 40 mmol) of a Hunig base in a round bottom flask. About 3.08 g (10 mmol) of dimethoxytrityl chloride (DMTX) was added to the tetraethylene glycol mixture, and the resulting DMTX-TEG mixture was kept under constant stirring at room temperature (about 25 ^° C.) for about 8 to 12 hours to form DMTEG.

 38b. Obtaining dimethoxytrityl tetraethylene glycol cyanophosphine (DMTTEGCF).

 6 g of DMTTEG obtained in stage (a) are mixed with 20 ml of dry dichloromethane. Approximately 6.2 ml of Hunig base is added to the resulting mixture, followed by the dropwise addition of a chlorophosphine mixture, resulting in DMTTEGCF. A chlorophosphine mixture is prepared by dissolving 1.67 g of 2-cyanoethyl-N, N-diisopropylchlorophosphoramidite in 5 ml of dry dichloromethane.

 38c. Obtaining a TEG-terminal DNA sequence of anti-RAS oncogen.

 The oligodeoxynucleotides of Table 3 are prepared by the modified phosphorimidite method of solid-phase synthesis (see GAIT above). These oligodeoxynucleotides are obtained from the 3'- towards the 5'-end.

Table 3
Sequence - Ref.

5 'X GGA GCT GGT GGC GTA X (A) 3' - 7
5 'XX GGA GCT GGT GGC GTA XX (A) 3' - 8
5 'X CCT CGA CCA CCG CAT X (A) 3' - 9
5 'XX CCT CGA CCA CCG CAT XX (A) 3' - 10
5 'CCT CGA CCA CCG CAT 3' - 11
X is TEG;
A, C, G, and T are deoxy-adenosine, cytidine, guanosine, and thymidine, respectively.

 Both adenosine (7, 8, 9, 10) and thymidine (II) are attached to this glass substrate using a succinate bond (see GAIT above). The synthesis of thymidine (II) proceeds according to traditional methods of solid-phase synthesis using a forphoramidite reagent. In sequences 7, 8, 9, and 10, the synthesis proceeds according to methods for modifying the phosphoramidite reagent. 5'-hydroxyl attached adenosine is reacted with trichloroacetic acid to deprotect 5'-hydroxyl. After deprotection, the attached adenosine is reacted with an activator, in particular tetrazole and a phosphoramidite reagent containing dimethoxytrityl tetraethylene glycol cyanophosphine (DMTTEGCF) obtained in the above steps a and b. The activation step is accompanied by capping of unreacted 5′-hydroxyls using acetic anhydride and N-methylimidazole. The resulting phosphorus bond is then oxidized with iodine in accordance with standard techniques. In sequences 8 and 10 containing two tetraethylene glycol residues (TEG), the above steps of deprotection, activation, capping and oxidation are repeated. The chain growth then proceeds by carrying out the usual sequential deprotection, activation, capping and oxidation operations according to the procedure described above, but with the modification that the phosphoramidite reagent in the target nucleoside is replaced with DMTTEGCF during the activation stage. After attachment of the terminal target nucleoside, either or each of the two TEG residues is attached to the 5'-end, similarly to the binding of the TEG to the 3'-end.

At the end of chain assembly, the DNA strand is removed from a glass-based support with controlled porosity using a concentrated solution of ammonium hydroxide. The resulting solution is then treated at a temperature of 55 ^o C for about 8 to 15 hours to remove all protective groups from exocyclic amino groups of the bases.

 Example 39

 Obtaining the hexaethylene glycol terminal (GEG) DNA sequence of the anti-RAS oncogen.

 The hexaethylene glycol terminal DNA sequence of the anti-RAS-oncogene was prepared according to the methods of Example 38. The GEG was reacted with DMTX (DMTC1) to form DMTEGEG (DMTHEG). DMTEGEG is then reacted with a cyanophosphine compound to form DMTHEGCP (DMTHEGCP), which is used in the modified solid-state synthesis method according to the phosphoramidate mechanism according to Example 38 (c) to obtain the GEG-terminal DNA sequence of the anti-RAS oncogen. The sequences of these oligonucleotides are shown in the following table 4.

Table 4
Sequence
5 'X GGA GCT GGT GGC GTA X (A) 3'
5 'XX GGA GCT GGT GGC GTA XX (A) 3'
5 'X CCT CGA CCA CCG CAT X (A) 3'
5 'XX CCT CGA CCA CCG CAT XX (A) 3'
5 'CCT CGA CCA CCG CAT 3'
where X is a GEG;
A, C, G, and T are deoxy-adenosine, cytidine, guanosine, and thymididine, respectively.

 Example 40

 Resistance to nuclease of the TEG-terminal DNA sequence of the anti-RAS oncogen.

 The resulting oligonucleotides shown in table 4 are dissolved in water. DNA concentrations are then determined by measuring the absorption band of the samples at a wavelength of 260 nm (on a Perkin Elmer Lambda 4C spectrophotometer at room temperature) using calculated extinction coefficients (Cantor-Warsaw method, CRC Handbook of Biochemistry and Molecular Biology, 3rd Vol. 1, CRC Press, p. 589, 1975).

Oligonucleotides are incubated for 2 hours at a temperature of 37 ^o C at a total concentration of the entire DNA chain of about 6 or 7 μmol in a cell culture medium containing RPMI 1 640; 20 mmol of N- (2-hydroxyethyl) piperazine-N '- (2-ethanesulfonic acid), pH 7.4; and 10% fetal calf serum (FCS) (GIBCO Laboratories. Grand Islands, NY). FCS is inactivated by heating at a temperature of 56 ^o C for 0.5 hours before its use. The samples are then placed on ice and deprotected five times with a solvent mixture of chloroform: isopropyl alcohol, 24: 1. The treated samples are then either stored frozen at -20 ^° C or immediately placed in a cooled (4 ^° C) HPLC chromatograph auto-injector WISP (Waters).

The degree of hydrolysis of oligonucleotides is evaluated by the released amount of the starting compound. Oligonucleotides (isolated from the reaction mixture) were separated on an LKB Ultrachrome dual-injection chromatographic system equipped with a fixed wavelength detector (260 nm) and an integrator recorder using a GenPak FAX (Waters) anion exchange resin column balanced in buffer A (1 mM EDTA; 15 mM sodium phosphate, pH 8.5). The column temperature was held at 60 ^° C. using a Waters thermostat. Use 50 μl dispensers to inject samples. Oligonucleotides elute using a linear gradient of 0% relative to 100% buffer A (buffer A contains a 0.5 M NaCl solution) over a period of 60 minutes. The flow rate of the buffer solution is 1 ml / min.

 After incubation (2 hours) in the presence of an FCS-linked exonuclease, no degradation of compounds 7 or 10 was noted (table 5, at the end of the description, see% decomposition of the main peak). During the same incubation period, 87.0% and 82% of sample 9 and 8 are not hydrolyzed, respectively. In contrast, only 24.7% of oligomer II remains on the carrier after a similar incubation period.

 All 4 TEG-attached oligomers are resistant to the hydrolysis of FCS-bound exonucleases. Bis-diTEG-attached oligomers (7 and 10), as noted, are completely resistant to nuclease hydrolysis. TEG-derived oligodeoxynucleotides show a significant increase in resistance to exonuclease hydrolysis compared to unmodified compounds.

 Example 41

 The ability of TEG antisense oligomers to inhibit protein expression and growth in human tumor cell lines and to stimulate PHA of peripheral lymphocytes.

 It has been demonstrated by others (Heikkila, R., et al., Nature, 328, pp. 445–449) that unmodified antisense oligonucleotides associated with the initiation region of the c-myc codon of the oncogen can inhibit the expression of c-myc protein with PHA -stimulated peripheral blood lymphocytes (PBL), thus leading to blocking cell progression in the direction of the S-phase of the cell cycle. C-myc-directed antisense DNA has also been shown to inhibit human in vitro erythroleukemia cells HL-60 (Wickstrom TL et al., Proc. Natl. Acad. Sci. USA, 85, pp. 1,028-1,032 1988). The sequences shown in table 6 were obtained and investigated by the methods of Examples 41a and 41b.

Table 6
5 'AAC GNN GAG GGG CFT 3'
5 'XX FFC GNN GAG GGG CAT XX A 3'
(X = TEG)
41a. Comparative studies of the effect of modified (TEG) and unmodified C-myc antisense DNA on the progression of PHA-stimulated peripheral lymphocytes in the S phase of the cell cycle.

 Human peripheral lymphocytes stimulated PHA for 48 hours in the presence or absence of the antisense oligonucleotide sequences shown in Table 6. The percentage of the cell population in each treated group in the S phase of the cell cycle compared to the control was determined using standard flow cytometry methods. The results are shown in table 7 (see the end of the description).

 These data indicate that the presence of TEG at both 3'- and 5'-ends does not affect the inhibitory effect of antisense DNA.

 41b. A comparative study of the effect of modified (in the presence of TEG) unmodified C-myc antisense DNA on protein expression in T-cell-mediated human MOLT-4 leukemia cells.

 Asynchronous exponentially growing Molt 4 cells are incubated for 8 hours with or without 60 µm c-myc-mediated antisense DNA. The cells are then incubated for 45 minutes with the addition of 35 S-methionine, and the protein content is determined by the immune precipitation method using c-myc antibody. The results are shown in table 8. (see the end of the description).

 TEG-containing antisense DNA is slightly more effective than unmodified antisense DNA.

 41c. A comparative study of the effect of modified (in the presence of TEG) and unmodified c-myc antisense DNA on the inhibition of inhibition of growth factor CCRF-CEM T-cell-mediated leukemia in vitro.

 Asynchronous exponentially growing CCRF-CEM cells are incubated for 48 hours in the presence or absence of antisense DNA, after which the cytolytic index in each treated group is determined. After that, the concentration of antisense DNA necessary for 50% inhibition of cell growth (IC 50) is determined. Both the modified and unmodified antisense DNAs shown in Table 5 show approximately the same IR 50 40 μmol.

 These data indicate that the presence of TEG at the 3'- and 5'-end of antisense DNA does not affect the ability of such antisense DNA to hybridize with target nucleic acids and inhibit their function.

 Example 42

 Exonuclease-resistant oligonucleotide dimers.

 The exonuclease-resistant oligonucleotide dimers shown in table 9, obtained by the method of Example 38.

 It is clear to those skilled in the art that various variations and modifications may take place within the scope and spirit of the invention. (01) (19) (11) (21) (51) 6 (54) (57) (08) (09) (00)

Claims (8)

1. Nuclease Resistant Oligonucleoside Containing a Sequence of General Formula

where each Z means independently R ¹ or

where W is —DDD, where each D is independently CHR, NR ⁶ , where R is independently hydrogen, OH, R ⁶ is hydrogen, provided that only one of D is an NR ⁶ group;
W 'means independently W or

R ¹ independently is OH, SH, Nr ² R ³ , where R ² and R ^{3 are} independently H or C ₁ -C ₆ alkyl;
each R ⁵ - independently means H or C ₁ - C ₁₂ alkyl;
each Y means independently H or OH, each B means independently adenine, cytosine, guanine, thymine, uracil or their modifications;
each e and f is independently an integer from 0 to 50, provided that at least one of e and f takes at least 1,
j is an integer from 1 to about 200;
k is 0 or an integer from 1 to about 197;
each m and n are independently an integer from 1 to 200;
each P is independently a number from 2 to 4;
q is 0 or an integer from 1 to about 197, provided that the sum j + k + q is from about 5 to 200.  2. The oligonucleoside according to claim 1, characterized in that it contains a diol at any or each of the ends.  3. The oligonucleoside according to claim 1, characterized in that tetraethylene glycol is used as the diol. 4. Nuclease-resistant oligonucleoside containing the sequence of the General formula

where W - means DDD, where each D is independently CHR, NR ⁶ , where R is independently hydrogen, OH, R ⁶ is hydrogen, provided that only one of D is an NR ⁶ group, each W 'is W or

each R ¹ independently is OH, SH, NR ² R ³ , where R ² and R ^{3 are} independently H or C ₁ -C ₆ alkyl;
each Y is independently H or OH;
each B is independently adenine, cytosine, uracil, or their modifications;
J is an average of 1 to 200;
k is 0 or an average of 1 to 197;
q is 0 or the average is from 1 to 197, provided j + k + q is from 4 to 200. 5. A method for producing oligonucleoside sequences resistant to nuclease degradation, comprising the preparation of oligonucleoside sequences of the general formula

where W is -DDD-, in which each radical D is independently CHR, oxygen or an NR ⁶ group, where R is independently hydrogen, OH, SH or NH ₂ , R ^{6 is} hydrogen or C ₁ -C _{2 is} alkyl, provided that only one of D is oxygen or an NR ⁶ group;
each W ¹ means independently W or

R ¹ independently OH, SH, NR ² R ³ , where R ² and R ³ independently hydrogen or C ₁ - C ₆ -alkyl, or NHR ⁴ , where R ⁴ - C ₁ - C ₁₂ acyl;
each Y is independently H or OH;
each B independently adenine, cytosine, uracil or their modifications;
j is an integer from 1 to about 200;
k is 0 or an integer from 1 to about 197;
q is 0 or an integer from 1 to about 197, provided that the sum j + k + q is 4-200.  6. The method according to claim 5, characterized in that to stabilize the oligonucleoside sequences, a polyalkylene glycol radical is attached to one or each of the ends of the indicated oligonucleoside sequence.  7. Nuclease-resistant oligonucleoside according to any one of claims 1 to 5, having the ability to inhibit gene expression in mammals. 8. Nuclease Resistant Nucleoside Dimer of General Formula

where B, W, Y have the meanings indicated in claim 1;
R ^{7 is} OH or a phosphoramidate group;
R ⁸ is OH or tert-butyldimethylsilyloxy.

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