JP2014169239A - Nucleic acid derivative and production method thereof - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for producing a nucleic acid derivative with an oxygen atom on a ribose ring being substituted by a -S-S- group; and a nucleic acid derivative produced by the method.SOLUTION: The production method of the nucleic acid derivative according to the present invention is a method for producing a nucleic acid derivative having a dithiane ring, where a carboxylate compound is obtained from a thiolsulfinate compound by Pummerer rearrangement reaction before a nucleic acid base group is introduced by Vorbruggen reaction.

Description

  The present invention relates to a nucleic acid derivative and an efficient production method thereof.

  Nucleic acid derivatives are expected to be developed as pharmaceuticals and agricultural chemicals having target specificity and various activities. However, for example, oligo RNA has problems such as low stability, an antigen of an innate immune response, and a drug delivery system to a target has not been established, and it is actually not in practical use as a pharmaceutical .

  The present inventor has developed a 4′-thionucleic acid in which a sugar atom oxygen atom of a nucleoside is substituted with a sulfur atom in order to avoid the attack by a nucleolytic enzyme and improve the stability of a nucleic acid derivative or an oligomer thereof ( Non-patent documents 1 to 3).

Nucleic Acids Res. , 2012, 40, 5787 ChemBioChem, 2007, 8, 2133 FEBS Lett. , 2005, 579, 3115

  As described above, by devising nucleic acid derivatives and oligomers thereof, there is a possibility that the drawbacks of conventional nucleic acid medicines can be improved, or that new or better medicinal effects can be obtained.

  As a new nucleic acid derivative, the present inventors have devised a 1,2-dithiane derivative in which the oxygen atom of the ribose ring of the nucleic acid is a -S-S- group and started synthesis. However, the target compound could not be obtained by applying the conventional method.

  Therefore, an object of the present invention is to provide a method for producing a nucleic acid derivative in which the oxygen atom of the ribose ring is substituted with a -SS- group, and a nucleic acid derivative produced by the method.

  This inventor repeated earnest research in order to solve the said subject. First, the oxygen atom of the ribose ring of a nucleic acid cannot be directly substituted with a -S-S- group. Therefore, an attempt was made to directly introduce a nucleobase group by converting the 1,2-dithiane compound into a sulfinate and then modifying the Pummerer rearrangement reaction. However, the yield was low and only a few target compounds were obtained. Therefore, the target nucleic acid derivative can be obtained by once introducing an acetoxy group into the thiolsulfinate compound by reacting acetic anhydride using the Pummerer rearrangement reaction and further introducing a nucleobase group by the Vorbruggen reaction. And the present invention was completed.

  A method for producing a nucleic acid derivative according to the present invention is a method for producing a nucleic acid derivative represented by the following formula (I):

[Where:
R ¹ and R ² independently represent a hydrogen atom or a hydroxyl protecting group;
R ³ represents a hydrogen atom, a hydroxyl group, a protected hydroxyl group, a C _1-6 alkoxy group, a 2- (C _1-6 alkoxy) ethoxy group or a halogen atom;
B is any nucleobase represented by the following formula:

[Wherein R ⁴ represents a hydrogen atom or an amino-protecting group]
The process of obtaining the carboxylate compound represented by following formula (III) by making a carboxylic anhydride act on the thiol sulfinate compound represented by following formula (II):

[Wherein R ^{1 to} R ³ are as defined above; R ⁵ is a C _1-6 alkyl group or a C _1-6 halogenated alkyl group]; and carboxy in the presence of a silylating agent and a Lewis acid. It comprises a step of obtaining a nucleic acid derivative (I) by reacting a rate compound (III) with a nucleobase.

  In the method of the present invention, trimethylsilyl trifluoromethanesulfonate can be suitably used as the Lewis acid, and N, O-bis (trimethylsilyl) acetamide can be suitably used as the silylating agent.

  In the method of the present invention, as the step of obtaining the thiolsulfinate compound (II), the step of further oxidizing the dithiane compound (IV) to obtain the thiolsulfinate compound (II):

[Wherein R ^{1 to} R ³ are as defined above]
It is preferable to contain.

  The nucleic acid derivative according to the present invention is represented by the following formula (I).

[Where:
R ¹ and R ² independently represent a hydrogen atom or a hydroxyl protecting group;
R ³ represents a hydrogen atom, a hydroxyl group, a protected hydroxyl group, a C _1-6 alkoxy group, a 2- (C _1-6 alkoxy) ethoxy group or a halogen atom;
B is any nucleobase represented by the following formula:

[Wherein R ⁴ represents a hydrogen atom or an amino-protecting group]

  In the present invention, the “hydroxyl-protecting group” is not particularly limited as long as it is generally used in the art as a hydroxyl-protecting group, and examples thereof include a benzyl group, a p-methoxybenzyl group, and a 3,4-dimethoxybenzyl group. Benzyl protecting groups such as o- or p-nitrobenzyl group, p-halobenzyl group, 2,6-dichlorobenzyl group; trimethylsilyl group, triethylsilyl group, triisopropylsilyl group, t-butyldimethylsilyl group, triphenyl Silyl protecting group such as silyl group; Acyl protecting group such as acetyl group and benzoyl group; Methoxymethyl group, p-methoxybenzyloxymethyl group, t-butoxymethyl group, 2-methoxyethoxymethyl group, 1-methoxyethyl And alkoxymethyl protecting groups such as 1-ethoxyethyl group It can be. Further, as a protecting group capable of simultaneously protecting the 4-position and 5-position hydroxyl groups of the thiolsulfinate ring, a cyclic acetal-based protecting group such as a methylene acetal group, an ethylene acetal group, an acetonide group, or the like may be used.

  “Protected hydroxyl group” means a hydroxyl group protected by the above-described protecting group. For example, a benzyloxy group, a p-methoxybenzyloxy group, a trimethylsilyloxy group, a triisopropylsilyloxy group, a 1-methoxyethoxy group, and the like can be given.

The “C _1-6 alkoxy group” refers to a linear or branched monovalent aliphatic saturated hydrocarbon oxy group having 1 to 6 carbon atoms. For example, methoxy group, ethoxy group, n-propoxy group, isopropoxy group, n-butoxy group, isobutoxy group, s-butoxy group, t-butoxy group, n-pentyloxy group, n-hexyloxy group, etc. Can do. Of these, a C _1-4 alkoxy group is preferable, a C _1-2 alkoxy group is more preferable, and a methoxy group is particularly preferable.

“2- (C _1-6 alkoxy) ethoxy group” refers to a group in which the C _1-6 alkoxy group is substituted at the 2-position of the ethoxy group. For example, the 2-methoxyethoxy group has a structure of CH ₃ OCH ₂ CH ₂ O—.

Examples of the “halogen atom” include a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom. The halogen atom for R ³ is preferably a fluorine atom.

In addition, when R ³ is a C _1-6 alkoxy group, a 2- (C _1-6 alkoxy) ethoxy group or a halogen atom, the nucleic acid derivative according to the present invention or an oligomer containing the nucleic acid derivative is hardly decomposed in vivo. Stability is improved.

The “amino-protecting group” is not particularly limited as long as it is commonly used in the art as an amino-protecting group. For example, a methyl carbamate group, an ethyl carbamate group, a 9-fluorenylmethyl carbamate group, t -Carbamate protecting groups such as butyl carbamate groups; acyl protecting groups such as acetyl groups and benzoyl groups. In the -NHR ⁴ group, R ⁴ is a protecting group, for example, dimethylamino methylene group, a benzylidene group, and imine protecting groups such as diphenylmethylene group, a cyclic protecting group, -N = the form of R ⁴ In this case, the amino group may be protected.

  “Carboxylic anhydride” refers to a linear or branched carboxylic anhydride having 2 to 6 carbon atoms and optionally substituted with a halogen atom. For example, acetic anhydride, propionic anhydride, trifluoroacetic anhydride and the like can be mentioned.

The “C _1-6 alkyl group” refers to a linear or branched monovalent aliphatic saturated hydrocarbon group having 1 to 6 carbon atoms. For example, methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, isobutyl group, s-butyl group, t-butyl group, n-pentyl group, n-hexyl group and the like can be mentioned. Of these, a C _1-4 alkyl group is preferable, a C _1-2 alkyl group is more preferable, and a methyl group is particularly preferable.

“C _1-6 halogenated alkyl group” means a C _1-6 alkyl group substituted with at least one halogen atom. Examples thereof include a trifluoromethyl group, a trifluoroethyl group, a pentafluoroethyl group, a chloromethyl group, and a bromomethyl group.

  According to the method of the present invention, it is possible to produce a novel nucleic acid derivative in which the oxygen atom of the ribose ring is substituted with the —SS— group. In the nucleic acid derivative, the nucleic acid oligomer itself or at least a part of the nucleic acid derivative may overcome the shortcomings of conventional nucleic acid drugs, and may exhibit a new or superior medicinal effect. Therefore, the method of the present invention is industrially useful as an aid for providing a new nucleic acid drug.

  Hereinafter, the method of the present invention will be described in the order of execution. In any of the following steps, each protecting group may be changed to one suitable for each reaction at an appropriate time. Appropriate protecting groups and protecting and deprotecting reactions can be appropriately selected from known methods by those skilled in the art. For example, T.W. W. Green, P. G. M. Wuts, "PROTECTIVE GROUPS IN ORGANIC SYNTHESIS", JOHN WILEY & SONS, Inc. Please refer to.

  The absolute structures of the compounds (I) to (IV) and the like are not limited. In the following, description will be made with an example using D-ribose, which is a component of natural nucleic acid, as a starting material. Compounds having other steric configurations can also be synthesized by applying the following method. However, as the compounds (I) to (IV), those having the same steric structure as D-ribose are suitable.

(1) Production process of D-ribitol compound As described above, since the oxygen atom of the ribose ring of the nucleic acid cannot be directly substituted with the -SS- group, it is necessary to obtain a dithiane compound as a raw material intermediate. In this step, a D-ribitol compound, which is a synthetic intermediate for obtaining a dithian compound, is produced from D-ribose.

In this step, R ¹ and R ² are protective groups that can withstand nucleophilic substitution reactions and basic conditions, and R ³ is also a hydroxyl group protected by a protective group that can withstand nucleophilic substitution reactions and basic conditions. preferable. In the following, these R ^{1 to} R ³ may be appropriately converted into desired functional groups.

  A protecting group that can withstand a nucleophilic substitution reaction and basic conditions may be appropriately selected. For example, a benzyl group, a p-methoxybenzyl group, a 3,4-dimethoxybenzyl group, an o- or p-nitrobenzyl group, p And benzyl protecting groups such as -halobenzyl group and 2,6-dichlorobenzyl group.

For example, a ribitol compound in which R ¹ and R ² are p-methoxybenzyl groups and R ³ is a p-methoxybenzyloxy group is a known compound produced from D-ribose. Therefore, this process is described in, for example, Tetrahedronn, 2003, 59, pp. It can be implemented with reference to 1699-1702.

(2) Manufacturing process of dithiane compound Next, a dithiane compound (IV) is obtained by converting a hydroxyl group into -SAc group using a two-step nucleophilic substitution reaction and then ring-closing.

[In the above scheme, Hal represents a halogen atom serving as a leaving group such as a chlorine atom, a bromine atom or an iodine atom]

  In the first step of this process, after leaving the hydroxyl group to be a leaving group such as a methanesulfonyl group, the configuration of the secondary alcohol group of the ribitol compound is reversed by a nucleophilic substitution reaction, and the leaving group is a halogen group. Convert to atom.

  In the second step of this process, the halogen atom is nucleophilically substituted with a -SAc group using thioacetate. At this time, since the configuration is reversed, the configuration at the 4-position is the same as that of D-ribitol.

  In the third stage of this step, the ring is closed by hydrolysis in an oxygen atmosphere to obtain a dithiane compound (IV). The conditions for the hydrolysis are not particularly limited as long as the Ac group can be removed. For example, the hydrolysis may be performed in an aqueous solution of potassium hydroxide or sodium hydroxide. At this time, if the compound is difficult to dissolve, a water-miscible organic solvent such as an alcohol solvent may be added. The oxygen atmosphere is not particularly problematic as long as the reaction is performed in an open system in the atmosphere, but in addition, a gas containing oxygen such as air or oxygen itself may be bubbled into the reaction solution.

(3) Manufacturing process of thiol sulfinate compound Next, the thiol sulfinate compound (II) is obtained by oxidizing the dithiane compound (IV).

  In this step, an oxidizing agent is allowed to act on the dithian compound (IV) in a solvent.

  The solvent that can be used is not particularly limited as long as each compound can be appropriately dissolved and does not inhibit the reaction, and may be appropriately selected. For example, a halogenated hydrocarbon solvent such as dichloromethane or dichloroethane. A nitrile solvent such as acetonitrile; an alcohol solvent such as methanol and ethanol; an ether solvent such as diethyl ether and tetrahydrofuran; and a mixture of two or more of these.

  The oxidizing agent may be selected as appropriate, but it is preferable to oxidize the thiolsulfonate form with little difficulty in oxidizing the thiolsulfinate form. Examples thereof include metaperiodates such as metachloroperbenzoic acid and sodium periodate, hydrogen peroxide, peracetic acid, ozone, halogen, and N-halogen compounds.

  The concentration of the dithian compound (IV) in the reaction solution may be appropriately adjusted. For example, it may be about 0.01 g / mL or more and 0.5 g / mL or less.

  Regarding the amount of the oxidizing agent used, since it is necessary to sufficiently oxidize the dithian compound (IV) while suppressing sulfonation, it is 0.9 times mol or more with respect to the dithian compound (IV). It is preferably 5 times mol or less, more preferably 0.95 times mol or more and 1.2 times mol or less.

  The reaction temperature may be adjusted as appropriate, but in order to control the oxidation reaction, for example, it can be 10 ° C. or lower, preferably 0 ° C. or lower, more preferably −50 ° C. or lower. Specifically, since the sublimation point of dry ice is −79 ° C. and the temperature of liquid nitrogen is −196 ° C., these may be used. Further, the reaction time may be adjusted as appropriate, and may be determined until the dithian compound (IV) as the raw material compound is substantially undetectable, or by preliminary experiments, but is usually about 5 minutes to 1 hour. It can be.

  After the reaction is completed, general post-treatment may be performed. For example, a saturated aqueous sodium hydrogen carbonate solution may be added to decompose excess oxidant, extracted with an organic solvent, dried and concentrated, and then purified by chromatography or the like.

  In addition, since the sulfur atom of the sulfoxide group of the thiol sulfinate compound (II) has an unshared electron pair and exhibits chirality, the thiol sulfinate compound (II) has a diastereomer. Any isomer can be used in the present invention.

(4) Acetate step Next, the acetate compound (III) is obtained by reacting the thiolsulfinate compound (II) with a carboxylic acid anhydride.

  In this step, an acetoxy group is introduced into the thiolsulfinate compound (II) by reacting acetic anhydride using a Pummerer rearrangement reaction.

  In this step, acetate compound (III) can be easily obtained simply by heating using acetic anhydride as a solvent. The concentration of the thiolsulfinate compound (II) in the reaction solution may be appropriately adjusted. For example, the concentration may be about 0.01 g / mL or more and 1.0 g / mL or less.

  The reaction temperature may be appropriately adjusted. For example, the reaction temperature may be about 80 ° C. or higher and 180 ° C. or lower, and it is easy to heat and reflux. The reaction time may be adjusted as appropriate, and may be determined until the thiolsulfinate compound (II) as a raw material compound is substantially undetectable, or by preliminary experiments, etc., but usually 1 hour or more and 50 hours or less Can be about.

  After the reaction is completed, general post-treatment may be performed. For example, in order to treat excess acetic anhydride, a saturated aqueous solution of sodium bicarbonate is added while cooling, extracted with an organic solvent, dried and concentrated, and then purified by chromatography or the like.

  In the Pummerer rearrangement reaction, since it passes through a sulfonium ion, the three-dimensional structure of the product cannot usually be controlled, and the acetate compound (III) is obtained as a diastereomeric mixture. Any isomer can be used in the present invention.

(5) Vorbruggen reaction step Next, the nucleic acid derivative (I) as the target compound is obtained by reacting the acetate compound (III) with the nucleobase in the presence of a silylating agent and a Lewis acid.

  In this step, the acetate compound (III) and the nucleobase are mixed in a solvent, a silylating agent is further added, and then a Lewis acid is added to the reaction while cooling.

  The solvent that can be used is not particularly limited as long as each compound can be appropriately dissolved and does not inhibit the reaction, and may be appropriately selected. For example, nitrile solvents such as acetonitrile; dichloromethane, dichloroethane, and the like A halogenated hydrocarbon solvent of the above; aromatic hydrocarbon solvents such as benzene and toluene; a mixed solvent of two or more of these.

  The following are used as nucleobases. These nucleobases can be used if they are commercially available, or can be easily synthesized by selectively protecting amino groups from commercially available compounds.

[Wherein R ⁴ is as defined above]

  Examples of the silylating agent include N, O-bis (trimethylsilyl) acetamide, 1,1,1,3,3,3-hexamethyldisilazane and the like.

  Examples of the Lewis acid include trimethylsilyl trifluoromethanesulfonate, trimethylsilane iodide, tin chloride, zinc chloride, mercury chloride, and titanium tetrachloride. However, when the nucleic acid derivative according to the present invention is used for medicines and the like, it is not preferable that heavy metal remains. Therefore, it is desirable to use a substance that does not contain heavy metals such as trimethylsilyl trifluoromethanesulfonate and trimethylsilane iodide. Trimethylsilyl trifluoromethanesulfonate acts not only as a Lewis acid but also as a silylating agent.

  The acetate compound (III) and the nucleobase may theoretically be used in an equimolar amount, but it is preferable to use an excess of a nucleobase that is commercially available or that can be synthesized simply by protecting the commercially available product. For example, the nucleobase can be used in an amount of 1.0-fold mol or more and 3-fold mol or less, more preferably 2.5-fold mol or less, and further preferably 2.0-fold mol or less with respect to acetate compound (III). The amount of the silylating agent and Lewis acid used may be adjusted as appropriate, but for the same reason, the silylating agent is preferably used in an amount of 2.0 to 6 moles with respect to the acetate compound (III). The Lewis acid is preferably used in an amount of 2.0 to 5 times the mol of the acetate compound (III).

  The amount of the solvent used may be appropriately adjusted. For example, the total concentration of the acetate compound (III) and the nucleobase may be 10 mg / mL or more and 300 mg / mL or less.

  In this step, Lewis acid is usually added to a solution containing acetate compound (III), nucleobase and silylating agent. At this time, cooling may be performed in order to suppress the rapid progress of the reaction. Although the cooling temperature is not particularly limited, for example, the reactor may be ice-cooled.

  After adding the Lewis acid, it is preferable to raise the reaction temperature and allow the reaction to proceed sufficiently. The reaction temperature may be appropriately adjusted. For example, the reaction temperature may be about 40 ° C. or higher and 120 ° C. or lower, and it is easy to heat and reflux. What is necessary is just to adjust reaction time suitably, and it may be determined by the preliminary experiment etc. until the acetate compound (III) which is a raw material compound becomes substantially undetectable, but it is usually about 1 hour or more and 20 hours or less be able to.

  After the reaction is completed, general post-treatment may be performed. For example, in order to treat excess Lewis acid or the like, a saturated aqueous sodium hydrogen carbonate solution is added while cooling, extracted with an organic solvent, dried and concentrated, and then purified by chromatography or the like.

Thereafter, the obtained compound may be further subjected to a deprotection reaction, or the hydroxyl group of R ³ may be functionally converted to an alkoxy group or a halogen atom.

  According to the method of the present invention described above, a nucleic acid derivative in which the ribose moiety is changed to a 1,2-dithiane ring can be produced. The nucleic acid derivative and the nucleic acid oligomer containing the nucleic acid derivative can be expected to be used as a medicine or an agricultural chemical.

  EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited by the following examples, but may be appropriately modified within a range that can meet the purpose described above and below. Of course, it is possible to implement them, and they are all included in the technical scope of the present invention.

NMR was measured using FT-NMR AV500 and AV400 manufactured by Bruker. The chemical shifts of ¹ H NMR and ¹³ C NMR are tetramethylsilane 0.00 ppm ( ¹ H), CHCl ₃ 7.26 ppm ( ¹ H) and 77.0 ppm ( ¹³ C), or DMSO 2.50 ppm ( ¹ H) and 39 .5 ppm ( ¹³ C) was used as an internal standard. Values are expressed in ppm, and coupling constants are expressed in J values (Hz). The multiplicity of signals is shown using abbreviations of s: singlet, d: doublet, t: triplet, q: quartet, m: multiplet, br: broad. Signal assignment was performed based on the 1H-1H COSY spectrum.

  Mass spectrometry (MS) was performed using a Micromass LCT PREMIER manufactured by Waters.

  The reaction was performed using a commercially available anhydrous solvent under an argon atmosphere unless otherwise specified. As the solvent, acetonitrile and dichloromethane used were diphosphorus pentoxide, and pyridine used was distilled using potassium hydroxide.

  A silica gel 60F254 manufactured by Merck was used for TLC. Silica gel 60 (0.063-0.200 mesh) manufactured by Merck was used for silica gel column chromatography. For flash silica gel column chromatography, silica gel 60 (230-600 mesh ASTM) manufactured by Merck was used. For celite filtration, tesque Celite 545 manufactured by Nacalai was used.

  Example 1

(1) 2,3,5-tri-Op-methoxybenzyl-D-ribitol (compound 1)
D-ribose (12.0 g, 79.9 mmol) was suspended in allyl alcohol (250 mL), concentrated sulfuric acid (1.30 mL, 24.4 mmol) was added, and the mixture was stirred at room temperature for 22 hours. The reaction mixture was neutralized with solid sodium hydrogen carbonate, and the precipitated salt was filtered off through a celite pad and washed with methanol. The filtrate was concentrated under reduced pressure and azeotroped with methanol, followed by toluene azeotropy three times to obtain a crude allyl glycoside as a yellow oily substance. Sodium hydride (60% mineral oil suspension, 14.4 g, 360 mmol) was dissolved in tetrahydrofuran (140 mL), and a mixed solution of the above allyl glycoside and N, N-dimethylformamide (70 mL) was added thereto under ice cooling. After dropwise addition over 15 minutes, the mixture was stirred at room temperature for 1 hour and 30 minutes. A third of paramethoxybenzyl chloride (43.4 mL, 320 mmol) was added dropwise to the reaction mixture over 15 minutes under ice cooling, and then the remaining paramethoxybenzyl chloride was added dropwise while removing the ice bath and returning to room temperature. did. After dropping, the mixture was stirred at room temperature for 14 hours, and ice was added to the reaction mixture. The reaction mixture was partitioned between ethyl acetate and water, and the resulting organic layer was washed with water and saturated brine, and dried over anhydrous sodium sulfate. After the solution was filtered, the filtrate was concentrated under reduced pressure to obtain a crude paramethoxybenzyl protected product as a white yellow oily substance. The obtained substance was dissolved in chloroform (240 mL), water (160 mL) and palladium chloride (II value, 2.12 g, 12.0 mmol) were added, and the mixture was vigorously stirred at 50 ° C. for 20 hours in an oxygen atmosphere. The solid precipitated under ice cooling was filtered off through a celite pad and washed with ethyl acetate. The filtrate was concentrated under reduced pressure, and the resulting residue was partitioned between ethyl acetate and water. The organic layer was washed with water and saturated brine, and dried over anhydrous sodium sulfate. After the solution was filtered, the filtrate was concentrated under reduced pressure to obtain the crude product as a brown oily substance. The obtained substance was dissolved in methanol (400 mL), sodium borohydride was added under ice-cooling, the temperature was returned to room temperature, and the mixture was stirred for 10 minutes. The reaction mixture was concentrated under reduced pressure, methanol azeotropy was performed twice, and the resulting residue was partitioned between ethyl acetate and water. The organic layer was washed with water and saturated brine, and dried over anhydrous sodium sulfate. After filtration of the solution, the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (eluent: hexane / ethyl acetate = 3/1 to 2/1 to 1/1) to give compound 1 (29.3 g, 57 .2 mmol, total yield of 4 steps: 72%) was obtained as a pale yellow oil.
¹ H NMR (CDCl ₃ ); δ 7.21 (m, 6H, Ar), 6.85 (m, 6H, Ar), 4.52 (m, 6H, —OCH ₂ − × 3), 3.95 (m, 2H, H— 2), 3.80 (s, 9H, -OMe × 3), 3.71 (m, 4H, H-3 and H-4 and H-5), 3.52 (m, 2H, H-1), 2.70, 2.36 (each br s, each 1H, 1-OH and 5-OH)

(2) (2S, 3S, 4R) -2,5-dibromo-1,3,4-tris-p-methoxybenzyloxypentane (compound 2)
Compound 1 (27.9 g, 54.5 mmol) was dissolved in pyridine (45 mL), methanesulfonyl chloride (14.8 mL, 0.19 mol) was added under ice cooling, and the mixture was stirred for 30 minutes. Ice was added to the reaction mixture, and the mixture was partitioned between ethyl acetate and water. The organic layer was washed with saturated aqueous sodium hydrogen carbonate solution and saturated brine, and dried over anhydrous sodium sulfate. After the solution was filtered, the filtrate was concentrated under reduced pressure, and toluene azeotropy was performed 4 times to obtain a dimesyl compound as a yellow oily crude product. The obtained dimesyl compound was dissolved in methyl ethyl ketone (130 mL), lithium bromide (45.5 g, 0.52 mol) was added, and the mixture was heated to reflux for 5 hours. The reaction mixture was partitioned between ethyl acetate and water, and the organic layer was washed with water and saturated brine, and dried over anhydrous sodium sulfate. The solution was filtered, the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (eluent: hexane / ethyl acetate = 7/1 to 3/1) to give compound 2 (20.9 g, 32.7 mmol, 2 Total yield of process: 62%) was obtained as a yellow oil.
¹ H NMR (CDCl ₃ ); δ 7.32-7.21 (m, 6H, Ar), 6.86 (m, 6H, Ar), 4.68-4.46 (m, 6H, —O—CH ₂ — × 3), 4.40 ( m, 1H), 3.79 (s, 9H, -OCH ₃ × 3), 3.89 (m, 1H), 3.76-3.69 (m, 4H), 3.64 (m, 1H)

(3) (2R, 3S, 4R) -2,5-dithioacetyl-1,3,4-tris-p-methoxybenzyloxypentane (compound 3)
Compound 2 (20.8 g, 32.7 mmol) was dissolved in N, N-dimethylformamide (32.0 mL), potassium thioacetate (26.0 g, 0.2 mol) was added, and the mixture was stirred at 100 ° C. for 7 hours. did. The reaction mixture was partitioned between ethyl acetate and water, and the organic layer was washed with water and saturated brine, and dried over anhydrous sodium sulfate. After filtration of the solution, the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (eluent: hexane / ethyl acetate = 6/1 to 3/1) to obtain compound 3 (7.60 g, 12.1 mmol, yield). Ratio: 37%) was obtained as a reddish brown oil.
¹ H NMR (CDCl ₃ ); δ 7.22 (m, 6H, Ar), 6.84 (m, 6H, Ar), 4.53-4.39 (m, 6H, —O—CH ₂ − × 3), 3.90 (m, 1H, H-2), 3.84 (dd, 1H, H-3, J3,2 = 4.8, J3,5 = 5.5), 3.79 (s, 9H, -OCH ₃ × 3), 3.63 (m, 2H, H -5), 3.43 (dd, 1H, H-4, J4,3 = 5.5, J4,5 = 6.5), 3.33 (dd, 1H, H-1a J1a, 2 = 4.3, J1a, 1b = 14.1), 3.18 (Dd, 1H, H-1b J1b, 2 = 5.3, J1b, 1a = 14.1), 2.35 (s, 3H, SC (O) CH ₃ ), 2.30 (s, 3H, SC (O) CH ₃ )
¹³ C NMR (CDCl ₃ ); 30.01
ESIMS-LR m / z = 651 (MNa ⁺ )

(4) (3R, 4S, 5R) -4,5-bis-p-methoxybenzyloxy-3-p-methoxybenzyloxymethyl-1,2-dithiane (compound 4)
Compound 3 (7.60 g, 12.1 mmol) was dissolved in a mixed solution (84 mL) of 10% aqueous potassium hydroxide: methanol (1: 5) and stirred for 8 hours in an oxygen atmosphere. Under ice cooling, a saturated aqueous ammonium chloride solution was added to the reaction mixture, and the mixture was partitioned between ethyl acetate and water. The organic layer was washed with water and saturated brine, and dried over anhydrous sodium sulfate. After filtering the solution, the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (eluent: hexane / ethyl acetate = 6/1 to 3/1) to obtain compound 4 (4.79 g, 8.83 mmol). Rate: 73%) was obtained as a reddish brown oil.
¹ H NMR (CDCl ₃ ); δ7.23 (m, 6H, Ar), 6.86 (m, 6H, Ar), 4.56-4.41 (m, 6H, —O—CH ₂ − × 3), 3.89-3.75 ( m, 4H), 3.80 (s, 9H, -OCH ₃ × 3), 3.50 (m, 1H), 3.22 (m, 1H), 2.78 (m, 1H)
¹³ C NMR (CDCl ₃ ); δ 159.05, 129.48, 129.43, 129.18, 113.65, 113.62, 80.18, 78.94, 72.55, 71.27, 71.16, 68.19, 55.13, 46.99, 30.59
ESIMS-LR m / z = 565 (MNa ⁺ )

(5) (3R, 4S, 5R) -4,5-dihydroxy-3-hydroxymethyl-1,2-dithiane (compound 5)
The above compound 4 (4.79 g, 8.83 mmol) was dissolved in a 20% trifluoroacetic acid-dichloromethane mixed solution (20 mL) and stirred for 4 hours and 30 minutes. The reaction mixture was concentrated under reduced pressure and azeotroped with methanol three times. Methanol was added to the residue, the insoluble material was removed by filtration, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: chloroform / methanol = 19/1 to 17/3) to obtain Compound 5 (1.35 g, 7.42 mmol, yield: 84%) as a colorless transparent oily substance. Obtained.
¹ H NMR (DMSO-d ₆ ); δ 4.85 (br s, 3H, 4-OH, 5-OH, 7-OH exchangable with D ₂ O), 3.86 (m, 1H), 3.75-3.55 (m, 3H), 3.16 (m, 1H), 2.97 (m, 1H), 2.75 (m, 1H)
¹³ C NMR (DMSO-d ₆ ); δ 75.56, 73.89, 64.34, 52.18, 32.36
ESIMS-LR m / z = 205 (MNa ⁺ )

(6) (3R, 4S, 5R) -4,5-dihydroxy-3-triisopropylsiloxymethyl-1,2-dithiane (Compound 6)
Compound 5 (1.20 g, 6.58 mmol) was dissolved in N, N-dimethylformamide (16.0 mL), and imidazole (0.99 g, 14.5 mmol) and triisopropylsilyl chloride (1 .50 mL, 7.24 mmol) was sequentially added, and the mixture was stirred at room temperature for 5 hours. Ice was added to the reaction solution to stop the reaction, and the mixture was partitioned with ethyl acetate and water. The organic layer was washed with water and saturated brine, and dried over anhydrous sodium sulfate. After filtering the solution, the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (eluent: hexane / ethyl acetate = 5/1 to 1/1) to give compound 6 (2.0 g, 5.90 mmol, Yield: 91%) was obtained as a reddish brown oil.
¹ H NMR (DMSO-d ₆ ); δ 4.85 (br s, 2H, 4-OH, 5-OH, exchangable with D ₂ O), 4.12 (m, 1H), 3.92 (m, 1H), 3.80 ( m, 1H), 3.61 (m, 1H), 3.31 (m, 1H), 2.98 (m, 1H), 2.79 (m, 1H)
¹³ C NMR (CDCl ₃ ); δ 74.74, 67.07, 60.30, 48.19, 33.52, 17.78, 11.63
ESIMS-LR m / z = 361 (MNa ⁺ )

(7) (3R, 4S, 5R) -4,5-O- (1-methylethylidene) -3-triisopropylsiloxymethyl-1,2-dithiane (Compound 7)
Compound 6 (2.0 g, 5.90 mmol) was dissolved in acetone (20 mL), 2,2-dimethoxypropane (5.30 mL, 41.0 mmol) and p-toluenesulfonic acid hydrate (0.22 g, 1.20 mmol) was sequentially added, and the mixture was stirred at room temperature for 10 minutes. A saturated aqueous sodium hydrogen carbonate solution was added to the reaction solution under ice-cooling, and the mixture was partitioned between ethyl acetate and water. The organic layer was washed with water and saturated brine, and dried over anhydrous sodium sulfate. After filtering the solution, the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (eluent: hexane / ethyl acetate = 49/1 to 24/1) to give compound 7 (1.98 g, 5.23 mmol, Yield: 98%) was obtained as a reddish brown oil.
¹ H NMR (CDCl ₃ ); δ 4.33 (m, 1H, H-4), 4.22 (dd, 1H, H-5, J5,4 = 4.6, J5,6 = 8.7 Hz), 4.11 (dd, 1H , H-7a, J7,6 = 4.5, J7a, 7b = 10.4 Hz), 3.98 (dd, 1H, H-7b, J7b, 6 = 5.0, J7b, 7a = 10.4 Hz), 3.31 (dd, 1H, H -3a, J3a, 4 = 3.7, J3a, 3b = 14.6 Hz), 3.26 (m, 1H, H-6), 3.18 (dd, 1H, H-3b, J3b, 4 = 4.5, J3b, 3a = 14.6 Hz ), 1.52, 1.38 (each s, each 3H, isopropylidene), 1.07 (m, 21H, TIPS)
¹³ C NMR (CDCl ₃ ); δ 108.14, 72.73, 71.14, 62.96, 51.89, 36.58, 28.60, 26.86, 18.12, 12.04
ESIMS-LR m / z = 401 (MNa ⁺ )

(8) (3R, 4S, 5R) -4,5-O- (1-methylethylidene) -1-oxo-3-triisopropylsiloxymethyl-1,2-dithiane (Compound 8)
The compound 7 (1.98 g, 5.23 mmol) was dissolved in dichloromethane (26 mL), metachloroperbenzoic acid (1.42 g, 5.75 mmol) was added little by little at −78 ° C., and the mixture was stirred for 10 minutes. Saturated aqueous sodium hydrogen carbonate solution was added at −78 ° C., and then the reaction mixture was returned to room temperature and partitioned between ethyl acetate and water. The organic layer was washed with water and saturated brine, and dried over anhydrous sodium sulfate. After filtration of the solution, the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (eluent: hexane / ethyl acetate = 7/1 to 4/1) to give compound 8 (1.78 g, 4.50 mmol, Yield: 86%) was obtained as a diastereomeric mixture of white solid material. Two diastereomers were separated as analytical samples.

¹ H NMR of diastereomer A
¹ H NMR (CDCl ₃ ); δ 4.58 (ddd, 1H, H-4, J4, 3a = 3.5, J4, 5 = 5.5, J4, 3b = 7.8 Hz), 4.30 (dd, 1H, H-7a, J7a, 6 = 4.3, J7a, 7b = 10.5 Hz), 4.26 (dd, 1H, H-5, J5,4 = 5.5, J5,6 = 9.5 Hz), 4.13 (t, 1H, H-7, J7b, 6 = J7b, 7a = 10.5), 3.55 (dd, 1H, H-3a, J3a, 4 = 3.5, J3a, 3b = 13.6 Hz), 3.16 (dd, 1H, H-3b, J3b, 4 = 7.8, J3b , 3a = 13.6 Hz), 3.06 (ddd, 1H, H-6, J6,7a = 4.3, J6,5 = 9.5, J6,7b = 10.5 Hz), 1.50, 1.37 (each s, each 3H, isopropylidene), 1.07 (m, 21H, TIPS)
¹ H NMR of diastereomer B
δ4.71 (dd, 1H, H-5, J5,4 = 6.0, J5,6 = 10.3 Hz), 4.38 (dd, 1H, H-7a, J7a, 6 = 2.3, J7a, 7b = 10.5 Hz), 4.20 (ddd, 1H, H-4, J4,3a = 4.0, J4,5 = 6.0, J4,3b = 10.3 Hz), 4.09 (dd, 1H, H-7, J7b, 6 = 2.8, J7b, 7a = 10.5), 3.84 (dd, 1H, H-3a, J3a, 4 = 4.0, J3a, 3b = 13.3 Hz), 3.80 (m, 1H, H-6), 2.98 (dd, 1H, H-3b, J3b, 4 = 10.3, J3b, 3a = 13.3 Hz), 1.49, 1.36 (each s, each 3H, isopropylidene), 1.07 (m, 21H, TIPS).
¹³ C NMR of diastereomer A
¹³ C NMR (500 MHz, CDCl ₃ ) δ 109.25, 71.24, 70.18, 67.02, 61.64, 28.30, 27.83, 26.20, 18.30, 12.23
¹³ C NMR of diastereomer B
¹³ C NMR (500 MHz, CDCl ₃ ) δ 109.76, 72.85, 71.66, 62.25, 55.31, 43.29, 27.87, 25.32, 17.79, 11.73
ESIMS-LR m / z = 395 (MH ⁺ )

(9) (4R, 5S, 6R) -3-Acetoxy-4,5-O- (1-methylethylidene) -6-triisopropylsiloxymethyl-1,2-dithiane (Compound 9)
Compound 8 (1.75 g, 4.40 mmol) was dissolved in acetic anhydride (22 mL) and heated to reflux for 33 hours. Under ice-cooling, the reaction solution returned to room temperature was added little by little to a saturated aqueous sodium hydrogen carbonate solution, and the mixture was stirred until no foaming occurred. Thereafter, the reaction mixture was partitioned between ethyl acetate and water, and the organic layer was washed with saturated aqueous sodium hydrogen carbonate solution and saturated brine, and dried over anhydrous sodium sulfate. After filtration of the solution, the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (eluent: hexane / ethyl acetate = 97 / 3-23 / 2) to give compound 9 (371.0 mg, 0.85 mmol, Yield: 19%) was obtained as a diastereomeric mixture of orange oil.

¹ H NMR of diastereomer A
¹ H NMR (CDCl ₃ ); δ6.01 (d, 1H, H-3, J3,4 = 4.8 Hz), 4.48 (dd, 1H, H-5, J5,4 = 4.8, J5,6 = 7.5 Hz ), 4.16 (t, 1H, H-4, J4,3 = J4,5 = 4.8), 4.14 (dd, 1H, H-7a, J7a, 6 = 5.5, J7a, 7b = 10.5 Hz), 4.00 (dd , 1H, H-7b, J7b, 6 = 5.5, J7b, 7a = 10.5 Hz), 3.26 (dt, 1H, H-6, J6,7 = 5.5, J6,5 = 7.5 Hz), 2.16 (s, 3H , -OC (O) CH ₃ ), 1.52, 1.36 (each s, each 3H, isopropylidene), 1.07 (m, 21H, TIPS)
¹ H NMR of diastereomer B
¹ H NMR (CDCl ₃ ); δ 6.16 (d, 1H, H-3, J3,4 = 3.0 Hz), 4.46 (dd, 1H, H-5, J5,4 = 5.0, J5,6 = 9.0 Hz ), 4.41 (dd, 1H, H-4, J4,3 = 3.0, J4,5 = 5.0), 4.12 (dd, 1H, H-7a, J7a, 6 = 4.3, J7a, 7b = 10.5 Hz), 4.01 (Dd, 1H, H-7b, J7b, 6 = 4.3, J7b, 7a = 10.5 Hz), 3.25 (dt, 1H, H-6, J6,7 = 4.3, J6,5 = 9.0 Hz), 2.16 (s , 3H, -OC (O) CH 3), 1.54,1.39 (each s, each 3H, isopropylidene), 1.07 (m, 21H, TIPS)
¹³ C NMR of diastereomer A
¹³ C NMR (CDCl ₃ ); δ 169.15, 108.50, 74.60, 74.45, 73.56, 62.88, 50.27, 28.38, 26.73, 21.15, 18.08, 12.01
¹³ C NMR of diastereomer B
δ169.34, 109.86, 74.60, 74.45, 73.56, 62.41, 51.78, 27.93, 26.56, 21.19, 18.08, 12.01
ESIMS-LR m / z = 459 (MNa ⁺ )

(10) 1-[(3′R, 4′R, 5 ′S, 6′R) -1 ′, 2′-dithian-4 ′, 5′-O- (1-methylethylidene) -6′- Triisopropylsiloxymethyl-3′-yl] uracil (Compound 10)
Compound 9 (223 mg, 0.53 mmol) and uracil (120 mg, 1.07 mmol) were suspended in acetonitrile (2.60 mL), and N, O-bis (trimethylsilyl) acetamide (0.52 mL, 2.14 mmol) was added. In addition, a clear solution was obtained. Trimethylsilyl trifluoromethanesulfonate (0.24 mL, 1.30 mmol) was added thereto under ice cooling, and the mixture was heated to reflux for 5 hours. The reaction liquid which was returned to room temperature was added little by little to saturated sodium hydrogencarbonate aqueous solution under ice-cooling. Thereafter, the reaction mixture was partitioned between ethyl acetate and water, and the organic layer was washed with saturated aqueous sodium hydrogen carbonate solution and saturated brine, and dried over anhydrous sodium sulfate. After filtering the solution, the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (eluent: hexane / ethyl acetate = 9 / 1-3 / 2) to give compound 10 (94.8 mg, 0.19 mmol, Yield: 37%) was obtained as a brown oil.
¹ H NMR (CDCl ₃ ); δ8.18 (br s, 1H, NH, exchangable with D ₂ O), 7.39 (d, 1H, H-6, J6,5 = 8.2 Hz), 5.78 (d, 1H, H-3 ', J3', 4 '= 7.6 Hz), 5.75 (d, 1H, H-5, J5,6 = 8.2 Hz), 4.80 (t, 1H, H-5', J5,4 '= J5 , 6 '= 4.7 Hz), 4.38 (dd, 1H, H-4', J4 ', 5' = 4.7, J4 ', 3' = 7.6 Hz), 4.17 (dd, 1H, H-7'a, J7a ', 6' = 6.9, J7'a, 7'b = 10.4 Hz), 4.14 (dd, 1H, H-7'b, J7b, 6 '= 5.4, J7'b, 7'a = 10.4 Hz), 3.33 (ddd, 1H, H-6 ', J6', 5 '= 4.7, J6', 7'b = 5.4 J6 ', 7'a = 6.9 Hz), 1.57, 1.37 (each s, each 3H, isopropylidene) , 1.07 (m, 21H, TIPS)
ESIMS-LR m / z = 511 (MNa ⁺ )

(11) 1-[(3′R, 4′R, 5 ′S, 6′R) -4 ′, 5′-dihydroxy-1 ′, 2′-dithian-6′-hydroxymethyl-3′-yl ] Uracil (Compound 11)
A separately prepared 50% trifluoroacetic acid-dichloromethane mixed solution (1.5 mL) was added to the compound 10 (73.0 mg, 0.15 mmol), and the mixture was stirred for 25 hours. The reaction solution was concentrated under reduced pressure, azeotroped 3 times with methanol, and then azeotroped with toluene 3 times. The residue was purified by silica gel column chromatography (eluent: chloroform / methanol = 17/3 to 3/1) to obtain compound 11 (40 mg, 0.14 mmol, yield: 91%) as a brown solid substance.
¹ H NMR (500 MHz, DMSO-d ₆ , 70 ° C) δ7.63 (dd, 1H, H-6, J = 4.0, J = 8.2 Hz), 5.72 (d, 1H, H-3 ', J3' , 4 '= 9.8 Hz), 5.62 (dd, 1H, H-5, J = 1.6 J = 8.2 Hz), 5.34 (br s, 2H, 4'-OH, 5'-OH, exchangable with D ₂ O) , 5.06 (br s, 1H, 7'-OH, exchangable with D ₂ O), 4.28 (dd, 1H, H-5 ', J5', 4 '= 2.2, J5', 6 '= 3.5 Hz), 4.09 (Dd, 1H, H-4 ', J4', 5 '= 2.2, J4', 3 '= 9.8 Hz), 3.92 (dd, 1H, H-7'a, J7'a, 6' = 3.8, J7 'a, 7'b = 11.3 Hz), 3.91 (dd, 1H, H-7' b, J7'b, 6 '= 6.9, J7'b, 7'a = 11.3 Hz), 3.10 (ddd, 1H, H-6 ', J6', 5 '= 3.5 J6', 7'a = 3.8, J6 ', 7'b = 6.9 Hz)

Claims (5)

A method for producing a nucleic acid derivative represented by the following formula (I):

[Where:
R ¹ and R ² independently represent a hydrogen atom or a hydroxyl protecting group;
R ³ represents a hydrogen atom, a hydroxyl group, a protected hydroxyl group, a C _1-6 alkoxy group, a 2- (C _1-6 alkoxy) ethoxy group or a halogen atom;
B is any nucleobase represented by the following formula:

[Wherein R ⁴ represents a hydrogen atom or an amino-protecting group]
The process of obtaining the carboxylate compound represented by following formula (III) by making a carboxylic anhydride act on the thiol sulfinate compound represented by following formula (II):

[Wherein R ^{1 to} R ³ are as defined above; R ⁵ is a C _1-6 alkyl group or a C _1-6 halogenated alkyl group]; and carboxy in the presence of a silylating agent and a Lewis acid. A production method comprising a step of obtaining a nucleic acid derivative (I) by reacting a rate compound (III) with a nucleobase.   The production method according to claim 1, wherein trimethylsilyl trifluoromethanesulfonate is used as the Lewis acid.   The production method according to claim 1 or 2, wherein N, O-bis (trimethylsilyl) acetamide is used as the silylating agent. Furthermore, the step of oxidizing the dithiane compound (IV) to obtain the thiolsulfinate compound (II):

[Wherein R ^{1 to} R ³ are as defined above]
The manufacturing method in any one of Claims 1-3 containing. A nucleic acid derivative represented by the following formula (I).

[Where:
R ¹ and R ² independently represent a hydrogen atom or a hydroxyl protecting group;
R ³ represents a hydrogen atom, a hydroxyl group, a protected hydroxyl group, a C _1-6 alkoxy group, a 2- (C _1-6 alkoxy) ethoxy group or a halogen atom;
B is any nucleobase represented by the following formula:

[Wherein R ⁴ represents a hydrogen atom or an amino-protecting group]