Preparation method of S4-(2-cyanoethyl)-4-thiothymine deoxynucleoside monomer

Abstract

The invention discloses a preparation method of a S4-(2-cyanoethyl)-4-thiothymine deoxynucleoside monomer, which comprises the following steps: by taking 5 '-O-DMT-uridine as an initial raw material, firstly, reacting the 5'-O-DMT-uridine with a sulfonylation reagent, and carrying out sulfonylation reaction on carbonyl at the 4 position of the 5 '-O-DMT-uridine to obtain a compound II; reacting the compound II with 3-mercaptopropionitrile, and continuously performing substitution reaction on the fourth site to obtain a compound III; and finally, reacting the compound III with a phosphorus reagent to obtain an S4-(2-cyanoethyl)-4-thiothymine deoxynucleoside monomer. According to the method, 3 '-hydroxyl does not need to be protected, the reaction route is short, the reaction conditions are mild, and the synthesis efficiency is high.

Description

Technical Field

[0001] The invention belongs to the technical field of organic chemical synthesis, and specifically relates to a method for preparing a S4-(2-cyanoethyl)-4-thiothymidine deoxynucleoside monomer. Background Art

[0002] S4-(2-cyanoethyl)-4-thio-2'-deoxyuridine phosphoramidite monomer was used to synthesize 4-thio-2'-deoxyuridine (d S4 U) oligonucleotide sequence. After the synthesis is completed, the d S4 The sulfyl group of U was selectively and quantitatively modified with sulfhydryl-specific reagents, and this protocol represents a versatile post-synthetic modification approach to introduce a variety of functional groups at any base position in the oligo chain.

[0003] S4-(2-cyanoethyl)-4-thio-2'-deoxyuridine phosphoramidite is arranged in triplets to form oligonucleotides (TFOs). Due to the strong stacking effect of the thiocarbonyl group, the stability and base recognition ability of TFOs are increased, and they can selectively bind to complementary DNA double strands.

[0004] 5'-O-[bis(4-methoxyphenyl)phenylmethyl]-S4-(2-cyanoethyl)-4-thio-2'-deoxyuridine (1) is a key intermediate for the synthesis of the above-mentioned phosphoramidite monomers.

[0005] The currently known synthesis route of the key intermediate 1 is reported by Robert S. Coleman and Edward A. Kesicki, using 3',5'-di-O-TBS-dU (2) as the starting material, and obtaining intermediate 3 through sulfonylation of the 4-carbonyl group, 3 and 3-mercaptopropionitrile substitution to obtain intermediate 4, 4 is deprotected by acetic acid to obtain S4-(2-cyanoethyl)-4-thio-2'-deoxyuridine (5), and 5 is further reacted with DMTrCl in pyridine to obtain the target product 1. The reaction process of the synthesis method is shown in the following reaction formula:

[0006]

[0007] ; The reaction steps of this method are complicated, the synthesis route is long, and the synthesis efficiency is low. Summary of the invention

[0008] In order to solve the above technical problems, the present invention provides a method for preparing S4-(2-cyanoethyl)-4-thiothymidine deoxynucleoside monomer, which uses 5'-O-DMT-uridine as a starting material, does not require protection of the 3'-hydroxyl group, has a short reaction route, mild reaction conditions and high synthesis efficiency.

[0009] The technical solution adopted by the present invention is as follows:

[0010] A method for preparing S4-(2-cyanoethyl)-4-thiothymidine deoxynucleoside monomer, the preparation method comprising the following steps:

[0011] (1) Compound I reacts with a sulfonylating agent under the action of a base to obtain Compound II; the structural formula of Compound I is: Wherein, R1 is one of H, F, OCH3, OCH2CH2OCH3; R2 is one of H and CH3;

[0012] (2) Compound II undergoes a substitution reaction with 3-mercaptopropionitrile to obtain compound III;

[0013] (3) Compound III reacts with a phosphorus reagent to obtain S4-(2-cyanoethyl)-4-thiothymidine deoxynucleoside monomer.

[0014] Furthermore, the preparation method comprises the following steps:

[0015] (1) Compound I is dissolved in a solvent, a base, a phase transfer catalyst and a sulfonylating agent are added, and the mixture is stirred at room temperature for 1.5 to 2.5 hours. The reaction solution is post-treated to obtain Compound II;

[0016] (2) dissolving compound II in a solvent, adding a base and 3-mercaptopropionitrile in sequence, stirring at room temperature for 3.5 to 4.5 hours, and post-treating the reaction solution to obtain compound III;

[0017] (3) Compound III is dissolved in a solvent, a base and a phosphorus reagent are added, and the mixture is stirred at room temperature for 2.5 to 3.5 hours. The reaction solution is post-treated to obtain S4-(2-cyanoethyl)-4-thiothymidine deoxynucleoside monomer.

[0018] In step (1), the phase transfer catalyst is tetrabutylammonium bromide or 4-dimethylaminopyridine; the sulfonylating agent is one of 2,4,6-triisopropylbenzenesulfonyl chloride and p-toluenesulfonyl chloride.

[0019] In step (1), the molar ratio of compound I to sulfonyl chloride reagent is 1:1.0-1.5.

[0020] In step (1), the solvent is dichloromethane or a mixed solvent of dichloromethane and deionized water; and the base is sodium carbonate or triethylamine.

[0021] In step (1), the post-treatment method is: allowing the reaction solution to stand, separating the liquids, washing once with a saturated sodium bicarbonate aqueous solution, washing once with a saturated sodium chloride aqueous solution, drying the organic phase with anhydrous sodium sulfate for 10 minutes, and concentrating to dryness.

[0022] In step (2), the molar ratio of compound II to 3-mercaptopropionitrile is 1:2.5-3.5.

[0023] In step (2), the solvent is dichloromethane, or a mixed solvent of ethanol and deionized water; and the base is potassium carbonate or sodium carbonate.

[0024] In step (2), the post-treatment method is: adding CH2Cl2 to the reaction solution to dilute the reaction solution, then adding saturated sodium bicarbonate solution for washing, washing the organic phase once with saturated sodium sulfite, washing once with saturated brine, drying the organic phase with anhydrous sodium sulfate, concentrating the organic phase, and separating and purifying on a silica gel column with n-hexane:ethyl acetate in a volume ratio of 4:1 as the eluent, collecting the target product, and concentrating and drying.

[0025] In step (3), the solvent is dichloromethane; the base is one of 1-H-tetrazole, DCI, and diisopropylamine tetrazolium; and the phosphorus reagent is P-reagent.

[0026] In step (3), the molar ratio of compound III to the phosphorus reagent is 1:1.0-2.0.

[0027] In step (3), the post-treatment method is: adding CH2Cl2 to the reaction solution to dilute the reaction solution, then adding saturated sodium bicarbonate solution to wash, washing once with saturated brine, drying the organic phase with anhydrous sodium sulfate, concentrating the organic phase, and separating and purifying on a silica gel column with n-hexane:ethyl acetate in a volume ratio of 2:1 as the eluent, collecting the target product, and concentrating and drying.

[0028] The structural formula of the compound II is: The structural formula of the compound III is: The structural formula of the S4-(2-cyanoethyl)-4-thiothymidine deoxynucleoside monomer is: The group structure represented by CEPO- is: The group structure represented by DMTrO- is:

[0029] The invention provides a method for preparing a S4-(2-cyanoethyl)-4-thiothymidine deoxynucleoside monomer. The method uses 5'-O-DMT-uridine as a starting material, first reacts it with a sulfonylating agent, and a sulfonylating reaction occurs on the carbonyl group at position 4 thereof to obtain a compound II; then reacts the compound II with 3-mercaptopropionitrile, and a substitution reaction occurs on the carbonyl group at position 4 thereof to obtain a compound III; and finally reacts the compound III with a phosphorus reagent to obtain a S4-(2-cyanoethyl)-4-thiothymidine deoxynucleoside monomer. The method does not require protection of the 3'-hydroxyl group, has a short reaction route, mild reaction conditions, and high synthesis efficiency.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] The method for preparing S4-(2-cyanoethyl)-4-thiothymidine deoxynucleoside monomer provided by the invention avoids using multiple protecting groups, improves atom economy, shortens reaction steps, has wide substrate applicability, and has a total yield of more than 75%. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 The synthetic route of S4-(2-cyanoethyl)-4-thiothymidine deoxynucleoside monomer is shown in FIG.

[0033] Figure 2 This is the H NMR spectrum of compound III-A;

[0034] Figure 3 This is the H NMR spectrum of compound III-B;

[0035] Figure 4 This is the H NMR spectrum of compound III-C;

[0036] Figure 5 This is the H NMR spectrum of compound III-D;

[0037] Figure 6 is the H NMR spectrum of compound Ⅳ-A;

[0038] Figure 7 is the H NMR spectrum of compound Ⅳ-B;

[0039] Figure 8 This is the H NMR spectrum of compound IV-C;

[0040] Fig. 9 This is the H NMR spectrum of compound IV-D. DETAILED DESCRIPTION

[0041] The present invention is described in detail below with reference to the embodiments.

[0042] Example

[0043] A method for preparing S4-(2-cyanoethyl)-4-thiothymidine deoxynucleoside monomer, comprising the following steps:

[0044] (1) Compound I was dissolved in dichloromethane to a concentration of 10 mL / g, and Na2CO3 aqueous solution, tetrabutylammonium bromide, and 2,4,6-triisopropylbenzenesulfonyl chloride were added, and the molar ratio of compound I, Na2CO3, tetrabutylammonium bromide, and 2,4,6-triisopropylbenzenesulfonyl chloride was 1:4:0.5:1.3. The reaction was stirred for 2 h, and the reaction solution was separated into layers. The organic phase was washed once with saturated brine, dried with anhydrous sodium sulfate for 10 min, and then spin-dried to obtain compound II;

[0045] (2) Compound II was further dissolved in a mixed solution of EtOH and H2O in a volume ratio of 9:1, wherein the concentration of Compound II was 10 mL / g, K2CO3 and 3-mercaptopropionitrile were added in sequence, wherein the molar ratio of Compound II, K2CO3, and 3-mercaptopropionitrile was 1:1:3, and the reaction was stirred at room temperature for 4 h. An equal volume of dichloromethane was added to the reaction solution, and the solution was washed twice with a saturated sodium bicarbonate aqueous solution and once with saturated brine, dried over anhydrous sodium sulfate for 10 min, and then purified by a silica gel column after being spin-dried. The eluent was a mixed solution of n-hexane and ethyl acetate in a volume ratio of 4:1, and finally a yellow oily product, Compound III, was obtained with a molar yield of 85%.

[0046] (3) Compound III was dissolved in dichloromethane to a concentration of 5 mL / g, and 2-cyanoethyl N,N,N',N'-tetraisopropylphosphorodiamidite and 1-H-tetrazole were added. The reaction was stirred at room temperature for 3 h, and the molar ratio of compound III, P-reagent, and 1-H-tetrazole was 1:1.1:0.95; an equal volume of dichloromethane was added to the reaction solution, and the mixture was washed twice with a saturated sodium bicarbonate aqueous solution and once with a saturated saline solution, dried over anhydrous sodium sulfate for 10 min, and then purified by a silica gel column. The eluent was a mixture of n-hexane and ethyl acetate in a volume ratio of 2:1, and finally S4-(2-cyanoethyl)-4-thiothymidine deoxynucleoside monomer was obtained.

[0047] The raw materials, molar yield, purity and characterization data of the intermediate compound III containing different substituents are shown in Table 1.

[0048] Table 1

[0049]

[0050]

[0051] The molar yield, purity and characterization data of the S4-(2-cyanoethyl)-4-thiothymidine deoxynucleoside monomer prepared from the intermediate compound III containing different substituents are shown in Table 2.

[0052] Table 2

[0053]

[0054]

[0055] The detailed description of the preparation method of a S4-(2-cyanoethyl)-4-thiothymidine deoxynucleoside monomer with reference to the above-mentioned embodiment is illustrative rather than restrictive, and several embodiments can be listed according to the limited scope. Therefore, changes and modifications without departing from the overall concept of the present invention should fall within the scope of protection of the present invention.

Claims

1. A method for preparing S4-(2-cyanoethyl)-4-thiothymidine deoxynucleoside monomer, characterized in that: The preparation method comprises the following steps: (1) Compound I reacts with a sulfonylating agent under the action of a base to obtain Compound II; the structural formula of Compound I is: Wherein, R1 is one of H, F, OCH3, OCH2CH2OCH3; R2 is one of H and CH3; (2) Compound II undergoes a substitution reaction with 3-mercaptopropionitrile to obtain compound III; (3) Compound III reacts with a phosphorus reagent to obtain S4-(2-cyanoethyl)-4-thiothymidine deoxynucleoside monomer.

2. The preparation method according to claim 1, characterized in that: The preparation method comprises the following steps: (1) Compound I is dissolved in a solvent, a base, a phase transfer catalyst and a sulfonylating agent are added, and the mixture is stirred at room temperature for 1.5 to 2.5 hours. The reaction solution is post-treated to obtain Compound II; (2) dissolving compound II in a solvent, adding a base and 3-mercaptopropionitrile in sequence, stirring at room temperature for 3.5 to 4.5 hours, and post-treating the reaction solution to obtain compound III; (3) Compound III is dissolved in a solvent, a base and a phosphorus reagent are added, and the mixture is stirred at room temperature for 2.5 to 3.5 hours. The reaction solution is post-treated to obtain S4-(2-cyanoethyl)-4-thiothymidine deoxynucleoside monomer.

3. The preparation method according to claim 2, characterized in that: In step (1), the phase transfer catalyst is tetrabutylammonium bromide or 4-dimethylaminopyridine; the sulfonylating agent is one of 2,4,6-triisopropylbenzenesulfonyl chloride and p-toluenesulfonyl chloride.

4. The preparation method according to claim 2, characterized in that: In step (1), the molar ratio of compound I to sulfonyl chloride reagent is 1:1.0-1.

5.

5. The preparation method according to claim 2, characterized in that: In step (1), the solvent is dichloromethane or a mixed solvent of dichloromethane and deionized water; and the base is sodium carbonate or triethylamine.

6. The preparation method according to claim 2, characterized in that: In step (2), the molar ratio of compound II to 3-mercaptopropionitrile is 1:2.5-3.

5.

7. The preparation method according to claim 2, characterized in that: In step (2), the solvent is dichloromethane, or a mixed solvent of ethanol and deionized water; and the base is potassium carbonate or sodium carbonate.

8. The preparation method according to claim 2, characterized in that: In step (3), the solvent is dichloromethane; the base is one of 1-H-tetrazole, DCI, and diisopropylamine tetrazolium; and the phosphorus reagent is P-reagent.

9. The preparation method according to claim 2, characterized in that: In step (3), the molar ratio of compound III to the phosphorus reagent is 1:1.0-2.

0.

10. The preparation method according to any one of claims 2 to 9, characterized in that: The structural formula of the compound II is: The structural formula of the compound III is: The structural formula of the S4-(2-cyanoethyl)-4-thiothymidine deoxynucleoside monomer is:

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