A process for the preparation of S4-(2-cyanoethyl)-4-thiothymidine deoxynucleoside monomers

Abstract

The application discloses a preparation method of S4-(2-cyanoethyl)-4-thiothymine deoxyriboside monomer, and 5'-O-DMT-uridine is used as a starting material. First, the starting material is reacted with a sulfonylation reagent to generate a sulfonylation reaction on a carbonyl group at a 4 position of the starting material to obtain compound II. Then, the compound II is reacted with 3-mercapto propionitrile to continuously generate a substitution reaction on the 4 position to obtain compound III. Finally, the compound III is reacted with a phosphorus reagent to obtain the S4-(2-cyanoethyl)-4-thiothymine deoxyriboside monomer. The method does not need to protect a 3'-hydroxyl group, has a shorter reaction route, milder reaction conditions, and high synthesis efficiency.

Description

Technical Field

[0001] This invention belongs to the field of organic chemical synthesis technology, specifically relating to a method for preparing S4-(2-cyanoethyl)-4-thiothymidine deoxynucleotide monomer. Background Technology

[0002] S4-(2-cyanoethyl)-4-thio-2'-deoxyuridine phosphoramide monomer was used to synthesize 4-thio-2'-deoxyuridine (d S4 U) Oligonucleotide sequence. After synthesis, the d in the oligonucleotide sequence S4 The thioacryl group of U is selectively and quantitatively modified with a mercapto-specific reaction reagent. This approach represents a diverse post-synthetic modification method that can introduce various functional groups at any base position in the oligo chain.

[0003] S4-(2-cyanoethyl)-4-thio-2'-deoxyuridine phosphoramidamide, through the formation of oligonucleotide (TFO) arrangements in triplet, increases the stability and base recognition ability of TFOs due to the strong stacking effect of the thiocarbonyl group, enabling it to 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 in the synthesis of the above phosphoramidide monomer.

[0005] The known synthetic route for this key intermediate 1 is reported by Robert S. Coleman and Edward A. Kesicki, which uses 3',5'-di-O-TBS-dU (2) as the starting material, followed by sulfonation of the 4-carbonyl group to obtain intermediate 3, substitution of 3 with 3-mercaptopropionitrile to obtain intermediate 4, removal of the TBS protecting group from acetic acid to obtain S4-(2-cyanoethyl)-4-thio-2'-deoxyuridine (5), and further reaction of 5 with DMTrCl in pyridine to obtain the target product 1. The reaction process of the synthetic method is shown in the following reaction formula:

[0006]

[0007] This method involves complex reaction steps, a long synthetic route, and low synthetic efficiency. Summary of the Invention

[0008] To address the aforementioned technical problems, this invention provides a method for preparing S4-(2-cyanoethyl)-4-thiothymidine deoxynucleotide monomers. This method uses 5'-O-DMT-uridine as the starting material, does not require protection of the 3'-hydroxyl group, has a shorter reaction route, milder reaction conditions, and higher synthesis efficiency.

[0009] The technical solution adopted in this invention is as follows:

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

[0011] (1) Compound I reacts with a sulfonating agent under alkaline conditions to give compound II; the structural formula of compound I is: Wherein, R1 is one of H, F, OCH3, and OCH2CH2OCH3; R2 is one of H and CH3;

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

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

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

[0015] (1) Compound I was dissolved in a solvent, and a base, a phase transfer catalyst and a sulfonating agent were added. The mixture was stirred at room temperature for 1.5 to 2.5 h. The reaction solution was then post-treated to obtain compound II.

[0016] (2) Compound II was dissolved in a solvent, and alkali and 3-mercaptopropionitrile were added in sequence. The mixture was stirred at room temperature for 3.5 to 4.5 h. The reaction solution was then post-treated to obtain compound III.

[0017] (3) Dissolve compound III in a solvent, add alkali and phosphorus reagent, stir at room temperature for 2.5-3.5 h, and then post-process the reaction solution to obtain S4-(2-cyanoethyl)-4-thiothymidine deoxynucleotide monomer.

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

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

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

[0021] In step (1), the post-processing method is as follows: let the reaction solution stand, separate the liquids, wash once with saturated sodium bicarbonate aqueous solution, wash once with saturated sodium chloride aqueous solution, dry the organic phase with anhydrous sodium sulfate for 10 min, and concentrate and dry.

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

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

[0024] In step (2), the post-processing method is as follows: add CH2Cl2 to the reaction solution to dilute the reaction solution, then add saturated sodium bicarbonate solution to wash, wash the organic phase once with saturated sodium sulfite, wash once with saturated brine, dry the organic phase with anhydrous sodium sulfate, concentrate the organic phase, and then separate and purify it by silica gel column chromatography using n-hexane:ethyl acetate with a volume ratio of 4:1 as the eluent, collect the target product, and concentrate and dry it.

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

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

[0027] In step (3), the post-processing method is as follows: add CH2Cl2 to the reaction solution to dilute the reaction solution, then add saturated sodium bicarbonate solution to wash, wash once with saturated saline solution, dry the organic phase with anhydrous sodium sulfate, concentrate the organic phase, and then separate and purify it by silica gel column chromatography with a volume ratio of n-hexane:ethyl acetate of 2:1 as the eluent, collect the target product, and concentrate and dry it.

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

[0029] The present invention provides a method for preparing S4-(2-cyanoethyl)-4-thiothymidine deoxyribonucleoside monomer, using 5'-O-DMT-uridine as the starting material. First, it reacts with a sulfonating agent to undergo sulfonation at the carbonyl group at the 4-position to obtain compound II. Then, compound II reacts with 3-mercaptopropionitrile to undergo a substitution reaction at the 4-position to obtain compound III. Finally, compound III reacts with a phosphorus reagent to obtain the S4-(2-cyanoethyl)-4-thiothymidine deoxyribonucleoside monomer. This method does not require protection of the 3'-hydroxyl group, has a shorter reaction route, milder 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 deoxynucleotide monomer provided by this invention avoids the use of multiple protecting groups, improves atom economy, shortens reaction steps, has wide substrate applicability, and has an overall yield of over 75%. Attached Figure Description

[0032] Figure 1 Synthetic route diagram for S4-(2-cyanoethyl)-4-thiothymidine deoxynucleotide monomer;

[0033] Figure 2 The proton NMR spectrum of compound III-A;

[0034] Figure 3 The proton NMR spectrum of compound III-B;

[0035] Figure 4 The proton NMR spectrum of compound III-C;

[0036] Figure 5 The proton NMR spectrum of compound III-D;

[0037] Figure 6 The proton NMR spectrum of compound IV-A;

[0038] Figure 7 The proton NMR spectrum of compound IV-B;

[0039] Figure 8 The proton NMR spectrum of compound IV-C;

[0040] Figure 9 The NMR spectrum of compound IV-D is shown in the 1H NMR spectrum. Detailed Implementation

[0041] The present invention will now be described in detail with reference to the embodiments.

[0042] Example

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

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

[0045] (2) Compound II was further dissolved in a mixed solution of EtOH and H2O in a volume ratio of 9:1, with a concentration of 10 mL / g. K2CO3 and 3-mercaptopropionitrile were added sequentially, with a molar ratio of 1:1:3 for compound II, K2CO3, and 3-mercaptopropionitrile. The mixture was stirred at room temperature for 4 h. An equal volume of dichloromethane was added to the reaction solution. The mixture was washed twice with saturated sodium bicarbonate aqueous solution and once with saturated brine. After drying with anhydrous sodium sulfate for 10 min, the mixture was purified by silica gel column chromatography. The eluent was a mixture of n-hexane and ethyl acetate in a volume ratio of 4:1. The final product, compound III, was a yellow oil 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'-tetraisopropylphosphine diamine and 1-H-tetrazazole were added. The mixture was stirred at room temperature for 3 h, with a molar ratio of 1:1.1:0.95 for compound III, P-reagent, and 1-H-tetrazazole. An equal volume of dichloromethane was added to the reaction solution, and the mixture was washed twice with saturated sodium bicarbonate aqueous solution, once with saturated brine, dried over anhydrous sodium sulfate for 10 min, and purified by silica gel column chromatography after rotary evaporation. The eluent was a mixture of n-hexane and ethyl acetate in a volume ratio of 2:1, and the final product was S4-(2-cyanoethyl)-4-thiothymidine deoxynucleotide monomer.

[0047] The starting materials, molar yields, purity, and characterization data of 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 S4-(2-cyanoethyl)-4-thiothymidine deoxynucleotide monomers prepared from intermediate compound III containing different substituents are shown in Table 2.

[0052] Table 2

[0053]

[0054]

[0055] The above detailed description of a method for preparing an S4-(2-cyanoethyl)-4-thiothymidine deoxynucleotide monomer with reference to the embodiments is illustrative rather than limiting. Several embodiments may be listed within the defined scope. Therefore, variations and modifications without departing from the overall concept of the present invention should be within the protection scope of the present invention.

Claims

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

2. The preparation method according to claim 1, characterized in that, The preparation method includes the following steps: (1) Compound I was dissolved in a solvent, and a base, a phase transfer catalyst and a sulfonating agent were added. The mixture was stirred at room temperature for 1.5 to 2.5 h. The reaction solution was then post-treated to obtain compound II. (2) Compound II was dissolved in a solvent, and alkali and 3-mercaptopropionitrile were added in sequence. The mixture was stirred at room temperature for 3.5 to 4.5 h. The reaction solution was then post-treated to obtain compound III. (3) Dissolve compound III in a solvent, add alkali and phosphorus reagent, stir at room temperature for 2.5-3.5 h, and then post-process the reaction solution to obtain S4-(2-cyanoethyl)-4-thiothymidine deoxynucleotide 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 sulfonating agent is one of 2,4,6-triisopropylbenzenesulfonyl chloride and p-methylbenzenesulfonyl 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 to 1.

5.

5. The preparation method according to claim 2, characterized in that, In step (1), the solvent is dichloromethane or a mixture of dichloromethane and deionized water; 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 to 3.

5.

7. The preparation method according to claim 2, characterized in that, In step (2), the solvent is dichloromethane or a mixture of ethanol and deionized water; 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 diisopropylaminetetrazole.

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

0.

10. The preparation method according to any one of claims 2-9, characterized in that, The structural formula of compound II is: The structural formula of compound III is:

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