Synthesis method for pyrimidine locked nucleic acid

Abstract

The present invention relates to a synthesis method for a pyrimidine locked nucleic acid. In the present invention, when starting from readily available commercial raw materials, a pyrimidine locked nucleic acid can be obtained only by means of three reaction steps: oxidation, aldol condensation-reduction and cyclization. The method of the present invention does not comprise any protection / deprotection operation, which remarkably shortens the synthetic route. Moreover, conventional reagents are used in the process of each reaction step, and the reaction conditions are relatively mild. The method is good in applicability and beneficial to industrial production.

Description

A method for synthesizing pyrimidine-locked nucleic acid

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of Chinese Application No. 2023117397120, filed on December 18, 2023. Said application No. 2023117397120 is hereby incorporated by reference in its entirety. Technical Field

[0003] The present invention relates to the field of nucleic acid analogs, and more particularly to a method for synthesizing pyrimidine-locked nucleic acids. Background Art

[0004] After more than 40 years of development, oligonucleotide therapy technology has achieved tremendous clinical results. The efficacy of oligonucleotide drugs is due to the continuous modification of their overall chemical structure by researchers. By modifying the sugar ring, backbone and base, the oligonucleotide compound structure is given drugability (Kilanowska, A., & Studzińska, S.. In vivo and invitrostudies of antisense oligonucleotides – a review. RSC Advances, 10(57), 34501–34516.). So far, oligonucleotide modifications have developed into three generations: the first generation is backbone thiolation modification (PS); the second generation targets the 2' position modification of the ribose ring, including 2'-Ome, 2'-F and 2'-MOE; and the third generation focuses on locked nucleic acids, namely LNA (locked nucleic acids).

[0005] LNA, also known as 2',4'-methyleneoxy bridged nucleic acids (BNA), has stronger chemical stability. Structural studies have shown that LNA / LNA duplexes can enhance base stacking, thereby enhancing the binding ability of the duplex. Oligonucleotide sequences with the addition of LNA monomers can greatly improve the binding activity with paired complementary RNA or DNA chains. A single LNA monomer can increase the Tm value of the hybrid chain by 5-9 degrees Celsius (Koshkin, AA, et al. LNA (Locked Nucleic Acid): An RNA Mimic Forming Exceedingly Stable LNA: LNA Duplexes. Journal of the American Chemical Society, 120 (50), 13252–13253.). LNA has been widely used in the research and development of oligonucleotide drugs, and many related drugs have entered the clinical development stage (Crooke, ST, et al. Antisense technology: an overview and prospectus. Nature Reviews Drug Discovery, 20 (6), 427–453).

[0006] At present, the chemical synthesis methods of LNA mainly include linear synthesis and convergent synthesis (Kaur, H., et al. Perspectives on Chemistry and Therapeutic Applications of Locked Nucleic Acid (LNA). Chemical Reviews, 107 (11), 4672–4697.), but both methods use a long synthesis route, and the synthesis process involves the protection and deprotection of multiple hydroxyl groups. Since it is difficult to control the regioselective protection of the two hydroxymethyl groups, the overall synthesis efficiency of the route is low, the cost is high, and it is difficult to mass produce. Although the chemical-enzymatic reaction strategy (Sharma, VK, et al. Chemoenzymatic Convergent Synthesis of 2′-O, 4′-C-Methyleneribonucleosides. The Journal of Organic Chemistry, 79 (13), 6336–6341.) solves the regioselectivity problem to a certain extent, the enzymatic reaction has high requirements for the substrate, and the overall synthesis route still has not gotten rid of the problems of long steps and multiple protecting group operations, which also limits its application. In view of this, developing simpler and more economical synthesis methods has become a technical problem that needs to be solved urgently in this technical field.

[0007] Summary of the Invention

[0008] This invention develops a novel synthetic strategy that, starting from readily available commercial raw materials, yields the target product through a simple three-step reaction: oxidation, aldol condensation-reduction, and cyclization. This method, which does not involve any protection / deprotection steps, significantly shortens the synthetic route. Furthermore, each step utilizes conventional reagents and relatively mild conditions. This method is highly applicable and amenable to industrial production.

[0009] In one aspect, the present invention provides a method for preparing a pyrimidine-locked nucleic acid compound 4, comprising:

[0010] Step i: treating the compound of formula 1 in an oxidation system to obtain a compound of formula 2;

[0011] Step ii: condensing the compound of formula 2 with an aldehyde R2-CHO, followed by treatment with a reducing agent to obtain a compound of formula 3;

[0012] Step iii: treating the compound of formula 3 with a base to obtain compound 4;

[0013] Wherein, R1 and R2 are independently selected from hydrogen, halogen and optionally substituted: alkyl, alkenyl, alkynyl, cycloalkyl, aryl and aralkyl; wherein the substituents are independently selected from one or more of halogen, hydroxyl, cyano, nitro, amino, alkylamino, dialkylamino, arylamino, diarylamino, alkoxycarbonyl, amide, alkylamide, carboxyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylsulfinyl, arylcycloacyl and trialkylsilyl.

[0014] In the present invention, the alkyl group, alone or in a substituent, has 1 to 10 carbon atoms, preferably 1 to 6 carbon atoms, or 1 to 3 carbon atoms; the alkenyl group has 2 to 10 carbon atoms, preferably 2 to 6 carbon atoms, or 2 to 4 carbon atoms; the alkynyl group has 2 to 10 carbon atoms, preferably 2 to 6 carbon atoms, or 2 to 4 carbon atoms; the cycloalkyl group has 3 to 10 carbon atoms, preferably 3 to 6 carbon atoms; the aryl group, alone or in a substituent, has 6 to 14 carbon atoms, preferably 6 to 10 carbon atoms.

[0015] Preferably, R1 and R2 are independently selected from hydrogen, halogen, C1-3 alkyl;

[0016] Further preferably, R1 and R2 are independently selected from hydrogen or methyl;

[0017] More preferably, R1 is selected from hydrogen or methyl, and R2 is hydrogen.

[0018] Preferably, the oxidant system in step i is selected from any one of IBX, DCC / PPTS, oxalyl chloride / TEA / DMSO or DMP; and the reaction solvent in the oxidation reaction in step i is selected from any one of DMSO, DMSO / dichloromethane or dichloromethane.

[0019] More preferably, the conditions for the oxidation reaction in step i are one of the following:

[0020] a.DCC,PPTS,DMSO,rt;

[0021] b.(COCl)2,TEA,DMSO / DCM,-78℃ to rt;

[0022] c.IBX, DMSO, rt;

[0023] d.DMP,rt.

[0024] Preferably, the aldehyde in step ii is any one of formaldehyde, trioxymethylene or paraformaldehyde.

[0025] In step ii, a base is used for the condensation, preferably, the base used is any one of DIPEA, TEA or potassium carbonate;

[0026] The condensation in step ii is carried out in an organic solvent, preferably, the organic solvent used is any one of DMF, DMSO, DMA, HMPA or water / DMF;

[0027] The reducing agent in step ii is any one of NaBH4, LiBH4 or NaBH3CN;

[0028] Preferably, the base in step iii is selected from any one of NaH, t-BuOK, t-BuONa, LiHMDS, NaHMDS, sodium hydroxide, potassium hydroxide or potassium carbonate; more preferably, the base is NaH;

[0029] Preferably, the reaction solvent in step iii is any one of DMF, DMA, DMSO, HMPA, NMP, THF or dioxane; more preferably, the reaction solvent is DMF;

[0030] Preferably, the reaction temperature of step iii is 70-110°C, preferably 80-100°C.

[0031] In another aspect, the present invention provides a compound of formula 2,

[0032] wherein R1 is selected from hydrogen, halogen, and optionally substituted alkyl, alkenyl, alkynyl, cycloalkyl, aryl, and aralkyl; and wherein the substituent is selected from one or more of halogen, hydroxyl, cyano, nitro, amino, alkylamino, dialkylamino, arylamino, diarylamino, alkoxycarbonyl, amide, alkylamide, carboxyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylsulfinyl, arylcycloacyl, and trialkylsilyl.

[0033] Preferably, R1 is selected from hydrogen, halogen, C1-3 alkyl;

[0034] More preferably, R1 is selected from hydrogen or methyl.

[0035] In another aspect, the present invention provides a compound of formula 3,

[0036] wherein R1 and R2 are independently selected from hydrogen, halogen, and optionally substituted alkyl, alkenyl, alkynyl, cycloalkyl, aryl, and aralkyl groups; wherein the substituent is selected from one or more of halogen, hydroxyl, cyano, nitro, amino, alkylamino, dialkylamino, arylamino, diarylamino, alkoxycarbonyl, amide, alkylamide, carboxyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylsulfinyl, arylcycloacyl, and trialkylsilyl groups.

[0037] Preferably, R1 and R2 are independently selected from hydrogen, halogen, C1-3 alkyl;

[0038] Further preferably, R1 and R2 are independently selected from hydrogen or methyl;

[0039] More preferably, R1 is selected from hydrogen or methyl, and R2 is hydrogen. DETAILED DESCRIPTION

[0040] In order to make the technical solution of the present invention clearer and more specific, the present invention is further described in detail by the following examples. The following examples are only used to illustrate the specific embodiments of the present invention so that those skilled in the art can understand the present invention, but are not intended to limit the scope of protection of the present invention. In the specific embodiments of the present invention, the technical means or methods not specifically described are conventional technical means or methods in the art. The materials, reagents, etc. used in the examples, unless otherwise specified, can be obtained from commercial sources.

[0041] Table 1

[0042] Example 1 Preparation of Compounds 2a and 2b

[0043] Compound 1a (4.8 g, 0.02 mol) was added to a flask, followed by DMSO (20 mL) and IBX (8.4 g, 0.03 mol). The reaction was stirred at room temperature for 12 hours. TLC confirmed the reaction was complete. DMSO was removed by freeze drying, and water (50 mL) was added to the residue, stirred, and filtered. The filtrate was freeze-dried to obtain product 2a, which was used directly in the next reaction. MS (ESI) was calculated for C 10 H 10 N2O5[M+H2O+H] + 257.1,found 257.1.

[0044] Compound 1a was replaced with compound 1b to obtain compound 2b, which was used directly in the next step without purification. MS (ESI) was calculated for C9H8N2O5[M+H2O+H] + 243.1,found 243.1.

[0045] Example 2 Oxidative synthesis condition screening

[0046] Table 2

[0047] The screening conditions used in this example are shown in Table 2. All screening reactions used compound 1a, and the reactions were monitored and the product conversions were calculated using HPLC-MS. The results showed that the oxidation reaction of the present invention could be achieved using various oxidation systems.

[0048] Example 3 Preparation of Compounds 3a and 3b

[0049] The product 2a obtained in Example 1 was added to a flask, and DMF (80 mL), formalin (37% formaldehyde aqueous solution, 8.2 mL, 5 equiv.), and DIPEA (17.5 mL, 5 equiv.) were added. The mixture was stirred at 50°C for 24 hours, cooled to room temperature, and an ice bath was placed under the reaction flask. NaBH4 (1.0 g) was slowly added to the reaction flask. The reaction solution was stirred at room temperature for 30 minutes and concentrated under reduced pressure. The resulting residue was dissolved in water and purified by C18 reverse-phase silica gel column chromatography (water:methanol = 95:5) to obtain compound 3a. MS (ESI) was calculated for C 11 H 14 N2O6[M+H] + 271.1,found 271.1.

[0050] Compound 2a was replaced by compound 2b, and compound 3b was prepared by the same reaction and post-treatment conditions. MS (ESI) was calculated for C 10 H 12 N2O6[M+H] + 257.1,found 257.0.

[0051] Example 4 Aldol condensation-reduction synthesis condition screening

[0052] Table 3

[0053] a: The volume of water added is the same as the volume of formalin in Example 3

[0054] The screening conditions used in this example are shown in Table 3. All screening reactions used compound 2a. HPLC-MS was used to monitor the reactions and calculate the product conversion. The results showed that, although the yields varied, compound 3a was obtained using different reaction conditions.

[0055] Example 5 Preparation of Compounds 4a and 4b

[0056] Product 3a from Example 3 was added to a flask, followed by anhydrous DMF (100 mL) and stirring to dissolve. NaH (1.44 g) was then quickly added. The reaction was stirred at 90°C for 10 hours. After completion of the reaction as determined by LCMS, the reaction was quenched by adding water. The reaction solution was concentrated under pressure, and the residue was purified by silica gel column chromatography (dichloromethane:methanol = 15:1) to afford compound 4a (2.54 g, using the same conditions as in Examples 1 and 3, with a three-step total yield of 47%).

[0057] 1 H NMR (400MHz, DMSO-d6) δ11.33(s,1H),7.65(d,J=1.1Hz,1H),5.65(d,J=4.3Hz,1H),5.41(s,1H),5.19(t,J=5.6Hz,1H) ,4.13(s,1H),3.91(d,J=4.4Hz,1H),3.81(d,J=7.4Hz,1H),3.73(d,J=5.5Hz,2H),3.63(d,J=7.4Hz,1H),1.78(s,3H).. 13 C NMR(101MHz,DMSO-d6)δ164.3,150.6,134.8,106.2,89.0,86.1,79.4,71.2,68.4,56.3,12.6.MS(ESI)caled for C 11 H 14 N2O6[M+H] + 271.1,found 271.2.

[0058] Compound 3a was replaced by compound 3b, and compound 4b (0.72 g, according to the conditions of Examples 1 and 3, total yield of three steps 28%) was prepared through the same reaction and post-treatment conditions.

[0059] 1 H NMR (400MHz, DMSO-d6) δ11.35(s,1H),7.75(d,J=8.1Hz,1H),5.67(d,J=4.3Hz,1H) ,5.62(dd,J=1.9,8.1Hz,1H),5.41(s,1H),5.15(t,J=5.5Hz,1H),4.14(s,1H),3.87 (d,J=4.4Hz,1H),3.82(d,J=7.8Hz,1H),3.74(d,J=5.5Hz,2H),3.63(d,J=7.8Hz,1H). 13C NMR(101MHz,DMSO-d6)δ163.40,150.74,139.71,100.91,88.97,86.81,79.36,71.08,68.71,56.02.MS(ESI)caled for C 10 H 12 N2O6[M+H] + 257.1,found 257.0.

[0060] It should be noted that the present invention is particularly described using individual embodiments. However, without departing from the principles of the present invention, a person skilled in the art may make various improvements in form or detail to the present invention, and these improvements also fall within the scope of protection of the present invention.

Claims

1. A method for preparing a pyrimidine locked nucleic acid compound 4, comprising: Step i: treating the compound of formula 1 in an oxidation system to obtain a compound of formula 2; Step ii: using aldehyde R2-CHO to condense with the compound of formula 2, followed by treatment with a reducing agent to obtain a compound of formula 3; Step iii: treating the compound of formula 3 with a base to obtain compound 4; Wherein, R1 and R2 are independently selected from hydrogen, halogen and optionally substituted alkyl, alkenyl, alkynyl, cycloalkyl, aryl and aralkyl; wherein the substituent is independently selected from one or more of halogen, hydroxyl, cyano, nitro, amino, alkylamino, dialkylamino, arylamino, diarylamino, alkoxycarbonyl, amide, alkylamide, carboxyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylsulfinyl, arylcycloacyl and trialkylsilyl.

2. The preparation method according to claim 1, characterized in that R1 and R2 are independently selected from hydrogen, halogen, and C1-3 alkyl.

3. The preparation method according to claim 1, characterized in that R1 and R2 are independently selected from hydrogen or methyl; preferably, R1 is selected from hydrogen or methyl, and R2 is hydrogen.

4. The method for preparing the pyrimidine locked nucleic acid compound 4 according to claim 1, wherein the oxidant system described in step i is selected from any one of IBX, DCC / PPTS, oxalyl chloride / TEA / DMSO or DMP; and the reaction solvent of the oxidation reaction is selected from any one of DMSO, DMSO / dichloromethane, and dichloromethane.

5. The method for preparing the pyrimidine locked nucleic acid compound 4 according to claim 1, wherein the aldehyde in step ii is any one of formaldehyde, trioxymethylene or polyformaldehyde.

6. The method for preparing the pyrimidine locked nucleic acid compound 4 according to claim 1, wherein a base is used during the condensation in step ii, and preferably, the base used is any one of DIPEA, TEA or potassium carbonate; The condensation in step ii is carried out in an organic solvent. Preferably, the organic solvent used is any one of DMF, DMSO, DMA, HMPA or water / DMF.

7. The method for preparing the pyrimidine-locked nucleic acid compound 4 according to claim 1, wherein the reducing agent in step ii is any one of NaBH4, LiBH4 or NaBH3CN.

8. The method for preparing the pyrimidine locked nucleic acid compound 4 according to claim 1, wherein the base in step iii is selected from any one of NaH, t-BuOK, t-BuONa, LiHMDS, NaHMDS, sodium hydroxide, potassium hydroxide or potassium carbonate; preferably, the base is NaH; The reaction solvent of step iii is any one of DMF, DMA, DMSO, HMPA, NMP, THF or dioxane; preferably, the reaction solvent is DMF.

9. A compound of formula 2, wherein R1 is selected from hydrogen, halogen, and optionally substituted alkyl, alkenyl, alkynyl, cycloalkyl, aryl, and aralkyl; wherein the substituent is selected from one or more of halogen, hydroxyl, cyano, nitro, amino, alkylamino, dialkylamino, arylamino, diarylamino, alkoxycarbonyl, amide, alkylamide, carboxyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylsulfinyl, arylcycloacyl, and trialkylsilyl; Preferably, R1 is selected from hydrogen, halogen, C1-3 alkyl; Further preferably, R1 is selected from hydrogen or methyl.

10. A compound of formula 3, wherein R1 and R2 are independently selected from hydrogen, halogen, and optionally substituted alkyl, alkenyl, alkynyl, cycloalkyl, aryl and aralkyl; wherein the substituent is selected from halogen, hydroxyl, cyano, nitro, amino, alkylamino, dialkylamino, arylamino, diarylamino, alkoxycarbonyl, amide, alkylamide, carboxyl, alkenyl, alkynyl, alkoxy, One or more of alkylthio, alkylsulfinyl, arylcycloacyl, and trialkylsilyl; Preferably, R1 and R2 are independently selected from hydrogen, halogen, C1-3 alkyl; Further preferably, R1 and R2 are independently selected from hydrogen or methyl; More preferably, R1 is selected from hydrogen or methyl, and R2 is hydrogen.

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