Asymmetric catalytic synthesis of pentavalent oxythiophosphorane monomer and preparation method of intermediate of pentavalent oxythiophosphorane monomer

Abstract

The invention provides an asymmetric preparation method of a pentavalent oxythiophosphorane monomer. The structure of the pentavalent oxythiophosphorane monomer is shown as a formula 3, wherein R2 is any one of hydrogen, alkyl and halogen. Starting from a key initial raw material, under the catalysis of a cyclohexanediamine catalyst, the reaction is carried out; the three-dimensional pure pentavalent oxathiane monomer is synthesized through a coupling reaction with 5 '-OH of nucleoside, the synthesis method of the three-dimensional pure oxathiane monomer is expanded, and the obtained three-dimensional pure oxathiane monomer can smoothly realize solid-phase synthesis of chiral PS and PS / PO mixed skeleton oligonucleotide and synthesis of PS ring dinucleotide. The research and development of orphan drugs and anticancer drugs are greatly promoted.

Description

Technical Field

[0001] This invention relates to the field of chemical synthesis technology, specifically to an asymmetric catalytic synthesis of pentavalent oxythiophosphine monomers and a method for preparing intermediates thereof. Background Technology

[0002] Compared to common PO (phosphate ester) DNA, PS (phosphate thioester) DNA exhibits stronger antioxidant properties, protecting microorganisms from oxidants. Phosphothioester modification does not significantly affect the structure of nucleic acids; PS oligonucleotides can still form double helical structures with complementary DNA or RNA single strands. Furthermore, PS oligonucleotides can be better taken up by cells through dynamic covalent exchange with cellular thiols and disulfides, and also exhibit higher affinity for proteins within the cell. Therefore, PS modification is widely used in nucleic acid drugs, including antisense oligonucleotides (ASOs) and cyclic dinucleotides (CDNs).

[0003] Thio modification endows the originally achiral P atom of the phosphate diester with chirality, producing R p With S p Two stereoconfigurations. The naturally occurring PS modifications found in bacterial DNA are both R. p Configuration, in vitro studies have shown that (S) p )-PSDNA ratio (R p PSDNA has stronger antioxidant properties and higher stability against nucleases, therefore, the development of stereoselective PS oligonucleotide synthesis methods has attracted widespread attention.

[0004] Compared with trivalent phosphorus monomers, pentavalent oxythiophosphine monomers have better stability. They do not require strict control of anhydrous and oxygen-free conditions during monomer preparation, and do not require oxidative sulfidation during the synthesis of oligonucleotides and cyclic dinucleotides, making the synthesis cycle simpler. Developing pentavalent oxythiophosphine monomers with higher coupling activity for the preparation of stereopure PS oligonucleotides and cyclic dinucleotides is a research direction worthy of in-depth exploration.

[0005] The STEC research group has disclosed a method for preparing stereopure pentavalent oxothiophosphane monomers. Previously, diastereomeric substrates were obtained through cumbersome silica gel column chromatography or expensive preparative HPLC. The group then significantly reduced the difficulty of classifying stereopure monomers by introducing spirocyclic structures into the oxothiophosphane monomers, allowing for the acquisition of two diastereomers with different configurations through a single column separation. The synthesis of pentavalent oxothiophosphane monomers all begin with aldehydes, proceeding through multiple steps to synthesize a pentavalent oxothiophosphane chloride intermediate, followed by a coupling reaction with a nucleoside under basic conditions, and finally oxidative sulfidation to obtain the racemic pentavalent oxothiophosphane monomer. The specific synthetic route of this method is as follows:

[0006]

[0007] Although the above-mentioned method for synthesizing oxythiophosphine monomers can simultaneously yield two diastereomers, the trivalent phosphorus method involves a long preparation process, expensive and difficult-to-obtain raw materials, and the use of hazardous sulfur, which is environmentally unfriendly. The unavoidable configuration separation problem further limits the application of the trivalent phosphorus method in actual production. Summary of the Invention

[0008] To address the aforementioned problems, the present invention aims to provide an asymmetric catalytic synthesis of pentavalent oxythiophosphine monomers and a method for preparing intermediates therein, particularly a method for the asymmetric synthesis of pentavalent oxythiophosphine monomers that features readily available and inexpensive raw materials, a short reaction route, high overall yield, and environmental friendliness.

[0009] The objective of this invention is achieved through the following technical solution:

[0010] In a first aspect, the present invention provides an intermediate compound, the structure of which is shown in Formula 1 below:

[0011] Where X is O or S, and R1 is any one of hydrogen, alkyl, halogen, cyano, trifluoromethyl, or nitro.

[0012] As a preferred embodiment, when X is O, R1 is any one of hydrogen, alkyl, halogen, cyano, trifluoromethyl, and nitro;

[0013] When X is S, R1 is any one of hydrogen, alkyl, cyano, trifluoromethyl, bromine, and nitro.

[0014] As a further preferred embodiment, when R1 is an alkyl group, the alkyl group is selected from C1 to C10 straight-chain or branched alkyl groups.

[0015] In a second aspect, the present invention provides a method for preparing the intermediate compound according to the foregoing, comprising the following steps: reacting the compound shown in Formula 1a-1 with phosphorus pentasulfide under the presence of a base, and then cyclizing the compound shown in Formula 1a-2 with ethylene oxide under the action of trifluoroacetic acid to obtain the intermediate compound shown in Formula 1.

[0016] The structure of the compound of formula 1a-1 is as follows:

[0017] Wherein: X is O or S; R1 is one or more substituents on the benzene ring, and the substituents are any one of hydrogen, alkyl, halogen, cyano, trifluoromethyl, and nitro.

[0018] The structure of the compound of formula 1a-2 is as follows:

[0019] Wherein: X is O or S, and R1 is any one of hydrogen, alkyl, halogen cyano, trifluoromethyl, and nitro.

[0020] As a preferred embodiment, the molar ratio of the compound represented by Formula 1a-1 to phosphorus pentasulfide is 2:1.

[0021] As a preferred embodiment, the molar ratio of the compound represented by Formula 1a-1 to the base is 2:2.1.

[0022] As a preferred embodiment, the base is triethylamine.

[0023] As a preferred embodiment, the molar ratio of the compound represented by Formula 1a-2 to ethylene oxide is 1:1.5.

[0024] As a preferred embodiment, the molar ratio of the compound represented by Formula 1a-2 to trifluoroacetic acid is 1:1.5.

[0025] Thirdly, the present invention provides an intermediate compound, the structure of which is shown in Formula 2 below:

[0026] R2 can be any one of hydrogen, alkyl, or halogen.

[0027] Fourthly, the present invention provides a method for preparing the intermediate compound according to the foregoing, comprising the following steps: protecting the compound shown in Formula 2a-1 with TBSCl, attaching a DMTr group, and then removing the TBS protecting group from the compound shown in Formula 2a-2 to obtain the intermediate compound shown in Formula 2.

[0028] The structure of the compound represented by Formula 2a-1 is as follows:

[0029] Wherein: R2 is any one of hydrogen, alkyl, or halogen;

[0030] The structure of the compound represented by formula 2a-2 is as follows:

[0031] Wherein: R2 is any one of hydrogen, alkyl, or halogen.

[0032] As a further preferred embodiment, when R2 is an alkyl group, the alkyl group is selected from C1 to C10 straight-chain or branched alkyl groups.

[0033] As a preferred embodiment, the specific steps of the preparation method are as follows:

[0034] A1. Imidazole and TBSCl were added to the compound shown in Formula 2a-1, and the reaction yielded a TBS protected intermediate compound. DMTTrCl was then added to the TBS protected intermediate compound, and the reaction yielded the compound shown in Formula 2a-2.

[0035] A2. Add the compound shown in Formula 2a-2 to tetrabutylammonium fluoride to react and obtain the intermediate compound shown in Formula 2.

[0036] As a preferred embodiment, in step A1, the molar ratio of the compound represented by formula 2a-1 to TBSCl is 1:1.2, and the molar ratio of the compound represented by formula 2a-1 to imidazole is 1:1.5.

[0037] As a preferred embodiment, in step A1, the molar ratio of the compound represented by formula 2a-2 to tetrabutylammonium fluoride is 1:1.5.

[0038] Fifthly, the present invention provides a pentavalent oxythiophosphine monomer, the structure of which is shown in Formula 3 below:

[0039] R2 can be any one of hydrogen, alkyl, or halogen.

[0040] As a further preferred embodiment, when R2 is an alkyl group, the alkyl group is selected from C1 to C10 straight-chain or branched alkyl groups. Even more preferably, the alkyl group is methyl.

[0041] Sixthly, the present invention provides a method for preparing the aforementioned pentavalent oxythiophosphine monomer, comprising the following steps:

[0042] Under the catalysis of cyclohexanediamine catalyst, the intermediate compound shown in Formula 1 and the intermediate compound shown in Formula 2 undergo an asymmetric catalytic coupling reaction to generate a pentavalent oxothiophosphine monomer; the obtained pentavalent oxothiophosphine monomer is a stereopure pentavalent oxothiophosphine monomer.

[0043] As a preferred embodiment, the cyclohexanediamine catalyst has a compound structure having any of the following formulas (C1-C3):

[0044]

[0045] More preferably, the cyclohexanediamine catalyst has the compound structure shown in formula C1.

[0046] As a preferred embodiment, the molar ratio of the intermediate compound shown in Formula 1 to the intermediate compound shown in Formula 2 is 2-3:1; the amount of catalyst added is 10-20 mol of the total molar amount of the reactants.

[0047] As a preferred embodiment, the reaction is carried out in the presence of a solvent, wherein the solvent is at least one of toluene, fluorobenzene, anisole, and tetrahydrofuran; more preferably, the solvent is toluene.

[0048] As a preferred embodiment, the reaction temperature is -10℃ to 40℃; more preferably, the reaction temperature is 0℃ to 10℃.

[0049] In a seventh aspect, the present invention provides the use of the aforementioned pentavalent oxythiophosphine monomer in the preparation of PS cyclic dinucleotides or in the preparation of chiral PS and PS / PO mixed backbone oligonucleotides.

[0050] Compared with the prior art, the present invention has the following beneficial effects:

[0051] 1. The synthetic route of this invention can catalytically synthesize pentavalent oxythiophosphine monomers asymmetrically, which is not possible with other synthetic routes.

[0052] 2. The synthetic route of this invention avoids the cumbersome and expensive separation of racemic pentavalent oxothiophosphane monomers and installation of prosthetic groups, greatly shortens the reaction steps, avoids the use of toxic and odorous compounds, and can obtain pentavalent oxothiophosphane monomers with a ratio as high as 99:1 d.r. with only one catalytic step.

[0053] 3. The raw materials used in the synthesis method of this invention are inexpensive and readily available, the operation is simple, the reaction route is short, and the overall yield is high. Attached Figure Description

[0054] Other features, objects, and advantages of the present invention will become more apparent from the following detailed description of non-limiting embodiments with reference to the accompanying drawings:

[0055] Figure 1 This is the reaction route for the non-corresponding catalytic synthesis of pentavalent oxythiophosphine monomers in the embodiments of the present invention;

[0056] Figure 2 This serves as a verification of the reaction route used in Example 1 of this invention to prepare optically pure PS cyclic dinucleotides from pentavalent oxythiophosphine monomers. Detailed Implementation

[0057] The present invention will now be described in detail with reference to specific embodiments. These embodiments will help those skilled in the art to further understand the present invention, but do not limit the invention in any way. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention. These all fall within the scope of protection of the present invention.

[0058] In a specific embodiment of the present invention, an asymmetric preparation method for pentavalent oxythiophosphine monomers is provided, using key starting materials... (X is O or S, R1 is any one of hydrogen, alkyl, halogen, cyano, trifluoromethyl, nitro) undergoes nucleophilic substitution with a cyclohexanediamine catalyst (such as C1), activating formula 1; then the reactant formula 2 (R2 is any one of hydrogen, alkyl, or halogen) participates in the reaction, while another part of the quinoline acts as a hydrogen-extracting agent, promoting the departure of the 5'-OH proton in formula 2. The three are tightly linked by hydrogen bonds, jointly achieving the asymmetric synthesis of the pentavalent oxythiophosphine monomer. The synthetic mechanism is as follows:

[0059]

[0060] In a specific embodiment of the present invention, the cyclohexanediamine catalyst C1 is prepared by the following reaction formula and specific method:

[0061]

[0062] In a 100 mL round-bottom flask, (1S,2S)-cyclohexanediamine (1.10 g, 9.6 mmol, 1.0 equiv.), 2-chloro-8-methylquinoline (3.4 g, 19.2 mmol, 2.0 equiv.), sodium tert-butoxide (2.3 g, 24.0 mmol, 2.5 equiv.), Pd(dba)₂ (0.55 g, 0.96 mmol, 0.10 equiv.), and rac-BINAP (1.20 g, 1.92 mmol, 0.2 equiv.) were added. The mixture was purged with nitrogen three times, and toluene (50 mL) was added via syringe. The reaction mixture was placed in an oil bath (preheated to 80 °C) and stirred at this temperature for 12 hours. After cooling to room temperature, the reaction mixture was diluted with dichloromethane, the organic phase was washed twice with water, the solvent was removed by rotary evaporation under reduced pressure, and the product was purified by column chromatography to give a white solid product (3.4 g, 90% yield).

[0063] (1S,2S)-N1,N2-bis(8-methylquinolin-2-yl)cyclohexane-1,2-diamine(C1)

[0064] 1 H NMR (400MHz, CDCl3) δ7.59(d,J=8.8Hz,2H),7.47–7.40(m,4H),7.13(t,J=7.5Hz,2H),6.31(d,J=8.8Hz,2H),6.03(br s,2H),4.19–4.16(m,2H),2.78(s,6H),2.52–2.49(m,2H),1.91–1.86(m,2H),1.57–1.41(m,4H).

[0065] 13C NMR (101MHz, CD3OD) δ156.4,146.6,137.2,133.6,129.8,125.4,123.0,121.4,112.4,57.0,33.0,25.2,18.0.

[0066] HRMS calcd.for C 26 H 29 N4 + [M+H] + 397.2387 found 397.2389.

[0067] This invention can synthesize a series of pentavalent oxothiophosphine monomers with different bases. (R2 is any one of hydrogen, alkyl, or halogen), and these monomers were successfully applied to the solid-phase synthesis of chiral PS and PS / PO mixed backbone oligonucleotides and the synthesis of PS cyclic dinucleotides.

[0068] All reaction raw materials and reagents used in the following examples were obtained through conventional means or prepared using existing methods. This invention does not impose any particular limitations.

[0069] Example 1: Preparation of Compound 1

[0070] This embodiment provides a series of methods for preparing compounds of Formula 1, and the reaction routes are as follows:

[0071]

[0072] The specific steps are as follows:

[0073] 1) Under nitrogen protection, the compound shown in Formula 1a-1 (63.6 mmol, 2.0 equiv.) was added to a DCM (36 mL) solution of phosphorus pentasulfide (7.2 g, 32.9 mmol, 1.0 equiv.), and triethylamine (9.4 mL, 67.2 mmol, 2.1 equiv.) was slowly added dropwise over 30 minutes (Note: keep the reaction below 40 °C). After the addition was complete, the reaction was heated to 35 °C and stirred overnight. After the reaction was completed, the reaction was cooled to room temperature, and a mixture of methyl ether / n-hexane (v / v = 1:1, 60 mL) was added (Note: using n-hexane alone will cause the product to precipitate in an oily form). The mixture was washed with water (3 × 24 mL), and the organic phase was concentrated until the solid just precipitated. Methanol (18 mL) was added, and the mixture was further concentrated to 18 mL. Then n-hexane (9 mL) was added, followed by water (12 mL) slowly. After about 25 minutes, the resulting slurry was stirred for 1.5 h and filtered to obtain a white solid 1a-2.

[0074] 2) Trifluoroacetic acid (6.5 mL, 86.2 mmol, 1.5 equiv.) was added to a solution of 1a-2 (57.5 mmol, 1.0 equiv.) and ethylene oxide (28.7 mL, 86.2 mmol in 3 M THF, 1.5 equiv.) in 171 mL of dichloromethane. After reacting for 3 h, the resulting mixture was concentrated, and the residue was poured into 1 L of water while stirring vigorously. The precipitated solid was filtered and further slurried with 200 mL of water for 30 min, and then filtered again to obtain the final product, the compound shown in Formula 1. When the compound shown in Formula 1a-1 used in step 1) is... The final product obtained during preparation is as follows:

[0075] The compound shown in Formula 1-1:

[0076]

[0077] 2-(perfluorophenoxy)-1,3,2-oxathiaphospholane 2-sulfide

[0078] 1 H NMR (500MHz, CDCl3) δ4.72–4.54(m,2H),3.74–3.63(m,2H).

[0079] 13 C NMR(126MHz, CDCl3)δ142.8–142.6(m),140.8–140.6(m),140.3–140.1(m),139 .1–138.9(m),138.3–138.0(m),137.1–136.8(m),126.3–126.0(m),71.6,37.1.

[0080] 31 P NMR (202MHz, CDCl3): δ 105.10.

[0081] 19 F NMR (202MHz, CDCl3): δ-151.57–-151.64(m,2F),-158.48–-158.59(m,1F),-162.11–-162.21(m,2F).

[0082] HRMS calcd for C8H5F5O2PS2 + [M+H] + 322.9383 found 322.9385.

[0083] When the compound represented by formula 1a-1 used in step 1) is The final product obtained during preparation is as follows:

[0084] The compounds shown in Formula 1-2:

[0085]

[0086] 2-(p-tolyloxy)-1,3,2-oxathiaphospholane 2-sulfide

[0087] 1 H NMR (400MHz, CDCl3) δ7.16–7.13(m,2H),7.12–7.07(m,2H),4.57–4.40(m,2H),3.49–3.29(m,2H),2.33(d,J=1.8Hz,3H).

[0088] 13 C NMR (101MHz, CDCl3) δ148.7,148.6,135.62,135.60,130.3,130.2,121.44,121.39,71.2,71.1,36.80,36.78,21.0.

[0089] 31 P NMR (162MHz, CDCl3): δ 100.18.

[0090] HRMS calcd for C9H 12 O2PS2 + [M+H] + :247.0011found 247.0007.

[0091] When the compound represented by formula 1a-1 used in step 1) is The final product obtained is a compound represented by the following formulas 1-3:

[0092]

[0093] 2-phenoxy-1,3,2-oxathiaphospholane 2-sulfide

[0094] 1 H NMR (400MHz, CDCl3) δ7.39–7.34(m,2H),7.26–7.20(m,3H),4.58–4.42(m,2H),3.52–3.31(m,2H).

[0095] 13 C NMR (101MHz, CDCl3) δ150.9,150.8,129.77,129.75,125.91,125.89,121.8,121.7,71.18,71.16,36.80,36.78.

[0096] 31 P NMR (162MHz, CDCl3): δ 99.86.

[0097] HRMS calcd for C8H 10 O2PS2 + [M+H] + :232.9854found 232.9851.

[0098] When the compound represented by formula 1a-1 used in step 1) is The final product obtained is a compound represented by the following formulas 1-4:

[0099]

[0100] 2-(4-fluorophenoxy)-1,3,2-oxathiaphospholane 2-sulfide

[0101] 1 H NMR (400MHz, CDCl3) δ7.21–7.15(m,2H),7.08–7.02(m,2H),4.61–4.44(m,2H),3.54–3.36(m,2H).

[0102] 13 C NMR (101MHz, CDCl3) δ160.2 (dd, J=244.7, 2.8Hz), 146.6 (dd, J=10.7, 2.8Hz), 12 3.1(dd,J=8.4,4.7Hz),116.3(dd,J=23.6,2.2Hz),71.13,71.11,36.71,36.69.

[0103] 31 P NMR (162MHz, CDCl3): δ100.62 (d, J=4.8Hz).

[0104] 19 F NMR (202MHz, CDCl3): δ-116.60 (d, J = 4.1Hz)

[0105] HRMS calcd.for C8H9FO2PS2 + [M+H] + :250.9760found 250.9760.

[0106] When the compound represented by formula 1a-1 used in step 1) is The final product obtained during preparation is as follows:

[0107] The compounds shown in Formulas 1-5:

[0108]

[0109] 4-((2-sulfido-1,3,2-oxathiaphospholan-2-yl)oxy)benzonitrile

[0110] 1 H NMR (400MHz, CDCl3) δ7.69–7.66(m,2H),7.34–7.30(m,2H),4.66–4.48(m,2H),3.62–3.49(m,2H).

[0111] 13 C NMR (101MHz, CDCl3) δ154.0,153.9,134.01,134.00,122.71,122.66,118.19,118.18,109.70,109.67,71.4,71.3,36.82,36.80.

[0112] 31 P NMR (162MHz, CDCl3): δ 99.73.

[0113] HRMS calcd for C9H9NO2PS2 + [M+H] + 257.9807 found 257.9807.

[0114] When the compound represented by formula 1a-1 used in step 1) is The final product obtained is a compound represented by the following formulas 1-6:

[0115]

[0116] 2-(3,5-difluorophenoxy)-1,3,2-oxathiaphospholane 2-sulfide

[0117] 1 H NMR (400MHz, CDCl3) δ6.83–6.77(m,2H),6.74–6.68(m,1H),4.65–4.47(m,2H),3.61–3.48(m,2H).

[0118] 13 C NMR (101MHz, CDCl3) δ164.3 (dd, J=14.8, 2.0Hz), 161.9 (dd, J=14.8, 2.1Hz), 152.1–1 51.7(m),106.1–105.8(m),164.33(dd,J=14.8,2.0Hz),71.34,71.31,36.84,36.82.

[0119] 31 P NMR (162MHz, CDCl3): δ 99.81.

[0120] 19 F NMR (376MHz, CDCl3): δ-107.80 (d, J=2.0Hz)

[0121] HRMS calcd for C8H8F2O2PS2 + [M+H] + 268.9666 found 268.9667.

[0122] When the compound represented by formula 1a-1 used in step 1) is The final product obtained is a compound represented by the following formulas 1-7:

[0123]

[0124] 2-(3,5-bis(trifluoromethyl)phenoxy)-1,3,2-oxathiaphospholane 2-sulfide

[0125] 1 H NMR (400MHz, CDCl3) δ7.75(s,1H),7.67(s,2H),4.71–4.51(m,2H),3.64–3.58(m,2H).

[0126] 13C NMR (101MHz, CDCl3) 151.3 (d, J = 9.3Hz), 133.7–132.7 (m), 122.8 (q, J = 274.7Hz), 122.6–122.5 (m), 119.7–119.5 (m), 71.51, 71.49, 36.90, 36.88.

[0127] 31 P NMR (162MHz, CDCl3): δ 10 ≤ 0.74 ...

[0128] 19 F NMR (376MHz, CDCl3): δ-62.93.

[0129] HRMS calcd.for C 10 H8F6O2PS2 + [M+H] + 368.9602 found 368.9606.

[0130] When the compound represented by formula 1a-1 used in step 1) is The final product obtained during preparation is as follows:

[0131] The compounds shown in Formulas 1-8:

[0132]

[0133] 2-(2,4,6-tribromophenoxy)-1,3,2-oxathiaphospholane 2-sulfide

[0134] 1 H NMR (400MHz, CDCl3) δ7.71 (d, J = 0.9Hz, 2H), 4.69–4.55 (m, 2H), 3.74–3.60 (m, 2H).

[0135] 13 C NMR (101MHz, CDCl3)147.1,147.0,135.0,134.9,119.12,119.09,119.07,70.54,70.54,37.04,37.03.

[0136] 31 P NMR (162MHz, CDCl3): δ 101.60.

[0137] HRMS calcd for C8H7Br3O2PS2 + [M+H] +:466.7170found 466.7171.

[0138] When the compound represented by formula 1a-1 used in step 1) is The final product obtained is a compound represented by the following formulas 1-9:

[0139]

[0140] 2-(2-nitrophenoxy)-1,3,2-oxathiaphospholane 2-sulfide

[0141] 1 H NMR (400MHz, CDCl3) δ7.98 (d, J = 8.1Hz, 1H), 7.64–7.59 (m, 1H), 7.53–7.51 (m, 1H), 7.38–7.33 (m, 1H), 4.70–4.49 (m, 2H), 3.68–3.54 (m, 2H).

[0142] 13 C NMR (101MHz, CDCl3)143.6,143.5,142.2,134.43,134.41,126.2,126.1,125.94,125.92,124.83,124.79,71.39,71.37,36.87,36.85.

[0143] 31 P NMR (162MHz, CDCl3): δ 102.04.

[0144] HRMS calcd for C8H9NO4PS2 + [M+H] + 277.9705 found 277.9703.

[0145] When the compound represented by formula 1a-1 used in step 1) is The final product obtained during preparation is as follows:

[0146] The compounds shown in Formula 1-10:

[0147]

[0148] 2-(4-nitrophenoxy)-1,3,2-oxathiaphospholane 2-sulfide

[0149] 1H NMR (400MHz, CDCl3) δ8.27(d,J=8.8Hz,2H),7.37(d,J=8.5Hz,2H),4.70–4.51(m,2H),3.66–3.56(m,2H).

[0150] 13 C NMR (101MHz, CDCl3)155.5,155.4,145.3,125.63,125.61,122.43,122.38,71.5,71.4,36.89,36.88.

[0151] 31 P NMR (162MHz, CDCl3): δ 99.73.

[0152] HRMS calcd for C8H9NO4PS2 + [M+H] + 277.9705 found 277.9703.

[0153] When the compound represented by formula 1a-1 used in step 1) is The final product obtained during preparation is as follows:

[0154] The compounds shown in Formula 1-11:

[0155]

[0156] 2-((4-bromophenyl)thio)-1,3,2-oxathiaphospholane 2-sulfide

[0157] 1 H NMR (400MHz, CDCl3) δ7.56–7.47(m,4H),4.45–4.35(m,1H),4.26–4.15(m,1H),3.33–3.24(m,1H),2.73–2.65(m,1H).

[0158] 13 C NMR (101MHz, CDCl3)137.9,137.8,132.8,132.7,128.2,128.1,125.4,125.3,71.72,71.68,37.8.

[0159] 31 P NMR (162MHz, CDCl3): δ 111.86.

[0160] HRMS calcd for C8H8BrNaOPS3 + [M+Na] + 348.8550found 348.8551.

[0161] When the compound represented by formula 1a-1 used in step 1) is The final product obtained is a compound represented by the following formula 1-12:

[0162]

[0163] 2-((perfluorophenyl)thio)-1,3,2-oxathiaphospholane 2-sulfide

[0164] 1 H NMR (400MHz, CD3CN) δ7.56–7.47(m,4H),4.61–4.51(m,1H),4.48–4.34(m,1H),3.68–3.53(m,1H),3.33–3.23(m,1H).

[0165] 31 P NMR (162MHz, CD3CN): δ 109.6.

[0166] HRMS calcd for C8H4F5NaOPS3 + [M+Na] + 360.8974 found 360.8976.

[0167] Example 2: Preparation of compound of formula 2

[0168] This embodiment provides a series of methods for preparing compounds of formula 2, and the reaction routes are as follows:

[0169]

[0170] The specific steps are as follows:

[0171] 1) At 0 °C, imidazole (1.28 g, 18.6 mmol) and TBSCl (2.25 g, 14.9 mmol) were added to a DMF (23 mL) solution of the compound shown in Formula 2a-1 (12.5 mmol). After returning to room temperature and stirring for 8 h, water (120 mL) and ethyl acetate (100 mL) were added. The aqueous phase was extracted three times with ethyl acetate, the organic layers were combined, washed with saturated brine (10 mL), dried over anhydrous sodium sulfate, filtered and concentrated under vacuum, and purified by column chromatography to obtain the TBS protected intermediate compound.

[0172] 2) The TBS-protected intermediate compound (2.13 mmol) was dissolved in ultra-dry pyridine (15 mL) under an inert atmosphere, followed by the addition of DMTrCl (2.20 g, 6.49 mmol). After stirring at room temperature for 12 h, the reaction was complete by TLC. The solvent was removed, and the compound was purified by column chromatography to obtain the compound shown in formula 2a-2.

[0173] 3) The compound shown in Formula 2a-2 (22.7 mmol) was dissolved in anhydrous THF (40 mL), and TBAF (34.1 mL, 1 M in THF, 34.1 mmol) was added. The mixture was stirred at room temperature for 1 h, and THF was removed under reduced pressure. The crude product was dissolved in DCM, washed with water, and the organic layer was dried over anhydrous sodium sulfate and concentrated. The solid product, shown in Formula 2, was purified by column chromatography. When R2 in the compound shown in Formula 2a-1 used in step 1) is hydrogen, the final product obtained is the compound shown in Formula 2-1:

[0174]

[0175] 1-((2R,4S,5R)-4-(bis(4-methoxyphenyl)(phenyl)methoxy)-5-(hydroxymethyl)tetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione

[0176] 1 H NMR(500MHz, CDCl3)δ9.53(s,1H),7.56(d,J=8.1Hz,1H),7.45–7.43(m,2H),7.35–7.25(m,6H),7.23–7.19(m,1H),6.84–6.81(m,4H),6.22(dd,J=8 .0,6.4Hz,1H),5.61(d,J=8.1Hz,1H),4.36–4.34(m,1H),3.93–3.91(m,1 H),3.76(s,6H),3.63–3.60(m,1H),3.31–3.28(m,1H),1.83–1.79(m,2H).

[0177] 13C NMR(101MHz, CDCl3)163.8,158.7,150.4,145.1,141.4,136.31,136.27,130.3,130.2 ,128.3,128.0,127.1,113.4,113.3,102.4,87.3,86.9,86.8,74.4,62.4,55.3,39.3.

[0178] HRMS calcd.for C 30 H 30 N2NaO7 + [M+Na] + :553.1945found 553.1946.

[0179] When R2 is a methyl group in the compound represented by formula 2a-1 used in step 1), the final product obtained is as follows:

[0180] The compound shown in Formula 2-2:

[0181]

[0182] 1-((2R,4S,5R)-4-(bis(4-methoxyphenyl)(phenyl)methoxy)-5-(hydroxymethyl)tetrahydrofuran-2-yl)-5-methylpyrimidine-2,4(1H,3H)-dione

[0183] 1 H NMR(400MHz, Acetone-d6)δ10.00(s,1H),7.71–7.70(m,1H),7.53–7.50(m,2H) ),7.40–7.31(m,6H),7.26–7.22(m,1H),6.93–6.89(m,4H),6.37–6.33(m,1H) ,4.43(d,J=5.6Hz,1H),3.92–3.91(m,1H),3.78–3.77(m,6H),3.60–3.55(m,1 H),3.41–3.38(m,1H),1.87(ddd,J=13.1,9.3,5.8Hz,1H),1.75–1.70(m,4H).

[0184] 13C NMR (126MHz, Acetone-d6)164.4,159.9,151.6,146.7,137.5,137.2,131.3,129.3,1 28.9,127.9,114.23,114.21,110.9,88.2,87.7,86.0,76.2,63.2,55.7,40.0,12.7.

[0185] HRMS calcd.for C 31 H 32 N2NaO7 + [M+Na] + :567.2102found 567.2102.

[0186] When R2 is fluorine in the compound represented by formula 2a-1 used in step 1), the final product obtained is the compound represented by formula 2-3 below:

[0187]

[0188] 1-((2R,4S,5R)-4-(bis(4-methoxyphenyl)(phenyl)methoxy)-5-(hydroxymethyl)tetrahydrofuran-2-yl)-5-fluoropyrimidine-2,4(1H,3H)-dione

[0189] 1 H NMR(500MHz, Acetone-d6)δ8.13(dd,J=7.1,1.5Hz,1H),7.53–7.50(m,2H),7.40–7.32(m,6H),7.26–7.23(m,1H),6.93–6.90(m,4H), 6.38–6.34(m,1H),4.45–4.43(m,1H),3.90–3.88(m,1H),3.79–3.78(m,6H),3.60–3.57(m,1H),3.42–3.39(m,1H),1.88–1.85(m,2H).

[0190] 13C NMR(126MHz, Acetone-d6)159.9,157.7(d,J=27.5Hz),150.0,146.7,141.5(d,J=231.6Hz),137.4, 131.3,129.3,128.9,127.9,125.4(d,J=35.4Hz),114.2,88.2,88.0,86.4,76.3,63.1,55.7,40.5.

[0191] 19 F NMR (376MHz, DMSO): δ-166.56.

[0192] HRMS calcd.for C 30 H 29 FN2NaO7 + [M+Na] + :571.1851found 571.1849.

[0193] When R2 is bromine in the compound represented by formula 2a-1 used in step 1), the final product obtained is the compound represented by formula 2-4:

[0194]

[0195] 1-((2R,4S,5R)-4-(bis(4-methoxyphenyl)(phenyl)methoxy)-5-(hydroxymethyl)tetrahydrofuran-2-yl)-5-bromopyrimidine-2,4(1H,3H)-dione

[0196] 1 H NMR (500MHz, Acetone-d6) δ10.40(s,1H),8.35(s,1H),7.52–7.50(m,2H),7.40–7.32(m,6H),7.26–7.23(m,1H),6.93–6.90(m,4H),6.36–6. 33(m,1H),4.45–4.43(m,1H),3.91–3.90(m,1H),3.78(s,6H),3.58(dd,J=11.8,2.5Hz,1H),3.41(dd,J=11.9,2.5Hz,1H),1.90–1.88(m,2H).

[0197] 13C NMR (126MHz, Acetone-d6)160.0,159.6,150.8,146.7,141.3,137.4,131.3, 129.3,128.9,127.9,114.3,97.0,88.2,88.1,86.7,76.3,63.0,55.7,40.8.

[0198] HRMS calcd.for C 30 H 29 BrN2NaO7 + [M+Na] + :631.1050found 631.1051.

[0199] When R2 is iodine in the compound represented by formula 2a-1 used in step 1), the final product obtained is the compound represented by formula 2-5:

[0200]

[0201] 1-((2R,4S,5R)-4-(bis(4-methoxyphenyl)(phenyl)methoxy)-5-(hydroxymethyl)tetrahydrofuran-2-yl)-5-iodopyrimidine-2,4(1H,3H)-dione

[0202] 1 H NMR(500MHz, Acetone-d6)δ8.41(s,1H),7.51(d,J=7.8Hz,2H),7.40–7.32(m,6H),7.24(t,J=7.2Hz,1H),6.92–6.90(m,4H),6 .35–6.31(m,1H),4.45–4.44(m,1H),3.93–3.92(m,1H),3.78(s,6H),3.61–3.57(m,1H),3.43–3.39(m,1H),1.90–1.87(m,2H). 13 C NMR (126MHz, Acetone-d6)160.8,160.0,151.2,146.7,146.49,146.46,137.4,131.3 ,129.3,128.9,127.9,114.3,88.2,88.1,86.6,76.3,68.8,63.0,55.74,55.71,40.8.

[0203] HRMS calcd.for C 30 H29 IN2NaO7 + [M+Na] + :679.0912found 679.0909.

[0204] Example 3: Preparation of Compound 3

[0205] This embodiment provides a series of methods for preparing compounds shown in Formula 3 (e.g. Figure 1 As shown), the reaction route is as follows:

[0206]

[0207] The specific steps are as follows:

[0208] Catalyst C1 (20 mol%), the compound of Formula 1 (2.0 equiv.), and the compound of Formula 2 (0.1 mmol, 1.0 equiv.) were added to a reaction flask. Anhydrous PhMe (0.1 M) was added under nitrogen protection, and the reaction was carried out at 0 °C for 72 h. Toluene was removed by rotary evaporation, and the compound of Formula 3 was purified by column chromatography (using a thin-layer chromatography plate prepared with EtOAc / PE (4:1 (v / v))). The dr value was determined at 40 °C using a Daicel Chiralpak IE column. The yields reported here refer to the isolated yields. Using the compound of Formula 1-1 (64.4 mg, 0.2 mmol, 2.0 equiv.) and the compound of Formula 2-1 (53 mg, 0.1 mmol, 1.0 equiv.), the following compound of Formula 3-1 (38.1 mg, yield 57%, dr = 97:3) was prepared:

[0209]

[0210] 1-((2R,4S,5R)-4-(bis(4-methoxyphenyl)(phenyl)methoxy)-5-((((S)-2-sulfido-1,3,2-oxathiaphospholan-2-yl)oxy)methyl)tetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione

[0211] Physical state: White solid.

[0212] [α] 25 D = +9.6 (c = 1.3, CH3OH).

[0213] 1H NMR(500MHz,CDCl3):δ9.26(s,1H),7.52(d,J=8.2Hz,1H),7.45(dd,J=8.4,1.3Hz,2H),7.36–7.30(m,6H),7.26–7.22(m,1H),6.87–6.85(m,4H),6.40(dd,J=8.8,5.6Hz,1H),5.69(dd,J=8.3,2.0Hz,1H),4.38–4.31(m,3H),3.90–3.86(m,2H),3.80(s,6H),3.65–3.60(m,1H),3.48–3.33(m,2H),2.19(ddd,J=13.7,5.6,1.4Hz,1H),1.71(ddd,J=13.7,8.9,6.3Hz,1H).

[0214] 13 C NMR(126MHz,CDCl3):δ163.4,158.9,150.4,145.0,139.8,136.1,136.0,130.34,130.31,128.3,128.2,127.3,113.6,102.6,87.7,85.8,84.63,84.56,74.5,70.8,68.03,67.97,55.4,39.9,36.8.

[0215] 31 P NMR(162MHz,Acetone-d6):δ104.30.

[0216] HRMS calcd.for C 32 H 33 N2NaO8PS2 + [M+Na] + :691.1308found 691.1306.

[0217] HPLC(Daicel Chiralpak ADH Column,n-Hexane / i-PrOH=50 / 50,1.0mL / min,40℃,254nm),t R =17.746min(major):t R =22.334min(minor),97:3d.r.

[0218] Using the compound shown in Formula 1-1 (64.4 mg, 0.2 mmol, 2.0 equiv.) and the compound shown in Formula 2-3 (54.8 mg, 0.1 mmol, 1.0 equiv.), the following compound shown in Formula 3-2 (56.9 mg, yield 83%, dr = 99:1) was prepared:

[0219]

[0220] 1-((2R,4S,5R)-4-(bis(4-methoxyphenyl)(phenyl)methoxy)-5-((((S)-2-sulfido-1,3,2-oxathiaphospholan-2-yl)oxy)methyl)tetrahydrofuran-2-yl)-5-fluoropyrimidine-2,4(1H,3H)-dione

[0221] Physical state: White solid.

[0222] [α] 25 D = +16.4 (c = 1.1, CH3OH).

[0223] 1 H NMR (500MHz, Acetone-d6): δ10.48(s,1H),7.70(d,J=6.9Hz,1H),7.53–7.51(m,2H),7.42–7.39(m,4 H),7.36–7.33(m,2H),7.27–7.24(m,1H),6.95–6.91(m,4H),6.37(ddd,J=9.1,5.6,1.9Hz,1H),4.58– 4.50(m,1H),4.44–4.35(m,2H),4.06–4.02(m,1H),3.97(ddd,J=11.4,7.0,2.7Hz,1H),3.90–3.85(m ,1H),3.80–3.79(m,6H),3.68–3.61(m,1H),3.58–3.52(m,1H),2.05–2.01(m,1H),1.88–1.83(m,1H).

[0224] 13C NMR (101MHz, CDCl3): δ160.1, 157.6 (d, J = 26.7Hz), 149.9, 146.5, 140.9 (d, J = 252.1Hz), 137.2, 137.1, 131.4, 131.3, 129.2, 129. 0,128.0,124.6(d,J=34.3Hz),114.41,114.39,88.5,86.5,85.4,85.3,75.7,72.54,72.53,68.9,68.8,55.7,39.8,37.52,37.50.

[0225] 31 P NMR (202MHz, CDCl3): δ104.79,104.49.

[0226] 19 F NMR (376MHz, Acetone-d6): δ-167.66.

[0227] HRMS calcd.for C 32 H 32 FN2NaO8PS2 + [M+Na] + 709.1214 found 709.1212.

[0228] HPLC (Daicel Chiralpak ADH Column, n-Hexane / i-PrOH=50 / 50, 1.0mL / min, 40 ℃ ,254nm),t R =15.306min(major):t R =17.014min(minor),99:1d.r.

[0229] When using the compound shown in Formula 1-1 (64.4 mg, 0.2 mmol, 2.0 equiv.) and the compound shown in Formula 2-4 (60.8 mg, 0.1 mmol, 1.0 equiv.), the following compound (58.2 mg, yield 78%, dr = 99:1) was prepared:

[0230]

[0231] 1-((2R,4S,5R)-4-(bis(4-methoxyphenyl)(phenyl)methoxy)-5-((((S)-2-sulfido-1,3,2-oxathiaphospholan-2-yl)oxy)methyl)tetrahydrofuran-2-yl)-5-bromopyrimidine-2,4(1H,3H)-dione

[0232] Physical state: white solid.

[0233] [α] 25 D = -12.5 (c = 0.7, CH3OH).

[0234] 1 H NMR (400 MHz, Acetonitrile-d6): δ 9.47 (s, 1H), 7.73 (s, 1H), 7.48–7.46 (m, 2H), 7.37–7.31 (m, 6H), 7.27–7.23 (m, 1H), 6.90–6.88 (m, 4H), 6.19 (dd, J = 8.8, 5.6 Hz, 1H), 4.50–4.27 (m, 3H), 3.95–3.74 (m, 9H), 3.56–3.43 (m, 2H), 1.97–1.96 (m, 1H), 1.67 (ddd, J = 14.3, 8.9, 6.3 Hz, 1H).

[0235] 13 C NMR (101 MHz, Acetonitrile-d6): δ 159.6, 150.3, 146.0, 139.9, 136.8, 136.6, 130.9, 130.8, 128.72, 128.66, 127.7, 114.03, 114.02, 97.0, 88.0, 86.4, 84.9, 84.8, 75.1, 72.3, 68.4, 55.6, 39.7, 37.3.

[0236] 31 P NMR (202 MHz, CDCl3): δ 104.97, 104.79.

[0237] HRMS calcd. for C 32 H 32 BrN2NaO8PS2 + [M+Na] + : 769.0413 found 769.0411.

[0238] HPLC (Daicel Chiralpak IBN Column, n-Hexane / i-PrOH=40 / 60, 1.0mL / min, 40℃, 254nm), t R =33.222min(minor):t R =37.508min(major),99:1d.r.

[0239] When using the compound shown in Formula 1-1 (64.4 mg, 0.2 mmol, 2.0 equiv.) and the compound shown in Formula 2-5 (65.6 mg, 0.1 mmol, 1.0 equiv.), the following compound (65.9 mg, yield 83%, dr = 98:2) was prepared:

[0240]

[0241] 1-((2R,4S,5R)-4-(bis(4-methoxyphenyl)(phenyl)methoxy)-5-((((S)-2-sulfido-1,3,2-oxathiaphospholan-2-yl)oxy)methyl)tetrahydrofuran-2-yl)-5-iodopyrimidine-2,4(1H,3H)-dionecompound was obtained from 2-(perfluorophenoxy)-1,3,2-oxathiaphospholane 2-sulfide(64.4mg,0.2mmol,2.0equiv.)and1-((2R,4S,5R)-4-(bis(4-methoxyphenyl)(phenyl)methoxy)-5-(hy droxymethyl)tetrahydrofuran-2-yl)-5-iodopyrimidine-2,4(1H,3H)-dione(65.6mg,0.1mmol,1.0equiv.).The residue was purified by prepared thin layerchromatography plate eluting with EtOAc / PE(4:1(v / v))toafford 65.9mg of the title compound. (Yield=83%, dr=98:2).

[0242] Physical state: Colorless oil.

[0243] [α] 25 D =-26.1(c=1.0,CH3OH).

[0244] 1 H NMR(400MHz,Acetonitrile-d6):δ9.45(s,1H),7.78(s,1H),7.48–7.45(m,2H),7.37–7.30(m,6H),7.27–7.22(m,1H),6.91–6.87(m,4H),6.18(dd,J=8.9,5.5Hz,1H),4.52–4.41(m,1H),4.37–4.28(m,2H),3.95–3.75(m,9H),3.53–3.46(m,2H),1.93–1.90(m,1H),1.66(ddd,J=13.9,8.9,6.2Hz,1H).

[0245] 13 C NMR(101MHz,Acetone-d6):δ160.7,160.0,151.0,146.5,145.2,137.2,137.1,131.3,129.2,129.0,128.0,114.39,114.37,88.5,86.6,85.4,85.3,75.7,72.5,69.5,68.82,68.75,55.7,40.2,37.7.

[0246] 31 P NMR(202MHz,CDCl3):δ104.94,104.77.

[0247] HRMS calcd.for C 32 H 32 IN2NaO8PS2 + [M+Na] + :817.0275found 817.0275.

[0248] HPLC(Daicel Chiralpak IBN Column,n-Hexane / i-PrOH=40 / 60,1.0mL / min,40℃,254

[0249] nm),t R =41.394min(minor):t R =50.816min(major),98:2d.r.

[0250] When using the compound shown in Formula 1-1 (64.4 mg, 0.2 mmol, 2.0 equiv.) and the compound shown in Formula 2-2 (65.6 mg, 0.1 mmol, 1.0 equiv.), the following compound (54.5 mg, yield 74%, dr = 97:3) was prepared:

[0251]

[0252] 1-((2R,4S,5R)-4-(bis(4-methoxyphenyl)(phenyl)methoxy)-5-((((S)-2-sulfido-1,3,2-oxathiaphospholan-2-yl)oxy)methyl)tetrahydrofuran-2-yl)-5-methylpyrimidine-2,4(1H,3H)-dione

[0253] Physical state: White solid.

[0254] 1 H NMR (400MHz, Acetone-d6): δ10.04(s,1H),7.54–7.52(m,2H),7.43–7.39(m,5H) ,7.37–7.33(m,2H),7.27–7.23(m,1H),6.94–6.92(m,4H),6.41(dd,J=9.2,5.5Hz ,1H),4.59–4.48(m,1H),4.45–4.34(m,2H),4.02–3.94(m,2H),3.84–3.78(m,7H) ,3.67–3.52(m,2H),2.01–1.96(m,1H),1.90–1.84(m,1H),1.82(d,J=1.0Hz,3H).

[0255] 13 C NMR (101MHz, Acetone-d6): δ164.0,159.7,151.1,146.2,136.9,136.8,135.8,131.02,131.01,128.9,128.7,127. 7,114.07,114.05,111.1,88.1,85.6,84.7,84.6,75.4,72.10,72.08,68.8,68.7,55.4,39.3,37.43,37.41,12.5.

[0256] 31P NMR (162MHz, Acetone-d6): δ104.38,103.99.

[0257] HRMS calcd.for C 33 H 35 N2NaO8PS2 + [M+Na] + 705.1465 found 705.1467.

[0258] HPLC (Daicel Chiralpak IBN Column, n-Hexane / i-PrOH=40 / 60, 1.0mL / min, 40℃, 254nm), t R =31.579min(minor):t R =35.888min(major),97:3d.r.

[0259] Example 4: Preparation of Compound 3

[0260] This embodiment provides a method for preparing the compounds shown in Formulas 3-5, and the reaction route is as follows:

[0261]

[0262] The specific preparation steps are basically the same as those for the compounds shown in Formulas 3-5 in Example 3, except that the compounds shown in Formula 1-1 are replaced by the compounds shown in Formula 1-5 to Formula 1-8 and Formula 1-12 to Formula 1-15 shown in Table 1; wherein, Formulas 1-5 to Formula 1-8 and Formula 1-12 are the compounds prepared in Example 1, and the structures of the compounds shown in Formulas 1-13 to Formula 1-15 are as follows:

[0263]

[0264] The compounds shown in Formulas 1-13 to 1-15 were prepared using the same method as in Example 1, except that the compounds of Formula 1a used were respectively...

[0265] The yields and dr results of the compounds of formulas 3-5 prepared using compounds of formula 1 are shown in Table 1.

[0266] Table 1

[0267]

[0268]

[0269] Example 4

[0270] This embodiment provides a method for preparing compound 3-1. The specific steps are the same as in Example 3, except that: in experimental groups 1-4, the catalyst shown in Table 2 is used instead of the C1 catalyst; in experimental groups 5-8, the solvent shown in Table 2 is used instead of toluene; and in experimental groups 9-10, the reaction temperature shown in Table 2 is used instead of 0℃.

[0271] Table 2

[0272]

[0273]

[0274] As can be seen from the results in Table 2, using other catalysts, solvents, and reaction temperatures all led to a decrease in yield and / or dr value, or even no reaction.

[0275] Functional verification of pentavalent oxythiophosphine monomers:

[0276] Verification Example 1

[0277] This verification example provides a method for preparing optically pure PS cyclic dinucleotides, the specific reaction route of which is as follows: Figure 2 As shown. The specific preparation steps are as follows:

[0278] 1) Add the compound shown in Formula 4 (142.4 mg, 0.4 mmol, 2.0 equiv.), the compound shown in Formulas 3-5 (136.4 mg, 0.2 mmol, 1.0 equiv.), DBU (0.3 mL, 2.0 mmol, 2.0 equiv.), and acetonitrile (10 mL) to a 20 mL round-bottom flask equipped with a magnetic magnet. After stirring at room temperature for 30 min, purify by column chromatography (MeOH / DCM (6:1 (v / v) thin-layer chromatography plate) to obtain the desired product, the compound shown in Formula 5 (113.4 mg, yield 58%, dr = 97:3).

[0279]

[0280] O-((2R,3S,5R)-2-(((tert-butyldimethylsilyl)oxy)methyl)-5-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)tetrahydrofuran-3-yl)O-hydrogen(S)-phosphorothioate

[0281] Physical state: White solid.

[0282] [α] 25D =+2.9(c=0.3,CH3OH).

[0283] 1 H NMR(400MHz,DMSO):δ7.82(s,1H),7.45(s,1H),7.41–7.39(m,2H),7.34–7.27(m,6H),7.22(t,J=7.3Hz,1H),6.92–6.88(m,4H),6.29(dd,J=9.8,5.4Hz,1H),6.10(dd,J=9.0,5.5Hz,1H),4.80–4.76(m,1H),4.28(d,J=5.2Hz,1H),3.92–3.89(m,1H),3.73(s,6H),3.65–3.60(m,3H),3.42–3.37(m,2H),2.18(dd,J=13.0,5.5Hz,1H),1.96–1.87(m,2H),1.81–1.78(m,6H),1.68(dd,J=13.2,5.4Hz,1H),0.87(s,9H),0.06(d,J=3.6Hz,6H).

[0284] 13 C NMR(101MHz,CD3OD):δ163.9,163.8,158.4,158.3,150.7,150.5,145.3,136.1,136.09,136.0,135.2,130.0,129.9,128.1,127.9,127.0,113.5,110.3,109.5,86.8,85.6,84.7,84.6,84.2,83.8,75.5,63.6,55.1,40.2,39.9,39.8,39.6,39.4,39.0,38.7,38.2,25.9,18.1,12.4,12.2,-5.4.

[0285] 31 P NMR(202MHz,CD3OD):δ52.61.

[0286] HRMS calcd.for C 47 H 58 N4O 13 PSSi - [M-H] - :977.3233found 977.3238.

[0287] 2) Add TFA (116 mg, 1.0 mmol, 10 equiv.) to a DCM (10 mL) solution of the compound shown in Formula 5 (100 mg, 0.1 mmol) and stir overnight at room temperature. After completion, adjust the pH of the system to about 8 with TEA, concentrate under reduced pressure, purify by reversed-phase chromatography, collect the fraction containing the product, mix and concentrate to obtain the compound shown in Formula 6 as a white solid (40 mg, yield 78%).

[0288] 3) Add DBU (20 mg, 0.133 mmol, 15 equiv.) to a solution of compound 6 (5 mg, 0.0089 mmol, 1.0 equiv.) dissolved in DMF (1 mL), then slowly add a DMF (0.2 mL) solution of compound 7 (11.8 mg, 0.026 mmol, 3 equiv.) for 30 min. Stir at room temperature for 1 h, remove the solvent, dissolve the residue in methanol, and purify by reversed-phase chromatography using a 0.1% formic acid aqueous solution and acetonitrile elution system. Concentrate the fraction containing the product and freeze-dry to obtain an amorphous solid (S). p, R p )-CDNs (1.2mg, yield 21%, dr = 97:3).

[0289]

[0290] 1,1'-((2R,3aR,5R,7aR,9R,10aR,12S,14aR)-5,12-dimercapto-5,12-dioxido℃tahydro-2H,7H-difuro[3,2-d:3',2' -j][1,3,7,9]tetraoxa[2,8]diphosphacyclododecine-2,9-diyl)bis(5-methylpyrimidine-2,4(1H,3H)-dione)((S p ,R p CDNs

[0291] 31 P NMR (202MHz, CD3OD): δ 52.55.

[0292] HRMS calcd.for C 20 H 26 N4O 12 P2S2 - [MH] - 639.0386; found 639.0387.

[0293] LC-MS trace of Compound(Sp,Rp)-CDNs(Agilent EC-C18,H2O(0.1%formicacid) / CH3CN from 95 / 5to 30 / 70,0.3mL / min,25℃,254nm),t R = 3.491 min.

[0294] It should be noted that the other compounds of Formula 3 prepared in Example 3 of this invention can also be used to prepare corresponding optically pure PS cyclic dinucleotides, and will not be listed one by one in this invention.

[0295] Furthermore, the compounds of Formula 3 prepared in Example 3 of this invention can also be applied to the solid-phase synthesis of chiral PS and PS / PO mixed backbone oligonucleotides, which will not be listed in detail in this invention.

[0296] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the invention should be within the protection scope of the present invention.

Claims

1. An intermediate compound, characterized in that, The structure of the intermediate compound is shown in Formula 1 below: Where X is O or S, and R1 is one or more substituents on the benzene ring, each substituent being any one of hydrogen, alkyl, halogen, cyano, trifluoromethyl, or nitro.

2. A method for preparing the intermediate compound according to claim 1, characterized in that, Includes the following steps: The compound shown in Formula 1a-1 is reacted with phosphorus pentasulfide in the presence of a base, and the resulting compound shown in Formula 1a-2 is then cyclized with ethylene oxide in the presence of trifluoroacetic acid to obtain the intermediate compound shown in Formula 1. The structure of the compound of formula 1a-1 is as follows: Wherein: X is O or S; R1 is one or more substituents on the benzene ring, and the substituent is any one of hydrogen, methyl, fluorine, cyano, trifluoromethyl, bromine, or nitro. The structure of the compound of formula 1a-2 is as follows: Wherein: X is O or S, and R1 is any one of hydrogen, alkyl, halogen, cyano, trifluoromethyl, and nitro.

3. The method for preparing the intermediate compound according to claim 2, characterized in that, The molar ratio of the compound represented by Formula 1a-1 to phosphorus pentasulfide is 2:1; the molar ratio of the compound represented by Formula 1a-1 to the base is 2:2.1; The base is triethylamine; The molar ratio of the compound represented by Formula 1a-2 to ethylene oxide is 1:1.5; The molar ratio of the compound represented by Formula 1a-2 to trifluoroacetic acid is 1:1.

5.

4. An intermediate compound, characterized in that, The structure of the intermediate compound is shown in Formula 2 below: R2 can be any one of hydrogen, alkyl, or halogen.

5. A method for preparing the intermediate compound according to claim 4, characterized in that, Includes the following steps: After protecting the compound shown in Formula 2a-1 with TBSCl, attach the DMTr group to the compound shown in Formula 2a-2, and then remove the TBS protecting group from the compound to obtain the intermediate compound shown in Formula 2. The structure of the compound represented by Formula 2a-1 is as follows: Wherein: R2 is any one of hydrogen, alkyl, or halogen; The structure of the compound represented by formula 2a-2 is as follows: Wherein: R2 is any one of hydrogen, alkyl, or halogen.

6. The method for preparing the intermediate compound according to claim 5, characterized in that, The specific steps of the preparation method are as follows: A1. Imidazole and TBSCl were added to the compound shown in Formula 2a-1, and the reaction yielded a TBS protected intermediate compound. DMTTrCl was then added to the TBS protected intermediate compound, and the reaction yielded the compound shown in Formula 2a-2. A2. Add the compound shown in Formula 2a-2 to tetrabutylammonium fluoride to react and obtain the intermediate compound shown in Formula 2. The molar ratio of Formula 2a-1 to TBSCl is 1:1.2, and the molar ratio of Formula 2a-1 to imidazole is 1:1.

5. The molar ratio of Formula 2a-2 to tetrabutylammonium fluoride is 1:1.

5.

7. A pentavalent oxythiophosphine monomer, characterized in that, The structure of the pentavalent oxythiophosphine monomer is shown in Formula 3 below: R2 can be any one of hydrogen, alkyl, or halogen.

8. A method for preparing a pentavalent oxythiophosphine monomer according to claim 7, characterized in that, Includes the following steps: Under the catalysis of cyclohexanediamine catalyst, the intermediate compound of Formula 1 as described in claim 1 and the intermediate compound of Formula 2 as described in claim 4 undergo an asymmetric catalytic coupling reaction to generate a pentavalent oxythiophosphine monomer.

9. The method for preparing pentavalent oxythiophosphine monomer according to claim 8, characterized in that, The cyclohexanediamine catalyst has a compound structure having any of the following formulas (C1-C3): The molar ratio of the intermediate compound shown in Formula 1 to the intermediate compound shown in Formula 2 is 2-3:1; the amount of catalyst added is 10-20 mol% of the total molar amount of the reactants; The reaction is carried out in the presence of a solvent, wherein the solvent is at least one of toluene, fluorobenzene, anisole, and tetrahydrofuran; The reaction temperature is -10℃ to 40℃.

10. Use of the pentavalent oxythiophosphine monomer according to claim 7 in the preparation of PS cyclic dinucleotides or in the preparation of chiral PS and PS / PO mixed backbone oligonucleotides.

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