CN1709898A - Cyclic pyrophosphate compounds and preparation methods thereof - Google Patents

Abstract

This invention has disclosed a manufacturing method for circle pyrophosphate compound, the purpose is to offer the cADPR analogs with strong stability and high activity. This circle pyrophosphate pyrophosphoric acid compound has structure of general formula (I), thereinto, X is carbon or oxygen, Y is carbon or oxygen; Its preparation use N9-[( tertiary butyl dimethyl silicon base-oxygen-ethoxyl) -methyl]-hypoxanthine as raw materials. This cIDPRE is membrane permeability cADPR analog, it has simple and convenient synthesis method, little localization to substrate, result heavy; The result can send out fluorescence itself, the stability is good. This cADPR is a good pharmacology tool.

Description

Cyclic pyrophosphate compounds and preparation methods thereof

technical field

The invention relates to cyclic pyrophosphate compounds and a preparation method thereof.

Background technique

Cyclic adenosine diphosphate-ribose (cADPR) is an endogenous intracellular calcium ion agonist. Recent studies have shown that it can also act as a second messenger for intracellular signal transduction. Because cADPR is easy to acid and enzymatic hydrolysis in cells, there is an urgent need for cADPR analogs with strong stability and high activity as pharmacological tools to study the mechanism of cADPR in cells. Currently, there are three main methods for synthesizing cADPR analogues: enzymatic synthesis, biomimetic synthesis and chemical synthesis. Among them, enzymatic synthesis and biomimetic synthesis have relatively large limitations on substrates.

Contents of the invention

The object of the present invention is to provide a cyclic pyrophosphate compound with strong stability and high activity and a preparation method thereof.

The cyclic pyrophosphate compound provided by the present invention is a compound of general formula (I),

In the formula, X is C or O, and Y is C or O.

The preparation method of the cyclic pyrophosphate compound represented by the general formula (I) comprises the following steps: 1) N ⁹ -[(tert-butyldimethylsilyl-O-ethoxy)-methyl]-time Xanthine was dissolved in dichloromethane, after adding DBU, ethyl chloromethoxyacetate was added dropwise, stirred at room temperature for 0.5-1h, evaporated to dryness, and column chromatography gave N ¹ -[(acetyl-O-ethoxy )-methyl]-N ⁹ -(methyl-ethylene glycol)-hypoxanthine;

2) Dissolve N ¹ -[(acetyl-O-ethoxy)-methyl]-N ⁹ -[(tert-butyldimethylsilyl-O-ethoxy)-methyl]-hypoxanthine After THF, add the THF solution of TBAF, stir at room temperature for 1-3h, evaporate the solvent to dryness, and obtain N ¹ -[(acetyl-O-ethoxy)-methyl]-N ⁹ -(methyl- Ethylene glycol)-hypoxanthine;

3) Dissolve N ¹ -[(acetyl-O-ethoxy)-methyl]-N ⁹ -(methyl-ethylene glycol)-hypoxanthine in anhydrous pyridine, add 1-H tetrazole and (PhNH) ₂ POCl, stirred at room temperature for 44-52h, evaporated to dryness, and column chromatography gave N ¹ -[(acetyl-O-ethoxy)-methyl]-N ⁹ -[(diphenylamine phosphoryl-O -ethoxy)-methyl]-hypoxanthine;

4) Dissolve N ¹ -[(acetyl-O-ethoxy)-methyl]-N ⁹ -[(diphenylamine phosphoryl-O-ethoxy)-methyl]-hypoxanthine in anhydrous methanol Add sodium methoxide solution dropwise, stir at room temperature for 1-3h, filter and concentrate the filtrate to obtain N ¹ -[methyl-ethylene glycol]-N ⁹ -[(diphenylaminophosphoryl-O-ethoxy)-methanol Base]-hypoxanthine;

5) Dissolve N ¹ -[methyl-ethylene glycol]-N ⁹ -[(diphenylaminophosphoryl-O-ethoxy)-methyl]-hypoxanthine in anhydrous pyridine, add TPSCl (2 , 4,6-triisopropylbenzenesulfonyl chloride), 1-H tetrazole and PSS (S, S'-diphenylmercaptophosphorylcyclohexylamine salt), stirred at room temperature for 20-28h, evaporated to dryness, column layer N ¹ -[[(diphenylthio)phosphoryl-O-ethoxy]-methyl]-N ⁹ -[(diphenylaminophosphoryl-O-ethoxy)-methyl]-time xanthine;

6) N ¹ -[[(diphenylthio)phosphoryl-O-ethoxy]-methyl]-N ⁹ -[(diphenylaminophosphoryl-O-ethoxy)-methyl]- Dissolve hypoxanthine in pyridine-acetic acid-acetic anhydride, add isoamyl nitrite, stir at room temperature for 6-10 hours, evaporate the solvent and dissolve in anhydrous pyridine, add H ₃ PO ₂ and triethylamine and stir at room temperature for 10 hours -12h, evaporate the solvent to dryness, extract, column chromatography, and lyophilize to obtain N ¹ -[(phenylphosphorothioate-O-ethoxy)-methyl]-N ⁹ -[(phosphoryl-O- Triethylamine salt of ethoxy)-methyl]-hypoxanthine;

7) Put the activated 3 Å molecular sieve and I ₂ in the reaction flask, add anhydrous pyridine, stir at room temperature, and N ¹ -[(phenylphosphorothioate-O-ethoxy)-methyl] -N ⁹ -[(phosphoryl-O-ethoxy)-methyl]-hypoxanthine (formula (XII)) triethylamine salt in the above reaction flask, continue to stir for 1-3 hours, filter out molecular sieves, After extraction, column chromatography and lyophilization, N ¹ -[(phosphoryl-O-ethoxy)-methyl]-N ⁹ -[(phosphoryl-O-ethoxy)-methyl]-hypoxanthin was obtained Triethylamine salt of purine-cyclic pyrophosphate.

The N ⁹ -[(tert-butyldimethylsilyl-O-ethoxy)-methyl]-hypoxanthine is prepared according to the following steps: 1) Dissolving adenine in anhydrous pyridine, adding benzene Formyl chloride was refluxed for 1-2 hours, the solvent was evaporated to dryness, extracted, filtered and concentrated to obtain N ⁶ -benzoyl-adenine;

2) Dissolve N ⁶ -benzoyl-adenine in anhydrous 1,2 dichloroethane, add N, O-bistrimethylsilylacetamide (BSA), heat to 110-130°C, add Ethyl chloromethoxyacetate, stirred at 70-90°C for 1-2h, extracted and concentrated to obtain N ⁶ -benzoyl-N ⁹ -[(acetyl-O-ethoxy)-methyl]- adenine;

3) Dissolve N ⁶ -benzoyl-N ⁹ -[(acetyl-O-ethoxy)-methyl]-adenine in methanol-ammonia water, stir the reaction at room temperature for 20-28 hours, evaporate the solvent to dryness, and water Recrystallization to obtain N ⁹ -methyl ethylene glycol-adenine;

4) Dissolve N ⁹ -methylethylene glycol-adenine in glacial acetic acid, add sodium nitrite aqueous solution under stirring, stir at room temperature for 20-28 hours, evaporate the solvent to get N ⁹ -methylethylene glycol-hypoxanth Purine;

5) Dissolve N ⁹ -methylethylene glycol-hypoxanthine in anhydrous DMF, add imidazole and tert-butyldimethylsilyl chloride, stir at room temperature for 2-4 hours, precipitate, filter, and concentrate to obtain N ⁹ -[(tert-Butyldimethylsilyl-O-ethoxy)-methyl]-hypoxanthine.

The synthetic reaction formula of the present invention is as follows, the reaction formula (1) is the reaction formula of synthesizing N ⁹ -[(tert-butyldimethylsilyl-O-ethoxy)-methyl]-hypoxanthine from adenine; the reaction formula (2) Synthesis of N ¹ -[(phosphoryl-O-ethoxy)-methanol from N ⁹ -[(tert-butyldimethylsilyl-O-ethoxy)-methyl]-hypoxanthine Base]-N ⁹ -[(phosphoryl-O-ethoxy)-methyl]-hypoxanthine-cyclic pyrophosphate reaction formula.

Reagents: (a) BzCl, Py; (b) BSA, TMSOTf, DCE; (c) NH ₃ /CH ₃ OH; (d) NaNO ₂ , AcOH; (e) TBDMSCl, Imidazole, DMF.

Reagents: (a) DBU, ClCH ₂ CH ₂ OAC, CH ₂ Cl ₂ ; (b) TBAF, THF; (c) (PhNH) ₂ POCl, tetrazole, Py; (d) CH ₃ Ona, CH ₃ OH; ( e) PSS, TPSCl, tetrazole, Py; (f) i isoamyl nitrite, Py: ACOH: AC ₂ O (2: 1: 1), ii H ₃ PO ₂ , Et ₃ N, Py; (g) I ₂ , 3 Å MS, Py.

The cIDPRE of the present invention is a membrane-permeable cADPR analog, which has the characteristics of simple and convenient synthesis method, small limitation on the substrate, and large amount of synthetic product; the product itself can emit fluorescence, has good stability, and has the advantages of wide modification range, etc. cADPR mediates the release of calcium ions from intracellular calcium pools, and the intracellular calcium signal transduction involved in cADPR is an ideal pharmacological tool.

Detailed ways

Example 1, N ¹ -[(phosphoryl-O-ethoxy)-methyl]-N ⁹ -[(phosphoryl-O-ethoxy)-methyl]-hypoxanthine-cyclic pyrophosphate Synthesis of (cIDPDE, X=O in formula (I), Y=O)

All solvents, raw materials and reagents are analytically or chemically pure unless otherwise specified. The anhydrous treatment of the solvent is carried out according to a conventional method.

Instruments and methods for product separation and identification: thin-layer chromatography silica gel GF ₂₅₄ , column chromatography coarse-pore silica gel (200-300 mesh), and silica gel H are all produced by Qingdao Ocean Chemical Factory; Molybdic acid ethanol solution was used for color development; melting point was measured by XT-4A melting point apparatus, and the thermometer was not corrected; optical rotation was measured by Perkin-Elmer 243B polarimeter; infrared spectrum was measured by DE-983G infrared spectrometer, and potassium bromide was pressed into tablets; liquid for syrup Membrane method. FAB and MALDI-TOF were measured by VG-ZAB-HS and Bruker APEX ^TM II, HR-FAB was measured by Bruker BIFLEX ^TM III mass spectrometer; UV spectrum was measured by Pharmacia LKB Biochrom 4060 spectrophotometer. The nuclear magnetic resonance spectrum is determined by Varian VXR-500, JEOL AL300, Bruker Advance 300 nuclear magnetic resonance instrument, the hydrogen spectrum and carbon spectrum use TMS as the internal standard; the phosphorus spectrum uses 85% H ₃ PO ₄ as the external standard; the elemental analysis uses PE-240C Elemental analyzer determination. The syringe pump was model 74900 produced by Cole-Parmer Company. HPLC uses a Gilson high-performance liquid chromatograph and a Delter Paker C-18 semi-preparative column for separation.

1. Synthesis of N ⁶ -Benzyl-Adenosine (N ⁶ -Benzyl-Adenosine) (Formula (II))

Dissolve adenine (10g, 74mmol) in 50ml of anhydrous pyridine (Py), add dropwise benzoyl chloride (26g, 222mmol) under stirring, reflux for two hours after the dropwise addition, stop the reaction, evaporate the solvent to dryness, add saturated carbonic acid Sodium hydrogen was stirred for 30 min, the aqueous layer was extracted with chloroform, the organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated, and the resulting crude product was recrystallized with chloroform to obtain 14 g of white needle-like crystals (formula (II)), with a yield of 79%. , with a melting point of 248.5-249.5°C.

2. Synthesis of N ⁶ -benzoyl-N ⁹ -[(acetyl-O-ethoxy)-methyl]-adenine (formula (III))

N ⁶ -Benzyl-N ⁹ -[(Acetoxy-ethoxy)-methyl]-Adenosine

Dissolve N ⁶ -benzoyl-adenine (formula (II)) (5.9 g, 24.7 mmol) in 200 ml of anhydrous 1,2 dichloroethane, add N, O-bistrimethylsilylacetamide (BSA) (12.4ml, 49mmol), heated to 120°C, until the solid was completely dissolved (about 20min), added ethyl chloromethoxyacetate (4.9ml, 29.6mmol), stirred at 80°C for 1.5h, cooled to At room temperature, 200ml of ethyl acetate was added, washed with 100×2ml of saturated sodium bicarbonate, washed with 100ml of saturated brine, dried over anhydrous sodium sulfate, concentrated, and subjected to normal pressure column chromatography to obtain 3.1g of a white solid (formula (III)), the yield 35%.

¹ H NMR (300 MHz, DMSO) δ1.936 (s, 3H, CH ₃ CO-), 3.75-3.78 (m, 2H, H _5′ ), 4.08-4.11 (m, 2H, H _4′ ), 5.71 ( s, 2H, H _1' ), 7.53-7.68 (m, 5H, ArH), 8.64 (s, 1H, H ₂ ), 8.79 (s, 1H, H ₈ ), 11.24 (s, 1H, NHCO). Anal. Calcd for C ₁₇ H ₁₇ N ₅ O ₄ ·1/5 Et ₂ O: C 57.75, H 5.17, N 18.92; Found C 57.20, H 4.94, N 18.92.

The data indicated that the synthesized product was correct.

3. Synthesis of N ⁹ -methyl glycol-adenine (N ⁹ -(methyl-glycol)-Adenosine) (formula (IV))

N ⁶ -benzoyl-N ⁹ -[(acetyl-O-ethoxy)-methyl]-adenine (formula (III)) (3.1 g, 8.7 mmol) was dissolved in 300 ml of methanol-ammonia (v: v=1:1), stirred at room temperature for 24 h, evaporated the solvent, and recrystallized with water to obtain 2.1 g of a white solid (formula (IV)), with a yield of 99%.

¹ H NMR (300MHz, DMSO) δ3.44-3.50 (m, 4H, H _4' , H _5' ), 4.67 (t, 1H, J _{OH, CH2} = 5.1, OH), 5.55 (s, 2H, H _1' ), 7.28 (s, 2H, NH ₂ ), 8.17 (s, 1H, H ₂ ), 8.27 (s, 1H, H ₈ ).

The data indicated that the synthesized product was correct.

4. Synthesis of N ⁹ -methylglycol-hypoxantine (N ⁹ -(methyl-glycol)-Hypoxantine) (Formula (V))

Dissolve N ⁹ -methylethylene glycol-adenine (formula (IV)) (2.1g, 8.6mmol) in 140ml glacial acetic acid, add sodium nitrite under stirring to obtain an aqueous solution (5g+40mlH ₂ O), and emit reddish-brown gas, stirred at room temperature for 24 h, evaporated to dryness, and recrystallized with water to obtain 1.5 g of light yellow crystals (formula (V)), with a yield of 71%.

¹ H NMR (300MHz, DMSO) δ3.44-3.50(m, 4H, H _4′ , H _5′ ), 4.67(s, 1H, OH), 5.55(s, 2H, H _1′ ), 8.07(s , 1H, H ₂ ), 8.21 (s, 1H, H ₈ ), 12.35 (s, 1H, NH). Anal. Calcd for _C8H10N4O3 · ₁ / _2H2O : C _43.84 , H 5.06, N 25.56; Found: C _, 44.43; H, 4.774; N, 25.99.

The data indicated that the synthesized product was correct.

5. Synthesis of N ⁹ -[(tert-butyldimethylsilyl-O-ethoxy)-methyl]-hypoxanthine (formula (VI))

N ⁹ -[(TBDMS-O-ethoxy)-methyl]-Hypoxantine

Dissolve N ⁹ -methylethylene glycol-hypoxanthine (formula (V)) (942 mg, 3.86 mmol) in 25 ml of anhydrous N, N-dimethylformamide (DMF), add imidazole (806 mg, 5.79 mmol) and tert-butyldimethylsilyl chloride (TBDMSCl) (874mg, 5.79mmol), stirred at room temperature for 3h, stopped stirring, added 30ml H ₂ O, filtered, washed the filter cake with water, and dissolved the filter cake in dichloromethane , added anhydrous sodium sulfate to dry, filtered, and concentrated to obtain 912 mg of white solid (formula (VI)). The yield was 73%.

¹ H NMR (300 MHz, DMSO) δ0.011 (s, 6H, Si(CH ₃ ) ₂ ), 0.823 (s, 9H, C(CH ₃ ) ₃ ), 3.56-3.67 (m, 4H, H _4′ , H _5' ), 5.57(s, 2H, H _1' ), 8.10(s, 1H, H ₂ ), 8.24(s, 1H, H ₈ ), 12.40(s, 1H, H ₁ ); ¹³ C NMR ( 75MHz, DMSO) δ156.62, 148.49, 146.10, 140.68, 123.99, 72.80, 70.53, 61.85, 25.87, 17.90, -5.30. Anal _. Calcd for _C14H24N4O3Si : C _, 51.83; H, 7.46; N, 17.27; Found: C, 52.03 _; H, 7.48; N, 17.03.

The data indicated that the synthesized product was correct.

6. Synthesis of N ¹ -[(acetyl-O-ethoxy)-methyl]-N ⁹ -[(tert-butyldimethylsilyl-O-ethoxy)-methyl]-hypoxanthine ( Formula (VII))

N ¹ -[(Acetoxy-ethoxy)-methyl]-N ⁹ -[(TBDMS-O-ethoxy)-methyl]-Hypoxantine

Dissolve N ⁹ -methylethylene glycol-hypoxanthine (formula (VI)) (360mg, 1.10mmol) in 10ml of dichloromethane, add 1,8-diazabicyclo[5.4.0]undecane After -7-ene (DBU) (0.82ml, 5.50mmol), ethyl chloromethoxyacetate (1.65ml, 11.0mmol) was added dropwise, the reaction was exothermic, stirred at room temperature, and after 30min, TLC detected that the raw material disappeared completely. The solvent was evaporated to dryness, and column chromatography under normal pressure (200-300 mesh silica gel) yielded 67 mg of a yellow oily liquid (formula (VII)), with a yield of 61%.

¹ H NMR (300 MHz, DMSO) δ 0.084 (s, 6H, Si(CH ₃ ) ₂ ), 0.825 (s, 9H, C(CH ₃ ) ₃ ) 1.96 (s, 3H, OAc), 3.56-3.67 ( m, 4H, H _4′ , H _5′ ), 3.77(m, 2H, H _5′ ), 4.11(m, 2H, H _4′ ), 5.48(s, 2H, H _1′ ), 5.59(s, 2H, H _1′ ), 8.29 (s, 1H, H ₂ ), 8.52 (s, 1H, H ₈ ); ¹³ C NMR (75MHz, DMSO) δ170.20, 156.07, 149.11, 147.80, 141.37, 123.25, 74.77 , 72.70, 70.53, _{67.03 ,} 62.91, 61.83, 25.82 _, 20.55, 17.87, -5.34. Anal. Calcd for C19H32N4O6Si: C, 51.80 _; H, 7.32; N, 12.72; Found: C, 51.74 , H, 7.47; N, 12.42.

The data indicated that the synthesized product was correct.

7. Synthesis of N ¹ -[(acetyl-O-ethoxy)-methyl]-N ⁹ -(methyl-ethylene glycol)-hypoxanthine (formula (VIII))

N ¹ -[(Acetoxy-ethoxy)-methyl]-N ⁹ -(methyl-glycol)-Hypoxantine

N ¹ -[(acetyl-O-ethoxy)-methyl]-N ⁹ -[(tert-butyldimethylsilyl-O-ethoxy)-methyl]-hypoxanthine (formula ( VII)) (268mg, 0.609mmol) was dissolved in 10ml THF, 1M tert-butyl amine fluoride (TBAF) THF solution (1.25ml, 1.25mmol) was added, stirred at room temperature, TLC detected that the raw material disappeared completely after 2h, evaporated to dryness Solvent, normal pressure column chromatography (200-300 mesh silica gel), 178 mg of white foamy solid (formula (VIII)) was obtained, and the yield was 90%.

¹ H NMR (300MHz, DMSO) δ1.94(s, 3H, OAc), 3.44-3.53(m, 4H, H _4' , H _5' ), 3.76(m, 2H, H _5' ), 4.09(m , 2H, H _4' ), 4.69(t, 1H, J _{H5', OH} = 5.0Hz, OH), 5.46(s, 2H, H 1 _' ), 5.56(s, 2H, H _1' ), 8.28( s, 1H, H ₂ ), 8.50 (s, 1H, H ₈ ). Anal. Calcd for C ₁₃ H ₁₈ N ₄ O ₆ C, 47.85; H, 5.56; N, 17.17; Found: C, 48.00; H, 5.75; N, 16.98.

The data indicated that the synthesized product was correct.

8. Synthesis of N ¹ -[(acetyl-O-ethoxy)-methyl]-N ⁹ -[(diphenylamine phosphoryl-O-ethoxy)-methyl]-hypoxanthine (formula (IX) )

N ¹ -[(Acetoxy-ethoxy)-methyl]-N ⁹ -[(dianilinophosphoryl-O-ethoxy)-methyl]-Hypoxantine

Dissolve N ¹ -[(acetyl-O-ethoxy)-methyl]-N ⁹ -(methyl-ethylene glycol)-hypoxanthine (formula (VIII)) (250mg, 0.767mmol) in 5ml without Add 1-H tetrazole (161 mg, 2.30 mmol) and (PhNH) ₂ POCl (613 mg, 2.30 mmol) to water pyridine, stir at room temperature for 48 h, evaporate the solvent to dryness, and perform column chromatography under normal pressure (petroleum ether: acetone = 2 : 1) (200-300 mesh silica gel) to obtain 392 mg of yellow foam (formula (IX)), the yield was 92%.

¹ H NMR (300MHz, DMSO) δ1.93(s, 3H, OAc), 3.72-3.76(m, 4H, H _4' , H _5' ), 4.07-4.10(m, 4H, H _5' , H _{4 '} ), 5.46(s, 2H, H _1' ), 5.55(s, 2H, H _1' ), 6.77-7.16(m, 10H, ArH), 8.05(d, 2H, 2×NH), 8.25(s , 1H, H ₂ ), 8.49 (s, 1H, H ₈ ); ¹³ C NMR (75MHz, DMSO) δ170.28, 156.07, 149.23, 147.82, 141.40, 128.75, 123.26, 120.30, 117.23, 74.74, 72.45, 68.16 , 67.07, 63.58, 62.95, 20.59, 20.53; ³¹ P NMR (D ₂ O 81 MHz, decoupled with ¹ H) δ 3.07 (s). Anal _. Calcd for _C25H29N6O7P : C, 53.96; H, 5.25; N, 15.10; _Found : C, 53.95 _; H, 5.45; N, 14.82.

The data indicated that the synthesized product was correct.

9. Synthesis of N ¹ -[methyl-ethylene glycol]-N ⁹ -[(diphenylaminophosphoryl-O-ethoxy)-methyl]-hypoxanthine (formula (X))

N ¹ -[methyl-glycol]-N ⁹ -[(dianilinophosphoryl-O-ethoxy)-methyl]-Hypoxantine

N ¹ -[(acetyl-O-ethoxyl)-methyl]-N ⁹ -[(dianiline phosphoryl-O-ethoxyl)-methyl]-hypoxanthine (formula (IX)) ( 200mg, 0.36mmol) was dissolved in 10ml of anhydrous methanol, two drops of 5.4M sodium methoxide solution were dropped, stirred at room temperature for 2h, acidic cation exchange resin was added to adjust the pH to neutral, filtered, and the filtrate was concentrated to obtain the product (formula (X )) 180 mg.

10. Synthesis of N ¹ -[[(diphenylthio)phosphoryl-O-ethoxy]-methyl]-N ⁹ -[(diphenylaminophosphoryl-O-ethoxy)-methyl]- Hypoxanthine (formula (XI))

N ¹ -[[[Bis-(Phenylthio)phosphoryl]oxy-ethoxy]-methyl]-N ⁹ -[(dianilinophosphoryl-O-ethoxy)-methyl]-Hypoxantine

Dissolve N ¹ -[methyl-ethylene glycol]-N ⁹ -[(diphenylamine phosphoryl-O-ethoxy)-methyl]-hypoxanthine (formula (X)) (180mg, 0.36mmol) In 10ml of anhydrous pyridine, add 2,4,6-triisopropylbenzenesulfonyl chloride (TPSCl) (217mg, 0.72mmol), 1-H tetrazole, S,S'-diphenylmercaptophosphorylcyclohexylamine Salt (PSS) (410 mg, 1.08 mmol), stirred at room temperature for 24 h, evaporated to dryness, atmospheric column chromatography (200-300 mesh silica gel) to obtain 221 mg of the product (formula (XI)), the yield was 79%.

¹ H NMR (300MHz, DMSO) δ3.68-3.71(m, 2H, H _5' ), 3.81(m, 2H, H _4' ), 4.07(m, 2H, H _5' ), 4.31(m, 2H , H _4' ), 5.47(s, 2H, H _1' ), 5.55(s, 2H, H _1' ), 6.77-7.53(m, 20H, Ar H), 8.05(d, 2H, 2×NH) , 8.26(s, 1H, H ₂ ), 8.50(s, 1H, H ₈ ); ¹³ C NMR (75MHz, DMSO) δ156.09, 149.23, 147.84, 141.95, 135.09, 129.71, 128.75, 126.17, 125.59, 123.28 , 121.35, 120.31, 117.22, 74.76, 72.41, 68.14, 67.91, 67.00, 63.52; ³¹ P NMR (D ₂ O 81MHz, decoupled with ¹ H) δ 3.07(s), 49.36(s). Anal. Calcd for C ₃₅ H ₃₆ N ₆ O ₇ P ₂ S ₂ ·2acetone: C, 55.03; H, 5.41; N, 9.39; Found: C, 55.55; H, 5.62; N, 8.99.

The data indicated that the synthesized product was correct.

11. Synthesis of N ¹ -[(phenylphosphorothioate-O-ethoxy)-methyl]-N ⁹ -[(phosphoryl-O-ethoxy)-methyl]-hypoxanthine (formula (XII))

N ¹ -[[[(Phenylthio)phosphoryl]oxy-ethoxy]-methyl]-N ⁹ -[(phosphoryl-O-ethoxy)-methyl]-Hypoxantine

N ¹ -[[(diphenylthio)phosphoryl-O-ethoxy]-methyl]-N ⁹ -[(diphenylaminophosphoryl-O-ethoxy)-methyl]-hypoxanthin Purine (formula (XI)) (110mg, 0.14mmol) was dissolved in 4ml of pyridine-acetic acid-acetic anhydride (2:1:1, v:v:v), added isoamyl nitrite, and stirred at room temperature for 8h , evaporated the solvent to dryness, dried pyridine three times, dissolved in 3ml of anhydrous pyridine, added H ₃ PO ₂ and stirred at room temperature for 11h, then evaporated the solvent, added 5ml of H ₂ O and CH ₂ Cl ₂ to separate liquids, The aqueous phase was washed with 5×3 ml of CH ₂ Cl ₂ , and the aqueous phase was evaporated to dryness under reduced pressure (temperature less than 30° C.). 2 ml of 1M triethylamine bicarbonate (TEAB) buffer solution was added and evaporated to dryness. The above mixture was dissolved in 1ml of 0.1M TEAB buffer solution, separated by reverse C18 column HPLC, the gradient was 0-40% acetonitrile 0.1M TEAB within 30min, the flow rate was 5ml/min, the detection wavelength was 260nm, the main peak was collected for 31min, and after lyophilization 61 mg of the triethylamine salt of the product (formula (XII)) was obtained with a yield of 82%.

¹ H NMR (500MHz, DMSO) δ3.72(m, 2H, H _5' ), 3.86(m, 2H, H _4' ), 3.92(m, 2H, H _5' ), 4.11(m, 2H, H _4' ), 5.52(s, 2H, H _1' ), 5.65(s, 2H, H _1' ), 7.17-7.43(m, 5H, Ar H), 8.26(s, 1H, H ₂ ), 8.39( s, 1H, H ₈ ); ³¹ P NMR (D ₂ O 81 MHz, decoupled with ¹ H) δ 1.02(s), 17.85(s).

The data indicated that the synthesized product was correct.

12. Synthesis of N ¹ -[(phosphoryl-O-ethoxy)-methyl]-N ⁹ -[(phosphoryl-O-ethoxy)-methyl]-hypoxanthine-cyclic pyrophosphate ( Formula (XIII))

N ¹ -[(phosphoryl-O-ethoxy)-methyl]-N ⁹ -[(phosphoryl-O-ethoxy)-methyl]-Hypoxantine-cyclicpyrophosphate

Put activated 3 Å (1.5g) molecular sieves and I2 (159mg, 604μmol) into a round bottom flask, add 50ml of anhydrous pyridine, stir at room temperature, and N ¹ -[(phenylthiophosphoryl-O- Ethoxy)-methyl]-N ⁹ -[(phosphoryl-O-ethoxy)-methyl]-hypoxanthine (formula (XII)) triethylamine salt (25mg, 29.9μmol) was dissolved in Inject 5ml of anhydrous pyridine into the above-mentioned reaction bottle within 20 hours with a micro-injection pump. After the injection, continue to stir for 2 hours, filter out the molecular sieve with diatomaceous earth, wash the filter cake with a small amount of water, evaporate the filtrate to dryness, add water and chloroform Separate the liquid, wash the water phase with chloroform for 3 times, evaporate the water phase to dryness, add 2ml of 1M TEAB, evaporate to dryness, dissolve the above mixture in 1ml of 0.1M TEAB buffer solution, and separate by reverse C18 column HPLC with a gradient within 30min 0-80% acetonitrile, 0.1MTEAB, the flow rate is 5ml/min, the detection wavelength is 260nm, the main peak is collected for 30min, and after lyophilization, 6mg of triethylamine salt of (formula (XIII)) is obtained with a yield of 61%.

¹ H NMR (500MHz, DMSO) δ3.74(m, 2H, H _5′ ), 3.83(m, 2H, H _4′ ), 4.11(m, 4H, H _4′ , H _5′ ), 5.64(s , 2H, H _1′ ), 5.71(s, 2H, H _1′ ), 8.23(s, 1H, H ₂ ), 8.51(s, 1H, H ₈ ); ³¹ P NMR (D ₂ O 81MHz, decoupled with ^1H ) δ - 10.09 (s). HRMS (TOF, positive) for C ₁₃ H ₁₉ N ₅ O ₁₂ P ₂ Calcd, 427.0342 [(M+1) ⁻ ]; Found, 427.0435.

The data indicated that the synthesized product was correct.

Embodiment 2, the synthesis of cIDPDE (X=O in the formula (I), Y=C)

N ⁶ -benzoyl-adenine (formula (II)) (5.5 g, 22.5 mmol) was dissolved in 200 ml of anhydrous 1,2 dichloroethane, and BSA (11.2 ml, 45 mmol) was added under argon protection , heated to 80°C, until the solid was completely dissolved (about 20min), added ethyl chloromethoxyacetate (4.5ml, 25.6mmol), stirred at 80°C for 1.5h, cooled to room temperature, added 200ml ethyl acetate, and used Wash with 100×2ml saturated sodium bicarbonate, 100ml saturated brine, dry over anhydrous sodium sulfate, concentrate, and normal pressure column chromatography (ether-acetone=3:1) to give white solid N ⁶ -benzoyl-N ⁹ -[ (Acetyl-O-ethoxy)-methyl]-adenine (formula (III)) 2.9 g, yield 36%.

Other steps are the same as in Example 1 to obtain the compound of formula (I), wherein X=O, Y=C).

Embodiment 3, the synthesis of cIDPDE (X=C, Y=O in the formula (I))

Dissolve N ⁹ -methylethylene glycol-hypoxanthine (formula (VI)) (180mg, 0.55mmol) in 20ml of dichloromethane, add DBU (0.82ml, 5.5mmol), and then add chloromethoxy Ethyl acetate (0.41ml, 2.75mmol), the reaction was exothermic, stirred at room temperature, after 30min, TLC detected that the raw material disappeared completely, the solvent was evaporated to dryness, and column chromatography under normal pressure (petroleum ether: acetone = 5:1) gave a yellow Oily liquid N ¹ -[(acetyl-O-ethoxy)-methyl]-N ⁹ -[(diphenylamine phosphoryl-O-ethoxy)-methyl]-hypoxanthine (formula (IX)) 128 mg, 63% yield.

The other steps are the same as in Example 1 to obtain the compound of formula (I), wherein X=C, Y=O).

Embodiment 4, the synthesis of cIDPDE (X=C, Y=C in the formula (I))

N ⁶ -benzoyl-adenine (formula (II)) (5.5 g, 22.5 mmol) was dissolved in 200 ml of anhydrous 1,2 dichloroethane, and BSA (11.2 ml, 45 mmol) was added under argon protection , heated to 80°C, until the solid was completely dissolved (about 20min), added ethyl chloromethoxyacetate (4.5ml, 25.6mmol), stirred at 80°C for 1.5h, cooled to room temperature, added 200ml ethyl acetate, and used Wash with 100×2ml saturated sodium bicarbonate, 100ml saturated brine, dry over anhydrous sodium sulfate, concentrate, and normal pressure column chromatography (ether-acetone=3:1) to give white solid N ⁶ -benzoyl-N ⁹ -[ (Acetyl-O-ethoxy)-methyl]-adenine (formula (III)) 2.9 g, yield 36%.

Dissolve N ⁹ -methylethylene glycol-hypoxanthine (formula (VI)) (180mg, 0.55mmol) in 20ml of dichloromethane, add DBU (0.82ml, 5.5mmol), and then add chloromethoxy Ethyl acetate (0.41ml, 2.75mmol), the reaction was exothermic, stirred at room temperature, after 30min, TLC detected that the raw material disappeared completely, the solvent was evaporated to dryness, and column chromatography under normal pressure (petroleum ether: acetone = 5:1) gave a yellow Oily liquid N ¹ -[(acetyl-O-ethoxy)-methyl]-N ⁹ -[(diphenylamine phosphoryl-O-ethoxy)-methyl]-hypoxanthine (formula (IX)) 128 mg, 63% yield.

Other steps are the same as in Example 1 to obtain the compound of formula (I), wherein X=C, Y=C).

Claims (4)

1. Cyclic pyrophosphate compounds with general formula (I),

In the formula, X is C or O, and Y is C or O. 2. The cyclic pyrophosphate compound according to claim 1, wherein said X and Y are O. 3. The preparation method of the cyclic pyrophosphate compound represented by the general formula (I) of claim 1, comprising the following steps: 1) adding N ⁹ -[(tert-butyldimethylsilyl-O-ethoxy) -Methyl]-hypoxanthine was dissolved in dichloromethane, after adding DBU, ethyl chloromethoxyacetate was added dropwise, stirred at room temperature for 0.5-1h, evaporated to dryness, and column chromatography gave N ¹ -[(acetyl -O-ethoxy)-methyl]-N ⁹ -(methyl-ethylene glycol)-hypoxanthine; 2) Dissolve N ¹ -[(acetyl-O-ethoxy)-methyl]-N ⁹ -[(tert-butyldimethylsilyl-O-ethoxy)-methyl]-hypoxanthine After THF, add the THF solution of TBAF, stir at room temperature for 1-3h, evaporate the solvent to dryness, and obtain N ¹ -[(acetyl-O-ethoxy)-methyl]-N ⁹ -(methyl- Ethylene glycol)-hypoxanthine; 3) Dissolve N ¹ -[(acetyl-O-ethoxy)-methyl]-N ⁹ -(methyl-ethylene glycol)-hypoxanthine in anhydrous pyridine, add 1-H tetrazole and (PhNH) ₂ POCl, stirred at room temperature for 44-52h, evaporated to dryness, and column chromatography gave N ¹ -[(acetyl-O-ethoxy)-methyl]-N ⁹ -[(diphenylamine phosphoryl-O -ethoxy)-methyl]-hypoxanthine; 4) Dissolve N ¹ -[(acetyl-O-ethoxy)-methyl]-N ⁹ -[(diphenylamine phosphoryl-O-ethoxy)-methyl]-hypoxanthine in anhydrous methanol Add sodium methoxide solution dropwise, stir at room temperature for 1-3h, filter and concentrate the filtrate to obtain N ¹ -[methyl-ethylene glycol]-N ⁹ -[(diphenylaminophosphoryl-O-ethoxy)-methanol Base]-hypoxanthine; 5) Dissolve N ¹ -[methyl-ethylene glycol]-N ⁹ -[(diphenylaminophosphoryl-O-ethoxy)-methyl]-hypoxanthine in anhydrous pyridine, add 2,4 , 6-triisopropylbenzenesulfonyl chloride, 1-H tetrazole and S, S'-diphenylmercaptophosphorylcyclohexylamine salt, stirred at room temperature for 20-28h, evaporated to dryness, and obtained N ¹ -[ [(Diphenylthio)phosphoryl-O-ethoxy]-methyl]-N ⁹ -[(diphenylaminophosphoryl-O-ethoxy)-methyl]-hypoxanthine; 6) N ¹ -[[(diphenylthio)phosphoryl-O-ethoxy]-methyl]-N ⁹ -[(diphenylaminophosphoryl-O-ethoxy)-methyl]- Dissolve hypoxanthine in pyridine-acetic acid-acetic anhydride, add isoamyl nitrite, stir at room temperature for 6-10 hours, evaporate the solvent and dissolve in anhydrous pyridine, add H ₃ PO ₂ and stir at room temperature for 10-12 hours , evaporated to dryness, extraction, column chromatography, and lyophilization to obtain N ¹ -[(phenylphosphorothioate-O-ethoxy)-methyl]-N ⁹ -[(phosphoryl-O-ethoxy Base)-methyl]-triethylamine salt of hypoxanthine; 7) Put the activated 3 Å molecular sieve and I ₂ in the reaction flask, add anhydrous pyridine, stir at room temperature, and N ¹ -[(phenylphosphorothioate-O-ethoxy)-methyl] -N ⁹ -[(phosphoryl-O-ethoxy)-methyl]-hypoxanthine (formula (XII)) triethylamine salt in the above reaction flask, continue to stir for 1-3 hours, filter out molecular sieves, After extraction, column chromatography and lyophilization, N ¹ -[(phosphoryl-O-ethoxy)-methyl]-N ⁹ -[(phosphoryl-O-ethoxy)-methyl]-hypoxanthin was obtained Triethylamine salt of purine-cyclic pyrophosphate. 4. The preparation method according to claim 3, characterized in that the N ⁹ -[(tert-butyldimethylsilyl-O-ethoxy)-methyl]-hypoxanthine is prepared according to the following steps: 1) Dissolve adenine in anhydrous pyridine, add benzoyl chloride and then reflux for 1-2 hours, evaporate the solvent to dryness, extract, filter and concentrate to obtain N ⁶ -benzoyl-adenine; 2) Dissolve N ⁶ -benzoyl-adenine in anhydrous 1,2-dichloroethane, add N, O-bistrimethylsilylacetamide, heat to 110-130°C, add chloromethoxy Ethyl acetate, stirred at 70-90°C for 1-2h, extracted and concentrated to obtain N ⁶ -benzoyl-N ⁹ -[(acetyl-O-ethoxy)-methyl]-adenine; 3) Dissolve N ⁶ -benzoyl-N ⁹ -[(acetyl-O-ethoxy)-methyl]-adenine in methanol-ammonia water, stir the reaction at room temperature for 20-28 hours, evaporate the solvent to dryness, and water Recrystallization to obtain N ⁹ -methyl ethylene glycol-adenine; 4) Dissolve N ⁹ -methylethylene glycol-adenine in glacial acetic acid, add sodium nitrite aqueous solution under stirring, stir at room temperature for 20-28 hours, evaporate the solvent to get N ⁹ -methylethylene glycol-hypoxanth Purine; 5) Dissolve N ⁹ -methylethylene glycol-hypoxanthine in anhydrous DMF, add imidazole and tert-butyldimethylsilyl chloride, stir at room temperature for 2-4 hours, precipitate, filter, and concentrate to obtain N ⁹ -[(tert-Butyldimethylsilyl-O-ethoxy)-methyl]-hypoxanthine.