Preparation method of stable isotope labeled imiquimod metabolite

Abstract

The invention discloses a preparation method of a stable isotope-labeled imiquimod metabolite, the stable isotope-labeled imiquimod metabolite with higher purity can be obtained, the vacancy of the prior art is filled, the structure of the stable isotope-labeled imiquimod metabolite is verified to be correct through HNMR and MS characterization, the purity of the stable isotope-labeled imiquimod metabolite can reach 99% or above, a test sample is provided for the research on the metabolic mechanism of imiquimod, and the stable isotope-labeled imiquimod metabolite has good application prospects. The important application value is realized.

Description

Technical Field

[0001] The invention relates to the field of compound synthesis, and in particular to a method for preparing a stable isotope-labeled imiquimod metabolite. Background Art

[0002] Imiquimod, whose chemical name is 1-(2-methylpropyl)-4-amino-1H-imidazo[4,5-C]quinoline, is an economically valuable compound. Stable isotope-labeled imiquimod metabolites are the labeled form of metabolite S-27700. The synthesis and study of labeled imiquimod metabolites can provide samples for the study of the metabolic mechanism of imiquimod and further enhance the economic value of imiquimod.

[0003] The unlabeled imiquimod metabolite S-27700 has been reported in the literature J Clin Pharmacol 2012; 52, 828-836; Pediatric Dermatology Vol.26No.2 203–212, 2009; Primary Care Update for OBGYNS, Volume 5, Issue 4, July–August 1998, Page 151, etc., but there is no synthesis method. The synthesis method of the R-type unlabeled imiquimod metabolite S-27700 has been reported in the literature US6677348B2, but the reaction selectivity of this method is poor, the reaction products are relatively mixed, and the yield is low. At present, no literature has been found to report the synthesis method of the stable isotope-labeled imiquimod metabolite S-27700.

[0004] Therefore, it is necessary to develop a method for preparing stable isotope-labeled imiquimod metabolites to fill the gaps in the existing technology. Summary of the invention

[0005] Purpose of the invention: In view of the shortcomings and defects of the prior art, the present invention provides a method for preparing a stable isotope-labeled imiquimod metabolite, which can obtain a stable isotope-labeled imiquimod metabolite with high purity. The structure is verified to be correct by HNMR and MS characterization, and the purity can reach more than 99%, which provides a test sample for the study of the metabolic mechanism of imiquimod and has important application value.

[0006] Technical solution: A method for preparing a stable isotope-labeled imiquimod metabolite of the present invention is characterized by comprising the following steps:

[0007] 1) adding compound I into an organic solvent;

[0008]

[0009] 2) adding compound II to the organic solvent in step 1);

[0010] Where X is H or D;

[0011] 3) Adding a base to the mixed solution obtained in step 2) to react to obtain compound III:

[0012] Where X is H or D;

[0013] 4) Add compound III to a solvent, add a reducing agent, and react to obtain compound IV:

[0014] Where X is H or D;

[0015] 5) Compound IV is added to an organic solvent and subjected to a ring-closure reaction to obtain compound V:

[0016] Where X is H or D;

[0017] 6) Compound V is added to a solvent, and an oxidant is added to react to obtain compound VI:

[0018] Where X is H or D;

[0019] 7) Compound VI is added to a solvent and subjected to chlorination and ammoniolysis to obtain compound VII, i.e., a stable isotope-labeled imiquimod metabolite:

[0020] Wherein X is H or D.

[0021] Wherein, the organic solvent in step 1) and step 2) is ethanol, methanol, acetonitrile or DMF, and the volume ratio of the organic solvent to compound I is 5-30:1.

[0022] Preferably, the organic solvent is acetonitrile, and the volume ratio of the organic solvent to compound I is 10:1.

[0023] Wherein, the base in step 3) is triethylamine, N,N-diisopropylethylamine, potassium carbonate or cesium carbonate, and the molar ratio of the base to compound I is 0-5:1; the reaction temperature is 0-100°C, and the reaction time is 2h-24h.

[0024] Preferably, the base is N,N-diisopropylethylamine, the base is 3 equivalents, the reaction temperature is 50° C., and the reaction time is 2 h.

[0025] Wherein, the solvent in step 4) is acetic acid, hydrochloric acid, methanol, ethanol or tetrahydrofuran, and the volume ratio of the solvent to compound III is 5-30:1.

[0026] Preferably, the solvent is methanol, and the volume ratio of the solvent to compound III is 20:1.

[0027] Wherein, in said step 4), the reducing agent is iron powder, zinc powder, stannous chloride, hydrosulfite or sodium bisulfite, and the molar ratio of the reducing agent to compound III is 1-10:1; the reaction temperature is 0-120° C., and the reaction time is 2h-24h.

[0028] Preferably, the reducing agent is stannous chloride, the reducing agent is 5 equivalents, the reaction temperature is 70° C., and the reaction time is 5 hours.

[0029] Wherein, the organic solvent in step 5) is toluene, benzene, chloroform, tetrahydrofuran or dioxane; and the ring-closing reagent in the ring-closing reaction is trimethyl orthoformate, triethyl orthoformate, formic acid or formamidine acetate.

[0030] Preferably, the organic solvent is benzene and the ring-closing reagent is formic acid.

[0031] Wherein, in said step 5), the molar ratio of the ring-closing reagent to compound IV is 0.5-5:1; the reaction temperature is 0-150°C, and the reaction time is 0.5h-24h.

[0032] Preferably, the ring-closing reagent is 2 equivalents, the reaction temperature is 90° C., and the reaction time is 12 h.

[0033] Wherein, in said step 6), the solvent is dichloromethane, chloroform, tetrahydrofuran, methanol or ethyl acetate; and the oxidant is m-chloroperbenzoic acid, hydrogen peroxide, peracetic acid or tert-butyl hydroperoxide.

[0034] Preferably, the solvent is tetrahydrofuran and the oxidant is hydrogen peroxide.

[0035] Wherein, in the step 6), the molar ratio of the oxidant to the compound V is 0.5-5:1, the volume ratio of the solvent to the compound V is 5-30:1; the reaction temperature is 0-80°C, and the reaction time is 0.5h-24h.

[0036] Preferably, the oxidant is 2 equivalents, the volume ratio of the solvent to the compound V is 20:1, the reaction temperature is 40° C., and the reaction time is 5 h.

[0037] Wherein, in the step 7), the solvent is dichloromethane, chloroform, methanol or toluene, the chlorination reagent is p-toluenesulfonyl chloride, ethyl chloroformate or phosphorus oxychloride, and the ammonolysis reagent is ammonia water, ammonia-containing methanol or ammonia gas; the molar ratio of the chlorination reagent to compound VI is 0.5-10:1, and the volume ratio of the solvent to compound VI is 5-30:1; the reaction temperature is 0-100°C, and the reaction time is 0.5h-24h.

[0038] Preferably, the chlorination agent is phosphorus oxychloride, the ammonolysis agent is aqueous ammonia, the chlorination agent is 2 equivalents, the volume ratio of the solvent to compound VI is 20:1; the reaction temperature is 80° C., and the reaction time is 2 h.

[0039] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: the present invention can obtain a stable isotope-labeled imiquimod metabolite with high purity, and its structure is verified to be correct by HNMR and MS characterization, and the purity can reach more than 99%, providing a test sample for the study of the metabolic mechanism of imiquimod, which has important application value. The synthetic process of the present invention is reasonably designed, highly operable, and the reagents used in the preparation method are simple, easy to obtain, and low in cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 is a synthetic route flow chart of the present invention, wherein X is H or D;

[0041] Figure 2 This is the mass spectrum of compound VII obtained in an example of the present invention, i.e., a metabolite of imiquimod;

[0042] Figure 3 This is the NMR spectrum of compound VII obtained in an example of the present invention, i.e., a metabolite of imiquimod;

[0043] Figure 4 This is the liquid phase spectrum of compound VII obtained in the example of the present invention, i.e., the metabolite of imiquimod. DETAILED DESCRIPTION

[0044] The technical solution of the present invention is further described below in conjunction with the accompanying drawings and specific implementation methods.

[0045] The method for preparing the stable isotope-labeled imiquimod metabolite of the present invention comprises the following steps:

[0046] 1) adding compound I into an organic solvent;

[0047]

[0048] 2) adding compound II to the organic solvent in step 1);

[0049] Where X is H or D;

[0050] The organic solvent in step 1) and step 2) is ethanol, methanol, acetonitrile or DMF, and the volume ratio of the organic solvent to compound I is 5-30:1. Preferably, the organic solvent is acetonitrile, and the volume ratio of the organic solvent to compound I is 10:1.

[0051] 3) Adding a base to the mixed solution obtained in step 2) to react to obtain compound III:

[0052] Where X is H or D;

[0053] The base in step 3) is triethylamine, N,N-diisopropylethylamine, potassium carbonate or cesium carbonate, and the molar ratio of the base to compound I is 0-5:1; the reaction temperature is 0-100°C, and the reaction time is 2h-24h. Preferably, the base is N,N-diisopropylethylamine, the base is 3 equivalents, the reaction temperature is 50°C, and the reaction time is 2h.

[0054] 4) Add compound III to a solvent, add a reducing agent, and react to obtain compound IV:

[0055] Where X is H or D;

[0056] The solvent in step 4) is acetic acid, hydrochloric acid, methanol, ethanol or tetrahydrofuran, and the volume ratio of the solvent to compound III is 5-30: 1. Preferably, the solvent is methanol, and the volume ratio of the solvent to compound III is 20: 1.

[0057] In step 4), the reducing agent is iron powder, zinc powder, stannous chloride, hydrosulfite or sodium bisulfite, the molar ratio of the reducing agent to compound III is 1-10:1; the reaction temperature is 0-120°C, and the reaction time is 2h-24h. Preferably, the reducing agent is stannous chloride, 5 equivalents, the reaction temperature is 70°C, and the reaction time is 5h.

[0058] 5) Compound IV is added to an organic solvent and subjected to a ring-closure reaction to obtain compound V:

[0059] Where X is H or D;

[0060] In step 5), the organic solvent is toluene, benzene, chloroform, tetrahydrofuran or dioxane; the ring-closing reagent of the ring-closing reaction is trimethyl orthoformate, triethyl orthoformate, formic acid or formamidine acetate. Preferably, the organic solvent is benzene and the ring-closing reagent is formic acid.

[0061] In step 5), the molar ratio of the ring-closing reagent to compound IV is 0.5-5:1; the reaction temperature is 0-150°C, and the reaction time is 0.5h-24h. Preferably, the ring-closing reagent is 2 equivalents, the reaction temperature is 90°C, and the reaction time is 12h.

[0062] 6) Compound V is added to a solvent, and an oxidant is added to react to obtain compound VI:

[0063] Where X is H or D;

[0064] In step 6), the solvent is dichloromethane, chloroform, tetrahydrofuran, methanol or ethyl acetate; the oxidant is m-chloroperbenzoic acid, hydrogen peroxide, peracetic acid or tert-butyl hydroperoxide. Preferably, the solvent is tetrahydrofuran and the oxidant is hydrogen peroxide.

[0065] In step 6), the molar ratio of the oxidant to the compound V is 0.5-5:1, the volume ratio of the solvent to the compound V is 5-30:1, the reaction temperature is 0-80°C, and the reaction time is 0.5h-24h. Preferably, the oxidant is 2 equivalents, the volume ratio of the solvent to the compound V is 20:1, the reaction temperature is 40°C, and the reaction time is 5h.

[0066] 7) Compound VI is added to a solvent and subjected to chlorination and ammoniolysis to obtain compound VII, i.e., a stable isotope-labeled imiquimod metabolite:

[0067] Where X is H or D;

[0068] In step 7), the solvent is dichloromethane, chloroform, methanol or toluene, the chlorination reagent is p-toluenesulfonyl chloride, ethyl chloroformate or phosphorus oxychloride, and the ammonolysis reagent is ammonia water, ammonia-containing methanol or ammonia gas; the molar ratio of the chlorination reagent to compound VI is 0.5-10:1, and the volume ratio of the solvent to compound VI is 5-30:1; the reaction temperature is 0-100°C, and the reaction time is 0.5h-24h. Preferably, the chlorination reagent is phosphorus oxychloride, the ammonolysis reagent is ammonia water, the chlorination reagent is 2 equivalents, the volume ratio of the solvent to compound VI is 20:1; the reaction temperature is 80°C, and the reaction time is 2h.

[0069] (I) Preparation of Compound III:

[0070] Example 1: Compound I (5.00 g) was dissolved in 50.0 mL of acetonitrile, and 2.23 g of compound II and DIPEA (9.29 g) were added. The mixture was stirred at 50 degrees for 2 hours. The reaction was completed on a plate. After treatment and purification, 5.80 g of compound III was obtained with a yield of 91.20%.

[0071] Example 2: Compound I (4.00 g) was dissolved in 40.0 mL of acetonitrile, and 1.79 g of compound II and triethylamine (5.82 g) were added. The mixture was stirred at 50 degrees for 2 hours. The reaction was completed on a plate. After treatment and purification, 4.12 g of compound III was obtained with a yield of 81.10%.

[0072] Example 3: Compound I (4.00 g) was dissolved in 40.0 mL acetonitrile, 1.79 g of compound IIa and potassium carbonate (7.95 g) were added, and stirred at 50 degrees for 3 hours. The reaction was completed on a plate, and then purified to obtain 3.35 g of compound III, with a yield of 65.94%, MS: 226.2 [M+H]+ .

[0073] (II) Preparation of Compound IV:

[0074] Example 1: Compound III (5.50 g) was dissolved in 110.0 mL of methanol, stannous chloride (19.65 g) was added, and the mixture was stirred at 70 degrees for 5 hours. The reaction was completed on a plate, and the compound IVa (4.23 g) was obtained after treatment and purification, with a yield of 86.68%.

[0075] Example 2: Compound III (3.00 g) was dissolved in 60.0 mL of methanol, and sodium hydrosulfite (9.84 g) was added. The mixture was stirred at 70 degrees for 4 hours. The reaction was completed on a plate. The compound IV (1.90 g) was obtained after purification with a yield of 71.43%.

[0076] Example 3: Compound III (3.00 g) was dissolved in 60.0 mL methanol, sodium sulfite (7.13 g) was added, and stirred at 70 degrees for 8 hours. The reaction was completed on a plate, and then purified to obtain compound IV 1.56 g, with a yield of 58.65%, MS: 236.3 [M+H] + .

[0077] (III) Preparation of Compound V:

[0078] Example 1: Compound IV (4.00 g) was dissolved in 40.0 mL of benzene, formic acid (1.56 g) was added, and the mixture was stirred at 90 degrees for 12 hours. The reaction was completed on a plate, and the compound V (3.01 g) was obtained after purification with a yield of 72.18%.

[0079] Example 2: Compound IV (2.00 g) was dissolved in 20.0 mL of benzene, trimethyl orthoformate (0.90 g) was added, and the mixture was stirred at 90 degrees for 12 hours. The reaction was completed on a plate, and the compound V was purified to obtain 1.35 g of compound V with a yield of 64.90%.

[0080] Example 3: Compound IV (2.00 g) was dissolved in 20.0 mL of benzene, triethyl orthoformate (1.26 g) was added, and the mixture was stirred at 90 degrees for 12 hours. The reaction was completed by spotting. The compound V was purified by treatment to obtain 1.16 g of compound V with a yield of 55.77%. MS: 246.2 [M+H] + .

[0081] (IV) Preparation of Compound VI:

[0082] Example 1: Compound V (2.50 g) was dissolved in 50.0 mL of tetrahydrofuran, 30% hydrogen peroxide (2.31 g) was added, and the mixture was stirred at 40 degrees for 5 hours. The reaction was completed on a plate, and the compound VI was purified to obtain 2.35 g of compound VI with a yield of 88.34%.

[0083] Example 2: Compound V (1.20 g) was dissolved in 24.0 mL of tetrahydrofuran, and m-chloroperbenzoic acid (75%, 2.25 g) was added. The mixture was stirred at room temperature for 5 hours. The reaction was completed on a plate. The compound VI was purified by treatment to obtain 1.02 g of compound VI with a yield of 79.69%.

[0084] Example 3: Compound V (1.20 g) was dissolved in 24.0 mL of tetrahydrofuran, 70% tert-butyl hydroperoxide (1.26 g) was added, and the mixture was stirred at 40 degrees for 8 hours. The reaction was completed on a plate, and the compound VI was purified to obtain 0.77 g of the compound VI with a yield of 60.16%.

[0085] (V) Preparation of Compound VII:

[0086] Example 1: Compound VI (2.20 g) was dissolved in 44.0 mL toluene and 6.6 mL ammonia water, phosphorus oxychloride (2.58 g) was added, and stirred at 80 degrees for 2 hours. The reaction was completed on a plate, and the compound VII was purified by treatment to obtain 1.87 g, with a yield of 85.39%.

[0087] Example 2: Compound VI (1.00 g) was dissolved in 20.0 mL toluene and 3.0 mL ammonia water, p-toluenesulfonyl chloride (0.73 g) was added, and stirred at 80 degrees for 2 hours. The reaction was completed on a plate, and the compound VII was purified to obtain 0.74 g, with a yield of 74.75%.

[0088] Example 3: Compound VI (1.00 g) was dissolved in 20.0 mL toluene and 3.0 mL methanol containing ammonia, ethyl chloroformate (0.42 g) was added, and stirred at 80 degrees for 2 hours. The reaction was completed after spotting. After treatment and purification, 0.61 g of compound VII was obtained, with a yield of 61.62%, MS: 261.2 [M+H] + , deuterium abundance 99.30%, liquid phase purity 99.8508%.

Claims

1. A method for preparing a stable isotope-labeled imiquimod metabolite, characterized in that: The steps include: 1) adding compound I into an organic solvent; 2) adding compound II to the organic solvent in step 1); 3) Adding a base to the mixed solution obtained in step 2) to react to obtain compound III: Where X is H or D; 4) Add compound III to a solvent, add a reducing agent, and react to obtain compound IV: Where X is H or D; 5) Compound IV is added to an organic solvent and subjected to a ring-closure reaction to obtain compound V: Where X is H or D; 6) Compound V is added to a solvent, and an oxidant is added to react to obtain compound VI: Where X is H or D; 7) Compound VI is added to a solvent and subjected to chlorination and ammoniolysis to obtain compound VII, i.e., a stable isotope-labeled imiquimod metabolite: Wherein X is H or D.

2. The method for preparing a stable isotope-labeled imiquimod metabolite according to claim 1, characterized in that: The organic solvent in step 1) and step 2) is ethanol, methanol, acetonitrile or DMF, and the volume ratio of the organic solvent to compound I is 5-30:

1.

3. The method for preparing a stable isotope-labeled imiquimod metabolite according to claim 1, characterized in that: The base in step 3) is triethylamine, N,N-diisopropylethylamine, potassium carbonate or cesium carbonate, and the molar ratio of the base to compound I is 0-5:1; the reaction temperature is 0-100°C, and the reaction time is 2h-24h.

4. The method for preparing a stable isotope-labeled imiquimod metabolite according to claim 1, characterized in that: The solvent in step 4) is acetic acid, hydrochloric acid, methanol, ethanol or tetrahydrofuran, and the volume ratio of the solvent to compound III is 5-30:

1.

5. The method for preparing a stable isotope-labeled imiquimod metabolite according to claim 1, characterized in that: In the step 4), the reducing agent is iron powder, zinc powder, stannous chloride, hydrosulfite or sodium bisulfite, and the molar ratio of the reducing agent to compound III is 1-10:1; the reaction temperature is 0-120° C., and the reaction time is 2h-24h.

6. The method for preparing a stable isotope-labeled imiquimod metabolite according to claim 1, characterized in that: In the step 5), the organic solvent is toluene, benzene, chloroform, tetrahydrofuran or dioxane; and the ring-closing reagent for the ring-closing reaction is trimethyl orthoformate, triethyl orthoformate, formic acid or formamidine acetate.

7. The method for preparing a stable isotope-labeled imiquimod metabolite according to claim 6, characterized in that: In the step 5), the molar ratio of the ring-closing reagent to compound IV is 0.5-5:1; the reaction temperature is 0-150° C., and the reaction time is 0.5h-24h.

8. The method for preparing a stable isotope-labeled imiquimod metabolite according to claim 1, characterized in that: In the step 6), the solvent is dichloromethane, chloroform, tetrahydrofuran, methanol or ethyl acetate; and the oxidant is m-chloroperbenzoic acid, hydrogen peroxide, peracetic acid or tert-butyl hydroperoxide.

9. The method for preparing a stable isotope-labeled imiquimod metabolite according to claim 1, characterized in that: In the step 6), the molar ratio of the oxidant to the compound V is 0.5-5:1, the volume ratio of the solvent to the compound V is 5-30:1; the reaction temperature is 0-80°C, and the reaction time is 0.5h-24h.

10. The method for preparing a stable isotope-labeled imiquimod metabolite according to claim 1, characterized in that: In the step 7), the solvent is dichloromethane, chloroform, methanol or toluene, the chlorination reagent is p-toluenesulfonyl chloride, ethyl chloroformate or phosphorus oxychloride, and the ammonolysis reagent is ammonia water, ammonia-containing methanol or ammonia gas; the molar ratio of the chlorination reagent to compound VI is 0.5-10:1, and the volume ratio of the solvent to compound VI is 5-30:1; the reaction temperature is 0-100°C, and the reaction time is 0.5h-24h.

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