A process for the preparation of a ketoconazole metabolite

Abstract

The application discloses a preparation method of a ketoconazole metabolite. The ketoconazole metabolite is an aromatic ring oxide of ketoconazole, and no synthesis report is found at present. In-vitro synthesis of the drug has extremely important research significance for studying the metabolism of ketoconazole. The preparation method has reasonable process design and simple experiment operation, and lays a foundation for future industrial production. The prepared ketoconazole metabolite has high purity, and provides important scientific support for the research of the ketoconazole metabolite.

Description

Technical Field

[0001] This invention relates to a method for preparing ketoconazole metabolites, belonging to the field of medicinal chemistry. Background Technology

[0002] Ketoconazole (Nizoral), chemically named cis-1-acetyl-4-[4-[[2-(2,4-dichlorophenyl)-2-(1H-imidazol-1-ylmethyl)-1,3-dioxolane-4-yl]methoxy]phenyl]piperazine, CAS: 65277-42-1, has the following structural formula:

[0003]

[0004] Ketoconazole (Nizoral) is an imidazole dioxane metabolite antifungal drug first synthesized by Janssen Pharmaceuticals in Belgium in 1976. It was introduced into clinical use in 1978, included in the 21st edition of the United States Pharmacopeia in 1980, and classified as a Class A broad-spectrum antifungal drug by the US FDA. Due to its outstanding clinical performance, it was added to the Chinese Pharmacopoeia as a new drug in 2000. As an inhibitor of lanosterol 14-α demethylase (encoded by the ERG11 gene), ketoconazole interferes with ergosterol production and alters the composition of other lipid compounds in the cell membrane, thereby exerting its antifungal effect. Due to its high efficacy and low toxicity, and its good antifungal activity against many clinically pathogenic fungi, such as Candida, Aspergillus, Cryptococcus neoformans, Blastomyces, Coccidia, Histoplasma capsulatum, and other deep fungi and Trichophyton, it is widely used to treat superficial and deep fungal infections with good therapeutic effects. In recent years, research on ketoconazole and observation of its clinical efficacy have led to the development of many new applications, such as the treatment of advanced prostate cancer, precocious puberty in children, and hirsutism in women. Its metabolite, neoconazole, overcomes the limitation of ketoconazole in personal care products, possessing good water solubility and suitability for use in this field. It has been included in the European guidelines for personal care products and has a very broad market prospect. However, ketoconazole also faces challenges such as drug resistance and a narrow antibacterial spectrum. Therefore, designing and synthesizing novel ketoconazole metabolites that are highly effective, low in toxicity, and have a broad antibacterial spectrum has become a hot topic in antifungal drug research.

[0005] The compound name of the ketoconazole metabolite is Ketoeonazole Metabolite (M8), and its molecular formula is C8. 26 H 28 Cl2N4O5, the structural formula is as follows:

[0006]

[0007] This ketoconazole metabolite has not yet been reported in the literature for synthesis, and it is highly likely to have more unique therapeutic effects, making it significant for drug metabolite research. US Patent US20090227560A1 synthesized an important intermediate II, but this method uses an expensive palladium catalyst, which is not conducive to industrial production. Therefore, there is a need to develop a well-designed and highly operable preparation method. Summary of the Invention

[0008] Purpose of the invention: The purpose of this invention is to provide a method for preparing ketoconazole metabolites that has a reasonable process design and is highly operable.

[0009] Technical solution: The present invention provides a method for preparing a ketoconazole metabolite, wherein the molecular formula of the ketoconazole metabolite is C0. 26 H 28 Cl2N4O5, its structural formula is shown below:

[0010]

[0011] The synthetic route is shown below:

[0012]

[0013] Furthermore, the method for preparing the ketoconazole metabolite includes the following steps:

[0014] (1) Dissolve compound I in a solvent, add a brominating reagent, react, extract and concentrate, and purify to obtain compound II;

[0015] (2) Compound II was dissolved in dimethyl sulfoxide (DMSO), sodium methoxide was added, the reaction was carried out, the mixture was extracted and concentrated, and the compound III was purified.

[0016] (3) Dissolve compound III in a solvent, add alkali and compound IV, heat to react, concentrate under reduced pressure, extract and concentrate, and purify to obtain compound V;

[0017] (4) Dissolve compound V in a solvent, add thiol and alkali, heat to react, adjust pH, extract with ethyl acetate to remove impurities, adjust pH again, extract and concentrate, purify, and recrystallize to obtain the product.

[0018] Furthermore, in step (1), the solvent is selected from acetic acid, acetonitrile, or N,N-dimethylformamide (DMF).

[0019] Furthermore, in step (1), the brominating agent is N-bromosuccinimide (NBS).

[0020] Furthermore, in step (1), the mass ratio of compound I to the brominating reagent is 1:0.8 to 1.5, preferably 1:1.

[0021] Furthermore, in step (1), the reaction temperature is 0 to 25°C, preferably 25°C, and the reaction time is 1 to 24 hours, preferably 1 hour.

[0022] Furthermore, in step (2), the mass ratio of compound II to sodium methoxide is 1:1 to 5, preferably 1:3.

[0023] Furthermore, in step (2), the reaction temperature is 25–140°C, preferably 120°C, and the reaction time is 1–24 h, preferably 10 h.

[0024] Furthermore, in step (3), the solvent is selected from DMF, dimethyl sulfoxide (DMSO), tetrahydrofuran (THF) or 1,4-dioxane, preferably DMF.

[0025] Furthermore, in step (3), the alkali is selected from sodium hydride, potassium carbonate, cesium carbonate or sodium hydroxide, preferably potassium carbonate.

[0026] Furthermore, in step (3), the mass ratio of compound III to the base is 1:1 to 3, preferably 1:2.

[0027] Furthermore, in step (3), the mass ratio of compound III to compound IV is 1:1 to 2, preferably 1:1.

[0028] Furthermore, in step (3), the temperature of the heating reaction is 25 to 90°C, preferably 80°C, and the heating reaction time is 1 to 10 hours, preferably 8 hours.

[0029] Furthermore, in step (4), the solvent is selected from DMF or DMSO.

[0030] Furthermore, in step (4), the thiol is selected from n-dodecyl mercaptan, n-octyl mercaptan or ethanethiol, preferably n-dodecyl mercaptan.

[0031] Furthermore, in step (4), the alkali is selected from sodium methoxide, sodium ethoxide, potassium hydroxide or sodium hydroxide, preferably sodium methoxide.

[0032] Furthermore, in step (4), the mass ratio of compound V, thiol and base is 1:1 to 2:1 to 2, preferably 1:1.7:1.7.

[0033] Furthermore, in step (4), the temperature of the heating reaction is 25 to 100°C, preferably 100°C, and the heating reaction time is 4 to 8 hours, preferably 8 hours.

[0034] Furthermore, in step (4), the pH is first adjusted to alkaline with an alkaline solution to make the product form a salt, and then the impurities are removed by extraction with ethyl acetate before the pH is adjusted to 5-6.

[0035] Furthermore, in steps (1) to (4), the sample is extracted and concentrated with ethyl acetate or dichloromethane, and then purified by column chromatography.

[0036] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0037] (1) The method of the present invention synthesizes ketoconazole metabolites, and the purity can reach 98% by characterization by NMR, mass spectrometry, liquid chromatography, etc.

[0038] (2) The ketoconazole metabolite synthesized for the first time by the method of the present invention is of great significance for drug metabolism research and has great application value in clinical pharmacokinetic studies.

[0039] (3) The preparation method of the present invention has a reasonable process design and strong operability, providing necessary technical support for future industrial production. Attached Figure Description

[0040] Figure 1 This is the HNMR spectrum of the ketoconazole metabolite of this invention;

[0041] Figure 2 This is an MS chromatogram of the ketoconazole metabolite of the present invention;

[0042] Figure 3 This is an HPLC chromatogram of the ketoconazole metabolite of the present invention. Detailed Implementation

[0043] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0044] Compound I is 1-acetyl-4-(4-hydroxyphenyl)piperazine, CAS: 67914-60-7;

[0045] Compound IV is cis-[2-(2,4-dichlorophenyl)-2-(1H-imidazol-1-ylmethyl)-1,3-dioxolane-4-yl]methanol-toluenesulfonate, CAS: 134071-44-6.

[0046] Example 1

[0047] (1) The preparation route of compound II is shown below:

[0048]

[0049] Compound I (30.00 g, 0.136 mol) was dissolved in DMF (120.0 mL), and a DMF solution of NBS (24.24 g, 0.136 mol) (169.7 mL) was slowly added dropwise at room temperature. The mixture was stirred at room temperature for 1 hour, and the reaction was detected by thin-layer chromatography (TLC) to confirm completion. The solution was diluted with saturated sodium chloride solution (360.0 mL), extracted with ethyl acetate (360.0 mL), and the crude product was concentrated. The crude product was purified by column chromatography (300–400 mesh silica gel, dichloromethane:methanol, 10:0.05–10:0.2) to obtain compound II (30.7 g), with a yield of 75%.

[0050] The structure of compound II was obtained by 1H NMR and mass spectrometry analysis as follows:

[0051]

[0052] (2) The preparation route of compound III is shown below:

[0053]

[0054] Compound II (15.00 g, 0.050 mol) was dissolved in DMSO (120.0 mL), and sodium methoxide (8.13 g, 0.150 mol) was added. The mixture was stirred at 120 °C for 10 hours, and the reaction was monitored by TLC until completion. The solution was diluted with 360 mL of water, and the crude product was extracted and concentrated with ethyl acetate (360.0 mL). The crude product was then purified by column chromatography (300–400 mesh silica gel, dichloromethane:methanol, 10:0.1–10:0.25) to give compound III (11.67 g), with a yield of 93%.

[0055] The structure of compound III was obtained by 1H NMR and mass spectrometry analysis as follows:

[0056]

[0057] (3) The preparation route of compound V is shown below:

[0058]

[0059] Compounds III (10.00 g, 0.040 mol) and IV (21.24 g, 0.044 mol) were dissolved in DMF (100.0 mL), and potassium carbonate (11.04 g, 0.080 mol) was added. The reaction mixture was stirred at 80 °C for 8 hours, and the reaction was monitored by TLC until completion. The reaction solution was concentrated under reduced pressure at 45 °C to remove DMF (concentrated until no more condensate dripped from rotary evaporation), diluted with 200 mL of water, and the crude product was extracted and concentrated with dichloromethane (200.0 mL x 2). The crude product was purified by column chromatography (300–400 mesh silica gel, dichloromethane:methanol, 10:0.1–10:0.4) to give compound V (19.15 g), with a yield of 85%.

[0060] The structure of compound V was obtained by 1H NMR and mass spectrometry analysis as follows:

[0061]

[0062] (4) The preparation route of compound VI is shown below:

[0063]

[0064] Compound V (12.00 g, 0.021 mol) was dissolved in DMF (48.0 mL), followed by the addition of n-dodecyl mercaptan (7.4 mL) and sodium methoxide (1.96 g). The mixture was stirred at 100 °C for 8 hours, and the reaction was monitored by TLC until completion. The solution was diluted with 60 mL of water, and 10% NaOH solution (8.0 mL) was added dropwise under ice bath conditions. Impurities were removed by extraction with ethyl acetate (60.0 mL x 2) (confirmed by TLC). The aqueous phase was adjusted to pH 6 with dilute hydrochloric acid, and the crude product was concentrated by extraction with ethyl acetate (120.0 mL x 2). The crude product was purified by column chromatography (300–400 mesh silica gel, dichloromethane:methanol, 10:0.2–10:0.8), and then recrystallized from diethyl ether to obtain VI (11.03 g), with a yield of 94%.

[0065] The prepared compound VI was analyzed by 1H NMR and M NMR spectra, and the results are as follows: Figure 1-2 As shown, HNMR(DMSO-d6)δ8.88(s,1H),7.68(d,1H),7.57(d,1H),7.46(m,2H),6.99(s.1H),6.81(s,1H),6.76(d,1H),6.45(d,1H),6. 32(m,1H),4.54(m,2H),4.33(m,1H),3.86(m,1H),3.71(m,2H),3.54(m,4H),3.47(m,1H),2.99(m,2H),2.92(m,2H),2.03(s.3H).

[0066] The structure of compound VI was obtained by 1H NMR and mass spectrometry analysis as follows:

[0067]

[0068] The purity of compound VI was analyzed by HPLC, and the results are as follows: Figure 3 As shown, its purity is over 98%.

[0069] Example 2

[0070] A method for preparing a ketoconazole metabolite, the preparation process being the same as in Example 1, includes the following steps:

[0071] (1) The preparation route of compound II is shown below:

[0072]

[0073] Compound I (20.00 g, 0.091 mol) was dissolved in dry acetonitrile (100.0 mL). A dry acetonitrile solution of NBS (17.78 g, 0.100 mol) (711.1 mL) was slowly added dropwise at 10-20 °C. The reaction was stirred at 25 °C for 1.5 hours, and TLC was used to confirm the completion of the reaction. The solution was diluted with water (200.0 mL), and the crude product was extracted and concentrated with dichloromethane (240.0 mL). The crude product was purified by column chromatography (300-400 mesh silica gel, dichloromethane:methanol, 10:0.05-10:0.2). Analysis by 1H NMR and mass spectrometry yielded compound II (4.12 g), with a yield of 15%.

[0074] (2) The preparation route of compound III is shown below:

[0075]

[0076] Compound II (15.00 g, 0.050 mol) was dissolved in DMSO (120.0 mL), and sodium methoxide (13.54 g, 0.251 mol) was added. The reaction was stirred at 120 °C for 24 hours, and the reaction was confirmed by TLC. The product was diluted with 360 mL of water, and the crude product was extracted and concentrated with ethyl acetate (360.0 mL). The crude product was purified by column chromatography (300–400 mesh silica gel, dichloromethane:methanol, 10:0.1–10:0.25). The crude product was then analyzed by 1H NMR and mass spectrometry to obtain compound III (10.2 g), with a yield of 81%.

[0077] (3) The preparation route of compound V is shown below:

[0078]

[0079] Compounds III (10.00 g, 0.040 mol) and IV (20.28 g, 0.042 mol) were dissolved in DMF (200.0 mL), and potassium hydroxide (4.93 g, 0.088 mol) was added. The reaction was stirred at 70 °C for 8 hours, and the reaction was confirmed by TLC. The reaction solution was concentrated under reduced pressure at 45 °C to remove DMF, diluted with 200 mL of water, and the crude product was extracted and concentrated with dichloromethane (200.0 mL x 2). The crude product was purified by column chromatography (300–400 mesh silica gel, dichloromethane:methanol, 10:0.1–10:0.4). The crude product was identified as compound V (14.2 g) by 1H NMR and mass spectrometry, with a yield of 63%.

[0080] (4) The preparation route of compound VI is shown below:

[0081]

[0082] Compound V (8.00 g, 0.014 mol) was dissolved in DMF (32.0 mL) and n-dodecyl mercaptan (32 mL), and sodium methoxide (7.70 g) was added. The reaction was stirred at 80 °C for 5 hours, and the reaction was confirmed by TLC. The solution was diluted with 40 mL of water, and 10% NaOH solution (5.3 mL) was added dropwise under ice bath conditions. Impurities were removed by extraction with ethyl acetate (40.0 mL x 2) (confirmed by TLC). The aqueous phase was adjusted to pH 6 with dilute hydrochloric acid, and the crude product was extracted and concentrated with ethyl acetate (80.0 mL x 2). The crude product was purified by column chromatography (300–400 mesh silica gel, dichloromethane:methanol, 10:0.2–10:0.8), and then recrystallized from diethyl ether. Analysis by 1H NMR and mass spectrometry yielded compound VI (10.2 g), with a yield of 71%.

[0083] Example 3

[0084] A method for preparing a ketoconazole metabolite, the preparation process being the same as in Example 1, includes the following steps:

[0085] (1) The preparation route of compound II is shown below:

[0086]

[0087] Compound I (20.00 g, 0.091 mol) was dissolved in acetic acid (160.0 mL). A solution of NBS (17.78 g, 0.100 mol) in acetic acid (129.3 mL) was slowly added dropwise at 10-20 °C. The reaction was stirred at 25 °C for 1.5 hours, and TLC was used to confirm the completion of the reaction. The solution was diluted with water (240.0 mL), extracted with ethyl acetate (240.0 mL), and concentrated to obtain the crude product. The crude product was purified by column chromatography (300-400 mesh silica gel, dichloromethane:methanol, 10:0.05-10:0.2). Analysis by 1H NMR and mass spectrometry yielded compound II (17.7 g), with a yield of 65%.

[0088] (2) The preparation route of compound III is shown below:

[0089]

[0090] Compound II (15.00 g, 0.050 mol) was dissolved in DMSO (120.0 mL), and sodium methoxide (5.42 g, 0.100 mol) was added. The reaction was stirred at 100 °C for 10 hours, and the reaction was confirmed by TLC. The product was diluted with 360 mL of water, and the crude product was extracted and concentrated with ethyl acetate (360.0 mL). The crude product was purified by column chromatography (300–400 mesh silica gel, dichloromethane:methanol, 10:0.1–10:0.25). The crude product was then analyzed by 1H NMR and mass spectrometry to obtain compound III (8.30 g), with a yield of 66%.

[0091] (3) The preparation route of compound V is shown below:

[0092]

[0093] Compounds III (10.00 g, 0.040 mol) and IV (21.24 g, 0.044 mol) were dissolved in 1,4-dioxane (100.0 mL), and cesium carbonate (26.03 g, 0.080 mol) was added. The reaction mixture was stirred at 80 °C for 9 hours, and the reaction was confirmed by TLC. The reaction solution was concentrated under reduced pressure at 45 °C to remove DMF, diluted with 200 mL of water, and the crude product was extracted and concentrated with dichloromethane (200.0 mL x 2). The crude product was purified by column chromatography (300–400 mesh silica gel, dichloromethane:methanol, 10:0.1–10:0.4). The crude product was identified as compound V (10.8 g) by 1H NMR and mass spectrometry, with a yield of 48%.

[0094] (4) The preparation route of compound VI is shown below:

[0095]

[0096] Compound V (10.00 g, 0.018 mol) was dissolved in DMF (40.0 mL) and n-dodecyl mercaptan (6.1 mL), and sodium methoxide (1.64 g, 0.030 mol) was added. The reaction was stirred at 100 °C for 2 hours, and the reaction was confirmed by TLC. The solution was diluted with 60 mL of water, and 10% NaOH solution (6.6 mL) was added dropwise under ice bath conditions. Impurities were removed by extraction with ethyl acetate (50.0 mL x 2) (confirmed by TLC). The aqueous phase was adjusted to pH 6 with dilute hydrochloric acid, and the crude product was concentrated by extraction with ethyl acetate (50.0 mL x 2). The crude product was purified by column chromatography (300–400 mesh silica gel, dichloromethane:methanol, 10:0.2–10:0.8), and then recrystallized from diethyl ether. Analysis by 1H NMR and mass spectrometry yielded compound VI (7.82 g), with a yield of 80%.

[0097] The above description is merely an embodiment of the present invention and does not imply the patent scope of the present invention. Any equivalent structural or procedural transformations made using the content of the present invention specification, or direct or indirect applications in other related technical fields, are similarly included within the patent protection scope of the present invention.

Claims

1. A method for preparing a ketoconazole metabolite, characterized in that, The molecular formula of the ketoconazole metabolite is C. 26 H 28 Cl2N4O5, its structural formula is shown below: ； The synthetic route is shown below: ； Includes the following steps: (1) Dissolve compound I in a solvent, add a brominating reagent, react, extract and concentrate, and purify to obtain compound II; (2) Compound II was dissolved in DMSO, sodium methoxide was added, the reaction was carried out, the mixture was extracted and concentrated, and the compound III was purified. (3) Dissolve compound III in a solvent, add alkali and compound IV, heat to react, concentrate under reduced pressure, extract and concentrate, and purify to obtain compound V; (4) Dissolve compound V in a solvent, add thiol and alkali, heat to react, adjust pH, extract with ethyl acetate to remove impurities, adjust pH again, extract and concentrate, purify, and recrystallize to obtain the product.

2. The method for preparing ketoconazole metabolites according to claim 1, characterized in that, In step (1), the solvent is selected from acetic acid, acetonitrile or DMF, the brominating agent is NBS, the mass ratio of compound I to brominating agent is 1:0.8~1.5, the reaction temperature is 0~25°C, and the reaction time is 1~24h.

3. The method for preparing ketoconazole metabolites according to claim 1, characterized in that, In step (2), the mass ratio of compound II to sodium methoxide is 1:1~5, the reaction temperature is 25~140°C, and the reaction time is 1~24h.

4. The method for preparing ketoconazole metabolites according to claim 1, characterized in that, In step (3), the solvent is selected from DMF, DMSO, THF or 1,4-dioxane, and the base is selected from sodium hydride, potassium carbonate, cesium carbonate or sodium hydroxide.

5. The method for preparing ketoconazole metabolites according to claim 1, characterized in that, In step (3), the mass ratio of compound III to base is 1:1~3, the mass ratio of compound III to compound IV is 1:1~2, the heating temperature is 25~90°C, and the heating time is 1~10h.

6. The method for preparing ketoconazole metabolites according to claim 1, characterized in that, In step (4), the solvent is selected from DMF or DMSO, the thiol is selected from n-dodecyl mercaptan, n-octyl mercaptan or ethanethiol, and the base is selected from sodium methoxide, sodium ethoxide, potassium hydroxide or sodium hydroxide.

7. The method for preparing ketoconazole metabolites according to claim 1, characterized in that, In step (4), the mass ratio of compound V, thiol and base is 1:1~2:1~2, the heating temperature is 25~100°C, and the heating time is 4~8h.

8. The preparation method according to claim 1, characterized in that, In step (4), the pH is first adjusted to alkaline with an alkaline solution to form a salt in the product, and then the impurities are removed by extraction with ethyl acetate before the pH is adjusted to 5-6.

9. The preparation method according to claim 1, characterized in that, In steps (1) to (4), the sample is extracted and concentrated with ethyl acetate or dichloromethane, and then purified by column chromatography.

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