A high performance liquid chromatography detection method for 3α,7α-dihydroxy-6α-ethyl-5β-cholan-24-aldehyde

Abstract

The present invention relates to a method for preparing 3α,7α-dihydroxy-6α-ethyl-5β-cholane-24-aldehyde, which is simple and convenient, has high yield and content, can be conveniently used as a related substance reference substance for quality control of obeticholic acid or its derivatives, or can be used for impurity identification of obeticholic acid or its diffractors, and is beneficial to detection and control in the production process of obeticholic acid or its derivatives and quality control of medicines containing obeticholic acid or its derivatives.

Description

Technical Field

[0001] The invention belongs to the field of medicinal chemistry and relates to a preparation method of 3α,7α-dihydroxy-6α-ethyl-5β-cholan-24-aldehyde and application thereof. Background Art

[0002] Obeticholic acid (OCA, chemical name 6-ethylchenodeoxycholic acid) is a new generation of primary biliary cirrhosis (PBC) treatment drug developed by Intercept Pharmaceuticals in the United States. Its marketing application for the indication of non-alcoholic steatohepatitis (NASH) with grade 2 to 3 liver fibrosis is under review.

[0003]

[0004] During the research on obeticholic acid, our company found that the reduced impurity 3α,7α-dihydroxy-6α-ethyl-5β-cholan-24-aldehyde (such as the structure of Formula Ⅰ) would be produced during the synthesis of obeticholic acid. Since obeticholic acid and its related impurities have no ultraviolet absorption, conventional ultraviolet liquid detection methods cannot detect and quantify it.

[0005]

[0006] The method for preparing the obeticholic acid impurity formula (I) disclosed in US2016145295A1 is likely to produce multiple by-products such as dimers and alcohols in actual implementation. Since the product has no ultraviolet absorption, it is not conducive to purification. When the product prepared by this method is detected using a CAD detector, it is found that the content is relatively low. Summary of the invention

[0007] In order to overcome the deficiencies of the prior art, the present invention provides a novel method for preparing 3α,7α-dihydroxy-6α-ethyl-5β-cholan-24-aldehyde, which has a simple method and a high yield and content of the product.

[0008] In a first aspect of the present invention, a method for preparing 3α,7α-dihydroxy-6α-ethyl-5β-cholan-24-aldehyde (Formula I) is provided, characterized in that the compound shown in Formula II is subjected to a reduction reaction, and the reaction equation is as follows:

[0009]

[0010] Preferably, the method comprises the following steps: adding the compound of formula II and solvent E into a reactor, and adding reducing agent F to react at a temperature of -100 to -20°C under nitrogen protection.

[0011] More preferably, solvent E is selected from one or more of dichloromethane, chloroform, carbon tetrachloride, tetrahydrofuran, methyltetrahydrofuran, isopropyl ether, 1,2-dichloroethane, toluene, xylene, n-pentane, n-hexane, n-heptane, cyclohexane, cyclopentane, ether, and methyl tert-butyl ether; solvent E is preferably tetrahydrofuran or methyltetrahydrofuran.

[0012] More preferably, the reducing agent F is diisobutylaluminum hydride (DIBAL-H).

[0013] More preferably, the temperature is -70 to -60°C.

[0014] More preferably, the method may further include a post-treatment step, which includes but is not limited to: adding the reaction solution into an acid solution for separation, washing, drying and concentration;

[0015] Further preferably, the acid is acetic acid, and the concentration of the acid solution is 5 to 15% (w / w), such as 5% (w / w), 10% (w / w), 15% (w / w) acetic acid solution;

[0016] Further preferably, in the washing step, washing is performed with saturated sodium bicarbonate and / or saline;

[0017] Further preferably, the product can be further purified after drying and concentration, and the purification method is preferably recrystallization or column chromatography; the solvent used for the recrystallization is preferably selected from one or more of ethyl acetate, isopropyl acetate, methyl acetate, ethyl formate, ethanol, methanol, isopropanol, acetone, acetonitrile, toluene, and n-heptane; or, the column chromatography uses an ordinary silica gel column, a macroporous resin column or a gelatin column; the silica gel column preferably uses an elution solvent such as a mixed solvent of petroleum ether and ethyl acetate or a mixed solvent of n-hexane and ethyl acetate, more preferably a mixed solvent of petroleum ether and ethyl acetate with a volume ratio of 1:1 or a mixed solvent of n-hexane and ethyl acetate with a volume ratio of 1:1.

[0018] The present invention also provides a method for preparing the compound of formula II in claim 1, characterized in that it comprises the following steps: adding the compound of formula III and dimethylhydroxylamine hydrochloride to a reactor, adding solvent A, condensation agent B, condensation activator C and base D, and reacting to obtain the compound of formula II, wherein the reaction equation is as follows:

[0019]

[0020] Preferably, the solvent A is selected from one or more of dichloromethane, chloroform, carbon tetrachloride, tetrahydrofuran, methyltetrahydrofuran, isopropyl ether, 1,2-dichloroethane, toluene, xylene, N,N-dimethylformamide, N,N-dimethylacetamide, dimethyl sulfoxide, ethyl acetate, isopropyl acetate, acetonitrile, 1,4-dioxane, n-pentane, n-hexane, n-heptane, cyclohexane, cyclopentane, and water; more preferably, it is one or more of dichloromethane, methyltetrahydrofuran, and ethyl acetate.

[0021] Preferably, the condensing agent B is one or more of dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDCI) or its hydrochloride, HATU, HCTU, HAPyU, HBPyU, HBTU, TNTU, DPP-Cl, DECP, DPPA, MPTA, and BOP-Cl; more preferably, it is one or more of diisopropylcarbodiimide (DIC), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDCI) or its hydrochloride.

[0022] Preferably, the condensation activator C is one or more of DMAP, 4-PPY, 1-hydroxybenzotriazole (HOBt), 1-hydroxy-7-azabenzotriazole (HOAt), HOSu, NHPI, NHNI, and PFPOH; more preferably, it is one or more of 1-hydroxybenzotriazole (HOBt) and 1-hydroxy-7-azabenzotriazole (HOAt).

[0023] Preferably, the base D is one or more of DMAP, pyridine, triethylamine, N,N-diisopropylethylamine, potassium carbonate, sodium carbonate, sodium hydroxide, potassium hydroxide; more preferably, it is one or more of triethylamine and N,N-diisopropylethylamine.

[0024] Preferably, the reaction temperature is room temperature.

[0025] Preferably, the method further comprises post-processing steps, including but not limited to washing and concentration steps; the washing may be carried out in sequence with water, sodium bicarbonate solution, and saturated sodium chloride solution; and the concentration is preferably carried out under reduced pressure.

[0026] The present invention also provides an application of 3α,7α-dihydroxy-6α-ethyl-5β-cholan-24-aldehyde (Formula I), which is used as a related substance reference substance for quality control of obeticholic acid or its derivatives, or for impurity identification of obeticholic acid or its derivatives.

[0027] The present invention also provides a high performance liquid chromatography detection method for 3α,7α-dihydroxy-6α-ethyl-5β-cholan-24-aldehyde (Formula I) or obeticholic acid solution, wherein the chromatographic column is YMC Hydrosphere C18, the mobile phase A is 0.02-0.10% (V / V) TFA aqueous solution; the mobile phase B is acetonitrile, and the gradient elution is performed according to the following table:

[0028] Time (minutes) A％ B％ 0 50 50 2 40 60 10 35 65 20 25 75 22 0 100 25 0 100 25.1 50 50 30 50 50

[0029] Chromatographic conditions: column temperature: 30-40°C; flow rate: 0.5-1.0 ml / min; injection volume: 1-5 μl.

[0030] Preferably, the specification of the YMC Hydrosphere C18 is 4.6mm*150mm, 3μm; the mobile phase A is 0.05% (V / V) TFA aqueous solution; the column temperature is 35°C; the flow rate is 0.8ml / min; and the injection volume is 2μl.

[0031] Beneficial technical effects of the present invention:

[0032] The preparation method of 3α,7α-dihydroxy-6α-ethyl-5β-cholan-24-aldehyde disclosed in the present invention is simple and convenient, has high yield and content, is suitable for large-scale preparation, can be conveniently used as a reference substance for quality control of obeticholic acid or its derivatives, or for impurity identification of obeticholic acid or its diffractions, and is beneficial to the detection and control in the production process of obeticholic acid or its derivatives and the quality control of drugs containing obeticholic acid or its derivatives.

[0033] The present invention also provides a high-performance liquid phase detection method for 3α,7α-dihydroxy-6α-ethyl-5β-cholan-24-aldehyde or obeticholic acid, which can detect substances without ultraviolet response and ensure that each impurity has a good separation degree from the main component. It can effectively detect 3α,7α-dihydroxy-6α-ethyl-5β-cholan-24-aldehyde or obeticholic acid and related impurities that cannot be detected by ordinary ultraviolet liquid phase methods, thereby ensuring that the results obtained by the detection method are credible. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 : The compound of formula 1 1 H-NMR;

[0035] Figure 2 : The compound of formula 1 13 C-NMR;

[0036] Figure 3 : Liquid phase diagram of system solution of obeticholic acid. DETAILED DESCRIPTION

[0037] The present invention is further illustrated by the following examples, but the protection scope of the present invention is not limited thereto.

[0038] Example 1: Preparation of Compound of Formula II

[0039] 100.0 g of compound III and 29.3 g of dimethylhydroxylamine hydrochloride were added to a reaction bottle, and 2000 ml of dichloromethane was added, followed by 60.7 g of triethylamine, 42.2 g of 1-hydroxybenzotriazole and 59.8 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride. The mixture was reacted at room temperature. After HPLC confirmed that the remaining raw material was less than 2.0%, the mixture was washed with water, sodium bicarbonate solution and saturated sodium chloride solution in turn, and concentrated under reduced pressure to obtain 108.2 g of a light yellow solid compound of compound II.

[0040] Example 2: Preparation of Compounds of Formula II

[0041] 100.0 g of the compound of formula III and 29.3 g of dimethylhydroxylamine hydrochloride were added to a reaction bottle, and 2000 ml of methyltetrahydrofuran was added, followed by 77.6 g of diisopropylethylamine, 42.5 g of 1-hydroxy-7-azabenzotriazole and 59.8 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride. The mixture was reacted at room temperature. After HPLC confirmed that the remaining raw material was less than 2.0%, the mixture was washed with water, sodium bicarbonate solution and saturated sodium chloride solution in turn, and concentrated under reduced pressure to obtain 107.6 g of the off-white solid compound of the compound of formula II.

[0042] Example 3: Preparation of Compounds of Formula II

[0043] 100.0 g of the compound of formula III and 29.3 g of dimethylhydroxylamine hydrochloride were added to a reaction bottle, and 2000 ml of ethyl acetate was added, followed by 86.87 g of diisopropylethylamine, 40.8 g of 1-hydroxybenzotriazole and 37.8 g of diisopropylcarbodiimide. The mixture was reacted at room temperature. After HPLC confirmed that the remaining raw material was less than 2.0%, the mixture was washed with water, sodium bicarbonate solution and saturated sodium chloride solution in turn, and concentrated under reduced pressure to obtain 104.4 g of an off-white solid compound of the compound of formula II.

[0044] Example 4: Preparation of Compounds of Formula I

[0045] 100.0 g of the compound of formula II and 2000 ml of tetrahydrofuran were added to a reaction flask, stirred and dissolved, cooled to -75°C under nitrogen protection, and then 500 ml of DIBAL-H solution was slowly added dropwise. After the addition was completed, the reaction solution was reacted at -75°C. After the reaction was complete, the reaction solution was poured into a 10% acetic acid solution, extracted with ethyl acetate, washed with saturated sodium bicarbonate, washed with brine, dried and concentrated to obtain a crude product, and purified by column chromatography (silica gel, n-hexane / ethyl acetate = 1 / 1) to obtain 61.1 g of the compound of formula I as a white solid compound, with an HPLC content of 96.89% (CAD detector). 1 H-NMR Figure 1 , 13 C-NMR Figure 2 .

[0046] The structure of the compound was confirmed to be compound of formula I (ABDS055).

[0047] Nuclear magnetic resonance instrument: Bruker AV400MHz NMR (TMS as internal standard)

[0048] Determination solvent: deuterated chloroform

[0049]

[0050] H NMR spectrum data and attribution of compound Ⅰ

[0051]

[0052] Carbon NMR spectrum data and assignment of compound Ⅰ

[0053]

[0054] Example 5: Preparation of Compounds of Formula I

[0055] 100.0 g of compound II and 2000 ml of methyltetrahydrofuran were added to a reaction flask, stirred and dissolved, cooled to -70°C under nitrogen protection, and then 500 ml of DIBAL-H solution was slowly added dropwise. After the addition was completed, the reaction solution was reacted at -70°C. After the reaction was complete, the reaction solution was poured into 10% acetic acid solution, the organic layer was separated, washed with saturated sodium bicarbonate and brine, dried and concentrated to obtain a crude product, which was recrystallized from ethyl acetate-n-heptane to obtain 65.7 g of white solid compound of compound I with HPLC content of 99.14% (CAD detector).

[0056] Example 6: Preparation of Compounds of Formula I

[0057] 100.0 g of compound II and 2000 ml of tetrahydrofuran were added to a reaction flask, stirred and dissolved, cooled to -60°C under nitrogen protection, and then 500 ml of DIBAL-H solution was slowly added dropwise. After the addition was completed, the reaction solution was reacted at -60°C. After the reaction was complete, the reaction solution was poured into 10% acetic acid solution, the organic layer was separated, washed with saturated sodium bicarbonate and brine, dried and concentrated to obtain a crude product, and purified by column chromatography (silica gel, petroleum ether / ethyl acetate = 1 / 1) to obtain 68.3 g of white solid compound of compound I with HPLC content of 98.35% (CAD detector).

[0058] Example 7: High performance liquid chromatography detection method

[0059] 1. Preparation of system solution: Weigh about 2 mg of 3α,7α-dihydroxy-6α-ethyl-5β-cholan-24-aldehyde (compound of formula I), obeticholic acid (ABDS), 3α,7α-dihydroxy-6α-ethyl-5β-cholan-24-ol, 3α-hydroxy-6α-ethyl-7-keto-5β-cholan-24-acid, N-methoxy-N-methyl-3α-hydroxy-6α-ethyl-7-keto-5β-cholan-24-amide, N-methoxy-N-methyl-3α-hydroxy-6β-ethyl-7-keto-5β-cholan-24-amide and about 200 mg of N-methoxy-N-methyl-3α,7α-dihydroxy-6α-ethyl-5β-cholan-24-amide into a 100 ml volumetric flask, add acetonitrile to dissolve, and dilute to 100 ml for use.

[0060] 2. Detection method:

[0061] The prepared system solution was detected by high performance liquid chromatography, and the chromatographic conditions were as follows:

[0062] Instrument: High performance liquid chromatograph equipped with CAD detector

[0063] Chromatographic column: YMC Hydrosphere C18 4.6 mm*150 mm, 3 μm

[0064] Mobile phase: Phase A: 0.05% TFA aqueous solution; Phase B: acetonitrile, linear gradient elution according to the following table:

[0065] Time (minutes) A％ B％ 0 50 50 2 40 60 10 35 65 20 25 75 22 0 100 25 0 100 25.1 50 50 30 50 50

[0066] Chromatographic conditions: column temperature: 35°C; drift tube temperature: 50°C; frequency: 2Hz; filter: 5.0s; flow rate: 0.8ml / min; injection volume: 2μl

[0067] The liquid phase diagram of obeticholic acid system solution is shown in Figure 3It can be seen that this method can detect substances without UV response and ensure good separation between each impurity and the main component. It can effectively detect obeticholic acid and its related impurities that cannot be detected by ordinary UV liquid phase methods, ensuring that the obeticholic acid detection method obtains reliable results.

Claims

1. A high performance liquid chromatography separation method for 3α,7α-dihydroxy-6α-ethyl-5β-cholan-24-aldehyde and obeticholic acid solution, characterized in that: The chromatographic column is YMC Hydrosphere C18, mobile phase A: 0.02-0.10% TFA aqueous solution, V / V; mobile phase B: acetonitrile, gradient elution is performed as shown in the following table: Chromatographic conditions: column temperature: 30-40°C; flow rate: 0.5-1.0 ml / min; injection volume: 1-5 μl; CAD detector was used.

2. The high performance liquid chromatography separation method according to claim 1, characterized in that: The specification of the YMC HydrosphereC18 is 4.6 mm*150 mm, 3 μm; the mobile phase A is 0.05% TFA aqueous solution, V / V; the column temperature is 35° C.; the flow rate is 0.8 ml / min; and the injection volume is 2 μl.

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