Preparation method of menadione impurity A

Abstract

The invention belongs to the technical field of chemical synthesis, and particularly relates to a preparation method, a detection method and application of menadione cis-isomer impurities. The impurity is a menadione impurity A. The preparation method comprises the following steps: by taking farnesyl acetone as a raw material, carrying out Wittig-Horner reaction, reduction reaction, bromination reaction, substitution reaction and inverse Diels-Alder reaction to obtain the menadione impurity A. The invention provides a method for detecting the impurity A in a menadione bulk drug. The preparation method is simple in process, low in equipment requirement and good in reaction selectivity, and the detection method is simple and high in applicability and has guiding significance on quality control of menadione bulk drugs.

Description

Technical Field

[0001] The invention belongs to the technical field of chemical synthesis, and particularly relates to a preparation method, a detection method and an application of a menatetrenone impurity A. Background Art

[0002] Menatetrenone, chemical name: 2-methyl-3-[(2E,6E,10E)-3,7,11,15-tetramethyl-2,6,10,14-hexadecatetraenyl]-1,4-naphthoquinone, structural formula:

[0003]

[0004] Menatetrenone belongs to the vitamin K2 family, and its unique structure gives it many unique biological activities. Compared with other vitamin K2 subtypes, menatetrenone is more easily absorbed and utilized in the human body, which makes it have great potential in maintaining health and preventing diseases. In recent years, more and more studies have shown that menatetrenone plays a key role in bone health, cardiovascular health, intestinal health and many other aspects. Among them, the role of menatetrenone in promoting bone health is particularly noteworthy. Studies have found that menatetrenone can effectively promote the absorption of calcium, thereby helping the body maintain strong bones. At the same time, menatetrenone can also reduce the risk of fractures, which is especially important for high-risk groups with reduced bone density such as the elderly and menopausal women.

[0005] The chemical properties of tetraene methyl quinone are relatively unstable and sensitive to light and heat. Its structure contains multiple double bonds, which makes it easy to produce multiple isomers and other impurities during the synthesis process. For example, the 2-position double bond of the side chain of tetraene methyl quinone is easily changed under light conditions, thereby degrading to form a cis isomer, i.e., impurity A. In addition, impurity A will inevitably form in the synthesis process. These isomeric impurities are not only difficult to separate and purify, but also increase the difficulty and technical requirements of synthesis, which may affect the safety of the product. According to the quality standards of vitamin K drugs in the Chinese Pharmacopoeia, the cis isomer impurity A is a known important impurity, and it has been reported in many patents such as US20240082198A1 and EP0679394B1, which is a key indicator in drug quality control. However, there is no literature report on the preparation method and detection method of tetraene methyl quinone cis isomer impurities. Therefore, how to improve the quality control of tetraene methyl quinone raw materials and preparations has become an urgent problem to be solved. Summary of the invention

[0006] In view of this, the present invention provides a preparation method and a detection method of tetraene-methyl menatequinone impurity A. The preparation method has simple process, low equipment requirements, good reaction selectivity, simple detection method, strong applicability, and provides effective technical support for the quality control of tetraene-methyl menatequinone.

[0007] To achieve the above object, the technical solution of the present invention is:

[0008] In a first aspect, the present invention provides a method for preparing a menatetrenone impurity A, wherein the impurity has the following structural formula:

[0009]

[0010] The reaction route of the preparation method is as follows:

[0011]

[0012] The preparation method steps are as follows:

[0013] (1) Using the compound of formula 1 as a raw material and tetrahydrofuran as a solvent, adding a base, dripping a tetrahydrofuran solution of triethyl phosphonoacetate, stirring at room temperature for 1.5 hours, dripping a tetrahydrofuran solution of the compound of formula 1, after the reaction is completed, extracting, purifying by column chromatography, and concentrating under reduced pressure to dryness to obtain a compound of formula 2;

[0014] (2) dissolving the compound of formula 2 obtained in step (1) in tetrahydrofuran, reducing the ester group with a reducing agent, and obtaining a compound of formula 3 by extraction and column chromatography purification;

[0015] (3) dissolving the compound of formula 3 obtained in step (2) in tetrahydrofuran, adding phosphorus tribromide dropwise at below 0° C. to carry out bromination reaction, and obtaining the compound of formula 4 after extraction, washing and concentration;

[0016] (4) The compound of formula 5 is used as the starting material 2, dissolved in methanol, and then glacial acetic acid and cyclopentadiene are added thereto, and the temperature is raised to 50° C. for reaction. After the reaction is completed, the compound is concentrated, recrystallized, and dried to obtain a white solid, namely the compound of formula 6;

[0017] (5) controlling the temperature below 0° C., dissolving potassium tert-butoxide in a tetrahydrofuran solution, adding dropwise the tetrahydrofuran solution of the compound of formula 6 obtained in step (4), and reacting at this temperature for 20 to 30 minutes, then adding dropwise the tetrahydrofuran solution of the compound of formula 4 obtained in step (3), and reacting at this temperature, and then extracting, drying, concentrating, and column chromatography to obtain a compound of formula 7;

[0018] (6) Add toluene to the compound of formula 7 obtained in step (5), raise the temperature to 100° C. for reaction, and obtain the tetraene-methyl-menaquinone impurity A after concentration and column chromatography purification.

[0019] According to an embodiment of the present invention, the base used in step (1) is selected from one or more of sodium hydrogen sulfide, sodium methoxide, sodium ethoxide, and lithium diisopropylamide.

[0020] According to an embodiment of the present invention, the reducing agent used in step (2) is selected from one or more of dihydrogen bis(dimethoxyethoxy)aluminate sodium, lithium tri-sec-butylborohydride, lithium aluminum hydride, and diisobutylaluminum hydride.

[0021] According to an embodiment of the present invention, the eluent used for column chromatography separation in step (2) is a n-hexane-ethyl acetate solution, with n-hexane:ethyl acetate = 30:1.

[0022] According to an embodiment of the present invention, the eluent used for column chromatography separation in step (5) is a n-hexane-ethyl acetate solution, with n-hexane:ethyl acetate = 200:1 for eluting impurities and 100:1 for eluting the main product.

[0023] According to an embodiment of the present invention, the eluent used for column chromatography purification in step (6) is a n-hexane-ethyl acetate solution, with n-hexane:ethyl acetate=100:1.

[0024] In a second aspect, the present invention also provides application of the preparation method in drug process research, which can be used for the study of menatetrenone impurities.

[0025] In a third aspect, the present invention provides a method for detecting impurity A in menatetrenone raw materials, which specifically comprises the following steps:

[0026] (1) Instrument and chromatographic conditions:

[0027] Chromatographic column: Silica gel column with amylose-tris(3,5-dichlorophenylcarbamate) covalently bonded to the surface IE, specifications are 4.6mm×250mm, 5μm;

[0028] Mobile phase: a mixed solution of n-hexane and ethanol;

[0029] Column temperature: 35°C;

[0030] Flow rate: 1.0 mL / min;

[0031] Injection volume: 10 μL;

[0032] Detection wavelength: 270nm,

[0033] (2) Sample configuration:

[0034] Preparation of test solution: Take an appropriate amount of menatetrenone, weigh accurately, dissolve and dilute with diluent to make a solution containing about 0.5 mg per 1 mL;

[0035] Preparation of reference solution: Take an appropriate amount of menatetrenone reference substance, weigh accurately, dissolve with solvent and quantitatively dilute to make a solution containing about 0.75 μg per 1 mL;

[0036] Preparation of system suitability solution: take 2.5 mg of PZA, accurately weigh it, place it in a 25 mL volumetric flask, add diluent to dissolve and dilute to the mark, shake well; accurately measure 2 mL, place it in a 20 mL volumetric flask, dilute to the mark with diluent, shake well, as the stock solution; take about 10 mg of tetraene methyl quinone reference substance, accurately weigh it, place it in a 20 mL volumetric flask, accurately add 1.5 mL of stock solution, add diluent to dissolve and dilute to the mark, shake well, and the solution is ready;

[0037] (3) Detection and calculation:

[0038] Accurately measure 20 μL of the system suitability solution and inject it into the liquid chromatograph. Then accurately measure 10 μL each of the control solution and the test solution and inject them into the liquid chromatograph respectively, and record the chromatogram.

[0039] According to an embodiment of the present invention, the diluent and the mobile phase are a mixed solution of n-hexane and ethanol, with a volume ratio of 98:2.

[0040] The beneficial effects of the present invention are:

[0041] (1) The present invention provides a novel method for preparing tetraene menaquinone impurity A, which can effectively prepare high-purity impurity A (purity 95.38%), and has significant significance for improving the safety and reliability of the final product.

[0042] (2) The preparation method of the present invention has a simple process, low requirements on equipment, and good reaction selectivity. By adopting this technology, the product quality can be controlled more accurately, which has important application value in guiding and optimizing the overall production process of tetraene menaquinone.

[0043] (3) Strengthening drug monitoring and research: The present invention not only achieves structural confirmation of the impurity, but also discloses an HPLC detection method for detecting the content of impurity A in tetraenylmethyl quinone raw materials. The implementation of this method helps to more comprehensively control the quality of tetraenylmethyl quinone and ensure the purity and efficacy of the drug. In addition, this technology has important research and practical value for studying and controlling quality problems that may occur in the drug production process and adverse reactions of drug receptors. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 HPLC spectrum for the detection of related substances of crude menatetrenone.

[0045] Figure 2 The HPLC spectrum of impurity A of menatetrenone.

[0046] Figure 3 This is a schematic diagram of the atomic numbering of the tetraene menaquinone impurity A.

[0047] Figure 4 This is the H NMR spectrum of the impurity A of tetraene-menatetraquinone.

[0048] Figure 5 This is the carbon NMR spectrum of the impurity A of tetraene menaquinone.

[0049] Figure 6 This is the HPLC spectrum for the detection of related substances of pure menatetrenone. DETAILED DESCRIPTION

[0050] The present invention is further described in detail below in conjunction with specific examples and drawings. The following examples are only illustrative and not restrictive, and the protection scope of the present invention cannot be limited thereto. Unless otherwise specified, the raw materials used can be obtained from the market or homemade.

[0051] During the process of developing the synthesis of tetraenylmethyl quinone, we found that tetraenylmethyl quinone is easily degraded to form 2-cis isomer (impurity A) under light conditions. In addition, impurity A is inevitably produced in the synthesis process. The content of this impurity in the crude tetraenylmethyl quinone is 2.81%. The HPLC test results of the crude tetraenylmethyl quinone related substances are shown in Table 1, and the corresponding test spectrum is shown in Table 1. Figure 1 .

[0052] Table 1 HPLC test results of related substances in crude menatetraenequinone

[0053] name batch number Impurity A(%) Retention time (min) Menatetrenone C1-24031401 2.81 83.492

[0054] The present invention uses farnesyl acetone as a raw material, and obtains the tetraene menaquinone impurity A through Wittig-Horner, reduction, bromination, substitution, and reverse Diels-Alder reactions, and after structural confirmation, its chemical name is named as follows:

[0055] 2-Methyl-3-[(2Z,6E,10E)-3,7,11,15-tetramethyl-2,6,10,14-hexadecatetraenyl]-1,4-naphthoquinone

[0056] The structural formula is:

[0057]

[0058] The present invention aims to protect the preparation method and detection method of impurity A, which are used for quality control of menatetrenone to ensure the product quality and medication safety of menatetrenone.

[0059] Example 1 Preparation of Tetetrazolone Impurity A

[0060] Step 1: Preparation of compound of formula 2

[0061] Reaction:

[0062]

[0063] 6.9g of sodium hydrogen was freed with 150mL of tetrahydrofuran and added to a 1L reaction bottle. 64.2g of triethyl phosphonoacetate was dissolved in 150mL of tetrahydrofuran, and the temperature was controlled below 0°C after nitrogen replacement protection and slowly dripped into the reaction solution. After the dripping was completed, 30.0g of formula 1 was dissolved in 150mL of tetrahydrofuran and slowly dripped into the reaction solution under nitrogen protection and temperature control below 0°C. After the dropwise addition was completed, the temperature was raised to 20°C for reaction, and TLC was monitored until the reaction of the raw materials was complete. 200mL of ice water was added to quench the reaction, 200mL of n-hexane was used for extraction, 200mL of saturated brine was used for washing, an appropriate amount of anhydrous sodium sulfate was dried, and then concentrated to dryness under reduced pressure, and then purified by column chromatography (n-hexane was used to moisten the column, wet loading, and the product was eluted with n-hexane) and concentrated to dryness under reduced pressure to obtain 28.0g of formula 2 compound, with a yield of 73.6%.

[0064] Step 2: Preparation of compound of formula 3

[0065] Reaction:

[0066]

[0067] Dissolve 20.0g of the compound of formula 2 in 150mL of tetrahydrofuran, add it to a 500mL reaction bottle, and cool it to -10°C. Dilute 21.0g of sodium dihydrobis(dimethoxyethoxy)aluminate with 50mL of tetrahydrofuran and slowly drop it into the reaction solution, keeping the temperature below 0°C during the dropwise addition. After the dropwise addition is completed, move to room temperature for reaction, monitor by TLC until the reaction of the raw material is complete, add 100mL of saturated aqueous ammonium chloride to quench the reaction, filter with suction, extract with 200mL of n-hexane, wash with 200mL of saturated brine, dry with an appropriate amount of anhydrous sodium sulfate, and concentrate to dryness under reduced pressure, and then purify by column chromatography (n-hexane moistening column, wet loading, eluting the product with n-hexane: ethyl acetate = 30:1) and concentrate to dryness under reduced pressure to obtain 11.3g of pure compound of formula 3, with a yield of 64.7%.

[0068] Step 3: Preparation of compound of formula 4

[0069] Reaction:

[0070]

[0071] 9.5 g of the compound of formula 3 was dissolved in 50 mL of tetrahydrofuran and added to a 200 mL reaction bottle. 4.0 g of phosphorus tribromide was dissolved in 50 mL of tetrahydrofuran and then slowly dripped into the reaction solution under nitrogen protection at a temperature of -10°C to -5°C. After the dripping, the reaction was kept warm and shaded, and TLC was monitored until the reaction of the raw materials was complete. 50 mL of ice water was added to quench the reaction, and 150 mL of n-hexane was used for extraction, 100 mL of saturated sodium chloride solution was used for washing, and an appropriate amount of anhydrous sodium sulfate was used for drying. After concentration under reduced pressure, 10.8 g of the compound of formula 4 was obtained, with a yield of 93.5%.

[0072] Step 4: Preparation of compound of formula 6

[0073] Reaction:

[0074]

[0075] Take 40.0g of compound 5, add it to a 1L reaction bottle and dissolve it with 200mL of methanol, then add 60mL of glacial acetic acid and 60.2g of cyclopentadiene, heat to 55°C for reaction, monitor by TLC until the reaction of the raw materials is complete, concentrate the solvent under reduced pressure to dryness, then add 200mL of ethyl acetate to the concentrate to dissolve it, and add 200mL of water to wash, after stratification, wash the ethyl acetate layer with 200mL of saturated sodium bicarbonate solution, and then wash with 200mL of saturated sodium chloride solution. After drying with an appropriate amount of anhydrous sodium sulfate, the solvent is concentrated under reduced pressure to dryness. The concentrate is dispersed with 120mL of methanol and transferred to a 500mL reaction bottle, heated to 50-60°C for dissolution, slowly cooled to -10°C for crystallization for 1h, filtered, and dried to obtain 45.0g of white solid, i.e., compound 6, with a yield of 81.3%.

[0076] Step 5: Preparation of compound of formula 7

[0077] Reaction:

[0078]

[0079] 7.9g potassium tert-butoxide was dissolved in 150mL tetrahydrofuran and added to a 500mL reaction bottle. 5.6g of compound 6 was dissolved in 50mL tetrahydrofuran and placed in a dropping funnel. Under nitrogen protection, the temperature was controlled to be below 0°C and slowly added. After the drop was completed, the reaction was kept warm for 20-30min. After that, 50mL tetrahydrofuran solution of formula 4 (10.8g) was slowly added to the reaction solution under the conditions of light protection and nitrogen protection. After the drop was completed, the reaction was kept warm. TLC was monitored until the reaction of the raw material was completed. 100mL saturated ammonium chloride aqueous solution was added to quench the reaction, 200mL n-hexane was extracted, 100mL saturated salt water was washed, and an appropriate amount of anhydrous sodium sulfate was dried and concentrated under reduced pressure to obtain 10.2g compound 7, with a yield of 85.0%.

[0080] Step 6: Preparation of Menatetrenone Impurity A

[0081] Reaction:

[0082]

[0083] Add 100 mL of toluene to 8.3 g of formula 7, and heat to 100 °C for reaction. Monitor by TLC until the reaction of the raw material is complete, and concentrate the reaction solution under reduced pressure. Purify by column chromatography to obtain 6.3 g of impurity A, with a yield of 87.1%. HPLC purity: 95.38%, HPLC spectrum: Figure 2 , the atomic number of the menatetrenone impurity A is shown in Figure 3 , H NMR spectrum see Figure 4 , NMR carbon spectrum Figure 5 , [M+Na] + The m / z of is 467.33. The data are shown in Tables 2 and 3.

[0084] Table 2 Results of H NMR spectroscopy

[0085]

[0086]

[0087] Table 3 Results of carbon NMR spectrometry

[0088]

[0089]

[0090] Example 2 Detection of Impurity A in Menatetrenone

[0091] A method for detecting impurity A in menatetrenone raw materials can effectively separate impurity A and other impurities, and specifically comprises the following steps:

[0092] (1) Sample configuration:

[0093] Preparation of test solution: Take an appropriate amount of menatetrenone, weigh accurately, dissolve with solvent and quantitatively dilute to make a solution containing about 0.5 mg per 1 mL;

[0094] Preparation of reference solution: Take an appropriate amount of menatetrenone reference substance, weigh accurately, dissolve with solvent and quantitatively dilute to make a solution containing about 0.75 μg per 1 mL;

[0095] Preparation of system suitability solution: Take 2.5 mg of impurity A, accurately weigh it, place it in a 25 mL volumetric flask, add diluent to dissolve and dilute to the mark, shake well; accurately measure 2 mL, place it in a 20 mL volumetric flask, dilute to the mark with diluent, shake well, as the stock solution. Take about 10 mg of tetraene menadione reference substance, accurately weigh it, place it in a 20 mL volumetric flask, accurately add 1.5 mL of stock solution, add diluent to dissolve and dilute to the mark, shake well, and the solution is ready.

[0096] (2) Instruments and chromatographic conditions: Chromatographic column: Silica gel column with amylose-tris(3,5-dichlorophenylcarbamate) covalently bonded to the surface IE, specification: 4.6mm×250mm, 5μm; column temperature: 35℃; flow rate: 1.0mL / min; mobile phase: n-hexane-ethanol-diethylamine (98:2).

[0097] (3) Detection and calculation:

[0098] Accurately measure 20 μL of the system suitability solution and inject it into the liquid chromatograph. Then accurately measure 10 μL each of the control solution and the test solution and inject them into the liquid chromatograph respectively, and record the chromatogram.

[0099] The results are as follows Figure 6 It shows that the test impurity A and other impurities are well separated from the main peak, and the sensitivity and response are good.

Claims

1. A method for preparing a menatetrenone impurity A, wherein the impurity is a menatetrenone impurity A, and the structural formula thereof is as follows: It is characterized in that The reaction route of the preparation method is as follows:

2. The preparation method according to claim 1, characterized in that: The preparation method steps are as follows: (1) Using the compound of formula 1 as a raw material and tetrahydrofuran as a solvent, adding a base, dripping a tetrahydrofuran solution of triethyl phosphonoacetate, stirring at room temperature for 1.5 hours, dripping a tetrahydrofuran solution of the compound of formula 1, after the reaction is completed, extracting, purifying by column chromatography, and concentrating under reduced pressure to dryness to obtain a compound of formula 2; (2) dissolving the compound of formula 2 obtained in step (1) in tetrahydrofuran, reducing the ester group with a reducing agent, and obtaining a compound of formula 3 by extraction and column chromatography purification; (3) dissolving the compound of formula 3 obtained in step (2) in tetrahydrofuran, adding phosphorus tribromide dropwise at below 0° C. to carry out bromination reaction, and obtaining the compound of formula 4 after extraction, washing and concentration; (4) The compound of formula 5 is used as raw material 2, dissolved in methanol, and then glacial acetic acid and cyclopentadiene are added thereto, and the temperature is raised to 50° C. for reaction. After the reaction is completed, the compound is concentrated, recrystallized, and dried to obtain a white solid, namely the compound of formula 6; (5) controlling the temperature below 0° C., dissolving potassium tert-butoxide in a tetrahydrofuran solution, adding dropwise the tetrahydrofuran solution of the compound of formula 6 obtained in step (4), and reacting at this temperature for 20 to 30 minutes, then adding dropwise the tetrahydrofuran solution of the compound of formula 4 obtained in step (3), and reacting at this temperature, and then extracting, drying, concentrating, and column chromatography to obtain a compound of formula 7; (6) Add toluene to the compound of formula 7 obtained in step (5), raise the temperature to 100° C. for reaction, and obtain the tetraene-methyl-menaquinone impurity A after concentration and column chromatography purification.

3. The preparation method according to claim 2, characterized in that: The base used in step (1) is selected from one or more of sodium hydrogen sulfide, sodium methoxide, sodium ethoxide, and lithium diisopropylamide.

4. The preparation method according to claim 2, characterized in that: The reducing agent used in step (2) is selected from one or more of dihydrogen bis(dimethoxyethoxy)aluminate sodium, lithium tri-sec-butylborohydride, lithium aluminum hydride, and diisobutylaluminum hydride.

5. The preparation method according to claim 2, characterized in that: The eluent used for column chromatography separation in step (2) is a n-hexane-ethyl acetate solution, with n-hexane:ethyl acetate = 30:

1.

6. The preparation method according to claim 2, characterized in that: In step (5), the eluent used for column chromatography separation is n-hexane-ethyl acetate solution, with n-hexane:ethyl acetate = 200:1 to elute impurities and 100:1 to elute the main product.

7. The preparation method according to claim 2, characterized in that: In step (6), the eluent used for column chromatography purification is n-hexane-ethyl acetate solution, n-hexane:ethyl acetate=100:

1.

8. Application of the preparation method according to any one of claims 1 to 7 in drug process research, which can be used for the study of menatetrenone impurities.

9. A method for detecting impurity A in menatetrenone bulk drug, comprising the following steps: (1) Instrument and chromatographic conditions: Chromatographic column: Silica gel column with amylose-tris(3,5-dichlorophenylcarbamate) covalently bonded to the surface IE, specifications are 4.6mm×250mm, 5μm; Mobile phase: a mixed solution of n-hexane and ethanol; Column temperature: 35°C; Flow rate: 1.0 mL / min; Injection volume: 10 μL; Detection wavelength: 270nm, (2) Sample configuration: Preparation of test solution: Take an appropriate amount of menatetrenone, weigh accurately, dissolve and dilute with diluent to make a solution containing about 0.5 mg per 1 mL; Preparation of reference solution: Take an appropriate amount of menatetrenone reference substance, weigh accurately, dissolve with solvent and quantitatively dilute to make a solution containing about 0.75 μg per 1 mL; Preparation of system suitability solution: take 2.5 mg of impurity A, accurately weigh it, place it in a 25 mL volumetric flask, add diluent to dissolve and dilute to the scale, shake well; accurately measure 2 mL, place it in a 20 mL volumetric flask, dilute to the scale with diluent, shake well, as the stock solution; take about 10 mg of tetraene methyl quinone reference substance, accurately weigh it, place it in a 20 mL volumetric flask, accurately add 1.5 mL of stock solution, add diluent to dissolve and dilute to the scale, shake well, and obtain; (3) Detection and calculation: Accurately measure 20 μL of the system suitability solution and inject it into the liquid chromatograph. Then accurately measure 10 μL each of the control solution and the test solution and inject them into the liquid chromatograph respectively, and record the chromatogram.

10. The method according to claim 9, characterized in that The diluent and the mobile phase are a mixed solution of n-hexane and ethanol, with a volume ratio of 98:2.

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