Preparation method of menadione impurity B

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 B. According to the preparation method, (5E, 9Z)-farnesyl acetone is taken as a raw material, and the menadione impurity B is obtained through Wittig-Horner, reduction, bromination, substitution and reverse Diels-Alder reaction. 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 B. Background Art

[0002] Menatetrenone, chemical name: 2-methyl-3-[(2E,6E,10E)-3,7,11,15-tetramethylhexadecanedetraenyl]-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] However, the chemical properties of tetraene methyl quinone are relatively unstable and sensitive to light and heat. It contains multiple double bonds in its structure, which makes it easy to produce multiple isomers and other impurities during the synthesis process. For example, the 6-position double bond of the side chain of tetraene methyl quinone changes under light conditions, thereby degrading to form a cis isomer, i.e., impurity B. In addition, impurity B will inevitably be formed in the synthesis process. These isomer 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 Pharmacopoeia of my country, cis isomers are known important impurities and are key indicators 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 a 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 quinone impurity B. The preparation method has the advantages of simple process, low equipment requirements, good reaction selectivity, etc., and the detection method is simple and has strong applicability, which provides effective technical support for the quality control of tetraene methyl quinone.

[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 B, 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-methylmenaquinone impurity B 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, and n-hexane:ethyl acetate=100:1 is used to elute the main product.

[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 B 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 impurity B, 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;

[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) Improving impurity removal efficiency and product safety: The present invention provides a novel method for preparing tetraene methyl quinone impurity B, which can effectively prepare impurity B with high liquid phase purity (purity 97.40%), and can significantly improve the impurity removal efficiency in tetraene methyl quinone crude products, thereby reducing safety hazards in the product, which is of great significance for improving the safety and reliability of the final product.

[0042] (2) Optimizing the production process and ensuring quality control: The preparation method of the present invention is simple in process, has low requirements on equipment, and has good reaction selectivity, which provides a more effective and economical production solution for the manufacturing process of tetraene methyl quinone. 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 methyl quinone.

[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 B 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 It is the HPLC spectrum of the related impurity B of menatetrenone.

[0045] Figure 2 This is a schematic diagram of the atomic numbering of the tetraene menaquinone impurity B.

[0046] Figure 3 This is the hydrogen nuclear magnetic resonance spectrum of the related impurity B of tetraene menaquinone.

[0047] Figure 4 This is the carbon NMR spectrum of the impurity B of tetraene menaquinone.

[0048] Figure 5 HPLC spectrum for the detection of related substances of pure menatetrenone. DETAILED DESCRIPTION

[0049] 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.

[0050] During the process of developing the synthesis of menatetrenone, we found that menatetrenone was easily degraded under light conditions to generate the 6-cis isomer (impurity B). In addition, impurity B is inevitably produced in the synthesis process. This impurity is difficult to remove by traditional purification methods such as column chromatography and recrystallization, and cannot be completely removed during the reaction process, which will bring hidden dangers to product safety.

[0051] The present invention uses (5E, 9Z)-farnesyl acetone as a raw material, and obtains the tetraene menaquinone impurity B through Wittig-Horner, reduction, bromination, substitution, and reverse Diels-Alder reactions, and the structure is confirmed. The structural formula and name are as follows:

[0052] 2-Methyl-3-[(2E,6Z,10E)-3,7,11,15-tetramethylhexadeca-2,6,10,14-hexadecatetraenyl]-1,4-naphthoquinone.

[0053]

[0054] The present invention aims to protect the preparation method and detection method of impurity B, which are used for quality control of menatetrenone to ensure the product quality and medication safety of menatetrenone.

[0055] Example 1 Preparation of Tetetrazolone Impurity B

[0056] Step 1: Preparation of compound of formula 2

[0057] Reaction:

[0058]

[0059] Dissolve 6.5g of sodium hydride in 50ml of dry tetrahydrofuran and add it to a 500ml reaction bottle. Dissolve 21.5g of triethyl phosphonoacetate in 50ml of dry tetrahydrofuran and slowly drip it into the reaction solution at a temperature below 0°C after nitrogen replacement. After dripping, move to room temperature and stir for 1.5h, then dissolve 10.0g of formula 1 in 50ml of dry tetrahydrofuran and slowly drip it into the reaction solution at a temperature below 0°C under nitrogen protection. After the addition was completed, the temperature was raised to 20°C for reaction. TLC was monitored until the reaction of the raw materials was completed. 100 ml of ice water was added to quench the reaction, and 200 ml of n-hexane was used for extraction. The mixture was washed with 200 ml of saturated brine, dried with an appropriate amount of anhydrous sodium sulfate, and concentrated to dryness under reduced pressure. The mixture was purified by silica gel column chromatography (n-hexane was used to moisten the column, wet loading was performed, impurities were eluted with n-hexane, and the product was eluted with n-hexane: ethyl acetate = 30:1) and concentrated to dryness under reduced pressure to obtain 11.5 g of the compound of formula 2 with a yield of 90.7%.

[0060] Step 2: Preparation of compound of formula 3

[0061] Reaction:

[0062]

[0063] Dissolve 10.0g of compound 2 in 50ml dry tetrahydrofuran, add to a 300ml reaction bottle, and cool to -10°C. Dilute 10.5g sodium dihydrobis(dimethoxyethoxy)aluminate with 50ml dry tetrahydrofuran and slowly drop 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 saturated aqueous ammonium chloride solution to quench the reaction, filter and extract with 200ml n-hexane, wash with 200ml saturated brine, dry with an appropriate amount of anhydrous sodium sulfate, and concentrate to dryness under reduced pressure, and then purify by silica gel column chromatography (n-hexane moistening column, wet loading, n-hexane: ethyl acetate = 30:1 to elute impurities, n-hexane: ethyl acetate = 10:1 to elute products) and concentrate to dryness under reduced pressure to obtain 6.2g of pure compound 3, with a yield of 71.2%.

[0064] Step 3: Preparation of compound of formula 4

[0065] Reaction:

[0066]

[0067] 3.5g of the compound of formula 3 was dissolved in 25ml of dry tetrahydrofuran, added to a 200ml reaction bottle, a little pyridine was added thereto, and 1.5g of phosphorus tribromide was dissolved in 25ml of dry tetrahydrofuran, and then the temperature was controlled at -10°C to below -5°C under nitrogen protection, and then slowly dripped into the reaction solution. After the dripping, the reaction was kept warm and shaded, and TLC was monitored until the reaction of the raw materials was completed, 50ml of ice water was added to quench the reaction, and 150ml of n-hexane was used for extraction, 100ml of saturated sodium chloride solution was used for washing, and an appropriate amount of anhydrous sodium sulfate was used for drying, and 4.0g of the compound of formula 4 was obtained after concentration under reduced pressure, with a yield of 93.9%.

[0068] Step 4: Preparation of compound of formula 6

[0069] Reaction:

[0070]

[0071] Take 40.0g of compound 5, add it to a 1L reaction bottle and dissolve it with 200ml methanol, then add 60ml glacial acetic acid and 60.2g cyclopentadiene, heat to 55℃ to react, monitor by TLC until the reaction of the raw materials is complete, concentrate the solvent under reduced pressure to dryness, then add 200ml ethyl acetate to the concentrate to dissolve it, and add 200ml water to wash, after stratification, wash the ethyl acetate layer with 200ml saturated sodium bicarbonate solution, and then wash with 200ml 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 methanol and transferred to a 500ml reaction bottle, heated to 50-60℃ to dissolve, slowly cooled to -10℃ to crystallize for 1h, filtered, and the filter cake is dried to obtain 45.0g of white solid, i.e. compound 6, with a yield of 81.3%.

[0072] Step 5: Preparation of compound of formula 7

[0073] Reaction:

[0074]

[0075] 2.9g potassium tert-butoxide was dissolved in 50ml tetrahydrofuran and added to a 200ml reaction bottle. Another 2.0g compound of formula 6 was dissolved in 25ml tetrahydrofuran and placed in a dropping funnel. Under nitrogen protection, the temperature was controlled to be below 0°C and slowly added dropwise. After the dropwise addition, the reaction was kept warm for 20-30min. After that, 25ml tetrahydrofuran solution of formula 4 (4.0g) was slowly added dropwise to the reaction solution under the conditions of light protection and nitrogen protection. After the dropwise addition, the reaction was kept warm. TLC was monitored until the reaction of the raw materials was complete. 100ml saturated aqueous ammonium chloride solution was slowly added dropwise to quench the reaction. 200ml n-hexane was extracted, 100ml saturated brine was washed, and an appropriate amount of anhydrous sodium sulfate was dried and concentrated under reduced pressure to obtain 3.9g compound of formula 7, with a yield of 91.0%.

[0076] Step 6: Preparation of Menatetetrazolone Impurity B

[0077] Reaction:

[0078]

[0079] Add 50 ml of toluene to 3.9 g of formula 9, heat to 100 °C and react. Monitor by TLC until the reaction of the raw material is complete, concentrate the reaction solution under reduced pressure, and purify it by silica gel column chromatography (n-hexane column, wet loading, n-hexane: ethyl acetate = 100:1 to elute the product) to obtain 2.5 g of impurity B, with a yield of 73.7%. HPLC purity: 97.40%, HPLC spectrum: Figure 1 , the atom number of the menatetrenone impurity B is shown in Figure 2 , H NMR spectrum see Figure 3 , NMR carbon spectrum Figure 4 , [MH] + The m / z of 443.35 is shown in Tables 1 and 2.

[0080] Table 1 Results of H NMR spectroscopy

[0081]

[0082]

[0083] Table 2 Results of carbon NMR spectrometry

[0084]

[0085]

[0086] Example 2 Detection of Impurity B in Menatetrenone

[0087] A method for detecting impurity B in menatetrenone raw materials can effectively separate impurity B and other impurities, and specifically comprises the following steps:

[0088] (1) Sample configuration:

[0089] 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;

[0090] 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;

[0091] Preparation of system suitability solution: Take 2.5 mg of impurity B, 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.

[0092] (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).

[0093] (3) Detection and calculation:

[0094] 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.

[0095] The results are as follows Figure 5 It shows that the test impurity B 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 B, wherein the impurity is a menatetrenone impurity B, and the structural formula is: 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 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; (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-methylmenaquinone impurity B 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, and n-hexane:ethyl acetate=100:1 is used to elute the main product.

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 B 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 B, 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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