Quality control method of dotegravir
Detection of dotelavir and dimer impurity I through high performance liquid chromatography solved the problem of insufficient research on ring-opening impurities in dotelavir production, achieved stable and controllable drug quality, provided high-purity dimer impurity I standard products, and ensured the safety and effectiveness of the drug.
Patent Information
- Application Number
- CN202410916069.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-07-09
- Publication Date
- 2025-08-26
AI Technical Summary
In the prior art, there are insufficient research on ring-opening impurities in the production process of dotelavir, resulting in unstable drug quality and difficulty in achieving precise control.
High performance liquid chromatography was used to detect dotelavir and dimer impurity I. The combination of gradient elution and specific mobile phases was combined with a phenylsilane-bonded silica gel chromatography column. The detection wavelength was 256-260 nm, the flow rate was 0.8-1.2 ml/min, and the column temperature was 25-35°C. The dotelavir and dimer impurity I were accurately separated.
The quality control of the entire process of dotetravir preparation is achieved, ensuring the safety and effectiveness of the drug, and providing high-purity dimer impurity I standard products for impurity control and detection.
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Figure CN120539296A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of drug analysis, and in particular to a quality control method for dolutegravir. Background Art
[0002] HIV-1 integrase inhibitors are the third class of anti-HIV drugs discovered after nucleoside / non-nucleoside reverse transcriptase inhibitors and protease inhibitors. Based on extensive clinical use, they have demonstrated excellent efficacy and safety and are now recommended as the preferred option in many international HIV treatment guidelines.
[0003] Dolutegravir is a new-generation HIV integrase strand transfer inhibitor jointly developed by GlaxoSmithKline (GSK) and Shionogi & Co., Ltd. of Japan. It was approved by the FDA in 2013 for a 50mg oral dose once daily under the trade name Tivicay. Several combination preparations with dolutegravir as the core drug have been launched, including JULUCA, which contains the active ingredients dolutegravir and rilpivirine and was launched on November 21, 2017. Trimeq, which contains three ingredients: abacavir, lamivudine, and dolutegravir, was launched in China in 2018. In 2019, the World Health Organization recommended dolutegravir as the first-line treatment option for all HIV patients. It was approved in March 2021 for the treatment of adult HIV-infected patients for the first time. The expansion of its indication to include stable transitioning adults and adolescents will provide more optimized treatment options for a wider range of HIV-infected patients at different treatment stages.
[0004] The chemical name of Dolutegravir is (4R,12aS)-9-{[(2,4-difluorophenyl)methyl]carbamoyl}-4-methyl-6,8-dioxo-3,4,6,8,12,12a-hexahydro-2H-pyrido[1',2':4,5]pyrazino[2,1-b][1,3]oxazine-7-enol, and its structural formula is as follows:
[0005]
[0006] Patent CN102933080 discloses a preparation route for dolutegravir. A methyl acetoacetate derivative is used as a raw material for condensation, substitution, cyclization, and deprotection reactions to generate 1-(2,2-dimethoxyethyl)-5-methoxy-6-(methoxycarbonyl)-4-oxo-1,4-dihydropyridine-3-carboxylic acid. This is followed by a deprotection reaction, cyclization with (R)-3-aminobutanol, amide condensation, and demethylation to synthesize dolutegravir in five steps. The synthetic route is shown below:
[0007]
[0008] Dolutegravir has a multifunctional tricyclic core structure. Its oxazine ring is prone to ring-opening under acidic conditions to generate ring-opening impurities. Acidic reagents are inevitably used in the production process. Therefore, the study of ring-opening impurities is essential for the quality research of dolutegravir. Currently, the known literature reports the generation mechanism and preparation method of degradation ring-opening impurities (Fan Yinbo, Wang Zhongmei, Li Xiang, et al. Synthesis and characterization of dolutegravir-related substances [J]. Zhejiang Chemical Industry, 52, 3 (2021), 7-10):
[0009]
[0010] However, this study was limited to open-ring impurities themselves and did not address specific impurities generated by derivatization of key reagents and materials. In reality, such impurities may undergo multiple transfer and derivatization processes during process transfer, hindering the study of drug quality.
[0011] In drug development, demonstrating drug safety and efficacy should be a constant concern for researchers. Stable and controllable drug quality is the prerequisite and foundation for ensuring safety and efficacy. If a drug's quality cannot be stable and controllable, it cannot remain safe and effective during use and, therefore, cannot be approved for marketing. To ensure stable and controllable drug quality, drug purity is crucial. Therefore, during the R&D process, comprehensive research on impurities in drugs is essential, ensuring that these impurities are completely and accurately controlled within a reasonable range.
[0012] Therefore, it is necessary to develop a stable and reliable method for detecting impurities derived from open-ring impurities in order to achieve the goal of accurately controlling product quality. Summary of the Invention
[0013] To solve the above problems, the present invention provides a quality control method for dolutegravir. The quality control method has accurate and reliable detection results and is applicable to the quality control of the entire process of dolutegravir preparation, raw materials and preparations, which is conducive to ensuring the effectiveness and safety of dolutegravir.
[0014] The present invention also provides a dolutegravir dimer impurity I and a preparation method and application thereof, which solves the problem of lack of types, structures and synthesis methods of impurities in dolutegravir raw materials in the prior art, thereby better controlling the quality of dolutegravir raw materials.
[0015] A first aspect of the present invention provides a quality control method for dolutegravir, comprising the following steps:
[0016] S1. Prepare standard reference solutions of dolutegravir and dimer impurity I;
[0017] S2. Prepare the test solution of dolutegravir;
[0018] S3. Chromatographic results of standard reference substances and test samples were detected by high performance liquid chromatography; wherein, mobile phase A was EDTA-2Na salt solution, mobile phase B was an organic solvent, and the elution procedure was gradient elution;
[0019] S4. Determine the content of dolutegravir and dimer impurity I in the test sample based on the chromatographic results of the standard reference substance and the test sample;
[0020] Wherein, the dimer impurity I is 2,2'-((2R,2'R)-oxybis(butane-4,2-diyl))bis(N-(2,4-difluorobenzyl)-9-hydroxy-1,8-dioxo-1,8-dihydro-2H-pyrido[1,2-a]pyrazine-7-carboxamide, and its structure is shown in the following formula I:
[0021]
[0022] The dimer impurity I is prepared by the following steps:
[0023] A. Under acidic conditions, compound A undergoes a ring-opening etherification reaction to obtain compound B;
[0024] B. Compound B undergoes a demethylation reaction to obtain Compound I, i.e., dimer impurity I;
[0025]
[0026] In another preferred embodiment, the mobile phase A is 0.1-0.5 g / L EDTA-2Na buffered saline solution.
[0027] In another preferred embodiment, the mobile phase A is 0.15-0.20 g / L EDTA-2Na buffered saline solution.
[0028] In a preferred embodiment, the pH of the mobile phase A used is 2-5.
[0029] In another preferred embodiment, the mobile phase B is a mixed solution of methanol and tetrahydrofuran.
[0030] In another preferred embodiment, the mobile phase B is a mixed solution of methanol and tetrahydrofuran, wherein the volume ratio of methanol to tetrahydrofuran is (5-10):1; preferably (8-10):1.
[0031] In a preferred embodiment, the gradient elution program comprises: based on the volume ratio of mobile phase A, 0-30 minutes: decreasing from 60% to 50%, 30-40 minutes: decreasing from 50% to 30%, 40-60 minutes: maintaining at 30%, 60-62 minutes: increasing from 30% to 55-65%, 62-70 minutes: maintaining at 55-65%.
[0032] In another preferred embodiment, the gradient elution procedure comprises:
[0033]
[0034] In a preferred embodiment, the high performance liquid chromatography method uses a phenylsilane bonded silica gel column, a detection wavelength of 256 to 260 nm, a mobile phase flow rate of 0.8 to 1.2 ml / min, and a column temperature of 25 to 35°C.
[0035] In another preferred embodiment, the high performance liquid chromatography method is a pentafluorophenylsilane bonded silica gel chromatography column.
[0036] In another preferred embodiment, the chromatographic column is Supelco Ascentis Express F5, 4.6mm×250mm, 5μm.
[0037] In another preferred embodiment, the detection wavelength is 258 nm, the flow rate of the mobile phase is 0.8 ml / min, and the column temperature is 30°C.
[0038] In a preferred embodiment, the ring-opening etherification reaction and the demethylation reaction are carried out in the presence of solvent A and solvent B, respectively, and the solvent A and solvent B are independently selected from the following group: tetrahydrofuran, acetonitrile, methanol, ethanol, isopropanol, or a combination thereof.
[0039] In another preferred embodiment, the solvent A and solvent B are independently selected from the following group: tetrahydrofuran, acetonitrile, or a combination thereof.
[0040] In a preferred embodiment, in step A, the acidic condition is provided by an acidic reagent, and the acidic reagent is selected from the following group: hydrochloric acid, sulfuric acid, methanesulfonic acid, glacial acetic acid, formic acid, or a combination thereof.
[0041] In another preferred embodiment, in step A, the acidic reagent is selected from the group consisting of hydrochloric acid, methanesulfonic acid, or a combination thereof.
[0042] In a preferred embodiment, the molar ratio of the acidic reagent to compound A is 1:(500-1000); preferably 1:(700-1000); more preferably 1:(900-1000).
[0043] In another preferred embodiment, in step A, the temperature of the ring-opening etherification reaction is 60-80°C; preferably 70-80°C; more preferably 75-80°C.
[0044] In another preferred embodiment, in step A, the ring-opening etherification reaction time is 20 to 30 hours, preferably 20 to 26 hours.
[0045] In a preferred embodiment, in step B, the demethylation agent is selected from the group consisting of lithium bromide, magnesium bromide, sodium bromide, potassium bromide, or a combination thereof.
[0046] In another preferred embodiment, in the step B, the demethylation agent is lithium bromide.
[0047] In a preferred embodiment, in the step B, the molar ratio of the compound B to the demethylation agent is 1:(1-10); preferably 1:(2-8); more preferably 1:(3-6).
[0048] In another preferred embodiment, in the step B, the temperature of the demethylation reaction is 40-66°C; preferably 50-66°C; more preferably 65-66°C.
[0049] In another preferred embodiment, in the step B, the demethylation reaction time is 1 to 9 hours; preferably 2 to 7 hours; more preferably 2 to 4 hours.
[0050] In another preferred embodiment, the step B comprises the following steps:
[0051] B-1. In the presence of a demethylating agent, compound B is subjected to a demethylation reaction to obtain a crude solution of compound I;
[0052] B-1. Solvent C was added to the crude solution obtained in step B-1, the liquids were separated, concentrated under reduced pressure, recrystallized, stirred, filtered, and dried to obtain compound I, ie, dimer impurity I;
[0053] The solvent C is selected from the following group: a mixed solution of glacial acetic acid and 5-15% NaOH, a mixed solution of formic acid and 5-15% sodium bicarbonate, or a combination thereof.
[0054] In another preferred embodiment, in step B, the mass volume ratio of the glacial acetic acid to 5-15% NaOH is 1:(10-35) g / mL; preferably 1:(20-30) g / mL; more preferably 1:(23-27) g / mL.
[0055] In another preferred embodiment, in the step B, the solvent used for the recrystallization is selected from the group consisting of ethanol, water, methanol, isopropanol, acetonitrile, or a combination thereof.
[0056] In a preferred example, the detection limit of the dimer impurity I is 0.046 μg / ml.
[0057] In another preferred embodiment, the dolutegravir test sample is a dolutegravir solid preparation or a dolutegravir liquid preparation.
[0058] In another preferred embodiment, the standard reference solution and the test solution are both prepared with an acetonitrile aqueous solution having a volume fraction of 30% to 50%.
[0059] In another preferred embodiment, the mass concentration of the test solution is 0.2-1.0 mg / mL; preferably 0.2-0.6 mg / mL.
[0060] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one. DETAILED DESCRIPTION
[0061] After extensive and lengthy research and extensive screening, the inventors have developed a quality control method for dolutegravir. This method, which yields accurate and reliable results, is applicable to the entire dolutegravir production process, including quality control of APIs and formulations, and helps ensure the effectiveness and safety of dolutegravir. Furthermore, the inventors have developed a novel, previously unreported route for preparing high-purity dolutegravir dimer impurity I. Based on this, the applicants have completed the present invention.
[0062] the term
[0063] As used herein, "dolutegravir dimer impurity I" and "impurity I" are synonymous and refer to the compound of formula I, i.e., compound 2,2'-((2R,2'R)-oxybis(butane-4,2-diyl))bis(N-(2,4-difluorobenzyl)-9-hydroxy-1,8-dioxo-1,8-dihydro-2H-pyrido[1,2-a]pyrazine-7-carboxamide).
[0064]
[0065] Preparation method of standard reference substance of dolutegravir dimer impurity I
[0066] In the present invention, the synthesis steps of the target product compound of formula I include:
[0067] A. Under acidic conditions, compound A undergoes a ring-opening etherification reaction to obtain compound B;
[0068] B. Compound B undergoes a demethylation reaction to obtain Compound I, i.e., dimer impurity I;
[0069]
[0070] The ring-opening etherification reaction and the demethylation reaction are carried out in the presence of solvent A and solvent B, respectively. The solvent A and solvent B are independently selected from the following group: tetrahydrofuran, acetonitrile, methanol, ethanol, isopropanol, or a combination thereof.
[0071] In another preferred embodiment, in step A, the acidic condition is provided by an acidic reagent, which is not particularly limited and can be preferably selected from the following group: hydrochloric acid, sulfuric acid, methanesulfonic acid, glacial acetic acid, formic acid, or a combination thereof.
[0072] In another preferred embodiment, in step A, the temperature of the ring-opening etherification reaction is 60-80°C.
[0073] In another preferred embodiment, in step A, the ring-opening etherification reaction time is 20 to 30 hours, preferably 20 to 26 hours.
[0074] In another preferred embodiment, in step B, the demethylation agent is selected from the group consisting of lithium bromide, magnesium bromide, sodium bromide, potassium bromide, or a combination thereof.
[0075] In another preferred embodiment, in the step B, the temperature of the demethylation reaction is 40-66°C; preferably 50-66°C; more preferably 65-66°C.
[0076] In another preferred embodiment, in the step B, the demethylation reaction time is 1 to 9 hours; preferably 2 to 7 hours; more preferably 2 to 4 hours.
[0077] In another preferred embodiment, the step B comprises the following steps:
[0078] B-1. In the presence of a demethylating agent, compound B is subjected to a demethylation reaction to obtain a crude solution of compound I;
[0079] B-1. Solvent C was added to the crude solution obtained in step B-1, the liquids were separated, concentrated under reduced pressure, recrystallized, stirred, filtered, and dried to obtain compound I, ie, dimer impurity I;
[0080] The solvent C is selected from the following group: a mixed solution of glacial acetic acid and 5-15% NaOH, a mixed solution of formic acid and 5-15% sodium bicarbonate, or a combination thereof.
[0081] In another preferred embodiment, in step B, the mass volume ratio of the glacial acetic acid to 5-15% NaOH is 1:(10-35) g / mL; preferably 1:(20-30) g / mL; more preferably 1:(23-27) g / mL.
[0082] In another preferred embodiment, in the step B, the solvent used for the recrystallization is selected from the group consisting of ethanol, water, methanol, isopropanol, acetonitrile, or a combination thereof.
[0083] Preferably, the application of the dolutegravir dimer impurity I in the field of quality control includes the preparation of impurity reference substances and the analysis and detection of dolutegravir raw materials and preparations, wherein the dolutegravir preparations include but are not limited to solid preparations and liquid preparations, and the solid preparations are preferably tablets.
[0084] The main advantages of the present invention are:
[0085] 1. The quality control method of the present invention has accurate and reliable detection results and is applicable to the quality control of the entire process of dolutegravir preparation, raw materials and preparations, which is beneficial to the process research and quality control of dolutegravir and has great significance for ensuring the safety of patient medication and enterprise production.
[0086] 2. The dolutegravir dimer impurity I provided by the present invention has a novel structure, and its preparation method is simple, efficient, easy to operate, and the raw materials are readily available.
[0087] 3. The dimer impurity I prepared according to the preparation method of the present invention has a high purity and can be used as a reference substance or standard for the impurity of dolutegravir sodium, thereby better controlling the quality of the dolutegravir sodium raw material.
[0088] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention and are not intended to limit the scope of the invention. The experimental methods in the following examples, for which no specific conditions are specified, are generally carried out under conventional conditions or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are percentages by weight and parts by weight.
[0089] Unless otherwise specified, the detection conditions of high performance liquid chromatography (HPLC) were the same as those in Example 1.
[0090] Example 1. Quality Control Method for Dolutegravir
[0091] 1.1 Preparation of Dolutegravir Dimer Impurity I Standard Reference Substance
[0092]
[0093] (1) Preparation of Compound B
[0094] In a reaction flask, (4R,12aS)-N-(2,4-difluorobenzyl)-3,4,6,8,12,12a-hexahydro-7-methoxy-4-methyl-6,8-dioxo-2H-pyrido[1',2':4,5]pyrazino[2,1-b][1,3]oxazine-9-carboxamide (40.0 g, 92.29 mol), 200 ml of tetrahydrofuran and 200 ml of acetonitrile were added, and hydrochloric acid (8 ml, 0.09 6 mol), refluxed (75-80 ° C) for 24 h, concentrated under reduced pressure, purified by column chromatography, and eluted with dichloromethane / methanol (volume ratio of 30:1) to obtain 2.1 g of 2,2'-((2R, 2'R)-oxybis(butane-4,2-diyl))bis(N-(2,4-difluorobenzyl)-9-methoxy-1,8-dioxo-1,8-dihydro-2H-pyrido[1,2-a]pyrazine-7-carboxamide), i.e., compound B (2.55 mmol).
[0095] H NMR spectrum: 1 H NMR (400MHz, DMSO-d6) δ10.60(t,J=5.9Hz,1H),8.88(s,1H),7.48(d,J=6.2Hz,1H),7.47-7.3 9(m,1H),7.27(ddd,J=10.6,9.3,2.6Hz,1H),7.09(tdd,J=8.5,2.6,1.0Hz,1H),7.03(d,J=6.3 Hz,1H),5.03-4.91(m,1H),4.57(d,J=5.9Hz,2H),3.81(s,3H),3.60(qt,J=11.1,6.7Hz,2H), 2.20(ddt,J=15.2,9.0,6.4Hz,1H), 2.06(dtd,J=14.4,7.1,5.6Hz,1H), 1.29(d,J=6.9Hz,3H).
[0096] C-NMR spectrum: 13 C NMR (101MHz, DMSO-d6) δ172.30,163.65,163.29,161.84,160.85,159.51,154.17,152.49,138.59,131. 50,130.07,122.67,120.07,115.53,112.36,112.03,104.61,60.51,47.89,42.35,36.92,36.40,18.77.
[0097] Mass spectrum: [M+MeOH+Na] + =903.31.
[0098] (2) Preparation of Dolutegravir Dimer Impurity I
[0099] Compound B (0.2 g, 0.24 mmol) and 3 ml of tetrahydrofuran were added to a reaction flask and stirred to dissolve. Lithium bromide (0.08 g, 0.92 mmol) was then added and the mixture was brought to reflux (60-66°C). After reacting for 30 h, 0.04 g of glacial acetic acid and 1 ml of 10% NaOH were added. The layers were separated and the organic layer was concentrated under reduced pressure. 1 ml of ethanol and 5 ml of water were added, the mixture was stirred for 1 h, filtered, and dried to obtain 0.19 g (0.23 mmol) of dolutegravir dimer impurity I. HPLC purity was 96.32%.
[0100] H NMR spectrum: 1 H-NMR(400MHz,Chloroform-d)δ12.03(s,1H),10.59(s,1H),8.60(s,1H),7.37( td,J=8.7,6.3Hz,1H),6.86(d,J=6.4Hz,1H),6.85-6.77(m,2H),6.32(d,J=6.3Hz ,1H),5.05-4.93(m,1H),4.67(d,J=5.9Hz,2H),3.64-3.44(m,2H),2.27(ddt,J= 15.0, 9.1, 6.0Hz, 1H), 2.13 (ddt, J=14.7, 7.5, 5.9Hz, 1H), 1.45 (d, J=6.9Hz, 3H).
[0101] C-NMR spectrum: 13 C-NMR(101MHz,Chloroform-d)δ169.42,163.86,162.04,161.04,159.57,154.65,134.92,130.4 5,121.44,118.33,116.73,114.26,113.38,111.36,104.07,49.80,40.83,36.90,28.37,18.63.
[0102] Mass spectrum: [M+MeOH+Na] + =875.21.
[0103] 1.2 Detection of Dolutegravir and Dimer Impurity I by HPLC
[0104] HPLC detection conditions:
[0105] Filling column: Pentafluorophenylsilane bonded silica gel chromatography column (Supelco Ascentis Express F5, 4.6 mm × 250 mm, 5 μm or chromatographic column of equivalent performance).
[0106] Mobile phase A: EDTA-2Na buffered saline solution (weigh 0.186 g of EDTA-2Na, dissolve in 1 L of water, and adjust the pH to 3.0 with phosphoric acid).
[0107] Mobile phase B: a mixed solution of methanol and tetrahydrofuran, wherein methanol / tetrahydrofuran = 9:1 (v / v).
[0108] Perform gradient elution according to the elution program described in the table below:
[0109]
[0110] Detection wavelength: 258nm.
[0111] Flow rate: 0.8 ml per minute.
[0112] Column temperature: 30℃.
[0113] Injection volume: 10 μl.
[0114] Diluent: acetonitrile aqueous solution, wherein acetonitrile / water = 40:60 (v / v).
[0115] Experimental methods:
[0116] S1. Preparation of standard reference solutions of dolutegravir and dolutegravir dimer impurity I
[0117] The dolutegravir dimer impurity I standard reference substance (i.e., impurity I prepared in Example 1.1) was dissolved in a diluent to prepare a solution containing approximately 0.5 μg per 1 ml, which was used as the dolutegravir dimer impurity I standard reference substance solution.
[0118] Take approximately 25 mg of dolutegravir reference substance, accurately weigh it, place it in a 50 ml volumetric flask, add 25 ml of diluent to dissolve it and dilute it to the scale, shake well to obtain a solution containing approximately 0.5 mg of this product per 1 ml.
[0119] S2. Preparation of Dolutegravir Test Solution
[0120] Take approximately 25 mg of the dolutegravir test sample, accurately weigh it, place it in a 50 ml volumetric flask, add 25 ml of diluent to dissolve it and dilute it to the scale, shake well to obtain a solution containing approximately 0.5 mg of the product per 1 ml.
[0121] S3. Use the above-mentioned HPLC detection conditions to detect the chromatographic results of the standard reference substance and the test substance.
[0122] Experimental results:
[0123] The HPLC results of the dolutegravir dimer impurity I standard reference solution are shown below, wherein the dolutegravir dimer impurity I retention time is 43.42 min.
[0124]
[0125] The HPLC results of the dolutegravir standard reference solution are shown below, wherein the retention time of dolutegravir is 34.24 min.
[0126]
[0127] The HPLC results of the test solution are shown below, wherein Peak 4 is the peak of dolutegravir and Peak 5 is the peak of impurity I.
[0128]
[0129] From the above results, it can be seen that dolutegravir is well separated from impurity I, and this method can effectively detect impurity I in dolutegravir.
[0130] Experimental conclusion:
[0131] Under the liquid phase conditions described in the present invention, the main peak (dolutegravir) and the impurity peak (dolutegravir impurity I) can be completely separated, indicating that the quality control method of the present invention can effectively detect the dolutegravir dimer impurity I, and also indicating that the quality control method of the present invention is a suitable condition for detecting the dolutegravir dimer impurity I.
[0132] Example 2. Residue of Dimer Impurity I in Dolutegravir
[0133] The residual levels of dimer impurity I in multiple batches of dolutegravir and dolutegravir mother liquor were tested using the method in Example 1. The test results are as follows:
[0134]
[0135] # The quantification limit here is the percentage of the concentration of impurity I when the signal-to-noise ratio (S / N) is not less than 10 to the concentration of the main component, that is, the quantification limit concentration of dimer impurity I / main component concentration = 0.046 μg / ml / 500 μg / ml = 0.0092%.
[0136] The test results showed that the dimer impurity I of dolutegravir was not detected in multiple batches of dolutegravir (20103-230501, 20103-230502, and 20103-230503). However, the dimer impurity I (0.12%) of dolutegravir was detected in the dolutegravir mother liquor (20103-230503-MY). This indicates that the dimer impurity I of dolutegravir is an unavoidable impurity produced during the process. In order to ensure the quality of the drug, it is necessary to study this impurity.
[0137] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the claims appended hereto.
Claims
1. A quality control method for dolutegravir, characterized in that: The method comprises the following steps: S1. Prepare standard reference solutions of dolutegravir and dimer impurity I; S2. Prepare the test solution of dolutegravir; S3. Chromatographic results of standard reference substances and test samples were detected by high performance liquid chromatography; wherein, mobile phase A was EDTA-2Na salt solution, mobile phase B was an organic solvent, and the elution procedure was gradient elution; S4. Determine the content of dolutegravir and dimer impurity I in the test sample based on the chromatographic results of the standard reference substance and the test sample; Wherein, the dimer impurity I is 2,2'-((2R,2'R)-oxybis(butane-4,2-diyl))bis(N-(2,4-difluorobenzyl)-9-hydroxy-1,8-dioxo-1,8-dihydro-2H-pyrido[1,2-a]pyrazine-7-carboxamide, and its structure is shown in the following formula I: The dimer impurity I is prepared by the following steps: A. Under acidic conditions, compound A undergoes a ring-opening etherification reaction to obtain compound B; B. Compound B undergoes a demethylation reaction to obtain Compound I, i.e., dimer impurity I; 2. The quality control method according to claim 1, wherein: The pH of the mobile phase A used was 2-5.
3. The quality control method according to claim 1, wherein: The gradient elution program includes: based on the volume ratio of mobile phase A, 0-30 minutes: reducing from 60% to 50%, 30-40 minutes: reducing from 50% to 30%, 40-60 minutes: maintaining at 30%, 60-62 minutes: increasing from 30% to 55-65%, 62-70 minutes: maintaining at 55-65%.
4. The quality control method according to claim 1, wherein: The high performance liquid chromatography method adopts a phenylsilane bonded silica gel chromatography column, a detection wavelength of 256-260 nm, a mobile phase flow rate of 0.8-1.2 ml / min, and a column temperature of 25-35°C.
5. The quality control method according to claim 1, wherein: The ring-opening etherification reaction and the demethylation reaction are carried out in the presence of solvent A and solvent B, respectively. The solvent A and solvent B are independently selected from the following group: tetrahydrofuran, acetonitrile, methanol, ethanol, isopropanol, or a combination thereof.
6. The quality control method according to claim 1, wherein: In step A, the acidic condition is provided by an acidic reagent, and the acidic reagent is selected from the following group: hydrochloric acid, sulfuric acid, methanesulfonic acid, glacial acetic acid, formic acid, or a combination thereof.
7. The quality control method according to claim 4, wherein: The molar ratio of the acidic reagent to compound A is 1:(500-1000); preferably 1:(700-1000); more preferably 1:(900-1000).
8. The quality control method according to claim 1, wherein: In step B, the demethylation agent is selected from the group consisting of lithium bromide, magnesium bromide, sodium bromide, potassium bromide, or a combination thereof.
9. The quality control method according to claim 1, wherein: In the step B, the molar ratio of the compound B to the demethylation agent is 1:(1-10); preferably 1:(2-8); more preferably 1:(3-6).
10. The quality control method according to claim 1, wherein: The detection limit of the dimer impurity I is 0.046 μg / ml.