WO2024060271A1 - 一种恩格列净原料药或制剂中的特征杂质及其应用 - Google Patents

一种恩格列净原料药或制剂中的特征杂质及其应用 Download PDF

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WO2024060271A1
WO2024060271A1 PCT/CN2022/121230 CN2022121230W WO2024060271A1 WO 2024060271 A1 WO2024060271 A1 WO 2024060271A1 CN 2022121230 W CN2022121230 W CN 2022121230W WO 2024060271 A1 WO2024060271 A1 WO 2024060271A1
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compound
impurity
empagliflozin
formula
preparation
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French (fr)
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汪海波
梅光耀
金辉
徐陈力
喻丽霞
肖扬
白旭刚
万子康
陈安途
潘英伟
蒙海平
许肖阳
张凤
席俊清
吴于娟
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浙江宏元药业股份有限公司
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D309/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only ring hetero atom, not condensed with other rings
    • C07D309/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only ring hetero atom, not condensed with other rings having no double bonds between ring members or between ring members and non-ring members
    • C07D309/08—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only ring hetero atom, not condensed with other rings having no double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D309/10—Oxygen atoms
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/351—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom not condensed with another ring
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H7/00—Compounds containing non-saccharide radicals linked to saccharide radicals by a carbon-to-carbon bond
    • C07H7/04—Carbocyclic radicals
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/26—Conditioning of the fluid carrier; Flow patterns
    • G01N30/28—Control of physical parameters of the fluid carrier
    • G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/62—Detectors specially adapted therefor
    • G01N30/72—Mass spectrometers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/62—Detectors specially adapted therefor
    • G01N30/72—Mass spectrometers
    • G01N30/7233—Mass spectrometers interfaced to liquid or supercritical fluid chromatograph

Definitions

  • the invention belongs to the technical fields of pharmacy and analytical chemistry, and relates to characteristic impurities in empagliflozin raw materials or preparations and their applications. More specifically, the present invention relates to an empagliflozin dimer impurity and its application in the quality control of empagliflozin raw materials or preparations.
  • Diabetes is a group of metabolic diseases characterized by hyperglycemia.
  • the International Diabetes Federation (IDF) predicts that the number of diabetic patients worldwide will reach nearly 6 billion by 2035.
  • IDF International Diabetes Federation
  • diabetes is clinically divided into two types, namely insulin-dependent diabetes (type I diabetes) and non-insulin-dependent diabetes (type II diabetes), of which type II diabetes patients account for more than 90%.
  • Empagliflozin is a sodium-glucose co-transporter 2 (SGLT-2) inhibitor developed by Boehringer Ingelheim. It was first approved for the treatment of type 2 diabetes in May 2014 and has become a mainstream drug in the field of diabetes treatment.
  • SGLT-2 sodium-glucose co-transporter 2
  • Impurities in empagliflozin as a human drug have been studied for many years.
  • the main characteristic process impurities in the synthesis process of empagliflozin API include five-membered ring impurities (Impurity A ), ring-opening impurities (Impurity B), dimer impurities (Impurity C), etc.
  • Impurities A ⁇ Impurities C are unique impurities produced during the process and can be clearly observed in the crude empagliflozin API, so most empagliflozin API suppliers will Characteristic impurities are listed in the quality standards of empagliflozin API for quality control.
  • (S)-3-hydroxytetrahydrofuran the key starting material in the synthesis process of empagliflozin. Research.
  • (S)-3-Oxytetrahydrofuran is a significant structural fragment in the empagliflozin molecule, which is introduced through the substitution reaction of related intermediates with (S)-3-hydroxytetrahydrofuran.
  • There are two main mature industrial synthesis processes for (S)-3-hydroxytetrahydrofuran namely the synthesis route using malic acid as raw material and the synthesis route using (S)-4-chloro-3-hydroxybutyric acid ethyl ester as raw material.
  • Tadon et al. first proposed a synthesis route for chiral (S)-3-hydroxytetrahydrofuran (J.Org.Chem, 1983, 48, 2767-2769). Later, many published documents optimized and improved Tadon's (S)-3-hydroxytetrahydrofuran synthesis route, such as patents CN107935971, CN107235937A and CN109503523. These routes use L-malic acid as a starting material, generate L-malic acid dimethyl ester through methyl esterification, then reduce it to chiral 1,2,4-butanetriol, and finally dehydrate and cyclize under acidic conditions to obtain the product (S)-3-hydroxytetrahydrofuran.
  • Patent documents CN102477019, CN107098872, WO2008093955, WO2000063199, EP761663, etc. report using optically pure (S)-4-chloro-3-hydroxybutyric acid ethyl ester as raw material and obtaining (S)-4-chloro-1 through ester reduction. , 3-butanediol, and then cyclized to obtain the target product (S)-3-hydroxytetrahydrofuran.
  • Impurity control is the lifeline of quality control of raw materials or preparations. Comprehensive monitoring and control of impurities is of great significance to the safety and effectiveness of raw materials and preparations. In view of the fact that there are few literatures focusing on the risk of impurities introduced by 1,4-butanediol in (S)-3-hydroxytetrahydrofuran, and considering that the detection capabilities of analytical methods are limited, impurities in empagliflozin APIs may not be detected. Condition.
  • the technical personnel of the present invention conducted a comprehensive and thorough analysis and research on the impurities that may be produced during the synthesis process of empagliflozin raw material drug, and developed effective analysis and detection methods for sample testing, and unexpectedly discovered that empagliflozin New dimer impurities generated during the synthesis of Gliflozin API.
  • the technicians of the present invention further conducted in-depth research on the in vitro pharmacological activity and in vitro cytotoxicity of this new impurity, which provided an important basis for the control of this impurity in empagliflozin raw materials or preparations.
  • the present invention provides characteristic impurities in empagliflozin raw materials or preparations and their applications.
  • the impurities play a very important role in the quality control of empagliflozin raw materials or preparations.
  • the compound described in the present invention is characterized by being used for quality control of empagliflozin raw materials or preparations.
  • the compound described in the present invention is characterized in that the content of the compound represented by formula I in the empagliflozin raw material drug is not more than 0.15% or the content in the empagliflozin preparation is not more than 0.2%.
  • the compound described in the present invention is characterized in that the content of the compound represented by formula I in the empagliflozin raw material or preparation can be detected by liquid chromatography or liquid mass spectrometry.
  • the compound described in the present invention is characterized in that when liquid chromatography or liquid mass spectrometry is used to detect the content of the compound represented by formula I in the empagliflozin raw material or preparation, the chromatographic detection conditions are: chromatographic column stationary phase It is carbon 18 silica gel, the column specification is 4.6 ⁇ 150mm ⁇ 3 ⁇ m, the mobile phase is methanol and water, the mobile phase flow rate is 0.8 ⁇ 1.0mL/min, the injection volume is 10 ⁇ 20 ⁇ L, the column temperature is 30 ⁇ 40°C, the wavelength is 225nm , running time 70min.
  • the compound described in the present invention is characterized in that when liquid chromatography or liquid mass spectrometry is used to detect the content of the compound represented by formula I in the empagliflozin raw material or preparation, when the wavelength of the ultraviolet detector is 225nm, the formula
  • the correction factor of the compound shown in I relative to empagliflozin is 0.8 to 1.2.
  • the compound described in the present invention is characterized in that when liquid chromatography or liquid mass spectrometry is used to detect the content of the compound represented by formula I in the empagliflozin raw material or preparation, the ratio of methanol:water in the chromatographic mobile phase is in order For: 0 ⁇ 10 minutes, 40:60; 10 ⁇ 15 minutes, 40:60 ⁇ 52:48; 15 ⁇ 35 minutes, 52:48; 35 ⁇ 50 minutes, 52:48 ⁇ 80:20; 50 ⁇ 60 minutes , 80:20; 60 ⁇ 60.1 minutes, 80:20 ⁇ 40:60; 60.1 ⁇ 70 minutes, 40:60.
  • X is a halogen atom, selected from bromine atoms and iodine atoms;
  • Step 1 Compound II undergoes reduction reaction to prepare compound III;
  • Step 2 Compound III and Compound IV form a carbon glycoside compound, which is then combined with methanol to form a methoxy glycoside compound, and finally is reduced to obtain Compound V;
  • Step 3 Compound V undergoes a substitution reaction with 1,4-butanediol to prepare target compound I.
  • Step 1 compound II is reduced under the action of triethylsilane and aluminum chloride to obtain compound III;
  • Step 2 Compound III and Compound IV form a carbon glycoside compound under the action of n-butyllithium at a temperature of -75 ⁇ -85°C, and form a methoxy glycoside compound with methanol under the catalysis of methanesulfonic acid at 10 ⁇ 40°C. Finally, compound V is obtained by reduction with triethylsilane and aluminum trichloride at 10-40°C;
  • Step 3 Compound V and 1,4-butanediol are heated and refluxed in tetrahydrofuran solvent under the action of potassium tert-butoxide to prepare compound I.
  • the compound of formula I described in the present invention is characterized by having a significant inhibitory effect on sodium-glucose cotransporter 2.
  • the invention relates to the use of the compound of formula I in the preparation of a drug for treating diabetes.
  • a pharmaceutical composition comprising an effective dose of the compound of formula I or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, excipient or a combination thereof.
  • the compound of formula I described in the present invention has almost the same SGLT2 inhibitory activity as empagliflozin, which means that the compound of formula I may have less impact on the effectiveness of empagliflozin API or preparation.
  • the compound of formula I according to the present invention has higher toxicity to human liver cells and kidney cells than empagliflozin. Therefore, the compound of formula I may affect the safety of empagliflozin to a great extent.
  • the compound of formula I described in the present invention has obvious SGLT2 inhibitory activity. Therefore, the compound of formula I can be prepared into any form of drug targeting the SGLT2 target. Any form of pharmaceutical composition of a compound of Formula I is deemed to be disclosed and protected by this invention.
  • the impurities mentioned in the present invention refer to starting materials, intermediates, polymers, side reaction products brought in during the product production process, as well as degradation products during storage, etc.
  • the characteristic impurities mentioned in the present invention refer to impurities that appear repeatedly in each batch of the product under specific process conditions and consistent raw material quality, and are impurities that are determined to be contained in the product.
  • Triethylsilane (16.7 g, 142.6 mmol, 1.5 eq) and 2-methyltetrahydrofuran (150 ml) were added to a 500 ml four-necked flask, cooled to 0-10 ° C, and anhydrous aluminum chloride (19.1 g, 143.6 mmol, 1.5 eq) was added in batches.
  • Compound II (30.0 g, 95.7 mmol) was dissolved in 2-methyltetrahydrofuran (90 ml), and the resulting solution was added dropwise to a mixture of sodium borohydride and aluminum chloride, and stirred at 80 ° C overnight.
  • reaction solution was cooled to 0-10 ° C, and water (120 ml) was added dropwise to quench the reaction.
  • the organic layer was separated and concentrated under reduced pressure at 40-50 ° C.
  • Ethanol 90 ml was added to the obtained slurry and stirred at room temperature for crystallization. After the solid was precipitated, water (15 ml) was added for slurrying, and filtered and dried to obtain 25.3 g of compound III-1, with a yield of 88.2%.
  • reaction solution is cooled to 0-10°C, and water (120 ml) is added dropwise to quench the reaction.
  • the organic layer was separated and concentrated under reduced pressure at 40-50°C.
  • the obtained slurry was stirred and crystallized by adding ethanol (90 ml) at room temperature. After the solid was precipitated, water (15 ml) was added to make a slurry, filtered and dried to obtain 26.1 g of compound III-2, with a yield of 90.4%.
  • Example 6 System adaptability and specificity test of liquid phase or liquid-mass chromatography detection method
  • the separation effect of the analysis method is judged by measuring the separation between two adjacent peaks.
  • System adaptability solution preparation Take 18.9 mg of impurity A, 18.7 mg of impurity B, 18.5 mg of impurity C, and 18.7 mg of the compound of formula I, accurately weigh and place it in a 25 mL volumetric flask, dissolve it in 50% methanol water and dilute it to the mark with 50% methanol water. , mix well, which is the impurity stock solution. Accurately weigh 49.7 mg of empagliflozin working reference substance, put it in a 100 mL volumetric flask, add 50% methanol water to dissolve it, transfer into 1.00 ml impurity stock solution, and dilute to the mark with 50% methanol water.
  • Needles 1-6 in the above table are system adaptability solutions, and needles 7-12 are program control needles.
  • the blank solution has no interference with the detection of empagliflozin, impurity A, impurity B, impurity C and the compound of formula I.
  • the RSD of the peak area of empagliflozin in the system adaptability solution with repeated injection of 6 injections is 0.0%, and the RSD of the peak area of impurity A is 0.0%.
  • the peak area RSD of impurity B is 0.3%, the peak area RSD of impurity C is 0.3%, and the peak area RSD of the compound of formula I is 0.1%; the maximum RSD of empagliflozin for 1-12 needles after adding programmable needles is 0.7 %, the maximum RSD of impurity A is 1.0%, the maximum RSD of impurity B is 1.0%, the maximum RSD of impurity C is 1.2%, and the maximum RSD of the compound of formula I is 0.6%.
  • the separation between empagliflozin and impurity A in the system suitability solution is 13.2
  • the separation between empagliflozin and impurity B is 6.8
  • the separation between impurity A and impurity C is 43.5
  • the separation between impurity C is 43.5
  • the resolution from the compound of formula I is 1.7.
  • Example 7 Quantitative limit and detection limit test of liquid phase or liquid-mass chromatography detection method
  • the limit of detection (LOD) and limit of quantitation (LOQ) of impurity A, impurity B, impurity C, and the compound of formula I are determined based on the signal-to-noise ratio. Solutions diluted to different concentrations are measured using the dilution method. Each quantitative When the limit is less than the acceptance standard and the signal-to-noise ratio is not less than 10, it is the quantification limit concentration. When the detection limit is diluted 3 times by the LOQ test solution and the signal-to-noise ratio is not less than 3, it is the detection limit concentration.
  • Quantitative limit impurity A, impurity B, impurity C, formula I compound mixed solution preparation take impurity A 18.9mg, impurity B 18.7mg, impurity C 18.5mg, formula I compound 18.7mg, accurately weigh and place in a 25mL volumetric flask, use 50 Dissolve % methanol water and dilute to the mark with 50% methanol water, and mix well; accurately transfer 1.00 mL to a 100 mL volumetric flask, dilute to the mark with 50% methanol water, and mix well; then accurately transfer 1.00 mL to a 100 mL volumetric flask. Dilute to volume with 50% methanol water and mix well.
  • Preparation of the mixed solution of quantitative limit impurity A, impurity B, impurity C, and compound of formula I Precisely transfer 3.0 mL of the mixed solution of quantitative limit impurity A, impurity B, impurity C, and compound of formula I into a 10 mL volumetric flask, and use 50% methanol water Dilute to volume and mix.
  • the above quantitative limit test results show that the 3-pin signal-to-noise ratio of impurity A LOQ is a minimum signal-to-noise ratio of 10.1 and the RSD value is 2.0%.
  • the 3-pin signal-to-noise ratio of impurity B LOQ is a minimum signal-to-noise ratio of 17.6 and the RSD value is 4.6%
  • the 3-pin signal-to-noise ratio of the impurity C LOQ is a minimum signal-to-noise ratio of 36.3
  • the RSD value is 5.5%.
  • the 3-pin signal-to-noise ratio of the compound of formula I LOQ is a minimum signal-to-noise ratio of 42.8, and the RSD value is 3.9%.
  • the above detection limit test results show that the 3-pin signal-to-noise ratio of impurity A LOD is a minimum signal-to-noise ratio of 3.9, the 3-pin signal-to-noise ratio of impurity B LOD is a minimum signal-to-noise ratio of 6.0, and the 3-pin signal-to-noise ratio of impurity C LOD is 6.0.
  • the minimum signal-to-noise ratio is 10.4
  • the 3-pin LOD of the compound of formula I has a minimum signal-to-noise ratio of 14.3.
  • Example 8 Investigation of the linear range of liquid phase or liquid mass chromatography detection methods
  • Preparation of a mixed solution of linear impurity A, impurity B, impurity C, and compound of formula I Take 18.9 mg of impurity A, 18.7 mg of impurity B, 18.5 mg of impurity C, and 18.7 mg of compound of formula I, accurately weigh and place it in a 25 mL volumetric flask, and use 50% Dissolve methanol in water and dilute to volume with 50% methanol water, and mix well; accurately transfer 1.00mL to a 100mL volumetric flask, dilute with 50% methanol water to volume, and mix well; then accurately pipette 1.00ml, 3.0mL, 5.0mL, 10.0mL, 15.0mL, and 20.0mL were placed in six 100mL volumetric flasks, diluted to volume with 50% methanol water, and mixed; LOQ, 0.225, 0.375, 0.75, 1.125, and 1.5 ⁇ g/mL linear solutions were obtained; The linear solution concentration is the abscissa, the peak
  • Example 8 Impurity correction factors for liquid phase or liquid-mass chromatography detection methods
  • the correction factor was determined by comparing the peak areas of impurity A, impurity B, impurity C and the compound of formula I at a concentration of 7.5 ⁇ g/mL with that of empagliflozin at the same concentration.
  • test solutions for impurity A, impurity B, impurity C and compound of formula I the same as the preparation of empagliflozin test solution.
  • correction factor of the impurity A test solution is 1.00, between 0.8 and 1.2; the correction factor of the impurity B test solution is 1.07, between 0.8 and 1.2; the correction factor of the impurity C test solution is 1.13, between 0.8 and 1.2 between; the correction factor of the test solution of the compound of formula I is 0.93 and is between 0.8 and 1.2.
  • Chromatographic conditions chromatographic column CAPCELL PAKC18 4.6mm ⁇ 150mm ⁇ 3 ⁇ m; flow rate 0.9mL/min; injection volume 20 ⁇ L; column temperature 40°C; wavelength 225nm; running time 70min; the ratio of methanol: water in the chromatographic mobile phase is: 0 ⁇ 10 minutes, 40:60; 10 ⁇ 15 minutes, 40:60 ⁇ 52:48; 15 ⁇ 35 minutes, 52:48; 35 ⁇ 50 minutes, 52:48 ⁇ 80:20; 50 ⁇ 60 minutes, 80:20; 60 ⁇ 60.1 minutes, 80:20 ⁇ 40:60; 60.1 ⁇ 70 minutes, 40:60.
  • Preparation of impurity solutions of impurity A, impurity B, impurity C, and compound of formula I weigh 18.9 mg of impurity A working reference substance, 18.7 mg of impurity B working reference substance, 18.5 mg of impurity C working reference substance, and 18.7 mg of formula I compound working reference substance. Dissolve in 25 mL volumetric flask with 50% methanol water, dilute to volume with 50% methanol water, and mix well.
  • System adaptability solution preparation Weigh 49.7 mg of the empagliflozin working reference substance into a 100 mL volumetric flask, transfer 1 mL of the impurity solution, dissolve it in 50% methanol water, dilute it to the mark with 50% methanol water, and mix.
  • test solution Accurately weigh 50 mg of crude empagliflozin raw material for test, put it into a 100 mL volumetric flask, dissolve it in 50% methanol water, and dilute to volume with 50% methanol water, and shake well.
  • Preparation of blank solution In a 100mL volumetric flask, add methanol + 50mL water to make the volume to the mark, and shake well.
  • Injection procedure 2 injections of blank, 6 injections of system suitability solution, 2 injections of test solution, and 1 injection of system suitability solution.
  • the compound of formula I is 0.09%;
  • the test results of crude empagliflozin API Z1258-211202 are impurity A is 1.00%, impurity B is 0.09%, impurity C is 0.14%, compound of formula I is 0.11%; empagliflozin raw material
  • the test results of the crude drug Z1259-220101 showed that impurity A was 0.96%, impurity B was 0.08%, impurity C was 0.16%, and the compound of formula I was 0.13%.
  • the typical HPLC spectrum detected for crude empagliflozin API Z1258-211201 is shown in Figure 7 of the instruction manual.
  • Chromatographic conditions chromatographic column CAPCELL PAKC18 4.6mm ⁇ 150mm ⁇ 3 ⁇ m; flow rate 0.8mL/min; injection volume 10 ⁇ L; column temperature 30°C; wavelength 225nm; running time 70min; the ratio of methanol to water in the chromatographic mobile phase is: 0 ⁇ 10 minutes, 40: 60; 10 ⁇ 15 minutes, 40: 60 ⁇ 52: 48; 15 ⁇ 35 minutes, 52: 48; 35 ⁇ 50 minutes, 52: 48 ⁇ 80: 20; 50 ⁇ 60 minutes, 80: 20; 60 ⁇ 60.1 minutes, 80: 20 ⁇ 40: 60; 60.1 ⁇ 70 minutes, 40: 60.
  • Example 9 For the preparation of relevant solutions, refer to Example 9.
  • the compound of formula I is 0.03%; the quality empagliflozin raw material medicine Z1259-220302 test results impurity A is not detected, the impurity B is not detected, the impurity C is not detected, the compound of formula I is 0.01%; the raw material of empagliflozin The test results of the high-quality drug Z1259-220303 showed that no impurities were detected, impurity B was not detected, impurity C was not detected, and the compound of formula I was 0.05%.
  • Example 11 SGLT2 inhibitory activity test of empagliflozin and characteristic impurities
  • Example 12 Toxicity test of empagliflozin and characteristic impurities
  • Daunorubicin was used as the control compound to test the in vitro cytotoxicity of empagliflozin, impurity A, impurity B, impurity C, and the compound of formula I against human hepatocytes HEPG2 and kidney cells HK-2. HepG2 and HK-2 cells were seeded in a 96-well plate in a certain number. The compound was added to the corresponding cell wells according to the concentration gradient. After a certain period of time, there was a dose-effect dependence between the compound concentration and the cell absorbance value OD450.
  • HEPG2 and HK-2 cells digested with 0.25% trypsin were centrifuged at 1000 rpm for 10 minutes, the supernatant was discarded, HEPG2 and HK-2 cells were resuspended with DMEM and MEM complete medium, respectively, the cell density was adjusted to 30,000 cells/ml after counting, 100 ⁇ l was inoculated into each well of a 96-well plate, B well (only medium) and Control well (cell inoculation without compound) were set, and PBS or sterile water was added to the side wells.
  • the dosage per well is 150 ⁇ l.
  • R represents the positive control
  • B represents the blank
  • S represents the test compound
  • the test was administered according to the intention shown above. Each compound solution was added in order from high concentration to low concentration in columns 2-11, and DMSO culture medium with the same volume as the high-dose compound solution was added in column 12. Place the cell culture plate into a 37°C, 5% CO2 incubator for 3 days.

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Abstract

一种恩格列净原料药或制剂中的特征杂质和应用，并提供了该杂质的检测方法、合成方法、体外药理活性和体外毒性，为恩格列净原料药或制剂的质量控制提供了重要依据，对恩格列净原料药或制剂的质量控制具有重要意义。

Description

一种恩格列净原料药或制剂中的特征杂质及其应用 技术领域
本发明属于药学和分析化学技术领域，涉及一种恩格列净原料药或制剂中的特征杂质及其应用。更具体地说，本发明涉及一种恩格列净二聚体杂质及其在恩格列净原料药或制剂质量控制中的应用。
背景技术
糖尿病是一组以高血糖为特征的代谢性疾病，国际糖尿病协会(IDF)预计到2035年全球糖尿病患者数量将达到近60亿。目前，临床上糖尿病分为两种，即胰岛素依赖型糖尿病(Ⅰ型糖尿病)和非胰岛素依赖型糖尿病(Ⅱ型糖尿病)，其中Ⅱ型糖尿病患者约占90％以上。
恩格列净是由勃林格殷格翰开发的一款钠-葡萄糖协同转运蛋白2(SGLT-2)抑制剂，于2014年5月首次获批上市用于II型糖尿病的治疗，已成为糖尿病治疗领域的主流药物。
Figure PCTCN2022121230-appb-000001
恩格列净作为人用药物的杂质研究已有多年，根据原研专利文献WO2005092877和WO2006120208中公开的技术方案，恩格列净原料药合成过程中主要特征工艺杂质包括了五元环杂质(杂质A)、开环杂质(杂质B)、二聚体杂质(杂质C)，等。
Figure PCTCN2022121230-appb-000002
Figure PCTCN2022121230-appb-000003
这些特征工艺杂质(杂质A～杂质C)均是在工艺过程中产生，并在恩格列净原料药粗品中能明显观察到的特有杂质，所以大部分恩格列净原料药供应商会将这些特征杂质列于恩格列净原料药的质量标准中用于其质量的控制。但是在对恩格列净原料药或制剂杂质研究的相关报道中，鲜有文献对恩格列净合成过程中的关键起始物料(S)-3-羟基四氢呋喃引入的杂质进行详细并透彻的研究。
(S)-3-氧基四氢呋喃是恩格列净分子中的显著结构片段，其通过相关中间体与(S)-3-羟基四氢呋喃发生取代反应引入。(S)-3-羟基四氢呋喃成熟的产业化合成工艺主要有两个，即苹果酸为原料的合成路线和(S)-4-氯-3-羟基丁酸乙酯为原料的合成路线。
早在1983年，Tadon等人就首次提出手性(S)-3-羟基四氢呋喃的合成路(J.Org.Chem,1983,48,2767-2769),后来许多已公开的文献对Tadon的(S)-3-羟基四氢呋喃合成路线进行优化和改进，如专利CN107935971、CN107235937A和CN109503523。这些路线以L-苹果酸为起始原料，通过甲酯化生成L-苹果酸二甲酯，再还原成手性1,2,4-丁三醇，最后在酸性条件下脱水关环得产物(S)-3-羟基四氢呋喃。
Figure PCTCN2022121230-appb-000004
专利文献CN102477019、CN107098872、WO2008093955、WO2000063199、EP761663等报道了以光学纯的(S)-4-氯-3-羟基丁酸乙酯为原料，经酯基 还原得到(S)-4-氯-1,3-丁二醇，再经环合得到目标产物(S)-3-羟基四氢呋喃。
Figure PCTCN2022121230-appb-000005
上述两条(S)-3-羟基四氢呋喃的产业化合成路线中，都会产生一个特征杂质，即1,4-丁二醇。苹果酸路线中，L-苹果酸中往往含有丁二酸，丁二酸在参与工艺过程中的反应后得到1,4-丁二醇；同样，(S)-4-氯-3-羟基丁酸乙酯中通常含有4-氯丁酸乙酯，4-氯丁酸乙酯在参与工艺过程中的反应后得到1,4-丁二醇。所以，(S)-3-羟基四氢呋喃中通常含有一定量的1,4-丁二醇。
Figure PCTCN2022121230-appb-000006
而(S)-3-羟基四氢呋喃中的1,4-丁二醇会进一步参与到恩格列净原料药合成工艺中的各步反应，形成对应的杂质难以在结晶过程中清除，直至带入最终的原料药中，并形成了新的二聚体杂质。
Figure PCTCN2022121230-appb-000007
Figure PCTCN2022121230-appb-000008
杂质控制是原料药或制剂质量控制的生命线，做到全面监测和控制杂质对于原料药和制剂的安全性、有效性都具有非常重要的意义。鉴于鲜有文献关注(S)-3-羟基四氢呋喃中的1,4-丁二醇引入的杂质风险，并考虑因分析方法检测能力有限，可能导致恩格列净原料药中杂质检测不出来的情况。本发明的技术人员对恩格列净原料药合成程中可能产生的杂质进行了全面、透彻的分析和研究，并针对性的开发了有效的分析检测方法进行了样品检测，意外地发现了恩格列净原料药合成过程中产生的新二聚体杂质。本发明技术人员进一步深入研究了该新杂质的体外药理活性和体外细胞毒性，为恩格列净原料药或制剂中该杂质的控制提供了重要的依据。
发明内容
本发明提供一种恩格列净原料药或制剂中的特征杂质和应用，该杂质对于恩格列净原料药或制剂的质量控制具有非常重要的作用。
本发明中所述的恩格列净原料药或制剂中的特征杂质和应用，包括以下内容：
如式I所示化合物：
Figure PCTCN2022121230-appb-000009
本发明中所述的化合物，其特征在于，用于恩格列净原料药或制剂的 质量控制。
本发明中所述的化合物，其特征在于，式I所示化合物在恩格列净原料药中的含量不大于0.15％或在恩格列净制剂中的含量不大于0.2％。
本发明中所述的化合物，其特征在于，式I所示化合物在恩格列净原料药或制剂中的含量可用液相色谱或液质联用色谱检测。
本发明中所述的化合物，其特征在于，用液相色谱或液质联用色谱检测恩格列净原料药或制剂中式I所示化合物的含量时，其色谱检测条件为：色谱柱固定相为碳十八硅胶，色谱柱规格为4.6×150mm×3μm，流动相为甲醇和水，流动相流速为0.8～1.0mL/min，进样量10～20μL，柱温30～40℃，波长225nm，运行时间70min。
本发明中所述的化合物，其特征在于，用液相色谱或液质联用色谱检测恩格列净原料药或制剂中式I所示化合物的含量时，紫外检测器波长为225nm条件下，式I所示化合物的相对于恩格列净的校正因子为0.8～1.2。
本发明中所述的化合物，其特征在于，用液相色谱或液质联用色谱检测恩格列净原料药或制剂中式I所示化合物的含量时，色谱流动相中甲醇：水的比例依次为：0→10分钟，40：60；10→15分钟，40：60→52：48；15→35分钟，52：48；35→50分钟，52：48→80：20；50→60分钟，80：20；60→60.1分钟，80：20→40：60；60.1→70分钟，40：60。
本发明中所述的化合物，其特征在于，制备方法包括如下步骤：
Figure PCTCN2022121230-appb-000010
其中，X为卤素原子，选自溴原子和碘原子；
步骤1，化合物II经还原反应制得化合物III；
步骤2，化合物III和化合物IV形成碳苷化合物，再与甲醇形成甲氧苷化合物，最后经还原制得化合物V；
步骤3，化合物V与1,4-丁二醇发生取代反应制得目标化合物I。
本发明中所述的化合物，其特征在于，制备方法的条件如下：
步骤1，化合物II在三乙基硅烷和三氯化铝的作用下还原制得化合物III；
步骤2，化合物III和化合物IV在正丁基锂作用下于-75～-85℃温度下形成碳苷化合物，并在甲磺酸催化作用下于10～40℃与甲醇形成甲氧苷化合物，最后经三乙基硅烷和三氯化铝于10～40℃还原制得化合物V；
步骤3，化合物V和1,4-丁二醇在叔丁醇钾作用下，在四氢呋喃溶剂中加热回流制得化合物I。
本发明中所述的式I化合物，其特征在于对钠-葡萄糖协同转运蛋白2具有明显的抑制作用。
本发明中所述的式I化合物在制备用于治疗糖尿病的药物中的用途。
一种药物组合物，所述的药物组合物含有有效剂量的本发明所述的式I化合物或其药学可接受的盐，及可药用的载体、赋形剂或它们的组合。
需要说明的是，原料药或药物制剂中的杂质在一定程度上影响到药物的安全性和有效性。所以，对特征杂质的控制和监测在药品的质量控制中具有非常重要的意义。本发明中所述的式I化合物具有与恩格列净几乎等效的SGLT2抑制活性，也就是说式I化合物对恩格列净原料药或制剂的有效性影响可能较小。但需要进一步强调的是，本发明所述的式I化合物相对于恩格列净，对人员肝细胞和肾细胞具有更高的毒性。所以，式I化合物可能在很大程度上影响到恩格列净药物的安全性。
需要进一步说明的是，本发明中所述的式I化合物具有明显的SGLT2抑制活性活性。所以，式I化合物可制备成任何形式针对SGLT2靶点的药物。任何形式的式I化合物药物均被视为本发明所公开并保护。
需要强调和说明的是，本发明中所述的式I化合物的液相或液质联用色谱检测方法，使用的色谱柱和色谱条件均是最优方案，任何在本发明所述的检测方法基础上进行简单调整和概念变换均被视为包括在本发明范围之内，均被视为侵犯了本发明。
术语：
本发明中所述的杂质是指产品生产过程中带入的起始物料、中间体、聚合物、副反应产物，以及贮藏过程中的降解产物等。本发明中所述的特征杂质是指产品在特定的工艺条件和原料质量一致的情况下每批次重复出现的杂质，是产品中确定含有的杂质。
附图说明
图1式I化合物核磁共振氢谱
图2式I化合物核磁共振碳谱
图3杂质A线性方程标准曲线
图4杂质B线性方程标准曲线
图5杂质C线性方程标准曲线
图6式I化合物线性方程标准曲线
图7恩格列净原料药粗品杂质检测HPLC谱图
图8式I化合物对SGLT2靶点抑制活性的IC 50曲线图
图9式I化合物肝细胞毒性和肾细胞毒性IC 50曲线图
具体实施方式
下面具体实施例可以使本专业技术人员全面理解本发明，但不以任何方式限制本发明。
在下列实施例中，除非另有指明，所有温度为摄氏温度；除非另有指明，所述的室温均为20～30℃；除非另有指明，各种起始原料和试剂均来自市售，均不经进一步纯化直接使用；除非另有指明，合成反应中使用的各种溶剂均为工业级溶剂，不经进一步处理直接使用；除非另有指明，合成反应中使用的各种溶剂和试剂均为市售厂家包括但不限于杭州化学试剂、国药试剂等；除非另有指明，分析检测过程中使用的各种溶剂均为色谱级溶剂，不经进一步处理直接使用，市售厂家包括但不限于Honeywell、Sigma等；除非另有说明，药理活性检测使用的相关试剂均来自市售。
实施例1：化合物Ⅲ-1的合成
Figure PCTCN2022121230-appb-000011
将三乙基硅烷(16.7g，142.6mmol，1.5eq)和2-甲基四氢呋喃(150ml)加入至500ml四口烧瓶，降温至0～10℃，分批加入无水三氯化铝(19.1g，143.6mmol，1.5eq)。将化合物Ⅱ(30.0g，95.7mmol)溶于2-甲基四氢呋喃(90ml)中，所得溶液滴加到硼氢化钠和三氯化铝混合液中，加入至80℃搅拌过夜。TLC检测反应完毕后，反应液降温至0～10℃，滴加水(120ml)淬灭反应。分出有机层，并于40～50℃减压浓缩。所得浆状物加入乙醇(90ml)于室温搅拌析晶，析出固体后加入水(15ml)打浆，过滤干燥，得25.3g化合物Ⅲ-1，收率88.2％。EI-MS:m/z 298.9[M+H] +； 1H-NMR(600MHz,DMSO-d 6):δ7.82(s,1H),7.82(d,J＝8.2Hz,1H),7.49(m,3H),7.16(m,2H),3.93(s,2H)； 13C-NMR(150MHz,DMSO-d 6):δ162.1,160.4,114.6,139.0,138.9,131.4,131.2,130.4,128.9,120.3,115.0,114.9,70.0。
实施例2：化合物Ⅲ-2的合成
Figure PCTCN2022121230-appb-000012
将三乙基硅烷(14.5g，125.0mmol，1.5eq)和2-甲基四氢呋喃(150ml)加入至500ml四口烧瓶，降温至0～10℃，分批加入无水三氯化铝(16.7g，125.0mmol，1.5eq)。将化合物Ⅱ-2(30.0g，83.3mmol)溶于2-甲基四氢呋喃(90ml)中，所得溶液滴加到硼氢化钠和三氯化铝混合液中，加入至80℃搅拌过夜。TLC检测反应完毕后，反应液降温至0～10℃，滴加水(120ml)淬灭反应。分出有机层，并于40～50℃减压浓缩。所得浆状物加入乙醇(90ml)于室温搅拌析晶，析出固体后加入水(15ml)打浆，过滤干燥，得26.1g化合物Ⅲ-2，收率90.4％。EI-MS:m/z 346.96[M+H] +； 1H-NMR(600MHz,DMSO-d 6):δ7.86(s,1H),7.83(d,J＝8.2Hz,1H), 7.52(m,3H),7.19(m,2H),3.96(s,2H)。
实施例3：化合物Ⅴ的合成
Figure PCTCN2022121230-appb-000013
将化合物Ⅲ-1(20.0g,66.8mmol)、四氢呋喃(40ml)和甲苯(120ml)加入至500ml四口烧瓶中，氮气保护，降温至-75～-85℃。滴加2.5M正丁基锂溶液(40ml,100.2mmol,1.5eq),保温-75～-85℃反应1～2h。滴加化合物Ⅳ(46.9g,100.2mmol,1.5eq)的甲苯(40ml)溶液，保温-75～-85℃反应1～2h。加入水(80ml)淬灭反应液，分出有机层，并于50～60℃减压浓缩。所得浆状物加入甲醇(160ml)和甲磺酸(6.4g，66.8mmol，1.0eq)，室温搅拌过夜。TLC检测反应完毕后，加入饱和碳酸氢钠溶液调节PH值到7～8，于40～50℃减压浓缩脱溶。浓缩结束加入二氯甲烷(60ml)萃取，分出有机层，水相用二氯甲烷(60ml×2)反提取两次。合并二氯甲烷层，并于30～40℃减压浓缩，浆状物加入甲苯(60ml)升温到60～70℃减压浓缩夹带水分，得黄色浆状物密封备用。将无水三氯化铝(17.8g，133.6mmol，2.0eq)和二氯甲烷(40ml)加入到500ml四口瓶中，降温到0～10℃，滴加乙腈(40ml)，搅拌溶清后加入三乙基硅烷(11.7g,100.2mmol，1.5eq)。向备用的黄色浆状物加入二氯甲烷(40ml)和乙腈(40ml)溶解，滴加到反应液中室温反应3～4h。加入水(80ml)淬灭，将混合液于40～50℃减压浓缩脱溶，加入乙酸异丙酯(80ml)夹带。浓缩结束加入乙酸异丙酯(80ml)于室温打浆3～4h，过滤干燥，得14.1g化合物Ⅴ，收率为55.3％。EI-MS:m/z 383.81[M+H] +； 1H-NMR(600MHz,DMSO-d 6):δ7.84(s,1H),7.80(d,J＝8.2Hz,1H),7.44(m,3H),7.18(m,2H),4.96(s,1H)3.95(s,2H),3.79(m,2H),3.73(m,1H),3.64(m,1H),,3.58(br s,2H),3.48(m,1H),3.40(m,1H)。
实施例4：化合物Ⅴ的合成
Figure PCTCN2022121230-appb-000014
将化合物Ⅲ-2(20.0g,57.7mmol)、四氢呋喃(40ml)和甲苯(120ml)加入至500ml四口烧瓶中，氮气保护，降温至-75～-85℃。滴加2.5M正丁基锂溶液(34.5ml,86.3mmol,1.5eq),保温-75～-85℃反应1～2h。滴加化合物Ⅳ(40.3g,86.3mmol,1.5eq)的甲苯(40ml)溶液，保温-75～-85℃反应1～2h。加入水(80ml)淬灭反应液，分出有机层，并于50～60℃减压浓缩。所得浆状物加入甲醇(160ml)和甲磺酸(5.5g，57.7mmol，1.0eq)，室温搅拌过夜。TLC检测反应完毕后，加入饱和碳酸氢钠溶液调节PH值到7～8，于40～50℃减压浓缩脱溶。浓缩结束加入二氯甲烷(60ml)萃取，分出有机层，水相用二氯甲烷(60ml×2)反提取两次。合并二氯甲烷层，并于30～40℃减压浓缩，浆状物加入甲苯(60ml)升温到60～70℃减压浓缩夹带水分，得黄色浆状物密封备用。将无水三氯化铝(15.4g，115.4mmol，2.0eq)和二氯甲烷(40ml)加入到500ml四口瓶中，降温到0～10℃，滴加乙腈(40ml)，搅拌溶清后加入三乙基硅烷(10.1g,86.6mmol，1.5eq)。向备用的黄色浆状物加入二氯甲烷(40ml)和乙腈(40ml)溶解，滴加到反应液中室温反应3～4h。加入水(80ml)淬灭，将混合液于40～50℃减压浓缩脱溶，加入乙酸异丙酯(80ml)夹带。浓缩结束加入乙酸异丙酯(80ml)于室温打浆3～4h，过滤干燥，得13.0g化合物Ⅴ，收率为58.7％。
实施例5：化合物的Ⅰ合成
Figure PCTCN2022121230-appb-000015
将化合物Ⅴ(10.0g,26.1mmol)和四氢呋喃(50ml)加入到100ml三口烧瓶中，再加入1,4-丁二醇(1.2g，13.1mmol，0.5eq)、叔丁醇钾(11.7g，104.4mmol，4eq)和四丁基溴化铵(0.8g,2.6mmol,0.1eq)后升温到60～70℃，保温反应过夜。TLC检测反应完毕后,降温至室温，加入水(20ml)淬灭反应液。分出有机层，并于40～50℃减压浓缩，得黄色浆状物。经过柱状层析(流动相为二氯甲烷：甲醇＝10：1)得到3.6g化合物Ⅰ，收率为16.9％。HR-ESI-MS:m/z 837.2404[M+Na] +， 1H-NMR(600MHz,DMSO-d 6):δ7.37(d,J＝8.2Hz,2H),7.32(d,J＝2.1Hz，2H)，7.23(dd,J＝8.2,2.1Hz,2H),7.09(d,J＝8.6Hz,2H),6.84(d,J＝8.6Hz,2H),4.95(d,J＝5.8Hz,2H),4.94(d,J＝5.8Hz,2H),4.82(d,J＝5.8Hz,2H),4.44(d,J＝5.8Hz,2H),4.00(m,2H),3.99(m,2H),3.97(m,4H),3.95(m,2H),3.69(ddd,J＝11.8,5.8,2.0Hz,2H),3.44(ddd,J＝11.8,5.8,2.0Hz,2H),3.26(td,J＝9.1,5.8Hz,2H),3.21(dt,J＝5.8,2.0Hz,2H),3.16(td,J＝9.1,5.8Hz,2H),3.11(td,J＝9.1,5.8Hz,2H),1.82(m,4H)； 13C-NMR(150MHz,DMSO-d 6):δ157.5,140.2,138.3,132.4,131.8,131.3,130.0,129.1,127.8,114.8,81.7,81.2,78.8,75.2,70.8,67.5,61.8,38.1,26.0。
实施例6：液相或液质连用色谱检测方法系统适应性、专属性试验
(1)试验简述
通过测定相邻两峰之间的分离度而判断该分析方法的分离效果。
(2)溶液配制
系统适应性溶液配制：取杂质A 18.9mg、杂质B 18.7mg、杂质C 18.5mg、式I化合物18.7mg，精密称定置25mL容量瓶中，用50％甲醇水溶解并用50％甲醇水稀释至刻度，混匀，即杂质储备液。准确称取恩格列净工作对照品49.7mg，于100mL容量瓶中，加50％甲醇水溶解，移入1.00ml杂质储备液，用50％甲醇水定容至刻度。
(3)进样程序
进样空白(50％甲醇水)2针、系统适应性溶液6针、为避免系统错误随机进样。每隔6个供试品溶液进1针程序控制溶液，并在序列最后进1针程序控制溶液
表1系统适应性测定结果
Figure PCTCN2022121230-appb-000016
Figure PCTCN2022121230-appb-000017
备注：上表中1-6针为系统适应性溶液，7-12针为程序控制针。
空白溶液对恩格列净、杂质A、杂质B、杂质C和式I化合物检测无干扰，重复进样6针系统适应性溶液中恩格列净峰面积RSD为0.0％，杂质A峰面积RSD为0.3％，杂质B峰面积RSD为0.3％，杂质C峰面积RSD为0.3％，式I化合物峰面积RSD为0.1％；加程序控制针后1-12针恩格列净的最大RSD为0.7％,杂质A的最大RSD为1.0％,杂质B的最大RSD为1.0％，杂质C的最大RSD为1.2％,式I化合物的最大RSD为0.6％。
表2分离度测定结果
Figure PCTCN2022121230-appb-000018
系统适应性溶液中恩格列净与杂质A之间的分离度为13.2，恩格列净与杂质B之间的分离度为6.8，杂质A与杂质C之间的分离度为43.5，杂质C与式I化合物之间的分离度为1.7。
实施例7：液相或液质连用色谱检测方法定量限和检测限试验
(1)试验简述
方法中杂质A、杂质B、杂质C、式I化合物的检测限(LOD)和定量限(LOQ)是根据信噪比来确定的，用稀释法对稀释成不同浓度的溶液进行测定，各定量限小于接受标准且信噪比不小于10时即为定量限浓度，检测限是通过LOQ测试溶液稀释3倍且信噪比不小于3时即为检测限浓度。
(2)溶液配制
定量限杂质A、杂质B、杂质C、式I化合物混合溶液配制：取杂质A18.9mg、杂质B 18.7mg、杂质C 18.5mg、式I化合物18.7mg，精密称定置25mL容量瓶中，用50％甲醇水溶解并用50％甲醇水稀释至刻度，混匀；精密移入1.00mL于100mL容量瓶中，用50％甲醇水稀释至刻度，混匀；再精密移取1.00mL于100mL容量瓶中，用50％甲醇水稀释至刻度，混匀。
定量限杂质A、杂质B、杂质C、式I化合物混合溶液配制：精密移取定量限杂质A、杂质B、杂质C、式I化合物混合溶液3.0mL于10mL容量瓶中，用50％甲醇水稀释至刻度，混匀。
表3定量限测定结果
Figure PCTCN2022121230-appb-000019
以上定量限试验结果表明，杂质A LOQ的3针信噪比为最小信噪比为10.1，RSD值为2.0％，杂质B LOQ的3针信噪比为最小信噪比为17.6，RSD值为4.6％，杂质C LOQ的3针信噪比为最小信噪比为36.3，RSD值为5.5％，式I化合物LOQ的3针信噪比为最小信噪比为42.8，RSD值为3.9％。
表4检测限测定结果
Figure PCTCN2022121230-appb-000020
Figure PCTCN2022121230-appb-000021
以上检测限试验结果表明，杂质A LOD的3针信噪比为最小信噪比为3.9，杂质B LOD的3针信噪比为最小信噪比为6.0，杂质C LOD的3针信噪比为最小信噪比为10.4，式I化合物LOD的3针信噪比为最小信噪比为14.3。
实施例8：液相或液质连用色谱检测方法线性范围考察
(1)试验简述
在LOQ～200％的浓度范围内比较均匀的6个浓度点，以浓度为横坐标，峰面积为纵坐标画一条曲线，计算该条曲线的线性回归系数R 2的数值。
(2)溶液配制
线性杂质A、杂质B、杂质C、式I化合物混合溶液配制：取杂质A18.9mg、杂质B 18.7mg、杂质C 18.5mg、式I化合物18.7mg，精密称定置25mL容量瓶中，用50％甲醇水溶解并用50％甲醇水稀释至刻度，混匀；精密移入1.00mL于100mL容量瓶中，用50％甲醇水稀释至刻度，混匀；再精密移取1.00ml、3.0mL、5.0mL、10.0mL、15.0mL、20.0mL，分别置于6个100mL容量瓶中，用50％甲醇水稀释至刻度，混匀；得到LOQ、0.225、0.375、0.75、1.125、1.5μg/mL线性溶液；以线性溶液浓度为横坐标，峰面积为纵坐标，绘制线性方程标准曲线。
(3)进样程序
进样空白(50％甲醇水)2针、系统适应性溶液6针、每个线性溶液进 样3针、最后回系统适应性溶液(1针)，为避免系统错误随机进样。每隔6个样品溶液进1针系统适应性溶液。
表5杂质A线性测定结果
Figure PCTCN2022121230-appb-000022
Figure PCTCN2022121230-appb-000023
以上线性试验结果表明，空白溶液对杂质A的检测无干扰；杂质A线性回归系数R 2＝0.9991；杂质A测试溶液回收率在93.9％～103.5％；同一浓度各三针质谱峰面积杂质A最大RSD值为3.9％，杂质A的回收精密度RSD为2.7％。杂质A线性方程标准曲线见说明书附图3。
表6杂质B线性测定结果
Figure PCTCN2022121230-appb-000024
Figure PCTCN2022121230-appb-000025
以上线性试验结果表明，空白溶液对杂质B的检测无干扰；杂质B线性回归系数R 2＝0.9998；杂质B测试溶液回收率在98.6％～103.2％；同一浓度各三针质谱峰面积杂质B最大RSD值为4.6％，杂质B的回收精密度RSD为1.7％。杂质B线性方程标准曲线见说明书附图4。
表7杂质C线性测定结果
Figure PCTCN2022121230-appb-000026
Figure PCTCN2022121230-appb-000027
以上线性试验结果表明，空白溶液对杂质C的检测无干扰；杂质C线性回归系数R 2＝0.9999；杂质C测试溶液回收率在95.8％～100.5％；同一浓度各三针质谱峰面积杂质C最大RSD值为5.5％，杂质C的回收精密度RSD为1.6％。杂质C线性方程标准曲线见说明书附图5。
表8式I化合物线性测定结果
Figure PCTCN2022121230-appb-000028
Figure PCTCN2022121230-appb-000029
以上线性试验结果表明，空白溶液对式I化合物的检测无干扰；式I化合物线性回归系数R 2＝0.9988；式I化合物测试溶液回收率在97.9％～100.6％；同一浓度各三针质谱峰面积式I化合物最大RSD值为3.9％，式I化合物的回收精密度RSD为0.6％。式I化合物的线性方程标准曲线见说明书附图6。
实施例8：液相或液质连用色谱检测方法杂质校正因子
(1)试验简述
杂质A、杂质B、杂质C、式I化合物在7.5μg/mL的浓度时通过与相同浓度的恩格列净比较峰面积来确定校正因子。
(2)溶液配制
校正因子恩格列净工作对照品、杂质A、杂质B、杂质C、式I化合物混合溶液配制：取恩格列净工作对照品18.7mg、杂质A 18.9mg、杂质 B 18.7mg、杂质C 18.5mg、式I化合物18.7mg，精密称定置25mL容量瓶中，用50％甲醇水溶解并用50％甲醇水稀释至刻度，混匀；精密移入1.00mL于100mL容量瓶中，用50％甲醇水稀释至刻度，混匀。
恩格列净测试溶液配制：取杂质A 18.7mg，精密称定置25mL容量瓶中，用50％甲醇水溶解并用50％甲醇水稀释至刻度，混匀；精密移入1.00mL于100mL容量瓶中，用50％甲醇水稀释至刻度，混匀；得到7.48μg/mL测试溶液。
杂质A、杂质B、杂质C、式I化合物测试溶液配制：与恩格列净测试溶液配制相同。
表9校正因子测定结果
Figure PCTCN2022121230-appb-000030
以上校正因子试验结果表明，杂质A测试溶液校正因子为1.00在0.8～1.2之间；杂质B测试溶液校正因子为1.07在0.8～1.2之间；杂质C测试溶液校正因子为1.13在0.8～1.2之间；式I化合物测试溶液校正因子为0.93在0.8～1.2之间。
实施例9：恩格列净原料药粗品样品中杂质A、杂质B、杂质C、式I化合物杂质的检测
色谱条件：色谱柱CAPCELL PAKC18 4.6mm×150mm×3μm；流速0.9mL/min；进样量20μL；柱温40℃；波长225nm；运行时间70min；色谱流动相中甲醇：水的比例依次为：0→10分钟，40：60；10→15分钟，40：60→52：48；15→35分钟，52：48；35→50分钟，52：48→80：20；50→60分钟，80：20；60→60.1分钟，80：20→40：60；60.1→70分钟，40：60。
杂质A、杂质B、杂质C、式I化合物杂质溶液配制：称杂质A工作对照品18.9mg,杂质B工作对照品18.7mg,杂质C工作对照品18.5mg,式I化合物工作对照品18.7mg置于25mL容量瓶中，用50％甲醇水溶解，并用50％甲醇水稀释至刻度，混匀。
系统适应性溶液配制：称取恩格列净工作对照品49.7mg置于100mL容量瓶中，移取1mL杂质溶液，用50％甲醇水溶解，并用50％甲醇水稀释至刻度，混匀。
供试品溶液配制：准确称取恩格列净原料药粗品供试品50mg，于100mL容量瓶中，用50％甲醇水溶解，并用50％甲醇水定容至刻度，摇匀。
空白溶液配制：100mL的容量瓶中，用甲醇+50mL水定容至刻度线，摇匀。
进样程序：进样空白2针、系统适应性溶液(6针)、供试品溶液(2针)、系统适应性溶液(1针)。
按面积归一化结果计算杂质A、杂质B、杂质C、式I化合物含量，恩格列净原料药粗品Z1258-211201检测结果杂质A为0.93％、杂质B为0.08％、杂质C为0.13％、式I化合物为0.09％；恩格列净原料药粗品Z1258-211202检测结果杂质A为1.00％、杂质B为0.09％、杂质C为0.14％、式I化合物为0.11％；恩格列净原料药粗品Z1259-220101检测结果杂质A为0.96％、杂质B为0.08％、杂质C为0.16％、式I化合物为0.13％。恩格列净原料药粗品Z1258-211201检测典型HPLC谱图见说明书附图7。
实施例10：恩格列净原料药精品样品中杂质A、杂质B、杂质C、式I化合物杂质的检测
色谱条件：色谱柱CAPCELL PAKC18 4.6mm×150mm×3μm；流速0.8mL/min；进样量10μL；柱温30℃；波长225nm；运行时间70min；色谱流动相中甲醇：水的比例依次为：0→10分钟，40：60；10→15分钟，40：60→52：48；15→35分钟，52：48；35→50分钟，52：48→80：20；50→60分钟，80：20；60→60.1分钟，80：20→40：60；60.1→70分钟，40：60。相关溶液配制参照实施例9。
按面积归一化结果计算杂质A、杂质B、杂质C、式I化合物含量，恩格列净原料药精品Z1259-220301检测结果杂质A未检出、杂质B未检出、杂质C未检出、式I化合物为0.03％；恩格列净原料药精品Z1259-220302 检测结果杂质A未检出、杂质B未检出、杂质C未检出、式I化合物为0.01％；恩格列净原料药精品Z1259-220303检测结果杂质未检出、杂质B未检出、杂质C未检出、式I化合物为0.05％。
实施例11：恩格列净及特征杂质的SGLT2抑制活性试验
以恩格列净做对照化合物，测试杂质A、杂质B、杂质C、式I化合物对人源钠葡萄糖协同转运蛋白2(SGLT2)抑制活性。主要试验步骤如下：
(1)化合物溶液制备：阳性对照恩格列净溶液起始浓度500nM,5倍稀释。其他化合物起始浓度500nM,100％DMSO溶解,6倍稀释，8个浓度点。以DMSO为高对照，500nM恩格列净溶液为低对照；
(2)取出人源SGLT2/CHO细胞培养液，用150μL试验缓冲液清洗细胞1次。每孔加入49uL检测缓冲液；
(3)在各孔中加入1μL待测化合物各浓度的溶液。在高对照孔，加入DMSO1μL；在低对照孔加恩格列净各浓度的溶液；
(4)在各孔中加入50μL的6μM  14C-甲基-α-D-吡喃葡萄糖苷(终浓度为3μM)；
(5)将人源SGLT2/CHO细胞于37℃孵育2小时；
(6)除去待测化合物缓冲液，用冷检测缓冲液冲洗孔板3次；
(7)向每孔再加入50μL 10％NaOH使人源SGLT2/CHO细胞充分裂解；
(8)将所有孔的裂解液分别转移到闪烁管中，在每个管中再加入2ml闪烁混合液并充分混合；
(9)用Tri-Carb 2910TR型号的液体闪烁计数仪检测孔板的对应的数据。
表10各化合物对SGLT2抑制活性的IC 50值汇总表
Figure PCTCN2022121230-appb-000031
实施例12：恩格列净及特征杂质毒性试验
以柔红霉素做对照化合物，测试恩格列净、杂质A、杂质B、杂质C、式I化合物对人源肝细胞HEPG2、肾细胞HK-2的体外细胞毒性。HepG2及HK-2细胞按一定数量接种于96孔板，化合物按照浓度梯度加入到对应的细胞孔，作用一定时间后，化合物浓度与细胞吸光度值OD450间出现量效依赖关系。
(1)DMEM完全培养基
DMEM                      500ml
FBS                       50ml
配制后2-8℃保存，有效期为3个月。
RPMI-1640                 500ml
FBS                       50ml
配制后2-8℃保存，有效期为3个月。
(2)细胞接种
取0.25％胰酶消化后的HEPG2、HK-2细胞以1000rpm离心10分钟，弃上清，分别用DMEM和MEM完全培养基重悬HEPG2和HK-2细胞，计数后调整细胞密度至30000个/ml，96孔板每孔接种100μl，设B孔(只加培养基)和Control孔(接种细胞不加化合物)，边孔加PBS或者无菌水。
(3)化合物溶液的制备(用完全培养基稀释)
①将化合物稀释至首孔50μM，柔红霉素稀释至首浓1μM，再各3倍浓度梯度稀释，共9个浓度，各浓度设2个复孔；
②每孔给药量为150μl。
(4)细胞给药
  1 2 3 4 5 6 7 8 9 10 11 12  
A                          
B   R R R R R R R R R R B  
C   R R R R R R R R R R B  
D   S S S S S S S S S S B  
E   S S S S S S S S S S B  
F   S S S S S S S S S S B  
G   S S S S S S S S S S B  
H                          
备注：上表中R代表阳性对照，B代表空白，S代表测试化合物。
试验按上表示意图给药，从2-11列依次由高浓度到低浓度加入各化 合物溶液，12列加入同高剂量化合物溶液等体积的DMSO的培养基。将细胞培养板放入37℃、5％CO 2培养箱中培养3天。
(5)显色
每孔加入现配的40μl CTG显色液，室温10min内测试。
(6)检测
将细胞培养板放入微孔板检测系统中，选择吸光度检测模式，读取吸光值OD值，应用GraphPad Prism8软件进行数据分析，以浓度为横坐标，对应的化学发光值为纵坐标，拟合四参数方程曲线，方程为y＝(A-D)/(1+(X/C)^B)+D，其中B代表斜率，C代表IC 50(单位：μM)。
(7)毒性试验结果及分析
根据上述试验，所得数据经分析，相对于阳性对照柔红霉素，恩格列净及其特征杂质对人源肝细胞HepG2和肾细胞HK-2毒性均较弱。但其中，式I化合物表现出了一定的HepG2肝细胞毒性和HK-2肾细胞毒性。相关IC 50值见下表10，式I化合物IC 50计算曲线图见说明书附图9。
表11肝毒性和肾毒性IC 50值汇总表
Figure PCTCN2022121230-appb-000032
本发明的方法通过较佳实施例进行了描述，相关领域人员明显能在本发明内容和范围内对本发明中所述的方法和应用在有必要的地方稍加适当常识性的调整、改动和组合，来实现和应用本发明技术。本领域人员也可以借鉴本发明内容，通过适当改进工艺参数实现。特别需要指出的是，所有类似的改进和调整对本领域技术人员来说是显而易见的，都应被视为包括在本发明之内。

Claims (12)

  1. 如式I所示化合物：
    Figure PCTCN2022121230-appb-100001
  2. 如权利要求1中所述的化合物，其特征在于，用于恩格列净原料药或制剂的质量控制。
  3. 如权利要求1或2中所述的化合物，其特征在于，式I所示化合物在恩格列净原料药中的含量不大于0.15％或在恩格列净制剂中的含量不大于0.2％。
  4. 如权利要求1-3中任意一项所述的化合物，其特征在于，式I所示化合物在恩格列净原料药或制剂中的含量可用液相色谱或液质联用色谱检测。
  5. 如权利要求1-4中任意一项所述的化合物，其特征在于，用液相色谱或液质联用色谱检测恩格列净原料药或制剂中式I所示化合物的含量时，其色谱检测条件为：色谱柱固定相为碳十八硅胶，色谱柱规格为4.6×150mm×3μm，流动相为甲醇和水，流动相流速为0.8～1.0mL/min，进样量10～20μL，柱温30～40℃，波长225nm，运行时间70min。
  6. 如权利要求1-5中任意一项所述的化合物，其特征在于，用液相色谱或液质联用色谱检测恩格列净原料药或制剂中式I所示化合物的含量时，紫外检测器波长为225nm条件下，式I所示化合物的相对于恩格列净的校正因子约为0.8～1.2。
  7. 如权利要求5或6所述的化合物，其特征在于，用液相色谱或液质联用色谱检测恩格列净原料药或制剂中式I所示化合物的含量时，色谱流动相中甲醇：水的比例依次为：0→10分钟，40：60；10→15分钟，40：60 →52：48；15→35分钟，52：48；35→50分钟，52：48→80：20；50→60分钟，80：20；60→60.1分钟，80：20→40：60；60.1→70分钟，40：60。
  8. 如权利要求1所述的化合物，其特征在于，制备方法包括如下步骤：
    Figure PCTCN2022121230-appb-100002
    其中，X为卤素原子，选自溴原子和碘原子；
    步骤1，化合物II经还原反应制得化合物III；
    步骤2，化合物III和化合物IV形成碳苷化合物，再与甲醇形成甲氧苷化合物，最后经还原制得化合物V；
    步骤3，化合物V与1,4-丁二醇发生取代反应制得目标化合物I。
  9. 如权利要求8所述的化合物，其特征在于，制备方法的条件如下：
    步骤1，化合物II在三乙基硅烷和三氯化铝的作用下还原制得化合物III；
    步骤2，化合物III和化合物IV在正丁基锂作用下于-75～-85℃温度下形成碳苷化合物，并在甲磺酸催化作用下于10～40℃与甲醇形成甲氧苷化合物，最后经三乙基硅烷和三氯化铝于10～40℃还原制得化合物V；
    步骤3，化合物V和1,4-丁二醇在叔丁醇钾作用下，在四氢呋喃溶剂中加热回流制得化合物I。
  10. 如权利要求1中所述的化合物，其特征在于对钠-葡萄糖协同转运蛋白2具有明显的抑制作用。
  11. 如权利要求1中所述的化合物在制备用于治疗糖尿病的药物中的用途。
  12. 一种药物组合物，所述的药物组合物含有有效剂量的权利要求1所述的化合物或其药学可接受的盐，及可药用的载体、赋形剂或它们的组合。
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CN113372315A (zh) * 2021-08-12 2021-09-10 北京惠之衡生物科技有限公司 一种c-糖苷类衍生物的杂质的合成方法
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CN113372315A (zh) * 2021-08-12 2021-09-10 北京惠之衡生物科技有限公司 一种c-糖苷类衍生物的杂质的合成方法

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