Quality control method and impurity compound of thiatipiperidine

Abstract

The invention provides a quality control method and impurity compounds of thiatipine, and particularly, the impurity compounds comprise a compound 3, a compound 4, a compound 5 and a compound 7. The impurity compound can be used as a standard reference substance in a synthesis process of thiatipiperidine, and the purity of a product is detected, so that the medicine quality is further improved.

Description

Technical Field

[0001] The invention relates to the field of pharmaceutical chemistry, and in particular to a quality control method for thiotepa and an impurity compound thereof. Background Art

[0002] Thiotepa (ThioTEPA or thiotepa, also known as thiotepa, thiotepa), chemical full name N, N', N"-triethylenethiophosphoramide, is an alkylating agent used to treat cancer. Its structure is shown below:

[0003]

[0004] Thiotepa is an anti-tumor chemical drug and a representative of ethyleneimine alkylating agents. It is effective against a variety of solid tumors. Its mechanism of action is similar to that of nitrogen mustard. The active alkylating group is the ethyleneimine group produced in the body. It is a non-specific drug for the cell cycle. Experimental studies have shown that it has a significant inhibitory effect on a variety of tumors. The drug is mainly used for intracavitary injection of breast cancer, ovarian cancer, cancerous body cavity effusions, local perfusion of bladder cancer, etc. It can also be used for gastrointestinal tumors, etc. Thiotepa was first synthesized by the American Cyanamid Company in the early 1950s and reported in 1953. People began to use this drug in the 1960s. Thiotepa was certified as one of the optional orphan drugs before bone marrow stem cell transplantation by the European Medicines Agency (EMA) and the US Food and Drug Administration (FDA) on January 29, 2007 and April 2, 2007, respectively. At present, thiotepa for this purpose is mainly produced by the Italian company ADIENNE Pharma & Biotech under the brand name "TEPADINA". On March 19, 2024, the National Medical Products Administration (NMPA) issued an announcement approving Hengrui Medicine's products (Chinese and English generic name: Thiotepa for Injection) was launched in China. Its first indication is the conditioning treatment for children with severe β-thalassemia (<18 years old) undergoing allogeneic hematopoietic stem cell transplantation (allo-HSCT). It is the first thiotepa product in China approved for transplant conditioning-related indications, which will truly benefit Chinese children with severe β-thalassemia.

[0005] There are few literature reports on the research of related substances of thiotepa APIs and preparations. Among the pharmacopoeias of various countries, the European Pharmacopoeia does not include the drug; the Japanese Pharmacopoeia included the drug in version 16, but did not include related substance detection items; the Chinese Pharmacopoeia included the drug but did not include related substance detection items. Only the United States Pharmacopoeia includes related substance detection items, but only includes one impurity "thiotepa chloroethyl analogue", the structure of which is shown below:

[0006]

[0007] Thiotepa is a drug that has been marketed and widely used at home and abroad. There are currently few reports on the synthesis of its related process impurities and intermediate impurities (see related common impurities Figure 1 ). Impurity research is an important part of the drug development process, especially the impurities of intermediates. If the impurities carried by the intermediates in the drug synthesis process cannot be effectively identified, these impurities will enter the active pharmaceutical ingredients (API) of the drug through chemical reactions, or be derivatized into the API of the drug, which will eventually affect the quality of the drug and may also have a huge impact on the health of patients.

[0008] Therefore, it is very necessary to conduct further research on the synthesis process of impurities in the preparation of thiotepa, so that the relevant impurities have standard reference substances, and can effectively identify drug intermediate impurities and accurately control their content in the subsequent preparation of thiotepa related drugs, which is of great significance for controlling drug quality. Summary of the invention

[0009] The purpose of the present invention is to provide a quality control method for thiotepa and an impurity compound thereof.

[0010] A first aspect of the present invention provides a quality control method for thiotepa, the method comprising the following steps:

[0011] S1. Prepare standard reference solutions of thiotepa and impurities;

[0012] S2. Prepare a test solution containing thiotepa;

[0013] S3. The chromatographic results of the standard reference substance and the test substance were detected by high performance liquid chromatography; wherein the mobile phase A was water, the mobile phase B was acetonitrile, and the elution procedure was gradient elution;

[0014] S4. Determine the content of thiotepa and impurities in the test sample based on the chromatographic results of the standard reference substance and the test sample;

[0015] Wherein, the impurities are selected from the following group:

[0016] In another preferred embodiment, the impurities are of formula 3 and formula 4, and the preparation method of the impurities of formula 3 and formula 4 comprises the steps of:

[0017]

[0018] (1) In an inert solvent, in the presence of a catalyst, compound 1 reacts with compound 2 to obtain compound 3 and compound 4.

[0019] In another preferred embodiment, the preparation method further has one or more characteristics selected from the following group:

[0020] (1) The inert solvent in said step is selected from the group consisting of dichloromethane, 1,2-dichloroethane, chloroform, chlorobenzene and nitrobenzene;

[0021] (2) The catalyst in said step is selected from the group consisting of triethylamine, DBU, pyridine, 2,6-lutidine and 2,4,6-trimethylpyridine:

[0022] (3) The molar equivalent ratio of compound 1 to compound 2 in the step is 1:(1.6-2.5);

[0023] (4) The molar equivalent ratio of compound 1 to the catalyst in the step is 1:(2.0-2.4).

[0024] In another preferred embodiment, the reaction time of the step is 1 to 10 hours.

[0025] In another preferred embodiment, the reaction temperature of the step is -80 to 0°C.

[0026] In another preferred embodiment, the molar ratio of compound 1 to compound 2 in the step is 1:1.8-2.2.

[0027] In another preferred embodiment, the molar ratio of compound 1 to the catalyst in the step is 1:2.0-2.4.

[0028] In another preferred embodiment, the inert solvent in the step is dichloromethane.

[0029] In another preferred embodiment, the catalyst in the step is triethylamine.

[0030] In another preferred embodiment, the steps are also performed under the protection of an inert gas.

[0031] In another preferred embodiment, the inert gas is nitrogen.

[0032] In another preferred embodiment, the catalyst in the step is added to the reaction solution in a dropwise manner.

[0033] In another preferred embodiment, the reaction time in the step is 1-4h.

[0034] In another preferred embodiment, the reaction temperature in the step is -50 to -30°C.

[0035] In another preferred embodiment, the first step of the steps is to obtain a mixed solution A in which compound 1 is dissolved in an inert solvent, the second step is to add a catalyst to the mixed solution, and the third step is to add compound 2 to the mixed solution obtained in the second step.

[0036] In another preferred embodiment, the step further comprises cooling the mixed solution A to -80 to 0°C, preferably -40°C.

[0037] In another preferred embodiment, the step further includes introducing an inert gas into the mixed solution A before the catalyst is added to the mixed solution A.

[0038] In another preferred embodiment, the step further includes separating compound 3 and compound 4 from the reaction mixture.

[0039] In another preferred embodiment, the steps include: dissolving compound 1 in dichloromethane, stirring, cooling to -40°C, sealing and introducing nitrogen, then slowly adding triethylamine dropwise thereto, and finally adding raw material 2, and keeping the temperature for reaction for 2 hours.

[0040] In another preferred embodiment, the step further includes: after the reaction is completed, the reaction mixture is subjected to rotary evaporation to remove the solvent, and then purified by silica gel column chromatography (PE: CH2Cl2 = 8:1) to obtain compound 3 and compound 4.

[0041] In another preferred embodiment, the impurity is of Formula 5, and the preparation method of the impurity of Formula 5 comprises the steps of:

[0042]

[0043] (2) Compound 3 is reacted with ethanolamine in an inert solvent to obtain compound 5.

[0044] In another preferred embodiment, the preparation method further has one or more characteristics selected from the following group:

[0045] (1) The inert solvent in said step is selected from the group consisting of dichloromethane, 1,2-dichloroethane, chloroform, chlorobenzene and nitrobenzene;

[0046] (2) The molar equivalent ratio of compound 3 to ethanolamine in the step is 1:(1-4).

[0047] In another preferred embodiment, the reaction time in the step is 1 to 10 hours;

[0048] In another preferred embodiment, the reaction temperature in the step is 15-45°C;

[0049] In another preferred embodiment, the inert solvent in the step is dichloromethane.

[0050] In another preferred embodiment, the reaction time in the step is 1 to 4 hours.

[0051] In another preferred embodiment, the reaction temperature in the step is 20-28°C.

[0052] In another preferred example, the steps include: using compound 3 as a raw material, using dichloromethane as a reaction solvent, adding ethanolamine, reacting at room temperature for 2 hours, and separating and purifying to obtain compound 5.

[0053] In another preferred embodiment, the impurity is of Formula 5, and the preparation method of the impurity of Formula 5 comprises the steps of:

[0054]

[0055] (1) Compound 1 reacts with Compound 2 in an inert solvent in the presence of a catalyst to form a mixed solution C;

[0056] (2) Add ethanolamine to the mixed solution C obtained in step (1) to obtain compound 5.

[0057] In another preferred embodiment, the preparation method further has one or more characteristics selected from the following group:

[0058] (1) The inert solvent in step (1) is selected from the group consisting of dichloromethane, 1,2-dichloroethane, chloroform, chlorobenzene and nitrobenzene;

[0059] (2) The catalyst in step (1) is selected from the group consisting of triethylamine, DBU, pyridine, 2,6-dimethylpyridine and 2,4,6-trimethylpyridine:

[0060] (3) The molar equivalent ratio of compound 1 to compound 2 in step (1) is 1:(1-6);

[0061] (4) The inert solvent in step (2) is selected from the group consisting of dichloromethane, 1,2-dichloroethane, chloroform, chlorobenzene, and nitrobenzene;

[0062] (5) The molar equivalent ratio of compound 1 to ethanolamine in step (2) is 1:(0.8-1.2).

[0063] In another preferred embodiment, the reaction time in the step (1) is 1 to 10 hours.

[0064] In another preferred embodiment, the reaction temperature in step (1) is -80 to 0°C.

[0065] In another preferred embodiment, the reaction time in step (2) is 1 to 10 hours.

[0066] In another preferred embodiment, the reaction temperature in step (2) is 15-45°C.

[0067] In another preferred embodiment, the impurity is of Formula 7, and the preparation method of the impurity of Formula 7 comprises the steps of:

[0068]

[0069] (1) Compound 1 reacts with compound 2 in an inert solvent to obtain compound 7.

[0070] In another preferred embodiment, the preparation method further has one or more characteristics selected from the following group:

[0071] (1) The inert solvent in said step is selected from the group consisting of dichloromethane, 1,2-dichloroethane, chloroform, chlorobenzene and nitrobenzene;

[0072] (2) The catalyst in said step is selected from the group consisting of triethylamine, DBU, pyridine, 2,6-lutidine and 2,4,6-trimethylpyridine:

[0073] (3) The molar equivalent ratio of compound 1 to compound 2 in the step is 1:(0.5-1.5).

[0074] (4) The molar equivalent ratio of compound 1 to the catalyst in the step is 1:(0.8-1.4).

[0075] In another preferred embodiment, the reaction time in the step is 1 to 10 hours.

[0076] In another preferred embodiment, the reaction temperature in the step is -80 to 0°C.

[0077] In another preferred embodiment, the molar ratio of compound 1 to compound 2 in the step is 1:(0.9-1.1).

[0078] In another preferred embodiment, the molar ratio of compound 1 to the catalyst in the step is 1:(0.9-1.2).

[0079] In another preferred embodiment, the inert solvent in the step is dichloromethane.

[0080] In another preferred embodiment, the catalyst in the step is triethylamine.

[0081] In another preferred embodiment, the steps are also performed under the protection of an inert gas.

[0082] In another preferred embodiment, the inert gas is nitrogen.

[0083] In another preferred embodiment, the catalyst in the step is added to the reaction solution in a dropwise manner.

[0084] In another preferred embodiment, the reaction time in the step is 1 to 4 hours.

[0085] In another preferred embodiment, the reaction temperature in the reaction is -50 to -30°C.

[0086] In another preferred embodiment, the first step of the steps is to obtain a mixed solution A in which compound 1 is dissolved in an inert solvent, the second step is to add a catalyst to the mixed solution, and the third step is to add compound 2 to the mixed solution obtained in the second step.

[0087] In another preferred embodiment, the step further comprises cooling the mixed solution A to -80 to 0°C, preferably -50 to -30°C.

[0088] In another preferred embodiment, the step further includes introducing an inert gas into the mixed solution A before the catalyst is added to the mixed solution A.

[0089] In another preferred embodiment, the step further comprises isolating compound 7 from the reaction mixture solution.

[0090] In another preferred embodiment, the steps include: dissolving compound 1 in dichloromethane, stirring, cooling to -40°C, sealing and introducing nitrogen, then slowly adding triethylamine dropwise thereto, and finally adding raw material 2, and keeping the temperature for reaction for 2 hours.

[0091] In another preferred embodiment, the step further includes: after the reaction is completed, the reaction mixture is evaporated to remove the solvent, and then purified by silica gel column chromatography (PE: CH2Cl2 = 20:1) to obtain compound 7.

[0092] In another preferred embodiment, the chromatographic column in the high performance liquid chromatography is a C18 chromatographic column; the volume percentage of the mobile phase A is 40-100%, and the volume percentage of the mobile phase B is 0-60%.

[0093] In another preferred embodiment, the C18 chromatographic column is a C18 chromatographic column that can tolerate 100% aqueous phase.

[0094] In another preferred embodiment, the volume percentage of each component in the mobile phase of the gradient elution is as follows:

[0095] Time (minutes) water Acetonitrile 0 90-98％ 2-10％ 7-10 2-10％ 90-98％ 10-15 2-10％ 90-98％

[0096] In another preferred embodiment, the volume percentage of each component in the mobile phase of the gradient elution is as follows:

[0097] Time (minutes) water Acetonitrile 0 95％ 5％ 7 5％ 95％ 10 5％ 95％

[0098] In another preferred embodiment, the high performance liquid chromatography method has one or more characteristics selected from the following group:

[0099] (1) The detection wavelength is 210-280nm;

[0100] (2) Column temperature is 25-40°C;

[0101] (3) Flow rate is 0.5 to 1.0 mL per minute;

[0102] (4) Injection volume is 10 to 30 μL

[0103] In another preferred example, the detection wavelength is 210 nm.

[0104] In another preferred embodiment, the column temperature is 30°C.

[0105] In another preferred embodiment, the flow rate is 0.8 mL per minute;

[0106] In another preferred example, the injection volume is 20 μL.

[0107] 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 specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0108] Figure 1 It is a representative impurity of thiotepa sold on the market.

[0109] Figure 2 This is the H NMR spectrum of compound 3.

[0110] Figure 3 The NMR carbon spectrum of compound 3

[0111] Figure 4 This is the H NMR spectrum of compound 4.

[0112] Figure 5 This is the C NMR spectrum of compound 4.

[0113] Figure 6 This is the H NMR spectrum of compound 5.

[0114] Figure 7 is the mass spectrum of compound 5.

[0115] Figure 8 This is the HPLC chart of compound 5.

[0116] Fig. 9 This is the H NMR spectrum of compound 6.

[0117] Fig.10 This is the C NMR spectrum of compound 6.

[0118] Fig.11 This is the H NMR spectrum of compound 7.

[0119] Fig.12 This is the C NMR spectrum of compound 7.

[0120] Fig.13 HPLC charts of impurities 3, 5 and thiotepa 6.

[0121] Fig.14 HPLC charts of impurity 4 and thiotepa 6.

[0122] Fig.15 This is the HPLC chart of thiotepa 6.

[0123] Fig.16 This is the HPLC chart of impurity 4. DETAILED DESCRIPTION

[0124] The inventors have conducted extensive and in-depth research, and through a large number of screenings and tests, have provided a quality control method for thiotepa and its impurity compounds. The quality control method solves the problem of impurities that are easily generated in the existing thiotepa synthesis process and have a key impact on the quality of thiotepa products, greatly improves the quality of the drug, and further ensures the safety of the patient's medication. The present invention was completed on this basis.

[0125] the term

[0126] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0127] As used herein, when used in reference to a specific recited numerical value, the term "about" means that the value may vary by no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0128] As used herein, the term "comprising" or "including (comprising)" may be open, semi-closed and closed. In other words, the term also includes "consisting essentially of" or "consisting of".

[0129] As used herein, the term "room temperature" or "normal temperature" refers to a temperature of 4-40°C, preferably, 25±5°C.

[0130] As used herein, the terms "Compound 2" and "ethyleneimine" are used interchangeably and both refer to a compound having a structure as shown in the following formula.

[0131]

[0132] As used herein, the terms "impurity compound 3", "compound 3" and "impurity 3" are used interchangeably and refer to a compound having a structure such as The compounds shown.

[0133] As used herein, the terms "impurity compound 4", "compound 4" and "impurity 4" are used interchangeably and refer to a compound having a structure such as The compounds shown.

[0134] As used herein, the terms "impurity compound 5", "compound 5" and "impurity 5" are used interchangeably and refer to a compound having a structure such as The compounds shown.

[0135] As used herein, the terms "thiotepa compound 6", "compound 6" and "thiotepa 6" are used interchangeably and refer to a compound having a structure such as The compounds shown.

[0136] As used herein, the terms "impurity compound 7", "compound 7" and "impurity 7" are used interchangeably and refer to a compound having a structure such as The compounds shown.

[0137] As used herein, the term "TLC" refers to thin layer chromatography.

[0138] As used herein, the term "TEA" refers to triethylamine.

[0139] As used herein, the term "PE" refers to petroleum ether.

[0140] As used herein, the term "DCM" refers to dichloromethane.

[0141] As used herein, the term "EtOAc" refers to ethyl acetate.

[0142] The main advantages of the present invention include:

[0143] The present invention provides three structural formulae of impurity compounds and four impurity compounds, which can be used independently or in any combination as reference substances in the quality control of the thiotepa synthesis process, and are of great significance for controlling product quality and ensuring drug safety.

[0144] The present invention will be further described below in conjunction with specific implementation. It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples without specifying specific conditions are usually based on conventional conditions or the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

[0145] Unless otherwise defined, all professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein can be applied to the methods of the present invention. The preferred implementation methods and materials described herein are for demonstration purposes only.

[0146] General Implementation Methods

[0147] Preparation of compound 1

[0148]

[0149] Take 196g of sulfur powder and 18g of anhydrous aluminum chloride, put them in a 3000mL flask, install a reflux condenser on the flask, and add 500mL of phosphorus trichloride (PCl3). The reaction liquid immediately boils violently and is quickly cooled with an ice water bath. When the boiling stops, the sulfur powder is completely dissolved. Remove the ice water bath, replace it with a water bath and heat it to 95℃, continue the reaction for 2h, cool it, and wash it with ice water twice. Separate the milky liquid. Dry it with anhydrous calcium chloride. Distill bp.124-126℃, yield 86%.

[0150] Preparation of compound 2 ethyleneimine

[0151]

[0152] 100mL of sulfuric acid (102.9g, 1.05mol) aqueous solution was slowly added to a reaction bottle containing 60mL of ethanolamine (61.1g, 1.0mol) aqueous solution. After all the sulfuric acid aqueous solution was added, the water was removed by vacuum distillation at 100°C until it was concentrated to form a solid. At this time, the solid product was ethanolamine sulfate. After cooling to room temperature, 233mL of 30% NaOH solution was added to ethanolamine sulfate and heated to 100°C for 3h to hydrolyze it to obtain a solution. Then, the solution was distilled at 120°C to obtain a mixture of ethyleneimine and distilled water. After that, NaOH particles were added to the mixed solution, and pure ethyleneimine (35.1g, yield 81.5%) was obtained after extraction and calcium hydride. The raw material was unstable and needed to be stored at low temperature.

[0153] Example 1 Synthesis of Compound 3 and Compound 4

[0154]

[0155] Weigh compound 1 (3.39 g, 20 mmol, 1.0 equiv.) into a 50 mL Shrek tube, measure 20 mL of dichloromethane and place it in a bottle, stir it magnetically, place it in a low-temperature stirrer and cool it to -40 ° C, seal the bottle mouth and protect it with nitrogen, then slowly add triethylamine (4.45 g, 44 mmol, 2.2 equiv.) dropwise with a syringe, and finally add raw material 2 (1.72 g, 40 mmol, 2.0 equiv.) into the reaction bottle, and keep the temperature for 2 hours. Monitor the reaction by TLC (a mixed solvent of petroleum ether: dichloromethane = 2:1 as the developing solvent).

[0156] TLC detected that the raw material point disappeared and two new product points were generated in the reaction solution. The solvent dichloromethane was evaporated using a rotary evaporator to obtain a transparent liquid, which was then separated and purified by column chromatography (PE: CH2Cl2 = 8:1) to obtain impurity compound 3 (0.445 g, yield: 12.2%) and impurity compound 4 (0.317 g, yield; 10.1%).

[0157] Impurity compound 3:

[0158] 1 H NMR (400MHz, CDCl3): δ (ppm) = 2.41 (s, 4H), 2.35 (s, 4H).

[0159] 13 C NMR (151MHz, CDCl3): δ (ppm) = 27.40, 27.34.

[0160] Impurity compound 4:

[0161] 1 H NMR (400MHz, CDCl3): δ (ppm) = 4.59 (s, 4H), 3.75 (t, J = 5.5Hz, 2H), 3.61 (dq, J = 17.9, J = 5.9Hz, 2H).

[0162] 13 C NMR (151MHz, CDCl3): δ (ppm) = 45.23, 45.21, 43.94, 43.88.

[0163] Example 2 Synthesis of Impurity Compound 5

[0164]

[0165] Compound 3 obtained in Example 1 was used as a raw material, dichloromethane was used as a reaction solvent, and an equivalent amount of ethanolamine was added. After reacting at room temperature for 2 hours, the raw material was basically reacted completely according to the spot plate detection. Impurity compound 5 was separated and purified, and the yield was 78.5%.

[0166] Impurity compound 5:

[0167] 1 H NMR (400MHz, CDCl3): δ (ppm) = 3.73-3.66 (m, 2H), 3.62-3.51 (m, 3H), 3.31-3.23 (m, 2H), 2.07-2.01 (m, 4H), 1.96-1.89 (m, 4H).

[0168] 13C NMR (151MHz, CDCl3): δ (ppm) = 63.49, 63, 47, 44.17, 44.14, 24.06, 24.02.

[0169] [M+H + ] + (ESI): m / z = 208.25

[0170] Example 3 Synthesis of Compound 5

[0171]

[0172] At -40°C, dichloromethane (40 mL) was added as the reaction solvent into the Shrek tube, the reaction bottle was filled with nitrogen for protection, and then compound 1 (3.39 g, 20 mmol, 1 equiv.), triethylamine (4.45 g, 44 mmol, 2.2 equiv.), and compound 2 (1.72 g, 40 mmol, 2 equiv.) were added in sequence with a syringe. After 2 hours of low temperature reaction, the raw materials were basically reacted completely by spot plate detection, the temperature was raised to 0°C, and then an equivalent amount of ethanolamine (1.22 g, 20 mmol, 1 equiv.) was added to the reaction bottle, and the reaction was monitored by TLC until the raw materials reacted completely. The reaction solution was washed with saturated sodium bicarbonate, dried over anhydrous sodium sulfate, filtered, concentrated, and the crude product was separated and purified by column chromatography to obtain a colorless oily impurity compound 5 (2.59 g, yield: 62.5%).

[0173] Example 4 Synthesis of Compound 3

[0174]

[0175] Compound 1 (3.39 g, 20 mmol, 1 equiv) was weighed and added to a 100 mL Shrek tube, 30 mL of dichloromethane was measured and placed in a bottle, magnetically stirred, placed in a low temperature stirrer and cooled to -40 ° C, the bottle mouth was sealed and protected with nitrogen, and then triethylamine (6.68 g, 66 mmol, 3.3 equiv) was slowly added dropwise with a syringe, and finally compound 2 (2.58 g, 60 mmol, 3 equiv.) was added to the reaction bottle, and the reaction was kept warm for 2 hours. The reaction was monitored by TLC, and it was detected that the raw material was basically reacted completely, and two product points were produced, namely compound 3 and thiotepa 6. The solvent dichloromethane was evaporated using a rotary evaporator, and then the treated mixture was separated and purified by column chromatography (PE: EtOAc = 8: 1) to obtain compound 6 thiotepa (0.783 g, yield: 20.7%) and impurity compound 3 (0.712 g, yield: 19.5%).

[0176] Compound 6

[0177] 1 H NMR (400MHz, CDCl3): δ (ppm) == 2.13 (d, J = 16.6Hz, 12H).

[0178] 13 C NMR (151MHz, CDCl3): δ (ppm) = 24.30, 24.23.

[0179] Example 5 Synthesis of Impurity Compound 4 and Impurity Compound 7

[0180]

[0181] Weigh compound 1 (3.39 g, 20 mmol, 1.0 equiv.) into a 50 mL Shrek tube, measure 20 mL of dichloromethane and place it in a bottle, stir it magnetically, place it in a low-temperature stirrer and cool it to -40 ° C, seal the bottle mouth and protect it with nitrogen, then slowly add triethylamine (2.23 g, 22 mmol, 1.1 equiv.) dropwise into it with a syringe, and finally add raw material 2 (0.86 g, 20 mmol, 1.0 equiv.) into the reaction bottle, and keep the temperature for 2 hours. The reaction was monitored by TLC (a mixed solvent of petroleum ether: dichloromethane = 2:1 as a developing solvent), and it was detected that the raw material was basically reacted completely, and two product points were produced, namely impurity 4 and impurity 7. The solvent dichloromethane was evaporated using a rotary evaporator, and the treated mixture was then separated and purified by column chromatography (PE: CH2Cl2 = 20:1) to obtain impurity compound 7 (0.535 g, yield: 15.2%) and impurity compound 4 (0.423 g, yield: 13.1%).

[0182] Compound 7:

[0183] 1 H NMR (400MHz, CDCl3): δ (ppm) = 2.57 (s, 2H), 2.51 (s, 2H)..

[0184] 13 C NMR (151MHz, CDCl3): δ (ppm) = 30.13, 30.05.

[0185] Compound structure list

[0186]

[0187] Example 6 High Performance Liquid Chromatography Detection Method for Impurity Compound 5

[0188] 6.1. Instruments and equipment

[0189] Agilent 1260 Infinity II liquid chromatography system;

[0190] Agilent InfinityLab poroshell 120EC-C18 column (specifications: 4.6*100mm, 2.7μm);

[0191] One hundred thousandth electronic balance (SECURA225D-1CN, Sartorius);

[0192] Acetonitrile was of HPLC grade and water was deionized.

[0193] 6.2. Chromatographic conditions

[0194] Octadecylsilane bonded silica gel was used as filler (InfinityLab poroshell 120EC-C18, 4.6*100mm, 2.7μm);

[0195] Deionized water was used as mobile phase A, acetonitrile was used as mobile phase B, and gradient elution was performed according to the following table:

[0196] Table 1 Gradient elution conditions

[0197]

[0198]

[0199] The detection wavelength was 210 nm; the flow rate was 0.8 mL per minute; the column temperature was 30°C; and the injection volume was 2 μL.

[0200] 6.3. Sample preparation

[0201] Weigh 3.481 mg of homemade impurity 5 into a 10 mL volumetric flask, add mobile phase B to dissolve and dilute to the scale, shake well, and set aside.

[0202] 6.4 Results Analysis

[0203] The analytical test chromatogram of impurity compound 5 is shown in Figure 8 , the specific data are shown in Table 2 below, the retention time is 7.542 minutes, the peak area is 7280.56738, the peak shape is symmetrical, and it can be well analyzed and detected by HPLC.

[0204] Table 2 Liquid chromatography data of impurity compound 5

[0205]

[0206] Example 7 High Performance Liquid Chromatography Detection Method for Impurity 3, Impurity 5 and Thiotepa 6

[0207] 7.1 Instruments and Equipment

[0208] Agilent 1260 Infinity II liquid chromatography system;

[0209] Agilent InfinityLab poroshell 120EC-C18 column (specifications: 4.6*100mm, 2.7μm);

[0210] One hundred thousandth electronic balance (SECURA225D-1CN, Sartorius);

[0211] Acetonitrile was of HPLC grade and water was deionized.

[0212] 7.2 Chromatographic conditions

[0213] Octadecylsilane bonded silica gel was used as filler (InfinityLab poroshell 120EC-C18, 4.6*100mm, 2.7μm);

[0214] Deionized water was used as mobile phase A, acetonitrile was used as mobile phase B, and gradient elution was performed according to the following table:

[0215] Table 3 Gradient elution conditions

[0216] Time (minutes) water Acetonitrile 0 95％ 5％ 10 5％ 95％ 15 5％ 95％

[0217] The detection wavelength was 210 nm; the flow rate was 0.8 mL per minute; the column temperature was 30°C; and the injection volume was 2 μL.

[0218] 7.3 Sample preparation

[0219] Weigh 3.785 mg of homemade impurity 3, 35.633 mg of homemade thiotepa 6 and 3.282 mg of homemade impurity 5 into a 100 mL volumetric flask, add mobile phase B to dissolve and dilute to the scale, shake well and set aside.

[0220] 6.4 Results Analysis

[0221] The analytical test chromatograms of impurity 3, impurity 5 and thiotepa 6 are shown in Fig.13 Specific data are shown in Table 4 below. The retention time of impurity 3 is 9.341 minutes, and the peak area is 644.72687; the retention time of thiotepa 6 is 11.216 minutes, and the peak area is 11638.8; the retention time of impurity 5 is 12.079 minutes, and the peak area is 961.78644. The peak shapes of the three compounds are symmetrical and can be well analyzed and detected by HPLC.

[0222] Table 4 Liquid chromatography data of impurities 3, 5 and thiotepa 6

[0223]

[0224] Total amount: 1.32453e42099.58110

[0225] Example 8 High Performance Liquid Chromatography Detection Method for Impurity 4 and Thiotepa 6

[0226] 8.1 Instruments and Equipment

[0227] Agilent 1260 Infinity II liquid chromatography system;

[0228] Agilent InfinityLab poroshell 120EC-C18 column (specifications: 4.6*100mm, 2.7μm);

[0229] One hundred thousandth electronic balance (SECURA225D-1CN, Sartorius);

[0230] Acetonitrile was of HPLC grade and water was deionized.

[0231] 8.2 Chromatographic conditions

[0232] Octadecylsilane bonded silica gel was used as filler (InfinityLab poroshe1l 120EC-C18, 4.6*100mm, 2.7μm);

[0233] Deionized water was used as mobile phase A, acetonitrile was used as mobile phase B, and gradient elution was performed according to the following table:

[0234] Table 5 Gradient elution conditions

[0235] Time (minutes) water Acetonitrile 0 95％ 5％ 7 5％ 95％ 10 5％ 95％

[0236] The detection wavelength was 210 nm; the flow rate was 0.8 mL per minute; the column temperature was 30°C; and the injection volume was 2 μL.

[0237] 8.3 Sample preparation

[0238] Weigh 6.528 mg of homemade impurity 4 and 18.723 mg of homemade thiotepa 6 into a 100 mL volumetric flask, add mobile phase B to dissolve and dilute to the scale, shake well, and set aside.

[0239] 8.4 Results Analysis

[0240] The analytical test chromatograms of impurity 4 and thiotepa 6 are shown in Fig.14 Specific data are shown in Table 6 below. The retention time of impurity 4 is 6.677 minutes, and the peak area is 823.28589; the retention time of thiotepa 6 is 7.198 minutes, and the peak area is 5438.32373. The peak shapes of the two compounds are symmetrical and can be well analyzed and detected by HPLC.

[0241] Table 6 Liquid chromatography data of impurity 4 and thiotepa 6

[0242]

[0243] Total amount: 6415.04307 1164.21264

[0244] Example 9 High-performance liquid phase detection method for thiotepa 6

[0245] 9.1 Instruments and Equipment

[0246] Agilent 1260 Infinity II liquid chromatography system;

[0247] Agilent InfinityLab poroshe11120EC-C18 column (specifications: 4.6*100mm, 2.7μm);

[0248] One hundred thousandth electronic balance (SECURA225D-1CN, Sartorius);

[0249] Acetonitrile was of HPLC grade and water was deionized.

[0250] 9.2 Chromatographic conditions

[0251] Octadecylsilane bonded silica gel was used as filler (InfinityLab poroshell 120EC-C18, 4.6*100mm, 2.7μm);

[0252] Deionized water was used as mobile phase A, acetonitrile was used as mobile phase B, and gradient elution was performed according to the following table:

[0253] Table 7 Gradient elution conditions

[0254] Time (minutes) water Acetonitrile 0 95％ 5％ 7 5％ 95％ 10 5％ 95％

[0255] The detection wavelength was 210 nm; the flow rate was 0.8 mL per minute; the column temperature was 30°C; and the injection volume was 2 μL.

[0256] 9.3 Sample preparation

[0257] Weigh 6.573 mg of homemade thiotepa 6 into a 100 mL volumetric flask, add mobile phase B to dissolve and dilute to the scale, shake well, and set aside.

[0258] 9.4 Results Analysis

[0259] The analytical test chromatogram of Thiotepa 6 is shown in Fig.15Specific data are shown in Table 8 below. The retention time of thiotepa 6 is 7.200 minutes, and the peak area is 1716.06360. The peak shape of the compound is symmetrical and can be well analyzed and detected by HPLC.

[0260] Table 8 Liquid chromatography data of Thiotepa 6

[0261]

[0262] Total amount: 1749.07432 322.06625

[0263] Example 10 High Performance Liquid Chromatography Detection Method for Impurity 4

[0264] 10.1 Instruments and Equipment

[0265] Agilent 1260 Infinity II liquid chromatography system;

[0266] Agilent InfinityLab poroshell 120EC-C18 column (specifications: 4.6*100mm, 2.7μm);

[0267] One hundred thousandth electronic balance (SECURA225D-1CN, Sartorius);

[0268] Acetonitrile was of HPLC grade and water was deionized.

[0269] 10.2 Chromatographic conditions

[0270] Octadecylsilane bonded silica gel was used as filler (InfinityLab poroshell 120EC-C18, 4.6*100mm, 2.7μm);

[0271] Deionized water was used as mobile phase A, acetonitrile was used as mobile phase B, and gradient elution was performed according to the following table:

[0272] Table 9 Gradient elution conditions

[0273] Time (minutes) water Acetonitrile 0 95％ 5％ 7 5％ 95％ 10 5％ 95％

[0274] The detection wavelength was 210 nm; the flow rate was 0.8 mL per minute; the column temperature was 30°C; and the injection volume was 2 μL.

[0275] 10.3 Sample preparation

[0276] Weigh 42.618 mg of homemade impurity 4 into a 100 mL volumetric flask, add mobile phase B to dissolve and dilute to the scale, shake well, and set aside.

[0277] 10.4 Results Analysis

[0278] The analytical test chromatogram of impurity 4 is shown in Fig.16 , the specific data are shown in Table 10 below, the retention time of impurity 4 is 6.664 minutes, and the peak area is 10548.6; the peak shape of the compound is symmetrical and can be well analyzed and detected by HPLC.

[0279] Table 10 Liquid chromatography data of impurity 4

[0280]

[0281] Total amount: 1.06298e4 1837.26563

[0282] In summary, the liquid phase detection method described in the present application has good separation for various impurity compounds in thiotepa, and can be applied to industrial production to improve the purity of thiotepa and improve the quality of drugs.

[0283] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A quality control method for thiotepa, characterized in that: The method comprises the following steps: S1. Prepare standard reference solutions of thiotepa and impurities; S2. Prepare a test solution containing thiotepa; S3. The chromatographic results of the standard reference substance and the test substance were detected by high performance liquid chromatography; wherein the mobile phase A was water, the mobile phase B was acetonitrile, and the elution procedure was gradient elution; S4. Determine the content of thiotepa and impurities in the test sample based on the chromatographic results of the standard reference substance and the test sample; Wherein, the impurity is selected from the following group: Formula 3 Formula 4 Formula 5 Formula 7 2. The method according to claim 1, characterized in that The impurities are of formula 3 and formula 4, and the preparation methods of the impurities of formula 3 and formula 4 include the steps of: (1) In an inert solvent, in the presence of a catalyst, compound 1 reacts with compound 2 to obtain compound 3 and compound 4.

3. The method according to claim 2, characterized in that The preparation method also has one or more characteristics selected from the following group: (1) The inert solvent in said step is selected from the group consisting of dichloromethane, 1,2-dichloroethane, chloroform, chlorobenzene and nitrobenzene; (2) The catalyst in said step is selected from the group consisting of triethylamine, DBU, pyridine, 2,6-lutidine and 2,4,6-trimethylpyridine: (3) The molar equivalent ratio of compound 1 to compound 2 in the step is 1:(1.6-2.5); (4) The molar equivalent ratio of compound 1 to the catalyst in the step is 1:(2.0-2.4).

4. The method according to claim 1, characterized in that The impurity is of Formula 5, and the preparation method of the impurity of Formula 5 comprises the steps of: (2) Compound 3 is reacted with ethanolamine in an inert solvent to obtain compound 5.

5. The method according to claim 4, characterized in that The preparation method also has one or more characteristics selected from the following group: (1) The inert solvent in said step is selected from the group consisting of dichloromethane, 1,2-dichloroethane, chloroform, chlorobenzene and nitrobenzene; (2) The molar equivalent ratio of compound 3 to ethanolamine in the step is 1:(1-4).

6. The method according to claim 1, characterized in that The impurity is of Formula 5, and the preparation method of the impurity of Formula 5 comprises the steps of: (1) Compound 1 reacts with Compound 2 in an inert solvent in the presence of a catalyst to form a mixed solution C; (2) Add ethanolamine to the mixed solution C obtained in step (1) to obtain compound 5.

7. The method according to claim 6, characterized in that The preparation method also has one or more characteristics selected from the following group: (1) The inert solvent in step (1) is selected from the group consisting of dichloromethane, 1,2-dichloroethane, chloroform, chlorobenzene and nitrobenzene; (2) The catalyst in step (1) is selected from the group consisting of triethylamine, DBU, pyridine, 2,6-dimethylpyridine and 2,4,6-trimethylpyridine: (3) The molar equivalent ratio of compound 1 to compound 2 in step (1) is 1:(1-6); (4) The inert solvent in step (2) is selected from the group consisting of dichloromethane, 1,2-dichloroethane, chloroform, chlorobenzene, and nitrobenzene; (5) The molar equivalent ratio of compound 1 to ethanolamine in step (2) is 1:(0.8-1.2).

8. The method according to claim 1, characterized in that The impurity is of Formula 7, and the preparation method of the impurity of Formula 7 comprises the steps of: (1) Compound 1 reacts with compound 2 in an inert solvent to obtain compound 7.

9. The method according to claim 8, characterized in that The preparation method also has one or more characteristics selected from the following group: (1) The inert solvent in said step is selected from the group consisting of dichloromethane, 1,2-dichloroethane, chloroform, chlorobenzene and nitrobenzene; (2) The catalyst in said step is selected from the group consisting of triethylamine, DBU, pyridine, 2,6-lutidine and 2,4,6-trimethylpyridine: (3) The molar equivalent ratio of compound 1 to compound 2 in the step is 1:(0.5-1.5); (4) The molar equivalent ratio of compound 1 to the catalyst in the step is 1:(0.8-1.4).

10. The method according to claim 1, characterized in that The chromatographic column in the high performance liquid chromatography is a C18 chromatographic column; the volume percentage of water in the mobile phase is 40-100%, and the volume percentage of acetonitrile is 0-60%.

11. The method according to claim 1, characterized in that The volume percentages of the components in the mobile phase of the gradient elution are as follows: 。 12. The method according to claim 1, characterized in that The high performance liquid chromatography method has one or more characteristics selected from the group consisting of: (1) The detection wavelength is 210-280nm; (2) Column temperature is 25-40°C; (3) Flow rate is 0.5 to 1.0 mL per minute; (4) The injection volume is 10 to 30 μL.

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