Raltitrexed starting material reference substance impurity as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of pharmaceutical chemistry, and particularly discloses a raltitrexed starting material reference substance impurity and a preparation method and application thereof.The chemical name of the impurity is (3S)-3-aminotetrahydropyran-2, 6-diketone, and the impurity is prepared through the following steps that N-[(benzyloxy) carbonyl]-L-glutamic acid serves as a raw material, N-[(benzyloxy) carbonyl]-L-glutamic acid is added into a reaction kettle, the reaction kettle is heated, and the reaction kettle is cooled to room temperature to obtain the raltitrexed starting material reference substance impurity. The preparation method comprises the following steps: carrying out condensation in a solvent under the action of acetic anhydride, carrying out secondary column chromatography to obtain a compound 1, adding the compound 1 and an acid-binding agent into ultra-dry tetrahydrofuran, completely dissolving, and carrying out heating and pressurizing under the catalysis of Pd / C to remove carbobenzoxy so as to obtain a compound 2. The process route is relatively simple, the operation is simple and convenient, the yield is high, the synthesized impurity can be used as a reference substance for controlling the quality of a raltitrexed starting material, and the method has certain use value and popularization value.

Description

Technical Field

[0001] The invention belongs to the technical field of pharmaceutical chemistry, relates to the preparation and quality control of a raw material drug, raltitrexed, and particularly relates to an impurity of a raltitrexed starting raw material reference substance, a preparation method and an application thereof. Background Art

[0002] Raltitrexed is a water-soluble thymidine synthase inhibitor that can inhibit thymidylate synthase, leading to DNA breakage and cell death. It is used to treat patients with advanced colorectal cancer. Its chemical name is: (5-(methyl((2-methyl-4-oxo-3,4-dihydroquinazolin-6-yl)methyl)amino)thiophene-2-carbonyl)-L-glutamic acid, and its structural formula is:

[0003]

[0004] In the prior art, most studies have focused on the derivatized impurities or degradation impurities of raltitrexed during the preparation of raltitrexed. A key starting material is involved in the synthesis of raltitrexed: L-glutamic acid diethyl ester hydrochloride, which has the structural formula:

[0005]

[0006] L-glutamic acid diethyl ester hydrochloride is a key starting material in the synthesis of raltitrexed. The quality of this material is closely related to the quality research of raltitrexed. In order to further improve the quality of this intermediate, it is necessary to minimize the content of unknown impurities in this material. Currently, there are no reports on the control of impurities in the reference substance of the starting material of raltitrexed. Further identification, research and structural confirmation of impurities introduced by the starting material of raltitrexed are of great significance to further improve the level of quality control and safety of raltitrexed. Summary of the Invention

[0007] In view of the shortcomings of the prior art, the present invention aims to provide an impurity of a reference substance of a raltitrexed starting material. The present invention clarifies the structure of the impurity and provides a preparation method capable of obtaining the impurity in large quantities. The present invention also provides the use of the impurity as a reference substance in the detection of raltitrexed starting materials.

[0008] The present invention is achieved through the following technical solutions:

[0009] An impurity in the reference substance of the starting material of raltitrexed, whose chemical name is (3S)-3-aminotetrahydropyran-2,6-dione, and whose chemical structure is shown below:

[0010]

[0011] During the analysis of the initial raw materials for the synthesis of raltitrexed, technicians discovered the presence of a stable and high-content impurity. The structure of the impurity was determined by LC-MS and HPLC. After analysis, it was found that the impurity may undergo the following transformation process. Therefore, the quality research of this impurity (Compound 2) is extremely important for the starting material L-glutamic acid diethyl ester hydrochloride.

[0012]

[0013] A further improvement of the present invention is:

[0014] A method for preparing impurities of a raltitrexed starting material reference substance comprises the following steps:

[0015] (1) N-[(benzyloxy)carbonyl]-L-glutamic acid is used as a raw material, dissolved in a solvent, and heated under the action of a dehydrating agent to carry out an intramolecular carboxyl condensation ring reaction to obtain compound 1;

[0016] (2) Compound 1 and an acid-binding agent are added to a solvent and dissolved, and Pd / C is used as a catalyst, and the benzyloxycarbonyl group is removed by heating and pressurizing to obtain Compound 2, which is an impurity of the reference substance of the starting material of raltitrexed;

[0017] The chemical equation is as follows:

[0018]

[0019] Furthermore, the solvent in step (1) is one or a mixture of two or more of benzene, toluene, xylene or N,N-dimethylformamide.

[0020] Preferably, the solvent is toluene.

[0021] Furthermore, the dehydrating agent in step (1) is one or a mixture of two or more of acetic anhydride, trifluoroacetic anhydride, maleic anhydride, phthalic anhydride, propionic anhydride, acetyl chloride, phosphorus oxychloride, phosphorus pentoxide or phosphorus pentachloride.

[0022] Preferably, the dehydrating agent is acetic anhydride.

[0023] Furthermore, in step (1), the volume ratio of the N-[(benzyloxy)carbonyl]-L-glutamic acid to the dehydrating agent is 1:5 to 50;

[0024] Furthermore, in step (1), the reaction temperature is 80-120° C., and the reaction time is 3-18 h.

[0025] Furthermore, in step (2), the acid binding agent is one or a mixture of two or more of ammonium formate, ammonium acetate, ammonium propionate, ammonium valerate, ammonium sulfate, ammonium chloride, ammonium sulfite, ammonium thiocyanate, triammonium citrate, dimethylamine, pyridine or triethylamine;

[0026] Preferably, the acid binding agent is ammonium formate.

[0027] Furthermore, in step (2), the solvent is one or a mixture of two or more of tetrahydrofuran, toluene, ethanol or acetonitrile.

[0028] Preferably, the solvent is tetrahydrofuran.

[0029] Furthermore, in step (2), the molar ratio of Pd / C to compound 1 is 1:20-100;

[0030] Preferably, the molar ratio of Pd / C to compound 1 is 1:30-60;

[0031] And / or, the reaction temperature is 30-80° C., the reaction time is 3-18 h; and the reaction pressure is 0-10 MPa.

[0032] Preferably, the reaction temperature is 40-60° C., and the reaction time is 8-16 h.

[0033] A further improvement of the present invention is:

[0034] The use of the above impurities as reference substances in the detection of raltitrexed starting materials specifically comprises the following steps:

[0035] Step A: Preparation of reference solution: Accurately weigh the impurity reference substance described in claim 1, dilute to the mark with purified water, and shake well to make 0.03 mg / ml;

[0036] Step B: Test solution: Accurately weigh the test sample, dissolve it in purified water by ultrasonication, dilute to the mark, and shake well to make 15 mg / ml;

[0037] Step C, aspirating the reference solution and the test solution and injecting them into the HPLC;

[0038] The chromatographic conditions are:

[0039] Injection volume: 20 μL; column temperature: 30°C; sample chamber temperature: 10°C; detection wavelength: 210 nm;

[0040] The chromatographic column is a Waters X-Bridge shield RP18, 150 × 4.6 mm, 3.5 μm, or a column of equivalent performance;

[0041] Mobile phase A: 10 mmol potassium dihydrogen phosphate solution and 10 mmol dipotassium hydrogen phosphate solution; mobile phase B: acetonitrile;

[0042] gradient:

[0043]

[0044]

[0045] The beneficial effects of the present invention are:

[0046] The invention discloses a new reference substance impurity of a starting material of raltitrexed and confirms the structure of the impurity, which is of great significance for further improving the level of quality control and safety of raltitrexed.

[0047] The present invention uses N-[(benzyloxy)carbonyl]-L-glutamic acid as a raw material, condenses the carboxyl group in the molecule into a ring under the action of acetic anhydride to generate compound 1, and removes the benzyloxycarbonyl group under the conditions of Pd / C and an acid-binding agent to obtain high-purity compound 2. The method has a short route, relatively simple operation, is environmentally friendly, has a mild reaction, and is easy to be detected by common detection methods such as HPLC, TLC, and HNMR, thus filling the synthesis gap of this impurity compound. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 Shown is the HPLC spectrum of L-glutamic acid diethyl ester hydrochloride, the starting material of raltitrexed;

[0049] Figure 2 Shown is the HPLC spectrum of (3S)-3-aminotetrahydropyran-2,6-dione isolated in Example 1;

[0050] Figure 3 The (3S)-3-aminotetrahydropyran-2,6-dione isolated in Example 1 is shown. 1 H-NMR spectrum;

[0051] Figure 4 The (3S)-3-aminotetrahydropyran-2,6-dione isolated in Example 1 is shown. 13 C-NMR spectrum;

[0052] Figure 5 Shown is the HPLC spectrum of (3S)-3-aminotetrahydropyran-2,6-dione synthesized in Example 2. DETAILED DESCRIPTION

[0053] The present invention is described in detail below with reference to specific embodiments.

[0054] Example 1: Confirmation of impurities in the reference substance of raltitrexed starting material

[0055] During the analysis of the initial raw materials for the synthesis of raltitrexed, technicians discovered the presence of a stable and high-content impurity that was difficult to remove. The results are as follows:

[0056]

[0057] After column chromatography separation and purification, the impurity was determined to be (3S)-3-aminotetrahydropyran-2,6-dione, with the following structure: 1 H-NMR (400MHz, DMSO) δ4.07-4.05(m,1H),2.35-2.29(m,1H),2.16-2.11(m,2H),1.99-1.98(m,1H).

[0058] Example 2: Preparation of impurities in the starting material reference substance of raltitrexed

[0059]

[0060] 30g of N-[(benzyloxy)carbonyl]-L-glutamic acid was added to 300ml of acetic anhydride and toluene solution, heated to 110°C for 12h, cooled, concentrated to dryness, and subjected to column chromatography using DCM:MeOH=50:1 to obtain intermediate 1; intermediate 1 and ammonium formate were added to 360ml of tetrahydrofuran and dissolved completely, 1.2g of 10% Pd / C was removed by tetrahydrofuran, and after removal, the water was added to a hydrogenation kettle, heated to 50°C, pressurized to 2.0MPa, reacted for 12h, cooled to room temperature, filtered, concentrated to dryness, and then 20ml of tetrahydrofuran was added to slurry at room temperature for 20min, filtered and dried to obtain compound 2.

[0061] Example 3: Preparation of impurities in the starting material reference substance of raltitrexed

[0062] 15g of N-[(benzyloxy)carbonyl]-L-glutamic acid was added to 150ml of acetic anhydride and toluene solution, heated to 100°C for 8h, cooled, concentrated to dryness, and column chromatography was performed using DCM:MeOH = 50:1 to obtain compound 1; compound 1 and ammonium formate were added to 360ml of tetrahydrofuran and dissolved completely, 0.8g of 10% Pd / C was removed by tetrahydrofuran, and after removal, the water was added to a hydrogenation kettle, heated to 40°C, pressurized to 2.0MPa, and reacted for 8h. The mixture was cooled to room temperature, filtered, concentrated to dryness, and 20ml of tetrahydrofuran was added to slurry at room temperature for 20min, filtered and dried to obtain compound 2.

[0063] Example 4: Detection of impurities related to the starting material L-glutamic acid diethyl ester hydrochloride

[0064] Standard: The impurity (3S)-3-aminotetrahydropyran-2,6-dione shall not exceed 0.5%, and other individual impurities shall not exceed 0.3%.

[0065] Reference solution: Accurately weigh approximately 150 mg of the impurity reference substance (batch: GR3304-SM2) prepared according to the method of Example 2 or 3, place it in a 10 mL volumetric flask, add diluent, ultrasonically dissolve and dilute to the mark, shake well. Accurately pipette 1.0 mL, place it in a 10 mL volumetric flask, dilute to the mark with diluent, shake well. Accurately pipette 1.0 mL, place it in a 50 mL volumetric flask, dilute to the mark with diluent, shake well. (approximately 0.03 mg / mL)

[0066] Test solution: Accurately weigh about 150 mg of the raw material L-glutamic acid diethyl ester hydrochloride test sample, place it in a 10 mL volumetric flask, add diluent, ultrasonically dissolve and dilute to the scale, and shake well. (about 15 mg / mL)

[0067] Chromatographic conditions: Chromatographic column Waters X-Bridge shield RP18, 150×4.6mm, 3.5μm or chromatographic column with equivalent performance. Column temperature: 30℃. Sample chamber temperature: 10℃. Mobile phase A: 10mmol potassium dihydrogen phosphate solution and 10mmol dipotassium hydrogen phosphate solution (for example: accurately weigh 1.36g of potassium dihydrogen phosphate and 1.74g of dipotassium hydrogen phosphate, add 1000mL of purified water, stir to dissolve), filter with 0.45μm water filter membrane, and ultrasonically degas. Mobile phase B: acetonitrile. Flow rate: 0.8mL / min. Injection volume: 20μL. Detector wavelength: UV210 nm. Run time: 22min. Needle washing reagent: 50% methanol. The retention time of L-glutamic acid diethyl ester hydrochloride, the main raw material of raltitrexed, is approximately 12.1min, the retention time of the impurity is approximately 2.3min, and the gradient:

[0068]

[0069] If there are impurity peaks in the chromatogram of the limit test solution, the impurity (3S)-3-aminotetrahydropyran-2,6-dione shall not be greater than 0.5% by peak area normalization method, and other individual impurities shall not be greater than 0.3%.

[0070] The above embodiments are intended only to illustrate the technical concepts and features of the present invention. Their purpose is to enable those skilled in the art to understand the contents of the present invention and implement them accordingly. They are not intended to limit the scope of protection of the present invention. Any equivalent changes or modifications made in accordance with the spirit of the present invention are intended to be covered by the scope of protection of the present invention.

Claims

1. A raltitrexed starting material reference substance impurity, characterized in that, The chemical name is (3S)-3-aminotetrahydropyran-2,6-dione, and the chemical structure is as follows:

2. The method for preparing a raltitrexed starting material reference substance impurity as claimed in claim 1, characterized in that: The following steps are involved: (1) N-[(benzyloxy)carbonyl]-L-glutamic acid is used as a raw material, dissolved in a solvent, and heated under the action of a dehydrating agent to carry out an intramolecular carboxyl condensation ring reaction to obtain compound 1; (2) Compound 1 and an acid-binding agent are added to a solvent and dissolved, and Pd / C is used as a catalyst, and the benzyloxycarbonyl group is removed by heating and pressurizing to obtain Compound 2, which is an impurity of the reference substance of the starting material of raltitrexed; The chemical equation is as follows:

3. The method for preparing a raltitrexed starting material reference substance impurity according to claim 2, characterized in that: The solvent in step (1) is one or a mixture of two or more of benzene, toluene, xylene or N,N-dimethylformamide.

4. The method for preparing a raltitrexed starting material reference substance impurity according to claim 2, characterized in that: The dehydrating agent in step (1) is one or a mixture of two or more of acetic anhydride, trifluoroacetic anhydride, maleic anhydride, phthalic anhydride, propionic anhydride, acetyl chloride, phosphorus oxychloride, phosphorus pentoxide or phosphorus pentachloride.

5. The method for preparing a raltitrexed starting material reference substance impurity according to claim 2, characterized in that: In step (1), the volume ratio of the N-[(benzyloxy)carbonyl]-L-glutamic acid to the dehydrating agent is 1:5 to 50.

6. The method for preparing a raltitrexed starting material reference substance impurity according to claim 2, characterized in that: In step (1), the reaction temperature is 80-120° C. and the reaction time is 3-18 h.

7. The method for preparing a raltitrexed starting material reference substance impurity according to claim 2, characterized in that: In step (2), the acid binding agent is one or a mixture of two or more of ammonium formate, ammonium acetate, ammonium propionate, ammonium valerate, ammonium sulfate, ammonium chloride, ammonium sulfite, ammonium thiocyanate, triammonium citrate, dimethylamine, pyridine or triethylamine.

8. The method for preparing a raltitrexed starting material reference substance impurity according to claim 2, characterized in that: In step (2), the solvent is one or a mixture of two or more of tetrahydrofuran, toluene, ethanol or acetonitrile.

9. The method for preparing a raltitrexed starting material reference substance impurity according to claim 2, characterized in that: In step (2), the molar ratio of Pd / C to compound 1 is 1:20-100; And / or, the reaction temperature is 30-80° C., the reaction time is 3-18 h; and the reaction pressure is 0-10 MPa.

10. Use of the impurity as claimed in claim 1 as a reference substance in the detection of raltitrexed starting materials, characterized in that: The following steps are involved: Step A: Preparation of reference solution: Accurately weigh the impurity reference substance of claim 1, dilute to scale with purified water, and shake well to make 0.03 mg / ml; Step B: Test solution: accurately weigh the test sample, add purified water to dissolve and dilute to the scale by ultrasonic, and shake well to make 15 mg / ml; Step C: draw the reference solution and the test solution and inject them into the HPLC; The chromatographic conditions are: Injection volume: 20 μL; column temperature: 30°C; sample chamber temperature: 10°C; detection wavelength: 210 nm; The chromatographic column is Waters X-Bridge shield RP18, 150×4.6mm, 3.5μm or a column with equivalent performance; Mobile phase A: 10 mmol potassium dihydrogen phosphate solution and 10 mmol potassium hydrogen phosphate solution; Mobile phase B: acetonitrile; gradient:

Images