Raltitrexed degradation impurity and preparation method thereof

Abstract

The invention provides the preparation method of the raltitrexed degradation impurity, and the structure and the preparation route of the degradation impurity are defined. According to the present invention, the synthesis route of the raltitrexed degradation impurity is researched, such that the degradation impurity can be stably and massively obtained, and the structure of the degradation impurity can be clarified so as to provide the basis for the subsequent toxicological performance research, such that the raltitrexed degradation impurity is prevented from being obtained through the raltitrexed acid degradation manner, and the obtaining difficulty of the raltitrexed degradation impurity is reduced.

Description

Technical Field

[0001] The present invention relates to the technical field of drug intermediate synthesis, and in particular to a raltitrexed degradation impurity and a preparation method thereof. Background Art

[0002] Raltitrexed, chemical name is N-[5-[N-methyl-N-(2-methyl-4-oxo-3,4-dihydroquinazoline-6-methyl)amino]thiophene-2-carbonyl]-L-glutamic acid, the structural formula is:

[0003]

[0004] Raltitrexed is a quinazoline folate analog. Currently, Raltitrexed is a new type of water-soluble thymidylate synthase inhibitor that can cause DNA breakage and cell death by inhibiting thymidylate synthase. Compared with 5-fluorouracil, Raltitrexed has stronger selectivity and can replace fluorouracil in the treatment of advanced colorectal cancer. It has become a first-line drug for the treatment of advanced colorectal cancer.

[0005] Impurities in APIs mainly come from the degradation and manufacturing process of the API itself. Process impurities include unreacted raw materials, intermediates, reaction by-products, derivatives of impurities contained in raw materials or intermediates, solvents, reagents, catalyst residues used in the reaction process or impurities involved in the reaction, API degradation products, etc.

[0006] In a series of studies by the inventors, it was found that there is an unknown degradation impurity in raltitrexed, which has been detected in both the raw materials and commercially available preparations, but has not been reported in existing literature and has the following structural formula.

[0007]

[0008] Therefore, further identification, research and structural confirmation of the impurities of raltitrexed are of great significance to further improve the level of quality control and safety of raltitrexed. The impurity can be degraded by raltitrexed acid degradation, but the degradation content is not high, and the impurity itself is extremely unstable, and separation and purification are extremely difficult, so it is extremely difficult to obtain, let alone subsequent research on it. In view of the shortcomings of the prior art, the inventors have systematically studied the unknown impurities of raltitrexed, invented a directional synthesis method, and clarified the structure of the key impurity through preparation and separation, and then used for purity, impurities and quantitative analysis of raltitrexed to control product quality. Summary of the invention

[0009] The technical problem to be solved by the present invention is to overcome the defects of the prior art. The present invention proposes a degradation impurity of raltitrexed and a preparation method thereof, clarifies the structure of the degradation impurity, and provides a synthetic route that can obtain the impurity in large quantities.

[0010] In order to solve the above technical problems, the technical solution adopted by the present invention is: a method for preparing raltitrexed degraded impurities, using 5-[(tert-butyloxycarbonyl)methylamino]thiophene-2-carboxylic acid methyl ester as a reaction raw material [hereinafter referred to as QS(A)], and the reaction route of the preparation process is as follows:

[0011]

[0012] The steps include:

[0013] S1, deprotection: dissolve QS (A) in dichloromethane, cool the solution and drop acid, wherein the acid may be hydrochloric acid and / or trifluoroacetic acid, add n-heptane after the temperature reaction is completed, cool the solution and crystallize, and filter to obtain the impurity intermediate-1;

[0014] S2, substitution: After the impurity intermediate 1 and compound A are dissolved in N,N-dimethylformamide, a base is added thereto. The added base may be an organic base or an inorganic base, including one or more of 2,6-lutidine, pyridine, triethylamine, N,N-diisopropylethylamine, sodium bicarbonate, potassium carbonate, sodium carbonate, and potassium hydroxide, preferably 2,6-lutidine. After the temperature reaction is completed, dichloromethane and purified water are added for extraction, and the obtained organic phase is concentrated to obtain the impurity intermediate-2;

[0015] S3, hydrolysis: dissolve the impurity intermediate-2 in purified water. In this process, the weight ratio of the impurity intermediate-2 to the purified water is 1:8-15, preferably 1:10. After cooling, sodium hydroxide solution is added dropwise for reaction. After the temperature reaction is completed, the temperature is cooled again and an acid solution is added dropwise. The acid used above can be hydrochloric acid and / or tartaric acid, preferably tartaric acid. The weight ratio of the impurity intermediate-2 to tartaric acid and purified water is 1:1-1.5:25-30. After crystallization and suction filtration, the impurity intermediate-3 is obtained;

[0016] S4, decarboxylation: dissolve the impurity intermediate-3 with N, N-dimethylformamide, add quinoline and copper powder and heat to react, add purified water and dichloromethane to extract after the reaction, concentrate the organic phase, and separate by column chromatography to obtain a crude product of degraded impurity A;

[0017] S5. Separation and purification: The crude product of degradation impurity A is dissolved in acetonitrile and filtered through a filter membrane. The obtained solution is separated by liquid phase using high performance liquid chromatography.

[0018] Further, in S1, after dissolving, the temperature is controlled to 0-10°C and acid is added dropwise. The reaction temperature is 10-30°C, preferably 20-25°C. The reaction time is 1-3h, preferably 2h. The cooling and crystallization process is completed at 0-5°C.

[0019] Furthermore, the weight ratio of QS (A) to dichloromethane and n-heptane is 1:5-8:5-10, preferably 1:6:8.

[0020] Furthermore, in S2, the reaction temperature is 40-60°C, and the reaction time is 5-10h.

[0021] Furthermore, the weight ratio of the impurity intermediate-1 to compound A, N,N-dimethylformamide and organic base is 1:0.5-1:8-12:0.2-0.5, preferably 1:0.74:9.40:0.35.

[0022] Furthermore, during the extraction process, the weight ratio of the impurity intermediate-1 to dichloromethane and purified water is 1:10-15:10-15, preferably 1:13.50:13.50.

[0023] Further, in S3, the temperature is controlled at 0-10°C, sodium hydroxide solution is added dropwise, and then the temperature is controlled at 20-40°C for reaction for 2-5h, preferably at 20-30°C for reaction for 3h, and then the temperature is controlled to 0-10°C, acid solution is added dropwise, and then crystallization is carried out at 0-10°C, and impurity intermediate-3 is obtained by suction filtration.

[0024] Furthermore, in the sodium hydroxide solution, the weight ratio of sodium hydroxide to purified water is 0.3-0.5:3-6, preferably 0.35:5; the weight ratio of the impurity intermediate-2 to the purified water and sodium hydroxide used for dissolution is 1:8-15:0.3-0.5, preferably 1:10:0.35.

[0025] Further, in S4, quinoline and copper powder are added and the temperature is controlled at 100-150° C. to react for 10-20 hours.

[0026] Furthermore, the weight ratio of the impurity intermediate-3 to N,N-dimethylformamide, quinoline and copper powder is 1:3-8:1-5:0.2-0.5.

[0027] Furthermore, in the extraction process of S4, the weight ratio of the impurity intermediate-3 to dichloromethane and purified water is 1:8-12:8-12, preferably 1:10:10.

[0028] Furthermore, in the column chromatography separation process of S4, the eluent used is composed of dichloromethane and methanol, and the polarity of the eluent is dichloromethane, dichloromethane:methanol=80:1, and dichloromethane:methanol=60:1.

[0029] Furthermore, in S5, the following steps are used to separate and purify the degradation impurities:

[0030] S51: Dissolve the crude product of raltitrexed degradation impurity A in acetonitrile.

[0031] S52: Filter the dissolved solution using a filter membrane.

[0032] S53: The filtered solution is subjected to preparative liquid phase separation, and the preparation conditions are as follows:

[0033] Chromatographic conditions: octadecylsilane bonded silica gel was used as filler; purified water was used as mobile phase A, acetonitrile was used as mobile phase B, and gradient elution was performed according to Table 1 below; the flow rate was 300 mL / min; the detection wavelength was 226 nm; and the injection volume was 2.5 g.

[0034] Table 1 Gradient elution table

[0035]

[0036]

[0037] Compared with the prior art, the beneficial effects of the present invention include: by studying the structure and synthesis route of raltitrexed degradation impurities, the purity, impurities and quantitative analysis of raltitrexed are used to control the product quality, and it is applied to the subsequent optimization of the preparation route of raltitrexed to improve the purity and yield of raltitrexed.

[0038] Compared with the prior art, the beneficial effects of the present invention include: by studying the synthetic route of the degradation impurity of raltitrexed, the degradation impurity can be stably obtained in large quantities, and its structure can be clarified, thereby providing a basis for subsequent toxicological performance research, avoiding the degradation of the impurity by raltitrexed acid degradation, and reducing the difficulty of obtaining it. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] The disclosure of the present invention is described with reference to the accompanying drawings. It should be understood that the drawings are for illustrative purposes only and are not intended to limit the scope of protection of the present invention. In the accompanying drawings, the same reference numerals are used to refer to the same components. Among them:

[0040] Figure 1 This is a reaction flow chart of Example 1 of the present application;

[0041] Figure 2 This is the HPLC detection spectrum of the degradation impurity A prepared in Example 1 of the present application;

[0042] Figure 3 This is the mass spectrum of the degradation impurity A prepared in Example 1 of the present application;

[0043] Figure 4 This is the NMR spectrum of the degradation impurity A prepared in Example 1 of the present application;

[0044] Figure 5 This is the HPLC spectrum of the degradation impurity A degraded in the comparative example of this application. DETAILED DESCRIPTION

[0045] It is easy to understand that according to the technical solution of the present invention, without changing the essential spirit of the present invention, a person skilled in the art can propose a variety of interchangeable structural modes and implementation modes. Therefore, the following specific implementation modes and drawings are only exemplary descriptions of the technical solution of the present invention, and should not be regarded as the whole of the present invention or as a limitation or restriction to the technical solution of the present invention.

[0046] Embodiment 1:

[0047] A method for preparing a raltitrexed intermediate impurity degradation impurity A, the reaction process is shown in Figure 1 ;

[0048] Follow the steps below:

[0049] S1 (deprotection): dissolve 20.0g QS (A) in 120.0g dichloromethane, cool to 0-10°C, add 50.4g trifluoroacetic acid dropwise, raise the temperature to 20-25°C and react for 2 hours, add 160.0g n-heptane after the reaction, cool to 0-5°C for crystallization, filter and dry to obtain 14.2g impurity intermediate-1;

[0050] S2 (substitution): 14.2g of impurity intermediate-1 and 10.5g of compound A were dissolved in 133.5g of N,N-dimethylformamide, 5.0g of 2,6-lutidine was added, and the temperature was raised to 50-55°C for reaction for 6 hours. After the reaction was completed, 192.0g of dichloromethane and 192.0g of purified water were added for extraction, and the organic phase was concentrated to obtain 17.5g of impurity intermediate-2;

[0051] S3 (hydrolysis): dissolve 17.5 g of impurity intermediate-2 in 175.0 g of purified water, cool to 0-10 ° C, add sodium hydroxide solution [6.13 g of sodium hydroxide dissolved in 87.5 g of purified water] dropwise, heat to 20-30 ° C and react for 3 hours. After the reaction is completed, cool to 0-10 ° C, add tartaric acid solution [21 g of tartaric acid dissolved in 490 g of purified water] dropwise, crystallize for 1 hour, and filter to obtain 12.4 g of impurity intermediate-3;

[0052] S4 (decarboxylation): 12.4 g of impurity intermediate-3 was dissolved in 62.0 g of N, N-dimethylformamide, 37.2 g of quinoline and 3.72 g of copper powder were added, and the temperature was raised to 120°C for reaction for 15 hours. After the reaction, 124 g of purified water and 124 g of dichloromethane were added for extraction, and the organic phase was concentrated and separated by column chromatography to obtain 2.5 g of crude degradation impurity impurity A;

[0053] S5 (separation and purification): 2.5 g of crude impurity A was heated to 40° C. with 30 ml of acetonitrile and stirred to dissolve. After the reaction solution was filtered through a membrane, the sample was loaded and eluted according to the following preparative liquid phase method. The target peak was at 22.6 minutes. The target eluate was concentrated and extracted twice with dichloromethane. The dichloromethane phase was concentrated to obtain 85 mg of impurity A with a purity of 91.04%. The HPLC spectrum is shown in Figure 2 , impurity A mass spectrum see Figure 3 , impurity A NMR Figure 4 .

[0054] Chromatographic conditions: octadecylsilane bonded silica gel was used as filler; purified water was used as mobile phase A, acetonitrile was used as mobile phase B, and gradient elution was performed according to Table 1 below; the flow rate was 300 mL / min; the detection wavelength was 226 nm; and the injection volume was 2.5 g.

[0055] Comparative Example

[0056] Dissolve 5g of raltitrexed finished product in tetrahydrofuran, add 10ml of 1mol dilute hydrochloric acid, stir at 100℃ overnight, take a sample for inspection, and the HPLC spectrum is shown in Figure 5 As can be seen from the figure, RT = 39.5min is the peak time of impurity A. Although this impurity can be directly degraded by the finished product, the degradation content is low, and there are many impurity peaks around it, making separation and purification extremely difficult and hard to achieve.

[0057] Therefore, the raltitrexed degradation impurity obtained by the preparation method of raltitrexed degradation impurity can clarify its structure through the impurity, and obtain the degradation impurity with high purity in a stable and large amount, providing a research basis for subsequent toxicological performance research and reducing the difficulty of obtaining it.

[0058] The technical scope of the present invention is not limited to the contents in the above description. Those skilled in the art can make various deformations and modifications to the above embodiments without departing from the technical idea of ​​the present invention, and these deformations and modifications should all fall within the protection scope of the present invention.

Claims

1. A method for preparing raltitrexed by degrading impurities, characterized in that: Using 5-[(tert-butyloxycarbonyl)methylamino]thiophene-2-carboxylic acid methyl ester as the reaction raw material [hereinafter referred to as QS(A)], the reaction route of the preparation process is as follows: The steps include: S1, deprotection: dissolve QS (A) in dichloromethane, add acid to the solution after cooling, add n-heptane after the temperature reaction is completed, cool and crystallize, and filter to obtain the impurity intermediate-1; S2, substitution: the impurity intermediate 1 and compound A are dissolved in N, N-dimethylformamide, and then a base is added thereto. After the temperature reaction is completed, dichloromethane and purified water are added for extraction, and the obtained organic phase is concentrated to obtain the impurity intermediate-2; S3, hydrolysis: dissolve the impurity intermediate-2 with purified water, cool it down and then drop a sodium hydroxide solution to react, cool it down again after the temperature reaction is completed and drop an acid solution, and obtain the impurity intermediate-3 after crystallization and suction filtration; S4, decarboxylation: dissolve the impurity intermediate-3 with N, N-dimethylformamide, add quinoline and copper powder and heat to react, add dichloromethane and purified water to extract after the reaction, concentrate the organic phase, and separate by column chromatography to obtain a crude product of degraded impurity A; S5. Separation and purification: The crude product of degradation impurity A is dissolved in acetonitrile and filtered through a filter membrane. The obtained solution is separated by liquid phase using high performance liquid chromatography.

2. The method for preparing raltitrexed degraded impurities according to claim 1, characterized in that: In S1, after the solution is clear, the temperature is controlled to 0-10°C and the acid is added dropwise. The reaction temperature is 10-30°C, the reaction time is 1-3h, and the cooling and crystallization process is completed at 0-5°C.

3. The method for preparing raltitrexed degraded impurities according to claim 2, characterized in that: The weight ratio of QS(A) to dichloromethane and n-heptane is 1:5-8:5-10.

4. The method for preparing raltitrexed degraded impurities according to claim 1, characterized in that: In S2, the reaction temperature is 40-60°C and the reaction time is 5-10h.

5. The method for preparing raltitrexed degraded impurities according to claim 4, characterized in that: The weight ratio of the impurity intermediate-1 to compound A, N,N-dimethylformamide and organic base is 1:0.5-1:8-12:0.2-0.

5.

6. The method for preparing raltitrexed degraded impurities according to claim 1, characterized in that: In S3, add sodium hydroxide solution at 0-10°C, then control the temperature to 20-40°C for reaction for 2-5h, then control the temperature to 0-10°C, add acid solution, crystallize at 0-10°C, and filter to obtain impure intermediate-3.

7. The method for preparing raltitrexed degraded impurities according to claim 6, characterized in that: In the sodium hydroxide solution, the weight ratio of sodium hydroxide to purified water is 0.3-0.5:3-6; The weight ratio of the impurity intermediate-2 to the purified water used for dissolution and the sodium hydroxide is 1:8-15:0.3-0.

5.

8. The method for preparing raltitrexed degraded impurities according to claim 1, characterized in that: In S4, add quinoline and copper powder and control the temperature to 100-150°C for reaction for 10-20h.

9. The method for preparing raltitrexed degraded impurities according to claim 8, characterized in that: The weight ratio of the impurity intermediate-3 to N,N-dimethylformamide, quinoline and copper powder is 1:3-8:1-5:0.2-0.

5.

10. A raltitrexed degradation impurity, characterized in that: The invention is prepared according to the method for preparing raltitrexed degradation impurities according to any one of claims 1 to 9.

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