A method for detecting the purity of tert-butyl 1-methyl-1,4,6,7-tetrahydro-5H-imidazo[4,5-c]pyridine-5-carboxylate

Abstract

The present application relates to a kind of 1-methyl-1,4,6,7-tetrahydro-5H-imidazo [4,5-C] pyridine-5-carboxylic acid tert-butyl ester purity detection method, belong to analytical chemistry field.The present application 1-methyl-1,4,6,7-tetrahydro-5H-imidazo [4,5-C] pyridine-5-carboxylic acid tert-butyl ester detection method, the method is determined using liquid chromatography, wherein chromatographic column is Waters Shield RP18, 150*4.6mm, 3.5um or equivalent chromatographic column of performance;The method can effectively separate the intermediate 2a and intermediate 3a impurities present in the sample to be measured, and can accurately quantify the purity detection of 1-methyl-1,4,6,7-tetrahydro-5H-imidazo [4,5-C] pyridine-5-carboxylic acid tert-butyl ester sample.

Description

Technical Field

[0001] This invention relates to the field of analytical chemistry, specifically to a method for determining the purity of tert-butyl 1-methyl-1,4,6,7-tetrahydro-5H-imidazo[4,5-C]pyridine-5-carboxylic acid. Background Technology

[0002] PD-1 inhibitors have shown good efficacy in the immunotherapy of various cancers and other immune-related diseases. CN114650993A discloses a novel class of PD-1 inhibitors, one of which has the following chemical formula:

[0003]

[0004] Among them, 1-methyl-1,4,6,7-tetrahydro-5H-imidazo[4,5-C]pyridine-5-carboxylic acid tert-butyl ester can be used as a raw material to produce this type of compound.

[0005] Patent application CN202211572219X discloses a method for preparing 2(1-methylimidazolium-5-yl)ethylamine analogs with high efficiency and high yield. The synthetic route is as follows:

[0006]

[0007] In the prior art, this 2(1-methylimidazolium-5-yl)ethylamine analog can be used to further prepare 1-methyl-1,4,6,7-tetrahydro-5H-imidazo[4,5-C]pyridine-5-carboxylic acid tert-butyl ester (compound 6a), and the synthetic route is as follows:

[0008]

[0009] The 1-methyl-1,4,6,7-tetrahydro-5H-imidazo[4,5-C]pyridine-5-carboxylic acid tert-butyl ester prepared by these two synthetic routes inevitably contains a certain amount of intermediates 2a and 3a, which reduces the purity of the product. Therefore, it is necessary to effectively monitor and separate intermediates 2a and 3a in the product of this synthetic route.

[0010] However, existing technologies typically involve purifying crude 1-methyl-1,4,6,7-tetrahydro-5H-imidazo[4,5-C]pyridine-5-carboxylic acid tert-butyl ester samples using various methods to improve product purity and better promote PD-1 synthesis, as illustrated by Chinese patent CN202211605594X. However, no method has been reported that can effectively separate impurities from the crude sample to determine the purity of 1-methyl-1,4,6,7-tetrahydro-5H-imidazo[4,5-C]pyridine-5-carboxylic acid tert-butyl ester.

[0011] Therefore, it is necessary to further investigate a precise and effective method for detecting the purity of tert-butyl 1-methyl-1,4,6,7-tetrahydro-5H-imidazo[4,5-C]pyridine-5-carboxylic acid. Summary of the Invention

[0012] In view of the defects and deficiencies of the existing technology, the present invention provides a method for effectively detecting the purity of tert-butyl 1-methyl-1,4,6,7-tetrahydro-5H-imidazo[4,5-C]pyridine-5-carboxylic acid.

[0013] The first objective of this invention is to provide a method for detecting tert-butyl 1-methyl-1,4,6,7-tetrahydro-5H-imidazo[4,5-C]pyridine-5-carboxylic acid, wherein the method employs liquid chromatography to determine the tert-butyl 1-methyl-1,4,6,7-tetrahydro-5H-imidazo[4,5-C]pyridine-5-carboxylic acid sample; the structural formula of the tert-butyl 1-methyl-1,4,6,7-tetrahydro-5H-imidazo[4,5-C]pyridine-5-carboxylic acid is:

[0014] The liquid chromatography method includes the following chromatographic conditions: the chromatographic column is a Waters column. Use a Shield RP18 column, 150*4.6mm, 3.5um or equivalent performance; mobile phase A is 0.008-0.05 vol% ammonia solution, mobile phase B is acetonitrile, and the elution mode is gradient elution with an elution time of 16 min-30 min. The first time interval is 0 min-(4.0-8.0) min, and the volume ratio of mobile phase A to mobile phase B is (95-98):(2-5); the second time interval is (7.0-13.0) min-(11.0-21.0) min, and the volume ratio of mobile phase A to mobile phase B is (2-5):(95-98); the third time interval is (11.1-21.1) min-(15.0-30.0) min, and the volume ratio of mobile phase A to mobile phase B is (95-98):(2-5).

[0015] In one embodiment, the gradient elution program is as follows: the first time period is 0 min to 5 min, and the volume ratio of mobile phase A to mobile phase B is 95:5; the second time period is 10 min to 15 min, and the volume ratio of mobile phase A to mobile phase B is 5:95; the third time period is 15.1 min to 20 min, and the volume ratio of mobile phase A to mobile phase B is 95:5.

[0016] In one embodiment, the detector used in the liquid chromatography is an ultraviolet detector with a detection wavelength of 210 nm to 250 nm; preferably 210 nm.

[0017] In one embodiment, the flow rate of the liquid chromatography is 0.5 mL / min to 1.5 mL / min; preferably 1.0 mL / min.

[0018] In one embodiment, the column temperature of the chromatographic column is 25°C-40°C; preferably 30°C.

[0019] In one embodiment, the mobile phase A is a 0.008–0.02 vol% ammonia solution, preferably a 0.01 vol% ammonia solution.

[0020] In one embodiment, the 1-methyl-1,4,6,7-tetrahydro-5H-imidazo[4,5-C]pyridine-5-carboxylic acid tert-butyl ester sample contains intermediates 2a and 3a.

[0021] In one embodiment, the preparation process of the 1-methyl-1,4,6,7-tetrahydro-5H-imidazo[4,5-C]pyridine-5-carboxylic acid tert-butyl ester sample is as follows:

[0022] Water, compound 4a, and paraformaldehyde (CH2O) were added to the reaction vessel. n After stirring, methanesulfonic acid was added, and the reaction was carried out under nitrogen protection. The pH of the reaction solution was adjusted to 7-8, MeOH and K2CO3 were added, and Boc2O was added dropwise. The temperature was controlled at 20±5℃ and the reaction was continued to obtain the reaction solution. The reaction solution was filtered, concentrated, extracted, and dried to obtain 1-methyl-1,4,6,7-tetrahydro-5H-imidazo[4,5-C]pyridine-5-carboxylic acid tert-butyl ester compound 6a. The reaction formula is as follows:

[0023]

[0024] In one embodiment, the preparation of compound 4a is carried out in accordance with CN202211572219X, and the specific synthetic route is as follows:

[0025]

[0026] A second object of the present invention is to provide the application of the above-described method in the preparation of tert-butyl 1-methyl-1,4,6,7-tetrahydro-5H-imidazo[4,5-C]pyridine-5-carboxylic acid.

[0027] In one embodiment, the application includes monitoring or separation of the product during or after the preparation of 1-methyl-1,4,6,7-tetrahydro-5H-imidazo[4,5-C]pyridine-5-carboxylic acid tert-butyl ester.

[0028] A third objective of this invention is to provide a method for determining the purity of tert-butyl 1-methyl-1,4,6,7-tetrahydro-5H-imidazo[4,5-C]pyridine-5-carboxylic acid, the method comprising the following steps:

[0029] (1) Construct a quantitative relationship model;

[0030] (2) Purity determination of the tert-butyl 1-methyl-1,4,6,7-tetrahydro-5H-imidazo[4,5-C]pyridine-5-carboxylic acid sample.

[0031] Dissolve the sample to be tested in a diluent solution and perform high performance liquid chromatography to determine the purity of 1-methyl-1,4,6,7-tetrahydro-5H-imidazo[4,5-C]pyridine-5-carboxylic acid tert-butyl ester in the sample to be tested according to the quantitative relationship model constructed in step (1).

[0032] The liquid chromatography method includes the following chromatographic conditions: the chromatographic column is a Waters column. Use a Shield RP18 column, 150*4.6mm, 3.5um or equivalent performance; mobile phase A is 0.008-0.05 vol% ammonia solution, mobile phase B is acetonitrile, the elution mode is gradient elution, the elution time is 16min-30min, the first time period is 0min-(4.0~8.0)min; the volume ratio of mobile phase A to mobile phase B is (95-98):(2-5); the second time period is (7.0~13.0)min-(11.0~21.0)min, the volume ratio of mobile phase A to mobile phase B is (2-5):(95-98); the third time period is (11.1~21.1)min-(15.0~30.0)min, the volume ratio of mobile phase A to mobile phase B is (95-98):(2-5).

[0033] In one embodiment, the construction of the quantitative relationship model in step (1) specifically involves: preparing a series of standard sample solutions of 1-methyl-1,4,6,7-tetrahydro-5H-imidazo[4,5-C]pyridine-5-carboxylic acid tert-butyl ester at various concentrations, and then performing high performance liquid chromatography on the standard solution; using the concentration of 1-methyl-1,4,6,7-tetrahydro-5H-imidazo[4,5-C]pyridine-5-carboxylic acid tert-butyl ester standard sample as the abscissa and the corresponding peak area as the ordinate to construct a quantitative relationship model.

[0034] In one embodiment, the concentration of the 1-methyl-1,4,6,7-tetrahydro-5H-imidazo[4,5-C]pyridine-5-carboxylic acid tert-butyl ester standard sample solution is 1.0 mg / mL to 2.0 mg / mL.

[0035] In one embodiment, the diluent in step (2) is selected from acetonitrile, methanol, or a mixture of acetonitrile and water (such as a mixture of acetonitrile and water in a volume ratio of 7:3).

[0036] In one embodiment, the gradient elution is as follows: the first time period is 0 min to 5 min, and the volume ratio of mobile phase A to mobile phase B is 95:5; the second time period is 10 min to 15 min, and the volume ratio of mobile phase A to mobile phase B is 5:95; the third time period is 15.1 min to 20 min, and the volume ratio of mobile phase A to mobile phase B is 95:5.

[0037] In one embodiment, the detector used in the liquid chromatography is an ultraviolet detector with a detection wavelength of 210 nm to 250 nm; preferably 210 nm.

[0038] In one embodiment, the flow rate of the liquid chromatography is 0.5 mL / min to 1.5 mL / min; preferably 1.0 mL / min.

[0039] In one embodiment, the column temperature of the chromatographic column is 25°C-40°C; preferably 30°C.

[0040] In one embodiment, the ammonia solution is a 0.008 to 0.02 vol% ammonia solution, preferably a 0.01 vol% ammonia solution.

[0041] In one embodiment, the specific process for preparing the 1-methyl-1,4,6,7-tetrahydro-5H-imidazo[4,5-C]pyridine-5-carboxylic acid tert-butyl ester sample to be tested is as follows:

[0042] Water, compound 4a, and paraformaldehyde (CH2O) were added to the reaction vessel. n After stirring, methanesulfonic acid was added, and the reaction was carried out under nitrogen protection. The pH of the reaction solution was adjusted to 7-8, MeOH and K2CO3 were added, and Boc2O was added dropwise. The temperature was controlled at 20±5℃ and the reaction was continued to obtain the reaction solution. The reaction solution was filtered, concentrated, extracted, and dried to obtain 1-methyl-1,4,6,7-tetrahydro-5H-imidazo[4,5-C]pyridine-5-carboxylic acid tert-butyl ester compound 6a. The reaction formula is as follows:

[0043]

[0044] In one embodiment, the preparation of compound 4a is carried out in accordance with CN202211572219X, and the specific synthetic route is as follows:

[0045]

[0046] The beneficial effects of this invention are:

[0047] The purity detection method for 1-methyl-1,4,6,7-tetrahydro-5H-imidazo[4,5-C]pyridine-5-carboxylic acid tert-butyl ester involved in this invention, through liquid chromatography combined with specific liquid chromatography and elution conditions, can effectively separate intermediates 2a and 3a impurities present in the 1-methyl-1,4,6,7-tetrahydro-5H-imidazo[4,5-C]pyridine-5-carboxylic acid tert-butyl ester sample, and detect the purity of 1-methyl-1,4,6,7-tetrahydro-5H-imidazo[4,5-C]pyridine-5-carboxylic acid tert-butyl ester. Attached Figure Description

[0048] Figure 1 The liquid chromatogram of the 1-methyl-1,4,6,7-tetrahydro-5H-imidazo[4,5-C]pyridine-5-carboxylic acid tert-butyl ester sample in Example 1 of the present invention is shown.

[0049] Figure 2 A linear equation graph constructed for Embodiment 1 of the present invention;

[0050] Figure 3 The liquid chromatogram of 1-methyl-1,4,6,7-tetrahydro-5H-imidazo[4,5-C]pyridine-5-carboxylic acid tert-butyl ester sample under liquid phase condition 1 provided in Example 5 of the present invention;

[0051] Figure 4 The liquid chromatogram of the 1-methyl-1,4,6,7-tetrahydro-5H-imidazo[4,5-C]pyridine-5-carboxylic acid tert-butyl ester sample under liquid phase condition 2 provided in Example 5 of the present invention;

[0052] Figure 5 The liquid chromatogram of the 1-methyl-1,4,6,7-tetrahydro-5H-imidazo[4,5-C]pyridine-5-carboxylic acid tert-butyl ester sample under liquid phase condition 3 provided in Example 5 of the present invention. Detailed Implementation

[0053] To make the technical means, creative features, objectives and effects of this invention easy to understand, the invention will be specifically described below in conjunction with embodiments and accompanying drawings.

[0054] In the following examples, unless otherwise stated, the mobile phase, diluent, injection washing solution, etc. used are all commercially available chromatographically pure products.

[0055] The purity of the 1-methyl-1,4,6,7-tetrahydro-5H-imidazo[4,5-C]pyridine-5-carboxylic acid tert-butyl ester involved in this invention is greater than 98%, and it was purchased from Shanghai Maclean Biochemical Technology Co., Ltd.

[0056] In the following examples, unless otherwise stated, the 2(1-methylimidazolium-5-yl)ethylamine analog (compound 4a) sample was prepared by the method described in Chinese patent application CN202211572219X, and the synthetic route is as follows:

[0057]

[0058] The 1-methyl-1,4,6,7-tetrahydro-5H-imidazo[4,5-C]pyridine-5-carboxylic acid tert-butyl ester sample involved in the embodiments of the present invention was prepared by the following method:

[0059] The reaction formula is as follows:

[0060]

[0061] 1335 mL of water, 267.2 g of compound 4a (1.65 mol, 1 eq), and 128.1 g of paraformaldehyde (CH2O) were added to a 5 L reaction vessel. n After stirring, 6.2 g of methanesulfonic acid (0.06 mol, 0.36 eq) was added, and the reaction was carried out at 57 °C for 14 hours under nitrogen protection. The reaction solution was then cooled to 20 °C, and K2CO3 was added in portions to adjust the pH to 7-8. 1340 mL of MeOH and 324.8 g of K2CO3 were added, and 465.9 g of Boc2O (2.1 mol, 1.3 eq) was added dropwise. The temperature was controlled at 20 ± 5 °C and the reaction was carried out for 10 hours to obtain the reaction solution. The reaction solution was filtered, concentrated, extracted, and dried to obtain 221 g of pure compound 6a, with a yield of 56.4%.

[0062] Example 1

[0063] The detection method for 1-methyl-1,4,6,7-tetrahydro-5H-imidazo[4,5-C]pyridine-5-carboxylic acid tert-butyl ester includes the following:

[0064] Take 30 mg of the prepared compound 6a into a 20 mL volumetric flask, dilute and dissolve it with acetonitrile, make up to volume, shake well, filter through an organic filter membrane, and place in a sample vial for high performance liquid chromatography (HPLC) determination. Specific chromatographic conditions are shown in Table 1. The HPLC chromatogram of the detection results is shown below. Figure 1 As shown:

[0065] Table 1 Chromatographic conditions

[0066]

[0067] like Figure 1As shown: the retention time of intermediate 2a was 13.174 min, the retention time of intermediate 3a was 12.657 min, and the product retention time was 10.140 min. The product and intermediates were well separated and there was no interference from other impurities. This method can achieve the quantitative analysis of tert-butyl 1-methyl-1,4,6,7-tetrahydro-5H-imidazo[4,5-C]pyridine-5-carboxylic acid.

[0068] Example 2

[0069] The purity determination method for 1-methyl-1,4,6,7-tetrahydro-5H-imidazo[4,5-C]pyridine-5-carboxylic acid tert-butyl ester includes the following:

[0070] (1) Prepared standard sample solutions of 1-methyl-1,4,6,7-tetrahydro-5H-imidazo[4,5-C]pyridine-5-carboxylic acid tert-butyl ester (commercially available product, purity greater than 98%) with volume fractions of 0.02%, 0.05%, 0.1%, 0.4%, and 0.6%, respectively. Then, the standard samples of each concentration were filtered through an organic filter membrane and placed in sample vials for high-performance liquid chromatography (HPLC) determination. The chromatographic conditions recorded in Table 1 were used for detection. The test results are shown in Table 2 and... Figure 2 As shown:

[0071] (2) Purity determination of tert-butyl 1-methyl-1,4,6,7-tetrahydro-5H-imidazo[4,5-C]pyridine-5-carboxylic acid sample

[0072] Take 30 mg of the compound 6a prepared above into a 20 ml volumetric flask, dilute and dissolve it with acetonitrile and make up to volume, shake well, filter through an organic filter membrane, and put it into a sample bottle for high performance liquid chromatography determination. The determination is carried out according to the detection method described above, and the purity of 1-methyl-1,4,6,7-tetrahydro-5H-imidazo[4,5-C]pyridine-5-carboxylic acid tert-butyl ester in the sample is calculated based on the quantitative relationship model constructed in step (1).

[0073] From Table 2 and Figure 2 It is known that 1-methyl-1,4,6,7-tetrahydro-5H-imidazo[4,5-C]pyridine-5-carboxylic acid tert-butyl ester, in the range of 0.00032 mg / mL to 0.01621 mg / mL, R 2 =0.999≥0.990, maintaining good linearity, the linear equation is: y=3291.3x-0.1252; the purity of the sample to be tested is 98.273%.

[0074] Table 2. Linear Range Data Table

[0075]

[0076] Example 3 System Adaptability Test

[0077] The preparation method of the sample reference solution is as follows: Accurately weigh 32.42 mg of reference standard (commercially available 1-methyl-1,4,6,7-tetrahydro-5H-imidazo[4,5-C]pyridine-5-carboxylic acid tert-butyl ester, purity greater than 98%), place it in a 20 ml volumetric flask, dissolve it in acetonitrile, sonicate, dilute to the mark, and shake well to obtain STD-1;

[0078] Accurately weigh 31.12 mg of reference standard, place it in a 20 ml volumetric flask, dissolve it with diluent, sonicate, dilute to the mark, and shake well to obtain STD-2.

[0079] Transfer STD-1 and STD-2 solutions into vials, and inject STD-1 six times consecutively and STD-2 once, following the detection method in Example 1. The test results are shown in Table 3.

[0080] Table 3. System Applicability Test Results

[0081]

[0082] As shown in Table 3, the RSD of product peak retention time is 0.02% (≤2%), and the RSD of peak area is 0.13% (≤2%). The RF value of STD-2 injection 1 needle / the average RF value of STD01 injection 6 needles is 99.23% (98-102%), indicating that the detection method provided in Example 1 has good system applicability.

[0083] Example 4: Limit of Detection and Limit of Quantification

[0084] LOD and LOQ solutions were prepared separately and tested according to the method in Example 1:

[0085] The preparation method of the sample LOQ solution is as follows: accurately transfer 1 ml of STD-1 solution from Example 2 into a 100 ml volumetric flask, dilute with acetonitrile, bring to volume, and shake well to obtain LOQ-stock-1; accurately transfer 1 ml of the above LOQ-stock-1 solution into a 20 ml volumetric flask, dilute with diluent, bring to volume, and shake well to obtain LOQ.

[0086] The LOD solution is prepared as follows: Accurately transfer 1 ml of the above LOQ-stock-1 solution into a 50 ml volumetric flask, dilute with diluent, bring to volume, shake well, and label as LOD.

[0087] The test results are shown in Table 4:

[0088] Table 4. Results of Limit of Detection and Limit of Quantitation

[0089]

[0090] As shown in Table 4, the S / N of the main peak in the detection limit solution is 9 (S / N≥3); the S / N of the main peak in the quantitation limit solution is 27 (S / N≥10); and for the quantitation limit solution, after 6 consecutive injections, the RSD of the main peak area is 1.13 (≤2%).

[0091] Example 5: Screening of Liquid Phase Conditions

[0092] Based on Example 1, the liquid chromatography conditions were screened. The specific liquid chromatography conditions were the same as those described in Example 1, except for the data in Table 5 below. The specific chromatographic conditions for this example are shown in Table 5:

[0093] Table 5. Screening of liquid phase conditions

[0094]

[0095] As shown in Table 5, liquid phase condition 4 was used to detect tert-butyl 1-methyl-1,4,6,7-tetrahydro-5H-imidazo[4,5-C]pyridine-5-carboxylic acid. The results showed that the target product could be effectively separated from intermediates 2a and 3a.

[0096] According to the method for detecting the content of 1-methyl-1,4,6,7-tetrahydro-5H-imidazo[4,5-C]pyridine-5-carboxylic acid tert-butyl ester involved in the above embodiments, liquid chromatography was used with linear gradient elution of the mobile phase. Specific liquid phase conditions were selected and different mobile phase ratios were used at different elution times. This method can not only effectively separate 1-methyl-1,4,6,7-tetrahydro-5H-imidazo[4,5-C]pyridine-5-carboxylic acid tert-butyl ester and intermediate impurities, but also quantitatively analyze 1-methyl-1,4,6,7-tetrahydro-5H-imidazo[4,5-C]pyridine-5-carboxylic acid tert-butyl ester.

[0097] The above embodiments are preferred embodiments of the present invention and are not intended to limit the scope of protection of the present invention.

Claims

1. A method for detecting tert-butyl 1-methyl-1,4,6,7-tetrahydro-5H-imidazo[4,5-C]pyridine-5-carboxylic acid, characterized in that, The structural formula of the 1-methyl-1,4,6,7-tetrahydro-5H-imidazo[4,5-C]pyridine-5-carboxylic acid tert-butyl ester is as follows: ； The sample of 1-methyl-1,4,6,7-tetrahydro-5H-imidazo[4,5-C]pyridine-5-carboxylic acid tert-butyl ester was prepared by the following method: The reaction formula is as follows: ； 1335 mL of water, 267.2 g of compound 4a, and 128.1 g of paraformaldehyde were added to a 5 L reaction vessel. After stirring, 6.2 g of methanesulfonic acid was added, and the reaction was carried out at 57 °C for 14 hours under nitrogen protection. The reaction solution was then cooled to 20 °C, and K2CO3 was added in portions to adjust the pH to 7-8. 1340 mL of MeOH and 324.8 g of K2CO3 were added, and 465.9 g of Boc2O was added dropwise. The reaction was carried out at 20 ± 5 °C for 10 hours to obtain the reaction solution. The reaction solution was filtered, concentrated, extracted, and dried to obtain 221 g of pure compound 6a. The synthetic route for compound 4a is as follows: The specific preparation process includes the following: 184 mL of dichloromethane and 50.5 g of triethylamine were added to a reactor vessel and stirred. Under nitrogen protection, the temperature was controlled at 0–10 °C. 18.4 g of compound 1a was added and the temperature was controlled at 5 °C. 69.7 g of triphenylchloromethane was added in three batches. After the addition was complete, the reaction temperature was raised to 25 °C and the reaction was carried out for 15 h. After the reaction was completed, the mixture was extracted, concentrated under reduced pressure, and filtered to obtain 58.8 g of compound 2a. 140 mL of toluene and 28.0 g of compound 2a were added to a reaction vessel and stirred. 8.9 g of dimethyl sulfate was added, and the reaction was heated to 60 °C for 3 h. After the reaction was completed, the reaction solution was cooled to 20 ± 5 °C, filtered, and the filter cake was washed with toluene. The filter cake was dried to obtain 32.3 g of compound 3a. 68.9 mL of methanol and 14.4 g of compound 3a were added to a reaction vessel and stirred. 6.9 mL of concentrated hydrochloric acid was slowly added, and the temperature was raised to 60 °C and heated for 3 h. After the reaction was completed, the reaction solution was cooled to room temperature, and 51.7 mL of toluene was added. The mixture was stirred for 15 min until the solid was completely dissolved. The methanol was then concentrated and extracted, and the aqueous phase was retained. 60 mL of isopropanol was added to the aqueous phase, and the mixture was stirred for 2 h. The mixture was filtered, and the filter cake was washed with isopropanol. After drying under vacuum, the filter cake was evaporated to dryness to obtain 2.7 g of compound 4a. Its characteristic is that liquid chromatography is used to detect the sample; The sample is soluble in a diluent; the diluent is selected from any one of acetonitrile, methanol, or a mixture of acetonitrile and water. The liquid chromatography method used a UV detector with a detection wavelength of 210nm-250nm; The liquid chromatography method includes the following chromatographic conditions: the chromatographic column is a Waters Xbridge® Shield RP18, 150*4.6mm, 3.5um; the column temperature is 30℃; Mobile phase A is a 0.008-0.05 vol% ammonia solution, and mobile phase B is acetonitrile. The elution method is gradient elution, specifically: 0-5 min, mobile phase A remains at 95%, and mobile phase B remains at 5%; 5-10 min, mobile phase A decreases from 95% to 5%, and mobile phase B decreases from 5% to 95%; 10-15 min, mobile phase A remains at 5%, and mobile phase B remains at 95%; 15-15.10 min, mobile phase A decreases from 5% to 95%, and mobile phase B decreases from 95% to 5%; 15.10-20 min, mobile phase A remains at 95%, and mobile phase B remains at 5%.

2. The method according to claim 1, characterized in that, The mobile phase A is a 0.008~0.02 vol% ammonia solution.

3. The method according to claim 1, characterized in that, The flow rate of the liquid chromatography was 0.5 mL / min to 1.5 mL / min.

4. A method for determining the purity of tert-butyl 1-methyl-1,4,6,7-tetrahydro-5H-imidazo[4,5-C]pyridine-5-carboxylic acid, characterized in that, The sample of 1-methyl-1,4,6,7-tetrahydro-5H-imidazo[4,5-C]pyridine-5-carboxylic acid tert-butyl ester was prepared by the following method: The reaction formula is as follows: ； 1335 mL of water, 267.2 g of compound 4a, and 128.1 g of paraformaldehyde were added to a 5 L reaction vessel. After stirring, 6.2 g of methanesulfonic acid was added, and the reaction was carried out at 57 °C for 14 hours under nitrogen protection. The reaction solution was then cooled to 20 °C, and K2CO3 was added in portions to adjust the pH to 7-8. 1340 mL of MeOH and 324.8 g of K2CO3 were added, and 465.9 g of Boc2O was added dropwise. The reaction was carried out at 20 ± 5 °C for 10 hours to obtain the reaction solution. The reaction solution was filtered, concentrated, extracted, and dried to obtain 221 g of pure compound 6a. The synthetic route for compound 4a is as follows: The specific preparation process includes the following: 184 mL of dichloromethane and 50.5 g of triethylamine were added to a reactor vessel and stirred. Under nitrogen protection, the temperature was controlled at 0–10 °C. 18.4 g of compound 1a was added and the temperature was controlled at 5 °C. 69.7 g of triphenylchloromethane was added in three batches. After the addition was complete, the reaction temperature was raised to 25 °C and the reaction was carried out for 15 h. After the reaction was completed, the mixture was extracted, concentrated under reduced pressure, and filtered to obtain 58.8 g of compound 2a. 140 mL of toluene and 28.0 g of compound 2a were added to a reaction vessel and stirred. 8.9 g of dimethyl sulfate was added, and the reaction was heated to 60 °C for 3 h. After the reaction was completed, the reaction solution was cooled to 20 ± 5 °C, filtered, and the filter cake was washed with toluene. The filter cake was dried to obtain 32.3 g of compound 3a. 68.9 mL of methanol and 14.4 g of compound 3a were added to a reaction vessel and stirred. 6.9 mL of concentrated hydrochloric acid was slowly added, and the temperature was raised to 60 °C and heated for 3 h. After the reaction was completed, the reaction solution was cooled to room temperature, and 51.7 mL of toluene was added. The mixture was stirred for 15 min until the solid was completely dissolved. The methanol was then concentrated and extracted, and the aqueous phase was retained. 60 mL of isopropanol was added to the aqueous phase, and the mixture was stirred for 2 h. The mixture was filtered, and the filter cake was washed with isopropanol. After drying under vacuum, the filter cake was evaporated to dryness to obtain 2.7 g of compound 4a. The purity detection method includes the following steps: (1) Construct a quantitative relationship model; (2) Purity determination of the tert-butyl 1-methyl-1,4,6,7-tetrahydro-5H-imidazo[4,5-C]pyridine-5-carboxylic acid sample. Dissolve the sample to be tested in a diluent solution and perform high performance liquid chromatography to determine the purity of 1-methyl-1,4,6,7-tetrahydro-5H-imidazo[4,5-C]pyridine-5-carboxylic acid tert-butyl ester in the sample to be tested according to the quantitative relationship model constructed in step (1). The diluent is selected from any one of acetonitrile, methanol, or a mixture of acetonitrile and water; The liquid chromatography method used a UV detector with a detection wavelength of 210nm-250nm; The liquid chromatography method includes the following chromatographic conditions: the chromatographic column is a Waters Xbridge® Shield RP18, 150*4.6mm, 3.5um; the column temperature is 30℃; Mobile phase A is a 0.008-0.05 vol% ammonia solution, and mobile phase B is acetonitrile. The elution method is gradient elution, specifically: 0-5 min, mobile phase A remains at 95%, and mobile phase B remains at 5%; 5-10 min, mobile phase A decreases from 95% to 5%, and mobile phase B decreases from 5% to 95%; 10-15 min, mobile phase A remains at 5%, and mobile phase B remains at 95%; 15-15.10 min, mobile phase A decreases from 5% to 95%, and mobile phase B decreases from 95% to 5%; 15.10-20 min, mobile phase A remains at 95%, and mobile phase B remains at 5%.

Images