Preparation method and detection method of key intermediate of dihydropyrido [4, 3-d] pyrimidone analogue

Abstract

The invention provides a preparation method and a detection method of a dihydropyrido [4, 3-d] pyrimidone analogue key intermediate, and belongs to the technical field of medicine preparation and detection.The preparation method comprises the steps that 3, 6-dichloro-4-isopropyl pyridazine and 2, 6-dichloro-4-aminophenol are taken to react under the action of fine powder potassium carbonate, after the reaction is completed, alcohol recrystallization is conducted, and the dihydropyrido [4, 3-d] pyrimidone analogue key intermediate is obtained. And the dihydropyrido [4, 3-d] pyrimidone analogue key intermediate is obtained after the reaction is completed. The detection method comprises the following steps: preparing a test solution from the key intermediate of the dihydropyrido [4, 3-d] pyrimidone analogue, and carrying out liquid chromatography detection through gradient elution so as to detect impurities in the key intermediate of the dihydropyrido [4, 3-d] pyrimidone analogue. The preparation method and the refining method have the advantages of simplicity in operation, high yield, low cost, suitability for industrial production and the like. The detection method provided by the invention can effectively separate, accurately detect and quantify various impurities.

Description

Technical Field

[0001] This invention relates to drug preparation and detection techniques, and more particularly to a method for preparing and detecting a key intermediate of a dihydropyrido[4,3-d]pyrimidinone analogue. Background Technology

[0002] Resmetirom, originally developed by Roche, received accelerated approval from the FDA in March 2024 for the treatment of non-alcoholic steatohepatitis (MASH) with liver fibrosis. Its chemical name is 2-(3,5-dichloro-4-((5-isopropyl-6-oxo-1,6-dihydropyridazin-3-yl)oxy)phenyl)-3,5-dioxy-2,3,4,5-tetrahydro-1,2,4-triazin-6-onitrile, and its structural formula is as follows: , Globally, 10-30% of adult MASLD patients are MASH patients, and the prevalence of MASLD is increasing year by year. Therefore, the demand for this drug will continue to grow in the future.

[0003] Chinese invention patent CN101228135B discloses a method for preparing resmetiro, wherein compound 7 reacts with 2,6-dichloro-4-aminophenol to generate compound 24, which is hydrolyzed to obtain compound 25, and reacts with ethyl (2-cyanoacetyl)carbamate and metal nitrite in the presence of an alkaline environment to obtain compound 30. Compound 30 is cyclized in an acidic environment to obtain a crude product, and the crude product is purified to obtain resmetiro.

[0004] , The crude product obtained from this route was dissolved in acetonitrile, decolorized with activated carbon, filtered, concentrated, and then purified twice with hot acetonitrile to obtain resmetiro, with a yield of 61%.

[0005] The above route uses a key intermediate 24, namely Its chemical name is 3,5-dichloro-4-((6-chloro-5-isopropylpyridazin-3-yl)oxo)aniline, and its CAS number is 920509-27-9.

[0006] Currently, the mainstream routes for preparing this compound can be broadly classified into the following four types based on different acid-binding agents: Route 1: The preparation method of this compound is reported in literature such as CN109574995, US2021 / 292304, CN111592528, and WO2019 / 240938. The reaction is carried out in dimethyl sulfoxide with cuprous iodide and potassium carbonate as acid-binding agents and then purified by column chromatography. The yield is 52-67%, which is not suitable for industrial production. Patent CN119504714A reports recrystallization using ethanol, which increases the yield to 78.5%. However, the price of cuprous iodide is high, resulting in high material costs, which is still not suitable for industrial production.

[0007] Route 2: WO2007 / 9913 reported the preparation of this compound using potassium tert-butoxide as an acid-binding agent. In this operation, the compound was not purified but directly used in the next step, which is not conducive to the quality control of the intermediate and is not suitable for industrial production.

[0008] Route 3: CN111909137 and US2025 / 206725 reported the preparation of this compound using cesium carbonate as an acid-binding agent, with yields of 69% and 32%, respectively. This method has a low yield and uses expensive cesium carbonate, resulting in high costs and making it unsuitable for industrial production.

[0009] Route 4: CN117843621 reported the preparation of this compound using potassium carbonate as an acid-binding agent, but the post-processing of this method still requires column chromatography purification, which is not suitable for industrial production.

[0010] Given the promising market prospects of resimetiro, it is necessary to find a green, environmentally friendly, simple, high-yield, and low-cost method for preparing this key intermediate, and it is also necessary to develop a method for detecting impurities in this key intermediate in order to better control its quality. Summary of the Invention

[0011] To address the above problems, this invention provides a method for preparing key intermediates of dihydropyrido[4,3-d]pyrimidinone analogs and a method for detecting them.

[0012] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A method for preparing a key intermediate of a dihydropyrido[4,3-d]pyrimidinone analogue, wherein 3,6-dichloro-4-isopropylpyridazine and 2,6-dichloro-4-aminophenol are reacted in the presence of fine potassium carbonate, and after the reaction is completed, the mixture is recrystallized from alcohol to obtain the key intermediate of the dihydropyrido[4,3-d]pyrimidinone analogue. The key intermediate of the dihydropyrido[4,3-d]pyrimidinone analogue is 3,5-dichloro-4-((6-chloro-5-isopropylpyridazin-3-yl)oxo)aniline.

[0013] Furthermore, the alcohol used for recrystallization is a C1 to C4 alcohol solvent, specifically methanol, ethanol, isopropanol, or tert-butanol. The amount of alcohol used is 5 to 15 times the amount of the product obtained after the reaction is complete, preferably 10 times.

[0014] Furthermore, the particle size D50 of fine potassium carbonate is 50~200μm.

[0015] Furthermore, the molar ratio of 3,6-dichloro-4-isopropylpyridazine to 2,6-dichloro-4-aminophenol is 1:1.0~2.0; the molar ratio of 3,6-dichloro-4-isopropylpyridazine to finely powdered potassium carbonate is 1:2.0~5.0.

[0016] Furthermore, the reaction temperature is 80~140℃; the reaction time is 6~24h; The solvent for the reaction is N,N-dimethylacetamide, N,N-dimethylformamide, N-methylpyrrolidone, or 1,3-dimethyl-2-imidazolinone.

[0017] A method for detecting key intermediates of dihydropyrido[4,3-d]pyrimidinone analogues, wherein the method involves preparing a test solution by adding a solvent to the key intermediate of the dihydropyrido[4,3-d]pyrimidinone analogue prepared by the above-mentioned preparation method, and then using a mixture of phosphate buffer and acetonitrile with a volume ratio of 90:10 as mobile phase A and a mixture of phosphate buffer and acetonitrile with a volume ratio of 20:80 as mobile phase B, followed by gradient elution and liquid chromatography detection to detect impurities in the key intermediate of the dihydropyrido[4,3-d]pyrimidinone analogue; The elution conditions for the gradient elution are as follows: 0~15min, 70%→45% mobile phase A, 30%→55% mobile phase B; 15~30 min, 45%→15% mobile phase A, 55%→85% mobile phase B; 30~48 min, 15% mobile phase A, 85% mobile phase B; 48~50 min, 15%→70% mobile phase A, 85%→30% mobile phase B; 50-60 min, 70% mobile phase A, 30% mobile phase B; The impurities include impurity 1, impurity 2, impurity 5, impurity 6, impurity 11, and impurity 12, with the following specific structure: , .

[0018] Furthermore, the detection method includes the following specific steps: Take the key intermediate of dihydropyrido[4,3-d]pyrimidinone analogue and add solvent to prepare a test solution; Impurity 1, impurity 2, impurity 5, impurity 6, impurity 11 and impurity 12 were respectively prepared into corresponding impurity positioning solutions; The test solution and the localization solutions of each impurity were subjected to liquid chromatography to detect each impurity in the key intermediate of dihydropyridino[4,3-d]pyrimidinone analogue.

[0019] Furthermore, the method also includes using liquid chromatography to detect the obtained chromatogram and calculating the content of key intermediates and impurities of dihydropyridino[4,3-d]pyrimidinone analogs by area normalization or standard curve method.

[0020] Furthermore, the solvent is obtained by mixing mobile phase A and mobile phase B in a volume ratio of 70:30; Phosphate buffer is prepared by dissolving ammonium dihydrogen phosphate in water, adding triethylamine, adjusting the pH to 6.00±0.05 with phosphoric acid, and shaking well.

[0021] Furthermore, the chromatographic column uses octadecylsilane-bonded silica gel as the packing material; the detection wavelength is 205~220nm; the flow rate is 0.8~1.2mL / min; and the column temperature is 25~35℃.

[0022] The beneficial effects of the preparation method and detection method of a key intermediate of a dihydropyrido[4,3-d]pyrimidinone analogue of the present invention are as follows: This invention addresses the problems of complex preparation, difficult operation, and low yield of key intermediates of resimetiro. It explores a method for preparing and purifying key intermediates of resimetiro, solving the aforementioned problems and successfully obtaining a low-cost, high-purity key intermediate of resimetiro—a dihydropyrido[4,3-d]pyrimidinone analog. The preparation and purification methods of this invention have advantages such as simple operation, high yield, low cost, and suitability for industrial production. This invention explores a detection method for key intermediates of dihydropyrido[4,3-d]pyrimidinone analogs, which can effectively separate, accurately detect and quantify all impurities contained therein, thereby better controlling the quality of the intermediate. The detection method of this invention has the advantages of strong anti-interference ability and good adaptability, and can obtain stable and reliable results under different instruments, different analysts and different dates, ensuring the reproducibility and consistency of the detection results. Attached Figure Description

[0023] Figure 1 The chromatogram of the mixed solution in Example 8; Figure 2 This is a standard curve diagram of impurity 1 in Example 9; Figure 3 This is a standard curve diagram of impurity 12 in Example 9; Figure 4 This is a standard curve diagram of impurity 2 in Example 9; Figure 5 This is a standard curve of the key intermediate of the dihydropyrido[4,3-d]pyrimidinone analogue in Example 9; Figure 6 This is a standard curve diagram of impurity 5 in Example 9; Figure 7 This is a standard curve diagram of impurity 11 in Example 9; Figure 8 This is the standard curve of impurity 6 in Example 9. Detailed Implementation

[0024] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Many specific details are set forth in the following description to provide a thorough understanding of the present invention. However, the present invention may also be implemented in other ways different from those described herein. Those skilled in the art can make similar extensions without departing from the spirit of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0025] Example 1: A method for preparing a key intermediate of a dihydropyrido[4,3-d]pyrimidinone analogue. This embodiment describes a method for preparing a key intermediate of a dihydropyridano[4,3-d]pyrimidinone analogue, specifically including the following steps: Ordinary potassium carbonate is pulverized using a conventional pulverizer to obtain fine potassium carbonate powder with a D50 of 150 μm.

[0026] 2,6-Dichloro-4-aminophenol (46.6 g, 0.26 mol) was added to 250 mL of N,N-dimethylacetamide, followed by 3,6-dichloro-4-isopropylpyridazine (50 g, 0.26 mol) and finely powdered potassium carbonate (72.3 g, 0.52 mol). The resulting system was heated to 100–110 °C with stirring and reacted for 12 h. After the reaction was complete, the mixture was cooled to room temperature, and 1000 mL of water was added to the reaction solution. The mixture was stirred for 0.5 h, filtered, and isopropanol (250 mL) was added to the collected filter cake. The mixture was heated to dissolve the crystals, cooled to allow crystallization, filtered, and dried to obtain 69.7 g of a key intermediate of the dihydropyrido[4,3-d]pyrimidinone analogue, namely 3,5-dichloro-4-((6-chloro-5-isopropylpyridazine-3-yl)oxo)aniline, with a yield of 80.2% and a purity of 99.6%.

[0027] The specific chemical reaction formula for preparing the key intermediate (hereinafter referred to as the key intermediate) of the dihydropyrido[4,3-d]pyrimidinone analog in this embodiment is as follows: .

[0028] Example 2: A method for preparing a key intermediate of a dihydropyrido[4,3-d]pyrimidinone analogue. This embodiment describes a method for preparing a key intermediate of a dihydropyridano[4,3-d]pyrimidinone analogue, specifically including the following steps: Ordinary potassium carbonate is pulverized using a conventional pulverizer to obtain fine potassium carbonate powder with a D50 of 200 μm.

[0029] 2,6-Dichloro-4-aminophenol (186.9 g, 1.04 mol) was added to 1500 mL of N,N-dimethylformamide, followed by 3,6-dichloro-4-isopropylpyridazine (100 g, 0.52 mol) and finely powdered potassium carbonate (216.6 g, 1.57 mol). The resulting system was heated to 110–120 °C with stirring and reacted for 8 h. After the reaction was complete, the mixture was cooled to room temperature, and 3000 mL of water was added to the reaction solution. The mixture was stirred for 0.5 h, filtered, and tert-butanol (1000 mL) was added to the collected filter cake. The mixture was heated to dissolve the crystals, cooled to allow crystals to precipitate, filtered, and dried to obtain 144.8 g of a key intermediate of a dihydropyrido[4,3-d]pyrimidinone analogue, with a yield of 83.2% and a purity of 99.2%.

[0030] Example 3: A method for preparing a key intermediate of a dihydropyrido[4,3-d]pyrimidinone analogue. This embodiment describes a method for preparing a key intermediate of a dihydropyridano[4,3-d]pyrimidinone analogue, specifically including the following steps: Ordinary potassium carbonate is pulverized twice using a conventional pulverizer to obtain fine potassium carbonate powder with a D50 of 50 μm.

[0031] 2,6-Dichloro-4-aminophenol (55.5 g, 0.31 mol) was added to 250 mL of N-methylpyrrolidone, followed by 3,6-dichloro-4-isopropylpyridazine (50 g, 0.26 mol) and finely powdered potassium carbonate (107.6 g, 0.78 mol). The resulting system was heated to 110–120 °C with stirring and reacted for 8 h. After the reaction was complete, the mixture was cooled to room temperature, and 500 mL of water was added to the reaction solution. The mixture was stirred for 0.5 h, filtered, and ethanol (350 mL) was added to the collected filter cake. The mixture was heated to dissolve the crystals, cooled to allow crystallization, filtered, and dried to obtain 74.3 g of a key intermediate of a dihydropyrido[4,3-d]pyrimidinone analogue, with a yield of 85.4% and a purity of 99.4%.

[0032] Example 4: A method for preparing a key intermediate of a dihydropyrido[4,3-d]pyrimidinone analogue. This embodiment describes a method for preparing a key intermediate of a dihydropyridano[4,3-d]pyrimidinone analogue, specifically including the following steps: Ordinary potassium carbonate is pulverized twice using a conventional pulverizer to obtain fine potassium carbonate powder with a D50 of 80 μm.

[0033] 2,6-Dichloro-4-aminophenol (69.4 g, 0.39 mol) was added to 250 mL of 1,3-dimethyl-2-imidazolinone, followed by 3,6-dichloro-4-isopropylpyridazine (50 g, 0.26 mol) and finely powdered potassium carbonate (179.4 g, 1.3 mol). The resulting system was heated to 130–140 °C with stirring and reacted for 6 h. After the reaction was complete, the mixture was cooled to room temperature, and 1200 mL of water was added to the reaction solution. The mixture was stirred for 1 h, filtered, and methanol (150 mL) was added to the collected filter cake. The mixture was heated to dissolve the crystals, cooled to allow crystallization, filtered, and dried to obtain 70.0 g of a key intermediate of a dihydropyrido[4,3-d]pyrimidinone analogue, with a yield of 80.5% and a purity of 99.0%.

[0034] Example 5: A method for preparing a key intermediate of a dihydropyrido[4,3-d]pyrimidinone analogue. This embodiment describes a method for preparing a key intermediate of a dihydropyridano[4,3-d]pyrimidinone analogue, specifically including the following steps: Ordinary potassium carbonate is pulverized using a conventional pulverizer to obtain fine potassium carbonate powder with a D50 of 100 μm.

[0035] 2,6-Dichloro-4-aminophenol (55.5 g, 0.31 mol) was added to 500 mL of N,N-dimethylacetamide, followed by 3,6-dichloro-4-isopropylpyridazine (50 g, 0.26 mol) and finely powdered potassium carbonate (89.7 g, 0.65 mol). The resulting system was heated to 80–90 °C with stirring and reacted for 24 h. After the reaction was complete, the mixture was cooled to room temperature, and 1500 mL of water was added to the reaction solution. The mixture was stirred for 1 h, filtered, and isopropanol (200 mL) was added to the collected filter cake. The mixture was heated to dissolve the crystals, cooled to allow crystallization, filtered, and dried to obtain 74.4 g of a key intermediate of a dihydropyrido[4,3-d]pyrimidinone analogue, with a yield of 85.5% and a purity of 99.2%.

[0036] Example 6: A method for preparing a key intermediate of a dihydropyrido[4,3-d]pyrimidinone analogue. This embodiment describes a method for preparing a key intermediate of a dihydropyridano[4,3-d]pyrimidinone analogue, specifically including the following steps: Ordinary potassium carbonate is pulverized using a conventional pulverizer to obtain fine potassium carbonate powder with a D50 of 100 μm.

[0037] 2,6-Dichloro-4-aminophenol (22.36 kg, 125.6 mol) was added to 100 kg of N,N-dimethylacetamide, followed by 3,6-dichloro-4-isopropylpyridazine (20 kg, 104.6 mol) and finely powdered potassium carbonate (28.87 kg, 209.2 mol). The resulting system was heated to 90-100 °C with stirring and reacted for 18 h. After the reaction was complete, the mixture was cooled to room temperature, and 300 kg of purified water was added to the reaction solution. The mixture was stirred for 1 h and then centrifuged. Isopropanol (100 kg) was added to the collected filter cake, and the mixture was heated to dissolve the crystals. The crystals were then cooled to allow them to crystallize, centrifuged, and dried to obtain 30.57 kg of a key intermediate of a dihydropyrido[4,3-d]pyrimidinone analogue, with a yield of 87.8% and a purity of 99.5%.

[0038] Example 7: A method for preparing a key intermediate of a dihydropyrido[4,3-d]pyrimidinone analogue. This embodiment describes a method for preparing a key intermediate of a dihydropyrido[4,3-d]pyrimidinone analogue disclosed in patent CN117843621, specifically including the following steps: At 25 °C, 2,6-dichloro-4-aminophenol (13.8 g, 78 mmol) was added to N,N-dimethylformamide, followed by 3,6-dichloro-4-isopropylpyridazine (15.4 g, 78 mmol) and potassium carbonate (32.4 g, 234 mmol). The resulting system was heated to 90 °C and reacted for 18 h. Subsequently, the system was cooled to 25 °C and poured into a mixture of ice water and 1N hydrochloric acid aqueous solution (150 mL). The pH was adjusted to 7 with 1N hydrochloric acid aqueous solution, and the mixture was extracted twice with ethyl acetate (1000 mL). The organic phase was washed with saturated sodium chloride aqueous solution (500 mL), dried over magnesium sulfate, filtered, concentrated under reduced pressure, and the residue was subjected to silica gel column chromatography to give a key intermediate of dihydropyrido[4,3-d]pyrimidinone analogues in 64% yield.

[0039] The reaction process mainly produces impurities 1, 2, 5, 6, 11, and 12. The purpose of column chromatography is to remove these six impurities. ,

[0040] In response to the aforementioned impurities, this invention develops a method for detecting key intermediates of dihydropyrido[4,3-d]pyrimidinone analogs, which improves the separation of various impurities and enables better control over the quality of the intermediates.

[0041] Example 8: A method for detecting key intermediates of dihydropyrido[4,3-d]pyrimidinone analogs This embodiment describes a method for detecting a key intermediate of a dihydropyrido[4,3-d]pyrimidinone analogue, determined by liquid chromatography (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 0512), specifically including the following steps: S1, Solution Preparation Solvents: Mobile phase A was a mixture of phosphate buffer (2.3 g ammonium dihydrogen phosphate dissolved in 1000 mL of water, 2 mL of triethylamine added, and pH adjusted to 6.00 ± 0.05 with phosphoric acid, then shaken well) and acetonitrile at a volume ratio of 90:10; mobile phase B was a mixture of phosphate buffer (2.3 g ammonium dihydrogen phosphate dissolved in 1000 mL of water, 2 mL of triethylamine added, and pH adjusted to 6.00 ± 0.05 with phosphoric acid, then shaken well) and acetonitrile at a volume ratio of 20:80; and mobile phase B was a mixture of mobile phase A and mobile phase B at a volume ratio of 70:30, which was also used as a blank solvent.

[0042] Impurity localization solution: Take appropriate amounts of each impurity (impurity 1, impurity 2, impurity 5, impurity 6, impurity 11 and impurity 12), accurately weigh them, dissolve and dilute them with solvent to prepare an impurity localization solution containing approximately 20 μg per 1 ml.

[0043] Test solution: Weigh approximately 20 mg of the key intermediate of the dihydropyrido[4,3-d]pyrimidinone analog prepared in this invention, place it in a 100 mL volumetric flask, dissolve it in solvent and make up to volume, shake well to obtain the test solution.

[0044] Mixed sample solution: Accurately measure an appropriate amount of the key intermediate of dihydropyrido[4,3-d]pyrimidinone analogue and each impurity (impurity 1, impurity 2, impurity 5, impurity 6, impurity 11 and impurity 12), dissolve and dilute with solvent to prepare a mixed sample solution containing 0.2 mg of the key intermediate of dihydropyrido[4,3-d]pyrimidinone analogue and 2 μg of each impurity per 1 mL.

[0045] S2, Measurement Accurately measure 20 μL each of blank solution, impurity localization solution, test solution, and mixed solution, inject them into the liquid chromatograph, record the chromatograms, and calculate the content of key intermediates and impurities of dihydropyrido[4,3-d]pyrimidinone analogues by area normalization method. The results are shown in Table 1.

[0046] The liquid chromatography conditions are as follows: The chromatographic column uses octadecylsilane-bonded silica gel as the packing material (Venusil MP). A C18 column (4.6 mm × 250 mm, 5 μm or equivalent performance) was used. Mobile phase A consisted of a 90:10 mixture of phosphate buffer (2.3 g ammonium dihydrogen phosphate dissolved in 1000 mL of water, 2 mL triethylamine added, pH adjusted to 6.00 ± 0.05 with phosphoric acid, and shaken well) and acetonitrile. Mobile phase B consisted of a 20:80 mixture of phosphate buffer (2.3 g ammonium dihydrogen phosphate dissolved in 1000 mL of water, 2 mL triethylamine added, pH adjusted to 6.00 ± 0.05 with phosphoric acid, and shaken well) and acetonitrile. The detection wavelength was 205–220 nm, the flow rate was 0.8–1.2 mL / min, and the column temperature was 25–35 °C (in this example, the detection wavelength was 220 nm, the flow rate was 1.0 mL / min, and the column temperature was 30 °C). Gradient elution was used; the elution program was as follows: 0~15min, 70%→45% mobile phase A, 30%→55% mobile phase B; 15~30 min, 45%→15% mobile phase A, 55%→85% mobile phase B; 30~48 min, 15% mobile phase A, 85% mobile phase B; 48~50 min, 15%→70% mobile phase A, 85%→30% mobile phase B; 50-60 min, 70% mobile phase A, 30% mobile phase B.

[0047] Table 1. Determination results of key intermediates and impurities of dihydropyrido[4,3-d]pyrimidinone analogs in mixed solutions (area normalization method)

[0048] Table 2. Results of determination of key intermediates and impurities of dihydropyrido[4,3-d]pyrimidinone analogs prepared in Example 1 (area normalization method).

[0049] The chromatogram of the mixed solution of this invention is as follows: Figure 1 As shown in Tables 1 and 2, the detection method of the present invention can not only effectively separate and determine the types of impurities (impurity 1, impurity 2, impurity 5, impurity 6, impurity 11 and impurity 12) in the key intermediate of dihydropyrido[4,3-d]pyrimidinone analogs, but also determine the content of each impurity in the key intermediate of dihydropyrido[4,3-d]pyrimidinone analogs.

[0050] Example 9 Validation of the detection method This embodiment conducts a methodological investigation of the detection method in Example 8. During the methodological investigation, the chromatographic conditions for liquid chromatography detection are the same as those in Example 8.

[0051] I. System Applicability Accurately measure the blank solvent, impurity localization solution, test solution, and mixed solution, and perform the detection according to the method in Example 1. Record the chromatogram. It can be seen that the blank solvent does not interfere with the determination of related substances; the main component and each impurity can achieve good baseline separation, and the system has good applicability.

[0052] II. Solution Stability Accurately measure an appropriate amount of the key intermediate of the dihydropyrido[4,3-d]pyrimidinone analog prepared in this invention and the positioning solution of each impurity, dissolve and dilute with blank solvent to prepare a solution containing approximately 0.2 mg of the key intermediate of the dihydropyrido[4,3-d]pyrimidinone analog and 2 μg of each impurity per 1 mL. The stability of the solution was tested at 0, 2, 4, 8, 12, 18 and 24 h according to the method in Example 8. The results are shown in Table 3.

[0053] Table 3. Results of the stability study of the test sample solutions (peak area)

[0054] As shown in Table 3, the solution exhibits good stability within 24 hours.

[0055] III. Limit of Detection and Limit of Quantification Reference stock solution: Take an appropriate amount of key intermediates of dihydropyrido[4,3-d]pyrimidinone analogues and reference standards of each impurity, accurately weigh them, dissolve and dilute them with blank solvent to prepare a solution containing 200 μg per 1 mL, which is used as the corresponding reference stock solution.

[0056] Accurately measure an appropriate amount of each reference standard stock solution, dilute it with blank solvent to an appropriate concentration, and inject it. Using a signal-to-noise ratio (S / N) of 10 as an indicator, determine the limit of quantitation (prepare 6 parallel samples). The results are shown in Table 4. Using a signal-to-noise ratio (S / N) of 3 as an indicator, determine the limit of detection (LOD). The results are shown in Table 5.

[0057] Table 4 Detection Limit Test Data

[0058] Table 5. Limit of Quantitation Test Data

[0059] As shown in Tables 4 and 5, the detection limit (LOD) for impurity 2 is 0.1013 ng (0.0001%), the LOD is 0.2026 ng (0.0002%), and the peak area RSD is 8.2%; the LOD for impurity 1 is 0.0982 ng (0.0001%), the LOD is 0.1964 ng (0.0002%), and the peak area RSD is 12.4%; the LOD for impurity 12 is 0.1000 ng (0.0001%), the LOD is 0.2000 ng (0.0002%), and the peak area RSD is 10.7%; and the LOD for the main component is 0.1012 ng (0.0001%). The limit of quantitation (LOQ) for impurity 5 was 0.2024 ng (0.0002%), with a peak area RSD of 3.4%. The LQ for impurity 11 was 0.1007 ng (0.0001%), with a LOQ of 0.2013 ng (0.0002%) and a peak area RSD of 6.5%. The LQ for impurity 6 was 0.2114 ng (0.0002%), with a LOQ of 0.4228 ng (0.0004%) and a peak area RSD of 7.4%. This demonstrates that the present invention has good detection capabilities, and the RSDs of the detection results for key intermediates and various impurities in dihydropyrido[4,3-d]pyrimidinone analogues are all less than 15%, meeting the specified requirements.

[0060] IV. Linearity and Range Take appropriate amounts of the key intermediates and impurities of the dihydropyrido[4,3-d]pyrimidinone analogue, accurately weigh them, dissolve them in blank solvent and quantitatively dilute them to prepare solutions of different concentrations. The specific concentrations are shown in Table 5.

[0061] Accurately measure 20 μL of each of the above solutions and detect them according to the chromatographic conditions of Example 8. Record the chromatogram and the main peak area. Plot a standard curve with concentration as the abscissa (x) and peak area as the ordinate (y). The results are shown in Table 6.

[0062] Table 6 Standard Curve Data

[0063] As shown in Table 6, impurity 1 exhibits a good linear relationship between peak area and concentration in the range of 0.010–3.928 μg / mL, with the linear equation y = 79795x - 1606.9, R0. 2 =0.9998, see Figure 2 Impurity 12 showed a good linear relationship between peak area and concentration in the range of 0.010~3.841µg / mL, with the linear equation y=87681x-1331, R0. 2 =0.9998, see Figure 3Impurity 2 showed a good linear relationship between peak area and concentration in the range of 0.015~4.095 μg / mL, with the linear equation y = 102627x - 2697.5, R0. 2 =0.9997, see Figure 4 The main component showed a good linear relationship between peak area and concentration in the range of 0.010–4.032 µg / mL, with the linear equation y = 111522x + 290.8, R0. 2 =1, see Figure 5 Impurity 5 showed a good linear relationship between peak area and concentration in the range of 0.010~3.879µg / mL, with the linear equation y=116059x-290.9, R0. 2 =9999, see Figure 6 Impurity 11 showed a good linear relationship between peak area and concentration in the range of 0.010~4.026µg / mL, with the linear equation y=102143x-1652.2, R0. 2 =9999, see Figure 7 Impurity 6 showed a good linear relationship between peak area and concentration in the range of 0.011–4.329 µg / mL, with the linear equation y = 79241x - 285.96 and R0. 2 =9999, see Figure 8 .

[0064] Given the aforementioned standard curve, this invention can also calculate the content of key intermediates and impurities of dihydropyridino[4,3-d]pyrimidinone analogs directly using the standard curve method by preparing a test solution, performing liquid chromatography detection, and then using the peak area in the obtained chromatogram and the standard curve.

[0065] V. Accuracy Weigh an appropriate amount of the key intermediate of dihydropyrido[4,3-d]pyrimidinone analogue, accurately measure it, and dilute it with blank solvent to prepare a solution containing approximately 0.2 mg per 1 mL.

[0066] Take appropriate amounts of each impurity, weigh them accurately, dissolve and dilute them with blank solvent to prepare impurity stock solutions containing 10 μg of each impurity per 1 mL.

[0067] 50% test solution: Take an appropriate amount of the key intermediate of the dihydropyrido[4,3-d]pyrimidinone analogue, accurately weigh it, add an appropriate amount of each impurity stock solution, and dilute with blank solvent to prepare a solution containing approximately 0.2 mg of the key intermediate of the dihydropyrido[4,3-d]pyrimidinone analogue and 0.3 μg of each impurity per 1 mL (prepare 3 parallel portions).

[0068] 100% test solution: Take an appropriate amount of the key intermediate of the dihydropyrido[4,3-d]pyrimidinone analogue, accurately weigh it, add an appropriate amount of each impurity stock solution, and dilute with blank solvent to prepare a solution containing approximately 0.2 mg of the key intermediate of the dihydropyrido[4,3-d]pyrimidinone analogue and 0.6 μg of each impurity per 1 mL (prepare 3 parallel portions).

[0069] 150% test solution: Take an appropriate amount of the key intermediate of the dihydropyrido[4,3-d]pyrimidinone analogue, accurately weigh it, add an appropriate amount of each impurity stock solution, and dilute with blank solvent to prepare a solution containing approximately 0.2 mg of the key intermediate of the dihydropyrido[4,3-d]pyrimidinone analogue and 0.9 μg of each impurity per 1 ml (prepare 3 parallel portions).

[0070] Accurately measure 20 μL of each test solution and inject it into the high performance liquid chromatograph. Detect it according to the chromatographic conditions in Example 8 and record the chromatogram. The results are shown in Tables 7 to 12.

[0071] Table 7. Accuracy Test Results for Impurity 1

[0072] Table 8. Accuracy test results for impurity 2

[0073] Table 9. Accuracy Test Results (Miscellaneous)

[0074] Table 10 Results of Accuracy Test for Impurity 6

[0075] Table 11 Accuracy Test Results for Impurity 11

[0076] Table 12 Accuracy Test Results for Impurity 12

[0077] As shown in Tables 7-12, the recovery rate of impurity 1 was 95.9%, with an RSD of 1.8%; the recovery rate of impurity 2 was 99.3%, with an RSD of 1.3%; the recovery rate of impurity 5 was 101.6%, with an RSD of 1.3%; the recovery rate of impurity 6 was 104.0%, with an RSD of 1.2%; the recovery rate of impurity 11 was 98.7%, with an RSD of 4.2%; and the recovery rate of impurity 12 was 103.5%, with an RSD of 2.6%.

[0078] VI. Instrument Precision Accurately measure an appropriate amount of the key intermediate of the dihydropyrido[4,3-d]pyrimidinone analogue prepared in Example 8 and the positioning solution of each impurity. Dissolve and dilute with blank solvent to prepare a solution containing approximately 0.2 mg of the key intermediate of the dihydropyrido[4,3-d]pyrimidinone analogue and 2 μg of each impurity per 1 mL. Use this solution as the test solution and determine it according to the method in Example 8. Perform the determination 6 times consecutively and record the chromatogram. The results are shown in Table 13.

[0079] Table 13 Results of Instrument Precision Test

[0080] As can be seen from Table 13, this method has good instrument precision.

[0081] VII. Repetitiveness Weigh an appropriate amount of the key intermediate of the dihydropyrido[4,3-d]pyrimidinone analogue accurately, add an appropriate amount of impurity stock solution, and dilute with blank solvent to prepare a solution containing approximately 0.2 mg of the key intermediate of the dihydropyrido[4,3-d]pyrimidinone analogue and 2 μg of each impurity per 1 mL. This solution is used as the test solution (prepare 6 parallel solutions). Accurately inject 20 μL of each of the above solutions into the liquid chromatograph for detection, record the chromatogram, and the results are shown in Table 14.

[0082] Table 14 Repeatability Test Results

[0083] Table 14 shows that the method has good repeatability.

[0084] Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

Claims

1. A method for preparing a key intermediate of a dihydropyrido[4,3-d]pyrimidinone analogue, characterized in that, The preparation method is to react 3,6-dichloro-4-isopropylpyridazine with 2,6-dichloro-4-aminophenol in the presence of fine potassium carbonate. After the reaction is complete, the mixture is recrystallized from alcohol to obtain the key intermediate of the dihydropyrido[4,3-d]pyrimidinone analogue. The key intermediate of the dihydropyrido[4,3-d]pyrimidinone analogue is 3,5-dichloro-4-((6-chloro-5-isopropylpyridazin-3-yl)oxo)aniline.

2. The method for preparing the key intermediate of the dihydropyrido[4,3-d]pyrimidinone analog according to claim 1, characterized in that, The alcohol to be recrystallized is a C1 to C4 alcohol solvent; The amount of alcohol used is 5 to 15 times the amount of the product obtained after the reaction is complete, preferably 10 times.

3. The method for preparing the key intermediate of the dihydropyrido[4,3-d]pyrimidinone analog according to claim 1 or 2, characterized in that, The particle size D50 of fine potassium carbonate is 50~200μm.

4. The method for preparing the key intermediate of the dihydropyrido[4,3-d]pyrimidinone analog according to claim 1 or 2, characterized in that, The molar ratio of 3,6-dichloro-4-isopropylpyridazine to 2,6-dichloro-4-aminophenol is 1:1.0~2.0; the molar ratio of 3,6-dichloro-4-isopropylpyridazine to finely powdered potassium carbonate is 1:2.0~5.

0.

5. The method for preparing the key intermediate of the dihydropyrido[4,3-d]pyrimidinone analog according to claim 1 or 2, characterized in that, The reaction temperature is 80~140℃; the reaction time is 6~24h; The solvent for the reaction is N,N-dimethylacetamide, N,N-dimethylformamide, N-methylpyrrolidone, or 1,3-dimethyl-2-imidazolinone.

6. A method for detecting key intermediates of dihydropyrido[4,3-d]pyrimidinone analogs, characterized in that, The detection method involves taking the key intermediate of the dihydropyrido[4,3-d]pyrimidinone analogue prepared by any one of the preparation methods described in claims 1-5, adding a solvent to prepare a test solution, and then using a mixture of phosphate buffer and acetonitrile with a volume ratio of 90:10 as mobile phase A and a mixture of phosphate buffer and acetonitrile with a volume ratio of 20:80 as mobile phase B, performing gradient elution and liquid chromatography detection to detect impurities in the key intermediate of the dihydropyrido[4,3-d]pyrimidinone analogue. The elution conditions for the gradient elution are as follows: 0~15min, 70%→45% mobile phase A, 30%→55% mobile phase B; 15~30 min, 45%→15% mobile phase A, 55%→85% mobile phase B; 30~48 min, 15% mobile phase A, 85% mobile phase B; 48~50 min, 15%→70% mobile phase A, 85%→30% mobile phase B; 50-60 min, 70% mobile phase A, 30% mobile phase B; The impurities include impurity 1, impurity 2, impurity 5, impurity 6, impurity 11, and impurity 12, with the following specific structure: ， 。 7. The method for detecting key intermediates of dihydropyrido[4,3-d]pyrimidinone analogs according to claim 6, characterized in that, The detection method includes the following specific steps: Take the key intermediate of dihydropyrido[4,3-d]pyrimidinone analogue and add solvent to prepare a test solution; Impurity 1, impurity 2, impurity 5, impurity 6, impurity 11 and impurity 12 were respectively prepared into corresponding impurity positioning solutions; The test solution and the localization solutions of each impurity were subjected to liquid chromatography to detect each impurity in the key intermediate of dihydropyridino[4,3-d]pyrimidinone analogue.

8. The method for detecting the key intermediate of the dihydropyrido[4,3-d]pyrimidinone analogue according to claim 7, characterized in that, The method also includes using liquid chromatography to detect the chromatogram obtained, and calculating the content of key intermediates and impurities of dihydropyridino[4,3-d]pyrimidinone analogs by area normalization or standard curve method.

9. The method for detecting key intermediates of dihydropyrido[4,3-d]pyrimidinone analogs according to any one of claims 6-8, characterized in that, The solvent is obtained by mixing mobile phase A and mobile phase B in a volume ratio of 70:30; Phosphate buffer is prepared by dissolving ammonium dihydrogen phosphate in water, adding triethylamine, adjusting the pH to 6.00±0.05 with phosphoric acid, and shaking well.

10. The method for detecting key intermediates of dihydropyrido[4,3-d]pyrimidinone analogs according to any one of claims 6-8, characterized in that, The chromatographic column was packed with octadecylsilane-bonded silica gel; the detection wavelength was 205-220 nm; the flow rate was 0.8-1.2 mL / min; and the column temperature was 25-35 °C.

Images