Preparation method for fused ring nitrogen-containing compound and intermediate thereof

Abstract

Disclosed in the present invention is a preparation method for a fused ring nitrogen-containing compound and an intermediate thereof. The present invention provides a preparation method for a compound represented by formula 13, comprising the following steps: in a solvent, carrying out an amidation reaction on a compound represented by formula 11 and a compound represented by formula 12 in the presence of a condensing agent to obtain the compound represented by formula 13. The preparation method of the present invention has one or more of the following advantages: (1) the yield is high; (2) the cost is low; (3) the method is green and environment-friendly; and (4) the industrial production is facilitated.

Description

A method for preparing cyclic nitrogen-containing compounds and their intermediates

[0001] This application claims priority to Chinese patent application 2024105006830, filed on April 24, 2024. The entire contents of the aforementioned Chinese patent application are incorporated herein by reference. Technical Field

[0002] This invention relates to a method for preparing cyclic nitrogen-containing compounds and their intermediates. Background Technology

[0003] DNA polymerase theta (Pol theta, or Polθ) is a key protein in the microhomological end joining (MMEJ) repair pathway. It is a unique multifunctional polymerase composed of an N-terminal helicase domain, a central domain, and a C-terminal polymerase domain. Basic research has revealed that the polymerase domain is essential for DNA elongation at the DSB damage repair site, while the helicase and central domains play crucial roles in the recognition and binding of Polθ to substrates. Polθ can deactivate the interaction between DNA and the damage repair complex (e.g., competitive binding of single-stranded DNA to RAD51), inhibiting the HR repair pathway. Furthermore, the helicase domain of Polθ is involved in DNA replication arrest; its loss of function leads to increased replication pressure in tumor cells, resulting in apoptosis.

[0004] Polθ is not expressed or is expressed at low levels in normal tissues and cells, but it is highly expressed in various tumors, including lung cancer, breast cancer, HR-deficient ovarian cancer, gastric cancer, and colon cancer, and is associated with poor prognosis. In particular, over 70% of breast cancers show Polθ overexpression. These phenomena suggest that Polθ may play an important role in these cancers and is a potential tumor-specific target.

[0005] Studies knocking down or knocking out Polθ in tumor cells have revealed that Polθ deficiency can sensitize these cells to radiation, induce DSB production, enhance replication fork instability, and sensitize tumor cells to genotoxic agents, potentially enhancing the efficacy of radiotherapy and chemotherapy, making it a potential drug target. Research has also found a combined lethal effect between Polθ and HR deficiency; its small-molecule inhibitors can kill HR-deficient tumor cells in vitro and in vivo. Particularly in HR-deficient reversion mutation-resistant tumors resistant to PARP inhibitors (such as Olaparib), Polθ inhibitors can resensitize cells to PARP inhibitors, providing valuable therapeutic opportunities. Furthermore, given that Polθ is a key protein in the MMEJ pathway, its functional deficiency can also lead to increased genomic instability in cancer cells, increasing somatic mutations and facilitating the production of neoantigens. In addition, Polθ has been reported to participate in cGAS-STING-mediated immune activation; therefore, targeting Polθ also has the potential to enhance immunotherapy.

[0006] In summary, Polθ is a highly promising target for cancer treatment. Designing ATPase activity inhibitors targeting the Polθ protein to inhibit intracellular MMEJ, and using them alone or in combination with other chemotherapy, radiotherapy, antibody therapy, immunotherapy, etc., can kill tumor cells and has great potential in the treatment of tumors such as lung cancer, breast cancer, HR-deficient ovarian cancer, gastric cancer, colon cancer, prostate cancer, and pancreatic cancer.

[0007] Developing new preparation processes for Polθ inhibitors is essential for promoting their research and commercialization. Summary of the Invention

[0008] This invention provides a method for preparing cyclic nitrogen-containing compounds and their intermediates. The preparation method of this invention has high yield, low cost, is environmentally friendly, and is conducive to industrial production.

[0009] The present invention solves the above-mentioned technical problems through the following technical solutions.

[0010] This invention provides a method for preparing a compound as shown in Formula 13, comprising the following steps: in a solvent, in the presence of a condensing agent, an amidation reaction is carried out between a compound as shown in Formula 11 and a compound as shown in Formula 12 to obtain a compound as shown in Formula 13.

[0011] In some embodiments, the solvent used in the amidation reaction can be of the type conventional for such reactions in the art, and the solvent is a nitrogen-containing compound solvent, preferably acetonitrile or DMF.

[0012] In some embodiments, the amount of solvent used in the amidation reaction can be conventional for such reactions in the art, with the mass-to-volume ratio of the compound as shown in Formula 11 to the solvent being 0.03-0.3 g / mL, preferably 0.06-0.25 g / mL, for example 0.067-0.2 g / mL.

[0013] In some embodiments, the condensing agent in the amidation reaction may be a combination of N,N,N',N'-tetramethylchloroformamidin hexafluorophosphate (TCFH) and N-methylimidazole, or a combination of T4P and DIPEA.

[0014] In some embodiments, during the amidation reaction, the molar ratio of the compound shown in Formula 11 to N,N,N',N'-tetramethylchloroformamidine hexafluorophosphate (TCFH) is 1:(2-6), preferably 1:(3-5), for example 1:4.5.

[0015] In some embodiments, during the amidation reaction, the molar ratio of the compound shown in Formula 11 to the N-methylimidazole is 1:(5-12), preferably 1:(7-10), for example 1:9.

[0016] In some embodiments, the molar ratio of the compound of Formula 11 to T4P in the amidation reaction is 1:(1-4), preferably 1:(2-3), for example 1:2.5.

[0017] In some embodiments, in the amidation reaction, the molar ratio of the compound as shown in Formula 11 to the DIPEA is 1:(2-6), preferably 1:(3-5), for example 1:4.

[0018] In some embodiments, the molar ratio of the compound shown in Formula 11 to the compound shown in Formula 12 is 1:(0.08-1.5), preferably 1:(1-1.2), for example 1:(1.1-1.18).

[0019] In some embodiments, the amidation reaction is carried out at a temperature that is conventional for such reactions in the art, ranging from 40 to 100°C, preferably from 50 to 80°C, for example from 60 to 75°C.

[0020] In some embodiments, the progress of the amidation reaction can be detected using methods conventional in the art (e.g., TLC, GC, HPLC, or NMR). Generally, the endpoint is defined as the disappearance of the compound shown in Formula 11 or the cessation of the increase of the compound shown in Formula 13 by HPLC. The reaction time is 1-10 h, preferably 2-5 h, for example 3-4 h.

[0021] In some embodiments, the amidation reaction further includes post-treatment steps, such as crystallization, filtration, washing, and drying.

[0022] In some embodiments, the method for preparing the compound shown in Formula 13 further includes the following step: neutralizing the compound shown in Formula 11' with an aqueous inorganic base solution to obtain the compound shown in Formula 11.

[0023] In some embodiments, in the neutralization reaction, the compound shown in Formula 11' is neutralized with an inorganic alkaline aqueous solution to obtain the compound shown in Formula 11.

[0024] In some embodiments, the inorganic alkaline aqueous solution in the neutralization reaction is an aqueous sodium hydroxide solution.

[0025] In some embodiments, the sodium hydroxide aqueous solution has a mass fraction of 20-30%, preferably 25%, in the neutralization reaction.

[0026] In some embodiments, the neutralization reaction further includes post-processing steps, such as filtration, washing, recrystallization, filtration, washing, and drying.

[0027] In some embodiments, the method for preparing the compound shown in Formula 13 further includes the following step: in a solvent, in the presence of an acid, the compound shown in Formula 10 undergoes a deprotection reaction to obtain the compound shown in Formula 11'.

[0028] In some embodiments, the solvent in the deprotection reaction is an inorganic solvent or a mixture of an inorganic and an organic solvent, preferably water.

[0029] In some embodiments, in the deprotection reaction, the organic solvent is an alcohol solvent, an ether solvent, or a sulfur-containing compound solvent. Preferably, the alcohol solvent is methanol, the ether solvent is THF, and the sulfur-containing compound solvent is DMSO.

[0030] In some embodiments, during the deprotection reaction, the mass-to-volume ratio of the compound as shown in Formula 10 to the solvent is 0.05-0.2 g / mL, preferably 0.08-0.15 g / mL, for example 0.09-0.12 g / mL.

[0031] In some embodiments, the acid in the deprotection reaction is an inorganic acid, preferably concentrated hydrochloric acid or concentrated sulfuric acid, and more preferably concentrated hydrochloric acid.

[0032] In some embodiments, during the deprotection reaction, the mass-to-volume ratio of the compound as shown in Formula 10 to the concentrated hydrochloric acid is 0.1-1 g / mL, preferably 0.3-0.7 g / mL, for example 0.47-0.6 g / mL.

[0033] In some embodiments, the deprotection reaction is carried out at a temperature of 40-100°C, preferably 70-95°C, for example, 70-90°C.

[0034] In some embodiments, the progress of the deprotection reaction can be detected using methods conventional in the art (e.g., TLC, GC, HPLC, or NMR). Generally, the endpoint is defined as the disappearance of the compound shown in Formula 10 or the cessation of the increase of the compound shown in Formula 11 by HPLC. The reaction time is 2-12 hours, preferably 3-6 hours, for example 3-5.5 hours.

[0035] In some embodiments, the method for preparing the compound shown in Formula 13 further includes the following step: in a solvent, under palladium catalyst conditions, a coupling reaction is carried out between the compound shown in Formula 8 and the compound shown in Formula 9 to obtain the compound shown in Formula 10.

[0036] Wherein, R is H, Bu4Sn, ClZn or ClMg;

[0037] When R is H, it also includes phosphine ligands and bases.

[0038] In some embodiments, the solvent in the coupling reaction is an aromatic solvent or an epoxy solvent; the aromatic solvent may be toluene or xylene; the epoxy solvent may be dioxane; for example, the solvent is toluene, xylene, or dioxane.

[0039] In some embodiments, in the coupling reaction, the mass-to-volume ratio of the compound as shown in Formula 8 to the solvent is 0.02-0.2 g / mL, preferably 0.04-0.17 g / mL, for example 0.05-0.1 g / mL.

[0040] In some embodiments, the palladium catalyst in the coupling reaction is one or more of palladium chloride, palladium di(acetonitrile)chloride, palladium dichlorobis(triphenylphosphine)dichloride, palladium trifluoroacetate, tridibenzylacetone dipalladium, (1,1'-bis(diphenylphosphine)ferrocene)dichloride, [n-butyldi(1-adamantyl)phosphine](2-amino-1,1'-biphenyl-2-yl)palladium(II), palladium dichlorobis(triphenylphosphine), palladium tetra(triphenylphosphine)tetra(triphenylphosphine) or palladium acetate, preferably palladium tetra(triphenylphosphine), palladium dichlorobis(triphenylphosphine), or palladium acetate.

[0041] In some embodiments, the molar ratio of the compound as shown in Formula 8 to the palladium catalyst in the coupling reaction is 1:(0.1-0.5), preferably 1:(0.15-0.4), for example 1:(0.15-0.3).

[0042] In some embodiments, the phosphine ligand in the coupling reaction is one or more of tri-tert-butylphosphine, tricyclohexylphosphine, 4,5-bis(diphenylphosphine)-9,9-dimethyloxanthracene or n-butyldi(1-adamantyl)phosphine, preferably n-butyldi(1-adamantyl)phosphine.

[0043] In some embodiments, the molar ratio of the compound as shown in Formula 8 to the phosphine ligand in the coupling reaction is 1:(0.2-2), preferably 1:(0.3-1.5), for example 1:(0.4-1.3).

[0044] In some embodiments, the base in the coupling reaction is an inorganic base, preferably one or more of sodium bicarbonate, sodium carbonate, sodium acetate, potassium carbonate, potassium acetate, potassium phosphate, or cesium carbonate, more preferably potassium acetate or potassium phosphate.

[0045] In some embodiments, in the coupling reaction, the molar ratio of the compound as shown in Formula 8 to the base is 1:(1-10), preferably 1:(1.5-6), for example 1:(1.5-5).

[0046] In some embodiments, the molar ratio of the compound shown in Formula 8 to the compound shown in Formula 9 in the coupling reaction is 1:(0.3-2), preferably 1:(0.4-1.7), for example 1:(0.43-1.5).

[0047] In some embodiments, the coupling reaction is carried out at a temperature of 90-160°C, preferably 100-150°C, such as 100-110°C, 110-120°C or 150°C.

[0048] In some embodiments, the progress of the coupling reaction can be detected using methods conventional in the art (e.g., TLC, GC, HPLC, or NMR). The reaction endpoint is generally defined as the disappearance of the compound shown in Formula 8 or the cessation of the increase of the compound shown in Formula 10 by HPLC. The reaction time is 1-24 hours, preferably 3-20 hours, for example, 5-16 hours.

[0049] In some embodiments, the coupling reaction further includes a post-processing step, such as one or more of filtration, elution, extraction, column chromatography, concentration, crystallization, or drying.

[0050] In some embodiments, the preparation method of the compound shown in Formula 13 further includes the following step: in a solvent, in the presence of an organic acid, the compound shown in Formula 7 undergoes a cyclization reaction with acetone-acetone to obtain the compound shown in Formula 8.

[0051] In some embodiments, the solvent in the cyclization reaction is, for example, an aromatic solvent or a hydrocarbon solvent, such as toluene or cyclohexane.

[0052] In some embodiments, in the cyclization reaction, for example, the mass-to-volume ratio of the compound as shown in Formula 7 to the solvent is 0.02-0.1 g / mL, preferably 0.04-0.06 g / mL, for example 0.05 g / mL.

[0053] In some embodiments, in the cyclization reaction, the organic acid is one or more of acetic acid, methanesulfonic acid, benzenesulfonic acid, or p-toluenesulfonic acid. Preferably, the organic acid is toluenesulfonic acid, and more preferably, the toluenesulfonic acid is toluenesulfonic acid monohydrate.

[0054] In some embodiments, in the cyclization reaction, the molar ratio of the compound as shown in Formula 7 to the organic acid is 1:(0.1-0.5), preferably 1:(0.15-0.3), for example 1:0.19 or 1:0.2.

[0055] In some embodiments, in the cyclization reaction, the molar ratio of the compound as shown in Formula 7 to the acetone-based acetone is 1:(1-4), preferably 1:(1.5-3), for example 1:2.

[0056] In some embodiments, the cyclization reaction is carried out at a temperature of 90-130°C, preferably 100-120°C, for example 100-110°C.

[0057] In some embodiments, the progress of the cyclization reaction can be detected using methods conventional in the art (e.g., TLC, GC, HPLC, or NMR). The reaction endpoint is generally defined as the disappearance of the compound shown in Formula 7 or the cessation of the increase of the compound shown in Formula 8 by HPLC. The reaction time is 1-24 hours, preferably 5-10 hours, for example, 9 hours.

[0058] In some embodiments, the cyclization reaction further includes post-processing steps, such as washing, concentration, and column chromatography purification.

[0059] In some embodiments, the method for preparing the compound shown in Formula 13 further includes the step of: hydrolyzing the compound shown in Formula 6 in a solvent under acidic conditions to obtain the compound shown in Formula 7.

[0060] In some embodiments, the solvent in the hydrolysis reaction is an inorganic solvent or a mixture of an inorganic and an organic solvent, preferably water.

[0061] In some embodiments, the organic solvent in the hydrolysis reaction is an alcohol solvent, an ether solvent, or a sulfur-containing compound solvent. Preferably, the alcohol solvent is methanol, the ether solvent is THF, and the sulfur-containing compound solvent is DMSO.

[0062] In some embodiments, during the hydrolysis reaction, the mass-to-volume ratio of the compound as shown in Formula 6 to the solvent is 0.1-0.4 g / mL, preferably 0.2-0.3 g / mL, for example 0.25 g / mL or 0.26 g / mL.

[0063] In some embodiments, the acid in the hydrolysis reaction is an inorganic acid, preferably concentrated hydrochloric acid.

[0064] In some embodiments, during the hydrolysis reaction, the mass-to-volume ratio of the compound as shown in Formula 6 to the concentrated hydrochloric acid is 0.1-0.4 g / mL, preferably 0.2-0.3 g / mL, for example 0.25 g / mL or 0.26 g / mL.

[0065] In some embodiments, the hydrolysis reaction is carried out at a temperature of 40-100°C, preferably 90-100°C, for example 95-100°C.

[0066] In some embodiments, the progress of the hydrolysis reaction can be detected using methods conventional in the art (e.g., TLC, GC, HPLC, or NMR). Generally, the endpoint of the reaction is defined as the disappearance of the compound shown in Formula 6 or the cessation of the increase of the compound shown in Formula 7 by HPLC. The reaction time is 0.5-5 h, preferably 0.5-2 h, for example, 1 h.

[0067] In some embodiments, the hydrolysis reaction further includes post-processing steps such as extraction, neutralization, filtration, rinsing, and drying.

[0068] In some embodiments, the method for preparing the compound shown in Formula 13 further includes the following step: in a solvent, in the presence of a base, the compound shown in Formula 5 undergoes a cyclization reaction to obtain the compound shown in Formula 6.

[0069] In some embodiments, the solvent in the cyclization reaction is an amide solvent, preferably DMF.

[0070] In some embodiments, in the cyclization reaction, the mass-to-volume ratio of the compound as shown in Formula 5 to the solvent is 0.1-0.4 g / mL, preferably 0.15-0.2 g / mL, for example 0.17 g / mL or 0.18 g / mL.

[0071] In some embodiments, the base in the cyclization reaction is an inorganic base, preferably one or more of potassium carbonate, sodium carbonate, sodium hydroxide, or potassium hydroxide, more preferably potassium carbonate.

[0072] In some embodiments, in the cyclization reaction, the molar ratio of the compound as shown in Formula 5 to the base is 1:(1-2), preferably 1:(1.1-1.5), for example 1:1.1 or 1:1.2.

[0073] In some embodiments, the cyclization reaction is carried out at a temperature of 60-100°C; preferably 70-90°C, for example 80-85°C.

[0074] In some embodiments, the progress of the cyclization reaction can be detected using methods conventional in the art (e.g., TLC, GC, HPLC, or NMR). The reaction endpoint is generally defined as the disappearance of the compound shown in Formula 5 or the cessation of the increase of the compound shown in Formula 6 by HPLC. The reaction time is 1-10 hours, preferably 2-5 hours, for example, 3.5 hours.

[0075] In some embodiments, the cyclization reaction further includes post-processing steps, such as filtration, rinsing, neutralization, and filtration.

[0076] In some embodiments, the method for preparing the compound shown in Formula 13 further includes the following step: in a solvent, the compound shown in Formula 4 undergoes a condensation reaction with acetyl chloride and ammonium thiocyanate to prepare the compound shown in Formula 4.

[0077] In some embodiments, the solvent in the condensation reaction is a ketone solvent, preferably acetone.

[0078] In some embodiments, in the condensation reaction, the mass-to-volume ratio of the compound as shown in Formula 4 to the solvent is 0.02-0.1 g / mL, preferably 0.05-0.09 g / mL, for example 0.07 g / mL or 0.08 g / mL.

[0079] In some embodiments, in the condensation reaction, the molar ratio of the compound as shown in Formula 4 to the ammonium thiocyanate is 1:(0.8-2), preferably 1:(1-1.5), for example 1:1.1 or 1:1.2.

[0080] In some embodiments, the molar ratio of the compound as shown in Formula 4 to the acetyl chloride in the condensation reaction is 1:(0.8-2), preferably 1:(1-1.2), for example 1:1 or 1:1.1.

[0081] In some embodiments, the condensation reaction is carried out at a temperature of 40-80°C, preferably 50-70°C, for example 50-60°C.

[0082] In some embodiments, the progress of the condensation reaction can be detected using methods conventional in the art (e.g., TLC, GC, HPLC, or NMR). Generally, the endpoint is defined as the disappearance of the compound shown in Formula 5 or the cessation of the increase of the compound shown in Formula 6 by HPLC. The reaction time is 10-24 hours, preferably 15-20 hours, for example, 19 hours.

[0083] In some embodiments, the condensation reaction can be performed without post-processing steps and can be directly used for the next reaction; further, the condensation reaction can be directly used to prepare the compound shown in Formula 6.

[0084] The present invention also provides a method for preparing the compound shown in Formula 10, comprising the following steps: in a solvent, under palladium catalyst conditions, a coupling reaction is carried out between the compound shown in Formula 8 and the compound shown in Formula 9 to obtain the compound shown in Formula 10.

[0085] In the preparation method of the compound shown in Formula 10, each reaction condition and operation is independently as described in any one of the present invention.

[0086] The present invention also provides a method for preparing the compound shown in Formula 6, comprising the following steps: in a solvent, in the presence of a base, the compound shown in Formula 5 undergoes a cyclization reaction to obtain the compound shown in Formula 6.

[0087] In the preparation method of the compound shown in Formula 6, the reaction conditions and operations are independently as described in any one of the present invention.

[0088] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0089] The reagents and raw materials used in this invention are all commercially available.

[0090] The positive and progressive effects of this invention are as follows: by preparing the compound shown in Formula 13 and the intermediates shown in Formula 5 and Formula 6, this invention achieves high yield, is environmentally friendly, convenient in experimental operation, and simple in post-processing. Detailed Implementation

[0091] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0092] List of abbreviations

[0093] Example 1

[0094] Method 1:

[0095] Add acetonitrile (3.6 L), 6-(1,4-dimethyl-1H-1,2,3-triazol-5-yl)-7-fluorothiazo[4,5-c]pyridine-2-amine hydrochloride (240.0 g, 711.7 mmol), 2'-chloro-5'-methoxy-6-methyl-[4,4'-bipyridine]-3-carboxylic acid (234.0 g, 839.6 mmol), and N-methylimidazolium (525.3 g, 6.40 mol) to a three-necked flask, and stir at room temperature for 10 minutes. Add N,N,N,N-Tetramethylchloromethanemid hexafluorophosphate (897.6 g, 3.20 mol), heat to 60-70 °C, maintain the temperature for 3 hours, cool to room temperature, transfer the reaction solution to a 100 L reactor, add water (9 L) dropwise to the reaction solution, stir, filter, rinse the filter cake twice with water (960 ml), then rinse with ethyl acetate (1 L), and dry the filter cake at 40-45 °C for 16-24 hours to obtain compound 13 (301.6 g, yield 72.0%). 1 H NMR(400MHz,DMSO-d6)δ13.64(s,1H),9.14(d,J=1.7Hz,1H),8.91(s,1H),8.18(s,1H),7.62(s,1H),7 .51(s,1H),4.03(s,3H),3.62(s,3H),2.63(s,3H),2.27(d,J=1.4Hz,3H).LC / MS(ESI)m / z:525.2[M+H] + .

[0096] Method 2:

[0097] Under nitrogen protection, 25 mL of ACN, 6-(1,4-dimethyl-1H-1,2,3-triazol-5-yl)-7-fluorothiazo[4,5-c]pyridine-2-amine (5.00 g, 18.92 mmol), DIPEA (9.8 g, 75.83 mmol), and 2'-chloro-5'-methoxy-6-methyl-[4,4'-bipyridine]-3-carboxylic acid (5.8 g, 20.81 mmol) were added sequentially to the reaction flask. Then, T4P (34.08 g, 47.33 mmol) was added dropwise to the reaction flask. After the addition was complete, the mixture was stirred at 65-75 °C for 4 hours. 45 mL of purified water was added to the reaction solution, and the mixture was stirred at 65-75 °C for 1 hour. The mixture was then cooled to room temperature, filtered, and the filter cake was washed with 5 mL of water. The product was dried under reduced pressure to obtain a white solid product 13 (9.4 g, yield 94.64%). LC / MS (ESI) m / z: 525.2 [M+H] + .

[0098] Example 2

[0099] Method 1:

[0100] Dissolve 940 mL of concentrated hydrochloric acid in 4.66 L of water, stir at room temperature, add 6-(1,4-dimethyl-1H-1,2,3-triazol-5-yl)-2-(2,5-dimethyl-1H-pyrrolo-1-yl)-7-fluorothiazo[4,5-c]pyridine (450 g, 1.31 mol), heat to 80-85 °C, and maintain the reaction temperature for 5.5 hours. Cool to room temperature, filter, wash the filter cake with 500 mL of water, concentrate the filtrate under reduced pressure, and beat the resulting pale yellow solid with 1.85 L of acetonitrile at room temperature for 2 hours. Filter, wash the filter cake with a small amount of acetonitrile, and dry the filter cake to obtain a yellow powder solid compound 11' (361.6 g, yield 81.5%). 1 HNMR(400MHz,DMSO-d6)δ11.11(brs,2H),8.62(d,J=2.0Hz,1H),8.48(s,1H),3.98(s,3H),2.22(d,J=1.6Hz,3H).LC / MS(ESI)m / z:265.0[M+H] + .

[0101] Method 2:

[0102] Add 14.70 kg of water and 3.48 kg of 12N hydrochloric acid to the reactor. At 10-30℃, add compound 10 (1.75 kg, 5.11 mol) to the reactor and stir at 70-90℃ for 4-6 hours. Adjust the temperature to 40-60℃ and add 1.75 kg of thiourea resin to the reaction solution, stirring for 1 hour. Filter, and wash the filter cake with 1.75 kg of process water. Add another 1.75 kg of thiourea resin to the filtrate and stir for 1 hour. Filter again, and wash the filter cake with 1.75 kg of process water. Combine the filtrates and wash once with 5.25 kg of MTBE. Maintain the temperature of the filtrate at 0-10℃ and add a 25% sodium hydroxide aqueous solution dropwise to adjust the pH of the aqueous phase to 12. Filter again, and wash the filter cake with 3.50 kg of process water. Add 7.00 kg of acetonitrile to the filter cake and stir at 40-50℃ for 2 hours. The sample was filtered, and the filter cake was washed with 1.40 kg of acetonitrile. The wet product was dried under reduced pressure for 24 hours to obtain 11 (0.96 kg, purity 98.8%, yield 80.9%) of off-white solid powder. ¹H NMR (400 MHz, DMSO-d⁶) δ 8.62 (d, J = 2.4 Hz, 1H), 8.29 (s, 2H), 3.98 (s, 3H), 2.22 (d, J = 2.0 Hz, 3H). LC / MS (ESI) m / z: 265.0 [M+H] + .

[0103] Example 3

[0104] Method 1:

[0105] Toluene (11.3 L), potassium acetate (1.31 kg, 13.3 mol), palladium acetate (119.5 g, 0.53 mol), n-butyldi(1-adamantyl)phosphine (381.8 g, 1.06 mol), 6-chloro-7-fluorothiazole[4,5-c]pyridine-2-amine (750 g, 2.66 mol), and 1,4-dimethyl-1H-1,2,3-triazole (284.4 g, 2.93 mol) were added to the reaction vessel. The mixture was substituted with N2 three times, heated to 100-110 °C, and reacted for 16 hours. The mixture was then cooled, filtered, and the filter cake was washed with ethyl acetate. Saturated sodium chloride solution (15 L) was added to the filtrate, and the mixture was stirred for 20 minutes. The mixture was allowed to stand, separated, and the organic phase was collected. The aqueous phase was extracted again with ethyl acetate (5 L). The organic phases were combined, and saturated sodium chloride solution (15 L) was added. The mixture was stirred for 20 minutes, allowed to stand, separated, and the organic phase was collected. The aqueous phase was extracted again with ethyl acetate (5 L). The mixture was separated, and the organic phases were combined. The mixture was stirred with concentrated silica gel and subjected to column chromatography with petroleum ether / ethyl acetate at a ratio of 20:1 to 3:1 to obtain a white solid compound 10 (480 g, yield 52.6%). 1H NMR (400MHz, DMSO-d6) δ9.35(d,J=1.9Hz,1H),6.05(s,2H),4.06(s,3H),2.40(s,6H),2.29(d,J=2.0Hz,3H).LC / MS(ESI)m / z:343.0[M+H] + .

[0106] Method 2:

[0107] At 10-30℃, dioxane (19.24 kg), potassium phosphate (2.86 kg, 10.2 mol), palladium acetate (226.1 g, 1.01 mol), n-butyldi(1-adamantyl)phosphine (0.79 kg, 8.13 mol), 6-chloro-7-fluorothiazole[4,5-c]pyridine-2-amine (1.90 kg, 6.74 mol), and 1,4-dimethyl-1H-1,2,3-triazole (284.4 g, 2.93 mol) were added to the reactor, and N2 was used for three replacements. The temperature was raised to 110-120℃ and the reaction was maintained for 15 hours. The reaction mixture was cooled, and 0.95 kg of thiourea resin was added. The mixture was stirred at 40-60 °C for 1.5 hours, cooled, and filtered. The filter cake was washed with ethyl acetate (9.5 L, 5V). The mixture was concentrated under reduced pressure, and 9.5 L and 9.5 L of ethyl acetate were added to the concentrate. The mixture was allowed to stand and separated. The aqueous phase was extracted with ethyl acetate. The organic phases were combined and concentrated under reduced pressure to 2-4V. 19 L of n-heptane was added and concentrated under reduced pressure to 2-4V. 19 L of n-heptane and 1 L of methyl tert-butyl were added. The mixture was combined, stirred, filtered, and dried under reduced pressure to give a light brown solid compound 10 (4.27 kg, yield 82.5%). 1H NMR (400MHz, DMSO-d6) δ9.35(d,J=1.9Hz,1H),6.05(s,2H),4.06(s,3H),2.40(s,6H),2.29(d,J=2.0Hz,3H).LC / MS(ESI)m / z:343.0[M+H] + .

[0108] Method 3:

[0109] To a xylene (30 mL) solution of 6-chloro-7-fluorothiazolyl[4,5-c]pyridine-2-amine (1.5 g, 5.32 mmol), 1,4-dimethyl-5-(tributyltinyl)-1H-1,2,3-triazole (3.08 g, 7.99 mmol), tetra(triphenylphosphine)palladium (1.85 g, 1.60 mmol) was added, and the mixture was stirred at 150 °C for 5 hours under nitrogen protection. The mixture was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate = 3, V / V) to give compound 10 (1.25 g, 68.7% yield), a pale yellow solid. LC / MS (ESI) m / z: 343.1 [M+H] + .

[0110] Example 4

[0111] A mixture of 6-chloro-7-fluorothiazo[4,5-c]pyridine-2-amine (1.05 kg, 5.16 mol), acetone-based acetone (1.18 kg, 10.34 mol), and p-toluenesulfonic acid monohydrate (196 g, 1.03 mol) was refluxed in toluene (21.0 L) at 100–110 °C for 9 hours to remove water. The reaction mixture was cooled to room temperature, washed with an aqueous sodium bicarbonate solution, washed with saturated brine, concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate = 10, V / V) to give a yellow solid compound 8 (752 g, yield 51.8%). 1 H NMR(400MHz, CDCl3)δ8.89(s,1H),5.99(s,2H),2.41(s,6H).LC / MS(ESI)m / z:282.0[M+H] + .

[0112] Example 5

[0113] Water (11 L), concentrated hydrochloric acid (11 L), and N-(6-chloro-7-fluorothiazolyl[4,5-c]pyridin-2-yl)acetamide (2.8 kg, 11.4 mol) were added to a reaction vessel. Stirring was started, and the temperature was raised to 95-100 °C. The reaction was carried out for 1 hour, then cooled to room temperature. Ethyl acetate (8 L) was added for extraction, and the layers were separated. The pH of the aqueous phase was adjusted to 6-7 with sodium hydroxide solution. The mixture was filtered, and the filter cake was washed with water and dried under reduced pressure at 50 °C to obtain compound 7 (brownish-yellow solid, 1.06 kg, yield 45.7%). The HPLC purity was 86.09%. 1 H NMR(400MHz,DMSO-d6)δ8.27-8.25(m,3H).LC / MS(ESI)m / z:204.1[M+H] + .

[0114] Example 6

[0115] N-((4-bromo-6-chloro-5-fluoropyridin-3-yl)aminomethylthio)acetamide (4.38 kg, 10.2 mol) was dissolved in DMF (25 L), and potassium carbonate (3.0 kg, 12.2 mol) was added. The mixture was heated to 80 ± 3 °C and reacted for 3.5 hours. The mixture was then cooled to 30-35 °C, filtered, and the filter cake was washed with DMF (2 L). A saturated ammonium chloride solution (25 L) was added to the filtrate, and the mixture was stirred at room temperature for 0.5 hours to precipitate a solid. The solid was then filtered, and the filter cake was washed with water (1 L) to obtain compound 6 (2.8 kg, crude product yield 89.6%) with an HPLC purity of 93.28%, which was directly used in the next reaction. 1 H NMR(400MHz,DMSO-d6)δ12.99(s,1H),8.70(s,1H),2.26(s,3H).LC / MS(ESI)m / z:246.0[M+H] + .

[0116] Example 7

[0117] Ammonium thiocyanate (900 g, 12.2 mol) was dissolved in 30 L of acetone and stirred at room temperature until dissolved. Acetyl chloride (0.8 L, 11.2 mol) was slowly added, and the mixture was heated to 50-60 °C and maintained at this temperature for 0.6 h. Then, a solution of 4-bromo-6-chloro-5-fluoropyridine-3-amine (2.3 kg, 10.2 mol) in 4 L of acetone was added, and the mixture was stirred at 50-60 °C for 19 h. The mixture was then concentrated under reduced pressure at 40-50 °C to obtain a brown solid compound 5 (4.38 kg, crude product yield 76.3%) with an HPLC purity of 75.25%. The product was used directly in the next reaction without further processing. LC / MS (ESI) m / z: 325.91 [M+H] + .

[0118] Effect Example

[0119] Experimental methods:

[0120] 1. Biochemical activity assay of Polθ inhibitors (ADP Glo assay)

[0121] The N-terminus of the Polθ protein contains a helicase domain with ATPase activity, capable of hydrolyzing ATP into ADP, which can be detected by the ADP-Glo ​​assay kit (ADP-Glo). TM The Kinase Assay (Promega, V9102) was used to analyze the ATPase activity of Polθ protein and the inhibitory effect of small molecule compounds on the protein. The specific detection method is as follows:

[0122] 1) The helicase domain of the Polθ protein was purified using an insect system to obtain protein with a purity >90%. ssDNA (SEQ ID NO.1:5'-CCAGTGAATTGTTGCTCGGTACCTGCTAAC-3', Hangzhou Youkang Biotechnology Co., Ltd.) was ordered as the substrate for the Polθ protein; a reaction buffer containing 10 mM DTT (dithiothreitol), 20 mM MgCl2, Tris-HCl, and pH 7.5 was prepared.

[0123] 2) Prepare 2x ssDNA-Polθ premix and perform the reaction in a 384-well plate. Set up a control group with only buffer and add the premix to the other groups. Add the compound using an automated pipette (Thermo, Multidrop 8), starting at a concentration of 10 μM and diluting by 1 / 3. Set up a total of 9 detection sites, with 2 replicates per group.

[0124] 3) Prepare a 2x ATP solution. Add an equal volume of 2x ATP solution to all wells and let stand at room temperature for 60 minutes.

[0125] 4) Following the Promega reagent instructions, add ADP-Glo ​​Detection Reagent to the reaction system and let it stand at room temperature for 60 minutes;

[0126] 5) Following the Promega reagent instructions, add Kinase Detection Reagent to the reaction system, let it stand at room temperature for 60 minutes, and use a microplate reader (Thermo, Varioskan LUX) to detect the chemiluminescence signal. Set the reading time interval for each well to 1000 milliseconds.

[0127] Data analysis was performed on the measurement results: the CV% of the control group test results should be less than 10%, and the z' value should be greater than 0.5. Data meeting the above quality control results were used to calculate the inhibition rate of the compound (inhibition rate (%) = 100 * (control group average - experimental group) / (control group average - buffer group average). A nonlinear regression was used to fit the inhibition rate curve of the compound and the IC50 value was obtained.

[0128] 2. Cell viability assay for Polθ inhibitors

[0129] The in vitro efficacy of the inhibitor was assessed using a cell viability assay, specifically the commonly used CTG assay, with CellTiter Glo reagent (Promega, G7573). The specific assay method is as follows:

[0130] 1) Culture DLD1 and DLD1- / - cells. One day before the assay, digest the cells with trypsin (0.025% Trypsin-EDTA, Hyclone), centrifuge at 1000 rpm for 3 minutes, and collect the cells. Count the cells using a cell counter (Shanghai Mengwei Biomedical Technology Co., Ltd., SmartCell600A.SC1006), and seed them into 96-well white culture plates at an appropriate seeding ratio.

[0131] 2) Drug treatment was performed 24 hours after cell seeding, marked as Day 0. The compound was added using an automated pipette (Thermo, Multidrop 8). The plate was set as a 96-well plate with an initial concentration of 30 μM. The cells were diluted by 1 / 3 of the initial concentration, and a total of 9 detection points were set up, with 2 replicates per group. The cells were incubated at 37°C in a 5% CO2 incubator.

[0132] 3) Perform two solution changes on Day 3 and Day 7 respectively, and add the compound as in step 2);

[0133] 4) On Day 10, remove the cell culture plate and add an equal volume of CTG reagent (the CTG reagent needs to be brought back to temperature and premixed according to the reagent instructions). Gently mix for 10 minutes (speed 300) on a constant temperature mixer (Hangzhou Ausen Instrument Co., Ltd., MSC-100). Perform chemiluminescence detection using an ELISA reader (Thermo, Varioskan LUX).

[0134] 5) Data analysis of the measurement results: The CV% of the control group should be less than 20%, and the z' value should be greater than 0.5. Data meeting the above quality control results were used to calculate cell viability (inhibition rate (%) = 100 * (control group average - experimental group) / (control group average - culture medium control group average). GraphPad Prism8 was used to perform nonlinear regression fitting of the compound's inhibition rate curve and derive the IC50. 50 value.

[0135] Data list:

[0136] Specific data from biochemical experiments and cell viability testing experiments:

[0137] 3. Solubility Assay

[0138] 1) Prepare 0.1M Na2PO4 buffer (pH 7.4):

[0139] Add 11g Na2HPO4 (FW: 141.96) and 3.5g NaH2PO4_2H2O (FW: 156.03) to 1L of Mili-Q water, and adjust the pH to 7.4 with phosphoric acid or sodium hydroxide;

[0140] 2) Take 10 μL of the test compound (concentration: 10 mM in DMSO) and add it to 990 μL of the Na2PO4 buffer prepared in step 1 (final DMSO concentration: 1%).

[0141] 3) Shake the sample tube at room temperature for 2 hours (1000 rpm);

[0142] 4) Preparation of calibration curve:

[0143] a) Preparation of 300 μM spiking solution (SS):

[0144] Add 6 μL (10 mM in DMSO) of the test compound stock solution to 194 μL MeOH / ACN (4:1);

[0145] b) Plot the standard curve:

[0146] 5) Centrifuge the sample (10 minutes, 12,000 rpm) to precipitate undissolved particles. Filter the supernatant through a 0.22 μm filter membrane and then transfer the supernatant to a new centrifuge tube.

[0147] 6) Dilute the supernatant 10 times with 100mM buffer.

[0148] Add the supernatant (10 μL) to the buffer (100 mM, 90 μL) and dilute 10 times;

[0149] 7) Sample preparation for LC-MS / MS (API 4000) detection

[0150] Add 10 μL of sample (10-fold dilution) and standard curve sample to 400 μL of solution (MeOH:ACN = 1:1), perform instrument detection, and calculate the solubility value based on the standard curve.

[0151] Experimental results:

[0152] 4. Metabolic stability of mouse liver microsomes assay

[0153] 1) Preparation of experimental reagents

[0154] a) Phosphate-buffered saline (PBS): 100 mM, pH = 7.4 ± 0.1.

[0155] Dissolve 73.21g of dipotassium hydrogen phosphate trihydrate K2HPO4·3H2O (MW=228.22) and 10.78g of potassium dihydrogen phosphate KH2PO4 (MW=136.09) in 4000mL of pure water, and adjust the pH to 7.4±0.1 with phosphoric acid or potassium hydroxide.

[0156] b) Microparticle working solution; 1.0 mg / mL.

[0157] The purchased mouse liver microsomes (IPHASE, Cat. No.: 0121E1.01, Lot. No.: 22F001) were 20 mg / mL (10 mg, 0.5 mL). 225 μL of the solution was added to 4275 μL of PBS to prepare a 1.0 mg / mL microsome working solution.

[0158] c) NADPH regeneration system working solution: a mixture of 2 mM NADPH solution and 6 mM magnesium chloride solution

[0159] I. Weigh 6.8 mg of NADPH (MW = 833.35, 98%) powder and add it to 2000 μL of PBS solution to obtain a NADPH stock solution with a concentration of 40 mM.

[0160] II. Weigh 24.64 mg of magnesium chloride hexahydrate (MV = 203.3, 99%) solid, dissolve it in 10 mL of PBS solution to prepare a 12 mM magnesium chloride stock solution. (Note whether precipitation occurs.)

[0161] III. Take 200 μL of 40 mM NADPH stock solution, 2.0 mL of 12 mM magnesium chloride stock solution, and 1.80 mL of PBS solution to prepare a working solution for the NADPH regeneration system containing a mixture of 2 mM NADPH solution and 6 mM magnesium chloride solution.

[0162] d) Termination solution: Tolbutamide acetonitrile solution containing 200 ng / mL

[0163] I. Weigh 6.006 mg of tolbutamide (MV = 270.35, 99.9%) solid and add 30 mL of acetonitrile to prepare a 200 μg / mL tolbutamide stock solution.

[0164] II. Take 500 μL of 200 μg / mL tolbutamide stock solution and add 500 mL of acetonitrile to obtain a 200 ng / mL tolbutamide acetonitrile solution.

[0165] e) Working solution of test sample / reference standard: 100 μM working solution of test sample / reference standard

[0166] I. Purchase 1 mL of a 1.0 mg / mL testosterone (MV = 288.42) solution and add 155.8 μL ACN to prepare a 3 mM testosterone stock solution.

[0167] II. Take 50 μL of 3 mM testosterone stock solution and add 1450 μL of acetonitrile to prepare a working solution containing 100 μM testosterone.

[0168] III. Weigh 6.98 mg propafenone hydrochloride and dissolve it in 1.8433 mL of acetonitrile; weigh 5.67 mg diclofenac sodium and dissolve it in 1.783 mL of acetonitrile. Prepare 10 mM stock solutions of propafenone and diclofenac, respectively.

[0169] IV. Take 10 μL of propafenone and diclofenac stock solutions respectively, add 990 μL of acetonitrile, and prepare working solutions of propafenone and diclofenac respectively containing 100 μM.

[0170] 2) Experimental Procedure

[0171] a) Prepare 8 incubation plates and name them T0, T5, T15, T30, T45, T60, Blank60 and NCF60 respectively.

[0172] b) Add 100 μL of microparticle working solution to each of T0, T5, T15, T30, T45, T60, Blank60 and NCF60. Add 2 μL of test sample (reference) working solution to each of T0, T5, T15, T30, T45, T60 and NCF60. Add 2 μL of acetonitrile to Blank60.

[0173] c) Place T5, T15, T30, T45, T60, and Blank60 in a 37°C water bath for pre-incubation for 10 minutes.

[0174] d) Add 600 μL of stop solution to the T0 sample to terminate the reaction; add 98 μL of phosphate buffer to NCF60, and use the NCF60 sample without the NADPH generation system as a negative control.

[0175] e) Add 98 μL of NADPH regeneration working solution to the T0 sample after the previous step is terminated. After the pre-incubation at T5, T15, T30, T45, T60, and Blank60, add 98 μL of NADPH regeneration working solution to start the reaction. Therefore, the total incubation system is 200 μL, the test sample (control) concentration is 1 μM, the microsomal concentration is 0.5 mg / mL, and the NADPH regeneration solution concentration is 1 mM.

[0176] f) Place the above T5, T15, T30, T45, T60, Blank60 and NCF60 samples into a 37℃ water bath and incubate for the corresponding times: 5 minutes, 15 minutes, 30 minutes, 45 minutes, 60 minutes, 60 minutes and 60 minutes respectively.

[0177] g) After incubation for the corresponding time, 600 μL of stop solution was added to the T5, T15, T30, T45, T60, Blank60 and NCF60 samples respectively to terminate the reaction.

[0178] h) After termination, the samples T0, T5, T15, T30, T45, T60, Blank60, and NCF60 were shaken well and centrifuged at 14000 rpm for 5 minutes at 4°C. 100 μL of the supernatant was then analyzed by LC-MS / MS.

[0179] 3. Data Processing:

[0180] (1) The formula for calculating half-life is as follows:

[0181] Experimental results:

[0182] 5. Mouse PK Assay

[0183] The purpose of this experiment was to conduct a pharmacokinetic study on the test compound in male ICR mice (18-22g, 4-6 weeks old, Vital River Pharmaceuticals, Beijing) after oral administration. The experimental procedure is as follows:

[0184] 1) Dissolve the compound in 5% DMSO + 40% PEG 400 + 20% (10% TPGS) in water + 35% water to a concentration of 0.5 mg / mL, and then administer orally (5 mL / Kg);

[0185] 2) Collect blood samples: 0.03 mL at 0.25, 0.5, 1, 2, 4, 8, and 24 hours, and add EDTA-K2. CD1 mice: Collect approximately 0.03 mL of blood at each time point. Centrifuge at 4000g for 5 minutes at 4℃ to collect plasma. Aliquot plasma samples into clean polyethylene microcentrifuge tubes and store at -75±15℃ until analysis.

[0186] 3) Sample Analysis: The concentration of compounds in plasma samples will be analyzed using LC-MS / MS. Pharmacokinetic parameters will be calculated from the plasma assay results using WinNonlin (Phoenix™, version 8.3) or other similar software.

[0187] Experimental results:

[0188] While specific embodiments of the present invention have been described above, those skilled in the art should understand that these are merely illustrative examples, and various changes or modifications can be made to these embodiments without departing from the principles and essence of the present invention. Therefore, the scope of protection of the present invention is defined by the appended claims.

Claims

1. A method for preparing a compound as shown in Formula 13, characterized in that, It includes the following steps: In a solvent and in the presence of a condensing agent, the compound shown in Formula 11 undergoes an amidation reaction with the compound shown in Formula 12 to give the compound shown in Formula 13.

2. The method for preparing the compound as shown in Formula 13 according to claim 1, characterized in that, The amidation reaction satisfies one or more of the following conditions: (1) In the amidation reaction, the solvent is a nitrogen-containing compound solvent, preferably acetonitrile or DMF; (2) In the amidation reaction, the mass-to-volume ratio of the compound as shown in Formula 11 to the solvent is 0.03-0.3 g / mL, preferably 0.06-0.25 g / mL, for example 0.067-0.2 g / mL; (3) In the amidation reaction, the condensing agent may be a combination of N,N,N',N'-tetramethylchloromethamine hexafluorophosphate and N-methylimidazole or a combination of T4P and DIPEA; (4) The molar ratio of the compound shown in Formula 11 to the compound shown in Formula 12 is 1:(0.08-1.5), preferably 1:(1-1.2), for example 1:(1.1-1.18); (5) In the amidation reaction, the reaction temperature is 40-100℃, preferably 50-80℃, for example 60-75℃; (6) In the amidation reaction, the reaction time is 1-10 h, preferably 2-5 h, for example 3-4 h; and (7) The amidation reaction further includes a post-treatment step, such as crystallization, filtration, washing, and drying; preferably, the amidation reaction satisfies one or more of the following conditions: (1) The molar ratio of the compound shown in Formula 11 to the N,N,N',N'-tetramethylchloromethanemidazone hexafluorophosphate is 1:(2-6), preferably 1:(3-5), for example 1:4.5; (2) In the amidation reaction, the molar ratio of the compound shown in Formula 11 to the N-methylimidazole is 1:(5-12), preferably 1:(7-10), for example 1:9; (3) In the amidation reaction, the molar ratio of the compound as shown in Formula 11 to T4P is 1:(1-4), preferably 1:(2-3), for example 1:2.5; and (4) In the amidation reaction, the molar ratio of the compound shown in Formula 11 to DIPEA is 1:(2-6), preferably 1:(3-5), for example 1:

4.

3. The method for preparing the compound as shown in Formula 13 according to claim 2, characterized in that, It also includes the following steps: neutralizing the compound shown in Formula 11' with an aqueous inorganic base solution to obtain the compound shown in Formula 11. For example, the neutralization reaction satisfies one or more of the following conditions: (1) In the neutralization reaction, the compound shown in Formula 11' is neutralized with an inorganic alkaline aqueous solution to obtain the compound shown in Formula 11; (2) In the neutralization reaction, the inorganic alkaline aqueous solution is an aqueous solution of sodium hydroxide; (3) In the neutralization reaction, the mass fraction of the sodium hydroxide aqueous solution is 20-30%, preferably 25%; and (4) The neutralization reaction also includes post-processing steps, such as filtration, washing, recrystallization, filtration, washing and drying; Preferably, the method for preparing the compound shown in Formula 13 further includes the following step: in a solvent, in the presence of an acid, the compound shown in Formula 10 undergoes a deprotection reaction to obtain the compound shown in Formula 11'. More preferably, the deprotection reaction satisfies one or more of the following conditions: (1) In the deprotection reaction, the solvent is an inorganic solvent or a mixture of an inorganic solvent and an organic solvent; the solvent is preferably water; the organic solvent is an alcohol solvent, an ether solvent or a sulfur-containing compound solvent, preferably, the alcohol solvent can be methanol, the ether solvent is THF, and the sulfur-containing compound solvent is DMSO; (2) In the deprotection reaction, the mass-to-volume ratio of the compound as shown in Formula 10 to the solvent is 0.05-0.2 g / mL, preferably 0.08-0.15 g / mL, for example 0.09-0.12 g / mL; (3) In the deprotection reaction, the acid is an inorganic acid, preferably concentrated hydrochloric acid or concentrated sulfuric acid, and more preferably concentrated hydrochloric acid; (4) In the deprotection reaction, the mass-to-volume ratio of the compound shown in Formula 10 to the concentrated hydrochloric acid is 0.1-1 g / mL, preferably 0.3-0.7 g / mL, for example 0.47-0.6 g / mL; (5) In the deprotection reaction, the reaction temperature is 40-100℃, preferably 70-95℃, for example 70-90℃; and (6) In the deprotection reaction, the reaction time is 2-12h, preferably 3-6h, for example 3-5.5h.

4. The method for preparing the compound as shown in Formula 13 according to claim 3, characterized in that, It also includes the following steps: in a solvent, under palladium catalytic conditions, a coupling reaction is carried out between the compound shown in Formula 8 and the compound shown in Formula 9 to obtain the compound shown in Formula 10. Wherein, R is H, Bu4Sn, ClZn or ClMg; when R is H, it also includes phosphine ligands and bases; Preferably, the coupling reaction satisfies one or more of the following conditions: (1) In the coupling reaction, the solvent is an aromatic solvent or an epoxy solvent; the aromatic solvent may be toluene or xylene; the epoxy solvent may be dioxane; for example, the solvent is toluene, xylene or dioxane; (2) In the coupling reaction, the mass-to-volume ratio of the compound as shown in Formula 8 to the solvent is 0.02-0.2 g / mL, preferably 0.04-0.17 g / mL, for example 0.05-0.1 g / mL; (3) In the coupling reaction, the phosphine ligand is one or more of tritert-butylphosphine, tricyclohexylphosphine, 4,5-bis(diphenylphosphine)-9,9-dimethyloxanthracene or n-butyldi(1-adamantyl)phosphine, preferably n-butyldi(1-adamantyl)phosphine. (4) In the coupling reaction, the molar ratio of the compound shown in Formula 8 to the phosphine ligand is 1:(0.2-2), preferably 1:(0.3-1.5), for example 1:(0.4-1.3); (5) In the coupling reaction, the palladium catalyst is one or more of palladium chloride, bis(acetonitrile) palladium chloride, dichlorobis(triphenylphosphine) palladium, trifluoroacetate palladium, tridibenzylacetone dipalladium, (1,1'-bis(diphenylphosphine)ferrocene) dichloride, methanesulfonic acid [n-butylbis(1-adamantyl)phosphine](2-amino-1,1'-biphenyl-2-yl)palladium(II), dichlorobis(triphenylphosphine) palladium, tetra(triphenylphosphine) palladium or palladium acetate, preferably tetra(triphenylphosphine) palladium, dichlorobis(triphenylphosphine) palladium or palladium acetate; (6) In the coupling reaction, the molar ratio of the compound shown in Formula 8 to the palladium catalyst is 1:(0.1-0.5), preferably 1:(0.15-0.4), for example 1:(0.15-0.3); (7) In the coupling reaction, the base is an inorganic base, preferably one or more of sodium bicarbonate, sodium carbonate, sodium acetate, potassium carbonate, potassium acetate, potassium phosphate or cesium carbonate, more preferably potassium acetate or potassium phosphate; (8) In the coupling reaction, the molar ratio of the compound as shown in Formula 8 to the base is 1:(1-10), preferably 1:(1.5-6), for example 1:(1.5-5); (9) In the coupling reaction, the molar ratio of the compound shown in Formula 8 to the compound shown in Formula 9 is 1:(0.3-2), preferably 1:(0.4-1.7), for example 1:(0.43-1.5); (10) In the coupling reaction, the reaction temperature is 90-160℃, preferably 100-150℃, for example 100-110℃, 110-120℃ or 150℃; (11) In the coupling reaction, the reaction time is 1-24 h, preferably 3-20 h, for example 5 h-16 h; and (12) The coupling reaction further includes a post-processing step, such as one or more of the following: filtration, rinsing, extraction, column chromatography, concentration, crystallization or drying.

5. The method for preparing the compound as shown in Formula 13 according to claim 4, characterized in that, It also includes the following steps: in a solvent, in the presence of an organic acid, the compound shown in Formula 7 undergoes a cyclization reaction with acetone-acetone to obtain the compound shown in Formula 8. Preferably, the cyclization reaction satisfies one or more of the following conditions: (1) In the cyclization reaction, for example, the solvent is an aromatic solvent or a hydrocarbon solvent, such as toluene or cyclohexane; (2) In the cyclization reaction, for example, the mass-to-volume ratio of the compound as shown in Formula 7 to the solvent is 0.02-0.1 g / mL, preferably 0.04-0.06 g / mL, for example 0.05 g / mL; (3) In the cyclization reaction, the organic acid is one or more of acetic acid, methanesulfonic acid, benzenesulfonic acid or p-toluenesulfonic acid, preferably, the organic acid is toluenesulfonic acid, more preferably, the toluenesulfonic acid is toluenesulfonic acid monohydrate; (4) In the cyclization reaction, the molar ratio of the compound shown in Formula 7 to the organic acid is 1:(0.1-0.5), preferably 1:(0.15-0.3), for example 1:0.19 or 1:0.2; (5) In the cyclization reaction, the molar ratio of the compound shown in Formula 7 to the acetone-acetone is 1:(1-4), preferably 1:(1.5-3), for example 1:2; (6) In the cyclization reaction, the reaction temperature is 90-130℃, preferably 100-120℃, for example 100-110℃; (7) In the cyclization reaction, the reaction time is 1-24 h, preferably 5-10 h, for example 9 h; and (8) The cyclization reaction also includes post-processing steps, such as washing, concentration and column chromatography purification.

6. The method for preparing the compound as shown in Formula 13 according to claim 5, characterized in that, It also includes the following step: in a solvent, under acidic conditions, the compound shown in Formula 6 undergoes a hydrolysis reaction to obtain the compound shown in Formula 7. Preferably, the hydrolysis reaction satisfies one or more of the following conditions: (1) In the hydrolysis reaction, the solvent is an inorganic solvent or a mixture of an inorganic solvent and an organic solvent, preferably water; the organic solvent is an alcohol solvent, an ether solvent or a sulfur-containing compound solvent, preferably methanol, THF, or DMSO. (2) In the hydrolysis reaction, the mass-to-volume ratio of the compound shown in Formula 6 to the solvent is 0.1-0.4 g / mL, preferably 0.2-0.3 g / mL, for example 0.25 g / mL or 0.26 g / mL; (3) In the hydrolysis reaction, the acid is an inorganic acid, preferably concentrated hydrochloric acid; (4) In the hydrolysis reaction, the mass-to-volume ratio of the compound shown in Formula 6 to the concentrated hydrochloric acid is 0.1-0.4 g / mL, preferably 0.2-0.3 g / mL, for example 0.25 g / mL or 0.26 g / mL; (5) In the hydrolysis reaction, the reaction temperature is 40-100℃, preferably 90-100℃, for example 95-100℃; (6) In the hydrolysis reaction, the reaction time is 0.5-5 h, preferably 0.5-2 h, for example 1 h; and (7) The hydrolysis reaction also includes post-processing steps, such as extraction, neutralization, filtration, rinsing and drying.

7. The method for preparing the compound as shown in Formula 13 according to claim 6, characterized in that, It also includes the following steps: in a solvent, in the presence of a base, the compound shown in Formula 5 undergoes a cyclization reaction to obtain the compound shown in Formula 6. Preferably, the cyclization reaction satisfies one or more of the following conditions: (1) In the cyclization reaction, the solvent is an amide solvent, preferably DMF; (2) In the cyclization reaction, the mass-to-volume ratio of the compound shown in Formula 5 to the solvent is 0.1-0.4 g / mL, preferably 0.15-0.2 g / mL, for example 0.17 g / mL or 0.18 g / mL; (3) In the cyclization reaction, the base is an inorganic base, preferably one or more of potassium carbonate, sodium carbonate, sodium hydroxide or potassium hydroxide, more preferably potassium carbonate; (4) In the cyclization reaction, the molar ratio of the compound shown in Formula 5 to the base is 1:(1-2), preferably 1:(1.1-1.5), for example 1:1.1 or 1:1.2; (5) In the cyclization reaction, the reaction temperature is 60-100℃; preferably 70-90℃, for example 80-85℃; (6) In the cyclization reaction, the reaction time is 1-10 h, preferably 2-5 h, for example 3.5 h; and (7) The cyclization reaction also includes post-processing steps, such as filtration, rinsing, neutralization and filtration.

8. The method for preparing the compound as shown in Formula 13 according to claim 6, characterized in that, It also includes the following steps: In a solvent, the compound shown in Formula 4 undergoes a condensation reaction with acetyl chloride and ammonium thiocyanate to prepare the compound shown in Formula 4. Preferably, the method for preparing the compound as shown in Formula 13 satisfies one or more of the following conditions: (1) In the condensation reaction, the solvent is a ketone solvent, preferably acetone; (2) In the condensation reaction, the mass-to-volume ratio of the compound as shown in Formula 4 to the solvent is 0.02-0.1 g / mL, preferably 0.05-0.09 g / mL, for example 0.07 g / mL or 0.08 g / mL; (3) In the condensation reaction, the molar ratio of the compound shown in Formula 4 to the ammonium thiocyanate is 1:(0.8-2), preferably 1:(1-1.5), for example 1:1.1 or 1:1.2; (4) In the condensation reaction, the molar ratio of the compound shown in Formula 4 to the acetyl chloride is 1:(0.8-2), preferably 1:(1-1.2), for example 1:1 or 1:1.1; (5) In the condensation reaction, the reaction temperature is 40-80℃, preferably 50-70℃, for example 50-60℃; and (6) In the condensation reaction, the reaction time is 10-24h, preferably 15-20h, for example 19h.

9. A method for preparing a compound as shown in Formula 10, characterized in that, It includes the following steps: In a solvent, under palladium catalytic conditions, the compound shown in Formula 8 and the compound shown in Formula 9 undergo a coupling reaction to give the compound shown in Formula 10. The reaction conditions and operating conditions of the method for preparing the compound as shown in Formula 10 are as described in claim 4.

10. A method for preparing a compound as shown in Formula 6, characterized in that, It includes the following steps: In a solvent and in the presence of a base, the compound shown in Formula 5 undergoes a cyclization reaction to give the compound shown in Formula 6. The reaction conditions and operating conditions of the method for preparing the compound as shown in Formula 6 are as described in claim 7.

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