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. Provided in the present invention is a preparation method for a compound as represented by formula 13. The method comprises the following step: using a compound as represented by formula 3-1 as a raw material, and implementing a reaction process shown in the formula below, so as to obtain the compound as 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 environmentally friendly; and (4) the method is favorable for industrial production.

Description

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

[0001] This application claims priority to Chinese patent application 2024105006845, 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 method involves a one-pot preparation of the compound shown in Formula 7 from the compound shown in Formula 3-1, achieving high yield, convenient experimental operation, and simple post-processing. The method for preparing cyclic nitrogen-containing compounds and their intermediates 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:

[0011] (1) In a solvent, the compound shown in Formula 3-1 was reacted under inorganic acid conditions and then condensed with KSCN to prepare the compound shown in Formula 7.

[0012] (2) In a solvent and in the presence of an organic acid, the compound shown in Formula 7 undergoes a cyclization reaction with acetone-acetone to prepare the compound shown in Formula 8.

[0013] (3) In a solvent, under the condition of a palladium catalyst, the compound shown in Formula 8 and the compound shown in Formula 9 were coupled to prepare the compound shown in Formula 10.

[0014] (4) In a solvent and in the presence of acid, the compound shown in Formula 10 undergoes a deprotection reaction to prepare the compound shown in Formula 11'.

[0015] (5) In a solvent, in the presence of a condensing agent, the compound shown in Formula 11' and the compound shown in Formula 12 are subjected to an amidation reaction to prepare the compound shown in Formula 13.

[0016] Alternatively, (4') in a solvent, in the presence of an acid, the compound shown in Formula 10 undergoes a deprotection reaction to prepare the compound shown in Formula 11', and then undergoes a neutralization reaction with an inorganic alkaline aqueous solution to obtain the compound shown in Formula 11.

[0017] (5') In a solvent, in the presence of a condensing agent, the compound shown in Formula 11 is subjected to an amidation reaction with the compound shown in Formula 12 to prepare the compound shown in Formula 13.

[0018] Wherein, R is H, Bu4Sn, ClZn or ClMg;

[0019] When R is H, it also includes phosphine ligands, palladium catalysts, and bases.

[0020] In some embodiments, in step (1), the solvent may be a conventional solvent for such reactions in the art, and the solvent may be a protic solvent, preferably water.

[0021] In some embodiments, in step (1), the amount of solvent used can be conventional for such reactions in the art, and the mass-to-volume ratio of the compound as shown in Formula 3-1 to the solvent is 1-5 g / mL, preferably 1.5-3 g / mL, for example 2 g / mL.

[0022] In some embodiments, in step (1), the acid is an inorganic acid or an organic acid, preferably hydrochloric acid or sulfuric acid, and preferably p-toluenesulfonic acid.

[0023] In some embodiments, in step (1), the concentration of hydrochloric acid is the conventional concentration of hydrochloric acid in the art, preferably 2-12 mol / L.

[0024] In some embodiments, in step (1), the mass-to-volume ratio of the compound as shown in Formula 3-1 to concentrated hydrochloric acid is 0.1-0.5 g / mL, preferably 0.15-0.3 g / mL, for example 0.2 g / mL.

[0025] In some embodiments, in step (1), the reaction temperature is the conventional reaction temperature for such reactions in the art, which is 60-100°C, preferably 70-100°C, for example 70-80°C.

[0026] In some embodiments, the progress of the reaction in step (1) can be detected using methods conventional in the art (e.g., TLC, GC, HPLC, or NMR), and the reaction endpoint is generally defined as the disappearance of the compound shown in Formula 3-1 detected by HPLC. The reaction time is 1-10 h, preferably 1.5-5 h, for example 2 h.

[0027] In some embodiments, in the condensation reaction of step (1), the molar ratio of the compound shown in Formula 3-1 to the KSCN is 1:(2-8), preferably 1:(3-7), for example 1:4, 1:5, 1:6 or 1:7.

[0028] In some embodiments, the reaction temperature in the condensation reaction of step (1) is 50-100°C, preferably 60-100°C, for example 60-85°C.

[0029] In some embodiments, the progress of the condensation reaction in step (1) can be detected using methods conventional in the art (e.g., TLC, GC, HPLC, or NMR), generally with the endpoint of the reaction being the point at which the compound shown in Formula 7 no longer increases, detected by HPLC. The reaction time is 1-40 h, preferably 1-31 h, for example 2 h.

[0030] In some embodiments, the condensation reaction in step (1) further includes post-processing, such as filtration, washing, neutralization, filtration, washing and drying.

[0031] In some embodiments, in the cyclization reaction of step (2), the solvent is an aromatic solvent or a hydrocarbon solvent, such as toluene or cyclohexane.

[0032] In some embodiments, in the cyclization reaction of step (2), 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.

[0033] In some embodiments, in the cyclization reaction of step (2), 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.

[0034] In some embodiments, in the cyclization reaction of step (2), 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.

[0035] In some embodiments, in the cyclization reaction of step (2), 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.

[0036] In some embodiments, the cyclization reaction in step (2) is carried out at a temperature of 90-130°C, preferably 100-120°C, for example 100-110°C.

[0037] In some embodiments, the progress of the cyclization reaction in step (2) 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 h, preferably 5-10 h, for example 9 h.

[0038] In some embodiments, the cyclization reaction in step (2) further includes post-processing steps, such as washing, concentration and column chromatography purification.

[0039] In some embodiments, in the coupling reaction of step (3), 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.

[0040] In some embodiments, in the coupling reaction of step (3), 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.

[0041] In some embodiments, in the coupling reaction of step (3), the palladium catalyst is one or more of palladium chloride, palladium di(acetonitrile) chloride, palladium dichlorobis(triphenylphosphine)palladium trifluoromethanesulfonate, palladium trifluoroacetate, tridibenzylacetone dipalladium, (1,1'-bis(diphenylphosphine)ferrocene)palladium dichloride, [n-butyldi(1-adamantyl)phosphine](2-amino-1,1'-biphenyl-2-yl)palladium(II), palladium dichlorobis(triphenylphosphine), palladium tetra(triphenylphosphine) or palladium acetate, preferably palladium tetra(triphenylphosphine), palladium dichlorobis(triphenylphosphine), or palladium acetate.

[0042] In some embodiments, in the coupling reaction of step (3), the molar ratio of the compound as 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).

[0043] In some embodiments, in the coupling reaction of step (3), the phosphine ligand 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.

[0044] In some embodiments, in the coupling reaction of step (3), the molar ratio of the compound as 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).

[0045] In some embodiments, in the coupling reaction of step (3), 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).

[0046] In some embodiments, in the coupling reaction of step (3), 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.

[0047] In some embodiments, in the coupling reaction of step (3), 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).

[0048] In some embodiments, the reaction temperature in the coupling reaction of step (3) is 90-160°C, preferably 100-150°C, for example 100-110°C, 110-120°C or 150°C.

[0049] In some embodiments, the progress of the substitution reaction in step (3) 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 8 or the cessation of the increase of the compound shown in Formula 10 by HPLC. The reaction time is 1-24 h, preferably 3-20 h, for example 15 h or 16 h.

[0050] In some embodiments, the coupling reaction in step (3) further includes a post-processing step, such as one or more of filtration, rinsing, extraction, column chromatography, concentration, crystallization or drying.

[0051] In some embodiments, in the deprotection reaction of step (4), the solvent is an inorganic solvent or a mixture of an inorganic solvent and an organic solvent; preferably water.

[0052] In some embodiments, in the deprotection reaction of step (4), the organic solvent is an alcohol solvent, an ether solvent, or a sulfoxide solvent. Preferably, the alcohol solvent is methanol, the ether solvent is THF, and the sulfoxide solvent is DMSO.

[0053] In some embodiments, in the deprotection reaction of step (4), 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.

[0054] In some embodiments, in the deprotection reaction of step (4), the acid is an inorganic acid, preferably concentrated hydrochloric acid or concentrated sulfuric acid, more preferably concentrated hydrochloric acid.

[0055] In some embodiments, in the deprotection reaction of step (4), 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.

[0056] In some embodiments, the deprotection reaction in step (4) is carried out at a temperature of 40-100°C, preferably 70-95°C, for example 70-90°C.

[0057] In some embodiments, the progress of the deprotection reaction in step (4) 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 10 or the cessation of the increase of the compound shown in Formula 11' by HPLC. The reaction time is 2-12 h, preferably 3-6 h, for example 3-5.5 h.

[0058] In some embodiments, the deprotection reaction in step (4) further includes post-processing steps, such as filtration, rinsing, concentration, pulping, filtration, rinsing and drying.

[0059] In some embodiments, in the deprotection reaction of step (4'), the solvent is an inorganic solvent or a mixture of an inorganic solvent and an organic solvent; preferably water.

[0060] In some embodiments, in the deprotection reaction of step (4'), the organic solvent is an alcohol solvent, an ether solvent, or a sulfoxide solvent. Preferably, the alcohol solvent is methanol, the ether solvent is THF, and the sulfoxide solvent is DMSO.

[0061] In some embodiments, in the deprotection reaction of step (4'), 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.

[0062] In some embodiments, in the deprotection reaction of step (4'), the acid is an inorganic acid, preferably concentrated hydrochloric acid or concentrated sulfuric acid, more preferably concentrated hydrochloric acid.

[0063] In some embodiments, in the deprotection reaction of step (4'), 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.

[0064] In some embodiments, the deprotection reaction in step (4') is carried out at a temperature of 40-100°C, preferably 70-95°C, for example 70-90°C.

[0065] In some embodiments, the progress of the deprotection reaction in step (4') 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 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.

[0066] In some embodiments, the deprotection reaction in step (4') further includes post-processing steps, such as filtration, extraction, neutralization, rinsing, filtration and drying.

[0067] In some embodiments, the inorganic alkaline aqueous solution in the neutralization reaction of step (4') is an aqueous sodium hydroxide solution.

[0068] In some embodiments, in the neutralization reaction of step (4'), the mass fraction of the sodium hydroxide aqueous solution is 20-30%, preferably 25%.

[0069] In some embodiments, in the amidation reaction of step (5), the solvent is a nitrile solvent or an amide solvent, preferably acetonitrile or DMF.

[0070] In some embodiments, in the amidation reaction of step (5), 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.

[0071] In some embodiments, the condensing agent in the amidation reaction of step (5) may be a combination of N,N,N',N'-tetramethylchloroformamidine hexafluorophosphate (TCFH) and 1-methylimidazole.

[0072] In some embodiments, in the amidation reaction of step (5), the molar ratio of the compound as 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.

[0073] In some embodiments, in the amidation reaction of step (5), the molar ratio of the compound as shown in Formula 11' to the N-methylimidazole is 1:(5-12), preferably 1:(7-10), for example 1:9.

[0074] In some embodiments, in the amidation reaction of step (5), 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).

[0075] In some embodiments, the reaction temperature in step (5) of the amidation reaction is 40-100°C, preferably 50-80°C, for example 60-75°C.

[0076] In some embodiments, the progress of the amidation reaction in step (5) 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 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.

[0077] In some embodiments, the amidation reaction in step (5) further includes post-processing steps, such as crystallization, filtration, washing and drying.

[0078] In some embodiments, in the amidation reaction of step (5'), the solvent is a nitrile solvent or an amide solvent, preferably acetonitrile or DMF.

[0079] In some embodiments, in the amidation reaction of step (5'), 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.

[0080] In some embodiments, the condensing agent in the amidation reaction of step (5') may be a combination of T4P and DIPEA.

[0081] In some embodiments, in the amidation reaction of step (5'), the molar ratio of the compound as shown in Formula 11 to the T4P is 1:(1-4), preferably 1:(2-3), for example 1:2.5.

[0082] In some embodiments, in the amidation reaction of step (5'), 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.

[0083] In some embodiments, in the amidation reaction of step (5'), 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.

[0084] In some embodiments, the reaction temperature in step (5') of the amidation reaction is 40-100°C, preferably 50-80°C, for example 60-75°C.

[0085] In some embodiments, the progress of the amidation reaction in step (5') 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.

[0086] In some embodiments, the amidation reaction in step (5') further includes post-treatment steps, such as crystallization, filtration, washing, and drying.

[0087] The present invention also provides a method for preparing the compound shown in Formula 7, comprising the following steps: reacting the compound shown in Formula 3-1 under inorganic acid conditions in a solvent, followed by a condensation reaction with KSCN to prepare the compound shown in Formula 7.

[0088] In some embodiments, the conditions and operating steps of the preparation method of the compound shown in Formula 7 are as described in step (1) above.

[0089] The present invention also provides a method for preparing the compound shown in Formula 8, which includes the following steps:

[0090] (1) In a solvent, the compound shown in Formula 3-1 was reacted under inorganic acid conditions and then condensed with KSCN to prepare the compound shown in Formula 7.

[0091] (2) The compound shown in Formula 7 was prepared by cyclization reaction of the compound shown in Formula 7 with acetone-acetone in a solvent and in the presence of an organic acid to obtain the compound shown in Formula 8.

[0092] In some embodiments, the conditions and operating steps of the preparation method of the compound shown in Formula 8 are as described in steps (1) and (2) above.

[0093] The present invention also provides a method for preparing a compound as shown in Formula 10, comprising the following steps:

[0094] (1) In a solvent, the compound shown in Formula 3-1 was reacted under inorganic acid conditions and then condensed with KSCN to prepare the compound shown in Formula 7.

[0095] (2) The compound shown in Formula 7 was prepared by cyclization reaction of the compound shown in Formula 7 with acetone-acetone in a solvent and in the presence of an organic acid to obtain the compound shown in Formula 8.

[0096] (3) In a solvent, under the condition of a palladium catalyst, the compound shown in Formula 8 and the compound shown in Formula 9 were subjected to a substitution reaction to prepare the compound shown in Formula 10.

[0097] In some embodiments, the conditions and operating steps of the preparation method of the compound shown in Formula 10 are as described in steps (1), (2) and (3) above.

[0098] The present invention also provides a method for preparing a compound as shown in Formula 11 or 11', comprising the following steps:

[0099] (1) In a solvent, the compound shown in Formula 3-1 was reacted under inorganic acid conditions and then condensed with KSCN to prepare the compound shown in Formula 7.

[0100] (2) The compound shown in Formula 7 was prepared by cyclization reaction of the compound shown in Formula 7 with acetone-acetone in a solvent and in the presence of an organic acid to obtain the compound shown in Formula 8.

[0101] (3) In a solvent, under the condition of a palladium catalyst, the compound shown in Formula 8 and the compound shown in Formula 9 were subjected to a substitution reaction to prepare the compound shown in Formula 10.

[0102] (4) In a solvent, in the presence of an acid, the compound shown in Formula 10 is subjected to a deprotection reaction to prepare the compound shown in Formula 11 or 11'.

[0103] In some embodiments, the conditions and operating steps of the preparation method of the compound shown in Formula 11 are as described in steps (1), (2), (3) and (4) above.

[0104] 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.

[0105] The reagents and raw materials used in this invention are all commercially available.

[0106] The positive and progressive effects of this invention are as follows: by preparing the compound as shown in Formula 13 and the intermediate as shown in Formula 7, this invention achieves high yield, is environmentally friendly, convenient in experimental operation, and simple in post-processing. Detailed Implementation

[0107] 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.

[0108] List of abbreviations

[0109] Example 1

[0110] Method 1:

[0111] 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] + .

[0112] Method 2:

[0113] 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 mixture, 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, compound 13 (9.4 g, yield 94.64%). LC / MS (ESI) m / z: 525.2 [M+H] + .

[0114] Example 2

[0115] Method 1:

[0116] 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] + .

[0117] Method 2:

[0118] 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 a white solid powder, compound 11 (0.96 kg, purity 98.8%, yield 80.9%). 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] + .

[0119] Example 3

[0120] Method 1:

[0121] 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 compound 10 (white solid, 480 g, yield 52.6%). 1 H 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] + .

[0122] Method 2:

[0123] 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 compound 10 (light brown solid, 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] + .

[0124] Method 3:

[0125] 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, yield 68.7%) as a pale yellow solid. LC / MS (ESI) m / z: 343.1 [M+H] + .

[0126] Example 4

[0127] 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 compound 8 (yellow solid, 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] + .

[0128] Example 5

[0129] Under nitrogen protection, tert-butyl (4-bromo-6-chloro-5-fluoropyridine)-3-carbamate (200.5 g, 538.2 mmol) was added to 100 mL of H₂O and 1000 mL of HCl (12N), and the reaction temperature was adjusted to 70-80 °C and stirred for 2 hours. KSCN (261.2 g, 2.69 mol) was added to the reaction flask, and the reaction was maintained at 70-80 °C for 2 hours. The reaction solution was cooled to 50-60 °C and filtered. The filter cake was washed with 1 L of water. The filtrate was kept at 0-10 °C, and 6N NaOH solution was added dropwise to adjust the pH to 7. The solution was then filtered, and the filter cake was washed with 1.5 L of water. The filter cake was dried under reduced pressure at 50 °C to obtain a yellow solid compound 7 (304 g, purity 91.8%, crude product yield 138.7%), which was directly used in the next reaction step. 1 H NMR(400MHz,DMSO-d6)δ8.27-8.25(m,3H).LC / MS(ESI)m / z:204.1[M+H] + .

[0130] The selection criteria for experimental conditions are shown in the table below:

[0131] Effect Example

[0132] Experimental methods:

[0133] 1. Biochemical activity assay of Polθ inhibitors (ADP Glo assay)

[0134] 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:

[0135] 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.

[0136] 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.

[0137] 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.

[0138] 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;

[0139] 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.

[0140] 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.

[0141] 2. Cell viability assay for Polθ inhibitors

[0142] 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:

[0143] 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.

[0144] 2) Drug treatment was performed 24 hours after cell seeding, which was recorded as Day 0. The compound was added using an automated pipette (Thermo, Multidrop8). 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.

[0145] 3) Perform two solution changes on Day 3 and Day 7 respectively, and add the compound as in step 2);

[0146] 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).

[0147] 5) Perform data analysis on the measurement results: The CV% of the control group should be less than 20%, and the z' value should be greater than 0.5. For data meeting the above quality control results, calculate cell viability (inhibition rate (%) = 100 * (control group average - experimental group) / (control group average - culture medium control group average). Use GraphPad Prism8 to perform nonlinear regression fitting of the compound's inhibition rate curve and obtain the IC50 value.

[0148] Data list:

[0149] Specific data from biochemical experiments and cell viability testing experiments:

[0150] 3. Solubility Assay

[0151] 1) Prepare 0.1M Na2PO4 buffer (pH 7.4):

[0152] 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;

[0153] 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%).

[0154] 3) Shake the sample tube at room temperature for 2 hours (1000 rpm);

[0155] 4) Preparation of calibration curve:

[0156] a) Preparation of 300 μM spiking solution (SS):

[0157] Add 6 μL (10 mM in DMSO) of the test compound stock solution to 194 μL MeOH / ACN (4:1);

[0158] b) Plot the standard curve:

[0159] 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.

[0160] 6) Dilute the supernatant 10 times with 100mM buffer.

[0161] Add the supernatant (10 μL) to the buffer (100 mM, 90 μL) and dilute 10 times;

[0162] 7) Sample preparation for LC-MS / MS (API 4000) detection

[0163] 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.

[0164] Experimental results:

[0165] 4. Metabolic stability of mouse liver microsomes assay

[0166] 1) Preparation of experimental reagents

[0167] a) Phosphate-buffered saline (PBS): 100 mM, pH = 7.4 ± 0.1.

[0168] 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.

[0169] b) Microparticle working solution; 1.0 mg / mL.

[0170] 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.

[0171] c) NADPH regeneration system working solution: a mixture of 2 mM NADPH solution and 6 mM magnesium chloride solution

[0172] 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.

[0173] 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.)

[0174] 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.

[0175] d) Termination solution: Tolbutamide acetonitrile solution containing 200 ng / mL

[0176] 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.

[0177] 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.

[0178] e) Test / Reference Working Solution: 100 μM test / reference working solution

[0179] 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.

[0180] 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.

[0181] 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.

[0182] 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.

[0183] 2) Experimental Procedure

[0184] a) Prepare 8 incubation plates and name them T0, T5, T15, T30, T45, T60, Blank60 and NCF60 respectively.

[0185] 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.

[0186] c) Place T5, T15, T30, T45, T60, and Blank60 in a 37°C water bath for pre-incubation for 10 minutes.

[0187] 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.

[0188] 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.

[0189] 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.

[0190] 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.

[0191] 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.

[0192] 3. Data Processing:

[0193] (1) The formula for calculating half-life is as follows:

[0194] Experimental results:

[0195] 5. Mouse PK Assay

[0196] 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:

[0197] 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);

[0198] 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.

[0199] 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.

[0200] Experimental results:

[0201] 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: (1) In a solvent, the compound shown in Formula 3-1 was reacted under inorganic acid conditions and then condensed with KSCN to prepare the compound shown in Formula 7. (2) In a solvent and in the presence of an organic acid, the compound shown in Formula 7 undergoes a cyclization reaction with acetone-acetone to prepare the compound shown in Formula 8. (3) In a solvent, under the condition of a palladium catalyst, the compound shown in Formula 8 and the compound shown in Formula 9 were coupled to prepare the compound shown in Formula 10. The steps also include: (4) in a solvent, in the presence of an acid, the compound shown in Formula 10 undergoes a deprotection reaction to prepare the compound shown in Formula 11'; (5) In a solvent, in the presence of a condensing agent, the compound shown in Formula 11' and the compound shown in Formula 12 are subjected to an amidation reaction to prepare the compound shown in Formula 13. Or (4') in a solvent, in the presence of an acid, the compound shown in Formula 10 undergoes a deprotection reaction to prepare the compound shown in Formula 11, and then undergoes a neutralization reaction with an inorganic alkaline aqueous solution to obtain the compound shown in Formula 11. (5') In a solvent, in the presence of a condensing agent, the compound shown in Formula 11 is subjected to an amidation reaction with the compound shown in Formula 12 to prepare the compound shown in Formula 13. Wherein, R is H, Bu4Sn, ClZn or ClMg; when R is H, step (3) also includes a phosphine ligand, a palladium catalyst and a base.

2. The method for preparing the compound as shown in Formula 13 according to claim 1, characterized in that, Step (1) satisfies one or more of the following conditions: (1) In step (1), the solvent is a protic solvent, preferably water; (2) In step (1), the mass-volume ratio of the compound as shown in Formula 3-1 to the solvent is 1-5 g / mL, preferably 1.5-3 g / mL, for example 2 g / mL; (3) In step (1), the acid is an inorganic acid or an organic acid, preferably hydrochloric acid or sulfuric acid, and preferably p-toluenesulfonic acid; (4) In step (1), the concentration of hydrochloric acid is 2-12 mol / L; (5) In step (1), the mass-to-volume ratio of the compound as shown in Formula 3-1 to concentrated hydrochloric acid is 0.1-0.5 g / mL, preferably 0.15-0.3 g / mL, for example 0.2 g / mL; (6) In step (1), the reaction temperature is 60-100℃, preferably 70-100℃, for example 70-80℃; (7) In step (1), the reaction time is 1-10h, preferably 1.5-5h, for example 2h; (8) In the condensation reaction of step (1), the molar ratio of the compound shown in Formula 3-1 to the KSCN is 1:(2-8), preferably 1:(3-7), for example 1:4, 1:5, 1:6 or 1:7; (9) In the condensation reaction of step (1), the reaction temperature is 50-100℃, preferably 60-100℃, for example 60-85℃; (10) In the condensation reaction of step (1), the reaction time is 1-40 h, preferably 1-31 h, for example 2 h; and (11) The condensation reaction in step (1) also includes post-processing, such as filtration, washing, neutralization, filtration, washing and drying.

3. The method for preparing the compound as shown in Formula 13 according to claim 1, characterized in that, Step (2) satisfies one or more of the following conditions: (1) In the cyclization reaction of step (2), the solvent is an aromatic solvent or a hydrocarbon solvent, such as toluene or cyclohexane; (2) In the cyclization reaction of step (2), the mass-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 of step (2), 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 of step (2), 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 of step (2), 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 of step (2), the reaction temperature is 90-130℃, preferably 100-120℃, for example 100-110℃; (7) In the cyclization reaction of step (2), the reaction time is 1-24 hours, preferably 5-10 hours, for example 9 hours; and (8) The cyclization reaction in step (2) also includes post-processing steps, such as washing, concentration and column chromatography purification.

4. The method for preparing the compound as shown in Formula 13 according to claim 1, characterized in that, Step (3) satisfies one or more of the following conditions: (1) In the coupling reaction of step (3), 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 of step (3), the mass-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 of step (3), 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 of step (3), 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 of step (3), the palladium catalyst is one or more of palladium chloride, palladium di(acetonitrile) chloride, palladium dichlorobis(triphenylphosphine) palladium trifluoromethanesulfonate, 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) or palladium acetate, preferably palladium tetra(triphenylphosphine), palladium dichlorobis(triphenylphosphine) or palladium acetate; (6) In the coupling reaction of step (3), 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 of step (3), 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 of step (3), the molar ratio of the compound 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 of step (3), 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 of step (3), the reaction temperature is 90-160℃, preferably 100-150℃, for example 100-110℃, 110-120℃ or 150℃. (11) In the coupling reaction of step (3), the reaction time is 1-24 h, preferably 3-20 h, for example 15 h or 16 h; and (12) The coupling reaction in step (3) further includes a post-processing step, such as one or more of filtration, rinsing, extraction, column chromatography, concentration, crystallization or drying.

5. The method for preparing the compound as shown in Formula 13 according to claim 1, characterized in that, Step (4) satisfies one or more of the following conditions: (1) In the deprotection reaction of step (4), 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 sulfoxide solvent, preferably, the alcohol solvent can be methanol, the ether solvent is THF, and the sulfoxide solvent is DMSO; (2) In the deprotection reaction of step (4), the mass-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 of step (4), the acid is an inorganic acid, preferably concentrated hydrochloric acid or concentrated sulfuric acid, more preferably concentrated hydrochloric acid; (4) In the deprotection reaction of step (4), 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; (5) In the deprotection reaction of step (4), the reaction temperature is 40-100℃, preferably 70-95℃, for example 70-90℃; (6) In the deprotection reaction of step (4), the reaction time is 2-12 hours, preferably 3-6 hours, for example 3-5.5 hours; and (7) The deprotection reaction in step (4) also includes post-processing steps, such as filtration, rinsing, concentration, pulping, filtration, rinsing and drying. Alternatively, the deprotection reaction in step (4') may satisfy one or more of the following conditions: (1) In the deprotection reaction of step (4'), 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 sulfoxide solvent, preferably, the alcohol solvent can be methanol, the ether solvent is THF, and the sulfoxide solvent is DMSO; (2) In the deprotection reaction of step (4'), the mass-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 of step (4'), the acid is an inorganic acid, preferably concentrated hydrochloric acid or concentrated sulfuric acid, more preferably concentrated hydrochloric acid; (4) In the deprotection reaction of step (4'), 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; (5) In the deprotection reaction of step (4'), the reaction temperature is 40-100℃, preferably 70-95℃, for example 70-90℃; (6) In the deprotection reaction of step (4'), the reaction time is 2-12 hours, preferably 3-6 hours, for example 3-5.5 hours; and (7) The deprotection reaction in step (4') also includes post-processing steps, such as filtration, extraction, neutralization, rinsing, filtration and drying; Preferably, the neutralization reaction in step (4') satisfies one or more of the following conditions: (1) In the neutralization reaction of step (4'), the inorganic alkaline aqueous solution is an aqueous solution of sodium hydroxide; (2) In the neutralization reaction of step (4'), the mass fraction of the sodium hydroxide aqueous solution is 20-30%, preferably 25%.

6. The method for preparing the compound as shown in Formula 13 according to claim 1, characterized in that, Step (5) satisfies one or more of the following conditions: (1) In the amidation reaction of step (5), the solvent is a nitrile solvent or an amide solvent, preferably acetonitrile or DMF; (2) In the amidation reaction of step (5), the mass-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 of step (5), the condensing agent is a combination of N,N,N',N'-tetramethylchloromethamine hexafluorophosphate and 1-methylimidazole; (4) In the amidation reaction of step (5), 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 of step (5), the reaction temperature is 40-100℃, preferably 50-80℃, for example 60-75℃; (6) In the amidation reaction of step (5), the reaction time is 1-10 h, preferably 2-5 h, for example 3-4 h; and (7) The amidation reaction in step (5) also includes a post-treatment step, such as crystallization, filtration, washing and drying; Alternatively, step (5') satisfies one or more of the following conditions: (1) In the amidation reaction of step (5'), the solvent is a nitrile solvent or an amide solvent, preferably acetonitrile or DMF; (2) In the amidation reaction of step (5'), the mass-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 of step (5'), the condensing agent is a combination of T4P and DIPEA; (4) In the amidation reaction of step (5'), 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 of step (5'), the reaction temperature is 40-100℃, preferably 50-80℃, for example 60-75℃; (6) In the amidation reaction of step (5'), the reaction time is 1-10 h, preferably 2-5 h, for example 3-4 h; and (7) The amidation reaction in step (5') also includes a post-treatment step, such as crystallization, filtration, washing and drying; Preferably, the amidation reaction in step (5) satisfies one or more of the following conditions: (1) In the amidation reaction of step (5), the molar ratio of the compound as shown in Formula 11' to N,N,N',N'-tetramethylchloromethanemidane hexafluorophosphate (TCFH) is 1:(2-6), preferably 1:(3-5), for example 1:4.5; and (2) In the amidation reaction of step (5), the molar ratio of the compound as shown in Formula 11' to N-methylimidazole is 1:(5-12), preferably 1:(7-10), for example 1:9; Alternatively, the amidation reaction in step (5') may satisfy one or more of the following conditions: (1) In the amidation reaction of step (5'), 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 (2) In the amidation reaction of step (5'), the molar ratio of the compound as shown in Formula 11 to DIPEA is 1:(2-6), preferably 1:(3-5), for example 1:

4.

7. A method for preparing a compound as shown in Formula 7, characterized in that, It includes the following steps: In a solvent, the compound shown in Formula 3-1 reacts under inorganic acid conditions, followed by a condensation reaction with KSCN to prepare the compound shown in Formula 7. Preferably, the reaction conditions and operations of the method for preparing the compound as shown in Formula 7 are the same as those described in claim 2.

8. A method for preparing a compound as shown in Formula 8, characterized in that, It includes the following steps: (1) The compound shown in Formula 3-1 was reacted in a solvent under inorganic acid conditions and then condensed with KSCN to prepare the compound shown in Formula 7. (2) The compound shown in Formula 7 was prepared by cyclization reaction of the compound shown in Formula 7 with acetone-acetone in a solvent and in the presence of an organic acid to obtain the compound shown in Formula 8. Preferably, the reaction conditions and operating steps of step (1) are as described in claim 2; and the reaction conditions and operating steps of step (2) are as described in claim 3.

9. A method for preparing a compound as shown in Formula 10, characterized in that, It includes the following steps: (1) The compound shown in Formula 3-1 was reacted in a solvent under inorganic acid conditions and then condensed with KSCN to prepare the compound shown in Formula 7. (2) The compound shown in Formula 7 was prepared by cyclization reaction of the compound shown in Formula 7 with acetone-acetone in a solvent and in the presence of an organic acid to obtain the compound shown in Formula 8. (3) In a solvent, under the condition of a palladium catalyst, the compound shown in Formula 8 and the compound shown in Formula 9 were subjected to a substitution reaction to prepare the compound shown in Formula 10. Preferably, the reaction conditions and operating steps of step (1) are as described in claim 2; the reaction conditions and operating steps of step (2) are as described in claim 3; and the reaction conditions and operating steps of step (3) are as described in claim 4.

10. A method for preparing a compound as shown in Formula 11 or 11', characterized in that, It includes the following steps: (1) The compound shown in Formula 3-1 was reacted in a solvent under inorganic acid conditions and then condensed with KSCN to prepare the compound shown in Formula 7. (2) The compound shown in Formula 7 was prepared by cyclization reaction of the compound shown in Formula 7 with acetone-acetone in a solvent and in the presence of an organic acid to obtain the compound shown in Formula 8. (3) In a solvent, under the condition of a palladium catalyst, the compound shown in Formula 8 and the compound shown in Formula 9 were subjected to a substitution reaction to prepare the compound shown in Formula 10. (4) In a solvent, in the presence of an acid, the compound shown in Formula 10 is deprotected to prepare the compound shown in Formula 11 or 11'. Preferably, the conditions and operating steps of step (1) are as described in claim 2; the conditions and operating steps of step (2) are as described in claim 3; the conditions and operating steps of step (3) are as described in claim 4; and the conditions and operating steps of step (4) are as described in claim 5.

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