Pyrazolo [4, 3-d] pyrimidone derivative as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of medicines, and particularly discloses a tricyclic pyrazole [4, 3-d] pyrimidone derivative as well as a preparation method and application thereof. The method comprises the following steps: reacting 1-methyl-4-aminopyrazole-3-methyl formate serving as a raw material with phosphorus oxychloride serving as a catalyst, then reacting with equal amount of N-bromosuccinimide in an acetonitrile solution, then reacting with equal amount of 3, 4, 5-trimethoxybenzaldehyde and equal amount of p-toluenesulfonic acid in xylene, and finally reacting with an organic solvent, thereby obtaining the 4-aminopyrazole-3-methyl formate. And finally reacting with different boric acids in 1, 4-dioxane and water under the action of a catalytic amount of tetrakis (triphenylphosphine) palladium and excessive potassium phosphate to obtain different substituted derivatives (C1-C28). The 28 compounds are investigated, and results show that 8 compounds have certain inhibitory activity on human cervical cancer cells Hela, 17 compounds have certain inhibitory activity on colon cancer cells HT-29, and 4 compounds have certain inhibitory activity on gastric cancer cells HGC-27.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to a tricyclic pyrazole [4,3-d]pyrimidinone derivative, its preparation method, and its uses. Background Technology

[0002] Cancer, commonly known as malignant tumors, is a group of diseases that seriously threaten human life and health. The incidence and mortality rates of cancer are showing a continuous upward trend. Lung cancer remains the leading cause of cancer death, followed by colorectal cancer, liver cancer, stomach cancer, and breast cancer. Cancer has become the second leading cause of death after cardiovascular disease, placing enormous pressure on global public health systems. Therefore, the search for highly effective new anti-tumor drugs has become a very important and urgent task in current cancer treatment research.

[0003] Small molecule targeted antitumor agents, characterized by high targeting and significant efficacy, can improve patients' quality of life and have become a highly sought-after category in clinical drug use. In the medical field, saturated heterocyclic compounds, represented by piperidine and pyrimidine, are well-known and are currently the most representative saturated heterocyclic systems among all small molecule therapeutics approved by the US FDA. In specific experiments, introducing non-purely aromatic or isoaromatic cyclic systems such as thiophene, pyrrole, pyrazole, pyrimidine, and pyridine into molecules often yields unexpected active effects. Furthermore, this not only increases molecular diversity but also further enriches the structural novelty of these compounds. Therefore, the structure of drug molecules is gradually shifting towards the introduction of heterocycles, and saturated ring systems, represented by heterocycles, are attracting increasing attention.

[0004] Pyrimidine or pyrimidinone fragments, due to their unique structures, can form a wide variety of bases, making them an indispensable class of molecules in the life sciences. Combining pyrimidines with five-membered heterocycles can provide a broader range of nitrogen-containing groups in drug design and development.

[0005] Studies have shown that pyrazole[4,3-d]pyrazolopyrimidine ones possess antitumor activity. The search for and exploration of the synthetic methods and bioactivities of pyrazole[4,3-d]pyrazolopyrimidine ones has significant theoretical and practical value for the discovery of new drugs and lead compounds. Based on the analysis of relevant domestic and international patents and literature, and combined with the previous research work of our group, this invention modifies and transforms the structure of pyrazole[4,3-d]pyrazolopyrimidine ones by introducing different types of substituent groups to enhance their antitumor activity and drug-likeness. The inhibitory activity of these compounds against human cervical cancer cells (HeLa), colon cancer cells (HT-29), and gastric cancer cells (HGC-27) was investigated to discover highly effective and low-toxicity antitumor drug candidates.

[0006] This invention utilizes drug design principles to modify the substituents of pyrazole[4,3-d]pyrazolopyrimidinone compounds and further investigates their antitumor activity. Activity screening results showed that 8 compounds exhibited inhibitory activity against human cervical cancer cells (HeLa), 17 compounds exhibited inhibitory activity against human colon cancer cells (HT-29), and 4 compounds exhibited inhibitory activity against human gastric cancer cells (HGC-27). Summary of the Invention

[0007] The present invention aims to provide a tricyclic pyrazol[4,3-d]pyrimidinone derivative, its preparation method, and its uses. The compound is prepared by reacting methyl 1-methyl-4-aminopyrazol-3-carboxylate with 2-azhexanecycloone under phosphorus oxychloride catalysis to obtain 2-methyl-5,6,7,8-tetrahydropyrazolo[4,3-d]pyridin[1,2-a]pyrimidin-10(2H)-one (A), followed by overnight incubation with an equimolar amount of N-bromosuccinimide in acetonitrile solution at 80°C to obtain a monosubstituted 3-bromo-2-methyl-5,6 7,8-Tetrahydropyrazolo[4,3-d]pyrido[1,2-a]pyrimidin-10(2H)-one (B) was then refluxed with an equimolar amount of p-toluene in xylene to give (E)-3-bromo-2-methyl-5-(3,4,5-trimethoxybenzyl)-5,6,7,8-tetrahydropyrazolo[4,3-d]pyrido[1,2-a]pyrimidin-10(2H)-one (C). Finally, it was reacted with equimolar amounts of different types of borate compounds under triphenylphosphine palladium and potassium phosphate catalysis to give compounds C1-C28. The antitumor activity of the above compounds was investigated. The results showed that 8 compounds exhibited inhibitory activity against human cervical cancer cells (HeLa), 17 compounds exhibited inhibitory activity against human colon cancer cells (HT-29), and 4 compounds exhibited inhibitory activity against human gastric cancer cells (HGC-27).

[0008] The results showed that, using doxorubicin (DOX) as a positive control, a total of 8 compounds had IC50 values. 50 <50 μM, exhibiting inhibitory effects on human cervical cancer cells HeLa, with compounds C-5 and C-16 showing the best activity, IC50. 50 The concentrations were 29.17 μM and 32.36 μM, respectively; a total of 17 compounds had IC50 values. 50 <50μM, exhibiting inhibitory effects on human colon cancer cells HT-29, with compounds C-4, C-14, and C-24 showing the best activity, IC50 <50μM. 50 The concentrations were 15.64 μM, 17.11 μM, and 12.85 μM, respectively; there were a total of 4 compounds with IC50 values. 50 <50μM, exhibiting inhibitory effects on human gastric cancer cells HGC-27, with compound C-24 showing the best activity, IC50 <50μM.50 The value is 32.27 μM.

[0009] To achieve the above objectives, the basic solution provided by this invention is: a tricyclic pyrazole [4,3-d]pyrimidinone derivative, the structural formula of which is as follows: in: Compound C1 is (E)-2-methyl-3-(o-tolyl)-5-(3,4,5-trimethoxybenzylidene)-5,6,7,8-tetrahydropyrazolo[4,3-d]pyridolo[1,2-a]pyrimidin-10(2H)-one; Compound C2 is (E)-2-methyl-3-(m-tolyl)-5-(3,4,5-trimethoxybenzylidene)-5,6,7,8-tetrahydropyrazolo[4,3-d]pyrido[1,2-a]pyrimidin-10(2H)-one; Compound C3 is (E)-3-(4-methoxyphenyl)-2-methyl-5-(3,4,5-trimethoxybenzyl)-5,6,7,8-tetrahydropyrazolo[4,3-d]pyrido[1,2-a]pyrimidine-10(2H)-one; Compound C4 is (E)-2-methyl-3-(naphth-1-yl)-5-(3,4,5-trimethoxybenzylidene)-5,6,7,8-tetrahydropyrazolo[4,3-d]pyrido[1,2-a]pyrimidin-10(2H)-one; Compound C5 is (E)-2-methyl-3-(4-phenoxyphenyl)-5-(3,4,5-trimethoxybenzyl)-5,6,7,8-tetrahydropyrazolo[4,3-d]pyrido[1,2-a]pyrimidine-10(2H)-one; Compound C6 is (E)-3-(4-(tert-butyl)phenyl)-2-methyl-5-(3,4,5-trimethoxybenzyl)-5,6,7,8-tetrahydropyrazolo[4,3-d]pyrido[1,2-a]pyrimidin-10(2H)-one; Compound C7 is (E)-2-methyl-3-phenyl-5-(3,4,5-trimethoxybenzyl)-5,6,7,8-tetrahydropyrazolo[4,3-d]pyrido[1,2-a]pyrimidin-10(2H)-one; Compound C8 is (E)-3-(2-methoxynaphth-1-yl)-2-methyl-5-(3,4,5-trimethoxybenzylmethyl)-5,6,7,8-tetrahydropyrazolo[4,3-d]pyridolo[1,2-a]pyrimidin-10(2H)-one; Compound C9 is (E)-2-methyl-5-(3,4,5-trimethoxybenzyl)-3-vinyl-5,6,7,8-tetrahydropyrazolo[4,3-d]pyridolo[1,2-a]pyrimidin-10(2H)-one; Compound C10 is (E)-2-methyl-3-(p-tolyl)-5-(3,4,5-trimethoxybenzylidene)-5,6,7,8-tetrahydropyrazolo[4,3-d]pyrido[1,2-a]pyrimidin-10(2H)-one; Compound C11 is (E)-3-(3-chlorophenyl)-2-methyl-5-(3,4,5-trimethoxybenzyl)-5,6,7,8-tetrahydropyrazolo[4,3-d]pyrido[1,2-a]pyrimidin-10(2H)-one; Compound C12 is (E)-3-(3-chloro-4-fluorophenyl)-2-methyl-5-(3,4,5-trimethoxybenzyl)-5,6,7,8-tetrahydropyrazolo[4,3-d]pyrido[1,2-a]pyrimidin-10(2H)-one; Compound C13 is (E)-3-(2-aminophenyl)-2-methyl-5-(3,4,5-trimethoxybenzyl)-5,6,7,8-tetrahydropyrazolo[4,3-d]pyrido[1,2-a]pyrimidine-10(2H)-one; Compound C14 is (E)-3-(2-bromophenyl)-2-methyl-5-(3,4,5-trimethoxybenzyl)-5,6,7,8-tetrahydropyrazolo[4,3-d]pyrido[1,2-a]pyrimidine-10(2H)-one; Compound C15 is (E)-3-(4-isopropylphenyl)-2-methyl-5-(3,4,5-trimethoxybenzyl)-5,6,7,8-tetrahydropyrazolo[4,3-d]pyrido[1,2-a]pyrimidin-10(2H)-one; Compound C16 is (E)-3-(3-acetylphenyl)-2-methyl-5-(3,4,5-trimethoxybenzyl)-5,6,7,8-tetrahydropyrazolo[4,3-d]pyrido[1,2-a]pyrimidine-10(2H)-one; Compound C17 is (E)-3-(4-acetylphenyl)-2-methyl-5-(3,4,5-trimethoxybenzyl)-5,6,7,8-tetrahydropyrazolo[4,3-d]pyrido[1,2-a]pyrimidin-10(2H)-one; Compound C18 is (E)-3-(5-chloro-2-methoxyphenyl)-2-methyl-5-(3,4,5-trimethoxybenzyl)-5,6,7,8-tetrahydropyrazolo[4,3-d]pyrido[1,2-a]pyrimidine-10(2H)-one; Compound C19 is (E)-3-(2-chlorophenyl)-2-methyl-5-(3,4,5-trimethoxybenzyl)-5,6,7,8-tetrahydropyrazolo[4,3-d]pyrido[1,2-a]pyrimidine-10(2H)-one; Compound C20 is (E)-2-methyl-3-(4-nitrophenyl)-5-(3,4,5-trimethoxybenzyl)-5,6,7,8-tetrahydropyrazolo[4,3-d]pyrido[1,2-a]pyrimidine-10(2H)-one; Compound C21 is (E)-2-methyl-3-(pyridin-4-yl)-5-(3,4,5-trimethoxybenzyl)-5,6,7,8-tetrahydropyrazolo[4,3-d]pyridolo[1,2-a]pyrimidin-10(2H)-one; Compound C22 is (E)-4-(2-methyl-10-oxo-5-(3,4,5-trimethoxybenzyl)-2,5,6,7,8,10-hexahydropyrazolo[4,3-d]pyridolo[1,2-a]pyrimidin-3-yl)benzamide; Compound C23 is (E)-3-(3,5-dimethoxyphenyl)-2-methyl-5-(3,4,5-trimethoxybenzyl)-5,6,7,8-tetrahydropyrazolo[4,3-d]pyridolo[1,2-a]pyrimidin-10(2H)-one; Compound C24 is (E)-2-methyl-3-(2-(trifluoromethyl)phenyl)-5-(3,4,5-trimethoxybenzylidene)-5,6,7,8-tetrahydropyrazolo[4,3-d]pyrido[1,2-a]pyrimidin-10(2H)-one; Compound C25 is (E)-3-(4-cyclohexylphenyl)-2-methyl-5-(3,4,5-trimethoxybenzyl)-5,6,7,8-tetrahydropyrazolo[4,3-d]pyridolo[1,2-a]pyrimidin-10(2H)-one; Compound C26 is (E)-N-(4-(2-methyl-10-oxo-5-(3,4,5-trimethoxybenzylmethyl)-2,5,6,7,8,10-hexahydropyrazolo[4,3-d]pyrido[1,2-a]pyrimidin-3-yl)phenyl)acetamide; Compound C27 is (E)-3-(4-hydroxyphenyl)-2-methyl-5-(3,4,5-trimethoxybenzyl)-5,6,7,8-tetrahydropyrazolo[4,3-d]pyrido[1,2-a]pyrimidin-10(2H)-one; Compound C28 is (E)-3-(isoquinolin-6-yl)-2-methyl-5-(3,4,5-trimethoxybenzylmethyl)-5,6,7,8-tetrahydropyrazolo[4,3-d]pyrido[1,2-a]pyrimidin-10(2H)-one.

[0010] Use of compounds C2, C5, C9, C12, C16, C20, C24 or C26 of a tricyclic pyrazolopyrimidine ketone derivative in the treatment of human cervical cancer cells HeLa.

[0011] Use of compounds C1, C2, C4, C6, C7, C8, C10, C11, C12, C13, C14, C15, C19, C20, C23, C24 or C25 of a tricyclic pyrazolopyrimidine ketone derivative in the treatment of HT-29 colon cancer cells.

[0012] Use of compounds C7, C8, C13 or C24 from a tricyclic pyrazolopyrimidinone derivative in the treatment of gastric cancer cells HGC-27.

[0013] A method for preparing a tricyclic pyrazole [4,3-d]pyrimidinone derivative, comprising the following steps: Preparation of compound A: 5.0 g (32.2 mmol) of methyl 1-methyl-4-aminopyrazole-3-carboxylate was dissolved in 64 mL of anhydrous 1,4-dioxane. Then, 6.0 mL (64.5 mmol) of phosphorus oxychloride was slowly added dropwise. The mixture was heated to 80 °C and stirred for 30 min. Then, 6.4 g (64.5 mmol) of 2-azhexanecycloone was added, and the mixture was heated to 110 °C and refluxed for 12 h. After the TLC reactants were fully reacted, the reaction solution was cooled to room temperature and then quenched in an ice bath with 60 mL of water. NaOH aqueous solution was slowly added to adjust the pH to 7, followed by extraction with dichloromethane (30 mL × 3). The combined organic phases were washed with 50 mL of saturated brine, dried over anhydrous sodium sulfate, concentrated, and the dried product was slurried with 80 mL of petroleum ether and filtered. The product was washed with a mixed solution at -20 °C (PE:EA = 30:1) to obtain 6.07 g of pale yellow solid A (2-methyl-5,6,7,8-tetrahydropyrazolo[4,3-d]pyridin[1,2-a]pyrimidine-10(2H)-one), with a yield of 92.4%.

[0014] Preparation of compound B: 5.0 g (24.5 mmol) of 2-methyl-5,6,7,8-tetrahydropyrazolo[4,3-d]pyrido[1,2-a]pyrimidin-10(2H)-one (compound A) was dissolved in 50 mL of 1,2-dichloroethane. 4.4 g (24.7 mmol) of NBS was added in portions, and the mixture was heated to 80 °C and reacted for 6 h. After the reaction was complete by TLC, 50 mL of water was added, followed by dichloromethane (30 mL × 3). The mixture was extracted separately, and the combined organic phases were washed with 50 mL of saturated brine, dried over anhydrous sodium sulfate, concentrated, and dried further. The mixture was then slurried in 50 mL of -20 °C tetrahydrofuran and filtered to obtain 6.72 g of a grayish-white solid B (3-bromo-2-methyl-5,6,7,8-tetrahydropyrazolo[4,3-d]pyrido[1,2-a]pyrimidin-10(2H)-one), with a yield of 97%.

[0015] Preparation of compound C: Compound B (6.0 g, 21.2 mmol of 3-bromo-2-methyl-5,6,7,8-tetrahydropyrazolo[4,3-d]pyrido[1,2-a]pyrimidin-10(2H)-one), 8.3 g, 42.4 mmol of 3,4,5-trimethoxybenzaldehyde, and 4.1 g, 21.2 mmol of p-toluenesulfonic acid were added to 40 mL of xylene and refluxed at 140 °C for 12 h. After the TLC reaction was complete, 40 mL of water and 20 mL of dichloromethane were added to completely dissolve the reactants. Then, 30 mL of dichloromethane (30 mL × 3) was added for extraction. The combined organic phases were washed with 50 mL of saturated brine, dried over anhydrous sodium sulfate, concentrated, and dried. After drying, 50 mL of -20℃ methanol was added and the mixture was stirred. The mixture was then filtered to obtain a yellow solid. The solid was purified by silica gel column chromatography (DCM:MeOH = 40:1) to obtain 8.80 g of a pale yellow solid C ((E)-3-bromo-2-methyl-5-(3,4,5-trimethoxybenzyl)-5,6,7,8-tetrahydropyrazolo[4,3-d]pyrido[1,2-a]pyrimidin-10(2H)-one, with a yield of 90.0%.

[0016] Preparation of compounds C1-C28: The prepared (E)-3-bromo-2-methyl-5-(3,4,5-trimethoxybenzyl)-5,6,7,8-tetrahydropyrazolo[4,3-d]pyridolo[1,2-a]pyrimidin-10(2H)-one (compound C) was added sequentially to a 25 mL dry reaction tube. Argon gas was purged 2-3 times, and 1.2 mL of a 1,4-dioxane:water mixture was added. The reaction was carried out at 100 °C for 8 h. After the reactants were completely reacted by TLC, the mixture was directly evaporated to dryness and then subjected to silica gel column chromatography (DCM:MeOH = 120:1) to give yellow or pale yellow solids C1-C28, with yields ranging from 40% to 90%.

[0017] The present invention discloses a tricyclic pyrazol[4,3-d]pyrimidinone derivative, wherein the compound is prepared by reacting methyl 1-methyl-4-aminopyrazol-3-carboxylate with 2-azhexanecycloone under phosphorus oxychloride catalysis to obtain 2-methyl-5,6,7,8-tetrahydropyrazolo[4,3-D]pyridin[1,2-a]pyrimidin-10(2H)-one (A), followed by reaction with an equimolar amount of N-bromosuccinimide in acetonitrile solution to obtain monosubstituted 3-bromo-2-methyl-5,6,7,8-tetrahydropyrazolo[4,3-D]pyridin[1,2-a]pyrimidin-10(2H)-one. [4,3-d]pyrido[1,2-a]pyrimidin-10(2H)-one (B) was then refluxed with an equimolar amount of p-toluene in xylene to give (E)-3-bromo-2-methyl-5-(3,4,5-trimethoxybenzyl)-5,6,7,8-tetrahydropyrazolo[4,3-d]pyrido[1,2-a]pyrimidin-10(2H)-one (C), which was then reacted with equimolar amounts of different types of borate compounds under triphenylphosphine palladium and potassium phosphate catalysis to give compounds C1-C28. The synthetic route is shown below: In vitro activity screening results for this class of compounds showed that, using doxorubicin (DOX) as a positive control, a total of 8 compounds at 10 μM were effective. <IC 50 <50 μM, exhibiting inhibitory effects on human cervical cancer cells HeLa; a total of 17 compounds at 10 μM... <IC 50 <50 μM, exhibiting inhibitory effects on human colon cancer cells HT-29; a total of 4 compounds at 10 μM. <IC 50 <50μM, exhibiting inhibitory effect on human gastric cancer cells HGC-27. Detailed Implementation

[0018] The present invention will be further described in detail below through specific embodiments: Reagents: All reagents were commercially available analytical grade.

[0019] Example 1 5.0 g (32.2 mmol) of methyl 1-methyl-4-aminopyrazole-3-carboxylate was dissolved in 64 mL of anhydrous 1,4-dioxane. Then, 6.0 mL (64.5 mmol) of phosphorus oxychloride was slowly added dropwise. The mixture was heated to 80 °C and stirred for 30 min. Then, 6.4 g (64.5 mmol) of 2-azhexanecycloone was added, and the mixture was heated to 110 °C and refluxed for 12 h. After the TLC reactants were fully reacted, the reaction solution was cooled to room temperature and then quenched in an ice bath with 60 mL of water. NaOH aqueous solution was slowly added to adjust the pH to 7, followed by extraction with dichloromethane (30 mL × 3). The combined organic phases were washed with 50 mL of saturated brine, dried over anhydrous sodium sulfate, concentrated, and the dried product was slurried with 80 mL of petroleum ether and filtered. The product was washed with a mixed solution at -20 °C (PE:EA = 30:1) to obtain 6.07 g of pale yellow solid A (2-methyl-5,6,7,8-tetrahydropyrazolo[4,3-D]pyridin[1,2-a]pyrimidine-10(2H)-one), yield 92.4%, melting point: 228.3-229.1 °C.

[0020] 1 H NMR (400 MHz, CDCl3)δ 7.68 (s, 1H), 4.09 (m, 5H), 2.88 (t, J = 5.2Hz, 2H), 2.00 - 1.81 (m, 4H). HRMS (ESI) m / z: 205.2410.

[0021] Example 2 5.0 g (24.5 mmol) of 2-methyl-5,6,7,8-tetrahydropyrazolo[4,3-D]pyridin[1,2-a]pyrimidin-10(2H)-one (compound A) was dissolved in 50 mL of 1,2-dichloroethane. 4.4 g (24.7 mmol) of NBS was added in portions, and the mixture was heated to 80 °C and reacted for 6 h. After the TLC feedstock was completely reacted, 50 mL of water was added, followed by dichloromethane (30 mL × 3). The mixture was extracted separately, and the combined organic phases were washed with 50 mL of saturated brine, dried over anhydrous sodium sulfate, concentrated, and dried further. After slurrying with 50 mL of tetrahydrofuran at -20℃, the mixture was filtered to obtain 6.72 g of grayish-white solid B (3-bromo-2-methyl-5,6,7,8-tetrahydropyrazolo[4,3-d]pyrido[1,2-a]pyrimidine-10(2H)-one), yield 97%, melting point: 170.5-172.6℃.

[0022] 1H NMR (400 MHz, CDCl3) δ 4.10 (s, 5H), 2.96 (q, J = 6.6 Hz, 2H), 2.00- 1.85 (m, 4H). HRMS (ESI) m / z: 285.1451.

[0023] Example 3 Compound B (6.0 g, 21.2 mmol of 3-bromo-2-methyl-5,6,7,8-tetrahydropyrazolo[4,3-d]pyrido[1,2-a]pyrimidin-10(2H)-one), 8.3 g, 42.4 mmol of 3,4,5-trimethoxybenzaldehyde, and 4.1 g, 21.2 mmol of p-toluenesulfonic acid were added to 40 mL of xylene and refluxed at 140 °C for 12 h. After the TLC reaction was complete, 40 mL of water and 20 mL of dichloromethane were added to completely dissolve the reactants. Then, 30 mL of dichloromethane (30 mL × 3) was added for extraction. The combined organic phases were washed with 50 mL of saturated brine, dried over anhydrous sodium sulfate, concentrated, and dried again. After slurrying with 50 mL of -20℃ methanol, the mixture was filtered to obtain a yellow solid. The solid was purified by silica gel column chromatography (DCM:MeOH = 40:1) to obtain 8.80 g of a pale yellow solid C ((E)-3-bromo-2-methyl-5-(3,4,5-trimethoxybenzyl)-5,6,7,8-tetrahydropyrazolo[4,3-d]pyridolo[1,2-a]pyrimidin-10(2H)-one, yield 90.0%, melting point: 182.5-185.2℃.

[0024] 1 H NMR (400 MHz, CDCl3) δ 7.99 (s, 1H), 6.68 (s, 2H), 4.19 - 4.09 (m,5H), 3.89 (d, 9H), 2.94 (td, J = 6.7, 2.2 Hz, 2H), 1.98 (m, 2H). HRMS (ESI)m / z:463.0743.

[0025] Example 4 The prepared (E)-3-bromo-2-methyl-5-(3,4,5-trimethoxybenzyl)-5,6,7,8-tetrahydropyrazolo[4,3-d]pyridolo[1,2-a]pyrimidin-10(2H)-one (compound C) 30.0 mg, 0.065 mmol; tetrakis(triphenylphosphine)-palladium phosphate 3.8 mg, 3.25 μmol; potassium phosphate 27.5 mg, 0.13 mmol; and 2-methylphenylboronic acid (10.6 mg, 0.078 mmol) were sequentially added to a 25 mL dry reaction tube. Argon gas was purged 2-3 times, and 1.2 mL of a 1,4-dioxane:water = 1:1 mixed solvent was added. The reaction was carried out at 100 °C for 8 h. After the TLC reaction was complete, the sample was directly evaporated to dryness and then subjected to silica gel column chromatography (DCM:MeOH = 120:1) to give 25.7 mg of a pale yellow solid C1(E)-2-methyl-3-(o-tolyl)-5-(3,4,5-trimethoxybenzyl)-5,6,7,8-tetrahydropyrazolo[4,3-d]pyrido[1,2-a]pyrimidine-10(2H)-one, yield 84.0%, melting point: 244.1-245.4℃.

[0026] 1 H NMR (400 MHz, CDCl3) δ 7.80 (d, J = 2.2 Hz, 1H), 7.46 - 7.37 (m,2H), 7.37 - 7.29 (m, 2H), 6.60 (s, 2H), 4.30 (m, 1H), 4.09 (m, 1H), 3.95 (s, 3H), 3.86 (d, J = 5.8 Hz, 9H), 2.91 (td, J = 6.6, 2.1 Hz, 2H), 2.27 (s, 3H), 2.01 (m, 2H). HRMS (ESI) m / z: 473.2188.

[0027] Example 5 The prepared (E)-3-bromo-2-methyl-5-(3,4,5-trimethoxybenzyl)-5,6,7,8-tetrahydropyrazolo[4,3-d]pyridolo[1,2-a]pyrimidin-10(2H)-one (compound C) 30.0 mg, 0.065 mmol; tetrakis(triphenylphosphine)-palladium phosphate 3.8 mg, 3.25 μmol; potassium phosphate 27.5 mg, 0.13 mmol; and 3-methylphenylboronic acid (10.6 mg, 0.078 mmol) were sequentially added to a 25 mL dry reaction tube. Argon gas was purged 2-3 times, and 1.2 mL of a 1,4-dioxane:water = 1:1 mixed solvent was added. The reaction was carried out at 100 °C for 8 h. After the TLC reaction was complete, the sample was directly evaporated to dryness and then subjected to silica gel column chromatography (DCM:MeOH = 120:1) to give 25.1 mg of a pale yellow solid C2(E)-2-methyl-3-(m-tolyl)-5-(3,4,5-trimethoxybenzyl)-5,6,7,8-tetrahydropyrazolo[4,3-d]pyridolo[1,2-a]pyrimidine-10(2H)-one, yield 82.0%, melting point: 205.6-208.5℃.

[0028] 1 H NMR (400 MHz, CDCl3) δ7.89 (d, J = 2.1 Hz, 1H), 7.51 (d, J = 1.7Hz, 1H), 7.49 - 7.41 (m, 2H), 7.32 - 7.27 (m, 1H), 6.63 (s, 2H), 4.23 - HRMS (ESI) m / z:473.2183.

[0029] Example 6 The prepared (E)-3-bromo-2-methyl-5-(3,4,5-trimethoxybenzyl)-5,6,7,8-tetrahydropyrazolo[4,3-d]pyridolo[1,2-a]pyrimidin-10(2H)-one (compound C) 30.0 mg, 0.065 mmol; tetrakis(triphenylphosphine)-palladium phosphate 3.8 mg, 3.25 μmol; potassium phosphate 27.5 mg, 0.13 mmol; and 4-methoxyphenylboronic acid (11.9 mg, 0.078 mmol) were sequentially added to a 25 mL dry reaction tube. Argon gas was purged 2-3 times, and 1.2 mL of a 1,4-dioxane:water = 1:1 mixed solvent was added. The reaction was carried out at 100 °C for 8 h. After the reactants were completely reacted by TLC, the solution was directly evaporated to dryness and then subjected to silica gel column chromatography (DCM:MeOH = 120:1) to give 22.8 mg of a pale yellow solid C3(E)-3-(4-methoxyphenyl)-2-methyl-5-(3,4,5-trimethoxybenzyl)-5,6,7,8-tetrahydropyrazolo[4,3-d]pyridolo[1,2-a]pyrimidine-10(2H)-one, yield 72%, melting point: 217.6-219.5℃.

[0030] 1 H NMR (400 MHz, CDCl3) δ 7.87 (d, J = 2.3 Hz, 1H), 7.62 (d, J = 8.6Hz, 2H), 7.12 - 7.05 (m, 2H), 6.63 (s, 2H), 4.22 - 4.17 (m, 2H), 4.16 (s,3H), 3.91 - 3.85 (m, 12H), 2.92 (s, 2H), 2.01 (p, J = 6.3 Hz, 2H).HRMS (ESI)m / z:489.2131.

[0031] Example 7 The prepared (E)-3-bromo-2-methyl-5-(3,4,5-trimethoxybenzyl)-5,6,7,8-tetrahydropyrazolo[4,3-d]pyridolo[1,2-a]pyrimidin-10(2H)-one (compound C) 30.0 mg, 0.065 mmol; tetra-triphenylphosphine palladium 3.8 mg, 3.25 μmol; potassium phosphate 27.5 mg, 0.13 mmol; and 1-naphthoboric acid (13.5 mg, 0.078 mmol) were sequentially added to a 25 mL dry reaction tube. Argon gas was purged 2-3 times, and 1.2 mL of a 1,4-dioxane:water = 1:1 mixed solvent was added. The reaction was carried out at 100 °C for 8 h. After the TLC reaction was complete, the sample was directly evaporated to dryness and then subjected to silica gel column chromatography (DCM:MeOH = 120:1) to give 22.5 mg of a pale yellow solid, C4(E)-2-methyl-3-(naphth-1-yl)-5-(3,4,5-trimethoxybenzyl)-5,6,7,8-tetrahydropyrazolo[4,3-d]pyrido[1,2-a]pyrimidin-10(2H)-one, yield 68.1%, melting point: 195.3-197.8℃.

[0032] 1 H NMR (400 MHz, CDCl3) δ 8.04 (d, J = 8.0 Hz, 1H), 7.97 (d, J = 8.0Hz, 1H), 7.69 (d, J = 2.2 Hz, 1H), 7.66 - 7.58 (m, 2H), 7.58 - 7.48 (m, 3H), 6.53 (s, 2H), 4.29 (s, 1H), 4.16 (s, 1H), 3.91 (s, 3H), 3.83 (d, J = 12.3 Hz, 9H), 2.90 (s, 2H), 2.02 (dt, J = 9.0, 5.2 Hz, 2H).HRMS (ESI) m / z:509.2185.

[0033] Example 8 The prepared (E)-3-bromo-2-methyl-5-(3,4,5-trimethoxybenzyl)-5,6,7,8-tetrahydropyrazolo[4,3-d]pyridolo[1,2-a]pyrimidin-10(2H)-one (compound C) 30.0 mg, 0.065 mmol; tetratriphenylphosphine palladium 3.8 mg, 3.25 μmol; potassium phosphate 27.5 mg, 0.13 mmol; and 4-phenoxyphenylboronic acid (16.7 mg, 0.078 mmol) were sequentially added to a 25 mL dry reaction tube. Argon gas was purged 2-3 times, and 1.2 mL of a 1,4-dioxane:water = 1:1 mixed solvent was added. The reaction was carried out at 100 °C for 8 h. After the TLC reaction was complete, the sample was directly evaporated to dryness and then subjected to silica gel column chromatography (DCM:MeOH = 120:1) to give 20.0 mg of a pale yellow solid, C5(E)-2-methyl-3-(4-phenoxyphenyl)-5-(3,4,5-trimethoxybenzyl)-5,6,7,8-tetrahydropyrazolo[4,3-d]pyrido[1,2-a]pyrimidine-10(2H)-one, yield 56%, melting point: 167.5-169.1℃.

[0034] 1 H NMR (400 MHz, CDCl3)δ 7.86 (d, J = 2.1 Hz, 1H), 7.69 - 7.62 (m,2H), 7.43 - 7.35 (m, 2H), 7.21 - 7.08 (m, 5H), 6.63 (s, 2H), 4.19 (s, 5H), 3.88 (d, J = 5.1 Hz, 9H), 2.96 - 2.88 (m, 2H), 2.01 (p, J = 6.4 Hz, 2H). HRMS(ESI) m / z: 551.2289.

[0035] Example 9 The prepared (E)-3-bromo-2-methyl-5-(3,4,5-trimethoxybenzyl)-5,6,7,8-tetrahydropyrazolo[4,3-d]pyridolo[1,2-a]pyrimidin-10(2H)-one (compound C) 30.0 mg, 0.065 mmol; tetrakis(triphenylphosphine)-palladium phosphate 3.8 mg, 3.25 μmol; potassium phosphate 27.5 mg, 0.13 mmol; and 4-tert-butylphenylboronic acid (13.8 mg, 0.078 mmol) were sequentially added to a 25 mL dry reaction tube. Argon gas was purged 2-3 times, and 1.2 mL of a 1,4-dioxane:water = 1:1 mixed solvent was added. The reaction was carried out at 100 °C for 8 h. After the TLC reaction was complete, the sample was directly evaporated to dryness and then subjected to silica gel column chromatography (DCM:MeOH = 120:1) to give 25.1 mg of a pale yellow solid C6(E)-3-(4-(tert-butyl)phenyl)-2-methyl-5-(3,4,5-trimethoxybenzyl)-5,6,7,8-tetrahydropyrazolo[4,3-d]pyridolo[1,2-a]pyrimidin-10(2H)-one, yield 75.1%, melting point: 205.1-205.7℃.

[0036] 1 H NMR (400 MHz, CDCl3)δ7.87 (d, J = 2.2 Hz, 1H), 7.67 - 7.61 (m, 2H), 7.60 - 7.54 (m, 2H), 6.63 (s, 2H), 4.22 - 4.18 (m, 5H), 3.88 (d, J = 3.7 Hz, 9H), 3.70 (d, J = 0.5 Hz, 3H), 2.91 (td, J = 6.6, 2.1 Hz, 2H), 2.01 (p, J =6.4 Hz, 2H), 1.38 (s, 9H). HRMS (ESI) m / z: 515.2653.

[0037] Example 10 The prepared (E)-3-bromo-2-methyl-5-(3,4,5-trimethoxybenzyl)-5,6,7,8-tetrahydropyrazolo[4,3-d]pyridolo[1,2-a]pyrimidin-10(2H)-one (compound C) 30.0 mg, 0.065 mmol; tetrakis(triphenylphosphine)-palladium phosphate 3.8 mg, 3.25 μmol; potassium phosphate 27.5 mg, 0.13 mmol; and phenylboronic acid (9.5 mg, 0.078 mmol) were sequentially added to a 25 mL dry reaction tube. Argon gas was purged 2-3 times, and 1.2 mL of a 1,4-dioxane:water = 1:1 mixed solvent was added. The reaction was carried out at 100 °C for 8 h. After the TLC reaction was complete, the sample was directly evaporated to dryness and then subjected to silica gel column chromatography (DCM:MeOH = 120:1) to give 26.9 mg of a pale yellow solid, C7(E)-2-methyl-3-phenyl-5-(3,4,5-trimethoxybenzyl)-5,6,7,8-tetrahydropyrazolo[4,3-d]pyrido[1,2-a]pyrimidine-10(2H)-one, with a yield of 90.4% and a melting point of 261.8-263.1 °C.

[0038] 1 H NMR (400 MHz, CDCl3)δ 7.90 - 7.86 (m, 1H), 7.73 - 7.66 (m, 2H), 7.59 - 7.52 (m, 2H), 7.51 - 7.45 (m, 1H), 6.63 (s, 2H), 4.19 (s, 5H), 3.87(d, J = 4.9 Hz, 9H), 2.92 (td, J = 6.6, 2.1 Hz, 2H), 2.06 - 1.94 (m, 2H).HRMS(ESI) m / z:459.2024.

[0039] Example 11 The prepared (E)-3-bromo-2-methyl-5-(3,4,5-trimethoxybenzyl)-5,6,7,8-tetrahydropyrazolo[4,3-d]pyridolo[1,2-a]pyrimidin-10(2H)-one (compound C) 30.0 mg, 0.065 mmol; tetratriphenylphosphine palladium 3.8 mg, 3.25 μmol; potassium phosphate 27.5 mg, 0.13 mmol; 2-methoxy-1-naphthylboronic acid (15.7 mg, 0.078 mmol) were sequentially added to a 25 mL dry reaction tube, purged with argon gas 2-3 times, and then 1.2 mL of a 1,4-dioxane:water = 1:1 mixed solvent was added. The reaction was carried out at 100 °C for 8 h. After the TLC reaction was complete, the sample was directly evaporated to dryness and then subjected to silica gel column chromatography (DCM:MeOH = 120:1) to give 31.9 mg of a pale yellow solid C8(E)-3-(2-methoxynaphth-1-yl)-2-methyl-5-(3,4,5-trimethoxybenzylmethyl)-5,6,7,8-tetrahydropyrazolo[4,3-d]pyrido[1,2-a]pyrimidin-10(2H)-one, yield 91.3%, melting point: 106.9-109.4℃.

[0040] 1 H NMR (400 MHz, CDCl3)δ 8.04 (d, J = 9.1 Hz, 1H), 7.87 (dd, J = 7.4,1.7 Hz, 1H), 7.60 - 7.56 (m, 1H), 7.45 - 7.34 (m, 4H), 6.49 (s, 2H), HRMS (ESI) m / z:539.2289.

[0041] Example 12 The prepared (E)-3-bromo-2-methyl-5-(3,4,5-trimethoxybenzyl)-5,6,7,8-tetrahydropyrazolo[4,3-d]pyridolo[1,2-a]pyrimidin-10(2H)-one (compound C) 30.0 mg, 0.065 mmol; tetrakis(triphenyl)phosphine-palladium 3.8 mg, 3.25 μmol; potassium phosphate 27.5 mg, 0.13 mmol; and vinylboronic acid pinacol ester (12.0 mg, 0.078 mmol) were sequentially added to a 25 mL dry reaction tube. Argon gas was purged 2-3 times, and 1.2 mL of a 1,4-dioxane:water = 1:1 mixed solvent was added. The reaction was carried out at 100 °C for 8 h. After the TLC reaction was complete, the sample was directly evaporated to dryness and then subjected to silica gel column chromatography (DCM:MeOH = 120:1) to give 12.6 mg of a pale yellow solid, C9(E)-2-methyl-5-(3,4,5-trimethoxybenzyl)-3-vinyl-5,6,7,8-tetrahydropyrazolo[4,3-d]pyrido[1,2-a]pyrimidine-10(2H)-one, yield 47.4%, melting point: 158.7-159.8 °C.

[0042] 1 H NMR (400 MHz, CDCl3)δ 7.98 (d, J = 2.2 Hz, 1H), 6.83 (dd, J = 17.5,11.4 Hz, 1H), 6.72 - 6.63 (m, 3H), 5.69 (dd, J = 11.4, 1.7 Hz, 1H), 4.22 -4.14 (m, 2H), 4.13 (s, 3H), 3.90 (d, J = 1.0 Hz, 9H), 2.94 (td, J = 6.5, 1.9Hz, 2H), 2.06 - 1.96 (m, 2H).HRMS (ESI) m / z:409.1871.

[0043] Example 13 The prepared (E)-3-bromo-2-methyl-5-(3,4,5-trimethoxybenzyl)-5,6,7,8-tetrahydropyrazolo[4,3-d]pyridolo[1,2-a]pyrimidin-10(2H)-one (compound C) 30.0 mg, 0.065 mmol; tetrakis(triphenylphosphine)-palladium phosphate 3.8 mg, 3.25 μmol; potassium phosphate 27.5 mg, 0.13 mmol; and 4-methylphenylboronic acid (10.6 mg, 0.078 mmol) were sequentially added to a 25 mL dry reaction tube. Argon gas was purged 2-3 times, and 1.2 mL of a 1,4-dioxane:water = 1:1 mixed solvent was added. The reaction was carried out at 100 °C for 8 h. After the TLC reaction was complete, the sample was directly evaporated to dryness and then subjected to silica gel column chromatography (DCM:MeOH = 120:1) to give 26.9 mg of a pale yellow solid C10(E)-2-methyl-3-(p-tolyl)-5-(3,4,5-trimethoxybenzyl)-5,6,7,8-tetrahydropyrazolo[4,3-d]pyrido[1,2-a]pyrimidine-10(2H)-one, yield 87.9%, melting point: 169.5-171.7℃.

[0044] 1 H NMR (400 MHz, CDCl3)δ 7.80 (d, J = 2.2 Hz, 1H), 7.54 - 7.44 (m,2H), 7.13 (td, J = 7.5, 1.0 Hz, 1H), 7.07 (d, J = 8.3 Hz, 1H), 6.60 (s, 2H),4.22 - 4.15 (m, 2H), 3.98 (s, 3H), 3.89 - 3.83 (m, 12H), 2.90 (td, J = 6.6,2.2 Hz, 2H), 2.02 - 1.94 (m, 2H).HRMS (ESI) m / z:489.2131.

[0045] Example 14 The prepared (E)-3-bromo-2-methyl-5-(3,4,5-trimethoxybenzyl)-5,6,7,8-tetrahydropyrazolo[4,3-d]pyridolo[1,2-a]pyrimidin-10(2H)-one (compound C) 30.0 mg, 0.065 mmol; tetrakis(triphenylphosphine)-palladium phosphate 3.8 mg, 3.25 μmol; potassium phosphate 27.5 mg, 0.13 mmol; and 3-chlorophenylboronic acid (12.2 mg, 0.078 mmol) were sequentially added to a 25 mL dry reaction tube. Argon gas was purged 2-3 times, and 1.2 mL of a 1,4-dioxane:water = 1:1 mixed solvent was added. The reaction was carried out at 100 °C for 8 h. After the TLC reaction was complete, the sample was directly evaporated to dryness and then subjected to silica gel column chromatography (DCM:MeOH = 120:1) to give 16.7 mg of a pale yellow solid C11(E)-3-(3-chlorophenyl)-2-methyl-5-(3,4,5-trimethoxybenzyl)-5,6,7,8-tetrahydropyrazolo[4,3-d]pyrido[1,2-a]pyrimidine-10(2H)-one, yield 52.3%, melting point: 242.4-243.4℃.

[0046] 1 H NMR (400 MHz, CDCl3)δ 7.93 (d, J = 2.2 Hz, 1H), 7.78 (t, J = 1.8Hz, 1H), 7.58 (dt, J = 7.5, 1.5 Hz, 1H), 7.54 - 7.42 (m, 2H), 6.65 (s, 2H), 4.20 (s, 5H), 3.88 (d, J = 3.4 Hz, 9H), 2.97 - 2.90 (m, 2H), 2.06 - 1.96 (m, 2H). HRMS (ESI) m / z: 493.1638.

[0047] Example 15 The prepared (E)-3-bromo-2-methyl-5-(3,4,5-trimethoxybenzyl)-5,6,7,8-tetrahydropyrazolo[4,3-d]pyridolo[1,2-a]pyrimidin-10(2H)-one (compound C) 30.0 mg, 0.065 mmol; tetrakis(triphenylphosphine)-palladium phosphate 3.8 mg, 3.25 μmol; potassium phosphate 27.5 mg, 0.13 mmol; and 3-chloro-4-fluorophenylboronic acid (13.6 mg, 0.078 mmol) were sequentially added to a 25 mL dry reaction tube. Argon gas was purged 2-3 times, and 1.2 mL of a 1,4-dioxane:water = 1:1 mixed solvent was added. The reaction was carried out at 100 °C for 8 h. After the reactants were completely reacted by TLC, the solution was directly evaporated to dryness and then subjected to silica gel column chromatography (DCM:MeOH = 120:1) to give 21.6 mg of a pale yellow solid C12(E)-3-(3-chloro-4-fluorophenyl)-2-methyl-5-(3,4,5-trimethoxybenzyl)-5,6,7,8-tetrahydropyrazolo[4,3-d]pyrido[1,2-a]pyrimidine-10(2H)-one, yield 65.3%, melting point: 243.8-245.8℃.

[0048] 1 H NMR (400 MHz, CDCl3)δ 7.93 - 7.88 (m, 1H), 7.83 (dd, J = 7.0, 2.2Hz, 1H), 7.57 (s, 1H), 7.34 (t, J = 8.6 Hz, 1H), 6.65 (s, 2H), 4.23 - 4.16(m, 5H), 3.88 (d, J = 3.8 Hz, 9H), 2.94 (td, J = 6.7, 2.1 Hz, 2H), 2.04 -1.99 (m, 2H). HRMS (ESI) m / z: 511.1546.

[0049] Example 16 The prepared (E)-3-bromo-2-methyl-5-(3,4,5-trimethoxybenzyl)-5,6,7,8-tetrahydropyrazolo[4,3-d]pyridolo[1,2-a]pyrimidin-10(2H)-one (compound C) 30.0 mg, 0.065 mmol; tetrakis(triphenyl)phosphine, palladium phosphate 3.8 mg, 3.25 μmol; potassium phosphate 27.5 mg, 0.13 mmol; and 2-aminophenylboronic acid (10.7 mg, 0.078 mmol) were sequentially added to a 25 mL dry reaction tube. Argon gas was purged 2-3 times, and 1.2 mL of a 1,4-dioxane:water = 1:1 mixed solvent was added. The reaction was carried out at 100 °C for 8 h. After the reactants were completely reacted by TLC, the solution was directly evaporated to dryness and then subjected to silica gel column chromatography (DCM:MeOH = 120:1) to give 26.1 mg of a pale yellow solid C13(E)-3-(2-aminophenyl)-2-methyl-5-(3,4,5-trimethoxybenzyl)-5,6,7,8-tetrahydropyrazolo[4,3-d]pyrido[1,2-a]pyrimidine-10(2H)-one, yield 84.6%, melting point: 250.2-251.6 °C.

[0050] 1 H NMR (400 MHz, CDCl3)δ7.77 (d, J = 2.1 Hz, 1H), 7.31 - 7.23 (m, 1H),7.15 (dd, J = 7.7, 1.6 Hz, 1H), 6.88 (td, J = 7.5, 1.1 Hz, 1H), 6.77 (dd, J =8.1, 1.2 Hz, 1H), 6.63 (s, 2H), 4.23 - 4.13 (m, 4H), 3.98 (s, 3H), 3.87 (d, J= 3.0 Hz, 9H), 2.99 - 2.91 (m, 2H), 2.04 (p, J = 6.2 Hz, 2H).HRMS (ESI) m / z:472.2139.

[0051] Example 17 The prepared (E)-3-bromo-2-methyl-5-(3,4,5-trimethoxybenzyl)-5,6,7,8-tetrahydropyrazolo[4,3-d]pyridolo[1,2-a]pyrimidin-10(2H)-one (compound C) 30.0 mg, 0.065 mmol; tetratriphenylphosphine palladium 3.8 mg, 3.25 μmol; potassium phosphate 27.5 mg, 0.13 mmol; and 2-bromophenylboronic acid (15.7 mg, 0.078 mmol) were sequentially added to a 25 mL dry reaction tube. Argon gas was purged 2-3 times, and 1.2 mL of a 1,4-dioxane:water = 1:1 mixed solvent was added. The reaction was carried out at 100 °C for 8 h. After the TLC reaction was complete, the sample was directly evaporated to dryness and then subjected to silica gel column chromatography (DCM:MeOH = 120:1) to give 19.4 mg of a pale yellow solid C14(E)-3-(2-bromophenyl)-2-methyl-5-(3,4,5-trimethoxybenzyl)-5,6,7,8-tetrahydropyrazolo[4,3-d]pyridolo[1,2-a]pyrimidine-10(2H)-one, yield 55.6%, melting point: 238.6-240.4℃.

[0052] 1 H NMR (400 MHz, CDCl3)δ 7.81 - 7.75 (m, 2H), 7.54 - 7.36 (m, 3H), 6.60 (s, 2H), 4.28 - 4.07 (m, 2H), 3.98 (s, 3H), 3.86 (d, J = 6.2 Hz, 9H), 2.91 (m, 2H), 2.02 (m, 2H). HRMS (ESI) m / z: 537.1132.

[0053] Example 18 The prepared (E)-3-bromo-2-methyl-5-(3,4,5-trimethoxybenzyl)-5,6,7,8-tetrahydropyrazolo[4,3-d]pyridolo[1,2-a]pyrimidin-10(2H)-one (compound C) 30.0 mg, 0.065 mmol; tetrakis(triphenylphosphine)-palladium phosphate 3.8 mg, 3.25 μmol; potassium phosphate 27.5 mg, 0.13 mmol; and 2-isopropylphenylboronic acid (12.8 mg, 0.078 mmol) were sequentially added to a 25 mL dry reaction tube. Argon gas was purged 2-3 times, and 1.2 mL of a 1,4-dioxane:water = 1:1 mixed solvent was added. The reaction was carried out at 100 °C for 8 h. After the TLC reaction was complete, the sample was directly evaporated to dryness and then subjected to silica gel column chromatography (DCM:MeOH = 120:1) to give 27.5 mg of a pale yellow solid C15(E)-3-(4-isopropylphenyl)-2-methyl-5-(3,4,5-trimethoxybenzyl)-5,6,7,8-tetrahydropyrazolo[4,3-d]pyridolo[1,2-a]pyrimidin-10(2H)-one, yield 84.4%, melting point: 235.8-237.4℃.

[0054] 1 H NMR (400 MHz, CDCl3)δ 7.87 (s, 1H), 7.62 (d, J = 7.9 Hz, 2H), 7.41 (d, J = 7.9 Hz, 2H), 6.63 (s, 2H), 4.18 (s, 5H), 3.87 (d, J = 3.8 Hz, 9H), 2.99 (p, J = 6.9 Hz, 1H), 2.94 - 2.89 (m, 2H), 2.01 (m, 2H), 1.31 (d, J = 6.9Hz, 6H). HRMS (ESI) m / z: 501.2496.

[0055] Example 19 The prepared (E)-3-bromo-2-methyl-5-(3,4,5-trimethoxybenzyl)-5,6,7,8-tetrahydropyrazolo[4,3-d]pyridolo[1,2-a]pyrimidin-10(2H)-one (compound C) 30.0 mg, 0.065 mmol; tetrakis(triphenyl)phosphine, palladium phosphate 3.8 mg, 3.25 μmol; potassium phosphate 27.5 mg, 0.13 mmol; and 3-acetylboronic acid (12.8 mg, 0.078 mmol) were sequentially added to a 25 mL dry reaction tube. Argon gas was purged 2-3 times, and 1.2 mL of a 1,4-dioxane:water = 1:1 mixed solvent was added. The reaction was carried out at 100 °C for 8 h. After the TLC reaction was complete, the sample was directly evaporated to dryness and then subjected to silica gel column chromatography (DCM:MeOH = 120:1) to give 31.3 mg of a pale yellow solid C16(E)-3-(3-acetylphenyl)-2-methyl-5-(3,4,5-trimethoxybenzyl)-5,6,7,8-tetrahydropyrazolo[4,3-d]pyridolo[1,2-a]pyrimidin-10(2H)-one, yield 96.3%, melting point: 181.6-183.0℃.

[0056] 1 H NMR (400 MHz, CDCl3)δ 8.45 (s, 1H), 8.04 (d, J = 7.6 Hz, 1H), 7.98- 7.88 (m, 2H), 7.67 (t, J = 7.6 Hz, 1H), 6.67 (s, 2H), 4.23 (d, J = 9.5 Hz, 5H), 3.88 (d, J = 2.0 Hz, 9H), 2.95 (s, 2H), 2.68 (s, 3H), 2.04 (d, J = 5.9Hz, 2H). HRMS (ESI) m / z: 501.2133.

[0057] Example 20 The prepared (E)-3-bromo-2-methyl-5-(3,4,5-trimethoxybenzyl)-5,6,7,8-tetrahydropyrazolo[4,3-d]pyridolo[1,2-a]pyrimidin-10(2H)-one (compound C) 30.0 mg, 0.065 mmol; tetrakis(triphenylphosphine)-palladium phosphate 3.8 mg, 3.25 μmol; potassium phosphate 27.5 mg, 0.13 mmol; and 4-acetylphenylboronic acid (12.8 mg, 0.078 mmol) were sequentially added to a 25 mL dry reaction tube. Argon gas was purged 2-3 times, and 1.2 mL of a 1,4-dioxane:water = 1:1 mixed solvent was added. The reaction was carried out at 100 °C for 8 h. After the reactants were completely reacted by TLC, the solution was directly evaporated to dryness and then subjected to silica gel column chromatography (DCM:MeOH = 120:1) to give 22.4 mg of a pale yellow solid C17(E)-3-(4-acetylphenyl)-2-methyl-5-(3,4,5-trimethoxybenzyl)-5,6,7,8-tetrahydropyrazolo[4,3-d]pyridolo[1,2-a]pyrimidin-10(2H)-one, yield 68.7%, melting point: 276.0-277.0 °C.

[0058] 1 H NMR (400 MHz, CDCl3)δ 8.16 - 8.10 (m, 2H), 7.89 (d, J = 2.1 Hz,1H), 7.86 - 7.82 (m, 2H), 6.62 (s, 2H), 4.22 (s, 3H), 4.21 - 4.16 (m, 2H),3.87 (d, J = 3.5 Hz, 9H), 2.92 (m, 2H), 2.66 (s, 3H), 2.01 (m, 2H).HRMS (ESI)m / z:501.2132.

[0059] Example 21 The prepared (E)-3-bromo-2-methyl-5-(3,4,5-trimethoxybenzyl)-5,6,7,8-tetrahydropyrazolo[4,3-d]pyridolo[1,2-a]pyrimidin-10(2H)-one (compound C) 30.0 mg, 0.065 mmol; tetratriphenylphosphine palladium 3.8 mg, 3.25 μmol; potassium phosphate 27.5 mg, 0.13 mmol; and 5-chloro-2-methoxyphenylboronic acid (14.6 mg, 0.078 mmol) were sequentially added to a 25 mL dry reaction tube. Argon gas was purged 2-3 times, and 1.2 mL of a 1,4-dioxane:water = 1:1 mixed solvent was added. The reaction was carried out at 100 °C for 8 h. After the reactants were completely reacted by TLC, the solution was directly evaporated to dryness and then subjected to silica gel column chromatography (DCM:MeOH = 120:1) to give 29.1 mg of a pale yellow solid C18(E)-3-(5-chloro-2-methoxyphenyl)-2-methyl-5-(3,4,5-trimethoxybenzyl)-5,6,7,8-tetrahydropyrazolo[4,3-d]pyrido[1,2-a]pyrimidin-10(2H)-one, yield 85.6%, melting point: 203.8-205.1℃.

[0060] 1 H NMR (400 MHz, CDCl3)δ7.85 (d, J = 2.1 Hz, 1H), 7.48 (d, J = 2.6 Hz,1H), 7.44 (dd, J = 8.8, 2.7 Hz, 1H), 6.99 (d, J = 8.9 Hz, 1H), 6.63 (s, HRMS (ESI) m / z:523.1745.

[0061] Example 22 The prepared (E)-3-bromo-2-methyl-5-(3,4,5-trimethoxybenzyl)-5,6,7,8-tetrahydropyrazolo[4,3-d]pyridolo[1,2-a]pyrimidin-10(2H)-one (compound C) 30.0 mg, 0.065 mmol; tetrakis(triphenylphosphine)-palladium phosphate 3.8 mg, 3.25 μmol; potassium phosphate 27.5 mg, 0.13 mmol; and 2-chlorophenylboronic acid (12.2 mg, 0.078 mmol) were sequentially added to a 25 mL dry reaction tube. Argon gas was purged 2-3 times, and 1.2 mL of a 1,4-dioxane:water = 1:1 mixed solvent was added. The reaction was carried out at 100 °C for 8 h. After the reactants were completely reacted by TLC, the solution was directly evaporated to dryness and then subjected to silica gel column chromatography (DCM:MeOH = 120:1) to give 20.8 mg of a pale yellow solid C19(E)-3-(2-chlorophenyl)-2-methyl-5-(3,4,5-trimethoxybenzyl)-5,6,7,8-tetrahydropyrazolo[4,3-d]pyrido[1,2-a]pyrimidine-10(2H)-one, yield 65.1%, melting point: 244.4-246.1℃.

[0062] 1 H NMR (400 MHz, CDCl3)δ 7.78 (d, J = 2.2 Hz, 1H), 7.61 - 7.57 (m,1H), 7.51 - 7.41 (m, 3H), 6.60 (s, 2H), 4.19 (m, 2H), 3.99 (s, 3H), 3.86 (d,J = 6.4 Hz, 9H), 2.91 (m, 2H), 2.01 (p, J = 6.5 Hz, 2H). HRMS (ESI) m / z: 493.1638.

[0063] Example 23 The prepared (E)-3-bromo-2-methyl-5-(3,4,5-trimethoxybenzyl)-5,6,7,8-tetrahydropyrazolo[4,3-d]pyridolo[1,2-a]pyrimidin-10(2H)-one (compound C) 30.0 mg, 0.065 mmol; tetrakis(triphenylphosphine)-palladium phosphate 3.8 mg, 3.25 μmol; potassium phosphate 27.5 mg, 0.13 mmol; and 4-nitrophenylboronic acid (13.1 mg, 0.078 mmol) were sequentially added to a 25 mL dry reaction tube. Argon gas was purged 2-3 times, and 1.2 mL of a 1,4-dioxane:water = 1:1 mixed solvent was added. The reaction was carried out at 100 °C for 8 h. After the TLC reaction was complete, the sample was directly evaporated to dryness and then subjected to silica gel column chromatography (DCM:MeOH = 120:1) to give 30.4 mg of a yellow solid C20(E)-2-methyl-3-(4-nitrophenyl)-5-(3,4,5-trimethoxybenzyl)-5,6,7,8-tetrahydropyrazolo[4,3-d]pyrido[1,2-a]pyrimidine-10(2H)-one, yield 93.1%, melting point: 254.0-257.2℃.

[0064] 1 H NMR (400 MHz, CDCl3)δ 8.44 - 8.36 (m, 2H), 7.99 - 7.92 (m, 2H),7.89 (d, J = 2.1 Hz, 1H), 6.63 (s, 2H), 4.25 (s, 3H), 4.23 - 4.15 (m, 2H), 3.88 (d, J = 3.0 Hz, 9H), 2.93 (m, 2H), 2.07 - 1.96 (m, 2H). HRMS (ESI) m / z: 504.1877.

[0065] Example 24 The prepared (E)-3-bromo-2-methyl-5-(3,4,5-trimethoxybenzyl)-5,6,7,8-tetrahydropyrazolo[4,3-d]pyridolo[1,2-a]pyrimidin-10(2H)-one (compound C) 30.0 mg, 0.065 mmol; tetrakis(triphenylphosphine)-palladium phosphate 3.8 mg, 3.25 μmol; potassium phosphate 27.5 mg, 0.13 mmol; and pyridine-4-boronic acid (9.6 mg, 0.078 mmol) were sequentially added to a 25 mL dry reaction tube. Argon gas was purged 2-3 times, and 1.2 mL of a 1,4-dioxane:water = 1:1 mixed solvent was added. The reaction was carried out at 100 °C for 8 h. After the TLC reaction was complete, the sample was directly evaporated to dryness and then subjected to silica gel column chromatography (DCM:MeOH = 120:1) to give 20.5 mg of a pale yellow solid C21(E)-2-methyl-3-(pyridin-4-yl)-5-(3,4,5-trimethoxybenzyl)-5,6,7,8-tetrahydropyrazolo[4,3-d]pyridolo[1,2-a]pyrimidine-10(2H)-one, yield 68.7%, melting point: 268.6-269.9℃.

[0066] 1 H NMR (400 MHz, CDCl3)δ 8.83 - 8.77 (m, 2H), 7.91 (d, J = 2.1 Hz,1H), 7.73 - 7.67 (m, 2H), 6.64 (s, 2H), 4.26 (s, 3H), 4.21 - 4.15 (m, 2H), 3.88 (d, J = 2.7 Hz, 9H), 2.93 (td, J = 6.7, 2.1 Hz, 2H), 2.07 - 1.96 (m, 2H). HRMS (ESI) m / z: 460.1979.

[0067] Example 25 The prepared (E)-3-bromo-2-methyl-5-(3,4,5-trimethoxybenzyl)-5,6,7,8-tetrahydropyrazolo[4,3-d]pyridolo[1,2-a]pyrimidin-10(2H)-one (compound C) 30.0 mg, 0.065 mmol; tetrakis(triphenylphosphine)-palladium phosphate 3.8 mg, 3.25 μmol; potassium phosphate 27.5 mg, 0.13 mmol; and 4-carbamoylphenylboronic acid (12.9 mg, 0.078 mmol) were sequentially added to a 25 mL dry reaction tube. Argon gas was purged 2-3 times, and 1.2 mL of a 1,4-dioxane:water = 1:1 mixed solvent was added. The reaction was carried out at 100 °C for 8 h. After the reactants were completely reacted by TLC, the solution was directly evaporated to dryness and then subjected to silica gel column chromatography (DCM:MeOH = 120:1) to give 19.3 mg of a pale yellow solid C22(E)-4-(2-methyl-10-oxo-5-(3,4,5-trimethoxybenzyl)-2,5,6,7,8,10-hexahydropyrazolo[4,3-d]pyridolo[1,2-a]pyrimidin-3-yl)benzamide, yield 59.3%, melting point: 303.8-304.4 °C.

[0068] 1 H NMR (400 MHz, CDCl3)δ 8.01 (d, J = 7.9 Hz, 2H), 7.88 (s, 1H), 7.82 (d, J = 8.0 Hz, 2H), 6.63 (s, 2H), 4.21 (s, 5H), 3.88 (d, J = 4.5 Hz, 9H),2.97 - 2.89 (m, 2H), 2.02 (q, J = 6.2 Hz, 2H).HRMS (ESI) m / z:502.2084.

[0069] Example 26 The prepared (E)-3-bromo-2-methyl-5-(3,4,5-trimethoxybenzyl)-5,6,7,8-tetrahydropyrazolo[4,3-d]pyridolo[1,2-a]pyrimidin-10(2H)-one (compound C) 30.0 mg, 0.065 mmol; tetrakis(triphenylphosphine)-palladium phosphate 3.8 mg, 3.25 μmol; potassium phosphate 27.5 mg, 0.13 mmol; and 2,4-dimethoxyphenylboronic acid (14.2 mg, 0.078 mmol) were sequentially added to a 25 mL dry reaction tube. Argon gas was purged 2-3 times, and 1.2 mL of a 1,4-dioxane:water = 1:1 mixed solvent was added. The reaction was carried out at 100 °C for 8 h. After the reactants were completely reacted by TLC, the solution was directly evaporated to dryness and then subjected to silica gel column chromatography (DCM:MeOH = 120:1) to give 26.7 mg of a pale yellow solid C23(E)-3-(3,5-dimethoxyphenyl)-2-methyl-5-(3,4,5-trimethoxybenzyl)-5,6,7,8-tetrahydropyrazolo[4,3-d]pyridolo[1,2-a]pyrimidine-10(2H)-one, yield 79.3%, melting point: 208.2-210.3℃.

[0070] 1 H NMR (400 MHz, CDCl3)δ 7.80 (d, J = 2.1 Hz, 1H), 7.38 (d, J = 8.4Hz, 1H), 6.65 (dd, J = 8.4, 2.3 Hz, 1H), 6.60 (d, J = 3.1 Hz, 3H), 4.21 -4.14 HRMS (ESI) m / z:519.2241.

[0071] Example 27 The prepared (E)-3-bromo-2-methyl-5-(3,4,5-trimethoxybenzyl)-5,6,7,8-tetrahydropyrazolo[4,3-d]pyridolo[1,2-a]pyrimidin-10(2H)-one (compound C) 30.0 mg, 0.065 mmol; tetrakis(triphenylphosphine)-palladium phosphate 3.8 mg, 3.25 μmol; potassium phosphate 27.5 mg, 0.13 mmol; and 2-trifluoromethylphenylboronic acid (14.8 mg, 0.078 mmol) were sequentially added to a 25 mL dry reaction tube. Argon gas was purged 2-3 times, and 1.2 mL of a 1,4-dioxane:water = 1:1 mixed solvent was added. The reaction was carried out at 100 °C for 8 h. After the TLC reaction was complete, the sample was directly evaporated to dryness and then subjected to silica gel column chromatography (DCM:MeOH = 120:1) to give 28.8 mg of a pale yellow solid C24(E)-2-methyl-3-(2-(trifluoromethyl)phenyl)-5-(3,4,5-trimethoxybenzyl)-5,6,7,8-tetrahydropyrazolo[4,3-d]pyrido[1,2-a]pyrimidine-10(2H)-one, yield 84.2%, melting point: 225.5-226.9℃.

[0072] 1 H NMR (400 MHz, CDCl3)δ 7.90 (dd, J = 7.5, 1.8 Hz, 1H), 7.75 - 7.66(m, 4H), 7.48 - 7.42 (m, 1H), 6.57 (s, 2H), 4.34 (m, 1H), 4.02 (m, 1H), 3.87(s, 3H), 3.86 (s, 3H), 3.84 (s, 6H), 2.90 (m, 2H), 2.12 - 1.89 (m, 2H).HRMS(ESI) m / z:527.1904.

[0073] Example 28 The prepared (E)-3-bromo-2-methyl-5-(3,4,5-trimethoxybenzyl)-5,6,7,8-tetrahydropyrazolo[4,3-d]pyridolo[1,2-a]pyrimidin-10(2H)-one (compound C) 30.0 mg, 0.065 mmol; tetrakis(triphenylphosphine)-palladium phosphate 3.8 mg, 3.25 μmol; potassium phosphate 27.5 mg, 0.13 mmol; and 4-cyclohexylphenylboronic acid (16.0 mg, 0.078 mmol) were sequentially added to a 25 mL dry reaction tube. Argon gas was purged 2-3 times, and 1.2 mL of a 1,4-dioxane:water = 1:1 mixed solvent was added. The reaction was carried out at 100 °C for 8 h. After the reactants were completely reacted by TLC, the solution was directly evaporated to dryness and then subjected to silica gel column chromatography (DCM:MeOH = 120:1) to give 29.6 mg of a pale yellow solid C25(E)-2-methyl-3-(4-(cyclohexyl-1-yl)phenyl)-5-(3,4,5-trimethoxybenzyl)-5,6,7,8-tetrahydropyrazolo[4,3-d]pyrido[1,2-a]pyrimidin-10(2H)-one, yield 84.3%, melting point: 241.3-243.0 °C.

[0074] 1 H NMR (400 MHz, CDCl3)δ 7.87 (d, J = 2.1 Hz, 1H), 7.64 - 7.58 (m,2H), 7.42 - 7.35 (m, 2H), 6.63 (s, 2H), 4.20 - 4.16 (m, 5H), 3.87 (d, J = 3.8Hz, 9H), 2.91 (td, J = 6.6, 2.1 Hz, 2H), 2.63 - 2.52 (m, 1H), 2.06 - 1.96 (m,2H), 1.96 - 1.83 (m, 4H), 1.77 (m, 1H), 1.54 - 1.34 (m, 4H), 1.34 - 1.21 (m,1H).HRMS (ESI) m / z:541.2814.

[0075] Example 29 The prepared (E)-3-bromo-2-methyl-5-(3,4,5-trimethoxybenzyl)-5,6,7,8-tetrahydropyrazolo[4,3-d]pyridolo[1,2-a]pyrimidin-10(2H)-one (compound C) 30.0 mg, 0.065 mmol; tetrakis(triphenylphosphine)-palladium phosphate 3.8 mg, 3.25 μmol; potassium phosphate 27.5 mg, 0.13 mmol; and 4-acetamidophenylboronic acid (14.0 mg, 0.078 mmol) were sequentially added to a 25 mL dry reaction tube. Argon gas was purged 2-3 times, and 1.2 mL of a 1,4-dioxane:water = 1:1 mixed solvent was added. The reaction was carried out at 100 °C for 8 h. After the reactants were completely reacted by TLC, the solution was directly evaporated to dryness and then subjected to silica gel column chromatography (DCM:MeOH = 120:1) to give 22.0 mg of a pale yellow solid C26(E)-N-(4-(2-methyl-10-oxo-5-(3,4,5-trimethoxybenzylmethyl)-2,5,6,7,8,10-hexahydropyrazolo[4,3-d]pyrido[1,2-a]pyrimidin-3-yl)phenyl)acetamide, yield 65.7%, melting point: 269.5-270.9℃.

[0076] 1 H NMR (400 MHz, CDCl3)δ7.84 (d, J = 2.1 Hz, 1H), 7.76 (s, 1H), 7.72(d, J = 8.4 Hz, 2H), 7.63 (d, J = 8.6 Hz, 2H), 6.61 (s, 2H), 4.22 - 4.14 (m,2H), 4.12 (s, 3H), 3.86 (d, J = 7.8 Hz, 9H), 2.94 - 2.87 (m, 2H), 2.23 (s,3H), 2.00 (m, 2H). HRMS (ESI) m / z:516.2247.

[0077] Example 30 The prepared (E)-3-bromo-2-methyl-5-(3,4,5-trimethoxybenzyl)-5,6,7,8-tetrahydropyrazolo[4,3-d]pyridolo[1,2-a]pyrimidin-10(2H)-one (compound C) 30.0 mg, 0.065 mmol; tetrakis(triphenylphosphine)-palladium phosphate 3.8 mg, 3.25 μmol; potassium phosphate 27.5 mg, 0.13 mmol; and 4-hydroxyphenylboronic acid (10.8 mg, 0.078 mmol) were sequentially added to a 25 mL dry reaction tube. Argon gas was purged 2-3 times, and 1.2 mL of a 1,4-dioxane:water = 1:1 mixed solvent was added. The reaction was carried out at 100 °C for 8 h. After the reactants were completely reacted by TLC, the solution was directly evaporated to dryness and then subjected to silica gel column chromatography (DCM:MeOH = 120:1) to give 15.8 mg of a pale yellow solid C27(E)-3-(4-hydroxyphenyl)-2-methyl-5-(3,4,5-trimethoxybenzyl)-5,6,7,8-tetrahydropyrazolo[4,3-d]pyrido[1,2-a]pyrimidine-10(2H)-one, yield 51.3%, melting point: 294.7-295.8℃.

[0078] 1 H NMR (400 MHz, CDCl3)δ 7.85 (s, 1H), 7.54 (d, J = 8.5 Hz, 2H), 7.00 (d, J = 8.5 Hz, 2H), 6.63 (s, 2H), 4.20 (t, J = 5.9 Hz, 2H), 4.14 (s, 3H), 3.87 (d, J = 6.9 Hz, 9H), 2.93 (t, J = 6.7 Hz, 2H), 2.02 (m, 2H). HRMS (ESI)m / z: 475.1976.

[0079] Example 31 The prepared (E)-3-bromo-2-methyl-5-(3,4,5-trimethoxybenzyl)-5,6,7,8-tetrahydropyrazolo[4,3-d]pyridolo[1,2-a]pyrimidin-10(2H)-one (compound C) 30.0 mg, 0.065 mmol; tetratriphenylphosphine palladium 3.8 mg, 3.25 μmol; potassium phosphate 27.5 mg, 0.13 mmol; and 6-quinoline borate pinacol ester (19.9 mg, 0.078 mmol) were sequentially added to a 25 mL dry reaction tube. Argon gas was purged 2-3 times, and 1.2 mL of a 1,4-dioxane:water = 1:1 mixed solvent was added. The reaction was carried out at 100 °C for 8 h. After the TLC reaction was complete, the sample was directly evaporated to dryness and then subjected to silica gel column chromatography (DCM:MeOH = 120:1) to give 27.9 mg of a pale yellow solid C28(E)-3-(isoquinolin-6-yl)-2-methyl-5-(3,4,5-trimethoxybenzylmethyl)-5,6,7,8-tetrahydropyrazolo[4,3-d]pyrido[1,2-a]pyrimidin-10(2H)-one, yield 84.3%, melting point: 299.4-300.4℃.

[0080] 1 H NMR (400 MHz, CDCl3)δ 8.99 (dt, J = 4.3, 1.4 Hz, 1H), 8.31 - 8.22(m, 2H), 8.14 (d, J = 1.9 Hz, 1H), 8.07 (dd, J = 8.7, 2.0 Hz, 1H), 7.89 (d, J= 2.1 Hz, 1H), 7.48 (ddd, J = 8.3, 4.3, 1.1 Hz, 1H), 6.61 (s, 2H), 4.25 (s,3H), 4.23 - 4.17 (m, 2H), 3.87 (s, 3H), 3.84 (s, 6H), 2.92 (td, J = 6.6, 2.1Hz, 2H), 2.01 (p, J = 6.6 Hz, 2H). HRMS (ESI) m / z:510.2139.

[0081] Example 32 The tricyclic pyrazole [4,3-d]pyrimidinone derivatives obtained in Examples 4-31 were screened for in vitro antitumor activity: MTT assay for cell viability: Cells growing in the logarithmic growth phase were aspirated from the culture medium, washed once with PBS, digested with trypsin, and culture medium was added to stop the digestion. Cells were gently pipetted, counted, and seeded at the appropriate cell density in 96-well plates (100 μL / well) and cultured overnight. Compounds were added (20 μL / well), with concentration gradients for each compound and three replicates for each concentration. Cells were incubated at 37°C with CO2 for 48 hours. The old culture medium was discarded, 100 μL of MTT was added, and the cells were incubated for another 2 hours at 37°C. The absorbance (OD) at 570 nm was measured using an MB microplate reader. Calculation formula: Cell viability percentage % = (Compound OD - Blank OD / Control Group OD - Blank OD) × 100% Cell inhibition rate % = 1 - Cell viability % = [1 - (Compound OD - Blank OD / Control Group OD - Blank OD)] × 100%. The IC50 value is obtained by fitting the formula using GraphPad. 50 ; Sample preparation: The sample was dissolved in dimethyl sulfoxide (DMSO) and stored at low temperature. The concentration of DMSO in the final system was controlled within a range that would not affect the detection activity. Data processing and results explanation: Initial screening was conducted under single-concentration conditions, such as a monomeric compound concentration of 50 μM and an extract concentration of 50 μg / μL, to test the activity of the samples. For samples exhibiting activity under certain conditions, such as an inhibition rate greater than 50%, the dose-dependent relationship of activity, i.e., IC50, was further tested. 50 The value is obtained by nonlinearly fitting the sample activity to the sample concentration, and the software used for calculation is Graphpad Prism 4; typically, each sample is tested with duplicate wells (n≥3).

[0082] Table 1. Results of in vitro antitumor bioactivity of derivatives C1-C28 The table shows that, using doxorubicin (DOX) as a positive control, 8 out of 28 compounds exhibited some inhibitory activity against human cervical cancer cells (HeLa) (IC50). 50 <50μM), of which C-5 and C-16 show better activity, IC50 50 The concentrations were 29.17 ± 1.58 μM and 32.36 ± 1.81 μM, respectively; 17 compounds showed inhibitory activity against human colon cancer cells HT-29 (IC50). 50The soluble solids concentrations (C-4, C-14, and C-24) were <50 μM, with C-4 at 15.64 ± 0.78 μM, C-14 at 17.11 ± 0.86 μM, and C-24 at 12.85 ± 0.62 μM, respectively. All four compounds exhibited inhibitory activity against human gastric cancer cells HGC-27, with compound C-24 showing the best activity (IC50 ± 0.50 μM). 50 The value was 32.27 ± 1.65 μM.

[0083] The above descriptions are merely embodiments of the present invention, and common knowledge regarding specific structures and characteristics is not elaborated upon here. It should be noted that those skilled in the art can make various modifications and improvements without departing from the structure of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications and improvements will not affect the effectiveness of the present invention or the practicality of the patent. The scope of protection claimed in this application should be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.

Claims

1. A tricyclic pyrazole [4,3-d]pyrimidinone derivative, characterized in that, The structural formula of the derivative is as follows: in: Compound C1 is (E)-2-methyl-3-(o-tolyl)-5-(3,4,5-trimethoxybenzylidene)-5,6,7,8-tetrahydropyrazolo[4,3-d]pyridolo[1,2-a]pyrimidin-10(2H)-one; Compound C2 is (E)-2-methyl-3-(m-tolyl)-5-(3,4,5-trimethoxybenzylidene)-5,6,7,8-tetrahydropyrazolo[4,3-d]pyrido[1,2-a]pyrimidin-10(2H)-one; Compound C3 is (E)-3-(4-methoxyphenyl)-2-methyl-5-(3,4,5-trimethoxybenzyl)-5,6,7,8-tetrahydropyrazolo[4,3-d]pyrido[1,2-a]pyrimidine-10(2H)-one; Compound C4 is (E)-2-methyl-3-(naphth-1-yl)-5-(3,4,5-trimethoxybenzylidene)-5,6,7,8-tetrahydropyrazolo[4,3-d]pyrido[1,2-a]pyrimidin-10(2H)-one; Compound C5 is (E)-2-methyl-3-(4-phenoxyphenyl)-5-(3,4,5-trimethoxybenzyl)-5,6,7,8-tetrahydropyrazolo[4,3-d]pyrido[1,2-a]pyrimidine-10(2H)-one; Compound C6 is (E)-3-(4-(tert-butyl)phenyl)-2-methyl-5-(3,4,5-trimethoxybenzyl)-5,6,7,8-tetrahydropyrazolo[4,3-d]pyrido[1,2-a]pyrimidin-10(2H)-one; Compound C7 is (E)-2-methyl-3-phenyl-5-(3,4,5-trimethoxybenzyl)-5,6,7,8-tetrahydropyrazolo[4,3-d]pyrido[1,2-a]pyrimidin-10(2H)-one; Compound C8 is (E)-3-(2-methoxynaphth-1-yl)-2-methyl-5-(3,4,5-trimethoxybenzylmethyl)-5,6,7,8-tetrahydropyrazolo[4,3-d]pyridolo[1,2-a]pyrimidin-10(2H)-one; Compound C9 is (E)-2-methyl-5-(3,4,5-trimethoxybenzyl)-3-vinyl-5,6,7,8-tetrahydropyrazolo[4,3-d]pyridolo[1,2-a]pyrimidin-10(2H)-one; Compound C10 is (E)-2-methyl-3-(p-tolyl)-5-(3,4,5-trimethoxybenzylidene)-5,6,7,8-tetrahydropyrazolo[4,3-d]pyrido[1,2-a]pyrimidin-10(2H)-one; Compound C11 is (E)-3-(3-chlorophenyl)-2-methyl-5-(3,4,5-trimethoxybenzyl)-5,6,7,8-tetrahydropyrazolo[4,3-d]pyrido[1,2-a]pyrimidin-10(2H)-one; Compound C12 is (E)-3-(3-chloro-4-fluorophenyl)-2-methyl-5-(3,4,5-trimethoxybenzyl)-5,6,7,8-tetrahydropyrazolo[4,3-d]pyrido[1,2-a]pyrimidin-10(2H)-one; Compound C13 is (E)-3-(2-aminophenyl)-2-methyl-5-(3,4,5-trimethoxybenzyl)-5,6,7,8-tetrahydropyrazolo[4,3-d]pyrido[1,2-a]pyrimidine-10(2H)-one; Compound C14 is (E)-3-(2-bromophenyl)-2-methyl-5-(3,4,5-trimethoxybenzyl)-5,6,7,8-tetrahydropyrazolo[4,3-d]pyrido[1,2-a]pyrimidine-10(2H)-one; Compound C15 is (E)-3-(4-isopropylphenyl)-2-methyl-5-(3,4,5-trimethoxybenzyl)-5,6,7,8-tetrahydropyrazolo[4,3-d]pyrido[1,2-a]pyrimidin-10(2H)-one; Compound C16 is (E)-3-(3-acetylphenyl)-2-methyl-5-(3,4,5-trimethoxybenzyl)-5,6,7,8-tetrahydropyrazolo[4,3-d]pyrido[1,2-a]pyrimidine-10(2H)-one; Compound C17 is (E)-3-(4-acetylphenyl)-2-methyl-5-(3,4,5-trimethoxybenzyl)-5,6,7,8-tetrahydropyrazolo[4,3-d]pyrido[1,2-a]pyrimidin-10(2H)-one; Compound C18 is (E)-3-(5-chloro-2-methoxyphenyl)-2-methyl-5-(3,4,5-trimethoxybenzyl)-5,6,7,8-tetrahydropyrazolo[4,3-d]pyrido[1,2-a]pyrimidine-10(2H)-one; Compound C19 is (E)-3-(2-chlorophenyl)-2-methyl-5-(3,4,5-trimethoxybenzyl)-5,6,7,8-tetrahydropyrazolo[4,3-d]pyrido[1,2-a]pyrimidine-10(2H)-one; Compound C20 is (E)-2-methyl-3-(4-nitrophenyl)-5-(3,4,5-trimethoxybenzyl)-5,6,7,8-tetrahydropyrazolo[4,3-d]pyrido[1,2-a]pyrimidine-10(2H)-one; Compound C21 is (E)-2-methyl-3-(pyridin-4-yl)-5-(3,4,5-trimethoxybenzyl)-5,6,7,8-tetrahydropyrazolo[4,3-d]pyridolo[1,2-a]pyrimidin-10(2H)-one; Compound C22 is (E)-4-(2-methyl-10-oxo-5-(3,4,5-trimethoxybenzyl)-2,5,6,7,8,10-hexahydropyrazolo[4,3-d]pyridolo[1,2-a]pyrimidin-3-yl)benzamide; Compound C23 is (E)-3-(3,5-dimethoxyphenyl)-2-methyl-5-(3,4,5-trimethoxybenzyl)-5,6,7,8-tetrahydropyrazolo[4,3-d]pyridolo[1,2-a]pyrimidin-10(2H)-one; Compound C24 is (E)-2-methyl-3-(2-(trifluoromethyl)phenyl)-5-(3,4,5-trimethoxybenzylidene)-5,6,7,8-tetrahydropyrazolo[4,3-d]pyrido[1,2-a]pyrimidin-10(2H)-one; Compound C25 is (E)-3-(4-cyclohexylphenyl)-2-methyl-5-(3,4,5-trimethoxybenzyl)-5,6,7,8-tetrahydropyrazolo[4,3-d]pyridolo[1,2-a]pyrimidin-10(2H)-one; Compound C26 is (E)-N-(4-(2-methyl-10-oxo-5-(3,4,5-trimethoxybenzylmethyl)-2,5,6,7,8,10-hexahydropyrazolo[4,3-d]pyrido[1,2-a]pyrimidin-3-yl)phenyl)acetamide; Compound C27 is (E)-3-(4-hydroxyphenyl)-2-methyl-5-(3,4,5-trimethoxybenzyl)-5,6,7,8-tetrahydropyrazolo[4,3-d]pyrido[1,2-a]pyrimidin-10(2H)-one; Compound C28 is (E)-3-(isoquinolin-6-yl)-2-methyl-5-(3,4,5-trimethoxybenzylmethyl)-5,6,7,8-tetrahydropyrazolo[4,3-d]pyrido[1,2-a]pyrimidin-10(2H)-one.

2. The method for preparing a tricyclic pyrazolopyrimidinone derivative according to claim 1, characterized in that, S1. Preparation of compound A: 1-methyl-4-aminopyrazole-3-carboxylic acid methyl ester was dissolved in anhydrous 1,4-dioxane, then phosphorus oxychloride was added dropwise and heated and stirred, then 2-azhexanecycloone was added and heated to react. When the raw materials reacted completely, the temperature of the reaction solution was lowered to room temperature and placed in an ice bath. NaOH aqueous solution was slowly added to adjust the pH value to 7, then dichloromethane was added for separation and extraction. The organic phases were combined and washed with saturated brine, dried with anhydrous sodium sulfate, and concentrated. The dried product was then slurried with petroleum ether and filtered, and washed with a mixed solution at -20℃ to obtain compound A. The chemical formula of compound A is 2-methyl-5,6,7,8-tetrahydropyrazolo[4,3-d]pyridin[1,2-a]pyrimidin-10(2H)-one. S2. Preparation of compound B: Dissolve compound A obtained in S1 in 1,2-dichloroethane, then add NBS in batches for heating reaction. After the raw material reaction is complete, add water, then add dichloromethane for extraction by liquid-liquid extraction. Combine the organic phases and wash them with saturated brine, dry them with anhydrous sodium sulfate, concentrate them, and then add tetrahydrofuran at -20℃ to the dried product. After slurrying and filtration, obtain grayish-white solid compound B. The chemical formula of compound B is 3-bromo-2-methyl-5,6,7,8-tetrahydropyrazolo[4,3-d]pyrido[1,2-a]pyrimidin-10(2H)-one. S3. Preparation of compound C: Compound B, 3,4,5-trimethoxybenzaldehyde and p-toluenesulfonic acid were added to xylene and heated to reflux. After the reaction of the raw materials was complete, water and dichloromethane were added to dissolve them. After complete dissolution, dichloromethane was added for extraction by separation. The organic phases were combined and washed with saturated brine, dried with anhydrous sodium sulfate, and concentrated. Then, methanol at -20℃ was added to the dried product for slurrying. After slurrying, the product was filtered to obtain a yellow solid. The yellow solid was then purified by silica gel column chromatography to obtain a pale yellow solid compound C. The chemical formula of compound C is (E)-3-bromo-2-methyl-5-(3,4,5-trimethoxybenzyl)-5,6,7,8-tetrahydropyrazolo[4,3-d]pyrido[1,2-a]pyrimidin-10(2H)-one. S4. Preparation of compounds C1-C28: The obtained compound C, tetra-triphenylphosphine palladium and potassium phosphate were combined with different types of boric acid compounds and then added to a dry reaction tube. Argon gas was replaced 2-3 times, and then a mixed solvent of 1,4-dioxane:water = 1:1 was added. The mixture was then reacted at 100℃ for 8 h. After the reactants were completely reacted, the mixture was directly evaporated to dryness and then subjected to silica gel column chromatography (DCM:MeOH = 120:1) to obtain yellow or pale yellow solid compounds C1-C28.

3. The use of compounds C2, C5, C9, C12, C16, C20, C24 or C26 of a tricyclic pyrazolopyrimidine one derivative according to claim 1 in the treatment of human cervical cancer cells HeLa.

4. The use of compounds C1, C2, C4, C6, C7, C8, C10, C11, C12, C13, C14, C15, C19, C20, C23, C24 or C25 of a tricyclic pyrazolopyrimidine ketone derivative according to claim 1 in the treatment of HT-29 colon cancer cells.

5. The use of compound C7, C8, C13 or C24 of a tricyclic pyrazolopyrimidinone derivative according to claim 1 in the treatment of gastric cancer cells HGC-27.

Images