Pyrimidopyrazole derivatives and their use in medicine

Abstract

The application discloses a kind of pyrimidopyrazole derivatives and its medical purposes.The pyrimidopyrazole derivatives are the compounds shown in general formula I, its pharmaceutically acceptable salt or solvate, the compound of the application can be used as ALKBH5 protein small molecule inhibitor, inhibits RNA demethylase at the level of protease, and can be used for treating acute leukemia, lung cancer, breast cancer, colon cancer, prostate cancer, pancreatic cancer, gastric cancer, liver cancer, ovarian cancer, kidney cancer, neuroglioma, melanoma, myeloma, lymphoma, bladder cancer or cervical cancer.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a class of pyrimidinepyrazole derivatives and their uses in medicine. Background Technology

[0002] Dynamic RNA modification plays a crucial role in many physiological processes in living organisms, such as cell cycle regulation, cell proliferation, and differentiation. For eukaryotes, the main RNA modifications include N... 6 Methyladenosine (N) 6 -methyladenosine, m 6 A) 5-methylcytosine (m) 5 C) and N 1 Methyladenosine (N) 1 -methyladenosine, m 1 A), and m 6 A is the most common and conserved modification of messenger RNA (mRNA), and it is also one of the current research hotspots. 6 The dynamic balance of A is crucial in the body. Once this balance is disrupted, physiological and even pathological changes can occur, leading to diseases such as breast cancer, colon cancer, and leukemia. It is also associated with many cardiovascular and cerebrovascular diseases, bone-related diseases, diabetes, and some viral infections.

[0003] ALKBH5 is an mRNA demethylase that can modify and regulate mRNA. 6 Level A. Studies have shown that ALKBH5 is abnormally expressed in acute myeloid leukemia. Therefore, designing small molecule inhibitors to inhibit the enzymatic catalytic activity of ALKBH5 and thus block tumor occurrence and development is of positive significance. Summary of the Invention

[0004] The purpose of this invention is to provide a class of pyrimidopyrazole derivatives that can act as small molecule inhibitors of the ALKBH5 protein, inhibiting RNA demethylases at the protease level, and thus be used to treat RNA demethylase-related diseases.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] Compounds represented by formula I, their pharmaceutically acceptable salts, or solvates,

[0007]

[0008] R1 and R2 may be the same as or different from each other, and each is independently selected from hydrogen, amino, nitro, hydroxyl, halogen, cyano, trifluoromethyl, C1-C6 alkyl, C3-C6 cycloalkyl, C1-C6 alkoxy or C1-C6 alkoxy-substituted C1-C6 alkyl.

[0009] R3 is selected from hydrogen, hydroxyl, halogen, COOR4, COR4, OCOR4, CONHR4, COOL 1 R4, COL 1 R4, OCOL 1 R4 and CONHL 1 R4; wherein: R4 is selected from hydrogen, C1-C6 alkyl, C3-C7 cycloalkyl, nitrogen- or oxygen-containing three- to seven-membered heterocycles, phenyl or substituted phenyl; L 1 Selectable from C1-C3 saturated carbon chains;

[0010] L is selected from C1-C6 saturated carbon chains, C1-C6 unsaturated carbon chains, carbonyl chains, amide bonds, ester bonds, oxy ethers, ketone carbonyl groups, and thioethers.

[0011] W, X, and Y may be the same as or different from each other, and each may be independently selected from carbon, nitrogen, oxygen, or sulfur.

[0012] Furthermore, R1 is selected from hydrogen, hydroxyl, halogen, cyano, C1-C6 alkyl, and C1-C6 alkoxy.

[0013] R2 is selected from hydrogen, hydroxyl, halogen, C1-C6 alkyl, C3-C6 cycloalkyl, and C1-C6 alkoxy.

[0014] R3 is selected from hydrogen, hydroxyl, halogen, COOR4, COR4, OCOR4, CONHR4, CONHL 1 R4 and COOL 1 R4; wherein: R4 is selected from hydrogen, C1-C6 alkyl, C3-C7 cycloalkyl, nitrogen- or oxygen-containing three- to seven-membered heterocycles, phenyl or substituted phenyl; L 1 Selected from C1-C3 saturated carbon chains;

[0015] L is selected from amide bond, ester bond, oxy ether or ketone carbonyl group;

[0016] W, X, and Y may be the same as or different from each other, and each may be independently selected from carbon or nitrogen.

[0017] This invention also includes pharmaceutically acceptable salts of the compounds represented by general formula I, and their solvates, all of which have the same pharmacological effects as the compounds.

[0018] The present invention also discloses a method for preparing the relevant compound, such as method a, b or c shown below.

[0019] Method a includes the following steps:

[0020]

[0021] Method b includes the following steps:

[0022]

[0023] Method c includes the following steps:

[0024]

[0025] Method d includes the following steps:

[0026]

[0027] The compounds of this invention can be used as small molecule inhibitors of the ALKBH5 protein, inhibiting RNA demethylases at the protease level, and can be used to treat acute leukemia, lung cancer, breast cancer, colon cancer, prostate cancer, pancreatic cancer, gastric cancer, liver cancer, ovarian cancer, kidney cancer, glioma, melanoma, myeloma, lymphoma, bladder cancer, or cervical cancer. Detailed Implementation

[0028] The preferred embodiments of the present invention will now be described in detail with reference to specific examples. It should be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the invention. Those skilled in the art can make various modifications and substitutions to the present invention without departing from its spirit and essence.

[0029] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0030] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0031] The target compound was synthesized using the following method in the following examples:

[0032] Option 1

[0033]

[0034] Under ice bath conditions, 5.79 mmol of substituted 2'-hydroxy-ethyl ketone was dissolved in 10 mL of N,N-dimethylformamide. The air in the reaction flask was purged with nitrogen, and 3.18 mL of phosphorus oxychloride was slowly added dropwise over half an hour. The ice bath was removed, and the mixture was allowed to return to room temperature naturally for 12 hours. The reaction solution was then slowly diluted in 100 mL of an ice-water mixture, and stirred under ice bath conditions for 2 hours, resulting in the gradual precipitation of a yellow solid. The crude product was filtered, and recrystallized from it using a petroleum ether / ethyl acetate system to obtain the pure target product.

[0035] Option 2

[0036]

[0037] Under ice bath conditions, 6.49 mmol of 2'-fluoro-6'-hydroxyacetophenone was dissolved in 10 mL of glacial acetic acid, and 0.3 mL of fuming nitric acid was slowly added dropwise over half an hour. The ice bath was then removed, and the mixture was allowed to return to room temperature naturally for 12 hours. The reaction solution was then slowly diluted in 100 mL of an ice-water mixture. Under ice bath conditions, saturated NaHCO3 solution was added to adjust the pH to 4-5, and the mixture was stirred for 2 hours. Finally, the solution was extracted with ethyl acetate to obtain the pure target product.

[0038] Option 3

[0039]

[0040] At room temperature, 4.92 mmol of methyl 4-(cyanoacetyl)benzoate was added to a 50 mL reaction flask. 3 mL of a mixture of methanol and tetrahydrofuran was added, followed by 7 mL of 1 mol / L NaOH solution. The mixture was stirred at room temperature for 8 h, concentrated under reduced pressure until a small amount of water remained, and then 5 mL of water was added. The pH was adjusted to 1-2 with dilute hydrochloric acid, and a white solid was observed to precipitate.

[0041] Option 4

[0042]

[0043] 1.52 mmol of 4-(cyanoacetyl)benzoic acid, 1.52 mmol of substituted amine, and 1.82 mmol of N,N,N',N'-tetramethylchloromethylammonium hexafluorophosphate were dissolved in 10 mL of acetonitrile solution. 0.5 mL of N-methylimidazolium was added dropwise, and the mixture was stirred at room temperature for 16 h. The solvent acetonitrile was removed by vacuum distillation. The crude product was purified by silica gel column chromatography (elution: petroleum ether / ethyl acetate, gradient 0-20%) to obtain the target product.

[0044] Option 5

[0045]

[0046] 6 mmol of the substituted benzoyl acetonitrile was dissolved in 20 mL of ethanol, and 1 mL of 80% hydrazine hydrate aqueous solution was added. The air in the reaction flask was replaced with nitrogen, and the reaction solution was heated at 80 °C for 3–8 h. The solvent was removed by vacuum distillation, and the crude product was purified by silica gel column chromatography (eluent: dichloromethane / methanol, gradient 0–5%) to obtain the target product.

[0047] Option Six

[0048]

[0049] 0.456 mol of substituted chromone-3-carboxaldehyde was dissolved in 10 mL of ethanol, and the mixture was heated to 80 °C. After the starting material was completely dissolved, 0.456 mmol of substituted 5-phenyl-1H-pyrazole-3-amine was added, and the mixture was heated at 80 °C for 1-2 h. A yellow solid gradually precipitated out. The reaction solution was cooled to room temperature to allow crystals to crystallize. The crystals were filtered, washed with ice-cold ethanol, and dried. The crude product was slurried using a dichloromethane / petroleum ether system to obtain the pure target product.

[0050] Option 7

[0051]

[0052] 5.64 mmol of substituted 5-phenyl-1H-pyrazole-3-amine was dissolved in 50 mL of ethanol, and 711.7 μL of ethyl 2-formyl-3-oxopropionate was added dropwise. The air in the reaction flask was purged with nitrogen, and the color of the reaction solution gradually deepened. The reaction solution was heated at 80 °C for 3 h, and a solid gradually precipitated out. The reaction solution was cooled to room temperature and allowed to stand to crystallize. The crystals were filtered, washed with ice-cold ethanol, and dried to obtain the target product solid, which can be directly used for the next reaction without purification.

[0053] Option 8

[0054]

[0055] Dissolve 9 mmol of 60% sodium hydrogen in 20 mL of anhydrous tetrahydrofuran and stir at room temperature until no more bubbles are generated. Replace the air in the reaction flask with argon gas, and slowly add 6 mmol of substituted acetonitrile dropwise under ice bath conditions (0°C). After stirring for half an hour, add 3 mmol of the starting material methyl ester in batches. After the addition is complete, remove the ice bath and allow the mixture to return to room temperature for 2-6 hours. Quench the reaction with 20 mL of water, remove the tetrahydrofuran by vacuum distillation, and adjust the pH of the residual aqueous phase to 2 with 1 mol / L dilute hydrochloric acid. A solid gradually precipitates out; filter, wash with ice water, and dry. The target product is obtained and can be directly used in the next step without further purification.

[0056] Option Nine

[0057]

[0058] 0.28 mmol of substituted N-(2-methoxy-5-nitrophenyl)-2-phenylpyrazolo[1,5-a]pyrimidine-6-carboxamide was added to 3 mL of anhydrous DMF, along with 0.864 mmol of LiCl powder. The mixture was heated to 150 °C and refluxed for 8 h. After heating was stopped and the mixture was cooled to room temperature, 20 mL of water was added, and a solid precipitated. Filtering yielded 32 mg of a yellow solid, with a yield of 27.58%.

[0059] Option 10

[0060]

[0061] 1.52 mmol of 4-(cyanoacetyl)benzoic acid, 1.52 mmol of substituted benzyl alcohol, 3.04 mmol of 4-dimethylaminopyridine, and 1.52 mmol of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride were dissolved in 10 mL of dichloromethane solution, and the mixture was stirred at room temperature for 10 h. The solvent dichloromethane was removed by vacuum distillation, and the crude product was purified by silica gel column chromatography (elution: petroleum ether / ethyl acetate, gradient 0-20%) to obtain the target product.

[0062] Example 1

[0063] 4-(6-(2-hydroxy-5-nitrobenzoyl)pyrazol[1,5-a]pyrimidin-2-yl)-N-(4-(trifluoromethyl)benzyl)benzamide

[0064]

[0065] The compound was prepared using Scheme Six, starting with 0.14 mmol of 6-nitrochrome-3-carboxaldehyde and 0.14 mmol of 4-(3-amino-1H-pyrazol-5-yl)-N-[2-(trifluoromethyl)benzyl]benzamide, yielding 44 mg of the target compound as a yellow solid, with a yield of 57.25%. 1 H NMR (300MHz, DMSO-d6) δ11.92(s,1H),9.50(d,J=2.2Hz,1H),9.021(t,J=5.8 Hz,1H),8.90(d,J=2.2Hz,1H),8.37(d,J=7.6Hz,2H),8.23(d,J=8.1Hz,2H),8 .10(d,J=8.1Hz,2H),7.77(d,J=7.8Hz,1H),7.69(t,J=7.6Hz,1H),7.58(d,J =4.5Hz,2H),7.50(t,J=7.6Hz,1H),7.25–7.14(m,1H),4.72(d,J=5.6Hz,2H).

[0066] Preparation of 4-(2-cyanoacetyl)benzoic acid

[0067] The compound was prepared using Scheme 3, starting with 9.21 mmol of methyl 4-(2-cyanoacetyl)benzoate, yielding 1.6 g of the target compound as a white solid with a yield of 91.86%.

[0068] Preparation of 4-(2-cyanoacetyl)-N-[4-(trifluoromethyl)benzyl]benzamide

[0069] The compound was prepared using scheme four, with 1.59 mmol of 4-(2-cyanoacetyl)benzoic acid as the starting material. After the reaction was completed by TLC monitoring, the reaction solution was concentrated under reduced pressure to obtain 730 mg of the target crude product, a red oily liquid, which was directly added to the next step without any further treatment.

[0070] Preparation of 4-(3-amino-1H-pyrazole-5-yl)-N-[4-(trifluoromethyl)benzyl]benzamide

[0071] The compound was prepared using Scheme 5, using the crude product of 4-(2-cyanoacetyl)-N-[4-(trifluoromethyl)benzyl]benzamide from the previous step as a starting material to obtain 240 mg of the target compound as a white solid. The two-step yield was 41.89%.

[0072] Preparation of 6-nitrochrome-3-carboxaldehyde

[0073] The compound was prepared using Scheme 1, starting with 11.04 mmol of 1-(2-hydroxy-5-nitrophenyl)ethane-1-one, yielding 1.8 g of the target compound as a pale yellow solid. The yield was 74.39%.

[0074] Example 2

[0075] 4-(6-(2-hydroxy-5-nitrobenzoyl)pyrazol[1,5-a]pyrimidin-2-yl)-N-(3-(trifluoromethyl)benzyl)benzamide

[0076]

[0077] The preparation was carried out using Scheme 6, with 0.14 mmol of 6-nitrochrome-3-carboxaldehyde and 0.14 mmol of 4-(3-amino-1H-pyrazol-5-yl)-N-(3-(trifluoromethyl)benzyl)benzamide as starting materials, yielding 56 mg of the target compound as a yellow solid, in a yield of 72.86%. mp: 1 H NMR (300MHz, DMSO-d6) δ11.92(s,1H),9.50(d,J=2.2Hz,1H),9.29(t,J=6.0Hz,1H),8.90(d,J=2.2Hz,1H),8.43–8.3 4(m,2H),8.22(d,J=8.2Hz,2H),8.07(d,J=8.4Hz,2H),7.75–7.57(m,5H),7.24–7.15(m,1H),4.62(d,J=5.8Hz,2H).

[0078] Preparation of 4-(2-cyanoacetyl)-N-[3-(trifluoromethyl)benzyl]benzamide

[0079] The compound was prepared using scheme four, with 1.59 mmol of 4-(2-cyanoacetyl)benzoic acid as the starting material. After the reaction was completed by TLC monitoring, the reaction solution was concentrated under reduced pressure to obtain 694 mg of the target crude product, a red oily liquid, which was directly added to the next step without any further treatment.

[0080] Preparation of 4-(3-amino-1H-pyrazole-5-yl)-N-[3-(trifluoromethyl)benzyl]benzamide

[0081] The compound was prepared using Scheme 5, using the crude product of 4-(2-cyanoacetyl)-N-[3-(trifluoromethyl)benzyl]benzamide from the previous step as a starting material to obtain 230 mg of the target compound as a white solid. The two-step yield was 40.14%.

[0082] Example 3

[0083] Preparation of 4-(6-(2-hydroxy-5-nitrobenzoyl)pyrazol[1,5-a]pyrimidin-2-yl)-N-(2-(trifluoromethyl)benzyl)benzamide

[0084]

[0085] The preparation method of Scheme 6 was used, with 0.14 mmol of 6-nitrochrome-3-carboxaldehyde and 0.14 mmol of 4-(3-amino-1H-pyrazol-5-yl)-N-[2-(trifluoromethyl)benzyl]benzamide as raw materials, to obtain 44 mg of the target compound as a yellow solid, with a yield of 57.25%. mp:1H NMR (300MHz, DMSO-d6) δ11.92(s,1H),9.50(d,J=2.2Hz,1H),9.021(t,J=5.8 Hz,1H),8.90(d,J=2.2Hz,1H),8.37(d,J=7.6Hz,2H),8.23(d,J=8.1Hz,2H),8 .10(d,J=8.1Hz,2H),7.77(d,J=7.8Hz,1H),7.69(t,J=7.6Hz,1H),7.58(d,J =4.5Hz,2H),7.50(t,J=7.6Hz,1H),7.25–7.14(m,1H),4.72(d,J=5.6Hz,2H).

[0086] Preparation of 4-(2-cyanoacetyl)-N-[2-(trifluoromethyl)benzyl]benzamide

[0087] The compound was prepared using scheme four, with 1.59 mmol of 4-(2-cyanoacetyl)benzoic acid as the starting material. After the reaction was completed by TLC monitoring, the reaction solution was concentrated under reduced pressure to obtain 629 mg of the target crude product, a red oily liquid, which was directly added to the next step without any further treatment.

[0088] Preparation of 4-(3-amino-1H-pyrazole-5-yl)-N-[2-(trifluoromethyl)benzyl]benzamide

[0089] The compound was prepared using Scheme 5, using the crude product of 4-(2-cyanoacetyl)-N-[2-(trifluoromethyl)benzyl]benzamide from the previous step as a starting material to obtain 240 mg of the target compound as a white solid. The two-step yield was 41.89%.

[0090] Example 4

[0091] Preparation of N-(4-cyanobenzyl)-4-(6-(2-hydroxy-5-nitrobenzoyl)pyrazolo[1,5-a]pyrimidin-2-yl)benzamide

[0092]

[0093] The preparation method of Scheme 6 was used, with 0.14 mmol of 6-nitrochrome-3-carboxaldehyde and 0.14 mmol of 4-(3-amino-1H-pyrazol-5-yl)-N-(4-cyanobenzyl)benzamide as raw materials, to obtain 36 mg of the target compound as a yellow solid, with a yield of 50.72%. mp:1H NMR (300MHz, DMSO-d6) δ11.93(s,1H),9.50(d,J=2.2Hz,1H),9.30(t,J=6.0Hz,1H),8.90(d,J=2.2Hz,1H),8.42–8.32(m,2H),8 .22(d,J=8.1Hz,2H),8.07(d,J=8.2Hz,2H),7.84(d,J=8.2Hz,2H),7.61–7.51(m,3H),7.25–7.16(m,1H),4.61(d,J=5.8Hz,2H).

[0094] Preparation of 4-(2-cyanoacetyl)-N-(4-cyanobenzyl)benzamide

[0095] The compound was prepared using scheme four, with 1.59 mmol of 4-(2-cyanoacetyl)benzoic acid as the starting material. After the reaction was completed by TLC monitoring, the reaction solution was concentrated under reduced pressure to obtain 610 mg of the target crude product, a red oily liquid, which was directly added to the next step without any further treatment.

[0096] Preparation of 4-(3-amino-1H-pyrazole-5-yl)-N-(4-cyanobenzyl)benzamide

[0097] The compound was prepared using Scheme 5, using the crude product of 4-(2-cyanoacetyl)-N-(4-cyanobenzyl)benzamide from the previous step as a starting material to obtain 190 mg of the target compound as a white solid. The two-step yield was 37.65%.

[0098] Example 5

[0099] Preparation of N-(3-cyanobenzyl)-4-(6-(2-hydroxy-5-nitrobenzoyl)pyrazolo[1,5-a]pyrimidin-2-yl)benzamide

[0100]

[0101] The preparation method of Scheme 6 was used, with 0.14 mmol of 6-nitrochrome-3-carboxaldehyde and 0.14 mmol of 4-(3-amino-1H-pyrazol-5-yl)-N-(3-cyanobenzyl)benzamide as starting materials, to give 48 mg of the target compound as a yellow solid, with a yield of 67.63%. mp: 158.1–160.8 °C. 1 H NMR (300MHz, DMSO-d6) δ11.91(s,1H),9.50(d,J=2.2Hz,1H),9.25(t,J=6.0Hz,1H),8.90(d,J=2.2Hz,1H),8.40–8.32(m,2H), 8.22(d,J=8.2Hz,2H),8.07(d,J=8.2Hz,2H),7.83–7.67(m,3H),7.63–7.54(m,2H),7.27–7.14(m,1H),4.58(d,J=5.8Hz,2H). 13 C NMR(75MHz,DMSO-d6)δ190.24,166.36,162.34,150.14,141.79,140.28,140.18,132.77,131.32,131 .20,130.14,129.12,128.58,127.17,126.84,125.60,119.54,119.36,118.20,111.74,95.96,42.64.

[0102] Preparation of 4-(2-cyanoacetyl)-N-(3-cyanobenzyl)benzamide

[0103] The compound was prepared using scheme four, with 1.59 mmol of 4-(2-cyanoacetyl)benzoic acid as the starting material. After the reaction was completed by TLC monitoring, the reaction solution was concentrated under reduced pressure to obtain 620 mg of the target crude product, a red oily liquid, which was directly added to the next step without any further treatment.

[0104] Preparation of 4-(3-amino-1H-pyrazole-5-yl)-N-(3-cyanobenzyl)benzamide

[0105] The compound was prepared using Scheme 5, using the crude product of 4-(2-cyanoacetyl)-N-(3-cyanobenzyl)benzamide from the previous step as a starting material to obtain 240 mg of the target compound as a white solid. The two-step yield was 35.28%.

[0106] Example 6

[0107] Preparation of N-(cyclohexylmethyl)-4-[6-(2-hydroxy-5-nitrobenzoyl)pyrazol[1,5-a]pyrimidin-2-yl]benzamide

[0108]

[0109] The preparation was carried out using Scheme 6, with 0.14 mmol of 6-nitrochrome-3-carboxaldehyde and 0.14 mmol of 4-(3-amino-1H-pyrazol-5-yl)-N-(cyclohexylmethyl)benzamide as starting materials, yielding 54 mg of the target compound as a yellow solid, with a yield of 78.79%. mp: 1 H NMR (300MHz, DMSO-d6) δ11.92(s,1H),9.49(d,J=2.2Hz,1H),8.89(d,J=2.2Hz,1H),8.56(t,J=5.8Hz,1H),8.43–8.30(m,2H),8.18(d,J=8.1Hz,2H ),8.00(d,J=8.2Hz,2H),7.56(s,1H),7.28–7.15(m,1H),3.15(t,J=6.3H z,2H),1.83–1.50(m,6H),1.20(t,J=10.4Hz,3H),0.97(t,J=11.6Hz,2H).

[0110] Preparation of 4-(2-cyanoacetyl)-N-(cyclohexylmethyl)benzamide

[0111] The compound was prepared using scheme four, with 1.59 mmol of 4-(2-cyanoacetyl)benzoic acid as the starting material. After the reaction was completed by TLC monitoring, the reaction solution was concentrated under reduced pressure to obtain 750 mg of the target crude product, a red oily liquid, which was directly added to the next step without any further treatment.

[0112] Preparation of 4-(3-amino-1H-pyrazole-5-yl)-N-(cyclohexylmethyl)benzamide

[0113] The compound was prepared using Scheme 5, using the crude 4-(2-cyanoacetyl)-N-(cyclohexylmethyl)benzamide product from the previous step as a starting material to obtain 302 mg of the target compound as a white solid. The two-step yield was 63.65%.

[0114] Example 7

[0115] Preparation of N-(cyclopentylmethyl)-4-[6-(2-hydroxy-5-nitrobenzoyl)pyrazol[1,5-a]pyrimidin-2-yl]benzamide

[0116]

[0117] The preparation was carried out using Scheme 6, with 0.14 mmol of 6-nitrochrome-3-carboxaldehyde and 0.14 mmol of 4-(3-amino-1H-pyrazol-5-yl)-N-(cyclopentylmethyl)benzamide as starting materials, yielding 45 mg of the target compound as a yellow solid, with a yield of 67.71%. mp: 1 H NMR (300MHz, DMSO-d6) δ11.93(s,1H),9.49(d,J=2.2Hz,1H),8.89(d,J=2.2H z,1H),8.61(t,J=5.8Hz,1H),8.36(d,J=8.2Hz,2H),8.18(d,J=8.1Hz,2H),8 .00(d,J=8.2Hz,2H),7.55(s,1H),7.19(d,J=8.8Hz,1H),3.28–3.19(m,2H), 2.17(h,J=7.3Hz,1H),1.81–1.44(m,6H),1.28(dq,J=14.7,5.1,3.2Hz,2H).

[0118] Preparation of 4-(2-cyanoacetyl)-N-(cyclopentylmethyl)benzamide

[0119] The compound was prepared using scheme four, with 1.59 mmol of 4-(2-cyanoacetyl)benzoic acid as the starting material. After the reaction was completed by TLC monitoring, the reaction solution was concentrated under reduced pressure to obtain 650 mg of the target crude product, a dark red oily liquid, which was directly added to the next step without any further treatment.

[0120] Preparation of 4-(3-amino-1H-pyrazol-5-yl)-N-(cyclopentylmethyl)benzamide

[0121] The compound was prepared using Scheme 5, using the crude 4-(2-cyanoacetyl)-N-(cyclopentylmethyl)benzamide product from the previous step as a starting material to obtain 220 mg of the target compound as a white solid. The two-step yield was 48.66%.

[0122] Example 8

[0123] Preparation of N-(cyclobutylmethyl)-4-[6-(2-hydroxy-5-nitrobenzoyl)pyrazol[1,5-a]pyrimidin-2-yl]benzamide

[0124]

[0125] The preparation was carried out using Scheme 6, with 0.14 mmol of 6-nitrochrome-3-carboxaldehyde and 0.14 mmol of 4-(3-amino-1H-pyrazol-5-yl)-N-(cyclobutylmethyl)benzamide as starting materials, yielding 38 mg of the target compound as a yellow solid, in a yield of 58.88%. mp: 1 H NMR (300MHz, DMSO-d6) δ11.91(s,1H),9.49(d,J=2.2Hz,1H),8.89(d,J=2.2Hz, 1H),8.57(t,J=5.7Hz,1H),8.37(d,J=7.2Hz,2H),8.18(d,J=8.1Hz,2H),7.99( d,J=8.2Hz,2H),7.55(s,1H),7.34–7.10(m,1H),3.34(t,J=6.4Hz,2H),2.58(q ,J=7.4Hz,1H),2.09–1.96(m,2H),1.81(dddd,J=28.9,17.8,10.3,7.2Hz,4H).

[0126] Preparation of 4-(2-cyanoacetyl)-N-(cyclobutylmethyl)benzamide

[0127] The compound was prepared using scheme four, with 1.59 mmol of 4-(2-cyanoacetyl)benzoic acid as the starting material. After the reaction was completed by TLC monitoring, the reaction solution was concentrated under reduced pressure to obtain 668 mg of the target crude product, an orange-red oily liquid, which was directly added to the next step without any further treatment.

[0128] Preparation of 4-(3-amino-1H-pyrazole-5-yl)-N-(cyclobutylmethyl)benzamide

[0129] The compound was prepared using Scheme 5, using the crude 4-(2-cyanoacetyl)-N-(cyclobutylmethyl)benzamide product from the previous step as a starting material to obtain 167 mg of the target compound as a white solid. The two-step yield was 38.85%.

[0130] Example 9

[0131] N-(cyclopropylmethyl)-4-[6-(2-hydroxy-5-nitrobenzoyl)pyrazol[1,5-a]pyrimidin-2-yl]benzamide

[0132]

[0133] The preparation was carried out using Scheme 6, with 0.14 mmol of 6-nitrochrome-3-carboxaldehyde and 0.14 mmol of 4-(3-amino-1H-pyrazol-5-yl)-N-(cyclobutylmethyl)benzamide as starting materials, yielding 34 mg of the target compound as a yellow solid, in a yield of 54.30%. mp: 1 H NMR (300MHz, DMSO-d6) δ11.93(s,1H),9.49(d,J=2.2Hz,1H),8.89(d,J=2.2H z,1H),8.70(t,J=5.8Hz,1H),8.37(d,J=7.6Hz,2H),8.18(d,J=8.1Hz,2H),8 .01(d,J=8.1Hz,2H),7.56(s,1H),7.28–7.13(m,1H),3.19(t,J=6.2Hz,2H), 1.06(td,J=7.5,4.1Hz,1H), 0.46(dt,J=8.5,2.9Hz,2H), 0.33–0.19(m,2H).

[0134] Preparation of 4-(2-cyanoacetyl)-N-(cyclopropylmethyl)benzamide

[0135] The compound was prepared using scheme four, with 1.59 mmol of 4-(2-cyanoacetyl)benzoic acid as the starting material. After the reaction was completed by TLC monitoring, the reaction solution was concentrated under reduced pressure to obtain 702 mg of the target crude product, a dark red oily liquid, which was directly added to the next step without any further treatment.

[0136] Preparation of 4-(3-amino-1H-pyrazole-5-yl)-N-(cyclopropylmethyl)benzamide

[0137] The compound was prepared using Scheme 5, using the crude 4-(2-cyanoacetyl)-N-(cyclopropylmethyl)benzamide product from the previous step as a starting material to obtain 244 mg of the target compound as a white solid. The two-step yield was 59.87%.

[0138] Example 10

[0139] N-(bicyclo[1.1.1]pentan-1-yl)-4-(6-(2-hydroxy-5-nitrobenzoyl)pyrazolo[1,5-a]pyrimidin-2-yl)benzamide

[0140]

[0141] The preparation was carried out using Scheme 6, with 0.14 mmol of 6-nitrochrome-3-carboxaldehyde and 0.14 mmol of 4-(3-amino-1H-pyrazol-5-yl)-N-(bicyclo[1.1.1]pentan-1-ylmethyl)benzamide as starting materials, yielding 34 mg of the target compound as a yellow solid, with a yield of 54.30%. mp: 1 H NMR (300MHz, DMSO-d6) δ9.49(d,J=2.2Hz,1H),9.09(s,1H),8.89(d,J=2.2Hz,1H),8.37(d,J=7.3Hz,2H),8 .17(d,J=8.1Hz,2H),7.99(d,J=8.2Hz,2H),7.56(s,1H),7.19(d,J=9.0Hz,1H),2.49(s,1H),2.13(s,6H).

[0142] Preparation of N-(bicyclo[1.1.1]pentane)-4-(2-cyanoacetyl)benzamide

[0143] The compound was prepared using scheme four, with 1.59 mmol of 4-(2-cyanoacetyl)benzoic acid as the starting material. After the reaction was completed by TLC monitoring, the reaction solution was concentrated under reduced pressure to obtain 802 mg of the target crude product, a dark red oily liquid, which was directly added to the next step without any further treatment.

[0144] Preparation of 4-(3-amino-1H-pyrazole-5-yl)-N-(bicyclo[1.1.1]pentan-1-yl)benzamide

[0145] The compound was prepared using Scheme 5, using the crude product of N-(bicyclo[1.1.1]pentane-1-methyl)-4-(2-cyanoacetyl)benzamide from the previous step as a starting material, yielding 210 mg of the target compound as a white solid. The two-step yield was 46.78%.

[0146] Example 11

[0147] Preparation of 4-[6-(2-hydroxy-5-nitrobenzoyl)pyrazol[1,5-a]pyrimidin-2-yl]phenyl}(piperidin-1-yl)methyl ketone

[0148]

[0149] The preparation was carried out using Scheme 6, starting with 0.14 mmol of 6-nitrochrome-3-carboxaldehyde and 0.14 mmol of [4-(3-amino-1H-pyrazol-5-yl)phenyl](piperidin-1-yl)methyl ketone, yielding 40 mg of the target compound as a yellow solid, in a yield of 61.98%. mp: 1H NMR (300MHz, DMSO-d6) δ11.92(s,1H),9.49(d,J=2.2Hz,1H),8.88(d,J=2.2Hz,1H),8.44–8.34(m,2H),8.15(d,J =8.2Hz,2H),7.60–7.43(m,3H),7.33–7.06(m,1H),3.61(s,2H),3.38(d,J=021.0Hz,2H),1.59(d,J=29.4Hz,6H).

[0150] Preparation of 3-oxo-3-[4-(piperidin-1-carbonyl)phenyl]propionitrile

[0151] The compound was prepared using scheme four, with 1.59 mmol of 4-(2-cyanoacetyl)benzoic acid and piperidine as raw materials. After the reaction was completed by TLC monitoring, the reaction solution was concentrated under reduced pressure to obtain 750 mg of the target crude product, a pale yellow liquid, which was directly added to the next step without any treatment.

[0152] Preparation of [4-(3-amino-1H-pyrazol-5-yl)phenyl](piperidin-1-yl)methyl ketone (21a)

[0153] The compound was prepared using Scheme 5, using the crude product of [4-(3-amino-1H-pyrazol-5-yl)phenyl](piperidin-1-yl)methyl ketone from the previous step as a starting material, yielding 205 mg of the target compound as a white solid. The two-step yield was 47.69%.

[0154] Example 12

[0155] Preparation of (4,4-dimethylpiperidin-1-yl){4-[6-(2-hydroxy-5-nitrobenzoyl)pyrazol[1,5-a]pyrimidin-2-yl]phenyl} ketone

[0156]

[0157] The preparation was carried out using Scheme 6, starting with 0.14 mmol of 6-nitrochrome-3-carboxaldehyde and 0.14 mmol of [4-(3-amino-1H-pyrazol-5-yl)phenyl](4,4-dimethylpiperidin-1-yl)methyl ketone, yielding 33 mg of the target compound as a yellow solid, with a yield of 46.80%. mp: 1H NMR (300MHz, DMSO-d6) δ9.47(d,J=2.2Hz,1H),8.88(d,J=2.1Hz,1H),8.44–8.29(m,2H),8.15(d,J=8 .2Hz,2H),7.53(dd,J=8.2,1.7Hz,3H),7.14(d,J=9.0Hz,1H),3.64(s,2H),1.36(s,4H),0.99(s,6H).

[0158] Preparation of 3-[4-(4,4-dimethylpiperidin-1-carbonyl)phenyl]-3-oxopropionitrile

[0159] The compound was prepared using scheme four, with 1.59 mmol of 4-(2-cyanoacetyl)benzoic acid as the starting material. After the reaction was completed by TLC monitoring, the reaction solution was concentrated under reduced pressure to obtain 650 mg of the target crude product, an orange-red oily liquid, which was directly added to the next step without any further treatment.

[0160] Preparation of [4-(3-amino-1H-pyrazol-5-yl)phenyl](4,4-dimethylpiperidin-1-yl)methyl ketone

[0161] The compound was prepared using Scheme 5, using the crude product of 3-[4-(4,4-dimethylpiperidin-1-carbonyl)phenyl]-3-oxypropionitrile from the previous step as a starting material, yielding 152 mg of the target compound as a white solid. The two-step yield was 32.04%.

[0162] Example 13

[0163] Preparation of (4-(6-(2-hydroxy-5-nitrobenzoyl)pyrazolo[1,5-a]pyrimidin-2-yl)phenyl)(pyrrolidine-1-yl)methyl ketone

[0164]

[0165] The preparation was carried out using Scheme 6, starting with 0.14 mmol of 6-nitrochrome-3-carboxaldehyde and 0.14 mmol of [4-(5-amino-1H-pyrazol-3-yl)phenyl](pyrrolidine-1-yl)methyl ketone, yielding 32 mg of the target compound as a yellow solid, with a yield of 68.29%. mp: 1H NMR (300MHz, DMSO-d6) δ12.43(s,1H),11.90(s,1H),9.48(d,J=2.2Hz,1H),9.20(s,1H),8.89(d,J=2.2Hz,1H),8 .37(d,J=7.3Hz,2H),8.18(d,J=8.2Hz,2H),7.99(d,J=8.4Hz,2H),7.56(s,1H),7.27–7.13(m,1H),2.33(s,6H).

[0166] Preparation of 3-oxo-3-(4-(pyrrolidine-1-carbonyl)phenyl)propionitrile

[0167] The compound was prepared using scheme four, with 1.59 mmol of 4-(2-cyanoacetyl)benzoic acid as the starting material. After the reaction was completed by TLC monitoring, the reaction solution was concentrated under reduced pressure to obtain 740 mg of the target crude product, an orange-red oily liquid, which was directly added to the next step without any further treatment.

[0168] Preparation of (4-(3-amino-1H-pyrazol-5-yl)phenyl)(pyrrolidine-1-yl)methyl ketone

[0169] The compound was prepared using Scheme 5, using the crude product of 3-oxo-3-(4-(pyrrolidine-1-carbonyl)phenyl)propionitrile (20c) from the previous step as a starting material, yielding 182 mg of the target compound as a white solid. The two-step yield was 44.66%.

[0170] Example 14

[0171] Preparation of azirmonobutane-1-yl{4-[6-(2-hydroxy-5-nitrobenzoyl)pyrazol[1,5-a]pyrimidin-2-yl]phenyl} methyl ketone (DDO-02161)

[0172]

[0173] The preparation was carried out using Scheme 6, starting with 0.14 mmol of 6-nitrochrome-3-carboxaldehyde and 0.14 mmol of [4-(3-amino-1H-pyrazol-5-yl)phenyl](azacyclobutane-1-yl)methyl ketone, yielding 32 mg of the target compound as a yellow solid, in a yield of 57.66%. mp: 1H NMR (300MHz, DMSO-d6) δ11.91(d,J=12.8Hz,1H),9.49(d,J=2.2Hz,1H),8.89(d,J=2.2Hz,1H),8.37(d,J=7.3Hz,2H),8.16(d,J=8.1 Hz,2H),7.87–7.71(m,2H),7.54(s,1H),7.19(d,J=9.1Hz,1H),4.37(t,J=7.7Hz,2H),4.09(t,J=7.7Hz,2H),2.30(p,J=7.7Hz,2H).

[0174] Preparation of 3-[4-(azacyclobutane-1-carbonyl)phenyl]-3-oxopropionitrile

[0175] The compound was prepared using scheme four, with 1.59 mmol of 4-(2-cyanoacetyl)benzoic acid as the starting material. After the reaction was completed by TLC monitoring, the reaction solution was concentrated under reduced pressure to obtain 780 mg of the target crude product, a brownish-yellow oily liquid, which was directly added to the next step without any further treatment.

[0176] Preparation of [4-(3-amino-1H-pyrazol-5-yl)phenyl](azacyclobutane-1-yl)methyl ketone

[0177] The compound was prepared using Scheme 5, using the crude product of 3-[4-(azacyclobutyl-1-carbonyl)phenyl]-3-oxopropionitrile from the previous step as a starting material, yielding 92 mg of the target compound as a white solid. The two-step yield was 23.88%.

[0178] Example 15

[0179] Preparation of 3,3-difluoroazacyclobutane-1-yl){4-[6-(2-hydroxy-5-nitrobenzoyl)pyrazol[1,5-a]pyrimidin-2-yl]phenyl} methyl ketone

[0180]

[0181] The preparation was carried out using Scheme 6, starting with 0.14 mmol of 6-nitrochrome-3-carboxaldehyde and 0.14 mmol of [4-(3-amino-1H-pyrazol-5-yl)phenyl](3,3-difluoroazacyclobutane-1-yl) methyl ketone, yielding 31 mg of the target compound as a yellow solid, with a yield of 49.05%. mp: 1H NMR (300MHz, DMSO-d6) δ11.94(s,1H),9.53(d,J=2.2Hz,1H),8.93(d,J=2.2Hz,1H),8.45–8.37(m,2H) ,8.22(d,J=8.3Hz,2H),7.89(d,J=8.4Hz,2H),7.61(s,1H),7.33–7.12(m,1H),4.73(d,J=86.9Hz,4H).

[0182] Preparation of 3-[4-(3,3-difluoroazacyclobutane-1-carbonyl)phenyl]-3-oxopropionitrile

[0183] The compound was prepared using scheme four, with 1.59 mmol of 4-(2-cyanoacetyl)benzoic acid as the starting material. After the reaction was completed by TLC monitoring, the reaction solution was concentrated under reduced pressure to obtain 590 mg of the target crude product, a red oily liquid, which was directly added to the next step without any further treatment.

[0184] Preparation of [4-(3-amino-1H-pyrazol-5-yl)phenyl](3,3-difluoroazacyclobutane-1-yl)methyl ketone

[0185] The compound was prepared using Scheme 5, using the crude product of 3-[4-(3,3-difluoroazacyclobutane-1-carbonyl)phenyl]-3-oxopropionitrile from the previous step as a starting material, yielding 152 mg of the target compound as a white solid. The two-step yield was 34.36%.

[0186] Example 16

[0187] Preparation of (2-(4-(2-oxa-6-azaspiro[3.3]heptane-6-carbonyl)phenyl)pyrazolo[1,5-a]pyrimidin-6-yl)(2-hydroxy-5-nitrophenyl)methyl ketone

[0188]

[0189] The preparation was carried out using Scheme 6, with 0.14 mmol of 6-nitrochrome-3-carboxaldehyde and 0.14 mmol of (4-(3-amino-1H-pyrazol-5-yl)phenyl)(2-oxa-6-azaspiro[3.3]heptane-6-yl) methyl ketone as starting materials, yielding 41 mg of the target compound as a yellow solid, in a yield of 56.78%. mp: 1H NMR (300MHz, DMSO-d6) δ11.94(s,1H),9.53(d,J=2.2Hz,1H),8.93(d,J=2.2Hz,1H),8.45–8.37(m,2H) ,8.22(d,J=8.3Hz,2H),7.89(d,J=8.4Hz,2H),7.61(s,1H),7.33–7.12(m,1H),4.73(d,J=86.9Hz,4H).

[0190] Preparation of 3-(4-(2-oxo-6-azaspiro[3.3]heptane-6-carbonyl)phenyl)-3-oxopropionitrile

[0191] The compound was prepared using scheme four, with 1.59 mmol of 4-(2-cyanoacetyl)benzoic acid as the starting material. After the reaction was completed by TLC monitoring, the reaction solution was concentrated under reduced pressure to obtain 590 mg of the target crude product, a red oily liquid, which was directly added to the next step without any further treatment.

[0192] Preparation of 4-(3-amino-1H-pyrazol-5-yl)phenyl)(2-oxa-6-azaspiro[3.3]heptane-6-yl)methyl ketone

[0193] The compound was prepared using Scheme 5, using the crude product of 3-(4-(2-oxo-6-azaspiro[3.3]heptane-6-carbonyl)phenyl)-3-oxopropionitrile from the previous step as a starting material, yielding 166 mg of the target compound as a white solid. The two-step yield was 36.72%.

[0194] Example 17

[0195] Preparation of 4-(6-(2,6-dihydroxy-3-nitrobenzoyl)pyrazolo[1,5-a]pyrimidin-2-yl)-N-(4-(trifluoromethyl)benzyl)benzamide

[0196]

[0197] The compound was prepared using method six, with 0.13 mmol of 5-hydroxy-6-nitrochrome-3-carboxaldehyde and 0.13 mmol of 4-(3-amino-1H-pyrazol-5-yl)-N-[4-(trifluoromethyl)benzyl]benzamide as starting materials, yielding 32 mg of the target compound as a yellow solid, with a yield of 43.44%. 1H NMR (300MHz, DMSO-d6) δ11.76(s,2H),10.96(s,1H),9.65(dd,J=36.1,2.3Hz,1H),9.30(t,J=6.0Hz,1H),8.99(dd,J=5.1,2.2Hz,1H),8 .33–8.13(m,3H),8.07(d,J=8.1Hz,2H),7.73(d,J=8.1Hz,2H),7.66–7.54(m,3H),6.85(dd,J=87.7,9.3Hz,1H),4.61(d,J=5.7Hz,2H).

[0198] 5-Hydroxy-6-nitro-4-oxo-4H-methylene-3-carboxaldehyde

[0199] The compound was prepared using method five. Starting with 1.00 mmol of 2'-hydroxy-5'-nitro-6'-fluoroacetophenone, 150 mg of a pale yellow solid was obtained, with a yield of 63.5%. m / z (EI-MS): 236.2 [M] +

[0200] 2'-Hydroxy-5'-nitro-6'-fluoroacetophenone

[0201] Using the preparation method of Scheme 2, starting with 6.49 mmol of 2'-hydroxy-6'-fluoroacetophenone, 480 mg of the pale yellow target product was obtained, with a yield of 37.15%. m / z (EI-MS): 198.1 [M] -

[0202] Example 18

[0203] Preparation of 4-(6-(2,6-dihydroxy-3-nitrobenzoyl)pyrazolo[1,5-a]pyrimidin-2-yl)-N-(3-(trifluoromethyl)benzyl)benzamide

[0204]

[0205] The compound was prepared using method six, starting with 0.13 mmol of 5-hydroxy-6-nitrochrome-3-carboxaldehyde and 0.13 mmol of 4-(3-amino-1H-pyrazol-5-yl)-N-[4-(trifluoromethyl)benzyl]benzamide, yielding 27 mg of the target compound as a yellow solid, with a yield of 36.65%. 1H NMR (300MHz, DMSO-d6) δ9.71(d,J=2.2Hz,1H),9.31(t,J=6.0Hz,1H),9.00(d,J=2.2Hz,1H),8.39–8 .16(m,3H),8.07(d,J=8.1Hz,2H),7.77–7.53(m,5H),7.01(d,J=9.3Hz,1H),4.61(d,J=5.8Hz,2H).

[0206] Example 18

[0207] Preparation of 4-(6-(2,6-dihydroxy-3-nitrobenzoyl)pyrazolo[1,5-a]pyrimidin-2-yl)-N-(2-(trifluoromethyl)benzyl)benzamide

[0208]

[0209] The compound was prepared using method six, starting with 0.13 mmol of 5-hydroxy-6-nitrochrome-3-carboxaldehyde and 0.13 mmol of 4-(3-amino-1H-pyrazol-5-yl)-N-[4-(trifluoromethyl)benzyl]benzamide, yielding 36 mg of the target compound as a yellow solid, mp: 1 H NMR (300MHz, DMSO-d6) δ9.71(d,J=2.2Hz,1H),9.31(t,J=6.0Hz,1H),9.00(d,J=2.2Hz,1H),8.39–8 .16(m,3H),8.07(d,J=8.1Hz,2H),7.77–7.53(m,5H),7.01(d,J=9.3Hz,1H),4.61(d,J=5.8Hz,2H).

[0210] Example 19

[0211] Preparation of N-(3-cyanobenzene)-4-[6-(2-hydroxy-5-nitrobenzoyl)pyrazol[1,5-a]pyrimidin-2-yl]benzamide

[0212]

[0213] The compound was prepared using method six, starting with 0.13 mmol of 5-hydroxy-6-nitrochrome-3-carboxaldehyde and 0.13 mmol of 4-(5-amino-1H-pyrazol-3-yl)-N-(3-cyanophenyl)carboxamide, yielding 51 mg of the target compound as a yellow solid, with a yield of 55.89%. 1H NMR (300MHz, DMSO-d6) δ11.77(s,1H),10.91(s,1H),9.58(d,J=2.2Hz,1H),9.25(t,J=6.0Hz,1H),8.99(dd,J=5.3,2.2Hz,1H) ,8.30–8.13(m,3H),8.06(d,J=8.1Hz,2H),7.84–7.67(m,3H),7.64–7.51(m,2H),6.70(d,J=9.4Hz,1H),4.57(d,J=5.8Hz,2H).

[0214] Example 20

[0215] Preparation of N-(4-cyanobenzene)-4-[6-(2-hydroxy-5-nitrobenzoyl)pyrazol[1,5-a]pyrimidin-2-yl]benzamide

[0216]

[0217] The compound was prepared using method six, starting with 0.13 mmol of 5-hydroxy-6-nitrochrome-3-carboxaldehyde and 0.13 mmol of 4-(3-amino-1H-pyrazol-5-yl)-N-(4-cyanobenzyl)benzamide, yielding 51 mg of the target compound as a yellow solid, with a yield of 55.89%. 1 H NMR (300MHz, DMSO-d6) δ11.35(d,J=227.8Hz,1H),9.59(s,1H),9.30(t,J=6.0Hz,1H),8.99(t,J=2.7Hz,1H),8.25–8.1 4(m,3H),8.07(d,J=8.1Hz,2H),7.84(d,J=8.1Hz,2H),7.65–7.47(m,3H),6.70(d,J=9.4Hz,1H),4.60(d,J=5.8Hz,2H). 13 CNMR(75MHz,DMSO-d6)δ189.28,166.38,163.36,158.24,154.45,150.31,149.37,145.98,140.48,135.37, 134.85,132.81,129.27,128.56,128.25,1021.85,119.47,119.43,115.06,110.06,109.90,96.23,43.04.

[0218] Example 21

[0219] Preparation of N-(cyclohexylmethyl)-4-[6-(2,6-dihydroxy-3-nitrobenzoyl)pyrazol[1,5-a]pyrimidin-2-yl]benzamide

[0220]

[0221] The compound was prepared using method six, starting with 0.13 mmol of 5-hydroxy-6-nitrochrome-3-carboxaldehyde and 0.13 mmol of 4-(3-amino-1H-pyrazol-5-yl)-N-(cyclohexylmethyl)benzamide, yielding 29 mg of the target compound as a yellow solid, with a yield of 44.09%. 1 H NMR (300MHz, DMSO-d6) δ11.21(s,1H),9.57(s,1H),8.99(d,J=4.9Hz,1H),8.56(t,J=5.8Hz,1H),8.23–8.13(m,3H),8.00(d,J=8.2Hz,2H ),7.59(d,J=6.9Hz,1H),6.70(d,J=9.4Hz,1H),3.15(t,J=6.3Hz,2H),1.83–1.51(m,6H),1.18(d,J=8.9Hz,3H),0.94(q,J=11.7Hz,2H).

[0222] Example 22

[0223] Preparation of N-(cyclopentylmethyl)-4-[6-(2,6-dihydroxy-3-nitrobenzoyl)pyrazol[1,5-a]pyrimidin-2-yl]benzamide

[0224]

[0225] The compound was prepared using method six, starting with 0.13 mmol of 5-hydroxy-6-nitrochrome-3-carboxaldehyde and 0.13 mmol of 4-(3-amino-1H-pyrazol-5-yl)-N-(cyclopentylmethyl)benzamide, yielding 32 mg of the target compound as a yellow solid, with a yield of 50.02%. 1H NMR (300MHz, DMSO-d6) δ11.68(s,1H),10.95(s,1H),9.58(s,1H),8.98(s,1H),8.57(t,J=5.8Hz,1H),8.18(d,J=8.6Hz,3H),8.00(d,J=8.1H z,2H),7.58(s,1H),6.70(d,J=9.4Hz,1H),3.46(q,J=6.9Hz,1H),3.34(s,3H),2.58(d,J=7.4Hz,1H),2.11–1.95(m,2H),1.94–1.67(m,4H).

[0226] Example 23

[0227] Preparation of N-(cyclobutylmethyl)-4-(6-(2,6-dihydroxy-3-nitrobenzoyl)pyrazolo[1,5-a]pyrimidin-2-yl)benzamide

[0228]

[0229] The compound was prepared using method six, starting with 0.13 mmol of 5-hydroxy-6-nitrochrome-3-carboxaldehyde and 0.13 mmol of 4-(3-amino-1H-pyrazol-5-yl)-N-(cyclobutylmethyl)benzamide, yielding 28 mg of the target compound as a yellow solid, with a yield of 45.02%. 1 H NMR (300MHz, DMSO-d6) δ11.37(s,1H),9.57(s,1H),8.98(s,1H),8.57(t,J=5.8Hz,1H),8.29–8.15(m,3H),8.00(d,J=8.4Hz,2H),7.59(d,J =6.6Hz,1H),6.69(dd,J=9.3,2.2Hz,1H),3.34(d,J=12.9Hz,3H),2.02(tt,J=12.1,4.6Hz,2H),1.82(dddd,J=29.0,17.9,10.3,7.1Hz,4H).

[0230] Example 24

[0231] Preparation of N-(cyclopropylmethyl)-4-[6-(2,6-dihydroxy-3-nitrobenzoyl)pyrazol[1,5-a]pyrimidin-2-yl]benzamide

[0232]

[0233] The compound was prepared using method six, starting with 0.13 mmol of 5-hydroxy-6-nitrochrome-3-carboxaldehyde and 0.13 mmol of 4-(3-amino-1H-pyrazol-5-yl)-N-(cyclopropylmethyl)benzamide, yielding 35 mg of the target compound as a yellow solid, with a yield of 57.95%. 1 H NMR (300MHz, DMSO-d6) δ11.50(s,2H),9.58(s,1H),8.99(d,J=4.6Hz,1H),8.70(t,J=5.7Hz,1H),8.23–8.14(m,3H),8.02(d,J=8.5H z,2H),7.60(d,J=6.6Hz,1H),6.71(d,J=9.4Hz,1H),3.19(t,J=6.2Hz,2H),1.15–1.00(m,1H),0.57–0.42(m,2H),0.35–0.20(m,2H).

[0234] Example 25

[0235] Preparation of N-(bicyclo[1.1.1]pentan-1-yl)-4-[6-(2,6-dihydroxy-3-nitrobenzoyl)pyrazol[1,5-a]pyrimidin-2-yl]benzamide

[0236]

[0237] The compound was prepared using method six, starting with 0.13 mmol of 5-hydroxy-6-nitrochrome-3-carboxaldehyde and 0.13 mmol of 4-(5-amino-1H-pyrazol-3-yl)-N-(bicyclo[1.1.1]pentan-1-yl)benzamide, yielding 21 mg of the target compound as a yellow solid, with a yield of 41.98%. 1 H NMR (300MHz, DMSO-d6) δ11.38(s,1H),9.54(s,1H),9.08(s,1H),8.97(d,J=6.8Hz,1H),8.22–8.11(m ,3H),7.99(d,J=8.1Hz,2H),7.58(d,J=8.7Hz,1H),6.68(d,J=9.5Hz,1H),2.49(s,1H),2.12(s,6H).

[0238] Example 26

[0239] (4-(6-(2,6-dihydroxy-3-nitrobenzoyl)pyrazolo[1,5-a]pyrimidin-2-yl)phenyl)(pyrrolidine-1-yl)methyl ketone

[0240]

[0241] The compound was prepared using method six, starting with 0.13 mmol of 5-hydroxy-6-nitrochromene-3-carboxaldehyde and 0.13 mmol of (4-(6-(2-hydroxy-5-nitrobenzoyl)pyrazolo[1,5-a]pyrimidin-2-yl)phenyl)(pyrrolidine-1-yl)methyl ketone, yielding 48 mg of the target compound as a yellow solid, with a yield of 79.47%. 1 H NMR(300MHz,DMSO-d6)δ9.58(s,1H),8.98(s,1H),8.16(t,J=9.4Hz,4H),7.78–7.55(m ,4H),6.70(d,J=9.4Hz,1H),3.46(q,J=6.6,5.1Hz,4H),1.87(dq,J=13.2,6.9Hz,5H).

[0242] Example 27

[0243] Azacyclobutane-1-yl(4-(6-(2,6-dihydroxy-3-nitrobenzoyl)pyrazolo[1,5-a]pyrimidin-2-yl)phenyl) methyl ketone

[0244]

[0245] The compound was prepared using method six, starting with 0.13 mmol of 5-hydroxy-6-nitrochrome-3-carboxaldehyde and 0.13 mmol of [4-(3-amino-1H-pyrazol-5-yl)phenyl](azacyclobutane-1-yl)methyl ketone, yielding 39 mg of the target compound as a yellow solid with a yield of 66.54%. 1 H NMR(500MHz,DMSO-d6)δ9.24(d,J=1.6Hz,1H),8.00(d,J=7.5Hz,1H),7.94–7.88(m,2H),7.62–7.56(m,2H ),7.06(s,1H),6.97(d,J=7.5Hz,1H),3.66(t,J=7.1Hz,2H),3.60(t,J=7.1Hz,2H),1.99(p,J=7.1Hz,2H).

[0246] Example 28

[0247] Preparation of (3,3-difluoroazacyclobutane-1-yl){4-[6-(2-6-dihydroxy-5-nitrobenzoyl)pyrazol[1,5-a]pyrimidin-2-yl]phenyl} ketone

[0248]

[0249] The compound was prepared using method six, starting with 0.13 mmol of 5-hydroxy-6-nitrochromene-3-carboxaldehyde and 0.13 mmol of [4-(3-amino-1H-pyrazol-5-yl)phenyl](3,3-difluoroazacyclobutane-1-yl) methyl ketone, yielding 31 mg of the target compound as a yellow solid, with a yield of 49.05%. 1 H NMR(300MHz,DMSO-d6)δ11.45(s,1H),9.58(s,1H),9.03–8.94(m,1H),8.18(dt,J=9.5,2.6Hz ,3H),7.91–7.82(m,2H),7.61(d,J=7.0Hz,1H),6.70(d,J=9.4Hz,1H),4.70(d,J=87.1Hz,4H).

[0250] Example 29

[0251] Preparation of (2-(4-(2-oxo-6-azaspiro[3.3]heptane-6-carbonyl)phenyl)pyrazolo[1,5-a]pyrimidin-6-yl)(2,6-dihydroxy-3-nitrophenyl) methyl ketone

[0252]

[0253] The compound was prepared using method six, starting with 0.13 mmol of 5-hydroxy-6-nitrochromene-3-carboxaldehyde and 0.13 mmol of (4-(3-amino-1H-pyrazol-5-yl)phenyl)(2-oxa-6-azaspiro[3.3]heptane-6-yl) methyl ketone, yielding 27 mg of the target compound as a yellow solid, with a yield of 42.20%. 1 H NMR (300MHz, DMSO-d6) δ11.37(s,1H),9.58(s,1H),8.98(d,J=6.7Hz,1H),8.16(dd,J=9.0,4.0Hz,3H),7. 77(d,J=8.0Hz,2H),7.59(d,J=8.3Hz,1H),6.69(d,J=9.2Hz,1H),4.71(s,4H),4.55(s,2H),4.25(s,2H).

[0254] Example 30

[0255] Preparation of (2-(4-chlorophenyl)-3-ethylpyrazol[1,5-a]pyrimidin-6-yl)(2-hydroxy-5-nitrophenyl) methyl ketone

[0256]

[0257] The compound was prepared using method six, starting with 0.37 mmol of 6-nitrochrome-3-carboxaldehyde and 0.37 mmol of 3-(4-chlorophenyl)-4-ethyl-1H-pyrazole-5-amine, yielding 110 mg of the target compound as a yellow solid, with a yield of 71.27%. 1 H NMR (300MHz, DMSO-d6) δ11.91(s,1H),9.42(d,J=2.2Hz,1H),8.86(d,J=2.2Hz,1H),8.36(dd,J=6.5,2.9Hz, 2H),7.93–7.82(m,2H),7.75–7.57(m,2H),7.31–7.17(m,1H),3.00(q,J=7.5Hz,2H),1.25(t,J=7.4Hz,3H). 13 C NMR(75MHz,DMSO-d6)δ190.21,166.20,162.29,158.08,150.10,140.22,136.03,134.48,12 9.09,128.41,127.15,126.70,125.58,119.46,118.17,95.88,44.94,35.27,25.78,18.32.

[0258] Preparation of 3-(4-chlorophenyl)-4-ethyl-1H-pyrazole-5-amine

[0259] The compound was prepared using method five, starting with 4.33 mmol of 2-(4-chlorobenzoyl)butyronitrile, yielding 680 mg of the target compound as a white solid. The yield was 70.77%.

[0260] Preparation of 2-(4-chlorobenzoyl)butyronitrile

[0261] The compound was prepared using method eight, with 5.68 mmol of methyl p-chlorobenzoate and 11.72 mmol of butyronitrile as raw materials, yielding 960 mg of the target compound as a green oily liquid with a yield of 78.86%.

[0262] Example 31

[0263] Preparation of (2-(4-chlorophenyl)-3-isopropylpyrazole[1,5-a]pyrimidin-6-yl)(2-hydroxy-5-nitrophenyl) methyl ketone

[0264]

[0265] The compound was prepared using method six, starting with 0.37 mmol of 6-nitrochrome-3-carboxaldehyde and 0.37 mmol of 3-(4-chlorophenyl)-4-isopropyl-1H-pyrazole-5-amine, yielding 124 mg of the target compound as a yellow solid, with a yield of 77.76%. 1 H NMR (300MHz, DMSO-d6) δ11.93(s,1H),9.42(d,J=2.2Hz,1H),8.85(d,J=2.2Hz,1H),8.44–8.2 9(m,2H),7.78–7.53(m,4H),7.021–7.15(m,1H),3.29(p,J=6.9Hz,1H),1.48(d,J=6.9Hz,6H). 13 C NMR(75MHz,DMSO-d6)δ190.23,162.19,156.30,148.08,146.62,140.20,139.97,134.39,131.87 ,130.91,129.28,128.99,127.07,125.55,118.89,118.10,115.33,31.46,24.76,22.93,22.56.

[0266] Preparation of 3-(4-chlorophenyl)-4-isopropyl-1H-pyrazole-5-amine

[0267] The compound was prepared using method five, starting with 4.33 mmol of 2-(4-chlorobenzoyl)-3-methylbutyronitrile, yielding 680 mg of the target compound as a white solid. The yield was 70.77%.

[0268] Preparation of 2-(4-chlorobenzoyl)-3-methylbutyronitrile

[0269] The compound was prepared using method eight, with 5.68 mmol of methyl p-chlorobenzoate and 11.72 mmol of isovaleronitrile as raw materials, yielding 880 mg of the target compound as a green oily liquid with a yield of 69.17%.

[0270] Example 32

[0271] Preparation of (2-(4-chlorophenyl)-3-cyclopropylpyrazole[1,5-a]pyrimidin-6-yl)(2-hydroxy-5-nitrophenyl) methyl ketone (DDO-02117)

[0272]

[0273] The compound was prepared using method six, starting with 0.19 mmol of 6-nitrochrome-3-carboxaldehyde and 0.19 mmol of 3-(4-chlorophenyl)-4-cyclopropyl-1H-pyrazole-5-amine, yielding 65 mg of the target compound as a yellow solid, with a yield of 77.63%. 1 H NMR 11.91(300MHz, DMSO-d6)δ(s,1H),9.39(d,J=2.2Hz,1H),8.82(d,J=2.2Hz,1H),8.45–8.30(m,2H),8. 18–8.01(m,2H),7.78–7.61(m,2H),7.35–7.13(m,1H),2.05(tt,J=7.4,5.8Hz,1H),1.10–0.96(m,5H).

[0274] Preparation of 3-(4-chlorophenyl)-4-cyclopropyl-1H-pyrazole-5-amine

[0275] The compound was prepared using method five, starting with 4.33 mmol of 3-(4-chlorophenyl)-2-cyclopropyl-3-oxypropionitrile, yielding 605 mg of the target compound as a white solid. The yield was 47.39%.

[0276] Preparation of 3-(4-chlorophenyl)-2-cyclopropyl-3-oxopropionitrile

[0277] The compound was prepared using method eight, with 5.68 mmol of methyl p-chlorobenzoate and 11.72 mmol of cyclopropylacetonitrile as raw materials, yielding 1.2 g of the target compound as a green oily liquid, with a yield of 84.72%.

[0278] Example 33

[0279] Preparation of (2-(4-chlorophenyl)-3-cyclohexylpyrazol[1,5-a]pyrimidin-6-yl)(2-hydroxy-5-nitrophenyl) methyl ketone

[0280]

[0281] The compound was prepared using method six, starting with 0.23 mmol of 6-nitrochrome-3-carboxaldehyde and 0.23 mmol of 3-(4-chlorophenyl)-4-cyclohexyl-1H-pyrazole-5-amine, yielding 80 mg of the target compound as a yellow solid, with a yield of 82.93%. 1H NMR (300MHz, DMSO-d6) δ11.90(s,1H),9.40(d,J=2.2Hz,1H),8.85(d,J=2.2Hz,1H),8.40–8.29(m,2H),7.74–7.61(m ,4H),7.29–7.14(m,1H),2.87(t,J=12.2Hz,1H),2.14(d,J=11.7Hz,2H),1.81(d,J=10.5Hz,4H),1.41–1.23(m,4H).

[0282] Preparation of 3-(4-chlorophenyl)-4-cyclohexyl-1H-pyrazole-5-amine

[0283] The compound was prepared using method five, starting with 4.33 mmol of 2-(4-chlorobenzoyl)-3-methylbutyronitrile, yielding 160 mg of the target compound as a white solid. The yield was 18.98%.

[0284] Preparation of 3-(4-chlorophenyl)-2-cyclohexyl-3-oxopropionitrile

[0285] The compound was prepared using method eight, with 5.68 mmol of methyl p-chlorobenzoate and 11.72 mmol of cyclohexylacetonitrile as raw materials, yielding 925 mg of the target compound as a green oily liquid, with a yield of 62.29%.

[0286] Example 34

[0287] Preparation of (2-(4-chlorophenyl)-3-methoxypyrazole[1,5-a]pyrimidin-6-yl)(2-hydroxy-5-nitrophenyl) methyl ketone (DDO-02119)

[0288]

[0289] The compound was prepared using method six, starting with 0.23 mmol of 6-nitrochrome-3-carboxaldehyde and 0.23 mmol of 3-(4-chlorophenyl)-4-methoxy-1H-pyrazole-5-amine, yielding 65 mg of the target compound as a yellow solid, with a yield of 77.63%. 1 H NMR (300MHz, DMSO-d6) δ9.30(d,J=2.1Hz,1H),8.74(d,J=2.1Hz,1H),8.37(dd,J=6.6,3. 0Hz,2H),8.13(d,J=8.5Hz,2H),7.62(d,J=8.5Hz,2H),7.23–7.14(m,1H),4.021(s,3H).

[0290] Preparation of 3-(4-chlorophenyl)-4-methoxy-1H-pyrazole-5-amine

[0291] The compound was prepared using method five, starting with 5.87 mmol of crude 3-(4-chlorophenyl)-2-methoxy-3-oxopropionitrile, yielding 520 mg of the target compound as a white solid. The yield was 39.62%.

[0292] Preparation of 3-(4-chlorophenyl)-2-methoxy-3-oxopropionitrile

[0293] The compound was prepared using method eight, with 5.68 mmol of methyl p-chlorobenzoate and 11.72 mmol of methoxyacetonitrile as raw materials, yielding 1.2 g of crude product of the target compound, which was a green oily liquid.

[0294] Example 35

[0295] Preparation of 2-(4-chlorophenyl)-N-(5-cyano-2-hydroxyphenyl)pyrazole[1,5-a]pyrimidine-6-carboxamide

[0296]

[0297] The compound was prepared using Scheme 9, starting with 0.28 mmol of 2-(4-chlorophenyl)-N-(5-cyano-2-methoxyphenyl)pyrazolo[1,5-a]pyrimidine-6-carboxamide, yielding 32 mg of a yellow solid with a yield of 27.58%. mp: 223.9-225.1 °C. 1 H NMR (300MHz, DMSO-d6) δ10.16(s,1H),9.74(d,J=2.2Hz,1H),8.99(d,J=2.2Hz,1H),8.80(d,J=2.9Hz,1H ),8.23–8.09(m,2H),8.06(dd,J=9.0,2.9Hz,1H),7.69–7.56(m,2H),7.47(s,1H),7.12(d,J=9.0Hz,1H).

[0298] Preparation of 2-(4-chlorophenyl)-N-(5-cyano-2-methoxyphenyl)pyrazolo[1,5-a]pyrimidine-6-carboxamide

[0299] The compound was prepared using Scheme 4, with 0.73 mmol of 2-(4-chlorophenyl)pyrazole[1,5-a]pyrimidine-6-carboxylic acid and 1.10 mmol of 3-amino-4-methoxybenzonitrile as raw materials, yielding 140 mg of yellow solid, with a yield of 47.44%.

[0300] Preparation of 2-(4-chlorophenyl)pyrazole[1,5-a]pyrimidine-6-carboxylic acid

[0301] The compound was prepared using Scheme 3, starting with 0.93 mmol of ethyl 2-(4-chlorophenyl)pyrazole[1,5-a]pyrimidine-6-carboxylate, yielding 230 mg of white solid with a yield of 90.56%.

[0302] Preparation of ethyl 2-(4-(chlorophenyl)pyrazole[1,5-a]pyrimidine-6-carboxylate

[0303] The compound was prepared using method seven, starting with 1.03 mmol of 5-(4-chlorophenyl)-1H-pyrazole-3-amine, yielding 288 mg of a white solid with a yield of 92.41%.

[0304] Preparation of 5-(4-chlorophenyl)-1H-pyrazole-3-amine

[0305] The compound was prepared using method five, with 5.57 mol of p-chlorobenzoylacetonitrile as the starting material, yielding 630 mg of white solid, with a yield of 58.43%.

[0306] Example 36

[0307] Preparation of 2-(4-chlorophenyl)-N-(5-nitro-2-hydroxyphenyl)pyrazole[1,5-a]pyrimidine-6-carboxamide

[0308]

[0309] The compound was prepared using Scheme 9, starting with 0.28 mmol of 2-(4-chlorophenyl)-N-(2-methoxy-5-nitrophenyl)pyrazole[1,5-a]pyrimidine-6-carboxamide, yielding 32 mg of a yellow solid with a yield of 27.58%. mp: 223.9-225.1 °C. 1 H NMR (300MHz, DMSO-d6) δ10.16(s,1H),9.74(d,J=2.2Hz,1H),8.99(d,J=2.2Hz,1H),8.80(d,J=2.9Hz,1H ),8.23–8.09(m,2H),8.06(dd,J=9.0,2.9Hz,1H),7.69–7.56(m,2H),7.47(s,1H),7.12(d,J=9.0Hz,1H).

[0310] Preparation of 2-(4-chlorophenyl)-N-(2-methoxy-5-nitrophenyl)pyrazolo[1,5-a]pyrimidine-6-carboxamide

[0311] The compound was prepared using Scheme 4, with 0.73 mmol of 2-(4-chlorophenyl)pyrazole[1,5-a]pyrimidine-6-carboxylic acid and 1.10 mmol of 2-methoxy-5-nitroaniline as raw materials, yielding 140 mg of yellow solid, with a yield of 47.44%.

[0312] Example 37

[0313] 4-(6-(2-hydroxy-5-nitrobenzoyl)pyrazolo[1,5-a]pyrimidin-2-yl)benzoic acid 4-(trifluoromethyl)benzyl ester

[0314]

[0315] The compound was prepared using Scheme 6, using 0.14 mmol of 6-nitrochrome-3-carboxaldehyde and 0.14 mmol of 4-(3-amino-1H-pyrazol-5-yl)benzoic acid 4-(trifluoromethyl)benzyl ester as raw materials, yielding 44 mg of the target compound as a yellow solid with a yield of 57.25%. 1 H NMR (300MHz, DMSO-d6) δ8.84(d,J=1.5Hz,1H),8.60(d,J=1.5Hz,1H),8.09(dd,J=7.5,1.5Hz,1H),8.04–7.98(m,2H),7.6 9–7.63(m,2H),7.60–7.55(m,2H),7.52(dt,J=7.5,1.1Hz,2H),7.12(d,J=7.5Hz,1H),7.06(s,1H),5.27(t,J=1.0Hz,2H).

[0316] Preparation of 4-(2-cyanoacetyl)benzoic acid 4-(trifluoromethyl)benzyl ester

[0317] The compound was prepared using method ten, with 1.59 mmol of 4-(2-cyanoacetyl)benzoic acid and 4-(trifluoromethyl)benzyl alcohol as raw materials. After the reaction was completed by TLC monitoring, the reaction solution was concentrated under reduced pressure to obtain 730 mg of the target crude product, a yellow oily liquid, which was directly added to the next step without any further treatment.

[0318] Preparation of 4-(3-amino-1H-pyrazole-5-yl)benzoic acid 4-(trifluoromethyl)benzyl ester

[0319] The compound was prepared using method five, using the crude product of 4-(2-cyanoacetyl)benzoic acid 4-(trifluoromethyl)benzyl ester from the previous step as a starting material to obtain 240 mg of the target compound as a white solid. The two-step yield was 41.89%.

[0320] Example 38

[0321]

[0322] The compound was prepared using Scheme 6, using 0.14 mmol of 5-hydroxy-6-nitrochrome-3-carboxaldehyde and 0.14 mmol of 4-(3-amino-1H-pyrazol-5-yl)benzoic acid 4-(trifluoromethyl)benzyl ester as raw materials, yielding 44 mg of the target compound as a yellow solid with a yield of 57.25%. 1 H NMR(300MHz,DMSO-d6)δ11.29(d,J=130.7Hz,1H),9.75–9.56(m,1H),9.08–8.96(m,1H),8.3 7–8.13(m,5H),7.89–7.71(m,4H),7.64(d,J=5.9Hz,1H),6.72(d,J=9.3Hz,1H),5.52(s,2H).

[0323] Example 39

[0324]

[0325] The compound was prepared using Scheme 6, using 0.14 mmol of 6-nitrochrome-3-carboxaldehyde and 0.14 mmol of benzyl 4-(3-amino-1H-pyrazol-5-yl)benzoate as starting materials, yielding 44 mg of the target compound as a yellow solid with a yield of 57.25%. 1 HNMR(300MHz,DMSO-d6))δ8.81(d,J=1.5Hz,1H),8.58(d,J=1.5Hz,1H),8.09(dd,J=7.5,1.5Hz,1H),8.07–8 .02(m,2H),7.60–7.54(m,2H),7.38–7.27(m,5H),7.12(d,J=7.5Hz,1H),7.06(s,1H),5.22(d,J=1.0Hz,2H).

[0326] Preparation of benzyl 4-(2-cyanoacetyl)benzoate

[0327] The compound was prepared using method ten, with 1.59 mmol of 4-(2-cyanoacetyl)benzoic acid and benzyl alcohol as raw materials. After the reaction was completed by TLC monitoring, the reaction solution was concentrated under reduced pressure to obtain 730 mg of the target crude product, a yellow oily liquid, which was directly added to the next step without any treatment.

[0328] Preparation of benzyl 4-(3-amino-1H-pyrazole-5-yl)benzoate

[0329] The compound was prepared using method five, using the crude product of 4-(2-cyanoacetyl)benzoate from the previous step as a starting material to obtain 240 mg of the target compound as a white solid. The two-step yield was 41.89%.

[0330] Example 40

[0331]

[0332] The compound was prepared using Scheme 6, using 0.14 mmol of 6-nitrochrome-3-carboxaldehyde and 0.14 mmol of 4-(3-amino-1H-pyrazol-5-yl)benzoic acid 4-(trifluoromethyl)benzyl ester as raw materials, yielding 44 mg of the target compound as a yellow solid with a yield of 57.25%. 1 H NMR (300MHz, DMSO-d6) δ8.84(d,J=1.5Hz,1H),8.60(d,J=1.5Hz,1H),8.09(dd,J=7.5,1.5Hz,1H),8.04–7.98(m,2H),7 .77(p,J=1.0Hz,1H),7.60–7.53(m,4H),7.48–7.42(m,1H),7.12(d,J=7.5Hz,1H),7.06(s,1H),5.25(t,J=1.0Hz,2H).

[0333] Preparation of 3-(trifluoromethyl)benzyl 4-(2-cyanoacetyl)benzoic acid

[0334] The compound was prepared using method ten, with 1.59 mmol of 4-(2-cyanoacetyl)benzoic acid and 3-(trifluoromethyl)benzyl alcohol as raw materials. After the reaction was completed by TLC monitoring, the reaction solution was concentrated under reduced pressure to obtain 730 mg of the target crude product, a yellow oily liquid, which was directly added to the next step without any further treatment.

[0335] Preparation of 3-(trifluoromethyl)benzyl ester of 4-(3-amino-1H-pyrazol-5-yl)benzoic acid

[0336] The compound was prepared using method five, using the crude product of 3-(trifluoromethyl)benzyl 4-(2-cyanoacetyl)benzoic acid from the previous step as a starting material to obtain 240 mg of the target compound as a white solid. The two-step yield was 41.89%.

[0337] Example 41

[0338]

[0339] The compound was prepared using Scheme 6, starting with 0.14 mmol of 5-hydroxy-6-nitrochrome-3-carboxaldehyde and 0.14 mmol of 3-(trifluoromethyl)benzyl 4-(3-amino-1H-pyrazol-5-yl)benzoic acid, yielding 44 mg of the target compound as a yellow solid with a yield of 57.25%. 1 H NMR(300MHz,DMSO-d6)δ9.02(d,J=1.5Hz,1H),8.05–7.98(m,3H),7.77(h,J=1.0Hz,1H),7.6 0–7.53(m,4H),7.48–7.42(m,1H),7.06(s,1H),6.92(d,J=7.5Hz,1H),5.25(t,J=1.0Hz,2H).

[0340] Example 42

[0341]

[0342] The compound was prepared using Scheme 6, using 0.14 mmol of 6-nitrochrome-3-carboxaldehyde and 0.14 mmol of 4-(3-amino-1H-pyrazol-5-yl)benzoic acid 4-(trifluoromethyl)benzyl ester as raw materials, yielding 44 mg of the target compound as a yellow solid with a yield of 57.25%. 1 H NMR (300MHz, DMSO-d6) δ8.83(d,J=1.5Hz,1H),8.58(d,J=1.5Hz,1H),8.09(dd,J=7.4,1.6Hz,1H),8.07–8.02(m,2H),7.6 0–7.54(m,2H),7.19–7.14(m,2H),7.12(d,J=7.5Hz,1H),7.08–7.01(m,3H),5.25(t,J=1.0Hz,2H),2.34(d,J=0.8Hz,3H).

[0343] Preparation of 4-(2-cyanoacetyl)benzoic acid 4-methyl benzyl ester

[0344] The compound was prepared using method ten, with 1.59 mmol of 4-(2-cyanoacetyl)benzoic acid and 4-methylbenzyl alcohol as raw materials. After the reaction was completed by TLC monitoring, the reaction solution was concentrated under reduced pressure to obtain 730 mg of the target crude product, a yellow oily liquid, which was directly added to the next step without any further treatment.

[0345] Preparation of 4-(3-amino-1H-pyrazole-5-yl)benzoic acid 4-methyl benzyl ester

[0346] The compound was prepared using method five, using the crude product of 3-(trifluoromethyl)benzyl 4-(2-cyanoacetyl)benzoic acid from the previous step as a starting material to obtain 240 mg of the target compound as a white solid. The two-step yield was 41.89%.

[0347] This invention constructs a fluorescent molecular probe based on a segment of ssDNA that binds to ALKBH5 (the fluorescent probe used is m 6 A-ssDNA-FAM was used as a method to study the competitive binding of this class of compounds to ALKBH5 and ssDNA. The inhibition rate of this class of compounds under different concentration conditions was measured, and then the IC50 was calculated. 50 The experimental procedure was as follows: Add 20 μL of ALKBH5 protein, 20 μL of fluorescent probe, and 20 μL of compounds at different concentration gradients to each well of a 384-well plate. After incubation for 0.5 hours, use a multi-mode microplate reader to read the fluorescence at an excitation wavelength of 485 nm and an emission wavelength of 535 nm. Calculate the mP value, calculate the inhibition rate using the following formula, and then calculate the IC50 using GraphPad software. 50 The values ​​are shown in Table 1.

[0348] Competition combined m 6 The A binding site inhibits the demethylase activity of ALKBH5, thereby regulating the downregulation of TACC3 expression and inhibiting the proliferation of leukemia cells.

[0349] Table 1. IC50 of the compounds of the present invention against ALKBH5 protein 50 value

[0350]

[0351]

[0352] The results above show that the compounds of the present invention can act as small molecule inhibitors of ALKBH5 protein, inhibiting RNA demethylases at the protease level.

Claims

1. A compound as shown below: ###0001### or a pharmaceutically acceptable salt thereof. 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 。 2. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein, The pharmaceutically acceptable salt is a sodium salt, a potassium salt, a lithium salt, a calcium salt, a zinc salt, a magnesium salt, a meglumine salt, a tromethamine salt, a diethylamine salt, or a ethanolamine salt of the compound.

3. A pharmaceutical composition, characterized by, A pharmaceutical composition comprising the compound of claim 1 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

4. Use of the compound of claim 1 or a pharmaceutically acceptable salt thereof in the preparation of a therapeutic drug for a disease related to the function of ALKBH5 protein.

5. Use according to claim 4, characterized in that, The disease related to the function of ALKBH5 protein is acute leukemia, lung cancer, breast cancer, colon cancer, prostate cancer, pancreatic cancer, gastric cancer, liver cancer, ovarian cancer, kidney cancer, glioma, melanoma, myeloma, lymphoma, bladder cancer, or cervical cancer.

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