7-difluoromethyl-5-arylpyrazolopyrimidines, preparation and use thereof

Abstract

The present application relates to a kind of 7-difluoromethyl-5-aryl pyrazolopyrimidine compound and preparation and application, the structural general formula of the compound is as shown in formula a, it is a kind of compound with inhibiting HIF-2 alpha activity, can block HIF-2 alpha / ARNT dimerization and play the role of HIF-2 transcription inhibition.The present application is proved by multiple experiments, the synthesized compound all has excellent HIF-2 transcription inhibition effect, in human renal clear cell carcinoma cell strain 786-O, show good VEGF protein inhibitory activity.The compound described in the present application can be applied in the preparation of hypoxia-inducible factor-2 alpha (HIF-2 alpha) inhibitor for treating VHL deletion or / and HIF-2 alpha abnormal expression disease.This kind of compound can inhibit the overexpression and activation of HIF-2 alpha, to achieve the treatment and prevention of target disease, such as VHL deletion or / and HIF-2 alpha abnormal expression disease.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of medicine, and particularly relates to a 7-difluoromethyl-5-aryl pyrazolopyrimidine compound and a preparation method and application thereof in preparation of a hypoxia inducible factor-2 alpha inhibitor drug. BACKGROUND

[0002] Renal cell carcinoma (RCC) is also known as renal adenocarcinoma, which is a malignant tumor originating from the renal tubular epithelial system. In recent years, the incidence of renal cell carcinoma has been increasing, and about 134,000 patients die from it every year worldwide. The pathological types of renal cell carcinoma mainly include clear cell renal cell carcinoma (ccRCC), papillary renal cell carcinoma (pRCC), chromophobe renal cell carcinoma (chRCC), etc. Among them, ccRCC is the most common form of renal cancer (85%), which is characterized by the inactivation of tumor suppressor von Hippel-Lindau (VHL) due to genetic predisposition, somatic mutation or methylation. VHL acts as a substrate recognition subunit of E3 ubiquitin ligase complex, which targets the degradation of hypoxia induced factors (HIF) and regulates the stress response of cells to hypoxia. The loss of VHL function will cause HIF to accumulate in cancer cells and transcribe downstream target genes, promoting tumor proliferation and metastasis.

[0003] At present, the targeted therapeutic drugs for advanced ccRCC are mainly represented by VEGFR inhibitors such as sorafenib and sunitinib, but these drugs mainly act on downstream factors promoting neovascularization, have strong cardiotoxicity, and are prone to drug resistance. Studies have shown that HIF-2 can regulate cell proliferation, metabolism, angiogenesis, and tumor metastasis and other physiological processes, and is associated with the poor prognosis of various cancers. In particular, in VHL-deficient clear cell renal cell carcinoma (ccRCC), HIF-2 is considered to be a key driver of tumor growth, and therefore, HIF-2 has become a new target for the treatment of clear cell renal cell carcinoma. - / -

[0004] ​Currently, small molecule inhibitors targeting HIF-2α / ARNT dimerization can specifically bind to the cavity unique to the PAS-B domain of HIF-2α, and have high selectivity for HIF-2. For example, PT-2385 (J. Med. Chem. 2018, 61, 9691), Belzutifan (MK-6482, PT-2977) (J. Med. Chem. 2019, 62, 6876), THS-044 (J. Am. Chem. Soc. 2009, 131, 17647), 0X3 (J. Med. Chem. 2013, 56, 1739), and tetrazolohydropyrimidine derivative (S, R)-37 (J. Med. Chem. 2015, 58, 5930), etc. Among them, Belzutifan, as a selective HIF-2α / ARNT dimerization inhibitor, was approved for marketing on August 31, 2021, and is used for the treatment of recurrent glioblastoma, and renal clear cell carcinoma related to von Hippel-Lindau syndrome.

[0005] Therefore, it can be seen that the HIF-2α / ARNT dimerization small molecule inhibitor has good clinical application prospects, but at present, the structural types thereof are few, and therefore, it is of important clinical significance to find and discover a novel skeleton HIF-2 inhibitor for the treatment of renal clear cell carcinoma and other tumors.

[0006] The reported HIF-2α inhibitor has the following structural formula:

[0007] SUMMARY

[0008] In view of the fact that the structural types of the existing HIF-2 inhibitors are few, the purpose of the present application is to provide a 7-difluoromethyl-5-aryl pyrazolopyrimidine compound having the structure of general formula a:

[0009]

[0010] In formula a:

[0011] R0 and R1 are independently selected from hydrogen, nitro, halogen, cyano, hydroxyl, amino, methyl, methoxy, difluoromethyl, trifluoromethyl, difluoromethoxy and trifluoromethoxy;

[0012] R2 is selected from hydrogen, C 1-6 alkyl, C 1-6 fluoroalkyl;

[0013] R3 is selected from hydrogen, cyano, aldehyde, C 1-6 alkyl, C 1-6 fluoroalkyl, C 1-3 alkylsulfone, C 3-8cycloalkyl, -CH2OR c , -CH2NR c R d , -COR c , -COOR c , -CONR c R d , wherein R c , R d are each independently selected from hydrogen, amino, hydroxyl, C 1-6 alkyl, C 1-6 fluoroalkyl, C 1-6 alkoxy.

[0014] Further, the preferred 7-difluoromethyl-5-arylpyrazolopyrimidine compounds of the present application, R0, R1 are each selected from hydrogen, fluorine, chlorine, bromine, cyano, methyl, methoxy, difluoromethyl, trifluoromethyl; R2 is selected from hydrogen, methyl; R3 is selected from hydrogen, cyano, aldehyde, methylsulfone, cyclopropyl,

[0015] In particular, the preferred compounds of the 7-difluoromethyl-5-arylpyrazolopyrimidine derivatives of the present application of the general structure a are selected from one of the following:

[0016]

[0017]

[0018] A second object of the present application is to provide a method for the preparation of said compounds, the 7-difluoromethyl-5-arylpyrazolopyrimidine derivatives of the general structure a are prepared by the following steps, but not limited to the following method.

[0019] 1. Synthesis method of the compound IA series:

[0020]

[0021] The specific reaction process can be:

[0022] The substituted acetophenone Ia is condensed with ethyl 2,2-difluoroacetate to obtain the 1-substituted phenyl-4,4-difluorobutane-1,3-dione intermediate Ib;

[0023] The intermediate Ib is subjected to cyclization reaction with substituted 3-aminopyrazole Ic in the presence of glacial acetic acid to obtain the target compound IA of the IA series.

[0024] 2. Synthesis method of the compound IB series:

[0025]

[0026] The specific reaction process can be:

[0027] The intermediate IA is subjected to hydrolysis reaction under alkaline conditions to obtain the intermediate IBa of pyrazolopyrimidine-3-carboxylic acid;

[0028] The intermediate IBa is subjected to nucleophilic substitution reaction with an amine compound to obtain the target compound of the IB series.

[0029] 3. Synthesis method of the compound II series:

[0030]

[0031] The specific reaction process can be:

[0032] The 3-amino-1H-pyrazole-4-carboxylic acid ethyl ester is subjected to condensation reaction with 4,4-difluoro-3-oxobutanoic acid ethyl ester under acidic conditions to obtain the intermediate II-b;

[0033] The intermediate II-b is subjected to hydrolysis reaction under the action of lithium hydroxide to obtain the chloro intermediate II-c;

[0034] The intermediate II-c is subjected to hydrolysis reaction under the action of lithium hydroxide to obtain the chloro intermediate II-c;

[0035] The intermediate II-d is dissolved in dichloromethane with methylamine hydrochloride and a condensing agent, and is stirred at room temperature overnight to obtain the amide intermediate II-e;

[0036] The intermediate II-e is subjected to Suziki coupling reaction with different substituted phenylboronic acids to obtain the target compound of the II series.

[0037] 4. Synthesis method of the compound III series:

[0038]

[0039] The specific reaction process can be:

[0040] The compound MY-32 is reduced by using LiAlH4 system to obtain the target compound III-1;

[0041] The compound III-1 is dissolved in anhydrous dichloromethane with DAST, and is stirred at room temperature overnight to obtain the target compound III-2.

[0042] 5. Synthesis method of the compound IV series:

[0043]

[0044] The specific reaction process can be:

[0045] The compound IA-7 is reacted with phosphorus oxychloride to obtain the target compound IV-1;

[0046] The compound IV-1 is subjected to reductive amination with methylamine hydrochloride to obtain the target compound IV-2;

[0047] The compound IV-1 is dissolved in anhydrous dichloromethane with DAST, and stirred at room temperature overnight to obtain the target compound IV-3.

[0048] 6. A method for synthesizing the compound V series:

[0049]

[0050] The specific reaction process can be:

[0051] The compound IA-10 is dissolved in anhydrous dichloromethane with DAST, and stirred at room temperature overnight to obtain the target compound V-1;

[0052] The compound IA-10 is dissolved in anhydrous tetrahydrofuran with TMSCF3 and TBAF, and stirred at room temperature to obtain the target compound V-2.

[0053] Still another object of the present application is to provide the use of the 7-difluoromethyl-5-aryl pyrazolopyrimidine compound in the preparation of a hypoxia-inducible factor-2α (HIF-2α) inhibitor for treating a VHL deletion or / and HIF-2α abnormal expression disease. The compound can specifically bind to HIF-2α, inhibit the overexpression and activation of HIF-2α, so as to achieve the treatment and prevention of the target disease, such as a VHL deletion or / and HIF-2α abnormal expression disease.

[0054] The disease includes a tumor, an autoimmune disease, inflammation, and an iron overload disease.

[0055] The present application also relates to a pharmaceutical composition comprising the HIF-2α inhibitor or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient.

[0056] The "pharmaceutically acceptable carrier" in the present application refers to a conventional pharmaceutical carrier in the pharmaceutical field, including conventional diluents, excipients such as water, fillers such as starch, binders such as cellulose derivatives and gelatin, wetting agents such as glycerol, disintegrants such as agar, calcium carbonate, absorption promoters such as quaternary ammonium compounds, surfactants such as cetyl alcohol, adsorption carriers such as kaolin and soap clay, lubricants such as talc, and if necessary, flavoring agents, sweeteners and the like.

[0057] The pharmaceutical preparation is suitable for administration by any appropriate route, such as oral (including buccal or sublingual administration), rectal, nasal, topical (including buccal, sublingual or transdermal), vaginal or parenteral (including subcutaneous, intramuscular, intravenous or intradermal) administration. These preparations can be prepared by any method known in the art of pharmacy, such as by a method of mixing the active ingredient with a carrier or excipient.

[0058] In the present application, "pharmaceutically acceptable salt" refers to a salt prepared by a conventional method, including but not limited to organic acid salt, inorganic acid salt, organic base salt, inorganic base salt. The organic acid salt includes but is not limited to oxalate, lactate, p-toluenesulfonate, malate, citrate, fumarate, camphorsulfonate, methanesulfonate, etc.; the inorganic acid salt includes but is not limited to nitrate, sulfate, hydrohalide, phosphate, etc. The organic base salt includes but is not limited to sodium methoxide, potassium methoxide, sodium ethoxide, potassium ethoxide, triethylamine, tert-butylamine, etc. The inorganic base salt includes but is not limited to sodium hydroxide, potassium hydroxide, lithium hydroxide, etc.

[0059] Specifically, the present application relates to the use of the above-mentioned 7-difluoromethyl-5-aryl pyrazolopyrimidine HIF-2α inhibitor in the treatment of tumors.

[0060] As a preferred embodiment of the above technical solution of the present application, the tumor is selected from VHL-deficient solid tumors, including renal cancer, liver cancer, colorectal cancer, lung cancer, gastric cancer, breast cancer, ovarian cancer, cervical cancer, skin cancer, glioma, lymphoma and neuroblastoma.

[0061] In the treatment of tumors, in addition to the use of the above-mentioned compound alone, other anti-tumor drugs can also be used in combination, including but not limited to mitotic inhibitors (such as vinblastine, vindesine, etc.), microtubulin inhibitors (such as taxol), antimetabolites (such as 5-fluorouracil, methotrexate and cytarabine, etc.), alkylating agents (such as cisplatin, carboplatin and cyclophosphamide, etc.), intercalating antibiotics (such as adriamycin, mitomycin and bleomycin, etc.), enzymes (such as asparaginase, etc.), topoisomerase inhibitors (such as etoposide and camptothecin, etc.).

[0062] Specifically, the present application also relates to the use of the above-mentioned 7-difluoromethyl-5-aryl pyrazolopyrimidine HIF-2α inhibitor in the preparation of a medicament for treating autoimmune diseases and inflammation.

[0063] As a preferred embodiment of the above technical solution of the present application, the autoimmune disease and inflammation is selected from asthma, nephritis, pneumonia, enteritis, dermatitis, arthritis, vasculitis, pancreatitis and traumatic infection.

[0064] In particular, the present application also relates to the use of the above-mentioned 7-difluoromethyl-5-arylpyrazolopyrimidine HIF-2a inhibitors in the manufacture of a medicament for treating iron overload diseases.

[0065] As a preferred technical solution of the present application, the iron overload disease is selected from the group consisting of hemochromatosis, polycythemia, von Hippel-Lindau syndrome and beta-thalassemia.

[0066] Compared with the prior art, the present application has the following technical effects:

[0067] The above-mentioned compound provided by the present application has the activity of antagonizing HIF-2 transcription, and shows VEGF protein inhibitory activity in human renal clear cell carcinoma cell line 786-O. Overexpression and activation of HIF-2a is the main cause of VHL- / - clear cell renal cell carcinoma. Therefore, the above-mentioned compound can be applied to the preparation of a medicament for treating VHL deletion or HIF-2a abnormal expression diseases including cancer, inflammation, autoimmune diseases and iron overload related diseases. BRIEF DESCRIPTION OF DRAWINGS

[0068] Figure 1 is a virtual screening process based on the fusion of pharmacophore model-molecular docking. DETAILED DESCRIPTION

[0069] The specific examples and drawings contained herein are intended to be illustrative only and should not be construed as limiting the scope of the present application. Furthermore, it should be understood that various modifications and changes can be made to the present application by those skilled in the art reading the teachings provided herein without departing from the scope of the application. Such equivalent forms are intended to fall within the scope of the claims appended hereto.

[0070] Example 1: Virtual screening based on the fusion of pharmacophore model-molecular docking

[0071] See Figure 1The pharmacophore model of the Belzutifan small molecule was constructed using the Pharmacophore module of Discovery studio. Thirteen features of the Belzutifan small molecule were selected, and six pharmacophores were generated. In the pharmacophore screening stage, the created pharmacophore model was loaded, the number of matching pharmacophores (≥5) was selected, the volume effect was excluded in the screening, and the screening based on the pharmacophore model was performed. Through the evaluation and differentiation of the ability of 20 HIF-2α / ARNT protein crystal structures by using re-docking and cross-docking strategies, 5UFP and 6X37 crystals were selected as the best crystal proteins for subsequent molecular docking. About 1.5 million data of 5UFP and 6X37 crystal proteins and small molecule commercial databases Chemdiv and specs were screened by Glide program in SP mode, and the top 20w molecules were selected according to the scoring function evaluation. Then the overlapping small molecules in the two groups of 20w database were selected, indicating that these structures had good docking effect on the two proteins, and about 13w small molecules were obtained. The above 13w small molecule database was screened by pharmacophore matching, and 12k small molecules were matched, and then high-precision docking (XP docking) with 5UFP crystal protein was carried out. The top 2000 compounds were retained, and MM / GBSA molecular Gibbs free energy calculation was performed, K-means clustering method was used to obtain 25 classifications, combined with the threshold of ΔG<-66kcal / mol, more than 30 small molecule compounds were selected for purchase, and the VEGF Elisa Assay was used to evaluate the VEGF protein inhibition activity of the compounds on human renal clear cell carcinoma cell line 786-O. A small molecule compound MY-32 with obvious HIF-2 transcription inhibition activity was found, and a series of 7-difluoromethyl-5-aryl pyrazolopyrimidine derivatives were obtained by structure modification and optimization.

[0072] Example 2: Synthesis of IA series of compounds

[0073]

[0074] Step 1: 1-(3,4-dimethylphenyl)-4,4-difluorobutan-1,3-dione (MY-32b)

[0075] Dissolve 60% sodium hydride (502 mg, 12.5 mmol) in anhydrous THF (10 mL), add 3,4-dimethylacetophenone (1.48 mL, 10 mmol) under ice bath, stir at room temperature for 30 min. Add ethyl 2,2-difluoroacetate (1.32 mL, 12.5 mmol) dropwise under ice bath, react at room temperature for 4 h. Add 2N HCl to the system, adjust pH = 7, extract with ethyl acetate (30 mL x 3), combine the organic layers and wash with saturated brine, dry over anhydrous sodium sulfate, and concentrate under reduced pressure to obtain a yellow solid 2.214 g in yield: 98%. ESI-MS: m / z = 227 [M+H] + .

[0076] 1-(2-bromophenyl)-4,4-difluorobutane-1,3-dione (IAb-1)

[0077] Synthesis method is the same as MY-32b, replace 3,4-dimethylacetophenone with 2-bromoacetophenone (2 g, 10 mmol), obtain a yellow solid 2.709 g in yield: 98%. ESI-MS: m / z = 277 [M+H] + .

[0078] 1-(3-bromophenyl)-4,4-difluorobutane-1,3-dione (IAb-2)

[0079] Synthesis method is the same as MY-32b, replace 3,4-dimethylacetophenone with 3-bromoacetophenone (1.33 mL, 10 mmol), obtain a yellow solid 2.612 g in yield: 94%. ESI-MS: m / z = 277 [M+H] + .

[0080] 1-(4-bromophenyl)-4,4-difluorobutane-1,3-dione (IAb-3)

[0081] Synthesis method is the same as MY-32b, replace 3,4-dimethylacetophenone with 4-bromoacetophenone (2 g, 10 mmol), obtain a yellow solid 2.655 g in yield: 96%. ESI-MS: m / z = 277 [M+H] + .

[0082] 4,4-difluoro-1-(2-methoxyphenyl)butane-1,3-dione (IAb-4)

[0083] Synthesis method is the same as MY-32b, replace 3,4-dimethylacetophenone with 2-methoxyacetophenone (1.37 mL, 10 mmol), obtain a yellow solid 2.166 g in yield: 95%. ESI-MS: m / z = 229 [M+H] + .

[0084] 4,4-difluoro-1-(3-methoxyphenyl)butane-1,3-dione (IAb-5)

[0085] Synthesis method same as MY-32b, replace 3,4-dimethylacetophenone with 3- methoxyacetophenone (1.37 mL, 10 mmol), get yellow solid 2.167 g, yield: 95%. ESI-MS: m / z = 229 [M+H] + .

[0086] 4,4-difluoro-1-(4-methoxyphenyl)butane-1,3-dione (IAb-6)

[0087] Synthesis method same as MY-32b, replace 3,4-dimethylacetophenone with 4- methoxyacetophenone (1.5 g, 10 mmol), get yellow solid 2.218 g, yield: 97%. ESI-MS: m / z = 229 [M+H] + .

[0088] Step 2: 4-(methylsulfonyl)-1H-pyrazol-3-amine (IAc-9)

[0089] The system of 2-(methylsulfonyl)acetonitrile (93 μL, 1 mmol), triethyl orthoformate (1.16 mL, 7 mmol) and acetic anhydride (657 μL, 7 mmol) was heated to 140 °C, after the reaction was complete, water (5 mL) was added to the system to quench, extracted with ethyl acetate (5 mL x 3), combined organic layer and washed with saturated brine, dried over anhydrous sodium sulfate, concentrated under reduced pressure to get yellow oil 155 mg; ESI-MS: m / z = 176 [M+H] + .

[0090] The intermediate was dissolved in ethanol (2 mL), 50% hydrazine aqueous solution (90 μL, 1 mmol) was added dropwise, the system was warmed to reflux for 6 h. The system was rotary dried, the crude product was purified by column chromatography to get colorless oil 85 mg, two-step yield 53%; ESI-MS: m / z = 162 [M+H] + . 1 H NMR (500 MHz, DMSO-d6) δ 7.50 (s, 1H), 6.30 (s, 2H), 3.19 (s, 3H).

[0091] 1-(3-amino-1H-pyrazol-4-yl)ethan-1-one (IAc-10)

[0092] Synthesis method same as IAc-10, replace 2-(methylsulfonyl)acetonitrile with 3- oxobutanenitrile (85 μL, 1 mmol), get colorless oil 70 mg, two-step yield 56%. 1H NMR (500 MHz, DMSO-d6) δ 7.99 (s, 1H), 6.30 (s, 2H), 2.44 (s, 3H).

[0093] 4-cyclopropyl-lH-pyrazole-3-amine (IAc-11)

[0094] 2-cyclopropylacetonitrile (420 μL, 4.55 mmol) and ethyl formate (800 μL, 10 mmol) were added dropwise to a solution of potassium tert-butoxide (1.23 mL, 10 mmol) in THF (10 mL) and the reaction was allowed to proceed at room temperature overnight. Water (20 mL) was added to the system, the pH was adjusted to 4 with 2N HC1, and the product was extracted with dichloromethane (20 mL x 3). The organic layers were combined and washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give a yellow oil 150 mg.

[0095] The intermediate was dissolved in ethanol (3 mL), a 50% aqueous hydrazine solution (885 μL, 9.1 mmol) and glacial acetic acid (416 μL, 7.28 mmol) were added dropwise, and the system was heated to reflux and allowed to react overnight. The system was dried by evaporation, a saturated sodium carbonate solution was added to adjust the pH to 7, and the product was extracted with dichloromethane (20 mL x 3). The organic layers were combined and washed with saturated brine, dried over anhydrous sodium sulfate, and the crude product was purified by column chromatography to give a colorless oil 275 mg, 49% yield over two steps. 1 H NMR (500 MHz, DMSO-d6) δ 7.52 (s, 1H), 6.30 (s, 2H), 2.39 (p, J = 7.0 Hz, 1H), 1.12-1.02 (m, 2H), 0.82-0.72 (m, 2H).

[0096] 3-amino-5-methyl-lH-pyrazole-4-carboxylic acid methyl ester (IAc-12)

[0097] Methyl 2-cyanoacetate (1 mL, 10 mmol) and triethyl orthoacetate (2 mL, 11 mmol) were dissolved in acetic anhydride (6 mL) and heated to reflux for 3 h. The system was poured into ice water, filtered, and the precipitate was washed with water to give the crude product 338 mg, 20% yield; ESI-MS: m / z = 170 [M+H] + .

[0098] The intermediate was dissolved in glacial acetic acid (3 mL), a 50% aqueous hydrazine solution (389 μL, 4 mmol) was added dropwise, and the system was heated to 110°C and allowed to react for 1 h. The system was dried by evaporation, a saturated sodium carbonate solution was added to adjust the pH to 7, and the product was extracted with dichloromethane (20 mL x 3). The organic layers were combined and washed with saturated brine, dried over anhydrous sodium sulfate, and the crude product was purified by column chromatography to give a white solid 169 mg, 55% yield; ESI-MS: m / z = 156 [M+H] + .1 H NMR (500 MHz, DMSO-d6) δ 6.30 (s, 1H), 3.88 (s, 2H), 2.54 (s, 2H).

[0099] Step 3: 7-Difluoromethyl-5-(3,4-dimethylphenyl)pyrazolo[l,5-a]pyrimidine-3- carboxylic acid methyl ester (MY-32)

[0100] Compound MY-32b (113 mg, 0.5 mmol) and methyl 3-aminopyrazole-4-carboxylate (IAc-l, 71 mg, 0.5 mmol) were dissolved in glacial acetic acid (4 mL) and warmed to reflux overnight. Water (10 mL) was added to the system, extracted with ethyl acetate (10 mL x 3), the organic layers were combined and washed with saturated brine, dried over anhydrous sodium sulfate, and the crude product was purified by column chromatography to give a white solid 131 mg, yield: 79%; ESI-MS: m / z = 332 [M+H] + . 1 H NMR (500 MHz, DMSO-d6) δ 8.56 (s, 1H), 7.80 (s, 1H), 7.74-7.55 (t, J = 50.0 Hz, 1H), 7.62 (d, J = 2.0 Hz, 1H), 7.36 (dd, J = 7.5, 2.0 Hz, 1H), 7.20 (d, J = 7.5 Hz, 1H), 3.88 (s, 3H), 2.30 (s, 3H), 2.23 (s, 3H).

[0101] 5-(2-Bromophenyl)-7-difluoromethylpyrazolo[l,5-a]pyrimidine-3-carboxylic acid methyl ester (IA-l)

[0102] Synthesis method is the same as MY-32, compound MY-32b is replaced by IAb-l (139 mg, 0.5 mmol), white solid 127 mg, yield: 67%. ESI-MS: m / z = 382 [M+H] + . 1 H NMR (500 MHz, DMSO-d6) δ 8.84 (s, 1H), 7.86 (dd, J = 7.5, 1.0 Hz, 1H), 7.83 (s, 1H), 7.82-7.62 (t, J = 50.0 Hz, 1H), 7.70 (dd, J = 7.5, 2.0 Hz, 1H), 7.61 (td, J = 7.5, 1.0 Hz, 1H), 7.53 (td, J = 7.5, 2.0 Hz, 1H), 3.84 (s, 3H).

[0103] 5-(3-Bromophenyl)-7-difluoromethylpyrazolo[l,5-a]pyrimidine-3-carboxylic acid methyl ester (IA-2)

[0104] Synthetic procedure similar to MY-32, replacing compound MY-32b with IAb-2 (139 mg, 0.5 mmol). White solid, 115 mg, yield: 60%. ESI-MS: m / z = 382 [M+H] + . 1 H NMR (500 MHz, DMSO-d6) δ 8.79 (s, 1H), 8.60 (t, J = 2.0 Hz, 1H), 8.42 (dt, J = 8.0, 1.0 Hz, 1H), 8.27 (s, 1H), 7.84 (ddd, J = 8.0, 2.0, 1.0 Hz, 1H), 7.78 - 7.58 (t, J = 50.0 Hz, 1H), 7.59 (t, J = 8.0 Hz, 1H), 3.89 (s, 3H).

[0105] 5-(4-bromophenyl)-7-difluoromethylpyrazolo[l,5-a]pyrimidine-3-carboxylic acid methyl ester (IA-3)

[0106] Synthetic procedure similar to MY-32, replacing compound MY-32b with IAb-3 (139 mg, 0.5 mmol). White solid, 130 mg, yield: 68%. ESI-MS: m / z = 382 [M+H] + . 1 H NMR (500 MHz, DMSO-d6) δ 8.77 (s, 1H), 8.38 - 8.33 (m, 2H), 8.20 (s, 1H), 7.85 - 7.82 (m, 2H), 7.78 - 7.57 (t, J = 50.0 Hz, 1H), 3.88 (s, 3H).

[0107] 7-difluoromethyl-5-(2-methoxyphenyl)pyrazolo[l,5-a]pyrimidine-3-carboxylic acid methyl ester (IA-4)

[0108] Synthetic procedure similar to MY-32, replacing compound MY-32b with IAb-4 (114 mg, 0.5 mmol). White solid, 127 mg, yield: 76%. ESI-MS: m / z = 334 [M+H] + . 1H NMR (500 MHz, DMSO-d6) δ 8.75 (s, 1H), 8.02 (s, 1H), 7.98 (dd, J = 7.5, 2.0 Hz, 1H), 7.79 - 7.59 (t, J = 50.0 Hz, 1H), 7.59 (ddd, J = 8.5, 7.5, 2.0 Hz, 1H), 7.28 (dd, J = 8.5, 1.0 Hz, 1H), 7.19 (td, J = 7.5, 1.0 Hz, 1H), 3.94 (s, 3H), 3.86 (s, 3H).

[0109] 7-Difluoromethyl-5-(3-methoxyphenyl)pyrazolo[l,5-a]pyrimidine-3-carboxylic acid methyl ester (IA-5)

[0110] Synthetic method is the same as MY-32, replace compound MY-32b with IA b-5 (114 mg, 0.5 mmol), get white solid 123 mg, yield: 74%. ESI-MS: m / z = 334 [M+H] + . 1 H NMR (500 MHz, DMSO-d6) δ 8.76 (s, 1H), 8.19 (s, 1H), 7.98 (ddd, J = 8.0, 2.0, 1.0 Hz, 1H), 7.93 (dd, J = 2.5, 2.0 Hz, 1H), 7.78 - 7.57 (t, J = 50.0 Hz, 1H), 7.54 (t, J = 8.0 Hz, 1H), 7.21 (ddd, J = 8.0, 2.5, 1.0 Hz, 1H), 3.90 (s, 3H), 3.88 (s, 3H).

[0111] 7-Difluoromethyl-5-(4-methoxyphenyl)pyrazolo[l,5-a]pyrimidine-3-carboxylic acid methyl ester (IA-6)

[0112] Synthetic method is the same as MY-32, replace compound MY-32b with IA b-6 (114 mg, 0.5 mmol), get white solid 117 mg, yield: 70%; ESI-MS: m / z = 334 [M+H] + . 1 H NMR (500 MHz, DMSO-d6) δ 8.71 (s, 1H), 8.42 - 8.37 (m, 2H), 8.11 (s, 1H), 7.75 - 7.54 (t, J = 50.0 Hz, 1H), 7.18 - 7.13 (m, 2H), 3.89 (s, 3H), 3.87 (s, 3H).

[0113] 7-Difluoromethyl-5-(3,4-dimethylphenyl)pyrazolo[l,5-a]pyrimidine (IA-7)

[0114] Synthetic procedure similar to MY-32, replacing compound IAc-1 with 1H- pyrazol-3-amine (41 μL, 0.5 mmol) to afford 93 mg of white solid, yield: 68%; ESI-MS: m / z = 274 [M+H] + . 1 H NMR (500 MHz, DMSO-d6) δ 7.82 (d, J = 7.5 Hz, 1H), 7.74 (s, 1H), 7.72 - 7.52 (t, J = 50.0 Hz, 1H), 7.60 (d, J = 2.0 Hz, 1H), 7.31 (dd, J = 7.5, 2.0 Hz, 1H), 7.18 (d, J = 7.5 Hz, 1H), 6.33 (d, J = 7.5 Hz, 1H), 2.29 (s, 3H), 2.23 (s, 3H).

[0115] 7-difluoromethyl-5-(3,4-dimethylphenyl)pyrazolo[l,5-a]pyrimidine-3-carbonitrile (IA-8)

[0116] Synthetic procedure similar to MY-32, replacing compound IAc-1 with methyl 3- cyano-lH-pyrazole-4-carboxylate (54 mg, 0.5 mmol) to afford 91 mg of white solid, yield: 61%; ESI-MS: m / z = 298 [M+H] + . 1 H NMR (500 MHz, DMSO-d6) δ 8.91 (s, 1H), 8.22 (s, 1H), 8.19 (d, J = 2.0 Hz, 1H), 8.13 (dd, J = 8.0, 2.0 Hz, 1H), 7.76 - 7.56, 7.39 (d, J = 8.0 Hz, 1H), 2.37 (s, 4H), 2.34 (s, 3H).

[0117] 7-difluoromethyl-5-(3,4-dimethylphenyl)-3-(methylsulfonyl)pyrazolo[l,5-a]pyrimidine (IA-9)

[0118] Synthetic procedure similar to MY-32, replacing compound IAc-1 with IAc-9 (81 mg, 0.5 mmol) to afford 113 mg of white solid, yield: 64%; ESI-MS: m / z = 352 [M+H] + . 1H NMR(500MHz,DMSO-d6)δ8.03(s,1H),7.84(s,1H),7.72-7.51(t,J=50.0Hz,1H),7.61(d,J=2.0 ,1H),7.29(dd,J=7.5,2.0Hz,1H),7.21(d,J=7.5,1H),3.31(s,3H),2.30(s,3H),2.23(s,3H).

[0119] 1-(7-Difluoromethyl-5-(3,4-dimethylphenyl)pyrazolo[1,5-a]pyrimidin-3-yl)ethane-1-one (IA-10)

[0120] The synthesis method was the same as MY-32, except that compound IAc-1 was replaced with IAc-10 (63 mg, 0.5 mmol), yielding 99 mg of a white solid, yield: 63%; ESI-MS: m / z = 316 [M+H] + . 1 H NMR (500MHz, DMSO-d6) δ8.44(s,1H),7.82(s,1H),7.73-7.52(t,J=50.0Hz,1H),7.62(d,J=2.0H z,1H),7.31(dd,J=7.5,2.0Hz,1H),7.21(d,J=7.5,1H),2.61(s,3H),2.30(s,3H),2.23(s,3H).

[0121] 3-Cyclopropyl-7-difluoromethyl-5-(3,4-dimethylphenyl)pyrazolo[1,5-a]pyrimidine (IA-11)

[0122] The synthesis method was the same as MY-32, except that compound IAc-1 was replaced with IAc-11 (62 mg, 0.5 mmol), yielding 117 mg of a white solid, yield: 76%; ESI-MS: m / z = 314 [M+H] + . 1 H NMR(500MHz, DMSO-d6)δ8.09(s,1H),8.06(d,J=2.0Hz,1H),8.00(dd,J=8.0,2.0Hz,1H),7.80(s,1H),7.69-7.48( t,J=50.0Hz,1H),7.33(d,J=8.0Hz,1H),2.35(s,3H),2.31(s,3H),2.15(tt,J=8.5,5.0Hz,1H),1.04-0.92(m,4H).

[0123] 7-difluoromethyl-5-(3,4-dimethylphenyl)-2-methylpyrazolo[1,5-a]pyrimidine-3- carboxylic acid methyl ester (IA-12)

[0124] Synthetic method is the same as MY-32, replace compound IAc-1 with IAc-14 (78 mg, 0.5 mmol), 122 mg of white solid, yield: 71%. ESI-MS: m / z = 346 [M+H] + . 1 H NMR (500 MHz, DMSO-d6) δ 8.18 (d, J = 2.0 Hz, 1H), 8.12 (dd, J = 8.0, 2.0 Hz, 1H), 8.05 (s, 1H), 7.73-7.53 (t, J = 50.0 Hz, 1H), 7.36 (d, J = 8.0 Hz, 1H), 3.89 (s, 3H), 2.65 (s, 3H), 2.36 (s, 3H), 2.33 (s, 3H).

[0125] Example 3: Synthesis of compounds in IB series

[0126]

[0127] Step 1: 7-difluoromethyl-5-(3,4-dimethylphenyl)pyrazolo[1,5-a]pyrimidine-3- carboxylic acid (IBa-1)

[0128] Compound MY-32 (690 mg, 2 mmol) and sodium hydroxide (240 mg, 6 mmol) were dissolved in 3 mL THF, 3 mL methanol and 3 mL water, and the reaction was heated to 50 °C. After the reaction was completed by TLC detection, it was spin-dried, the pH was adjusted to 2 with 2N hydrochloric acid solution, a solid was precipitated, suction filtration was performed, and drying was performed to obtain 550 mg of a white solid, with a yield of 87%. ESI-MS: m / z = 318 [M+H] + .

[0129] 7-difluoromethyl-5-(3,4-dimethylphenyl)-2-methylpyrazolo[1,5-a]pyrimidine-3- carboxylic acid (IBa-11)

[0130] Synthetic method is the same as IBa-1, replace MY-32 with IA-12 (690 mg, 2 mol), 370 mg of white solid, yield: 56%. ESI-MS: m / z = 332 [M+H] + .

[0131] Step 2: 7-difluoromethyl-5-(3,4-dimethylphenyl)pyrazolo[1,5-a]pyrimidine-3- carboxamide (IB-1)

[0132] Compound IBa-1 (63 mg, 0.2 mmol) was dissolved in thionyl chloride (1.5 mL) and a catalytic amount of anhydrous DMF was added, heated to reflux until the starting material was completely consumed. The thionyl chloride was evaporated under anhydrous conditions to give a yellow oil, the acyl chloride was dissolved in anhydrous dichloromethane (3 mL) for later use. Meanwhile, aqueous ammonia (1 mL) was diluted in anhydrous dichloromethane (5 mL) and slowly added to the acyl chloride prepared above through a constant pressure dropping funnel under an ice-water bath. After no white smoke was generated, the reaction was allowed to proceed at room temperature until the starting material was completely consumed. The crude product was purified by column chromatography to give 28 mg of white solid, yield: 45%. ESI-MS: m / z = 317 [M+H] + . 1 H NMR (500 MHz, DMSO-d6) δ 8.64 (s, 1H), 8.19 (s, 1H), 8.15-8.11 (m, 1H), 8.08 (s, 1H), 7.77-7.56 (t, J = 50.0 Hz, 1H), 7.62 (s, 1H), 7.59 (s, 1H), 7.38 (d, J = 8.0 Hz, 1H), 2.37 (s, 3H), 2.33 (s, 3H).

[0133] 7-Difluoromethyl-5-(3,4-dimethylphenyl)-N-methylpyrazolo[l,5-a]pyrimidine-3- carboxamide (IB-2)

[0134] Compound IBa-1 (63 mg, 0.2 mmol), methylamine hydrochloride (27 mg, 0.4 mmol), EDCI (115 mg, 0.6 mmol) and HOBT (81 mg, 0.6 mmol) were dissolved in DMF (2 mL), N-methylmorpholine (220 μL, 2 mmol) was added dropwise, and the reaction was allowed to proceed at room temperature overnight. Water (10 mL) was added to the system, and the product was extracted with ethyl acetate (10 mL x 3). The organic layer was washed with water (10 mL x 3) and saturated brine (10 mL), dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the crude product was purified by column chromatography to give 46 mg of white solid, yield: 69%; ESI-MS: m / z = 331 [M+H] + . 1 H NMR (500 MHz, DMSO-d6) δ 8.64 (s, 1H), 8.19 (s, 1H), 8.15-8.11 (m, 1H), 8.08 (s, 1H), 7.77-7.56 (t, J = 50.0 Hz, 1H), 7.62 (s, 1H), 7.59 (s, 1H), 7.38 (d, J = 8.0 Hz, 1H), 2.37 (s, 3H), 2.33 (s, 3H).

[0135] 7-difluoromethyl-5-(3,4-dimethylphenyl)-N-ethylpyrazolo[1,5-a]pyrimidine-3- carboxamide (IB-3)

[0136] Synthetic method is the same as IB-2, replace methanamine hydrochloride with ethanamine hydrochloride (33 mg, 0.4 mmol), white solid 39 mg, yield: 57%; ESI-MS: m / z = 345 [M+H] + . 1 H NMR (500 MHz, DMSO-d6) δ 8.63 (s, 1H), 8.17 (d, J = 2.0 Hz, 1H), 8.12 (dd, J = 8.0, 2.0 Hz, 1H), 8.07 (s, 1H), 8.03 (t, J = 5.5 Hz, 1H), 7.76 - 7.55 (t, J = 50.0 Hz, 1H), 7.37 (d, J = 8.0 Hz, 1H), 3.44 (qd, J = 7.0, 5.5 Hz, 2H), 2.36 (s, 3H), 2.32 (s, 3H), 1.25 (t, J = 7.0 Hz, 3H).

[0137] 7-difluoromethyl-5-(3,4-dimethylphenyl)-N,N-diethylpyrazolo[1,5-a]pyrimidine-3- carboxamide (IB-4)

[0138] Synthetic method is the same as IB-2, replace methanamine hydrochloride with diethanamine hydrochloride (46 mg, 0.4 mmol), white solid 50 mg, yield: 67%; ESI-MS: m / z = 373 [M+H] + . 1 H NMR (500 MHz, DMSO-d6) δ 8.47 (s, 1H), 8.11 (d, J = 2.0 Hz, 1H), 8.03 (dd, J = 8.0, 2.0 Hz, 1H), 8.00 (s, 1H), 7.76 - 7.55 (t, J = 50.0 Hz, 1H), 7.36 (d, J = 8.0 Hz, 1H), 3.51 (s, 4H), 2.35 (s, 3H), 2.32 (s, 3H), 1.15 (s, 6H).

[0139] 7-difluoromethyl-5-(3,4-dimethylphenyl)-pyrazolo[1,5-a]pyrimidine-3-carboxylic acid hydrazide (IB-5)

[0140] Synthetic method is the same as IB-2, replace methanamine hydrochloride with hydrazine hydrochloride (27 mg, 0.4 mmol), white solid 50 mg, yield: 67%; ESI-MS: m / z = 332 [M+H] + . 1H NMR (500 MHz, DMSO-d6) δ 10.59 (s, 1H), 8.77 (s, 1H), 8.15 (d, J = 2.0 Hz, 1H), 8.07 (s, 1H), 8.05 (dd, J = 8.0, 2.0 Hz, 1H), 7.79 - 7.58 (t, J = 50.0 Hz, 1H), 7.69 (s, 2H), 7.38 (d, J = 8.0 Hz, 1H), 4.48 (s, 2H), 2.29 (s, 3H), 2.23 (s, 3H).

[0141] 7-Difluoromethyl-5-(3,4-dimethylphenyl)-N-hydroxy-N-methylpyrazolo[1,5- a]pyrimidine-3-carboxamide (IB-7)

[0142] Synthesis method same as IB-1, replace ammonia water with hydroxylamine hydrochloride (28 mg, 0.4 mmol) and triethylamine (83 μL, 0.6 mmol), white solid 36 mg, yield: 54%; ESI-MS: m / z = 333 [M+H] + H NMR (500 MHz, DMSO-d6) δ 10.06 (s, 1H), 8.61 (s, 1H), 8.15 (d, J = 2.0 Hz, 1H), 8.10 (dd, J = 8.0, 2.0 Hz, 1H), 8.04 (s, 1H), 7.76 - 7.55 (t, J = 50.0 Hz, 1H), 7.37 (d, J = 8.0 Hz, 1H), 2.36 (s, 3H), 2.33 (s, 3H).

[0143] 7-Difluoromethyl-5-(3,4-dimethylphenyl)-N-hydroxy-N-methylpyrazolo[1,5- a]pyrimidine-3-carboxamide (IB-7)

[0144] Synthesis method same as IB-2, replace methylamine hydrochloride with N- methylhydroxylamine hydrochloride (33 mg, 0.4 mmol), white solid 44 mg, yield: 63%; ESI-MS: m / z = 347 [M+H] + . 1 H NMR (500 MHz, DMSO-d6) δ 10.06 (s, 1H), 8.61 (s, 1H), 8.15 (d, J = 2.0 Hz, 1H), 8.10 (dd, J = 8.0, 2.0 Hz, 1H), 8.04 (s, 1H), 7.76 - 7.55 (t, J = 50.0 Hz, 1H), 7.37 (d, J = 8.0 Hz, 1H), 2.36 (s, 3H), 2.33 (s, 3H).

[0145] 7-difluoromethyl-5-(3,4-dimethylphenyl)-N-methyloazolo[l,5-a]pyrimidine-3- carboxamide (IB-8)

[0146] Synthetic method is the same as IB-2, replace methanamine hydrochloride with methoxyamine hydrochloride (33 mg, 0.4 mmol), white solid 51 mg, yield: 73%; ESI-MS: m / z = 347 [M+H] + . 1 H NMR (500 MHz, DMSO-d6) δ 10.98 (s, 1H), 8.66 (s, 1H), 8.23 (d, J = 2.0 Hz, 1H), 8.20 (dd, J = 8.0, 2.0 Hz, 1H), 8.08 (s, 1H), 7.76 - 7.56 (t, J = 50.0 Hz, 1H), 7.38 (d, J = 8.0 Hz, 1H), 3.80 (s, 3H), 2.37 (s, 3H), 2.33 (s, 3H).

[0147] 7-difluoromethyl-5-(3,4-dimethylphenyl)-N,2-dimethylpyrazolo[l,5-a]pyrimidine-3- carboxamide (IB-9)

[0148] Synthetic method is the same as IB-2, replace compound IBa-1 with IBa-11 (66 mg, 0.2 mmol), white solid 46 mg, yield: 67%; ESI-MS: m / z = 345 [M+H] + . 1 H NMR (500 MHz, DMSO-d6) δ 8.70 (s, 1H), 7.79 (s, 1H), 7.70 (t, J = 50.0 Hz, 1H), 7.60 (d, J = 2.0 Hz, 1H), 7.27 (dd, J = 7.5, 2.0 Hz, 1H), 7.20 (d, J = 7.5 Hz, 1H), 2.89 (s, 3H), 2.66 (s, 3H), 2.30 (s, 3H), 2.23 (s, 3H).

[0149] Example 4: Synthesis of 7-difluoromethyl-5-(3,4-difluorophenyl)-N-methylpyrazolo[l,5- a]pyrimidine-3-carboxamide

[0150]

[0151] Step 1: 7-difluoromethyl-5-oxo-4,5-dihydropyrazolo[l,5-a]pyrimidine-3-carboxylic acid ethyl ester (II-b)

[0152] Ethyl 3-amino pyrazole-4-carboxylate (3.1 g, 0.02 mol) and ethyl 4,4- difluoroacetoacetate (4.06 g, 0.022 mol) were dissolved in AcOH (5 mL) and heated to reflux for 3 h. Cooled to room temperature, spin dry, saturated aqueous NaHCO3solution (15 mL) was added to the system, extracted with ethyl acetate (15 mL x 3) and washed with saturated brine (15 mL), dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain a hydrate. The intermediate was dissolved in TFA (15 mL) and the mixture was refluxed for 24 h. Cooled to room temperature, spin dry, the system was poured into ice water, the pH was adjusted to 7 with saturated aqueous NaHCO3solution, extracted with ethyl acetate (30 mL x 3) and washed with saturated brine (30 mL), dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain a solid 1.43 g, yield: 26%; ESI-MS: m / z = 258 [M+H] + . 1 H NMR (500 MHz, DMSO-d6) δ 8.27 (s, 1H), 6.71 (s, 1H), 6.65-6.42 (t, J = 50.0 Hz, 1H), 4.24 (q, J = 8.0 Hz, 2H), 1.30 (t, J = 8.0 Hz, 3H).

[0153] Step 2: Ethyl 5-(2-chlorophenyl)-3-methylisoxazole-4-carboxylate (II-c)

[0154] A mixture of compound II-b (1.4 g, 5 mmol) and POCl3(10 mL, 0.1 mol) was refluxed overnight. Cooled to room temperature, the system was poured into saturated aqueous NaHCO3solution (20 mL), extracted with ethyl acetate (30 mL x 3) and washed with saturated brine (30 mL), dried over anhydrous sodium sulfate and concentrated under reduced pressure, the crude product was purified by column chromatography to obtain a white solid 1 g, yield 68%; ESI-MS: m / z = 276 [M+H] + . 1 H NMR (500 MHz, DMSO-d6) δ 8.27 (s, 1H), 6.71 (s, 1H), 6.65-6.42 (t, J = 50.0 Hz, 1H), 4.24 (q, J = 8.0 Hz, 2H), 1.30 (t, J = 8.0 Hz, 3H).

[0155] Step 3: 5-(2-chlorophenyl)-3-methylisoxazole-4-carboxylic acid (II-d)

[0156] Compound II-c (1 g, 3.64 mmol) and sodium hydroxide (240 mg, 10.9 mmol) were dissolved in 5 mL THF, 5 mL methanol and 5 mL water, and the reaction was heated to 50 °C. After the reaction was completed by TLC detection, it was spin-dried, the pH was adjusted to 2 with a 2N hydrochloric acid solution, a solid was precipitated, suction filtration was performed, and drying was performed to obtain a white solid 647 mg with a yield of 72%. ESI-MS: m / z = 248 [M+H] + .

[0157] Step 4: 5-(2-chlorophenyl)-3-methylisoxazole-4-carboxamide (II-e)

[0158] Compound II-d, methylamine hydrochloride (351 mg, 5.2 mmol), EDCI (1.5 g, 7.8 mmol) and HOBT (1.05 g, 7.8 mmol) were dissolved in DMF (20 mL), N-methylmorpholine (2.86 mL, 26 mmol) was added dropwise, and the reaction was allowed to stand at room temperature overnight. Water (100 mL) was added to the system, and extraction was performed with ethyl acetate (100 mL x 3), the organic layer was washed with water (100 mL x 3) and saturated brine (100 mL), dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the crude product was purified by column chromatography to obtain a white solid 385 mg with a yield of 57%; ESI-MS: m / z = 261 [M+H] + . 1 H NMR (500 MHz, DMSO-d6) δ 8.25 (s, 1H), 7.92 (s, 1H), 7.91 (s, 1H), 7.62 (t, J = 55 Hz, 1H), 2.89 (s, 3H).

[0159] Step 5: 7-difluoromethyl-5-(3,4-difluorophenyl)-N-methylpyrazolo[1,5-a]pyrimidine-3- carboxamide (II-1)

[0160] Compound II-e (52 mg, 0.2 mmol), 3,4-difluorophenylboronic acid (35 mg, 0.22 mmol) and Pd(PPh3)2Cl2(7 mg, 0.01 mmol) were dissolved in DMF (2 mL), nitrogen protection was performed, 1M K2CO3 aqueous solution (210 μL, 0.21 mmol) was added, the system was heated to 90 °C and allowed to stand overnight. After cooling to room temperature, water (10 mL) was added to the system, extraction was performed with ethyl acetate (10 mL x 3), the organic layer was washed with water (10 mL x 3) and saturated brine (10 mL), dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the crude product was purified by column chromatography to obtain a white solid 47 mg with a yield of 69%; ESI-MS: m / z = 339 [M+H] + . 1H NMR (500 MHz, DMSO-d6) δ 8.69 (s, 1H), 8.65 (ddd, J = 12.0, 8.0, 2.5 Hz, 1H), 8.17 (s, 1H), 7.94 (d, J = 4.5 Hz, 1H), 7.78 - 7.57 (t, J = 50.0 Hz, 1H), 7.72 - 7.66 (m, 1H), 2.95 (d, J = 4.5 Hz, 3H).

[0161] Example 5: Synthesis of compounds II-2~II-13

[0162] Using compound II-e as raw material, 3-chloro-4-fluoroboric acid, 3-trifluoromethyl-4- fluoroboric acid, 3-cyano-4-fluoroboric acid, 3-difluoromethyl-4-fluoroboric acid, 3,5- difluoroboric acid, 3-chloro-5-fluoroboric acid, 3-trifluoromethyl-5-fluoroboric acid, 3- cyano-5-fluoroboric acid, 3-difluoromethyl-5-fluoroboric acid, 4-fluoroboric acid, 4- cyanoboric acid, 4-trifluoromethylboric acid were replaced by 3,4-difluoroboric acid, respectively, to prepare compounds II-2~II-13.

[0163] The target molecules of this series are shown in Table 1 below.

[0164]

[0165] Example 6: Synthesis of compounds of series III

[0166]

[0167] Step 1: (7-difluoromethyl-5-(3,4-dimethylphenyl)pyrazolo[1,5-a]pyrimidin-3-yl)methanol (III-1)

[0168] Compound MY-32 (345 mg, 1 mmol) and lithium aluminum hydride (114 mg, 3 mmol) were dissolved in anhydrous THF (5 mL) under ice bath, and the system was warmed to room temperature and stirred for 2 h. After rotary evaporation, the residue was added to water (10 mL), extracted with ethyl acetate (10 mL x 3) and washed with saturated brine (10 mL), dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the crude product was purified by column chromatography to obtain 233 mg of white solid with a yield of 77%; ESI-MS: m / z = 304 [M+H] + . 1H NMR (500 MHz, DMSO-d6) δ 8.40 (d, J = 2.0 Hz, 1H), 7.82 (s, 1H), 7.38 (d, J = 2.0 Hz, 1H), 7.31 (dd, J = 8.0, 2.0 Hz, 1H), 7.25 (d, J = 8.0 Hz, 1H), 6.46-6.25 (t, J = 50.0 Hz, 1H), 5.56-5.47 (m, 1H), 5.14 (dd, J = 4.0, 2.0 Hz, 1H), 4.28-4.19 (m, 2H), 2.29 (s, 3H), 2.27 (s, 3H).

[0169] Step 2: 7-difluoromethyl-5-(3,4-dimethylphenyl)-3-(fluoromethyl)pyrazolo[l,5- a]pyrimidine (III-2)

[0170] Compound III-1 (61 mg, 0.2 mmol) was dissolved in anhydrous dichloromethane (2.0 mL) under nitrogen protection, diethylamine sulfide (79 μL, 0.6 mmol) was added dropwise under ice bath, stirred at room temperature overnight. Water was added dropwise under ice bath to make no bubbles, the organic solvent was removed under reduced pressure, extracted with ethyl acetate (5 mL x 3), the organic layers were combined, washed with saturated brine, dried over anhydrous sodium sulfate, concentrated and purified by silica gel column chromatography to give 31 mg of white solid, with a yield of 51%; ESI-MS: m / z = 306 [M+H] + . 1 H NMR (500 MHz, DMSO-d6) δ 7.75 (s, 1H), 7.74 (s, 1H), 7.72-7.50 (t, J = 50 Hz, 1H), 7.60 (d, J = 2.0 Hz, 1H), 7.28 (dd, J = 7.5, 2.0 Hz, 1H), 7.21-7.17 (m, 1H), 5.35 (d, J = 45 Hz, 2H), 2.29 (s, 3H), 2.23 (s, 3H).

[0171] Example 7: Synthesis of compounds in series IV

[0172]

[0173] Step 1: 7-difluoromethyl-5-(3,4-dimethylphenyl)pyrazolo[l,5-a]pyrimidine-3- carboxaldehyde (IV-1)

[0174] Phosphorus oxychloride (400 μL) was added dropwise into anhydrous DMF (3 mL) under ice-bath, stirred at room temperature for 1 h, the system was transferred to ice-bath, IA-7 (273 mg, 1 mmol) was added, stirred at room temperature for 1 h, then warmed to 80 °C for 4 h. After cooling to room temperature, quenched with ice-water and neutralized with 1 N NaOH solution, extracted with ethyl acetate (5 mL x 3), the organic layer was washed with water (5 mL x 3) and saturated brine (5 mL), dried over anhydrous sodium sulfate, concentrated and purified by silica gel column chromatography to give white solid 198 mg with a yield of 66%. 1 H NMR (500 MHz, DMSO-d6) δ 9.96 (s, 1H), 8.41 (s, 1H), 7.82 (s, 1H), 7.75-7.52 (t, J = 50 Hz, 1H), 7.49-7.47 (m, 1H), 7.45 (dd, J = 7.5, 2.0 Hz, 1H), 7.23-7.19 (m, 1H), 2.30 (s, 3H), 2.23 (d, s, 3H).

[0175] Step 2: 1-(7-difluoromethyl-5-(3,4-dimethylphenyl)pyrazolo[l,5-a]pyrimidin-3-yl)-N- methylmethanamine (IV-2)

[0176] Compound IV-1 (60 mg, 0.2 mmol), methylamine hydrochloride (20 mg, 0.3 mmol) and triethylamine (31 μL, 0.22 mmol) were dissolved in acetonitrile (2 mL), stirred at room temperature for 1 h, sodium triacetoxyborohydride (85 mg, 0.4 mmol) and glacial acetic acid (34 μL, 0.6 mmol) were added, and the reaction was carried out at room temperature overnight. After concentration under reduced pressure, water (5 mL) was added to the residue, which was extracted with ethyl acetate (5 mL x 3) and saturated brine (5 mL), dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the crude product was purified by column chromatography to give white solid 47 mg with a yield of 74%; ESI-MS: m / z = 317 [M+H] + . 1 H NMR (500 MHz, DMSO-d6) δ 8.27 (s, 1H), 8.09 (d, J = 2.0 Hz, 1H), 8.02 (dd, J = 8.0, 2.0 Hz, 1H), 7.84 (s, 1H), 7.73-7.52 (t, J = 50 Hz, 1H), 7.33 (d, J = 8.0 Hz, 1H), 3.92 (s, 2H), 2.35 (s, 3H), 2.34 (s, 3H), 2.31 (s, 3H).

[0177] 3,7-bis(difluoromethyl)-5-(3,4-dimethylphenyl)pyrazolo[l,5-a]pyrimidine (IV-3)

[0178] Compound IV-1 (60 mg, 0.2 mmol) was dissolved in anhydrous dichloromethane (2.0 mL), protected by nitrogen, and diethylamine sulfide (79 μL, 0.6 mmol) was added dropwise under ice bath. After stirring overnight at room temperature, saturated sodium bicarbonate solution was added dropwise under ice bath to quench the reaction, and extracted with ethyl acetate (5 mL x 3). The combined organic layers were washed with saturated brine, dried over anhydrous sodium sulfate, concentrated, and purified by silica gel column chromatography to give 29 mg of white solid with a yield of 45%; ESI-MS: m / z = 324 [M+H] + . 1 H NMR (500 MHz, DMSO-d6) δ 8.60 (s, 1H), 8.16 (d, J = 2.0 Hz, 1H), 8.09 (dd, J = 8.0, 2.0 Hz, 1H), 8.06 (s, 1H), 7.76-7.56 (t, J = 50 Hz, 1H), 7.55-7.34 (t, J = 50 Hz, 1H), 7.36 (d, J = 8.0 Hz, 1H),, 2.36 (s, 3H), 2.32 (s, 3H).

[0179] Example 8: Synthesis of compounds in series V

[0180]

[0181] 3-(1,1-difluoroethyl)-7-difluoromethyl-5-(3,4-dimethylphenyl)pyrazolo[1,5-a]pyrimidine (V-1)

[0182] Synthesis method same as IV-3, replace IV-1 with IA-10 (63 mg, 0.2 mmol), give 37 mg of white solid, yield: 55%; ESI-MS: m / z = 338 [M+H] + . 1 H NMR (500 MHz, DMSO-d6) δ 7.74 (s, 1H), 7.73 (s, 1H), 7.72-7.48 (m, 2H), 7.30 (dd, J = 7.5, 2.0 Hz, 1H), 7.19 (d, J = 7.5 Hz, 1H), 2.29 (s, 3H), 2.23 (sz, 3H), 2.06 (t, J = 20.0 Hz, 3H).

[0183] 2-(7-difluoromethyl-5-(3,4-dimethylphenyl)pyrazolo[1,5-a]pyrimidin-3-yl)-1,1,1- trifluoropropan-2-ol (V-2)

[0184] To a solution of compound IA-10 (63 mg, 0.2 mmol) and TMSCF3 (35 mg, 0.24 mmol) in anhydrous THF (5 mL) was added TBAF (0.6 mg, 0.002 mmol) at 0 °C. The mixture was stirred at room temperature for 2 h. To the mixture was added appropriate amount of 1 M HC1 and stirred at room temperature for 30 min. The mixture was extracted with ethyl acetate (10 mL x 3) and washed with saturated brine (10 mL), dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude product was purified by column chromatography to give 55 mg of white solid with a yield of 72%; ESI-MS: m / z = 386 [M+H] + . 1 H NMR (500 MHz, DMSO-d6) δ 7.87 (s, 1H), 7.72 (s, 1H), 7.70-7.48 (t, J = 50.0 Hz, 1H), 7.58 (m, 1H), 7.31 (dd, J = 7.5, 2.0 Hz, 1H), 7.19 (d, J = 7.5 Hz, 1H), 5.19 (s, 1H), 2.29 (s, 3H), 2.23 (s, 3H), 1.64 (s, 3H).

[0185] Example 9: VEGF Elisa activity test of the compounds (7-difluoromethyl-5-aryl pyrazolopyrimidines) of the present application

[0186] In this part, M1002 was used as a positive control to evaluate the VEGF protein inhibitory activity of the above-mentioned compounds on human renal clear cell carcinoma cell line 786-0 by VEGF Elisa Assay. Other compounds of the present application have similar beneficial effects as the following listed compounds, but this should not be interpreted as the compounds of the present application only have the following beneficial effects.

[0187] 786-O cells in log phase were seeded in 96-well plates (Fisher Scientific) at 7500 cells per well (180 μL / well) and incubated for 8 hours. Twenty microliters of compound stock solution (DMSO dissolved, concentration of 10 mmol / L) was added to each well to make the final concentration of 10 μM, and three parallel wells were set up. After about 24 hours, the culture medium was removed by suction and 180 μL of growth medium was provided to each well. Twenty microliters of freshly prepared 10x test compound stock solution was added to each well. Incubation was carried out under hypoxic conditions (1% oxygen + 5% carbon dioxide + 94% nitrogen) for 24 hours, and the cell culture medium was removed. The VEGF concentration was determined by using an ELISA kit purchased from R&D Systems. The reaction was terminated by adding 50 μL of Celltiter Glo reagent to each well, and the termination reaction was carried out by gently shaking the enzyme-labeled plate. The Celltiter-Glo luminescent cell viability assay (Promega) was performed on the plate seeded with cells, and then the light absorption value of each well was immediately measured at a wavelength of 450 nm by using an enzyme-labeled instrument. The data were analyzed by GraphPad Prism using a dose-response-inhibition (four-parameter) equation, and the EC50values were calculated. The results are shown in Table 2. 50

[0188] Table 2 VEGF protein inhibitory activity of 7-difluoromethyl-5-aryl pyrazolopyrimidine compounds

[0189] Compound EC 50 (μM) Compound EC 50 (μM) MY-32 ++ IA-5 ++ IA-6 ++ IA-8 ++ IA-9 ++ IA-11 ++ IB-1 ++ IB-2 ++++ IB-3 +++ IB-4 ++ IB-5 ++ IB-6 +++ IB-7 +++ IB-8 ++ IV-2 ++ IV-3 ++ PT2385 ++++

[0190] "++++" represents < 5 μM; "+++" represents 5-10 μM; "++" represents 10-20 μM; and "+" represents > 20 μM.

[0191] From the experimental results in Table 2, it can be seen that most of the compounds exhibit moderate VEGF protein expression inhibitory activity, and have good application prospects.

[0192] Example 8: Luciferase assay of the compounds (7-difluoromethyl-5-aryl pyrazolopyrimidines) of the present application

[0193] ​The luciferase LUC gene was stably transfected into 786-O cells (purchased from ATCC) using Lipofectamine 3000 transfection reagent (purchased from Invitrogen) to construct HIF2α reporter gene cells (786-O-HIF2α-Luc cells). When the 786-O-HIF2α-Luc cells were in the logarithmic growth phase, the culture medium (RPMI MEDIUM 1640, purchased from Invitrogen) was discarded, and the cells were washed with PBS three times; Trypsin (TrypLE, purchased from Invitrogen) was added to digest the cells, and the cells were washed with culture medium, 10% fetal bovine serum, and 1% penicillin and streptomycin to stop the digestion. The cells were collected by centrifugation and washed with PBS twice to remove the phenol red in the culture medium. The cells were resuspended to an appropriate concentration, and the cell density and viability were detected to ensure that the cell viability was greater than 90% before use in the experiment.

[0194] Gradient concentrations of compounds were transferred to 384-well plates using Echo550 (non-contact acoustic liquid handling system, purchased from Labcyte), 25 nL / well; the cells were seeded into 384-well plates, 4500 cells / well, 25 uL of medium, so that the final concentration of the compounds was 10000, 3333, 1111, 370, 123, 41.1, 13.7, 4.6, 1.5, 0.5 nM, respectively. The cells were incubated at 37°C in a 5% CO2 environment for 18-20 h; Steady-GloTM luciferase assay system (purchased from Promega) was added to the 384-well plate, 25 uL / well; and Envision was used to detect the luminescence value. The inhibition rate % was calculated according to the RLU (Record Luminescence) signal value of each well, and then the IC 50 of the corresponding compound was calculated by Graphpad 8.0 fitting. The results are shown in Table 3.

[0195] Table 3 HIF 2α protein inhibition activity of related compounds

[0196] Compound IC 50 (μM) Compound IC 50 (μM) MY-32 ++ IA-8 ++ IA-9 ++ IB-1 ++ IB-2 ++++ IB-3 +++ IB-4 ++ IB-5 ++ IB-6 +++ IB-7 +++ IB-8 ++ IV-2 ++ PT2385 ++++

[0197] “++++” represents <5 μM; “+++” represents 5-10 μM; “++” represents 10-20 μM; and “+” represents >20 μM.

[0198] As can be seen from the experimental results in Table 3, most of the compounds exhibit moderate HIF 2α expression inhibition activity, which has the potential for further development.

[0199] The raw materials and equipment used in the present application are conventional raw materials and equipment in the art unless otherwise specified; and the methods used in the present application are conventional methods in the art unless otherwise specified.

[0200] The above is only the preferred embodiment of the present application, and does not limit the present application, and any simple modification, change and equivalent transformation of the above embodiment according to the technical essence of the present application still belongs to the protection scope of the technical scheme of the present application.

Claims

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