A hydrazide-based HIF-2α inhibitor and its application

Abstract

The present invention relates to the field of pharmaceutical biotechnology, and discloses a hydrazide HIF-2α inhibitor and its application. The hydrazide HIF-2α inhibitor provided by the present invention can block HIF-2α / ARNT dimerization, thereby exerting the effect of HIF-2 transcription inhibition. The HIF-2α inhibitor compound has the activity of antagonizing HIF-2 transcription, and shows VEGF protein inhibitory activity in human renal clear cell carcinoma cell line 786-O. Therefore, the compound has the prospect of being used in the preparation of drugs for treating diseases with VHL deficiency or abnormal expression of HIF-2α, including cancer, inflammation, autoimmune diseases, and iron overload-related diseases.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical biotechnology, and in particular to a hydrazide hydrazone HIF-2α inhibitor and application thereof. Background Art

[0002] Hypoxia-induced factor (HIF) is a key nuclear transcriptional regulator of cells under hypoxia. In a hypoxic environment, the oxygen-dependent functional subunit HIF-α accumulates and forms a heterodimer complex with the structural subunit HIF-β. The dimeric complex further interacts with the transcriptional coactivator p300 protein and binds to the hypoxia response element HRE on the DNA of downstream target genes, thereby regulating a series of protein transcription processes, such as angiogenesis, mitochondrial metabolism, cell proliferation, metastasis, etc., to help the body cope with the stress caused by hypoxia.

[0003] Abnormal activity of the HIF pathway is closely related to a variety of diseases such as cancer, inflammation, anemia and pulmonary hypertension. Among them, the overexpression and activation of HIF-2α is the main cause of VHL. - / - The main cause of clear cell renal cell carcinoma (ccRCC). Studies have shown that more than 90% of ccRCC patients have VHL inactivation, and VHL acts as a substrate recognition subunit of the E3 ubiquitin ligase complex, targeting the degradation of HIF proteins to regulate cellular stress to hypoxia. VHL functional loss causes HIF-2α to accumulate in cancer cells and transcriptionally activate downstream target genes, promoting tumor cell proliferation and metastasis. Therefore, HIF-2 has become a new target for the treatment of clear cell renal carcinoma.

[0004] In the prior art, small molecule inhibitors targeting HIF-2α / ARNT dimerization have been reported in the literature, such as PT-2385 (J.Med.Chem.2018,61,9691), MK-6482 (PT-2977) (J.Med.Chem.2019,62,6876), THS-044 (J.Am.Chem.Soc.2009,131,17647), OX3 (J.Med.Chem.2013,56,1739) and tetrazolylhydropyrimidine derivative (S,R)-37 (J.Med.Chem.2015,58,5930). The structures of PT-2385, MK-6482, THS-044, OX3 and (S,R)-37 are shown below.

[0005] The above molecules target HIF-2α / ARNT dimerization and are highly selective for HIF-2 by specifically binding to the unique cavity of the PAS-B domain of HIF-2α. Among them, Belzutifan (PT-2977) was approved for marketing on August 31, 2021 for the treatment of Hippel-Lindau syndrome-related renal clear cell carcinoma, recurrent glioblastoma, etc. Small molecule inhibitors targeting HIF-2α / ARNT dimerization have received increasing attention, but there are not many structural types.

[0006] Therefore, it is of great clinical significance to search for and discover HIF-2α inhibitors with different skeletons for the treatment of diseases such as renal clear cell carcinoma. Summary of the invention

[0007] In order to solve the above technical problems, the present invention provides a hydrazide hydrazone HIF-2α inhibitor and application thereof.

[0008] The specific technical scheme of the present invention is:

[0009] In one aspect, the present invention provides a hydrazide-based HIF-2α inhibitor. The hydrazide-based HIF-2α inhibitor can specifically bind to HIF-2α to inhibit the overexpression and activation of HIF-2α. Specifically, the structural formula of the HIF-2α inhibitor is:

[0010]

[0011] Wherein, G is -O- or -NH-;

[0012] R1 is selected from hydrogen, halogen, hydroxy, amino, nitro, cyano, C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Alkylamino, C 1-6 Dialkylamino, C 1-6 Fluorinated alkyl, C 3-8 Cycloalkyl, C 2-6 Unsaturated aliphatic chain hydrocarbon group;

[0013] R2 is selected from hydrogen, C 1-6 Alkyl, C 3-6 Cycloalkyl, C 1-6 Fluorinated alkyl, C 2-6 Unsaturated aliphatic chain hydrocarbon group;

[0014] R3 is selected from hydrogen, C 1-6 Alkyl, C 3-6 Cycloalkyl, C 1-6 Fluorinated alkyl, C 2-6 Unsaturated aliphatic chain hydrocarbon group;

[0015] R4 is selected from hydrogen, halogen, cyano, C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Fluorinated alkyl, C 1-6 Fluorinated alkoxy, C 3-8 Cycloalkyl, C 2-6 Unsaturated aliphatic chain hydrocarbon group;

[0016] R5 is selected from hydrogen, halogen, cyano, C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Fluorinated alkyl, C 1-6 Fluorinated alkoxy, C 3-8 Cycloalkyl, C 2-6 Unsaturated aliphatic chain hydrocarbon group.

[0017] The researchers of this team have found that the above-mentioned hydrazide HIF-2α inhibitor provided by the present invention can block HIF-2α / ARNT dimerization, thereby exerting the effect of HIF-2 transcription inhibition. The above-mentioned compound 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-2α are the causes of VHL - / - The main cause of clear cell renal cell carcinoma. Therefore, the above compounds can be used to prepare drugs for treating diseases with VHL deficiency or abnormal HIF-2α expression, including cancer, inflammation, autoimmune diseases, and iron overload-related diseases.

[0018] As a preferred embodiment of the above technical scheme of the present invention, R1 is selected from hydrogen, fluorine, chlorine, bromine, hydroxyl, cyano, methyl, and methoxy; R2 is selected from hydrogen, methyl, and ethyl; R3 is selected from hydrogen, methyl, and ethyl; R4 is selected from fluorine, chlorine, bromine, cyano, methyl, methoxy, trifluoromethyl, trifluoromethoxy, and difluoromethoxy; R5 is selected from fluorine, chlorine, bromine, cyano, methyl, methoxy, trifluoromethyl, trifluoromethoxy, and difluoromethoxy.

[0019] When R1 to R5 are selected from the above groups, the compound has excellent antagonistic activity of HIF-2 transcription, and shows strong VEGF protein inhibitory activity in human renal clear cell carcinoma cell line 786-O.

[0020] As a preferred embodiment of the above technical solution of the present invention, the HIF-2α inhibitor is selected from compounds YA-1 to YA-38, YB-1 to YB-28, and the structural formulas of the compounds YA-1 to YA-38, YB-1 to YB-28 are:

[0021]

[0022]

[0023]

[0024] Compounds YA-1 to YA-38 and YB-1 to YB-28 have excellent HIF-2 transcription antagonism activity and show strong VEGF protein inhibitory activity in human renal clear cell carcinoma cell line 786-O.

[0025] As the preferred technical solution of the present invention, the HIF-2α inhibitor is selected from compounds YA-1, YA-6, YA-7, YA-13, YA-15, YA-18, YA-19, YA-21, YA-23, YA-25, YA-28, YA-29, YA-32, YA-34, YA-35, YA-36, YB-1, YB-3, YB-5, YB-6, YB-7, YB-1 0. Chiral isomers of YB-13, YB-17, YB-18, and YB-19, that is, the HIF-2α inhibitor is selected from the group consisting of compounds R-YA-1, R-YA-6, R-YA-7, and R-YA-1 3. R-YA-15, R-YA-18, R-YA-19, R-YA-21, R-YA-23, R-YA-25, R-YA-28, R-YA-29, R-YA-32, R-Y A-34, R-YA-35, R-YA-36, R-YB-1, R-YB-3, R-YB-5, R-YB-6, R-YB-7, R-YB-10, R-YB-13, R-YB -17, R-YB-18, R-YB-19, the compounds R-YA-1, R-YA-6, R-YA-7, R-YA-13, R-YA-15, R-YA-18, R-YA-19 , R-YA-21, R-YA-23, R-YA-25, R-YA-28, R-YA-29, R-YA-32, R-YA-34, R-YA-35, R-YA-36, R-Y The structural formulas of B-1, R-YB-3, R-YB-5, R-YB-6, R-YB-7, R-YB-10, R-YB-13, R-YB-17, R-YB-18 and R-YB-19 are:

[0026]

[0027]

[0028] Specifically, the present invention also provides a method for preparing a hydrazide-based HIF-2α inhibitor as follows:

[0029] (1) When G is -NH-, the reaction flow is as follows:

[0030]

[0031] The reaction process is:

[0032] ① The substituted aniline (compound I-1) is dissolved in a solvent and subjected to a reductive amination reaction with C-2 substituted ethyl carboxylate under weakly acidic conditions (pH 5-7) to obtain 2-phenylaminoacetic acid ester intermediate I-2. The acid used includes glacial acetic acid, hydrochloric acid, sulfuric acid, and the solvent used includes acetonitrile, DMF, DMSO, etc.;

[0033] ② The intermediate I-2 undergoes hydrazine hydrolysis reaction with hydrazine hydrate to obtain 2-phenylaminoacetylhydrazine intermediate I-3, and the solvent used includes ethanol, THF, dioxane, acetone, etc.;

[0034] ③Finally, the intermediate I-3 is subjected to condensation reaction with substituted 2-hydroxybenzaldehyde or 2-hydroxy-naphthalene-1-aldehyde under weakly acidic conditions (pH 5-7) to prepare the target compounds YA-1 to YA-38. The acid used is glacial acetic acid, and the solvent used is ethanol, acetonitrile, THF, etc.

[0035] (2) When G is -O-, the reaction flow is as follows:

[0036]

[0037] The reaction process is:

[0038] ① Substituted phenol (compound II-1) undergoes nucleophilic reaction with ethyl 2-bromoacetate substituted with different alkyl groups under alkaline conditions to obtain 2-phenoxyacetic acid ester intermediate II-2. Alkaline reagents include potassium carbonate, sodium carbonate, cesium carbonate, and solvents include acetonitrile, acetone, THF, DMF, DMSO, etc.;

[0039] ② Intermediate II-2 undergoes hydrazine hydrolysis reaction with hydrazine hydrate to obtain 2-phenoxyacetylhydrazine intermediate II-3, and the solvent used includes ethanol, THF, dioxane, acetone, etc.;

[0040] ③Finally, the intermediate II-3 is subjected to condensation reaction with substituted 2-hydroxybenzaldehyde or 2-hydroxy-naphthalene-1-aldehyde under weakly acidic conditions (pH 5-7) to prepare the target compounds YB-1 to YB-28. The acid used is glacial acetic acid, and the solvent used is selected from ethanol, acetonitrile, THF, etc.

[0041] On the other hand, the present invention also relates to a pharmaceutical composition, which comprises the above-mentioned HIF-2α inhibitor or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient.

[0042] The present invention provides a hydrazide hydrazone HIF-2α inhibitor, i.e., a hydrazide hydrazone compound, by taking HIF-2 as a target. The compound has the effect of inhibiting the activity of HIF-2α to achieve the treatment and prevention of target diseases, such as cancer, inflammation, autoimmune diseases, and iron overload-related diseases.

[0043] The "pharmaceutically acceptable carrier" mentioned in the present invention refers to conventional drug carriers in the pharmaceutical field, including conventional diluents and excipients such as water, fillers such as starch, binders such as cellulose derivatives, 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 bentonite, lubricants such as talc, etc., and flavoring agents, sweeteners, etc. may be added if necessary.

[0044] The pharmaceutical preparations are suitable for administration by any appropriate route, such as oral (including buccal or sublingual administration), rectal administration, nasal administration, topical administration (including buccal, sublingual or transdermal administration), vaginal administration or parenteral administration (including subcutaneous injection, intramuscular injection, intravenous injection or intradermal injection). These preparations can be prepared by any method known in the field of pharmacy, such as a method by mixing the active ingredient with a carrier or excipient.

[0045] In the present invention, "pharmaceutically acceptable salts" refer to salts prepared by conventional methods, including but not limited to organic acid salts, inorganic acid salts, organic alkali salts, and inorganic alkali salts. The organic acid salts include but are not limited to oxalates, lactates, p-toluenesulfonates, malates, citrates, fumarates, camphorsulfonates, methanesulfonates, etc.; the inorganic acid salts include but are not limited to nitrates, sulfates, hydrohalides, phosphates, etc. The organic alkali salts include but are not limited to sodium methoxide, potassium methoxide, sodium ethoxide, potassium ethoxide, triethylamine, tert-butylamine, etc. The inorganic alkali salts include but are not limited to sodium hydroxide, potassium hydroxide, lithium hydroxide, etc.

[0046] Specifically, the present invention relates to the use of the above-mentioned hydrazide hydrazone HIF-2α inhibitor in the preparation of drugs for treating diseases with VHL deficiency and / or abnormal HIF-2α expression.

[0047] Specifically, the present invention also relates to the use of the above-mentioned hydrazide hydrazone HIF-2α inhibitor in the preparation of drugs for preventing and / or treating tumors.

[0048] As a preferred embodiment of the above technical scheme of the present invention, the tumor is selected from solid tumors, including renal cancer, liver cancer, lung cancer, gastric cancer, sarcoma, colorectal cancer, head cancer, neck cancer, thyroid cancer, breast cancer, ovarian cancer, cervical cancer, prostate cancer, testicular cancer, bladder cancer, nasopharyngeal cancer, cheek cancer, oral cancer, skin cancer, gastrointestinal stromal tumor, multiple myeloma, glioma, lymphoma and neuroblastoma.

[0049] It is worth noting that when preparing drugs for preventing and / or treating tumors, other anti-tumor drugs can be selected and used in combination with the hydrazide hydrazone HIF-2α inhibitor compounds or pharmaceutically acceptable salts thereof provided by the present invention. Other anti-tumor drugs include, but are not limited to, mitotic inhibitors (such as vinblastine, vindesine, etc.), microtubule protein degradation inhibitors (such as taxol), antimetabolites (such as 5-fluorouracil, methotrexate and cytarabine, etc.), alkylating agents (such as cisplatin, carboplatin and cyclophosphamide, etc.), insertable antibiotics (such as alexin, mitomycin and bleomycin, etc.), enzymes (such as aspartase, etc.), topoisomerase inhibitors (such as etoposide and camptothecin, etc.).

[0050] Specifically, the present invention also relates to the use of the above-mentioned hydrazide hydrazone HIF-2α inhibitor in the preparation of drugs for preventing and / or treating autoimmune diseases.

[0051] Specifically, the present invention also relates to the use of the above-mentioned hydrazide hydrazone HIF-2α inhibitor in the preparation of drugs for preventing and / or treating inflammation.

[0052] As a preferred embodiment of the above technical scheme of the present invention, the autoimmune disease and inflammation are selected from allergy, asthma, rheumatoid arthritis, osteoarthritis, allergic contact dermatitis, pancreatitis, renal failure, ulcerative colitis, Crohn's disease, fibromyalgia, lupus erythematosus, chronic obstructive pulmonary disease, psoriasis, multiple sclerosis and traumatic infection.

[0053] Specifically, the present invention also relates to the use of the above-mentioned hydrazide hydrazone HIF-2α inhibitor in the preparation of drugs for preventing and / or treating iron overload diseases.

[0054] As a preferred embodiment of the above technical solution of the present invention, the iron overload disease is selected from hemochromatosis, polycythemia, Partau syndrome and β-thalassemia.

[0055] Compared with the prior art, the present invention has the following technical effects:

[0056] The present invention provides a hydrazide hydrazone HIF-2α inhibitor, which can block HIF-2α / ARNT dimerization and thus exert the effect of HIF-2 transcription inhibition. The hydrazide hydrazone HIF-2α inhibitor compound has excellent antagonistic activity of HIF-2 transcription and shows strong VEGF protein inhibition activity in human renal clear cell carcinoma cell line 786-O. DETAILED DESCRIPTION

[0057] The present invention will be further described below in conjunction with the embodiments.

[0058] The HIF-2α inhibitors described in the examples of the present invention include compounds YA-1 to YA-38 and YB-1 to YB-28. The structural formulas of compounds YA-1 to YA-38 and YB-1 to YB-28 are shown in Table 1.

[0059] Table 1

[0060]

[0061]

[0062]

[0063]

[0064]

[0065] Example 1 Preparation of target molecules YA-1 to YA-17

[0066] 1. Preparation of N'-(2-hydroxybenzylidene)-2-((3-fluorophenyl)amino)propanoylhydrazide (Compound YA-1)

[0067]

[0068] (1) Preparation of (3-fluorophenyl)alanine ethyl ester (Compound I-2-1)

[0069] Dissolve 3-fluoroaniline (I-1-1, 222 mg, 2 mmol) and ethyl pyruvate (255 mg, 2.2 mmol) in 10 mL DMF, add glacial acetic acid to pH 5-7, stir at room temperature for 2 h, add sodium cyanoborohydride (188 mg, 6 mmol) twice, stir at room temperature for 8 h, add water, extract with ethyl acetate, combine the organic layers, wash with saturated NaCl, dry with anhydrous Na2SO4, concentrate and purify by silica gel column chromatography to obtain a white solid. The yield is 55%; ESI-MS: m / z=212.2[M+H] + ; 1 HNMR(δ,DMSO-d6):7.07(m,1H),6.48(m,1H),6.39(m,1H),6.15(m,1H),5.95(s,1H),4.19 (m,1H),4.09(m,1H),3.97(q,1H,J=6.8Hz),1.50(d,3H,J=6.8Hz),1.18(t,3H,J=8.0Hz).

[0070] (2) Preparation of 2-((3-fluorophenyl)amino)propanoylhydrazide (Compound I-3-1)

[0071] Dissolve (3-fluorophenyl)alanine ethyl ester (I-2-1, 1.0 mmol) and 50% N2H4.H2O (10.0 mmol) in 10 mL EtOH and react at 80 °C for 12 h. After removing the solvent by vacuum distillation, a white solid was obtained by silica gel column chromatography. Yield: 96%; ESI-MS: 198.2 [M+H] + ; 1 HNMR(δ,DMSO-d6):7.07(m,1H),6.54(s,1H),6.48(m,1H),6.37(m,2H),6.19(m,1H),4.14(q,1H,J=6.8Hz,),2.39(s,2H),1.53(d,3H,J=6.8Hz).

[0072] (3) Preparation of N'-(2-hydroxybenzylidene)-2-((3-fluorophenyl)amino)propanoylhydrazide (Compound YA-1)

[0073] Compound I-3-1 (100 mg) was dissolved in 10 mL EtOH, 2-hydroxybenzaldehyde (61 mg, 0.5 mmol) was added, and the mixture was reacted at 80 ° C for 2 h. After cooling to room temperature, a solid was precipitated. The filter cake was recrystallized with ethanol and dried in vacuo to obtain a white solid. Yield: 73%; ESI-MS: 301.1 [M+H] + ; 1 HNMR (δ, DMSO-d6): 11.77 (s, 1H), 11.05 (s, 1H), 8.47 (s, 1H), 7.50 (dd, 1H, J1 = 7.8, J2 = 1.6 Hz), 7.28 (m, 1H), 7.09 (q, 1H, J = 7.9 Hz), 6.90 (d, 2H, J = 8.1 Hz), 6.43 (dd, 1H, J1 = 8.2, J2 = 2.1 Hz), 6.34 (m, 3H), 3.97 (p, 1H, J = 6.9 Hz), 1.39 (d, 3H, J = 6.9 Hz). 2. Preparation of (R)-N'-(2-hydroxybenzylidene)-2-((3-fluorophenyl)amino)propanoylhydrazide (Compound R-YA-1)

[0074] The compound YA-1 prepared in 1. is separated by a chiral column to obtain a chiral isomer R-YA-1.

[0075] 3. Preparation of N'-(4-fluoro-2-hydroxybenzylidene)-2-((3-fluorophenyl)amino)propanoylhydrazide (Compound YA-2)

[0076] Compound YA-1 was prepared with reference to 1. 2-Hydroxybenzaldehyde was replaced with 4-fluoro-2-hydroxybenzaldehyde, and the amount ratio of the substances used in the reaction process was the same as that of compound YA-1 prepared in 1. A yellow solid was obtained. The yield was 67%; ESI-MS: m / z = 320.3 [M+H] + .

[0077] 4. Preparation of N'-(5-fluoro-2-hydroxybenzylidene)-2-((3-fluorophenyl)amino)propanoylhydrazide (Compound YA-3)

[0078] Compound YA-1 was prepared with reference to 1. 2-Hydroxybenzaldehyde was replaced with 5-fluoro-2-hydroxybenzaldehyde, and the amount ratio of the substances used in the reaction process was the same as that of compound YA-1 prepared in 1. A yellow solid was obtained. The yield was 63%; ESI-MS: m / z = 320.3 [M+H] + .

[0079] 5. Preparation of N'-(3-chloro-2-hydroxybenzylidene)-2-((3-fluorophenyl)amino)propanoylhydrazide (Compound YA-4)

[0080] Compound YA-1 was prepared with reference to 1. 2-Hydroxybenzaldehyde was replaced with 3-chloro-2-hydroxybenzaldehyde, and the amount ratio of the substances used in the reaction process was the same as that of compound YA-1 prepared in 1. A white solid was obtained. The yield was 58%; ESI-MS: m / z = 336.1 [M+H] + .

[0081] 6. Preparation of N'-(4-chloro-2-hydroxybenzylidene)-2-((3-fluorophenyl)amino)propanoylhydrazide (Compound YA-5)

[0082] Compound YA-1 was prepared with reference to 1. 2-Hydroxybenzaldehyde was replaced with 4-chloro-2-hydroxybenzaldehyde, and the amount ratio of the substances used in the reaction process was the same as that of compound YA-1 prepared in 1. A white solid was obtained. The yield was 61%; ESI-MS: m / z = 336.1 [M+H] + .

[0083] 7. Preparation of N'-(5-chloro-2-hydroxybenzylidene)-2-((3-fluorophenyl)amino)propanoylhydrazide (Compound YA-6)

[0084] Compound YA-1 was prepared with reference to 1. 2-Hydroxybenzaldehyde was replaced with 5-chloro-2-hydroxybenzaldehyde, and the amount ratio of the substances used in the reaction process was the same as that of compound YA-1 prepared in 1. A white solid was obtained. The yield was 69%; ESI-MS: m / z = 336.1 [M+H] + .

[0085] 8. Preparation of Compound R-YA-6

[0086] The compound YA-6 prepared in 7. is separated by a chiral column to obtain the chiral isomer R-YA-6.

[0087] 9. Preparation of N'-(6-chloro-2-hydroxybenzylidene)-2-((3-fluorophenyl)amino)propanoylhydrazide (Compound YA-7)

[0088] Compound YA-1 was prepared with reference to 1. 2-Hydroxybenzaldehyde was replaced with 6-chloro-2-hydroxybenzaldehyde, and the amount ratio of the substances used in the reaction process was the same as that of compound YA-1 prepared in 1. A white solid was obtained. The yield was 65%; ESI-MS: m / z = 336.1 [M+H] + .

[0089] 10. Preparation of Compound R-YA-7

[0090] The compound YA-7 prepared in 9. is separated by a chiral column to obtain the chiral isomer R-YA-7.

[0091] 11. Preparation of N'-(3-bromo-2-hydroxybenzylidene)-2-((3-fluorophenyl)amino)propanoylhydrazide (Compound YA-8)

[0092] Compound YA-1 was prepared with reference to 1. 2-Hydroxybenzaldehyde was replaced with 3-bromo-2-hydroxybenzaldehyde, and the amount ratio of the substances used in the reaction process was the same as that of compound YA-1 prepared in 1. A white solid was obtained. The yield was 72%; ESI-MS: m / z = 381.2 [M+H] + .

[0093] 12. Preparation of N'-(4-bromo-2-hydroxybenzylidene)-2-((3-fluorophenyl)amino)propanoylhydrazide (Compound YA-9)

[0094] Compound YA-1 was prepared with reference to 1. 2-Hydroxybenzaldehyde was replaced with 4-bromo-2-hydroxybenzaldehyde, and the amount ratio of the substances used in the reaction process was the same as that of compound YA-1 prepared in 1. A white solid was obtained. The yield was 75%; ESI-MS: m / z = 381.2 [M+H] + .

[0095] 13. Preparation of N'-(5-bromo-2-hydroxybenzylidene)-2-((3-fluorophenyl)amino)propanoylhydrazide (Compound YA-10)

[0096] Compound YA-1 was prepared with reference to 1. 2-Hydroxybenzaldehyde was replaced with 5-bromo-2-hydroxybenzaldehyde, and the amount ratio of the substances used in the reaction process was the same as that of compound YA-1 prepared in 1. A white solid was obtained. The yield was 77%; ESI-MS: m / z = 381.2 [M+H]+ .

[0097] 14. Preparation of N'-(6-bromo-2-hydroxybenzylidene)-2-((3-fluorophenyl)amino)propanoylhydrazide (Compound YA-11)

[0098] Compound YA-1 was prepared with reference to 1. 2-Hydroxybenzaldehyde was replaced with 6-bromo-2-hydroxybenzaldehyde, and the amount ratio of the substances used in the reaction process was the same as that of compound YA-1 prepared in 1. A white solid was obtained. The yield was 74%; ESI-MS: m / z = 381.2 [M+H] + .

[0099] 15. Preparation of N'-(5-cyano-2-hydroxybenzylidene)-2-((3-fluorophenyl)amino)propanoylhydrazide (Compound YA-12)

[0100] Compound YA-1 was prepared with reference to 1. 2-Hydroxybenzaldehyde was replaced with 3-formyl-4-hydroxybenzonitrile, and the amount ratio of the substances used in the reaction process was the same as that of compound YA-1 prepared in 1. A white solid was obtained. The yield was 76%; ESI-MS: m / z = 327.1 [M+H] + .

[0101] 16. Preparation of N'-(3-methyl-2-hydroxybenzylidene)-2-((3-fluorophenyl)amino)propanoylhydrazide (Compound YA-13)

[0102] Compound YA-1 was prepared with reference to 1. 2-Hydroxybenzaldehyde was replaced with 3-methyl-2-hydroxybenzaldehyde, and the amount ratio of the substances used in the reaction process was the same as that of compound YA-1 prepared in 1. A white solid was obtained. The yield was 82%; ESI-MS: m / z = 316.1 [M+H] + .

[0103] 17. Preparation of Compound R-YA-13

[0104] The compound YA-13 prepared in 16. is separated by a chiral column to obtain the chiral isomer R-YA-13.

[0105] 18. Preparation of N'-(5-methyl-2-hydroxybenzylidene)-2-((3-fluorophenyl)amino)propanoylhydrazide (Compound YA-14)

[0106] Compound YA-1 was prepared with reference to 1. 2-Hydroxybenzaldehyde was replaced with 5-methyl-2-hydroxybenzaldehyde, and the amount ratio of the substances used in the reaction process was the same as that of compound YA-1 prepared in 1. A white solid was obtained. The yield was 86%; ESI-MS: m / z = 316.1 [M+H] + .

[0107] 19. Preparation of N'-(5-methoxy-2-hydroxybenzylidene)-2-((3-fluorophenyl)amino)propanoylhydrazide (Compound YA-15)

[0108] Compound YA-1 was prepared with reference to 1. 2-Hydroxybenzaldehyde was replaced with 5-methoxy-2-hydroxybenzaldehyde, and the amount ratio of the substances used in the reaction process was the same as that of compound YA-1 prepared in 1. A white solid was obtained. The yield was 83%; ESI-MS: m / z = 332.3 [M+H] + .

[0109] 20. Preparation of Compound R-YA-15

[0110] The compound YA-15 prepared in 19. is separated by a chiral column to obtain the chiral isomer R-YA-15.

[0111] 21. Preparation of N'-(2,3-dihydroxybenzylidene)-2-((3-fluorophenyl)amino)propanoylhydrazide (Compound YA-16)

[0112] Compound YA-1 was prepared with reference to 1. 2-Hydroxybenzaldehyde was replaced with 2,3-dihydroxybenzaldehyde, and the amount ratio of the substances used in the reaction process was the same as that of compound YA-1 prepared in 1. A white solid was obtained. The yield was 69%; ESI-MS: m / z = 318.3 [M+H] + .

[0113] 22. Preparation of N'-(2,6-dihydroxybenzylidene)-2-((3-fluorophenyl)amino)propanoylhydrazide (Compound YA-17)

[0114] Compound YA-1 was prepared with reference to 1. 2-Hydroxybenzaldehyde was replaced with 2,6-dihydroxybenzaldehyde, and the amount ratio of the substances used in the reaction process was the same as that of compound YA-1 prepared in 1. A light red solid was obtained. The yield was 75%; ESI-MS: m / z = 318.3 [M+H] + .

[0115] Example 2 Preparation of Compounds YA-18 to YA-27

[0116] 1. Preparation of N'-(2-hydroxybenzylidene)-2-((3-fluorophenyl)amino)butyric acid hydrazide (Compound YA-18)

[0117] Compound YA-1 was prepared by referring to 1. of Example 1. Ethyl pyruvate was replaced by ethyl 2-ketobutyrate. The amount ratio of the substances used in the reaction process was the same as that of compound YA-1 prepared in 1. to obtain a white solid. The yield was 80%; ESI-MS: m / z = 315.1 [M+H] + .

[0118] 2. Preparation of Compound R-YA-18

[0119] The compound YA-18 prepared in 1. of Example 2 was separated by a chiral column to obtain the chiral isomer R-YA-18.

[0120] 3. Preparation of N'-(5-fluoro-2-hydroxybenzylidene)-2-((3-fluorophenyl)amino)butyric acid hydrazide (Compound YA-19) Compound YA-18 was prepared with reference to 1. of Example 2. 2-Hydroxybenzaldehyde was replaced with 5-fluoro-2-hydroxybenzaldehyde. The amount ratio of the substances used in the reaction process was the same as that of Compound YA-18 prepared in 1. of Example 2 to obtain a yellow solid. The yield was 73%; ESI-MS: m / z=333.1 [M+H] + .

[0121] 4. Preparation of Compound R-YA-19

[0122] The compound YA-19 prepared in 3. of Example 2 was separated by a chiral column to obtain the chiral isomer R-YA-19.

[0123] 5. Preparation of N'-(5-chloro-2-hydroxybenzylidene)-2-((3-fluorophenyl)amino)butyric acid hydrazide (Compound YA-20) Compound YA-18 was prepared with reference to 1. of Example 2. 2-Hydroxybenzaldehyde was replaced with 5-chloro-2-hydroxybenzaldehyde. The amount ratio of the substances used in the reaction process was the same as that of Compound YA-18 prepared in 1. of Example 2 to obtain a white solid. The yield was 76%; ESI-MS: m / z=349.1 [M+H] + .

[0124] 6. Preparation of N'-(6-chloro-2-hydroxybenzylidene)-2-((3-fluorophenyl)amino)butyric acid hydrazide (Compound YA-21) Compound YA-18 was prepared with reference to 1. of Example 2. 2-Hydroxybenzaldehyde was replaced with 6-chloro-2-hydroxybenzaldehyde. The amount ratio of the substances used in the reaction process was the same as that of Compound YA-18 prepared in 1. of Example 2 to obtain a white solid. The yield was 74%; ESI-MS: m / z=349.1 [M+H] + .

[0125] 7. Preparation of Compound R-YA-21

[0126] The compound YA-21 prepared in 6. of Example 2 was separated by a chiral column to obtain the chiral isomer R-YA-21.

[0127] 8. Preparation of N'-(5-bromo-2-hydroxybenzylidene)-2-((3-fluorophenyl)amino)butyric acid hydrazide (Compound YA-22) Compound YA-18 was prepared with reference to 1. of Example 2. 2-Hydroxybenzaldehyde was replaced with 5-bromo-2-hydroxybenzaldehyde. The amount ratio of the substances used in the reaction process was the same as that of Compound YA-18 prepared in 1. of Example 2 to obtain a white solid. The yield was 81%; ESI-MS: m / z=393.0 [M+H] + .

[0128] 9. Preparation of N'-(3-methyl-2-hydroxybenzylidene)-2-((3-fluorophenyl)amino)butyric acid hydrazide (Compound YA-23) Compound YA-18 was prepared with reference to 1. of Example 2. 2-Hydroxybenzaldehyde was replaced with 3-methyl-2-hydroxybenzaldehyde. The amount ratio of the substances used in the reaction process was the same as that of Compound YA-18 prepared in 1. of Example 2 to obtain a white solid. The yield was 80%; ESI-MS: m / z=329.2 [M+H] + .

[0129] 10. Preparation of Compound R-YA-23

[0130] The compound YA-23 prepared in 9. of Example 2 was separated by a chiral column to obtain the chiral isomer R-YA-23.

[0131] 11. Preparation of N'-(5-methyl-2-hydroxybenzylidene)-2-((3-fluorophenyl)amino)butyric acid hydrazide (Compound YA-24) Compound YA-18 was prepared with reference to 1. of Example 2. 2-Hydroxybenzaldehyde was replaced with 5-methyl-2-hydroxybenzaldehyde. The amount ratio of the substances used in the reaction process was the same as that of Compound YA-18 prepared in 1. of Example 2 to obtain a white solid. The yield was 78%; ESI-MS: m / z=329.2 [M+H] + .

[0132] 12. Preparation of N'-(5-methoxy-2-hydroxybenzylidene)-2-((3-fluorophenyl)amino)butyric acid hydrazide (Compound YA-25) Compound YA-18 was prepared with reference to 1. of Example 2. 2-Hydroxybenzaldehyde was replaced with 5-methoxy-2-hydroxybenzaldehyde. The amount ratio of the substances used in the reaction process was the same as that of Compound YA-18 prepared in 1. of Example 2 to obtain a white solid. The yield was 84%; ESI-MS: m / z=345.1 [M+H] + .

[0133] 13. Preparation of Compound R-YA-25

[0134] The compound YA-25 ​​prepared in 12. of Example 2 was separated by a chiral column to obtain the chiral isomer R-YA-25.

[0135] 14. Preparation of N'-(2,3-dihydroxybenzylidene)-2-((3-fluorophenyl)amino)butyric acid hydrazide (Compound YA-26) Compound YA-18 was prepared with reference to 1. of Example 2. 2-Hydroxybenzaldehyde was replaced with 2,3-dihydroxybenzaldehyde, and the amount ratio of the substances used in the reaction process was the same as that of Compound YA-18 prepared in 1. of Example 2 to obtain a white solid. The yield was 76%; ESI-MS: m / z=331.1 [M+H] + .

[0136] 15. Preparation of N'-(2,6-dihydroxybenzylidene)-2-((3-fluorophenyl)amino)butyric acid hydrazide (Compound YA-27) Compound YA-18 was prepared with reference to 1. of Example 2. 2-Hydroxybenzaldehyde was replaced with 2,6-dihydroxybenzaldehyde, and the amount ratio of the substances used in the reaction process was the same as that of Compound YA-18 prepared in 1. of Example 2 to obtain a pink solid. The yield was 75%; ESI-MS: m / z=331.1 [M+H] + .

[0137] Example 3 Preparation of Compounds YA-28 to YA-36

[0138] 1. Preparation of N'-(3-methyl-2-hydroxybenzylidene)-2-((3-cyano-5-fluorophenyl)amino)butyric acid hydrazide (Compound YA-28) Compound YA-23 was prepared with reference to 9. of Example 2. 3-Fluoroaniline was replaced with 3-fluoro-5-aminobenzonitrile, and the amount ratio of the substances used in the reaction process was the same as that of compound YA-23 prepared in 9. of Example 2 to obtain a white solid. The yield was 71%; ESI-MS: m / z=354.1 [M+H] + .

[0139] 2. Preparation of Compound R-YA-28

[0140] The compound YA-28 prepared in 1. of Example 3 was separated by a chiral column to obtain the chiral isomer R-YA-28.

[0141] 3. Preparation of N'-(3-methyl-2-hydroxybenzylidene)-2-((3-chlorophenyl)amino)butyric acid hydrazide (Compound YA-29) Compound YA-23 was prepared with reference to 9. of Example 2. 3-fluoroaniline was replaced with 3-chloroaniline, and the amount ratio of the substances used in the reaction process was the same as that of compound YA-23 prepared in 9. of Example 2 to obtain a white solid. The yield was 74%; ESI-MS: m / z=345.1 [M+H] + .

[0142] 4. Preparation of Compound R-YA-29

[0143] The compound YA-29 prepared in 3. of Example 3 was separated by a chiral column to obtain the chiral isomer R-YA-29.

[0144] 5. Preparation of N'-(3-methyl-2-hydroxybenzylidene)-2-((4-chlorophenyl)amino)butyric acid hydrazide (Compound YA-30) Compound YA-23 was prepared with reference to 9. of Example 2. 3-Fluoroaniline was replaced with 4-chloroaniline, and the amount ratio of the substances used in the reaction process was the same as that of compound YA-23 prepared in 9. of Example 2 to obtain a white solid. The yield was 75%; ESI-MS: m / z=345.1 [M+H] + .

[0145] 6. Preparation of N'-(3-methyl-2-hydroxybenzylidene)-2-((3-methylphenyl)amino)butyric acid hydrazide (Compound YA-31) Compound YA-23 was prepared with reference to 9. of Example 2. 3-Fluoroaniline was replaced with 3-methylaniline, and the amount ratio of the substances used in the reaction process was the same as that of compound YA-23 prepared in 9. of Example 2 to obtain a white solid. The yield was 68%; ESI-MS: m / z=325.2 [M+H] + .

[0146] 7. Preparation of N'-(3-methyl-2-hydroxybenzylidene)-2-((3-methyl-5-cyanophenyl)amino)butyric acid hydrazide (Compound YA-32) Compound YA-23 was prepared with reference to 9. of Example 2. 3-Fluoroaniline was replaced with 3-methyl-5-aminobenzonitrile, and the amount ratio of the substances used in the reaction process was the same as that of compound YA-23 prepared in 9. of Example 2 to obtain a white solid. The yield was 76%; ESI-MS: m / z=350.2 [M+H] + .

[0147] 8. Preparation of Compound R-YA-32

[0148] The compound YA-32 prepared in 7. of Example 3 was separated by a chiral column to obtain the chiral isomer R-YA-32.

[0149] 9. Preparation of N'-(3-methyl-2-hydroxybenzylidene)-2-((3-methoxyphenyl)amino)butyric acid hydrazide (Compound YA-33) Compound YA-23 was prepared with reference to 9. of Example 2. 3-Fluoroaniline was replaced with 3-methoxyaniline, and the amount ratio of the substances used in the reaction process was the same as that of compound YA-23 prepared in 9. of Example 2 to obtain a white solid. The yield was 72%; ESI-MS: m / z=341.2 [M+H] +.

[0150] 10. Preparation of N'-(3-methyl-2-hydroxybenzylidene)-2-((3-trifluoromethoxyphenyl)amino)butyric acid hydrazide (Compound YA-34) Compound YA-23 was prepared with reference to 9. of Example 2. 3-Fluoroaniline was replaced with 3-trifluoromethoxyaniline, and the amount ratio of the substances used in the reaction process was the same as that of compound YA-23 prepared in 9. of Example 2 to obtain a white solid. The yield was 81%; ESI-MS: m / z=395.1 [M+H] + .

[0151] 11. Preparation of Compound R-YA-34

[0152] The compound YA-34 prepared in 10. of Example 3 was separated by a chiral column to obtain the chiral isomer R-YA-34.

[0153] 12. Preparation of N'-(3-methyl-2-hydroxybenzylidene)-2-((3-difluoromethoxyphenyl)amino)butyric acid hydrazide (Compound YA-35) Compound YA-23 was prepared with reference to 9. of Example 2. 3-Fluoroaniline was replaced with 3-difluoromethoxyaniline, and the amount ratio of the substances used in the reaction process was the same as that of compound YA-23 prepared in 9. of Example 2 to obtain a white solid. The yield was 82%; ESI-MS: m / z=377.2 [M+H] + .

[0154] 13. Preparation of Compound R-YA-35

[0155] The compound YA-35 prepared in 12. of Example 3 was separated by a chiral column to obtain the chiral isomer R-YA-35.

[0156] 14. Preparation of N'-(3-methyl-2-hydroxybenzylidene)-2-((3-cyanophenyl)amino)butyric acid hydrazide (Compound YA-36) Compound YA-23 was prepared with reference to 9. of Example 2. 3-Fluoroaniline was replaced with 3-cyanoaniline, and the amount ratio of the substances used in the reaction process was the same as that of compound YA-23 prepared in 9. of Example 2 to obtain a white solid. The yield was 80%; ESI-MS: m / z=336.1 [M+H] + .

[0157] 15. Preparation of Compound R-YA-36

[0158] The compound YA-36 prepared in 14. of Example 3 was separated by a chiral column to obtain the chiral isomer R-YA-36.

[0159] Example 4 Preparation of Compounds YA-37 to YA-38

[0160] 1. Preparation of N'-(2-hydroxybenzylidene)-2-((3-fluorophenyl)amino)acetylhydrazine (Compound YA-37)

[0161] Compound YA-1 was prepared with reference to 1. of Example 1. Ethyl pyruvate was replaced with ethyl glyoxylate, and the amount ratio of the substances used in the reaction process was the same as that of compound YA-1 prepared in 1. of Example 1 to obtain a white solid. The yield was 85%; ESI-MS: m / z = 287.1 [M+H] + .

[0162] 2. Preparation of N'-(3-methyl-2-hydroxybenzylidene)-2-((3-fluorophenyl)amino)acetohydrazide (Compound YA-38) Compound YA-37 was prepared with reference to 1. of Example 4. 2-Hydroxybenzaldehyde was replaced with 3-methyl-2-hydroxybenzaldehyde. The amount ratio of the substances used in the reaction process was the same as that of Compound YA-37 prepared in 1. of Example 4 to obtain a white solid. The yield was 83%; ESI-MS: m / z=301.1 [M+H] + .

[0163] Example 5 Preparation of Compounds YB-1 to YB-9

[0164] 1. Preparation of N'-(2-hydroxybenzylidene)-2-(3-fluorophenoxy)propanoylhydrazide (Compound YB-1)

[0165]

[0166] (1) Preparation of ethyl 2-(3-fluorophenoxy)propionate (Compound II-2-1)

[0167] Dissolve 3-fluorophenol II-1-1 (336 mg, 3.0 mmol) and potassium carbonate (829 mg, 6.0 mmol) in 10 mL of acetonitrile, stir at room temperature for 20 min, add ethyl 2-bromopropionate (255 mg, 3.0 mmol), react at 50°C for 18 h, remove potassium carbonate by suction filtration, concentrate the filtrate and purify by silica gel column chromatography to obtain a white oily liquid. The yield is 89%; ESI-MS: m / z = 212.2 [M + H] +; 1 HNMR(δ,DMSO-d6):7.21(m,1H),6.69(m,1H),6.65(m,1H),6.60(m,1H),4.72(q,1 H, J=6.8Hz), 4.23 (q, 2H, J=7.1Hz), 1.62 (d, 3H, J=6.8Hz), 1.26 (t, 3H, J=7.1Hz).

[0168] (2) Preparation of 2-(3-fluorophenoxy)propanoylhydrazide (Compound II-3-1)

[0169] Dissolve ethyl 2-(3-fluorophenoxy)propionate (II-2-1, 2.0 mmol) and 50% N2H4.H2O (20.0 mmol) in 15 mL EtOH and react at 80 °C for 12 h. After removing the solvent by vacuum distillation, a white solid was obtained by column chromatography purification. Yield: 50%, ESI-MS: 199.2 [M+H] + ; 1 HNMR(δ,DMSO-d6):7.28(m,1H),6.90(m,1H),6.84(m,1H),6.69(m,1H),6.38(s,1H),4.58(q,1H,J=6.8Hz),2.44(s,2H),1.60(d,3H,J=6.8Hz).

[0170] (3) Preparation of N'-(2-hydroxybenzylidene)-2-(3-fluorophenoxy)propanoylhydrazide (Compound YB-1)

[0171] Compound II-3-1 (100 mg) was dissolved in 10 mL EtOH, 2-hydroxybenzaldehyde (61 mg, 0.5 mmol) was added, and the mixture was reacted at 80 ° C for 2 h. After cooling to room temperature, a solid was precipitated. The filter cake was recrystallized from ethanol and dried in vacuo to obtain a white solid. Yield: 76%, ESI-MS: 417.1 [M+H] + ; 1 HNMR(δ,DMSO-d6):8.34(d,1H,J=1.1Hz),7.76(s,1H),7.51(ddd,1H,J1=7.5Hz,J2=2.0Hz,J3 =1.0Hz),7.33(m,2H),6.91(m,3H),6.76(m,2H),4.62(q,1H,J=6.8Hz),1.61(d,3H,J=6.9Hz).

[0172] 2. Preparation of R-N'-(2-hydroxybenzylidene)-2-(3-fluorophenoxy)propionylhydrazide (compound R-YB-1)

[0173] Compound YB-1 obtained in 1. of Example 5 was separated by a chiral column to obtain chiral isomer R-YB-1.

[0174] 3. Preparation of N'-(5-fluoro-2-hydroxybenzylidene)-2-(3-fluorophenoxy)propanoylhydrazide (Compound YB-2) Compound YB-1 was prepared with reference to 1. of Example 5. 2-Hydroxybenzaldehyde was replaced with 5-fluoro-2-hydroxybenzaldehyde, and the amount ratio of the substances used in the reaction was the same as that of Compound YB-1 prepared in 1. of Example 5 to obtain a yellow solid. Yield 80%; ESI-MS: 320.1 [M+H]+ .

[0175] 4. Preparation of N'-(6-chloro-2-hydroxybenzylidene)-2-(3-fluorophenoxy)propanoylhydrazide (Compound YB-3)

[0176] Compound YB-1 was prepared with reference to 1. of Example 5. 2-Hydroxybenzaldehyde was replaced with 6-chloro-2-hydroxybenzaldehyde, and the amount ratio of the substances used in the reaction was the same as that of compound YB-1 prepared in 1. of Example 5 to obtain a white solid. Yield: 78%; ESI-MS: 336.1 [M+H] + .

[0177] 5. Preparation of Chiral Compound R-YB-3

[0178] Compound YB-3 obtained in 4. of Example 5 was separated by a chiral column to obtain chiral isomer R-YB-3.

[0179] 6. Preparation of N'-(5-chloro-2-hydroxybenzylidene)-2-(3-fluorophenoxy)propanoylhydrazide (Compound YB-4) Compound YB-1 was prepared with reference to 1. of Example 5. 2-Hydroxybenzaldehyde was replaced with 5-chloro-2-hydroxybenzaldehyde, and the amount ratio of the substances used in the reaction was the same as that of Compound YB-1 prepared in 1. of Example 5 to obtain a white solid. Yield 79%; ESI-MS: 336.1 [M+H] + ;

[0180] 7. Preparation of N'-(5-bromo-2-hydroxybenzylidene)-2-(3-fluorophenoxy)propanoylhydrazide (Compound YB-5)

[0181] Compound YB-1 was prepared with reference to 1. of Example 5. 2-Hydroxybenzaldehyde was replaced with 5-bromo-2-hydroxybenzaldehyde. The amount ratio of the substances used in the reaction was the same as that of compound YB-1 prepared in 1. of Example 5 to obtain a white solid. Yield: 83%; ESI-MS: 380.0 [M+H] + .

[0182] 8. Preparation of Chiral Compound R-YB-5

[0183] The compound YB-5 obtained in 7. of Example 5 was separated by a chiral column to obtain the chiral isomer R-YB-5.

[0184] 9. Preparation of N'-(3-methyl-2-hydroxybenzylidene)-2-(3-fluorophenoxy)propanoylhydrazide (Compound YB-6) Compound YB-1 was prepared with reference to 1. of Example 5. 2-Hydroxybenzaldehyde was replaced with 3-methyl-2-hydroxybenzaldehyde, and the amount ratio of the substances used in the reaction was the same as that of Compound YB-1 prepared in 1. of Example 5 to obtain a white solid. Yield 75%; ESI-MS: 316.1 [M+H] + .

[0185] 10. Preparation of Chiral Compound R-YB-6

[0186] The compound YB-6 obtained in 9. of Example 5 was separated by a chiral column to obtain the chiral isomer R-YB-6.

[0187] 11. Preparation of N'-(5-methoxy-2-hydroxybenzylidene)-2-(3-fluorophenoxy)propanoylhydrazide (Compound YB-7) Compound YB-1 was prepared with reference to 1. of Example 5. 2-Hydroxybenzaldehyde was replaced with 5-methoxy-2-hydroxybenzaldehyde, and the amount ratio of the substances used in the reaction was the same as that of Compound YB-1 prepared in 1. of Example 5 to obtain a white solid. Yield 77%; ESI-MS: 332.1 [M+H] + .

[0188] 12. Preparation of Chiral Compound R-YB-7

[0189] The compound YB-7 obtained in 11. of Example 5 was separated by a chiral column to obtain the chiral isomer R-YB-7.

[0190] 13. Preparation of N'-(2,3-dihydroxybenzylidene)-2-(3-fluorophenoxy)propanoylhydrazide (Compound YB-8) Compound YB-1 was prepared with reference to 1. of Example 5. 2-Hydroxybenzaldehyde was replaced with 2,3-dihydroxybenzaldehyde, and the amount ratio of the substances used in the reaction was the same as that of 1. of Example 5 to prepare compound YB-1, and a white solid was obtained. Yield 78%; ESI-MS: 318.1 [M+H] + .

[0191] 14. Preparation of N'-(2,6-dihydroxybenzylidene)-2-(3-fluorophenoxy)propanoylhydrazide (Compound YB-9) Compound YB-1 was prepared with reference to 1. of Example 5. 2-Hydroxybenzaldehyde was replaced with 2,6-dihydroxybenzaldehyde, and the amount ratio of the substances used in the reaction was the same as that of Compound YB-1 prepared in 1. of Example 5 to obtain a pink solid. Yield 79%; ESI-MS: 318.1 [M+H] + .

[0192] Example 6 Preparation of Compounds YB-10 to YB-19

[0193] 1. Preparation of N'-(2-hydroxybenzylidene)-2-(3-fluorophenoxy)butyric acid hydrazide (Compound YB-10)

[0194] Compound YB-1 was prepared with reference to 1. of Example 5. Ethyl 2-bromopropionate was replaced with ethyl 2-bromobutyrate, and the amount ratio of the substances used in the reaction was the same as that of compound YB-1 prepared in 1. of Example 5 to obtain a white solid. Yield 72%; ESI-MS: 316.1 [M+H] + .

[0195] 2. Preparation of the chiral compound R-YB-10

[0196] The compound YB-10 obtained in 1. of Example 6 was separated by a chiral column to obtain the chiral isomer R-YB-10.

[0197] 3. Preparation of N'-(3-methyl-2-hydroxybenzylidene)-2-(3-fluorophenoxy)butyric acid hydrazide (Compound YB-11) Compound YB-10 was prepared with reference to 1. of Example 6. 2-Hydroxybenzaldehyde was replaced with 3-methyl-2-hydroxybenzaldehyde, and the amount ratio of the substances used in the reaction was the same as that of Compound YB-10 prepared in 1. of Example 6 to obtain a white solid. Yield 81%; ESI-MS: 330.1 [M+H] + .

[0198] 4. Preparation of Chiral Compound R-YB-11

[0199] The compound YB-11 obtained in 3. of Example 6 was separated by a chiral column to obtain the chiral isomer R-YB-11.

[0200] 5. Preparation of N'-(3-methyl-2-hydroxybenzylidene)-2-(3-cyano-5-fluorophenoxy)butyric acid hydrazide (Compound YB-12) Compound YB-11 was prepared with reference to 3. of Example 6. 3-Fluorophenol was replaced with 3-fluoro-5-hydroxybenzonitrile, and the amount ratio of the substances used in the reaction was the same as that of Compound YB-11 prepared in 3. of Example 6 to obtain a white solid. Yield 85%; ESI-MS: 355.1 [M+H] + .

[0201] 6. Preparation of Chiral Compound R-YB-12

[0202] The compound YB-12 obtained in 5. of Example 6 was separated by a chiral column to obtain the chiral isomer R-YB-12.

[0203] 7. Preparation of N'-(3-methyl-2-hydroxybenzylidene)-2-(3-chlorophenoxy)butyric acid hydrazide (Compound YB-13) Compound YB-11 was prepared with reference to 3. of Example 6. 3-Fluorophenol was replaced with 3-chlorophenol, and the amount ratio of the substances used in the reaction was the same as that of compound YB-11 prepared in 3. of Example 6 to obtain a white solid. Yield 86%; ESI-MS: 346.1 [M+H] + ; 8. Preparation of compound R-YB-13

[0204] Compound YB-13 obtained in 7. of Example 6 was separated by a chiral column to obtain chiral isomer R-YB-13.

[0205] 9. Preparation of N'-(3-methyl-2-hydroxybenzylidene)-2-(3-methylphenoxy)butyric acid hydrazide (Compound YB-14) Compound YB-11 was prepared with reference to 3. of Example 6. 3-Fluorophenol was replaced with 3-methylphenol, and the amount ratio of the substances used in the reaction was the same as that of compound YB-11 prepared in 3. of Example 6 to obtain a white solid. Yield 74%; ESI-MS: 326.2 [M+H] + . 10. Preparation of N'-(3-methyl-2-hydroxybenzylidene)-2-(3-cyano-5-methylphenoxy)butyric acid hydrazide (Compound YB-15) Compound YB-11 was prepared by referring to 3. of Example 6. 3-Fluorophenol was replaced with 3-methyl-5-hydroxybenzonitrile, and the amount ratio of the substances used in the reaction was the same as that of Compound YB-11 prepared in 3. of Example 6 to obtain a white solid. Yield 75%; ESI-MS: 351.1 [M+H] + .

[0206] 11. Preparation of N'-(3-methyl-2-hydroxybenzylidene)-2-(3-methoxyphenoxy)butyric acid hydrazide (Compound YB-16) Compound YB-11 was prepared with reference to 3. of Example 6. 3-Fluorophenol was replaced with 3-methoxyphenol, and the amount ratio of the substances used in the reaction was the same as that of compound YB-11 prepared in 3. of Example 6 to obtain a white solid. Yield 71%; ESI-MS: 342.1 [M+H] + .

[0207] 12. Preparation of N'-(3-methyl-2-hydroxybenzylidene)-2-(3-trifluoromethoxyphenoxy)butyric acid hydrazide (Compound YB-17) Compound YB-11 was prepared with reference to 3. of Example 6. 3-Fluorophenol was replaced with 3-trifluoromethoxyphenol, and the amount ratio of the substances used in the reaction was the same as that of compound YB-11 prepared in 3. of Example 6 to obtain a white solid. Yield 76%; ESI-MS: 396.1 [M+H] + .

[0208] 13. Preparation of Compound R-YB-17

[0209] The compound YB-17 obtained in 12. of Example 6 was separated by a chiral column to obtain the chiral isomer R-YB-17.

[0210] 14. Preparation of N'-(3-methyl-2-hydroxybenzylidene)-2-(3-difluoromethoxyphenoxy)butyric acid hydrazide (Compound YB-18) Compound YB-11 was prepared with reference to 3. of Example 6. 3-Fluorophenol was replaced with 3-difluoromethoxyphenol, and the amount ratio of the substances used in the reaction was the same as that of compound YB-11 prepared in 3. of Example 6 to obtain a white solid. Yield 75%; ESI-MS: 378.1 [M+H] + .

[0211] 15. Preparation of compound R-YB-18

[0212] The compound YB-18 obtained in 14. of Example 6 was separated by a chiral column to obtain the chiral isomer R-YB-18.

[0213] 16. Preparation of N'-(3-methyl-2-hydroxybenzylidene)-2-(3-cyanophenoxy)butyric acid hydrazide (Compound YB-19) Compound YB-11 was prepared by referring to 3. of Example 6. 3-Fluorophenol was replaced with 3-hydroxybenzonitrile, and the amount ratio of the substances used in the reaction was the same as that of Compound YB-11 prepared in 3. of Example 6 to obtain a white solid. Yield 80%; ESI-MS: 337.1 [M+H] + .

[0214] 17. Preparation of compound R-YB-19

[0215] The compound YB-19 obtained in 16. of Example 6 was separated by a chiral column to obtain the chiral isomer R-YB-19.

[0216] Example 7 Preparation of Compounds YB-20 to YB-25

[0217] 1. Preparation of N'-(2-hydroxybenzylidene)-2-(3-fluorophenoxy)-2-methylpropanoic acid hydrazide (Compound YB-20) Compound YB-1 was prepared with reference to 1. of Example 5. Ethyl 2-bromopropionate was replaced with ethyl 2-bromo-2-methylpropionate, and the amount ratio of the substances used in the reaction was the same as that of compound YB-1 prepared in 1. of Example 5 to obtain a white solid. Yield 52%; ESI-MS: 316.1 [M+H] + .

[0218] 2. Preparation of N'-(3-methyl-2-hydroxybenzylidene)-2-(3-fluorophenoxy)-2-methylpropanoylhydrazide (Compound YB-21) Compound YB-20 was prepared with reference to 1. of Example 7. 2-Hydroxybenzaldehyde was replaced with 3-methyl-2-hydroxybenzaldehyde, and the amount ratio of the substances used in the reaction was the same as that of Compound YB-20 prepared in 1. of Example 7 to obtain a white solid. Yield 50%; ESI-MS: 330.1 [M+H] + .

[0219] 3. Preparation of N'-(3-methyl-2-hydroxybenzylidene)-2-(3-chlorophenoxy)-2-methylpropanoylhydrazide (Compound YB-22) Compound YB-21 was prepared with reference to 2. of Example 7. 3-Fluorophenol was replaced with 3-chlorophenol, and the amount ratio of the substances used in the reaction was the same as that of Compound YB-21 prepared in 2. of Example 7 to obtain a white solid. Yield 55%; ESI-MS: 346.1 [M+H] + .

[0220] 4. Preparation of N'-(3-methyl-2-hydroxybenzylidene)-2-(3-trifluoromethoxyphenoxy)-2-methylpropanoylhydrazide (Compound YB-23) Compound YB-21 was prepared with reference to 2. of Example 7. 3-Fluorophenol was replaced with 3-trifluoromethoxyphenol, and the amount ratio of the substances used in the reaction was the same as that of Compound YB-21 prepared in 2. of Example 7 to obtain a white solid. Yield 58%; ESI-MS: 396.1 [M+H] + .

[0221] 5. Preparation of N'-(3-methyl-2-hydroxybenzylidene)-2-(3-difluoromethoxyphenoxy)-2-methylpropanoylhydrazide (Compound YB-24)

[0222] Compound YB-21 was prepared with reference to 2. of Example 7. 3-Fluorophenol was replaced with 3-difluoromethoxyphenol, and the amount ratio of the substances used in the reaction was the same as that of compound YB-21 prepared in 2. of Example 7 to obtain a white solid. Yield 56%; ESI-MS: 378.1 [M+H] + .

[0223] 6. Preparation of N'-(3-methyl-2-hydroxybenzylidene)-2-(3-cyanophenoxy)-2-methylpropanoylhydrazide (Compound YB-25)

[0224] Compound YB-21 was prepared with reference to 2. of Example 7. 3-Fluorophenol was replaced with 3-hydroxybenzonitrile, and the amount ratio of the substances used in the reaction was the same as that of compound YB-21 prepared in 2. of Example 7 to obtain a white solid. Yield 57%; ESI-MS: 337.1 [M+H] + .

[0225] Example 8 Preparation of Compounds YB-26 to YB-28

[0226] 1. Preparation of N'-(3-methyl-2-hydroxybenzylidene)-2-(3-fluorophenoxy)acetohydrazide (Compound YB-26)

[0227] Compound YB-6 was prepared with reference to 9. of Example 5. Ethyl 2-bromopropionate was replaced with ethyl 2-bromoacetate, and the amount ratio of the substances used in the reaction was the same as that of compound YB-6 prepared in 9. of Example 5 to obtain a white solid. Yield 85%; ESI-MS: 302.1 [M+H] + .

[0228] 2. Preparation of N'-(3-methyl-2-hydroxybenzylidene)-2-(3-chlorophenoxy)acetohydrazide (Compound YB-27)

[0229] Compound YB-26 was prepared with reference to 1. of Example 8. 3-Fluorophenol was replaced with 3-chlorophenol, and the amount ratio of the substances used in the reaction was the same as that of compound YB-26 prepared in 1. of Example 8 to obtain a white solid. Yield 86%; ESI-MS: 318.1 [M+H] + .

[0230] 3. Preparation of N'-(3-methyl-2-hydroxybenzylidene)-2-(3-cyanophenoxy)acetohydrazide (Compound YB-28)

[0231] Compound YB-26 was prepared with reference to 1. of Example 8. 3-Fluorophenol was replaced with 3-hydroxybenzonitrile, and the amount ratio of the substances used in the reaction was the same as that of compound YB-26 prepared in 1. of Example 8 to obtain a white solid. Yield 87%; ESI-MS: 309.1 [M+H] + .

[0232] Example 9 VEGF Elisa activity test of the compounds of the present invention

[0233] PT-2385 was used as a positive control, and VEGF Elisa Assay was used to evaluate the VEGF protein inhibitory activity of some of the above compounds on the human renal clear cell carcinoma cell line 786-O. Other compounds of the present invention have similar beneficial effects to the compounds listed below, but this should not be understood as the compounds of the present invention only have the following beneficial effects.

[0234] 786-O cells in logarithmic growth phase were seeded in 96-well plates (Fisher Scientific) with 7500 cells per well (180 μL / well). After 4 hours of culture, 20 μL of different concentrations of compound stock solutions were added to each well to give the following final concentrations (μM): 20, 6.67, 2.22, 0.74, 0.25, 0.082, 0.027, 0.009. Approximately 20 hours later, the culture medium was removed by aspiration and 180 μL of growth medium was provided to each well. 20 μL of freshly prepared 10x stock solutions of the compounds to be tested were added to each well at the same concentrations as above. After 24 hours of culture under hypoxic conditions (1% oxygen + 5% carbon dioxide + 94% nitrogen), the cell culture medium was removed. VEGF concentrations were determined using an ELISA kit purchased from R&D Systems. The reaction was terminated by adding 50 μL CelltiterGlo reagent to each well, and the ELISA plate was gently shaken to allow the termination reaction to proceed fully. The plate inoculated with cells was subjected to Celltiter-Glo luminescent cell viability assay (Promega), and then the light absorption value of each well was measured at a wavelength of 450 nm using a microplate reader. The data were analyzed by GraphPadPrism using the dose-response-inhibition (four-parameter) formula to calculate the EC50 value, and the results are shown in Table 2.

[0235] Table 2 VEGF protein inhibitory activity of related compounds

[0236] Compound EC50 (μM) Compound EC50 (μM) YA-1 ++ YA-4 ++ YA-13 +++ YA-15 ++ YA-18 +++ YA-21 ++ YA-23 +++ YA-28 ++ YA-34 ++ YB-3 ++ YB-6 +++ YB-7 ++ YB-10 ++ YB-11 +++ YB-12 ++ YB-17 +++ YB-21 ++ R-YA-13 ++++ R-YA-15 +++ R-YA-23 ++++ R-YA-34 +++ R-YB-10 +++ R-YB-11 ++++ R-YB-17 ++++ PT-2385 ++++

[0237] “++++” represents <5 μM; “+++” represents 5-10 μM; “++” represents 10-20 μM; “+” represents >20 μM.

[0238] From the experimental results in Table 2, it can be seen that most compounds exhibit strong VEGF protein expression inhibitory activity and have good application prospects.

[0239] Example 10 Luciferase assay of the compounds of the present invention

[0240] The luciferase LUC gene was stably transferred into 786-O cells (purchased from ATCC) using Lipofectamine3000 transfection reagent (purchased from Invitrogen) to construct HIF2α reporter gene cells (786-O-HIF2α-Luc cells). The experiment was carried out when the 786-O-HIF2α-Luc cells were in the logarithmic growth phase, the culture medium (RPMIMEDIUM1640, purchased from Invitrogen) was discarded, and PBS was rinsed 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, 1% penicillin, and streptomycin to terminate the digestion. The cells were collected by centrifugation, washed twice with PBS, the phenol red in the culture medium was removed, and the cells were resuspended to an appropriate concentration, and the cell density and viability were detected to ensure that the cell viability was above 90% before it could be used for the experiment.

[0241] Use Echo550 (non-contact sonic pipetting system, purchased from Labcyte) to transfer the gradient concentration compound to 384 wells, 25nl / well; plant the cells in 384-well plates, 4500 cells / well, 25pul culture medium, so that the final concentration of the compound is 10000, 3333, 1111, 370, 123, 41.1, 13.7, 4.6, 1.5, 0.5nM. Culture the cells at 37°C, 5% CO2 environment for 18-20h; add Steady-GloTM luciferase assay system (purchased from Promega) to 384-well plates, 25ul / well; use Envision to detect the luminescence value. Calculate the inhibition rate % based on the RLU (Record Luminescence) signal value of each well, and then calculate the corresponding compound IC by fitting through Graphpad8.0 50 The results are shown in Table 3.

[0242] Table 3 HIF2α protein inhibitory activity of related compounds

[0243] Compound IC50 (μM) Compound IC50 (μM) YA-13 +++ YA-15 ++ YA-18 +++ YA-23 +++ YA-34 ++ YB-3 ++ YB-6 +++ YB-10 ++ YB-11 +++ YB-17 +++ YB-21 ++ R-YA-13 ++++ R-YA-15 +++ R-YA-23 ++++ R-YA-34 +++ R-YB-10 +++ R-YB-11 ++++ R-YB-17 ++++ PT-2385 ++++

[0244] “++++” represents <5 μM; “+++” represents 5-10 μM; “++” represents 10-20 μM; “+” represents >20 μM.

[0245] The raw materials and equipment used in the present invention, unless otherwise specified, are all commonly used raw materials and equipment in the art; the methods used in the present invention, unless otherwise specified, are all conventional methods in the art.

[0246] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change and equivalent transformation made to the above embodiment based on the technical essence of the present invention still falls within the protection scope of the technical solution of the present invention.

Claims

1. Use of a hydrazide-based HIF-2α inhibitor in the preparation of a drug for treating diseases with VHL deficiency or / and abnormal HIF-2α expression, characterized in that: The structural formula of the HIF-2α inhibitor is: Wherein, G is -O- or -NH-; R1 is selected from hydrogen, halogen, hydroxyl, C 1-6 Alkyl, C 1-6 Alkoxy; R2 is selected from hydrogen, C 1-6 alkyl; R3 is selected from hydrogen, C 1-6 alkyl; R4 is selected from hydrogen, halogen, cyano, C 1-6 Alkyl, C 1-6 Alkoxy; R5 is selected from hydrogen, halogen, cyano, C 1-6 Alkyl, C 1-6 Alkoxy.

2. The hydrazide hydrazone HIF-2α inhibitor according to claim 1, characterized in that: The R1 is selected from hydrogen, fluorine, chlorine, bromine, hydroxyl, methyl, and methoxy; the R2 is selected from hydrogen, methyl, and ethyl; the R3 is selected from hydrogen, methyl, and ethyl; the R4 is selected from fluorine, chlorine, bromine, cyano, methyl, and methoxy; the R5 is selected from fluorine, chlorine, bromine, cyano, methyl, and methoxy.

3. A hydrazide-based HIF-2α inhibitor, characterized in that: The HIF-2α inhibitor is selected from the following compounds:

4. A hydrazide-based HIF-2α inhibitor, characterized in that: The HIF-2α inhibitor is selected from the following compounds:

5. A pharmaceutical composition, characterized in that The invention comprises the HIF-2α inhibitor or a pharmaceutically acceptable salt thereof according to any one of claims 3 to 4, and a pharmaceutically acceptable carrier or excipient.

6. Use of the hydrazide hydrazone HIF-2α inhibitor according to any one of claims 3 to 4 in the preparation of a drug for treating diseases of VHL deficiency and / or abnormal HIF-2α expression.

7. The use according to claim 6, characterized in that: The disease is a tumor.

8. The use according to claim 6, characterized in that: The disease is an autoimmune disease.

9. The use according to claim 6, characterized in that: The disease is inflammation.

10. The use according to claim 6, characterized in that: The disease is an iron overload disease.

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