Phenylpyrazoles and their use

Abstract

This invention discloses a phenylpyrazole compound. It also discloses the application of the aforementioned compound in the preparation of drugs for treating prostate cancer. The phenylpyrazole compound provided by this invention exhibits significant antagonistic activity against androgen receptors, offering a new avenue for drug development in prostate cancer.

Description

Technical Field

[0001] This invention belongs to the field of medicine, specifically relating to a phenylpyrazole compound as an androgen receptor antagonist and its application in the preparation of drugs for the treatment of prostate cancer. Technical Background

[0002] Prostate cancer is one of the most common malignant tumors in men. More than 70 years ago, Charles Huggins demonstrated that prostate cancer is androgen-dependent and that orchiectomy could induce tumor regression. Therefore, androgen deprivation therapy (ADT) became a primary treatment method. While symptoms can be alleviated to some extent in the early stages of treatment, the vast majority of patients eventually develop castration-resistant prostate cancer (CRPC). In CRPC, although androgen levels are reduced to extremely low levels, the tumor continues to survive, and patients typically die within 2-4 years. Studies have shown that CRPC growth is still AR-dependent; therefore, targeting the AR signaling pathway remains important for the treatment of prostate cancer.

[0003] The androgen receptor (AR) belongs to the nuclear receptor superfamily. It is a ligand-dependent transcription factor with similar structure and function to other nuclear receptors, regulating the expression of certain genes. The AR protein contains 919 amino acids and consists of four important domains: the N-terminal domain (NTD), the DNA-binding domain (DBD), and the C-terminal ligand-binding domain (LBD). The DBD and LBD are connected by a hinge region. In men, androgens such as testosterone are mainly synthesized in the testes. Once synthesized, testosterone binds to sex hormone-binding globulin and albumin in the serum. Free testosterone enters prostate cells and, under the catalysis of 5α-reductase, is converted into the more potent dihydrotestosterone (DHT). Under normal circumstances, the androgen receptor binds to heat shock proteins. When androgens bind to AR, a conformational change occurs, inducing AR to dissociate from heat shock proteins and subsequently be transported into the cell nucleus. AR homodimers are formed in the cell nucleus, recognize androgen response elements in the promoter regions of target genes, and recruit more co-regulatory proteins to promote the transcription of AR target genes.

[0004] Androgen receptor antagonists, or antiandrogens, inhibit AR activity by directly binding to or blocking the AR ligand binding domain, thus preventing androgens from exerting their corresponding biological activities.

[0005] Based on their chemical structures, antiandrogens can be divided into steroidal and non-steroidal types. Steroidal antiandrogens like cyproterone acetate have limited clinical application due to their poor oral bioavailability and selectivity, which easily cause adverse reactions. Currently marketed non-steroidal antiandrogens include first-generation steroids such as flutamide, nilutamide, and bicalutamide, and second-generation steroids such as enzalutamide, apalutamide, and darolutamide. These antagonists share essentially the same mechanism of action against AR (alanine globulin). They all act on the ligand-binding pocket (LBP) of the AR LBD. Specifically, they bind to the AR LBP, causing a conformational change in the AR protein, thereby preventing AR activation. Although these antiandrogens can inhibit tumor growth in the early stages of treatment, drug resistance inevitably develops with long-term treatment, and there is even a possibility that the antagonists may transform into agonists, thus promoting tumor growth. For example, existing literature reports that long-term use of the second-generation antagonist enzalutamide can lead to the F876L missense mutation in AR LBD, resulting in enzalutamide resistance and causing it to change from an antagonist to an agonist. Therefore, the development of novel, safe, and effective AR antagonists is urgently needed, as it has significant clinical implications for providing treatment strategies for prostate cancer and improving patient survival and quality of life. Summary of the Invention

[0006] The purpose of this invention is to provide a novel compound with androgen receptor antagonistic activity and to apply it in anti-prostate cancer drugs.

[0007] This invention provides a phenylpyrazole compound or a pharmaceutically acceptable salt thereof with the structural formula shown in formula (I):

[0008]

[0009] R1 is selected from hydrogen, halogen, cyano, and nitro;

[0010] R2 is selected from hydrogen, trifluoromethyl, halogen, cyano, nitro, C 1-6 Alkyl, halogen-substituted C 1-6 alkyl;

[0011] R3 is selected from hydrogen, halogens, and C.1-6 Alkyl, halogen-substituted C 1-6 Alkyl, aryl;

[0012] Ring A is selected from C 5-12 Carbon rings, heterocycles, aromatic rings, aromatic heterocycles, and benzo[a]heterocycles, wherein the carbon atoms on the ring can be replaced by one or more heteroatoms;

[0013] R4 and R5 are independently selected from hydrogen, halogen, and C, respectively. 1-6 Alkyl, C 1-6 alkoxy- or halogen-substituted C 1-6 Alkyl, hydroxyl substituted C 1-6 Alkyl, cyano, nitro, amide, ester, aromatic ring, aromatic heterocycle;

[0014] n is 0, 1, 2, or 3;

[0015] X is selected from –NHC(O)–, –NHS(O)2–, –NHC(O)NH–, and –C(O)NH–;

[0016] Terminology explanation: The aforementioned C 1-6 Alkyl groups are straight-chain or branched hydrocarbon chains containing 1 to 6 carbon atoms. Examples include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, and substituted alkyl groups.

[0017] The alkoxy group refers to an -O-alkyl group, wherein the alkyl group is as described above. Examples include, but are not limited to, methoxy, ethoxy, n-propoxy, isopropoxy, tert-butoxy, and substituted alkoxy groups.

[0018] The aromatic ring refers to a monocyclic or fused polycyclic group with 5-12 carbon atoms, possessing a fully conjugated π-electron system. Examples include benzene rings, naphthalene rings, and anthracene rings.

[0019] The aromatic heterocycles referred to are non-all-carbon monocyclic or fused polycyclic groups with 5-12 carbon atoms, possessing a fully conjugated π-electron system. Examples include, but are not limited to, pyridine, imidazole, pyrazole, furan, thiazole, indole, and thiophene.

[0020] The halogens referred to are fluorine, chlorine, bromine, or iodine.

[0021] Preferably, R1 is selected from cyano or nitro; R2 is selected from chloro or trifluoromethyl; and R3 is hydrogen or methyl.

[0022] As a preferred option, ring A is selected from...

[0023]

[0024] Preferably, R4 and R5 are independently selected from hydrogen, fluorine, chlorine, methyl, methoxy, trifluoromethyl, 2-hydroxy-prop-2-yl, cyano, nitro, and carbamoyl, respectively.

[0025] As a preferred option, X is selected from...

[0026] As a further preferred embodiment, R1 is cyano; R2 is trifluoromethyl or chlorine; R3 is hydrogen; n is 1; X is –C(O)NH–; ring A is a pyridine ring or a benzene ring; R4 and R5 are independently selected from hydrogen, F, cyano or nitro.

[0027] Preferably, the compound has the following structural formula.

[0028]

[0029]

[0030]

[0031] Preferably, the pharmaceutically acceptable salt is a hydrochloride, sulfate, acetate, benzenesulfonate, phosphate, maleate, tartrate, or fumarate.

[0032] The present invention demonstrates that the compound exhibits significant androgen receptor transcriptional repression activity and can significantly inhibit the proliferation of prostate cancer cells without significant cytotoxicity.

[0033] This invention provides the use of the said compound or a pharmaceutically acceptable salt thereof in the preparation of androgen receptor antagonists.

[0034] The present invention also provides the use of the said compound or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating prostate cancer.

[0035] The present invention also provides a method for preparing the said compound or a pharmaceutically acceptable salt thereof, the reaction route of which is shown below:

[0036] The intermediate is synthesized according to the following route:

[0037]

[0038] or

[0039]

[0040] or

[0041]

[0042] or

[0043]

[0044] n is 0, 1, 2, or 3;

[0045] Compounds 1 and 2 were prepared according to the following route:

[0046]

[0047] Compound 3-24 was prepared according to the following route:

[0048]

[0049] Compounds 25-27 were prepared according to the following route:

[0050]

[0051] Compounds 28-48 were prepared according to the following route:

[0052]

[0053] R 1 R 2 R 3 R 4 R 5 As described above. Attached Figure Description

[0054] Figure 1 The results show the inhibitory effects of compounds 33, 40, and 47 on LNCaP cell proliferation at a series of concentration gradients.

[0055] Figure 2 The results show the effects of compounds 33, 40, and 47 on the proliferation inhibition of 22Rv1 cells at a series of concentration gradients.

[0056] Figure 3 The results show the inhibitory effects of compounds 33, 40, and 47 on the proliferation of DU145 cells at a series of concentration gradients.

[0057] Figure 4 The results show the experimental effects of compounds 33, 40, and 47 on the proliferation inhibition of PC3 cells at a series of concentration gradients.

[0058] Figure 5 The results show the effects of compounds 33, 40, and 47 on the proliferation inhibition of GES-1 cells at a series of concentration gradients.

[0059] Figure 6 The results show the experimental effects of compounds 33, 40, and 47 on the proliferation inhibition of Chang cells at a series of concentration gradients.

[0060] Figure 7The results show the inhibition of prostate-specific antigen (PSA) protein levels in LNCaP cells by compounds 33, 40, and 47 at a series of concentration gradients. Detailed Implementation

[0061] The following description, in conjunction with specific embodiments, further illustrates the present invention. These specific embodiments are for illustrative purposes only and are not intended to limit the invention in any way.

[0062] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0063] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0064] Example 1: Synthesis of 1-(1-(4-cyano-3-trifluoromethylphenyl)-1H-pyrazol-3-yl)-3-(4-fluorophenyl)urea (1)

[0065] a) Intermediate methyl 1-(4-cyano-3-trifluoromethylphenyl)-1H-pyrazole-3-carboxylic acid ester

[0066]

[0067] In a 100 mL two-necked round-bottom flask, 4-bromo-2-trifluoromethylbenzonitrile (7.50 g, 30 mmol), methyl 3-pyrazolecarboxylate (4.54 g, 36 mmol), cuprous iodide (1.14 g, 6 mmol), L-proline (1.38 g, 12 mmol), and potassium carbonate (8.29 g, 60 mmol) were added. After argon exchange, 50 mL of dimethyl sulfoxide was added, and the mixture was heated to 100 °C for 3 h. After the reaction was complete as monitored by TLC, the mixture was cooled to room temperature, and ethyl acetate and water were added and stirred for 30 min. The mixture was filtered to remove insoluble matter, and the filtrate was extracted three times with ethyl acetate. The combined organic layers were washed with copious amounts of water and twice with saturated sodium chloride. The mixture was dried over anhydrous sodium sulfate, concentrated, and the crude product was recrystallized from PE / EA and filtered to give a white solid (6.20 g, 70%).

[0068] b) Intermediate 1-(4-cyano-3-trifluoromethylphenyl)-1H-pyrazole-3-carboxylic acid

[0069]

[0070] In a 100 mL single-necked round-bottom flask, 4.43 g (15 mmol) of the product from the previous step was weighed and dissolved in 30 mL of tetrahydrofuran. Lithium hydroxide (1.08 g, 45 mmol) and 6 mL of water were then added, and the mixture was stirred at room temperature for 2 h. After the reaction was complete as monitored by TLC, 2N HCl solution was added to the reaction mixture to adjust the pH to 3–4. The mixture was extracted three times with ethyl acetate, and the combined organic layers were washed twice with saturated sodium chloride, dried over anhydrous sodium sulfate, and concentrated to give a white solid (4.01 g, 95%).

[0071] c) Intermediate (1-(4-cyano-3-trifluoromethylphenyl)-1H-pyrazole-3-yl)tert-butyl carbamate

[0072]

[0073] Weigh the carboxylic acid product (2.81 g, 10 mmol) from the previous step into a 100 mL two-necked round-bottom flask. After argon exchange, add 20 mL of tert-butanol and 20 mL of dioxane to dissolve it. Then add diphenyl azidophosphate (2.59 mL, 3.30 g, 12 mmol) and triethylamine (1.67 mL, 1.21 g, 12 mmol), respectively, and heat to 100 °C overnight. After the reaction is complete as monitored by TLC, cool to room temperature, add water, and extract three times with ethyl acetate. Combine the organic layers, wash twice with saturated sodium chloride, dry to anhydrous sodium sulfate, concentrate, and separate by silica gel column chromatography using PE / EA (v / v, 5 / 1) as the developing solvent to obtain a yellow liquid (2.22 g, 63%).

[0074] d) Intermediate 4-(3-amino-1H-pyrazol-1-yl)-2-trifluoromethylbenzonitrile

[0075]

[0076] Weigh the product from the previous step (1.76 g, 5 mmol) into a 50 mL single-necked round-bottom flask, dissolve it in 20 mL of dichloromethane, then add trifluoroacetic acid (1.91 mL, 2.85 g, 25 mmol), and heat under reflux for 5 h. After the reaction was complete as monitored by TLC, the residual trifluoroacetic acid was removed by vortexing, saturated sodium bicarbonate was added, and the mixture was extracted three times with ethyl acetate. The organic layers were combined, washed twice with saturated sodium chloride, dried over anhydrous sodium sulfate, concentrated, and separated by silica gel column chromatography using DCM / MeOH (v / v, 100 / 1) as the developing solvent to obtain a yellow solid (1.07 g, 85%).

[0077] e)1-(1-(4-cyano-3-trifluoromethylphenyl)-1H-pyrazol-3-yl)-3-(4-fluorophenyl)urea(1)

[0078]

[0079] Weigh the amine product (126 mg, 0.5 mmol) from the previous step into a 25 mL two-necked round-bottom flask. After argon exchange, dissolve it in anhydrous tetrahydrofuran. Cool to 0 °C, add 85 μL (103 mg, 0.75 mmol) of 4-fluorophenyl isocyanate dropwise, followed by triethylamine (104 μL, 76 mg, 0.75 mmol). After the addition is complete, allow the mixture to return to room temperature and react overnight. Monitor the reaction by TLC. Once the reaction is complete, add water, filter out the insoluble solid, wash with water, and separate the crude product by silica gel column chromatography using DCM / MeOH (v / v, 30 / 1) as the developing solvent.

[0080] White solid; yield: 82%; mp 315.6-316.7℃; 1 H NMR(500MHz,DMSO–d6)δ9.53(br,1H),8.87(br,1H),8.75(d,J=2.5Hz,1H),8.32–8. 21 (m, 3H), 7.48 (dd, J = 9.0, 5.0 Hz, 2H), 7.14 (t, J = 9.0 Hz, 2H), 6.80 (d, J = 2.5 Hz, 1H).

[0081] Example 2: Synthesis of 1-((1-(4-cyano-3-trifluoromethylphenyl)-1H-pyrazol-3-yl)methyl)-3-(4-fluorophenyl)urea (2)

[0082] a) Intermediate 4-(3-hydroxymethyl-1H-pyrazole-1-yl)-2-trifluoromethylbenzonitrile

[0083]

[0084] In a 100 mL two-necked round-bottom flask, 4.22 g (15 mmol) of the intermediate 1-(4-cyano-3-trifluoromethylphenyl)-1H-pyrazole-3-carboxylic acid obtained in Preparation Example 1(b) was added. After argon exchange, 15 mL of anhydrous tetrahydrofuran was added, and the mixture was cooled to -5 °C. 75 mL (75 mmol) of a 1.0 mol / L borane tetrahydrofuran complex was slowly added dropwise. After the addition was complete, the mixture was stirred at low temperature for 5 h. After the reaction was complete as monitored by TLC, 10 mL of methanol was slowly added dropwise to quench the reaction mixture. The mixture was then moved to room temperature and stirred for 30 min. The solvent was evaporated, water was added, and the mixture was extracted three times with ethyl acetate. The combined organic layers were washed twice with saturated sodium chloride, dried over anhydrous sodium sulfate, concentrated, and separated by silica gel column chromatography using DCM / MeOH (v / v, 50 / 1) as the developing solvent to obtain a white solid (3.49 g, 87%).

[0085] b) Intermediate (1-(4-cyano-3-trifluoromethylphenyl)-1H-pyrazole-3-yl)methyl-4-methylbenzenesulfonate

[0086]

[0087] Weigh 2.67 g (10 mmol) of the product from the previous step and 0.12 g (1 mmol) of 4-dimethylaminopyridine into a 100 mL single-necked round-bottom flask. Add triethylamine (2.02 g, 20 mmol) and dissolve in 20 mL of anhydrous dichloromethane. Cool to 0 °C. Then dissolve p-toluenesulfonyl chloride (1.91 g, 10 mmol) in 10 mL of anhydrous dichloromethane and slowly add it dropwise to the round-bottom flask at 0 °C. After the addition is complete, maintain the reaction at low temperature for 2 h. After the reaction is complete as monitored by TLC, add water, extract three times with ethyl acetate, combine the organic layers, wash twice with saturated sodium chloride, dry to anhydrous sodium sulfate, concentrate to obtain a crude white solid, and directly add to the next step.

[0088] c) Intermediate 4-(3-azidomethyl-1H-pyrazol-1-yl)-2-trifluoromethylbenzonitrile

[0089]

[0090] The product obtained in the previous step was placed in a 100 mL single-necked round-bottom flask, dissolved in 20 mL of acetone, and stirred at room temperature. Sodium azide (1.63 g, 25 mmol) was slowly added to the round-bottom flask, and the mixture was heated to 60 °C and refluxed for 3 h. After the reaction was complete as monitored by TLC, the acetone was removed by vortexing, water was added, and the mixture was extracted three times with ethyl acetate. The organic layers were combined, washed twice with saturated sodium chloride, dried over anhydrous sodium sulfate, concentrated, and separated by silica gel column chromatography using PE / EA (v / v, 4 / 1) as eluent to obtain a yellow solid (2.63 g, 90%).

[0091] d) Intermediate 4-(3-aminomethyl-1H-pyrazol-1-yl)-2-trifluoromethylbenzonitrile

[0092]

[0093] The product obtained in the previous step (2.63 g, 9 mmol) was placed in a 100 mL single-necked round-bottom flask, dissolved in 20 mL of tetrahydrofuran, and stirred at room temperature. Triphenylphosphine (2.60 g, 9.9 mmol) and 5 mL of water were added slowly in sequence, and the reaction was carried out at room temperature for 12 h. After the reaction was complete as monitored by TLC, the pH was adjusted to acidic by adding 2N HCl solution, and the mixture was extracted with ethyl acetate. The organic layer was discarded, and the aqueous layer was adjusted to alkaline by adding sodium hydroxide. The mixture was extracted three times with ethyl acetate, and the organic layers were combined. The mixture was washed twice with saturated sodium chloride, dried over anhydrous sodium sulfate, concentrated, and the crude product was recrystallized from PE / EA and filtered to give a white solid (1.96 g, 82%).

[0094] e)1-((1-(4-cyano-3-trifluoromethylphenyl)-1H-pyrazol-3-yl)methyl)-3-(4-fluorophenyl)urea(2)

[0095] The intermediate 4-(3-aminomethyl-1H-pyrazol-1-yl)-2-trifluoromethylbenzonitrile and 4-fluorophenyl isocyanate were reacted using the method described in Example 1(e).

[0096]

[0097] White solid; yield: 85%; mp 264.6-265.5℃; 1 H NMR(500MHz,DMSO–d6)δ8.77(d,J=2.0Hz,1H),8.70(br,1H),8.38(s,1H),8.34–8.28(m,2H),7.41(dd,J=8 .5,5.0Hz,2H),7.06(t,J=8.5Hz,2H),6.65(t,J=5.5Hz,1H),6.58(d,J=2.0Hz,1H),4.37(d,J=5.5Hz,2H).

[0098] Example 3: Synthesis of N-(1-(4-cyano-3-trifluoromethylphenyl-1H-pyrazol-3-yl)-4-fluorobenzamide (3)

[0099]

[0100] In a 25 mL two-necked round-bottom flask, 4-fluorobenzoic acid (84 mg, 0.6 mmol) and HATU (228 mg, 0.6 mmol) were added separately. After argon exchange, 5 mL of anhydrous dichloromethane was added to dissolve them. Then, N,N-diisopropylethylamine (105 μL, 77 mg, 0.6 mmol) was added, and the mixture was stirred at room temperature for 10 min. The intermediate 4-(3-amino-1H-pyrazol-1-yl)-2-trifluoromethylbenzonitrile (126 mg, 0.5 mmol) obtained in Example 1(d) was added to the reaction system, and the reaction was carried out overnight at room temperature. After the reaction was complete as monitored by TLC, water was added, and the mixture was extracted three times with ethyl acetate. The organic layers were combined, washed successively with saturated sodium bicarbonate and saturated sodium chloride, dried over anhydrous sodium sulfate, concentrated, and separated by silica gel column chromatography using PE / EA (v / v, 2 / 1) as the developing solvent to obtain a white solid.

[0101] White solid; yield: 90%; mp 205.4-206.5℃; 1H NMR (400MHz, Acetone–d6) δ10.35(br,1H),8.62(d,J=2.5Hz,1H),8.34(s,1H),8.29–8.17(m,4H),7.31(t,J=8.8Hz,2H),7.22(d,J=2.5Hz,1H).

[0102] Example 4: Synthesis of N-((1-(4-cyano-3-trifluoromethylphenyl)-1H-pyrazol-3-yl)methyl)-4-fluorobenzamide (4)

[0103] The intermediate 4-(3-aminomethyl-1H-pyrazol-1-yl)-2-trifluoromethylbenzonitrile and 4-fluorobenzoic acid were prepared by condensation reaction using the method described in Example 3.

[0104]

[0105] White solid; yield: 90%; mp 200.1-201.6℃; 1 H NMR(500MHz, Acetone–d6)δ8.60(d,J=2.5Hz,1H),8.41–8.37(m,1H),8.36–8.29(m,2H),8.19(d, J=8.5Hz,1H),8.06–8.00(m,2H),7.28–7.18(m,2H),6.65(d,J=2.5Hz,1H),4.68(d,J=6.0Hz,2H).

[0106] Example 5: Synthesis of N-((1-(4-cyano-3-trifluoromethylphenyl)-1H-pyrazol-3-yl)methyl)-4-chlorobenzamide (5)

[0107]

[0108] The intermediate 4-(3-aminomethyl-1H-pyrazol-1-yl)-2-trifluoromethylbenzonitrile and 4-chlorobenzoic acid were prepared by condensation reaction using the method described in Example 3.

[0109] Pale yellow solid; yield: 89%; mp 183.9-184.7℃; 1H NMR(500MHz, Acetone–d6)δ8.58(d,J=2.6Hz,1H),8.41–8.36(m,2H),8.32–8.28(m,1H),8.18(d,J=8 .5Hz, 1H), 7.97 (d, J = 9.0Hz, 2H), 7.50 (d, J = 9.0Hz, 2H), 6.64 (d, J = 2.6Hz, 1H), 4.69 (d, J = 6.0Hz, 2H).

[0110] Example 6: Synthesis of N-((1-(4-cyano-3-trifluoromethylphenyl)-1H-pyrazol-3-yl)methyl)-4-trifluoromethylbenzamide (6)

[0111]

[0112] The intermediate 4-(3-aminomethyl-1H-pyrazol-1-yl)-2-trifluoromethylbenzonitrile and 4-trifluoromethylbenzoic acid were prepared by condensation reaction using the method described in Example 3.

[0113] White solid; yield: 87%; mp 218.9-220.6℃; 1 H NMR (500MHz, Acetone–d6) δ8.60(d,J=2.5Hz,1H),8.53(br,1H),8.39(d,J=2.0Hz,1H),8.31(dd,J=8.5,2.0Hz,1H) ,8.18(d,J=8.5Hz,1H),8.15(d,J=8.0Hz,2H),7.83(d,J=8.0Hz,2H),6.66(d,J=2.5Hz,1H),4.72(d,J=6.0Hz,2H).

[0114] Example 7: Synthesis of N-((1-(4-cyano-3-trifluoromethylphenyl)-1H-pyrazol-3-yl)methyl)-4-cyanobenzamide (7)

[0115]

[0116] The intermediate 4-(3-aminomethyl-1H-pyrazol-1-yl)-2-trifluoromethylbenzonitrile and 4-cyanobenzoic acid were prepared by condensation reaction using the method described in Example 3.

[0117] White solid; yield: 82%; mp 205.8-207.1℃; 1H NMR (500MHz, Acetone–d6) δ8.60(d,J=3.0Hz,1H),8.55(br,1H),8.38(d,J=2.0Hz,1H),8.31(dd,J=8.5,2.0Hz,1H) ,8.19(d,J=8.5Hz,1H),8.12(d,J=8.5Hz,2H),7.90(d,J=8.6Hz,2H),6.66(d,J=3.0Hz,1H),4.71(d,J=5.5Hz,2H).

[0118] Example 8: Synthesis of N-((1-(4-cyano-3-trifluoromethylphenyl)-1H-pyrazol-3-yl)methyl)-4-nitrobenzamide (8)

[0119]

[0120] The intermediate 4-(3-aminomethyl-1H-pyrazol-1-yl)-2-trifluoromethylbenzonitrile and 4-nitrobenzoic acid were prepared by condensation reaction using the method described in Example 3.

[0121] White solid; yield: 80%; mp 209.7-210.8℃; 1 H NMR (500MHz, Acetone–d6) δ8.63(t,J=6.0Hz,1H),8.60(d,J=2.5Hz,1H),8.38(d,J=2.0Hz ,1H),8.34–8.29(m,3H),8.21–8.17(m,3H),6.67(d,J=2.5Hz,1H),4.73(d,J=6.0Hz,2H).

[0122] Example 9: Synthesis of N-((1-(4-cyano-3-trifluoromethylphenyl)-1H-pyrazol-3-yl)methyl)-4-(2-hydroxypropyl-2-yl)benzamide (9)

[0123]

[0124] The intermediate 4-(3-aminomethyl-1H-pyrazol-1-yl)-2-trifluoromethylbenzonitrile and 4-(2-hydroxypropyl-2-yl)benzoic acid were prepared by condensation reaction using the method described in Example 3.

[0125] White solid; yield: 64%; mp 181.3-182.7℃; 1H NMR (500MHz, DMSO–d6) δ8.57(d,J=2.5Hz,1H),8.39(d,J=2.0Hz,1H),8.31(dd,J=8.5,2.1Hz,1H),8.25(t,J=6.0Hz,1H),8.17(d,J =8.5Hz, 1H), 7.91 (d, J = 8.5Hz, 2H), 7.62 (d, J = 8.5Hz, 2H), 6.64 (d, J = 2.5Hz, 1H), 4.69 (d, J = 6.0Hz, 2H), 4.19 (s, 1H), 1.53 (s, 6H).

[0126] Example 10: N 4 -((1-(4-cyano-3-trifluoromethylphenyl)-1H-pyrazole-3-yl)methyl)-2-fluoro-N 1 Synthesis of methyl terephthalamide (10)

[0127]

[0128] The intermediate 4-(3-aminomethyl-1H-pyrazol-1-yl)-2-trifluoromethylbenzonitrile and 4-(methylcarbamoyl)-3-fluorobenzoic acid were prepared by condensation reaction using the method described in Example 3.

[0129] White solid; yield: 57%; mp 261.4-262.8℃; 1 H NMR(500MHz, DMSO–d6)δ9.26(t,J=5.5Hz,1H),8.78(d,J=2.5Hz,1H),8.40–8.35(m,2H),8.34–8. 28(m,2H),7.80–7.68(m,3H),6.61(d,J=2.5Hz,1H),4.56(d,J=5.5Hz,2H),2.78(d,J=4.5Hz,3H).

[0130] Example 11: Synthesis of N-((1-(4-cyano-3-trifluoromethylphenyl)-1H-pyrazol-3-yl)methyl)-5-(1-hydroxyethyl)-1H-pyrazol-3-carboxamide (11)

[0131]

[0132] The intermediate 4-(3-aminomethyl-1H-pyrazol-1-yl)-2-trifluoromethylbenzonitrile and 5-(1-hydroxyethyl)-1H-pyrazol-3-carboxylic acid were prepared by condensation reaction using the method described in Example 3.

[0133] White solid; yield: 63%; mp 188.0-189.8℃;1 H NMR (500MHz, Acetone–d6) δ12.32(br,1H),8.57(d,J=2.5Hz,1H),8.39(d,J=2.0Hz,1H),8.30(dd,J=8.5,2.0Hz,2H),8.18(d, J=8.5Hz,1H),8.01(br,1H),6.67–6.57(m,2H),5.03–4.96(m,1H),4.66(d,J=6.0Hz,2H),4.60(br,1H),1.50(d,J=6.5Hz,5H).

[0134] Example 12: Synthesis of N-((1-(4-cyano-3-trifluoromethylphenyl)-1H-pyrazol-3-yl)methyl)-6-fluoropyridineamide (12)

[0135]

[0136] The intermediate 4-(3-aminomethyl-1H-pyrazol-1-yl)-2-trifluoromethylbenzonitrile and 6-fluoronicotinic acid were prepared by condensation reaction using the method described in Example 3.

[0137] White solid; yield: 92%; mp 226.4-227.6℃; 1 H NMR (400MHz, DMSO–d6) δ9.31 (br, 1H), 8.83–8.69 (m, 2H), 8.47–8.26 (m, 4H), 7.32 (d, J = 7.2Hz, 1H), 6.63 (s, 1H), 4.57 (d, J = 4.8Hz, 2H).

[0138] Example 13: Synthesis of N-((1-(4-cyano-3-trifluoromethylphenyl)-1H-pyrazol-3-yl)methyl)-5-fluoropyridineamide (13)

[0139]

[0140] The intermediate 4-(3-aminomethyl-1H-pyrazol-1-yl)-2-trifluoromethylbenzonitrile and 5-fluoro-2-pyridinecarboxylic acid were prepared by condensation reaction using the method described in Example 3.

[0141] White solid; yield: 88%; mp 220.7-221.8℃; 1H NMR (400MHz, DMSO–d6) δ9.30(t,J=6.0Hz,1H),8.75(d,J=2.2Hz,1H),8.66(d,J=2.4Hz,1H),8.35(s,1H),8.32–8. 25(m,2H),8.13(dd,J=8.8,4.8Hz,1H),7.91(td,J=8.8,2.4Hz,1H),6.57(d,J=2.2Hz,1H),4.57(d,J=6.0Hz,2H).

[0142] Example 14: Synthesis of N-((1-(4-cyano-3-trifluoromethylphenyl)-4-methyl-1H-pyrazol-3-yl)methyl)-4-fluorobenzamide (14)

[0143]

[0144] The intermediate 4-(3-(aminoethyl)-4-methyl-1H-pyrazol-1-yl)-2-trifluoromethylbenzonitrile was prepared by replacing methyl 3-pyrazol carboxylate with methyl 4-methyl-1H-pyrazol-3-carboxylate using the methods described in Examples 1(ab) and 2(ad), and then prepared by condensation reaction of the intermediate 4-(3-(aminoethyl)-4-methyl-1H-pyrazol-1-yl)-2-trifluoromethylbenzonitrile and 4-fluorobenzoic acid using the method described in Example 3.

[0145] White solid; yield: 84%; mp 208.2-209.4℃; 1 H NMR(500MHz,DMSO–d6)δ8.99(t,J=5.5Hz,1H),8.58(s,1H),8.33–8.21(m,3H),7.9 6(dd,J=8.5,5.5Hz,2H), 7.29(t,J=8.8Hz,2H), 4.54(d,J=5.5Hz,2H), 2.08(s,3H).

[0146] Example 15: Synthesis of N-((1-(4-cyano-3-trifluoromethylphenyl)-4-methyl-1H-pyrazol-3-yl)methyl)-4-cyanobenzamide (15)

[0147]

[0148] The intermediate 4-(3-(aminoethyl)-4-methyl-1H-pyrazol-1-yl)-2-trifluoromethylbenzonitrile and 4-cyanobenzoic acid were prepared by condensation reaction using the method described in Example 3.

[0149] White solid; yield: 82%; mp 237.1-238.1℃;1 H NMR (500MHz, DMSO–d6) δ9.24(br,1H),8.59(s,1H),8.34–8.20(m,3H),8.03(d,J=8.0Hz,2H),7.96(d,J=8.0Hz,2H),4.56(d,J=4.5Hz,2H),2.09(s,3H).

[0150] Example 16: Synthesis of N-((1-(4-cyano-3-trifluoromethylphenyl)-4-methyl-1H-pyrazol-3-yl)methyl)-4-nitrobenzamide (16)

[0151]

[0152] The intermediate 4-(3-(aminoethyl)-4-methyl-1H-pyrazol-1-yl)-2-trifluoromethylbenzonitrile and 4-nitrobenzoic acid were prepared by condensation reaction using the method described in Example 3.

[0153] White solid; yield: 81%; mp 249.8-250.5℃; 1 H NMR(500MHz,DMSO–d6)δ9.33(t,J=5.5Hz,1H),8.59(s,1H),8.33–8.30(m,3H),8.27(d,J=8.5H z, 1H), 8.23 ​​(dd, J = 8.5, 2.0Hz, 1H), 8.11 (d, J = 9.0Hz, 2H), 4.57 (d, J = 5.5Hz, 2H), 2.10 (s, 3H).

[0154] Example 17: Synthesis of N-((1-(4-cyano-3-trifluoromethylphenyl)-4-trifluoromethyl-1H-pyrazol-3-yl)methyl)-4-fluorobenzamide (17)

[0155]

[0156] The intermediate 4-(3-aminomethyl-4-trifluoromethyl)-1H-pyrazol-1-yl)-2-trifluoromethylbenzonitrile was prepared by replacing methyl 3-pyrazol carboxylate with methyl 4-trifluoromethyl-1H-pyrazol-3-carboxylate using the methods described in Examples 1 (ab) and 2 (ad), and then prepared by condensation reaction of the intermediate 4-(3-aminomethyl-4-trifluoromethyl)-1H-pyrazol-1-yl)-2-trifluoromethylbenzonitrile and 4-fluorobenzoic acid using the method described in Example 3.

[0157] White solid; yield: 86%; mp 192.4-193.5℃; 1H NMR(500MHz,DMSO–d6)δ9.50(s,1H),9.09(t,J=5.5Hz,1H),8.48(s,1H),8.39–8.3 5(m,2H),7.94(dd,J=9.0,5.5Hz,2H),7.32(t,J=9.0Hz,2H),4.66(d,J=5.5Hz,2H).

[0158] Example 18: Synthesis of N-((1-(4-cyano-3-trifluoromethylphenyl)-4-phenyl-1H-pyrazol-3-yl)methyl)-4-fluorobenzamide (18)

[0159]

[0160] The intermediate 4-(3-aminomethyl-4-phenyl-1H-pyrazole-1-yl)-2-trifluoromethylbenzonitrile was prepared by replacing methyl 3-pyrazolecarboxylate with methyl 4-phenyl-1H-pyrazole-3-carboxylate using the methods described in Examples 1(ab) and 2(ad), and then prepared by condensation reaction of the intermediate 4-(3-aminomethyl-4-phenyl-1H-pyrazole-1-yl)-2-trifluoromethylbenzonitrile and 4-fluorobenzoic acid using the method described in Example 3.

[0161] White solid; yield: 79%; mp 207.0-208.2℃; 1 H NMR(500MHz,DMSO–d6)δ9.14(s,1H),9.01(t,J=5.5Hz,1H),8.46–8.43(m,1H),8.37–8.31(m,2H),7.87(dd,J=9.0,6.0H z, 2H), 7.61 (d, J = 7.5Hz, 2H), 7.44 (t, J = 7.5Hz, 2H), 7.33 (t, J = 7.5Hz, 1H), 7.27 (t, J = 9.0Hz, 2H), 4.73 (d, J = 5.5Hz, 2H).

[0162] Example 19: Synthesis of N-(2-(1-(4-cyano-3-trifluoromethylphenyl)-1H-pyrazol-3-yl)ethyl)-4-fluorobenzamide (19)

[0163] a) Intermediate 4-(3-formyl-1H-pyrazole-1-yl)-2-trifluoromethylbenzonitrile

[0164]

[0165] The intermediate 4-(3-hydroxymethyl-1H-pyrazol-1-yl)-2-trifluoromethylbenzonitrile (2.67 g, 10 mmol) obtained in Example 2(a) was placed in a 100 mL single-necked round-bottom flask, dissolved in 20 mL of dichloromethane, and then manganese dioxide (4.35 g, 50 mmol) was added. The mixture was heated to 45 °C and refluxed for 5 h. After the reaction was complete as monitored by TLC, the reaction solution was filtered through diatomaceous earth to remove manganese dioxide, washed with ethyl acetate, and the filtrate was concentrated and separated by silica gel column chromatography using DCM / MeOH (v / v, 100 / 1) as the developing solvent to obtain a white solid (2.44 g, 92%).

[0166] b) Intermediate 4-(3-vinyl-1H-pyrazole-1-yl)-2-trifluoromethylbenzonitrile

[0167]

[0168] 4.82 g (13.5 mmol) of methyltriphenylphosphine bromide and 0.86 g (36 mmol) of 60% sodium hydride were weighed into a 100 mL two-necked round-bottom flask. Argon exchange was performed, and the flask was cooled to -20 °C. 15 mL of anhydrous tetrahydrofuran was added to dissolve the aldehyde, and the mixture was stirred for 30 min. The mixture was then transferred to room temperature and stirred for 2 h. The reaction solution was recooled to 0 °C. The aldehyde product (2.39 g, 9 mmol) from the previous step was dissolved in 15 mL of anhydrous tetrahydrofuran and slowly added dropwise to the round-bottom flask. The mixture was then transferred to room temperature and reacted overnight. After the reaction was complete as monitored by TLC, saturated ammonium chloride was added to quench the reaction. The mixture was extracted three times with ethyl acetate, and the combined organic layers were washed twice with saturated sodium chloride, dried over anhydrous sodium sulfate, concentrated, and separated by silica gel column chromatography using PE / EA (v / v, 4 / 1) as the developing solvent to obtain a white solid (2.66 g, 75%).

[0169] c) Intermediate 4-(3-(2-(4,4,5,5-tetramethyl-1,3,2-dioxacyclopentabor-2-yl)ethyl)-1H-pyrazol-1-yl)-2-trifluoromethylbenzonitrile

[0170]

[0171] Weigh the product from the previous step (1.32 g, 5 mmol), pinacol diborate (1.90 g, 7.5 mmol), and cesium carbonate (4.07 g, 12.5 mmol) into a 100 mL two-necked round-bottom flask. After argon exchange, add 15 mL of dioxane to dissolve the product, followed by methanol (1.01 mL, 0.8 g, 25 mmol). Heat to 100 °C and react overnight. Then, dilute the reaction solution with ethyl acetate, filter through diatomaceous earth to remove insoluble matter, wash the filter residue with ethyl acetate, concentrate the filtrate to obtain a crude yellow solid, and directly add it to the next reaction step.

[0172] d) Intermediate 4-(3-(2-hydroxyethyl)-1H-pyrazol-1-yl)-2-trifluoromethylbenzonitrile

[0173]

[0174] The crude product obtained in the previous step was placed in a 50 mL single-necked round-bottom flask, dissolved in 15 mL of tetrahydrofuran, and cooled to 0 °C. Sodium hydroxide (1.0 g, 25 mmol) was weighed and added to the flask, followed by the slow addition of 30% hydrogen peroxide (2.55 mL, 2.83 g, 25 mmol). After the addition was complete, the reaction was kept at a low temperature for 2 h. After the reaction was complete as monitored by TLC, water was added to quench the reaction, and the product was extracted three times with ethyl acetate. The organic layers were combined, washed twice with saturated sodium chloride, dried over anhydrous sodium sulfate, concentrated, and separated by silica gel column chromatography using DCM / MeOH (v / v, 100 / 1) as the developing solvent to obtain a white solid (703 mg, 50%).

[0175] e) Intermediate 4-(3-(2-aminoethyl)-1H-pyrazole-1-yl)-2-trifluoromethylbenzonitrile

[0176]

[0177] Using the method described in Example 2(bd), the intermediate 4-(3-(2-hydroxyethyl)-1H-pyrazol-1-yl)-2-trifluoromethylbenzonitrile obtained in the previous step was used as a raw material to prepare the product.

[0178] f)N-(2-(1-(4-cyano-3-trifluoromethylphenyl)-1H-pyrazol-3-yl)ethyl)-4-fluorobenzamide (19)

[0179]

[0180] The intermediate 4-(3-(2-aminoethyl)-1H-pyrazol-1-yl)-2-trifluoromethylbenzonitrile and 4-fluorobenzoic acid were prepared by condensation reaction using the method described in Example 3.

[0181] White solid; yield: 75%; mp 165.4-166.7℃; 1 H NMR(500MHz, DMSO–d6)δ8.74(d,J=2.5Hz,1H),8.64(t,J=5.5Hz,1H),8.36–8.33(m,1H),8.32–8.26(m,2H),7.8 9(dd,J=8.8,5.5Hz,2H),7.28(t,J=8.8Hz,2H),6.58(d,J=2.5Hz,1H),3.63–3.55(m,2H),2.94(t,J=7.5Hz,2H).

[0182] Example 20: Synthesis of N-(2-(1-(4-cyano-3-(trifluoromethyl)phenyl)-1H-pyrazol-3-yl)ethyl)-4-cyanobenzamide (20)

[0183]

[0184] The intermediate 4-(3-(2-aminoethyl)-1H-pyrazol-1-yl)-2-trifluoromethylbenzonitrile and 4-cyanobenzoic acid were prepared by condensation reaction using the method described in Example 3.

[0185] White solid; yield 74%; mp 236.7-237.9℃; 1 H NMR(500MHz,DMSO–d6)δ8.88(t,J=6.0Hz,1H),8.75(d,J=2.5Hz,1H),8.33(s,1H),8.31–8.2 6(m,2H),7.99–7.93(m,4H),6.58(d,J=2.5Hz,1H),3.64–3.58(m,2H),2.95(t,J=7.5Hz,2H).

[0186] Example 21: Synthesis of N-(2-(1-(4-cyano-3-trifluoromethylphenyl)-1H-pyrazol-3-yl)ethyl)-4-nitrobenzamide (21)

[0187]

[0188] The intermediate 4-(3-(2-aminoethyl)-1H-pyrazol-1-yl)-2-trifluoromethylbenzonitrile and 4-nitrobenzoic acid were prepared by condensation reaction using the method described in Example 3.

[0189] White solid; yield: 71%; mp 206.0-207.5℃; 1 H NMR(500MHz, DMSO–d6)δ8.96(t,J=5.5Hz,1H),8.75(d,J=2.5Hz,1H),8.35–8.33(m,1H),8.32–8. 27(m,4H),8.05(d,J=9.0Hz,2H),6.59(d,J=2.5Hz,1H),3.66–3.60(m,2H),2.96(t,J=7.0Hz,2H).

[0190] Example 22: Synthesis of N-(3-(1-(4-cyano-3-trifluoromethylphenyl)-1H-pyrazol-3-yl)propyl)-4-fluorobenzamide (22)

[0191] a) Intermediate (E)-3-(1-(4-cyano-3-trifluoromethylphenyl)-1H-pyrazole-3-yl)acrylic acid

[0192]

[0193] Malonic acid (1.56 g, 15 mmol) was weighed and placed in a 50 mL single-necked round-bottom flask. 5 mL of pyridine and 0.5 mL of piperidine were added to dissolve the flask, and the mixture was heated to 85 °C. The intermediate 4-(3-formyl-1H-pyrazole-1-yl)-2-trifluoromethylbenzonitrile (2.65 g, 10 mmol) obtained in Example 19(a) was slowly added to the round-bottom flask, and the reaction mixture was heated for another 6 h. After the reaction was complete as monitored by TLC, the mixture was cooled to room temperature, and the reaction mixture was neutralized with 2N HCl solution, precipitating a white solid. The solid was filtered, washed three times with water, and recrystallized from the residue with ethanol to obtain a white solid (2.76 g, 90%).

[0194] b) Intermediate 3-(1-(4-cyano-3-trifluoromethylphenyl)-1H-pyrazol-3-yl)propionic acid

[0195]

[0196] In a 100 mL two-necked round-bottom flask, the product from the previous step (2.76 g, 9 mmol) and Pd / C (276 mg, 10% Pd) were added, dissolved in 15 mL of methanol, and reacted at room temperature in a hydrogen atmosphere for 8 h. After the reaction was complete as monitored by TLC, ethyl acetate was added for dilution, and the mixture was filtered through diatomaceous earth to remove insoluble matter. The solution was washed with ethyl acetate, and the filtrate was concentrated and separated by silica gel column chromatography using DCM / MeOH (v / v, 50 / 1) as the developing solvent to obtain a white solid (2.48 g, 89%).

[0197] c) Intermediate 4-(3-(3-aminopropyl)-1H-pyrazole-1-yl)-2-trifluoromethylbenzonitrile

[0198]

[0199] Using the method described in Example 2(ad), the intermediate 3-(1-(4-cyano-3-trifluoromethylphenyl)-1H-pyrazole-3-yl)propionic acid obtained in the previous step was used as a raw material to prepare the product.

[0200] d)N-(3-(1-(4-cyano-3-trifluoromethylphenyl)-1H-pyrazole-3-yl)propyl)-4-fluorobenzamide (22)

[0201]

[0202] The intermediate 4-(3-(3-aminopropyl)-1H-pyrazol-1-yl)-2-trifluoromethylbenzonitrile and 4-fluorobenzoic acid were prepared by condensation reaction using the method described in Example 3.

[0203] White solid; yield: 77%; mp 151.9-153.0℃; 1 H NMR (400MHz, Acetone–d6) δ8.56(d,J=2.4Hz,1H),8.37(d,J=2.0Hz,1H),8.29(dd,J=8.4,2.0Hz,1H),8.17(d,J=8.4Hz,1H),7.95(dd,J =8.8,5.6Hz,2H),7.86(br,1H),7.19(t,J=8.8Hz,2H),6.56(d,J=2.4Hz,1H),3.55–3.47(m,2H),2.90–2.79(m,2H),2.09–2.04(m,2H).

[0204] Example 23: Synthesis of N-(3-(1-(4-cyano-3-trifluoromethylphenyl)-1H-pyrazol-3-yl)propyl)-4-cyanobenzamide (23)

[0205]

[0206] The intermediate 4-(3-(3-aminopropyl)-1H-pyrazol-1-yl)-2-trifluoromethylbenzonitrile and 4-cyanobenzoic acid were prepared by condensation reaction using the method described in Example 3.

[0207] White solid; yield: 75%; mp 200.2-201.1℃; 1 H NMR(400MHz, DMSO–d6)δ8.76(t,J=5.2Hz,1H),8.72(d,J=2.4Hz,1H),8.34–8.25(m,3H),8.00–7 .91(m,4H),6.57(d,J=2.4Hz,1H),3.40–3.33(m,2H),2.72(t,J=7.6Hz,2H),1.99–1.89(m,2H).

[0208] Example 24: Synthesis of N-(3-(1-(4-cyano-3-trifluoromethylphenyl)-1H-pyrazol-3-yl)propyl)-4-nitrobenzamide (24)

[0209]

[0210] The intermediate 4-(3-(3-aminopropyl)-1H-pyrazol-1-yl)-2-trifluoromethylbenzonitrile and 4-nitrobenzoic acid were prepared by condensation reaction using the method described in Example 3.

[0211] White solid; yield: 69%; mp 170.3-171.8℃; 1 H NMR(400MHz, Acetone–d6)δ8.55(d,J=2.4Hz,1H),8.36(d,J=1.6Hz,1H),8.30–8.25(m,3H),8.1 9–8.08(m,4H),6.56(d,J=2.6Hz,1H),3.59–3.52(m,2H),2.90–2.80(m,2H),2.12–2.06(m,2H).

[0212] Example 25: Synthesis of N-((1-(4-cyano-3-trifluoromethylphenyl)-1H-pyrazol-3-yl)methyl)-4-fluorobenzenesulfonamide (25)

[0213]

[0214] The intermediate 4-(3-aminomethyl-1H-pyrazol-1-yl)-2-trifluoromethylbenzonitrile (133 mg, 0.5 mmol) obtained in Example 2(d) was placed in a 25 mL single-necked round-bottom flask, dissolved in 5 mL of anhydrous tetrahydrofuran, followed by the addition of triethylamine (83 μL, 61 mg, 0.6 mmol). The mixture was cooled to 0 °C, and 4-fluorobenzenesulfonyl chloride (117 mg, 0.6 mmol) was slowly added. After the addition was complete, the mixture was allowed to return to room temperature for 2 h. After the reaction was complete as monitored by TLC, water was added, and the mixture was extracted three times with ethyl acetate. The combined organic layers were washed successively with saturated sodium bicarbonate and saturated sodium chloride, dried over anhydrous sodium sulfate, concentrated, and separated by silica gel column chromatography using PE / EA (v / v, 2 / 1) as the developing solvent to obtain the product.

[0215] White solid; yield: 91%; mp 196.7-198.1℃; 1 H NMR (500MHz, Acetone–d6) δ8.51(d,J=2.5Hz,1H),8.30(d,J=2.0Hz,1H),8.24(dd,J=8.5,2.0Hz,1H),8.18(d,J=8.5Hz,1H ), 7.92 (dd, J = 8.8, 5.0 Hz, 2H), 7.26 (t, J = 8.8 Hz, 2H), 7.15 (t, J = 6.0 Hz, 1H), 6.51 (d, J = 2.5 Hz, 1H), 4.30 (d, J = 6.0 Hz, 2H).

[0216] Example 26: Synthesis of N-((1-(4-cyano-3-trifluoromethylphenyl)-1H-pyrazol-3-yl)methyl)-4-trifluoromethylbenzenesulfonamide (26)

[0217]

[0218] It was prepared by replacing 4-fluorobenzenesulfonyl chloride with 4-trifluoromethylbenzenesulfonyl chloride using the method described in Example 25.

[0219] White solid; yield: 88%; mp 165.9-167.9℃; 1 H NMR(500MHz, Acetone–d6)δ8.48(d,J=2.5Hz,1H),8.25(d,J=2.0Hz,1H),8.21–8.16(m,2H),8.03(d,J= 8.0Hz, 2H), 7.80 (d, J = 8.5Hz, 2H), 7.39 (t, J = 6.0Hz, 1H), 6.51 (d, J = 2.5Hz, 1H), 4.38 (d, J = 6.0Hz, 2H).

[0220] Example 27: Synthesis of N-((1-(4-cyano-3-trifluoromethylphenyl)-1H-pyrazol-3-yl)methyl)-4-cyanobenzenesulfonamide (27)

[0221]

[0222] It was prepared by replacing 4-fluorobenzenesulfonyl chloride with 4-cyanobenzenesulfonyl chloride using the method described in Example 25.

[0223] White solid; yield: 78%; mp 212.4-213.5℃; 1 H NMR(500MHz, Acetone–d6)δ8.50(d,J=2.5Hz,1H),8.26(s,1H),8.23–8.17(m,2H),7.99(d,J=8.5H z, 2H), 7.88 (d, J = 8.5Hz, 2H), 7.44 (t, J = 6.0Hz, 1H), 6.51 (d, J = 2.5Hz, 1H), 4.38 (d, J = 6.0Hz, 2H).

[0224] Example 28: Synthesis of 1-(4-cyano-3-trifluoromethylphenyl)-N-(4-fluorophenyl)-1H-pyrazole-3-carboxamide (28)

[0225]

[0226] In a 25 mL two-necked round-bottom flask, the intermediates 1-(4-cyano-3-trifluoromethylphenyl)-1H-pyrazole-3-carboxylic acid (140 mg, 0.5 mmol) and HATU (228 mg, 0.6 mmol) obtained in Example 1(b) were added separately. After argon exchange, 5 mL of anhydrous dichloromethane was added to dissolve them. N,N-diisopropylethylamine (105 μL, 77 mg, 0.6 mmol) was added, and the mixture was stirred at room temperature for 10 min. 4-fluoroaniline (57 μL, 67 mg, 0.6 mmol) was weighed and added to the reaction system, and the reaction was carried out overnight at room temperature. After the reaction was complete as monitored by TLC, water was added, and the mixture was extracted three times with ethyl acetate. The organic layers were combined, washed successively with saturated sodium bicarbonate and saturated sodium chloride, dried over anhydrous sodium sulfate, concentrated, and separated by silica gel column chromatography using PE / EA (v / v, 2 / 1) as the developing solvent to obtain the product.

[0227] White solid; yield: 82%; mp 229.3-230.0℃; 1 H NMR (400MHz, DMSO–d6) δ10.39(br,1H),8.98(d,J=2.4Hz,1H),8.65(d,J=1.5Hz,1H),8.56(dd,J=8.4,1.5 Hz, 1H), 8.43 (d, J = 8.4Hz, 1H), 7.83 (dd, J = 8.8, 4.8Hz, 2H), 7.22 (t, J = 8.9Hz, 2H), 7.14 (d, J = 2.4Hz, 1H).

[0228] Example 29: Synthesis of 1-(4-cyano-3-trifluoromethylphenyl)-N-(4-cyanophenyl)-1H-pyrazole-3-carboxamide (29)

[0229]

[0230] It was prepared by replacing 4-fluoroaniline with 4-cyanoaniline using the method described in Example 28.

[0231] Yellow solid; yield: 60%; mp 30 1.2-30 2.3℃; 1 H NMR (400MHz, DMSO–d6) δ10.69(br,1H),9.00(d,J=2.4Hz,1H),8.65(d,J=2.0Hz,1H),8.57(dd,J=8.8, 2.0Hz, 1H), 8.43 (d, J = 8.8Hz, 1H), 8.05 (d, J = 8.8Hz, 2H), 7.85 (d, J = 8.8Hz, 2H), 7.18 (d, J = 2.4Hz, 1H).

[0232] Example 30: Synthesis of 1-(4-cyano-3-trifluoromethylphenyl)-N-(6-fluoropyridin-3-yl)-1H-pyrazole-3-carboxamide (30)

[0233]

[0234] It was prepared by replacing 4-fluoroaniline with 2-fluoro-5-aminopyridine using the method described in Example 28.

[0235] White solid; yield: 79%; mp 200.3-201.0℃; 1 H NMR(400MHz, DMSO–d6)δ10.64(br,1H),9.00(d,J=2.4Hz,1H),8.66–8.62(m,2H),8.56(dd,J=8.8,2.0 Hz,1H),8.44(d,J=8.8Hz,1H),8.39–8.33(m,1H),7.24(dd,J=8.8,3.2Hz,1H),7.16(d,J=2.4Hz,1H).

[0236] Example 31: Synthesis of 1-(4-cyano-3-trifluoromethylphenyl)-N-(5-fluoropyridin-2-yl)-1H-pyrazole-3-carboxamide (31)

[0237]

[0238] It was prepared by replacing 4-fluoroaniline with 2-amino-5-fluoropyridine using the method described in Example 28.

[0239] White solid; yield: 70%; mp 218.5-219.9℃; 1 H NMR (400MHz, DMSO–d6) δ10.71(br,1H),8.97(d,J=2.4Hz,1H),8.71(d,J=2.0Hz,1H),8.60(dd,J=8.4,2.0Hz,1H),8 .43(d,J=2.8Hz,1H),8.40(d,J=8.4Hz,1H),8.23(dd,J=9.2,4.4Hz,1H),7.87–7.80(m,1H),7.20(d,J=2.4Hz,1H).

[0240] Example 32: Synthesis of 1-(4-cyano-3-trifluoromethylphenyl)-N-(pyridin-4-yl)-1H-pyrazole-3-carboxamide (32)

[0241]

[0242] It was prepared by replacing 4-fluoroaniline with 4-aminopyridine using the method described in Example 28.

[0243] White solid; yield: 81%; mp 236.8-237.2℃; 1 H NMR (400MHz, DMSO–d6) δ10.65(br,1H),9.00(d,J=2.4Hz,1H),8.65(d,J=2.0Hz,1H),8.57(dd,J=8 .8,2.0Hz,1H),8.53–8.49(m,2H),8.44(d,J=8.4Hz,1H),7.87–7.83(m,2H),7.19(d,J=2.4Hz,1H).

[0244] Example 33: Synthesis of 2-(1-(4-cyano-3-trifluoromethylphenyl)-1H-pyrazol-3-yl)-N-(4-fluorophenyl)acetamide (33)

[0245] a) Intermediate 2-(1-(4-cyano-3-trifluoromethylphenyl)-1H-pyrazol-3-yl)acetic acid

[0246]

[0247] Weigh the intermediates 4-(3-(2-hydroxyethyl)-1H-pyrazol-1-yl)-2-trifluoromethylbenzonitrile (844 mg, 3 mmol) and 2,2,6,6-tetramethylpiperidine oxide (47 mg, 0.3 mmol) obtained in Example 19(d) and place them in a 50 mL single-necked round-bottom flask. Add 10 mL of acetonitrile and 10 mL of phosphate buffer solution (pH = 6.7) to dissolve them, and heat to 35 °C. Weigh 1.36 g (15 mmol) of 80% sodium chlorite and add it to the round-bottom flask in five batches, each 30 min apart. Add a few drops of sodium hypochlorite solution (6-14% active chlorine) to the reaction solution to maintain the wine-red color of the reaction solution. After the reaction was complete as monitored by TLC, sodium hydroxide solution was added to adjust the pH to alkaline, and the mixture was extracted with ethyl acetate. The organic layer was discarded, and the aqueous layer was adjusted to acidic pH with 1N HCl solution. The mixture was extracted three times with ethyl acetate, and the organic layers were combined. The mixture was washed twice with saturated sodium chloride, dried over anhydrous sodium sulfate, concentrated, and separated by silica gel column chromatography using DCM / MeOH (v / v, 50 / 1) as the developing solvent to obtain a white solid (646 mg, 73%).

[0248] b) 2-(1-(4-cyano-3-trifluoromethylphenyl)-1H-pyrazole-3-yl)-N-(4-fluorophenyl)acetamide (33)

[0249]

[0250] In a 25 mL two-necked round-bottom flask, the product from the previous step (148 mg, 0.5 mmol) and HATU (228 mg, 0.6 mmol) were added separately. After argon exchange, 5 mL of anhydrous dichloromethane was added to dissolve the product. N,N-diisopropylethylamine (105 μL, 77 mg, 0.6 mmol) was added, and the mixture was stirred at room temperature for 10 min. 4-fluoroaniline (57 μL, 67 mg, 0.6 mmol) was weighed and added to the reaction system, and the reaction was allowed to proceed overnight at room temperature. After the reaction was complete as monitored by TLC, water was added, and the mixture was extracted three times with ethyl acetate. The combined organic layers were washed successively with 1 N HCl solution, saturated sodium bicarbonate, and saturated sodium chloride solution, dried over anhydrous sodium sulfate, concentrated, and separated by silica gel column chromatography using PE / EA (v / v, 2 / 1) as the developing solvent to obtain the product.

[0251] White solid; yield: 82%; mp 227.5-228.6℃; 1 H NMR(400MHz,DMSO–d6)δ10.30(br,1H),8.77(d,J=2.4Hz,1H),8.37(s,1H),8.33–8.27(m,2 H), 7.62 (dd, J = 8.8, 5.2Hz, 2H), 7.15 (t, J = 8.8Hz, 2H), 6.64 (d, J = 2.4Hz, 1H), 3.78 (s, 2H).

[0252] Example 34: Synthesis of 2-(1-(4-cyano-3-trifluoromethylphenyl)-1H-pyrazol-3-yl)-N-(4-cyanophenyl)acetamide (34)

[0253]

[0254] It was prepared by replacing 4-fluoroaniline with 4-cyanoaniline using the method described in Example 33.

[0255] Yellow solid; yield: 81%; mp 215.4-216.4℃; 1 H NMR (400MHz, DMSO–d6) δ10.67(br,1H),8.78(d,J=2.0Hz,1H),8.37(s,1H),8.34–8.26(m,2H),7.82–7.74(m,4H),6.65(d,J=2.0Hz,1H),3.86(s,2H).

[0256] Example 35: Synthesis of 2-(1-(4-cyano-3-trifluoromethylphenyl)-1H-pyrazol-3-yl)-N-(4-nitrophenyl)acetamide (35)

[0257]

[0258] It was prepared by replacing 4-fluoroaniline with 4-nitroaniline using the method described in Example 33.

[0259] Yellow solid; yield: 79%; mp 227.4-228.9℃; 1 H NMR(400MHz,DMSO–d6)δ10.86(br,1H),8.79(d,J=2.0Hz,1H),8.37(s,1H),8 .32–8.20(m,4H),7.85(d,J=8.8Hz,2H),6.66(d,J=2.0Hz,1H),3.89(s,2H).

[0260] Example 36: Synthesis of 2-(1-(4-cyano-3-trifluoromethylphenyl)-1H-pyrazol-3-yl)-N-(4-methylphenyl)acetamide (36)

[0261]

[0262] It was prepared by replacing 4-fluoroaniline with 4-methylaniline using the method described in Example 33.

[0263] White solid; yield: 86%; mp 214.2-215.6℃; 1 H NMR(400MHz,DMSO–d6)δ10.13(br,1H),8.77(d,J=2.4Hz,1H),8.37(s,1H),8.33–8.27(m,2H), 7.48(d,J=8.4Hz,2H), 7.10(d,J=8.4Hz,2H), 6.63(d,J=2.4Hz,1H), 3.77(s,2H), 2.24(s,3H).

[0264] Example 37: Synthesis of 2-(1-(4-cyano-3-trifluoromethylphenyl-1H-pyrazol-3-yl)-N-(4-methoxyphenyl)acetamide (37)

[0265]

[0266] It was prepared by replacing 4-fluoroaniline with 4-methoxyaniline using the method described in Example 33.

[0267] Off-white solid; yield: 93%; mp 208.9-210.4℃; 1H NMR(400MHz, DMSO–d6)δ10.08(br,1H),8.77(d,J=2.4Hz,1H),8.37(s,1H),8.33–8.27(m,2H), 7.51(d,J=9.0Hz,2H), 6.88(d,J=9.0Hz,2H), 6.63(d,J=2.4Hz,1H), 3.75(s,2H), 3.71(s,3H).

[0268] Example 38: Synthesis of 2-(1-(4-cyano-3-trifluoromethylphenyl)-1H-pyrazol-3-yl)-N-(3,4-difluorophenyl)acetamide (38)

[0269]

[0270] It was prepared by replacing 4-fluoroaniline with 3,4-difluoroaniline using the method described in Example 33.

[0271] White solid; yield: 83%; mp 183.6-184.7℃; 1 H NMR (400MHz, DMSO–d6) δ10.48 (br, 1H), 8.75 (d, J = 2.4Hz, 1H), 8.35 (s, 1H), 8.31–8. 24(m,2H),7.81–7.73(m,1H),7.42–7.26(m,2H),6.63(d,J=2.4Hz,1H),3.79(s,2H).

[0272] Example 39: Synthesis of 2-(1-(4-cyano-3-trifluoromethylphenyl)-1H-pyrazol-3-yl)-N-(3-cyano-4-fluorophenyl)acetamide (39)

[0273]

[0274] It was prepared by replacing 4-fluoroaniline with 5-amino-2-fluorobenzonitrile using the method described in Example 33.

[0275] White solid; yield: 81%; mp 238.6-239.7℃; 1 H NMR(400MHz, DMSO–d6)δ10.62(br,1H),8.78(d,J=2.4Hz,1H),8.37(s,1H),8.33–8.27(m,2H),8.12( dd,J=5.6,2.4Hz,1H),7.88–7.82(m,1H),7.50(t,J=8.8Hz,1H),6.65(d,J=2.4Hz,1H),3.82(s,2H).

[0276] Example 40: Synthesis of 2-(1-(4-cyano-3-trifluoromethylphenyl)-1H-pyrazol-3-yl)-N-(6-fluoropyridin-3-yl)acetamide (40)

[0277]

[0278] It was prepared by replacing 4-fluoroaniline with 2-fluoro-5-aminopyridine using the method described in Example 33.

[0279] Pale yellow solid; yield: 78%; mp 232.8-233.9℃; 1 H NMR(400MHz, DMSO–d6)δ10.54(br,1H),8.78(d,J=2.4Hz,1H),8.44–8.36(m,2H),8.34–8.27 (m,2H),8.19–8.13(m,1H),7.16(dd,J=8.8,3.2Hz,1H),6.65(d,J=2.4Hz,1H),3.83(s,2H).

[0280] Example 41: Synthesis of 2-(1-(4-cyano-3-trifluoromethylphenyl-1H-pyrazol-3-yl)-N-(5-fluoropyridin-2-yl)acetamide (41)

[0281]

[0282] It was prepared by replacing 4-fluoroaniline with 2-amino-5-fluoropyridine using the method described in Example 33.

[0283] White solid; yield: 72%; mp 205.1-205.9℃; 1 H NMR(400MHz, DMSO–d6)δ10.86(br,1H),8.77(d,J=2.4Hz,1H),8.38–8.26(m,4H),8.10( dd, J=9.2, 4.0Hz, 1H), 7.73 (td, J=8.8, 3.2Hz, 1H), 6.64 (d, J=2.4Hz, 1H), 3.89 (s, 2H).

[0284] Example 42: Synthesis of 2-(1-(4-cyano-3-trifluoromethylphenyl)-1H-pyrazol-3-yl)-N-(pyridin-4-yl)acetamide (42)

[0285]

[0286] It was prepared by replacing 4-fluoroaniline with 4-aminopyridine using the method described in Example 33.

[0287] Pale yellow solid; yield: 83%; mp 232.8-233.3℃; 1 H NMR(400MHz,DMSO–d6)δ10.62(br,1H),8.78(d,J=2.6Hz,1H),8.43(d,J=6.0Hz,2H),8.3 8–8.36(m,1H),8.33–8.27(m,2H),7.59–7.55(m,2H),6.65(d,J=2.6Hz,1H),3.85(s,2H).

[0288] Example 43: Synthesis of N-(4-fluorophenyl)-2-(1-(4-nitro-3-trifluoromethylphenyl)-1H-pyrazol-3-yl)acetamide (43)

[0289] a) Intermediate 1-(4-nitro-3-trifluoromethylphenyl)-1H-pyrazole-3-carboxylic acid

[0290]

[0291] The preparation was carried out by replacing the starting material 4-bromo-2-trifluoromethylbenzonitrile with 5-bromo-2-nitrotrifluorotoluene using the method described in Example 1(ab).

[0292] b) Intermediate (1-(4-nitro-3-trifluoromethylphenyl)-1H-pyrazole-3-yl)methanol

[0293]

[0294] The intermediate 1-(4-nitro-3-trifluoromethylphenyl)-1H-pyrazole-3-carboxylic acid was prepared using the method described in Example 2(a).

[0295] c) Intermediate 2-(1-(4-nitro-3-trifluoromethylphenyl)-1H-pyrazole-3-yl)ethanol-1-ol

[0296]

[0297] The intermediate (1-(4-nitro-3-trifluoromethylphenyl)-1H-pyrazole-3-yl)methanol was prepared using the method described in Example 19(ad).

[0298] d) Intermediate 2-(1-(4-nitro-3-trifluoromethylphenyl)-1H-pyrazol-3-yl)acetic acid

[0299]

[0300] The intermediate 2-(1-(4-nitro-3-trifluoromethylphenyl)-1H-pyrazol-3-yl)ethanol-1-ol was prepared using the method described in Example 33(a).

[0301] e)N-(4-fluorophenyl)-2-(1-(4-nitro-3-trifluoromethylphenyl)-1H-pyrazole-3-yl)acetamide (43)

[0302]

[0303] The intermediate 2-(1-(4-nitro-3-trifluoromethylphenyl)-1H-pyrazol-3-yl)acetic acid and 4-fluoroaniline were prepared by condensation reaction using the method described in Example 33(b).

[0304] Pale yellow solid; yield: 84%; mp 196.9-198.4℃; 1 H NMR(500MHz,DMSO–d6)δ10.30(br,1H),8.78(d,J=2.5Hz,1H),8.38(s,1H),8.35–8.33(m,2 H), 7.62 (dd, J = 9.0, 5.5Hz, 2H), 7.15 (t, J = 9.0Hz, 2H), 6.65 (d, J = 2.5Hz, 1H), 3.79 (s, 2H).

[0305] Example 44: Synthesis of N-(6-fluoropyridin-3-yl)-2-(1-(4-nitro-3-trifluoromethylphenyl)-1H-pyrazol-3-yl)acetamide (44)

[0306]

[0307] The intermediate 2-(1-(4-nitro-3-trifluoromethylphenyl)-1H-pyrazol-3-yl)acetic acid and 2-fluoro-5-aminopyridine were prepared by condensation reaction using the method described in Example 33(b).

[0308] White solid; yield: 80%; mp 199.8-201.2℃; 1 H NMR(500MHz,DMSO–d6)δ10.56(br,1H),8.79(d,J=2.5Hz,1H),8.44–8.42(m,1H),8.38(s,1H),8.3 6–8.33(m,2H),8.19–8.14(m,1H),7.17(dd,J=9.0,3.0Hz,1H),6.66(d,J=2.5Hz,1H),3.84(s,2H).

[0309] Example 45: Synthesis of N-(5-fluoropyridin-2-yl)-2-(1-(4-nitro-3-trifluoromethylphenyl)-1H-pyrazol-3-yl)acetamide (45)

[0310]

[0311] The intermediate 2-(1-(4-nitro-3-trifluoromethylphenyl)-1H-pyrazol-3-yl)acetic acid and 2-amino-5-fluoropyridine were prepared by condensation reaction using the method described in Example 33(b).

[0312] White solid; yield: 84%; mp 181.5-182.4℃; 1 H NMR(500MHz,DMSO–d6)δ10.88(br,1H),8.77(d,J=2.5Hz,1H),8.37(s,1H),8.36–8.32(m ,3H),8.11(dd,J=9.0,4.0Hz,1H),7.77–7.71(m,1H),6.65(d,J=2.5Hz,1H),3.89(s,2H).

[0313] Example 46: Synthesis of 2-(1-(3-chloro-4-cyanophenyl)-1H-pyrazol-3-yl)-N-(4-fluorophenyl)acetamide (46)

[0314] a) Intermediate 1-(3-chloro-4-cyanophenyl)-1H-pyrazole-3-carboxylic acid

[0315]

[0316] The preparation was carried out by replacing the starting material 4-bromo-2-trifluoromethylbenzonitrile with 4-bromo-2-chlorobenzonitrile using the method described in Example 1(ab).

[0317] b) Intermediate 2-chloro-4-(3-hydroxymethyl-1H-pyrazole-1-yl)benzonitrile

[0318]

[0319] The intermediate 1-(3-chloro-4-cyanophenyl)-1H-pyrazole-3-carboxylic acid was prepared using the method described in Example 2(a).

[0320] c) Intermediate 2-chloro-4-(3-(2-hydroxyethyl)-1H-pyrazole-1-yl)benzonitrile

[0321]

[0322] The intermediate 2-chloro-4-(3-hydroxymethyl-1H-pyrazol-1-yl)benzonitrile was prepared using the method described in Example 19(ad).

[0323] d) Intermediate 2-(1-(3-chloro-4-cyanophenyl)-1H-pyrazol-3-yl)acetic acid

[0324]

[0325] The intermediate 2-chloro-4-(3-(2-hydroxyethyl)-1H-pyrazol-1-yl)benzonitrile was prepared using the method described in Example 33(a).

[0326] e)2-(1-(3-chloro-4-cyanophenyl)-1H-pyrazole-3-yl)-N-(4-fluorophenyl)acetamide (46)

[0327]

[0328] The intermediate 2-(1-(3-chloro-4-cyanophenyl)-1H-pyrazol-3-yl)acetic acid and 4-fluoroaniline were prepared by condensation reaction using the method described in Example 33(b).

[0329] White solid; yield: 90%; mp 191.3-193.0℃; 1 H NMR(500MHz,DMSO–d6)δ10.28(br,1H),8.66(d,J=3.0Hz,1H),8.21(d,J=2.0Hz,1H),8.09(d,J=8.5Hz,1H),8.0 0 (dd, J = 8.5, 2.0 Hz, 1H), 7.62 (dd, J = 9.0, 5.0 Hz, 2H), 7.14 (t, J = 9.0 Hz, 2H), 6.61 (d, J = 3.0 Hz, 1H), 3.76 (s, 2H).

[0330] Example 47: Synthesis of 2-(1-(3-chloro-4-cyanophenyl)-1H-pyrazol-3-yl)-N-(6-fluoropyridin-3-yl)acetamide (47)

[0331]

[0332] The intermediate 2-(1-(3-chloro-4-cyanophenyl)-1H-pyrazol-3-yl)acetic acid and 2-fluoro-5-aminopyridine were prepared by condensation reaction using the method described in Example 33(b).

[0333] White solid; yield: 86%; mp 238.2-239.1℃; 1H NMR(500MHz,DMSO–d6)δ10.53(br,1H),8.67(d,J=2.5Hz,1H),8.44–8.41(m,1H),8.21(d,J=2.5Hz,1H),8.18–8.14(m,1 H), 8.09 (d, J = 9.0Hz, 1H), 8.00 (dd, J = 9.0, 2.0Hz, 1H), 7.16 (dd, J = 9.0, 3.0Hz, 1H), 6.62 (d, J = 2.5Hz, 1H), 3.81 (s, 2H).

[0334] Example 48: Synthesis of 2-(1-(3-chloro-4-cyanophenyl)-1H-pyrazol-3-yl)-N-(5-fluoropyridin-2-yl)acetamide (48)

[0335]

[0336] The intermediate 2-(1-(3-chloro-4-cyanophenyl)-1H-pyrazol-3-yl)acetic acid and 2-amino-5-fluoropyridine were prepared by condensation reaction using the method described in Example 33(b).

[0337] White solid; yield: 84%; mp 212.6-213.6℃; 1 H NMR (500MHz, DMSO–d6) δ10.86(br,1H),8.66(d,J=2.5Hz,1H),8.34(d,J=3.0Hz,1H),8.21(d,J=2.5Hz,1H),8. 13–8.07(m,2H),7.99(dd,J=8.5,2.0Hz,1H),7.74(td,J=8.5,3.0Hz,1H),6.61(d,J=2.5Hz,1H),3.87(s,2H).

[0338] Biological Experiment Example 1: Androgen Receptor Antagonism Assessment Test

[0339] Detection Principle: The androgen receptor (AR), as a transcription factor, requires binding to a specific sequence to exert its transcriptional activity. In the detection method of this invention, an enhanced green fluorescent protein (EGFP) reporter gene controlled by the ARR2PB promoter is introduced into androgen receptor-dependent prostate cancer cells (LNCaP), causing LNCaP cells to express an androgen receptor-regulated EGFP prostate cancer cell line (LNCaP-ARR2PB-EGFP). After treatment with different concentration gradient test compounds, the expression level of EGFP in LNCaP-ARR2PB-EGFP cells is detected, thereby determining the strength of the compound's antagonistic ability against the androgen receptor.

[0340] Detection procedure: LNCaP-ARR2PB-eGFP cells were starved in androgen-free complete culture medium for 3-5 days. Background fluorescence values ​​were measured, and once they decreased to a low level, they were analyzed at 3.5 × 10⁻⁶ cells / mL. 4 Cells were seeded at a density of 1:1 in 96-well plates and incubated at 37°C for 24 hours in a 5% CO2 incubator. After the cells were stably attached, 5 nM DHT and different concentrations of the test compound (50, 16.667, 5.556, 1.852, 0.617, 0.206, 0.069, and 0.023 μM) were added to each well in triplicate, and the plates were incubated for another 72 hours. The fluorescence intensity near 530 nm was measured using a Synergy H1 (BioTek) microplate reader under 485 nm excitation light to calculate the AR antagonistic activity of the test compound at the cellular level.

[0341] Test results: As shown in Table 1, IC 50 Compounds with concentrations less than 0.2 μM included compounds 33, 34, 35, 38, 40, 47, and 48. Since these compounds all exhibited good androgen receptor antagonistic activity, their inhibitory activity against prostate cancer cell proliferation was further investigated.

[0342] Table 1. Inhibitory activity of compounds on LNCaP cell transcription levels.

[0343]

[0344]

[0345] Biological Experiment Example 2: Detection of the proliferation inhibitory activity of the compound in various prostate cancer cell lines by the 3-(4,5-dimethylthiazol-2)-2,5-diphenyltetrazolium bromide (MTT) method.

[0346] Detection principle: The basic principle of this experiment is that succinate dehydrogenase in the mitochondria of living cells can reduce exogenous MTT to water-insoluble blue-purple crystals formazan, which are deposited in the cells. Dead cells do not have this function. Buffer solution is added to dissolve the formazan formed in the cells, and its light absorbance value is measured at a wavelength of 490nm using an enzyme-linked immunosorbent assay (ELISA) instrument, which can quantitatively reflect the number of living cells.

[0347] Detection steps: Select cells in the logarithmic growth phase, and arrange them at a density of 3–8 × 10⁶ cells per well. 3Cells were seeded into 96-well plates and incubated at 37°C for 24 hours in a 5% CO2 incubator. Culture medium containing different concentrations of the test compound was added, and the plates were incubated for another 72 hours. Then, 10 μL of 5 mg / mL MTT solution was added to each well, and the plates were incubated for another 3–5 hours. Following this, 100 μL of LDS-HCl-PBS triple buffer was added, and the plates were incubated overnight at 37°C. The absorbance was measured at 570 nm using a microplate reader.

[0348] Test results: such as Figure 1 As shown, compounds 33, 40, 47, and enzalutamide all dose-dependently inhibited androgen receptor-dependent LNCaP cells. Compounds 33, 40, and 47 also exhibited significant inhibitory activity against proliferation in 22Rv1 cells expressing the androgen receptor splice variant, while enzalutamide showed almost no inhibitory effect on 22Rv1 cells. Figure 2 In androgen receptor-independent brain metastases of prostate cancer cells (DU145), compounds 33, 40, and 47 exhibited significant inhibitory activity; in bone metastases of prostate cancer cells (PC3), compounds 33, 40, and 47 showed weaker inhibitory activity, while enzalutamide only showed a slight inhibitory effect at high concentrations. Figure 3 and Figure 4 ).

[0349] Biological Experiment Example 3: MTT Assay for Compound Safety

[0350] Detection principle: In order to further clarify that the inhibitory activity of the compound is not caused by the toxicity of the compound, this invention uses two commonly used normal cells, including human gastric mucosal epithelial cells (GES-1) and human Zhang hepatocytes (Chang), to perform toxicity detection to determine its safety.

[0351] Detection steps: Use complete culture medium at 5×10 3 GES-1 and Chang cells were seeded at a density of cells / well in 96-well plates. After cell adhesion, the cells were incubated at 37°C for 24 hours, followed by treatment with different concentrations of compounds and incubation for another 72 hours. Then, 10 μL of 5 mg / ml MTT was added to each well, and the cells were incubated for 4 hours. Next, 100 μL of SDS-HCl-PBS triple buffer was added to each well, and the cells were incubated overnight at 37°C. Finally, the absorbance at 570 nm was measured using a microplate reader and converted to the viability percentage.

[0352] Experimental results: such as Figures 5-6 As shown, compounds 33, 40, and 47 exhibited low toxicity to both GES-1 and Chang cells.

[0353] Biological Experiment Example 4: Detection of Prostate-Specific Antigen (PSA) Expression

[0354] Detection principle: PSA is one of the key indicators for clinical detection of prostate cancer, and its expression is positively correlated with the transcriptional level of androgen receptor.

[0355] Detection steps: After the LNCaP-ARR2PB-eGFP cell detection is completed, the supernatant of the sample is extracted and used... PSA levels were measured using the 2000XPi immunoassay system.

[0356] Test results: such as Figure 7 As shown, compounds 33, 40, and 47 can inhibit PSA expression at the protein level in a dose-dependent manner. Among them, compound 47 showed the best performance, with an inhibitory activity approximately half that of enzalutamide.

[0357] The above results demonstrate that the compounds of the present invention have good androgen receptor antagonistic activity and high safety.

Claims

1. A phenylpyrazole compound or a pharmaceutically acceptable salt thereof, characterized in that, The phenylpyrazole compound mentioned is one of the following compounds:

2. The phenylpyrazole compound or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that, The pharmaceutically acceptable salts are hydrochloride, sulfate, phosphate, acetate, benzenesulfonate, maleate, tartrate, or fumarate.

3. The phenylpyrazole compound or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that, The phenylpyrazole compound mentioned is one of the following compounds:

4. The use of the phenylpyrazole compound or a pharmaceutically acceptable salt thereof as described in any one of claims 1-3 in the preparation of androgen receptor antagonists.

5. The application as described in claim 4, characterized in that: The androgen receptor includes mutants of the androgen receptor.

6. The application according to claim 4, characterized in that, The compound is used to prepare drugs for treating prostate cancer, benign prostatic hyperplasia, hirsutism, hair loss, breast cancer, and male sexual dysfunction.

7. The use of the phenylpyrazole compound or a pharmaceutically acceptable salt thereof as described in any one of claims 1-3 in the preparation of a medicament for treating prostate cancer.

8. The use of the phenylpyrazole compound or a pharmaceutically acceptable salt thereof as described in any one of claims 1-3 in the preparation of a therapeutic agent for metastatic prostate cancer.

Images