A bis(pyrazolyl)methane derivative and use thereof

Abstract

The application discloses a kind of double (pyrazolyl) methane derivatives and application thereof.The double (pyrazolyl) methane derivatives of the application are a kind of effective IGF2BP2 small molecule inhibitors, have obvious inhibitory activity to IGF2BP2, inhibit RNA reader on the level of protease, modify and regulate m 6 A level, can be used for treating diseases related to IGF2BP2, such as breast cancer, colorectal cancer, pancreatic cancer, hepatocellular carcinoma, acute myeloid leukemia, T-cell acute lymphoblastic leukemia, nasopharyngeal carcinoma and various tumors.

Description

Technical Field

[0001] This invention belongs to the field of medicinal chemistry technology, specifically relating to a class of bis(pyrazolyl)methane derivatives and their applications. Background Technology

[0002] N 6 -Methyladenosine (m 6 A) is the most abundant modification in eukaryotic RNA. It is a dynamic and reversible RNA modification that includes writeons, erasers, and readers. 6 A modification plays a promoting or inhibiting role in cell cycle regulation, cell differentiation, changes in cell state, and stress response. Increasing research has found that m 6 Insulin-like growth factor 2 (IGF2) modification plays an increasingly important regulatory role in the development and progression of tumors and cancers, and has the potential to become a biomarker for cancer. IGF2BP2 is an RNA-binding protein that regulates a variety of biological processes. In recent years, studies have revealed numerous roles of IGF2BP2 in various biological processes, particularly in tumors, and have speculated on the mechanisms by which it exerts its anti-cancer activity. Furthermore, targeting IGF2BP2 or its downstream mechanisms has received widespread attention as an effective treatment for different types of tumors.

[0003] IGF2BP2 plays a crucial role in various tumors, including breast cancer, colorectal cancer, pancreatic cancer, hepatocellular carcinoma, acute myeloid leukemia, T-cell acute lymphoblastic leukemia, and nasopharyngeal carcinoma. Therefore, utilizing the enzymatic catalytic activity of small molecule inhibitors of IGF2BP2 could be used to treat indications associated with IGF2BP2 protein dysfunction.

[0004] Currently, there are no reports of highly active small molecule inhibitors of IGF2BP2. Summary of the Invention

[0005] This invention designs and synthesizes a series of derivatives based on substituted bis(pyrazolyl)methane lead compounds and conducts preliminary structure-activity relationship discussions, ultimately obtaining a novel small molecule inhibitor of IGF2BP2 with relatively high activity. In vitro, this inhibitor can significantly inhibit the effect of IGF2BP2 on m... 6 The activity of methylation reading of A is expected to become a promising candidate anti-tumor drug.

[0006] One object of the present invention is to provide a compound as shown in Formula I or a pharmaceutically acceptable salt thereof:

[0007]

[0008] R 1Selected from H, substituted or unsubstituted C1-C10 alkyl, substituted or unsubstituted 3-8 membered cycloalkyl or heterocyclic group, substituted or unsubstituted 5-8 membered heteroaryl or aryl;

[0009] R 3 and R 4 Independently selected from H, substituted or unsubstituted C1-C10 alkyl;

[0010] R 2 and R 5 Independently selected from H, substituted or unsubstituted C1-C10 alkyl, substituted or unsubstituted 3-8 membered cycloalkyl or heterocyclic group, substituted or unsubstituted 5-8 membered heteroaryl or aryl or a combination of two rings;

[0011] R 6 and R 7 Independently selected from H, substituted or unsubstituted C1-C10 alkyl, containing oxygen.

[0012] In some preferred embodiments,

[0013] R 1 Selected from H, substituted or unsubstituted C1-C7 alkyl, substituted or unsubstituted 5-8 membered heteroaryl or aryl;

[0014] R 3 and R 4 Independently selected from H, substituted or unsubstituted C1-C7 alkyl;

[0015] R 2 and R 5 Independently selected from substituted or unsubstituted five- or six-membered nitrogen-containing heterocycles or aromatic rings, or combinations of five- or six-membered heteroaromatic rings;

[0016] R 6 and R 7 Independently selected from H, substituted or unsubstituted C1-C5 alkyl, containing oxygen.

[0017] In some further preferred embodiments,

[0018] R 1 Selected from H, methyl, ethyl, isopropyl, tert-butyl,

[0019] R 3 and R 4 Selected from H, difluoromethyl, trifluoromethyl, methyl, and ethyl;

[0020] R 2 and R 5 Selected from

[0021] R6 and R 7 Selected from H, hydroxyl, methoxy, ethoxy;

[0022] In R 1 R 2 and R 5 In the middle, R a It can be monosubstituted or polysubstituted, and the substituent is selected from H, halogen, hydroxyl, alkyl, nitro, amino, methoxy, trifluoromethyl, difluoromethyl, carboxyl, difluoromethoxy, trifluoromethoxy, mercapto, or cyano.

[0023] The compounds of general formula I mentioned above in this invention can also exist in the form of their salts, which are converted into compounds of formula I in vivo. For example, within the scope of this invention, the compounds of this invention are converted into pharmaceutically acceptable salt forms according to processes known in the art, and used in salt form.

[0024] A second objective of this invention is to provide a pharmaceutical composition in which the active ingredient comprises a compound of formula I or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0025] A third objective of this invention is to provide the use of compounds of Formula I or pharmaceutically acceptable salts thereof in the preparation of medicaments for treating diseases associated with IGF2BP2 protein dysfunction. These diseases include various tumors such as breast cancer, colorectal cancer, pancreatic cancer, hepatocellular carcinoma, acute myeloid leukemia, T-cell acute lymphoblastic leukemia, and nasopharyngeal carcinoma.

[0026] Beneficial effects: The compounds of formula I or their pharmaceutically acceptable salts provided by this invention are effective small molecule inhibitors of IGF2BP2, exhibiting significant inhibitory activity against IGF2BP2, inhibiting RNA readers at the protease level, and modifying and regulating m 6 Level A can be used to treat diseases associated with IGF2BP2, such as breast cancer, colorectal cancer, pancreatic cancer, hepatocellular carcinoma, acute myeloid leukemia, T-cell acute lymphoblastic leukemia, nasopharyngeal carcinoma, and many other tumors. Detailed Implementation

[0027] The preferred embodiments of the present invention will now be described in detail with reference to specific examples. It should be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the invention. Those skilled in the art can make various modifications and substitutions to the present invention without departing from its spirit and essence.

[0028] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0029] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0030] In some embodiments of the present invention, the compound is selected from the following compounds:

[0031]

[0032]

[0033]

[0034] Example 1

[0035] Preparation of 4,4'-(4-bromophenyl)methylenebis(1-(4-(2-hydroxyphenyl)thiazolyl)-3-methyl-1H-pyrazole-5-ol)1

[0036]

[0037] Step 1: Synthesize compounds 1-2

[0038] Compound 1-1 (500 mg, 2.33 mmol) was dissolved in 15 mL of 1,4-dioxane, and thiourea (211.89 mg, 2.33 mmol) was added. The mixture was reacted at room temperature for 12 h. The reaction was monitored by TLC until completion, and then purified by column chromatography. The resulting gray-green solid 1-2 was obtained by silica gel column chromatography (PE:EA = 3:1), with a yield of 70.22%. 1 H NMR (300MHz, DMSO-d6) δ10.62(s,1H),8.62(t,J=3.4Hz,1H),7.66(dd,J=8.7,1.2Hz,1H),7.31-7.20(m, 1H), 7.13 (s, 1H), 7.06 (ddd, J = 9.0, 7.7, 1.4Hz, 1H), 6.94 (dd, J = 8.2, 1.4Hz, 1H), 4.88 (d, J = 3.3Hz, 2H).

[0039] Step 2: Synthesize compound 1

[0040] Compounds 1-2 (150 mg, 723.76 μmol) were dissolved in 10 mL of glacial acetic acid, and ethyl acetoacetate (94.19 mg, 723.76 μmol) was added. The reaction was carried out at 80 °C for 6 h. After the reaction was completed by TLC monitoring, the solvent was evaporated, and 15 mL of anhydrous ethanol was added to dissolve the compound. Then, p-bromobenzaldehyde (66.96 mg, 361.88 μmol) was added, and the reaction was carried out at 78 °C for 4 h. A white solid precipitated out. The solid was filtered and dried to give white solid 1, with a yield of 60.22%. 1H NMR(300MHz,DMSO-d6)δ12.33(s,1H),10.45(s,1H),8.11(d,J=7.8Hz,2H),7.87(s,2H) ,7.53(d,J=8.0Hz,2H),7.31-7.14(m,4H),7.03-6.86(m,4H),5.12(s,1H),2.17(s,6H).

[0041] Example 2

[0042] Preparation of 4,4'-(4-bromophenyl)methylene)bis(3-methyl-1-(4-phenylthiazol-2-yl)-1H-pyrazole-5-ol)2

[0043]

[0044] Step 1: Synthesize compound 2-2

[0045] Compound 2-1 (500 mg, 2.33 mmol) was dissolved in 15 mL of 1,4-dioxane, and thiourea (211.89 mg, 2.33 mmol) was added. The mixture was reacted at room temperature for 12 h. The reaction was monitored by TLC until completion, and then purified by column chromatography. The resulting gray-green solid, 2-2, was obtained by silica gel column chromatography (PE:EA = 3:1), with a yield of 60.25%. 1 H NMR (300MHz, DMSO-d6) δ8.53 (t, J = 3.4 Hz, 1H), 7.82 (dd, J = 7.5, 1.6 Hz, 2H), 7.54-7.43 (m, 2H), 7.47-7.36 (m, 1H), 7.13 (s, 1H), 4.88 (d, J = 3.4Hz, 2H).

[0046] Step 2: Synthesize compound 2

[0047] Compound 2-2 (150 mg, 723.76 μmol) was dissolved in 10 mL of glacial acetic acid, and ethyl acetoacetate (94.19 mg, 723.76 μmol) was added. The reaction was carried out at 80 °C for 6 h. After the reaction was completed by TLC, the solvent was evaporated, and 15 mL of anhydrous ethanol was added to dissolve it. Then p-bromobenzaldehyde (66.96 mg, 361.88 μmol) was added, and the reaction was carried out at 78 °C for 4 h. A white solid precipitated out. The solid was filtered and dried to give white solid 2, with a yield of 70.23%. 1H NMR(500MHz,Chloroform-d)δ7.95(t,J=3.4Hz,1H),7.85-7.79(m,2H),7.53-7.45(m,2H) ,7.44-7.37(m,1H),7.14(s,1H),4.56(dd,J=8.3,3.4Hz,1H),4.49(dd,J=8.3,3.4Hz,1H).

[0048] Example 3

[0049] Preparation of 4,4'-(4-bromophenyl)methylenebis(1-(4-(2-methoxyphenyl)thiazolyl-2-yl)-3-methylpyrazole-5-ol)3

[0050]

[0051] Step 1: Synthesize compound 3-2

[0052] The preparation method described in Example 1 was followed for synthesis. Compound 3-2 was obtained as a grayish-black solid with a yield of 70.12%. 1 H NMR (300MHz, DMSO-d6) δ8.50 (t, J=3.4Hz, 1H), 7.77 (dd, J=8.5, 1.3Hz, 1H), 7.38 (td, J= 7.6,1.3Hz,1H),7.31-7.11(m,2H),6.90(dd,J=7.7,1.4Hz,1H),4.88(d,J=3.4Hz,2H).

[0053] Step 2: Synthesize compound 3

[0054] The synthesis was performed according to the preparation method in Example 1. Compound 3 was obtained as a grayish-white solid with a yield of 75.45%. ¹H NMR (300 MHz, DMSO-d6) δ 12.20 (s, 1H), 8.27 (d, J = 8.7 Hz, 2H), 7.78 (s, 2H), 7.51 (d, J = 8.5 Hz, 2H), 7.38–7.29 (m, 2H), 7.25 (d, J = 8.4 Hz, 2H), 7.14 (d, J = 8.0 Hz, 2H), 7.06 (t, J = 7.5 Hz, 2H), 5.08 (s, 1H), 3.93 (s, 6H), 2.12 (s, 6H).

[0055] Example 4

[0056] Preparation of 4-(bis(5-hydroxy-1-(4-(3-methoxyphenyl)thiazolyl-2-yl)-3-methyl-1H-pyrazole-4-yl)methyl)benzoic acid

[0057]

[0058] Step 1: Synthesize compound 4-2

[0059] The preparation method described in Example 1 was followed for synthesis. Compound 4-2 was obtained as a grayish-black solid with a yield of 71.26%. 1 H NMR (300MHz, DMSO-d6) δ8.49(t,J=3.4Hz,1H),7.71-7.53(m,1H),7.42-7.30(m,2H),7.20(s,1H),7.05-6.88(m,1H),4.88(d,J=3.4Hz,2H).

[0060] Step 2: Synthesize compound 4

[0061] The synthesis was carried out according to the preparation method of Example 1. The difference was that p-bromobenzaldehyde was replaced with p-carboxybenzaldehyde. Compound 4 was obtained as a grayish-white solid with a yield of 72.21%. 1 H NMR (300MHz, DMSO-d6) δ12.39(s,1H),10.63(s,2H),7.99-7.90(m,2H),7.64(ddd,J=8.7,2.1,1.2Hz ,2H),7.43-7.28(m,8H),6.96(ddd,J=8.0,2.1,1.2Hz,2H),4.98(s,1H),3.82(s,6H),2.27(s,6H).

[0062] Example 5

[0063] Preparation of 4,4'-((4-chlorophenyl)methylene)bis(1-(4-(4-methoxyphenyl)thiazolyl-2-yl)-3-methyl-1H-pyrazole-5-ol)5

[0064]

[0065] Step 1: Synthesize compound 5-2

[0066] The preparation method described in Example 1 was followed for synthesis. Compound 5-2 was obtained as a light green solid with a yield of 71.89%. 1 H NMR (300MHz, DMSO-d6) δ8.53 (t, J = 3.4Hz, 1H), 7.78-7.63 (m, 2H), 7.15 (s, 1H), 7.01-6.79 (m, 2H), 4.88 (d, J = 3.4Hz, 2H).

[0067] Step 2: Synthesize compound 5

[0068] The synthesis was carried out according to the preparation method of Example 1. The difference was that p-bromobenzaldehyde was replaced with p-chlorobenzaldehyde. Compound 5 was obtained as a grayish-white solid with a yield of 74.36%. 1 H NMR (300MHz, DMSO-d6) δ12.20 (s, 1H), 7.93 (d, J = 8.8Hz, 4H), 7.70-7.45 (m, 4H), 7. 24(d,J=8.1Hz,2H),7.01(d,J=8.9Hz,4H),5.08(s,1H),3.80(s,6H),2.11(s,6H).

[0069] Example 6

[0070] Preparation of 4,4'-(4-bromophenyl)methylenebis(1-(4-(2-chlorophenyl)thiazol-2-yl)-3-methyl-1H-pyrazole-5-ol)6

[0071]

[0072] Step 1: Synthesize compound 6-2

[0073] The preparation method described in Example 1 was followed for synthesis. Compound 6-2 was obtained as a black solid with a yield of 68.89%. 1 HNMR (300MHz, DMSO-d6) δ8.52(t,J=3.4Hz,1H),7.72-7.61(m,1H),7.52-7.33(m,3H),7.26(s,1H),4.88(d,J=3.4Hz,2H).

[0074] Step 2: Synthesize compound 6

[0075] The preparation method of Example 1 was followed for synthesis. Compound 6 was obtained as a grayish-white solid with a yield of 66.34%. 1 HNMR(300MHz,DMSO-d6)δ12.30(s,2H),7.97(d,J=7.5Hz,2H),7.76(s,2H),7.48 (ddd,J=34.8,17.6,7.6Hz,8H),7.24(d,J=8.1Hz,2H),5.09(s,1H),2.10(s,6H).

[0076] Example 7

[0077] Preparation of 4,4'-(4-bromophenyl)methylenebis(1-(4-(3,4-dichlorophenyl)thiazol-2-yl)-3-methylpyrazole-5-ol)7

[0078]

[0079] Step 1: Synthesize compound 7-2

[0080] The preparation method of Example 1 was followed for synthesis. Compound 7-2 was obtained as a grayish-black solid with a yield of 69.09%. 1 H NMR(300MHz,DMSO-d6)δ8.50(t,J=3.3Hz,1H),8.04(d,J=1.9Hz,1H),7.83(dd ,J=8.7,1.9Hz,1H),7.58(d,J=8.7Hz,1H),7.22(s,1H),4.88(d,J=3.3Hz,2H).

[0081] Step 2: Synthesize compound 7

[0082] The preparation method described in Example 1 was followed for synthesis. Compound 7 was obtained as a grayish-white solid with a yield of 77.01%. 1 HNMR(300MHz,DMSO-d6)δ12.28(s,1H),8.23(d,J=2.1Hz,2H),7.99-7.91(m,4H),7.69 (d,J=8.5Hz,2H),7.51-7.43(m,2H),7.19(d,J=8.2Hz,2H),5.04(s,1H),2.09(s,6H).

[0083] Example 8

[0084] Preparation of 4,4'-(4-bromophenyl)methylenebis(1-(4-chlorophenyl)thiazolyl)-3-methyl-1H-pyrazole-5-ol)8

[0085]

[0086] Step 1: Synthesize compound 8-2

[0087] The preparation method described in Example 1 was followed for synthesis. Compound 8-2 was obtained as a dark green solid with a yield of 67.78%. 1 H NMR (300MHz, DMSO-d6) δ8.53 (t, J = 3.3Hz, 1H), 7.70 (d, J = 8.5Hz, 2H), 7.53-7.43 (m, 2H), 7.16 (s, 1H), 4.88 (d, J = 3.3Hz, 2H).

[0088] Step 2: Synthesize compound 8

[0089] The preparation method described in Example 1 was followed for synthesis. Compound 8 was obtained as a grayish-white solid with a yield of 78.44%. 1HNMR (300MHz, DMSO-d6) δ12.27 (s, 2H), 8.07-7.99 (m, 4H), 7.84 (s, 2H), 7.56-7.48 (m, 6H), 7.24 (d, J = 8.1Hz, 2H), 5.08 (s, 1H), 2.12 (s, 6H).

[0090] Example 9

[0091] Preparation of 4,4'-(4-bromophenyl)methylenebis(1-(4-(3-chlorophenyl)thiazol-2-yl)-3-methyl-1H-pyrazole-5-ol)9

[0092]

[0093] Step 1: Synthesize compound 9-2

[0094] The preparation method described in Example 1 was followed for synthesis. Compound 9-2 was obtained as a light green solid with a yield of 73.11%. 1 H NMR (300MHz, DMSO-d6) δ8.50 (t, J = 3.4Hz, 1H), 7.72-7.59 (m, 2H), 7.43-7.30 (m, 2H), 7.21 (s, 1H), 4.88 (d, J = 3.4Hz, 2H).

[0095] Step 2: Synthesize compound 9

[0096] The preparation method described in Example 1 was followed for synthesis. Compound 9 was obtained as a grayish-white solid with a yield of 69.04%. 1 HNMR (300MHz, DMSO-d6) δ12.28 (s, 2H), 8.10 (s, 2H), 8.02-7.88 (m, 4H), 7.55-7.39 (m, 6H), 7.25 (d, J = 8.1Hz, 2H), 5.09 (s, 1H), 2.13 (s, 6H).

[0097] Example 10

[0098] Preparation of 4,4'-(4-bromophenyl)methylenebis(1-(4-hydroxyphenyl)thiazolyl)-3-methyl-1H-pyrazole-5-ol)10

[0099]

[0100] Step 1: Synthesize compound 10-2

[0101] The preparation method described in Example 1 was followed for synthesis. Compound 10-2 was obtained as a light green solid with a yield of 77.79%. 1H NMR (300MHz, DMSO-d6) δ9.67 (s, 1H), 9.03 (s, 1H), 8.53 (t, J = 3.4Hz, 1H), 7.67-7.57 (m, 2H), 7.16 (s, 1H), 6.94-6.84 (m, 2H), 4.88 (s, 1H).

[0102] Step 2: Synthesize compound 10

[0103] The preparation method described in Example 1 was followed for synthesis. Compound 10 was obtained as a grayish-white solid with a yield of 59.89%. 1 HNMR(300MHz,DMSO-d6)δ12.18(s,1H),9.64(s,2H),7.81(d,J=8.2Hz,4H),7.51(d,J =9.7Hz, 4H), 7.24 (d, J = 8.0Hz, 2H), 6.82 (d, J = 8.3Hz, 4H), 5.07 (s, 1H), 2.10 (s, 6H).

[0104] Example 11

[0105] Preparation of 4,4'-(4-bromophenyl)methylene)bis(3-methyl-1-(4-(4-trifluoromethylphenyl)thiazo-2-yl)-1H-pyrazole-5-ol)11

[0106]

[0107] Step 1: Synthesize compound 11-2

[0108] The preparation method described in Example 1 was followed for synthesis. Compound 11-2 was obtained as a grayish-white solid with a yield of 77.78%. 1 H NMR (300MHz, DMSO-d6) δ 8.53 (t, J = 3.4 Hz, 1H), 7.81 (d, J = 11.3 Hz, 2H), 7.69 (d, J = 11.4 Hz, 2H), 7.16 (s, 1H), 4.88 (d, J = 3.3 Hz, 2H).

[0109] Step 2: Synthesize compound 11

[0110] The preparation method described in Example 1 was followed for synthesis. Compound 11 was obtained as a grayish-white solid with a yield of 73.34%. 1HNMR(300MHz,DMSO-d6)δ12.34(s,2H),8.23(d,J=8.1Hz,4H),8.02(s,2H),7.84(d,J =8.2Hz, 4H), 7.52 (d, J = 8.2Hz, 2H), 7.25 (d, J = 8.1Hz, 2H), 5.11 (s, 1H), 2.14 (s, 6H).

[0111] Example 12

[0112] Preparation of 4,4'-(4-bromophenyl)methylenebis(1-(4-(3-hydroxyphenyl)thiazol-2-yl)-3-methyl-1H-pyrazole-5-ol)12

[0113]

[0114] Step 1: Synthesize compound 12-2

[0115] The preparation method described in Example 1 was followed for synthesis. Compound 12-2 was obtained as a grayish-black solid with a yield of 70.47%. 1 H NMR (300MHz, DMSO-d6) δ9.40 (s, 1H), 8.49 (t, J = 3.4Hz, 1H), 7.59-7.49 (m, 1H), 7. 28(t,J=8.4Hz,1H),7.23-7.12(m,2H),6.87-6.77(m,1H),4.88(d,J=3.3Hz,2H).

[0116] Step 2: Synthesize compound 12

[0117] The preparation method described in Example 1 was followed for synthesis. Compound 12 was obtained as a grayish-white solid with a yield of 88.87%. 1 HNMR(300MHz,DMSO-d6)δ12.19(s,1H),9.53(s,2H),7.67(s,2H),7.51(d,J=8.4Hz,2H) ,7.44-7.38(m,4H),7.23(t,J=7.9Hz,4H),6.79-6.71(m,2H),5.08(s,1H),2.12(s,6H).

[0118] Example 13

[0119] Preparation of 4,4'-(4-bromophenyl)methylenebis(3-methyl-1-(4-(3-trifluoromethylphenyl)thiazolyl-2-yl)-1H-pyrazole-5-ol)13

[0120]

[0121] Step 1: Synthesize compound 13-2

[0122] The preparation method described in Example 1 was followed for synthesis. Compound 13-2 was obtained as a grayish-black solid with a yield of 67.43%. 1 H NMR (300MHz, DMSO-d6) δ8.50 (t, J = 3.3Hz, 1H), 8.28 (q, J = 1.3Hz, 1H), 7.75-7.63 (m, 3H), 7.19 (s, 1H), 4.88 (d, J = 3.4Hz, 2H).

[0123] Step 2: Synthesize compound 13

[0124] The preparation method described in Example 1 was followed for synthesis. Compound 13 was obtained as a grayish-white solid with a yield of 64.57%. 1 HNMR(300MHz,DMSO-d6)δ10.63(s,2H),8.27(q,J=1.5Hz,2H),7.73-7.65(m, 8H), 7.51 (d, J = 8.1Hz, 2H), 7.20 (d, J = 8.0Hz, 2H), 4.97 (s, 1H), 2.12 (s, 6H).

[0125] Example 14

[0126] Preparation of 4,4'-(4-bromophenyl)methylenebis(3-methyl-1-(4-(2-trifluoromethylphenyl)thiazo-2-yl)-1H-pyrazole-5-ol)14

[0127]

[0128] Step 1: Synthesize compound 14-2

[0129] The preparation method described in Example 1 was followed for synthesis. Compound 14-2 was obtained as a black solid with a yield of 71.85%. 1 HNMR(300MHz,DMSO-d6)δ8.67(t,J=3.4Hz,1H),7.89-7.81(m,1H),7.67-7.55 (m, 2H), 7.48 (ddd, J = 7.9, 4.7, 3.4Hz, 1H), 7.27 (s, 1H), 4.88 (d, J = 3.4Hz, 2H).

[0130] Step 2: Synthesize compound 14

[0131] The preparation method described in Example 1 was followed for synthesis. Compound 14 was obtained as a grayish-white solid with a yield of 78.51%. 1HNMR (300MHz, DMSO-d6) δ 10.63 (s, 2H), 7.88-7.80 (m, 2H), 7.67-7.56 (m, 6H), 7.49 (ddt, J = 11.1, 7.9, 2.4Hz, 4H), 7.20 (d, J = 8.0Hz, 2H), 4.97 (s, 1H).

[0132] Example 15

[0133] Preparation of 4,4'-(4-bromophenyl)methylenebis(1-(4-(2-fluorophenyl)thiazol-2-yl)-3-methyl-1H-pyrazole-5-ol)15

[0134]

[0135] Step 1: Synthesize compound 15-2

[0136] The preparation method described in Example 1 was followed for synthesis. Compound 15-2 was obtained as a reddish-brown solid with a yield of 75.49%. 1 H NMR(300MHz, DMSO-d6)δ8.62(t,J=3.4Hz,1H),7.72-7.63(m,1H),7.34(ddd,J=9.9, 5.3, 2.0Hz, 1H), 7.33-7.22 (m, 2H), 7.15 (s, 1H), 4.88 (d, J = 3.3Hz, 2H), 2.12 (s, 3H).

[0137] Step 2: Synthesize compound 15

[0138] The preparation method described in Example 1 was followed for synthesis. Compound 15 was obtained as a grayish-white solid with a yield of 87.90%. 1 HNMR(300MHz,DMSO-d6)δ10.63(s,2H),7.68(d,J=1.3Hz,1H),7.65(s,1H),7.56-7.47(m,2H),7.34(ddd, J=8.5,5.9,3.0Hz,2H),7.33-7.24(m,6H),7.20(d,J=8.0Hz,2H),4.97(s,1H),2.57(s,3H),1.93(s,3H).

[0139] Example 16

[0140] Preparation of 4,4'-(4-bromophenyl)methylenebis(3-methyl-1-(4-o-tolyl)thiazolyl-2-yl)-1H-pyrazole-5-ol)16

[0141]

[0142] Step 1: Synthesize compound 16-2

[0143] The preparation method described in Example 1 was followed for synthesis. Compound 16-2 was obtained as a white solid with a yield of 71.54%. 1 HNMR (300MHz, DMSO-d6) δ8.72 (t, J = 3.4Hz, 1H), 7.84 (ddd, J = 9.6, 5.0, 1.3Hz, 1H), 7.47 (dddd, J = 8. 8,7.7,5.0,1.3Hz,1H),7.30(ddd,J=9.8,8.5,1.6Hz,1H),7.25-7.14(m,2H),4.88(d,J=3.4Hz,2H).

[0144] Step 2: Synthesize compound 16

[0145] The preparation method described in Example 1 was followed for synthesis. Compound 16 was obtained as a grayish-white solid with a yield of 77.89%. 1 HNMR(300MHz,DMSO-d6)δ10.63(s,2H),7.82(ddd,J=9.5,5.0,1.3Hz,2H),7.65( d,J=2.0Hz,2H),7.55-7.42(m,4H),7.35-7.16(m,6H),4.97(s,1H),1.93(s,6H).

[0146] Example 17

[0147] Preparation of 4,4'-(4-bromophenyl)methylenebis(1-(4-(3-fluorophenyl)thiazol-2-yl)-3-methyl-1H-pyrazole-5-ol)17

[0148]

[0149] Step 1: Synthesize compound 17-2

[0150] The preparation method described in Example 1 was followed for synthesis. Compound 17-2 was obtained as a pale yellow-green solid with a yield of 69.38%. 1 H NMR (300MHz, DMSO-d6) δ8.50(t,J=3.4Hz,1H),7.78(dt,J=8.4,1.5Hz,1H),7.65(dt,J=8.0,1.9Hz,1H ), 7.48 (td, J = 8.1, 5.0Hz, 1H), 7.22 (s, 1H), 7.17 (ddt, J = 9.4, 7.9, 1.4Hz, 1H), 4.88 (d, J = 3.4Hz, 2H).

[0151] Step 2: Synthesize compound 17

[0152] The preparation method described in Example 1 was followed for synthesis. Compound 17 was obtained as a grayish-white solid with a yield of 79.95%. 1 HNMR(300MHz,DMSO-d6)δ10.63(s,2H),7.81-7.75(m,2H),7.61(dt,J=8.0,2.1Hz, 2H),7.55-7.43(m,4H),7.40(s,2H),7.24-7.12(m,4H),4.97(s,1H),2.31(s,6H).

[0153] Example 18

[0154] Preparation of 3,3'-((4-bromophenyl)methylene)bis(5-hydroxy-3-methyl-1H-pyrazole-4,1-diyl)bis(thiazole-2,4-diyl))dibenzoic acid 18

[0155]

[0156] Step 1: Synthesize compound 18-2

[0157] The preparation method described in Example 1 was followed for synthesis. Compound 18-2 was obtained as a reddish-brown solid with a yield of 88.89%. 1 H NMR (300MHz, DMSO-d6) δ8.50(t,J=3.3Hz,1H),8.32(t,J=1.9Hz,1H),7.98(dt,J=8.0,1.5H z, 1H), 7.80 (dt, J = 8.8, 1.5Hz, 1H), 7.70-7.58 (m, 1H), 7.19 (s, 1H), 4.88 (d, J = 3.4Hz, 2H).

[0158] Step 2: Synthesize compound 18

[0159] The preparation method described in Example 1 was followed for synthesis. Compound 18 was obtained as a yellow solid with a yield of 80.52%. 1 HNMR(300MHz,DMSO-d6)δ10.63(s,2H),8.22(t,J=2.1Hz,2H),8.03-7.94(m,2H),7.84-7.76( m,2H),7.69-7.59(m,4H),7.55-7.47(m,2H),7.20(d,J=8.0Hz,2H),4.97(s,1H),2.31(s,6H).

[0160] Example 19

[0161] Preparation of 3,3'-((4-bromophenyl)methylene)bis(5-hydroxy-3-methyl-1H-pyrazole-4,1-diyl)bis(thiazole-2,4-diyl))dibenzonitrile 19

[0162]

[0163] Step 1: Synthesize compound 19-2

[0164] The preparation method described in Example 1 was followed for synthesis. Compound 19-2 was obtained as a dark red solid with a yield of 88.89%. 1 H NMR (300MHz, DMSO-d6) δ8.50(t,J=3.4Hz,1H),8.07(t,J=1.9Hz,1H),7.83(dt,J=7.7,1.5Hz,1H ),7.70(dt,J=6.5,1.5Hz,1H),7.58(dd,J=7.6,6.4Hz,1H),7.19(s,1H),4.88(d,J=3.3Hz,2H).

[0165] Step 2: Synthesize compound 19

[0166] The preparation method described in Example 1 was followed for synthesis. Compound 19 was obtained as a grayish-white solid with a yield of 64.77%. 1 HNMR (300MHz, DMSO-d6) δ10.63 (s, 2H), 8.04 (t, J = 2.1Hz, 2H), 7.87-7.79 (m, 2H), 7. 74-7.64(m,4H),7.63-7.46(m,4H),7.20(d,J=8.0Hz,2H),4.97(s,1H),2.30(s,6H).

[0167] Example 20

[0168] Preparation of 3-(bis(5-hydroxy-1-(4-(2-hydroxyphenyl)thiazo-2-yl)-3-methyl-1H-pyrazole-4-yl)methyl)benzoic acid 20

[0169]

[0170] Step 1: Synthesize compound 20-2

[0171] The preparation method described in Example 1 was followed for synthesis. Compound 20-2 was obtained as a reddish-brown solid with a yield of 69.14%. 1H NMR (300MHz, DMSO-d6) δ10.62(s,1H),8.62(t,J=3.4Hz,1H),7.66(dd,J=8.7,1.2Hz,1H),7.31-7.20(m, 1H), 7.13 (s, 1H), 7.06 (ddd, J = 9.0, 7.7, 1.4Hz, 1H), 6.94 (dd, J = 8.2, 1.4Hz, 1H), 4.88 (d, J = 3.3Hz, 2H).

[0172] Step 2: Synthesize compound 20

[0173] The preparation method described in Example 1 was followed for synthesis. The difference was that p-bromobenzaldehyde in the second step was replaced with m-carboxybenzaldehyde, yielding compound 20 as a grayish-white solid with a yield of 85.20%. 1 H NMR (300MHz, DMSO-d6) δ12.57(s,1H),10.62(d,J=1.4Hz,4H),8.01(t,J=2.3Hz,1H),7.90(ddd,J=7.9,2.3,1.3Hz,1H),7.62-7.50(m,3H),7.45( dt,J=7.7,1.9Hz,1H),7.32(s,2H),7.30-7.20(m,2H),7.06(ddd,J=9.0, 7.7,1.4Hz,2H),6.94(dd,J=8.2,1.4Hz,2H),4.99(s,1H),2.12(s,6H).

[0174] Example 21

[0175] Preparation of 4,4'-(4-(trifluoromethyl)phenyl)methylenebis(1-(4-(2-hydroxyphenyl)thiazolyl)-3-methylpyrazole-5-ol)21

[0176]

[0177] Step 1: Synthesize compound 21-2

[0178] The preparation method described in Example 21 was followed for synthesis. Compound 21-2 was obtained as a reddish-brown solid with a yield of 70.42%. 1H NMR (300MHz, DMSO-d6) δ10.62(s,1H),8.62(t,J=3.4Hz,1H),7.66(dd,J=8.7,1.2Hz,1H),7.31-7.20(m, 1H), 7.13 (s, 1H), 7.06 (ddd, J = 9.0, 7.7, 1.4Hz, 1H), 6.94 (dd, J = 8.2, 1.4Hz, 1H), 4.88 (d, J = 3.3Hz, 2H).

[0179] Step 2: Synthesize compound 21

[0180] The preparation method described in Example 1 was followed. The difference was that p-bromobenzaldehyde in the second step was replaced with p-trifluoromethylbenzaldehyde, yielding compound 21 as a grayish-white solid with a yield of 67.50%. 1 H NMR(300MHz,DMSO-d6)δ12.33(s,1H),10.45(s,1H),8.11(d,J=7.8Hz,2H),7.87(s,2H) ,7.53(d,J=8.0Hz,2H),7.31-7.14(m,4H),7.03-6.86(m,4H),5.12(s,1H),2.17(s,6H).

[0181] Example 22

[0182] Preparation of 4,4'-p-Tolylmethylenebis(1-(4-(2-hydroxyphenyl)thiazolyl)-3-methylpyrazole-5-ol)22

[0183]

[0184] Step 1: Synthesize compound 22-2

[0185] The preparation method described in Example 21 was followed for synthesis. Compound 22-2 was obtained as a reddish-brown solid with a yield of 70.47%. 1 H NMR (300MHz, DMSO-d6) δ10.62(s,1H),8.62(t,J=3.4Hz,1H),7.66(dd,J=8.7,1.2Hz,1H),7.31-7.20(m, 1H), 7.13 (s, 1H), 7.06 (ddd, J = 9.0, 7.7, 1.4Hz, 1H), 6.94 (dd, J = 8.2, 1.4Hz, 1H), 4.88 (d, J = 3.3Hz, 2H).

[0186] Step 2: Synthesize compound 22

[0187] The preparation method described in Example 1 was followed. The difference was that p-bromobenzaldehyde in the second step was replaced with p-methylbenzaldehyde, yielding compound 22 as a grayish-white solid with a yield of 74.33%. 1 H NMR (300MHz, DMSO-d6) δ12.33(s,1H),10.45(s,1H),8.11(d,J=7.8Hz,2H),7.87(s,2H),7.53( d,J=8.0Hz,2H),7.31-7.14(m,4H),7.03-6.86(m,4H),5.12(s,1H),2.19(s,3H),2.17(s,6H).

[0188] Example 23

[0189] Preparation of 4,4'-(4-bromophenyl)methylenebis(3-methyl-1-phenylpyrazole-5-ol)23

[0190]

[0191] The synthesis was carried out according to the preparation method of Example 1. The difference was that the raw materials purchased in the first step yielded compound 23 as a grayish-white solid with a yield of 78.45%. 1 H NMR (300MHz, DMSO-d6) δ7.78 (dd, J = 7.3, 1.4Hz, 6H), 7.50 (dd, J = 7.7, 6.1Hz, 6H), 7.43-7.32 (m, 2H), 5.48 (d, J = 8.0Hz, 1H), 1.93 (s, 6H).

[0192] Example 24

[0193] Preparation of 4,4'-(4-bromophenyl)methylenebis(3-methyl-1-phenylpyrazole-5-ol)24

[0194]

[0195] The synthesis was carried out according to the preparation method of Example 1. The difference was that the raw materials purchased in the first step yielded compound 23 as a grayish-white solid and compound 24 as a grayish-white solid, with a yield of 64.35%. 1 H NMR (300MHz, DMSO-d6) δ7.78 (dd, J = 7.3, 1.4Hz, 6H), 7.50 (dd, J = 7.7, 6.1Hz, 6H), 7.43-7.32 (m, 2H), 5.48 (d, J = 8.0Hz, 1H), 1.93 (s, 6H).

[0196] Example 25

[0197] Preparation of 4,4'-((2-chlorophenyl)methylene)bis(1-(4-(2-hydroxyphenyl)thiazolyl)-3-methyl-1H-pyrazole-5-ol)25

[0198]

[0199] Step 1: Synthesize compound 25-2

[0200] The preparation method described in Example 1 was followed for synthesis. Compound 25-2 was obtained as a reddish-brown solid with a yield of 69.14%. 1 H NMR (300MHz, DMSO-d6) δ10.62(s,1H),8.62(t,J=3.4Hz,1H),7.66(dd,J=8.7,1.2Hz,1H),7.31-7.20(m, 1H), 7.13 (s, 1H), 7.06 (ddd, J = 9.0, 7.7, 1.4Hz, 1H), 6.94 (dd, J = 8.2, 1.4Hz, 1H), 4.88 (d, J = 3.3Hz, 2H).

[0201] Step 2: Synthesize compound 25

[0202] The preparation method described in Example 1 was followed for synthesis. The difference was that p-bromobenzaldehyde was replaced with o-chlorobenzaldehyde, yielding compound 25 as a white solid with a yield of 67.39%. 1 H NMR (300MHz, DMSO-d6) δ10.62(d,J=1.4Hz,4H),7.57(ddd,J=7.4,4.5,1.5Hz,3H),7.44(dd,J=7.4,1.7Hz,1H) ,7.36-7.19(m,6H),7.06(ddd,J=9.0,7.7,1.4Hz,2H),6.94(dd,J=8.2,1.5Hz,2H),5.37(s,1H),2.11(s,6H).

[0203] Example 26

[0204] Preparation of 4,4'-((4-fluorophenyl)methylene)bis(1-(4-(2-hydroxyphenyl)thiazolyl)-3-(trifluoromethyl)-1H-pyrazole-5-ol)26

[0205]

[0206] Step 1: Synthesize compound 26-2

[0207] The preparation method described in Example 1 was followed for synthesis. Compound 26-2 was obtained as a reddish-brown solid with a yield of 69.14%. 1H NMR (300MHz, DMSO-d6) δ10.62(s,1H),8.62(t,J=3.4Hz,1H),7.66(dd,J=8.7,1.2Hz,1H),7.31-7.20(m, 1H), 7.13 (s, 1H), 7.06 (ddd, J = 9.0, 7.7, 1.4Hz, 1H), 6.94 (dd, J = 8.2, 1.4Hz, 1H), 4.88 (d, J = 3.3Hz, 2H).

[0208] Step 2: Synthesize compound 26

[0209] The preparation method described in Example 1 was followed for synthesis. The difference was that ethyl acetoacetate was replaced with ethyl 4,4,4-trifluoro-3-oxobutyrate, and p-bromobenzaldehyde was replaced with p-fluorobenzaldehyde, yielding compound 26 as a white solid with a yield of 73.97%. 1 HNMR(300MHz,DMSO-d6)δ10.62(s,2H),10.34(s,2H),7.63-7.45(m,4H),7.39-7.13 (m, 6H), 7.06 (ddd, J = 9.0, 7.7, 1.4Hz, 2H), 6.94 (dd, J = 8.2, 1.5Hz, 2H), 5.21 (s, 1H).

[0210] Example 27

[0211] Preparation of 4,4'-(4-bromophenyl)methylenebis(3-methyl-1-(4-(pyridin-3-yl)thiazo-2-yl)-1H-pyrazole-5-ol)27

[0212]

[0213] Step 1: Synthesize compound 27-2

[0214] The preparation method described in Example 1 was followed for synthesis. Compound 27-2 was obtained as a light green solid with a yield of 59.98%. 1 H NMR (300MHz, DMSO-d6) δ8.99(t,J=2.0Hz,1H),8.73(t,J=3.3Hz,1H),8.63(dt,J=4.9,1.9Hz,1H ), 8.20 (dt, J = 8.5, 2.2Hz, 1H), 7.44 (dd, J = 8.5, 4.8Hz, 1H), 7.16 (s, 1H), 4.88 (d, J = 3.4Hz, 2H).

[0215] Step 2: Synthesize compound 27

[0216] The preparation method described in Example 1 was followed for synthesis. Compound 27-2 was obtained as a green solid with a yield of 63.26%. 1 HNMR(300MHz,DMSO-d6)δ10.63(s,2H),8.99-8.87(m,2H),8.71-8.59(m,2H),8.20(dt, J=8.5,2.1Hz,2H),7.58-7.39(m,6H),7.20(d,J=8.0Hz,2H),4.97(s,1H),2.31(s,6H).

[0217] Example 28

[0218] Preparation of 4,4'-((4-bromophenyl)methylene)bis(5-hydroxy-3-methyl-1H-pyrazole-4,1-diyl)bis(thiazole-2,4-diyl)dibenzoic acid 28

[0219]

[0220] Step 1: Synthesize compound 28-2

[0221] The preparation method described in Example 1 was followed for synthesis. Compound 28-2 was obtained as a reddish-brown solid with a yield of 60.82%. 1 H NMR (300MHz, DMSO-d6) δ12.71 (s, 1H), 8.53 (t, J = 3.4Hz, 1H), 7.95 (d, J = 8.9Hz, 2H), 7.86 (d, J = 8.9Hz, 2H), 7.16 (s, 1H), 4.88 (d, J = 3.4Hz, 2H).

[0222] Step 2: Synthesize compound 28

[0223] The preparation method described in Example 1 was followed for synthesis. Compound 28 was obtained as a yellow solid with a yield of 72.57%. 1 HNMR (300MHz, DMSO-d6) δ11.40(s,2H),10.63(s,2H),7.89(q,J=9.0Hz,8H),7.62-7.46(m,2H),7.40-7.13(m,4H),4.97(s,1H),2.25(s,1H).

[0224] Example 29

[0225] Preparation of 4,4'-((4-bromophenyl)methylene)bis(5-hydroxy-3-methyl-1H-pyrazole-4,1-diyl)bis(thiazole-2,4-diyl))dibenzonitrile 29

[0226]

[0227] Step 1: Synthesize compound 29-2

[0228] The preparation method described in Example 1 was followed for synthesis. Compound 29-2 was obtained as a grayish-black solid with a yield of 74.37%. 1 H NMR (300MHz, DMSO-d6) δ8.53 (t, J = 3.3Hz, 1H), 7.89 (d, J = 7.2Hz, 2H), 7.78-7.69 (m, 2H), 7.16 (s, 1H), 4.88 (d, J = 3.3Hz, 2H).

[0229] Step 2: Synthesize compound 29

[0230] The preparation method described in Example 1 was followed for synthesis. Compound 29 was obtained as a grayish-white solid with a yield of 71.59%. 1 HNMR (300MHz, DMSO-d6) δ11.40 (s, 2H), 7.89 (q, J = 9.0Hz, 8H), 7.62-7.46 (m, 2H), 7.40-7.13 (m, 4H), 4.97 (s, 1H), 2.25 (s, 1H).

[0231] Example 30

[0232] Preparation of 4,4'-(4-bromophenyl)methylenebis(3-methyl-1-(4-p-tolyl)thiazo-2-yl)-1H-pyrazole-5-ol)30

[0233]

[0234] Step 1: Synthesize compound 30-2

[0235] The preparation method of Example 1 was followed for synthesis. Compound 30-2 was obtained as a white solid with a yield of 74.29%. 1 HNMR (300MHz, DMSO-d6) δ8.53(t,J=3.4Hz,1H),7.82-7.73(m,2H),7.27(d,J=7.2Hz,2H),7.15(s,1H),4.88(d,J=3.4Hz,2H),2.34(s,1H).

[0236] Step 2: Synthesize compound 30

[0237] The preparation method described in Example 1 was followed for synthesis. Compound 30 was obtained as a white solid with a yield of 78.94%. 1HNMR (300MHz, DMSO-d6) δ11.40(s,2H),7.89(q,J=9.0Hz,8H),7.62-7.46(m,2H),7.40-7.13(m,4H),4.97(s,1H),2.34(s,1H),2.25(s,1H).

[0238] Example 31

[0239] Preparation of 4,4'-(4-bromophenyl)methylenebis(1-(4-fluorophenyl)thiazolyl)-3-methylpyrazole-5-ol)31

[0240]

[0241] Step 1: Synthesize compound 31-2

[0242] The preparation method described in Example 1 was followed for synthesis. Compound 31-2 was obtained as a light green solid with a yield of 77.20%. 1 H NMR (300MHz, DMSO-d6) δ8.53 (t, J = 3.4 Hz, 1H), 7.93 (dd, J = 8.6, 5.0 Hz, 2H), 7.24 (t, J = 8.4 Hz, 2H), 7.16 (s, 1H), 4.88 (d, J = 3.4 Hz, 2H).

[0243] Step 2: Synthesize compound 31

[0244] The preparation method of Example 1 was followed for synthesis. Compound 31 was obtained as a white solid with a yield of 74.29%. 1 HNMR (300MHz, DMSO-d6) δ11.40 (s, 2H), 7.89 (q, J = 9.0Hz, 8H), 7.62-7.46 (m, 2H), 7.40-7.13 (m, 4H), 4.97 (s, 1H), 2.25 (s, 6H).

[0245] Example 32

[0246] Preparation of 4,4'-(2,2-dimethylpropane-1,1-diyl)bis(3-methyl-1-pyridin-2-yl)-1H-pyrazole-5-ol)32

[0247]

[0248] The synthesis was carried out according to the preparation method of Example 1. The difference is that the product of the first step was a purchased raw material, yielding compound 32 as a gray solid with a yield of 45.52%. 1H NMR (300MHz, DMSO-d6) δ10.50(s,2H),8.47(dd,J=3.4,1.7Hz,2H),7.97(td,J=6.8,1.7Hz,2H),7.7 2(dd,J=7.0,1.4Hz,2H),7.37(ddd,J=6.8,3.3,1.4Hz,2H),4.55(s,1H),2.25(s,6H),1.07(s,9H).

[0249] Example 33

[0250] Preparation of 4,4'-(4-bromophenyl)methylenebis(3-methyl-1-pyridin-3-yl)-1H-pyrazole-5-ol)33

[0251]

[0252] The synthesis was carried out according to the preparation method of Example 1. The difference is that the product of the first step was a purchased raw material; compound 33 was a pale yellow solid with a yield of 70.58%. 1 H NMR (300MHz, DMSO-d6) δ8.73-8.66(m,2H),8.47-8.38(m,2H),7.87(dt,J=7.2,2.1Hz,2H),7. 56-7.47(m,2H),7.41(dd,J=7.2,3.7Hz,2H),7.20(d,J=8.0Hz,2H),4.94(s,1H),2.25(s,6H).

[0253] Example 34

[0254] Preparation of 4,4'-((4-bromophenyl)methylene)bis(3-methyl-1-(pyridin-4-yl)-1H-pyrazole-5-ol)34

[0255]

[0256] The synthesis was carried out according to the preparation method of Example 1. The difference is that the product of the first step was a purchased raw material; compound 34 was a white-orange solid with a yield of 76.32%. 1 H NMR (300MHz, DMSO-d6) δ8.65-8.40(m,4H),7.51(d,J=8.1Hz,2H),7.45-7.30(m,4H),7.20(d,J=8.0Hz,2H),4.94(s,1H),2.25(s,6H).

[0257] Example 35

[0258] Preparation of 4,4'-((4-bromophenyl)methylene)bis(3-methyl-1-(pyrimidin-2-yl)-1H-pyrazole-5-ol)35

[0259]

[0260] The synthesis was carried out according to the preparation method of Example 1. The difference is that the product of the first step was a purchased raw material, and compound 35 was a pink solid with a yield of 79.82%. 1 H NMR (300MHz, DMSO-d6) δ11.12 (s, 2H), 8.76 (d, J = 3.3Hz, 4H), 7.58-7.45 (m, 2H), 7.30 (t, J = 3.3Hz, 2H), 7.20 (d, J = 8.0Hz, 2H), 5.00 (s, 1H), 2.25 (s, 6H).

[0261] Example 36

[0262] Preparation of (((4-bromophenyl)methylene)bis(5-hydroxy-3-methyl-1H-pyrazole-4,1-diyl))bis(pyridin-2-yl ketone)36

[0263]

[0264] The synthesis was carried out according to the preparation method of Example 1. The difference is that the product of the first step was a purchased raw material; compound 36 was a light orange solid with a yield of 65.38%. 1 H NMR(300MHz,DMSO-d6)δ11.51(s,2H),8.74(dd,J=4.3,1.7Hz,2H),8.05(dd,J=6.7,1.5Hz,2H), 7.94(td,J=7.0,1.7Hz,2H),7.60-7.47(m,4H),7.20(d,J=8.0Hz,2H),5.01(s,1H),2.25(s,6H).

[0265] Example 37

[0266] Preparation of 4-((4-bromophenyl)(1-(4-(4-chlorophenyl)thiazol-2-yl)-5-hydroxy-3-methyl-1H-pyrazol-4-yl)methyl)-1-(4-(tetrafluorophenyl)thiazol-2-yl)-3-methyl-1H-pyrazol-5-ol 37

[0267]

[0268] Step 1: Synthesize compound 37-3

[0269] The synthesis was carried out according to the preparation method of Example 1.

[0270] The difference lies in step two. After the reaction with ethyl acetoacetate is added and monitored by TLC, the solvent is evaporated and the solution is purified by column chromatography. The resulting product, 37-3, is a light green solid, purified by silica gel column chromatography (DCM:MeOH = 100:1), with a yield of 70.22%. 1 H NMR (300MHz, DMSO-d6) δ11.95(s,1H),7.93(dd,J=8.6,5.0Hz,2H),7.30(s,1H),7.24(t,J=8.3Hz,2H),5.38(s,1H),2.30(s,3H).

[0271] Step 2: Synthesize compound 37-4

[0272] Compound 37-3 (50.00 mg, 181.62 μmol), p-bromobenzaldehyde (84.01 mg, 454.05 μmol), and a few drops of piperidine were added to 15 mL of isopropanol. The mixture was refluxed at 60 °C for 4 hours. After the reaction was completed by TLC, the mixture was filtered and dried to obtain compound 37-4, which was light green in color, with a yield of 68.72%. 1 H NMR (300MHz, DMSO-d6) δ7.92(dd,J=8.6,5.0Hz,2H),7.81(d,J=8.6Hz,2H),7.59(dd,J=7.3,1.5Hz,3H),7.46(s,1H),7.24(t,J=8.3Hz,2H),1.93(s,3H).

[0273] Step 3: Synthesize compound 37-5

[0274] Following the synthetic methods described in Examples 37-1 to 37-3, compound 37-5 was obtained as a light green solid in yield of 78.35%. 1 H NMR (300MHz, DMSO-d6) δ11.95 (s, 1H), 7.61 (d, J = 8.5Hz, 2H), 7.52-7.43 (m, 2H), 7.30 (s, 1H), 5.38 (s, 1H), 2.31 (s, 3H).

[0275] Step 4: Synthesize compound 37

[0276] Compound 37-4 (50.00 mg, 112.53 μmol) and a similar compound 37-5 (49.25 mg, 168.80 μmol) synthesized according to the first two steps of Example 37 were added to anhydrous ethanol and refluxed at 78 °C for 6 hours. After the reaction was completed by TLC monitoring, the mixture was filtered and dried to obtain compound 37 as a white solid with a yield of 68.79%. 1H NMR (300MHz, DMSO-d6) δ 10.63 (s, 2H), 7.97-7.88 (m, 2H), 7.61 (d, J = 8.5Hz, 2H), 7.54-7.44 (m, 4H), 7.34-7.16 (m, 6H), 4.97 (s, 1H), 2.25 (s, 6H).

[0277] Example 38

[0278] Preparation of 4-(2-(4-(4-bromophenyl)(1-(4-(4-bromophenyl)thiazol-2-yl)-5-hydroxy-3-methyl-1H-pyrazol-4-yl)methyl)-5-hydroxy-3-methyl-1H-pyrazol-1-yl)thiazol-4-yl)benzoic acid 38

[0279]

[0280] Step 1: Synthesize compound 38

[0281] The preparation method of Example 37 was used for synthesis.

[0282] Compound 38-3 was obtained as a pale yellow solid with a yield of 74.28%. 1 H NMR (300MHz, DMSO-d6) δ11.95 (s, 1H), 7.72 (d, J = 8.7Hz, 2H), 7.64-7.55 (m, 2H), 7.30 (s, 1H), 5.38 (s, 1H), 2.31 (s, 3H).

[0283] Compound 38-4 was obtained as a white solid with a yield of 83.92%. 1 H NMR (300MHz, DMSO-d6) δ7.76 (dd, J = 25.8, 8.6Hz, 3H), 7.64-7.55 (m, 4H), 7.39 (s, 1H), 7.34 (s, 1H), 2.23 (s, 3H).

[0284] Compound 38-5 was obtained as a white solid with a yield of 83.92%. 1 H NMR (300MHz, DMSO-d6) δ12.73 (s, 1H), 11.95 (s, 1H), 7.89 (q, J = 9.0Hz, 4H), 7.30 (s, 1H), 5.38 (s, 1H), 2.18 (s, 3H).

[0285] Compound 38 was obtained as a white solid with a yield of 76.89%. 1H NMR (300MHz, DMSO-d6) δ10.63(s,2H),7.89(q,J=8.9Hz,4H),7.72(d,J=8.6Hz,2H),7.60(d,J=8.6 Hz, 2H), 7.54-7.47 (m, 2H), 7.30 (d, J = 0.8Hz, 2H), 7.20 (d, J = 8.0Hz, 2H), 4.97 (s, 1H), 2.25 (s, 6H).

[0286] Example 39

[0287] Preparation of 4-((4-chlorophenyl)(5-hydroxy-1-(4-(2-hydroxyphenyl)thiazo-2-yl)-3-methyl-1H-pyrazol-4-yl)methyl)-3-methyl-1H-pyrazol-4-yl)methyl)-3-methyl-1-(4-(p-tolyl)thiazo-2-yl)-1H-pyrazol-5-ol 39

[0288]

[0289] Step 1: Synthesize compound 39

[0290] The preparation method of Example 37 was used for synthesis.

[0291] Compound 39-3 was obtained as a white solid with a yield of 63.23%. 1 H NMR (300MHz, DMSO-d6) δ11.95(s,1H),7.78-7.69(m,2H),7.33-7.22(m,3H),5.38(s,1H),2.40(s,3H),2.23(s,3H).

[0292] Compound 39-4 was obtained as a white solid with a yield of 68.71%. 1 H NMR (300MHz, DMSO-d6) δ7.79 (dd, J = 12.8, 8.0 Hz, 3H), 7.59 (dd, J = 7.2, 1.4 Hz, 2H), 7.40 (s, 4H), 7.27 (d, J = 7.2 Hz, 1H), 2.40 (s, 6H).

[0293] Compound 39-5 was obtained as a pink solid with a yield of 68.71%. 1H NMR (300MHz, DMSO-d6) δ11.95(s,1H),10.62(s,1H),7.57(dd,J=8.7,1.3Hz,1H),7.32(s,1H),7.25(td,J= 8.0, 1.3Hz, 1H), 7.06 (ddd, J=9.0, 7.7, 1.5Hz, 1H), 6.94 (dd, J=8.2, 1.5Hz, 1H), 5.38 (s, 1H), 2.20 (s, 3H).

[0294] Compound 39 was obtained as a white solid with a yield of 57.83%. 1 H NMR (300MHz, DMSO-d6) δ10.62(d,J=1.5Hz,3H),7.73(d,J=7.4Hz,2H),7.61-7.47(m,3H),7.34-7.17(m,7 H), 7.06 (ddd, J=9.0, 7.7, 1.5Hz, 1H), 6.94 (dd, J=8.2, 1.5Hz, 1H), 4.97 (s, 1H), 2.4 (s, 3H), 2.25 (s, 6H).

[0295] Example 40

[0296] Preparation of 4-((4-bromophenyl)(1-(4-(3-chlorophenyl)thiazol-2-yl)-5-hydroxy-3-methyl-1H-pyrazole-4-yl)methyl)-1-(4-(2-hydroxyphenyl)thiazol-2-yl)-3-methyl-1H-pyrazole-5-ol 40

[0297]

[0298] Step 1: Synthesize compound 40

[0299] The preparation method of Example 37 was used for synthesis.

[0300] Compound 40-3 was obtained as a white solid with a yield of 78.37%. 1 H NMR (300MHz, DMSO-d6) δ11.95(s,1H),7.72-7.59(m,2H),7.43-7.31(m,3H),5.38(s,1H),2.25(s,3H).

[0301] Compound 40-4 was obtained as a white solid with a yield of 83.84%. 1H NMR (300MHz, DMSO-d6) δ7.81 (d, J = 8.6Hz, 2H), 7.71-7.61 (m, 1H), 7.66-7.55 (m, 4H), 7.51 (s, 1H), 7.43-7.31 (m, 2H), 2.21 (s, 3H).

[0302] Compound 40-5 was obtained as a pink solid with a yield of 75.27%. 1 H NMR (300MHz, DMSO-d6) δ11.95(s,1H),10.62(s,1H),7.57(dd,J=8.7,1.3Hz,1H),7.32(s,1H),7.25(td,J= 8.0, 1.3Hz, 1H), 7.06 (ddd, J=9.0, 7.7, 1.5Hz, 1H), 6.94 (dd, J=8.2, 1.5Hz, 1H), 5.38 (s, 1H), 2.20 (s, 3H).

[0303] Compound 40 was obtained as a white solid with a yield of 45.87%. 1 H NMR(300MHz,DMSO-d6)δ10.62(d,J=1.5Hz,3H),7.70-7.47(m,6H),7.41-7.16(m,6H),7 .06(ddd,J=9.0,7.7,1.5Hz,1H), 6.94(dd,J=8.2,1.6Hz,1H), 4.97(s,1H), 2.25(s,6H).

[0304] Example 41

[0305] Preparation of 4-((4-bromophenyl)(5-hydroxy-1-(4-(2-hydroxyphenyl)thiazo-2-yl)-3-methyl-1H-pyrazol-4-yl)methyl)-3-methyl-1-(pyridin-2-yl)-1H-pyrazin-5-ol 41

[0306]

[0307] Step 1: Synthesize compound 41-1

[0308] The preparation method of Example 37 was used for synthesis.

[0309] Compound 41-3 was obtained as a pink solid with a yield of 48.35%. 1H NMR (300MHz, DMSO-d6) δ11.95(s,1H),10.62(s,1H),7.57(dd,J=8.7,1.3Hz,1H),7.32(s,1H),7.25(td,J= 8.0, 1.3Hz, 1H), 7.06 (ddd, J=9.0, 7.7, 1.5Hz, 1H), 6.94 (dd, J=8.2, 1.5Hz, 1H), 5.38 (s, 1H), 2.20 (s, 3H).

[0310] Compound 41-4 was obtained as an orange-yellow solid with a yield of 74.38%. 1 H NMR (300MHz, DMSO-d6) δ10.62(s,1H),7.81(d,J=8.6Hz,2H),7.58(td,J=6.8,1.3Hz,4H),7.52(s,1H),7 .25(td,J=7.9,1.3Hz,1H), 7.06(ddd,J=9.0,7.7,1.5Hz,1H), 6.94(dd,J=8.2,1.5Hz,1H), 2.21(s,3H).

[0311] The difference from Example 37 is that compound 41-5 was reacted directly with ethyl acetoacetate using purchased raw materials to produce compound 41-5 as a white solid with a yield of 57.29%. 1 H NMR(300MHz,DMSO-d6)δ10.96(s,1H),8.46(dd,J=3.4,1.7Hz,1H),7.96(td,J=6.9,1.7Hz,1 H), 7.73 (dd, J=7.0, 1.5Hz, 1H), 7.37 (ddd, J=6.8, 3.3, 1.5Hz, 1H), 5.43 (s, 1H), 2.15 (s, 3H).

[0312] Compound 41 was obtained as a white solid with a yield of 57.28%. 1H NMR (300MHz, DMSO-d6) δ11.21(s,1H),10.62(d,J=1.5Hz,2H),8.47(dd,J=3.3,1 .7Hz,1H),7.97(td,J=6.9,1.7Hz,1H),7.72(dd,J=7.0,1.5Hz,1H),7.60-7.48(m ,3H),7.37(ddd,J=6.8,3.3,1.5Hz,1H),7.32(s,1H),7.29-7.17(m,3H),7.06(dd d, J=9.0, 7.7, 1.5Hz, 1H), 6.94 (dd, J=8.3, 1.6Hz, 1H), 4.98 (s, 1H), 2.25 (s, 6H).

[0313] Example 42

[0314] Preparation of 4-((4-bromophenyl)(5-hydroxy-1-(4-(2-hydroxyphenyl)thiazo-2-yl)-3-methyl-1H-pyrazol-4-yl)methyl)-3-methyl-1-(4-phenylthiazo-2-yl)-1H-pyrazol-5-ol 42

[0315]

[0316] Step 1: Synthesize compound 42

[0317] The preparation method of Example 41 was used for synthesis.

[0318] Compound 42-3 was obtained as a pink solid with a yield of 48.35%. 1 H NMR (300MHz, DMSO-d6) δ11.95(s,1H),10.62(s,1H),7.57(dd,J=8.7,1.3Hz,1H),7.32(s,1H),7.25(td,J= 8.0, 1.3Hz, 1H), 7.06 (ddd, J=9.0, 7.7, 1.5Hz, 1H), 6.94 (dd, J=8.2, 1.5Hz, 1H), 5.38 (s, 1H), 2.20 (s, 3H).

[0319] Compound 42-4 was obtained as an orange-yellow solid with a yield of 74.38%. 1H NMR (300MHz, DMSO-d6) δ10.62(s,1H),7.81(d,J=8.6Hz,2H),7.58(td,J=6.8,1.3Hz,4H),7.52(s,1H),7 .25(td,J=7.9,1.3Hz,1H), 7.06(ddd,J=9.0,7.7,1.5Hz,1H), 6.94(dd,J=8.2,1.5Hz,1H), 2.21(s,3H).

[0320] Compound 42-5 was obtained as a white solid with a yield of 57.29%. 1 H NMR (300MHz, DMSO-d6) δ11.95(s,1H),7.81(dd,J=7.5,1.6Hz,2H),7.54-7.43(m,2H),7.47-7.36(m,1H),7.30(s,1H),5.38(s,1H),2.13(s,6H).

[0321] Compound 42 was obtained as a white solid with a yield of 48.57%. 1 H NMR (300MHz, DMSO-d6) δ10.62(d,J=1.5Hz,3H),7.81(dd,J=7.5,1.6Hz,2H),7.57(dd,J=8.7,1.2Hz,1H),7.54-7.38( m,5H),7.34-7.15(m,5H),7.06(ddd,J=8.9,7.7,1.4Hz,1H),6.94(dd,J=8.2,1.4Hz,1H),4.97(s,1H),2.12(s,6H).

[0322] Example 43

[0323] Preparation of 4-((3-chlorophenyl)(1-(4-(3-fluorophenyl)thiazol-2-yl)-5-hydroxy-3-methyl-1H-pyrazole-4-yl)methyl)-1-(4-(2-hydroxyphenyl)thiazol-2-yl)-3-methyl-1H-pyrazole-5-ol 43

[0324]

[0325] Step 1: Synthesize compound 43

[0326] The preparation method of Example 41 was used for synthesis.

[0327] Compound 43-3 was obtained as a pink solid with a yield of 48.35%. 1H NMR (300MHz, DMSO-d6) δ11.95(s,1H),10.62(s,1H),7.57(dd,J=8.7,1.3Hz,1H),7.32(s,1H),7.25(td,J= 8.0, 1.3Hz, 1H), 7.06 (ddd, J=9.0, 7.7, 1.5Hz, 1H), 6.94 (dd, J=8.2, 1.5Hz, 1H), 5.38 (s, 1H), 2.20 (s, 3H).

[0328] Compound 43-4 was obtained as an orange-yellow solid with a yield of 74.38%. 1 H NMR (300MHz, DMSO-d6) δ10.62(s,1H),7.98-7.86(m,1H),7.79(t,J=1.6Hz,1H),7.65-7.49(m,3H),7.46-7.34( m, 2H), 7.25 (td, J = 7.9, 1.3Hz, 1H), 7.06 (ddd, J = 9.0, 7.7, 1.5Hz, 1H), 6.94 (dd, J = 8.2, 1.5Hz, 1H), 2.23 (s, 3H).

[0329] Compound 43-5 was obtained as a white solid with a yield of 76.21%. 1 H NMR (300MHz, DMSO-d6) δ11.95(s,1H),7.78(dt,J=8.4,1.6Hz,1H),7.61(dt,J=8.0,1.9Hz, 1H),7.48(td,J=8.0,5.0Hz,1H),7.40(s,1H),7.23-7.11(m,1H),5.38(s,1H),2.13(s,6H). .

[0330] Compound 43 was obtained as a white solid with a yield of 48.36%. 1 H NMR (300MHz, DMSO-d6) δ10.62(d,J=1.4Hz,2H),7.78(dt,J=8.3,1.6Hz,1H),7.66-7. 11(m,11H),7.17-7.00(m,1H),6.94(dd,J=8.2,1.4Hz,1H),5.03(s,1H),2.25(s,6H).

[0331] Example 44

[0332] Preparation of 3-(2-(4-(4-bromophenyl)(5-hydroxy-3-methyl-1-(pyrimidin-2-yl)-1H-pyrazol-4-yl)methyl)-5-hydroxy-3-methyl-1H-pyrazin-1-yl)thiazolyl)benzyl nitrile 44

[0333]

[0334] Step 1: Synthesize compound 44

[0335] The preparation method of Example 41 was used for synthesis.

[0336] Compound 44-3 was obtained as a white solid with a yield of 76.37%. 1 H NMR (300MHz, DMSO-d6) δ11.95(s,1H),8.04(t,J=1.9Hz,1H),7.83(dt,J=7.7,1.5 Hz, 1H), 7.75-7.64 (m, 2H), 7.58 (dd, J = 7.6, 6.4Hz, 1H), 5.38 (s, 1H), 2.31 (s, 3H).

[0337] Compound 44-4 was obtained as a grayish-white solid with a yield of 65.07%. 1 H NMR (300MHz, DMSO-d6) δ8.04(t,J=1.9Hz,1H),7.82(dd,J=8.0,6.3Hz,3H),7.70(dt,J=6.5,1.5Hz,1H),7.64-7.52(m,4H),7.50(s,1H),2.28(s,3H).

[0338] Compound 44-5 was obtained as a dark red solid with a yield of 88.89%. 1 H NMR (300MHz, DMSO-d6) δ11.71 (s, 1H), 8.76 (d, J = 3.2Hz, 2H), 7.29 (t, J = 3.3Hz, 1H), 5.50 (s, 1H), 2.31 (s, 3H).

[0339] Compound 44 was obtained as a white solid with a yield of 51.47%. 1 H NMR (300MHz, DMSO-d6) δ11.12(s,1H),10.63(s,1H),8.76(d,J=3.3Hz,2H),8.04(t,J=1.9Hz,1H),7.83(dt,J=7.7,1.6 Hz, 1H), 7.73-7.65 (m, 2H), 7.61-7.47 (m, 3H), 7.30 (t, J = 3.3Hz, 1H), 7.20 (d, J = 8.0Hz, 2H), 5.01 (s, 1H), 2.25 (s, 6H).

[0340] Example 45

[0341] Preparation of 4-((4-bromophenyl)(1-(4-(3-bromophenyl)thiazo-2-yl)-5-hydroxy-3-methyl-1H-pyrazol-4-yl)methyl)-3-methyl-1-(pyridin-4-yl)-1H-pyrazin-5-ol 45

[0342]

[0343] Step 1: Synthesize compound 45

[0344] The preparation method of Example 37 was used for synthesis.

[0345] Compound 45-3 was obtained as a grayish-white solid with a yield of 75.32%. 1 H NMR(300MHz,DMSO-d6)δ11.95(s,1H),7.90(t,J=1.9Hz,1H),7.72(dt,J=8.5,1.6Hz,1H), 7.67(s,1H),7.58(dt,J=8.0,1.5Hz,1H),7.40(t,J=8.2Hz,1H),5.38(s,1H),2.13(s,3H).

[0346] Compound 45-4 was obtained as a grayish-white solid with a yield of 71.29%. 1 H NMR (300MHz, DMSO-d6) δ7.90(t,J=1.9Hz,1H),7.81(d,J=8.6Hz,2H),7.72(dt,J=8.6,1.5H z,1H),7.59(ddt,J=7.0,5.5,1.6Hz,4H),7.50(s,1H),7.40(t,J=8.2Hz,1H),2.11(s,3H).

[0347] Compound 45-5 was obtained as a yellow solid with a yield of 68.38%. 1 H NMR (300MHz, DMSO-d6) δ11.66(s,1H),8.56-8.48(m,2H),7.45-7.37(m,2H),5.46(s,1H),2.12(s,3H).

[0348] Compound 45 was obtained as a white solid with a yield of 53.20%. 1H NMR (300MHz, DMSO-d6) δ10.63(s,1H),8.58-8.48(m,2H),7.90(t,J=1.9Hz,1H),7.75-7.65(m,2H),7.58(dt ,J=8.0,1.5Hz,1H),7.53-7.48(m,2H),7.44-7.36(m,3H),7.20(d,J=8.0Hz,2H),4.97(s,1H),2.25(s,6H).

[0349] Example 46

[0350] Preparation of 3-(2-(4-(4-bromophenyl)(5-hydroxy-3-methyl-1-(pyrimidin-2-yl)-1H-pyrazol-4-yl)methyl)-5-hydroxy-3-methyl-1H-pyrazin-1-yl)thiazolyl)benzyl nitrile 46

[0351]

[0352] Step 1: Synthesize compound 46

[0353] The preparation method of Example 37 was used for synthesis.

[0354] Compound 46-3 was obtained as a white solid with a yield of 76.37%. 1 H NMR (300MHz, DMSO-d6) δ11.95(s,1H),8.04(t,J=1.9Hz,1H),7.83(dt,J=7.7,1.5 Hz, 1H), 7.75-7.64 (m, 2H), 7.58 (dd, J = 7.6, 6.4Hz, 1H), 5.38 (s, 1H), 2.31 (s, 3H).

[0355] Compound 46-4 was obtained as a grayish-white solid with a yield of 65.07%. 1 H NMR (300MHz, DMSO-d6) δ8.04(t,J=1.9Hz,1H),7.82(dd,J=8.0,6.3Hz,3H),7.70(dt,J=6.5,1.5Hz,1H),7.64-7.52(m,4H),7.50(s,1H),2.28(s,3H).

[0356] Compound 46-5 was obtained as a white solid with a yield of 57.29%. 1H NMR (300MHz, DMSO-d6) δ11.53(s,1H),8.76(dd,J=2.0,1.0Hz,1H),8.41(ddd,J=3.7,1.9,1. 0Hz, 1H), 7.88 (dt, J=7.2, 1.9Hz, 1H), 7.41 (dd, J=7.2, 3.7Hz, 1H), 5.47 (s, 1H), 2.12 (s, 3H).

[0357] Compound 46 was obtained as a white solid with a yield of 57.63%. 1 H NMR(300MHz,DMSO-d6)δ12.30(s,1H),10.63(s,1H),8.73-8.66(m,1H),8.43 (dt,J=3.8,1.9Hz,1H),8.04(t,J=1.9Hz,1H),7.85(ddt,J=10.7,7.6,1.9Hz ,2H),7.75-7.64(m,2H),7.58(dd,J=7.6,6.4Hz,1H),7.51(d,J=8.2Hz,2H), 7.41 (dd, J=7.2, 3.7Hz, 1H), 7.20 (d, J=8.0Hz, 2H), 4.97 (s, 1H), 2.12 (s, 6H).

[0358] Example 47

[0359] Preparation of (3-bromophenyl)(4-((4-bromophenyl)5-hydroxy-3-methyl-1-(pyrimidin-2-yl)-1H-pyrazol-4-yl)methyl)-5-hydroxy-3-methyl-1H-pyrazin-1-yl)methyl ketone 47

[0360]

[0361] Step 1: Synthesize compound 47

[0362] The preparation method of Example 37 was used for synthesis.

[0363] Compound 47-2 was a purchased raw material, and compound 47-3 was obtained as a white solid with a yield of 63.27%. 1 H NMR(300MHz,DMSO-d6)δ11.64(s,1H),8.07(t,J=1.9Hz,1H),7.94-7.85(m,1 H),7.74-7.64(m,1H),7.45(dd,J=8.1,7.0Hz,1H),6.08(s,1H),2.14(s,3H).

[0364] Compound 47-4 was obtained as a white solid with a yield of 67.38%.1 H NMR(300MHz,DMSO-d6)δ8.11(t,J=1.9Hz,1H),8.02-7.92(m,1H),7.84(d,J=8.6Hz, 2H),7.74-7.65(m,1H),7.65-7.55(m,3H),7.44(dd,J=8.1,7.0Hz,1H),2.13(s,3H).

[0365] Compound 47-5 was obtained as a dark red solid with a yield of 88.89%. 1 H NMR (300MHz, DMSO-d6) δ11.71 (s, 1H), 8.76 (d, J = 3.2Hz, 2H), 7.29 (t, J = 3.3Hz, 1H), 5.50 (s, 1H), 2.31 (s, 3H).

[0366] Compound 47 was obtained as a white solid with a yield of 54.49%. 1 H NMR (300MHz, DMSO-d6) δ11.64(s,1H),11.12(s,1H),8.76(d,J=3.3Hz,2H),8.07(t,J=1.9Hz,1H),7.95-7.85(m,1H),7.74-7.64 (m,1H),7.56-7.47(m,2H),7.45(dd,J=8.1,7.0Hz,1H),7.30(t,J=3.3Hz,1H),7.20(d,J=8.0Hz,2H),5.01(s,1H),2.25(s,6H).

[0367] Example 48

[0368] Preparation of 4-((4-bromophenyl)(5-hydroxy-3-methyl-1-(pyridin-2-yl)-1H-pyrazol-4-yl)methyl)-3-methyl-3-(pyrimidin-2-yl)-1H-pyrazol-5-ol 48

[0369]

[0370] Step 1: Synthesize compound 48

[0371] The preparation method of Example 37 was used for synthesis.

[0372] Compound 48-3 was obtained as a white solid with a yield of 57.29%. 1H NMR(300MHz,DMSO-d6)δ10.96(s,1H),8.46(dd,J=3.4,1.7Hz,1H),7.96(td,J=6.9,1.7Hz,1 H), 7.73 (dd, J=7.0, 1.5Hz, 1H), 7.37 (ddd, J=6.8, 3.3, 1.5Hz, 1H), 5.43 (s, 1H), 2.15 (s, 3H).

[0373] Compound 48-4 was obtained as a white solid with a yield of 65.38%. 1 H NMR (300MHz, DMSO-d6) δ8.36(dd,J=3.4,1.7Hz,1H),8.04(dd,J=7.0,1.5Hz,1H),7.81(d,J=8.6Hz,2H),7.6 6(td,J=6.9,1.7Hz,1H),7.59(d,J=8.7Hz,2H),7.53(s,1H),7.14(ddd,J=6.8,3.3,1.4Hz,1H),2.13(s,3H).

[0374] Compound 48-5 was obtained as a dark red solid with a yield of 88.89%. 1 H NMR (300MHz, DMSO-d6) δ11.71 (s, 1H), 8.76 (d, J = 3.2Hz, 2H), 7.29 (t, J = 3.3Hz, 1H), 5.50 (s, 1H), 2.31 (s, 3H).

[0375] Compound 48 was obtained as a white solid with a yield of 53.72%. 1 H NMR (300MHz, DMSO-d6) δ11.21(s,1H),11.12(s,1H),8.76(d,J=3.3Hz,2H),8.47(dd,J=3.4,1.7Hz,1H),7.97(td,J=6.8,1.7Hz,1H),7.72(dd,J= 7.0,1.3Hz,1H),7.51(d,J=8.1Hz,2H),7.37(ddd,J=6.8,3.3,1.3Hz,1H) ,7.30(t,J=3.3Hz,1H),7.20(d,J=8.0Hz,2H),5.01(s,1H),2.23(s,6H).

[0376] Example 49

[0377] Preparation of 4-((4-bromophenyl)(5-hydroxy-3-methyl-1-(pyridin-3-yl)-1H-pyrazol-4-yl)methyl)-3-methyl-3-(pyrimidin-2-yl)-1H-pyrazol-5-ol 49

[0378]

[0379] Step 1: Synthesize compound 49

[0380] The preparation method of Example 37 was used for synthesis.

[0381] Compound 49-3 was obtained as a white solid with a yield of 57.29%. 1 H NMR (300MHz, DMSO-d6) δ11.53(s,1H),8.76(dd,J=2.0,1.0Hz,1H),8.41(ddd,J=3.7,1.9,1. 0Hz, 1H), 7.88 (dt, J=7.2, 1.9Hz, 1H), 7.41 (dd, J=7.2, 3.7Hz, 1H), 5.47 (s, 1H), 2.12 (s, 3H).

[0382] Compound 49-4 was obtained as a white solid with a yield of 59.29%. 1 H NMR (300MHz, DMSO-d6) δ8.88(dd,J=2.0,1.0Hz,1H),8.51(ddd,J=3.7,1.9,1.0Hz,1H),8.08(dt,J=7.2, 1.9Hz, 1H), 7.82 (d, J = 8.6Hz, 2H), 7.59 (dd, J = 7.3, 1.5Hz, 3H), 7.46 (dd, J = 7.2, 3.7Hz, 1H), 2.13 (s, 3H).

[0383] Compound 49-5 was obtained as a dark red solid with a yield of 88.89%. 1 H NMR (300MHz, DMSO-d6) δ11.71 (s, 1H), 8.76 (d, J = 3.2Hz, 2H), 7.29 (t, J = 3.3Hz, 1H), 5.50 (s, 1H), 2.31 (s, 3H).

[0384] Compound 49 was obtained as a white solid with a yield of 59.24%. 1H NMR (300MHz, DMSO-d6) δ11.12(s,1H),8.76(d,J=3.3Hz,2H),8.70(dd,J=2.0,1.0Hz,1H),8.43(ddd,J=3.5,2.2,1.0Hz,1H),7.87(dt,J= 7.2,2.1Hz,1H),7.56-7.47(m,2H),7.41(dd,J=7.2,3.7Hz,1H),7.30(t,J=3.3Hz,1H),7.20(d,J=8.0Hz,2H),4.98(s,1H),2.24(s,6H).

[0385] Example 50

[0386] Preparation of 4-(1-(5-hydroxy-1-(4-(2-hydroxyphenyl)thiazolyl-2-yl)-3-(trifluoromethyl)-1H-pyrazol-4-yl)-2,2-dimethylpropyl)-3-methyl-1-(pyrimidin-2-yl)-1H-pyrazol-5-ol 50

[0387]

[0388] Step 1: Synthesize compound 50

[0389] The preparation method of Example 37 was used for synthesis.

[0390] Compound 50-3 was obtained as a white solid with a yield of 65.36%. 1 H NMR(300MHz,DMSO-d6)δ11.98(s,1H),10.62(s,1H),7.57(dd,J=8.7,1.3Hz,1H),7.34(s,1H),7.25 (td,J=7.9,1.3Hz,1H),7.06(ddd,J=9.0,7.7,1.5Hz,1H),6.94(dd,J=8.2,1.5Hz,1H),6.33(s,1H).

[0391] Compound 50-4 was obtained as a white solid with a yield of 62.36%. 1 H NMR (300MHz, DMSO-d6) δ10.62(s,1H),7.62-7.50(m,2H),7.25(td,J=7.9,1.3Hz,1H),7.06(ddd,J=9 .1,7.7,1.5Hz,1H),6.94(dd,J=8.2,1.5Hz,1H),6.59(td,J=2.1,1.1Hz,1H),1.10(d,J=1.0Hz,9H).

[0392] Compound 50-5 was obtained as a dark red solid with a yield of 88.89%. 1 H NMR (300MHz, DMSO-d6) δ11.71 (s, 1H), 8.76 (d, J = 3.2Hz, 2H), 7.29 (t, J = 3.3Hz, 1H), 5.50 (s, 1H), 2.31 (s, 3H).

[0393] Compound 50 was obtained as a white solid with a yield of 57.49%. 1 H NMR (300MHz, DMSO-d6) δ10.62(s,1H),10.42(s,1H),9.95(s,1H),8.76(d,J=3.3Hz,2H),7.57(dd,J=8.7,1.3Hz,1H),7 .37-7.20(m,3H),7.06(ddd,J=9.0,7.7,1.5Hz,1H),6.94(dd,J=8.2,1.5Hz,1H),4.6(s,1H),2.26(s,3H),1.07(s,9H).

[0394] Test case

[0395] 1. Expression and purification of IGF2BP2 protein

[0396] The IGF2BP2 gene plasmid was purchased from Genscript Biotech.

[0397] Experimental steps:

[0398] Escherichia coli BL21(DE3) strain was transfected with a recombinant plasmid, and the cells were revived in sterile LB medium at 37°C. Single colonies were picked and transferred to 10 mL of LB liquid medium (containing 50 μg / mL kanamycin, Amp), and cultured overnight at 37°C with shaking (220 rpm). The culture was then transferred to 1 L of LB liquid medium (containing 50 μg / mL kanamycin, Amp), and cultured at 37°C with shaking (220 rpm) for 6-8 hours until the OD600 reached 0.6-0.8. The culture was then cooled to 12°C, and 1 mMIPTG (Merck) was added to induce expression for 16 hours (180 rpm). The cells were then harvested and stored at -80°C for later use.

[0399] Add 4g of the bacterial sludge from the previous step to 40mL of lysis buffer, mix well, then add PMSF (Beyotime), and sonicate for 40 minutes. The lysed mixture is then centrifuged at low temperature and high speed (10000rpm, 20min, 4℃). The supernatant is filtered (0.4μm microporous membrane) and purified using an AKTA pure25 (GE Healthcare, Life Sciences) His column (equilibration buffer: 20mM pH 8.0 Tris-HCl, 300mM NaCl, 10mM imidazole; elution buffer: 20mM pH 8.0 Tris-HCl, 300mM NaCl, 500mM imidazole). 10% SDS-PAGE is used to confirm the molecular weight and purity of the bands. Dialysis is performed overnight (20mM pH 8.0 Tris-HCl, 300mM NaCl). The obtained protein concentration is determined by BCA and stored at -80℃ for later use.

[0400] 2. Determination of the inhibitory activity of compounds against IGF2BP2 based on fluorescence polarization (FP)

[0401] This invention is based on a fluorescent molecular probe that binds to IGF2BP2, used to study the competition between this class of compounds and IGF2BP2. 6 A nucleic acid binding method was used to determine the inhibition rate of this type of compound under different concentration conditions, and then the IC50 was calculated. 50 value.

[0402] The equipment and reagents used are as follows:

[0403] The instrument used in this experiment was a SpectraMax Paradigm Multi-Mode Microplate Reader (Molecular Devices). The protein used was IGF2BP2 (a protein expressed and purified in our laboratory, sequence shown below). The probe used was a fluorescently labeled m... 6 All A-ssRNA (sequence) test compounds were prepared as 10 mM stock solutions in DMSO. The 384-well blackboard used in the experiment was manufactured by Corning.

[0404] Experimental steps:

[0405] The final test volume was 60 μL. The following solutions were added to each well in the following order: 20 μL of different concentrations of compound (each compound was serially diluted 10-14 times, with an initial concentration of 100 μM), 20 μL of IGF2BP2 protein (300 nM, final concentration 100 nM), and 20 μL of fluorescent probe (300 nM, final concentration 10 nM). A blank control (40 μL pH 7.5 Tris-HCl buffer + 20 μL 10 nM fluorescent probe) and a negative control (20 μL pH 7.5 Tris-HCl buffer + 20 μL IGF2BP2 protein + 20 μL 10 nM fluorescent probe) were also included in each experiment. After adding the sample, the 384-well plate was covered with aluminum foil and incubated on a shaker at room temperature for 1 hour. Fluorescence was read using a SpectraMax Paradigm Multi-Mode Microplate Reader at an excitation wavelength of 485 nm and an emission wavelength of 535 nm. The mP value was calculated, and the inhibition rate was calculated using the following formula. Finally, the IC50 was calculated using GraphPadPrism 5.0. 50 value.

[0406] Inhibition rate = (mP value of compound group - mP value of blank group) / (mP value of negative control group - mP value of blank group) × 100%

[0407] The experimental results are shown in Table 1. Using unlabeled m 6 A-ssRNA was used as a positive control, with the compound shown in the following formula as a lead compound, and its activity data as a control.

[0408]

[0409] Table 1. IC50 of the compounds of the present invention against IGF2BP2 protein 50 value

[0410]

[0411]

[0412] As shown in the table above, the compound obtained in this invention has significant inhibitory activity against IGF2BP2 and can be used as a small molecule inhibitor of IGF2BP2 protein to inhibit the binding of IGF2BP2 to mRNA.

[0413] In summary, the substituted bis(pyrazolyl)methane derivative compounds provided by this invention exhibit significant inhibitory activity against IGF2BP2, thus representing an effective IGF2BP2 inhibitor. Therefore, drugs containing the above-mentioned compounds as active ingredients can be used to prepare remedies for clinical conditions related to IGF2BP2.

[0414] As described above, although specific preferred embodiments have been referenced and the invention has been shown and described, this should not be construed as limiting the invention itself. Various changes in form and detail may be made without departing from the spirit and scope of the invention as defined in the appended claims.

Claims

1. The following compounds or their pharmaceutically acceptable salts: 、 、 、 、 。 2. Use of the compound of claim 1 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating diseases associated with IGF2BP2 protein dysfunction.

3. A pharmaceutical composition, characterized in that, The active ingredient of the pharmaceutical composition includes the compound of claim 1 or a pharmaceutically acceptable salt thereof.

4. The pharmaceutical composition according to claim 3, characterized in that, The pharmaceutical composition also includes a pharmaceutically acceptable carrier.

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