3-amino-5-phenyl-pyrazole derivatives as microtubulin inhibitors and their preparation and medical use

Abstract

This invention provides a 3-amino-5-phenyl-pyrazole derivative microtubule inhibitor, its preparation, and its pharmaceutical uses. The structure of the 3-amino-5-phenyl-pyrazole derivative is shown in formula (I): In formula (I), R1 is independently selected from 3,4,5-trimethoxyphenyl, 3,4-dimethoxyphenyl, or dioxolane[1,3-d]phenyl; R2 and R3 are independently selected from methyl, methoxy, fluorine, chlorine, or bromine. This type of compound not only possesses strong antitumor activity but also anti-angiogenic effects, effectively inhibiting the proliferation of tumor cells and can be used to prepare tumor proliferation inhibitors. Furthermore, it has a strong ability to inhibit microtubule aggregation, providing a novel microtubule inhibitor for inhibiting tumor cell proliferation.

Description

Technical Field

[0001] This invention belongs to the pharmaceutical field, specifically relating to 3-amino-5-phenyl-pyrazole derivative microtubule inhibitors, their preparation, and pharmaceutical uses. Background Technology

[0002] Malignant tumors are among the most serious diseases threatening human health. Their incidence rate is second only to cardiovascular and cerebrovascular diseases, making them the second leading cause of death worldwide. Furthermore, their mortality rate surpasses that of cardiovascular and cerebrovascular diseases, ranking first among all diseases. Therefore, finding and developing new drugs to treat tumors is a major challenge. Because microtubules play a crucial role in the proliferation and division of tumor cells, tubulin has become a relatively ideal target for anti-tumor drugs.

[0003] Microtubules play crucial roles in various cellular processes, including spindle formation, cell shape maintenance, and intracellular transport. Their function in mitosis makes them attractive targets for anticancer drugs. Microtubule-targeting agents disrupt microtubule formation, thereby inhibiting cancer cells from entering the G2 / M phase and ultimately leading to apoptosis. Therefore, tubulin inhibitors are widely used in cancer treatment. Currently, all marketed tubulin inhibitors bind to paclitaxel or vincristine binding sites on tubulin. These compounds have the advantage of high antitumor activity and effectiveness against a variety of cancers. However, these compounds also have significant toxic side effects and are prone to inducing drug resistance.

[0004] Microtubule inhibitors based on colchicine binding sites can overcome the aforementioned drawbacks and possess therapeutic advantages over those based on taxane and vinca alkaloid binding sites. For example, they have good water solubility and can be administered orally. Furthermore, they are less prone to multidrug resistance. To date, many microtubule inhibitors based on colchicine binding sites have been discovered as effective anticancer agents. Some of these have already reached clinical trials, demonstrating the great potential of colchicine-based antitumor drug analogs. 3-Amino-5-phenyl-pyrazole derivatives, based on the structure-activity relationship of colchicine-based microtubule inhibitors, introduce a five-membered heterocyclic pyrazole ring and hold promise for the treatment of various cancers. Summary of the Invention

[0005] The purpose of this invention is to provide a microtubule inhibitor that can inhibit the proliferation of tumor cells at the cellular level and inhibit the growth of xenografts in nude mice at the animal level, and can be used to treat various cancers.

[0006] The first object of the present invention is to provide 3-amino-5-phenyl-pyrazole derivatives, the structures of which are shown in formula (I):

[0007]

[0008] In formula (I), R1 is independently selected from 3,4,5-trimethoxyphenyl, 3,4-dimethoxyphenyl or dioxolane[1,3-d]phenyl; R2 and R3 are independently selected from hydrogen, methyl, methoxy, fluorine, chlorine or bromine.

[0009] A second object of the present invention is to provide a method for preparing the aforementioned 3-amino-5-phenyl-pyrazole derivatives, the method comprising the following steps:

[0010]

[0011] Furthermore, the method specifically includes the following steps:

[0012] At room temperature, NH2NH2·H2O and CH3SO3H were added to a suspension of substituted benzoylacetonitrile 1 and ethanol, and the mixture was heated under reflux for 1.5 h.

[0013] After evaporating the solvent, the residue was dissolved in dichloromethane, washed with saturated NaHCO3 solution, dried with anhydrous sodium sulfate, and purified by silica gel chromatography to obtain compound 2.

[0014] The substituted benzoic acid 3 was slowly added to SOCl2 at 0°C and stirred. The mixture was heated under reflux for 8 hours and dried under vacuum. The resulting solid 4 was not further processed and was immediately used as the raw material for the next reaction.

[0015] Compound 2 was dissolved in dichloromethane, and triethylamine was added. Then, a dichloromethane solution of compound 4 was slowly added. After stirring thoroughly at room temperature, the mixture was washed with saturated NaHCO3 and purified by silica gel chromatography to obtain the 3-amino-5-phenyl-pyrazole derivative shown in formula (I).

[0016] A third object of the present invention is to provide a pharmaceutical composition comprising a 3-amino-5-phenyl-pyrazole derivative or a pharmaceutically acceptable salt thereof as shown in formula (I), or a solvate thereof.

[0017] Furthermore, the pharmaceutically acceptable salts include hydrochloride, hydrobromide, sulfate, phosphate, methanesulfonate, acetate, lactate, succinate, fumarate, maleate, citrate, benzoate, p-toluenesulfonate, or alkaline metal cation salts.

[0018] Furthermore, the pharmaceutical composition further includes pharmaceutically acceptable excipients, preferably selected from one or more pharmaceutically acceptable carriers, diluents, or excipients.

[0019] A fourth object of the present invention is to provide the use of the aforementioned 3-amino-5-phenyl-pyrazole derivatives or the aforementioned pharmaceutical compositions in the preparation of microtubule inhibitors.

[0020] A fifth object of the present invention is to provide the use of the aforementioned 3-amino-5-phenyl-pyrazole derivatives or the aforementioned pharmaceutical compositions in the preparation of medicaments for treating tumors.

[0021] Compared with the prior art, the technical solution of the present invention has the following beneficial effects:

[0022] This invention synthesizes a class of 3-amino-5-phenyl-pyrazole derivatives. These compounds not only possess strong antitumor activity, exhibiting nanomolar-level IC50 activity against tumor cells, but also demonstrate anti-angiogenic effects, effectively inhibiting tumor cell proliferation and making them suitable for preparing tumor proliferation inhibitors. Furthermore, they exhibit a strong ability to inhibit tubulin aggregation, providing a novel tubulin inhibitor for suppressing tumor cell proliferation. Attached Figure Description

[0023] Figure 1 The reaction route diagram for synthesizing the compounds of this invention;

[0024] Figure 2 This is a statistical graph showing the growth inhibitory activity of MCF-7 breast cancer cells on nude mouse xenografts, where A represents the change in xenograft volume and B represents the tumor inhibition rate. Detailed Implementation

[0025] The following examples illustrate specific implementation methods to further describe the content of the present invention in detail. However, this should not be construed as limiting the scope of the above-mentioned subject matter of the present invention to the following examples. All technologies that can be implemented in the art based on the above-mentioned content of the present invention should be included in the content of the present invention.

[0026] In the following examples, "room temperature" means approximately 20°C to approximately 35°C. The proportions of the mixed solvents are volume mixing proportions; unless otherwise stated, % refers to wt%.

[0027] Example 1

[0028] (3-Amino-5-(3-Fluorophenyl)-1H-pyrazol-1-yl)(3,4,5-trimethoxyphenyl)methyl ketone (5a)

[0029]

[0030] Step 1:

[0031] At room temperature, NH₂NH₂·H₂O (0.6 mL, 12 mmol) and CH₃SO₃H (0.1 mL, 1 mmol) were added to a suspension of 3-fluorobenzoylacetonitrile (compound 1, 0.5 g, 3.4 mmol) and ethanol (20 mL), and the mixture was heated to 70 °C and refluxed for 1.5 h. After evaporating the solvent, the residue was dissolved in dichloromethane, washed three times (20 mL × 3) with saturated NaHCO₃ solution, dried over anhydrous sodium sulfate, and purified by silica gel chromatography to obtain compound 2.

[0032] Step Two:

[0033] 3,4,5-Trimethoxybenzoic acid (compound 3, 500 mg, 1.1 mmol) was slowly added to 10 mL of SOCl2 at 0 °C with stirring. The mixture was heated to 80 °C, refluxed for 8 h, and dried under vacuum. The resulting solid 4 was not further processed and was immediately used as the starting material for the next reaction.

[0034] Step 3:

[0035] Compound 2 was dissolved in dichloromethane (20 mL), and 1 mL of triethylamine was added. Then, a dichloromethane solution of compound 4 was slowly added. The mixture was stirred thoroughly at room temperature for 10 h, washed three times with saturated NaHCO3 (20 mL × 3), and purified by silica gel chromatography to obtain compound 5a. It was a yellow powder with a yield of 35.7%.

[0036] 1 H NMR(500MHz,Chloroform-d)δ7.65(s,2H),7.60–7.54(m,1H),7.52(dt,J=10.0,2.0Hz,1H),7.39(t d,J=8.0,5.8Hz,1H),7.08(td,J=8.4,2.7Hz,1H),5.86(s,1H),5.75(s,2H),3.98(d,J=14.3Hz,9H). 13 C NMR (126MHz, DMSO) δ168.48,153.76,153.04,150.11,149.24,147.92,126. 34,125.16,119.34,115.24,112.19,111.10,109.50,85.17,56.22,55.96.

[0037] Example 2

[0038] (3-Amino-5-(p-Tolyl)-1H-pyrazole-1-yl)(3,4,5-trimethoxyphenyl)methyl ketone (5b)

[0039]

[0040] The preparation method was the same as in Example 1, except that 3-fluorophenylacetonitrile in Example 1 was replaced with 4-methylbenzoylacetonitrile. Compound 5b was a pale yellow powder with a yield of 45.9%. 1 H NMR(500MHz,Chloroform-d)δ7.71(d,J=8.1Hz,2H),7.67(s,2H),7.23(d,J=7.8 Hz, 2H), 5.86 (s, 1H), 5.72 (s, 2H), 5.37 (t, J = 4.9Hz, 3H), 3.97 (d, J = 13.0Hz, 9H). 13 C NMR (75MHz, DMSO) δ168.67,154.22,153.82,152.40,141.63,139.00,129.84,129.15,128.14,126.22,109.61,85.08,60.68,56.47,21.38.

[0041] Example 3

[0042] (3-Amino-5-(4-fluorophenyl)-1H-pyrazol-1-yl)(3,4,5-trimethoxyphenyl) methyl ketone (5c)

[0043]

[0044] The preparation method was the same as in Example 1, except that 3-fluorophenylacetonitrile in Example 1 was replaced with 4-fluorobenzoylacetonitrile. Compound 5c was a yellow powder with a yield of 35.2%. 1 H NMR (600MHz, DMSO-d6) δ7.87–7.82(m,2H),7.53(s,2H),7.28(t,J=8.8Hz,2H),6.87(s,2H),5.91(s,1H),3.83(d,J=35.1Hz,9H). 13 C NMR (75MHz, DMSO) δ168.77,153.95,153.29,152.42,141.64,129.15,128.39,128.09,116.32,116.03,109.53,85.17,60.68,56.50.

[0045] Example 4

[0046] (3-Amino-5-(3-methoxyphenyl)-1H-pyrazole-1-yl)(3,4,5-trimethoxyphenyl) methyl ketone (5d)

[0047]

[0048] The preparation method was the same as in Example 1, except that 3-fluorophenylacetonitrile in Example 1 was replaced with 3-methoxybenzoylacetonitrile. Compound 5d was a yellow powder with a yield of 50.2%. 1 H NMR (500MHz, DMSO-d6) δ7.59(s,2H),7.42–7.29(m,3H),6.98(ddd,J=7.6,4.7,2.7Hz,1H),6.84(s,2H),5.93(s,1H),3.89–3.78(m,12H). 13 C NMR (126MHz, DMSO) δ168.63,159.97,154.14,153.88,152.43,141.85,134.05, 130.34,127.99,118.85,115.27,111.36,109.80,85.48,60.70,56.52,55.55.

[0049] Example 5

[0050] (3-Amino-5-(3-Chlorophenyl)-1H-pyrazole-1-yl)(3,4,5-trimethoxyphenyl)methyl ketone (5e)

[0051]

[0052] The preparation method was the same as in Example 1, except that 3-fluorophenylacetonitrile in Example 1 was replaced with 3-chlorobenzoylacetonitrile. Compound 5e was a white powder with a yield of 30.9%. 1 H NMR(500MHz,DMSO-d6)δ7.84(d,J=2.1Hz,1H),7.77(dt,J=7.0,1.8Hz,1H),7.5 6(s,2H),7.49–7.45(m,2H),6.90(s,2H),5.98(s,1H),3.84(d,J=30.5Hz,9H). 13 CNMR(126MHz,DMSO)δ168.70,154.03,152.80,152.44,141.82,134.79,133 .99,131.23,129.25,127.90,125.87,124.98,109.70,85.36,60.71,56.49.

[0053] Example 6

[0054] (3-Amino-5-(4-chlorophenyl)-1H-pyrazol-1-yl)(3,4,5-trimethoxyphenyl) methyl ketone (5f)

[0055]

[0056] The preparation method was the same as in Example 1, except that 3-fluorophenylacetonitrile in Example 1 was replaced with 4-chlorobenzoylacetonitrile. Compound 5f was a pale yellow powder with a yield of 35.9%. 1 H NMR (500MHz, DMSO-d6) δ7.82(d,J=8.4Hz,2H),7.53(s,2H),7.50(d,J=8.3Hz,2H),6.87(s,2H),5.93(s,1H),3.83(d,J=26.7Hz,9H). 13 C NMR (126MHz, DMSO) δ168.78,154.00,153.08,152.44,141.74,134.01,131.52,129.30,128.04,128.02,109.59,85.24,60.69,56.53.

[0057] Example 7

[0058] (3-Amino-5-(4-bromophenyl)-1H-pyrazole-1-yl)(3,4,5-trimethoxyphenyl)methyl ketone (5g)

[0059]

[0060] The preparation method was the same as in Example 1, except that 3-fluorophenylacetonitrile in Example 1 was replaced with 4-bromobenzoylacetonitrile. 5 g of the compound was a yellow powder, with a yield of 34.7%. 1 H NMR (500MHz, DMSO-d6) δ7.75(d,J=8.4Hz,2H),7.64(d,J=8.5Hz,2H),7.53(s,2H),6.87(s,2H),5.93(s,1H),3.83(d,J=26.2Hz,9H). 13 C NMR (126MHz, DMSO) δ168.77,154.01,153.13,152.45,141.81,132.21,131.89,128.31,128.00,122.69,109.65,85.23,60.69,56.55.

[0061] Example 8

[0062] (3-Amino-5-(3-bromophenyl)-1H-pyrazole-1-yl)(3,4,5-trimethoxyphenyl) methyl ketone (5h)

[0063]

[0064] The preparation method was the same as in Example 1, except that 3-fluorophenylacetonitrile in Example 1 was replaced with 3-bromobenzoylacetonitrile. The compound was thawed for 5 hours, resulting in a white powder with a yield of 37.0%. 1 H NMR(500MHz,DMSO-d6)δ7.99(d,J=1.8Hz,1H),7.85–7.77(m,1H),7.65–7.57(m,1H), 7.57(s,2H),7.41(t,J=7.8Hz,1H),6.91(s,2H),5.97(s,1H),3.84(d,J=33.0Hz,9H). 13 C NMR (126MHz, DMSO) δ168.60,160.45,159.16,153.82,153.01,148.04,128. 91,128.19,126.36,125.24,115.40,113.98,112.73,84.30,55.89,55.55.

[0065] Example 9

[0066] (3-Amino-5-(4-methoxyphenyl)-1H-pyrazole-1-yl)(3,4,5-trimethoxyphenyl)methyl ketone (5i)

[0067]

[0068] The preparation method was the same as in Example 1, except that 3-fluorophenylacetonitrile in Example 1 was replaced with 4-methoxybenzoylacetonitrile. Compound 5i was a pale yellow powder with a yield of 40.9%. 1 H NMR (500MHz, DMSO-d6) δ7.73(d,J=8.6Hz,2H),7.56(s,2H),7.00(d,J=8.7Hz,2H),6.81(s,2H),5.85(s,1H),3.88–3.78(m,12H). 13 C NMR (126MHz, DMSO) δ168.60,160.45,154.10,153.81,152.41,141.70,128.21,127.72,125.14,114.62,109.69,84.97,60.68,56.53,55.67.

[0069] Example 10

[0070] (3-Amino-5-m-Tolyl-1H-Pyrazol-1-yl)(3,4,5-Trimethoxyphenyl)methyl ketone (5j)

[0071]

[0072] The preparation method was the same as in Example 1, except that 3-fluorophenylacetonitrile in Example 1 was replaced with 4-methoxybenzoylacetonitrile. Compound 5j was a pale yellow powder with a yield of 47.9%. 1 H NMR(500MHz,DMSO-d6)δ7.64(s,1H),7.59(d,J=8.2Hz,3H),7.33(t,J=7.5H z,1H),7.25–7.18(m,2H),6.84(s,2H),5.90(s,1H),3.84(d,J=32.5Hz,9H). 13 C NMR (126MHz, DMSO) δ168.61,154.28,153.85,152.42,141.74,138.32,132.56, 130.15,129.13,128.05,126.82,123.51,109.74,85.23,60.69,56.47,21.49.

[0073] Example 11

[0074] (3-Amino-5-(3,4-dimethoxyphenyl)-1H-pyrazole-1-yl)(3,4,5-trimethoxyphenyl) methyl ketone (5k)

[0075]

[0076] The preparation method was the same as in Example 1, except that 3-fluorophenylacetonitrile in Example 1 was replaced with 3,4-dimethoxybenzoylacetonitrile. Compound 5j was a white powder with a yield of 43.4%. 1 H NMR(500MHz,DMSO-d6)δ7.66–7.57(m,2H),7.35(d,J=6.0Hz,2H),7.05–6.96( m,1H),6.84(s,2H),5.93–5.85(m,1H),3.90–3.84(m,6H),3.82–3.75(m,10H). 13 C NMR (126MHz, DMSO) δ168.41,154.35,153.86,152.42,150.18,149.25,141.76,128 .06,125.31,119.43,112.21,109.79,109.31,85.17,60.70,56.49,56.00,55.84.

[0077] Example 12

[0078] (3-Amino-5-(3,4-Dimethoxyphenyl)-1H-pyrazole-1-yl)(benzo[d][1,3]dioxolane-5-yl)methyl ketone (5l)

[0079]

[0080] The preparation method was the same as in Example 1, except that 3-fluorophenylacetonitrile was replaced with 3,4-dimethoxybenzoylacetonitrile, and 3,4,5-trimethoxybenzoic acid was replaced with piperic acid. Compound 5L was a pale yellow powder with a yield of 33.4%. 1 H NMR (500MHz, DMSO-d6) δ7.95(s,1H),7.88(dd,J=8.5,2.0Hz,1H),7.35(d,J=7.7Hz,2H),7.14(d,J=8.6 Hz,1H),7.01(d,J=8.3Hz,1H),6.79(s,2H),5.88(s,1H),3.86(d,J=17.2Hz,6H),3.79(d,J=1.8Hz,6H). 13 CNMR(126MHz,DMSO)δ168.48,154.05,153.76,153.04,150.11,149.24,147.92,126.34,12 5.43,125.16,119.34,115.24,112.19,111.10,109.50,85.17,56.20,56.00,55.96,55.92.

[0081] Example 13

[0082] (3-Amino-5-(4-fluorophenyl)-1H-pyrazole-1-yl)(benzo[d][1,3]dioxolane-5-yl)methyl ketone (5m)

[0083]

[0084] The preparation method was the same as in Example 1, except that 3-fluorophenylacetonitrile was replaced with 4-fluorobenzoylacetonitrile, and 3,4,5-trimethoxybenzoic acid was replaced with piperic acid. Compound 5m was a yellow powder with a yield of 37.2%. ¹H NMR (500MHz, DMSO-d6) δ 7.82 (dd, J = 8.6, 5.7Hz, 2H), 7.79–7.74 (m, 1H), 7.69 (d, J = 1.8Hz, 1H), 7.27 (t, J = 8.8Hz, 2H), 7.10 (d, J = 8.2Hz, 1H), 6.82 (s, 2H), 6.18 (s, 2H), 5.89 (s, 1H). ¹³C NMR (126MHz, DMSO) δ168.48,153.86,153.17,151.44,147.22,129.22,128.56,127.87,126.82,116.18,116.00,111.52,108.20,102.51,85.27.

[0085] Example 13

[0086] (3-Amino-5-(4-Chlorophenyl)-1H-pyrazole-1-yl)(benzo[d][1,3]dioxolane-5-yl)methyl ketone(5n)

[0087]

[0088] The preparation method was the same as in Example 1, except that 3-fluorophenylacetonitrile was replaced with 4-chlorobenzoylacetonitrile, and 3,4,5-trimethoxybenzoic acid was replaced with piperic acid. Compound 5n was a white powder with a yield of 31.2%. ¹H NMR (600 MHz, DMSO-d6) δ 7.79 (d, J = 8.5 Hz, 2H), 7.77 (dd, J = 8.2, 1.8 Hz, 1H), 7.68 (d, J = 1.7 Hz, 1H), 7.50 (d, J = 8.6 Hz, 2H), 7.10 (d, J = 8.2 Hz, 1H), 6.84 (s, 2H), 6.18 (s, 2H), 5.91 (s, 1H). ¹³C NMR (151MHz, DMSO) δ168.50,153.90,152.95,151.47,147.23,133.98,131.53,129.22,128.12,127.87,126.75,111.50,108.22,102.52,85.33.

[0089] Example 14

[0090] (3-Amino-5-(3-Methoxyphenyl)-1H-pyrazole-1-yl)(benzo[d][1,3]dioxolane-5-yl)methyl ketone (5o)

[0091]

[0092] The preparation method was the same as in Example 1, except that 3-fluorophenylacetonitrile was replaced with 3-methoxybenzoylacetonitrile and 3,4,5-trimethoxybenzoic acid was replaced with piperic acid. Compound 5O was a pale yellow powder with a yield of 31.2%. ¹H NMR (600 MHz, DMSO-d6) δ 7.80 (dd, J = 8.2, 1.8 Hz, 1H), 7.70 (d, J = 1.8 Hz, 1H), 7.37–7.33 (m, 2H), 7.31–7.28 (m, 1H), 7.10 (d, J = 8.3 Hz, 1H), 7.00–6.96 (m, 1H), 6.80 (s, 2H), 6.18 (s, 2H), 5.90 (s, 1H), 3.80 (s, 3H). ¹³C NMR(75MHz,DMSO)δ168.73,153.94,153.09,152.79,148.07,133.94,133.29,131.6 0,129.30,129.25,128.05,126.40,126.37,125.00,114.88,111.04,85.22,56.00.

[0093] Example 15

[0094] (3-Amino-5-p-methylphenyl-1H-pyrazole-1-yl)(benzo[d][1,3]dioxolane-5-yl)methyl ketone (5p)

[0095]

[0096] The preparation method was the same as in Example 1, except that 3-fluorophenylacetonitrile was replaced with 4-methylbenzoylacetonitrile, and 3,4,5-trimethoxybenzoic acid was replaced with piperic acid. Compound 5p was a yellow powder with a yield of 41.3%. ¹H NMR (600 MHz, DMSO-d6) δ 7.79 (dd, J = 8.2, 1.8 Hz, 1H), 7.70 (d, J = 1.7 Hz, 1H), 7.66 (d, J = 7.9 Hz, 2H), 7.24 (d, J = 7.8 Hz, 2H), 7.10 (d, J = 8.2 Hz, 1H), 6.79 (s, 2H), 6.18 (d, J = 1.5 Hz, 2H), 5.86 (s, 1H), 2.33 (s, 3H). ¹³C NMR (126MHz, DMSO) δ167.78,163.09,139.42,138.36,136.27,133.94,131. 11,130.19,129.69,128.92,128.06,126.91,112.91,107.13,86.28,21.19.

[0097] Example 16

[0098] (3-Amino-5-(4-fluorophenyl)-1H-pyrazole-1-yl)(3,4-dimethoxyphenyl)methyl ketone (5q)

[0099]

[0100] The preparation method was the same as in Example 1, except that 3-fluorophenylacetonitrile was replaced with 4-fluorobenzoylacetonitrile, and 3,4,5-trimethoxybenzoic acid was replaced with 3,4-dimethoxybenzoic acid. Compound 5q was a white powder with a yield of 40.2%. 1 H NMR(500MHz,DMSO-d6)δ7.90(dd,J=8.3,2.1Hz,1H),7.88–7.80(m,2H),7.28(t,J=8 .6Hz,2H),7.15(d,J=8.5Hz,2H),6.84(s,2H),5.91(s,1H),3.87(d,J=25.2Hz,6H). 13 C NMR (126MHz, DMSO) δ168.49,153.74,153.03,150.10,149.22,147.91,126. 35,125.40,119.35,115.20,112.19,111.09,109.46,85.20,55.99,55.91.

[0101] Example 17

[0102] (3-Amino-5-(4-chlorophenyl)-1H-pyrazole-1-yl)(3,4-dimethoxyphenyl) methyl ketone (5r)

[0103]

[0104] The preparation method was the same as in Example 1, except that 3-fluorophenylacetonitrile was replaced with 4-chlorobenzoylacetonitrile, and 3,4,5-trimethoxybenzoic acid was replaced with 3,4-dimethoxybenzoic acid. Compound 5r was a yellow powder with a yield of 30.9%. 1 H NMR(500MHz,DMSO-d6)δ7.91–7.88(m,1H),7.83(d,J=8.0Hz,2H),7.81(s,1H),7.51(d, J=8.1Hz,2H),7.15(d,J=8.5Hz,1H),6.85(s,2H),5.93(s,1H),3.87(d,J=26.9Hz,6H). 13C NMR (126MHz, DMSO) δ168.74,153.96,153.12,152.80,148.10,133.95,131. 63,129.25,128.06,126.38,125.02,114.94,111.07,85.24,56.23,56.02.

[0105] Example 18

[0106] (3-Amino-5-(3-methoxyphenyl)-1H-pyrazole-1-yl)(3,4-dimethoxyphenyl) methyl ketone (5S)

[0107]

[0108] The preparation method was the same as in Example 1, except that 3-fluorophenylacetonitrile was replaced with 4-chlorobenzoylacetonitrile, and 3,4,5-trimethoxybenzoic acid was replaced with 3,4-dimethoxybenzoic acid. Compound 5S was a white powder with a yield of 33.9%. 1 H NMR (500MHz, DMSO-d6) δ7.94–7.89(m,1H),7.87–7.84(m,1H),7.74(d,J=8.1Hz,2H),7.15(d ,J=8.4Hz,1H),7.01(d,J=8.2Hz,2H),6.80(s,2H),5.85(d,J=3.6Hz,1H),3.95–3.73(m,9H). 13 C NMR (126MHz, DMSO) δ168.60,160.39,153.82,153.76,153.01,148.04,127.73,127.46, 126.36,125.24,115.40,115.03,114.57,113.98,111.04,84.94,56.21,56.01,55.67.

[0109] Example 19

[0110] (3-Amino-5-(p-Tolyl)-1H-pyrazole-1-yl)(3,4-Dimethoxyphenyl)methyl ketone (5t)

[0111]

[0112] The preparation method was the same as in Example 1, except that 3-fluorophenylacetonitrile was replaced with 4-methylbenzoylacetonitrile, and 3,4,5-trimethoxybenzoic acid was replaced with 3,4-dimethoxybenzoic acid. 5 tons of the compound were produced as a pale yellow powder, with a yield of 38.3%. 1H NMR(500MHz,DMSO-d6)δ7.90(dd,J=8.5,2.1Hz,1H),7.85(d,J=2.1Hz,1H),7.69(d,J=8.1Hz,2H),7.25 (d,J=7.8Hz,2H),7.15(d,J=8.6Hz,1H),6.80(s,2H),5.87(s,1H),3.86(d,J=25.5Hz,6H),2.34(s,3H). 13 C NMR(151MHz,DMSO)δ167.44,154.41,153.70,151.37,150.17,149.76,149.21,129 .74,125.50,125.27,119.32,113.28,112.21,109.81,84.94,56.01,55.55,28.08.

[0113] Example 20

[0114] (3-Amino-5-(4-bromophenyl)-1H-pyrazole-1-yl)(3,4-dimethoxyphenyl)methyl ketone (5u)

[0115]

[0116] The preparation method was the same as in Example 1, except that 3-fluorophenylacetonitrile was replaced with 4-bromobenzoylacetonitrile, and 3,4,5-trimethoxybenzoic acid was replaced with 3,4-dimethoxybenzoic acid. Compound 5u was a pale yellow powder with a yield of 41.3%. 1 H NMR(500MHz,DMSO-d6)δ7.88(dd,J=8.6,2.0Hz,1H),7.80(d,J=2.1Hz,1H),7.75(d,J=8.4Hz,2H) ,7.64(d,J=8.4Hz,2H),7.14(d,J=8.5Hz,1H),6.84(s,2H),5.92(s,1H),3.86(d,J=27.7Hz,6H). 13 CNMR(126MHz,DMSO)δ164.06,153.74,153.03,150.10,149.22,147.91,126 .35,125.14,119.35,115.20,112.19,111.09,109.46,85.20,56.19,55.91.

[0117] Example 21

[0118] (3-Amino-5-(3-Chlorophenyl)-1H-pyrazole-1-yl)(3,4-dimethoxyphenyl) methyl ketone (5v)

[0119]

[0120] The preparation method was the same as in Example 1, except that 3-fluorophenylacetonitrile was replaced with 3-chlorobenzoylacetonitrile, and 3,4,5-trimethoxybenzoic acid was replaced with 3,4-dimethoxybenzoic acid. Compound 5V was a white powder with a yield of 31.3%. 1 H NMR(500MHz,DMSO-d6)δ7.88(dd,J=8.5,2.1Hz,1H),7.84(d,J=2.3Hz,2H),7.77(dt,J=7.0,1.8Hz ,1H),7.51–7.41(m,2H),7.16(d,J=8.6Hz,1H),6.86(s,2H),5.97(s,1H),3.87(d,J=22.0Hz,6H). 13 C NMR (126MHz, DMSO) δ167.65,153.92,153.44,151.31,149.66,129.72,129.14,129. 12,128.52,125.37,119.35,119.18,116.20,116.03,113.32,85.24,56.85,56.81.

[0121] Example 22

[0122] (3-Amino-5-(m-Tolyl)-1H-Pyrazol-1-yl)(3,4-Dimethoxyphenyl)methyl ketone (5w)

[0123]

[0124] The preparation method was the same as in Example 1, except that 3-fluorophenylacetonitrile was replaced with 3-methylbenzoylacetonitrile, and 3,4,5-trimethoxybenzoic acid was replaced with 3,4-dimethoxybenzoic acid. Compound 5w was a white powder with a yield of 36.1%. 1 H NMR(500MHz,DMSO-d6)δ7.89(d,J=8.7Hz,2H),7.62(s,1H),7.59(d,J=7.8Hz,1H),7.32(t,J=7.6Hz,1H),7 .21(d,J=7.5Hz,1H),7.15(d,J=8.3Hz,1H),6.81(s,2H),5.89(s,1H),3.87(d,J=19.5Hz,6H),2.35(s,3H). 13C NMR(75MHz,DMSO)δ168.67,154.22,153.82,152.40,141.63,138.63,131.74,129.20, 128.40,128.38,126.86,126.22,122.23,113.20,109.61,85.08,60.68,56.47,21.36.

[0125] Example 23 Anti-proliferation experiment on tumor cells

[0126] The antiproliferative activity of the compound described in this invention against five tumor cell lines: MDA-MB-231, MCF-7, A549, HT-29, and HeLa was tested. Methods: Cells were digested, counted, and prepared to a concentration of 5 × 10⁻⁶. 4 Add 100 μL of cell suspension per well to a 96-well plate (3-5 × 10⁶ cells / mL). 3 (cells). 96-well plates were incubated at 37°C in a 5% CO2 incubator for 24 hours. The drug was diluted to the required concentration with complete culture medium, and 100 μL of the corresponding drug-containing medium was added to each well. The 96-well plates were incubated at 37°C in a 5% CO2 incubator for 48 hours. 20 μL of MTT (5 mg / mL) was added to each well, and the plates were incubated for another 4 hours. The culture medium was discarded, and 150 μL of DMSO was added to each well to dissolve the drug. The plates were gently mixed by shaking for 10 minutes. The OD value of each well was read using a microplate reader at λ = 570 nm. The test results were analyzed using a Graphpad Prism 6. The test results for representative compounds are shown in Table 1.

[0127] Table 1. Anti-cell proliferation activity of compounds

[0128]

[0129]

[0130] These results indicate that the compounds of this invention have a significant inhibitory effect on tumor cells, and are superior to the positive control drugs paclitaxel (PTX) and Combretastatin A-4 (CA-4), suggesting their potential use in cancer treatment.

[0131] Example 24: Inhibition of Tubulin Polymerization

[0132] The inhibitory activity of compound 5b on tubulin polymerization was tested.

[0133] Methods: The compound was prepared in five concentration gradients (2, 10, 50, 250, and 1250 nM), with each concentration biologically replicated three times. 2 mg / mL tubulin (cytoskeleton) was resuspended in PEM buffer [80 mM PIPES (pH 6.9), 0.5 mM EGTA, 2 mM MgCl2] and 15% glycerol, and then pre-incubated on ice with the compound or solvent DMSO for 5 minutes. Before detecting the tubulin polymerization reaction, PEG containing GTP (guanosine triphosphate) was added to a final concentration of 3 mg / mL. Fluorescence was detected once per minute at 360 nm using a multi-functional fluorescent microplate reader, with continuous fluorescence detection at 450 nm for 60 minutes. A blank control group (0.1% DMSO + PEM) was set up. The test results were analyzed using Graphpad Prism 6. The test results are shown in Table 2.

[0134] The results show that compound 5b in this invention can effectively inhibit the aggregation of tubulin, and its inhibitory activity is comparable to that of the positive control drug CA-4, further indicating that the target of compound 5b is the colchicine site of tubulin.

[0135] Table 2. Inhibition of microtubule polymerization by the compounds

[0136]

[0137] Example 25: Inhibitory activity of compound 5b of the present invention against the growth of MCF-7 breast cancer cell xenografts in nude mice.

[0138] Seventy-two four-week-old female Balb / c nude mice, weighing 17-19g, were provided by Jiangsu Huachuang Xinno Pharmaceutical Technology Co., Ltd. Cultured TNBC breast cancer MDA-MB-231 cells were collected, centrifuged, resuspended in PBS, and counted. An equal volume of the PBS-resuspended cells was mixed with an ABW (Absorbent Polymerase Injection) mixture to adjust the cell suspension concentration to 3 × 10⁻⁶ cells / mL. 7 0.2 mL of the tumor cells were injected subcutaneously into the right axilla of each nude mouse. The long and short diameters of the xenograft tumor in the nude mice were measured using calipers, and the tumor volume was calculated as (V = length (mm) × width (mm) × width (mm) / 2). Fourteen days after tumor cell inoculation, the tumors grew to 100 mm. 3At approximately 2000 mmol / L, nude mice were randomly divided into 8 groups of 9 mice each. Paclitaxel 5b was dissolved in a mixture of 10% DMSO, 2% Tween 80, 10% PEG300, and 78% saline. The model group received an equal volume of the solvent intraperitoneally every two days for 21 days. The positive control group received 10 mg / kg paclitaxel intraperitoneally every two days for 21 days. The experimental groups received 5 mg / kg, 10 mg / kg, and 20 mg / kg of compound 5b intraperitoneally every two days for 21 days. Tumor volume and mouse weight were recorded every two days. After 21 days of treatment, the nude mice were sacrificed, and the tumors were surgically removed and weighed. The tumor growth inhibition rate (%) was calculated, and the results were analyzed using Graphpad Prism 6. Statistical analysis between groups was performed using a t-test.

[0139] Experimental results showed that compound 5b effectively inhibited tumor growth in nude mice, significantly reducing tumor volume. Figure 2 In the drug A), at doses of 5 mg / kg, 10 mg / kg, and 20 mg / kg, the tumor inhibition rates of compound 5b were 27.01%, 36.07%, and 68.95%, respectively. Figure 2 (B in the middle).

Claims

1. 3-Amino-5-phenyl-pyrazole derivatives, characterized in that, The 3-amino-5-phenyl-pyrazole derivatives are as follows: (a) (3-Amino-5-(3-fluorophenyl)-lH-pyrazol-l-yl)(3,4,5-trimethoxyphenyl)methanone (b) (3-Amino-5-(p-tolyl)-lH-pyrazol-l-yl)(3,4,5-trimethoxyphenyl)methanone (c) (3-Amino-5-(4-fluorophenyl)-lH-pyrazol-l-yl)(3,4,5-trimethoxyphenyl)methanone (d) (3-Amino-5-(3-methoxyphenyl)-lH-pyrazol-l-yl)(3,4,5-trimethoxyphenyl)methanone (e) (3-Amino-5-(3-chlorophenyl)-lH-pyrazol-l-yl)(3,4,5-trimethoxyphenyl)methanone (f) (3-Amino-5-(4-chlorophenyl)-lH-pyrazol-l-yl)(3,4,5-trimethoxyphenyl)methanone (g) (3-Amino-5-(4-bromophenyl)-lH-pyrazol-l-yl)(3,4,5-trimethoxyphenyl)methanone (h) (3-Amino-5-(3-bromophenyl)-lH-pyrazol-l-yl)(3,4,5-trimethoxyphenyl)methanone (i) (3-Amino-5-(4-methoxyphenyl)-lH-pyrazol-l-yl)(3,4,5-trimethoxyphenyl)methanone (j) (3-Amino-5-m-tolyl-lH-pyrazol-l-yl)(3,4,5-trimethoxyphenyl)methanone (k) (3-Amino-5-(3,4-dimethoxyphenyl)-lH-pyrazol-l-yl)(3,4,5-trimethoxyphenyl)methanone (l) (3-Amino-5-(3,4-dimethoxyphenyl)-lH-pyrazol-l-yl)(benzo[d][l,3]dioxol-5-yl)methanone (m) (3-Amino-5-(4-fluorophenyl)-lH-pyrazol-l-yl)(benzo[d][l,3]dioxol-5-yl)methanone (n) (3-Amino-5-(4-chlorophenyl)-lH-pyrazol-l-yl)(benzo[d][l,3]dioxol-5-yl)methanone (p) (3-Amino-5-p-tolyl-lH-pyrazol-l-yl)(benzo[d][l,3]dioxol-5-yl)methanone (r) (3-Amino-5-(4-chlorophenyl)-lH-pyrazol-l-yl)(3,4-dimethoxyphenyl)methanone (s) (3-Amino-5-(3-methoxyphenyl)-lH-pyrazol-l-yl)(3,4-dimethoxyphenyl)methanone (t) (3-Amino-5-(p-tolyl)-lH-pyrazol-l-yl)(3,4-dimethoxyphenyl)methanone (u) (3-Amino-5-(4-bromophenyl)-lH-pyrazol-l-yl)(3,4-dimethoxyphenyl)methanone (v) (3-Amino-5-(3-chlorophenyl)-lH-pyrazol-l-yl)(3,4-dimethoxyphenyl)methanone (w) (3-Amino-5-(m-tolyl)-lH-pyrazol-l-yl)(3,4-dimethoxyphenyl)methanone 2. A pharmaceutical composition, characterized by, The pharmaceutical composition comprises the 3-amino-5-phenyl-pyrazole derivative or pharmaceutically acceptable salt according to claim 1.

3. The pharmaceutical composition of claim 2, wherein, The pharmaceutically acceptable salt is selected from the group consisting of hydrochloride, hydrobromide, sulfate, phosphate, methanesulfonate, acetate, lactate, succinate, fumarate, maleate, citrate, benzoate, p-toluenesulfonate or alkali metal cation salt.

4. The pharmaceutical composition of claim 2, wherein, The pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

5. The pharmaceutical composition of claim 4, wherein, The pharmaceutically acceptable excipient is selected from one or more of pharmaceutically acceptable carriers, diluents or excipients.

6. Use of the 3-amino-5-phenyl-pyrazole derivative according to claim 1 or the pharmaceutical composition according to claim 2 for the manufacture of a tubulin inhibitor.

7. Use of the 3-amino-5-phenyl-pyrazole derivative according to claim 1 or the pharmaceutical composition according to claim 2 for the manufacture of a medicament for the treatment of a tumor.

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