Pyrazole derivatives with antitumor activity and preparation method and application thereof

Abstract

The present invention relates to the field of biomedicine technology, and more specifically to pyrazole derivatives with anti-tumor activity, their preparation methods, and applications. In the present invention, a novel pyrazole derivative is prepared that has the property of effectively inhibiting HIF-2α activity and exhibits good anti-colorectal cancer cell proliferation effects in animals, providing a safe and effective candidate drug molecule for the targeted treatment of colorectal cancer. The preparation process of the pyrazole derivative is simple and easy, with low raw material costs and good functional group compatibility.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to pyrazole derivatives with anti-tumor activity, and preparation methods and applications thereof. Background Art

[0002] Traditional treatments for colorectal cancer (surgery, chemotherapy, and radiotherapy) have limitations: surgery is difficult to remove tiny lesions, chemotherapy drugs (such as 5-FU and oxaliplatin) are prone to cause systemic toxicity and low response rates, and targeted therapy is only effective for patients with specific genotypes (KRAS, NRAS, BRAF wild type) and is prone to drug resistance.

[0003] Hypoxia is a core characteristic of the solid tumor microenvironment. The hypoxia-inducible factor (HIF-2α) promotes angiogenesis, invasion, and metastasis in colorectal cancer by upregulating genes such as VEGF. Clinical studies have shown that high HIF-2α expression is closely associated with tumor malignancy, lymph node metastasis, and poor prognosis. The mechanisms involved in this include angiogenesis regulation and tumor metabolic remodeling, leading to treatment resistance.

[0004] Although drugs targeting HIF-2α (such as Belzutifan) have been approved for the treatment of renal cancer, their suitability for colorectal cancer remains unclear. Certain pyrazole derivatives have demonstrated potent inhibitory activity against HIF-2α, blocking its signaling pathway and inhibiting tumor progression, offering new avenues for colorectal cancer treatment. However, the field still needs to develop safe and effective HIF-2α inhibitors to meet clinical needs. Summary of the Invention

[0005] In order to promote the therapeutic effect of colorectal cancer, the present invention provides a pyrazole derivative with anti-tumor activity and a preparation method and application thereof.

[0006] The first aspect of the present invention provides a pyrazole derivative or a pharmaceutically acceptable salt thereof, wherein the pyrazole derivative has a structure shown in Formula 6:

[0007] ;

[0008] Wherein, R is one of the following groups:

[0009] 、 、 、

[0010] 、 、 、

[0011] 、 、 ;

[0012] Wherein, n is 1, 2, 3 or 4.

[0013] The pyrazole derivative represented by Formula 6 or a pharmaceutically acceptable salt thereof has the property of inhibiting HIF-2α activity and is a HIF-2α inhibitor. The pharmaceutically acceptable salt of the pyrazole derivative represented by Formula 6 can be easily prepared using the pyrazole derivative represented by Formula 6 as a raw material. For example, the hydrochloride salt of the pyrazole derivative represented by Formula 6 can be prepared by reacting the pyrazole derivative represented by Formula 6 with hydrochloric acid.

[0014] In some optional embodiments, the pyrazole derivative has a structure shown in one of Formulas 6a to 6k:

[0015] 、 、

[0016] 、 、

[0017] 、 、

[0018] 、 、

[0019] 、 、

[0020] .

[0021] The second aspect of the present invention provides a method for preparing a pyrazole derivative represented by Formula 6, which comprises the following steps:

[0022] In the presence of a palladium catalyst, the compound represented by Formula 1 is subjected to a hydrogenation reaction with hydrogen to obtain a compound represented by Formula 2;

[0023] The compound represented by Formula 2 and the compound represented by Formula 3 undergo an amide condensation reaction to obtain a compound represented by Formula 4;

[0024] In the presence of a base, the compound represented by Formula 4 is hydrolyzed, and the hydrolysis product is reacted with the compound represented by Formula 5 to obtain the pyrazole derivative represented by Formula 6;

[0025] 、 、 、

[0026] 、 、 ;

[0027] Wherein, R is one of the following groups:

[0028] 、 、 、

[0029] 、 、 、

[0030] 、 、 ;

[0031] Wherein, n is 1, 2, 3 or 4.

[0032] In some optional embodiments, the palladium catalyst is palladium carbon.

[0033] In some optional embodiments, the amide condensation reaction is carried out in the presence of N,N-dimethylformamide.

[0034] In some optional embodiments, the base is lithium hydroxide.

[0035] In some optional embodiments, the hydrolysis product reacts with the compound shown in Formula 5 in the presence of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 1-hydroxybenzotriazole and N,N-diisopropylethylamine.

[0036] The third aspect of the present invention provides the use of a pyrazole derivative represented by Formula 6 or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating and / or preventing colorectal cancer.

[0037] A fourth aspect of the present invention provides a drug for treating and / or preventing colorectal cancer, comprising a pyrazole derivative represented by Formula 6 or a pharmaceutically acceptable salt thereof. The pyrazole derivative represented by Formula 6 or a pharmaceutically acceptable salt thereof is the main active ingredient (primary drug) in the drug for treating and / or preventing colorectal cancer.

[0038] In some optional embodiments, the drug for treating and / or preventing colorectal cancer further comprises an excipient. The drug for treating and / or preventing colorectal cancer can be in any pharmaceutically acceptable dosage form. The excipient is stable, has no incompatibility with the main drug, does not produce side effects, does not affect efficacy, is not easily deformed, cracked, or moldy at room temperature, and is harmless to the human body.

[0039] In some optional embodiments, the excipient is at least one of gum arabic, syrup, lanolin, and starch.

[0040] The technical solution of the embodiment of the present invention has the following beneficial effects: a new pyrazole derivative is prepared, which has the property of effectively inhibiting HIF-2α activity and exhibits good anti-colorectal cancer cell proliferation effect at the animal level, providing a safe and efficient candidate drug molecule for the targeted treatment of colorectal cancer; the preparation process of the pyrazole derivative is simple and easy, the raw material cost is low, and the functional group compatibility is good. DETAILED DESCRIPTION

[0041] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0042] In the following examples, the pyrazole derivatives shown in Formula 6 were synthesized according to the following route:

[0043]

[0044] The definition of R is the same as above.

[0045] Example 1

[0046] The compound represented by formula 6a was synthesized according to the following route:

[0047] Step 1: In a 250 mL three-necked flask, methyl 4-nitro-1H-pyrazole-3-carboxylate (3.0 g, 17.53 mmol, 1.0 eq) of Formula 1 was dissolved in 150 mL of methanol, and palladium on carbon (10% w / w, 400 mg) was added. The reaction apparatus was purged with argon three times and hydrogen three times, and hydrogen was injected. The reaction was allowed to react at room temperature for 8 h. The reaction progress was monitored by thin layer chromatography (TLC). After the reaction was complete, the reaction solution was filtered through diatomaceous earth and concentrated to obtain the compound of Formula 2 (white solid, 2.42 g, 98%).

[0048] The compound shown in Formula 2 is methyl 4-amino-1H-pyrazole-3-carboxylate, and its characterization results are as follows: 1 H NMR (400MHz, DMSO-d6) δ 12.89 (s, 1H), 7.15 (s, 1H), 4.84 (s, 2H), 3.78 (s, 3H). 13 CNMR (100 MHz, DMSO-d6) δ 160.9, 137.5, 128.9, 116.1, 51.4. HRMS (ESI)calculated for C5H8N3O2 +[M+H] + : 142.1300, found: 142.1303.

[0049] Step 2: In a 250 mL round-bottom flask, the compound represented by Formula 2 (2.4 g, 17.01 mmol, 1.0 eq) was dissolved in 150 mL of dichloromethane. A catalytic amount of N,N-dimethylformamide was added dropwise. Finally, 2,6-dichlorobenzoyl chloride represented by Formula 3 (3.52 g, 17.01 mmol, 1.0 eq) was added dropwise to the above system. The reaction progress was monitored by thin layer chromatography (TLC). After the reaction was complete, the mixture was concentrated and purified by silica gel column chromatography (PE:EA = 1:1) to obtain the compound represented by Formula 4 (white solid, 4.06 g, 76%).

[0050] The compound shown in Formula 4 is methyl 4-(((2,6-dichlorophenyl)carbonyl)amino)-1H-pyrazole-3-carboxylate, and its characterization results are as follows: 1 H NMR (400 MHz, CDCl3) δ 12.59 (s, 1H), 9.21 (s, 1H), 7.57-7.27 (m, 3H), 4.04 (s, 3H). 13 C NMR (100 MHz, CDCl3) δ 163.8, 161.8, 135.2, 132.6,131.3, 129.9, 128.3, 124.1, 122.8, 52.5. HRMS (ESI) calculated for C 12 H 10 C l2 N3O3 + [M+H] + : 315.1220, found: 315.1224.

[0051] Step 3: In a 50 mL round-bottom flask, the compound of formula 4 (200 mg, 0.64 mmol, 1.0 eq) was dissolved in 10 mL of a 1:1 mixture of tetrahydrofuran and water. Lithium hydroxide hydrate (161 mg, 3.84 mmol, 6.0 eq) was added and the reaction progress was monitored by thin layer chromatography (TLC). After the reaction was complete, 1N HCl was added to adjust the pH to 4. The mixture was extracted with ethyl acetate (2×20 mL). The organic phase was collected, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The concentrated product was dissolved in 10 mL of N,N-dimethylformamide and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI, 147 mg, 0.77 mmol, 1.2 eq), 1-hydroxybenzotriazole (HOBt, 104 mg, 0.77 mmol, 1.2 eq) and N,N-diisopropylethylamine (DIEA, 165 mg, 1.28 mmol, 2.0 eq), and finally the compound represented by Formula 5a (104 mg, 0.64 mmol, 1.0 eq) were added; the reaction progress was monitored by thin layer chromatography (TLC). After the reaction was complete, saturated NaCl solution (20 mL) was added, and the mixture was extracted with ethyl acetate (2×20 mL). The organic phase was collected, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by silica gel column chromatography to obtain the pyrazole derivative represented by Formula 6a (white solid, 146 mg, 52%).

[0052]

[0053] The compound represented by formula 5a is 4-(pyrrolidin-1-yl)aniline.

[0054]

[0055] The characterization results of the pyrazole derivative represented by Formula 6a are as follows: Mp 170.2-172.3 °C; IR (KBr): 3689, 3283, 3107, 2175, 1915, 1782, 1368, 1094, 920 cm -1 . 1 H NMR (400 MHz, CDCl3) δ10.46 (s, 1H), 9.96 (s, 1H), 8.54 (s, 1H), 8.49 (s, 1H), 7.41 (d, J = 8.9 Hz,2H), 7.38-7.28 (m, 3H), 6.53 (d, J = 8.9 Hz, 2H), 3.27 (q, J = 6.3, 4.6 Hz, 4H), 2.52-1.56 (m, 4H).13 C NMR (100 MHz, CDCl3) δ 161.8, 161.2, 135.4, 134.1,132.8, 132.6, 131.0, 128.2, 122.7, 122.4, 121.5, 111.7, 47.8, 25.5. HRMS(ESI) calculated for C 21 H 20 Cl2N5O2 + [M+H] + : 445.3160, found: 445.3164.

[0056] Example 2

[0057] The compound shown in formula 6b was synthesized according to the following route:

[0058] Step 1: Same as step 1 in Example 1.

[0059] Step 2: Same as step 2 in Example 1.

[0060] Step 3: Substitute the compound of Formula 5a in Step 3 of Example 1 with the compound of Formula 5b, and perform other operations unchanged to obtain the pyrazole derivative of Formula 6b (white solid, 169 mg, 60%).

[0061]

[0062] The compound represented by formula 5b is 4-(pyrrol-1-yl)aniline.

[0063]

[0064] The characterization results of the pyrazole derivative represented by Formula 6b are as follows: Mp 176.8-177.5 ℃; IR (KBr): 3686, 3281, 3102, 2182, 1920, 1786, 1372, 1096, 922 cm -1 . 1 H NMR (400 MHz, CDCl3) δ10.24 (s, 1H), 9.79 (s, 1H), 8.70 (s, 1H), 8.60 (s, 1H), 7.68 (d, J = 8.8 Hz, 2H), 7.39 (d, J = 8.8 Hz, 2H), 7.38-7.30 (m, 3H), 7.06 (t, J = 2.2 Hz, 2H), 6.34 (t, J = 2.2 Hz, 2H). 13C NMR (100 MHz, DMSO-d6) δ 162.3, 161.2, 136.3,136.1, 136.0, 132.3, 132.1, 131.8, 131.7, 128.8, 122.2, 122.1, 119.9, 119.4,110.7. HRMS (ESI) calculated for C 21 H 16 C l2 N5O2 + [M+H] + : 441.2840, found:441.2842.

[0065] Example 3

[0066] The compound represented by formula 6c was synthesized according to the following route:

[0067] Step 1: Same as step 1 in Example 1.

[0068] Step 2: Same as step 2 in Example 1.

[0069] Step 3: Substitute the compound of Formula 5a in Step 3 of Example 1 with the compound of Formula 5c, and perform other operations unchanged to obtain the pyrazole derivative of Formula 6c (white solid, 171 mg, 58%).

[0070]

[0071] The compound represented by formula 5c is N 1 -(2-(dimethylamino)ethyl)benzene-1,4-diamine.

[0072]

[0073] The characterization results of the pyrazole derivative represented by Formula 6c are as follows: Mp 189.3-190.7 ℃; IR (KBr): 3684, 3280, 2982, 2112, 1915, 1669, 1375, 1101, 921 cm -1 . 1 H NMR (400 MHz, CD3OD) δ8.39 (s, 1H), 7.52-7.45 (m, 3H), 7.44 (d, J = 8.8 Hz, 2H), 6.72 (d, J = 8.8Hz, 2H), 3.48 (t, J = 6.0 Hz, 2H), 3.27 (t, J = 6.0 Hz, 2H), 2.86 (s, 6H).13 CNMR (100 MHz, CD3OD) δ 163.1, 162.0, 145.1, 135.3, 132.1, 131.6, 131.4,128.2, 128.1, 122.5, 121.8, 121.3, 113.0, 56.6, 42.5, 38.9. HRMS (ESI)calculated for C 21 H 23 Cl2N6O2 + [M+H] + : 462.3470, found: 462.3474.

[0074] Example 4

[0075] The compound represented by formula 6d was synthesized according to the following route:

[0076] Step 1: Same as step 1 in Example 1.

[0077] Step 2: Same as step 2 in Example 1.

[0078] Step 3: Substitute the compound of Formula 5a in Step 3 of Example 1 with the compound of Formula 5d, and perform other operations unchanged to obtain the pyrazole derivative of Formula 6d (white solid, 166 mg, 54%).

[0079]

[0080] The compound represented by formula 5d is N 1 -(2-(dimethylamino)ethyl)-N 1 -methylbenzene-1,4-diamine.

[0081]

[0082] The characterization results of the pyrazole derivative represented by Formula 6d are as follows: Mp 197.3-198.8 ℃; IR (KBr): 3687, 3282, 2985, 2120, 1913, 1701, 1379, 1098, 923 cm -1 . 1H NMR (400 MHz, CDCl3) δ9.91 (s, 1H), 8.56 (s, 1H), 8.48 (s, 1H), 7.40 (d, J = 8.8 Hz, 2H), 7.36-7.23(m, 3H), 6.72 (d, J = 8.8 Hz, 2H), 3.56 (t, J = 7.4 Hz, 2H), 2.89 (s, 3H), 2.72 (t, J = 7.4 Hz, 2H), 2.47 (s, 6H). 13 C NMR (100 MHz, CDCl3) δ 161.7,161.7, 146.5, 135.5, 133.3, 132.6, 131.0, 128.2, 126.8, 122.6, 122.3, 121.8,113.0, 55.6, 50.3, 45.1, 38.9. HRMS (ESI) calculated for C 22 H 25 Cl2N6O2 + [M+H] + :476.3740, found: 476.3742.

[0083] Example 5

[0084] The compound represented by formula 6e was synthesized according to the following route:

[0085] Step 1: Same as step 1 in Example 1.

[0086] Step 2: Same as step 2 in Example 1.

[0087] Step 3: Substitute the compound of Formula 5e for the compound of Formula 5a in Step 3 of Example 1, and perform other operations unchanged to obtain the pyrazole derivative of Formula 6e (white solid, 186 mg, 61%).

[0088]

[0089] The compound represented by formula 5e is 4-(3-(dimethylamino)propoxy)aniline.

[0090]

[0091] The characterization results of the pyrazole derivative represented by Formula 6e are as follows: Mp 197.80-198.6 °C; IR (KBr): 3682, 3279, 2973, 2109, 1914, 1706, 1382, 1103, 922 cm-1 . 1 H NMR (400 MHz, CDCl3) δ 9.86 (s, 1H), 8.54 (s, 2H), 7.36 (d, J = 8.7 Hz, 2H), 7.35-7.26 (m,3H), 6.79 (d, J = 8.7 Hz, 2H), 3.97 (t, J = 6.1 Hz, 2H), 2.85 (t, J = 6.4 Hz, 2H), 2.57 (s, 6H), 2.13 (m, J = 6.4 Hz, 2H). 13 C NMR (100 MHz, CDCl3) δ 161.7,161.6, 155.7, 135.4, 133.0, 132.6, 131.1, 130.2, 128.2, 122.7, 122.2, 122.1,114.7, 65.5, 55.9, 44.4, 26.1. HRMS (ESI) calculated for C 22 H 24 Cl2N5O3 + [M+H] + :477.3580, found: 477.3582.

[0092] Example 6

[0093] The compound represented by formula 6f was synthesized according to the following route:

[0094] Step 1: Same as step 1 in Example 1.

[0095] Step 2: Same as step 2 in Example 1.

[0096] Step 3: Substitute the compound of Formula 5a in Step 3 of Example 1 with the compound of Formula 5f and perform other operations unchanged to obtain the pyrazole derivative of Formula 6f (white solid, 176 mg, 57%).

[0097]

[0098] The compound represented by formula 5f is 4-(2-(pyrrolidin-1-yl)ethoxy)aniline.

[0099]

[0100] The characterization results of the pyrazole derivative represented by Formula 6f are as follows: Mp 196.2-197.3 °C; IR (KBr): 3684, 3281, 2975, 2112, 1913, 1715, 1376, 1095, 921 cm -1 . 1 H NMR (400 MHz, CDCl3) δ9.80 (s, 1H), 8.55 (s, 1H), 8.47 (s, 1H), 7.43 (d, J = 8.9 Hz, 2H), 7.38-7.29(m, 3H), 6.83 (d, J = 8.9 Hz, 2H), 4.42-4.23 (m, 2H), 3.37-3.28 (m, 2H), 3.23 (s, 4H), 2.09 (s, 4H). 13 C NMR (100 MHz, CDCl3) δ 161.6, 154.6, 135.5, 132.6,132.5, 131.0, 131.0, 128.2, 128.2, 122.7, 122.1, 114.8, 64.3, 54.8, 54.4,23.3. HRMS (ESI) calculated for C 23 H 24 Cl2N5O3 + [M+H] + : 489.3690, found:489.3694.

[0101] Example 7

[0102] The compound shown in formula 6g was synthesized according to the following route:

[0103] Step 1: Same as step 1 in Example 1.

[0104] Step 2: Same as step 2 in Example 1.

[0105] Step 3: Substitute the compound of Formula 5a in Step 3 of Example 1 with the compound of Formula 5g, and perform other operations unchanged to obtain the pyrazole derivative of Formula 6g (white solid, 181 mg, 55%).

[0106]

[0107] The compound represented by formula 5g is 4-(2-(4-methylpiperazin-1-yl)ethoxy)aniline.

[0108]

[0109] The characterization results of the pyrazole derivative represented by Formula 6g are as follows: Mp 197.5-198.6 °C; IR (KBr): 3681, 3278, 2971, 2108, 1916, 1721, 1382, 1108, 924 cm -1 . 1 H NMR (400 MHz, CDCl3) δ9.84 (s, 1H), 8.61 (s, 1H), 8.55 (s, 1H), 7.43 (d, J = 8.6 Hz, 2H), 7.38-7.27(m, 3H), 6.81 (d, J = 8.6 Hz, 2H), 4.09 (t, J = 4.9 Hz, 2H), 2.98-2.84 (m,10H), 2.58 (s, 3H). 13 C NMR (100 MHz, CD3OD) δ 162.0, 161.9, 155.7, 135.2,133.3, 132.0, 131.4, 130.8, 128.1, 122.2, 121.9, 121.4, 114.4, 65.3, 56.6,54.0, 52.3, 44.2. HRMS (ESI) calculated for C 24 H 27 Cl2N6O3 + [M+H] + : 518.4110,found: 519.4113.

[0110] Example 8

[0111] The compound represented by formula 6h was synthesized according to the following route:

[0112] Step 1: Same as step 1 in Example 1.

[0113] Step 2: Same as step 2 in Example 1.

[0114] Step 3: Substitute the compound of Formula 5a in Step 3 of Example 1 with the compound of Formula 5h, and perform other operations unchanged to obtain the pyrazole derivative of Formula 6h (white solid, 191 mg, 58%).

[0115]

[0116] The compound represented by formula 5h is N-(2-(4-methylpiperazin-1-yl)ethyl)benzene-1,4-diamine.

[0117]

[0118] The characterization results of the pyrazole derivative represented by Formula 6h are as follows: Mp 198.1-199.3 °C; IR (KBr): 3680, 3281, 2969, 2110, 1917, 1730, 1391, 1110, 921 cm -1 . 1 H NMR (400 MHz, CDCl3) δ9.94 (s, 1H), 8.68 (s, 1H), 8.49 (s, 1H), 7.34 (d, J = 8.4 Hz, 2H), 7.34-7.23(m, 3H), 6.56 (d, J = 8.4 Hz, 2H), 3.15 (t, J = 5.9 Hz, 2H), 2.80-2.64 (m,10H), 2.47 (s, 3H). 13 C NMR (100 MHz, CDCl3) δ 161.7, 161.6, 145.7, 135.4,133.2, 132.5, 131.1, 128.2, 127.2, 122.6, 122.5, 121.9, 113.1, 56.3, 54.3,51.5, 45.0, 40.4. HRMS (ESI) calculated for C 24 H 28 Cl2N7O2 + [M+H] + : 517.4270,found: 517.4271.

[0119] Example 9

[0120] The compound represented by formula 6i was synthesized according to the following route:

[0121] Step 1: Same as step 1 in Example 1.

[0122] Step 2: Same as step 2 in Example 1.

[0123] Step 3: Substitute the compound of Formula 5a in Step 3 of Example 1 with the compound of Formula 5i, and perform other operations unchanged to obtain the pyrazole derivative of Formula 6i (white solid, 185 mg, 62%).

[0124]

[0125] The compound represented by formula 5i is 4-(2-(dimethylamino)ethoxy)aniline.

[0126]

[0127] The characterization results of the pyrazole derivative represented by Formula 6i are as follows: Mp 190.1-191.70 °C; IR (KBr): 3687, 3280, 2971, 2077, 1913, 1653, 1463, 1103, 922 cm -1 . 1 H NMR (400 MHz, CDCl3) δ 9.81 (s, 1H), 8.55 (s, 1H), 8.44 (s, 1H), 7.45 (d, J = 8.7 Hz, 2H), 7.40-7.30 (m, 3H), 6.88 (d, J = 8.8 Hz, 2H), 4.21 (t, J = 5.1 Hz, 2H), 2.98 (s, 2H), 2.56 (s, 6H). 13 C NMR (100 MHz, CDCl3) δ 161.7, 161.6, 154.9, 135.5,133.1, 132.6, 132.6, 131.1, 130.8, 128.2, 122.7, 122.0, 114.9, 64.4, 57.6,44.8. HRMS (ESI) calculated for C 21 H 22 Cl2N5O3 + [M+H] + : 463.3310, found:463.3312.

[0128] Example 10

[0129] The compound of formula 6j was synthesized according to the following route:

[0130] Step 1: Same as step 1 in Example 1.

[0131] Step 2: Same as step 2 in Example 1.

[0132] Step 3: Substitute the compound of Formula 5a in Step 3 of Example 1 with the compound of Formula 5j, and perform other operations unchanged to obtain the pyrazole derivative of Formula 6j (white solid, 157 mg, 53%).

[0133]

[0134] The compound represented by formula 5j is 2-(2-(dimethylamino)ethoxy)aniline.

[0135]

[0136] The characterization results of the pyrazole derivative represented by Formula 6j are as follows: Mp 188.4-189.3 °C; IR (KBr): 3686, 3278, 2975, 2078, 1913, 1648, 1428, 1109, 921 cm -1 . 1 H NMR (400 MHz, CDCl3) δ9.83 (s, 1H), 9.52 (s, 1H), 8.49 (s, 1H), 8.25 (d, J = 7.8 Hz, 1H), 7.41-7.29(m, 3H), 7.08 (t, J = 7.9 Hz, 1H), 7.00 (t, J = 7.8 Hz, 1H), 6.93 (d, J = 8.1Hz, 1H), 4.31 (t, J = 4.6 Hz, 2H), 3.10 (t, J = 4.6 Hz, 2H), 2.63 (s, 6H). 13 CNMR (100 MHz, CDCl3) δ 161.7, 147.6, 135.4, 133.1, 132.6, 131.1, 128.2,127.2, 124.5, 122.6, 122.1, 121.8, 121.0, 111.8, 65.4, 57.5, 44.9. HRMS (ESI)calculated for C 21 H 22 Cl2N5O3 + [M+H] + : 463.3310, found: 463.3313.

[0137] Example 11

[0138] The compound represented by formula 6k was synthesized according to the following route:

[0139] Step 1: Same as step 1 in Example 1.

[0140] Step 2: Same as step 2 in Example 1.

[0141] Step 3: Substitute the compound of Formula 5a in Step 3 of Example 1 with the compound of Formula 5k, and perform other operations unchanged to obtain the pyrazole derivative of Formula 6k (white solid, 176 mg, 59%).

[0142]

[0143] The compound represented by formula 5k is 3-(2-(dimethylamino)ethoxy)aniline.

[0144]

[0145] The characterization results of the pyrazole derivative represented by Formula 6k are as follows: Mp 189.6-190.3 ℃; IR (KBr): 3687, 3269, 2970, 2074, 1915, 1688, 1435, 1107, 925 cm -1 . 1 H NMR (400 MHz, CDCl3) δ9.74 (s, 1H), 8.73 (s, 1H), 8.50 (s, 1H), 7.36 (d, J = 2.7 Hz, 1H), 7.35-7.28(m, 3H), 7.17 (t, J = 8.1 Hz, 1H), 7.07 (d, J = 8.1 Hz, 1H), 6.60 (d, J = 5.5Hz, 1H), 4.23 (t, J = 5.0 Hz, 2H), 3.05 (t, J = 5.0 Hz, 2H), 2.62 (s, 6H). 13 CNMR (100 MHz, CDCl3) δ 161.7, 161.7, 158.6, 138.4, 135.4, 132.6, 131.1,129.9, 128.2, 122.7, 122.1, 113.0, 110.5, 106.7, 64.4, 57.7, 45.0. HRMS (ESI)calculated for C 21 H 22 Cl2N5O3 + [M+H] + : 463.3310, found: 463.3315.

[0146] Example 12

[0147] The in vitro antiproliferative activity of the pyrazole derivatives represented by Formulas 6a to 6k against colorectal cancer cell lines HCT116 and SW480 was evaluated using a CCK-8 assay, with 5-FU serving as a control group. The experimental procedure is as follows:

[0148] The highest concentration of 5-FU and the pyrazole derivative to be tested was set to 10 μM, and the dilution was performed in a 3-fold gradient, with the lowest concentration set to 0 μM. A total of 10 concentration gradients were used to incubate HCT116 and SW480 cells for 72 h. Cell viability was detected by CCK-8, and data were processed using GraphPad Prism 9 to calculate the IC 50 value.

[0149] The results are shown in Table 1. The pyrazole derivatives represented by Formula 6a to Formula 6k showed different in vitro antiproliferative activities against colorectal cancer cell lines.

[0150] Table 2 Results of screening for antiproliferative activity of pyrazole derivatives on cells

[0151]

[0152] In Table 1, + represents greater than 1000 nM; ++ represents 1000-100 nM; and +++ represents less than 100 nM.

[0153] Example 13

[0154] The HIF-2α inhibition rate of the pyrazole derivatives represented by Formula 6a to Formula 6f and Formula 6h to Formula 6k was tested respectively according to the following method:

[0155] HCT116 cells were seeded into 6-well plates and allowed to adhere overnight. 10 nM of the pyrazole derivative to be tested was added and the cells were incubated in a 37°C incubator for 24 hours. The cells were harvested and disrupted with lysis buffer to release protein. The protein concentration was determined by the BCA assay. SDS loading buffer was added and the proteins were completely denatured by boiling. Equal amounts of protein sample and molecular weight marker were added to the gel wells, and the proteins were separated at constant pressure in electrophoresis buffer. After transfer and blocking, the membrane was incubated with primary antibody (hypoxia-inducible factor 2α / EPAS1 antibody) and incubated overnight at 4°C. The membrane was washed three times with TBST and then incubated with secondary antibody (HRP-conjugated goat anti-rabbit IgG (H+L) antibody, dilution 1:5000) at room temperature for 2 hours. ECL luminescent solution was evenly applied to the membrane, and the signal was detected using a gel imager or chemiluminescence instrument. The inhibitory effect of pyrazole derivatives on HIF-2α protein in HCT116 cells was evaluated by Western blotting.

[0156] The results are shown in Table 2, which shows that some pyrazole derivatives have good inhibitory ability on HIF-2α protein.

[0157] Table 2 Determination of the inhibitory activity of pyrazole derivatives on HIF-2α

[0158]

[0159] In Table 2, +++ represents an inhibition rate greater than 80%, ++ represents an inhibition rate between 50% and 80%, and + represents an inhibition rate less than 50%.

[0160] Example 14

[0161] Animal experiments were conducted using two colorectal cancer cell xenograft models (HCT116 and SW480) in BALB / c mice to evaluate the anti-tumor activity of pyrazole derivatives in vivo. Subcutaneous tumor-bearing mouse models were established to evaluate the anti-tumor effects of pyrazole derivatives represented by Formula 6a, Formula 6c, Formula 6d, Formula 6e, Formula 6h, Formula 6i, and Formula 6j in BALB / c mice. The following procedures were used:

[0162] HCT116 and SW480 were cultured in a 37°C, 5% carbon dioxide incubator. All mice were housed under standard specific pathogen-free (SPF) conditions. 6- to 8-week-old BALB / c mice were subcutaneously injected with HCT116 and SW480 cells. The mice were examined daily for tumor formation to determine tumor progression. When the tumors grew to a certain size, the mice were randomly divided into a control group and a treatment group, with 5 mice in each group. The treatment group received an intraperitoneal injection of 50 mpk of the drug. Tumor diameters were measured daily using a vernier caliper and mouse weights were recorded. Tumor tissue was removed 21 days later, and the compound's anti-tumor effect was evaluated by changes in tumor volume.

[0163] The results are shown in Table 3. The pyrazole derivatives represented by Formula 6a, Formula 6c, Formula 6d, Formula 6e, Formula 6h, Formula 6i and Formula 6j all have excellent in vivo anti-proliferative activity.

[0164] Table 3 Animal experiment results

[0165]

[0166] In Table 3, + represents a tumor inhibition rate of less than 50%, ++ represents a tumor inhibition rate of 50% to 80%, and +++ represents a tumor inhibition rate of more than 80%.

[0167] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form or substance. It should be pointed out that ordinary technicians in this technical field can make several improvements and supplements without departing from the method of the present invention. These improvements and supplements should also be regarded as the scope of protection of the present invention. Any equivalent changes, modifications and evolutions made by technicians familiar with this profession without departing from the spirit and scope of the present invention by using the technical content disclosed above are all equivalent embodiments of the present invention; at the same time, any equivalent changes, modifications and evolutions made to the above embodiments based on the essential technology of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. Use of a pyrazole derivative or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating and / or preventing colon cancer, characterized in that: The pyrazole derivative has a structure shown in Formula 6a, Formula 6c, Formula 6d or Formula 6h: 、 、 、 。 2. A drug for treating and / or preventing colon cancer, characterized in that: The invention comprises a pyrazole derivative or a pharmaceutically acceptable salt thereof, wherein the pyrazole derivative has a structure shown in Formula 6a, Formula 6c, Formula 6d or Formula 6h: 、 、 、 。 3. The drug for treating and / or preventing colon cancer according to claim 2, characterized in that The drug for treating and / or preventing colon cancer further comprises an excipient.

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