Pyrazole derivative with anti-tumor activity as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biological medicines, in particular to a pyrazole derivative with antitumor activity and a preparation method and application thereof. In the invention, a novel pyrazole derivative is prepared, and the pyrazole derivative has the property of efficiently inhibiting the activity of HIF-2alpha and shows a good effect of resisting the proliferation of colorectal cancer cells at an animal level, so that a safe and efficient candidate drug molecule is provided for targeted therapy of colorectal cancer; the pyrazole derivative is simple and feasible in preparation process, low in raw material cost and good in functional group compatibility.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly to pyrazole derivatives with anti-tumor activity, their preparation methods and applications. Background Art

[0002] Traditional treatment methods for colorectal cancer (surgery, chemotherapy, radiotherapy) have limitations: surgery is difficult to remove tiny lesions, chemotherapy drugs (such as 5-FU, 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 feature of the solid tumor microenvironment, in which hypoxia-inducible factor HIF-2α promotes angiogenesis, invasion and metastasis of colorectal cancer by upregulating genes such as VEGF. Clinical studies have shown that high expression of HIF-2α is closely related to tumor malignancy, lymph node metastasis and poor prognosis, and its mechanism involves 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 has not been clarified. Certain pyrazole derivatives exhibit high inhibitory activity against HIF-2α, can block its signaling pathway and inhibit tumor progression, providing a new direction for the treatment of colorectal cancer. However, there is still a need to develop safe and effective HIF-2α inhibitors in this field to meet clinical needs. Summary of the Invention

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

[0006] In the first aspect of the present invention, there is provided a pyrazole derivative or a pharmaceutically acceptable salt thereof, and the above pyrazole derivative has the structure shown in Formula 6: ; Wherein, R is one of the following groups: , , , , , , , , ; Wherein, n is 1, 2, 3 or 4.

[0007] The pyrazole derivative represented by Formula 6 or a pharmaceutically acceptable salt thereof has the property of inhibiting HIF-2α activity and is an HIF-2α inhibitor. The pharmaceutically acceptable salt of the pyrazole derivative represented by Formula 6 can be easily prepared from the pyrazole derivative represented by Formula 6. 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.

[0008] In some alternative embodiments, the above pyrazole derivative has a structure represented by one of Formulas 6a to 6k: , , , , , , , , , , .

[0009] The second aspect of the present invention provides a method for preparing the pyrazole derivative represented by Formula 6, which includes the following steps: Under the participation of a palladium catalyst, the compound represented by Formula 1 is subjected to a hydrogenation reaction with hydrogen to obtain the compound represented by Formula 2; The compound represented by Formula 2 is subjected to an amide condensation reaction with the compound represented by Formula 3 to obtain the compound represented by Formula 4; Under the participation 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; , , , , , ; Wherein, R is one of the following groups: , , , , , , , , ; Wherein, n is 1, 2, 3 or 4.

[0010] In some alternative embodiments, the above-mentioned palladium catalyst is palladium on carbon.

[0011] In some alternative embodiments, the above-mentioned amide condensation reaction is carried out under the condition of participation of N,N-dimethylformamide.

[0012] In some alternative embodiments, the above-mentioned base is lithium hydroxide.

[0013] In some alternative embodiments, the above-mentioned hydrolysis product reacts with the compound shown in Formula 5 under the conditions of participation of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 1-hydroxybenzotriazole and N,N-diisopropylethylamine.

[0014] The third aspect of the present invention provides the use of the pyrazole derivative shown in Formula 6 or its pharmaceutically acceptable salt in the preparation of a drug for treating and / or preventing colorectal cancer.

[0015] The fourth aspect of the present invention provides a drug for treating and / or preventing colorectal cancer, which comprises the pyrazole derivative shown in Formula 6 or its pharmaceutically acceptable salt. The pyrazole derivative shown in Formula 6 or its pharmaceutically acceptable salt is the main active ingredient (main drug) in the above-mentioned drug for treating and / or preventing colorectal cancer.

[0016] In some alternative embodiments, the above-mentioned drug for treating and / or preventing colorectal cancer further comprises an excipient. The dosage form of the above-mentioned drug for treating and / or preventing colorectal cancer is any pharmaceutically acceptable dosage form. The excipient has stable properties, no incompatibility with the main drug, no side effects, does not affect the efficacy, is not easily deformed, cracked or mildewed at room temperature, and is harmless to the human body.

[0017] In some alternative embodiments, the above-mentioned excipient is at least one of gum arabic, syrup, lanolin, and starch.

[0018] The technical solution of the embodiment of the present invention has the following beneficial effects: A novel pyrazole derivative is prepared. This pyrazole derivative has the property of efficiently inhibiting the activity of HIF-2α and shows a good effect of anti-proliferation of colorectal cancer cells at the animal level, providing a safe and efficient candidate drug molecule for targeted treatment of colorectal cancer; the preparation process of this pyrazole derivative is simple and easy to implement, with low raw material cost and good functional group compatibility. Detailed Embodiments

[0019] Next, in combination with the embodiments of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts belong to the scope of protection of the present invention.

[0020] In the following embodiments, the pyrazole derivative shown in Formula 6 is synthesized according to the following route:

[0021] Among them, the definition of R is the same as that described above.

[0022] Example 1 The compound shown in Formula 6a is synthesized according to the following route: Step 1: In a 250 mL three-necked flask, dissolve methyl 4-nitro-1H-pyrazole-3-carboxylate (3.0 g, 17.53 mmol, 1.0 eq) shown in Formula 1 in 150 mL of methanol, and add palladium on carbon (10% w / w, 400 mg); displace the reaction device three times with argon, three times with hydrogen, inject hydrogen, and react at room temperature for 8 h; monitor the reaction process by thin-layer chromatography (TLC). After the reaction is complete, filter the reaction solution through diatomaceous earth and concentrate it to obtain the compound shown in Formula 2 (white solid, 2.42 g, 98%).

[0023] 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-d 6 ) δ 12.89 (s, 1H), 7.15 (s, 1H), 4.84 (s, 2H), 3.78 (s, 3H). 13 CNMR (100 MHz, DMSO-d 6 ) δ 160.9, 137.5, 128.9, 116.1, 51.4. HRMS (ESI)calculated for C 5 H 8 N 3 O 2 + [M+H] + : 142.1300, found: 142.1303. Step 2: In a 250 mL round-bottom flask, dissolve the compound shown in Formula 2 (2.4 g, 17.01 mmol, 1.0 eq) in 150 mL of dichloromethane, add a catalytic amount of N,N-dimethylformamide dropwise, and finally add 2,6-dichlorobenzoyl chloride shown in Formula 3 (3.52 g, 17.01 mmol, 1.0 eq) dropwise to the above system; monitor the reaction progress by thin-layer chromatography (TLC). After the reaction is complete, concentrate the mixture and purify it by silica gel column chromatography (PE:EA = 1:1) to obtain the compound shown in Formula 4 (white solid, 4.06 g, 76%).

[0024] 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, CDCl 3 ) δ 12.59 (s, 1H), 9.21 (s, 1H), 7.57 - 7.27 (m, 3H), 4.04 (s, 3H). 13 C NMR (100 MHz, CDCl 3 ) 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 N 3 O 3 + [M+H] + : 315.1220, found: 315.1224. Step 3: In a 50 mL round-bottom flask, dissolve the compound shown in Formula 4 (200 mg, 0.64 mmol, 1.0 eq) in a 1:1 mixture of 10 mL of tetrahydrofuran and water. Add lithium hydroxide hydrate (161 mg, 3.84 mmol, 6.0 eq). Monitor the reaction progress by thin-layer chromatography (TLC). After the reaction is complete, adjust the pH of the reaction solution to 4 by adding 1N HCl; extract with ethyl acetate (2×20 mL), collect the organic phase, dry over anhydrous sodium sulfate, filter, and concentrate under reduced pressure; dissolve the concentrated product in 10 mL of N,N-dimethylformamide, add 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 add the compound shown in Formula 5a (104 mg, 0.64 mmol, 1.0 eq); monitor the reaction progress by thin-layer chromatography (TLC). After the reaction is complete, add saturated NaCl solution (20 mL), extract with ethyl acetate (2×20 mL), collect the organic phase, dry over anhydrous sodium sulfate, filter, concentrate under reduced pressure, and purify by silica gel column chromatography to obtain the pyrazole derivative shown in Formula 6a (white solid, 146 mg, 52%).

[0025]

[0026] The compound shown in Formula 5a is 4-(pyrrolidin-1-yl)aniline.

[0027]

[0028] The characterization results of the pyrazole derivative shown in Formula 6a are as follows: M.p. 170.2 - 172.3 °C; IR (KBr): 3689, 3283, 3107, 2175, 1915, 1782, 1368, 1094, 920 cm -1 . 1 H NMR (400 MHz, CDCl 3) 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, CDCl 3 ) 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 Cl 2 N 5 O 2 + [M+H] + : 445.3160, found: 445.3164. Example 2 The compound shown in Formula 6b was synthesized according to the following route: Step 1: The same as Step 1 in Example 1.

[0029] Step 2: The same as Step 2 in Example 1.

[0030] Step 3: Replace the compound shown in Formula 5a in Step 3 of Example 1 with the compound shown in Formula 5b, and keep other operations unchanged to obtain the pyrazole derivative shown in Formula 6b (white solid, 169 mg, 60%).

[0031]

[0032] The compound shown in Formula 5b is 4-(pyrrol-1-yl)aniline.

[0033]

[0034] The characterization results of the pyrazole derivative shown in Formula 6b are as follows: M.p. 176.8 - 177.5 ℃; IR (KBr): 3686,3281, 3102, 2182, 1920, 1786, 1372, 1096, 922 cm -1 . 1 H NMR (400 MHz, CDCl3 ) δ 10.24 (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). 13 C NMR (100 MHz, DMSO - d 6 ) 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 N 5 O 2 + [M + H] + : 441.2840, found: 441.2842. Example 3 The compound shown in Formula 6c was synthesized according to the following route: Step 1: The same as Step 1 in Example 1.

[0035] Step 2: The same as Step 2 in Example 1.

[0036] Step 3: Replace the compound shown in Formula 5a in Step 3 of Example 1 with the compound shown in Formula 5c, and keep other operations unchanged to obtain the pyrazole derivative shown in Formula 6c (white solid, 171 mg, 58%).

[0037]

[0038] The compound shown in Formula 5c is N 1 -(2-(dimethylamino)ethyl)benzene - 1,4 - diamine.

[0039]

[0040] The characterization results of the pyrazole derivative shown in Formula 6c are as follows: M.p. 189.3 - 190.7 °C; IR (KBr): 3684, 3280, 2982, 2112, 1915, 1669, 1375, 1101, 921 cm-1 . 1 1H NMR (400 MHz, CD 3 OD) 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 13C NMR (100 MHz, CD 3 OD) 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 17 23 1 2 2 6 1 2 + [M+H] + : 462.3470, found: 462.3474. Example 4 The compound shown in Formula 6d was synthesized according to the following route: Step 1: The same as Step 1 in Example 1.

[0041] Step 2: The same as Step 2 in Example 1.

[0042] Step 3: Replace the compound shown in Formula 5a in Step 3 of Example 1 with the compound shown in Formula 5d, and keep other operations unchanged to obtain the pyrazole derivative shown in Formula 6d (white solid, 166 mg, 54%).

[0043]

[0044] The compound shown in Formula 5d is N 1 -(2-(dimethylamino)ethyl)-N 1 -methylbenzene-1,4-diamine.

[0045]

[0046] The characterization results of the pyrazole derivative shown in Formula 6d are as follows: M.p. 197.3 - 198.8 °C; IR (KBr): 3687, 3282, 2985, 2120, 1913, 1701, 1379, 1098, 923 cm -1 . 1 H NMR (400 MHz, CDCl 3 ) 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, CDCl 3 ) 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 Cl 2 N 6 O 2 + [M+H] + : 476.3740, found: 476.3742. Example 5 The compound shown in Formula 6e was synthesized according to the following route: Step 1: The same as Step 1 in Example 1.

[0047] Step 2: The same as Step 2 in Example 1.

[0048] Step 3: Replace the compound shown in Formula 5a in Step 3 of Example 1 with the compound shown in Formula 5e, and keep other operations unchanged to obtain the pyrazole derivative shown in Formula 6e (white solid, 186 mg, 61%).

[0049]

[0050] The compound shown in Formula 5e is 4-(3-(dimethylamino)propoxy)aniline.

[0051]

[0052] The characterization results of the pyrazole derivative shown in Formula 6e are as follows: M.p. 197.80 - 198.6 °C; IR (KBr): 3682, 3279, 2973, 2109, 1914, 1706, 1382, 1103, 922 cm -1 . 1 H NMR (400 MHz, CDCl 3 ) 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, CDCl 3 ) 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 Cl 2 N 5 O 3 + [M + H] + : 477.3580, found: 477.3582. Example 6 Synthesize the compound shown in Formula 6f according to the following route: Step 1: The same as Step 1 in Example 1.

[0053] Step 2: The same as Step 2 in Example 1.

[0054] Step 3: Replace the compound shown in Formula 5a in Step 3 of Example 1 with the compound shown in Formula 5f, and keep other operations unchanged to obtain the pyrazole derivative shown in Formula 6f (white solid, 176 mg, 57%).

[0055]

[0056] The compound shown in Formula 5f is 4-(2-(pyrrolidin-1-yl)ethoxy)aniline.

[0057]

[0058] The characterization results of the pyrazole derivative shown in Formula 6f are as follows: M.p. 196.2 - 197.3 °C; IR (KBr): 3684, 3281, 2975, 2112, 1913, 1715, 1376, 1095, 921 cm -1 . 1 1H NMR (400 MHz, CDCl 3 ) δ 9.80 (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 13C NMR (100 MHz, CDCl 3 ) 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 1H 24 1Cl 2 1N 5 1O 3 + [M + H] + : 489.3690, found: 489.3694. Example 7 The compound shown in Formula 6g was synthesized according to the following route: Step 1: The same as Step 1 in Example 1.

[0059] Step 2: The same as Step 2 in Example 1.

[0060] Step 3: Replace the compound shown in Formula 5a in Step 3 of Example 1 with the compound shown in Formula 5g, and keep other operations unchanged to obtain the pyrazole derivative shown in Formula 6g (white solid, 181 mg, 55%).

[0061]

[0062] The compound shown in Formula 5g is 4-(2-(4-methylpiperazin-1-yl)ethoxy)aniline.

[0063]

[0064] The characterization results of the pyrazole derivative shown in Formula 6g are as follows: M.p. 197.5 - 198.6 °C; IR (KBr): 3681, 3278, 2971, 2108, 1916, 1721, 1382, 1108, 924 cm -1 . 1 H NMR (400 MHz, CDCl 3 ) 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, CD 3 OD) 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 Cl 2 N 6 O 3 + [M+H] + : 518.4110,found: 519.4113. Example 8 The compound shown in Formula 6h was synthesized according to the following route: Step 1: The same as Step 1 in Example 1.

[0065] Step 2: The same as Step 2 in Example 1.

[0066] Step 3: Replace the compound shown by Formula 5a in Step 3 of Example 1 with the compound shown by Formula 5h, and keep other operations unchanged to obtain the pyrazole derivative shown by Formula 6h (white solid, 191 mg, 58%).

[0067]

[0068] The compound shown by Formula 5h is N-(2-(4-methylpiperazin-1-yl)ethyl)benzene-1,4-diamine.

[0069]

[0070] The characterization results of the pyrazole derivative shown by Formula 6h are as follows: M.p. 198.1 - 199.3 °C; IR (KBr): 3680, 3281, 2969, 2110, 1917, 1730, 1391, 1110, 921 cm -1 . 1 H NMR (400 MHz, CDCl 3 ) 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, CDCl 3 ) 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 Cl 2 N 7 O 2 + [M+H] + : 517.4270,found: 517.4271. Example 9 Synthesize the compound shown by Formula 6i according to the following route: Step 1: The same as Step 1 in Example 1.

[0071] Step 2: The same as step 2 in Example 1.

[0072] Step 3: Replace the compound shown in Formula 5a in step 3 of Example 1 with the compound shown in Formula 5i, and keep other operations unchanged to obtain the pyrazole derivative shown in Formula 6i (white solid, 185 mg, 62%).

[0073]

[0074] The compound shown in Formula 5i is 4-(2-(dimethylamino)ethoxy)aniline.

[0075]

[0076] The characterization results of the pyrazole derivative shown in Formula 6i are as follows: M.p. 190.1 - 191.70 °C; IR (KBr): 3687, 3280, 2971, 2077, 1913, 1653, 1463, 1103, 922 cm -1 . 1 H NMR (400 MHz, CDCl 3 ) 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, CDCl 3 ) 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 Cl 2 N 5 O 3 + [M + H] + : 463.3310, found: 463.3312. Example 10 Synthesize the compound shown in Formula 6j according to the following route: Step 1: The same as step 1 in Example 1.

[0077] Step 2: The same as Step 2 in Example 1.

[0078] Step 3: Replace the compound shown by Formula 5a in Step 3 of Example 1 with the compound shown by Formula 5j, and keep other operations unchanged, to obtain the pyrazole derivative shown by Formula 6j (white solid, 157 mg, 53%).

[0079]

[0080] The compound shown by Formula 5j is 2-(2-(dimethylamino)ethoxy)aniline.

[0081]

[0082] The characterization results of the pyrazole derivative shown by Formula 6j are as follows: M.p. 188.4 - 189.3 °C; IR (KBr): 3686, 3278, 2975, 2078, 1913, 1648, 1428, 1109, 921 cm -1 . 1 H NMR (400 MHz, CDCl 3 ) 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, CDCl 3 ) 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 Cl 2 N 5 O 3 + [M+H] +: 463.3310, found: 463.3313. Example 11 The compound shown in Formula 6k was synthesized according to the following route: Step 1: The same as Step 1 in Example 1.

[0083] Step 2: The same as Step 2 in Example 1.

[0084] Step 3: Replace the compound shown in Formula 5a in Step 3 of Example 1 with the compound shown in Formula 5k, and keep other operations unchanged to obtain the pyrazole derivative shown in Formula 6k (white solid, 176 mg, 59%).

[0085]

[0086] The compound shown in Formula 5k is 3-(2-(dimethylamino)ethoxy)aniline.

[0087]

[0088] The characterization results of the pyrazole derivative shown in Formula 6k are as follows: M.p. 189.6 - 190.3 °C; IR (KBr): 3687, 3269, 2970, 2074, 1915, 1688, 1435, 1107, 925 cm -1 . 1 H NMR (400 MHz, CDCl 3 ) 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, CDCl 3 ) 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 H22 Cl 2 N 5 O 3 + [M+H] + : 463.3310, found: 463.3315. Example 12 The in vitro anti-proliferative activities of the pyrazole derivatives shown in Formulas 6a - 6k against the colorectal cancer cell lines HCT116 and SW480 were evaluated by the CCK-8 assay, with 5-FU as the control group. The experimental procedure was as follows: The highest concentrations of 5-FU and the pyrazole derivatives to be tested were set at 10 μM and serially diluted by a factor of 3, with the lowest concentration set at 0 μM, resulting in a total of 10 concentration gradients. These were co-incubated with HCT116 and SW480 cells for 72 h, and the cell viability was detected by CCK-8. Data processing was performed using GraphPad Prism 9 to calculate the IC 50 value.

[0089] The results are shown in Table 1. The pyrazole derivatives shown in Formulas 6a - 6k exhibited different in vitro anti-proliferative activities against the colorectal cancer cell lines.

[0090] Table 2 Screening Results of the Anti-proliferative Activities of Pyrazole Derivatives against Cells

[0091] In Table 1, + represents greater than 1000 nM; ++ represents 1000 - 100 nM; +++ represents less than 100 nM.

[0092] Example 13 The HIF-2α inhibition rates of the pyrazole derivatives shown in Formulas 6a - 6f and Formulas 6h - 6k were tested respectively according to the following method: HCT116 cells were seeded into 6-well plates. After the cells adhered overnight, 10 nM of the pyrazole derivative to be tested was added, and the cells were cultured in an incubator at 37 °C for 24 hours; the cells were collected, the samples were lysed using a lysis buffer to release proteins, the protein concentration was determined by the BCA method, SDS loading buffer was added, and the proteins were completely denatured by boiling. Equal amounts of protein samples and molecular weight markers were added to the gel wells, and the proteins were separated at a constant voltage in an electrophoresis buffer; after membrane transfer and blocking, the primary antibody (hypoxia-inducible factor 2α / EPAS1 antibody) was added and incubated overnight at 4 °C. After washing the membrane 3 times with TBST, the secondary antibody (HRP-conjugated goat anti-rabbit IgG(H+L) antibody, diluted 1:5000) was added and incubated at room temperature for 2 hours; the ECL luminescent solution was evenly dropped onto the membrane, and the signal was detected using a gel imager or chemiluminescence detector; the inhibitory effect of the pyrazole derivative on HIF-2α protein in HCT116 cells was evaluated by Western Blot experiment.

[0093] As shown in Table 2, it can be seen that some pyrazole derivatives have good inhibitory ability on HIF-2α protein.

[0094] Table 2 Determination results of the inhibitory activity of pyrazole derivatives on HIF-2α

[0095] 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%.

[0096] Example 14 An animal experiment on a xenograft tumor model of two colorectal cancer cells (HCT116 and SW480) in BALB / c mice was carried out to evaluate the anti-tumor ability of the pyrazole derivative in vivo. A subcutaneous tumor-bearing mouse model was constructed, and the anti-tumor effects of the pyrazole derivatives shown in Formula 6a, Formula 6c, Formula 6d, Formula 6e, Formula 6h, Formula 6i, and Formula 6j on BALB / c mice were evaluated respectively. The procedure is as follows: HCT116 and SW480 were cultured in an incubator at 37 °C and 5% carbon dioxide; all mice were raised under standard specific pathogen-free (SPF) conditions. HCT116 and SW480 cells were subcutaneously injected into 6-8-week-old BALB / c mice; the mice were examined for tumor formation every day to determine the development of the tumors; when the tumors grew to a certain size, the mice were randomly divided into a control group and a drug administration group, with 5 mice in each group. The drug administration group was given intraperitoneal injection, and the dosage was 50 mpk; the tumor diameter was measured using a vernier caliper every day and the body weight of the mice was recorded; after 21 days, the tumor tissues were excised, and the tumor inhibitory effect of the compound was evaluated based on the change in tumor volume.

[0097] 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-proliferation activities.

[0098] Table 3 Results of Animal Experiments

[0099] In Table 3, + represents that the tumor inhibition rate is less than 50%, ++ represents that the tumor inhibition rate is between 50% and 80%; +++ represents that the tumor inhibition rate is greater than 80%.

[0100] As mentioned above, only the preferred embodiments of the present invention are described, and there is no limitation in any form and substance to the present invention. It should be pointed out that for those of ordinary skill in the art in the technical field, without departing from the method of the present invention, several improvements and supplements can still be made, and these improvements and supplements should also be regarded as the protection scope of the present invention. All those who are familiar with the professional technology, without departing from the spirit and scope of the present invention, when making some changes, modifications and evolutions of equivalent changes by using the technical content disclosed above, are all equivalent embodiments of the present invention; at the same time, any changes, modifications and evolutions of equivalent changes made to the above embodiments according to the substantial technology of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. A pyrazole derivative or a pharmaceutically acceptable salt thereof, characterized in that: The pyrazole derivative has a structure shown in Formula 6: ； Wherein, R is one of the following groups: 、 、 、 、 、 、 、 、 ； Wherein, n is 1, 2, 3 or 4.

2. The pyrazole derivative or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: The pyrazole derivative has a structure shown in one of Formula 6a to Formula 6k: 、 、 、 、 、 、 、 、 、 、 。 3. A method for preparing a pyrazole derivative represented by formula 6, characterized in that: The following steps are involved: 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; Allowing the compound represented by Formula 2 to undergo an amide condensation reaction with the compound represented by Formula 3 to obtain a compound represented by Formula 4; 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; 、 、 、 、 、 ； Wherein, R is one of the following groups: 、 、 、 、 、 、 、 、 ； Wherein, n is 1, 2, 3 or 4.

4. The method according to claim 3, characterized in that The palladium catalyst is palladium carbon.

5. The method according to claim 3, characterized in that: The amide condensation reaction is carried out under the condition of participation of N,N-dimethylformamide.

6. The method according to claim 3, characterized in that The base is lithium hydroxide.

7. The method according to claim 3, characterized in that The hydrolysis product reacts with the compound shown in Formula 5 under the conditions of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 1-hydroxybenzotriazole and N,N-diisopropylethylamine.

8. Use of the pyrazole derivative or a pharmaceutically acceptable salt thereof according to claim 1 in the preparation of a medicament for treating and / or preventing colorectal cancer.

9. A drug for treating and / or preventing colorectal cancer, characterized in that: The invention comprises the pyrazole derivative according to claim 1 or 2 or a pharmaceutically acceptable salt thereof.

10. The drug for treating and / or preventing colorectal cancer according to claim 9, characterized in that: The drug for treating and / or preventing colorectal cancer further comprises an excipient.

Images