CN104725319A - 1H-indazole-3-aminobphenyl urea compound with anti-tumor activity as well as preparation method and application of 1H-indazole-3-aminobphenyl urea compound - Google Patents

Abstract

The present invention provides a 1H-indazole-3-aminobiphenylurea compound with anti-tumor activity and its preparation method and application. The structural formula of the compound is Wherein _R1 is hydrogen, alkane group, halogenated alkane or halogen, _R2 is hydrogen, alkane group, alkoxyl group, halogenated alkane, halogen or tertiary amine moiety, A ring is benzene ring, pyridine ring, thiazole ring or benzodiazepines The oxolene ring and the urea structure are located at the meta or para position of the benzene ring. The compound can be prepared through a four-step organic synthesis reaction, and has the advantages of simple operation of the reaction process, readily available raw materials, mild reaction conditions, cheap reagents, etc., and is suitable for large-scale production of pharmaceutical companies. The compound has good inhibitory activity on VEGFR-2 kinase, and can inhibit the proliferation activity of tumor cells, can be used for the preparation of antitumor drugs and drugs for inhibiting VEGFR-2 kinase activity, and has good application prospects and scientific research value.

Description

A kind of 1H-indazole-3-aminobiphenylurea compound with antitumor activity and its preparation method and application

technical field

The invention relates to the technical field of biomedicine, relates to an anti-tumor compound, in particular to a 1H-indazole-3-aminobiphenylurea compound with anti-tumor activity and its preparation method and application.

Background technique

Malignant tumors, as one of the largest public health problems in the world, have greatly endangered human health and will become the number one killer of human beings in the new century. Malignant tumors are no longer just a serious disease in developed industrial countries, and developing countries are facing a greater burden of disease.

Chemotherapy, as one of the important means of treating tumors, has undergone tremendous development and progress in the past three decades, and a large number of clinical antitumor drugs with different mechanisms of action have been obtained. However, antineoplastic drugs also have many adverse reactions, such as hair loss, vomiting, bone marrow suppression, rapid drug resistance, etc., which lead to the inability of chemical drugs to achieve the expected therapeutic effect. Therefore, the research and development of new antitumor drugs is one of the hot and difficult issues in the field of pharmacy.

Contents of the invention

The object of the present invention is to provide a 1H-indazole-3-aminobiphenylurea compound with antitumor activity and its preparation method and application. The compound exhibits good antitumor activity in vitro and can be applied to antitumor Preparation of tumor drugs.

The present invention is achieved through the following technical solutions:

A 1H-indazole-3-aminobiphenylurea compound with antitumor activity, its structural formula is as follows:

Wherein _R1 is hydrogen, alkane group, halogenated alkane or halogen, _R2 is hydrogen, alkane group, alkoxyl group, halogenated alkane, halogen or tertiary amine moiety, A ring is benzene ring, pyridine ring, thiazole ring or benzodiazepines The oxolene ring and the urea structure are located at the meta or para position of the benzene ring.

The tertiary amine segment is dimethylaminomethyl, diethylaminomethyl, piperidinylmethyl or dimethylaminoethoxy substituted with fluorine on the benzene ring.

The R ₁ is H, 4-F, 4-CH ₃ , 6-CH ₃ , 4-F, 4-Cl, 5-CF ₃ or 3-F.

The R ₂ is 2-F, 3-F, H, 2-Cl, 3-CH(CH ₃ ) ₂ , 3-CF ₃ , 3-OCH ₃ , 2-CH ₃ , 3-Cl, 4- 4- 4- or 4-

A preparation method of 1H-indazole-3-aminobiphenylurea compounds with antitumor activity, comprising the following steps:

1) 2-fluoro-6-iodobenzonitrile reacts with hydrazine hydrate under the action of sodium bicarbonate to obtain 4-iodo-1H-indazol-3-amine;

2) reacting 3-aminophenylboronic acid with pinacol under the catalysis of magnesium sulfate to obtain 3-aminophenylboronic acid pinacol ester;

3) forming an isocyanate with aniline containing a substituent group and triphosgene, and then condensing with 3-aminophenylboronic acid pinacol ester to obtain a urea compound containing phenylboronic acid pinacol ester;

4) Under the catalysis of tetrakis(triphenylphosphine) palladium, the urea compound containing phenylboronic acid pinacol ester and 4-iodo-1H-indazol-3-amine were reacted by Suzuki to generate antitumor active 1H-indazole-3-aminobiphenylurea compounds.

Application of the 1H-indazole-3-aminobiphenylurea compound with antitumor activity in the preparation of a drug for inhibiting VEGFR-2 kinase activity.

Application of 1H-indazole-3-aminobiphenylurea compounds with antitumor activity in the preparation of antitumor drugs.

The anti-tumor drugs are drugs targeting VEGFR-2 kinase, including anti-lung cancer, anti-liver cancer, anti-gastric cancer, anti-leukemia, anti-nerve cancer, anti-colon cancer, anti-breast cancer, anti-cervical cancer and anti-pancreatic cancer Drug.

The antitumor drug is a drug that inhibits tumor cell proliferation.

The antitumor drug is a drug for inhibiting the proliferative activity of lung cancer cell A549 and liver cancer cell SMCC-7721.

Compared with the prior art, the present invention has the following beneficial effects:

The 1H-indazole-3-aminobiphenylurea compound with antitumor activity provided by the present invention is a novel compound with antitumor activity obtained through chemical synthesis, which has a good effect on VEGFR-2 kinase Inhibitory activity, and can inhibit the proliferative activity of tumor cells. Scientific research has found that angiogenesis is closely related to the occurrence, development and migration of tumors. Inhibiting the formation of angiogenesis can effectively inhibit the growth and migration of tumors. Many growth factors are involved in the regulation of angiogenesis, among which VEGFR-2 It is the strongest positive regulator known. The 1H-indazole-3-aminobiphenylurea compound with anti-tumor activity provided by the present invention can inhibit the proliferation and migration of tumor cells by inhibiting the activity of VEGFR-2 kinase and blocking the signal pathway induced by it. The 1H-indazole-3-aminobiphenylurea compound with antitumor activity provided by the invention can be used in the preparation of antitumor drugs and drugs for inhibiting VEGFR-2 kinase activity, and has good application prospects and scientific research value.

The preparation method of 1H-indazole-3-aminobiphenylurea compounds with anti-tumor activity provided by the present invention can obtain the target compound through four steps of conventional organic synthesis reactions, and has the advantages of simple operation in the reaction process and easy raw materials It has the advantages of mild reaction conditions, cheap reagents, etc., and is suitable for large-scale production in pharmaceutical companies.

In addition, the 1H-indazole-3-aminobiphenylurea compounds with antitumor activity provided by the present invention can treat tumor cells including human non-small cell lung cancer cells (A549) and liver cancer cells (SMCC-7721). Inhibiting its cell proliferation activity can be applied to the preparation of antitumor drugs.

Description of drawings

Fig. 1 is a synthetic route diagram of 1H-indazole-3-aminobiphenylurea compounds with antitumor activity;

Wherein compound 1 is 2-fluoro-6-iodobenzonitrile, compound 2 is 4-iodo-1H-indazol-3-amine, compound 3 is 3-aminophenylboronic acid, compound 4 is 3-aminophenylboronic acid pina alcohol ester; compound 5 is a urea compound containing pinacol phenylboronic acid ester, and compound 6 is a 1H-indazole-3-aminobiphenylurea compound with antitumor activity. Specifically marked in the figure are: a: EtOH, NaHCO ₃ , NH ₂ NH ₂ ·H ₂ O; b: MgSO ₄ , THF; c: BTC, Et ₃ N, DCM: d: Pd(PPh ₃ ) ₄ , Na ₂ CO ₃ , H ₂ O, dioxane.

Detailed ways

The invention provides a 1H-indazole-3-aminobiphenylurea compound with antitumor activity. The 1H-indazole-3-aminobiphenylurea compound exhibits antitumor activity in vitro and can be used in antitumor Preparation of tumor drugs.

The present invention will be described in detail below in conjunction with the accompanying drawings and embodiments, which are explanations rather than limitations of the present invention.

The 1H-indazole-3-aminobiphenylurea compound with antitumor activity provided by the present invention has a chemical structural formula of:

Wherein, R ₁ and R ₂ are respectively one of hydrogen, alkane group, alkoxy group, halogenated alkane, halogen or tertiary amine segment, and ring A is a benzene ring, pyridine ring, thiazole ring or benzodioxol ring, The urea structure is located at the meta or para position of the benzene ring. The tertiary amine segment is dimethylaminomethyl, diethylaminomethyl, piperidinylmethyl or dimethylaminoethoxy substituted with fluorine on the benzene ring.

Specifically, R ₁ is H, 4-F, 4-CH ₃ , 6-CH ₃ , 4-F, 4-Cl, 5-CF ₃ or 3-F, R ₂ is 2-F, 3-F, H, 2-Cl, 3-CH(CH ₃ ) ₂ , 3-CF ₃ , 3-OCH ₃ , 2-CH ₃ , 3-Cl, 4- 4- 4- or 4-

The preparation method and activity screening method of the candidate drug 1H-indazole-3-aminobiphenylurea compound with anti-tumor activity of the present invention will be described in detail below in conjunction with the synthetic route shown in Figure 1 and specific synthetic examples .

Example 1

In the structural formula of this compound, ring A is a benzene ring, _R1 is 4-fluoro, _R2 is hydrogen, and the urea structure is located at the meta-position of the benzene ring, which is prepared by the following steps (see Figure 1):

1) Preparation of compound 4-iodo-1H-indazol-3-amine (compound 2) from 2-fluoro-6-iodobenzonitrile (compound 1)

Dissolve 10g of 2-fluoro-6-iodobenzonitrile (compound 1) and 5.2g of sodium bicarbonate in 60ml of ethanol, add 12.5ml of hydrazine hydrate, heat to reflux for 8 hours, cool to 25°C, add 60ml of water, and stir at 25°C for 2 hours , stirred at 0°C for 2 hours. The reaction solution was suction-filtered and dried to obtain 8.6 g of yellow-white solid compound 4-iodo-1H-indazol-3-amine (compound 2), yield: 83%;

2) Preparation of compound 3-aminophenylboronic acid pinacol ester (compound 4) by reacting 3-aminophenylboronic acid (compound 3) with pinacol

Add 20g of 3-aminophenylboronic acid (compound 3), 19.2g of pinacol, and 46.6g of anhydrous magnesium sulfate into a 500ml round bottom flask, add 250ml of tetrahydrofuran, stir at room temperature for 5 hours, filter, and use saturated sodium chloride for the organic phase Wash twice and dry over anhydrous sodium sulfate. After spin-drying the liquid, 21.5 g of yellowish-brown crude product 3-aminophenylboronic acid pinacol ester (compound 4) was obtained, with a yield of 76%;

3) Preparation of 1-(4-fluorophenyl)-3-(3-(4,4,5,5-tetramethyl) by condensation reaction of 3-aminophenylboronic acid pinacol ester (compound 4) with p-fluoroaniline -1,3,2-dioxaborolan-2-yl)phenyl)urea (Compound 5)

Under ice-bath condition, dissolve 0.80g (2.7mmol) bis(trichloromethyl)carbonate (BTC) with 20mL redistilled dichloromethane and stir for 5min, then slowly add 0.76g (6.8mmol) p-fluoroaniline dropwise After the dropwise addition, stir for 15 minutes, continue to drop 10 mL of 0.83 mL (8.2 mmol) of triethylamine in dichloromethane to the turbid solution, continue to stir for 15 minutes after the dropwise addition, and then add dropwise to the reaction solution 1.50g (6.8mmol) 3-aminophenylboronic acid pinacol ester (compound 4) and 0.83mL (8.2mmol) triethylamine dichloromethane solution 10mL, after the dropwise addition, continue to stir for 20min, the reaction solution is sequentially washed with saturated Sodium bicarbonate solution, washed with saturated brine, dried over anhydrous sodium sulfate, and spin-dried to obtain a red residue, which was separated by chromatography to obtain a light yellow solid 1-(4-fluorophenyl)-3-(3-(4, 4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)urea (compound 5) 1.06g, yield 43.3%;

4) Compound 4-iodo-1H-indazol-3-amine (compound 2) and 1-(4-fluorophenyl)-3-(3-(4,4,5,5-tetramethyl-1, 3,2-Dioxaborolan-2-yl)phenyl)urea (compound 5) to prepare 1-(3-(3-amino-1H-indazol-4-yl)phenyl) by Suzuki coupling reaction -3-(4-Fluorophenyl)urea (Compound 6)

1-(4-fluorophenyl)-3-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)urea (compound 5) 1.0 g (2.8mmol), 4-iodo-1H-indazol-3-amine (compound 2) 0.7g (2.7mmol), sodium carbonate 0.7g (6.6mmol), tetrakis (triphenylphosphine) palladium 0.3g (0.26 mmol) was dissolved in 20mL 1,4-dioxane and 5ml water, and reacted overnight at 100°C under nitrogen protection. Cool to room temperature, filter with suction, wash the filter cake with 1,4-dioxane, collect the filtrate, and spin dry to obtain a yellow residue, which is separated by column chromatography (the elution solvent is petroleum ether: ethyl acetate = 1:1) Obtained 0.56 g of white solid 1-(3-(3-amino-1H-indazol-4-yl)phenyl)-3-(4-fluorophenyl)urea (compound 6), with a yield of 56%;

The compound structure obtained is shown in the following formula:

Physical and chemical properties: mp: 204～206℃, MS(ESI)[MH] ⁺ :m/z＝360.10

Proton NMR data: ¹ H NMR (400MHz, DMSO) δ11.77 (s, 1H), 8.85 (s, 1H), 8.77 (s, 1H), 7.62 (s, 1H), 7.48 (dd, J =5.7,3.5Hz,1H),7.45(dd,J=6.8,2.6Hz,1H),7.41(d,J=8.1Hz,1H),7.30(d,J=3.9Hz,2H),7.12(s ,1H),7.10(d,J=3.6Hz,1H),6.84–6.80(m,1H),4.48(s,2H).

Example 2

In the structural formula of the compound, ring A is a benzene ring, R ₁ is hydrogen, R ₂ is 3-isopropyl, and the urea structure is located at the meta-position of the benzene ring.

Steps 1) to 3) are the same as steps 1) to 3) in Example 1, except that in step 3), the raw material p-fluoroaniline is replaced by 3-isopropylaniline, that is, from the starting compound 2-fluoro-6-iodo Preparation of compound 4-iodo-1H-indazol-3-amine (compound 2) from benzonitrile (compound 1) and compound 1-(3-isopropylphenyl) from 3-aminophenylboronic acid (compound 3) -3-(3-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)urea (Compound 5).

4) 1-(3-isopropylphenyl)-3-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl ) urea (compound 5) and 4-iodo-1H-indazol-3-amine (compound 2) to prepare compound 1-(3-(3-amino-1H-indazol-4-yl)benzene by Suzuki coupling reaction base)-3-(3-isopropylphenyl)urea (compound 6), the specific operation steps are:

1-(3-isopropylphenyl)-3-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl) Urea (compound 5) 0.9g (1.9mmol), 4-iodo-1H-indazol-3-amine (compound 2) 0.5g (1.9mmol), sodium carbonate 0.42g (3.9mmol), tetrakis (triphenylphosphine ) Palladium 0.3g (0.15mmol) was dissolved in 20mL 1,4-dioxane and 5ml water, and reacted overnight at 100°C under nitrogen protection. Cool to room temperature, filter with suction, wash the filter cake with 1,4-dioxane, collect the filtrate, and spin dry to obtain a yellow residue, which is separated by column chromatography (the elution solvent is petroleum ether: ethyl acetate = 1:1) 0.47 g of white solid 1-(3-(3-amino-1H-indazol-4-yl)phenyl)-3-(3-isopropylphenyl)urea (compound 6) was obtained, with a yield of 52%;

The compound structure obtained is shown in the following formula:

Physical and chemical properties: mp: 197～198℃, MS(ESI)[MH] ⁺ :m/z＝384.20.

Proton NMR data are: ¹ H NMR (400MHz, DMSO) δ11.77(s, 1H), 8.81(s, 1H), 8.68(s, 1H), 7.63(d, J=1.8Hz, 1H), 7.46–7.40(m,2H),7.35(d,J=1.7Hz,1H),7.30–7.28(m,2H),7.27–7.25(m,1H),7.19(t,J=7.8Hz,1H) ,7.09–7.06(m,1H),6.86(d,J＝7.5Hz,1H),6.84–6.81(m,1H),4.45(s,2H),2.88–2.81(m,1H),1.19(d ,J=6.9Hz,6H).

Example 3

In the structural formula of the compound, ring A is a benzene ring, _R1 is 5-trifluoromethyl, _R2 is 3-trifluoromethyl, and the urea structure is located at the meta-position of the benzene ring.

Steps 1) to 3) are the same as steps 1) to 3) in Example 1, except that in step 3), the raw material p-fluoroaniline is replaced by 3,5-bis(trifluoromethyl)aniline, that is, from the starting compound 2 -Fluoro-6-iodobenzonitrile (compound 1) prepares compound 4-iodo-1H-indazol-3-amine (compound 2), and simultaneously prepares compound 1-(3, 5-bis(trifluoromethyl)phenyl)-3-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl) Urea (Compound 5).

4) 1-(3,5-bis(trifluoromethyl)phenyl)-3-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolane- 2-yl)phenyl)urea (compound 5) and 4-iodo-1H-indazol-3-amine (compound 2) were prepared by Suzuki coupling reaction compound 1-(3-(3-amino-1H-indazole -4-yl)phenyl)-3-(3,5-bis(trifluoromethyl)phenyl)urea (compound 6), the specific operation steps are:

1-(3,5-bis(trifluoromethyl)phenyl)-3-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolane-2 -yl)phenyl)urea (compound 5) 0.9g (1.9mmol), 4-iodo-1H-indazol-3-amine (compound 2) 0.5g (1.9mmol), sodium carbonate 0.42g (3.9mmol), Tetrakis(triphenylphosphine)palladium 0.3g (0.15mmol) was dissolved in 20mL 1,4-dioxane and 5ml water, and reacted overnight at 100°C under nitrogen protection. Cool to room temperature, filter with suction, wash the filter cake with 1,4-dioxane, collect the filtrate, and spin dry to obtain a yellow residue, which is separated by column chromatography (the elution solvent is petroleum ether: ethyl acetate = 1:1) 0.47 g of white solid 1-(3-(3-amino-1H-indazol-4-yl)phenyl)-3-(3,5-bis(trifluoromethyl)phenyl)urea (compound 6) was obtained , yield 53%;

The compound structure obtained is shown in the following formula:

Physical and chemical properties: mp: 203～204℃, MS(ESI)[MH] ⁺ :m/z＝478.10

Proton NMR data: ¹ H NMR (400MHz, DMSO) δ11.78(s,1H),9.47(s,1H),9.19(s,1H),8.15(s,2H),7.65(t,J ＝4.3Hz, 2H), 7.53–7.51(m, 1H), 7.45(t, J＝4.3Hz, 1H), 7.31(t, J＝4.3Hz, 2H), 7.13(d, J＝7.5Hz, 1H ),6.85–6.82(m,1H),4.43(s,2H).

Example 4

In the structural formula of the compound, ring A is a pyridine ring, _R1 is 4-methyl, _R2 is hydrogen, and the urea structure is located at the para position of the benzene ring.

Steps 1) to 3) are the same as steps 1) to 3) in Example 1, except that in step 3), the raw material p-fluoroaniline is replaced by 2-amino 4-picoline, that is, from the starting compound 2-fluoro- Compound 4-iodo-1H-indazol-3-amine (compound 2) was prepared from 6-iodobenzonitrile (compound 1), while compound 1-(4-picoline was prepared from 3-aminophenylboronic acid (compound 3) -2-yl)-3-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)urea (Compound 5).

4) 1-(4-methylpyridin-2-yl)-3-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl) Phenyl)urea (compound 5) and 4-iodo-1H-indazol-3-amine (compound 2) prepared by Suzuki coupling reaction compound 1-(4-(3-amino-1H-indazol-4-yl ) phenyl)-3-(4-methylpyridin-2-yl)urea (compound 6), the specific operation steps are:

1-(4-methylpyridin-2-yl)-3-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzene base) urea (compound 5) 1.5g (4.2mmol), 4-iodo-1H-indazol-3-amine (compound 2) 1.2g (4.6mmol), sodium carbonate 1.1g (10.3mmol), tetrakis (triphenyl Dissolve 0.5g (0.43mmol) of palladium (0.43mmol) in 20mL 1,4-dioxane and 5ml water, and react overnight at 100°C under nitrogen protection. Cool to room temperature, filter with suction, wash the filter cake with 1,4-dioxane, collect the filtrate, and spin dry to obtain a yellow residue, which is separated by column chromatography (the elution solvent is petroleum ether: ethyl acetate = 1:1) 0.79 g of white solid 1-(4-(3-amino-1H-indazol-4-yl)phenyl)-3-(4-methylpyridin-2-yl)urea (compound 6) was obtained, yield 52.5 %;

The resulting compound is shown in the following formula

Physical and chemical properties: mp: 228～229℃, MS(ESI)[MH] ⁺ :m/z＝357.10

Proton NMR data are: ¹ H NMR (400MHz, DMSO) δ11.74(s, 1H), 10.89(s, 1H), 9.50(s, 1H), 8.17(d, J=5.2Hz, 1H), 7.68(d, J=8.5Hz, 2H), 7.43(d, J=8.5Hz, 2H), 7.30(d, J=5.2Hz, 2H), 7.27(s, 1H), 6.88(d, J=5.0 Hz,1H),6.81-6.79(m,1H),4.35(s,2H),2.31(s,3H).

Example 5

In the structural formula of the compound, ring A is a benzodioxol ring, _R1 is hydrogen, _R2 is hydrogen, and the urea structure is located at the para position of the benzene ring.

Steps 1) to 3) are the same as steps 1) to 3) in Example 1, except that the raw material p-fluoroaniline is replaced by That is, the preparation of compound 4-iodo-1H-indazol-3-amine (compound 2) from the starting compound 2-fluoro-6-iodobenzonitrile (compound 1) and the preparation of 3-aminophenylboronic acid (compound 3) Compound 1-(Benzo[d](1,3)dioxolan-5-yl)-3-(4-(4,4,5,5-tetramethyl-1,3,2-di Oxaborolan-2-yl)phenyl)urea (Compound 5).

4) 1-(Benzo[d](1,3)dioxolan-5-yl)-3-(4-(4,4,5,5-tetramethyl-1,3,2- Compound 1-(4-(3- Amino-1H-indazol-4-yl)phenyl)-3-(benzo[d](1,3)dioxolan-5-yl)urea (compound 6), the specific operation steps are:

1-(Benzo[d](1,3)dioxolan-5-yl)-3-(4-(4,4,5,5-tetramethyl-1,3,2-di Oxaborolan-2-yl)phenyl)urea (compound 5) 0.75g (1.9mmol), 4-iodo-1H-indazol-3-amine (compound 2) 0.56g (2.1mmol), sodium carbonate 0.52 g (4.7mmol), tetrakis(triphenylphosphine)palladium 0.23g (0.20mmol) was dissolved in 20mL 1,4-dioxane and 5ml water, and reacted overnight at 100°C under nitrogen protection. Cool to room temperature, filter with suction, wash the filter cake with 1,4-dioxane, collect the filtrate, and spin dry to obtain a yellow residue, which is separated by column chromatography (the elution solvent is petroleum ether: ethyl acetate = 1:1) 1-(4-(3-Amino-1H-indazol-4-yl)phenyl)-3-(benzo[d](1,3)dioxolan-5-yl)urea was obtained as a white solid (Compound 6) 0.41g, yield 55%;

The resulting compound is shown in the following formula

Physical and chemical properties: mp: 150～152℃, MS(ESI)[MH] ⁺ :m/z＝386.10

Proton NMR data: ¹ H NMR (400MHz, DMSO) δ11.73(s, 1H), 8.77(s, 1H), 8.63(s, 1H), 7.58(d, J=8.6Hz, 2H), 7.39(d, J=8.5Hz, 2H), 7.27(d, J=8.6Hz, 2H), 7.24(d, J=2.1Hz, 1H), 6.58(d, J=8.6Hz, 1H), 6.80– 6.78(m,2H),5.99(s,2H),4.35(s,2H).

Example 6

In the structural formula of the compound, ring A is a benzene ring, _R1 is hydrogen, _R2 is diethylaminomethyl, and the urea structure is located at the para-position of the benzene ring.

Steps 1) to 3) are the same as steps 1) to 3) in Example 1, except that in step 3), the raw material p-fluoroaniline is replaced with 4-diethylaminomethylaniline, that is, the starting compound 2-fluoro-6 -Iodobenzonitrile (compound 1) prepares compound 4-iodo-1H-indazol-3-amine (compound 2), and simultaneously prepares compound 1-(4-((diethyl Amino)methyl)phenyl)-3-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)urea (Compound 5 ).

4) 1-(4-((diethylamino)methyl)phenyl)-3-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolane- 2-yl)phenyl)urea (compound 5) and 4-iodo-1H-indazol-3-amine (compound 2) were prepared by Suzuki coupling reaction compound 1-(4-(3-amino-1H-indazole -4-base) phenyl)-3-(4-((diethylamino) methyl) phenyl) urea (compound 6), the specific operation steps are:

1-(4-((diethylamino)methyl)phenyl)-3-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolane-2 -yl)phenyl)urea (compound 5) 0.8g (1.9mmol), 4-iodo-1H-indazol-3-amine (compound 2) 0.61g (2.3mmol), sodium carbonate 0.65g (6.1mmol), Tetrakis(triphenylphosphine)palladium 0.24g (0.20mmol) was dissolved in 20mL 1,4-dioxane and 5ml water, and reacted overnight at 100°C under nitrogen protection. Cool to room temperature, filter with suction, wash the filter cake with 1,4-dioxane, collect the filtrate, and spin dry to obtain a yellow residue, which is separated by column chromatography (the elution solvent is petroleum ether: ethyl acetate = 1:1) 0.46 g of white solid 1-(4-(3-amino-1H-indazol-4-yl)phenyl)-3-(4-((diethylamino)methyl)phenyl)urea (compound 6) was obtained , yield 58%;

The resulting compound is shown in the following formula

Physical and chemical properties: mp: 200～202℃, MS(ESI)[MH] ⁺ :m/z＝427.50

Proton NMR data: ¹ H NMR (400MHz, DMSO) δ11.74(s,1H),8.83(s,1H),8.72(s,1H),7.60(d,J＝8.5Hz,2H), 7.41(m,4H),7.27(d,J=5.8Hz,2H),7.22(d,J=8.4Hz,2H),6.79(dd,J=5.9,1.8Hz,1H),4.36(s,2H ),3.47(s,2H),2.48–2.42(m,4H),0.98(t,J＝7.1Hz,6H).

Example 7

In the structural formula of the compound, ring A is a thiazole ring, _R1 is hydrogen, _R2 is hydrogen, and the urea structure is located at the para-position of the benzene ring.

Steps 1) to 4) are the same as steps 1) to 4) in Example 1, except that in step 3), the raw material p-fluoroaniline is replaced by 2-aminothiazole, that is, from the starting compound 2-fluoro-6-iodobenzene Compound 4-iodo-1H-indazol-3-amine (compound 2) was prepared from forminonitrile (compound 1), while compound 6 was prepared from 3-aminophenylboronic acid (compound 3). The obtained compound 6 is shown in the following formula

Angiogenesis is closely related to the occurrence, development and migration of tumors. Inhibiting the formation of new blood vessels can effectively inhibit the growth of tumors. Many growth factors are involved in the regulation of angiogenesis, among which VEGFR-2 is the strongest known positive regulator. By inhibiting the activity of VEGFR-2 kinase and blocking the signal pathway induced by it, the proliferation and migration of tumor cells can be inhibited, thereby achieving the purpose of treating tumors.

The antitumor activity experiment of the 1H-indazole-3-aminobiphenylurea compounds provided by the present invention with antitumor activity is carried out below.

1. Screening of the inhibitory activity of 1H-indazole-3-aminobiphenylurea compounds with anti-tumor activity on VEGFR-2 kinase

The kinase VEGFR-2 and the substrate Abltide were purchased from Signal-Chem, and the ADP-GlobTM Kinase Assays kit from Promega was used to detect the inhibitory activity of the target compound, and the operation method was carried out according to the kit instructions.

ATP (1mM) was diluted 80 times with buffer (2×) (Tris 80mM, MgCl ₂ 20mM, BSA 0.2mg/mL, DTT 2mM) to prepare ATP (125μM) buffer (2×) solution; 125μM ATP solution and The volume of Abltide solution was mixed at 1:1 to make a mixed solution of ATP (62.5 μM)-Abltide (0.5 μg/μl) for later use; buffer (1×) (Tris 40 mM, MgCl ₂ 10 mM, BSA 0.1 mg/μl) was used for VEGFR-2 kinase solution mL, DTT 1mM) was diluted 100 times to prepare the buffer (1×) solution of VEGFR-2 (10ng/μl) for use; the target compound and the positive control drug (Sorafinib) were prepared into 6×10 ^{- 5} mol/L, 6×10 ^-6 mol/L, 6×10 ^-7 mol/L, 6×10 ^-8 mol/L, 6×10 ^-9 mol/L, 6×10 ^-10 mol/L Gradient sample solution, add 2 μL ATP-Abltide mixed solution, 1 μL sample solution, and 2 μL enzyme solution to each well of the 384-well plate; add 3 μL buffer and 2 μL ATP-Abltide mixed solution to the blank well; Abltide mixed solution, 1 μL buffer, 2 μL enzyme solution, after adding, incubate at 30°C for 60 min; add 5 μL of ADP-Glo reagent, incubate at 25°C for 40 min; add 10 μL of Kinase detection reagent, incubate at 25°C for 30 min.

The luminescence value of each hole was measured by the chemiluminescent module of the PerkinElmer multifunctional microplate reader, and the inhibition rate and IC of the 1H-indazole-3-aminobiphenylurea compounds with anti-tumor activity provided by the present invention were calculated for VEGFR-2. ₅₀ .

The results obtained are shown in Table 1:

Table 1 IC ₅₀ of 1H-indazole-3-aminobiphenylurea compounds on VEGFR-2 kinase

Among them, the urea structural fragments of compounds S1-S16 are located at the meta-position of the benzene ring, and the urea structural fragments of compounds S17-S22 are located at the para-position of the benzene ring.

It can be seen from Table 1 that individual compounds in the 1H-indazole-3-aminobiphenylurea compounds with antitumor activity provided by the present invention have good activity of inhibiting VEGFR-2 kinase, and can inhibit VEGFR-2 in the preparation of Drugs with kinase activity.

2. Antitumor activity screening of 1H-indazole-3-aminobiphenylurea compounds with antitumor activity

MTT method was used to test the growth inhibitory activity of 1H-indazole-3-aminobiphenylurea compounds on tumor cells:

The 1H-indazole-3-aminobiphenylurea compound with anti-tumor activity provided by the present invention has anti-tumor effect and has anti-proliferation activity on tumor cells in vitro. The cell line has the activity of inhibiting the proliferation of tumor cells and can be used for the treatment of these cancers; compared with the active drug Sorafinib (Sorafinib), individual compounds show higher activity of inhibiting the proliferation of tumor cells.

Digest lung cancer cells (A549) and liver cancer cells (SMCC-7721) in the logarithmic growth phase with 0.25% trypsin for 3 to 5 minutes, pipette evenly and dilute to a concentration of 1×10 ⁴ to 2×10 ⁴ /mL single-cell suspension, inoculated in parallel in 96-well culture plate, each hole inoculation volume was 180 μL; cultivated in 37 ℃, 5% CO ₂ incubator for 24 hours; with 0.25% DMSO (N,N- The aqueous solution of dimethyl sulfoxide) was used as a negative control, and Sorafenib was used as a positive control, and 4 different concentrations of 1H-indazole-3-aminobiphenylurea compounds (8×10 ^-7 mol /L, 4×10 ^-6 mol/L, 2×10 ^-5 mol/L, 1×10 ^-4 mol/L), set 3 replicate wells for each concentration, continue culturing for 48 hours; then add 22 μL of 5 mg/mL MTT working solution, mix well, take out after incubating at 37°C for 4 hours, carefully aspirate and discard the culture solution, add 150 μL DMSO to each well, shake for 10 min, and measure the ultraviolet absorption at 490nm of each well with an enzyme-linked immunosorbent assay value (OD value), then calculate the cell inhibition rate, and calculate the IC ₅₀ value based on the inhibition rate. The formula for calculating the cell inhibition rate is:

Inhibition rate% = (OD value of positive control group - OD value of medication group) / OD value of negative control cells × 100%

The test results showed that compared with the negative control group, the 1H-indazole-3-aminobiphenylurea compounds had different degrees of inhibitory effects on the above two tumor cells in vitro, and the results are shown in Table 2.

Table 2.1 IC ₅₀ (μM) of H-indazole-3-aminobiphenylurea compounds on different cell lines

The results showed that the inhibition of some compounds on tumor cells was equivalent to that of the positive control drug Sorafenib, and some of them even had higher activity than the positive control. Illustrate that the 1H-indazole-3-aminobiphenylurea compound with antitumor activity provided by the present invention has a more obvious inhibitory effect on the proliferation of tumor cells (lung cancer cell A549 and liver cancer cell SMCC-7721), and can be used in the preparation of antitumor drugs in the application.

Due to the growth effect of VEGFR-2 kinase on tumor cells, and the inhibitory effect of the 1H-indazole-3-aminobiphenylurea compound with anti-tumor activity provided by the invention on VEGFR-2 kinase, the invention provides 1H-indazole-3-aminobiphenylurea compounds with anti-tumor activity can be used in anti-tumor drugs targeting VEGFR-2 kinase, including not only the above-mentioned anti-lung cancer and anti-liver cancer drugs, but also Anti-gastric cancer, anti-leukaemia, anti-nerve cancer, anti-colon cancer, anti-breast cancer, anti-cervical cancer and anti-pancreatic cancer can be included.

Claims (10)

1. A 1H-indazole-3-aminobiphenylurea compound with antitumor activity is characterized in that its structural formula is as follows: Wherein _R1 is hydrogen, alkane group, halogenated alkane or halogen, _R2 is hydrogen, alkane group, alkoxyl group, halogenated alkane, halogen or tertiary amine moiety, A ring is benzene ring, pyridine ring, thiazole ring or benzodiazepines The oxolene ring and the urea structure are located at the meta or para position of the benzene ring. 2. The 1H-indazole-3-aminobiphenylurea compound with antitumor activity according to claim 1, characterized in that: the tertiary amine segment is dimethylaminomethyl, diethylaminomethyl , piperidinylmethyl or dimethylaminoethoxy substituted with fluorine on the benzene ring. 3. The 1H-indazole-3-aminobiphenylurea compound with antitumor activity according to claim 1, characterized in that: said R ₁ is H, 4-F, 4-CH ₃ , 6 _-CH3 , 4-F, 4-Cl, ₅ -CF3 or 3-F. 4. the 1H-indazole-3-aminobiphenylurea compound with antitumor activity according to claim 1, is characterized in that: described R ₂ is 2-F, 3-F, H, 2- Cl, 3-CH(CH ₃ ) ₂ , 3-CF ₃ , 3-OCH ₃ , 2-CH ₃ , 3-Cl, 5. the preparation method of the 1H-indazole-3-aminobiphenylurea compound with antitumor activity described in any one of claims 1-4, is characterized in that, comprises the following steps: 1) 2-fluoro-6-iodobenzonitrile reacts with hydrazine hydrate under the action of sodium bicarbonate to obtain 4-iodo-1H-indazol-3-amine; 2) reacting 3-aminophenylboronic acid with pinacol under the catalysis of magnesium sulfate to obtain 3-aminophenylboronic acid pinacol ester; 3) forming an isocyanate with aniline containing a substituent group and triphosgene, and then condensing with 3-aminophenylboronic acid pinacol ester to obtain a urea compound containing phenylboronic acid pinacol ester; 4) Under the catalysis of tetrakis(triphenylphosphine) palladium, the urea compound containing phenylboronic acid pinacol ester and 4-iodo-1H-indazol-3-amine were reacted by Suzuki to generate antitumor active 1H-indazole-3-aminobiphenylurea compounds. 6. The use of the 1H-indazole-3-aminobiphenylurea compound with antitumor activity according to any one of claims 1-4 in the preparation of a drug for inhibiting VEGFR-2 kinase activity. 7. The application of the 1H-indazole-3-aminobiphenylurea compound with antitumor activity described in any one of claims 1-4 in the preparation of antitumor drugs. 8. The application according to claim 7, characterized in that: the antineoplastic drug is a drug targeting VEGFR-2 kinase, including anti-lung cancer, anti-liver cancer, anti-gastric cancer, anti-leukaemia, anti-nerve cancer, Drugs against colon, breast, cervix, and pancreas cancers. 9. The application according to claim 7, characterized in that: the antitumor drug is a drug that inhibits tumor cell proliferation. 10. The application according to claim 9, characterized in that: the antitumor drug is a drug that inhibits the proliferative activity of lung cancer cell A549 and liver cancer cell SMCC-7721.