CN115286589A

CN115286589A - Cinnamoyl tetrazine compound and preparation and application thereof

Info

Publication number: CN115286589A
Application number: CN202210880694.7A
Authority: CN
Inventors: 赵鹏程; 徐晓亮; 饶国武; 靳浩
Original assignee: Zhejiang University of Technology ZJUT
Current assignee: Zhejiang University of Technology ZJUT
Priority date: 2022-07-25
Filing date: 2022-07-25
Publication date: 2022-11-04
Anticipated expiration: 2042-07-25
Also published as: CN115286589B

Abstract

The invention discloses a cinnamoyl tetrazine compound and preparation and application thereof. The cinnamoyl tetrazine compound has the following structural formula (I), wherein n =1, R is H, 2, 4-dichloro, 2, 4-dimethyl, 2-methoxy, 2-methyl, 3, 5-dimethyl, 3-methyl, 4-F, 3, 4-dimethyl, 3, 4-dimethoxy, 2-Cl, 4-methoxy, 4-Cl or 3-Cl; or n =0, R is H, 4-F, 4-CH ₃ 、4‑Cl、2‑Cl、4‑OCH ₃ 2-OH or 3-Br. The invention provides an application of the cinnamoyl tetrazine compound or the pharmaceutically acceptable salt thereof in preparing a medicine for treating or preventing VEGFR-2 mediated diseases or a medicine for inhibiting VEGFR-2, and the cinnamoyl tetrazine compound or the pharmaceutically acceptable salt thereof shows good inhibitory activity.

Description

Cinnamoyl tetrazine compound and preparation and application thereof

Technical Field

The invention relates to a cinnamoyl tetrazine compound, a preparation method thereof and application of the compound as a VEGFR-2 inhibitor in preparing a medicine for treating or preventing VEGFR-2 mediated diseases or a medicine for inhibiting VEGFR-2.

Background

The tetrazine compound has many better physical properties, spectral properties and higher reaction activities, and particularly some tetrazine derivatives with special structures have obvious antitumor activity and antiviral activity and can be used as pesticides and insecticides. For example, two varieties of pesticides (clofentezine and flutenzine) are on the market, and one variety of medicines (temozolomide, an anti-tumor medicine) is on the market.

In 1978, the literature reported that 3, 6-diphenylalkynyl-hexahydro-1, 2,4, 5-tetrazine had antitumor activity (see Eremeev, A.V.; tikhomirova, D.A.; tyusheva, V.A.; liepns, F.Khim. Getertsikl. Soedin,1978, 753), which is the first reported potential antitumor activity of the 1,2,4, 5-tetrazine class of compounds. Thereafter, some 1,2,4, 5-tetrazines were reported to have antitumor activity, such as 3, 6-bis (2 '-hydroxy-5' -chlorophenyl) -1,2,4, 5-tetrazine having antitumor activity (see Rao, g. -w.; hu, w. -x.bioorg.med.chem.lett.2006,16 (14), 3702). Of course most 1,2,4, 5-tetrazines do not have anti-tumor activity.

Disclosure of the invention

The first object of the present invention is to provide a novel cinnamoyl tetrazine compound having inhibitory activity against VEGFR-2 mediated diseases and VEGFR-2.

The second purpose of the invention is to provide a preparation method of the cinnamoyl tetrazine compound, which is simple and convenient, easy to operate, easy to obtain raw materials, low in production cost and suitable for industrial application.

The third purpose of the invention is to provide an application of the cinnamoyl tetrazine compound or the pharmaceutically acceptable salt thereof in preparing a medicine for treating or preventing VEGFR-2 mediated diseases or a medicine for inhibiting VEGFR-2, wherein the VEGFR-2 mediated diseases are cancers, the cancers are human lung cancers or human liver cancers, and cancer cells of the cancers are A549, huh7 or HepG2 cells.

The technical solution adopted by the present invention is specifically explained below.

In a first aspect, the present invention provides a cinnamoyl tetrazine compound having the following structural formula (i):

in the formula (I), n =1, R is H, 2, 4-dichloro, 2, 4-dimethyl, 2-methoxy, 2-methyl, 3, 5-dimethyl, 3-methyl, 4-F, 3, 4-dimethyl, 3, 4-dimethoxy, 2-Cl, 4-methoxy, 4-Cl or 3-Cl;

or n =0, R is H, 4-F, 4-CH ₃ 、4-Cl、2-Cl、4-OCH ₃ 2-OH or 3-Br.

The cinnamoyl tetrazine compound is particularly preferably selected from one of the following compounds:

in a second aspect, the invention provides a preparation method of cinnamoyl tetrazine compounds represented by formula (I), wherein the preparation method comprises the following steps:

(1) Adding triphosgene into an organic solvent A, dropwise adding an organic solvent A solution containing 3, 6-dimethyl-1, 6-dihydro-1, 2,4, 5-tetrazine shown in a formula (II) and an alkaline catalyst a under the stirring condition of-10-12 ℃, heating the reaction solution to room temperature after the dropwise adding is finished, stirring and reacting at room temperature for 0.5-50 hours, introducing nitrogen into the reaction system until tail gas absorption liquid (NaOH aqueous solution) does not generate white mist, and directly using the generated product for the next reaction;

(2) Adding a compound shown in a formula (III) and a basic catalyst B into an organic solvent B, stirring for dissolving, dropwise adding the reaction liquid obtained in the step (1) under the stirring condition of-10-12 ℃, heating the reaction liquid to room temperature after dropwise adding, stirring for reacting for 0.5-50 hours at room temperature, and separating and purifying the reaction liquid to obtain a cinnamoyl tetrazine compound shown in a formula (I);

the basic catalyst a and the basic catalyst b are one of the following: triethylamine, 4-Dimethylaminopyridine (DMAP), pyridine or sodium hydroxide;

in the formula (III), R and n are as defined in the formula (I).

The reaction for preparing the cinnamoyl tetrazine compound (I) is shown in the following reaction formula, and the following reaction formula is not reported in the literature:

further, the ratio of the amount of the compound (ii) to the amount of the substance charged with the basic catalyst a, the basic catalyst b, the triphosgene and the compound (iii) is 1: 0.1 to 3, and preferably the ratio of the amount of the compound (ii) to the amount of the substance charged with the basic catalyst a, the basic catalyst b, the triphosgene and the compound (iii) is 1: 0.1 to 2.

Further, the organic solvent A and the organic solvent B are each independently selected from one of the following: dichloromethane, chloroform or toluene. The organic solvent a may be used in an amount capable of dissolving triphosgene, the compound (ii), and the basic catalyst a, and the organic solvent B may be used in an amount capable of dissolving the basic catalyst B and the compound (iii). It is preferable that the total volume of the organic solvent A is 3 to 18mL/mmol based on the amount of the compound represented by the formula (II), the volume of the organic solvent A for dissolving triphosgene is 1 to 10mL/mmol based on the amount of the compound represented by the formula (II), and the total volume of the organic solvent B is 2 to 20mL/mmol based on the amount of the compound represented by the formula (II).

Further, in the step (1), an organic solvent A solution containing 3, 6-dimethyl-1, 6-dihydro-1, 2,4, 5-tetrazine represented by the formula (II) and a basic catalyst a is added dropwise under stirring at-10 to 5 ℃.

Further, in the step (2), the reaction liquid after the reaction in the step (1) is dropwise added under the condition of stirring at-10 to 5 ℃.

Further, the reaction processes of the steps (1) and (2) are followed by TLC (a developing solvent is a mixed solution of petroleum ether and ethyl acetate in a volume ratio of 0.5-20.

Further, the separation and purification in the step (2) adopts the following steps: and (3) after the reaction is finished, washing the reaction solution with water, separating out an organic phase, evaporating to remove the solvent, and carrying out column chromatography on residues to obtain the cinnamoyl tetrazine compound shown in the formula (I).

Furthermore, the column chromatography is specifically performed by the following steps: taking the residue after the solvent is removed by evaporation, adding an organic solvent C into the residue in a single-mouth bottle, dissolving the residue to obtain a dissolved solution, adding column chromatography silica gel (preferably 300-400 meshes of coarse pore (zcx.II) type column chromatography silica gel) with the amount of 1-2 times of the residue into the dissolved solution, uniformly mixing, removing the solvent by evaporation to obtain a mixture of the dried residue and the silica gel, loading the mixture into a column, and then loading the mixture into a column according to the volume ratio of 0.5-20: 1 as an eluent, eluting, tracking and detecting by TLC (a developing solvent is a mixed solution of petroleum ether and ethyl acetate with the volume ratio of 0.5-20; the organic solvent C is one of the following: petroleum ether, dichloromethane, chloroform or ethyl acetate; the organic solvent C is used in an amount capable of dissolving the residue.

The organic solvent A, the organic solvent B and the organic solvent C are all indicated as organic solvents for reaction or column chromatography, and the letters in the invention do not refer to certain organic solvents, but are used for convenience of expression and clarity and are used for distinguishing the organic solvents appearing in different steps. The organic solvent A may be the same organic solvent, and the organic solvents A, B, or C may be the same or different solvents.

In a third aspect, the invention provides an application of cinnamoyl tetrazine compound shown in formula (I) or pharmaceutically acceptable salt thereof in preparing a medicine for treating or preventing VEGFR-2 mediated diseases or inhibiting VEGFR-2.

In a specific embodiment of the invention, the VEGFR-2 mediated disease is cancer. Such cancers include, but are not limited to: non-small cell lung cancer, lung adenocarcinoma, breast cancer, liver cancer. In a preferred embodiment, the cancer is VEGFR-2 mediated non-small cell lung cancer or liver cancer, and the cancer cells of the cancer are A549, huh7 or HepG2 cells.

Preferably, the cancer cell is an A549 cell, and the cinnamoyl tetrazine compound represented by formula (I) is compound (ie).

Preferably, the cancer cell is Huh7 cell, and the cinnamoyl tetrazine compound represented by formula (i) is compound (ih).

Preferably, the cancer cell is a HepG2 cell, and the cinnamoyl tetrazine compound represented by formula (I) is a compound (Id), (Ig) (ih) or (iS).

The term "pharmaceutically acceptable" means in this application: the compounds are compatible chemically and/or toxicologically, with the other ingredients comprising the formulation and/or with the human or mammal with which the disease or condition is being prevented or treated.

The term "pharmaceutically acceptable salts" refers to the relatively non-toxic, inorganic or organic acid addition salts of the compounds of the present invention. See, e.g., S.M.Berge et al, "Pharmaceutical Salts", J.pharm.Sci.1977, 66,1-19. Among them, inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, nitric acid, or the like; organic acids such as formic acid, acetic acid, acetoacetic acid, pyruvic acid, trifluoroacetic acid, propionic acid, butyric acid, caproic acid, heptanoic acid, undecanoic acid, lauric acid, benzoic acid, salicylic acid, 2- (4-hydroxybenzoyl) -benzoic acid, camphoric acid, cinnamic acid, cyclopentanepropionic acid, diglucosic acid, 3-hydroxy-2-naphthoic acid, nicotinic acid, pamoic acid, pectinic acid, 3-phenylpropionic acid, picric acid, pivalic acid, 2-hydroxyethanesulfonic acid, itaconic acid, sulfamic acid, trifluoromethanesulfonic acid, dodecylsulfuric acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, methanesulfonic acid, 2-naphthalenesulfonic acid, naphthalenedisulfonic acid, camphorsulfonic acid, citric acid, tartaric acid, stearic acid, lactic acid, oxalic acid, malonic acid, succinic acid, malic acid, adipic acid, alginic acid, maleic acid, fumaric acid, D-gluconic acid, mandelic acid, ascorbic acid, glucoheptonic acid, glycerophosphoric acid, aspartic acid, sulfosalicylic acid, and the like.

Compared with the prior art, the invention has the following beneficial effects: (1) A novel 1,2,4, 5-tetrazine compound having excellent anticancer activity (especially human lung cancer or human liver cancer) is provided; (2) The preparation method of the 1,2,4, 5-tetrazine compound is provided, the preparation method is simple and easy to operate, the raw materials are easy to obtain, the production cost is lower, and the preparation method is suitable for practicality and is expected to be applied to the preparation of the medicines for preventing or treating the tumor diseases; (3) Provides the application of a novel 1,2,4, 5-tetrazine compound or pharmaceutically acceptable salt thereof in preparing medicaments for treating or preventing VEGFR-2 mediated diseases or medicaments for inhibiting VEGFR-2.

(IV) detailed description of the preferred embodiments

The invention is further illustrated by reference to specific examples, which are intended to illustrate the invention, but not to limit it in any way.

3, 6-dimethyl-1, 6-dihydro-1, 2,4, 5-tetrazine (II) by the method described in Synthetic Communications,2003,33 (16), 2769-2775.

The following compounds (iiin) to (iiiu) were prepared by the methods described in the references (Journal of molecular Structure,2022,1254,132422, indian Pat. appl.,2010de00068,15jul 2011, chemistry &bioreversity, 2007,4 (2), 203-214).

The following compounds (IIIa) to (IIIm) and (IIIv) were prepared by methods described in ACS Catalysis,2022,12 (8), 4688-4695, journal of pharmacy and Pharmacology,2020, 72 (7), 916-926, journal of medicinal chemistry,2014,57 (12), 5333-5347.

The compounds (IIIa) to (IIIm) and (IIIv) are prepared by the following reaction schemes, in which R ¹ The structures of the compounds (IIIa) to (IIIm) and (IIIv) are as follows:

example 1: preparation of Compound (IIIa)

7.4g (50 mmol) of cinnamic acid and 100mL of anhydrous dichloromethane are added into a three-neck flask, 100mL of anhydrous dichloromethane containing 70.0g (551 mmol) of oxalyl chloride is added dropwise under ice bath condition, 2.0g of DMF is added after dropwise addition, reaction is carried out for 12 hours at room temperature, and organic solvent is distilled off, so that 4.8g (29 mmol) of cinnamoyl chloride which is light yellow oily substance is obtained.

4.8g (29 mmol) of cinnamoyl chloride and 100mL of anhydrous dichloromethane are added into a three-neck flask, 100mL of dichloromethane solution containing 5.5g (40 mmol) of m-nitroaniline and 4.2g (42 mmol) of triethylamine is added dropwise under the ice bath condition, reaction is carried out for 10 hours at room temperature after dropwise addition, organic solvent is removed by evaporation, 100mL of diluted hydrochloric acid with the pH value of 1 is added, filtration is carried out, 100mL multiplied by 3 water is used for washing a filter cake, and drying is carried out to obtain 5.6g (21 mmol) of N- (3-nitrophenyl) cinnamamide.

A three-necked flask was charged with 5.4g (20 mmol) of N- (3-nitrophenyl) cinnamamide, 15.2g (80.0 mmol) of tin dichloride and 1.1g (21 mmol) of ammonium chloride, followed by addition of 200mL of anhydrous ethanol, reflux-reacting for 1.5 hours under magnetic stirring, cooling to room temperature, adjusting the pH of the reaction solution to weak alkalinity with saturated aqueous sodium bicarbonate solution, extracting with ethyl acetate (100 mL. Times.3), combining the organic phases, drying over anhydrous magnesium sulfate, filtering, and distilling off the organic solvent to obtain 3.2g (13 mmol) of the compound (IIIa).

Preparation of Compounds (IIIb) to (IIIm) and Compound (IIIv): compounds (IIIb) to (IIIm) and (IIIv) were prepared by the method described in example 1 except that cinnamic acid was replaced with the corresponding substituted cinnamic acid, and the details thereof are omitted.

Example 2: preparation of Compound (Ia)

Dissolving 0.297g (1.0 mmol) of triphosgene in 10mL of chloroform, dropwise adding 20mL of chloroform solution containing 1.121g (10.0 mmol) of 3, 6-dimethyl-1, 6-dihydro-1, 2,4, 5-tetrazine (II) and 0.122g (1.0 mmol) of DMAP under the condition of magnetic stirring at the temperature of-10 ℃, heating the reaction solution to room temperature after dropwise adding, reacting for 50 hours at the room temperature (the reaction process adopts TLC tracking detection, and a developing agent is a mixed solution of petroleum ether and ethyl acetate in a volume ratio of 1.

Dissolving 2.383g (10.0 mmol) of compound (IIIa) and 0.122g (1.0 mmol) of DMAP in 60mL of chloroform, dropwise adding the reaction solution obtained in the previous step under the condition of magnetic stirring at the temperature of-10 ℃, heating the reaction solution to room temperature after dropwise adding, reacting at the room temperature for 10 hours (the reaction process is detected by tracking TLC, a developing agent is a mixed solution of petroleum ether and ethyl acetate with a volume ratio of 1: 2), washing the reaction solution with (50 mL x 3) water, separating an organic phase, evaporating the solvent, performing column chromatography on residues, namely adding 10mL of petroleum ether solvent into the residues after solvent evaporation to dissolve the residues to obtain a dissolved solution, then adding 2.0g of silica gel (300-400 meshes of coarse pore (zcx.II) type column chromatography silica gel into the dissolved solution, uniformly mixing, evaporating the solvent to obtain a mixture of dried residues and the silica gel, filling the mixture into a column, and then mixing the mixture in a volume ratio of 1:2 (eluent is a mixed solution of petroleum ether and ethyl acetate in a volume ratio of 1. ¹ H NMR(500MHz,DMSO-d ₆ )δ10.27(s,1H),9.32(s,1H),9.04(s,1H),8.03(s,1H),7.70(d,J＝7.6Hz,2H),7.59-7.54(m,1H),7.53(d,J＝15.7Hz,1H),7.37-7.31(m,3H),7.26(d,J＝7.7Hz,1H),7.06(t,J＝7.4Hz,1H),6.82(d,J＝15.7Hz,1H),2.21(s,3H),1.95(s,3H).

Example 3: preparation of Compound (Ia)

Dissolving 8.903g (30.0 mmol) of triphosgene in 100mL of dichloromethane, dropwise adding 80mL of dichloromethane solution containing 1.121g (10.0 mmol) of 3, 6-dimethyl-1, 6-dihydro-1, 2,4, 5-tetrazine (II) and 2.373g (30.0 mmol) of pyridine under the condition of magnetic stirring at 12 ℃, heating the reaction solution to room temperature after dropwise adding, reacting for 0.5 hour at the room temperature (the reaction process adopts TLC tracking detection, and a developing agent is a mixed solution of petroleum ether and ethyl acetate in a volume ratio of 20), introducing nitrogen into the reaction system, absorbing gas by using a tail gas absorbing device (the tail gas absorbing solution is 10 NaOH aqueous solution), and generating no white fog in the tail gas absorbing solution, wherein the generated product is directly used for the next reaction.

Dissolving 7.149g (30.0 mmol) of the compound (IIIa) and 2.373g (30.0 mmol) of pyridine in 200mL of dichloromethane, dropwise adding the reaction solution obtained in the previous step under the condition of magnetic stirring at 12 ℃, heating the reaction solution to room temperature after completing dropwise addition, reacting at room temperature for 0.5 hour (the reaction process is detected by TLC tracking, a developing agent is a mixed solution of petroleum ether and ethyl acetate with a volume ratio of 20:1 is used as eluent, elution is carried out, TLC tracking detection is carried out (the developing solvent is the mixed solution of petroleum ether and ethyl acetate with the volume ratio of 20. ¹ H NMR as in example 2.

Example 4: preparation of Compound (Ia)

Dissolving 5.935g (20.0 mmol) of triphosgene in 40mL of toluene, dropwise adding 20mL of toluene solution containing 1.121g (10.0 mmol) of 3, 6-dimethyl-1, 6-dihydro-1, 2,4, 5-tetrazine (II) and 2.024g (20.0 mmol) of triethylamine under the condition of magnetic stirring at 0 ℃, heating the reaction solution to room temperature after the dropwise adding is finished, reacting for 3 hours at the room temperature (the reaction process adopts TLC tracking detection, and a developing agent is a mixed solution of petroleum ether and ethyl acetate with a volume ratio of 10), introducing nitrogen into the reaction system, absorbing gas by using a tail gas absorbing device (the tail gas absorbing solution is 10 NaOH aqueous solution), and generating no white mist in the tail gas absorbing solution, wherein the generated product is directly used for the next reaction.

50mDissolving 4.766g (20.0 mmol) of compound (IIIa) and 2.024g (20.0 mmol) of triethylamine in L toluene, dropwise adding the reaction solution obtained in the previous step under the condition of magnetic stirring at 0 ℃, heating the reaction solution to room temperature after the dropwise addition, reacting at room temperature for 8 hours (the reaction process adopts TLC tracking detection, a developing agent is a mixed solution of petroleum ether and ethyl acetate with a volume ratio of 10): 1 is used as eluent, elution is carried out, tracking detection is carried out by TLC (a developing solvent is a mixed solution of petroleum ether and ethyl acetate with the volume ratio of 10. ¹ H NMR as in example 2.

Example 5: preparation of Compound (Ia)

Dissolving 1.484g (5.0 mmol) of triphosgene in 20mL of toluene, dropwise adding 50mL of toluene solution containing 1.121g (10.0 mmol) of 3, 6-dimethyl-1, 6-dihydro-1, 2,4, 5-tetrazine (II) and 0.200g (5.0 mmol) of sodium hydroxide under the condition of magnetic stirring at 5 ℃, heating the reaction solution to room temperature after dropwise adding, reacting for 12 hours at room temperature (the reaction process adopts TLC tracking detection, and a developing agent is a mixed solution of petroleum ether and ethyl acetate with a volume ratio of 5), introducing nitrogen into the reaction system, absorbing gas by using a tail gas absorbing device (the tail gas absorbing solution is 10 NaOH aqueous solution) until no white mist is generated in the tail gas absorbing solution, and directly using the generated product for the next reaction.

Dissolving 0.238g (1.0 mmol) of the compound (IIIa) and 0.200g (5.0 mmol) of sodium hydroxide in 20mL of chloroform, dropwise adding the reaction solution obtained in the previous step under the condition of magnetic stirring at 5 ℃, heating the reaction solution to room temperature after dropwise adding, and reacting at room temperature for 50 hours (the reaction process adopts TLC tracking detection, and a developing agent is petroleum ether with a volume ratio of 5Ethyl acetate mixed solution), washing the reaction solution with (50 mL × 3) water, separating out an organic phase, evaporating to remove the solvent, performing column chromatography on the residue, namely adding 10mL of chloroform solvent into the residue after the solvent is evaporated to dissolve the residue to obtain a dissolved solution, adding 0.2 g of silica gel (300-400 meshes of coarse pore (zcx.ii) type column chromatography silica gel) into the dissolved solution, uniformly mixing, evaporating to remove the solvent to obtain a mixture of the dried residue and the silica gel, filling the mixture into a column, and then mixing the mixture in a volume ratio of 5:1 is used as eluent, elution is carried out, TLC tracking detection is carried out (a developing solvent is a mixed solution of petroleum ether and ethyl acetate with the volume ratio of 5. ¹ H NMR was the same as in example 2.

Example 6: preparation of Compound (Ia)

1.484g (5.0 mmol) of triphosgene is dissolved in 20mL of toluene, 50mL of toluene solution containing 1.121g (10.0 mmol) of 3, 6-dimethyl-1, 6-dihydro-1, 2,4, 5-tetrazine (II) and 0.200g (5.0 mmol) of sodium hydroxide is added dropwise under magnetic stirring at 5 ℃, the reaction solution is warmed to room temperature after completion of the addition, the reaction is carried out for 12 hours at room temperature (the reaction process is followed by TLC, and the developer is a mixed solution of petroleum ether and ethyl acetate in a volume ratio of 5), nitrogen is introduced into the reaction system, and gas is absorbed by a tail gas absorption device (the tail gas absorption solution is 10 NaOH aqueous solution), until white mist is not generated in the tail gas absorption solution, and the generated product is directly used for the next reaction.

Dissolving 2.383g (10.0 mmol) of the compound (IIIa) and 1.012g (10.0 mmol) of triethylamine in 60mL of dichloromethane, dropwise adding the reaction solution obtained in the previous step under the condition of magnetic stirring at-5 ℃, heating the reaction solution to room temperature after dropwise adding, reacting for 9 hours at room temperature (the reaction process adopts TLC tracking detection, a developing agent is a mixed solution of petroleum ether and ethyl acetate with a volume ratio of 5)(300-400 mesh coarse pore (zcx. II) type column chromatography silica gel), mixing, evaporating the solvent to obtain a mixture of a dry residue and the silica gel, loading the mixture into a column, and then mixing the mixture in a volume ratio of 5: the mixed solution of petroleum ether and ethyl acetate of 1 is used as eluent to elute, TLC tracking detection is carried out (a developing solvent is the mixed solution of petroleum ether and ethyl acetate with the volume ratio of 5. ¹ H NMR was the same as in example 2.

Example 7: preparation of Compound (Ib)

Dissolving 2.968g (10.0 mmol) of triphosgene in 40mL of toluene, dropwise adding 20mL of toluene solution containing 1.121g (10.0 mmol) of 3, 6-dimethyl-1, 6-dihydro-1, 2,4, 5-tetrazine (II) and 1.012g (10.0 mmol) of triethylamine under the condition of magnetic stirring at 0 ℃, heating the reaction solution to room temperature after dropwise adding, reacting for 8 hours at room temperature (the reaction process adopts TLC tracking detection, and a developing agent is a mixed solution of petroleum ether and ethyl acetate in a volume ratio of 1).

Dissolving 3.072g (10.0 mmol) of compound (IIIb) and 1.012g (10.0 mmol) of triethylamine in 40mL of toluene, dropwise adding the reaction solution obtained in the previous step under the condition of magnetic stirring at 0 ℃, heating the reaction solution to room temperature after dropwise adding, reacting at room temperature for 11 hours (the reaction process adopts TLC tracking detection, a developing agent is a mixed solution of petroleum ether and ethyl acetate with a volume ratio of 3:1, eluting by using a mixed solution of petroleum ether and ethyl acetate as an eluent, and performing TLC tracking detection (a developing solvent comprises petroleum ether and ethyl acetate in a volume ratio of 3Ethyl acetate mixed solution), collecting the eluent containing the compound shown in the formula (Ib) according to TLC detection, evaporating the solvent from the collected eluent, and drying to obtain a white solid product, namely the compound (Ib), with the yield of 60% and the melting point of 189-191 ℃. ¹ H NMR(500MHz,DMSO-d ₆ )δ9.84(s,1H),9.31(s,1H),9.03(s,1H),8.03-96(m,2H),7.69-7.54(m,2H),7.52(d,J＝15.7Hz,1H),7.43(dd,J＝8.8,2.4Hz,1H),7.35(t,J＝7.9Hz,1H),7.28(d,J＝7.7Hz,1H),7.09(d,J＝15.7Hz,1H),2.21(s,3H),1.95(s,3H)； ¹³ C NMR(125MHz,DMSO-d ₆ )δ164.0,154.8,149.2,141.2,141.2,139.2,134.9,134.2,129.2,129.0,128.9,127.5,126.5,126.4,123.2,121.6,121.5,118.1,17.7,15.5.HRMS(ESI)m/z[M+Na] ⁺ calcd for C ₂₀ H ₁₈ Cl ₂ N ₆ NaO ₂ :467.0766,found:467.0761.

Example 8: preparation of Compound (ic)

Dissolving 2.968g (10.0 mmol) of triphosgene in 40mL of toluene, dropwise adding 20mL of toluene solution containing 1.121g (10.0 mmol) of 3, 6-dimethyl-1, 6-dihydro-1, 2,4, 5-tetrazine (II) and 1.012g (10.0 mmol) of triethylamine under the condition of magnetic stirring at 0 ℃, heating the reaction solution to room temperature after completing the dropwise adding, reacting for 8 hours at the room temperature (the reaction process adopts TLC tracking detection, a mixed solution of petroleum ether and ethyl acetate with a developing agent of volume ratio 1), introducing nitrogen into the reaction system, absorbing gas by using a tail gas absorbing device (the tail gas absorbing solution is 10 NaOH aqueous solution) until white fog is not generated in the tail gas absorbing solution, and directly using the generated product for the next reaction.

Dissolving 2.663g (10.0 mmol) of compound (IIIc) and 1.012g (10.0 mmol) of triethylamine in 40mL of toluene, dropwise adding the reaction solution obtained in the previous step under the condition of magnetic stirring at 0 ℃, heating the reaction solution to room temperature after completing dropwise adding, reacting for 9 hours at room temperature (the reaction process adopts TLC tracking detection, a developer is a mixed solution of petroleum ether and ethyl acetate with a volume ratio of 3Evaporating the solvent to obtain a mixture of the dried residue and silica gel, loading the mixture into a column, and then mixing the dried residue and the silica gel in a volume ratio of 3: eluting with a mixed solution of petroleum ether and ethyl acetate as an eluent, tracking and detecting by TLC (a developing solvent is a mixed solution of petroleum ether and ethyl acetate with a volume ratio of 3. ¹ H NMR(500MHz,DMSO-d ₆ )δ9.47(s,1H),9.32(s,1H),9.03(s,1H),8.01(s,1H),7.60(d,J＝8.8Hz,1H),7.52(d,J＝15.7Hz,1H),7.46(d,J＝8.0Hz,1H),7.35(d,J＝7.9Hz,1H),7.27(d,J＝7.6Hz,1H),7.03(s,1H),6.98(d,J＝15.7Hz,1H),6.96(s,1H),2.24(s,3H),2.22(s,3H),2.21(s,3H),1.97(s,3H)； ¹³ C NMR(125MHz,DMSO-d ₆ )δ163.7,155.1,149.5,141.5,140.0,139.4,135.4,134.2,134.0,131.2,131.0,129.4,126.6,124.7,123.2,122.6,121.4,118.2,20.7,18.1,17.9,15.7.HRMS(ESI)m/z[M+Na] ⁺ calcd for C ₂₂ H ₂₄ N ₆ NaO ₂ :427.1858,found:427.1860.

Example 9: preparation of Compound (Id)

Dissolving 2.683g (10.0 mmol) of the compound (IIId) and 1.012g (10.0 mmol) of triethylamine in 40mL of toluene, dropwise adding the reaction solution obtained in the previous step under the condition of magnetic stirring at 0 ℃, heating the reaction solution to room temperature after dropwise adding, reacting at room temperature for 9 hours (the reaction process adopts TLC tracking detection, and the developing agent is a mixed solution of petroleum ether and ethyl acetate with a volume ratio of 3X 3) water washing, separating an organic phase, evaporating to remove a solvent, performing residue column chromatography, namely adding 10ml of ethyl acetate solvent into the residue after the solvent is evaporated to dissolve the residue to obtain a dissolved solution, adding 4.0 g of silica gel (300-400 meshes of coarse (zcx.II) type column chromatography silica gel) into the dissolved solution, uniformly mixing, evaporating to remove the solvent to obtain a mixture of dry residue and silica gel, filling the mixture into a column, and then performing volume ratio of 3: eluting with a mixed solution of petroleum ether and ethyl acetate as eluent, tracking and detecting by TLC (the developing solvent is a mixed solution of petroleum ether and ethyl acetate with a volume ratio of 3. ¹ H NMR(500MHz,DMSO-d ₆ )δ9.44(s,1H),9.31(s,1H),9.00(s,1H),8.15(d,J＝7.8Hz,1H),7.94(s,1H),7.65-7.55(m,1H),7.49(d,J＝15.6Hz,1H),7.34(t,J＝7.8Hz,1H),7.27(d,J＝7.7Hz,1H),7.18(d,J＝15.6Hz,1H),7.11-7.06(m 2H),6.99-6.90(m,1H),3.86(s,3H),2.20(s,3H),1.95(s,3H)； ¹³ CNMR(125MHz,DMSO-d ₆ )δ163.7,154.9,149.4,149.3,141.3,140.0,139.2,135.3,129.2,127.6,124.3,123.0,122.9,121.6,121.3,120.3,118.4,111.1,55.7,17.8,15.6.HRMS(ESI)m/z[M+Na] ⁺ calcd for C ₂₁ H ₂₂ N ₆ NaO ₃ :429.1651,found:429.1649.

Example 10: preparation of Compound (ie)

40mL of toluene were dissolved in 2.523g (10.0 mmol) of the compound (IIIe) and 1.012g (10.0 mmol) of tris (hydroxymethyl) phosphonium chlorideEthylamine, under the condition of magnetic stirring at 0 ℃, dropwise adding the reaction solution obtained in the previous step, heating the reaction solution to room temperature after dropwise adding, reacting at room temperature for 9 hours (the reaction process adopts TLC tracking detection, a developing agent is a mixed solution of petroleum ether and ethyl acetate with a volume ratio of 3:1 is used as eluent to elute, TLC tracking detection is carried out (the developing solvent is the mixed solution of petroleum ether and ethyl acetate with the volume ratio of 3. ¹ H NMR(500MHz,DMSO-d ₆ )δ9.53(s,1H),9.32(s,1H),9.03(s,1H),8.01(s,1H),7.60-7.57(m,2H),7.54(d,J＝15.7Hz,1H),7.36(t,J＝7.8Hz,1H),7.28-7.13(m,3H),7.08(d,J＝15.7Hz,1H),7.06-7.01(m,1H),2.26(s,3H),2.22(s,3H),1.96(s,3H)； ¹³ C NMR(125MHz,DMSO-d ₆ )δ163.7,154.9,149.4,141.3,140.1,139.3,136.5,135.3,131.1,130.4,129.3,126.0,125.0,124.5,123.1,122.4,121.3,118.1,18.0,17.8,15.6.HRMS(ESI)m/z[M+Na] ⁺ calcd for C ₂₁ H ₂₂ N ₆ NaO ₂ :413.1702,found:413.1702.

Example 11: preparation of Compound (if)

Dissolving 2.663g (10.0 mmol) of compound (IIIf) and 1.012g (10.0 mmol) of triethylamine in 40mL of toluene, dropwise adding the reaction solution obtained in the previous step under the condition of magnetic stirring at 0 ℃, heating the reaction solution to room temperature after dropwise adding, reacting for 9 hours at room temperature (the reaction process adopts TLC tracking detection, a developing agent is a mixed solution of petroleum ether and ethyl acetate with a volume ratio of 3: the mixed solution of petroleum ether and ethyl acetate of 1 is used as eluent to elute, TLC tracking detection is carried out (a developing solvent is the mixed solution of petroleum ether and ethyl acetate with the volume ratio of 3. ¹ H NMR(500MHz,DMSO-d ₆ )δ10.12(s,1H),9.32(s,1H),9.04(s,1H),8.02(s,1H),7.58-7.55(m,1H),7.51(d,J＝15.6Hz,1H),7.37-7.33(m,3H),7.26(d,J＝7.7Hz,1H),6.82(d,J＝15.6Hz,1H),6.71(s,1H),2.24(s,6H),2.22(s,3H),1.96(s,3H)； ¹³ C NMR(125MHz,DMSO-d ₆ )δ163.3,154.8,149.2,141.2,139.9,139.2,139.1,137.7,135.0,129.2,124.9,123.0,122.4,121.2,117.8,116.9,21.1,17.7,15.5.HRMS(ESI)m/z[M+Na] ⁺ calcdfor C ₂₂ H ₂₄ N ₆ NaO ₂ :427.1858,found:427.1859.

Example 12: preparation of Compound (Ig)

Dissolving 2.523g (10.0 mmol) of compound (III g) and 1.012g (10.0 mmol) of triethylamine in 40mL of toluene, dropwise adding the reaction solution obtained in the previous step under the condition of magnetic stirring at 0 ℃, heating the reaction solution to room temperature after the dropwise addition, reacting for 9 hours at room temperature (the reaction process adopts TLC tracking detection, a developing agent is a mixed solution of petroleum ether and ethyl acetate with a volume ratio of 3: the mixed solution of petroleum ether and ethyl acetate of 1 is used as eluent to elute, TLC tracking detection is carried out (a developing solvent is the mixed solution of petroleum ether and ethyl acetate with the volume ratio of 3. ¹ H NMR(500MHz,DMSO-d ₆ )δ10.18(s,1H),9.31(s,1H),9.03(s,1H),8.02(s,1H),7.62-7.55(m,1H),7.53(d,J＝15.7Hz,1H),7.51-7.48(m,2H),7.35(t,J＝7.9Hz,1H),7.27-7.22(m,1H),7.18(d,J＝15.7Hz,1H),6.88-6.85(m,2H),2.29(s,3H),2.20(s,3H),1.95(s,3H)； ¹³ C NMR(125MHz,DMSO-d ₆ )δ163.3,154.8,149.2,141.2,140.0,139.2,139.1,137.8,135.0,129.1,128.5,124.0,123.0,122.3,121.2,119.6,117.8,116.3,21.1,17.7,15.4.

Example 13: preparation of Compound (ih)

Dissolving 2.563g (10.0 mmol) of compound (IIIh) and 1.012g (10.0 mmol) of triethylamine in 40mL of toluene, dropwise adding the reaction solution obtained in the previous step under the condition of magnetic stirring at 0 ℃, heating the reaction solution to room temperature after dropwise adding, reacting at room temperature for 10 hours (the reaction process adopts TLC tracking detection, a developing agent is a mixed solution of petroleum ether and ethyl acetate with a volume ratio of 3:1 is used as eluent, elution is carried out, TLC tracking detection is carried out (the developing solvent is the mixed solution of petroleum ether and ethyl acetate with the volume ratio of 3. ¹ H NMR(500MHz,DMSO-d ₆ )δ10.34(s,1H),9.32(s,1H),9.04(s,1H),8.04(s,1H),7.75-7.71(m,2H),7.61-7.55(m,1H),7.53(d,J＝15.7Hz,1H),7.36(t,J＝7.8Hz,1H),7.27(d,J＝7.7Hz,1H),7.18(dd,J＝8.8,4.1Hz,2H),6.80(d,J＝15.7Hz,1H),2.22(s,3H),1.96(s,3H)； ¹³ C NMR(125MHz,DMSO-d ₆ )δ163.5,159.2,157.2,155.0,149.4,141.4,140.4,139.4,135.1,129.4,123.3,121.1,121.0,118.0,115.6,115.5,17.9,15.7.HRMS(ESI)m/z[M+Na] ⁺ calcd for C ₂₀ H ₁₉ FN ₆ NaO ₂ :417.1451,found:417.1433.

Example 14: preparation of Compound (II)

Dissolving 2.663g (10.0 mmol) of compound (IIIi) and 1.012g (10.0 mmol) of triethylamine in 40mL of toluene, dropwise adding the reaction solution obtained in the previous step under the condition of magnetic stirring at 0 ℃, heating the reaction solution to room temperature after dropwise adding, reacting for 9 hours at room temperature (the reaction process adopts TLC tracking detection, a developing agent is a mixed solution of petroleum ether and ethyl acetate with a volume ratio of 3: eluting with a mixed solution of petroleum ether and ethyl acetate as eluent, tracking and detecting by TLC (the developing solvent is a mixed solution of petroleum ether and ethyl acetate with a volume ratio of 3. ¹ H NMR(500MHz,DMSO-d ₆ )δ9.34(s,1H),9.02(s,1H),8.20(s,1H),8.04-7.92(m,2H),7.81-7.78(m,2H),7.62-7.57(m,1H),7.54(d,J＝15.7Hz,1H),7.41(t,J＝7.9Hz,1H),7.06-7.02(m,1H),6.99(d,J＝15.7Hz,1H),2.25(s,3H),2.22(s,3H),2.21(s,3H),1.97(s,3H)； ¹³ C NMR(125MHz,DMSO-d ₆ )δ163.2,154.8,149.2,141.2,139.8,139.2,137.0,136.3,135.1,131.1,129.6,129.2,123.0,122.5,121.2,120.3,117.8,116.7,19.6,18.8,17.7,15.5.

Example 15: preparation of Compound (Ij)

Dissolving 2.727g (10.0 mmol) of compound (IIIj) and 1.012g (10.0 mmol) of triethylamine in 40mL of toluene, dropwise adding the reaction solution obtained in the previous step under the condition of magnetic stirring at 0 ℃, heating the reaction solution to room temperature after dropwise adding, reacting at room temperature for 11 hours (the reaction process adopts TLC tracking detection, a developing agent is a mixed solution of petroleum ether and ethyl acetate with a volume ratio of 3: eluting with a mixed solution of petroleum ether and ethyl acetate as an eluent, tracking and detecting by TLC (a developing solvent is a mixed solution of petroleum ether and ethyl acetate in a volume ratio of 3. ¹ H NMR(500MHz,DMSO-d ₆ )δ9.78(s,1H),9.32(s,1H),9.03(s,1H),8.01(s,1H),7.93-7.88(m,1H),7.62-7.54(m,2H),7.52(d,J＝15.7Hz,1H),7.38-7.33(m,2H),7.29(d,J＝7.7Hz,1H),7.20(td,J＝7.9,1.6Hz,1H),7.08(d,J＝15.7Hz,1H),2.22(s,3H),1.95(s,3H)； ¹³ C NMR(125MHz,DMSO-d ₆ )δ163.9,154.8,149.2,141.2,140.8,139.2,135.0,135.0,129.4,129.2,127.4,126.0,125.8,125.6,123.1,121.8,121.4,118.1,17.7,15.5.HRMS(ESI)m/z[M+Na] ⁺ calcd for C ₂₀ H ₁₉ ClN ₆ NaO ₂ :433.1156,found:433.1153.

Example 16: preparation of Compound (Ik)

Dissolving 2.683g (10.0 mmol) of compound (IIIk) and 1.012g (10.0 mmol) of triethylamine in 40mL of toluene, dropwise adding the reaction solution obtained in the previous step under the condition of magnetic stirring at 0 ℃, heating the reaction solution to room temperature after completing dropwise addition, reacting at room temperature for 9 hours (the reaction process is detected by TLC tracking, a developing agent is a mixed solution of petroleum ether and ethyl acetate with a volume ratio of 3: eluting with a mixed solution of petroleum ether and ethyl acetate as an eluent, tracking and detecting by TLC (a developing solvent is a mixed solution of petroleum ether and ethyl acetate with a volume ratio of 3. ¹ H NMR(500MHz,DMSO-d ₆ )δ10.16(s,1H),9.32(s,1H),9.04(s,1H),8.03(s,1H),7.64(d,J＝8.5Hz,2H),7.58(d,J＝8.1Hz,1H),7.52(d,J＝15.7Hz,1H),7.36(t,J＝7.8Hz,1H),7.26(d,J＝7.6Hz,1H),6.92(d,J＝9.0Hz,2H),6.80(d,J＝15.7Hz,1H),3.74(s,3H),2.22(s,3H),1.96(s,3H)； ¹³ C NMR(125MHz,DMSO-d ₆ )δ163.0,155.3,154.9,149.3,141.3,139.7,139.2,135.2,132.6,129.2,123.1,122.5,121.3,120.6,117.8,114.0,55.2,17.8,15.6.

Example 17: preparation of Compound (il)

Dissolving 2.727g (10.0 mmol) of compound (III) and 1.012g (10.0 mmol) of triethylamine in 40mL of toluene, dropwise adding the reaction solution obtained in the previous step under the condition of magnetic stirring at 0 ℃, heating the reaction solution to room temperature after dropwise adding, reacting at room temperature for 11 hours (the reaction process adopts TLC tracking detection, a developing agent is a mixed solution of petroleum ether and ethyl acetate with a volume ratio of 3:1 as eluent, eluting, tracking and detecting by TLC (a developer is a mixed solution of petroleum ether and ethyl acetate with a volume ratio of 3The solvent was evaporated and dried to give the compound (il) as a white solid in 74% yield with a melting point of 169-171 ℃. ¹ H NMR(500MHz,DMSO-d ₆ )δ10.42(s,1H),9.33(s,1H),9.05(s,1H),8.05(s,1H),7.76-7.72(m,2H),7.64-7.57(m,1H),7.54(d,J＝15.7Hz,1H),7.41-7.34(m,3H),7.28(d,J＝7.7Hz,1H),6.80(d,J＝15.7Hz,1H),2.22(s,3H),1.96(s,3H).

Example 18: preparation of Compound (im)

Dissolving 2.727g (10.0 mmol) of compound (IIIm) and 1.012g (10.0 mmol) of triethylamine in 40mL of toluene, dropwise adding the reaction solution obtained in the previous step under the condition of magnetic stirring at 0 ℃, heating the reaction solution to room temperature after completing dropwise addition, reacting at room temperature for 11 hours (the reaction process is detected by TLC tracking, a developing agent is a mixed solution of petroleum ether and ethyl acetate with a volume ratio of 3:1 is used as eluent, elution is carried out, TLC tracking detection is carried out (the developing solvent is the mixed solution of petroleum ether and ethyl acetate with the volume ratio of 3150～152℃。 ¹ H NMR(500MHz,DMSO-d ₆ )δ9.32(s,1H),9.29(s,1H),9.02(s,1H),8.14(d,J＝7.8Hz,1H),7.94(s,1H),7.66-7.59(m,1H),7.51(d,J＝15.6Hz,1H),7.38-7.29(m,2H),7.17(d,J＝15.6Hz,1H),7.07(dd,J＝5.8,2.0Hz,2H),6.94-6.91(m,1H),2.21(s,3H),1.96(s,3H).

Example 19: preparation of Compound (ln)

Dissolving 2.233g (10.0 mmol) of compound (IIIn) and 0.122g (1.0 mmol) of DMAP in 60mL of chloroform, dropwise adding the reaction solution obtained in the previous step under the condition of magnetic stirring at-10 ℃, heating the reaction solution to room temperature after completing dropwise addition, reacting at room temperature for 10 hours (the reaction process is detected by tracking TLC, a developing agent is a mixed solution of petroleum ether and ethyl acetate with a volume ratio of 1: 2), washing the reaction solution with (50 mL x 3) water, separating out an organic phase, evaporating the solvent, performing column chromatography on residues, namely adding 10mL of petroleum ether solvent into the residues after solvent evaporation to dissolve the residues to obtain a dissolved solution, then adding 2.0g of silica gel (300-400 mesh coarse pore (zcx.II) type column chromatography silica gel into the dissolved solution, uniformly mixing, evaporating the solvent to obtain a mixture of dried residues and silica gel, filling the mixture into a column, and then mixing the mixture in a volume ratio of 1: and (2) eluting by using a mixed solution of petroleum ether and ethyl acetate as an eluent, tracking and detecting by TLC (a developing solvent is a mixed solution of petroleum ether and ethyl acetate with a volume ratio of 1. ¹ H NMR(500MHz,DMSO-d ₆ )δ9.32(s,1H),9.00(s,1H),8.18-8.10(m,2H),7.99(s,1H),7.86(d,J＝15.6Hz,1H),7.78(dd,J＝8.1,1.3Hz,1H),7.71-7.66(m,2H),7.58-7.55(m,2H),7.53(d,J＝15.6Hz,1H),7.38(t,J＝7.9Hz,1H),2.20(s,3H),1.95(s,3H)； ¹³ C NMR(125MHz,DMSO-d ₆ )δ189.2,154.9,149.2,144.0,141.2,139.1,137.5,134.9,133.1,129.1,128.8,128.4,123.6,122.2,122.1,120.0,17.7,15.5.

Example 20: preparation of Compound (ln)

Dissolving 6.698g (30.0 mmol) of compound (IIIn) and 2.373g (30.0 mmol) of pyridine in 200mL of dichloromethane, dropwise adding the reaction solution obtained in the previous step under the condition of magnetic stirring at 12 ℃, heating the reaction solution to room temperature after dropwise adding, reacting at room temperature for 0.5 hour (the reaction process adopts TLC tracking detection, a developing agent is a mixed solution of petroleum ether and ethyl acetate with a volume ratio of 20:1 as eluent, eluting, tracking and detecting by TLC (the developing solvent is a mixed solution of petroleum ether and ethyl acetate with a volume ratio of 20(ln), yield 72%, melting point 136-138 ℃. ¹ H NMR and ¹³ c NMR was the same as in example 19.

Example 21: preparation of Compound (ln)

Dissolving 4.465g (20.0 mmol) of compound (IIIn) and 2.024g (20.0 mmol) of triethylamine in 50mL of toluene, dropwise adding the reaction solution obtained in the previous step under the condition of magnetic stirring at 0 ℃, heating the reaction solution to room temperature after dropwise adding, reacting at room temperature for 8 hours (the reaction process adopts TLC tracking detection, a developing agent is a mixed solution of petroleum ether and ethyl acetate with a volume ratio of 10:1 is used as eluent, elution is carried out, tracking detection is carried out by TLC (a developing solvent is a mixed solution of petroleum ether and ethyl acetate with the volume ratio of 10. ¹ H NMR and ¹³ c NMR was the same as in example 19.

Example 22: preparation of Compound (ln)

Dissolving 0.223g (1.0 mmol) of compound (IIIn) and 0.200g (5.0 mmol) of sodium hydroxide in 20mL of chloroform, dropwise adding the reaction solution obtained in the previous step under the condition of magnetic stirring at 5 ℃, heating the reaction solution to room temperature after dropwise adding, reacting at room temperature for 50 hours (the reaction process adopts TLC tracking detection, a developing agent is a mixed solution of petroleum ether and ethyl acetate with a volume ratio of 5: the mixed solution of petroleum ether and ethyl acetate of 1 is used as eluent to elute, TLC tracing detection is carried out (a developing solvent is the mixed solution of petroleum ether and ethyl acetate with the volume ratio of 5. ¹ H NMR and ¹³ c NMR was the same as in example 19.

Example 23: preparation of Compound (ln)

Dissolving 2.233g (10.0 mmol) of compound (IIIn) and 1.012g (10.0 mmol) of triethylamine in 60mL of dichloromethane under the condition of magnetic stirring at-5 ℃, dropwise adding the reaction solution obtained in the previous step, heating the reaction solution to room temperature after completing dropwise addition, reacting at room temperature for 9 hours (the reaction process is detected by TLC tracking, a developing agent is a mixed solution of petroleum ether and ethyl acetate with a volume ratio of 5:1 is used as eluent, elution is carried out, TLC tracking detection is carried out (the developing solvent is the mixed solution of petroleum ether and ethyl acetate with the volume ratio of 5. ¹ H NMR and ¹³ c NMR was the same as in example 19.

Example 24: preparation of Compound (Io)

Dissolving 2.413g (10.0 mmol) of compound (IIIo) and 1.012g (10.0 mmol) of triethylamine in 40mL of toluene, dropwise adding the reaction solution obtained in the previous step under the condition of magnetic stirring at 0 ℃, heating the reaction solution to room temperature after completing dropwise addition, reacting at room temperature for 11 hours (the reaction process is detected by TLC tracking, a developer is a mixed solution of petroleum ether and ethyl acetate with a volume ratio of 5: 1), washing the reaction solution with (50 mL x 3) water, separating out an organic phase, evaporating the solvent, performing column chromatography on the residue, namely adding 10mL of ethyl acetate solvent into the residue after solvent evaporation to dissolve the residue to obtain a dissolved solution, then adding 3.5 g of silica gel (300-400 mesh coarse pore (zcx.II) type column chromatography silica gel into the dissolved solution, uniformly mixing, evaporating the solvent to obtain a mixture of dried residue and silica gel, packing the mixture into a column, and then mixing the volume ratio of the mixture is 5:1 is used as eluent, elution is carried out, TLC tracking detection is carried out (the developing solvent is the mixed solution of petroleum ether and ethyl acetate with the volume ratio of 5. ¹ H NMR(500MHz,DMSO-d ₆ )δ9.38(s,1H),9.29(s,1H),8.14(d,J＝8.9Hz,2H),7.98(dd,J＝8.7,5.6Hz,2H),7.93(d,J＝15.6Hz,1H),7.84(d,J＝8.9Hz,2H),7.72(d,J＝15.6Hz,1H),7.30(t,J＝8.8Hz,2H),2.21(s,3H),1.96(s,3H)； ¹³ C NMR(125MHz,DMSO-d ₆ )δ187.5,162.4,155.2,149.0,143.5,142.1,141.1,131.9,131.6,131.3,131.2,129.8,122.0,119.0,116.1,115.9,17.8,15.6.

Example 25: preparation of Compound (IP)

Dissolving 2.373g (10.0 mmol) of compound (IIIp) and 1.012g (10.0 mmol) of triethylamine in 40mL of toluene, dropwise adding the reaction solution obtained in the previous step under the condition of magnetic stirring at 0 ℃, heating the reaction solution to room temperature after the completion of the dropwise addition, reacting at room temperature for 10 hours (the reaction process is detected by TLC tracking, a developer is a mixed solution of petroleum ether and ethyl acetate with a volume ratio of 5): the mixed solution of petroleum ether and ethyl acetate of 1 is used as eluent to elute, TLC tracking detection is carried out (a developing solvent is the mixed solution of petroleum ether and ethyl acetate with the volume ratio of 5. ¹ H NMR(500MHz,DMSO-d ₆ )δ9.33(s,1H),9.00(s,1H),8.05(d,J＝8.2Hz,2H),7.99(s,1H),7.86(d,J＝15.6Hz,1H),7.79(dd,J＝8.1,1.2Hz,1H),7.67(d,J＝15.6Hz,1H),7.55(d,J＝7.7Hz,1H),7.39(s,2H),7.37(s,1H),2.40(s,3H),2.22(s,3H),1.97(s,3H)； ¹³ C NMR(125MHz,DMSO-d ₆ )δ188.5,154.8,149.2,143.6,142.5,141.2,139.1,135.0,129.3,129.1,128.8,128.6,123.5,122.1,122.0,119.9,21.1,17.7,15.5.HRMS(ESI)m/z[M+Na] ⁺ calcd for C ₂₁ H ₂₁ N ₅ NaO ₂ :398.1593,found:398.1587.

Example 26: preparation of Compound (Iq)

Dissolving 2.577g (10.0 mmol) of compound (IIIq) and 1.012g (10.0 mmol) of triethylamine in 40mL of toluene, dropwise adding the reaction solution obtained in the previous step under the condition of magnetic stirring at 0 ℃, heating the reaction solution to room temperature after dropwise adding, reacting at room temperature for 12 hours (the reaction process adopts TLC tracking detection, a developing agent is a mixed solution of petroleum ether and ethyl acetate with a volume ratio of 5: the mixed solution of petroleum ether and ethyl acetate of 1 is used as an eluent to elute, TLC tracking detection is carried out (a developing solvent is the mixed solution of petroleum ether and ethyl acetate with the volume ratio of 5. ¹ H NMR(500MHz,DMSO-d ₆ )δ9.31(s,1H),9.01(s,1H),7.92(s,1H),7.83-7.76(m,1H),7.58-7.55(m,3H),7.51-7.48(m,1H),7.43(d,J＝7.8Hz,1H),7.37(d,J＝8.1Hz,1H),7.34(d,J＝16.1Hz,1H),7.18(d,J＝16.1Hz,1H),2.18(s,3H),1.94(s,3H)； ¹³ C NMR(125MHz,DMSO-d ₆ )δ193.3,154.9,149.3,146.4,141.3,139.3,138.6,134.4,131.9,130.1,129.9,129.3,129.2,127.5,122.4,119.8,17.8,15.5,14.0.HRMS(ESI)m/z[M+Na] ⁺ calcdfor C ₂₀ H ₁₈ ClN ₅ NaO ₂ :418.1047,found:418.1051.

Example 27: preparation of Compound (ir)

Dissolving 2.577g (10.0 mmol) of compound (IIIr) and 1.012g (10.0 mmol) of triethylamine in 40mL of toluene, dropwise adding the reaction solution obtained in the previous step under the condition of magnetic stirring at 0 ℃, heating the reaction solution to room temperature after dropwise adding, reacting at room temperature for 12 hours (the reaction process adopts TLC tracking detection, a developing agent is a mixed solution of petroleum ether and ethyl acetate with a volume ratio of 5:1 is used as eluent to elute, TLC tracking detection is carried out (the developing solvent is the mixed solution of petroleum ether and ethyl acetate with the volume ratio of 5. ¹ H NMR(500MHz,DMSO-d ₆ )δ9.32(s,1H),9.02(s,1H),7.93(s,1H),7.78(dd,J＝8.1,1.1Hz,1H),7.62-7.55(m,3H),7.50(td,J＝7.3,1.4Hz,1H),7.44(d,J＝7.8Hz,1H),7.38-7.33(m,1H),7.32(d,J＝16.1Hz,1H),7.19(d,J＝16.1Hz,1H),2.20(s,3H),1.95(s,3H)； ¹³ C NMR(125MHz,DMSO-d ₆ )δ193.3,154.9,149.3,146.4,141.3,139.3,138.7,134.4,131.9,130.1,129.9,129.3,129.2,127.5,126.4,123.5,122.4,119.8,17.8,15.6.HRMS(ESI)m/z[M+Na] ⁺ calcd for C ₂₀ H ₁₈ ClN ₅ NaO ₂ :418.1047,found:418.1048.

Example 28: preparation of Compound (Is)

Dissolving 2.533g (10.0 mmol) of compound (IIIs) and 1.012g (10.0 mmol) of triethylamine in 40mL of toluene, dropwise adding the reaction solution obtained in the previous step under the condition of magnetic stirring at 0 ℃, heating the reaction solution to room temperature after the dropwise addition, reacting at room temperature for 10 hours (the reaction process adopts TLC tracking detection, a developing agent is a mixed solution of petroleum ether and ethyl acetate with a volume ratio of 5): 1 is used as eluent, elution is carried out, TLC tracking detection is carried out (the developing solvent is the mixed solution of petroleum ether and ethyl acetate with the volume ratio of 5. ¹ H NMR(500MHz,DMSO-d ₆ )δ9.33(s,1H),9.00(s,1H),8.18-8.14(m,2H),7.98(s,1H),7.87(d,J＝15.6Hz,1H),7.78(dd,J＝8.1,1.2Hz,1H),7.65(d,J＝15.6Hz,1H),7.55(d,J＝7.7Hz,1H),7.38(t,J＝7.9Hz,1H),7.11-7.08(m,2H),3.87(s,3H),2.21(s,3H),1.97(s,3H)； ¹³ C NMR(125MHz,DMSO-d ₆ )δ187.4,163.4,155.0,149.4,143.2,141.4,139.2,135.2,131.0,130.5,129.3,123.6,122.3,122.0,120.1,114.2,55.7,17.8,15.7.

Example 29: preparation of Compound (IT)

Dissolving 2.393g (10.0 mmol) of compound (IIIt) and 1.012g (10.0 mmol) of triethylamine in 40mL of toluene, dropwise adding the reaction solution obtained in the previous step under the condition of magnetic stirring at 0 ℃, heating the reaction solution to room temperature after the dropwise addition, reacting at room temperature for 10 hours (the reaction process adopts TLC tracking detection, a developing agent is a mixed solution of petroleum ether and ethyl acetate with a volume ratio of 5): 1 is used as eluent, elution is carried out, TLC tracking detection is carried out (the developing solvent is the mixed solution of petroleum ether and ethyl acetate with the volume ratio of 5. ¹ H NMR(500MHz,DMSO-d ₆ )δ12.41(s,1H),9.34(s,1H),9.02(s,1H),8.20(dd,J＝8.3,1.6Hz,1H),8.04-7.92(m,2H),7.79(d,J＝8.4Hz,2H),7.62-7.57(m,1H),7.54(d,J＝15.6Hz,1H),7.41(t,J＝7.9Hz,1H),7.06-7.02(m,1H),7.01(d,J＝15.6Hz,1H),2.21(s,3H),1.97(s,3H).

Example 30: preparation of Compound (iu)

Dissolving 3.022g (10.0 mmol) of compound (IIIu) and 1.012g (10.0 mmol) of triethylamine in 40mL of toluene, dropwise adding the reaction solution obtained in the previous step under the condition of magnetic stirring at 0 ℃, heating the reaction solution to room temperature after dropwise adding, reacting at room temperature for 12 hours (the reaction process adopts TLC tracking detection, a developing agent is a mixed solution of petroleum ether and ethyl acetate with a volume ratio of 5: the mixed solution of petroleum ether and ethyl acetate of 1 is used as eluent to elute, TLC tracking detection is carried out (a developing solvent is the mixed solution of petroleum ether and ethyl acetate with the volume ratio of 5. ¹ H NMR(500MHz,DMSO-d ₆ )δ9.33(s,1H),9.00(s,1H),8.27-8.22(m,2H),7.99(s,1H),7.88(d,J＝15.6Hz,1H),7.78(dd,J＝8.1,1.3Hz,1H),7.69(d,J＝15.6Hz,1H),7.57(d,J＝7.8Hz,1H),7.43-7.37(m,3H),2.20(s,3H),1.96(s,3H).

Example 30: preparation of Compound (iv)

Dissolving 2.983g (10.0 mmol) of compound (IIIv) and 1.012g (10.0 mmol) of triethylamine in 40mL of toluene, dropwise adding the reaction solution obtained in the previous step under the condition of magnetic stirring at 0 ℃, heating the reaction solution to room temperature after completing dropwise addition, reacting at room temperature for 9 hours (the reaction process is detected by TLC tracking, a developing agent is a mixed solution of petroleum ether and ethyl acetate with a volume ratio of 3: eluting with a mixed solution of petroleum ether and ethyl acetate as eluent, tracking and detecting by TLC (the developing solvent is a mixed solution of petroleum ether and ethyl acetate with a volume ratio of 3. ¹ H NMR(500MHz,DMSO-d ₆ )δ10.16(s,1H),9.32(s,1H),9.04(s,1H),8.02(s,1H),7.57(dd,J＝8.1,1.1Hz,1H),7.50(d,J＝15.7Hz,1H),7.42(d,J＝2.3Hz,1H),7.35(t,J＝7.9Hz,1H),7.27-7.19(m,2H),6.91(d,J＝8.8Hz,1H),6.79(d,J＝15.7Hz,1H),3.76(s,3H),3.74(s,3H),2.21(s,3H),1.95(s,3H).

Example 31: preparation of Compound (Ia)

Dissolving 2.383g (10.0 mmol) of compound (IIIa) and 1.012g (10.0 mmol) of triethylamine in 40mL of toluene, dropwise adding the reaction solution obtained in the previous step under the condition of magnetic stirring at 0 ℃, heating the reaction solution to room temperature after the dropwise addition, reacting for 9 hours at room temperature (the reaction process adopts TLC tracking detection, a developing agent is a mixed solution of petroleum ether and ethyl acetate with a volume ratio of 3: eluting with a mixed solution of petroleum ether and ethyl acetate as an eluent, tracking and detecting by TLC (a developing solvent is a mixed solution of petroleum ether and ethyl acetate with a volume ratio of 3. ¹ H NMR as in example 2.

Example 32: preparation of Compound (ln)

Dissolving 2.233g (10.0 mmol) of compound (IIIn) and 1.012g (10.0 mmol) of triethylamine in 40mL of toluene, dropwise adding the reaction solution obtained in the previous step under the condition of magnetic stirring at 0 ℃, heating the reaction solution to room temperature after the dropwise addition, reacting at room temperature for 10 hours (the reaction process adopts TLC tracking detection, a developing agent is a mixed solution of petroleum ether and ethyl acetate with a volume ratio of 5): eluting with a mixed solution of petroleum ether and ethyl acetate as an eluent, tracking and detecting by TLC (a developing solvent is a mixed solution of petroleum ether and ethyl acetate with a volume ratio of 5. ¹ H NMR and ¹³ c NMR was the same as in example 19.

Example 33: in vitro kinase Activity assay

Kinase-Lumi ^TM A chemical luminescence method kinase activity detection reagent kit is a reagent kit for quantitatively detecting the kinase activity by measuring the residual amount of ATP in a solution after kinase reaction by a chemical luminescence method. Kinase-Lumi is adopted in the experiment ^TM The compounds (Ib), (ie), (ih), (ij), (ik) and (io) prepared in the above examples were tested for their inhibitory activities against EGFR, PEGFR beta and VEGFR-2 kinase, respectively, at room temperature using a chemiluminescence method kinase activity assay kit (Beyotime).

The following is a 96-well plate recommended detection system.

(1) Preparation of ATP Standard Curve

The reaction buffer was prepared with 1mM manganese dichloride, 5mM magnesium dichloride, 1mM Dithiothreitol (DTT).

Set 0, 0.03, 0.07, 0.15, 0.3, 0.6, 1.25, 2.5, 5, 10 μ MATP standard wells (all ATP concentrations above are final concentrations of the substance when the total volume in the standard wells reaches 100 μ L). For preparation, 50. Mu.L of ATP was first diluted with reaction buffer. Then 50 mu Lkinase-Lumi is added ^TM And (3) mixing the chemiluminescence kinase detection reagent and the mixture. After reaction at room temperature (about 25 ℃) for 10 minutes, chemiluminescence detection was carried out using a multifunctional microplate reader, and an ATP standard curve was prepared.

(2) Sample detection

Sample wells were configured to contain 0.1. Mu.g/mL polyglutamic acid and tyrosine (4) kinase substrates, 5. Mu.MATP and 10. Mu.g/L kinase (EGFR, PEGFR. Beta. Or VEGFR-2), and different concentrations (50, 100, 200, 400, 800, 1000 nM) of compounds (Ib), (ie), (ih), (ij), (ik), (io) when the total volume per well reached 100. Mu.L. When in preparation, polyglutamic acid, tyrosine (4), a kinase substrate, ATP, kinase (EGFR, PEGFR beta or VEGFR-2) and compounds (Ib), (ie), (ih), (ij), (ik) and (Io) are added into each hole, and reaction buffer is added to dilute the mixture until the total volume is 50 mu L; then 50 mu Lkinase-Lumi is added ^TM And (3) mixing the chemiluminescence kinase detection reagent and the mixture. The reaction was carried out at room temperature (about 25 ℃ C.) for 10 minutes. Then, chemiluminescence detection is carried out by using a multifunctional microplate reader. The amount of ATP remaining in the sample wells was calculated from the standard curve, and then the enzyme activity was calculated according to the definition of enzyme activity. Finally calculate the IC ₅₀ The value is obtained.

In vitro kinase Activity assay results are shown in Table 1, which shows data on the activity of compounds (Ib), (ie), (ih), (ij), (ik), (io) on EGFR, PEGFR β and VEGFR-2 kinase. It can be seen from the data in the table that compounds (Ib), (ie), (ih), (ij), (ik), (io) have different degrees of inhibitory effect on the activity of EGFR, PEGFR β and VEGFR-2, and that the inhibitory activity of compounds (Ib), (ie), (ih), (ij), (ik) and (io) on VEGFR-2 kinase is overall superior to that of EGFR and PDGFR β.

TABLE 1 tyrosine kinase inhibitory Activity of Compound (I)

Example 20: in vitro test for anti-cancer Activity

The prepared compounds (Ia) to (IV) were tested for the biological activities of human lung cancer cell A549, human hepatoma cell Huh7 and human hepatoma cell HepG2, respectively, and the compounds (Ia) and (In) were prepared by the methods of example 31 and example 32, respectively.

The test method comprises the following steps: tetrazolium salt reduction (MTT process).

Cell lines: human lung cancer cell A549, human liver cancer cell Huh7 and human liver cancer cell HepG2. The cell lines are purchased from cell banks of Shanghai Life sciences academy of sciences of China.

The experimental procedure was as follows:

(1) Culture of tumor cells

The cell generation number used in the experiment is within 5 generations. The consumables and reagents used for cell culture are subjected to strict sterilization procedures, and all experimental procedures are performed in a sterile operating platform.

(a) Resuscitation of tumor cells

Before the experiment, the articles to be used, such as a culture bottle, a centrifuge tube, a liquid-transferring gun, a gun head, a waste liquid barrel and the like, are placed in a super clean bench, an ultraviolet lamp is turned on for sterilization, and the reagents used for cell culture are placed in a 37 ℃ constant-temperature water bath kettle for preheating. And after all the work is ready, the ultraviolet lamp is turned off, and the super-clean bench fluorescent lamp and the ventilating fan are turned on. Taking out the frozen tumor cells from a refrigerator at the temperature of minus 80 ℃, shaking the tumor cells in a water bath at the temperature of 37 ℃ to quickly thaw the tumor cells, spraying alcohol to the tumor cells and putting the tumor cells into a super clean bench under the condition that a small part of frozen stock solution in the frozen tube is still unfrozenImmediately, the cells in the cryopreserved tube were transferred to a 15mL centrifuge tube containing 1640 culture medium, and gently pipetted and mixed by using a pipette gun. Placing the centrifugal tube in a centrifuge, centrifuging at 1000rpm for 5min, and sucking off the supernatant in a super clean bench. Sucking 2mL1640 culture solution into a centrifuge tube containing cell sediment by using a pipette to prepare cell suspension, blowing, beating and uniformly mixing the cell suspension, and transferring the cell suspension to a bottle with the bottom area of 25cm ² The air-permeable flask of (4) was supplemented with 1640 medium solution 4mL, the cells were mixed by gently shaking the flask, and the flask was charged to 5% CO ₂ And culturing in a constant-temperature incubator at 37 ℃.

(b) Passage of tumor cells

When the cell growth state is good and the bottom of the culture bottle is paved by 70-80%, the cell can be passaged. The passage operation steps are all completed in a super clean bench, and the passage can be started after the preparation work is done. Firstly, sucking the original culture solution in a culture bottle off by a pipette gun and beating the culture solution into a waste solution barrel, adding 2mL of PBS for repeated washing for several times, then adding about 600-700 mu L of trypsin (containing 0.02% of EDTA, phenol red and 0.25% of pancreatin) to ensure that the bottom of the bottle is completely covered by the trypsin, slightly shaking the culture bottle, putting the culture bottle into a constant-temperature incubator at 37 ℃ for incubation for 1-2 min, observing the cell shedding condition under a microscope, and slightly beating the bottle wall by using a finger belly if a small amount of cells are still attached to the wall until most of the cells can shed from the bottom of the bottle. After the digestion is stopped by adding 2mL of 1640 culture solution, the cells are gently blown out by a pipette, the cells are all blown off from the bottom of the bottle, and the cell suspension is transferred to a 15mL centrifuge tube and centrifuged at 1000rpm for 5min. The supernatant is aspirated off, diluted with 1640 medium and blown evenly, then distributed evenly into 2-3 flasks and continuously treated with 5% CO ₂ And culturing in a constant-temperature incubator at 37 ℃.

(c) Cryopreservation of tumor cells

A cell culture solution was prepared in advance in a volume ratio of FBS (fetal bovine serum): DMSO =9, and placed in a refrigerator at 4 ℃ for use. And (4) freezing and storing operation steps are finished in the super clean bench, and freezing and storing can be started after preparation work is done. The original culture solution in the culture bottle is firstly sucked off, 2mL of PBS buffer solution is added for repeated washing and pouring off for several times, then trypsin (containing 0.02 percent of EDTA, phenol red and 0.25 percent of pancreatin) of about 600-700 mu L is added, the culture bottle is gently shaken to ensure that the pancreatin can cover the bottom of the culture bottle, the culture bottle is put into a constant-temperature incubator at 37 ℃ for incubation for 1-2 min, the cell shedding condition is observed under a microscope, if a small amount of cells are still attached to the wall, the bottle wall can be lightly knocked by using a finger belly until most of the cells can shed from the bottle bottom. After the digestion was terminated by adding 2mL of 1640 medium, the cells were gently pipetted using a pipette gun, the cells were completely blown off the bottom of the flask, and the cell suspension was transferred to a 15mL centrifuge tube and centrifuged at 1000rpm for 5min. The supernatant was decanted off. 1mL of the cryopreservation solution just taken out of the refrigerator at 4 ℃ was added, and the mixture was blown up uniformly to form a cell suspension, which was transferred into a cryopreservation tube. Standing at 4 deg.C for 30min after labeling cell types, cell generation number and freezing storage date, standing at-20 deg.C for 1 hr, and storing in-80 deg.C ultra-low temperature refrigerator.

(2) MTT experimental method

(a) Cell counting: digesting and centrifuging tumor cells with good growth state in a culture flask, then re-suspending with 4mL1640 culture solution, taking 10 mu L cell suspension to a cell counting plate, counting, diluting B16F10 cells to 5 multiplied by 10 ⁴ one/mL.

(b) Plate paving: taking a 96-well plate, adding 100 mu L of diluted cell suspension into an experimental well, adding 100 mu L of 1640 culture solution into a blank well, supplementing 100 mu L of LPBS buffer solution around the blank well, and adding CO ₂ Culturing in a constant temperature incubator.

(c) Preparing a compound and adding medicine: the compound prepared by DMSO in 10. Mu. Mol/mL and the positive control drug Sorafenib were diluted to the specified concentrations in 1640 culture medium, 40. Mu.M, 20. Mu.M, 10. Mu.M, 5. Mu.M, 2.5. Mu.M and 1.25. Mu.M, respectively, and applied by the solution change method. Discarding the original culture solution, adding 100 μ L1640 culture solution containing compound or positive control drug into the experimental well, adding 100 μ L1640 culture solution into the control group and blank well, adding the drug, and adding the 96-well plate into 5% CO ₂ And continuously culturing in a constant-temperature incubator at 37 ℃.

(d) Adding MTT: after 48 hours, the 96-well plate was taken out and placed in a clean bench, and 10. Mu.L of 5mg/mL MTT solution was added to each well in the dark, followed by 5% CO ₂ And continuously culturing in a constant-temperature incubator at 37 ℃.

(e) And (3) detection: the 96-well plate added with MTT is incubated in the incubator for 3.5-4 h and taken out, the solution in each well is carefully sucked out, 150 mu L of DMSO is added into each well to dissolve generated formazan, then the formazan is placed on a flat plate oscillator to vibrate for 20min, and an enzyme-labeling instrument is used for detecting the absorbance value under 490 nm.

(f) And (3) processing experimental data: the cell viability was calculated according to the following formula, and the value of 50% of the cell viability was IC ₅₀ 。

Cell survival (%) = [ (As-Ab)/(Ac-Ab) ]. Times.100%

As assay well (cell-containing culture solution, MTT, toxic substance)

Ac control well (cell culture, MTT, no toxic substance)

Ab blank well (MTT-containing, cell and toxic free medium culture).

The results of the tests are shown in tables 2 and 3:

TABLE 2 inhibitory Effect of Compound (I) on cancer cell growth

TABLE 3 inhibitory Effect of Compound (I) on cancer cell growth

Claims

1. A cinnamoyl tetrazine compound is characterized in that: the cinnamoyl tetrazine compound has the following structural formula (I):

or n =0, R is H, 4-F, 4-CH ₃ 、4-Cl、2-Cl、4-OCH ₃ 2-OH or 3-Br.

2. A method of preparing the cinnamoyl tetrazine compound of claim 1, comprising: the preparation method comprises the following steps:

(1) Adding triphosgene into an organic solvent A, dropwise adding an organic solvent A solution containing 3, 6-dimethyl-1, 6-dihydro-1, 2,4, 5-tetrazine shown in a formula (II) and an alkaline catalyst a under the stirring condition of-10-12 ℃, heating the reaction solution to room temperature after the dropwise adding is finished, stirring and reacting at room temperature for 0.5-50 hours, introducing nitrogen into the reaction system until tail gas absorption liquid does not generate white mist, and directly using the generated product for the next reaction;

the basic catalyst a and the basic catalyst b are one of the following: triethylamine, 4-dimethylaminopyridine, pyridine or sodium hydroxide;

in the formula (III), R and n are as defined in the formula (I).

3. The method of claim 2, wherein: the ratio of the amount of the compound (II) to the amount of the alkaline catalyst a, the amount of the alkaline catalyst b, the amount of the triphosgene and the amount of the compound (III) to be fed is 1: 0.1 to 3.

4. The method of claim 2, wherein: the ratio of the amount of the compound (II) to the amount of the alkaline catalyst a, the amount of the alkaline catalyst b, the amount of the triphosgene and the amount of the compound (III) to be fed is 1: 0.1 to 2.

5. The method of claim 2, wherein: the organic solvent A and the organic solvent B are respectively and independently selected from one of the following: dichloromethane, chloroform or toluene.

6. The method of claim 2, wherein: in the step (1), under the condition of stirring at the temperature of minus 10 to 5 ℃, dropwise adding an organic solvent A solution containing 3, 6-dimethyl-1, 6-dihydro-1, 2,4, 5-tetrazine shown in a formula (II) and an alkaline catalyst a;

in the step (2), the reaction solution obtained in the step (1) is dropwise added under the condition of stirring at a temperature of between 10 ℃ below zero and 5 ℃.

7. The use of a cinnamoyl tetrazine, or a pharmaceutically acceptable salt thereof, according to claim 1, in the preparation of a medicament for treating or preventing a VEGFR-2 mediated disease or a medicament for inhibiting VEGFR-2, wherein the VEGFR-2 mediated disease is cancer, and the cancer cells of the cancer are a549, huh7, or HepG2 cells.

8. The use of claim 7, wherein: the cancer cells are A549 cells, and the cinnamoyl tetrazine compound shown in the formula (I) is a compound (ie);

9. the use of claim 7, wherein: the cancer cell is Huh7 cell, and the cinnamoyl tetrazine compound shown in the formula (I) is a compound (ih);

10. the use of claim 7, wherein: the cancer cell is a HepG2 cell, and the cinnamoyl tetrazine compound shown in the formula (I) is a compound (id), (ig), (ih) or (iS);