CN117752661A

CN117752661A - Application of dimethyl tetrazine formamide compound in preparation of medicines for treating or preventing cervical cancer

Info

Publication number: CN117752661A
Application number: CN202311088022.3A
Authority: CN
Inventors: 郑翔; 李鑫; 饶国武; 艾晶燕
Original assignee: Zhejiang University of Technology ZJUT
Current assignee: Zhejiang University of Technology ZJUT
Priority date: 2023-08-28
Filing date: 2023-08-28
Publication date: 2024-03-26

Abstract

The invention discloses application of a dimethyl tetrazine formamide compound in preparation of a medicament for treating or preventing cervical cancer. The structural formula of the dimethyl tetrazine formamide compound is shown as formula (I), R is 4-CF ₃ 、2‑CF ₃ 2-F-4-Br, 2-Cl, 2-Br-4-Cl or 4-Cl. The dimethyl tetrazine formamide compound shows good cervical cancer resistance activity.

Description

Application of dimethyl tetrazine formamide compound in preparation of medicines for treating or preventing cervical cancer

Technical Field

The invention relates to application of a dimethyl tetrazine formamide compound in preparing a medicament for treating or preventing cervical cancer.

Background

Tetrazine compounds have good physical properties, spectral properties and high reactivity, and especially tetrazine derivatives with special structures have obvious anti-tumor activity and antiviral activity, and can be used as pesticides and insecticides. For example, two varieties of pesticides (clofentezine and flufenzine) are on the market, and one variety of drugs (temozolomide) is on the market.

In 1978, the literature reported that 3, 6-diphenyl alkynyl-hexahydro-1, 2,4, 5-tetrazine had antitumor activity (see Eremeev, a.v.; tikhomirova, d.a.; tyrushiva, v.a.; liekins, f.khim. Geotsikl.soedin, 1978,753), which was the first reported that 1,2,4, 5-tetrazine compounds may have potential antitumor activity. After that, some 1,2,4, 5-tetrazine compounds have been reported to have antitumor activity, for example, 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-tetrazine compounds do not have antitumor activity.

Disclosure of Invention

The invention aims to provide an application of a dimethyl tetrazine formamide compound in preparing a medicament for treating or preventing cervical cancer, which shows good anticancer activity.

The technical scheme adopted by the invention is specifically described below.

The invention provides an application of a dimethyl tetrazine formamide compound shown in a formula (I) or a pharmaceutically acceptable salt thereof in preparing a medicament for treating or preventing cervical cancer;

in the formula (I), R is 4-CF ₃ 、2-CF ₃ 2-F-4-Br, 2-Cl, 2-Br-4-Cl or 4-Cl. Namely, the compound (I-2), (I-9), (I-12), (I-15), (I-16) or (I-17) in the specific embodiment.

In a specific embodiment of the invention, the cancer cells of cervical cancer are human cervical cancer cells Hela.

Preferably, in formula (I), R is 2-CF ₃ Or 4-Cl. Namely, the compound (I-9) or (I-17) in the specific embodiment.

The invention provides a preparation method of a dimethyl tetrazine formamide compound shown in a formula (I), which 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 a reaction liquid to room temperature after the dripping is finished, stirring at room temperature for 0.5-50 hours, introducing nitrogen into a reaction system, absorbing tail gas by a tail gas absorbing device until white fog is not generated in an alkaline tail gas absorbing liquid (NaOH aqueous solution), and directly using the generated product in the next reaction;

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

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

in the formula (III), R is defined as in the formula (I).

The reaction for preparing the dimethyl tetrazine formamide compound (I) is shown in the following reaction formula:

further, the ratio of the amount of the 3, 6-dimethyl-1, 6-dihydro-1, 2,4, 5-tetrazine (II) to the amount of the basic catalyst a, the basic catalyst b, triphosgene, and the amount of the compound (III) to be fed is 1:0.1 to 3:0.1 to 3, and preferably the ratio of the amount of the 3, 6-dimethyl-1, 6-dihydro-1, 2,4, 5-tetrazine (II) to the amount of the basic catalyst a, the basic catalyst b, triphosgene, and the amount of the compound (III) to be fed is 1:0.1 to 2:0.1 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 amount of the organic solvent A is required to be capable of dissolving triphosgene, 3, 6-dimethyl-1, 6-dihydro-1, 2,4, 5-tetrazine (II) and the alkaline catalyst a, and the amount of the organic solvent B is required to be capable of dissolving the alkaline catalyst B and the compound (III). Preferably, the total volume of the organic solvent A is 3-18mL/mmol based on the amount of 3, 6-dimethyl-1, 6-dihydro-1, 2,4, 5-tetrazine (II), wherein the triphosgene is dissolved in an amount of 1-10mL/mmol based on the amount of 3, 6-dimethyl-1, 6-dihydro-1, 2,4, 5-tetrazine (II), and the total volume of the organic solvent B is 2-20mL/mmol based on the amount of 3, 6-dimethyl-1, 6-dihydro-1, 2,4, 5-tetrazine (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 dropwise added under stirring at-10 to 5 ℃.

In the step (2), the reaction liquid after the reaction in the step (1) is dripped under the stirring condition of minus 10 to 5 ℃.

Further, the reaction process of steps (1) and (2) is followed by TLC (developing agent is petroleum ether and ethyl acetate mixed solution with volume ratio of 0.5-20:1) to determine the reaction end point, and the reaction time is generally 0.5-50 hours.

Further, the separation and purification in the step (2) adopts the following steps: after the reaction is finished, the reaction liquid is washed with water, an organic phase is separated, and after the solvent is distilled off, the residue is subjected to column chromatography to obtain the dimethyl tetrazine formamide compound shown in the formula (I).

Further, the operation steps of the column chromatography are specifically as follows: taking residues after solvent evaporation in a single-mouth bottle, adding an organic solvent C to dissolve the residues to obtain a dissolution solution, adding column chromatography silica gel (preferably 300-400 mesh coarse pore (zcx.II) column chromatography silica gel) with the mass of 1-2 times of the residues into the dissolution solution, uniformly mixing, evaporating the solvent to obtain a mixture of the dried residues and the silica gel, loading the mixture into a column, and loading the sample into a sample with the volume ratio of 0.5-20: 1, eluting with petroleum ether and ethyl acetate mixed solution as eluent, performing TLC tracking detection (developing agent is petroleum ether and ethyl acetate mixed solution with volume ratio of 0.5-20:1), collecting eluent containing the compound shown in formula (I) according to TLC detection, concentrating and drying the eluent to obtain the compound shown in formula (I); 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 to dissolve the residue.

The organic solvents A, B and C in the present invention are all organic solvents used for reaction or column chromatography, and letters are not meant to designate some organic solvents, but are used for clarity of expression to distinguish the organic solvents appearing in different steps. The organic solvent A, B, C may be the same solvent or different solvents.

In the present invention, the term "pharmaceutically acceptable" means: the compounds are chemically and/or toxicologically compatible with the other ingredients comprising the formulation and/or with the human or mammal with which the disease or condition is to be prevented or treated.

The term "pharmaceutically acceptable salt" refers to the relatively non-toxic, inorganic or organic acid addition salts of the compounds of the present invention. See, for example, S.M. Bere 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 or nitric acid, etc.; 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, digluconic acid, 3-hydroxy-2-naphthoic acid, nicotinic acid, pamoic acid, pectate 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, glycerophosphate, aspartic acid, sulfosalicylic acid, and the like.

Compared with the prior art, the invention has the beneficial effects that: (1) Provides a novel 1,2,4, 5-tetrazine compound with good anti-cervical cancer activity; (2) The preparation method of the 1,2,4, 5-tetrazine compound is simple, easy to operate, easy to obtain raw materials, low in production cost, applicable to practicality and expected to be applied to preparation of medicines for preventing or treating tumor diseases; (3) Provides the application of the novel 1,2,4, 5-tetrazine compound or the pharmaceutically acceptable salt thereof in preparing medicaments for treating or preventing cervical cancer, and shows good anticancer activity.

Detailed Description

The invention is further illustrated by reference to specific examples, which are given below to illustrate the invention and are not to be construed as limiting the invention in any way.

The specific conditions are not noted in the examples of the present invention, and are carried out according to conventional conditions or conditions suggested by the manufacturer. The reagents or apparatus used are conventional products, which are available by conventional technical means or commercially available, without the manufacturer's knowledge.

Preparation of 3, 6-dimethyl-1, 6-dihydro-1, 2,4, 5-tetrazine (II) reference (Synthetic Communications,2003,33 (16), 2769-2775).

The preparation reaction formulas of the compounds (III-1) to (III-17) are shown below, wherein the structure of R in the reaction formulas is shown in the structures of the compounds (III-1) to (III-17).

The structural formula of the dimethyltetrazine formamide compound prepared by the embodiment of the invention is specifically shown as follows:

example 1: preparation of Compound (III-1)

M-nitroacetophenone (2.48 g,15.0 mmol) and potassium hydroxide (1.01 g,18.0 mmol) are sequentially added into a three-necked flask, 40mL of absolute ethyl alcohol and 10mL of distilled water are used for dissolution, benzaldehyde (1.59 g,15.0 mmol) is added under the ice bath condition after 10min, stirring is carried out for 2-6H, TLC (developing agent is a mixed solution of petroleum ether and ethyl acetate with the volume ratio of 1:1) is used for detecting that the reaction is complete, stirring is stopped, the reaction solution is poured into 100mL of ice water, the pH is regulated to be acidic by dilute hydrochloric acid (pH=1), a large amount of solids are precipitated, the solid is filtered, a filter cake is washed twice by water and petroleum ether in sequence, the solids are dried, and the compound IV (R=H) is obtained by recrystallization by ethanol, and the yield is 75.5%.

In a three-necked flask, compound IV (R=H) (1.52 g,6.00 mmol) is added, 48mL of absolute methanol is added for stirring and dissolution, then 5% palladium-carbon (0.064 g,0.6 mmol) is added, hydrogen is introduced into the three-necked flask, stirring is carried out at normal temperature for 1-2H, TLC (developing agent is petroleum ether and ethyl acetate mixed solution with volume ratio of 1:1) detects that the reaction is complete, palladium-carbon is filtered off, filter cakes are washed by absolute methanol (10 mL multiplied by 3), the filtrate is distilled under reduced pressure to remove the solvent, and column chromatography (petroleum ether and ethyl acetate mixed solution with volume ratio of 2:1 is used as eluent) is carried out to purify and obtain 1.07g of compound (III-1), and the yield is 79.1%.

Preparation of Compounds (III-2) to (III-17): the corresponding compounds (III-2) to (III-17) were prepared by substituting benzaldehyde with the corresponding substituted benzaldehyde by the method of example 1, and will not be described here.

Example 2: preparation of Compound (I-1)

10mL of chloroform was dissolved in 0.297g (1.0 mmol) of triphosgene, a solution of 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 in 20mL of chloroform was added dropwise under magnetic stirring at-10℃to react at room temperature for 50 hours (the reaction process was followed by TLC, the developing solvent was a mixed solution of petroleum ether and ethyl acetate in a volume ratio of 1:2), nitrogen was introduced into the reaction system, and the gas was absorbed by a tail gas absorbing device (the tail gas absorbing solution was a 10% aqueous NaOH solution) until no more white fog was generated in the tail gas absorbing solution, and the resultant product was directly used for the next reaction.

Dissolving 2.253g (10.0 mmol) of compound (III-1) and 0.122g (1.0 mmol) of DMAP in 60mL of chloroform, dropwise adding the reaction solution after the completion of the reaction under magnetic stirring at-10 ℃, heating the reaction solution to room temperature after the completion of the dropwise adding, reacting for 10 hours at room temperature (the reaction process adopts TLC tracking detection, the developing agent is a petroleum ether and ethyl acetate mixed solution with the volume ratio of 1:2), washing the reaction solution with (50 mL multiplied by 3), separating an organic phase, evaporating the solvent, and then carrying out residue column chromatography, namely adding 10mL of petroleum ether solvent into the residue after the solvent is evaporated to dissolve the residue to obtain a solution, then adding 2.0 g of silica gel (300-400 meshes of coarse pore (zcx.II) column chromatography silica gel) into the solution, uniformly mixing, evaporating the solvent to obtain a mixture of dried residue and the silica gel, and filling the mixture into a column, and then carrying out the column chromatography with the solvent according to the volume ratio of 1:2 as eluent, TLC tracking detection (developing agent is petroleum ether and ethyl acetate mixed solution with volume ratio of 1:2), collecting eluent containing compound shown in formula (I-1) according to TLC detection, evaporating solvent from the collected eluent, and drying to obtain white solid product, namely compound (I-1), with yield of 31% (calculated by 3, 6-dimethyl-1, 2,4, 5-tetrazine substance, except for example 5, same below), melting point of-107 ℃. ¹ H NMR(500MHz,Chloroform-d)δ8.51(s,1H),7.95(t,J＝1.8Hz,1H),7.89-7.87(m,1H),7.65(dt,J＝8.0,1.0Hz,1H),7.41(t,J＝7.8Hz,1H),7.32-7.29(m,2H),7.27-7.25(m,2H),7.22-7.19(m,1H),3.31(t,J＝7.5Hz,2H),3.07(t,J＝7.8Hz,2H),2.34(s,3H),2.03(s,3H).

Example 3: preparation of Compound (I-1)

100mL of methylene chloride is dissolved into 8.903g (30.0 mmol) of triphosgene, under the condition of magnetic stirring at 12 ℃, 80mL of methylene chloride 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 is dropwise added, the reaction solution is warmed to room temperature after the dropwise addition, the reaction is carried out for 0.5 hour (the reaction process is tracked and detected by TLC, the developing agent is a petroleum ether and ethyl acetate mixed solution with the volume ratio of 20:1), nitrogen is introduced into the reaction system, a tail gas absorbing device is used for absorbing gas (the tail gas absorbing solution is 10 percent NaOH aqueous solution) until white fog is not generated in the tail gas absorbing solution, and the generated product is directly used for the next reaction.

200mL of dichloromethane is dissolved into 6.759g (30.0 mmol) of compound (III-1) and 2.373g (30.0 mmol) of pyridine, the reaction solution after the completion of the reaction is dripped under the condition of magnetic stirring at 12 ℃, the reaction solution is warmed to room temperature after the dripping is completed, the reaction is carried out for 0.5 hour at room temperature (the reaction process is detected by TLC tracking, the developing agent is a petroleum ether and ethyl acetate mixed solution with the volume ratio of 20:1), the reaction solution is washed by (50 mL multiplied by 3), an organic phase is separated, the solvent is distilled off, the residue is subjected to column chromatography, namely, the residue after the solvent is distilled off is taken and added into 10 mL of dichloromethane solvent to dissolve the residue to obtain a dissolving solution, 3.0 g of silica gel (300-400-mesh coarse pore (zcx.II) column chromatography silica gel) is added into the dissolving solution, after the solvent is distilled off, the mixture of the dried residue and the silica gel is filled into a column, and then the mixture is prepared according to the volume ratio of 20:1 as eluent, TLC tracking detection (developing solvent is petroleum ether and ethyl acetate mixed solution with volume ratio of 20:1), collecting eluent containing compound shown in formula (I-1) according to TLC detection, evaporating solvent from the collected eluent, and drying to obtain white solid product, namely compound (I-1), with yield of 45% and melting point of 104-107 ℃. ¹ H NMR was as in example 2.

Example 4: preparation of Compound (I-1)

40mL of toluene was dissolved in 5.935g (20.0 mmol) of triphosgene, a solution of 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 in 20mL of toluene was added dropwise under magnetic stirring at 0℃to react at room temperature for 3 hours (the reaction process was followed by TLC, the developing solvent was a mixed solution of petroleum ether and ethyl acetate in a volume ratio of 10:1), nitrogen was introduced into the reaction system, and the gas was absorbed by an off-gas absorbing device (the off-gas absorbing solution was a 10% aqueous NaOH solution) until no more white fog was produced in the off-gas absorbing solution, and the resultant product was directly used for the next reaction.

50mL of toluene was dissolved in 4.506g (20.0 mmol) of Compound (III-1) and 2.024g (20.0 mmol) of triethylamine, and the reaction was completed in the above step by dropwise addition under magnetic stirring at 0 ℃After the reaction solution is dripped, the reaction solution is warmed to room temperature, the room temperature is reacted for 8 hours (TLC tracking detection is adopted in the reaction process, the developing agent is petroleum ether and ethyl acetate mixed solution with the volume ratio of 10:1), the reaction solution is washed with (50 mL multiplied by 3), an organic phase is separated, the solvent is distilled off, the residue is subjected to column chromatography, namely, 10 mL of ethyl acetate solvent is added into the residue after the solvent is distilled off to dissolve the residue, so as to obtain a dissolving solution, then 2.5 g of silica gel (300-400 meshes of coarse pore (zcx.II) column chromatography silica gel) is added into the dissolving solution, after the mixing is carried out, the solvent is distilled off, so as to obtain a mixture of the dried residue and the silica gel, the mixture is packed into a column, and then the mixture is subjected to the volume ratio of 10:1 as eluent, TLC tracking detection (developing solvent is mixed solution of petroleum ether and ethyl acetate in volume ratio of 10:1), collecting eluent containing compound shown in formula (I-1) according to TLC detection, evaporating solvent from the collected eluent, and drying to obtain white solid product, namely compound (I-1), with yield of 39% and melting point of 104-107 ℃. ¹ H NMR was as in example 2.

Example 5: preparation of Compound (I-1)

20mL of toluene is dissolved into 1.484g (5.0 mmol) of triphosgene, under the condition of magnetic stirring at 5 ℃, 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 dropwise added, the reaction solution is warmed to room temperature after the dropwise addition, the reaction is carried out for 12 hours (TLC tracking detection is adopted in the reaction process, the developing agent is a petroleum ether and ethyl acetate mixed solution with the volume ratio of 5:1), nitrogen is introduced into the reaction system, and a tail gas absorbing device is used for absorbing gas (the tail gas absorbing solution is 10 percent NaOH aqueous solution) until white fog is not generated in the tail gas absorbing solution, and the generated product is directly used for the next reaction.

Dissolving 0.225g (1.0 mmol) of compound (III-1) and 0.200g (5.0 mmol) of sodium hydroxide in 20mL of chloroform, dropwise adding the reaction solution after the completion of the reaction under the condition of magnetic stirring at 5 ℃, heating the reaction solution to room temperature, reacting for 50 hours at room temperature (the reaction process adopts TLC tracking detection, the developing agent is a mixed solution of petroleum ether and ethyl acetate in a volume ratio of 5:1), washing the reaction solution with (50 mL multiplied by 3), separating an organic phase, evaporating the solvent, and performing residue column chromatography, namely taking and evaporating Adding 10 ml of chloroform solvent into the residue after the solvent to dissolve the residue to obtain a dissolution solution, adding 0.3 g of silica gel (300-400 mesh coarse pore (zcx.II) column chromatography silica gel) into the dissolution solution, uniformly mixing, steaming the solvent to obtain a mixture of the dried residue and the silica gel, filling the mixture into a column, and then carrying out the process according to the volume ratio of 5:1 as eluent, TLC tracking detection (developing solvent is mixed solution of petroleum ether and ethyl acetate in volume ratio of 5:1), collecting eluent containing the compound shown in formula (I-1) according to TLC detection, evaporating solvent from the collected eluent, and drying to obtain white solid product, namely compound (I-1), wherein the yield is 43% (based on the amount of compound III-1) and the melting point is 104-107 ℃. ¹ H NMR was as in example 2.

Example 6: preparation of Compound (I-1)

Dissolving 2.253g (10.0 mmol) of compound (III-1) and 1.012g (10.0 mmol) of triethylamine in 60mL of methylene chloride, dropwise adding the reaction solution after the completion of the reaction under magnetic stirring at-5 ℃, heating the reaction solution to room temperature after the completion of the dropwise adding, reacting for 9 hours at room temperature (the reaction process adopts TLC tracking detection, the developing agent is a mixed solution of petroleum ether and ethyl acetate in a volume ratio of 5:1), washing the reaction solution with (50 mL multiplied by 3), separating an organic phase, evaporating the solvent, performing column chromatography on the residue, namely adding 10 mL of chloroform solvent into the residue after the solvent is evaporated to dissolve the residue to obtain a solution, adding 2.5 g of silica gel (300-400 mesh coarse pore (zcx.II) column chromatography silica gel) into the solution, uniformly mixing, evaporating the solvent to obtain a mixture of the dried residue and the silica gel,the mixture was packed in a column and then subjected to a volume ratio of 5:1 as eluent, TLC tracking detection (developing solvent is mixed solution of petroleum ether and ethyl acetate in volume ratio of 5:1), collecting eluent containing compound shown in formula (I-1) according to TLC detection, evaporating solvent from the collected eluent, and drying to obtain white solid product, namely compound (I-1), with yield of 37% and melting point of 104-107 ℃. ¹ H NMR was as in example 2.

Example 7: preparation of Compound (I-2)

40mL of toluene is dissolved in 2.968g (10.0 mmol) of triphosgene, under the condition of magnetic stirring at 0 ℃, 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 is dropwise added, the reaction solution is warmed to room temperature after the dropwise addition, the reaction is carried out for 8 hours (TLC tracking detection is adopted in the reaction process, the developing agent is a petroleum ether and ethyl acetate mixed solution with the volume ratio of 1:1), nitrogen is introduced into the reaction system, and a tail gas absorbing device is used for absorbing gas (the tail gas absorbing solution is 10 percent NaOH aqueous solution) until white fog is not generated in the tail gas absorbing solution, and the generated product is directly used for the next reaction.

Dissolving 2.933g (10.0 mmol) of compound (III-2) and 1.012g (10.0 mmol) of triethylamine in 40mL of toluene, dropwise adding a reaction solution obtained after the completion of the reaction under the condition of magnetic stirring at 0 ℃, heating the reaction solution to room temperature after the completion of the dropwise adding, reacting for 11 hours at room temperature (the reaction process adopts TLC tracking detection, the developing agent is a petroleum ether and ethyl acetate mixed solution with the volume ratio of 3:1), washing the reaction solution with (50 mL multiplied by 3), separating an organic phase, evaporating the solvent, and then performing column chromatography on the residue, namely adding 10 mL of ethyl acetate solvent into the residue obtained after the solvent evaporation to dissolve the residue to obtain a solution, adding 5.0 g of silica gel (300-400-mesh coarse pore (zcx.II) column chromatography silica gel), uniformly mixing, evaporating the solvent to obtain a mixture of dried residue and the silica gel, and filling the mixture into a column according to the volume ratio of 3:1 as eluent, eluting, TLC tracking and detecting (developing agent is mixed solution of petroleum ether and ethyl acetate in volume ratio of 3:1), collecting eluent containing compound shown in formula (I-2) according to TLC detection, and steaming the collected eluent Removing solvent, drying to obtain white solid product, namely compound (I-2), the yield is 62%, and the melting point is 85-88 ℃. ¹ H NMR(500MHz,Chloroform-d)δ8.52(s,1H),8.01(t,J＝2.0Hz,1H),7.83-7.81(m,1H),7.65(dt,J＝8.0,1.0Hz,1H),7.54(d,J＝8.0Hz,2H),7.41(t,J＝8.0Hz,1H),7.37(d,J＝8.0Hz,2H),6.82(s,1H),3.33(t,J＝7.5Hz,2H),3.13(t,J＝7.5Hz,2H),2.34(s,3H),2.02(s,3H)；MS(ESI)m/z[M+H] ⁺ calcd for C ₂₁ H ₂₁ F ₃ N ₅ O ₂ :432.2,found432.2；HPLC:t _R ＝29.777min(>98％).

Example 8: preparation of Compound (I-3)

Dissolving 2.853g (10.0 mmol) of compound (III-3) and 1.012g (10.0 mmol) of triethylamine in 40mL of toluene, dropwise adding a reaction solution obtained after the completion of the reaction under the condition of magnetic stirring at 0 ℃, heating the reaction solution to room temperature after the completion of the dropwise adding, reacting for 9 hours at room temperature (the reaction process adopts TLC tracking detection, the developing agent is a petroleum ether and ethyl acetate mixed solution with the volume ratio of 3:1), washing the reaction solution with (50 mL multiplied by 3), separating an organic phase, evaporating the solvent, and then performing column chromatography on the residue, namely adding 10 mL of ethyl acetate solvent into the residue obtained after the solvent evaporation to dissolve the residue to obtain a solution, adding 5.0 g of silica gel (300-400-mesh coarse pore (zcx.II) column chromatography silica gel), uniformly mixing, evaporating the solvent to obtain a mixture of dried residue and the silica gel, and filling the mixture into a column with the volume ratio of 3:1 as eluent, and TLC tracking detection (developing agent is petroleum ether and ethyl acetate in volume ratio of 3:1) Mixed solution), the eluent containing the compound shown in the formula (I-3) is collected according to TLC detection, the collected eluent is distilled to remove the solvent, and the white solid product is obtained after drying, namely the compound (I-3), the yield is 61 percent, and the melting point is 133-136 ℃. ¹ H NMR(500MHz,Chloroform-d)δ8.52(s,1H),7.98(t,J＝1.8Hz,1H),7.87-7.84(m,1H),7.66(dt,J＝7.5,1.5Hz,1H),7.41(t,J＝8.0Hz,1H),6.80-6.78(m,4H),3.87(s,3H),3.86(s,3H),3.29(t,J＝7.8Hz,2H),3.01(t,J＝7.8Hz,2H),2.34(s,3H),2.03(s,3H)； ¹³ C NMR(125MHz,Chloroform-d)δ199.2,153.2,149.4,148.9,147.4,143.2,138.5,137.6,133.9,129.3,124.1,123.1,120.3,118.9,111.2,111.4,56.0,55.9,40.89,29.9,18.19,16.5；MS(ESI)m/z[M+H] ⁺ calcd for C ₂₂ H ₂₆ N ₅ O ₄ :424.2,found 424.2；HPLC:t _R ＝11.899min(>98％).

Example 9: preparation of Compound (I-4)

Dissolving 3.042g (10.0 mmol) of compound (III-4) and 1.012g (10.0 mmol) of triethylamine in 40mL of toluene, dropwise adding the reaction solution after completion of the reaction under magnetic stirring at 0 ℃, heating the reaction solution to room temperature, reacting for 9 hours at room temperature (the reaction process adopts TLC tracking detection, the developing agent is a mixed solution of petroleum ether and ethyl acetate in a volume ratio of 3:1), washing the reaction solution with (50 mL multiplied by 3), separating an organic phase, evaporating the solvent, performing column chromatography on the residue, namely taking the residue after evaporating the solvent, adding 10 mL of ethyl acetate solvent to dissolve the residue to obtain a solution, and then adding 6.0 g of silica gel (300-400 meshes of coarse pore (zcx.II) column chromatography silica gel) into the solution After mixing evenly, evaporating the solvent to obtain a mixture of the dried residue and silica gel, filling the mixture into a column, and then, carrying out the following steps in a volume ratio of 3:1 as eluent, TLC tracking detection (developing solvent is mixed solution of petroleum ether and ethyl acetate in volume ratio of 3:1), collecting eluent containing compound shown in formula (I-4) according to TLC detection, evaporating solvent from the collected eluent, and drying to obtain white solid product, namely compound (I-4), the yield is 71%, and the melting point is 117-120 ℃. IR (KBr, cm) ^-1 ):3356,3332,3152,2925,1699,1682,1650,1606,1520,1488,1385,1290,993,776； ¹ H NMR(500MHz,Chloroform-d)δ8.52(s,1H),7.98(t,J＝1.8Hz,1H),7.85-7.83(m,1H),7.64(dt,J＝8.0,1.0Hz,1H),7.42-7.39(m,3H),7.13(d,J＝8.5Hz,2H),6.74(s,1H),3.28(t,J＝7.5Hz,2H),3.02(t,J＝7.5Hz,2H),2.34(s,3H),2.03(s,3H)；MS(ESI)m/z[M+H] ⁺ calcd for C ₂₀ H ₂₁ BrN ₅ O ₂ :442.1,found442.2；HPLC:t _R ＝14.722min(>98％).

Example 10: preparation of Compound (I-5)

Dissolving 2.433g (10.0 mmol) of compound (III-5) and 1.012g (10.0 mmol) of triethylamine in 40mL of toluene, dropwise adding the reaction solution after completion of the reaction under magnetic stirring at 0 ℃, heating the reaction solution to room temperature, reacting for 9 hours at room temperature (the reaction process adopts TLC tracking detection, the developing agent is a mixed solution of petroleum ether and ethyl acetate in a volume ratio of 3:1), washing the reaction solution with (50 mL multiplied by 3), separating out an organic phase, evaporating the solvent, and performing column chromatography on residues, namely taking the distilled solventAdding 10 ml of ethyl acetate solvent into the residue to dissolve the residue to obtain a dissolution solution, adding 4.5 g of silica gel (300-400 mesh coarse pore (zcx.II) column chromatography silica gel) into the dissolution solution, uniformly mixing, steaming the solvent to obtain a mixture of the dried residue and the silica gel, filling the mixture into a column, and then carrying out the process according to the volume ratio of 3:1 as eluent, TLC tracking detection (developing solvent is mixed solution of petroleum ether and ethyl acetate in volume ratio of 3:1), collecting eluent containing compound shown in formula (I-5) according to TLC detection, evaporating solvent from the collected eluent, and drying to obtain white solid product, namely compound (I-5), yield 63%, melting point 129-131 ℃. ¹ H NMR(500MHz,Chloroform-d)δ8.52(s,1H),7.98(t,J＝2.0Hz,1H),7.85-7.83(m,1H),7.64(dt,J＝8.0,1.5Hz,1H),7.41(t,J＝8.0Hz,1H),7.20(dd,J＝10.0,5.0Hz,2H),6.97(t,J＝8.8Hz,2H),6.81(s,1H),3.28(t,J＝7.8Hz,2H),3.04(t,J＝7.5Hz,2H),2.34(s,3H),2.02(s,3H)；MS(ESI)m/z[M+H] ⁺ calcd for C ₂₀ H ₂₁ FN ₅ O ₂ :382.2,found 382.2；HPLC:t _R ＝11.062min(>98％).

Example 11: preparation of Compound (I-6)

40mL of toluene is dissolved with 2.942g (10.0 mmol) of compound (III-6) and 1.012g (10.0 mmol) of triethylamine, the reaction solution after the completion of the reaction is dripped under the condition of magnetic stirring at 0 ℃, the reaction solution is warmed to room temperature after the dripping is completed, the reaction is carried out for 9 hours at room temperature (TLC tracking detection is adopted in the reaction process, the developing agent is petroleum ether and ethyl acetate mixed solution with the volume ratio of 3:1), and the reaction is carried outWashing the applied solution (50 mL multiplied by 3) with water, separating out an organic phase, steaming to remove a solvent, performing column chromatography on the residue, namely, taking the residue after steaming to remove the solvent, adding 10 milliliters of ethyl acetate solvent to dissolve the residue to obtain a dissolved solution, adding 5.5 grams of silica gel (300-400-mesh coarse pore (zcx.II) column chromatography silica gel) into the dissolved solution, uniformly mixing, steaming to remove the solvent to obtain a mixture of the dried residue and the silica gel, filling the mixture into a column, and then filling the column with a volume ratio of 3:1 as eluent, TLC tracking detection (developing solvent is petroleum ether and ethyl acetate mixed solution with volume ratio of 3:1), collecting eluent containing compound shown in formula (I-6) according to TLC detection, evaporating solvent from the collected eluent, and drying to obtain white solid product, namely compound (I-6), with yield of 68% and melting point of 116-119 ℃. ¹ H NMR(500MHz,Chloroform-d)δ8.52(s,1H),7.97(t,J＝2.0Hz,1H),7.87-7.85(m,1H),7.66(dt,J＝8.0,1.0Hz,1H),7.41(t,J＝8.0Hz,1H),7.33(dd,J＝8.0,1.5Hz,1H),7.23(dd,J＝8.0,1.5Hz,1H),7.13(t,J＝7.8Hz,1H),6.82(s,1H),3.32(d,J＝7.5Hz,2H),3.21(d,J＝7.5Hz,2H),2.33(s,3H),2.03(s,3H)；MS(ESI)m/z[M+H] ⁺ calcd for C ₂₀ H ₂₀ Cl ₂ N ₅ O ₂ :432.1,found432.2；HPLC:t _R ＝17.325min(>95％).

Example 12: preparation of Compound (I-7)

40mL of toluene was dissolved in 2.553g (10.0 mmol) of Compound (III-7) and 1.012g (10.0 mmol) of triethylamine, and the reaction mixture after completion of the reaction was stirred magnetically at 0℃and then the reaction mixture was allowed to rise toAfter reacting for 9 hours at room temperature (TLC tracking detection is adopted in the reaction process, the developing agent is petroleum ether and ethyl acetate mixed solution with the volume ratio of 3:1), the reaction solution is washed with (50 mL multiplied by 3), the organic phase is separated, the solvent is distilled off, the residue is subjected to column chromatography, namely, 10 mL ethyl acetate solvent is added to the residue after the solvent is distilled off to dissolve the residue, so as to obtain a dissolving solution, then 5.0 g silica gel (300-400 meshes of coarse pore (zcx.II) column chromatography silica gel) is added to the dissolving solution, after uniform mixing, the solvent is distilled off, so as to obtain a mixture of the dried residue and the silica gel, the mixture is packed into a column, and then the volume ratio of the mixture is 3:1 as eluent, TLC tracking detection (developing solvent is mixed solution of petroleum ether and ethyl acetate in volume ratio of 3:1), collecting eluent containing compound shown in formula (I-7) according to TLC detection, evaporating solvent from the collected eluent, and drying to obtain white solid product, namely compound (I-7), yield is 69%, and melting point is 138-141 ℃. ¹ H NMR(500MHz,Chloroform-d)δ8.52(s,1H),7.96(t,J＝2.0Hz,1H),7.87(d,J＝8.0Hz,1H),7.65(d,J＝7.5Hz,1H),7.40(t,J＝7.8Hz,1H),7.21(t,J＝8.0Hz,1H),6.84(t,J＝3.0Hz,2H),6.81(t,J＝2.0Hz,1H),6.75(dd,J＝8.0,2.0Hz,1H),3.80(s,3H),3.30(t,J＝7.8Hz,2H),3.04(t,J＝7.8Hz,2H),2.33(s,3H),2.02(s,3H)；MS(ESI)m/z[M+H] ⁺ calcdfor C ₂₁ H ₂₄ N ₅ O ₃ :394.2,found 394.4.

Example 13: preparation of Compound (I-8)

40mL of toluene was dissolved in 2.393g (10.0 mmol) of compound (III-8) and 1.012g (10.0 mmol) of triethylammoniumUnder the condition of magnetically stirring at the temperature of 0 ℃, dropwise adding reaction liquid after finishing the reaction, heating the reaction liquid to room temperature, reacting for 10 hours at room temperature (TLC tracking detection is adopted in the reaction process, the developing agent is petroleum ether and ethyl acetate mixed solution with the volume ratio of 3:1), washing the reaction liquid with (50 mL multiplied by 3), separating out an organic phase, evaporating a solvent, and then performing column chromatography on residues, namely adding 10 milliliters of ethyl acetate solvent into residues after evaporating the solvent to dissolve the residues to obtain a dissolving liquid, adding 4.5 grams of silica gel (300-400 meshes of coarse pore (zcx.II) column chromatography silica gel) into the dissolving liquid, uniformly mixing, evaporating the solvent to obtain a mixture of dried residues and the silica gel, loading the mixture into a column, and then carrying out the column chromatography according to the volume ratio of 3:1 as eluent, TLC tracking detection (developing solvent is mixed solution of petroleum ether and ethyl acetate in volume ratio of 3:1), collecting eluent containing compound shown in formula (I-8) according to TLC detection, evaporating solvent from the collected eluent, and drying to obtain white solid product, namely compound (I-8), yield 66%, melting point 103-106 ℃. ¹ H NMR(500MHz,Chloroform-d)δ8.51(s,1H),7.94(t,J＝1.8Hz,1H),7.87(dt,J＝9.5,1.0Hz,1H),7.64(dt,J＝8.0,1.5Hz,1H),7.40(t,J＝8.0Hz,1H),7.14(d,J＝8.0Hz,2H),7.10(d,J＝7.5Hz,2H),6.87(s,1H),3.28(t,J＝7.8Hz,2H),3.02(t,J＝7.8Hz,2H),2.33(s,3H),2.31(s,3H),2.01(s,3H)；MS(ESI)m/z[M+H] ⁺ calcd for C ₂₁ H ₂₄ N ₅ O ₂ :378.2,found 378.4；HPLC:t _R ＝13.287min(>98％).

Example 14: preparation of Compound (I-9)

Dissolving 2.933g (10.0 mmol) of compound (III-9) and 1.012g (10.0 mmol) of triethylamine in 40mL of toluene, dropwise adding a reaction solution obtained after the completion of the reaction under the condition of magnetic stirring at 0 ℃, heating the reaction solution to room temperature after the completion of the dropwise adding, reacting for 9 hours at room temperature (the reaction process adopts TLC tracking detection, the developing agent is a petroleum ether and ethyl acetate mixed solution with the volume ratio of 3:1), washing the reaction solution with (50 mL multiplied by 3), separating an organic phase, evaporating the solvent, and then performing column chromatography on the residue, namely adding 10 mL of ethyl acetate solvent into the residue obtained after the solvent evaporation to dissolve the residue to obtain a solution, adding 4.5 g of silica gel (300-400-mesh coarse pore (zcx.II) column chromatography silica gel), uniformly mixing, evaporating the solvent to obtain a mixture of dried residue and the silica gel, and filling the mixture into a column according to the volume ratio of 3:1 as eluent, TLC tracking detection (developing solvent is mixed solution of petroleum ether and ethyl acetate in volume ratio of 3:1), collecting eluent containing compound shown in formula (I-9) according to TLC detection, evaporating solvent from the collected eluent, and drying to obtain white solid product, namely compound (I-9), yield 55%, melting point 124-127 ℃. ¹ H NMR(500MHz,Chloroform-d)δ8.52(s,1H),7.95(t,J＝2.0Hz,1H),7.90-7.88(m,1H),7.66-7.65(m,1H),7.65-7.64(m,1H),7.48(t,J＝7.5Hz,1H),7.43-7.39(m,2H),7.32(t,J＝7.8Hz,1H),6.66(s,1H),3.30(t,J＝7.3Hz,2H),3.24(t,J＝7.3Hz,2H),2.33(s,3H),2.04(s,3H).

Example 15: preparation of Compound (I-10)

40mL of toluene was dissolvedSolution 2.393g (10.0 mmol) of compound (III-10) and 1.012g (10.0 mmol) of triethylamine are added dropwise to the reaction solution after completion of the reaction under magnetic stirring at 0 ℃, the reaction solution is warmed to room temperature after completion of the dropwise addition, the reaction is carried out for 11 hours at room temperature (TLC tracking detection is adopted in the reaction process, the developing agent is a mixed solution of petroleum ether and ethyl acetate in a volume ratio of 3:1), the reaction solution is washed with (50 mL×3), an organic phase is separated, the solvent is distilled off, and then the residue is subjected to column chromatography, namely, the residue after the solvent is distilled off is taken and added with 10 mL of ethyl acetate solvent to dissolve the residue to obtain a solution, 4.5 g of silica gel (300-400 mesh coarse pore (zcx.II) column chromatography silica gel) is added to the solution, after the solvent is distilled off, the mixture of dried residue and silica gel is packed, and then the mixture is packed into a column according to a volume ratio of 3:1 as eluent, TLC tracking detection (developing solvent is mixed solution of petroleum ether and ethyl acetate in volume ratio of 3:1), collecting eluent containing compound shown in formula (I-10) according to TLC detection, evaporating solvent from the collected eluent, and drying to obtain white solid product, namely compound (I-10), with yield of 67% and melting point of 112-115 ℃. IR (KBr, cm) ^-1 ):3362,3336,3123,2924,1698,1683,1651,1604,1522,1487,1419,1408,1296,992,779； ¹ H NMR(500MHz,Chloroform-d)δ8.52(s,1H),7.95(t,J＝1.8Hz,1H),7.90-7.88(m,1H),7.66(dt,J＝7.5,1.5Hz,1H),7.42(t,J＝8.0Hz,1H),7.20-7.11(m,4H),6.68(s,1H),3.25(t,J＝7.8Hz,2H),3.05(t,J＝8.0Hz,2H),2.35(s,3H),2.34(s,3H),2.03(s,3H).

Example 16: preparation of Compound (I-11)

Dissolving 2.693g (10.0 mmol) of compound (III-11) and 1.012g (10.0 mmol) of triethylamine in 40mL of toluene, dropwise adding a reaction solution obtained after the completion of the reaction under the condition of magnetic stirring at 0 ℃, heating the reaction solution to room temperature after the completion of the dropwise adding, reacting for 9 hours at room temperature (the reaction process adopts TLC tracking detection, the developing agent is a petroleum ether and ethyl acetate mixed solution with the volume ratio of 3:1), washing the reaction solution with (50 mL multiplied by 3), separating an organic phase, evaporating the solvent, and then performing column chromatography on the residue, namely adding 10 mL of ethyl acetate solvent into the residue obtained after the solvent evaporation to dissolve the residue to obtain a solution, adding 5.0 g of silica gel (300-400-mesh coarse pore (zcx.II) column chromatography silica gel), uniformly mixing, evaporating the solvent to obtain a mixture of dried residue and the silica gel, and filling the mixture into a column according to the volume ratio of 3:1 as eluent, TLC tracking detection (developing solvent is mixed solution of petroleum ether and ethyl acetate in volume ratio of 3:1), collecting eluent containing compound shown in formula (I-11) according to TLC detection, evaporating solvent from the collected eluent, and drying to obtain white solid product, namely compound (I-11), with yield of 67% and melting point of 135-138 ℃. ¹ H NMR(500MHz,Chloroform-d)δ8.51(s,1H),7.94(t,J＝1.8Hz,1H),7.89-7.87(m,1H),7.65(dt,J＝8.0,1.0Hz,1H),7.41(t,J＝8.0Hz,1H),7.16(d,J＝8.5Hz,2H),6.83(d,J＝9.0Hz,2H),6.72(s,1H),4.01(q,J＝7.0Hz,2H),3.27(t,J＝7.8Hz,2H),3.00(t,J＝7.5Hz,2H),2.34(s,3H),2.03(s,3H),1.40(t,J＝7.0Hz,3H).

Example 17: preparation of Compound (I-12)

Dissolving 3.222g (10.0 mmol) of compound (III-12) and 1.012g (10.0 mmol) of triethylamine in 40mL of toluene, dropwise adding a reaction solution obtained after the completion of the reaction under the condition of magnetic stirring at 0 ℃, heating the reaction solution to room temperature after the completion of the dropwise adding, reacting for 11 hours at room temperature (the reaction process adopts TLC tracking detection, the developing agent is a petroleum ether and ethyl acetate mixed solution with the volume ratio of 3:1), washing the reaction solution with (50 mL multiplied by 3), separating an organic phase, evaporating the solvent, and then performing residue column chromatography, namely adding 10 mL of ethyl acetate solvent into the residue obtained after the solvent evaporation to dissolve the residue to obtain a solution, adding 4.5 g of silica gel (300-400 meshes of coarse pore (zcx.II) column chromatography silica gel), uniformly mixing, evaporating the solvent to obtain a mixture of dried residue and the silica gel, and filling the mixture into a column, and then performing the column chromatography according to the volume ratio of 3:1 as eluent, TLC tracking detection (developing solvent is petroleum ether and ethyl acetate mixed solution with volume ratio of 3:1), collecting eluent containing compound shown in formula (I-12) according to TLC detection, evaporating solvent from the collected eluent, and drying to obtain white solid product, namely compound (I-12), with yield of 52% and melting point of 134-137 ℃. ¹ H NMR(500MHz,Chloroform-d)δ8.52(s,1H),7.96(t,J＝1.8Hz,1H),7.87-7.85(m,1H),7.64(dt,J＝7.5,1.5Hz,1H),7.41(t,J＝7.8Hz,1H),7.20(d,J＝8.5Hz,2H),7.16(t,J＝8.0Hz,1H),6.67(s,1H),3.29(t,J＝7.5Hz,2H),3.05(t,J＝7.5Hz,2H),2.34(s,3H),2.03(s,3H).

Example 18: preparation of Compound (I-13)

40mL of toluene was dissolved in 2.942g (10.0 mmol) of compound (III-13) and 1.012g (10.0 mmol) of triethylamine, dropwise adding a reaction solution after completion of the reaction at a temperature of 0 ℃ under magnetic stirring, heating the reaction solution to room temperature, reacting for 11 hours at room temperature (TLC tracking detection is adopted in the reaction process, a developing agent is a petroleum ether and ethyl acetate mixed solution with a volume ratio of 3:1), washing the reaction solution with (50 mL multiplied by 3), separating an organic phase, evaporating a solvent, and then carrying out column chromatography on a residue, namely adding 10 mL of ethyl acetate solvent into the residue after evaporation of the solvent to dissolve the residue to obtain a solution, then adding 4.0 g of silica gel (300-400 mesh coarse pore (zcx.II) column chromatography silica gel) into the solution, uniformly mixing, evaporating the solvent to obtain a dried mixture of the residue and the silica gel, and filling the mixture into a column according to a volume ratio of 3:1 as eluent, TLC tracking detection (developing solvent is petroleum ether and ethyl acetate mixed solution with volume ratio of 3:1), collecting eluent containing compound shown in formula (I-13) according to TLC detection, evaporating solvent from the collected eluent, and drying to obtain white solid product, namely compound (I-13), with yield of 51% and melting point of 133-136 ℃. ¹ H NMR(500MHz,Chloroform-d)δ8.52(s,1H),8.01(t,J＝2.0Hz,1H),7.84-7.82(m,1H)),7.64(dt,J＝8.0,1.5Hz,1H),7.42(t,J＝8.0Hz,1H),7.36-7.34(m,2H),7.10(dd,J＝8.0,2.0Hz,1H),6.66(s,1H),3.29(t,J＝7.3Hz,2H),3.03(t,J＝7.5Hz,2H),2.34(s,3H),2.04(s,3H).

Example 19: preparation of Compound (I-14)

40mL of toluene was dissolved in 2.533g (10.0 mmol) of Compound (III-14) and 1.012g (10.0 mmol) of triethylamine under magnetic stirring at 0 ℃,dropping the reaction solution after the completion of the reaction, heating the reaction solution to room temperature after the completion of the dropping, reacting for 10 hours at room temperature (TLC tracking detection is adopted in the reaction process, the developing agent is a mixed solution of petroleum ether and ethyl acetate in a volume ratio of 5:1), washing the reaction solution with water (50 mL multiplied by 3), separating an organic phase, steaming out a solvent, and then carrying out column chromatography on the residue, namely, taking the residue after steaming out the solvent, adding 10 mL of ethyl acetate solvent to dissolve the residue to obtain a dissolution solution, then adding 5.5 g of silica gel (300-400 meshes of coarse pore (zcx.II) type column chromatography silica gel) into the dissolution solution, uniformly mixing, steaming out the solvent to obtain a mixture of the dried residue and the silica gel, loading the mixture into a column, and then carrying out the column chromatography according to a volume ratio of 5:1 as eluent, TLC tracking detection (developing solvent is mixed solution of petroleum ether and ethyl acetate in volume ratio of 5:1), collecting eluent containing compound shown in formula (I-14) according to TLC detection, evaporating solvent from the collected eluent, and drying to obtain white solid product, namely compound (I-14), yield 72%, melting point 103-106 ℃. ¹ H NMR(500MHz,Chloroform-d)δ8.51(s,1H),7.94(t,J＝2.0Hz,1H),7.90-7.87(m,1H),7.65(dt,J＝8.0,1.0Hz,1H),7.41(t,J＝8.0Hz,1H),7.18(d,J＝8.0Hz,2H),7.13(d,J＝8.0Hz,2H),6.69(s,1H),3.29(t,J＝7.8Hz,2H),3.03(t,J＝7.8Hz,2H),2.62(q,J＝7.5Hz,2H),2.34(s,3H),2.03(s,3H),1.23(t,J＝7.5Hz,3H).

Example 20: preparation of Compound (I-15)

40mL of toluene was dissolved in 2.597g (10.0 mmol) of Compound (III-15) and 1.012g (10.0 mmol) of triethylamine, and the reaction was carried out dropwise with magnetic stirring at 0 ℃The reaction solution to be completed is warmed to room temperature after dripping, the reaction is carried out for 11 hours (TLC tracking detection is adopted in the reaction process, the developing agent is petroleum ether and ethyl acetate mixed solution with the volume ratio of 5:1), the reaction solution is washed by (50 mL multiplied by 3), the organic phase is separated, the solvent is distilled off, namely, 10 mL ethyl acetate solvent is added to the residue after the solvent is distilled off to dissolve the residue, so as to obtain a dissolving solution, 4.0 g silica gel (300-400 meshes coarse pore (zcx.II) type column chromatography silica gel) is added to the dissolving solution, after uniform mixing, the solvent is distilled off, so as to obtain a dry mixture of the residue and the silica gel, the mixture is packed into a column, and then the volume ratio of the mixture is 5:1 as eluent, TLC tracking detection (developing solvent is mixed solution of petroleum ether and ethyl acetate in volume ratio of 5:1), collecting eluent containing compound shown in formula (I-15) according to TLC detection, evaporating solvent from the collected eluent, and drying to obtain white solid product, namely compound (I-15), yield is 53%, melting point is 146-149 ℃. ¹ H NMR(500MHz,Chloroform-d)δ8.51(s,1H),7.94(t,J＝1.8Hz,1H),7.90-7.88(m,1H),7.67(dt,J＝8.0,1.0Hz,1H),7.41(t,J＝8.0Hz,1H),7.36(dd,J＝7.5,1.0Hz,1H),7.31(dd,J＝7.0,2.0Hz,1H),7.21-7.14(m,2H),6.73(s,1H),3.31(t,J＝7.8Hz,2H),3.17(t,J＝7.5Hz,2H),2.33(s,3H),2.03(s,3H)； ¹³ C NMR(125MHz,Chloroform-d)δ198.9,153.3,149.4,143.1,138.7,138.5,137.4,133.9,130.8,129.6,129.3,127.7,127.0,124.2,123.1,118.9,38.6,28.3,18.2,16.4；MS(ESI)m/z[M+H] ⁺ calcd for C ₂₀ H ₂₁ ClN ₅ O ₂ :398.2,found 398.2；HPLC:t _R ＝7.678min(>99％).

Example 21: preparation of Compound (I-16)

Dissolving 3.386g (10.0 mmol) of a compound (III-16) and 1.012g (10.0 mmol) of triethylamine in 40mL of toluene, dropwise adding a reaction solution obtained after the completion of the reaction under the condition of magnetic stirring at 0 ℃, heating the reaction solution to room temperature after the completion of the dropwise adding, reacting for 10 hours at room temperature (the reaction process adopts TLC tracking detection, a developing agent is a mixed solution of petroleum ether and ethyl acetate in a volume ratio of 5:1), washing the reaction solution with (50 mL multiplied by 3), separating an organic phase, evaporating a solvent, and then performing residue column chromatography, namely adding 10 mL of ethyl acetate solvent into the residue obtained after the solvent is evaporated to obtain a solution, adding 5.0 g of silica gel (300-400 meshes of coarse pore (zcx.II) column chromatography silica gel) into the solution, uniformly mixing, evaporating the solvent to obtain a mixture of dried residue and the silica gel, and filling the mixture into a column, and then performing a volume ratio of 5:1 as eluent, TLC tracking detection (developing solvent is mixed solution of petroleum ether and ethyl acetate in volume ratio of 5:1), collecting eluent containing compound shown in formula (I-16) according to TLC detection, evaporating solvent from the collected eluent, and drying to obtain white solid product, namely compound (I-16), with yield of 55% and melting point of 114-117 ℃. ¹ H NMR(400MHz,Chloroform-d)δ8.52(s,1H),7.96(t,J＝2.4Hz,1H),7.89-7.86(m,1H),7.66(d,J＝7.6Hz,1H),7.56(t,J＝1.2Hz,1H),7.42(t,J＝7.8Hz,1H),7.25-7.21(m,2H),6.64(s,1H),3.30(t,J＝7.4Hz,2H),3.15(t,J＝7.4Hz,2H),2.34(s,3H),2.04(s,3H).

Example 22: preparation of Compound (I-17)

Dissolving 2.597g (10.0 mmol) of a compound (III-17) and 1.012g (10.0 mmol) of triethylamine in 40mL of toluene, dropwise adding a reaction solution obtained after the completion of the reaction under the condition of magnetic stirring at 0 ℃, heating the reaction solution to room temperature after the completion of the dropwise adding, reacting for 12 hours at room temperature (the reaction process adopts TLC tracking detection, the developing agent is a petroleum ether and ethyl acetate mixed solution with the volume ratio of 5:1), washing the reaction solution with (50 mL multiplied by 3), separating an organic phase, evaporating the solvent, and then performing column chromatography on the residue, namely adding 10 mL of ethyl acetate solvent into the residue obtained after the solvent is evaporated to obtain a solution, adding 4.0 g of silica gel (300-400-mesh coarse pore (zcx.II) column chromatography silica gel) into the solution, uniformly mixing, evaporating the solvent to obtain a mixture of dried residue and the silica gel, and filling the mixture into a column, and then carrying out column chromatography according to the volume ratio of 5:1 as eluent, TLC tracking detection (developing solvent is mixed solution of petroleum ether and ethyl acetate in volume ratio of 5:1), collecting eluent containing compound shown in formula (I-17) according to TLC detection, evaporating solvent from the collected eluent, and drying to obtain white solid product, namely compound (I-17), yield 54%, melting point 126-129 ℃. ¹ H NMR(400MHz,DMSO-d ₆ )δ9.32(s,1H),9.14(s,1H),8.24(s,1H),7.91(d,J＝8.0Hz,1H),7.64(d,J＝8.0Hz,1H),7.43(t,J＝6.0Hz,1H),7.33(s,4H),3.34(t,J＝8.0Hz,2H),2.93(t,J＝8.0Hz,2H),2.20(s,3H),1.95(s,3H).

Example 23: preparation of Compound (I-1)

Dissolving 2.253g (10.0 mmol) of compound (III-1) and 1.012g (10.0 mmol) of triethylamine in 40mL of toluene, dropwise adding a reaction solution obtained after the completion of the reaction under the condition of magnetic stirring at 0 ℃, heating the reaction solution to room temperature after the completion of the dropwise adding, reacting for 9 hours at room temperature (the reaction process adopts TLC tracking detection, the developing agent is a petroleum ether and ethyl acetate mixed solution with the volume ratio of 3:1), washing the reaction solution with (50 mL multiplied by 3), separating an organic phase, evaporating the solvent, and then performing residue column chromatography, namely adding 10 mL of ethyl acetate solvent into the residue obtained after the solvent evaporation to dissolve the residue to obtain a solution, adding 4.5 g of silica gel (300-400 meshes of coarse pore (zcx.II) column chromatography silica gel), uniformly mixing, evaporating the solvent to obtain a mixture of dried residue and the silica gel, and filling the mixture into a column, and then performing the column chromatography according to the volume ratio of 3:1 as eluent, TLC tracking detection (developing solvent is petroleum ether and ethyl acetate mixed solution with volume ratio of 3:1), collecting eluent containing compound shown in formula (I-1) according to TLC detection, evaporating solvent from the collected eluent, and drying to obtain white solid product, namely compound (I-1), with yield of 68% and melting point of 104-107 ℃. ¹ H NMR was as in example 2.

Example 24: in vitro test of anticancer Activity

The prepared compounds (I-1) to (I-17) are respectively subjected to biological activity tests of human hepatoma cells HepG2, human lung carcinoma cells A549, human cervical carcinoma cells Hela, human breast duct carcinoma cells T-47D, human breast carcinoma cells MDA-MB-231, human colorectal adenocarcinoma cells SW620 and human colorectal adenocarcinoma cells HCT116, wherein the compound (I-1) is prepared by the method of example 23.

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

Cell lines: human liver cancer cells HepG2, human lung cancer cells A549, human cervical cancer cells Hela, human breast ductal cancer cells T-47D, human breast cancer cells MDA-MB-231, human colorectal adenocarcinoma cells SW620 and human colorectal adenocarcinoma cells HCT116. The cell lines are purchased from cell banks of Shanghai Seisakusho of China academy of sciences.

The experimental procedure was as follows:

(1) Culture of tumor cells

The cell algebra used in the experiment is within 5 generations. The consumables and reagents used for cell culture were subjected to a strict sterilization procedure, and all experimental procedures were performed in a sterile operating table.

(a) Resuscitation of tumor cells

Before the experiment starts, articles to be used, such as a culture bottle, a centrifuge tube, a pipette, a gun head, a waste liquid barrel and the like, are placed into an ultra-clean bench to be sterilized by turning on an ultraviolet lamp, and reagents used for cell culture are placed into a constant-temperature water bath kettle at 37 ℃ to be preheated. 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 frozen tumor cells from a refrigerator at the temperature of minus 80 ℃, shaking in a water bath at the temperature of 37 ℃ to quickly defrost, spraying alcohol into an ultra-clean bench under the condition that a small part of frozen liquid in a frozen tube is still unfrozen, immediately transferring all the cells in the frozen tube into a 15mL centrifuge tube added with 1640 culture solution, and lightly blowing and uniformly mixing by a pipetting gun. The centrifuge tube was placed in a centrifuge and centrifuged at 1000rpm for 5min, and the supernatant was aspirated off in a super clean bench. Sucking 2mL1640 culture solution into a centrifuge tube containing cell sediment by a pipette to prepare a cell suspension, blowing and uniformly mixing the cell suspension, and transferring the cell suspension to a bottle bottom with an area of 25cm ² 4mL of 1640 culture solution is added into the breathable culture flask, the culture flask is gently shaken to mix cells, and the culture flask is put into 5% CO ₂ Culturing in a constant temperature incubator at 37 ℃.

(b) Passage of tumor cells

When the cell growth state is good and 70% -80% of the bottom of the culture flask is paved, the cell can be passaged. The steps of the passage operation are all completed in an ultra-clean bench, and the passage can be started after the preparation work is finished. Firstly, sucking the original culture solution in a culture bottle into a waste liquid barrel by a liquid transferring gun, adding 2mL of PBS, repeatedly washing for several times, sucking, then adding about 600-700 mu L of trypsin (containing 0.02% EDTA, phenol red and 0.25% pancreatin), ensuring that the bottle bottom is completely covered by trypsin, slightly shaking the culture bottle, placing 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 if a small number of cells still adhere to the wall, lightly beating the wall of the bottle by using a finger belly until most of the cells can shed from the bottle bottom. Adding inAfter 2mL of 1640 culture solution had been digested, the cells were gently blown off with a pipette, the cells were all blown off from the bottom of the flask, and the cell suspension was transferred to a 15mL centrifuge tube, 1000rpm, and centrifuged for 5min. Sucking the supernatant, diluting with 1640 culture solution, blowing uniformly, and evenly distributing into 2-3 culture bottles to continue in 5% CO ₂ Culturing in a constant temperature incubator at 37 ℃.

(c) Cryopreservation of tumor cells

Cell cryopreservation solutions were prepared in advance according to the volume ratio of FBS (fetal bovine serum): dmso=9:1 and placed in a refrigerator at 4 ℃ for later use. The freezing operation steps are completed in the super clean bench, and the frozen storage can be started after the preparation work is finished. Firstly, sucking the original culture solution in a culture flask, adding 2mL of PBS buffer solution, repeatedly washing for several times, pouring, then adding about 600-700 mu L of trypsin (containing 0.02% EDTA, phenol red and 0.25% pancreatin), gently shaking the culture flask, ensuring that the pancreatin can cover the bottom of the culture flask, placing the culture flask into a constant-temperature incubator at 37 ℃ for incubation for 1-2 min, observing the cell shedding condition under a microscope, if a small number of cells still cling to the wall, lightly beating the wall of the bottle by using a finger belly until the cells can be mostly shed from the bottom of the bottle. After the end of digestion by adding 2mL of 1640 culture solution, the cells were gently blown off with a pipette, the cells were all blown off from the bottom of the flask, and the cell suspension was transferred to a 15mL centrifuge tube, 1000rpm, and centrifuged for 5min. The supernatant was decanted. 1mL of frozen stock solution just taken out from a refrigerator at 4 ℃ is added, and is blown to be uniform to form cell suspension, and the cell suspension is transferred into a frozen stock tube. Placing the labeled cell types, cell algebra and frozen date into a refrigerator with ultralow temperature of-80 ℃ for preservation after placing the labeled cell types, cell algebra and frozen date into the refrigerator with ultralow temperature of-20 ℃ for standing for 1 hour at-4 ℃.

(2) MTT experimental method

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

(b) And (3) paving: taking 96-well plate, adding 100 μl diluted cell suspension into experimental well, adding 100 μl1640 culture solution into blank well, adding 100 μl PBS buffer solution around, adding CO ₂ Culturing in a constant temperature incubator.

(c) Compounding and adding a compound: the compound 10. Mu. Mol/mL in DMSO and the positive control LLY-507 were diluted to the indicated concentrations in 1640 medium, and the drug was applied by liquid exchange method at 20. Mu.M, 10. Mu.M, 5. Mu.M, 2.5. Mu.M, and 1.25. Mu.M, respectively. Discarding original culture solution, adding 100 μl of 1640 culture solution containing compound or positive control drug into experimental hole, adding 100 μl of 1640 culture solution into control group and blank hole, adding 96-well plate into 5% CO after adding drug ₂ Continuously culturing in a constant temperature incubator at 37 ℃.

(d) Adding MTT: after 48 hours, the 96-well plate is taken out and put into an ultra clean bench, 10 mu L of MTT solution with concentration of 5mg/mL is added into each well under the condition of light shielding, and then 5% CO is added ₂ Continuously culturing in a constant temperature incubator at 37 ℃.

(e) And (3) detection: incubating the MTT-added 96-well plate in an incubator for 3.5-4 h, taking out, carefully sucking out the solution in each well, adding 150 mu L of DMSO into each well to dissolve formazan generated by dissolution, then placing the formazan on a flat-plate oscillator to oscillate for 20min, and detecting the absorbance value at 490nm by using an enzyme-labeled instrument.

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

Cell viability (%) = [ (As-Ab)/(Ac-Ab) ] ×100%

As: experiment well (cell-containing culture solution, MTT, toxic substance (i.e., compound (I))

Ac control wells (cell-containing culture medium, MTT, without toxic substances)

Ab-blank wells (MTT-containing, cell and toxic material free culture broth).

The results of the test are shown in table 1 and below:

TABLE 1 inhibition of cancer cell growth by Compound (I)

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Claims

1. Application of dimethyl tetrazine formamide compound shown in formula (I) or pharmaceutically acceptable salt thereof in preparing medicament for treating or preventing cervical cancer;

in the formula (I), R is 4-CF ₃ 、2-CF ₃ 2-F-4-Br, 2-Cl, 2-Br-4-Cl or 4-Cl.

2. The use according to claim 1, wherein: the cancer cells of the cervical cancer are human cervical cancer cells Hela.

3. The use according to claim 2, wherein: in the formula (I), R is 2-CF ₃ Or 4-Cl.