CN115286589B - Cinnamoyl tetrazine compound, and preparation and application thereof - Google Patents
Cinnamoyl tetrazine compound, and preparation and application thereof Download PDFInfo
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- CN115286589B CN115286589B CN202210880694.7A CN202210880694A CN115286589B CN 115286589 B CN115286589 B CN 115286589B CN 202210880694 A CN202210880694 A CN 202210880694A CN 115286589 B CN115286589 B CN 115286589B
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- 238000002360 preparation method Methods 0.000 title claims abstract description 53
- -1 Cinnamoyl tetrazine compound Chemical class 0.000 title claims abstract description 33
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- BJHCYTJNPVGSBZ-YXSASFKJSA-N 1-[4-[6-amino-5-[(Z)-methoxyiminomethyl]pyrimidin-4-yl]oxy-2-chlorophenyl]-3-ethylurea Chemical compound CCNC(=O)Nc1ccc(Oc2ncnc(N)c2\C=N/OC)cc1Cl BJHCYTJNPVGSBZ-YXSASFKJSA-N 0.000 claims abstract description 21
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- 238000006243 chemical reaction Methods 0.000 claims description 336
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 claims description 246
- 150000001875 compounds Chemical class 0.000 claims description 177
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 claims description 159
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- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 123
- LKQNEFXKCOFFCO-UHFFFAOYSA-N 3,6-dimethyl-1,6-dihydro-1,2,4,5-tetrazine Chemical compound CC1NN=C(C)N=N1 LKQNEFXKCOFFCO-UHFFFAOYSA-N 0.000 claims description 73
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 68
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- VHYFNPMBLIVWCW-UHFFFAOYSA-N 4-Dimethylaminopyridine Chemical compound CN(C)C1=CC=NC=C1 VHYFNPMBLIVWCW-UHFFFAOYSA-N 0.000 claims description 14
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- WBYWAXJHAXSJNI-UHFFFAOYSA-N methyl p-hydroxycinnamate Natural products OC(=O)C=CC1=CC=CC=C1 WBYWAXJHAXSJNI-UHFFFAOYSA-N 0.000 description 4
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- AWUCVROLDVIAJX-GSVOUGTGSA-N sn-glycerol 3-phosphate Chemical compound OC[C@@H](O)COP(O)(O)=O AWUCVROLDVIAJX-GSVOUGTGSA-N 0.000 description 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D257/00—Heterocyclic compounds containing rings having four nitrogen atoms as the only ring hetero atoms
- C07D257/02—Heterocyclic compounds containing rings having four nitrogen atoms as the only ring hetero atoms not condensed with other rings
- C07D257/08—Six-membered rings
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Pharmacology & Pharmacy (AREA)
- Life Sciences & Earth Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
Abstract
The invention discloses cinnamoyl tetrazine compounds, 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 application of cinnamoyl tetrazine compounds or pharmaceutically acceptable salts thereof in preparing medicines for treating or preventing VEGFR-2 mediated diseases or medicines for inhibiting VEGFR-2, and the cinnamoyl tetrazine compounds or pharmaceutically acceptable salts thereof show good inhibition activity.
Description
Technical Field
The invention relates to cinnamoyl tetrazine compounds and a preparation method thereof, and application of the compounds as VEGFR-2 inhibitors in preparing medicines for treating or preventing VEGFR-2 mediated diseases or medicines for inhibiting VEGFR-2.
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
It is a first object of the present invention 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 object of the present invention is to provide an application of the cinnamoyl tetrazine compound or a pharmaceutically acceptable salt thereof in preparing a medicament for treating or preventing VEGFR-2 mediated diseases or a medicament for inhibiting VEGFR-2, wherein the VEGFR-2 mediated diseases are cancers, the cancers are human lung cancer or human liver cancer, and cancer cells of the cancers are A549, huh7 or HepG2 cells.
The technical scheme adopted by the invention is specifically described 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.
Particularly preferred cinnamoyl tetrazines of the invention are selected from one of the following:
in a second aspect, the present invention provides a preparation method of a cinnamoyl tetrazine compound represented by formula (I), the preparation method comprising:
(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 solution to room temperature after the dripping is finished, stirring at room temperature for reaction for 0.5-50 hours, and introducing nitrogen into a tail gas absorption solution (NaOH aqueous solution) to prevent white fog from being generated, wherein the generated product is directly used for 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 the cinnamoyl tetrazine compound shown in the 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 defined as 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 basic catalyst a, the basic catalyst b, the triphosgene, and the amount of the compound (III) to be fed is 1:0.1 to 3:0.1 to 3, and the ratio of the amount of the compound (II) to the amount of the basic catalyst a, the basic catalyst b, the triphosgene, and the amount of the compound (III) to be fed is preferably 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 may be an amount capable of dissolving triphosgene, the compound (ii) and the basic catalyst a, and the amount of the organic solvent B may be an amount capable of dissolving the basic catalyst B and the compound (iii). Preferably, the total volume of the organic solvent A is 3-18mL/mmol based on the amount of the compound represented by the formula (II), wherein the volume of the organic solvent A used for dissolving triphosgene is 1-10mL/mmol based on the amount of the compound represented by the formula (II), and the total volume of the organic solvent B is 2-20mL/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 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 cinnamoyl tetrazine compound shown in the formula (I) is obtained through the column chromatography of residues.
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 may be the same organic solvent, and the organic solvent A, B or C may be the same solvent or different solvents.
In a third aspect, the invention provides application of cinnamoyl tetrazine compounds shown in formula (I) or pharmaceutically acceptable salts thereof in preparing medicines for treating or preventing VEGFR-2 mediated diseases or medicines for inhibiting VEGFR-2.
In a specific embodiment of the present invention, the VEGFR-2 mediated disorder is cancer. Such cancers include, but are not limited to: non-small cell lung cancer, lung adenocarcinoma, breast cancer, and 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 cells are a549 cells, and the cinnamoyl tetrazine compound represented by formula (i) is compound (ie).
Preferably, the cancer cells are Huh7 cells, and the cinnamoyl tetrazine compound represented by formula (i) is a compound (ih).
Preferably, the cancer cell is a HepG2 cell, and the cinnamoyl tetrazine compound represented by the formula (I) is a compound (Id), (Ig) (ih) or (is).
The term "pharmaceutically acceptable" in this application 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 anticancer (especially human lung cancer or liver 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 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.
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.
Preparation of 3, 6-dimethyl-1, 6-dihydro-1, 2,4, 5-tetrazine (II) reference (Synthetic Communications,2003,33 (16), 2769-2775).
The following compounds (IIIn) to (IIIu) are prepared by the process described in reference (Journal ofMolecular Structure,2022,1254,132422;Indian Pat.Appl, 2010DE00068,15Jul 2011;Chemistry&Biodiversity,2007,4 (2), 203-214).
The following compounds (IIIa) to (IIIm) and (IIIv) are prepared by the methods described in the references (ACS Catalysis,2022,12 (8), 4688-4695;Journal ofPharmacy and Pharmacology,2020, 72 (7), 916-926;Journal ofMedicinal Chemistry,2014,57 (12), 5333-5347).
The preparation of the compounds (IIIa) to (IIIm) and (IIIv) are shown in the following reaction formula, wherein R 1 The structures of the above compounds (IIIa) to (IIIm) and (IIIv):
example 1: preparation of Compound (IIIa)
7.4g (50 mmol) of cinnamic acid and 100mL of anhydrous dichloromethane are added into a three-necked flask, 100mL of anhydrous dichloromethane solution containing 70.0g (551 mmol) of oxalyl chloride is dropwise added under the ice bath condition, 2.0g of DMF is added after the dropwise addition, the reaction is carried out for 12 hours at room temperature, and the organic solvent is distilled off, so that 4.8g (29 mmol) of light yellow oily cinnamoyl chloride is obtained.
Into a three-necked flask, 4.8g (29 mmol) of cinnamoyl chloride and 100mL of anhydrous dichloromethane were added, 100mL of a dichloromethane solution containing 5.5g (40 mmol) of m-nitroaniline and 4.2g (42 mmol) of triethylamine was added dropwise under ice bath conditions, the reaction was carried out at room temperature for 10 hours after the addition, the organic solvent was distilled off, 100mL of diluted hydrochloric acid with pH of 1 was added, the mixture was filtered, and the filter cake was washed with 100mL of 3 water and dried to obtain 5.6g (21 mmol) of N- (3-nitrophenyl) cinnamide.
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 are added into a three-necked flask, 200mL of absolute ethyl alcohol is added, the mixture is reacted for 1.5 hours under magnetic stirring, the mixture is cooled to room temperature, the pH of the reaction solution is adjusted to be slightly alkaline by a saturated sodium bicarbonate aqueous solution, ethyl acetate is used for extraction (100 mL multiplied by 3), an organic phase is combined, anhydrous magnesium sulfate is dried, filtered, and the organic solvent is distilled off to obtain 3.2g (13 mmol) of a compound (IIIa).
Preparation of Compounds (IIIb) to (IIIm) and Compound (IIIv): the corresponding substituted cinnamic acids were replaced with cinnamic acids, and the compounds (IIIb) to (IIIm) and (IIIv) were prepared by the method described in example 1, and are not described in detail herein.
Example 2: preparation of Compound (Ia)
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 2.383g (10.0 mmol) of compound (IIIa) and 0.122g (1.0 mmol) of DMAP in 60mL of chloroform, dropwise adding a reaction solution obtained 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 is detected by TLC tracking, 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 performing column chromatography on the residue, namely adding 10 mL of petroleum ether solvent into the residue obtained after the solvent evaporation to dissolve the residue to obtain a solution, adding 2.0 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 1:2, using petroleum ether and ethyl acetate mixed solution as eluent, washingThe reaction was carried out by TLC, the eluent containing the compound represented by the formula (Ia) was collected by TLC, the solvent was distilled off from the collected eluent, and the product was dried to give a white solid, namely the compound (Ia) in a yield of 35% (the same as below except for example 5 and example 22 in terms of the amount of 3, 6-dimethyl-1, 6-dihydro-1, 2,4, 5-tetrazine substance), having a melting point of 147 to 149 ℃. 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)
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 7.149g (30.0 mmol) of compound (IIIa) 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 obtained, the mixture is filled into a column, and then the mixture is carried out according to the volume ratio of 20: 1 as eluent, and performing TLC tracking detection (the developing solvent is the mixed solution of petroleum ether and ethyl acetate in a volume ratio of 20:1), collecting eluent containing the compound shown in the formula (Ia) according to TLC detection, evaporating the collected eluent to remove the solvent, and drying to obtain a white solid product, namely the compound (Ia), wherein the yield is 57%, and the melting point is 147-149 ℃. 1 H NMR was as in example 2.
Example 4: preparation of Compound (Ia)
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 is dissolved with 4.766g (20.0 mmol) of compound (IIIa) and 2.024g (20.0 mmol) of triethylamine, a reaction liquid after completion of the reaction is dripped under the condition of magnetic stirring at 0 ℃, the reaction liquid is warmed to room temperature after the dripping is completed, the reaction is carried out for 8 hours at room temperature (the reaction process is detected by TLC tracking, the developing agent is a mixed solution of petroleum ether and ethyl acetate in a volume ratio of 10:1), the reaction liquid is washed with (50 mL multiplied by 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 solvent to obtain a solution, then 2.0 g of silica gel (300-400-mesh coarse (zcx.II) column chromatography silica gel) is added into the solution, the solvent is distilled off after the solution is uniformly mixed, and a mixture of dried residue and the silica gel is packed into a column, and then the mixture is prepared according to a volume ratio of 10:1 as eluent, performing TLC tracking detection (the developing solvent is the mixed solution of petroleum ether and ethyl acetate in a volume ratio of 10:1), collecting the eluent containing the compound shown in the formula (Ia) according to TLC detection, evaporating the collected eluent to remove the solvent, drying to obtain a white solid product, Namely, the compound (Ia) is obtained in a yield of 46% and a melting point of 147-149 ℃. 1 H NMR was as in example 2.
Example 5: preparation of Compound (Ia)
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 0.238g (1.0 mmol) of compound (IIIa) and 0.200g (5.0 mmol) of sodium hydroxide in 20mL of chloroform, dropwise adding a reaction solution obtained after completion of the reaction under the condition of magnetic stirring at 5 ℃, heating the reaction solution to room temperature after completion of the dropwise adding, reacting for 50 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 carrying out column chromatography on the residue, namely adding 10 mL of chloroform solvent into the residue obtained after the solvent evaporation to dissolve the residue to obtain a solution, adding 0.2 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 loading 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 the compound shown in formula (Ia) according to TLC detection, evaporating solvent from the collected eluent, and drying to obtain white solid product, namely compound (Ia), wherein the yield is 41% (based on the mass of compound IIIa), and the melting point is 147-149 ℃. 1 H NMR was as in example 2.
Example 6: preparation of Compound (Ia)
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 2.383g (10.0 mmol) of compound (IIIa) and 1.012g (10.0 mmol) of triethylamine in 60mL of methylene chloride, dropwise adding a reaction solution obtained 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 is detected by TLC tracking, 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 the solvent, and then performing column chromatography on the residue, namely adding 10 mL of chloroform solvent into the residue obtained after the solvent evaporation to dissolve the residue to obtain a solution, adding 2.0 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 in a volume ratio of 5:1 as eluent, and performing TLC tracking detection (the developing solvent is the mixed solution of petroleum ether and ethyl acetate in a volume ratio of 5:1), collecting eluent containing the compound shown in the formula (Ia) according to TLC detection, evaporating the collected eluent to remove the solvent, and drying to obtain a white solid product, namely the compound (Ia), wherein the yield is 44%, and the melting point is 147-149 ℃. 1 H NMR was as in example 2.
Example 7: preparation of Compound (Ib)
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 3.072g (10.0 mmol) of compound (IIIb) 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 3.5 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 volume ratio of 3:1 as eluent, and performing TLC tracking detection (the developing solvent is the mixed solution of petroleum ether and ethyl acetate in a volume ratio of 3:1), collecting eluent containing the compound shown in the formula (Ib) according to TLC detection, evaporating the collected eluent to remove the solvent, and drying to obtain a white solid product, namely the compound (Ib), wherein the yield is 60%, and the melting point is 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)
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 2.663g (10.0 mmol) of compound (IIIc) 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 is detected by TLC tracking, a 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 carrying out 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 3.5 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 loading the mixture into a column 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 (ic) according to TLC detection, evaporating solvent from the collected eluent, and drying to obtain white solid product, namely compound (ic), with yield of 67% and melting point of 144-146 ℃. 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)
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 2.683g (10.0 mmol) of compound (IIId) 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 is detected by TLC tracking, a 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 carrying out 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 4.0 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 loading 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 the compound shown in formula (Id) according to TLC detection, evaporating the collected eluent to remove solvent, and drying to obtain white solid product, namely the compound (Id), wherein the yield is 73%, and the melting point is 198-200 ℃. 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 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 2.523g (10.0 mmol) of compound (IIIe) 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 4.0 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, TLC tracking detection (developing solvent is petroleum ether and ethyl acetate mixed solution with volume ratio of 3:1), collecting eluent containing the compound shown in formula (ie) according to TLC detection, evaporating the collected eluent to remove solvent, and drying to obtain white solid product, namely compound (ie), with yield of 65% and melting point of 145-147 ℃. 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)
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 2.663g (10.0 mmol) of compound (IIIf) 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 is detected by TLC tracking, a 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 carrying out 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 4.0 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 loading the mixture into a column according to 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), collecting the eluent containing the compound shown in the formula (if) according to TLC detection, evaporating the solvent from the collected eluent, and drying to obtain a white solid product, namely the compound (if), wherein the yield is 70%, and the melting point is 184-186 ℃. 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)
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 2.523g (10.0 mmol) of compound (III) and 1.012g (10.0 mmol) of triethylamine in 40mL of toluene, dropwise adding the reaction solution after completion of the reaction at 0 ℃ under magnetic stirring, 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, adding 3.0 g of silica gel (300-400-mesh coarse pore (zcx.II) column chromatography silica gel) into the solution, uniformly mixing, and evaporating the solvent to obtain a dry residue and silicon Glue mixture, column the mixture, then in volume ratio 3:1 as eluent, and performing TLC tracking detection (the developing solvent is the mixed solution of petroleum ether and ethyl acetate in a volume ratio of 3:1), collecting eluent containing the compound shown in the formula (Ig) according to TLC detection, evaporating the collected eluent to remove the solvent, and drying to obtain a white solid product, namely the compound (Ig), wherein the yield is 60%, and the melting point is 141-143 ℃. 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)
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 2.563g (10.0 mmol) of compound (III h) and 1.012g (10.0 mmol) of triethylamine in 40mL of toluene, dropwise adding the reaction solution after completion of the reaction at 0 ℃ under magnetic stirring, heating the reaction solution to room temperature, reacting at room temperature for 10 hours (the reaction process is detected by TLC tracking, 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, distilling off the solvent, performing column chromatography on the residue, namely taking the residue after distilling off the solvent, adding 10 mL of ethyl acetate solvent to dissolve the residue to obtain a solution, and adding 4.0 g of the solution Silica gel (300-400 mesh coarse pore (zcx.II) type column chromatography silica gel), evenly mixing, evaporating solvent to obtain a mixture of dry residues and silica gel, loading the mixture into a column, and then carrying out volume ratio of 3:1 as eluent, and performing TLC tracking detection (the developing solvent is the mixed solution of petroleum ether and ethyl acetate in a volume ratio of 3:1), collecting eluent containing the compound shown in the formula (ih) according to TLC detection, evaporating the collected eluent to remove the solvent, and drying to obtain a white solid product, namely the compound (ih), wherein the yield is 64%, and the melting point is 168-170 ℃. 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)
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 2.663g (10.0 mmol) of compound (III i) and 1.012g (10.0 mmol) of triethylamine in 40mL of toluene, dropwise adding the reaction solution after the completion of the reaction at the temperature of 0 ℃ under magnetic stirring, 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 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 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 3.0 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 performing column separation on the mixture 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 the compound shown in formula (ii) according to TLC detection, evaporating the collected eluent to remove solvent, and drying to obtain white solid product, namely compound (ii), with yield of 66% and melting point of 152-154 ℃. 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)
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 2.727g (10.0 mmol) of compound (III j) and 1.012g (10.0 mmol) of triethylamine in 40mL of toluene, dropwise adding the reaction solution after the completion of the reaction under magnetic stirring at 0 ℃, and reacting at room temperature for 11 hoursAfter that (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 water (50 mL multiplied by 3), an organic phase is separated, the solvent is distilled off, and then 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, 3.5 g of silica gel (300-400-mesh coarse pore (zcx.II) column chromatography silica gel) is added into 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 columns, and then the volume ratio of the mixture is 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 the compound shown in formula (Ij) according to TLC detection, evaporating the collected eluent to remove solvent, and drying to obtain white solid product, namely the compound (Ij), wherein the yield is 59%, and the melting point is 161-163 ℃. 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)
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 2.683g (10.0 mmol) of compound (IIIk) 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 is detected by TLC tracking, a 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 carrying out 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 4.0 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 loading the mixture into a column according to the volume ratio of 3:1 as eluent, and performing TLC tracking detection (the developing solvent is the mixed solution of petroleum ether and ethyl acetate in a volume ratio of 3:1), collecting eluent containing the compound shown in the formula (Ik) according to TLC detection, evaporating the collected eluent to remove the solvent, and drying to obtain a white solid product, namely the compound (Ik), wherein the yield is 68%, and the melting point is 194-196 ℃. 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)
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 2.727g (10.0 mmol) of compound (III) 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 is detected by TLC tracking, a 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 carrying out 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 4.0 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 loading the mixture into a column, and then carrying out 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 the compound shown in formula (il) according to TLC detection, evaporating the collected eluent to remove solvent, and drying to obtain white solid product, namely compound (il), with yield of 74% and 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)
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 2.727g (10.0 mmol) of compound (IIIm) 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 is detected by TLC tracking, a 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 carrying out 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 3.0 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 loading the mixture into a column 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 (im) according to TLC detection, evaporating solvent from the collected eluent, and drying to obtain white solid product, namely compound (im), yield of 53%, melting point of 150-152 ℃. 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 (In)
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.
60mL of chloroform was usedDissolving 2.233g (10.0 mmol) of compound (IIIn) and 0.122g (1.0 mmol) of DMAP, dropwise adding a reaction solution after completion of the reaction under magnetic stirring at-10 ℃, heating the reaction solution to room temperature, reacting at room temperature for 10 hours (TLC tracking detection is adopted in the reaction process, a 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 performing residue column chromatography, namely taking the residue after evaporation of the solvent, adding 10mL of petroleum ether solvent to dissolve the residue to obtain a solution, adding 2.0 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 the volume ratio of 1:2 as eluent, and performing TLC tracking detection (the developing solvent is the mixed solution of petroleum ether and ethyl acetate in a volume ratio of 1:2), collecting eluent containing the compound shown in the formula (In) according to TLC detection, evaporating the collected eluent to remove the solvent, and drying to obtain a white solid product, namely the compound (In), wherein the yield is 44%, and the melting point is 136-138 ℃. 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 (In)
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 6.698g (30.0 mmol) of compound (IIIn) 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 obtained, the mixture is filled into a column, and then the mixture is carried out according to the volume ratio of 20:1 as eluent, and performing TLC tracking detection (the developing solvent is the mixed solution of petroleum ether and ethyl acetate in a volume ratio of 20:1), collecting eluent containing the compound shown in the formula (In) according to TLC detection, evaporating the collected eluent to remove the solvent, and drying to obtain a white solid product, namely the compound (In), wherein the yield is 72%, and the melting point is 136-138 ℃. 1 H NMR 13 C NMR was as in example 19.
Example 21: preparation of Compound (In)
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 4.465g (20.0 mmol) of compound (IIIn) and 2.024g (20.0 mmol) of triethylamine, and the reaction mixture after completion of the reaction was allowed to drop under magnetic stirring at 0℃and the reaction mixture was allowed to warm to room temperatureAfter 8 hours of reaction 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 10:1), the reaction solution is washed with (50 mL multiplied by 3), an organic phase is separated, the solvent is distilled off, then the residue is subjected to column chromatography, namely, 10 mL of 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, 3.0 g of 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 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 the compound shown in formula (In) according to TLC detection, evaporating the collected eluent to remove solvent, and drying to obtain white solid product, namely the compound (In), wherein the yield is 59%, and the melting point is 136-138 ℃. 1 H NMR 13 C NMR was as in example 19.
Example 22: preparation of Compound (In)
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 0.223g (1.0 mmol) of compound (IIIn) and 0.200g (5.0 mmol) of sodium hydroxide in 20mL of chloroform, dropwise adding the reaction solution after the completion of the reaction under the condition of magnetic stirring at 5 ℃, heating the reaction solution to room temperature after the completion of the dropwise adding, 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 column chromatography on residues, namely taking residues after the solvent is evaporated Adding 10 ml of chloroform solvent to dissolve the mixture 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, 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 steps of: 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 (In) according to TLC detection, evaporating solvent from the collected eluent, and drying to obtain white solid product, namely compound (In), yield 54% (based on the amount of compound III n) and melting point 136-138 ℃. 1 H NMR 13 C NMR was as in example 19.
Example 23: preparation of Compound (In)
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 2.233g (10.0 mmol) of compound (IIIn) and 1.012g (10.0 mmol) of triethylamine in 60mL of dichloromethane, dropwise adding the reaction solution after completion of the reaction at-5 ℃ under magnetic stirring, heating the reaction solution to room temperature, reacting for 9 hours at room temperature (the reaction process is detected by TLC tracking, 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 taking the residue after evaporating the solvent, adding 10 mL of chloroform solvent to dissolve the residue to obtain a solution, adding 3.0 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 the dried residue and the silica gel, and carrying outThe 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 the compound shown in formula (In) according to TLC detection, evaporating the collected eluent to remove solvent, and drying to obtain white solid product, namely the compound (In), wherein the yield is 65%, and the melting point is 136-138 ℃. 1 H NMR 13 C NMR was as in example 19.
Example 24: preparation of Compound (I o)
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 2.413g (10.0 mmol) of compound (III o) 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 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 evaporation to dissolve the residue to obtain a solution, adding 3.5 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 with the column according to the volume ratio of 5:1 as eluent, eluting, and collecting the eluent containing the compound shown in formula (I o) according to TLC detection, wherein TLC tracking detection is carried out (the developing agent is the mixed solution of petroleum ether and ethyl acetate in a volume ratio of 5:1) The solvent is distilled off and the white solid product is obtained after drying, namely the compound (I o) with the yield of 68 percent and the melting point of 128-130 ℃. 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)
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 2.373g (10.0 mmol) of compound (IIIp) 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 is detected by TLC tracking, 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 the solvent, and then carrying out 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 3.0 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 loading the mixture into a column in a volume ratio of 5:1 as eluent, eluting, and performing TLC tracking detection (developing solvent is mixed solution of petroleum ether and ethyl acetate in volume ratio of 5:1), according to TLC Detecting and collecting the eluent containing the compound shown in the formula (ip), evaporating the solvent from the collected eluent, and drying to obtain a white solid product, namely the compound (ip), wherein the yield is 64%, and the melting point is 100-102 ℃. 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)
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 2.577g (10.0 mmol) of compound (III q) 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 magnetic stirring at 0 ℃, heating the reaction solution to room temperature, reacting for 12 hours at room temperature (the reaction process is detected by TLC tracking, 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 the solvent, 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 3.0 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 dry residue and the silica gel The mixture was packed in a column and then in 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 the compound shown in formula (Iq) according to TLC detection, evaporating the collected eluent to remove solvent, and drying to obtain white solid product, namely the compound (Iq), wherein the yield is 61%, and the melting point is 133-135 ℃. 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)
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 2.577g (10.0 mmol) of compound (IIIr) and 1.012g (10.0 mmol) of triethylamine in 40mL of toluene, dropwise adding the reaction solution after completion of the reaction at 0 ℃ under magnetic stirring, heating the reaction solution to room temperature, reacting at room temperature for 12 hours (the reaction process is detected by TLC tracking, 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, steaming the solvent, and then performing column chromatography on the residue, namely taking the residue after steaming the solvent, adding 10 mL of the residue Dissolving the mixture in ethyl acetate solvent to obtain a dissolving solution, adding 3.0 g of silica gel (300-400 mesh coarse pore (zcx.II) type column chromatography silica gel) into the dissolving solution, 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 volume ratio of 5:1 as eluent, and performing TLC tracking detection (the developing solvent is the mixed solution of petroleum ether and ethyl acetate in a volume ratio of 5:1), collecting eluent containing the compound shown in the formula (ir) according to TLC detection, evaporating the collected eluent to remove the solvent, and drying to obtain a white solid product, namely the compound (ir), wherein the yield is 60%, and the melting point is 122-124 ℃. 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)
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.
40mL of toluene was dissolved in 2.533g (10.0 mmol) of Compound (IIIs) and 1.012g (10.0 mmol) of triethylamine, and the reaction mixture after completion of the reaction was allowed to drop under magnetic stirring at 0℃and then was allowed to warm to room temperature, after which the reaction mixture was allowed to react at room temperature for 10 hours (reaction procedure was followed byDetection was performed by TLC tracking with a volume ratio of developing reagent of 5: 1) washing the reaction solution with (50 mL x 3), separating out an organic phase, steaming out a solvent, performing residue column chromatography, namely taking the residue after steaming out the solvent, adding 10 mL of ethyl acetate solvent to dissolve the residue to obtain a dissolved solution, adding 3.0 g of silica gel (300-400-mesh coarse pore (zcx.II) column chromatography silica gel) into the dissolved solution, uniformly mixing, steaming out the solvent to obtain a mixture of the dried residue and the silica gel, filling the mixture into a column, and then filling the mixture into a column with 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 the compound shown in formula (is) according to TLC detection, evaporating the collected eluent to remove solvent, and drying to obtain white solid product, namely compound (is), the yield is 55%, and the melting point is 120-122 ℃. 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)
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.
40mL of toluene was dissolved in 2.393g (10.0 mmol) of compound (IIIt) and 1.012g (10.0 mmol) of triethylamine, and the mixture was added dropwise under magnetic stirring at 0 ℃After the reaction is completed, the reaction solution is warmed to room temperature, after the reaction is completed, the room temperature is reacted for 10 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), an organic phase is separated, the solvent is removed by distillation, namely, the residue after the solvent is removed by distillation is taken and added with 10 milliliters of ethyl acetate solvent to dissolve the residue, so as to obtain a dissolving solution, 3.0 grams of silica gel (300-400 meshes of coarse pore (zcx.II) type column chromatography silica gel) is added into the dissolving solution, after the mixing is carried out, the solvent is removed by distillation, 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, and performing TLC tracking detection (the developing solvent is the mixed solution of petroleum ether and ethyl acetate in a volume ratio of 5:1), collecting eluent containing the compound shown in the formula (It) according to TLC detection, evaporating the collected eluent to remove the solvent, and drying to obtain a white solid product, namely the compound (It), wherein the yield is 60%, and the melting point is 130-132 ℃. 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 (I u)
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.
40mL of toluene was dissolved in 3.022g (10.0 mmol) of Compound (IIIu) and 1.012g (10.0 mmol) of triethylamine, and the reaction mixture after completion of the reaction was stirred magnetically at 0℃and was then allowed to rise to the chamberAfter reacting for 12 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 5:1), the reaction solution is washed with (50 mL multiplied by 3), an organic phase is separated, the solvent is distilled off, then the residue is subjected to column chromatography, namely, 10 mL of 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, 3.0 g of 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 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 u) according to TLC detection, evaporating the collected eluent to remove solvent, and drying to obtain white solid product, namely the compound (I u), wherein the yield is 55%, and the melting point is 129-131 ℃. 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)
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.
2.983g (10.0 mmol) of compound (III v) and 1.012g (10.0 mmol) of triethylamine are dissolved in 40mL of toluene, 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, and after the reaction is carried out for 9 hours at room temperature (TLC tracking detection is adopted in the reaction process, and the volume ratio of the developing agent is 3:1)Washing the reaction solution with (50 mL x 3), separating out an organic phase, evaporating the solvent, performing residue column chromatography, namely taking the residue after evaporating the solvent, adding 10 mL of ethyl acetate solvent to dissolve the residue to obtain a dissolved solution, adding 4.0 g of silica gel (300-400-mesh coarse pore (zcx.II) column chromatography silica gel) into the dissolved solution, uniformly mixing, evaporating 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 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 the compound shown in formula (iv) according to TLC detection, evaporating the collected eluent to remove solvent, and drying to obtain white solid product, namely the compound (iv), with yield of 72% and melting point of 175-177 ℃. 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)
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 2.383g (10.0 mmol) of compound (IIIa) and 1.012g (10.0 mmol) of triethylamine in 40mL of toluene, dropwise adding the reaction solution after the completion of the reaction under magnetic stirring at 0 ℃, and heating the reaction solution to room temperature after the completion of the dropwise adding, reacting for 9 hours at room temperature (TLC tracking detection is adopted in the reaction process, and the developing agent is petroleum ether and ethyl acetate in a volume ratio of 3:1 are mixed)Solution), washing the reaction solution with (50 mL×3), separating out an organic phase, steaming to remove the solvent, performing column chromatography on the residue, namely, taking the residue after steaming to remove the solvent, adding 10 mL of ethyl acetate solvent to dissolve the residue to obtain a solution, adding 3.0 g of silica gel (300-400-mesh coarse pore (zcx.II) column chromatography silica gel) into the 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 performing column separation 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 (Ia) according to TLC detection, evaporating solvent from the collected eluent, and drying to obtain white solid product, namely compound (Ia), with yield of 59% and melting point of 147-149 ℃. 1 H NMR was as in example 2.
Example 32: preparation of Compound (In)
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 2.233g (10.0 mmol) of compound (IIIn) 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 magnetic stirring at 0 ℃, heating the reaction solution to room temperature, reacting for 10 hours at room temperature (the reaction process is detected by TLC tracking, 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, distilling off the solvent, performing column chromatography on the residue, namely adding 10 mL of ethyl acetate solvent into the residue obtained after distilling off the solvent to dissolve the residue to obtain a solution, adding 3.5 g of silica gel (300-400 meshes of coarse pore (zcx.II) column chromatography silica gel) into the solution, After mixing, the solvent was distilled off to obtain a mixture of dried residue and silica gel, the mixture was packed in a column, and then the volume ratio was 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 (In) according to TLC detection, evaporating the collected eluent to remove solvent, and drying to obtain white solid product, namely the compound (In), wherein the yield is 74%, and the melting point is 136-138 ℃. 1 H NMR 13 C NMR was as in example 19.
Example 33: in vitro kinase Activity assay
Kinase-Lumi TM The chemiluminescence kinase activity detection kit is a kit for quantitatively detecting kinase activity by measuring the residual amount of ATP in a solution after a kinase reaction by a chemiluminescence method. The experiment uses Kinase-Lumi TM The compounds (Ib), (ie), (ih), (ij), (ik), (io) prepared in the above examples were tested for their inhibitory activity on EGFR, PEGFRbeta and VEGFR-2 kinase, respectively, under room temperature conditions using a chemiluminescent kinase activity assay kit (Beyotide).
The following is a 96-well plate recommended assay system.
(1) Preparation of ATP standard curve
The reaction buffer was prepared with 1mM manganese dichloride, 5mM magnesium dichloride, 1mM Dithiothreitol (DTT).
Wells of 0, 0.03, 0.07, 0.15, 0.3, 0.6, 1.25, 2.5, 5, 10 μmatp standards were set (all ATP concentrations above are final concentrations of the material when the total volume in the standard wells reaches 100 μl). For preparation, 50. Mu.L of ATP was diluted with reaction buffer per well. Then 50 mu L of Kinase-Lumi was added TM And (5) a chemiluminescent kinase detection reagent, and uniformly mixing. The reaction was carried out at room temperature (about 25 ℃) for 10 minutes, and then chemiluminescent detection was carried out by a multifunctional microplate reader to prepare an ATP standard curve.
(2) Sample detection
Sample wells were configured to contain 0.1. Mu.g/mL polyglutamic acid and tyrosine (4:1) kinase substrate, 5. Mu. MATP and 10. Mu.g/L kinase (EGFR, PEGFRbeta or VEGFR-2) at a total volume of 100. Mu.L per wellConcentration (50, 100, 200, 400, 800, 1000 nM) of compound (Ib), (ie), (ih), (ij), (ik), (io). During preparation, polyglutamic acid and tyrosine (4:1) kinase substrates, ATP, kinase (EGFR, PEGFR beta or VEGFR-2) and compounds (Ib), (ie), (ih), (ij), (ik) and (io) are added into each hole, and a reaction buffer is added to dilute the mixture to 50 mu L in total volume; then 50 mu L of Kinase-Lumi was added TM And (5) a chemiluminescent kinase detection reagent, and uniformly mixing. The reaction was carried out at room temperature (about 25 ℃) for 10 minutes. And then performing chemiluminescence detection by using a multifunctional enzyme-labeled instrument. The amount of ATP remaining in the sample wells was calculated from the standard curve, followed by calculation of the enzyme activity from the definition of enzyme activity. Final calculation of IC 50 Values.
The results of the in vitro kinase activity assay are shown in Table 1, and Table 1 shows the activity data of compounds (Ib), (ie), (ih), (ij), (ik), (io) against EGFR, PEGFRbeta and VEGFR-2 kinases. As can be seen from the data in the table, compounds (Ib), (ie), (ih), (ij), (ik), (io) have different degrees of inhibition on EGFR, PEGFR and VEGFR-2 activities, wherein the inhibition of VEGFR-2 kinase by compounds (Ib), (ie), (ih), (ij), (ik), (io) is overall better than EGFR, PDGFR.
TABLE 1 inhibitory Activity of Compound (I) against tyrosine kinase
Example 20: in vitro test of anticancer Activity
The prepared compounds (Ia) - (iv) are respectively subjected to biological activity tests of human lung cancer cells A549, human liver cancer cells Huh7 and human liver cancer cells HepG2, wherein the compounds (Ia) and (Id) are respectively prepared by the methods of example 31 and example 32.
The testing method comprises the following steps: tetrazolium salt reduction method (MTT method).
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 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, and then adding about 600 to 700 mu L of trypsin(containing 0.02% EDTA, phenol red and 0.25% pancreatin) to ensure that the bottle bottom is completely covered by trypsin, gently shaking the culture flask, placing the flask in a constant temperature incubator at 37 ℃ for incubation for 1-2 min, observing the cell shedding condition under a microscope, if a small amount of cells still adhere to the wall, lightly beating the wall of the flask with a finger belly until most of the cells can shed from the bottle bottom. 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. 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, and taking 10 mu L of fine tumor cellsCell suspension to cell counting plate, after counting, B16F10 cells were diluted to 5X 10 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 LPBS buffer around, adding CO 2 Culturing in a constant temperature incubator.
(c) Compounding and adding a compound: the compound 10. Mu. Mol/mL prepared in DMSO and the positive control Sorafenib were diluted to the indicated concentrations in 1640 medium, and the drug was applied by the liquid exchange method at 40. Mu.M, 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 hole (culture solution containing cells, MTT, toxic substance)
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 tests are shown in tables 2 and 3:
TABLE 2 inhibition of cancer cell growth by Compound (I)
TABLE 3 inhibition of cancer cell growth by Compound (I)
Claims (9)
1. A cinnamoyl tetrazine compound or a pharmaceutically acceptable salt thereof, characterized in that: the cinnamoyl tetrazine compound has 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.
2. A process for the preparation of cinnamoyl tetrazine compounds according to claim 1, 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 a reaction solution to room temperature after the dripping is finished, stirring at room temperature for reaction for 0.5-50 hours, and introducing nitrogen into a reaction system until no white fog is generated in a tail gas absorption solution, wherein the generated product is directly used for 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 the cinnamoyl tetrazine compound shown in the formula (I);
the basic catalyst a and the basic catalyst b are one of the following: triethylamine, 4-dimethylaminopyridine, pyridine or sodium hydroxide; the organic solvent A and the organic solvent B are each independently selected from one of the following: dichloromethane, chloroform or toluene;
In the formula (III), R and n are defined as in the formula (I).
3. The method of manufacturing as claimed in claim 2, wherein: the ratio of the amount of the compound (II) to the amount of the basic catalyst a, the basic catalyst b, the triphosgene and the compound (III) is 1:0.1-3:0.1-3.
4. The method of manufacturing as claimed in claim 2, wherein: the ratio of the amount of the compound (II) to the amount of the basic catalyst a, the basic catalyst b, the triphosgene and the compound (III) is 1:0.1-2:0.1-2.
5. The method of manufacturing as claimed in claim 2, wherein: in the step (1), under the stirring condition 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 liquid obtained in the step (1) is dropwise added under the stirring condition of minus 10-5 ℃.
6. The use of a cinnamoyl tetrazine compound or a pharmaceutically acceptable salt thereof according to claim 1 in the preparation of a medicament for treating or preventing VEGFR-2 mediated diseases or a medicament for inhibiting VEGFR-2, wherein the VEGFR-2 mediated diseases are cancers and the cancer cells of the cancers are a549, huh7 or HepG2 cells.
7. The use according to claim 6, wherein: the cancer cells are A549 cells, and the cinnamoyl tetrazine compound shown in the formula (I) is a compound (ie);
8. the use according to claim 6, wherein: the cancer cells are Huh7 cells, and the cinnamoyl tetrazine compound shown in the formula (I) is a compound (ih);
9. the use according to claim 6, wherein: the cancer cells are HepG2 cells, and the cinnamoyl tetrazine compound shown in the formula (I) is a compound (Id), (Ig), (ih) or (is);
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0252682A2 (en) * | 1986-07-02 | 1988-01-13 | May & Baker Limited | Tetrazines |
| CN105949140A (en) * | 2016-05-06 | 2016-09-21 | 浙江工业大学 | Diphenyl tetrazine dicarboamide compound, preparation and application |
| CN105949139A (en) * | 2016-05-06 | 2016-09-21 | 浙江工业大学 | Sec-butyl diphenyl tetrazine formamide compound, preparation and application |
| CN105968064A (en) * | 2016-05-06 | 2016-09-28 | 浙江工业大学 | Bis(m-methylphenyl) tetrazine dicarboxamide compound as well as preparation and application thereof |
| EP3342424A1 (en) * | 2016-12-29 | 2018-07-04 | Marius Yildiz | A system of collaborative modifiers and binders |
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Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0252682A2 (en) * | 1986-07-02 | 1988-01-13 | May & Baker Limited | Tetrazines |
| CN105949140A (en) * | 2016-05-06 | 2016-09-21 | 浙江工业大学 | Diphenyl tetrazine dicarboamide compound, preparation and application |
| CN105949139A (en) * | 2016-05-06 | 2016-09-21 | 浙江工业大学 | Sec-butyl diphenyl tetrazine formamide compound, preparation and application |
| CN105968064A (en) * | 2016-05-06 | 2016-09-28 | 浙江工业大学 | Bis(m-methylphenyl) tetrazine dicarboxamide compound as well as preparation and application thereof |
| EP3342424A1 (en) * | 2016-12-29 | 2018-07-04 | Marius Yildiz | A system of collaborative modifiers and binders |
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