CN102702120A - New compound o-hydroxyphenyl tetrazine dicarbonamide, preparation and application thereof - Google Patents

Abstract

The invention discloses N ¹ , N ⁴ -bis(2-hydroxyphenyl)-3,6-dimethyl-1,2,4,5-tetrazine-1 represented by formula (I), 4-dicarboxamide novel compound and its preparation method and application, the preparation of the compound is 3,6-dimethyl-1,6-dihydro-1,2,4,5-tetrazine, bis(trichloro Methyl)carbonate is used as a raw material and prepared by reacting with 2-hydroxyaniline in an organic solvent under the action of a basic catalyst. The compound of the present invention is used in the preparation of medicines for treating and preventing human lung cancer. The present invention has a reasonable design and a preparation method Simple, easy to operate, easy to obtain raw materials and low production cost, suitable for industrial application, and provides a new variety for the screening of anti-tumor drugs;

Description

A new compound o-hydroxyphenyltetrazine dicarboxamide and its preparation and application

(1) Technical field

The present invention relates to a new compound o-hydroxyphenyltetrazine dicarboxamide (i.e. N ¹ , N ⁴ -bis(2-hydroxyphenyl)-3,6-dimethyl-1,2,4,5-tetra oxazine-1,4-dicarboxamide) and its preparation method, and the application of said compound in the preparation of drugs for preventing or treating tumor diseases.

(2) Background technology

Tetrazine compounds have many good physical properties, spectral properties and high reactivity, especially some tetrazine derivatives with special structures have obvious antiviral activity, antitumor activity, and can be used as pesticides and insecticides. For example, there are currently two varieties of pesticides (tetrafenazine and flufenazine) on the market, and one variety of medicine (the antineoplastic drug temozolomide) has been on the market. the

In 1978, it was reported that 3,6-diphenynyl-hexahydro-1,2,4,5-tetrazine had antitumor activity (see Eremeev, AV; Tikhomirova, DA; Tyusheva, VA; Liepins, F.Khim .Geterotsikl.Soedin, 1978, 753.), this is the first report of 1,2,4,5-tetrazine compounds that may have potential anti-tumor activity. After that, it was successively reported that some 1,2,4,5-tetrazine compounds with specific structures had antitumor activity, such as 3,6-bis(2-hydroxyl-5-chlorophenyl)-1 , 2,4,5-tetrazine (see Rao, G.-W.; Hu, W.-X.Bioorg.Med.Chem.Lett.2006, 16(14), 3702.), 1-isobutyryl -3,6-diphenyl-1,4-dihydro-1,2,4,5-tetrazine (see Rao, G.-W.; Hu, W.-X.Bioorg.Med.Chem.Lett .2005, 15(12), 3714.) and N ¹ , N ⁴ -bis(m-methylphenyl)-3,6-dimethyl-1,2,4,5-tetrazine-1,4- Diformamide (see Hu, W.-X.; Rao, G.-W.; Sun, Y.-Q.Bioorg.Med.Chem.Lett.2004, 14(5), 1177.) etc., but large Most 1,2,4,5-tetrazine compounds do not have antitumor activity.

(3) Contents of the invention

The object of the present invention is to provide a new compound o-hydroxyphenyl tetrazine dicarboxamide with anticancer activity and its preparation method, that is, N ¹ , N ⁴ -bis(2-hydroxyphenyl)-3,6-bis The methyl-1, 2, 4, 5-tetrazine-1, 4-dicarboxamide and its preparation method provide a new variety for the screening of antitumor drugs. The compound has a certain inhibitory rate on the human lung cancer cell line A-549 at a certain dose (its IC ₅₀ on the A-549 cancer cell line is 21.9 μg/mL). The invention has reasonable design, simple and convenient preparation method, easy operation, readily available raw materials and low production cost, and is suitable for industrial application.

The technical scheme that the present invention adopts is as follows:

A new compound o-hydroxyphenyltetrazine dicarboxamide provided by the present invention is N ¹ , N ⁴ -bis(2-hydroxyphenyl)-3,6-dimethyl-1,2,4,5- Tetrazine-1,4-dicarboxamide compound, it has following structural formula (I):

N ¹ , N ⁴ -bis(2-hydroxyphenyl)-3,6-dimethyl-1,2,4,5-tetrazine-1,4-dicarboxamide (I) described in the present invention The preparation method is as follows: 3,6-dimethyl-1,6-dihydro-1,2,4,5-tetrazine, bis(trichloro Add methyl) carbonate (triphosgene, BTC) and basic catalyst into organic solvent A. After adding, heat and reflux for 4 to 12 hours. After the reaction, cool the reaction solution to about 5°C, usually to 4 ~6°C, blow nitrogen, then add dropwise the solution of 2-hydroxyaniline in organic solvent B, after the drop, heat and reflux for 12 to 48 hours, after the reaction is complete, evaporate the solvent, recrystallize the residue or obtain by column chromatography N ¹ , N ⁴ -bis(2-hydroxyphenyl)-3,6-dimethyl-1,2,4,5-tetrazine-1,4-dicarboxamide compound represented by formula (I), The basic catalyst is one of the following: pyridine, triethylamine, quinoline, potassium hydroxide, sodium hydroxide, N,N-xylidine, 4-dimethylaminopyridine or 4-pyrrolidinylpyridine;

Preparation of N ¹ , N ⁴ -bis(2-hydroxyphenyl)-3,6-dimethyl-1,2,4,5-tetrazine-1,4-dicarboxamide (I) described in the present invention The reaction is shown in the following reaction formula:

3,6-dimethyl-1,6-dihydro-1,2,4,5-tetrazine (II) and bis(trichloromethyl)carbonate, basic catalyst, 2- The ratio of the amount of hydroxyaniline feed material is 1:0.67～2:0.1～1:2～6. The total volume consumption of the organic solvent A and the organic solvent B is 13 to 13 based on the mass of 3,6-dimethyl-1,6-dihydro-1,2,4,5-tetrazine shown in formula (II). 75ml/g. the

The organic solvent A and the organic solvent B used in the reaction of the present invention are each independently selected from one of the following: dichloromethane, chloroform, toluene, methanol, ethanol or tetrahydrofuran. the

Specifically, the present invention prepares N ¹ , N ⁴ -bis(2-hydroxyphenyl)-3,6-dimethyl-1,2,4,5-tetrazine-1,4-dicarboxamide (I) The method recommendation is carried out as follows: 3,6-dimethyl-1,6-dihydro-1,2,4,5-tetrazine and basic catalyst shown in the dissolution formula (II) are used in organic solvent A1 to obtain Containing 3,6-dimethyl-1,6-dihydro-1,2,4,5-tetrazine and a solution of a basic catalyst, bis(trichloromethyl)carbonate and organic solvent A2 are placed in the reaction In a container, stir at -10～0°C, slowly drop the solution containing 3,6-dimethyl-1,6-dihydro-1,2,4,5-tetrazine and basic catalyst , 10-80 minutes after feeding, heating and reflux for 4-12 hours, TLC tracking detection, after the reaction, the temperature of the reaction solution is lowered to about 5°C, usually to 4-6°C, blowing nitrogen, and then adding 2-hydroxyaniline solution dropwise. In the solution of organic solvent B, after dripping, heat and reflux for 12 to 48 hours, TLC follow-up detection, after the reaction is completed, after the solvent is evaporated, the residue is recrystallized or column chromatographed to obtain N ¹ represented by formula (I), N ⁴ -bis(2-hydroxyphenyl)-3,6-dimethyl-1,2,4,5-tetrazine-1,4-dicarboxamide compound, the basic catalyst is one of the following : pyridine, triethylamine, quinoline, potassium hydroxide, sodium hydroxide, N, N-xylidine, 4-dimethylaminopyridine or 4-pyrrolidinylpyridine, the organic solvent A1 and organic solvent A2 Be the same organic solvent, organic solvent B and organic solvent A1 are the same or different organic solvents, organic solvent A1, organic solvent A2 are uniformly selected from one of the following: methylene chloride, chloroform, toluene, methanol, ethanol or tetrahydrofuran; The organic solvent B is selected from one of the following: methylene chloride, chloroform, toluene, methyl alcohol, ethanol or tetrahydrofuran; the total volume consumption of the organic solvent A1 and the organic solvent A2 is represented by formula (II) 3,6-di The mass of methyl-1,6-dihydro-1,2,4,5-tetrazine is 10-50ml/g; the volumetric amount of the organic solvent B is represented by formula (II) 3,6-dimethyl The mass of base-1,6-dihydro-1,2,4,5-tetrazine is 3-25ml/g, and the amount of the organic solvent A1 is enough to dissolve 3,6-dimethyl-1,6-di Hydrogen-1,2,4,5-tetrazine and a basic catalyst are enough, the 3,6-dimethyl-1,6-dihydro-1,2,4,5-tetrazine (II) The ratio with the amount of bis(trichloromethyl)carbonate, basic catalyst and 2-hydroxyaniline feed material is 1:0.67～2:0.1～1:2～6.

The recrystallization of the residue described in the method of the present invention is carried out as follows: take the residue after distilling off the solvent in the reaction flask, add the recrystallization solvent, stir and heat, reflux for 5 to 10 minutes, and filter while hot Remove the insolubles, cool the filtrate, precipitate solids, filter again, and dry the filter cake to obtain N ¹ , N ⁴ -bis(2-hydroxyphenyl)-3,6-dimethyl-1 shown in formula (I), 2,4,5-tetrazine-1,4-dicarboxamide compound; the recrystallization solvent is one of the following: methanol, ethanol, petroleum ether, ethyl acetate, dichloromethane, chloroform or tetrahydrofuran, preferably methanol , ethanol, ethyl acetate, dichloromethane, chloroform or tetrahydrofuran.

The residue column chromatography described in the method of the present invention is carried out according to the following steps: take the residue after distilling off the solvent in a one-necked bottle, add an organic solvent C to dissolve it, obtain a lysate, and then dissolve Add column chromatography silica gel of 1.5 to 2 times the mass of the residue in the solution, mix well, and evaporate the solvent to obtain a mixture of the dry residue and silica gel, and pack the mixture into a column, and then use a volume ratio of 0.5 to 10: 1 petroleum ether and ethyl acetate mixed solution are eluent, directly carry out elution, TLC tracking detects, detects and collects the eluate containing the compound shown in formula (I) according to TLC, collects liquid drying, obtains formula (I) ) represented by N ¹ , N ⁴ -bis(2-hydroxyphenyl)-3,6-dimethyl-1,2,4,5-tetrazine-1,4-dicarboxamide compound; the organic Solvent C is one of the following: methanol, ethanol, ethyl acetate, dichloromethane, chloroform, tetrahydrofuran, or dimethylsulfoxide.

Organic solvent A (organic solvent A1 and organic solvent A2) of the present invention, organic solvent B, what represented all refers to the organic solvent that is used for reaction, and the letter here does not refer to the implication of some organic solvents in particular, The letters are for clarity only and are used to distinguish these organic solvents appearing in different reaction steps. the

The N ¹ , N ⁴ -bis(2-hydroxyphenyl)-3,6-dimethyl-1,2,4,5-tetrazine-1,4-dicarboxamide (I) of the present invention can be It is applied to the preparation of medicines for preventing or treating tumor diseases, and particularly relates to the application in the preparation of medicines for preventing or treating human lung cancer diseases, and the human lung cancer cell line is human lung cancer cell line A-549.

The compound provided by the present invention has a certain inhibitory rate on human lung cancer cell line A-549 at a certain dose (the concentration of the compound of the present invention is 100 μg/mL, 10 μg/mL, 1 μg/mL), and it has a certain inhibitory rate on the A-549 cancer cell line The _IC50 of the compound is 21.9 μg/mL, and the compound of the present invention also has a certain inhibitory rate to the human breast cancer cell line MCF-7 at a certain dose (the concentration of the compound of the present invention is 100 μg/mL, 10 μg/mL, 1 μg/mL) , while compounds synthesized with other amines as substrates that are structurally similar to the compounds of the present invention do not necessarily have anticancer activity or have low anticancer activity.

The beneficial effects of the present invention are mainly reflected in: (1) The present invention provides a novel tetrazine compound with good anticancer activity, namely N ¹ , N ⁴ -bis(2- Hydroxyphenyl)-3,6-dimethyl-1,2,4,5-tetrazine-1,4-dicarboxamide compound; New species is provided for the screening of cancer drugs; (2) the present invention provides The preparation method of the tetrazine compound is simple, easy to operate, easy to obtain raw materials and low production cost, suitable for practical use, and is expected to be applied to the preparation of drugs for preventing or treating tumor diseases.

(4) Description of drawings

Compound N ¹ , N ⁴ -bis(4-ethoxyphenyl)-3,6-dimethyl-1,2,4,5-tetrazine-1,4-dicarboxamide in Example 9 in Figure 1 (Ⅲ), N ¹ , N ⁴ -dimethyl-3,6-dimethyl-1,2,4,5-tetrazine-1,4-dicarboxamide (Ⅳ) and N ¹ , N ⁴ - The chemical reaction scheme of bis(3-nitrophenyl)-3,6-dimethyl-1,2,4,5-tetrazine-1,4-dicarboxamide (Ⅴ).

(5) Specific implementation methods

The present invention is further described in conjunction with specific examples, and the following examples illustrate the present invention, rather than limit the present invention in any way. the

3,6-Dimethyl-1,6-dihydro-1,2,4,5-tetrazine (II) preparation reference (Sun, Y.Q.; Hu, W.X.; Yuan, Q.Synth.Commun.2003 , 33, 2769.) prepared by the method. the

Example 1: Preparation of N ¹ , N ⁴ -bis(2-hydroxyphenyl)-3,6-dimethyl-1,2,4,5-tetrazine-1,4-dicarboxamide (I)

Dissolve 2.00 grams (17.8 mmol) of 3,6-dimethyl-1,6-dihydro-1,2,4,5-tetrazine (II) and 2.16 grams (17.8 mmol) of N,N-xylidine Prepare a preliminary solution in 50 milliliters of dichloromethane solution, (that is, the solution containing 3,6-dimethyl-1,6-dihydro-1,2,4,5-tetrazine and basic catalyst), 250 10.60 g (35.7 mmol) of bis(trichloromethyl)carbonate and 20 ml of dichloromethane were sequentially added to a three-necked flask, stirred at 0° C., and the preliminary solution was slowly added dropwise. After the addition was completed within 10 minutes, TLC followed and detected, and the reaction was refluxed for 4 hours. Then the temperature was lowered to 5° C., nitrogen gas was blown, and a solution obtained by dissolving 3.90 g (35.7 mmol) of 2-hydroxyaniline in 20 ml of dichloromethane was added dropwise. After dripping, TLC tracking detection, heated to reflux for 48 hours, dichloromethane was evaporated, residue column chromatography, that is, the residue after the solvent was evaporated was added to 20 milliliters of methanol solvent to dissolve it, and the solution was obtained, and then dissolved Add 2.2 grams of silica gel (300-400 mesh coarse-pore (zcx.II) type column chromatography silica gel) to the liquid, mix well, and evaporate the solvent to obtain a mixture of dry residue and silica gel. The mixture is packed into a column, and then The mixed solution of petroleum ether and ethyl acetate with a volume ratio of 1:2 is the eluent, eluted, followed by TLC detection, and the eluate containing the compound shown in the formula (I) is collected according to the TLC detection, and the collected liquid is dried to obtain a white 0.34 grams of solid, namely N ¹ , N ⁴ -bis(2-hydroxyphenyl)-3,6-dimethyl-1,2,4,5-tetrazine-1,4-dicarboxamide (I), The yield is 5.0% (based on the amount of 3,6-dimethyl-1,6-dihydro-1,2,4,5-tetrazine of formula (II), the same below), and the melting point is 246-248°C. ¹ H NMR (500MHz, CDCl ₃ ) δ: 2.42 (s, 6H, CH ₃ ), 6.80-6.83 (m, 2H, C ₆ H ₄ ), 6.89-6.95 (m, 4H, C ₆ H ₄ ), 7.98 (dd, 2H, J=1.2, 6.8 Hz, _C6H4 ), 9.02 ₍ s, 2H), 10.16 (s, 2H).

Example 2: Preparation of N ¹ , N ⁴ -bis(2-hydroxyphenyl)-3,6-dimethyl-1,2,4,5-tetrazine-1,4-dicarboxamide (I)

Dissolve 2.00 g (17.8 mmol) of 3,6-dimethyl-1,6-dihydro-1,2,4,5-tetrazine (II) and 0.98 g (12.4 mmol) of pyridine in 10 ml of methanol to prepare To form a preparatory solution, 3.54 g (11.9 mmol) of bis(trichloromethyl)carbonate and 10 ml of methanol were sequentially added to a 50 ml three-necked flask, stirred at -5°C, and the preparatory solution was slowly added dropwise. After the addition was completed within 10 minutes, TLC followed and detected, and the reaction was refluxed for 10 hours. Then the temperature was lowered to 6°C, nitrogen gas was blown, and a solution obtained by dissolving 5.84 g (53.5 mmol) of 2-hydroxyaniline in 6 ml of ethanol was added dropwise. After dripping, TLC tracking detection, heating and reflux for 27 hours, distilled off methanol and ethanol, added 20 ml of chloroform to the residue, stirred and heated, refluxed for 5-10 minutes, filtered while hot to remove insoluble matter, cooled the filtrate, precipitated solids, filtered, filtered The cake was dried at 50°C to obtain 0.78 g of white solid, namely N ¹ , N ⁴ -bis(2-hydroxyphenyl)-3,6-dimethyl-1,2,4,5-tetrazine-1,4 - Diformamide (I), yield 11.4%, melting point 246-248°C. ¹ H NMR is the same as in Example 1.

Example 3: Preparation of N ¹ , N ⁴ -bis(2-hydroxyphenyl)-3,6-dimethyl-1,2,4,5-tetrazine-1,4-dicarboxamide (I)

Dissolve 2.00 g (17.8 mmol) of 3,6-dimethyl-1,6-dihydro-1,2,4,5-tetrazine (II) and 0.91 g (7.4 mmol) of 4-dimethylaminopyridine in Prepare a preparatory solution in 40 ml of chloroform solution, add 4.30 g (14.5 mmol) of bis(trichloromethyl)carbonate and 20 ml of chloroform to a 100 ml three-necked flask successively, stir at -10°C, and slowly add the preparatory solution dropwise. After the addition was completed within 40 minutes, TLC followed and detected, and the reaction was refluxed for 8 hours. Then the temperature was lowered to 6° C., nitrogen gas was blown, and a solution obtained by dissolving 7.77 g (71.2 mmol) of 2-hydroxyaniline in 10 ml of chloroform was added dropwise. After dripping, TLC tracking detection, heating to reflux for 12 hours, distilling off chloroform, and recrystallizing the residue with 20 ml of ethanol, other operations were the same as in Example 2, to obtain 0.49 g of white solid, namely N ¹ , N ⁴ -bis(2- Hydroxyphenyl)-3,6-dimethyl-1,2,4,5-tetrazine-1,4-dicarboxamide (I), yield 7.2%, melting point 246-248°C. ¹ H NMR is the same as in Example 1.

Example 4: Preparation of N ¹ , N ⁴ -bis(2-hydroxyphenyl)-3,6-dimethyl-1,2,4,5-tetrazine-1,4-dicarboxamide (I)

Dissolve 2.00 g (17.8 mmol) of 3,6-dimethyl-1,6-dihydro-1,2,4,5-tetrazine (II) and 1.75 g (13.5 mmol) of quinoline in 40 ml of THF Prepare a preparatory solution in a 250 ml three-necked flask, add 9.06 g (30.5 mmol) of bis(trichloromethyl)carbonate and 30 ml tetrahydrofuran in sequence, stir at -8°C, and slowly add the preparatory solution dropwise. After the addition was completed within 30 minutes, TLC followed and detected, and the reaction was refluxed for 12 hours. Then the temperature was lowered to 4° C., nitrogen gas was blown, and a solution obtained by dissolving 9.72 g (89.1 mmol) of 2-hydroxyaniline in 30 ml of chloroform was added dropwise. After the drop was completed, TLC followed detection, heated to reflux for 28 hours, distilled off THF and chloroform, and recrystallized the residue with 20 ml of ethyl acetate. Other operations were the same as in Example 2 to obtain 1.39 g of a white solid, namely N ¹ , N ⁴ - Bis(2-hydroxyphenyl)-3,6-dimethyl-1,2,4,5-tetrazine-1,4-dicarboxamide (I), yield 20.4%, melting point 246-248°C. ¹ H NMR is the same as in Example 1.

Example 5: Preparation of N ¹ , N ⁴ -bis(2-hydroxyphenyl)-3,6-dimethyl-1,2,4,5-tetrazine-1,4-dicarboxamide (I)

2.00 g (17.8 mmol) of 3,6-dimethyl-1,6-dihydro-1,2,4,5-tetrazine (II) and 1.60 g (10.8 mmol) of 4-pyrrolidinopyridine were dissolved in Prepare a preparatory solution in 80 ml of ethanol solution, add 8.00 g (27.0 mmol) of bis(trichloromethyl)carbonate and 20 ml of ethanol successively into a 250 ml three-necked flask, stir at 0° C., and slowly add the preparatory solution dropwise. After the addition was completed within 80 minutes, TLC followed and detected, and the reaction was refluxed for 6 hours. Then the temperature was lowered to 5° C., nitrogen gas was blown, and a solution obtained by dissolving 11.67 g (106.9 mmol) of 2-hydroxyaniline in 40 ml of ethanol was added dropwise. After dripping, TLC tracking detection, heated to reflux for 36 hours, distilled off ethanol, the residue was recrystallized with 20 ml of tetrahydrofuran, other operations were the same as in Example 2, and 1.03 g of white solid was obtained, namely N ¹ , N ⁴ -bis(2- hydroxyphenyl)-3,6-dimethyl-1,2,4,5-tetrazine-1,4-dicarboxamide (I). The yield is 15.1%, and the melting point is 246-248°C. ¹ H NMR is the same as in Example 1.

Example 6: Preparation of N ¹ , N ⁴ -bis(2-hydroxyphenyl)-3,6-dimethyl-1,2,4,5-tetrazine-1,4-dicarboxamide (I)

Dissolve 2.00 g (17.8 mmol) of 3,6-dimethyl-1,6-dihydro-1,2,4,5-tetrazine (II) and 0.18 g (1.78 mmol) of triethylamine in 50 ml of toluene Prepare a preparatory solution from the solution, add 10.60 g (35.7 mmol) of bis(trichloromethyl)carbonate and 50 ml toluene to a 250 ml three-necked flask in sequence, stir at -10°C, and slowly add the preparatory solution dropwise. After the addition was completed within 70 minutes, TLC followed and detected, and the reaction was refluxed for 12 hours. Then the temperature was lowered to 5° C., nitrogen gas was blown, and a solution obtained by dissolving 5.84 g (53.5 mmol) of 2-hydroxyaniline in 50 ml of toluene was added dropwise. After dripping, TLC tracking detection, 18 hours under reflux, evaporated toluene, the residue was recrystallized with 20 milliliters of dichloromethane, and other operations were the same as in Example 2 to obtain 1.22 grams of white solid shown in formula (I), i.e. N ¹ , N ⁴ -bis(2-hydroxyphenyl)-3,6-dimethyl-1,2,4,5-tetrazine-1,4-dicarboxamide (I), yield 17.9%, melting point 246～248℃. ¹ H NMR is the same as in Example 1.

Example 7: Preparation of N ¹ , N ⁴ -bis(2-hydroxyphenyl)-3,6-dimethyl-1,2,4,5-tetrazine-1,4-dicarboxamide (I)

Dissolve 2.00 g (17.8 mmol) of 3,6-dimethyl-1,6-dihydro-1,2,4,5-tetrazine (II) and 0.36 g (9.0 mmol) of sodium hydroxide in 50 ml of toluene Prepare a preparatory solution from the solution, add 10.60 g (35.7 mmol) of bis(trichloromethyl)carbonate and 50 ml toluene to a 250 ml three-necked flask in sequence, stir at -10°C, and slowly add the preparatory solution dropwise. After the addition was completed within 70 minutes, TLC followed and detected, and the reaction was refluxed for 12 hours. Then the temperature was lowered to 5° C., nitrogen gas was blown, and a solution obtained by dissolving 5.84 g (53.5 mmol) of 2-hydroxyaniline in 50 ml of toluene was added dropwise. After dripping, TLC tracking detection, heating to reflux for 18 hours, distilling off toluene, the residue was recrystallized with 20 ml of methanol, other operations were the same as in Example 2, to obtain 0.22 g of white solid shown in formula (I), i.e. N ¹ , N ⁴ -bis(2-hydroxyphenyl)-3,6-dimethyl-1,2,4,5-tetrazine-1,4-dicarboxamide (I), yield 3.2%, melting point 246～ 248°C. ¹ H NMR is the same as in Example 1.

Example 8: Preparation of N ¹ , N ⁴ -bis(2-hydroxyphenyl)-3,6-dimethyl-1,2,4,5-tetrazine-1,4-dicarboxamide (I)

Dissolve 2.00 g (17.8 mmol) of 3,6-dimethyl-1,6-dihydro-1,2,4,5-tetrazine (II) and 0.20 g (3.6 mmol) of potassium hydroxide in 40 ml of tetrahydrofuran Prepare a preparatory solution from the solution, add 9.06 g (30.5 mmol) of bis(trichloromethyl)carbonate and 30 ml tetrahydrofuran to a 250 ml three-necked flask in sequence, stir at -8°C, and slowly add the preparatory solution dropwise. After the addition was completed within 30 minutes, TLC followed and detected, and the reaction was refluxed for 12 hours. Then the temperature was lowered to 4° C., nitrogen gas was blown, and a solution obtained by dissolving 9.72 g (89.1 mmol) of 2-hydroxyaniline in 30 ml of chloroform was added dropwise. After dripping, TLC tracking detection, heated to reflux for 29 hours, distilled off tetrahydrofuran and chloroform, residue column chromatography (eluent is petroleum ether: ethyl acetate=10:1 (volume ratio)), the residue was washed with 20 ml Dichloromethane was dissolved, and other operations were the same as in Example 1 to obtain 0.35 g of a white solid, namely N ¹ , N ⁴ -bis(2-hydroxyphenyl)-3,6-dimethyl-1,2,4,5- Tetrazine-1,4-dicarboxamide (I), yield 5.1%, melting point 246-248°C. ¹ H NMR is the same as in Example 1.

Example 9: In vitro test of anticancer activity

1) The compound (I) prepared in Example 1 was tested for biological activity of human breast cancer and human lung cancer cell lines. The results show that compound (I) has good anticancer activity against human lung cancer cell line A-549. the

Test method: tetrazolium salt (Methyl-Thiazol-Tetrozolium, MTT) reduction method

Cell lines: human breast cancer cell line MCF-7 and human lung cancer cell line A-549

Action time: 72 hours

The compound (I) prepared in Example 1 is dissolved with DMSO (dimethyl sulfoxide) (the amount of DMSO can dissolve the compound) to configure the concentrated solution, and then the concentrated solution is cultivated with a medium (DMEM produced by Gibco Company) base; medium preparation: each 1000mL medium contains 800,000 units of penicillin, 1.0g streptomycin, 10% inactivated fetal calf serum) diluted to the use concentration (100μg/mL, 10μg/mL and 1μg/mL), Then the human breast cancer cell line MCF-7 and the human lung cancer cell line A-549 were tested for their anticancer activity. The test results are shown in Table 1 below:

Table 1 compound (I) inhibits the growth of MCF-7 and A-549 cancer cells

2) According to Example 1, 2-hydroxyaniline was replaced by 4-ethoxyaniline, methylamine or 3-nitroaniline, and other operations were the same as in Example 1, and compounds N ¹ , N ⁴ -bis( 4-ethoxyphenyl)-3,6-dimethyl-1,2,4,5-tetrazine-1,4-dicarboxamide (III), N ¹ , N ⁴ -dimethyl-3 , 6-Dimethyl-1,2,4,5-tetrazine-1,4-dicarboxamide (IV) and N ¹ , N ⁴ -bis(3-nitrophenyl)-3,6-bis Methyl-1,2,4,5-tetrazine-1,4-dicarboxamide (V), the reaction scheme is shown in Figure 1.

According to the above method, the prepared compounds (III) and (IV) were tested for biological activity of human breast cancer cell lines, and the test results showed that compounds (III) and (IV) had no inhibitory effect on human breast cancer cell line MCF-7. Poor, the compound (III), (IV) and (V) that make have been carried out human lung cancer cell line biological activity test, test result shows that compound (III), (IV) and (V) to human lung cancer cell line A The inhibitory effect of -549 was poor, and the anticancer activity of compounds (III), (IV) and (V) on human lung cancer cell line A-549 was far inferior to that of compound (I). The specific results are shown in Table 2 below:

Table 2 compounds inhibit the growth of MCF-7 and A-549 cancer cells

The above experiments show that the compound (I) has significant inhibitory effect on the growth of human lung cancer cell line A-549, but not obvious inhibitory effect on the growth of human breast cancer cell line MCF-7. While other two structurally similar compounds (III) and (IV) have no obvious inhibitory effect on the growth of human breast cancer cell line MCF-7, other three structurally similar compounds (III), (IV) and (V) The inhibitory effect on the growth of human lung cancer cell line A-549 is not obvious. the

Claims (9)

1. A N ¹ , N ⁴ -bis(2-hydroxyphenyl)-3,6-dimethyl-1,2,4,5-tetrazine-1,4- as shown in formula (I) Diformamide compound:

2. A method for preparing N ¹ as claimed in claim 1, N ⁴ -bis(2-hydroxyphenyl)-3,6-dimethyl-1,2,4,5-tetrazine-1,4- The method of diformamide (I), is characterized in that the method is: under the condition of -10～0°C, 3,6-dimethyl-1,6-dihydro-1 represented by formula (II) , 2,4,5-tetrazine, bis(trichloromethyl)carbonate and basic catalyst were added to the organic solvent A, after the addition was completed, the reaction was heated under reflux for 4 to 12 hours, and then the reaction solution was cooled to 4 to 6 ℃, blow nitrogen, then dropwise add the solution of organic solvent B of 2-hydroxyaniline, after dripping, heat and reflux for 12 to 48 hours, the reaction is complete, after the solvent is evaporated, the residue is recrystallized or column chromatographed to obtain the formula ( I) N ¹ , N ⁴ -bis(2-hydroxyphenyl)-3,6-dimethyl-1,2,4,5-tetrazine-1,4-dicarboxamide compound, said The basic catalyst is one of the following: pyridine, triethylamine, quinoline, potassium hydroxide, sodium hydroxide, N,N-xylidine, 4-dimethylaminopyridine or 4-pyrrolidinylpyridine;

3. the method for claim 2 is characterized in that described 3,6-dimethyl-1,6-dihydro-1,2,4,5-tetrazine (II) and two (trichloro The ratio of the amount of methyl) carbonate, basic catalyst, 2-hydroxyaniline feed material is 1: 0.67～2: 0.1～1: 2～6, and the total volume consumption of described organic solvent A and organic solvent B is expressed by formula The mass of 3,6-dimethyl-1,6-dihydro-1,2,4,5-tetrazine shown in (II) is 13-75 ml/g. 4. The method according to claim 2, wherein said organic solvent A and organic solvent B are each independently selected from one of the following: methylene chloride, chloroform, toluene, methanol, ethanol or tetrahydrofuran. 5. The method according to claim 2, characterized in that said method is carried out as follows: 3,6-dimethyl-1,6-dihydro-1 shown in organic solvent A1 dissolution formula (II) , 2,4,5-tetrazine and basic catalyst obtain the solution containing 3,6-dimethyl-1,6-dihydro-1,2,4,5-tetrazine and basic catalyst, bis( Trichloromethyl)carbonate and organic solvent A2 are placed in a reactor, stirred at -10 to 0°C, and slowly dropwise added the 3,6-dimethyl-1,6-dihydro-1, The solution of 2,4,5-tetrazine and basic catalyst, after the feeding is completed, heat and reflux for 4-12 hours, TLC tracking detection, after the reaction, cool the reaction solution to 4-6°C, blow nitrogen, and then add 2- Hydroxyaniline is dissolved in the solution of organic solvent B, after dripping, heat and reflux for 12 to 48 hours, TLC tracking detection, after the reaction is completed, after the solvent is evaporated, the residue is recrystallized or column chromatographed to obtain the compound shown in formula (I). N ¹ , N ⁴ -bis(2-hydroxyphenyl)-3,6-dimethyl-1,2,4,5-tetrazine-1,4-dicarboxamide compound, the organic solvent A1 and Organic solvent A2 is the same organic solvent, organic solvent B and organic solvent A1 are the same or different organic solvents, and described organic solvent A1 and organic solvent B are each independently selected from one of the following: methylene chloride, chloroform, toluene, Methanol, ethanol or tetrahydrofuran; The total volume consumption of described organic solvent A1 and A2 is shown in formula (II) 3,6-dimethyl-1,6-dihydro-1,2,4,5-tetrazine quality Calculated as 10-50ml/g; the volume of the organic solvent B is based on the mass of 3,6-dimethyl-1,6-dihydro-1,2,4,5-tetrazine shown in formula (II) 3-25ml/g. 6. The method according to claim 2 or 5, characterized in that the recrystallization of the residue described in the method is carried out as follows: get the residue after steaming the solvent and add the recrystallization solvent, stirring and heating, Reflux for 5 to 10 minutes, filter while hot to remove insoluble matter, cool the filtrate, precipitate solids, filter, and dry the filter cake to obtain N ¹ , N ⁴ -bis(2-hydroxyphenyl)-3 , 6-dimethyl-1,2,4,5-tetrazine-1,4-dicarboxamide compound; the recrystallization solvent is one of the following: methanol, ethanol, petroleum ether, ethyl acetate, dichloro Methane, chloroform or tetrahydrofuran. 7. The method as claimed in claim 2 or 5, characterized in that the residue column chromatography described in the method is carried out as follows: get the residue after steaming solvent removal and add organic solvent C to make it Dissolve to obtain a solution, then add column chromatography silica gel in an amount of 1.5 to 2 times the residue mass to the solution, mix well, evaporate the solvent to obtain a mixture of dry residue and silica gel, and pack the mixture into a column, Then take sherwood oil and ethyl acetate mixed solution of volume ratio 0.5～10: 1 as eluent, directly carry out elution, TLC tracking detects, detects and collects the eluate containing the compound shown in formula (I) according to TLC detection, The collected solution was dried to obtain N ¹ , N ⁴ -bis(2-hydroxyphenyl)-3,6-dimethyl-1,2,4,5-tetrazine-1,4- Diformamide compound; the organic solvent C is one of the following: methanol, ethanol, ethyl acetate, dichloromethane, chloroform, tetrahydrofuran or dimethyl sulfoxide. 8. N ¹ as claimed in claim 1, N ⁴ -bis(2-hydroxyphenyl)-3,6-dimethyl-1,2,4,5-tetrazine-1,4-dicarboxamide (1) Application in the preparation of medicaments for preventing or treating tumor diseases, characterized in that the tumor is human lung cancer. 9. N ¹ as claimed in claim 1, N ⁴ -bis(2-hydroxyphenyl)-3,6-dimethyl-1,2,4,5-tetrazine-1,4-dicarboxamide (1) Application in the preparation of drugs for preventing or treating tumor diseases, characterized in that the human lung cancer cell line is A-549.

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