CN103044326A - 5-bromo oxoisoaporphine, and synthesis method and application thereof - Google Patents
5-bromo oxoisoaporphine, and synthesis method and application thereof Download PDFInfo
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Abstract
The invention discloses a 5-bromo oxoisoaporphine, and a synthesis method and application thereof. The synthesis route of the 5-bromo oxoisoaporphine comprises the following steps: reducing 4-bromobenzylcyanide used as an initial raw material to obtain 4-bromobenzylethylamine, reacting the 4-bromobenzylethylamine with phthalic anhydride, and performing Friedel-Crafts acylation cyclization reaction twice, thus obtaining the 5-bromo oxoisoaporphine through synthesis. The applicant surveys the proliferation inhibition activity of the 5-bromo oxoisoaporphine on multiple human tumor cell strains, and results show that the 5-bromo oxoisoaporphine has remarkable in-vitro antitumor activity, has good potential pharmaceutical values and is hopefully used for the preparation of various antitumor medicaments. The chemical structural formula of the 5-bromo oxoisoaporphine is shown as below in the specification.
Description
Technical field
The present invention relates to medical technical field, be specifically related to the different aporphine of 5-bromine oxidation and synthetic method and application.
Background technology
The people such as nineteen eighty-two Kunitomo separate from the menispermaceous plants yellow parilla and identify to get the different aporphine alkaloid menisporphine of first oxidation, and the investigator successively isolated again the alkaloid of 4 same types from this plant afterwards.Up to the present existing tens kinds of Oxoisoaporphines are separated, and mainly comprise: bianfugecine alkali, bianfugenine alkali, the luxuriant and rich with fragrance alkali of yellow parilla, yellow parilla Fino woods alkali etc.
Aspect organic synthesis, the parent nucleus of the different aporphine alkaloid of oxidation, i.e. 1-azepine benzanthrone have just been synthesized as far back as nineteen fifty-three King; But just being regarded dyestuff at that time, its pharmacologically active is not conducted a research.1984, Iwashima etc. carried out organic complete synthesis by the synthetic method of Simple fast more to 1-azepine benzanthrone.Nineteen eighty-three, the discoverer Kunitomo of the different aporphine alkaloid of oxidation is complete synthesis to have obtained the luxuriant and rich with fragrance alkali (menisporphine) of yellow parilla, and this type of alkaloidal biosynthesizing route is analyzed.
At present, carry out also fewerly for the different aporphine alkaloid pharmacologically active of oxidation or bioactivity research.The amino different aporphine derivative of oxidation that replaces of a series of 9-has been synthesized in the designs such as Tang Huang, and select 3 kinds of tumor cell lines: the HepG2(human hepatoma cell strain), the NCI-H460(human lung carcinoma cell line), the strain of MCF(human breast cancer cell) adopt mtt assay to test the anti tumor activity in vitro (Tang H, et al.Eur.J.Med.Chem.2008) of the different aporphine derivative of oxidation that is synthesized.Also selected simultaneously HKESC-1(esophageal carcinoma cell line-1), use the MTS method to test the cytotoxicity of the different aporphine alkali derivant of oxidation to them, experimental result shows that the half-inhibition concentration of part of compounds is IC
50Value reaches 2.0 μ mol/L, and the cytotoxicity of this analog derivative is by the Intercalation between the adjacent base pair of DNA is suppressed the propagation that copies block cell of DNA, thereby shows good cytotoxicity.Cotton etc. have carried out cytotoxicity experiment (Cotton S.A. to the four kinds of different aporphine alkaloids of oxidation that obtain that extract from Rhizoma Menispermi, et al.Polyhedron, 2006), found that wherein two kinds of compounds have more intense toxicity, its IC to human breast cancer cell strain (MCF-7) and mouse leukemia cell strain (p388)
50Value is 6～30 μ mol/L.But in the prior art and there are no the relevant report of the different aporphine of relevant 5-bromine oxidation and synthetic method and application.
Summary of the invention
The technical problem to be solved in the present invention provides a kind of different aporphine alkaloid derivative of new oxidation that synthesizes by complete synthesis method---the different aporphine of 5-bromine oxidation, and its synthetic method and application.
The different aporphine of 5-bromine of the present invention oxidation, its structural formula is shown below:
The different aporphine of 5-bromine of the present invention oxidation namely is the assorted benzanthrone of 1-nitrogen-5-bromine, and molecular formula is C
16H
8BrNO, molecular weight are 310g/mol.
The synthetic thinking of the different aporphine of 5-bromine oxidation of the present invention is: take the 4-bromobenzylcyanide as starting raw material; at first obtain the 4-Bretylium Tosylate through reduction; again with itself and phthalic anhydride; and through twice friedel-crafts acylation ring closure reaction; synthetic obtain the different aporphine of 5-bromine oxidation, synthetic route is as follows:
Concrete synthetic method may further comprise the steps:
1) synthetic (being equivalent to a step in the synthetic route) of intermediate 1:
Mol ratio by 0.5～2.0:1 takes by weighing Lithium Aluminium Hydride and 4-bromobenzylcyanide, be dissolved in respectively in the anhydrous diethyl ether, stirring reaction is to complete under cryosel bath condition, then be neutralized to without Bubble formation with saturated metabisulfite solution, the reaction solution suction filtration, the chloroform washing, steaming desolventizes, obtain compound intermediate 1, productive rate is about 20～40%;
2) synthetic (being equivalent to the b step in the synthetic route) of intermediate 2:
Mol ratio by 1～1.2:1 takes by weighing Tetra hydro Phthalic anhydride and intermediate 1, mixes to be dissolved in the ethanol, and back flow reaction is extremely complete, the reaction solution cooling crystallization, and suction filtration, the crystal washing namely obtains compound intermediate 2, and productive rate is about 50～80%;
3) synthetic (being equivalent to the c step in the synthetic route) of intermediate 3:
Mol ratio by 5～8:1 takes by weighing aluminum trichloride (anhydrous) and intermediate 2, aluminum trichloride (anhydrous) and an amount of fusing assistant are mixed and heated to melting, again to wherein adding intermediate 2, be warming up to 180～220 ℃, the insulated and stirred reaction is to complete, reactant places mortar to grind, and obtains compound intermediate 3, and productive rate is about 60～80%;
4) synthetic (being equivalent to the d step in the synthetic route) of the different aporphine of 5-bromine oxidation:
Intermediate 3 is joined in the vitriol oil of a certain amount of heat, be heated to 200～240 ℃, the insulated and stirred reaction is poured in the frozen water after the reaction solution cooling to fully, separate out a large amount of black solids, suction filtration, washing, drying obtains the different aporphine crude product of 5-bromine oxidation, and productive rate is about 40～60%.
In order to improve the purity of the different aporphine of 5-bromine oxidation, preferably, after obtaining the different aporphine crude product of 5-bromine oxidation, carry out again purification step, concrete purification step is: with the different aporphine crude product of 5-bromine oxidation chloroform extraction, collected organic layer, washing, anhydrous sodium sulfate drying, removal of solvent under reduced pressure, silica gel column chromatography on the gained residue, using by volume ratio is the sherwood oil of 20～100:1 and the mixed solvent wash-out that ethyl acetate forms, the elutriant solvent evaporated, namely get the different aporphine of 5-bromine oxidation, productive rate is about 20～40% (pressing intermediate 3 calculates).After testing, the purity of the different aporphine of 5-bromine oxidation that obtains of purified step 〉=99.5%.
Above-mentioned synthetic method, step 1) in, the synthetic method of intermediate 1 is more specifically: the mol ratio by 0.5～2.0:1 takes by weighing Lithium Aluminium Hydride and 4-bromobenzylcyanide, be dissolved in respectively in the anhydrous diethyl ether, under cryosel bath condition, slowly drip the anhydrous ether solution of 4-bromobenzylcyanide in the anhydrous ether solution of Lithium Aluminium Hydride, stirring reaction is to complete under the normal temperature condition, then be neutralized to without Bubble formation with saturated metabisulfite solution, the reaction solution suction filtration, filter residue washs with chloroform, removes solvent under reduced pressure, obtains compound intermediate 1.Wherein the consumption of anhydrous diethyl ether can be determined as required, and generally, the Lithium Aluminium Hydride of 0.1mol dissolves with 25～125mL anhydrous diethyl ether, and the 0.1mol4-bromobenzylcyanide dissolves with 10～25mL anhydrous diethyl ether.
Step 2) in, the temperature of back flow reaction is preferably 80～100 ℃, and the gained crystal is usually with anhydrous diethyl ether or washing with acetone.
Step 3) in, described fusing assistant preferably adopts sodium-chlor, and the mol ratio of its add-on and intermediate 3 is preferably 1:1.5～3.Normally mixed aluminum trichloride (anhydrous) and fusing assistant are heated to that insulation makes their meltings more than 150 ℃.In this step, product places step that mortar grinds just in order to be beneficial to follow-up intermediate 3 and strong sulfuric acid response, and the step that also can place mortar to grind product is carried out before being placed on follow-up adding sulfuric acid.
Step 4) in, the consumption of the vitriol oil gets final product for dissolving intermediate 3, and common consumption is the hot concentrated sulfuric acid dissolving of 10～50 milliliters of every mmole intermediate 3 usefulness; The temperature of hot concentrated sulfuric acid is generally 50～70 ℃, and temperature is difficult for too high, otherwise causes carbonization easily.In this step, when intermediate 3 is joined hot concentrated sulfuric acid, for avoiding reaction too violent, preferably intermediate is added in batches.The consumption that is used for the frozen water that reaction solution separates out is determined as required.
In the above-mentioned synthetic method, whether various reactions can adopt thin-layer chromatography to follow the tracks of is fully detected, under above-mentioned qualifications, and step 1) in, the stirring at normal temperature reaction is to fully approximately needing 2～8h; Step 2) in, back flow reaction is to fully approximately needing 2～12h; Step 3) in, stirring reaction is to fully approximately needing 1～6h; Step 4) in, stirring reaction is to fully approximately needing 2～6h.
In the purification step of the different aporphine crude product of 5-bromine oxidation, the mixed solvent that is used for wash-out is sherwood oil and the ethyl acetate that 20～50:1 forms by volume ratio preferably.
The present invention also comprises the application of the different aporphine of above-mentioned 5-bromine oxidation in the preparation antitumor drug.
The present invention also comprises the antitumor drug of the preparation take the different aporphine of above-mentioned 5-bromine oxidation as effective constituent.
Compared with prior art, the different aporphine alkaloid derivative of new oxidation that the present invention synthesizes by complete synthesis method---the different aporphine of 5-bromine oxidation, and investigated the proliferation inhibition activity of the different aporphine of 5-bromine oxidation to the various human tumor cell line, the result shows that it has significant anti tumor activity in vitro, have preferably potential pharmaceutical use, be expected to the preparation for various antitumor drugs.
Description of drawings
Fig. 1 is the infrared spectrum of the final product that makes of the embodiment of the invention 1;
Fig. 2 is the proton nmr spectra spectrogram of the final product that makes of the embodiment of the invention 1;
Fig. 3 is the X ray single crystal diffraction spectrum spectrogram of the final product that makes of the embodiment of the invention 1.
Embodiment
The invention will be further described with specific embodiment for the below, but the present invention is not limited to these embodiment.
1) intermediate 1 (4-Bretylium Tosylate) is synthetic:
The Lithium Aluminium Hydride of getting 0.2mol is dissolved in the anhydrous diethyl ether of 200mL, 0.2mol the 4-bromobenzylcyanide be dissolved in the 50mL anhydrous diethyl ether, under cryosel bath condition, slowly drip the anhydrous ether solution of 4-bromobenzylcyanide in the anhydrous ether solution of Lithium Aluminium Hydride, add complete after, stirring reaction is 6 hours under the normal temperature condition, be neutralized to without Bubble formation with saturated metabisulfite solution, the reaction solution suction filtration, filter residue washs with chloroform, underpressure distillation desolventizes, obtain intermediate 1 (the 4-Bretylium Tosylate liquid of colourless slightly thickness), productive rate 40%;
2) intermediate 2 is synthetic:
Getting the Tetra hydro Phthalic anhydride of 0.1mol and intermediate 1 mixing of 0.1mol is dissolved in the ethanol of 200mL, 90 ℃ of back flow reaction 8 hours, reaction solution is cooled to room temperature, separate out clear crystal, suction filtration is isolated crystal, and crystal is washed with anhydrous diethyl ether, obtain compound intermediate 2 (for the white plates crystal), productive rate 80%;
3) intermediate 3 is synthetic:
Getting the aluminum trichloride (anhydrous) of 0.5mol and the sodium-chlor of 0.25mol mixes, and be heated to 180 ℃ of constant temperature to the solid melting, again to the intermediate 2 that wherein adds 0.1mol in batches, add and be warming up to 200 ℃ after complete, insulated and stirred reaction 4 hours, reactant is poured in the mortar, grinds while cool off, obtain compound intermediate 3, productive rate 80%;
4) the different aporphine of 5-bromine oxidation (1-nitrogen-5-bromine mix benzanthrone) is synthetic
Get in the vitriol oil that the above-mentioned intermediate that makes 3 of 1mmol joins 20mL heat in batches (temperature is 60 ℃), add and be warming up to again 220 ℃ after complete, insulated and stirred reaction 4 hours, cooling is poured in the frozen water of about 200mL at last, separate out a large amount of black solids, suction filtration is isolated solid, the washing of gained solid, drying obtains crude product (the different aporphine crude product of 5-bromine oxidation); Crude product is repeatedly extracted on a small quantity with chloroform, merge organic layer, washing, anhydrous sodium sulfate drying removes solvent under reduced pressure, passes through silica gel column chromatography on the gained residue, using by volume ratio is the sherwood oil of 50:1 and the mixed solvent wash-out that ethyl acetate forms, the elutriant solvent evaporated obtains yellow solid product 4, productive rate about 40%.
Above-mentioned gained yellow solid product 4 is carried out infrared spectra, proton nmr spectra and single crystal diffraction analysis, and concrete spectral characteristic is as follows:
(1) infrared spectrum, as shown in Figure 1: IR (KBr): 3060,1667,1595,1396,1283,1190,1092,897,705,642,595cm
-1
(2) proton nmr spectra, as shown in Figure 2:
1H NMR (500MHz, CDCl3) δ 8.94 (d, J=7.9Hz, 1H, 2-H-Ar), (8.82 d, J=5.6Hz, 1H, 11-H-Ar), 8.60 (s, 1H, 4-H-Ar), 8.44 (d, J=7.7Hz, 1H, 8-H-Ar), 8.16 (s, 1H, 6-H-Ar), 7.86 (t, J=7.5Hz, 1H, 10-H-Ar), 7.70 (m, 2H, 8-H-Ar and9-H-Ar);
(3) X ray single crystal diffraction spectrum spectrogram, as shown in Figure 3.
Therefore, the yellow solid product 4 that can determine gained is the different aporphine of 5-bromine oxidation (1-nitrogen-5-bromine mix benzanthrone), and its chemical structural formula is as follows:
Embodiment 2
1) intermediate 1 (4-Bretylium Tosylate) is synthetic:
The Lithium Aluminium Hydride of getting 0.1mol is dissolved in the anhydrous diethyl ether of 100mL, 0.2mol the 4-bromobenzylcyanide be dissolved in the 50mL anhydrous diethyl ether, under cryosel bath condition, slowly drip the anhydrous ether solution of 4-bromobenzylcyanide in the anhydrous ether solution of Lithium Aluminium Hydride, add complete after, stirring reaction is 8 hours under the normal temperature condition, be neutralized to without Bubble formation with saturated metabisulfite solution, the reaction solution suction filtration, filtrate is washed with chloroform, underpressure distillation desolventizes, obtain intermediate 1 (the 4-Bretylium Tosylate liquid of colourless slightly thickness), productive rate 20%;
2) intermediate 2 is synthetic:
Getting the Tetra hydro Phthalic anhydride of 0.1mol and intermediate 1 mixing of 0.1mol is dissolved in the ethanol of 50mL, 95 ℃ of back flow reaction 2 hours, suction filtration while hot, filtrate is cooled to room temperature, separates out clear crystal, suction filtration, isolate crystal, crystal is washed with anhydrous diethyl ether, obtain compound intermediate 2 (for the white plates crystal), productive rate 50%;
3) intermediate 3 is synthetic:
Getting the aluminum trichloride (anhydrous) of 0.5mol and the sodium-chlor of 0.2mol mixes, and be heated to 180 ℃ and be incubated to the solid melting, again to the intermediate 2 that wherein adds 0.1mol in batches, add and be warming up to 180 ℃ after complete, insulated and stirred reaction 6 hours, reactant is poured in the mortar while hot, grinds while cool off, obtain compound intermediate 3, productive rate 60%;
4) the different aporphine of 5-bromine oxidation (1-nitrogen-5-bromine mix benzanthrone) is synthetic
The above-mentioned intermediate that makes 3 of 1mmol is joined in batches in the vitriol oil of 10mL heat (temperature is 50 ℃), add and be warming up to again 200 ℃ after complete, insulated and stirred reaction 6 hours, cooling is poured in the frozen water of about 200mL at last, separate out a large amount of black solids, suction filtration is isolated solid, the washing of gained solid, drying obtains the different aporphine crude product of 5-bromine oxidation; The different aporphine crude product of 5-bromine oxidation is repeatedly extracted on a small quantity with chloroform, merge organic layer, washing, anhydrous sodium sulfate drying removes solvent under reduced pressure, passes through silica gel column chromatography on the gained residue, using by volume ratio is the sherwood oil of 20:1 and the mixed solvent wash-out that ethyl acetate forms, the elutriant solvent evaporated obtains the yellow different aporphine of 5-bromine oxidation, productive rate about 30%.
Embodiment 3
1) intermediate 1 (4-Bretylium Tosylate) is synthetic:
The Lithium Aluminium Hydride of getting 0.4mol is dissolved in the anhydrous diethyl ether of 300mL, 0.2mol the 4-bromobenzylcyanide be dissolved in the 50mL anhydrous diethyl ether, under cryosel bath condition, slowly drip the anhydrous ether solution of 4-bromobenzylcyanide in the anhydrous ether solution of Lithium Aluminium Hydride, add complete after, stirring reaction is 2 hours under the normal temperature condition, be neutralized to without Bubble formation with saturated metabisulfite solution, the reaction solution suction filtration, filtrate is washed with chloroform, underpressure distillation desolventizes, obtain intermediate 1 (the 4-Bretylium Tosylate liquid of colourless slightly thickness), productive rate 30%;
2) intermediate 2 is synthetic:
Getting the Tetra hydro Phthalic anhydride of 0.12mol and intermediate 1 mixing of 0.1mol is dissolved in the ethanol of 50mL, 100 ℃ of back flow reaction 12 hours, suction filtration while hot, filtrate is cooled to room temperature, separates out clear crystal, suction filtration, isolate crystal, crystal is washed with anhydrous diethyl ether, obtain compound intermediate 2 (for the white plates crystal), productive rate 65%;
3) intermediate 3 is synthetic:
Getting the aluminum trichloride (anhydrous) of 0.6mol and the sodium-chlor of 0.2mol mixes, and be heated to 200 ℃ and be incubated to the solid melting, again to the intermediate 2 that wherein adds 0.1mol in batches, add and be warming up to 220 ℃ after complete, insulated and stirred reaction 1 hour, reactant is poured in the mortar while hot, grinds while cool off, obtain compound intermediate 3, productive rate 70%;
4) the different aporphine of 5-bromine oxidation (1-nitrogen-5-bromine mix benzanthrone) is synthetic
Get in the vitriol oil that the above-mentioned intermediate that makes 3 of 1mmol joins 25mL heat in batches (temperature is 70 ℃), add and be warming up to again 240 ℃ after complete, insulated and stirred reaction 2 hours, cooling is poured in the frozen water of about 200mL at last, separate out a large amount of black solids, suction filtration is isolated solid, the washing of gained solid, drying obtains the different aporphine crude product of 5-bromine oxidation; The different aporphine crude product of 5-bromine oxidation is repeatedly extracted on a small quantity with chloroform, merge organic layer, washing, anhydrous sodium sulfate drying removes solvent under reduced pressure, passes through silica gel column chromatography on the gained residue, using by volume ratio is the sherwood oil of 40:1 and the mixed solvent wash-out that ethyl acetate forms, the elutriant solvent evaporated obtains the yellow different aporphine of 5-bromine oxidation, productive rate about 20%.
Embodiment 4
1) intermediate 1 (4-Bretylium Tosylate) is synthetic:
The Lithium Aluminium Hydride of getting 0.3mol is dissolved in the anhydrous diethyl ether of 200mL, 0.2mol the 4-bromobenzylcyanide be dissolved in the 50mL anhydrous diethyl ether, under cryosel bath condition, slowly drip the anhydrous ether solution of 4-bromobenzylcyanide in the anhydrous ether solution of Lithium Aluminium Hydride, add complete after, stirring reaction is 4 hours under the normal temperature condition, be neutralized to without Bubble formation with saturated metabisulfite solution, the reaction solution suction filtration, filtrate is washed with chloroform, underpressure distillation desolventizes, obtain intermediate 1 (the 4-Bretylium Tosylate liquid of colourless slightly thickness), productive rate 35%;
2) intermediate 2 is synthetic:
Getting the Tetra hydro Phthalic anhydride of 0.11mol and intermediate 1 mixing of 0.1mol is dissolved in the ethanol of 50mL, 80 ℃ of back flow reaction 5 hours, suction filtration while hot, filtrate is cooled to room temperature, separates out clear crystal, suction filtration, isolate crystal, crystal is washed with anhydrous diethyl ether, obtain compound intermediate 2 (for the white plates crystal), productive rate 70%;
3) intermediate 3 is synthetic:
Getting the aluminum trichloride (anhydrous) of 0.8mol and the sodium-chlor of 0.5mol mixes, and be heated to 160 ℃ and be incubated to the solid melting, again to the intermediate 2 that wherein adds 0.1mol in batches, add and be warming up to 190 ℃ after complete, insulated and stirred reaction 3 hours, reactant is poured in the mortar while hot, grinds while cool off, obtain compound intermediate 3, productive rate 65%;
4) the different aporphine of 5-bromine oxidation (1-nitrogen-5-bromine mix benzanthrone) is synthetic
Get in the vitriol oil that the above-mentioned intermediate that makes 3 of 1mmol joins 50mL heat in batches (temperature is 50 ℃), add and be warming up to again 230 ℃ after complete, insulated and stirred reaction 5 hours, cooling is poured in the frozen water of about 200mL at last, separate out a large amount of black solids, suction filtration is isolated solid, the washing of gained solid, drying obtains the different aporphine crude product of 5-bromine oxidation; The different aporphine crude product of 5-bromine oxidation is repeatedly extracted on a small quantity with chloroform, merge organic layer, washing, anhydrous sodium sulfate drying removes solvent under reduced pressure, passes through silica gel column chromatography on the gained residue, using by volume ratio is the sherwood oil of 100:1 and the mixed solvent wash-out that ethyl acetate forms, the elutriant solvent evaporated obtains the yellow different aporphine of 5-bromine oxidation, productive rate about 35%.
For the purposes of the different aporphine of 5-bromine of the present invention oxidation in pharmacy is described, the applicant has carried out the anti-tumor activity experiment to the different aporphine of 5-bromine oxidation.
One, the different aporphine of 5-bromine oxidation is tested the proliferation inhibition activity of various human tumor cell line:
1, cell strain and cell cultures
5 kinds of human tumor cell lines such as liver cancer cell BEL-7404, cervical cancer cell HeLa 229, Proliferation of Human Ovarian Cell cisplatin resistance strain SK-OV-3/DDP, stomach cancer cell MGC80-3 and lung carcinoma cell NCI-H460 are selected in this experiment.
The all cells strain is all cultivated in the RPMI-1640 nutrient solution that contains the little ox blood of 10wt%, 100U/mL penicillin, 100U/mL Streptomycin sulphate, puts 37 ℃ and contains volumetric concentration 5%CO
2Cultivate in the incubator.
2, the preparation of testing compound
The purity of the used different aporphine of 5-bromine oxidation 〉=95% (being made by the embodiment of the invention 1), with its DMSO liquid storage with the whole solution that is mixed with 20 μ mol/L after the physiological buffer dilution, the final concentration of solubility promoter DMSO≤1% is wherein tested under this concentration compound to the inhibition degree of various growth of tumour cell.
3, cell growth inhibition test (mtt assay)
The tumour cell of (1) taking the logarithm vegetative period, behind tryptic digestion, be mixed with the cell suspension that concentration is 5000/mL with the nutrient solution that contains 10% calf serum, be inoculated in 96 well culture plates with every hole 190 μ L, make cell density to 1000 to be measured～10000 holes (marginal pore is filled with aseptic PBS);
(2) 5%CO
2, hatch 24h for 37 ℃, be paved with the hole to cell monolayer at the bottom of, every hole adds the medicine 10 μ L of finite concentration gradient, each concentration gradient is established 4 multiple holes;
(3) 5%CO
2, hatched 48 hours, and observed under the inverted microscope for 37 ℃;
(4) every hole adds the MTT solution (5mg/mL PBS, i.e. 0.5%MTT) of 10 μ L, continues to cultivate 4h;
(5) stop cultivating, carefully suck nutrient solution in the hole, the DMSO that every hole adds 150 μ L fully dissolves first a ceremonial jade-ladle, used in libation precipitation, behind the vibrator mixing, is 570nm at the microplate reader wavelength, and reference wavelength is the optical density value that 450nm measures each hole;
(6) zeroing hole (substratum, MTT, DMSO), control wells (the medicine dissolution medium of cell, same concentrations, nutrient solution, MTT, DMSO) are set simultaneously.
(7) according to the optical density value (OD value) that records, judge viable cell quantity, the OD value is larger, and cytoactive is stronger.Utilize formula:
Calculate medicine to the inhibiting rate of growth of tumour cell, calculate respectively each test-compound to the IC of several tumor cell lines with the Bliss method again
50Value.Its result is as shown in the following Table 1:
The different aporphine of table 1:5-bromine oxidation is to the IC of five kinds of human tumor cell lines
50Value (μ M)
Experimental result shows, the different aporphine of 5-bromine oxidation all shows in various degree inhibited proliferation to the growth of various human tumor cell line, under same experimental level, its suppress activity all be higher than clinical in cancer therapy drug-cis-platinum, IC
50All less than the cis-platinum.Except the IC to MGC80-3
50Value (20.45 ± 0.02 μ M) is greater than beyond the 20 μ M, to the IC of other 4 kinds of tumor cell lines
50Value all is lower than 20 μ M, especially human cervical carcinoma cell HeLa229 is shown the highest proliferation inhibition activity, its IC
50Value only is 7.67 ± 0.40 μ M; Simultaneously, the different aporphine of 5-bromine oxidation is to the IC of human ovarian cancer cisplatin resistance strain SK-OV-3/DDP and lung carcinoma cell NCI-H460
50Value (being respectively 13.49 ± 0.17 μ M, 15.31 ± 1.05 μ M) also is starkly lower than cis-platinum (IC
50Value is respectively 65.97 ± 1.53 μ M, 66.10 ± 0.06 μ M).In addition, this compound is also very remarkable to the restraining effect of liver cancer cell BEL-7404, its IC
50Value is 16.56 ± 1.63 μ M.
Can be found out that by the above results the different aporphine aggregate performance of 5-bromine of the present invention oxidation has gone out significantly and the anti tumor activity in vitro of wide spectrum has preferably potential pharmaceutical use, be expected to the preparation for various antitumor drugs.
Claims (10)
1.5-the different aporphine of bromine oxidation, its structural formula is shown below:
2. the synthetic method of the different aporphine of 5-bromine oxidation claimed in claim 1 is characterized in that may further comprise the steps:
1) intermediate 1 is synthetic:
Mol ratio by 0.5～2.0:1 takes by weighing Lithium Aluminium Hydride and 4-bromobenzylcyanide, is dissolved in respectively in the anhydrous diethyl ether, and stirring reaction is to complete under cryosel bath condition, then be neutralized to without Bubble formation reaction solution suction filtration, chloroform washing with saturated metabisulfite solution, steaming desolventizes, and obtains compound intermediate 1;
2) intermediate 2 is synthetic:
Mol ratio by 1～1.2:1 takes by weighing Tetra hydro Phthalic anhydride and intermediate 1, mixes to be dissolved in the ethanol, and back flow reaction is extremely complete, the reaction solution cooling crystallization, and suction filtration, the crystal washing namely obtains compound intermediate 2;
3) intermediate 3 is synthetic:
Mol ratio by 5～8:1 takes by weighing aluminum trichloride (anhydrous) and intermediate 2, aluminum trichloride (anhydrous) and an amount of fusing assistant are mixed and heated to melting, again to wherein adding intermediate 2, be warming up to 180～220 ℃, the insulated and stirred reaction is to complete, reactant places mortar to grind, and obtains compound intermediate 3;
4) the different aporphine of 5-bromine oxidation is synthetic:
Intermediate 3 is joined in the vitriol oil of a certain amount of heat, be heated to 200～240 ℃, the insulated and stirred reaction is poured in the frozen water after the reaction solution cooling to fully, separates out a large amount of black solids, suction filtration, and washing, drying obtains the different aporphine crude product of 5-bromine oxidation.
3. the synthetic method of the different aporphine of 5-bromine oxidation according to claim 2, it is characterized in that: the purification step that also comprises the different aporphine of 5-bromine oxidation, concrete purification step is: with the different aporphine crude product of 5-bromine oxidation chloroform extraction, collected organic layer, washing, anhydrous sodium sulfate drying, removal of solvent under reduced pressure, silica gel column chromatography on the gained residue, using by volume ratio is the sherwood oil of 20～100:1 and the mixed solvent wash-out that ethyl acetate forms, the elutriant solvent evaporated namely gets the different aporphine of 5-bromine oxidation.
4. according to claim 2 or the synthetic method of the different aporphine of 3 described 5-bromine oxidations, it is characterized in that: step 1) in, the synthetic method of intermediate 1 is specially:
Mol ratio by 0.5～2.0:1 takes by weighing Lithium Aluminium Hydride and 4-bromobenzylcyanide, be dissolved in respectively in the anhydrous diethyl ether, under cryosel bath condition, slowly drip the anhydrous ether solution of 4-bromobenzylcyanide in the anhydrous ether solution of Lithium Aluminium Hydride, then stirring reaction is neutralized to without Bubble formation with saturated metabisulfite solution to fully under the normal temperature condition, the reaction solution suction filtration, the chloroform washing removes solvent under reduced pressure, obtains compound intermediate 1.
5. according to claim 2 or the synthetic method of the different aporphine of 3 described 5-bromine oxidations, it is characterized in that: step 2) in, the gained crystal is with anhydrous diethyl ether or washing with acetone.
6. according to claim 2 or the synthetic method of the different aporphine of 3 described 5-bromine oxidations, it is characterized in that: step 3) in, described fusing assistant is sodium-chlor, the mol ratio of its add-on and intermediate 3 is 1:1～3.
7. according to claim 2 or the synthetic method of the different aporphine of 3 described 5-bromine oxidations, it is characterized in that: step 4) in, the hot concentrated sulfuric acid dissolving that every mmole intermediate 3 usefulness are 10～50 milliliters.
8. the synthetic method of the different aporphine of 5-bromine oxidation according to claim 3 is characterized in that: be used for the mixed solvent of wash-out, the volume ratio of sherwood oil and ethyl acetate is 20～50:1.
9. the application of the different aporphine of 5-bromine claimed in claim 1 oxidation in the preparation antitumor drug.
10. the antitumor drug for preparing take the different aporphine of right 1 described 5-bromine oxidation as effective constituent.
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