Sertraline side chain amino structure derivative and preparation method and application thereof
阅读说明：本技术 一种舍曲林侧链氨基结构衍生物及其制备方法和应用 (Sertraline side chain amino structure derivative and preparation method and application thereof ) 是由 欧阳勤 母超 古晶 郭春伶 于 2021-01-07 设计创作，主要内容包括：本发明公开了一类舍曲林侧链氨基结构衍生物,结构式如式Ⅰ或式Ⅱ所示,其中,R～1为H或环丙烷基,R～2选自烷基、萘基、烷基胺、环丙烷基、苯基,或者,3号位和/或4号位被烷基、甲氧烷基、卤代烷基、卤基取代的苯基；R～3为氢基或1-卤代烷基。本发明还提供了所述舍曲林衍生物的制备方法,及其在制备用于治疗胃癌的药物中的应用。本发明提供的舍曲林衍生物对于耐药胃癌细胞有明显的致敏作用。(The invention discloses a sertraline side chain amino structure derivative with a structural formula shown as a formula I or a formula II, wherein R 1 Is H or cyclopropylalkyl, R 2 Selected from alkyl groups, Naphthyl, alkylamine, cyclopropane, phenyl, or phenyl substituted with alkyl, methoxyalkyl, haloalkyl, halo at the 3-and/or 4-positions; r 3 Is hydrogen radical or 1-halogenated alkyl. The invention also provides said sertraline derivativesA preparation method of the compound and application thereof in preparing medicaments for treating gastric cancer. The sertraline derivative provided by the invention has obvious sensitization effect on drug-resistant gastric cancer cells.)
1. A sertraline side chain amino structure derivative is characterized in that the structural formula is shown as a formula I or a formula II,
wherein R is1Is H or cyclopropylalkyl, R2Selected from alkyl groups, Any one of naphthyl, alkylamine, cyclopropane, phenyl, or phenyl substituted with alkyl, methoxyalkyl, haloalkyl or halo at the 3-and/or 4-positions; r3Is hydrogen radical or 1-halogenated alkyl.
2. The sertraline side chain amino structure derivative of claim 1, wherein when formula i, R is2Is a straight chain alkyl or branched alkyl.
3. The sertraline side chain amino structure derivative of claim 1, wherein when formula i, R is2Is selected from- (CH)2)nCH3N-1, 2 or 4, -CH (CH)3) m, m is 2, 3, 4, 5, 6 …, or- (CH)2)xC6H5、x＝0、1、2、3…。
4. A process for the preparation of a sertraline side chain amino structure derivative according to any one of claims 1 to 3, comprising:
1) dissolving a first intermediate compound in an organic solvent, adding a modifying group compound and tetraisopropyl titanate to react at 70-100 ℃ in an inert atmosphere;
2) step 1), adding sodium cyanoborohydride to react at 70-100 ℃ after full reaction;
3) adding a quenching agent for quenching after the reaction in the step 2) is completed;
4) extracting, removing water, and purifying by chromatography to obtain a first product, wherein the first product is a sertraline side chain amino structure derivative shown in a structural formula I;
wherein the equivalent ratio of the first intermediate compound to the modifying group compound, tetraisopropyl titanate and sodium cyanoborohydride is 1: 2-1: 3.375-1: 2.5;
the structural formula of the first intermediate compound is shown as a formula III,
the structural formula of the modifying group is shown as a formula V,
wherein R is1Is H or cyclopropylalkyl, R2Selected from alkyl groups, Naphthyl, alkylamine, cyclopropane, phenyl, or phenyl substituted with alkyl, methoxyalkyl, haloalkyl, halo at the 3-and/or 4-positions;
alternatively, the preparation method further comprises: preparing a sertraline side chain amino derivative having the general structural formula II by a process comprising the steps of:
5) weighing a proper amount of a second intermediate compound IV, mixing the second intermediate compound IV with a proper amount of dichloromethane in a container protected by an inert atmosphere, adding triethylamine after ice bath to 0 ℃, uniformly stirring, then dropwise adding a compound with a structural formula VI, stirring at room temperature until the reaction is complete, adding a quenching agent for quenching, extracting by using an extracting agent, and purifying by using column chromatography to obtain a compound of a formula II; wherein, R is3Is hydrogen radical or 1-halogenated alkyl, and the second intermediate compoundThe structural formula of the compound is shown as formula IV;
preferably, the molar ratio of the second intermediate compound, triethylamine and the compound of formula vi is 1: 2: 1.5.
5. the method according to claim 4, wherein the organic solvent in step 1) is selected from isopropanol and methanol.
6. The method according to claim 4, wherein the quenching agent in step 3) is a saturated sodium bicarbonate solution, a saturated sodium bicarbonate solution or a saturated ammonium chloride solution.
7. The method of claim 4, wherein the extractant used in step 4) is methylene chloride.
8. The method according to claim 4, wherein the column chromatography leaf eluate in step 4) is a mixture of 10-20: 1, or a mixture of petroleum ether and ethyl acetate in a volume ratio of 100:1 dichloromethane and triethylamine.
9. The method of claim 4, wherein the first intermediate compound in step 1) is prepared by a method comprising the steps of:
mixing the compound 1 with aluminum trichloride, adding the compound 2 in an inert atmosphere, heating to 100 ℃ for reaction, cooling to room temperature after complete reaction, quenching by using a mixture of 1N hydrochloric acid and ice, extracting, dewatering, spin-drying, and performing column chromatography separation to obtain a first intermediate compound;
wherein the structural formula of the compound 1 isThe structural formula of the compound 2 is
10. Use of sertraline side chain amino structure derivatives according to claims 1-3 for the preparation of a medicament for the treatment of gastric cancer.
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to a sertraline side chain amino structure derivative and a preparation method and application thereof.
Background
In addition to its use in depression, sertraline is a selective serotonin reuptake inhibitor, and reports of its value in tumor therapy have increased dramatically in recent years. For example, sertraline has been reported to induce ER calcium release in human prostate cancer cell PC 3. Furthermore, sertraline interacts with and reduces the cellular levels of translationally controlled tumor proteins, resulting in decreased migration properties and colony forming ability of melanoma cells. The Xiufeng Pang subject group in 2018 identified the effect of sertraline in the treatment of non-small cell lung cancer (NSCLC) in "Reutilizationof sertraline sensitizes non-small cell lung cancer cells to erlotinib by inducing autophagy" demonstrating that sertraline inhibits the viability of NSCLC cells, showing a synergistic effect of erlotinib. They further studied the mechanism and demonstrated that sertraline enhances the therapeutic effect of erlotinib in NSCLC cells by targeting the AMPK/mTOR signaling pathway. The veltchedrinberg group demonstrated that sertraline in combination with a nano-drug can modulate the multidrug resistance of cancer.
According to the global cancer statistics published by the american cancer society of 2019, gastric cancer has high incidence (5.7% of new cancers, the fifth) and mortality (8.2% of deaths caused by cancer, the third) worldwide. The treatment modalities for gastric cancer typically include surgery, chemotherapy, or targeted drug therapy. However, it is difficult to achieve satisfactory results with conventional therapeutic means because of low response rate and high susceptibility to drug resistance. Therefore, the development of a more effective novel anti-gastric cancer medicament has important significance for improving the cure rate of gastric cancer and prolonging the life of a patient. The development of new anti-gastric cancer drugs mainly needs to solve the key problem of tumor drug resistance.
Disclosure of Invention
The invention aims to provide a sertraline derivative prepared by modifying a sertraline side chain, and the sertraline derivative has a sensitization effect when being used for drug-resistant gastric cancer cells so as to solve the problem that tumor cells have drug resistance to chemotherapeutic drugs in the current gastric cancer treatment.
The invention firstly provides a sertraline side chain amino structure derivative, the structural formula is shown as formula I or formula II,
wherein R is1Is H or cyclopropylalkyl, R2Selected from alkyl groups, Naphthyl, alkylamine, cyclopropane, phenyl, or phenyl substituted with alkyl, methoxyalkyl, haloalkyl, halo at the 3-and/or 4-positions; r3Is hydrogen radical or 1-halogenated alkyl.
In one embodiment according to the invention, when the structural formula of the sertraline side chain amino structure derivative is formula I, R is2Is a straight chain alkyl or branched alkyl.
In one embodiment according to the invention, when the structural formula of the sertraline derivative is formula I, R is2Is selected from- (CH)2)nCH3N-1, 2 or 4, -CH (CH)3) m, m is an integer of 2 or more, such as 2, 3, 4, 5, and 6 …, or- (CH)2)xC6H5And x is an integer such as 0, 1, 2, 3 …, etc.
The invention also provides a preparation method of the sertraline side chain amino structure derivative, which comprises the following steps:
1) dissolving a first intermediate compound in an organic solvent, adding a modifying group compound and tetraisopropyl titanate to react at 70-100 ℃ in an inert atmosphere;
2) step 1), adding sodium cyanoborohydride to react at 70-100 ℃ after full reaction;
3) adding a quenching agent for quenching after the reaction in the step 2) is completed;
4) extracting, removing water, and purifying by chromatography to obtain a first product, wherein the first product is a sertraline derivative shown in a structural formula I;
wherein the equivalent ratio of the first intermediate compound to the modifying group compound, tetraisopropyl titanate and sodium cyanoborohydride is 1: 2-1: 3.375-1: 2.5;
the structural formula of the first intermediate is shown as a formula III,
the structural general formula of the modifying group is shown as a formula V,
wherein R is1Is H or cyclopropylalkyl, R2Selected from alkyl groups, Naphthyl, alkylamine, cyclopropane, phenyl, or phenyl substituted with alkyl, methoxyalkyl, haloalkyl, halo at the 3-and/or 4-positions.
In one embodiment according to the present invention, further comprising: the preparation method of the sertraline side chain amino derivative with the structural general formula as formula II comprises the following steps:
5) weighing a proper amount of second intermediate compound, mixing the second intermediate compound with a proper amount of dichloromethane in a container protected by an inert atmosphere, adding triethylamine after ice bath to 0 ℃, uniformly stirring, then dropwise adding a compound with a structural formula VI, stirring at room temperature until the reaction is complete, quenching with a quenching agent, extracting with an extracting agent, and purifying by column chromatography to obtain a compound of a formula II; wherein the structural formula of the second intermediate compound is shown as formula IV, and R is shown as the specification3Is hydrogen radical or1-haloalkyl;
preferably, the molar ratio of the second intermediate compound, triethylamine and the compound of formula vi is 1: 2: 1.5.
in one embodiment according to the present invention, the organic solvent in step 1) is isopropanol or methanol.
In one embodiment according to the invention, the quenching agent is a saturated sodium bicarbonate solution, saturated sodium bicarbonate or saturated ammonium chloride.
In one embodiment according to the present invention, the extractant is dichloromethane.
In one embodiment according to the invention, the column chromatography leaf eluate is a mixture of 10-20: 1 of petroleum ether and ethyl acetate, or the volume ratio of 1: 100 parts of a mixed solution of dichloromethane and triethylamine.
In one embodiment according to the present invention, the first intermediate compound in step 1) is prepared by a process comprising the steps of:
mixing the compound 1 with aluminum trichloride, adding the compound 2 in an inert atmosphere, heating to 100 ℃ for reaction, cooling to room temperature after complete reaction, quenching by using a mixture of 1N hydrochloric acid and ice, extracting, dewatering, spin-drying, and performing column chromatography separation to obtain a first intermediate compound;
wherein, the compound 1 has a structural formulaThe structural formula of the compound 2 is
In one embodiment according to the invention, the second intermediate is achieved by a process comprising the steps of:
mixing a proper amount of first intermediate compound and ammonium acetate, adding a proper amount of isopropanol in an inert atmosphere, fully reacting at 70-100 ℃, adding sodium borohydride for continuous reaction until the reaction is complete, cooling to room temperature, quenching with a quenching agent, extracting with an extracting agent, and purifying by column chromatography to obtain a second intermediate compound; preferably, the molar ratio of the first intermediate compound to ammonium acetate is 1: 2;
the invention further provides the application of the sertraline derivative in preparing a medicine for treating gastric cancer.
The derivatives of the synthetic sertraline structure in the invention are original and have no literature report;
compared with the prior art, the derivative with the sertraline structure synthesized by the invention has the following characteristics that part of the compounds can sensitize drug-resistant gastric cancer cells:
the invention discloses that the sertraline derivative provided by the invention has obvious sensitization effect on gastric cancer cells, can be used for sensitization of drug-resistant gastric cancer cells, or can be used for improving the inhibition effect of chemotherapeutic drugs on tumor cells.
Drawings
FIG. 1 is a schematic diagram of the synthetic route for a side chain amino structure derivative of sertraline in accordance with the present invention;
FIG. 2 is a NMR spectrum of Compound 5 a;
FIG. 3 is a NMR spectrum of Compound 6 a;
FIG. 4 is a NMR spectrum of Compound 5 k;
FIG. 5 is a graph showing the results of an experiment on the proliferation of compound 6a for inhibiting human gastric cancer cell resistant strain SGC-7901/DDP;
FIG. 6 is a data chart showing the results of experiment for inhibiting proliferation of human gastric cancer cell resistant strain SGC-7901/DDP of Compound 6 b;
FIG. 7 is a data chart showing the results of experiments on the proliferation of compound 6c inhibiting human gastric cancer cell resistant strain SGC-7901/DDP;
FIG. 8 is a data chart of the results of experiments on compounds 5t and 6d for inhibiting the proliferation of human gastric cancer cell resistant strain SGC-7901/DDP.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be described in further detail below with reference to the accompanying drawings and examples. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Unless otherwise specified, all reagents used in this example were analytical grade, and all chemical reaction progress was checked by thin layer chromatography.
Example 1: synthesis of Compounds
The synthetic process of the sertraline side chain amino structure derivative according to the invention is shown in figure 1, and the compound 1 and aluminum trichloride are weighed in a three-neck flask in N2Adding the compound 2 into the mixture under the protection condition, heating the mixture to 100 ℃ for reaction for 2.5 hours, cooling the mixture to room temperature after the reaction is completed, quenching the mixture by using a mixture of 1N hydrochloric acid and ice, extracting the mixture by using dichloromethane, then carrying out conventional water removal operation, and carrying out column chromatography separation after spin drying to obtain an intermediate compound; the equivalent ratio of the compound 1 to the aluminum trichloride to the compound 2 is as follows: 1: 8.157～1：1：2.5。
1. Synthesis of sertraline derivatives of formula I
Dissolving the intermediate compound III in isopropanol solution, N2Adding a modifying group compound and tetraisopropyl titanate into the mixture under the protection condition, reacting for two hours at 85 ℃, adding sodium cyanoborohydride into the mixture, reacting at 85 ℃ until the reaction is complete, quenching the mixture by using a saturated sodium bicarbonate solution after the reaction is complete, extracting the mixture by using two paths of methane, performing conventional water removal operation, and performing column chromatography separation after the reaction is finished to obtain a sertraline derivative with the structural formula shown in the formula I; the equivalent ratio of the intermediate compound to the modifying group compound, tetraisopropyl titanate and sodium cyanoborohydride is 1: 2～1：3.375～1。
The structural formula of the modifying group is shown as a formula V.
Wherein R is1Is H or cyclopropylalkyl, R2Selected from alkyl groups, Naphthyl, alkylamine, cyclopropane, phenyl, or phenyl substituted with alkyl, methoxyalkyl, haloalkyl, halo at the 3-and/or 4-positions.
2. Synthesis of sertraline derivatives of formula II
As shown in FIG. 1, after obtaining intermediate compound III, sertraline compound derivatives represented by formula II can be further synthesized.
Intermediate compound III (583.9mg (2mmol)) and compound ammonium acetate (311.4mg (4mmol)) were weighed out in a three-necked flask and degassed with N2Protecting, adding an appropriate amount of isopropanol, heating to 80 ℃, reacting for 2 hours, adding a compound sodium borohydride (756.6mg (20mmol)) to react at 80 ℃ for 16 hours, cooling to room temperature, quenching with a saturated sodium bicarbonate solution, extracting with dichloromethane, drying, spin-drying, performing column chromatography separation with an eluent of PE: EA ═ 20:1, and spin-drying to obtain an intermediate compound IV.
The intermediate compound IV (200mg (0.68mmol)) was weighed out in a flask with purging and nitrogen blanketing and the appropriate amount of dichloromethane was added. Adding triethylamine (137.7mg (1.36mmol)) into the mixture after the mixture is cooled to 0 ℃ in an ice bath, stirring the mixture for 5 minutes, then dropwise adding a compound (1.03mmol) with a structural formula VI into the mixture, stirring the mixture at room temperature until the reaction is completed, then quenching the mixture by using a saturated sodium bicarbonate solution, extracting the mixture by using dichloromethane, drying the mixture by using anhydrous sodium sulfate, and separating the mixture by using a spin-dry column chromatography to obtain the sertraline side chain amino derivative with the structural formula II.
The list of partially synthesized sertraline derivatives of the present invention is shown in table 1:
TABLE 1 display of sertraline derivatives prepared according to the invention
EXAMPLE 2 Synthesis of No.1 Sertraline derivative
Intermediate compound III (583.9mg (2mmol)) was weighed into a dry three-neck round bottom flask with a stirrer, treated anhydrous and oxygen-free, after which n-propylamine (0.33ml (4mmol)) was added and stirred for 5 minutes, tetraisopropyl titanate (2ml (6.75mmol)) was added thereto and refluxed in an oil bath at 85 ℃ for 2 hours, after which it was cooled to room temperature, sodium cyanoborohydride (314.2mg (5mmol)) was added thereto and heated to 85 ℃ and refluxed until the reaction was complete. After the reaction is completed, cooling the reaction liquid to room temperature, adding a proper amount of saturated sodium bicarbonate solution into the reaction liquid for quenching, then extracting the reaction liquid by using dichloromethane, drying the reaction liquid by using anhydrous sodium sulfate, performing suction filtration and spin drying to obtain a yellow oily substance; then carrying out column chromatography separation and eluting agent, wherein the eluting agent is petroleum ether: ethyl acetate 20:1, the eluent was collected and concentrated to dryness to give the product as a colourless oil in 47% yield.
Determining the structural formula of the compound 5a by using a 600MHz nuclear magnetic hydrogen spectrum and high resolution:1H NMR(600MHz,CDCl3),δ7.31(d,J＝7.5Hz,1H),7.27(d,J＝8.2Hz,1H),7.19(d,J＝2.1Hz,1H),7.13(t,J＝7.4Hz,1H),7.03(t,J＝7.5Hz,1H),6.92(dd,J＝8.2,1.5Hz,1H),6.72(d,J＝7.7Hz,1H),3.91(dd,J＝9.0,5.7Hz,1H),3.76(s,1H),2.69–2.57(m,2H),2.08–1.99(m,1H),1.96–1.88(m,2H),1.80–1.72(m,1H),1.50(dq,J＝14.2,7.1Hz,2H),0.89(t,J＝7.4Hz,3H)；HRMS(ESI+):m/z[M+H]+calcd for C19H22Cl2N:334.1124,found 334.1124.
EXAMPLE 3 Synthesis of No.22 sertraline derivative
Intermediate compound iii (583.9mg (2mmol)) was weighed into a dry three necked round bottom flask with a stirrer, treated anhydrous and oxygen free, to which Boc protected ethylenediamine (0.65ml (4mmol)) was added and stirred for 5 minutes before tetraisopropyl titanate (2ml (6.75mmol)) was added and refluxed in an oil bath at 85 ℃ for 2 hours, after which it was cooled to room temperature after 2 hours, sodium cyanoborohydride (314.2mg (5mmol)) was added and heated to 85 ℃ and refluxed to completion. After the reaction is completed, cooling the reaction liquid to room temperature, adding a proper amount of saturated sodium bicarbonate solution into the reaction liquid for quenching, then extracting the reaction liquid by using dichloromethane, drying the reaction liquid by using anhydrous sodium sulfate, performing suction filtration and spin drying to obtain a yellow oily substance; then separating by column chromatography, wherein an eluent is petroleum ether with the volume ratio of 10: 1: ethyl acetate, collecting the eluent, concentrating until the product is dry to obtain a colorless oil, weighing the colorless oil in a stirring flask, and adding proper amounts of trifluoroacetic acid and dichloromethane into the colorless oil, wherein the ratio of trifluoroacetic acid: and (2) stirring the mixture at normal temperature for 3.5 hours, quenching the mixture by using a saturated sodium bicarbonate solution after the reaction is completed, extracting the mixture by using dichloromethane, drying the mixture by using anhydrous sodium sulfate, performing suction filtration and spin drying, and performing column chromatography separation, wherein an eluent is dichloromethane: triethylamine is 100:1, eluent is collected and concentrated to be dry, and the yield of the product which is colorless oil is 83%;
the compound (code 6a) structural formula was determined using 600MHz nuclear magnetic hydrogen spectroscopy and high resolution:1H NMR(600MHz,CDCl3)δ7.39(d,J＝7.7Hz,1H),7.32(d,J＝8.2Hz,1H),7.24(s,1H),7.18(t,J＝7.4Hz,1H),7.08(t,J＝7.4Hz,1H),6.96(d,J＝8.2Hz,1H),6.78(d,J＝7.7Hz,1H),4.01–3.94(m,1H),3.81(d,J＝4.0Hz,1H),2.91–2.77(m,4H),2.06(dd,J＝21.2,10.1Hz,1H),2.02–1.91(m,2H),1.81(dd,J＝17.1,7.4Hz,1H)；HRMS(ESI+):m/z[M+H]+calcd for C18H21Cl2N2:335.1076,found 335.1083.
the chemical reaction equation is as follows:
EXAMPLE 4 Synthesis of Sertraline derivative No.11
Intermediate compound III (583.9mg (2mmol)) was weighed into a dry three-neck round bottom flask with a stirrer, treated anhydrous and oxygen-free, after which p-methoxybenzylamine (0.52ml (4mmol)) was added and stirred for 5 minutes, tetraisopropyl titanate (2ml (6.75mmol)) was added thereto and refluxed in an oil bath at 85 ℃ for 2 hours, after which it was cooled to room temperature, sodium cyanoborohydride (314.2mg (5mmol)) was added thereto and heated to 85 ℃ and refluxed until the reaction was complete. Cooling the reaction liquid to room temperature after the reaction is completed, then adding a proper amount of saturated sodium carbonate solution into the reaction liquid for quenching, then extracting the reaction liquid by using dichloromethane, then drying the reaction liquid by using anhydrous sodium sulfate, carrying out suction filtration and spin drying to obtain a yellow oily substance; then carrying out column chromatography separation and eluting agent, wherein the eluting agent is petroleum ether: ethyl acetate 20:1, collecting the eluent, and concentrating to dryness to obtain a colorless oil with a yield of 27%;
compound (code 5k) structural formula was determined using 600MHz nuclear magnetic hydrogen spectroscopy and high resolution:1H NMR(600MHz,CDCl3)δ7.34(dd,J＝16.1,6.8Hz,4H),7.29(s,1H),7.19(t,J＝7.4Hz,1H),7.11(t,J＝7.4Hz,1H),7.01(d,J＝8.2Hz,1H),6.91(d,J＝8.2Hz,2H),6.80(d,J＝7.7Hz,1H),4.00(dd,J＝8.6,5.0Hz,1H),3.93(d,J＝13.0Hz,1H),3.87(s,1H),3.81(d,J＝12.3Hz,4H),2.18(dd,J＝20.7,11.2Hz,1H),2.06–1.99(m,2H),1.85(td,J＝11.5,4.2Hz,1H)；HRMS(ESI+):m/z[M+H]+calcd for C24H24Cl2NO:412.1229,found412.1234.
the chemical reaction equation is as follows:
EXAMPLE 5 experiment of inhibition of proliferation of human gastric cancer cell-resistant strain SGC-7901/DDP by Compounds
(1) Cell collection
A. Gastric cancer cell-resistant strain SGC-7901/DDP (purchased from American Type Culture Collection (ATCC)) was cultured in complete medium (RPMI-1640 medium containing 10% by volume of fetal bovine serum and 1% by volume of a cyan chain double antibody) at 37 ℃ in 5% CO2Culturing for 48 hours under the condition, observing the growth state of the cells by a microscope, and taking out the cells from an incubator when the fusion degree of the cells reaches 80-90 percent;
B. removing the culture medium by a vacuum pump, washing the cells once by using PBS buffer solution, sucking out the PBS, adding 1mL of pancreatin digestive juice preheated to room temperature into a culture dish, and digesting for 2 minutes in an incubator at 37 ℃;
C. adding 5mL of complete culture medium preheated to 37 ℃ to terminate digestion, and repeatedly and softly blowing the bottom of the culture dish by using a pipette gun to ensure that cells fall off to form cell suspension;
D. the cell suspension was transferred to a 15mL centrifuge tube, centrifuged at 800rpm for 5min using a low speed centrifuge, the supernatant was aspirated off, and 10mL complete medium was added to resuspend the cells.
(2) Cell plating
A. Taking a 96-well plate, adding PBS buffer solution into 36 holes at the periphery of the 96-well plate, respectively, and adding 90 mu L of the cell suspension into 60 holes at the center of the 96-well plate, wherein the number of cells in each hole is 5000 per hole;
B. the 96-well plate inoculated with the cells was returned to the 37 ℃ incubator to continue the culture overnight for the cells to adhere to the wall.
(3) Adding chemicals for treatment
A. Precisely weighing compounds No.22, 23, 24 and 25, preparing into 50mmol drug mother liquor with dimethyl sulfoxide (DMSO), and diluting with complete culture medium (RPMI-1640 culture medium containing 10% fetal calf serum and 1% cyan chain double antibody) to obtain 10, 20, 40, 80, 120, 160, 200 and 240 μmol/L series concentration gradient drug solutions;
B. adding the diluted liquid medicine into corresponding cell inoculation holes according to the dosage of 10 mu L/hole, setting 3 multiple holes (the final concentration of the compound in each hole is 1, 2, 4, 8, 12, 16, 20 and 24 mu mol/L) at each concentration, and simultaneously setting a negative control hole (adding a dimethyl sulfoxide complete culture medium solution with the volume fraction of 0.1%) and a blank control hole (not containing cells and liquid medicine);
C. after the medicine is added, the hole plate is sufficiently shaken to ensure that the liquid medicine is sufficiently and uniformly mixed, and then the mixture is placed back to the incubator at 37 ℃ to be continuously cultured for 24 hours.
(4) Cell proliferation viability assay
A. Adding 10 μ L of CCK-8 solution (Cell Counting Kit-8, available from Biyun, C0039) into each well, shaking the well plate sufficiently, placing the well plate back into a 37 ℃ incubator, and culturing for 2 h;
B. taking out the 96-well plate, and reading the OD value of each well at the wavelength of 450nm by using an enzyme-labeling instrument;
C. the half inhibitory concentration (IC50) of each compound for inhibiting the proliferation of gastric cancer cell-resistant strain SGC-7901/DDP was calculated.
The experimental results are shown in fig. 5-8: the synthesized compound can exert the biological activity of inhibiting the proliferation of the gastric cancer cell drug-resistant strain SGC-7901/DDP.
The above summary and the detailed description are intended to demonstrate the practical application of the technical solutions provided by the present invention, and should not be construed as limiting the scope of the present invention. Various modifications, equivalent substitutions, or improvements may be made by those skilled in the art within the spirit and principles of the invention. The scope of the invention is to be determined by the appended claims.