CN115141200A - M-methyl benzylidene tetrahydropyrazole [3,4-D ] pyridine [1,2-A ] pyrimidone compound B and application thereof - Google Patents
M-methyl benzylidene tetrahydropyrazole [3,4-D ] pyridine [1,2-A ] pyrimidone compound B and application thereof Download PDFInfo
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Abstract
The invention relates to m-methyl benzylidene tetrahydropyrazole [3,4-d]Pyridine [1,2-a]The pyrimidone compound B is prepared by using 1-methyl-5-amino-4-pyrazolecarboxylate as an initial raw material and reacting with valerolactam to form a ring under the action of phosphorus oxychloride to obtain 1-methyltetrahydropyrazole [3,4-d]Pyridine [1,2-a]Pyrimidine-4 (1)H) -ketone (A) followed by reaction with 3-methylbenzaldehyde under ethanolic sodium hydroxide conditions to form: (E) -1-methyl-9- (3-methylbenzylidene) -6,7,8,9-tetrahydropyrazol [3,4-d]Pyridine [1,2-a]A pyrimidinone compound (B). The compound B can be used for treating 16 human tumor cells such as colon cancer HCT-115, HCT-8, caco-2, HT-29, HCT-116, breast cancer MDA-MB-231, MCF-7, T47-D, liver cancer HepG-2, huh-7, hep3-B, gastric cancer HGC-27, cervical cancer HeLa, endometrial cancer ISK, and lung cancer A54 in vitro cell test9 and the prostate cancer cell PC-3 show good proliferation inhibition effect. Half inhibition rate (IC) 50 Value) between 0.01 and 5.3. Mu.M. Among them, heLa and HGC-27 cells showed the most prominent inhibitory activity and half-inhibitory rate (IC) 50 Value) were 0.032. Mu.M (HeLa) and 0.016. Mu.M (HGC-27), respectively.
Description
Technical Field
The invention relates to m-methyl benzylidene tetrahydropyrazole [3,4-d ] pyridine [1,2-a ] pyrimidone compound B and application thereof.
Background
Cancer is one of the most prevalent diseases with morbidity and mortality,
the global burden of cancer is continuously increasing, and thus, the situation of tumor prevention and treatment is becoming more severe, and how to effectively treat tumors is becoming a priority.
Currently, the approaches to treat cancer are roughly classified into four categories: surgical treatment, radiation treatment, chemotherapy, immunotherapy, and the like. Among them, chemotherapy is one of the important methods for treating cancer by inhibiting the growth of cancer cells by allowing a drug to reach a focal site through blood circulation. Although the traditional chemotherapy achieves certain treatment effect, the traditional chemotherapy still has the defects of poor treatment effect, poor prognosis, serious toxic and side effects, easy generation of drug resistance and the like. Therefore, the search for highly effective and safe antitumor drugs will remain the focus of research in this field for a long time.
The research of antitumor drugs started in the 40 th 20 th century, and has been 70 years old so far. At present, the traditional cytotoxic drugs (such as paclitaxel and docetaxel) are still the main subjects for tumor drug therapy, and the main defects are that the curative effect on solid tumors is poor and the side effect is large. The focus of research and development of tumor drugs gradually shifts from traditional cytotoxic drugs to novel targeted drugs based on molecular mechanisms, and the molecular targeted therapy of tumors achieves obvious curative effects clinically.
Therefore, the research and development of novel high-efficiency and low-toxicity antitumor drugs aiming at molecular targets is a key scientific and technological problem which is urgently solved by tumor treatment and must bring huge social and economic benefits.
Heterocyclic compounds, represented by piperidine and pyrimidine, have long been known in the medical field, and are the most representative saturated heterocyclic systems of all small molecule drugs listed in the FDA orange book of the united states (Taylor et al, 2014).
The heterocyclic compound is adapted to the requirements of future medicine and pesticide development by virtue of flexible and changeable structure, high activity and low toxicity, and lays a foundation for the research and development of new heterocyclic medicines. Among heterocyclic compounds, pyrimidine heterocyclic compounds have various biological activities, such as anticancer, antiviral, antibacterial, etc.
To date, 147 pyrimidine heterocyclic compounds are in different clinical stages, 57 pyrimidine heterocyclic compounds have been approved by FDA for clinical treatment of various diseases, and 22 pyrimidine heterocyclic compound drugs have been used for treatment of different cancers. Recently wang et al reported the synthetic pathways and antitumor activities of 22 pyrimidine heterocyclic compounds approved by FDA, among which the quinazoline fragment-containing drugs are: gefitinib (Gefitinib), erlotinib (Erlotinib), afatinib (Afatinib), dacomitinib (Dacomitinib), tocatinib (tucatetinib), lapatinib (Lapatinib); among the drugs containing thienyl, pyrrolyl, pyrazolyl, imidazolyl, pyrazolyl and pyridyl pyrimidine moieties are: imatinib (Olmutinib), ribociclib (Ribociclib), ibrutinib (Ibutinib), du Weili sibu (Duvelisib) and Palbociclib (Palbociclib), (Wang et al, 2021).
Pyrazolo [3,4-d ] pyrimidine ring is an important heterocyclic compound and can be used as a skeleton with a plurality of pharmacological activities. Pyrazolopyrimidinones are composed of a pyrazole ring and a ketone, and their structures mainly include, for example, pyrazole [5,1-b ] pyrimidine (Kiessling et al, 2007), pyrazole [3,4-d ] pyrimidine (Schenone et al, 2014, hassan et al, 2011), pyrazole [1,5-a ] pyrimidine (Kiselyov and Smith II, 2006), pyrazole [4,3-d ] pyrimidine (raslad et al, 2011). They are considered to be important heterocyclic systems and have been extensively studied by researchers from a chemical and pharmacological perspective.
The subject group is dedicated to the research of heterocyclic pyrimidine compounds, and according to the skeleton transition principle in drug design, a heterocyclic ring is used for replacing a benzene ring structure in deoxyvasicidone alkali, and a plurality of novel molecular skeletons such as tricyclic thienopyrimidine, tricyclic oxazolopyrimidine, tricyclic triazolopyrimidine and the like are designed and synthesized, and are subjected to systematic and deep research, so that the structure-activity relationship is preliminarily clarified (Zeng et al, 2022.
On the basis of related research of a subject group, a pyrazole ring replaces a benzene ring structure in deoxyvasicinone base to synthesize a novel tricyclic pyrazolopyrimidine molecular skeleton, and an active methylene segment exists at the ortho position of a carbon-nitrogen double bond of pyrimidine, so that the pyrimidine and 3-methylbenzaldehyde react to synthesize the (E) -1-methyl-9- (3-methylbenzylidene) -6,7,8,9-tetrahydropyrazolyl [3,4-d ] pyridine [1,2-a ] pyrimidone compound (B).
Therefore, the pyrazole [3,4-d ] pyrimidinone compound has great potential in the research and development of antitumor drugs and has wide market prospect. According to the biological activities of the oxazole pyrimidine compounds and the thiazole pyrimidine compounds, the method for searching and exploring the compounds has larger theoretical and application values for searching lead compounds such as new medicines, and the like, lays a foundation for further optimizing the structure of the compounds, and accumulates data and experience for the development of the new medicines.
On the basis of comprehensive analysis of relevant patents and documents at home and abroad, pyrazolo [3,4-d ] pyrrolo [1,2-a ] pyrimidone arylene compounds are completely synthesized, and are simply transformed and modified, and 3-methylbenzaldehyde is introduced into pyrazolo [3,4-d ] pyrrolo [1,2-a ] pyrimidone arylene compounds to improve the drug potency of the compounds.
The invention researches the inhibition effect of the compound B on the proliferation and growth of 16 human tumor cells, such as colon cancer HCT-115, HCT-8, caco-2, HT-29, HCT-116, breast cancer MDA-MB-231, MCF-7, T47-D, liver cancer HepG-2, huh-7, hep3-B, stomach cancer HGC-27, cervical cancer HeLa, endometrial cancer ISK, lung cancer A549 and prostate cancer cell PC-3 cell models, and detects the proliferation inhibition effect of the compound B on the tumor cells. The experimental results show that: the compound B shows obvious inhibition activity and half inhibition rate (IC) on different tumor cell proliferations 50 Value) 0.01-5.3 μ MAnd (3) removing the solvent. Among them, the inhibitory activity against HeLa and HGC-27 cells was most prominent, IC 50 The values were 0.032. Mu.M (HeLa) and 0.016. Mu.M (HGC-27), respectively.
The dose-effect and time-effect relationship of compound B on the proliferation inhibitory effects of both cancer cells were further evaluated. The experimental result shows that the inhibitory activity of the compound B is obviously improved along with the prolonging of the action time under a certain drug concentration, and the inhibitory activity is improved along with the increasing of the compound B concentration under a certain action time, which shows that the proliferation inhibitory action of the compound B to two kinds of cancer cells shows obvious time dependence and concentration dependence.
According to the obtained activity result of the antitumor cells, the antitumor biological activity and action mechanism of the compound B are further researched.
Disclosure of Invention
The invention aims to provide m-methyl benzylidene tetrahydropyrazole [3,4-d]Pyridine [1,2-a]Pyrimidone compound B and application thereof, wherein the name of the compound B is (E) -1-methyl-9- (3-methylbenzylidene) -6,7,8,9-tetrahydropyrazolyl [3,4-d]Pyridine [1,2-a]Pyrimidine-4 (1H) -ketone, which takes 1-methyl-5 amino-4 pyrazole ethyl formate as initial raw material to react with valerolactam to form ring under the action of phosphorus oxychloride to obtain 1-methyl tetrahydropyrazole [3,4-d]Pyridine [1,2-a]Pyrimidine-4 (1H) -ketone (A) is reacted with 3-methyl benzaldehyde under the condition of ethanol sodium hydroxide to generate (E) -1-methyl-9- (3-methyl benzylidene) -6,7,8,9-tetrahydropyrazole [3,4-d]Pyridine [1,2-a]A pyrimidinone compound (B); the anti-tumor action mechanism of the compound B is researched, and the compound B has good inhibition effect on 16 human tumor cell series, such as colon cancer (HCT-115, HCT-8, caco-2, HT-29 and HCT-116), breast cancer (MDA-MB-231, MCF-7 and T47-D), liver cancer (HepG-2, huh-7 and Hep 3-B), stomach cancer (HGC-27), cervical cancer (HeLa), endometrial cancer (ISK), lung cancer (A549) and prostate cancer cell (PC-3) cell models, and the proliferation inhibition effect of the compound B on the tumor cells is detected. The experimental results show that: the compound B shows obvious inhibition activity and half inhibition rate (IC) on the proliferation of different tumor cells 50 Value) between 0.01 and 5.3. Mu.M. Among them, heLa and HGC-27 cells were most remarkably inhibited, and half-inhibition rate (IC) was obtained 50 Values) are respectively0.032 μ M (HeLa) and 0.016 μ M (HGC-27).
The invention relates to a compound B of m-methyl benzylidene tetrahydropyrazole [3,4-D ] pyridine [1,2-A ] pyrimidone, wherein the structural formula (I) of the compound B is as follows:
wherein:
the compound B is named as (E) -1-methyl-9- (3-methylbenzylidene) -6,7,8,9-tetrahydropyrazol [3,4-d ] pyridine [1,2-a ] pyrimidin-4 (1H) -one.
The compound B is used for preparing antitumor drugs for inhibiting the growth of human colon cancer cell strains HCT-115, HCT-8, caco-2, HT-29 and HCT-116.
The compound B is used for preparing antitumor drugs for inhibiting the growth of human breast cancer cell strains MDA-MB-231, MCF-7 and T47-D.
The compound B is used for preparing antitumor drugs for inhibiting the growth of human hepatoma cell strains HepG-2, huh-7 and Hep 3-B.
The application of the compound B in preparing an anti-tumor medicament for inhibiting the growth of a human gastric cancer cell strain HGC-27.
The compound B is used for preparing antitumor drugs for inhibiting growth of human cervical cancer HeLa and endometrial cancer ISK cell strains.
The compound B is used for preparing an antitumor drug for inhibiting the growth of a human lung cancer A549 cell strain.
The compound B is used for preparing an anti-tumor medicament for inhibiting the growth of a human prostate cancer cell PC-3 cell strain.
The invention relates to a m-methyl benzylidene tetrahydropyrazole [3,4-d ] pyridine [1,2-a ] pyrimidone compound B and application thereof, wherein the preparation method of the compound B comprises the steps of taking 1-methyl-5-amino-4-ethyl pyrazolecarboxylate as an initial raw material, reacting with valerolactam to form a ring under the action of phosphorus oxychloride to obtain 1-methyl tetrahydropyrazole [3,4-d ] pyridine [1,2-a ] pyrimidine-4 (1H) -one (A), then reacting with 3-methylbenzaldehyde under the condition of ethanol sodium hydroxide to generate (E) -1-methyl-9- (3-methyl benzylidene) -6,7,8,9-tetrahydropyrazole [3,4-d ] pyridine [1,2-a ] pyrimidone compound (B), and specifically operating according to the following steps:
preparation of compound a:
dissolving 1.69g and 10mmoL of compound 1-methyl-5 amino-4 pyrazole ethyl formate in 20mL of anhydrous dioxane, adding 1.5g and 15mmoL of valerolactam, slowly dropwise adding 3.8g and 25mmoL of phosphorus oxychloride, refluxing for reaction until all raw materials disappear, filtering and concentrating reaction liquid, performing gradient elution by adopting forward silica gel column chromatography, wherein an eluent is petroleum ether and ethyl acetate in a volume ratio of 1:1, and obtaining a compound A which is 1-methyltetrahydropyrazole [3,4-d ] pyridine [1,2-a ] pyrimidine-4 (1H) -one;
preparation of compound B:
dissolving the obtained compound A, namely 1-methyltetrahydropyrazole [3,4-d ] pyridine [1,2-a ] pyrimidine-4 (1H) -ketone, in 20mL of mixed solution of anhydrous ethanol and sodium hydroxide, namely, 1.2g and 30mmoL, then slowly dropwise adding 3-methylbenzaldehyde and 15mmoL, refluxing to react until all raw materials disappear, filtering and concentrating the reaction solution, performing gradient elution by adopting forward silica gel column chromatography, and obtaining a compound B, namely (E) -1-methyl-9- (3-methylbenzylidene) -6,7,8,9-tetrahydropyrazole [3,4-d ] pyridine [1,2-a ] pyrimidine ketone by using pure ethyl acetate as an eluent to obtain a compound B, namely (E) -1-methyl-9- (3-methylbenzylidene) -6,7,8,9-tetrahydropyrazole [3,4-d ] pyridine [1,2-a ] pyrimidine ketone
The invention relates to a m-methyl benzylidene tetrahydropyrazole [3,4-d ] pyridine [1,2-a ] pyrimidone compound B and application thereof, wherein the synthetic route is as follows:
the invention relates to a m-methyl benzylidene tetrahydropyrazole [3,4-d ] pyridine [1,2-a ] pyrimidone compound B and application thereof,
two cancer cell models, cervical cancer (HeLa) and gastric cancer (HGC-27), were selected for study:
firstly, it was observed that compound B at different concentrations acted on cells for 24h and significantly affected the morphological changes of both HeLa and HGC-27 cancer cells.
To further confirm the effect of compound B on cell proliferation potency and cell population dependence, the effect of compound on cell clonogenic potency was examined using in vitro plate clonogenic experiments, showing that the number of cell clones gradually decreased with increasing concentration of compound B administered, the clonogenic rates of cervical cancer (HeLa) and gastric cancer (HGC-27) cell control groups were 94% and 83%, respectively, and the clonogenic rates of cervical cancer (HeLa) and gastric cancer (HGC-27) in the 0.1 μ M administration group were 15% and 11%.
Further performing scratch and Transwell migration experiments, and showing that after the compound B acts on cells, the cell migration links of cervical cancer (HeLa) and gastric cancer (HGC-27) cancer cells are obviously inhibited.
The lumen generation experiment shows that the compound B acts on a human venous vascular endothelial cell model (HUVEC), and the angiogenesis link has an obvious inhibiting effect.
Examining the influence of the compound B on cell cycle distribution (DNA content) after acting on cells shows that different concentrations of (0, 0.025,0.05,0.1 mu M) compound B act on the cells for 24h to cause the cells to block in the G2/M phase, for example, the proportion of cervical cancer (HeLa) cell control group in the G2/M phase cells is 9.42%, while the proportion of 0.1 mu M compound B group in the G2/M phase cells is increased to 45.75%; the ratio of cells in the gastric cancer (HGC-27) cell control group in the G2/M phase was 15.26%, while the ratio of cells in the compound B group at 0.1. Mu.M in the G2/M phase was increased to 51.6%;
in view of the relationship between ROS and apoptosis, the change of intracellular oxidation environment is analyzed by a flow cytometer, and the ROS content in HeLa and HGC-27 cells is rapidly and remarkably increased along with the increase of the concentration of the compound B.
Further, a flow cytometer is adopted to detect the cancer cell apoptosis condition after the compound B is administrated, which shows that the apoptosis of the cells is caused after the compound B with different concentrations (0, 0.025,0.05,0.1 mu M) acts on the cells for 24 hours, for example, the proportion of early and late apoptotic cells in a cervical cancer (HeLa) cell control group is respectively 4.19% and 10.9%, and the proportion of early and late apoptotic cells in a 0.1 mu M compound B group is increased to 7.15% and 55%; also, the control group of gastric cancer (HGC-27) cells occupied early and late apoptotic cell ratios were 10.1% and 12.4%, respectively, while the 0.1 μ M compound B group occupied early and late apoptotic cell ratios increased to 20.6% and 30.9%, indicating that the proliferation inhibitory activity of compound B on cervical cancer (HeLa) and gastric cancer (HGC-27) cells may be due to its ability to induce apoptosis.
The invention also detects the change condition of various protein expression levels playing an important role in the apoptosis regulation and control channel after the compound B treats the cells through an immunoblotting (Western blot) experiment, and the experimental result shows that the compound B causes the cleavage of polymerase (PARP) in two cell lines after the compound B acts on the cells at different concentrations of 0 and 0.2,0.6,1.5 mu M, thereby proving the apoptosis promotion activity of the compound B. In addition, the expression of the anti-apoptotic protein Bcl-2 and pro-apoptotic Bak was also studied and it was found that the magnitude of the decrease in Bcl-2 expression and the expression of the pro-apoptotic protein Bak increased in a dose and time dependent manner in both cell lines, indicating that compound B induced the activation of polymerase (PARP) and decreased the expression of the anti-apoptotic protein, which in turn induced apoptosis in the cells.
Drawings
FIG. 1 is a graph showing the proliferation inhibitory activity of Compound B of the present invention on various tumor cells;
FIG. 2 is a graph of the dose versus time relationship of the inhibitory effect of Compound B of the present invention on Hela and HGC-27 cell proliferation;
FIG. 3 is a graph showing the change of morphology of Hela, HGC-27 cells after treatment with Compound B of the present invention;
FIG. 4 is a graph showing that Compound B of the present invention inhibits clonogenic activity of Hela, HGC-27 cells;
FIG. 5 is a graph showing the effect of Compound B of the present invention on healing of scratches on Hela, HGC-27 cells after treatment;
FIG. 6 is a graph showing the inhibitory effect of Compound B of the present invention on cell migration of Hela and HGC-27;
FIG. 7 is a graph showing the inhibitory effect of Compound B of the present invention on HUVEC cell lumen production;
FIG. 8 is a graph showing that Compound B of the present invention causes block of G2/M phase in the cell phase of Hela and HGC-27;
FIG. 9 is a graph showing the change in ROS induced in Hela and HGC-27 cells by Compound B of the present invention;
FIG. 10 is a graph showing flow analysis of Hela, HGC-27 cell apoptosis induced by Compound B of the present invention;
FIG. 11 is a photograph showing the analysis of the immunoblotting test in which Compound B of the present invention induces apoptosis of cervical cancer (HeLa) and gastric cancer (HGC-27).
Detailed Description
The present invention is further illustrated by the following examples, but is not limited thereto;
reagent: all reagents were commercially available analytical grade;
example 1
Preparation of compound a:
dissolving 2.3g and 10mmoL of compound 1-methyl-5 amino-4 pyrazole ethyl formate in 20mL of anhydrous dioxane, adding 1.5g and 15mmoL of valerolactam, then slowly dropwise adding 3.8g and 25mmoL of phosphorus oxychloride, refluxing for reaction until all raw materials disappear, filtering and concentrating reaction liquid, and performing gradient elution by adopting forward silica gel column chromatography, wherein an eluent is petroleum ether and ethyl acetate in a volume ratio of 1:2 to obtain a compound A, namely 1-methyl-6,7,8,9-tetrahydropyrazole [3,4-d ] pyridine [1,2-a ] pyrimidine-4 (1H) -one, and the yield is as follows: 88%, white solid, melting point: 166-167 ℃;
1 H NMR(CDCl 3 with 0.05％v/v TMS,400MHz):δ H 8.04(1H,s,H3),4.05(2H,dd, J＝6.7,5.8Hz,H6),3.96(3H,s,NCH 3 ),2.98(2H,t,J＝6.7Hz,H9),1.96(4H,m, H7,H8)。
example 2
Preparation of compound B:
dissolving the compound A obtained in example 1, namely 1-methyl-6,7,8,9-tetrahydropyrazol [3,4-d ] pyridine [1,2-a ] pyrimidine-4 (1H) -ketone, 2.0g and 10mmoL in 20mL of mixed solution of absolute ethyl alcohol and sodium hydroxide, 1.2g and 30mmoL, slowly dropwise adding 3-methylbenzaldehyde, 1.8g and 15mmoL, refluxing until all raw materials disappear, filtering reaction liquid, concentrating, performing forward silica gel column chromatography gradient elution, and obtaining the compound B, namely (E) -1-methyl-9- (3-methylbenzylidene) -6,7,8,9-tetrahydropyrazol [3,4-d ] pyridine [1,2-a ] pyrimidine-4 (1H) -ketone, wherein the yield is as follows: 79%, light yellow solid, melting point: 251 to 252 ℃;
1 H NMR(CDCl 3 with 0.05％v/v TMS,400MHz):δ H 1 H NMR(400MHz,cdcl 3 )δ8.17(s, 1H),8.03(s,1H),7.31(d,J＝10.8Hz,1H),7.25(d,J＝9.2Hz,2H),7.15(d,J＝ 7.3Hz,1H),4.12(m,2H),4.00(s,3H),2.91(m,2H),2.38(s,3H),1.97(m,2H)。
example 3
And (3) analyzing the anti-tumor activity of the obtained compound B in cell models of human colon cancer, breast cancer, liver cancer, gastric cells, cervical cancer, lung cancer, prostatic cancer and endometrial cancer:
preparing a reagent: preparing compound B stock solution into 10mM or 30mM mother solution with DMSO, subpackaging with EP tube after completely dissolving, storing at-40 deg.C, and diluting with complete culture medium (DMEM culture solution + serum + double antibody) to desired concentration when using; MTT stock solution is prepared into 5mg/mL concentration by 1 Xphosphate buffered saline (PBS), and is stored at-40 ℃ after being subpackaged by an EP tube, and is diluted to 0.5mg/mL by a culture solution (DMEM culture solution) when in use;
cells used for the experiments: human colon cancer cells HCT-115, HCT-8, caco-2, HT-29, HCT-116, breast cancer cells MDA-MB-231, MCF-7, T47-D, liver cancer cells HepG-2, huh-7, hep3-B, stomach cancer cells HGC-27, cervical cancer cells HeLa, endometrial cancer cells ISK, lung cancer cells A549 and prostate cancer cells PC-3;
cell recovery: heating the frozen cells in warm water at 37 deg.C while shaking the tube, sucking the cells into a pre-prepared pre-heated culture solution when the cells are completely dissolved, centrifuging at 4 deg.C at 1000rpm/3min, discarding the supernatant, suspending the cells in a complete culture medium, placing in a cell culture flask, and incubating at 37 deg.C and 5% CO 2 Culturing under the condition of overnight adherence;
cell culture: HT-29 cells (McCoy 5A +10% FBS + double antibody); HUVEC cells (DMEM high glucose + growth factor ECG +10% serum + double antibody); all other cells were treated with medium (DMEM high sugar +10% serum + diabody) or (RPMI 1640+10% serum + diabody) at a temperature of 37 ℃,5% 2 Culturing in a constant-temperature incubator;
cell passage: when the cells occupy 85% of the culture bottles, carrying out cell passage and continuing culture, adjusting the density of the cells during the cell passage according to the growth speed and the size of the cells, directly carrying out centrifugal counting on the suspension cells and then carrying out the passage, wherein the adherent cells and the semi-adherent cells can be subjected to the passage only after being digested by pancreatin;
freezing and storing cells: when the cells grow and the fusion degree reaches about 90 percent, the cells are digested, the cell sediment is collected, the frozen solution (10 percent of DMSO +90 percent of serum) is directly put into a freezing box for freezing, the frozen solution is put at 4 ℃ for 30min, the temperature is minus 20 ℃ for 1h, the temperature is minus 80 ℃ overnight, the frozen solution is transferred to a liquid nitrogen tank for long-term storage the next day.
Example 4
The experimental method comprises the following steps: MTT method, MTT is a powdered chemical reagent, which is fully called 3- (4,5) -dimethylthiohia azo (-z-y 1) -3,5-di-phenylyttrazolumromide, and the Chinese chemical name is 3- (4,5-dimethylthiazole-2) -2,5-diphenyltetrazolium bromide;
the MTT method comprises the following specific operation steps: the cells were incubated with complete medium (DMEM medium + serum + diabodies) at 37 ℃ with 5% CO 2 Culturing in incubator, selecting cells in logarithmic growth phase, digesting, centrifuging, re-suspending, counting, and diluting to density (1 × 10) 4 -5×10 4 mL) were inoculated in 96-well plates (100 μ L/well) and incubated in an incubator; sucking out the original culture medium in each well, adding 0,0.001,0.003,0.009,0.027,0.081,0.243,0.729,2.187,6.561 and 19.683 mu M compounds B to be tested and positive control drugs with different concentrations, 100 mu L/well, setting blank control and zero setting wells (100 mu L/well culture solution), and setting 6 multiple wells for each concentration; putting a 96-well plate into a cell culture box, incubating for 24 hours or 72 hours, adding 0.5mg/ml MTT (100 mu L/well) for incubating for 4 hours, sucking out the culture solution in each well, adding dimethyl sulfoxide (DMSO) into the culture solution, performing oscillation for 100-120 mu L/well, shaking the micropore plate for 3min to fully dissolve blue crystals, reading the OD value of each well under the OD570 nm wavelength by using a microplate reader, calculating the cell proliferation inhibition rate after deducting the OD value of a zero-adjustment group, and obtaining the cell survival rate or the inhibition rate by using Excel software; IC calculation Using GraphPad prism software 50 A value; the experiment was repeated three times, and the data were in IC 50 Values ± standard deviation SD format list; cell survival (%) = OD assay/OD DMSO assay × 100%;
observation of cell morphology: observing the change of the growth state and the morphology of the HeLa and HGC-27 cells by the compound B; cells were seeded into 12-well plates and after overnight adherence, varying concentrations of 0,0.04,0.06,0.08 μ M compound B were allowed to act on the cells for 24h, observed with an inverted fluorescence microscope and photographed.
Example 5
Plate clone formation experiment: inoculating cells with good growth state to a 6-well plate, incubating in an incubator, adding drugs after cell adherence, adding compounds B with different concentrations of 0.001,0.025,0.005 and 0.1 mu M into a dimethyl sulfoxide (DMSO) control group and a drug administration group, setting 3 multiple holes in each group, continuing culturing, regularly observing the formation condition of clone colonies, terminating culturing after approximately 2 weeks, taking a picture by using an inverted fluorescence microscope (DMi 8-Leica-Germany), sucking away a culture solution, washing for 2 times by using Phosphate Buffer Saline (PBS), fixing for 15min by using 4% formaldehyde, washing for 2 times by using Phosphate Buffer Saline (PBS), adding a Piemax staining solution for staining for 15min, washing off the staining solution, and drying; photographs were taken with a stereo microscope (SMZ 25-Nikon-Japan); quantitative analysis was performed using Image J software to obtain the number of clones, and the number of clones was calculated, with a clone formation rate (%) = (number of clones formed/number of inoculated cells) × 100%;
cell scoring (round Healing) experiment: inoculating cervical cancer (HeLa) and gastric cancer (HGC-27) cells to a 6-well plate at a proper density, when the cell growth fusion degree is 80-90%, scratching the bottom of the 6-well plate by using a 10 mu L gun head, adding a fresh DMEN culture solution (containing 1% serum), adding a compound B with different concentrations of 0,0.005,0.1 mu M, observing and shooting the scratching state of the cells for 0h under an inverted microscope, marking the shooting point, continuously culturing for 12h and 24h, and shooting and recording the scratching change at the same marking point of 0 h; measuring the scratch width by using Image J software, obtaining the cell scratch width of each hole at different time points and calculating the scratch healing rate, wherein the healing rate is% = (0 h scratch width-12 h and 24h scratch width)/0 h scratch width multiplied by 100%;
cell migration assay (Transwell chamber): the specific experimental steps are as follows: cervical cancer (HeLa) and gastric cancer (HGC-27) cell digestion, preparing cell suspension with serum-free medium, ready for use; DMEN medium (500. Mu.L/well) containing 10% serum was added to the lower Transwell chamber, dimethyl sulfoxide (DMSO) (1.0. Mu.L/well) to the control wells, compound B (1.0. Mu.L/well) to the experimental wells, the previously prepared cell suspension (150. Mu.L/well) to the upper chamber, and the chamber was incubated for 24h; washing the small chamber with Phosphate Buffered Saline (PBS), fixing with 4% paraformaldehyde for 15min, dyeing with 0.5% crystal violet for 15min, washing off the dye, and drying; taking images in a stereoscopic microscope (SMZ 25-Nikon-Japan); 10% acetic acid (100. Mu.L/well) was added to the lower chamber of the Transwell, and after 10min of extraction, the plate was pipetted into a 96-well plate and OD600 nm was measured with a microplate reader. Migration inhibition (%) = (OD control well-OD dosing well)/OD control well × 100%;
vascular endothelial cell lumen neogenesis experiment: before the experiment, a sterile 96-well cell culture plate and a sterile gun head are placed at-80 ℃ for freezing for 1h, and matrigel gel is placed at 4 ℃ overnight; the next day, adding Matrigel gel (50-100 μ L/hole) into the pre-cooled 96-well cell culture plate with the pre-cooled gun head, uniformly coating the whole hole to ensure that no bubbles are generated, and standing at 37 ℃ for 1h to solidify; selecting Human Umbilical Vein Endothelial Cells (HUVEC) within three generations and in a logarithmic phase of growth, seeding the cells in a 96-well plate (6000 cells/well), simultaneously adding 0,0.025,0.1,0.5 MuM compound B with different concentrations, placing in an incubator at 37 ℃ for 6-12h, observing by using a microscope and taking pictures; quantitative analysis (number of nodes, main connections and grids, grid area, branch length and line segment length) is carried out on the pictures by using Image J software;
cell cycle assay: inoculating cervical cancer (HeLa) and gastric cancer (HGC-27) cells into a 6-well plate at an appropriate density, treating the cells with 0,0.025,0.1,0.5. Mu.M compound B at different concentrations for 24h after the cells adhere, and continuing incubation; according to the kit manual (550825, BD Pharmingen TM ) Carrying out an experiment; sucking and discarding the cell culture solution, collecting the cells in a centrifuge tube, centrifuging at 4 ℃, and discarding the supernatant at 1000rpm/5 min; adding Phosphate Buffered Saline (PBS) to resuspend the cells, then dropwise adding absolute ethyl alcohol, standing overnight at-20 ℃, and washing the fixed cells with precooled Phosphate Buffered Saline (PBS) for 2 times for centrifugation before detection; take 1X 10 6 Cells, propidium iodide/RNase staining buffer, 15mi staining at room temperaturen; filtering, loading on a machine, analyzing with a flow cytometer (Mo Flo × DP; beckman Coulter), and analyzing the percentage of each population with summit V5.4.0.16584 software; 2 ten thousand cells were collected per data point and analyzed using Mod Fit LT 4.1 software;
assay for the detection of intracellular Reactive Oxygen Species (ROS): the active Oxygen detection Kit (Reactive Oxygen specifices Assay Kit) is a Kit for detecting active Oxygen by using a fluorescent probe DCFH-DA;
cervical cancer (HeLa) and gastric cancer (HGC-27) cells were cultured at 2X 10 5 Seeding in 6-well plates at a density of/mL and culturing the cells overnight; compound B treated cells and incubated for 24h; the experiment was performed according to the kit manual, the cells were collected in a centrifuge tube, centrifuged, the supernatant was discarded, and the cells were resuspended at 2X 10 in Phosphate Buffered Saline (PBS) 6 Adding a probe at a concentration of 10 mu M, taking a cell which is not added with the probe and is only added with Phosphate Buffered Saline (PBS) as a negative control tube, taking a cell suspension added with the probe, simultaneously adding 20 mu M of active oxygen hydrogen donor to induce the cell to be a positive control tube, incubating at 37 ℃ for 30min, and analyzing on a flow cytometer;
an Annexin V-FITC/PI double staining method apoptosis detection experiment: detecting apoptosis on a flow cytometer by using an FITC annexin V detection kit; inoculating cervical cancer (HeLa) and gastric cancer (HGC-27) cells into a 6-well plate, and incubating in an incubator; adding a compound B with different concentrations of 0,0.025,0.1,0.5 mu M, continuously incubating for 24h, and collecting samples after acting on a specified time point; performing experiment according to kit manual, digesting cells with pancreatin without ethylenediaminetetraacetic acid (EDTA), mixing with supernatant culture medium, centrifuging at 4 deg.C at 1000rpm/3min, washing cells with ice Phosphate Buffered Saline (PBS) once, centrifuging at 4 deg.C at 1000rpm/3min, removing supernatant, and collecting precipitate; cells were washed with cold Phosphate Buffered Saline (PBS) and were washed at 1X 10 6 cells/mL were suspended in 1 Xbuffer; transferring 100 μ L of the solution, adding FITC Annexin V (5 μ L/tube) and PI (5 μ L/tube), setting blank control group, PI single staining group and Annexin V-FITC single staining group, and incubating at room temperature for 15min; 1 Xbuffer solution (400. Mu.L) was supplemented; sample detection on computer as soon as possibleApoptosis, preventing fluorescence quenching; collecting at least 1.0 ten thousand cells from each group of samples, analyzing the percentage of each group by using Summit V5.4.0.16584 software, analyzing sample data by using Flow JO software, and drawing an apoptosis scatter diagram of sample cells;
an experiment for detecting apoptosis by a Western blot method with protein immunoblotting: inoculating cervical cancer (HeLa) and gastric cancer (HGC-27) cells in a logarithmic growth phase into a 6-well plate, adding a compound B with a corresponding concentration after the cells are attached overnight, and incubating for 24h and 48h;
protein quantification experiments with Bradford method: discarding culture solution in the hole, washing cells with cold Phosphate Buffered Saline (PBS), collecting the cells in a 1.5mL EP tube, centrifuging at 4 ℃,1000rpm/3min, discarding supernatant, adding RIPA lysate cell lysate (all cells with 5-7 times of volume), gently shaking for 5min, placing on ice for 30min for lysis, centrifuging at 4 ℃,12000rpm/20min, sucking upper cell lysate for later use, transferring to a new EP tube, simultaneously taking 3uL protein quantitative detection reagent BCA, measuring protein concentration, subpackaging protein samples, and storing at-80 ℃;
protein isolation and immunodetection were performed using a fully automated capillary electrophoresis Wes system: specific antibody, description of 12-230kda protein isolation module, purchased from CST corporation for Wes (fully automated protein expression quantitative analysis system) analysis; the instrument sets default parameters, the Compass for SW 4.0Mac software is used to visualize the electropherograms and analyze the area under the curve (AUC) for the associated peak, the identified peak for the associated molecular weight due to the capillary to capillary slight variations in the molecular weight readings, allowed to be within 10% of the molecular weight (automatically set by the Wes system).
Example 6
Proliferation inhibition of tumor cells by compounds:
in order to comprehensively evaluate the tumor activity and selectivity of the compound B for cell proliferation experiments, 16 human tumor cell models, such as colon cancer HCT-115, HCT-8, caco-2, HT-29 and HCT-116, are selected; gastric cancer HGC-27, breast cancer MDA-MB-231, MCF-7, T47-D, liver cancer HepG-2, huh-7, hep3-B, cervical cancer HeLa, endometrial cancer ISK, lung cancer A549 and pre-carcinomaThe proliferation inhibition effect of the compound B and a positive control drug Doxorubicin (DOX) on tumor cells is examined by adopting an MTT method for the PC-3 of the prostate cancer cells, and IC is calculated 50 A value;
the results show that the compound B shows obvious inhibitory activity and half inhibition rate (IC) on the proliferation of different tumor cells 50 Value) of 0.01-5.3 mu M, which shows that the anti-tumor effect of the compound B has broad-spectrum characteristics, wherein the cervical cancer has the most outstanding inhibitory activity on HeLa and gastric cancer HGC-27 cells and the half-inhibition rate (IC) 50 Values) were 0.032. Mu.M (HeLa) and 0.016. Mu.M (HGC-27), respectively, as shown in FIG. 1.
Example 7
Dose-effect and time-dependent relationship of compound B to inhibition of cell proliferation:
the dose and duration of action of the compound may reflect the affinity of the drug for the target and ultimately the efficacy of the drug:
the result of the inhibitory activity of the compound B on 16 tumor cells shows that IC 50 The difference of the sensitivity of the compound B to different cells is shown from 0.01 mu M to 5.0 mu M, wherein the compound B is most sensitive to the cervical cancer cell Hela and the gastric cancer cell HGC-27, and the IC is 50 The values were 0.01. Mu.M and 0.03. Mu.M, respectively; therefore, cervical cancer (Hela) cells and gastric cancer (HGC-27) cells are selected as research objects in subsequent research, and the detection is carried out after the different concentrations of the compound B act on the cells for 24 hours, 48 hours and 72 hours respectively;
the experimental result shows that the inhibitory activity of the compound B is obviously improved along with the prolonging of the action time under the drug concentrations of 0,0.001,0.003,0.009,0.027,0.081,0.243,0.729,2.187,6.561 and 19.683 mu M of different concentrations, and the inhibitory activity is improved along with the increasing of the concentration of the compound B under the action times of 24 hours, 48 hours and 72 hours, which indicates that the proliferation inhibitory effects of the compound B on the cervical cancer cell Hela and the gastric cancer cell HGC-27 both show obvious time dependence and concentration dependence, and the figure 2 shows that the compound B has obvious time dependence and concentration dependence.
Example 8
Compound B caused a morphological change in cancer cells:
it was further observed whether compound B at different concentrations of 0,0.04,0.06,0.08 μ M acted on cells for 24h and changed morphology in cervical cancer (Hela), gastric cancer (HGC-27) cells:
after 24 hours of cell administration treatment, the cervical cancer (Hela) cell control group is observed to be in a normal elliptical shape, the intercellular connection is tight, the cell membrane structure is complete, the cells of the compound B treatment group can be separated from the surrounding cells and suspended, the cytoplasm is turbid, more cell fragments are in the culture solution, and the morphological characteristics are more obvious along with the increase of the concentration of the compound B; the gastric cancer (HGC-27) cell control group is normal in shape, firm in adhesion, tight in intercellular connection and regular in arrangement, presents a multi-antenna state, can disorderly arrange cells in the compound B treatment group, increases the intercellular space, gradually disappears the antenna state, gradually reduces the cell shape and becomes round, and some cells basically lose the original shape and are in a floating state along with the increase of the concentration of the compound B; shows that the compound B acts on the cells to obviously influence the morphological states of the cervical cancer (Hela) cells and the gastric cancer (HGC-27) cells, and is shown in figure 3.
Example 9
Compound B inhibits cancer cell clonogenic:
in order to further confirm the influence of the compound B on the cell proliferation capacity and the cell population dependence, an in-vitro plate clone formation experiment is utilized, and the clone formation experiment can reflect the influence of a medicament on the tumor cell proliferation by measuring the size and the number of clones; compound B was administered to cervical cancer (Hela) and gastric cancer (HGC-27) cells at 0.001,0.025,0.005,0.1. Mu.M at different concentrations for two weeks, and the effect on the clonality of the cells was observed.
The experimental result shows that the cell clone number is gradually reduced along with the increase of the administration concentration of the compound B, the clone formation rates of the cervical cancer (HeLa) and the gastric cancer (HGC-27) cell control group are respectively 94% and 83%, the clone formation rates of the cervical cancer (HeLa) and the gastric cancer (HGC-27) of the 0.1 mu M administration group are respectively 15%, the statistical difference is extremely significant (P < 0.001), and the experimental result shows that: the compound B obviously inhibits the in vitro clone forming capability of cervical cancer (Hela) and gastric cancer (HGC-27) cells, possibly blocks the colony forming capability of the cancer cells, thereby reducing the proliferation capability of the cancer cells and playing an important role in inducing apoptosis, as shown in figure 4.
Example 10
Compound B inhibits cancer cell scratch repair (healing):
the invention utilizes a wound healing experiment to investigate the inhibitory effect of compound B on the migration capacity of cervical cancer (HeLa) and gastric cancer (HGC-27) cells. Considering that the healing of the cell scratch is the result of the combined action of cell migration and cell propagation, the experiment uses a culture solution containing 1% of serum to culture and observe the healing and migration of the cell;
as shown in fig. 5, the healing rates of the cellular wounds were 50% and 78% after 12h and 24h of cervical cancer (HeLa) cell control groups, 35% and 50% after 12h and 24h of compound B administration at 0.1 μ M, 60% and 83% after 12h and 24h of HGC-27 cell control groups, and 20% and 35% after 12h and 24h of compound B administration, respectively, from which it can be concluded that compound B inhibits the wound healing rates of cervical cancer (HeLa) and gastric cancer (HGC-27) cells in a concentration-and time-dependent manner.
Example 11
Compound B ability to inhibit cancer cell migration:
in order to further verify the results of the previous scratch experiments, the invention adopts a Transwel experiment to investigate whether the compound B has influence on the cell migration of cervical cancer (Hela) and gastric cancer (HGC-27), the compound B with different concentrations of 0,0.025,0.05,0.2 mu M acts on the cells for 24h, the cells are fixed and dyed, and the influence of the compound on the cell migration rate can be obtained by photographing, counting the cells entering the lower chamber and comparing with a control group;
the experimental results showed that a large number of cervical cancer (HeLa) and gastric cancer (HGC-27) cells in the control group migrated to the other side of the membrane through the pores, and that compound B acted on the cells for 24 hours, and the number of migrated cells appeared to be decreased in a clear gradient, and the quantitative analysis results showed that the number of cells penetrating the membrane gradually decreased to exhibit concentration dependence, and that the cell mobility of the cervical cancer (HeLa) and gastric cancer (HGC-27) control group was 63% and 78%, respectively, and the cell mobility of the 0.2 μ M compound B administration group was 19% (HeLa) and 11% (HGC-27), respectively, from which it can be concluded that compound B inhibited the migration rate of cervical cancer (HeLa) and gastric cancer (HGC-27) cells in concentration and time dependence, as shown in fig. 6.
Example 12
Compound B has inhibitory effect on vascular endothelial cell (HUVEC) lumenal formation:
in the later stage of angiogenesis, endothelial cells finish steps of proliferation, migration and the like, the endothelial cells are further arranged to form a long-strip-shaped lumen structure, and then the potential influence of the compound B on angiogenesis is further evaluated on Human Umbilical Vein Endothelial Cells (HUVEC);
experimental results it was observed that endothelial cell (HUVEC) control group formed well-aligned attached and distinct network structures on matrigel, but part of the network tubes of compound B treated group were disconnected, and even vascular cells whose network structures became gradually incomplete and sparse, standard image analysis was performed to obtain commonly evaluated parameters: results of nodes, main connections, segment lengths, total branch lengths, grid numbers, grid areas, segmentation, total grid areas and grid numbers show that the compound B has a dose-dependent effect, and the compound B has obvious activity of destroying new vessels, as shown in FIG. 7.
Example 13
Compound B blocks cervical cancer (HeLa) and gastric cancer (HGC-27) cells in the G2/M phase:
the invention adopts a flow cytometry analysis method, takes cervical cancer (HeLa) and gastric cancer (HGC-27) as research objects, and investigates and measures the influence of the compound B on the cell cycle distribution of the cervical cancer (HeLa) and the gastric cancer (HGC-27); compound B treatment in both cervical (HeLa) and gastric (HGC-27) cancer cells caused significant accumulation of cells in the G2/M phase in a concentration-dependent manner, as shown by Propidium Iodide (PI) staining; for example, the ratio of the cervical cancer (HeLa) cell control group to the G2/M phase cells is 9.42%, while the ratio of the compound B group with 0.1 mu M to the G2/M phase cells is increased to 45.75%; the control group of gastric cancer (HGC-27) cells accounted for 15.26% of the cells in the G2/M phase, while the group of compound B at 0.1. Mu.M accounted for 51.6% of the cells in the G2/M phase, as shown in FIG. 8.
Example 14
Compound B caused an increase in cervical cancer (HeLa) and gastric cancer (HGC-27) Reactive Oxygen Species (ROS) levels:
in view of the relationship between ROS and apoptosis, in order to verify whether the addition of different concentrations of compound B can affect the expression level of active oxygen in cervical cancer (HeLa) and gastric cancer (HGC-27) cells, the change condition of the oxidation environment in the cells is analyzed by respectively adopting an inverted fluorescence microscope method. Compound B was applied to cervical cancer (HeLa) and gastric cancer (HGC-27) cells at varying concentrations of 0,0.025,0.05,0.1. Mu.M for 24h and photographs taken by fluorescence microscopy.
The results show that with the increase of the concentration of the compound B, the ROS amount in the cells of the cervical cancer (HeLa) and the gastric cancer (HGC-27) is rapidly and remarkably increased, and the ROS amount is increased to be more than 4 times of the original ROS level, which is remarkably higher than the ROS level of a control group, and the result is shown in figure 9.
Example 15
Compound B induced apoptosis in Hela, HGC-27 cells:
in order to further explore the mechanism behind the anti-cell proliferation effect of the compound B, in view of the fact that the compound B can cause human cervical cancer (HeLa) and gastric cancer (HGC-27) to generate G2/M phase block, and cycle block is often accompanied with apoptosis, the compound B is used for administering to cervical cancer (HeLa) and gastric cancer (HGC-27) cells, and the apoptosis condition after administration is detected;
in order to research the relationship between apoptosis and administration concentration, the compound B with different concentrations of 0,0.025,0.05,0.1 mu M acts on cervical cancer (HeLa) and gastric cancer (HGC-27) cells for 24 hours, and a flow cytometer detects the apoptosis degree after administration; the experimental results show that: for example, the control group of cervical cancer (HeLa) cells accounted for 4.19% and 10.9% of early and late apoptotic cells, respectively, while the group of Compound B at 0.1. Mu.M accounted for 7.15% and 55% of early and late apoptotic cells; the control group of gastric cancer (HGC-27) cells accounted for 10.1% and 12.4% of early and late apoptotic cells, respectively, while the 0.1 μ M group of compound B accounted for 20.6% and 30.9% of early and late apoptotic cells, indicating that the antiproliferative activity of compound B on cervical cancer (HeLa) and gastric cancer (HGC-27) cells may be due to its ability to induce apoptosis, see fig. 10;
in order to further confirm the apoptosis effect of the compound B on cervical cancer (HeLa) and gastric cancer (HGC-27) cells, the change condition of various proteins playing an important role in an apoptosis regulation pathway after the compound B is treated is detected through a western blot experiment, such as PARP; the cleavage of ADP ribose polymerase (PARP) during apoptosis induced by compound B, cervical cancer (HeLa) and gastric cancer (HGC-27) cells treated for 24h and 48h with increasing concentration of compound B, which caused PARP cleavage in both cell lines in a dose and time dependent manner, was evaluated, confirming its pro-apoptotic activity; in addition, the expression of the anti-apoptotic protein Bcl-2 and the apoptosis-promoting Bak were also studied, and the experimental results showed that the expression level of Bcl-2 protein in cervical cancer (HeLa) and gastric cancer (HGC-27) cells was reduced after 24 hours in the 0.6 and 1.5 μ M groups, the decrease in Bcl-2 expression was even greater in both cervical cancer (HeLa) and gastric cancer (HGC-27) cell lines after 48 hours, and that the expression of the pro-apoptotic protein Bak was increased in a dose-and time-dependent manner after treatment with compound B at a concentration of 1.0 and 1.5 μ M for 24 and 48 hours, confirming that compound B induces the activation of polymerase (PARP) and reduces the expression of the anti-apoptotic protein to induce apoptosis of the cells, see fig. 11.
Claims (8)
1. A compound B of m-methyl benzylidene tetrahydropyrazol [3,4-D ] pyridine [1,2-A ] pyrimidinone, characterized in that the structural formula (I) of the compound B is:
wherein:
the compound B is named as (E) -1-methyl-9- (3-methylbenzylidene) -6,7,8,9-tetrahydropyrazole [3,4-d ] pyridine [1,2-a ] pyrimidin-4 (1H) -one.
2. The use of the compound B according to claim 1 in the preparation of an antitumor medicament for inhibiting the growth of human colon cancer cell lines HCT-115, HCT-8, caco-2, HT-29, HCT-116.
3. The use of the compound B according to claim 1 in the preparation of an antitumor medicament for inhibiting the growth of human breast cancer cell lines MDA-MB-231, MCF-7 and T47-D.
4. The use of the compound B as claimed in claim 1 in the preparation of antitumor drugs for inhibiting the growth of human hepatoma cell lines HepG-2, huh-7 and Hep 3-B.
5. The use of the compound B according to claim 1 for the preparation of an antitumor medicament for inhibiting the growth of human gastric cancer cell line HGC-27.
6. The use of compound B according to claim 1 in the preparation of an anti-tumor medicament for inhibiting the growth of human cervical cancer HeLa and endometrial cancer ISK cell lines.
7. The use of the compound B according to claim 1 in the preparation of an antitumor medicament for inhibiting the growth of a human lung cancer a549 cell line.
8. The use of compound B according to claim 1 in the preparation of an anti-tumor medicament for inhibiting the growth of PC-3 cell lines of human prostate cancer cells.
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| ZUKELA RUZI等: "Novel pyrazolo[3, 4-d]pyrimidines as potential anticancer agents: Synthesis, VEGFR-2 inhibition, and mechanisms of action", 《BIOMEDICINE & PHARMACOTHERAPY 》, vol. 156, no. 113948, pages 1 - 27 * |
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