CN108685921B

CN108685921B - Application of quinoline derivative of N-isostere tectoridin in anti-liver cancer drugs

Info

Publication number: CN108685921B
Application number: CN201810659356.4A
Authority: CN
Inventors: 陈鲲; 马咏诗; 黎裕麟; 马祥; 樊静; 张继匀; 刘琼玉; 胡佳钦; 徐丹; 朱海亮
Original assignee: Guangzhou University
Current assignee: Guangzhou University
Priority date: 2017-12-19
Filing date: 2018-06-22
Publication date: 2020-01-17
Anticipated expiration: 2038-06-22
Also published as: CN108685921A; CN107669676A

Abstract

The invention discloses an application of quinoline derivatives of N-isosteric tectoridin in anti-liver cancer drugs, and the quinoline derivatives of the N-isosteric tectoridin have the chemical structural formula as follows:the invention relates to application of a quinoline derivative New Tecotorigenin of N-isostere tectoridin in preparation of a drug for inhibiting liver cancer HepG2 cells. The invention inhibits the proliferation of the liver cancer HepG2 cells by inhibiting RAF/MEK/ERK pathway and JAK/STAT pathway. The anti-liver cancer drug has obvious treatment effect and is not easy to generate drug resistance and adverse reaction. Meanwhile, the invention also discloses a preparation method of the quinoline derivative of N-isostere tectoridin, which has higher yield; the New Tecotorigenin has obvious effect of inhibiting the transplanted tumor.

Description

Application of quinoline derivative of N-isostere tectoridin in anti-liver cancer drugs

Technical Field

The invention relates to tectorigenin isostere, in particular to application of quinoline derivatives of N-isostere tectoridin in anti-liver cancer drugs.

Background

Primary Hepatic Carcinoma (PHC) refers to a cancer that occurs in parenchymal hepatic cells or intrahepatic bile duct epithelial cells, and is a malignant tumor with high malignancy, rapid progression, poor prognosis and short survival time, and the survival rate is only 3% -5%. Worldwide, the morbidity and mortality of primary liver cancer ranks the 7 th and 3 rd sites of all tumors, respectively. China is a large disease-causing country of liver cancer, and the disease rate accounts for 45 percent of the whole world. In recent years, the incidence of liver cancer in China is on the rise, and the fatality rate is also on the rise, which accounts for the third position of the national cancer mortality rate. The national census population in 2010 of China is 13.4 hundred million people (not included in Taiwan), and according to the liver cancer incidence and mortality level in 2003-2007, 36 million people with liver cancer incidence and 35 million people with liver cancer death in each year of China are estimated.

According to the latest statistical data of 2015 in the country and Guangdong province, the Guangdong is one of the highest incidence areas in the world of liver cancer, and the Guangdong is prone to the younger population at high risk.

The etiology of primary liver cancer is not completely clear up to now, and the current research shows that the primary liver cancer may be related to factors such as viral hepatitis, aflatoxin, cirrhosis, drinking water pollution, heredity, obesity, smoking, drinking, and the like. Especially, the coastal area has hot and humid climate, which creates conditions for the breeding of carcinogen aflatoxin and increases the risk of suffering from liver cancer. Secondly, some rural residents who drink waste or polluted water also have an increased risk of liver cancer. For northeast, drinking wine is an important cause of liver cancer, and long-term drinking of wine can cause repeated steatosis, necrosis and regeneration of liver cells, so that liver cirrhosis is caused and finally converted into liver cancer, and the proportion of liver cirrhosis converted into liver cancer is as high as 70%. In recent years, the clinical treatment method for liver cancer is biological treatment and molecular targeted drug treatment besides traditional radical resection, liver transplantation, radiotherapy and chemotherapy and the like. In the 80's of the 20 th century, the new concept of biotherapy of tumors was proposed by the doctor Olidham in the united states. As a fourth major auxiliary treatment mode after primary liver cancer surgical resection, radiotherapy and chemotherapy, the compound can enhance the defense capacity of an organism to tumors by regulating the biological response of the organism, thereby inhibiting and/or delaying the tumor growth, improving the survival rate and reducing the recurrence rate. The pathogenesis of liver cancer is known to be very complex, the occurrence, development and transfer of the liver cancer are closely related to mutation of various genes, cell signaling pathways, angiogenesis abnormity and the like, and a plurality of key links exist, which are the theoretical basis and potential targets for molecular targeted therapy.

In 2007, the first targeted drug tyrosine kinase inhibitor is successful in treating advanced liver cancer, and the treatment of liver cancer enters a new targeted treatment era. Therefore, the molecular targeting drug for treating liver cancer has gradually attracted attention and is becoming a new research hotspot. In recent years, some molecular targeted drugs have made certain progress in liver cancer treatment, such as: epidermal growth factor receptor inhibitory drugs: gefitinib, erlotinib, lapatinib; multi-kinase inhibitory drugs: sorafenib, sunitinib; MEK/ERK class of inhibitors: A2D6244, and the like. However, each of the above molecularly targeted drugs has its limitations.

At present, the research is necessary to be started from the perspective of the traditional Chinese medicine in China, and a new molecular target medicine which is suitable for large-scale popularization, benefits the masses of the civilian, has easy preparation process, high efficiency and small side effect and can directionally treat liver cancer is developed from the traditional Chinese medicinal materials commonly used for treating liver diseases in the folk.

The genetic material of the mouse is very similar to that of the human, and the cost for constructing the mouse model is low,The period is shorter, and the gene modification technology is easier to realize, so the mouse is a better model animal for the research of human liver cancer^[1]. The construction of the mouse model which can accurately simulate the human liver cancer symptoms, is easy to obtain and is economically applicable not only contributes to the deep exploration of the molecular mechanism of liver cancer occurrence and pathogenesis, but also can be used for detecting and evaluating new liver cancer treatment methods and means, thereby promoting the aim of transforming the liver cancer research results in a laboratory into clinical application^[2]。

Reference documents:

[1]Mou H，Kennedy Z，Anderson DG，et al.Precision cancermouse modelsthrough genome editing with CRISPR-Cas9[J].Genome Med.2015，7:53；

[2] li shun, Chenlixiang, Pengxuhua, Jiangjiang and Zhou Xiaohui, research progress of liver cancer model in mice [ J ]. Chinese Experimental animal bulletin, 2016,4,24(2): 213-214.

Disclosure of Invention

The invention aims to solve the problems and provide application of a quinoline derivative of N-isosteric tectoridin in an anti-liver cancer medicament, which has remarkable medicinal effect and is difficult to generate medicament resistance.

In order to achieve the purpose, the invention adopts the following technical scheme: the invention relates to application of quinoline derivatives of N-isostere tectoridin in anti-liver cancer drugs.

Further, the quinoline derivative of the N-isostere tectoridin has a chemical structural formula as follows:

further, said quinoline derivatives of N-isosterin inhibit hepatoma cell proliferation by inhibiting RAF/MEK/ERK pathway and JAK/STAT pathway; the quinoline derivative of the N-isosterin promotes the apoptosis of the liver cancer cells by inhibiting a P13K/AKT/mTOR pathway; the quinoline derivative of the N-isostere tectoridin promotes the apoptosis of the liver cancer cell by up-regulating an anti-apoptosis signal P53 and an anti-apoptosis signal PTEN gene.

Furthermore, the quinoline derivative of the N-isosterin can improve the generation of ROS, increase the permeability of a mitochondrial membrane and cause the combination of excessive stress of endoplasmic reticulum by up-regulating the expression level of an anti-apoptosis signal p53 so as to induce the apoptosis of the liver cancer cell; the quinoline derivative of N-isosterin can prevent the migration of liver cancer cells by regulating the expression of related genes of a angiogenesis abnormal hyperplasia pathway.

Further, the quinoline derivative of N-isosterin can inhibit the proliferation of hepatoma carcinoma cells by down-regulating the expression levels of AKT, ERK and pERK; the quinoline derivative of N-isosterin tectoridin can improve the sensitivity of tumor to drugs and reduce drug resistance in the process of treating liver cancer.

The invention relates to application of quinoline derivatives of N-isostere tectoridin in preparation of drugs or preparations for inhibiting RAF/MEK/ERK pathway, JAK/STAT pathway and P13K/AKT/mTOR pathway, promoting apoptosis of liver cancer cells and inhibiting liver cancer.

The invention relates to application of quinoline derivatives of N-isostere tectoridin in preparation of drugs or preparations for down-regulating anti-apoptosis signals P53 and PTEN genes so as to promote apoptosis of liver cancer cells and inhibit liver cancer.

The quinoline derivative of N-isosterin disclosed by the invention is applied to preparation of a medicine or a preparation for up-regulating an anti-apoptosis signal p53, down-regulating the expression levels of AKT, ERK and pERK, improving the generation of ROS, increasing the permeability of a mitochondrial membrane and combining with the phenomenon of causing excessive stress of endoplasmic reticulum so as to induce apoptosis of liver cancer cells and inhibit liver cancer.

The invention relates to application of quinoline derivatives of N-isostere tectoridin in preparation of drugs or preparations for preventing liver cancer cell migration and inhibiting liver cancer invasion and metastasis by regulating and controlling the expression of cancer suppressor genes P53, ERK1/2 gene and AKT gene of a angiogenesis abnormal proliferation pathway.

The quinoline derivative of N-isostere tectoridin is applied to preparation of a medicament or a preparation for improving sensitivity of liver cancer to the medicament and reducing drug resistance of the liver cancer.

Has the advantages that: the anti-liver cancer drug has obvious treatment effect and is not easy to generate drug resistance and adverse reaction.

In another aspect, the present invention provides a process for the preparation of the above quinoline derivatives of N-isosteric tectoridin in higher yield comprising the steps of:

(1) dissolving 1, 3-dibromo-2-methoxyl-5-nitrobenzene in ethanol, and then adding Na₂S₂O₄Stirring the aqueous solution at 50-60 ℃ for 3-7 hours, cooling to room temperature, purifying with ethyl acetate, washing, drying, and decompressing to remove the ethyl acetate to obtain white powdery 3, 5-dibromo-4-methoxyaniline;

(2) dissolving 3, 5-dibromo-4-methoxyaniline in CH (OMe)₃In (c), stirring to remove excess CH (OMe)₃Then, white powdery (cis) -N-3, 5-dibromo-4-methoxyphenyl formylimine is obtained;

(3) dissolving (cis) -N-3, 5-dibromo-4-methoxyphenyl carboximide in anhydrous MeOH, then adding NaOMe and stirring to be uniform, adding ethyl 2- (4-methoxyphenyl) acetate to remove excess MeOH, then adding water and stirring to be uniform, and filtering under reduced pressure to obtain white powdery ethyl (trans) -3- (3, 5-dibromo-4-methoxyphenyl amino) -2- (4-methoxyphenyl) acrylate;

(4) dissolving ethyl (trans) -3- (3, 5-dibromo-4-methoxyphenyl amino) -2- (4-methoxyphenyl) acrylate in ethanol, and adding H₂SO₄Stirring for 4-10 hours at 50-70 ℃, cooling to room temperature, adding water, stirring, and filtering under reduced pressure to obtain white powdered 5, 7-dibromo-6-methoxy-3- (4-methoxyphenyl) quinolin-4 (1H) -one;

(5) dissolving 5, 7-dibromo-6-methoxy-3- (4-methoxyphenyl) quinolin-4 (1H) -one in DMF and Et₂O, then adding PCl dropwise₃Stirring for 8-16 hours, adding water, stirring uniformly, extracting the obtained mixture with AcOEt, washing, and drying to remove the AcOEt to obtain white powdery 5, 7-dibromo-4-chloro-6-methoxy-3- (4-methoxyphenyl) quinolone, namely the quinoline derivative of the N-isostere tectoridin.

Wherein the equivalent weight of the ethanol in the step (1) is preferably 10-20; na (Na)₂S₂O₄The mass concentration of the aqueous solution is preferably 10-30%; na (Na)₂S₂O₄The equivalent weight of the aqueous solution is preferably 2-6; when ethyl acetate is used for purification, preferably three times of purification is carried out, and the volume of the ethyl acetate is 2-4 times that of the ethyl acetate; the total extract purified with ethyl acetate was washed with saturated salt solution and MgSO₄Drying;

in the step (2), CH (OMe)₃The equivalent weight of (A) is preferably 10-20, and the stirring time is preferably 6-12 hours;

in the step (3), the equivalent weight of the anhydrous MeOH is preferably 10-20; the equivalent weight of NaOMe is preferably 1-4, and the stirring time is preferably 15-45 minutes; the equivalent weight of the 2- (4-methoxyphenyl) ethyl acetate is preferably 1-3, the stirring time is preferably 4-10 hours, then the water is added with the same volume, and the stirring is carried out for 10-20 minutes;

in the step (4), the equivalent weight of ethanol is preferably 10-20; h₂SO₄The equivalent of (A) is preferably 0.5 to 1; cooling to room temperature, adding water with the volume preferably 5-10 times that of the mixture, and stirring for 30-50 minutes;

in the step (5), PCl is added dropwise₃The temperature is preferably 0 ℃; the equivalent weight of DMF is preferably 20 to 40, Et₂The equivalent weight of O is preferably 20-40, the water adding amount is preferably 2-6 times, the AcOEt extraction frequency is preferably 3, the volume of each extraction is 2-4 times, and the saturated salt solution is preferably adopted for washing and MgSO (MgSO) is preferably used₄And (5) drying.

In still another aspect, the present invention provides a use of the above 5, 7-dibromo-4-chloro-6-methoxy-3- (4-methoxyphenyl) quinolone for preparing a medicament for inhibiting a transplant tumor. Wherein, the transplantation tumor is preferably a mouse transplantation tumor; the source of the transplantable tumor is preferably HEPG2 cells, H22 cells or Hepa1-6 cells. The graft tumor may be an orthotopic graft tumor or an ectopic graft tumor.

In still another aspect, the present invention provides a tumor-suppressing drug comprising 5, 7-dibromo-4-chloro-6-methoxy-3- (4-methoxyphenyl) quinolone. Wherein, the tumor can be a primary tumor or a transplantation tumor, and more preferably, the transplantation tumor; most preferably a transplantation tumor of HEPG2 cell, H22 cell or Hepa1-6 cell origin. More preferably, when the tumor is a Hepa1-6 or H22 cell transplantation tumor, the concentration of 5, 7-dibromo-4-chloro-6-methoxy-3- (4-methoxyphenyl) quinolone is 7 to 14 mg/kg; when the tumor is HEPG2 cell transplantation tumor, the concentration of 5, 7-dibromo-4-chloro-6-methoxy-3- (4-methoxyphenyl) quinolone is 8-32 μ M.

Compared with the prior art, the invention has the following advantages:

(1) the invention improves the generation of ROS by culturing HEPG2 cells in vitro and regulating the expression level of an anti-apoptosis signal p53 by New Tectrorigenin, increases the permeability of a mitochondrial membrane and combines the induction of endoplasmic reticulum excessive stress, thereby inducing HepG2 cell apoptosis. The proliferation of HepG2 cells is also inhibited by down-regulating the expression level of AKT, ERK and pERK. The NewTecorgenin can be used as an anti-liver cancer drug to prevent and treat primary liver cancer and other diseases;

(2) the method for preparing the quinoline derivative of N-isosterin tectoridin is adopted to synthesize Newtectorigenin, the yield is obviously improved, and the yield of the Newtectorigenin can be improved by 10 percent.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to these drawings without creative efforts.

FIG. 1 is a graph showing the effect of New tectorigenin at various concentrations (0. mu.M, 4. mu.M, 8. mu.M, 16. mu.M, 24. mu.M and 32. mu.M) on HepG2 cells;

FIG. 2 is a graph showing the results of viability of Newtectorigenin-treated HepG2 cells at 24h and 48h at different concentrations (0. mu.M, 4. mu.M, 8. mu.M, 16. mu.M, 24. mu.M and 32. mu.M) measured by M T colorimetry;

FIG. 3 is a graph showing the effect of New Tectrorigenin at various concentrations (0. mu.M, 4. mu.M, 8. mu.M, 16. mu.M, 24. mu.M and 32. mu.M) on HepG2 karyotype;

FIG. 4 is a graph showing the effect of New Tectrorigenin at various concentrations (0. mu.M, 4. mu.M, 8. mu.M, 16. mu.M, 24. mu.M and 32. mu.M) on Mitochondrial Membrane Potential (MMP) in HepG2 cells;

FIG. 5 is a graph showing the results of the effect of New Tectrorigenin at various concentrations (0. mu.M, 4. mu.M, 8. mu.M, 16. mu.M, 24. mu.M and 32. mu.M) on the subcellular localization of Calnexin;

FIG. 6 is a graph showing the effect of New Tectrorigenin at various concentrations (0. mu.M, 4. mu.M, 8. mu.M, 16. mu.M, 24. mu.M and 32. mu.M) on ROS in HepG2 cells;

FIG. 7 is a graph showing the effect of Western blot to detect the effect of NewTecoriginin at different concentrations (0. mu.M, 4. mu.M, 8. mu.M, 16. mu.M, 24. mu.M and 32. mu.M) on the expression of p53, Caspase-3, Bax and Bcl-2 proteins;

FIG. 8 is a graph showing the effect of Western blot to detect the effect of NewTecorigigenin at different concentrations (0. mu.M, 4. mu.M, 8. mu.M, 16. mu.M, 24. mu.M and 32. mu.M) on Akt and PI3K protein expression;

FIG. 9 is a graph showing the effect of Western blot to detect the effect of NewTecorigigenin at different concentrations (0. mu.M, 4. mu.M, 8. mu.M, 16. mu.M, 24. mu.M and 32. mu.M) on ERK and pERK protein expression.

FIG. 10 is a graph showing the change in tumor size of normal mice, model group mice, low concentration New Tecotorigenin and high concentration New Tecotorigenin group H22 tumor-bearing mice;

FIG. 11 is a graph of the tumor sizes at week 3 of normal mice, model group mice, low concentration New Tecotorigenin and high concentration New Tecotorigenin group H22 tumor-bearing mice;

FIG. 12 is a graph of mouse in vivo imaging and tumor size at the end of the experiment in normal mice, model group mice, low concentration New Tecoriginin and high concentration New Tecoriginin group mice;

FIG. 13 is a graph of H & E staining (. times.200) of tumor tissues of model group mice, low concentration New Tecotorigenin and high concentration New Tecotorigenin group H22 tumor-bearing mice;

FIG. 14 is an immunohistochemical microscopic view (X100) of tumor tissues Arg-1, AFP, CA125, GPC3 of a model group mouse, a low concentration New Tectrorigenin and a high concentration New Tectrorigenin group H22 tumor-bearing mouse;

FIG. 15 is a graph of tumor in vivo imaging of model group mice, low concentration New Tecotorigenin and high concentration New Tecotorigenin group Hepa1-6 tumor-bearing mice at different times;

FIG. 16 is a graph showing the tumor sizes of model group mice, low concentration New Tecotorigenin and high concentration New Tecotorigenin group Hepa1-6 tumor-bearing mice at different times;

FIG. 17 is a graph of mouse in vivo imaging and tumor size at the end of the experiment in the model group mouse, the low concentration New Tectrorigenin and the high concentration New Tectrorigenin group mouse;

FIG. 18 is a graph of H & E staining (. times.200) of tumor tissues of a model group mouse, a low concentration New Tecotorigenin and a high concentration New Tecotorigenin group Hepa1-6 tumor-bearing mouse;

FIG. 19 is a graph of immunohistochemical microscopic observations (. times.100) of tumor tissues Arg-1, AFP, CA125, and GPC3 of model group mice, low concentration New Tectrorigenin, and high concentration New Tectrorigenin group Hepa1-6 tumor-bearing mice.

Detailed Description

The technical method in the embodiment of the invention will be clearly and completely described below with reference to the accompanying drawings in the embodiment of the invention. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention. Unless otherwise specified, the concentrations of the reagents in the present application are mass concentrations.

Example 1

One embodiment of the process for the preparation of the N-isosteric tectoridin quinoline derivative of the invention comprises the steps of:

(1) dissolving 1, 3-dibromo-2-methoxy-5-nitrobenzene in 10 equivalents of ethanol at room temperature; to the resulting solution was added 6 equivalents of 10% Na₂S₂O₄Stirring the aqueous solution at 50 ℃ for 7 hours; when the reaction mixture was cooled to room temperature, it was extracted three times with AcOEt (ethyl acetate), 2-4 volumes each; the total extract was washed with saturated salt solution and MgSO₄Drying; removing AcOEt under reduced pressure to obtain white powdery 3, 5-diBromo-4-methoxyaniline;

(2) dissolving 3, 5-dibromo-4-methoxyaniline in 10 equivalents of CH (OMe) at room temperature₃Stirring for 6-12 hours; removal of excess CH (OMe)₃Then obtaining white powdery (cis) -N-3, 5-dibromo-4-methoxyphenyl formyl imine;

(3) (cis) -N-3, 5-dibromo-4-methoxyphenylcarboximide was dissolved in 20 equivalents of anhydrous MeOH at room temperature; adding 1 equivalent of NaOMe (sodium methoxide) into the mixture, and stirring for 15-45 minutes; then adding 1 equivalent of 2- (4-methoxyphenyl) ethyl acetate, and stirring for 4-10 hours; after removal of excess MeOH, add equal volume of water and stir for 10-20 minutes; the resulting mixture was filtered under reduced pressure to give ethyl (trans) -3- (3, 5-dibromo-4-methoxyphenylamino) -2- (4-methoxyphenyl) acrylate as a white powder;

(4) dissolving ethyl (trans) -3- (3, 5-dibromo-4-methoxyphenyl amino) -2- (4-methoxyphenyl) acrylate in 10 equivalents of ethanol at room temperature; to the resulting mixture was added 0.5 equivalent of H₂SO₄Stirring for 4-10 hours at 50-70 ℃; when the reaction mixture is cooled to room temperature, 5 times the volume of water is added and stirred for 30-50 minutes; the resulting mixture was filtered under reduced pressure to give 5, 7-dibromo-6-methoxy-3- (4-methoxyphenyl) quinolin-4 (1H) -one as a white powder;

(5) 5, 7-dibromo-6-methoxy-3- (4-methoxyphenyl) quinolin-4 (1H) -one was dissolved in a mixture of 40 equivalents of DMF (N, N-dimethylformamide) and 20 equivalents of Et2O (diethyl ether) at 0 ℃ and then PCl was added dropwise₃Stirring for 8-16 hours; 6 volumes of water were added and vigorously stirred, and the resulting mixture was extracted 3 times with 4 volumes of AcOEt, washed with saturated salt solution and MgSO₄Drying; AcOEt was removed to give 5, 7-dibromo-4-chloro-6-methoxy-3- (4-methoxyphenyl) quinolone as a white powder.

The corresponding synthetic route of the preparation method is as follows:

example 2

(1) dissolving 1, 3-dibromo-2-methoxy-5-nitrobenzene in 15 equivalents of ethanol at room temperature; to the resulting solution was added 2 equivalents of 20% Na₂S₂O₄Stirring the aqueous solution at 55 ℃ for 5 hours; when the reaction mixture was cooled to room temperature, it was extracted three times with AcOEt, 2-4 volumes each time; the total extract was washed with saturated salt solution and MgSO₄Drying; removing AcOEt under reduced pressure to obtain white powdery 3, 5-dibromo-4-methoxyaniline;

(2) 3, 5-dibromo-4-methoxyaniline was dissolved in 16 equivalents of CH (OMe) at room temperature₃Stirring for 6-12 hours; removal of excess CH (OMe)₃Then obtaining white powdery (cis) -N-3, 5-dibromo-4-methoxyphenyl formyl imine;

(3) (cis) -N-3, 5-dibromo-4-methoxyphenylcarboximide was dissolved in 14 equivalents of anhydrous MeOH at room temperature; adding 2 equivalents of NaOMe into the mixture, and stirring for 15-45 minutes; then adding 2 equivalents of 2- (4-methoxyphenyl) ethyl acetate, and stirring for 4-10 hours; after removal of excess MeOH, add equal volume of water and stir for 10-20 minutes; the resulting mixture was filtered under reduced pressure to give ethyl (trans) -3- (3, 5-dibromo-4-methoxyphenylamino) -2- (4-methoxyphenyl) acrylate as a white powder;

(4) dissolving ethyl (trans) -3- (3, 5-dibromo-4-methoxyphenyl amino) -2- (4-methoxyphenyl) acrylate in 15 equivalents of ethanol at room temperature; to the resulting mixture was added 0.8 equivalent of H₂SO₄Stirring for 4-10 hours at 50-70 ℃; when the reaction mixture was cooled to room temperature, 7 times the volume of water was added and stirred for 30-50 minutes; the resulting mixture was filtered under reduced pressure to give 5, 7-dibromo-6-methoxy-3- (4-methoxyphenyl) quinolin-4 (1H) -one as a white powder;

(5) 5, 7-dibromo-6-methoxy-3- (4-methoxyphenyl) quinolin-4 (1H) -one was dissolved in 20 equivalents of DMF and 40 equivalents of Et at 0 deg.C₂O, then adding PCl dropwise₃Stirring for 8-16 hours;2 volumes of water were added and vigorously stirred, and the resulting mixture was extracted 3 times with 3 volumes of AcOEt, washed with saturated salt solution and MgSO₄Drying; AcOEt was removed to give 5, 7-dibromo-4-chloro-6-methoxy-3- (4-methoxyphenyl) quinolone as a white powder.

Example 3

(1) dissolving 1, 3-dibromo-2-methoxy-5-nitrobenzene in 20 equivalents of ethanol at room temperature; to the resulting solution was added 4 equivalents of 30% Na₂S₂O₄Stirring the aqueous solution at 60 ℃ for 3 hours; when the reaction mixture was cooled to room temperature, it was extracted three times with AcOEt, 2-4 volumes each time; the total extract was washed with saturated salt solution and MgSO₄Drying; removing AcOEt under reduced pressure to obtain white powdery 3, 5-dibromo-4-methoxyaniline;

(2) dissolving 3, 5-dibromo-4-methoxyaniline in 20 equivalents of CH (OMe) at room temperature₃Stirring for 6-12 hours; removal of excess CH (OMe)₃Then obtaining white powdery (cis) -N-3, 5-dibromo-4-methoxyphenyl formyl imine;

(3) (cis) -N-3, 5-dibromo-4-methoxyphenylcarboximide was dissolved in 10 equivalents of anhydrous MeOH at room temperature; adding 4 equivalents of NaOMe into the mixture, and stirring for 15-45 minutes; then adding 3 equivalents of 2- (4-methoxyphenyl) ethyl acetate, and stirring for 4-10 hours; after removal of excess MeOH, add equal volume of water and stir for 10-20 minutes; the resulting mixture was filtered under reduced pressure to give ethyl (trans) -3- (3, 5-dibromo-4-methoxyphenylamino) -2- (4-methoxyphenyl) acrylate as a white powder;

(4) dissolving ethyl (trans) -3- (3, 5-dibromo-4-methoxyphenyl amino) -2- (4-methoxyphenyl) acrylate in 20 equivalents of ethanol at room temperature; to the resulting mixture was added 1 equivalent of H₂SO₄Stirring for 4-10 hours at 50-70 ℃; when the reaction mixture is cooled to room temperature, 10 times the volume of water is added and stirred for 30-50 minutes; filtering the resulting mixture under reduced pressure to obtain 5, 7-bis (methylene terephthalate) as a white powderBromo-6-methoxy-3- (4-methoxyphenyl) quinolin-4 (1H) -one;

(5) 5, 7-dibromo-6-methoxy-3- (4-methoxyphenyl) quinolin-4 (1H) -one was dissolved in 30 equivalents of DMF and 30 equivalents of Et at 0 deg.C₂O, then adding PCl dropwise₃Stirring for 8-16 hours; 4 volumes of water were added and vigorously stirred, and the resulting mixture was extracted 3 times with 2 volumes of AcOEt, washed with saturated salt solution and MgSO₄Drying; AcOEt was removed to give 5, 7-dibromo-4-chloro-6-methoxy-3- (4-methoxyphenyl) quinolone as a white powder.

Example 4

The invention relates to application of quinoline derivatives of N-isostere tectoridin in anti-liver cancer drugs.

The quinoline derivative of N-isosterin (New Tecotorigenin, herein) has the chemical structural formula:

the quinoline derivative of the N-isosterin can inhibit the proliferation of the liver cancer cells by inhibiting RAF/MEK/ERK pathway and JAK/STAT pathway; the quinoline derivative of the N-isosterin promotes the apoptosis of the liver cancer cells by inhibiting a P13K/AKT/mTOR pathway; the quinoline derivative of the N-isostere tectoridin promotes the apoptosis of the liver cancer cell by up-regulating an anti-apoptosis signal P53 and an anti-apoptosis signal PTEN gene.

The quinoline derivative of the N-electron isostere tectoridin can improve the generation of ROS by up-regulating the expression level of an anti-apoptosis signal p53, increase the permeability of a mitochondrial membrane and cause the combination of endoplasmic reticulum overstress, thereby inducing the apoptosis of the liver cancer cell; the quinoline derivative of N-isosterin can prevent the migration of liver cancer cells by regulating the expression of related genes of a angiogenesis abnormal hyperplasia pathway.

The quinoline derivative of the N-isosterin can inhibit the proliferation of the liver cancer cells by down-regulating the expression levels of AKT, ERK and pERK; the quinoline derivative of N-isosterin tectoridin can improve the sensitivity of tumor to drugs and reduce drug resistance in the process of treating liver cancer.

The quinoline derivative of N-isostere tectoridin is applied to the preparation of medicines or preparations for improving the sensitivity of liver cancer to medicines, reducing the drug resistance of the liver cancer and improving the immunity of organisms in the liver cancer treatment process.

The reagent drugs used in the examples were purchased from conventional biochemical reagents, unless otherwise specified; the New compound studied was provided by professor zhahui, university of beijing.

The New compound New Tecotorigenin researched by the invention is an N-isostere Tectorigenin derivative synthesized by taking the Tectorigenin structural formula as a basis and optimizing the structure. Tectorigenin, 5,7, 4-trihydroxy-6-methoxyisoflavone, is the main ingredient of iris plants, and is commonly found in rhizoma Belamcandae and iris. It has biological activity and pharmacological action of estrogen-like, antioxidant, liver protecting, anticancer, etc.

In vitro experiments further show that tectorigenin has strong inhibitory action on HL-60 cells (leukemia cells) and has a certain inhibitory action on the proliferation of BGC cells (human gastric adenocarcinoma cells). Animal experiments of Thelen P et al also show that irigenin has an obvious tumor-inhibiting effect, and is found to be a drug for preventing and treating human prostate cancer. The research of Saratikov, A.S.et al, shows that iridofloxacin and other polyhydroxy compounds can inhibit the necrosis of liver cells, prevent the imbalance of intrahepatic fat and protein, normalize the activities of transaminase and glutamyltransferase, prevent the development of hepatic fibrosis, and play roles in protecting liver and restoring liver function.

However, the tectorigenin has low content in the traditional Chinese medicine blackberry lily, and is difficult to separate and purify and difficult to obtain. Therefore, the project group of Nanjing university combines the technical advantages thereof, utilizes the thought of isostere design to carry out total synthesis research on tectorigenin, and designs and synthesizes a series of more than 200N isostere tectorigenin derivatives based on the research.

In the series of Tectorigenin derivatives, New Tecotorigenin has the strongest activity and simultaneously has good safety and stability. However, New tecorigenin as an Tectorigenin derivative has not been reported in the aspects of inhibiting the proliferation of liver cancer cells, protecting the liver and functioning. Therefore, the research aims to research the anti-liver cancer activity of the New Tectrorigenin and the anti-liver cancer molecular action target thereof through experiments.

Test 1

Inhibition effect of New tecorigenin on mouse liver cancer model

1.1 mouse liver cancer model establishment

1.1.1 Diethylnitrosamine (DEN) compound induction method

60 healthy female mice were randomly divided into normal control group, DEN group, New tectorigenin and DEN feeding group, every 20 mice. The normal control group was given normal feed, the DEN group and the New tectorigenin and DEN feeding group, gavaged with 0.25% DEN aqueous solution at 10mg/kg body weight once a week, and the rest time 0.025% DEN aqueous solution was freely drunk therefrom, while the Newtectorigenin and DEN feeding group was fed with New tectorigenin (100mg/kg) for about 4 months.

1.1.2 transplantation method

70 healthy Kunming mice are randomly divided into a control group, a New tectorigenin group, a model group, a positive control group and a high, medium and low dose N e w te c t o r ig e N feeding model group, wherein each group comprises 10 mice and each half of the mice are male and female. Except for the control group and the New tectorigenin group, 2X 10 subcutaneous injections were injected into the axilla of the right anterior limb of each mouse⁷0.2mL of HepG2 cell suspension. Beginning on the next day of inoculation, the New tecorigenin group, the high, medium and low dose feeding New tecorigenin group are subjected to intragastric administration for 1 time every day, and the administration is continuously carried out for 10 days; the positive drug group is administrated by intragastric administration for 1 time each day; the control and model groups were gavaged with equal volumes of 0.9% sodium chloride solution.

Weighing the mass of the mouse body before administration and the day after the last administration, cutting off liver cancer tissues of each group, weighing the mass of tumor mass tissues, and researching the influence on the mass of the mouse body and the influence on the tumor mass by calculating the tumor inhibition rate.

1.2 frozen sections:

the mice were sacrificed to take their livers, and the livers were calculated by observation under a dissecting microscope and photographed, and then frozen sectioned. Fixing liver tissue in fixative, washing with water, and cleaning, and preferably fixing with formaldehyde. The tissue is washed with water, immersed in a 12.5% gelatin aqueous solution (prepared with a 1% carbolic acid aqueous solution), and immersed in an incubator at 37 ℃ for 6-24 hours. Transferring to 25% gelatin solution, and soaking for 6-24 hr. Embedding with 25% gelatin, placing into refrigerator together with the embedding device to rapidly solidify gelatin, taking out, and evaporating in air for 10 min. Placing into tissue fixing solution for fixing gelatin block for 1-2 days. Washing with distilled water, and slicing with a freezing slicer or a sliding slicer. The sections were stored in 10% formalin. Staining with different reagents, without ethanol dehydration and xylene clearing after staining, and blocking with gelatin solution or other similar gelatin solutions: 26g of fructose, 1.1g of gelatin, 0.75g of potassium alum and 1.15ml of thymol aqueous solution.

Test 2

Inhibition of New tectorigenin on HepG2 cells

2.1 resuscitating HepG2 cells

Taking out the frozen HepG2 cells from a liquid nitrogen tank, quickly placing the cells in a constant-temperature water bath kettle at 38 ℃, quickly shaking, transferring cell suspension in a super-clean workbench after the cells are ablated, centrifuging for 5min at 800rpm and 25 ℃, collecting cell precipitates, adding a proper amount of culture solution (using low-sugar DMEM culture solution containing 2% 100u/mL double antibody and 10% FBS), blowing, uniformly mixing, transferring to a culture bottle, and placing in an incubator for culture. The culture conditions are as follows: 5% CO₂And saturated humidity at 37 ℃. After 12h of culture, the effect of resuscitating HepG2 cells was observed using an inverted phase contrast microscope, nonadherent floating as dead cells or cell debris, adherent or differentiated as live cells.

2.2New tectorigenin treatment of HepG2 cells

When the cell adhesion of HepG2 is more than 80%, washing with PBS buffer solution for 2 times, hydrolyzing with 0.25% Trypsin for 3 mlin, and finally hydrolyzing with about 1.0 × 10⁵The density of each m L was divided into six-well plates, the experimental groups were supplemented with different doses of N ew Tetorigenin (to make the final concentrations 2. mu. mol/L, 4. mu. mol/L, 8. mu. mol/L, 12. mu. mol/L, 16. mu. mol/L, respectively), the control groups were replaced with the same amount of DMEM medium, and the cells were cultured in an incubator for 24h, and the growth of HepG2 cells was observed under an inverted microscope and recorded by photography. The experiment was repeated three times under the same treatment conditions.

As shown in fig. 1, as the concentration of New Tectorigenin increased, the number and density of cells gradually decreased, and the edges of the cells gradually became less conspicuous, or were stacked into clumps, or were shrunken into circles, or were broken into fragments, as compared to the control group. When the concentration of N e wteltorigenin is 4 mu M, the reduction of the number and density of cells is not obvious; when the concentration of the New tectorigenin is 8 mu M, the number and the density of HepG2 cells are obviously reduced relative to the control group, and cell fragments and cells which shrink into a circular shape begin to appear; when the New tectorigenin concentration reaches 32. mu.M, most HepG2 cells die, leaving only the apoptotic cells that crinkle into circles and cell debris that die by cell lysis. Therefore, experiments preliminarily show that the New tectorigenin can inhibit the proliferation and growth of HepG2 cells.

Test 3

MTT colorimetric method for detecting HepG2 cell activity

In order to further explore the inhibition effect of New Tetorigenin on HepG2 cells, the cell survival rate is detected by an MTT colorimetric method, and the inhibition effect of New Tetorigenin on HepG2 cells can be further shown by specific data.

The specific method comprises the following steps: culturing HepG2 in vitro until the growth logarithmic phase, hydrolyzing with 0.25% Trypsin enzyme to obtain single cell suspension, and separating into 96-well plates with density of 1.0 × 10⁵And (4) culturing the cells per mL in an incubator for 24 h. After the cells are attached to the wall, adding New Tectrorigenin according to the designed dose, setting 5 multiple holes for each concentration, adding the same amount of sterile PBS into a control group, and continuously culturing for 24h and 48 h. After the culture, 20. mu.L of MTT was added to each well, the culture was incubated in an incubator for 4 hours, the stock culture was aspirated, 150. mu.L of DMSO was added to each well, and the mixture was shaken with a micro-shaker for 10min to sufficiently dissolve formazan. The wells were then subjected to dry-zeroing on a microplate reader with a blank control, and the OD values of the wells were measured at 490nm wavelength and the experiment was repeated three times. Calculating the survival rate and the growth inhibition rate of the cells according to the OD value:

equation 1 calculation equation for cell viability

Equation 2 formula for calculating the cell growth inhibition rate

As shown in FIG. 2, after HepG2 cells were treated with different doses of NewTecorgenin for 24h and 48h, the activity of HepG2 cells was significantly reduced, the growth inhibition rate was significantly increased, and the inhibition effect was greater for 48h than 24 h. The N e wTecorgenin has certain cytotoxicity, the growth inhibition rate of the N e wTecorgenin on HepG2 cells is gradually increased along with the increase of the dose of the N e Tecorgenin, and the N e Tecorgenin has a dose effect and a time course effect, and the difference is significant (P < 0.05).

Test of the Effect of 4New Tectrorigenin on Nuclear karyotype

The experimental result shows that the NewTecorgenin can influence the activity of HepG2 cells in a certain concentration range, inhibit the growth of HepG2 cells and trigger the apoptosis of HepG2 cells. In order to further observe the apoptosis characteristics, the change of the HepG2 cell karyotype after the medicine is added is observed by a Hochest33258 fluorescent staining method.

The specific method comprises the following steps: culturing HepG2 in vitro until the growth logarithmic phase, hydrolyzing with 0.25% Trypsin enzyme to obtain single cell suspension, and separating into six-well plates with density of 1.0 × 10⁵And (4) culturing the cells in an incubator. After the cells are attached to the wall, New Tectrorigenin is added according to the designed dosage, and the control group is replaced by the same amount of DMEM culture solution. After 48h incubation in the incubator, the stock culture was aspirated, washed 3 times with PBS, and fixed for 20min by adding 0.5mL of fixative (methanol: ice CH3 COOH: 3:1, used as prepared) to each well. Then, the fixation solution is discarded, the cells are washed for 3 times in 3min by PBS (phosphate buffer solution), 0.5mL of Hochest33258 staining solution is added into each well in the dark, the cells are incubated for 15min at room temperature, the staining solution is absorbed, the cells are washed for 3 times in 3min by PBS, the cells are air-dried in the dark, and then the cells are subjected to color development observation by a fluorescence microscope and photographed and recorded.

As a result, as shown in FIG. 3, the nuclei of the blank control group without NewTecortigenin treatment exhibited diffuse and uniform fluorescence, indicating that most of them were living cells. The cell nucleus of the HepG2 cell treated by the NewTecorgenin for 48 hours in the experimental group shows densely-stained and compact fluorescence, the nucleolus is cracked and is in a particle block shape, and the apoptotic bodies appear. With the increase of the concentration of the NewTecorgenin, the apoptosis characteristics of HepG2 cells are more obvious, and a certain dosage effect is achieved.

Test 5 Effect of New Tectrorigenin on cell Mitochondrial Membrane Potential (MMP)

The above experiment shows the influence of New Tecorigigenin on H e p G2 cell nucleus, and in order to further explore the influence of New Tecorigigenin on mitochondrial membrane potential, the group performed rhodamine 123 staining test.

The specific method comprises the following steps: treating HepG2 cells according to the dosing method, culturing for 24h, carefully sucking out original culture solution, washing for 1 time by PBS, adding 1mL of rhodamine 123 working dye solution into each hole, and placing in CO₂Incubate in incubator for 30 min. Then the staining solution was aspirated, the cells in the wells were washed 2 times with normal low-sugar medium, the morphology and distribution of mitochondria were observed under a fluorescence microscope, and the fluorescence intensities of each experimental group and the control group were compared. The rhodamine 123 staining solution can enable mitochondria in living cells to present green fluorescence and is in a short rod shape or a granular shape.

As shown in fig. 4, when HepG2 cells were treated with different concentrations of newtecorigenin, the degree of fluorescence in the cells was reduced compared to the control group, and the greater the concentration, the weaker the fluorescence, and the concentration effect was exhibited. The results suggest that newtectorigen leads to increased permeability of the mitochondrial membrane, leading to decreased MMP.

Test the Effect of 6New Tectrorigenin on the subcellular localization of Calnexin

To observe the effect of newtecorigenin on the endoplasmic reticulum of HepG2 cells, cells were stained with Calnexin antibody.

The specific method comprises the following steps: HepG2 cells were treated by the above dosing method, cultured for 24h, aspirated away the culture medium in the six-well plate, carefully added with the now-prepared fixative covering the surface of the wells, and fixed for 40 min. After washing 2 times with PBS once for 5min, 1ml of 5% BSA was added to each well and the cells were blocked slowly on a shaker. After 1h the well was aspirated, primary anti-diluent (containing Calnexin) was added, incubated on a shaker for 1h at slow speed and then placed in a refrigerator at 4 ℃ overnight. Sucking away liquid in the holes next day, washing for 3 times at 5min by PBS, adding rabbitit anti-mouse secondary antibody diluent, continuously incubating for 1h on a shaking table, washing for 3 times at 5min by PBS after finishing, adding DAB color development working solution, developing for about 3min, re-dyeing for 30S by hematoxylin dye solution, dehydrating, air drying, sealing, and observing the dyeing condition of endoplasmic reticulum under a microscope.

As shown in fig. 5, the staining of the experimental group after the treatment of newtectorgenin was lighter than that of the control group, and the color gradually became lighter with increasing concentration of newtectorgenin, which has a concentration effect. This indicates that the newly synthesized protein has reduced binding to Calnexin and misfolded or unassembled protein subunits are retained within the endoplasmic reticulum, resulting in a lighter color. Newtectorgenin causes excessive endoplasmic reticulum stress, thereby inducing apoptosis of HEPG2 cells.

Test 7 Effect of New Tectrorigenin on ROS in HepG2 cells

ROS can produce oxidative effects on cell membrane structures, nucleic acid and protein molecules, causing their structural and functional disorders. Increased ROS causes Oxidative Stress (Oxidative Stress) in the body to cause cell damage, which is involved in a variety of pathological mechanisms. To determine the effect of New tecorigenin on intracellular ROS content, reactive oxygen species detection was performed using the fluorescent probe DCFH-DA.

The specific method comprises the following steps: culturing HepG2 cells, inoculating in 6-well plate at 3-4 × 10 density⁵The cells are subjected to debridement for 12 hours after being attached to the wall; and (3) changing the culture medium containing the serum to continue culturing for 3-6h, and then adding New Tectrorigenin for treatment. Collecting cells after 24h treatment of Newtecorigenin, adding a culture medium containing serum to terminate digestion, centrifuging to remove supernatant, and adding 400ul PBS to resuspend the cells; ultrasonic cracking for 40-60w, each time for 3-4 times, total 3 times (operation on ice; centrifugation at 4 degree 12000 r, 15 min.) adding reagent of kit (dichlorofluorescein active oxygen detection kit), and determining od value of each group with enzyme labeling instrument according to the method of the instruction.

From the staining results of fig. 6, it can be seen that the drug-administered group after the New Tectorigenin treatment stained more deeply than the control group, and the color gradually deepened and the area was larger with the increase of the New Tectorigenin concentration, which has a concentration effect.

Experiment 8Westernblot to detect the influence of New Tectrorigenin on PI3K, Akt, Caspase-3, p53, Bax and Bcl-2 protein expression

The experimental results show that NewTecorgenin has obvious inhibition effect on the growth of HepG2 cells, and in order to explore the action mechanism of apoptosis, the inventor detects the expression of PI3K, Akt, Caspase-3, p53, Bax and Bcl-2 proteins in HepG2 cells after administration.

The specific method comprises the following steps: HepG2 cells were treated accordingly as described above and cultured for 48 h. The procedure was performed on ice, the stock culture in the six well plates was discarded, washed 3 times with pre-cooled PBS, 80. mu.L of lysis buffer (lysbuffer) was added to each well to lyse the cells, adherent cells were scraped off in the same direction with a cell scraper, the cells were collected separately in EP tubes and lysed on a shaker for at least 30min on ice. Then, the mixture was centrifuged at 13200rpm at 4 ℃ for 10min, and the supernatant was collected. And (3) measuring the concentration of each group of proteins by a Coomassie brilliant blue G-250 method, measuring the absorbance at 595nm by a visible spectrophotometer, drawing a protein standard curve, respectively calculating the concentration of each group of proteins, and calculating the volume of the protein to be added according to the protein concentration and the protein amount to be added.

The determined proteins were loaded in equal amounts and subjected to polyacrylamide gel electrophoresis (SDS-PAGE). After electrophoresis, the proteins were transferred to nitrocellulose membranes (PVDF). Sealing with 5% skimmed milk powder on shaking table for 1 hr. Primary antibodies such as PI3K, Akt, ERK, p-ERK (1:1000), etc. were incubated on ice for 1h with shaking and placed in a refrigerator at 4 ℃ overnight. Washing the membrane with TBS × 1 for 3 times, each time for 5min, slowly incubating with a coat anti-rabbit (1:1000) secondary antibody in a shaking table at room temperature for 1h, washing the membrane with TBS × 1 for 5 times, each time for 5min, performing ECL detection, developing, fixing, scanning and observing.

As shown in FIG. 7, first, the increased expression of p53 induced the expression of Caspase-3, which is an apoptosis factor in HepG2 cells, to induce programmed cell death. Meanwhile, the expression level of a pro-apoptotic protein Bax in a mitochondrial apoptotic pathway is increased, and the expression level of an anti-apoptotic protein Bcl-2 is reduced, so that the combined actions result in the apoptosis of HepG2 cells. Secondly, as shown in fig. 8, newtectorgenin has inhibitory effect on PI3K, Akt protein expression and concentration effect. Activated AKT can directly phosphorylate precursor apoptotic proteins and produce short-term effects to prevent activation of apoptotic pathways leading to cell death. PI3K and AKT expression is reduced, and HepG2 cell proliferation is inhibited.

Experiment 9Western blot for detecting influence of New Tectrorigenin on ERK1/2 protein expression

To further explore the mechanism by which newtectorigen inhibits HepG2 cell proliferation, the inventors also examined ERK and pERK protein expression in HepG2 cells following administration.

The specific experimental method is as described in experiment 8.

The experimental result is shown in FIG. 9, the NewTecoriginin has inhibition effect on ERK protein expression and concentration effect, and the Ras/Raf/MEK/ERK signal transduction pathway plays a crucial role in the physiological processes of cells, such as proliferation, survival, differentiation, apoptosis, movement and metabolism. The expression of ERK is reduced, the expression of related downstream proteins is also reduced, and the growth and proliferation of HepG2 cells are inhibited.

Example 5 inhibition of New Tectrorigenin against H22 liver cancer transplanted tumors

(1) Ectopic transplantation

First, H22 cell recovery

Taking out the freezing tube filled with H22 cells from liquid nitrogen, shaking rapidly in water bath at 37 deg.C, immediately transferring the melted freezing solution into a centrifuge tube in sterile environment when ice in the freezing tube is almost completely melted, centrifuging at 1000rpm and 25 deg.C for 3min, discarding supernatant, blowing slightly with new culture solution to disperse cells, transferring into a culture bottle and supplementing culture solution to 3ml, and adding 5% CO at 37 deg.C₂Culturing in an incubator with saturated humidity.

II, establishing a model of H22 subcutaneous transplanted tumor

When H22 cells grow to log phase, H22 cells are centrifuged at 1000rpm and 25 ℃ for 3min, the culture solution is discarded, PBS is washed three times, and the cells are diluted to 1x10 by PBS⁶。

Intraperitoneal injection of 0.2ml 1x10 into 3 nude mice⁶H22 cells, and feeding for about 7 days under the following feeding conditions: the basic feed is freely taken and eaten, the air humidity is 50 +/-5%, and the temperature is 20-25 ℃.

After 7 days, the mice were aseptically washed with opalescent ascites in PBS three times, diluted to 1X10⁷。

About 0.2mL of sterile H22 liver cancer cell suspension (containing 2.0X 10) is injected subcutaneously into the right forelimb of each nude mouse⁶Individual cells), measuring the longest diameter and the shortest diameter of the tumor body by using a ruler every 2 days after inoculation, calculating the tumor volume,

tumor length (L) and width (W)

V＝1/2*LW²

After 4 days, the tumor volume is increased to about 0.2cm³Post-randomizationThe test samples were divided into 3 groups, namely a model group, a low concentration group (7mg/kg), and a high concentration group (14 mg/kg). The model group was gavaged daily with 0.2ml of physiological saline, low concentration with 0.2ml of New Tecorgenin (7 mg/ml), low concentration with 0.2ml of New Tecorgenin (14 mg/ml). The treatment was continued for 14 days.

Nude mice were sacrificed on day 2 after the last dose. Separating tumor mass, and calculating the tumor inhibition rate (%) (control group tumor mass-experimental group tumor mass)/control group tumor mass x 100%.

Tumor tissues and organs were collected after the nude mice were sacrificed.

Third, H & E staining of tumor tissue

The tumor tissue is taken down and immediately soaked in 10 percent formaldehyde solution, the tumor tissue is placed at 4 ℃ for fixing for 24 hours, tissue blocks with the size of about 1cm multiplied by 0.5cm are taken, and the tissue blocks are dehydrated, transparent, waxed and embedded by paraffin wax. Placing on a microtome for continuous slicing, wherein the thickness of the slices is 5 μm, spreading on a glass slide in warm water, and storing at room temperature for later use. After 7 minutes of hematoxylin staining solution staining, the excess staining solution was slowly rinsed with long running tap water until the effluent water was colorless (about 6 minutes), slightly dried, stained with eosin staining solution for 1 minute, and subjected to conventional dehydration, transparent, neutral gum sealing. The pathomorphology observation was performed under an optical microscope and photographed.

Fourthly, immunohistochemical determination of expression levels of Arg-1 (arginase-1), AFP (alpha fetoprotein), CA125 (glycoprotein) and GPC3 (phosphatidylinositol proteoglycan 3) in tumor tissues

The paraffin-embedded tumor tissues obtained in the above experiment were taken, sectioned conventionally, dewaxed and dehydrated conventionally, and then treated with 3% H₂O₂Sealing at room temperature for 10min, washing with PBS 3 times; sucking water, and sealing with goat serum sealing liquid at room temperature for 20 min; dropping primary antibody to cover the tissue, and standing at 4 deg.C overnight; the next day, the mixture was left to stand at room temperature and washed 3 times with PBS; adding appropriate amount of streptavidin-peroxidase solution dropwise, incubating at room temperature for 1h, and washing with PBS 3 times; adding DAB dropwise for color development until dyeing is proper, washing with PBS for 3 times, counterstaining with lignum sappan, differentiating with 1% hydrochloric acid alcohol, dehydrating, sealing with neutral gum, and taking pictures under microscope.

Fifth, ELISA method for measuring blood CEA content

Strictly according to the kit instructions. The collected blood was centrifuged at 3000 rpm for 30 minutes, and the supernatant was collected. The standard sample is added according to 50 mu L of each hole, the sample hole is 10 mu L of the sample to be detected, the sample diluent is added according to 40 mu L, and the blank hole is not added. Add 100. mu.L of the detection antibody solution to the standard wells and sample wells, seal with a sealing membrane, and incubate for 60min in a 37 ℃ incubator. Removing all liquid, drying on absorbent paper, adding 1X washing solution into each hole, standing for 1min, removing washing solution, drying on absorbent paper, and repeating for 5 times. 50. mu.L of substrate A, B was added to each well and incubated at 37 ℃ for 15min in the absence of light. 50. mu.L of a reaction terminator was added to each well, and the OD value of each well was measured at a wavelength of 450 nm. Drawing a standard curve: and (4) drawing a linear regression curve by taking the concentration as an abscissa and the OD value as an ordinate, and calculating the concentration value of each sample according to a curve equation.

(2) In situ transplantation

Performing abdominal anesthesia on mice with 5% of hydralic aldehyde 0.1ml/10g, checking whether the mice stop moving after 2-3min, but breathing, taking supine position and fixing limbs on the experimental plate, and keeping the ratio of 10% after anesthesia is completedRemoving body hair with sodium sulfide, sterilizing with 73% ethanol, opening abdomen layer by layer along the white line of abdomen, exposing abdominal cavity of mouse, slightly pressing thoracic cavity of mouse, making liver jump out of abdominal cavity, and selecting liver leaf closest to body surface for planting tumor. The injection needle head is obliquely inserted into the needle, the needle point forms 20 degrees with the horizontal plane, the needle head penetrates into the liver by about 1cm, the needle core of the injector is slightly pushed, and 0.05mL of cell suspension is slowly injected, about 10⁶And H22 cells. Slowly withdrawing the needle, lightly burning the needle hole immediately by a red iron wire, lightly delivering the needle to the liver and returning the needle to the abdominal cavity, and closing the abdominal cavity layer by layer.

(3) The experimental results are as follows:

first, H22 tumor size change in tumor-bearing mice

As shown in fig. 10, the tumor size was visually observed on day 7, and the tumor volume growth rate increased from day 11 and the model tumor volume increased greatly from day 17 in all groups, but the tumor volume increase was more gradual in the low-concentration and high-concentration groups than in the model group, and the tumor size increased gradually from day 21 to day 26 as shown in fig. 11. At the end of the experiment, the tumor size is shown in fig. 12, the mean tumor volume of the low-concentration and high-concentration groups is significantly different compared with the model group (p <0.05), and the high-concentration effect is better than the low-concentration effect. Therefore, the New Tectrorigenin is preliminarily shown to have a remarkable inhibiting effect on H22 tumor-bearing mouse tumors.

Secondly, the influence of New Tectrorigenin on visceral index and tumor inhibition rate of H22 tumor-bearing mice

As can be seen from table 1, after the experiment is finished, the spleen index of the model group mouse is significantly increased (p <0.05) compared with that of the blank group mouse, while the spleen index of the low-concentration and high-concentration New Tectorigenin is significantly decreased (p <0.05), and the spleen index of the low-concentration and high-concentration New Tectorigenin is closer to that of the blank group. Compared with the model group, the liver indexes of the normal group, the low-concentration group and the high-concentration group of New Tectrorigenin have no obvious difference, which indicates that the New Tectrorigenin has no toxic or side effect on the liver. In addition, the tumor mass of the mice with low and high concentration New Tectrorigin group is obviously reduced (p <0.05) compared with that of the model group, and the tumor mass is in a concentration-dependent effect. The results show that the New Tectrorigenin not only can obviously inhibit the growth of mouse tumors, but also has a certain protective effect on immune organs.

TABLE 1 influence of New Tectrorigenin on organ index and tumor inhibition rate of H22 mice

Note: x ± s, n ═ 4, compare to model group, P < 0.05.

Thirdly, the change of the CEA content of the H22 tumor-bearing mice

CEA (carcinoemergencigenic antigen), a broad-spectrum tumor marker, which reflects the existence of multiple tumors. The experimental results show (table 2) that the CEA content of the model group, the low-concentration group and the high-concentration group is significantly increased relative to the blank group (p <0.05), the CEA content of the low-concentration group and the high-concentration group is significantly reduced relative to the model group (p <0.05), and it is proved that New Tectorigenin can inhibit the growth of the tumor of the H22 tumor-bearing mouse by reducing the CEA generation, and the reduction amplitude of the high-concentration group is larger than that of the low-concentration group (p <0.05) and is in a concentration-dependent effect.

TABLE 2 influence of New Tectrorigenin on CEA content in H22 mice

Note: x ± s, n ═ 4, compare to model group, P < 0.05.

H & E staining of tumor tissue of tumor-bearing mouse of H22

As can be seen from fig. 13, the tumor cells in the model group are arranged regularly, the gaps between the cells are small, the nuclei with normal cell morphology are clear and complete, and the New capillaries in the intercellular spaces are abundant, while the cells in the New tectorigen low-concentration group are sparse, have different sizes, are damaged, and have the cell membrane with the shrunken nuclei and the typical apoptosis characteristics, the area of the apoptotic cells in the New tectorigen high-concentration group is increased, the tumor tissue is looser, and the number of capillaries is reduced. The result shows that the New Tectroriginin has obvious effects of inhibiting the growth of tumor blood vessels and inducing tumor apoptosis, and the inhibition effect of high-concentration is obviously different from that of low-concentration, thus showing a certain dose-dependent effect.

Fifth, immunohistochemical determination of the expression level of Arg-1, AFP, CA125 and GPC3 in tumor tissue of H22 tumor-bearing mice

The expression of a liver cancer marker Arg-1 in tumor tissues of various groups of H22 tumor-bearing mice is further detected by an immunohistochemical method. SABC (strept avidin-biotin complex), which is one of the immunohistochemical staining methods, shows a brownish yellow substance in the cells. As shown in FIG. 14, Arg-1 was expressed in both cytoplasm and nucleus, the model group had fewer tan regions, fewer positive cells, lower Arg-1 expression, and higher Arg-1 expression in both low and high concentration groups of NewTecortignin. The areas of the model group positive regions of AFP, CA125 and GPC3 are higher than that of New Tectrorigenin at low concentration and high concentration, and the high concentration is lower than that of the low concentration positive regions, so that the model group positive regions have certain dosage effect. The results show that New Tectrorigenin can increase the expression of Arg-1 in tumor cells and reduce the expression of AFP, CA125 and GPC 3.

The liver cancer solid animal model is an important way for discussing the drug effect and development of the pre-clinical anti-liver cancer drug, is efficient and accurate, and can promote the transformation of the laboratory achievements. However, the New Tecotorigenin serving as a New anti-liver cancer drug has not been discussed to play a role in inhibiting a solid animal liver cancer model.

In this example, H22 cells were cultured at 1X10⁶Injecting the cells/ml, 0.2ml into abdominal cavity of nude mouse, collecting ascites of nude mouse 7 days later, washing with PBS 3 times, diluting with PBS to 1 × 10⁷The cells/ml are injected into the subcutaneous part of the right forelimb of a nude mouse in an amount of 0.2ml, when the nude mouse has a tumor visible with naked eyes on the right forelimb, the mice with the same tumor growth vigor and size are randomly divided into 3 groups, namely a model group, a low-concentration group (7mg/kg) and a high-concentration group (14mg/kg), the model group is perfused with 0.2ml of physiological saline every day, the low-concentration group is perfused with 0.2ml of 7mg/ml New Tecorgenin, and the low-concentration group is perfused with 0.2ml of 14mg/ml New Tecorgenin. After 14 days of intragastric administration by the above method, the patient is killed by cervical dislocation, the tumor is removed, the patient is preserved in 10% formaldehyde solution, and paraffin section is performed within 2 days. Then H is carried out&E staining and tissue exemptionStaining and microscopic observation.

Analysis of the sizes of the tumors in each group revealed that the size of the mouse liver cancer tumor decreased with the increase of the concentration of New Tectorigenin. H & E staining found an increase in the area of apoptotic cells with increasing New Tectorigenin concentration. And the blood CEA detection finds that New Tecotorigenin can reduce the generation of CEA. Immunohistological staining revealed that NewTecorigigenin increased Arg-1 expression in tumor cells and decreased AFP, CA125, GPC3 expression. Therefore, the NewTecorgenin can obviously inhibit the proliferation of the H22 liver cancer transplantation tumor, and has important significance for the development of anti-liver cancer drugs.

Example 6 inhibition of New Tectrorigenin on Hepa1-6 liver cancer transplantation tumor

(1) Ectopic transplantation

First, Hepa1-6 cell recovery

Taking out the tube filled with Hepa1-6 cells from liquid nitrogen, shaking rapidly in water bath at 37 deg.C, immediately transferring the thawed frozen solution into a centrifuge tube in sterile environment when ice in the tube is almost completely thawed, centrifuging at 25 deg.C at 1000rpm for 3min, discarding supernatant, blowing with newly dispensed culture solution to disperse cells, transferring into a culture bottle and supplementing culture solution to 3ml, and adding 5% CO at 37 deg.C₂Culturing in an incubator with saturated humidity.

Second, establishing model of Hepa1-6 subcutaneous transplantation tumor

When the Hepa1-6 cells grow to logarithmic phase, centrifuging Hepa1-6 cells at 1000rpm at 25 deg.C for 3min, discarding the culture solution, washing with PBS three times, diluting with PBS to 1X10⁶。

Intraperitoneal injection of 0.2ml 1x10 into 3 nude mice⁶Hepa1-6 cells were fed for about 7 days under the following conditions: the basic feed is freely taken and eaten, the air humidity is 50 +/-5%, and the temperature is 20-25 ℃.

About 0.2mL of sterile Hepa1-6 liver cancer cell suspension (containing 2.0X 10 liver cancer cells) is injected subcutaneously into the right forelimb of each nude mouse⁶Individual cells), measuring the longest diameter and the shortest diameter of the tumor body by using a ruler every 2 days after inoculation, calculating the tumor volume,

tumor length (L) and width (W)

V＝1/2*LW²

After 4 days, the tumor volume is increased to about 0.2cm³The samples were then randomly divided into 3 groups, i.e., a model group, a low concentration group (7mg/kg), and a high concentration group (14 mg/kg). The model group was gavaged daily with 0.2ml of physiological saline, low concentration with 0.2ml of New Tecorgenin (7 mg/ml), low concentration with 0.2ml of New Tecorgenin (14 mg/ml). The treatment was continued for 14 days.

Tumor tissues and organs were collected after the nude mice were sacrificed.

Third, H & E staining of tumor tissue

The paraffin-embedded tumor tissues obtained in the above experiment were taken, sectioned conventionally, dewaxed and dehydrated conventionally, and then treated with 3% H₂O₂Sealing at room temperature for 10min, washing with PBS 3 times; sucking water, and sealing with goat serum sealing liquid at room temperature for 20 min; dropping primary antibody to cover the tissue, and standing at 4 deg.C overnight; the next day, the mixture was left to stand at room temperature and washed 3 times with PBS; adding appropriate amount of streptavidin-peroxidase solution dropwise, incubating at room temperature for 1h, and washing with PBS 3 times;adding DAB dropwise for color development until dyeing is proper, washing with PBS for 3 times, counterstaining with lignum sappan, differentiating with 1% hydrochloric acid alcohol, dehydrating, sealing with neutral gum, and taking pictures under microscope.

Fifth, ELISA method for measuring blood CEA content

(2) In situ transplantation

Carrying out abdominal anesthesia on a mouse by using 0.1ml/10g of 5% hydralic aldehyde, checking whether the movement of the mouse stops or not after 2-3min, but breathing, taking a supine position and fixing four limbs on an experimental board after complete anesthesia, removing body hair by using 10% sodium sulfide, disinfecting by using 73% ethanol, opening the abdomen layer by layer along a white abdominal line, exposing the abdominal cavity of the mouse, slightly pressing the chest cavity of the mouse, jumping out of the abdominal cavity by using the liver, and selecting the liver lobe closest to the body surface to plant the tumor. The injection needle head is obliquely inserted into the needle, the needle point forms 20 degrees with the horizontal plane, the needle head penetrates into the liver by about 1cm, the needle core of the injector is slightly pushed, and 0.05mL of cell suspension is slowly injected, about 10⁶And each Hepa1-6 cells. Slowly withdrawing the needle, lightly burning the needle hole immediately by a red iron wire, lightly delivering the needle to the liver and returning the needle to the abdominal cavity, and closing the abdominal cavity layer by layer.

(3) The experimental results are as follows:

first, Hepa1-6 tumor-bearing mouse model

The experimental results are shown in fig. 15 and 16, and at the end of the experiment, the tumor volumes of the low concentration and the high concentration of the New Tectorigenin are smaller than those of the model group (p < 0.05). Therefore, the New Tectroriginin is preliminarily shown to have the inhibiting effect on the tumor of a mouse with the Hepa1-6 tumor.

Second, the tumor inhibition rate of New Tecotorigenin on Hepa1-6 tumor-bearing mice

As can be seen from Table 3 and FIG. 17, the tumor mass of the low-concentration group and the high-concentration group of New Tecoriginin is obviously lighter than that of the model group (p <0.05), and the tumor inhibition rate reaches 43.97% and 70.07%, so that the New Tecoriginin has statistical significance. Therefore, the NewTecorgenin can obviously inhibit the growth of the tumor of a mouse with Hepa1-6 tumor.

TABLE 3 influence of New Tecorgenin on the tumor suppression rate of Hepa1-6 mice

Note: x ± s, n ═ 4, compare to model group, P < 0.05.

Thirdly, the change of the CEA content of the mouse with the Hepa1-6 tumor

As shown in table 4, the CEA content in the model group was significantly increased compared to the blank group (p <0.05), the CEA content in the New tecoriginin low-concentration group was lower than that in the high-concentration group, the CEA content in the low-concentration group was not significantly different from that in the model group, but the CEA content in the high-concentration group was significantly different from that in the model group (p < 0.05). This indicates that New Tecotorigenin can also inhibit the growth of tumor in Hepa1-6 tumor-bearing mice by reducing the production of CEA.

TABLE 4 Effect of New Tectrorigenin on CEA content in Hepa1-6 mice

Note: x ± s, n ═ 4, compare to model group, P < 0.05.

H & E staining of tumor tissue of mouse with tumor of tetrapa 1-6

As shown in FIG. 18, the tumor tissue cells in the model group were intact, and the nuclei were clearly visible and closely arranged. While the low-concentration group and the high-concentration group of NewTecorgenin can see a certain necrotic area, cells in the necrotic area are loose, and staining is light. This shows that New Tecotorigenin has certain inhibiting effect on tumor tissue cells of mouse with Hepa1-6 tumor.

Fifthly, immunohistochemical detection of expression levels of tumor tissues Arg-1, AFP, CA125 and GPC3 of Hepa1-6 tumor-bearing mice

As can be seen from FIG. 19, the yellowish brown positive area of the Arg-1 model group was smaller than that of the New Tecorgenin low concentration group and the high concentration group, while the high concentration positive area was significantly larger than that of the low concentration group, and had a certain dose-dependent effect. The positive area of the CA125 model group is larger than that of the low concentration and the high concentration, and more positive cells have obvious difference. But the low concentration and the high concentration have no obvious difference. The AFP and GPC3 model group showed a significant increase in positive cells compared to the low concentration, high concentration group. Therefore, the New Tectroriginin can up-regulate the expression of Arg-1 and inhibit the expression of AFP, CA125 and GPC 3.

This embodiment is generally applicableHepa1-6 cells were cultured at 1X10⁶Injecting the cells/ml, 0.2ml into abdominal cavity of nude mouse, collecting ascites of nude mouse 7 days later, washing with PBS 3 times, diluting with PBS to 1 × 10⁷The cells/ml are injected into the subcutaneous part of the right forelimb of a nude mouse in an amount of 0.2ml, when the naked-eye tumor grows on the right forelimb of the nude mouse, the nude mouse with the same tumor growth vigor and size is randomly divided into 3 groups, namely a model group, a low-concentration group (7mg/kg) and a high-concentration group (14mg/kg), wherein the model group is perfused with 0.2ml of physiological saline every day, the low-concentration group is perfused with 0.2ml of 7mg/ml New Tetorigenin, and the low-concentration group is perfused with 0.2ml of 14mg/ml New Tetorigenin. After 14 days of intragastric administration by the above method, the patient is killed by cervical dislocation, the tumor is removed, the patient is preserved in 10% formaldehyde solution, and paraffin section is performed within 2 days. Then H is carried out&E staining and tissue immunostaining microscopic observation.

Analysis of the sizes of the tumors in each group revealed that the size of the mouse liver cancer tumor decreased with the increase of the concentration of New Tectorigenin. H & E staining found an increase in the area of apoptotic cells with increasing New Tectorigenin concentration. And the blood CEA detection finds that New Tecotorigenin can reduce the generation of CEA. Immunohistological staining revealed that NewTecorigigenin increased Arg-1 expression in tumor cells and decreased AFP, CA125, GPC3 expression. Therefore, the NewTecorgenin can obviously inhibit the proliferation of the Hepa1-6 liver cancer transplantation tumor, and has important significance for the development of anti-liver cancer drugs.

The foregoing shows and describes the general principles, essential features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the foregoing description only for the purpose of illustrating the principles of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the invention as defined by the appended claims, specification, and equivalents thereof.

Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.

Claims

1. An application of N-isostere tectoridin quinoline derivative in preparing anti-liver cancer medicine; the quinoline derivative of the N-isostere tectoridin has a chemical structural formula as follows:

。

2. use according to claim 1, characterized in that: the quinoline derivative of the N-isosterin can inhibit the proliferation of the liver cancer cells by inhibiting RAF/MEK/ERK pathway and JAK/STAT pathway; the quinoline derivative of the N-isosterin promotes the apoptosis of the liver cancer cells by inhibiting a P13K/AKT/mTOR pathway; the quinoline derivative of the N-isostere tectoridin promotes the apoptosis of the liver cancer cell by up-regulating an anti-apoptosis signal P53 and an anti-apoptosis signal PTEN gene.

3. Use according to claim 1, characterized in that: the quinoline derivative of the N-electron isostere tectoridin can improve the generation of ROS by up-regulating the expression level of an anti-apoptosis signal p53, increase the permeability of a mitochondrial membrane and cause the combination of endoplasmic reticulum overstress, thereby inducing the apoptosis of the liver cancer cell.

4. Use according to claim 1, characterized in that: the quinoline derivative of N isosterin can inhibit the proliferation of hepatoma carcinoma cells by down-regulating the expression levels of AKT, ERK and pERK.