CN108685921A

CN108685921A - A kind of application of the quinoline of N isosteres iridin in medicines resistant to liver cancer

Info

Publication number: CN108685921A
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: 2018-10-23
Anticipated expiration: 2038-06-22
Also published as: CN107669676A; CN108685921B

Abstract

The invention discloses a kind of application of quinoline of N isosteres iridin in medicines resistant to liver cancer, a kind of quinoline of N isosteres iridin of the invention, chemical structural formula is:The quinoline New Tectorigenin of N isosteres iridin of the present invention are preparing the application in inhibiting hepatoma Hep G 2 cells drug.The present invention is by inhibiting RAF/MEK/ERK accesses and JAK/STAT accesses to inhibit proliferation of hepatocellular carcinoma HepG 2 cell line.The medicines resistant to liver cancer therapeutic effect of the present invention is notable, is not likely to produce drug resistance and adverse reaction.Meanwhile the invention also discloses the higher preparation methods of yield of the quinoline of N isostere iridins;New Tectorigenin inhibit the significant effect of transplantable tumor.

Description

A kind of quinoline of N isosteres iridin is in medicines resistant to liver cancer Using

Technical field

The present invention relates to Roofirisrhizome flavin non polar isosteres, and in particular to a kind of quinoline derivatives of N isosteres iridin Application of the object in medicines resistant to liver cancer.

Background technology

Primary carcinoma of liver (primary hepatic carcinoma, PHC), which refers to, betides hepatic parenchymal cells or stones in intrahepatic bile duct The cancer of epithelial cell, is the malignant tumour that a kind of grade malignancy is high, progress is fast, poor prognosis, life cycle are short, and survival rate is only 3%~5%.In the world, the morbidity and mortality of primary carcinoma of liver arrange the 7th and the 3rd of all tumours respectively Position.China is the morbidity big country of liver cancer, and incidence accounts for the 45% of the whole world.In recent years, the incidence of China's liver cancer is in rising trend, Case fatality rate also rises therewith, accounts for the third position of the Nattonal Cancer death rate.China's whole nation generaI investigation population in 2010 is 13.4 hundred million people's (platforms Gulf area does not include), according to 2003~2007 years onset of liver cancer rates and mortality level, the annual onset of liver cancer in estimation China 360,000 People, dead 350,000 people.

According to country and Guangdong Province's recent statistics data in 2015, Guangdong is one of liver cancer whole world most hotspot, and The tendency of high risk group rejuvenation is presented.

The cause of disease of primary carcinoma of liver is not fully understood so far, current study show that may be with virus hepatitis, aspergillus flavus The factors such as toxin, hepatic sclerosis, drinking water pollution, heredity, obesity, tobacco and wine are related.Especially coastal area hot climate, humidity, Growth for carcinogenic substance aflatoxins creates condition, increases the risk for suffering from liver cancer.Secondly, some rural residents drink Gou Tangshui Or contaminated water, it can also increase and suffer from liver cancer risk.For northeast, it is a major incentive for leading to liver cancer to drink, long Phase, which drinks, can make liver cell repeated steatosis, necrosis and regeneration, lead to hepatic sclerosis, be eventually converted into liver cancer, and by liver The ratio that hardening is converted to liver cancer is up to 70%.In recent years, the method for clinical treatment liver cancer in addition to traditionally radical excision, Liver transfer operation, radiotherapy chemotherapy etc., also biological therapy and molecular targeted agents cure.In the 1980s, U.S. Olidham is rich Scholar proposes the biological therapy new concept of tumour.It is as after Surgical Resection of Hepatic Carcinoma, radiotherapy, chemotherapy The fourth-largest auxiliary treatment pattern, by adjusting the biologically of body itself, to enhance defence capability of the body to tumour, from And inhibits and (or) delay tumour growth, improve survival rate, reduce recurrence rate.The pathogenesis of known liver cancer is sufficiently complex, Generation, development and transfer and the mutation of several genes, Cell signal propagation pathways and neovascularization resulting exception etc. are closely related, Wherein there is multiple the key links, exactly carry out the theoretical foundation of molecular targeted therapy and potential target spot.

2007, first targeted drug tyrosine kinase inhibitor late succeeded on liver cancer treatment, liver cancer Treatment enters the targeted therapy new era.Therefore, molecular targeted agents treatment liver cancer gradually draws attention, is becoming new and is grinding Study carefully hot spot.In recent years, some molecular targeted agents make some progress in terms of liver cancer treatment, such as:Epidermal growth factor Receptor depressant class:Gefitinib, Tarceva, Lapatinib;More kinase inhibition medicine classes:Sorafenib, Sutent;MEK/ ERK class depressants:A2D6244 etc..However above-mentioned molecular targeted agents respectively also have its limitation.

Currently, it is necessary to set about studying from the angle of China's traditional Chinese medicine, from the civil Chinese medicine for being usually used in treating hepatopathy A kind of suitable large-scale promotion of middle exploitation, to benefit the common people public, that production process is easier to, efficient, Small side effects, calmly To the molecular target novel drugs for the treatment of liver cancer.

The inhereditary material of mouse and people are closely similar, and structure mouse model cost is relatively low, the period is shorter, genetic modification skill Art is easier to realize, therefore mouse is to can be used for human liver cancer to study preferable animal pattern^[1].Structure can the accurate simulation mankind Liver cancer symptom but be easily obtained, economic and practical mouse model not only facilitate deeply probe into liver cancer occur and morbidity molecule Mechanism, it may also be used for the new liver cancer treatment ways and means of detect and assess, and then promote the liver cancer research achievement in laboratory The target being transformed into clinical application^[2]。

Bibliography:

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

[2]Li Shun, Chen Lixiang, Peng Xiuhua, all Jiang Ming, Zhou Xiaohui rat liver cancer model progress;J]Chinese experimental Animal journal, 2016,4,24 (2):213-214.

Invention content

Regarding the issue above, the present invention provides a kind of drug effect is notable, it is not likely to produce a kind of N electronics of drug resistance Application of the quinoline of isostere iridin in medicines resistant to liver cancer.

In order to achieve the above objectives, present invention employs following technical proposals:A kind of N isosteres iridin of the present invention Application of the quinoline in medicines resistant to liver cancer.

Further, the quinoline of the N isosteres iridin, chemical structural formula are:

Further, the quinoline of the N isosteres iridin is by inhibiting RAF/MEK/ERK accesses Inhibit hepatoma cell proliferation with JAK/STAT accesses;The quinoline of the N isosteres iridin is by inhibiting P13K/ AKT/mTOR accesses promote hepatoma cell apoptosis;The quinoline of the N isosteres iridin is by raising anti-cell Apoptotic signal P53 and anti-apoptotic signal PTEN genes are to promote hepatoma cell apoptosis.

Further, the quinoline of the N isosteres iridin is by raising anti-apoptotic signal p53's Expression improves the generation of ROS, and increasing the permeability of mitochondrial membrane stress be combined to induce with causing endoplasmic reticulum excessively Hepatoma cell apoptosis;The quinoline of the N isosteres iridin is by regulating and controlling new vessels paraplasm access The expression of related gene prevents fucosylation.

Further, the expression that the quinoline of the N isosteres iridin passes through downward AKT, ERK, pERK Level, to inhibit the proliferation of liver cancer cells;The quinoline of the N isosteres iridin is during treating liver cancer Improve sensibility, reduction drug resistance of the tumour to drug.

A kind of quinoline of N isosteres iridin of the present invention prepare inhibit RAF/MEK/ERK accesses, JAK/STAT accesses and P13K/AKT/mTOR accesses promote hepatoma cell apoptosis and inhibit the drug of liver cancer or answering in preparation With.

A kind of quinoline of N isosteres iridin of the present invention is preparing downward anti-apoptotic signal P53 With PTEN genes to promote hepatoma cell apoptosis and inhibit the application in the drug or preparation of liver cancer.

A kind of quinoline of N isosteres iridin of the present invention is preparing up-regulation anti-apoptotic signal p53, The expression for lowering AKT, ERK, pERK, improves the generation of ROS, increases the permeability of mitochondrial membrane and causes endoplasmic reticulum excessive It stress be combined to liver cancer apoptosis reducing and the drug for inhibiting liver cancer or the application in preparation.

A kind of quinoline of N isosteres iridin of the present invention is logical in preparation regulation and control new vessels paraplasm The expression of the suppressor gene p53, ERK1/2 genes, AKT genes on road prevents fucosylation and liver cancer is inhibited to invade and shift Application in drug or preparation.

A kind of quinoline of N isosteres iridin of the present invention is preparing the sensitivity for improving liver cancer to drug Property, reduce liver cancer drug resistance drug or the application in preparation.

Advantageous effect:The medicines resistant to liver cancer therapeutic effect of the present invention is notable, is not likely to produce drug resistance and adverse reaction.

On the other hand, the present invention provides the yield highers of the quinoline of above-mentioned N isosteres iridin Preparation method, include the following steps:

(1) 1,3-, bis- bromo- 2- methoxyl groups -5- nitrobenzenes are dissolved in ethyl alcohol, Na is then added₂S₂O₄Aqueous solution, in 50~ It is stirred 3~7 hours at 60 DEG C, is cooled to room temperature, is purified with ethyl acetate, it is washed later, dry, it depressurizes to remove acetic acid second Ester obtains 3,5-, the bis- bromo- 4- aminoanisoles of white powder;

(2) bis- bromo- 4- aminoanisoles of 3,5- are dissolved in CH (OMe)₃In, it stirs to remove excessive CH (OMe)₃ Afterwards, the bis- bromo- 4- methoxyphenyls carboximides of (suitable)-N-3,5- of white powder are obtained;

(3) by (suitable)-N-3, bis- bromo- 4- methoxyphenyls carboximides of 5- are dissolved in anhydrous MeOH, are then added NaOMe is simultaneously stirred until homogeneous, and adds 2- (4- methoxyphenyls) ethyl acetate to remove excessive MeOH, later plus water stirs It mixes to uniform, is filtered under diminished pressure to obtain (anti-) -3- (3,5- bis- bromo- 4- Methoxyphenylaminos) -2- (4- first of white powder Phenyl) ethyl acrylate;

(4) (anti-) -3- (bis- bromo- 4- Methoxyphenylaminos of 3,5-) -2- (4- methoxyphenyls) ethyl acrylate is molten Then H is added in ethyl alcohol in solution₂SO₄, stirred 4-10 hours at 50 DEG C -70 DEG C, be cooled to room temperature, water is added to stir, depressurized Filter obtains bis- bromo- 6- methoxyl groups -3- (4- methoxyphenyls) quinoline -4 (1H) -one of 5,7- of white powder;

(5) bis- bromo- 6- methoxyl groups -3- (4- methoxyphenyls) quinoline -4 (1H) -one of 5,7- is dissolved in DMF and Et₂O Mixture in, PCl is then added dropwise₃, stir 8~16 hours, later plus water is stirred until homogeneous, and gained mixture is used AcOEt is extracted, then washed, dry to remove AcOEt, obtains the chloro- 6- methoxyl groups-of 5,7-, bis- bromo- 4- of white powder 3- (4- methoxyphenyls) quinolone, the i.e. quinoline of the N isostere iridins.

Wherein, in the step (1) ethyl alcohol equivalent preferably 10~20;Na₂S₂O₄The mass concentration of aqueous solution preferably 10~ 30%;Na₂S₂O₄The equivalent of aqueous solution preferably 2~6;It when being purified with ethyl acetate, preferably purifies three times, every time with 2~4 times of bodies Product;Total extract after being purified with ethyl acetate is washed with saturated salt solution and uses MgSO₄It is dry;

In the step (2), CH (OMe)₃Equivalent preferably 10~20, mixing time preferably 6~12 hours;

In the step (3), the equivalent preferably 10~20 of anhydrous MeOH;The equivalent of NaOMe preferably 1~4, mixing time It is preferred that 15~45 minutes;The equivalent preferably 1~3 of 2- (4- methoxyphenyls) ethyl acetate, mixing time preferably 4~10 hours, The preferred equal volume of amount of water later stirs 10~20 minutes;

In the step (4), the equivalent preferably 10~20 of ethyl alcohol;H₂SO₄Equivalent preferably 0.5~1;After being cooled to room temperature Amount of water preferably 5~10 times of volumes stir 30~50 minutes;

In the step (5), PCl is added dropwise₃When preferably 0 DEG C of temperature;The equivalent of DMF preferably 20~40, Et₂O's Equivalent preferably 20~40, preferably 2~6 times of amount of water, AcOEt extraction times are preferably 3, and the dosage extracted every time is 2~4 times of bodies Product, it is preferred to use saturated salt solution washs and uses MgSO₄It is dry.

In yet another aspect, the present invention provides the chloro- 6- methoxyl groups -3- of bis- bromo- 4- of above-mentioned 5,7- (4- methoxyphenyls) Application of the quinolone in preparing the drug for inhibiting transplantable tumor.Wherein, transplantable tumor is preferably mice-transplanted tumor;Transplantable tumor source is excellent It is selected as HEPG2 cells, H22 cells or Hepa1-6 cells.The transplantable tumor can be orthotopic transplantation tumor, can also be that dystopy is moved Plant tumor.

In yet another aspect, the present invention provides a kind of drug inhibiting tumour, it is bromo- to contain 5,7- bis- in the drug 4- chloro- 6- methoxyl groups -3- (4- methoxyphenyls) quinolone.Wherein, the tumour can be primary tumo(u)r, can also be transplanting Tumor, more preferably transplantable tumor;The most preferably transplantable tumor of HEPG2 cells, H22 cells or Hepa1-6 cell origins.More preferably Ground, when tumour is Hepa1-6 or H22 cell transplantation tumors, 5, the 7- bis- bromo- chloro- 6- methoxyl groups -3- of 4- (4- methoxyphenyls) A concentration of 7~14mg/kg of quinolone;When tumour is HEPG2 cell transplantation tumors, 5, the 7- bis- bromo- chloro- 6- methoxyl groups -3- of 4- A concentration of 8~32 μM of (4- methoxyphenyls) quinolone.

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

(1) present invention is by vitro culture HEPG2 cells, and New Tectorigenin are by raising anti-apoptotic signal The expression of p53 improves the generation of ROS, and increasing the permeability of mitochondrial membrane stress be combined with causing endoplasmic reticulum excessively, and Induce HepG2 Apoptosis.Also by lowering the expression of AKT, ERK, pERK, inhibit the proliferation of HepG2 cells. NewTectorigenin can be used as a kind of medicines resistant to liver cancer and apply to prevent the diseases such as primary carcinoma of liver;

(2) preparation method of the quinoline of N isosteres iridin using the present invention synthesizes New Tectorigenin, yield significantly improve, and the yield of New tectorigenin can be improved 10%.

Description of the drawings

In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below There is attached drawing needed in technology description to be briefly described, it should be apparent that, the accompanying drawings in the following description is only this Some embodiments of invention without having to pay creative labor, may be used also for those of ordinary skill in the art With obtain other attached drawings according to these attached drawings.

Fig. 1 is the New tectorigenin of various concentration (0 μM, 4 μM, 8 μM, 16 μM, 24 μM and 32 μM) to HepG2 The influence result figure of cell;

Fig. 2 is the New of M T T colorimetric determinations various concentration (0 μM, 4 μM, 8 μM, 16 μM, 24 μM and 32 μM) Tectorigenin handle HepG2 cells for 24 hours with the survival results figure of 48h;

Fig. 3 is the New Tectorigenin of various concentration (0 μM, 4 μM, 8 μM, 16 μM, 24 μM and 32 μM) to HepG2 The influence result figure of cell caryogram;

Fig. 4 is the New Tectorigenin of various concentration (0 μM, 4 μM, 8 μM, 16 μM, 24 μM and 32 μM) to HepG2 The influence result figure of mitochondrial membrane potential in anoxic (MMP);

Fig. 5 is the New Tectorigenin couple of various concentration (0 μM, 4 μM, 8 μM, 16 μM, 24 μM and 32 μM) The influence result figure of the subcellular localization of Calnexin;

Fig. 6 is the New Tectorigenin of various concentration (0 μM, 4 μM, 8 μM, 16 μM, 24 μM and 32 μM) to HepG2 The influence result figure of intracellular ROS;

Fig. 7 is the New that Western blot detect various concentration (0 μM, 4 μM, 8 μM, 16 μM, 24 μM and 32 μM) Influence result figures of the Tectorigenin to p53, Caspase-3, Bax, Bcl-2 protein expression;

Fig. 8 is the New that Western blot detect various concentration (0 μM, 4 μM, 8 μM, 16 μM, 24 μM and 32 μM) Influence result figures of the Tectorigenin to Akt and PI3K protein expressions;

Fig. 9 is the New that Western blot detect various concentration (0 μM, 4 μM, 8 μM, 16 μM, 24 μM and 32 μM) Influence result figures of the Tectorigenin to ERK and pERK protein expressions.

Figure 10 is normal mouse, model group mouse, low concentration New Tectorigenin and high concentration New Tectorigenin group H22 tumor-bearing mice tumor size variation diagrams;

Figure 11 is normal mouse, model group mouse, low concentration New Tectorigenin and high concentration New The 3rd week tumor size figure of Tectorigenin group H22 tumor-bearing mices;

Figure 12 is normal mouse, model group mouse, low concentration New Tectorigenin and high concentration New Mouse living imaging and tumor size figure at the end of Tectorigenin group mouse experiments;

Figure 13 is model group mouse, low concentration New Tectorigenin and high concentration New Tectorigenin groups H22 Tumor-bearing mice tumor tissues H&E dyes (× 200) figure;

Figure 14 is model group mouse, low concentration New Tectorigenin and high concentration New Tectorigenin groups H22 Tumor-bearing mice tumor tissues Arg-1, AFP, CA125, GPC3 immunohistochemistry microexamination figure (× 100);

Figure 15 is model group mouse, low concentration New Tectorigenin and high concentration New Tectorigenin groups Hepa1-6 tumor-bearing mice different time tumour living imaging figures;

Figure 16 is model group mouse, low concentration New Tectorigenin and high concentration New Tectorigenin groups Hepa1-6 tumor-bearing mice different time tumor size figures;

Figure 17 is that model group mouse, low concentration New Tectorigenin and high concentration New Tectorigenin groups are small Mouse living imaging and tumor size figure at the end of mouse is tested;

Figure 18 is model group mouse, low concentration New Tectorigenin and high concentration New Tectorigenin groups Hepa1-6 tumor-bearing mice tumor tissues H&E dyes (× 200) figure;

Figure 19 is model group mouse, low concentration New Tectorigenin and high concentration New Tectorigenin groups Hepa1-6 tumor-bearing mice tumor tissues the microexamination of Arg-1, AFP, CA125, GPC3 immunohistochemistry (× 100) figure.

Specific implementation mode

Following will be combined with the drawings in the embodiments of the present invention, is carried out to the technical method in the embodiment of the present invention clear, complete Site preparation describes.Obviously, described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.It is based on Embodiment in the present invention, those of ordinary skill in the art are obtained every other without creative efforts Embodiment shall fall within the protection scope of the present invention.Unless otherwise instructed, the reagent concentration in the application is mass concentration.

Embodiment 1

A kind of embodiment of the preparation method of the quinoline of the N isostere iridins of the present invention, including it is as follows Step:

(1) bis- bromo- 2- methoxyl groups -5- nitrobenzenes of 1,3- are dissolved in the ethyl alcohol of 10 equivalents at room temperature;To acquired solution 10% Na of 6 equivalents is added₂S₂O₄Aqueous solution stirs 7 hours at 50 DEG C;When reaction mixture is cooled to room temperature, use AcOEt (ethyl acetate) is extracted three times, every time with 2-4 times of volume;Total extract is washed with saturated salt solution and uses MgSO₄It is dry It is dry;Decompression removal AcOEt, obtains 3,5-, the bis- bromo- 4- aminoanisoles of white powder;

(2) bis- bromo- 4- aminoanisoles of 3,5- are dissolved in the CH (OMe) of 10 equivalents at room temperature₃In, stirring 6-12 is small When;Remove excessive CH (OMe)₃The two bromo- 4- methoxyphenyls carboximides of (suitable)-N-3,5- of white powder are obtained afterwards;

(3) two bromo- 4- methoxyphenyls carboximides of (suitable)-N-3,5- are dissolved in the anhydrous MeOH of 20 equivalents at room temperature In;The NaOMe (sodium methoxide) of 1 equivalent is added to said mixture, stirs 15-45 minutes;Add 2- (the 4- methoxies of 1 equivalent Base phenyl) ethyl acetate, is stirred 4-10 hours;After removing excessive MeOH, the water of equal volume is added and stirs 10-20 Minute;It is filtered under diminished pressure gained mixture, obtains (anti-) -3- (3,5- bis- bromo- 4- Methoxyphenylaminos) -2- of white powder (4- methoxyphenyls) ethyl acrylate;

(4) at room temperature by (anti-) -3- (bis- bromo- 4- Methoxyphenylaminos of 3,5-) -2- (4- methoxyphenyls) acrylic acid Ethyl ester is dissolved in the ethyl alcohol of 10 equivalents;The H of 0.5 equivalent is added to gained mixture₂SO₄, 4-10 is stirred at 50 DEG C -70 DEG C Hour;When reaction mixture is cooled to room temperature, the water of 5 times of volumes of addition simultaneously stirs 30-50 minutes;It is filtered under diminished pressure gained mixing Object obtains 5,7-, bis- bromo- 6- methoxyl groups -3- (4- methoxyphenyls) quinoline -4 (1H) -one of white powder;

At (5) 0 DEG C, 5,7-, bis- bromo- 6- methoxyl groups -3- (4- methoxyphenyls) quinoline -4 (1H) -one is dissolved in 40 and is worked as In the mixture of the DMF (n,N-Dimethylformamide) of amount and the Et2O (ether) of 20 equivalents, PCl is then added dropwise₃, stirring 8-16 hours;The water of 6 times of volumes is added and firmly stirs, the AcOEt of 4 times of volumes of gained mixture is extracted 3 times, uses saturated salt Solution washs and uses MgSO₄It dries;AcOEt is removed, the chloro- 6- methoxyl groups -3- (4- of 5,7-, bis- bromo- 4- of white powder are obtained Methoxyphenyl) quinolone.

The above-mentioned corresponding synthetic route of preparation method is as follows:

Embodiment 2

(1) bis- bromo- 2- methoxyl groups -5- nitrobenzenes of 1,3- are dissolved in the ethyl alcohol of 15 equivalents at room temperature;To acquired solution 20% Na of 2 equivalents is added₂S₂O₄Aqueous solution stirs 5 hours at 55 DEG C;When reaction mixture is cooled to room temperature, use AcOEt is extracted three times, every time with 2-4 times of volume;Total extract is washed with saturated salt solution and uses MgSO₄It is dry;Decompression removal AcOEt obtains 3,5-, the bis- bromo- 4- aminoanisoles of white powder;

(2) bis- bromo- 4- aminoanisoles of 3,5- are dissolved in the CH (OMe) of 16 equivalents at room temperature₃In, stirring 6-12 is small When;Remove excessive CH (OMe)₃The two bromo- 4- methoxyphenyls carboximides of (suitable)-N-3,5- of white powder are obtained afterwards;

(3) two bromo- 4- methoxyphenyls carboximides of (suitable)-N-3,5- are dissolved in the anhydrous of 14 equivalents at room temperature In MeOH;The NaOMe of 2 equivalents is added to said mixture, stirs 15-45 minutes;Add 2- (the 4- methoxybenzenes of 2 equivalents Base) ethyl acetate, is stirred 4-10 hours;After removing excessive MeOH, the water of equal volume is added and stirs 10-20 minutes; It is filtered under diminished pressure gained mixture, obtains (anti-) -3- (3,5- bis- bromo- 4- Methoxyphenylaminos) -2- (4- of white powder Methoxyphenyl) ethyl acrylate;

(4) at room temperature by (anti-) -3- (bis- bromo- 4- Methoxyphenylaminos of 3,5-) -2- (4- methoxyphenyls) acrylic acid Ethyl ester is dissolved in the ethyl alcohol of 15 equivalents;The H of 0.8 equivalent is added to gained mixture₂SO₄, 4-10 is stirred at 50 DEG C -70 DEG C Hour;When reaction mixture is cooled to room temperature, the water of 7 times of volumes of addition simultaneously stirs 30-50 minutes;It is filtered under diminished pressure gained mixing Object obtains 5,7-, bis- bromo- 6- methoxyl groups -3- (4- methoxyphenyls) quinoline -4 (1H) -one of white powder;

At (5) 0 DEG C, 5,7-, bis- bromo- 6- methoxyl groups -3- (4- methoxyphenyls) quinoline -4 (1H) -one is dissolved in 20 and is worked as The Et of the DMF of amount and 40 equivalents₂In the mixture of O, PCl is then added dropwise₃, stir 8-16 hours;The water of 2 times of volumes is added And firmly stir, the AcOEt of 3 times of volumes of gained mixture is extracted 3 times, is washed with saturated salt solution and is used MgSO₄It dries; AcOEt is removed, 5,7-, bis- bromo- 4- chloro- 6- methoxyl groups -3- (4- methoxyphenyls) quinolone of white powder is obtained.

Embodiment 3

(1) bis- bromo- 2- methoxyl groups -5- nitrobenzenes of 1,3- are dissolved in the ethyl alcohol of 20 equivalents at room temperature;To acquired solution 30% Na of 4 equivalents is added₂S₂O₄Aqueous solution stirs 3 hours at 60 DEG C;When reaction mixture is cooled to room temperature, use AcOEt is extracted three times, every time with 2-4 times of volume;Total extract is washed with saturated salt solution and uses MgSO₄It is dry;Decompression removal AcOEt obtains 3,5-, the bis- bromo- 4- aminoanisoles of white powder;

(2) bis- bromo- 4- aminoanisoles of 3,5- are dissolved in the CH (OMe) of 20 equivalents at room temperature₃In, stirring 6-12 is small When;Remove excessive CH (OMe)₃The two bromo- 4- methoxyphenyls carboximides of (suitable)-N-3,5- of white powder are obtained afterwards;

(3) two bromo- 4- methoxyphenyls carboximides of (suitable)-N-3,5- are dissolved in the anhydrous of 10 equivalents at room temperature In MeOH;The NaOMe of 4 equivalents is added to said mixture, stirs 15-45 minutes;Add 2- (the 4- methoxybenzenes of 3 equivalents Base) ethyl acetate, is stirred 4-10 hours;After removing excessive MeOH, the water of equal volume is added and stirs 10-20 minutes; It is filtered under diminished pressure gained mixture, obtains (anti-) -3- (3,5- bis- bromo- 4- Methoxyphenylaminos) -2- (4- of white powder Methoxyphenyl) ethyl acrylate;

(4) at room temperature by (anti-) -3- (bis- bromo- 4- Methoxyphenylaminos of 3,5-) -2- (4- methoxyphenyls) acrylic acid Ethyl ester is dissolved in the ethyl alcohol of 20 equivalents;The H of 1 equivalent is added to gained mixture₂SO₄, it is small that 4-10 is stirred at 50 DEG C -70 DEG C When;When reaction mixture is cooled to room temperature, the water of 10 times of volumes of addition simultaneously stirs 30-50 minutes;It is filtered under diminished pressure gained mixing Object obtains 5,7-, bis- bromo- 6- methoxyl groups -3- (4- methoxyphenyls) quinoline -4 (1H) -one of white powder;

At (5) 0 DEG C, 5,7-, bis- bromo- 6- methoxyl groups -3- (4- methoxyphenyls) quinoline -4 (1H) -one is dissolved in 30 and is worked as The Et of the DMF of amount and 30 equivalents₂In the mixture of O, PCl is then added dropwise₃, stir 8-16 hours;The water of 4 times of volumes is added And firmly stir, the AcOEt of 2 times of volumes of gained mixture is extracted 3 times, is washed with saturated salt solution and is used MgSO₄It dries; AcOEt is removed, 5,7-, bis- bromo- 4- chloro- 6- methoxyl groups -3- (4- methoxyphenyls) quinolone of white powder is obtained.

Embodiment 4

A kind of application of the quinoline of N isosteres iridin of the present invention in medicines resistant to liver cancer.

The quinoline (New Tectorigenin i.e. herein) of the N isosteres iridin, chemistry Structural formula is:

The quinoline of the N isosteres iridin is logical by inhibiting RAF/MEK/ERK accesses and JAK/STAT Road inhibits hepatoma cell proliferation;The quinoline of the N isosteres iridin is by inhibiting P13K/AKT/mTOR accesses Promote hepatoma cell apoptosis;The quinoline of the N isosteres iridin by raise anti-apoptotic signal P53 and Anti-apoptotic signal PTEN genes are to promote hepatoma cell apoptosis.

The quinoline of the N isosteres iridin by raise anti-apoptotic signal p53 expression, The generation for improving ROS, increasing the permeability of mitochondrial membrane stress be combined to induce liver cancer cells with causing endoplasmic reticulum excessively Apoptosis;The related gene that the quinoline of the N isosteres iridin passes through regulation and control new vessels paraplasm access Expression prevent fucosylation.

The quinoline of the N isosteres iridin by lower AKT, ERK, pERK expression, to Inhibit the proliferation of liver cancer cells;The quinoline of the N isosteres iridin improves tumour during treating liver cancer Sensibility, reduction drug resistance to drug.

A kind of quinoline of N isosteres iridin of the present invention is preparing the sensitivity for improving liver cancer to drug Property, reduce liver cancer drug resistance, application during treat liver cancer while during the drug or preparation of immunity of organisms can be improved.

Reagent chemicals used in embodiment unless otherwise specified, are purchased from conventional biochemical reagent company;That is studied is new Compound N ew Tectorigenin are provided by Nanjing University professor Zhu Hailiang.

The noval chemical compound New Tectorigenin that the present invention is studied are the warps based on the structural formula of tectorigenin Structure optimization and the N isostere tectorigenin derivatives synthesized.Tectorigenin i.e. 5,7, the 4- different Huangs of trihydroxy -6- methoxyl groups Ketone is the main component of iris, is common in Chinese medicine blackberry lily and iris.Its with oestrogen-like hormone, anti-oxidant, liver protection, The bioactivity such as anticancer and pharmacological action.

Experiment in vitro further demonstrates that tectorigenin has very strong inhibiting effect to HL-60 cells (leukaemia cell), right The proliferation of BGC cells (adenocarcinoma of stomach) also has a degree of inhibiting effect.The zoopery of Thelen P et al. It also indicates that, tectorigenin plays the role of significantly inhibiting tumour, it is found that it can be as the prostate cancer of prevention and treatment people Drug.Saratikov, A.S.et al.'s studies have shown that polyols such as tectorigenin can inhibit the necrosis of liver cell, The unbalance of intrahepatic fat and protein is prevented, makes transaminase and the active normalization of glutamyl transferase, it is therefore prevented that liver fiber The development of change is played the role of protecting liver, restores liver function.

But since content of the tectorigenin in Chinese medicine blackberry lily is relatively low, and the difficulty isolated and purified is larger, is not easy to obtain. Therefore, seminar of Nanjing University combines self-technique advantage, and the thought designed using isostere has carried out full tectorigenin Study on the synthesis, and based on this is studied, design, synthesized a N isosteres tectorigenin derivative more than a series of 200.

In this serial tectorigenin derivative, the activity of New Tectorigenin is most strong, while having good peace Full property and stability.However, New Tectorigenin are inhibiting hepatoma cell proliferation, protection as tectorigenin derivative It is had not been reported in terms of liver, function.Therefore, object of this investigation is through Experimental Research, the anti-livers of New Tectorigenin The molecular action target spot of cancer activity and its anti-liver cancer and anti-.

Experiment 1

Inhibiting effect of the New tectorigenin to rat liver cancer model

1.1 rat liver cancer models are established

1.1.1 diethylnitrosamine (diethylnitrosamine, DEN) compound revulsion

By 60 Healthy female mouse be randomly divided into Normal group, DEN groups, New tectorigenin and DEN feed Foster group, every 20.Normal group gives chow diet, DEN groups and New tectorigenin and DEN nursing groups, uses 0.25%DEN aqueous solutions once a week by 10mg/kg weight gavage, by it freely drunk by remaining time 0.025%DEN aqueous solutions With, and Newtectorigenin adds hello New tectorigenin (100mg/kg) with DEN nursing groups, feeds about 4 months.

1.1.2 grafting

70 healthy Kunming mouses are randomly divided into control group, New tectorigenin groups, model group, positive control Group and high, medium and low dosage N e w te c t o r ig e n i n nursing model groups, every group 10, half male and half female.Except pair It is outer according to group, New tectorigenin groups, it is subcutaneously injected 2 × 10 in the right fore armpit of every mouse⁷HepG2 cells it is outstanding Liquid 0.2mL.Inoculation starts for second day, and New tectorigenin groups, high, medium and low dosage feed New tectorigenin groups Daily gastric infusion 1 time, successive administration 10 days;The daily gastric infusion of positive drug group 1 time;The bodies such as control group and model group gavage 0.9% long-pending sodium chloride solution.

Next day weighs mouse weight before administration and after the last administration, cuts off each group liver cancer tissue, weighs tumor mass tissue matter Amount studies the influence to mouse weight and studies the influence of tumor quality by calculating tumour inhibiting rate.

1.2 frozen section:

It puts to death mouse and takes its liver, observed under disecting microscope and calculate liver size, and taken pictures, then carried out frost and cut Piece.Liver organization is fixed in fixer, is fully washed after fixed, cleans, fixer containing formaldehydes should not be selected.Tissue washing After immerse 12.5% aqueous gelatin solution (being prepared with 1% phenolated water solution), soak 6-24h in 37 DEG C of incubators.Move to 25% gelatin 6-24h is soaked in solution.25% gelatin embedding, being put into refrigerator together with embedding device makes gelatin quick solidification, after taking-up in air Evaporate 10min.It is put into tissue fixative solution and fixes gelatin block 1-2 days.Again with after distillation washing, sets freezing microtome or slidingtype is cut Piece machine-cut piece.Slice is stored in 10% formalin.It is saturating without ethanol dehydration and dimethylbenzene after dyeing with different reagent dyeings It is bright, and with gelatin solution or other similar gelatin solution mountings:Fructose 26g, gelatin 1.1g, arcanite 0.75g, thymol water Solution 1.15ml.

Experiment 2

Inhibiting effect of the New tectorigenin to HepG2 cells

2.1 recovery HepG2 cells

The HepG2 cells frozen are taken out from liquid nitrogen container, are immediately placed in 38 DEG C of thermostat water baths, quickly shakes, waits for After it is melted, centrifuge tube metastatic cells suspension is used in superclean bench, with 800rpm, after 25 DEG C of pelleted by centrifugation 5min, is received Collect cell precipitation, adds appropriate culture solution (low sugar DMEM culture solutions being used, containing 2%100u/mL is dual anti-and 10%FBS) and blow and beat Culture bottle is gone to after mixing, is put into incubator and is cultivated.Condition of culture:5%CO₂, 37 DEG C of saturated humidities.After cultivating 12h, use Inverted phase contrast microscope observes the effect of recovery HepG2 cells, and not adherent floating is dead cell or cell fragment, it is adherent or Differentiated is living cells.

2.2New tectorigenin handle HepG2 cells

It when the adherent length of HepG2 cells is to 80% or more, is cleaned 2 times with PBS buffer solution, then with 0.25% Trypsin enzymes 3m i n are solved, finally with about 1.0 × 10⁵The density of a/m L is assigned in six orifice plates, and experimental group adds the N e of various dose W Tectorigenin (so that final concentration is respectively 2 μm of ol/L, 4 μm of ol/L, 8 μm of ol/L, 12 μm of ol/L, 16 μm of ol/L) are right It is then replaced with DMEM culture solution equivalent according to group, is placed in incubator and cultivates for 24 hours, it is thin to observe HepG2 respectively under inverted microscope The growing state of born of the same parents simultaneously photographs to record.It is tested in triplicate under the conditions of same treatment.

As shown in Figure 1, with the rising of New Tectorigenin concentration, compared with the control group, cell quantity and density It gradually decreases, the gradual unobvious of cell edges, or accumulation is agglomerating, or is crimped to circle, or fracture fragmentates.As N e w When tectorigenin is 4 μM a concentration of, cell quantity and density reduce unobvious;A concentration of 8 μM as New tectorigenin When, it is opposite to be significantly reduced with control group HepG2 cell quantities and density, and start cell fragment occur and be crimped to circular Cell;And when New tectorigenin concentration reaches 32 μM, most HepG2 cell deaths are only remaining to be crimped to circle Shape is by the cell fragment of the cell of apoptosis and cell cracking death.Therefore experiment tentatively shows New tectorigenin energy Inhibit proliferation and the growth of HepG2 cells.

Experiment 3

MTT colorimetric determination HepG2 cell activity

It is thin with MTT colorimetric determinations in order to further probe into inhibiting effect of the New Tectorigenin to HepG2 cells Born of the same parents' survival rate more can be shown that inhibiting effect of the New Tectorigenin to HepG2 cells by specific data.

The specific method is as follows:In vitro culture HepG2 cells to growing logarithmic phase, quantity up to after 90% with 0.25% Trypsin is digested into single cell suspension, is assigned in 96 orifice plates, and density is 1.0 × 10⁵A/mL, is placed in incubator and cultivates for 24 hours. After cell is adherent, New Tectorigenin are added by the dosage that front is designed, each concentration sets 5 multiple holes, and control group adds Enter the sterile PBS of equivalent, continues culture for 24 hours and 48h.20 μ L MTT are added after culture per hole, are incubated 4h in the incubator, original is trained Nutrient solution sops up, and 150 μ L DMSO are added in each hole, shakes 10min with micro oscillator, first a ceremonial jade-ladle, used in libation is made fully to dissolve.In microplate reader It is carried out with blank control group withered, the OD values in each hole is measured under 490nm wavelength, are tested in triplicate.Cell is calculated according to OD values Survival rate and growth inhibition ratio:

The calculation formula of 1 cell survival rate of formula

The calculation formula of 2 inhibitory rate of cell growth of formula

As shown in Fig. 2, handling HepG2 cells for 24 hours with after 48h with the NewTectorigenin of various dose, HepG2 is thin The activity of born of the same parents is substantially reduced, and growth inhibition ratio is significantly raised, and the inhibiting effect of 4 8h is bigger than 2 4h.Illustrate N e w Tectorigenin has certain cytotoxicity, and with the increase of NewTectorigenin dosage, to HepG2 cells Growth inhibition ratio is gradually increased, and there is dosage effect and time-effect, difference to have conspicuousness (P < 0.05).

Test influences of the 4New Tectorigenin to nucleus caryogram

It is above-mentioned the experimental results showed that NewTectorigenin is within the scope of a certain concentration can influence the activity of HepG2 cells, Inhibit HepG2 cell growths, causes HepG2 Apoptosis.It is glimmering using Hochest33258 to further look at its apoptosis feature Light decoration method, after observing dosing, the variation of HepG2 cell caryogram.

The specific method is as follows:In vitro culture HepG2 cells to growing logarithmic phase, quantity up to after 90% with 0.25% Trypsin is digested into single cell suspension, is assigned in six orifice plates, and density is 1.0 × 10⁵A/mL, is placed in incubator and cultivates.It waits for After cell is adherent, the dosage that designs by front adds New Tectorigenin, control group then with the DMEM culture solutions of equivalent come Instead of.After cultivating 48h in the incubator, exhaust original fluid, is cleaned 3 times with PBS, and 0.5mL fixer (first is added in each hole Alcohol:Ice CH3COOH=3:1, now match and use) fix 20min.Fixer is then abandoned, once cleans 3 times with PBS with 3min, often Hole is protected from light 33258 dyeing liquors of addition 0.5mL Hochest, is incubated at room temperature 15min, siphons away dyeing liquor, primary with 3min with PBS Cleaning 3 times is protected from light air-dried i.e. usable fluorescence microscope and carries out colour developing observation, and photographs to record.

The results are shown in Figure 3, and the nucleus presentation disperse of the blank control group without NewTectorigenin processing is uniform Fluorescence, this explanation mostly be all living cells.And HepG2 cell of the experimental group after NewTectorigenin handles 48h The fine and close fluorescence of dense dye is presented in nucleus, and kernel cracking is blocky in particle, apoptotic body occurs.With NewTectorigenin The feature of the increasing of concentration, HepG2 Apoptosis is more obvious, has certain dosage effect.

Test influences of the 5New Tectorigenin to mitochondrial membrane potential in anoxic (MMP)

Above-mentioned description of test influences of the New Tectorigenin to 2 nucleus of H e p G, in order to further probe into Influences of the NewTectorigenin to mitochondrial membrane potential, this team have carried out Rhodamine 123 dye test.

The specific method is as follows:It is careful that former culture is sucked out after culture for 24 hours by above-mentioned adding method thereof respective handling HepG2 cells Liquid, after cleaning 1 time with PBS, each hole is separately added into 1mL Rhodamine 123s work dye liquor, is placed in CO₂It is incubated in incubator 30min.It inhales later and abandons dye liquor, cleaned 2 times with normal low sugar culture medium device to hole inner cell, mitochondria is observed under fluorescence microscope Form and distribution, and the fluorescence intensity of more each experimental group and control group.Rhodamine 123 dye liquor can make the line grain in living cells Green fluorescence is presented in body, is corynebacterium or graininess.

As shown in figure 4, with after the NewTectorigenin processing HepG2 cells of various concentration, compared with the control group, carefully The fluorescence degree of intracellular decreases, and concentration is bigger, and fluorescence is weaker, has concentration effect.As a result it prompts, NewTectorigenin causes the permeability of mitochondrial membrane to increase, and MMP is made to reduce.

Test influences of the 6New Tectorigenin to the subcellular localization of Calnexin

Influence for observation NewTectorigenin to HepG2 endocytoplasmic reticulums, is carried out with Calnexin antibody on cell Dyeing.

The specific method is as follows:By above-mentioned adding method thereof respective handling HepG2 cells, after culture for 24 hours, training in six orifice plates is siphoned away Nutrient solution, then it is carefully added into the surface for now matching fixer covering hole, fixed 40min.After once cleaning 2 times with PBS with 5min, often 1ml 5%BSA are added in hole, are put on shaking table and carry out cell closing at a slow speed.Liquid in hole is siphoned away after 1h, and primary antibody dilution is added (containing Calnexin) is placed in 4 DEG C of refrigerator overnights after being incubated 1h at a slow speed on shaking table.Siphon away liquid in hole within second day, with PBS with 5min once cleaning 3 times, be added rabbit anti-mouse secondary antibody diluents, continuation 1h is incubated on shaking table, after use PBS is once cleaned 3 times with 5min, adds DAB colour developing working solutions, and colour developing 3min or so is finally redyed with haematoxylin dye liquor 30S, dehydration is aerial to air-dry, mounting, in the staining conditions of microscopically observation endoplasmic reticulum.

The results are shown in Figure 5, and NewTectorigenin treated experimental group dyeing compares control group and wants shallow, and with The increasing of NewTectorigenin concentration, color gradually become shallower as, and have concentration effect.This show newly synthesized albumen with Calnexin, which is combined, to be reduced, and false folding or unassembled protein protomer are trapped in endoplasmic reticulum, lead to lighter. NewTectorigenin cause endoplasmic reticulum excessively stress, to induce HEPG2 Apoptosis.

Test the influence of 7New Tectorigenin ROSs intracellular to HepG2

ROS can generate oxidation to membrane structure, nucleic acid and protein molecular, cause its structure and function obstacle.ROS Increasing causes body response to oxidative stress (Oxidative Stress) to cause cellular damage, participates in a variety of pathomechanisms.In order to Influences of the New Tectorigenin to the content of intracellular ROS is measured, active oxygen inspection is carried out using fluorescence probe DCFH-DA It surveys.

The specific method is as follows:HepG2 cells are cultivated, connect 6 orifice plates, inoculum density is in 3-4 × 10⁵/ ml, after waiting cells adherent 1 2h of serum deprivationization;The culture medium containing serum is changed to continue to add New Tectorigenin processing after cultivating 3-6h.New Tectorigenin collects cell after handling for 24 hours, and the culture medium containing serum is added to terminate digestion, and centrifugation abandons supernatant, adds 400ul PBS suspension cells again;Ultrasound cracking 40-60w, it is 3-4 times super every time, it (operates on ice for 3 times in total;4 degree of centrifugations 12000 Turn, 15min.The reagent of kit (dichlorofluorescein active oxygen detection kit), the method microplate reader of by specification is added Measure every group of od values.

It can be seen that New Tectorigenin treated that control group is compared in administration group dyeing from the coloration result of Fig. 6 It is deep, and with the increasing of New Tectorigenin concentration, color gradually deepens area bigger, has concentration effect.

Test 8Westernblot detection New Tectorigenin to PI3K, Akt, Caspase-3, p53, Bax, The influence of Bcl-2 protein expressions

It is above-mentioned the experimental results showed that, NewTectorigenin significantly inhibits the growth of HepG2 cells, To probe into the mechanism of action of its apoptosis, inventor have detected the intracellular PI3K, Akt of HepG2 after administration, Caspase-3, p53, Bax, Bcl-2 protein expression.

The specific method is as follows:By above-mentioned adding method thereof respective handling HepG2 cells, after continuous culture 48h.It grasps on ice Make, discard the original fluid in six orifice plates, cleaned 3 times with the PBS of precooling, 80 μ L lysates (lysis are added per hole Buffer) lytic cell scrapes attached cell toward same direction with cell scraper, cell is collected into EP pipes respectively, sets ice In at least cracking 30min on shaking table.Later under conditions of 13200rpm, 4 DEG C, 10min is centrifuged, collects supernatant.With examining Mas bright blue G-250 methods measure the concentration per histone matter, and absorbance is measured under 595nm wavelength with visible spectrophotometer, Protein standard curve is drawn, the concentration of every histone matter is calculated separately, according to albumen concentration and sample-adding protein content, calculates sample-adding egg Lean type is accumulated.

By the albumen equivalent loading after measurement, polyacrylamide gel electrophoresis (SDS-PAGE) electrophoresis is carried out.Electrophoresis terminates It afterwards, will be in albumen transferring film to nitrocellulose membrane (PVDF).With 5% skimmed milk power in closing 1h on shaking table.With PI3K, Akt, ERK, p-ERK(1:Etc. 1000) primary antibodies are placed in shaking table on ice and are incubated 1h, are placed in 4 DEG C of refrigerator overnights.Film is washed with TBS × 13 times, every time 5min, with goat anti-rabbit (1:1000) secondary antibody is incubated at a slow speed 1h in room temperature shaker, and film is washed 5 times in TBS × 1, every time 5min, then ECL detections are carried out, develop, fixing, scanning.

Experimental result is as shown in fig. 7, first, p53 expression increases, and will induce the intracellular antiapoptotic factors Caspase-3 of HepG2 Expression, so that cell is entered programmed cell death.Meanwhile the pro apoptotic protein Bax expressions in mitochondrial apoptotic pathway rise, Press down apoptotic proteins Bcl-2 expressions to decline, above-mentioned effect leads to HepG2 Apoptosis jointly.Next, as shown in figure 8, NewTectorigenin is inhibited to PI3K, Akt protein expression, and has concentration effect.The AKT of activation can be straight Connecing makes precursor apoptotic proteins phosphorylation and generates short-term effect to prevent activation from leading to the apoptosis pathway of cell death.PI3K,AKT Expression is reduced, and inhibits HepG2 cell Proliferations.

Test influences of the 9Western blot detections New Tectorigenin to ERK1/2 protein expressions

HepG2 cell proliferation machineries, inventor is inhibited also to have detected administration in order to further probe into NewTectorigenin The intracellular ERK and pERK protein expressions of HepG2 afterwards.

Specific experiment method is the same as described in experiment 8.

Experimental result is as shown in figure 9, NewTectorigenin is inhibited to ERK protein expressions, and has concentration Effect, Ras/Raf/MEK/ERK signal transduction pathways cell physiology course such as proliferation, survival, differentiation, apoptosis, movement and Vital effect has been played in terms of metabolism.ERK expression is reduced, and causes the GAP-associated protein GAP in downstream also to express reduction, HepG2 is thin The growing multiplication of born of the same parents is suppressed.

Inhibiting effect of the embodiment 5New Tectorigenin to H22 liver cancer transplantation tumors

(1) heterotopic transplantation

One, H22 cell recoveries

It is taken out from liquid nitrogen equipped with H22 cell cryopreservation tubes, is quickly shaken in 37 DEG C of water-bath, wait for the ice cube in cryopreservation tube Close to when melting completely, the frozen stock solution melted is transferred in centrifuge tube in gnotobasis at once, 1000rpm, 25 DEG C of centrifugations 3min abandons supernatant, is gently blown and beaten with the culture solution newly matched and dissipates cell, is then transferred in culture bottle and adds culture solution and arrives 3ml, at 37 DEG C, 5%CO₂The incubator culture of saturated humidity.

Two, H22 subcutaneous transplantation knurl models are established

When H22 cells grow to logarithmic phase, H22 cells 1000rpm, 25 DEG C of centrifugation 3min are abandoned into culture solution, PBS washes three It is secondary, it is diluted to 1x10 with PBS⁶。

In 3 nude mice abdominal cavity injection 0.2ml 1x10⁶H22 cells are raised 7 days or so, and rearing conditions are:Basal feed, Free feeding, air humidity 50 ± 5%, 20~25 DEG C of temperature.

Aseptic condition takes mouse milky ascites, PBS to wash three times after 7 days, and PBS is diluted to 1x10⁷。

Every nude mice right fore is subcutaneously injected the sterile H22 liver cancer cells suspensions of about 0.2mL and (contains 2.0 × 10⁶A cell), it connects Knurl longest diameter and shortest diameter are measured with ruler per 2d after kind, calculates gross tumor volume,

Tumour is grown (L) and wide (W)

V=1/2*LW²

Wait for that gross tumor volume is grown to about 0.2cm after 4 days³After be randomly divided into 3 groups:Respectively model group, low concentration group (7mg/ Kg), high concentration group (14mg/kg).Model group is daily with the physiological saline gavage of 0.2ml, and low concentration is with 0.2ml l7mg/ml's New Tectorigenin gavages, low concentration is with the New Tectorigenin gavages of 0.2ml 14mg/ml.Continuous processing 14 It.

Nude mice was put to death in the 2nd day after last medication.Separation tumour claims quality, calculates tumour inhibiting rate:Tumour inhibiting rate (%)=(right According to a group tumor quality-experimental group tumor quality)/control group tumor quality × 100%.

Nude mice takes tumor tissues and internal organs after putting to death.

Three, tumor tissues H&E is dyed

Tumor tissues are dipped into after removing in 10% formalin immediately, are placed and are fixed 24 h of tumor tissues at 4 DEG C, are taken big The tissue block of small about 1cm × 1cm × 0.5cm, the paraffin embedding after conventional dehydration, transparent, waxdip.It is placed on slicer and continuously cuts Piece, 5 μm of slice thickness, is deployed in warm water on glass slide, room temperature preservation is spare.It is dyed, is used by 7 minutes haematoxylin dyeing liquid Long stream tap water slowly rinses extra dyeing liquor, slight to dry until the water colorless of outflow (about 6 minutes), then Yihong Dyeing liquor dyes 1 minute, then carries out conventional dehydration, transparent, neutral gum mounting.Pathomorphology is carried out under an optical microscope Observation, and take pictures.

Four, immunohistochemistry survey tumor tissues Arg-1 (arginase -1), AFP (alpha-fetoprotein), CA125 (glycoprotein), GPC3 (glypican-3) expression quantity

The tumor tissues for taking the paraffin embedding of above-mentioned experiment good, conventional section, then through conventional dewaxing and dehydration, use 3%H₂O₂ Room temperature closes 10min, and PBS is washed 3 times;Suck dry moisture closes 20min with lowlenthal serum confining liquid room temperature;Primary antibody is added dropwise to cover Tissue puts 4 DEG C overnight;It places within second day to room temperature, PBS is washed 3 times;Appropriate amount streptomycete antibiotin-peroxide is added dropwise Enzyme solutions, are incubated at room temperature 1h, and PBS is washed 3 times;DAB colour developings are added dropwise, until until dyeing is suitable, PBS washings 3 times, bush are multiple Dye, the differentiation of 1% hydrochloride alcohol, dehydration, neutral gum mounting, microscopically observation are taken pictures.

Five, ELISA method surveys blood CEA contents

It is carried out in strict accordance with kit specification.Taken blood is centrifuged under the conditions of 3000 turns, 30 minutes, takes supernatant. Standard items sample is added by 50 μ L of each hole, sample aperture then presses 10 μ L of sample to be tested, and 40 μ L of Sample dilution are added, and blank well is not Add.100 μ L detection antibody-solutions are added to standard sample wells with sample aperture, is closed, is placed in 37 DEG C of insulating boxs with sealing plate film It is incubated 60min.All liq is removed, is patted dry on blotting paper, 1X cleaning solutions are added per hole, 1min is placed, gets rid of cleaning solution, and It pats dry, is repeated 5 times on blotting paper.Substrate A, each 50 μ L of B are added per hole, 37 DEG C are protected from light incubation 15min.Reaction is added per hole eventually Only 50 μ L of liquid measure the OD values in each hole at 450nm wavelength.Draw standard curve:Concentration makees abscissa, and OD values make ordinate, Linear regression curves are drawn, each sample concentration value is calculated by curvilinear equation.

(2) orthotopic transplantation

Mouse peritoneal anesthesia is carried out with the amount of the sharp aldehyde 0.1ml/10g of 5% hydration, after 2-3min, whether checks mouse activity Stop, but also have and breathe, after anesthesia completely, takes dorsal position and fixing limbs are on experimental plate, body is sloughed with 10% vulcanized sodium Mao, after 73% ethanol disinfection, abdomen is successively opened along hunter's line, exposure mouse peritoneal is light that mouse thoracic cavity, liver is pressed just to jump out abdominal cavity, Choose the lobe of the liver implantation tumor closest to body surface.Injection needle diagonal inserting needle, needle point are pierced into liver about 1cm horizontal by 20 ° Left and right, touches syringe needle core, is slowly injected into cell suspension 0.05mL, about 10⁶A H22 cell.The slow withdraw of the needle, and use immediately Burn red iron wire gently calcination pin hole, then liver is gently sent also to receive abdominal cavity, successively closes abdomen.

(3) experimental result:

One, H22 tumor-bearing mices tumor size changes

If Figure 10 experimental results are shown, it is inoculated with and the visible big little tumour of naked eyes, all groups of tumours from 11 days occurs within the 7th day Volume rate of rise increases, and significantly rises pattern drawing gross tumor volume since 17 days, but New Tectorigenin are low dense Degree and high concentration group gross tumor volume amplification are gentle compared with model group, and the 21st day tumor size such as Figure 11 became to 26 days all groups of amplification In gentle.At the end of experiment, tumor size is shown in that Figure 12, low concentration and high concentration group mean tumor volume have compared with model group Significant difference (p<0.05), and high concentration effect is more preferable than low concentration effect.Therefore preliminary display New Tectorigenin There is significant inhibiting effect to H22 tumor-bearing mice tumours.

Two, influences of the New Tectorigenin to H22 tumor-bearing mice internal organ indexes and tumour inhibiting rate

As it can be seen from table 1 after experiment, model group mouse significantly increases (p&lt compared with naive mice spleen index; 0.05), and compared with model group mouse spleen index, low concentration and high concentration New Tectorigenin spleen indexs significantly reduce (p <0.05), low, high concentration New Tectorigenin spleen indexs and blank group are closer.Normal group compared with model group, Low, high concentration New Tectorigenin group liver index no significant differences illustrate New Tectorigenin to liver without poison Side effect.In addition, low, high concentration New Tectorigenin group mouse tumor quality significantly reduces (p&lt compared to model group; 0.05) it is in concentration dependant effect.It these results suggest that, New Tectorigenin can not only significantly inhibit the life of mouse tumor It is long, also there is certain protective effect to immune organ.

Influences of the table 1New Tectorigenin to H22 mice organs index and tumour inhibiting rate

Note:X ± s, n=4, compared with model group, * P < 0.05.

Three, the variation of H22 tumor-bearing mices CEA contents

CEA (carcinoembryonicantigen) carcinomebryonic antigen, carcinomebryonic antigen are a broad spectrum activity tumor markers, It can reflect the presence of kinds of tumors.Experimental result shows (table 2), model group, low concentration group and high concentration group CEA contents (p&lt is increased relative to blank group conspicuousness;0.05), low concentration group is significantly reduced with high concentration group relative to model group CEA contents (p<0.05), it was demonstrated that New Tectorigenin can inhibit the growth of H22 tumor-bearing mice tumours by reducing the generation of CEA, And big (the p&lt of Amplitude Ratio low concentration group that high concentration group reduces;0.05), it is in concentration dependant effect.

Influences of the 2 New Tectorigenin of table to H22 mouse CEA contents

Note:X ± s, n=4, compared with model group, * P < 0.05.

Four, H22 tumor-bearing mices tumor tissues H&E is dyed

As seen from Figure 13, model group tumour cell overall alignment is neat, and gap is smaller between cell, and cellular morphology is just Normal nucleus complete display, and new capillary vessel is abundant in cytoplasm, and compared with model group, New Tectorigenin Low concentration group cell is more sparse, not of uniform size, cell membrane breakage occurs, and the thin of typical apoptosis feature is presented in nucleus shrinkage The area of born of the same parents, New Tectorigenin high concentration group apoptosis cells increase, and tumor tissues are more loose, capillary vessel number Amount is reduced.As a result illustrate that New Tectorigenin have apparent inhibition tumor vascular growth and induction tumor death effect, and High concentration concentration inhibition has notable difference compared with low concentration, and certain dose-dependent effect is presented.

Five, immunohistochemistry surveys H22 tumor-bearing mice tumor tissues Arg-1, AFP, CA125, GPC3 expression quantity

Liver cancer marker Arg-1 is further detected in each group H22 tumor-bearing mice tumor tissues with the method for immunohistochemistry Expression.SABC (strept avidin-biotin complex), i.e. Streptavidin-biotin composite, are immune groups Change one of colouring method, positive cell shows as occurring pale brown color substance into the cell.As shown in figure 14, Arg-1 cytoplasm with There is expression in nucleus, model group brown color region is less, and positive cell is less, and Arg-1 expresses relatively low, New Tectorigenin low concentrations are then expressed higher with Arg-1 in high concentration group.And the model group hot spot of AFP, CA125, GPC3 Domain area ratio New Tectorigenin low concentrations and high concentration are high, and high concentration is lower than low concentration positive region, have one Fixed dosage effect.The above result shows that New Tectorigenin can increase the expression of Arg-1 in tumour cell, and reduce The expression of AFP, CA125, GPC3.

Liver cancer entity animal model is the important channel for inquiring into preclinical medicines resistant to liver cancer drug effect and exploitation, efficiently and accurate Really, the conversion of laboratory result can be promoted.And New Tectorigenin do not inquire into it also and are inhibiting as new medicines resistant to liver cancer The effect of entity animal liver cancer model.

The present embodiment is by cultivating H22 cells, with 1 × 10⁶The amount of a cell/ml, 0.2ml are injected into the abdominal cavity of nude mice, and 7 It takes nude mice ascites, PBS to wash after it 3 times, 1 × 10 is diluted to PBS⁷A cell/ml is injected into nude mice mouse with the amount of 0.2ml Right fore is subcutaneous, waits for that nude mice right fore grows macroscopic tumour, and tumour growing way mouse identical with size is randomly divided into 3 Group, respectively model group, low concentration group (7mg/kg), high concentration group (14mg/kg), model group is daily with the physiology salt of 0.2ml Water gavage, low concentration is with the New Tectorigenin gavages of 0.2ml 7mg/ml, and low concentration is with the New of 0.2ml 14mg/ml Tectorigenin gavages.After method described above gavage 14 days, cervical approach of breaking is put to death, and strips tumour, and 10% formalin preserves, and 2 Paraffin section is carried out in it.H&amp is carried out again;E is dyed dyes microexamination with histogenic immunity.

Analysis to each group tumor size finds the increase with New Tectorigenin concentration, rat liver cancer tumour Size reduce.H&E dyeing finds to increase with the area of the increase apoptosis cells of New Tectorigenin concentration.And Blood CEA detections find that New Tectorigenin can reduce the generation of CEA.Immuning tissue's dyeing finds New Tectorigenin can increase the expression of Arg-1 in tumour cell, and reduce the expression of AFP, CA125, GPC3.Therefore, New Tectorigenin can significantly inhibit the proliferation of H22 liver cancer transplantation tumors, have great importance to the exploitation of medicines resistant to liver cancer.

Inhibiting effect of the embodiment 6New Tectorigenin to Hepa1-6 liver cancer transplantation tumors

(1) heterotopic transplantation

One, Hepa1-6 cell recoveries

It is taken out from liquid nitrogen equipped with Hepa1-6 cell cryopreservation tubes, quickly shakes, waited in cryopreservation tube in 37 DEG C of water-bath The frozen stock solution melted is transferred in centrifuge tube in gnotobasis, 1000rpm, 25 DEG C by ice cube at once close to when melting completely 3min is centrifuged, supernatant is abandoned, is gently blown and beaten with the culture solution newly matched and dissipates cell, be then transferred in culture bottle and add culture solution To 3ml, at 37 DEG C, 5%CO₂The incubator culture of saturated humidity.

Two, Hepa1-6 subcutaneous transplantation knurl models are established

When Hepa1-6 cells grow to logarithmic phase, Hepa1-6 cells 1000rpm, 25 DEG C of centrifugation 3min are abandoned into culture solution, PBS is washed three times, and 1x10 is diluted to PBS⁶。

In 3 nude mice abdominal cavity injection 0.2ml 1x10⁶Hepa1-6 cells are raised 7 days or so, and rearing conditions are:It raises on basis Material, free feeding, air humidity 50 ± 5%, 20~25 DEG C of temperature.

Every nude mice right fore is subcutaneously injected the sterile Hepa1-6 liver cancer cells suspensions of about 0.2mL and (contains 2.0 × 10⁶It is a thin Born of the same parents), knurl longest diameter and shortest diameter are measured with ruler per 2d after inoculation, calculates gross tumor volume,

Tumour is grown (L) and wide (W)

V=1/2*LW²

Nude mice takes tumor tissues and internal organs after putting to death.

Three, tumor tissues H&E is dyed

Five, ELISA method surveys blood CEA contents

(2) orthotopic transplantation

Mouse peritoneal anesthesia is carried out with the amount of the sharp aldehyde 0.1ml/10g of 5% hydration, after 2-3min, whether checks mouse activity Stop, but also have and breathe, after anesthesia completely, takes dorsal position and fixing limbs are on experimental plate, body is sloughed with 10% vulcanized sodium Mao, after 73% ethanol disinfection, abdomen is successively opened along hunter's line, exposure mouse peritoneal is light that mouse thoracic cavity, liver is pressed just to jump out abdominal cavity, Choose the lobe of the liver implantation tumor closest to body surface.Injection needle diagonal inserting needle, needle point are pierced into liver about 1cm horizontal by 20 ° Left and right, touches syringe needle core, is slowly injected into cell suspension 0.05mL, about 10⁶A Hepa1-6 cell.The slow withdraw of the needle, exists side by side I.e. with the red iron wire gently calcination pin hole that burns, then liver is gently sent also to receive abdominal cavity, successively closes abdomen.

(3) experimental result:

One, Hepa1-6 bearing mouse models

Experimental result such as Figure 15,16 displays, at the end of experiment, New Tectorigenin low concentrations and high concentration tumour body Small (p&lt of the product compared with model group;0.05).Therefore preliminary display New Tectorigenin are equally to Hepa1-6 tumor-bearing mice tumours It is inhibited.

Two, tumour inhibiting rates of the New Tectorigenin to Hepa1-6 tumor-bearing mices

New Tectorigenin low concentration groups are can be seen that from table 3 and Figure 17 with high concentration group tumor quality obviously to compare Light (the p&lt of model group;0.05), inhibitory rate has statistical significance to 43.97% and 70.07%.Therefore, New Tectorigenin can significantly inhibit the growth of Hepa1-6 tumor-bearing mice tumours.

Influences of the 3 New Tectorigenin of table to Hepa1-6 mouse tumour inhibiting rates

Note:X ± s, n=4, compared with model group, * P < 0.05.

Three, the variation of Hepa1-6 tumor-bearing mices CEA contents

As shown in table 4, model group CEA contents significantly increase (p&lt compared with blank group;0.05),New Tectorigenin Low concentration group and high concentration group CEA contents are lower than model group, and low concentration group CEA contents do not have significant difference with model group, but High concentration group CEA contents and model group ratio, have significant difference (p<0.05).This illustrates that New Tectorigenin are same It can inhibit the growth of Hepa1-6 tumor-bearing mice tumours by reducing the generation of CEA.

Influences of the 4 New Tectorigenin of table to Hepa1-6 mouse CEA contents

Note:X ± s, n=4, compared with model group, * P < 0.05.

Four, Hepa1-6 tumor-bearing mices tumor tissues H&E is dyed

As shown in figure 18, model group tumor tissue cell is complete, and nucleus is high-visible, and arrangement is close.And New Tectorigenin low concentration groups certain necrotic zone visible with high concentration group, necrotic zone cell is loose, and dyeing is shallower.This table Bright New Tectorigenin have certain inhibiting effect to Hepa1-6 tumor-bearing mices tumor tissue cell.

Five, immunohistochemistry surveys Hepa1-6 tumor-bearing mice tumor tissues Arg-1, AFP, CA125, GPC3 expression quantity

As can be seen from Figure 19, Arg-1 model groups brown color positive region compared with New Tectorigenin low concentration groups with Wanting for high concentration group is small, and high concentration positive region is obviously bigger than low concentration, has certain dose-dependent effect.And CA125 model groups positive region will be big than low concentration and high concentration, and positive cell is more, has notable difference.But it is low dense Degree and high concentration no significant difference.AFP with GPC3 model groups compared with low concentration, high concentration group, positive cell showed increased.Cause This New Tectorigenin can raise the expression of Arg-1, inhibit the expression of AFP, CA125, GPC3.

The present embodiment is by cultivating Hepa1-6 cells, with 1 × 10⁶The amount of a cell/ml, 0.2ml are injected into the abdomen of nude mice Chamber takes nude mice ascites, PBS to wash 3 times, 1 × 10 is diluted to PBS after 7 days⁷A cell/ml is injected into nude mice with the amount of 0.2ml The right fore of mouse is subcutaneous, waits for that nude mice right fore grows macroscopic tumour, and tumour growing way mouse identical with size is random It is divided into 3 groups, respectively model group, low concentration group (7mg/kg), high concentration group (14mg/kg), model group is daily with the life of 0.2ml Brine gavage is managed, low concentration is with the New Tectorigenin gavages of 0.2ml 7mg/ml, and low concentration is with 0.2ml 14mg/ml's New Tectorigenin gavages.After method described above gavage 14 days, cervical approach of breaking is put to death, and strips tumour, and 10% formalin is protected It deposits, paraffin section is carried out in 2 days.H&amp is carried out again;E is dyed dyes microexamination with histogenic immunity.

Analysis to each group tumor size finds the increase with New Tectorigenin concentration, rat liver cancer tumour Size reduce.H&E dyeing finds to increase with the area of the increase apoptosis cells of New Tectorigenin concentration.And Blood CEA detections find that New Tectorigenin can reduce the generation of CEA.Immuning tissue's dyeing finds New Tectorigenin can increase the expression of Arg-1 in tumour cell, and reduce the expression of AFP, CA125, GPC3.Therefore, New Tectorigenin can significantly inhibit the proliferation of Hepa1-6 liver cancer transplantation tumors, have to the exploitation of medicines resistant to liver cancer important Meaning.

The basic principles, main features and advantages of the present invention have been shown and described above.The technology of the industry Personnel are it should be appreciated that the present invention is not limited to the above embodiments, and the above embodiments and description only describe this The principle of invention, without departing from the spirit and scope of the present invention, various changes and improvements may be made to the invention, the present invention Claimed range is delineated by the appended claims, the specification and equivalents thereof from the appended claims.

Finally, it should be noted that the above embodiments are merely illustrative of the technical solutions of the present invention rather than is protected to the present invention The limitation of range is protected, although being explained in detail to the present invention with reference to preferred embodiment, those skilled in the art should Understand, technical scheme of the present invention can be modified or replaced equivalently, without departing from the essence of technical solution of the present invention And range.

Claims

1. a kind of application of quinoline of N isosteres iridin in medicines resistant to liver cancer.

2. application according to claim 1, it is characterised in that:The quinoline of the N isosteres iridin, Chemical structural formula is:

3. application according to claim 2, it is characterised in that:The quinoline of the N isosteres iridin is logical It crosses inhibition RAF/MEK/ERK accesses and JAK/STAT accesses inhibits hepatoma cell proliferation;The quinoline of the N isosteres iridin Quinoline derivant is by inhibiting P13K/AKT/mTOR accesses to promote hepatoma cell apoptosis;The quinoline of the N isosteres iridin Derivative is by raising anti-apoptotic signal P53 and anti-apoptotic signal PTEN genes to promote hepatoma cell apoptosis.

4. application according to claim 3, it is characterised in that:The quinoline of the N isosteres iridin is logical The expression for crossing up-regulation anti-apoptotic signal p53, improves the generation of ROS, increases the permeability of mitochondrial membrane and causes interior Matter net stress excessively be combined to liver cancer apoptosis reducing;The quinoline of the N isosteres iridin passes through Regulating and controlling the expression of the related gene of new vessels paraplasm access prevents fucosylation.

5. application according to claim 4, it is characterised in that:The quinoline of the N isosteres iridin is logical The expression for lowering AKT, ERK, pERK is crossed, to inhibit the proliferation of liver cancer cells;The quinoline of the N isosteres iridin Quinoline derivant improves sensibility, reduction drug resistance of the tumour to drug during treating liver cancer.

6. a kind of quinoline of N isosteres iridin is preparing inhibition RAF/MEK/ERK accesses, JAK/STAT accesses Promote hepatoma cell apoptosis with P13K/AKT/mTOR accesses and inhibits the application in the drug or preparation of liver cancer.

7. a kind of quinoline of N isosteres iridin is preparing downward anti-apoptotic signal P53 and PTEN gene To promote hepatoma cell apoptosis and inhibit the application in the drug or preparation of liver cancer.

8. a kind of quinoline of N isosteres iridin prepare raise anti-apoptotic signal p53, lower AKT, The expression of ERK, pERK improve the generation of ROS, and the permeability for increasing mitochondrial membrane excessively stress be tied mutually with endoplasmic reticulum is caused It closes to liver cancer apoptosis reducing and the drug for inhibiting liver cancer or the application in preparation.

9. a kind of quinoline of N isosteres iridin is in the suppression cancer base for preparing regulation and control new vessels paraplasm access Because the expression of P53, ERK1/2 gene, AKT genes prevents fucosylation and inhibits liver cancer invasion and diversion medicaments or system Application in agent.

10. a kind of quinoline of N isosteres iridin is preparing sensibility, the reduction liver cancer for improving liver cancer to drug The drug of drug resistance or the application in preparation.

11. the preparation method of the quinoline of the N isostere iridins in claim 1~10, which is characterized in that packet Include following steps:

(1) 1,3-, bis- bromo- 2- methoxyl groups -5- nitrobenzenes are dissolved in ethyl alcohol, Na is then added₂S₂O₄Aqueous solution, in 50~60 DEG C Lower stirring 3~7 hours, is cooled to room temperature, is purified with ethyl acetate, washed later, dry, depressurizes to remove ethyl acetate, Obtain the bis- bromo- 4- aminoanisoles of 3,5- of white powder;

(2) bis- bromo- 4- aminoanisoles of 3,5- are dissolved in CH (OMe)₃In, it stirs to remove excessive CH (OMe)₃Afterwards, it obtains The two bromo- 4- methoxyphenyls carboximides of (suitable)-N-3,5- of white powder;

(3) by (suitable)-N-3, bis- bromo- 4- methoxyphenyls carboximides of 5- are dissolved in anhydrous MeOH, and NaOMe then is added simultaneously It is stirred until homogeneous, adds 2- (4- methoxyphenyls) ethyl acetate to remove excessive MeOH, later plus water is stirred until homogeneous, It is filtered under diminished pressure to obtain (anti-) -3- (bis- bromo- 4- Methoxyphenylaminos of 3,5-) -2- (4- methoxyphenyls) third of white powder Olefin(e) acid ethyl ester;

(4) (anti-) -3- (bis- bromo- 4- Methoxyphenylaminos of 3,5-) -2- (4- methoxyphenyls) ethyl acrylate is dissolved in In ethyl alcohol, H is then added₂SO₄, stirred 4-10 hours at 50 DEG C -70 DEG C, be cooled to room temperature, water is added to stir, be filtered under diminished pressure To bis- bromo- 6- methoxyl groups -3- (4- methoxyphenyls) quinoline -4 (1H) -one of 5,7- of white powder;

(5) bis- bromo- 6- methoxyl groups -3- (4- methoxyphenyls) quinoline -4 (1H) -one of 5,7- is dissolved in DMF and Et₂The mixing of O In object, PCl is then added dropwise₃, it stirs 8~16 hours, later plus water is stirred until homogeneous, and gained mixture is extracted with AcOEt, Then washed, drying obtains chloro- 6- methoxyl groups -3- (the 4- methoxyl groups of 5,7-, bis- bromo- 4- of white powder to remove AcOEt Phenyl) quinolone, the i.e. quinoline of the N isostere iridins.

12. the chloro- 6- methoxyl groups -3- of bis- bromo- 4- of 5,7- (4- methoxyphenyls) quinolones prepared by claim 11 are preparing suppression Application in the drug of transplantable tumor processed.

13. a kind of drug inhibiting tumour, which is characterized in that contain the chloro- 6- methoxyl groups -3- of 5,7-, bis- bromo- 4- in the drug (4- methoxyphenyls) quinolone.