CN103130760B

CN103130760B - Novel targeting antineoplastic drug and manufacture method thereof and application thereof

Info

Publication number: CN103130760B
Application number: CN201110386523.0A
Authority: CN
Inventors: 杨凌; 艾纯芝; 葛广波; 于洋; 夏杨柳; 刘兆明
Original assignee: Dalian Institute of Chemical Physics of CAS
Current assignee: Zhangjiagang Institute Of Industrial Technology Dalian Institute Of Chemical Physics China Academy Of Sciences
Priority date: 2011-11-29
Filing date: 2011-11-29
Publication date: 2015-06-17
Anticipated expiration: 2031-11-29
Also published as: CN103130760A

Abstract

The invention provides a novel targeting antineoplastic drug and a manufacture method thereof and application thereof. The antineoplastic drug is a tyrosine kinase inhibitor, a novel core section of the drug is a catechol part and the left side is a hydroxide radical etherification part. The manufacture method of the drug is that 2, 4, 5-trihydroxy acetophenone reacts with methylclhlorofonmate; through an aldol reaction, a benzo dihydropyran structure is generated in a mode of cyclization through the aldol reaction; then the benzo dihydropyran structure reacts with interhalogen aniline or acetylene aniline to generate 4-interhalogen aniline-6, 7-dyhydroxy coumarin or 4-acetylene aniline-6, 7-dyhydroxy coumarin; methyl formate of benzene dihydroxy is removed; etherification is performed; and purification and crystallization are performed through flash chromatography to obtain the target product. The drug is used for treating people with locally advanced or metastatic non-small cell lung cancer and receiving chemical treatment before.

Description

A kind of New Target tropism antitumor drug and preparation method thereof and application

Technical field

The invention belongs to new drug design and medicinal chemistry art, be specifically related to a kind of New Target tropism antitumor drug and preparation method thereof and application.

Background technology

Protein tyrosine kinase (PTK) is key enzyme system in cell signalling process that is the most active in one group of tumour generating process, that make fast progress, the phosphate on ATP is made to transfer on the tyrosine residues of much important protein by catalysis, make its residue phosphorylation, thus activate various substrates enzymes, affect the growth of cell, propagation and differentiation by series reaction.The active abnormal rising of most tumors cell PTK, the oncogene of nearly 80% all contains Tyrosylprotein kinase coding.Therefore PTK is an extremely important and valuable antitumor target spot.Suppress tyrosine kinase receptor effectively can control the phosphorylation of downstream signal, thus the growth of inhibition tumor cell.Based on the new drug of Tyrosylprotein kinase target spot exploitation, for the difference between normal cell and tumour cell, highly selective, hypotoxic result for the treatment of can be reached, thus overcome the shortcoming such as poor selectivity, strong, the easy generation resistance of toxic side effect of conventional cell cytotoxic drug.Tyrosine kinase receptor is divided into EGF-R ELISA (EGFR), vascular endothelial growth factor receptor (VEGFR), platelet-derived growth factor receptor (PDGFR) etc., and what the tyrosine kinase inhibitor for various acceptor had developed listing at present is mainly epidermal growth factor recipient tyrosine kinase (EGFR-TK) inhibitor, vascular endothelial growth factor receptor Tyrosylprotein kinase (VEGFR-TK) inhibitor and platelet-derived growth factor receptor Tyrosylprotein kinase (PDGFR-TK) inhibitor etc.Its epidermal growth factor receptor (EGFR) high expression level in nonsmall-cell lung cancer, its inhibitor obtains extraordinary curative effect at the nonsmall-cell lung cancer for the treatment of chemotherapy failure and transfer in late period.Tens kinds of tyrosine kinase inhibitors have been had to be approved for the clinical trial of the antitumor smelting treatment of targeting in the U.S. and the Western European countries, comprising Gefitinib, the inhibitor (inhibitors) of the EGFR such as Erlotinib, and Lapatinib and its Her2 family closely-related with it.This three classes inhibitor for best core skeleton, is design based on the ATP binding pocket of target spot EGFR with 4-aniline quinazoline.4-aniline quinazoline class inhibitor can produce resistance because bringing out receptor mutation in application process, and the skeleton that therefore very necessary searching is new is the tyrosine kinase epidermal growth factor receptor inhibitors of core.This kind of protein tyrosine kinase inhibitor is all developed by external pharmaceutical manufacturer on the other hand, price is very high, picture Te Kailuo, the every box of imatinib mesylate (150mg × 30 slice) price are at 23000 yuan, compatriots are difficult to afford, and are also necessary very much to develop the tyrosine kinase inhibitor series antineoplastic medicament with our independent intellectual property right.

Research thinks that the hydrogen bond donor in 1 aromatic nitrogen of 4-aniline quinazoline and EGFR acceptor forms hydrogen bond, and the water molecules in 3 aromatic nitrogens and acceptor forms hydrogen bond, and the Thr790 in this water molecules and acceptor forms hydrogen bond, such formation stable in conjunction with conformation, phenylalanine simultaneously in the aromatic substituent that connects of aniline and acceptor is superimposed in π-π mode, and therefore 4-aniline quinazoline class EGFR inhibitor shows good pharmacologically active.Find the new core skeleton suitable with 4-aniline quinazoline class inhibitor activity, need the constitutional features based on target EGFR, and the Interactions Mode of acceptor and 4-aniline quinazoline, from the beginning design, verify, synthesize, evaluate with new skeleton the derivative being core.

The present invention will with Tyrosylprotein kinase if EGF-R ELISA be for target spot, based on the avtive spot information of EGF-R ELISA, computation chemical process and model, design and synthesize the EGFR inhibitor of the new core skeleton suitable with 4-phenylamino quinazoline ditosylate salt inhibitor activity, through cell-based assay, new core skeleton inhibitor directly acts on EGFR, and demonstrate the good pharmacologically active such as anti-nonsmall-cell lung cancer, prostate cancer, carry out by inside and outside model the assessment that it becomes efficacy in addition.

Summary of the invention

The object of this invention is to provide a kind of New Target tropism antitumor drug and preparation method thereof and application, according to the Three Dimensions Structure of the tyrosine kinase epidermal growth factor receptors (EGFR) of high expression level in tumour, from the beginning computation chemical process designs EGFR inhibitor, and preparation synthesis, pass through cell-based assay, display has good EGFR inhibit activities, thus reaches antitumous effect, can be used for the treatment for the treatment of through chemotherapy failure and Metastatic Nsclc.

The invention provides a kind of New Target tropism antitumor drug, this medicine has new core skeleton, and concrete structure general formula is as follows:

Wherein: R ₁and R ₂for the one in methyl, methyl alcohol ethyl ester, 2-morpholine-4-ethyl, R ₃for halogenic substituent Br or Cl or ethynyl, R ₄for H, F, to the one in F benzene substituting group.

Antitumor drug provided by the invention, described antitumor drug is tyrosine kinase inhibitor, and relative to the drug molecular structure gone on the market, this medicine has new core skeleton catechol, and left side is etherification of hydroxyl groups part.

Antitumor drug provided by the invention, described antitumor drug can be made into tablet, capsule or injection.

Antitumor drug provided by the invention, described antitumor drug effect is the EGF-R ELISA (EGFR) of high expression level in Tumor suppression, reaches antineoplastic object.

Present invention also offers the preparation method of antitumor drug, the method is with 2, 4, 5-trihydroxy-acetophenone and methyl-chloroformate are starting raw material, take salt of wormwood as acid binding agent, the product after obtaining phenolic hydroxyl group protection for 1 hour is reacted at-20 DEG C, then in anhydrous system, close ring by the catalysis of sodium Metal 99.5 by aldol reaction and generate chroman scaffold intermediate, react at 60 DEG C and chroman intermediate and a halobenzene amine effect are generated halogen (or acetylene) aniline-6 between 4-, 7-dihydroxycoumarin, then at 20 DEG C by bromo-ethyl-methyl ether-acetonitrile solution by 4-between halogen (or acetylene) aniline-6, 7-dihydroxycoumarin intermediate etherificate, reactant is by purification by flash chromatography and obtain target product after crystallization.

Antitumor drug provided by the invention, is applied to treatment and previously accepted chemotherapeutical Locally Advanced or Metastatic Nsclc (NSCLC).

Antitumor drug provided by the invention, be the new drug candidate with antitumor action, the new core skeleton series compound of this medicine, through cell and Enzyme activity assay, all has good anti-tumor activity, experiment in vitro shows, and this skeleton has the good pharmacologically active acting on EGFR; This series compound can be used as the drug candidates such as anti-nonsmall-cell lung cancer, anti-prostate cancer, anti-breast cancer.

Antitumor drug EGFR inhibitor provided by the invention shows good activity in the detection of tumor cell line level, and the cell strain detected comprises prostate cell strain lincap, PC3, Non-small Cell Lung Cancer A 549 strain, the IC of detection ₅₀value and positive control drug erlotinib, Gefitinib compares, and has carried out EGF simultaneously induce and carry out Cytotoxicity tests cell strain, result display, and this series compound is by acting on EGFR and inhibition of cell proliferation; Done the tumour modeling experiment in the body of rat, result shows simultaneously, and this series compound can the effectively propagation of Tumor suppression and transfer, and effectively alleviates the physiological signs of trouble mouse, has and has suitable pharmacologically active with marketed drug erlotinib and Gefitinib.

The present invention, compared with the antitumor drug EGFR inhibitor of going on the market, has the following advantages:

1, there is the new core skeleton of independent intellectual property right, alternative existing 4-aniline quinazoline core skeleton;

2, idiosyncrasy toxicity is as lower in probability of occurrence such as liver toxicity, LD ₅₀higher than front other medicines;

3, human body metabolism's product process is clear and definite, and such as this series compound only can by I phase metabolic enzyme P450 enzymes metabolism, and not by II phase metabolic enzyme as glucuronyl transferase, catechol oxygen transferase, institute's metabolism such as sulfonated enzyme.

Accompanying drawing explanation

Fig. 1 is the nucleus magnetic hydrogen spectrum figure of EGFR inhibitor ERI-09;

Fig. 2 is the nucleus magnetic hydrogen spectrum figure of EGFR inhibitor ERI-13.

Embodiment

Following examples will be further described the present invention, but not thereby limiting the invention.

Embodiment 1: the synthesis of epidermal growth factor receptor inhibitor (ERI-09)

1.68 grams of raw material A 2 are added in 25 ml single necked round bottom flask, 4,5-trihydroxy-acetophenone (10 mmol), under whipped state, add 6.9 grams, salt of wormwood (50 mmol) after dissolving raw material with 10 ml acetonitriles, at-20 DEG C, slowly drip methyl-chloroformate-acetonitrile solution (32 mmol).Whether transform completely, continue stirring and filter after 0.5 hour, revolve after steaming removing acetonitrile and obtain 3.2 grams of product B after raw material A conversion completely if reacting TLC detection raw material after 1 hour.Redissolve B with dry toluene, add sodium Metal 99.5 after reflux, there is condensation reaction and obtain 3.3 grams of crude products containing product C in B under the catalysis of sodium Metal 99.5.After chromatographic column is separated, acquisition 1.65 grams of product C sterlings, add 1.0 grams of m-chloro anilines after C being dissolved in acetonitrile, react 4-8 hour after stirring lower dropping NaOH solution at 60 DEG C, and TLC detects the rear 25% formic acid termination reaction of product C disappearance.Obtain 1.5 grams of product D after extraction into ethyl acetate, under agitation add 3.5 grams of salt of wormwood (25 mmol) with after acetonitrile redissolution product D, at 20 DEG C, slowly drip bromo-ethyl-methyl ether-acetonitrile solution (20 mmol).Whether transform completely, transform by 25% formic acid termination reaction completely until D, product is through extraction into ethyl acetate, dry, obtains 1.2 grams of finished product E after revolving steaming if reacting TLC detection compound D after 1 hour.Concrete synthetic route is as follows:

Embodiment 2: the synthesis of epidermal growth factor receptor inhibitor (ERI-13)

Under agitation add 3.5 grams of salt of wormwood (25 mmol) after 1.5 grams of product D are dissolved in acetonitrile, at 20 DEG C, slowly drip monobromomethane-acetonitrile solution (20 mmol).Whether transform completely, transform by 25% formic acid termination reaction completely until D, product is through extraction into ethyl acetate, dry, obtains 1.1 grams of finished product F after revolving steaming if reacting TLC detection compound D after 1 hour.Synthetic route is as follows:

The detection of embodiment 3 EGFR inhibitor (ERI-09) Non-small Cell Lung Cancer A 549 level

Non-small cell lung carcinoma cell line A549 is incubated at containing 10% fetal bovine serum (FBS, Hyclone, USA) HAM'S/F-12 substratum (HyClone, USA), and in this substratum, add 100ng/ml epidermal growth factor (EGF, Sigma, USA), above-mentioned cell, purchased from Shanghai Inst. of Life Science, CAS cellular resources center, is placed in CO ₂constant incubator, cultivates after 3-5 days for experiment.

Collect the cell being in logarithmic phase, be inoculated in (6500 cells/well/100 ul in 96 orifice plates respectively, RPMI1640 containing 100ng/ml EGF), after cultivating 24 h, add the compound (500,250,100,50,25,12.5,6.25,1 μMs/L) of gradient concentration respectively; The RPMI1640 of same volume 100ng/ml EGF is added 1% dimethyl sulfoxide (DMSO) (Dimethyl sulfoxide, DMSO) and be set to experiment contrast group; Only having nutrient solution but not adding cell is blank group.Each density component does not establish 3 parallel holes.After continuing cultivation 48 h, srb assay detects the survival rate of cell.Every hole adds the trichoroacetic acid(TCA) (Trichloroacetic acid, TCA) of the precooling (4 DEG C) of 50 μ l 10%, fixes 1 hour through 4 DEG C, after tap water cleans 5 times, add the SRB solution (Sigma, USA) of certain volume after abundant drying, put 37 DEG C of lucifuges and to dye 30 min.1% acetic acid wash for several times, dries, and the Tris solution (10 mM, pH 10.5) adding appropriate volume fully dissolves rearmounted microplate reader and measures absorbancy (A570 nm).According to following formulae discovery growth inhibition ratio (IR): IR (%)=[1-(experimental group A value-blank group A value)/(control group A value-blank group A value)] × 100 %, and adopt Origin 6.0 computed in software IC ₅₀value.The A549 cell strain of EGFR inhibitor ERI09 does not add the IC of EGF induction ₅₀value is 15 μm of ol, adds the IC of EGF induction ₅₀be 0.48 μm of ol, illustrate and by acting on EGFR, antitumous effect is produced to new core skeleton compound.

Embodiment 4 EGFR inhibitor acts on the Activity determination of artificial tumor model in Mice Body

Use 6-8 male C57BL/6 system mouse in age in week, be divided into trial drug group, model group, cyclophosphamide-a control group, often organize six, the EDTA Digestive system of Lewis lung cancer cells strain through 2.5g/L pancreatin+0.2g/L is digested, get single cell suspension, be injected in right fore oxter with every 0.2mL, observe tumor formation rate.Drug effect observation index is represented with the heavy inhibition percentage of knurl with the curative effect of solid tumor, test medicine EGFR inhibitor and negative control (physiological saline) and positive control (erlotinib) was given before bringing out, EGFR inhibitor demonstrates the restraining effect depending on dosage, and its ED ₅₀for 12mg/Kg, suitable with positive control drug erlotinib (10mg/Kg), the validity of new core skeleton EGFR inhibitor is described.

The metabolic activity of embodiment 5 EGFR inhibitor detects

The metabolism spectrum of application recombination human source I phase II phase metabolic enzyme system to EGFR inhibitor ERI-9 carries out identification and analysis, find that ERI-9 is only by the slow metabolism of I phase metabolic enzyme P450 enzyme, people's liver particle system transformation period is minute, 10% is extended than similar marketed drug, illustrate that the EGFR inhibitor of new skeleton has suitable metabolic stability, druggability is good.

Claims

1. a targeting antineoplastic medicine thing, is characterized in that: the core skeleton of this medicine, and concrete structure general formula is as follows:

R ₁and R ₂be the one in methyl, methoxyethyl simultaneously, R ₃for halogenic substituent Cl, R ₄for H;

The preparation method of this medicine is as follows: with 2, 4, 5-trihydroxy-acetophenone and methyl-chloroformate are starting raw material, take salt of wormwood as acid binding agent, the product after obtaining phenolic hydroxyl group protection for 1 hour is reacted at-20 DEG C, then in anhydrous system, close ring by the catalysis of sodium Metal 99.5 by aldol reaction and generate chroman scaffold intermediate, react at 60 DEG C and chroman intermediate and m-chloro aniline effect are generated 4-m-chloro aniline-6, 7-dihydroxycoumarin, then at 20 DEG C by bromo-ethyl-methyl ether-acetonitrile solution or monobromomethane-acetonitrile solution by 4-m-chloro aniline-6, 7-dihydroxycoumarin intermediate etherificate, reactant is by purification by flash chromatography and obtain target product after crystallization.

2. according to targeting antineoplastic medicine thing according to claim 1, it is characterized in that, described antitumor drug is tyrosine kinase inhibitor, and relative to the drug molecular structure gone on the market, this medicine has core skeleton catechol, and left side is etherification of hydroxyl groups part.

3. according to targeting antineoplastic medicine thing according to claim 1, it is characterized in that, described antitumor drug can be made into tablet, capsule or injection.

4. according to targeting antineoplastic medicine thing according to claim 1, it is characterized in that, described antitumor drug effect is the EGF-R ELISA of high expression level in Tumor suppression, reaches antineoplastic object.