CN104151376B - Anti-tumor medicament as well as synthetic method and application thereof - Google Patents

Abstract

The invention relates to an anti-tumor medicament namely an amino acid-doxorubicin derivative, a preparation method of the anti-tumor medicament, and an application of the anti-tumor medicament. Specifically, the invention relates to an amino acid-doxorubicin derivative with a structure shown in a formula I, pharmaceutically-acceptable salts of the amino acid-doxorubicin derivative, a medicament composition adopting the compound and the pharmaceutically-acceptable salts of the compound as active and effective components, a preparation method of the amino acid-doxorubicin derivative, the pharmaceutically-acceptable salts and the medicament composition, and an application of the amino acid-doxorubicin derivative, the pharmaceutically-acceptable salts and the medicament composition in treating cancers (liver cancer, acute leukemia, malignant lymphoma, breast cancer, ovarian cancer, soft tissue sarcoma, osteogenic sarcoma, rhabdomyosarcoma, kidney cancer, colon cancer, bladder cancer, thyroid cancer, prostatic cancer, testicular cancer, gastric cancer and the like), and preferentially treating the cancers (liver cancer, colon cancer, breast cancer, prostatic cancer and the like) with over-expressed L-type amino acid transporters (LATs).

Description

A kind of antitumor drug and its preparation method and use

Technical field

The present invention relates to the targeted delivery systems of a kind of doxorubicin that can be used in treating cancer and its analog, Particularly a kind of combination aspartic acid and doxorubicin and its analog and design compound, pharmaceutical composition, Its preparation method, medical usage, belong to medicinal chemistry art.

Background technology

One of difficult problem for the treatment of of cancer is to cancerous tissue or cancerous cell target administration, to reduce the damage of normal tissue.

And traditional anti-tumor medicine doxorubicin and its analog be just be found the second half in 19th century can be wide General for treating various tumors, including leukemia, bladder cancer, breast carcinoma, gastric cancer, pulmonary carcinoma, ovarian cancer, thyroid carcinoma, myeloma Etc. more effective cancer therapy drug.

Although numerous patent documentations all report substantial amounts of for improveing doxorubicin, and obtain analog (US4268451A、US4107423A、US4322412A、GB2067552A、FR2520365A1).But this kind of compound all exists The selection toxicity to cancerous cell poor, also larger injury being caused to normal cell while killing cancerous cell, thus leading More serious side effect, and the defect that the half-life is shorter are caused.Even some medicines also do not reach focus after being absorbed by the body Position will be metabolized, and bioavailability is low, and this makes pharmacokinetic property not ideal enough, bring larger to treatment of cancer Difficult.

And scientific research personnel is to solve the above problems, it has been also carried out the effort of hardships, made the work of a large amount of creativeness, such as It is prepared for the dosage forms such as the polyoxyl 40 stearate plastid of doxorubicin.But the problems referred to above are not still thoroughly solved, the controlling of cancer Treat there is still a need for new breakthrough mouth is found on the root problem of compound structure design.

Amino acid transporter (LATs) is widely present in the liver of mammal, bone marrow, brain, Placenta Hominiss, heart, testis etc. In tissue, and only there is expression in Placenta Hominiss and cerebral tissue, in malignant tumor such as colorectal cancer, gastric cancer, breast carcinoma, renal carcinoma in human body Etc. all there being specific high expression in kinds of tumor cells, its effect is to intracellular special picked-up L-type neutral amino acid (such as Leucine, histidine, methionine, Phenylalanine etc.) or the molecule containing similar structures.Experiment in vitro shows, LATs centering The transport activity of the molecule (as L-3,4 dihydroxyphenylalanine, melphalan etc.) of acidic amino acid or similar structures exceed 10 times of normal cell it was demonstrated that LATs is a new tumor diagnosis and therapy target spot.

And also not had both at home and abroad at present based on LATs transporter is the inventive concept of shot design medical compoundss.This Bright exactly as starting point, using tumor cell membrane overexpression LATs albumen as the basis of target administration, design is prepared and is obtained To a kind of brand-new targeting antineoplastic medicine compounds, improve the pharmacokinetic property of traditional antineoplastic, fill up domestic The outer blank based on drug transporter LATs design medicine compound field.

Content of the invention

In view of this, the chemical combination designing to seek new combination aspartic acid and doxorubicin and its analog Thing, pharmaceutical composition, its preparation method, medical usage, present inventor has performed further investigation, are paying substantial amounts of creativeness After work, thus completing the present invention.

Specifically, technical scheme and content are related to four aspects：In conjunction with aspartic acid and doxorubicin and Its analog and the compound that designs and its pharmaceutically acceptable salt, its preparation method, medical usage, drug regimen Thing.

In a first aspect, the present invention relates to a kind of combination aspartic acid and doxorubicin and its analog and the change that designs Compound and its pharmaceutically acceptable salt, shown in structural formula of compound such as following formula (I).

Wherein, R₁-R₉Each identical or different, and independently selected from H, hydroxyl, amino, sulfydryl, halogen, C₁-C₆Alkyl, C₁-C₆Alkoxyl, C₁-C₆Alkylamino, C₁-C₆Alkylthio group；

In the present invention, unless otherwise prescribed, from start to finish, C₁-C₆The implication of alkyl refers to there is 1-6 carbon atom Straight or branched alkyl, that includes C₁Alkyl, C₂Alkyl, C₃Alkyl, C₄Alkyl, C₅Alkyl or C₆Alkyl, example in non-limiting manner As being methyl, ethyl, n-pro-pyl, isopropyl, normal-butyl, sec-butyl, isobutyl group, the tert-butyl group, n-pentyl, isopentyl or just own Base etc..

In the present invention, unless otherwise prescribed, from start to finish, C₁-C₆Alkoxyl, C₁-C₆Alkylamino, C₁-C₆Alkylthio group is Refer to " C defined above₁-C₆Alkyl " be connected with O, N, S atom after group.

In the present invention, unless otherwise prescribed, from start to finish, the implication of halogen refers to halogen, non-exclusively for example Can be F, Cl, Br or I.

As a kind of preferred implementation, R₁-R₉Each identical or different, and independently selected from H, hydroxyl, amino, sulfydryl, Halogen, C₁-C₃Alkyl, C₁-C₃Alkoxyl, C₁-C₃Alkylamino, C₁-C₃Alkylthio group.

As a kind of preferred implementation, R₁-R₃For H or C₁-C₃Alkyl, R₄For C₁-C₃Alkoxyl, R₅-R₇For hydroxyl or C₁-C₃Alkoxyl, R₈For C₁-C₃Alkyl, R₉For hydroxyl or C₁-C₃Alkoxyl.

As a kind of preferred implementation, R₁-R₃For H, R₄For C₁-C₃Alkoxyl, R₅-R₇For hydroxyl, R₈For C₁-C₃Alkyl, R₉For hydroxyl.

As a kind of preferred implementation, R₁-R₃For H, R₄For methoxyl group, R₅-R₇For hydroxyl, R₈For methyl, R₉For hydroxyl, It is the structure as shown in following formula (II).

(II) doxorubicin-aspartic acid (Doxorubicin-Asp)

Second aspect, the present invention relates to the synthetic method of formula (I) structural compounds and its pharmaceutically acceptable salt.Pass through Method representative below is realized, wherein, unless specified otherwise herein, R₁-R₉There is implication any one of defined above. Corresponding reaction raw materials and reaction reagent can be obtained by technology well known in the art.

Methods described includes following route：

Methods described step is：The doxorubicin analog of formula (III) structure (can adopt skill well known in the art Art obtains, such as the preparation side in US4268451A, US4107423A, US4322412A, GB2067552A, FR2520365A1 Method) and the Fmoc-Asp-OALL of formula (IV) structure (aspartic acid with Fmoc and pi-allyl protection well known in the art, CAS steps on Mark 144120-53-6), in DIPEA (DIEA), 1- hydroxy benzo triazole (HOBt), 2- (7- diphenyl diimide And triazole)-tetramethylurea hexafluorophosphoric acid ester (HATU, CAS registration number 148893-10-1) participation under, in a solvent, described Solvent is one or more of dichloromethane, chloroform, oxolane, DMF or toluene, reacts under the conditions of room temperature, lucifuge Prepared compound V；V is again in PdCl afterwards₂(PPh₃)₂And Bu₃Under the catalysis of SnH, and in the presence of AcOH with dichloromethane it is Solvent, reacts under room temperature condition and compound VI is obtained；It is molten with dichloromethane, chloroform, oxolane, DMF or toluene again Matchmaker, with organic bases triethylamine, pyridine or hexahydropyridine as acid binding agent, reacts under room temperature condition and compounds I is obtained.

In the described synthetic method of the present invention, the post processing after reaction terminates can be using any in organic synthesis field Known conventional treatment means, such as any one of crystallization, recrystallization, chromatography over CC, extraction etc. processing means or many Plant the combination of processing means.As a kind of exemplary post processing means, for example, can be：The mixture obtaining after reaction is terminated inclines Enter in organic media such as ethyl acetate, dichloromethane etc., then sequentially use saturation NaHCO₃Aqueous solution and salt water washing, water layer is used After the extraction such as conventional organic media such as ethyl acetate, benzene, chloroform or oxolane, merge organic layer and (merge having after washing Machine layer and the organic layer being obtained by extraction), use anhydrous Na HSO₄Or anhydrous MgSO₄It is dried, negative pressure evaporation removes solvent, residue leads to Cross quick chromatography over CC, obtain target product, and using the compound structure such as nuclear-magnetism, mass spectrum confirmation handss known to organic field Section, confirms compound structure.

Above-mentioned product I can be made corresponding pharmaceutically acceptable salt using technology known to organic field.As A kind of exemplary salifying method, above-mentioned for gained product I is dissolved in Deca in DMF, acetone, methanol, ethanol or DMSO equal solvent Mineral acid or organic acid make pharmaceutically acceptable salt.Specifically by compound be dissolved in ether, DMF, acetone, methanol, ethanol, One of ethyl acetate or DMSO, under ice-water bath, Deca ethereal HCI, to pH=2, makes hydrochlorate；Or by variousization Compound is dissolved in one of ether, DMF, acetone, methanol, ethanol, ethyl acetate or DMSO, and under ice-water bath, Deca concentrated sulphuric acid is extremely PH=3, makes sulfate.

The third aspect, the present invention relates to the compound of formula (I) and/or its pharmaceutically acceptable salt are anti-swollen in preparation treatment Purposes in tumor medicine.Described tumor includes but is not limited to hepatocarcinoma, acute leukemia, carcinoma of prostate, breast carcinoma, colon cancer, testis Ball cancer, gastric cancer malignant lymphoma, lung bronchogenic carcinoma, ovarian cancer, nephroblastoma, bladder cancer, thyroid carcinoma etc. are wherein preferably Hepatocarcinoma, breast carcinoma, colon cancer, carcinoma of prostate etc. are capable of the cancer of overexpression LATs transporter.

Fourth aspect, the present invention relates to comprise the compound of formula (I) and/or the drug regimen of its pharmaceutically acceptable salt Thing.The more particularly, to compound of formula (I) and/or its pharmaceutically acceptable salt and pharmaceutically acceptable carrier and/or other The pharmaceutical composition of antitumor drug composition.Described pharmaceutical composition can be using technology preparation known to organic field.

As follows as a kind of preparation method of exemplary pharmaceutical composition：Make can connect on the compounds of this invention and galenic pharmacy The solid being subject to or liquid-carrier combine, and are allowed to arbitrarily be combined preparation with adjuvant acceptable on galenic pharmacy and excipient Become microgranule or microsphere.Solid dosage formss include tablet, discrete particles, capsule, slow releasing tablet, slow-release micro-pill etc..Solid carrier is permissible At least one material, its can serve as diluent, flavouring agent, solubilizing agent, lubricant, suspending agent, binding agent, disintegrating agent and Coating agent.Inert solid carrier includes magnesium phosphate, magnesium stearate, smoothers sugar, Lactose, pectin, propylene glycol, polyoxyethylene sorbitan monoleate, paste Essence, starch, gelatin, cellulose substances such as methylcellulose, Microcrystalline Cellulose, low melt point paraffin, Polyethylene Glycol, manna Alcohol, cocoa butter etc..Liquid dosage form includes solvent, suspension such as injection, powder etc..

The amount of the active ingredient (the compounds of this invention) containing in pharmaceutical composition and unit dosage form can be according to patient The state of an illness, the situation of diagnosis be specifically applied, the amount of compound used or concentration are in a wider scope Adjust, generally, the amount scope of reactive compound is the 0.5%-90% (weight) of compositionss.Another preferred scope is 0.5%- 70%.

Brief description

Fig. 1 is the compound of formula (I) of the present invention；

Fig. 2 is the HPCL detection of the compound doxorubicin-aspartic acid (Doxorubicin-Asp) of formula (II) of the present invention Feature；

Fig. 3 is the mass spectrum table of the compound doxorubicin-aspartic acid (Doxorubicin-Asp) of formula (II) of the present invention Levy；

Fig. 4 is medicine DOX and Asp-DOX of the present invention to hepatoma H22 cells (a) cytotoxicity result；

Fig. 5 is the cytotoxicity result to hepatoma cell strain HCT116 for medicine DOX and Asp-DOX of the present invention；

Fig. 6 is the cytotoxicity result to breast carcinoma cell strain MCF7 for medicine DOX and Asp-DOX of the present invention；

Fig. 7 is the cytotoxicity result to lung cancer cell line H1299 for medicine DOX and Asp-DOX of the present invention；

Fig. 8 is the Drug-time curve of DOX and Asp-DOX of the present invention；

Fig. 9 is comparison medicine DOX of the present invention and target medicine Asp-DOX organizes in nude mice lotus human liver cancer (HepG2) model and divides Cloth situation；

Figure 10 is comparison medicine DOX of the present invention and target medicine Asp-DOX tissue distribution feelings in nude mice lotus human colon carcinoma model Condition；

Figure 11 is that DOX and Asp-DOX tail vein injections of the present invention are administered 3 weeks nude mice lotus human liver cancer Model Tumor volumes Change.

Specific embodiment

Below by specific embodiment, the present invention is described in detail, but the purposes of these exemplary embodiments and Purpose is only used for enumerating the present invention, and not the real protection scope of the present invention is constituted with any type of any restriction, more non-general Protection scope of the present invention is confined to this.

Embodiment 1

MS AB SCIEX company 4000LC/MS/MS type instrument measures, and is EI source in addition to special indicating (70ev)；Before use all through redistillation, the anhydrous solvent being used is all to obtain by standard method dried to all solvents ?；Unless otherwise specified, all reactions carry out and carry out TLC tracking at room temperature, and the purification of product is equal unless otherwise specified Using silica gel (300-400 mesh) column chromatography；Wherein silica gel (300-400 mesh) is produced by Haiyang Chemical Plant, Qingdao, GF254 thin layer Silica gel plate is produced by Yantai Jiang You silica gel development corporation, Ltd.；Prepared product is confirmed through HPLC and MS.

The preparation of Formula II structural compounds (doxorubicin-aspartic acid/Doxorubicin-Asp/Asp-DOX), preparation Route is as follows：

Preparation method be doxorubicin and Fmoc-Asp-OALL in the presence of DIEA, HOBt, HATU, with dichloromethane, Chloroform, oxolane, DMF or toluene etc. are solvent, react compound Fmoc-Asp- is obtained under the conditions of room temperature, lucifuge (OALL)-DOX；Fmoc-Asp- (OALL)-DOX is again in PdCl afterwards₂(PPh₃)₂And Bu₃Under the catalysis of SnH, and AcOH's With dichloromethane as solvent under participation, react under room temperature condition and compound Fmoc-Asp-DOX is obtained；Again with dichloromethane, trichlorine Methane, oxolane, DMF or toluene are solvent, with organic bases triethylamine, pyridine or hexahydropyridine as acid binding agent, room temperature condition Lower reaction is obtained compound (II) doxorubicin-aspartic acid/Doxorubicin-Asp/Asp-DOX.

Comprise the following steps that：

1), Fmoc-Asp-OALL (2.32g, 4mmol) is placed in 100mL reaction bulb, adds cold DMF (20mL) stirring So that it is dissolved, add HATU (2.52g, 6.58mmol), stirring makes it sequentially add HOBt (0.9g, 6.58mmol) after dissolving With DIEA (218 μ l, 13.16mmol) stirring a moment reaction system uniformly mix, be eventually adding intermediate doxorubicin (2g, 5mmol), finish reaction system and be changed into peony, magnetic stirring apparatuss react 16h, TLC detection display reaction terminates (developing solvent Ethyl acetate/petroleum ether).After adding 50ml CH2Cl2 dilution, with the water soluble ingredient in 50ml water extractive reaction system, extract Take 3 times, be filtered to remove impurity, merge organic layer, anhydrous sodium sulfate drying, avoid light place is overnight.Filter, evaporated under reduced pressure solvent, Obtain red powder.Products therefrom silica gel column chromatography is purified, and obtains Fmoc-Asp- (OALL)-DOX pink solid 2.92g.Pure Degree 98.2% (HPLC normalization method), yield 80.60%.

2), take 2mmol Fmoc-Asp- (OALL)-DOX, add 20ml CH₂Cl₂It is completely dissolved product, sequentially add 0.08mmol PdCl₂(PPh₃)₂, 5mmol glacial acetic acid (former compound be completely dissolved after add under a kind of compound), afterwards It is rapidly added 4mmol Bu under magnetic stirring₃SnH, after on magnetic stirring apparatuss, lucifuge reacts 10～30min, TLC detection display Reaction terminates (developing solvent ethyl acetate/petroleum ether=1/1, v/v), by evaporation and concentration after reacting liquid filtering to 2ml, products therefrom Purified with silica gel column chromatography, obtain pink solid 1.362g of intermediate product Fmoc-Asp-DOX, (HPLC returns purity 95.5% One change method), yield 84.1%.

3), take 9ml DMF dissolving 2) in gained intermediate product Fmoc-Asp-DOX (1mmol), stirring be completely dissolved after, plus Enter 1ml hexahydropyridine, be added dropwise over while stirring, stopped reaction after 30min, evaporated under reduced pressure solvent, dissolved with 2-5ml methanol, Products therefrom silica gel column chromatography purification obtains the aubergine solid 0.52g of product doxorubicin-aspartic acid, purity 98.9% (HPLC normalization method), yield 80.0%.ESI-MS：659.41.

Embodiment 2

Compound doxorubicin-aspartic acid hydrochloric acid salt：Take compound purple solid product 1.5g, be dissolved in 10mL anhydrous Ethanol.Ice-water bath is cooled to 5 DEG C, and Deca 11.1% ethanol solution hydrochloride to pH is 2, continues at stir about 1h under ice-water bath.Cross Filter, vacuum drying, obtain violet solid powder.

Embodiment 3

Compound doxorubicin-aspartic acid becomes taurate：Take purple solid product 2.0g of compound, be dissolved in 10mL Acetone.Add equimolar taurine after being heated to backflow, continue at stirred at reflux reaction about 1.5h.Reaction finishes, in room temperature Lower standing 24h.Separate out purple crystal, filter, vacuum drying, that is, obtain the taurate of I.

Embodiment 4

Compound doxorubicin-aspartic acid becomes sulfate：Take compound purple solid product 1.2g, be dissolved in 15mL first Alcohol.Ice-water bath is cooled to 0 DEG C, and Deca 10% methanolic solution to pH is 3, continues at stir about 1h under ice-water bath.Filter, obtain Dark red solid.

Embodiment 5

The preparation of injection：

The hydrochlorate 200mg of doxorubicin-aspartic acid；Propylene glycol 100mg；Polysorbate 0.3～0.5%；Distilled water 300mL

Preparation method：Take active component to be added in solubilized solution Polysorbate and the water for injection of propylene glycol, add medicinal Alkali adjusts pH value makes it dissolve to 4～8.Add activated carbon, stirring and adsorbing 30min, carbon removal, fine straining, embedding, sterilizing.

Embodiment 6

The preparation of injection lyophilized powder：

The taurate 100mg of doxorubicin-aspartic acid；Medicinal basic 0.1-7.0%；Mannitol 55-85%.

Preparation method：Active component is taken to add water for injection, adjusting pH value with medicinal basic makes it dissolve to 4-8.Add Mannitol, carries out autoclaving by the requirement of injection, adds activated carbon, using filtering with microporous membrane, filtrate carries out subpackage, adopts With freeze-drying, loose block, sealing are obtained, obtain final product.

Biological analysiss

Used by this experiment, JEG-3 HepG2, HCT116, MCF7 and H1299 cell strain are purchased from Chinese Academy of Sciences typical case Culture collection committee cell bank, and through Shanghai Inst. of Life Science, CAS cell resource center certification.Its Middle tri- kinds of cell strains of HepG2, HCT116 and MCF7 are LATs positive cell strain, and H1299 is the cell strain not expressing LATs.

1st, LATs positive tumor cell cellulotoxic experiment

HepG2 cell model

Choose LATs- positive tumor cell HepG2, detect the cytotoxicity of medicine by the method for MTT, cytotoxicity The strong and weak inhibitory action (IR) passing through drug on tumor cell growth is characterized and is made with LATs- negative tumor cells H1299 For negative control.

Medicine DOX and Asp-DOX is as shown in Figure 4 to hepatoma H22 cells (a) cytotoxicity result.

HCT116 cell model

Choose LATs- positive tumor cell HCT116, detect the cytotoxicity of medicine, cytotoxicity by the method for MTT Power carry out characterizing concrete outcome following medicine DOX and Asp- by the inhibitory action (IR) of drug on tumor cell growth DOX is as shown in Figure 5 to the cytotoxicity result of hepatoma cell strain HCT116.

MCF7 cell model

Choose LATs- positive tumor cell MCF7 and to detect the cytotoxicity of medicine, cytotoxicity by the method for MTT Power carry out characterizing concrete outcome by the inhibitory action (IR) of drug on tumor cell growth as follows：

Medicine DOX and Asp-DOX is as shown in Figure 6 to the cytotoxicity result of breast carcinoma cell strain MCF7.

H1299 cell model

Choose the negative tumor cells H1299 not expressing LATs and to detect the cytotoxicity of medicine by the method for MTT, It is as follows that the power of cytotoxicity carries out sign concrete outcome by the inhibitory action (IR) of drug on tumor cell growth

Medicine DOX and Asp-DOX is as shown in Figure 7 to the cytotoxicity result of lung cancer cell line H1299；

Medicine shows to cancerous cell toxicity data, aspartic acid-doxorubicin (Asp-DOX) to HepG2, HCT116, The inhibitory action of the growth of MCF7 is significantly higher than doxorubicin (DOX).Compared with DOX, Asp-DOX is positive to LATs swollen for this explanation Oncocyte has significance inhibitory action, and weaker to the inhibitory action of LATs negative tumor cells.Thus also illustrate that Asp- The targeted therapy effect of the cancer to LATs overexpression for the DOX, and the injury to the tissue not expressing LATs transporter is less.

2nd, Absorption test

Test-compound aspartic acid-doxorubicin (Asp-DOX) weighs appropriate compound before use and is dissolved in DPBS buffering In saline solution, then obtain being diluted to 10,20,50 μ g/ml.Matched group gives the solvent of same volume.

Subject cell is the positive hepatoma H22 cells of LATs and the lung cancer cell line H1299 of LATs feminine gender.Before administration Two kinds of cells are seeded in 24 porocyte culture plates under sterile conditions, 24 porocyte culture plates are positioned over constant temperature culture In case, 5%CO₂, 37 DEG C, under the conditions of culture 48h about cover with hole to cell.Siphon away old culture medium afterwards, be subsequently adding and contain There is DMEM (containing the 10%FBS) culture medium of 0,10,20 and 50ug/ml doxorubicin and aspartic acid-doxorubicin, continue culture Cleaned 3 times with cold DPBS buffer solution after 2 hours, terminate administration.

Take drug treating 2 hours and terminate the tumor cell after being administered, every hole adds medicine in 400ul methanol extraction cell Content, supersound process 15-30min, be centrifuged (10000r/min, 5min), remove precipitation, take supernatant 40ul to carry out HPLC analysis Measure medicament contg, result is as shown in the table：

^**, P ＜ 0.01：ND.not detected

HPLC analysis result shows, 3 dosage Asp-DOX processed LATs positive tumor cell strain HepG2, cell extraction In thing, the content of medicine is all remarkably higher than the HepG2 cell that DOX was processed；However, in LATs negative cells H1299, Asp- The extract drug content of the cell that DOX was processed then is substantially less than the cell that DOX was processed.After this means that Asp modifies DOX be Asp-DOX, to LATs-, positive cell targeted significance improves, and to the targeting of the cell not expressing LATs relatively Weak.

3rd, pharmacokinetic studies

(tested ALB/c-nu nude mouse, male, SPF level, 18～20g, buy and tie up tonneau in Beijing to take 80 kunming mices Magnificent laboratory animal Technology Co., Ltd.) it is divided into two big groups one group injection DOX, another group of injection Asp-DOX, each big group is divided into 8 Individual group, gives the dosage of 5mg/kg respectively by every group 5, and administering mode is tail vein injection.Two administration groups arrange 8 and take Blood point：2min,5min,10min,15min,30min,60min,120min,240min.Blood mode is taken to take blood for eye frame.Centrifugation, 3000r/min, 4 DEG C, 15min, take serum 200ul, be separately added into 1ml methanol and 50ul internal standard (alpha-Naphthol, 2mg/ml), vortex Concussion 1min, takes supernatant to dry up organic solvent in Nitrogen evaporator.Addition 160ul mobile phase afterwards, gravity treatment extract, then short Temporarily centrifugation goes supernatant 40ul HPLC to analyze after going precipitation.Chromatographic condition is：C8 pillar (Angelent, 200*4.6mm, 0.45um), mobile phase is methanol/0.01M ammonium acetate/H₃PO₄=145/155/0.16 (v/v).Pharmacokinetic results are as follows：

The Drug-time curve of DOX and Asp-DOX is as shown in Figure 8.

Understand that the Asp-DOX half-life of removing in blood significantly extends by original by pharmacokinetic studies result 19.1min extends to 49.3min, and area under the drug-time curve also dramatically increases (being doxorubicin more than 8 times).

4th, tissue distribution experiment

Human liver cancer (HepG2) model

Tested ALB/c-nu nude mouse, male, SPF level, 18～20g, buys in Beijing dimension tonneau China laboratory animal technology Company limited.Cage for animal is raised, and normal feedstuff is experimental mouse full-valence pellet feed, purchased from Beijing dimension tonneau China laboratory animal technology Company limited, free water, humidity 60-80%, the light and shade cycle of natural lighting and about 12h.First carry out one week before experiment Convalescent period observes, and is divided into 3 to organize every group 10, by the tumor to nude mice front left limb axillary fossa subcutaneous injection 0.1～0.2ml afterwards Cell suspension (1*10⁷Cell/ml) method, set up nude mice lotus human liver cancer (HepG2) model, HepG2 is LATs positive cell Strain.When gross tumor volume reaches 100mm³During left and right to three kinds of tumor bearing nude mices respectively tail vein injections with the DOX of 5mg/kg and Asp-DOX (wherein DOX is comparison), every kind of medicine does 5 animal subjects.After administration 1h, cervical dislocation puts to death animal subject, point Tumor, the heart, liver, spleen, lung, kidney, brain and 8 kinds of tissues of testis of not taking every animal subject do tissue homogenate, and in tissue Medicine is extracted, and by the method for HPLC, the medicament contg in each tissue is analyzed with (unit, mg/g organizes) and carries out medicine The tissue distribution research of thing.Tissue distribution results are as follows：

Comparison medicine DOX and target medicine Asp-DOX tissue distribution patterns such as Fig. 9 in nude mice lotus human liver cancer (HepG2) model Shown.

Nude mice lotus people's tumor model tissue distribution results display Asp-DOX administration group medicine is in LATs- positive tumor HepG2 Content significance be higher than content in tumor tissues for the DOX administration group medicine, but DOX administration group medicine in other organs and Content in tissue is higher than then Asp-DOX administration group.This explanation Asp-DOX significantly improves to the targeting of LATs- positive tumor.

Human colon carcinoma (HCT116) model

Tested ALB/c-nu nude mouse, male, SPF level, 18～20g, buys in Beijing dimension tonneau China laboratory animal technology Company limited.Cage for animal is raised, and normal feedstuff is experimental mouse full-valence pellet feed, purchased from Beijing dimension tonneau China laboratory animal technology Company limited, free water, humidity 60-80%, the light and shade cycle of natural lighting and about 12h.First carry out one week before experiment Convalescent period observes, and is divided into 3 to organize every group 10, by the tumor to nude mice front left limb axillary fossa subcutaneous injection 0.1～0.2ml afterwards Cell suspension (1*10⁷Cell/ml) method, set up nude mice lotus human colon carcinoma (HCT116) model and nude mice lotus human lung cancer (H1299) model, wherein HCT116 are LATs positive cell strains, and H1299 is LATs negative cells strain.When gross tumor volume reaches 100mm³During left and right, to three kinds of tumor bearing nude mices difference tail vein injections, with DOX and Asp-DOX of 5mg/kg, (wherein DOX is right According to), every kind of medicine does 5 animal subjects.After administration 1h, cervical dislocation puts to death animal subject, takes every animal subject respectively Tumor, the heart, liver, spleen, lung, kidney, brain and 8 kinds of tissues of testis do tissue homogenate, and the medicine in tissue is extracted, and pass through The method of HPLC is analyzed the tissue distribution research that (unit, mg/g organizes) carries out medicine to the medicament contg in each tissue. Tissue distribution results are as follows：Tissue distribution patterns are such as in nude mice lotus human colon carcinoma model for comparison medicine DOX and target medicine Asp-DOX Shown in Figure 10.

Nude mice lotus people's tumor model tissue distribution results display Asp-DOX administration group medicine is in LATs- positive tumor HCT116 In content significance be higher than content in tumor tissues for the DOX administration group medicine, but DOX administration group medicine is in other organs It is higher than then Asp-DOX administration group with the content in tissue.

5th, anti-tumor in vivo effect experiment

Tested ALB/c-nu nude mouse, male, SPF level, 18～20g, buys in Beijing dimension tonneau China laboratory animal technology Company limited.Cage for animal is raised, and normal feedstuff is experimental mouse full-valence pellet feed, purchased from Beijing dimension tonneau China laboratory animal technology Company limited, free water, humidity 60-80%, the light and shade cycle of natural lighting and about 12h.First carry out one week before experiment Convalescent period observes, and is divided into 2 to organize every group 5, by the tumor to nude mice front left limb axillary fossa subcutaneous injection 0.1～0.2ml afterwards Cell suspension (1*10⁷Cell/ml) method, set up nude mice lotus human liver cancer (HepG2) model, in gross tumor volume hypertrophy extremely 100mm³When, respectively two groups of animal subjects are given with same dose (5mg/kg, 2 times/week, 3 weeks, tail vein injections were administered) DOX and Asp-DOX folk prescription is treated, and measures weekly gross tumor volume (1/2 tumor long * tumor width²).Experimental result is as follows：

DOX and Asp-DOX tail vein injections are administered change such as Figure 11 institute of 3 weeks nude mice lotus human liver cancer Model Tumor volumes Show.

Understand that Asp-DOX shows to the growth inhibition effect of tumor in nude mice lotus people's tumor model by internal pharmacodynamic experiment result Work is better than positive control drug DOX.

In sum, can clearly be found out by above-mentioned all embodiments, present invention obtains brand-new transports egg based on LATs The antitumor drug with excellent anti-tumor activity and pharmacokineticss behavior designing, has paid substantial amounts of creativeness in vain Work, the design for the treatment of cancer, medical compoundss molecule provides brand-new design.

It should be appreciated that the purposes of these embodiments is merely to illustrate the present invention and is not intended to limit the protection model of the present invention Enclose.Additionally, it will also be appreciated that after the technology contents having read the present invention, those skilled in the art can make each to the present invention Plant and change, change and/or modification, all these equivalent form of value equally falls within the guarantor that the application appended claims are limited Within the scope of shield.

Claims (7)

1. a kind of compound or its pharmaceutically acceptable salt, shown in its structural formula of compound such as following formula (I)：

Wherein：R₁-R₃For H or C₁-C₃Alkyl, R₄For C₁-C₃Alkoxyl, R₅-R₇For hydroxyl or C₁-C₃Alkoxyl, R₈For C₁-C₃Alkane Base, R₉For hydroxyl or C₁-C₃Alkoxyl.

2. compound as claimed in claim 1 or its pharmaceutically acceptable salt it is characterised in that：R₁-R₃For H, R₄For C₁-C₃ Alkoxyl, R₅-R₇For hydroxyl, R₈For C₁-C₃Alkyl, R₉For hydroxyl.

3. the compound as described in any one in claim 1 to 2 or its pharmaceutically acceptable salt it is characterised in that：R₁- R₃For H, R₄For methoxyl group, R₅-R₇For hydroxyl, R₈For methyl, R₉For hydroxyl, the as structure as shown in following formula (I I),

.

4. the synthetic method of compound as claimed in claim 1 or its pharmaceutically acceptable salt is it is characterised in that include following Route：

Wherein, R₁-R₉As the corresponding definition in claim 1.

5. synthetic method as claimed in claim 4 it is characterised in that：Methods described step is the how soft ratio of formula (III) structure Star analog and the Fmoc-Asp-OALL of formula (IV) structure, diisopropylethylamine, hydroxybenzotriazole, HATU ginseng With under, in a solvent, described solvent is one or more of dichloromethane, chloroform, oxolane, DMF or toluene, room Under the conditions of temperature, lucifuge, reaction is obtained compound V；V is again in PdCl afterwards₂(PPh₃)₂And Bu₃Under the catalysis of SnH, and AcOH's With dichloromethane as solvent under participation, react under room temperature condition and compound VI is obtained；Again with dichloromethane, chloroform, tetrahydrochysene Furan, DMF or toluene are solvent, with organic bases triethylamine, pyridine or hexahydropyridine as acid binding agent, react and be obtained under room temperature condition Compound (I).

6. the compound any one of claim 1-3 or its pharmaceutically acceptable salt are in preparation treatment pulmonary carcinoma, hepatocarcinoma Or the purposes in colon cancer drug.

7. comprise the pharmaceutical composition of compound any one of claim 1-3 or its pharmaceutically acceptable salt.