CN104151376A - Anti-tumor medicament as well as synthetic method and application thereof - Google Patents
Anti-tumor medicament as well as synthetic method and application thereof Download PDFInfo
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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
Technical field
The present invention relates to a kind for the treatment of Dx of cancer and target administration system of analog thereof of can be used in, the compound, pharmaceutical composition, its preparation method, the medicinal use that relate to especially a kind of combination aspartic acid and Dx and analog thereof and design, belong to pharmaceutical chemistry field.
Background technology
One of difficult problem of cancer therapy is to cancerous tissue or cancer cells target administration, to reduce the damage of normal tissue.
And traditional antineoplastic thing Dx and analog thereof are the just found various tumours for the treatment of that can the be widely used in second half in 19th century, comprise the comparatively effectively cancer therapy drug of leukemia, bladder cancer, mammary cancer, cancer of the stomach, lung cancer, ovarian cancer, thyroid carcinoma, myelomatosis etc.
Although it is improvement Dx that numerous patent documentations have all been reported a large amount of, and obtains analog (US4268451A, US4107423A, US4322412A, GB2067552A, FR2520365A1).But it is poor that this compounds all exists the selection toxicity of cancer cells, also normal cell caused to larger injury in killing cancer cells, thereby caused comparatively severe side effect, and shorter defect of transformation period.Even some medicine does not also arrive lesions position and will be fallen by metabolism after being absorbed by the body, and bioavailability is low, and this makes pharmacokinetic property not ideal enough, brings larger difficulty to cancer therapy.
And scientific research personnel is for addressing the above problem, also carry out hard effort, make a large amount of creationary work, as prepared the formulations such as the Macrogol Ester plastid of Dx.But the problems referred to above are not still solved thoroughly, the treatment of cancer still need to be found new breakthrough mouth on the root problem of compound structure design.
Amino acid transport body (LATs) is extensively present in the tissues such as mammiferous liver, marrow, brain, placenta, heart, testis, and in human body, only in placenta and cerebral tissue, have an expression, in malignant tumour as large bowel cancer, cancer of the stomach, mammary cancer, in the kinds of tumor cells such as kidney, all have specific high expression level, its effect is to special picked-up L-type neutral amino acids (as leucine, Histidine, methionine(Met), phenylalanine etc.) in cell or the molecule that contains similar structures.Experiment in vitro demonstration, the transport activity of the molecule (as L-3,4 dihydroxyphenylalanine, melphalan etc.) of LATs centering amino acid or similar structures exceeds 10 times of normal cells, proves that LATs is a new tumor diagnosis and therapy target spot.
Be the inventive concept of shot design medical compounds and also do not had based on LATs transporter both at home and abroad at present.The present invention is just as starting point, cross and express the basis of LATs albumen as target administration using tumor cell membrane, design is prepared and is obtained a kind of brand-new targeting antineoplastic medicine compounds, improve the pharmacokinetic property of traditional antineoplastic, fill up the blank based on drug transporter LATs designer drug compounds field both at home and abroad.
Summary of the invention
In view of this, the compound, pharmaceutical composition, its preparation method, the medicinal use that design in order to seek new combination aspartic acid and Dx and analog thereof, the inventor conducts in-depth research, and is paying after a large amount of creative works, thereby is completing the present invention.
Particularly, technical scheme of the present invention and content relate to four aspects: in conjunction with aspartic acid and Dx and analog thereof and the compound designing and pharmacy acceptable salt thereof, its preparation method, medicinal use, pharmaceutical composition.
First aspect, the present invention relates to a kind of combination aspartic acid and Dx and analog thereof and the compound and the pharmacy acceptable salt thereof that design, and structural formula of compound is as shown in the formula shown in (I).
Wherein, R1-R9 is identical or different separately, and independently selected from H, hydroxyl, amino, sulfydryl, halogen, C
1-C
6alkyl, C
1-C
6alkoxyl group, C
1-C
6alkylamino, C
1-C
6alkylthio;
In the present invention, unless otherwise prescribed, from start to finish, C
1-C
6the implication of alkyl refers to the straight or branched alkyl with 1-6 carbon atom, and it has comprised C
1alkyl, C
2alkyl, C
3alkyl, C
4alkyl, C
5alkyl or C
6alkyl, for example can be to indefiniteness methyl, ethyl, n-propyl, sec.-propyl, normal-butyl, sec-butyl, isobutyl-, the tertiary butyl, n-pentyl, isopentyl or n-hexyl etc.
In the present invention, unless otherwise prescribed, from start to finish, C
1-C
6alkoxyl group, C
1-C
6alkylamino, C
1-C
6alkylthio refers to " C defined above
1-C
6alkyl " group after being connected with O, N, S atom.
In the present invention, unless otherwise prescribed, from start to finish, the implication of halogen refers to haloid element, non-exclusively for example can be F, Cl, Br or I.
As a kind of preferred implementation, R1-R9 is identical or different separately, and independently selected from H, hydroxyl, amino, sulfydryl, halogen, C
1-C
3alkyl, C
1-C
3alkoxyl group, C
1-C
3alkylamino, C
1-C
3alkylthio.
As a kind of preferred implementation, R1-R3 is H or C
1-C
3alkyl, R4 are C
1-C
3alkoxyl group, R5-R7 are hydroxyl or C
1-C
3alkoxyl group, R8 are C
1-C
3alkyl, R9 are hydroxyl or C
1-C
3alkoxyl group.
As a kind of preferred implementation, R1-R3 is that H, R4 are C
1-C
3alkoxyl group, R5-R7 are that hydroxyl, R8 are C
1-C
3alkyl, R9 are hydroxyl.
As a kind of preferred implementation, R1-R3 is that H, R4 are that methoxyl group, R5-R7 are that hydroxyl, R8 are that methyl, R9 are hydroxyl, is as shown in the formula the structure shown in (II).
[0019](II) Dx-aspartic acid (Doxorubicin-Asp)
Second aspect, the present invention relates to the synthetic method of formula (I) structural compounds and pharmacy acceptable salt thereof.Realize by following representative method, wherein, unless specified otherwise herein, R1-R9 has any implication in definition above.Can obtain corresponding reaction raw materials and reaction reagent by technology well known in the art.
Described method comprises following route:
Described method steps is: the Dx analog of formula (III) structure (can adopt technology well known in the art to obtain, for example US4268451A, US4107423A, US4322412A, GB2067552A, preparation method in FR2520365A1) and the Fmoc-Asp-OALL of formula (IV) structure (aspartic acid with Fmoc and allyl group protection well known in the art, CAS registration number 144120-53-6), at N, N-diisopropylethylamine (DIEA), 1-hydroxy benzo triazole (HOBt), 2-(7-azo benzotriazole)-tetramethyl-urea phosphofluoric acid ester (HATU, CAS registration number 148893-10-1) participation under, in solvent, described solvent is methylene dichloride, trichloromethane, tetrahydrofuran (THF), one or more in DMF or toluene, room temperature, under lucifuge condition, reaction makes compound V, V is again at PdCl afterwards
2(PPh
3)
2and Bu
3under the catalysis of SnH, and in the presence of AcOH taking methylene dichloride as solvent, under room temperature condition, reaction makes compound VI, taking methylene dichloride, trichloromethane, tetrahydrofuran (THF), DMF or toluene as solvent, taking organic bases triethylamine, pyridine or hexahydropyridine as acid binding agent, under room temperature condition, reaction makes chemical compounds I again.
In described synthetic method of the present invention, aftertreatment after reaction finishes can adopt any known conventional processing means in organic synthesis field, such as, any processing means in crystallization, recrystallization, column chromatography purification, extraction etc. or the combination of multiple processing means.As the exemplary aftertreatment means of one, for example, can be: the mixture impouring organic medium obtaining after reaction is finished, as in ethyl acetate, methylene dichloride etc., is then used saturated NaHCO in turn
3the aqueous solution and salt water washing, water layer with conventional organic medium as the extraction such as ethyl acetate, benzene, chloroform or tetrahydrofuran (THF) after, merge organic layer (merging the organic layer that organic layer after washing and extraction obtain), use anhydrous Na HSO
4or anhydrous MgSO
4dry, negative pressure evaporation is except desolventizing, and residue is purified by flash column chromatography, obtains target product, and adopts the compound structure confirmation means such as the known nuclear-magnetism in organic field, mass spectrum, confirms compound structure.
Can adopt the known technology in organic field to make corresponding pharmacy acceptable salt above-mentioned product I.As a kind of exemplary salifying method, above-mentioned gained product I is dissolved in and in DMF, acetone, methyl alcohol, ethanol or DMSO equal solvent, drips mineral acid or organic acid is made pharmacy acceptable salt.Specifically compound is dissolved in to the one in ether, DMF, acetone, methyl alcohol, ethanol, ethyl acetate or DMSO, under ice-water bath, drips salt acid ether to pH=2, make hydrochloride; Or various compounds are dissolved in to the one in ether, DMF, acetone, methyl alcohol, ethanol, ethyl acetate or DMSO, under ice-water bath, drip the vitriol oil to pH=3, make vitriol.
The third aspect, the compound and/or its pharmacy acceptable salt that the present invention relates to formula (I) are treated the purposes in antitumor drug in preparation.Described tumour includes but not limited to liver cancer, and acute leukemia, prostate cancer, mammary cancer, colorectal carcinoma, carcinoma of testis, cancer of the stomach malignant lymphoma, lung bronchogenic carcinoma, ovarian cancer, the nephroblastoma, bladder cancer, thyroid carcinoma etc. are wherein preferably liver cancer, mammary cancer, colorectal carcinoma, prostate cancer etc. can cross the cancer of expressing LATs transporter.
Fourth aspect, the present invention relates to the compound of contained (I) and/or the pharmaceutical composition of its pharmacy acceptable salt.Relate in particular to the compound of formula (I) and/or the pharmaceutical composition of its pharmacy acceptable salt and pharmaceutically acceptable carrier and/or other antitumor drug composition.Described pharmaceutical composition can adopt the known technology preparation in organic field.
Preparation method as a kind of exemplary pharmaceutical composition is as follows: the compounds of this invention acceptable solid or liquid vehicle on technology of pharmaceutics are combined, and make it at random on technology of pharmaceutics acceptable auxiliary and vehicle and be combined and be prepared into particulate or microballoon.Solid dosage comprises tablet, discrete particles, capsule, slow releasing tablet, sustained release pellet etc.Solid carrier can be at least one material, and it can serve as thinner, flavouring agent, solubilizing agent, lubricant, suspension agent, tackiness agent, disintegrating agent and coating agent.Inert solid carrier comprises trimagnesium phosphate, Magnesium Stearate, smoothers sugar, lactose, pectin, propylene glycol, Polysorbate 80, dextrin, starch, gelatin, cellulose substances such as methylcellulose gum, Microcrystalline Cellulose, low melt point paraffin, polyoxyethylene glycol, N.F,USP MANNITOL, theobroma oil etc.Liquid dosage form comprises solvent, suspension for example injection, pulvis etc.
The amount of the active ingredient (the compounds of this invention) containing in pharmaceutical composition and unit dosage form can specifically be applied according to the situation of patient's the state of an illness, diagnosis, the amount of compound used or concentration regulate in a wider scope, conventionally the 0.5%-90%(weight that, the weight range of active compound is composition).Another preferred scope is 0.5%-70%.
Brief description of the drawings
Fig. 1 is the compound of formula of the present invention (I);
Fig. 2 is that the HPCL of the compound Dx-aspartic acid (Doxorubicin-Asp) of formula of the present invention (II) detects feature;
Fig. 3 is the mass spectral characteristi of the compound Dx-aspartic acid (Doxorubicin-Asp) of formula of the present invention (II);
Fig. 4 is that medicine DOX of the present invention and Asp-DOX are to hepatoma cell strain HepG2(a) cytotoxicity result;
Fig. 5 is medicine DOX of the present invention and the cytotoxicity result of Asp-DOX to hepatoma cell strain HCT116;
Fig. 6 is medicine DOX of the present invention and the cytotoxicity result of Asp-DOX to breast carcinoma cell strain MCF7;
Fig. 7 is medicine DOX of the present invention and the cytotoxicity result of Asp-DOX to lung cancer cell line H1299;
Curve when Fig. 8 is the medicine of DOX of the present invention and Asp-DOX;
Fig. 9 is that the present invention contrasts medicine DOX and target medicine Asp-DOX tissue distribution situation in nude mice lotus people liver cancer (HepG2) model;
Figure 10 is that the present invention contrasts medicine DOX and target medicine Asp-DOX tissue distribution situation in nude mice lotus human colon carcinoma model;
Figure 11 is the variation of DOX of the present invention and 3 weeks nude mice lotus people liver cancer model gross tumor volumes of Asp-DOX tail vein drug administration by injection.
embodiment
Below by specific embodiment, the present invention is described in detail; but the purposes of these exemplary embodiments and object are only used for exemplifying the present invention; not real protection scope of the present invention is formed to any type of any restriction, more non-protection scope of the present invention is confined to this.
embodiment 1
MS measures with AB SCIEX company 4000 QTRAP LC/MS/MS type instrument, except special indicating, is EI source (70ev); All solvents all pass through vapor enrichment before use, and the anhydrous solvent using all obtains by standard method drying treatment; Except specified otherwise, institute responds and at room temperature carries out and carry out TLC tracking, and the purifying of product all uses silica gel (300-400 order) column chromatography except specified otherwise; Wherein silica gel (300-400 order) is produced by Haiyang Chemical Plant, Qingdao, and GF254 thin-layer silicon offset plate is produced by Yantai Jiang You silica gel development corporation, Ltd.; Prepared product is through HPLC and MS confirmation.
The preparation of formula II structural compounds (Dx-aspartic acid/Doxorubicin-Asp/ Asp-DOX), syntheti c route is as follows:
Preparation method is that Dx and Fmoc-Asp-OALL are in the presence of DIEA, HOBt, HATU, taking methylene dichloride, trichloromethane, tetrahydrofuran (THF), DMF or toluene etc. as solvent, under room temperature, lucifuge condition, reaction makes compound F 17-hydroxy-corticosterone moc-Asp-(OALL)-DOX; Fmoc-Asp-(OALL)-DOX is again at PdCl afterwards
2(PPh
3)
2and Bu
3under the catalysis of SnH, and in the presence of AcOH taking methylene dichloride as solvent, under room temperature condition, reaction makes compound F 17-hydroxy-corticosterone moc-Asp-DOX; Again taking methylene dichloride, trichloromethane, tetrahydrofuran (THF), DMF or toluene as solvent, taking organic bases triethylamine, pyridine or hexahydropyridine as acid binding agent, under room temperature condition, reaction makes compound (II) Dx-aspartic acid/Doxorubicin-Asp/ Asp-DOX.
Concrete steps are as follows:
1), Fmoc-Asp-OALL(2.32 g, 4 mmol) be placed in 100 mL reaction flasks, add cold DMF(20 mL) stir and make its dissolving, add again HATU(2.52 g, 6.58 mmol), stirring adds HOBt (0.9 g after it is dissolved successively, 6.58 mmol) and DIEA (218 μ l, 13.16 mmol) stir a moment reaction system evenly mix, finally add intermediate Dx (2 g, 5 mmol), finish reaction system and become scarlet, on magnetic stirring apparatus, react 16 h, the reaction of TLC detection display finishes (developping agent ethyl acetate/petroleum ether).Add after 50 ml CH2Cl2 dilutions, with the water soluble component in 50 ml water extractive reaction systems, extract 3 times, remove by filter impurity, merge organic layer, anhydrous sodium sulfate drying, lucifuge is placed and is spent the night.Filter, evaporated under reduced pressure solvent, obtains red powder.Products therefrom purification by silica gel column chromatography, obtains Fmoc-Asp-(OALL)-DOX pink solid 2.92 g.Purity 98.2 %(HPLC normalization methods), yield 80.60%.
2), get 2 mmol Fmoc-Asp-(OALL)-DOX, add 20 ml CH
2cl
2lysate, adds 0.08 mmol PdCl successively completely
2(PPh
3)
2, 5 mmol Glacial acetic acid (front a kind of compound adds lower a kind of compound after dissolving completely), under magnetic agitation, add rapidly afterwards 4 mmol Bu
3snH, on magnetic stirring apparatus, lucifuge is reacted after 10～30 min, the reaction of TLC detection display finishes (developping agent ethyl acetate/petroleum ether=1/1, v/v), by evaporation concentration to 2 ml after reacting liquid filtering, products therefrom purification by silica gel column chromatography, obtains pink solid 1.362 g of intermediate product Fmoc-Asp-DOX, purity 95.5%(HPLC normalization method), yield 84.1%.
3), get 9 ml DMF and dissolve 2) middle gained intermediate product Fmoc-Asp-DOX (1 mmol), stir after dissolving completely, add 1 ml hexahydropyridine, dropwise add while stirring, stopped reaction after 30 min, evaporated under reduced pressure solvent, with 2-5 ml dissolve with methanol, products therefrom obtains red-purple solid 0.52 g of product Dx-aspartic acid with purification by silica gel column chromatography, purity 98.9%(HPLC normalization method), yield 80.0%.ESI-MS：659.41。
embodiment 2
Compound Dx-aspartic acid becomes hydrochloride: get compound purple solid product 1.5 g, be dissolved in 10 mL dehydrated alcohols.Ice-water bath is cooled to 5 DEG C, drip 11.1% ethanol solution hydrochloride to pH be 2, continue at stir about 1 h under ice-water bath.Filter, vacuum-drying, obtains purple pressed powder.
embodiment 3
Compound Dx-aspartic acid becomes taurate: get purple solid product 2.0 g of compound, be dissolved in 10 mL acetone.After being heated to reflux, adding and wait mole taurine, continue at time stirring reaction approximately 1.5 h of refluxing.React complete, under room temperature, leave standstill 24 h.Separate out purple crystal, filter, vacuum-drying, obtains the taurate of I.
embodiment 4
Compound Dx-aspartic acid becomes vitriol: get compound purple solid product 1.2 g, be dissolved in 15 mL methyl alcohol.Ice-water bath is cooled to 0 DEG C, drip 10 % sulfuric acid methanol solutions to pH be 3, continue at stir about 1 h under ice-water bath.Filter, obtain dark red solid.
embodiment 5
The preparation of injection liquid:
Hydrochloride 200 mg of Dx-aspartic acid; Propylene glycol 100 mg; Polysorbate 0.3 ~ 0.5%; Distilled water 300 mL
Preparation method: get activeconstituents and join in the water for injection that dissolves polysorbate and propylene glycol, add medicinal basic to regulate pH value to 4 ~ 8 to make its dissolving.Add gac, whip attachment 30 min, carbon removal, smart filter, embedding, sterilizing.
embodiment 6
The preparation of injection lyophilized powder:
Taurate 100 mg of Dx-aspartic acid; Medicinal basic 0.1-7.0%; N.F,USP MANNITOL 55-85%.
Preparation method: get activeconstituents and add water for injection, regulate pH value to make its dissolving to 4-8 with medicinal basic.Add N.F,USP MANNITOL again, carry out autoclaving by the requirement of injection, add gac, adopt filtering with microporous membrane, filtrate is carried out packing, adopts freeze-drying, makes loose block, and sealing, to obtain final product.
biological analysis
This tests JEG-3 HepG2 used, HCT116, MCF7 and H1299 cell strain all purchased from typical case's culture collection council of Chinese Academy of Sciences cell bank, and authenticates through Shanghai Inst. of Life Science, CAS cell resource center.Wherein tri-kinds of cell strains of HepG2, HCT116 and MCF7 are the strain of LATs positive cell, and H1299 is the cell strain of not expressing LATs.
1, LATs positive tumor cell cellulotoxic experiment
hepG2 cell model
Choose LATs-positive tumor cell HepG2, the cytotoxicity of the method detection of drugs by MTT, by medicine, the restraining effect to growth of tumour cell (IR) characterizes and using the negative tumour cell H1299 of LATs-as negative control Cytotoxic power.
Medicine DOX and Asp-DOX are to hepatoma cell strain HepG2(a) cytotoxicity result is as shown in Figure 4.
HCT116 cell model
Choose LATs-positive tumor cell HCT116, the cytotoxicity of the method detection of drugs by MTT, Cytotoxic power by medicine the restraining effect to growth of tumour cell (IR) to characterize concrete outcome as follows:
Medicine DOX and Asp-DOX are to the cytotoxicity result of hepatoma cell strain HCT116 as shown in Figure 5.
MCF7 cell model
Choose LATs-positive tumor cell MCF7 the cytotoxicity with the method detection of drugs by MTT, Cytotoxic power by medicine the restraining effect to growth of tumour cell (IR) to characterize concrete outcome as follows:
Medicine DOX and Asp-DOX are to the cytotoxicity result of breast carcinoma cell strain MCF7 as shown in Figure 6.
H1299 cell model
Choose and do not express the negative tumour cell H1299 of LATs the cytotoxicity with the method detection of drugs by MTT, Cytotoxic power by medicine the restraining effect to growth of tumour cell (IR) to characterize concrete outcome as follows:
Medicine DOX and Asp-DOX are to the cytotoxicity result of lung cancer cell line H1299 as shown in Figure 7;
Medicine is to the demonstration of cancer cells toxicity result, and the restraining effect of the growth of aspartic acid-Dx (Asp-DOX) to HepG2, HCT116, MCF7 is significantly higher than Dx (DOX).This illustrates that Asp-DOX has significance restraining effect to LATs positive tumor cell compared with DOX, and to the restraining effect of the negative tumour cell of LATs a little less than.Thereby Asp-DOX crosses the cancer of expression targeted therapy effect to LATs has also been described, and less to not expressing the injury of tissue of LATs transporter.
2, Absorption test
Test-compound aspartic acid-Dx (Asp-DOX) takes before use appropriate compound and is dissolved in DPBS buffer salt solution, then obtains being diluted to 10,20,50 μ g/ml.Control group gives the solvent of same volume.
Subject cell is the hepatoma cell strain HepG2 of the LATs positive 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 aseptic condition, 24 porocyte culture plates are positioned in constant incubator to 5% CO
2, 37 DEG C, under condition, cultivate 48 h left and right to cells and cover with hole.Siphon away afterwards old substratum, then add the DMEM(that contains 0,10,20 and 50 ug/ml Dxs and aspartic acid-Dx containing 10%FBS) substratum, continue to cultivate after 2 hours and clean 3 times with cold DPBS buffered soln, stop administration.
Get drug treating 2 hours and stop the tumour cell after administration, every hole adds the content of 400 ul methanol extraction cell Chinese traditional medicines, supersound process 15-30 min, centrifugal (10000 r/min, 5min), remove precipitation, get supernatant 40 ul and carry out HPLC analysis mensuration medicament contg, result is as shown in the table:
The demonstration of HPLC analytical results, 3 dosage Asp-DOX processed LATs positive tumor cell strain HepG2, and the content of cell extract Chinese traditional medicine is all significantly higher than the HepG2 cell that DOX processed; But, in LATs negative cells H1299, the cell that the extract Chinese traditional medicine content of the cell that Asp-DOX processed was significantly processed lower than DOX.This means that the DOX after Asp modifies is Asp-DOX, the positive cell targeted significance of LATs-improved, and to do not express LATs cell targeting a little less than.
3, pharmacokinetics experiment
Get 80 kunming mices (tested ALB/c-nu nude mouse, male, SPF level, 18～20g, buys in Beijing Vital River Experimental Animals Technology Co., Ltd.) be divided into the two large injection DOX that organize, another group is injected Asp-DOX, each large component is 8 groups, every group 5, give respectively the dosage of 5mg/kg, administering mode is tail vein injection.Two administration groups arrange 8 and get blood point: 2 min, 5 min, 10 min, 15 min, 30 min, 60 min, 120 min, 240 min.Getting blood mode is that an eye frame is got blood.Centrifugal, 3000 r/min, 4 DEG C, 15 min, get serum 200 ul, add respectively mark (α-naphthols, 2 mg/ml) in 1 ml methyl alcohol and 50 ul, and eddy current shakes 1 min, dries up organic solvent after getting supernatant in Nitrogen evaporator.Add afterwards 160 ul moving phases, gravity treatment extract, then of short duration centrifugal going goes supernatant 40 ul HPLC to analyze after precipitation.Chromatographic condition is: C8 pillar (Angelent, 200*4.6 mm, 0.45 um), moving phase is methyl alcohol/0.01 M ammonium acetate/H
3pO
4=145/155/0.16 (v/v).Pharmacokinetic results is as follows:
When the medicine of DOX and Asp-DOX, curve as shown in Figure 8;
Removing transformation period significant prolongation by the known Asp-DOX of pharmacokinetics experimental result in blood extends to 49.3min by 19.1 original min, and area under the drug-time curve also significantly increases (being more than 8 times of Dx).
4, tissue distribution experiment
People's liver cancer (HepG2) model
Tested ALB/c-nu nude mouse, male, SPF level, 18～20g, buys in Beijing Vital River Experimental Animals Technology Co., Ltd..Cage for animal is raised, and basal feed is experimental mouse full-valence pellet feed, purchased from Beijing Vital River Experimental Animals Technology Co., Ltd., freely drink water, and humidity 60-80%, natural lighting and approximately the light and shade cycle of 12h.The decubation of first carrying out before experiment one week observes, and is divided into afterwards 10 every group of 3 groups, by the tumour cell suspension (1*10 to nude mice front left limb armpit subcutaneous injection 0.1 ~ 0.2 ml
7cell/ml) method, set up nude mice lotus people liver cancer (HepG2) model, HepG2 is the strain of LATs positive cell.When gross tumor volume reaches 100 mm
3when the left and right to three kinds of tumor bearing nude mices respectively tail vein injection taking the DOX of 5 mg/kg and Asp-DOX(wherein DOX as contrast), every kind of medicine does 5 animal subjects.After administration 1h, animal subject is put to death in cervical vertebra dislocation, tumour, the heart, liver, spleen, lung, kidney, brain and 8 kinds of tissues of testis of getting respectively every animal subject do tissue homogenate, and the medicine in tissue is extracted, by the method for HPLC, the medicament contg in each tissue is analyzed to (unit, mg/g tissue) and carry out the tissue distribution research of medicine.Tissue distribution result is as follows:
Tissue distribution situation is as shown in Figure 9 in nude mice lotus people liver cancer (HepG2) model for contrast medicine DOX and target medicine Asp-DOX.
Nude mice lotus people knurl model organize distribution results show the content significance of Asp-DOX administration group medicine in LATs-positive tumor HepG2 higher than DOX administration group medicine the content in tumor tissues, but the content of DOX administration group medicine in other Organ and tissues is higher than Asp-DOX administration group.This explanation Asp-DOX significantly improves 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 Vital River Experimental Animals Technology Co., Ltd..Cage for animal is raised, and basal feed is experimental mouse full-valence pellet feed, purchased from Beijing Vital River Experimental Animals Technology Co., Ltd., freely drink water, and humidity 60-80%, natural lighting and approximately the light and shade cycle of 12h.The decubation of first carrying out before experiment one week observes, and is divided into afterwards 10 every group of 3 groups, by the tumour cell suspension (1*10 to nude mice front left limb armpit subcutaneous injection 0.1 ~ 0.2 ml
7cell/ml) method, set up nude mice lotus human colon carcinoma (HCT116) model and nude mice lotus people lung cancer (H1299) model, wherein HCT116 is the strain of LATs positive cell, H1299 is the strain of LATs negative cells.When gross tumor volume reaches 100 mm
3when the left and right to three kinds of tumor bearing nude mices respectively tail vein injection taking the DOX of 5 mg/kg and Asp-DOX(wherein DOX as contrast), every kind of medicine does 5 animal subjects.After administration 1h, animal subject is put to death in cervical vertebra dislocation, tumour, the heart, liver, spleen, lung, kidney, brain and 8 kinds of tissues of testis of getting respectively every animal subject do tissue homogenate, and the medicine in tissue is extracted, by the method for HPLC, the medicament contg in each tissue is analyzed to (unit, mg/g tissue) and carry out the tissue distribution research of medicine.Tissue distribution result is as follows:
Tissue distribution situation is as shown in figure 10 in nude mice lotus human colon carcinoma model for contrast medicine DOX and target medicine Asp-DOX.
Nude mice lotus people knurl model organize distribution results show the content significance of Asp-DOX administration group medicine in LATs-positive tumor HCT116 higher than DOX administration group medicine the content in tumor tissues, but the content of DOX administration group medicine in other Organ and tissues is higher than Asp-DOX administration group.
5, anti-tumor in vivo effect experiment
Tested ALB/c-nu nude mouse, male, SPF level, 18～20g, buys in Beijing Vital River Experimental Animals Technology Co., Ltd..Cage for animal is raised, and basal feed is experimental mouse full-valence pellet feed, purchased from Beijing Vital River Experimental Animals Technology Co., Ltd., freely drink water, and humidity 60-80%, the light and shade cycle of natural lighting and about 12 h.The decubation of first carrying out before experiment one week observes, and is divided into afterwards 5 every group of 2 groups, by the tumour cell suspension (1*10 to nude mice front left limb armpit subcutaneous injection 0.1 ~ 0.2 ml
7cell/ml) method, set up nude mice lotus people liver cancer (HepG2) model, at gross tumor volume hyperplasia to 100 mm
3time, respectively two groups of animal subjects are given DOX and the treatment of Asp-DOX folk prescription of same dose (5 mg/kg, 2 times/week, 3 weeks, tail vein drug administration by injection), (the long * tumour of 1/2 tumour is wide to measure weekly gross tumor volume
2).Experimental result is as follows:
The variation of 3 weeks nude mice lotus people liver cancer model gross tumor volumes of DOX and Asp-DOX tail vein drug administration by injection as shown in figure 11.
By the known Asp-DOX of pharmacodynamic experiment result in body, the growth-inhibiting effect of tumour in nude mice lotus people knurl model is significantly better than to positive control drug DOX.
In sum, can clearly be found out by above-mentioned all embodiment, the present invention has obtained the brand-new antitumor drug with good anti-tumor activity and pharmacokinetics behavior designing based on LATs translocator, pay a large amount of creative works, provide brand-new design for the treatment of cancer, the design of medical compounds molecule.
The purposes that should be appreciated that these embodiment only limits the scope of the invention for the present invention being described but not being intended to.In addition; also should understand; after having read technology contents of the present invention, those skilled in the art can make various changes, amendment and/or modification to the present invention, within these all equivalent form of values fall within the protection domain that the application's appended claims limits equally.
Claims (9)
1. compound or its pharmacy acceptable salt, its structural formula of compound is as shown in the formula shown in (I):
Wherein: R1-R9 is identical or different separately, and independently selected from H, hydroxyl, amino, sulfydryl, halogen, C
1-C
6alkyl, C
1-C
6alkoxyl group, C
1-C
6alkylamino, C
1-C
6alkylthio.
2. compound as claimed in claim 1, is characterized in that: R1-R9 is identical or different separately, and independently selected from H, hydroxyl, amino, sulfydryl, halogen, C
1-C
3alkyl, C
1-C
3alkoxyl group, C
1-C
3alkylamino, C
1-C
3alkylthio.
3. compound as claimed in claim 1 or 2, is characterized in that: R1-R3 is H or C
1-C
3alkyl, R4 are C
1-C
3alkoxyl group, R5-R7 are hydroxyl or C
1-C
3alkoxyl group, R8 are C
1-C
3alkyl, R9 are hydroxyl or C
1-C
3alkoxyl group.
4. the compound as described in any one in claims 1 to 3, is characterized in that: R1-R3 is that H, R4 are C
1-C
3alkoxyl group, R5-R7 are that hydroxyl, R8 are C
1-C
3alkyl, R9 are hydroxyl.
5. the compound as described in any one in claim 1 to 4, is characterized in that: R1-R3 is that H, R4 are that methoxyl group, R5-R7 are that hydroxyl, R8 are that methyl, R9 are hydroxyl, is as shown in the formula the structure shown in (II).
6. the synthetic method of compound or its pharmacy acceptable salt as claimed in claim 1, is characterized in that comprising following route:
Wherein, R1-R9 is as the corresponding definition in claim 1.
7. synthetic method as claimed in claim 6, it is characterized in that: described method steps is the Dx analog of formula (III) structure and the Fmoc-Asp-OALL of formula (IV) structure, at N, N-diisopropylethylamine (DIEA), 1-hydroxy benzo triazole (HOBt), under the participation of 2-(7-azo benzotriazole)-tetramethyl-urea phosphofluoric acid ester (HATU), in solvent, described solvent is methylene dichloride, trichloromethane, tetrahydrofuran (THF), one or more in DMF or toluene, room temperature, under lucifuge condition, reaction makes compound V, V is again at PdCl afterwards
2(PPh
3)
2and Bu
3under the catalysis of SnH, and in the presence of AcOH taking methylene dichloride as solvent, under room temperature condition, reaction makes compound VI, taking methylene dichloride, trichloromethane, tetrahydrofuran (THF), DMF or toluene as solvent, taking organic bases triethylamine, pyridine or hexahydropyridine as acid binding agent, under room temperature condition, reaction makes chemical compounds I again.
8. the compound described in any one or its pharmacy acceptable salt purposes in preparation treatment antitumor drug in claim 1-5.
9. comprise the pharmaceutical composition of the compound described in any one in claim 1-5 or its pharmacy acceptable salt.
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| CN108864221A (en) * | 2018-06-08 | 2018-11-23 | 中国科学院上海有机化学研究所 | Aclacinomycin analog and its preparation method and purposes |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108864221A (en) * | 2018-06-08 | 2018-11-23 | 中国科学院上海有机化学研究所 | Aclacinomycin analog and its preparation method and purposes |
| CN108864221B (en) * | 2018-06-08 | 2022-04-01 | 中国科学院上海有机化学研究所 | Aclacinomycin analogue and preparation method and application thereof |
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