CN105601903A - High polymer compound with anticancer activity, and preparing method and application thereof - Google Patents

Abstract

The invention belongs to the technical field of high polymer coupling anticancer medicines, and in particular relates to a high polymer compound PEG (A+G) with an anticancer activity, and a preparing method and application thereof. The anticancer medicine and a Chk1 inhibitor arrive at a focus at the same time according to certain ratio by a polyethylene glycol copolymer nanometer targeted carrier, thereby reducing the toxicity of the anticancer medicine, enhancing the treatment effect of the anticancer medicine, and solving the problem of unified use of the anticancer medicine and the Chk1 inhibitor clinically; an experiment shows that the PEG (A+G) has obvious inhibiting action on Colo-205 naked mouse transplantation tumor and has application prospect in preparing new anticancer medicine.

Description

A kind of macromolecular compound, its preparation method and application with active anticancer

Technical field

The invention belongs to macromolecule coupling anticarcinogen technical field, be specifically related to a kind of have active anticancer by poly-second twoMacromolecular compound, its preparation method and the application of alcohol nano target carrier coupling anticarcinogen and Chk1 inhibitor.

Background technology

Chemotherapy is the important means for the treatment of cancer, but traditional chemotherapy exists the problems such as toxic and side effect is large, curative effect is low. Previous generationRecord latter stage, can be used for medicine transmission to lysosome, Ringsdorf along with Trouet and DeDuve find cell endocytic approachProposed macromolecule coupling drug model and Maeda and found " strengthen infiltration and retain " (EPR) effect, nanometer technology is in healthIn science, be used widely gradually, expedited the emergence of this new subject of nanosecond medical science, main research object is Nano medication transmissionSystem (NDDS), comprises lipidosome cream, nanoparticle suspension thing, high molecular nanometer particle, macromolecule treatment and for gene deliveryThe direction such as nano particle, can improve significantly bioavilability and the pharmacokinetics of medicine, have high medicinalWith commercial value. Wherein, macromolecule treatment is the most successfully field of nanosecond medical science, the past two during the last ten years, in this fieldHave 11 macromolecule coupling drugs to be come into the market to sell by FDA approval, wherein 10 are gathered epoxy second with polyethylene glycol (PEG) or titleAlkane (PEO) is carrier, and 5 is macromolecule coupling anticarcinogen, also has in addition multiple macromolecule coupling anticarcinogens to enter clinical testing.A large amount of preclinical test and clinical testings show: macromolecule coupling anticarcinogen nano target medicine can reduce anticancer significantlyThe toxicity of medicine, significantly improves its curative effect, is the breakthrough to traditional medicine, has become the new direction of medicament research and development.

About polyethylene glycol has had much as the research of medicine macromolecule carrier. Other macromolecule carrier is as N-relatively(2-hydroxypropyl) methacrylamide copolymer, polyglutamic acid etc., polyethylene glycol carrier is supervised by U.S. food medicineCommercialization use is carried out in office (FDA) approval, has generally acknowledged safety indexes, has obtained huge success. The people such as ZhaoOnce synthesized single armed polyethylene glycol conjugation daunorubicin, four arm polyethylene glycol conjugation SN-38 and bifurcated polyethylene glycol conjugation four poly-Or eight poly-cytarabines etc., improve all significantly solubility, half-life and the bioavilability of medicine, significantly reduce medicineThe toxicity of thing, has improved the anticancer live body curative effect of medicine.

Most of treatments of cancer damage to kill cancer cell by inducing DNA and realize. By anticarcinogen chemotherapy orUnder the gene poison stress situation that radiation therapy causes, in order to maintain the integrality of gene and the existence of cell, comprise thin at oneThe complex network of the multiple signal path of born of the same parents' circular test point, DNA reparation, transducer and cell suicide etc. has occurred so-called" DNA damage reaction " (DDR), causes cell cycle to stagnate temporarily, makes the free DNA plerosis damage of cell. Chk1(CheckpointKinase1: checkpoint kinases 1) is the core composition of DNA damage reaction (DDR), and suppressing Chk1 can be specialSurely strengthen the function of the selectively killing of DNA damage reagent to the defective cancer cell of p53.

In mammiferous normal cell, regulate the G1 causing because of DNA damage to stagnate by caused by tumor suppressor p 53, because ofAnd normal cell is mainly stagnated in G1 under gene poison stress situation; Most of tumour cells have p53 and RB pathway deficiency, andThe defective tumour cell of p53 is at G1 checkpoint defectiveness, stagnates and depend critically upon S or G2 checkpoint to repair damageDNA and be the integrality of basic existence maintainer gene, thereby, suppress Chk1, eliminating the special circumstances of S and G2 checkpointUnder, normal cell still can be stuck in G1 and damage with DNA plerosis; And there is no the tumour cell of G1 checkpoint, will be through having silk point all through the agesSplit disaster, thereby cause cell suicide. Obviously, tumor cell ratio normal cell more depends on Chk1 kinases to maintain cell cycleStagnate, therefore, suppress Chk1 and can be used to the specifically selection of enhanced sensitivity DNA damage reagent (chemotherapy or radiation therapy) to cancer cellProperty is killed. In a variety of cancers, based on p53 approach, can set up treatment window to increase with one or more Chk1 inhibitorStrong treatment of cancer. Therefore, research and development Chk1 inhibitor can be used as the new way for the treatment of cancer.

Existing many companies have carried out a large amount of research work to Chk1 inhibitor. AbbottLaboratories is syntheticUrea series compound if A-690002 etc. is as Chk1 inhibitor, clinical front animal experiment shows this compounds significantlyGround has strengthened the curative effect of the anticarcinogens such as adriamycin, camptothecine, Irinotecan, and toxicity is faint. And AstraZeneca and PfizerThe scientist of company has synthesized AZD7762 and PF-477736 etc., in preclinical test, replaces respectively with gemcitabine, Yi LiThe various anticarcinogen couplings such as health, Docetaxel, carboplatin, have strengthened live body curative effect significantly. But there is not yet up to now,The report that Chk1 inhibitor phase iii clinical trial succeeds, reason may be many-sided, as anticarcinogen and Chk1 inhibitorToxic and side effect, anticarcinogen and Chk1 inhibitor be difficult to " and by a certain percentage " arrive cancer location performance drug effect etc. simultaneously.

Summary of the invention

For solving the technical scheme that above-mentioned clinical testing problem adopts be:

By anticarcinogen and Chk1 inhibitor by amino acid connection chain graft on polyethylene glycol nano-carrier simultaneously withSynthesizing polyethylene glycol coupling (anticarcinogen and Chk1 inhibitor), obtain a class make anticarcinogen and Chk1 inhibitor be integrated into one wholeThe nano target medicine of body, reaches and reduces significantly the toxic and side effect of anticarcinogen and Chk1 inhibitor and significantly improve its curative effectEffect, can make again anticarcinogen and Chk1 inhibitor " simultaneously and by a certain percentage " arrive cancer location, to form very simultaneously" therapeutic alliance " in positive meaning, solves anticarcinogen and a Chk1 inhibitor difficult problem for coupling clinically, makes live body curative effect againSignificantly improve, thereby start a class PTS.

The macromolecular compound with active anticancer of the present invention, its structural formula is as shown in XI:

The present invention also discloses the preparation method of above-mentioned macromolecular compound XI, it is characterized in that: comprise the following steps,

HBTU, HOBT, DIEA and solvent DMF, 0 DEG C under room temperature condition, by Boc-Gly and Chk1 inhibitorAZD7762 carries out amidatioon connection, after TFA goes Boc protection, and the glutamic acid of the Gly-AZD7762 of gained and Fmoc protection-The 5-tert-butyl ester carries out amidatioon connection under PyAOP and 2,4,6-trimethylpyridine, 0 DEG C of condition, then goes with triethylamine and DMFFmoc protection, then under PyAOP and 2,4,6-trimethylpyridine, 0 DEG C of condition, carry out acid amides with the 6-aminocaprolc acid of Fmoc protectionChange connect, and then with triethylamine and DMF condition go Fmoc protect, gained micromolecular compound intermediate and quadrifurcate poly-secondGlycol macromolecule carrier (4ARM-SCM-40K) carries out substituted amide connection, and gained macromolecular compound carries out uncle through TFA againButyl ester goes protection, and the hydroxy-acid group of gained macromolecule intermediate is again through N-hydroxy-succinamide, DCC, DMAP and CH₂Cl₂ConditionActivate, finally under pyridine condition, carry out coupling with gemcitabine, obtain final products XI.

The invention also discloses above-mentioned macromolecular compound XI in the application of preparing in anticarcinogen. According to anti-human knot rectumThe embodiment experimental result of cancerous cell line Colo-205 shows, the using dosage of described macromolecular compound XI is 700～When 800mg/kg, experiment terminal relative tumor proliferation rate T/C (%) is 60% left and right, can show significant antitumor workWith.

Brief description of the drawings

Fig. 1: the proton nmr spectra of midbody compound VII (¹H-NMR)。

Fig. 2: the proton nmr spectra of intermediate macromolecular compound VIII (¹H-NMR)。

Fig. 3: the proton nmr spectra of intermediate macromolecular compound IX (¹H-NMR)。

Proton nmr spectra (XI) of Fig. 4: PEG (A+G) (¹H-NMR)。

Fig. 5: PEG (A+G) (XI), the tumour of small-molecule drug gemcitabine and the coupling of Chk1 inhibitor and physiological saline is rawLong curve.

Detailed description of the invention

Following non-limiting example can make the present invention of those of ordinary skill in the art's comprehend, but not withAny mode limits the present invention.

Embodiment 1

Ethylene glycol copolymer coupling (anticarcinogen gemcitabine and Chk1 inhibitor AZD7762) (for convenience of statement, is write a Chinese character in simplified formFor PEG (A+G)) synthetic

By Boc-Gly (265.8mg, 1.5176mmol, self-control), AZD7662 (500mg, 1.3796mmol, the auspicious poem in ShanghaiChemical Co., Ltd.), HBTU (776.6mg, 2.0694mmol), HOBT (279.6mg, 2.0694mmol) joins 250mL'sIn flask, and with DMF (15mL) dissolving, mixed solution is transferred under 0 DEG C of condition and stirred cooling 20 minutes, then slowly dripDIEA (1.08mL, 6.2082mmol). After 2 hours, move to stirring at room temperature, spend the night. Reaction stops, and reactant liquor is poured into saturatedNaHCO₃In solution (200mL), be extracted with ethyl acetate (100mL × 4), then use saturated NaHCO₃Solution (150mL) cleans to be hadMachine phase, then use saturated NaCl solution (100mL) to clean. The anhydrous MgSO of final organic phase₄Dry, filter, then reduce pressure denseContracting is by solution evaporate to dryness. Silica gel column chromatography, with 5% MeOH/CH₂Cl₂Wash-out, collects product, and evaporated under reduced pressure obtains product (for convenienceStatement, is abbreviated as Compound I) 530.7mg, productive rate 74.1%.¹H-NMR(500MHz,DMSO-d₆)δ10.05-9.55(m,1H),8.45-8.22(m,1H),8.15-7.98(m,1H),7.65-7.38(m,1H),7.34-7.18(m,1H),6.95-6.28(m,3H),4.45-4.12(m,1H),3.90-3.68(m,3H),3.65-3.55(m,1H),3.05-2.92(m,1H),2.65-2.52(m,1H),1.98-1.84(m,1H),1.82-1.56(m,2H),1.54-1.28(m,10H)；ITMS+cESI：[M+Na⁺]542.2173

Get Compound I (500mg, 0.9630mmol) and be placed in 25mL round-bottomed flask, add carrene (5mL), more slowThe slow TFA (0.074mL, 9.630mmol) that drips, stirs and spends the night under room temperature. Be spin-dried for, thick product dissolves with methyl alcohol, adds bicarbonateSodium (211.2mg, 2.5137mmol) neutralizes unnecessary TFA. Solids removed by filtration impurity, adds silica white (3g) to filtrate,Be spin-dried for and make solid solution, dry method loading is crossed silicagel column purifying. First use 5%MeOH/CH₂Cl₂Wash-out, then use 4%NH₃·H₂O/8％MeOH/CH₂Cl₂To 5%NH₃·H₂O/10％MeOH/CH₂Cl₂Gradient elution. Collect product, concentrate drying obtains product and (isConvenient statement, is abbreviated as intermediate product II) 403.5mg, productive rate 100%.¹H-NMR(500MHz,DMSO-d₆)δ10.05-9.8(m,1H),8.32-8.24(m,1H),8.22-8.02(m,1H),7.55-7.44(m,3H),7.28-7.22(m,1H),6.84-6.48(m,2H),4.44-4.10(m,1H),4.01-3.68(m,3H),3.65-3.58(m,1H),3.15-2.90(m,1H),2.82-2.60(m,1H),1.98-1.84(m,1H),1.84-1.75(m,1H),1.74-1.58(m,1H),1.56-1.35(m,1H)；MALDI-TOFMS:[M+Na⁺]442.1302

By intermediate product II (403.7mg, 0.9630mmol), PyAOP (702.9mg, 1.3482mmol), Fmoc-L-Glu-OH (5-Bu-t) (573.6mg, 1.3482mmol) adds in the flask of 250mL, and with DMF (15mL) dissolving, will mixSolution is transferred under 0 DEG C of condition and is stirred cooling 20min, then slowly drip 2,4,6-trimethylpyridine (0.127mL,0.9630mmol). After reaction 4h, move under 3 DEG C of conditions and stir, spend the night. Pour reactant liquor into saturated NaHCO₃(200mL) solutionIn, be extracted with ethyl acetate (100mL × 10). Then use saturated NaHCO₃Solution (150mL) drip washing 1 time, molten with saturated NaClLiquid (100mL) drip washing 1 time. Merge organic phase, then reduced pressure concentration, by liquid evaporate to dryness, with ethyl acetate and methyl alcohol dissolving, addsProper amount of silicon rubber powder, dry method loading. Silica gel column chromatography, first uses 200mlCH₂Cl₂Wash-out, then use 0.5%MeOH/CH₂Cl₂To 2%MeOH/CH₂Cl₂Wash-out, finally uses 5%NH₃·H₂O/10％MeOH/CH₂Cl₂Wash-out. Collect product evaporate to dryness and obtain product (for convenienceStatement, is abbreviated as intermediate product III) 558.9mg (70.1%).¹H-NMR(500MHz,DMSO-d₆)δ10.20-9.90(m,1H),8.35-8.25(m,1H),8.08-8.02(m,1H),7.98-7.94(m,2H),7.92-7.88(m,2H),7.80-7.70(m,2H),7.65-7.58(m,1H),7.55-7.40(m,5H),7.38-7.32(m,2H),7.28-7.25(m,1H),6.78-6.60(m,1H),4.30-4.28(m,1H),4.26-4.20(m,3H),4.03-3.96(m,1H),3.94-3.88(m,1H),3.86-3.70(m,2H),2.98-2.92(m,1H),2.70-2.55(m,1H),2.40-2.22(m,2H),2.05-1.85(m,2H),1.70-1.60(m,2H),1.52-1.30(m,11H)；MALDI-TOFMS:[M+Na⁺]849.2960,[M+K⁺]865.2725

By intermediate product III (550.0mg, 0.6764mmol), triethylamine (68mg, 6.764mmol) joins 150mL'sIn flask, with DMF (10mL) dissolving, reaction 3h, stops reaction. Pour reactant liquor into saturated NaHCO₃(200mL), in solution, useEthyl acetate extraction (100mL × 4). Then use saturated NaHCO₃Solution (150mL) drip washing 1 time, with saturated NaCl solution(100mL) drip washing 2 times. Dry with anhydrous MgSO4, suction filtration, adds (3g) silica white in filtrate, dry method loading, and silica gel column chromatography,First use 100mlCH₂Cl₂Wash-out, then use 2%MeOH/CH₂Cl₂To 6%MeOH/CH₂Cl₂Wash-out, finally uses 5%NH₃·H₂O/10％MeOH/CH₂Cl₂Wash-out. Collection product, evaporate to dryness obtain IV intermediate product 312.8mg, productive rate 76.7%.¹H-NMR(500MHz，DMSO-d₆)δ10.05-9.95(m,1H),8.40-8.22(m,1H),8.15-7.82(m,2H),7.62-7.36(m,3H),7.35-7.20(m,1H),6.90-6.45(m,2H),4.45-4.26(m,1H),4.25-4.10(m,1H),4.00-3.90(m,2H),3.88-3.64(m,2H),3.25-3.15(m,1H),2.98-2.90(m,1H),2.72-2.55(m,1H),2.35-2.22(m,2H),1.98-1.80(m,2H),1.68-1.55(m,2H),1.51-1.49(m,1H),1.48-1.40(m,9H)；MALDI-TOF-MS:[M+H⁺]605.3076，[M+Na⁺]627.2876，[M+K⁺]643.2742

6-aminocaprolc acid (2.3047g, 17.57mmol) is joined in the flask of 500mL, then add 150mL (75/75) THF/H₂O stirs mixed solution cooling 20 minutes at 0 DEG C, adds Na₂CO₃Solid (3.2475g,35.14mmol). After dripping off, the THF solution (10mL) that drips Fmoc-Cl (5.0g, 19.327mmol) after 20 minutes moves to room temperatureStir 1h, with 10% lemon acid for adjusting pH value to 3. Reactant liquor is transferred in the separatory funnel of 1L, is extracted with ethyl acetate(100mL × 4 time), with 1NHCl drip washing (120mL × 2). Merge organic phase, use anhydrous MgSO₄Dry, suction filtration, then decompressionConcentrate liquid evaporate to dryness, use 5mL acetic acid ethyl dissolution, add while stirring n-hexane (300mL) sedimentation, crude product is analysed with PowderedGo out. By crude product acetic acid ethyl dissolution, add proper amount of silicon rubber powder, make solid solution, dry method loading, column chromatography, uses 20% secondAcetoacetic ester/benzinum to 80% ethyl acetate/petroleum ether is carried out gradient elution. Collect product, in the middle of reduced pressure concentration evaporate to dryness obtains, produceThing V6.209g, productive rate 99.0%.¹H-NMR(500MHz，DMSO-d₆)δ12.5-11.75(m,1H),7.99-7.80(m,2H),7.78-7.60(m,2H),7.49-7.38(m,2H),7.37-7.29(m,2H),7.28-7.18(m,1H),4.45-4.20(m,3H),2.20-2.15(m,2H),2.10-2.00(m,2H),1.50-1.40(m,3H),1.40-1.35(m,3H)

By intermediate product IV (550mg, 0.9095mmol), intermediate product V (482.2mg, 1.3643mmol), EDCI(261.53mg, 1.3643mmol), HOBT (221.2mg, 1.6371mmol) joins in the flask of 250mL, uses CH₂Cl₂(15mL) dissolve, solution is stirred cooling 20 minutes under 0 DEG C of condition, then slowly drip DIEA (0.285mL,1.6371mmol). After reaction 4h, move to stirring at room temperature, spend the night, stop reaction, directly evaporate to dryness. By crude product CH₂Cl₂Dissolve, wetMethod loading, column chromatography, first uses 200mlCH₂Cl₂Wash-out, then use 2%MeOH/CH₂Cl₂To 5%MeOH/CH₂Cl₂Wash-out, lastUse 2%NH₃·H₂O/4％MeOH/CH₂Cl₂Wash-out. Collection product evaporate to dryness obtains product and (for convenience of statement, is abbreviated as intermediate productVI) 855.2mg, productive rate 100%.¹H-NMR(500MHz，DMSO-d₆)δ10.00-9.78(m，1H),8.88-8.54(m,1H),8.28-8.16(m,1H),7.98-7.86(m,2H),7.84-7.74(m,3H),7.65-7.56(m,2H),7.48-7.31(m,3H),7.30-7.28(m,2H),7.28-7.22(m,2H),7.18-7.12(m,2H),6.68-6.51(m,1H),4.35-4.22(m,1H),4.21-4.17(m,2H),4.15-4.08(m,1H),3.94-3.80(m,2H),3.78-3.58(m,2H),3.57-3.48(m,3H),2.98-2.90(m,2H),2.88-2.84(m,2H),2.58-2.45(m,1H),2.25-2.13(m,2H),2.09-1.93(m,2H),1.88-1.74(m,2H),1.70-1.48(m,4H),1.46-1.35(m,2H),1.35-1.22(m,10H)；MALDI-TOFMS:[M+Na⁺]962.2350,[M+K⁺]978.2200

By intermediate product VI (855.0mg, 1.2120mmol), triethylamine (1.27mL, 12.12mmol) joins 150mLFlask in, use CH₂Cl₂(15mL) dissolve. spend the night, solution becomes muddiness and adds triethylamine (2mL) to reacting completely, and stops reaction,Directly evaporate to dryness, uses CH₂Cl₂Dissolve, wet method loading, column chromatography, first uses 200mlCH₂Cl₂Wash-out, then use 2%MeOH/CH₂Cl₂Arrive4％MeOH/CH₂Cl₂Wash-out, finally uses 5%NH₃·H₂O/10％MeOH/CH₂Cl₂To 7.5%NH₃·H₂O/15％MeOH/CH₂Cl₂Wash-out. Collect product evaporate to dryness and obtain product (for convenience of statement, being abbreviated as intermediate product VII) 885.1mg, productive rate 98.7%.Proton nmr spectra is as Fig. 1:¹H-NMR(500MHz,DMSO-d₆)δ8.35-8.20(m,1H),8.15-7.78(m,3H),7.55-7.35(m,4H),7.30-7.15(m,1H),6.85-6.42(m,2H),4.43-4.34(m,1H),4.33-4.21(m,1H),4.20-4.12(m,1H),3.85-3.75(m,2H),3.74-3.64(m,2H),2.98-2.85(m,2H),2.64-2.53(m,1H),2.28-2.18(m,2H),2.15-2.06(m,2H),1.93-1.84(m,2H),1.77-1.52(m,4H),1.50-1.41(m,3H),1.40-1.35(m,12H)；MALDI-TOFMS:[M+H⁺]718.3108，[M+Na⁺]740.2903

By intermediate product VII (360.0mg, 0.5020mmol), 4AMR-SCM-40K (2.510g, 0.06276mmol, northJing Jiankai Science and Technology Ltd.) join in 100mL flask, use CH₂Cl₂(5ml) dissolve, under room temperature condition, slowly stir two weeks,Be transferred in 200mL flask and react again 7 days with DMF. Concentrated by rotary evaporation, then adds absolute ether sedimentation, filters to obtain thick product; SoAfter thick product is dissolved with carrene and methyl alcohol, add ether sedimentation, filtration drying obtains VIII intermediate product 2.0578g, producesRate 78.8%. Proton nmr spectra is as Fig. 2:¹H-NMR(500MHz,DMSO-d₆)δ10.20-9.90(m,4H),8.40-8.20(m,4H),8.10-7.91(m,8H),7.90-7.78(m,4H),7.65-7.58(m,4H),7.55-7.40(m,12H),7.31-7.20(m,4H),6.82-6.51(m,4H),4.48-4.37(m,4H),4.34-4.27(m,4H),4.25-4.15(m,4H),4.05-3.91(m,12H),3.85-3.60(m,24H),3.70-3.35(m,4199H),3.15-3.05(m,8H),3.03-2.85(m,4H),2.35-2.20(m,8H),2.18-2.05(m,8H),1.96-1.84(m,8H),1.82-1.58(m,12H),1.54-1.45(m,8H),1.44-1.31(m,44H)。

In 30mL round-bottomed flask, add intermediate product VIII (2.4g, 57.69mmol), and use CH₂Cl₂(20mL) dissolve,Then add TFA (1mL) room temperature slowly to stir, stop reaction after two weeks, directly evaporate to dryness, with carrene (2mL) dissolving, thenAdd absolute ether precipitation. Filter, filter cake vacuum drying obtains intermediate product IX1.6697g, productive rate 62.2%. Hydrogen nuclear magnetic resonanceCompose as Fig. 3:¹H-NMR(500MHz,CDCl₃)δ9.98-9.82(m,4H),8.25-8.10(m,4H),7.78-7.55(m,4H),7.48-7.20(m,8H),7.15-6.50(m,20H),6.20-6.10(m,4H),6.30-6.10,4.59-4.45(m,4H),4.45-4.23(m,4H),4.23-4.02(m,12H),3.98-3.90(m,12H),3.85-3.40(m,4353H),2.54-2.32(m,24H),2.28-2.06(m,16H),2.05-1.93(m,8H),1.86-1.70(m,8H),1.68-1.36(m,32H)。

(1) by intermediate product IX (1.6697g, 0.0384mmol), succinamide alcohol (132.7mg, 1.1528mmol),DMAP (4.7mg, 0.0384mmol) joins in 100ml round-bottomed flask, uses CH₂Cl₂(400ml) dissolve, then by hybrid reactionLiquid is placed under-5 DEG C of conditions and stirs 20min, adds DCC (237.9mg, 1.1528mmol) in batches, adds after complete 1 hour and moves toRoom temperature, slowly stirring reaction two weeks. Stop reaction, filter concentrated filtrate evaporate to dryness is obtained to intermediate product X (1.6908g).

(2) by intermediate product X (1.6908g, 0.0384mmol) and GEMCITABINE HYDROCHLORIDE (368.4mg,1.2288mmol) be dissolved in 100mL flask with pyridine (10mL), room temperature slowly stirs two weeks, stops reaction, directly evaporate to dryness,Then dissolve by dichloromethane/ethyl acetate, then evaporate to dryness 3 times repeatedly. Finally use CH₂Cl₂Dissolve, wet method loading, uses MeOH/CH₂Cl₂Wash-out successively, collects product point, and reduced pressure concentration vacuum drying obtains thick product, then dissolves with 2mL carrene, addsAbsolute ether makes its precipitation, filters, and vacuum drying obtains finished product XI1.2953g, productive rate 73.7%. For convenience of statement, by this eventuallyProduct XI called after compound PEG (A+G). Proton nmr spectra is as Fig. 4:¹H-NMR(500MHz,DMSO-d₆)δ10.01-9.9(m,4H),8.50-8.18(m,8H),8.16-7.95(m,8H),7.94-7.84(m,4H),7.82-7.70(m,8H),7.68-7.60(m,4H),7.59-7.40(m,16H),7.38-7.20(m,4H),6.81-6.54(m,4H),6.38-6.21(m,4H),6.00-5.30(m,4H),4.97-4.68(m,4H),4.65-4.35(m,8H),3.90-3.88(m,24H),3.85-3.42(m,4263H),3.15-3.01(m,8H),2.87-2.72(m,16H),2.35-2.07(m,16H),2.05-1.83(m,16H),1.80-1.58 (m, 8H), 1.57-1.32 (m, 24H); MALDI-TOFMS is at 37500-47500 scope, top42254.5313. The theoretical molecular of compound PEG (A+G): 44497, wherein the theoretical medicine carrying amount of AZDD7762 is 3.257%,And the theoretical medicine carrying amount of gemcitabine is 2.366%.

Embodiment 2

1. experiment reagent and experimental animal

On the nude mice by subcutaneous transplantable tumor model of p53 sudden change, investigate the compound PEG (A+G) that embodiment 1 obtains, compoundThe antitumor drug effect of A and G coupling.

Main agents

RPMI-1640: Hyclone, article No. SH30809.01B;

FBS:GIBCO, article No. 10099-141;

100* penicillin streptomycin is dual anti-: Invitrogen, article No. 10378-016;

PEG (A+G): light yellow translucent viscous solid, carry little molecule A medicine 3.257%, carry little molecule G medicine 2.366%,The about 1.3g of sample size;

A:AZD7762, molecular weight 362.4, white crystal;

G: gemcitabine, molecular weight 263.2, GEMCITABINE HYDROCHLORIDE (G hydrochloride) 299.65, white crystal;

The compound method of polyethylene glycol conjugation (gemcitabine and AZD7762) (being abbreviated as PEG (A+G) for convenience of statement):Take appropriate tested medicine, use SPSS to dissolve, disperse, mix laggard row animals administer, now join existingWith.

A compound method: take appropriate tested medicine, use HP-β-CD of 12% to dissolve, disperse (suspension), mixedClose even laggard row animals administer.

G compound method: take appropriate tested medicine, with physiological saline solution.

Animal used as test

Strain: Balb/c nude mice, SPF level; Source: Ling Chang bio tech ltd, Shanghai; Age in week: 5-6 week; Sex:Male.

2. experimental technique

PEG (A+G) tolerance studies

Under selected prescription, when dosage is 1540mg/kg, the tolerance situation of medicine is studied; Totally 4 nakedMouse, i.p., administration 1 time, observes 4-5 days;

Colo-205 nude mice by subcutaneous transplantable tumor model is set up

A) recover and increase Colo-205 cell;

B) to be amplified to enough cells, collecting cell, be made into concentration with 1640 culture mediums containing serum and be 2 ×10⁷The cell suspension of cells/ml;

C) nude mice right side subcutaneous vaccination, only, every nude inoculation cell number is 2 × 10 to 0.1ml/⁶Individual; Inoculate 27.

Observe: observe animal state 2 times weekly, go out knurl situation, treat that tumor growth is to average external volume 100-200mm³Left and rightTime the administration of dividing into groups.

Grouping and administration: from 27 animals, select 18 animals that gross tumor volume is close, divide at random according to gross tumor volumeBe 3 groups, be respectively physiological saline control group, PEG (A+G) group, small-molecule drug gemcitabine and the (letter of Chk1 inhibitor coupling groupBe written as A+G coupling), every group of 6 animals. Grouping was designated as Day1 the same day, started administration the same day. Grouping and former dosage regimen are in table 1:

The grouping of table 1. animal and administration

* the dosage of G is in free form

Day2 is observed in administration, and A+G organizes 1#, 2#, and 3#, 6# animal dead is chosen 4 animals and is added to this from animal for subsequent useGroup, makes the tumor average volume of this group suitable with physiological saline control group; And former dosage regimen is revised, specifically seeTable 2:

Table 2: animal grouping and administration adjustment

* the dosage of G is in free form

Observe and detect: measure weekly 3-4 gross tumor volume and the weight of animals, measure observation 14 days.

A+G coupling (the first i.p. administration of G, A is i.p. administration after 4h), according to former dosage regimen, (50+36.3) mg/kg, givesThere is 4/6 animal dead next day in medicine; Therefore dosage is adjusted into (25+18.1) mg/kg, and according to the changes of weight of animalAdministration A, G dosing interval are adjusted into 48h (the first i.p. administration of G, A is i.p. administration after 48h). And PEG (A+G) is with 1540mg/After kg administration 1 time, Body weight loss is obvious, thus dosage is adjusted into 770mg/kg, and by dosing interval be adjusted into every 4 days toMedicine 1 time.

3. detect index and calculating, statistical analysis technique

Gross tumor volume (tumorvolume, TV)

TV＝1/2×a×b², wherein a, b represent respectively length and width.

Relative tumour volume (relativetumorvolume, RTV)

RTV＝TVt/TV₁Wherein TV₁(d1) gross tumor volume during for point cage administration, TVt is the tumour while measuring each timeVolume.

Tumor proliferation rate T/C (%) relatively

$T/C\left(\%\right)=\frac{T_{RTV}}{C_{RTV}}\×100$

T_RTV: treatment group RTV; C_RTV: blank group RTV.

Statistical analysis: when experiment terminal, each group of relative tumour volume (RTV) (1) carried out to the Levene inspection of homogeneity of varianceTest, if homogeneity of variance is analyzed nonsignificance, i.e. P 0.05, adopt one-way analysis of variance, when variance analysis has remarkable meaningJustice, i.e. P≤0.05, select Dunnett inspection control group and other processed group are organized between comparison. (2) if Levene inspectionP≤0.05 of testing, i.e. heterogeneity of variance, carries out number conversion the initial data that does not contain negative value, and the data after conversion are carried out varianceThe Levene inspection of homogeneous, if homogeneity of variance is analyzed nonsignificance, i.e. P > 0.05, adopt one-way analysis of variance, the side of working asDifference analysis significance, i.e. P≤0.05, then select Dunnett inspection control group and other processed group are organized between comparison.

4. experimental result

The tolerance studies of PEG (A+G)

Under selected prescription, when dosage is 1540mg/kg, Body weight loss in 2 days after animal intraperitoneal injection,Have subsequently recovery trend, through the observation of 1 week, as table 3, medicine, comprising without impact the general state of animal: hair indifference;Changes of weight no significant difference; Without nausea,vomiting,diarrhea, appetite, indigestion, dry skin, fash, red swelling of the skin, scratchItch, the situation such as pain, backache, expiratory dyspnea, tired and insomnia occurs. Intracorporeal organ naked eyes are watched indifference. Mice sleep andEnliven situation indifference; Mouse anus and defecation situation indifference, do not have diarrhoea situation.

The impact (body weight and general state) of table 3PEG (A+G) on animal

*-represent not administration on the same day

Nude mice body weight and gross tumor volume result are as table 4:

Table 4PEG (A+G), A+G coupling on Colo-205 tumor bearing nude mice body weight impact (data acquisition represents with Mean ± SEM,n＝6)

A: dosage regimen is specifically in table 2;

B: administration the 3rd day, 2# animal dead

C: administration the 2nd day, 1#, 2#, 3#, 6# animal dead is chosen 4 animals and is added to this group from animal for subsequent use;

When this experiment grouping, in the group of each group gross tumor volume, RSD is all less than 20%, physiological saline contrast when experiment terminalGroup gross tumor volume reaches 1333.6mm³, while grouping, gross tumor volume has increased approximately 6.4 times, meets transplanted tumor in nude mice drug efficacy studyGeneral Requirements.

Table 5PEG (A+G), the inhibitory action of A+G coupling to Colo-205 transplanted tumor in nude mice growth (Mean for data acquisition ±SEM represents, n=6)

A: dosage regimen is specifically in table 2;

B:vs physiological saline control group, * p < 0.05, * * p < 0.01

According to the dosage regimen after above adjustment, administration was observed after 14 days, and PEG (A+G) administration group shows significant anti-Function of tumor, experiment terminal relative tumor proliferation rate T/C (%) is 59.9%; And the experiment terminal of A+G coupling administration group is relativeTumor proliferation rate T/C (%) is 72.0%. Fig. 5 is the tumor growth of PEG (A+G) group, A+G coupling group and physiological saline control groupCurve.

The result of above-described embodiment 2 shows: physiological saline control animals body weight is on a declining curve (with institute's lotus tumour phaseClose), and administration treated animal Body weight loss is relatively more, administration on the general state of animal without impact. To sum up, at Human colorectal carcinomaOn clone Colo-205 Nude Mouse Model, under dosage regimen of the present invention, PEG (A+G) is to Colo-205 nude mouseTransplantable tumor has remarkable inhibitory action.

Claims (3)

1. have a macromolecular compound for active anticancer, its structural formula is as shown in XI:

2. the preparation method of macromolecular compound as claimed in claim 1, is characterized in that: comprises the following steps,

HBTU, HOBT, DIEA and solvent DMF, 0 DEG C under room temperature condition, Boc-Gly and Chk1 inhibitor AZD7762 are enteredRow amidatioon connects, after TFA goes Boc protection, and glutamic acid-5-tert-butyl ester of the Gly-AZD7762 of gained and Fmoc protectionUnder PyAOP and 2,4,6-trimethylpyridine, 0 DEG C of condition, carry out amidatioon connection, then go Fmoc to protect with triethylamine and DMFProtect, then under PyAOP and 2,4,6-trimethylpyridine, 0 DEG C of condition, carry out amidatioon connection with the 6-aminocaprolc acid of Fmoc protection,And then going Fmoc protection by triethylamine and DMF condition, gained micromolecular compound intermediate and quadrifurcate polyethylene glycol are highMolecular vehicle 4ARM-SCM-40K carries out substituted amide connection, and gained macromolecular compound carries out the tert-butyl ester through TFA again and goes to protectProtect, the hydroxy-acid group of gained macromolecule intermediate is again through N-hydroxy-succinamide, DCC, DMAP and CH₂Cl₂Condition is livedChange, finally under pyridine condition, carry out coupling with gemcitabine, obtain final products XI.

3. macromolecular compound as claimed in claim 1 is in the application of preparing in anticarcinogen.