CN105601903B - A kind of high-molecular compound with active anticancer, its preparation method and application - Google Patents

Abstract

The invention belongs to the technical field of polymer coupling anticancer drugs, and specifically relates to a polymer compound PEG (A+G) with anticancer activity, its preparation method and application. The carrier enables anticancer drugs and Chk1 inhibitors to reach the cancer lesion "simultaneously and in a certain proportion", reduces the toxicity of anticancer drugs, improves the efficacy of anticancer drugs, and can solve the problem of clinical combination of anticancer drugs and Chk1 inhibitors The experiment proves that the above-mentioned PEG (A+G) has a significant inhibitory effect on Colo-205 nude mouse transplanted tumors, and has application prospects in the preparation of new anticancer drugs.

Description

A polymer compound with anticancer activity, its preparation method and application

technical field

The invention belongs to the technical field of macromolecule coupling anticancer drugs, and in particular relates to a macromolecular compound having anticancer activity coupled with anticancer drugs and Chk1 inhibitors by a polyethylene glycol nano-targeting carrier, its preparation method and application .

Background technique

Chemotherapy is an important means of treating cancer, but traditional chemotherapy has problems such as high toxicity and side effects and low curative effect. At the end of the last century, as Trouet and De Duve discovered that the endocytic pathway can be used to transport drugs to lysosomes, Ringsdorf proposed a polymer coupled drug model and Maeda discovered the "enhanced penetration and retention" (EPR) effect, nano Technology has gradually been widely used in health sciences, which has given birth to the new discipline of nanomedicine. The main research objects are nano drug delivery systems (NDDS), including liposome creams, nano suspensions, polymer nanoparticles, and polymers. Nanoparticles for treatment and gene delivery can greatly improve the bioavailability and pharmacokinetics of drugs, and have extremely high medicinal and commercial value. Among them, polymer therapy is the most successful field of nanomedicine. In the past two decades, 11 polymer-conjugated drugs have been approved by the FDA to enter the market, and 10 of them are based on polyethylene glycol (PEG). Or called polyethylene oxide (PEO) as the carrier, 5 are polymer-conjugated anticancer drugs, and several polymer-conjugated anticancer drugs have entered clinical trials. A large number of preclinical and clinical trials have shown that nano-targeted drugs coupled with polymer anticancer drugs can greatly reduce the toxicity of anticancer drugs and significantly improve their efficacy. It is a breakthrough in traditional medicine and has become a new research and development of drugs. direction.

There have been many studies on polyethylene glycol as a drug polymer carrier. Compared with other polymer carriers such as N-(2-hydroxypropyl) methacrylamide copolymer, polyglutamic acid, etc., polyethylene glycol carrier is approved by the US Food and Drug Administration (FDA) for commercial use , with well-recognized security metrics, has achieved great success. Zhao et al. have synthesized single-arm polyethylene glycol-coupled daunorubicin, four-arm polyethylene glycol-coupled SN-38, and bifurcated polyethylene glycol-coupled tetrameric or octarabinocytarabine, etc. Both greatly improved the solubility, half-life and bioavailability of the drug, significantly reduced the toxicity of the drug, and improved the anticancer efficacy of the drug in vivo.

Most cancer treatments work by inducing DNA damage to kill cancer cells. To maintain gene integrity and cell survival under genotoxic stress induced by anticancer drug chemotherapy or radiation therapy, multiple signaling pathways including cell cycle checkpoints, DNA repair, transcriptional programs, and cell suicide A complex network of cells undergoes the so-called "DNA damage response" (DDR), causing a temporary arrest of the cell cycle, allowing the cell time to repair DNA damage. Chk1 (Checkpoint Kinase 1: Checkpoint Kinase 1) is the core component of the DNA damage response (DDR), and inhibition of Chk1 can specifically enhance the selective killing of p53-deficient cancer cells by DNA damaging agents.

In mammalian normal cells, the G1 arrest caused by DNA damage is regulated by the tumor suppressor gene p53, so normal cells mainly arrest in G1 under genotoxic stress; most tumor cells have p53 and RB pathway defects, and p53 Defective tumor cells are deficient in the G1 checkpoint, stalled and heavily dependent on the S or G2 checkpoints to repair damaged DNA and maintain genetic integrity for essential survival, thus inhibiting Chk1 and eliminating S and G2 checkpoints In the special case of the G1 checkpoint, normal cells can still stall at G1 to repair DNA damage; while tumor cells without the G1 checkpoint will experience mitotic catastrophe, leading to cell suicide. Evidently, tumor cells are more dependent on Chk1 kinase than normal cells to maintain cell cycle arrest, therefore, inhibition of Chk1 can be used to specifically sensitize DNA damaging agents (chemotherapy or radiation therapy) to the selective killing of cancer cells. In many cancers, one or more Chk1 inhibitors can be used to create a therapeutic window to enhance cancer therapy based on the p53 pathway. Therefore, the development of Chk1 inhibitors may serve as a new way to treat cancer.

Many companies have done a lot of research work on Chk1 inhibitors. Abbott Laboratories has synthesized urea series compounds such as A-690002 as Chk1 inhibitors. Preclinical animal experiments have shown that such compounds can greatly enhance the efficacy of anticancer drugs such as doxorubicin, camptothecin, and irinotecan, and the toxicity weak. Scientists from AstraZeneca and Pfizer synthesized AZD7762 and PF-477736, etc., which were combined with various anticancer drugs such as gemcitabine, irinotecan, docetaxel, and carboplatin in preclinical tests, which significantly enhanced the anticancer effect in vivo. curative effect. However, so far, there have been no reports of successful phase III clinical trials of Chk1 inhibitors. The reasons may be various, such as the toxic and side effects of anticancer drugs and Chk1 inhibitors, and the difficulty of "simultaneously and in accordance with the schedule" of anticancer drugs and Chk1 inhibitors. A certain proportion" reaches the cancer site to exert the drug effect and so on.

Contents of the invention

The technical solutions adopted to solve the above clinical trial problems are:

Anticancer drugs and Chk1 inhibitors are simultaneously grafted on polyethylene glycol nanocarriers through amino acid linking chains to synthesize polyethylene glycol coupling (anticancer drugs and Chk1 inhibitors), and a class of anticancer drugs and Chk1 inhibitors is obtained. Chk1 inhibitors are integrated into a whole nano-targeted drug, which can greatly reduce the toxic and side effects of anticancer drugs and Chk1 inhibitors and significantly improve their curative effect. At the same time, anticancer drugs and Chk1 inhibitors can be "simultaneously and According to a certain proportion” to reach the cancer site, to form a real “combined therapy”, to solve the problem of clinical combination of anticancer drugs and Chk1 inhibitors, so that the curative effect of the living body can be greatly improved again, thus creating a new class of anticancer drugs.

The polymer compound with anticancer activity described in the present invention has a structural formula as shown in XI:

The present invention also discloses the preparation method of the above-mentioned polymer compound XI, which is characterized in that it comprises the following steps,

Under the condition of HBTU, HOBT, DIEA and solvent DMF, 0°C to room temperature, Boc-Gly was linked with Chk1 inhibitor AZD7762 by amidation, and after de-Boc protection by TFA, the obtained Gly-AZD7762 and Fmoc-protected glutamic acid -5-tert-butyl ester was amidated with PyAOP and 2,4,6-collidine at 0°C, then de-Fmoc-protected with triethylamine and DMF, and then combined with Fmoc-protected 6-aminocaproic acid Perform amidation connection with PyAOP and 2,4,6-collidine at 0°C, and then remove Fmoc protection with triethylamine and DMF. Alcohol polymer carrier (4ARM-SCM-40K) is used for substitution and amidation connection, the obtained polymer compound is deprotected by TFA with tert-butyl ester, and the carboxylic acid group of the obtained polymer intermediate is then treated with N-hydroxysuccinimide , DCC, DMAP and CH ₂ Cl ₂ conditions for activation, and finally coupled with gemcitabine under pyridine conditions to obtain the final product XI.

The present invention also discloses the application of the above polymer compound XI in the preparation of anticancer drugs. According to the experimental results of the examples of the anti-human colorectal cancer cell line Colo-205, when the dosage of the polymer compound XI is 700-800 mg/kg, the relative tumor proliferation rate T/C (%) of the experimental endpoint is 60 %, it can show significant anti-tumor effect.

Description of drawings

Figure 1: Proton Nuclear Magnetic Resonance Spectrum ( ¹ H-NMR) of Intermediate Compound VII.

Fig. 2: Proton nuclear magnetic resonance spectrum ( ¹ H-NMR) of the intermediate polymer compound VIII.

Fig. 3: Proton nuclear magnetic resonance spectrum ( ¹ H-NMR) of the intermediate polymer compound IX.

Figure 4: Proton nuclear magnetic resonance spectrum ( ¹ H-NMR) of PEG(A+G)(XI).

Figure 5: Tumor growth curves of PEG(A+G)(XI), small molecule drug gemcitabine and Chk1 inhibitor combined with normal saline.

detailed description

The following non-limiting examples can enable those skilled in the art to understand the present invention more fully, but do not limit the present invention in any way.

Example 1

Synthesis of polyethylene glycol copolymer coupling (anticancer drug gemcitabine and Chk1 inhibitor AZD7762) (abbreviated as PEG(A+G) for convenience of description)

Boc-Gly (265.8mg, 1.5176mmol, self-made), AZD7662 (500mg, 1.3796mmol, Shanghai Ruishi Chemical Co., Ltd.), HBTU (776.6mg, 2.0694mmol), HOBT (279.6mg, 2.0694mmol) were added to 250mL The flask was dissolved with DMF (15 mL), and the mixed solution was transferred to 0° C. and cooled with stirring for 20 minutes, and then DIEA (1.08 mL, 6.2082 mmol) was slowly added dropwise. After 2 hours, move to room temperature and stir overnight. The reaction was stopped, and the reaction solution was poured into saturated NaHCO solution (200mL), extracted with ethyl acetate (100mL× ₄ ), and then the organic phase was washed with saturated NaHCO solution (150mL), and then washed with saturated _NaCl solution (100mL) . The final organic phase was dried over anhydrous _MgSO4 , filtered, then concentrated under reduced pressure to evaporate the solution to dryness. Silica gel column chromatography, eluting with 5% MeOH/CH ₂ Cl ₂ , collected the product, and evaporated to dryness under reduced pressure to obtain 530.7 mg of the product (abbreviated as compound I for convenience), with a yield of 74.1%. ¹ H-NMR (500MHz,DMSO-d ₆ )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+c ESI: [M+Na ⁺ ]542.2173

Compound I (500 mg, 0.9630 mmol) was placed in a 25 mL round bottom flask, dichloromethane (5 mL) was added, TFA (0.074 mL, 9.630 mmol) was slowly added dropwise, and stirred overnight at room temperature. After spinning to dryness, the crude product was dissolved in methanol, and sodium bicarbonate (211.2 mg, 2.5137 mmol) was added to neutralize excess TFA. Filter to remove solid impurities, add silica gel powder (3g) to the filtrate, spin dry to make a solid solution, and dry load the sample through a silica gel column for purification. Elution with 5% MeOH/CH ₂ Cl ₂ , then 4% NH ₃ ·H ₂ O/8% MeOH/CH ₂ Cl ₂ to 5% NH ₃ ·H ₂ O/10% MeOH/CH ₂ Cl ₂ Gradient elution. The product was collected, concentrated and dried to obtain 403.5 mg of product (abbreviated as intermediate product II for convenience of description), with a yield of 100%. ¹ H-NMR (500MHz,DMSO-d ₆ )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 MALDI -TOF MS: [M+Na ⁺ ]442.1302

The intermediate product II (403.7mg, 0.9630mmol), PyAOP (702.9mg, 1.3482mmol), Fmoc-L-Glu-OH (5-Bu-t) (573.6mg, 1.3482mmol) was added to a 250mL flask and washed with DMF (15 mL) was dissolved, and the mixed solution was transferred to 0° C. and cooled with stirring for 20 min, and then 2,4,6-collidine (0.127 mL, 0.9630 mmol) was slowly added dropwise. After 4 hours of reaction, move to 3°C and stir overnight. The reaction solution was poured into saturated NaHCO ₃ (200 mL) solution, extracted with ethyl acetate (100 mL×10). Then rinse with saturated NaHCO ₃ solution (150 mL) once and with saturated NaCl solution (100 mL) once. The organic phases were combined, then concentrated under reduced pressure to evaporate the liquid to dryness, dissolved in ethyl acetate and methanol, added an appropriate amount of silica gel powder, and loaded the sample by dry method. Silica gel column chromatography, first eluted with 200ml CH ₂ Cl ₂ , then with 0.5% MeOH/CH ₂ Cl ₂ to 2% MeOH/CH ₂ Cl ₂ , and finally with 5% NH ₃ ·H ₂ O/10% MeOH/ _{CH2Cl2 eluted} . The collected products were evaporated to dryness to obtain 558.9 mg (70.1%) of the product (abbreviated as intermediate product III for convenience of description). ¹ H-NMR (500MHz,DMSO-d ₆ )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-TOF MS: [M+Na ⁺ ]849.2960, [M+K ⁺ ]865.2725

Add intermediate product III (550.0 mg, 0.6764 mmol) and triethylamine (68 mg, 6.764 mmol) into a 150 mL flask, dissolve with DMF (10 mL), react for 3 h, and stop the reaction. The reaction solution was poured into saturated NaHCO ₃ (200 mL) solution, extracted with ethyl acetate (100 mL×4). Then rinse with saturated NaHCO ₃ solution (150 mL) once and with saturated NaCl solution (100 mL) twice. Dry with anhydrous MgSO4, filter with suction, add (3g) silica gel powder to the filtrate, apply dry method, silica gel column chromatography, first elute with 100ml _CH2Cl2 , then use ₂ % MeOH/ _CH2Cl2 to ₆ % MeOH/ _CH2Cl2 and finally ₅ % _NH3 - _H2O / ₁₀ % MeOH/ _CH2Cl2 . The products were collected and evaporated to dryness to obtain 312.8 mg of IV intermediate product with a yield of 76.7%. ¹ H-NMR (500MHz, DMSO-d ₆ )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

Add 6-aminocaproic acid (2.3047g, 17.57mmol) into a 500mL flask, then add 150mL (75/75) THF/H ₂ O, stir and cool the mixed solution at 0°C for 20 minutes, add Na _{2 CO3} solid (3.2475 g, 35.14 mmol). After 20 minutes, a THF solution (10 mL) of Fmoc-Cl (5.0 g, 19.327 mmol) was added dropwise, then moved to room temperature and stirred for 1 h, and the pH value was adjusted to 3 with 10% citric acid. The reaction solution was transferred to a 1L separatory funnel, extracted with ethyl acetate (100 mL×4 times), and rinsed with 1N HCl (120 mL×2). The organic phases were combined, dried with anhydrous MgSO ₄ , filtered with suction, then concentrated under reduced pressure to evaporate the liquid to dryness, dissolved in 5 mL of ethyl acetate, added n-hexane (300 mL) to settle while stirring, and the crude product was precipitated as a powder. The crude product was dissolved in ethyl acetate, and an appropriate amount of silica gel powder was added to make a solid solution. The sample was dry-loaded and subjected to column chromatography, and gradient elution was performed with 20% ethyl acetate/petroleum ether to 80% ethyl acetate/petroleum ether. The collected products were concentrated under reduced pressure and evaporated to dryness to obtain 6.209 g of intermediate product V with a yield of 99.0%. ¹ H-NMR (500MHz, DMSO-d ₆ ) 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)

Intermediate product IV (550 mg, 0.9095 mmol), intermediate product V (482.2 mg, 1.3643 mmol), EDCI (261.53 mg, 1.3643 mmol), HOBT (221.2 mg, 1.6371 mmol) were added to a 250 mL flask and washed with CH ₂ Cl ₂ (15 mL) was dissolved, the solution was stirred and cooled at 0°C for 20 minutes, and then DIEA (0.285 mL, 1.6371 mmol) was slowly added dropwise. After reacting for 4h, move to room temperature and stir overnight, stop the reaction, and directly evaporate to dryness. The crude product was dissolved in CH ₂ Cl ₂ , wet-loaded, and column chromatographed, first eluted with 200 ml CH ₂ Cl ₂ , then eluted with 2% MeOH/CH ₂ Cl ₂ to 5% MeOH/CH ₂ Cl ₂ , Finally eluted with ₂ % _NH3 - _H2O /4% MeOH/ _CH2Cl2 . The collected products were evaporated to dryness to obtain 855.2 mg of product (abbreviated as intermediate product VI for convenience of description), with a yield of 100%. ¹ H-NMR (500MHz, DMSO-d ₆ ) 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- TOF MS:[M+Na ⁺ ]962.2350,[M+K ⁺ ]978.2200

The intermediate product VI (855.0mg, 1.2120mmol), triethylamine (1.27mL, 12.12mmol) was added to a 150mL flask, and dissolved with CH ₂ Cl ₂ (15mL). Overnight, the solution became cloudy and triethylamine ( 2mL) until the reaction is complete, stop the reaction, directly evaporate to dryness, dissolve with CH ₂ Cl ₂ , apply the wet method, and perform column chromatography, first elute with 200ml CH ₂ Cl ₂ , then use 2% MeOH/CH ₂ Cl ₂ 4% MeOH/ _CH2Cl2 eluting, finally 5% _NH3 · _H2O / ₁₀ % MeOH/ _CH2Cl2 to 7.5% _{NH3 · H2O} / ₁₅ % MeOH/ _CH2Cl2 . The collected products were evaporated to dryness to obtain 885.1 mg of product (abbreviated as intermediate VII for convenience of description), with a yield of 98.7%. The hydrogen nuclear magnetic resonance spectrum is shown in Figure 1: ¹ H-NMR (500MHz, DMSO-d ₆ ) 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-TOF MS: [M+H ⁺ ]718.3108, [M+ Na ⁺ ]740.2903

The intermediate product VII (360.0mg, 0.5020mmol), 4AMR-SCM-40K (2.510g, 0.06276mmol, Beijing Jiankai Technology Co., Ltd.) was added to a 100mL flask, dissolved in CH ₂ Cl ₂ (5ml), at room temperature Stir slowly for two weeks, transfer to a 200mL flask with DMF and react for another 7 days. Concentrate by rotary evaporation, then add anhydrous ether for precipitation, and filter to obtain a crude product; then dissolve the crude product with dichloromethane and methanol, add ether for precipitation, filter and dry to obtain 2.0578 g of VIII intermediate product, yield 78.8%. The H NMR spectrum is shown in Figure 2: ¹ H-NMR (500MHz, DMSO-d ₆ ) 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).

Add intermediate product VIII (2.4g, 57.69mmol) into a 30mL round bottom flask, and dissolve it with CH ₂ Cl ₂ (20mL), then add TFA (1mL) and stir slowly at room temperature, stop the reaction after two weeks, directly evaporate to dryness, and use two Chloromethane (2 mL) was dissolved, and anhydrous ether was added to precipitate. After filtration, the filter cake was vacuum-dried to obtain 1.6697 g of intermediate product IX with a yield of 62.2%. The H NMR spectrum is shown in Figure 3: ¹ H-NMR (500MHz, CDCl ₃ ) 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) Intermediate product IX (1.6697g, 0.0384mmol), succinamide alcohol (132.7mg, 1.1528mmol), DMAP (4.7mg, 0.0384mmol) were added to a 100ml round bottom flask, and CH ₂ Cl ₂ (400ml) After dissolving, the mixed reaction solution was stirred at -5°C for 20 min, and DCC (237.9 mg, 1.1528 mmol) was added in batches. After the addition was completed, it was moved to room temperature for 1 hour, and the reaction was stirred slowly for two weeks. Stop the reaction, filter and concentrate the filtrate to dryness to obtain intermediate product X (1.6908g).

(2) Dissolve intermediate product X (1.6908g, 0.0384mmol) and gemcitabine hydrochloride (368.4mg, 1.2288mmol) in a 100mL flask with pyridine (10mL), stir slowly at room temperature for two weeks, stop the reaction, and directly evaporate to dryness, Then dissolve with dichloromethane/ethyl acetate, and evaporate to dryness repeatedly 3 times. Finally, dissolve with CH ₂ Cl ₂ , load the sample by wet method, elute with MeOH/CH ₂ Cl ₂ sequentially, collect the product points, concentrate under reduced pressure and dry in vacuum to obtain the crude product, then dissolve with 2 mL of dichloromethane, add anhydrous ether It was precipitated, filtered, and vacuum-dried to obtain 1.2953 g of the final product XI, with a yield of 73.7%. For convenience of presentation, the final product XI is named compound PEG(A+G). The H NMR spectrum is shown in Figure 4: ¹ H-NMR (500MHz, DMSO-d ₆ ) 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-TOF MS is in the range of 37500-47500, the highest peak is 42254.5313. The theoretical molecular weight of the compound PEG (A+G): 44497, wherein the theoretical drug loading of AZDD7762 is 3.257%, and the theoretical drug loading of gemcitabine is 2.366%.

Example 2

1. Experimental reagents and experimental animals

The compound PEG (A+G) obtained in Example 1, and the antitumor efficacy of compound A and G combined were investigated on p53 mutant nude mouse subcutaneous xenograft tumor model.

main reagent

1640 culture medium: Hyclone, product number SH30809.01B;

FBS: GIBCO, Cat. No. 10099-141;

100* penicillin and streptomycin double antibody: Invitrogen, catalog number 10378-016;

PEG(A+G): light yellow translucent viscous solid, loaded with small molecule drug A 3.257%, loaded with small molecule drug G 2.366%, sample size about 1.3g;

A: AZD7762, molecular weight 362.4, white crystal;

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

The preparation method of polyethylene glycol coupling (gemcitabine and AZD7762) (abbreviated as PEG(A+G) for convenience): Weigh an appropriate amount of the test drug, dissolve and disperse it with sterile saline, mix it evenly, and carry out the animal test. Administration, ready to use now.

A preparation method: Weigh an appropriate amount of the test drug, dissolve and disperse it (suspension) with 12% HP-β-CD, and administer it to animals after mixing evenly.

G Preparation method: Weigh an appropriate amount of the test drug and dissolve it with physiological saline.

experimental animals

Strain: Balb/c nude mice, SPF grade; source: Shanghai Lingchang Biotechnology Co., Ltd.; age: 5-6 weeks; sex: male.

2. Experimental method

PEG(A+G) Tolerance Study

Under the selected prescription, when the dosage is 1540mg/kg, the drug tolerance is studied; a total of 4 nude mice, i.p., administered once, observed for 4-5 days;

Establishment of Colo-205 nude mouse subcutaneous xenograft tumor model

a) recovery and expansion of Colo-205 cells;

b) After sufficient cells are amplified, the cells are collected, and a cell suspension with a concentration of 2×10 ⁷ cells/ml is prepared with serum-free 1640 medium;

c) Subcutaneously inoculate the right side of nude mice, 0.1ml/mouse, that is, the number of inoculated cells per nude mouse is 2×10 ⁶ ; inoculate 27 nude mice.

Observation: Observe the state of the animals twice a week and the appearance of tumors. When the tumors grow to an average volume of about 100-200 mm ³ , they are divided into groups for administration.

Grouping and administration: 18 animals with similar tumor volumes were selected from 27 animals and randomly divided into 3 groups according to the tumor volumes, namely normal saline control group, PEG(A+G) group, small molecule drug gemcitabine and Chk1 Inhibitor combination group (abbreviated as A+G combination), 6 animals in each group. The day of grouping was recorded as Day1, and the administration began on that day. The grouping and original dosage regimen are shown in Table 1:

Table 1. Animal grouping and dosing

*Dose of G is in free form

On Day 2 of drug administration observation, 1#, 2#, 3#, 6# animals in A+G group died, and 4 animals were selected from the spare animals to supplement this group, so that the average tumor volume of this group was equivalent to that of the normal saline control group; And the original dosage regimen was revised, see Table 2 for details:

Table 2: Animal grouping and administration adjustment

*Dose of G is in free form

Observation and detection: The tumor volume and animal body weight were measured 3-4 times a week for 14 days.

A+G combination (G first i.p. administration, A i.p. administration after 4h), according to the original dosage regimen, (50+36.3) mg/kg, 4/6 animals died the next day after administration; The dosage was adjusted to (25+18.1) mg/kg, and the interval between the administration of A and G was adjusted to 48h according to the weight change of the animal (G was administered i.p. first, and A was administered i.p. after 48h). However, after administration of PEG (A+G) once at 1540 mg/kg, the body weight decreased significantly, so the dosage was adjusted to 770 mg/kg, and the dosing interval was adjusted to once every 4 days.

3. Detection indicators and calculation and statistical analysis methods

Tumor volume (TV)

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

Relative tumor volume (RTV)

RTV=TVt/TV ₁ where TV ₁ is the tumor volume at the time of cage administration (ie d1), and TVt is the tumor volume at each measurement.

Relative tumor proliferation rate T/C (%)

T _RTV : RTV of the treatment group; C _RTV : RTV of the blank control group.

Statistical analysis: At the end of the experiment, the Levene test for homogeneity of variance was performed on the relative tumor volume (RTV) (1) of each group. If the homogeneity of variance analysis was not significant, that is, P>0.05, a one-way analysis of variance was used. When the analysis of variance Significant, that is, P≤0.05, the Dunnett test was used to compare the control group with other treatment groups. (2) If the P≤0.05 of the Levene test, that is, the variance is not homogeneous, logarithmic transformation is performed on the original data without negative values, and the transformed data is subjected to the Levene test of the homogeneity of variance. If the analysis of the homogeneity of variance is not significant, That is, P>0.05, using one-way analysis of variance, when the analysis of variance is significant, that is, P≤0.05, and then using the Dunnett test to compare the control group with other treatment groups.

4. Experimental results

Tolerance Study of PEG(A+G)

Under the selected prescription, when the dosage was 1540mg/kg, the body weight of the animal decreased within 2 days after intraperitoneal injection, and then there was a recovery trend. After 1 week of observation, as shown in Table 3, the drug has no effect on the general state of the animal. Including: no difference in hair; no significant difference in weight change; no nausea, vomiting, diarrhea, appetite, indigestion, dry skin, rash, skin redness, itching, pain, back pain, dyspnea, fatigue and insomnia. There is no difference in internal organs when viewed with the naked eye. There was no difference in the sleep and activity of the mice; there was no difference in the anus and defecation of the mice, and there was no diarrhea.

Table 3 Effects of PEG (A+G) on animals (body weight and general state)

*-indicates that no drug was administered that day

The results of body weight and tumor volume of nude mice are shown in Table 4:

Table 4 PEG (A+G), the effect of A+G combination on the body weight of Colo-205 tumor-bearing nude mice (data are represented by Mean±SEM, n=6)

a: See Table 2 for details of the dosage regimen;

b: On the 3rd day of administration, 2# animal died

c: On the second day of administration, 1#, 2#, 3#, 6# animals died, and 4 animals were selected from the spare animals to supplement this group;

In this experiment, the RSD of the tumor volume in each group was less than 20%. At the end of the experiment, the tumor volume of the normal saline control group reached 1333.6mm ³ , which was about 6.4 times higher than that of the grouping, which was in line with the efficacy of xenograft tumors in nude mice. General Requirements for Research.

Table 5 PEG (A+G), the inhibitory effect of A+G combination on the growth of Colo-205 nude mouse xenograft tumor (data are represented by Mean±SEM, n=6)

a: See Table 2 for details of the dosage regimen;

b: vs normal saline control group, *p<0.05, **p<0.01

According to the above adjusted dosing regimen, after administration and observation for 14 days, the PEG (A+G) administration group showed a significant anti-tumor effect, and the relative tumor proliferation rate T/C (%) of the experimental endpoint was 59.9%; while A The relative tumor proliferation rate T/C (%) of the experimental endpoint of the +G combined administration group was 72.0%. Fig. 5 is the tumor growth curves of the PEG (A+G) group, the A+G combination group and the normal saline control group.

The results of Example 2 above show that the body weight of the animals in the normal saline control group showed a downward trend (related to the tumor), while the body weight of the animals in the administration group decreased relatively more, and the administration had no effect on the general state of the animals. In summary, in the human colorectal cancer cell line Colo-205 nude mouse xenograft tumor model, under the administration regimen of the present invention, PEG (A+G) has a significant inhibitory effect on the Colo-205 nude mouse xenograft tumor.

Claims (3)

1. a kind of high-molecular compound with active anticancer, its structural formula is as shown in XI：

2. the preparation method of high-molecular compound as claimed in claim 1, it is characterised in that：Comprise the following steps,

Under HBTU, HOBT, DIEA and solvent DMF, 0 DEG C to room temperature condition, Boc-Gly and Chk1 inhibitor AZD7762 is entered Row amidatioon is connected, and is gone through TFA after Boc protections, the glutamic acid -5- tert-butyl esters of Gly-AZD7762 and the Fmoc protection of gained In PyAOP and 2,4,6- trimethylpyridines, amidatioon connection is carried out under the conditions of 0 DEG C, then go Fmoc to protect with triethylamine and DMF Shield, then the 6-aminocaprolc acid protected with Fmoc is in PyAOP and 2,4,6- trimethylpyridines, carries out amidatioon connection under the conditions of 0 DEG C, Then Fmoc is gone to protect with triethylamine and DMF conditions again, gained micromolecular compound intermediate is high with quadrifurcate polyethylene glycol Molecular vehicle 4ARM-SCM-40K carries out substituted amide connection, and gained high-molecular compound carries out the tert-butyl ester through TFA again and goes to protect Shield, the hydroxy-acid group of gained aromatic polymer intermediates is again through n-hydroxysuccinimide, DCC, DMAP and CH₂Cl₂Condition is lived Change, be finally coupled under the conditions of pyridine with gemcitabine, obtain final products XI.

3. application of the high-molecular compound as claimed in claim 1 in anticarcinogen is prepared.