CN109422799B - Docetaxel liver cancer-resisting targeted prodrug and medicinal application thereof - Google Patents
Docetaxel liver cancer-resisting targeted prodrug and medicinal application thereof Download PDFInfo
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Abstract
The invention belongs to the field of biological medicines, and particularly relates to a novel docetaxel targeted anti-liver cancer prodrug represented by a general formula (1) and medicinal application thereof. The compounds of the invention have pharmacological research value and can be used as anticancer prodrugs targeting matrix metalloproteinase MMP-2 or MMP-9. Can be used for treating cancers with high specificity expression of MMP-2 or MMP-9, and specifically, the compound of the invention can be used for treating at least one disease selected from the following diseases: liver cancer, ovarian cancer, breast cancer, non-small cell lung cancer, colon cancer, and the like.
In the formula (1), R 1 Substrate polypeptide sequences specifically identified and hydrolyzed by matrix metalloproteinases MMP-2 and MMP-9 which are specifically and highly expressed in liver cancer tissues; r 2 Is methyl, trifluoromethyl, etc.; r 3 Hydrogen atom, fluorine atom, etc.
Description
Technical Field
The invention belongs to the field of biological medicines, and particularly relates to a novel anti-liver cancer targeted docetaxel prodrug and medicinal application thereof.
Background
Data show that liver cancer is one of the most common malignant tumors, and more than 70 million new cases and death cases are generated every year in the world. As a big liver disease country, China has a rising trend that the number of new cases accounts for 50.5% of the total liver cancer diseases worldwide, and the number of dead cases accounts for 51.4% of the total liver cancer deaths worldwide. According to the evaluation criteria of the size and the number of liver tumors, whether extrahepatic metastasis occurs, the liver function grade, whether vascular infiltration exists and the like, the clinical treatment scheme for liver cancer mainly comprises surgical treatment (hepatectomy, liver transplantation, local ablation treatment and the like, and is suitable for patients at the early stage of liver cancer), hepatic artery interventional chemotherapy (the first treatment scheme for liver cancer at the middle and late stages) and targeted drug sorafenib treatment (auxiliary drug for liver cancer at the middle and late stages). Because the liver cancer is occult in pathogenesis, more than 80 percent of patients cannot be treated by surgery, the main treatment scheme of most liver cancer patients adopts the interventional therapy of chemotherapeutic drugs (comprising 10-hydroxycamptothecin, 5-fluorouracil, cisplatin, adriamycin, methotrexate and the like) and the auxiliary therapy of sorafenib targeted drugs. However, due to the hypofunction of drug metabolism caused by the loss of liver function, chemotherapy drugs can cause more serious toxic and side effects to liver cancer patients, including cardiotoxicity, nephrotoxicity, bone marrow suppression, serious gastrointestinal reactions, urinary system diseases and the like. Sorafenib, as a first-line anti-liver cancer drug approved by FDA, EMEA and SFDA in the united states, has certain clinical defects including severe gastrointestinal reactions, limb weakness, hand-foot syndrome and the like, and is expensive and is a practical economic problem which cannot be ignored. In the face of the growing liver cancer patient population in China, the development of a novel efficient anti-liver cancer medicament with the independent intellectual property rights in China faces huge opportunities and challenges and is also a difficult point to be urgently solved clinically.
As the most successful antitumor drugs in the modern medical history of human beings, paclitaxel and the second-generation taxane anticancer drug docetaxel are widely applied to the clinical treatment of cancers such as ovarian cancer, breast cancer, non-small cell lung cancer and the like, and the global accumulated sales has broken through $ 300 billion so far. Despite the lack of alternatives in clinical efficacy, there are still considerable disadvantages, such as metabolic instability, poor water solubility, susceptibility to drug resistance and toxic side effects. In recent years, in view of the above-mentioned shortcomings of paclitaxel drugs, scientists have focused on methods such as structure optimization and formulation modification of various strategies, and have obtained a series of research results in the development of new generation of taxane antitumor drugs, including: (1) for the problem of drug resistance of paclitaxel, the carbazitaxel (Cabazitaxel) is subjected to methylation structure optimization at the C-7 hydroxyl and the C-10 hydroxyl simultaneously, and is approved by FDA on the market in 2010;
(2) ornaxel (Orataxel) is optimized aiming at the defect of low oral bioavailability of paclitaxel, and phase II clinical experiments are carried out by Spectrum company at present, so that the Ornaxel has 400 times stronger toxicity to multidrug-resistant cancer cells than the paclitaxel and also shows the same excellent inhibitory activity to drug-sensitive cells; (3) the paclitaxel liposome-likukosu for injection in 2004 is marketed in China, the technology successfully overcomes the technical problem that paclitaxel is difficult to dissolve in water and various medicinal solvents, and radically solves the adverse reaction and hypersensitive reaction caused by a surfactant; (4) in 005, the first non-solvent type nano albumin combined chemotherapeutic drug Abraxane appeared on the market in the United states, which mainly improves the clinical defect of poor water solubility of taxol compounds and has the capacity of being combined with specific protein on the surface of tumor cells; (5) paclitaxel-targeted prodrug Opaxio developed by Cell Therapeutics, which was approved by FDA in 2012 to be marketed in the united states, has improved water solubility and tumor targeting to a greater extent than paclitaxel. Although the research and development of new generation taxane anticancer drugs have advanced sufficiently, the clinical defects of poor metabolic stability and large system toxicity caused by drug heterogeneity existing in all the paclitaxel drugs, especially in the aspect of expanding new indications of the paclitaxel drugs on the liver cancer resistance, are not reported in the drugs on the market at present.
The prodrug design (prodrug design) strategy is deeply and widely applied to the research and development of new paclitaxel drugs. The prodrug compound has the greatest characteristic that specific structural modification is carried out on a parent drug group, so that specific clinical defects of the parent drug group are effectively improved; the active configuration of the parent drug group is sealed by adopting carrier molecules, so that the system toxicity brought by the parent drug is reduced, and the metabolic stability of the drug is improved; the specific carrier (monoclonal antibody, polypeptide, etc.) is specifically identified by tumor tissue, so that the targeting ability of the medicine is improved.
Matrix Metalloproteinases (MMPs) are a restriction enzyme family with high homology, the expression level of the MMPs in a healthy organism is very low, and the expression level of the MMPs in a tumor part is remarkably improved due to stimulation of various over-expressed cytokines, wherein MMP-2 and MMP-9 are proteolytic enzymes which are discovered so far and have the closest relationship with tumor invasion and metastasis, have the most important effects in tumor infiltration and invasion, and are also considered as two most important tumor markers. It has a common characteristic that polypeptide molecules which can recognize a section of amino acid residues with a specific sequence can be specifically hydrolyzed; but also has the characteristics of high distribution and high expression in malignant tumor cells and low expression in certain normal or inflammatory tissues of a human body.
The design strategy of the prodrug is one of the most important means for reducing the toxicity of the medicine, takes substrate polypeptide which is identified and hydrolyzed by specific high-expression mechanism metalloprotease MMPs in liver cancer tissues as a targeting carrier group, takes docetaxel with better anticancer activity as a parent drug, and connects the substrate polypeptide and the polyene taxane through a bridge chain molecule with a self-degradation function to form the prodrug which is an unreported compound. The research and development of the targeted prodrug molecules mainly aim at the defects of poor selectivity and high systemic toxicity of the docetaxel, and expand the application of the drugs in the anti-liver cancer tumor treatment.
Disclosure of Invention
The invention aims to provide a novel docetaxel anti-liver cancer targeted prodrug and medicinal application thereof.
The invention comprises a novel polyene taxane targeting anti-liver cancer prodrug represented by the following general formula (1). The polyene taxane targeted prodrug is covalently coupled with substrate polypeptide which is identified and hydrolyzed by protease with high specificity expression in liver cancer tissues in taxane compound molecules through bridge molecules, and can obviously improve the targeting property of the medicine to liver cancer.
In the formula (1), R 1 Including but not limited to substrate polypeptide sequences specifically recognized and hydrolyzed by matrix metalloproteinase MMP-2 such as Gly-Pro-Gln-Gly-Met-Ala-Gly-Gln, Gly-Pro-Gln-Gly-Ile-Ala-Ser-Gln, Gly-Pro-Gln-Gly-Ser-Ala-Gly-Gln, Gly-Pro-Gln-Gln-Ile-Ala-Gly-Gln, Gly-Pro-Gln-Gly-Ile-Trp-Gly-Gln, Gly-Pro-Gln-Gly-Ile-Hyp-Gly-Gln, etc., including but not limited to substrate polypeptide sequences specifically recognized and hydrolyzed by matrix metalloproteinase MMP-9 such as Gly-Pro-Gln-Phe-Ile-Ala-Gly-Gln, Ala-Ser-Gly-Pro-Ala-Gly-Pro, Gly-Pro-Gln-Gly-Ile-Ala-Gly-Gln, Gly-Pro-Gln-Gly-Tyr-Ala-Gly-Gln, Gly-Asn-Gln-Gly-Ile-Ala-Gly-Gln, etc. R 2 Including but not limited to methyl, trifluoromethyl, and the like; r 3 Including but not limited to hydrogen atoms, fluorine atoms, and the like.
The invention also provides a synthesis method of the novel docetaxel targeted anti-liver cancer prodrug, which comprises the steps of introducing a corresponding bridging group into a C-2' hydroxyl group of a docetaxel compound to synthesize a derivative of the docetaxel compound; then mixing the derivative containing the bridging group with the target polypeptide, and stirring and coupling at 5 ℃ under the action of a condensing agent to obtain the docetaxel targeted prodrug.
The compounds of the invention have pharmacological research value and can be used as anticancer prodrugs targeting matrix metalloproteinase MMP-2 or MMP-9. Can treat cancers with high specificity expression of enzymes MMP-2 or MMP-9, and specifically, the compound can treat at least one disease selected from the following diseases: liver cancer, ovarian cancer, breast cancer, non-small cell lung cancer, colon cancer, and the like.
The invention provides the following data for such anti-liver cancer targeted prodrugs: the inhibitory activity on HepG2 and SMMC-7721 liver cancer cells; toxicity to HL7702 normal liver cells and HEK296 kidney cells.
In one embodiment of the invention, the synthesis route of the docetaxel or the tetrafluorodocetaxel through the coupling prodrug of the bridging group Leu-PABC and the liver cancer targeting polypeptide A1-B11 is shown as Scheme 1.
Synthetic route of Scheme 1 liver cancer targeting polypeptide prodrug
Compared with the prior art, the invention has the beneficial effects that:
aiming at the defects of high system toxicity and lack of tumor targeting capability of docetaxel drugs, the invention designs and synthesizes a prodrug compound with targeting and inhibiting effects on liver cancer by adopting a prodrug design strategy and taking tetrafluorodocetaxel and docetaxel with good anti-liver cancer activity as parent drugs and taking an amino acid sequence which can be specifically identified and hydrolyzed by MMP-2 or MMP-9 as a targeting carrier group, preferably taking PABC with a self-degradation function as a bridge chain molecule according to the characteristics that high-expression matrix metalloproteinases MMP-2 and MMP-9 exist in liver cancer tumors and can specifically identify and hydrolyze polypeptides consisting of 8 amino acids according to literature reports. Compared with pure polyene taxane compounds, the polyene taxane compound has the advantages that the polyene taxane compound greatly improves the targeting ability to tumors and can effectively reduce the toxicity to normal cells while retaining most anti-liver cancer activity. In addition, the preparation method of the prodrug compound is simple and easy to implement, does not need harsh reaction conditions, is easy to realize large-scale production, and reduces the cost of the medicament.
Detailed Description
The present invention is further illustrated below with reference to examples, which are by no means intended to limit the scope of the invention.
Example 1 preparation of a prodrug of docetaxel or tetrafluorodocetaxel coupled to a liver cancer-targeting polypeptide A1-B11 via a bridging group Leu-PABOH
The synthesis route of the taxane targeting prodrug prepared by coupling paclitaxel (DTX) or tetrafluorodocetaxel (4FDT) with liver cancer targeting polypeptides A1-B11 comprises the following steps:
1) synthesis and activation modification of bridging group Leu-PABOH
A100 mL round-bottomed flask was charged with 800mg (2.27mmol) of Fmoc-L-leucine, 300mg (2.5mmol) of aminobenzol, 50mL of anhydrous DCM was added, and the mixture was stirred at room temperature for 10 min. Then, 610mg (2.5mmol) of 2-ethoxy-1-ethoxycarbonyl-1, 2-dihydroquinoline was added thereto, and the reaction was stirred at room temperature. After 24h the reaction was monitored on a TLC plate (dichloromethane: methanol ═ 35:1) and, after disappearance of Fmoc-L-leucine, the reaction was stopped by addition of water, washed with saturated sodium chloride solution and extracted twice with ethyl acetate. Drying with anhydrous sodium sulfate, vacuum filtering, spin-drying the organic solvent to obtain crude product, purifying with silica gel column chromatography, eluting with DCM/MeOH 60:1-40:1 to obtain compound 1 as white solid 945mg, 91% yield.
A50 mL two-necked bottle is taken, 600mg (1.3mmol) of the compound 1 is added, 790mg (2.6mmol) of bis (p-nitrophenyl) carbonate is added, 20mL of anhydrous DMF is added under the protection of nitrogen, the mixture is stirred for 10min at the temperature of 0 ℃ in ice bath, 664 mu l (3.9mmol) of diisopropylethylamine is slowly added dropwise, the mixture is stirred for 15min at the temperature of 0 ℃ after the dropwise addition is completed, and then the mixture is moved to room temperature for reaction for 12 h. Monitoring the reaction condition by a TLC plate (normal hexane: acetone ═ 4:1), adding water to stop the reaction when the compound 1 is completely reacted, separating out a solid, recrystallizing the solid obtained by suction filtration by normal hexane/ethyl acetate to obtain a crude product, purifying by silica gel column chromatography, and obtaining the compound 2 as a white solid 952mg with the yield of 84% by adopting the normal hexane: acetone ═ 10:1-6:1 as an elution system.
Experimental methods see the Bakhet Elsadek et al, development of novel design of novel and in vivo evaluation test, European Journal of Cancer,2010,46(18):3434-
2) Synthesis of docetaxel C-2' position intermediate 3a/3b
A50 mL two-necked flask was charged with 710mg (0.81mmol) of tetrafluorodocetaxel, 500mg (0.81mmol) of compound 20, and 25mL of anhydrous dichloromethane, and stirred at room temperature for 10 min. Then, 92mg (0.81mmol) of 4-dimethylaminopyridine was added thereto, and the mixture was stirred at room temperature. After 8h the reaction was monitored by TLC plate (DCM: MeOH ═ 40:1), after the starting material had reacted, the reaction was quenched with water, washed with saturated sodium chloride solution and extracted twice with ethyl acetate. Drying with anhydrous sodium sulfate, vacuum filtering, spin-drying the organic solvent to obtain crude product, purifying with silica gel column chromatography, eluting with dichloromethane and methanol at a ratio of 100:1-80:1-40:1 to obtain intermediate 3a as white solid 662mg with a yield of 60%.
Compound 3a data are as follows:
mp:172.1-175.3℃；1H NMR(400MHz,Acetone-d6):δ9.43(s,1H),8.12(d,J＝7.6Hz,2H),7.86(d,J＝7.6Hz,2H),7.76-7.64(m,5H),7.58(t,J＝7.6Hz,2H),7.51(d,J＝7.6Hz,2H),7.46-7.35(m,6H),7.34-7.25(m,3H),7.04(d,J＝9.6Hz,1H),6.83(d,J＝8.0Hz,1H),6.11(d,J＝8.4Hz,1H),5.68(d,J＝7.2Hz,1H),5.40(d,J＝9.2Hz,1H),5.30(d,J＝5.2Hz,1H),5.25(s,1H),4.97(d,J＝8.8Hz,1H),4.39-4.30(m,5H),4.24(t,J＝7.0HZ,2H),4.17(s,2H),3.93(d,J＝6.8Hz,1H),3.68(s,1H),2.83(s,1H),2.45(d,J＝17.6Hz,4H),2.38-2.28(m,1H),2.12(d,J＝8.4Hz,1H),1.93-1.77(m,5H),1.75-1.65(m,5H),1.45-1.25(m,20H),1.17(d,J＝12.4Hz,6H),0.96(dd,J＝10.8,6.6Hz,6H),0.88(t,J＝6.6Hz,1H).ESI-MS:m/z 1292.4[M+H]+,1314.2[M+Na]+；C72H81N3O19:HRMS calcd.1314.5356[M+Na]+,found 1314.5366.
compound 3b was synthesized analogously to 3a in 64% yield with the following data:
mp:167.4-169.8℃；1H NMR(400MHz,Acetone-d6):δ9.46(s,1H),7.95(d,J＝7.6Hz,1H),7.86(d,J＝7.6Hz,2H),7.79(d,J＝9.6Hz,1H),7.71(dd,J＝13.2,7.2Hz,6H),7.67-7.61(m,1H),7.52(t,J＝13.6Hz,3H),7.47-7.35(m,7H),7.30(dd,J＝15.2,7.8Hz,3H),6.84(d,J＝8.0Hz,1H),6.10(t,J＝9.2Hz,1H),5.66(d,J＝6.8Hz,1H),5.40-5.34(m,1H),5.32(d,J＝5.6Hz,1H),5.24(s,1H),5.20(d,J＝12.0Hz,1H),5.12(d,J＝12.0Hz,1H),4.98(d,J＝9.2Hz,1H),4.41-4.28(m,7H),4.24(t,J＝6.8Hz,1H),4.17(q,J＝8.0Hz,3H),3.92(d,J＝7.2Hz,1H),3.79(s,1H),2.52-2.42(m,5H),2.26(dd,J＝15.6,8.6Hz,2H),2.02-1.95(m,1H),1.90-1.75(m,7H),1.73-1.63(m,6H),1.57(d,J＝3.6Hz,7H),1.34-1.24(m,5H),1.17(s,4H),0.95(dd,J＝10.4,6.6Hz,7H).ESI-MS:m/z 1364.2[M+H]+,1386.2[M+Na]+；C72H77F4N3O19:HRMS calcd.1386.4980[M+Na]+,found 1386.5001.
3) synthesis of intermediate 4a/4b
A10 mL two-necked flask was charged with 30mg (0.022mmol) of Compound 3a and 3mL of anhydrous DMF, and the mixture was stirred at room temperature for 10 min. mu.L of piperidine (0.055mmol) was added dropwise quickly and stirred at room temperature for 45 min. The reaction was monitored by TLC plate (dichloromethane: methanol ═ 20:1) and starting material 21a was reacted completely, water was added to stop the reaction, dichloromethane was extracted 3 times, the organic phase was collected, water was pumped off, then oil pump was pumped for 30min to obtain crude compound 4a with a purity of about 80% which was used in the next reaction without purification. Compound 4b was synthesized analogously to 4a, in 79% yield.
4) Synthesis of liver cancer targeted prodrug compounds 5a/5 b-15 a/15b
DTX-PABC-Leu-A1(5a)
Taking a 25ml two-necked bottle, N 2 Adding 0.01mmol of compound 4a, 0.015mmol of targeting carrier polypeptide A1(Gly-Pro-Gln-Gly-Met-Ala-Gly-Gln-Fmoc), 0.06mmol of 1-hydroxybenzotriazole (HOBt) and 1.5mL of N, N-Dimethylformamide (DMF) under protection, after fully dissolving at 0 ℃, adding 0.04mmol of N-methylmorpholine, reacting for 15min, adding 0.08mmol of N, N' -Diisopropylcarbodiimide (DIC), heating to 5 ℃, continuing the reaction, monitoring the reaction by HPLC, supplementing 0.02-0.03mmol of targeting carrier polypeptide A1 every other day, and supplementing for 2 times. The reaction can be completed in about 72 hours; after the reaction is finished, the solvent N, N-Dimethylformamide (DMF) is removed as much as possible by a vacuum oil pump at room temperature, a small amount of dimethyl sulfoxide (DMSO) is added for dissolution, and the preparation and purification are carried out at reverse medium pressure. The mobile phase adopts an acetonitrile/water system, and the specific method comprises the following steps: 0% acetonitrile + 100% water, 20min (removing DMSO), 0% -40% acetonitrile + 100% -60% water, 20min, 40% -65% acetonitrile + 60% -35% water, 40 min. 5a generating a peak in 53% acetonitrile gradient, collecting the product peak, and concentrating under reduced pressure to minimumVolume, freeze-drying afforded 5a as a white solid in 35% yield. The remaining prodrug compounds were synthesized.
1 H NMR(400MHz,DMSO-d 6 )δ9.97(s,1H),8.37-8.04(m,5H),8.04-7.87(m,6H),7.83(d,J＝7.5Hz,2H),7.75-7.52(m,8H),7.50-7.15(m,14H),7.10(t,J＝7.1Hz,1H),6.94-6.61(m,2H),5.71(t,J＝8.4Hz,1H),5.31(t,J＝17.0Hz,1H),5.16-4.89(m,7H),4.84(d,J＝9.8Hz,1H),4.33(m,4H),4.20(m,6H),4.12-4.05(m,1H),3.96(m,3H),3.89-3.45(m,8H),3.45-3.37(m,1H),2.33(t,J＝19.7Hz,2H),2.19(m,5H),2.06(m,5H),1.93(t,J＝9.6Hz,5H),1.90-1.75(m,7H), 1.75-1.62(m,8H),1.57(d,J＝11.3Hz,4H),1.39(d,J＝44.0Hz,6H),1.34-1.22(m,10H),1.17(s,9H),0.95(m,7H),0.82(m,8H).ESI-MS:2040.3[M+Na] + ；C 101 H 127 N 13 O 29 S:HRMS calcd.2040.8476[M+Na] + ,found 2040.8595.
4FDT-PABC-Leu-A1(5b)
The synthesis method is the same as 5a, white solid, and the yield is 32%.
1 H NMR(400MHz,DMSO-d 6 )δ9.97(s,1H),8.50-8.45(m,4H),8.19(s,2H),8.10(d,J＝18.1Hz,8H),8.02-7.89(m,7H),7.86-7.80(m,7H),7.78-7.74(m,2H),7.65(t,J＝7.3Hz,9H),7.59(d,J＝7.9Hz,11H),7.44(s,1H),7.36(t,J＝7.5Hz,14H),7.24(d,J＝21.9Hz,29H),7.11(s,2H),6.93(d,J＝11.8Hz,0H),6.77(d,J＝19.2Hz,4H),5.13-4.99(m,10H),4.97-4.89(m,3H),4.88-4.83(m,1H),4.48(d,J＝8.5Hz,0H),4.17(d,J＝27.1Hz,13H),4.07(d,J＝10.2Hz,1H),4.02-3.91(m,3H),3.78-3.60(m,4H),3.49(s,0H),2.36(d,J＝6.1Hz,6H),2.18(m,10H),2.02(br,29H),1.94(br,12H),1.84(brs,17H),1.66(brs,25H),1.60-1.52(m,10H),1.50-1.41(m,37H),1.17(brs,31H),0.92(brs,19H),0.83(m,24H).ESI-MS:m/z 2112.3[M+Na] + ；C 101 H 123 F 4 N 13 O 29 S:HRMS calcd.2112.8099[M+Na] + ,found 2112.8194.
DTX-PABC-Leu-A2(6a)
The synthesis was performed as in 5a, white solid, 29% yield.
1 H NMR(400MHz,CDCl 3 )δ＝9.86(s,1H),8.34-8.36(m,3H),8.16-8.18(m,2H),8.09-8.13 (m,5H),8.00-8.01(m,1H),7.58-7.70(m,5H),7.24-7.39(m,10H),7.76(d,J＝2.6Hz,2H),4.84-5.11(m,8H),3.50-4.41(m,12H),2.06-2.19(m,10H),1.82(m,9H),1.67(m,12H),1.46(m,6H),1.29(m,11H),1.18(d,J＝1.7Hz,5H),0.93(s,1H),0.76-0.86(m,13H).ESI-MS:m/z 1872.7[M+Na] + ；C 90 H 123 N 13 O 29 :HRMS calcd.1872.8506[M+Na] + ,found 1872.8587.
4FDT-PABC-Leu-A2(6b)
The synthesis was performed as in 5a, white solid, 37% yield.
1 H NMR(400MHz,DMSO-d 6 )δ9.82(s,1H),8.44(s,1H),8.07(s,4H),7.95(d,J＝2.5Hz,2H),7.86(s,2H),7.73(s,2H),7.56(s,5H),7.24(s,10H),7.08(s,1H),6.72(s,2H),5.67(s,1H),5.28(s,1H),4.95(m,11H),4.45(m,2H),4.18(m,8H),3.91(m,5H),3.60(m,9H),2.14(m,5H),2.02(m,6H),1.76(m,12H),1.62(m,12H),1.42(m,18H),1.13(m,10H),0.79(m,26H).ESI-MS:1944.8[M+Na] + ；C 90 H 119 F 4 N 13 O 29 :HRMS calcd.1944.8065[M+Na] + ,found1944.8081.
DTX-PABC-Leu-A3(7a)
The synthesis method is the same as 5a, white solid, and the yield is 36%.
1 H NMR(400MHz,CDCl 3 )δ＝9.94(s,1H),8.19-8.23(m,2H),8.08-8.09(m,2H),7.90-8.00(m,8H),7.58-7.68(m,5H),7.25-7.39(m,8H),7.10-7.13(m,1H),7.78(d,J＝3.4Hz,2H),5.34-5.36(m,1H),4.95-5.10(m,8H),4.86(d,J＝2.4Hz,1H),3.33-4.41(m,20H),2.20(m,4H),2.05-2.08(m,5H),1.82(m,8H),1.71(m,6H),1.46(m,5H),1.29(m,9H),1.18-1.20(m,6H),0.93(m,6H),0.62-0.87(m,7H).ESI-MS:1816.4[M+Na] + ；C 86 H 115 N 13 O 29 :HRMS calcd.1816.7684[M+Na] + ,found1816.7702.
4FDT-PABC-Leu-A3(7b)
The synthesis method is the same as 5a, white solid, and the yield is 30%.
1 H NMR(400MHz,DMSO-d 6 )δ9.95(s,1H),8.43(dd,J＝45.0,8.5Hz,1H),8.30-8.15(m,2H),8.09(d,J＝6.4Hz,2H),7.96(dt,J＝15.6,5.5Hz,4H),7.77(d,J＝7.8Hz,1H),7.60(d,J＝8.2Hz,5H),7.44-7.16(m,8H),7.12(t,J＝7.1Hz,1H),6.78(m,2H),5.72(m,1H),5.30(m,1H),5.18-4.98(m,6H),4.97(s,2H),4.86(d,J＝9.9Hz,1H),4.50(s,1H),4.44-4.14(m,6H),4.14-4.06(m,1H),3.95(m,4H),3.67(m,5H),3.63-3.51(m,3H),3.51(m,2H),3.45-3.36(m,2H),2.18(s,4H),2.15-1.95(m,6H),1.81(m,8H),1.68(m,7H),1.63-1.51(m,4H),1.51-1.33(m,11H),1.18(brs,5H),0.92(brs,7H),0.86(m,4H),0.82(d,J＝6.1Hz,3H).ESI-MS:m/z 1888.7[M+Na] + ；C 86 H 111 F 4 N 13 O 29 :HRMS calcd.1888.7439[M+Na] + ,found 1888.7462.
DTX-PABC-Leu-A4(8a)
The synthesis method is the same as 5a, white solid, and the yield is 32%.
1 H NMR(400MHz,DMSO-d 6 )δ9.95(s,1H),8.38-8.02(m,J＝44.2,31.5,13.1Hz,6H),8.02-7.88(m,J＝16.9,7.0Hz,4H),7.88-7.55(m,7H),7.45-7.16(m,9H),7.11(t,J＝7.2Hz,1H),6.80(d,J＝12.5Hz,3H),5.72(t,J＝8.9Hz,1H),5.32(t,J＝12.8Hz,1H),5.17-4.91(m,7H),4.86(d,J＝9.7Hz,1H),4.38(m,2H),4.19(m,4H),4.13-4.04(m,2H),3.97(d,J＝10.2Hz,4H),3.85-3.37(m,6H),2.20(m,4H),2.02(m,8H),1.96-1.77(m,10H),1.77-1.53(m,13H),1.46(m,6H),1.31(m,10H),1.17(m,8H),0.97(m,8H),0.84(m,7H),0.74(m,7H).ESI-MS:m/z 1913.8[M+Na] + ；C 92 H 126 N 14 O 29 :HRMS calcd.1913.8707[M+Na] + ,found 1913.8703.
4FDT-PABC-Leu-A4(8b)
The synthesis was performed as in 5a, white solid, 29% yield.
1 H NMR(400MHz,CDCl 3 )δ＝10.13(10.21)(s,1H),7.09-8.50(m,23H),5.76(s,3H),5.31(s,1H),5.07(d,J＝2.0Hz,2H),4.88-4.92(m,2H),3.94-4.16(m,10H),3.51-3.76(m,5H),2.18(s,3H),2.04(bs,7H),1.79(s,9H),1.65(s,8H),1.60(s,6H),1.43-1.48(m,10H),1.16(s,9H),0.71-1.11(m,20H).ESI-MS:m/z 1985.7[M+Na] + ；C 92 H 122 F 4 N 14 O 29 :HRMS calcd.1985.9326[M+Na] + ,found 1985.9347.
DTX-PABC-Leu-A5(9a)
The synthesis method is the same as 5a, white solid, and the yield is 15%.
1 H NMR(400MHz,CDCl 3 )δ＝10.73(s,1H),9.97(s,1H),8.10-8.13(m,4H),7.99(s,2H),7.89-7.94(m,4H),7.59-7.67(m,5H),7.49(d,J＝2.0Hz,1H),6.79-7.38(m,15H),5.71(d,J＝0.4Hz,1H),5.34(d,J＝1.6Hz,1H),4.84-5.07(m,7H),3.40-4.45(m,17H),3.11(m,3H),2.19(m,4H),2.07(m,4H),1.80(m,8H),1.67(m,6H),1.56(m,4H),1.45(m,5H),1.28(m,10H),1.17(m,2H),0.92(m,7H),0.81-0.86(m,6H),0.68(m,5H).ESI-MS:m/z 1957.5[M+Na] + ；C 97 H 126 N 14 O 28 :HRMS calcd.1957.8659[M+Na] + ,found 1957.8673.
4FDT-PABC-Leu-A5(9b)
The synthesis method is the same as 5a, white solid, and the yield is 16%.
1 H NMR(400MHz,DMSO-d 6 )δ10.75(s,1H),9.97(d,J＝17.5Hz,1H),8.49(d,J＝9.1Hz,1H),8.37(d,J＝7.9Hz,0H),8.26-8.06(m,5H),8.00(s,2H),7.90(s,1H),7.77(s,2H),7.59(d,J＝8.4Hz,5H),7.50(d,J＝7.8Hz,1H),7.40-7.23(m,8H),7.18(d,J＝9.2Hz,0H),7.10(s,2H),6.99(t,J＝7.5Hz,1H),6.92-6.86(m,1H),6.80(s,2H),5.72(t,J＝8.9Hz,1H),5.38-5.23(m,1H),5.13-4.83(m,7H),4.51(m,3H),4.22(m,2H),4.17(m,2H),3.96(m,4H),3.69(m,5H),3.43(m,2H),3.09(m,1H),2.90(m,1H),2.19(m,4H),2.13-2.01(m,4H),1.80(m,9H),1.69(m,6H),1.60(m,4H),1.53-1.33(m,11H),1.18(m,5H),0.93(m,7H),0.84(m,6H),0.68(d,J＝6.9Hz,6H).ESI-MS:1026.3[M+2Na] 2+ ；C 97 H 122 F 4 N 14 O 28 :HRMS calcd.1944.8065[M+Na] + ,found 1944.8081.
DTX-PABC-Leu-A6(10a)
The synthesis method is the same as 5a, white solid, and the yield is 34%.
1 H NMR(400MHz,DMSO-d 6 )δ9.93(d,J＝12.6Hz,1H),8.41(d,J＝13.2Hz,1H),8.38-8.30(m,-1H),8.07(s,3H),7.97(s,2H),7.93(d,J＝7.9Hz,8H),7.84(t,J＝10.4Hz,2H),7.66(d,J＝7.0Hz,1H),7.60(m,8H),7.35(m,4H),7.30(m,10H),7.23(m,2H),7.13-7.07(m,1H),6.83(d,J＝12.4Hz,1H),6.73(d,J＝8.2Hz,1H),5.11(m,4H),5.02(m,6H),4.94(m,3H), 4.87-4.81(m,1H),4.41(m,2H),4.29(m,6H),4.21(m,2H),4.09(m,7H),3.95(m,5H),3.77(dd,J＝15.1,8.5Hz,1H),3.59(m,10H),3.11(m,15H),2.19(m,6H),2.05(m,7H),1.95(m,2H),1.83(m,21H),1.66(m,17H),1.45(m,11H),1.28(m,19H),1.17(m,5H),0.92(m,14H),0.87-0.76(m,20H),0.73(s,6H).ESI-MS:m/z 1885.8[M+Na] + ；C 91 H 122 N 12 O 30 :HRMS calcd.1026.4137[M+Na] + ,found 1026.4145.
4FDT-PABC-Leu-A6(10b)
The synthesis method is the same as 5a, white solid, and the yield is 31%.
1 H NMR(400MHz,DMSO-d 6 )δ9.96(s,1H),8.53-8.39(m,2H),8.33(d,J＝8.0Hz,1H),8.08(s,2H),8.04-7.91(m,2H),7.86(dd,J＝12.6,6.5Hz,2H),7.77(s,1H),7.62(t,J＝8.4Hz,5H),7.41-7.17(m,8H),7.12(t,J＝7.1Hz,1H),6.80(d,J＝42.1Hz,2H),5.72(t,J＝8.8Hz,1H),5.37-5.24(m,1H),5.18-4.91(m,8H),4.87(d,J＝10.1Hz,1H),4.51(s,1H),4.30(d,J＝8.1Hz,8H),3.96(s,4H),3.85-3.45(m,10H),3.45-3.38(m,1H),3.11(t,J＝5.8Hz,0H),2.19(s,4H),2.16-2.03(m,5H),1.94(s,2H),1.83(dd,J＝15.3,5.6Hz,10H),1.72-1.55(m,10H),1.49(dd,J＝21.0,10.3Hz,14H),1.18(s,4H),0.93(s,8H),0.88-0.77(m,10H),0.73(t,J＝7.3Hz,3H).ESI-MS:m/z 1957.3[M+Na] + ；C 91 H 118 F 4 N 12 O 30 :HRMS calcd.1957.7905[M+Na] + ,found 1957.7877.
DTX-PABC-Leu-B7(11a)
The synthesis method is the same as 5a, white solid, and the yield is 11%.
1 H NMR(400MHz,DMSO-d 6 )δ10.02-9.91(s,1H),8.32-8.22(s,1H),8.10(d,J＝6.0Hz,11H),8.03-7.87(m,17H),7.83(t,J＝10.4Hz,1H),7.73(t,J＝7.2Hz,1H),7.66(d,J＝7.1Hz,2H),7.60(d,J＝7.5Hz,12H),7.35(d,J＝7.2Hz,5H),7.32-7.24(m,16H),7.22(s,3H),7.12(m,18H),6.86-6.71(m,3H),5.02(m,16H),4.40(m,2H),4.33(m,2H),4.20(m,9H),3.95(m,8H),3.87-3.77(m,2H),3.65(m,3H),3.57(m,1H),3.41(d,J＝7.0Hz,1H),2.44(m,9H),2.18(m,10H),2.05(m,9H),1.94(d,J＝7.1Hz,3H),1.76(m,24H),1.68(m,24H),1.59(m,10H),1.45(m,16H),1.37(m,2H),1.27(m,29H),1.17(m,16H),1.01(m,2H),0.92(m,19H),0.84(m,9H),0.81-0.57(m,27H).ESI-MS:m/z 1932.8[M+Na] + ；C 96 H 127 N 13 O 28 :HRMS calcd.1932.8806[M+Na] + ,found 1932.8842.
4FDT-PABC-Leu-B7(11b)
The synthesis method is the same as 5a, white solid, and the yield is 12%.
1 H NMR(400MHz,DMSO-d 6 )δ9.96(s,1H),8.48(d,J＝9.0Hz,1H),8.32-8.03(m,5H),7.98(d,J＝4.3Hz,3H),7.88-7.72(m,3H),7.72-7.52(m,5H),7.32(ddd,J＝22.8,14.9,7.7Hz,7H),7.22(s,1H),7.16-7.04(m,6H),6.78(d,J＝16.2Hz,2H),5.72(m,1H),5.35-5.23(m,1H),5.17-4.89(m,7H),4.86(d,J＝9.6Hz,1H),4.56-4.41(m,2H),4.36(s,1H),4.25-4.09(m,4H),4.05-3.90(m,4H),3.90-3.77(m,2H),3.71(m,2H),3.58(d,J＝6.9Hz,1H),3.49(s,1H),3.46-3.35(m,1H),2.96(m,1H),2.81-2.66(m,1H),2.29-2.13(m,4H),2.13-1.98(m,4H),1.94(m,3H),1.87-1.75(m,6H),1.75-1.53(m,13H),1.53-1.34(m,14H),1.18(m,11H),1.01(m,2H),0.92(s,6H),0.79(m,14H).ESI-MS:2004.5[M+Na] + ；C 96 H 123 F 4 N 13 O 28 :HRMS calcd.2004.8429[M+Na] + ,found 2004.8456.
DTX-PABC-Leu-B8(12a)
The synthesis method is the same as 5a, white solid, and the yield is 30%.
1 H NMR(400MHz,DMSO-d 6 )δ10.20(d,J＝3.9Hz,0H),9.80(d,J＝10.5Hz,1H),8.43(d,J＝14.9Hz,-1H),8.35(d,J＝13.9Hz,-1H),8.06(dd,J＝22.9,10.9Hz,4H),7.95(d,J＝11.3Hz,3H),7.91(t,J＝8.6Hz,12H),7.81(t,J＝15.7Hz,13H),7.66(t,J＝7.4Hz,11H),7.59(m,14H),7.50(m,2H),7.35(m,16H),7.28(m,23H),7.08(m,2H),5.36-5.29(m,1H),5.08(s,3H),5.00(m,10H),4.96-4.87(m,8H),4.87-4.80(m,2H),4.38(d,J＝9.1Hz,2H),4.35-4.12(m,19H),4.07(m,2H),3.94(m,11H),3.89-3.78(m,2H),3.55(m,3H),3.43(m,8H),2.18(m,15H),2.01(m,16H),1.79(m,17H),1.65(m,21H),1.44(m,17H),1.27(m,35H),1.17(m,32H),0.91(m,22H),0.85(m,12H),0.80(m,11H).ESI-MS:m/z 1851.4[M+Na] + ；C 95 H 116 N 10 O 27 :HRMS calcd.1851.7904[M+Na] + ,found 1851.7979.
4FDT-PABC-Leu-B8(12b)
The synthesis method is the same as 5a, white solid, and the yield is 25%.
1 H NMR(400MHz,DMSO-d 6 )δ9.87(d,J＝7.3Hz,1H),8.48(d,J＝9.1Hz,3H),8.10(m, 5H),8.00(d,J＝10.3Hz,1H),7.83(m,18H),7.77(m,3H),7.68(m,11H),7.64-7.50(m,20H),7.36(m,20H),7.29(m,24H),7.14-7.08(m,2H),6.98(m,3H),5.34-5.30(m,2H),5.25(d,J＝5.5Hz,1H),5.13-5.01(m,15H),4.93(m,9H),4.85m,2H),4.27(m,7H),4.17(m,9H),4.10(m,4H),3.96(m,12H),3.83(m,1H),3.57(m,4H),3.51(m,24H),2.99(m,3H),2.92(m,9H),2.67-2.61(m,2H),2.41-2.38(m,5H),2.32-2.21(m,8H),2.18(m,15H),2.03(m,2H),1.94(m,8H),1.81(m,14H),1.70(m,4H),1.66(m,10H),1.60(m,20H),1.51-1.42(m,51H),1.41-1.36(m,4H),1.18(m,75H),0.92(m,24H),0.83(m,27H).ESI-MS:m/z 1923.7[M+Na] + ；C 95 H 112 F 4 N 10 O 27 :HRMS calcd.1923.7527[M+Na] + ,found 1923.7520.
DTX-PABC-Leu-B9(13a)
The synthesis method is the same as 5a, white solid, and the yield is 24%.
1 H NMR(400MHz,DMSO-d 6 )δ9.96(d,J＝9.5Hz,1H),8.33(d,J＝7.8Hz,0H),8.28-8.24(m,0H),8.21-8.01(m,10H),7.92(m,14H),7.84(m,6H),7.77(d,J＝8.2Hz,1H),7.67(m,8H),7.60(m,11H),7.43(d,J＝6.2Hz,2H),7.35(m,13H),7.28(m,21H),7.21(s,1H),7.16-7.09(m,5H),6.76(m,3H),5.70(d,J＝8.4Hz,2H),5.37-5.32(m,1H),5.09(m,3H),5.02(m,8H),4.93(m,3H),4.85(t,J＝6.9Hz,1H),4.44-4.31(m,2H),4.29-4.09(m,18H),3.93(m,5H),3.72(m,8H),2.19(m,9H),2.09-2.02(m,9H),1.83(m,10H),1.67(m,20H),1.58(m,8H),1.46(m,13H),1.28(m,27H),1.17(m,16H),0.99(d,J＝6.2Hz,1H),0.92(m,17H),0.84(m,18H),0.78-0.69(m,17H).ESI-MS:m/z 2022.9[M+Na] + ；C 102 H 129 N 13 O 29 :HRMS calcd.2022.8911[M+Na] + ,found 2022.8878.
4FDT-PABC-Leu-B9(13b)
The synthesis was performed as in 5a, white solid, 29% yield.
1 H NMR(400MHz,DMSO-d 6 )δ9.97(s,1H),8.49(d,J＝9.2Hz,1H),8.34(d,J＝7.3Hz,1H),8.25-8.03(m,5H),7.93(dd,J＝11.4,4.9Hz,2H),7.85(d,J＝7.5Hz,2H),7.78(d,J＝7.1Hz,2H),7.73-7.52(m,7H),7.48-7.24(m,12H),7.21(s,1H),6.82-6.73(m,2H),5.37-5.22(m,1H),5.18-4.98(m,5H),4.98-4.92(m,2H),4.87(d,J＝9.8Hz,1H),4.49(d,J＝14.0Hz,1H),4.20(m,8H),3.97(m,3H),3.89-3.54(m,7H),3.49(s,1H),3.47-3.37(m,2H),2.19(m,4H),2.08(m,4H),1.90(t,J＝70.8,25.4Hz,7H),1.69(d,J＝14.3Hz,11H),1.52-1.26(m,13H),1.17(d,J＝6.9Hz,8H),0.93(m,7H),0.84(m,7H),0.80-0.64(m,7H).ESI-MS:m/z 2094.3[M+Na] + ；C 102 H 125 F 4 N 13 O 29 :HRMS calcd.2094.6817[M+Na] + ,found 2094.6863
DTX-PABC-Leu-B10(14a)
The synthesis method is the same as 5a, white solid, and the yield is 17%.
1 H NMR(400MHz,DMSO-d 6 )δ9.96(s,1H),9.15(s,1H),8.37-8.17(m,2H),8.10(d,J＝6.3Hz,2H),8.06-7.77(m,8H),7.69-7.51(m,5H),7.32(m,8H),7.12(t,J＝7.0Hz,1H),6.98(d,J＝8.1Hz,2H),6.79(m,2H),6.58(d,J＝8.0Hz,2H),5.73(t,J＝8.6Hz,1H),5.35(d,J＝6.8Hz,1H),5.13-4.91(m,7H),4.86(d,J＝9.8Hz,1H),4.41(d,J＝9.0Hz,3H),4.18(m,4H),3.94(m,3H),3.86-3.74(m,1H),3.68(m,3H),3.61-3.36(m,4H),2.86(d,J＝17.1Hz,1H),2.68(s,1H), 2.57(t,J＝11.5Hz,1H),2.20(m,4H),2.11-1.98(m,4H),1.92-1.75(m,9H),1.65(m,10H),1.46(m,5H),1.31(m,9H),1.18(m,7H),0.97(m,7H),0.84(m,7H).ESI-MS:m/z 1892.8[M+Na] + ；C 92 H 119 N 13 O 29 :HRMS calcd.1892.8129[M+Na] + ,found 1892.8114.
4FDT-PABC-Leu-B10(14b)
The synthesis method is the same as 5a, white solid, and the yield is 12%.
1 H NMR(400MHz,DMSO-d 6 )δ10.03-9.94(m,1H),9.14(s,2H),8.47(t,J＝7.8Hz,1H),8.33-8.19(m,1H),8.09(d,J＝7.2Hz,3H),7.98(br,8H),7.84(s,1H),7.76(t,J＝6.5Hz,1H),7.60(m,9H),7.35(m,4H),7.29(m,10H),7.20(d,J＝17.7Hz,2H),7.11(m,1H),6.97(m,3H),6.81(d,J＝8.2Hz,1H),6.73(d,J＝12.3Hz,1H),6.58(m,4H),5.70(d,J＝16.0Hz,1H),5.32(t,J＝7.9Hz,1H),5.10(m,2H),5.05(m,6H),4.97(m,3H),4.85(t,J＝8.8Hz,1H),4.50(s,1H),4.35(m,3H),4.18(m,4H),3.95(m,5H),3.67(m,4H),3.58(m,2H),3.47(m,1H),2.84(s,2H),2.68(s,1H),2.19(m,6H),2.14(s,1H),2.05(m,8H),1.95(t,J＝10.0Hz,1H),1.83(m,5H),1.80(m,10H),1.68(m,13H),1.62-1.52(m,6H),1.50-1.41(m,21H),1.40(m,2H),1.17(m,8H),0.93(m,12H),0.83(m,13H).ESI-MS:m/z 1964.3[M+Na] + ；C 92 H 115 F 4 N 13 O 29 :HRMS calcd.1964.7752[M+Na] + ,found 1964.7779.
DTX-PABC-Leu-B11(15a)
The synthesis method is the same as 5a, white solid, and the yield is 31%.
1 H NMR(400MHz,DMSO-d 6 )δ9.97(s,1H),8.31-7.99(m,8H),7.94(dd,J＝16.6,8.4Hz,4H),7.68(t,J＝8.1Hz,2H),7.60(m,4H),7.48-7.32(m,3H),7.29(m,5H),7.20(s,1H),7.12(t,J ＝7.3Hz,1H),6.96(d,J＝19.0Hz,1H),6.74(t,J＝19.9Hz,2H),5.73(t,J＝8.8Hz,1H),5.30(dd,J＝35.8,9.0Hz,1H),5.10(m,2H),5.08-4.91(m,5H),4.86(d,J＝10.2Hz,1H),4.52-4.43(m,1H),4.39(m,2H),4.27-4.04(m,4H),3.98(m,3H),3.78-3.51(m,7H),3.12(d,J＝5.2Hz,1H),2.84(s,1H),2.68(m,1H),2.57-2.47(m,1H),2.43-2.35(m,1H),2.20(m,4H),2.14-1.98(m,5H),1.98-1.83(m,3H),1.81(m,4H),1.78-1.52(m,11H),1.46(m,5H),1.31(m,10H),1.18(m,7H),1.07-0.90(m,7H),0.85(m,6H),0.75(m,6H).ESI-MS:m/z 1859.8[M+Na] + ；C 88 H 120 N 14 O 29 :HRMS calcd.1859.8238[M+Na] + ,found 1859.8199.
4FDT-PABC-Leu-B11(15b)
The synthesis was performed as in 5a, white solid, yield 27%.
1 H NMR(400MHz,dmso)δ10.17(s,1H),8.71-8.36(m,5H),8.36-8.08(m,5H),7.93-7.70(m,3H),7.70-7.46(m,5H),7.46-7.22(m,7H),7.20-7.05(m,0H),6.92(d,J＝15.2Hz,1H),6.72(t,J＝16.0Hz,2H),5.70(t,J＝8.5Hz,1H),5.36-5.22(m,1H),5.18-4.99(m,5H),4.96-4.79(m,2H),4.45(m,2H),4.32(s,1H),4.11(m,5H),4.01-3.86(m,3H),3.73-3.53(m,7H),3.07(d,J＝17.3Hz,1H),2.29-2.09(m,4H),1.98(m,4H),1.86(m,2H),1.76(m,3H),1.74-1.51(m,13H),1.51-1.40(m,8H),1.31(m,3H),1.16(m,6H),1.05-0.87(m,7H),0.77(m,12H).ESI-MS:m/z 1931.7[M+Na] + ；C 88 H 116 F 4 N 14 O 29 :HRMS calcd.1931.7861[M+Na] + ,found 1931.7872.。
Example 2: (tetrafluoro) docetaxel anti-hepatoma target prodrug has proliferation inhibition activity on human hepatoma cell lines HepG2 and SMMC-7721 in vitro and toxicity on normal liver cell lines HL7702 and normal kidney cell lines HEK293
The experimental method comprises the following steps: in vitro cell assays were performed using the MTT method. Sorafenib and 10-hydroxycamptothecin are used as positive control, docetaxel and tetrafluorodocetaxel are used as mother drug control, the inhibition of the drugs on cell growth under different concentrations is observed, and the half inhibition rate (IC) of the drugs is calculated 50 Value) to evaluate its anti-hepatoma activity in vitro and toxicity to normal cells.
HepG2 cells and HEK293 cells were cultured in DMEM medium containing 10% fetal bovine serum, and SMMC-7721 and HL-7702 cells were cultured in 1640 medium containing 10% fetal bovine serum. When the cells are in the division phase, pancreatin is added to digest and collect the cells, and the cell density is adjusted to 5X 10 4 one/mL, 100 μ L of cell suspension was seeded into 96 wells, 5000 cells per well. Place 96-well plate in CO 2 Culturing in a cell culture box under the following culture conditions: constant temperature of 37 ℃ and 5% CO 2 And the humidity is more than 95 percent. After 24h, observing and confirming the cell adherence condition under a microscope, discarding the culture medium, adding 200 mu L of culture medium containing the tested drugs with different concentrations, and setting the concentrations as follows: for liver tumor cell strains HepG2 and SMMC-7721, the concentration of the tested drug is set to be 5 x 10 -6 、1×10 -6 、2×10 -7 、4×10 -8 、8×10 -9 、1.6×10 -9 、3.2×10 -10 mol/L. Setting the concentration of the tested medicine to be 3.2 multiplied by 10 for the normal liver cell HL-7702 and the normal kidney cell -4 、1.6×10 -4 、8×10 -5 、4×10 -5 、2×10 -5 、1×10 -5 、5×10 -6 mol/L. Blank medium without cells served as a blank control, and medium with cells and 0.2% DMSO served as a cell control. Duplicate wells were set for each group as a parallel control. Place 96-well plate in CO 2 Culturing in a cell culture box. After 72h, 20. mu.L (5mg/mL) of freshly prepared MTT solution was added to each well and incubated at 37 ℃ for 4 h. The wells were discarded, 150. mu.L of DMSO was added to each well, shaken for 5min, and the OD was measured at 490nm detection wavelength. The inhibition rate of each concentration of the drug on cells is calculated according to the following formula:
the inhibition rate is [1- (drug group OD value-blank control group 0D value)/(cell control group OD value-blank control group 0D value) ] × 100%
IC50 values were calculated using SPSS software. The above experiment was repeated three times to calculate IC 50 Values and standard deviations.
The experimental results show that all prodrug compounds have certain differential selectivity on tumor cells and normal tissue cells. The toxicity to normal cells is obviously reduced while most of the anti-liver cancer activity is kept, and the feasibility of a prodrug design strategy is proved: the active parent drug is modified, so that the active parent drug has a killing effect on liver cancer cells and has low toxicity on normal cells, and the aim of designing a prodrug is achieved.
Table 1 shows the in vitro cell assay data for the anti-liver cancer targeted prodrug.
TABLE 1
Claims (7)
1. The docetaxel targeted anti-liver cancer prodrug is characterized by having a structure shown in a formula (1):
wherein R is 1 Is a peptide substrate 8 with the sequence Gly-Pro-Gln-Gly-Met-Ala-Gly-Gln, Gly-Pro-Gln-Gly-Ile-Ala-Ser-Gln, Gly-Pro-Gln-Gly-Ser-Ala-Gly-Gln, Gly-Pro-Gln-Gln-Ile-Ala-Gly-Gln, Gly-Pro-Gln-Gly-Ile-Ala-Gly-Gl, Gly-Asn-Gln-Gly-Ile-Ala-Gly-Gln, or Gly-Asn-Gln-Gly-Ile-Ala-Gly-Gln, respectively;
R 2 is methyl or trifluoromethyl;
R 3 is a hydrogen atom or a fluorine atom.
2. The docetaxel targeted anti-hepatoma prodrug of claim 1, wherein the target of the docetaxel targeted anti-hepatoma prodrug is specifically and highly expressed matrix metalloproteinase MMP-2 or MMP-9 in liver cancer tissues.
3. The docetaxel targeted anti-hepatoma prodrug of claim 2, wherein the MMP-2 specifically recognizes and is easy to hydrolyze the docetaxel targeted anti-hepatoma prodrug, and the structure of the prodrug is selected from the group consisting of:
4. the docetaxel targeted anti-hepatoma prodrug of claim 2, wherein the MMP-9 specifically recognizes and is easy to hydrolyze the docetaxel targeted anti-hepatoma prodrug, and the structure of the prodrug is selected from the group consisting of:
5. use of the docetaxel targeted anti-liver cancer prodrug as claimed in any one of claims 1, 3 and 4 in preparation of a medicament for treating liver cancer.
6. The use of claim 5, characterized by significant safety against at least one of normal liver and kidney cells while having an inhibitory effect on the growth of liver cancer cells.
7. The use of claims 5 and 6, wherein the docetaxel targeted anti-hepatoma prodrug for treating hepatoma comprises improving the anti-hepatoma activity of the parent drug and significantly reducing the systemic toxicity in vivo.
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