CN109420179B - Docetaxel targeted prodrug and anti-colon cancer medicinal application thereof - Google Patents

Abstract

The invention belongs to the field of biological medicine, in particular to a novel docetaxel targeted prodrug and medicinal application thereof, and provides a novel docetaxel targeted prodrug represented by a general formula (1) and medicinal application thereof for resisting colon cancer: the compound of the invention has pharmacological research value, and can be used as an anticancer prodrug of protease which targets the specific high expression of the tumor site of the colon cancer. Can be used for treating one or more colon cancer diseases with high specificity expression, such as matrix metalloproteinase MMP-7, cysteine cathepsin B, fibroblast activation protein alpha, and the like.

In the formula (1), A is a substrate polypeptide sequence which is specifically recognized and hydrolyzed and comprises, but is not limited to, matrix metalloproteinase MMP-7, cysteine cathepsin B, fibroblast activator protein alpha and the like which are obviously and highly expressed in colon cancer tissues; b is a degradable bridging group selected from (a-i), and is connected with the carbon end or the nitrogen end of A through amido bond; r ₁ Including but not limited to fluorine atoms, hydrogen atoms, and the like.

Description

Docetaxel targeted prodrug and anti-colon cancer medicinal application thereof

Technical Field

The invention belongs to the field of biological medicines, and particularly relates to a novel docetaxel targeted prodrug and medicinal application thereof in resisting colon cancer.

Background

The prior art discloses that colon cancer is one of common malignant tumors, the morbidity and the mortality of colon cancer are in the third place of the malignant tumors in developed countries in Europe and America, the morbidity and the mortality of colon cancer in China are increased from the fifth place and the sixth place in 80 years to the fourth place and the fifth place, and about 20 ten thousand people die of colon cancer every year. Clinical surgical resection is the most basic treatment, and adjuvant chemotherapy is the conventional therapy after surgery for patients with colon cancer stage II and III. Currently, colon cancer adjuvant chemotherapy drugs fall into two categories: one is a chemical small molecular drug, mainly comprises 5-fluorouracil, capecitabine, tegafur, irinotecan, oxaliplatin and the like, can be used independently or jointly, but inevitably generates toxic and side effects such as nausea, stomatitis, vomiting and the like; another class is biological drugs with molecular targeting, such as epidermal growth factor receptor inhibitor (EGFR) cetuximab and vascular endothelial growth factor receptor inhibitor (VEGF) bevacizumab, but because of the increasing resistance of EGFR and VEGF, this class of drugs is poorly effective, does not show long-term benefit for progression-free and overall survival of the disease in adjuvant therapy modalities, and is expensive to use. Therefore, the research and development of anti-colon cancer small molecule drugs with more effectiveness, less toxic and side effects and low price is a major problem to be solved urgently.

Among the anticancer drugs discovered so far, paclitaxel isolated from natural yew is the first-line drug for clinical treatment of breast cancer, ovarian cancer and non-small cell lung cancer, and its Me-beta second-generation paclitaxel drug, docetaxel, is considered to be the most effective antitumor drug discovered so far by human. Aiming at the existing many clinical defects, such as multidrug resistance, low oral bioavailability, anaphylactic reaction, poor water solubility, poor metabolic stability, large systemic toxicity side effect caused by drug heterogeneity and the like, the development of the third-generation high-activity paclitaxel antitumor drug is greatly progressed, for example, in 2010, Sanofi-Aventis methylates the hydroxyl groups at the C-7 position and the C-10 position of docetaxel to obtain a new compound, namely Cabazitaxel (trade name, Jtanaa), and the compound is approved by FDA on the market; secondly, aiming at the defect of low oral bioavailability, Oraxol (Orataxel) which is mainly developed by Spectrum company is currently in phase II clinical experiments, and the compound has the same excellent cytotoxic activity on drug sensitive cells and multidrug resistant cells except that the oral bioavailability is greatly improved; thirdly, aiming at the defect of anaphylactic reaction, paclitaxel liposome, namely the lipusu for injection, which is successfully developed by encapsulating paclitaxel by liposome in the green leaf cisco pharmaceutical industry, is approved by SFDA in 2004 to be marketed; aiming at the defect of poor water solubility, the first non-solvent nano albumin combined chemotherapeutic drug Abraxane successfully developed by Abraxis company is approved by FDA to be on the market in 2005; cell Therapeutics company connects C-2' OH of paclitaxel with poly-L-glutamic acid to obtain prodrug Xyotax/Opaxio, which has higher water solubility and tumor targeting than paclitaxel, and currently carries out phase III clinical test. However, the existing medicines on the market are not reported in terms of poor metabolic stability and large systemic toxicity, especially in terms of development of new indications of the colon cancer resistance.

Studies have shown that there are many specific proteases present on the surface of tumor cells or at the margins of tumor sites, which play a crucial role in tumor growth, infiltration and metastasis. In particular, Matrix Metalloproteinases (MMPs), cysteine Cathepsin B (Catherin B) and Fibroblast activation protein alpha (FAP alpha) similar to MMPs and the like are specifically distributed at the cell site of colon cancer, are easy to over-express, and on the contrary, are low-expressed in normal tissues or inflammatory tissues, so that the Matrix Metalloproteinases (MMPs) are very important colon cancer tumor markers and treatment targets.

The design strategy of the prodrug is one of the most important means for reducing the toxicity of the medicine, a substrate polypeptide which is identified and hydrolyzed by a protease (such as mechanism metalloprotease MMP-7, cysteine cathepsin B, fibroblast activation protein alpha and the like) which is specifically and highly expressed in colon cancer tissues is taken as a targeting carrier group, a polyene taxane cytotoxic small molecular compound with better anticancer activity is taken as a parent drug, and the polyene taxane cytotoxic small molecular compound and the fibroblast activation protein alpha are connected through a bridge chain molecule with a 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 polyene taxane cytotoxic small molecular compounds, and expand the application of the compounds in the treatment of colon cancer tumors.

Disclosure of Invention

The invention aims to provide a novel docetaxel targeted prodrug and medicinal application thereof in resisting colon cancer.

The invention provides a novel docetaxel targeted prodrug represented by the following general formula (1), which is covalently coupled with substrate polypeptide which is specifically and highly expressed and recognized by protease and hydrolyzed in colon cancer tissues in taxane compound molecules through bridge chain molecules, and can obviously improve the targeting property of a medicament to colon cancer.

In the formula (1), A is a specifically recognized and hydrolyzed substrate polypeptide sequence including, but not limited to, MMP-7, cysteine cathepsin B, fibroblast activation protein alpha, and the like, which are significantly highly expressed in colon cancer tissues, such as Fmoc-Gln-Gly-Ala-Ile-Gly-Leu-Pro-Gly, Ac-Gln-Gly-Ala-Ile-Gly-Met-Pro-Gly, Ac-Gln-Gly-Ala-Ile-Ala-Gln-Pro-Gly, Ac-Gln-Met-Ile-Gly-Gln-Pro-Gly, Ac-Gln-Gly-Ala-Leu-Gly-Gln-Pro-Gly, Gly-Leu-Phe-Gly, and A, Gly-Phe-Leu-Gly, Ala-Leu-Ala-Leu, Mca-Ala-Ser-Gly-Pro-Ala-Gly-Ala-Pro-Dnp, Mca-Glu-Pro-Gly-Pro-Ala-Dnp, Mca-Asp-Arg-Gly-Glu-Thr-Gly-Pro-Ala-Dnp, Mca-Val-Gly-Pro-Ala-Gly-Lys-Dnp, Mca-Asp-Lys-Gly-Glu-Ser-Gly-Pro-Ala, Mca-Ala-Pro-Gly-Ser-Lys-Gly-Asp-Ala, and the like;

b is a degradable bridging group including, but not limited to (a-i)

The structure is the same, and the structure is connected with the carbon end or the nitrogen end of the polypeptide sequence A through amido bond;

R ₁ including but not limited to fluorine atoms, hydrogen atoms, and the like.

The preparation method of the novel docetaxel targeted prodrug comprises the following steps: firstly, 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 invention provides the in vitro proliferation inhibition activity of the docetaxel targeted prodrug on human colon cancer cell strains HCT116 and SW620 and the toxicity on tissue normal colon cell strains CCD18Co and kidney cell strains HEK 293.

The compound has pharmacological research value, and can be used as an anticancer prodrug of protease which targets colon cancer tumor sites and is specifically and highly expressed. Can be used for treating colon cancer diseases with high specificity expression, such as matrix metalloproteinase MMP-7, cysteine cathepsin B, fibroblast activation protein alpha, etc.

The novel docetaxel targeted prodrug can be prepared into various preparations containing safe and effective dose of the novel docetaxel targeted prodrug and medicinal carriers, and is used for treating colon cancer diseases.

The safe and effective amount of the invention refers to: the amount of the compound is sufficient to significantly improve the condition without causing serious side effects. The safe and effective amount is determined according to the age, condition, course of treatment, etc. of the subject.

Examples of pharmaceutically acceptable carrier moieties described herein are sugars (e.g., glucose, sucrose, lactose, etc.), starches (e.g., corn starch, potato starch, etc.), celluloses and derivatives thereof (e.g., sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (e.g., stearic acid, magnesium stearate), calcium sulfate, vegetable oils (e.g., soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (e.g., propylene glycol, glycerol, lactose, etc.), sugars (e.g., sucrose, lactose, etc.), sugars (e.g., corn starch, potato starch, etc.), celluloses and derivatives thereof (e.g., sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (e.g., stearic acid, magnesium stearate), calcium sulfate, vegetable oils (e.g., soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (e.g., propylene glycol, glycerol, propylene glycol, and the like,Mannitol, sorbitol, etc.), emulsifiers (e.g., sorbitol, etc.)

) Wetting agents (e.g., sodium lauryl sulfate), coloring agents, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, and the like.

Compared with the modern technology, the invention has the following beneficial effects:

1) compared with a positive control drug 5-fluorouracil (5-FU) for clinically treating colon cancer, the taxane targeted prodrug has better colon cancer resisting activity, for example, the inhibition activity on colon cancer cells HCT116 can reach 2.7nmol, shows better colon cancer resisting potential and has good patent drug prospect;

2) compared with docetaxel compounds, the targeted polypeptide in the prodrug can specifically convey taxane micromolecules to the colon cancer tumor tissue part, the killing power to normal cells is reduced, and degradation products of the targeted polypeptide in the cells are amino acids, so that the prodrug has lower tissue system toxicity and higher biocompatibility.

Drawings

FIG. 1 is a scheme showing the synthesis scheme of a prodrug of docetaxel or tetrafluorodocetaxel coupled to a colon cancer targeting polypeptide A1-A5 via a bridging group Leu-PABC (a) in example 1.

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 docetaxel or docetaxel coupled prodrug with colon cancer targeting polypeptide A1-A5 through bridging group Leu-PABOH (a)

The taxane targeted prodrug is prepared by coupling paclitaxel (DTX) or tetrafluorodocetaxel (4FDT) with colon cancer targeted polypeptide A1-A5, and the synthetic route is shown in figure 1, and comprises the following steps:

1) synthesis and activation modification of bridging group Leu-PABOH

Reference is made to the literature (Elsadek B, Graeser R, Esser N, et al. development of a novel prodrug of paclitaxel which is distinct by state-specific antigen: An in vitro and in vivo evaluation determination [ J]The synthesis and activated modification of the bridging group Leu-PABOH was accomplished in a two-step reaction using the method employed in European Journal of Cancer,2010,46(18): 3434-. Namely: in a 25mL single neck flask, 1mmol of Fmoc-L-Leu (1), 2mmol of p-aminobenzyl alcohol (PABOH) and 2mmol of the condensing agent 2-ethoxy-1-ethoxycarbonyl-1, 2-dihydroquinoline (EEDQ) were added, 14mL of methanol and 7mL of dichloromethane were added as reaction solvents, the reaction was carried out at room temperature in the dark for about 48h, TLC (DCM: MeOH ═ 55:1) was monitored, after the reaction was completed, the organic solvent was removed by concentration under reduced pressure, and the reaction solution was purified by dichloromethane: purifying a methanol (100: 1-70: 1) system to obtain a light yellow intermediate Fmoc-Leu-PABOH (2), wherein the yield is 73%; taking a two-necked flask, adding 1mmol of intermediate Fmoc-Leu-PABOH (2), 2mmol of di (p-nitrophenyl) carbonate and N ₂ Protecting, adding 10mL of anhydrous N, N-Dimethylformamide (DMF) to dissolve a sample, slowly adding 1.5mmol of Diisopropylethylamine (DIPEA) under the condition of zero centigrade, moving to room temperature after 5min, monitoring the reaction by TLC (PE: ACE ═ 3:1), and reacting completely for about 8-12 h; after the reaction is finished, adding a large amount of saturated salt (NaCl) aqueous solution to terminate the reaction, carrying out layered extraction on Ethyl Acetate (EA) for three times, combining organic phases, separating out a relatively pure white solid when concentrating under reduced pressure, filtering, combining the residual organic phases, purifying by a silica gel column (PE: ACE ═ 10:1-7:1) and recrystallizing to obtain a white solid Fmoc-Leu-PABC-PNP (3), wherein the total yield is 82%. ¹ H NMR(400MHz,CDCl ₃ ):δ8.27(d,J＝8.0Hz,2H),7.76(d,J＝8.0Hz,2H),7.61-7.49(m,4H),7.49-7.44(m,1H),7.41-7.35(m,6H),7.30-7.28(m,1H),5.24(s,2H),4.51-4.45(m,2H),4.29(s,1H),4.21(dd,J＝8.0Hz,12.0Hz,1H),1.80-1.68(m,2H),1.04(d,J＝4.0Hz,1H),0.98-0.95(m,6H)；ESI-MS:m/z 623.6[M+H]+,646.6[M+Na]+；C ₃₅ H ₃₃ N ₃ O ₈ :HRMS calcd.646.2160[M+Na]+,found 646.2162.

2) Synthesis of intermediate 4 at C-2' position of docetaxel

Adding 1mmol of Fmoc-Val-Cit-PABC-PNP (3) and 1mmol of Docetaxel (DTX) into a two-neck bottle, protecting by N2, adding 80mL of anhydrous dichloromethane solution, adding 1mmol of condensing agent 4-Dimethylaminopyridine (DMAP) at zero centigrade, moving to room temperature after 5min, reacting for about 48h, monitoring the reaction by TLC (DCM: MeOH ═ 25:1), concentrating under reduced pressure after the reaction is finished, removing the organic solvent dichloromethane, and separating and purifying by silica gel column (DCM: MeOH ═ 100:1) to obtain an off-white solid 4 with the yield of about 50%. ¹ H NMR(400MHz,Acetone-d ₆ ):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),5.19(d,J＝11.80Hz,1H),5.11(d,J＝11.80Hz,1H),4.97(d,J＝8.8Hz,1H),4.39-4.30(m,5H),4.27-4.21(m,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] ⁺ ；C ₇₂ H ₈₁ N ₃ O ₁₉ :HRMS calcd.1314.5356[M+Na] ⁺ ,found1314.5366.

3) Synthesis of intermediate 5 at C-2' position of tetrafluoropolyene taxol

The synthesis was as for intermediate 4, off white solid, 54% yield. Off white solid, 54% yield. ¹ H NMR(400MHz,Acetone-d ₆ ):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),7.75-7.67(m,5H),7.67-7.61(m,1H),7.56-7.47(m,3H),7.47-7.35(m,7H),7.34-7.25(m,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(brs,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(dd,J ₁ ＝6.8Hz,8.0Hz,1H),4.20-4.12(m,2H),3.92(d,J＝7.2Hz,1H),3.79(brs,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),1.62-1.50(m,7H),1.44-1.34(m,1H),1.34-1.24(m,5H),1.17(brs,4H),1.00-0.84(m,7H).ESI-MS:m/z 1364.2[M+H] ⁺ ,1386.2[M+Na] ⁺ ；C ₇₂ H ₇₇ F4N ₃ O ₁₉ :HRMS calcd.1386.4980[M+Na] ⁺ ,found 1386.5001.

4) Synthesis of derivative 6 (or 7)

Taking a 25mL single-neck bottle, adding 15mg of intermediate 4 (or 5) and 0.9mL of N, N-Dimethylformamide (DMF), fully dissolving, adding 3.6 mu L of piperidine at zero centigrade, reacting for 5min, moving to room temperature, and monitoring by TLC (DCM: MeOH 15:1), wherein about 36min can completely react; after the reaction was completed, the solvent N, N-Dimethylformamide (DMF) was removed as much as possible at room temperature by a vacuum oil pump, the residual DMF was removed by saturated aqueous NaCl solution, dichloromethane (100mL) was extracted three times, and the organic phase was concentrated under reduced pressure rapidly to give a pale yellow solid 6 (or 7) which was used directly in the next reaction.

5) Synthesis of targeted polypeptide prodrugs

DTX-PABC-Leu-A1(8a)

Taking a 25mL single-neck bottle, adding 0.01mmol of intermediate 4, 0.012mmol of targeting carrier polypeptide A1(Gly-Pro-Leu-Gly-Ile-Ala-Gly-Gln-Fmoc), 0.03mmol of 1-Hydroxybenzotriazole (HOBT) and 1.5mL of N, N-Dimethylformamide (DMF), fully dissolving at 0 deg.C, adding 0.04mmol of N-methylmorphineReacting for 15min, adding N, N' -Diisopropylcarbodiimide (DIC), heating to 5 ℃, continuing to react, and monitoring the reaction by HPLC (method: water/acetonitrile is mobile phase, acetonitrile is 5% -100%, and 30min), wherein the reaction can be completed in about 72 h; after the reaction was completed, the solvent N, N-Dimethylformamide (DMF) was removed as much as possible by a vacuum oil pump at room temperature, a small amount of dimethyl sulfoxide (DMSO) was added to dissolve it, and the mixture was purified by a reverse phase column (water/acetonitrile was a mobile phase: the DMSO solution in the sample was removed by sequentially eluting with 100% water for 15 min; acetonitrile 0-85%, 40min) to obtain a white solid 8a in 17% yield. ¹ H NMR(400MHz,DMSO-d ₆ ):δ9.95(s,1H),8.16-8.05(m,4H),7.99(d,J＝8Hz,1H),7.94(d,J＝6.4Hz,3H),7.90(d,J＝8Hz,2H),7.85(d,J＝7.6Hz,2H),7.68(d,J＝7.6Hz,3H),7.61(d,J＝7.6Hz,3H),7.38(t,J＝7.6Hz,6H),7.29(dd,J＝14.4,7.2Hz,7H),7.13(t,J＝7.2Hz,1H),6.78(s,1H),5.74(t,J＝8.4Hz,1H),5.36(d,J＝7.2Hz,1H),5.11(s,2H),5.04(d,J＝7.2Hz,3H),4.99(d,J＝7.2Hz,2H),4.93(s,1H),4.86(d,J＝10.4Hz,1H),4.45-4.34(m,3H),4.28(d,J＝8.0Hz,1H),4.19(dd,J＝14.8,7.2Hz,9H),4.03-3.92(m,4H),3.88-3.77(m,1H),3.76-3.71(m,2H),3.69(s,3H),3.60(d,J＝6.8Hz,2H),3.44-3.37(m,1H),2.20(s,4H),2.10-2.04(m,2H),1.87-1.79(m,3H),1.68(s,3H),1.62-1.56(m,4H),1.47(s,6H),1.30(s,9H),1.18(d,J＝8Hz,3H),0.94(s,6H),0.87(d,J＝6Hz,3H),0.84-0.81(m,6H),0.80-0.71(m,12H).ESI-MS:m/z 1015.4[M/2+Na] ⁺ ；C ₁₀₃ H ₁₃₂ N ₁₂ O ₂₈ :HRMS calcd.2007.9166[M+Na] ⁺ ,found 2007.9246.

4FDT-PABC-Leu-A1(8b)

The synthesis was performed as in 8a, white solid, 22% yield. ¹ H NMR(400MHz,DMSO-d ₆ ):δ10.00(s,1H),8.51(d,J＝8.0Hz,1H),8.28–8.07(m,4H),8.05–7.93(m,3H),7.90(d,J＝8.0Hz,2H),7.82(d,J＝8.0Hz,2H),7.72(d,J＝8.0Hz,3H),7.65(d,J＝8.0Hz,3H),7.49-7.37(m,7H),7.33(d,J＝4.0Hz,5H),7.19(t,J＝8.0,4.0Hz,1H),6.82(s,1H),5.78(t,16,8.0Hz,1H),5.38(d,J＝4.0Hz,1H),5.22-5.08(m,4H),5.06-4.96(m,3H),4.91(d,J＝8.0Hz,1H),4.54(s,1H),4.41(s,1H),4.34-4.17(m,7H),4.12(d,J＝4.0Hz,6H),4.01(s,3H),3.90-3.49(m,5H),3.17(d,J＝4.6Hz,14H),2.89(s,3H),2.73(s,3H),2.24(s,4H),1.72(s,5H),1.57-1.49(m,10H),1.23(s,5H),0.98(s,5H),0.91(d,J＝8.0Hz,3H),0.87(s,5H),0.80(d,J＝8.0Hz,6H).ESI-MS:m/z 1029.4[M/2+H] ⁺ ；C ₁₀₃ H ₁₂₈ F ₄ N ₁₂ O ₂₈ :HRMS calcd.2079.8789[M+Na] ⁺ ,found 2079.8879.

DTX-PABC-Leu-A2(9a)

The synthesis was performed as in 8a, white solid, 21% yield. ¹ H NMR(400MHz,DMSO-d ₆ ):δ9.99(s,1H),8.19(d,J＝7.9Hz,1H),8.13-7.09(m,3H),8.05-7.92(m,6H),7.86-7.79(m,1H),7.75-7.69(m,1H),7.65(d,J＝7.2Hz,4H),7.41(t,J＝7.2Hz,2H),7.38-7.31(m,4H),7.29(s,1H),7.17(t,J＝7.2Hz 1H),6.82(s,1H),5.78(t,J＝9.6Hz,1H),5.40(d,J＝6.9Hz,1H),5.15(s,2H),5.08(d,J＝8.3Hz,2H),5.03(d,J＝7.3Hz,2H),4.96(s,1H),4.90(d,J＝9.2Hz,1H),4.5-4.37(m,3H),4.33-4.17(m,5H),4.12-4.96(m,4H),3.95-3.89(m,1H),3.87-3.78(m,2H),3.78-3.67(m,4H),3.64(d,J＝6.4Hz,1H),3.53(s,1H),2.24(s,5H),2.15-2.08(m,2H),2.03(s,4H),1.86(s,5H),1.84(s,2H),1.72(s,4H),1.51(s,6H),1.34(s,10H),1.23(d,J＝6.0Hz,6H),0.98(s,7H),0.89(dd,J＝16.4,6.0Hz,7H),0.80(dd,J＝15.0,7.2Hz,7H).ESI-MS:m/z 1843.8[M+Na] ⁺ ；C ₈₉ H ₁₂₂ N ₁₂ O ₂₇ S:HRMS calcd.1845.8155[M+Na] ⁺ ,found 1845.8219.

4FDT-PABC-Leu-A2(9b)

The synthesis was as for 8a, white solid, 23% yield. ¹ H NMR(400MHz,DMSO-d ₆ ):δ10.00(s,1H),8.52(d,J＝8.0Hz,1H),8.11(d,J＝8.0Hz,2H),8.06-7.93(m,3H),7.82(dd,J＝8.0,4.0Hz,2H),7.78-7.61(m,5H),7.47-7.39(m,3H),7.37-7.29(m,4H),7.18(t,J＝7.0Hz,1H),6.82(s,1H),5.78(t,J＝8.0Hz,1H),5.38(d,J＝8.0Hz,1H),5.35-5.29(m,1H),5.21-5.07(m,4H),5.05(d,J＝4.0Hz,1H),5.01(s,2H),4.92(d,J＝8.0Hz,1H),4.54(s,1H),4.51-3.66(m,13H),3.66-3.38(m,1H),3.29-2.96(m,4H),2.89(s,1H),2.73(s,1H),2.24(s,3H),2.15-2.08(m,2H),2.03(s,4H),1.85(d,J＝8.0Hz,5H),1.79-1.62(m,9H),1.55(s,3H),1.52(d,J＝8.0Hz,7H),1.23(s,9H),0.98(s,6H),0.91(d,J＝8.0Hz,3H),0.87(d,J＝8.0Hz,4H),0.83-0.75(m,7H).ESI-MS:m/z948.6[M/2+H] ⁺ ；C ₈₉ H ₁₁₈ F ₄ N ₁₂ O ₂₇ S:HRMS calcd.1917.7778[M+Na] ⁺ ,found 1917.7831.

DTX-PABC-Leu-A3(10a)

The synthesis was as for 8a, white solid, 18% yield. ¹ H NMR(400MHz,DMSO-d ₆ ):δ9.97(s,1H),8.22-8.10(m,4H),8.07-7.95(m,6H),7.79-7.61(m,7H),7.47-7.27(m,8H),6.81(s,2H),5.86-5.72(m,1H),5.45-5.36(m,1H),5.17-5.01(m,6H),4.93(d,J＝27.6Hz,2H),4.44(s,2H),4.26(s,4H),4.18-4.11(m,2H),4.07-3.98(m,3H),3.90(s,2H),3.74(s,3H),3.58-3.47(m,2H),2.29-2.20(m,4H),2.17-2.06(m,5H),1.91-1.79(m,8H),1.76-1.68(m,6H),1.67-1.61(m,3H),1.52(s,5H),1.33(s,9H),1.24(s,9H),1.01-0.95(m,6H),0.92-0.87(m,6H),0.83-0.77(m,6H).ESI-MS:m/z 940.2[M/2+Na] ⁺ ；C ₉₀ H ₁₂₃ N ₁₃ O ₂₈ :HRMS calcd.1856.8493[M+Na] ⁺ ,found 1856.8486.

4FDT-PABC-Leu-A3(10b)

The synthesis was performed as in 8a, white solid, 21% yield. ¹ H NMR(400MHz,DMSO-d ₆ ,rotamers):δ9.97(d,J＝12.0Hz,1H),8.52(d,J＝12.0Hz,1H),8.23-7.97(m,12.1Hz,7H),7.82(d,J＝8.0Hz,2H),7.76(d,J＝8.0Hz,1H),7.74-7.68(m,2H),7.65(d,J＝8.0Hz,2H),7.40(d,J＝8.0Hz,3H),7.37-7.25(m,5H),7.18(d,J＝4.0Hz,1H),6.81(d,J＝8.0Hz,2H),5.77(t,J＝16.0,8.0Hz,1H),5.38(d,J＝8.0Hz,1H),5.22-4.95(m,7H),4.91(d,J＝8.0Hz,1H),4.54(s,1H),4.40(s,1H),4.34-4.18(m,4H),4.12(s,7H),4.01(s,2H),3.90(s,1H),3.73(s,1H),3.66-3.46(m,1H),3.17(s,5H),2.88(s,1H),2.72(s,1H),2.23(s,5H),2.16-2.05(m,5H),1.91-1.82(m,6H),1.72(s,6H),1.56-1.48(m,10H),1.22(s,7H),0.98(s,5H),0.88(dd,J＝16.0,4.0Hz,6H),0.84-0.73(m,7H).ESI-MS:m/z954.0[M/2+H] ⁺ ；C ₉₀ H ₁₁₉ F ₄ N ₁₃ O ₂₈ :HRMS calcd.1928.8116[M+Na] ⁺ ,found 1928.8183.

DTX-PABC-A4(11a)

The synthesis was as for 8a, white solid, 24% yield. ¹ H NMR(400MHz,DMSO-d ₆ ,rotamers):δ10.03(s,1H),8.29-8.15(m,3H),8.12-7.91(m,8H),7.84-7.61(m,6H),7.47-7.16(m,8H),6.80(s,2H),5.90-5.68(m,1H),5.40(t,J＝4Hz,1H),5.21-4.84(m,8H),4.55-4.37(m,3H),4.35-4.14(m,6H),4.07-3.96(m,3H),3.89-3.54(m,8H),2.24(s,4H),2.18-2.07(m,5H),2.06-2.00(m,3H),1.95-1.82(m,9H),1.77-1.63(m,9H),1.51(s,6H),1.34(s,9H),1.27-1.16(m,6H),1.03-0.95(m,6H),0.93-0.85(m,6H),0.83-0.75(m,6H).ESI-MS:m/z 969.8[M/2+Na] ⁺ ；C ₉₂ H ₁₂₇ N ₁₃ O ₂₈ S:HRMS calcd.1916.8526[M+Na] ⁺ ,found 1916.8535.

4FDT-PABC-A4(11b)

The synthesis was as for 8a, white solid, 25% yield. ¹ H NMR(400MHz,DMSO-d ₆ ,rotamers)δ10.01(d,J＝20.8Hz,1H),8.47(d,J＝8.4Hz,1H),8.14(d,J＝6.4Hz,2H),8.08-7.98(m,3H),7.95-7.91(m,2H),7.78(q,J＝16.4,12.4Hz,2H),7.71-7.66(m,1H),7.66-7.57(m,4H),7.37(t,J＝7.6Hz,2H),7.32(d,J＝7.6Hz,2H),7.29(d,J＝8.6Hz,2H),7.24(s,2H),7.14(t,J＝8.0Hz,1H),6.76(s,2H),5.74(t,J＝8.0Hz,1H),5.34(d,J＝8.0Hz,1H),5.08(dd,J＝16,12Hz,4H),5.01(d,J＝8.0Hz,1H),4.97(d,J＝8.0Hz,2H),4.87(d,J＝8.0Hz,1H),4.43-4.34(m,1H),4.39(s,1H),4.18(ddd,J＝23.7,17.2,9.0Hz,6H),3.98(d,J＝9.0Hz,3H),3.86(dd,J＝16.0,8.0Hz,1H),3.80-3.64(m,2H),3.62-3.42(m,2H),3.34(s,1H),2.85(s,3H),2.69(s,3H),2.43(s,2H),2.20(s,3H),2.16-2.03(m,4H),1.98(s,3H),1.87(s,3H),1.83(d,J＝4.0Hz,4H),1.76-1.69(m,4H),1.68(s,3H),1.65-1.57(m,4H),1.51(s,3H),1.49(s,3H),1.47(d,J＝4.0Hz,3H),1.21-1.14(m,6H),0.94(s,6H),0.87(d,J＝6.2Hz,3H),0.83(d,J＝6.2Hz,3H),0.76(dd,J＝6.4,3.6Hz,6H).ESI-MS:m/z984.2[M/2+H] ⁺ ；C ₉₂ H ₁₂₃ F ₄ N ₁₃ O ₂₈ S:HRMS calcd.1988.8150[M+Na] ⁺ ,found 1988.8169.

DTX-PABC-A5(12a)

The synthesis was performed as in 8a, white solid, 24% yield. ¹ H NMR(400MHz,DMSO-d ₆ ):δ9.96(s,1H),8.14(d,J＝28.0Hz,5),8.03(s,1H),7.95(d,J＝21.6Hz,5H),7.68(dd,J＝24.4,6.4Hz,5H),7.41(d,J＝6.0Hz,2H),7.31(d,J＝26.4Hz,6H),7.17(s,1H),6.81(d,J＝14.8Hz,2H),5.78(t,J＝4Hz,1H),5.39(d,J＝6.0Hz,1H),5.15(s,3H),5.04(dd,J＝22.8,7.2Hz,3H),4.96(s,1H),4.90(d,J＝9.2Hz,1H),4.47-4.37(m,2H),4.35-4.12(m,5H),4.10-3.89(m,4H),3.88-3.77(m,1H),3.72(s,3H),3.67-3.60(m,1H),3.59-3.46(m,2H),3.28(s,1H),2.24(s,3H),2.15-2.02(m,5H),1.94-1.88(m,2H),1.86(s,3H),1.85-1.73(m,3H),1.68-1.53(m,6H),1.51(s,3H),1.47-1.42(m,2H),1.33(s,9H),1.25-1.19(m,4H),0.98(s,6H),0.92-0.82(m,13H).ESI-MS:m/z 1842.8[M+Na] ⁺ ；C ₈₉ H ₁₂₁ N ₁₃ O ₂₈ :HRMS calcd.1842.8336[M+Na] ⁺ ,found 1842.8347.

4FDT-PABC-A5(12b)

The synthesis was performed as in 8a, white solid, 22% yield. ¹ H NMR(400MHz,DMSO-d ₆ ):δ9.97(s,1H),8.52(d,J＝8Hz,1H),8.17(d,J＝8.0Hz,2H),8.11(dd,J＝6.0,4.0Hz,3H),8.04(t,J＝8Hz,4H),7.97(d,J＝8.0Hz,1H),7.91(dd,J＝8.0,4.0Hz,1H),7.82(d,J＝8.0Hz,1H),7.78–7.58(m,5H),7.43-7.28(m,9H),7.18(t,J＝8.0Hz,1H),6.81(d,J＝16Hz,2H),5.78(t,J＝8.0Hz,1H),5.38(d,J＝8.0Hz,1H),5.21-5.08(m,4H),5.07-4.96(m,3H),4.92(d,J＝8.0Hz,1H),4.54(s,1H),4.51-3.85(m,10H),3.84-3.68(m,4H),3.64(d,J＝8.0Hz,1H),3.60-3.35(m,2H),3.34(s,1H),2.36-2.19(m,4H),2.17-1.99(m,5H),1.98-1.82(m,7H),1.80-1.57(m,10H),1.55(s,3H),1.52(d,J＝4Hz,7H),1.48-1.31(m,4H),1.23(d,J＝4.0Hz,3H),0.98(s,5H),0.91(d,J＝4Hz,3H),0.89-0.85(m,6H),0.94-0.77(m,4H).ESI-MS:m/z 947.2[M/2+H] ⁺ ；C ₈₉ H ₁₁₇ F ₄ N ₁₃ O ₂₈ :HRMS calcd.1914.7959[M+Na] ⁺ ,found 1914.7963.。

Example 2: proliferation inhibition activity of targeted anti-colon cancer prodrug on human colon cancer cell strains HCT116 and SW620 in vitro and toxicity evaluation on tissue normal colon cell strains CCD18Co and kidney cell strain HEK293

The experimental method comprises the following steps: 5-fluorouracil, parent drugs docetaxel and tetrafluorodocetaxel are used as positive controls, and the in vitro antitumor activity of the prodrug compound on HCT116 and SW620 and the toxicity of the prodrug compound on normal colon cell strains CCD18Co and kidney cell strains HEK293 are determined by adopting an MTT method. The inhibition of the drug on the growth of tumor cells and normal cells under different concentrations is observed, and the half inhibition rate (IC50 value) is calculated to evaluate the antitumor activity and the toxicity on normal tissues in vitro.

Taking well-conditioned cells in the logarithmic growth phase, adding a proper amount of pancreatin (purchased from GIBCO) to digest the cells, then removing the pancreatin by aspiration, collecting and resuspending the cells by flushing with a serum-containing culture solution, counting, and adjusting the cell density. Taking cell suspension for inoculationSeeded on 96-well plates (100. mu.L, 4000-. After confirming that the cells were attached to the surface, the culture medium was aspirated, and the prodrug compound solution (concentration: 10) was dissolved in the culture medium ^-4 、10 ^-5 、10 ^-6 、10 ^-7 、10 ^-8 、10 ^-9 、10 ^-10 、10 ^-11 mol/L) were added to the cells (200. mu.L/well), respectively, as an experimental group. Media containing 0.2% DMSO was added to the wells (200 μ L/well) as a media control. Culture broth was added to wells that were not seeded with cells as a blank. Each concentration was set with 6 wells. After the cells were incubated at 37 ℃ under 5% CO2 and 95% air for 48 or 72h, MTT/PBS solution (20. mu.L, 5mg/mL) was added to each well and incubation was continued for 4 h. The liquid was carefully aspirated off and 150. mu.L of DMSO was added to dissolve completely. The sample was shaken at medium speed for 5 minutes using a microplate reader (Thermo Fisher) to measure the absorbance (OD) at 490 nm. The inhibition rate of the compound on cell proliferation was calculated according to the following formula:

the inhibition rate (1- [ (mean OD value of administration group-mean OD value of blank group)/(mean OD value of culture solution control group-mean OD value of blank group) ] was calculated by using SPSS software to calculate an IC50 value. The above experiment was repeated three times, and the average value and standard deviation of the experimental IC50 were calculated three times.

The experimental results are as follows: the proliferation inhibition activity of the targeted anti-colon cancer prodrug on human colon cancer cell strains HCT116 is stronger than that of a positive control drug 5-fluorouracil, and is equal to or slightly lower than that of respective corresponding parent drugs (docetaxel or tetrafluorodocetaxel); has lower toxicity to normal colon cell line CCD18Co and kidney cell line HEK293 and is lower than the corresponding mother drugs.

TABLE 14 in vitro anti-colon cancer cell Activity test results of FDT/DTX-PABC-polypeptide prodrugs

TABLE 24 results of in vitro toxicity test of FDT/DTX-PABC-polypeptide prodrugs to human Normal cells

。

Claims (2)

1. The docetaxel targeted colon cancer resistant prodrug is characterized by having a structure selected from:

。

2. the use of the docetaxel targeted anti-colon cancer prodrug as claimed in claim 1 in the preparation of a medicament for treating colon cancer.

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