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

Abstract

式(1)中，A为包含但不限于结肠癌组织中显著高表达的基质金属蛋白酶MMP‑7、半胱氨酸组织蛋白酶B、成纤维细胞激活蛋白α等特异识别并水解的底物多肽序列；B为选自(a～i)的可降解的桥接基团，且与A的碳端或氮端通过酰胺键相接；R₁为包含但不限于氟原子、氢原子等。

The invention belongs to the field of biomedicine, and in particular relates to a novel targeted prodrug of docetaxel and its medicinal use. The invention provides a novel targeted prodrug of docetaxel represented by the general formula (1). And its medicinal use against colon cancer: the compound of the present invention has pharmacological research value, and can be used as an anticancer prodrug targeting a protease that is specifically and highly expressed in colon cancer tumor sites. It can treat colon cancer diseases with high specific expression of one or more of matrix metalloproteinase MMP-7, cysteine cathepsin B, fibroblast activation protein alpha, etc.

In formula (1), A is a substrate polypeptide that is specifically recognized and hydrolyzed, including but not limited to matrix metalloproteinase MMP-7, cysteine cathepsin B, fibroblast activation protein α, etc., which are significantly highly expressed in colon cancer tissue sequence; B is a degradable bridging group selected from (a～i), and is connected to the carbon end or nitrogen end of A through an amide bond; R ₁ is including but not limited to fluorine atom, hydrogen atom, etc.

Description

Targeted prodrug of docetaxane and its medicinal use against colon cancer

technical field

The invention belongs to the field of biomedicine, and in particular relates to a novel targeted prodrug of docetaxel and its medicinal use against colon cancer.

Background technique

The prior art discloses that colon cancer is one of the common malignant tumors, and its morbidity and mortality ranks third among malignant tumors in developed countries in Europe and America. Five and sixth moved up to fourth and fifth with about 200,000 deaths from colon cancer each year. Clinical surgical resection is the most basic treatment method, and adjuvant chemotherapy is the routine therapy for patients with colon cancer stage II and III after surgery. At present, adjuvant chemotherapy drugs for colon cancer are divided into two categories: one is chemical small molecule drugs, mainly 5-fluorouracil, capecitabine, tegafur, irinotecan, oxaliplatin, etc. They can be used alone or Combination drugs, but all inevitably produce toxic side effects such as nausea, stomatitis, vomiting; the other type 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 due to the increasing resistance to EGFR and VEGF, this class of drugs has low efficacy, and it is not effective for disease progression-free survival and overall survival in the adjuvant treatment mode No long-term benefits appear and are expensive to use. Therefore, the development of more effective, less toxic and side effects and low-cost anti-colon cancer small molecule drugs has become a major issue that needs to be solved urgently.

Among the many anticancer drugs discovered so far, paclitaxel isolated from natural Taxus chinensis is the first-line drug for clinical treatment of breast cancer, ovarian cancer and non-small cell lung cancer. , is considered to be the most effective antitumor drug discovered by humans so far. In view of their many clinical defects, such as multidrug resistance, low oral bioavailability, allergic reactions, poor water solubility, poor metabolic stability, systemic toxicity and side effects caused by drug miscellaneous, the third-generation highly active The research and development of paclitaxel antitumor drugs has made great progress. For example, in view of the shortcomings of multidrug resistance, in 2010, Sanofi-Aventis carried out methylation on the C-7 and C-10 hydroxyl groups of docetaxel. A new compound, Cabazitaxel (trade name Jevtana), was obtained and approved by the FDA; ② In view of its low oral bioavailability, Spectrum's focus on the development of Orataxel is currently undergoing Phase II clinical trials. , In addition to the greatly improved oral bioavailability, the compound has the same excellent cytotoxic activity against drug-susceptible cells and multidrug-resistant cells; The paclitaxel liposome for injection, Lipusu, was successfully developed by encapsulating paclitaxel in the body and was approved by the SFDA in 2004; ④ In view of its poor water solubility, Abraxis successfully developed the first non-solvent-based nanoalbumin-binding chemotherapy The drug Abraxane was approved by the FDA in 2005; Cell Therapeutics linked the C-2′ OH of paclitaxel to poly-L-glutamic acid to obtain a prodrug Xyotax with greater water solubility and better tumor targeting than paclitaxel /Opaxio, currently undergoing Phase III clinical trials. However, in view of the shortcomings of poor metabolic stability and high systemic toxicity, especially in the expansion of new anti-colon cancer indications, there have been no reports on the drugs that have been marketed.

Studies have shown that there are many specific proteases on the surface of tumor cells or at the edge of the tumor site, which play a pivotal role in tumor growth, infiltration and metastasis. In particular, matrix metalloproteinases (MMPs), as well as MMPs-like cysteine cathepsin B (Cathepsin B, Cat B) and fibroblast activation protein α (FAPα), all have specific distribution. It is easily overexpressed in the cell site of colon cancer. On the contrary, it is underexpressed in normal tissues or inflammatory tissues. It is an important colon cancer tumor marker and therapeutic target.

The "prodrug" design strategy is one of the most important means to reduce drug toxicity. Proteases (such as: mechanistic metalloproteinase MMP-7, cysteine cathepsin B, fibroblast activation protein, and fibroblast activation protein) are specifically highly expressed in colon cancer tissue. α, etc.) recognized and hydrolyzed substrate polypeptides are used as targeting carrier groups, and docetaxel cytotoxic small molecule compounds with good anticancer activity are used as parent drugs. The prodrug formed by the linker is an unreported compound. The research and development of such targeted prodrug molecules is mainly aimed at the disadvantages of poor selectivity and high systemic toxicity of docetaxel cytotoxic small molecule compounds, and to expand the application of such compounds in anti-colon cancer tumor therapy.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a novel targeted prodrug of docetaxel and its medicinal use against colon cancer.

The present invention provides a novel docetaxane targeted prodrug represented by the following general formula (1), the docetaxel targeted prodrug covalently passes through a bridge molecule in the taxane molecule Coupling the substrate polypeptides recognized and hydrolyzed by specific and highly expressed proteases in colon cancer tissue can significantly improve the targeting of drugs to colon cancer.

In formula (1), A is a substrate polypeptide that is specifically recognized and hydrolyzed, including but not limited to matrix metalloproteinase MMP-7, cysteine cathepsin B, fibroblast activation protein α, etc., which are significantly highly expressed in colon cancer tissue Sequences 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-Ala-Ile-Gly-Gln-Pro-Gly, Ac-Gln-Gly-Ala-Leu-Gly-Gln-Pro--Gly, Gly-Leu-Phe -Gly, Gly-Phe-Leu-Gly, Ala-Leu-Ala-Leu, Mca-Ala-Ser-Gly-Pro-Ala-Gly-Ala--Pro-Dnp, Mca-Glu-Pro-Gly-Pro- 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, etc.;

B is a degradable bridging group, including but not limited to (a～i)

Isostructure, and connected with the carbon terminal or nitrogen terminal of polypeptide sequence A through an amide bond;

R ₁ includes, but is not limited to, a fluorine atom, a hydrogen atom, and the like.

The preparation method of the above-mentioned novel docetaxane targeting prodrug of the present invention comprises the following steps: firstly introducing a corresponding bridging group into the hydroxyl group at the C-2' position of the docetaxane compound, and synthesizing the docetaxane compound Derivative of the compound; then the derivative containing the bridging group is mixed with the targeting polypeptide, and under the action of a condensing agent, the mixture is stirred at 5° C. for coupling to obtain the docetaxane-based targeting prodrug.

The invention provides the proliferation inhibitory activity of such docetaxane targeted prodrugs on human colon cancer cell lines HCT116 and SW620 in vitro, and the toxicity to normal tissue colon cell line CCD18Co and kidney cell line HEK293.

The compounds of the present invention have pharmacological research value, and can be used as anticancer prodrugs targeting the protease that is specifically and highly expressed in colon cancer tumor sites. It can treat colon cancer diseases with specific high expression of matrix metalloproteinase MMP-7, cysteine cathepsin B, and fibroblast activation protein alpha.

The novel docetaxane targeted prodrug of the present invention can be prepared into various preparations comprising a safe and effective amount of the novel docetaxane prodrug and a pharmaceutical carrier, and is used for the treatment of colon cancer.

The "safe and effective amount" in the present invention refers to: the amount of the compound is sufficient to significantly improve the condition without causing serious side effects. The safe and effective dose is determined according to the age, disease condition, course of treatment, etc. of the object to be treated.

)、润湿剂(如十二烷基硫酸钠)，着色剂，调味剂，稳定剂，抗氧化剂，防腐剂，无热原水等。Examples of the pharmaceutically acceptable carrier part of the present invention include sugar (such as glucose, sucrose, lactose, etc.), starch (such as corn starch, potato starch, etc.), cellulose and its derivatives (such as sodium carboxymethylcellulose) , ethyl cellulose sodium, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.) , polyols (such as propylene glycol, glycerol, mannitol, sorbitol, etc.), emulsifiers (such as

), wetting agents (such as sodium lauryl sulfate), colorants, flavors, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

Compared with modern technology, the beneficial effects of the present invention are as follows:

1) Compared with 5-fluorouracil (5-FU), a positive control drug for clinical treatment of colon cancer, the taxane targeted prodrugs of the present invention have better anti-colon cancer activity, for example, on colon cancer cells HCT116 The inhibitory activity can reach 2.7nmol, showing good anti-colon cancer potential, and has a good drug prospect;

2) Compared with docetaxanes, the targeting peptides in the prodrugs can specifically transport small taxane molecules to the tumor tissue of colon cancer, reduce the lethality to normal cells, and target peptides. The intracellular degradation products are amino acids, so it has lower tissue system toxicity and higher biocompatibility.

Description of drawings

Fig. 1 is a synthetic route diagram of the prodrugs of docetaxel or tetrafluorodocetaxel coupled with colon cancer targeting polypeptides A1-A5 through a bridging group Leu-PABC(a) in Example 1.

Detailed ways

The present invention will be further described below in conjunction with the examples, but these examples are by no means any limitation to the present invention.

Example 1: Preparation of docetaxel or tetrafluorodocetaxel coupled with colon cancer targeting polypeptides A1-A5 via bridging group Leu-PABOH(a) prodrugs

Taxane targeted prodrugs are prepared by coupling paclitaxel (DTX) or tetrafluorodocetaxel (4FDT) with colon cancer targeting polypeptides A1-A5. The synthetic route is shown in Figure 1, including the following steps:

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

References (Elsadek B, Graeser R, Esser N, et al. Development of a novelprodrug of paclitaxel that is cleaved by prostate-specific antigen: An invitro and in vivo evaluation study [J]. European Journal of Cancer, 2010, 46 ( 18): The method adopted in 3434-3444) completes the synthesis and activation modification of the bridging group Leu-PABOH through a two-step reaction. Namely: in 25mL single-neck flask, add the Fmoc-L-Leu (1) of 1mmol, the p-aminobenzyl alcohol (PABOH) of 2mmol and the condensing agent 2-ethoxy-1-ethoxycarbonyl-1 of 2mmol, 2-Dihydroquinoline (EEDQ) was added with 14 mL of methanol and 7 mL of dichloromethane as the reaction solvent, and the reaction was carried out for about 48 h at room temperature in the dark, monitored by TLC (DCM:MeOH=55:1). The organic solvent was removed by concentrating under pressure, and purified by the system of dichloromethane:methanol (100:1—70:1) to obtain a pale yellow intermediate Fmoc-Leu-PABOH (2) with a yield of 73%; take a two-necked flask, add 1 mmol The intermediate Fmoc-Leu-PABOH (2), 2 mmol of bis(p-nitrobenzene) carbonate, _N2 protected, after adding 10 mL of anhydrous N,N-dimethylformamide (DMF) to dissolve the sample, at zero Under the condition of degrees Celsius, slowly add 1.5mmol of diisopropylethylamine (DIPEA), move to room temperature after 5min, monitor the reaction by TLC (PE:ACE=3:1), and the reaction is complete in about 8-12h; after the reaction, add A large amount of saturated sodium chloride (NaCl) aqueous solution terminated the reaction, ethyl acetate (EA) was layered and extracted three times, the organic phases were combined, a relatively pure white solid was precipitated during concentration under reduced pressure, filtered, and the remaining organic phases were combined and purified by silica gel column ( PE:ACE=10:1-7:1) and recrystallization to obtain white solid Fmoc-Leu-PABC-PNP(3) with a total yield of 82%. ¹ H NMR (400 MHz, CDCl ₃ ): δ 8.27 (d, J=8.0 Hz, 2H), 7.76 (d, J=8.0 Hz, 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 : HRMScalcd.646.2160[M+Na]+, found 646.2162.

2) Synthesis of Docetaxel C-2' Intermediate 4

In a two-necked flask, add 1 mmol of Fmoc-Val-Cit-PABC-PNP (3), 1 mmol of docetaxel (DTX), N2 protection, add 80 mL of anhydrous dichloromethane solution, at zero degrees Celsius, add 1 mmol of The condensing agent 4-dimethylaminopyridine (DMAP) was moved to room temperature after 5 minutes, and the reaction was carried out for about 48 hours. The reaction was monitored by TLC (DCM:MeOH=25:1). After the reaction was completed, it was concentrated under reduced pressure and the organic solvent dichloromethane was removed. Silica gel column separation and purification (DCM:MeOH=100:1) gave off-white solid 4 with a yield of about 50%. ¹ H NMR (400 MHz, Acetone-d ₆ ): δ 9.43 (s, 1H), 8.12 (d, J=7.6 Hz, 2H), 7.86 (d, J=7.6 Hz, 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] ⁺ ; _C72H81N3O19 : _{HRMScalcd.1314.5356} [M+Na] ⁺ ,found1314.5366.

3) Synthesis of Intermediate 5 at C-2′ Position of Tetrafluorodocetaxel

The synthetic method is the same as that of intermediate 4, off-white solid, yield 54%. Off-white solid, 54% yield. ¹ H NMR (400 MHz, Acetone-d ₆ ): δ 9.46 (s, 1H), 7.95 (d, J=7.6 Hz, 1H), 7.86 (d, J=7.6 Hz, 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)

Take a 25mL single-neck flask, add 15mg of intermediate 4 (or 5) and 0.9mL of N,N-dimethylformamide (DMF), after fully dissolving, add 3.6μL of piperidine at zero degrees Celsius, and react Move to room temperature after 5min, monitor by TLC (DCM:MeOH=15:1), the reaction can be completed in about 36min; after the reaction, immediately remove the solvent N,N-dimethylformamide (DMF) as much as possible at room temperature with a vacuum oil pump , and the remaining residual DMF was removed with saturated aqueous NaCl solution, extracted three times with dichloromethane (100 mL), and the organic phase was rapidly concentrated under reduced pressure to obtain pale yellow solid 6 (or 7), which was directly used in the next reaction.

5) Synthesis of targeted polypeptide prodrugs

DTX-PABC-Leu-A1(8a)

Take a 25mL single-neck flask, add 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-Hydroxybenzone Triazole (HOBT), and 1.5 mL of N,N-dimethylformamide (DMF) were fully dissolved at 0 °C, and 0.04 mmol of N-methylmorpholine was added. After the reaction was performed for 15 min, N,N was added. '-Diisopropylcarbodiimide (DIC), warmed up to 5°C, continued the reaction, monitored the reaction by HPLC (method: water/acetonitrile as mobile phase, acetonitrile: 5%-100%, 30min), about 72h can react Complete; after the reaction, remove the solvent N,N-dimethylformamide (DMF) as much as possible with a vacuum oil pump at room temperature, add a small amount of dimethyl sulfoxide (DMSO) to dissolve, and purify by reverse-phase column (water/acetonitrile is flowing Phase: successively eluted with 100% water for 15min to remove the DMSO solution in the sample; acetonitrile 0-85%, 40min) to obtain a white solid 8a with a yield of 17%. 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=7.2 Hz, 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=10.4 Hz,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 : HRMScalcd.2007.9166[M+Na] ⁺ ,found 2007.9246.

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

Synthesis method is the same as 8a, white solid, yield 22%. 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)

Synthesis method is the same as 8a, white solid, yield 21%. 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] ⁺ , found1845.8219.

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

Synthesis method is the same as 8a, white solid, yield 23%. 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] ⁺ ; _{C89H118F4N12O27S} : _{HRMScalcd.1917.7778} [M+Na] ⁺ ,found _1917.7831 .

DTX-PABC-Leu-A3(10a)

Synthesis method is the same as 8a, white solid, yield 18%. 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] ⁺ ; _{C90H123N13O28} :HRMS _{calcd.1856.8493} [M+Na] ⁺ ,found 1856.8486.

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

Synthesis method is the same as 8a, white solid, yield 21%. ¹ H NMR (400MHz, DMSO-d ₆ , rotamers): δ9.97 (d, J=12.0 Hz, 1H), 8.52 (d, J=12.0 Hz, 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=8.0 Hz, 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 : HRMScalcd.1928.8116[M+Na] ⁺ , found 1928.8183.

DTX-PABC-A4(11a)

Synthesis method is the same as 8a, white solid, yield 24%. 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)

Synthesis method is the same as 8a, white solid, yield 25%. ¹ H NMR (400MHz, DMSO-d ₆ , rotamers) δ 10.01 (d, J=20.8 Hz, 1H), 8.47 (d, J=8.4 Hz, 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)

Synthesis method is the same as 8a, white solid, yield 24%. 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] ⁺ ; _{C89H121N13O28} :HRMS _{calcd.1842.8336} [M+Na] ⁺ , found _1842.8347 .

4FDT-PABC-A5(12b)

Synthesis method is the same as 8a, white solid, yield 22%. 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 F4N13O28} : HRMS calcd. _1914.7959 [M+Na] ⁺ , found _1914.7963 ..

Example 2: In vitro proliferation inhibitory activity of targeted anti-colon cancer prodrugs on human colon cancer cell lines HCT116 and SW620 and toxicity evaluation on normal colon cell line CCD18Co and kidney cell line HEK293

Experimental methods: With 5-fluorouracil and parent drugs docetaxel and tetrafluorodocetaxel as positive controls, the in vitro antitumor activities of the prodrug compounds on HCT116 and SW620, and their effects on normal colon cell lines were determined by MTT assay. Toxicity of CCD18Co and the kidney cell line HEK293. The inhibition of the growth of tumor cells and normal cells at different concentrations was observed, and its half-inhibition rate (IC50 value) was calculated to evaluate its anti-tumor activity in vitro and its toxicity to normal tissues.

Take the cells in logarithmic growth phase and in good condition, add an appropriate amount of trypsin (purchased from GIBCO) to digest the cells, then remove the trypsin, rinse with serum-containing culture medium to collect and resuspend the cells, count and adjust the cell density. The cell suspension was seeded on a 96-well plate (100 μL, 4000-5000 cells/well), and cultured for 12 h at 37° C. in a 5% CO2 and 95% air atmosphere. After confirming that the cells adhered, the culture medium was aspirated, and the prodrug compound solution (concentration: ^10-4 , ^10-5 , ^10-6 , ^10-7 , ^10-8 , ^10-9 , 10- ⁾ dissolved in the culture medium was added. ¹⁰ and 10 ^-11 mol/L) were added to the cells (200 μL/well), respectively, as the experimental group. The medium containing 0.2% DMSO was added to the cell wells (200 μL/well) as a medium control group. The culture medium was added to the wells that were not seeded with cells as a blank group. Six replicate wells were set for each concentration. After the cells were incubated for 48 or 72 h at 37° C., 5% CO2 and 95% air atmosphere, MTT/PBS solution (20 μL, 5 mg/mL) was added to each well, and the incubation was continued for 4 h. Carefully aspirate the liquid and add 150 μL of DMSO to dissolve completely. Shake at medium speed with a microplate reader (Thermo Fisher) for 5 minutes, and measure the absorbance (OD) at 490 nm. The inhibition rate of the compound on cell proliferation was calculated according to the following formula:

Inhibition rate=1-[(average OD value of administration group-average OD value of blank group)/(average OD value of culture medium control group-average OD value of blank group)] IC50 value was calculated by SPSS software. The above experiments were repeated three times, and the average and standard deviation of IC50 of the three experiments were calculated.

Experimental results: The proliferation inhibitory activity of targeted anti-colon cancer prodrugs on human colon cancer cell line HCT116 was stronger than that of the positive control drug 5-fluorouracil, and was equivalent to or slightly lower than that of the corresponding parent drugs (docetaxel or tetracycline). Fluorinated docetaxel); the toxicity to normal colon cell line CCD18Co and kidney cell line HEK293 is lower and lower than that of their respective parent drugs.

Table 1 In vitro anti-colon cancer cell activity test results of 4FDT/DTX-PABC-polypeptide prodrugs

Table 2 In vitro test results of 4FDT/DTX-PABC-polypeptide prodrugs on human normal cytotoxicity

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Claims (2)

1. a docetaxane class targeting anti-colon cancer prodrug, is characterized in that, its structure is selected from:

. 2. The use of the docetaxane-targeted anti-colon cancer prodrug according to claim 1 in the preparation of a medicament for the treatment of colon cancer.

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