Paclitaxel molecular umbrella delivery system prodrug as well as preparation method and application thereof

Abstract

The invention provides a paclitaxel molecular umbrella delivery system prodrug as well as a preparation method and application thereof. The paclitaxel molecular umbrella delivery system prodrug comprises a molecular umbrella target head part which is formed by connecting two surface amphiphilic molecules through spermidine; a paclitaxel drug moiety; and a linking group that links the molecular umbrella target head portion to the paclitaxel drug portion through a disulfide bond; wherein the surface amphiphilic molecule is selected from cholic acid, deoxycholic acid or sodium sulfonate thereof. Compared with paclitaxel, the paclitaxel molecular umbrella delivery system prodrug has the advantages that the water solubility is obviously improved, the toxicity to normal cells is lower than that of free paclitaxel, and the paclitaxel molecular umbrella delivery system prodrug has obviously improved safety; the paclitaxel molecular umbrella delivery system prodrug has the following characteristics in a physiological environment: an active drug is released in a high GSH / ROS microenvironment of tumor cells; and maintaining the prodrug state in normal cells.

Description

Technical Field

[0001] The present application relates to the field of medicine, and in particular to a paclitaxel molecular umbrella delivery system prodrug and a preparation method and application thereof. Background Art

[0002] Any discussion of the prior art throughout the specification should not be considered as an admission that such prior art is widely known or forms part of the common general knowledge in the field.

[0003] Cancer is still one of the diseases with the highest mortality rate in the world. The main treatment methods for cancer are still radiotherapy and chemotherapy, and chemotherapy is currently the most effective treatment method. However, chemotherapy drugs usually have poor selectivity and biocompatibility, and some chemotherapy drugs may even develop drug resistance, causing serious toxic side effects to patients.

[0004] Paclitaxel is a microtubule stabilizer that is currently widely used in clinical practice, mainly for the treatment of breast cancer. However, its water solubility and selectivity are poor, which poses challenges to its clinical application.

[0005]

[0006] The prodrug theory is currently widely used in chemotherapy drugs. It reduces the toxicity of chemotherapy drugs by blocking the active essential groups, thereby reducing their toxic side effects and increasing their targeting. Therefore, it is of great clinical significance to design and synthesize paclitaxel prodrug molecules to increase the water solubility and targeting of paclitaxel and overcome the drug resistance of paclitaxel. Summary of the invention

[0007] The purpose of the present invention is to overcome the technical problems of poor water solubility, low selectivity, and large toxic and side effects of existing paclitaxel drugs, and provide a paclitaxel molecular umbrella delivery system prodrug and its preparation method. The present invention constructs a molecular umbrella carrier by connecting two surface amphiphilic molecules (cholic acid, deoxycholic acid and its sodium sulfonate) through spermidine, and uses a disulfide bond to connect the paclitaxel 2'-OH site with the molecular umbrella target head, and designs and synthesizes a series of intelligent responsive prodrug molecules. The prodrug can selectively release the active drug paclitaxel under the high GSH / ROS microenvironment of tumor cells, while maintaining the prodrug state in normal cells. The experimental results show that the prodrug of the present invention significantly improves the water solubility of paclitaxel (more than 5500 times), while maintaining good anti-tumor activity and reducing the toxicity to normal cells; in vivo experiments confirm its anti-tumor effect, especially some compounds can achieve the anti-tumor effect of high-dose paclitaxel at low doses, and the safety is significantly improved. The present invention provides a new drug delivery strategy for improving the clinical application effect of paclitaxel drugs.

[0008] Specifically, the present invention provides the following technical solutions.

[0009] In the first aspect of the present invention, a paclitaxel molecular umbrella delivery system prodrug is provided, comprising:

[0010] The target head of the molecular umbrella is composed of two surface amphiphilic molecules connected by spermidine;

[0011] the paclitaxel drug portion; and

[0012] A linking group, which connects the molecular umbrella target head portion to the paclitaxel drug portion via a disulfide bond;

[0013] Wherein, the surface amphiphilic molecules are selected from at least one of the following compounds: cholic acid, sodium cholic acid sulfonate, deoxycholic acid and sodium deoxycholic acid sulfonate. The two surface amphiphilic molecules are the same or different, preferably the same.

[0014] In an embodiment of the present invention, the paclitaxel drug moiety is linked to the linker group via the 2'-OH site.

[0015] In an embodiment of the present invention, the linking group has a structure shown in Formula III:

[0016]

[0017] In some embodiments of the present invention, the paclitaxel molecular umbrella delivery system prodrug has one of the following structures:

[0018]

[0019] Wherein, R1 is selected from -OH and -H; R2 is selected from -OSO3Na and -H.

[0020] In some embodiments of the present invention, the paclitaxel molecular umbrella delivery system prodrug is selected from the following compounds:

[0021]

[0022] In the second aspect of the present invention, a method for preparing the paclitaxel molecular umbrella delivery system prodrug described in the first aspect is provided, which comprises:

[0023] (1) allowing mercaptopropionic acid and 2,2-dipyridyldisulfide to undergo a substitution reaction to generate 3-(pyridin-2-yldisulfide)propionic acid (i.e., compound I-3);

[0024]

[0025] (2) esterifying 3-(pyridin-2-yldisulfide)propionic acid (i.e., compound I-3) with the 2'-OH of paclitaxel to obtain compound I-4;

[0026]

[0027] (3) Compound I-4 is subjected to a substitution reaction with dithiothreitol to obtain compound I-5;

[0028]

[0029] (4) ester condensing cholic acid (R1 in I-6 is -OH) or deoxycholic acid (R1 in I-6 is -H) with NHS to obtain compound I-7;

[0030]

[0031] (5) Compound I-7 is subjected to a substitution reaction with spermidine to obtain compound I-8;

[0032]

[0033] (6) esterifying 3-hydroxy-1,2,3-benzotriazine-4(3H)-one (i.e., compound I-10, also known as HOOBt) with compound I-3 to obtain compound I-11;

[0034]

[0035] (7) subjecting compound I-8 and compound I-11 to a substitution reaction to obtain compound I-9;

[0036]

[0037] (8) Compound I-9 is oxidized with sulfur trioxide pyridine to obtain compound I-12;

[0038]

[0039] (9) subjecting Compound I-5 and Compound I-9 or Compound I-12 to a nucleophilic substitution reaction to obtain a paclitaxel molecular umbrella delivery system prodrug;

[0040]

[0041] Wherein, R1 is selected from -OH and -H; R2 is selected from -OSO3Na and -H.

[0042] In one embodiment of the present invention, step (1) is carried out under the conditions of acetic acid and methanol.

[0043] In one embodiment of the present invention, step (2) is carried out under DCC and dichloromethane conditions.

[0044] In one embodiment of the present invention, step (3) is carried out under dichloromethane conditions.

[0045] In one embodiment of the present invention, step (4) is carried out under DCC and tetrahydrofuran conditions.

[0046] In one embodiment of the present invention, step (5) is carried out under DCM / DIPEA conditions.

[0047] In one embodiment of the present invention, step (6) is carried out under DCC and dichloromethane conditions.

[0048] In one embodiment of the present invention, step (7) is carried out under DIEA and DMF conditions.

[0049] In one embodiment of the present invention, step (8) is carried out under DMF conditions.

[0050] In one embodiment of the present invention, step (9) is carried out under methanol conditions.

[0051] In the third aspect of the present invention, a pharmaceutical composition is provided, which comprises the paclitaxel molecular umbrella delivery system prodrug described in the first aspect.

[0052] The pharmaceutical composition can be used to treat tumor diseases such as breast cancer, ovarian cancer, pancreatic cancer, lung cancer, head and neck cancer, etc. Since the prodrug of the present invention has good water solubility and stability, the prescription design can be significantly simplified. In some embodiments, it can be combined with other anti-tumor drugs according to clinical needs to provide a more flexible treatment plan.

[0053] In the fourth aspect of the present invention, a pharmaceutical preparation is provided, which comprises the paclitaxel molecular umbrella delivery system prodrug described in the first aspect or the pharmaceutical composition described in the third aspect, and at least one pharmaceutically acceptable excipient or carrier.

[0054] In some embodiments, the pharmaceutical preparation may be an injectable dosage form such as an injection, an infusion or a lyophilized powder injection, or may be an oral preparation such as a tablet or a capsule.

[0055] In some embodiments, the pharmaceutically acceptable excipients or carriers can be selected as needed, for example, including: water for injection, physiological saline, glucose injection or phosphate buffer used as solvents or carriers; antioxidants, pH adjusters or buffers used as stabilizers; sodium chloride, glucose or mannitol used as isotonicity adjusters; and preservatives such as benzyl alcohol or parabens, etc. that can be added when necessary.

[0056] Since the prodrug of the present invention has significantly improved water solubility, no complicated solubilization system is required in the formulation design, and a simple preparation process can be adopted, while ensuring a high bioavailability, and having the characteristics of being easy to use and having good safety.

[0057] In the fifth aspect of the present invention, there is provided use of the paclitaxel molecular umbrella delivery system prodrug described in the first aspect above, or a pharmaceutical composition or a pharmaceutical preparation comprising the same, in the preparation of an anti-tumor drug.

[0058] In some embodiments of the present invention, the anti-tumor drug is used to treat the following cancers: breast cancer, ovarian cancer, pancreatic cancer, lung cancer, and head and neck cancer.

[0059] In some embodiments of the present invention, the paclitaxel molecular umbrella delivery system prodrug has the following characteristics under physiological conditions:

[0060] Release active drugs in the high GSH / ROS microenvironment of tumor cells;

[0061] Remains in the prodrug state in normal cells.

[0062] In some embodiments of the present invention, the paclitaxel molecular umbrella delivery system prodrug has the following characteristics:

[0063] Water solubility is not less than 50 mg / mL;

[0064] The toxicity to normal cells is much lower than that of free paclitaxel.

[0065] Compared with the prior art, the advantages of the present invention include:

[0066] The paclitaxel molecular umbrella delivery system prodrug provided by the present invention has significant technical advantages. First, the prodrug system significantly improves the water solubility of paclitaxel, wherein the water solubility of I-14a and I-14b reaches 243 mg / mL and 90 mg / mL respectively (equivalent paclitaxel is 82 mg / mL and 33 mg / mL respectively), which is more than 5500 times higher than the 6 μg / mL solubility of the paclitaxel original drug, fundamentally solving the problem of poor water solubility of paclitaxel in drug administration.

[0067] Secondly, the prodrug system achieves intelligent responsive release. Experiments have shown that the prodrug can effectively release paclitaxel in both a reducing environment (10mM DTT) and an oxidative environment (10mM H2O2), cleverly utilizing the microenvironmental characteristics of high GSH / ROS in tumor cells to achieve targeted release at the tumor site while maintaining the prodrug state in normal cells, thereby significantly reducing systemic toxicity.

[0068] In terms of anti-tumor activity, in vitro experiments have shown that the prodrug exhibits good inhibitory activity against a variety of tumor cell lines (including MCF7, PC3, HepG2, H460, A549, etc.), while significantly reducing toxicity to normal cells (HUVEC, HBE, Beas-2b). In vivo experiments have further confirmed its excellent efficacy and safety. For example, I-14a has comparable tumor inhibition effects to equal-dose paclitaxel but is safer; in particular, I-14b can achieve the tumor inhibition effect of high-dose paclitaxel at a low dose, while significantly reducing toxicity to normal cells, reflecting a good safety advantage. PCNA staining results also confirmed that the inhibitory effect of low-dose I-14b is better than that of equal-dose paclitaxel.

[0069] In addition, the prodrug of the present application also has good advantages in preparation process. Its synthetic route is clear, the reaction conditions are mild, the yield is good, and it is convenient for large-scale production. In terms of molecular design, it provides good water solubility through the molecular umbrella structure, uses disulfide bonds to achieve intelligent response release, and the structure has good modifiability, which is conducive to further optimization and improvement. These advantages together constitute the innovative value of the prodrug of the present application, and provide a new option for the clinical application of paclitaxel. BRIEF DESCRIPTION OF THE DRAWINGS

[0070] The drawings constituting part of the present application are used to provide a further understanding of the present application. The illustrative embodiments and descriptions of the present application are used to explain the present application and do not constitute an improper limitation on the present application. The implementation scheme of the present application is described in detail below in conjunction with the drawings, wherein:

[0071] Figure 1 : Standard curve of the content of compound I-14a.

[0072] Figure 2 : Standard curve of the content of compound I-14b.

[0073] Figure 3 : Paclitaxel standard curve.

[0074] Figure 4 : Release curve of paclitaxel under 10 mM DTT condition.

[0075] Figure 5 : Release curve of paclitaxel under 10 mM H2O2 condition.

[0076] Figure 6 : In vitro antiproliferative activities of paclitaxel and four prodrugs in different tumor cells (A) MCF7, (B) PC3, (C) HepG2, (D) H460, (E) A549.

[0077] Figure 7:In vitro antiproliferative activities of paclitaxel and four prodrugs in different normal cells (A) HUVEC, (B) HBE, (C) Beas-2b.

[0078] Figure 8 : Dosage regimen for tumor-bearing mice in in vivo tumor inhibition experiment.

[0079] Fig. 9 : Body weight changes of mice in each group in the in vivo tumor inhibition experiment.

[0080] Fig.10 : Changes in tumor volume in tumor-bearing mice after injection of PBS, PTX (10 mg / kg), PTX (20 mg / kg), I-14a (equivalent to 10 mg / kg PTX), I-14a (equivalent to 20 mg / kg PTX), I-14b (equivalent to 10 mg / kg PTX) and I-14b (equivalent to 20 mg / kg PTX) in the in vivo tumor inhibition experiment.

[0081] Fig.11 : Tumor weight of mice in each group in the in vivo tumor inhibition experiment.

[0082] Fig.12 : Tumor photos of mice in each group in the in vivo tumor inhibition experiment (the dotted circle represents the complete disappearance of the tumor, and the cross represents the death of the mouse) Statistical significance: *p<0.05, **p<0.01, ***p<0.001.

[0083] Fig.13 : PCNA staining results of tumor tissue. DETAILED DESCRIPTION

[0084] The present application is further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present application and are not intended to limit the scope of the present application. The experimental methods in the following examples without specifying specific conditions are usually carried out under conventional conditions or according to the conditions recommended by the manufacturer.

[0085] Unless otherwise defined, all professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. The reagents or raw materials used in this application can be purchased through conventional channels. Unless otherwise specified, the reagents or raw materials used in this application are used in a conventional manner in the art or in accordance with the product instructions. In addition, any method and material similar to or equivalent to the content described herein can be applied to the method of this application. The preferred implementation methods and materials described in the text are for demonstration purposes only.

[0086] Example 1 Preparation of Compound I-3

[0087]

[0088] Compound I-1 (200 mg, 0.91 mmol) and acetic acid (0.5 mL, 10.625 mmol) were placed in a reaction flask, dissolved in methanol, and then a methanol solution of mercaptopropionic acid (39.55 μL, 0.454 mmol) was added dropwise under an ice bath, and reacted at room temperature. After TLC detection of the reaction was complete, the reaction solution was concentrated under reduced pressure and column chromatography was performed with petroleum ether: ethyl acetate = 50:1-25:1-10:1 to obtain a transparent oil (70 mg, 72%). 1 H NMR (400MHz, Chloroform-d) δ8.48(d,J=4.9Hz,1H),7.71–7.60(m,2H),7.15(ddd,J=6.6,5.0,2.0Hz,1H),3.06(t,J=6.8Hz,2H),2.80(t,J=6.8Hz,2H). 13 C NMR (101MHz, CDCl3) δ175.98,159.31,149.43,137.53,121.27,120.55,34.10,33.82.

[0089] Example 2 Preparation of Compound I-4

[0090]

[0091] 3-(Pyridin-2-yldisulfanyl)propionic acid (70 mg, 0.325 mmol) was dissolved in dichloromethane (10 mL), and paclitaxel (185 mg, 0.217 mmol) and DCC (268.64 mg, 1.3 mmol) were added under ice bath conditions. The reaction was carried out at room temperature for 16 h. The precipitate was filtered and washed with a small amount of dichloromethane. The filtrate was concentrated under reduced pressure and column chromatography was performed with DCM:MeOH=100:1 to obtain a white solid (66.8 mg, 30%). 1HNMR(400MHz,Chloroform-d)δ8.45(d,J=5.2Hz,1H),8.14(d,J=7.6Hz,2H),7.76(d,J=7.5Hz,2H),7.65–7.36(m,13H),7.11–7.07(m,1 H),6.29(s,1H),6.25(d,J=8.1Hz,1H),5.98(dd,J=8.9,3.4Hz,1H),5.68(d,J=7.0Hz,1H),5.54(d,J=3.5Hz,1H),4.98(d,J=7.9Hz,1H) ,4.46–4.41(m,1H),4.32(d,J=8.3Hz,1H),4.21(d,J=8.6Hz,1H),3.81(d,J=6.3Hz,1H),3.01(t,J=6.7Hz,2H),2.92–2.86(m,2H),2.57 (d,J=5.1Hz,1H),2.45(s,3H),2.37–2.32(m,1H),2.22(s,3H),2.16–2.11(m,1H),1.91(s,3H),1.69(s,3H),1.23(s,3H),1.14(s,3H). 13 CNMR(101MHz, CDCl3)δ203.85,171.29,170.73,169.80,167.89,167.08,159.29,156.70,149.82,142 .75,137.21,136.87,133.74,133.53,132.79,132.06,130.25,129.16,128.78,128.75,128.57,127.1 6,126.55,121.01,119.88,84.46,81.07,75.61,75.06,74.36,72.16,71.90,58.53,53.47,52.76,49.17,45.60,43.18,35.55,33.97,33.37,32.99,26.83,25.63,24.97,22.73,22.12,20.87,14.87,9.62.

[0092] Example 3 Preparation of Compound I-5

[0093]

[0094] Compound I-4 (100 mg, 0.095 mmol) was dissolved in dichloromethane, and dithiothreitol (146.86 mg, 0.95 mmol) was added. The mixture was reacted at room temperature for 6 h. The reaction solution was washed with water three times, and the organic phase was dried and concentrated under reduced pressure. Then, column chromatography (DCM: MeOH = 100: 1) was performed to obtain a white solid (76.4 mg, 85.4%). 1 H NMR(400MHz,Chloroform-d)δ8.14(d,J=7.6Hz,2H),7.75(d,J=7.8Hz,2H),7.60(d,J=7.9Hz,1H),7.52(t,J=7.6Hz,3H),7.45–7.35(m,7H) ,6.96(d,J=8.6Hz,1H),6.30(s,1H),6.26–6.21(m,1H),5.99–5.95(m,1H),5.68(d,J=7.0Hz,1H),5.56(d,J=3.4Hz,1H),4.98(d,J=9.0Hz,1 H),4.44(dd,J=11.2,4.9Hz,1H),4.32(d,J=8.6Hz,1H),4.20(d,J=8.6Hz,1H),3.81(d,J=6.8Hz,1H),2.96–2.92(m,2H),2.71–2.67(m,2H), 2.52–2.49(m,1H),2.44(s,3H),2.36–2.31(m,1H),2.23(s,3H),2.15 (d,J=6.3Hz,1H),1.93(s,3H),1.68(s,3H),1.23(s,3H),1.14(s,3H). 13C NMR (101MHz, CDCl3) δ203.79,171.20,170.93,170.67,169.85,169.81,168.07,168.02,167.31,167 .21,167.01,166.98,156.98,153.58,149.48,142.64,136.96,136.85,136.04,133.78,133.67,133 .64,132.96,132.91,132.89,132.05,131.96,130.23,129.28,129.26,129.14,129.07,129.05,128.74,128.71,128.66,128.56,127.26,127.19,127.16,127.13,126.79,126.66,126.55,122.50,119. 67,84.46,81.10,81.07,79.12,79.08,76.45,75.61,75.15,74.43,74.28,72.10,71.97,71.90,58.52,58.50,53.03,52.96,52.81,50.13,49.18,45.63,43.20,38.06,35.94,35.59,35.10,34.38,33.8 9,33.43,32.67,32.51,31.89,30.98,29.77,29.69,29.51,29.31,29.24,27.22,26.82,26.79,26.13,25.61,25.55,25.41,25.29,24.93,24.71,22.71,22.12,20.82,19.53,14.83,14.81,14.11,9.63.

[0095] Example 4 Preparation of active esters of cholic acid and deoxycholic acid I-7a and I-7b

[0096]

[0097] Cholic acid (500 mg, 1.22 mmol) was dissolved in tetrahydrofuran, NHS (140.4 mg, 1.22 mmol) and DCC (303.3 mg, 1.47 mmol) were added at 0°C, and the mixture was reacted overnight at room temperature. The solid was filtered, and the filtrate was concentrated under reduced pressure. Column chromatography with DCM: MeOH = 50: 1 gave I-7a and I-7b, both of which were white solids. The yield of I-7a was 96%, and the yield of I-7b was 69%. The compound was directly used for the next step reaction.

[0098] Example 5 Preparation of compounds I-8a and I-8b

[0099]

[0100] Compound I-7a or I-7b (100 mg, 0.2 mmol) was dissolved in DCM (5 mL), spermidine (14.525 μL, 0.1 mmol) and DIPEA (52.52 μL, 0.3 mmol) were added, and stirred at room temperature for 16 h. A saturated NaHCO3 solution was added to produce a white precipitate, which was filtered and dissolved in methanol, concentrated under reduced pressure, and column chromatography was performed with DCM: MeOH: NH3·H2O = 5: 1: 0.1 to obtain I-8a or I-8b, both of which were white powders.

[0101] Compound I-8a: white powder, yield 79%. 1 H NMR (400MHz, Methanol-d4) δ3.95(s,2H),3.80(s,2H),3.26(t,J=6.5Hz,2H),3.20(t,J=6.6Hz ,2H),2.84(q,J=6.8Hz,4H),2.28–2.22(m,4H),1.93–1.00(m,58H),0.92(s,6H),0.71(s,6H). 13 C NMR(101MHz,MeOD)δ175.98,175.48,72.63,71.48,67.66,46.62,46.11,45.69,41.80,41.63,39.63,39.09,38.43,36.14,35 .59,35.14,34.53,32.78,32.69,31.99,29.83,28.24,27.61,27.38,26.53,26.50,24.88,22.90,21.86,16.44,16.42,11.71.

[0102] Compound I-8b: white powder, yield 47%. 1 H NMR(400MHz, Methanol-d4)δ3.96(s,2H),3.52(s,2H),3.23–3.16(m,4H),2.58(t,J=7.0Hz,4H),2.28–2.21(m,2H),2.14– 2.07(m,2H),1.91–1.66(m,16H),1.63–1.27(m,32H),1.24–1.11(m,4H),1.02(d,J=6.3Hz,8H),0.93(s,6H),0.71(s,6H). 13C NMR(101MHz,MeOD)δ177.25,173.90,72.63,71.17,46.75,46.26,46.23,46. 20,42.25,36.52,36.11,36.08,35.83,35.53,35.37,35.11,33.97,33.95,33 .44,32.89,31.74,31.02,30.90,30.51,29.70,29.46,29.15,28.56,27.30,27.08,26.13,24.93,23.55,22.49,22.43,16.43,16.28,13.18,12.03,11.93.

[0103] Example 6 Preparation of Compound I-11

[0104]

[0105] 3-(Pyridin-2-yldisulfanyl)propionic acid (200 mg, 0.93 mmol) was dissolved in dichloromethane, HOOBt (182.67 mg, 1.12 mmol) was added, and DCC (231.1 mg, 1.12 mmol) was added at 0°C. The mixture was reacted at room temperature for 6 h, and the precipitate was filtered and washed with a small amount of dichloromethane. The filtrate was concentrated under reduced pressure and column chromatography was performed with DCM:MeOH=100:1 to obtain I-11 as a white solid (247 mg, 73.7%). 1 H NMR(400MHz,Chloroform-d)δ8.53(d,J=4.9Hz,1H),8.38(d,J=8.0Hz,1H),8.24(d,J=8.2Hz,1H),8.02(t,J= 7.7Hz,1H),7.86(t,J=7.6Hz,1H),7.69(d,J=8.6Hz,2H),7.13(dd,J=6.6,4.7Hz,1H),3.25(d,J=3.1Hz,4H). 13 C NMR (101MHz, CDCl3) δ174.93,167.81,159.16,150.22,149.82,149.41,144.33,137.51,137.46,135 .54,132.86,129.07,125.81,122.24,121.28,121.18,120.64,120.19,34.21,34.11,32.84,31.28.

[0106] Example 7 Preparation of Compound I-9a-b

[0107]

[0108] Dissolve I-8a or I-8b (95 mg, 0.1 mmol) in 5 mL DMF, add compound I-11 (37 mg, 0.1 mmol) and DIPEA (70 μL, 0.4 mmol) to the reaction bottle, stir at room temperature for 12 h, concentrate under reduced pressure, and column chromatography DCM:MeOH=5:1 to obtain I-9a or I-9b, both of which are white powders.

[0109] Compound I-9a: white powder, yield 46%. H NMR (400MHz, Methanol-d4) δ8.41 (d, J = 4.9 Hz, 1H), 7.88–7.79 (m, 2H), 7.24 (td, J = 6.1, 5.5, 2.8 Hz, 1H), 3.94 (s, 2H), 3.79 (s, 2H), 3.35 (s, 2H), 3.28–3.05 (m, 8H), 2.81 (q, J = 6.3 Hz, 2H), 2.30 (s, 2H), 1.83–0.90 (m, 66H), 0.70 (s, 6H). 13 CNMR(101MHz,MeOD)δ175.38,149.10,137.85,121.07,119.83,72.63,71.49,67.64,54.43,46.67,46.12,41.80, 41.61,39.63,39.07,35.53,35.12,34.52,32.85,32.05,29.81,28.22,27.38,26.49,22.89,21.84,16.42,11.73.

[0110] Compound I-9b: white powder, yield 54%. 1 H NMR(400MHz,Chloroform-d)δ8.45(d,J=4.7Hz,1H),7.75(d,J=7.2Hz,1H),7.68(t,J =7.6Hz,1H),7.12(t,1H),3.96(s,2H),3.65–3.54(m,2H),3.42–3.15(m,8H),3.07(q ,J=6.9Hz,2H),2.77(t,J=6.6Hz,2H),2.29–2.21(m,2H),2.18–2.07(m,2H),1.86–1. 66(m,16H),1.55–1.21(m,32H),1.16–1.03(m,4H),0.99–0.86(m,14H),0.65(s,6H).

[0111] Example 8Preparation of compounds I-12a and I-12b

[0112]

[0113] I-9a or I-9b (68 mg, 0.06 mmol) was dissolved in DMF, cooled to 0°C, sulfur trioxide pyridine (385.28 mg, 2.42 mmol) was added, and the mixture was heated to room temperature and stirred. After reacting for 24 hours, a small amount of water was added to the reaction flask, saturated sodium bicarbonate was added to adjust the pH to 10, and the mixture was concentrated under reduced pressure at low temperature. Column chromatography was performed with DCM: MeOH = 5:1-2:1 to obtain I-12a or I-12b, both of which were yellow solids.

[0114] Compound I-9a: white powder, yield 62%. 1 H NMR (400MHz, Methanol-d4) δ8.41(d,J=4.9Hz,1H),7.89–7.78(m,2H),7.25(t,J=5.3Hz,1H),4.66(s,2H),4.45(s,2H),4.14(dt,J =11.7,6.7Hz,3H),3.38–3.34(m,2H),3.23–3.06(m,8H),2.86–2.77(m,2H),2.17–1.47(m,49H),1.10–1.04(m,10H),0.76(s,8H). 13 C NMR(101MHz,MeOD)δ175.78,175.61,175.57,171.41,171.20,159.91,159.82,149.17,149.14,138.0 7,127.74,121.29,121.27,119.95,81.14,79.87,76.72,46.11,46.00,45.89,45.50,43.58,42.21,41 .85,39.23,38.58,38.42,36.65,36.42,35.67,35.42,35.07,34.37,34.12,32.82,31.95,31.81,30.33,28.46,27.54,27.37,27.17,26.37,25.83,24.58,22.56,21.80,16.97,16.92,11.63,11.61,11.57.

[0115] Compound I-9b: white powder, yield 45%. 1H NMR(400MHz, Methanol-d4)δ8.41(d,J=4.9Hz,1H),7.89–7.81(m,2H),7.28 –7.22(m,1H),4.66(s,2H),4.28–4.23(m,2H),3.29–3.06(m,10H),2.81(td ,J=7.3,6.9,4.0Hz,2H),2.29–2.24(m,4H),1.90–1.78(m,16H),1.51–1.19 (m,32H),1.18–1.14(m,2H),1.09–1.02(m,10H),0.93(s,6H),0.75(s,6H).

[0116] Example 9 Preparation of Compound I-13a or I-13b

[0117]

[0118] Dissolve compound I-9a or I-9b (50 mg, 0.044 mmol) in methanol, add compound I-5 (42 mg, 0.044 mmol), react at room temperature for 12 h, concentrate the reaction solution under reduced pressure, and column chromatography DCM:MeOH=10 / 1-8 / 1-5 / 1 to obtain I-13a or I-13b, both of which are white powders.

[0119] Compound I-13a: white powder, yield 60%. 1 H NMR (400MHz, Methanol-d4) δ8.12(d,J=7.6Hz,2H),7.83(d,J=7.6Hz,2H),7.74–7.42(m,10H),7.27(t,J=7.4Hz,1H),6.45(s,1H ),6.06(t,J=9.3Hz,1H),5.85(d,J=6.6Hz,1H),5.64(d,J=7.2Hz,1H),5.51(d,J=6.6Hz,1H),5.00(d,J=9.8Hz,1H),4.57(s,1H) ,4.38–4.31(m,1H),4.19(s,2H),3.94(s,2H),3.83–3.77(m,3H),3.25–3.12(m,5H),3.02–2.87(m,7H),2.55–2.46(m,1H),2.40 (s,3H),2.32–2.26(m,3H),2.17(s,3H),2.13–2.10(m,1H),1.93(s,3H),1.82–0.95(m,72H),0.91(s,6H),0.69(d,J=2.1Hz,6H). 13C NMR(101MHz,MeOD)δ205.15,176.93,176.73,173.13,172.87,172.59,171. 56,171.27,170.43,170.38,167.62,142.31,138.29,135.53,134.87,134.6 5,132.95,131.37,131.21,130.13,129.74,129.60,128.67,85.87,82.22,78.97,77.44,76.80,76.21,76.13,74.00,72.98,72.85,72.30,69.02,59.2 1,55.81,55.34,48.05,47.86,47.49,44.57,43.15,42.98,40.97,40.44,39.76,37.90,37.53,36.90,36.49,35.89,35.22,34.44,34.24,33.68,33.41 ,31.18,30.74,29.58,28.76,27.85,27.36,27.00,26.04,24.27,23.38,23 .24,22.39,20.90,17.81,15.10,13.14,13.11,10.50.HRMS(ESI)m / z:Calcd for C 108 H 152 N4O 24 S2[M+H] + :1954.03107,Found:1954.03137.[M+Na] + :1976.01301,Found:1976.01440.

[0120] Compound I-13b: white powder, yield 33%. 1H NMR(400MHz,Methanol-d4)δ8.12(d,J=7.7Hz,2H),7.82(d,J=7.6Hz,2H),7.72–7.42(m,10H),7.28(t,J=7.4Hz,1H),6.45(s,1H),6.07(t,J=9.2Hz,1H),5.85(d,J=6.5Hz,1H),5.64(d,J=7.2Hz,1H),5.51(d,J=6.6Hz,1H),5.00(d,J=9.5Hz,1H),4.60(s,1H),4.39–4.31(m,1H),4.19(s,2H),3.95(s,2H),3.81(d,J=7.1Hz,1H),3.57–3.48(m,2H),3.42–3.33(m,4H),3.25–3.12(m,4H),3.03–2.87(m,6H),2.81–

[0121] 2.68(m,2H),2.48(dt,J=15.2,8.6Hz,1H),2.40(s,3H),2.37–2.19(m,3H),2.17(s,6H),1.94–1.66(m,22H),1.64–1.28(m,36H),1.16–1.10(m,7H),1.06–0.92(m,14H),0.69(s,6H). 13CNMR(101MHz,MeOD)δ205.09,176.85,176.70,176.65,173.06,172.79,172.53,171.4 9,171.24,170.35,170.32,167.58,142.31,138.28,135.51,134.83,134.63,132.93, 131.36,131.20,130.12,129.73,129.58,128.71,128.66,85.83,82.20,78.95,77.41,76.78,76.21,76.12,73.94,72.94,72.49,72.28,59.20,55.30,49.85,48.07,48.02 ,47.81,47.55,46.95,44.87,44.55,43.56,39.88,39.75,37.88,37.52,37.40,37.19,36.82,36.44,35.28,35.22,34.77,34.45,34.22,33.67,33.56,33.39,31.06,30.08 ,29.91,28.72,28.59,28.42,27.84,27.48,27.35,27.03,26.03,24.91,23.82,23.38 ,22.43,20.94,17.81,17.77,17.76,15.11,13.37,13.35,10.53.HRMS(ESI)m / z:Calcd for C 108 H 152 N4O 22 S2[M+H] + :1922.04124,Found:1922.04175.[M+Na] + :1944.02318,Found:1944.02307.

[0122] Example 10 Preparation of Compound I-14a or I-14b

[0123]

[0124] Compound I-12a or I-12b (30 mg, 0.01773 mmol) was dissolved in methanol, and compound I-5 (16.3 mg, 0.01773 mmol) was added. The reaction was carried out at room temperature for 12 h. The reaction solution was concentrated under reduced pressure and column chromatography was performed with DCM:MeOH = 2:1 to obtain I-14a or I-14b, both of which were white powders.

[0125] Compound I-14a: white powder, yield 38%. 1 H NMR (400MHz, Methanol-d4) δ8.11(d,J=7.7Hz,2H),7.83(d,J=7.7Hz,2H),7.72–7.43(m,10H),7.27(t,J=7.4Hz,1H),6.44(s,1H),6. 04(t,J=9.2Hz,1H),5.82(d,J=7.0Hz,1H),5.63(d,J=7.1Hz,1H),5.50(s,1H),5.01(d,J=9.5Hz,1H),4.68(s,2H),4.47(s,2H),4.38 –4.08(m,6H),3.80(d,J=7.0Hz,1H),3.43–3.39(m,2H),3.28–3.17(m,4H),3.07–2.82(m,8H),2.82–2.73(m,2H),2.53–2.45(m,1H), 2.39(s,3H),2.32–2.28(m,3H),2.17(s,3H),2.07–2.06(m,1H),1.92(s,3H),1.88–1.02(m,67H),0.94(s,6H),0.76(d,J=4.4Hz,6H). 13C NMR(101MHz,MeOD)δ203.93,175.66,171.27,170.30,170.17,169.18,169. 07,166.34,141.09,136.90,134.05,133.36,131.69,129.93,129.84,128.8 1,128.46,128.43,128.31,128.27,127.44,127.33,127.19,125.59,84.52,81.17,80.76,79.89,77.63,76.78,75.53,74.85,71.57,71.00,57.82,54. 16,46.45,46.07,45.91,43.21,43.18,42.25,41.87,39.21,36.66,36.12,35.69,35.46,35.08,34.14,33.81,33.36,33.08,32.84,32.57,32.29,31.8 5,31.68,30.36,29.38,27.58,27.41,26.41,26.03,25.62,24.61,22.55,22.10,21.81,21.04,19.59,16.93,13.77,13.43,13.14,11.65,11.60,9.15.

[0126] Compound I-14b: white powder, yield 41%. 1H NMR(400MHz,Methanol-d4)δ8.11(d,J=7.6Hz,2H),7.87–7.81(m,2H),7.70–7.45(m,10H),7.27(t,J=7.4Hz,1H),6.44(s,1H),6.05(t,J=9.2Hz,1H),5.83(d,J=6.7Hz,1H),5.63(d,J=7.1Hz,1H),5.49(d,J=6.5Hz,1H),5.00(d,J=9.4Hz,1H),4.69(s,2H),4.35–4.27(m,3H),4.18(s,2H),3.80(d,J=7.1Hz,1H),3.40(s,4H),3.26–3.17(m,4H),3.00–2.89(m,6H),2.77(t,J=6.8Hz,2H),2.52–2.44(m,1H),2.39(s,3H),2.31–2.25(m,3H),2.19–2.12(m,6H),1.93–1.79(m,19H),1.65(s,3H),1.54–1.28(m,36H),1.14–1.07(m,12H),0.99–0.92(m,9H),0.75(s,6H). 13CNMR(101MHz,MeOD)δ203.91,175.74,175.57,173.17,171.78,171.61,171.24,170.53, 170.27,170.17,169.17,169.00,166.31,141.12,138.56,136.92,134.04,133.44,133.3 5,131.70,129.95,129.85,128.83,128.44,128.32,128.27,127.75,127.49,127.37,127.24,84.50,81.49,80.75,79.47,77.63,76.10,75.55,74.87,73.62,71.55,71.02,70.81 ,57.86,57.82,54.20,46.43,46.10,45.95,43.21,43.18,42.36,38.55,36.75,36.14,35.86,35.72,35.15,34.94,33.98,33.85,33.66,33.34,33.12,32.78,32.57,32.35,31.77 ,30.34,30.24,29.40,28.58,27.49,27.34,27.06,26.40,26.11,25.66,24.79,23.56,22.67,22.40,22.14,22.00,21.08,19.63,19.55,16.91,13.80,13.46,11.73,11.70,9.16.

[0127] Embodiment 11 Solubility determination of molecular umbrella compounds

[0128] This method uses ultraviolet spectrophotometry to determine the solubility of I-14a and I-14b.

[0129] Selection of measurement wavelength: First, prepare a low concentration of I-14a I-14b solution, place it in a UV spectrophotometer, measure its absorption curve, select a wavelength with better absorption for subsequent experiments, and finally determine the absorption wavelength to be 230nm.

[0130] Drawing of I-14a standard curve: accurately prepare the mother solution of I-14a, dilute it step by step to obtain standard solutions of 10.4, 15.6, 20.8, 26, 31.2, 52, 104, and 208 μg / mL, place it in an ultraviolet spectrophotometer, measure its absorbance at a wavelength of 230 nm, and draw a standard curve with absorbance as the ordinate and concentration as the abscissa, and calculate the regression equation.

[0131] Table 1 Standard curve determination of compound I-14a

[0132]

[0133] The linear equation between absorbance and I-14a concentration was established, and we got y=0.0084x+0.0386, R 2 =0.9958, indicating that the linear relationship between absorbance and concentration is good within the concentration range of 10.2μg / mL to 208μg / mL, which meets the test requirements. The standard curve is shown in Figure 1 .

[0134] Drawing of I-14b standard curve: accurately prepare the mother solution of I-14b, dilute it step by step to get standard solutions of 12.3, 18.45, 24.6, 30.75, 36.9, 61.5, 123, and 246 μg / mL, place it in a UV spectrophotometer, measure its absorbance at a wavelength of 230 nm, and draw a standard curve with absorbance as the ordinate and concentration as the abscissa, and calculate the regression equation.

[0135] Table 2 Standard curve determination of compound I-14b

[0136]

[0137] The linear equation between absorbance and I-14b concentration was established, and we got y=0.0099x+0.1665, R 2 =0.9993, indicating that the linear relationship between absorbance and concentration is good within the concentration range of 12.2μg / mL to 244μg / mL, which meets the test requirements. The standard curve is shown in Figure 2 .

[0138] Determination of accurate solubility of I-14a b: put an excess of I-14a b into 0.5 mL of distilled water until solids precipitate in the solution and the solids do not dissolve after ultrasound, filter to obtain a high-concentration mother solution, dilute the mother solution step by step until the absorbance of the solution is within the range of the standard curve, and measure the absorbance to calculate the solubility. The solubility of I-14a was determined to be 243 mg / mL, equivalent to 82 mg / mL of paclitaxel; the solubility of I-14b was 90 mg / mL, equivalent to 33 mg / mL of paclitaxel. The solubility of both prodrugs in water is greater than 50 mg / mL, which is more than 5500 times higher than the 6 μg / mL of paclitaxel.

[0139] Example 12 In vitro GSH release assay of prodrug compounds

[0140] The HPLC method was used to determine the content of paclitaxel in the release medium solution through the standard curve method to reflect the degradation of the molecular prodrug. The relative release rate of the drug (%) = paclitaxel content in the release medium solution * 100% / theoretical release amount of paclitaxel.

[0141] Standard curve establishment: accurately prepare the standard mother solution of paclitaxel, and dilute it into 100.00, 50.00, 25.00, 12.50, 6.25, 3.12, 1.56μg / mL standard solution in sequence. Take 50μL for determination in HPLC, and draw the standard curve with the peak area as the ordinate and the concentration as the abscissa, and calculate the regression equation. The liquid phase conditions are Agilent 1260 high performance liquid chromatograph; chromatographic column: Agilent, Eclipse XDB-C18, 5μm, 4.6x 250mm; mobile phase: MeOH:H2O=75:25; detection wavelength: 230nm; flow rate: 0.8ml / min.

[0142] Table 3 Paclitaxel standard curve determination

[0143]

[0144] A linear equation was established between the peak area and the concentration of paclitaxel, and y = 69.018x-79.924R was obtained. 2 =0.9998, indicating that the peak area and concentration have a good linear relationship in the concentration range of 1.5625μg / mL to 200μg / mL, which meets the test requirements. The standard curve is shown in Figure 3 .

[0145] Paclitaxel release curve establishment: Prepare 10mM DTT PBS solution (containing 30% methanol V / V), add 1.31mg I-14a or 1.26mg I-14b, and react in a 37°C water bath. Take 200μL samples at 0.5h, 1h, 2h, 3h, 4h, 6h, 8h, 10h, 12h, 24h, and 48h, add 200μL chromatographic ethanol to the sample, filter, and perform real-time liquid phase monitoring (liquid phase conditions are the same as those for drawing the standard curve). Calculate the paclitaxel content in the solution according to the standard curve equation, and draw the paclitaxel release time curve, see Figure 4 .

[0146] from Figure 4It can be seen that in the reducing environment of 10mM DTT, both prodrugs showed controllable paclitaxel release behavior. Among them, I-14a showed a trend of sustained slow release within 48 hours, and the final release rate was about 60%; while I-14b showed a faster initial release rate in the first 8 hours, reaching about 30% and then tending to stabilize, and the cumulative release rate at 48 hours was about 35%. The two prodrugs showed different release kinetics: I-14a had a sustained sustained release effect, while I-14b showed a plateau after rapid release. This result verified the rationality of the prodrug molecular design and confirmed that the disulfide bond could be broken in a reducing environment to release paclitaxel; it showed that the prodrug system could respond to reducing stimulation, which matched the high GSH microenvironment in tumor cells and provided a basis for achieving selective drug release at the tumor site; the different release behaviors of the two prodrugs provided options for clinical applications: I-14a is suitable for situations where continuous administration is required, while I-14b is suitable for treatments that require rapid onset. The feasibility and superiority of the prodrug system of the present invention as a paclitaxel delivery carrier were further confirmed by the study of release behavior.

[0147] Embodiment 13 In vitro ROS release assay of prodrug compounds

[0148] The HPLC method was used to determine the content of paclitaxel in the release medium solution through the standard curve method to reflect the degradation of the molecular prodrug. The relative release rate of the drug (%) = paclitaxel content in the release medium solution * 100% / theoretical release amount of paclitaxel.

[0149] Standard curve establishment: same as Example 11

[0150] Paclitaxel release curve establishment: Prepare 10mM H2O2 PBS solution (containing 30% methanol V / V), add 1.26mg I-14a or 1.40mg I-14b, and react in a 37°C water bath. Take 200μL samples at 0.5h, 1h, 2h, 3h, 4h, 6h, 8h, 10h, 12h, and 24h, add 200μL chromatographic ethanol to the sample, filter, and monitor the sample in real time by liquid phase (liquid phase conditions are the same as those for drawing the standard curve). Calculate the paclitaxel content in the solution according to the standard curve equation, and draw the paclitaxel release time curve, see Figure 5 .

[0151] like Figure 5As shown in the figure, both prodrugs showed high paclitaxel release efficiency in the oxidative environment of 10mM H2O2. Among them, I-14a and I-14b showed rapid release characteristics in the first 2 hours, with a release rate of about 40%; then continued to release stably within 2-24 hours, and finally the cumulative release rate of I-14a reached about 90%, and I-14b reached about 80%. This result confirms that the prodrug can effectively release paclitaxel in an oxidative environment, and verifies the responsiveness of the molecular design to the high ROS microenvironment in tumor cells; both prodrugs can achieve a high cumulative release rate (80-90%) under oxidative conditions, which is significantly higher than the release rate under a reducing environment (35-60%), indicating that oxidative stress may be a more effective triggering mechanism for drug release; the prodrug is responsive to both reducing (GSH) and oxidative (ROS) stimulation, and this synergistic effect is conducive to more efficient drug release in the tumor microenvironment.

[0152] Combination Figure 4 and Figure 5 The results fully confirmed that the prodrug system designed in the present invention can respond to the characteristic stimulation of the tumor microenvironment (high GSH and high ROS) and achieve targeted drug release, which provides an important basis for improving the therapeutic effect of paclitaxel and reducing systemic toxicity.

[0153] Embodiment 14 Evaluation of the anti-cell proliferation activity of prodrug compounds in vitro

[0154] The MTT method was used to evaluate the anti-cell proliferation activity of the four prepared molecular umbrella prodrugs (I-13a, I-13b, I-14a, I-14b). The cells in the logarithmic growth phase were configured as a suspension with a cell density of 50000 / ml, 100μL per well, and evenly inoculated in a 96-well plate. After addition, it was placed in a 5% CO2, 37°C cell culture incubator for incubation. After the cells grew attached to the wall, the cells were treated with different concentrations of compounds, and a DMSO negative control group was set up for 72h. The culture was terminated, and 10μL of 5% MTT solution was added to each well and continued to be cultured for 4h, then the supernatant was carefully discarded, 150μL DMSO was added, and it was placed on a shaker for full shaking to fully dissolve the crystals. The absorbance OD of each well was measured at 570nm by an enzyme label analyzer, the cell survival rate was calculated, and the cell viability curve was drawn according to the drug concentration.

[0155] Cell viability % = (OD sample ―OD blank ) / (OD control ―OD blank )

[0156] like Figure 6 , Figure 7As shown in the figure, different concentrations of paclitaxel and prodrug molecules were co-incubated with tumor cells human large cell lung cancer cells (H460), human prostate cancer cells (PC3), human non-small cell lung cancer cells (A549), human liver cancer cells (HepG2), human breast cancer cells (MCF-7), normal cells human bronchial epithelial cells (HBE), human intravenous endothelial cells (HUVEC), and bronchial epithelial cells (Beas-2b) for 72 hours, and the cell survival rate was determined by MTT method. The experimental results showed that the anti-cell proliferation activity of paclitaxel and the four prodrugs showed concentration dependence, and for tumor cells, the efficacy of the molecular umbrella prodrug was slightly lower than that of paclitaxel, but the difference was not significant, indicating that the prodrug design did not significantly affect the anti-tumor activity of the drug. In three normal cells HUVEC (human intravenous endothelial cells), HBE (human bronchial epithelial cells) and Beas-2b (bronchial epithelial cells), the toxicity of the four prodrugs to cells was significantly lower than that of free paclitaxel. At the same concentration, the cell survival rate of the prodrug treatment group was significantly higher than that of the PTX group, especially at higher concentrations. The above results confirm that the prodrug system can maintain the anti-tumor activity of paclitaxel while significantly reducing the toxicity to normal cells, achieving the goal of selective enhancement.

[0157] Embodiment 15 In vivo tumor inhibition experiments of molecular umbrella prodrugs I-14a and I-14b

[0158] Female nude mice aged 6-8 weeks and weighing 18-20 g were used.

[0159] (1) 0.1 mL of human breast cancer cell (MCF-7) suspension (1*10^7 cells / mL) was inoculated under the right forelimbs of two female nude mice. When the tumor grew to a sufficient size, the mice were killed by cervical dislocation to remove the tumor tissue, which was then crushed into pieces of approximately 2 mm*2 mm.

[0160] (2) Tumor fragments were implanted in the right forelimb of female nude mice. 3 At around 24 hours, the tumor-bearing mice were randomly divided into 7 groups, with 6 mice in each group, and the drugs were administered through the tail vein.

[0161] (3) At the time of drug administration, the mice in the blank group were injected with 100 μL PBS solution through the tail vein, the mice in the low-dose paclitaxel group were injected with 100 μL paclitaxel solution (10 mg / kg, 3 mg PTX dissolved in 75 μL DMSO, 675 μL PEG, 750 μL PBS solution) through the tail vein, the mice in the low-dose paclitaxel group were injected with 100 μL paclitaxel solution (20 mg / kg, 6 mg PTX dissolved in 75 μL DMSO, 675 μL PEG, 750 μL PBS solution) through the tail vein, the mice in the low-dose I-14a group were injected with 100 μL I-14a solution (30 mg / kg, 9 mg dissolved in 1.5 mL PBS solution) through the tail vein, and the mice in the high-dose I-14a group were injected with 100 μL I-14a solution (60 mg / kg, 18 mg dissolved in 1.5 mL PBS solution), I-14b low-dose group mice tail vein injection of I-14b solution (27.3mg / kg, 8.19mg dissolved in 1.5mL PBS solution) and I-14a high-dose group mice tail vein injection of I-14b solution (54.6mg / kg, 16.5mg dissolved in 1.5mL PBS solution), once every four days, three times in a row. The survival of the mice was observed every day, and the tumor volume and mouse weight were measured on days 0, 4, 8, 12, 13, 14, and 16. The calculation formula for tumor volume is: V = L (long diameter) * W 2 (short diameter) / 2.

[0162] (4) After the experiment, the tumor-bearing mice were killed by dislocating the neck. The tumor tissues of the mice were removed, photographed, and fixed with 4% paraformaldehyde solution. The fixed tissues were sliced, stained with PCNA, and observed and photographed under a microscope.

[0163] pass Figures 8 to 12 It can be seen that compared with the control group, both high-dose and low-dose paclitaxel and I-14a and I-14b showed tumor growth inhibition activity. The prodrug group I-14a had the same tumor inhibition effect as the equal-dose paclitaxel group, but its safety was significantly higher than that of the paclitaxel group; the prodrug group I-14b had the same tumor inhibition efficiency as the high-dose paclitaxel group at low doses, but the toxicity was very small; at high doses, although the tumor inhibition effect was significantly reduced, its toxicity was also greatly increased. In vivo tumor inhibition experiments have shown that molecular umbrellas can be used as paclitaxel delivery bodies to improve the selectivity of paclitaxel, and the tumor inhibition effect is equivalent to that of paclitaxel, and can be used as candidate drugs for further research.

[0164] To further demonstrate the anti-tumor effect and safety of prodrugs I-14a and I-14b, tissue sections were prepared from the tumor tissues of mice and immunohistochemical analysis was performed. Proliferating cell nuclear antigen (PCNA) staining also showed that the proportion of PCNA-positive (brown) cells in the tumor tissues of the paclitaxel group and the prodrug group was significantly reduced compared with the saline group ( Fig.13 ), and the PCNA brown color of low-dose I-14b was significantly lower than that of equal-dose PTX and I-14a, indicating that the anti-tumor effect of I-14b was indeed improved. Therefore, the above results all show that the obtained prodrugs I-14a and I-14b have significant anti-tumor activity and low toxic side effects.

[0165] The above in vivo experimental results fully prove that the paclitaxel molecular umbrella prodrug developed by the present invention, especially I-14b, significantly reduces systemic toxicity while maintaining good anti-tumor activity, has excellent safety, and provides a new choice for the clinical application of paclitaxel.

[0166] The above description is only the preferred embodiment of the present application and is not intended to limit the present application. Although the present application is described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. A paclitaxel molecular umbrella delivery system prodrug, characterized in that: include: The target head of the molecular umbrella is composed of two surface amphiphilic molecules connected by spermidine; Paclitaxel drug portion; as well as A linking group, which connects the molecular umbrella target head portion to the paclitaxel drug portion via a disulfide bond; Wherein, the surface amphiphilic molecule is selected from at least one of the following compounds: cholic acid, sodium cholic acid sulfonate, deoxycholic acid and sodium deoxycholic acid sulfonate.

2. The paclitaxel molecular umbrella delivery system prodrug according to claim 1, characterized in that: The paclitaxel drug moiety is linked to the linker group via the 2'-OH site.

3. The paclitaxel molecular umbrella delivery system prodrug according to claim 1, characterized in that: The linking group has a structure shown in Formula III:

4. The paclitaxel molecular umbrella delivery system prodrug according to any one of claims 1 to 3, characterized in that: It has one of the following structures: Wherein, R1 is selected from -OH and -H; R2 is selected from -OSO3Na and -H.

5. The paclitaxel molecular umbrella delivery system prodrug according to any one of claims 1 to 4, characterized in that: Selected from the following compounds:

6. A method for preparing the paclitaxel molecular umbrella delivery system prodrug according to any one of claims 1 to 4, characterized in that: include: (1) causing mercaptopropionic acid and 2,2-dipyridyldisulfide to undergo a substitution reaction to generate 3-(pyridin-2-yldisulfide)propionic acid; (2) allowing 3-(pyridin-2-yldisulfide)propionic acid to undergo an esterification reaction with the 2'-OH group of paclitaxel to obtain compound I-4; (3) Compound I-4 is subjected to a substitution reaction with dithiothreitol to obtain compound I-5; (4) ester condensing cholic acid or deoxycholic acid with NHS to obtain compound I-7; (5) Compound I-7 is subjected to a substitution reaction with spermidine to obtain compound I-8; (6) esterifying 3-hydroxy-1,2,3-benzotriazine-4(3H)-one with 3-(pyridin-2-yldisulfide)propionic acid to obtain compound I-11; (7) subjecting compound I-8 and compound I-11 to a substitution reaction to obtain compound I-9; (8) Compound I-9 is oxidized with sulfur trioxide pyridine to obtain compound I-12; (9) subjecting Compound I-5 and Compound I-9 or Compound I-12 to a nucleophilic substitution reaction to obtain a paclitaxel molecular umbrella delivery system prodrug; Among them, the structures of compounds I-4, I-5, I-7, I-8, I-9, I-11, and I-12 are shown below: Wherein, R1 is selected from -OH and -H.

7. A pharmaceutical composition, characterized in that A paclitaxel molecular umbrella delivery system prodrug comprising any one of claims 1 to 5.

8. A pharmaceutical preparation, characterized in that The invention comprises the paclitaxel molecular umbrella delivery system prodrug according to any one of claims 1 to 5 or the pharmaceutical composition according to claim 7, and at least one pharmaceutically acceptable excipient or carrier.

9. Use of the paclitaxel molecular umbrella delivery system prodrug according to any one of claims 1 to 5 or the pharmaceutical composition according to claim 7 in the preparation of anti-tumor drugs.

10. The use according to claim 7, characterized in that: The anti-tumor drug is used to treat the following cancers: breast cancer, ovarian cancer, pancreatic cancer, lung cancer, and head and neck cancer; Preferably, the prodrug has the following properties under physiological conditions: Release active drugs in the high GSH / ROS microenvironment of tumor cells; Remains in the prodrug state in normal cells.

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