Paclitaxel molecular umbrella delivery system prodrug, and preparation method and application thereof

Abstract

The application provides a paclitaxel molecular umbrella delivery system prodrug, a preparation method and application thereof. The paclitaxel molecular umbrella delivery system prodrug comprises a molecular umbrella target head part composed of two surface amphiphilic molecules connected by spermidine, a paclitaxel drug part, and a connecting group connecting the molecular umbrella target head part and the paclitaxel drug part through a disulfide bond; wherein the surface amphiphilic molecule is selected from cholic acid, deoxycholic acid or a sodium sulfonate thereof. Compared with paclitaxel, the paclitaxel molecular umbrella delivery system prodrug has significantly improved water solubility, lower toxicity to normal cells than free paclitaxel, and significantly improved safety; and the paclitaxel molecular umbrella delivery system prodrug has the following characteristics in a physiological environment: releasing active drugs in the high GSH / ROS microenvironment of tumor cells; and maintaining a prodrug state in normal cells.

Description

Technical Field

[0001] This application relates to the pharmaceutical field, specifically to a prodrug of a paclitaxel molecular umbrella delivery system, its preparation method, and its application. Background Technology

[0002] Any discussion of prior art throughout the specification should not be construed as an admission that such prior art is well-known or constitutes part of common general knowledge in the art.

[0003] Cancer remains one of the leading causes of death worldwide. Currently, the main treatments for cancer are radiotherapy and chemotherapy, with chemotherapy being the most effective. However, chemotherapy drugs often have poor selectivity and biocompatibility, and some can even lead to drug resistance, causing serious toxic side effects for patients.

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

[0005]

[0006] The prodrug theory is currently widely used in chemotherapy drugs. By blocking the essential active groups, the toxicity of chemotherapy drugs is reduced, thereby decreasing their side effects and increasing their targeting. Therefore, designing and synthesizing paclitaxel prodrug molecules to increase the water solubility and targeting of paclitaxel, and overcoming paclitaxel resistance, has significant clinical implications. Summary of the Invention

[0007] The purpose of this invention is to overcome the technical problems of existing paclitaxel drugs, such as poor water solubility, low selectivity, and significant toxicity, and to provide a paclitaxel molecular umbrella delivery system prodrug and its preparation method. This invention constructs a molecular umbrella carrier by linking two surface amphiphilic molecules (cholic acid, deoxycholic acid, and their sodium sulfonate salts) through spermidine, and uses disulfide bonds to connect the 2'-OH site of paclitaxel to the molecular umbrella target, designing and synthesizing a series of intelligent responsive prodrug molecules. This prodrug selectively releases the active drug paclitaxel in the high GSH / ROS microenvironment of tumor cells, while maintaining the prodrug state in normal cells. Experimental results show that the prodrug of this invention significantly improves the water solubility of paclitaxel (by more than 5500 times), reduces toxicity to normal cells while maintaining good antitumor activity; in vivo experiments have confirmed its antitumor effect, especially that some compounds can achieve the antitumor effect of high-dose paclitaxel at low doses, and significantly improve safety. This invention provides a new drug delivery strategy for improving the clinical application of paclitaxel drugs.

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

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

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

[0011] Paclitaxel drug portion; and

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

[0013] The surface amphiphilic molecule is selected from at least one of the following compounds: cholic acid, sodium cholate, deoxycholic acid, and sodium deoxycholate. The two surface amphiphilic molecules may be the same or different, preferably the same.

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

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

[0016]

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

[0018]

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

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

[0021]

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

[0023] (1) A substitution reaction is made between mercaptopropionic acid and 2,2-dipyridine disulfide to generate 3-(pyridin-2-yldithio)propionic acid (i.e., compound I-3);

[0024]

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

[0026]

[0027] (3) Compound I-4 was substituted with dithiothreitol to obtain compound I-5;

[0028]

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

[0030]

[0031] (5) Compound I-7 was substituted with spermidine to obtain compound I-8;

[0032]

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

[0034]

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

[0036]

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

[0038]

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

[0040]

[0041] 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 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 performed 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 performed under DIEA and DMF conditions.

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

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

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

[0052] This pharmaceutical composition can be used to treat tumors such as breast cancer, ovarian cancer, pancreatic cancer, lung cancer, and head and neck cancer. Because the prodrug of this invention has good water solubility and stability, it can significantly simplify formulation design. In some embodiments, it can be used in combination with other antitumor drugs according to clinical needs, providing a more flexible treatment plan.

[0053] In a fourth aspect of the invention, a pharmaceutical formulation is provided comprising 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, infusion, or lyophilized powder for injection, or an oral dosage form such as a tablet or capsule.

[0055] In some embodiments, the pharmaceutically acceptable excipients or carriers may be selected as needed, including, for example: water for injection, physiological saline, glucose injection or phosphate buffer as a solvent or carrier; antioxidants, pH adjusters or buffers as stabilizers; sodium chloride, glucose or mannitol as isotonic adjusters; and preservatives such as benzyl alcohol or parabens may be added if necessary.

[0056] Because the prodrug of this invention has significantly improved water solubility, there is no need for a complex solubilization system in the formulation design, a simple preparation process can be used, and high bioavailability is ensured. It is convenient to use and has good safety.

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

[0058] In some embodiments of the present invention, the antitumor 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 prodrug of the paclitaxel molecular umbrella delivery system has the following characteristics under physiological conditions:

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

[0061] It maintains the prodrug state in normal cells.

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

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

[0064] Its toxicity to normal cells is far lower than that of free paclitaxel.

[0065] Compared to existing technologies, the advantages of this invention include:

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

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

[0068] Regarding antitumor activity, in vitro experiments showed that this prodrug exhibited good inhibitory activity against various tumor cell lines (including MCF7, PC3, HepG2, H460, A549, etc.), while significantly reducing toxicity to normal cells (HUVEC, HBE, Beas-2b). In vivo experiments further confirmed its excellent efficacy and safety. For example, I-14a showed comparable tumor-suppressing effects to an equivalent dose of paclitaxel but with higher safety; in particular, I-14b at low doses achieved the same tumor-suppressing effect as high doses of paclitaxel, while significantly reducing toxicity to normal cells, demonstrating a good safety advantage. PCNA staining results also confirmed that the inhibitory effect of low-dose I-14b was superior to that of an equivalent dose of paclitaxel.

[0069] Furthermore, the prodrug of this application also possesses significant advantages in its preparation process. Its synthetic route is clear, the reaction conditions are mild, the yield is good, and it is suitable for large-scale production. In terms of molecular design, the molecular umbrella structure provides excellent water solubility, disulfide bonds enable intelligent responsive release, and the structure exhibits good modifiability, facilitating further optimization and improvement. These advantages collectively constitute the innovative value of the prodrug of this application, providing a new option for the clinical application of paclitaxel. Attached Figure Description

[0070] The accompanying drawings, which form part of this application, are used to provide a further understanding of this application. The illustrative embodiments and descriptions of this application are used to explain this application and do not constitute an undue limitation of this application. The embodiments of this application will be described in detail below with reference to the accompanying drawings, wherein:

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

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

[0073] Figure 3 : Paclitaxel standard curve.

[0074] Figure 4 Paclitaxel release curve under 10mM DTT conditions.

[0075] Figure 5 Paclitaxel release curve under 10mM H2O2 conditions.

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

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

[0078] Figure 8 Dosing regimen for tumor-bearing mice in in vivo tumor suppression experiments.

[0079] Figure 9 Changes in body weight of mice in each group during in vivo tumor suppression experiments.

[0080] Figure 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 an in vivo tumor suppression experiment.

[0081] Figure 11 Tumor weight of mice in each group during in vivo tumor suppression experiments.

[0082] Figure 12 : Tumor images of mice in each group during the in vivo tumor suppression experiment (dashed circles represent complete tumor disappearance, crosses represent mouse death). Statistical significance: *p<0.05, **p<0.01, ***p<0.001.

[0083] Figure 13 PCNA staining results in tumor tissue. Detailed Implementation

[0084] The present application is further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the application. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions or as recommended by the manufacturer.

[0085] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of skill in the art. All reagents and materials used in this application are readily available through conventional means, and unless otherwise specified, they are used in accordance with conventional methods in the art or as per the product instructions. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to the methods of this application. The preferred embodiments and materials described herein are for illustrative 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 ice bath conditions. The reaction was allowed to proceed at room temperature. After the reaction was confirmed to be complete by TLC, the reaction solution was concentrated under reduced pressure and subjected to column chromatography with petroleum ether:ethyl acetate ratios of 50:1–25:1–10:1 to obtain a clear oily substance (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-yldithio)propionic acid (70 mg, 0.325 mmol) was dissolved in dichloromethane (10 mL). Paclitaxel (185 mg, 0.217 mmol) and DCC (268.64 mg, 1.3 mmol) were added under ice bath conditions. The mixture was reacted at room temperature for 16 h. The precipitate was filtered off, washed with a small amount of dichloromethane, and the filtrate was concentrated under reduced pressure. Column chromatography with DCM:MeOH = 100:1 yielded a white solid (66.8 mg, 30%). 1H NMR (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 three times with water, the organic phase was dried, concentrated under reduced pressure, and then subjected to column chromatography (DCM:MeOH = 100:1) to give 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 cholic acid and deoxycholic acid active esters 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 off, and the filtrate was concentrated under reduced pressure. Column chromatography (DCM:MeOH = 50:1) yielded I-7a and I-7b, both white solids. The yield of I-7a was 96%, and the yield of I-7b was 69%. The compounds were directly added to the next 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), and spermidine (14.525 μL, 0.1 mmol) and DIPEA (52.52 μL, 0.3 mmol) were added. The mixture was stirred at room temperature for 16 h. A white precipitate was formed upon addition of saturated NaHCO3 solution. The white precipitate was filtered, dissolved in methanol, concentrated under reduced pressure, and subjected to column chromatography (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-yldithio)propionic acid (200 mg, 0.93 mmol) was dissolved in dichloromethane, and HOOBt (182.67 mg, 1.12 mmol) was added. DCC (231.1 mg, 1.12 mmol) was added at 0 °C, and the mixture was reacted at room temperature for 6 h. The precipitate was filtered, washed with a small amount of dichloromethane, and the filtrate was concentrated under reduced pressure. Column chromatography (DCM:MeOH = 100:1) yielded 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] I-8a or I-8b (95 mg, 0.1 mmol) was dissolved in 5 mL of DMF. Compound I-11 (37 mg, 0.1 mmol) and DIPEA (70 μL, 0.4 mmol) were added to the reaction flask. The mixture was stirred at room temperature for 12 h, concentrated under reduced pressure, and column chromatography (DCM:MeOH = 5:1) was used to obtain I-9a or I-9b, both of which were white powders.

[0109] Compound I-9a: white powder, yield 46%. ¹H NMR (400 MHz, methanol-d⁴): δ 8.41 (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] Dissolve I-9a or I-9b (68 mg, 0.06 mmol) in DMF, cool to 0 °C, add pyridine trioxide (385.28 mg, 2.42 mmol), raise to room temperature and stir. After reacting for 24 h, add a small amount of water to the reaction flask, add saturated sodium bicarbonate to adjust the pH to 10, concentrate under reduced pressure at low temperature, and perform column chromatography (DCM:MeOH = 5:1-2:1) to obtain I-12a or I-12b, both of which are 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 compounds I-13a or I-13b

[0117]

[0118] Compound I-9a or I-9b (50 mg, 0.044 mmol) was dissolved in methanol, and compound I-5 (42 mg, 0.044 mmol) was added. The mixture was reacted at room temperature for 12 h. The reaction solution was concentrated under reduced pressure and column chromatography was performed to obtain I-13a or I-13b, both of which were 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] + Found: 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 compounds 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 mixture was reacted at room temperature for 12 h. The reaction solution was concentrated under reduced pressure and column chromatography (DCM:MeOH = 2:1) was used 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] Example 11 Determination of the solubility of molecular umbrella compounds

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

[0129] Selection of wavelength for measurement: First, prepare low-concentration I-14a and I-14b solutions, place them in an ultraviolet spectrophotometer, measure their absorption curves, select the wavelength with better absorption for subsequent experiments, and finally determine the absorption wavelength as 230 nm.

[0130] Plotting the I-14a standard curve: The stock solution of I-14a was precisely prepared and serially diluted to obtain standard solutions of 10.4, 15.6, 20.8, 26, 31.2, 52, 104, and 208 μg / mL. The absorbance was measured at 230 nm using a UV spectrophotometer. A standard curve was plotted with absorbance on the ordinate and concentration on the abscissa, and the regression equation was calculated.

[0131] Table 1. Determination of the standard curve for compound I-14a

[0132]

[0133] A linear equation was established between absorbance and I-14a concentration, yielding y = 0.0084x + 0.0386, R0 2 =0.9958, indicating a good linear relationship between absorbance and concentration within the concentration range of 10.2 μg / mL to 208 μg / mL, which meets the testing requirements. The standard curve is shown below. Figure 1 .

[0134] Plotting the I-14b standard curve: The stock solution of I-14b was precisely prepared and serially diluted to obtain standard solutions of 12.3, 18.45, 24.6, 30.75, 36.9, 61.5, 123, and 246 μg / mL. The absorbance was measured at 230 nm using a UV spectrophotometer. A standard curve was plotted with absorbance on the ordinate and concentration on the abscissa, and the regression equation was calculated.

[0135] Table 2. Determination of the standard curve for compound I-14b

[0136]

[0137] A linear equation was established between absorbance and I-14b concentration, yielding y = 0.0099x + 0.1665, R0 2 =0.9993, indicating a good linear relationship between absorbance and concentration within the concentration range of 12.2 μg / mL to 244 μg / mL, which meets the testing requirements. The standard curve is shown below. Figure 2 .

[0138] Determination of accurate solubility of I-14a and I-14b: Excess I-14a and I-14b were added to 0.5 mL of distilled water until solid precipitated and did not dissolve after sonication. The solution was filtered to obtain a high-concentration mother liquor. The mother liquor was serially diluted until the absorbance of the solution was within the range of the standard curve. The absorbance was measured 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 was greater than 50 mg / mL, representing an increase of more than 5500 times compared to the 6 μg / mL solubility of paclitaxel.

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

[0140] The degradation of the molecular umbrella prodrug was reflected by determining the content of paclitaxel in the release medium solution using HPLC and the standard curve method. The relative drug release rate (%) = paclitaxel content in the release medium solution * 100% / theoretical paclitaxel release amount.

[0141] Standard curve establishment: A precise standard stock solution of paclitaxel was prepared and sequentially diluted to 100.00, 50.00, 25.00, 12.50, 6.25, 3.12, and 1.56 μg / mL standard solutions. 50 μL of each solution was analyzed in HPLC, and a standard curve was plotted with peak area on the ordinate and concentration on the abscissa. The regression equation was calculated. The HPLC conditions were: Agilent 1260 HPLC system; column: Agilent Eclipse XDB-C18, 5 μm, 4.6 x 250 mm; mobile phase: MeOH:H2O = 75:25; detection wavelength: 230 nm; flow rate: 0.8 mL / min.

[0142] Table 3. Determination of Paclitaxel Standard Curve

[0143]

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

[0145] Establishment of paclitaxel release curve: Prepare 10 mM DTT PBS solution (containing 30% methanol v / v), add 1.31 mg I-14a or 1.26 mg I-14b, and react in a 37℃ water bath. Take 200 μL samples at 0.5 h, 1 h, 2 h, 3 h, 4 h, 6 h, 8 h, 10 h, 12 h, 24 h, and 48 h, respectively. Add 200 μL of chromatographic ethanol to the sample, filter, and perform real-time HPLC monitoring (HPLC conditions are the same as for standard curve plotting). Calculate the paclitaxel content in the solution according to the standard curve equation, and plot the paclitaxel release time curve. See [reference needed]. Figure 4 .

[0146] from Figure 4As can be seen, both prodrugs exhibited controllable paclitaxel release behavior under the reducing environment of 10 mM DTT. I-14a showed a sustained, slow release trend over 48 hours, with a final release rate of approximately 60%; while I-14b showed a faster initial release rate in the first 8 hours, reaching approximately 30% before stabilizing, with a cumulative release rate of approximately 35% at 48 hours. The two prodrugs exhibited different release kinetics: I-14a had a sustained-release effect, while I-14b showed a plateau phase after rapid release. These results validate the rationality of the prodrug molecule design, confirming that disulfide bonds can break under reducing conditions to release paclitaxel; indicating that the prodrug system can respond to reducing stimuli, which matches the high GSH microenvironment within tumor cells, providing a basis for selective drug release at tumor sites; the different release behaviors of the two prodrugs provide a choice for clinical application: I-14a is suitable for situations requiring continuous administration, while I-14b is suitable for treatment regimens requiring rapid onset of action. The study of release behavior further confirmed the feasibility and superiority of the prodrug system of this invention as a paclitaxel delivery carrier.

[0147] Example 13 In vitro ROS release assay of prodrug compound

[0148] The degradation of the molecular umbrella prodrug was reflected by determining the content of paclitaxel in the release medium solution using HPLC and the standard curve method. The relative drug release rate (%) = paclitaxel content in the release medium solution * 100% / theoretical paclitaxel release amount.

[0149] Standard curve establishment: Same as in Example 11

[0150] Establishment of paclitaxel release curve: Prepare 10 mM H2O2 PBS solution (containing 30% methanol v / v), add 1.26 mg I-14a or 1.40 mg I-14b, and react in a 37℃ water bath. Take 200 μL samples at 0.5 h, 1 h, 2 h, 3 h, 4 h, 6 h, 8 h, 10 h, 12 h, and 24 h respectively, add 200 μL of chromatographic ethanol to the sample, filter, and perform real-time HPLC monitoring (HPLC conditions are the same as for standard curve plotting). Calculate the paclitaxel content in the solution according to the standard curve equation, and plot the paclitaxel release time curve, see [reference needed]. Figure 5 .

[0151] like Figure 5As shown, both prodrugs exhibited high paclitaxel release efficiency under an oxidizing environment of 10 mM H2O2. Specifically, I-14a and I-14b showed rapid release characteristics in the first 2 hours, with a release rate of approximately 40%; subsequently, stable release continued over 2-24 hours, with I-14a ultimately reaching a cumulative release rate of approximately 90% and I-14b approximately 80%. These results confirm that the prodrugs can effectively release paclitaxel under oxidizing conditions, validating the responsiveness of this molecular design to the high ROS microenvironment in tumor cells. Both prodrugs achieved high cumulative release rates (80-90%) under oxidizing conditions, significantly higher than the release rates under reducing conditions (35-60%), indicating that oxidative stress may be a more effective drug release triggering mechanism. The prodrugs showed responsiveness to both reducing (GSH) and oxidizing (ROS) stimuli, and this synergistic effect facilitates more efficient drug release in the tumor microenvironment.

[0152] Combination Figure 4 and Figure 5 The results fully demonstrate that the prodrug system designed in this invention can respond to the characteristic stimuli 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] Example 14 Evaluation of the in vitro anti-cell proliferation activity of prodrug compounds

[0154] The anti-cell proliferation activity of four prepared molecular umbrella prodrugs (I-13a, I-13b, I-14a, and I-14b) was evaluated using the MTT assay. Logarithmic growth phase cells were prepared into a suspension with a cell density of 50,000 / ml, and 100 μL was evenly seeded into each well of a 96-well plate. After addition, the plates were incubated in a 5% CO2 incubator at 37°C. After cell adhesion and growth, cells were treated with different concentrations of the compounds, with a DMSO negative control group included. The cells were cultured for 72 h. After termination of culture, 10 μL of 5% MTT solution was added to each well, and the cells were cultured for another 4 h. The supernatant was then carefully discarded, and 150 μL of DMSO was added. The plates were shaken thoroughly to dissolve any crystals. The absorbance (OD) of each well was measured at 570 nm using a microplate reader, and cell viability was calculated. Cell viability curves were plotted based on drug concentrations.

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

[0156] like Figure 6 , Figure 7As shown, different concentrations of paclitaxel and prodrug molecules were co-incubated with tumor cells (H460, PC3, A549, HepG2, MCF-7) and normal cells (HBE, HUVEC, and Beas-2b) for 72 hours. Cell viability was then determined by the MTT assay. The results showed that the anti-cell proliferation activity of paclitaxel and the four prodrugs was concentration-dependent. For tumor cells, the efficacy of the prodrugs 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 drugs. In the three normal cell types (HUVEC, HBE, and Beas-2b), the cytotoxicity of all four prodrugs was significantly lower than that of free paclitaxel. At the same concentration, the cell viability of the prodrug-treated group was significantly higher than that of the PTX group, especially at higher concentrations. These results confirm that the prodrug system can maintain the antitumor activity of paclitaxel while significantly reducing its toxicity to normal cells, achieving the goal of selective enhancement.

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

[0158] Female nude mice aged 6-8 weeks and weighing 18-20 grams 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 forelimb axilla of two female nude mice. After the tumor grew to a sufficient size, the mice were euthanized by cervical dislocation and the tumor tissue was removed. The tumor tissue was then crushed into fragments of about 2 mm*2 mm.

[0160] (2) The tumor fragments were implanted into the axilla of the right forelimb of a female nude mouse. The tumor was allowed to grow to 100 mm. 3 At approximately 10:00 AM, tumor-bearing mice were randomly divided into 7 groups of 6 mice each, and administered the drug via tail vein.

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

[0162] (4) After the experiment, the tumor-bearing mice were euthanized by cervical dislocation. The tumor tissue of the mice was removed, photographed, and fixed with 4% paraformaldehyde solution. The fixed tissue was sectioned, stained with PCNA, and observed and photographed under a microscope.

[0163] pass Figures 8 to 12 It was found that, compared with the control group, both high-dose and low-dose paclitaxel and I-14a and I-14b exhibited tumor growth inhibitory activity. The prodrug group I-14a showed the same tumor-suppressing effect as the equal-dose paclitaxel group, but its safety profile was significantly higher than that of the paclitaxel group. The prodrug group I-14b, at low doses, showed the same tumor-suppressing efficiency as the high-dose paclitaxel group, but with very low toxicity; at high doses, although the tumor-suppressing effect was significantly reduced, its toxicity was also greatly increased. In vivo tumor inhibition experiments demonstrated that the molecular umbrella can serve as a delivery system for paclitaxel, achieving improved selectivity, and its tumor-suppressing effect is comparable to that of paclitaxel, making it a potential candidate drug for further research.

[0164] To further demonstrate the antitumor efficacy and safety of prodrugs I-14a and I-14b, tumor tissue from mice was sectioned and subjected to immunohistochemical analysis. Proliferating cell nuclear antigen (PCNA) staining also showed that, compared with the saline group, the proportion of PCNA-positive (brown) cells in the tumor tissues of the paclitaxel group and the prodrug group was significantly reduced. Figure 13 Furthermore, the PCNA brown color of low-dose I-14b was significantly lower than that of equal doses of PTX and I-14a, indicating that the antitumor effect of I-14b was indeed improved. Therefore, all the above results show that the obtained prodrugs I-14a and I-14b have significant antitumor activity and low toxicity.

[0165] The above in vivo experimental results fully demonstrate that the paclitaxel molecular umbrella prodrug developed in this invention, especially I-14b, maintains good antitumor activity while significantly reducing systemic toxicity and has excellent safety, providing a new option for the clinical application of paclitaxel.

[0166] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A prodrug for a paclitaxel molecular umbrella delivery system, characterized in that, It has one of the following structures: R1 is selected from -OH and -H; R2 is selected from -OSO3Na and -H.

2. The prodrug of the paclitaxel molecular umbrella delivery system according to claim 1, characterized in that, Selected from the following compounds: 。 3. A method for preparing a prodrug of the paclitaxel molecular umbrella delivery system according to any one of claims 1-2, characterized in that, include: (1) A substitution reaction is made between mercaptopropionic acid and 2,2-dipyridine disulfide to generate 3-(pyridin-2-yldithio)propionic acid; (2) The esterification reaction of 3-(pyridin-2-yldithio)propionic acid with the 2'-OH of paclitaxel yields compound I-4; (3) Compound I-4 was substituted with dithiothreitol to obtain compound I-5; (4) Ester condensation of cholic acid or deoxycholic acid with NHS yields compound I-7; (5) Compound I-7 was subjected to a substitution reaction with spermidine to obtain compound I-8; (6) The esterification of 3-hydroxy-1,2,3-benzotriazine-4(3H)-one with 3-(pyridin-2-yldithio)propionic acid yields compound I-11; (7) Compound I-8 and compound I-11 were subjected to a substitution reaction to obtain compound I-9; (8) Compound I-9 was oxidized with pyridine trioxide to obtain compound I-12; (9) Compound I-5 and compound I-9 or compound I-12 undergo a nucleophilic substitution reaction to obtain a prodrug for the paclitaxel molecular umbrella delivery system; The structures of compounds I-4, I-5, I-7, I-8, I-9, I-11, and I-12 are shown below: ； R1 is selected from -OH and -H.

4. A pharmaceutical composition, characterized in that, A prodrug comprising the paclitaxel molecular umbrella delivery system according to any one of claims 1 to 2.

5. A pharmaceutical preparation, characterized in that, The drug comprises a prodrug of the paclitaxel molecular umbrella delivery system according to any one of claims 1 to 2 or the pharmaceutical composition according to claim 4, and at least one pharmaceutically acceptable excipient or carrier.

6. The use of the paclitaxel molecular umbrella delivery system prodrug of any one of claims 1 to 2 or the pharmaceutical composition of claim 4 in the preparation of an antitumor drug; said antitumor drug for the treatment of the following cancers: breast cancer, ovarian cancer, pancreatic cancer, lung cancer, and head and neck cancer.

7. The application according to claim 6, characterized in that, The prodrug has the following properties under physiological conditions: Release of active drugs in the high GSH / ROS microenvironment of tumor cells; It maintains the prodrug state in normal cells.

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