Hypoxia response paclitaxel prodrug, nano preparation and preparation method and application thereof

Abstract

The invention relates to a hypoxia response paclitaxel prodrug, a nano preparation as well as a preparation method and application thereof. The paclitaxel prodrug is a paclitaxel prodrug small molecule modified by a nitro derivative; the preparation method comprises the following steps: preparing the nano-drug by self-assembly of the paclitaxel prodrug, co-assembly of the paclitaxel prodrug and the photosensitizer or co-assembly of the paclitaxel prodrug and the polymer distearoyl phosphatidyl ethanolamine methoxy polyethylene glycol, so as to realize efficient loading and delivery of the drug and responsive release of the focus site. The prodrug obtained by the invention has a chemical structure for optimizing a connecting bond, can reduce the toxic and side effects of paclitaxel and quickly respond to a hypoxic microenvironment, and realizes quick and efficient release of an active drug in a tumor microenvironment.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and nanomedicine technology, specifically relating to a paclitaxel prodrug, nanoformulations, their preparation methods, and applications. More specifically, this invention relates to a hypoxia-responsive paclitaxel prodrug, nanoformulations based on this prodrug, their preparation methods, and their applications in antitumor therapy. Background Technology

[0002] Paclitaxel is a highly effective, broad-spectrum natural anticancer drug; however, its poor water solubility (<0.03 mg / mL) and strong crystallinity pose significant challenges to formulation development. For example, commercially available Taxol uses large amounts of polyoxyethylene castor oil and ethanol as solubilizers and co-solvents. This solubilization system easily causes severe allergic reactions, neurotoxicity, and other side effects, greatly limiting its clinical application. Furthermore, paclitaxel lacks tumor targeting specificity; its widespread distribution in the body, while killing tumor cells, also causes severe damage to normal tissues (such as bone marrow, digestive tract, and peripheral nerves). This results in a narrow therapeutic window, significant side effects, severely limiting its efficacy and reducing patient tolerance. Therefore, improving the adverse properties of paclitaxel, enhancing delivery efficiency, and achieving tumor-specific release are urgent clinical challenges that need to be addressed.

[0003] To overcome the aforementioned shortcomings, prodrug strategies have been extensively studied and considered an effective solution. The core of this strategy lies in the specific and reversible chemical modification of paclitaxel molecules to prepare prodrugs that are inactive or have low activity in vitro, thereby reducing the toxic side effects of chemotherapy drugs. In the tumor microenvironment, prodrugs can be activated by specifically expressed enzymes, a slightly acidic environment, etc., to release the active drug and exert its effect. The hypoxic microenvironment of malignant tumors is an important characteristic of solid tumors, stemming from their rapid proliferation leading to abnormal vascular structures and insufficient blood supply. This microenvironment not only promotes malignant progression and metastasis of tumors but also leads to resistance to traditional radiotherapy and chemotherapy. However, this pathological feature, significantly different from normal tissues, provides an ideal target for the design of chemotherapeutic drug prodrugs.

[0004] Based on this, nitro-aromatic compounds or azobenzene structures are often used as triggering units for hypoxia response in the highly reducing microenvironment unique to hypoxic tumor regions to prepare prodrugs. In tissues with normal oxygen partial pressure, the prodrugs remain stable. However, in hypoxic tumor tissues, nitroreductases overexpressed by cells preferentially catalyze the reduction of nitro or azo bonds. Nitro groups typically undergo multiple reduction steps to generate reactive intermediates such as hydroxylamine and amino groups, while azo bonds are directly reduced and cleaved to generate two amino groups, thereby triggering specific cleavage of the chemical bonds attached to the drug molecule, achieving precise release of the active drug at the target site. Currently reported hypoxia-responsive paclitaxel prodrugs based on azobenzene or p-nitrobenzyl alcohol (DOI:10.1021 / acsnano.2c05341; DOI: 10.1016 / j.nantod.2024.102395) effectively reduce the toxic side effects of paclitaxel in normal tissues and increase its loading rate. However, our further research found that the release rate of active paclitaxel prodrugs modified with azobenzene or p-nitrobenzyl alcohol was too slow or incomplete after responding to hypoxia signals, failing to reach an effective therapeutic concentration at the target site and affecting the final antitumor effect. Summary of the Invention

[0005] The purpose of this invention is to address the shortcomings of existing technologies by providing a hypoxia-responsive paclitaxel prodrug, nano-formulation, its preparation method, and its application. A nitro derivative-modified paclitaxel prodrug small molecule was designed and synthesized. Nanomedicines were prepared through methods such as self-assembly of the paclitaxel prodrug, co-assembly of the paclitaxel prodrug with a photosensitizer, or co-assembly of the paclitaxel prodrug with the polymer distearate-phosphatidylethanolamine methoxy polyethylene glycol (DSPE-mPEG), achieving efficient drug loading and delivery and responsive release at the lesion site. The objective of this invention is to provide a rapidly hypoxia-responsive paclitaxel prodrug, nano-formulation, its preparation method, and its application. The prodrug obtained by this invention has an optimized chemical structure of linkage bonds, enabling rapid response to the hypoxic microenvironment and achieving rapid and efficient release of the active drug in the tumor microenvironment.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: A hypoxia-responsive paclitaxel prodrug, the prodrug having one of the following three structural formulas:

[0007] The method for preparing the hypoxia-responsive paclitaxel prodrug includes the following steps: (1) Dissolve the nitro derivative in a first organic solvent, add triethylamine to obtain a mixed solution, and then add 4-nitrophenyl chloroformate dropwise to the mixed solution under ice bath conditions; then react for 3 to 5 hours, filter and purify to obtain the intermediate compound; The nitro derivative is 5-nitrofurfuryl alcohol (NFA), (3-fluoro-4-nitrophenyl)methanol (FNPC) or ((1-methyl-5-nitro-1H-imidazol-2-yl)methanol (HMMNI). The molar ratio of nitro derivative, triethylamine and 4-nitrophenyl chloroformate is 1:(1-2):(1-2); (2) Dissolve the intermediate compound obtained above with paclitaxel in a first organic solvent, add 4-dimethylaminopyridine (DMAP), 10-30 o C. React under stirring for 4-24 h, and then purify to obtain a white product; The molar ratio of paclitaxel, intermediate compound and DMAP is 1:(1-2):(0.05-0.4). The first organic solvent in steps (1) and (2) may be the same or different, and may be one of tetrahydrofuran, dichloromethane, ethyl acetate, or N,N-dimethylformamide.

[0008] The nanoformation of paclitaxel prodrug is one of the following three: The first type is a nano-formulation of paclitaxel prodrug self-assembled. The preparation method includes: dissolving paclitaxel prodrug in a second organic solvent to obtain a paclitaxel prodrug solution; adding the paclitaxel prodrug solution dropwise into deionized water under magnetic stirring; and dialysis to obtain a nano-formulation based on paclitaxel prodrug. The concentration of the paclitaxel prodrug solution is (1-10) mg / mL, and the volume ratio of the second organic solvent to water is 1:(1-10). The second type is a nano-formulation co-assembled with paclitaxel prodrug and photosensitizer. Its preparation method includes: dissolving paclitaxel prodrug and photosensitizer in a second organic solvent to obtain a mixed solution, adding the mixed solution dropwise into deionized water under magnetic stirring, and then dialyzing to obtain a nano-formulation based on paclitaxel prodrug. The concentration of the paclitaxel prodrug mixture is (0.1–10) mg / mL; the mass ratio of the paclitaxel prodrug to the photosensitizer is 1:(0.1–20); and the volume ratio of the second organic solvent to water is 1:(1–10). The photosensitizer is one of methylene blue (MB), indocyanine green (ICG), dihydroporphyrin e6 (Ce6), rhein, vitamin B2, iridium complex (PC-Ir), and curcumin (Cur); The third method involves co-assembling a nano-formulation with a paclitaxel prodrug, a photosensitizer, and a DSPE-mPEG polymer. The preparation method includes: dissolving the paclitaxel prodrug, the photosensitizer, and the polymer DSPE-mPEG in a second organic solvent to obtain a mixed solution; adding the obtained mixed solution dropwise to deionized water, stirring, and dialyzing to obtain the nano-formulation. The mass ratio of the paclitaxel prodrug to the photosensitizer is 1:(0.1-20); the mass ratio of the paclitaxel prodrug, photosensitizer, and polymer is 1:(0.1-20):(1-99). The concentration of the paclitaxel mixed solution is (0.1–10) mg / mL, and the volume ratio of the second organic solvent to water is 1:(1–10).

[0009] The second organic solvent of the nano-formulation of the first, second or third paclitaxel prodrug is the same or different, and is methanol, ethanol, tetrahydrofuran, acetone, dimethyl sulfoxide (DMSO) or N,N-dimethylformamide (DMF). The aforementioned nano-formulation of paclitaxel prodrug is used as an injectable therapeutic agent in the treatment of breast cancer, ovarian cancer, pancreatic cancer, or non-small cell lung cancer.

[0010] The essential features of this invention are: In current technology, azobenzene and p-nitrobenzyl alcohol are commonly used as triggering units for hypoxia response in the preparation of prodrugs. Density functional theory (DFT) calculations show that the lowest unoccupied molecular orbital (LUMO) levels of 5-nitrofurfuryl alcohol (NFA), (3-fluoro-4-nitrophenyl)methanol (FNPC), and ((1-methyl-5-nitro-1H-imidazol-2-yl)methanol (HMMNI) involved in this invention are -4.58 eV (NFA), -4.49 eV (FNPC), and -4.71 eV (HMMNI), respectively. In comparison, the LUMO levels of azobenzene and p-nitrobenzyl alcohol are -4.44 eV and -0.78 eV, respectively. The eV indicates that NFA, FNP, and HMMNI have lower LUMO energy levels than azobenzene and p-nitrobenzyl alcohol. Therefore, azobenzene and p-nitrobenzyl alcohol have relatively higher reduction potentials, resulting in lower activation efficiency and slower reduction cleavage rates of azobenzene or p-nitrobenzyl alcohol-based paclitaxel prodrugs (PTX-Azo and PTX-NB) in the hypoxic environment of tumors. This inherent characteristic directly leads to delayed and incomplete release of active paclitaxel, making it difficult to rapidly reach an effective therapeutic concentration window within tumor cells, thus weakening its anticancer efficacy.

[0011] In contrast, the nitroaromatic derivatives (NFA, FNPC, and HMMNI) involved in this invention, as hypoxia-responsive groups, have greater kinetic advantages. NFA, FNPC, and HMMNI have lower reduction potentials and faster reaction kinetics than azobenzene and p-nitrobenzyl alcohol, demonstrating significant advantages in achieving efficient and rapid drug release, and are expected to fundamentally solve the aforementioned technical bottlenecks of poor release kinetics.

[0012] The beneficial effects of this invention are as follows: This invention designs and synthesizes a class of nitro derivative paclitaxel prodrugs that rapidly respond to hypoxic microenvironments. Compared to the azobenzene paclitaxel prodrugs (PTX-Azo) and p-nitrobenzyl alcohol hypoxia-responsive groups (PTX-NB) reported in the literature, the PTX-NFA, PTX-FNPC, or PTX-HMMNI involved in this invention have lower reduction potentials and faster reaction kinetics. Under the same concentration of Na2S2O4, they exhibit rapid responsiveness and high efficiency in releasing the active drug (PTX). Figure 5 It demonstrates significant advantages in achieving efficient and rapid drug release, and is expected to fundamentally solve the aforementioned technical bottleneck of poor release kinetics.

[0013] Under simulated hypoxia (2 mM Na2S2O4) conditions, the series of nitro derivative paclitaxel prodrugs (PTX-NFA, PTX-FNPC, and PTX-HMMNI) synthesized in this invention exhibit faster hypoxia activation and higher paclitaxel release efficiency compared to the two previously reported prodrugs, PTX-NB and PTX-Azo. Under the same conditions, the degradation rates of PTX-NFA, PTX-FNPC, PTX-HMMNI, PTX-NB, and PTX-Azo were 56.2%, 37.5%, 30.6%, 19.5%, and 14.8%, respectively; the corresponding paclitaxel release rates were 54.3%, 25.4%, 21.2%, 17.4%, and 9.9%, respectively. Figure 6 By further increasing the concentration of Na2S2O4, the three prodrugs involved in this invention (PTX-NFA, PTX-FNPC, PTX-HMMNI) can be rapidly cleaved, completely releasing active PTX. Attached Figure Description

[0014] Figure 1 The paclitaxel-5-nitrofurfural prodrug (PTX-NFA) of Example 1 1 HNMR spectrum; Figure 2 The paclitaxel-(3-fluoro-4-nitrophenyl)methanol prodrug (PTX-FNPC) of Example 2 1 HNMR spectrum; Figure 3 The paclitaxel-((1-methyl-5-nitro-1H-imidazol-2-yl)methanol prodrug (PTX-HMMNI) of Example 3 1 HNMR spectrum; Figure 4 The high-performance liquid chromatograms of the paclitaxel prodrugs PTX-NFA, PTX-FNPC and PTX-HMMNI tested in Examples 4-6 under simulated hypoxia conditions in Na2S2O4 are shown. Figure 5 The graph shows a comparison of the prodrug cleavage and paclitaxel release efficiency of the paclitaxel prodrugs PTX-NFA, PTX-FNPC, and PTX-HMMNI tested in Examples 4-6 with that of the current technology's PTX-NB and PTX-Azo prodrugs. Figure 6 The bar chart shows the comparison of prodrug cleavage and paclitaxel release efficiency of the paclitaxel prodrugs PTX-NFA, PTX-FNPC, and PTX-HMMNI tested in Examples 4-6 under 2 mM Na2S2O4 conditions with the current technology prodrugs PTX-NB and PTX-Azo. Figure 7 The particle size distribution and stability diagram of the nanomedicine prepared in Example 8; Figure 8 The structural formula, transmission electron microscope image, particle size distribution, and stability diagram of the polymer photosensitizer of the nano-formulation in Example 10 are shown below. Figure 9 These are confocal images and flow cytometry results of the nano-formulation in Example 11; Figure 10 The in vitro cytotoxicity of Taxol, PTX-NFA and nano-formulation as determined in Example 12 is shown in the figure. Figure 11 In vivo imaging images of the nano-formulation measured in Example 13; Figure 12 The image shows the tumor inhibition effects of PBS, Taxol, and nano-formulation as determined in Example 14. Detailed Implementation

[0015] The synthetic route for the compound PTX-NFA is as follows:

[0016] The specific preparation process is as follows: NFA was dissolved in a first organic solvent, and triethylamine was added to obtain a mixed solution. 4-Nitrophenyl chloroformate (NPC) was added dropwise to this mixed solution under ice bath conditions. After reacting for 3-5 hours, the solid residue was removed by filtration, and the mixture was purified by silica gel column chromatography to obtain intermediate compound 1.

[0017] The first organic solvent is one of tetrahydrofuran, dichloromethane, ethyl acetate, and N,N-dimethylformamide; the molar ratio of NFA, triethylamine and NPC is 1:(1-2):(1-2).

[0018] Compound 1 and paclitaxel were dissolved in an organic solvent, DMAP was added, and the reaction was stirred until complete. After the reaction, the mixture was purified to obtain the white product PTX-NFA. The reaction temperature was 10–30 °C. oC, the reaction time is 4–24 h; the molar ratio of paclitaxel, compound 1 and DMAP is 1:(1–2):(0.05–0.4). The organic solvent is one of tetrahydrofuran, dichloromethane, ethyl acetate, and N,N-dimethylformamide.

[0019] The synthetic route for the compound PTX-FNPC is as follows: The specific process is as follows: FNPC was dissolved in a first organic solvent, and triethylamine was added to obtain a mixed solution. 4-Nitrophenyl chloroformate (NPC) was added dropwise to this mixed solution under ice bath conditions. After reacting for 3-5 hours, the solid residue was removed by filtration, and the mixture was purified by silica gel column chromatography to obtain intermediate compound 2.

[0020] The first organic solvent is one of tetrahydrofuran, dichloromethane, ethyl acetate, and N,N-dimethylformamide; the molar ratio of FNPC, triethylamine and NPC is 1:(1-2):(1-2).

[0021] Compound 2 and paclitaxel were dissolved in an organic solvent, DMAP was added, and the reaction was stirred until complete. After the reaction, the mixture was purified to obtain the white product PTX-NFA. The reaction temperature was 10–30 °C. o C, the reaction time is 4–24 h; the molar ratio of paclitaxel, compound 2 and DMAP is 1:(1–2):(0.05–0.4). The organic solvent is one of tetrahydrofuran, dichloromethane, ethyl acetate, and N,N-dimethylformamide.

[0022] The synthetic route for the compound PTX-HMMNI is as follows:

[0023] The specific process is as follows: HMMNI was dissolved in a first organic solvent, and triethylamine was added to obtain a mixed solution. 4-Nitrophenyl chloroformate (NPC) was added dropwise to this mixed solution under ice bath conditions. After reacting for 3-5 hours, the solid residue was removed by filtration, and the mixture was purified by silica gel column chromatography to obtain intermediate compound 3.

[0024] The first organic solvent is one of tetrahydrofuran, dichloromethane, ethyl acetate, and N,N-dimethylformamide; the molar ratio of HMMNI, triethylamine, and NPC is 1:(1-2):(1-2).

[0025] Compound 3 was dissolved in an organic solvent with paclitaxel, DMAP was added, and the reaction was stirred until complete. After the reaction, the product was purified to obtain a white product, PTX-NFA. The reaction temperature was 10–30 °C. o C, the reaction time is 4–24 h; the molar ratio of paclitaxel, compound 3 and DMAP is 1:(1–2):(0.05–0.4). The organic solvent is one of tetrahydrofuran, dichloromethane, ethyl acetate, and N,N-dimethylformamide.

[0026] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments and accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention. The technical solutions of this invention will be further described in detail below by way of embodiments, but this does not limit the invention to the scope of the described examples.

[0027] Example 1: Synthesis of 5-nitroconol-modified paclitaxel prodrug (PTX-NFA) (1) 5-Nitrofurfuryl alcohol (500 mg, 3.5 mmol) was dissolved in anhydrous dichloromethane (15 mL), followed by the addition of triethylamine (707 mg, 7 mmol). Finally, 4-nitrophenyl chloroformate (845 mg, 4.2 mmol) was added dropwise to the anhydrous dichloromethane solution under ice bath conditions. After reacting for 4 hours, the solid residue was removed by filtration, and the mixture was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 6:1) to obtain a light yellow solid intermediate compound 1. The structure of intermediate compound 1 was determined by proton nuclear magnetic resonance spectroscopy, indicating that intermediate compound 1 was successfully synthesized. 1 ¹H NMR (400 MHz, CDCl₃) δ 8.34 (d, J = 8.6 Hz, 2H), 7.44 (d, J = 8.7 Hz, 2H), 7.36 (s, 1H), 6.81 (d, J = 3.7 Hz, 1H), 5.36 (s, 2H). (400 mg, yield: 37%)

[0028] (2) Paclitaxel (150 mg, 0.18 mmol) and intermediate compound 1 (81 mg, 0.26 mmol) were then dissolved in anhydrous dichloromethane (10 mL), followed by the addition of anhydrous dichloromethane solution (2 mL) of DMAP (6.5 mg, 0.05 mmol). The reaction was carried out overnight at room temperature. The crude product was purified by silica gel column chromatography (eluent: dichloromethane: tetrahydrofuran = 20:1) to obtain the white product PTX-NFA (116 mg, yield: 64%).

[0029] The structure of PTX-NFA in Example 1 was determined using proton nuclear magnetic resonance spectroscopy, such as... Figure 1 As shown. 1 H NMR (400MHz, CDCl3) δ 8.18 (d, J = 7.6 Hz, 2H), 7.76 (d, J = 7.6 Hz, 2H), 7.65 (t, J = 7.3 Hz, 1H), 7.54 (q, J = 7.0 Hz, 3H), 7.44 (q, J = 7.2 Hz, 7H), 6.91 (d, J = 9.3 Hz, 1H), 6.70 (d, J = 3.7 Hz, 1H), 6.33 (d, J = 10.1 Hz, 2H), 6.05 (d, J = 9.3 Hz, 1H), 5.73 (d, J = 7.0 Hz, 1H), 5.47 (s, 1H), 5.20 (s, 2H), 5.01(d, J = 9.4 Hz, 1H), 4.48 (dd, J = 10.9, 6.6 Hz, 1H), 4.36 (d, J = 8.5 Hz, 1H), 4.25 (d, J = 8.5 Hz, 1H), 3.86 (d, J = 7.0 Hz, 1H), 2.60 (t, J= 6.9 Hz,1H), 2.50 (s, 3H), 2.47 – 2.41 (m, 1H), 2.28 (s, 4H), 1.97 (s, 4H), 1.73 (s,3H), 1.28 (s, 3H), 1.19 (s, 3H). The chemical shift at δ 5.20 ppm corresponds to the methylene group (-CH2-O-COO-) in PTX-NFA. The calculation of the integral area of ​​the chemical shift indicates that the hypoxia-responsive group NFA was successfully linked to the chemotherapeutic drug PTX, and the target product PTX-NFA was obtained.

[0030] Example 2: Synthesis of a paclitaxel prodrug modified with (3-fluoro-4-nitrophenyl)methanol (PTX-FNPC) (1) (3-fluoro-4-nitrophenyl)methanol (500 mg, 2.9 mmol) was dissolved in anhydrous dichloromethane (20 mL), followed by the addition of triethylamine (443 mg, 4.35 mmol). Finally, 4-nitrophenyl chloroformate (705 mg, 3.5 mmol) was added dropwise to the anhydrous dichloromethane solution under ice bath conditions. After reacting for 4 hours, the solid was removed by filtration, and the mixture was purified by silica gel column chromatography (eluent: petroleum ether: ethyl acetate = 4:1) to obtain a light yellow solid intermediate compound 2. The structure of intermediate compound 2 was determined by proton nuclear magnetic resonance spectroscopy, indicating that intermediate compound 2 was successfully synthesized. 1 ¹H NMR (400 MHz, CDCl₃) δ 8.33 – 8.26 (m, 2H), 8.12 (t, J = 8.0 Hz, 1H), 7.43 – 7.33 (m, 4H), 5.36 (s, 2H), (330 mg, yield: 34%).

[0031] (2) Paclitaxel (130 mg, 0.15 mmol) and compound 2 (100.5 mg, 0.3 mmol) were dissolved in anhydrous dichloromethane (15 mL), and then an anhydrous dichloromethane solution of DMAP (6.5 mg, 0.05 mmol) (2 mL) was added. The reaction was carried out overnight at room temperature. The crude product was purified by silica gel column chromatography (dichloromethane:tetrahydrofuran = 20:1) to obtain the white product PTX-FNPC (97 mg, yield: 62%).

[0032] The structure of the prodrug PTX-FNPC in Example 2 was determined using proton nuclear magnetic resonance spectroscopy, such as... Figure 2 As shown. 1 H NMR (400 MHz, CDCl3) δ 8.15 (d,J = 7.6 Hz, 2H), 8.05 (t, J = 7.9 Hz, 1H), 7.73 (d, J = 7.6 Hz, 2H), 7.61 (t, J = 7.4 Hz, 1H), 7.53 – 7.49 (m, 3H), 7.41 (dq, J = 14.1, 7.0 Hz, 7H), 7.23 (d, J = 8.6 Hz, 1H), 6.86 (d, J = 9.4 Hz, 1H), 6.29 (d, J = 4.6 Hz, 2H), 6.03 (dd, J = 9.4, 2.8 Hz, 1H), 5.70 (d, J = 7.1Hz, 1H), 5.47 (d, J = 2.8 Hz, 1H), 5.26 – 5.18 (m, 2H), 4.97 (dd, J = 9.7, 2.4 Hz, 1H), 4.44 (dd, J = 11.0, 6.6 Hz, 1H), 4.32 (d, J = 8.5 Hz, 1H), 4.21(d, J = 8.5 Hz, 1H), 3.82 (d, J = 7.0 Hz, 1H), 2.56 (td, J = 9.4, 4.8 Hz,1H), 2.47 (s, 3H), 2.43 – 2.39 (m, 1H), 2.23 (s, 4H), 1.92 (s, 4H), 1.69 (s,3H), 1.25 (d, J = 7.4 Hz, 3H), 1.15 (s, 3H). The chemical shift at δ 5.18-5.26 ppm corresponds to the methylene group (-CH2-O-COO-) in PTX-FNPC. The calculation of the integral area of ​​the chemical shift indicates that the hypoxia-responsive group FNPC was successfully linked to the chemotherapeutic drug PTX, and the target product PTX-FNPC was obtained.

[0033] Example 3: Synthesis of 1-methyl-5-nitro-2-hydroxymethylimidazol-modified paclitaxel prodrug (PTX-HMMNI) (1) 1-Methyl-5-nitro-2-hydroxymethylimidazolium (500 mg, 3.2 mmol) was dissolved in anhydrous dichloromethane (20 mL), followed by the addition of triethylamine (644 mg, 6.4 mmol). Finally, 4-nitrophenyl chloroformate (769 mg, 3.8 mmol) was added dropwise to the anhydrous dichloromethane solution under ice bath conditions. After reacting for 4 hours, the solid was removed by filtration, and the mixture was purified by silica gel column chromatography (eluent: dichloromethane: ethyl acetate = 100:1) to obtain a light yellow solid intermediate compound 3. The structure of intermediate compound 3 was determined by proton nuclear magnetic resonance spectroscopy, indicating the successful synthesis of intermediate compound 3. 1 ¹H NMR (400 MHz, CDCl₃) δ 8.39 – 8.29 (m, 2H), 8.06 (s, 1H), 7.48 – 7.40 (m, 2H), 5.46 (s, 2H), 4.13 (s, 3H). (450 mg, yield: 44%)

[0034] (2) Paclitaxel (150 mg, 0.18 mmol) and compound 3 (68 mg, 0.21 mmol) were dissolved in anhydrous dichloromethane (15 mL), and then an anhydrous dichloromethane solution of DMAP (6.5 mg, 0.05 mmol) (3 mL) was added. The reaction was carried out overnight at room temperature. The crude product was purified by silica gel column chromatography (eluent: dichloromethane: ethyl acetate = 5:1) to obtain the white product PTX-HMMNI (105 mg, yield: 58%).

[0035] The structure of the prodrug PTX-HMMNI in Example 3 was determined using proton nuclear magnetic resonance spectroscopy, such as... Figure 3 As shown. 1H NMR (400 MHz, CDCl3) δ 8.19 – 8.11 (m, 2H), 7.81 (s, 1H), 7.74 (d, J = 7.7 Hz, 2H), 7.63 – 7.57 (m, 1H), 7.51 (dt, J = 9.9, 4.8 Hz, 3H), 7.43 – 7.34 (m,7H), 7.09 (d, J = 9.2 Hz, 1H), 6.33 – 6.25 (m, 2H), 6.08 – 6.01 (m, 1H), 5.70 (d, J = 7.0 Hz, 1H), 5.47 (d, J = 2.7 Hz, 1H), 5.28 (q, J = 13.6 Hz, 2H), 4.97 (d, J = 9.5 Hz, 1H), 4.44 (dd, J = 10.9, 6.7 Hz, 1H), 4.32 (d, J = 8.6Hz, 1H), 4.21 (d, J = 8.5 Hz, 1H), 3.96 (d, J = 1.9 Hz, 3H), 3.82 (d, J = 7.0Hz, 1H), 2.56 (ddd, J = 15.5, 9.6, 6.8 Hz, 1H), 2.46 (d, J = 1.9 Hz, 3H), 2.43 – 2.37 (m, 1H), 2.24 (d, J = 1.9 Hz, 3H), 2.22 (s, 1H), 1.93 (s, 4H), 1.69 (d, J = 1.9 Hz, 3H), 1.25 (s, 3H), 1.15 (s, 3H). The chemical shifts at δ 5.25–5.31 ppm correspond to the methylene group (-CH2-O-COO-) in PTX-HMMNI; the chemical shift at δ 3.96 ppm corresponds to the methyl group (-CH3) on the HMMNI group. Based on the chemical shift integral area calculation, the hypoxia-responsive group HMMNI was successfully linked to the chemotherapeutic drug PTX, yielding the target product PTX-HMMNI.

[0036] Example 4: High-performance liquid chromatography determination of prodrug (PTX-NFA) cleavage and paclitaxel release under simulated hypoxia conditions Weigh 2 mg of the prodrug PTX-NFA obtained in Example 1 and dissolve it in 5 mL of chromatographic grade acetonitrile to obtain an acetonitrile solution of PTX-NFA (0.4 mg / mL). Mix equal volumes (250 µL each) of PBS solution containing sodium dithionite (Na₂S₂O₄) at different concentrations (0–5 mM) (pH 7.4) with the above prodrug solution and incubate at 37°C for 2 hours. After incubation, add 1 mL of chromatographic grade acetonitrile to terminate the reaction. Filter the final mixture through a 0.22 µm filter membrane and analyze by high-performance liquid chromatography (HPLC). The test results are as follows: Figure 4 As shown in Figure A, with the increase of Na2S2O4 concentration, the elution peak of PTX-NFA at 7.9 min gradually decreased, while the HPLC elution peak of paclitaxel at 6.5 min gradually increased, indicating that PTX-NFA gradually cleaved in the reducing microenvironment and could completely release the active drug PTX.

[0037] Example 5: High-performance liquid chromatography determination of prodrug (PTX-FNPC) cleavage and paclitaxel release under simulated hypoxia conditions Weigh 4.2 mg of the prodrug PTX-FNPC obtained in Example 2 and dissolve it in 10 mL of chromatographic grade acetonitrile to obtain an acetonitrile solution of PTX-FNPC (0.42 mg / mL). Mix equal volumes (250 µL each) of PBS solution containing sodium dithionite (Na₂S₂O₄) at different concentrations (0–5 mM) (pH 7.4) with the above prodrug solution and incubate at 37°C for 2 hours. After incubation, add 1 mL of chromatographic grade acetonitrile to terminate the reaction. Filter the final mixture through a 0.22 µm filter membrane and analyze by high-performance liquid chromatography (HPLC). The test results are as follows: Figure 4 As shown in Figure B, with the increase of Na2S2O4 concentration, the elution peak of PTX-FNPC at 8.9 min gradually decreased, while the HPLC elution peak of paclitaxel at 6.5 min gradually increased, indicating that PTX-FNPC gradually cleaved in the reducing microenvironment and could completely release the active drug PTX.

[0038] Example 6: High-performance liquid chromatography determination of prodrug (PTX-HMMNI) cleavage and paclitaxel release under simulated hypoxia conditions Weigh 4.05 mg of the prodrug PTX-HMMNI obtained in Example 3 and dissolve it in 10 mL of chromatographic grade acetonitrile to obtain an acetonitrile solution of PTX-HMMNI (0.405 mg / mL). Mix equal volumes (250 µL each) of PBS solution containing sodium dithionite (Na₂S₂O₄) at different concentrations (0–5 mM) (pH 7.4) with the above prodrug solution and incubate at 37°C for 2 hours. After incubation, add 1 mL of chromatographic grade acetonitrile to terminate the reaction. Filter the final mixture through a 0.22 µm filter membrane and analyze by high-performance liquid chromatography (HPLC). The test results are as follows: Figure 4 As shown in C, with the increase of Na2S2O4 concentration, the elution peak of PTX-HMMNI at 7.2 min gradually decreased, while the HPLC elution peak of paclitaxel at 6.5 min gradually increased, indicating that PTX-HMMNI gradually cleaved in the reducing microenvironment and could completely release the active drug PTX.

[0039] The content of PTX-NFA, PTX-FNPC, PTX-HMMNI, and PTX in solution can be calculated from the integrated area of ​​the elution peak in high-performance liquid chromatography (HPLC). Figure 5 It can be seen that under 4 mM Na2S2O4 conditions, PTX-NFA, PTX-FNPC, and PTX-HMMNI can be almost completely cleaved, releasing PTX. The rates of PTX-Azo prodrug cleavage and paclitaxel release are significantly lower than those of the PTX-NFA, PTX-FNPC, and PTX-HMMNI prodrugs involved in this invention.

[0040] To further compare the degradation rate and paclitaxel release efficiency of several prodrugs, the degradation rate and paclitaxel release rate of several prodrugs in Examples 4-6 under 2mM Na2S2O4 conditions were plotted as a bar chart to visually demonstrate the differences among the prodrugs. Figure 6 As shown in Figure A, the prodrug cleavage rate is: PTX-NFA > PTX-FNPC > PTX-HMMNI > PTX-NB > PTX-Azo. The paclitaxel release rate is: PTX-NFA > PTX-FNPC > PTX-HMMNI > PTX-NB > PTX-Azo. Figure 6 (B in the middle).

[0041] Example 7: Preparation of nanomedicines by self-assembly of paclitaxel prodrug PTX-FNPC Accurately weighed PTX-FNPC (1.0 mg) was dissolved in 200 μL of methanol to form a homogeneous, clear organic solution. This solution was then slowly added dropwise to deionized water (900 µL) under stirring at room temperature. The resulting nanoparticle suspension was dialyzed (molecular weight cutoff: 3.5 kDa) for 12 hours to remove unencapsulated drug and organic solvent, ultimately yielding PTX-FNPC nanoparticles.

[0042] Example 8: Preparation of nanomedicines by self-assembly of paclitaxel prodrug and photosensitizer Ce6 Precisely weighed PTX-NFA (1.0 mg) and photosensitizer Ce6 (1.0 mg) were dissolved together in 200 μL of DMSO to form a homogeneous and clear organic solution. This solution was then slowly added dropwise to deionized water (900 µL) under stirring at room temperature. Through rapid permeation into the aqueous phase, the hydrophobic paclitaxel prodrug and the somewhat amphiphilic Ce6 molecules spontaneously aggregated and aligned themselves through intermolecular forces such as π-π stacking and hydrophobic interactions, forming nanoparticles with the paclitaxel prodrug as the core and Ce6 partially embedded / encapsulated on the surface. The mixture was continuously stirred overnight to ensure complete self-assembly. The resulting nanoparticle suspension was then dialyzed (molecular weight cutoff: 3.5 kDa) for 12 hours to remove unencapsulated drug and organic solvent, finally yielding Ce6@PTX-NFA nanoparticles. The particle size and particle size distribution were determined using dynamic light scattering, and the results are shown below. Figure 7 As shown. Figure 7 As shown in A, the particle size of Ce6@PTX-NFA nanoparticles ranges from 180 nm to 210 nm. Figure 7 As shown in B, the particle size of Ce6@PTX-NFA nanoformulation remained stable within 7 days.

[0043] Example 9: Preparation of nanomedicines by self-assembly of paclitaxel prodrug and photosensitizer ICG Precisely weighed PTX-HMMNI (1.0 mg) and photosensitizer ICG (1.0 mg) were dissolved together in 200 µL of DMSO to form a homogeneous, clear organic solution. This solution was then slowly added dropwise to deionized water (900 µL) under stirring at room temperature. The resulting mixture was stirred overnight to ensure complete self-assembly. The resulting nanoparticle suspension was then dialyzed (molecular weight cutoff: 3.5 kDa) for 12 hours to remove unencapsulated drug and organic solvent, ultimately yielding ICG@PTX-HMMNI nanoparticles.

[0044] Example 10: Preparation of nanomedicines by self-assembly of paclitaxel prodrug and polymer photosensitizer PC-Ir First, the prodrug PTX-NFA (1 mg) and the photosensitizing polymer PC-Ir (12.5 mg) were co-dissolved in DMSO (300 µL) to form a homogeneous organic phase. Then, under stirring at room temperature, this solution was slowly added dropwise to deionized water (900 µL), allowing for spontaneous nanoparticle formation through hydrophobic interactions. The mixture was stirred overnight to ensure complete self-assembly. The resulting nanoparticle suspension was then dialyzed (molecular weight cutoff: 3.5 kDa) for 12 hours to remove unencapsulated drug and organic solvents, ultimately yielding the PC-Ir@PTX-NFA nanoparticle formulation. PC-Ir nanoparticles without PTX-NFA were prepared as a control formulation following the same procedure. The structural formula of the polymer PC-Ir used is shown below. Figure 8 As shown in A in the diagram. Transmission electron microscopy revealed that they all possess a spherical structure, such as... Figure 8 As shown in Figure B, the particle size and particle size distribution were determined using dynamic light scattering method, and the results are as follows: Figure 8 As shown in C, the nano-formulations assembled with polymer photosensitizers (PC-Ir NPs) and those co-assembled with paclitaxel prodrug and polymer photosensitizers (PC-Ir@PTX-NFA NPs) both have particle sizes of 40-50 nm, appear as orange-yellow transparent solutions, and exhibit uniform particle size distribution. Figure 8 As can be seen from D, the particle size of the nano-formulations PC-Ir NPs and PC-Ir@PTX-NFA NPs remained stable within 7 days.

[0045] Example 11: Endocytosis The uptake of PC-Ir@PTX-NFA nanoparticles prepared in Example 10 by 4T1 cells was studied using confocal laser scanning microscopy and flow cytometry. 4T1 cells were cultured at 1 × 10⁶ cells per well. 5 Cells were seeded at a density of [number] cells / well in 6-well plates and cultured in DMEM medium containing 10% fetal bovine serum and 1% antibiotics under standard culture conditions (5% CO2, 37°C) for 24 hours. PC-Ir@PTX-NFA nanoparticles (5 μg / mL) were then added to the cells, and incubation was performed for predetermined times (2, 4, and 6 hours). After incubation, the cells were washed three times with PBS, fixed with 4% paraformaldehyde for 10 minutes at room temperature, and the nuclei were stained with DAPI (10 μg / mL) for 5 minutes. Finally, the cells were observed using a confocal laser scanning microscope. Figure 9 As shown in Figure A, the fluorescence intensity within the cells increases with increasing incubation time, reaching its maximum after 6 hours, indicating that the cells can effectively take up PC-Ir@PTX-NFA nanoparticles.

[0046] Further quantitative analysis of cellular uptake of PC-Ir@PTX-NFA was conducted using flow cytometry: 4T1 cells were loaded at a density of 1 × 10⁶ cells per well.5 Cells were seeded at a density of [number] cells / well in 6-well plates and incubated with PC-Ir@PTX-NFA (5 μg / mL) for 2, 4, and 6 hours, respectively. After incubation, cells were washed with PBS and then digested with EDTA-free trypsin to collect the cells. The collected cells were resuspended in PBS, centrifuged, and the supernatant was discarded. This washing process was repeated three times. Finally, the cells were resuspended in PBS and analyzed by flow cytometry. Figure 9 As shown in B, the image shifts to the right as the incubation time increases, and the average fluorescence intensity of the cell population increases, indicating that the cells can effectively take up PC-Ir@PTX-NFA nanoformulation.

[0047] Example 12: Cytotoxicity The toxic effects of different formulations on 4T1 cells were detected using the CCK-8 assay. 4T1 cells were cultured at 1 × 10⁶ cells per well. 4 Cells were seeded at a density of [number] cells / well in 96-well plates and incubated at 37°C for 24 hours to allow cell adhesion. After 24 hours, the culture medium was removed and replaced with fresh culture medium (100 μL per well) containing different formulations (paclitaxel injection, PTX-NFA, PC-Ir, PC-Ir@PTX-NFA). After culturing for another 48 hours, the old culture medium containing the drugs was removed, and diluted CCK-8 indicator (CCK-8 to culture medium volume ratio 1:9) was added to each well. After incubation at 37°C for another 40 minutes, the absorbance at 450 nm was measured using a microplate reader. For the light-illuminated experimental group, cells were exposed to a 660 nm laser (power density 0.2 W / cm²) after 4 hours of drug incubation. 2 Irradiate for 5 minutes, then continue culturing for 48 hours, and finally detect cell viability using the CCK-8 assay as described above. Cytotoxicity results are as follows: Figure 10 As shown in the left image, under dark conditions (left panel), even at high concentrations (64 μg / mL PC-Ir nanoparticles), PC-Ir NP and PC-Ir@PTX-NFANP exhibited inhibition rates exceeding 90% against 4T1 cells, demonstrating their excellent biocompatibility. In contrast, after 5 minutes of laser irradiation (660 nm, 0.2 W / cm²), the inhibition rate was significantly lower. 2 Following this, both formulations (PC-Ir NP and PC-Ir@PTX-NFA) exhibited concentration-dependent cytotoxicity against 4T1 cells. Figure 10 As shown in the right figure, at an equivalent PC-Ir concentration, cell viability decreased to 23.7% after PC-Ir@PTX-NFA NP+ light treatment, indicating that the nano-formulation prepared by co-assembling paclitaxel prodrug and polymer photosensitizer has a significant inhibitory effect on tumor cells.

[0048] Example 13: In vivo imaging in animals To investigate the specific accumulation of the nanoformulation prepared in Example 10 at the tumor site, a 4T1 tumor-bearing mouse model was constructed. When the tumor volume reached 150 mm²... 3 At that time, tumor-bearing mice were injected with PC-Ir@PTX-NFA (10 mg / kg) via tail vein. The in vivo distribution of the nanoparticles was monitored at preset time points (0, 2, 4, 8, 12, 24, and 48 hours after injection) using an IVIS in vivo imaging system (excitation / emission wavelength = 751 nm / 810 nm). The results are as follows: Figure 11 As shown, within 2 hours of injection, the fluorescence signal was present throughout the mouse body. The fluorescence signal in normal tissues gradually weakened over time, while the fluorescence signal at the tumor site showed a clear time-dependent aggregation, exhibiting an initial enhancement followed by a weakening trend, reaching its peak at 12 hours post-injection. This indicates that the prepared paclitaxel prodrug and polymer photosensitizer nanoformulation can selectively aggregate at the tumor site, which is beneficial in reducing toxic side effects on normal organs.

[0049] Example 14: Tumor Suppression Effect To verify the tumor-inhibiting effect of the nano-formulation prepared in Example 10, a 4T1 tumor-bearing BALB / c mouse model was constructed. When the tumor volume was approximately 75 mm... 3 Mice were randomly divided into three groups (PBS, Taxol, PC-Ir@PTX-NFA NPs + L). 200 μL of saline or nanomedicine formulation was administered via tail vein on days 1, 4, and 7 of treatment. Based on in vivo fluorescence imaging results, 12 h after tail vein administration, PC-Ir@PTX-NFA NPs + L were irradiated for 5 min (660 nm, 190 mW / cm²). 2 Compared with the PBS control group, PC-Ir@PTX-NFA NPs + L significantly inhibited tumor growth. Results are as follows... Figure 12 As shown, the nano-formulation prepared from paclitaxel prodrug and polymer photosensitizer exhibits superior therapeutic efficacy against mouse breast cancer compared to commercially available Taxol formulations. This demonstrates that the hypoxia-responsive prodrug based on nitro derivatives designed in this invention can conveniently integrate photosensitizers into the same delivery system, constructing a combined treatment regimen with synergistic effects of chemotherapy and photodynamic therapy.

[0050] In summary, the paclitaxel prodrug based on NFA, FNPC, and HMMNI of this invention can achieve faster hypoxia activation, more complete drug release, and stronger antitumor efficacy. Furthermore, the hypoxia-responsive prodrug platform based on nitro derivatives designed in this invention has good scalability, allowing for the convenient integration of chemotherapeutic drugs with other mechanisms of action, photosensitizers, or immunomodulators into the same delivery system through similar strategies to construct synergistic combination therapy regimens. This is particularly suitable for reducing the toxic side effects of chemotherapy drugs or treating advanced solid tumors, demonstrating significant platform technology value.

[0051] The foregoing detailed examples of the present invention are merely preferred embodiments and should not be construed as limiting the scope of the invention. All equivalent variations and modifications made within the scope of the present invention should still fall within the patent coverage of the present invention.

[0052] Matters not covered in this invention are common knowledge.

Claims

1. An oxygen-responsive paclitaxel prodrug, characterized in that, The structural formula of the prodrug is one of the following three: 。 2. The method of preparing a hypoxia-responsive paclitaxel prodrug according to claim 1, wherein, The method comprises the following steps: (1) dissolving the nitro derivative in a first organic solvent, adding triethylamine to obtain a mixed solution, and then adding 4-nitrophenyl chloroformate dropwise to the mixed solution under ice bath condition; then reacting for 3-5 hours, and filtering and purifying to obtain an intermediate compound; The nitro derivative is 5-nitrofurfuryl alcohol NFA, (3-fluoro-4-nitrophenyl)methanol FNPC, or ((1-methyl-5-nitro-1H-imidazol-2-yl)methanol HMMNI; The molar ratio of the nitro derivative, triethylamine, and 4-nitrophenyl chloroformate is 1:(1-2):(1-2); (2) dissolving the intermediate compound obtained in the above step (1) and paclitaxel in a first organic solvent, adding 4-dimethylaminopyridine (DMAP), and reacting for 4-24 hours under stirring at room temperature, and then purifying to obtain a white product; The molar ratio of paclitaxel, the intermediate compound, and DMAP is 1:(1-2):(0.05-0.4).

3. The method of preparing a hypoxia-responsive paclitaxel prodrug according to claim 2, wherein, The first organic solvent in step (1) and step (2) is the same or different, and is one of tetrahydrofuran, dichloromethane, ethyl acetate, and N,N-dimethylformamide.

4. Nanofomulation of paclitaxel prodrug, characterized in that, The structural formula of the prodrug is one of the following three: The first kind is a nano-preparation of a paclitaxel prodrug self-assembled, and the preparation method comprises the following steps: dissolving the paclitaxel prodrug in claim 1 in a second organic solvent to obtain a paclitaxel prodrug solution, and then adding the paclitaxel prodrug solution dropwise into deionized water under magnetic stirring, and then dialyzing to obtain a nano-preparation based on the paclitaxel prodrug; The concentration of the paclitaxel prodrug solution is (1-10) mg / mL, and the volume ratio of the second organic solvent to water is 1:(1-10); Alternatively, the second kind is a nano-preparation of a paclitaxel prodrug and a photosensitizer co-assembled, and the preparation method comprises the following steps: dissolving the paclitaxel prodrug and the photosensitizer in claim 1 in a second organic solvent to obtain a mixed solution, and then adding the mixed solution dropwise into deionized water under magnetic stirring, and then dialyzing to obtain a nano-preparation based on the paclitaxel prodrug; The concentration of the paclitaxel prodrug mixed solution is (0.1-10) mg / mL; the mass ratio of the paclitaxel prodrug to the photosensitizer is 1:(0.1-20); and the volume ratio of the second organic solvent to water is 1:(1-10); The photosensitizer is one of methylene blue, indocyanine green, chlorin e6, rhein, vitamin B2, iridium complex, and curcumin; Alternatively, the third kind is a nano-preparation of a paclitaxel prodrug, a photosensitizer, and a polymer DSPE-mPEG co-assembled, and the preparation method comprises the following steps: dissolving the paclitaxel prodrug, the photosensitizer, and the polymer DSPE-mPEG in claim 1 in a second organic solvent to obtain a mixed solution, and then adding the obtained mixed solution dropwise into deionized water, and then stirring and dialyzing to obtain the nano-preparation; The mass ratio of the paclitaxel prodrug to the photosensitizer is 1:(0.1-20); and the mass ratio of the paclitaxel prodrug, the photosensitizer, and the polymer is 1:(0.1-20):(1-99). The concentration of the paclitaxel mixed solution is (0.1-10) mg / mL, and the volume ratio of the second organic solvent to water is 1:(1-10).

5. The nanofomulation of paclitaxel prodrug of claim 4, wherein the nanofonnulation is a liposome. The second organic solvent of the nano-preparation of the first, second or third paclitaxel prodrug is the same or different, and is methanol, ethanol, tetrahydrofuran, acetone or N,N-dimethylformamide.

6. The use of a nanofomulation of a paclitaxel prodrug according to claim 4, wherein the nanofonnulation is administered in combination with a chemotherapeutic agent. Injection for breast cancer, ovarian cancer, pancreatic cancer or non-small cell lung cancer therapy as a therapeutic drug.

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