Thermosensitive nanogel system based on tirapazamine derivative as well as preparation method and application of thermosensitive nanogel system

Abstract

The invention discloses a temperature-sensitive nanogel system based on a tirapazamine derivative, the temperature-sensitive nanogel system is composed of RTPZ, PIB nanogel and normal saline, the concentration of PIB is 25-40 mg / mL, and the concentration of RTPZ is 2-6 mg / mL. The preparation method comprises the following steps: preparing a tirapazamine derivative RTPZ; dissolving a tirapazamine derivative RTPZ and PIB nanogel in absolute ethyl alcohol, stirring until the materials are uniformly mixed, spin-drying to remove ethyl alcohol, and drying in a vacuum drying oven to obtain a mixture of PIB and RTPZ; and dissolving a mixture of PIB and RTPZ in normal saline to prepare the RTPZ temperature-sensitive nanogel system. The temperature-sensitive nanogel system disclosed by the invention is a novel embolism treatment delivery system, integrates the characteristics of novel temperature-sensitive nanogel PIB, hypoxia activation prodrug and slow release, and constructs a local, slow-release and targeted hypoxia tumor treatment system for transcatheter arterial chemoembolism.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical chemistry, and in particular to a thermosensitive nanogel system based on tirazamine derivatives, its preparation method, and its application. Background Technology

[0002] Transcatheter arterial chemoembolization (TACE), a common interventional treatment for liver cancer, is a combination of TAI and TAE therapies. A catheter is selectively or superselectively inserted into the target artery supplying the tumor via an artery or vein, and an appropriate amount of chemoembolic drugs are injected at a suitable rate to occlude the tumor, causing ischemic necrosis of the tumor tissue. However, the clinical outcomes of TACE remain unsatisfactory, typically achieving partial remission in only 15%–55% of patients and extending median survival from 16 months to 20 months. This poor outcome may be partly due to low tumor drug delivery efficiency. Furthermore, chemotherapy drugs often fail to effectively target tumor stem cells, a fundamental reason for tumor recurrence after conventional TACE treatment, as their cytotoxic effects may be inhibited in the hypoxic environment created by embolization.

[0003] The hypoxic prodrug terazamine (3-amino-1,2,4-benzotriazine-1,4-dioxide, TPZ) has attracted considerable attention due to its ability to selectively damage the DNA of hypoxic cells within solid tumors. In the tumor hypoxic microenvironment (TME), TPZ can be bioreduced to a transient free radical intermediate (·TPZ), accompanied by the generation of reactive oxygen species (ROS, such as superoxide anions and hydroxyl radicals), and further reduced to the cytotoxic product benzotriazine (BTZ), which can damage DNA and ultimately induce apoptosis. TPZ exhibits approximately 200 times higher cytotoxicity in hypoxic environments compared to normoxic environments, making it an ideal candidate drug for interventional therapy of liver cancer, particularly in the hypoxic tumor microenvironment created after transcatheter arterial chemoembolization (TACE). However, the practical clinical application of terazamine faces the following key challenges: (1) Tirazamine has limited hypoxic cytotoxicity and insufficient evidence of efficacy: In a phase III clinical trial for locally advanced cervical cancer, the addition of tirazamine to the standard chemoradiotherapy regimen (cisplatin + radiotherapy) failed to significantly improve progression-free survival (PFS) and overall survival (OS) in patients.

[0004] (2) Safety challenges exist: In the above studies, the interim safety assessment found that the initial regimen was poorly tolerated, and the combination therapy regimen was only made tolerable after the initial dose of telatazamine was reduced.

[0005] (3) As a drug that needs to be activated in a hypoxic environment of the tumor, conventional intravenous administration may not be able to achieve the ideal concentration in the tumor. Current research is exploring new methods such as local arterial administration of drug-loaded microspheres, but these are still in the preclinical or early research stages.

[0006] Therefore, it is crucial to establish a new embolization delivery system that integrates novel thermosensitive embolic agents, hypoxia-selective formulations, and sustained-release properties to construct a local, sustained-release, targeted hypoxic tumor treatment system for transcatheter arterial chemoembolization. Summary of the Invention

[0007] To address the shortcomings of existing technologies, this invention provides a thermosensitive nanogel system based on teirazamine derivatives, its preparation method, and its applications.

[0008] To achieve the above objectives, the present invention adopts the following technical solution: A thermosensitive nanogel system based on terazamine derivative, wherein the thermosensitive nanogel system is composed of terazamine derivative RTPZ, PIB nanogel and physiological saline, wherein the concentration of PIB nanogel is 25~40 mg / mL and the concentration of terazamine derivative RTPZ is 2~6 mg / mL. The structural formula of the tirazamine derivative RTPZ is: ; Where R is C 1-17 Straight-chain or branched alkyl groups, C 3-6 cycloalkyl, C 1-7 One of perfluoroalkyl, vinyl, ethynyl, substituted benzyl, 1-piperidinyl, and 4-morpholinoyl, wherein the benzyl group has a C-substituent on the benzene ring. 1-2 Alkyl, C 1-2 Alkoxy, trifluoromethyl.

[0009] Preferably, the concentration of the terazamine derivative RTPZ is 2 mg / mL, 4 mg / mL, or 6 mg / mL.

[0010] Preferably, the structural formula of R is: .

[0011] This invention also provides a method for preparing the above-mentioned thermosensitive nanogel system based on tirazamine derivatives, comprising the following steps: S1. Dissolve terazamine and an alkaline substance in a solvent and stir, then add alkyl acyl chloride to react, and continue stirring during the reaction; the molar ratio of terazamine, alkaline substance and alkyl acyl chloride is 1:(2-4):(1-1.5); After the reactions in S2 and S1 are completed, the solvent is removed by rotary evaporation, then dichloromethane is added for dissolution, followed by washing with saturated brine, drying with anhydrous sodium sulfate, and finally evaporation to remove the solvent, yielding the crude product. S3. The crude product was purified by column chromatography to obtain the terazamine derivative RTPZ; S4. Dissolve the terazamine derivative RTPZ and PIB nanogel in anhydrous ethanol, stir until homogeneous, remove the ethanol by rotary evaporation, and dry in a vacuum drying oven to obtain a mixture of PIB and RTPZ. S5. Prepare an RTPZ thermosensitive nanogel system by dissolving a mixture of PIB and RTPZ in physiological saline, wherein the concentration of PIB is 25~40 mg / mL and the concentration of RTPZ is 2~6 mg / mL.

[0012] The terazamine derivative in the thermosensitive nanogel system of this invention was prepared through molecular structure alkylation modification. The molecular structure alkylation modification mainly includes: (1) Alkyl / cycloalkyl chain: Introducing alkyl chains or cycloalkyl chains, etc., to enhance hydrophobicity; (2) Halogen atoms: Introducing halogen atoms (especially fluorine and chlorine). Fluorine atoms are "universal modifiers" in medicinal chemistry, which can moderately increase lipid solubility and affect metabolism, electrical properties and conformation. (3) Aromatic rings / heterocyclic aromatic rings: Introduce hydrophobic ring systems such as phenyl, piperidine, and morpholine.

[0013] The reaction formula is: .

[0014] Preferably, the amount of terazamine is 1 eq, the amount of alkaline substance is 2-4 eq, the amount of alkyl acyl chloride is 1-1.5 eq, the amount of solvent is 30-50 mL, the amount of dichloromethane is 30-60 mL, the amount of saturated saline solution is 30-60 mL, and the amount of anhydrous sodium sulfate is 15-30 g.

[0015] Preferably, the alkaline substance is one or a combination of sodium hydroxide, triethylamine, pyridine, and dimethylpyridine.

[0016] Preferably, the solvent is one or a combination of tetrahydrofuran, dichloromethane, chloroform, N,N-dimethylformamide, acetonitrile, dimethyl ether, benzene, toluene, ethanol and methanol.

[0017] Preferably, in S1, the alkyl acyl chloride is added under an argon atmosphere, the reaction temperature is 0~30℃, and the reaction time is 2~12h.

[0018] Preferably, in S1, after the addition of alkyl acyl chloride, the reaction is monitored by thin-layer chromatography until the raw material is completely converted.

[0019] Preferably, in S3, the eluent for column chromatography purification is dichloromethane and ethyl acetate, wherein the volume ratio of dichloromethane to ethyl acetate is (4-6):1.

[0020] This invention also discloses the application of the above-mentioned thermosensitive nanogel system based on teirazamine derivatives in the preparation of drugs for treating tumors.

[0021] Compared with the prior art, the present invention has the following beneficial effects: (1) The thermosensitive nanogel system of the present invention is a novel embolization therapy delivery system that integrates a novel thermosensitive nanogel PIB, a hypoxia-activating prodrug, and sustained-release properties to construct a local, sustained-release, targeted hypoxic tumor therapy system for transcatheter arterial chemoembolization. The tirazamine derivative RTPZ in the thermosensitive nanogel system of the present invention achieved significantly better antitumor efficacy than the original drug TPZ in a 3D cell spheroid model.

[0022] (2) The hypoxia prodrug telatazamine derivative RTPZ in the thermosensitive nanogel system of the present invention has better hypoxia cytotoxicity than the original drug TPZ, indicating that as a prodrug it can be more effectively activated in the hypoxic microenvironment of tumors or has stronger killing power itself, achieving a much better anti-tumor effect than the original drug telatazamine.

[0023] (3) The terazamine derivative RTPZ in the thermosensitive nanogel system of the present invention can achieve stronger DNA damage and mitochondrial damage, thereby leading to stronger cell apoptosis.

[0024] (4) The preparation process of the temperature-sensitive nanogel system provided by the present invention is simple, avoids complex carrier systems, and is suitable for industrial production. Attached Figure Description

[0025] Figure 1 These are fluorescence images of DNA damage in A-7 prepared according to the present invention under normal oxygen (21% O2) and hypoxia (1% O2) conditions; Figure 2 These are fluorescence images of mitochondrial membrane potential changes of A-7 prepared in this invention under normoxic (21% O2) and hypoxic (1% O2) conditions; Figure 3 These are images showing the cell viability of A-7 cells prepared according to this invention under normoxic (21% O2) and hypoxic (1% O2) conditions; Figure 4 These are images showing the inhibition of 3D cell spheroid growth of A-7 cells prepared according to this invention under normoxic (21% O2) and hypoxic (1% O2) conditions; Figure 5This is a schematic diagram showing the changes in G' and G'' of PIB nanogel itself and its thermosensitive nanogel systems with different contents of A-7 as a function of temperature. Detailed Implementation

[0026] The present invention will be further described below with reference to embodiments. However, it should be noted that the embodiments do not constitute a limitation on the scope of protection of the present invention.

[0027] Unless otherwise defined, the technical or scientific terms used in this invention shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0028] This invention discloses a thermosensitive nanogel system based on teirazamine derivatives. The thermosensitive nanogel system is composed of teirazamine derivative RTPZ, PIB nanogel and physiological saline, wherein the concentration of PIB nanogel is 25~40 mg / mL and the concentration of teirazamine derivative RTPZ is 2~6 mg / mL.

[0029] The structural formula of the tirazamine derivative RTPZ is: ; Where R is C 1-17 Straight-chain or branched alkyl groups, C 3-6 cycloalkyl, C 1-7 One of perfluoroalkyl, vinyl, ethynyl, substituted benzyl, 1-piperidinyl, and 4-morpholinoyl, wherein the benzyl group has a C-substituent on the benzene ring. 1-2 Alkyl, C 1-2 Alkoxy, trifluoromethyl.

[0030] Preferably, the concentration of the terazamine derivative RTPZ is 2 mg / mL, 4 mg / mL, or 6 mg / mL.

[0031] In some embodiments, the structure of R is: .

[0032] The above-mentioned method for preparing a thermosensitive nanogel system based on tirazamine derivatives includes the following steps: S1. Dissolve terazamine and an alkaline substance in a solvent and stir, then add alkyl acyl chloride to react, and continue stirring during the reaction; the molar ratio of terazamine, alkaline substance and alkyl acyl chloride is 1:(2-4):(1-1.5); After the reactions in S2 and S1 are completed, the solvent is removed by rotary evaporation, then dichloromethane is added for dissolution, followed by washing with saturated brine, drying with anhydrous sodium sulfate, and finally evaporation to remove the solvent, yielding the crude product. S3. The crude product was purified by column chromatography to obtain the terazamine derivative RTPZ; S4. Dissolve the terazamine derivative RTPZ and PIB nanogel in anhydrous ethanol, stir until homogeneous, remove the ethanol by rotary evaporation, and dry in a vacuum drying oven to obtain a mixture of PIB and RTPZ. S5. Prepare an RTPZ thermosensitive nanogel system by dissolving a mixture of PIB and RTPZ in physiological saline, wherein the concentration of PIB is 25~40 mg / mL and the concentration of RTPZ is 2~6 mg / mL.

[0033] This invention also discloses the application of the above-mentioned thermosensitive nanogel system based on teirazamine derivatives in the preparation of drugs for treating tumors.

[0034] RTPZ was prepared through the following Examples 1-22: Example 1 This invention provides a method for preparing tirazamine, comprising the following steps: S1. First, add 40 mL of tetrahydrofuran to the reaction flask, then add 1 eq of teirazamine and 3 eq of triethylamine, and stir with a stirrer; under an argon atmosphere, add 1.3 eq of 5,5-dimethylhexanoyl chloride to react, continue stirring during the reaction, and monitor the reaction by thin-layer chromatography until the starting material is completely converted. After the reactions in S2 and S1 are completed, 50 mL of dichloromethane is added to dissolve the product, then it is washed with 50 mL of saturated saline solution, dried with 20 g of anhydrous sodium sulfate, and finally the solvent is removed by rotary evaporation to obtain the crude product. S3. The crude product was purified by column chromatography using dichloromethane and ethyl acetate as eluents in a volume ratio of 5:1, yielding a yellow product, terazamine derivative RTPZ, namely 3-(5,5-dimethylhexanoylamino)-1,2,4-benzotriazine-1,4-diazoxide derivative, denoted as A-5, with a yield of 84.3% and a solubility of 13.20 mg / mL in iodized oil at room temperature.

[0035] The A-5 prepared in Example 1 was characterized by proton nuclear magnetic resonance spectroscopy and electrospray ionization mass spectrometry. 1 HNMR(600 MHz, CDCl3) δ 9.63 (s, 1H), 8.41-8.34 (m, 2H), 7.99-7.88 (m, 1H), 7.72-7.62 (m, 1H), 2.71-2.61 (m, 2H), 1.51-1.46 (m, 2H), 1.34-1.22(m, 2H),0.91-0.79 (s, 9H). ESI-MS, m / z:305.16[M+H]+ .

[0036] Example 2 A method for preparing terazamine is the same as in Example 1, except that 3,5,5-trimethylhexanoyl chloride is used instead of 5,5-dimethylhexanoyl chloride. Column chromatography purification yielded a yellow solid, namely a 3-(3,5,5-trimethylhexanoylamino)-1,2,4-benzotriazine-1,4-diazepine derivative, denoted as A-7. Yield: 79.3%, solubility in iodized oil at room temperature: 14.85 mg / mL.

[0037] The A-7 prepared in Example 2 was characterized by proton nuclear magnetic resonance spectroscopy and electrospray ionization mass spectrometry. 1 HNMR(600 MHz, CDCl3) δ 9.73 (s, 1H), 8.51-8.45 (m, 2H), 8.05-8.00 (m, 1H), 7.82-7.75 (m, 1H), 2.63-2.54 (m, 1H), 1.86-1.78 (m, 2H), 1.42-1.38 (m, 2H), 0.92-0.83 (m, 12H). ESI-MS, m / z:319.17[M+H] + .

[0038] Example 3 A method for preparing terazamine is the same as in Example 1, except that neodecanoyl chloride is used instead of 5,5-dimethylhexanoyl chloride. Column chromatography purification yields a yellow solid, namely a 3-neodecanamido-1,2,4-benzotriazine-1,4-diazepine derivative, denoted as A-9. Yield: 80.2%, solubility in iodized oil at room temperature: 16.32 mg / mL.

[0039] The A-9 prepared in Example 3 was characterized by proton nuclear magnetic resonance spectroscopy and electrospray ionization mass spectrometry. 1 HNMR(600 MHz, CDCl3) δ 9.84 (s, 1H), 8.61-8.53 (m, 2H), 8.14-8.08 (m, 1H), 7.93-7.82 (m, 1H), 2.64-2.30 (m, 2H), 1.83-1.73 (m, 2H), 1.52-1.43 (m, 2H), 1.33-1.21 (m, 2H), 1.12-1.03 (m, 2H), 0.91-0.82 (s, 9H). ESI-MS, m / z:333.27[M+H] + .

[0040] Example 4 A method for preparing terazamine is the same as in Example 1, except that 2-tert-butyl-3,3-dimethylbutyryl chloride is used instead of 5,5-dimethylhexanoyl chloride. Column chromatography purification yielded a yellow solid, namely a 3-(2-tert-butyl-3,3-dimethylbutyrylamino)-1,2,4-benzotriazine-1,4-diazepine derivative, denoted as A-10. Yield: 74.2%, solubility in iodized oil at room temperature: 18.30 mg / mL.

[0041] The A-10 prepared in Example 4 was characterized by proton nuclear magnetic resonance spectroscopy and electrospray ionization mass spectrometry. 1 HNMR(600 MHz, CDCl3) δ 9.79 (s, 1H), 8.65-8.55 (m, 2H), 8.14-8.03 (m, 1H), 7.83-7.74 (m, 1H), 1.72-1.63 (m, 1H), 0.99-0.87 (s, 18H). ESI-MS, m / z:333.23[M+H] + .

[0042] Example 5 A method for preparing terazamine is the same as in Example 1, except that oleoyl chloride is used instead of 5,5-dimethylhexanoyl chloride. Column chromatography purification yielded a yellow solid, namely a 3-oleamido-1,2,4-benzotriazine-1,4-diazepine derivative, denoted as A-12. Yield: 74.8%, solubility in iodized oil at room temperature: 8.97 mg / mL.

[0043] The A-12 prepared in Example 5 was characterized by proton nuclear magnetic resonance spectroscopy and electrospray ionization mass spectrometry. 1 HNMR(600 MHz, CDCl3) δ 9.64 (s, 1H), 8.51-8.41 (m, 2H), 8.04-7.98 (m, 1H), 7.79-7.72 (m, 1H), 5.43-5.29 (m, 2H), 2.79-2.70 (m, 2H), 2.11-1.93 (m, 4H),1.83-1.72(m, 2H), 1.39-1.22 (m, 20H), 0.91-0.85 (m, 3H).ESI-MS, m / z:443.30[M+H] + .

[0044] Example 6 A method for preparing terazamine is the same as in Example 1, except that cyclopropionyl chloride is used instead of 5,5-dimethylhexanoyl chloride. Column chromatography purification yields a yellow solid, namely a 3-cyclopropionylamino-1,2,4-benzotriazine-1,4-diazepine derivative, denoted as A-13. Yield: 50.2%, solubility in iodized oil at room temperature: 0.60 mg / mL.

[0045] The A-13 prepared in Example 6 was characterized by proton nuclear magnetic resonance spectroscopy and electrospray ionization mass spectrometry. 1 HNMR(600 MHz, CDCl3) δ 9.88 (s, 1H), 8.63-8.49 (m, 2H), 8.21-8.13 (m, 1H), 7.35-7.22 (m, 1H), 1.42-1.33 (m, 2H), 0.88-0.78 (m, 4H). ESI-MS, m / z:247.31[M+H] + .

[0046] Example 7 A method for preparing terazamine is the same as in Example 1, except that cyclobutyryl chloride is used instead of 5,5-dimethylhexanoyl chloride. Column chromatography purification yielded a yellow solid, namely a 3-cyclobutyrylamino-1,2,4-benzotriazine-1,4-diazepine derivative, denoted as A-14. Yield: 53.2%, solubility in iodized oil at room temperature: 0.95 mg / mL.

[0047] The A-14 prepared in Example 7 was characterized by proton nuclear magnetic resonance spectroscopy and electrospray ionization mass spectrometry. 1 HNMR(600 MHz, CDCl3) δ 9.86 (s, 1H), 8.51-8.43 (m, 2H), 8.24-8.18 (m, 1H), 7.95-7.84 (m, 1H), 3.14-3.03 (m, H), 2.01-1.76 (m, 4H), 1.60-1.51 (m, 2H).ESI-MS, m / z:261.56[M+H] + .

[0048] Example 8 A method for preparing terazamine is the same as in Example 1, except that cyclohexanoyl chloride is used instead of 5,5-dimethylhexanoyl chloride. Column chromatography purification yields a yellow solid, namely a 3-cyclohexanoylamino-1,2,4-benzotriazine-1,4-diazepine derivative, denoted as A-15. Yield: 67.6%, solubility in iodized oil at room temperature: 1.50 mg / mL.

[0049] The A-15 prepared in Example 8 was characterized by proton nuclear magnetic resonance spectroscopy and electrospray ionization mass spectrometry. 1 HNMR(600 MHz, CDCl3) δ 9.86 (s, 1H), 8.67-8.55 (m, 2H), 8.12-8.02 (m, 1H), 7.93-7.85 (m, 1H), 2.38 (m, 1H), 1.82-1.53 ​​(m, 4H), 1.53-1.41 (m, 4H), 1.43-1.41 (m, 2H). ESI-MS, m / z:289.56[M+H] + .

[0050] Example 9 A method for preparing terazamine is the same as in Example 1, except that trifluoroacetyl chloride is used instead of 5,5-dimethylhexanoyl chloride. Column chromatography purification yielded a yellow solid, namely a 3-trifluoroacetamido-1,2,4-benzotriazine-1,4-diazepine derivative, denoted as A-16. Yield: 34.3%, solubility in iodized oil at room temperature: 0.90 mg / mL.

[0051] The A-16 prepared in Example 9 was characterized by proton nuclear magnetic resonance spectroscopy and electrospray ionization mass spectrometry. 1 HNMR (600 MHz, CDCl3) δ 9.94 (s, 1H), 8.60-8.23 (m, 2H), 8.14-8.04 (m, 1H), 7.98-7.86 (m, 1H). ESI-MS, m / z:275.01[M+H] + .

[0052] Example 10 A method for preparing terazamine is the same as in Example 1, except that perfluorobutyryl chloride is used instead of 5,5-dimethylhexanoyl chloride. Column chromatography purification yielded a yellow solid, namely a 3-perfluorobutyrylamino-1,2,4-benzotriazine-1,4-diazepine derivative, denoted as A-17. Yield: 21.1%, solubility in iodized oil at room temperature: 13.50 mg / mL.

[0053] The A-17 prepared in Example 10 was characterized by proton nuclear magnetic resonance spectroscopy and electrospray ionization mass spectrometry. 1 HNMR (600 MHz, CDCl3) δ 9.94 (s, 1H), 8.68-8.55 (m, 2H), 8.24-8.08 (m, 1H), 7.83-7.65 (m, 1H). ESI-MS, m / z:375.27[M+H] + .

[0054] Example 11 A method for preparing terazamine is the same as in Example 1, except that perfluorobutyryl chloride is used instead of 5,5-dimethylhexanoyl chloride. Column chromatography purification yielded a yellow solid, namely a 3-perfluorohexanoylamino-1,2,4-benzotriazine-1,4-diazepine derivative, denoted as A-18. Yield: 22.1%, solubility in iodized oil at room temperature: 17.28 mg / mL.

[0055] The A-18 prepared in Example 11 was characterized by proton nuclear magnetic resonance spectroscopy and electrospray ionization mass spectrometry. 1 HNMR (600 MHz, CDCl3) δ 9.94 (s, 1H), 8.62-8.43 (m, 2H), 8.24-8.08 (m, 1H), 7.92-7.72 (m, 1H). ESI-MS, m / z:474.02[M+H] + .

[0056] Example 12 A method for preparing terazamine is the same as in Example 1, except that perfluorooctanoyl chloride is used instead of 5,5-dimethylhexanoyl chloride. Column chromatography purification yielded a yellow solid, namely a 3-perfluorooctanoylamino-1,2,4-benzotriazine-1,4-diazepine derivative, denoted as A-19. Yield: 27.7%, solubility in iodized oil at room temperature: 18.43 mg / mL.

[0057] The A-19 prepared in Example 12 was characterized by proton nuclear magnetic resonance spectroscopy and electrospray ionization mass spectrometry. 1 HNMR (600 MHz, CDCl3) δ 9.89 (s, 1H), 8.63-8.43 (m, 2H), 8.19-8.02 (m, 1H), 7.95-7.72 (m, 1H). ESI-MS, m / z:575.07[M+H] + .

[0058] Example 13 A method for preparing terazamine is the same as in Example 1, except that acryloyl chloride is used instead of 5,5-dimethylhexanoyl chloride. Column chromatography purification yields a yellow solid, namely a 3-acrylamido-1,2,4-benzotriazine-1,4-diazepine derivative, denoted as A-20. Yield: 72.9%, solubility in iodized oil at room temperature: 0.60 mg / mL.

[0059] The A-20 prepared in Example 13 was characterized by proton nuclear magnetic resonance spectroscopy and electrospray ionization mass spectrometry. 1HNMR(600 MHz, CDCl3) δ 9.84 (s, 1H), 8.61-8.53 (m, 2H), 8.14-8.08 (m, 1H), 7.93-7.82 (m, 1H), 6.64-6.49 (m, 1H), 6.03-5.93 (m, 1H), 5.72-5.63 (m, 1H).ESI-MS, m / z:233.02[M+H] + .

[0060] Example 14 A method for preparing terazamine is the same as in Example 1, except that propyneyl chloride is used instead of 5,5-dimethylhexanoyl chloride. Column chromatography purification yielded a yellow solid, 3-propyneylamino-1,2,4-benzotriazine-1,4-diazepine derivative, designated A-21. Yield: 78.2%, solubility in iodized oil at room temperature: 0.56 mg / mL.

[0061] The A-21 prepared in Example 14 was characterized by proton nuclear magnetic resonance spectroscopy and electrospray ionization mass spectrometry. 1 HNMR (600 MHz, CDCl3) δ 9.94 (s, 1H), 8.73-8.53 (m, 2H), 8.24-8.09 (m, 1H), 7.93-7.81 (m, 1H), 2.84-2.73 (m, 1H). ESI-MS, m / z:231.11[M+H] + .

[0062] Example 15 A method for preparing terazamine is the same as in Example 1, except that phenylacetyl chloride is used instead of 5,5-dimethylhexanoyl chloride. Column chromatography purification yielded a yellow solid, 3-phenylacetamido-1,2,4-benzotriazine-1,4-diazepine derivative, designated A-22. Yield: 68.8%, solubility in iodized oil at room temperature: 0.10 mg / mL.

[0063] The A-22 prepared in Example 15 was characterized by proton nuclear magnetic resonance spectroscopy and electrospray ionization mass spectrometry. 1 HNMR(600 MHz, CDCl3) δ 9.99 (s, 1H), 8.91-8.79 (m, 2H), 8.54-8.48 (m, 1H), 8.09-7.98 (m, 1H), 7.54-7.40 (m, 2H), 7.20-7.05 (m, 3H), 3.92-3.83 (m, 1H).ESI-MS, m / z:297.09 [M+H] + .

[0064] Example 16 A method for preparing terazamine is the same as in Example 1, except that p-methylphenylacetyl chloride is used instead of 5,5-dimethylhexanoyl chloride. Column chromatography purification yielded a yellow solid, namely a 3-p-methylphenylacetylamino-1,2,4-benzotriazine-1,4-diazepine derivative, denoted as A-23. Yield: 72.2%, solubility in iodized oil at room temperature: 1.49 mg / mL.

[0065] The A-23 prepared in Example 16 was characterized by proton nuclear magnetic resonance spectroscopy and electrospray ionization mass spectrometry. 1 ¹H NMR (600 MHz, CDCl₃) δ 9.94 (s, 1H), 8.81–8.69 (m, 2H), 8.34–8.28 (m, 1H), 7.99–7.88 (m, 1H), 7.44–7.31 (m, 2H), 7.13–7.01 (m, 3H), 3.99–3.88 (m, 1H), 2.23–2.11 (m, 1H). Melting point: 142.7 ℃. ESI-MS, m / z: 311.07 [M+H] + .

[0066] Example 17 A method for preparing terazamine is the same as in Example 1, except that p-ethylphenylacetyl chloride is used instead of 5,5-dimethylhexanoyl chloride. Column chromatography purification yielded a yellow solid, namely a 3-p-ethylphenylacetylamino-1,2,4-benzotriazine-1,4-diazepine derivative, denoted as A-24. Yield: 69.2%, solubility in iodized oil at room temperature: 1.30 mg / mL.

[0067] The A-24 prepared in Example 17 was characterized by proton nuclear magnetic resonance spectroscopy and electrospray ionization mass spectrometry. 1 HNMR(600 MHz, CDCl3) δ 9.94 (s, 1H), 8.81-8.73 (m, 2H), 8.22-8.18 (m, 1H), 7.99-7.85 (m, 1H), 7.44-7.29 (m, 2H), 7.05-6.93 (m, 2H), 3.92-3.82 (m, 2H), 2.72-2.58 (m, 2H), 1.18-0.99 (m, 3H). ESI-MS, m / z:325.09[M+H] + .

[0068] Example 18 A method for preparing terazamine is the same as in Example 1, except that 5,5-dimethylhexanoyl chloride is used instead of 4-methoxyphenylacetyl chloride. Column chromatography purification yields a yellow solid, namely a 3-(4-methoxyphenylacetamido)-1,2,4-benzotriazine-1,4-diazepine derivative, denoted as A-25. Yield: 80.2%, solubility in iodized oil at room temperature: 0.73 mg / mL.

[0069] The A-25 prepared in Example 18 was characterized by proton nuclear magnetic resonance spectroscopy and electrospray ionization mass spectrometry. 1 HNMR(600 MHz, CDCl3) δ 9.94 (s, 1H), 8.87-8.73 (m, 2H), 8.14-8.02 (m, 1H), 7.92-7.72 (m, 1H), 7.34-7.20 (m, 2H), 6.83-6.70 (m, 2H), 3.92-3.73 (m, 2H), 3.63-3.41 (m, 3H). ESI-MS, m / z:327.12[M+H] + .

[0070] Example 19 A method for preparing terazamine is the same as in Example 1, except that 4-ethoxyphenylacetyl chloride is used instead of 5,5-dimethylhexanoyl chloride. Column chromatography purification yields a yellow solid, namely a 3-(4-ethoxyphenylacetamido)-1,2,4-benzotriazine-1,4-diazepine derivative, designated A-26. Yield: 80.2%, solubility in iodized oil at room temperature: 0.94 mg / mL.

[0071] The A-26 prepared in Example 19 was characterized by proton nuclear magnetic resonance spectroscopy and electrospray ionization mass spectrometry. 1 HNMR(600 MHz, CDCl3) δ 9.91 (s, 1H), 8.54-8.33 (m, 2H), 8.10-7.98 (m, 1H), 7.79-7.62 (m, 1H), 7.34-7.20 (m, 2H), 6.83-6.73 (m, 2H), 4.02-3.93 (m, 2H), 3.83-3.71 (m, 2H), 1.42-1.23 (m, 3H). ESI-MS, m / z:341.26[M+H] + .

[0072] Example 20 A method for preparing terazamine is the same as in Example 1, except that 5,5-dimethylhexanoyl chloride is replaced with p-2-(4-(trifluoromethyl)phenylacetyl chloride. Column chromatography purification yields a yellow solid, namely a 3-(2-(4-(trifluoromethyl)phenyl)acetamido)-1,2,4-benzotriazine-1,4-diazepine derivative, designated A-27. Yield: 60.2%, solubility in iodized oil at room temperature: 1.50 mg / mL.

[0073] The A-27 prepared in Example 20 was characterized by proton nuclear magnetic resonance spectroscopy and electrospray ionization mass spectrometry. 1 HNMR(600 MHz, CDCl3) δ 9.94 (s, 1H), 8.60-8.51 (m, 2H), 8.24-8.18 (m, 1H), 7.95-7.83 (m, 1H), 7.54-7.40 (m, 2H), 7.23-7.11 (m, 2H), 3.82-3.67 (m, 2H).ESI-MS, m / z:365.09 [M+H] + .

[0074] Example 21 A method for preparing terazamine is the same as in Example 1, except that 1-piperidinyl chloride is used instead of 5,5-dimethylhexanoyl chloride. Column chromatography purification yielded a yellow solid, namely a 3-(1-piperidinylamino)-1,2,4-benzotriazine-1,4-diazepine derivative, designated A-28. Yield: 54.8%, solubility in iodized oil at room temperature: 0.95 mg / mL.

[0075] The A-28 prepared in Example 21 was characterized by proton nuclear magnetic resonance spectroscopy and electrospray ionization mass spectrometry. 1 HNMR(600 MHz, CDCl3) δ 9.64 (s, 1H), 8.60-8.52 (m, 2H), 8.12-8.01 (m, 1H), 7.83-7.70 (m, 1H), 3.64-3.30 (m, 4H), 1.82-1.63 (m, 4H), 1.52-1.42 (m, 2H).ESI-MS, m / z:290.10[M+H] + .

[0076] Example 22 A method for preparing terazamine is the same as in Example 1, except that 4-morpholinocarbonyl chloride is used instead of 5,5-dimethylhexanoyl chloride. Column chromatography purification yields a yellow solid, namely a 3-(4-morpholinocarbonylamide)-1,2,4-benzotriazine-1,4-diazepine derivative, designated A-29. Yield: 48.9%, solubility in iodized oil at room temperature: 0.63 mg / mL.

[0077] The A-29 prepared in Example 22 was characterized by proton nuclear magnetic resonance spectroscopy and electrospray ionization mass spectrometry. 1 HNMR(600 MHz, CDCl3) δ 9.68 (s, 1H), 8.64-8.55 (m, 2H), 8.18-8.03 (m, 1H), 7.97-7.79 (m, 1H), 3.65-3.47 (m, 4H), 3.33-3.13 (m, 4H). ESI-MS, m / z:292.08[M+H] + .

[0078] When the alkyl acyl chlorides are acetyl chloride, butyryl chloride, hexanoyl chloride, octanoyl chloride, nonanoyl chloride, decanoyl chloride, and dodecanoyl chloride, the operating steps as described in Example 1 are used, and the corresponding products are 3-acetamido-1,2,4-benzotriazine-1,4-diazoxide (denoted as A-1); 3-butyrylamido-1,2,4-benzotriazine-1,4-diazoxide (denoted as A-2); 3-hexanoylamido-1,2,4-benzotriazine-1,4-diazoxide (denoted as A-2); and 3-hexanoylamido-1,2,4-benzotriazine-1,4-diazoxide (denoted as A-2). Nitrogen oxides (denoted as A-3); 3-octanoylamino-1,2,4-benzotriazine-1,4-diazoxide (denoted as A-4); 3-nonanoylamino-1,2,4-benzotriazine-1,4-diazoxide (denoted as A-6); 3-decanoylamino-1,2,4-benzotriazine-1,4-diazoxide (denoted as A-8); 3-dodecanoylamino-1,2,4-benzotriazine-1,4-diazoxide (denoted as A-11).

[0079] DNA damage test The effects of TPZ and its derivative RTPZ on DNA damage in tumor cells were detected by γH2AX immunofluorescence assay. Figure 1As shown, the results indicate that under hypoxic conditions, tumor cells treated with either TPZ or RTPZ exhibited stronger green fluorescence signals than those under normoxic conditions, suggesting that both can induce more significant DNA double-strand breaks in a hypoxic environment. Further comparison revealed that the fluorescence intensity of the RTPZ-treated group was significantly higher than that of the TPZ-treated group under hypoxic conditions (1% O2), indicating that RTPZ had a stronger damaging effect on HepG-2 cell DNA than TPZ under hypoxic conditions (1% O2). These results suggest that RTPZ, obtained through structural modification of TPZ, can more effectively enhance its hypoxia-specific toxicity to tumor cells, providing experimental evidence for further development of targeted therapeutic strategies for hypoxic tumor regions.

[0080] Mitochondrial damage detection The effects of TPZ and RTPZ on mitochondrial function in HepG-2 cells were further evaluated by detecting mitochondrial membrane potential (ΔΨm) using the JC-1 fluorescent probe method. Figure 2 As shown in the figure, the results indicate that under hypoxic conditions (1% O2), the decrease in mitochondrial membrane potential in the RTPZ-treated group was significantly greater than that in the TPZ-treated group, manifested by a sharp decrease in the ratio of JC-1 polymers (red fluorescence) to monomers (green fluorescence), suggesting that RTPZ can more effectively disrupt the electrochemical gradient of the mitochondrial inner membrane. Corresponding to the DNA damage results, the mitochondrial membrane potential collapse and ROS burst induced by both TPZ and RTPZ were particularly pronounced under hypoxic conditions (1% O2), while only slight changes were observed under normoxic conditions (21% O2), further confirming the hypoxia selectivity of both treatments. Therefore, RTPZ may further enhance its DNA damage efficacy through its "mitochondrial oxidative stress amplifier" effect, which may be an important reason why its toxicity is significantly stronger than that of TPZ.

[0081] HepG-2 Liver Cancer Cell Detection Further qualitative and semi-quantitative analysis of the viability of HepG-2 cells was performed using a Calcein-AM and PI double staining kit and laser confocal microscopy. Microscopic observation revealed (…). Figure 3 Under hypoxic conditions (1% O2), scattered PI red fluorescence (dead cells) was observed in the TPZ-treated group, while the intensity of Calcein-AM green fluorescence (live cells) was reduced. The A7-treated group, however, exhibited more significant morphological changes, with a marked increase in the number of strongly PI-positive (red) cells in the field of view, clustered together, while the number of surviving green fluorescent cells decreased sharply, clearly indicating widespread cell death. This direct morphological evidence corroborates the high apoptosis rate detected by flow cytometry.

[0082] A7's growth inhibition detection on 3D cell spheres The A7 assay was used to evaluate the growth inhibition of 3D cell spheroids. The specific procedure was as follows: 1000 cells / well were seeded in a 96-well ultra-low adsorption cell culture plate to prepare a 3D cell spheroid model. After 3 days of culture, 3D cell spheroids with a size of approximately 200 μm were obtained. Subsequently, the 3D cell spheroids in the normoxic group were incubated in a medium containing 10% FBS and the drug, and then placed in a normoxic incubator (21% O2). The 3D cell spheroids in the hypoxic group were incubated in a medium containing 10% FBS and the drug, and then placed in a hypoxic incubator (1% O2). The cells were removed at 0, 2, and 4 days for photography, and the growth inhibition of the 3D cell spheroids was statistically analyzed using ImageJ software. Figure 4 As shown, compared to the continuous growth of tumor cell spheroids in the PBS-treated group from day 0 to day 4, TPZ and A7 showed significantly stronger tumor inhibition in 1% O2 than in 21% O2. Furthermore, since A7 is more toxic than TPZ, its hypoxic cytotoxicity showed a significantly stronger inhibitory effect on day 4.

[0083] Rheological testing The rheological properties of the A7-based thermosensitive nanogel system were tested, and the specific procedures are as follows: Thermosensitive nanogel systems with different A7 contents were prepared: composed of A7, PIB nanogel and physiological saline, with A7 contents of 2 mg / mL, 4 mg / mL and 6 mg / mL, respectively; the concentration of PIB nanogel was 25 mg / mL.

[0084] Thermosensitive nanogel systems with different A7 contents were tested using an advanced rotational rheometer to measure the temperature variations of G' and G''. The temperature range was 20-50 ℃, the heating rate was 1.0 ℃ / min, the frequency was 1 Hz, the shear stress was 0.1 Pa, the fixture was a 50 mm plate, and the plate spacing was 0.5 mm. Figure 5 It can be seen that as the A7 content increases, the storage modulus of G' and G'' gradually decreases at 37℃, while the gelation temperature (CGT) gradually increases, which is attributed to the destruction of the PIB gel network structure by the relatively hydrophobic properties of A7.

[0085] The thermosensitive nanogel system of the present invention is a novel embolization therapy delivery system that integrates thermosensitive nanogel embolization agent, hypoxia-activating prodrug, and sustained-release properties to construct a local, sustained-release, targeted hypoxic tumor treatment system for transcatheter arterial chemoembolization.

[0086] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A thermosensitive nanogel system based on tirazamine derivatives, characterized in that, The thermosensitive nanogel system consists of terazamine derivative RTPZ, PIB nanogel and physiological saline, wherein the concentration of PIB nanogel is 25~40 mg / mL and the concentration of terazamine derivative RTPZ is 2~6 mg / mL. The structural formula of the tirazamine derivative RTPZ is: ； Where R is C 1-17 Straight-chain or branched alkyl groups, C 3-6 cycloalkyl, C 1-7 One of perfluoroalkyl, vinyl, ethynyl, substituted benzyl, 1-piperidinyl, and 4-morpholinoyl, wherein the benzyl group has a C-substituent on the benzene ring. 1-2 Alkyl, C 1-2 Alkoxy, trifluoromethyl.

2. The thermosensitive nanogel system based on tirazamine derivatives according to claim 1, characterized in that, The concentration of the terazamine derivative RTPZ is 2 mg / mL, 4 mg / mL or 6 mg / mL.

3. The thermosensitive nanogel system based on tirazamine derivatives according to claim 1, characterized in that, The structural formula for R: 。 4. A method for preparing a thermosensitive nanogel system based on tirazamine derivatives as described in any one of claims 1 to 3, characterized in that, Includes the following steps: S1. Dissolve terazamine and an alkaline substance in a solvent and stir, then add alkyl acyl chloride to react, and continue stirring during the reaction; the molar ratio of terazamine, alkaline substance and alkyl acyl chloride is 1:(2-4):(1-1.5); After the reactions in S2 and S1 are completed, the solvent is removed by rotary evaporation, then dichloromethane is added for dissolution, followed by washing with saturated brine, drying with anhydrous sodium sulfate, and finally evaporation to remove the solvent, yielding the crude product. S3. The crude product was purified by column chromatography to obtain the terazamine derivative RTPZ; S4. Dissolve the terazamine derivative RTPZ and PIB nanogel in anhydrous ethanol, stir until homogeneous, remove the ethanol by rotary evaporation, and dry in a vacuum drying oven to obtain a mixture of PIB and RTPZ. S5. Prepare an RTPZ thermosensitive nanogel system by dissolving a mixture of PIB and RTPZ in physiological saline, wherein the concentration of PIB is 25~40 mg / mL and the concentration of RTPZ is 2~6 mg / mL.

5. The method for preparing a thermosensitive nanogel system based on tirazamine derivatives according to claim 4, characterized in that, The dosage of terazamine is 1 eq, the dosage of the alkaline substance is 2-4 eq, the dosage of the alkyl acyl chloride is 1-1.5 eq, the dosage of the solvent is 30-50 mL, the dosage of dichloromethane is 30-60 mL, the dosage of saturated saline is 30-60 mL, and the dosage of anhydrous sodium sulfate is 15-30 g.

6. The method for preparing a thermosensitive nanogel system based on tirazamine derivatives according to claim 4, characterized in that, The alkaline substance is one or a combination of sodium hydroxide, triethylamine, pyridine, and dimethylpyridine.

7. The method for preparing a thermosensitive nanogel system based on tirazamine derivatives according to claim 4, characterized in that, The solvent is one or a combination of tetrahydrofuran, dichloromethane, chloroform, N,N-dimethylformamide, acetonitrile, dimethyl ether, benzene, toluene, ethanol and methanol.

8. The method for preparing a thermosensitive nanogel system based on tirazamine derivatives according to claim 4, characterized in that, In S1, alkyl acyl chloride is added under an argon atmosphere, the reaction temperature is 0~30℃, and the reaction time is 2~12h. After the addition of alkyl acyl chloride, the reaction is monitored by thin-layer chromatography until the raw material is completely converted.

9. The method for preparing a thermosensitive nanogel system based on tirazamine derivatives according to claim 4, characterized in that, In S3, the eluent for column chromatography purification is dichloromethane and ethyl acetate, with a volume ratio of (4-6):

1.

10. The use of a thermosensitive nanogel system based on teirazamine derivatives as described in any one of claims 1 to 3 in the preparation of drugs for treating tumors.

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