Polypeptide-coupled tetravalent platinum prodrug as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biochemistry, and discloses a polypeptide-coupled tetravalent platinum prodrug and a preparation method and application thereof.The tetravalent platinum prodrug can specifically recognize tumor cell surface receptors by being coupled with a functional polypeptide with tumor cell recognition capacity or a derivative of the functional polypeptide, so that the tumor cell surface receptors can be specifically recognized; therefore, the prodrug is endowed with the targeting ability to tumor cells. Based on the targeting effect, the accumulation of the drug in a tumor target region can be improved, and the distribution of the drug in non-targeting tissues is reduced, so that the occurrence of adverse reactions is reduced. The polypeptide-coupled tetravalent platinum prodrug has good stability under physiological conditions, and can be converted into a bivalent platinum drug with cytotoxic activity under the action of a reducing environment in tumor cells so as to play an anti-tumor role. In addition, the tetravalent platinum prodrug can be self-assembled in a solution to form a nanoparticle structure to entrap the drug, so that the solubility and the stability of the drug are improved.

Description

Technical Field

[0001] This invention belongs to the field of biochemical technology, and in particular relates to a polypeptide-conjugated tetravalent platinum prodrug, its preparation method and application. Background Technology

[0002] Nanotechnology is a discipline that studies matter or structures at the 1–100 nm scale. It refers to the manipulation of atoms, molecules, or molecular clusters through specific microfabrication methods to rearrange and recombine them, forming novel substances or structures with nanoscale characteristics, and studying their properties and practical applications. Since its inception, nanotechnology has shown broad application prospects in many fields, including materials science, chemical engineering, biomedicine, environmental remediation, and food.

[0003] In the field of drug research, the continuous development and penetration of nanotechnology has driven the transformation of drug delivery systems, and nanomedicines have gradually become a research hotspot. Nanomedicines typically refer to formulations formed by combining drugs with nanoscale carrier materials, such as polymeric nanoparticles, nanospheres, or nanocapsules, through physical encapsulation or chemical coupling. Their particle size is generally in the nanometer range. Nanoparticle technology can improve the solubility, stability, and in vivo distribution of drugs to a certain extent.

[0004] However, most existing nanomedicine systems rely on exogenous nanocarriers, whose preparation processes are typically complex, involving multiple assembly or encapsulation steps, limiting process reproducibility and yield. Furthermore, some nanocarriers exhibit insufficient stability in vivo, easily undergoing dissociation or non-specific release, affecting the controlled release of drugs. In addition, the carrier materials themselves may introduce potential biosafety issues, limiting their further applications.

[0005] Platinum-based chemotherapy drugs are commonly used in clinical practice and play an important role in the treatment of various solid tumors. However, their clinical application still faces problems such as poor selectivity and significant systemic toxicity. Tetravalent platinum prodrugs, due to their relatively high stability and ability to be reduced within tumor cells to divalent platinum drugs with cytotoxic activity, are considered a promising form of platinum-based drug. However, existing tetravalent platinum prodrugs generally lack active targeting capabilities against tumor cells; their in vivo distribution still relies on passive accumulation, making it difficult to effectively distinguish between tumor tissue and normal tissue.

[0006] Therefore, how to simplify the preparation process while introducing functional structural units with tumor cell recognition capabilities to endow tetravalent platinum prodrugs with good targeting, stability and controllable release performance remains a technical problem that urgently needs to be solved in this field.

[0007] Based on the above analysis, the problems and shortcomings of the existing technology are as follows: Existing tetravalent platinum prodrugs generally lack the ability to actively target tumor cells, and their distribution in vivo still relies on passive enrichment, making it difficult to effectively distinguish between tumor tissue and normal tissue. Summary of the Invention

[0008] To address the problems existing in the prior art, this invention provides a polypeptide-conjugated tetravalent platinum prodrug, its preparation method, and its application.

[0009] This invention is achieved as follows: a polypeptide-conjugated tetravalent platinum prodrug structure is shown in general formula I:

[0010] Formula I.

[0011] Another object of the present invention is to provide a class of polypeptide derivative-conjugated tetravalent platinum prodrugs of general formula I comprising:

[0012] Formula II;

[0013] Formula III;

[0014] Formula IV;

[0015] Formula V;

[0016] Formula VI;

[0017] Formula VII.

[0018] Another object of the present invention is to provide a method for preparing a polypeptide-conjugated tetravalent platinum prodrug as follows: Step 1, Pt IV Synthesis of -OH: Cisplatin was suspended in water; the mixture was stirred overnight at 40°C to form a clear solution; after cooling to room temperature, a large number of needle-like crystals precipitated; the product was washed several times with acetone and dried using a desiccator to separate CisPt. IV -OH; Step 2, Mal-Pt IV Synthesis of -OH: CisPt IV-OH was suspended in anhydrous DMSO; then 5-maleimide valerate (NHS) was added to the mixture, and the mixture was stirred at 60°C for 48 hours; the filtrate was evaporated under reduced pressure, and the crude product was dispersed in methanol precipitated in diethyl ether, and then dried under vacuum to give a white solid, Mal-Pt. IV -OH; Step 3, Mal-Pt IV Synthesis of -C16: Mal-Pt IV -OH was suspended in 10 mL of anhydrous DMF; then hexadecyl isocyanate was added; the reaction mixture was stirred overnight at 75 °C until a clear yellow solution was obtained; the solvent was removed under reduced pressure, and Mal-Pt was recrystallized from MeOH. IV -C16, yields a yellow solid; Step 4, RGD-Pt IV Synthesis: RGD-Pt IV Originating from Mal-Pt IV The addition reaction of -C16 and peptide, firstly, Mal-Pt IV -C16 was dissolved in anhydrous DMF; the peptide was added to the mixture; the reaction was then carried out for 48 hours; the solvent in the reaction solution was evaporated to dryness; methanol was then added to the mixture and shaken; after centrifugation to remove insoluble matter, recrystallization was performed; finally, RGD-Pt was obtained by preparative liquid chromatography. IV The product; Step 5, AAG-Pt IV Synthesis: AAG-Pt IV Originating from Mal-Pt IV The addition reaction of -C16 and peptide, firstly, Mal-Pt IV -C16 was dissolved in anhydrous DMF; the peptide was added to the mixture; the reaction was then carried out for 48 hours; the solvent in the reaction solution was evaporated to dryness; methanol was then added to the mixture and shaken; after centrifugation to remove insoluble matter, recrystallization was performed; finally, AAG-Pt was obtained by preparative liquid chromatography. IV The product; Step 6, Pt IV -COOH synthesis: Pt IV -OH groups were suspended in anhydrous DMF; then succinic anhydride was added to the mixture, and the mixture was stirred at room temperature for 48 hours; the filtrate was evaporated under reduced pressure, and the crude product was dispersed in methanol precipitated in diethyl ether, and then dried under vacuum to give CisPt as a white solid. IV -COOH; Step 7, C16-PtIV Synthesis of -COOH: Pt IV -COOH was suspended in 10 mL of anhydrous DMF; then hexadecyl isocyanate was added; the reaction mixture was stirred overnight at 75 °C until a clear yellow solution was obtained; the solvent was removed under reduced pressure, and C16-Pt(IV)-COOH was recrystallized from MeOH to give a yellow solid; Step 8, Pt IV -DG synthesis: C16-CisPt IV -COOH, DCC and 2-DG were suspended in 10 mL of anhydrous DMF; the mixture was stirred in ice water for half an hour, and then DMAP was added; the reaction was continued at room temperature for 2 days, and after evaporating the DMF in the mixture, MeOH was added to the mixture; the precipitate was removed by centrifugation, and C16-Pt(IV)-COOH was recrystallized with MeOH and freeze-dried to give a white product; Step 9, Synthesis of Biotin-N-hydroxysuccinimide ester: Biotin, EDC, and NHS were dissolved in 15 mL of anhydrous DMF; the solution was stirred at room temperature for 24 h, and then added to 100 mL of an ice-water mixture; the precipitate was collected, washed twice with water and methanol, and then air-dried to obtain a white powder of Biotin-N-hydroxysuccinimide ester. Step 10, OPt IV Synthesis of -OH: Oxaliplatin was added to a round-bottom flask containing 3 ml of H2O2 and stirred at room temperature for 4 h. The H2O2 mixture was then transferred to a 250 mL flask. The liquid remaining on the walls of the smaller flask was rinsed off with DMF and then transferred to the 250 mL flask. Subsequently, about 100 mL of DMF was added to the flask and stirred. The mixture was filtered and the precipitate was collected. The filter cake was washed three times with ice-cold ether and dried to obtain a white product. Step 11, OPt IV -BT synthesis: OPt IV -OH and Biotin-N-hydroxysuccinimide ester were dissolved in 5 mL of DMSO, and the mixture was stirred at 60°C for 12 h; unreacted OPt was separated by centrifugation. IV -OH; then add 1 mL of methanol, add 30 mL of ice-cold diethyl ether to disperse and wash the above product; after centrifugation, collect the precipitate, freeze-dry to obtain a white product; Step 12, Lipo-OPt IV -BT synthesis: Weigh out OPt IV -BT and hexadecyl isocyanate were added to a round-bottom flask containing 10 mL DMF and heated and stirred at 75°C for 8 h. Subsequently, DMF in the reaction system was removed by rotary evaporation, and the remaining residue was recrystallized from methanol. After freeze-drying, a white product was obtained.

[0019] Furthermore, the Pt IV The chemical formula for -OH is: Cl2H8N2O2Pt; Mal-Pt IV The chemical formula for -OH is: C9H 17 Cl2N3O5Pt; Mal-Pt IV The chemical formula for -C16 is: C 26 H 50 Cl2N4O6Pt; RGD-Pt IV The chemical formula is: C 56 H 95 Cl2N 13 O 14 Pt; AAG-Pt IV The chemical formula is: C 40 H 76 Cl2N8O 11 Pt; Pt IV The chemical formula for -COOH is: C4H 12 Cl2N2O5Pt; C16-Pt IV The chemical formula for -COOH is: C 21 H 44 Cl2N3O6Pt; Pt IV The chemical formula of -DG is: C 27 H 55 Cl2N3O 10 Pt; The chemical formula of biotin-N-hydroxysuccinimide ester is: C 14 H 19 N3O5S; OPt IV The chemical formula for -OH is: C8H 16 N2O6PtS; OPt IV The chemical formula of -BT is: C 18 H 30 N4O8PtS; Lipo-OPtIV The chemical formula of -BT is: C 35 H 63 N5O9PtS.

[0020] Another object of the present invention is to provide the application of a polypeptide-conjugated tetravalent platinum prodrug in the preparation of a cancer cell inhibitory drug.

[0021] Based on the above technical solutions and the technical problems solved, the advantages and positive effects of the technical solution to be protected by this invention are as follows: This invention creatively proposes an active-targeting, self-assembled tetravalent platinum prodrug nanosystem based on integrated molecular design. This approach integrates a tumor-specific targeting ligand, a microenvironment-responsive release unit, and a tetravalent platinum core into a single molecule, achieving a simplified preparation process (one-step self-assembly, high drug loading) while endowing the nanodrug with multiple functions including active targeting recognition, blood circulation stabilization, and tumor-specific activation and release. Research data show that this system significantly improves drug accumulation in tumor tissues and efficiently releases the active drug in the tumor microenvironment, thus exhibiting potent anti-tumor effects in in vitro and in vivo models. Simultaneously, systemic toxicity is significantly reduced, successfully solving the technical challenge of synergistically improving targeting, controlled release, and biosafety. This provides a novel strategy for platinum-based drug delivery that combines innovation with clinical application potential.

[0022] The tetravalent platinum prodrug conjugated with a polypeptide derivative provided by this invention introduces a functional polypeptide or its derivative at the axial position of the tetravalent platinum, thereby enabling the tetravalent platinum prodrug to possess both good stability and tumor cell recognition ability. The functional polypeptide can interact with relevant receptors on the surface of tumor cells, thus endowing the tetravalent platinum prodrug with the ability to target tumor cells. This is beneficial for increasing drug accumulation in the tumor target area, reducing its distribution in non-target tissues, and consequently reducing the occurrence of adverse reactions.

[0023] Furthermore, the tetravalent platinum prodrug conjugated with the polypeptide derivative described in this invention can self-assemble into nanoparticle structures in solution, encapsulating drug molecules and improving drug solubility and system stability. Compared with existing delivery systems that rely on exogenous nanocarriers, this tetravalent platinum prodrug can form a nanoscale structure without introducing additional carriers. Its preparation method is relatively simple, reproducible, and yields a highly stable product, showing promising application prospects.

[0024] (1) The expected benefits and commercial value of the technical solution of this invention after transformation are as follows: This technical solution successfully overcomes the core defects of existing platinum drugs, such as poor targeting, complex preparation, and high toxicity, by constructing an integrated, actively targeted, self-assembled tetravalent platinum prodrug nanosystem. Its expected benefits and commercial value after transformation are enormous: First, with its significant "synergistic and toxicity-reducing" advantages, this product is expected to quickly establish a high-end position in the vast platinum drug market, gaining a significant market share and pricing power. Second, its simplified "one-step" self-assembly process significantly reduces production costs and development risks. More importantly, this technology builds a strong patent barrier and has the potential to be scalable into a universal delivery platform for different targets and drugs, which can not only foster a series of product pipelines but also generate revenue early through licensing partnerships. Therefore, this invention not only represents a highly competitive new anticancer drug but also an innovative platform capable of generating sustainable commercial value and leading the direction of precision drug delivery, possessing extremely high investment return potential and strategic significance.

[0025] (2) The technical solution of this invention fills a technical gap in the industry both domestically and internationally: The technical solution of this invention, for the first time, achieves a single-molecule integrated design, enabling a tetravalent platinum prodrug to simultaneously possess active targeting, tumor microenvironment-responsive release, and stable self-assembly capabilities without the need for an exogenous carrier. This successfully overcomes the key bottleneck of the difficulty in synergistically optimizing "process complexity," "functional comprehensiveness," and "clinical safety," providing a new technical paradigm and a practical solution for developing next-generation highly effective and low-toxicity platinum-based chemotherapy drugs.

[0026] (3) Whether the technical solution of the present invention solves the technical problem that people have long wanted to solve but have never been able to solve successfully: This invention fundamentally solves a long-standing contradiction in the field of platinum-based drug delivery: how to simultaneously achieve efficient active targeting, stable in vivo delivery, and precise, controllable release of drugs at tumor sites without relying on complex exogenous carriers. This challenge has long constrained the development of this field because past technologies often only made progress in one aspect, failing to achieve multifunctional synergy while simplifying the process and ensuring safety. This invention, through an integrated design paradigm of "drug self-carrier," systematically overcomes this series of mutually exclusive challenges, truly achieving the long-desired clinical goal of "enhanced efficacy and reduced toxicity." Attached Figure Description

[0027] Figure 1 This is a flowchart of the preparation method of the polypeptide-conjugated tetravalent platinum prodrug provided in the embodiments of the present invention.

[0028] Figure 2 The tumor-targeting nanomicelles (RGD-Pt) provided in this embodiment of the invention IV Particle size distribution diagram.

[0029] Figure 3 This is a diagram illustrating the cancer cell inhibition effect of tumor-targeting nanomicelles provided in an embodiment of the present invention.

[0030] Figure 4 This is a graph showing the relationship between the effects of different platinum complexes on cell activity and platinum concentration, as provided in the embodiments of the present invention. Detailed Implementation

[0031] 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. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0032] The structure of the polypeptide-conjugated tetravalent platinum prodrug provided in this embodiment of the invention is shown in general formula I:

[0033] Formula I.

[0034] The present invention provides a class of polypeptide derivative-conjugated tetravalent platinum prodrugs of general formula I, comprising:

[0035] Formula II;

[0036] Formula III;

[0037] Formula IV;

[0038] Formula V;

[0039] Formula VI;

[0040] Formula VII.

[0041] like Figure 1 As shown in the embodiment of the present invention, a method for preparing a polypeptide-conjugated tetravalent platinum prodrug is as follows: S101, Pt IV Synthesis of -OH: Cisplatin was suspended in water; the mixture was stirred overnight at 40°C to form a clear solution; after cooling to room temperature, a large number of needle-like crystals precipitated; the product was washed several times with acetone and dried using a desiccator to separate CisPt. IV -OH; S102, Mal-Pt IV Synthesis of -OH: CisPt IV -OH was suspended in anhydrous DMSO; then 5-maleimide valerate (NHS) was added to the mixture, and the mixture was stirred at 60°C for 48 hours; the filtrate was evaporated under reduced pressure, and the crude product was dispersed in methanol precipitated in diethyl ether, and then dried under vacuum to give a white solid, Mal-Pt. IV -OH; S103, Mal-Pt IV Synthesis of -C16: Mal-Pt IV -OH was suspended in 10 mL of anhydrous DMF; then hexadecyl isocyanate was added; the reaction mixture was stirred overnight at 75 °C until a clear yellow solution was obtained; the solvent was removed under reduced pressure, and Mal-Pt was recrystallized from MeOH. IV -C16, yields a yellow solid; S104, RGD-Pt IV Synthesis: RGD-Pt IV Originating from Mal-Pt IV The addition reaction of -C16 and peptide, firstly, Mal-Pt IV -C16 was dissolved in anhydrous DMF; the peptide was added to the mixture; the reaction was then carried out for 48 hours; the solvent in the reaction solution was evaporated to dryness; methanol was then added to the mixture and shaken; after centrifugation to remove insoluble matter, recrystallization was performed; finally, RGD-Pt was obtained by preparative liquid chromatography. IV The product; S105, AAG-Pt IV Synthesis: AAG-Pt IV Originating from Mal-Pt IV The addition reaction of -C16 and peptide, firstly, Mal-Pt IV -C16 was dissolved in anhydrous DMF; the peptide was added to the mixture; the reaction was then carried out for 48 hours; the solvent in the reaction solution was evaporated to dryness; methanol was then added to the mixture and shaken; after centrifugation to remove insoluble matter, recrystallization was performed; finally, AAG-Pt was obtained by preparative liquid chromatography. IV The product; S106, Pt IV -COOH synthesis: Pt IV -OH groups were suspended in anhydrous DMF; then succinic anhydride was added to the mixture, and the mixture was stirred at room temperature for 48 hours; the filtrate was evaporated under reduced pressure, and the crude product was dispersed in methanol precipitated in diethyl ether, and then dried under vacuum to give CisPt as a white solid. IV-COOH; S107, C16-Pt IV Synthesis of -COOH: Pt IV -COOH was suspended in 10 mL of anhydrous DMF; then hexadecyl isocyanate was added; the reaction mixture was stirred overnight at 75 °C until a clear yellow solution was obtained; the solvent was removed under reduced pressure, and C16-Pt(IV)-COOH was recrystallized from MeOH to give a yellow solid; S108, Pt IV -DG synthesis: C16-CisPt IV -COOH, DCC and 2-DG were suspended in 10 mL of anhydrous DMF; the mixture was stirred in ice water for half an hour, and then DMAP was added; the reaction was continued at room temperature for 2 days, and after evaporating the DMF in the mixture, MeOH was added to the mixture; the precipitate was removed by centrifugation, and C16-Pt(IV)-COOH was recrystallized with MeOH and freeze-dried to give a white product; Synthesis of S109, Biotin-N-hydroxysuccinimide ester: Biotin, EDC, and NHS were dissolved in 15 mL of anhydrous DMF; the solution was stirred at room temperature for 24 h, and then added to 100 mL of an ice-water mixture; the precipitate was collected, washed twice with water and methanol, and then air-dried to obtain a white powder of Biotin-N-hydroxysuccinimide ester. S110, OPt IV Synthesis of -OH: Oxaliplatin was added to a round-bottom flask containing 3 ml of H2O2 and stirred at room temperature for 4 h. The H2O2 mixture was then transferred to a 250 mL flask. The liquid remaining on the walls of the smaller flask was rinsed off with DMF and then transferred to the 250 mL flask. Subsequently, about 100 mL of DMF was added to the flask and stirred. The mixture was filtered and the precipitate was collected. The filter cake was washed three times with ice-cold ether and dried to obtain a white product. S111,OPt IV -BT synthesis: OPt IV -OH and Biotin-N-hydroxysuccinimide ester were dissolved in 5 mL of DMSO, and the mixture was stirred at 60°C for 12 h; unreacted OPt was separated by centrifugation. IV -OH; then add 1 mL of methanol, add 30 mL of ice-cold diethyl ether to disperse and wash the above product; after centrifugation, collect the precipitate, freeze-dry to obtain a white product; S112, Lipo-OPt IV -BT synthesis: Weigh out OPt IV -BT and hexadecyl isocyanate were added to a round-bottom flask containing 10 mL DMF and heated and stirred at 75°C for 8 h. Subsequently, DMF in the reaction system was removed by rotary evaporation, and the remaining residue was recrystallized from methanol. After freeze-drying, a white product was obtained.

[0042] The Pt provided in the embodiments of the present invention IV The chemical formula for -OH is: Cl2H8N2O2Pt; Mal-Pt IV The chemical formula for -OH is: C9H 17 Cl2N3O5Pt; Mal-Pt IV The chemical formula for -C16 is: C 26 H 50 Cl2N4O6Pt; RGD-Pt IV The chemical formula is: C 56 H 95 Cl2N 13 O 14 Pt; AAG-Pt IV The chemical formula is: C 40 H 76 Cl2N8O 11 Pt; Pt IV The chemical formula for -COOH is: C4H 12 Cl2N2O5Pt; C16-Pt IV The chemical formula for -COOH is: C 21 H 44 Cl2N3O6Pt; Pt IV The chemical formula of -DG is: C 27 H 55 Cl2N3O 10 Pt; The chemical formula of biotin-N-hydroxysuccinimide ester is: C 14 H 19 N3O5S; OPt IV The chemical formula for -OH is: C8H 16 N2O6PtS; OPt IV The chemical formula of -BT is: C 18 H30 N4O8PtS; Lipo-OPt IV The chemical formula of -BT is: C 35 H 63 N5O9PtS.

[0043] The application of the polypeptide-conjugated tetravalent platinum prodrug provided in this invention in the preparation of cancer cell inhibitory drugs.

[0044] like Figure 2 As shown, in this embodiment of the invention, tumor-targeting nanomicelles (RGD-Pt) IV Particle size distribution diagram; like Figure 3 The diagram shows the cancer cell inhibition effect of tumor-targeting nanomicelles in an embodiment of the present invention.

[0045] like Figure 4 The graph shown illustrates the relationship between the effect of different platinum complexes on cell viability and platinum concentration in embodiments of the present invention. Example 1 (corresponding formula II: RGD-Pt) IV ) This embodiment provides a tetravalent platinum prodrug conjugated to an RGD peptide. Mal-Pt IV -C16 was dissolved in anhydrous DMF, and a thiol-containing cyclic RGD peptide was added. The mixture was reacted at room temperature, allowing the maleimide group to undergo an addition reaction with the thiol group in the peptide to form a thioether bond. After the reaction, the solvent was removed under reduced pressure, and the mixture was recrystallized from methanol. The product was then centrifuged, dried, and purified by preparative liquid chromatography to obtain a pale yellow solid, RGD-Pt. IV .

[0046] The obtained RGD-Pt IV It exhibits good dispersibility in PBS buffer and can gradually release cisplatin in a simulated reducing environment (containing glutathione). It shows significant selective inhibition of tumor cells expressing αvβ3 integrin, indicating that the structure can balance stability and targeting, making it suitable for tumor-targeted chemotherapy applications.

[0047] Example 2 (corresponding formula III: AAG-Pt) IV ) This embodiment provides a tetravalent platinum prodrug conjugated with an AAG peptide. Mal-Pt IV- C16 was dissolved in anhydrous DMF, and a thiol-modified AAG peptide was added. The mixture was stirred at room temperature under nitrogen protection for 48 hours, allowing the peptide to covalently link the thiol groups to the maleimide groups. After the reaction was complete, the solvent was evaporated, methanol was added to precipitate the peptide, and the mixture was washed and recrystallized to obtain a white solid, AAG-Pt. IV .

[0048] This product is stable in the aqueous phase and can release active platinum centers in a reducing environment. It has an inhibitory effect on a variety of cancer cell lines. At the same time, because the AAG peptide has an affinity for the tumor microenvironment, it can enhance the drug’s accumulation ability in tumor tissue, thereby improving efficacy and reducing toxicity to normal tissues.

[0049] Example 3 (corresponding to formula IV: another configuration AAG-Pt) IV ) This embodiment provides an AAG peptide conjugated with a tetravalent platinum prodrug with a different structural configuration. It employs a Pt-based prodrug with oxaliplatin oxide as the core. IV The -OH intermediate is reacted with succinic anhydride to introduce a carboxyl group, followed by grafting of an ester chain with hexadecyl isocyanate. Subsequently, it is activated by maleimide and coupled with a thiolated AAG peptide to obtain esterified -AAG-Pt. IV derivative.

[0050] The obtained compound is both lipid-soluble and peptide-targeting, and can enter tumor cells through a dual mechanism of passive diffusion across the cell membrane and receptor-mediated endocytosis. It then reduces and releases active platinum ions within the cell, exhibiting strong cytotoxic activity and a long in vivo circulation time.

[0051] Example 4 (corresponding formula V: DG-Pt) IV ) This embodiment provides a 2-deoxy-D-glucose-conjugated tetravalent platinum prodrug. C16-Pt IV -COOH reacts with DCC, DMAP, and 2-DG in anhydrous DMF to form an ester bond structure. After the reaction, the solvent is removed and the mixture is recrystallized from methanol to give a white solid, DG-Pt. IV .

[0052] This product can enter cells by taking advantage of the high glucose uptake characteristics of tumor cells, and release platinum active centers in a reducing environment, thereby showing a preferential killing effect on high metabolic tumor cells and improving drug selectivity.

[0053] Example 5 (corresponding formula VI: Biotin-Pt) IV ) This embodiment provides a biotin-conjugated tetravalent platinum prodrug. First, a biotin-NHS activated ester is prepared, then combined with OPt... IV -OH reacts in DMSO to form amide bonds linked to OPt. IV -BT, then grafted with lipid chains via hexadecyl isocyanate, yields esterified biotin-Pt. IV product.

[0054] This product can bind to and be endocytosed by tumor cells that highly express biotin receptors, enhancing the drug uptake efficiency in tumor tissues. At the same time, the introduction of lipid chains improves membrane permeability and in vivo stability.

[0055] Example 6 (corresponding formula VII: Biotin-lipidized Pt) IV ) This embodiment provides a tetravalent platinum prodrug containing both biotin and lipid chain modification. OPt IV -BT reacts with hexadecyl isocyanate to give Lipo-OPt IV -BT was purified by recrystallization to obtain the target product.

[0056] This product exhibits good plasma stability and strong tumor-targeting enrichment ability, and shows higher inhibitory effects than unmodified platinum drugs in in vitro tumor cell models.

[0057] Example 7 (Comparative Structure Coverage: Different Lipid Chain Lengths) Replace hexadecyl isocyanate with dodecyl isocyanate, and prepare the corresponding peptides or small molecule conjugated Pt according to a similar method as in Examples 1-6. IV derivative.

[0058] The obtained compounds showed that lipid chain length can regulate the lipid solubility and cellular uptake efficiency of the drug, thus further demonstrating the good scalability of this technical route.

[0059] Example 8 (In vitro stability verification) Dissolve any of the above products in PBS (pH 7.4) and a buffer containing 5 mM glutathione, incubate at 37°C, and monitor their stability and reductive release behavior by HPLC.

[0060] The results show that this type of tetravalent platinum prodrug is stable in a neutral environment and gradually transforms into an active divalent platinum form in a reducing environment, which is consistent with the prodrug design purpose.

[0061] Example 9 (Cell Inhibition Experiment) The above products were added to various cancer cell line culture systems, and changes in cell viability were measured.

[0062] The results showed that this type of polypeptide or small molecule conjugated tetravalent platinum prodrug had a significant inhibitory effect on cancer cells and low toxicity to normal cells.

[0063] Example 10 (Application Verification) Any of the above products was used in an in vitro cancer cell inhibition model to observe the proportion of apoptosis and the degree of cell proliferation inhibition.

[0064] The results demonstrate that these compounds are suitable as antitumor active ingredients in cancer-suppressing formulations.

[0065] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any modifications, equivalent substitutions, and improvements made by those skilled in the art within the scope of the technology disclosed in the present invention, and within the spirit and principles of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A polypeptide-conjugated tetravalent platinum prodrug, characterized in that, The tetravalent platinum prodrug comprises a tetravalent platinum center, two axial ligands, and a polypeptide molecule covalently linked together, wherein at least one of the axial ligands contains a maleimide group or a carboxylic acid ester group, and the polypeptide is linked to the tetravalent platinum center by an addition or condensation reaction with the axial ligands via a thiol or amino group.

2. The polypeptide-conjugated tetravalent platinum prodrug as described in claim 1, characterized in that, The tetravalent platinum center is derived from the tetravalent platinum structure obtained by oxidizing cisplatin or oxaliplatin.

3. A targeted polypeptide-conjugated tetravalent platinum prodrug, characterized in that, The polypeptide is a polypeptide that recognizes receptors on the surface of tumor cells. The polypeptide is linked to an axial ligand at the center of a tetravalent platinum via a thioether bond or an amide bond, thereby forming a tetravalent platinum prodrug with targeted recognition capability.

4. The targeted peptide-conjugated tetravalent platinum prodrug as described in claim 3, characterized in that, The polypeptide is a polypeptide containing arginine, glycine and aspartic acid sequences or a polypeptide containing alanine, alanine and glycine sequences.

5. A tetravalent platinum prodrug conjugated with an esterified polypeptide, characterized in that, The tetravalent platinum prodrug is further linked to an aliphatic alkyl chain on the axial ligand, so that the resulting tetravalent platinum prodrug has both a lipophilic structure and a polypeptide targeting structure.

6. The tetravalent platinum prodrug conjugated with an esterified polypeptide as described in claim 5, characterized in that, The aliphatic alkyl chain has 16 carbon atoms.

7. A glycosyl-modified tetravalent platinum prodrug, characterized in that, The axial ligand of the tetravalent platinum prodrug is linked to a glucose derivative via an ester bond, giving the resulting tetravalent platinum prodrug tumor metabolic targeting properties.

8. A method for preparing a tetravalent platinum prodrug, characterized in that, Includes the following steps: The divalent platinum drug is converted into a tetravalent platinum hydroxyl intermediate under oxidative conditions; The tetravalent platinum hydroxyl intermediate is reacted with a compound containing an active ester group or an isocyanate group to form an axially modified tetravalent platinum intermediate; The axially modified tetravalent platinum intermediate is subjected to an addition or condensation reaction with a polypeptide containing a thiol or amino group to obtain a polypeptide-conjugated tetravalent platinum prodrug.

9. The preparation method according to claim 8, characterized in that, The molar amount of the added polypeptide is the same as the molar amount of the tetravalent platinum intermediate.

10. A pharmaceutical composition comprising the tetravalent platinum prodrug as described in any one of claims 1 to 7, characterized in that, The pharmaceutical composition is used to inhibit the growth of cancer cells or induce apoptosis in cancer cells.

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