A class of prodrug structures with ROS signal response properties, their preparation methods and applications

Abstract

This invention relates to the field of biomedicine, specifically to a class of prodrug structures with ROS signal-responsive properties, their preparation methods, and applications. The prodrug molecules with ROS signal-responsive properties developed in this invention can rapidly release bioactive molecules in ROS-rich environments, achieving targeted therapeutic effects. This method has good universality and has been verified to be able to prodrugize various molecules, including four approved drug molecules, improving drug solubility, biocompatibility, reducing cytotoxicity, and enhancing targeted therapeutic effects. Based on the structural characteristics of these molecules, they can also complex with PVA hydrogels to further achieve responsive and controllable drug release.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to an aromatic quaternary ammonium salt prodrug with ROS signal response bond breaking function, its preparation method, and its application. Background Technology

[0002] The information disclosed in this background section is intended only to enhance some understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that the information constitutes prior art known to those skilled in the art.

[0003] Reactive oxygen species (ROS) are a series of chemically reactive oxygen-containing molecules, including hydrogen peroxide (H₂O₂) and superoxide anion (O₂). 2- ), hydroxyl radical (·OH), hypochlorite ion (OCl) - ) and singlet oxygen ( 1 Reactive oxygen species (ROS), such as O2, are intermediate products of cellular metabolism in organisms. The normal balance of ROS concentrations plays a crucial role in cellular activities such as cell signaling, bacterial elimination, inflammation reduction, and protein function regulation. Normal cells generally maintain a stable, low ROS concentration. In contrast, in tissues with inflammation and tumors, ROS concentrations abnormally increase to high levels; for example, ROS concentrations in tumor cells exceed 100 μM, more than 100 times that of normal cells. Diseases related to ROS concentration imbalances are rapidly spreading globally, evolving into a pressing problem that seriously threatens human health and sustainable socio-economic development. Therefore, developing drugs and releasing substances targeting ROS concentration differences has significant scientific and social value.

[0004] ROS-responsive prodrugs are an effective strategy for treating these diseases that has emerged in recent years. Drugs can be chemically modified to obtain pharmacologically inert prodrugs that are inactive or have low activity in vitro. These prodrugs then undergo chemical reactions in vivo to release the active drug and exert their therapeutic effect. Prodrugs offer advantages such as increased bioavailability, enhanced targeting, improved drug stability, and reduced toxicity. Furthermore, prodrugs are based on modifications of existing drugs, resulting in a lower risk of failure, lower development costs, and a relatively shorter development cycle, which aligns with the actual needs of drug research and application in my country. Based on ROS-sensitive chemical bonds, responsive prodrugs are designed so that they are specifically activated by high concentrations of ROS at the lesion site, releasing the active drug to selectively kill cells at tumor or inflammatory sites, achieving precise therapeutic effects. Chemical structures used to prepare ROS-responsive prodrugs include phenylboronic acid (esters), sulfur / selenoethers, thiazolidinones, peroxisomalates, and aminoacrylic acids. Among these, phenylboronic acid (esters) are the most commonly used structure in ROS-responsive prodrug design due to their ease of preparation, rapid response, and lack of toxic byproducts. In the past five years, drugs or natural products modified with phenylboronic acid structures, both domestically and internationally, include evodiamine, tacrine, 5-fluorouracil, doxorubicin, tamoxifen, and coumarin-quinazolinone derivatives. In the prodrugs mentioned above, carbonates and carbamates are primarily used as linking points between the chemical structure and the drug to control covalent bond breaking and thus release the drug. Summary of the Invention

[0005] According to existing technologies, benzyl phenylboronic acid can also be directly linked to a tertiary amine structure to form a quaternary ammonium salt without introducing a linkage site. Figure 1 However, this structure has not yet been utilized for modifying ROS-responsive prodrugs. This invention discovers that aromatic quaternary ammonium salts formed using this structure can be cleaved by ROS, thereby releasing the drug (mechanism see [link to mechanism]). Figure 2 This invention investigated the structures of various aromatic tertiary amines containing different substituents and found that aromatic quaternary ammonium salts with different structures exhibit significant differences in their response efficiency to ROS. Based on this, the invention utilizes these ROS-responsive aromatic quaternary ammonium salts for prodrug modification of various drugs. Figure 12 This includes FDA-approved drugs such as nicotinamide, nicotinic acid, milrinone, and abiraterone. Figure 3 Furthermore, due to the unique structure of the prodrug containing phenylboronic acid, it can be dynamically covalently linked to the PVA hydrogel, achieving ROS-responsive drug release. The method developed in this invention has significant universality and practicality, and is of great value for drug redevelopment and utilization.

[0006] The purpose of this invention is to provide a class of prodrugs based on phenylboronic acid with ROS-responsive release properties, including their structure, preparation method, and usage method. This method can quaternize and modify biologically active drugs, offering advantages such as improved water solubility and compatibility, increased bioavailability, reduced toxicity, and enhanced targeting.

[0007] The technical solution adopted in this invention is as follows:

[0008] In a first aspect of the invention, a class of bioactive molecules with ROS-responsive properties is provided, having structures as shown in general formula I and the following structural formulas:

[0009]

[0010] Where R is -H, -COOH, -COOCH3, or -CONH2.

[0011] The bioactive molecules with ROS-responsive properties of this invention are generally aromatic quaternary ammonium salt structures with N-substituted benzyl phenylboronic acid, constructed by aromatic tertiary amine molecules and nucleophilic phenylboronic acid molecules. These molecules can rapidly release bioactive molecules to the lesion site to exert their effects in the presence of ROS.

[0012] In a second aspect of the invention, a method for preparing a class of bioactive molecules with ROS-responsive properties is provided, the method comprising the following steps:

[0013] (1) In a nitrogen-protected environment, 4-bromomethylphenylboronic acid or 4-chloromethylphenylboronic acid and the active ingredient molecule - aromatic tertiary amine are completely dissolved in the solvent;

[0014] (2) Heat and stir the solution from step (1) for 12 to 36 hours;

[0015] (3) Thin-layer chromatography (TLC) was used to detect that the reaction of the raw materials was complete and that a clear product was formed;

[0016] (4) After the mixed solution is brought back to room temperature, ethyl acetate is added to precipitate the product. The precipitate is purified by vacuum filtration or centrifugation and washed with ethyl acetate to obtain a pure product.

[0017] (5) For compounds that cannot be purified by centrifugation or precipitation, column chromatography is required to purify them into pure prodrug products.

[0018] (6) The compound obtained in step (4) or step (5) is dried to obtain a dried product.

[0019] In one or more embodiments of the present invention, in step (1), the molar ratio of 4-bromomethylphenylboronic acid or 4-chloromethylphenylboronic acid to an aromatic tertiary amine is (0.5-1.5):(0.5-1.5).

[0020] Preferably, the molar ratio of 4-bromomethylphenylboronic acid or 4-chloromethylphenylboronic acid to the aromatic tertiary amine is 1:1. Depending on the specific compound, the ratio can be adjusted appropriately to facilitate purification so that one of the raw materials reacts completely.

[0021] In one or more embodiments of the present invention, in step (1), the aromatic tertiary amines include, but are not limited to, nicotinamide, nicotinic acid, milrinone, abirateroneacetate, camptothecin, pyridoxine, nicorandil, Piroxicam, riseronate, papaverine, pranoprofen, tipranavir, pioglitazone, omeprazole, etc.

[0022] In one or more embodiments of the present invention, in step (1), the solvent is generally an inert organic solvent that can dissolve the raw material, such as anhydrous acetonitrile.

[0023] In one or more embodiments of the present invention, in step (2), the heating temperature is 5–100°C. The reaction is carried out under heating conditions to increase the reaction rate and yield; a slow reaction can also be carried out at room temperature, with the heating temperature generally set to 50°C to 80°C. For raw materials with poor stability, a lower temperature is required for the reaction.

[0024] In one or more embodiments of the present invention, in step (3), the thin-layer chromatography method used generally employs a mixed solution of methanol and dichloromethane as the developing solvent, with a volume ratio of methanol to dichloromethane generally between 1:10 and 1:1.

[0025] In one or more embodiments of the present invention, in step (4), the solvent for the method of precipitation by blending solvent is a nonpolar or aprotic solvent, generally a nonpolar or aprotic solvent such as ethyl acetate or n-hexane.

[0026] In step (4), the centrifuge speed is 5500 r / min and the centrifugation time is 5 min.

[0027] In a third aspect of the invention, the use of the bioactive molecules described in the first aspect and / or the bioactive molecules prepared by the method described in the second aspect in the preparation of products having ROS signal response performance is provided.

[0028] In one or more embodiments of the present invention, the product is a prodrug.

[0029] In one or more embodiments of the present invention, the ROS signal response performance can be reflected in improved solubility, biocompatibility, reduced biotoxicity, and improved targeting, and can be used to prepare drugs with clinical value.

[0030] In a fourth aspect of the invention, a drug having ROS signal response properties is provided, the drug comprising the bioactive molecule described in the first aspect and / or the bioactive molecule prepared by the method described in the second aspect, and a PVA hydrogel for loading the bioactive molecule.

[0031] In this invention, bioactive molecules (prodrugs) can be linked into PVA hydrogels via phenylboronic acid ester structures to achieve ROS-triggered drug release.

[0032] In a fifth aspect of the invention, a method for preparing a drug having ROS signal response performance as described in the fourth aspect is provided, the method comprising the following steps:

[0033] Polyvinyl alcohol (PVA) and the bioactive molecule are dissolved in a mixed solution, heated and stirred to obtain a clear and transparent homogeneous solution. The obtained solution is cooled and then frozen, then thawed until completely thawed, and then frozen and thawed until completely thawed again. The freezing and thawing process is repeated at least three times to allow the bioactive molecule to be fully cross-linked with PVA through dynamic covalent bonds of phenylboronic acid and PVA, thus preparing a drug-loaded hydrogel, which is the drug with ROS signal response performance.

[0034] In one or more embodiments of the present invention, the polyvinyl alcohol (PVA) in the mixed solution has a mass fraction of 3-8%; preferably 5%.

[0035] The type of polyvinyl alcohol is not particularly limited, and those skilled in the art will know that polyvinyl alcohol self-crosslinking can form hydrogels.

[0036] In one or more embodiments of the present invention, the mixed solution is composed of PBS and water; the volume ratio of PBS to water is 1:1.

[0037] In one or more embodiments of the present invention, the heating temperature is 70-90°C and the heating time is 0.5-1.5 h, more preferably 80°C for 1 h.

[0038] In one or more embodiments of the present invention, the feeding ratio of the bioactive molecule and the mixed solution is (0.5-1.5):(0.5-1.5).

[0039] In a sixth aspect of the invention, a method for releasing a drug having ROS signal response performance is provided, the method comprising the following steps:

[0040] Strongly oxidizing reactive oxygen species attack phenylboronic acid, which has reducing properties, and undergo a Baeyer-Villiger-like rearrangement to generate a borate intermediate. This intermediate is then hydrolyzed to generate a phenol intermediate. The covalent bond is broken via a 1,6-elimination reaction (quinone elimination), which causes the link between phenylboronic acid and the drug to disappear, and the bioactive molecule decomposes into the drug prototype.

[0041] Compared with the related technologies known to the inventors, one of the technical solutions of the present invention has the following beneficial effects:

[0042] (1) Advantages of this invention: It provides a novel method for preparing ROS-responsive prodrugs. This synthetic method is mainly targeted at drugs containing aromatic tertiary amine structures, and is particularly suitable for drugs without other modification sites. This invention provides prodrug modification for a variety of FDA-approved drugs, demonstrating its excellent versatility. Quaternary ammonium salt prodrugs modified using this method can significantly improve the water solubility of the drug, reduce biotoxicity, and improve bioavailability.

[0043] (2) The ROS-responsive prodrug constructed in this invention can undergo a bond-breaking reaction under the influence of high ROS in disease-causing organisms, thereby releasing the drug and avoiding release of the drug at healthy sites, thus reducing the toxic effects of the drug on healthy tissues. At the same time, by utilizing the phenylboronic acid structural characteristics of this prodrug, a dynamic covalent bond is formed with PVA, which has an ortho-diol structure, to load the drug into the hydrogel, further improving the ability of controlled drug release. Attached Figure Description

[0044] The accompanying drawings, which form part of this specification, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0045] Figure 1 The basic structure and reaction of the preparation and release of aromatic quaternary ammonium salt ROS-responsive prodrugs.

[0046] Figure 2 Mechanism of drug release from ROS-responsive prodrugs of aromatic quaternary ammonium salts.

[0047] Figure 3The molecular structures for constructing aromatic quaternary ammonium salt ROS-responsive prodrugs include four FDA-approved drug structures (4, 5, 7, 8).

[0048] Figure 4 Real-time nuclear magnetic resonance (NMR) 1 ROS response performance was detected by ¹H NMR. (A) The structure of the prodrug and the reaction that occurred in the test; (B) The change of the NMR signal peak of the nicotinamide prodrug over time; (C) A graph showing the change of product formation over time based on the NMR detection results; (D) A graph showing the change of product 2 formation over time.

[0049] Figure 5 The sensitivity of the prodrug response in the presence of ROS was determined by ultraviolet absorption spectroscopy (UV-VIS).

[0050] Figure 6 Mass spectrometry analysis of ROS-responsive aromatic quaternary ammonium salts.

[0051] Figure 7 Nuclear magnetic resonance spectral analysis of representative compounds. (A) Prodrug with pyridine structure; (B) Prodrug with pyrimidine structure; (C) Prodrug with nicotinamide; (D) Prodrug with milrinone; (E) Prodrug with nicotinic acid; (F) Prodrug with abiraterone.

[0052] Figure 8 Solubility of abiraterone prodrug and active ingredient in different solvents.

[0053] Figure 9 ROS-responsive release of phenylboronic acid-modified prodrug from PVA hydrogel. (A) Schematic diagram of drug loading on PVA and drug release mechanism; (B) Real-time UV detection of drug release from PVA hydrogel under ROS triggering.

[0054] Figure 10 Cell compatibility (L929) assay of ROS-responsive phenylboronic acid-modified quaternary ammonium salt prodrugs. (A) Nicotinamide prodrug; (B) Milrinone prodrug; (C) Abiraterone prodrug.

[0055] Figure 11 Cellular experiments on the antitumor effects of abiraterone prodrug. Anticancer effects of abiraterone prodrug on different tumor cells: 4T1(A), PC3(C), A549(E), SW48(G); Anticancer effects of abiraterone prodrug on different tumor cells after the addition of 0 μM, 0.1 μM, and 0.4 μM H2O2: 4T1(B), PC3(D), A549(F), SW48(H).

[0056] Figure 12(a) Commonly used structures based on phenylboronic acid in previous ROS response studies; (b) Prodrug structure of the present invention; (c) FDA-approved drug structure modified by this method. Detailed Implementation

[0057] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0058] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments of the present invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, and / or combinations thereof.

[0059] This invention proposes a method for preparing a prodrug based on phenylboronic acid that responds to ROS signals and exhibits aromatic quaternary ammonium salt characteristics, as well as a method for controlled release. To make the advantages and technical solutions of this invention clearer and more explicit, the invention will be described in detail below with reference to specific embodiments.

[0060] First, a detailed description of the main raw materials and experimental equipment required for this invention will be provided.

[0061] Experimental apparatus:

[0062] Electronic balance, oil bath stirrer, pH meter, cuvette, vacuum oven, magnetic stirrer, high-speed centrifuge, -80℃ medical freezer, general medical freezer, ultraviolet-visible spectrophotometer, Fourier transform infrared spectrometer, liquid chromatograph-mass spectrometer, nuclear magnetic resonance spectroscopy, freeze dryer, vacuum drying oven, ultrasonic cleaner, water purification system.

[0063] Experimental drugs:

[0064] Pyrimidine, 4-pyridinecarboxamide, nicotinic acid, pyridine, pyrazine, milrinone, polyvinyl alcohol 1788 (Mw = 44.06 kDa, degree of alcoholysis 87-89%), nicotinamide, and acetonitrile were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., and 4-bromomethylphenylboronic acid was purchased from Anaiji Chemical Co., Ltd. Abiraterone, hydrogen peroxide (3%), sodium carbonate, sodium bicarbonate, sodium hydroxide, and methanol were purchased from Maclean Biochemical Technology Co., Ltd.

[0065] The present invention will now be described in detail with reference to specific embodiments.

[0066] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0067] Example 1: Preparation and purification of nicotinamide prodrug

[0068] Nicotinamide and 4-bromomethylphenylboronic acid were placed in a thick-walled pressure-resistant tube at a molar ratio of 1:1.2. Acetonitrile (ACN) solution was added until dissolved (1-2 mL of methanol can be added as a co-solvent). A magnetic stir bar was added, and the oil bath stirrer was adjusted to 80°C. The reaction was carried out for 24 hours under oil bath stirring to ensure complete drug reaction. The reaction product was rotary evaporated, and the resulting solid was added to ethyl acetate and ultrasonically cleaned until uniformly dispersed. After centrifugation at 10000 r / min for 10 min, the supernatant was removed, and the product was washed with ethyl acetate. This process was repeated 2-3 times to remove impurities. The product was then dried in a vacuum drying oven to obtain pure nicotinamide prodrug.

[0069] Example 2: Preparation and purification of nicotinic acid prodrug

[0070] Nicotinic acid and 4-bromomethylphenylboronic acid were placed in a thick-walled pressure-resistant tube at a molar ratio of 1:1.2. Acetonitrile (ACN) solution was added until dissolved (1-2 mL of methanol could be added as a co-solvent). A magnetic stir bar was added, and the mixture was stirred in an oil bath at 80°C for 24 hours to ensure complete drug reaction. After the reaction, an appropriate amount of ethyl acetate was added to the reaction solution to precipitate the product, which was then purified by filtration using vacuum filtration and repeated washing with ethyl acetate. The resulting crude solid product was purified by column chromatography (methanol:dichloromethane = 1:10 → 1:4, volume ratio). The solvent was removed by rotary evaporation of the resulting product solution, and the product was dried in a vacuum drying oven to obtain pure nicotinic acid prodrug.

[0071] The preparation steps and conditions for milrinone prodrug are the same as in Example 1.

[0072] The preparation steps and conditions for abiraterone prodrug are the same as in Example 1.

[0073] Other prodrug structures can be obtained by referring to the preparation method in Example 1, and will not be described again here.

[0074] Example 3 1 H-NMR structural characterization of prodrug-based drugs

[0075] Deuterated methanol was selected as the solvent, and the solid prodrug powders prepared in the above examples were dissolved in the solvent to prepare a 5-7 mg / mL solution, which was then placed in a clean and dry NMR tube to prepare the test sample. Figure 7 The spectral density was obtained by nuclear magnetic resonance (NMR) hydrogen spectroscopy, and the purity, molecular structure, and linkage mode of the sample were analyzed. The measurement results are as follows: Figure 7As shown, the proton NMR spectrum confirmed the successful synthesis of the prodrug product. In the phenylboronic acid, the proton signal in the aromatic ring generally shows distinct doublets around δ = 7.8 ppm and δ = 7.7 ppm, while the methylene group shows a distinct singlet at δ = 5.88 ppm.

[0076] Example 4: Real-time reaction of nicotinamide prodrug in the presence of ROS 1 Detection of drug release by H-NMR

[0077] The nicotinamide prodrug was prepared into a 1 mM solution using deuterated PBS solution (PBS powder dissolved in deuterated water). 1 The structure of the solution was analyzed by 1H-NMR, and the spectrum was recorded as time 0h. 1mM H2O2 solution was added to the nicotinamide prodrug, and NMR was performed rapidly. The time at which the NMR test ended was recorded as the spectrum at that time. Measurements were then performed every 0.5h thereafter, and the results were recorded. The NMR spectra were integrated to quantify the decrease of the reactant peak and the formation of the product peak, and a graph showing the change in product formation over time was plotted. Figure 4 ).

[0078] Example 5: Real-time monitoring of the ROS response performance of nicotinamide prodrug using ultraviolet absorption spectroscopy (UV-VIS).

[0079] Nicotinamide and other prodrug molecules were dissolved in sodium phosphate buffer (pH 7.4) to prepare a 0.1 mM solution. This solution was added to a 1 cm quartz cuvette, and the UV absorption spectrum was measured after 0 h of reaction. Then, H₂O₂ (0.1 mM or 1 mM) was added, and continuous UV absorption measurements were immediately performed. Finally, the reaction rate curve was obtained by analyzing the changes in reaction time and absorption spectra. Figure 5 ).

[0080] The ROS response performance was evaluated using a pseudo-first-order reaction rate equation. The fitted equation is as follows:

[0081] rate = -k[A t [B] t (eq.2)

[0082] ln([A t ] / [A0])–ln([B t ] / [B0])=k([A0]–[B0])t (eq.3)

[0083] When [A0] >> [B0]:

[0084] [B t ] / [B0]=exp(-k[A0]t)(eq.4)

[0085] final:

[0086] ln([B t ] / [B0])=-k[A0]t(eq.5)

[0087] Based on this, [B] was made. t The slope of the linear equation ] / [B0] changes over time is used to calculate the reaction rate constant k, which is then used to evaluate the ROS response rate.

[0088] The test results are as follows Figure 5 As shown, prodrug structures such as nicotinic acid prodrug and nicotinamide prodrug all exhibit excellent response properties.

[0089] Example 6: LC-MS characterization of the prodrug

[0090] Molecular weight determination of prodrugs: Nicotinamide prodrug, nicotinic acid prodrug, milrinone prodrug, and abiraterone prodrug were dissolved in 1 mL of methanol solution to prepare 0.1 mM solutions, and the molecular weights of the prodrug molecules were characterized. Figure 6 ).

[0091] Example 7: Solubility measurement of abiraterone prodrug in different solvents

[0092] Weigh 1 mg of abiraterone prodrug sample and add it to a glass bottle. Add different solvents (H₂O, PBS, 10% glucose solution), shake well, and dissolve using sonication until the sample is completely dissolved to form a clear, transparent solution. Record the volume of the solution at this point and calculate the solubility. (Centrifugation (10,000 rpm) can be used to help determine if the sample is completely dissolved.) Simultaneously measure the solubility of abiraterone technical grade in distilled water as a control. Figure 8 As shown, the pre-drugated abiraterone prodrug has a solubility in water that is more than 1600 times higher than that of the original drug.

[0093] Example 8: Nicotinamide prodrug loaded into PVA hydrogel in response to ROS release

[0094] Take 5% polyvinyl alcohol (PVA) by mass and 1.67 × 10⁻⁶ -6 The mol / L nicotinamide prodrug was placed in a screw-top glass bottle and dissolved in 1 mL of a PBS:H2O = 1:1 (volume ratio) mixed solution. A magnetic stir bar was added, and the oil bath stirrer was set to 80°C and 300 r / min. The mixture was heated and stirred in the oil bath for 1 h to form a clear and transparent homogeneous solution from the polymer and solvent mixture.

[0095] After cooling the obtained homogeneous solution to room temperature, it was added to a 96-well plate for shaping and placed in a medical freezer at -20°C for 24 hours to cool and shape. The plate was then removed, thawed at room temperature, and placed back into the -20°C medical freezer. This freeze-thaw cycle was repeated at least three times to ensure the prodrug is fully cross-linked with PVA via dynamic covalent bonds between phenylboronic acid and PVA. Figure 9 A drug-loaded hydrogel was prepared. Buffer solutions were prepared using Na₂CO₃ and NaHCO₃. 1 mol / L Na₂CO₃ and NaHCO₃ solutions were mixed in a 1:1 volume ratio, and NaOH solution was added to adjust the pH to >10 using a pH meter, resulting in an alkaline buffer solution. The obtained hydrogel was then immersed in the prepared alkaline buffer solution for at least 12 hours to obtain a drug-loaded hydrogel with low background release and superior mechanical strength.

[0096] The model drug is a ROS-responsive prodrug, consisting of three parts: a ROS-responsive moiety, a pharmacodynamic moiety, and a chain connecting the first two parts that can spontaneously break. The release mechanism is as follows: Figure 3 Strongly oxidizing reactive oxygen species attack phenylboronic acid, which has reducing properties, undergoing a Baeyer-Villiger-like rearrangement to generate a borate intermediate. This is followed by hydrolysis to form a phenol intermediate, which is then broken via a 1,6-elimination reaction (quinone elimination), resulting in the loss of the link between the drug and phenylboronic acid, and the bioactive molecule decomposes into the drug prototype. After the model drug was loaded into the hydrogel, ROS response testing was performed. Hydrogen peroxide was used as a ROS stimulus to mimic ROS conditions in cancer cells, and the effect of reactive oxygen species on drug release from the hydrogel was detected by UV spectroscopy, specifically including:

[0097] The prepared drug-loaded hydrogel was rinsed three times with PBS (pH=7.4) solution to prevent the acid and alkaline environment from affecting the experiment and causing errors.

[0098] Add 3 mL of PBS (pH = 7.4) to two 10 mm double-lens cuvettes and divide them into a control group and an experimental group. The experimental group was given 3 μL of 3% hydrogen peroxide solution that had been brought to room temperature, while the control group was not treated.

[0099] Add the same size of pre-washed drug-loaded hydrogel to cuvettes. Set the UV spectrophotometer path length to 200–800 nm and scan the entire wavelength range. Measure continuously for 5 hours using different time gradients. Shake the contents well before each full-wavelength scan to prevent uneven drug distribution in the solution caused by the hydrogel releasing the drug from the PBS, which could lead to measurement errors.

[0100] Data analysis was performed after the measurement. Data from a wavelength of 230nm was selected for plotting and comparison, and the results are as follows: Figure 9As shown in (A), the drug release 5 hours after the addition of hydrogen peroxide was approximately three times that under conditions without reactive oxygen species, indicating almost complete release. Furthermore, as... Figure 9 As shown in (B), if the control group was given the same concentration and amount of hydrogen peroxide as the experimental group 2 hours after drug release, the release rate increased rapidly, and the final release amount was almost identical to that of the experimental group. Clearly, the drug release effect is related to the presence of reactive oxygen species (ROS), indicating that the drug-loaded hydrogel has good ROS-sensitive drug release energy, which is a substrate for self-accelerated drug release in the cellular environment.

[0101] Example 9: Cell compatibility assay of aromatic quaternary ammonium salts based on phenylboronic acid

[0102] Milrinone technical, milrinone prodrug, nicotinamide technical, and nicotinamide prodrug were prepared at 1 mg / mL. Using DMEM complete medium as the solvent, the standard solutions were serially diluted to obtain solutions of 1 mg / mL, 0.5 mg / mL, 0.25 mg / mL, and 0.125 mg / mL, and added to each culture well. Due to their poor solubility, abiraterone technical and abiraterone prodrug were prepared using DMSO (<5% for dissolution) to achieve concentrations of 106.25, 53.125, 26.5625, 13.28125, 6.640625, and 3.320313 μg / mL. Only an equal volume of DMEM complete medium was added as a control group. Each group had three replicate wells. The treated cells were incubated in a CO2 incubator for 24 hours. After culture, discard the old culture medium, wash twice with PBS, add 110 μL of DMEM medium containing 10% CCK-8 solution to each well, and continue incubation for 2 hours. After incubation, aspirate 100 μL of supernatant from each well into a new 96-well plate, avoiding air bubbles, and set up a blank group (DMEM medium containing only 10% CCK-8 solution). Measure the absorbance (OD value) of each well at 450 nm using a microplate reader. Convert the OD values ​​of each well to relative cell viability, and calculate the cell survival rate of each group against the control group. By comparing cell viability at different concentrations, evaluate the effect of phenylboronic acid-modified miridone prodrug and nicotinamide prodrug on the activity of L929 cells. Calculate cell viability using the formula:

[0103] Cell viability = [(As-Ab) / (Ac-Ab)] × 100%

[0104] in:

[0105] As: Absorbance of experimental wells (containing cell culture medium, CCK-8, and the test drug); Ac: Absorbance of control wells (containing cell culture medium, CCK-8, and no test drug); Ab: Absorbance of blank wells (containing no cells, test drug, or CCK-8).

[0106] Within the defined concentration range, the phenylboronic acid-modified miridone and nicotinamide prodrugs exhibited good cytocompatibility with their respective parent drugs in L929 cells. Compared to the control group, except for nicotinamide and the prodrug at a concentration of 1 mg / mL which had some impact on cell viability, cell viability at different drug concentrations in other experimental groups exceeded 80%. Furthermore, the differences in cell bioactivity between the parent drug and the prodrug at the same concentration were not significant. However, for the highly toxic parent drug abiraterone, the prodrug modified using this strategy showed a significant improvement in biocompatibility. In particular, the cytotoxicity of the abiraterone prodrug was significantly reduced compared to the parent drug, maintaining a 75% survival rate even at 106 μg / mL. At this point, the survival rate of the parent abiraterone was only about 31%. This demonstrates that the quaternary ammonium salt prodrug strategy based on phenylboronic acid has excellent biocompatibility and can significantly reduce the cytotoxicity of the parent drug. Figure 10 Example 10: Detection of the antitumor effect of phenylboronic acid-modified abiraterone prodrug on different tumor cells.

[0107] Cancer cells were digested and resuspended, and seeded at a density of 5 × 10^3 cells per well in 96-well plates. The cells were then cultured at 37°C and 5% CO2 for 24 hours to allow for cell adhesion and achieve the desired density. Abiraterone prodrug was dissolved in 9 g / L glucose solution to prepare an initial solution of 850 μg / mL. This solution was then diluted with 9 g / L glucose solution and mixed with an equal volume of DMEM sugar-free medium (HEPES-free) to form abiraterone prodrug solutions with concentrations of 425, 212.5, 106.25, 53.125, 26.5625, 13.28125, 6.640625, and 3.320313 μg / mL. 100 μL of each solution was added to the respective well. A control group was established, consisting of 100 μL of a mixture of 9 g / L glucose solution and DMEM sugar-free medium (HEPES-free). Each group had three replicate wells. The treated cells were incubated in a CO2 incubator for 24 hours. After incubation, the old culture medium was discarded, the cells were washed twice with PBS, and 110 μL of DMEM medium containing 10% CCK-8 solution was added to each well, followed by incubation for another 2 hours. After incubation, 100 μL of supernatant was transferred from each well to a new 96-well plate, avoiding air bubbles, to create a blank control group (DMEM medium containing only 10% CCK-8 solution). The absorbance (OD value) of each well was measured at 450 nm using a microplate reader. The OD values ​​of each well were converted to relative cell viability, and cell survival rate was calculated for each group against the control group. The effect of phenylboronic acid-modified abiraterone prodrug on the activity of 22RV1 cells was evaluated by comparing cell viability at different concentrations. Cell viability was calculated using the following formula:

[0108] Cell viability = [(As-Ab) / (Ac-Ab)] × 100%

[0109] in:

[0110] As: Absorbance of experimental wells (containing cell culture medium, CCK-8, and the test drug); Ac: Absorbance of control wells (containing cell culture medium, CCK-8, and no test drug); Ab: Absorbance of blank wells (containing no cells, test drug, or CCK-8).

[0111] Within the defined effective concentration range, the phenylboronic acid-modified abiraterone prodrug exhibited inhibitory activity in various tumor cell lines, including: breast cancer cell line (4T1), human prostate cancer bone metastasis cells (PC3), lung cancer basal epithelial cells (A549), and human colon adenocarcinoma cells (SW48). Figure 11 Of particular importance, abiraterone prodrug did not show significant anticancer activity at low concentrations, but exhibited significant inhibitory activity when used in combination with H2O2, especially in human prostate cancer bone metastases and human colon adenocarcinoma cells. This indicates that abiraterone prodrug can release active drug in response to ROS to exert its effect.

[0112] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A class of prodrug molecules with ROS signal response properties, characterized by: The prodrug molecule is any one of the following structures: 、 、 、 。 2. A method for preparing the prodrug molecule of claim 1, characterized in that, The method includes the following steps: (1) In a nitrogen-protected environment, 4-bromomethylphenylboronic acid and an aromatic tertiary amine are completely dissolved in anhydrous acetonitrile; the aromatic tertiary amine is nicotinamide, nicotinic acid, milrinone, or abiraterone acetate; the molar ratio of 4-bromomethylphenylboronic acid to the aromatic tertiary amine is (0.5~1.5):(0.5~1.5); (2) Heat and stir the solution from step (1) for 12 to 36 hours; (3) Thin-layer chromatography (TLC) was used to detect that the reaction of the raw materials was complete and that a clear product was formed; (4) After the reaction is complete, use the method of precipitation with a blended solvent, and use vacuum filtration or centrifugation to purify the product to obtain a pure product; (5) For compounds that cannot be purified by centrifugation or precipitation, column chromatography is required to purify them into pure prodrug products. (6) Dry the compound obtained in step (4) or step (5) to obtain the dried product.

3. The method as described in claim 2, characterized in that, In step (2), the heating temperature is 5~100℃.

4. The method as described in claim 2, characterized in that, In step (3), the thin-layer chromatography method used is a mixed solution of methanol and dichloromethane as the developing solvent.

5. The use of the prodrug molecule of claim 1 and / or the prodrug molecule prepared by any one of claims 2 to 4 in the preparation of products with ROS signal response performance.

6. A drug exhibiting ROS signal response properties, characterized in that, The drug comprises the prodrug molecule of claim 1 and / or the prodrug molecule prepared by the method of any one of claims 2 to 4, and a hydrogel for loading the prodrug molecule.

7. The method for preparing a drug with ROS signal response performance as described in claim 6, characterized in that, The method includes the following steps: Polyvinyl alcohol (PVA) and the prodrug molecule are dissolved in a mixed solution, heated and stirred to obtain a clear and transparent homogeneous solution. The obtained solution is cooled and then frozen, then thawed until completely thawed, and then frozen and thawed until completely thawed again. The freezing and thawing process is repeated at least three times to allow the prodrug molecule to be fully cross-linked with PVA through dynamic covalent bonds of phenylboronic acid and PVA, thus preparing a drug-loaded hydrogel, which is the drug with ROS signal response performance.

8. The preparation method according to claim 7, characterized in that, The polyvinyl alcohol (PVA) in the mixed solution has a mass fraction of 3-8%. The mixed solution consists of PBS and water; The heating temperature is 70~90℃, and the heating time is 0.5~1.5h; The feeding ratio of the prodrug molecule and the mixed solution is (0.5~1.5):(0.5~1.5).

9. A method for releasing a drug with ROS signal response performance as described in claim 6, characterized in that, The method includes the following steps: Strongly oxidizing reactive oxygen species attack phenylboronic acid, which has reducing properties, and undergo a Baeyer-Villiger-like rearrangement to generate a borate intermediate. This intermediate is then hydrolyzed to generate a phenol intermediate, which is broken via a 1,6-elimination reaction. This breaks the covalent bond between the phenylboronic acid and the drug, causing the prodrug molecule to decompose and release the corresponding active drug.

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