Prodrug structure with ROS signal response performance and preparation method and application thereof

Abstract

The invention relates to the field of biological medicine, in particular to a prodrug structure with ROS signal response performance and a preparation method and application thereof. The prodrug molecule with ROS signal response performance developed by the invention can quickly release living active molecules in a disease signal ROS-enriched environment, and plays a role in targeted therapy. The method has good universality, prodrug modification can be carried out on multiple molecules, four kinds of drug molecules approved to appear on the market are included, and it is verified that the solubility and biocompatibility of drugs can be improved, cytotoxicity can be reduced, and the effect of targeted therapy can be improved. Based on the structural characteristics of the molecules, the molecules can also be complexed with PVA hydrogel, and responsive controllable release of the medicine is further achieved.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to an aromatic quaternary ammonium salt prodrug with a ROS signal response bond-breaking function, and a preparation method and application thereof. Background Art

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

[0003] Reactive oxygen species (ROS) are a series of chemically active oxygen-containing molecules, including hydrogen peroxide (H2O2), superoxide anion (O 2- ), hydroxyl radical (·OH), hypochlorite (OCl - ) and singlet oxygen ( 1 O2), etc. Reactive oxygen species are intermediate products of cellular metabolism in organisms. The normal balance of ROS concentration plays an important role in cellular activities such as cell signaling, eliminating bacteria, clearing inflammation, and regulating protein function. The ROS concentration in normal cells is generally stable and maintained at a low level. In contrast, in tissues with diseases such as inflammation and tumors, the ROS concentration abnormally increases to a higher level. For example, the ROS concentration in tumor cells exceeds 100μM, which is more than 100 times that of normal cells. Currently, diseases related to imbalanced ROS concentration levels are spreading rapidly around the world and have evolved into an urgent problem that seriously endangers human health and sustainable social and economic development. Therefore, the development of drugs and their release based on the difference in ROS concentrations has great scientific research value and social significance.

[0004] ROS-responsive prodrugs (prodrugs) have been developed in recent years as an effective strategy for treating such diseases. Chemically modified drugs can produce pharmacologically inert prodrugs that are inactive or minimally active in vitro. These prodrugs, converted to active drugs in vivo through chemical reactions, exert their efficacy. Prodrugs offer advantages such as increased bioavailability, enhanced targeting, improved stability, and reduced toxic side effects. Furthermore, prodrugs are modified from existing drugs, resulting in a low risk of failure, low R&D costs, and a relatively short development cycle, meeting the practical needs of drug development and application in my country. Prodrugs designed based on ROS-sensitive chemical bonds are specifically activated by high concentrations of ROS at the lesion site, releasing the active drug to selectively kill cells in tumors or inflammatory areas, achieving a precise therapeutic effect. Chemical structures used to prepare ROS-responsive prodrugs include phenylboronic acid (esters), thio / selenoethers, thiazolidinones, peroxyoxalate esters, and aminoacrylic acid. Phenylboronic acid (esters) are currently 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 and natural products modified with phenylboronic acids, both domestically and internationally, include evodiamine, tacrine, 5-fluorouracil, doxorubicin, tamoxifen, and coumarin-quinazolinone derivatives. Among these prodrugs, carbonates and carbamates are primarily used as the connection point between the chemical structure and the drug, controlling the cleavage of the covalent bond and thus releasing the drug. Summary of the Invention

[0005] According to the prior art, benzyl phenylboronic acid can also be directly connected to the tertiary amine structure to form a quaternary ammonium salt without introducing a connection point ( Figure 1 ), however, this structure has not yet been used to modify ROS-responsive prodrugs. This invention discovered that the aromatic quaternary ammonium salt formed by this structure can be broken by ROS, thereby releasing the drug (see the mechanism Figure 2 ). The present invention studies various aromatic tertiary amine structures containing different substituents and finds that aromatic quaternary ammonium salts with different structures have very different response efficiencies to ROS. Based on this, the present invention utilizes this ROS-responsive aromatic quaternary ammonium salt to perform prodrug modification on various drugs ( Figure 12 ), including FDA-approved drugs such as nicotinamide, niacin, milrinone, and abiraterone ( Figure 3 Furthermore, due to the unique structure of phenylboronic acid, the prodrug can be dynamically covalently linked to the PVA hydrogel, enabling ROS-responsive drug release. This method has broad applicability and practicality, and holds great value for drug redevelopment and utilization.

[0006] The present invention aims to provide a class of phenylboronic acid-based prodrugs with ROS-responsive release properties, including their structure, preparation method, and use. This method can be used to quaternize biologically active drugs, with advantages such as increased water solubility and compatibility, improved bioavailability, reduced toxic side effects, and enhanced targeting.

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

[0008] In the first aspect of the present invention, a class of bioactive molecules having ROS responsive properties is provided, the structure of which is shown in Formula I and the following structural formula:

[0009]

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

[0011] The bioactive molecules with ROS responsiveness of the present invention are generally aromatic quaternary ammonium salt structures of N-substituted phenylboronic acid benzyl groups, which are constructed by aromatic tertiary amine molecules and nucleophilic phenylboronic acid molecules. Such 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 present invention, a method for preparing a class of bioactive molecules having 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 original drug molecule - aromatic tertiary amine are completely dissolved in the solvent;

[0014] (2) heating and stirring the solution in step (1) for 12 to 36 hours;

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

[0016] (4) The mixed solution is returned to room temperature, ethyl acetate is added to precipitate the product, the precipitate is purified by vacuum filtration or centrifugation, and the purified product is obtained by washing with ethyl acetate;

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

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

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

[0020] Preferably, the molar ratio of 4-bromomethylphenylboric acid or 4-chloromethylphenylboric acid to the aromatic tertiary amine is 1:1. The ratio can be appropriately adjusted to allow one of the raw materials to react completely for the convenience of purification according to the specific compound.

[0021] In one or some embodiments of the present invention, in step (1), the aromatic tertiary amine includes but is not limited to nicotinamide, nicotinic acid, milrinone, abiraterone acetate, camptothecin, pyridoxine, nicorandil, piroxicam, risedronate, papaverine, pranoprofen, tipranavir, pioglitazone, omeprazole, etc.

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

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

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

[0025] In one or some embodiments of the present invention, in step (4), the solvent of the co-solvent precipitation method is a non-polar or aprotic solvent, generally a non-polar or aprotic solvent such as ethyl acetate and n-hexane.

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

[0027] In a third aspect of the present invention, there is provided use of the bioactive molecule described in the first aspect and / or the bioactive molecule prepared by the method described in the second aspect in preparing a product having ROS signal responsiveness.

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

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

[0030] In a fourth aspect of the present invention, a drug having ROS signal response performance is provided, which includes 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 the present invention, bioactive molecules (prodrugs) can be linked to PVA hydrogels via phenylboronic acid ester structures to achieve ROS-triggered drug release.

[0032] In a fifth aspect of the present invention, a method for preparing the drug having ROS signal response performance according to 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 uniform solution. The obtained solution is cooled and then frozen, thawed until completely melted, and then frozen and thawed until completely melted. The freezing and thawing until completely melted steps are performed at least three times to fully cross-link the bioactive molecule with PVA through the dynamic covalent bond between phenylboronic acid and PVA, thereby preparing a drug-loaded hydrogel, namely the drug with ROS signal responsiveness.

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

[0035] The type of polyvinyl alcohol is not particularly limited. Those skilled in the art generally know that polyvinyl alcohol can form a hydrogel by self-crosslinking.

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

[0037] In one or some 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 some 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 present invention, a method for releasing a drug having ROS signal responsiveness is provided, the method comprising the following steps:

[0040] Reactive oxygen species with strong oxidizing effects attack phenylboronic acid with reducing function, causing a Baeyer-Villiger-like rearrangement to generate a boronate intermediate, which is then hydrolyzed to generate a phenol intermediate. The covalent bond is broken through a 1,6-elimination reaction (quinone elimination), thereby eliminating the link between phenylboronic acid and the drug, and the bioactive molecule is decomposed into the drug prototype.

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

[0042] (1) Advantages of the present invention: A method for preparing a novel ROS-responsive prodrug is provided. This synthesis method is mainly targeted at drugs containing aromatic tertiary amine structures, and is particularly suitable for drugs without other modification sites. The invention provides prodrug modifications for a variety of FDA-approved drugs, demonstrating its excellent universality. The quaternary ammonium salt prodrug modified using this method can significantly improve the water solubility of the drug, reduce biological toxicity, and improve bioavailability.

[0043] (2) The ROS-responsive prodrug constructed in the present invention can undergo a bond-breaking reaction under the action of high ROS in disease-prone tissues, thereby releasing the drug, which can avoid the release of the drug in healthy locations and reduce the toxic effects of the drug on healthy tissues. At the same time, the phenylboronic acid structural characteristics of the prodrug are utilized to form a dynamic covalent bond with PVA having a vicinal diol structure, thereby loading the drug into the hydrogel, further improving the ability of controlled drug release. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] The accompanying drawings, which constitute a part of the specification of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

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

[0046] Figure 2 .Drug release mechanism of ROS-responsive prodrugs of aromatic quaternary ammonium salts.

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

[0048] Figure 4 .Real-time NMR ( 1 H NMR (H NMR) testing of ROS responsiveness. (A) Prodrug structure and the reaction that occurred; (B) Changes in the NMR signal peak of the nicotinamide prodrug over time; (C) Data graph showing the change in product production over time based on NMR results; (D) Data graph showing the change in product 2 production over time.

[0049] Figure 5 .Ultraviolet absorption spectroscopy (UV-vis) was used to detect the response sensitivity of the prodrug in the presence of ROS.

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

[0051] Figure 7 NMR spectra of representative compounds. (A) Pyridine prodrug; (B) Pyrimidine prodrug; (C) Nicotinamide prodrug; (D) Milrinone prodrug; (E) Nicotinic acid prodrug; (F) Abiraterone prodrug.

[0052] Figure 8 .Solubility of abiraterone prodrug and original drug in different solvents.

[0053] Figure 9 ROS-responsive release of phenylboronic acid-modified prodrugs from PVA hydrogels. (A) Schematic diagram of PVA drug loading and drug release mechanism; (B) Real-time UV detection of drug release from PVA hydrogels triggered by ROS.

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

[0055] Figure 11 Cell-based studies on the antitumor effects of abiraterone prodrugs. Anticancer effects of abiraterone prodrugs on different tumor cells: 4T1 (A), PC3 (C), A549 (E), and SW48 (G); Anticancer effects of abiraterone prodrugs in the presence of 0μM, 0.1μM, and 0.4μM H2O2 on different tumor cells: 4T1 (B), PC3 (D), A549 (F), and SW48 (H).

[0056] Figure 12(a) Common structures based on phenylboronic acid used in previous ROS response studies; (b) Prodrug structures of the present invention; (c) Structures of FDA-approved drugs modified by this method. DETAILED DESCRIPTION

[0057] It should be noted that the following detailed descriptions are exemplary and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs.

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

[0059] The present invention provides a method for preparing a prodrug based on phenylboronic acid that responds to ROS signals and has the characteristics of an aromatic quaternary ammonium salt and a controlled release method. In order to make the advantages and technical solutions of the present invention clearer and more specific, the present invention is described in detail below with reference to specific embodiments.

[0060] First, the main raw materials and experimental equipment required for the present invention are described in detail.

[0061] Experimental instruments:

[0062] Electronic balance, oil bath stirrer, pH meter, cuvette, vacuum oven, magnetic stirrer, high-speed centrifuge, -80℃ medical freezer, ordinary medical refrigerator, UV-visible spectrophotometer, Fourier transform infrared spectrometer, liquid chromatograph-mass spectrometer, nuclear magnetic resonance hydrogen spectrometer, freeze dryer, vacuum drying oven, ultrasonic cleaning machine, 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 is described in detail below with reference to specific embodiments.

[0066] In order to enable those skilled in the art to more clearly 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 are placed in a thick-walled pressure-resistant tube at a molar ratio of 1:1.2. Acetonitrile (ACN) solution is added until dissolved (1-2 mL of methanol can be added as a cosolvent). A magnetic stirrer is added and the oil bath agitator is set to 80°C. The mixture is stirred in the oil bath for 24 hours to allow for full reaction. The reaction product is rotary evaporated, and the resulting solid is added with ethyl acetate and ultrasonically dispersed using an ultrasonic cleaner. After centrifugation at 10,000 r / min for 10 minutes, the supernatant is removed and washed with ethyl acetate. This process is repeated two to three times to remove impurities from the raw materials and dry in a vacuum oven. Pure nicotinamide prodrug is obtained after drying.

[0069] Example 2: Preparation and purification of niacin 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 cosolvent). A magnetic stirrer was added, and the mixture was stirred in an oil bath at 80°C for 24 hours to allow for full reaction. After the reaction, an appropriate amount of ethyl acetate was added to the reaction solution to precipitate the product. The product was then filtered and purified using suction filtration and repeatedly washed with ethyl acetate. The resulting crude solid product was purified by column chromatography (methanol:dichloromethane = 1:10 → 1:4, volume ratio). The resulting solution was rotary evaporated to remove the solvent and dried in a vacuum oven to yield pure nicotinic acid prodrug.

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

[0072] The preparation steps and conditions of Abiraterone prodrug are the same as those in Example 1.

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

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

[0075] Deuterated methanol was selected as the solvent, and each solid prodrug powder prepared in the above examples was dissolved in the solvent to make a 5-7 mg / mL solution and placed in a clean and dry NMR tube to prepare a test sample ( Figure 7 ). The spectrum is obtained by nuclear magnetic resonance hydrogen spectrum scanning and the sample purity, molecular structure and connection mode are analyzed. The measurement results are as follows Figure 7As shown, the successful synthesis of the prodrug product was confirmed by H NMR spectroscopy, wherein the proton signal of the aromatic ring of phenylboronic acid generally appears at δ = 7.8 ppm, with a clear doublet peak around δ = 7.7 ppm, and the methylene group appears at δ = 5.88 ppm.

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

[0077] Nicotinamide prodrug was prepared into a 1 mM solution using deuterated PBS solution (PBS powder dissolved in deuterated water). 1 H-NMR was used to test the structure of the solution and record the spectrum at time 0h. 1mM H2O2 solution was added to the nicotinamide prodrug and the NMR test was performed immediately. The time when the NMR test ended was recorded as the spectrum at that time. Thereafter, measurements were taken every 0.5h and recorded. The NMR spectrum was integrated to quantify the reduction of the raw material peak and the generation of the product peak, and a data graph of the product generation amount over time was plotted ( Figure 4 ).

[0078] Example 5: Real-time monitoring of ROS response performance of nicotinamide prodrug by ultraviolet absorption spectroscopy (UV-vis)

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

[0080] The ROS response performance was evaluated by using the pseudo-first-order reaction rate equation. The fitting 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] then:

[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 t The straight line of ] / [B0] changing with time was plotted, and the reaction rate constant k was calculated based on the slope to evaluate the ROS response rate.

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

[0089] Example 6. LC-MS Characterization of Prodrug

[0090] Prodrug molecular weight detection: Nicotinamide prodrug, nicotinic acid prodrug, milrinone prodrug, abiraterone prodrug, etc. were dissolved in 1 mL of methanol solution to prepare 0.1 mM solution, and the molecular weight of the prodrug molecule was characterized ( Figure 6 ).

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

[0092] Weigh 1 mg of abiraterone prodrug sample into a glass bottle. Add different solvents (H2O, PBS, 10% glucose solution), shake well, and dissolve with ultrasound until the added solvent completely dissolves the sample 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 assist in determining whether the sample is completely dissolved. At the same time, measure the solubility of the original abiraterone drug in distilled water as a control. Figure 8 As shown in Figure 3, the solubility of the prodrug of abiraterone in water 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) and 1.67×10 -6 mol / L nicotinamide prodrug was placed in a screw-capped glass bottle and dissolved in 1 mL of a mixed solution of PBS:H2O=1:1 (volume ratio). A magnetic bar was added and the oil bath stirrer was adjusted to 80°C, 300 rpm, and the oil bath was heated and stirred for 1 h to allow the polymer and solvent mixture to form a clear and transparent homogeneous solution.

[0095] The obtained homogeneous solution was cooled to room temperature, added to a 96-well plate for setting, and placed in a medical freezer at -20°C for 24 hours to set. The plate was then taken out and thawed at room temperature. After returning to room temperature and completely thawing, it was placed in a -20°C medical freezer. This process was repeated for at least three times to allow the prodrug to be fully cross-linked with PVA through the dynamic covalent bond between phenylboronic acid and PVA ( Figure 9 ) to prepare a drug-loaded hydrogel. A buffer solution was prepared using Na2CO3 and NaHCO3. A 1 mol / L Na2CO3 and NaHCO3 solution was prepared, each mixed in a 1:1 volume ratio. NaOH solution was added and adjusted to a pH > 10 using a pH meter to obtain an alkaline buffer solution. The resulting hydrogel was added to the prepared alkaline buffer solution and soaked for at least 12 hours to obtain a drug-loaded hydrogel with low background release and improved mechanical strength.

[0096] The model drug is a ROS-responsive prodrug consisting of three parts: a ROS-responsive part, a pharmacodynamic part, and a chain connecting the first two parts that can break spontaneously. Figure 3 , reactive oxygen species with strong oxidizing effects attack phenylboronic acid with reducing function, undergoing a rearrangement similar to Baeyer-Villiger to generate a boronate intermediate, which is then hydrolyzed to generate a phenol intermediate. The covalent bond is broken through a 1,6-elimination reaction (quinone elimination), thereby eliminating the link between phenylboronic acid and the drug, and the bioactive molecule is decomposed into a drug prototype. After the model drug is loaded into the hydrogel, an ROS response test is performed. Hydrogen peroxide is used as a ROS stimulus to simulate the ROS conditions in cancer cells, and the effect of reactive oxygen species on the release of hydrogel drugs is detected by ultraviolet spectroscopy, which specifically includes:

[0097] The prepared drug-loaded hydrogel was first rinsed repeatedly with PBS (pH=7.4) solution for 3 times to prevent the acid-base environment from affecting the experiment and causing errors.

[0098] 3 mL of PBS (PH=7.4) was added to two 10 mm two-way light cuvettes respectively, and the cells were divided into a control group and an experimental group. 3 uL of 3% hydrogen peroxide solution restored to room temperature was added to the experimental group, and the control group was not treated.

[0099] Add pre-rinsed drug-loaded hydrogels of equal size to the cuvettes. Set the UV spectrophotometer to a pathlength of 200-800 nm and scan the entire wavelength range. Measure continuously for 5 hours at varying time gradients. Be sure to shake the contents thoroughly before each full-wavelength scan to prevent uneven distribution of the drug released from the hydrogel in PBS, which could cause measurement errors.

[0100] After the measurement, the data was analyzed. The data at a wavelength of 230nm was selected for plotting and comparison. The results are as follows: Figure 9As shown in (A), the drug release at 5 h after the addition of hydrogen peroxide was about three times that in the absence of reactive oxygen species, almost achieving complete release. Figure 9 As shown in (B), when the control group was treated with hydrogen peroxide at the same concentration and volume as the experimental group 2 hours after drug release, the release rate increased rapidly, and the final release amount was nearly identical to that of the experimental group. Clearly, the drug release effect is related to the presence of reactive oxygen species, indicating that the drug-loaded hydrogel has excellent ROS-sensitive drug release properties, which serves as the basis for self-accelerated drug release in the cellular environment.

[0101] Example 9: Cytocompatibility testing of aromatic quaternary ammonium salts based on phenylboronic acid

[0102] Milrinone stock, milrinone prodrug, nicotinamide stock, and nicotinamide prodrug were prepared at 1 mg / mL each. 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, which were then added to each culture well. Due to their poor solubility, abiraterone stock and abiraterone prodrug were prepared using DMSO (<5% to aid dissolution) at concentrations of 106.25, 53.125, 26.5625, 13.28125, 6.640625, and 3.320313 μg / mL, respectively. An equal volume of DMEM complete medium alone was added to serve as a control group. Three replicate wells were set up for each group. The treated cells were incubated in a CO2 incubator for 24 hours. After the culture is completed, the old culture medium is discarded, the plate is washed twice with PBS, 110 μL of DMEM culture medium containing 10% CCK-8 solution is added to each well, and the incubation is continued for 2 hours. After the incubation is completed, 100 μL of supernatant from each well is aspirated into a new 96-well plate to avoid bubbles, and a blank group (DMEM culture medium containing only 10% CCK-8 solution) is set up. The absorbance value (OD value) of each well is measured at 450 nm using a microplate reader. The OD value of each well is converted into relative cell viability, and the cell survival rate of each group is calculated based on the control group. By comparing the cell survival rates at different concentrations, the effects of phenylboronic acid-modified mirinone prodrug and nicotinamide prodrug on the activity of L929 cells were evaluated. Cell viability was calculated according to the formula:

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

[0104] in:

[0105] As: absorbance of the experimental wells (culture medium containing cells, CCK-8, and test drug), Ac: absorbance of the control wells (culture medium containing cells, CCK-8, and no test drug), Ab: absorbance of the blank wells (culture medium without cells or test drug, CCK-8).

[0106] Within the set action concentration range, the phenylboronic acid-modified miridone prodrug and nicotinamide prodrug showed good cell compatibility with their respective original drugs in L929 cells. Compared with the control group, except for nicotinamide and the prodrug at a concentration of 1 mg / mL, which had a certain degree of effect on cell activity, the cell activity of the drugs at different concentrations in other experimental groups exceeded >80%. At the same time, there was no significant difference in the biological activity of the cells between the original drug and the prodrug at the same concentration. However, for the original drug abiraterone, which has a relatively high toxicity, the biocompatibility of the prodrug modified using this strategy has been greatly improved. In particular, the cytotoxicity of the abiraterone prodrug is greatly reduced compared to the original drug, and there is still a 75% survival rate at 106 ug / mL. At this time, the survival rate of the abiraterone original drug is only about 31%. This proves that the quaternary ammonium salt prodrug strategy based on phenylboronic acid has good biocompatibility and can significantly reduce the cytotoxicity of the original drug ( Figure 10 Example 10: Detection of the anti-tumor effect of phenylboronic acid-modified abiraterone prodrug on different tumor cells

[0107] Cancer cells were digested and resuspended, seeded in 96-well plates at a cell count of 5 × 10^3 per well, and cultured at 37°C in a 5% CO2 environment for 24 hours to allow the cells to adhere and reach a suitable density. Abiraterone prodrug was dissolved in 9g / L glucose water to prepare an initial solution of 850μg / mL, which was diluted with 9g / L glucose water and then mixed with equal volumes of DMEM (no HEPES) to form abiraterone prodrug solution with concentrations of 425, 212.5, 106.25, 53.125, 26.5625, 13.28125, 6.640625, and 3.320313μg / mL. 100μL of each solution was added to each culture well. A control group was set up with 100uL of an equal volume mixture of 9g / L glucose water and DMEM (no HEPES). Three replicate wells were set up for each group. The treated cells continued to be incubated in a CO2 incubator for 24 hours. After the culture was completed, the old culture medium was discarded, washed twice with PBS, and 110 μL of DMEM culture medium containing 10% CCK-8 solution was added to each well and incubated for another 2 hours. After the incubation was completed, 100 μL of supernatant from each well was aspirated into a new 96-well plate to avoid bubbles, and a blank group (DMEM culture medium containing only 10% CCK-8 solution) was set up. The absorbance value (OD value) of each well was measured at 450 nm using a microplate reader. The OD value of each well was converted into relative cell viability, and the cell survival rate of each group was calculated based on the control group. By comparing the cell survival rates at different concentrations, the effect of phenylboronic acid-modified abiraterone prodrug on the activity of 22RV1 cells was evaluated. Cell viability was calculated according to the calculation formula:

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

[0109] in:

[0110] As: absorbance of the experimental well (culture medium containing cells, CCK-8, and test drug); Ac: absorbance of the control well (culture medium containing cells, CCK-8, and no test drug); Ab: absorbance of the blank well (culture medium without cells or test drug, CCK-8).

[0111] Within the set action concentration range, phenylboronic acid modified abiraterone prodrug showed inhibitory activity in different tumor cells. Including: breast tumor cell line (4T1), human prostate cancer bone metastasis tumor cells (PC3), lung cancer basal epithelial cells (A549), human colon adenocarcinoma cells (SW48) ( Figure 11 Importantly, the abiraterone prodrug showed no significant anticancer activity at low concentrations, but exhibited significant inhibitory activity when combined with H₂O₂, particularly in human prostate cancer bone metastasis cells and human colon adenocarcinoma cells. This suggests that the abiraterone prodrug can release active drug in response to ROS.

[0112] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A class of bioactive molecules with ROS-responsive properties, characterized by: The structure is shown in the general formula I and the following structural formula: Where R is -H, -COOH, -COOCH3, -CONH2, 2. A method for preparing a class of bioactive molecules with ROS responsive properties, characterized in that: The method comprises the following steps: (1) In a nitrogen atmosphere, 4-bromomethylphenylboronic acid or 4-chloromethylphenylboronic acid and an aromatic tertiary amine are completely dissolved in anhydrous acetonitrile; (2) heating and stirring the solution in step (1) for 12 to 36 hours; (3) Thin layer chromatography (TLC) was used to detect that the reaction of the raw material was complete and a clear product was generated; (4) After the reaction is completed, the product is purified by using a co-solvent precipitation method and vacuum filtration or centrifugation to obtain a pure product; (5) For compounds that cannot be extracted by centrifugation or precipitation to obtain pure products, column chromatography is required to purify them to obtain pure prodrug products; (6) Drying the compound obtained in step (4) or step (5) to obtain a dried product.

3. The method for preparing a class of bioactive molecules with ROS responsiveness according to claim 2, characterized in that: In step (1), the molar ratio of 4-bromomethylphenylboric acid or 4-chloromethylphenylboric acid to the aromatic tertiary amine is (0.5-1.5): (0.5-1.5).

4. The method for preparing a class of bioactive molecules with ROS responsiveness according to claim 2, wherein: In step (1), the aromatic tertiary amine is nicotinamide, nicotinic acid, milrinone, abiraterone acetate, camptothecin, pyridoxine, nicorandil, piroxicam, risedronate, papaverine, pranoprofen, tipranavir, pioglitazone, or omeprazole.

5. The method for preparing a class of bioactive molecules with ROS responsiveness according to claim 2, characterized in that: In step (2), the heating temperature is 5 to 100° C.; In step (3), thin layer chromatography is used, and the developing solvent is selected as a mixed solution of methanol and dichloromethane.

6. Use of the bioactive molecule according to claim 1 and / or the bioactive molecule prepared by the method according to any one of claims 2 to 5 in the preparation of a product having ROS signal responsiveness.

7. A drug having ROS signal response performance, characterized in that: The drug comprises the bioactive molecule according to claim 1 and / or the bioactive molecule prepared by the method according to any one of claims 2 to 5, and a hydrogel for loading the bioactive molecule.

8. The method for preparing the drug having ROS signal response performance according to claim 7, characterized in that: The method comprises the following steps: Polyvinyl alcohol (PVA) and the bioactive molecule are dissolved in a mixed solution, heated and stirred to obtain a clear and transparent uniform solution. The obtained solution is cooled and then frozen, thawed until completely melted, and then frozen and thawed until completely melted. The freezing and thawing until completely melted steps are performed at least three times to fully cross-link the bioactive molecule with PVA through the dynamic covalent bond between phenylboronic acid and PVA, thereby preparing a drug-loaded hydrogel, namely the drug with ROS signal responsiveness.

9. The method for preparing a drug having ROS signal response performance according to claim 8, wherein: The mass fraction of the polyvinyl alcohol (PVA) in the mixed solution is 3 to 8%; The mixed solution is composed of PBS and water; The heating temperature is 70-90°C and the heating time is 0.5-1.5h; The feeding ratio of the bioactive molecule and the mixed solution is (0.5-1.5): (0.5-1.5).

10. A method for releasing a drug having ROS signal response performance according to claim 7, characterized in that: The method comprises the following steps: Reactive oxygen species with strong oxidizing effects attack phenylboronic acid with reducing function, causing a Baeyer-Villiger-like rearrangement to generate a borate ester intermediate, which is then hydrolyzed to generate a phenol intermediate. The covalent bond is broken through a 1,6-elimination reaction (quinone elimination), thereby eliminating the link between phenylboronic acid and the drug, and the bioactive molecule is decomposed into the drug prototype.

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