Fluorescent probe based on sulindac structure and preparation method thereof

Abstract

The invention relates to the field of novel application materials, in particular to a fluorescent probe based on a sulindac structure and a preparation method of the fluorescent probe. The method comprises the following steps: dissolving DHUCu-1 and alkali in an aprotic solvent, dropwise adding an intermediate dissolved by the aprotic solvent in an ice-water bath, and reacting in nitrogen after dropwise adding; the reaction progress is monitored using thin layer chromatography. And after the reaction is finished, purifying by silica gel column chromatography to obtain a white solid product. The innovative strategy provides a new thought for mechanism exploration of sulindac indications, and has wide clinical application prospects and conversion values.

Description

Technical Field

[0001] The present invention relates to the field of novel application materials, and in particular to the design and preparation of a functional hypochlorous acid fluorescent probe based on a sulindac structure and its application in hypochlorous acid detection. Background Art

[0002] Reactive oxygen species (ROS) play an important role in maintaining normal cell signaling and metabolic balance in organisms. However, abnormal overexpression of ROS can disrupt redox balance and trigger oxidative stress, leading to a variety of pathological conditions such as inflammation, cancer, neurodegenerative diseases, and cardiovascular diseases. Among these ROS, hypochlorous acid (HOCl) has attracted widespread attention due to its specific generation and significant role in pathological processes. Hypochlorous acid is mainly generated by myeloperoxidase in neutrophils and is involved in pathological damage and immune defense. Excessive concentrations may lead to cell apoptosis, tissue damage, and disease progression. Therefore, efficient detection of hypochlorous acid is of great significance for inflammation research, disease diagnosis, and the formulation of treatment strategies.

[0003] Although existing detection technologies (such as chromatography, colorimetry, and electrochemical sensors) show certain advantages in sensitivity and quantitative ability, these methods often rely on complex operating procedures and expensive equipment, such as precise laboratory conditions and skilled operators. This not only increases the time and economic cost of detection, but also limits its practicality in on-site detection and dynamic monitoring. Therefore, fluorescent probes have become a research hotspot due to their outstanding sensitivity, excellent real-time imaging capabilities, and simple operation methods. Fluorescent probes can accurately reflect the presence and concentration of specific target molecules through changes in optical signals, and are particularly suitable for monitoring in complex environments in biological systems. However, the existing hypochlorous acid fluorescent probes are relatively simple in functional design and usually only have the detection function for hypochlorous acid. This singleness limits its applicability in complex life systems.

[0004] The design of functional fluorescent probes is of great value in solving the above problems. Through reasonable chemical modification, functional probes can combine specific pathological features in the body (such as local overexpression of hypochlorous acid) to achieve highly sensitive and selective responses. In particular, the probe design strategy based on drug molecules can not only give the probes unique biological functions, but also significantly improve the biocompatibility and application potential of the probes. For example, sulindac, as a non-steroidal anti-inflammatory drug (NSAID) with anti-inflammatory and anti-tumor activity, has specific groups in its structure that provide ideal reaction sites for the chemical modification of the probe. At the same time, the development of sulindac derivatives can give the probes potential drug therapeutic functions. Therefore, the functional hypochlorous acid fluorescent probe based on the sulindac structure can not only accurately detect the hypochlorous acid level in the lesion area, but may also develop into an integrated diagnosis and treatment tool with dual functions in the future. Summary of the invention

[0005] In order to solve the problem that the existing hypochlorous acid fluorescent probes have a relatively single function and often only have a detection function, the present invention proposes a method for preparing a hypochlorous acid fluorescent probe based on the structure of sulindac. The method uses sulindac as a basic raw material, combines modern prodrug design concepts, and modifies its structure by introducing a reactive oxygen species (ROS) response mechanism, thereby developing a hypochlorous acid fluorescent probe with high specificity and selectivity.

[0006] A fluorescent probe based on sulindac structure has the structure shown in Formula 1:

[0007]

[0008] A method for preparing a fluorescent probe based on the structure of sulindac, the specific process is as follows:

[0009] DHUCu-1 and a base are dissolved in an aprotic solvent, and the intermediate dissolved in the aprotic solvent is added dropwise in an ice-water bath. After the addition is completed, the reaction is carried out under nitrogen. The reaction progress is monitored by thin layer chromatography. After the reaction is completed, silica gel column chromatography is used for purification to obtain a white solid product.

[0010] An in vitro detection solution for detection by the fluorescent probe of the present invention,

[0011] The preparation process is as follows: dissolving the compound of formula I in an organic solvent to prepare a 1-5 mM stock solution, then diluting it into a buffer solvent to make the final concentration of the prodrug 5 μM or 10 μM, and then testing the activation behavior, response time, and sulindac release;

[0012] The detection steps are as follows: During the test, first use a fluorescence spectrometer, an absorption spectrometer, and a high-performance liquid chromatography to evaluate the changes in fluorescence and absorption properties of the prodrug alone and the prodrug after adding different concentrations of active oxygen.

[0013] The compound with the structure of formula I of the present invention can be used for the detection of hypochlorous acid in the indications of sulindac, such as arthritis-related diseases (rheumatoid arthritis, osteoarthritis (degenerative joint disease), ankylosing spondylitis), acute gouty arthritis, musculoskeletal pain, soft tissue injury (such as sprain, strain), low back pain or neck pain, postoperative and other acute pain, cancer, etc.

[0014] The effects of the present invention are as follows:

[0015] 1. This invention proposes for the first time a fluorescent probe construction scheme based on the structure of sulindac and verifies it in an in vitro model;

[0016] 2. The fluorescent probe based on the structure of sulindac of the present invention can block the carboxyl group of the active site of sulindac, thereby avoiding the interference of other types of substances in the biological system with the carboxyl group;

[0017] 3. The fluorescent probe based on the structure of sulindac of the present invention can block the carboxyl group of the active site of sulindac, making it more difficult for the probe to carry charge, and thus not easily interfered by charged substances in the biological system;

[0018] 4. The fluorescent probe based on the sulindac structure of the present invention has good response performance to active oxygen and a fast response time.

[0019] 5. The fluorescent probe based on the sulindac structure of the present invention has good application in the indications of sulindac. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 It is the H NMR spectrum of the compound having the structure of formula I.

[0021] Figure 2 It is a high-resolution mass spectrum of the compound having the structure of formula I.

[0022] Figure 3 The fluorescence spectra of the compound having the structure of Formula I before and after the response to hypochlorous acid are shown in Figure 1. The concentration of I is 5 μM, the concentration of hypochlorous acid is 0-15 μM (from bottom to top, 0, 1, 2, 5, 10, 12, 15 μM, respectively), and the excitation wavelength is 620 nm.

[0023] Figure 4 The kinetic curve of the response of compound I with the structure of formula I of the present invention to hypochlorous acid (fluorescence intensity is 686nm). The concentration of I is 5μM, and the concentration of hypochlorous acid is 15μM (I is first added to the system, and hypochlorous acid is added after it is stable for nearly 30 seconds).

[0024] Figure 5 The HPLC chart of compound I with the structure of formula I according to the present invention releasing sulindac after reacting with hypochlorous acid.

[0025] Figure 6 This is an imaging diagram of the detection of hypochlorous acid in a tumor by compound I having a structure of formula I involved in the present invention. DETAILED DESCRIPTION

[0026] In order to enable those skilled in the art to better understand the technical solutions in the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments, and therefore cannot limit the present invention. Any changes and adjustments within the equivalent meaning and scope of the present invention should be considered to be within the scope of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0028] definition

[0029] Sulindac

[0030] Sulindac, CAS No. 38194-50-2, is a nonsteroidal anti-inflammatory drug (NSAID) belonging to the aromatic acetic acid class of compounds. Its chemical structure contains a pyrrole ring and a methylthiophenyl group. Its molecular formula is: C 20 H 17 F0 3 S, molecular weight is 356.42g / mol. As a prodrug, sulindac is metabolized in the body to form active metabolites, which inhibit cyclooxygenase (COX-1 and COX-2), reduce the production of prostaglandins, and exert anti-inflammatory, analgesic and antipyretic effects. Its main indications include rheumatoid arthritis, osteoarthritis, ankylosing spondylitis, acute gouty arthritis, soft tissue injury and postoperative pain. In addition, sulindac shows potential application value in the field of cancer chemoprevention (such as familial adenomatous polyposis). Common adverse reactions include gastrointestinal discomfort, liver and kidney dysfunction and cardiovascular risks. It should be used under the guidance of a doctor and relevant indicators should be monitored regularly to ensure safety. The structural formula contains a carboxyl group, which will interfere with its dissolution behavior and cellular uptake behavior in actual application. Therefore, modifying it into a prodrug with the help of a prodrug strategy and activating the release of sulindac by the active factors highly expressed in the disease area can greatly expand its application space.

[0031] Reactive oxygen species

[0032] Reactive oxygen species (ROS) are a class of oxidative species commonly found in disease areas, including hypochlorous acid (HOCl), hydrogen peroxide (H 2 O 2) and hydroxyl radicals (·OH), etc. In certain disease states, the body's biochemical balance is disrupted, causing an increase in oxidative stress and generating a large amount of ROS, such as hypochlorous acid (HOCl), superoxide anions (O 2 - ), hydrogen peroxide (H 2 O 2 ) and hydroxyl radicals (·OH). Therefore, reactive oxygen species are closely related to various diseases related to oxidative stress, such as arthritis, cancer, neurodegenerative diseases, liver damage, and kidney damage.

[0033] The present invention relates to a new type of fluorescent probe based on sulindac structure, wherein the compound has a structure shown in Formula 1:

[0034]

[0035] The preparation method of the intermediate SL-Cl of the present invention is as follows:

[0036]

[0037] Sulindac is dissolved in an aprotic solvent, and a small amount of N,N-dimethylformamide (DMF) is added to promote the reaction. Oxalyl chloride diluted with an aprotic solvent is added dropwise in an ice-water bath. The reaction is carried out under nitrogen protection to form an orange-yellow solution. Finally, the reaction solution is placed in a rotary evaporator and concentrated to remove excess oxalyl chloride and solvent to obtain the reaction intermediate SL-Cl.

[0038] In the preparation stage of the acyl chloride intermediate SL-Cl of the compound having the structure of formula I as shown, the aprotic solvent can be aprotic solvents such as dichloromethane and tetrahydrofuran. The amount of solvent used is 3-20 mL for 1 g of sulindac; the amount of DMF used is 0.01-0.5 mL; the amount of oxalyl chloride used is calculated by molar amount, and the molar ratio with sulindac is 1:1-5:1, preferably 1.2:1. The reaction temperature of the system is between 0 and 50°C, preferably 0°C.

[0039] The present invention relates to a method for preparing a compound having a structure of formula I, as follows:

[0040]

[0041] The synthesis of DHUCu-1 is referenced in Dyes Pigm.2022,204,110472.DOI:10.1016 / j.dyepig.2022.110472. DHUCu-1 (1.0 eq) and a base were dissolved in an aprotic solvent, and the intermediate dissolved in the aprotic solvent was added dropwise under an ice-water bath. After the addition was completed, the reaction was carried out under nitrogen. Thin layer chromatography (TLC) was used to monitor the reaction progress. After the reaction was completed, silica gel column chromatography was used for purification to obtain a white solid product.

[0042] In the preparation stage of the fluorescent probe based on the structure of sulindac of the present invention, the aprotic solvent may be dichloromethane, tetrahydrofuran, etc., and the amount of the aprotic solvent used is 3-20 mL for 1 g of DHUCu-1.

[0043] In the preparation stage of the fluorescent probe based on the sulindac structure of the present invention, the amount of SL-Cl is calculated by molar amount, and the molar ratio of DHUCu-1 to is 1:1 to 5:1, preferably 1.2:1.

[0044] In the preparation stage of the fluorescent probe based on sulindac structure of the present invention, the base can be carbonate, bicarbonate inorganic salts or triethylamine, dimethylaminopyridine (DMAP) and other organic bases, and the amount of the base is calculated by molar amount, and the molar ratio of the base to DHUCu-1 is 1:1 to 5:1.

[0045] In the preparation stage of the fluorescent probe based on sulindac structure of the present invention, the reaction time is 1 to 10 hours and the reaction temperature is between 0 and 50°C.

[0046] In the preparation stage of the fluorescent probe based on the sulindac structure of the present invention, the eluent used in the silica gel column chromatography can be a combination of ethyl acetate and petroleum ether, ethyl acetate and n-hexane, or methanol and dichloromethane.

[0047] The fluorescent probe based on the sulindac structure of the present invention has good response behavior and can respond and release in a short time.

[0048] The preparation and detection steps of the in vitro detection solution are as follows: dissolve the compound of formula I in an organic solvent (such as methanol, ethanol, N,N-dimethylformamide) to prepare a 1-5mM mother solution, then dilute it into a buffer solvent so that the final concentration of the prodrug is 5μM or 10μM, and then perform related tests such as activation behavior, response time, and sulindac release. During the test, first use a fluorescence spectrometer, an absorption spectrometer, a high-performance liquid chromatography, etc. to evaluate the changes in the fluorescence, absorption, and other properties of the prodrug alone and the prodrug after adding different concentrations of active oxygen.

[0049] The fluorescent probe based on the sulindac structure involved in the present invention can be used for the detection of hypochlorous acid in the indications of sulindac, such as arthritis-related diseases (rheumatoid arthritis, osteoarthritis (degenerative joint disease), ankylosing spondylitis), acute gouty arthritis, musculoskeletal pain, soft tissue injury (such as sprains, strains), low back pain or neck pain, postoperative and other acute pain, cancer, etc.

[0050] Example 1: Preparation of intermediate SL-Cl having a structural compound of formula I

[0051] Sulindac (0.75g, 2.29mmol, 1eq) and 0.02mL are added to a 100mL eggplant-shaped bottle of dry and clean, dichloromethane (DCM, 15mL) is added to dissolve, and a magnetic son is put into it and cooled with an ice-water bath. Subsequently, oxalyl chloride (560μL, 0.834g, 6.57mmol, 3eq) is added to a constant pressure dropping funnel with a pipette, and dissolved with DCM (5mL), and oxalyl chloride solution is dripped into the eggplant-shaped bottle under nitrogen protection, while stirring is maintained. Continue to stir after completion of the dropwise addition, and the reaction is completed within 10 minutes to form an orange-yellow solution. The reaction solution is placed in a rotary evaporator, and excess oxalyl chloride and solvent DCM are concentrated to obtain a reaction intermediate SL-Cl. The intermediate is directly used in the next step without purification.

[0052] Example 2: Preparation of a compound having the structure of formula I

[0053] Take a dry and clean 100mL eggplant-shaped bottle, add DHUCu-1 (1.20g, 3.34mmol, 1eq) and DMAP (0.82g, 6.68mmol, 2eq), place a magnetic bar and dissolve it with 10mL dichloromethane (DCM), and place the eggplant-shaped bottle in an ice-water bath to cool. Dissolve the SL-Cl obtained in the previous step with 10mL DCM, transfer it to a constant pressure dropping funnel, drip it into the reaction system drop by drop under nitrogen protection, and start stirring at the same time. Use thin layer chromatography (TLC) to monitor the reaction process, continue stirring the reaction for 3 hours, and the solution gradually turns yellow-green. After the reaction is completed, the solution is dried and the residue is dissolved with 400mL ethyl acetate. Subsequently, the organic phase is extracted with saturated brine, the extracted solution is dried, and then dissolved with DCM and loaded on the column. Purification is performed by silica gel column chromatography, and the initial mobile phase is ethyl acetate: petroleum ether = 1:5, and the ratio is gradually adjusted to 1:3, 1:1. 0.3g of white solid product is obtained with a yield of 19%. The NMR identification spectra and high-resolution identification spectra of the compounds are as follows: Figure 1 and Figure 2 As shown, the identification was shown as the target compound.

[0054] Example 3: Response performance of the compound of formula I to specific reactive oxygen species

[0055] This example studies the response performance of the compound having the structure of Formula I to a specific active oxygen species (hypochlorous acid, HOCl).

[0056] like Figure 3 As shown, at room temperature, 5 μM of compound 1 was prepared in 10 mM PBS buffer solution, and different concentrations of hypochlorous acid (0, 1, 2, 5, 10, 12, 15 μM) were added to the system. After reacting for 30 minutes, the fluorescence spectrometer was used for testing, and the excitation wavelength was 620 nm.

[0057] The experimental results show that when hypochlorous acid (0 μM) is not added to the system, compound I has no fluorescence signal; however, as the concentration of hypochlorous acid increases, sulindac and the fluorophore methylene blue are released from the system, resulting in a significant increase in fluorescence emission in the range of 640-850 nm. In particular, after adding 15 μM hypochlorous acid, the fluorescence intensity at 686 nm is enhanced by more than 1000 times compared to the system without hypochlorous acid.

[0058] The experimental results show that the compound I of formula I exhibits excellent response performance to hypochlorous acid.

[0059] Example 4: Reaction Kinetic Analysis of the Response of the Compound of Formula I to Active Oxygen Species

[0060] This example further analyzes the reaction kinetics of the compound of formula I in response to hypochlorous acid.

[0061] like Figure 5 As shown, 5 μM of compound 1 was prepared in 10 mM PBS buffer solution, 15 μM hypochlorous acid was added to the system, and the change in fluorescence intensity at 686 nm was monitored in real time.

[0062] During the experiment, 5 μM compound I was first added to the buffer solution, at which time the fluorescence intensity was low. After 50 seconds, hypochlorous acid was added, and the fluorescence intensity was observed to increase rapidly, and reached equilibrium at 150 seconds, of which the fluorescence intensity was close to the maximum within 100 seconds. This result shows that compound I has a fast response speed and good kinetic properties.

[0063] In summary, compound I having the structure of formula I responds sensitively and quickly to hypochlorous acid and has potential practical application value.

[0064] Example 5: Release behavior of sulindac after the compound having the structure of formula I reacts with HOCl

[0065] like Figure 5As shown, at room temperature, 20 μM sulindac was prepared in acetonitrile solution and 50 μM compound of formula I and 150 μM HOCl were prepared in 10 mM PBS buffer solution for 30 min. Then, PBS was extracted with DCM, filtered with a filter membrane, and loaded for HPLC test.

[0066] The experimental results show that after the reaction of the compound of formula I with HOCl, it can be clearly seen that its HPLC spectrum is consistent with the peak position of the sulindac standard sample, which indicates that the compound of formula I with HOCl can effectively release sulindac.

[0067] Example 6: Compounds having the structure of Formula I are used for imaging studies in tumors

[0068] Modeling: Mouse breast cancer cells (4T1 cells) were placed in a 37°C, 5% CO 2 Incubator, cultured in high-glucose DMEM medium containing 10% fetal bovine serum (FBS) and 1% streptomycin / penicillin. Balb / c mice were housed in an SPF-grade pathogen-free environment with a temperature of 20±3°C, a relative humidity of 40% to 70%, and a light / dark cycle of 12 hours. To establish a cancer model, mice were first fixed on a fixed plate and injected with 1% pentobarbital at a dose of 0.01 mL per gram of mouse body weight, and 1×10 6 4T1 cells were injected into the right axilla.

[0069] The compound having the structure of Formula I was injected into the tumor part, and the mouse was placed under a live imaging device for detection. It was found that the tumor area showed significant fluorescence, and the results were as follows: Figure 6 It is shown that the compound having the structure of formula 1 can detect hypochlorous acid in tumors.

[0070] The above description is not limited to the above examples. Technical features not described in the present invention can be achieved by or by adopting existing technologies, which will not be described herein. The above embodiments are only used to illustrate the technical scheme of the present invention and are not limitations of the present invention. Changes, modifications, additions or substitutions made by ordinary technicians in this technical field within the essential scope of the present invention do not depart from the purpose of the present invention and should also fall within the scope of protection of the claims of the present invention.

Claims

1. A fluorescent probe based on the structure of sulindac, characterized in that Having the structure shown in formula I:

2. The fluorescent probe based on the sulindac structure according to claim 1, characterized in that Including the intermediate SL-Cl, the preparation process is: Sulindac is dissolved in an aprotic solvent, and a small amount of N,N-dimethylformamide is added to promote the reaction; oxalyl chloride diluted in an aprotic solvent is added dropwise in an ice-water bath; the reaction is carried out under nitrogen protection to form an orange-yellow solution; finally, the reaction solution is placed in a rotary evaporator, and the excess oxalyl chloride and solvent are concentrated to obtain a reaction intermediate SL-Cl.

3. The fluorescent probe based on the sulindac structure according to claim 2, characterized in that The aprotic solvent is one of dichloromethane and tetrahydrofuran; the amount of the aprotic solvent is 3-20 mL for 1 g of sulindac; the amount of N,N-dimethylformamide is 0.01-0.5 mL; the amount of oxalyl chloride is calculated based on the molar amount, and the molar ratio with sulindac is 1:1-5:1; the reaction temperature is between 0 and 50°C.

4. A method for preparing a fluorescent probe based on the sulindac structure according to any one of claims 1 to 3, characterized in that The specific process is as follows: DHUCu-1 and a base are dissolved in an aprotic solvent, and the intermediate dissolved in the aprotic solvent is added dropwise in an ice-water bath. After the addition is completed, the reaction is carried out under nitrogen. The reaction progress is monitored by thin layer chromatography. After the reaction is completed, silica gel column chromatography is used for purification to obtain a white solid product.

5. The preparation method according to claim 4, characterized in that The aprotic solvent is one of dichloromethane and tetrahydrofuran; the amount of the aprotic solvent used is 3-20 mL for 1 g of DHUCu-1.

6. The preparation method according to claim 4, characterized in that The amount of SL-Cl used was calculated based on molar amount, and the molar ratio of DHUCu-1 to SL-Cl was 1:1 to 5:

1.

7. The preparation method according to claim 4, characterized in that The base is an inorganic salt or an organic base. The amount of the base is calculated based on the molar amount, and the molar ratio of the base to DHUCu-1 is 1:1 to 5:

1.

8. The preparation method according to claim 4, characterized in that The reaction time is between 1 and 10 hours, and the reaction temperature is between 0 and 50°C.

9. The preparation method according to claim 4, characterized in that The eluent used for silica gel column chromatography is a combination of ethyl acetate and petroleum ether or ethyl acetate and n-hexane or methanol and dichloromethane.

10. An in vitro detection solution for the fluorescent probe according to claim 1, characterized in that: The preparation process is as follows: dissolving the compound of formula I in an organic solvent to prepare a 1-5 mM stock solution, then diluting it into a buffer solvent to make the final concentration of the prodrug 5 μM or 10 μM, and then testing the activation behavior, response time, and sulindac release; The detection steps are as follows: During the test, first use a fluorescence spectrometer, an absorption spectrometer, and a high-performance liquid chromatography to evaluate the changes in fluorescence and absorption properties of the prodrug alone and the prodrug after adding different concentrations of active oxygen.

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