Application of ONOO- and / or Na2S2 fluorescent probe reagents for early diagnosis of tumors

Abstract

The present application provides an application for preparing ONOO‑ and / or Na2S2 signal fluorescent probe reagents for early diagnosis of tumors, characterized in that it includes a fluorescent probe 4‑chloro‑11‑ethylpyrano[2,3‑b]phenothiazine‑2(11H)‑one having the general structural formula: #imgabs0# The probe is used in the preparation of ONOO‑ and / or Na2S2 biomolecule detection reagents. The preparation method comprises: adding 2-methoxyphenothiazine and iodoethane to a mixed solution of dichloromethane and acetonitrile for reaction, and then performing post-treatment to obtain 10-ethyl-2-methoxy-10H-phenothiazine; adding boron tribromide to 10-ethyl-2-methoxy-10H-phenothiazine under inert gas protection conditions, reacting under ice bath conditions, and then performing post-treatment to obtain 10-ethyl-10H-phenothiazine-2-ol; mixing 10-ethyl-10H-phenothiazine-2-ol, malonic acid, zinc chloride, and phosphorus oxychloride for reaction, and then performing post-treatment to obtain a multi-signal fluorescent probe. The multi-signal fluorescent probe provided in the present application can simultaneously detect ONOO by ratio differentiation. ‑ and / or Na2S2.

Description

Technical Field

[0001] This application is a divisional application of the invention patent application with the application date of April 25, 2022, application number 202210441449.6, and invention name “Multi-signal fluorescent probe for early diagnosis of tumors, preparation method and application thereof”.

[0002] The present application relates to the field of analytical chemistry, and in particular to an application of an ONOO- and / or Na2S2 fluorescent probe reagent for preparing early diagnosis of tumors. Background Art

[0003] Reactive oxygen species (ROS) and reactive sulfur species (RNS) play important roles in a variety of physiological processes. They are also involved in various pathophysiological processes, including cancer, aging, signal transduction, cellular redox homeostasis, and defense against pathogen invasion. Peroxynitrite (ONOO - ) is an important reactive nitrogen that plays an important role in many physiological and pathological processes. There is evidence that excessive ONOO - It is associated with various human diseases, such as hypertension, gastric cancer, neurodegenerative diseases, and inflammatory diseases; in addition, hydrogen sulfide (Na2S2) plays an important role in regulating the intracellular redox state and basic signal transduction processes. Therefore, it is necessary to develop efficient detection methods / techniques for the simultaneous detection of ONOO - and Na2S2 are of great significance for the early diagnosis of diseases.

[0004] Fluorescence imaging technology based on small molecule fluorescent probes has become an effective method for visualizing the spatiotemporal distribution of biomolecules in biological specimens due to its real-time, sensitive, and non-invasive characteristics. Currently, many small molecule fluorescent probes have been developed for the detection of reactive oxygen species (ONOO - ), but some single / dual fluorescence channel fluorescent probes have also been developed for detecting ONOO - / Na2S2. However, the reported fluorescent probes cannot simultaneously distinguish and detect ONOO - and Na2S2, two biological molecules, especially endogenous ONOO - And Na2S2 dual-channel ratiometric fluorescence imaging analysis detection. Summary of the invention

[0005] The purpose of the present application is to provide a multi-signal fluorescent probe for early diagnosis of tumors and its preparation method and application to solve the above problems.

[0006] To achieve the above objectives, this application adopts the following technical solutions:

[0007] A multi-signal fluorescent probe for early diagnosis of tumors, the general structural formula of which is:

[0008]

[0009] The present application also provides a method for preparing the multi-signal fluorescent probe for early diagnosis of tumors, comprising:

[0010] Adding 2-methoxyphenothiazine and ethyl iodide to a mixed solution of dichloromethane and acetonitrile, performing a first reaction, and then performing a first post-treatment to obtain 10-ethyl-2-methoxy-10H-phenothiazine;

[0011] Under inert gas protection, adding boron tribromide to 10-ethyl-2-methoxy-10H-phenothiazine, performing a second reaction in an ice bath, and then performing a second post-treatment to obtain 10-ethyl-10H-phenothiazine-2-ol;

[0012] 10-ethyl-10H-phenothiazine-2-ol, malonic acid, zinc chloride and phosphorus oxychloride are mixed, subjected to a third reaction, and then subjected to a third post-treatment to obtain the multi-signal fluorescent probe for early diagnosis of tumors.

[0013] Preferably, the temperature of the first reaction is 65-75°C and the time is 8-12h.

[0014] Preferably, the first post-processing comprises:

[0015] The reaction system is separated by column chromatography to obtain the 10-ethyl-2-methoxy-10H-phenothiazine.

[0016] Preferably, the second reaction is carried out for 4-6 hours under light-shielding and stirring conditions.

[0017] Preferably, the second post-processing comprises:

[0018] The red solution obtained by the reaction was dropped into ice water, and the pH of the system was adjusted to 6-7; then, it was extracted with dichloromethane, and the solvent was removed from the organic layer to obtain the 10-ethyl-10H-phenothiazine-2-ol.

[0019] Preferably, the third reaction is carried out under reflux stirring at 80° C. for 24 hours.

[0020] Preferably, the third post-processing comprises:

[0021] The brown viscous solution obtained by the reaction is dropped into ice water, and the pH of the system is adjusted to 6-7; then it is extracted with dichloromethane, the solvent is removed from the organic layer, and then the multi-signal fluorescent probe for early diagnosis of tumors is obtained by column chromatography.

[0022] Preferably, the pH is adjusted using 20% ​​aqueous sodium hydroxide solution.

[0023] The present application also provides an application of a multi-signal fluorescent probe for early diagnosis of tumors, which is used to detect ONOO in cells. - and Na2S2.

[0024] Compared with the prior art, the beneficial effects of this application include:

[0025] The multi-signal fluorescent probe for early diagnosis of tumors provided in the present application emits red light of 610nm at an excitation wavelength of 410nm, and ONOO - After the reaction, it emits 492nm green light at 380nm excitation wavelength, and after reacting with Na2S2, it emits 548nm yellow light at 410nm excitation wavelength. It emits red fluorescence by itself and can react with ONOO under the same detection conditions. - Different chemical reactions occur with Na2S2 to generate different fluorescent substances, which emit green and yellow fluorescence at specific excitation wavelengths, achieving simultaneous ratio-differentiated detection of ONOO - and Na2S2, and can be widely used in analytical testing, early medical diagnosis and other fields. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings required for use in the embodiments will be briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present application and therefore should not be regarded as limiting the scope of the present application.

[0027] Figure 1 This is the nuclear magnetic resonance hydrogen spectrum of the fluorescent probe obtained in Example 1 of the present application.

[0028] Figure 2 This is the fluorescence spectrum of the fluorescent probe obtained in Example 1 of the present application for detecting peroxynitrite and hydrogen sulfide. DETAILED DESCRIPTION

[0029] As used herein:

[0030] "Prepared from" is synonymous with "comprising." As used herein, the terms "comprising," "including," "having," "containing," or any other variation thereof, are intended to cover a non-exclusive inclusion. For example, a composition, process, method, article, or apparatus that comprises the listed elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such composition, process, method, article, or apparatus.

[0031] The conjunction "consisting of excludes any unspecified element, step, or component. If used in a claim, this phrase renders the claim closed-ended so that it does not include materials other than those described, except for conventional impurities associated therewith. When the phrase "consisting of" appears in a clause of the body of a claim rather than immediately following the subject matter, it limits only the elements described in that clause; other elements are not excluded from the claim as a whole.

[0032] When an amount, concentration, or other value or parameter is expressed as a range, a preferred range, or a range defined by a series of upper preferred values ​​and lower preferred values, this should be understood as specifically disclosing all ranges formed by any pairing of any range upper limit or preferred value with any range lower limit or preferred value, regardless of whether the range is disclosed separately. For example, when a range of "1 to 5" is disclosed, the described range should be interpreted as including ranges "1 to 4", "1 to 3", "1 to 2", "1 to 2 and 4 to 5", "1 to 3 and 5", etc. When a numerical range is described in this article, unless otherwise stated, the range is intended to include its end values ​​and all integers and fractions within the range.

[0033] In these examples, parts and percentages are by mass unless otherwise indicated.

[0034] "Parts by mass" refers to the basic unit of measurement for expressing the mass ratio of multiple components. 1 part can represent any unit mass, such as 1g or 2.689g. If we say that the mass of component A is a parts and the mass of component B is b parts, it means the ratio of the mass of component A to the mass of component B is a:b. Alternatively, it means that the mass of component A is aK and the mass of component B is bK (K is an arbitrary number, indicating a multiple factor). It should not be misunderstood that, unlike the mass parts, the sum of the mass of all components is not limited to 100 parts.

[0035] "And / or" is used to indicate that one or both of the stated situations may occur, for example, A and / or B includes (A and B) and (A or B).

[0036] A multi-signal fluorescent probe for early diagnosis of tumors, the general structural formula of which is:

[0037]

[0038] The present application also provides a method for preparing the multi-signal fluorescent probe for early diagnosis of tumors, comprising:

[0039] Adding 2-methoxyphenothiazine and ethyl iodide to a mixed solution of dichloromethane and acetonitrile, performing a first reaction, and then performing a first post-treatment to obtain 10-ethyl-2-methoxy-10H-phenothiazine;

[0040] Under inert gas protection, adding boron tribromide to 10-ethyl-2-methoxy-10H-phenothiazine, performing a second reaction in an ice bath, and then performing a second post-treatment to obtain 10-ethyl-10H-phenothiazine-2-ol;

[0041] 10-ethyl-10H-phenothiazine-2-ol, malonic acid, zinc chloride, and phosphorus oxychloride are mixed, subjected to a third reaction, and then subjected to a third post-treatment to obtain the multi-signal fluorescent probe.

[0042] In an optional embodiment, the temperature of the first reaction is 65-75°C and the time is 8-12h.

[0043] Optionally, the temperature of the first reaction can be 65°C, 70°C, 75°C or any value between 65-75°C, and the time can be 8h, 10h, 12h or any value between 8-12h.

[0044] In an optional embodiment, the first post-processing includes:

[0045] The reaction system is separated by column chromatography to obtain the 10-ethyl-2-methoxy-10H-phenothiazine.

[0046] In an optional embodiment, the second reaction is carried out for 4-6 hours under light-shielding and stirring conditions.

[0047] In an optional embodiment, the second post-processing comprises:

[0048] The red solution obtained by the reaction was dropped into ice water, and the pH of the system was adjusted to 6-7; then, it was extracted with dichloromethane, and the solvent was removed from the organic layer to obtain the 10-ethyl-10H-phenothiazine-2-ol.

[0049] In an optional embodiment, the third reaction is carried out under reflux stirring at 80-90° C. for 24-36 hours.

[0050] Optionally, the temperature of the third reaction can be 80°C, 85°C, 90°C or any value between 80-90°C, and the time can be 24h, 30h, 36h or any value between 24-36h.

[0051] In an optional embodiment, the third post-processing includes:

[0052] The brown viscous solution obtained by the reaction is dropped into ice water, and the pH of the system is adjusted to 6-7; then it is extracted with dichloromethane, the solvent is removed from the organic layer, and then the multi-signal fluorescent probe for early diagnosis of tumors is obtained by column chromatography.

[0053] In an alternative embodiment, the pH is adjusted using 20% ​​aqueous sodium hydroxide solution.

[0054] The present application also provides an application of a multi-signal fluorescent probe for early diagnosis of tumors, which is used to detect ONOO in cells. - and Na2S2.

[0055] The embodiments of the present application will be described in detail below in conjunction with specific examples, but it will be appreciated by those skilled in the art that the following examples are only used to illustrate the present application and should not be considered as limiting the scope of the present application. If specific conditions are not specified in the examples, they are carried out according to normal conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified in the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0056] First, the synthesis route of the multi-signal fluorescent probe provided in this application is described as follows:

[0057]

[0058] Example

[0059] Step 1: Synthesis of 10-ethyl-2-methoxy-10H-phenothiazine

[0060] a. Add 2.0 g (8.72 mmol) of 2-methoxyphenothiazine and 3.26 g (20.93 mmol) of ethyl iodide to a mixed solution of 20 mL of dichloromethane and acetonitrile and react at 65 ° C for 8-12 hours.

[0061] b. After the reaction mixture was cooled to room temperature, column chromatography was performed to obtain 616 mg of 10-ethyl-2-methoxy-10H-phenothiazine as a white solid with a yield of 27.5%.

[0062] Step 2: Synthesis of 10-ethyl-10H-phenothiazin-2-ol

[0063] I. Under nitrogen protection, 5.84 g (23.31 mmol) of boron tribromide (BBr3) was slowly added to a dichloromethane solution of 1.0 g (3.89 mmol) of 10-ethyl-2-methoxy-10H-phenothiazine, and the mixture was stirred in an ice bath in the dark for 4-6 hours to obtain a red solution.

[0064] II. The reaction solution was slowly dripped into ice water and the pH was adjusted to 6-7 with 20% NaOH solution.

[0065] III. The substance in the ice water was extracted with dichloromethane (DCM), and the extraction was repeated several times. The organic layer obtained by extraction was spin-dried to obtain 600 mg of light green solid 10-ethyl-10H-phenothiazine-2-ol with a yield of 63.46%.

[0066] Step 3: Synthesis of multi-signal fluorescent probe 4-chloro-11-ethylpyrano[2,3-b]phenothiazine-2(11H)-one

[0067] i. 10-ethyl-10H-phenothiazine-2-ol, malonic acid and zinc chloride were mixed in a phosphorus oxychloride solution and stirred at 80°C under air reflux for 24 hours to obtain a brown viscous solution.

[0068] ii. The reaction solution was slowly dripped into ice water and the pH was adjusted to 6-7 with 20% NaOH solution.

[0069] iii. The substance in the ice water was extracted with dichloromethane (DCM), and the extraction was repeated several times. The organic layer obtained by the extraction was spin-dried and purified by column chromatography to obtain a yellow solid 4-chloro-11-ethylpyrano[2,3-b]phenothiazine-2(11H)-one with a yield of 30.3%.

[0070] The H spectrum of the obtained multi-signal fluorescent probe 4-chloro-11-ethylpyrano[2,3-b]phenothiazine-2(11H)-one is shown in Figure 1 shown.

[0071] Differentiate and detect ONOO produced in organisms - and Na2S2 fluorescent probes (unless otherwise specified, the operation steps for other probes are similar). The multi-signal fluorescent molecular probe spectral property experiment provided in this application: the probe is dissolved in dimethyl sulfoxide (DMSO) to prepare a probe solution with a concentration of 1mM, and ONOO - The specific test method is: take 20μL of 1mM probe solution, 980μL of analytical pure CH3CN, the required amount of 1mM ONOO - / Na2S2 aqueous solution and the required amount of PBS buffer aqueous solution were placed in a 2 mL sample tube. The volume ratio of the organic phase to the aqueous phase was kept at 5:5 for all tests (the total volume of each test sample was 2 mL). For example, when ONOO - When the concentration was 20 μM, the probe and ONOO - Fluorescence intensity after the reaction. The sample preparation was as follows: 20 μL of 1 mM probe solution, 980 μL of analytical grade CH3CN, 20 μL of 1 mM ONOO - The aqueous solution and 980 μL of PBS buffer solution were placed in a 2 mL sample tube and shaken at room temperature for 15 minutes before measuring the fluorescence emission intensity at an excitation wavelength of 350 nm. Other test operations were similar to the above steps. -The detection limits of Na2S2 and Na2S2 are as low as 42.12nM and 38.45nM respectively (the fluorescence spectra of peroxynitrite and hydrogen sulfide detected by fluorescent probes are shown in Figure 2). Figure 2 Therefore, the multi-signal fluorescent molecular probe disclosed in the present invention can achieve high-sensitivity distinguishing detection of the two.

[0072] The present invention distinguishes and detects ONOO produced in the organism - and Na2S2 fluorescent probes, and developed a class of efficient and simple multi-channel detection probes for oxidized and reduced substances, which can be used to simultaneously distinguish and detect ONOO - and Na2S2, based on the same probe and the same detection conditions using the probe and ONOO - Different chemical reactions occur with Na2S2 to generate different fluorescent substances, which emit green, yellow and three colors of fluorescence at specific excitation wavelengths, achieving the purpose of simultaneous differentiation and detection, and successfully realizing dual-channel simultaneous fluorescence imaging of endogenous ONOO in cells. - and Na2S2. We hope that this will provide some ideas for the future development of multi-signal biological fluorescent probes.

[0073] The multi-signal fluorescent probe provided in this application belongs to a class of probes that utilize dual-channel fluorescent signals to simultaneously distinguish and detect ONOO - The fluorescent molecular probes of Na2S2 provide some analytical detection methods and ideas for the fields of analytical detection, early medical diagnosis, etc.

[0074] The multi-signal fluorescent probe provided in the present application simultaneously distinguishes and detects ONOO - Usage of Na2S2: Unless otherwise specified, the probe molecule is usually dissolved in an environment with a volume ratio of 5:5 between the organic phase and the aqueous phase at room temperature for analysis and detection. The organic phase is acetonitrile (CH3CN), and the aqueous phase is a phosphate buffer solution (PBS) with a pH of 7.4 and an aqueous solution of the analyte.

[0075] The specific characteristics of the multi-signal active oxygen fluorescence probe provided in the present application are as follows: the molecular fluorescent probe is dissolved in dimethyl sulfoxide (DMSO), the probe molecule is dissolved in an organic phase and an aqueous phase (5:5, v / v) solution, and the probe is mixed with ONOO - After the reaction, it emits 492nm green light at 380nm excitation wavelength, and after reacting with Na2S2, it emits 548nm yellow light at 410nm excitation wavelength. Therefore, it can detect specific analytes by specific excitation and fluorescence emission signals, and the two can be well distinguished by different excitation and fluorescence emission signals. The above fluorescent molecular probe can simultaneously distinguish and detect ONOO under the same detection conditions. - and Na2S2, to OH,t-BuO, 1 O2,NO,ONOO -,O2· - ,GSH,Cys,Hcy,SO2,NAC,H2S,F-,Cl-,Br - ,I - ,NAC,Gly,Ala,His,Met,Thr,Lys,Asp,Glu,Pro,Ser,NaHS,NaHSO3,EtSH,PhSH,n-Butylamine,aniline and other amino acids, sulfur-containing derivatives, amine derivatives had no obvious response, and ONOO - The detection limits of 2,4-dihydrogen 2-acid and 2,4-dihydrogen 2-acid are as low as 42.12 nM and 38.45 nM, respectively. Therefore, the multi-signal fluorescent molecular probe provided by the present application can realize the high-sensitivity distinguishing detection of the two.

[0076] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present application, rather than to limit it. Although the present application has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present application.

[0077] In addition, those skilled in the art will appreciate that, although some embodiments herein include certain features included in other embodiments but not other features, the combination of features of different embodiments is meant to be within the scope of the present application and form different embodiments. For example, in the above claims, any one of the claimed embodiments may be used in any combination. The information disclosed in this background technology section is intended only to deepen the understanding of the overall background technology of the present application and should not be regarded as an admission or in any form of implication that the information constitutes prior art known to those skilled in the art.

Claims

1. Use of a fluorescent probe in the preparation of ONOO- and / or Na2S2 signal fluorescent probe reagents for early diagnosis of tumors, characterized in that: The fluorescent probe is 4-chloro-11-ethylpyrano[2,3-b]phenothiazine-2(11H)-one, and the fluorescent probe is used to prepare ONOO- and / or Na2S2 biomolecule detection reagents, wherein the fluorescent probe structural formula is:

Images