MULTI-SIGNAL FLUORESCENT PROBE FOR EARLY DIAGNOSIS OF TUMORS, AND PREPARATION AND USE THEREOF

Abstract

A multi-signal fluorescent probe, represented by:

##STR00001##

A method for preparing the multi-signal fluorescent probe includes: (a) adding 2-methoxyphenothiazine and ethyl iodide into a mixture of dichloromethane (DCM) and acetonitrile followed by a first reaction and a first post-treatment to obtain 10-ethyl-2-methoxy-10H-phenothiazine; (b) adding boron tribromide into the 10-ethyl-2-methoxy-10H-phenothiazine under an inert gas followed by a second reaction under an ice bath and a second post-treatment to obtain 10-ethyl-10H-phenothiazin-2-ol; and (c) mixing the 10-ethyl-10H-phenothiazin-2-ol, malonic acid, zinc chloride and phosphorus oxychloride followed by a third reaction and a third post-treatment to obtain the multi-signal fluorescent probe. A use of the multi-signal fluorescent probe in the detection of intracellular ONOO.sup.- and Na.sub.2S.sub.2 is also provided.

Claims

1. A multi-signal fluorescent probe for early diagnosis of tumors, wherein a structural formula of the multi-signal fluorescent probe is shown as follows: ##STR00005## .

2. A method for preparing the multi-signal fluorescent probe of claim 1, comprising: adding 2-methoxyphenothiazine and ethyl iodide into a mixture of dichloromethane (DCM) and acetonitrile followed by a first reaction to obtain a reaction mixture; and subjecting the reaction mixture to a first post-treatment to obtain 10-ethyl-2-methoxy-10H-phenothiazine; adding boron tribromide into the 10-ethyl-2-methoxy-10H-phenothiazine under an inert gas followed by a second reaction under an ice bath and a second post-treatment to obtain 10-ethyl-10H-phenothiazin-2-ol; and mixing the 10-ethyl-10H-phenothiazin-2-ol, malonic acid, zinc chloride and phosphorus oxychloride followed by a third reaction and a third post-treatment to obtain the multi-signal fluorescent probe.

3. The method of claim 2, wherein the first reaction is performed at 65-75° C. for 8-12 h.

4. The method of claim 2, wherein the first post-treatment comprises: subjecting the reaction mixture to column chromatography to obtain the 10-ethyl-2-methoxy-10H-phenothiazine.

5. The method of claim 2, wherein the second reaction is performed under stirring in a dark environment for 4-6 h.

6. The method of claim 2, wherein the second post-treatment comprises: dropwise adding a red reaction solution resulted from the second reaction into ice water followed by pH adjustment to 6-7 and extraction with DCM to collect an organic layer; and concentrating the organic layer to obtain the 10-ethyl-10H-phenothiazin-2-ol.

7. The method of claim 2, wherein the third reaction is performed under reflux and stirring at 80-90° C. for 24-36 h.

8. The method of claim 2, wherein the third post-treatment comprises: dropwise adding a brown viscous solution resulted from the third reaction into ice water followed by pH adjustment to 6-7 and extraction with DCM to collect an organic layer; and subjecting the organic layer to concentration and column chromatography to obtain the multi-signal fluorescent probe.

9. The method of claim 6, wherein the pH adjustment is performed with a 20 wt.% sodium hydroxide aqueous solution.

10. The method of claim 8, wherein the pH adjustment is performed with a 20 wt.% sodium hydroxide aqueous solution.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to explain the technical solutions of the present disclosure more clearly, the accompanying drawings needed in the description of the embodiments of the present disclosure will be briefly described below. Obviously, presented in the accompany drawings are merely some embodiments of the present disclosure, which are not intended to limit the disclosure.

[0029] FIG. 1 shows a proton nuclear magnetic resonance (.sup.1H-NMR) spectrum of a multi-signal fluorescent probe according to an embodiment of the present disclosure; and

[0030] FIG. 2 shows a fluorescence spectrum of the multi-signal fluorescent probe according to an embodiment of the present disclosure in the presence of peroxynitrite and sodium disulfide.

DETAILED DESCRIPTION OF EMBODIMENTS

[0031] It should be noted that “prepared by...” is synonymous with “include”. In addition, the term “include”, “have”, “contain” or any other variant thereof is intended to cover non-exclusive inclusion. For example, a process, method, article, or apparatus/device need not be limited to those listed elements, but may include other elements that are not explicitly listed or inherent to the process, method, article or device. The term “consist of” excludes any unspecified elements or components. This term has a closed meaning when used in claims, indicating the exclusion of other unspecified materials except for conventional impurities associated therewith. When the phrase “consist of” appears in a clause in the body of a claim rather than immediately following the subject matter, it is only to limit the element described in such clause; other elements are not excluded from the claim as a whole.

[0032] When 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, it should be understood as specifically disclosing all ranges formed by any pairing of any upper limit or preferred value with any lower limit or preferred value, regardless of whether such range is separately disclosed. For example, a range “1-5” should be interpreted to include ranges of “1-4”, “1-3”, “1-2”, “1-2 and 4-5”, and “1-3 and 5”. Unless otherwise indicated, the numerical range described herein is intended to include its endpoints and all integers and fractions within the range.

[0033] In this application, the parts and percentages are expressed by weight unless otherwise indicated.

[0034] The phrase “part by weight” refers to a basic measurement unit indicating the mass ratio relationship of multiple components. For example, 1 part can represent any unit of mass, such as 1 g and 2.689 g. If it is specified that there are a parts by weight of component A and b parts by weight of component B, it means a mass ratio of A to B is a:b, or indicates that the mass of component A is aK and the mass of component B is bK (K is an arbitrary value, representing a multiplication factor). It should be noted that the sum of the parts by weight of all the components is not limited to 100 parts.

[0035] The term “and/or” indicates that one or both of the specified conditions may occur, for example, “A and/or B” includes “A”, “B”, and “a combination of A and B”.

[0036] Provided is a multi-signal fluorescent probe for early diagnosis of a tumor, where structural formula of the multi-signal fluorescent probe is shown as follows:

##STR00003##

[0037] A method for preparing the multi-signal fluorescent probe is also provided, including: [0038] adding 2-methoxyphenothiazine and ethyl iodide into a mixture of DCM and acetonitrile followed by a first reaction to obtain a reaction mixture; and subjecting the reaction mixture to a first post-treatment to obtain 10-ethyl-2-methoxy-10H-phenothiazine; [0039] adding boron tribromide into the 10-ethyl-2-methoxy-10H-phenothiazine under an inert gas, followed by a second reaction under an ice bath and a second post-treatment to obtain 10-ethyl-10H-phenothiazin-2-ol; and [0040] mixing the 10-ethyl-10H-phenothiazin-2-ol, malonic acid, zinc chloride and phosphorus oxychloride followed by a third reaction and a third post-treatment to obtain the multi-signal fluorescent probe.

[0041] In an embodiment, the first reaction is performed at 65-75° C. for 8-12 h.

[0042] In an embodiment, the first reaction is performed at 65° C., 70° C., 75° C. or any temperature between 65-75° C. for 8 h, 10 h, 12 h or any duration between 8-12 h.

[0043] In an embodiment, the first post-treatment includes:

[0044] subjecting the reaction mixture to column chromatography to obtain the 10-ethyl-2-methoxy-10H-phenothiazine.

[0045] In an embodiment, the second reaction is performed under stirring in a dark environment for 4-6 h.

[0046] In an embodiment, the second post-treatment includes: [0047] dropwise adding a red solution resulted from the second reaction into ice water followed by pH adjustment to 6-7 and extraction with DCM to collect an organic layer; and [0048] concentrating the organic layer to obtain the 10-ethyl-10H-phenothiazin-2-ol.

[0049] In an embodiment, the third reaction is performed under reflux and stirring at 80-90° C. for 24-36 h.

[0050] In an embodiment, the third reaction is performed at 80° C., 85° C., 90° C. or any temperature within 80-90° C. for 24 h, 30 h, 36 h or any duration within 24-36 h.

[0051] In an embodiment, the third post-treatment includes: [0052] dropwise adding a brown viscous solution resulted from the third reaction into ice water followed by pH adjustment to 6-7 and extraction with DCM to collect an organic layer; and [0053] subjecting the organic layer to concentration and column chromatography to obtain the multi-signal fluorescent probe.

[0054] In an embodiment, the pH adjustment is performed with a 20 wt.% sodium hydroxide aqueous solution.

[0055] The present disclosure also provides a use of the multi-signal fluorescent probe in the detection of ONOO.sup.- and Na.sub.2S.sub.2 in cells.

[0056] The present disclosure will be described in detail below with reference to the embodiments. Obviously, described below are merely some embodiments of this disclosure, and are not intended to limit the disclosure. Unless otherwise specified, the materials and reagents used in the following embodiments are available commercially, and the experiments are carried out using conventional methods.

[0057] A synthesis route of the multi-signal fluorescent probe is illustrated as follows:

##STR00004##

EXAMPLE

(S1) Synthesis of 10-Ethyl-2-Methoxy-10H-Phenothiazine

[0058] (1a) 2.0 g (8.72 mmol) of 2-methoxyphenothiazine and 3.26 g (20.93 mmol) of ethyl iodide were added into 20 mL of DCM-acetonitrile mixture. The reaction mixture was reacted at 65° C. for 8-12 h.

[0059] (1b) After cooled to room temperature, the reaction mixture was subjected to column chromatography to obtain 616 mg of white solid as 10-ethyl-2-methoxy-10H-phenothiazine (yield: 27.5%).

(S2) Synthesis of 10-Ethyl-10H-Phenothiazin-2-ol

[0060] (2a) 5.84 g (23.31 mmol) of boron tribromide (BBr.sub.3) was slowly added into a solution of the 10-ethyl-2-methoxy-10H-phenothiazine (1.0 g, 3.89 mmol) in DCM under the protection of nitrogen gas. The reaction mixture was stirred on an ice bath in the dark for 4-6 h to obtain a red solution.

[0061] (2b) The red solution was slowly dropwise added into ice water, and the resultant mixture was adjusted to pH 6-7 with a 20 wt.% sodium hydroxide aqueous solution.

[0062] (2c) The reaction mixture was subjected to multiple extractions with DCM, and the resultant organic layers were collected, combined and subjected to rotary evaporation to obtain 600 mg of a light green solid as 10-ethyl-10H-phenothiazin-2-ol (yield: 63.46%).

(S3) Synthesis of 4-Chloro-11-Ethylpyrano[2,3-b]Phenothiazin-2(11H)-One (the Multi-Signal Fluorescent Probe)

[0063] (3a) The 10-ethyl-10H-phenothiazin-2-ol, malonic acid and zinc chloride were mixed in a phosphorus oxychloride solution, and reacted under stirring and air reflux at 80° C. for 24 h to obtain a brown viscous solution.

[0064] (3b) The brown viscous solution was slowly dropwise added into ice water, and the resultant mixture was adjusted to pH 6-7 with a 20 wt.% sodium hydroxide aqueous solution.

[0065] (3c) The reaction mixture was subjected to multiple extractions with DCM, and the resultant organic layers were collected, combined, and subjected to rotary evaporation and column chromatography to obtain a yellow solid as 4-chloro-11-ethylpyrano[2,3-b]phenothiazin-2(11H)-one (yield: 30.3%)

[0066] The .sup.1H-NMR spectrum of the multi-signal fluorescent probe was shown in FIG. 1.

[0067] The multi-signal fluorescent probe synthesized herein was capable of distinguishing and detecting ONOO.sup.- and Na.sub.2S.sub.2 produced in organisms. Unless otherwise specified, the experimental procedures were similar to those of other probes.

[0068] Spectral properties of the multi-signal fluorescent probe were investigated as follows.

[0069] The multi-signal fluorescent probe was dissolved with dimethyl sulfoxide (DMSO) to obtain a 1 mM probe solution. A 1 mM ONOO.sup.- aqueous solution and a 1 mM Na.sub.2S.sub.2 aqueous solution were prepared.

[0070] 20 .Math.L of the 1 mM probe solution, 980 .Math.L of analytically pure CH.sub.3CN, the required amount of the 1 mM ONOO.sup.- or Na.sub.2S.sub.2 aqueous solution and the required amount of phosphate buffered saline (PBS) were added into a 2 mL sample tube. A volume ratio of an organic phase to an aqueous phase was kept at 5:5 for all tests (a total volume of each test sample was 2 mL).

[0071] For example, when it was required to explore the fluorescence intensity of the multi-signal fluorescent probe after reacted with 20 mM ONOO.sup.-, the experiment was formulated as follows.

[0072] 20 .Math.L of the 1 mM probe solution, 980 .Math.L of analytically pure CH.sub.3CN, 20 .Math.L of the 1 mM ONOO.sup.- aqueous solution and 980 .Math.L of PBS were added into a 2 mL sample tube. The reaction mixture was mixed evenly under shaking at room temperature for 15 min. Then, the fluorescence emission intensity of the reaction mixture could be measured under the 350 nm excitation wavelength. Other steps were performed as above. The limit of detection (LOD) for ONOO.sup.- was 42.12 nM, and the LOD for Na.sub.2S.sub.2 was 38.45 nM. Fluorescence spectra of the multi-signal fluorescent probe in the detection of peroxynitrite and sodium disulfide were shown in FIG. 2. Accordingly, the highly-sensitive discrimination and detection for ONOO.sup.- and Na.sub.2S.sub.2 were realized by using the multi-signal fluorescent probe provided herein.

[0073] The multi-signal fluorescent probe provided herein is efficient and simple for the detection of oxidative and reductive substances, and can be used to discriminate and detect ONOO.sup.- and Na.sub.2S.sub.2 simultaneously. The multi-signal fluorescent probe can undergo different reactions respectively with ONOO.sup.- and Na.sub.2S.sub.2 under the same condition to generate different fluorescent matters which will emit green fluorescence and yellow fluorescence under specific wavelength. Therefore, ONOO.sup.- and Na.sub.2S.sub.2 can be simultaneously distinguished and detected, and the simultaneous dual-channel fluorescence imaging of endogenous ONOO.sup.- and Na.sub.2S.sub.2 in cells can be realized, which will facilitate the development of multi-signal bio-fluorescent probes.

[0074] The multi-signal fluorescent probe provided herein utilizes a dual-channel fluorescent signal to discriminate and detect ONOO.sup.- and Na.sub.2S.sub.2 simultaneously, developing the fields of analysis and detection and medical early diagnosis.

[0075] Regarding a method for detecting ONOO.sup.- and Na.sub.2S.sub.2 in cells, unless otherwise specified, a probe molecule is dissolved at room temperature for analytical detection, where a volume ratio of an organic phase to an aqueous phase is 5:5. The organic phase is acetonitrile (CH.sub.3CN), and the aqueous phase is formed by PBS (pH=7.4) and an aqueous solution of the analyte.

[0076] The multi-signal fluorescent probe for reactive oxygen was dissolved in a solution, in which a volume ratio of DMSO to the aqueous phase was 5:5. The multi-signal fluorescent probe emitted 492 nm green light under 380 nm excitation wavelength after reacted with ONOO.sup.-, and emitted 548 nm yellow light under 410 nm excitation wavelength after reacted with Na.sub.2S.sub.2. Accordingly, a specific analyte can be detected by the specific excitation and fluorescence emission signal. The ONOO.sup.- and Na.sub.2S.sub.2 can be distinguished by using different excitation wavelengths and fluorescence emission signals. The multi-signal fluorescent probe can detect the ONOO.sup.- and Na.sub.2S.sub.2 simultaneously, and has no significant response to amino acids, sulfur-containing derivatives and amine derivatives, such as •OH, t-BuO, .sup.1O.sub.2, NO, O.sub.2.sup..-, GSH, Cys, Hcy, SO.sub.2, NAC, H.sub.2S, F-, Cl-, Br-, I.sup.-, NAC, Gly, Ala, His, Met, Thr, Lys, Asp, Glu, Pro, Ser, NaHS, NaHSO.sub.3, EtSH, PhSH, n-Butylamine, and aniline. The LOD for ONOO.sup.- is 42.12 nM, and the LOD for Na.sub.2S.sub.2 is 38.45 nM. In summary, the multi-signal fluorescent probe can realize the high-sensitivity discrimination and detection of ONOO.sup.- and Na.sub.2S.sub.2.

[0077] It should be noted that described above are merely illustrative of the disclosure, and are not intended to limit the disclosure. Although the disclosure has been illustrated and described in detail above, it should be understood that those skilled in the art could still make modifications and replacements to the embodiments of the disclosure. Those modifications and replacements made by those skilled in the art based on the content disclosed herein without departing from the scope of the disclosure shall fall within the scope of the present disclosure defined by the appended claims.

[0078] In addition, the features of various embodiments may be combined in the absence of contradiction. The contents in the background are merely for better understanding of the general background of the application, and should not be considered as admitting or in any way implying that the content belongs to the prior art known to those skilled in the art.