CN109678888B

CN109678888B - Oxazine compound and application thereof

Info

Publication number: CN109678888B
Application number: CN201811635409.5A
Authority: CN
Inventors: 樊江莉; 姚起超; 杜健军; 孙文; 龙飒然; 邵堃; 王静云; 彭孝军
Original assignee: Dalian Kerong Biotechnology Co ltd; Dalian University of Technology
Current assignee: Dalian Kerong Biotechnology Co ltd; Dalian University of Technology
Priority date: 2018-12-29
Filing date: 2018-12-29
Publication date: 2021-06-18
Anticipated expiration: 2038-12-29
Also published as: CN109678888A

Abstract

An oxazine compound and application thereof, wherein the oxazine compound has a structure shown in a general formula F. The oxazine compound with the general formula F has specific response to mitochondria, can rapidly enter cells, is rapidly combined with the mitochondria and emits fluorescence with strong signals. The method shows better specificity recognition marks for mitochondria in both fixed cell experiments and living cell experiments. Furthermore, the compound of the invention is a kind of light/sound dynamic active organic molecule which has near infrared absorption emission and has photosensitive and sound sensitive characteristics. The compound has maximum absorption and emission wavelengths of more than 690nm and high triplet state conversion rate, can generate active oxygen species at high efficiency under the condition of illumination or ultrasound, has good killing effect on cancer cells and cancer tissues, and hardly increases toxic and side effects on normal tissues while achieving photodynamic therapy on tumors.

Description

Oxazine compound and application thereof

Technical Field

The invention relates to a novel oxazine compound, synthesis and new application thereof in biospecific recognition and detection. And belongs to the field of anti-cancer medicine design, synthesis and application. In particular to synthesis of a novel oxazine light/sound sensitive agent, specific identification of tumors and new application in diagnosis and treatment.

Background

Cells (cells) are protoplasm which are surrounded by membranes and can independently reproduce, are the most basic structural and functional units of human body, an adult is formed by about 100 trillion cells, and various physiological and pathological processes of the human body are closely related to cell reproduction and metabolism. Since the 20 th century, scientists have focused more on organelle research as cellular and molecular biology research continues. The organelles are morphological structural units with independent physiological functions and certain chemical compositions in cytoplasm, and take animal cells as an example, the organelles mainly comprise cell nucleus, mitochondria, lysosome, endoplasmic reticulum, Golgi body and the like

Mitochondria (Mitochondrion), which was first discovered in animal cells by Altmann, a german scientist 1894, is a cellular organelle of large volume that can be seen under a light microscope, is permeable, swells in hypotonic solution, and shrinks in hypertonic solution. Since the form is a thread or a granule, it is named as a mitochondrion. Studies have shown that many serious diseases are associated with abnormal mitochondrial behaviour. Most mitochondrial localization dyes are currently based on the property of mitochondrial negative membrane potential, and many other functions and mechanisms of mitochondria are not known. Thus, studies on mitochondria, such as mitochondrial DNA (mtDNA) imaging, reactive oxygen species response, ion (Ca)²⁺，Cu⁺Etc.), the role of mitochondria in apoptosis, etc., remain a focus of attention of scientists. If the morphology and physiological process of mitochondria become abnormal, it will bring about extreme healthTherefore, the real-time monitoring of mitochondria is realized, and the method has practical significance for preventing and treating various major diseases.

Fluorescent probes play a great role in the fields of biology, medicine, and the like, and are becoming popular for people to study. Numerous research results have been achieved over two hundred years since the time fluorescent dye molecules were discovered. The developed mature fluorescent dyes comprise cyanine dyes, BODIPY, coumarin, rhodamine, fluorescein, Nile blue and the like, and people connect a series of functional groups on the parent ring of the fluorescent dyes for modification to synthesize a plurality of fluorescent probe molecules with a detection function, so that the fluorescent probe molecules are used for realizing visual monitoring of target molecules. Fluorescent dye molecules have been used in a variety of fields such as luminescent materials and sensors, environmental pollution detection, biological research, and medical diagnosis. It is worth mentioning that the fluorescent probe plays an important role in medical health. The cell-specific marker can enter cells, identify and position various subcellular structures in the cells, and visually distinguish normal cells and diseased cells, so that the root cause of diseases is determined, and the targeted treatment of diseases is facilitated.

To date, efforts have been made in this direction, and some mitochondrial fluorescent probes and their outstanding advantages for imaging in cells have been developed. However, the currently reported probes generally have the defects of great staining concentration, long incubation time, great probe cytotoxicity and the like which are yet to be improved.

The photodynamic action refers to the action of photosensitizers, under the action of light, the function or the morphological change of organism cells or biomolecules is caused, and the cell damage and the necrosis are caused when the cell damage and the cell necrosis are serious. Photodynamic therapy is a new disease treatment means based on the interaction of light, photosensitizer and oxygen, and the research of photosensitizer (photodynamic therapy medicine) is the key point influencing the prospect of photodynamic therapy. Photosensitizers are special chemical substances whose essential function is to transfer energy, which can absorb a photon to be excited, and which rapidly transfer the absorbed light energy to a molecule of another component, causing it to be excited and the photosensitizer itself to return to the ground state. With the approval of the first photosensitizer Porfimer Sodium to be marketed in 1993-1997 in the united states, canada, the european union, japan and korea, research, development and application in the field of PDT have been rapidly activated. With the successful development of new photodynamic therapy drugs and the improvement of laser equipment technology, PDT has yet to be developed. Internationally, there are nearly ten new photosensitizers approved for marketing or in clinical research. Meanwhile, PDT is also used for treating non-tumor diseases, such as condyloma acuminatum, psoriasis, nevus flammeus, rheumatoid arthritis, fundus macular degeneration, restenosis after angioplasty, and the like. The representative domestic photosensitizers mainly comprise: ila (5-ALA, topical aminolevulinic acid hydrochloride powder) developed and produced by shanghai fudan zhangjiang biomedicine gmbh.

At present, the light/sound sensitizers applied to clinical use are mainly represented by porphyrin and phthalocyanine compounds, and although the compounds have achieved great success in tumor treatment, many defects still exist, such as: the composition proportion of a treatment system is unstable, the metabolism in a body is slow, the maximum excitation wavelength is short, and the toxic and side effects are easy to occur. These deficiencies seriously affect the practical efficacy and clinical use of photodynamic therapy. For the preparation and application research of the photosensitizer, a certain guiding thought can play a guiding role internationally, but for the preparation and application research of the sound-sensitive agent, no relevant theoretical guidance exists, and few sound-sensitive agents can be applied to clinic. Therefore, the light/sound sensitive agent is designed to have a great propulsion effect on tumor diagnosis and treatment.

Disclosure of Invention

The invention aims to provide a fluorescent probe molecule which has light/sound sensitive characteristics and can specifically identify mitochondria. For this purpose, the invention firstly provides an oxazine compound with a structure of a general formula F:

in the general formula F, the compound is represented by the general formula,

a is selected from oxygen (O), sulfur (S), selenium (Se) and tellurium (Te);

said R₁、R₂And R₃Each independently selected from hydrogen, a group of formula i-x, C_1-12Alkyl group, and C_1-12Substituted alkyl of (a);

the substituted alkyl is optionally substituted by the following groups: halogen, -OH, -COOH, -NO₂、-SO₃H (sulfonic group), -OCH₃、-OC₂H₅、-OC₃H₇、-OC₄H₉、-COOCH₃、-COOC₂H₅、-COOC₃H₇、-COOC₄H₉、-COOC₅H₁₁、-COOC₆H₁₃、-NR₄R₅or-CONR₆R₇(ii) a Wherein R is₄、R₅、R₆、R₇Each independently selected from hydrogen and C_1-6An alkyl group;

and X is selected from dihydrogen phosphate, hydrogen sulfate, nitrate radical, chlorine anion, bromine anion, iodine anion or perchlorate radical.

The oxazine compound with the general formula F has specific response to mitochondria, can rapidly enter cells, is rapidly combined with the mitochondria and emits fluorescence with strong signals. The method shows better specificity recognition marks for mitochondria in both fixed cell experiments and living cell experiments. Further through a series of performance tests, the probe molecule is found to have a maximum absorption wavelength (about 690nm) and a maximum emission wavelength (about 710nm) of near infrared in an aqueous system, the light energy of the excitation and emission wavelengths of long wavelength is lower, the damage to tissue cells is smaller, the light transmission is good, and the interference of autofluorescence of the tissue cells to the tissue cells is smaller. And the fluorescence quantum yields in the various organic solvents correspond to their corresponding fluorescence intensities. The compounds have certain water solubility, good cell membrane permeability and low biotoxicity, phototoxicity and photobleaching property. The spectral range of which is sufficiently different from the spectral range of the biological sample. Moreover, the compounds have better light stability, can stably exist under the condition of physiological pH value, and are beneficial to the application of the compounds in organisms to play the function of fluorescent probes.

Therefore, the invention also aims to provide the application of the oxazine compound in preparing a near-infrared fluorescent probe, wherein the near-infrared fluorescent probe is a mitochondrion targeted fluorescent probe. The prepared near-infrared fluorescent probe can be used for fluorescence imaging of mitochondria in cells or tissues, can monitor the morphology of mitochondria in real time, and can be applied to a super-resolution mode.

On the other hand, the oxazine compound is a photo/acoustic dynamic active organic molecule which has near infrared absorption and emission and photosensitive and acoustic sensitivity. The compound has maximum absorption and emission wavelengths of more than 690nm and high triplet state conversion rate, can generate active oxygen species at high efficiency under the condition of illumination or ultrasound, has good killing effect on cancer cells and cancer tissues, and hardly increases toxic and side effects on normal tissues while achieving photodynamic therapy on tumors. Therefore, the invention also provides the application of the oxazine compound in the preparation of the light/sound sensitive agent. The light/sound sensitive agent is a near-infrared long-wavelength fluorescent probe and is used for marking tumor cells. The oxazine compound can be used for micromolecule administration operation, can also be self-assembled into nanoparticles or coated into nano ions by other materials for administration operation, and has the nanoscale effective range of 1-1000 nm.

Drawings

The invention is illustrated in figure 13:

FIG. 1 is a structural general formula F of the oxazine compound.

FIG. 2 is a graph showing the results of an MCF-7 labeling assay for probe compound F-1 in breast cancer cells, in which: FIG. 2(a) shows an F-1 channel; FIG. 2(b) is a cell brightfield map; FIG. 2(c) shows the mixing channels (a) and (b).

FIG. 3 is a graph showing the results of experiments on mitochondrial co-localization of probe compound F-1 in breast cancer cells MCF-7, in which: FIG. 3(a) is a MitoTracker GreenFM channel, a commercial dye; FIG. 3(b) shows the F-1 channel; FIG. 3(c) shows the mixing channels (a) and (b); FIG. 3(d) is the intracellular signal profile and co-localization coefficient.

FIG. 4 is a graph showing the results of an experiment for lysosomal co-localization of probe compound F-1 in breast cancer cells MCF-7, in which: FIG. 4(a) is a commercial dye LysoTracker GreenFM channel; FIG. 4(b) shows the F-1 channel; FIG. 4(c) shows the mixing channels (a) and (b); FIG. 4(d) is the intracellular signal profile and co-localization coefficient.

FIG. 5 is a graph showing the results of an experiment for labeling a probe in a living Kunming mouse.

FIG. 6 is a graph showing the results of an MCF-7 labeling experiment for a probe compound F-2 in breast cancer cells. Wherein FIG. 6(a) the F-2 channel; FIG. 6(b) is a cell brightfield map; fig. 6(c) shows the mixing channels (a) and (b).

FIG. 7 is a graph showing the results of mitochondrial co-localization experiments in breast cancer cells MCF-7 using probe compound F-2. Wherein, fig. 7(a) is a commercial dye MitoTracker GreenFM channel; FIG. 7(b) shows an F-2 channel; FIG. 7(c) shows the mixing channels (a) and (b); FIG. 7(d) is the intracellular signal profile and co-localization coefficient.

FIG. 8 is a graph showing the results of an experiment of lysosomal co-localization of probe compound F-2 in breast cancer cells MCF-7. Wherein: FIG. 8(a) is a commercial lysosomal dye LysoTracker GreenFM channel; FIG. 8(b) shows an F-2 channel; FIG. 8(c) shows the mixing channels (a) and (b); FIG. 8(d) is the intracellular signal profile and co-localization coefficient.

FIG. 9 is a graph showing the results of experiments on the mitochondrial morphology of the probe compound F-2 in breast cancer cells MCF-7. Wherein FIG. 9(a) is normal filamentous mitochondria; FIG. 9(b) is a graph showing that most of the mitochondrial morphology remained filamentous with slight mitochondrial damage; FIG. 9(c) shows that when the damage is increased and the mitochondria begin to swell, the morphology gradually changes to a round point shape; FIG. 9(d) is a diagram showing that when the damage is very serious, the mitochondrial morphology is vacuolated; FIG. 9(A) is a partially enlarged view of (a); FIG. 9B is a partial enlarged view of (B); FIG. 9(C) is a partial enlarged view of (C); fig. 9(D) is a partially enlarged view of (D).

FIG. 10 is a graph showing the results of a labeling experiment of probe compound F-2 in a live Kunming mouse.

FIG. 11 is a graph showing the results of experiments on the mitochondrial morphology of probe compounds in MCF-7 breast cancer cells.

FIG. 12 is a graph showing the results of an experiment for measuring the amount of singlet oxygen produced by the probe compound F-3 under light irradiation.

FIG. 13 is a graph showing the results of in vitro cellular anticancer assays performed under light conditions with probe compound F-3.

Detailed Description

Unless otherwise indicated, the terms used herein have the following meanings.

The term "alkyl" as used herein includes straight chain and branched chain alkyl groups. Where not described as "substituted alkyl," the term "alkyl" generally refers to unsubstituted alkyl. Reference to a single alkyl group, such as "propyl", is intended to refer only to straight chain alkyl groups, and reference to a single branched alkyl group, such as "isopropyl", is intended to refer only to branched alkyl groups. For example, "C_1-6Alkyl "includes C_1-4Alkyl radical, C_1-3Alkyl, methyl, ethyl, n-propyl, isopropyl and tert-butyl. Similar rules apply to other groups used in this specification.

The term "halogen" as used herein includes fluorine, chlorine, bromine and iodine.

The invention firstly relates to an oxazine compound with a structure of a general formula F, as shown in figure 1:

in the general formula F, A is selected from oxygen (O), sulfur (S), selenium (Se) and tellurium (Te).

Said R₁、R₂And R₃The selectable groups are wide in range, and the selection within the range does not have substantial influence on the charge property and the hydrophilic-lipophilic coefficient of the molecule. Said R₁、R₂And R₃Each independently selected from hydrogen, a group of formula i-x, an alkyl or substituted alkyl group of 1 to 12 carbon atoms; the number of carbon atoms of the alkyl group or the substituted alkyl group mentioned therein is preferably 1 to 8, more preferably 1 to 6.

The substituted alkyl is optionally substituted by the following groups: halogen, -OH, -COOH, -NO₂、-SO₃H (sulfonic group), -OCH₃、-OC₂H₅、-OC₃H₇、-OC₄H₉、-COOCH₃、-COOC₂H₅、-COOC₃H₇、-COOC₄H₉、-COOC₅H₁₁、-COOC₆H₁₃、-NR₄R₅or-CONR₆R₇(ii) a Wherein R is₄、R₅、R₆、R₇Each independently selected from hydrogen and C_1-6An alkyl group. Preferably, said R is₄、R₅、R₆、R₇Are all hydrogen.

In a specific embodiment, R₁And R₂Wherein one of the groups is H; further preferably, R is₃Also referred to as H.

In some embodiments, R₁And R₂One of which is H and the other is a group selected from the formula i-x. The compounds have tumor recognition function due to the group of the formula i-x. Therefore, the compound can be used for positioning tumor cells and can be used as a light/sound sensitive agent.

In the general formula F, X is selected from dihydrogen phosphate, hydrogen sulfate, nitrate radical, chlorine anion, bromine anion, iodine anion or perchlorate radical.

Based on the screening and comparison of the mitochondria specific recognition capability of each compound, the most preferred embodiment provided by the invention is that the oxazine compound is selected from F-1, F-2, F-3, F-4, F-5, F-6, F-7, F-8, F-9, F-10, F-11 or F-12:

the oxazine compound has specific response to mitochondria, can quickly enter cells, is quickly combined with mitochondria in the cell nucleus and emits fluorescence with strong signals. The method shows better specificity identification marks for mitochondria in both fixed cell experiments and living cell experiments. Further through a series of performance tests, the probe molecule is found to have the maximum absorption wavelength (about 691nm) and the maximum emission wavelength (about 711nm) of near infrared in a water system, the light energy of the excitation wavelength and the emission wavelength of long wavelength is lower, the damage to tissue cells is smaller, the light transmission is good, and the interference of the autofluorescence of the tissue cells to the tissue cells is smaller. And the fluorescence quantum yields in the various organic solvents correspond to their corresponding fluorescence intensities. The compounds have certain water solubility, good cell membrane permeability and low biotoxicity, phototoxicity and photobleaching property. The spectral range of which is sufficiently different from the spectral range of the biological sample. Moreover, the compounds have better light stability, can stably exist under the condition of physiological pH value, and are beneficial to the application of the compounds in organisms to play the function of fluorescent probes. The excellent light stability enables the compound to have better super-resolution microscope working capacity.

Based on the above, the specific technical scheme of the application of the oxazine compound is that the oxazine compound is used for preparing near-infrared fluorescent probe products of mitochondria-targeted fluorescent probes and is used for fluorescence imaging of mitochondria in cells or tissues. The near-infrared fluorescent probe is used for specifically marking and detecting mitochondria in aqueous solution and buffer solution.

The compounds of formula F, as mentioned above, and the preparation thereof are disclosed for the first time, and those skilled in the art should be able to combine the technical information of the related art with the basic theory and techniques of organic synthesis to achieve the compounds of the present invention. The following methods for preparing oxazine compounds described in the present specification provide a specific scheme for the synthesis of such compounds, but should not be construed as limiting thereof.

The oxooxazine compound is synthesized by the following method: the target oxazine dye is prepared by condensing an azo compound formed by anthracene or derivatives thereof and 8-hydroxy julolidine in acid-containing DMF. The synthesis method has simple process and high conversion rate. More specifically, the synthetic route of the compound F of the general formula of the present invention can be represented as follows:

the preparation method of the compound of the general formula F represented by the above route comprises the following steps:

(1) in a hydrochloric acid acidification system, p-nitrodiazobenzene chloride reacts with a compound shown in a formula I according to a molar ratio of 1:1 at 25-35 ℃ for 0.5-2 hours to prepare a compound shown in a formula II;

(2) and (3) reacting the compound shown in the formula II with 8-hydroxy-n-tirlidine in acidic DMF at the temperature of 135-145 ℃ for 2-4 hours according to the molar ratio of 1:1 to prepare the compound shown in the general formula F.

The sulfur selenium tellurium substituted oxazine compound is synthesized by the following method: the target oxazine dye is prepared by condensing an azo compound formed by arylamine or derivatives thereof and 8-hydroxy julolidine in acid-containing DMF. The synthesis method has simple process and high conversion rate. More specifically, the synthetic route of the compound F with the general formula is as follows:

The near-infrared fluorescent probe taking oxazine as a matrix has the following advantages:

the compound has a certain level of water solubility and simultaneously has good cell membrane permeability.

The compound has specific and specific recognition on mitochondria;

the compound has excellent fluorescence property, has low biological photobleaching, photodamage and biological toxicity when being applied to biological sample imaging, and the generated fluorescence signal can penetrate deeper biological tissues;

the fluorescence emission wavelength of partial molecules of the compound is more than 700nm, and the compound can be used for in vivo imaging of animals;

the compound is used for marking tumors, tumor cells and tissues, can realize good mitochondrial marking, and can avoid the interference of external environmental factors on fluorescence intensity;

the compounds can be applied to super-resolution microscopy imaging;

the compound has the advantages of small toxicity and side effects, easily obtained raw materials, simple structure, easy preparation and easy industrialization;

therefore, the near-infrared fluorescent probe compound can be used for marking tumor and non-tumor cells and tissues. In addition to being used directly for the staining of tumor and non-tumor cells and tissues in the forms described herein, compositions containing the near-infrared fluorescent probe compounds of the present invention can also be used for the staining of tumor cells and tissues. The composition should include an effective amount of one of the two-photon fluorescent probe compounds provided by the present invention. In addition, other components required for staining the biological sample, such as a solvent, a pH adjuster, and the like, may be included. These components are all known in the art. The compositions described above may be presented as aqueous solutions or may be presented in other suitable forms for constitution with water as a solution prior to use.

The present invention also provides a method for labeling tumor cells and tissue biological samples using the near-infrared fluorescent probe compound of the present invention described above, which comprises the step of contacting the compound with the biological sample. The term "contacting" as used herein may include contacting in solution or in a solid phase.

The following non-limiting examples are presented to enable those of ordinary skill in the art to more fully understand the present invention and are not intended to limit the invention in any way.

Example 1: preparation of Probe Compound F-1

(1) Synthesis of intermediates 1-II

In a hydrochloric acid acidification system, p-nitrodiazobenzene chloride and a compound of 1-I react for 0.5-2 hours at 25-35 ℃ according to a molar ratio of 1:1, after the reaction is finished, a brick red solid powder crude product is obtained after suction filtration and washing operations, and the compound of formula 1-II is obtained with a yield of 95%.

(2) Synthesis of Compound F-1

The intermediate 1-II prepared in the above reaction (1) and Se-substituted julolidine were added to a round-bottomed flask containing DMF, and 1mL of perchloric acid solution was added dropwise. After the dropwise addition, the reaction was stopped after the system was stirred for 2.5 hours, and the target probe compound F-1 was purified by silica gel column chromatography (dichloromethane: methanol: 8: 1) to give a metallic blue-green needle crystal with a yield of 78.2%.

¹H NMR(400MHz,DMSO-d₆)δ9.69(s,1H),9.18(s,1H),9.09(s,1H),8.29(d,J＝7.9Hz,1H),8.11(d,J＝8.1Hz,1H),7.84–7.71(m,2H),7.39(s,1H),6.73(s,1H),3.57–3.44(m,3H),2.85(s,2H),2.80–2.72(m,2H),1.95(s,3H).HRMS(ESI,HRDFMagSec)m/z 456.0980(caled for C26H22N3Se+:456.0978)

Example 2: labeling experiment of Probe Compound F-1 in Breast cancer cells (MCF-7)

Using compound F-1 synthesized in example 1,

the F-1-DMSO solution was added to MCF-7 cells containing 2mL of medium, shaken, and imaged with a confocal laser microscope. Representative areas were selected and observed with an oil-glass (60 ×), which was repeated three times. The results are shown in FIG. 2, where: FIG. 2(a) the F-1 channel; FIG. 2(b) is a cell brightfield map; FIG. 2(c) shows the mixing channels (a) and (b). It can be seen that the F-1 molecule is capable of staining cells.

Example 3: mitochondrial Co-localization assay for Probe Compound F-1 in Breast cancer cells (MCF-7)

Using the compound F-1 synthesized in example 1,

the F-1-DMSO solution was added to MCF-7 cells containing medium (cells were pre-incubated at 37 ℃ C. and 5% CO)₂The commercial dye MitoTracker Green will be added^FMIncubate in medium for 20 minutes. Then, PBS was rinsed for 5min × 3 with shaking, and then cell culture medium was added) and shaken, and imaged with a laser confocal microscope. Representative areas were selected and observed with an oil-glass (60 ×), which was repeated three times. The results are shown in FIG. 3, FIG. 3(a) is a commercial dye MitoTracker Green^FMA channel; FIG. 3(b) shows the F-1 channel; FIG. 3(c) shows the mixing channels (a) and (b); FIG. 3(d) is the intracellular signal profile and co-localization coefficient. It can be seen that the F-1 molecule is distributed mainly within the cell mitochondria.

Example 4: test for lysosomal Co-localization of Probe Compound F-1 in Breast cancer cells (MCF-7)

Using the compound F-1 synthesized in example 1, a F-1-DMSO solution was added to MCF-7 cells containing a medium (cells were previously cultured at 37 ℃ C. and 5% CO)₂Next, lysosome dye LysoTracker Green, a commercial dye, was added^FMIncubate in medium for 20 minutes. Then, PBS was rinsed for 5min × 3 with shaking, and then cell culture medium was added) and shaken, and imaged with a laser confocal microscope. Representative areas were selected and observed with an oil-glass (60 ×), which was repeated three times. The results are shown in FIG. 4, in which FIG. 4(a) shows the commercial dye LysoTracker Green^FMA channel; FIG. 4(b) shows the F-1 channel; FIG. 4(c) shows the mixing channels (a) and (b); FIG. 4(d) is the intracellular signal profile and co-localization coefficient. It can be seen that the F-1 molecule is not distributed in the cell lysosome.

Example 5: labeling experiment of Probe Compound F-1 in Living Kunming mouse

The method comprises the steps of injecting 10% chloral hydrate (10mg/Kg) into a live Kunming mouse for anesthesia, inhaling a proper amount of isoflurane to deepen anesthesia and slightly inhibit respiration (motion and respiration artifacts are reduced to the minimum), and injecting an F-1-DMSO solution into the abdomen of the live Kunming mouse after diluting the solution by 1000 times with pure PBS. The living Kunming mouse is placed in a small animal imager and is placed on a fixed plate in a supine position for imaging. As a result, as shown in FIG. 5, it can be seen that the F-1 molecule can be used for color imaging of a living biological sample.

Example 6: preparation of Probe Compound F-2

(1) Synthesis of intermediates 2-II

In a hydrochloric acid acidification system, p-nitrodiazobenzene chloride and a compound of 2-I react for 0.5-2 hours at 25-35 ℃ according to a molar ratio of 1:1, after the reaction is finished, a purplish red solid powder crude product is obtained after suction filtration and washing operation to obtain the compound of formula 2-II, wherein the yield is 95%.

(2) Synthesis of Compound F-2

The intermediate 2-II prepared in the reaction (1) and 8-hydroxy julolidine are added into a round-bottom flask containing DMF, and 1mL perchloric acid solution is dripped. After the dropwise addition, the reaction was stopped after the system was stirred for 2.5 hours, and the reaction mixture was purified by silica gel column chromatography (dichloromethane: methanol: 8: 1) to obtain a target probe compound F-2 as a green needle crystal having metallic luster, with a yield of 65.4%.

¹H NMR(400MHz,DMSO-d₆)δ9.81(s,1H),9.14(s,1H),9.07(s,1H),8.25(d,J＝7.8Hz,1H),8.10(d,J＝7.5Hz,1H),7.80–7.72(m,2H),7.39(s,1H),6.88(s,1H),3.74(s,1H),3.55–3.46(m,3H),2.87(s,2H),2.77(s,2H),1.98(m,5H),1.43(t,J＝7.1Hz,3H).HRMS(ESI,HRDFMagSec)m/z 420.2063(caled for C26H22N3O+:420.2070)

Example 7: labeling experiment of Probe Compound F-2 in Breast cancer cells (MCF-7)

Using the compound F-2 synthesized in example 6, the F-2-DMSO solution was added to MCF-7 cells containing 2mL of the medium, shaken, and imaged with a confocal laser microscope. Representative areas were selected and observed with an oil-glass (60 ×), which was repeated three times. The results are shown in FIG. 6, where: FIG. 6(a) channel F-2; FIG. 6(b) is a cell brightfield map; fig. 6(c) shows the mixing channels (a) and (b). It can be seen that the F-2 molecule is capable of staining cells.

Example 8: mitochondrial Co-localization assay for Probe Compound F-2 in Breast cancer cells (MCF-7)

Using the compound F-2 synthesized in example 6, a F-2-DMSO solution was added to MCF-7 cells containing a medium (cells were previously cultured at 37 ℃ C. and 5% CO)₂The commercial dye MitoTracker Green will be added^FMIncubate in medium for 20 minutes. Then, PBS was rinsed for 5min × 3 with shaking, and then cell culture medium was added) and shaken, and imaged with a laser confocal microscope. Representative areas were selected and observed with an oil-glass (60 ×), which was repeated three times. The results are shown in FIG. 7, FIG. 7(a) is a commercial dye MitoTracker Green^FMA channel; FIG. 7(b) shows an F-2 channel; FIG. 7(c) shows the mixing channels (a) and (b); FIG. 7(d) is the intracellular signal profile and co-localization coefficient. It can be seen that the F-2 molecule is distributed mainly within the cell mitochondria.

Example 9: test for lysosomal Co-localization of Probe Compound F-2 in Breast cancer cells (MCF-7)

Using the compound F-2 synthesized in example 6, a F-2-DMSO solution was added to MCF-7 cells containing a medium (cells were previously cultured at 37 ℃ C. and 5% CO)₂Next, a commercially available dye, LysoTracker Green, will be added^FMIncubate in medium for 20 minutes. Then, PBS was rinsed for 5min × 3 with shaking, and then cell culture medium was added) and shaken, and imaged with a laser confocal microscope. Representative areas were selected and observed with an oil-glass (60 ×), which was repeated three times. The results are shown in FIG. 8, FIG. 8(a) is a commercial lysosomal dye LysoTracker Green^FMA channel; FIG. 8(b) shows an F-2 channel; FIG. 8(c) shows the mixing channels (a) and (b); FIG. 8(d) is the intracellular signal profile and co-localization coefficient. It can be seen that the F-2 molecule is not distributed in the cell lysosome.

Example 10: mitochondrial morphology display experiment of Probe Compound F-2 in Breast cancer cells (MCF-7)

Using the compound F-2 synthesized in example 6, the F-2-DMSO solution was added to MCF-7 cells containing medium and irradiated with laser for various lengths of time to cause different degrees of mitochondrial damage in the cells. Representative areas were selected and observed with an oil-glass (60 ×), which was repeated three times. The results are shown in FIG. 9, in which FIG. 9(a) shows normal filamentous mitochondria; FIG. 9(b) is a graph showing that most of the mitochondrial morphology remained filamentous with slight mitochondrial damage; FIG. 9(c) shows that when the damage is increased and the mitochondria begin to swell, the morphology gradually changes to a round point shape; FIG. 9(d) is a diagram showing that when the damage is very serious, the mitochondrial morphology is vacuolated; FIG. 9(A) is a partially enlarged view of (a); FIG. 9B is a partial enlarged view of (B); FIG. 9(C) is a partial enlarged view of (C); fig. 9(D) is a partially enlarged view of (D). It can be seen that the F-2 molecule can monitor the mitochondrial morphology in real time within the cell.

Example 11: labeling experiment of Probe Compound F-2 in Living Kunming mouse

The method comprises the steps of injecting 10% chloral hydrate (10mg/Kg) into a live Kunming mouse for anesthesia, inhaling a proper amount of isoflurane to deepen anesthesia and slightly inhibit respiration (motion and respiration artifacts are reduced to the minimum), and injecting an F-2-DMSO solution into the abdomen of the live Kunming mouse after diluting the solution by 1000 times with pure PBS. The living Kunming mouse is placed in a small animal imager and is placed on a fixed plate in a supine position for imaging. As a result, as shown in FIG. 10, it can be seen that the F-2 molecule can be used for color imaging of a living biological sample.

Example 12: experiment for super-resolution observation of intracellular mitochondria by using probe compound F-2

Using the compound F-2 synthesized in example 6, the elongated mitochondrial morphology was clearly observed by adding a F-2-DMSO solution to MCF-7 cells containing a medium and observing the mixture with a super-resolution microscope. As a result, as shown in FIG. 11, it was found that the F-2 molecule has a function of super-resolving the morphology of mitochondria in cells. It can be seen that the F-2 molecule can monitor the mitochondrial morphology in real time within the cell.

Example 13: preparation of Probe Compound F-3

(1) Synthesis of intermediate 3-II

In a hydrochloric acid acidification system, p-nitrodiazobenzene chloride and a compound of 2-I react for 0.5-2 hours at 25-35 ℃ according to a molar ratio of 1:1, after the reaction is finished, a purplish red solid powder crude product is obtained after suction filtration and washing operation to obtain the compound of formula 3-II, wherein the yield is 94%.

(2) Synthesis of Compound F-3

The intermediate 3-II prepared in the above reaction (1) and thiojulolidine were added to a round-bottomed flask containing DMF, and 1mL of perchloric acid solution was added dropwise. After the dropwise addition, the reaction was stopped after the system was stirred for 2.5 hours, and the target probe compound F-3 was purified by silica gel column chromatography (dichloromethane: methanol: 8: 1) to obtain a green plate layered crystal having a metallic luster, with a yield of 55.4%.

¹H NMR(400MHz,DMSO-d₆)δ8.83(s,1H),8.05–7.98(m,3H),7.62-7.58(m,2H),6.75(s,1H),6.22(s,1H),3.64(s,1H),3.55–3.46(m,8H),2.87(s,2H),2.77(s,2H),1.96(m,5H),.HRMS(ESI,HRDFMagSec)m/z 436.1841(caled for C28H26N3S+:436.1842)

Example 14: singlet oxygen yield determination experiment of compound F-3 under illumination condition

Using the compound F-3 synthesized in example 13, an F-3-DMSO solution was added to methanol and mixed uniformly, 1, 3-diphenyl isobenzofuran (DPBF) was added, the DPBF concentration was adjusted to have an absorbance value of about 1.0, and then a xenon light source (adjusted by grating filter) having a wavelength of 700nm was used for irradiation, and the uv-visible absorption curve of the system was measured at equal time intervals. According to the change of the absorbance of the DPBF at the wavelength of 411nm, a correlation curve of the absorbance and the time is drawn, methylene blue is used as a reference, and the singlet oxygen quantum yield of the compound F-3 is calculated, and the result is shown in figure 12. The figure shows the change of the ultraviolet visible absorption spectrum of the mixed system along with the illumination time, and the singlet oxygen quantum yield of the compound F-3 is about 0.03 according to a related formula.

Example 15: in vitro cellular anti-cancer assay under Compound F-3 Lighting conditions

MCF-7 (human breast cancer cells) was seeded at 5000 cell densities per well in 96-well culture plates and cultured in a cell incubator for 24 hours (37 ℃, 5% CO)₂) Using the compound F-3 synthesized in example 13, the F-3-DMSO solution was added to DMEM containing 10% fetal bovine serum to prepare solutions of different concentrations, and the prepared solutions were added to a 96-well plate, incubated in a cell incubator for 30 minutes, and then irradiated with red light of 700nm wavelength for a certain period of time. After irradiation, the 96-well plate was placed in a cell incubator for further incubation for 12 hours. Then, 100. mu.l of a medium containing 5mg/ml MTT was added to each well, and the mixture was placed in a cell incubator and incubated for 4 hours. The culture medium in the wells of the plate was removed, 100. mu.l DMSO was added to each well to sufficiently dissolve the MTT oxidation product, and then the absorbance at 570nm and 630nm was measured in each well using a microplate reader, and the cell viability was calculated as shown in FIG. 13.

As can be seen from FIG. 13, the killing effect of compound F-3 on cells in the absence of light was very small with little toxicity; under the condition of illumination, the compound F-3 can generate obvious killing effect on cells, and the phototoxicity of the compound F-3 is obviously improved along with the enhancement of illumination energy density.

Claims

1. An oxazine compound having the structure of formula F:

in the general formula F, the compound is represented by the general formula,

a is selected from oxygen, sulfur, selenium and tellurium;

said R₁、R₂And R₃Each independently selected from hydrogen and C_1-12Alkyl and C_1-12And R is substituted alkyl, and₁and R₂Not H at the same time;

the substituted alkyl is optionally substituted by the following groups: halogen, -OH, -OCH₃、-OC₂H₅、-OC₃H₇、-OC₄H₉Or NH₂；

2. The oxazine compound of claim 1, wherein R₃Is hydrogen.

3. The oxazine compound of claim 1, wherein R₁And R₂Each independently selected from hydrogen and C_1-6Alkyl and C_1-6Substituted alkyl groups of (1).

4. An oxazine compound according to claim 1, characterised in that it is selected from F-1, F-2, F-3, F-4, F-5 or F-6:

5. the use of oxazine compounds of claim 1 in the preparation of near-infrared fluorescent probes, said near-infrared fluorescent probes being mitochondrial-targeted fluorescent probes.

6. Use of an oxazine compound of claim 1 in the preparation of a photosensitizer.