一种有机染料化合物及其制备方法和应用

Organic dye compound and preparation method and application thereof

文档序号：2516 发布日期：2021-09-17 浏览：67次中文

1. A compound of formula I:

or a biologically acceptable salt, analog thereof; wherein the content of the first and second substances,

R₁、R₂each independently is H, methyl, ethyl, butyl, phenyl, 3-propanoic acid or 2-ethanesulfonic acid; or R₁Is carboxylic acid butyryl, 4-succinimidyl ester butyryl or 2- (N, N, N-trimethyl ammonium bromide) ethyl, R₂Is H; or R₁And R₂Bound as a propylidene or diazo group;

R₃is H, or with R₁、R₂Combined as a propylene group;

R₄is H or methoxy;

R₅、R₆each independently is H; or R₅、R₆Are combined into 3, 4-ethylenedioxy or 3, 4-ethylenediimine; or R₅Is 2- (2- (2-methoxyethoxy) ethoxy, R₆Is H;

R₇is H or F;

R₈any one of structures selected from formula A to formula I:

wherein R is₉Is methyl, 2-acetoxy, 2-acetic acid succinimide ester group, 2-ethanesulfonic acid group or N, N, N-trimethyl ammonium bromide methyl;

R₁₀is H or a sulfonic acid group.

2. The compound of formula I according to claim 1, wherein the compound of formula I is selected from any one of the following compounds or a biologically acceptable salt, solvate thereof:

3, 3-dimethyl-1- (2-trimethylammoniobromide ethyl) -2- (trans-2- (7- (trans-2- (5- (trans-2- (2-methoxy-4-dimethylaminophenyl) vinyl) thiophen-2-yl) vinyl) -2,1, 3-benzothiadiazol-4-yl) vinyl) -5, 7-disulfonic acid-3H-benzo [ e ] indole,

3, 3-dimethyl-1- (3-propanesulfonic acid) -2- (trans-2- (7- (trans-2- (5- (trans-2- (2-methoxy-4-dimethylaminophenyl) vinyl) thiophen-2-yl) vinyl) -2,1, 3-benzothiadiazol-4-yl) vinyl) -5, 7-disulfonic acid-3H-benzo [ e ] indole,

3, 3-dimethyl-1- (3-propanoyl) -2- (trans-2- (7- (trans-2- (5- (trans-2- (2-methoxy-4-dimethylaminophenyl) vinyl) thiophen-2-yl) vinyl) -2,1, 3-benzothiadiazol-4-yl) vinyl) -5, 7-disulfonyl-3H-benzo [ e ] indole,

3, 3-dimethyl-1- (3-propanesulfonic acid) -2- (trans-2- (7- (trans-2- (5- (trans-2- (4-dibutylaminophenyl) vinyl) thiophen-2-yl) vinyl) -2,1, 3-benzothiadiazol-4-yl) vinyl) -5, 7-disulfonic acid-3H-benzo [ e ] indole,

3, 3-dimethyl-1- (3-propanoyl) -2- (trans-2- (7- (trans-2- (5- (trans-2- (4-dibutylaminophenyl) vinyl) thiophen-2-yl) vinyl) -2,1, 3-benzothiadiazol-4-yl) vinyl) -5, 7-disulfonyl-3H-benzo [ e ] indole,

3, 3-dimethyl-1- (3-propanoyl) -2- (trans-2- (7- (trans-2- (5- (trans-2- (4-bis (2-ethanesulfonic acid) aminophenyl) vinyl) thiophen-2-yl) vinyl) -2,1, 3-benzothiadiazol-4-yl) vinyl) -5, 7-disulfonic acid-3H-benzo [ e ] indole,

3, 3-dimethyl-1-ethyl-2- (trans-2- (7- (trans-2- (5- (trans-2- (2-methoxy-4-dimethylaminophenyl) vinyl) thiophen-2-yl) vinyl) -2,1, 3-benzothiadiazol-4-yl) vinyl) -3H-benzo [ e ] indole,

3, 3-dimethyl-1- (2-trimethylammoniobromide ethyl) -2- (trans-2- (7- (trans-2- (4- (2- (2- (2-methoxyethoxy) ethoxy) -5- (trans-2- (2-methoxy-4-dimethylaminophenyl) vinyl) thiophen-2-yl) vinyl) -2,1, 3-benzothiadiazol-4-yl) vinyl) -5, 7-disulfonic acid-3H-benzo [ e ] indole,

3, 3-dimethyl-1- (2-trimethylammoniobromin ethyl) -2- (trans-2- (7- (trans-2- (5- (trans-2- (2-methoxy-4-dimethylaminophenyl) vinyl) -3, 4-ethylenedioxythiophen-2-yl) vinyl) -2,1, 3-benzothiadiazol-4-yl) vinyl) -5, 7-disulfonic acid-3H-benzo [ e ] indole,

3, 3-dimethyl-1- (3-propanesulfonic acid) -2- (trans-2- (7- (trans-2- (5- (trans-2- (2-methoxy-4-dimethylaminophenyl) vinyl) -3, 4-ethylenedioxythiophen-2-yl) vinyl) -2,1, 3-benzothiadiazol-4-yl) vinyl) -5, 7-disulfonic acid-3H-benzo [ e ] indole,

3, 3-dimethyl-1- (3-propanoyl) -2- (trans-2- (7- (trans-2- (5- (trans-2- (2-methoxy-4-dimethylaminophenyl) vinyl) -3, 4-ethylenedioxythiophen-2-yl) vinyl) -2,1, 3-benzothiadiazol-4-yl) vinyl) -5, 7-disulfonyl-3H-benzo [ e ] indole,

3, 3-dimethyl-1- (3-propanesulfonic acid group) -2- (trans-2- (7- (trans-2- (5- (trans-2- (4-diphenylaminophenyl) vinyl) -3, 4-ethylenedioxythiophen-2-yl) vinyl) -2,1, 3-benzothiadiazol-4-yl) vinyl) -5, 7-disulfonic acid-3H-benzo [ e ] indole,

3, 3-dimethyl-1- (2-trimethylammoniobromin ethyl) -2- (trans-2- (7- (trans-2- (5- (trans-2- (4-aminophenyl) vinyl) -3, 4-ethylenedioxythiophen-2-yl) vinyl) -2,1, 3-benzothiadiazol-4-yl) vinyl) -5, 7-disulfonic acid-3H-benzo [ e ] indole,

3, 3-dimethyl-1- (3-propanoyl) -2- (trans-2- (7- (trans-2- (5- (trans-2- (4-azidophenyl) vinyl) -3, 4-ethylenedioxythiophen-2-yl) vinyl) -2,1, 3-benzothiadiazol-4-yl) vinyl) -5, 7-disulfonyl-3H-benzo [ e ] indole,

3, 3-dimethyl-1- (2-trimethylammoniobromide ethyl) -2- (trans-2- (7- (trans-2- (5- (trans-2- (4-cyclobutylaminophenyl) vinyl) -3, 4-ethylenedioxythiophen-2-yl) vinyl) -2,1, 3-benzothiadiazol-4-yl) vinyl) -5, 7-disulfonyl-3H-benzo [ e ] indole,

3, 3-dimethyl-1- (2-trimethylammoniobromide ethyl) -2- (trans-2- (7- (trans-2- (5- (trans-2- (4-bis (3-propionyloxy) aminophenyl) vinyl) -3, 4-ethylenedioxythiophen-2-yl) vinyl) -2,1, 3-benzothiadiazol-4-yl) vinyl) -5, 7-disulfonic acid-3H-benzo [ e ] indole,

3, 3-dimethyl-1- (3-propanoyl) -2- (trans-2- (7- (trans-2- (5- (trans-2- (4- (2-trimethylammoniumbromide) ethylaminophenyl) ethenyl) -3, 4-ethylenedioxythiophen-2-yl) ethenyl) -2,1, 3-benzothiadiazol-4-yl) ethenyl) -5, 7-disulfonic acid-3H-benzo [ e ] indole,

3, 3-dimethyl-1- (3-propanoic acid succinimide ester group) -2- (trans-2- (7- (trans-2- (5- (trans-2- (4- (2-trimethylammoniumbromide) ethylaminophenyl) ethenyl) -3, 4-ethylenedioxythiophen-2-yl) ethenyl) -2,1, 3-benzothiadiazol-4-yl) ethenyl) -5, 7-disulfonic acid-3H-benzo [ e ] indole,

3, 3-dimethyl-1- (2-trimethylammoniobromide ethyl) -2- (trans-2- (7- (trans-2- (5- (trans-2- (4- (4-carboxylic acid butyrylamino) phenyl) vinyl) -3, 4-ethylenedioxythiophen-2-yl) vinyl) -2,1, 3-benzothiadiazol-4-yl) vinyl) -5, 7-disulfonic acid-3H-benzo [ e ] indole,

3, 3-dimethyl-1- (2-trimethylammoniobromin ethyl) -2- (trans-2- (7- (trans-2- (5- (trans-2- (4- (4-succinimidyl butyrylamino) phenyl) ethenyl) -3, 4-ethylenedioxythiophen-2-yl) ethenyl) -2,1, 3-benzothiadiazol-4-yl) ethenyl) -5, 7-disulfonic acid-3H-benzo [ e ] indole,

3- (2-Trimethylaminobromoethyl) -2- (trans-2- (7- (trans-2- (5- (trans-2- (4- (2-trimethylammoniobromoethyl) aminophenyl) vinyl) -3, 4-ethylenedioxythiophen-2-yl) vinyl) -2,1, 3-benzothiadiazol-4-yl) vinyl) -benzo [ d ] thiazole,

3-ethyl-2- (trans-2- (7- (trans-2- (5- (trans-2- (4- (2-trimethylammonioethyl) aminophenyl) amino) phenyl) vinyl) -3, 4-ethylenedioxythiophen-2-yl) vinyl) -2,1, 3-benzothiadiazol-4-yl) vinyl) -benzo [ d ] thiazole,

1- (3-propanesulfonic acid) -2- (trans-2- (7- (trans-2- (5- (trans-2- (2-methoxy-4-dimethylaminophenyl) vinyl) -3, 4-ethylenedioxythiophen-2-yl) vinyl) -2,1, 3-benzothiadiazol-4-yl) vinyl) -3H-benzo [ c, d ] indole,

2, 6-diphenyl-4- (trans-2- (7- (trans-2- (5- (trans-2- (2-methoxy-4-dimethylaminophenyl) vinyl) -3, 4-ethylenedioxythiophen-2-yl) vinyl) -2,1, 3-benzothiadiazol-4-yl) vinyl) thiopyrylium,

2-phenyl-7-dimethylamino-4- (trans-2- (7- (trans-2- (5- (trans-2- (2-methoxy-4-dimethylaminophenyl) vinyl) -3, 4-ethylenedioxythiophen-2-yl) vinyl) -2,1, 3-benzothiadiazol-4-yl) vinyl) benzo [ b ] pyrylium,

2- (4-methoxyphenyl) -6-diethylamino-4- (trans-2- (7- (trans-2- (5- (trans-2- (2-methoxy-4-dimethylaminophenyl) vinyl) -3, 4-ethylenedioxythiophen-2-yl) vinyl) -2,1, 3-benzothiadiazol-4-yl) vinyl) benzo [ b ] thiopyrylium,

9-diethylamino-2- (trans-2- (7- (trans-2- (5- (trans-2- (2-methoxy-4-dimethylaminophenyl) vinyl) -3, 4-ethylenedioxythiophen-2-yl) vinyl) -2,1, 3-benzothiadiazol-4-yl) methylene) dibenzo [ b, e ] pyrylium,

3, 3-dimethyl-1- (2-trimethylammoniobromide ethyl) -2- (trans-2- (7- (trans-2- (5- (trans-2- (9-julolidinyl) vinyl) thiophen-2-yl) vinyl) -2,1, 3-benzothiadiazol-4-yl) vinyl) -5, 7-disulfonyl-3H-benzo [ e ] indole,

3, 3-dimethyl-1- (2-trimethylammoniobromin ethyl) -2- (trans-2- (7- (trans-2- (2-methoxy-4-dimethylaminophenyl) vinyl) -thieno [3,4-b ] pyrazin-2-yl) vinyl) -2,1, 3-benzothiadiazol-4-yl) vinyl) -5, 7-disulfonic acid-3H-benzo [ e ] indole,

3, 3-dimethyl-1- (2-trimethylammoniobromin ethyl) -2- (trans-2- (5, 6-difluoro-7- (trans-2- (5- (trans-2- (2-methoxy-4-dimethylaminophenyl) vinyl) thiophen-2-yl) vinyl) -2,1, 3-benzothiadiazol-4-yl) vinyl) -5, 7-disulfonic acid-3H-benzo [ e ] indole,

1-ethyl-4- (trans-2- (7- (trans-2- (5- (trans-2- (2-methoxy-4-dimethylaminophenyl) vinyl) thiophen-2-yl) vinyl) -2,1, 3-benzothiadiazol-4-yl) vinyl) quinoline,

10-ethyl-9- (trans-2- (7- (trans-2- (5- (trans-2- (2-methoxy-4-dimethylaminophenyl) vinyl) thiophen-2-yl) vinyl) -2,1, 3-benzothiadiazol-4-yl) vinyl) acridine,

1- (3-propanesulfonic acid) -4- (trans-2- (7- (trans-2- (5- (trans-2- (2-methoxy-4-dimethylaminophenyl) vinyl) thiophen-2-yl) vinyl) -2,1, 3-benzothiadiazol-4-yl) vinyl) quinoline,

10- (3-propanesulfonic acid) -9- (trans-2- (7- (trans-2- (5- (trans-2- (2-methoxy-4-dimethylaminophenyl) vinyl) thiophen-2-yl) vinyl) -2,1, 3-benzothiadiazol-4-yl) vinyl) acridine.

3. A process for the preparation of a compound of formula I according to claim 1 or 2, characterized in that: the method comprises the following steps:

s1: a compound of formula II:

with a compound of formula III:

the intermediate product IV is obtained by reacting under the existence of tri (o-methylphenyl) phosphine, palladium acetate and triethylamine:

s2: reacting a compound of formula IV with methyl triphenyl phosphonium iodide in the presence of an organic base in an organic solvent to provide intermediate V:

s3: a compound of formula V with a compound of formula VI:

the reaction is carried out in the presence of tri (o-methylphenyl) phosphine, palladium acetate and triethylamine to obtain an intermediate product VII:

s4: contacting a compound of formula VII with any one of compounds of formulae a to i:

carrying out a reaction in an organic solvent to obtain a compound shown in the formula I or an intermediate product IX thereof;

wherein R is₁、R₂Each independently is H, methyl, ethyl, butyl, phenyl; or R₁Is tert-butyloxycarbonyl, benzyloxycarbonyl or 9-fluorenylmethoxycarbonyl, R₂Is H; r₃～R₇、R₉～R₁₀Is as defined in claim 1.

4. A process for the preparation of a compound of formula I according to claim 3, characterized in that: the organic base is selected from any one of potassium tert-butoxide and butyl lithium.

5. A process for the preparation of a compound of formula I according to claim 3, characterized in that: the organic solvent is any one of tetrahydrofuran, acetic anhydride and N, N-dimethylformamide.

6. A process for the preparation of a compound of formula I according to claim 3, characterized in that: the preparation method of the compound of the formula I comprises the following steps: and deprotecting the intermediate product IX under an acidic condition, and reacting the intermediate product IX with any one of bromopropionic acid, chloroethanesulfonic acid, 2-bromoethyl trimethyl ammonium bromide, succinic anhydride or sodium azide in a reaction solvent to obtain a compound shown in the formula I or an intermediate product X thereof.

7. The process for the preparation of compounds of formula I according to claim 6, characterized in that: the preparation method of the compound of the formula I comprises the following steps: and reacting the intermediate product X with N-hydroxysuccinimide in an organic solvent in the presence of dicyclohexylcarbodiimide or 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride to obtain the compound shown in the formula I.

8. An organic dye characterized by: comprising a compound of formula I according to claim 1 or 2 or a compound of formula I obtainable by a process for the preparation of a compound of formula I according to any one of claims 3 to 7.

9. A fluorescent probe, characterized in that: comprising a compound of formula I according to claim 1 or 2 or a compound of formula I obtainable by a process for the preparation of a compound of formula I according to any one of claims 3 to 7 or an organic dye according to claim 8.

10. Use of a compound of formula I according to claim 1 or 2 or a compound of formula I prepared by a process for the preparation of a compound of formula I according to any one of claims 3 to 7 or an organic dye according to claim 8 or a fluorescent probe according to claim 9 for the detection of cell membrane potential.

Background

Understanding the function of individual neurons and their connections during high-speed information processing in the brain is a final goal of brain science research. This requires rapid recording of neuronal action potentials at the cellular as well as sub-cellular scale with millisecond time resolution. Because the neuron activity can be recorded in real time in a high-flux manner, fluorescence imaging is distinguished in a plurality of in-vivo imaging modes and becomes an important technical means for neuroscience research. In recent years, a large number of fluorescent probes responding to nerve signals (such as membrane potential, calcium ions, glutamate, etc.) have been reported in the literature. However, the fluorescence emitted by these dyes is mainly concentrated in the visible region (400-.

The near infrared fluorescence imaging utilizes the advantages of long near infrared wavelength, low scattering and large penetration depth in biological tissues, thereby realizing the imaging of the physiological process deep in the organism under the non-invasive condition. Compared with multi-photon imaging, the wide-field imaging is used, the imaging speed is high, and therefore real-time recording of some rapid physiological processes in the living body can be achieved. If it is intended to record changes in neuronal membrane potential using near-infrared fluorescence imaging, it is necessary to use near-infrared fluorescent probes whose emission wavelengths are in the near-infrared region, and such probes have not been reported.

Disclosure of Invention

The present invention is directed to solving at least one of the problems of the prior art described above. To this end, the invention proposes, in a first aspect, a compound of formula I, whose emission wavelength is in the near infrared band, which can be inserted into the cell membrane.

In a second aspect, the present invention provides a process for the preparation of a compound of formula I as defined above.

In a third aspect, the present invention provides an organic dye comprising a compound of formula I as described above or a compound of formula I as prepared by a process for the preparation of a compound of formula I as described above.

In a fourth aspect, the present invention provides a fluorescent probe comprising the compound of formula I or the compound of formula I prepared by the preparation method of the compound of formula I or the organic dye.

In a fifth aspect, the present invention provides a compound of formula I, or a compound of formula I prepared by the preparation method of the compound of formula I, or an organic dye, or a fluorescent probe.

According to a first aspect of the present invention, there is provided a compound of formula I:

or a biologically acceptable salt, analog thereof; wherein the content of the first and second substances,

R₃is H, or with R₁、R₂Combined as a propylene group;

R₄is H or methoxy;

R₅、R₆each independently is H; or R₅、R₆Are combined into 3, 4-ethylenedioxy or 3, 4-ethylenediimine; or R₅Is 2- (2- (2-methoxyethoxy) ethoxy, R₆Is H;

R₇is H or F;

R₈any one of structures selected from formula A to formula I:

wherein R is₉Is methyl, 2-acetoxy, 2-acetic acid succinimide ester group, 2-ethanesulfonic acid group or N, N, N-trimethyl ammonium bromide methyl;

R₁₀is H or a sulfonic acid group.

In the present invention, the compound of formula I, wherein the phrase "biologically acceptable salt" is used herein, refers to a salt which is, within the scope of sound biological judgment, suitable for use in contact with living cells, laboratory animals without undue toxicity, irritation, and the like, commensurate with a reasonable benefit/risk ratio. The salts may be prepared in situ during the final isolation and purification of the compounds of the invention or separately by reacting the free basic functional groups of the compounds of the invention with a suitable inorganic or organic acid. Representative acid addition salts include, but are not limited to: hydrochloride, hydrobromide, hydroiodide, perchlorate and tetrafluoroborate. Such salts may be prepared in situ during the final isolation and purification of the compounds of the present invention by reacting the carboxylic acid, sulfonic acid containing moieties of the compounds of the present invention with a suitable base, including, but not limited to, cations based on alkali or alkaline earth metals such as lithium, sodium, potassium, calcium, magnesium, and aluminum salts, and the like.

The present invention relates to compounds of formula I, wherein the word "analogue" includes isomers, racemates, enantiomers, diastereomers, enantiomeric enrichments, solvates and esters thereof; the compounds of formula I of the present invention and their isomers, racemates, enantiomers, diastereomers, enantiomeric enrichments, solvates and esters may also form solvates, e.g. hydrates, alcoholates and the like; the selection and preparation of appropriate analogs is well known to those skilled in the art. Generally, for the purposes of the present invention, the solvate forms are equivalent to the non-solvate forms with biologically acceptable solvents such as water, ethanol, and the like. And such changes do not depart from the spirit and scope of the corresponding technical solutions of the embodiments of the present invention.

All compound name terms used in the present invention have general meanings well known to those skilled in the art, and refer to the organic compound naming principle published by the committee for naming of organic compounds of china, 2017 edition.

In some embodiments of the invention, the compound of formula I is selected from any one of the following compounds, or a biologically acceptable salt, solvate thereof:

3, 3-dimethyl-1-ethyl-2- (trans-2- (7- (trans-2- (5- (trans-2- (2-methoxy-4-dimethylaminophenyl) vinyl) thiophen-2-yl) vinyl) -2,1, 3-benzothiadiazol-4-yl) vinyl) -3H-benzo [ e ] indole,

2, 6-diphenyl-4- (trans-2- (7- (trans-2- (5- (trans-2- (2-methoxy-4-dimethylaminophenyl) vinyl) -3, 4-ethylenedioxythiophen-2-yl) vinyl) -2,1, 3-benzothiadiazol-4-yl) vinyl) thiopyrylium,

1-ethyl-4- (trans-2- (7- (trans-2- (5- (trans-2- (2-methoxy-4-dimethylaminophenyl) vinyl) thiophen-2-yl) vinyl) -2,1, 3-benzothiadiazol-4-yl) vinyl) quinoline,

10-ethyl-9- (trans-2- (7- (trans-2- (5- (trans-2- (2-methoxy-4-dimethylaminophenyl) vinyl) thiophen-2-yl) vinyl) -2,1, 3-benzothiadiazol-4-yl) vinyl) acridine,

1- (3-propanesulfonic acid) -4- (trans-2- (7- (trans-2- (5- (trans-2- (2-methoxy-4-dimethylaminophenyl) vinyl) thiophen-2-yl) vinyl) -2,1, 3-benzothiadiazol-4-yl) vinyl) quinoline,

10- (3-propanesulfonic acid) -9- (trans-2- (7- (trans-2- (5- (trans-2- (2-methoxy-4-dimethylaminophenyl) vinyl) thiophen-2-yl) vinyl) -2,1, 3-benzothiadiazol-4-yl) vinyl) acridine.

According to a second aspect of the present invention, there is provided a process for the preparation of a compound of formula I, comprising the steps of:

s1: a compound of formula II:

with a compound of formula III:

in tris (o-methylphenyl) phosphine, palladium acetate (Pd (OAc)₂) And Triethylamine (TEA) to obtain an intermediate product IV:

s2: reacting a compound of formula IV with methyl triphenyl phosphonium iodide (CH)₃PPh₃I) The reaction is carried out in an organic solvent in the presence of an organic base to obtain an intermediate product V:

s3: a compound of formula V with a compound of formula VI:

the reaction is carried out in the presence of tris (o-methylphenyl) phosphine, Pd (OAc)2 and TEA to give intermediate VII:

s4: contacting a compound of formula VII with any one of compounds of formulae a to i:

carrying out a reaction in an organic solvent to obtain a compound shown in the formula I or an intermediate product IX thereof;

wherein R is₁、R₂Each independently is H, methyl, ethyl, butyl, phenyl; or R₁Is tert-butyloxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or 9-fluorenylmethoxycarbonyl (Fmoc), R₂Is H; r₃～R₇、R₉～R₁₀As defined above.

In some embodiments of the invention, the organic base is selected from any one of potassium tert-butoxide (tBuOK), butyl lithium (nBuLi).

In some preferred embodiments of the present invention, the organic solvent may be any one of Tetrahydrofuran (THF), acetic anhydride, N-Dimethylformamide (DMF).

In some more preferred embodiments of the present invention, the process for preparing the compound of formula I comprises the steps of: deprotection of the intermediate IX under acidic conditions and reaction with bromopropionic acid, chloroacetic acid, 2-bromoethyltrimethylammonium bromide, succinic anhydride or sodium azide (NaN)₃) And reacting under appropriate conditions to obtain the compound of the formula I or an intermediate product X thereof.

In some more preferred embodiments of the invention, the reaction solvent comprises a base, which can be Triethylamine (TEA), Diisopropylethylamine (DIPEA), 4-Dimethylaminopyridine (DMAP), and an organic solvent for bromopropionic acid, chloroacetic acid, 2-bromoethyltrimethylammonium bromide, or succinic anhydride; the reaction solvent comprises hydrochloric acid, sodium nitrite and water solvent for sodium azide.

In some more preferred embodiments of the present invention, the process for preparing the compound of formula I comprises the steps of: and reacting the intermediate product X with N-hydroxysuccinimide (NHS) in an organic solvent in the presence of Dicyclohexylcarbodiimide (DCC) or 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) to obtain the compound shown in the formula I.

In some more preferred embodiments of the invention, some of the compounds of formulae III and VI are commercially available, and others of formulae III and VI, and compounds of formulae II and VIII are prepared by those skilled in the art in accordance with known techniques. In an exemplary process, the compounds of formula II, VIII can be prepared by reference to the literature, for example, see: michael j.patrick, et al.org.biomol.chem.,2007,5, 3347-.

The intermediates, raw materials, reagents, reaction conditions, etc. used in the above reaction schemes may be appropriately changed according to the instructions of those skilled in the art, or some or all of the technical features may be substituted with polar equivalents, and such changes or substitutions do not depart from the scope of the technical schemes of the embodiments of the present invention.

According to a third aspect of the present invention, there is provided an organic dye comprising a compound of formula I as described above or a compound of formula I prepared by a process for the preparation of a compound of formula I as described above. The intensity of the fluorescence emitted by the organic dye changes with the change of the cell membrane potential.

According to a fourth aspect of the present invention, there is provided a fluorescent probe comprising a compound of formula I as described above or a compound of formula I prepared by a process for the preparation of a compound of formula I as described above or an organic dye as described above.

According to a fifth aspect of the present invention, there is provided a use of the compound of formula I, or the compound of formula I prepared by the process for preparing the compound of formula I, or the organic dye, or the fluorescent probe in cell membrane potential detection.

In some embodiments of the present invention, the application includes using the compound of formula I or the organic dye prepared by the method for preparing the compound of formula I, by solid powder, aqueous solution, organic phase solution, or surfactant encapsulation, adding the compound of formula I or the organic dye into a cell culture system at any concentration, soaking an isolated tissue, an organ, or directly injecting the cell, the isolated tissue, the organ, or the living animal into the living animal, and imaging the cell, the isolated tissue, the organ, or the living animal in the near infrared band under the irradiation of excitation light.

In some preferred embodiments of the invention, the applications include deep optical imaging for ex vivo cultured cells, ex vivo tissues, organs, small live animals, including but not limited to ex vivo cultured cells, ex vivo brain tissue derived from experimental animals, cardiac muscle tissue, ex vivo heart, brain, heart, brain of in vivo experimental animals. The method specifically comprises the following steps: the compound of the formula I or the compound of the formula I prepared by the preparation method of the compound of the formula I or the organic dye is added into a cell culture system at a certain concentration through solid powder, aqueous phase solution, organic phase solution or surfactant entrapment, and is soaked in isolated tissues and organs or is directly injected into a living experimental animal body, and the cells, the isolated tissues and organs or the living experimental animal body are imaged in a near infrared band (>800nm) under the irradiation of exciting light.

The invention has the beneficial effects that:

1. the fluorescent probe formed by the compound of the formula I emits fluorescence with the wavelength in the near infrared band, can be inserted into a cell membrane and emits fluorescence intensity which is responsive to transmembrane potential of the cell membrane.

2. The fluorescent probe containing the compound of the formula I can detect the cell membrane potential in a near infrared band, overcomes the defect of poor penetration depth of the traditional fluorescent imaging method in biological tissues, and can further realize real-time recording of nerve signals of in-vivo experimental animals under a non-invasive condition. Compared with a multi-photon imaging technology, the method has the advantages of high imaging speed by more than 10 times, low requirement on equipment and capability of simultaneously recording nerve signals of a plurality of neurons.

Drawings

The invention is further described with reference to the following figures and examples, in which:

FIG. 1 shows an absorption spectrum and a fluorescence emission spectrum of Compound 1 of example 1 of the present invention.

FIG. 2 is a graph showing the response of a molecular probe formed from Compound 1 of example 1 of the present invention to the transmembrane potential of a cell membrane.

FIG. 3 shows the result of recording the membrane potential of neurons by PBS solution of infrared probe formed by compound 1 of example 1 of the present invention.

Detailed Description

The concept and technical effects of the present invention will be clearly and completely described below in conjunction with the embodiments to fully understand the objects, features and effects of the present invention. It is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments, and those skilled in the art can obtain other embodiments without inventive effort based on the embodiments of the present invention, and all embodiments are within the protection scope of the present invention.

Table 1 gives 34 specific compounds of formula I: a compound of formula I:

R₈any one of structures selected from formula A to formula I:

TABLE 1

Correspondingly, specific compounds of the compounds of formula I are:

3, 3-dimethyl-1-ethyl-2- (trans-2- (7- (trans-2- (5- (trans-2- (2-methoxy-4-dimethylaminophenyl) vinyl) thiophen-2-yl) vinyl) -2,1, 3-benzothiadiazol-4-yl) vinyl) -3H-benzo [ e ] indole (Compound 7)

3, 3-dimethyl-1- (2-trimethylammoniobromide ethyl) -2- (trans-2- (7- (trans-2- (5- (trans-2- (2-methoxy-4-dimethylaminophenyl) vinyl) -3, 4-ethylenedioxythiophen-2-yl) vinyl) -2,1, 3-benzothiadiazol-4-yl) vinyl) -5, 7-disulfonic acid-3H-benzo [ e ] indole (Compound 9)

3, 3-dimethyl-1- (3-propanesulfonic acid) -2- (trans-2- (7- (trans-2- (5- (trans-2- (4-diphenylaminophenyl) vinyl) -3, 4-ethylenedioxythiophen-2-yl) vinyl) -2,1, 3-benzothiadiazol-4-yl) vinyl) -5, 7-disulfonic acid-3H-benzo [ e ] indole (Compound 12)

3, 3-dimethyl-1- (2-trimethylammoniobrominate ethyl) -2- (trans-2- (7- (trans-2- (5- (trans-2- (4-aminophenyl) vinyl) -3, 4-ethylenedioxythiophen-2-yl) vinyl) -2,1, 3-benzothiadiazol-4-yl) vinyl) -5, 7-disulfonic acid-3H-benzo [ e ] indole (Compound 13)

3, 3-dimethyl-1- (3-propanoic acid succinimidyl ester) -2- (trans-2- (7- (trans-2- (5- (trans-2- (4- (2-trimethylammoniumbromide) ethylaminophenyl) ethenyl) -3, 4-ethylenedioxythiophen-2-yl) ethenyl) -2,1, 3-benzothiadiazol-4-yl) ethenyl) -5, 7-disulfonic acid-3H-benzo [ e ] indole (Compound 18)

3, 3-dimethyl-1- (2-trimethylammoniobromide ethyl) -2- (trans-2- (7- (trans-2- (5- (trans-2- (4- (4-succinimidyl butyrylamino) phenyl) ethenyl) -3, 4-ethylenedioxythiophen-2-yl) ethenyl) -2,1, 3-benzothiadiazol-4-yl) ethenyl) -5, 7-disulfonic acid-3H-benzo [ e ] indole (Compound 20)

3-Ethyl-2- (trans-2- (7- (trans-2- (5- (trans-2- (4- (2-trimethylammonioethyl) aminophenyl) vinyl) -3, 4-ethylenedioxythiophen-2-yl) vinyl) -2,1, 3-benzothiadiazol-4-yl) vinyl) -benzo [ d ] thiazole (Compound 22)

1- (3-Propanesulfonic acid) -2- (trans-2- (7- (trans-2- (5- (trans-2- (2-methoxy-4-dimethylaminophenyl) vinyl) -3, 4-ethylenedioxythiophen-2-yl) vinyl) -2,1, 3-benzothiadiazol-4-yl) vinyl) -3H-benzo [ c, d ] indole (Compound 23)

2, 6-Diphenyl-4- (trans-2- (7- (trans-2- (5- (trans-2- (2-methoxy-4-dimethylaminophenyl) vinyl) -3, 4-ethylenedioxythiophen-2-yl) vinyl) -2,1, 3-benzothiadiazol-4-yl) vinyl) thiopyrylium (Compound 24)

3, 3-dimethyl-1- (2-trimethylammoniobromide ethyl) -2- (trans-2- (7- (trans-2- (2-methoxy-4-dimethylaminophenyl) vinyl) -thieno [3,4-b ] pyrazin-2-yl) vinyl) -2,1, 3-benzothiadiazol-4-yl) vinyl) -5, 7-disulfonic acid-3H-benzo [ e ] indole (Compound 29)

3, 3-dimethyl-1- (2-trimethylammoniobromide ethyl) -2- (trans-2- (5, 6-difluoro-7- (trans-2- (5- (trans-2- (2-methoxy-4-dimethylaminophenyl) vinyl) thiophen-2-yl) vinyl) -2,1, 3-benzothiadiazol-4-yl) vinyl) -5, 7-disulfonic acid-3H-benzo [ e ] indole (Compound 30)

1-Ethyl-4- (trans-2- (7- (trans-2- (5- (trans-2- (2-methoxy-4-dimethylaminophenyl) vinyl) thiophen-2-yl) vinyl) -2,1, 3-benzothiadiazol-4-yl) vinyl) quinoline (Compound 31)

10-Ethyl-9- (trans-2- (7- (trans-2- (5- (trans-2- (2-methoxy-4-dimethylaminophenyl) vinyl) thiophen-2-yl) vinyl) -2,1, 3-benzothiadiazol-4-yl) vinyl) acridine (compound 32)

1- (3-Propanesulfonic acid) -4- (trans-2- (7- (trans-2- (5- (trans-2- (2-methoxy-4-dimethylaminophenyl) vinyl) thiophen-2-yl) vinyl) -2,1, 3-benzothiadiazol-4-yl) vinyl) quinoline (Compound 33)

10- (3-Propanesulfonic acid) -9- (trans-2- (7- (trans-2- (5- (trans-2- (2-methoxy-4-dimethylaminophenyl) vinyl) thiophen-2-yl) vinyl) -2,1, 3-benzothiadiazol-4-yl) vinyl) acridine (compound 34)

The following shows the preparation methods of compound 1, compound 14, compound 19 and compound 20, and the preparation methods of other compounds are similar to those of compound 1, compound 14, compound 19 and compound 20.

Example 1

Synthesis of 3, 3-dimethyl-1- (2-trimethylammoniobrominethyl) -2- (trans-2- (7- (trans-2- (5- (trans-2- (2-methoxy-4-dimethylaminophenyl) vinyl) thiophen-2-yl) vinyl) -2,1, 3-benzothiadiazol-4-yl) vinyl) -5, 7-disulfonyl-3H-benzo [ e ] indole (compound 1):

s1: synthesis of 5- (trans-2- (2-methoxy-4-dimethylaminophenyl) vinyl) thiophene-2-carbaldehyde:

0.89g of 2-methoxy-4-dimethylaminophenylethylene (5.0mmol), 0.955g of 5-bromo-2-thienylformaldehyde (5.0mmol), 20mg of Pd (OAc) were added to a thick-walled pressure-resistant tube₂40mg of tri (o-tolyl) phosphine and 5mL of dried TEA, freeze-thaw degassed three times, reacted at 114 ℃ for 72h, cooled to room temperature, and added 50mL of CHCl₃And 50mL of water, separating, and using CHCl for the water phase₃Washing twice, combining organic phases, washing with saturated NaCl solution and MgSO₄Drying, suction filtration and evaporation of the solvent gave 1.84g of crude product as an orange-brown oil. Performing column chromatography with CH₂Cl₂: a mixed solvent of 0.4:0.7 hexane was used as an eluent, to obtain 0.86g of a dark orange oily product in a yield of 59.9%.

S2: synthesis of 2-vinyl-5- (trans-2- (2-methoxy-4-dimethylaminophenyl) vinyl) thiophene:

4.3g of 5- (trans-2- (2-methoxy-)4-dimethylaminophenyl) vinyl) thiophene-2-carbaldehyde (15.0mmol), 12.2g of CH₃PPh₃I (30.0mmol) and 3.5g of tBuOK (31.5mmol) are dissolved in 40mL of Tetrahydrofuran (THF), heated to 66 ℃ for 3h, cooled to room temperature and filtered off with suction. The filter cake was separated by column chromatography using n-hexane as eluent to give 3.55g of a yellow solid in 82.9% yield.

S3: synthesis of 7- (trans-2- (5- (trans-2- (2-methoxy-4-dimethylaminophenyl) vinyl) thiophen-2-yl) vinyl) -2,1, 3-benzothiadiazole-4-carbaldehyde:

3.91g of 7-bromo-2, 1, 3-benzothiadiazole-4-carbaldehyde (16.7mmol), 3.82g of 2-vinyl-5- (trans-2- (2-methoxy-4-dimethylaminophenyl) vinyl) thiophene (13.4mmol), 30.9mg of Pd (OAc)₂84.7mg of tri (o-tolyl) phosphine were dissolved in 6.15mL of TEA, placed in a thick-walled pressure-resistant tube, subjected to freeze-thaw and degassing three times, heated at 120 ℃ for 24 hours for reaction, cooled to room temperature, stirred overnight, and added with 50mL of CH₂Cl₂And 50mL of water, separating, and using 40mL of CH for the aqueous phase₂Cl₂Washing twice, combining organic phases and MgSO₄Drying, rotary steaming and concentrating. Column chromatography was carried out using a mixed solvent of ethyl acetate and n-hexane as an eluent to obtain 3.51g of a purple solid product in 58.5% yield.

S4: synthesis of 3, 3-dimethyl-1- (2-trimethylammoniobrominethyl) -2- (trans-2- (7- (trans-2- (5- (trans-2- (2-methoxy-4-dimethylaminophenyl) vinyl) thiophen-2-yl) vinyl) -2,1, 3-benzothiadiazol-4-yl) vinyl) -5, 7-disulfonyl-3H-benzo [ e ] indole (compound 1):

0.45g of 7- (trans-2- (5- (trans-2- (2-methoxy-4-dimethylaminophenyl) vinyl) thiophen-2-yl) vinyl) -2,1, 3-benzothiadiazole-4-carbaldehyde (1.0mmol) and 0.52g of 2,3, 3-trimethyl-1- (2-trimethylammoniumbromide ethyl) -5, 7-disulfonic acid group-3H-benzo [ e]Indole (1.0mmol) was dissolved in 4.0mL acetic anhydride (Ac)₂O), heating to 110 ℃ in an atmosphere of N2 and maintaining for 46min, cooling to room temperature, adding 8.0mL of isopropanol, cooling at-20 ℃ overnight, evaporating off the solvent, dissolving the crude product in CHCl₃Column chromatography, with methanol: CHCl₃Mixed solvent 0.3:0.6 as eluent, dark blue-green solid product 0.42g was obtained, yield 47.5%.

¹H-NMR(600MHz,DMSO-d₆,δppm):9.07(d,1H,J＝1.5Hz),8.49(d,1H,J＝1.5Hz),8.02(d,1H,J＝7.5Hz),7.97(d,1H,J＝7.5Hz),7.75(d,1H,J＝7.5Hz),7.65(d,1H,J＝7.5Hz),7.22(d,1H,J＝15.1Hz),7.16(d,1H,J＝7.5Hz),7.08(d,2H,J＝7.5Hz),6.99(d,1H,J＝15.1Hz),6.95(d,1H,J＝15.1Hz),6.82(d,1H,J＝15.1Hz),6.79(d,1H,J＝15.1Hz),6.45(dd,1H,J＝1.5Hz,J＝7.5Hz),6.32(d,1H,J＝1.5Hz),5.67(d,1H,J＝15.1Hz),4.47(t,2H,J＝7.1Hz),3.90(s,3H),3.30(m,11H),2.90(s,6H),1.57(s,6H).

Example 2

Synthesis of 3, 3-dimethyl-1- (3-propanoyl) -2- (trans-2- (7- (trans-2- (5- (trans-2- (4-azidophenyl) vinyl) -3, 4-ethylenedioxythiophen-2-yl) vinyl) -2,1, 3-benzothiadiazol-4-yl) vinyl) -5, 7-disulfonyl-3H-benzo [ e ] indole (Compound 14):

the steps S1-S4 are similar to those in example 1 above, and only schematic diagrams of the synthesis steps are shown here:

of note, in step S4, is Ac₂And O is used as a solvent for reaction, and a product without the protection of the tert-butyloxycarbonyl group can be directly obtained.

S5: synthesis of 3, 3-dimethyl-1- (3-propanoyl) -2- (trans-2- (7- (trans-2- (5- (trans-2- (4-azidophenyl) vinyl) -3, 4-ethylenedioxythiophen-2-yl) vinyl) -2,1, 3-benzothiadiazol-4-yl) vinyl) -5, 7-disulfonyl-3H-benzo [ e ] indole (Compound 14):

44mg of NaNO₂(0.6mmol) was dissolved in 1.4mL of water, cooled in an ice-water bath, and 488mg of 3, 3-dimethyl-1- (3-propanoyl) -2- (trans-2- (7- (trans-2- (5- (trans-2- (4-aminophenyl) vinyl) -3, 4-ethylenedioxythiophen-2-yl) vinyl) -2,1, 3-benzothiadiazol-4-yl) vinyl) -5, 7-disulfonic acid-3H-benzo [ e]Indole (0.5mmol) was dissolved in 7.8mL of 4M HCl solution and the NaNO mentioned above was added dropwise₂And continuing stirring in the ice-water bath to react for 30 min. Then 42mg of NaN is added₃(0.6mmol) was dissolved in 1.7mL of water, added dropwise to the solution, and the reaction was continued for 30min with stirring in an ice-water bath and allowed to return to room temperature overnight. Extracted with ethyl acetate and then with saturated NaHCO₃Washing with solution, MgSO₄Drying, evaporating solvent to dryness to obtain product 0.25g with 50.7% yield.

¹H-NMR(600MHz,DMSO-d₆,δppm):8.30(d,1H,J＝1.5Hz),7.97(d,1H,J＝7.5Hz),7.77(dd,2H,J＝1.5Hz,J＝7.5Hz),7.71(d,1H,J＝1.5Hz),7.65(d,1H,J＝7.5Hz),7.51(d,2H,J＝7.5Hz),7.19(dd,2H,J＝1.5Hz,J＝7.5Hz),6.99-6.95(m,4H),6.79(d,1H,J＝15.1Hz),5.67(d,1H,J＝15.1Hz),4.30(t,2H,J＝7.1Hz),4.28(t,4H,J＝7.1Hz),2.30(t,2H,J＝7.1Hz),1.57(s,6H)

Example 3

Synthesis of 3, 3-dimethyl-1- (2-trimethylammoniobromoethyl) -2- (trans-2- (7- (trans-2- (5- (trans-2- (4- (4-succinimidyl butyrylamino) phenyl) vinyl) -3, 4-ethylenedioxythiophen-2-yl) vinyl) -2,1, 3-benzothiadiazol-4-yl) vinyl) -5, 7-disulfonic acid-3H-benzo [ e ] indole (Compound 20):

the S1-S4 steps are similar to those in example 1, and only schematic diagrams of the synthesis steps are shown here:

s5: synthesis of 3, 3-dimethyl-1- (2-trimethylammoniobromoethyl) -2- (trans-2- (7- (trans-2- (5- (trans-2- (4- (4-carboxylic acid butyrylamino) phenyl) vinyl) -3, 4-ethylenedioxythiophen-2-yl) vinyl) -2,1, 3-benzothiadiazol-4-yl) vinyl) -5, 7-disulfonic acid-3H-benzo [ e ] indole (Compound 19):

177mg of 3, 3-dimethyl-1- (2-trimethylammoniumbromide ethyl) -2- (trans-2- (7- (trans-2- (5- (trans-2- (4-aminophenyl) vinyl) -3, 4-ethylenedioxythiophen-2-yl) vinyl) -2,1, 3-benzothiadiazol-4-yl) vinyl) -5, 7-disulfonic-3H-benzo [ e ] indole (0.2mmol) were dissolved in 5mL of DMF and 40.2mg of succinic anhydride (0.4mmol), 27mg of DMAP (0.2mmol) and 31. mu.L of TEA (0.2mmol) were added in that order, stirred at room temperature for 12H and the solvent was evaporated. The crude product was dissolved in a small amount of acetone and recrystallized by adding ether to give 0.18g of a brown solid product in 91.5% yield.

¹H-NMR(600MHz,DMSO-d₆,δppm):9.97(bs,1H),8.30(d,1H,J＝1.5Hz),7.97(d,1H,J＝7.5Hz),7.91(dd,2H,J＝1.5Hz,J＝7.5Hz),7.75(dd,2H,J＝1.5Hz,J＝7.5Hz),7.71(d,1H,J＝1.5Hz),7.65(d,1H,J＝7.5Hz),7.51(d,2H,J＝7.5Hz),6.99-6.95(m,4H),6.79(d,1H,J＝15.1Hz),5.67(d,1H,J＝15.1Hz),4.47(t,2H,J＝7.1Hz),4.28(t,4H,J＝7.1Hz),3.30(m,11H),2.70(t,2H,J＝7.1Hz),2.47(t,2H,J＝7.1Hz),1.57(s,6H).

S6: synthesis of 3, 3-dimethyl-1- (2-trimethylammoniobromoethyl) -2- (trans-2- (7- (trans-2- (5- (trans-2- (4- (4-succinimidyl butyrylamino) phenyl) vinyl) -3, 4-ethylenedioxythiophen-2-yl) vinyl) -2,1, 3-benzothiadiazol-4-yl) vinyl) -5, 7-disulfonic acid-3H-benzo [ e ] indole (Compound 20):

305mg of 3, 3-dimethyl-1- (2-trimethylammoniumbromide ethyl) -2- (trans-2- (7- (trans-2- (5- (trans-2- (4- (4-carboxylic acid butyrylamino) phenyl) vinyl) -3, 4-ethylenedioxythiophen-2-yl) vinyl) -2,1, 3-benzothiadiazol-4-yl) vinyl) -5, 7-disulfonic acid-3H-benzo [ e ] indole (0.3mmol) were dissolved in 5mL of DMF, after cooling in an ice-water bath, 44mg of NHS (0.4mmol) and 72mg of EDC (0.4mmol) were added in this order, the reaction was stirred at room temperature overnight, the solvent was evaporated and washed with ether to give 0.20g of product in 59.7% yield.

Test example 1

The test example characterizes the performance of the near-infrared fluorescent probe of the compound of the invention, and the specific process is as follows:

the compound 1,3, 3-dimethyl-1- (2-trimethylammoniobromoethyl) -2- (trans-2- (7- (trans-2- (5- (trans-2- (2-methoxy-4-dimethylaminophenyl) vinyl) thiophen-2-yl) vinyl) -2,1, 3-benzothiadiazol-4-yl) vinyl) -5, 7-disulfonyl-3H-benzo [ e ] obtained in the above example 1 was reacted with a solvent to obtain a mixture]Indole dissolved in PBS buffer solution to obtain 3.4 μ M solution, measuring its absorption spectrum and fluorescence emission spectrum under 785nm laser excitation as shown in figure 1, wherein in water solution, compound 1 has maximum absorption at 696nm and molar absorption coefficient of 40.7 × 10³M^-1cm^-1Two fluorescence emission peaks are provided, one is at 838nm, the other is at 965nm, the fluorescence emission is in a near infrared band (800-.

Table 2 also provides optical property characterization data for other compounds:

TABLE 2

And (4) supplementary notes: "+" indicates that the compound is water soluble; "-" indicates that the compound is poorly water soluble.

Test example 2

The experimental example tests the interaction between partial compounds and protein molecules and the enrichment of cell membranes, and the specific process is as follows:

because the compound of the invention has a zwitterion structure, the interaction between the compound and protein can be reduced, so that the probe can be directly injected into a living animal body without being influenced by the protein in the body. When HEK293T cells were cultured using the compounds of the invention, it was found that the dye was mainly concentrated on the cell membrane. The results are shown in Table 3:

TABLE 3

The experimental example also tests the response capability of the molecular probe formed by the compound of the invention to the transmembrane potential of the cell membrane, and the specific process is as follows:

when a stepwise pulse voltage was applied to a cell labeled with the dye of the compound of the present invention using patch clamp, it was observed that the fluorescence intensity of the cell increased with an increase in the applied voltage by 17% for every 100mV increase in the voltage, as shown in FIG. 2. The ability of molecular probes formed from other compounds to respond to the transmembrane potential of cell membranes is shown in Table 4:

TABLE 4

The PBS solution of the infrared probe formed by the compound 1 in the example 1 is injected into the brain of a mouse through brain stereotaxic injection, and neurons in the brain of the mouse can be imaged by directly penetrating through skin and the skull under the excitation of 785nm laser, so that the high-flux real-time recording of the single-neuron membrane potential of the mammal under the non-invasive condition is realized, and the result is shown in figure 3.

The embodiments of the present invention have been described in detail, but the present invention is not limited to the embodiments, and various changes can be made without departing from the gist of the present invention within the knowledge of those skilled in the art. Furthermore, the embodiments of the present invention and the features of the embodiments may be combined with each other without conflict.

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