A-π-D-π-A' type two-photon fluorescent compound and its synthesis method and application

Abstract

The present invention discloses an A-π-D-π-A' type two-photon fluorescent compound and its synthesis method and application. The compound is shown in formula (I). The present invention provides a synthesis method of the compound, comprising the following steps: (1) a compound of formula (II) undergoes a bromomethylation reaction with paraformaldehyde and hydrobromic acid to obtain a compound of formula (III); (2) the compound of formula (III) is first salified with hexamethylenetetramine, and then undergoes a hydrolysis reaction to obtain a compound of formula (IV); (3) the compound of formula (IV) undergoes a unilateral dehydration condensation reaction with 2-methylbenzoxazole to obtain a compound of formula (V); (4) the compound of formula (V) undergoes a dehydration condensation reaction with a compound of formula (VI) to obtain a compound of formula (I). The compound has a large two-photon fluorescence active absorption cross section and Stokes shift, good living cell penetrability and low cytotoxicity, and can be used for two-photon fluorescence imaging in living cells.

Description

Technical Field

[0001] The present invention relates to an A-π-D-π-A' type two-photon fluorescent compound, a synthesis method thereof, and application of the compound in preparing a two-photon fluorescent imaging agent in living cells. Background Art

[0002] The two-photon absorption theory was first proposed by The concept was first proposed by Mayer M. in 1931 and experimentally confirmed by Kaiser W. and Garrett C.B.G. in 1961. Two-photon absorption is a third-order nonlinear optical process in which, under the influence of strong light, a substance simultaneously absorbs two photons of equal or different energies through a virtual intermediate state, thereby completing a transition from the ground state to an excited state. If the excited state subsequently undergoes a radiative transition back to the ground state, the resulting frequency up-conversion fluorescence is called two-photon fluorescence.

[0003] Compared with single-photon absorption, two-photon absorption has the following characteristics: (1) Single-photon absorption is a linear absorption process that obeys the Stark-Einstein law, while two-photon absorption is a nonlinear absorption process; (2) In the single-photon absorption process, a substance molecule absorbs a high-energy, short-wavelength photon to reach an excited state, while in the two-photon absorption process, a substance molecule absorbs two low-energy, long-wavelength photons to reach an excited state; (3) In the two-photon absorption process, the absorption intensity and electron transition probability of the substance molecule are proportional to the square of the excitation light intensity; (4) For fluorescent molecules, the absorption cross section is generally used to represent the ability of the molecule to absorb photons. The larger the absorption cross section, the stronger the ability of the substance molecule to absorb photons. Usually, the single-photon absorption cross section is between 10 32 -10 33 GM range, so the required optical density is small, and the two-photon absorption cross section is generally between 1-10 4 GM range, so the probability of two-photon absorption of ordinary molecules is very small; (5) Two-photon absorption occurs at the focus λ 3 (λ is the excitation wavelength), while single-photon absorption occurs within the entire focused light path.

[0004] Based on the above characteristics, two-photon fluorescence imaging technology based on two-photon absorption has many advantages that single-photon imaging technology cannot match: (1) Single-photon fluorescence is short-wave excitation and long-wave emission, while two-photon fluorescence is long-wave excitation and short-wave emission. The wavelength used in the two-photon excitation process is red-shifted by nearly one-fold and is within the biological optical window (650-950nm), thus avoiding the UV-visible light damage that living systems cannot withstand, enabling the imaging of living cells and the direct monitoring of life processes. (2) Since the linear absorption and Rayleigh scattering of light in this wavelength band by biological tissues are relatively small, the light penetration is good, which greatly increases the penetration of the excitation light in the organism, thus enabling deep three-dimensional imaging of the organism. (3) The probability of electron transition induced by two-photons is proportional to the square of the incident light intensity. Under the condition of tight laser beam focusing, the range of material excitation is limited to a micro-area volume equivalent to the cube of the incident wavelength, while the light intensity at other locations is insufficient to cause two-photon absorption, thus making the excitation of the material highly spatially selective, which can greatly improve the axial resolution and contrast of imaging. (4) It can greatly reduce photobleaching and phototoxicity to the organism, and the background fluorescence interference is small, making it easy to observe. Therefore, two-photon fluorescence imaging technology has significant application advantages in the fields of life science and medicine.

[0005] A key parameter characterizing two-photon absorption properties is the two-photon absorption cross section (TACS), which indicates the probability of a substance absorbing two photons simultaneously to excite from its ground state to a higher energy state. A key parameter characterizing fluorescence properties is the fluorescence quantum yield (FQY), which is the ratio of the number of photons emitted to the number of photons absorbed. The TACS, the product of the FQY and the TACS, is one of the most important parameters for determining whether a fluorescent molecule can achieve two-photon fluorescence imaging. A small TACS value means that the excitation light intensity must be increased to obtain a good image during two-photon fluorescence imaging, potentially causing damage to the organism. Therefore, possessing a large TACS is fundamental to its application in life sciences and medicine.

[0006] Fluorescence emission is the reverse of absorption. In most cases, due to factors such as vibrational relaxation, changes in molecular configuration, and solvent effects, there is a certain amount of energy loss between light emission and absorption, resulting in a fluorescence emission wavelength greater than the absorption wavelength. The Stokes shift is defined as the difference between the emission wavelength and the absorption wavelength. Most fluorescent molecules exhibit a small Stokes shift, making them susceptible to the fluorescence inner filtering effect. A large Stokes shift can effectively reduce the overlap between the absorption and emission spectra, eliminating interference from fluorescence self-absorption, thereby significantly improving the sensitivity and accuracy of fluorescence detection and the signal-to-noise ratio of imaging.

[0007] In summary, designing and synthesizing new compounds with large two-photon fluorescence active absorption cross-section, large Stokes shift, good living cell penetrability and low cytotoxicity, and realizing their practical application in two-photon fluorescence imaging in living cells, has both theoretical and practical significance. Summary of the Invention

[0008] The primary purpose of the present invention is to provide an A-π-D-π-A' type compound, which has a large two-photon fluorescence active absorption cross section, a large Stokes shift, good living cell penetration and low cytotoxicity.

[0009] The second object of the present invention is to provide a method for synthesizing the compound.

[0010] The third object of the present invention is to provide the use of the compound in preparing a two-photon fluorescence imaging agent in living cells.

[0011] In order to achieve the above-mentioned object of the invention, the technical solution adopted by the present invention is as follows:

[0012] In the first aspect, the present invention provides a compound whose structure is shown in formula (I). The chemical name of the compound of formula (I) is 4-[(1E)-2-[4-[(1E)-2-(2-benzoxazolyl)vinyl]-2-butoxy-5-methoxyphenyl]vinyl]-1-(2-hydroxyethyl)pyridinium bromide:

[0013]

[0014] In a second aspect, the present invention provides a method for synthesizing the compound of formula (I) described in the first aspect, comprising the following steps:

[0015] (1) The compound of formula (II) undergoes a bromomethylation reaction with paraformaldehyde and hydrobromic acid to prepare 1,4-bis(bromomethyl)-2-butoxy-5-methoxybenzene, i.e., the corresponding compound of formula (III);

[0016]

[0017]

[0018] (2) The compound of formula (III) is first salified with hexamethylenetetramine and then hydrolyzed to obtain 2-butoxy-5-methoxy-1,4-benzenedicarboxaldehyde, i.e., the corresponding compound of formula (IV);

[0019]

[0020] (3) The compound of formula (IV) undergoes a unilateral dehydration condensation reaction with 2-methylbenzoxazole to obtain 4-[(1E)-2-(2-benzoxazolyl)vinyl]-2-butoxy-5-methoxybenzaldehyde, i.e., the corresponding compound of formula (V);

[0021]

[0022] (4) the compound of formula (V) undergoes a dehydration condensation reaction with the compound of formula (VI) to prepare the corresponding compound of formula (I);

[0023]

[0024] The bromomethylation reaction described in step (1) of the present invention is specifically carried out as follows: a compound of formula (II), paraformaldehyde, and acetic acid are added to a reaction flask, and then a 33% by mass hydrobromic acid acetic acid solution is added dropwise. After the addition is completed, the reaction is carried out at 40-118°C (preferably 70-90°C) for 5-12 hours (preferably 7-9 hours). After the reaction is completed, the resulting reaction mixture is separated and purified to obtain a compound of formula (III). The solvent used is acetic acid, and the molar amount of acetic acid is 110-250 times the molar amount of the compound of formula (II). The molar ratio of the compound of formula (II) to paraformaldehyde is 1:3-5. The molar ratio of the compound of formula (II) to hydrobromic acid is 1:2-6. After the reaction is completed, the separation and purification method is preferably as follows: the reaction mixture is cooled to room temperature, the reaction flask is filled with distilled water, and then refrigerated overnight, filtered, and the resulting solid is recrystallized from a mixed solvent of chloroform and methanol.

[0025] Preferably, the step (1) is implemented as follows:

[0026] Add the compound of formula (II), paraformaldehyde and acetic acid to the reaction flask, then add dropwise a 33% mass fraction of hydrobromic acid and acetic acid solution. After the addition is complete, react at 70-90°C for 7-9 hours, then cool to room temperature, fill the reaction flask with distilled water, refrigerate overnight, filter, and recrystallize the resulting solid from a mixed solvent of chloroform and methanol to obtain a compound of formula (III).

[0027] The salt formation followed by hydrolysis reaction described in step (2) of the present invention is specifically carried out as follows: the compound of formula (III), hexamethylenetetramine, and chloroform are added to a reaction flask, followed by reaction at 40-62°C (preferably reflux temperature) for 6-14 hours (preferably 8-10 hours). After the reaction, the mixture is cooled to room temperature, filtered, and then a 50-90% (preferably 80-90%) volume fraction of acetic acid aqueous solution is added to the filtered solid. The mixture is then reacted at 60-110°C (preferably reflux temperature) for 6-12 hours (preferably 8-10 hours). After the reaction, the resulting reaction mixture is separated and purified to obtain the compound of formula (IV). In the salt formation reaction, the solvent used is chloroform, and the molar amount of chloroform is 50-100 times the molar amount of the compound of formula (III). The molar ratio of the compound of formula (III) to hexamethylenetetramine is 1:2-4. In the hydrolysis reaction, the molar amount of acetic acid is 70-140 times the molar amount of the compound of formula (III). After the reaction is completed, the separation and purification method is preferably as follows: cooling the reaction mixture to room temperature, pouring the reaction solution into a large amount of distilled water, then refrigerating overnight, filtering, and recrystallizing the obtained solid with a mixed solvent of DMF and water.

[0028] Preferably, the step (2) is implemented as follows:

[0029] The compound of formula (III), hexamethylenetetramine and chloroform are added to the reaction flask, and then heated under reflux for 8 to 10 hours, then cooled to room temperature and filtered, and then an acetic acid aqueous solution with a volume fraction of 80 to 90% is added to the filtered solid, and then heated under reflux for 8 to 10 hours, and then cooled to room temperature. The reaction solution is poured into a large amount of distilled water, and then refrigerated overnight and filtered. The obtained solid is recrystallized from a mixed solvent of DMF and water to obtain a compound of formula (IV).

[0030] The unilateral dehydration condensation reaction described in step (3) of the present invention is specifically carried out as follows: the compound of formula (IV), 2-methylbenzoxazole, acetic anhydride and acetic acid are added to a reaction flask, and then reacted at 100-139° C. (preferably reflux temperature) for 20-30 hours (preferably 22-25 hours). After the reaction is completed, the resulting reaction mixture is separated and purified to obtain the compound of formula (V). The molar amount of acetic anhydride is 10-30 times the molar amount of 2-methylbenzoxazole. The molar amount of acetic acid is 5-20 times the molar amount of 2-methylbenzoxazole. The molar ratio of 2-methylbenzoxazole to the compound of formula (IV) is 1:1.1-2. After the reaction is completed, the separation and purification method is preferably as follows: cooling the reaction mixture to 0°C, adding concentrated hydrochloric acid to the reaction solution, wherein the molar amount of HCl contained in the concentrated hydrochloric acid is 15 to 25 times the molar amount of 2-methylbenzoxazole, stirring for 0.5 to 1 hour, filtering with suction, and then adding sodium hydroxide aqueous solution to the filtrate to adjust the pH value to 7-8. The precipitated solid is filtered and then separated and purified by silica gel column chromatography, and the elution reagent is petroleum ether and ethyl acetate (volume ratio of 5 to 30:1).

[0031] Preferably, the step (3) is implemented as follows:

[0032] The compound of formula (IV), 2-methylbenzoxazole, acetic anhydride and acetic acid were added to the reaction flask, and then heated under reflux for 22 to 25 hours. The mixture was then cooled to 0°C, concentrated hydrochloric acid was added to the reaction solution, stirred for 0.5 to 1 hour, and filtered. Aqueous sodium hydroxide solution was added to the filtrate to adjust the pH value to 7-8. The precipitated solid was filtered and then separated and purified by silica gel column chromatography (eluting reagents: petroleum ether and ethyl acetate) to obtain the compound of formula (V).

[0033] The dehydration condensation reaction described in step (4) of the present invention is specifically carried out as follows: the compound of formula (V), the compound of formula (VI), and a solvent are added to a reaction flask, stirred to dissolve, and then a base is added. The mixture is then reacted at 30-150°C (preferably reflux temperature) for 5-24 hours (preferably 10-15 hours). After the reaction is completed, the reaction mixture is separated and purified to obtain the target compound of formula (I). The base used is generally piperidine, triethylamine, or potassium hydroxide, and the molar amount of the base is 1.2-4 times the molar amount of the compound of formula (VI). The solvent is generally methanol, ethanol, chloroform, dichloromethane, acetonitrile, DMF, or a mixture thereof, and the molar amount of the solvent is 200-700 times the molar amount of the compound of formula (VI). The molar ratio of the compound of formula (VI) to the compound of formula (V) is 1:1-2. After the reaction is completed, the separation and purification method is preferably: the reaction mixture is cooled to room temperature, filtered, and the obtained solid is recrystallized from acetonitrile.

[0034] Preferably, the step (4) is implemented as follows:

[0035] Add the compound of formula (V), the compound of formula (VI) and ethanol to the reaction flask, stir to dissolve, then add piperidine, and then heat to reflux to react for 10 to 15 hours, then cool to room temperature, filter, and the obtained solid is recrystallized from acetonitrile to obtain the compound of formula (I).

[0036] In the present invention, the compounds represented by formula (II) and formula (VI) can be synthesized by methods reported in the literature. The recommended synthetic routes are as follows:

[0037]

[0038] The compound of formula (I) provided herein uses styrene, a π-conjugated bridge with excellent electron-transporting properties, with electron-donating butoxy and methoxy groups introduced at the 2 and 5 positions of the central phenyl ring, respectively. Electron-withdrawing benzoxazole and pyridinium groups are bonded to the two ends, respectively, resulting in an asymmetric A-π-D-π-A' configuration (A is an electron acceptor, D is an electron donor). Density functional theory calculations of the compound of formula (I) show that in the HOMO molecular orbital, the electron cloud is primarily concentrated on the alkoxy oxygen atom, the benzoxazole group, and the π-bridge. In the LUMO molecular orbital, the electron cloud density on the alkoxy oxygen atom and the benzoxazole group is significantly reduced, while the electron cloud density on the pyridinium acceptor is significantly increased, resulting in strong intramolecular charge transfer throughout the molecule. The compound of formula (I) not only possesses a large, coplanar π-conjugated system, but also significantly enhances electron cloud delocalization due to the embedded electron donor-acceptor structure, all of which contribute to its enhanced two-photon absorption properties. In addition, the two vinyl groups in the styrene divinyl π-bridge are in a trans configuration that is conducive to fluorescence enhancement, and the introduced alkoxy group is also a fluorescent auxochrome, which can in turn be conducive to the strong fluorescence emission of the compound of formula (I). As is well known, living cells live in an aqueous environment, and the Stokes shift of the compound of formula (I) in water is as high as 130nm, so the overlap between its absorption spectrum and emission spectrum is very small, which can effectively weaken the fluorescence inner filter effect and improve the signal-to-noise ratio of cell imaging. In addition, the inventors also synthesized a control compound with a symmetrical A-π-D-π-A configuration in which the 2 and 5 positions of the central benzene ring are both methoxy groups and both ends are pyridinium groups bonded to N-hydroxyethyl groups. It was found that the control compound was too hydrophilic to pass through the cell membrane well to achieve staining and fluorescence imaging of living cells. In the compound of formula (I), two identical methoxy groups are not introduced at the center, but one of the methoxy groups is replaced by a butoxy group. Two identical N-hydroxyethyl-bonded pyridinium groups are not introduced at both ends, but one of the N-hydroxyethyl-bonded pyridinium groups is replaced by a benzoxazole group. By lengthening the alkyl chain of the alkoxy group and reducing the strongly hydrophilic pyridinium cation and the hydroxyethyl group that easily forms hydrogen bonds with water, the compound is given suitable hydrophilic and lipophilic amphiphilic properties, which effectively adjusts the oil-water partition coefficient of the entire molecule, ultimately resulting in good permeability to the cell membrane composed of the phospholipid bilayer.

[0039] Therefore, in a third aspect, the present invention provides the use of the compound of formula (I) in the preparation of a two-photon fluorescence imaging agent in living cells.

[0040] Compared with the prior art, the present invention has the following advantages: the present invention provides a compound with a large two-photon fluorescence active absorption cross section, a large Stokes shift, good living cell penetrance and low cytotoxicity, which can be applied to two-photon fluorescence imaging in living cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 The absorption and fluorescence emission spectra of the compound of formula (I) in H2O are shown in Figure 1. The left ordinate represents absorbance, the right ordinate represents fluorescence intensity, and the abscissa represents wavelength.

[0042] Figure 2 The two-photon fluorescence emission spectrum of the compound of formula (I) under the optimal wavelength excitation is shown in FIG.

[0043] Figure 3 Two-photon fluorescence imaging of living OVCAR-8 cells by the compound of formula (I). (a) Cell bright field, (b) two-photon fluorescence imaging, (c) cell bright field and two-photon fluorescence imaging superposition, scale bar 20 μm. DETAILED DESCRIPTION

[0044] In order to make the purpose, technical solutions and advantages of the present invention more clear, the technical solutions will be further clearly and completely described below through embodiments, but the protection scope of the present invention is not limited thereto.

[0045] Materials, reagents, or instruments used in the examples without manufacturer's indication are all commercially available products. If specific conditions are not specified in the examples, conventional conditions or those recommended by the manufacturer were followed.

[0046] Example 1 Compound of formula (III)

[0047] To a reaction flask, 0.54 g (3 mmol) of the compound of formula (II), 0.36 g (12 mmol) of paraformaldehyde, and 30 mL of acetic acid were added dropwise, followed by the dropwise addition of 2 mL of a 33% hydrobromic acid-acetic acid solution. The reaction was allowed to proceed at 80°C for 8 h, followed by cooling to room temperature. The reaction flask was topped up with distilled water and refrigerated overnight. The resulting solid was then filtered and recrystallized from a mixed solvent of chloroform and methanol to yield 0.84 g of the white compound of formula (III). mp 106-107°C; 1 H NMR (CDCl3, 500MHz) δ: 6.88 (s, 1H), 6.87 (s, 1H), 4.55 (s, 2H), 4.54 (s, 2H), 4.01 (t, J= 6.4Hz, 2H), 3.88 (s, 3H), 1.79-1.85 (m, 2H), 1.52-1.59 (m, 2H), 1.01 (t, J = 7.4Hz, 3H).

[0048] Example 2 Compound of formula (IV)

[0049] 1.83 g (5 mmol) of the compound of formula (III), 2.10 g (15 mmol) of hexamethylenetetramine, and 30 mL of chloroform were added to a reaction flask, followed by heating under reflux for 8 h. The mixture was then cooled to room temperature and filtered. 30 mL of a 90% aqueous acetic acid solution was added to the filtered solid, followed by heating under reflux for 8 h. The mixture was then cooled to room temperature and poured into a large amount of distilled water. The mixture was refrigerated overnight and filtered. The resulting solid was recrystallized from a mixture of DMF and water to yield 0.42 g of the yellow compound of formula (IV). mp 81-82°C; 1 HNMR (CDCl3, 500MHz) δ: 10.53 (s, 1H), 10.50 (s, 1H), 7.45 (s, 2H), 4.10 (t, J = 6.4H z, 2H), 3.94 (s, 3H), 1.80-1.86 (m, 2H), 1.48-1.55 (m, 2H), 0.99 (t, J = 7.4Hz, 3H).

[0050] Example 3 Compound of formula (V)

[0051] 1.30 g (5.5 mmol) of the compound of formula (Ⅳ), 0.66 g (5 mmol) of 2-methylbenzoxazole, 9 mL of acetic anhydride and 4.5 mL of acetic acid were added to the reaction flask, and then heated under reflux for 24 h. The mixture was then cooled to 0 ° C. 7.5 mL of concentrated hydrochloric acid was added to the reaction solution, stirred for 1 h, and filtered. Aqueous sodium hydroxide solution was added to the filtrate to adjust the pH value to 7-8. The precipitated solid was filtered and then separated and purified by silica gel column chromatography (elution reagent was V 石油醚 :V 乙酸乙酯 =20:1), to give 0.89 g of yellow compound of formula (V). mp 98-100 ° C; 1 H NMR (DMSO-d6, 500MHz) δ: 10.38 (s, 1H), 8.04 (d, J = 16.5 Hz, 1H), 7.78 (dt, J1 = 7. 5Hz,J2=1.5Hz,2H), 7.77(s,1H), 7.67(d,J=16.5Hz,1H), 7.45(td,J1=7.7Hz,J2 =1.3Hz,1H), 7.41(td,J1=7.6Hz,J2=1.1Hz,1H), 7.31(s,1H), 4.24(t,J=6.5Hz ,2H), 3.93(s,3H), 1.76-1.82(m,2H), 1.47-1.54(m,2H), 0.98(t,J=7.4Hz,3H).

[0052] Example 4 Compound of formula (I)

[0053] To a reaction flask, 0.22 g (1 mmol) of the compound of formula (VI), 0.42 g (1.2 mmol) of the compound of formula (V), and 15 mL of ethanol were added and dissolved with stirring. 0.17 g (2 mmol) of piperidine was then added and the mixture was heated under reflux for 14 h. The mixture was then cooled to room temperature and filtered. The resulting solid was recrystallized from acetonitrile to obtain 0.24 g of the orange-red target compound of formula (I). mp 258-260°C; 1 H NMR (DMSO-d6, 500MHz) δ: 8.87 (d, J = 6.4Hz, 2H), 8.22 (d, J = 6.4Hz, 2H), 8.07 (d, J = 16.5Hz, 1H), 8.05 (d,J=16.4Hz,1H), 7.76(d,J=7.8Hz,2H), 7.70(d,J=16.5Hz,1H), 7.65(s,1H), 7.57(d,J=16.4Hz,1H ), 7.50(s,1H), 7.39-7.45(m,2H), 5.27(t,J=5.0Hz,1H), 4.58(t,J=4.6Hz,2H), 4.21(t,J=6.4Hz,2 H), 3.99 (s, 3H), 3.87 (q, J = 4.7Hz, 2H), 1.82-1.88 (m, 2H), 1.50-1.57 (m, 2H), 1.00 (t, J = 7.3Hz, 3H); 13 C NMR (DMSO-d6, 125MHz) δ: 162.60, 152.93, 151.86, 151.71, 149.82, 144.72, 141.77, 134.75, 132.95, 126.26, 126.13, 125 .56, 124.89, 124.75, 123.42, 119.60, 115.48, 111.97, 111.19, 110.59, 68.76, 62.06, 60.03, 56.34, 30.71, 18.86, 13.75.

[0054] Example 5 Absorption and fluorescence emission spectrum test

[0055] All cells in the body live in an aqueous environment, so it is very important to characterize the absorption and fluorescence emission spectra of the compound of formula (I) in H2O. The absorption spectrum was measured using a Shimadzu UV-2550 UV-visible spectrophotometer, and the fluorescence emission spectrum was measured using a RF-5301PC fluorescence spectrophotometer. The specific results are shown in Figure 1The Stokes shift of the compound of formula (I) in H2O is large, reaching 130 nm, so that its emission spectrum and absorption spectrum overlap very little. This can significantly reduce fluorescence self-absorption during biological imaging, increase the signal-to-noise ratio, and thus improve the accuracy and sensitivity of imaging.

[0056] Example 6 Two-photon fluorescence active absorption cross section test

[0057] The two-photon fluorescence active absorption cross section (fluorescence quantum yield Φ × two-photon absorption cross section δ) is an important parameter for characterizing whether a substance can achieve two-photon fluorescence imaging. The two-photon fluorescence emission spectrum of the compound of formula (I) was tested using the two-photon induced fluorescence method. During the test, a mode-locked titanium sapphire femtosecond laser (Chameleon Ultra II, 680-1080nm, 80MHz, 140fs) was used as the pump light source, and a full-spectrum spectrometer (USB4000-FLG) was used to detect the two-photon fluorescence signal. A 1mM solution of the sample in ethanol was placed in a four-sided quartz cuvette with an excitation wavelength of 680-910nm and an interval of 20nm. Figure 2 The two-photon fluorescence emission spectrum of the compound of formula (I) under excitation at its optimal wavelength. Due to the reabsorption effect, it produces strong two-photon fluorescence emission near 600 nm.

[0058] A 0.1M sodium hydroxide aqueous solution of fluorescein was selected as a reference, and the two-photon fluorescence activity absorption cross section of the compound of formula (I) was calculated as shown in formula (1):

[0059]

[0060] Where the subscripts s and r represent the physical quantities corresponding to the sample and reference, respectively. δ is the two-photon absorption cross section, F is the two-photon fluorescence integrated intensity, Φ is the fluorescence quantum yield, n is the solution refractive index, and c is the solution concentration.

[0061] The maximum two-photon fluorescence active absorption cross section of the compound of formula (I) calculated according to the above formula is 152GM, and this brightness can well realize two-photon fluorescence imaging.

[0062] Example 7 Cytotoxicity Test

[0063] Cytotoxicity was tested using the MTT colorimetric assay. Human ovarian cancer cells (OVCAR-8) were used as the study subjects. Cell culture was performed in RPMI 1640 medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. OVCAR-8 cells were seeded at a density of 10,000 cells per well in a 96-well culture plate (100 μL of culture medium per well) and incubated at 37°C, 5% CO₂ for 24 h. Compound (I) was then added in a gradient of 0-80 μM, and the OVCAR-8 cells were incubated for 24 h. 20 μL of MTT solution was then added to each well, and the cells were incubated at 37°C, 5% CO₂ for another 4 h. Finally, the culture medium was removed, and DMSO (100 μL per well) was added to dissolve the formazan crystals. The photon density (OD) value of each well was measured using a microplate reader, and the cell viability was calculated according to formula (2).

[0064]

[0065] According to the above cytotoxicity experiments, the compound of formula (I) has a cell survival rate of more than 90% at the imaging concentration used, and has low cytotoxicity.

[0066] Example 8 Two-photon fluorescence imaging in living cells

[0067] Human ovarian cancer cells (OVCAR-8) were seeded into imaging-specific culture dishes and cultured for 24 hours in RPMI 1640 medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. The compound of formula (I) was then added at 10 μM and incubated at 37°C, 5% CO₂ for 0.5 hours. The culture medium was then removed and the cells were washed 2-3 times with PBS buffer. Two-photon fluorescence imaging was performed using an Olympus BX61W1-FV1000 two-photon confocal laser scanning microscope with an excitation wavelength of 800 nm and fluorescence emission signal collection channels of 575-630 nm.

[0068] Figure 3 The results show that the compound of formula (I) has good penetrability in living cells and can successfully enter the living cells and generate bright two-photon fluorescence signals.

[0069] The above description is merely an embodiment of the present application and is not intended to limit the scope of protection of the present application. For those skilled in the art, various modifications and variations of the present application are possible. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present application shall be included in the scope of protection of the present application.

Claims

1. A compound having the structure shown in formula (I) and the chemical name 4-[(1E)-2-[4-[(1E)-2-(2-benzoxazolyl)vinyl]-2-butoxy-5-methoxyphenyl]vinyl]-1-(2-hydroxyethyl)pyridinium bromide:

2. A method for synthesizing the compound according to claim 1, characterized in that: The synthesis method comprises the following steps: (1) The compound of formula (II) undergoes a bromomethylation reaction with paraformaldehyde and hydrobromic acid to prepare 1,4-bis(bromomethyl)-2-butoxy-5-methoxybenzene, i.e., the corresponding compound of formula (III); (2) The compound of formula (III) is first salified with hexamethylenetetramine and then hydrolyzed to obtain 2-butoxy-5-methoxy-1,4-benzenedicarboxaldehyde, i.e., the corresponding compound of formula (IV); (3) The compound of formula (IV) undergoes a unilateral dehydration condensation reaction with 2-methylbenzoxazole to obtain 4-[(1E)-2-(2-benzoxazolyl)vinyl]-2-butoxy-5-methoxybenzaldehyde, i.e., the corresponding compound of formula (V); (4) the compound of formula (V) undergoes a dehydration condensation reaction with the compound of formula (VI) to prepare the corresponding compound of formula (I); 3. Use of the compound according to claim 1 in the preparation of a two-photon fluorescence imaging agent in living cells.

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