A, pi, D, pi, Aapos; two-photon fluorescent compound as well as synthesis method and application thereof

Abstract

The invention discloses an A-pi-D-pi-A 'type two-photon fluorescent compound as well as a synthesis method and application thereof. The compound is shown as a formula (I). The invention provides a synthesis method of the compound. The synthesis method comprises the following steps: (1) carrying out bromomethylation reaction on a compound shown in a formula (II), paraformaldehyde and hydrobromic acid to obtain a compound shown in a formula (III); (2) salifying the compound shown in the formula (III) with hexamethylenetetramine, and then carrying out hydrolysis reaction to obtain a compound shown in a formula (IV); (3) carrying out unilateral dehydration condensation reaction on the compound shown in the formula (IV) and 2-methylbenzoxazole to obtain a compound shown in a formula (V); and (4) carrying out dehydration condensation reaction on the compound shown in the formula (V) and a compound shown in the formula (VI) to obtain a compound shown in the formula (I). The compound has large two-photon fluorescence activity absorption cross section and stokes shift, good living cell penetrability and low cytotoxicity, and can be applied to two-photon fluorescence imaging in living cells. # imgabs0 #

Description

Technical Field

[0001] The 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 -Mayer M. proposed it in 1931, and later confirmed it experimentally by Kaiser W. and Garrett CGB in 1961. Two-photon absorption refers to a third-order nonlinear optical process in which a substance absorbs two photons of the same or different energy through a virtual intermediate state under the action of strong light, thereby completing the transition from the ground state to the excited state. If the substance in 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 in the range of 10 32 -10 33 GM range, so the required optical density is small, and the two-photon absorption cross section is generally 1-10 4 GM range, so the probability of two-photon absorption in 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 in 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 the living system cannot bear, enabling the imaging of living cells and the direct monitoring of life processes; (2) Since the linear absorption and Rayleigh scattering of this wavelength band of light in 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 focusing of the laser beam, the range of material excitation is limited to a micro-area volume equivalent to the cube of the incident wavelength, and the light intensity at other positions is insufficient to cause two-photon absorption, so that the excitation of the material has a high degree of spatial selectivity, which can greatly improve the axial resolution and contrast of the imaging; (4) It can greatly reduce the photobleaching and phototoxicity of the organism, and the background fluorescence interference is small, which is easy to observe. Therefore, two-photon fluorescence imaging technology has significant application advantages in the fields of life sciences and medicine.

[0005] An important parameter to characterize two-photon absorption performance is the two-photon absorption cross section, which indicates the probability that a substance absorbs two photons simultaneously to be excited from the ground state to a high-energy state; an important parameter to characterize fluorescence performance is the fluorescence quantum yield, which is the ratio of the number of photons emitted by a substance to the number of photons absorbed. The two-photon fluorescence active absorption cross section is the product of the fluorescence quantum yield and the two-photon absorption cross section, and it is one of the most important parameters for measuring whether a fluorescent molecule can achieve two-photon fluorescence imaging. A small value means that when performing two-photon fluorescence imaging, in order to obtain good imaging, the excitation light intensity must be increased, which causes damage to the organism. Therefore, a substance having a large two-photon fluorescence active absorption cross section is the basis for realizing its application in two-photon fluorescence imaging in the fields of life sciences and medicine.

[0006] Fluorescence emission is the reverse process of absorption. In most cases, due to factors such as vibrational relaxation, molecular configuration changes, and solvent effects, there is a certain amount of energy loss between the emission and absorption of light, so that the fluorescence emission wavelength is 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, which makes them susceptible to the fluorescence inner filter effect. A large Stokes shift can effectively reduce the overlap between the absorption spectrum and the emission spectrum, eliminate the interference of fluorescence self-absorption, and thus significantly improve 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 invention object, the technical solution adopted by the present invention is as follows:

[0012] In the first aspect, the present invention provides a compound, the structure of which is shown in formula (I), and 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 obtain 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-benzenedicarbaldehyde, 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 obtain the corresponding compound of formula (I);

[0023]

[0024] The bromomethylation reaction described in step (1) of the present invention is specifically implemented as follows: add the compound of formula (II), paraformaldehyde and acetic acid to the reaction flask, then drip a hydrobromic acid acetic acid solution with a mass fraction of 33%, and after dripping, react at 40-118°C (preferably 70-90°C) for 5-12h (preferably 7-9h). After the reaction is completed, the obtained reaction mixture is separated and purified to obtain the 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: cool the reaction mixture to room temperature, fill the reaction flask with distilled water, then refrigerate overnight, filter, and recrystallize the obtained solid with 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 dropwise add 33% by mass fraction of hydrobromic acid and acetic acid solution, react at 70-90°C for 7-9h, then cool to room temperature, fill the reaction flask with distilled water, then refrigerate overnight, filter, and recrystallize the obtained solid with a mixed solvent of chloroform and methanol to obtain the compound of formula (III).

[0027] The salt-forming and then hydrolysis reaction described in step (2) of the present invention is specifically implemented as follows: add the compound of formula (III), hexamethylenetetramine and chloroform to the reaction flask, then react at 40-62°C (preferably reflux temperature) for 6-14h (preferably 8-10h), after the reaction is completed, cool to room temperature, filter, add acetic acid aqueous solution with a volume fraction of 50-90% (preferably 80-90%) to the filtered solid, and then react at 60-110°C (preferably reflux temperature) for 6-12h (preferably 8-10h), after the reaction is completed, the obtained reaction mixture is separated and purified to obtain the compound of formula (IV). In the salt-forming 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: 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] Add the compound of formula (III), hexamethylenetetramine and chloroform to the reaction flask, then heat and reflux to react for 8 to 10 hours, then cool to room temperature, filter, add 80 to 90% by volume acetic acid aqueous solution to the filtered solid, then heat and reflux to react for 8 to 10 hours, then cool to room temperature, pour the reaction solution into a large amount of distilled water, then refrigerate overnight, filter, and 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 implemented as follows: add the compound of formula (IV), 2-methylbenzoxazole, acetic anhydride and acetic acid into a reaction flask, and then react at 100-139° C. (preferably reflux temperature) for 20-30 hours (preferably 22-25 hours). After the reaction is completed, the obtained 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: 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, filtering the precipitated solid and then separating and purifying it by silica gel column chromatography, and the eluting reagent is petroleum ether and ethyl acetate (volume ratio is 5 to 30:1).

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

[0032] Add the compound of formula (IV), 2-methylbenzoxazole, acetic anhydride and acetic acid to the reaction flask, then heat under reflux to react for 22 to 25 hours, then cool to 0°C, add concentrated hydrochloric acid to the reaction solution, stir for 0.5 to 1 hour, filter with suction, and then add aqueous sodium hydroxide solution to the filtrate to adjust the pH value to 7-8. After filtering the precipitated solid, separate and purify it by silica gel column chromatography (eluting reagents are 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 implemented as follows: add the compound of formula (V), the compound of formula (VI) and the solvent to the reaction flask, stir and dissolve, then add the base, and then react at 30-150°C (preferably reflux temperature) for 5-24h (preferably 10-15h). 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 of several 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: cooling the reaction mixture to room temperature, suction filtering, and recrystallizing the obtained solid 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, and the recommended synthetic routes are as follows:

[0037]

[0038] The compound of formula (I) provided by the present invention uses styrene with excellent electron transport ability as a π-conjugated bridge, and introduces electron-donating butoxy and methoxy groups at the 2 and 5 positions of the central benzene ring, respectively, and electron-withdrawing benzoxazole and pyridinium groups are bonded at both ends, respectively, so that the whole molecule constitutes an asymmetric A-π-D-π-A' configuration (A is an electron acceptor, and D is an electron donor). Density functional theory calculations of the compound of formula (I) show that: on the HOMO molecular orbital, the electron cloud is mainly concentrated on the oxygen atom of the alkoxy group, the benzoxazole group and the π-bridge, while on the LUMO molecular orbital, the electron cloud density on the oxygen atom of the alkoxy group and the benzoxazole group is significantly reduced, and the electron cloud density on the pyridinium acceptor is significantly increased, so that the whole molecule undergoes strong intramolecular charge transfer. The compound of formula (I) not only has a large π-conjugated system with good coplanarity, but also greatly enhances the degree of electron cloud delocalization due to the embedding of electron donor-acceptor, all of which are conducive to improving the two-photon absorption performance of the compound of formula (I). In addition, the two vinyl groups in the styrene 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 also be conducive to the strong fluorescence emission of the compound of formula (I). As is known to all, 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 there is very little overlap between its absorption spectrum and emission spectrum, which can well 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, resulting in it being unable 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 in 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 increasing 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 alkoxy group has suitable hydrophilic and lipophilic amphiphilic properties, which well 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 the 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 beneficial effect of the present invention is that the present invention provides a compound having a large two-photon fluorescence active absorption cross section, a large Stokes shift, good living cell permeability and low cytotoxicity, which can be applied to two-photon fluorescence imaging in living cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 The compound of formula (I) is 2 Absorption and fluorescence emission spectra in O. 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 excitation of its optimal wavelength, wherein the ordinate represents the two-photon fluorescence intensity and the abscissa represents the wavelength.

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

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

[0045] The materials, reagents or instruments used in the examples without indicating the manufacturer are all conventional products that can be purchased from the market. If no specific conditions are indicated in the examples, conventional conditions or conditions recommended by the manufacturer were followed.

[0046] Example 1 Compound of formula (III)

[0047] 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 to the reaction flask, and then 2 mL of 33% hydrobromic acid and acetic acid solution was added dropwise. After the addition was completed, the reaction was carried out at 80°C for 8 h, and then cooled to room temperature. The reaction flask was filled with distilled water, and then refrigerated overnight, filtered, and the obtained solid was recrystallized with a mixed solvent of chloroform and methanol to obtain 0.84 g of a white compound of formula (III). mp106-107°C; 1 H NMR (CDCl 3 ,500MHz)δ: 6.88(s,1H), 6.87(s,1H), 4.55(s,2H), 4.54(s,2H), 4.01(t,J=6.4H z, 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 the reaction flask, and then heated to reflux for 8 h, then cooled to room temperature, filtered, and then 30 mL of 90% acetic acid aqueous solution was added to the filtered solid, and then heated to reflux for 8 h, then cooled to room temperature, poured into a large amount of distilled water, and then refrigerated overnight, filtered, and the obtained solid was recrystallized from a mixed solvent of DMF and water to obtain 0.42 g of a yellow compound of formula (IV). mp81-82°C; 1 HNMR (CDCl 3 ,500MHz)δ: 10.53(s,1H), 10.50(s,1H), 7.45(s,2H), 4.10(t,J=6.4Hz,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, then cooled to 0 ° C, 7.5 mL of concentrated hydrochloric acid was added to the reaction solution, stirred for 1 h, filtered, and then sodium hydroxide aqueous 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 reagent is V 石油醚 :V 乙酸乙酯 =20:1), to obtain 0.89 g of yellow compound of formula (V). mp 98-100°C; 1 H NMR (DMSO-d 6 ,500MHz)δ: 10.38(s,1H), 8.04(d,J=16.5Hz,1H), 7.78(dt,J 1 =7.5Hz,J 2 =1.5Hz,2H), 7.77(s,1H), 7.67(d,J=16.5Hz,1H), 7.45(td,J 1 =7.7Hz,J 2 =1.3Hz,1H),7.41(td,J 1 =7.6Hz,J 2=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] 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 to the reaction flask, stirred to dissolve, and then 0.17 g (2 mmol) of piperidine was added, and then heated to reflux for 14 h, then cooled to room temperature, filtered, and the obtained 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-d 6 ,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), 7.5 0(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,2H), 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-d 6 ,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, 12 4.89, 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 organisms live in water environments, so the compound of formula (I) is characterized by H2 The absorption and fluorescence emission spectra of O are very important. Its absorption spectrum was measured by Shimadzu UV-2550 UV-visible spectrophotometer, and the fluorescence emission spectrum was measured by RF-5301PC fluorescence spectrophotometer. The specific results are shown in Figure 1 The compound of formula (I) is in H 2 The Stokes shift in O is very large, reaching 130nm, which makes its emission spectrum and absorption spectrum overlap very little. This can significantly reduce the fluorescence self-absorption phenomenon and increase the signal-to-noise ratio during biological imaging, thereby improving 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 to characterize whether a substance can achieve two-photon fluorescence imaging. The two-photon fluorescence emission spectrum of the compound of formula (Ⅰ) was tested by 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 sample ethanol solution 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] Selecting 0.1M sodium hydroxide aqueous solution of fluorescein as a reference, the two-photon fluorescence active absorption cross section calculation formula of the compound of formula (I) is shown in formula (1):

[0059]

[0060] In the formula, the subscripts s and r represent the physical quantities corresponding to the sample and the 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 test was performed using tetrazolium salt (MTT) colorimetric method. Human ovarian cancer cells (OVCAR-8) were used as the research object, and RPMI 1640 medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin was used for cell culture. OVCAR-8 cells were seeded in 96-well culture plates at a concentration of 10,000 cells per well (100 μL of culture medium per well) and incubated at 37°C and 5% CO 2 Then, 0-80 μM of the compound of formula (I) was added in a gradient manner and the OVCAR-8 cells were incubated for 24 h. Then, 20 μL of MTT solution was added to each well and the cells were incubated at 37°C and 5% CO. 2 The cells were incubated for 4 h under the same conditions. 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 an ELISA instrument, and the cell survival rate was calculated according to formula (2).

[0064]

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

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

[0067] Human ovarian cancer cells (OVCAR-8) were inoculated into imaging culture dishes and cultured for 24 h in RPMI 1640 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin. Then, 10 μM of the compound of formula (I) was added and incubated at 37°C, 5% CO 2 The cells were incubated under the same conditions for 0.5 h, and then the culture medium was removed and the cells were washed 2-3 times with PBS buffer solution. 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 a fluorescence emission signal collection channel of 575-630 nm.

[0068] Figure 3 The two-photon fluorescence imaging of the compound of formula (I) on OVCAR-8 living cells. The results show that the compound of formula (I) has good penetrability to living cells, can successfully enter the living cells and generate bright two-photon fluorescence signals.

[0069] The above description is only an embodiment of the present application and is not intended to limit the protection scope of the present application. For those skilled in the art, the present application may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A compound, whose structure is shown in formula (I), and whose chemical name is 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 obtain 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-benzenedicarbaldehyde, 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 obtain the corresponding compound of formula (I); 3. Use of the compound as claimed in claim 1 in the preparation of a two-photon fluorescence imaging agent in living cells.

Images