A fluorescent dye with an alkenylindole as a parent nucleus, and preparation and application thereof

Abstract

The application discloses a fluorescent dye with an alkenyl indole as a mother nucleus and a preparation and application thereof, and has a structure as shown in formula I, formula II or formula III. The application solves the problem that an indole conjugated skeleton cannot be effectively extended in the prior art, and the application of the indole in the field of fluorescent dyes is poor. The fluorescent dye with the alkenyl indole as the mother nucleus is prepared, raw materials are simple and easy to obtain, and an operation process is simple; the fluorescent dye with the alkenyl indole as the mother nucleus prepared by the application can cover a visible light region to a near infrared region, and the fluorescent dye with the structure as shown in formula III prepared by the application can realize specific detection on ONOO ‑ ; the fluorescent dye with the alkenyl indole as the mother nucleus prepared by the application is a new type of functional fluorescent dye, and the range of fluorescent dye types is widened.

Description

TECHNICAL FIELD

[0001] The present application relates to a kind of functional fluorescent dyes, and specifically to a kind of fluorescent dye with alkenyl indole as parent nucleus and its preparation and application. BACKGROUND

[0002] Due to the high sensitivity and strong selectivity of fluorescence analysis technology, it can also be used for non-invasive imaging in cells, therefore, fluorescent dyes have important application value in the fields of molecular fluorescent probes and biological imaging. Among them, small molecule organic fluorescent dyes have excellent luminescent performance and diversified structure, and their preparation has always been a hot spot in the field of fluorescence research. A series of fluorescent dyes based on a single parent structure not only can obtain adjustable emission light, even cover the full color spectrum, but also can reveal the structure-activity relationship between fluorophore and luminescent performance, so the design of a series of fluorescent dyes based on a single parent structure has attracted much attention. Since a series of dyes are prepared around a common fluorophore parent structure, compared with different fluorophores with different luminescence, the preparation cost and sampling error are reduced.

[0003] Indole is an aromatic structure widely existing in natural products - cheap and easy to obtain, and has good biocompatibility. Therefore, indole structure can be used as a parent nucleus of excellent performance fluorescent group. However, indole itself does not have fluorescence, and the traditional synthesis method has limited effect on the extension of indole conjugated skeleton, which leads to the application of indole in the field of fluorescent dyes is far less than that of coumarin, quinoline and other structures. SUMMARY

[0004] The purpose of the present application is to provide a fluorescent dye with alkenyl indole as parent nucleus and its preparation and application, which solves the problem that the existing technology cannot effectively extend the conjugated skeleton of indole, leading to poor application of indole in the field of fluorescent dyes, and provides a new full-spectrum covering fluorescent dye.

[0005] In order to achieve the above purpose, the present application provides a fluorescent dye with alkenyl indole as parent nucleus, which has the structure shown in formula I, formula II or formula III;

[0006]

[0007] Among them, R 1 is any one selected from H, methoxy, aldehyde group, methyl formate group and cyano group; R 2 is any one selected from aldehyde group, methoxy, ethyl ester group, cyano group and dimethyl amino group; R 3 is H or methyl formate group.

[0008] R 1 is a substituent of each position of indole.

[0009] Preferably, R1 The groups mainly located at 3, 5 and 6 positions of the indole ring can improve the fluorescence quantum yield and emission wavelength of the prepared fluorescent dye. The 3-position of the indole has high charge density, and R 1 When the group is located at the 3-position, the occurrence of photo-oxidation reaction can be reduced, thereby improving the fluorescence quantum yield of the prepared fluorescent dye; R 1 When the group is located at the 5-position or 6-position, the intramolecular charge transfer (ICT) effect can be maximized, thereby increasing the emission wavelength.

[0010] The application further provides a preparation method of the fluorescent dye with the alkenyl indole as the mother nucleus.

[0011]

[0012] (1) at 0°C, sodium hydride (NaH) is added to a solution of the indole derivative in anhydrous N, N-dimethylformamide (DMF) and stirred (at 0°C), and then warmed to room temperature; 2-chloropyrimidine is added, and the temperature is continuously increased to 150°C for reaction, to prepare an intermediate with formula A;

[0013] (2) the intermediate prepared in step (1), a phenylacetylene derivative, a first catalyst, a second catalyst, an additive and 1, 2-dichloroethane (DCE) are mixed and reacted at 25-60°C, to prepare the fluorescent dye with formula I;

[0014] When the fluorescent dye has formula II, the method comprises:

[0015]

[0016] (1) N-methyl-4-methylpyridine salt, anhydrous ethanol (EtOH) and the compound with formula B are mixed, and then piperidine is added;

[0017] (2) reaction at 60°C, to prepare the fluorescent dye with formula II;

[0018] When the fluorescent dye has formula III, the method comprises:

[0019]

[0020] (1) the compound with formula C, the compound with formula D and anhydrous EtOH are mixed, and then piperidine is added;

[0021] (2) reaction at 60°C, to prepare the fluorescent dye with formula III;

[0022] The compound having the structure as shown in formula B and the compound having the structure as shown in formula C are prepared by the method for preparing the fluorescent dye having the structure as shown in formula I.

[0023] Preferably, when the fluorescent dye has the structure as shown in formula I, in step (1) of the method, the molar ratio of the indole derivative, NaH and 2-chloropyrimidine is 1:1.5:1.5; the molar amount of the indole derivative to the volume of anhydrous DMF is 1 mmol:2.5 mL. Too large molar ratio of the indole derivative, NaH and 2-chloropyrimidine results in waste; too small molar ratio results in decreased conversion rate and reduced atom utilization. Too large volume of DMF results in waste; too small volume results in decreased yield and increased by-products.

[0024] Preferably, when the fluorescent dye has the structure as shown in formula I, in step (1) of the method, the stirring (0℃) time is 0.5 h, and the reaction time at 150℃ is 12 h. Because NaH has high reactivity, too high reaction temperature results in very intense reaction which is difficult to control; too low reaction temperature results in reduced reactivity, prolonged reaction time and incomplete reaction. Too short stirring time results in incomplete reaction and reduced yield; too long stirring time does not significantly improve the yield. Too short reaction time at 150℃ results in incomplete reaction and reduced yield; too long reaction time does not significantly improve the yield. If the reaction temperature at 150℃ is reduced to 130℃, the reaction time needs to be prolonged to 24 h.

[0025] Preferably, when the fluorescent dye has the structure as shown in formula I, in step (2) of the method, the molar ratio of the intermediate, the phenylacetylene derivative, the first catalyst, the second catalyst and the additive is 1:1:0.05:0.1:0.5; the molar amount of the intermediate to the volume of DCE is 1 mmol:5 mL. Too large molar ratio of the intermediate, the phenylacetylene derivative, the first catalyst, the second catalyst and the additive results in waste; too small molar ratio results in decreased conversion rate. If the DCE is replaced by other solvents, the yield is reduced, and even the reaction does not occur. Too large volume of DCE results in waste; too small volume results in decreased yield and increased by-products.

[0026] Preferably, when the fluorescent dye has the structure as shown in formula I, in step (2) of the method, the reaction time at 25-60℃ is 10 min-12 h; the first catalyst is Cp*Co(CO)I2, and the second catalyst is AgSbF6; the additive is trimethylacetic acid or sodium acetate. If other catalysts and additives are used in the present application, the yield is significantly reduced, and even the reaction does not occur.

[0027] Preferably, when the fluorescent dye has the structure shown in Formula II, in step (1) of the method, the molar ratio of the compound having the structure shown in Formula B to N-methyl-4-methylpyridinium salt is 1:1.1; the molar ratio of the compound B to anhydrous EtOH is 1 mmol:(10-15) mL. In step (2) of the method, the reaction time at 60°C is 10-12 h.

[0028] N-methyl-4-methylpyridinium salt is cheap and easy to obtain compared with the compound having the structure shown in Formula B, so it is slightly excessive to improve the yield of the product. Too much volume of the anhydrous EtOH will cause waste; too little volume will lead to insufficient dissolution and reduce the reaction yield. The reaction temperature (60°C) is too low, the solubility of the raw material is reduced, and the reaction yield is reduced; the temperature is too high, and more by-products are generated. The reaction time at 60°C is too short, and the reaction is not completely performed, and the yield is reduced; the reaction time is too long, and the yield is not obviously improved.

[0029] Preferably, when the fluorescent dye has the structure shown in Formula III, in step (1) of the method, the molar ratio of the compound having Formula C to the compound having Formula D is 1:1; the molar ratio of the compound having Formula C to anhydrous EtOH is 1 mmol:12.5 mL; in step (2) of the method, the reaction time at 60°C is 11 h. The reaction temperature (60°C) is too low, the solubility of the raw material is reduced, and the reaction yield is reduced; the temperature is too high, and more by-products are generated. The reaction time at 60°C is too short, and the reaction is not completely performed, and the yield is reduced; the reaction time is too long, and the yield is not obviously improved.

[0030] The application further provides application of the fluorescent dye having an alkenyl indole as a mother nucleus in a fluorescent probe.

[0031] Preferably, when the fluorescent dye has the structure shown in Formula III, the detection of ONOO- is specific.

[0032] The fluorescent dye having an alkenyl indole as a mother nucleus, the preparation and application thereof, solve the problem that the prior art cannot effectively extend the indole conjugated skeleton, leading to poor application of indole in the field of fluorescent dyes, and have the following advantages:

[0033] 1. The fluorescent dye having an alkenyl indole as a mother nucleus prepared by the application has simple and easily obtained raw materials and simple operation process.

[0034] 2. The fluorescent dye having an alkenyl indole as a mother nucleus prepared by the application can cover the visible light region to the near-infrared region, has good luminescent performance and large Stokes shift, and the fluorescent dye having the structure shown in Formula III prepared by the application can realize detection of ONOO-- specific detection.

[0035] 3. The alkenyl indole-based fluorescent dye prepared by the method is a new functional fluorescent dye, which widens the range of fluorescent dyes. BRIEF DESCRIPTION OF DRAWINGS

[0036] Figure 1 Absorption spectra of the fluorescent probe III of the present application before and after reacting with different active substances.

[0037] Figure 2 Fluorescence spectra of the fluorescent probe III of the present application before and after reacting with different active substances.

[0038] Figure 3 Fluorescence spectra of the fluorescent probe III of the present application before and after reacting with different metal ions.

[0039] Figure 4 Changes of the fluorescence spectra of the fluorescent probe III of the present application with the increase of ONOO.

[0040] Figure 5 A graph showing the changes of the fluorescence intensity of the fluorescent probe III of the present application at 635 nm with the concentration of ONOO.

[0041] Figure 6 Cytotoxicity of the fluorescent probe III of the present application.

[0042] Figure 7 Endogenous ONOO — Cell imaging. DETAILED DESCRIPTION

[0043] The technical solutions in the embodiments of the present application will be described clearly and completely below. Obviously, the described embodiments are only some of the embodiments of the present application, but not all the embodiments. Based on the embodiments in the present application, all the other embodiments obtained by those skilled in the art without creative work fall within the protection scope of the present application.

[0044] Embodiment 1

[0045] A synthesis method of an alkenyl indole-based fluorescent dye 2a, which comprises the following steps:

[0046]

[0047] (1) Indole and NaH (molar ratio of 1:1.5) were added to anhydrous DMF (molar ratio of indole derivative to anhydrous DMF was 1 mmol: 2.5 mL) at 0 °C under argon protection and stirred for 30 min (at 0 °C), and then slowly returned to room temperature; after adding 2-chloropyrimidine (molar ratio of indole to 2-chloropyrimidine was 1:1.5), the temperature was raised to 150 °C and stirring was continued for 12 h, and then cooled to room temperature; after extraction with ethyl acetate for 3 times, the organic phase was combined, and the solvent was removed under reduced pressure, and the residue was separated by silica gel column chromatography to obtain white solid 1a with a yield of 95%.

[0048] (2) N-pyrimidyl indole 1a synthesized in step (1) was stirred with 4-ethynylbenzonitrile (molar ratio of 1a to 4-ethynylbenzonitrile was 1:1) in the presence of 5.0 mol% Cp*Co(CO)I2, 10.0 mol% AgSbF6 and 0.5 equiv NaOAc in DCE (ratio of 1 mmol of 1a to 5 mL of DCE) at 60 °C for 12 h. The solvent was removed under reduced pressure, and the residue was separated by silica gel column chromatography to obtain ginger-colored solid 2a with a yield of 51%.

[0049] The nuclear magnetic resonance characterization results of the above-mentioned fluorescent dye 2a with alkenyl indole as the core are as follows:

[0050] 1 H NMR (600 MHz, DMSO-d6) δ 9.10-9.02 (m, 2H), 8.22 (d, J = 8.3 Hz, 1H), 7.91 (d, J = 16.2 Hz, 1H), 7.88-7.84 (m, 2H), 7.79 (d, J = 8.2 Hz, 2H), 7.71 (d, J = 7.7 Hz, 1H), 7.58-7.56 (m, 1H), 7.39 (d, J = 16.2 Hz, 1H), 7.34-7.30 (m, 2H), 7.27 (t, J = 7.4 Hz, 1H). 13 C NMR (151 MHz, DMSO-d6) δ 159.11, 156.83, 141.84, 137.66, 136.87, 132.61, 128.50, 127.31, 127.08, 123.89, 123.73, 122.25, 120.53, 119.04, 118.52, 113.68, 109.40, 105.34. HRMS (ES+): Calculated for C 21 H 15 N4 + [M+H] +:323.1291, Found: 323.1281.

[0051] The spectral properties of 2a in different solvents were detected, and the test results are shown in Table 1:

[0052] Table 1 Spectral properties of 2a in different solvents

[0053]

[0054] In Table 1: λ abs max is the maximum absorption wavelength; λ em max is the maximum emission wavelength; ε is the maximum molar absorption coefficient; Φ is the fluorescence quantum yield; Stokes shift is λ em max -λ abs max .

[0055] Example 2

[0056] A method for synthesizing a fluorescent dye 2b with an alkenyl indole as the parent nucleus is basically the same as that in Example 1, with the difference being that:

[0057]

[0058] (1) The indole is changed to 6-cyanoindole of the same equivalent, and after the same operation as in Example 1, white solid 1b is prepared, with a yield of 72%.

[0059] (2) 1a (N-pyrimidyl indole) is changed to 1b of the same equivalent, 4-ethynylbenzonitrile is changed to phenylacetylene of the same equivalent, and sodium acetate is changed to trimethyl acetic acid of the same equivalent, and after stirring at 60°C for 12h, after the same operation as in Example 1, light yellow solid 2b is prepared, with a yield of 60%.

[0060] The nuclear magnetic characterization results of the above fluorescent dye 2b with an alkenyl indole as the parent nucleus are as follows:

[0061] 1 H NMR (600 MHz, DMSO-d6) δ 9.05 (d, J = 4.9 Hz, 1H), 8.54 (s, 1H), 7.78 (d, J = 8.2 Hz, 1H), 7.71 (d, J = 16.3 Hz, 1H), 7.60 - 7.56 (m, 3H), 7.54 (dd, J = 8.2, 1.4 Hz, 1H), 7.41 - 7.37 (m, 3H), 7.33 - 7.29 (m, 2H). 13C NMR (151 MHz, DMSO-d6) δ 159.32, 156.31, 142.20, 136.61, 135.39, 132.04, 131.84, 128.79, 128.25, 126.78, 124.84, 121.23, 120.25, 119.20, 119.15, 118.18, 104.27, 103.96. HRMS (ES+): Calcd for C 21 H 15 N4 + [M+H] + : 323.1291, Found: 323.1317.

[0062] The spectral properties of the above 2b in different kinds of solvents were detected, and the test results are shown in Table 2:

[0063] Table 2 Spectral properties of the above 2b in different kinds of solvents

[0064]

[0065] In Table 2: λ abs max is the maximum absorption wavelength; λ em max is the maximum emission wavelength; ε is the maximum molar absorption coefficient; Φ is the fluorescence quantum yield; Stokes shift is λ em max - λ abs max .

[0066] Example 3

[0067] A method for synthesizing a fluorescent dye 2c with an alkenyl indole as the parent nucleus is basically the same as that in Example 1, with the difference being that:

[0068]

[0069] (1) The indole is changed to the same equivalent of indole-6-carboxaldehyde, and after the same operation as in Example 1, white solid 1c is prepared in a yield of 62%.

[0070] (2) 1a is changed to the same equivalent of 1c, and 4-ethynylbenzonitrile is changed to the same equivalent of 4-methoxyphenylacetylene, and after stirring at 60°C for 6h, the same operation as in Example 1 is performed to prepare yellow solid 2c in a yield of 72%.

[0071] The nuclear magnetic characterization results of the above fluorescent dye 2c with an alkenyl indole as the parent nucleus are as follows:

[0072] 1H NMR (600 MHz, DMSO-d6) δ 10.00 (s, 1H), 9.07 (d, J = 4.8 Hz, 2H), 8.59 (s, 1H), 7.77 (d, J = 8.1 Hz, 1H), 7.72 (dd, J = 8.2, 1.3 Hz, 1H), 7.59 (t, J = 4.8 Hz, 1H), 7.57 - 7.52 (m, 3H), 7.37 (d, J = 16.3 Hz, 1H), 7.25 (s, 1H), 6.96 (d, J = 8.8 Hz, 2H), 3.78 (s, 3H). 13 C NMR (151 MHz, DMSO-d6) δ 192.42, 159.42, 159.28, 156.47, 143.06, 136.09, 133.71, 131.57, 131.31, 129.30, 128.18, 122.65, 120.32, 119.12, 116.82, 116.19, 114.24, 103.17, 55.16. HRMS (ES+): Calcd for C 22 H 18 N3O2 + [M+H] + : 356.1394, Found: 356.1382.

[0073] The spectral properties of the above 2c in different kinds of solvents were detected, and the test results are shown in Table 3:

[0074] Table 3 Spectral properties of the above 2c in different kinds of solvents

[0075]

[0076] In Table 3: λ abs max is the maximum absorption wavelength; λ em max is the maximum emission wavelength; ε is the maximum molar absorption coefficient; Φ is the fluorescence quantum yield; Stokes shift is λ em max - λ abs max .

[0077] Example 4

[0078] A synthesis method of a fluorescent dye 2d with alkenyl indole as the parent nucleus is basically the same as that in Example 1, and the difference is that:

[0079]

[0080] (1) Change 1a to 1c of the same equivalent, 4-ethynylbenzonitrile to 4-ethynylbenzaldehyde of the same equivalent, stir at 60 °C for 1 h, and then the same operation as Example 1 to prepare orange-yellow solid 2d, yield 90%.

[0081] The nuclear magnetic characterization results of the above fluorescent dye 2d with alkenyl indole as the core are as follows:

[0082] 1 H NMR (600 MHz, DMSO-d6) δ 8.91 (s, 1H), 8.88 (s, 1H), 7.96 (d, J = 4.8 Hz, 2H), 7.56 (s, 1H), 6.82-6.76 (m, 3H), 6.71-6.67 (m, 3H), 6.63 (dd, J = 8.2, 1.3 Hz, 1H), 6.49 (t, J = 4.8 Hz, 1H), 6.38 (d, J = 16.3 Hz, 1H), 6.28 (s, 1H). 13 C NMR (151 MHz, DMSO-d6) δ 192.57, 192.36, 159.35, 156.40, 142.63, 141.94, 136.35, 135.34, 133.40, 131.94, 130.07, 130.02, 127.27, 122.83, 122.66, 120.90, 119.16, 116.61, 105.09. HRMS (ESI+): Calculated for C 22 H 15 N3O2Na + [M+Na] + : 376.1062, Found: 376.1049.

[0083] The spectral properties of the above 2d in different kinds of solvents were detected, and the test results are shown in Table 4:

[0084] Table 4 Spectral properties of the above 2d in different kinds of solvents

[0085]

[0086] Table 4: λ abs max is the maximum absorption wavelength; λ em max is the maximum emission wavelength; ε is the maximum molar absorption coefficient; Φ is the fluorescence quantum yield; Stokes shift is λ em max - λ abs max .

[0087] Example 5

[0088] The synthesis method of the fluorescent dye 2e with alkenyl indole as the parent nucleus is basically the same as that of Example 1, except that:

[0089]

[0090] (1) The indole is changed to the same equivalent of 6-methoxyindole, and after the same operation as Example 1, white solid 1d is prepared in a yield of 70%.

[0091] (2) 1a is changed to the same equivalent of 1d, and 4-ethynylbenzonitrile is changed to the same equivalent of 4-ethynylbenzaldehyde, and after stirring at 60°C for 3h, the same operation as Example 1 is performed to prepare orange solid 2e in a yield of 77%.

[0092] The nuclear magnetic resonance characterization results of the above-mentioned fluorescent dye 2e with alkenyl indole as the parent nucleus are as follows:

[0093] 1 H NMR (400 MHz, CDCl3) δ 9.96 (s, 1H), 8.85 (d, J = 4.6 Hz, 2H), 7.93 (s, 1H), 7.89-7.78 (m, 3H), 7.59 (d, J = 7.8 Hz, 2H), 7.48 (d, J = 8.6 Hz, 1H), 7.21 (t, J = 4.6 Hz, 1H), 7.09 (d, J = 16.2 Hz, 1H), 7.02 (s, 1H), 6.89 (d, J = 8.6 Hz, 1H), 3.88 (s, 3H). 13 C NMR (100 MHz, CDCl3) δ 191.62, 158.34, 157.82, 143.81, 138.52, 137.02, 134.95, 130.20, 126.76, 126.48, 124.26, 123.31, 121.11, 117.26, 111.85, 106.58, 98.50, 55.71. HRMS (ESI+): Calculated for C 22 H 16 N3O2Na + [M+Na] + : 378.1218, Found: 378.1213.

[0094] The spectral properties of the above-mentioned 2e in different kinds of solvents are detected, and the test results are shown in Table 5:

[0095] Table 5 Spectral properties of the above-mentioned 2e in different kinds of solvents

[0096]

[0097] Table 5: λ abs max λmaxis the maximum absorption wavelength; λ em max λmaxis the maximum emission wavelength; εmaxis the molar extinction coefficient; Φ is the fluorescence quantum yield; Stokes shift is λ em max λmaxis the maximum absorption wavelength; λ abs max .

[0098] Example 6

[0099] The synthesis method of the fluorescent dye 3a with alkenyl indole as the mother nucleus is basically the same as that of Example 1, except that:

[0100]

[0101] (1) 4-ethynylbenzonitrile is changed to 4-ethynylbenzaldehyde of the same equivalent, stirred at 60°C for 0.5h, and then the same operation as in Example 1 is performed to prepare yellow solid 2f with a yield of 92%.

[0102] (2) N-methyl-4-methylpyridine salt and 2f prepared in step (1) in a molar ratio of 1.1:1 are added to anhydrous EtOH, and the molar ratio of 2f to anhydrous EtOH is 1 mmol: 10 mL. A few drops of piperidine are added, and after reaction at 60°C for 10h, it is cooled to room temperature. After suction filtration, washing with DCM, the filter residue is separated by silica gel column chromatography to obtain orange-red solid 3a with a yield of 42%.

[0103] The nuclear magnetic resonance characterization results of the above fluorescent dye 3a with alkenyl indole as the mother nucleus are as follows:

[0104] 1 H NMR (600 MHz, DMSO-d6) δ 8.99 (d, J = 4.8 Hz, 2H), 8.81 (d, J = 6.8 Hz, 2H), 8.17 (d, J = 6.9 Hz, 2H), 8.15-8.09 (m, 1H), 7.97 (d, J = 16.3 Hz, 1H), 7.79-7.69 (m, 3H), 7.64-7.60 (m, 3H), 7.53-7.45 (m, 2H), 7.29 (d, J = 16.2 Hz, 1H), 7.25-7.13 (m, 3H), 4.21 (s, 3H). 13C NMR (151 MHz, DMSO-d6) δ 159.67, 157.45, 152.99, 145.58, 140.74, 139.78, 139.25, 138.69, 137.39, 134.90, 129.20, 129.02, 127.68, 123.98, 123.48, 122.75, 122.19, 120.92, 119.10, 114.13, 113.47, 105.67, 105.24, 47.43. HRMS (ES+): Calcd for C 28 H 23 N4 + [M] + : 415.1917, Found: 415.1931.

[0105] The spectral properties of the above 3a in different kinds of solvents were detected, and the test results are shown in Table 6:

[0106] Table 6 Spectral properties of the above 3a in different kinds of solvents

[0107]

[0108] Table 6: λ abs max is the maximum absorption wavelength; λ em max is the maximum emission wavelength; ε is the maximum molar absorption coefficient; Φ is the fluorescence quantum yield; Stokes shift is λ em max - λ abs max .

[0109] Example 7

[0110] A method for synthesizing a fluorescent dye 3b with alkenyl indole as the parent nucleus is basically the same as that in Example 1, with the difference being that:

[0111]

[0112] (1) The indole is changed to the same equivalent of indole-3-carboxylic acid methyl ester, which is prepared into a white solid 1e after the same operation as in Example 1, with a yield of 47%.

[0113] (2) 1a is changed to the same equivalent of 1e (prepared in step (1)), and 4-ethynylbenzonitrile is changed to the same equivalent of 4-ethynylbenzaldehyde, which is stirred at 60°C for 12h, and then prepared into a color solid C after the same operation as in Example 1, with a yield of 32%.

[0114] (3) The substance amount ratio of C to N-methyl-4-methylpyridinium salt is 1:1.1, which is added to anhydrous EtOH, the substance amount and volume ratio of C to anhydrous EtOH is 1 mmol: 15 mL, a few drops of piperidine are added, and after reaction at 60°C for 12 h, it is cooled to room temperature; after suction filtration, it is washed with DCM, and the filter residue is collected, which is separated by silica gel column chromatography to obtain orange solid 3b in a yield of 62%.

[0115] The nuclear magnetic resonance characterization results of the above fluorescent dye 3b with alkenyl indole as the core are as follows:

[0116] 1 H NMR (600 MHz, DMSO-d6) δ 9.07 (d, J = 4.9 Hz, 2H), 8.85 (d, J = 6.7 Hz, 2H), 8.21 (d, J = 6.8 Hz, 2H), 8.13-8.12 (m, 1H), 7.99-7.91 (m, 2H), 7.73 (d, J = 8.3 Hz, 2H), 7.69 (t, J = 4.9 Hz, 1H), 7.63-7.62 (m, 1H), 7.56-7.48 (m, 3H), 7.36-7.29 (m, 2H), 6.40 (d, J = 16.6 Hz, 1H), 4.26 (s, 3H), 3.94 (s, 3H). 13 C NMR (151 MHz, DMSO-d6) δ 165.34, 160.39, 157.21, 152.82, 145.58, 142.53, 140.40, 138.48, 137.62, 135.88, 135.61, 129.21, 127.85, 126.69, 125.18, 124.07, 124.05, 123.80, 122.06, 121.40, 119.37, 112.43, 108.54, 51.95, 47.47. HRMS (ES+): Calculated for C 30 H 25 N4O2 + [M] + : 473.1972, Found: 473.1963.

[0117] The spectral properties of the above 3b in different kinds of solvents are detected, and the test results are shown in Table 7:

[0118] Table 7 Spectral properties of the above 3b in different kinds of solvents

[0119]

[0120] Table 7: λ abs max is the maximum absorption wavelength; λem max Maximum emission wavelength; ε maximum molar absorption coefficient; Φ is the fluorescence quantum yield; Stokes shift is λ em max - λ abs max .

[0121] Example 8

[0122] A method for synthesizing a fluorescent probe III with an alkenyl indole as a parent nucleus, the method comprising:

[0123]

[0124] (1) Synthesize compound D according to a literature report (Analytical Chemistry, 2018, 90(17), 10152-1015).

[0125] (2) Add C (prepared in Example 9) and D (prepared in step (1)) in a molar ratio of 1:1 to anhydrous EtOH, the molar amount of C to the volume of anhydrous EtOH is 1 mmol: 12.5 mL, add a few drops of piperidine, cool to room temperature after reacting at 60°C for 11 h, and collect the filter residue after suction filtration. The filter residue is separated by silica gel column chromatography to obtain dark red solid III with a yield of 27%.

[0126] The nuclear magnetic resonance characterization results of the above fluorescent probe III are as follows:

[0127] 1 H NMR (600 MHz, DMSO-d6) δ 9.08-9.06 (m, 4H), 8.27 (d, J = 6.9 Hz, 2H), 8.13 (d, J = 3.6 Hz, 3H), 8.02 (d, J = 16.3 Hz, 1H), 7.94 (d, J = 16.8 Hz, 1H), 7.85 (d, J = 8.1 Hz, 2H), 7.74 (d, J = 8.4 Hz, 2H), 7.70 (t, J = 4.9 Hz, 1H), 7.64-7.62 (m, 1H), 7.57-7.50 (m, 3H), 7.47 (d, J = 8.1 Hz, 2H), 7.37-7.30 (m, 2H), 6.38 (d, J = 16.7 Hz, 1H), 5.77 (s, 2H), 3.94 (s, 3H). 13C NMR (151 MHz, DMSO-d6) δ 164.79, 159.84, 156.66, 153.16, 144.36, 141.97, 140.50, 138.07, 137.07, 136.15, 135.32, 135.05, 134.83, 128.77, 127.48, 127.32, 126.15, 124.64, 124.22, 123.51, 123.26, 121.52, 120.84, 118.91, 111.90, 108.02, 62.27, 51.40. HRMS (ES+): Calcd for C 36 H 30 BN4O4 + [M] + : 593.2355, Found: 593.2382.

[0128] Spectra of fluorescent probe III and different active substances

[0129] The absorption spectrum and fluorescence spectrum of fluorescent probe III before and after the action of different active substances, and the specific experimental process is as follows: the fluorescent probe III prepared in Example 10 is dissolved in a phosphate buffer solution (PBS, pH is 7.2-7.4, concentration is 10 mM) containing 40% EtOH, and is configured into a 10 μM fluorescent probe III stock solution, and is evenly divided into 14 parts; 10 times the amount of different active substances (i.e. the concentration of active substances is 100 μM, and the active substances include H2O2, O2 - , ·OH, SO3 2- , S2O3 2- , Hcy, GSH, ClO - , ONOO - , t BuOOH, S 2- , HSO3 - , S2O5 2- and Cys) are added to each stock solution, and the absorption spectrum and fluorescence emission spectrum are measured after mixing. The excitation wavelength of the fluorescence spectrum is 420 nm, and the excitation and emission slit width is 1.5 nm.

[0130] As Figure 1 shown, the absorption spectrum of the fluorescent probe III before and after the action of different active substances, wherein the abscissa is wavelength (nm); the ordinate is absorbance. As Figure 1 can be seen, when no other active substance is added, the maximum absorption wavelength of the fluorescent probe III is about 420 nm. When ClO - and ONOO -When other active substances were added, the absorption spectrum of fluorescent probe III did not show significant changes. However, the addition of ClO... - Afterwards, the intensity of the absorption peak at 412 nm in its absorption spectrum decreased significantly; while with the addition of ONOO - Subsequently, its maximum absorption peak shifted from 412 nm to 307 nm.

[0131] like Figure 2 The image shows the fluorescence spectra of the fluorescent probe III of this invention before and after interaction with different active substances, where the horizontal axis represents wavelength (nm) and the vertical axis represents relative intensity. Figure 2 It can be seen that when other active substances besides ONOO￣ are added, the fluorescence spectrum of fluorescent probe III does not show significant changes. However, when ONOO￣ is added, the intensity of its emission peak at 635 nm decreases significantly.

[0132] Experimental Example 2: Spectra of the interaction between fluorescent probe III and different metal ions

[0133] The fluorescence spectra of fluorescent probe III before and after interaction with different metal ions were determined. The specific experimental procedure was as follows: Fluorescent probe III prepared in Example 10 was dissolved in a phosphate buffer solution (PBS, pH 7.2-7.4, concentration 10 mM) containing 20% ​​EtOH to prepare a 10 μM stock solution of fluorescent probe III, which was then divided into 10 equal portions. Ten equivalent amounts of different metal ions (i.e., the metal ion concentration was 100 μM, including K+) were added to each portion of the stock solution. + Na + Ag + Hg 2+ Ba 2+ Ca 2+ Fe 3+ Mn 2+ Ni 2+ and Pb 2+ After mixing thoroughly, the fluorescence emission spectrum was measured. The excitation wavelength for measuring the fluorescence spectrum was 400 nm, and the width of both the excitation and emission slits was 1.5 nm.

[0134] like Figure 3 The image shows the fluorescence spectra of the fluorescent probe III of this invention before and after interaction with different metal ions, where the horizontal axis represents wavelength (nm) and the vertical axis represents relative intensity. Figure 3 It can be seen that the fluorescence spectrum of fluorescent probe III did not change significantly when different metal ions were added. However, when ONOO￣ was added, the intensity of its emission peak at 635 nm decreased significantly.

[0135] Experimental Example 3: Spectral response of fluorescent probe III to ONOO￣

[0136] The absorption spectrum and fluorescence spectrum of the fluorescent probe III before and after reacting with different active substances. The specific experimental process is as follows: the fluorescent probe III prepared in Example 10 is dissolved in a phosphate buffer solution (PBS, pH 7.2-7.4, concentration 10 mM) containing 20% EtOH to prepare a stock solution of 10 μM fluorescent probe III. Different amounts of ONOO- are added to each stock solution to make the concentration of ONOO- 0-140 μM, and the fluorescence emission spectrum is measured after mixing. The excitation wavelength for measuring the fluorescence spectrum is 420 nm, and the excitation and emission slit widths are both 1.5 nm.

[0137] As shown in Figure 4 , the change of the fluorescence spectrum of the fluorescent probe III of the present application with the increase of ONOO- is shown, wherein the abscissa is wavelength (nm); and the ordinate is relative intensity. It can be seen from Figure 4 that the emission peak intensity at 635 nm gradually decreases with the increase of the amount of ONOO- added.

[0138] As shown in Figure 5 , the change of the fluorescence intensity of the fluorescent probe III of the present application at 635 nm with the concentration of ONOO- is shown, wherein the abscissa is the concentration of ONOO- (μM); and the ordinate is the relative fluorescence emission intensity of the fluorescent probe III at 635 nm. It can be seen from Figure 5 that the fluorescence intensity of the fluorescent probe III at 635 nm is linearly related to the concentration of ONOO-.

[0139] Experimental Example 4: Investigation of the cytotoxicity of the fluorescent probe III

[0140] The specific experimental process of the cytotoxicity of the fluorescent probe III is as follows: Raw264.7 cells Figure 6 (a) and HeLa cells Figure 6 (b) are subcultured in DMEM medium containing 10% FBS (V / V) and placed in a cell incubator with 5% CO2, 95% air and 37°C for proliferation culture; they are transferred to a 96-well plate (8000 cells / well) for culture for 24 h to make the cells adhere; then, different concentrations of the fluorescent probe III are added to the cells (to make the concentration 0, 5, 10, 15, 20, 25, 30, 35, 40, 50 μM), and the cells are cultured for another 24 h; 1 h later, the absorbance is measured at 450 nm wavelength using an enzyme marker. The control group is cultured under the same conditions without the fluorescent probe III, and only CCK-8 solution (5.0 mg / mL, 10 μL) is added to each well.

[0141] As shown in Figure 6As shown, the cytotoxicity of the fluorescent probe III of this invention is illustrated, where the horizontal axis represents the concentration of the added fluorescent probe III; the vertical axis represents cell viability; a) cytotoxicity graph of Raw264.7 cells, b) cytotoxicity graph of HeLa cells. Figure 6 It is known that fluorescent probe III has low cytotoxicity.

[0142] Experimental Example 5

[0143] The specific experimental procedure for endogenous ONOO￣ cell imaging using fluorescent probe III was as follows: Morphologically sound HeLa cells were digested and placed in confocal dishes, allowing them to adhere and grow for 24 hours at a cell count of 200,000 cells / dish. The cells were then divided into three groups: Group 1 was a blank control, without fluorescent probe III; Group 2 was probe III group, with fluorescent probe III added alone at a concentration of 5 μM; and Group 3 was lipopolysaccharide + probe III group, with LPS added (at a concentration of 1 μg / mL). After 6 hours of further culture, the confocal dishes were removed, washed three times with PBS, and fluorescent probe III (at a concentration of 5 μM) was added, followed by 40 minutes of further culture and washing three times with PBS. Confocal imaging was performed on all three groups of cells, with an excitation wavelength of 488 nm and a receiving window of 570-620 nm.

[0144] like Figure 7 As shown, the endogenous ONOO of the present invention — Cell imaging. (a)-(c) are the blank group, without fluorescent probe III and LPS; (d)-(f) are the probe III group, incubated with 5 μM fluorescent probe III for 40 min; (g)-(i) are the lipopolysaccharide + probe III group, incubated with 1 μg / mL LPS for 6 h, then incubated with 5 μM fluorescent probe III for 40 min. Bright field channels are (a), (d), and (g); fluorescence channels are (b), (e), and (h); overlay images are (c), (f), and (i); λ ex =488nm, λ em =570-620nm, scale bar: 20μm. (From...) Figure 7 (a)-(c) No fluorescence was observed in the cells without the addition of fluorescent probe III. Figure 7 (d)-(f) show that when fluorescent probe III is added alone, the cells exhibit bright fluorescence. From Figure 7 (g)-(i) revealed that the fluorescence intensity of cells stimulated with LPS was significantly lower than that of cells stimulated with fluorescent probe III alone. This is because LPS stimulation induces the production of endogenous ONOO￣, which quenches the fluorescence of fluorescent probe III.

[0145] While the application has been described in detail and with reference to specific preferred embodiments thereof, it will be apparent to one skilled in the art that various modifications and alternatives can be employed without departing from the spirit and scope of the application. Accordingly, the scope of the application should be determined by the appended claims and their equivalents.

Claims

1. A fluorescent dye having an alkenylindole as a parent nucleus, characterized in that, The fluorescent dye has a structure as shown in formula III; 。 2. A process for the preparation of a fluorescent dye having an alkenylindole as a parent nucleus according to claim 1, characterized in that, The method comprises: ; (1) mixing a compound having formula C, a compound having formula D and anhydrous ethanol, and adding piperidine; (2) reacting at 60 DEG C to prepare the fluorescent dye having a structure as shown in formula III.

3. The preparation method according to claim 2, characterized in that, In step (1) of the method, the mass ratio of the compound having formula C and the compound having formula D is 1:1; the mass / volume ratio of the compound having formula C and anhydrous ethanol is 1 mmol:12.5 mL; in step (2) of the method, the reaction is carried out at 60 DEG C for 11 hours.

4. Use of the fluorescent dye having an alkenyl indole as a mother nucleus according to claim 1 in the preparation of a fluorescent probe.

5. Use according to claim 4, characterized in that, The fluorescent dye is specific for the detection of ONOO - .

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