Fluorescent probe as well as preparation method and application thereof

Abstract

The invention discloses a fluorescent probe as well as a preparation method and application thereof. The invention provides a series of fluorescent probes with novel structures, and the fluorescent probes take 2-(isoquinoline-1-yl) pyrrole boron difluoride as fluorophores, so that the fluorescence quantum yield of the probes is improved, the detection resolution of nitric oxide is increased, and the detection sensitivity is improved. The fluorescent probe disclosed by the invention has the characteristics of large Stokes displacement, high fluorescence quantum yield, good selectivity and short response time.

Description

Technical Field

[0001] This invention relates to the field of organic synthesis technology, and in particular to a fluorescent probe, its preparation method, and its application. Background Technology

[0002] Nitric oxide (NO), as a gaseous signaling molecule, plays a crucial role in various physiological processes in organisms, including inflammation regulation, vasodilation, and neurotransmission. In vivo, NO is primarily produced by three nitric oxide synthases: endothelial nitric oxide synthase (eNOS), neuronal nitric oxide synthase (nNOS), and inducible nitric oxide synthase (iNOS). Under pathological conditions, these three synthases can lead to abnormal NO levels, subsequently inducing the production of other reactive oxygen species (ROS) and reactive nitrogen species (RNS). These substances are closely related to tumors, arthritis, diabetes, and neurodegenerative diseases. Abnormal intracellular NO levels have been reported to be an important marker for various diseases, including diabetes, atherosclerosis, and Alzheimer's disease.

[0003] In recent years, fluorescent probes for nitric oxide (NO) have received continuous attention from researchers. Their response mechanisms include o-phenylenediamine cyclization, N-nitrosation of aromatic secondary amines, and oxidation of dihydropyridine. Based on these response mechanisms, researchers have designed and synthesized a series of fluorescence-enhanced nitric oxide fluorescent probes. However, these probes suffer from problems such as long response time and small Stokes shift in the detection process of nitric oxide (NO), which are not conducive to the in vitro and in vivo analysis and detection of nitric oxide (NO).

[0004] Therefore, it is urgent to develop a new type of fluorescent probe with high selectivity and high sensitivity for detecting intracellular NO levels. Summary of the Invention

[0005] The present invention aims to at least solve one of the technical problems existing in the prior art. To this end, the first aspect of the present invention provides a fluorescent probe with high Stokes shift, high sensitivity, and high selectivity.

[0006] A second aspect of the present invention also provides a method for preparing a fluorescent probe.

[0007] A third aspect of the present invention also provides an application of a fluorescent probe.

[0008] According to a first aspect of the present invention, a fluorescent probe is provided having a structural formula of formula (I) or formula (II):

[0009]

[0010] Wherein, R1 is H or selected from the following structural formulas:

[0011]

[0012] R2 is selected from the following structural formula:

[0013]

[0014] Among them, R3 and R6 are independently selected from H or C. 1～10 Alkyl groups;

[0015] R4 and R5 are independently selected from H and C. 1～10 Alkyl, phenyl or halogenated, C 1～6 alkyl, nitro, hydroxyl, C 1～6 Halogenated alkyl, C 1～6 alkoxy-substituted phenyl groups.

[0016] According to a preferred embodiment of the present invention, R4 and R5 are independently selected from H and C. 1～6 Alkyl, phenyl or halogenated, C 1～3 alkyl, C 1～3 Halogenated alkyl, C 1～3 alkoxy-substituted phenyl groups.

[0017] According to a preferred embodiment of the present invention, the R2 groups defined above can all recognize nitric oxide (NO), and therefore all have good NO fluorescence detection effect.

[0018] According to a preferred embodiment of the present invention, the fluorescent probe is selected from the following structural formulas:

[0019]

[0020] The fluorescent probe according to embodiments of the present invention has at least the following beneficial effects:

[0021] This invention provides a series of novel fluorescent probes that use 2-(isoquinoline-1-yl)pyrrole boron difluoride as a fluorophore to improve the fluorescence quantum yield of the probe and increase the detection resolution of nitric oxide. The fluorescent probes of this invention have the characteristics of large Stokes shift, high fluorescence quantum yield, good selectivity and short response time.

[0022] According to a second aspect of the present invention, a method for preparing a fluorescent probe as described in the first aspect of the present invention is provided, comprising the following steps:

[0023] The compound is obtained by reacting compound 1, boron trifluoride diethyl ether, an organic base and a solvent;

[0024] The structural formula of compound 1 is as follows:

[0025]

[0026] The definitions of R1 and R2 in this invention are the same as those in the foregoing.

[0027] According to a preferred embodiment of the present invention, the organic base includes at least one of anhydrous triethylamine, potassium carbonate, sodium carbonate, and cesium carbonate.

[0028] According to a preferred embodiment of the present invention, the solvent includes at least one of toluene, tetrahydrofuran, 1,4-dioxane, and N,N-dimethylformamide.

[0029] According to a preferred embodiment of the present invention, the reaction temperature is 30°C to 100°C.

[0030] According to a preferred embodiment of the present invention, the reaction time is 6h to 16h.

[0031] According to a preferred embodiment of the present invention, the molar ratio of compound 1 to boron trifluoride ether is 1:(1-12).

[0032] A third aspect of the present invention provides an application of the fluorescent probe described above in the detection of nitric oxide content.

[0033] Other features and advantages of the invention will be set forth in the description which follows, and will be apparent in part from the description, or may be learned by practicing the invention. Attached Figure Description

[0034] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which:

[0035] Figure 1 This is a high-resolution mass spectrum of probe Y-4 prepared in Example 1 of the present invention;

[0036] Figure 2 This is a high-resolution mass spectrum of probe Y-2-6 prepared in Example 2 of this invention;

[0037] Figure 3 This is the ultraviolet absorption spectrum of the probe Y-4 prepared in Example 1 of this invention in response to NO;

[0038] Figure 4 This is a time-dependent fluorescence intensity diagram of the probe Y-4 prepared in Example 1 of this invention in response to NO;

[0039] Figure 5 This is the UV absorption spectrum of the probe Y-2-6 prepared in Example 2 of this invention in response to NO;

[0040] Figure 6This is a time-dependent fluorescence intensity diagram of the probe Y-2-6 prepared in Example 2 of this invention in response to NO;

[0041] Figure 7 This is a selective fluorescence image of probe Y-4 prepared in Example 1 of this invention. Detailed Implementation

[0042] The following are specific embodiments of the present invention, and the technical solutions of the present invention will be further described in conjunction with the embodiments, but the present invention is not limited to these embodiments.

[0043] Unless otherwise specified, the reagents, methods and equipment used in this invention are all conventional reagents, methods and equipment in this technical field.

[0044] Example 1

[0045] This example provides a fluorescent probe, the reaction equation of which and its preparation method are as follows:

[0046]

[0047] (1) Synthesis of compound Y-1

[0048] 1,4-Dichloroisoquinoline (1.98 g, 10 mmol) and 1-Boc-pyrrole-2-boronic acid (2.53 g, 12 mmol) were dissolved in dry THF (50 mL). Then, 2 M K2CO3 (17 mL) and tetrakis(triphenylphosphine)palladium (0.35 g, 0.3 mmol) were added at room temperature, followed by nitrogen purging. The mixture was reacted at 80 °C for 12 h, extracted three times with CH2Cl2 and H2O, dried over anhydrous Mg2SO4, concentrated and dried, and then separated by column chromatography to obtain a light yellow solid Y-1 (2.6 g, 80%).

[0049] 1 H NMR(500MHz,Chloroform-d)δ8.59(s,1H),8.21(d,J=8.4Hz,1H),7.81-7.71(m,2H),7.57(t,J=7.3H z,1H),7.48(dd,J=1.6,3.3Hz,1H),6.47(dd,J=1.6,3.3Hz,1H),6.36(t,J=3.3Hz,1H),0.97(s,9H).

[0050] (2) Synthesis of compound Y-2

[0051] Compound Y-1 (164 mg, 0.5 mmol) and 4-(N-methylamino)phenylboronic acid pinacol ester (234 mg, 1 mmol) were dissolved in dry THF (10 mL). Then, 2 M K2CO3 (1 mL), palladium acetate (22.5 mg, 0.1 mmol), and 2-dicyclohexylphospho-2′,4′,6′-triisopropylbiphenyl (95.3 mg, 0.2 mmol) were added at room temperature. After purging with nitrogen, the mixture was reacted at 80 °C for 14 h. The mixture was extracted three times with CH2Cl2 and H2O, dried over anhydrous Mg2SO4, concentrated, and dried. The mixture was then separated by column chromatography to obtain a light yellow oil Y-2 (169 mg, 85%).

[0052] 1 H NMR(500MHz,Chloroform-d)δ8.51(s,1H),8.04(d,J=8.4Hz,1H),7.82(d,J=8.4Hz,1H),7.64(m,1H),7.55-7.50(m,2 H),7.36(d,J=8.4Hz,2H),6.77(d,J=8.4Hz,2H),6.55–6.52(m,1H),6.39(t,J=3.3Hz,1H),2.93(s,3H),0.96(s,9H).

[0053] (3) Synthesis of compound Y-3

[0054] Sodium methoxide (270 mg, 5 mmol) was dissolved in methanol (20 mL), followed by the addition of compound Y-2 (199 mg, 0.5 mmol) and THF (10 mL) at room temperature. After purging with nitrogen, the mixture was reacted at 50 °C for 2 h. The mixture was then extracted three times with saturated aqueous solutions of CH2Cl2 and NH4Cl, dried over anhydrous Mg2SO4, concentrated, and dried. The mixture was then separated by column chromatography to obtain a deep yellow solid Y-3 (135 mg, 90%).

[0055] 1 H NMR(500MHz,Chloroform-d)δ10.29(s,1H),8.85–8.80(m,1H),8.40(s,1H),8.07–8.03(m,1H),7.69– 7.61(m,2H),7.40–7.35(m,2H),7.09–7.02(m,2H),6.81–6.76(m,2H),6.48–6.44(m,1H),2.94(s,3H).

[0056] (4) Synthesis of probe Y-4

[0057] Compound Y-3 (210 mg, 0.7 mmol), boron trifluoride ether (994 mg, 7 mmol), and anhydrous triethylamine (424 mg, 4.2 mmol) were dissolved in toluene (15 mL), reacted at 30 °C for 14 h, extracted three times with CH2Cl2 and H2O, dried over anhydrous Mg2SO4, concentrated and dried, and the mixture was separated by column chromatography to obtain a deep yellow solid Y-4 (170 mg, 70%).

[0058] The proton and mass spectrometry data are as follows:

[0059] 1 ¹H NMR (500MHz, Chloroform-d) δ 8.61 (d, J = 8.5Hz, 1H), 8.08 (d, J = 8.5Hz, 1H), 8.02 (s, 1H), 7.87–7.83 (m, 1H), 7.80–7.76 (m, 1H), 7.35–7.32 (m, 2H), 7.31–7.29 (m, 1H), 7.13 (d, J = 3.3Hz, 1H), 6.79–6.75 (m, 2H), 6.50–6.48 (m, 1H), 2.95 (s, 3H). Its high-resolution mass spectrum is as follows: Figure 1 Shown: HRMS found m / z 347.1514,Calcd.for Y-4[M + ]347.1514.

[0060] Example 2

[0061] This example provides a fluorescent probe, the reaction equation of which and its preparation method are as follows:

[0062]

[0063] (1) Synthesis of compound Y-2-1

[0064] 1,4-Dichloroisoquinoline (1.98 g, 10 mmol) and 1-Boc-pyrrole-2-boronic acid (2.53 g, 12 mmol) were dissolved in dry THF (50 mL). Then, 2 M K2CO3 (17 mL) and tetrakis(triphenylphosphine)palladium (0.35 g, 0.3 mmol) were added at room temperature, followed by nitrogen purging. The mixture was reacted at 80 °C for 12 h, extracted three times with CH2Cl2 and H2O, dried over anhydrous Mg2SO4, concentrated and dried, and then separated by column chromatography to obtain a light yellow solid Y-2-1 (2.6 g, 80%).

[0065] 1H NMR(500MHz,Chloroform-d)δ8.59(s,1H),8.21(d,J=8.4Hz,1H),7.81-7.71(m,2H),7.57(t,J=7.3H z,1H),7.48(dd,J=1.6,3.3Hz,1H),6.47(dd,J=1.6,3.3Hz,1H),6.36(t,J=3.3Hz,1H),0.97(s,9H).

[0066] (2) Synthesis of compound Y-2-2

[0067] Y-2-1 (328 mg, 1.0 mmol) was dissolved in dry THF (20 mL), and NBS (188 mg, 1.05 mmol) was added in the dark. The mixture was stirred at -78 °C for 30 min, then heated to room temperature and stirred for 3.5 h. The mixture was extracted three times with CH2Cl2 and H2O, dried over anhydrous Mg2SO4, concentrated and dried, and then separated by chromatographic column chromatography to obtain a light yellow solid Y-2-2 (286 mg, 70%).

[0068] 1 H NMR(500MHz,Chloroform-d)δ8.59(s,1H),8.24(d,J=8.5Hz,1H),8.11(d,J=8.5Hz,1H),7.81(td,J=7 .5,1.3Hz,2H),7.64(td,J=7.5,1.3Hz,2H),6.50(d,J=3.6Hz,1H),6.43(d,J=3.6Hz,1H),1.10(s,9H);

[0069] (3) Synthesis of compound Y-2-3

[0070] Compound Y-2-2 (102 mg, 0.25 mmol) and triphenylamine 4-borate (145 mg, 0.5 mmol) were dissolved in dry THF (10 mL). Then, 2 M K2CO3 (1 mL) and tetrakis(triphenylphosphine)palladium (29 mg, 0.025 mmol) were added at room temperature. After purging with nitrogen, the mixture was reacted at 80 °C for 12 h. The mixture was extracted three times with CH2Cl2 and H2O, dried over anhydrous Mg2SO4, concentrated and dried, and then separated by column chromatography to obtain a light yellow oil Y-2-3 (140.2 mg, 98%).

[0071] 1H NMR(500MHz,Chloroform-d)δ8.56(s,1H),8.24-8.21(m,2H),7.79(td,J=7.5,1.3Hz,1H),7.62(td,J=7.5,1.3Hz,1H),7.31-7.28(m,2H ),7.26-7.22(m,4H),7.13-7.10(m,4H),7.08-7.05(m,2H),7.03-6.99(m,2H),6.55(d,J=3.6Hz,1H),6.31(d,J=3.6Hz,1H),1.07(s,9H);

[0072] (4) Synthesis of compound Y-2-4

[0073] Compound Y-2-3 (286.1 mg, 0.5 mmol) and pinacol 4-(N-methylamino)phenylboronic acid (234 mg, 1 mmol) were dissolved in dry THF (10 mL). Then, 2 M K2CO3 (1 mL), palladium acetate (22.5 mg, 0.1 mmol), and 2-dicyclohexylphospho-2′,4′,6′-triisopropylbiphenyl (95.3 mg, 0.2 mmol) were added at room temperature. After purging with nitrogen, the mixture was reacted at 80 °C for 12 h. The mixture was extracted three times with CH2Cl2 and H2O, dried over anhydrous Mg2SO4, concentrated, and dried. The mixture was then separated by column chromatography to obtain a brown oily substance Y-2-5 (276.4 mg, 86%).

[0074] 1 H NMR(500MHz,Chloroform-d)δ8.46(s,1H),8.20(d,J=8.5Hz,1H),8.03(d,J=8.5Hz,1H), ,7.60(td,J=7.5,1.3Hz,1H),7.53(td,J=7.5,1.3Hz,1H),7.38-7.34(m,2H),7.33-7.29( m,2H),7.26-7.21(m,4H),7.14-7.10(m,4H),7.08-7.05(m,2H),7.03-6.99(m,2H),6.75 (d,J=8.5Hz,2H),6.57(d,J=3.6Hz,1H),6.33(d,J=3.6Hz,1H),2.89(s,3H),1.07(s,9H);

[0075] (5) Synthesis of compound Y-2-5

[0076] Sodium methoxide (270 mg, 0.5 mmol) was dissolved in methanol (20 mL), followed by the addition of compound Y-2-4 (321.4 mg, 0.5 mmol) and THF (10 mL) at room temperature. The mixture was then purged with nitrogen and reacted at 50 °C for 2 h. After extraction three times with saturated aqueous solutions of CH2Cl2 and NH4Cl, the mixture was dried over anhydrous Mg2SO4, concentrated, and dried. The mixture was then separated by column chromatography to obtain a deep yellow solid Y-2-5 (244.2 mg, 90%).

[0077] 1 H NMR(500MHz,Chloroform-d)8.56(d,J=8.5Hz,1H),8.03(d,J=8.5Hz,1H),7.96(s,1H),7.87(d,J=8.5Hz,2H),7.81(t,J=7.3Hz,1H),7.74 (t,J=7.3Hz,1H),7.37-7.27(m,8H),7.20-7.13(m,7H),7.07(t,J=7.3Hz,2H),6.78(d,J=8.5Hz,2H),6.73(d,J=3.6Hz,1H),2.96(s,3H).

[0078] (6) Synthesis of probe Y-2-6

[0079] Compound Y-2-5 (434 mg, 0.8 mmol), boron trifluoride ether (1136 mg, 8 mmol), and anhydrous triethylamine (484 mg, 4.8 mmol) were dissolved in toluene (10 mL), reacted at 80 °C for 14 h, extracted three times with CH2Cl2 and H2O, dried over anhydrous Mg2SO4, concentrated and dried, and the mixture was separated by column chromatography to obtain a deep yellow solid Y-2-6 (297.4 mg, 60%).

[0080] The proton and mass spectrometry data are as follows:

[0081] 1 ¹H NMR (500MHz, Chloroform-d) δ 8.55 (d, J = 8.5Hz, 1H), 8.01 (d, J = 8.5Hz, 1H), 7.94 (s, 1H), 7.84 (d, J = 8.5Hz, 2H), 7.80 (t, J = 7.3Hz, 1H), 7.73 (t, J = 7.3Hz, 1H), 7.34–7.30 (m, 2H), 7.30–7.24 (m, 5H), 7.17–7.10 (m, 7H), 7.04 (t, J = 7.3Hz, 2H), 6.80–6.75 (m, 2H), 6.71 (d, J = 3.6Hz, 1H), 2.93 (s, 3H); its high-resolution mass spectra are as follows: Figure 2As shown; HRMS found m / z619.2473,Calcd.for Y-2-6[M + 619.2473.

[0082] Performance testing

[0083] Y-4 and Y-2-6 prepared in Examples 1 and 2 were dissolved in PBS (30% DMSO) solution to obtain probe stock solutions (10... -3 (mol / L), then diluted with PBS to obtain 10 -5 Add NO solution (1.9 × 10⁻⁶ mol / L) to a Y⁻⁴ or Y⁻²⁻⁶ solution. - 3 The UV spectrum of the sample (mol / L) was measured after 3 minutes of reaction. The results are as follows: Figure 3 and Figure 5 As shown, the UV absorption of probes Y-4 and Y-2-6 both showed a blue shift. Specifically, the absorption peak of Y-4 at 438 nm weakened after responding to NO, and a new absorption peak was generated at 416 nm. Similarly, the absorption peak of probe Y-2-6 at 510 nm weakened after responding to NO, and a new absorption peak was generated at 410 nm.

[0084] Further, the probes Y-4 or Y-2-6 from Examples 1 and 2 were dissolved in PBS (30% DMSO) solution to obtain probe stock solution (10 -3 (mol / L), then diluted with PBS to obtain 10 -5 Using a Y-4 or Y-2-6 solution at mol / L and an excitation wavelength of 416 nm or 410 nm, observe the effect of adding NO solution (1.9 × 10⁻⁶ mol / L). -3 The changes in fluorescence intensity of Y-4 or Y-2-6 before and after (mol / L) are shown in the figure. Figure 4 and Figure 6 It can be seen that after the addition of NO, the fluorescence of probe Y-4 at 510 nm increased by nearly 30 times within 2 minutes; the fluorescence of probe Y-2-6 at 630 nm increased by 2 times within 3 minutes.

[0085] Selective experiment

[0086] The selectivity experiment of probe Y-4 prepared in Example 1 of the present invention was carried out, and the steps were as follows: probe Y-4 was dissolved in PBS (30% DMSO) solution to obtain probe stock solution (10 -3 (mol / L), then diluted with PBS to obtain 10 -5 A solution of Y-4 at mol / L was mixed with 30 equivalents of different analytes, and the changes in fluorescence intensity at 510 nm were measured. The results are as follows: Figure 7 As shown, where, Figure 7The coordinates in the figure represent the types of analytes, which are (1) NO; (2) blank; (3) Na. + (4) Ca 2+ (5)K + (6)Mg 2+ (7)Zn 2+ (8)Fe 2+ (9)Fe 3+ (10)Cu 2+ (11) NO 2- (12) NO 3- ;(13)GSH;(14)Cys;(15)TBHP;(16)AA;(17)H2O2;(18) 1 O2; (19)MGO; (20)ONOO - (21)ClO - (22)HCHO.) This probe has excellent selectivity for NO.

[0087] The present invention uses the probes prepared in Examples 1 and 2 as examples to illustrate that they can have good fluorescence effects. Other probe molecules of the present invention also have similar effects to the fluorescent probes in Examples 1 and 2.

[0088] The present invention has been described in detail above with reference to the embodiments of the present invention. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention.

Claims

1. A fluorescent probe, characterized by, A compound having a structural formula of formula (I) or formula (II): wherein R1 is H or selected from the following structural formula: R2 is selected from the following structural formula: wherein R3, R6are independently selected from H or C 1～10 alkyl; R4, R5are independently selected from H, C 1～10 alkyl, phenyl or phenyl substituted by halogen, C 1～6 alkyl, nitro, hydroxy, C 1～6 haloalkyl, C 1～6 alkoxy-substituted phenyl.

2. The fluorescent probe according to claim 1, wherein R4, R5are independently selected from H, C 1～6 alkyl, phenyl or phenyl substituted by halogen, C 1～3 alkyl, C 1～3 haloalkyl, C 1～3 alkoxy-substituted phenyl.

3. The fluorescent probe according to claim 1 or 2, characterized by The fluorescent probe is selected from the following structural formula:

4. A method of preparing the fluorescent probe according to any one of claims 1 to 3, characterized in that, The method comprises the following steps: The compound 1, boron trifluoride ether, an organic base and a solvent are mixed to react to obtain the compound 2. The structural formula of the compound 1 is as follows:

5. The preparation method according to claim 4, characterized in that, The organic base comprises at least one of anhydrous triethylamine, potassium carbonate, sodium carbonate and cesium carbonate.

6. The preparation method according to claim 4, characterized in that, The solvent comprises at least one of toluene, tetrahydrofuran, 1,4-dioxane and N,N-dimethylformamide.

7. The preparation method according to claim 4, characterized in that, The temperature of the reaction is 30-100 DEG C.

8. The preparation method according to claim 4, characterized in that, The time of the reaction is 6-16 hours.

9. The preparation method according to claim 4, characterized in that, The molar ratio of the compound 1 to boron trifluoride ether is 1:(1-12).

10. Application of the fluorescent probe in claim 1-3 to detection of the content of nitric oxide.

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