Chiral self-assembly fluorescent probe with spirocoumarin characteristics and preparation method

Abstract

The preparation method comprises the following steps: reacting 7, 7 '-bis (methoxymethoxy)-1, 1'-spirobiindane with a configuration S or a configuration R with butyl lithium and ultra-dry DMF (Dimethyl Formamide), removing a protecting group from a crude product under the action of hydrochloric acid, and performing column chromatography to obtain the chiral self-assembled fluorescent probe with spirocoumarin characteristics. And carrying out condensation cyclization on the spirocoumarin with cyanoacetic acid, ammonium acetate and acetic acid to obtain the spirocoumarin chiral fluorescent probe with configuration S or configuration R. The chiral fluorescent probe with spirocoumarin characteristics is prepared for the first time, through the synergistic effect of spirochirality of 2, 2 ', 3, 3'-tetrahydro-1, 1 '-spirodi [1H-indene]-7, 7'-diol (SPINOL) and a coumarin fragment, not only is the selective recognition ability of the coumarin probe enhanced, but also the stability and sensitivity of the coumarin probe in a complex environment are improved, background noise is also reduced, and the fluorescent probe has good application prospects. Therefore, the detection sensitivity of the fluorescent probe is improved.

Description

Technical Field

[0001] The present invention relates to a chiral self-assembled fluorescent probe with spirolactone coumarin properties and a preparation method thereof, and specifically belongs to the technical field of drug molecule detection. Background Art

[0002] In recent years, chiral self-assembled fluorescent sensors have shown great application potential in the fields of chemical sensing and drug monitoring. Their unique chiral recognition ability and high sensitivity are widely used in drug analysis and detection. Coumarin, as an important fluorescent dye, its unique optical properties and chemical structure make it an ideal choice for fluorescent probes. The high quantum yield, good photostability and extensive modification of coumarin give it unique advantages in the design and application of fluorescent probes. The fluorescent properties of coumarin enable it to emit strong and stable light signals in complex biological environments, thus achieving highly sensitive detection of biomolecules. For example, by introducing different functional groups on the coumarin molecule, selective recognition and labeling of specific biomolecules can be achieved. In 2024, the present invention first reported the preparation and application of a self-assembled SPINOL chiral fluorescent probe, introducing thiophene at the 6,6'-positions of spirolbisphenol, which has excellent recognition for the chiral insecticide fipronil.

[0003] Indomethacin is a non-steroidal anti-inflammatory drug, but excessive intake will cause various adverse effects. Excessive indomethacin will over-inhibit the synthesis of prostaglandins in the gastric mucosa, weaken the protective barrier of the gastric mucosa, may lead to gastric and duodenal ulcers, and in severe cases, it will cause gastrointestinal bleeding, perforation, and symptoms such as hematemesis and melena. In addition, excessive indomethacin may affect the normal function of the central nervous system, resulting in symptoms such as headache, dizziness, and tinnitus. In severe cases, it may even cause anemia and liver function damage. Therefore, there is an urgent need to design a probe for detecting indomethacin. Summary of the Invention

[0004] In view of the above situation, the present invention proposes a chiral self-assembled fluorescent probe with spirolactone coumarin properties and a preparation method thereof, and applies it to the selective detection and recognition of the anti-inflammatory drug indomethacin.

[0005] The present invention uses 1,1'-spirobi[1H-indene]-7,7'-diol as a chiral skeleton unit, introduces a coumarin group, and utilizes the high quantum yield, good photostability and recognition ability of coumarin to prepare a chiral self-assembled fluorescent probe with spirolactone coumarin properties.

[0006] The chemical structural formula of a chiral self-assembled fluorescent probe with spirolactone coumarin properties in the present invention is shown as formula a, b, c or d:

[0007]

[0008] A method for preparing a chiral self-assembled fluorescent probe with coumarin characteristics comprises the following steps:

[0009] Step 1: Add butyllithium and anhydrous DMF to a super-dry tetrahydrofuran solution of 7,7'-bis(methoxymethoxy)-1,1'-spirodihydronaphthalene of configuration S or configuration R, stir and react at 0 °C, extract and dry after quenching; dissolve the crude product in a mixed solution of methanol and tetrahydrofuran, add hydrochloric acid and react at room temperature, after quenching the reaction, extract, dry, concentrate and separate by column chromatography to obtain 6-formyl-2,2',3,3'-tetrahydro-1,1'-spiro[indene]-7,7'-diol or 6,6'-bisformyl-2,2',3,3'-tetrahydro-1,1'-spiro[indene]-7,7'-diol of configuration S or configuration R;

[0010] Step 2: Add cyanoacetic acid, ammonium acetate and acetic acid to 6-formyl-2,2',3,3'-tetrahydro-1,1'-spiro[indene]-7,7'-diol or 6,6'-bisformyl-2,2',3,3'-tetrahydro-1,1'-spiro[indene]-7,7'-diol of configuration S or configuration R, heat and reflux in a pressure-resistant bottle, after the reaction is completed, extract, dry, concentrate and separate by column chromatography to obtain a chiral self-assembled fluorescent probe with coumarin characteristics.

[0011] In the said Step 1: When the molar ratio of 7,7'-bis(methoxymethoxy)-1,1'-spirodihydronaphthalene to butyllithium is 1:1 - 2, the main product is 6-formyl-2,2',3,3'-tetrahydro-1,1'-spiro[indene]-7,7'-diol; when the molar ratio of 7,7'-bis(methoxymethoxy)-1,1'-spirodihydronaphthalene to butyllithium is 1:2 - 6, the main product is 6,6'-bisformyl-2,2',3,3'-tetrahydro-1,1'-spiro[indene]-7,7'-diol.

[0012] In the said Step 2: The equivalent ratio of 6-formyl-2,2',3,3'-tetrahydro-1,1'-spiro[indene]-7,7'-diol or 6,6'-bisformyl-2,2',3,3'-tetrahydro-1,1'-spiro[indene]-7,7'-diol of configuration S or configuration R to cyanoacetic acid is 1:1.5 - 3, and the equivalent ratio of cyanoacetic acid to ammonium acetate is 1:1.

[0013] The reaction equation of the present invention is as follows:

[0014]

[0015] Advantages of the present invention:

[0016] This invention combines the helical chirality, rigid structure, and excellent chiral recognition capabilities of spirobiphenols with the excellent optical properties, high fluorescence quantum efficiency, and large Stokes shift of coumarin, creating the first chiral fluorescent probe with spirocoumarin characteristics. The synergistic effect of the helical chirality of 2,2',3,3'-tetrahydro-1,1'-spirobi[1H-indene]-7,7'-diol (SPINOL) and the coumarin fragment not only enhances the coumarin probe's selective recognition capabilities, but also improves its stability and sensitivity in complex environments, reduces background noise, and thus increases the detection sensitivity of the fluorescent probe. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 For preparing compound a in Example 2 of the present invention 1 HNMR;

[0018] Figure 2 For preparing compound a in Example 2 of the present invention 13 CNMR;

[0019] Figure 3 For Example 4 of the present invention, compound b is prepared 1 HNMR;

[0020] Figure 4 For Example 4 of the present invention, compound b is prepared 13 CNMR;

[0021] Figure 5 The fluorescence response of compound a to indomethacin in Example 2 of the present invention;

[0022] Figure 6 The fluorescence response of compound b to indomethacin in Example 4 of the present invention;

[0023] Figure 7 is the chemical structural formula of the chiral self-assembled fluorescent probe of the present invention. DETAILED DESCRIPTION

[0024] Example 1

[0025]

[0026] Under argon protection, (S)-7,7'-bis(methoxymethoxy)-1,1'-spirobiindane (2.01 g, 6 mmol) and 13 mL of ultradry tetrahydrofuran were added to a 100 mL three-necked flask, and stirred at 0 °C for 30 minutes. A solution of n-butyllithium (1.6 mol / L, 5.6 mL, 9 mmol) was slowly added dropwise, and the reaction was continued for 1.5 hours. Ultra-dry N,N-dimethylformamide (0.7 mL, 9 mmol) was slowly added dropwise through a constant pressure funnel. After the addition of N,N-dimethylformamide was complete, the reaction was stirred overnight. After the reaction was completed, saturated ammonium chloride solution was added and stirred for 1 hour to quench the unreacted n-butyllithium in the reaction system. After complete quenching, the mixture was transferred to a separatory funnel for liquid separation. The organic layer was extracted with dichloromethane three times, washed with saturated brine, and dried over anhydrous magnesium sulfate. The organic phase was filtered by suction and concentrated. Without further purification, the crude product was dissolved in a mixed solvent of methanol and tetrahydrofuran (3:2, 15 mL), hydrochloric acid (0.75 mL, 9 mmol) was added, and the mixture was stirred at room temperature for 4.5 hours. Saturated aqueous sodium carbonate solution was added to quench the reaction. Liquid separation was carried out, and the organic layer was extracted with dichloromethane three times, washed with saturated brine, and dried over anhydrous magnesium sulfate. The organic phase was filtered by suction and concentrated, and column chromatography was carried out using petroleum ether / ethyl acetate (30:1) as the eluent to obtain 1.11 g of white solid (S)-7,7'-bis(methoxymethoxy)-1,1'-spirobiindane, with a yield of about 66%. 1 HNMR(400MHz,CDCl3)δ11.10(s,1H),9.84(s,1H),7.46(d,J=7.8Hz,1H),7.12(t,J=7.7Hz,1H),6.98(d,J=7.7Hz,1H),6.88(d,J=7.5Hz,1H),6.60(d,J=8.1Hz,1H),4.34(s,1H),3.13–3.01(m,4H),2.46–2.16(m,4H). 13 C NMR(100MHz,CDCl3)δ196.44,159.06,155.84,152.66,145.60,134.71,134.19,133.48,129.07,120.32,117.71,117.34,114.04,58.36,38.11,37.93,32.66,31.74.

[0027] Example 2

[0028]

[0029] (S)-6-Formyl-2,2',3,3'-tetrahydro-1,1'-spiro[indene]-7,7'-diol (2.06 g, 7.4 mmol), cyanoacetic acid (0.94 g, 11.1 mmol) and ammonium acetate (0.86 g, 11.1 mmol) were added to a 35 mL pressure-resistant bottle. 3 mL of acetic acid was added, and the mixture was transferred to an oil bath at 120 °C and refluxed overnight. After cooling to room temperature, the reaction was quenched with saturated sodium bicarbonate aqueous solution, transferred to a separatory funnel, extracted 3 times with dichloromethane, washed with saturated brine, and dried over anhydrous magnesium sulfate. The organic phase was filtered by suction and concentrated, and column chromatography separation was carried out using petroleum ether / ethyl acetate (5:1) as the eluent to obtain 1.40 g of a pale yellow solid, namely product a, (S)-7'-hydroxy-2',3',7,8-tetrahydro-spiro[cyclopenta[c]chromene-9,1'-indene]-2-one, with a yield of about 62%. 1 H NMR(400MHz,Chloroform-d)δ7.64(d,J=9.5Hz,1H),7.34(d,J=7.8Hz,1H),7.20(d,J=7.8Hz,1H),7.09(t,J=7.7Hz,1H),6.95–6.86(m,1H),6.52(d,J=7.9Hz,1H),6.26(d,J=9.5Hz,1H),4.27(s,1H),3.27–3.19(m,2H),3.13(d,J=8.7Hz,1H),3.06(dd,J=16.1,8.4Hz,1H),2.54–2.46(m,1H),2.45–2.35(m,2H),2.27(m,J=12.1,8.3,3.0Hz,1H). 13 C NMR(100MHz,CDCl3)δ160.28,151.82,150.84,149.73,145.53,143.62,134.52,132.91,128.60,127.53,120.98,117.63,117.43,115.42,113.48,58.45,38.58,38.58,31.91,31.46.

[0030] Example 3

[0031]

[0032] Under argon protection, (S)-7,7'-bis(methoxymethoxy)-1,1'-spirobiindane (2.02 g, 6 mmol) and 13 mL of ultra-dry tetrahydrofuran were added to a 100 mL three-necked flask, and stirred at low temperature for 30 minutes at 0 °C. A solution of n-butyllithium (1.6 mol / L, 11.2 mL, 18 mmol) was slowly added dropwise, and the reaction was stirred for 1.5 hours. Ultra-dry DMF (1.4 mL, 18 mmol) was slowly added dropwise through a constant pressure funnel, and the reaction was stirred overnight. After the reaction was completed, saturated aqueous ammonium chloride solution was added and stirred for 1 hour. Extracted with dichloromethane three times, washed with saturated brine, and dried over anhydrous magnesium sulfate. The organic phase was filtered by suction and concentrated. The crude product was dissolved in a mixed solvent of methanol and tetrahydrofuran (3:2, 15 mL), and hydrochloric acid (0.75 mL, 9 mmol) was added, and then stirred at room temperature for 4.5 hours. The reaction was quenched by adding saturated aqueous sodium carbonate solution. Separated by liquid separation, extracted with dichloromethane three times, washed with saturated brine, and dried over anhydrous magnesium sulfate. The organic phase was filtered by suction and concentrated, and column chromatographed with petroleum ether / ethyl acetate (15:1) as the eluent to obtain 1.15 g of white solid 6,6'-bisformyl-2,2',3,3'-tetrahydro-1,1'-spiro[indene]-7,7'-diol, with a yield of about 62%.

[0033] 1 H NMR(400MHz,CDCl3)δ11.05(d,J=1.3Hz,2H),9.80(d,J=1.3Hz,2H),7.40(dd,J=7.7,1.3Hz,2H),6.94(d,J=7.8Hz,2H),3.24(m,J=17.0,9.2,4.0Hz,2H),3.06(m,J=16.8,8.2Hz,2H),2.53(m,J=12.8,9.2,7.8Hz,2H),2.32-2.18(m,2H). 13 CNMR(100MHz,CDCl3)δ196.24,196.24,158.62,158.62,155.16,155.16,135.17,135.17,133.64,133.64,119.74,119.74,116.58,116.58,58.02,38.56,38.56,32.62,32.62.

[0034] Example 4

[0035]

[0036] In a 35 mL pressure-resistant bottle, (S)-6,6'-bisformyl-2,2',3,3'-tetrahydro-1,1'-spiro[indene]-7,7'-diol (1.01 g, 3.3 mmol), cyanoacetic acid (0.84 g, 9.9 mmol) and ammonium acetate (0.76 g, 9.9 mmol) were added. 5 mL of acetic acid was added, and the mixture was transferred to an oil bath at 120 °C and refluxed overnight. After cooling to room temperature, the reaction was quenched with saturated sodium bicarbonate aqueous solution, transferred to a separatory funnel, extracted 3 times with dichloromethane, washed with saturated brine, and dried over anhydrous magnesium sulfate. The organic phase was filtered by suction and concentrated, and column chromatography separation was carried out with petroleum ether / ethyl acetate (4:1) as the eluent to obtain 1.36 g of a pale yellow solid, that is, product b, (S)-7,7',8,8'-tetrahydro-9,9'-spirobichromene-2,2'-dione, with a yield of about 58%. 1 H NMR (400 MHz, CDCl3) δ 7.62 (d, J = 9.6 Hz, 2H), 7.31 (d, J = 7.8 Hz, 2H), 7.24 (d, J = 7.8 Hz, 2H), 6.19 (d, J = 9.5 Hz, 2H), 3.64 (dd, J = 22.5, 9.1 Hz, 2H), 3.25–3.12 (m, 2H), 2.75–2.62 (m, 2H), 2.45–2.33 (m, 2H). 13 C NMR (100 MHz, CDCl3) δ 160.59, 160.59, 150.78, 150.78, 150.21, 150.21, 144.22, 144.22, 134.75, 134.75, 127.65, 127.65, 121.29, 121.29, 117.57, 117.57, 114.96, 114.96, 58.89, 40.14, 40.14, 32.60, 32.60.

[0037] Example 5

[0038]

[0039] Add (R)-6-formyl-2,2',3,3'-tetrahydro-1,1'-spiro[indene]-7,7'-diol (2.06 g, 7.4 mmol), cyanoacetic acid (0.94 g, 11.1 mmol) and ammonium acetate (0.86 g, 11.1 mmol) into a 35 mL pressure-resistant bottle. Add 3 mL of acetic acid, and transfer it to an oil bath at 120 °C for reflux reaction overnight. After cooling to room temperature, quench the reaction with saturated sodium bicarbonate aqueous solution, transfer it to a separatory funnel, extract with dichloromethane three times, wash with saturated brine, and dry with anhydrous magnesium sulfate. Filter the organic phase by suction and concentrate it. Column chromatography separation with petroleum ether / ethyl acetate (5:1) as the eluent gives 1.38 g of a pale yellow solid, which is the product c, (R)-7'-hydroxy-2',3',7,8-tetrahydro-spiro[cyclopenta[c]chromene-9,1'-indene]-2-one, with a yield of about 61%.

[0040] Example 6

[0041]

[0042] Add (R)-6,6'-bisformyl-2,2',3,3'-tetrahydro-1,1'-spiro[indene]-7,7'-diol (1.01 g, 3.3 mmol), cyanoacetic acid (0.84 g, 9.9 mmol) and ammonium acetate (0.76 g, 9.9 mmol) into a 35 mL pressure-resistant bottle. Add 5 mL of acetic acid, and transfer it to an oil bath at 120 °C for reflux reaction overnight. After cooling to room temperature, quench the reaction with saturated sodium bicarbonate aqueous solution, transfer it to a separatory funnel, extract with dichloromethane three times, wash with saturated brine, and dry with anhydrous magnesium sulfate. Filter the organic phase by suction and concentrate it. Column chromatography separation with petroleum ether / ethyl acetate (4:1) as the eluent gives 1.42 g of a pale yellow solid, which is the product d, (R)-7,7',8,8'-tetrahydro-9,9'-spirobi[chromene]-2,2'-dione, with a yield of about 60%.

[0043] Example 7

[0044] Accurately weigh 3.0 mg of the fluorescent probe a synthesized in Example 2 with a ten-thousandth analytical balance into a 10 mL volumetric flask. Add 3 mL of ethanol solution to the volumetric flask. After ultrasonic oscillation until the probe is completely dissolved, continue to add ethanol solution to make up the volume to 10 mL to obtain a stock solution with a concentration of 10 -3 mol / L. Before the fluorescence test, dilute the stock solution to a test solution with a concentration of 10 -5 mol / L with ethanol solution and a 100 mL volumetric flask for fluorescence spectrum test.

[0045] Accurately weigh 3.6 mg of the fluorescent probe b synthesized in Example 4 using an analytical balance with a precision of one ten-thousandth into a 10 mL volumetric flask. Add 3 mL of ethanol solution to the volumetric flask. After ultrasonic oscillation until the probe is completely dissolved, continue to add ethanol solution to make up the volume to 10 mL to obtain a stock solution with a concentration of 10 -3 mol / L. Before the fluorescence test, dilute the stock solution to a test solution with a concentration of 10 -5 mol / L using ethanol solution and a 100 mL volumetric flask for fluorescence spectrum testing.

[0046] Weigh accurately the drug molecules to be detected, indomethacin, ribavirin, ranitidine, pantoprazole, donepezil, and loratadine, using an analytical balance with a precision of one ten-thousandth, and add them to a 5 mL glass bottle. Then add 4 mL of ethanol solvent respectively to prepare test solutions with a concentration of 0.05 M (prepared and used immediately). Before the test, according to the specific dosage calculated, use a micro-precise pipette to take the test solution to be measured. After mixing the taken test solution to be measured with the test solution prepared from the probe a synthesized in Example 2 evenly, perform fluorescence spectrum testing. In the same way, after specific calculation, use a micro-precise pipette to take the test solution to be measured, and after mixing the taken test solution to be measured with the test solution prepared from the probe b synthesized in Example 4 evenly, perform fluorescence spectrum testing. The test results are as shown in Figure 5 and Figure 6 .

[0047] Figure 5 shows the fluorescence response of compound a in Example 2 of the present invention to indomethacin. It can be found from the figure that after adding the drug test solution with the same equivalent, indomethacin has an obvious fluorescence response to the probe a synthesized in Example 2, and its fluorescence is greatly quenched. According to the calculation, it can be found that the fluorescence quenching rate of indomethacin to the probe a synthesized in Example 2 reaches 73% ( Figure 5 ), and the quenching effect is excellent.

[0048] Figure 6 shows the fluorescence response of compound b in Example 4 of the present invention to indomethacin. It can be found from the figure that after adding the drug test solution with the same equivalent, indomethacin has a fluorescence response to the probe b synthesized in Example 4, and its fluorescence is significantly quenched. According to the calculation, it can be found that the fluorescence quenching rate of indomethacin to the probe b synthesized in Example 4 reaches 53% ( Figure 6 ), and the quenching effect is remarkable.

Claims

1. A chiral self-assembled fluorescent probe with spirocoumarin properties, characterized in that: The chemical structure of the chiral self-assembled fluorescent probe is shown in formula a, b, c or d:

2. A method for preparing a chiral self-assembled fluorescent probe with spirocoumarin properties, characterized in that: The preparation method comprises the following steps: Step 1: adding butyl lithium and anhydrous DMF to a super-dried tetrahydrofuran solution of 7,7'-bis(methoxymethoxy)-1,1'-spirodihydroindane of configuration S or configuration R, stirring the reaction at 0°C, quenching the reaction, extracting and drying the mixture; dissolving the crude product in a mixture of methanol and tetrahydrofuran, adding hydrochloric acid, reacting the mixture at room temperature, quenching the reaction, extracting, drying, concentrating and column separation to obtain 6-formaldehyde-2,2',3,3'-tetrahydro-1,1'-spiro[indene]-7,7'-diol of configuration S or configuration R or 6,6'-bisformaldehyde-2,2',3,3'-tetrahydro-1,1'-spiro[indene]-7,7'-diol; Step 2: Add cyanoacetic acid, ammonium acetate and acetic acid to 6-formaldehyde-2,2',3,3'-tetrahydro-1,1'-spiro[indene]-7,7'-diol or 6,6'-diformaldehyde-2,2',3,3'-tetrahydro-1,1'-spiro[indene]-7,7'-diol of configuration S or configuration R, heat and reflux in a pressure bottle, and after the reaction is completed, extract, dry, concentrate and separate by column to obtain a chiral self-assembled fluorescent probe with spirocoumarin characteristics.

3. The method for preparing a chiral self-assembled fluorescent probe having spirocoumarin characteristics according to claim 2, characterized in that: In the step 1, when the molar ratio of 7,7'-bis(methoxymethoxy)-1,1'-spirodihydroindene to butyl lithium is 1:1-2, the main product is 6-formaldehyde-2,2',3,3'-tetrahydro-1,1'-spiro[indene]-7,7'-diol; when the molar ratio of 7,7'-bis(methoxymethoxy)-1,1'-spirodihydroindene to butyl lithium is 1:2-6, the main product is 6,6'-bisformaldehyde-2,2',3,3'-tetrahydro-1,1'-spiro[indene]-7,7'-diol.

4. The method for preparing a chiral self-assembled fluorescent probe having spirocoumarin characteristics according to claim 2, wherein: In the step 2, the equivalent ratio of 6-formaldehyde-2,2',3,3'-tetrahydro-1,1'-spiro[indene]-7,7'-diol or 6,6'-bisformaldehyde-2,2',3,3'-tetrahydro-1,1'-spiro[indene]-7,7'-diol of configuration S or configuration R to cyanoacetic acid is 1:1.5-3, and the equivalent ratio of cyanoacetic acid to ammonium acetate is 1:1.

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