A nopylalkylcarbazole-based enhanced fluorescent probe for detecting viscosity, and a preparation method and application thereof

Abstract

The application discloses a norpinane alkyl carbazole enhanced fluorescent probe for detecting viscosity and a preparation method and application thereof. The application takes norpinone as raw material, and performs a cyclization reaction with phenylhydrazine hydrochloride to obtain a compound TC; the compound TC is subjected to N-alkylation reaction with N,N-dimethyl-3-chloropropylamine to obtain a compound TC-AP; the compound TC-AP is subjected to formylation reaction with phosphorus oxychloride to obtain a compound TC-AP-F; and the compound TC-AP-F is subjected to condensation reaction with 4-(cyanomethyl)-1-methylpyridin-1-yl iodide to obtain a fluorescent probe TC-AP-A. The probe TC-AP-A can specifically recognize viscosity, and the red fluorescence of the solution is gradually enhanced with the continuous increase of the viscosity of the system, and the minimum detection limit is 1.41 cP. The probe has the advantages of simple synthesis, good selectivity, high sensitivity, good biocompatibility, low toxicity and the like, and has a good application prospect.

Description

Technical Field

[0001] This invention belongs to the field of fine organic synthesis technology, and relates to nopinel carbazole-based enhanced fluorescent probes, their preparation methods, and applications. Background Technology

[0002] Mitochondria and lysosomes play crucial roles in various physiological processes in organisms. Mitochondria are the energy centers of eukaryotic cells, and mitochondrial dysfunction is associated with many diseases, such as mitochondrial encephalomyopathy, epilepsy, hypertension, and metabolic diseases. Lysosomes are widely distributed in eukaryotes and degrade and recycle exogenous and abnormal intracellular macromolecules, providing cells with nutrients such as amino acids and lipids to maintain cellular function. Lysosomal dysfunction is associated with diseases such as rheumatoid arthritis, shock, and tumors. Both organs maintain normal cellular metabolism through interactions between organelles. Changes in the cellular microenvironment can lead to cancer, autoimmune diseases, and neurodegenerative diseases. Viscosity, as an important microenvironmental parameter in cells and subcellular structures, promotes the interaction of intracellular biomolecules and chemical signals, participating in various physiological activities such as signal regulation, cell metabolism, and apoptosis. Abnormal changes in intracellular viscosity can lead to organelle damage and subsequently various diseases, such as lysosomal storage diseases, Alzheimer's disease, malignant cell tumors, hypertension, and diabetes. Changes in the viscosity level within the mitochondrial matrix cavity can affect cellular respiration and metabolism, causing mitochondrial damage and leading to diseases such as autophagy, cell carcinogenesis, and apoptosis.

[0003] In recent years, many fluorescent probes for viscosity determination have been reported. These probes are mainly designed and synthesized from traditional fluorophores such as coumarin, rhodamine, and naphthalenedicarboximide. The detection performance of these fluorescent probes is easily affected by external factors such as competing ions and reaction time, and they cannot simultaneously target two organelles (lysosomes and mitochondria). Therefore, designing and developing a fluorescent probe that can highly sensitively, selectively, quantitatively, and rapidly determine viscosity targeting lysosomes and mitochondria is of great significance. Summary of the Invention

[0004] Objective of the Invention: To address the shortcomings of existing technologies, the objective of this invention is to provide a nopinel carbazole-based fluorescence-enhanced probe for viscosity detection, meeting specific application requirements. Another objective of this invention is to provide a method for preparing and applying this nopinel carbazole-based fluorescence-enhanced probe for viscosity detection. A further objective of this invention is to provide applications for this probe.

[0005] Technical solution: To achieve the above-mentioned objectives, the technical solution adopted by this invention is as follows:

[0006] The nopinelide carbazole-based fluorescence-enhancing probe, TC-AP-A, has the following structural formula:

[0007]

[0008] The preparation method of the nopinel carbazole-based enhanced fluorescent probe includes the following steps:

[0009] 1) Using nopinene as a starting material, a cyclization reaction was carried out with phenylhydrazine hydrochloride to obtain compound TC;

[0010] 2) Compound TC undergoes an N-alkylation reaction with N,N-dimethyl-3-chloropropane to prepare compound TC-AP;

[0011] 3) Compound TC-AP undergoes a formylation reaction with phosphorus oxychloride to prepare compound TC-AP-F;

[0012] 4) Compound TC-AP-F undergoes a condensation reaction with 4-cyanomethyl-1-methylpyridin-1-yl iodide to prepare compound TC-AP-A.

[0013] In step 1), the specific preparation method of compound TC is as follows:

[0014] (1) Add 2-3 mmol of norpinone, 2-3 mmol of phenylhydrazine hydrochloride, 2-3 mmol of citric acid and 2-4 mmol of 1,3-dimethylurea to a 100 mL dry pressure-resistant tube in sequence, and react at 80-100 °C for 10-20 h, and monitor the reaction by TLC.

[0015] (2) After the reaction solution is cooled, ethyl acetate is added, and the mixture is washed with saturated brine until neutral. The organic phase is then dried with anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product of compound TC.

[0016] (3) The crude product of compound TC was separated by silica gel column chromatography (ethyl acetate / petroleum ether = 1 / 30, v / v) to obtain compound TC.

[0017] In step 2), the specific preparation method of compound TC-AP is as follows:

[0018] (1) Dissolve 3-3.5 mmol of compound TC in DMF and add it to a 50 mL dry three-necked flask. After cooling to 0°C, add 30-35 mmol of sodium hydride under a nitrogen atmosphere. Stir for 1 h and then slowly add 4-6 mmol of N,N-dimethyl-3-chloropropane. React at 40°C-80°C for 12-24 h.

[0019] (2) After the reaction solution was cooled to room temperature, distilled water was added under ice bath and extracted with ethyl acetate. The organic phase was dried with anhydrous sodium sulfate, filtered and concentrated to obtain the crude product of compound TC-AP.

[0020] (3) The crude TC-AP product was separated by silica gel column chromatography (dichloromethane / methanol = 50 / 1, v / v) to obtain the compound TC-AP.

[0021] In step 3), the specific preparation method of compound TC-AP-F is as follows:

[0022] (1) Add 2-2.5 mmol of compound TC-AP, a small amount of DMF and 20 mL of chloroform to a 50 mL dry three-necked flask, cool to 0℃ and add 3-4 mmol of phosphorus oxychloride, and react at 40℃-80℃ for 12-36 h;

[0023] (2) After removing chloroform by vacuum distillation, ethyl acetate was added to the reaction solution, and the solution was washed with saturated brine until neutral. The organic phase was dried with anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product of compound TC-AP-F.

[0024] (3) The crude product of TC-AP-F was separated by silica gel column chromatography (dichloromethane / methanol = 15 / 1, v / v) to obtain compound TC-AP-F.

[0025] In step 4), the specific preparation method of compound TC-AP-A is as follows:

[0026] (1) 1–1.5 mmol of compound TC-AP-F, 2–3 mmol of 4-cyanomethyl-1-methylpyridin-1-yl iodide, 10 mL of ethanol and 2 drops of piperidine were added sequentially to a 50 mL dry three-necked flask and reacted at 78 °C for 12–36 h.

[0027] (2) The reaction solution was cooled and filtered to obtain compound TC-AP-A.

[0028] The application of the compound TC-AP-A in fluorescent probes.

[0029] The compound TC-AP-A exhibits suppressed double bond rotation in a glycerol system, and under ultraviolet light irradiation at a wavelength of 375 nm, the fluorescence color of the solution changes from a weak red to a strong red fluorescence.

[0030] Beneficial effects: Compared with existing technologies, the compound TC-AP-A of this invention can specifically respond to viscosity and is not affected by other competing species, with a minimum detection limit of 1.41 cP. As a fluorescent probe for viscosity detection, it has many advantages such as convenient synthesis, good selectivity, high sensitivity, good biocompatibility, and low toxicity, and has good application prospects. Attached Figure Description

[0031] Figure 1It represents the fluorescence intensity of the probe TC-AP-A at different viscosities within the range of 0.9-10¹⁰ cP;

[0032] Figure 2 It refers to the fluorescence intensity of the probe TC-AP-A in different solvents;

[0033] Figure 3 It represents the fluorescence intensity of the probe TC-AP-A interacting with different ions in a 90% glycerol system. Detailed Implementation

[0034] The present invention will be further described below with reference to specific embodiments.

[0035] Example 1

[0036] Preparation of probe TC-AP-A

[0037] The preparation route of probe TC-AP-A is as follows:

[0038]

[0039] The specific steps include:

[0040] 1) Preparation of compound TC:

[0041] 2 mmol of norpinone, 2 mmol of phenylhydrazine hydrochloride, 2 mmol of citric acid, and 3 mmol of 1,3-dimethylurea were sequentially added to a 100 mL dry pressure-resistant tube. The reaction was carried out at 92 °C for 12 h, monitored by TLC, and stopped after the reaction was complete. After cooling, ethyl acetate was added, and the mixture was washed with saturated brine until neutral. The organic phase was then dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product of compound TC. The crude TC product was separated by silica gel column chromatography (ethyl acetate / petroleum ether = 1 / 30, v / v) to obtain compound TC with a yield of 31.5% and a purity of 98.3%. 1 H NMR (600 MHz, DMSO-d6) δ:10.79 (s, 1H), 7.33–7.21 (m, 2H), 6.94–6.89 (m, 2H), 2.86 (dd, J = 15.3, 3.0Hz, 1H), 2.79 (t, J = 5.3 Hz, 1H), 2.76–2.70 (m, 2H), 2.35 (tt, J = 5.8, 2.9Hz, 1H), 1.42 (d, J = 2.8 Hz, 3H), 1.40 (s, 1H), 0.61 (s, 3H). 13C NMR (150MHz, DMSO-d6) δ: 145.51, 135.54, 127.51, 119.16, 118.92, 117.09, 111.46,103.32, 42.01, 41.84, 41.59, 40.54, 40.42, 40.28, 40.15, 40.01, 39.87, 39.73,39.59, 33.74, 26.78, 25.66, 21.78. HRMS: m / z calculated for C 15 H 17 N [M+H] + ,212.1439; found, 212.1434.

[0042] 2) Preparation of compound TC-AP:

[0043] 3 mmol of compound TC was dissolved in DMF and added to a 50 mL dry three-necked flask. After cooling to 0 °C, 30 mmol of sodium hydride was added under a nitrogen atmosphere. The mixture was stirred for 1 h, followed by the slow addition of 4.5 mmol of N,N-dimethyl-3-chloropropane, and the reaction was carried out at 60 °C for 12 h. After cooling the reaction solution to room temperature, the sodium hydride was quenched with distilled water in an ice bath, and the mixture was extracted with ethyl acetate. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product TC-AP. The crude product TC-AP was separated by silica gel column chromatography (dichloromethane / methanol = 50 / 1, v / v) to obtain compound TC-AP with a yield of 80.7% and a purity of 97.7%. 1 H NMR(600 MHz, DMSO-d6) δ: 7.34 (t, J = 8.2 Hz, 2H), 6.97 (dt, J = 23.1, 7.6 Hz, 2H), 4.08 (td, J = 6.7, 2.2 Hz, 2H), 2.99 (d, J = 5.3 Hz, 1H), 2.87 (dd, J =15.4, 3.0 Hz, 1H), 2.79–2.70 (m, 2H), 2.35 (dq, J = 5.8, 2.8 Hz, 1H), 2.14(dd, J = 12.1, 7.1 Hz, 1H), 2.11 (s, 6H), 2.09–2.06 (m, 1H), 1.73–1.66 (m,2H), 1.43 (s, 3H), 1.40 (d, J = 9.0 Hz, 1H), 0.62 (s, 3H). 13C NMR (150 MHz, DMSO-d6) δ: 146.30, 136.03, 126.99, 119.34, 119.04, 117.38, 109.85, 103.61,56.26, 45.51, 41.94, 41.82, 40.55, 40.43, 40.29, 40.22, 40.15, 40.02, 39.88,39.74, 39.60, 33.80, 28.84, 26.83, 25.56, 21.79, 0.56. HRMS: m / z calculated for C 20 H 28 N2 [M+H] + , 297.2331; found, 297.2340.

[0044] 3) Preparation of compound TC-AP-F:

[0045] Two mmol of compound TC-AP, a small amount of DMF, and 20 mL of chloroform were sequentially added to a 50 mL dry three-necked flask. After cooling to 0 °C, 3 mmol of phosphorus oxychloride was added, and the mixture was reacted at 60 °C for 12 h. The reaction solution was subjected to vacuum distillation to remove chloroform, followed by the addition of ethyl acetate and washing with saturated brine until neutral. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product TC-AP-F. The crude product TC-AP-F was then separated by silica gel column chromatography (dichloromethane / methanol = 15 / 1, v / v) to obtain compound TC-AP-F with a yield of 76.6% and a purity of 98.1%. 1 H NMR (600 MHz, DMSO-d6) δ: 9.93 (s,1H), 8.00 (s, 1H), 7.53–7.46 (m, 2H), 4.23 (td, J = 6.7, 4.5 Hz, 2H), 3.11(t, J = 5.3 Hz, 1H), 2.90 (dd, J = 15.5, 3.0 Hz, 1H), 2.84–2.75 (m, 2H), 2.39(tt, J = 5.6, 2.9 Hz, 1H), 2.13 (d, J = 13.2 Hz, 1H), 2.10 (s, 6H), 2.09–2.05(m, 1H), 1.77–1.70 (m, 2H), 1.45 (s, 3H), 1.40 (d, J = 9.3 Hz, 1H), 0.62 (s, 3H). 13C NMR (150 MHz, DMSO-d6) δ: 192.46, 152.41, 135.54, 131.60, 128.77,120.09, 117.55, 113.78, 105.62, 57.68, 55.65, 44.79, 41.91, 41.45, 33.32,32.81, 26.64, 25.28, 24.88, 21.76. HRMS: m / z calculated for C 21 H 28 N2O [M+H] + ,325.2280; found, 325.2284.

[0046] 4) Preparation of compound TC-AP-A:

[0047] 1 mmol of compound TC-AP-F, 2 mmol of 4-cyanomethyl-1-methylpyridin-1-yl iodide, 10 mL of ethanol, and 2 drops of piperidine were sequentially added to a 50 mL dry three-necked flask, and the reaction was carried out at 78 °C for 12 h. After cooling, the reaction solution was filtered to obtain compound TC-AP-A. The yield was 80.8%, and the purity was 98.5%. 1 H NMR (600 MHz, DMSO-d6) δ: 8.99–8.95 (m,2H), 8.87 (s, 1H), 8.40 (d, J = 1.6 Hz, 1H), 8.38–8.36 (m, 2H), 7.97 (dd, J =8.5, 1.5 Hz, 1H), 7.59 (d, J = 8.5 Hz, 1H), 4.31 (s, 3H), 4.29 (s, 1H), 4.07(s, 1H), 3.99 (d, J = 3.0 Hz, 2H), 3.23 (t, J = 5.3 Hz, 1H), 3.17–3.14 (m,1H), 2.98 (d, J = 5.2 Hz, 1H), 2.96–2.91 (m, 2H), 2.86 (dt, J = 9.7, 6.0 Hz,2H), 2.83–2.78 (m, 2H), 1.66 (p, J = 5.8 Hz, 1H), 1.49 (d, J = 7.7 Hz, 4H),1.43 (t, J = 10.3 Hz, 2H), 0.68 (d, J = 13.5 Hz, 4H). 13C NMR (150 MHz, DMSO-d6) δ: 154.28, 153.68, 150.56, 145.80, 143.35, 135.60, 131.19, 124.05,122.19, 121.34, 118.34, 118.19, 116.72, 107.25, 98.88, 84.12, 54.49, 47.38,45.77, 42.65, 42.10, 41.28, 33.16, 31.41, 26.53, 25.28, 22.05, 21.84, 14.43.HRMS: m / z calculated for C 29 H 35 IN4 + [M] + , 439.2856; found, 439.2873.

[0048] Example 2

[0049] Compound TC-AP-A was dissolved in DMSO to prepare a 1 mM probe stock solution. Glycerol and PBS were mixed in different proportions to prepare solutions with viscosities ranging from 0.9 to 10¹⁰ cP. The probe stock solution was then added to bring the final concentration to 1 × 10⁻⁶. -5 M. The fluorescence emission spectra of compound TC-AP-A at different viscosities were measured using a fluorescence spectrophotometer, such as... Figure 1 As shown, the fluorescence emission intensity at 625 nm gradually increases with increasing solution viscosity, indicating that the probe can sensitively detect viscosity changes.

[0050] Example 3

[0051] Compound TC-AP-A was dissolved in various organic solvents to prepare a solution of 1×10⁻⁶. -5 The probe solution had a concentration of M. The fluorescence emission spectra of compound TC-AP-A in different solvents were measured using a fluorescence spectrophotometer, as shown below. Figure 2 As shown, the probe exhibits strong fluorescence emission at 625 nm in the glycerol system, but no significant fluorescence emission in other organic solvents. Compound TC-AP-A was dissolved in PBS / glycerol (v / v = 1 / 9) buffer to prepare a 1×10⁻⁶ solution. -5 A probe solution with concentration M was prepared, and other ions (K+) were added. + Na + , Cr 3+ Fe 3+ Hg 2+ , Pd 2+ HPO4 2–HSO3 2– HS – NO3 – SO4 2– (H2O2, Hcy, Cys, GSH). For example... Figure 3 As shown, the fluorescence emission intensity at 625 nm did not change significantly before and after the addition of competing ions. This indicates that the probe can specifically detect viscosity.

Claims

1. A nopinel carbazole-enhanced fluorescent probe for viscosity detection, its preparation method, and its application, characterized in that, The probe is TC-AP-A, and its structural formula is as follows: 。 2. The method for preparing the nopinel carbazole-based enhanced fluorescent probe according to claim 1, characterized in that, Includes the following steps: 1) Using nopinene as a starting material, a cyclization reaction was carried out with phenylhydrazine hydrochloride to obtain compound TC; 2) Compound TC undergoes an N-alkylation reaction with N,N-dimethyl-3-chloropropane to prepare compound TC-AP; 3) Compound TC-AP undergoes a formylation reaction with phosphorus oxychloride to prepare compound TC-AP-F; 4) Compound TC-AP-F undergoes a condensation reaction with 4-cyanomethyl-1-methylpyridin-1-yl iodide to prepare compound TC-AP-A.

3. The nopinel carbazole-based enhanced fluorescent probe and its preparation method according to claim 2, characterized in that, In step 1), the specific preparation method of compound TC is as follows: (1) Add 2-3 mmol of norpinone, 2-3 mmol of phenylhydrazine hydrochloride, 2-3 mmol of citric acid and 2-4 mmol of 1,3-dimethylurea sequentially to a 100 mL dry pressure-resistant tube, and react at 80-100 °C for 10-20 h, and monitor the reaction by TLC. (2) After the reaction solution is cooled, ethyl acetate is added, and the mixture is washed with saturated brine until neutral. The organic phase is then dried with anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product of compound TC. (3) The crude product of compound TC was separated by silica gel column chromatography (ethyl acetate / petroleum ether = 1 / 30, v / v) to obtain compound TC.

4. The nopinel carbazole-based enhanced fluorescent probe and its preparation method according to claim 2, characterized in that, In step 2), the specific preparation method of compound TC-AP is as follows: (1) Dissolve 3-4 mmol of compound TC in DMF and add it to a 50 mL dry three-necked flask. After cooling to 0 °C, add 30-35 mmol of sodium hydride under a nitrogen atmosphere. Stir for 1 h and then slowly add 4-6 mmol of N,N-dimethyl-3-chloropropane. React at 40-80 °C for 12-24 h. (2) After the reaction solution was cooled to room temperature, distilled water was added under ice bath and extracted with ethyl acetate. The organic phase was dried with anhydrous sodium sulfate, filtered and concentrated to obtain the crude product of compound TC-AP. (3) The crude TC-AP product was separated by silica gel column chromatography (dichloromethane / methanol = 50 / 1, v / v) to obtain the compound TC-AP.

5. The method for synthesizing a nopinel carbazole-based viscosity-type fluorescent probe according to claim 2, characterized in that, In step 3), the specific preparation method of compound TC-AP-F is as follows: (1) Add 2-2.5 mmol of compound TC-AP, a small amount of DMF and 20 mL of chloroform to a 50 mL dry three-necked flask, cool to 0 °C and add 3-4 mmol of phosphorus oxychloride, and react at 40 °C-80 °C for 12-36 h; (2) After removing chloroform by vacuum distillation, ethyl acetate was added to the reaction solution, and the solution was washed with saturated brine until neutral. The organic phase was dried with anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product of compound TC-AP-F. (3) The crude product of TC-AP-F was separated by silica gel column chromatography (dichloromethane / methanol = 15 / 1, v / v) to obtain compound TC-AP-F.

6. The method for synthesizing a nopinel carbazole-based viscosity-type fluorescent probe according to claim 2, characterized in that, In step 4), the specific preparation method of compound TC-AP-A is as follows: (1) 1–1.5 mmol of compound TC-AP-F, 2–3 mmol of 4-cyanomethyl-1-methylpyridin-1-yl iodide, 10 mL of ethanol and 2 drops of piperidine were added sequentially to a 50 mL dry three-necked flask and reacted at 78 °C for 12–36 h. (2) The reaction solution was cooled and filtered to obtain compound TC-AP-A.

7. The use of the compound TC-AP-A according to claim 1 in a fluorescent probe.

8. The application according to claim 7, characterized in that... When compound TC-AP-A is irradiated with ultraviolet light at a wavelength of 375 nm in a glycerol system, the fluorescence color of the solution changes from a weak red to a strong red fluorescence.

9. The application according to claim 7, characterized in that... Compound TC-AP-A can specifically identify viscosity and is not affected by other competing species, with a detection limit of 1.41 cP.

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