A 2-hydroxy-3-pinonyl pyrimidine pH-sensitive fluorescent probe and its preparation method and application

Abstract

The present invention discloses a 2-hydroxy-3-pinone-based pyrimidine pH-sensitive fluorescent probe and its preparation method and application. The present invention uses 2-hydroxy-3-pinone as a raw material, and carries out an aldol condensation reaction with 6-bromo-9-ethyl-9H-carbazole-3-carbaldehyde to obtain 4-((6-bromo-9-ethyl-9H-carbazole-3-yl)methylene)-2-hydroxy-2,6,6-trimethylbicyclo[3.1.1]heptan-3-one; 4-((6-bromo-9-ethyl-9H-carbazole-3-yl)methylene)-2-hydroxy-2,6,6-trimethylbicyclo[3.1.1]heptan-3-one and 4-hydroxybenzamidine hydrochloride to carry out a cyclization reaction to obtain 4-(6-bromo-9-ethyl-9H-carbazole-3-yl)-2-( 4-(9-ethyl-6-(pyridin-4-yl)-9H-carbazol-3-yl)-2-(4-hydroxyphenyl)-6,6,8-trimethyl-5,6,7,8-tetrahydro-5,7-methanoquinazoline-8-ol; 4-(6-bromo-9-ethyl-9H-carbazol-3-yl)-2-(4-hydroxyphenyl)-6,6,8-trimethyl-5,6,7,8-tetrahydro-5,7-methanoquinazoline-8-ol and 4-pyridineboronic acid are coupled to prepare 4-(9-ethyl-6-(pyridin-4-yl)-9H-carbazol-3-yl)-2-(4-hydroxyphenyl)-6,6,8-trimethyl-5,6,7,8-tetrahydro-5,7-methanoquinazoline-8-ol. 4-(9-ethyl-6-(pyridin-4-yl)-9H-carbazol-3-yl)-2-(4-hydroxyphenyl)-6,6,8-trimethyl-5,6,7,8-tetrahydro-5,7-methanoquinazoline-8-ol can specifically recognize acidic and alkaline pH. Under acidic conditions, the fluorescence color of the solution changes from blue to yellow as the pH value in the system continues to decrease; under alkaline conditions, the blue fluorescence of the solution gradually weakens as the pH value in the system continues to increase. It can be used as a fluorescent probe for measuring pH and has many advantages such as convenient synthesis, good selectivity, and high sensitivity, and has good application prospects.

Description

Technical Field

[0001] The present invention belongs to the technical field of fine organic synthesis, and relates to a 2-hydroxy-3-pinone-pyrimidine pH-sensitive fluorescent probe, a synthesis method and an application thereof. Background Art

[0002] Maintaining a stable intracellular environment is essential for maintaining normal physiological functions and, ultimately, for maintaining normal life activities. pH homeostasis plays a crucial role in biological growth and development. Lysosomes have a pH range of 4.5-5.5 and contain a variety of hydrolases, such as proteases, nucleases, and phosphatases, that break down biomolecules such as proteins, nucleic acids, and polysaccharides. Lysosomal function is influenced by lysosomal pH. Abnormal lysosomal pH can lead to cellular dysfunction, neurodegenerative diseases, and lysosomal storage disorders. The acidic environment of lysosomes is created by the pumping of protons into the lumen by ATP, which is produced by mitochondria. Mitochondria are present in most eukaryotic cells and play a vital role in cellular metabolism, such as regulating cellular redox status, generating reactive oxygen species (ROS) to degrade pathogens, regulating calcium homeostasis, participating in apoptosis, and regulating stem cells. Mitochondria are slightly alkaline, with a pH of approximately 8. Mitochondrial dysfunction can lead to changes in mitochondrial pH, a common finding in diabetes, cardiovascular disease, and neurodegenerative diseases.

[0003] In recent years, many fluorescent probes for measuring pH have been reported. These fluorescent probes are mainly designed and synthesized from traditional fluorophores such as coumarin, rhodamine, and naphthalimide. The detection effects of these fluorescent probes are not only easily interfered by external factors such as competitive ions and reaction time, but also all have a single detection mode and cannot specifically detect acidity and alkalinity. In particular, they cannot simultaneously target two organelles (lysosomes and mitochondria). Therefore, it is of great significance to design and develop a pH fluorescent probe targeting lysosomes and mitochondria that can simultaneously measure acidity and alkalinity with high sensitivity, high selectivity, and quantitative speed. Summary of the Invention

[0004] Objectives of the Invention: To address the shortcomings of the prior art, the present invention aims to provide a 2-hydroxy-3-pinono-pyrimidine compound that meets the needs of its intended use. Another objective of the present invention is to provide a method for synthesizing the aforementioned 2-hydroxy-3-pinono-pyrimidine compound. Yet another objective of the present invention is to provide applications of the compound.

[0005] Technical solution: To achieve the above-mentioned purpose, the technical solution adopted by the present invention is:

[0006] 2-Hydroxy-3-pinonyl pyrimidine compound, 4-(9-ethyl-6-(pyridin-4-yl)-9H-carbazol-3-yl)-2-(4-hydroxyphenyl)-6,6,8-trimethyl-5,6,7,8-tetrahydro-5,7-methanoquinazolin-8-ol, has the structural formula:

[0007]

[0008] The method for synthesizing 2-hydroxy-3-pinonyl pyrimidine compounds comprises the following steps:

[0009] 1) Using 2-hydroxy-3-pinone as a raw material, an aldol condensation reaction is carried out with 6-bromo-9-ethyl-9H-carbazole-3-carbaldehyde to obtain 4-((6-bromo-9-ethyl-9H-carbazole-3-yl)methylene)-2-hydroxy-2,6,6-trimethylbicyclo[3.1.1]heptan-3-one;

[0010] 2) cyclization reaction of 4-((6-bromo-9-ethyl-9H-carbazol-3-yl)methylene)-2-hydroxy-2,6,6-trimethylbicyclo[3.1.1]heptan-3-one with 4-hydroxybenzamidine hydrochloride to prepare 4-(6-bromo-9-ethyl-9H-carbazol-3-yl)-2-(4-hydroxyphenyl)-6,6,8-trimethyl-5,6,7,8-tetrahydro-5,7-methanoquinazolin-8-ol;

[0011] 3) 4-(6-bromo-9-ethyl-9H-carbazol-3-yl)-2-(4-hydroxyphenyl)-6,6,8-trimethyl-5,6,7,8-tetrahydro-5,7-methanoquinazolin-8-ol is coupled with 4-pyridineboronic acid to prepare 4-(9-ethyl-6-(pyridin-4-yl)-9H-carbazol-3-yl)-2-(4-hydroxyphenyl)-6,6,8-trimethyl-5,6,7,8-tetrahydro-5,7-methanoquinazolin-8-ol.

[0012] In step 1), 4-((6-bromo-9-ethyl-9H-carbazol-3-yl)methylene)-2-hydroxy-2,6,6-trimethylbicyclo[3.1.1]heptan-3-one is prepared by:

[0013] (1) 1.1-1.5 mmol of 2-hydroxy-3-pinene, 1-1.36 mmol of 6-bromo-9-ethyl-9H-carbazole-3-carbaldehyde, 3-4.08 mmol of potassium tert-butoxide, and 50-68 mL of tert-butanol were sequentially added into a 100 mL dry three-necked flask, and the mixture was refluxed for 12 h. The reaction was monitored by TLC and stopped after the reaction was complete.

[0014] (2) The reaction solution was distilled under reduced pressure to remove tert-butanol, and then ethyl acetate was added. The mixture was washed with saturated brine until neutral. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product of 4-((6-bromo-9-ethyl-9H-carbazol-3-yl)methylene)-2-hydroxy-2,6,6-trimethylbicyclo[3.1.1]heptan-3-one;

[0015] (3) The crude product of 4-((6-bromo-9-ethyl-9H-carbazol-3-yl)methylene)-2-hydroxy-2,6,6-trimethylbicyclo[3.1.1]heptan-3-one was separated by silica gel column (ethyl acetate / petroleum ether = 1 / 5, v / v) to obtain 4-((6-bromo-9-ethyl-9H-carbazol-3-yl)methylene)-2-hydroxy-2,6,6-trimethylbicyclo[3.1.1]heptan-3-one.

[0016] In step 2), the specific preparation method of 4-(6-bromo-9-ethyl-9H-carbazol-3-yl)-2-(4-hydroxyphenyl)-6,6,8-trimethyl-5,6,7,8-tetrahydro-5,7-methanoquinazolin-8-ol includes:

[0017] (1) 3-3.5 mmol 4-hydroxybenzamidine hydrochloride, 1.5-1.75 mL 30% sodium hydroxide solution, 1-1.17 mmol 4-((6-bromo-9-ethyl-9H-carbazol-3-yl)methylene)-2-hydroxy-2,6,6-trimethylbicyclo[3.1.1]heptan-3-one, and 6-7 mL dimethyl sulfoxide were added sequentially into a 50 mL dry three-necked flask and heated under reflux for 12 h.

[0018] (2) After the reaction solution was cooled to room temperature, ethyl acetate was added and the mixture was washed with saturated brine until neutral. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product of 4-(6-bromo-9-ethyl-9H-carbazol-3-yl)-2-(4-hydroxyphenyl)-6,6,8-trimethyl-5,6,7,8-tetrahydro-5,7-methanoquinazolin-8-ol;

[0019] (3) The crude product of 4-(6-bromo-9-ethyl-9H-carbazole-3-yl)-2-(4-hydroxyphenyl)-6,6,8-trimethyl-5,6,7,8-tetrahydro-5,7-methanoquinazolin-8-ol was separated by silica gel column (ethyl acetate / petroleum ether = 1 / 1, v / v) to obtain 4-(6-bromo-9-ethyl-9H-carbazole-3-yl)-2-(4-hydroxyphenyl)-6,6,8-trimethyl-5,6,7,8-tetrahydro-5,7-methanoquinazolin-8-ol.

[0020] In step 3), the specific preparation method of 4-(9-ethyl-6-(pyridin-4-yl)-9H-carbazol-3-yl)-2-(4-hydroxypropyl)-6,6,8-trimethyl-5,6,7,8-tetrahydro-5,7-methanoquinazolin-8-ol is as follows:

[0021] (1) 2-2.4 mmol 4-(6-bromo-9-ethyl-9H-carbazol-3-yl)-2-(4-hydroxyphenyl)-6,6,8-trimethyl-5,6,7,8-tetrahydro-5,7-methanoquinazolin-8-ol, 6-7.2 mmol 4-pyridineboronic acid, 0.2-0.24 mmol tetrakis(triphenylphosphine)palladium, 9-10.8 mmol potassium carbonate, and 50-60 mL 1,4-dioxane / water solution were sequentially added into a 100 mL dry three-necked flask and heated under reflux for 20 h under nitrogen protection;

[0022] (2) After the reaction solution was distilled under reduced pressure to remove 1,4-dioxane / water solution, ethyl acetate was added and the mixture was washed with saturated brine until neutral. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product of 4-(9-ethyl-6-(pyridin-4-yl)-9H-carbazol-3-yl)-2-(4-hydroxyphenyl)-6,6,8-trimethyl-5,6,7,8-tetrahydro-5,7-methanoquinazolin-8-ol;

[0023] (3) The crude product of 4-(9-ethyl-6-(pyridin-4-yl)-9H-carbazole-3-yl)-2-(4-hydroxyphenyl)-6,6,8-trimethyl-5,6,7,8-tetrahydro-5,7-methanoquinazolin-8-ol was separated by silica gel column (dichloromethane / methanol = 35 / 1, v / v) to obtain 4-(9-ethyl-6-(pyridin-4-yl)-9H-carbazole-3-yl)-2-(4-hydroxyphenyl)-6,6,8-trimethyl-5,6,7,8-tetrahydro-5,7-methanoquinazolin-8-ol.

[0024] Application of the 4-(9-ethyl-6-(pyridin-4-yl)-9H-carbazole-3-yl)-2-(4-hydroxyphenyl)-6,6,8-trimethyl-5,6,7,8-tetrahydro-5,7-methanoquinazolin-8-ol in fluorescent probes.

[0025] The compound 4-(9-ethyl-6(pyridin-4-yl)-9H-carbazol-3-yl)-2-(4-hydroxyphenyl)-6,6,8-trimethyl-5,6,7,8-tetrahydro-5,7-methanoquinazolin-8-ol, under acidic conditions, shows that as the pH value in the system continues to decrease, the fluorescence color of the solution changes from blue to yellow; under alkaline conditions, as the pH value in the system continues to increase, the blue fluorescence of the solution gradually weakens. This shows that the probe can sensitively detect pH values ​​and can be used as a fluorescent probe for pH determination.

[0026] Beneficial effects: Compared with the prior art, the 2-hydroxy-3-pinone-based pyrimidine compound 4-(9-ethyl-6-(pyridin-4-yl)-9H-carbazole-3-yl)-2-(4-hydroxyphenyl)-6,6,8-trimethyl-5,6,7,8-tetrahydro-5,7-methanoquinazolin-8-ol prepared by the present invention using 2-hydroxy-3-pinone as a raw material can specifically identify acidic pH and alkaline pH. Under acidic conditions, as the pH value in the system continues to decrease, the fluorescence color of the solution changes from blue to yellow; under alkaline conditions, as the pH value in the system continues to increase, the blue fluorescence of the solution gradually weakens. The present invention can be used as a fluorescent probe for measuring pH, has many advantages such as convenient synthesis, good selectivity, high sensitivity, etc., and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 is the fluorescence intensity of 4-(9-ethyl-6-(pyridin-4-yl)-9H-carbazol-3-yl)-2-(4-hydroxyphenyl)-6,6,8-trimethyl-5,6,7,8-tetrahydro-5,7-methanoquinazolin-8-ol in the presence of different pH values ​​under acidic conditions;

[0028] Figure 2 is the fluorescence intensity of 4-(9-ethyl-6-(pyridin-4-yl)-9H-carbazol-3-yl)-2-(4-hydroxyphenyl)-6,6,8-trimethyl-5,6,7,8-tetrahydro-5,7-methanoquinazolin-8-ol in response to different pH values ​​under alkaline conditions;

[0029] Figure 3 It is the fluorescence intensity of 4-(9-ethyl-6-(pyridin-4-yl)-9H-carbazol-3-yl)-2-(4-hydroxyphenyl)-6,6,8-trimethyl-5,6,7,8-tetrahydro-5,7-methanoquinazolin-8-ol interacting with different ions. DETAILED DESCRIPTION

[0030] The present invention will be further described below with reference to specific embodiments.

[0031] Example 1

[0032] Preparation of 4-(9-ethyl-6-(pyridin-4-yl)-9H-carbazol-3-yl)-2-(4-hydroxyphenyl)-6,6,8-trimethyl-5,6,7,8-tetrahydro-5,7-methanoquinazolin-8-ol

[0033] The preparation route of 4-(9-ethyl-6-(pyridin-4-yl)-9H-carbazol-3-yl)-2-(4-hydroxyphenyl)-6,6,8-trimethyl-5,6,7,8-tetrahydro-5,7-methanoquinazolin-8-ol is as follows:

[0034]

[0035] The specific steps include:

[0036] 1) Preparation of 4-((6-bromo-9-ethyl-9H-carbazol-3-yl)methylene)-2-hydroxy-2,6,6-trimethylbicyclo[3.1.1]heptan-3-one:

[0037] 1.1-1.5 mmol of 2-hydroxy-3-pinene, 1-1.36 mmol of 6-bromo-9-ethyl-9H-carbazole-3-carbaldehyde, 3-4.08 mmol of potassium tert-butoxide, and 50-68 mL of tert-butanol were added sequentially to a 100 mL dry three-necked flask. The mixture was refluxed for 12 hours and monitored by TLC. The reaction was stopped after completion. The reaction solution was distilled under reduced pressure to remove tert-butanol, and then ethyl acetate was added. The mixture was washed with saturated brine until neutral. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product of 4-((6-bromo-9-ethyl-9H-carbazole-3-yl)methylene)-2-hydroxy-2,6,6-trimethylbicyclo[3.1.1]heptan-3-one. The crude product of 4-((6-bromo-9-ethyl-9H-carbazol-3-yl)methylene)-2-hydroxy-2,6,6-trimethylbicyclo[3.1.1]heptan-3-one was separated on a silica gel column (ethyl acetate / petroleum ether = 1 / 5, v / v) to obtain 4-((6-bromo-9-ethyl-9H-carbazol-3-yl)methylene)-2-hydroxy-2,6,6-trimethylbicyclo[3.1.1]heptan-3-one with a yield of 62.5% and a purity of 98.3%. 1H NMR (600MHz, DMSO-d6) δ: 8.47 (d, J=1.8Hz, 1H), 8.28-8.19 (m, 1H), 7.69-7.62 (m, 2 H), 7.61-7.57 (m, 2H), 7.50 (dd, J=8.6, 1.7Hz, 1H), 5.35 (s, 1H), 4.43 (d, J=7.2Hz, 2H), 3.46 (d, J=6.1Hz, 1H), 2.58 (t, J=10.4, 6.0Hz, 1H), 2.10 (d, J=6.1Hz, 1H), 1.8 6 (d, J=10.4Hz, 1H), 1.42 (s, 3H), 1.37 (s, 3H), 1.29 (d, J=7.1Hz, 3H), 0.86 (s, 3H). 13 C NMR (150MHz, DMSO-d6) δ: 201.86, 140.22, 139.38, 139.15, 133.48, 128.94, 128.68, 126.57, 124.43, 123.72, 122.93 , 121.87, 111.88, 111.82, 110.04, 75.27, 51.05, 43.18, 40.40, 37.74, 28.97, 27.49, 25.74, 24.33, 14.13.HRMS: m / z calculated for C 25 H 26 BrNO2[M+Na] + , 474.1045; found, 474.1050.

[0038] 2) Preparation of 4-(6-bromo-9-ethyl-9H-carbazol-3-yl)-2-(4-hydroxyphenyl)-6,6,8-trimethyl-5,6,7,8-tetrahydro-5,7-methanoquinazolin-8-ol:

[0039] 3-3.5 mmol of 4-hydroxybenzamidine hydrochloride, 1.5-1.75 mL of 30% sodium hydroxide solution, 1-1.17 mmol of 4-((6-bromo-9-ethyl-9H-carbazol-3-yl)methylene)-2-hydroxy-2,6,6-trimethylbicyclo[3.1.1]heptan-3-one, and 6-7 mL of dimethyl sulfoxide were added sequentially to a 50 mL dry three-necked flask and heated under reflux for 12 hours. After cooling the reaction solution to room temperature, ethyl acetate was added and the mixture was washed several times with saturated brine. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated to yield the crude product 4-(6-bromo-9-ethyl-9H-carbazol-3-yl)-2-(4-hydroxyphenyl)-6,6,8-trimethyl-5,6,7,8-tetrahydro-5,7-methanoquinazolin-8-ol. The crude product of 4-(6-bromo-9-ethyl-9H-carbazol-3-yl)-2-(4-hydroxyphenyl)-6,6,8-trimethyl-5,6,7,8-tetrahydro-5,7-methanoquinazolin-8-ol was separated on a silica gel column (ethyl acetate / petroleum ether = 1 / 1, v / v) to obtain 4-(6-bromo-9-ethyl-9H-carbazol-3-yl)-2-(4-hydroxyphenyl)-6,6,8-trimethyl-5,6,7,8-tetrahydro-5,7-methanoquinazolin-8-ol in a yield of 38.7% and a purity of 98.4%. 1 H NMR (600MHz, DMSO-d6) δ: 9.84 (d, J=2.4Hz, 1H), 8.41-8.36 (m, 2H), 8.30-8.24 (m, 1H), 7.75 (dd, J=10.4, 8.5Hz, 1H ), 7.69-7.64 (m, 1H), 7.62-7.57 (m, 1H), 7.51 (ddd, J=8.3, 7.1, 1.2Hz, 1H), 7.25 (t, J=7.6Hz, 1H), 6.92-6.88 (m, 2H ), 5.29 (d, J = 3.9Hz, 1H), 4.52-4.48 (m, 2H), 3.17 (dt, J = 12.0, 5.8Hz, 1H), 2.61-2.56 (m, 1H), 2.21 (td, J = 6.0, 2.5H z, 1H), 1.91 (t, J = 9.5Hz, 1H), 1.68 (s, 3H), 1.45-1.43 (m, 3H), 1.35 (dt, J = 9.1, 7.1Hz, 3H), 0.78 (d, J = 14.0Hz, 3H). 13C NMR (150MHz, DMSO-d6) δ: 169.64, 169.58, 168.54, 162.97, 162.42, 162.21, 161.71, 161.68, 160.04, 160.02, 153.12, 140.55, 140.49, 140.23, 139.23, 134.97, 131.64, 131.57, 129.90, 129.87, 129.65, 129.49, 129.46, 129.36, 129.28, 128.95, 128.83, 128.21, 127.41, 126.64, 124.62, 123.82, 122.64, 122.54, 122.37, 121.73, 121.63, 121.12, 119.61, 115.72, 115.69, 111.85, 111.82, 109.82, 109.55, 109.24, 73.84, 73.82, 73.52, 53.11, 52.93, 52.90, 44.95, 44.54, 43.62, 42.93, 42.87, 40.41, 40.28 , 40.14, 40.00, 39.86, 39.72, 39.58, 37.79, 37.60, 31.53, 31.42, 27.57, 27.55, 27. 31, 26.76, 26.26, 24.33, 24.28, 24.13, 22.52, 14.42, 14.23, 14.18, 14.14.HRMS: m / z calculated for C 32 H 30 BrN3O2[M+H] + , 568.1601; found, 568.1600.

[0040] 3) Preparation of 4-(9-ethyl-6-(pyridin-4-yl)-9H-carbazol-3-yl)-2-(4-hydroxyphenyl)-6,6,8-trimethyl-5,6,7,8-tetrahydro-5,7-methanoquinazolin-8-ol:

[0041] 2-2.4 mmol 4-(6-bromo-9-ethyl-9H-carbazol-3-yl)-2(4-hydroxyphenyl)-6,6,8-trimethyl-5,6,7,8-tetrahydro-5,7-methanoquinazolin-8-ol, 6-7.2 mmol 4-pyridineboronic acid, 0.2-0.24 mmol tetrakis(triphenylphosphine)palladium, 9-10.8 mmol potassium carbonate, and 50-60 mL of 1,4-dioxane / water solution were added sequentially into a 100 mL dry three-necked flask and heated under reflux under nitrogen protection for 20 h. The reaction solution was distilled under reduced pressure to remove 1,4-dioxane / water solution, and ethyl acetate was added. The mixture was washed with saturated brine until neutral. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product of 4-(9-ethyl-6-(pyridin-4-yl)-9H-carbazol-3-yl)-2-(4-hydroxyphenyl)-6,6,8-trimethyl-5,6,7,8-tetrahydro-5,7-methanoquinazolin-8-ol. The crude product of 4-(9-ethyl-6-(pyridin-4-yl)-9H-carbazol-3-yl)-2-(4-hydroxyphenyl)-6,6,8-trimethyl-5,6,7,8-tetrahydro-5,7-methanoquinazolin-8-ol was separated on a silica gel column (dichloromethane / methanol = 35 / 1, v / v) to obtain 4-(9-ethyl-6-(pyridin-4-yl)-9H-carbazol-3-yl)-2-(4-hydroxyphenyl)-6,6,8-trimethyl-5,6,7,8-tetrahydro-5,7-methanoquinazolin-8-ol; the yield was 44.8% and the purity was 97.8%. 1 HNMR (600MHz, DMSO-d6) δ: 9.86 (s, 1H), 8.84 (s, 1H), 8.63 (d, J = 5.2Hz, 2H), 8.55 (s, 1H), 8.41 (d, J = 8.3Hz, 2H), 7.97 (d, J=8.8Hz, 1H), 7.87 (d, J=5.3Hz, 2H), 7.78 (q, J=8.5, 7.3Hz, 2H), 7.58 (d, J=8.5Hz, 1H), 6.92 ( d, J=8.3Hz, 2H), 5.32 (s, 1H), 4.54 (q, J=7.1Hz, 2H), 3.18 (t, J=5.7Hz, 1H), 2.60 (dt, J=10.8, 5.7Hz, 1H), 2 .21 (t, J=5.9Hz, 1H), 1.93 (d, J=9.8Hz, 1H), 1.70 (s, 3H), 1.43 (s, 3H), 1.38 (t, J=7.2Hz, 3H), 0.77 (s, 3H). 13C NMR (150MHz, DMSO-d6) δ: 169.62, 162.40, 161.74, 160.04, 150.56, 148.16 ,141.09,140.80,131.67,129.91,129.45,129.40,128.56,127.89,125.41 ,123.47,122.87,122.25,121.52,119.85,115.69,110.53,109.50,73.82 ,52.91,44.57,42.89,37.83,31.41,27.56,26.76,24.30,14.25.HRMS: m / z calculated for C 37 H 34 N4O2[M+H] + ,567.2762;found,567.2760.

[0042] Example 2

[0043] 4-(9-ethyl-6-(pyridin-4-yl)-9H-carbazol-3-yl)-2-(4-hydroxyphenyl)-6,6,8-trimethyl-5,6,7,8-tetrahydro-5,7-methanoquinazolin-8-ol was dissolved in PBS / DMF (v / v=9 / 1) buffer solution to prepare 1×10 - 5 A probe solution with a concentration of M was prepared by adding hydrochloric acid to a PBS / DMF (v / v = 9 / 1) buffer solution to prepare a solution with a pH of 1.35-12.57. The fluorescence emission spectra of 4-(9-ethyl-6-(pyridin-4-yl)-9H-carbazol-3-yl)-2-(4-hydroxyphenyl)-6,6,8-trimethyl-5,6,7,8-tetrahydro-5,7-methanoquinazolin-8-ol at different pH values ​​were measured using a standard titration method using a fluorescence spectrophotometer. Figure 1 、 Figure 2 The results show that under acidic conditions ( Figure 1 ), as the pH value in the system continues to decrease, the fluorescence emission intensity of the probe at 422nm weakens, and a new emission peak gradually increases at 514nm, and the solution changes from blue to yellow fluorescence. The pKa value of the probe is 4.28. Under alkaline conditions ( Figure 2 ), with the increasing pH value in the system, the fluorescence emission intensity of the probe at 422 nm gradually decreased, and no new emission peak appeared. The blue fluorescence of the solution gradually weakened, and the pKa value of the probe was 8.24. This shows that the probe can sensitively detect pH value.

[0044] Example 3

[0045] 4-(9-ethyl-6-(pyridin-4-yl)-9H-carbazol-3-yl)-2-(4-hydroxyphenyl)-6,6,8-trimethyl-5,6,7,8-tetrahydro-5,7-methanoquinazolin-8-ol was dissolved in PBS / DMF (v / v=9 / 1) buffer solution to prepare 1×10 - 5 The probe solution with a concentration of M was prepared by dissolving various ions in PBS / DMF (v / v=9 / 1) buffer to a concentration of 5×10 -4 M concentration solution, and adjust the pH to 3.5, 7.5, and 11.5. The fluorescence emission spectra of different ion pairs 4-(9-ethyl-6-(pyridin-4-yl)-9H-carbazol-3-yl)-2-(4-hydroxyphenyl)-6,6,8-trimethyl-5,6,7,8-tetrahydro-5,7-methanoquinazolin-8-ol were measured using a standard titration method under a fluorescence spectrophotometer, as shown in FIG. Figure 3 The results showed that the fluorescence of the probe solution changed significantly under different pH conditions, and the addition of other ions such as Al 3+ , Ca 2+ 、Cu 2+ 、Co 2+ 、Fe 3+ 、Ni + Mg 2+ 、Cd 2+ , K + 、Na + 、Zn 2+ 、La 3+ 、Mn 2+ 、Ba 2+ 、Fe 2+ 、HPO4 2 -、CrO7 2- 、SO3 2- 、SCN - PO4 3- 、NO2 - , HS - 、CO3 2- 、SO4 2- Br - 、F - , I - 、AcO - 、HCO3 - 、NO3 - etc., the fluorescence intensity ratio of the solution under acidic conditions (F 514nm / F 422nm ) and alkaline conditions, the fluorescence intensity of the solution at 422 nm did not change significantly, indicating that the probe can specifically detect pH.

Claims

1. A 2-hydroxy-3-pinonyl pyrimidine pH-sensitive fluorescent probe, characterized in that: The probe is 4-(9-ethyl-6-(pyridin-4-yl)-9H-carbazol-3-yl)-2-(4-hydroxyphenyl)-6,6,8-trimethyl-5,6,7,8-tetrahydro-5,7-methanoquinazolin-8-ol, and its structural formula is:

2. The method for preparing the 2-hydroxy-3-pinonyl pyrimidine pH-sensitive fluorescent probe according to claim 1, wherein: The steps include: 1) Using 2-hydroxy-3-pinone as a raw material, an aldol condensation reaction is carried out with 6-bromo-9-ethyl-9H-carbazole-3-carbaldehyde to obtain 4-((6-bromo-9-ethyl-9H-carbazole-3-yl)methylene)-2-hydroxy-2,6,6-trimethylbicyclo[3.1.1]heptan-3-one; 2) cyclization reaction of 4-((6-bromo-9-ethyl-9H-carbazol-3-yl)methylene)-2-hydroxy-2,6,6-trimethylbicyclo[3.1.1]heptan-3-one with 4-hydroxybenzamidine hydrochloride to prepare 4-(6-bromo-9-ethyl-9H-carbazol-3-yl)-2-(4-hydroxyphenyl)-6,6,8-trimethyl-5,6,7,8-tetrahydro-5,7-methanoquinazolin-8-ol; 3) 4-(6-bromo-9-ethyl-9H-carbazol-3-yl)-2-(4-hydroxyphenyl)-6,6,8-trimethyl-5,6,7,8-tetrahydro-5,7-methanoquinazolin-8-ol is coupled with 4-pyridineboronic acid to prepare 4-(9-ethyl-6-(pyridin-4-yl)-9H-carbazol-3-yl)-2-(4-hydroxyphenyl)-6,6,8-trimethyl-5,6,7,8-tetrahydro-5,7-methanoquinazolin-8-ol.

3. The method for preparing a 2-hydroxy-3-pinonyl pyrimidine pH-sensitive fluorescent probe according to claim 2, wherein: In step 1), the specific preparation method of 4-((6-bromo-9-ethyl-9H-carbazol-3-yl)methylene)-2-hydroxy-2,6,6-trimethylbicyclo[3.1.1]heptan-3-one is as follows: (1) 1.1-1.5 mmol of 2-hydroxy-3-pinene, 1-1.36 mmol of 6-bromo-9-ethyl-9H-carbazole-3-carbaldehyde, 3-4.08 mmol of potassium tert-butoxide, and 50-68 mL of tert-butanol were sequentially added into a 100 mL dry three-necked flask, and the mixture was refluxed for 12 h. The reaction was monitored by TLC and stopped after the reaction was complete. (2) The reaction solution was distilled under reduced pressure to remove tert-butanol, and then ethyl acetate was added. The mixture was washed with saturated brine until neutral. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product of 4-((6-bromo-9-ethyl-9H-carbazol-3-yl)methylene)-2-hydroxy-2,6,6-trimethylbicyclo[3.1.1]heptan-3-one; (3) The crude product of 4-((6-bromo-9-ethyl-9H-carbazol-3-yl)methylene)-2-hydroxy-2,6,6-trimethylbicyclo[3.1.1]heptan-3-one was separated by silica gel column with ethyl acetate / petroleum ether = 1 / 5, v / v to obtain 4-((6-bromo-9-ethyl-9H-carbazol-3-yl)methylene)-2-hydroxy-2,6,6-trimethylbicyclo[3.1.1]heptan-3-one.

4. The method for preparing a 2-hydroxy-3-pinonyl pyrimidine pH-sensitive fluorescent probe according to claim 2, wherein: In step 2), the specific preparation method of 4-(6-bromo-9-ethyl-9H-carbazol-3-yl)-2-(4-hydroxyphenyl)-6,6,8-trimethyl-5,6,7,8-tetrahydro-5,7-methanoquinazolin-8-ol is as follows: (1) 3-3.5 mmol 4-hydroxybenzamidine hydrochloride, 1.5-1.75 mL 30% sodium hydroxide solution, 1-1.17 mmol 4-((6-bromo-9-ethyl-9H-carbazol-3-yl)methylene)-2-hydroxy-2,6,6-trimethylbicyclo[3.1.1]heptan-3-one, and 6-7 mL dimethyl sulfoxide were added sequentially into a 50 mL dry three-necked flask and heated under reflux for 12 h. (2) After the reaction solution was cooled to room temperature, ethyl acetate was added and the mixture was washed with saturated brine until neutral. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product of 4-(6-bromo-9-ethyl-9H-carbazol-3-yl)-2-(4-hydroxyphenyl)-6,6,8-trimethyl-5,6,7,8-tetrahydro-5,7-methanoquinazolin-8-ol; (3) The crude product of 4-(6-bromo-9-ethyl-9H-carbazol-3-yl)-2-(4-hydroxyphenyl)-6,6,8-trimethyl-5,6,7,8-tetrahydro-5,7-methanoquinazolin-8-ol was separated on a silica gel column with ethyl acetate / petroleum ether = 1 / 1, v / v, to obtain 4-(6-bromo-9-ethyl-9H-carbazol-3-yl)-2-(4-hydroxyphenyl)-6,6,8-trimethyl-5,6,7,8-tetrahydro-5,7-methanoquinazolin-8-ol.

5. The method for preparing a 2-hydroxy-3-pinonyl pyrimidine pH-sensitive fluorescent probe according to claim 2, wherein: In step 3), the specific preparation method of 4-(9-ethyl-6-(pyridin-4-yl)-9H-carbazol-3-yl)-2-(4-hydroxypropyl)-6,6,8-trimethyl-5,6,7,8-tetrahydro-5,7-methanoquinazolin-8-ol is as follows: (1) 2-2.4 mmol 4-(6-bromo-9-ethyl-9H-carbazol-3-yl)-2-(4-hydroxyphenyl)-6,6,8-trimethyl-5,6,7,8-tetrahydro-5,7-methanoquinazolin-8-ol, 6-7.2 mmol 4-pyridineboronic acid, 0.2-0.24 mmol tetrakis(triphenylphosphine)palladium, 9-10.8 mmol potassium carbonate, and 50-60 mL 1,4-dioxane / water solution were sequentially added into a 100 mL dry three-necked flask and heated under reflux for 20 h under nitrogen protection; (2) After the reaction solution was distilled under reduced pressure to remove 1,4-dioxane / water solution, ethyl acetate was added and the mixture was washed with saturated brine until neutral. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product of 4-(9-ethyl-6-(pyridin-4-yl)-9H-carbazol-3-yl)-2-(4-hydroxyphenyl)-6,6,8-trimethyl-5,6,7,8-tetrahydro-5,7-methanoquinazolin-8-ol; (3) The crude product of 4-(9-ethyl-6-(pyridin-4-yl)-9H-carbazol-3-yl)-2-(4-hydroxyphenyl)-6,6,8-trimethyl-5,6,7,8-tetrahydro-5,7-methanoquinazolin-8-ol was separated on a silica gel column with dichloromethane / methanol = 35 / 1, v / v, to obtain 4-(9-ethyl-6-(pyridin-4-yl)-9H-carbazol-3-yl)-2-(4-hydroxyphenyl)-6,6,8-trimethyl-5,6,7,8-tetrahydro-5,7-methanoquinazolin-8-ol.

6. Use of the 4-(9-ethyl-6-(pyridin-4-yl)-9H-carbazol-3-yl)-2-(4-hydroxyphenyl)-6,6,8-trimethyl-5,6,7,8-tetrahydro-5,7-methanoquinazolin-8-ol according to claim 1 in the preparation of a fluorescent probe.

7. The use according to claim 6, characterized in that The compound 4-(9-ethyl-6-(pyridin-4-yl)-9H-carbazol-3-yl)-2-(4-hydroxyphenyl)-6,6,8-trimethyl-5,6,7,8-tetrahydro-5,7-methanoquinazolin-8-ol can specifically recognize acidic and alkaline pH. Under acidic conditions, the fluorescence color of the solution changes from blue to yellow as the pH value in the system continues to decrease; under alkaline conditions, the blue fluorescence of the solution gradually weakens as the pH value in the system continues to increase, making it suitable as a fluorescent probe for measuring pH.

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