Trigger type self-degradation polycoumarin monomer molecule and synthesis method thereof

Abstract

The invention discloses a trigger type self-degradation polycoumarin monomer molecule and a synthesis method thereof, the monomer molecule comprises a 7-hydroxy coumarin derivative and a 7-amino coumarin derivative, the structures of the 7-hydroxy coumarin derivative and the 7-amino coumarin derivative respectively contain polymerizable hydroxy / amino derivative groups, and the 7-hydroxy coumarin derivative and the 7-amino coumarin derivative are synthesized through multi-step functional group modification. Two types of coumarin derivative monomers are designed and synthesized by combining a classical small-molecular fluorophore with a novel trigger type self-degradable polymer through a high-molecular synthesis means, and a polycarbonate type polycoumarin structure is successfully prepared. The triggering type self-degradation process of polycoumarin macromolecules under the action of an irritant F <-> is explored, the target of a novel triggering type self-degradation polymer structure with classical fluorescent micromolecule coumarin as a main chain skeleton is preliminarily achieved, and the detection sensitivity of a micromolecule fluorescent probe to an analysis object is improved through a chemical method. The construction of a novel molecular amplification analysis method provides guidance and help.

Description

Technical Field

[0001] The present invention relates to the technical field of polymer synthesis and biomedical analysis, in particular to a triggered self-degradable polycoumarin monomer molecule and a synthesis method thereof. Background Art

[0002] In the field of biochemical analysis, fluorescence signal amplification technology is crucial for the detection of low-level analytes. Traditional signal amplification strategies, such as nucleic acid tool enzyme-assisted technology and nanomaterial technology, rely on biological macromolecules and have problems such as low economy, poor stability, and insufficient controllability. Triggered self-degradable polymers (SIPs) have become ideal carriers for signal amplification due to their "1:n" degradation mode. A single trigger can release n molecular signals without the need for exogenous tools. However, existing SIPs (SIFPs) with fluorescent molecules as the main chain skeleton face challenges such as harsh monomer design, short fluorescence wavelength (<510nm), and insufficient penetration depth, which limit their application in deep systems such as in vivo imaging. Therefore, it is necessary to design a triggered self-degradable polycoumarin monomer molecule and its synthesis method. Summary of the Invention

[0003] The object of the present invention is to provide a triggered self-degradable polycoumarin monomer molecule and a synthesis method thereof, so as to solve the problems raised in the above background technology.

[0004] To achieve the above objectives, the present invention provides the following technical solution: a triggered self-degradable polycoumarin monomer molecule, comprising a 7-hydroxycoumarin derivative monomer and a 7-aminocoumarin derivative monomer; the structural formula is as follows:

[0005]

[0006] Preferably, the synthesis pathway of the 7-hydroxycoumarin derivative monomer is as follows:

[0007]

[0008] Preferably, the synthesis steps are as follows:

[0009] Synthesis of compound 1b: In a round-bottom flask, 2,4-dihydroxybenzaldehyde and malononitrile were dissolved in aqueous sodium bicarbonate solution and vigorously stirred at room temperature for 4 hours. Subsequently, concentrated hydrochloric acid was added, and the mixture was stirred at 100°C for 2.5 hours. After cooling, the crude product was filtered and washed with cold water. It was further crystallized from a methanol-water mixed solvent to obtain an orange solid.

[0010] Synthesis of Compound 1c: Weighing method of Raney nickel: Place commercially available Raney nickel in a weighing bottle. After weighing, wash the nickel catalyst with ethanol and pour out the suspension again. Then, immediately disperse the weighed Raney nickel in formic acid and add it to the reaction system.

[0011] The specific synthesis steps of compound 1c are as follows: 3-hydroxy-7-cyanocoumarin is weighed and dissolved in hot formic acid. After stirring, the Raney nickel prepared by the above method is added; after the addition, the mixture is vigorously stirred at 80-90°C, and the reaction is monitored by TLC until the reaction is complete; after the reaction, the mixture is filtered through diatomaceous earth, and the filtrate is clear and transparent dark red. After half of the formic acid is removed by rotary evaporation at 50°C, the mixture is allowed to stand overnight to precipitate a yellow solid, which is then filtered and collected; the filtrate is then rotary evaporated again at 50°C to remove half of the volume, allowed to stand until a yellow solid precipitates, and filtered and collected. The solid is then combined to obtain a yellow solid.

[0012] Synthesis of Compound 1: Compound 1c was weighed and dissolved in methanol. Sodium borohydride was added in batches while stirring at room temperature. The mixture was stirred for 2 hours and the reaction was monitored by TLC. If the reaction was incomplete, a small amount of sodium borohydride was added in batches. After the reaction was completed, 1M dilute hydrochloric acid was slowly added dropwise to the reaction solution to adjust the pH of the reaction solution to neutral to slightly acidic. The solvent was removed by rotary evaporation at 45°C to obtain a yellow solid. The yellow solid was ultrasonically washed with a mixed solvent of ethyl acetate / methanol, and the supernatant was collected by centrifugation. The operation was repeated until no product was left in the supernatant. All the supernatant was collected and the solvent was removed under reduced pressure to obtain a yellow solid.

[0013] Synthesis of compound 1d: Compound 1 was weighed and dissolved in ultra-dry ethyl acetate. N,N-diisopropylethylamine was slowly added dropwise to the solution with stirring. p-nitrophenyl chloroformate was weighed and dissolved in ultra-dry ethyl acetate. The solution was slowly added dropwise to the reaction system at 0°C. After 30 minutes of reaction, the reaction progress was monitored by TLC. A white solid precipitated during the reaction and adhered to the wall of the reaction flask. After the reaction was completed, the white solid was collected by filtration, washed three times with saturated ammonium chloride solution, filtered, and dried in vacuo to obtain a white solid.

[0014] Preferably, the synthetic route of the 7-aminocoumarin derivative monomer is as follows:

[0015]

[0016] Preferably, the synthesis steps are as follows:

[0017] Synthesis of compound 7b: 3-Aminophenol, allyl bromide, and freshly distilled triethylamine were dissolved in N-methyl-2-pyrrolidone and stirred at 80°C for 3 days. During the reaction, allyl bromide and triethylamine were added to the mixture on the first and second days, respectively. After the reaction, the reaction solution was cooled to room temperature, poured into water, and extracted with diethyl ether. The organic phases were combined, washed with saturated brine, dried over anhydrous magnesium sulfate, and filtered. The solvent was evaporated under reduced pressure to obtain a crude oil, which was purified by silica gel column chromatography to obtain 7b as a light yellow oil.

[0018] Synthesis of Compound 7c: 3-(Diallylamino)phenol was dissolved in acetonitrile, and anhydrous magnesium chloride, freshly distilled triethylamine, and paraformaldehyde were added sequentially. The reaction mixture was stirred at reflux for 18 hours. After completion of the reaction, the solvent was evaporated under reduced pressure, and water and hydrochloric acid were added to the residue. After stirring and shaking for several minutes, the mixture was extracted with ethyl acetate. The combined organic phases were washed with saturated brine, dried over anhydrous magnesium sulfate, filtered, and the solvent was evaporated under reduced pressure. The crude product was purified by silica gel column chromatography to obtain a yellow oil.

[0019] Synthesis of Compound 7d: Diethyl malonate, 4-diallyl salicylaldehyde, and piperidine were dissolved in ethanol and reacted under reflux for 4 hours. After the reaction, the solvent was evaporated under reduced pressure, and a mixture of glacial acetic acid and dilute hydrochloric acid was added. The mixture was refluxed at 120°C for 13 hours. After cooling to room temperature, the reaction solution was poured into ice water and the pH was adjusted to approximately 5 by adding 40% sodium hydroxide solution. A brown precipitate was formed. The precipitate was filtered, washed four times with water, and then dried in vacuo to obtain a yellow-brown solid.

[0020] Synthesis of compound 7e: POCl3 was slowly added dropwise to ultra-dry DMF at 0°C under nitrogen. After stirring for 10 minutes, the temperature was raised to room temperature, followed by the addition of chloroform and 7-diallylaminocoumarin dissolved in ultra-dry DMF. The reaction solution was stirred at 60°C for 4 hours, cooled to room temperature, poured into crushed ice, and the pH was adjusted to 6 with sodium hydroxide solution. The reaction solution was then extracted with dichloromethane, and the combined organic phases were washed with saturated brine, dried over anhydrous magnesium sulfate, filtered, and the solvent removed under reduced pressure. The crude product was purified by silica gel column chromatography to obtain a bright orange crystalline product.

[0021] Synthesis of compound 7f: Compound 7e was dissolved in a mixed solution of tetrahydrofuran and methanol, and sodium borohydride was added portionwise at 0°C. The mixture was then stirred at 0°C for 2 hours, with the reaction progress monitored by TLC. After completion of the reaction, a saturated ammonium chloride solution was added to quench the reaction. The reaction mixture was warmed to room temperature, and the organic phase was evaporated under reduced pressure. Water was added to the residue, and the mixture was extracted with ethyl acetate. The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, and filtered. The solvent was removed under reduced pressure, and the crude product was purified by silica gel column chromatography to obtain 7f as a yellow oil.

[0022] Synthesis of Compound 7g: 3-Hydroxymethyl-7-diallylaminocoumarin and imidazole were dissolved in dichloromethane. Triisopropylsilyl chloride was slowly added dropwise at 0°C. The reaction solution was then warmed to room temperature and stirred for 16 hours. After the reaction, the mixture was filtered through celite, and the solvent was evaporated under reduced pressure. The crude product was purified by silica gel column chromatography to obtain a colorless oil.

[0023] Synthesis of compound 7h: Under nitrogen protection, compound 7g, 1,3-dimethylbarbituric acid and tetrakis(triphenylphosphine)palladium were dissolved in dichloromethane and stirred at 35°C for 16 hours. Dichloromethane was then added to the reaction solution, and the organic phase was washed with saturated sodium bicarbonate and saturated brine, dried over anhydrous magnesium sulfate, and filtered. The solvent was removed under reduced pressure, and the crude product was purified by silica gel column chromatography to obtain a white powder product.

[0024] Preferably, the synthesis of compound 7 is as follows: Compound 7h is dissolved in tetrahydrofuran, tetrabutylammonium fluoride trihydrate is added, and the reaction is stirred at room temperature for 1 hour. The progress of the reaction is monitored by TLC. After the reaction is completed, dichloromethane is added, and the organic phase is washed with water. The organic phases are combined, dried over anhydrous magnesium sulfate, and filtered. The solvent is evaporated under reduced pressure, and the crude product is purified by silica gel column chromatography to obtain a white powdery solid 7;

[0025] Synthesis of compound 7i: 3-Hydroxymethyl-7-aminocoumarin was dissolved in a mixed solvent of tetrahydrofuran / saturated sodium bicarbonate / water. Subsequently, phenyl chloroformate was added dropwise thereto. The reaction was stirred at room temperature and the progress of the reaction was monitored by TLC. After the reaction was completed, ethyl acetate was added and the organic phase was washed twice with saturated ammonium chloride. The solvent was evaporated under reduced pressure and the crude product was purified by silica gel column chromatography to obtain a white solid.

[0026] Beneficial effects:

[0027] (1) Based on the multi-reaction site characteristics of the coumarin skeleton, the present invention regulates the intramolecular charge transfer effect through structural modifications at positions 7, 1, and 4, so that the fluorescence emission wavelength covers the visible light to the near-infrared region, breaking through the limitation of the shorter wavelength of traditional technology, meeting the demand for long-wavelength fluorescence for deep tissue imaging, reducing tissue damage and increasing penetration depth.

[0028] (2) The present invention designs two types of monomers, 7-hydroxycoumarin and 7-aminocoumarin, to construct different skeleton polymers through condensation reaction. The polymers can be expanded to the fields of biosensing, drug delivery, etc. through modular modification, providing a general strategy for the design of functionalized polymers.

[0029] (3) In the present invention, the coumarin-based small molecule backbone has low toxicity and is compatible with the living environment, reducing the risk of bioaccumulation of nanomaterials. Furthermore, the degradation process directly releases fluorescent signals, eliminating the need for complex sample pretreatment. This makes it suitable for real-time in situ detection, improving analytical efficiency in clinical and environmental settings.

[0030] The above description is only an overview of the technical solutions of the embodiments of the present application. In order to more clearly understand the technical means of the embodiments of the present application, they can be implemented according to the contents of the specification. In addition, in order to make the above and other purposes, features and advantages of the embodiments of the present application more obvious and easy to understand, the specific implementation methods of the present application are specifically listed below. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is a schematic diagram of the design of a fluorescent molecular monomer based on coumarin derivatives of the present invention;

[0032] Figure 2 is the H NMR spectrum of compound 1b of the present invention;

[0033] Figure 3 is the H NMR spectrum of compound 1c of the present invention;

[0034] Figure 4 is the H NMR spectrum of compound 1 of the present invention;

[0035] Figure 5 is the H NMR spectrum of compound 1d of the present invention;

[0036] Figure 6 is the H NMR spectrum of compound 7b of the present invention;

[0037] Figure 7 is the H NMR spectrum of compound 7c of the present invention;

[0038] Figure 8 is the H NMR spectrum of compound 7d of the present invention;

[0039] Figure 9 is the H NMR spectrum of compound 7e of the present invention;

[0040] Figure 10 is the H NMR spectrum of compound 7f of the present invention;

[0041] Figure 11 This is the H NMR spectrum of compound 7g of the present invention;

[0042] Figure 12 is the H NMR spectrum of compound 7h of the present invention;

[0043] Figure 13 is the H NMR spectrum of compound 7 of the present invention;

[0044] Figure 14 is the H NMR spectrum of compound 7i of the present invention;

[0045] Figure 15 This is a characterization diagram of the structure and degradation process of the polycarbonate-type polycoumarin of the present invention. DETAILED DESCRIPTION

[0046] To make the purpose, technical solutions, and advantages of the embodiments of this application more clear, the technical solutions in the embodiments of this application will be clearly and completely described below in conjunction with the drawings in the embodiments of this application. Obviously, the described embodiments are part of the embodiments of this application, not all of the embodiments. Based on the embodiments in this application, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of this application.

[0047] In order to enable those skilled in the art to better understand the solution of the present application, the technical solution in the embodiments of the present application will be clearly and completely described below in conjunction with the accompanying drawings.

[0048] The present invention discloses a triggered self-degradable polycoumarin monomer molecule, comprising a 7-hydroxycoumarin derivative monomer and a 7-aminocoumarin derivative monomer; the structural formula is as follows:

[0049]

[0050] In the present invention, the synthesis path of 7-hydroxycoumarin derivative monomer is as follows:

[0051]

[0052] The synthesis steps are as follows:

[0053] Synthesis of Compound 1b: In a round-bottom flask, 1.38 g (10.0 mmol) of 2,4-dihydroxybenzaldehyde and 0.08 g (12.5 mmol) of malononitrile were dissolved in 50 mL of 0.05 M aqueous sodium bicarbonate solution and stirred vigorously at room temperature for 4 hours. Subsequently, 2.0 mL (24.0 mmol) of concentrated hydrochloric acid was added, and the mixture was stirred at 100°C for 2.5 hours. After cooling, the crude product was filtered and washed with cold water. It was further crystallized from a methanol-water mixture to obtain an orange solid (yield ~87%). 1 HNMR (DMSO-d6) δ: 11.35 (s, 1H), 8.79 (s, 1H), 7.65 (d, 1H, J = 8.6Hz), 6.90 (dd, 1H, J = 8.6, 2.2Hz), 6.79 (d, 1H, J = 2.2Hz).

[0054] Synthesis of compound 1c: Weighing method of Raney nickel: Place commercially available Raney nickel (water suspension) in a weighing bottle, pour out the water as much as possible, after weighing, wash the nickel catalyst with ethanol, and pour out the suspension again, then immediately disperse the weighed Raney nickel in formic acid and add it to the reaction system. The specific synthesis steps of compound 1c are as follows: Weigh 2.2g (11.7mmol) of 3-hydroxy-7-cyanocoumarin, dissolve it in hot (80-90℃) formic acid (40mL), stir and add Raney nickel prepared by the above method (3g, dispersed in 5mL formic acid), note that this process will produce hydrogen and a large amount of bubbles, so pay attention to safety and speed. After addition, the mixture is vigorously stirred at 80-90℃, and the reaction is monitored by TLC until the reaction is complete. After the reaction, the mixture was filtered through celite. The filtrate was a clear, translucent dark red color. Approximately half of the formic acid was removed by rotary evaporation at 50°C. The mixture was allowed to stand overnight, and a yellow solid precipitated. The solid was then collected by filtration. The filtrate was then rotary evaporated again at 50°C to remove approximately half of the volume. The mixture was allowed to stand until a yellow solid precipitated. The solid was then collected by filtration. The resulting product yielded 1.75 g of a yellow solid (70% yield). 1 HNMR (DMSO-d6) δ: 11.41 (s, 1H), 9.96 (s, 1H), 8.57 (s, 1H), 7.81 (d, 1H, J = 8.6Hz), 6.86 (d, 1H, J = 6.6Hz), 6.77 (s, 1H).

[0055] Synthesis of Compound 1: Weigh 1 g (5.2 mmol) of Compound 1c and dissolve it in 30 mL of methanol. With stirring, sodium borohydride was added portionwise to a final total of 95 mg (2.5 mmol). Stir at room temperature for 2 hours. Monitor the reaction by TLC. If the reaction is incomplete, add small amounts of sodium borohydride in batches. After the reaction is complete, slowly add 1 M dilute hydrochloric acid dropwise to the reaction solution to adjust the pH to neutral to slightly acidic. Remove the solvent by rotary evaporation at 45°C to obtain a yellow solid. Ultrasonicate the yellow solid with a mixture of ethyl acetate and methanol (10 / 1). Centrifuge and collect the supernatant. Repeat this process until the supernatant is free of product. Collect all the supernatant and remove the solvent under reduced pressure to obtain 0.5 g of a yellow solid (yield ~50%). 1 HNMR (DMSO-d6) δ: 10.73 (s, 1H), 7.84 (s, 1H), 7.54 (d, 1H, J = 8.4Hz), 6.83 (dd, 1H, J = 8.4, 2.3Hz), 6.80 (d, 1H, J = 2.3Hz), 4.29 (s, 2H).

[0056] Synthesis of Compound 1d: 192 mg (1 mmol) of compound 1 was dissolved in 10 mL of ultra-dry ethyl acetate. With stirring, 348 μL (2 mmol) of N,N-diisopropylethylamine was slowly added dropwise. 242 mg (1.2 mmol) of p-nitrophenyl chloroformate was dissolved in 10 mL of ultra-dry ethyl acetate and slowly added dropwise to the reaction system at 0°C. After 30 minutes of reaction, the reaction progress was monitored by TLC. A white solid precipitated during the reaction and adhered to the walls of the reaction flask. After completion of the reaction, the white solid was collected by filtration, washed three times with saturated ammonium chloride solution, filtered, and dried under vacuum to yield 250 mg of a white solid (yield ~70%). 1 H NMR (DMSO-d6) δ: 8.38 (d, 2H, J = 9.2Hz), 8.00 (s, 1H), 7.90 (d, 1H, J = 8.6Hz), 7.73 (d, 2H, J = 9. 3Hz), 7.60 (d, 1H, J = 2.4Hz), 7.44 (dd, 1H, J = 8.5, 2.3Hz), 5.75 (s, 1H), 4.37 (d, 2H, J = 1.8Hz).

[0057] In the present invention, the synthesis path of 7-aminocoumarin derivative monomer is as follows:

[0058]

[0059] The synthesis steps are as follows:

[0060] Synthesis of compound 7b: 5.0 g (46 mmol) of 3-aminophenol, 9.5 mL (110 mmol) of allyl bromide, and 30 mL of freshly distilled triethylamine (TEA) (using CaH2 as a drying agent) were dissolved in 40 mL of N-methyl-2-pyrrolidone and stirred at 80°C for 3 days. During the reaction, 4.5 mL of allyl bromide and 11 mL of triethylamine were added to the mixture on the first and second days, respectively. After the reaction, the reaction solution was cooled to room temperature, poured into 400 mL of water, and extracted with 3 × 150 mL of diethyl ether. The organic phases were combined, washed with 100 mL of saturated brine, dried over anhydrous magnesium sulfate, filtered, and the solvent was evaporated under reduced pressure. The crude product was an oil, which was purified by silica gel column chromatography to obtain 5.39 g of 7b as a light yellow oil (yield ~60%). 1 H NMR (CDCl3) δ: 7.09 (t, 1H, J = 8.1Hz), 6.30 (dd, 1H, J = 8.3, 1.6Hz), 6.24-6.10 (m, 2H), 5.90-5.75 (m, 2H), 5.25-5.10 (m, 4H), 3.90 (d, 2H, J = 4.8Hz).

[0061] Synthesis of Compound 7c: 5.39 g (28 mmol) of 3-(diallylamino)phenol (Compound 7b) was dissolved in 90 mL of acetonitrile. 6.65 g (69 mmol) of anhydrous magnesium chloride, 15 mL (108 mmol) of freshly distilled (as CaH2) triethylamine (TEA), and 5.92 g (197 mmol) of paraformaldehyde were added sequentially. The reaction mixture was stirred at reflux for 18 hours. After completion of the reaction, the solvent was evaporated under reduced pressure. 80 mL of water and 45 mL of 2M hydrochloric acid (pH ~6) were added to the residue. After stirring and shaking for several minutes, the mixture was extracted with 3 × 100 mL of ethyl acetate. The combined organic phases were washed with 100 mL of saturated brine, dried over anhydrous magnesium sulfate, filtered, and the solvent was evaporated under reduced pressure. The crude product was purified by silica gel column chromatography to obtain 3.21 g of a yellow oil (yield ~50%). 1 HNMR(CDCl3)δ:11.55(s,1H),9.51(s,1H),7.26(d,1H,J=8.9Hz),6.28(dd,1H,J=8.9,2.5Hz),6.12(d, 1H, J=2.5Hz), 5.88-5.75 (m, 2H), 5.21 (d, 2H, J=10.4Hz), 5.15 (d, 2H, J=17.1Hz), 3.97 (d, 4H, J=4.8Hz).

[0062] Synthesis of Compound 7d: 3.20 g (20 mmol) of diethyl malonate, 2.17 g (10 mmol) of 4-diallyl salicylaldehyde (Compound 7c), and 1.2 mL of piperidine were dissolved in 60 mL of ethanol and reacted under reflux for 4 hours. After the reaction, the solvent was evaporated under reduced pressure, and a mixture of 40 mL of glacial acetic acid and 40 mL of dilute hydrochloric acid was added. The mixture was refluxed at 120°C for 13 hours. After cooling to room temperature, the reaction solution was poured into 200 mL of ice water and the pH was adjusted to approximately 5 by adding 40% sodium hydroxide solution. A brown precipitate formed. The precipitate was filtered, washed four times with water, and then dried in vacuo to obtain 1.69 g of a yellow-brown solid (yield ~70%). 1 HNMR(CDCl3)δ:7.52(d,1H,J=9.3Hz),7.22(d,1H,J=8.7Hz),6.58(dd,1H,J=8.7,2.1Hz),6.53(s,1H),6.0 4 (d, 1H, J = 9.3), 5.87-5.78 (m, 2H), 5.20 (d, 2H, J = 11.4Hz), 5.16 (d, 2H, J = 18.6Hz), 3.97 (d, 4H, J = 4.2Hz).

[0063] Synthesis of compound 7e: 2.5 mL of POCl3 was slowly added dropwise to 3.4 mL of ultra-dry DMF at 0°C under nitrogen. After stirring for 10 minutes, the temperature was raised to room temperature. Subsequently, 2 mL of chloroform and 7-diallylaminocoumarin (compound 7d, 1.07 g, 4.43 mmol) dissolved in 3 mL of ultra-dry DMF were added. The reaction solution was stirred at 60°C for 4 hours, cooled to room temperature, poured into 100 g of crushed ice, and the pH was adjusted to approximately 6 with sodium hydroxide solution. The reaction solution was then extracted with 3 × 100 mL of dichloromethane. The combined organic phases were washed with 150 mL of saturated brine, dried over anhydrous magnesium sulfate, filtered, and the solvent removed under reduced pressure. The crude product was purified by silica gel column chromatography to obtain 0.96 g of bright orange crystals (yield ~80%). 1 H NMR (CDCl3) δ: 10.14 (s, 1H), 8.27 (s, 1H), 7.41 (d, 1H, J = 9.0Hz), 6.66 (dd, 1H, J = 8.9, 2.5Hz), 6.55 (d, 1H, J=2.6Hz), 5.90-5.77 (m, 2H), 5.26 (d, 2H, J=9.1Hz), 5.18 (d, 2H, J=16.0Hz), 4.04 (d, 4H, J=6.6Hz).

[0064] Synthesis of compound 7f: 910 mg (3.38 mmol) of compound 7e was dissolved in a mixture of 20 mL of tetrahydrofuran and 10 mL of methanol. Sodium borohydride (317 mg, 8.38 mmol) was added portionwise at 0°C. The mixture was then stirred at 0°C for 2 hours, with the reaction progress monitored by TLC. After completion, 2 mL of saturated ammonium chloride solution was added to quench the reaction. The mixture was allowed to warm to room temperature and the organic phase was evaporated under reduced pressure. 30 mL of water was added to the residue, and the mixture was extracted with 3 × 70 mL of ethyl acetate. The combined organic phases were washed with 100 mL of saturated brine, dried over anhydrous sodium sulfate, filtered, and the solvent removed under reduced pressure. The crude product was purified by silica gel column chromatography to afford 449 mg of 7f as a yellow oil (yield ~50%). 1 H NMR (CDCl3) δ: 7.56 (s, 1H), 7.26 (d, 1H, J = 8.7Hz), 6.61 (dd, 1H, J = 8.7, 2.6Hz), 5.90-5.75 (m, 2H), 5.21 (d, 2H, J = 10.5Hz), 5.17 (d, 2H, J = 17.2Hz).

[0065] Synthesis of Compound 7g: 249 mg (0.92 mmol) of 3-hydroxymethyl-7-diallylaminocoumarin (Compound 7f) and 186 mg (2.73 mmol) of imidazole were dissolved in 10 mL of dichloromethane. 353 mg (1.83 mmol) of triisopropylsilyl chloride was slowly added dropwise at 0°C. The reaction mixture was then warmed to room temperature and stirred for 16 hours. After completion of the reaction, the mixture was filtered through celite, the solvent was evaporated under reduced pressure, and the crude product was purified by silica gel column chromatography to obtain 367 mg of a colorless oil (yield ~90%). 1 H NMR(CDCl3)δ:7.71(s,1H),7.26(dd,1H,J=8.8Hz),6.58(dd,1H,J=8.7,2.3Hz),6.55(d,1H,J=2.0Hz),5.89- 5.74 (m, 2H), 5.22-5.10 (m, 4H), 4.67 (s, 2H), 3.95 (d, 4H, J = 4.5Hz), 1.22-1.14 (m, 3H), 1.09 and 1.08 (2×s, 18H).

[0066] Synthesis of compound 7h: Under nitrogen protection, 200 mg (0.47 mmol) of compound 7g, 438 mg (2.81 mmol) of 1,3-dimethylbarbituric acid and 14 mg (0.012 mmol) of tetrakis(triphenylphosphine)palladium were dissolved in 1.5 mL of dichloromethane and stirred at 35°C for 16 hours. Subsequently, 20 mL of dichloromethane was added to the reaction solution, and the organic phase was washed with saturated sodium bicarbonate (2×10 mL) and saturated brine (2×10 mL), dried over anhydrous magnesium sulfate, and filtered. The solvent was removed under reduced pressure, and the crude product was purified by silica gel column chromatography to obtain 133 mg of the product as a white powder (yield ~80%). 1 H NMR (CDCl3) δ: 7.72 (s, 1H), 7.24 (d, 1H, J = 8.4 Hz), 6.64 (s, 1H), 6.55 (d, 1H, J = 8.2 Hz), 4.66 (s, 2H), 4.23 (s, 2H), 1.22-1.12 (m, 3H), 1.09 and 1.07 (2×s, 18H).

[0067] Synthesis of compound 7: 347 mg (1 mmol) of compound 7h was dissolved in 10 mL of tetrahydrofuran, and 315 mg (1 mmol) of tetrabutylammonium fluoride trihydrate was added. The reaction was stirred at room temperature for 1 hour. The reaction progress was monitored by TLC. After the reaction was completed, 30 mL of dichloromethane was added, and the organic phase was washed with 3×10 mL of water. The organic phases were combined, dried over anhydrous magnesium sulfate, and filtered. The solvent was evaporated under reduced pressure, and the crude product was purified by silica gel column chromatography to obtain 7152 mg of a white powdery solid (yield ~80%). 1H NMR(DMSO-d6)δ:7.71(s,1H),7.34(d,1H,J=8.4Hz),6.53(dd,1H,J=8.3,2.1 Hz), 6.42 (s, 1H), 6.04 (s, 2H), 5.18 (t, 1H, J = 5.5Hz), 4.26 (d, 1H, J = 5.5Hz).

[0068] Synthesis of compound 7i: 191 mg (1 mmol) of 3-hydroxymethyl-7-aminocoumarin (compound 7) was dissolved in 10 mL of a mixed solvent of tetrahydrofuran / saturated sodium bicarbonate / water (volume ratio of 2:2:1). Subsequently, 150 μL (1.2 mmol) of phenyl chloroformate was added dropwise thereto. The reaction was stirred at room temperature and the progress of the reaction was monitored by TLC. After the reaction was completed, ethyl acetate was added and the organic phase was washed twice with saturated ammonium chloride. The solvent was evaporated under reduced pressure and the crude product was purified by silica gel column chromatography to obtain 248 mg of a white solid (yield ~80%). 1 H NMR(DMSO-d6)δ:10.70(s,1H),7.90(s,1H),7.72(d,2H,J=8.6Hz),7.60(s,1H),7 .50-7.39(m,3H),7.33-7.22(m,3H),5.40(t,1H,J=5.5Hz),4.34(d,2H,J=5.5Hz).

[0069] This study, based on coumarin derivative monomers 1d and 7i, allows the construction of polycarbonate- or polyurethane-type polycoumarin polymers through polycondensation. Using coumarin derivative 1d as the primary polymerization monomer, the present invention investigates the polymerization process and the triggered self-degradation properties of the resulting polycoumarin.

[0070] The synthesis process of polycarbonate-type polycoumarin polymer is as follows Figure 14 The specific synthesis process is as follows: A 10 mL Schlenk reaction tube was connected to a double-row tube and, under vacuum, flame-dried three times with a flame gun and then filled with argon. Under argon protection, 60 mg (0.17 mmol, 1 equiv) of monomer 1d and an appropriate amount of end-capping molecule c1 (2.02 mg, 0.008 mmol, 0.05 equiv) were added. Subsequently, 0.5 mL of ultra-dry toluene was added. The reaction system was degassed by freeze-drying three times and then filled with argon. Under argon protection, the reaction system was cooled to 0°C. Subsequently, 23 mg (0.19 mmol, 1.1 equiv) of 4-dimethylaminopyridine (DMAP) was added. After reacting for 30 minutes, the temperature was raised to room temperature and the reaction was continued for 1 hour. The reaction solution was extracted three times with dichloromethane and washed once with saturated ammonium chloride. The solvent was evaporated under reduced pressure to obtain the crude product (TBS-capped C-SIFP).

[0071]

[0072] By comparing the H NMR spectra of the coumarin derivative monomer 1d, the polymer TBS-capped C-SIFP and the polymer degradation product ( 1 H NMR) data can preliminarily verify the successful synthesis of the polymer and the occurrence of the self-degradation process. 1 As can be seen from the H NMR comparison diagram, after polymerization reaction occurs, the methylene characteristic peak in the hydroxymethyl in the monomer molecule moves to the low field region in the polymer, and is changed from a bimodal to a unimodal, indicating that the monomer molecule undergoes polymerization reaction to form a macromolecular main chain. Meanwhile, characteristic peaks of tert-butyl and methyl appear in the structure of the polymer, indicating that the end-capping molecule is successfully connected to the macromolecular main chain. By the integral ratio of tert-butyl and methylene hydrogen in the main chain, it can be preliminarily estimated that the macromolecular polymerization degree is about 8. In the present invention, the reaction solvent and the consumption of the alkali for the polymerization reaction of polycarbonate type polycoumarin macromolecule are also preliminarily optimized, but it is currently found that the efficiency of the polymerization reaction is relatively low, and the macromolecular product obtained is very few. In subsequent research work, by replacing a more suitable reaction solvent, increasing reactant concentration, increasing the optimization of reaction conditions such as temperature of reaction, or replacing the activated group of the coumarin molecule to make it easier to leave in the polymerization reaction, it is expected to obtain a more ideal macromolecular structure of polymerization degree.

[0073] From the fluorescence change, the polymer has almost no fluorescence in the solution, which is different from the stimulant F - After the action of fluorescein, the fluorescence was rapidly and significantly enhanced. In addition, the molecular weight distribution changes of the polymer before and after degradation were characterized by gel permeation chromatography (GPC). Figure 15 It can be clearly seen from the illustration that - After the action, the peak of the polymer disappeared, which preliminarily verified the feasibility of generating fluorescent signals by polymer degradation.

[0074] In summary, the present invention combines a classic small molecule fluorophore with a novel trigger-type self-degradable polymer, designs and synthesizes two types of coumarin derivative monomers by means of polymer synthesis, and successfully prepares a polycarbonate-type polycoumarin structure, which has a great influence on the bioactivity of the polycoumarin polymer in response to the stimulant F. - The triggered self-degradation process under the action of the chemical reaction was investigated, and the goal of a new triggered self-degradable polymer structure with the classic fluorescent small molecule coumarin as the main chain skeleton was preliminarily achieved. This provides guidance and assistance for improving the detection sensitivity of small molecule fluorescent probes to the analysis objects through chemical methods and constructing new methods for molecular amplification analysis.

[0075] As described above, the above embodiments are only used to illustrate the technical solutions of the present application, rather than to limit them. Although the present application has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. A triggered self-degradable polycoumarin monomer molecule, characterized by: It includes 7-hydroxycoumarin derivative monomer and 7-aminocoumarin derivative monomer; the structural formula is as follows:

2. A method for synthesizing a triggered self-degradable polycoumarin monomer molecule, characterized by: The synthetic pathway of the 7-hydroxycoumarin derivative monomer is as follows:

3. The method for synthesizing a triggered self-degradable polycoumarin monomer molecule according to claim 2, characterized in that: The synthesis steps are as follows: Synthesis of compound 1b: In a round-bottom flask, 2,4-dihydroxybenzaldehyde and malononitrile were dissolved in aqueous sodium bicarbonate solution and vigorously stirred at room temperature for 4 hours. Subsequently, concentrated hydrochloric acid was added, and the mixture was stirred at 100°C for 2.5 hours. After cooling, the crude product was filtered and washed with cold water. It was further crystallized from a methanol-water mixed solvent to obtain an orange solid. Synthesis of Compound 1c: Weighing method of Raney nickel: Place commercially available Raney nickel in a weighing bottle. After weighing, wash the nickel catalyst with ethanol and pour out the suspension again. Then, immediately disperse the weighed Raney nickel in formic acid and add it to the reaction system. The specific synthesis steps of compound 1c are as follows: 3-hydroxy-7-cyanocoumarin is weighed and dissolved in hot formic acid. After stirring, the Raney nickel prepared by the above method is added; after the addition, the mixture is vigorously stirred at 80-90°C, and the reaction is monitored by TLC until the reaction is complete; after the reaction, the mixture is filtered through diatomaceous earth, and the filtrate is clear and transparent dark red. After half of the formic acid is removed by rotary evaporation at 50°C, the mixture is allowed to stand overnight to precipitate a yellow solid, which is then filtered and collected; the filtrate is then rotary evaporated again at 50°C to remove half of the volume, allowed to stand until a yellow solid precipitates, and filtered and collected. The solid is then combined to obtain a yellow solid. Synthesis of Compound 1: Compound 1c was weighed and dissolved in methanol. Sodium borohydride was added in batches while stirring at room temperature. The mixture was stirred for 2 hours and the reaction was monitored by TLC. If the reaction was incomplete, a small amount of sodium borohydride was added in batches. After the reaction was completed, 1M dilute hydrochloric acid was slowly added dropwise to the reaction solution to adjust the pH of the reaction solution to neutral to slightly acidic. The solvent was removed by rotary evaporation at 45°C to obtain a yellow solid. The yellow solid was ultrasonically washed with a mixed solvent of ethyl acetate / methanol, and the supernatant was collected by centrifugation. The operation was repeated until no product was left in the supernatant. All the supernatant was collected and the solvent was removed under reduced pressure to obtain a yellow solid. Synthesis of compound 1d: Compound 1 was weighed and dissolved in ultra-dry ethyl acetate. N,N-diisopropylethylamine was slowly added dropwise to the solution with stirring. p-nitrophenyl chloroformate was weighed and dissolved in ultra-dry ethyl acetate. The solution was slowly added dropwise to the reaction system at 0°C. After 30 minutes of reaction, the reaction progress was monitored by TLC. A white solid precipitated during the reaction and adhered to the wall of the reaction flask. After the reaction was completed, the white solid was collected by filtration, washed three times with saturated ammonium chloride solution, filtered, and dried in vacuo to obtain a white solid.

4. The method for synthesizing a triggered self-degradable polycoumarin monomer molecule according to claim 2, characterized in that: The synthetic pathway of the 7-aminocoumarin derivative monomer is as follows:

5. The method for synthesizing a triggered self-degradable polycoumarin monomer molecule according to claim 4, characterized in that: The synthesis steps are as follows: Synthesis of compound 7b: 3-Aminophenol, allyl bromide, and freshly distilled triethylamine were dissolved in N-methyl-2-pyrrolidone and stirred at 80°C for 3 days. During the reaction, allyl bromide and triethylamine were added to the mixture on the first and second days, respectively. After the reaction, the reaction solution was cooled to room temperature, poured into water, and extracted with diethyl ether. The organic phases were combined, washed with saturated brine, dried over anhydrous magnesium sulfate, and filtered. The solvent was evaporated under reduced pressure to obtain a crude oil, which was purified by silica gel column chromatography to obtain 7b as a light yellow oil. Synthesis of Compound 7c: 3-(Diallylamino)phenol was dissolved in acetonitrile, and anhydrous magnesium chloride, freshly distilled triethylamine, and paraformaldehyde were added sequentially. The reaction mixture was stirred at reflux for 18 hours. After completion of the reaction, the solvent was evaporated under reduced pressure, and water and hydrochloric acid were added to the residue. After stirring and shaking for several minutes, the mixture was extracted with ethyl acetate. The combined organic phases were washed with saturated brine, dried over anhydrous magnesium sulfate, filtered, and the solvent was evaporated under reduced pressure. The crude product was purified by silica gel column chromatography to obtain a yellow oil. Synthesis of Compound 7d: Diethyl malonate, 4-diallyl salicylaldehyde, and piperidine were dissolved in ethanol and reacted under reflux for 4 hours. After the reaction, the solvent was evaporated under reduced pressure, and a mixture of glacial acetic acid and dilute hydrochloric acid was added. The mixture was refluxed at 120°C for 13 hours. After cooling to room temperature, the reaction solution was poured into ice water and the pH was adjusted to approximately 5 by adding 40% sodium hydroxide solution. A brown precipitate was formed. The precipitate was filtered, washed four times with water, and then dried in vacuo to obtain a yellow-brown solid. Synthesis of compound 7e: POCl3 was slowly added dropwise to ultra-dry DMF at 0°C under nitrogen. After stirring for 10 minutes, the temperature was raised to room temperature, followed by the addition of chloroform and 7-diallylaminocoumarin dissolved in ultra-dry DMF. The reaction solution was stirred at 60°C for 4 hours, cooled to room temperature, poured into crushed ice, and the pH was adjusted to 6 with sodium hydroxide solution. The reaction solution was then extracted with dichloromethane, and the combined organic phases were washed with saturated brine, dried over anhydrous magnesium sulfate, filtered, and the solvent removed under reduced pressure. The crude product was purified by silica gel column chromatography to obtain a bright orange crystalline product. Synthesis of compound 7f: Compound 7e was dissolved in a mixed solution of tetrahydrofuran and methanol, and sodium borohydride was added thereto in batches at 0°C; The mixture was then stirred at 0°C for 2 hours, with the reaction progress monitored by TLC. After completion of the reaction, saturated ammonium chloride solution was added to quench the reaction, and the reaction mixture was warmed to room temperature. The organic phase was evaporated under reduced pressure, and water was added to the residue, followed by extraction with ethyl acetate. The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, and filtered. The solvent was removed under reduced pressure, and the crude product was purified by silica gel column chromatography to obtain 7f as a yellow oil. Synthesis of Compound 7g: 3-Hydroxymethyl-7-diallylaminocoumarin and imidazole were dissolved in dichloromethane. Triisopropylsilyl chloride was slowly added dropwise at 0°C. The reaction solution was then warmed to room temperature and stirred for 16 hours. After the reaction, the mixture was filtered through celite, and the solvent was evaporated under reduced pressure. The crude product was purified by silica gel column chromatography to obtain a colorless oil. Synthesis of compound 7h: Under nitrogen protection, compound 7g, 1,3-dimethylbarbituric acid and tetrakis(triphenylphosphine)palladium were dissolved in dichloromethane and stirred at 35°C for 16 hours. Dichloromethane was then added to the reaction solution, and the organic phase was washed with saturated sodium bicarbonate and saturated brine, dried over anhydrous magnesium sulfate, and filtered. The solvent was removed under reduced pressure, and the crude product was purified by silica gel column chromatography to obtain a white powder product.

6. The method for synthesizing a triggered self-degradable polycoumarin monomer molecule according to claim 5, characterized in that: Synthesis of compound 7: Compound 7h was dissolved in tetrahydrofuran, and tetrabutylammonium fluoride trihydrate was added. The reaction was stirred at room temperature for 1 hour. The progress of the reaction was monitored by TLC. After the reaction was completed, dichloromethane was added, and the organic phase was washed with water. The organic phases were combined, dried over anhydrous magnesium sulfate, and filtered. The solvent was evaporated under reduced pressure, and the crude product was purified by silica gel column chromatography to obtain a white powdery solid 7. Synthesis of compound 7i: 3-Hydroxymethyl-7-aminocoumarin was dissolved in a mixed solvent of tetrahydrofuran / saturated sodium bicarbonate / water. Subsequently, phenyl chloroformate was added dropwise thereto. The reaction was stirred at room temperature and the progress of the reaction was monitored by TLC. After the reaction was completed, ethyl acetate was added and the organic phase was washed twice with saturated ammonium chloride. The solvent was evaporated under reduced pressure and the crude product was purified by silica gel column chromatography to obtain a white solid.

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