Acid-activated organic functional molecule and its preparation method and application

Abstract

The present invention discloses an acid-activated organic functional molecule and a preparation method and application, and belongs to the field of organic functional molecule technology. In view of the problem that most organic functional molecules have only a single light excitation property and cannot avoid interference with normal tissues, the present invention uses BOC-DL-leucine and p-aminobenzyl alcohol as raw materials, obtains 1-((4-(bromomethyl)phenyl)amino)-2-((tert-butylcarbonyl)amino)-4-methyl-1-oxopentane through amidation reaction and substitution reaction, and further obtains ASC-Le through substitution reaction with a semi-cyanine derivative. The organic functional molecule obtained by the present invention can simultaneously generate ROS and heat under 660nm laser irradiation, and is significantly enhanced under acidic conditions (pH5.5) to avoid interference with normal tissues. The preparation method adopted by the molecule is simple and the conditions are mild; it can specifically activate photothermal and photodynamic properties in a bacterial infection microenvironment and synergistically kill methicillin-resistant Staphylococcus aureus (MRSA) and its biofilm, and has a high application value in the treatment of drug-resistant bacteria.

Description

Technical Field

[0001] The invention belongs to the technical field of organic functional molecules, and in particular relates to an acid-activated organic functional molecule and a preparation method and application thereof. Background Art

[0002] Bacterial infections have become one of the leading causes of illness and death, posing a serious threat to global public health. The emergence of drug-resistant bacteria, in particular, has often left clinicians facing a shortage of available treatments. Therefore, there is an urgent need to develop novel therapeutic strategies and approaches to address this crisis. Optical therapy, with its advantages of being non-invasive, highly effective, and lacking resistance to drug resistance, has shown great promise in combating drug-resistant bacteria. Antimicrobial photothermal therapy (aPTT) and antimicrobial photodynamic therapy (aPDT) have been the predominant strategies used in recent years. aPTT primarily utilizes the physical pathway of photothermal conversion to kill bacteria. Under laser irradiation, photothermal molecules in an excited state return to their ground state via a nonradiative transition, generating heat that kills the bacteria. aPDT involves the generation of reactive oxygen species (ROS) by a photosensitizer under illumination, which can kill bacteria at the site of infection. Current applications have demonstrated that aPTT combined with aPDT can achieve enhanced therapeutic efficacy. This is because the heat generated by aPTT is transient and dissipates immediately after the light source is removed, whereas the ROS generated by aPDT has a relatively long retention time. Therefore, aPTT synergistically with aPDT can combine the advantages of both, using heat to increase the permeability of bacterial cell membranes, allowing the ROS generated by aPDT to better enter bacterial cells, achieving a stronger therapeutic effect. However, current research often uses the integration of multiple functional molecules, and this cannot avoid interference with normal tissue. Therefore, the purpose of the present invention is to obtain organic functional molecules that generate both ROS and heat under the same laser irradiation, and to utilize the acidic microenvironment of bacterial infection to activate aPTT and aPDT. Summary of the Invention

[0003] In response to the problem that most current organic functional molecules have only a single photoexcitation property and cannot avoid interference with normal tissues, the present invention provides an organic functional molecule and preparation method that is activated by the acidic microenvironment of bacterial infection and can synergistically fight MRSA and its biofilms with photothermal / photodynamic effects. The preparation method used for this molecule is simple to operate and has mild reaction conditions.

[0004] In order to achieve the above object, the present invention adopts the following technical solutions:

[0005] The present invention provides an acidic organic molecule ASC-Le that enhances photothermal and photodynamic properties, and its structural formula is:

[0006]

[0007] The present invention provides a method for preparing an acid-activated organic functional molecule ASC-Le, comprising the following steps:

[0008] 1. Under N2 protection, place BOC-DL-leucine, p-aminobenzyl alcohol, (7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU), and N,N-diisopropylethylamine (DIPEA) in a round-bottom flask with dichloromethane as the solvent. Stir and react at room temperature for 24 hours. After completion of the reaction, wash the organic phase with saturated NaCl, combine the organic phases, and dry over anhydrous Na2SO4. The crude product is filtered, concentrated, and purified by silica gel column chromatography to obtain a colorless oil. Subsequently, the colorless oil obtained in the previous step and phosphorus tribromide are weighed and dissolved in tetrahydrofuran and stirred at 0°C for 2 hours. After completion of the reaction, neutralize with saturated NaHCO3 solution in an ice-water bath and extract three times with ethyl acetate. Collect and combine the organic phases, dry over anhydrous MgSO4, and remove the solvent. The crude product was separated and purified by silica gel column chromatography to obtain the compound 1-((4-(bromomethyl)phenyl)amino)-2-((tert-butylcarbonyl)amino)-4-methyl-1-oxopentane;

[0009] 2. (E)-3-(2-(2-(6-hydroxy-2,3-dihydro-1H-thioxanthene-4-yl)vinyl)-3,3-dimethyl-3H-indolium-1-yl)propionate and K2CO3 were placed in a round-bottom flask, DMF was added, and the reaction was carried out at room temperature for 30 minutes. 1-((4-(bromomethyl)phenyl)amino)-2-((tert-butylcarbonyl)amino)-4-methyl-1-oxopentane was then added to the above mixture, and the reaction was continued for 10 hours. After the reaction was completed, the mixture was extracted with saturated brine and dichloromethane, dried over anhydrous Na2SO4, and the crude product was purified by silica gel column chromatography to obtain a dark blue solid. Under ice-water bath conditions, a mixture of trifluoroacetic acid and dichloromethane was added dropwise to the above dark blue solid compound. After the addition was complete, the reaction was stirred at room temperature for 2 hours. The solvent was removed, and the crude product was purified by silica gel column chromatography to obtain ASC-Le as a blue solid; the reaction formula is as follows:

[0010]

[0011] Compared with the prior art, the present invention has the following advantages:

[0012] ASC-Le, a functional molecule developed by this invention for highly effective bacterial killing, generates ROS and heat under the same laser irradiation. Its optical properties are further enhanced in the acidic microenvironment of bacterial infection. Compared to normal physiological conditions, the acidic microenvironment increases the temperature by 15°C and significantly increases ROS. The preparation method employed is simple and operates under mild conditions. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 The hydrogen nuclear magnetic resonance spectrum of the organic functional molecule ASC-Le of the present invention.

[0014] Figure 2 The carbon nuclear magnetic resonance spectrum of the organic functional molecule ASC-Le of the present invention.

[0015] Figure 3 Mass spectrum of the organic functional molecule ASC-Le of the present invention.

[0016] Figure 4 Ultraviolet absorption and fluorescence emission spectra of the organic functional molecule ASC-Le of the present invention in PBS at different pH values.

[0017] Figure 5 The photothermal temperature rise curves of the organic functional molecule ASC-Le at different concentrations and pH values ​​are shown in the figure. The wavelength of the laser is 660 nm and the optical power density is 0.75 W / cm 2 .

[0018] Figure 6 The killing performance of the organic functional molecule ASC-Le on MRSA was studied before and after 660nm laser irradiation under pH 5.5 and pH 7.4 conditions.

[0019] Figure 7 Live-dead staining of the organic functional molecule ASC-Le killing MRSA before and after 660nm laser irradiation under pH 5.5 and pH 7.4 conditions.

[0020] Figure 8 Plate images of the organic functional molecule ASC-Le of the present invention killing MRSA at pH 5.5 and pH 7.4 before and after 660nm laser irradiation and after incubation for different time periods.

[0021] Figure 9 The performance of the organic functional molecule ASC-Le of the present invention in generating ROS in MRSA before and after 660nm laser irradiation under pH 5.5 and pH 7.4 conditions was studied.

[0022] Figure 10 The killing performance of the organic functional molecule ASC-Le on MRSA biofilms was studied before and after 660nm laser irradiation under pH 5.5 and pH 7.4 conditions.

[0023] Figure 11 Crystal violet staining of the organic functional molecule ASC-Le of the present invention killing MRSA before and after 660nm laser irradiation at pH 5.5 and pH 7.4. DETAILED DESCRIPTION

[0024] Example 1

[0025] Synthesis steps of the organic functional molecule ASC-Le:

[0026] 1) Under N2 protection, BOC-DL-leucine (0.925 g, 4 mmol), HATU (1.9 g, 5 mmol), DIPEA (1.034 g, 8 mmol), and p-aminobenzyl alcohol (0.984 g, 8 mmol) were weighed into a three-necked flask and dissolved in 30 mL of dichloromethane. The mixture was allowed to react at room temperature for 24 h. After completion of the reaction, the organic phase was washed with saturated brine, combined, and dried over anhydrous Na2SO4. The solvent was removed, and the crude product was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 1:1, v / v) to obtain 1-((4-(hydroxymethyl)phenyl)amino)-2-((tert-butylcarbonyl)amino)-4-methyl-1-oxopentane (1.12 g, 82%) as a colorless oil. The resulting colorless oil (170 mg, 0.371 mmol) and phosphorus tribromide (52.3 μL, 0.556 mmol) were dissolved in 6 mL of tetrahydrofuran and stirred at 0°C for 2 h. After completion of the reaction, the mixture was neutralized with 2 mL of saturated NaHCO₃ solution under an ice-water bath, diluted with 20 mL of water, and extracted with ethyl acetate (3 × 10 mL). The organic phases were combined, dried over anhydrous MgSO₄, and the solvent was removed. The crude product was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 5:1, v / v) to yield 1-((4-(bromomethyl)phenyl)amino)-2-((tert-butylcarbonyl)amino)-4-methyl-1-oxopentane (0.559 g, 70%). 1 H NMR (400MHz, CDCl3, ppm) δ: 0.94 (dd, J = 10.6, 6.5Hz, 6H), 1.39 (s, 9H), 1.72-1.57 (m, 2H), 1.85-1.74 (m, 1H) ),4.42(s,3H),5.82(d,J=7.6Hz,1H),7.13(d,J=8.1Hz,2H),7.42(d,J=8.3Hz,2H),9.54(s,1H).MALDI-TOF for C 18 H 27 BrN2KO3 + :Calculated for437.088,439.104[M+K] + ;Foundfor437.086,439.064.

[0027] 2) In a 25 mL single-necked round-bottom flask equipped with a magnetic stirrer, (E)-3-(2-(2-(6-hydroxy-2,3-dihydro-1H-thioxanthen-4-yl)vinyl)-3,3-dimethyl-3H-indolium-1-yl)propionate (91.6 mg, 0.2 mmol), K2CO3 (55 mg, 0.4 mmol) and 5 mL of DMF were added. After stirring at room temperature for 30 minutes, 1-((4-(bromomethyl)phenyl)amino)-2-((tert-butylcarbonyl)amino)-4-methyl-1-oxopentane (80 mg, 0.2 mmol) was added to the above mixture and the reaction was continued for 10 hours. After completion of the reaction, the mixture was extracted with dichloromethane and saturated brine, the organic phases were combined and dried over anhydrous Na2SO4, and the solvent was removed. The crude product was purified by silica gel column chromatography (dichloromethane:methanol = 50:1 to 10:1, v / v) to obtain (E)-3-(2-(2-(6-((4-(2-((tert-Butoxycarbonyl)amino)-4-methylpentanamido)benzyl)oxy)-2,3-dihydro-1H-thioxanthen-4-yl)vinyl)-3,3-dimethyl-3H-indolium-1-yl)propionate (93 mg, 60%) as a dark blue solid. Under ice-water conditions, the dark blue solid compound (77.6 mg, 0.1 mmol) was mixed with 6 mL of a mixture of trifluoroacetic acid and dichloromethane (v / v = 1:3), stirred at room temperature for 2 h, and the reaction mixture was stirred. The solvent was removed, and the crude product was purified by silica gel column chromatography (dichloromethane:methanol = 8:1, v / v) to obtain ASC-Le as a blue solid (58.8 mg, 87%). 1 H NMR (400MHz, CD3OD, ppm) δ: 1.00 (d, J=5.9Hz, 6H), 1.63~1.73 (broad, 11H), 2.57 (s,2H),2.73(s,2H),2.89(s,2H),3.61(s,1H),4.40(s,2H),5.01(s,3H),6.09( t,J=25.8Hz,1H),6.82(dd,J=33.0,24.7Hz,2H),7.20(dd,J=17.1,8.0Hz,5H),7 .37(d,J=7.3Hz,2H),7.47(s,3H),7.55(d,J=7.8Hz,2H),7.98(d,J=13.1Hz,1H). 13C NMR (100MHz, CD3OD) δ173.10,171.84,170.98,170.25155.55,141.89,140.48,138.53,138.24,128.34,124.26,122.08,119 .68,112.28,110.06,99.07,66.26,53.38,43.10,39.54,31.81,31.55,27.73,25.87,24.44,22.12,20.94,20.71.MALDI-TOF for C 41 H 45 N3O4S + :Calculated for 676.3204[M+H] + ;Found for676.3204, see the results Figure 1 、 Figure 2 and Figure 3 .

[0028] Example 2

[0029] UV and fluorescence spectra of the organic functional molecule ASC-Le at different pH values:

[0030] A certain amount of ASC-Le was weighed and dissolved in methanol to prepare an ASC-Le mother solution with a concentration of 1.0 mM, and stored at -20°C for future use. PBS buffers with different pH values ​​(4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.4, 8.0, 9.0) were prepared and stored at 4°C for future use. 20 μL of ASC-Le mother solution (10 μM) was added to the PBS buffers with different pH values, mixed well, and the solution was transferred to a fluorescence cell. The UV-visible absorption spectrum was tested on a HITACHI UH5300 UV-visible spectrophotometer. The results are shown in the table. Figure 4 a. Under acidic conditions, the absorption will blue-shift and the absorption intensity will increase as the pH decreases. The fluorescence spectrum was analyzed on a HITACHI F-7100 fluorimeter with excitation and emission slits of 5nm and 10nm respectively, excitation wavelength of 725nm, and emission wavelength of 763nm. The test results are shown in Figure 4 b.

[0031] Example 3

[0032] Test of the photothermal properties of the organic functional molecule ASC-Le:

[0033] ASC-Le was prepared into solutions with concentrations of 0 μM, 5 μM, 10 μM, 20 μM, 40 μM, 60 μM, 80 μM and 100 μM in PBS (pH 5.5). The above solutions with different concentrations were placed in 500 μL centrifuge tubes and heated at 0.75 W / cm 2 The solution was irradiated with 660nm laser for 5 minutes, and the temperature of the solution was recorded every 30 seconds using an infrared thermometer and statistically analyzed. Figure 5 a, The temperature gradually increased with increasing ASC-Le concentration.

[0034] 12 μL of ASC-Le stock solution was added to PBS of different pH values ​​(5.0, 5.5, 6.5, 7.0, 7.4) to a final concentration of 60 μM. The total volume was 200 μL and placed in a 500 μL centrifuge tube. 2 The solution was irradiated with 660nm laser for 6 minutes, and the temperature of the solution was recorded every 30 seconds using an infrared thermometer and statistically analyzed. Figure 5 b, Under the same conditions, the photothermal temperature of ASC-Le under acidic conditions (pH 5.5 or 5.0) was 15°C higher than that under neutral conditions.

[0035] Example 4

[0036] Study on the antibacterial properties of organic functional molecule ASC-Le against MRSA planktonic bacteria:

[0037] 1) Bacterial culture: The antibacterial activity of ASC-Le was studied using methicillin-resistant Staphylococcus aureus (MRSA). In a clean bench, 10 mL of NB liquid was added to a clean 50 mL centrifuge tube and cultured. 10 μL of MRSA bacterial culture was then cultured at 37°C and 180 rpm for 8 to 10 hours. The bacterial culture was aspirated and centrifuged at 7200 rpm / min for 2.5 minutes. The resulting bacterial pellet was washed twice with PBS and diluted with PBS (pH 7.4 or 5.5). Finally, the OD value was adjusted. 600 Reserved for 1.0.

[0038] 2) Antibacterial test: 40 μL of the reserved bacterial solution and different volumes of ASC-Le were added to PBS (pH 7.4 or 5.5) to a final total volume of 200 μL. The concentrations of ASC-Le were 0, 0.25, 0.5, 1, 2, 4, and 8 μM. After mixing, the mixture was incubated at 37°C for 20 min. The dark-treated group was directly diluted 30,000 times with PBS (pH 7.4 or 5.5), and 100 μL was used to plate the plate. The light-treated group was illuminated at 660 nm (0.75 W / cm 2After 6 minutes of laser irradiation, the solution was diluted and plated. The plated plates were incubated at 37°C for 24 hours. After the incubation, the colonies were counted and the survival rate was calculated.

[0039] The bacterial survival rate was calculated as:

[0040] CR=X / Y×100%

[0041] Where X is the number of bacterial colonies in the experimental group treated with ASC-Le, and Y is the number of bacterial colonies in the control group without ASC-Le. Figure 6 a, Antibacterial results at pH 7.4 are shown in Figure 6 b, the corresponding flat plate diagram is shown in Figure 6 c. ASC-Le can kill almost all MRSA.

[0042] Example 5

[0043] SYTO-9 / PI live-dead staining assay:

[0044] After the antibacterial experiment, centrifuge at 10,000 rpm / min for 2.5 minutes, remove the supernatant, add 30 μL SYTO-9 and 10 μL PI, mix well, and incubate for 20 minutes. Centrifuge at 12,000 rpm / min for 2.5 minutes, discard the supernatant, add 10 μL PBS (pH 7.4 or 5.5), mix well, and take 5 μL for bacterial confocal imaging. Imaging conditions are SYTO-9: Ex, 488 nm; PI: E X , 561nm. The measurement results are shown in Figure 7 At pH 5.5, there is no green fluorescence in the SYTO-9 fluorescence channel, but there is red fluorescence in the PI channel, indicating that all the bacteria are dead; while in a pH 7.4 environment, there are both green fluorescence and red fluorescence, and they can overlap in the merged channel, indicating that the bacteria have not been completely dead.

[0045] Example 6

[0046] After the antibacterial experiment (4 μM) was completed, the cells were cultured in a 37°C incubator for 24 h, and photographed. The cells were then cultured again and photographed. Figure 8 After culturing for 36 hours, new colonies were found in the pH 7.4 group, but no new colonies were found in the pH 5.5 group.

[0047] Example 7

[0048] Detection of ROS in bacteria:

[0049] The commercial probe DCFH-DA was used to detect ROS. MRSA bacterial solution (1×10 8CFU / mL) were incubated with 10 μM DCFH-DA in 2 mL PBS (pH 5.5 or 7.4) solution for 0.5 h, and the DCFH-DA that did not enter the bacteria was removed by centrifugation. Subsequently, the bacteria were incubated with PBS (pH 5.5) containing 8 μM ASC-Le for 20 min and centrifuged at 8000 rpm / min for 5 min. After the incubation of the dark treatment group was completed, the bacteria were collected by centrifugation and resuspended in 200 μL PBS (pH 5.5 or 7.4). After the incubation of the light treatment group was completed, the bacteria were collected by centrifugation and resuspended in 200 μL PBS (pH 5.5 or 7.4) and irradiated with 0.75 W / cm 2 The treated bacterial solution was imaged using laser confocal microscopy at 488 nm for 5 minutes and 525 nm for 660 nm laser irradiation. Figure 9 a.

[0050] In vitro ROS detection: The commercial fluorescent probe DCFH-DA was used to detect ROS produced by ASC-Le under different pH conditions. The fluorescence spectrum results are shown in Figure 9 b and 9c, ASC-Le produces more ROS under acidic conditions.

[0051] Example 7

[0052] Antibacterial experiment on MRSA biofilm:

[0053] 1) Biofilm culture: The cultured bacteria were centrifuged, washed twice with PBS, and then resuspended in TSB medium containing 0.1% glucose and the OD was adjusted. 600 The concentration of the solution was 0.1, diluted 10-fold, and inoculated into a 96-well plate, 100 μL per well. Incubate at 37°C for 24 hours, and MRSA biofilm will form at the bottom of the wells.

[0054] 2) Anti-biofilm test: After the incubation period, the culture medium was aspirated and washed twice with PBS. ASC-Le solutions of different concentrations were prepared using PBS buffers at pH 7.4 and pH 5.5, respectively: 0, 20, 40, 60, 80, and 100 μM. 100 μL was then added to the prepared biofilms and incubated at 37°C for 20 min. The illumination group was treated with 0.75 W / cm 2 The cells were irradiated with a 660nm laser for 5 minutes. Ultrasonication was then performed for 20 minutes. The bacterial solution was aspirated and diluted 6000-fold, and 100 μL was plated. The dark group was directly ultrasonicated, diluted and plated, and colonies were counted after 24 hours of incubation. The bacterial survival rate was calculated as follows:

[0055] CR=X / Y×100%

[0056] Where X is the number of bacterial colonies in the experimental group treated with ASC-Le, and Y is the number of bacterial colonies in the control group without ASC-Le. Figure 10 a, Antibacterial results at pH 7.4 are shown in Figure 10 b, ASC-Le has better optical properties under acidic conditions and can effectively kill MRSA biofilms.

[0057] Example 8

[0058] Crystal violet staining test against MRSA biofilm:

[0059] The anti-MRSA biofilm experimental procedure was the same as described in Example 7, using a 48-well plate to culture bacterial biofilms. After the anti-MRSA biofilm experiment was completed, PBS was washed, 200 μL of crystal violet stain was added and allowed to stand for 15 minutes, the stain was removed, 200 μL of anhydrous ethanol was added, and the cells were incubated for 30 minutes. The results were recorded. Figure 11 , at pH 5.5, the MRSA biofilm was almost completely destroyed, while at pH 7.4, the MRSA biofilm remained almost intact.

[0060] Any matters not described in detail in this specification are prior art known to those skilled in the art. Although the above description of the present invention is based on specific embodiments to facilitate understanding of the present invention by those skilled in the art, it should be understood that the present invention is not limited to the scope of the specific embodiments. As long as various modifications are within the spirit and scope of the present invention as defined and determined by the appended claims, such modifications will be obvious to those skilled in the art, and all inventions and creations utilizing the concepts of the present invention are protected.

Claims

1. An acid-activated organic functional molecule, characterized in that: The structural formula is: ； The molecular formula is: C 41 H 45 N3O4S.

2. The method for preparing an acid-activated organic functional molecule according to claim 1, wherein: ( E )-3-(2-(2-(6-hydroxy-2,3-dihydro-1 H -thioxanthen-4-yl)vinyl)-3,3-dimethyl-3 H -indolylammonium-1-yl) propionate and a condensing agent are dissolved in DMF to react to obtain a mixture; 1-((4-(bromomethyl)phenyl)amino)-2-((tert-butylcarbonyl)amino)-4-methyl-1-oxopentane was added to the mixture, and the resulting crude product was purified by silica gel column chromatography to obtain a dark blue solid; The dark blue solid was deprotected and then purified by silica gel column chromatography to obtain a blue solid ( E )-3-(2-(2-(6-((4-(2-amino-4-methylpentanamido)benzyl)oxy)-2,3-dihydro-1 H -thioxanthen-4-yl)vinyl)-3,3-dimethyl-3 H -indolium-1-yl) propionate, ie ASC-Le.

3. The method for preparing an acid-activated organic functional molecule according to claim 2, wherein: The condensing agent is potassium carbonate.

4. The method for preparing an acid-activated organic functional molecule according to claim 2, wherein: The DMF dissolution reaction was carried out at room temperature for 30 min.

5. The method for preparing an acid-activated organic functional molecule according to claim 2, wherein: The deprotection solvent is a mixed solution of trifluoroacetic acid and dichloromethane in a volume ratio of 1:

3.

6. The method for preparing an acid-activated organic functional molecule according to claim 2, wherein: Column chromatography purification conditions were DCM:MeOH=50:1 ~ 10:1, v / v.

7. The method for preparing an acid-activated organic functional molecule according to claim 2, wherein: The deprotection condition was stirring at room temperature for 2 h.

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