Preparation method and application of perylene diimide-based bimacrocyclic compound

Abstract

The invention discloses a preparation method and application of a perylene diimide-based bimacrocyclic compound, comprising mixing an intermediate product A with TCBTA and a solvent, heating and stirring, washing with dichloromethane and water, and vacuum drying to obtain a red crude product B, mixing the intermediate product B with a hydrobromic acid solution, heating and refluxing, cooling, adding a NaHCO3 solution, filtering and washing, and drying to obtain a yellow powder C, mixing the intermediate product C with 4,4'-ethylene bipyridine or a derivative thereof, heating and stirring, centrifuging and precipitating, dissolving the precipitate in deionized water, adding an ammonium hexafluorophosphate solution, filtering, washing, and drying, and mixing with tetrabutylammonium chloride to obtain a perylene diimide-based bimacrocyclic compound. The present invention effectively prevents fluorescence quenching caused by chromophore aggregation, and simultaneously realizes PDI generated by reduction. ●– The compounds can be reduced in situ to PDI with near-infrared photothermal activity in the hypoxic microenvironment. ●– , has good near-infrared absorption properties and excellent photothermal conversion effect and biocompatibility.

Description

Technical Field

[0001] The present invention relates to the field of environmental pollutant sensing, detection and control, and in particular to a preparation method and application of a perylene diimide-based bimacrocyclic compound. Background Art

[0002] Macrocyclic compounds have become one of the main contents of supramolecular chemistry research due to their special cyclic molecular structure and excellent host-guest properties. In addition, they are increasingly valued in drug delivery / controlled release. Designing and developing new macrocyclic hosts with unique structures and good host-guest properties is a permanent and challenging topic in this field.

[0003] Hypoxia is an important feature of the microenvironment of many tumors and inflammatory tissues. Its presence often becomes one of the root causes of inducing local tumor recurrence and drug resistance. This environment often exhibits strong reductive properties. In this microenvironment, perylene diimide (PDI) and its derivatives can be reduced in situ to generate anionic free radical species (PDI ●– ), thus showing good near-infrared absorption properties and excellent photothermal conversion effect. However, due to the strong π-π stacking effect of PDI, ●– Severe aggregation in aqueous solution greatly reduces its concentration and seriously affects its therapeutic effect. Therefore, high concentration and highly stable PDI in aqueous solution is obtained. ●– However, it is still a great challenge to apply it in the biomedical field. Therefore, a preparation method and application of a perylene diimide-based bimacrocyclic compound are needed. Summary of the invention

[0004] The purpose of the present invention is to provide a water-soluble tetrachloroperylene diimide bimacrocyclic compound, a preparation method and an application thereof, so as to solve the problems existing in the above-mentioned prior art. The perylene diimide bimacrocyclic compound is reduced to anion free radicals (PDI) in the above-mentioned hypoxic environment. ●– ), and the PDI mother core is isolated through the double-sided ring formation strategy to prevent PDI ●– Aggregation greatly improves its stability. ●– It can absorb near-infrared light and convert it into heat, has a high photothermal conversion efficiency, and can achieve photothermal ablation of facultative anaerobic bacteria such as Escherichia coli. As a near-infrared photothermal agent, it has the application of photothermal sterilization.

[0005] To achieve the above object, the present invention is implemented according to the following technical solutions:

[0006] The first aspect of the present invention provides a method for preparing a perylene diimide-based bimacrocyclic compound, comprising the following steps:

[0007] 1) 1,6,7,12-tetrachloroperylene tetracarboxylic dianhydride, intermediate product A and solvent are mixed, wherein the molar volume ratio of 1,6,7,12-tetrachloroperylene tetracarboxylic dianhydride, intermediate product A and solvent is 1mmol:(2.0-8.0)mmol:(30-45)mL; after the reaction is completed by heating and stirring, the organic layer is washed with dichloromethane and water, and the solvent is removed in vacuo to obtain a red crude product, and the red crude product is separated by column chromatography to obtain a pure red solid powder intermediate product B, wherein the intermediate product A has a structure as shown in formula (I):

[0008]

[0009] 2) Mix the intermediate product B and the hydrobromic acid and acetic acid solution, wherein the mass volume ratio of the intermediate product B to the hydrobromic acid solution is 1 g: (40-70) mL, heat and stir under reflux, and after the reaction is completed, cool to room temperature, and then add NaHCO 3 The aqueous solution was filtered and washed with NaHCO 3 The precipitate was washed with the solution and deionized water for 3-5 times, and dried to obtain a yellow solid powder intermediate product C. The intermediate product B has a structure as shown in formula (II):

[0010]

[0011] 3) mixing the intermediate product C, 4,4'-ethylenedipyridine or its derivatives and acetonitrile, wherein the molar ratio of the intermediate product C to 4,4'-ethylenedipyridine or its derivatives is 1:(2.0-2.2); heating and stirring, cooling to room temperature after the reaction is completed, centrifuging the suspension after the reaction to obtain a precipitate, dissolving the precipitate in deionized water, adding a saturated aqueous solution of ammonium hexafluorophosphate to the supernatant, filtering, washing, drying, and mixing with tetrabutylammonium chloride to obtain a perylene diimide-based bimacrocyclic compound, wherein the intermediate product C has a structure as shown in formula (III):

[0012]

[0013] Furthermore, the chemical formula of the perylene diimide bimacrocyclic compound is C 80 H 54 Cl 8 N 12 O 4 , named GBox-3·4Cl, and its structure is shown in formula (IV):

[0014]

[0015] In the formula, A is a high hydration energy ion such as hexafluorophosphate ion and halogen.

[0016] Furthermore, in step 1), the heating temperature is 110-165° C. and the heating time is 12-48 hours.

[0017] Furthermore, the solvent includes propionic acid, acetic acid, butyric acid or N,N'-dimethylformamide.

[0018] Further, in step 2), the heating temperature is 150-180°C and the time is 24-60h;

[0019] Furthermore, in step 3), the heating temperature is 40-100° C. and the heating time is 24-80 hours.

[0020] On the other hand, a use of the perylene diimide-based bimacrocyclic compound in the preparation of near-infrared photothermal agents in biomedicine.

[0021] The perylene diimide-based bimacrocyclic compound can form a bacterial complex with facultative anaerobic bacteria Escherichia coli and Staphylococcus aureus, and is used to develop a near-infrared photothermal agent. The bacterial complex formed by the GBox-3·4Cl and the facultative anaerobic bacteria Escherichia coli and Staphylococcus aureus can be reduced under near-infrared light irradiation to generate PDI with photothermal activity ●– , which can absorb near-infrared light and convert it into heat to achieve bacterial photothermal ablation, and can be developed as a hypoxia-responsive near-infrared photothermal therapeutic preparation.

[0022] Compared with the prior art, the embodiments of the present invention have at least the following advantages or beneficial effects:

[0023] The perylene diimide bimacrocyclic compound of the present invention is a novel PDI compound with excellent water solubility. At the same time, due to the spatial isolation of the two-sided rings of the PDI mother core and the repulsion of the same charge, the PDI generated by reduction is effectively prevented. ●– Quenching due to aggregation.

[0024] The perylene diimide-based bimacrocyclic compound of the present invention can be reduced in situ in an oxygen-deficient microenvironment to a near-infrared photothermal active PDI. ●– , thus showing good near-infrared absorption properties and excellent photothermal conversion effects. This hypoxia-specific response can be used to integrate near-infrared photothermal treatment and diagnosis of tumors with hypoxia as an important characteristic, as well as near-infrared photothermal ablation of facultative anaerobic bacteria such as Escherichia coli that also live in a hypoxic environment.

[0025] The synthesis strategy of the present invention is novel and the raw materials are readily available, and it has good universality. It can design and synthesize bicyclic compounds with different functions without any template agent, providing a new way for the synthesis of water-soluble bicyclic macrocyclic compounds and promoting their potential application in the biomedical field. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The spectroscopic test results of aqueous solutions of different concentrations of the perylene diimide bimacrocyclic compound GBox-3·4Cl prepared by the present invention (200 μM to 2 mM, GBox-3·4Cl aqueous solution);

[0027] Wherein, a) is the UV-visible absorption spectrum; b) the molar absorption coefficient of GBox-3·4Cl;

[0028] Figure 2 This is a co-localization image of the perylene diimide bimacrocyclic compound GBox-3·4Cl prepared by the present invention and the lysosomal red fluorescent probe in RAW264.7 cells;

[0029] Among them, a) is the confocal microscopy image of GBox-3·4Cl and RAW264.7 cells after co-incubation for 2 h;

[0030] b) is a confocal microscopy image of RAW264.7 cells stained with a lysosomal red fluorescent probe;

[0031] c) is the superposition of a) and b);

[0032] Figure 3 The results of using the perylene diimide-based bimacrocyclic compound prepared by the present invention for photothermal antibacterial treatment;

[0033] Wherein, a) is a four-arm perylene diimide compound prepared in Example 2 (concentration is 0.1 mM) at 1 Wcm -2 Photothermal images of Escherichia coli treated at different times under 808nm near-infrared light;

[0034] b) Photothermal images of Staphylococcus aureus treated under the same irradiation conditions at different times;

[0035] c) is a photothermal image of Escherichia coli treated under the same irradiation conditions;

[0036] d) is a photothermal image of Staphylococcus aureus treated under the same irradiation conditions;

[0037] e) is the UV-visible absorption spectrum of the Escherichia coli solution treated with GBox-3·4Cl;

[0038] f) is the UV-visible absorption spectrum of Staphylococcus aureus solution treated with GBox-3·4Cl;

[0039] Figure 4 Schematic diagram of the temperature rise curve and photothermal image of a bacterial complex solution sample formed by co-incubating the perylene diimide bimacrocyclic compound GBox-3·4Cl prepared in the present invention with Escherichia coli and Staphylococcus aureus;

[0040] Figure 5 Schematic diagram of the temperature rise curve and photothermal image of the solid sample of the bacterial complex formed by co-incubating the perylene diimide bimacrocyclic compound GBox-3·4Cl prepared by the present invention with Escherichia coli and Staphylococcus aureus;

[0041] Figure 6 Schematic diagram of heating-cooling curves of bacterial complexes formed by the perylene diimide bimacrocyclic compound GBox-3·4Cl prepared in the present invention and Escherichia coli and Staphylococcus aureus, and the corresponding time-lnθ linear curves;

[0042] Figure 7 Schematic diagram of SEM images of the bacterial complex aqueous suspension formed by co-incubating the perylene diimide bimacrocyclic compound GBox-3·4Cl prepared in the present invention with Escherichia coli and Staphylococcus aureus before and after dispersion. DETAILED DESCRIPTION

[0043] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0044] The reaction equation is as follows:

[0045]

[0046] (1) 1,6,7,12-Tetrachloroperylenetetracarboxylic dianhydride (238.54 mg, 0.45 mmol), unpurified intermediate product A (800 mg, 2.7 mmol) and propionic acid solution (40 mL) were added to a round-bottom flask. The reaction mixture was stirred and refluxed at 155°C for 48 h under nitrogen protection. After the reaction was completed and cooled to room temperature, the organic layer was washed with dichloromethane and water, separated, and the solvent was removed in vacuo. The crude product was separated by column chromatography using dichloromethane / petroleum ether (5:1, V / V) as the eluent to obtain a red solid powder, namely, intermediate product B 0.228 g, with a yield of 38%.

[0047] The intermediate product B was analyzed by NMR and mass spectrometry, and the data are as follows:

[0048] 1 H NMR (60 MHz, DMSO-d 6,298K): δ (ppm) = 8.80 (s, 4H), 7.645 (d, J = 6.0Hz, 8H), 7.44 (d, J = 12.0Hz, 8H), 5.06 (s, 8H), 2.315 (q, J = 6.0Hz, 8H), 0.975 (t, J = 6.0Hz, 12H); 13 CNMR (100MHz, DMSO-d 6 ,298K): δ(ppm)=173.96,139.44,128.69,127.48,124.28,64.66,26.77,8.99.HRMS(ESI)m / z calcd for[B+H] + :130.2219,found:130.2224.

[0049] (2) 300 mg of intermediate B, HBr / acetic acid solution (33% w / w, 8 mL) and HBr / water solution (48% w / w, 16 mL) were added to a round-bottom flask and stirred under reflux at 110°C under nitrogen protection. The reaction was monitored by thin layer chromatography. The reaction was stopped after 18 h, cooled to room temperature, and a 5% sodium bicarbonate aqueous solution was added to precipitate a red precipitate.

[0050] The precipitate was filtered, washed with 5% aqueous sodium bicarbonate solution and deionized water (150 mL) for 3-5 times, respectively, and dried to obtain a yellow solid powder, namely, intermediate product C (200.3 mg), with a yield of 65.4%.

[0051] The data of nuclear magnetic resonance and mass spectrometry analysis of intermediate product C are as follows:

[0052] 1 H NMR (600 MHz, DMSO-d 6 , 298K): δ (ppm) = 8.77 (s, 4H), 7.615 (d, J = 6.0Hz, 8H), 7.9 (d, J = 12.0Hz, 8H), 4.63 (s, 8H); 13 C NMR (150 MHz, DMSO-d 6 ,298K): δ(ppm)=159.99,153.78,141.40,135.46,134.65,130.78,130.06,128.23,124.73,121.64,33.68.HRMS(ESI)m / z calcd for[C+H] + :1338.7651,found:1338.7717.

[0053] (3) Add intermediate product C (93.5 mg, 0.069 mmol), 4,4'-ethylenedipyridine (25.51 mg, 0.14 mmol) and acetonitrile solvent (160 mL 1:1) into a round-bottom flask, protect with nitrogen to ensure the reaction under oxygen-free conditions, and stir at 60°C for 72 h. After the reaction is completed, cool to room temperature. After the reaction, the suspension in the round-bottom flask is centrifuged to obtain a precipitate, which is dissolved in deionized water (40 mL). Saturated ammonium hexafluorophosphate aqueous solution (10 mL) is added to the supernatant to precipitate. The acetonitrile solution of the hexafluorophosphate salt of the compound is mixed with tetrabutylammonium chloride to precipitate. The precipitate is washed with acetonitrile 3-5 times and dried to obtain a light yellow solid powder, i.e., the target perylene diimide bimacrocyclic compound GBox-3·4Cl 19.3 mg, with a yield of 18%;

[0054] The data of GBox-3·4Cl NMR analysis are as follows:

[0055] 1 H NMR (600 MHz, D 2 O,298K):8.735(d,J=6.0Hz,8H),8.54(s,4H),8.875(d,J=6.0Hz,8H),7.665(d ,J=6.0Hz,8H),7.58(d,J=12.0Hz,8H),7.43(s,4H),5.75(s,8H); GBox-3·4Cl: 13 C NMR (150 MHz, DMSO-d 6 ,298K): δ (ppm) = 150.97, 146.16, 137.72, 134.44, 129.85, 128.95, 125.99, 125.95, 79.59, 62.99. HRMS (ESI) m / z calcd for [GBox-3·4Cl] 4+ :345.8297,found:345.5617; m / z calcd for[GBox-3·4Cl+Cl] 3+ :472.7511,found:472.4035.

[0056] like Figure 2The experimental process shown is as follows: an aqueous solution (855 μL) of Escherichia coli (OD600≈2.0) was incubated with a GBox-3·4Cl solution (45 μL, 2 mM) at 37°C for 10 h. The UV-visible absorption of the aqueous solution of the bacterial complex was tested, and the electron paramagnetic resonance (EPR) of the bacterial complex water suspension and the solid sample was tested, and the cyclic voltammetry (CV) of GBox-3·4Cl was scanned. Staphylococcus aureus + GBox-3·4Cl was similar to Escherichia coli + GBox-3·4Cl.

[0057] like Figure 3 Experimental procedure shown: RAW 264.7 macrophages (2×104 cells / mL, 500 μL) were seeded in each well of the confocal culture dish and cultured overnight. GBox-3·4Cl solution (30 μM in PBS, 25 μL) was added to each well and incubated for 2 hours. After incubation, cells were washed with PBS and stained with Lyso Tracker Red (lysosomal stain) at a 1:5000 dilution. Plated cells were imaged using a 63x oil immersion objective in a humidified chamber on a Leica TCS SP8 SR confocal microscope with light detectors and lasers: 488 nm diode laser for material and 552 nm HeNe laser for Lyso Tracker Red and Mito Tracker Red.

[0058] like Figure 4 The experimental process shown is as follows: Escherichia coli (OD600≈2.0) and Staphylococcus aureus aqueous solutions (855 μL) were incubated with GBox-3·4Cl solution (45 μL, 2 mM) at 37°C for 10 h. At the same time, 6 equivalents of the reducing agent Na 2 S 2 O 4 The solution was added to the aqueous solution of GBox-3·4Cl for 10 hours. The three groups of solutions and the GBox-3·4Cl solution were irradiated with 808nm near-infrared laser for 10 minutes. Staphylococcus aureus + GBox-3·4Cl was similar to Escherichia coli + GBox-3·4Cl.

[0059] like Figure 5 The experimental process shown: 808nm laser 1W / cm 2 The solid samples of GBox-3·4Cl+S. aureus and GBox-3·4Cl+E. coli with a concentration of 0.2 mM were irradiated with a power density of 5 min.

[0060] like Figure 6 The experimental process shown: 808nm laser 1W / cm 2The power density of 808 nm laser was used to irradiate the solid sample of GBox-3·4Cl+S.aureus with a concentration of 0.2 mM for 5 minutes, after which the laser was turned off and then cooled to the initial temperature (room temperature). 2 The solid samples of GBox-3·4Cl+S.aureus and GBox-3·4Cl+E.coli with a concentration of 0.2 mM were irradiated with a power density of 1000 nm, and then the laser was turned off and cooled to the initial temperature (room temperature). The laser was turned on again for the next irradiation, and the cycle was repeated 10 times.

[0061] like Figure 7 The experimental process shown: Figure 4 50% PEG-300+5% Tween-80+45% NaCl were added to the GBox-3·4Cl+S. aureus and GBox-3·4Cl+E. coli aqueous suspensions respectively.

[0062] from Figure 1 It can be seen that GBox-3·4Cl does not aggregate at a concentration as high as 2 mM and has high stability; it maintains its intrinsic fluorescence in water, indicating that GBox-3·4Cl has excellent photostability and has the potential to be used in biological imaging.

[0063] from Figure 2 It can be seen that GBox-3·4Cl was incubated with Escherichia coli and Staphylococcus aureus to form bacterial complexes, and their UV-visible spectra both produced PDI at 808nm. ●– The electron paramagnetic resonance (EPR) of aqueous solution and solid samples of the two bacterial complexes showed that there was PDI ●– The generation of PDI in the cyclic voltammetry curve (CV) ●– The characteristic potential of

[0064] like Figure 3 The image shown is a microscope image of the perylene diimide bimacrocyclic compound GBox-3·4Cl prepared by the present invention used for staining living cells. Figure 3 It can be seen that the colocalization coefficient is 0.948 and the Pearson relative coefficient is 0.937. GBox-3·4Cl is mainly localized in the lysosomes in the cells, showing good lysosomal targeting. It can be used as a fluorescent probe targeting lysosomes in living cell fluorescence imaging.

[0065] from Figure 4It can be seen that when the Escherichia coli and GBox-3·4Cl aqueous solution and the Staphylococcus aureus and GBox-3·4Cl aqueous solution were irradiated with 808nm laser, the temperature increased significantly, indicating that the bacterial complex can absorb near-infrared light in aqueous solution and convert it into heat, and can be used as a near-infrared photothermal agent for photothermal killing of bacteria.

[0066] from Figure 5 It can be seen that when the Escherichia coli and GBox-3·4Cl aqueous solution and the Staphylococcus aureus and GBox-3·4Cl solid samples were irradiated with 808nm laser, the temperature increased significantly, indicating that the bacterial complex solid can also absorb near-infrared light and convert it into heat, and can be used as a near-infrared photothermal agent for photothermal killing of bacteria.

[0067] from Figure 6 It can be seen that after the two bacterial complexes experienced 10 heating-cooling cycles, the photothermal temperature did not drop significantly, indicating that the generated free radicals had very good photothermal stability; in addition, the photothermal conversion efficiency of GBox-3·4Cl free radicals was calculated to be 57.58% through the time-lnθ linear curve and related formulas.

[0068] from Figure 7 It can be seen that both bacterial complexes achieved good dispersion effects.

[0069] The novel water-soluble tetrachloroperylene diimide-based bimacrocyclic compound of the present invention is a novel PDI compound with excellent water solubility. At the same time, due to the spatial isolation of the two-sided rings of the PDI mother core and the repulsion of the same charge, the fluorescence quenching caused by the aggregation of chromophores is effectively prevented, and the PDI generated by reduction is realized. ●– The novel water-soluble perylene diimide-based bimacrocyclic compound of the present invention has good fluorescence emission in water and can be used for fluorescence imaging of cells; at the same time, the compound can be reduced in situ in an oxygen-deficient microenvironment to PDI with near-infrared photothermal activity. ●– , thus showing good near-infrared absorption properties and excellent photothermal conversion effects, while also having good biocompatibility. This hypoxia-specific response can be used to effectively perform near-infrared photothermal ablation treatment on facultative anaerobic bacteria such as Escherichia coli and Staphylococcus aureus, which are characterized by hypoxia.

[0070] The above contents are merely examples and explanations of the structure of the present invention. The technicians in this technical field may make various modifications or additions to the specific embodiments described or replace them in a similar manner. As long as they do not deviate from the structure of the invention or exceed the scope defined by the claims, they should all fall within the protection scope of the present invention.

Claims

1. A method for preparing a perylene diimide bimacrocyclic compound, characterized in that: The steps include: 1) 1,6,7,12-tetrachloroperylene tetracarboxylic dianhydride, intermediate product A and propionic acid are mixed, wherein the molar volume ratio of 1,6,7,12-tetrachloroperylene tetracarboxylic dianhydride, intermediate product A and propionic acid is 1 mmol:(2.0-8.0) mmol:(30-45) mL; after the reaction is completed by heating and stirring, the organic layer is washed with dichloromethane and water, and the propionic acid is removed in vacuo to obtain a red crude product, and the red crude product is separated by column chromatography to obtain a pure red solid powder intermediate product B, wherein the intermediate product A has a structure as shown in formula (I): （I）； 2) Mixing the intermediate product B and hydrobromic acid acetic acid solution, wherein the mass volume ratio of the intermediate product B to the hydrobromic acid solution is 1 g: (40-70) mL, heating and stirring under reflux, after the reaction is completed, cooling to room temperature, adding NaHCO3 aqueous solution, filtering, washing the precipitate with NaHCO3 solution and deionized water 3-5 times, and drying to obtain a yellow solid powder intermediate product C, wherein the intermediate product B has a structure as shown in formula (II): (II); 3) mixing the intermediate product C, 4,4'-ethylenedipyridine or its derivatives and acetonitrile, wherein the molar ratio of the intermediate product C to 4,4'-ethylenedipyridine or its derivatives is 1:(2.0-2.2); heating and stirring, after the reaction is completed, cooling to room temperature, centrifuging the suspension after the reaction to obtain a precipitate, dissolving the precipitate in deionized water, adding a saturated aqueous solution of ammonium hexafluorophosphate to the supernatant, filtering, washing, drying, and mixing with tetrabutylammonium chloride to obtain a perylene diimide-based bimacrocyclic compound, wherein the intermediate product C has a structure as shown in formula (III): (III) .; The chemical formula of the perylene diimide bimacrocyclic compound is C 80 H 54 8N 12 O4, named GBox-3·4Cl, has a structure as shown in formula (IV): (IV) Wherein A is hexafluorophosphate ion.

2. The method for preparing the perylene diimide bimacrocyclic compound according to claim 1, characterized in that: In step 1), the heating temperature is 110-165° C. and the heating time is 12-48 hours.

3. The method for preparing the perylene diimide bimacrocyclic compound according to claim 1, characterized in that: In step 2), the heating temperature is 150-180° C. and the heating time is 24-60 h.

4. The method for preparing the perylene diimide bimacrocyclic compound according to claim 1, characterized in that: In step 3), the heating temperature is 40-100° C. and the heating time is 24-80 hours.

5. Use of the perylene diimide bimacrocyclic compound as claimed in claim 1 in preparing near-infrared photothermal preparations in biomedicine.

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