CN117683084A - Novel polypeptide coupled drug compound and preparation method and application thereof - Google Patents
Novel polypeptide coupled drug compound and preparation method and application thereof Download PDFInfo
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- CN117683084A CN117683084A CN202311701040.4A CN202311701040A CN117683084A CN 117683084 A CN117683084 A CN 117683084A CN 202311701040 A CN202311701040 A CN 202311701040A CN 117683084 A CN117683084 A CN 117683084A
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- 125000004435 hydrogen atom Chemical class [H]* 0.000 claims abstract description 3
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 claims abstract description 3
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 29
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 claims description 28
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 24
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Landscapes
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Abstract
The invention discloses a novel polypeptide coupled drug compound, a preparation method and application thereof. The structural general formula of the compound is as follows:wherein: r1 and R2 are selected from hydrogen, methyl, methoxy, chlorine, bromine and iodine; n is selected from 0,2,4,6 and 8. The compound utilizes the synergistic effect of the photodynamic sterilization effect of the antibacterial peptide and the photosensitive group to achieve the high-efficiency broad-spectrum antibacterial effect on gram-negative bacteria and gram-positive bacteria, and the MIC can reach at least 62.5nM. Meanwhile, the introduction of the photosensitive group also endows the compound with aggregation-induced luminescence characteristics, and near infrared wash-free imaging of bacteria is realized. The compound has wide application prospect in the aspects of bacterial imaging and bacterial infection resistance. The polypeptide coupled drug compound provided by the invention has the advantages of simple preparation method, cheap and easily available raw materials, simple and convenient synthesis process, easy separation and purification, and suitability for large-scale production and popularization and application.
Description
Technical Field
The invention belongs to the technical field of organic synthesis and biological medicine, and in particular relates to a novel polypeptide coupled drug compound and a preparation method and application thereof.
Background
Bacterial infections pose a serious threat to global health, causing millions of deaths each year. However, the development of new antibiotics is not stopped, and in recent years, few new antibiotics are marketed. Due to misuse and abuse of antibiotics, bacterial drug resistance is increasingly becoming more and more, and as more and more "superbacteria" are continuously present, people face a non-drug-usable environment, so that development of novel antibacterial drugs which are not easy to cause drug resistance is a very urgent task.
The antibacterial peptide (AMPs) has incomparable advantages with the traditional antibiotics, has unique antibacterial mechanism, rapid sterilization and difficult initiation of bacterial drug resistance, and is a very potential antibacterial drug. Its most widespread mechanism of action is to increase permeability or destroy the bacterial membrane, leading to extravasation of the bacterial content, leading to bacterial death. Patent CN 112625106B reports an antibacterial polypeptide compound modified by Sar based on antibacterial peptide polybia-MPI, which has the advantages of high stability and high antibacterial activity; patent CN 117024607a reports an antibacterial peptide obtained by coupling a cell penetrating peptide with an inflammation-inhibiting peptide, which can be used for common infections and some refractory infections; patent CN 117050148A reports an antimicrobial peptide MAP34-B, based on which the product prepared can inhibit a variety of microorganisms. However, these antimicrobial peptides lack the functionality to visualize bacterial detection and antimicrobial processes.
In addition, photodynamic antimicrobial therapy is emerging as an effective sterilization strategy. The main principle is that ROS generated by the photosensitizer after light irradiation is utilized to destroy bacterial cell membranes or irreversibly damage proteins and nucleic acids in bacteria, thereby achieving the aim of antibiosis. While photosensitizers typically also have a fluorescent imaging function. Most of the reported photodynamic antibacterial agents have good bactericidal effects on gram-positive bacteria, but have unsatisfactory bactericidal effects on protecting gram-negative bacteria and related drug-resistant bacteria with an inner and outer two-layer membrane structure. In addition, most photosensitizers are hydrophobic, which limits their range of application.
The polypeptide coupling medicine is a kind of conjugate obtained by connecting polypeptide with certain biological function and functional molecule through connecting wall. Therefore, it is of great significance to develop a novel polypeptide coupled drug compound with high-efficiency broad-spectrum antibacterial effect and simultaneously with the functions of visual bacteria detection and antibacterial process.
Disclosure of Invention
One of the purposes of the invention is to provide a novel polypeptide coupled drug compound with high photodynamic antibacterial activity, high biocompatibility and visual bacteria detection and antibacterial process functions.
The second object of the present invention is to provide a method for preparing the polypeptide-conjugated pharmaceutical compound.
It is a further object of the present invention to provide the use of said polypeptide conjugated pharmaceutical compounds.
In order to achieve the above purpose, the present invention provides the following technical solutions:
in a first aspect, the present invention provides a polypeptide conjugated drug compound (TPI-CysHHC 10) having a general structural formula shown in the following formula:
wherein R is 1 And R is 2 Are all selected from: hydrogen, methyl, methoxy, chloro, bromo, iodo; n is selected from 0,2,4,6 and 8.
Preferably, the structural formula of the polypeptide-conjugated drug compound is any one of the structural formulas shown in the following compounds 1 to 12:
in a second aspect, the present invention also provides a method for preparing the polypeptide-conjugated pharmaceutical compound, comprising the steps of:
(1) Synthesis of compound TPI: reacting a mixture of substituted 4-triphenylborate shown in a formula I, 4, 7-dibromo-2, 1, 3-benzothiadiazole, alkali, palladium catalyst and solvent under the protection of nitrogen, cooling to room temperature after the reaction is finished, pouring the reaction solution into water, and separating and purifying to obtain a compound TPI; the synthetic route is as follows:
(2) Synthesis of compound TPIP: reacting a mixture of a compound TPI, pyridine-4-boric acid, alkali, a palladium catalyst and a solvent under the protection of nitrogen, cooling to room temperature after the reaction is finished, pouring the reaction solution into water, and separating and purifying to obtain the compound TPIP; the synthetic route is as follows:
(3) Synthesis of Compound TPI-PN: dissolving a compound TPIP and bromoamine in a solvent, carrying out reflux reaction, cooling to room temperature after the reaction is finished, evaporating the solvent, and separating and purifying to obtain a compound TPI-PN; the synthetic route is as follows:
wherein n is selected from 0,2,4,6,8;
(4) Synthesis of Compound TPI-PA: adding a compound TPI-PN and an acid binding agent into a solvent for mixing, cooling the mixed system to 0-5 ℃, adding acryloyl chloride, stirring uniformly, reacting at room temperature, evaporating the solvent after TLC monitoring reaction is completed, and separating and purifying to obtain the compound TPI-PA; the synthetic route is as follows:
(5) Synthesis of Compound TPI-CysHHC 10: dissolving a CysHHC10 peptide in HEPES buffer solution, adding the HEPES buffer solution containing a compound TPI-PA into a reaction system, stirring at room temperature for reaction, and carrying out vacuum freeze-drying, separation and purification after the reaction is completed to obtain TPI-CysHHC10; the synthetic route is as follows:
preferably, in the step (1), the solvent may be tetrahydrofuran, methanol, 1, 4-dioxane, or other organic solvents; the base may be potassium carbonate, cesium carbonate, etc.; the palladium catalyst may be [1,1' -bis (diphenylphosphine) ferrocene ] palladium (II) dichloride, tetrakis (triphenylphosphine) palladium (0), or the like; the mol volume ratio of the substituted 4-triphenylamine borate to the solvent is 1 (3-4) mmol/mL, the feeding mol ratio of the substituted 4-triphenylamine borate to the 4, 7-dibromo-2, 1, 3-benzothiadiazole is 1 (1-2), the feeding mol ratio of the substituted 4-triphenylamine borate to the palladium catalyst is 1 (0.01-0.02), the feeding mol ratio of the substituted 4-triphenylamine borate to the alkali is 1 (1-1.5), the reaction temperature is 80-120 ℃, and the reaction time is 6-8 h.
Preferably, in step (1), the separation and purification comprises: first using CH 2 Cl 2 Extracting to obtain an organic layer, and then subjecting the organic layer to anhydrous Na 2 SO 4 Drying, spin-drying on a rotary evaporator, and separating and purifying by silica gel column chromatography; the eluent adopted in the separation and purification of the silica gel column chromatography is petroleum ether/methylene dichloride=20/1, v/v.
Preferably, in the step (2), the solvent may be tetrahydrofuran, methanol, 1, 4-dioxane, or other organic solvents; the base may be potassium carbonate, cesium carbonate, etc.; the palladium catalyst may be [1,1' -bis (diphenylphosphine) ferrocene ] palladium (II) dichloride, tetrakis (triphenylphosphine) palladium (0), or the like; the mol volume ratio of the compound TPI to the solvent is 1 (2.5-4) mmol/mL, the feeding mol ratio of the compound TPI to the pyridine-4-boric acid is 1 (1-2), the feeding mol ratio of the compound TPI to the palladium catalyst is 1 (0.01-0.02), the feeding mol ratio of the compound TPI to the alkali is 1 (1-1.5), the reaction temperature is 80-120 ℃, and the reaction time is 15-20 h.
Preferably, in step (2), the separation and purification includes: first using CH 2 Cl 2 Extracting to obtain an organic layer, and then subjecting the organic layer to anhydrous Na 2 SO 4 Drying, spin-drying on a rotary evaporator, and separating and purifying by silica gel column chromatography; the eluent adopted in the separation and purification of the silica gel column chromatography is dichloromethane.
Preferably, in the step (3), the molar volume ratio of the compound TPIP to the solvent is 1 (3-4) mmol/mL, and the feeding molar ratio of the compound TPIP to the bromoamine is 1 (1-2); the reflux reaction temperature is 80-120 ℃ and the reaction time is 4-6 h; the solvent is CH 3 A CN; the separation and purification adopts high performance liquid chromatography.
Preferably, in the step (4), the molar volume ratio of the compound TPI-PN to the solvent is 1 (15-20) mmol/mL, and the compound TPI-PN and the acryloyl chloride are fedThe molar ratio is 1 (2-3), and the feeding molar ratio of the compound TPI-PN to the acid binding agent is 1 (2-3); the reaction time is 8-12 h; the solvent is anhydrous CH 2 Cl 2 The method comprises the steps of carrying out a first treatment on the surface of the The separation and purification adopts a silica gel column chromatography method.
Further preferably, in the step (4), the acid binding agent is triethylamine, which is used for reducing the acidity of the reaction system.
Preferably, in the step (5), the feeding molar ratio of the compound TPI-PA to the CysHHC10 peptide is (1-1.5): 1; the amino acid sequence of the CysHHC10 peptide is shown as SEQ ID NO. 1, specifically CKRWWKWIRW-NH 2 The method comprises the steps of carrying out a first treatment on the surface of the The reaction time is 8-12 h; the separation and purification adopts high performance liquid chromatography.
In a third aspect, the invention also provides application of the polypeptide coupled pharmaceutical compound in bacteria wash-free imaging, bacteria aggregation induction, bacteria near infrared fluorescence imaging or as preparation of broad-spectrum antibacterial drugs.
The polypeptide coupled drug compound can be used for rapid washing-free imaging of bacteria, and has a clear imaging effect on bacterial membranes.
The polypeptide coupling drug compound can induce gram negative bacteria to generate aggregation under certain conditions, and the photodynamic antibacterial effect is enhanced.
The beneficial effects of the invention are as follows:
1. the invention covalently couples the photosensitizer and the antibacterial peptide through biological orthogonal reaction, skillfully fuses the photodynamic antibacterial effect of the photosensitive group and the antibacterial mechanism of the antibacterial peptide, realizes near infrared fluorescence imaging of drug-resistant bacteria, and simultaneously achieves broad-spectrum efficient antibacterial effect. The polypeptide coupled drug compound has the advantages of high antibacterial activity, good biocompatibility and the like.
2. The polypeptide coupled drug compound provided by the invention endows antibacterial peptide with fluorescent property through the introduction of photosensitive groups, can be used as a bacterial membrane staining reagent, and can be used for visually observing an antibacterial mechanism.
3. The polypeptide coupled drug compound provided by the invention also has aggregation-induced emission effect, can effectively avoid interference of self-absorption and biological sample background fluorescence, and can be used for near infrared fluorescence imaging of bacteria.
4. The polypeptide coupled drug compound provided by the invention utilizes the positive synergistic effect of the photodynamic sterilization effect of the antibacterial peptide and the photosensitive group, so that the antibacterial activity of the polypeptide coupled drug compound is higher than that of the photosensitive agent or the antibacterial peptide when the photosensitive agent or the antibacterial peptide is singly used. The polypeptide coupled drug compound not only has high-efficiency antibacterial activity on gram-positive bacteria, but also has effective destructiveness on outer membrane barriers of gram-negative bacteria, can inhibit bacterial biofilm growth and destroy and remove mature bacterial biofilm, has broad-spectrum antibacterial activity, and has MIC (many times of activity) on the gram-negative bacteria under illumination condition of at least 62.5nM.
5. The preparation method of the polypeptide coupled drug compound provided by the invention is simple, the raw materials are cheap and easy to obtain, the synthesis process is simple and convenient, the separation and purification are easy, the yield is high, and the preparation method is suitable for large-scale production and popularization and application.
Drawings
FIG. 1 is a chemical synthesis route of Compound 1.
FIG. 2 is a chart showing AIE effect spectrum of Compound 1.
FIG. 3 is an evaluation of singlet oxygen generating ability of Compound 1.
FIG. 4 is a fluorescence imaging of compound 1 for bacterial confocal.
FIG. 5 is an in vitro antimicrobial activity of Compound 1 on various bacteria using plate counting.
FIG. 6 is an in vitro antimicrobial activity of Compound 6 against various bacteria using plate counting.
FIG. 7 is an in vitro antibacterial activity of Compound 1 on various bacteria using bacterial dead-living staining.
FIG. 8 is a cytotoxicity evaluation of Compound 1.
FIG. 9 is a representation of the in vivo antibacterial activity of Compound 1.
FIG. 10 is a graph showing the aggregation rate characterization of different bacteria under the action of Compound 1.
FIG. 11 is a hemolysis evaluation of Compound 1.
FIG. 12 is a representation of the anti-biofilm activity of Compound 1.
Detailed Description
The invention will be further illustrated with reference to the following specific examples, but the invention is not limited to the following examples. The methods are conventional methods unless otherwise specified. The starting materials are available from published commercial sources unless otherwise specified.
In the following examples, the agent THF is tetrahydrofuran; TFA is trifluoroacetic acid; HEPES is 4-hydroxyethyl piperazine ethane sulfonic acid; ABDA is 9, 10-anthracenediyl-bis (methylene) bis-malonic acid.
Example 1 synthesis of compound 1, the synthetic scheme is shown in fig. 1, and specifically comprises the following steps:
the first step: synthesis of Compound TPI (1)
4- (Diphenylamino) phenylboronic acid (578 mg,2.00 mmol), 4, 7-dibromo-2, 1, 3-benzothiadiazole (588 mg,2.00 mmol), K 2 CO 3 (200 mg), [1,1' -bis (diphenylphosphine) ferrocene]A mixture of palladium (II) dichloride (20 mg), THF (5 mL) and MeOH (10 mL) was refluxed at 90℃for 8h under nitrogen protection, then cooled to room temperature, the reaction was poured into water, then with CH 2 Cl 2 Extraction, anhydrous Na for organic layer 2 SO 4 Drying and evaporating under reduced pressure, separating and purifying by silica gel column chromatography, eluting with petroleum ether and dichloromethane (20/1, v/v) to obtain compound TPI (1) (594 mg, yield 65%). 1 H NMR(400MHz,CDCl 3 )δppm 7.91(d,J＝7.6Hz,1H),7.83(d,J＝8.8Hz,2H),7.56(d,J＝7.6Hz,1H),7.37-7.29(m,4H),7.25-7.17(m,6H),7.11(dd,J＝11.5,4.2Hz,2H). 13 C NMR(101MHz,CDCl 3 )δppm 153.96,153.15,148.44,147.33,133.55,132.39,129.92,129.83,129.43,127.34,125.05,123.53,122.63,112.19.
And a second step of: synthesis of Compound TPIP (1)
Compound TPI (1) (910 mg,2.00 mmol), pyridine-4-boronic acid (369 mg,3.00 mmol), K 2 CO 3 (200 mg), [1,1' -bis (diphenylphosphine) ferrocene]A mixture of palladium (II) dichloride (20 mg) and THF (5 mL) was refluxed at 90℃for 20 hours under nitrogen, cooled to room temperature, poured into water, and then treated with CH 2 Cl 2 And (5) extracting. Anhydrous Na for organic layer 2 SO 4 Drying and evaporating under reduced pressure. Separating and purifying by silica gel column chromatography, eluting with dichloromethane to obtain the compoundTPIP (1) (565 mg, 62% yield). 1 H NMR(500MHz,CDCl 3 )δppm 8.79(d,J＝5.1Hz,2H),7.94(d,J＝5.1Hz,2H),7.90(d,J＝8.3Hz,2H),7.87-7.75(m,2H),7.31(t,J＝7.7Hz,4H),7.22(t,J＝8.7Hz,6H),7.10(t,J＝7.4Hz,2H). 13 C NMR(126MHz,CDCl 3 )δppm 154.03,153.60,150.17,148.49,147.32,144.70,134.65,130.13,130.10,129.46,129.17,128.98,126.87,125.10,123.58,123.50,122.56.
And a third step of: synthesis of Compound TPI-PN (1)
Compound TPIP (1) (455 mg,1.00 mmol) and 2-bromoethylamine (307 mg,1.50 mmol) were dissolved in CH 3 CN (4 mL) was stirred at 90℃for 6h. After cooling to room temperature, the CH is evaporated to dryness under reduced pressure 3 CN. By high performance liquid chromatography (solvent A: water with 0.1% TFA, solvent B: CH) 3 CN) the purified mixture was isolated and lyophilized to give compound TPI-PN (1) (yield 43%,250 mg). 1 H NMR(500MHz,DMSO-d 6 )δppm 9.08(dd,J＝102.2,6.6Hz,4H),8.52(d,J＝7.6Hz,1H),8.11(d,J＝7.6Hz,1H),8.06(d,J＝8.7Hz,2H),7.39(t,J＝7.8Hz,4H),7.13(dd,J＝18.2,8.3Hz,8H),4.92(t,J＝5.2Hz,3H),3.61(t,J＝5.3Hz,3H). 13 C NMR(126MHz,DMSO-d 6 )δppm 153.71,153.13,152.67,148.92,148.88,147.02,145.72,136.95,132.83,131.15,130.28,129.40,127.23,126.89,125.46,124.56,121.88,57.88,55.37.HRMS(m/z):calculated for C 31 H 26 BrN 5 S[M-Br] + :500.1904；found:500.2320.
Fourth step: synthesis of Compound TPI-PA (1)
Compound TPI-PN (1) (290 mg,0.50 mmol) and triethylamine (115. Mu.L, 1.00 mmol) were added to 10mL of anhydrous CH 2 Cl 2 Is a kind of medium. After the mixed system was cooled to 0 ℃, acryloyl chloride (80 μl,1.0 mmol) was added and stirred well. The reaction system was taken out of the ice-water bath and reacted at room temperature for 10 hours. After TLC was complete monitoring the reaction, the solvent was evaporated under reduced pressure, and purified by silica gel column chromatography to give compound TPI-PA (1) (184 mg, yield 58%). 1 H NMR(400MHz,DMSO-d 6 )δppm 9.19(d,J＝6.7Hz,2H),8.92(d,J＝6.5Hz,2H),8.66(t,1H),8.51(d,J＝7.6Hz,1H),8.14-8.01(m,3H),7.46-7.34(m,4H),7.22-7.08(m,8H),6.26-6.14(m,1H),6.04(dd,J＝17.1,2.1Hz,1H),5.61(dd,J＝10.1,2.1Hz,1H),4.77(t,J＝5.4Hz,2H),3.81(q,J＝5.6Hz,2H). 13 C NMR(101MHz,DMSO-d 6 )δppm 165.82,153.71,153.12,152.15,148.90,147.05,145.45,136.79,132.79,131.54,131.13,130.26,129.45,127.19,126.50,125.44,124.53,121.94,60.22,40.89.HRMS(m/z):calculated for C 34 H 28 BrN 5 OS[M-Br] + :554.2010；found:554.1982.
Fifth step: synthesis of Compound 1
CysHHC10 (30 mg,0.02 mmol) was dissolved in 3mL HEPES buffer (H 2 O dmso=1:1, 10mm, ph=8.4). To the reaction system was added 2mL of HEPES buffer containing the compound TPI-PA (1) (19 mg,0.03 mmol), and the mixture was stirred at room temperature for 10 hours. After the reaction was completed, the system was lyophilized and subjected to high performance liquid chromatography (solvent A: water containing 0.1% TFA, solvent B: CH 3 CN) was isolated and purified, and lyophilized to give compound 1 (13 mg, yield 32%). HRMS (m/z): calculated for C 111 H 135 BrN 28 O 11 S 2 [M-Br] + :2101.0335；found:[M-Br+H] 2+ :1051.0133,[M-Br+2H] 3+ :701.0117,[M-Br+3H] 4+ :526.0113,[M-Br+4H] 5+ :421.0105.
Example 2 synthesis of compound 2, specifically comprising the steps of:
the first step: synthesis of Compound TPI (2)
4-boronic acid 4',4' -Dimethyltrianiline (634 mg,2.00 mmol), 4, 7-dibromo-2, 1, 3-benzothiadiazole (588 mg,2.00 mmol), K 2 CO 3 (200 mg), [1,1' -bis (diphenylphosphine) ferrocene]A mixture of palladium (II) dichloride (20 mg), THF (5 mL) and MeOH (10 mL) was refluxed at 90℃for 8h under nitrogen, cooled to room temperature, and the reaction solution was poured into water and then taken up with CH 2 Cl 2 And (5) extracting. Anhydrous Na for organic layer 2 SO 4 Drying and evaporating under reduced pressure, separating and purifying by silica gel column chromatography, eluting with petroleum ether and dichloromethane (20/1, v/v) to obtain compound TPI (2) (584 mg, yield 60%). 1 H NMR(400MHz,CDCl 3 )δppm 7.90(d,J＝7.5Hz,1H),7.63-7.54(m,2H),7.28-7.23(m,2H),7.11-7.03(m,10H),2.35(s,6H).
And a second step of: synthesis of Compound TPIP (2)
Compound TPI (2) (972 mg,2.00 mmol), pyridine-4-boronic acid (369 mg,3.00 mmol), K 2 CO 3 (200 mg), [1,1' -bis (diphenylphosphine) ferrocene]A mixture of palladium (II) dichloride (20 mg) and THF (5 mL) was refluxed at 90℃for 20 hours under nitrogen, cooled to room temperature, poured into water, and then treated with CH 2 Cl 2 And (5) extracting. Anhydrous Na for organic layer 2 SO 4 Drying and evaporating under reduced pressure. Silica gel column chromatography was performed eluting with methylene chloride to give compound TPIP (2) (630 mg, 65% yield). 1 H NMR(500MHz,CDCl 3 )δppm 8.69-8.65(m,2H),7.84-7.82(m,2H),7.69-7.64(m,2H),7.63-7.57(m,2H),7.24-7.18(m,2H),7.10-7.00(m,9H),2.33(d,J＝0.7Hz,6H).
And a third step of: synthesis of Compound TPI-PN (2)
Compound TPIP (2) (455 mg,1.00 mmol) and 2-bromoethylamine (307 mg,1.50 mmol) were dissolved in CH 3 CN (4 mL) was stirred at 90℃for 6h. After cooling to room temperature, the CH is evaporated to dryness under reduced pressure 3 CN. By high performance liquid chromatography (solvent A: water with 0.1% TFA, solvent B: CH) 3 CN) the purified mixture was isolated and lyophilized to give compound TPI-PN (2) (322 mg, 53% yield). 1 H NMR(500MHz,DMSO-d 6 )δppm 9.34-9.28(m,2H),8.90-8.85(m,2H),7.96(d,J＝7.5Hz,1H),7.65-7.59(m,2H),7.29-7.24(m,2H),7.11-7.02(m,9H),5.05-4.99(m,2H),3.80(t,J＝7.9Hz,2H),3.56-3.46(m,2H),2.35(d,J＝0.8Hz,6H).
Fourth step: synthesis of Compound TPI-PA (2)
Compound TPI-PN (2) (290 mg,0.50 mmol) and triethylamine (115. Mu.L, 1.00 mmol) were added to 10mL of anhydrous CH 2 Cl 2 After the mixed system was cooled to 0 ℃, acryloyl chloride (80. Mu.L, 1.0 mmol) was added and stirred well. The reaction system was taken out of the ice-water bath and reacted at room temperature for 10 hours. After complete monitoring of the reaction by TLC, the solvent was evaporated under reduced pressure and purified by column chromatography on silica gel to give compound TPI-PA (2) (208 mg, yield 63%). 1 H NMR(400MHz,DMSO-d 6 )δppm 9.37-9.25(m,2H),8.86(t,J＝7.3Hz,1H),8.76-8.65(m,2H),7.96(d,J＝7.5Hz,1H),7.75(d,J＝7.5Hz,1H),7.62-7.58(m,2H),7.32-7.26(m,2H),7.10-7.02(m,9H),6.41-6.30(m,1H),5.84(dd,J＝10.1,3.1Hz,1H),5.77(dd,J＝16.7,3.1Hz,1H),5.23(t,J＝7.1Hz,2H),3.56(q,J＝7.1Hz,2H),2.26(d,J＝0.8Hz,6H).
Fifth step: synthesis of Compound 2
CysHHC10 (30 mg,0.02 mmol) was dissolved in 3mL HEPES buffer (H 2 O, dmso=1:1, 10mm, ph=8.4), 2mL of HEPES buffer containing the compound TPI-PA (2) (19 mg,0.03 mmol) was added to the reaction system, and stirred at room temperature for 10 hours. After the reaction was completed, the system was lyophilized and subjected to high performance liquid chromatography (solvent A: water containing 0.1% TFA, solvent B: CH 3 CN) was isolated and purified, and lyophilized to give a purple powder, compound 2 (13 mg, yield 30%). HRMS (m/z): calculated for C 113 H 139 BrN 28 O 11 S 2 [M-Br] + :2207.9836；found:[M-Br] + :2207.6327.
Example 3 synthesis of compound 3, comprising in particular the following steps:
the first step: synthesis of Compound TPI (3)
4-boronic acid 4',4' -Dimethoxytriphenylamine (699 mg,2.00 mmol), 4, 7-dibromo-2, 1, 3-benzothiadiazole (588 mg,2.00 mmol), K 2 CO 3 (200 mg), [1,1' -bis (diphenylphosphine) ferrocene]A mixture of palladium (II) dichloride (20 mg), THF (5 mL) and MeOH (10 mL) was refluxed at 90℃for 8h under nitrogen protection, then cooled to room temperature, the reaction was poured into water, then with CH 2 Cl 2 Extraction, anhydrous Na for organic layer 2 SO 4 Drying and evaporating under reduced pressure, separating and purifying by silica gel column chromatography, eluting with petroleum ether and dichloromethane (20/1, v/v) to obtain compound TPI (3) (584 mg, yield 60%). 1 H NMR(400MHz,CDCl 3 )δppm 7.92(d,J＝7.5Hz,1H),7.70(d,J＝7.5Hz,1H),7.59-7.55(m,2H),7.30-7.27(m,6H),6.91-6.85(m,4H),3.88(s,6H).
And a second step of: synthesis of Compound TPIP (3)
Compound TPI (3) (1037 mg,2.00 mmol), pyridine-4-boronic acid (369 mg,3.00 mmol), K 2 CO 3 (200 mg), [1,1' -bis (diphenylphosphine) ferrocene]Palladium (II) dichloride (20 mg)The mixture with THF (5 mL) was refluxed at 90℃for 20h under nitrogen, cooled to room temperature, poured into water and then treated with CH 2 Cl 2 And (5) extracting. Anhydrous Na for organic layer 2 SO 4 Drying and evaporating under reduced pressure, separating and purifying by silica gel column chromatography, eluting with dichloromethane to obtain compound TPIP (3) (640 mg, yield 62%). 1 H NMR(500MHz,CDCl 3 )δppm 8.72-8.58(m,2H),7.89(d,J＝1.1Hz,2H),7.81-7.71(m,2H),7.67-7.58(m,2H),7.33(dd,J＝7.1,1.0Hz,6H),6.92-6.86(m,4H),3.90(s,6H).
And a third step of: synthesis of Compound TPI-PN (3)
Compound TPIP (3) (516 mg,1.00 mmol) and 2-bromoethylamine (307 mg,1.50 mmol) were dissolved in CH 3 CN (4 mL) was stirred at 90℃for 6h, cooled to room temperature, and evaporated to dryness under reduced pressure 3 CN, high performance liquid chromatography (solvent A: water with 0.1% TFA, solvent B: CH) 3 CN) the purified mixture was isolated and lyophilized to give the compound TPI-PN (3) (416 mg, 65% yield). 1 H NMR(500MHz,DMSO-d 6 )δppm 9.51-9.38(m,2H),8.93-8.88(m,2H),8.15(d,J＝7.5Hz,1H),7.85(d,J＝7.5Hz,1H),7.74-7.66(m,2H),7.13-7.02(m,2H),6.92-6.75(m,4H),6.70-6.64(m,4H),5.02(t,J＝7.1Hz,2H),3.91(d,J＝2.4Hz,8H),3.42-3.31(m,2H).
Fourth step: synthesis of Compound TPI-PA (3)
Compound TPI-PN (3) (320 mg,0.50 mmol) and triethylamine (115. Mu.L, 1.00 mmol) were added to 10mL of anhydrous CH 2 Cl 2 After the mixed system was cooled to 0 ℃, acryloyl chloride (80 μl,1.0 mmol) was added and stirred uniformly, the reaction system was taken out from the ice water bath, reacted at room temperature for 10 hours, after the reaction was monitored completely by TLC, the solvent was evaporated under reduced pressure, and separated and purified by silica gel column chromatography to give the compound TPI-PA (3) (402 mg, yield 58%). 1 H NMR(400MHz,DMSO-d 6 )δppm 9.29-9.21(m,2H),8.85(t,J＝7.3Hz,1H),8.67-8.65(m,2H),7.73(d,J＝7.5Hz,1H),7.54(d,J＝7.5Hz,1H),7.41-7.2(m,2H),7.16-7.04(m,2H),6.98-6.74(m,4H),6.56-6.42(m,4H),6.22(dd,J＝16.8,10.1Hz,1H),5.85(dd,J＝10.1,3.1Hz,1H),5.63(dd,J＝16.7,3.1Hz,1H),5.13(t,J＝7.1Hz,2H),3.88(m,8H).
Fifth step: synthesis of Compound 3
CysHHC10 (30 mg,0.02 mmol) was dissolved in 3mL HEPES buffer (H 2 O, dmso=1:1, 10mm, ph=8.4), 2mL of HEPES buffer containing the compound TPI-PA (3) (21 mg,0.03 mmol) was added to the reaction system and stirred at room temperature for 10h. After the reaction was completed, the system was lyophilized and subjected to high performance liquid chromatography (solvent A: water containing 0.1% TFA, solvent B: CH 3 CN) was isolated and purified, and lyophilized to give a purple powder, compound 3 (16 mg, yield 35%). HRMS (m/z): calculated for C 113 H 139 BrN 28 O 13 S 2 [M-Br] + :2239.9735；found:[M-Br] + :2239.5627.
Example 4 synthesis of compound 4, specifically comprising the steps of:
the first step: synthesis of Compound TPI (4)
4-boronic acid 4',4' -dichlorotriphenylamine (716 mg,2.00 mmol), 4, 7-dibromo-2, 1, 3-benzothiadiazole (588 mg,2.00 mmol), K 2 CO 3 (200 mg), [1,1' -bis (diphenylphosphine) ferrocene]A mixture of palladium (II) dichloride (20 mg), THF (5 mL) and MeOH (10 mL) was refluxed at 90℃for 8h under nitrogen, cooled to room temperature, and the reaction solution was poured into water and then taken up with CH 2 Cl 2 Extraction, anhydrous Na for organic layer 2 SO 4 Drying and evaporating under reduced pressure. The mixture was purified by column chromatography on silica gel eluting with petroleum ether and methylene chloride (20/1, v/v) to give compound TPI (4) (727 mg, 69% yield). 1 H NMR(400MHz,CDCl 3 )δppm 8.21(d,J＝7.5Hz,1H),8.03(d,J＝7.5Hz,1H),7.52-7.46(m,2H),7.22-7.10(m,10H).
And a second step of: synthesis of Compound TPIP (4)
Compound TPI (4) (1054 mg,2.00 mmol), pyridine-4-boronic acid (369 mg,3.00 mmol), K 2 CO 3 (200 mg), [1,1' -bis (diphenylphosphine) ferrocene]A mixture of palladium (II) dichloride (20 mg) and THF (5 mL) was refluxed at 90℃for 20 hours under nitrogen, cooled to room temperature, poured into water, and then treated with CH 2 Cl 2 Extraction, anhydrous Na for organic layer 2 SO 4 Drying, evaporating under reduced pressure, separating and purifying by silica gel column chromatography, eluting with dichloromethane to obtain the final productTPIP (4) (578 mg, 55% yield). 1 H NMR(500MHz,CDCl 3 )δppm 8.87-8.81(m,2H),7.85(dd,J＝24.5,7.5Hz,2H),7.74-7.70(m,2H),7.69-7.63(m,2H),7.03-6.89(m,10H).
And a third step of: synthesis of Compound TPI-PN (4)
Compound TPIP (4) (525 mg,1.00 mmol) and 2-bromoethylamine (307 mg,1.50 mmol) were dissolved in CH 3 CN (4 mL) was stirred at 90℃for 6h, cooled to room temperature, and evaporated to dryness under reduced pressure 3 CN, high performance liquid chromatography (solvent A: water with 0.1% TFA, solvent B: CH) 3 CN) the purified mixture was isolated and lyophilized to give compound TPI-PN (4) (428 mg, 66% yield). 1 H NMR(500MHz,DMSO-d 6 )δppm 9.34-9.28(m,2H),9.15-9.10(m,2H),7.97(d,J＝7.5Hz,1H),7.69-7.58(m,2H),7.25-7.10(m,10H),5.05-4.95(m,2H),3.87(t,J＝7.9Hz,2H),3.53-3.46(m,2H).
Fourth step: synthesis of Compound TPI-PA (4)
Compound TPI-PN (4) (325 mg,0.50 mmol) and triethylamine (115. Mu.L, 1.00 mmol) were added to 10mL of anhydrous CH 2 Cl 2 After the mixed system was cooled to 0 ℃, acryloyl chloride (80 μl,1.0 mmol) was added and stirred uniformly, the reaction system was taken out from the ice water bath, reacted at room temperature for 10 hours, after complete monitoring of the reaction by TLC, the solvent was evaporated under reduced pressure, and separated and purified by silica gel column chromatography to give the compound TPI-PA (4) (218 mg, yield 62%). 1 H NMR(400MHz,DMSO-d 6 )δppm 9.30-9.25(m,2H),8.96(t,J＝7.3Hz,1H),8.46-8.41(m,2H),7.94(d,J＝7.3Hz,1H),7.85(d,J＝7.5Hz,1H),7.62-7.56(m,2H),7.21-7.11(m,10H),6.44(dd,J＝16.8,10.1Hz,1H),5.89(dd,J＝10.1,3.1Hz,1H),5.81(dd,J＝16.7,3.1Hz,1H),5.23(t,J＝7.1Hz,2H),3.67(q,J＝7.1Hz,2H).
Fifth step: synthesis of Compound 4
CysHHC10 (30 mg,0.02 mmol) was dissolved in 3mL HEPES buffer (H 2 O: DMSO=1:1, 10mM, pH=8.4), 2mL of HEPES buffer containing the compound TPI-PA (4) (21 mg,0.03 mmol) was added to the reaction system, and after completion of the reaction, the system was lyophilized, and subjected to high performance liquid chromatography (solvent A: water containing 0.1% TFA, solvent B: CH 3 CN) separating and purifying,after lyophilization, a purple powder was obtained, compound 4 (14 mg, yield 32%). HRMS (m/z): calculated for C 111 H 133 BrCl 2 N 28 O 11 S 2 [M-Br] + :2247.8744；found:[M-Br] + :2247.7327.
EXAMPLE 5 Synthesis of Compound 5
Synthesis of Compound 5 referring to example 1, 2-bromoethylamine in the third step was replaced with 6-bromohexylamine in equimolar amount, and the remaining reaction conditions and manner were the same as in example 1.HRMS (m/z): calculated for C 115 H 143 BrN 28 O 11 S 2 [M-Br] + :2236.0149；found:[M-Br] + :2236.1372.
EXAMPLE 6 Synthesis of Compound 6
Synthesis of Compound 6 referring to example 2, 2-bromoethylamine in the third step was replaced with 6-bromohexylamine in equimolar amount, and the remaining reaction conditions and manner were the same as in example 2.HRMS (m/z): calculated for C 117 H 147 BrN 28 O 11 S 2 [M-Br] + :2264.0462；found:[M-Br] + :2264.2931.
EXAMPLE 7 Synthesis of Compound 7
Synthesis of Compound 7 referring to example 3, 2-bromoethylamine in the third step was replaced with 6-bromohexylamine in equimolar amount, and the remaining reaction conditions and manner were the same as in example 3.HRMS (m/z): calculated for C 117 H 147 BrN 28 O 13 S 2 [M-Br] + :2296.0361；found:[M-Br] + :2296.6382.
EXAMPLE 8 Synthesis of Compound 8
Synthesis of Compound 8 referring to example 4, 2-bromoethylamine in the third step was replaced with 6-bromohexylamine in equimolar amount, and the remaining reaction conditions and manners were the same as in example 4.HRMS (m/z): calculated for C 115 H 141 BrCl 2 N 28 O 11 S 2 [M-Br] + :2303.9370；found:[M-Br] + :2303.8628.
EXAMPLE 9 Synthesis of Compound 9
Synthesis of Compound 9 reference example 1, in the third step2-bromoethylamine was replaced with an equimolar amount of 10-bromodecylamine, and the remaining reaction conditions and manners were the same as in example 1.HRMS (m/z): calculated for C 119 H 151 BrN 28 O 11 S 2 [M-Br] + :2292.0775；found:[M-Br] + :2292.1794.
EXAMPLE 10 Synthesis of Compound 10
Synthesis of Compound 10 referring to example 2, 2-bromoethylamine in the third step was replaced with 10-bromodecylamine in equimolar amount, and the remaining reaction conditions and manners were the same as in example 2.HRMS (m/z): calculated for C 121 H 155 BrN 28 O 11 S 2 [M-Br] + :2320.1088；found:[M-Br] + :2320.2619.
EXAMPLE 11 Synthesis of Compound 11
Synthesis of Compound 11 referring to example 3, 2-bromoethylamine in the third step was replaced with 10-bromodecylamine in equimolar amount, and the remaining reaction conditions and manners were the same as in example 3.HRMS (m/z): calculated for C 121 H 155 BrN 28 O 13 S 2 [M-Br] + :2352.0987；found:[M-Br] + :2352.4216.
EXAMPLE 12 Synthesis of Compound 12
Synthesis of Compound 12 referring to example 4, 2-bromoethylamine in the third step was replaced with 10-bromodecylamine in equimolar amount, and the remaining reaction conditions and manners were the same as in example 4.HRMS (m/z): calculated for C 119 H 149 BrCl 2 N 28 O 11 S 2 [M-Br] + :2359.9996；found:[M-Br] + :2359.8034.
EXAMPLE 13 AIE Effect verification of Compound 1
Compound 1 was dissolved in deionized water to prepare a mother liquor (10 mM). The mother liquor was diluted with deionized water to a concentration of 20 μm and scanned under an ultraviolet-visible spectrophotometer to obtain its absorption spectrum and its maximum absorption wavelength was measured. The mother liquor was diluted to a concentration of 10 μm with water and a 1, 4-dioxane mixed solvent (containing 0%,10%,20%,30%,40%,50%,60%,70%,80%,90%,95%, 99%) in different volume ratios, the fluorescence spectrum of each sample at the excitation wavelength of the maximum absorption wavelength was measured under a fluorescence spectrophotometer, and the maximum emission wavelength and the fluorescence intensity at the maximum emission wavelength were recorded. As shown in FIG. 2, as the proportion of the solvent 1, 4-dioxane increases, the fluorescence intensity of the compound 1 also increases. When the proportion of 1, 4-dioxane reached 99%, compound 1 was in an aggregated state, and the fluorescence intensity increased sharply, showing the apparent AIE properties of compound 1.
EXAMPLE 14 determination of the singlet oxygen production Capacity of Compound 1
Use of ABDA as a drug 1 O 2 Generating an indicator of efficiency, a mixed solution of ABDA and Compound 1 (containing ABDA: 50. Mu.M, compound 1: 10. Mu.M) was irradiated with white light (60 mW/cm) 2 ) After various times (0, 10, 20, 30, 40, 50, 60, 70, 80, 90 and 100 seconds), the ultraviolet absorption spectrum (300 to 700 nm) of each sample was measured. Estimated by comparing the degree of absorbance decrease at 378nm 1 O 2 Is not limited to the generation efficiency of the above. As shown in fig. 3, in the mixed solution of ABDA and compound 1, the absorption value of ABDA at 378nm gradually decreases with the increase of the light irradiation time, and the efficiency is superior to that of Rose Bengal (RB).
EXAMPLE 15 Compound 1 no-wash imaging experiments on bacteria
Bacteria were collected by centrifugation at 4000rpm for 3min and washed 3 times with sterile DW (deionized water). Then, bacterial pellet was resuspended with 300. Mu.L of Compound 1 (1 mM) solution and incubated for different times, followed by the addition of 10. Mu.g/mL Hoechst 33342 and incubation for another 30min. All steps were kept at room temperature. Bacteria were collected after incubation, washed with sterile DW and placed on a microscope slide, and fluorescence images were obtained with Confocal Laser Scanning Microscopy (CLSM). As shown in fig. 4, bacteria incubated with compound 1 and nucleic acid dye can be observed under CLSM, and all bacterial cell interiors showed blue fluorescence, indicating that nucleic acid inside bacteria was successfully stained, which can be used to determine the location of bacteria. In contrast, red fluorescence (compound 1) only appears on the bacterial wall, indicating that compound 1 does not enter the inside of the bacteria, but only binds to the bacterial surface to exert antibacterial effect.
EXAMPLE 16 evaluation of Compound 1-Compound 12 in vitro antibacterial Activity
Antibacterial tests were performed using gram positive bacteria including staphylococcus aureus (s. Aureus) and methicillin-resistant staphylococcus aureus (MRSA) and gram negative bacteria including escherichia coli (e.coli), multi-drug resistant escherichia coli (MDR e.coli). Single colonies were obtained by plate streaking, and 3-5 colonies were isolated from the agar plates, and cultured in a proper volume of the culture medium at 37℃and a rotation speed of 180rpm, respectively, for several hours. The bacterial concentration was monitored at 600nm with Optical Density (OD). The optical density of the bacterial solution at 600nm was adjusted to 1.0, and then a bacterial experiment was performed.
And (3) respectively measuring the antibacterial activity of the compounds 1 to 12 by adopting a traditional plate method. Bacteria were collected at 4000rpm for 5 minutes and then washed 3 times with sterile Deionized Water (DW). Then, the bacterial stock was resuspended in PBS buffer (OD 600 =1.0). The bacterial stock was diluted 1000-fold with PBS and then samples were prepared with equal volumes of compounds 1-12, respectively, for antimicrobial assays. Non-illuminated group: after the samples are incubated for 2 hours at 37 ℃ in the dark, 100 mu L of each sample is dripped on a flat plate and evenly spread; illumination group: the sample is incubated for 30min at 37 ℃ in the dark, white light irradiation is carried out for 15min, and after incubation for 75min at 37 ℃ in the dark, 100 mu L of each sample is dripped on a flat plate and evenly spread out. The viable count of each group of Colony Forming Units (CFU) was then calculated by viable count after incubation of each plate at 37 ℃ for 24h.
FIG. 5 shows the results of the in vitro antimicrobial activity of Compound 1 (light and non-light groups) against different bacteria. As shown in fig. 5, compound 1 had MIC's of 125nM, 62.5nM, and MDR e.coli for s.aureus, MRSA, e.coli, and MDR e.coli, respectively, under white light irradiation (light group), and also had better antibacterial activity against these bacteria than the conventional antibiotics (vancomycin or polymyxin B). FIG. 6 shows the results of the in vitro antimicrobial activity of compound 6 (light and non-light groups) against different bacteria. As shown in fig. 6, the MIC of compound 6 under white light irradiation (light group) for s.aureus, MRSA, e.coli and MDR e.coli was 62.5nM, 125nM, 250nM, respectively, demonstrating that compound 6 has excellent antibacterial activity against both gram-negative and gram-positive bacteria.
The results of the in vitro antimicrobial experiments of compounds 1-12 under white light irradiation (light group) are shown in Table 1.
Table 1 results of in vitro antibacterial experiments of Compounds 1 to 12 under white light irradiation (light group)
EXAMPLE 17 Compound 1 dead and alive staining experiment on bacteria
Illumination group: mu.L of Compound 1 stock solution (1 mM) was added to 1mL of 10-concentration 9 The CFU/mL of different bacteria (Escherichia coli, staphylococcus aureus and methicillin-resistant Staphylococcus aureus) were mixed with the antibacterial agent, incubated at 37deg.C for 30min, and white light (60 mW/cm) 2 ) After the completion of the irradiation, 1. Mu.L of Hoechst 33342 (10 mg/mL) and 1. Mu. LYO-PRO-1 (1 mM) were added and mixed uniformly, and after incubation for 10min, the slide was prepared and observed under a confocal fluorescence microscope. Non-illuminated group: mu.L of Compound 1 stock solution (1 mM) was added to 1mL of 10-concentration 9 The preparation method comprises the steps of obtaining a mixture of bacteria and an antibacterial agent in different bacteria (escherichia coli, staphylococcus aureus and methicillin-resistant staphylococcus aureus) of CFU/mL, incubating at 37 ℃ for 1 hour, adding 1 mu L of Hoechst 33342 (10 mg/mL) and 1 mu LYO-PRO-1 (1 mM), uniformly mixing, incubating for 10min, and preparing a slide for observation under a confocal fluorescence microscope. Hoechst 33342 stains all bacteria, whereas YO-PRO-1 stains only dead bacteria, so that a stronger fluorescence intensity of the YO-PRO-1 channel indicates a greater number of dead bacteria in the sample. As shown in FIG. 7, for the non-illuminated group, the green fluorescence (fluorescent signal of YO-PRO-1 for staining dead bacteria) was not well matched with the blue fluorescence (fluorescent signal of Hoechst 33342 by which all bacterial nucleic acids could be stained). In contrast, for the white light-irradiated group, almost all green fluorescent signals match well with blue fluorescent signals, indicating that ROS generated under light irradiation play a crucial role in the bacteriostatic activity of compound 1.
Example 18 in vitro cytotoxicity assay of Compound 1
HeLa cells were cultured in DMEM medium containing 10% fetal bovine serum, and the flask was placed in a 37℃incubator containing 5% carbon dioxide. When cell growth was observed to be near confluent, cells were isolated with 0.25% pancreatin and plated in 96-well plates (about 5X 10 per well) 3 Cells), placed in the above incubator for 24 hours. After removal of the medium, 1100 μl of compound dissolved in the medium at different concentrations was added to each well. MTT assay measures cell viability. mu.L of fresh medium containing 10. Mu.L of MTT stock solution (5 mg/mL) was added to each well and incubated at 37℃for 4h. After removal of the medium per well, 100. Mu.L of DMSO was added to dissolve crystal violet and the absorbance at 570nm was measured. Cell viability of each group was compared to untreated control group. As shown in FIG. 8, after 24 hours of drug treatment, hela cell viability remained above 90% when the concentration of Compound 1 reached 64. Mu.M.
EXAMPLE 19 in vivo antibacterial Activity assay of Compound 1
All animal experiments were approved by the institutional animal care and use committee of south-middle university and met the relevant ethical specifications. Female BALB/c mice (6 weeks) were purchased from the university of south China laboratory animal center. BALB/c mice were randomly divided into 6 groups: (1) a bacterial infection group without any treatment (control); (2) the bacterial infection group was irradiated with white Light (control+light); (3) Bacterial infection group (HHC 10) treated with HHC10 peptide alone, the amino acid sequence of HHC10 peptide is shown in SEQ ID NO. 2, specifically KRWWKWIRW; (4) Compound 1 alone treated bacterial infection group (compound 1); (5) Bacterial infection group compound 1 treatment with white light irradiation (compound 1+light); (6) Bacterial infection groups were treated with vancomycin alone (vancomycin) (n=4 per group). Mice were anesthetized with 1.5% sodium pentobarbital injection. Two full-thickness lesions (approximately 8 mm in diameter) were then created on each mouse. 20. Mu.L of the bacterial suspension (1.0X10) 9 CFU/mlrsa) to establish a bacterial infection wound model. 24h after infection, 20. Mu.L of physiological saline, compound 1, HHC10 and vancomycin (10. Mu.M) were added to the wound surface, and the mixture was allowed to stand for 30 minutes. Subsequently, white light (. About.60 mW/cm) 2 ) The wound was irradiated for 10 minutes or placed in the dark. The first day of medication was performed with or without white light irradiation for a total of 3 additional days.The wound healing process is dynamically monitored by measuring the size of the wound, and the wound is photographed. On day 8, tissues from all mice at the site of infection were collected for further evaluation. The bacterial content at the infected site was detected by agar plate counting. As shown in fig. 9, the wound surface area of each group gradually decreases. Compared with the day 1, the wound surface of each drug treatment group is basically healed on the day 8, the area of the wound surface of the compound 1+light group is only 2.2% of the area of the wound surface of the first day, and the compound 1+light group is the group with the best healing degree. The wounds of the control group and the control plus Light group are 13.1% and 11.4% of the first day respectively. At the same time, the body weight of each group of mice steadily increased over time. These results indicate that compound 1 is a good anti-infective and wound healing agent, useful in photodynamic treatment of wound infections, and does not affect the growth of mice.
EXAMPLE 20 Compound 1 induced bacterial aggregation sedimentation experiments
OD is set to 600 1.5mL of each of a bacterial suspension (E.coli, MDRE.coli, S.aureus and MRSA) was added to a measuring dish, and OD was measured and recorded 600 After 30. Mu.L of Compound 1 (1 mM) was added to each bacterial liquid, the mixture was allowed to stand and OD of each group was measured at a predetermined time point 600 . As a result, as shown in fig. 10, the gram-negative bacteria (e.coli and MDR e.coli) of the compound 1 treatment group showed a decrease in the supernatant optical density with no significant change in the control optical density, while the generation of flocculent precipitate was observed, indicating that the action of the compound 1 caused the bacteria to form aggregates, thereby causing acceleration of the precipitation. However, the decrease in optical density occurred in both the compound 1 treated group and the control group of MRSA, presumably due to sedimentation due to the nature of MRSA bacteria themselves, and not the effect of compound 1. Whereas S.aureus did not show sedimentation, indicating that the action of Compound 1 failed to form aggregates of gram-positive bacteria.
Example 21 in vitro hemolysis experiment
Arterial blood (heart blood) of mice was collected, and heparin was added for anticoagulation. The anticoagulants were centrifuged at 1500rpm for 5min and erythrocytes were collected. The resulting red blood cells were further washed twice with PBS and diluted. Compound 1 was prepared at 500 μl of different concentrations (128, 64, 32, 16, 8, 4, 2,1, 0.5 μΜ), with physiological saline as negative control, deionized water as positive control, and mixed with 500 μl of red blood cell suspension. Incubate at 37℃for 2h, centrifuge image, and determine absorbance of supernatant at 540 nm. The calculation formula of the hemolysis rate is as follows:
as shown in FIG. 11, the results of the hemolysis test indicate that it is safe for in vivo treatment. Compound 1 was not hemolytically active at a concentration of less than 32. Mu.M, and the hemolysis rate was still low (hemolysis rate: 7.9%) at a concentration of 64. Mu.M.
EXAMPLE 22 evaluation experiment of anti-biofilm Activity
The first step: biofilm growth inhibition assay
Diluting MRSA in logarithmic growth phase to 3×10 with LB medium 6 CFU/mL was used as the working suspension and added to confocal cuvettes, 2mL each. The cells were divided into a control group to which 20. Mu.L of PBS was added, a non-illuminated group to which 20. Mu.L of Compound 1 (1 mM) was added to each dish, and an illuminated group which was incubated in the dark for 1 hour and then irradiated with white light for 15 minutes. After incubating all confocal dishes at 37℃for 24h, 48h and 72h, the medium was gently removed and washed with PBS and MRSA biofilms stained with Hoechst 33342 and YO-PRO-1. After 10 minutes, the biofilm was imaged by confocal laser fluorescence microscopy at different wavelengths.
And a second step of: mature biofilm disruption assay
Diluting MRSA in logarithmic growth phase to 3×10 with LB medium 6 CFU/mL was used as the working suspension and added to confocal cuvettes, 2mL each. After all confocal dishes were incubated at 37℃for 24h, 48h and 72h to form mature biofilms at different stages, they were divided into a control group, a non-illuminated group and an illuminated group, 20. Mu.L of Compound 1 (1 mM) was added to the control group, the non-illuminated group and the illuminated group were added to each dish, then the non-illuminated group was incubated in the dark for 2h, and the illuminated group was first in the darkAfter 1h incubation, the mixture is placed under white light for 15min, and then placed in a dark group for 45min. The media was gently removed and washed with PBS and MRSA biofilms stained with Hoechst 33342 and YO-PRO-1. After 10 minutes, the biofilm was imaged by CLSM at different wavelengths.
As shown in fig. 12, the biofilm of the control group showed blue fluorescence, indicating that a biofilm filled with living bacteria was formed. After incubation with compound 1, only a few dead bacteria were observed in CLSM images, as indicated by the green fluorescent label. This demonstrates that compound 1 is effective in preventing the formation of a biofilm of staphylococcus aureus. Compound 1 at a concentration of 10 μm was used to co-incubate with biofilms at different maturation stages and split into light and non-light groups and co-stained with Hoechst 33342 and YO-PRO-1. It can be observed in CLSM images that bacterial biofilms at different maturation stages were destroyed to some extent without illumination, and notably that after 15min of illumination, biofilm destruction was more pronounced, as a result of the scattered green fluorescence, indicating that most of the biofilms were destroyed and bacteria were dead, indicating that compound 1 was effective in removing mature biofilms.
Claims (10)
1. A polypeptide conjugated pharmaceutical compound, characterized in that the compound has a general structural formula as shown in the following formula:
wherein, R1 and R2 are both selected from: hydrogen, methyl, methoxy, chloro, bromo, iodo; n is selected from 0,2,4,6 and 8.
2. The polypeptide-conjugated pharmaceutical compound according to claim 1, wherein the structural formula of the polypeptide-conjugated pharmaceutical compound is any one of the structural formulas shown in the following compounds 1 to 12:
3. a method of preparing the polypeptide conjugated pharmaceutical compound of claim 1, comprising the steps of:
(1) Synthesis of compound TPI: reacting a mixture of substituted 4-triphenylborate shown in a formula I, 4, 7-dibromo-2, 1, 3-benzothiadiazole, alkali, palladium catalyst and solvent under the protection of nitrogen, cooling to room temperature after the reaction is finished, pouring the reaction solution into water, and separating and purifying to obtain a compound TPI; the synthetic route is as follows:
(2) Synthesis of compound TPIP: reacting a mixture of a compound TPI, pyridine-4-boric acid, alkali, a palladium catalyst and a solvent under the protection of nitrogen, cooling to room temperature after the reaction is finished, pouring the reaction solution into water, and separating and purifying to obtain the compound TPIP; the synthetic route is as follows:
(3) Synthesis of Compound TPI-PN: dissolving a compound TPIP and bromoamine in a solvent, carrying out reflux reaction, cooling to room temperature after the reaction is finished, evaporating the solvent, and separating and purifying to obtain a compound TPI-PN; the synthetic route is as follows:
(4) Synthesis of Compound TPI-PA: adding a compound TPI-PN and an acid binding agent into a solvent for mixing, cooling the mixed system to 0-5 ℃, adding acryloyl chloride, stirring uniformly, reacting at room temperature, evaporating the solvent after TLC monitoring reaction is completed, and separating and purifying to obtain the compound TPI-PA; the synthetic route is as follows:
(5) Synthesis of Compound TPI-CysHHC 10: dissolving a CysHHC10 peptide in HEPES buffer solution, adding the HEPES buffer solution containing a compound TPI-PA into a reaction system, stirring at room temperature for reaction, and carrying out vacuum freeze-drying, separation and purification after the reaction is completed to obtain TPI-CysHHC10; the synthetic route is as follows:
4. the method of claim 3, wherein in step (1), the solvent comprises at least one of tetrahydrofuran, methanol, and 1, 4-dioxane; the alkali comprises at least one of potassium carbonate and cesium carbonate; the palladium catalyst comprises at least one of [1,1' -bis (diphenylphosphine) ferrocene ] palladium (II) dichloride and tetra (triphenylphosphine) palladium (0); the mol volume ratio of the substituted 4-triphenylamine borate to the solvent is 1 (3-4) mmol/mL, the feeding mol ratio of the substituted 4-triphenylamine borate to the 4, 7-dibromo-2, 1, 3-benzothiadiazole is 1 (1-2), the feeding mol ratio of the substituted 4-triphenylamine borate to the palladium catalyst is 1 (0.01-0.02), the feeding mol ratio of the substituted 4-triphenylamine borate to the alkali is 1 (1-1.5), the reaction temperature is 80-120 ℃, and the reaction time is 6-8 h.
5. The method of claim 3, wherein in step (2), the solvent comprises at least one of tetrahydrofuran, methanol, and 1, 4-dioxane; the alkali comprises at least one of potassium carbonate and cesium carbonate; the palladium catalyst comprises at least one of [1,1' -bis (diphenylphosphine) ferrocene ] palladium (II) dichloride and tetra (triphenylphosphine) palladium (0); the mol volume ratio of the compound TPI to the solvent is 1 (2.5-4) mmol/mL, the feeding mol ratio of the compound TPI to the pyridine-4-boric acid is 1 (1-2), the feeding mol ratio of the compound TPI to the palladium catalyst is 1 (0.01-0.02), the feeding mol ratio of the compound TPI to the alkali is 1 (1-1.5), the reaction temperature is 80-120 ℃, and the reaction time is 15-20 h.
6. The preparation method according to claim 3, wherein in the step (3), the molar volume ratio of the compound TPIP to the solvent is 1 (3-4) mmol/mL, and the feeding molar ratio of the compound TPIP to the bromoamine is 1 (1-2); the reflux reaction temperature is 80-120 ℃ and the reaction time is 4-6 h; the solvent is CH 3 A CN; the separation and purification adopts high performance liquid chromatography.
7. The preparation method according to claim 3, wherein in the step (4), the molar volume ratio of the compound TPI-PN to the solvent is 1 (15-20) mmol/mL, the molar ratio of the compound TPI-PN to the acryloyl chloride is 1 (2-3), and the molar ratio of the compound TPI-PN to the acid binding agent is 1 (2-3); the acid binding agent is triethylamine; the reaction time is 8-12 h; the solvent is anhydrous CH 2 Cl 2 The method comprises the steps of carrying out a first treatment on the surface of the The separation and purification adopts a silica gel column chromatography method.
8. The method of claim 3, wherein in step (5), the compound TPI-PA is added to CysHHC10 peptide in a molar ratio of 1-1.5:1; the amino acid sequence of the CysHHC10 peptide is shown as SEQ ID NO. 1, specifically CKRWWKWIRW-NH 2 The method comprises the steps of carrying out a first treatment on the surface of the The reaction time is 8-12 h; the separation and purification adopts high performance liquid chromatography.
9. Use of a polypeptide-conjugated pharmaceutical compound according to any one of claims 1-2 or a polypeptide-conjugated pharmaceutical compound prepared by a method according to any one of claims 3-8 in bacterial wash-free imaging, bacterial aggregation induction, bacterial near infrared fluorescence imaging or as a preparation for a broad-spectrum antibacterial drug.
10. An antibacterial agent comprising the polypeptide-conjugated drug compound according to any one of claims 1 to 2 or the polypeptide-conjugated drug compound produced by the production method according to any one of claims 3 to 8.
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