TB-double bond-pyridinium derivatives and their preparation methods and applications

Abstract

The present invention provides a class of TB-double-bond-pyridinium derivatives and their preparation methods and applications. 4-bromoaniline, paraformaldehyde, DMF, three pyridine derivatives and iodomethane are selected as raw materials and synthesized through a multi-step reaction to obtain: #imgabs0# The three TB-double-bond-pyridinium derivatives have excellent photophysical properties, pH applicable range, viscosity response, AIE properties and good biocompatibility. The DCHA probe detected that all three products can produce ROS, and under the same conditions, the ROS production of 14 is higher. The DHR123 probe and EPR detected that all three compounds can produce superoxide anions. The results of antibacterial experiments showed that 14 exhibited excellent aPDT activity against Staphylococcus aureus and methicillin-resistant Staphylococcus aureus (8μmol·L ‑1 The inhibition rates were 92% and 96% respectively. 14 had low dark toxicity but high phototoxicity to human bronchial epithelial cells, and both dark and phototoxicity were high to A459 cells. It also had a high safety factor SI, suggesting that it has the potential to be developed as a new anti-lung cancer drug.

Description

Technical Field

[0001] The present invention belongs to the field of chemical synthesis, and specifically relates to Base (TB)-double bond-pyridinium derivatives and their preparation methods and applications in photodynamic antibacterial (especially anti-drug-resistant bacteria) and photodynamic anti-tumor. Background Art

[0002] Antimicrobial photodynamic therapy (aPDT) is a new technology that uses light of a specific wavelength to irradiate a photosensitizer, triggering the production of reactive oxygen species (ROS) that damage the bacterial cytoplasm and nucleus, achieving antimicrobial efficacy. This oxidative stress-based antimicrobial approach is not compromised by bacterial evolution and therefore does not contribute to drug resistance, making it a key approach to addressing bacterial resistance.

[0003] The core element of aPDT is the antimicrobial efficacy of the photosensitizer, which primarily derives from its ability to generate ROS. The structure of the photosensitizer is the primary factor determining its ROS production capacity. Therefore, designing photosensitizers with high ROS production rates is a key research priority in this field.

[0004] Traditional photosensitizers are prone to aggregation in aqueous solutions, leading to aggregation-caused fluorescence quenching (ACQ) effect, which reduces the production of ROS. Photosensitizers with aggregation-induced emission (AIE) properties can avoid the ACQ effect and produce high concentrations of ROS after aggregation, thus solving this problem. However, most of the reported AIE photosensitizer molecules use flexible structures (such as triphenylamine or tetraphenylethylene, etc.) as electron donors (D), and their single bonds can rotate freely, resulting in an increase in non-radiative transitions, and more energy is lost in the form of rotational energy or vibrational energy, causing the excited state molecules to transfer from S1 to T n The probability and efficiency of the disease are greatly reduced. And so far there is no antibacterial photosensitizer that can be used in clinical practice, so there is a lot of room for research.

[0005] The base (TB) has a V-shaped non-planar rigid structure that can avoid intermolecular π-π stacking; it has 8 π electrons and two pairs of lone pairs of electrons, making it an excellent electron donor. Theoretical calculation results show that the TB skeleton has multiple triplet energy levels (T n), the possibility of intersystem crossing is high, and the probability of ROS generation is high. Given this, based on the molecular structure requirements for photosensitizers against drug-resistant bacteria, TB was used as an electron donor (D) and a rigid linker, and various groups were introduced to design and synthesize photosensitizers with an aPDT effect. Therefore, in theory, TB possesses both AIE properties and high ROS generation efficiency, making it an advantageous backbone for the design and synthesis of highly effective aPDT photosensitizers. However, to date, there have been no reports of aPDT photosensitizers with AIE properties using TB as a backbone.

[0006] In view of this, according to the requirements of the molecular structure of the anti-resistant bacteria photosensitizer, the present invention uses TB as the electron donor (D) and the rigid linker, and introduces different groups thereon to design and synthesize a new and efficient aPDT photosensitizer. Summary of the Invention

[0007] Technical problem: The purpose of the present invention is to provide a class of TB-double bond-pyridinium derivatives, their preparation method and application, using 4-bromoaniline, paraformaldehyde, DMF and iodomethane as raw materials, and synthesizing a class of TB-double bond-pyridinium derivatives, namely three target products, through multi-step reactions, and applying them in the fields of aPDT and photodynamic anticancer.

[0008] Technical solution: The structural formulas of a class of TB-double bond-pyridinium derivatives of the present invention are shown as the first derivative 13, the second derivative 14 and the third derivative 15 below:

[0009]

[0010] The preparation method of the TB-double bond-pyridinium derivative of the present invention comprises the following steps:

[0011] Step 1: The following p-bromoaniline 1 reacts with paraformaldehyde 2 to obtain the first intermediate 3. The reaction formula is as follows:

[0012]

[0013] Step 2: The first intermediate 3 reacts with DMF 4 to obtain the second intermediate 5. The reaction formula is as follows:

[0014]

[0015] Step 3: 4-methylpyridine 6, 4-methylquinoline 9 and 4-pyridineacetonitrile 11 react with iodomethane 7 to obtain the third intermediate 8, the fourth intermediate 10 and the fifth intermediate 12, respectively. The reaction formulas are as follows:

[0016]

[0017] Step 4: The second intermediate 5 reacts with the third intermediate 8 to obtain the first derivative 13. The reaction formula is as follows:

[0018]

[0019] Step 5: The second intermediate 5 reacts with the fourth intermediate 10 to obtain the second derivative 14. The reaction formula is as follows:

[0020]

[0021] Step 6: The second intermediate 5 reacts with the fifth intermediate 12 to obtain the third derivative 15. The reaction formula is as follows:

[0022]

[0023] Application of the TB-double bond-pyridinium derivative of the present invention, and application of the first derivative 13, the second derivative 14, and the third derivative 15 in the preparation of viscosity probes.

[0024] The application of the TB-double bond-pyridinium derivative of the present invention, and the application of the first derivative 13, the second derivative 14, and the third derivative 15 in the preparation of photodynamic antibacterial drugs.

[0025] The application of the TB-double bond-pyridinium derivative of the present invention, and the application of the first derivative 13, the second derivative 14, and the third derivative 15 in the preparation of cancer photodynamic therapy drugs.

[0026] The antibacterial agent is directed against the inhibition of Staphylococcus aureus, Escherichia coli and methicillin-resistant Staphylococcus aureus (MRSA).

[0027] The cancer photodynamic therapy is directed to the inhibition of human non-small cell lung cancer cells A549.

[0028] Beneficial effects:

[0029] 1. A class of TB-double bond-pyridinium derivatives was synthesized for the first time. The synthesis method is simple and the post-processing is convenient.

[0030] 2. The product has a large Stokes shift, excellent viscosity response ability and significant AIE properties; the product has a wide pH range of application and can be used in human physiological environments.

[0031] 3. The second derivative 14 showed good aPDT activity against Staphylococcus aureus and MRSA (8 μmol·L -1 The inhibition rates were 92% and 96% respectively).

[0032] 4. The second derivative 14 has low dark toxicity to HBE cells but high phototoxicity, and has high dark toxicity and phototoxicity to A459 cells. Its safety factor SI is the largest, showing excellent photodynamic anti-tumor activity.

[0033] 5. The product's excellent optical properties, aPDT activity against drug-resistant bacteria, and PDT anti-tumor activity make it have great development value in the fields of hospital wastewater treatment, non-invasive anti-tumor drugs, viscosity probes, cell imaging, and organelle localization. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 is the product derivative 13 in the embodiment 1 H NMR spectrum;

[0035] Figure 2 is the product derivative 13 in the embodiment 13 C NMR spectrum;

[0036] Figure 3 is the product derivative 14 in the embodiment 1 H NMR spectrum;

[0037] Figure 4 is the product derivative 14 in the embodiment 13 C NMR spectrum;

[0038] Figure 5 is the product derivative 15 in the embodiment 1 H NMR spectrum;

[0039] Figure 6 is the product derivative 15 in the embodiment 13 C NMR spectrum;

[0040] Figure 7 These are the ultraviolet absorption spectra and fluorescence emission spectra of the first derivative 13, the second derivative 14 and the third derivative 15 in different solvents in the embodiment, 13: (a) (b), 14: (c) (d), 15: (e) (f).

[0041] Figure 8 (a) UV absorption spectra and (b) fluorescence emission spectra of the first derivative 13, the second derivative 14, and the third derivative 15 in DMSO in the examples;

[0042] Figure 9 is the solid-state fluorescence emission spectrum of the first derivative 13, the second derivative 14, and the third derivative 15 in the embodiment;

[0043] Figure 10The fluorescence emission spectra and line graphs of the first derivative 13, the second derivative 14, and the third derivative 15 at different pH values ​​in the embodiment are shown in Figures 13 (a) (b), 14 (c) (d), and 15 (e) (f).

[0044] Figure 11 The fluorescence emission spectra and line graphs of the first derivative 13, the second derivative 14, and the third derivative 15 at different viscosities in the embodiment are shown in Figures 13 (a) (b), 14 (c) (d), and 15 (e) (f).

[0045] Figure 12 Fluorescence emission spectra and line graphs of the first derivative 13, the second derivative 14, and the third derivative 15 in different ratios of n-hexane / EtOH (v / v) of the embodiment, 13: (a) (b), 14: (c) (d), 15: (e) (f);

[0046] Figure 13 SEM of the first derivative 13 and the second derivative 14 in EtOH / n-hexane (v / v) = 9 / 1 and EtOH / n-hexane (v / v) = 3 / 7; 13: (a) (b), 14: (c) (d);

[0047] Figure 14 is the electron paramagnetic resonance spectrum (EPR) of the first derivative 13 in Example;

[0048] Figure 15 is the EPR of the second derivative 14 in the example;

[0049] Figure 16 is the EPR of the third derivative 15 in the example;

[0050] Figure 17 The first derivative 13, the second derivative 14 and the third derivative 15 in the embodiment and the blank are in the LED white light (2.93W·cm -2 ) Line graph of DCFH fluorescence intensity at 5, 10, 15, and 30 min of irradiation;

[0051] Figure 18 The first derivative 13, the second derivative 14, the third derivative 15 and the blank in the embodiment are irradiated with white light (2.93W·cm -2 ) DHR123 fluorescence intensity line graph;

[0052] Figure 19 The first derivative 13, the second derivative 14, the third derivative 15 and RB in the embodiment are in the LED white light (2.93W·cm -2) decomposition rate of ABDA under irradiation, where A0 and A are the absorbance of ABDA at 378 nm before and after irradiation, respectively;

[0053] Figure 20 The first derivative 13 with different concentrations in the embodiment is in the dark / LED white light (2.93W·cm -2 ) Bactericidal activity against (A) Staphylococcus aureus and (B) Escherichia coli after 20 min of irradiation (plate count method);

[0054] Figure 21 The second derivative 14 with different concentrations in the embodiment is in the dark / LED white light (2.93W·cm -2 ) Bactericidal activity against (A) Staphylococcus aureus and (B) Escherichia coli after 20 min of irradiation (plate count method);

[0055] Figure 22 The third derivative 15 with different concentrations in the embodiment is in the dark / LED white light (2.93W·cm -2 ) Bactericidal activity against (A) Staphylococcus aureus and (B) Escherichia coli after 20 min of irradiation (plate count method);

[0056] Figure 23 The first derivative 13, the second derivative 14 and the third derivative 15 in the embodiment are -2 ) Histogram of the antibacterial activity against (a) Staphylococcus aureus and (b) Escherichia coli under irradiation;

[0057] Attachment Figure 24 The first derivative 13 with different concentrations in the embodiment is in the dark / LED white light (2.93W·cm -2 ) Antibacterial activity against MRSA after 20 minutes of irradiation (plate count method);

[0058] Figure 25 The first derivative 13, the second derivative 14 and the third derivative 15 in the embodiment are -2 ) Histogram of antibacterial activity against MRSA under irradiation;

[0059] Figure 26 The second derivative 14 in the embodiment was combined with Staphylococcus aureus or Escherichia coli in the dark and light (LED white light, 2.93W·cm -2 , 20 min) co-incubated SEM;

[0060] Figure 27 is the zeta potential of the second derivative 14 in the embodiment before and after incubation with Staphylococcus aureus and Escherichia coli;

[0061] Figure 281 is an electron micrograph of the second derivative 14 in Example after incubation with A549 under dark / light conditions. DETAILED DESCRIPTION

[0062] The present invention will be further described below with reference to the embodiments.

[0063] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and are not to be construed as limiting the present invention. It will be understood by those skilled in the art that various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention.

[0064] TB-double bond-pyridinium derivative, its structural formula is as follows:

[0065]

[0066] Example 1 Synthesis of the First Derivative 13, the Second Derivative 14 and the Third Derivative 15

[0067] The present invention provides a method for preparing the novel TB-double bond-pyridinium derivative, comprising:

[0068] In this embodiment, the product is prepared by a multi-step reaction using p-bromoaniline, paraformaldehyde, DMF, and iodine salt of a pyridine derivative as raw materials. The steps include:

[0069] p-Bromoaniline 1 reacts with paraformaldehyde 2 to obtain a first intermediate 3, which reacts with DMF 4 to obtain a second intermediate 5, which reacts with a third intermediate 8, a fourth intermediate 10, and a fifth intermediate 12 through nucleophilic elimination reactions to obtain a first derivative 13, a second derivative 14, and a third derivative 15.

[0070] The first derivative 13, the second derivative 14 and the third derivative 15 in the example were prepared by the above-mentioned synthesis method:

[0071] 1. Synthesis of the first intermediate 3

[0072] 4-Bromoaniline (50.0 mmol) and paraformaldehyde (100.0 mmol) were added sequentially to a 200.0 mL round-bottom flask, which was placed in a low-temperature tank and adjusted to -15°C. Trifluoroacetic acid (100.0 mL, approximately 30 minutes) was slowly added dropwise to the flask with stirring. The mixture was allowed to react at room temperature for 7 days. After the reaction was complete (TLC tracking), the mixture was poured into ice water, adjusted to pH 9-10 with aqueous ammonia, cooled to room temperature, extracted with dichloromethane (50.0 mL x 3), and dried to obtain a crude product. Acetone was added and heated until the crude product was completely dissolved. The product was recrystallized at room temperature, filtered, and washed with acetone to obtain the first intermediate 3.

[0073]

[0074] 2. Synthesis of the Second Intermediate 5

[0075] The first intermediate 3 (5 mmol) was weighed and added to a 100 mL two-necked flask under argon protection. 20 mL of dried tetrahydrofuran was added, and the temperature was lowered to -78 ° C under tax-free and oxygen-free conditions. 2.5 mL of n-butyl lithium was slowly added dropwise (controlled within 1 h), and then 1.2 mL of dried N, N-dimethylformamide was added dropwise. After the addition, the mixture was moved to room temperature and reacted for 12 h. After the reaction was completed (TLC tracking), the mixture was quenched with ice water and extracted with dichloromethane. The crude product was purified by column chromatography (petroleum ether: ethyl acetate = 3:1) to obtain the second intermediate 5 (55%).

[0076]

[0077] 3. Synthesis of the third intermediate 8 and the fourth intermediate 10

[0078] Weigh 1.0 mmol of 4-methylpyridine 6 (or 4-methylquinoline 9) and add it to a 25 mL single-necked flask. After stirring in an ice-water bath for 0.5 h, slowly add 1.0 mmol of iodomethane dropwise. White solids are continuously produced during the process. After the addition is completed, continue the reaction for 3 h (a large amount of solids precipitate). Add an appropriate amount of ether, stir for 1-3 h, filter, wash the filter cake with ether, and dry to obtain the third intermediate 8 (or fourth intermediate 10) (yields are 92% and 95%, respectively).

[0079]

[0080] 4. Synthesis of the Fifth Intermediate 12

[0081] 1.0 mmol of 4-pyridineacetonitrile 11 was weighed and added to a single-necked flask containing 5 mL of DCM. After dissolution, the mixture was stirred in an ice-water bath for 0.5 h. 1.0 mmol of iodomethane was then slowly added dropwise, producing a white solid. The reaction was continued for 3 h. After the reaction, an appropriate amount of diethyl ether was added, stirred for 0.5 h, and filtered. The filter cake was washed with diethyl ether and dried to obtain the fifth intermediate 12 (yield 95%).

[0082]

[0083] 5. Synthesis of the first derivative 13, the second derivative 14, and the third derivative 15

[0084] Weigh 0.3 mmol of the third intermediate 8 (the fourth intermediate 10 or the fifth intermediate 12) and 0.3 mmol of the second intermediate 5, add them to a 25 mL three-necked flask under argon protection, and evacuate for 1 hour, during which the argon is replaced and the evacuation is performed three times each. Under argon protection, add 4 mL of toluene and 6 mL of methanol, heat to reflux under argon protection and keep warm for 24 hours (solid material can be seen precipitating during this period). After the reaction is completed, cool to room temperature, add 8 mL of ether, stir for 1-2 hours, filter, wash with ether, and dry for use (yields are: 85%, 89% and 91% respectively). (Note: If the product still has impurities, add 1-2 mL of methanol to the filter cake, stir with a spatula for about 3 minutes, filter, and wash with ether).

[0085]

[0086] Product first derivative 13

[0087] The chemical formula is: C 23 H 21 BrIN3

[0088] The Chinese name is: 4-(2-(8-bromo-6H,12H-5,11-methanoldibenzo[b,f][1,5]indol-2-yl)vinyl)-1-methylpyridine iodide

[0089] The English name is: (E)-4-(2-(8-bromo-6H,12H-5,11-methanodibenzo[b,f][1,5]diazocin-2-yl)vinyl)-1-methylpyridin-1-ium iodide

[0090] Appearance: khaki solid

[0091] Melting point: 192.8-193.6℃

[0092] H NMR spectrum: 1 H NMR (400MHz, DMSO-d6) δ8.81(d,J=6.2Hz,2H),8.25–8.09(m,2H),7.90(d,J=16.3Hz,1H),7.56(d,J=8.3Hz,1H ),7.48–7.28(m,3H),7.22(d,J=11.4Hz,2H),7.13(d,J=8.9Hz,1H),4.67(t,J=16.6Hz,2H),4.47-4.06(m,7H).

[0093] C NMR spectrum: 13C NMR(100MHz,DMSO-d6)δ153.00,150.84,147.64,145.43,140.77,131.28,130.95,130.37,12 9.93,128.96,127.50,127.45,125.84,123.64,122.30,115.69,66.32,58.48,58.25,47.29.

[0094] Mass spectrometry: HRMS (ESI) for: C 23 H 21 N3[M+H] + :calcd 340.1814, found 340.1793.

[0095] Product second derivative 14

[0096] The chemical formula is: C 27 H 23 BrIN3

[0097] The Chinese name is: (7) 4-(2-(8-bromo-6H,12H-5,11-methanoldibenzo[b,f][1,5]indol-2-yl)vinyl)-1-methylquinoline iodide

[0098] The English name is: (E)-4-(2-(8-bromo-6H,12H-5,11-methanodibenzo[b,f][1,5]diazocin-2-yl)vinyl)-1-methylquinolin-1-ium iodide

[0099] Appearance: brown solid

[0100] Melting point: 255-256℃,

[0101] H NMR spectrum: 1 H NMR (400MHz, DMSO-d6) δ9.28(d,J=6.5Hz,1H),8.93(d,J=8.6Hz,1H),8.46–8.33(m,3H),8.25(t,J=8.0Hz,1H),8.02(t,J=7.8Hz,1H),7.99–7.9 0(m,1H),7.72(d,J=3.6Hz,1H),7.55(q,J=6.3,5.4Hz,2H),7.36(s,1H) ,7.25–7.09(m,3H),4.75–4.61(m,2H),4.50(s,3H),4.32–4.13(m,7H).

[0102] C NMR spectrum:13 C NMR(100MHz,DMSO-d6)δ152.55,150.74,147.91,147.17,142.64,138.74,134.93,130.87,129.92,129.48,129.14,12 8.46,128.06,127.59,126.96,126.37,126.21,125.24,119.33,118.34,115.80,115.23,65.89,58.10,57.86,44.60.

[0103] Mass spectrometry: HRMS (ESI) for C 27 H 23 N3[M+H] + :calcd 390.1971,found:390.1961.

[0104] Product third derivative 15

[0105] The chemical formula is: C 24 H 20 BrIN4

[0106] The Chinese name is: (Z) 4-(2-(8-bromo-6H,12H-5,11-methanoldibenzo[b,f][1,5]indol-2-yl)cyanovinyl)-1-methylpiperidinium iodide

[0107] The English name is: (Z)-4-(2-(8-bromo-6H,12H-5,11-methanodibenzo[b,f][1,5]diazocin-2-yl)-1-cyanovinyl)-1-methylpyridin-1-ium iodide

[0108] Appearance: yellow solid

[0109] Melting point: 242-244.7℃

[0110] H NMR spectrum: 1 H NMR (400MHz, DMSO-d6) δ9.00(d,J=6.4Hz,1H),8.60(s,1H),8.35(d,J=6.3Hz,1H),7.97(d,J=8.5Hz,1H),7.73(s,1H),7.39( d,J=8.6Hz,1H),7.32(d,J=8.6Hz,0H),7.22(s,0H),7.14(d,J=8.7Hz,1H),4.72(dd,J=16.9,12.8Hz,1H),4.39–4.11(m,4H).

[0111] C NMR spectrum: 13 C(100MHz,DMSO-d6)δ153.44,150.98,149.08,146.88,145.71,130.72,130.48,130.02,129.46, 129.42,128.88,127.54,127.02,125.56,122.69,116.45,115.35,102.95,65.73,57.87,47.25.

[0112] Mass spectrometry: HRMS (ESI) for: C 24 H 20 N4[M+H] + :calcd 365.1767, found 365.1759.

[0113] Example 2 Solvation Effects of Compounds of the Invention

[0114] The first derivative 13, the second derivative 14 and the third derivative 15 were prepared with n-hexane (n-Hexane), toluene (Toluene), tetrahydrofuran (THF), ethyl acetate (EA), acetonitrile (MeCN), methanol (MeOH), N,N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO) and deionized water (H2O) to a concentration of 1×10 -5 mol·L -1 The UV absorption spectrum and fluorescence emission spectrum of the solution were tested. Figure 7 ).

[0115] Depend on Figure 7 It can be seen that in each solvent, the maximum absorption peak wavelength (λ abs ) is concentrated around 390nm, and the relative fluorescence intensity is stronger in medium polarity solution. abs The fluorescence intensity is concentrated around 438 nm, and is relatively high in polar solutions. The λ of the third derivative 15 abs It is concentrated around 355nm, and the relative fluorescence intensity is greater in highly polar and medium polar solutions.

[0116] Example 3 Optical properties of compounds 13-15 of the present invention

[0117] The photophysical properties of the first derivative 13, the second derivative 14 and the third derivative 15 were tested using DMSO as solvent ( Figure 8 ). The line spacing and indentation are changed here.

[0118] The solid powders of the first derivative 13, the second derivative 14 and the third derivative 15 were spread and pressed into tablets, and their solid-state fluorescence emission spectra were measured using a fluorescence spectrophotometer ( Figure 9 )

[0119] Table 2 Spectral data of compounds 13-15

[0120]

[0121] a UV absorption wavelength in solution; b Molar extinction coefficient ε=A / bC,1×10 5 L·mol -1 cm -1 ; c Fluorescence emission wavelength in solution; d Stokes shift in solution; e Fluorescence brightness, unit is L·mol -1 cm -1 ; f solid-state excitation wavelength; g solid-state fluorescence emission wavelength; h Solid-state Stokes shift

[0122] Depend on Figure 8-Figure 9 As shown in Table 2, compared with the first intermediate 3, the first derivative 13, the second derivative 14 and the third derivative 15 have the following characteristics: em All exhibited significant red-shifts into the yellow-red region, with a significant increase in the Stokes shift. This is likely due to the enhanced electron mobility resulting from the growth of the molecular conjugated chain and the introduction of positive ions. The first derivative 13, the second derivative 14, and the third derivative 15 all exhibited wide absorption peaks. Compared to the first and third derivatives 13 and 15, the second derivative 14, with its quinolinium structure, exhibited longer absorption and emission wavelengths, with the emission wavelength entering the red region, indicating that the conjugated system significantly influences the optical properties of the product.

[0123] The above results indicate that the introduction of double bonds and pyridinium cations into TB can optimize the photophysical properties of TB derivatives.

[0124] Example 4 pH response of compounds 13-15 of the present invention

[0125] To investigate whether the product can be used in a physiological environment, we evaluated its pH response. The first derivative 13, the second derivative 14, and the third derivative 15 were dissolved in 1 mL of DMSO, and the volume was adjusted to 25 mL with EtOH to prepare a concentration of 1×10 - 3 mol·L -10.1 mL of the working solution was taken and added to 8 10 mL volumetric flasks, and the volume was adjusted in sequence with a buffer solution with a pH value of 3-11 (citric acid / sodium citrate for pH 3-5, sodium dihydrogen phosphate / disodium hydrogen phosphate for pH 6-8, Tris / hydrochloric acid for pH 9, and sodium bicarbonate / sodium hydroxide for pH 10-11) to make the concentration of the compound 1×10 -5 mol·L -1 , and its fluorescence emission spectrum ( Figure 10 ).

[0126] Depend on Figure 10 It can be seen that the fluorescence intensity of the first derivative 13, the second derivative 14 and the third derivative 15 is stable at pH = 6-8 and is suitable for the human physiological environment.

[0127] Example 5 Viscosity Response of Compounds 13-15 of the Invention

[0128] The viscosity of abnormal cells is often greater than that of normal cells. The high viscosity within the cells will restrict the movement of chemical bonds, making molecules more likely to aggregate. Therefore, photosensitizers that are responsive to viscosity may also have AIE properties.

[0129] Take 1 mL of the above working solution and add it to 9 100 mL volumetric flasks, and dilute them to the volume with a mixed solution of glycerol: water with a volume ratio of 1:9 to 9:1, so that the concentration of the compound is 1×10 -5 mol·L -1 , the fluorescence intensity of each compound at different viscosities was tested ( Figure 11 ).

[0130] Depend on Figure 11 As can be seen, the fluorescence intensity of the first derivative 13, the second derivative 14, and the third derivative 15 increases with increasing viscosity. This is because the increase in viscosity restricts the movement of chemical bonds, reduces vibrational and rotational degrees of freedom, and transforms non-radiative energy decay into radiative decay. The level of response to viscosity suggests that these three products may exhibit AIE properties.

[0131] Example 6 AIE properties of compounds 13-15 of the present invention

[0132] The response level to viscosity indicates that the three products may have AIE properties, so we studied the AIE properties of the products. Take 1mL of the above working solution and place it in a 100mL volumetric flask. Then, dilute the volume with a mixed solution (EtOH:n-hexane in the ratio of 9:1 to 1:9) to make the concentration of each product 1×10 -5 mol·L -1 , and tested its fluorescence emission spectrum ( Figure 12 ).

[0133] Depend on Figure 12 As can be seen, the fluorescence intensity of the first derivative 13, the second derivative 14, and the third derivative 15 gradually increases with increasing n-hexane content, all exhibiting significant AIE properties. When the n-hexane content is 80%, the relative fluorescence intensity (RFI) of the first derivative 13 is the highest, increasing by 3.2 times; when the n-hexane content is 70%, the RFI of the second derivative 14 is the highest, increasing by 2.4 times; and when the n-hexane content is 80%, the RFI of the third derivative 15 is the highest, increasing by 1.6 times.

[0134] The morphologies of the first derivative 13 and the third derivative 15 in EtOH / n-hexane (v / v) = 9 / 1 and EtOH / n-hexane (v / v) = 2 / 8, and the second derivative 14 in EtOH / n-hexane (v / v) = 9 / 1 and EtOH / n-hexane (v / v) = 3 / 7 were observed by SEM observation ( Figure 13 )

[0135] Depend on Figure 13 As shown in (a) and (b), the first derivative 13 is an irregular flocculent structure in the EtOH / n-hexane (v / v) = 9 / 1 solution. As the n-hexane content increases, the first derivative 13 continues to aggregate. When the EtOH / n-hexane (v / v) = 2 / 8, the first derivative 13 forms a flake structure with a size of about 500 nm. Figure 13 (c) and (d) show that the second derivative 14 is irregular particles in the EtOH / n-hexane (v / v) = 9 / 1 solution, and forms block aggregates when the EtOH / n-hexane (v / v) = 3 / 7. Figure 13 (e) and (f) show that the third derivative compound 15 is irregular particles in the EtOH / n-hexane (v / v) = 9 / 1 solution. When EtOH / n-hexane (v / v) = 2 / 8, the third derivative 15 aggregates into elliptical particles of about 200 nm.

[0136] The above results also show that the first derivative 13, the second derivative 14 and the third derivative 15 have AIE properties. Example 7 Reactive oxygen species (ROS) generation ability of the first derivative 13, the second derivative 14 and the third derivative 15 of the present invention

[0137] The results of viscosity and AIE experiments showed that the first derivative 13, the second derivative 14, and the third derivative 15 all have viscosity responsiveness and AIE properties. The AIE property can enhance the ROS generation ability of the product. Therefore, we used electron paramagnetic resonance spectroscopy (EPR) and fluorescence probes to detect the ROS generated.

[0138] Electron paramagnetic resonance (EPR) spectroscopy

[0139] Depend on Figures 14 to 16 It can be seen that the first derivative 13, the second derivative 14 and the third derivative 15 have superoxide anions (O2 ·- ) signal, no singlet oxygen ( 1 O2) and hydroxyl radical (OH·), indicating that the first derivative 13, the second derivative 14 and the third derivative 15 can generate superoxide anions under light irradiation.

[0140] Fluorescent probes

[0141] Total ROS

[0142] 2,7-Dichlorodihydrofluorescein (DCFH) is easily oxidized by ROS to generate 2,7-dichlorofluorescein (DCF), which emits fluorescence at 532 nm under 488 nm light excitation.

[0143] White LED lamp (2.93W·cm -2 ) was used as the light source for irradiation, and the fluorescence emission spectra (excitation wavelength: 488 nm) of the solutions with irradiation times of 5, 10, 15, 20, and 30 min were detected in sequence.

[0144] Depend on Figure 17 It can be seen that the first derivative 13, the second derivative 14, and the third derivative 15 generate ROS under light exposure, and ROS production increases with increasing exposure time. Under the same conditions, the second derivative 14 produces the highest total ROS, likely due to the additional benzene ring in the second derivative 14, which results in the highest electron cloud density and electron mobility.

[0145] Superoxide anion (O2 ·- )

[0146] Detection of O2 using dihydrorhodamine 123 (DHR123) as a fluorescent probe ·- , in LED white light (2.93W·cm -2 ) was irradiated. Under 495nm excitation, the fluorescence signal around 534nm was measured. The fluorescence intensity around 534nm was recorded every 1 minute to represent O2 ·- The generation rate of irradiation for 8 min ( Figure 18 ).Depend on Figure 18 It can be seen that compounds 13-15 have O2 under LED white light irradiation. ·- The generation capacity of the second derivative 14 under the same conditions is O2 ·- The yield was the highest, probably because the onium ion of the second derivative 14 had an additional fused benzene ring compared to the other two compounds, and the longer conjugated system made its electronic transition ability stronger.

[0147] Singlet oxygen (1 O2)

[0148] Detection using 9,10-anthracenediyl-bis(methylene)dimalonic acid (ABDA) 1 O2: Under LED white light irradiation (2.93W·cm -1 ) were used to monitor the changes in absorbance at 378 nm of ABDA at different time intervals. The changes in absorbance at 378 nm were recorded every 1 minute of irradiation, indicating that 1 The generation rate of O2 was 6 min in the whole experiment.

[0149] Depend on Figure 19 It can be seen that under the irradiation of LED white light (2.93W·cm -2 , the system contains 25 μmol·L -1 ABDA, 5 μmol·L -1 RB or 5 μmol·L -1 Samples), 7 and 13 1 The O2 generation capacity is not as good as that of Rose Bengal (RB, a commercial 1 O2 dye), 10 1 The O2 generation capacity is comparable to that of RB.

[0150] Example 8 In vitro antibacterial activity of the first derivative 13, the second derivative 14 and the third derivative 15 of the present invention

[0151] The above experimental results show that the three products designed and synthesized have excellent ROS generation ability, so we used the microbroth dilution method and plate colony counting method to detect the antibacterial activity of each compound.

[0152] Dark Toxicity

[0153] Before conducting the antibacterial activity test, its dark toxicity to bacteria was first tested (Table 3).

[0154] Table 3 Minimum inhibitory concentrations (MIC) of the second intermediate 5, third intermediate 8, fourth intermediate 10, fifth intermediate 12, RB (Rhodamine B), first derivative 13, second derivative 14 and third derivative 15 against Escherichia coli, Staphylococcus aureus and Bacillus subtilis under dark conditions (μg mL -1 )

[0155]

[0156] As shown in Table 3, the MICs of the second intermediate 5, the third intermediate 8, the fourth intermediate 10, the fifth intermediate 12, RB (rhodamine B), the first derivative 13, the second derivative 14 and the third derivative 15 against Escherichia coli, Staphylococcus aureus and Bacillus subtilis all exceeded 64 μg·mL under dark conditions. -1 , indicating that the above compounds have no antibacterial activity under dark conditions and can be used as photosensitizers to further study their aPDT activity.

[0157] Example 9 aPDT activity of the first derivative 13, the second derivative 14, and the third derivative 15 of the present invention

[0158] The antibacterial activities of the first derivative 13, the second derivative 14 and the third derivative 15 were tested by plate colony counting method ( Figure 20-23 ).

[0159] Depend on Figure 20-23 It can be seen that the first derivative 13 and the third derivative 15 have weak inhibitory activity against Staphylococcus aureus under LED white light conditions and have no inhibitory activity against Escherichia coli. The second derivative 14 shows antibacterial activity against both Staphylococcus aureus and Escherichia coli under light conditions, and its aPDT ability increases with the increase of its concentration. The concentration of the second derivative 14 is 8 μmol·L -1 It has obvious antibacterial activity against Staphylococcus aureus, with an inhibition rate of 92% at a concentration of 40 μmol·L -1 The second derivative 14 has a stronger antibacterial activity against Staphylococcus aureus than E. coli. This is because E. coli has a double-layer membrane structure with a thicker membrane, which is more difficult to penetrate, and its binding force with positive ions is not as strong as that of Staphylococcus aureus.

[0160] Among the three products, the second derivative 14 had the greatest aPDT activity, which was consistent with the ROS detection results.

[0161] The main purpose of this paper is to design a new and efficient aPDT photosensitizer with inhibitory activity against drug-resistant bacteria. Therefore, the aPDT activity of the first derivative 13, the second derivative 14 and the third derivative 15 against methicillin-resistant Staphylococcus aureus (MRSA) was detected.

[0162] Depend on Figure 24 and Figure 25 It can be seen that under dark conditions, the concentration of the second derivative 14 is 8 μmol·L -1 Under light conditions, the second derivative 14 had no antibacterial effect on MRSA at 8 μmol·L -1 It showed strong antibacterial activity against MRSA, with an inhibition rate of 96%, which was greater than its inhibition rate against Staphylococcus aureus (92%).

[0163] The above results show that the second derivative 14 has a high inhibitory activity against Staphylococcus aureus and MRSA, which shows that the present invention is reasonable and has achieved the purpose of designing an inhibitory effect on drug-resistant bacteria.

[0164] Example 10 Antibacterial Mechanism of the First Derivative 13, the Second Derivative 14, and the Third Derivative 15 of the Present Invention Scanning Electron Microscopy (SEM)

[0165] The morphology of bacteria after the second derivative 14 was treated with L(-) / (L(+)) was observed by SEM. According to the above antibacterial test results, the concentration of the second derivative 14 against Staphylococcus aureus was selected to be 8 μmol·L -1 , the concentration of anti-E. coli is 40 μmol·L -1 ( Figure 26 ).

[0166] Depend on Figure 26 As can be seen, the surfaces of Staphylococcus aureus and Escherichia coli in the dark group were smooth and intact, while those in the light-exposed group exhibited wrinkled cell walls, ruptured cell membranes, and leaked cytoplasm. These appearance characteristics of dead bacteria are consistent with bacterial destruction caused by ROS. Therefore, we believe that under light exposure, the ROS produced by the second derivative 14 destroyed the bacterial structure, leading to bacterial death.

[0167] Zeta potential

[0168] From the electron microscope image ( Figure 27 ) It can be seen that the second derivative 14 destroyed the bacterial cell membrane. To explore how the second derivative 14 destroyed the bacterial cell membrane, we detected the Zeta potential of the second derivative 14 before and after incubation with Staphylococcus aureus and Escherichia coli.

[0169] Depend on Figure 27 It can be seen that the Zeta potential of both bacteria increased after being co-incubated with the second derivative 14, indicating that the second derivative 14 was bound to the bacterial cell membrane through electrostatic interaction, thereby destroying the bacteria and exerting an antibacterial effect.

[0170] The Zeta potential of the second derivative 14 increased more after co-incubation with Staphylococcus aureus, indicating that it bound more tightly to Staphylococcus aureus and had stronger inhibitory activity against Staphylococcus aureus, which was consistent with the aforementioned antibacterial activity results.

[0171] Example 11 PDT antitumor activity of the first derivative 13, the second derivative 14, and the third derivative 15 of the present invention

[0172] In view of the excellent ROS generation ability of the three products, A549 cells were selected to test their PDT anti-tumor activity and toxicity to HBE cells (light source: LED green light, irradiation time: 2 hours) and SI safety factor.

[0173] SI=IC 50 (HBE) / IC 50 (A549)

[0174] Table 2 IC values ​​of the first derivative 13, the second derivative 14 and the third derivative 15 on A549 cells and HBE cells at L(-) / L(+) 50 (μg·mL -1 ) and SI

[0175]

[0176] As shown in Table 2, the second derivative 14 has low dark toxicity to HBE cells but high phototoxicity, and has high dark and phototoxicity to A459 cells. In addition, its SI is the largest, indicating that the second derivative 14 has the potential to be developed as a new anti-lung cancer drug. This may be because the quinoline receptor in A459 cells binds more tightly. The specific reason needs further exploration.

[0177] from Figure 28 As can be seen, normal A459 cells have a spindle-shaped structure. However, after co-incubation with the second derivative 14 under L(-), some A459 cells ruptured and their structure changed. However, under L(+), A459 cells ablated and died. Therefore, the photosensitizer we synthesized not only has excellent antibacterial activity but also can serve as an excellent photodynamic anti-tumor photosensitizer.

Claims

1. A TB-double bond-pyridinium derivative, characterized in that: The structural formulas of the derivatives are shown as follows: the first derivative (13), the second derivative (14) and the third derivative (15):

2. A method for preparing the TB-double bond-pyridinium derivative according to claim 1, characterized in that: The preparation method comprises the following steps: Step 1: The following p-bromoaniline (1) reacts with paraformaldehyde (2) to obtain the first intermediate (3), and the reaction formula is as follows: Step 2: The first intermediate (3) reacts with DMF (4) to obtain the second intermediate (5). The reaction formula is as follows: Step 3: 4-Methylpyridine (6), 4-methylquinoline (9) and 4-pyridineacetonitrile (11) react with iodomethane (7) to obtain the third intermediate (8), the fourth intermediate (10) and the fifth intermediate (12), respectively. The reaction formulas are as follows: Step 4: The second intermediate (5) reacts with the third intermediate (8) to obtain the first derivative (13). The reaction formula is as follows: Step 5: The second intermediate (5) reacts with the fourth intermediate (10) to obtain the second derivative (14). The reaction formula is as follows: Step 6: The second intermediate (5) reacts with the fifth intermediate (12) to obtain the third derivative (15). The reaction formula is as follows:

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