TB-thiophene-pyridinium photodynamic bactericidal photosensitizer and its preparation and application

Abstract

The present invention provides a type of TB-thiophene-pyridinium photodynamic bactericidal photosensitizer and its preparation and application. 4-bromoaniline, paraformaldehyde, 5-aldehyde-2-thiopheneboronic acid and iodomethane are selected as raw materials and synthesized through multi-step reactions to obtain: #imgabs0# Three TB-thiophene-pyridinium derivatives have excellent photophysical properties, pH applicable range, viscosity response, AIE properties and good biocompatibility. DCHA probe detects that all three products can produce ROS, and under the same conditions, the ROS yield of 14 is higher. DHR123 probe and EPR detect that all three compounds can produce superoxide anions. The three products have low dark toxicity and phototoxicity to normal cells; they have low dark toxicity but high phototoxicity to A549 (human non-small cell lung cancer cells), and the IC of A549 cells after illumination is low. 50 The serum creatinine levels were significantly reduced from >100 to 0.31, 0.11, and 1.4 μg·mL, respectively. ‑1 , and has a high safety factor, making it an excellent PDT anti-tumor photosensitizer with great value for further research and development.

Description

Technical Field

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

[0002] Pathogenic bacteria pose one of humanity's greatest threats. Antibiotics have made tremendous contributions to combating bacteria, saving hundreds of millions of lives. However, the overuse of antibiotics has led to the emergence of bacterial resistance. The emergence of drug-resistant bacteria has significantly reduced the effectiveness of antibiotics, and humanity is once again facing the threat of pathogens. During the recently concluded World Antimicrobial Awareness Week, pharmaceutical experts and drug regulators warned that if we do not accelerate research into drugs and methods to combat drug-resistant bacteria, we will eventually run out of medicines.

[0003] Against this backdrop, antimicrobial photodynamic therapy (aPDT) has emerged. aPDT is a novel technology that uses light of a specific wavelength to irradiate a photosensitizer, triggering the production of reactive oxygen species (ROS) that damage the bacterial cytoplasmic membrane 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.

[0004] 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.

[0005] 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.

[0006] 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 ), with a high probability of intersystem crossing and a high probability of ROS generation. Therefore, theoretically, 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, no aPDT photosensitizers with AIE properties based on TB have been reported.

[0007] 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

[0008] Technical Problem: The present invention aims to provide a class of TB-thiophene-pyridinium photodynamic bactericidal photosensitizers, their preparation, and applications. Using 4-bromoaniline, paraformaldehyde, 5-formyl-2-thiopheneboronic acid, and iodomethane as raw materials, three target products are synthesized through a multi-step reaction, and their applications in aPDT and photodynamic anticancer treatment. The three products exhibit excellent photodynamic antitumor activity against normal cells, excellent optical properties, and aPDT and PDT antitumor activity against drug-resistant bacteria, making them promising for development in areas such as hospital wastewater treatment, non-invasive antitumor drugs, viscosity probes, cell imaging, and organelle localization.

[0009] Technical solution: The structural formula of the TB-thiophene-pyridinium photodynamic bactericidal photosensitizer of the present invention is shown as the first derivative 13, the second derivative 14 or the third derivative 15 as follows:

[0010]

[0011] The synthesis method of the TB-thiophene-pyridinium photodynamic bactericidal photosensitizer comprises the following steps:

[0012] Step 1: 4-bromoaniline 1 reacts with paraformaldehyde 2 to obtain the first intermediate 3. The reaction formula is as follows:

[0013]

[0014] Step 2: The first intermediate 3 reacts with DMF 4 to obtain the second intermediate 5. The reaction formula is as follows:

[0015]

[0016] 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 formula is as follows:

[0017]

[0018] Step 4: The second intermediate 5 reacts with the third intermediate 8 to obtain the first derivative 13. The reaction formula is as follows:

[0019]

[0020] Step 5: The second intermediate 5 reacts with the fourth intermediate 10 to obtain the second derivative 14. The reaction formula is as follows:

[0021]

[0022] Step 6: The second intermediate 5 reacts with the fifth intermediate 12 to obtain the third derivative 15. The reaction formula is as follows:

[0023]

[0024] Application of the first derivative 13, the second derivative 14, and the third derivative 15 in the preparation of viscosity probes.

[0025] Application of the first derivative 13, the second derivative 14, and the third derivative 15 in the preparation of photodynamic antibacterial drugs.

[0026] Application of the first derivative 13, the second derivative 14, and the third derivative 15 in the preparation of cancer photodynamic therapy drugs.

[0027] The antibacterial agent is directed against the inhibition of Staphylococcus aureus, Escherichia coli and methicillin-resistant Staphylococcus aureus (MRSA).

[0028] The cancer photodynamic therapy is directed to the inhibition of human non-small cell lung cancer cells A549.

[0029] Beneficial effects:

[0030] 1. TB-thiophene-pyridinium derivative photosensitizer was synthesized for the first time. The synthesis method is simple and the post-processing is convenient.

[0031] 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.

[0032] 3. The second derivative 14 showed good aPDT activity against Staphylococcus aureus and MRSA (2 μmol·L -1The antibacterial rates were 99% and 99.9% respectively).

[0033] 4. The three products have low dark toxicity and phototoxicity to normal cells (human bronchial epithelial cells, HBE); low dark toxicity but high phototoxicity to A549 (human non-small cell lung cancer cells). The IC values ​​of the second derivative 14 and the third derivative 15 on A549 cells after illumination are 50 The serum creatinine levels were significantly reduced from >100 to 0.11 and 1.4 μg·mL, respectively. -1 , and has a high safety factor (SI), showing excellent photodynamic antitumor activity.

[0034] 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

[0035] Figure 1 The first derivative 13 of the product in the embodiment 1 H NMR spectrum;

[0036] Figure 2 The first derivative 13 of the product in the embodiment 13 C NMR spectrum;

[0037] Figure 3 The second derivative 14 of the product in the embodiment 1 H NMR spectrum;

[0038] Figure 4 The second derivative 14 of the product in the embodiment 13 C NMR spectrum;

[0039] Figure 5 It is the third derivative 15 of the product in the embodiment 1 H NMR spectrum;

[0040] Figure 6 It is the third derivative 15 of the product in the embodiment 13 C NMR spectrum;

[0041] Figure 7 The UV absorption spectra and fluorescence emission spectra of the second intermediate 5, the first derivative 13, the second derivative 14, and the third derivative 15 in different solvents are as follows: the second intermediate 5: (a)(b), the first derivative 13: (a)(b), the second derivative 14: (a)(b), and the third derivative 15: (a)(b);

[0042] 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 DCM solvent;

[0043] Figure 9 is the solid-state fluorescence emission spectrum of the first derivative 13, the second derivative 14, and the third derivative 15;

[0044] Figure 10 Fluorescence emission spectra and line graphs of the first derivative 13, the second derivative 14, and the third derivative 15 at different pH values, the first derivative 13: (a)(b), the second derivative 14: (c)(d), the third derivative 15: (e)(f);

[0045] Figure 11 These are the fluorescence emission spectra and line graphs of the first derivative 13, the second derivative 14, and the third derivative 15 at different viscosities, the first derivative 13: (a)(b), the second derivative 14: (c)(d), the third derivative 15: (e)(f);

[0046] 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), the first derivative 13: (a) (b), the second derivative 14: (c) (d), the third derivative 15: (e) (f);

[0047] Figure 13 SEMs 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; the first derivative 13: (a) (b), the second derivative 14: (c) (d);

[0048] Figure 14 is the electron paramagnetic resonance spectrum (EPR) of the first derivative 13;

[0049] Figure 15 is the EPR of the second derivative 14;

[0050] Figure 16 is the EPR of the third derivative 15;

[0051] Figure 17 The first derivative 13, the second derivative 14, the third derivative 15 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;

[0052] Figure 18 is irradiated by white light (2.93W·cm-2 ) Line graph of DHR123 fluorescence intensity of the first derivative 13, the second derivative 14, the third derivative 15 and the blank;

[0053] Figure 19 It is LED white light (2.93W·cm -2 ) Decomposition rate of ABDA under irradiation of the first derivative 13, the second derivative 14, the third derivative 15, and RB, where A0 and A are the absorbance of ABDA at 378 nm before and after irradiation, respectively;

[0054] Figure 20 The first derivative 13 with different concentrations was detected in 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 21 The second derivative 14 with different concentrations was detected in 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 22 The third derivative 15 with different concentrations was observed in 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);

[0057] Figure 23 It is LED white light (2.93W·cm -2 ) Histogram of the antibacterial activity of 13-15 against (a) Staphylococcus aureus and (b) Escherichia coli under irradiation;

[0058] Figure 24 The first derivative 13 with different concentrations was detected in dark / LED white light (2.93W·cm -2 ) Antibacterial activity against MRSA after 20 minutes of irradiation (plate count method);

[0059] Figure 25 The second derivative 14 with different concentrations was detected in dark / LED white light (2.93W·cm -2 ) Antibacterial activity against MRSA after 20 minutes of irradiation (plate count method);

[0060] Figure 26 The third derivative 15 with different concentrations was observed in dark / LED white light (2.93W·cm -2 ) Antibacterial activity against MRSA after 20 minutes of irradiation (plate count method);

[0061] Figure 27 It is LED white light (2.93W·cm -2 ) Histogram of the antibacterial activity of the first derivative 13, the second derivative 14, and the third derivative 15 against MRSA under irradiation;

[0062] Figure 28 The second derivative 14 was tested for the interaction with Staphylococcus aureus or Escherichia coli in the dark and under light (LED white light, 2.93 W·cm -2 , 20 min) co-incubated SEM;

[0063] Figure 29 is the zeta potential of Staphylococcus aureus and Escherichia coli before and after incubation with the second derivative 14;

[0064] Figure 30 are electron micrographs of the second derivative 14 after incubation with A549 under dark / light conditions. DETAILED DESCRIPTION

[0065] The present invention will be further described below with reference to the embodiments.

[0066] 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.

[0067] TB-thiophene-pyridinium derivative, the structural formula of which is shown in Table 1 below:

[0068] Table 1. Structural formulas of 13-15

[0069]

[0070] Example 1 Synthesis of the First Derivative 13, the Second Derivative 14, and the Third Derivative 15

[0071] The present invention provides a method for preparing the novel TB-thiophene-pyridinium derivative, comprising:

[0072] In this embodiment, TB-thiophene-pyridinium derivatives were synthesized using 4-bromoaniline, paraformaldehyde, 5-formyl-2-thiopheneboronic acid, three pyridine derivatives, and iodomethane as raw materials through a multi-step reaction, including the following steps:

[0073] 1. Synthesis of the first intermediate 3

[0074] 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.

[0075]

[0076] 2. Synthesis of the Second Intermediate 5

[0077] Intermediate 3 (5 mmol), starting material 4 (5.5 mmol), PdCl2(dppf) (1 mmol), and K2CO3 (10 mmol) were weighed and added sequentially to a 100 mL two-necked flask under argon. 6 mL of methanol and 30 mL of toluene were then added. The mixture was heated to reflux under argon for 48 h. After completion of the reaction, deionized water was added to quench the reaction, followed by extraction with DCM (3 x 30 mL). The organic phase was washed with deionized water, dried over Na2SO4, and spin-dried to obtain the crude product. The crude product was purified by column chromatography (petroleum ether:ethyl acetate = 6:1 to 3:1) to obtain the second intermediate 5 (35%).

[0078]

[0079] 3. Synthesis of the third intermediate 8 and the fourth intermediate 10

[0080] Weigh 1.0 mmol of the raw material 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. During the process, white solids are continuously produced. 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).

[0081]

[0082] 4. Synthesis of the Fifth Intermediate 12

[0083] 1.0 mmol of the raw material 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%).

[0084]

[0085] 5. Synthesis of the first derivative 13, the second derivative 14, and the third derivative 15

[0086] Weigh 0.3 mmol of the third intermediate 8 (the fourth intermediate 10 or the fifth intermediate 12) and 0.3 mmol of the 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).

[0087]

[0088]

[0089] Product first derivative 13

[0090] The chemical formula is: C 27 H 23 BrIN3S

[0091] The Chinese name is: (E)-4-(2-(5-(8-bromo-6H,12H-5,11-methanedibenzo[b,f][1,5]diazooctan-2-yl)thiophen-2-yl)vinyl)-1-methylpyridinium iodide

[0092] The English name is: (E)-4-(2-(5-(8-bromo-6H,12H-5,11-methanodibenzo[b,f][1,5]diazocin-2-yl)thiophen-2-yl)vinyl)-1-methylpyridin-1-ium iodide

[0093] Appearance: red solid

[0094] Melting point: 237.2-238℃,

[0095] H NMR spectrum: 1 H NMR (400MHz, DMSO-d6) δ8.80(d,J=6.3Hz,2H),8.20(s,1H),8.16(d,J=5.6Hz,2H),7.51(s,1H),7.48(s,2H),7.33(s,1H ),7.21(d,J=8.2Hz,1H),7.14(s,2H),6.96(s,2H),4.67(dd,J=16.7,5.5Hz,2H),4.27(s,2H),4.22(s,3H),4.19(s,2H).

[0096] C NMR spectrum: 13 C(100MHz,DMSO-d6)δ152.13,149.08,147.91,147.09,144.82,138.94,133.69,133.42,129.03,128.05, 127.09,126.91,125.54,124.82,124.54,124.53,124.15,123.48,122.98,121.37,66.08,58.11,46.76.

[0097] Mass spectrometry: HRMS (ESI) for: C 27 H 23 N3S[M+H] + :calcd 4221692, found 422.1686.

[0098] Product second derivative 14

[0099] The chemical formula is: C 31 H 25 BrIN3S

[0100] The Chinese name is: E) 4-(2-(5-(8-bromo-6H,12H-5,11-methanedibenzo[b,f][1,5]diazooctan-2-yl)thiophen-2-yl)vinyl)1-methylquinolinium iodide

[0101] The English name is: (E)-4-(2-(5-(8-bromo-6H,12H-5,11-methanodibenzo[b,f][1,5]diazocin-2-yl)thiophen-2-yl)vinyl)-1-methylquinolin-1-ium iodide

[0102] Appearance: dark brown solid

[0103] Melting point: 222.6-24.5℃

[0104] H NMR spectrum: 1 HNMR(400MHz,DMSO-d6)δ9.28(d,J=6.5Hz,1H),8.93(d,J=8.6Hz,1H),8.47–8.3 3(m,3H),8.25(t,J=8.0Hz,1H),8.02(t,J=7.8Hz,1H),7.98–7.88(m,1H),7.72( d,J=3.6Hz,1H),7.55(q,J=6.3,5.4Hz,2H),7.34(d,J=21.5Hz,1H),7.27–7.10( m,3H),6.97(d,J=4.7Hz,1H),4.74–4.59(m,2H),4.50(s,3H),4.32–4.12(m,4H).

[0105] C NMR spectrum: 13 C NMR(100MHz,DMSO-d6)δ154.42,150.89,149.46,148.35,145.96,144.30,142.28,137.87,135.59,129.87,129.63,129.43,128.74,12 8.56,127.74,127.66,127.45,126.23,125.85,125.68,125.47,125.42,125.37,124.04,122.65,117.06,115.78,66.58,58.74,47.61.

[0106] Mass spectrometry: HRMS (ESI) for C 31 H 25 N3S[M+H] + :calcd 472.1841, found 472.1831.

[0107] Product third derivative 15

[0108] The chemical formula is: C 28 H 22 BrIN4S

[0109] The Chinese name is: (Z) 4-(2-(5-(8-bromo-6H,12H-5,11-methanedibenzo[b,f][1,5]diazooctan-2-yl)thiophen-2-yl)cyanovinyl)1-methylpyridinium iodide

[0110] The English name is: (Z)-4-(2-(5-(8-bromo-6H,12H-5,11-methanodibenzo[b,f][1,5]diazocin-2-yl)thiophen-2-yl)-1-cyanovinyl)-1-methylpyridin-1-ium iodide

[0111] Appearance: Brown

[0112] Melting point: 246.6-247.6℃

[0113] H NMR spectrum: 1 H NMR (400MHz, DMSO-d6): δ8.95(d,J=5.6Hz,3H),8.29(d,J=6.3Hz,2H),7.96(d,J=4.0Hz,1H),7.70(d,J=4.0Hz,1H),7.61(d,J=8.1Hz,1H ),7.44(s,1H),7.28(d,J=18.3Hz,1H),7.13(d,J=7.3Hz,2H),6.96(d,J=4.9Hz,1H),4.76–4.59(m,2H),4.29(s,3H),4.28–4.14(m,4H).

[0114] C NMR spectrum: 13 C(100MHz,DMSO-d6)δ152.13,149.08,147.91,147.09,144.82,138.94,133.70,133.42,129.03,128.05, 127.09,126.91,125.54,124.82,124.54,124.53,124.15,123.48,122.98,121.37,66.08,58.11,46.76.

[0115] Mass spectrometry: HRMS (ESI) for: C 28 H 22 N4S[M+H] + :calcd 447.1644, found 447.1622.

[0116] Example 2 Solvation Effects of Compounds of the Invention

[0117] The second intermediate 5, the first derivative 13, the second derivative 14, and the third derivative 15 were prepared respectively with n-hexane (n-Hexane), toluene (Toluene), tetrahydrofuran (THF), ethyl acetate (EA), acetonitrile (MeCN), ethanol (EtOH), N,N-dimethylformamide (DMF), and deionized water (H2O) to a concentration of 1×10 -5 mol·L -1 Solution, measure the UV-visible absorption spectrum and fluorescence emission spectrum of the compound in different solvents ( Figure 7 ).

[0118] Example 3 Optical Properties of the First Derivative 13, the Second Derivative 14, and the Third Derivative 15 of the Present Invention

[0119] The photophysical properties of the first derivative 13, the second derivative 14, and the third derivative 15 were tested using DCM as solvent. Figure 8 ).

[0120] 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 )

[0121] Table 2 Spectral data of compounds 13, 14 and 15

[0122]

[0123] 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

[0124] Depend on Figure 8-9 From Table 2, we can see that:

[0125] The first derivative 13, the second derivative 14, and the third derivative 15 are in solution and in solid state. abs All of them enter the visible light zone, and white light can be used as the light source for the photodynamic antibacterial experiment.em In the red light region, red light has a long range and strong penetrating ability, and can form bright fluorescent imaging.

[0126] Compared with the first derivative 13 and the third derivative 15, the second derivative 14 having a quinolinium structure has a longer emission wavelength, and the emission wavelength enters the red light region, indicating that the conjugated system has a great influence on the optical properties of the product.

[0127] The above results indicate that the introduction of thiophene and pyridinium cations on TB can optimize the photophysical properties of TB derivatives.

[0128] Example 4 pH Response of the First Derivative 13, the Second Derivative 14, and the Third Derivative 15 of the Present Invention

[0129] 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 -1 0.1 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 ).

[0130] 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.

[0131] Example 5 Viscosity Response of the First Derivative 13, the Second Derivative 14, and the Third Derivative 15 of the Present Invention

[0132] 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 ).

[0133] Depend on Figure 11As 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 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.

[0134] Example 6 AIE properties of the first derivative 13, the second derivative 14, and the third derivative 15 of the present invention

[0135] 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 ).

[0136] Depend on Figure 12 It can be seen that with the increase of n-hexane content, the fluorescence intensity of the first derivative 13 and the second derivative 14 gradually increases. The first derivative 13 and the second derivative 14 both show the strongest fluorescence, and the fluorescence intensity increases by 2.0 times and 2.6 times, respectively, both showing significant AIE properties.

[0137] The morphologies 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 were observed by SEM ( Figure 13 )

[0138] Depend on Figure 13 As shown in Figures (a)-(d), the first and second derivatives 13 and 14 exhibited a small number of particles in the EtOH / n-hexane (v / v) ratio of 9 / 1. However, at EtOH / n-hexane (v / v) of 3 / 7, the number of aggregated nanoparticles increased dramatically, demonstrating AIE properties. The third derivative 15 lacked significant AIE properties, possibly due to its low solubility in this solvent system. Further work will require screening for more suitable solvent systems.

[0139] 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

[0140] 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.

[0141] Electron paramagnetic resonance (EPR) spectroscopy

[0142] Depend on Figures 14 to 16 Under illumination, EPR detected signals of superoxide anions and singlet oxygen from the first derivative 13. The second derivative 14 detected signals of hydroxyl radicals, singlet oxygen, and superoxide anions. Photosensitizers that can simultaneously generate these three reactive oxygen species are rarely reported. The third derivative 15 detected signals of hydroxyl radicals and singlet oxygen. Therefore, all three compounds have the potential to be used as photosensitizers for aPDT.

[0143] Fluorescent probes

[0144] Total ROS

[0145] 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.

[0146] 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.

[0147] Depend on Figure 17 It can be seen that the first derivative 13, the second derivative 14, and the third derivative 15 generate ROS, and the ROS production increases with time. Under the same conditions, the second derivative 14 has the highest ROS production.

[0148] Superoxide anion (O2 ·- )

[0149] 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 ).

[0150] Depend on Figure 18It can be seen that under the irradiation of LED white light, the first derivative 13, the second derivative 14, and the third derivative 15 all have O2 ·- 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.

[0151] Singlet oxygen ( 1 O2)

[0152] 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.

[0153] Depend on Figure 19 It can be seen that under the irradiation of LED white light, the first derivative 13, the second derivative 14, and the third derivative 15 can all produce 1 O2, and under the same conditions 1 The yield of O2 is higher than that of commercial RB. 1 O2 production is the highest.

[0154] Example 8 In vitro antibacterial activity of the first derivative 13, the second derivative 14, and the third derivative 15 of the present invention

[0155] 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.

[0156] Dark Toxicity

[0157] Before conducting the antibacterial activity test, the dark toxicity against bacteria was first tested (Table 3). As shown in Table 3, under dark conditions, the MICs of the second intermediate 5, the third intermediate 8, the fourth intermediate 10, the fifth intermediate 12, and RB against Escherichia coli, Staphylococcus aureus, and Bacillus subtilis all exceeded 64 μg·mL. -1 , indicating that the above raw materials and commercial Bengal rose red have no antibacterial activity under dark conditions. The MICs of the first derivative 13, the second derivative 14, and the third derivative 15 against Escherichia coli all exceeded 64 mg·L -1 , did not show significant antibacterial activity, and the MICs against Staphylococcus aureus were 8, 4, and 16 mg·L -1, the MICs against Bacillus subtilis were 16, 2, and 16 mg·L -1 , showing certain antibacterial activity against Gram-positive bacteria. Among them, the second derivative 14 showed the strongest antibacterial effect against the two Gram-positive bacteria.

[0158] Table 3 Minimum inhibitory concentrations (MIC) of the second intermediate 5, the third intermediate 8, the fourth intermediate 10, the fifth intermediate 12, the first derivative 13, the second derivative 14, and the third derivative 15 of RB (rhodamine B) against Escherichia coli, Staphylococcus aureus, and Bacillus subtilis under dark conditions (μg mL -1 )

[0159]

[0160]

[0161] Example 9 aPDT activity of the first derivative 13, the second derivative 14, and the third derivative 15 of the present invention

[0162] The antibacterial activities of the first derivative 13, the second derivative 14, and the third derivative 15 were tested by plate colony counting method.

[0163] Table 4 Inhibitory rate of the first derivative 13, the second derivative 14, and the third derivative 15 against Staphylococcus aureus (%)

[0164]

[0165] Table 5 Antibacterial rate of the first derivative 13, the second derivative 14, and the third derivative 15 on Escherichia coli (%)

[0166]

[0167] Depend on Figure 20 (A) Figure 23 As shown in Table 3, the first derivative 13 under dark conditions -1 The first derivative 13 showed antibacterial activity against Staphylococcus aureus at 2 μmol·L -1 It shows obvious antibacterial activity. Figure 20 (B) Figure 23 As shown in Table 4, the first derivative 13 under dark conditions -1 It showed certain anti-Escherichia coli activity. Under light conditions, 20μmol·L -1 It shows strong antibacterial effect, with an inhibition rate of 100%.

[0168] Depend on Figure 21 (A) Figure 23As shown in Table 3, the second derivative 14 under dark conditions -1 It has no antibacterial activity against Staphylococcus aureus. Under light conditions, 2 μmol·L -1 It shows a strong antibacterial effect. Figure 21 (B) Figure 23 As shown in Table 4, the second derivative 14 under dark conditions -1 It has no anti-Escherichia coli activity, but under light conditions, 10 μmol·L -1 It shows strong antibacterial effect and the sterilization rate is 100%.

[0169] Depend on Figure 22 (A) Figure 23 As shown in Table 3, the third derivative 15 under dark conditions -1 It has a strong antibacterial effect on Staphylococcus aureus, and the concentration of 4 μmol·L -1 It showed antibacterial effect at 8μmol·L -1 The sterilization rate is 100%. Figure 22 (B) Figure 23 As shown in Table 3, the third derivative 15 under dark conditions -1 It showed certain anti-Escherichia coli activity, and under light conditions, 20 μmol·L -1 The antibacterial rate is 100%.

[0170] Among the three products, the second derivative 14 had the greatest aPDT activity, which was consistent with the ROS detection results.

[0171] 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.

[0172] Table 6 Inhibitory rate of the first derivative 13, the second derivative 14, and the third derivative 15 against MRSA (%)

[0173]

[0174] Depend on Figure 24-26 、 Figure 27 As shown in Table 6, the first derivative 13, the second derivative 14, and the third derivative 15 all have aPDT activity against MRSA. The second derivative 14 has the highest aPDT activity and exhibits antibacterial activity under both dark and light conditions, but the concentration used to exhibit antibacterial activity under light conditions is lower, 1 μmol·L -1 That is, it has strong antibacterial activity.

[0175] Example 10 Antibacterial Mechanism of the First Derivative 13, the Second Derivative 14, and the Third Derivative 15 of the Present Invention Scanning Electron Microscope (SEM)

[0176] 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 2 μmol·L -1 , the concentration of anti-E. coli is 10 μmol·L -1 ( Figure 28 ).

[0177] Depend on Figure 28 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.

[0178] Zeta potential

[0179] From the electron microscope image ( Figure 29 ) 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.

[0180] Depend on Figure 29 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.

[0181] 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.

[0182] Example 11 PDT antitumor activity of compounds 13-15 of the present invention

[0183] 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.

[0184] SI=IC 50 (HBE) / IC 50 (A549)

[0185] Table 7 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

[0186]

[0187] As shown in Table 7, the first derivative 13, the second derivative 14, and the third derivative 15 have low dark toxicity to A549 cells. 50 The serum creatinine levels were significantly reduced from >100 to 0.31, 0.11, and 1.4 μg·mL, respectively. -1 , with high photodynamic antibacterial activity. At the same time, the first derivative 13 and the second derivative 14 have high SI, making them excellent PDT anti-tumor photosensitizers, and their further research is of great value.

[0188] from Figure 30 It can be seen that normal A459 cells have a spindle-shaped structure, while A459 cells co-incubated with the second derivative 14 under light conditions rupture, their structure changes, and the cells ablate.

[0189] Therefore, the photosensitizer we synthesized not only has excellent antibacterial activity but can also serve as an excellent photodynamic anti-tumor photosensitizer.

Claims

1. A type of TB-thiophene-pyridinium photodynamic bactericidal photosensitizer, characterized in that: Its structural formula is shown as the first derivative (13), the second derivative (14) or the third derivative (15) below:

2. A method for synthesizing the TB-thiophene-pyridinium photodynamic bactericidal photosensitizer according to claim 1, characterized in that: The following steps are involved: Step 1: 4-bromoaniline (1) reacts with paraformaldehyde (2) to obtain the first intermediate (3). The reaction formula is as follows: Step 2: The first intermediate (3) reacts with compound (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 formula is 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:

3. A use of the TB-thiophene-pyridinium photodynamic bactericidal photosensitizer according to claim 1, characterized in that The first derivative (13), the second derivative (14), and the third derivative (15) are used in the preparation of photodynamic antibacterial drugs, wherein the antibacterial drugs are directed against Staphylococcus aureus and Escherichia coli.

4. A use of the TB-thiophene-pyridinium photodynamic bactericidal photosensitizer according to claim 1, characterized in that The first derivative (13), the second derivative (14), and the third derivative (15) are used in the preparation of cancer photodynamic therapy drugs, wherein the cancer photodynamic therapy is aimed at inhibiting human non-small cell lung cancer cells A549.

Images