Preparation method and application of a novel carboline quaternary ammonium salt compound with photodynamic antibacterial activity

Abstract

This invention discloses a method for preparing and applying a novel carboline quaternary ammonium salt compound with photodynamic antibacterial activity. The novel carboline quaternary ammonium salt compound of this invention, by introducing a triphenylamine group onto the carboline core, effectively reduces the fluorescence intensity of this type of compound. After illumination, energy is induced to generate ROS via intersystem crossing, thus improving the photodynamic effect of the carboline compound. Simultaneously, due to the presence of the quaternary ammonium salt group, the novel carboline quaternary ammonium salt compound of this invention can target the highly negatively charged surface of bacteria, effectively killing bacteria and drug-resistant bacteria after illumination. Therefore, this novel carboline compound of this invention can be applied in the pharmaceutical field for the preparation of antibacterial drugs.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to a method for preparing a novel carboline quaternary ammonium salt compound with photodynamic antibacterial activity, particularly its pharmaceutical use as a photosensitizer that undergoes a photosensitization reaction and generates reactive oxygen species after light irradiation, thereby exerting a photodynamic antibacterial therapeutic effect without inducing the development of drug-resistant bacteria, and its application in the preparation of antibacterial drugs. Background Technology

[0002] Bacterial infectious diseases are a significant public health problem worldwide. However, the rapid industrialization of antibiotics due to rising global economic levels has led to further reductions in production costs, accelerating the overuse of these antibiotics in healthcare institutions globally. This has resulted in antibiotic resistance in some pathogenic bacteria, reducing the effectiveness of antibiotic drugs. Drug-resistant bacteria can inactivate antibiotics in various ways. Besides producing inactivating enzymes, they can also alter the structure of certain proteins on their outer membrane, making it difficult for antibiotics to bind. Simultaneously, bacteria can enhance their resistance by altering membrane permeability, strengthening efflux pumps, and forming biofilms to prevent antibiotic entry. Methicillin-resistant Staphylococcus aureus (MRSA) is the most notorious superbug, extremely common in hospital and community infections, causing infections of the skin, lungs, blood, and joints. In recent years, nosocomial infections caused by MRSA have not only been a frequent and challenging problem for clinicians but also pose a significant threat to human health. Exploring novel antibacterial methods to address the resistance of drug-resistant bacteria to traditional antibiotics has become a new research direction.

[0003] Photodynamic therapy (PDT) utilizes appropriate excitation light to activate photosensitizers (PS), generating reactive oxygen species (ROS) that further oxidize adjacent biomolecules, thus killing target cells. Antimicrobial photodynamic therapy (PACT) is a derivative of PDT, capable of killing both susceptible and drug-resistant bacteria without inducing resistance. Its mechanism of action primarily involves the photosensitizer transitioning from its ground state (singlet state) to an excited singlet state under irradiation with visible light of a specific wavelength. The short-lived excited singlet state then rapidly transitions to an excited triplet state, which reacts with molecular oxygen in the environment via electron transfer or energy transfer, producing cytotoxic ROS. This effectively kills both drug-sensitive and resistant pathogens. Furthermore, the high reactivity of ROS and its ability to denature known antioxidant enzymes (superoxide dismutase and catalase) makes them difficult to defend against, meaning that repeated photosensitization of bacteria does not induce the development of resistant strains. Therefore, antimicrobial photodynamic therapy is a promising approach for treating MRSA.

[0004] Alkaloids have long been an important source of antibacterial drugs. β-Carboline is a class of naturally occurring alkaloids widely distributed in plants, marine organisms, insects, mammals, and human tissues and fluids. In recent years, research on β-carboline compounds has increasingly focused on marine organisms. Marine organisms, living in unique environments such as high pressure and hypoxia, produce and accumulate large amounts of substances with special chemical structures and unique physiological activities during their growth and metabolism. Studies have shown that these substances have broad biological activities, mainly including antitumor, antibacterial, antifungal, antiviral, antiparasitic, anti-inflammatory, diabetes treatment, and neuropharmacological activities. Therefore, the discovery of β-carboline, a natural alkaloid with antibacterial activity, is an important foundation for developing novel and highly effective antibacterial drugs.

[0005] Based on the unique chemical structure of β-carboline (a tricyclic pyridine-indole ring as the parent nucleus), its highly conjugated planar polyaryl structure, strong fluorescence, and numerous studies in recent years, β-carboline alkaloids have been shown to be potential antibacterial lead compounds. Among them, Harman A69, Canthin-6-one A72, and Eudistomin U A98 exhibit particularly outstanding antibacterial activity. Canthin-6-one A72 has an IC50 range of 8-64 μg / mL against Staphylococcus aureus, while Eudistomin U A98 has an IC50 range of [missing information]. 50 With a concentration of 6.4 μg / mL, its antibacterial activity is close to that of commercially available antibacterial drugs such as fosfomycin sodium, ampicillin sodium, and ciprofloxacin. Furthermore, to date, there are nine commercially available drugs with β-carboline as the core skeleton, including one of the world's top 100 drugs, further illustrating the importance of research on this type of compound and the feasibility of drug development. However, long-term use easily leads to drug resistance, especially in multidrug-resistant bacterial strains, significantly reducing the possibility of future widespread use. Therefore, we use antibacterial photodynamic therapy to overcome the drug resistance defect. We introduce a triphenylamine group and a quaternary ammonium salt bacterial targeting group onto the β-carboline fragment to develop a novel carboline quaternary ammonium salt photosensitizer, which can be used for photodynamic anti-drug-resistant bacteria. Summary of the Invention

[0006] Purpose of the invention: The purpose of this invention is to provide a method for preparing and applying a novel carboline quaternary ammonium salt compound with photodynamic antibacterial activity.

[0007] This invention develops a carboline-based quaternary ammonium salt photosensitizer for photodynamic antibacterial effects. The introduction of a triphenylamine group quenches the fluorescence of the carboline, increasing the efficiency of ROS generation. The introduction of the quaternary ammonium salt group allows the compound to target the highly negatively charged surface of bacteria, achieving bacterial targeting. Upon irradiation at 420 nm, the novel carboline quaternary ammonium salt compound generates destructive ROS, effectively killing bacteria and drug-resistant bacteria, thus expanding the clinical translational prospects of carboline photosensitizers in antibacterial therapy.

[0008] Technical solution: The objective of this invention is achieved through the following technical solution:

[0009] This invention provides a novel carboline quaternary ammonium salt compound having the structure shown in general formula I and possessing photodynamic antibacterial activity;

[0010]

[0011] in,

[0012] R1 is an alkyl, brominated alkyl, or quaternized alkyl;

[0013] R2 is a halogen.

[0014] In a preferred embodiment of the present invention, R1 is selected from -CH3, -CH2CH3, -CH2CH2CH3, -CH2CH2CH2CH3, -CH2CH2CH2CH2CH3, -CH2Br, -CH2CH2Br, or -CH2CH2CH2N. + (CH3)3Br - R2 is selected from I - or Br - .

[0015] In a preferred embodiment of the present invention, the novel carboline compound preferably comprises the following compounds, the structural formulas of which are shown in Table 1:

[0016] Table 1. Compound codes and corresponding structures for some compounds of general formula I.

[0017]

[0018]

[0019] I1: 6-(4-(diphenylamino)phenyl)-2-methyl-9H-pyridine[3,4-b]indole-2-iodide

[0020] I2: 6-(4-(diphenylamino)phenyl)-2-propyl-9H-pyridine[3,4-b]indole-2-iodide

[0021] I3: 2-(2-bromoethyl)-6-(4-(diphenylamino)phenyl)-9H-pyridine[3,4-b]indole-2-bromide

[0022] I4: 6-(4-(diphenylamino)phenyl)-2-(3-(trimethylammonium)propyl)-9H-pyridine[3,4-b]indole-2-bromine I5: 6-(4-(diphenylamino)phenyl)-2-ethyl-9H-pyridine[3,4-b]indole-2-iodide

[0023] I6: 2-Butyl-6-(4-(diphenylamino)phenyl)-9H-pyridine[3,4-b]indole-2-iodide

[0024] I7: 6-(4-(diphenylamino)phenyl)-2-pentyl-9H-pyridine[3,4-b]indole-2-iodide

[0025] I8: 2-(bromomethyl)-6-(4-(diphenylamino)phenyl)-9H-pyridine[3,4-b]indole-2-iodide

[0026] This invention also provides a method for preparing novel carboline compounds represented by general formula I, comprising the following steps:

[0027]

[0028] In some specific technical solutions, the method of the present invention is as follows:

[0029] (1) Using demethylhalman as a raw material, it undergoes a substitution reaction with N-bromosuccinimide to obtain intermediate compound 2;

[0030] (2) Intermediate compound 2 reacts with triphenylamine 4-borate and potassium carbonate to give compound 3;

[0031] (3) Finally, compound 3 reacts with R1-R2 to obtain the novel carboline compound shown in general formula I.

[0032] R1 and R2 are defined as in general formula I.

[0033] In step (1), the reaction temperature is room temperature, the reaction solvent is acetic acid, and the reaction time is 10-14 hours.

[0034] In step (2), the catalyst used in the reaction is tetrakis(triphenylphosphine)palladium, the reaction solvent is toluene, and the reaction temperature is reflux temperature; in step (3), the reaction temperature is 90-110℃, the reaction time is 10-14h, and the reaction solvent is acetonitrile.

[0035] In some more specific technical solutions, the molar ratio of norhalman and N-bromosuccinimide in step (1) is 1:1 to 2.

[0036] In some more specific technical solutions, the catalyst in step (2) is an intermediate compound 2, 4-boronic acid triphenylamine, potassium carbonate and tetratriphenylphosphine palladium in a molar ratio of 0.3-1:0.5-1.5:0.5-2:0.01-1.

[0037] In some more specific technical solutions, the molar ratio of compound 3 to R1-R2 in step (3) is 1:1 to 1.5.

[0038] All compounds of general formula I of this invention can be prepared by the methods described above or similarly described, with the appropriate starting materials selected according to the different substituents and their positions. Those skilled in the art should recognize that the above-described route helps in understanding this invention, but does not limit its scope; unless otherwise specified, variables are defined as mentioned in general formula I.

[0039] The present invention also provides a pharmaceutical composition comprising a novel carboline compound of formula I and a pharmaceutically acceptable carrier or excipient.

[0040] The pharmaceutical compositions of the present invention can be administered in various known ways, such as orally, parenterally, or via an implanted reservoir. The pharmaceutical compositions of the present invention can be administered alone or in combination with other drugs. Oral compositions can be any orally acceptable dosage form, including, but not limited to, tablets, capsules, emulsions, suspensions, dispersions, and solutions. Commonly used pharmaceutically acceptable carriers or excipients include stabilizers, diluents, surfactants, lubricants, antioxidants, binders, colorants, fillers, emulsifiers, etc.

[0041] Sterile injectable compositions may be formulated using suitable dispersants or wetting agents and suspending agents in accordance with techniques known in the art. Pharmaceutically acceptable carriers and solvents that may be used include water, mannitol, sodium chloride solution, etc.

[0042] The actual dosage level of the active ingredient in the pharmaceutical composition of the present invention can be varied to obtain an amount of active ingredient that is effective in achieving the desired therapeutic response for a particular patient, composition, and route of administration, and is non-toxic to the patient. The selected dosage level depends on a variety of factors, including the activity of the specific compound of the present invention used, the route of administration, the time of administration, the excretion rate of the specific composition used, the duration of treatment, other drugs, compounds, and / or materials used in combination with the specific composition used, the age, sex, weight, general health condition, and medical history of the patient being treated, and similar factors known in the medical field.

[0043] This invention also provides the application of the novel carboline compounds in the preparation of antibacterial drugs.

[0044] The novel carboline compounds described above have antibacterial photodynamic therapy effects. After being irradiated with excitation light, they can generate ROS, thereby effectively killing sensitive and drug-resistant bacteria in a non-specific manner. Furthermore, ROS is highly reactive and has the property of denaturing known antioxidant enzymes (superoxide dismutase and catalase), making it difficult to defend against. Therefore, it will not induce the emergence of drug-resistant strains.

[0045] The novel carboline compounds introduced with quaternary ammonium groups adsorb onto the surface of bacteria with a high negative charge, thereby achieving targeted photodynamic antibacterial effects.

[0046] By summarizing the antibacterial activities of natural and derived synthetic β-carboline alkaloids, we preliminarily concluded the structure-activity relationship as follows: (1) Fully aromatic β-carboline nuclei are stronger than hydrogenated nuclei, but hydrogenation and heterocyclization at positions 2 and 3 will improve activity; (2) Substitution at position 1 is beneficial to improving activity, with aromatic ring substitution being better than alkyl chain substitution, but steric hindrance should not be too large; (3) Quaternization at position 2 can significantly improve antibacterial activity; (4) Substitution at positions 3 or 9 is beneficial to improving activity, but alkyl chain substitution at position 9 should not be too long; (5) Substitution of the A ring of the carboline nucleus with methoxy, hydroxyl and halogen can improve activity, while ester substitution will reduce activity; (6) Chain-like linkage at position 1, heterocyclic linkage at positions 1 and 2, dimerization modification can significantly improve activity; direct linkage at position 9, dimerization will reduce activity; (7) Increasing the D ring can significantly improve antibacterial activity.

[0047] Based on structure-activity relationship and antibacterial photodynamic therapy research, we introduced a quaternary ammonium group at the 2-position of the β-carboline core, introducing a positive charge. This allows the compound molecule to adsorb onto the surface of bacteria with a high negative charge, thus achieving a targeted antibacterial effect. Furthermore, we introduced a triphenylamine group at the 6-position of the β-carboline core, reducing the fluorescence of the carboline core and increasing the photodynamic properties of the compound. The resulting novel carboline quaternary ammonium salt compound exhibits bacterial-targeted photodynamic therapy, which is of great significance for addressing the practical challenges of drug-resistant bacterial infections and for clinical translation.

[0048] Beneficial effects:

[0049] The novel carboline compounds provided by this invention exhibit strong fluorescence and photodynamic properties, generating reactive oxygen species (ROS) upon excitation light irradiation, effectively killing both sensitive and drug-resistant bacteria. Furthermore, the introduction of quaternary ammonium groups into the compound structure allows positively charged molecules to adsorb onto the highly negatively charged bacterial surface, achieving targeted antibacterial effects. Therefore, these novel carboline compounds can be used to prepare antibacterial drugs, showing broad application prospects in the treatment of bacterial infections, particularly those caused by drug-resistant bacteria (such as MRSA), through precise and efficient photodynamic therapy. Attached Figure Description

[0050] Figure 1 The UV spectrum of the compound (10 μM) of this invention in a deionized aqueous solution of 1% DMSO is shown.

[0051] in, Figure 1 A is the ultraviolet spectrum of compound I1 in a 1% DMSO deionized aqueous solution; Figure 1 B is the ultraviolet spectrum of compound I2 in a 1% DMSO deionized aqueous solution; Figure 1 C represents the ultraviolet spectrum of compound I3 in a 1% DMSO deionized aqueous solution; Figure 1 D is the UV spectrum of compound I4 in a 1% DMSO deionized aqueous solution. All compounds were at a concentration of 10 μM.

[0052] Figure 2 Fluorescence spectroscopy detection of the compound (10 μM) of this invention in a deionized aqueous solution of 1% DMSO.

[0053] in, Figure 2 A is the fluorescence spectrum of compound norhalman in a deionized aqueous solution of 1% DMSO; Figure 2 B is the fluorescence spectrum of compound I1 in a 1% DMSO deionized aqueous solution; Figure 2 C represents the fluorescence spectrum of compound I2 in a 1% DMSO deionized aqueous solution; Figure 2 D is the fluorescence spectrum of compound I3 in a 1% DMSO deionized aqueous solution; Figure 2 E represents the fluorescence spectrum of compound I4 in a 1% DMSO deionized aqueous solution; the excitation wavelength is 365 nm. Figure 3 The compound of this invention (10 μM) was subjected to 420 nm (60 mW cm⁻¹) in a 1% DMSO deionized aqueous solution. -2 The ROS results were generated after 60 seconds of illumination.

[0054] in, Figure 3 A represents the change in the fluorescence spectrum of the indicator DCFH (5μm) before and after irradiation at 420nm; Figure 3B represents the change in the fluorescence spectrum of DCFH (5μm) after RB is irradiated at 420nm; Figure 3 C represents the change in the fluorescence spectrum of compound I1 at DCFH (5 μm) after irradiation at 420 nm; Figure 3 D represents the change in the fluorescence spectrum of compound I2 at DCFH (5 μm) after irradiation at 420 nm; Figure 3 E represents the change in the fluorescence spectrum of compound I3 at DCFH (5 μm) after irradiation at 420 nm; Figure 3 F represents the fluorescence spectrum change of compound I4 after irradiation at 420 nm using DCFH (5 μm).

[0055] Figure 4 This is the result of the dark toxicity and photodynamic antibacterial treatment of compounds I1-I4 of the present invention.

[0056] in, Figure 4 A represents the viability of LO2 cells incubated with different concentrations of compounds I1-I4 under light-protected conditions, as determined by MTT assay. Figure 4 B is OD 600 Measurement and detection at 420nm (60mW / cm) 2 Viability of MRSA bacteria incubated with different concentrations of compounds I1-I4 under light irradiation (6 min). Detailed Implementation

[0057] The technical solution of the present invention will be described in detail below through specific embodiments, but the scope of protection of the present invention is not limited to the embodiments described.

[0058] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0059] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the experimental materials used in the following examples are commercially available products.

[0060] Example 1 Preparation of 6-(4-(diphenylamino)phenyl)-2-methyl-9H-pyridine[3,4-b]indole-2-iodide (I1)

[0061]

[0062] Dissolve norhalman (168 mg, 1.0 mmol) in acetic acid, add N-bromosuccinimide (267 mg, 1.5 mmol), and stir overnight at room temperature until the reaction is complete. Dilute with water, extract with ethyl acetate, wash the organic phase with saturated brine, dry with anhydrous sodium sulfate, concentrate under reduced pressure to obtain crude product, and purify the crude product by column chromatography to obtain 160 mg of pale yellow solid (2), with a yield of 65.0%.

[0063] ESI-MS (m / z): 248 [M+H] + ;

[0064] 1 H NMR (400MHz, DMSO-d6) δ (ppm) 11.81 (s, 1H), 8.96 (s, 1H), 8.53 (d, J = 2.0Hz, 1H), 8.38 (d, J = 4.8Hz, 1H), 8.17 (d, J = 5.2Hz, 1H), 7.69 -7.67(m,1H),8.58(d,J=8.8Hz,1H).

[0065] Compound 2 (160 mg, 0.65 mmol), triphenylamine 4-borate (283 mg, 0.98 mmol), and potassium carbonate (179 mg, 1.3 mmol) were dissolved in toluene. Under nitrogen protection, the catalyst tetra-triphenylphosphine palladium (81 mg, 0.07 mmol) was added, and the mixture was refluxed and stirred overnight until the reaction was complete. After dilution with water and extraction with ethyl acetate, the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the crude product. The crude product was purified by column chromatography to obtain a yellow solid (3) of 162 mg, with a yield of 60.5%.

[0066] ESI-MS (m / z): 412 [M+H] + ;

[0067] 1 H NMR (400MHz, DMSO-d6) δ (ppm) 11.67 (s, 1H), 8.92 (s, 1H), 8.54 (s, 1H), 8.36 (d, J = 4.4Hz, 1H), 8.17 (d,J=5.2Hz,1H),7.85-7.82(m,1H),7.72-7.70(m,3H),7.33(t,J=8.0Hz,4H),7.11-7.04(m,8H).

[0068] Compound 3 (162 mg, 0.39 mmol) was dissolved in anhydrous acetonitrile. Iodomethane (61 mg, 0.43 mmol) was slowly added dropwise under ice bath conditions. After the addition was complete, the mixture was refluxed and stirred overnight until the reaction was complete. The reaction was quenched with water, extracted with ethyl acetate, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the crude product. The crude product was purified by column chromatography to give 109 mg of a yellow solid (I1), with a yield of 50.8%.

[0069] ESI-MS (m / z): 553 [M+H] + ;

[0070] 1 H NMR(500MHz,DMSO-d6)δ(ppm)13.01(s,1H),9.60(s,1H),8.97(d,J=6.5Hz,1H),8.85(s,1H),8.83(d,J=6.5Hz,1H),8.1 3(d,J=8.5Hz,1H),7.94(d,J=8.5Hz,1H),7.76(d,J=8.0Hz,2H),7.36(t,J=8.0Hz,4H),7.16–7.08(m,8H),4.45(s,3H).

[0071] Example 2 Preparation of 6-(4-(diphenylamino)phenyl)-2-propyl-9H-pyridine[3,4-b]indole-2-iodide (I2)

[0072] Following the synthesis method of (I1) in Example 1, 1-iodopropane was used instead of iodomethane in the method, and 123 mg of yellow solid (I2) was finally obtained, with a yield of 54.3%.

[0073] ESI-MS (m / z): 581 [M+H] + ;

[0074] 1 H NMR(500MHz,DMSO-d6)δ(ppm)12.99(s,1H),9.63(s,1H),8.89(d,J=6.5Hz,1H),8.86(s,1H),8.84(d,J=7.0Hz,1H),8.15(d,J=8.5Hz,1H),7.97( d,J=9.0Hz,1H),7.77(d,J=8.0Hz,2H),7.37(t,J=8.0Hz,4H),7.14–7.06 (m,8H),4.77(t,J=7.5Hz,2H),1.58-1.55(m,2H),1.02(t,J=7.5Hz,3H).

[0075] Example 3 Preparation of 2-(2-bromoethyl)-6-(4-(diphenylamino)phenyl)-9H-pyridine[3,4-b]indole-2-bromide (I3)

[0076] Following the synthesis method of (I1) in Example 1, 1,2-dibromoethane was used instead of iodomethane in the method, and 120 mg of yellow solid (I3) was finally obtained, with a yield of 51.5%.

[0077] ESI-MS (m / z): 599 [M+H] + ;

[0078] 1 H NMR(500MHz,DMSO-d6)δ(ppm)12.98(s,1H),9.61(s,1H),8.90(d,J=6.5Hz,1H),8.85(s,1H),8.82(d,J=6.5Hz,1H),8.13(d,J=8.5Hz,1 H),7.96(d,J=9.0Hz,1H),7.75(d,J=8.0Hz,2H),7.38(t,J=8.0Hz,4H),7.15–7.06(m,8H),4.80(t,J=7.0Hz,2H),3.50(t,J=7.0Hz,2H).

[0079] Example 4 Preparation of 6-(4-(diphenylamino)phenyl)-2-(3-(trimethylammonium)propyl)-9H-pyridine[3,4-b]indole-2-bromide (I4)

[0080] Following the synthesis method of (I1) in Example 1, (3-bromopropyl)trimethylammonium bromide was used instead of iodomethane in the method, and 130 mg of yellow solid (I4) was finally obtained, with a yield of 49.8%.

[0081] ESI-MS (m / z): 672 [M+H] + ;

[0082] 1H NMR(500MHz,DMSO-d6)δ(ppm)13.02(s,1H),9.59(s,1H),8.96(d,J=6.5Hz,1H),8.88(s,1H),8.84(d,J=6.5Hz,1H),8.14(d,J=8.5Hz,1H),7.92(d,J= 9.0Hz,1H),7.76(d,J=8.0Hz,2H),7.36(t,J=8.0Hz,4H),7.15–7.08(m,8H) ,4.88(t,J=7.5Hz,2H),3.51–3.47(m,2H),3.13(s,9H),2.59–2.56(m,2H).

[0083] Example 5 Preparation of 6-(4-(diphenylamino)phenyl)-2-ethyl-9H-pyridine[3,4-b]indole-2-iodide (I5)

[0084] Following the synthesis method described in Example 1 (I1), iodoethane was used instead of iodomethane in the method, resulting in 116 mg of a yellow solid (I5) with a yield of 52.6%.

[0085] ESI-MS (m / z): 567 [M+H] + .

[0086] 1 H NMR (500MHz, DMSO-d6) δ (ppm) 13.04 (s, 1H), 9.60 (s, 1H), 8.98 (d, J = 6.5Hz, 1H), 8.89 (s, 1H), 8.80 (d, J = 6.5Hz, 1H), 8.15 (d, J = 9.0Hz, 1H),7.95(d,J=9.0Hz,1H),7.77(d,J=8.0Hz,2H),7.37(t,J=8.0Hz,4H),7.15–7.07(m,8H),4.78-4.67(m,2H),1.60(t,J=7.0Hz,3H).

[0087] Example 6 Preparation of 2-Butyl-6-(4-(diphenylamino)phenyl)-9H-pyridine[3,4-b]indole-2-iodide (I6)

[0088] Following the synthesis method of (I1) in Example 1, iodomethane was replaced with 1-iodobutane, and 132 mg of yellow solid (I6) was finally obtained, with a yield of 56.7%.

[0089] ESI-MS (m / z): 595 [M+H] + .

[0090] 1 H NMR(500MHz,DMSO-d6)δ(ppm)12.97(s,1H),9.66(s,1H),8.90(d,J=6.5Hz,1H),8.87(s,1H),8.85(d,J=7.0Hz,1H),8.16(d,J=8.5Hz,1H),7.98( d,J=8.5Hz,1H),7.75(d,J=8.0Hz,2H),7.38(t,J=8.0Hz,4H),7.18–7.06 (m,8H),4.82(t,J=6.5Hz,2H),1.60-1.56(m,4H),1.10(t,J=7.0Hz,3H).

[0091] Example 7 Preparation of 6-(4-(diphenylamino)phenyl)-2-pentyl-9H-pyridine[3,4-b]indole-2-iodide (I7)

[0092] Following the synthesis method described in Example 1 (I1), 1-iodopentane was used instead of iodomethane in the method, resulting in 132 mg of a yellow solid (I7) with a yield of 55.5%.

[0093] ESI-MS (m / z): 609 [M+H] + .

[0094] 1 H NMR(500MHz,DMSO-d6)δ(ppm)12.98(s,1H),9.65(s,1H),8.96(d,J=6.5Hz,1H),8.90(s,1H),8.86(d,J=6.5Hz,1H),8.17(d,J=9.0Hz,1H),7.99( d,J=9.0Hz,1H),7.76(d,J=8.0Hz,2H),7.40(t,J=8.0Hz,4H),7.18–7.08 (m, 8H), 4.80 (t, J = 7.0Hz, 2H), 1.65-1.56 (m, 6H), 1.05 (t, J = 6.5Hz, 3H).

[0095] Example 8 Preparation of 2-(bromomethyl)-6-(4-(diphenylamino)phenyl)-9H-pyridine[3,4-b]indole-2-iodide (I8)

[0096] Following the synthesis method described in Example 1 (I1), bromoiodomethane was used instead of iodomethane in the method, resulting in 146 mg of a yellow solid (I8) with a yield of 59.2%.

[0097] ESI-MS (m / z): 632 [M+H] + .

[0098] 1 H NMR (500MHz, DMSO-d6) δ (ppm) 13.03 (s, 1H), 9.66 (s, 1H), 8.93 (d, J = 6.5Hz, 1H), 8.88 (s, 1H), 8.83 (d, J = 6.5Hz, 1H), 8.1 5(d,J=9.0Hz,1H),7.98(d,J=9.0Hz,1H),7.76(d,J=8.0Hz,2H),7.40(t,J=8.0Hz,4H),7.14–7.06(m,8H),4.85(s,2H).

[0099] Example 9: Ultraviolet spectral detection of the compounds of the present invention

[0100] The compounds of this invention were dissolved in deionized water containing 1% DMSO, and the concentration of the detection solution was 10 μM. Their ultraviolet spectra were measured using a UV spectrophotometer, collecting data from 200 to 700 nm.

[0101] Figure 1 This is the UV spectrum of the compound (10 μM) of this invention in a deionized aqueous solution of 1% DMSO. Figure 1 A is the ultraviolet spectrum of compound I1 in a 1% DMSO deionized aqueous solution; Figure 1 B is the ultraviolet spectrum of compound I2 in a 1% DMSO deionized aqueous solution; Figure 1 C represents the ultraviolet spectrum of compound I3 in a 1% DMSO deionized aqueous solution; Figure 1 D is the UV spectrum of compound I4 in a 1% DMSO deionized aqueous solution. All compounds were at a concentration of 10 μM.

[0102] The results showed that the longest ultraviolet absorption peak of the compound of the present invention in a 1% DMSO deionized aqueous solution was between 360-440 nm, which proved that the compound of the present invention can absorb photon energy in the ultraviolet-visible region.

[0103] Example 10: Fluorescence spectral detection of the compounds of the present invention

[0104] The compound of this invention (10 μM) was dissolved in a deionized aqueous solution containing 1% DMSO. The concentration of the detection solution was 10 μM. With normethylhalman as a control, the fluorescence properties were studied using 365 nm as the excitation wavelength. Fluorescence emission spectrometry data from 350–700 nm were collected using a fluorescence spectrometer. (See attached image.) Figure 2 .

[0105] in, Figure 2 A is the fluorescence spectrum of compound norhalman in a deionized aqueous solution of 1% DMSO; Figure 2B is the fluorescence spectrum of compound I1 in a 1% DMSO deionized aqueous solution; Figure 2 C represents the fluorescence spectrum of compound I2 in a 1% DMSO deionized aqueous solution; Figure 2 D is the fluorescence spectrum of compound I3 in a 1% DMSO deionized aqueous solution; Figure 2 E represents the fluorescence spectrum of compound I4 in a 1% DMSO deionized aqueous solution; the excitation wavelength is 365 nm.

[0106] The results showed that none of the compounds in this invention produced obvious fluorescence signals after excitation at 365 nm, and the fluorescence was quenched in solution. In contrast, the control compound, norhalman, had a stronger fluorescence intensity. This demonstrates that the triphenylamine group present in the compounds of this invention can effectively reduce the fluorescence of carbamoline, which is beneficial to improving the therapeutic effect of photodynamic therapy.

[0107] Example 11 ROS generation test of the compound of the present invention

[0108] The ability of the compounds of this invention to generate ROS was detected by fluorescence spectroscopy. The detection mechanism of the ROS indicator DCFH is as follows:

[0109]

[0110] The ROS indicator DCFH (Biode Pharmaceuticals) is oxidized to DCF when ROS is generated in the system, resulting in increased fluorescence intensity at 523 nm. The compound of this invention was dissolved in a 1% DMSO deionized water solution, with a detection concentration of 10 μM. Then, the indicator DCFH was added, and the fluorescence intensity was measured at 420 nm (60 mW cm⁻¹). -2 Irradiation was used to detect the fluorescence spectrum of the system from 500 nm to 600 nm; the control was Bengal Rose Red (RB, Bidex Pharmaceuticals).

[0111] The compound of this invention (10 μM) was analyzed at 420 nm (60 mW cm⁻¹). -2 The ROS generation results after 60 seconds of illumination are shown in [the original text]. Figure 3 .

[0112] in, Figure 3 A represents the change in the fluorescence spectrum of the indicator DCFH (5μm) before and after irradiation at 420nm; Figure 3 B represents the change in the fluorescence spectrum of DCFH (5μm) after RB is irradiated at 420nm; Figure 3 C represents the change in the fluorescence spectrum of compound I1 at DCFH (5 μm) after irradiation at 420 nm; Figure 3 D represents the change in the fluorescence spectrum of compound I2 at DCFH (5 μm) after irradiation at 420 nm; Figure 3 E represents the change in the fluorescence spectrum of compound I3 at DCFH (5 μm) after irradiation at 420 nm; Figure 3 F represents the fluorescence spectrum change of compound I4 after irradiation at 420 nm using DCFH (5 μm).

[0113] The results show that, Figure 3 As shown in Figure AF, the indicator DCFH showed a slight increase in autofluorescence intensity after irradiation at 420 nm for 60 seconds, while the control RB showed a significant increase in fluorescence intensity after irradiation, demonstrating the effective generation of ROS. The compounds I1-I4 of this invention showed significantly greater increases in fluorescence intensity after irradiation at 420 nm than the control RB, demonstrating that the ROS production of the compounds of this invention is higher than that of the control RB; furthermore, the ROS production of I4 is higher than that of I1-I3. Therefore, the carboline compounds of this invention, containing a triphenylamine group, can effectively generate ROS after irradiation and can be applied to antibacterial photodynamic therapy.

[0114] Example 12 In vitro photodynamic antibacterial experiment of the compound of the present invention

[0115] The compounds of this invention were first tested for dark toxicity using the MTT assay (tetramethylazolium blue colorimetric method). A flask of normal LO2 cells (Shanghai Institute of Cell Biology, China) in a healthy exponential growth phase was digested and prepared into 1×10⁻⁶ cells. 4 Cell suspension of cells / mL was seeded into 96-well plates and cultured at 37°C in the dark for 24 hours. Then, the compounds of this invention (0, 3, 5, 10, 15 and 30 μM) were added and cultured for another 24 hours. After adding MTT reaction for 4 hours, the supernatant was discarded, and 100 μL of DMSO was added. The absorbance at 570 nm was measured and the cell viability was calculated.

[0116] Cell survival rate formula:

[0117] Survival rate (%) = (OD value of experimental group - OD value of blank control group) / (OD value of control group - OD value of blank control group) × 100%

[0118] The OD value refers to the optical density value. The OD value of the experimental group is the absorbance value of the treated cells, the OD value of the control group is the absorbance value of the untreated cells, and the OD value of the blank control group is the absorbance value of the culture medium.

[0119] In vitro antibacterial experiments further evaluated the photodynamic therapeutic effect of the compounds of this invention. MRSA producing β-lactamase was isolated from human clinical specimens collected and identified by the Medical Laboratory Center of Zhongda Hospital Affiliated to Southeast University, and the enzyme type was identified by the Microbiology Laboratory of China Pharmaceutical University. MRSA was inoculated with NB solid medium and cultured in a 37°C incubator. Then, it was expanded using NB liquid medium (1×), and the culture was placed in a 37°C constant temperature water bath shaker for subsequent experiments.

[0120] In a 96-well plate, 10 μL of bacterial culture and 200 μL of liquid culture medium were added to each well. The plates were then incubated at 37°C for 30 minutes. The compound of this invention (30 μM) was then added, and the plates were incubated for another 30 minutes. The dark group was incubated in the dark, while the light group was incubated at 60 mW / cm². 2 Irradiate with 420nm light for 6 minutes, continue culturing for 4 hours, and then measure OD using a microplate reader. 600 The value was calculated. All results were replicated in six independent groups.

[0121] The results of the dark toxicity and photodynamic antibacterial treatment of compounds I1-I4 of this invention are shown in the figure. Figure 4 .in, Figure 4 A represents the viability of LO2 cells incubated with different concentrations of compounds I1-I4 under light-protected conditions, as determined by MTT assay. Figure 4 B is OD 600 Measurement and detection at 420nm (60mW / cm) 2 The viability of MRSA bacteria incubated with 30 μM compounds I1-I4 was detected under light irradiation for 6 min.

[0122] Experimental results showed that even at a concentration of 30 μM, the survival rate of LO2 cells remained above 80%, indicating low dark toxicity. Figure 4 A). Under light-protected conditions, incubation with 30 μM I1-I4 resulted in less than 20% bacterial mortality for MRSA bacteria, showing weak efficacy against drug-resistant bacteria alone. However, irradiation with 420 nm light effectively killed all MRSA bacteria. Figure 4 B), among which I4 has the most obvious antibacterial effect, proving that the compound of the present invention has no obvious cytotoxicity and can effectively target bacteria. It can be applied to photodynamic therapy of drug-resistant bacteria, providing a new method for the clinical treatment of drug-resistant bacterial infections, and has broad application prospects.

[0123] As described above, although the invention has been shown and described with reference to specific preferred embodiments, it should not be construed as limiting the invention itself. Various changes in form and detail may be made without departing from the spirit and scope of the invention as defined in the appended claims.

Claims

1. A class of bacterial-targeting carboline compounds with the structure shown in Formula I that exhibit photodynamic therapeutic activity: ； in, R1 is -CH3, -CH2CH3, -CH2CH2CH3, -CH2CH2CH2CH3, -CH2CH2CH2CH2CH3, -CH2Br, -CH2CH2Br, or -CH2CH2CH2N + (CH3)3Br - ; R2 is I - or Br - .

2. The carbamoline compound according to claim 1, characterized in that... Selected from: ； 。 3. A method for preparing a carbaline compound of formula I as claimed in claim 1, characterized in that, The approach of this method is as follows: 。 4. The method according to claim 3, characterized in that: The steps of this method are as follows: (1) Using norhalman as a raw material, it undergoes a substitution reaction with N-bromosuccinimide to obtain intermediate compound 2; (2) Intermediate compound 2 reacts with triphenylamine 4-borate and potassium carbonate to obtain compound 3; (3) Finally, compound 3 reacts with R1-R2 to obtain the novel carboline compound shown in general formula I.

5. The method according to claim 4, characterized in that: In step (1), the reaction temperature is room temperature, the reaction solvent is acetic acid, and the reaction time is 10~14h; The catalyst used in step (2) is tetraphenylphosphine palladium, the reaction solvent is toluene, and the reaction temperature is reflux temperature; In step (3), the reaction temperature is 90~110℃ and the reaction time is 10~14h; the reaction solvent is acetonitrile.

6. A pharmaceutical composition, characterized in that: This includes the carboline compounds of claim 1 and pharmaceutically acceptable carriers or excipients.

7. The use of the carboline compound of claim 1 in the preparation of anti-MRSA bacterial drugs.

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