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

Abstract

The invention discloses a preparation method and application of a novel carboline quaternary ammonium salt compound with a photodynamic antibacterial effect. According to the novel carboline quaternary ammonium salt compound disclosed by the invention, a triphenylamine group is introduced to a carboline parent nucleus, so that the fluorescence intensity of the compound is effectively reduced, after illumination, energy passes through systems to induce the generation of ROS, and the photodynamic effect of the carboline compound is improved. Meanwhile, the novel carboline quaternary ammonium salt compound disclosed by the invention can target the surface with high negative charges of bacteria due to the existence of quaternary ammonium salt groups, and bacteria and drug-resistant bacteria can be effectively killed after illumination is applied. Therefore, the novel carboline compound provided by the invention can be applied to the field of medicines and is used for preparing antibacterial medicines.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to a method for preparing a novel carboline quaternary ammonium salt compound with photodynamic antibacterial effect, in particular to its medical use as a photosensitizer that undergoes a photosensitization reaction and produces active oxygen after illumination, exerts a photodynamic antibacterial therapeutic effect without inducing the production of drug-resistant bacteria, and its application in the preparation of antibacterial drugs. Background Art

[0002] Bacterial infectious diseases are an important public health problem worldwide. However, due to the improvement of the economic level worldwide, the accelerated industrialization process of antibiotics has also led to a further decline in production costs, which has accelerated the abuse of such antibiotics by medical institutions around the world, making some pathogenic bacteria resistant to antibiotics and reducing the efficacy of antibiotics. Drug-resistant bacteria can make antibiotics ineffective in a variety of ways. In addition to relying on the production of inactivating enzymes that make antibiotics ineffective, they can also change certain protein structures on the outer membrane to make it difficult for them to bind to antibiotics. At the same time, bacteria can change the permeability of their own membranes, enhance the role of efflux pumps on the membrane, and form biofilms that resist the entry of antibiotics, which can enhance bacterial resistance. Methicillin-resistant Staphylococcus aureus (MRSA) is the most famous superbug, which is extremely common in hospital and community infections and can cause infections of the skin, lungs, blood and joints. In recent years, nosocomial infections caused by MRSA are not only a thorny problem that clinicians must often face, but also pose a great threat to the health of all mankind. In order to deal with the resistance of drug-resistant bacteria to traditional antibiotics, exploring new antibacterial methods has become a new research direction.

[0003] Photodynamic therapy (PDT) is a new treatment method for killing target cells by using appropriate excitation light to activate photosensitizers (PS) to generate reactive oxygen species (ROS) that further react with adjacent biomacromolecules for oxidation. Antimicrobial photodynamic therapy (PACT) is a derivative therapy of PDT that can kill sensitive and resistant bacteria without causing drug resistance. Its mechanism of action is mainly that the photosensitizer transitions from the ground state (singlet state) to the excited singlet state under the irradiation of visible light of a certain wavelength. The excited singlet state with a short life span generates an excited triplet state through rapid interpolar transition, and then reacts with molecular oxygen in the environment for electron transfer or energy transfer to generate cytotoxic ROS, which effectively kills pathogens that are sensitive to drugs and have tolerance. ROS are highly reactive and have the characteristics of denaturing known antioxidant enzymes (superoxide dismutase and catalase) and are difficult to defend against, which means that repeated photosensitization of bacteria will not induce the production of resistant strains. Therefore, antimicrobial photodynamic therapy is a promising way to treat MRSA.

[0004] Alkaloid compounds have always been an important source of antibacterial drugs. β-Carboline is a class of naturally occurring alkaloids that are widely distributed in plants, marine organisms, insects and mammals, as well as in human tissues and body fluids. In recent years, in the study of β-carboline compounds, people have gradually focused on marine organisms. Marine organisms have lived in special environments for a long time, and high pressure, hypoxia, etc. have caused them to produce and accumulate a large number of substances with special chemical structures and unique physiological activities during their growth and metabolism. Studies have shown that its biological activities are extensive, mainly including anti-tumor, anti-bacterial, anti-fungal, anti-viral, anti-parasitic, anti-inflammatory, diabetes treatment, and neuropharmacological activities. Therefore, the discovery of β-carboline, a natural alkaloid with antibacterial activity, is an important basis for the development of new and highly effective antibacterial drugs.

[0005] Based on the unique chemical structure of β-carboline (the mother core is a tricyclic pyridine-indole ring), it has a highly conjugated planar polyaryl structure, strong fluorescence, and a large number of studies in recent years have fully demonstrated that β-carboline alkaloids may become antibacterial lead compounds. Among them, harman A69, canthin-6-one A72 and eudistomin U A98 have particularly outstanding antibacterial activities. The MIC range of canthin-6-one A72 against Staphylococcus aureus is 8-64μg / mL, and the IC range of eudistomin U A98 against Staphylococcus aureus is 1. 50 The value is 6.4μg / mL, and its antibacterial activity is close to that of commercially available antibacterial drugs such as fosfomycin sodium, ampicillin sodium and ciprofloxacin. So far, there are 9 commercialized drugs with β-carboline as the core skeleton, including one of the world's TOP100 drugs, which further illustrates the importance of research on this type of compound and the feasibility of drug development. However, after long-term use, it is easy to develop drug resistance, especially multidrug-resistant bacterial strains, which greatly reduces the possibility of later promotion and use. Therefore, we use antibacterial photodynamic therapy to overcome the drug resistance defect, introduce triphenylamine groups and quaternary ammonium salt bacterial targeting groups on the β-carboline fragment, and develop a new type of carboline quaternary ammonium salt photosensitizer, which can be used for photodynamic anti-resistant bacteria. Summary of the invention

[0006] Purpose of the invention: The purpose of the present invention is to provide a preparation method and application of a novel carboline quaternary ammonium salt compound with photodynamic antibacterial effect.

[0007] The present invention develops a quaternary ammonium salt photosensitizer based on carboline, which can be used for photodynamic antibacterial action. The introduction of triphenylamine groups quenches the fluorescence of carboline and improves the generation efficiency of ROS. The introduction of quaternary ammonium salt groups enables the compound to target the highly negatively charged surface of bacteria, thereby achieving the purpose of bacterial targeting. After irradiation at 420nm, the novel carboline quaternary ammonium salt compound generates destructive ROS, which efficiently kills bacteria and drug-resistant bacteria, and will expand the clinical transformation vision of carboline photosensitizers in antibacterial treatment.

[0008] Technical solution: The purpose of the present invention is achieved through the following technical solution:

[0009] The present invention provides a novel carboline quaternary ammonium salt compound having a structure shown in general formula I and having photodynamic antibacterial effect;

[0010]

[0011] in,

[0012] R 1 is an alkyl group, a brominated alkyl group or a quaternized alkyl group;

[0013] R 2 It is a halogen.

[0014] A preferred embodiment of the present invention is that R 1 Selected from -CH 3 、-CH 2 CH 3 、-CH 2 CH 2 CH 3 、-CH 2 CH 2 CH 2 CH 3 、-CH 2 CH 2 CH 2 CH 2 CH 3 、-CH 2 Br, -CH 2 CH 2 Br or -CH 2 CH 2 CH 2 N + (CH 3 ) 3 Br - ; R 2 Selected from I - or Br - .

[0015] A preferred embodiment of the present invention is that the novel carboline compound is preferably the following compound, and its structural formula is shown in Table 1:

[0016] Table 1 Codes of some compounds of general formula I and their corresponding structures

[0017]

[0018]

[0019] I 1 :6-(4-(diphenylamino)phenyl)-2-methyl-9H-pyrido[3,4-b]indole-2-iodide

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

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

[0022] I 4 :6-(4-(diphenylamino)phenyl)-2-(3-(trimethylammonium)propyl)-9H-pyrido[3,4-b]indole-2-bromide I 5 :6-(4-(diphenylamino)phenyl)-2-ethyl-9H-pyrido[3,4-b]indole-2-iodide

[0023] I 6 :2-butyl-6-(4-(diphenylamino)phenyl)-9H-pyrido[3,4-b]indole-2-iodide

[0024] I 7 :6-(4-(diphenylamino)phenyl)-2-pentyl-9H-pyrido[3,4-b]indole-2-iodide

[0025] I 8 :2-(Bromomethyl)-6-(4-(diphenylamino)phenyl)-9H-pyrido[3,4-b]indole-2-iodide

[0026] The present invention also provides a method for preparing a novel carboline compound 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 norharman as a raw material, it undergoes a substitution reaction with N-bromosuccinimide to obtain an intermediate compound 2;

[0030] (2) the intermediate compound 2 reacts with 4-boric acid triphenylamine and potassium carbonate to obtain compound 3;

[0031] (3) Finally, compound 3 and R 1 -R 2 The reaction is carried out to obtain a novel carboline compound represented by the general formula I.

[0032] Among them, R 1 , R 2 As defined in Formula I.

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

[0034] The catalyst used in the reaction in step (2) is tetrakistriphenylphosphine palladium, the reaction solvent is toluene, and the reaction temperature is the reflux temperature; the reaction temperature in step (3) is 90-110° C., the reaction time is 10-14 h; and the reaction solvent is acetonitrile.

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

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

[0037] In some more specific technical solutions, in step (3), compound 3 and R 1 -R 2 The molar ratio is 1:1~1.5.

[0038] The compounds of the general formula I of the present invention can be prepared by the above or similar preparation methods, and the corresponding starting materials can be selected according to the different substituents and the different positions of the substituents. It should be appreciated by those skilled in the art that the above routes are helpful for understanding the present invention, but do not limit the content of the present invention. Unless otherwise specified, the variables are defined as mentioned in the general formula I.

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

[0040] The pharmaceutical composition of the present invention can be administered in various known ways, such as orally, parenterally, or via an implanted reservoir. The pharmaceutical composition of the present invention can be administered alone or in combination with other drugs. Oral compositions can be in 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, adhesives, colorants, fillers, emulsifiers, etc.

[0041] Sterile injectable compositions can be formulated using suitable dispersing agents or wetting agents and suspending agents according to techniques known in the art. Pharmaceutically acceptable carriers and solvents that can be used include water, mannitol, sodium chloride solution, and the like.

[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 the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. The dosage level selected 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 and previous medical history of the patient being treated, and similar factors well known in the medical field.

[0043] The present invention also provides the use of the novel carboline compound in the preparation of antibacterial drugs.

[0044] The novel carboline compound has an antibacterial photodynamic therapy effect and can generate ROS after being irradiated with excitation light, thereby effectively killing sensitive bacteria and drug-resistant bacteria in a non-specific manner. ROS is highly reactive and has the property of denaturing known antioxidant enzymes (superoxide dismutase and catalase) and is difficult to defend against, so it will not induce the generation of drug-resistant strains.

[0045] The novel carboline compound introduces a quaternary ammonium group and is adsorbed on the surface of bacteria with a high negative charge, thereby achieving a targeted photodynamic antibacterial effect.

[0046] We summarized the antimicrobial activities of natural and synthetic β-carboline alkaloids and preliminarily concluded the following structure-activity relationships: (1) The fully aromatic β-carboline nucleus is stronger than the hydrogenated nucleus, but hydrogenation and heterocyclization at the 2 and 3 positions will improve the activity; (2) Substitution at the 1 position is beneficial to improving the activity, and aromatic ring substitution is better than alkyl chain substitution, but the steric hindrance should not be too large; (3) Quaternization at the 2 position can greatly improve the antimicrobial activity; (4) Substitution at the 3 or 9 position is beneficial to improving the activity, and the alkyl chain substitution at the 9 position should not be too long; (5) Methoxy, hydroxyl and halogen substitutions on the A ring of the carboline nucleus can improve the activity, while ester substitutions will reduce the activity; (6) Chain connection at the 1 position and dimerization modification of heterocyclic connection at the 1 and 2 positions can significantly improve the activity; direct dimerization at the 9 position reduces the activity; (7) The increase of the D ring can significantly improve the antimicrobial activity.

[0047] Based on the study of structure-activity relationship and antibacterial photodynamic therapy, we introduced a quaternary ammonium group at the 2-position of the β-carboline nucleus to bring in positive charge, so that the compound molecule can be adsorbed on the surface of highly negatively charged bacteria, thus achieving a targeted antibacterial effect. In addition, a triphenylamine group was introduced at the 6-position of the β-carboline nucleus to reduce the fluorescence of the carboline nucleus and increase the photodynamic properties of the compound. The new carboline quaternary ammonium salt compound developed has a bacterial targeted photodynamic therapy effect, which is of great significance for the practical problem of coping with drug-resistant bacterial infections and clinical transformation in the future.

[0048] Beneficial effects:

[0049] The novel carboline compound structure provided by the present invention has strong fluorescence and photodynamic properties. It can generate ROS after irradiation with excitation light, effectively killing sensitive bacteria and drug-resistant bacteria. In addition, the introduction of quaternary ammonium groups in the compound structure can make the compound molecules with positive charges adsorbed on the surface of bacteria with high negative charges, achieving a targeted antibacterial effect. Therefore, the novel carboline compound of the present invention can be used to prepare antibacterial drugs, and has broad application prospects in antibacterial, especially drug-resistant bacteria (such as: MRSA) infection treatment through precise and efficient photodynamic therapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 The UV spectrum of the compound of the present invention (10 μM) in a 1% DMSO deionized water solution.

[0051] in, Figure 1 A is compound I 1 UV spectrum in 1% DMSO in deionized water; Figure 1 B is compound I 2 UV spectrum in 1% DMSO in deionized water; Figure 1 C is compound I 3 UV spectrum in 1% DMSO in deionized water; Figure 1 D is compound I 4 UV spectra in 1% DMSO in deionized water. The compound concentration was 10 μM.

[0052] Figure 2 The fluorescence spectrum of the compound of the present invention (10 μM) in a 1% DMSO deionized water solution was detected.

[0053] in, Figure 2 A is the fluorescence spectrum of the compound norharman in a 1% DMSO deionized water solution; Figure 2 B is compound I 1 Fluorescence spectrum in 1% DMSO deionized water solution; Figure 2 C is compound I 2 Fluorescence spectrum of 1% DMSO in deionized water solution; Figure 2 D is compound I 3 Fluorescence spectrum in 1% DMSO deionized water solution; Figure 2 E is compound I 4 Fluorescence spectrum of 1% DMSO in deionized water; the excitation wavelength is 365 nm. Figure 3 The compounds of the present invention (10 μM) were irradiated in a 1% DMSO solution in deionized water at 420 nm (60 mW cm -2 ) ROS production results after 60s of illumination.

[0054] in, Figure 3 A is the fluorescence spectrum change of indicator DCFH (5 μm) before and after irradiation with 420 nm; Figure 3 B is the fluorescence spectrum change of DCFH (5 μm) after RB was irradiated at 420 nm; Figure 3 C is compound I 1 Changes in the fluorescence spectrum of DCFH (5 μm) after irradiation at 420 nm; Figure 3 D is compound I 2 Changes in the fluorescence spectrum of DCFH (5 μm) after irradiation at 420 nm; Figure 3 E is compound I 3 Changes in the fluorescence spectrum of DCFH (5 μm) after irradiation at 420 nm; Figure 3 F is compound I 4 Changes in the fluorescence spectrum of DCFH (5 μm) after irradiation at 420 nm.

[0055] Figure 4 Compound I of the present invention 1 -I 4 Dark toxicity and the consequences of photodynamic antimicrobial therapy.

[0056] in, Figure 4 A is the MTT assay to detect the presence of different concentrations of compound I in the dark. 1 -I 4 Viability of incubated LO2 cells. Figure 4 B is OD 600 Determination detection 420nm (60mW / cm 2 , 6min) under light irradiation, with different concentrations of compound I 1 -I 4 Viability of incubated MRSA bacteria. DETAILED DESCRIPTION

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

[0058] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

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

[0060] Example 1 6-(4-(Diphenylamino)phenyl)-2-methyl-9H-pyrido[3,4-b]indole-2-iodide (I 1 )

[0061]

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

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

[0064] 1 H NMR (400 MHz, DMSO-d 6)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), 4-boronic acid triphenylamine (283 mg, 0.98 mmoL) and potassium carbonate (179 mg, 1.3 mmoL) were dissolved in toluene. Under nitrogen protection, tetrakistriphenylphosphine palladium (81 mg, 0.07 mmoL) was added as a catalyst, and the mixture was stirred under reflux overnight until the reaction was complete. The mixture was diluted with water and extracted with ethyl acetate. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a crude product. The crude product was purified by column chromatography to obtain 162 mg of a yellow solid (3) with a yield of 60.5%.

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

[0067] 1 H NMR (400 MHz, DMSO-d 6 )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=5.2H z,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, and iodomethane (61 mg, 0.43 mmol) was slowly added dropwise under ice bath conditions. After addition, the mixture was refluxed and stirred overnight until the reaction was complete. Water was added to quench the mixture, and the organic phase was extracted with ethyl acetate. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a crude product. The crude product was purified by column chromatography to obtain a yellow solid (I 1 )109 mg, yield 50.8%.

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

[0070] 1 H NMR (500 MHz, DMSO-d 6)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),8.13(d,J=8.5 Hz,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 6-(4-(Diphenylamino)phenyl)-2-propyl-9H-pyrido[3,4-b]indole-2-iodide (I 2 )

[0072] Referring to Example 1 (I 1 ), the methyl iodide in the method is replaced by 1-iodopropane, and finally a yellow solid (I 2 )123 mg, yield 54.3%.

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

[0074] 1 H NMR (500 MHz, DMSO-d 6 )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 2-(2-bromoethyl)-6-(4-(diphenylamino)phenyl)-9H-pyrido[3,4-b]indole-2-bromide (I 3 )

[0076] Referring to Example 1 (I 1 ), the iodomethane in the method is replaced by 1,2-dibromoethane, and finally a yellow solid (I 3 )120 mg, yield 51.5%.

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

[0078] 1 H NMR (500 MHz, DMSO-d 6 )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,1H),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 6-(4-(diphenylamino)phenyl)-2-(3-(trimethylammonium)propyl)-9H-pyrido[3,4-b]indole-2-bromide (I 4 )preparation

[0080] Referring to Example 1 (I 1 ), the methyl iodide in the method is replaced by (3-bromopropyl) trimethylammonium bromide, and finally a yellow solid (I 4 )130 mg, yield 49.8%.

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

[0082] 1 H NMR (500 MHz, DMSO-d 6 )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 6-(4-(Diphenylamino)phenyl)-2-ethyl-9H-pyrido[3,4-b]indole-2-iodide (I 5 )

[0084] Referring to Example 1 (I1 ), the methyl iodide in the method is replaced by ethyl iodide, and finally a yellow solid (I 5 )116 mg, yield 52.6%.

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

[0086] 1 H NMR (500 MHz, DMSO-d 6 )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 2-Butyl-6-(4-(diphenylamino)phenyl)-9H-pyrido[3,4-b]indole-2-iodide (I 6 )

[0088] Referring to Example 1 (I 1 ), the methyl iodide in the method is replaced by 1-iodobutane, and finally a yellow solid (I 6 )132 mg, yield 56.7%.

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

[0090] 1 H NMR (500 MHz, DMSO-d 6 )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 6-(4-(Diphenylamino)phenyl)-2-pentyl-9H-pyrido[3,4-b]indole-2-iodide (I 7 )

[0092] Referring to Example 1 (I 1 ), the iodomethane in the method is replaced by 1-iodopentane, and finally a yellow solid (I 7 )132 mg, yield 55.5%.

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

[0094] 1 H NMR (500 MHz, DMSO-d 6 )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 2-(Bromomethyl)-6-(4-(diphenylamino)phenyl)-9H-pyrido[3,4-b]indole-2-iodide (I 8 )

[0096] Referring to Example 1 (I 1 ), the iodomethane in the method is replaced by bromoiodomethane, and finally a yellow solid (I 8 )146 mg, yield 59.2%.

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

[0098] 1 H NMR (500 MHz, DMSO-d 6)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),8.15(d,J=9.0 Hz,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 Spectrum Detection of the Compounds of the Invention

[0100] The compounds of the present invention were respectively dissolved in a deionized water solution containing 1% DMSO, and the concentration of the detection solution was 10 μM. The ultraviolet spectrum data was tested using an ultraviolet spectrophotometer, and the data at 200-700 nm were collected.

[0101] Figure 1 is the UV spectrum of the compound of the present invention (10 μM) in a 1% DMSO deionized water solution. Figure 1 A is compound I 1 UV spectrum in 1% DMSO in deionized water; Figure 1 B is compound I 2 UV spectrum in 1% DMSO in deionized water; Figure 1 C is compound I 3 UV spectrum in 1% DMSO in deionized water; Figure 1 D is compound I 4 UV spectra in 1% DMSO in deionized water. The compound concentration was 10 μM.

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

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

[0104] The compound of the present invention (10 μM) was dissolved in a deionized water solution containing 1% DMSO. The concentration of the test solution was 10 μM. Norharman was used as a control. The fluorescence properties were studied using 365 nm as an excitation wavelength. The fluorescence emission spectrum data at 350-700 nm were collected by a fluorescence spectrometer. Figure 2 .

[0105] in, Figure 2 A is the fluorescence spectrum of the compound norharman in a 1% DMSO deionized water solution; Figure 2 B is compound I1 Fluorescence spectrum in 1% DMSO deionized water solution; Figure 2 C is compound I 2 Fluorescence spectrum in 1% DMSO deionized water solution; Figure 2 D is compound I 3 Fluorescence spectrum in 1% DMSO deionized water solution; Figure 2 E is compound I 4 Fluorescence spectrum of 1% DMSO in deionized water; the excitation wavelength is 365 nm.

[0106] The results showed that after being excited at 365 nm, the compound series of the present invention did not produce obvious fluorescence signals, and the fluorescence was quenched in the solution, while the control norharman fluorescence intensity was strong, proving that the triphenylamine group present in the compounds of the present invention can effectively reduce the fluorescence of carboline, which is beneficial to improving the therapeutic effect of photodynamic therapy.

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

[0108] Fluorescence spectroscopy was used to detect the ability of the compounds of the present invention to generate ROS. The detection mechanism of the ROS indicator DCFH is as follows:

[0109]

[0110] ROS indicator DCFH (BiDe Pharmaceuticals) is oxidized to DCF when ROS is generated in the system, and the fluorescence intensity at 523nm increases. The compound of the present invention is dissolved in a deionized water solution containing 1% DMSO, and the concentration of the detection solution is 10μM. Then the indicator DCFH is added and the fluorescence intensity at 420nm (60mW cm -2 ) illumination, and the fluorescence spectrum of the detection system was from 500 nm to 600 nm; the control was Rose Bengal (RB, Bid Pharmaceuticals Co., Ltd.).

[0111] The compounds of the present invention (10 μM) were detected by 420 nm (60 mW cm -2 ) The ROS production results after 60s of illumination are shown in Figure 3 .

[0112] in, Figure 3 A is the fluorescence spectrum change of indicator DCFH (5 μm) before and after irradiation with 420 nm; Figure 3 B is the fluorescence spectrum change of DCFH (5 μm) after RB was irradiated at 420 nm; Figure 3 C is compound I 1 Changes in the fluorescence spectrum of DCFH (5 μm) after irradiation at 420 nm; Figure 3 D is compound I 2Changes in the fluorescence spectrum of DCFH (5 μm) after irradiation at 420 nm; Figure 3 E is compound I 3 Changes in the fluorescence spectrum of DCFH (5 μm) after irradiation at 420 nm; Figure 3 F is compound I 4 Changes in the fluorescence spectrum of DCFH (5 μm) after irradiation at 420 nm.

[0113] The results show that if Figure 3 As shown in AF, after the indicator DCFH was irradiated at 420 nm for 60 seconds, its own fluorescence intensity increased slightly, while the fluorescence intensity of the control RB increased greatly after illumination, proving the effective generation of ROS. 1 -I 4 After irradiation at 420 nm, the fluorescence intensity increase was significantly higher than that of the control RB, proving that the ROS production of the compounds of the present invention was higher than that of the control RB; and 4 The ROS production was higher than that of I 1 -I 3 Therefore, the carboline compounds of the present invention having a triphenylamine group can effectively generate ROS after irradiation with light and can be applied to antibacterial photodynamic therapy.

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

[0115] The dark toxicity of the compounds of the present invention was first tested by the in vitro toxicity test of methyl tetrazolium blue colorimetry (MTT). A bottle of normal LO2 cells (Shanghai Institute of Cell Biology, China) in a good exponential growth phase was taken, digested and prepared into 1×10 4 The cell suspension of cells / mL was spread on a 96-well plate and kept at 37°C in the dark for 24 hours. The compound of the present invention (0, 3, 5, 10, 15 and 30 μM) was then added and cultured for another 24 hours. MTT was added and reacted for 4 hours, the supernatant was discarded, 100 μL of DMSO was added, the absorbance at 570 nm was detected, and the cell survival rate 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] Among them, 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 therapy effect of the compounds of the present 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 culture medium and cultured in a 37°C incubator. Then, NB liquid culture medium (1×) was used for expansion culture, 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 solution and 200 μL of liquid culture medium were added to each well, and the plates were placed in a constant temperature incubator at 37°C for 30 minutes. The compounds of the present invention (30 μM) were then added and cultured for another 30 minutes. The dark group was cultured in the dark, while the light group was cultured with 60 mW / cm 2 The 420 nm light was illuminated for 6 minutes, and the culture was continued for 4 hours, and the OD was measured using an enzyme reader. 600 All results were obtained by six independent repeated experiments.

[0121] Compound I of the present invention 1 -I 4 Dark toxicity and the results of photodynamic antimicrobial therapy are shown in Figure 4 .in, Figure 4 A is the MTT assay to detect the presence of different concentrations of compound I in the dark. 1 -I 4 Viability of incubated LO2 cells. Figure 4 B is OD 600 Determination detection 420nm (60mW / cm 2 , 6min) under light irradiation, 30μM compound I was used for detection 1 -I 4 Viability of incubated MRSA bacteria.

[0122] The experimental results show that compound I of the present invention 1 -I 4 Even when the concentration reached 30 μM, the survival rate of LO2 cells was still over 80%, and the dark toxicity was low ( Figure 4 A) MRSA bacteria in dark conditions, 30μM I 1 -I 4 During incubation, the bacterial death rate did not exceed 20%, and the anti-resistant bacteria effect alone was weak, but after irradiation with 420nm light, MRSA bacteria were effectively killed ( Figure 4 B), where I 4 The antibacterial effect is the most obvious, 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 present invention has been shown and described with reference to specific preferred embodiments, it should not be construed as limiting the present invention itself. Various changes in form and details may be made without departing from the spirit and scope of the present invention as defined in the appended claims.

Claims

1. A novel bacterial-targeted carboline compound having a photodynamic therapy effect and having a structure shown in general formula I: in, R1 is an alkyl group, a brominated alkyl group or a quaternized alkyl group; R2 is halogen.

2. The novel carboline compound according to claim 1, characterized in that: R1 is -CH3, -CH2CH3, -CH2CH2CH3, -CH2CH2CH2CH3, -CH2CH2CH2CH3, -CH2CH2CH2CH2CH3, -CH2Br, -CH2CH2Br or -CH2CH2CH2N + (CH3)3Br - .

3. The novel carboline compound according to claim 1, characterized in that: R2 is I- or Br - .

4. The novel carboline compound according to claim 1, characterized in that Selected from:

5. A method for preparing the novel carboline compound represented by the general formula I of claim 1, characterized in that: The method's route is as follows:

6. The method according to claim 5, characterized in that: The steps of this method are as follows: (1) Using norharman as a raw material, it undergoes a substitution reaction with N-bromosuccinimide to obtain an intermediate compound 2; (2) the intermediate compound 2 reacts with 4-boric acid triphenylamine and potassium carbonate to obtain compound 3; (3) Finally, compound 3 reacts with R1-R2 to obtain a novel carboline compound represented by general formula I. Wherein, R1 and R2 are as defined in Formula I.

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

8. A pharmaceutical composition, characterized in that: The invention comprises the novel carboline compound according to any one of claims 1 to 3 and a pharmaceutically acceptable carrier or excipient.

9. Use of the novel carboline compound according to any one of claims 1 to 3 in the preparation of antibacterial drugs.

10. The use according to claim 8, characterized in that: The novel carboline compounds have antibacterial photodynamic therapy effects and can generate active oxygen after light irradiation, thereby effectively killing drug-resistant bacteria; quaternary ammonium groups are introduced into the novel carboline compounds and adsorbed on the surface of bacteria with high negative charges, thereby achieving cell-targeted antibacterial effects.

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