Quaternized norharman dimer, its pharmaceutical composition and antibacterial use

Abstract

The present invention relates to the field of antimicrobial agents, and in particular to quaternized norharman dimers, pharmaceutical compositions thereof, and antimicrobial uses. The present invention provides a quaternized norharman dimer, the MIC value of which against MRSA is 0.015 to 0.5 μg / mL, which is stronger than the clinical first-line drug vancomycin, and can be used to treat infectious diseases caused by MRSA, such as bacteremia, pneumonia, myocarditis, wound infection, bone and joint infection, etc. The quaternized norharman dimer is active against Gram-negative bacteria Escherichia coli, Klebsiella pneumoniae, and Acinetobacter baumannii at the same time, and has a broad-spectrum antibacterial characteristic.

Description

Technical Field

[0001] The invention relates to the field of antimicrobial agents, and in particular to a quaternized norharman dimer, a pharmaceutical composition thereof and antimicrobial use thereof. Background Art

[0002] Methicillin-resistant Staphylococcus aureus (MRSA) can cause pneumonia, sepsis and other diseases. Its occurrence and rapid spread pose a huge threat to human health. The large-scale and irrational use of antibiotics has accelerated the occurrence and development of MRSA resistance, resulting in reduced efficacy of multiple antibiotics against MRSA infections. People are about to face the dilemma of having no drugs available. At present, the development of antibiotics is seriously lagging behind, and the speed of bacterial resistance far exceeds the speed of new drug development. In order to protect human health, it is imperative to find new antibiotics.

[0003] Natural products and their derivatives have always been an important source for the discovery of antibacterial drugs, such as penicillin and vancomycin. It is reported that dimerization and quaternization modification can enhance the antibacterial activity of natural products. Norharman is a β-carboline compound with a wide range of pharmacological activities, such as anti-tumor, antiviral, etc., and 12 drugs based on this skeleton have been marketed. Most of the existing technologies directly utilize the properties of norharman, for example, norharman is used for combined medication. CN116270626A discloses that when norharman and azithromycin are used in combination, there is an obvious synergistic effect, which can inhibit the expression of bacterial-related virulence factors without inhibiting bacterial growth, thereby weakening the virulence of bacteria, and has a good antibacterial effect on drug-resistant Pseudomonas aeruginosa in vivo. CN115244067A discloses a synthetic peptide comprising an amino acid sequence XnYm, wherein X represents a positively charged amino acid, Y represents a hydrophobic amino acid, and Y can be selected from tetrahydronorharman-3-carboxylic acid. However, the antibacterial properties of norharman are limited, which limits its commercial value.

[0004] Quaternary ammonium salt compounds have the amphiphilic and positively charged properties of traditional antimicrobial peptides, can act on bacterial cell membranes, are not prone to drug resistance, and can also solve the defects of traditional antimicrobial peptides such as high production costs and easy inactivation by proteases. Natural products have always been an important source for the discovery of antimicrobial drugs due to the diversity of their chemical structures and targets. The inventors synthesized a quaternized β-carboline dimer compound in previous studies, which has high anti-MRSA activity, but there is still a gap compared with vancomycin, and further improvement is needed. The inventors found that when the β-carboline structure is a norharman structure, the anti-MRSA activity of the quaternized norharman dimer is greatly improved, achieving unexpected technical effects, and thus proposed the present invention. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides a quaternized norharman dimer, which has much stronger anti-MRSA activity than the clinical first-line drug vancomycin and has a broad spectrum, and can be used to treat infectious diseases caused by MRSA such as bacteremia, pneumonia, myocarditis, wound infection, bone and joint infection, etc., and can provide more options for solving the increasingly serious problem of MRSA resistance.

[0006] The first aspect of the present invention provides a quaternized nor-Harman dimer, the general structural formula of which is as follows: Wherein n is a positive integer greater than or equal to 9.

[0007] In some embodiments, n is a positive integer from 9 to 20. For example, n can be 9, 10, 11, 12, 13, 15, 15, 16, 17, 18, 19, 20, etc.

[0008] The second aspect of the present invention provides a method for preparing the quaternized nor-Harman dimer, which is prepared by the following reaction route:

[0009]

[0010] In some embodiments, the preparation method comprises the following steps:

[0011] (i) dissolving tryptamine in glacial acetic acid / methanol solution, adding formaldehyde aqueous solution, and heating to react to obtain compound 2;

[0012] (ii) Compound 2 is mixed with palladium on carbon and added into xylene, and heated to react to obtain Compound 3;

[0013] (iii) dissolving compound 3 in anhydrous N,N-dimethylformamide, adding sodium hydride in an ice bath and stirring, and then adding dibromo long-chain alkyl to react to obtain compound 4;

[0014] (iv) Compound 4 is dissolved in acetonitrile, iodomethane is added, and the mixture is heated and stirred to react to obtain Compound 5, which is the quaternized nor-Harman dimer.

[0015] The carbon chain length of the dibromocarbon chain alkyl is greater than or equal to C9, such as C9, C12, C20; the dibromocarbon chain alkyl is 1,9-dibromononane, 1,10-dibromodecane, 1,11-dibromonoundecane, 1,12-dibromodecane, etc.

[0016] The third aspect of the present invention provides the use of the quaternized nor-Harman dimer for preparing anti-MRSA (methicillin-resistant Staphylococcus aureus) drugs.

[0017] The fourth aspect of the present invention provides an anti-MRSA pharmaceutical composition comprising the quaternized norharman dimer and pharmaceutical excipients.

[0018] In some embodiments, the pharmaceutical composition can be used to treat infectious diseases caused by MRSA, such as bacteremia, pneumonia, myocarditis, wound infection, bone and joint infection, etc.

[0019] Compared with the prior art, the beneficial effects of the present invention include at least:

[0020] The quaternized norharman dimer involved in the present invention has a novel structure. Compared with the existing β-carboline compounds or quaternized β-carboline compounds, the anti-MRSA activity is greatly improved, and the MIC value is 0.015-1 μg / mL, which is much stronger than the clinical first-line drug vancomycin. The quaternized norharman dimer is active against Gram-negative bacteria Escherichia coli, Klebsiella pneumoniae and Acinetobacter baumannii at the same time, and has a broad-spectrum antibacterial characteristic. DETAILED DESCRIPTION

[0021] In order to make the purpose, technical scheme and beneficial effect of the present invention clearer, the present invention is further described in detail below in conjunction with specific embodiments. The examples of the embodiments are shown. It should be understood that the specific embodiments described in the following embodiments of the present invention are only exemplary descriptions of the specific embodiments of the present invention, and are intended to be used for explaining the present invention, but do not constitute a limitation of the present invention.

[0022] The endpoints of ranges and any values ​​disclosed herein are not limited to the exact range or value, and these ranges or values ​​should be understood to include approximations to these ranges.

[0023] Example 1 Preparation of quaternized nor-Harman dimer

[0024] The quaternized nor-Harman dimer was prepared by the following reaction route:

[0025]

[0026] The specific steps are as follows:

[0027] (i) 30 mmol of tryptamine was dissolved in 25 mL of glacial acetic acid / methanol (10:1), and then 30 mmol of aqueous formaldehyde solution was added. The reaction system was stirred at 80°C for 1 h and then cooled to room temperature. The pH value was adjusted to 9-10 with aqueous ammonia under ice bath stirring. After filtration and washing, the target compound 2 was obtained as a white solid with a yield of 96%.

[0028] (ii) 10 mmol of compound 2 and 186 mg of palladium on carbon (10%) were added to 50 mL of xylene, and then heated at 140° C. until compound 2 was completely converted. After filtration, concentration and silica gel column chromatography (petroleum ether / ethyl acetate system gradient elution), compound 3 was obtained as a white solid with a yield of 84%.

[0029] (iii) 1 mmol of compound 3 was dissolved in 5 mL of anhydrous N,N-dimethylformamide, and then 3 mmol of sodium hydride (60%) was added under ice bath, and 0.5 mmol of 1,9-dibromononane or 1,10-dibromodecane or 1,11-dibromonoundecane or 1,12-dibromodecane was added after stirring under ice bath for 30 min. After the reaction was completed by thin layer chromatography (TLC), the reaction was quenched with 1 mL of water, extracted with dichloromethane, dried over anhydrous sodium sulfate, and then subjected to silica gel column chromatography (gradient elution with petroleum ether / ethyl acetate system) to obtain a white solid, namely compound 4a (yield 77%) or 4b (yield 81%) or 4c (yield 73%) or 4d (yield 74%).

[0030] (iv) Dissolve 1 mmol of compound 4a-4d in 15 mL of acetonitrile, then add 2.2 mmol of iodomethane, heat and stir at 70°C. After the reaction is completed by TLC, cool the reaction solution to room temperature while stirring, and filter or perform silica gel column chromatography (gradient elution with dichloromethane / methanol system) to obtain the target compounds 5a-5d.

[0031] Compound 5a: 9,9'-(1,9-nonane)bis(2-methyl-norharman-2-iodine salt), a light yellow solid, a melting point of 242-243°C, and a yield of 87%; 1 H NMR(DMSO-d6)δ:9.70(s,2H),8.85(d,J=4.0Hz,2H),8.70(d,J=8.0Hz,2H),8.55(d,J=8.0Hz,2H),7.96(d,J=8.0Hz,2H), 7.86(t,J=8.0Hz,2H),7.50(t,J=8.0Hz,2H),4.58(t,J=8.0Hz,4H),4.51(s,6H),1.85-1.78(m,4H),1.34-1.15(m,10H); 13 C NMR(DMSO-d6)δ:144.46,135.72,133.97,132.46,131.85,129.96,124.31, 122.21,119.45,118.09,111.88,48.33,44.08,29.39,29.20,29.13,26.73.

[0032] Compound 5b: 9,9'-(1,10-decane)bis(2-methyl-norharman-2-iodine salt), a light yellow solid, melting point 281-283°C, yield 82%; 1 H NMR(DMSO-d6)δ:9.70(s,2H),8.86(d,J=8.0Hz,2H),8.70(d,J=4.0Hz,2H),8.55(d,J=8.0Hz,2H),7.97(d,J=8.0Hz,2H),7.87(t, J=8.0Hz,2H),7.50(t,J=8.0Hz,2H),4.58(t,J=8.0Hz,4H),4.51(s,6H),1.85-1.78(m,4H),1.33-1.21(m,8H),1.19-1.14(m,4H); 13 C NMR(DMSO-d6)δ:144.48,135.73,133.97,132.46,131.85,129.96,124.31, 122.21,119.45,118.09,111.89,48.34,44.08,29.39,29.20,29.14,26.76.

[0033] Compound 5c: 9,9'-(1,11-undecane)bis(2-methyl-norharman-2-iodine salt) is a light yellow solid with a melting point of 273-274°C and a yield of 79%; 1 H NMR(DMSO-d6)δ:9.70(s,2H),8.85(d,J=8.0Hz,2H),8.70(d,J=8.0Hz,2H),8.55(d,J=8.0Hz,2H),7.97(d,J=8.0Hz,2H),7.86(t, J=8.0Hz,2H),7.50(t,J=8.0Hz,2H),4.58(t,J=8.0Hz,4H),4.51(s,6H),1.86-1.79(m,4H),1.32-1.22(m,8H),1.18-1.14(m,6H); 13 C NMR(DMSO-d6)δ:144.48,135.73,133.97,132.46,131.85,129.96,124.31,122 .21,119.45,118.09,111.89,48.34,44.08,29.39,29.34,29.20,29.14,26.76.

[0034] Compound 5d: 9,9'-(1,12-dodecane)bis(2-methyl-norharman-2-iodine salt), a light yellow solid, melting point 264-266°C, yield 72%;1 H NMR(DMSO-d6)δ:9.70(s,2H),8.86(d,J=8.0Hz,2H),8.70(d,J=4.0Hz,2H),8.55(d,J=8.0Hz,2H),7.98(d,J=12.0Hz,2H),7.87(td,J= 8.0Hz,2.0Hz,2H),7.50(t,J=8.0Hz,2H),4.59(t,J=8.0Hz,4H),4.51(s,6H),1.87-1.80(m,4H),1.35-1.22(m,8H),1.18-1.12(m,8H); 13 C NMR(DMSO-d6)δ:144.48,135.74,133.97,132.46,131.86,129.97,124.30,122 .21,119.45,118.09,111.90,48.34,44.09,29.41,29.39,29.23,29.13,26.77.

[0035] Example 2 Antimicrobial Activity Test of Quaternized Norharman Dimer

[0036] The minimum inhibitory concentration (MIC) of the test compounds was determined by the two-fold dilution method to evaluate the antibacterial activity of the compounds. 50 mmol of the test compound (5a, 5b, 5c, 5d) stock solution was prepared using dimethyl sulfoxide (DMSO). The test strains were Staphylococcus aureus standard strain (ATCC 29213), MRSA standard strain (ATCC33591, CGMCC 1.12409), MRSA clinical isolates (M-13, M-14), Acinetobacter baumannii standard strain (ATCC 19606) and Klebsiella pneumoniae standard strain (ATCC10031). The compound and bacterial suspension were diluted with Mueller-Hinton broth liquid medium. The final concentration of the compound in the 96-well plate was 0.0078-128 μg / mL, and the final concentration of the bacterial suspension was 1×105 CFU / mL. The bacterial growth was observed after culturing at 37°C for 14 hours. The values ​​corresponding to the wells with no sterile growth and complete clarification under naked eye observation were the MIC values ​​of the compound against bacteria. Vancomycin was used as a positive control, and phosphate buffer containing the corresponding DMSO concentration was used as a negative control.

[0037] The results of three parallel experiments were averaged, and the antibacterial activity is shown in Table 1. The antibacterial activity test results show that the quaternized nor-harman dimer compound of Example 1 has strong antibacterial activity, and the MIC value of MRSA is 0.015-1 μg / mL, which is stronger than the clinical first-line drug vancomycin (1 μg / mL), and has the use of medicine for treating diseases related to MRSA infection. It is also active against Gram-negative bacteria Escherichia coli, Klebsiella pneumoniae and Acinetobacter baumannii, and has broad-spectrum antibacterial characteristics.

[0038] Table 1 Antibacterial activity of the tested compounds

[0039]

[0040] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention and do not constitute a limitation on the content of the present invention. Within the technical concept of the present invention, the technical solution of the present invention can be subjected to a variety of simple modifications, including the combination of various technical features in any other suitable manner, and these simple modifications and combinations should also be regarded as the contents disclosed by the present invention and belong to the protection scope of the present invention.

Claims

1. A use of a quaternized nor-Harman dimer in the preparation of an anti-MRSA drug, characterized in that: The general structural formula of the quaternized nor-Harman dimer is shown in Formula I, , where n is 9, 10, 11, or 12.

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