Use of pyrazoloquinoline derivatives and pharmaceutical compositions thereof

Abstract

This invention discloses an application of a pyrazolium-quinoline derivative and its pharmaceutical composition, belonging to the field of biomedical technology. Specifically, it discloses the application of a pyrazolium-quinoline derivative in the preparation of drugs for the prevention or treatment of tuberculosis. The structure of the pyrazolium-quinoline derivative is shown in general formula (I), wherein R1 is isopropyl or methyl, and R2 is 4-quinolinyl or 5-quinolinyl. This invention also discloses a pharmaceutical composition comprising the pyrazolium-quinoline derivative used in the above application. The pyrazolium-quinoline derivative of this invention exhibits good inhibitory activity against Mycobacterium tuberculosis, broadening the types of drugs for the treatment of tuberculosis.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to the application of a pyrazolium quinoline derivative in the preparation of drugs for the prevention or treatment of tuberculosis and pharmaceutical compositions thereof. Background Technology

[0002] Tuberculosis (TB) is a chronic infectious disease caused by Mycobacterium tuberculosis. It can invade various organs throughout the body, primarily affecting the lungs, hence the name pulmonary tuberculosis. Other forms include lymph node tuberculosis, bone tuberculosis, and kidney tuberculosis. Pulmonary tuberculosis consistently ranks among the top Class A and B infectious diseases in China, and the harm caused by multidrug-resistant pulmonary tuberculosis is becoming increasingly prominent.

[0003] Currently, commonly used first-line anti-tuberculosis drugs include streptomycin, isoniazid, rifampin, and ethambutol; second-line anti-tuberculosis drugs include sodium para-aminosalicylate, ofloxacin, kanamycin, and clarithromycin. However, studies show that the resistance rate of Mycobacterium tuberculosis isolates from sputum samples of pulmonary tuberculosis patients in my country to any one of the four first-line anti-tuberculosis drugs is 36.8%, and the multidrug resistance rate is 6.8%; the resistance rate to any one of the seven second-line anti-tuberculosis drugs is 24.6%, and the extensively drug-resistant rate is 2.1%; and the resistance rate to any one of the eleven first-line and second-line anti-tuberculosis drugs is 42.1%. It is evident that current technologies face a serious drug resistance problem in the prevention and control of tuberculosis.

[0004] Therefore, how to provide an active molecule and candidate drug that can effectively inhibit Mycobacterium tuberculosis and also has a significant inhibitory effect on drug-resistant Mycobacterium tuberculosis is a technical problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0005] The purpose of this invention is to provide an application of pyrazolium quinoline derivatives and pharmaceutical compositions thereof, in order to solve the problems existing in the prior art.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] The application of a pyrazolium quinoline derivative in the preparation of drugs for the prevention or treatment of tuberculosis, wherein the structure of the pyrazolium quinoline derivative is shown in general formula (I):

[0008]

[0009] Wherein, R1 is isopropyl or methyl;

[0010] R2 is 4-quinolino or 5-quinolino.

[0011] Preferably, the general formula (I) has the following structure:

[0012]

[0013] Compound 1 is described in reference 1 (Ao A, Hao J, Hopkins CR, et al. DMH1, a Novel BMPSmall Molecule Inhibitor, Increases Cardiomyocyte Progenitors and Promotes Cardiac Differentiation in Mouse Embryonic Stem Cells[J]. Plos One, 2012, 7.).

[0014] Compound 2 is described in reference 2 (Mohedas AH, Xing X, Armstrong KA, et al. Development of an ALK2-biased BMP type I receptor kinase inhibitor.[J].Acs Chemical Biology,2012,8(6):1291-1302.)

[0015] Preferably, the tuberculosis includes one or more of pulmonary tuberculosis, bone tuberculosis, lymph node tuberculosis, and renal tuberculosis.

[0016] A pharmaceutical composition comprising the pyrazoquinoline derivatives used in the above applications.

[0017] Preferably, the dosage form of the pharmaceutical composition includes tablets, capsules, granules, or injections.

[0018] This invention discloses the application of a pyrazolium-quinoline derivative and its pharmaceutical composition. The pyrazolium-quinoline derivative of this invention not only exhibits significant inhibitory effects on drug-sensitive Mycobacterium tuberculosis but also shows potent inhibitory effects on clinically multidrug-resistant strains. Therefore, this type of pyrazolium-quinoline derivative provides a candidate drug for the treatment of tuberculosis, especially drug-resistant tuberculosis. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 Compound 1 in Example 1 1 H NMR spectrum;

[0021] Figure 2 Compound 1 in Example 1 13 C NMR spectrum;

[0022] Figure 3 The HR-MS spectrum of compound 1 in Example 1 is shown below.

[0023] Figure 4 Compound 2 in Example 2 1 H NMR spectrum;

[0024] Figure 5 Compound 2 in Example 2 13 C NMR spectrum;

[0025] Figure 6 The HR-MS spectrum of compound 2 in Example 2;

[0026] Figure 7 The effect of compound 1 in Example 4 on the growth curve of Mycobacterium tuberculosis H37Ra;

[0027] Figure 8 The effect of compound 2 in Example 4 on the growth curve of Mycobacterium tuberculosis H37Ra;

[0028] Figure 9 Compound 1 in Example 5 was used to inhibit Mycobacterium tuberculosis H37Ra infection of RAW264.7 macrophages;

[0029] Figure 10 Compound 2 in Example 5 was used to inhibit Mycobacterium tuberculosis H37Ra infection of macrophages RAW264.7. Detailed Implementation

[0030] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0031] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0032] Example 1

[0033] A pyrazolium quinoline derivative has the following structure:

[0034]

[0035] like Figure 1-3 As shown, its spectral data is as follows:

[0036] HR-MS m / z 381.1694 [M+H] + . 1 H NMR (600MHz, DMSO-d6) δ9.56(1H,d,J=2.4Hz), 9.04(1H,d,J=2.4Hz), 8.96(1H,d,J=4.8Hz), 8.71(1H,s), 8.20(1H,d, J=7.8Hz), 8.11(1H,d,J=7.8Hz), 7.81(4H,m), 7.62(1H,m), 7.08(2H,d,J=7.2Hz), 4.71(1H,m), 1.30(6H,d,J=6.0Hz). 13 C NMR(150MHz,DMSO-d6)δ158.37,151.04,150.53,148.91,145.79,144.72,138.15,132.70,129 .99,129.83,128.78,127.09,126.53,125.63,122.84,122.29,116.72,106.72,69.80,22.28.

[0037] Example 2

[0038] A pyrazolium quinoline derivative has the following structure:

[0039]

[0040] like Figure 4-6 As shown, its spectral data is as follows:

[0041] HR-MS m / z 353.1381 [M+H] + . 1 H NMR (600MHz, DMSO-d6) δ9.54(1H,d,J=2.4Hz), 8.97(1H,d,J=2.4Hz), 8.95(1H,dd,J=4.8,1.2Hz), 8.57(1H,s), 8.41 (1H,d,J=8.4Hz), 8.07(1H,d,J=8.4Hz), 7.84(4H,m), 7.52(1H,dd,J=8.4,4.2), 7.10(2H,d,J=8.4Hz), 3.83(3H,s). 13C NMR(150MHz,DMSO-d6)δ160.07,150.88,150.41,148.68,145.63,144.53,134.91,132.50,130 .32,129.64,128.77,128.67,128.65,126.89,126.10,122.36,121.85,115.19,108.24,55.79.

[0042] Example 3

[0043] Inhibitory effects of compounds 1 and 2 on Mycobacterium tuberculosis:

[0044] The AlamarBlue assay based on microplates was used to detect the inhibition rates and minimum inhibitory concentrations (MICs) of compounds 1 and 2 against Mycobacterium tuberculosis. The specific experimental steps are as follows:

[0045] (1) Prepare a concentration of 10 6 A pathogen suspension of CFU / mL was added to a 48-well plate at a rate of 500 μL / well.

[0046] (2) Compound 1 or 2 was added to the 48-well plate containing bacterial solution obtained in step (1) by a twofold dilution method, with the concentration gradient set to 2 μM, 1 μM, 0.5 μM, 0.25 μM, 0.125 μM and 0.0625 μM; at the same time, wells treated with the anti-tuberculosis drug isoniazid (INH concentration of 0.4 μM) were set as positive control groups, and wells containing only bacterial solution were set as blank control groups;

[0047] (3) Seal the 48-well plate obtained in step (2) with sealing film, place it in a constant temperature incubator at 37°C, and incubate for 7 days. Then take it out and add 400 μL of azuril coloring solution for color development.

[0048] (4) During the color development period, observe the color change of the liquid in each well of the 48-well plate in real time until the color of the blank control group changes from blue to pink, then remove the 48-well plate;

[0049] (5) The fluorescence value of the liquid in each well of the 48-well plate was detected by a fluorescence microplate reader at excitation wavelength of 550 nm and emission wavelength of 600 nm. After removing the background of culture medium and resazurin, the activity inhibition rate was calculated. The activity inhibition rate was 1 - (fluorescence intensity of compound treatment group / fluorescence intensity of blank control group) × 100%, and the minimum inhibitory concentration was the concentration of the compound when the activity inhibition rate was 90%.

[0050] The results are shown in Table 1. Compound 1 and Compound 2 showed their inhibitory activity against susceptible strains H37Ra and H37Rv and resistant strains MDR35163 and MDR 35205, respectively. Table 1 indicates that, using the above method, Compound 1 and Compound 2 exhibit significant inhibitory activity against the aforementioned Mycobacterium tuberculosis. Furthermore, when the concentration of Compound 1 is above 0.5 μM or the concentration of Compound 2 is above 1 μM, their inhibitory activity is higher than that of the INH control group.

[0051] Table 1

[0052]

[0053] Example 4

[0054] The effects of compounds 1 and 2 on the growth curve of Mycobacterium tuberculosis H37Ra were investigated using the following experimental procedures:

[0055] The growth curve was determined using Erlenmeyer flasks in 25 mL of 7H9-S medium.

[0056] The H37Ra bacterial culture in the logarithmic growth phase was diluted to OD. 595 An absorbance of 0.1 was used as the initial bacterial concentration. The diluted H37Ra bacterial suspension was evenly dispensed into Erlenmeyer flasks, with different concentrations of compound treatment groups, a blank control group, and an isoniazid control group (INH concentration of 0.4 μM). In the compound 1 treatment group, the concentrations of compound 1 were 0.25 μM, 0.5 μM, and 2 μM, respectively; in the compound 2 treatment group, the concentrations of compound 2 were 0.5 μM and 2 μM, respectively. The Erlenmeyer flasks were incubated at 37°C with shaking. The OD of the bacterial suspension was measured on days 0, 1, 3, 5, and 7. 595 The absorbance values ​​were used to plot the growth curves for different experimental groups.

[0057] The results are as follows Figure 7 , 8 As shown, compounds 1 and 2 significantly inhibited the growth and proliferation of Mycobacterium tuberculosis H37Ra compared with the blank control group, achieving almost the same inhibitory effect as the INH control group on day 7.

[0058] Example 5

[0059] The effects of compounds 1 and 2 on Mycobacterium tuberculosis H37Ra infection of RAW264.7 macrophages were investigated using the following experimental procedures:

[0060] (1) After digesting and counting RAW264.7 cells, the cell concentration was adjusted to 2×10⁻⁶. 5Cells / mL were added to 24-well plates at a rate of 500 μL / well as the cell suspension for plating, and then incubated in a 37°C CO2 incubator for 12 h.

[0061] (2) After removing the 24-well plate from the constant temperature incubator in step (1), wash the cells twice with PBS buffer, add DMEM medium containing H37Ra to the 24-well plate according to the infection ratio of MOI=10:1, and incubate in a 37℃ CO2 constant temperature incubator for 4h for infection.

[0062] (3) Open the 24-well plate obtained in step (2) after 4 hours of infection, wash the cells three times with PBS buffer, add 500 μL of DMED (containing 10% fetal bovine serum) medium containing compound 1 or 2, and use the treatment well with a compound concentration of 0 as a blank control and set up the treatment well with the anti-tuberculosis drug isoniazid (INH concentration of 0.4 μM) as a positive control. Continue to culture in a 37℃ CO2 constant temperature incubator for 48 hours. In the DMED medium containing compound 1, the concentrations of compound 1 were 0 μM, 1.25 μM, 2.5 μM, 5 μM and 10 μM, respectively. In the compound 2 treatment group, the concentrations of compound 2 were 0 μM, 1.25 μM and 2.5 μM, respectively.

[0063] (4) Take out the 24-well plate obtained in step (3), carefully wash the cells three times with PBS buffer, add 200 μL of 0.05% SDS solution to each well to lyse the cells and release H37Ra from the macrophages.

[0064] (5) Inoculate the cell lysate obtained in step (4) onto 7H11-S solid plates and incubate them in a 37°C incubator for 3-4 weeks. Count the number of colonies carrying units (CFU) on the plates.

[0065] The results are as follows Figure 9 , Figure 10 As shown, compared with the bacterial load in the control group cells, compounds 1 and 2, when co-incubated with cells at a lower concentration of 1.25 μM, significantly reduced the number of pathogens in the cells, indicating that the compounds could inhibit the invasion and infection of macrophages by Mycobacterium tuberculosis H37Ra; at the same time, it also showed that compounds 1 and 2 could effectively kill pathogens in the cells.

[0066] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. The application of a pyrazoquinoline derivative in the preparation of a drug for inhibiting Mycobacterium tuberculosis, characterized in that, The structure of the pyrazolquinoline derivative is shown below: 。

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