Application of bis(morpholinothiocarbonyl) disulfide in the preparation of drugs for the prevention or treatment of tuberculosis

Abstract

This invention provides the application of bis(morpholine thiocarbonyl) disulfide in the preparation of drugs for the prevention or treatment of tuberculosis, belonging to the field of biomedicine. This invention provides a new use for bis(morpholine thiocarbonyl) disulfide, which can be used to prepare drugs for the prevention or treatment of tuberculosis. Bis(morpholine thiocarbonyl) disulfide exerts its anti-tuberculosis effect by inhibiting the methionine aminopeptidase of Mycobacterium tuberculosis, and it is targeted to Mycobacterium tuberculosis, while being non-toxic to other tissues, living cells or microorganisms.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to the application of a bis(morpholine thiocarbonyl) disulfide in the preparation of drugs for the prevention or treatment of tuberculosis. Background Technology

[0002] Tuberculosis (TB) is a systemic chronic infectious disease caused by Mycobacterium tuberculosis, transmitted through the respiratory tract. It can invade various organs throughout the body, primarily affecting the lungs, where it is known as pulmonary tuberculosis. It is one of the leading causes of infectious disease death worldwide. It is estimated that in 2020, there were 10 million people living with TB globally, and approximately 1.32 million died, an unexpected increase from 2019.

[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 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 extensive drug resistance 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, discovering 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 key problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention proposes the application of a bis(morpholine thiocarbonyl) disulfide in the preparation of drugs for the prevention or treatment of tuberculosis.

[0006] To achieve the above objectives, this invention provides the application of bis(morpholine thiocarbonyl) disulfide in the preparation of drugs with inhibitory activity against Mycobacterium tuberculosis, wherein the structural formula of bis(morpholine thiocarbonyl) disulfide is as follows, and bis(morpholine thiocarbonyl) disulfide is designated as JX06:

[0007]

[0008] The application of bis(morpholine thiocarbonyl) disulfide in the preparation of drugs for the prevention or treatment of tuberculosis, wherein the structural formula of bis(morpholine thiocarbonyl) disulfide is as follows:

[0009]

[0010] Furthermore, tuberculosis is a systemic chronic infectious disease caused by infection with Mycobacterium tuberculosis.

[0011] Furthermore, tuberculosis is a systemic chronic infectious disease mainly caused by Mycobacterium tuberculosis transmitted through the respiratory tract.

[0012] Furthermore, the tuberculosis mentioned is pulmonary tuberculosis caused by infection with Mycobacterium tuberculosis.

[0013] Furthermore, the tuberculosis mentioned is bone tuberculosis caused by Mycobacterium tuberculosis infection.

[0014] Furthermore, the tuberculosis mentioned is lymph node tuberculosis caused by Mycobacterium tuberculosis infection. That is, systemic chronic infectious diseases caused by Mycobacterium tuberculosis infection include pulmonary tuberculosis, bone tuberculosis, or lymph node tuberculosis.

[0015] Bis(morpholine thiocarbonyl) disulfide is targeted at Mycobacterium tuberculosis, but is non-toxic to other tissues, living cells or microorganisms.

[0016] Furthermore, in the process of preventing or treating tuberculosis with bis(morpholine thiocarbonyl) disulfide, bis(morpholine thiocarbonyl) disulfide exerts its anti-tuberculosis effect by inhibiting Mycobacterium tuberculosis methionine aminopeptidase.

[0017] Furthermore, the dosage forms of drugs for the prevention or treatment of tuberculosis include tablets, capsules, granules, or injections.

[0018] Compared with the prior art, the present invention has the following advantages and technical effects:

[0019] This invention provides a new use for bis(morpholine thiocarbonyl) disulfide, which can be used to prepare drugs for the prevention or treatment of tuberculosis. Bis(morpholine thiocarbonyl) disulfide exerts its anti-tuberculosis effect by inhibiting methionine aminopeptidase in Mycobacterium tuberculosis and is targeted at Mycobacterium tuberculosis, while being non-toxic to other tissues, living cells or microorganisms. Attached Figure Description

[0020] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings:

[0021] Figure 1 JX06 is a bis(morpholine thiocarbonyl) disulfide. 1 H NMR spectrum;

[0022] Figure 2 JX06 is a bis(morpholine thiocarbonyl) disulfide. 13 C NMR spectrum;

[0023] Figure 3 The effect of bis(morpholine thiocarbonyl) disulfide JX06 on Mycobacterium tuberculosis H37Ra;

[0024] Figure 4 The inhibitory effects of bis(morpholine thiocarbonyl) disulfide JX06 on Gram-negative and Gram-positive bacteria are shown in (a) and (b).

[0025] Figure 5 The effects of bis(morpholine thiocarbonyl) disulfide JX06 on living cells are shown in (a) for A549, (b) for RKO, and (c) for MCF-7.

[0026] Figure 6 The effects of bis(morpholine thiocarbonyl) disulfide JX06 on Mycobacterium tuberculosis cells are shown in the following images: (a) is a scanning electron microscope image showing the effect of JX06 on Mycobacterium tuberculosis cells, with the left image in (a) representing the blank control group and the right image in (a) representing the effect of JX06 on Mycobacterium tuberculosis cells; (b) is a transmission electron microscope image showing the effect of JX06 on Mycobacterium tuberculosis cells, with the left image in (b) representing the blank control group and the right image in (b) representing the effect of JX06 on Mycobacterium tuberculosis cells.

[0027] Figure 7 The effect of bis(morpholine thiocarbonyl) disulfide JX06 on methionine aminopeptidase derived from Mycobacterium tuberculosis. Detailed Implementation

[0028] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0029] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0030] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0031] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0032] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0033] In the embodiments of the present invention, the bis(morpholinothiocarbonyl) disulfide was synthesized according to the method described in the literature Sun W, Xie Z, Liu Y, et al. JX06 Selectively Inhibits Pyruvate Dehydrogenase Kinase PDK1 by a Covalent Cysteine ​​Modification[J]. Cancer Research, 2015:4923-4936.

[0034] Example 1

[0035] The structure of bis(morpholinothiocarbonyl) disulfide JX06 is as follows:

[0036]

[0037] like Figure 1-2 As shown, its spectral data is as follows:

[0038] 1 H NMR (600MHz, DMSO-d6) δ4.22 (8H,t,J=4.8Hz), 3.73 (8H,t,J=4.8Hz).

[0039] 13 C NMR (150MHz, DMSO-d6) δ192.46, 66.26, 54.38.

[0040] Example 2

[0041] The effect of bis(morpholine thiocarbonyl) disulfide JX06 against Mycobacterium tuberculosis H37Ra:

[0042] The AlamarBlue assay based on microplates was used to determine the minimum inhibitory concentration (MIC) of bis(morpholine thiocarbonyl) disulfide JX06 against Mycobacterium tuberculosis. The specific experimental steps are as follows:

[0043] (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.

[0044] (2) Bis(morpholine thiocarbonyl) disulfide JX06 was added to the 48-well plate containing bacterial solution obtained in step (1) by a two-fold dilution method, with the concentration gradient set as 10 μM, 5 μM, 2.5 μM, 1.25 μM, 1 μM, 0.5 μM, 0.25 μM, and 0.125 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;

[0045] (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 out 100 μL from each well and put it into a 96-well plate, and add 50 μL of azuril coloring solution for color development.

[0046] (4) During the color development period, observe the color change of the liquid in each well of the 96-well plate in real time until the color of the blank control group changes from blue to pink. Then, remove the 96-well plate, observe, and take pictures.

[0047] The results are as follows Figure 3 As shown, the inhibitory effect of bis(morpholine thiocarbonyl) disulfide JX06 on the sensitive strain H37Ra was observed. It was found that bis(morpholine thiocarbonyl) disulfide JX06 exhibited significant inhibitory activity against the aforementioned Mycobacterium tuberculosis, and when the concentration of bis(morpholine thiocarbonyl) disulfide JX06 was above 0.5 μM, its inhibitory activity was similar to that of the INH control group.

[0048] Example 3

[0049] Inhibitory effects of bis(morpholine thiocarbonyl) disulfide JX06 on Gram-negative and Gram-positive bacteria:

[0050] The AlamarBlue assay based on microplates was used to determine the minimum inhibitory concentration (MIC) of bis(morpholine thiocarbonyl) disulfide JX06 against Gram-negative bacteria (Escherichia coli) and Gram-positive bacteria (Staphylococcus aureus). The specific experimental steps are as follows:

[0051] (1) Prepare a concentration of 10 5A pathogen suspension of CFU / mL was added to a 96-well plate at a rate of 100 μL / well.

[0052] (2) Add bis(morpholine thiocarbonyl) disulfide JX06 to the 96-well plate containing bacterial solution obtained in step (1), with concentration gradients of 100 μM, 50 μM, 20 μM, 10 μM, and 1 μM; at the same time, wells treated with ampicillin (Amp concentration of 50 μg / mL) were set as positive control groups, and wells containing only bacterial solution were set as blank control groups;

[0053] (3) Seal the 96-well plate obtained in step (2) with sealing film, place it in a constant temperature incubator at 37°C, and incubate for 1 day. Then add 50 μL of azuril coloring solution for color development.

[0054] (4) During the color development period, observe the color change of the liquid in each well of the 96-well plate in real time until the color of the blank control group changes from blue to yellow (affected by the color of the culture medium). Then, remove the 96-well plate, observe, and take pictures.

[0055] The results are as follows Figure 4 As shown, the bis(morpholine thiocarbonyl) disulfide JX06 is non-toxic to both Gram-negative bacteria (Escherichia coli) and Gram-positive bacteria (Staphylococcus aureus), exhibiting a certain degree of selectivity in antibacterial activity.

[0056] The results of Examples 2 and 3 are summarized and shown in Table 1.

[0057] Table 1

[0058]

[0059]

[0060] Example 4

[0061] Effects of bis(morpholinothiocarbonyl) disulfide JX06 on living cells:

[0062] The cytotoxicity of bis(morpholine thiocarbonyl) disulfide JX06 was detected using a CCK-8 microplate assay to verify the drug's safety. The specific experimental steps are as follows:

[0063] (1) Three cell lines were selected for the experiment: A549 (human lung cancer cells), RKO (human colon cancer cells), and MCF-7 (human breast cancer cells) (all purchased from the American College of Cell Bank, USA). After digestion and counting, the cell concentration was adjusted to 5 × 10⁻⁶ cells / year. 4 Cells / mL were added to 96-well plates at a rate of 100 μL / well as the cell suspension for plating, and then incubated in a 37°C CO2 incubator for 12 h.

[0064] (2) Add bis(morpholine thiocarbonyl) disulfide JX06 to the 96-well plate obtained in step (1) with concentration gradients of 100 μM, 50 μM, 20 μM, 10 μM and 1 μM. At the same time, wells treated with paclitaxel (concentration of 10 μM) were set as positive control, wells containing only cells were set as control, and wells containing only culture medium were set as blank. Then, the plate was placed back into a 37℃ CO2 constant temperature incubator for 48 h.

[0065] (3) Remove the 96-well plate from the constant temperature incubator in step (2), discard the supernatant, protect it from light, add 100 μL of CCK-8 (10%), and then place it back into the 37℃ CO2 constant temperature incubator for incubation. Every 30 min, use a fluorescent microplate reader to detect the absorbance value of the liquid in each well of the 96-well plate at the emission wavelength of 450 nm. After removing the background of the culture medium and CCK-8, calculate the viability. The viability is calculated as follows: viability = (OD value of experimental group - OD value of blank group) / (OD value of control group - OD value of blank group) × 100%.

[0066] The results are as follows Figure 5 As shown, JX06 affects the IC50 of three cell types. 50 The concentration is >20 μM, which is greater than the anti-tuberculosis concentration. Therefore, it has a certain degree of safety for development as a clinical drug.

[0067] Example 5

[0068] Effects of bis(morpholine thiocarbonyl) disulfide JX06 on Mycobacterium tuberculosis cells:

[0069] The effects of bis(morpholine thiocarbonyl) disulfide JX06 on Mycobacterium tuberculosis cells were observed using electron microscopy. The specific experimental steps are as follows:

[0070] (1) Culture two bottles of M.tb H37Ra, 20 mL in each bottle, until the bacterial culture OD 595 When the value is around 0.3, one bottle of bacterial culture is added with bis(morpholine thiocarbonyl) disulfide JX06 to a final concentration of 2×MIC as the experimental group; the other bottle of bacterial culture is added with an equal volume of DMSO as the blank control group. The two bottles of bacterial culture are placed in a shaker at 37℃ and cultured for 24h.

[0071] (2) Divide the bacterial solution in step (1) into two equal portions, transfer them to 50mL sterile centrifuge tubes, centrifuge at 3000×g for 10min at 4℃, and collect the bacterial precipitate.

[0072] (3) Add 5 mL of PBS (0.1 M, pH 7.4) to the two precipitates obtained in step (2) to wash the bacterial precipitates. Wash 3 times, and finally add an appropriate volume (about 500 μL) of 2.5% glutaraldehyde. Fix overnight at 4°C for scanning electron microscopy.

[0073] (4) Add an appropriate volume (about 500 μL) of 2.5% glutaraldehyde to the two precipitates (experimental group and blank control group) obtained in step (2), fix overnight at 4°C, and then perform transmission electron microscopy.

[0074] (5) The bacterial cells obtained in steps (3) and (4) were examined by electron microscopy to observe the effect of bis(morpholine thiocarbonyl) disulfide JX06 on Mycobacterium tuberculosis cells.

[0075] The results are as follows Figure 6 As shown in (a), compared with the blank control group, the experimental group showed significantly shorter bacterial cell length, wrinkled and twisted surface, and even partial breakage with visible fragments; in addition, as Figure 6 As shown in (b), the bacterial cell wall integrity in the experimental group was disrupted, and the outline became blurred. These results indicate that JX06 exhibits anti-tuberculosis activity by disrupting the bacterial cell wall.

[0076] Example 6

[0077] Effect of bis(morpholine thiocarbonyl) disulfide JX06 on methionine aminopeptidase derived from Mycobacterium tuberculosis:

[0078] Based on in vitro metabolic experiments, the effect of bis(morpholine thiocarbonyl) disulfide JX06 on methionine aminopeptidase derived from Mycobacterium tuberculosis was investigated. The specific experimental steps are as follows:

[0079] (1) Based on the Mycobacterium tuberculosis H37Ra protein information Rv0734, the Mycobacterium tuberculosis-derived methionine aminopeptidase MtMET-AP1 was designed. BL21(DE3) competent cells were selected, and the purified protein was obtained by purifying with ITPG (1mM, induced at 16℃ for 24h) and nickel column.

[0080] (2) The inhibitory effect of bis(morpholine thiocarbonyl) disulfide JX06 was verified using the protein obtained in step (1). The reaction system (200 μL) included: Hepes (100 mM), NaCl (50 mM), CoCl2 (200 μM), MtMET-AP1 (11 μg / mL) and the fluorescent probe DDAN-MT (2.5 μM) for detecting methionine aminopeptidase MtMET-AP1 from Mycobacterium tuberculosis. The final inhibitor concentration (μM) was set as: 0.2, 0.5, 1, 1.5, 2, 3, 5, 8, 10. The volume of DMSO was controlled to be <1%, and the reaction was carried out at 37℃ for 30 min.

[0081] (3) Add 100 μL of acetonitrile to step (2) to terminate the experiment, centrifuge at 20000g for 20 min to remove protein. Take 200 μL of supernatant and analyze its fluorescence response value using an ELISA reader (excitation / emission wavelength: λex 600 / λem 668nm).

[0082] The results are as follows Figure 7 As shown in the results, the IC of JX06 can be identified. 50 The concentration was 1.88 μM, and it exhibited anti-tuberculosis activity by inhibiting the activity of methionine aminopeptidase in Mycobacterium tuberculosis.

[0083] The above are merely preferred embodiments of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. The application of bis(morpholine thiocarbonyl) disulfide in the preparation of drugs with inhibitory activity against Mycobacterium tuberculosis, characterized in that, The structural formula of bis(morpholine thiocarbonyl) disulfide is as follows:

2. The application of bis(morpholinothiocarbonyl) disulfide in the preparation of drugs for the prevention or treatment of tuberculosis, characterized in that, The structural formula of bis(morpholine thiocarbonyl) disulfide is as follows:

3. The application according to claim 2, characterized in that, Tuberculosis is a systemic chronic infectious disease caused by infection with Mycobacterium tuberculosis.

4. The application according to claim 3, characterized in that, The tuberculosis mentioned is pulmonary tuberculosis caused by infection with Mycobacterium tuberculosis.

5. The application according to claim 3, characterized in that, The tuberculosis mentioned is bone tuberculosis caused by infection with Mycobacterium tuberculosis.

6. The application according to claim 3, characterized in that, The tuberculosis mentioned is lymph node tuberculosis caused by infection with Mycobacterium tuberculosis.

7. The application according to claim 2, characterized in that, It exerts its anti-tuberculosis effect by inhibiting methionine aminopeptidase in Mycobacterium tuberculosis.

8. The application according to claim 2, characterized in that, Drug dosage forms include tablets, capsules, granules, or injections.

Images