Use of compound imd-0354 in the manufacture of a medicament for treating tuberculosis

Abstract

The application provides application of a compound IMD-0354 in preparation of a drug for treating tuberculosis, and belongs to the technical field of drugs.The compound IMD-0354 screened from a compound library has a significant inhibitory effect on multidrug-resistant Mycobacterium tuberculosis Mtb28 and Mtb1731 and Mycobacterium tuberculosis MtbH37Rv, and has a wide application prospect in preparation of the drug for treating tuberculosis.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to the application of compound IMD-0354 in the preparation of drugs for treating tuberculosis. Background Technology

[0002] Tuberculosis (TB) is a serious chronic infectious disease caused by Mycobacterium tuberculosis (MTB). Due to long treatment cycles, poor patient compliance, and the unique physiological characteristics of MTB, single-drug resistant, multi-drug resistant, and even extensively drug-resistant MTB strains are constantly emerging and spreading, leading to a continuous decline in the clinical efficacy of traditional anti-TB drugs. Furthermore, the protection provided by the BCG vaccine for newborns is time-limited and cannot prevent the recurrence of latent TB. Currently, effective anti-TB drugs include bedaquiline, delamanid, and pretomanid. However, these drugs have significant toxicity, and drug-resistant MTB strains have quickly emerged in clinical practice. Therefore, developing new anti-TB drugs to address the challenges in TB treatment has become an urgent priority for controlling the TB epidemic.

[0003] Currently, research into discovering novel compounds that inhibit drug-resistant Mycobacterium tuberculosis (Mtb) is progressing slowly, making screening from existing known compounds an effective approach. However, screening numerous compounds effective against Mtb requires significant effort and resources. In recent years, high-throughput inhibitor screening models at the molecular level, based on essential gene-encoded products, have yielded a large number of compounds with enzyme-inhibiting activity. However, the cell wall and cell membrane shielding effect of Mtb, along with its complex internal metabolic environment, renders these compounds lacking antibacterial activity, resulting in situations where screened compounds fail to achieve the desired antibacterial activity. Summary of the Invention

[0004] This invention provides the application of compound IMD-0354 in the preparation of drugs for treating tuberculosis. The compound IMD-0354, screened from a compound library, has a significant inhibitory effect on multidrug-resistant Mycobacterium tuberculosis Mtb28 and Mtb1731, as well as Mycobacterium tuberculosis MtbH37Rv.

[0005] To solve the above-mentioned technical problems, the present invention provides the following technical solutions:

[0006] This invention provides the use of compound IMD-0354 in the preparation of drugs for treating tuberculosis.

[0007] This invention provides the use of a pharmaceutical composition containing compound IMD-0354 in the preparation of a drug for treating tuberculosis.

[0008] Preferably, the pharmaceutical composition further includes at least one pharmaceutically acceptable carrier.

[0009] Preferably, the effective concentration of the compound IMD-0354 is 0.2–10 μM.

[0010] Preferably, the pathogens involved in the tuberculosis include one or more of Mycobacterium tuberculosis Mtb H37Rv, multidrug-resistant Mycobacterium tuberculosis Mtb 28, and multidrug-resistant Mycobacterium tuberculosis Mtb 1731.

[0011] Preferably, the dosage form of the drug includes powder, granules, tablets, pills, capsules, and injections.

[0012] Compared with the prior art, the present invention has the following beneficial effects:

[0013] The compound IMD-0354, screened from the compound library in this invention, has a significant inhibitory effect on multidrug-resistant Mycobacterium tuberculosis Mtb 28 and Mtb 1731 as well as Mycobacterium tuberculosis Mtb H37Rv, and has broad application prospects in the preparation of drugs for the treatment of tuberculosis.

[0014] This invention utilizes a high-throughput inhibitor screening model at the Mtb bacterial level. Through a high-throughput screening system, compound IMD-0354, which inhibits Mtb H37Rv, multidrug-resistant strains Mtb 28, and Mtb 1731, was screened from the compound library L4000 (containing 7285 compounds). This screening method is simple and easy to implement. Attached Figure Description

[0015] Figure 1 Growth curve of Mtb H37Rv over 10 days.

[0016] Figure 2 A schematic diagram of a partial 96-well plate design for compound screening (positive samples are marked at the marked areas). Detailed Implementation

[0017] This invention provides the application of compound IMD-0354 in the preparation of drugs for treating tuberculosis. The molecular formula of compound IMD-0354 described in this invention is C0354. 15 H8ClF6NO2, with a relative molecular weight of 383.67, is named N-[3,5-bis(trifluoromethyl)phenyl]-5-chloro-2-hydroxybenzamide, and its structural formula is shown in Formula I.

[0018]

[0019] In this invention, the Mycobacterium tuberculosis is MtbH37Rv, as well as multidrug-resistant strains Mtb28 and Mtb1731. The Mtb28 strain of this invention is resistant to isoniazid (INH), rifampin, and ethionamide, but sensitive to streptomycin, ethambutol, amikacin, and ciprofloxacin, and to sodium ammonia salicylate. The Mtb1731 strain is resistant to INH, rifampin, streptomycin, and ethionamide, but sensitive to ethambutol, amikacin, ciprofloxacin, and sodium ammonia salicylate.

[0020] This invention provides the use of a pharmaceutical composition containing IMD-0354 in the preparation of a drug for treating tuberculosis. The pharmaceutical composition of this invention comprises the compound IMD-0354 and at least one pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier of this invention includes, but is not limited to, one or more of wetting agents, diluents, disintegrants, lubricants, fillers, binders, etc. The pharmaceutically acceptable carrier of this invention does not affect the pharmaceutical activity of the compound IMD-0354.

[0021] In this invention, the effective concentration of compound IMD-0354 is 0.2–10 μM, preferably 0.5–8 μM. The effective concentration of compound IMD-0354 in this invention can significantly inhibit Mycobacterium tuberculosis MtbH37Rv and multidrug-resistant strains Mtb28 and Mtb1731.

[0022] In this invention, the drug includes powders, granules, tablets, pills, capsules, or injections. The drug of this invention also includes, but is not limited to, aqueous powder injections, powders, patches, suppositories, emulsions, creams, gels, aerosols, sprays, powder mists, sustained-release agents, or controlled-release agents. The pharmaceutical excipients used in the preparation of the drug in this invention are those conventionally used in various formulations, but not limited to isotonic agents, buffers, flavoring agents, excipients, fillers, binders, disintegrants, and lubricants; they can also be selected to be compatible with the compound, such as emulsifiers, solubilizers, antibacterial agents, analgesics, and antioxidants. These excipients can effectively improve the stability and solubility of the compound in the pharmaceutical formulation or change the release rate and absorption rate of the compound, thereby improving the metabolism of the compound in vivo and enhancing the drug delivery effect of the composition.

[0023] In this invention, unless otherwise specified, all raw material components are commercially available products well known to those skilled in the art.

[0024] The technical solutions of this invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0025] Example 1: Establishment and Evaluation of a High-Throughput Screening Model

[0026] 1. Compound sample

[0027] The compound library (L4000, containing 7285 compounds) was purchased from Shanghai Taosu Biotechnology Co., Ltd., with an initial concentration of 10 mM, and diluted to a final concentration of 1 mM using DMSO. Isoniazid (INH, purchased from Sigma) was dissolved in DMSO to prepare a 1 mM solution, stored at 4°C for later use.

[0028] 2. Seed culture

[0029] Using an inoculation loop, Mtb H37Rv, clinical multidrug-resistant strain Mtb28, or multidrug-resistant strain Mtb 1731 stored at -80℃ were streaked onto 7H10 solid medium (containing 10% OADC, purchased from BD) and incubated at 37℃ until single colonies grew. Single colonies were picked and inoculated into 5 mL of 7H9 medium (containing 10% OADC and 0.05% Tween 80, purchased from BD) and incubated statically for 10–14 days.

[0030] The Mtb H37Rv (ATCC27294), clinically multidrug-resistant strains Mtb 28 and Mtb 1731 were preserved by the Bacterial Immunology Laboratory of the Beijing Tuberculosis and Thoracic Tumor Research Institute at Beijing Chest Hospital, Capital Medical University.

[0031] 3. Monitoring of Mtb growth

[0032] Add 1 μL of 1 mM INH solution (positive control, p) to column 1 of a 96-well plate, 1 μL of dimethyl sulfoxide (DMSO) solvent (negative control, n) to column 2, 99 μL of 7H9 culture medium inoculated with logarithmic-phase Mtb H37Rv (final concentration A600 ≈ 0.1, obtained in step 2) to columns 1 and 2, 100 μL of 7H9 culture medium inoculated with logarithmic-phase Mtb H37Rv (blank control, b) to column 3, and 100 μL of 7H9 culture medium without bacterial culture (containing 10% OADC and 0.05% Tween 80, purchased from BD) to column 4. Seal the 96-well plate with plastic wrap and incubate at 37°C. Measure the A value of the strain within 10 days using a microplate reader (PerkinElmer Enspire 2300 Multiabel Reader). 600 To observe the changes in light absorption, a growth curve was created.

[0033] When establishing a high-throughput inhibitor screening model at the cellular level using the slow-growing Mtb, it is necessary to save time as much as possible while taking into account the sensitivity and reproducibility of the model. The growth of Mtb H37Rv was monitored over 10 days. Figure 1 As shown in the figure, the growth of Mtb H37Rv in 7H9 medium containing 1% (v / v) DMSO is consistent with that in normal medium, indicating that DMSO at this concentration does not affect the growth of the strain. Therefore, based on compound screening experience, the initial screening concentration of the compound sample was set at 10 μM (at this concentration, the initial screening positive rate is approximately 1%), and the concentration of the positive control sample INH was also set at 10 μM.

[0034] 4. Establishment of a high-throughput inhibitor screening model

[0035] In step 2, the A content of MtbH37Rv in 7H9 liquid medium at days 6, 8, and 10 in 96-well plates was measured for each group. 600 Light absorption value, statistical analysis of group A 600 The absorbance value was assessed using the Z' factorial evaluation system to evaluate the reliability of the high-throughput screening model. The calculation formula is as follows:

[0036]

[0037] SD represents A 600 The standard deviation of absorbance, U represents A 600 The average value of the absorbance.

[0038] Based on the above formula and the measured A 600The absorbance values ​​were calculated, and the corresponding Z' values ​​for days 6, 8, and 10 were 0.382, 0.723, and 0.819, respectively. The criterion is that only systems with Z' > 0.5 can meet the requirements for high-throughput screening. Therefore, observing the experimental results after incubating the screening system at 37℃ for 8 days ensures the reliability and sensitivity of the results.

[0039] Example 2: High-throughput screening of positive compounds that inhibit Mycobacterium tuberculosis

[0040] 1. Preliminary screening in the compound library

[0041] The screening model established in step 4 was used to screen positive compounds in 96-well plates. The initial and final concentration of the compound samples was 10 μM. Positive compound samples that completely inhibited the growth of MtbH37Rv were screened from the compound library L4000 (containing 7285 compounds). The screening system is shown in Table 1.

[0042] Table 1. High-throughput inhibitor screening system for MtbH37Rv

[0043]

[0044] According to the table above, 99 μL of 7H9 culture medium containing log-phase MtbH37Rv was seeded into 96-well plates (final concentration A). 600 ≈0.1), add 1 μL of compound sample (initial screening final concentration 10 μM), and simultaneously set up a negative control group (add 1 μL LDMSO) and a positive control group (add 1 μL 1 mM isoniazid). Seal the 96-well plate with plastic wrap and incubate at 37℃ for 8 days, and determine system A. 600 Absorbance. Compounds that completely inhibited MtbH37Rv growth at a concentration of 10 μM (visually clear and turbid) were defined as positive compounds. A total of 411 positive compounds were obtained. The design of some compounds in the 96-well plate during the screening process is as follows: Figure 2 .

[0045] 2. Determination of the antituberculosis activity of positive compounds

[0046] The minimum inhibitory concentration (MIC) of the positive compound sample obtained in step 1 against MtbH37Rv and clinically isolated MDR strains Mtb28 and Mtb1731 was determined in a 96-well plate using a two-fold serial dilution method.

[0047] The specific steps of the two-fold serial dilution method include: the highest final concentration of the positive compound is 10 μM, and it is successively diluted to 5 μM, 2.5 μM, 1.25 μM, 0.625 μM, 0.3125 μM, 0.156 μM, and 0.078 μM, with the lowest concentration being 0.078 μM. Each test is performed in triplicate. DMSO is used as a negative control, and INH is used as a positive control. 200 μL of 7H9 medium is added around the perimeter of a 96-well plate to prevent drying. Positive compound 1 is added to wells B2-D2, and positive compound 2 is added to wells E2-G2. The total amount of positive compound added is 2 μL. Then, 7H9 culture medium containing logarithmic MtbH37Rv, Mtb28, or Mtb1731 is added to wells B2-G2, with a final concentration of A. 600 ≈0.1)198 μL was added to bring the final concentration of the positive compound to 10 μM. 100 μL of bacterial culture medium (7H9 culture medium inoculated with logarithmic-phase MtbH37Rv, Mtb28, or Mtb1731) was added to the remaining 54 wells. Finally, 100 μL of the culture medium was transferred from wells B2 to G2 using a pipette to wells B3 to G3, mixed thoroughly, and then transferred to wells B4 to G4, and so on, until the final concentration was 0.078 μM. Other positive compounds, DMSO, and INH were determined in the same manner. The wells were sealed with sealing film and incubated at 37°C for 8 days, and the results were observed. The concentration of the positive compound that completely inhibited the growth of MtbH37Rv, Mtb28, and Mtb1731 (visible as clear to the naked eye) was defined as the minimum inhibitory concentration, or MIC value.

[0048] Based on the above results, it can be seen that among the 411 positive compound samples, compound IMD-0354 (CAS: 978-62-1, structural formula as shown in Formula I) showed a strong inhibitory effect on the MTB standard strain MtbH37Rv and the clinically isolated MDR strains Mtb28 and Mtb1731. The results are shown in Table 2.

[0049] Table 2. Anti-tuberculosis effects of the compounds

[0050]

[0051] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Use of the compound IMD-0354 for the manufacture of a medicament for the treatment of tuberculosis, characterized in that, The bacteria involved in the tuberculosis include one or more of Mycobacterium tuberculosis Mtb H37Rv, multi-drug resistant Mycobacterium tuberculosis Mtb 28, multi-drug resistant Mycobacterium tuberculosis Mtb 1731.

2. Use of a pharmaceutical composition containing compound IMD-0354 in the preparation of a medicament for treating tuberculosis.

3. The use according to claim 2, wherein The pharmaceutical composition further comprises at least one pharmaceutically acceptable carrier.

4. Use according to claim 1 or 2, characterized in that, The effective concentration of the compound IMD-0354 is 0.2-10 μM.

5. The use according to claim 1 or 2, characterized in that, The dosage form of the medicament includes powder, granule, tablet, pill, capsule, injection.

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