Use of bacterial division protein ftsz inhibitor db1 in preparation of drug against drug-resistant bacteria

Abstract

This invention discloses the application of the bacterial fission protein FtsZ inhibitor DB1 in the preparation of drugs against drug-resistant bacteria, belonging to the field of drug development technology. The inhibitor provided by this invention is compound DB1, named 3-(2,8-dibromo-10,11-dihydro-5H-dibenzo[b,f]azapheno-5-yl)-N,N-dimethylpropylamine hydrochloride, with the molecular formula C2. 19 H 24 Br2ClN2. This compound, DB1, exhibits significant inhibitory effects against various drug-resistant bacteria, particularly vancomycin-resistant enterococci and methicillin-resistant Staphylococcus aureus (MRSA), and can significantly improve the susceptibility of methicillin to MRSA. The discovery of compound DB1 provides new ideas and technical support for the preparation of drugs against drug-resistant bacteria.

Description

Technical Field

[0001] This invention relates to the field of drug development technology, and in particular to the application of the bacterial fission protein FtsZ inhibitor DB1 in the preparation of drugs against drug-resistant bacteria. Background Technology

[0002] Bacterial infections are primarily treated with antibiotics in clinical practice. However, with the widespread use of antibiotics, bacterial resistance has become an increasingly serious problem. For example, the emergence of methicillin-resistant Staphylococcus aureus (MRSA), multidrug-resistant Staphylococcus aureus (MDRSA), vancomycin-resistant enterococci (VRE), extended-spectrum β-lactamases (ESBLs), and NDM-1 (New Delhi metallo-β-lactamase 1) positive strains has caused significant challenges to clinical drug use, with some resistant strains even facing a situation where no drugs are available. Another worrying issue is the relatively slow development of new antibacterial drugs. The World Health Organization's 2020 report, "Clinical and Preclinical Development of Antimicrobial Agents," points out that almost all new antibiotics introduced to the market in recent decades are variants of antibiotic classes discovered in the 1980s, and bacterial resistance to these new agents is expected to emerge rapidly. Of the 43 antibiotics currently in clinical development, none can adequately address the drug resistance problem of the world's most dangerous bacteria, making the development of new drugs and strategies for combating bacterial infections an urgent priority.

[0003] Recent studies have shown that FtsZ—filamenting temperature-sensitive protein Z—is an indispensable protein in bacterial division. It plays a crucial role in bacterial division and proliferation, is present in almost all bacteria, and is morphologically highly conserved and resistant to variation. When its biological activity is interfered with, bacterial growth and reproduction are inhibited. Therefore, research on antimicrobial molecules targeting FtsZ has important reference value for the development of next-generation antimicrobial drugs. Currently, there are no reports of 3-(2,8-dibromo-10,11-dihydro-5H-dibenzo[b,f]azapyro-5-yl)-N,N-dimethylpropylamine hydrochloride targeting FtsZ protein to inhibit bacterial division and thus exert antimicrobial activity. Summary of the Invention

[0004] The purpose of this invention is to provide the application of the bacterial fission protein FtsZ inhibitor DB1 in the preparation of drugs against drug-resistant bacteria, thereby solving the problems existing in the prior art. This invention provides an FtsZ inhibitor DB1, which has a significant inhibitory effect on a variety of drug-resistant bacteria, and has important practical significance for the development of drugs against drug-resistant bacteria.

[0005] To achieve the above objectives, the present invention provides the following solution:

[0006] In a first aspect, the present invention provides the use of compound DB1 in the preparation of drugs against drug-resistant bacteria, wherein compound DB1 is named 3-(2,8-dibromo-10,11-dihydro-5H-dibenzo[b,f]azaphen-5-yl)-N,N-dimethylpropylamine hydrochloride, and has the molecular formula C 19 H 24 Br2ClN2, the structural formula is shown in formula (I):

[0007]

[0008] Formula (I).

[0009] Preferably, the drug-resistant bacteria are methicillin-resistant Staphylococcus aureus, multidrug-resistant Staphylococcus aureus, vancomycin-resistant Staphylococcus aureus, methicillin-resistant Staphylococcus epidermidis, multidrug-resistant Escherichia coli, or vancomycin-resistant Enterococcus faecalis.

[0010] Preferably, the anti-drug-resistant bacteria drug further comprises a pharmaceutically acceptable carrier or excipient.

[0011] Preferably, the dosage form of the anti-drug-resistant bacteria drug is selected from any one or more of the following: injection, tablet, pill, capsule, suspension or emulsion.

[0012] Secondly, the present invention also provides the use of compound DB1 in the preparation of anti-Bacillus subtilis drugs, wherein compound DB1 is named 3-(2,8-dibromo-10,11-dihydro-5H-dibenzo[b,f]azaphen-5-yl)-N,N-dimethylpropylamine hydrochloride, and has the molecular formula C 19 H 24 Br2ClN2, the structural formula is shown in formula (I):

[0013]

[0014] Formula (I).

[0015] Preferably, the anti-Bacillus subtilis drug further comprises a pharmaceutically acceptable carrier or excipient.

[0016] Preferably, the excipient is a pharmaceutical excipient, including any one or more of solvents, disintegrants, flavoring agents, preservatives, colorants, or binders.

[0017] Preferably, the dosage form of the anti-Bacillus subtilis drug is selected from any one or more of the following: injection, tablet, pill, capsule, suspension or emulsion.

[0018] Thirdly, this invention also provides the use of compound DB1 in the preparation of drugs that enhance the susceptibility of methicillin to methicillin-resistant Staphylococcus aureus. The compound DB1 is named 3-(2,8-dibromo-10,11-dihydro-5H-dibenzo[b,f]azaphen-5-yl)-N,N-dimethylpropylamine hydrochloride, and its molecular formula is C1. 19 H 24 Br2ClN2, the structural formula is shown in formula (I):

[0019]

[0020] Formula (I).

[0021] Preferably, the drug for improving the susceptibility of methicillin to methicillin-resistant Staphylococcus aureus further comprises a pharmaceutically acceptable carrier or excipient.

[0022] The present invention discloses the following technical effects:

[0023] (1) The FtsZ protein inhibitor DB1 of the present invention has a significant inhibitory effect on a variety of drug-resistant bacteria, especially on vancomycin-resistant enterococci and methicillin-resistant Staphylococcus aureus.

[0024] (2) The FtsZ protein inhibitor DB1 of the present invention can significantly improve the susceptibility of methicillin to methicillin-resistant Staphylococcus aureus. Attached Figure Description

[0025] 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.

[0026] Figure 1 The effect of blank control (A) and FtsZ protein inhibitor DB1 (B) on the growth morphology of Bacillus subtilis in Example 3 (40×).

[0027] Figure 2 This illustrates the effect of the FtsZ protein inhibitor DB1 on the dynamic polymerization of FtsZ protein in Example 4. 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 obvious 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] The synthetic route for the inhibitor DB1 3-(2,8-dibromo-10,11-dihydro-5H-dibenzo[b,f]azaphen-5-yl)-N,N-dimethylpropylamine hydrochloride can be found in the following literature:

[0034] A new method for bromination of carbazoles, β-carbolines andiminodibenzyls by use of N-bromosuccinimide and silica gel (Keith Smith, D.Martin James, Anil G. Mistry, Martin R. Bye, D.John Faulkner, Tetrahedron (IF 2.2) Pub Date: 4 September 1992, DOI: 10.1016 / s0040-4020(01)90362-x);

[0035] Fluorescent additive for estimation of compatibility of polyesterblend by imipramine-containing polymer (Hideki Hayashi, Masanobu Maeda, Taiki Naruo, Yukio Onouchi, Masaki Harada, Yuzo Ishigaki, International Journal of Polymer Analysis and Characterization (IF 1.6) Pub Date: 2017-08-25, DOI: 10.1080 / 1023666x.2017.1369272).

[0036] Example 1 Antibacterial activity test

[0037] In this embodiment, the minimum inhibitory concentration (MIC) (μg / mL) of the FtsZ protein inhibitor DB1 was determined using the microbroth dilution method. The minimum inhibitory concentration (MIC) of the test compound was determined according to the microbroth dilution procedure described in the Clinical and Laboratory Standards Institute (CLSI) guidelines.

[0038] The implementation steps are as follows:

[0039] (1) Preparation of culture medium and antibacterial drug stock solution: The prepared MH broth culture medium was autoclaved at 121℃ for 30 min and then cooled; the test compound was dissolved in DMSO to prepare a stock solution of 38.4 mg / mL and filtered through a 0.22 μm filter membrane for sterilization.

[0040] (2) Activation and expansion culture of the test bacteria: The frozen test bacteria were spread on TSB agar plates and activated at 37°C for 24 h; the single colonies obtained from the activation culture were inoculated into 5 mL of TSB medium and cultured overnight at 37°C with shaking.

[0041] (3) Seed plate preparation: The OD value was measured at a wavelength of 600 nm using an ELISA reader to calculate the bacterial concentration. The bacterial solution was then diluted with MH broth to a concentration of approximately 5 × 10⁻⁶. 5 Add CFU / mL blank culture medium to the outermost well of a 96-well plate, and add 100 μL of bacterial culture to each of the remaining wells. Set aside for use.

[0042] (4) Drug addition: The outermost blank culture medium of the 96-well plate is used as a blank control. 96 μL of bacterial culture is added to each well of the second column, followed by 4 μL of the compound stock solution prepared in step (1). After thorough mixing, 100 μL of the mixed solution is added to the third column. After thorough mixing again, 100 μL of the mixed solution is added to the fourth column. This process is repeated until the eleventh column, at which point 100 μL of the mixed solution is discarded. The drug is then added using the 2:1 dilution method. The final drug concentrations in each well of columns 2 to 11 are 64 μg / mL, 32 μg / mL, 16 μg / mL, 8 μg / mL, 4 μg / mL, 2 μg / mL, 1 μg / mL, 0.5 μg / mL, 0.25 μg / mL, and 0.125 μg / mL, respectively. A DMSO solvent negative control is set up, and ampicillin (AMP), vancomycin (VAN), methicillin (MET), and rifampin (RIF) are used as positive controls.

[0043] (5) Incubation: Place the 96-well plate in a 37℃ constant temperature incubator for 24 hours.

[0044] (6) Result judgment: The blank control wells showed no bacterial growth and were obviously clear. The lowest drug concentration that was completely clear and transparent in the wells was taken as the MIC value, that is, the clarity that could be seen with the naked eye was the inhibition of 90% bacterial growth.

[0045] Test bacteria: The bacteria used in the microbroth dilution method included *Staphylococcus aureus* ATCC 29213, methicillin-resistant *Staphylococcus aureus* ATCC BAA-41, methicillin-resistant *Staphylococcus aureus* ATCC 43300, multidrug-resistant *Staphylococcus aureus* ATCC BAA-44, vancomycin-resistant *Staphylococcus aureus* VISA, *Staphylococcus epidermidis* ATCC 12228, methicillin-resistant *Staphylococcus epidermidis* MRSE, *Bacillus subtilis* 168, *Escherichia coli* ATCC 25922, multidrug-resistant *Escherichia coli* BAA-2469, *Enterococcus faecium* ATCC 49624, and vancomycin-resistant *Enterococcus faecium* ATCC 49624. 700221.

[0046] The test results are shown in Table 1. The test compounds can inhibit the growth of a variety of drug-resistant bacteria in vitro and can be used to prepare antibiotics against drug-resistant bacteria.

[0047] Table 1. MIC values ​​(μg / mL) of the test compounds against the test bacteria.

[0048]

[0049] Example 2: FtsZ protein inhibitor DB1 enhances the susceptibility of methicillin to methicillin-resistant Staphylococcus aureus.

[0050] The experimental steps are as follows:

[0051] (1) The combination of the compound and methicillin to combat methicillin-resistant Staphylococcus aureus was tested using the checkerboard method, based on the determination of the MIC of the compound and methicillin.

[0052] (2) Methicillin was prepared into a stock solution of 50 mg / mL using DMSO, and then filtered and sterilized for later use.

[0053] (3) Recovery and expanded culture: Refer to the minimum inhibitory concentration (MIC) experiment.

[0054] (4) The distribution of drug concentrations is shown in Table 2 (the numbers outside the parentheses are the MIC multiples of the compounds, and the numbers inside the parentheses are the MIC multiples of methicillin).

[0055] Table 2. Dosage concentration distribution

[0056]

[0057] (5) Based on the above chessboard layout, first dilute the bacterial solution to 10. 6 CFU / mL, prepare bacterial suspensions containing six graded concentrations of compounds using diluted bacterial suspension from the dosing tank, and inoculate 100 μL into each well as shown in the table. Then add 4 μL of methicillin stock solution to the first column, performing a 2:1 dilution according to the minimum inhibitory concentration (MIC). After completing the inoculation, incubate the 96-well plate at 37°C for 24 h.

[0058] (6) Result Interpretation: The blank control wells showed no bacterial growth and were clearly clear. The lowest drug concentration at which the wells became completely clear and transparent was taken as the MIC value for the combined use of the FtsZ protein inhibitor DB1 and methicillin, i.e., visible clarity indicated 90% inhibition of bacterial growth. Fractional inhibitory concentration (FIC) was calculated using the formula: FIC = MIC in combination / MIC alone; Fractional inhibitory concentration index (FICI) = FIC DB1 +FIC MET .

[0059] Table 3 Synergistic effect of FtsZ protein inhibitor DB1 and methicillin against Staphylococcus aureus

[0060]

[0061] The results are shown in Table 3. The graded inhibition concentration index was 0.3125, indicating that DB1 can significantly improve the sensitivity of methicillin to methicillin-resistant Staphylococcus aureus, and the two have a synergistic effect.

[0062] Example 3: Bacterial Morphology Study

[0063] In this embodiment, the growth morphology of Bacillus subtilis under the action of the FtsZ protein inhibitor DB1 was observed using an Olympus IX71 inverted fluorescence microscope. The test bacterium was B. subtilis 168; since bacterial growth was effectively inhibited at the MIC concentration of the compound, an experimental concentration of 0.5 × MIC was used to obtain a bacterial suspension of a certain concentration for observation.

[0064] The experimental steps are as follows:

[0065] (1) Dilute the expanded bacterial culture with sterilized MH broth medium to a concentration of approximately 5 × 10⁻⁶. 5 CFU / mL.

[0066] (2) Add the diluted bacterial solution and compound DB1 stock solution to a sterilized 5mL centrifuge tube, with a total volume of 1mL. The final concentration of the compound is 0.5×MIC value for inhibiting B. subtilis 168, i.e., 2μg / mL. The solvent control group is the bacterial solution without drug treatment.

[0067] (3) Place the centrifuge tube from step (2) in a constant temperature shaker and incubate at 200 rpm and 37°C for 4-5 hours until visible bacterial growth and turbidity are observed. Observe the bacterial morphology under a 40× lens.

[0068] Experimental results are as follows Figure 1 As shown, compared with the solvent control group ( Figure 1 Compared to group A, the drug-added group ( Figure 1 The bacterial morphology of B) was significantly elongated, which initially suggests that the compound can act on the target FtsZ protein, causing the bacteria to elongate due to their inability to divide normally, and thus die.

[0069] Example 4: FtsZ protein inhibitor DB1 interferes with the dynamic polymerization of FtsZ protein.

[0070] (1) The in vitro polymerization of FtsZ protein was determined using a multifunctional microplate reader with a wavelength of 340 nm and an experimental temperature of 25 °C.

[0071] (2) Add 50 mM MOPS buffer (pH 6.5) and FtsZ protein to a 96-well plate with a final concentration of 6 μM. Add a series of concentrations of the compound (2 μg / mL, 4 μg / mL, 8 μg / mL) and incubate for 10 min. Use 1% DMSO as a negative control. Set up control wells without FtsZ protein to remove background.

[0072] (3) After adding 50mM KCl, 2mM MgCl2 and 1mM CaCl2, continue scanning for 5min.

[0073] (4) Add 1 mM GTP solution, continue scanning for 2000 s, and record A. 340 The nm scan data were analyzed and processed, and all scan data underwent appropriate background data subtraction.

[0074] Experimental results are as follows Figure 2 As shown, DB1 can promote the dynamic polymerization of FtsZ protein in a concentration-dependent manner.

[0075] 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 compound DB1 in the preparation of drugs against drug-resistant bacteria, characterized in that, The compound DB1 is named 3-(2,8-dibromo-10,11-dihydro-5H-dibenzo[b,f]azaphen-5-yl)-N,N-dimethylpropylamine hydrochloride, and its molecular formula is C1. 19 H 24 Br2ClN2, the structural formula is shown in formula (I): Formula (I); The drug-resistant bacteria are methicillin-resistant Staphylococcus aureus, multidrug-resistant Staphylococcus aureus, vancomycin-resistant Staphylococcus aureus, methicillin-resistant Staphylococcus epidermidis, multidrug-resistant Escherichia coli, or vancomycin-resistant Enterococcus faecalis.

2. The application according to claim 1, characterized in that, The drug for treating drug-resistant bacteria also contains pharmaceutically acceptable carriers or excipients.

3. The application according to claim 1, characterized in that, The dosage form of the anti-drug resistant bacteria drug is selected from any one or more of the following: injection, tablet, pill, capsule, suspension or emulsion.

4. The application of compound DB1 in the preparation of drugs against Bacillus subtilis, characterized in that, The compound DB1 is named 3-(2,8-dibromo-10,11-dihydro-5H-dibenzo[b,f]azaphen-5-yl)-N,N-dimethylpropylamine hydrochloride, and its molecular formula is C1. 19 H 24 Br2ClN2, the structural formula is shown in formula (I): Formula (I).

5. The application according to claim 4, characterized in that, The anti-Bacillus subtilis drug also contains pharmaceutically acceptable carriers or excipients.

6. The application according to claim 4, characterized in that, The excipients are pharmaceutical excipients, including any one or more of solvents, disintegrants, flavoring agents, preservatives, colorants, or binders.

7. The application according to claim 4, characterized in that, The dosage form of the anti-Bacillus subtilis drug is selected from any one or more of the following: injection, tablet, pill, capsule, suspension or emulsion.

8. The use of compound DB1 in the preparation of drugs that enhance the susceptibility of methicillin to methicillin-resistant Staphylococcus aureus, characterized in that, The compound DB1 is named 3-(2,8-dibromo-10,11-dihydro-5H-dibenzo[b,f]azaphen-5-yl)-N,N-dimethylpropylamine hydrochloride, and its molecular formula is C1. 19 H 24 Br2ClN2, the structural formula is shown in formula (I): Formula (I).

9. The application according to claim 8, characterized in that, The drug for improving the susceptibility of methicillin to methicillin-resistant Staphylococcus aureus also contains a pharmaceutically acceptable carrier or excipient.

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