Discovery of new dibenzodiazepine derivatives as antibacterials against intracellular bacteria

Abstract

The emergence and spread of multidrug-resistant bacteria underscore the critical need for novel antibacterial interventions. In our screening of 12 synthesized thienobenzodiazepines, pyridobenzodiazepines, and dibenzodiazepines, we successfully identified a small molecule compound SW33. Notably, SW33 demonstrated potent inhibitory activity against intracellular multidrug-resistant and fluoroquinolone-resistant strains of S. typhimurium in both macrophages and epithelial cells. Furthermore, SW33 was also effective against intramacrophagic Salmonella typhi, Yersinia enterocolitica, and Listeria monocytogenes. These significant findings suggest that SW33 possesses broad-spectrum activity against intracellular bacteria.

Scheme 1. The synthetic scheme of the thienobenzodiazepine derivatives (SW38, 39, 41, and 42). The yield of compounds in each step is represented by percent values.

Scheme 2. The synthetic scheme of the pyridobenzodiazepine derivatives (SW21, 27, 29, and 30). The yield of compounds in each step is represented by percent values.

Scheme 3. The synthetic scheme of the dibenzodiazepine derivatives (SW33, 35, 44, and 45). The yield of compounds in each step is represented by percent values.

Fig. 1. The building blocks and structure activity relationship (SAR) for SW compounds against Salmonella. (A) The lead compound, SW14 (structurally originated from loxapine), could be deconstructed into two components: the tricyclic core (circled in yellow) and lateral chain (circled in blue). The building blocks used for the tricyclic core and lateral chain are highlighted in yellow and blue rectangles, respectively. (B, left) In the SAR analysis of tricyclic cores, diazepine tricyclics demonstrated lower toxicity compared to oxazepane tricyclics. (B, right) In the SAR analysis of the lateral chain, the alkyl–aliphatic ring could be dissected into the alkyl spacer (AS) and aliphatic terminal (AT). For the AS, the 3-carbon spacer showed higher antibacterial potential than the 2-carbon spacer. For the AT, a single nitrogen atom within the terminal group exhibited greater potency in antibacterial activity compared to two nitrogen atoms.

Fig. 2. SW33 does not suppress Salmonella growth in broth and does not bind to recombinant D2Rs. (A) RAW264.7 cells were infected with S. typhimurium and then treated with various concentrations of SW33 in the presence of 20 mg L⁻¹ gentamicin for 24 h. The viability of intracellular bacteria was assayed using HCA or a CFU assay. The data are expressed as percentages relative to the mock (DMSO)-treated control and are presented as the means ± SDs (n = 3 replicates per group). (B) The viability of RAW264.7 cells treated with increasing concentrations of SW33 was assessed using HCA or an MTT cell viability assay. The data are expressed as percentages relative to the mock (DMSO)-treated control and are presented as the means ± SDs (n = 3 replicates per group). ns, not significant; **, P < 0.01; ****, P < 0.0001 for the comparison of the cytotoxicity of SW33 at each concentration with that of the mock control. (C) The growth of S. typhimurium in CAMH broth (CAMHB) or cell culture medium (CCM) containing 0, 0.5, or 64 μM SW33 was monitored by measuring the OD600 of each bacterial culture at designated time points over a 24 h period. The data are presented as the means ± SDs (n = 3 replicates per group). (D) The binding of isotope-labelled spiperone to recombinant dopamine D2 receptors in the presence of different concentrations of loxapine or SW33 was assessed. The data are expressed as percentages of inhibition relative to the mock (DMSO)-treated control and are presented as the means ± SDs (n = 3 replicates per group).

Fig. 3. SW33 exhibits a stronger inhibitory effect on S. typhimurium infection in phagocytic cells than in epithelial cells. (A–F) Salmonella-infected J774.1 cells (A), macrophage-like THP-1 cells (B), monocytic THP-1 cells (C), HT-29 cells (D), HCT116 cells (E), and INT-407 cells (F) were treated with different concentrations of SW33 in combination with 20 mg L⁻¹ gentamicin for 24 h. The survival of intracellular S. typhimurium (icSTM) was assessed using a CFU assay. Additionally, the viability of each cell line after exposure to SW33 combined with 20 mg L⁻¹ gentamicin for 24 h was measured using an MTT cell viability assay. The data are expressed as percentages relative to the mock (DMSO)-treated control and are presented as the means ± SDs (n = 3 replicates per group). ns, not significant; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 for the comparison of the anti-intracellular Salmonella activity and cytotoxicity of SW33 in individual cell lines with those in RAW264.7 cells.

Fig. 4. SW33 inhibits infection with MDR S. typhimurium and other pathogenic bacteria in macrophages. (A) RAW264.7 cells infected with the MDR isolate CGC18 or the ciprofloxacin-resistant isolate NL10 of S. typhimurium were treated with various concentrations of SW33 in combination with 20 mg L⁻¹ gentamicin for 24 h. Intracellular bacterial survival was determined using a CFU assay, and the results are expressed as percentages relative to the mock (DMSO)-treated control. The data are presented as the means ± SDs (n = 3 replicates per group). **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. (B–D) RAW264.7 cells infected with S. typhi (B), Y. enterocolitica (C), or L. monocytogenes (D) were treated with SW33 at concentrations ranging from 0 to 1 μM for 24 h. Subsequently, the number of viable intracellular bacteria was determined using a CFU assay. The data are presented as the means ± SDs (n = 3 replicates per group). ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001.