Design, synthesis, and biological evaluation of novel arylpyrrole derivatives with antibacterial activity /

Abstract

The increasing prevalence of antimicrobial resistance poses an enormous burden and threat to the global public health. Infections caused by multidrugresistant bacteria are associated with serious morbidity and mortality, with at least 1.27 million deaths recorded per year. Conventional antibiotics are becoming ineffective against various Gram-negative and Gram-positive bacterial strains, especially the gram-positive methicillin-resistant S. aureus (MRSA). Since 1990 and up till today, MRSA infections presented a stressing global problem, and the development of new antimicrobial agents to combat MRSA infections is of utmost importance. According to the WHO, it is necessary to introduce novel chemical scaffolds, target novel pathways, and demonstrate wide-spectrum activity versus multidrug-resistant strains. Our research objectives were to design, synthesize, and biologically evaluate new antibacterial leads with broad antibacterial activity and potential anti-MRSA activity. This thesis consists of two parts. Part One focuses on the design, synthesis, and biological evaluation of novel arylpyrrole derivatives with antibacterial activity. Upon investigating literature for active chemical moieties that exhibit potent antibacterial activity, we came across a highly active phenylthiazole scaffold that exhibited notable antibacterial activity against gram-positive bacteria, including 18 MRSA strains. Several rational modifications of the phenylthaizole lead compound were proposed, and three series of novel N-arylpyrrole derivatives were suggested (Hydrazinecarboximidamide, carbothioamide, and carboxamide). Molecular modelling studies were performed to preliminary evaluate our newly designed compounds using consecutive computer-aided drug design protocols. The 3D QSAR pharmacophore generation protocol was utilized to generate a valid predictive pharmacophore model; then, the designed compounds were mapped via the ligand pharmacophore mapping protocol to predict their activity. Furthermore, the designed compounds were docked into the binding site of their proposed target, Undecaprenyl diphosphate phosphatase (UPPP), using the CDOCKER protocol in Discovery Studio 4.1 software, which allowed studying their binding modes and affinities. The designed compounds were synthesized, purified, and structurally confirmed by different analytical and spectral techniques. The study involved the synthesis of the following unavailable reported intermediate: 1) 2,5-Dimethyl-1-phenyl-1H-pyrrole (IIIa). 2) 1-(4-Chlorophenyl)-2,5-dimethyl-1H-pyrrole (IIIb). 3) 1-([1,1'-Biphenyl]-4-yl)-2,5-dimethyl-1H-pyrrole (IIIc). 4) 2,5-Dimethyl-1-phenyl-1H-pyrrole-3-carbaldehyde (IVa). 5) 1-(4-Chlorophenyl)-2,5-dimethyl-1H-pyrrole-3-carbaldehyde (IVb). 6) 1-(2,5-Dimethyl-1-phenyl-1H-pyrrol-3-yl)ethanone (IVd). 7) 1-[1-(4-Chlorophenyl)-2,5-dimethyl-1H-pyrrol-3-yl]ethanone (IVe). Also, it comprised the synthesis of the following new intermediates: 1) 1-([1,1'-Biphenyl]-4-yl)-2,5-dimethyl-1H-pyrrole-3-carbaldehyde (IVc). Furthermore, the study involved the synthesis and characterization of the following new final compounds: 1) 2-((2,5-Dimethyl-1-phenyl-1H-pyrrol-3yl)methylene) hydrazinecarboximidamide (Va). 2) 2-((1-(4-Chlorophenyl)-2,5-dimethyl-1H-pyrrol-3yl)methylene) hydrazinecarboximidamide (Vb). 3) 2-((1-([1,1'-Biphenyl]-4-yl)-2,5-dimethyl-1H-pyrrol-3-yl)methylene) hydrazinecarboximidamide (Vc). 4) 2-(1-(2,5-Dimethyl-1-phenyl-1H-pyrrol-3-yl)ethylidene) hydrazinecarboximidamide (Vd). XIX 5) 2-(1-(1-(4-Chlorophenyl)-2,5-dimethyl-1H-pyrrol-3-yl)ethylidene) hydrazinecarboximidamide (Ve). 6) 2-((1-([1,1'-Biphenyl]-4-yl)-2,5-dimethyl-1H-pyrrol-3-yl)methylene) hydrazinecarbothioamide (VIc). 7) 2-(1-(2,5-Dimethyl-1-phenyl-1H-pyrrol-3-yl)ethylidene) hydrazinecarbothioamide (VId). 8) 2-(1-(1-(4-Chlorophenyl)-2,5-dimethyl-1H-pyrrol-3-yl)ethylidene) hydrazinecarbothioamide (VIe). 9) 2-((1-(4-Chlorophenyl)-2,5-dimethyl-1H-pyrrol-3-yl)methylene) hydrazinecarboxamide (VIIb). 10) 2-((1-([1,1'-Biphenyl]-4-yl)-2,5-dimethyl-1H-pyrrol-3-yl)methylene) hydrazinecarboxamide (VIIc). 11) 2-(1-(2,5-Dimethyl-1-phenyl-1H-pyrrol-3-yl)ethylidene) hydrazinecarboxamide (VIId). 12) 2-(1-(1-(4-Chlorophenyl)-2,5-dimethyl-1H-pyrrol-3-yl)ethylidene) hydrazinecarboxamide (VIIe). Also, it comprised the synthesis of the following reported final compounds: 1) 2-((2,5-Dimethyl-1-phenyl-1H-pyrrol3yl)methylene) hydrazinecarbothioamide (VIa). 2) 2-((1-(4-Chlorophenyl)-2,5-dimethyl-1H-pyrrol-3- yl)methylene)hydrazinecarbothioamide (VIb). 3) 2-((2,5-Dimethyl-1-phenyl-1H-pyrrol-3yl)methylene)hydrazinecarboxamide (VIIa). All the synthesized compounds were tested for their biological activity by determining their minimum inhibitory concentrations (MIC) against a panel of susceptible and drug-resistant gram-positive and gram-negative bacterial strains using the broth microdilution method. The gram-positive panel includes the methicillin-susceptible S. aureus (ATCC 29213) and clinical isolates of methicillinresistant S. aureus (MRSA). The gram-negative panel includes the enteric bacterial strains E. coli (ATCC 25922) and K. pneumoniae (ATCC 700603) and the nonenteric bacterial strains P. aeruginosa (ATCC 27853) and clinical isolates of A. baumannii. In addition, all compounds were tested for their antitubercular activity against clinical isolates of Mycobacterium phlei. The antimicrobial evaluation of the new N-aryl-pyrrole derivatives revealed that the hydrazinecarboximidamide series Va-e was the most active. Compounds Vb, Vc, and Ve outperformed our standard reference Levofloxacin inhibitory activity against MRSA (MIC= 8 μg/ml) with a MIC of 4 μg/ml, while Vd had a MIC of 8 μg/ml, which is equivalent to the MIC of Levofloxacin, thus revealing promising activity against MRSA. In addition, compound Vc had MIC values of 4 μg/ml and 8 μg/ml against E. coli and K. pneumoniae, respectively. Compound Vc also showed promising activity against A. baumannii with a MIC of 8 μg/ml, which is equivalent to the MIC of Levofloxacin when tested against the same strain, and the compound also displayed some activity against P. aeruginosa with a MIC of 32 μg/ml. Vc also showed good inhibitory activity against clinical isolates of Mycobacterium phlei with a MIC value of 8 μg/ml. Compound Vb displayed the same promising activity as compound Vc. However, Vb exhibited one-fold higher MIC values against E. coli (8 μg/ml), A. baumannii (16 μg/ml), and Mycobacterium phlei (16 μg/ml) and onefold lower MIC value against P. aeruginosa (16 μg/ml). Compound Ve displayed one-fold higher MIC values than Vb, except for the MIC value against MRSA, which remained the same (4 μg/ml). The hydrazinecarbothioamide, and hydrazinecarboxamide series VIa-e and VIIa-e mostly displayed minimal antimicrobial activity. The cytotoxicity of the four most potent compounds (Vb, Vc, Vd, Ve) was evaluated by MTT assay against normal mammalian cell line VERO (African Green Monkey Kidney cells). Compounds Vc and Vb manifested great selectivity for bacterial over mammalian cells (Vero) and demonstrated an excellent safety profile (non-toxic up to 15μg/ml). Compounds Vd and Ve displayed moderate to weak selectivity against MRSA with mean SI values of 0.27, 2.55, and CC50 values of 2.8 ± 0.35 and 10.33± 0.49 µg/ml, respectively. Part Two of the thesis focuses on the lead generation of UPPS inhibitors targeting MRSA using 3D QSAR pharmacophore Modelling, virtual screening, molecular docking, and molecular dynamic simulations. Undecaprenyl Pyrophosphate Synthase (UPPS) is a vital target enzyme in the early stages of bacterial cell wall biosynthesis. UPPS inhibitors have antibacterial activity against resistant strains such as MRSA and VRE. In this study, we used several consecutive computer-based protocols to identify novel UPPS inhibitors. The 3D QSAR pharmacophore model generation (HypoGen algorithm) protocol was used to generate a valid predictive pharmacophore model using a set of UPPS inhibitors with known reported activity. The developed model consists of four pharmacophoric features: one hydrogen bond acceptor, two hydrophobic, and one aromatic ring. It had a correlation coefficient of 0.86 and a null cost difference of 191.39, reflecting its high predictive power. Hypo1 was proven to be statistically significant using Fischer's randomization at a 95% confidence level. The validated pharmacophore model was used for the virtual screening of several databases. The resulting hits were filtered using SMART and Lipinski filters. The hits were docked into the binding site of the UPPS protein, affording 70 hits with higher docking affinities than the reference compound (6TC, -21.17 kcal/mol). The top five hits were selected through extensive docking analysis and visual inspection based on docking affinities, fit values, and key residue interactions with the UPPS receptor. Moreover, molecular dynamic simulations of the top hits were performed to confirm the stability of the protein-ligand complexes, yielding five promising novel UPPS inhibitors.