Natural Product-Based 1,2,3-Triazole/Sulfonate Analogues as Potential Chemotherapeutic Agents for Bacterial Infections

Abstract

Despite the vast availability of antibiotics, bacterial infections remain a leading cause of death worldwide. In an effort to enhance the armamentarium against resistant bacterial strains, 1,2,3-triazole (5a-x) and sulfonate (7a-j) analogues of natural bioactive precursors were designed and synthesized. Preliminary screening against two Gram-positive (Streptococcus pneumoniae and Enterococcus faecalis) and four Gram-negative bacterial strains (Pseudomonas aeruginosa, Salmonella enterica, Klebsiella pneumoniae, and Escherichia coli) was performed to assess the potency of these analogues as antibacterial agents. Among all triazole analogues, 5e (derived from carvacrol) and 5u (derived from 2-hydroxy 1,4-naphthoquinone) bearing carboxylic acid functionality emerged as potent antibacterial agents against S. pneumoniae (IC₅₀: 62.53 and 39.33 μg/mL), E. faecalis (IC₅₀: 36.66 and 61.09 μg/mL), and E. coli (IC₅₀: 15.28 and 22.57 μg/mL). Furthermore, 5e and 5u also demonstrated moderate efficacy against multidrug-resistant E. coli strains and were therefore selected for further biological studies. Compound 5e in combination with ciprofloxacin displayed a synergistic effect on multidrug-resistant E. coli MRA11 and MRC17 strains, whereas compound 5u was selective against E. coli MRA11 strain. Growth kinetic studies on S. pneumoniae and E. coli treated with 5e and 5u showed an extended lag phase. 5e and 5u did not show significant cytotoxicity up to 100 μg/mL concentration on human embryonic kidney (HEK293) cells. Transmission electron microscopic (TEM) analysis of bacterial cells (S. pneumoniae and E. coli) exposed to 5e and 5u clearly showed morphological changes and damaged cell walls. Moreover, these compounds also significantly inhibited biofilm formation in S. pneumoniae and E. coli strains, which was visualized by scanning electron microscopic (SEM) analysis. Treatment of larvae of Galleria mellonella (an in vivo model for antimicrobial studies) with 5e and 5u did not cause an alteration in the hemocyte density, thereby indicating lack of an immune response, and were nontoxic up to a concentration of 2.5 mg/mL.

Figure 1

Natural product-based 1,2,3-triazole analogues as anti-biofilm agents.

Scheme 1. Synthesis of 1,2,3-Triazole Derivatives of Natural Precursors

Scheme 2. Synthesis of Sulfonic Esters of Natural Precursors

Figure 2

(A) XP diagram of compound 5c with atomic labeling scheme (50% probability level of thermal ellipsoids); (B) excerpt of the packing diagram of 5c representing the chain formation along the a-axis via CH···N interactions; (C) XP diagram of compound 5f with atomic labeling scheme (50% probability level of thermal ellipsoids); and (D) linear double-layer chain formation through CH···N interactions along the b-axis in 5f.

Figure 3

(A) XP diagram of compound 7g with atomic labeling scheme (50% probability level of thermal ellipsoids); (B) wave chain along the b-axis formed via π···π and CH···O interactions in the packing diagram of 7g; (C) XP diagram of compound 7h with atomic labeling scheme (50% probability level of thermal ellipsoids); and (D) linear chain of dimeric units along ab-diagonal formed via CH···O and π···π interactions in the packing diagram of 7h.

Figure 4

In vitro antibacterial activity of 5e, 5u, and CIP (μg/mL) against multidrug-resistant E. coli strains: MRA11, MRC17, MRC24, MRAE26, MRAE32, MRAE33, MRAE44, and MROB11. The numbers written above the respective columns correspond to the MIC concentrations of 5e, 5u, and CIP.

Figure 5

Growth kinetic studies under different concentrations of test compounds. (A,B) treated with 5e and (C,D) treated with 5u.

Figure 6

Representative transmission electron micrographs of (A) E. coli and (B) S. pneumoniae cells exposed to 5e (62.5 and 125 μg/mL concentrations, respectively) and 5u (125 μg/mL concentration) at their respective MICs.

Figure 7

Percentage of biofilm inhibition in E. coli and S. pneumoniae on treatment with 5e and 5u using (A) XTT assay and (B) crystal violet assay. The numbers (125 and 250) written above the respective columns correspond to the 2MIC concentrations of 5e and 5u, respectively.

Figure 8

Biomass determination in E. coli and S. pneumoniae at OD₅₉₀.

Figure 9

SEM images showing biofilm inhibition in E. coli.

Figure 10

(A) Cell viability assay on HEK293 cell line and (B) hemolytic assay for compounds 5e, 5u, and CIP on hRBCs.

Figure 11

Percentage viability of G. mellonella larvae in the presence of (A) 5e; (B) 5u; (C) dimethyl sulfoxide (DMSO); (D) hemocyte densities of larvae.