B-24

To discover novel natural-product-based insecticide candidates, herein, a variety of osthole-derived N-benzoylthioureas were synthesized and assessed for their insecticidal activities against three insect pests. An insecticidal assay showed that most of the target osthole-derived N-benzoylthioureas displayed a more potent and broad-spectrum insecticidal effect than the parent osthole after the introduction of N-benzoylthioureas on the C-3' position of osthole. Compound B24 displayed the most potent growth inhibitory (GI) effect on Mythimna separata Walker, with a final corrected mortality rate of 82.1% when treated with a concentration of 1 mg/mL, which was 1.64- and 1.53-fold higher in comparison to osthole and the botanical insecticide toosendanin, respectively. Compounds B22, B23, and B25 displayed a more promising aphicidal effect on Myzus persicae Sulzer, and their LD₅₀ values were 0.015, 0.017, and 0.019 μg/larvae, respectively, superior to the commercially available insecticide rotenone (0.024 μg/larvae). Derivatives B19, B20, B23, and B25 displayed more potent larvicidal activity against Plutella xylostella Linnaeus, with LC₅₀ values of 0.22, 0.26, 0.15, and 0.30 mg/mL, respectively, exceeding that of rotenone (0.37 mg/mL). Furthermore, both compounds B19 and B23 against P. xylostella were found to be more effective than rotenone in a control efficacy assay under greenhouse conditions. The structure-activity relationship (SAR) suggested that osthole-derived N-benzoylthioureas are more active in most cases when the R group is an electron-withdrawing group than when it is an electron-donating group, especially for halogenated groups. Additionally, the potent compounds B19 and B23 possessed good selectivity and were less toxic to non-target organisms. This study suggests that these osthole-derived N-benzoylthioureas could be further studied in depth as eco-friendly natural product pesticides in crop protection.

Dihydrofolate reductase is one of the important enzymes for thymidylate and purine synthesis in micro-organisms. A large number of drugs have been designed to inhibit microbial DHFR but over the period of time, some drugs have developed resistance and cross reactivity towards the enzyme. Over the past few decades, benzimidazoles, triazoles and their derivatives have been grabbing the attention of the synthetic chemists for their wide gamut of antibacterial and antifungal activities targeting microbial protein DHFR.

Our goal behind present investigation is to explore benzimidazoles class of drugs as microbial DHFR inhibitors by studying ligand-receptor binding interactions, in vitro enzyme inhibition assay and confirmation of anti-microbial activity against selected pathogenic microorganisms.

A library containing thirty novel 2,6-disubstituted 1H-benzimidazoles was synthesized by one pot condensation of o-nitro aniline or 2,4-dinitro aniline with series of aldehydes or acetophenones using Na2S2O4 or SnCl2 respectively and reflux for 5-6hr. Structures of compounds have been confirmed by spectroscopic methods as 1H and 13C NMR, FT-IR and MS. In vitro DHFR inhibition study was performed by using Epoch microplate reader and IC50 of the test compounds was compared with Trimethoprim. In vitro antimicrobial activity was performed against selected clinical pathogens by agar disk diffusion method and MIC (µg/mL) was reported.

Moderate to good level of DHFR inhibition was observed with IC50 values in the range of 7-23 µM. Compounds B1, B19, B22, B24 and B30 expressed 1.1 to 1.4 folds more prominent DHFR inhibitory activity as compared to standard Trimethoprim. Remarkable antimicrobial activity was exhibited by B1, B19, B22, B24 and B30. Molecular docking study revealed perfect binding of test ligands with key amino acids of DHFR as Phe31, Ile94, Ile5, Asp27, Gln32 and Phe36.

Nature of 1H-benzimidazole substituents at position 2 and 6 had influence over magnitude and type of molecular binding and variation in the biological activity. The present series of 1H-benzimidazoles could be considered promising broad-spectrum antimicrobial candidates that deserve in future for preclinical antimicrobial evaluation and development of newer antimicrobial agents targeting microbial DHFR.