1. Eur J Med Chem. 2020 Aug 15;200:112444. doi: 10.1016/j.ejmech.2020.112444. Epub 
2020 May 18.

Pharmacomodulations of the benzoyl-thiosemicarbazide scaffold reveal 
antimicrobial agents targeting d-alanyl-d-alanine ligase in bacterio.

Ameryckx A(1), Pochet L(2), Wang G(3), Yildiz E(1), Saadi BE(1), Wouters J(2), 
Van Bambeke F(3), Frédérick R(4).

Author information:
(1)Medicinal Chemistry Research Group (CMFA), Louvain Drug Research Institute 
(LDRI), Université catholique de Louvain (UCLouvain), 73 Avenue Mounier, 
B1.73.10, 1200, Bruxelles, Belgium.
(2)Namur Medicine & Drug Innovation Center (NAMEDIC), Namur Research Institute 
for Life Sciences (NARILIS), University of Namur, Namur, Belgium.
(3)Pharmacologie cellulaire et moléculaire (FACM), Louvain Drug Research 
Institute (LDRI), Université catholique de Louvain (UCLouvain), 73 Avenue 
Mounier, B1.73.05, 1200, Bruxelles, Belgium.
(4)Medicinal Chemistry Research Group (CMFA), Louvain Drug Research Institute 
(LDRI), Université catholique de Louvain (UCLouvain), 73 Avenue Mounier, 
B1.73.10, 1200, Bruxelles, Belgium. Electronic address: 
raphael.frederick@uclouvain.be.

d-Alanyl-d-alanine ligase (Ddl) is a validated and attractive target among the 
bacterial enzymes involved in peptidoglycan biosynthesis. In the present work, 
we investigated the pharmacomodulations of the benzoylthiosemicarbazide scaffold 
to identify new Ddl inhibitors with antibacterial potency. Five novel series of 
thiosemicarbazide analogues, 1,2,4-thiotriazole-3-thiones, 1,3,4-thiadiazoles, 
phenylthiosemicarbazones, diacylthiosemicarbazides and thioureas were 
synthesized via straightforward procedures, then tested against Ddl and on 
susceptible or resistant bacterial strains. Among these, the thiosemicarbazone 
and thiotriazole were identified as the most promising scaffolds with Ddl 
inhibition potency in the micromolar range. Antimicrobial evaluation of 
salicylaldehyde-4(N)-(3,4-dichlorophenyl) thiosemicarbazone 33, one of the best 
compounds in our study, revealed interesting antimicrobial activities with 
values of 3.12-6.25 μM (1.06-2.12 μg/mL) against VRE strains and 12.5-25.0 μM 
(4.25-8.50 μg/mL) towards MRSA and VRSA strains. A detailed mechanistic study 
was conducted on the Ddl inhibitors 
4-(3,4-dichlorophenyl)-5-(2-hydroxyphenyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione 
20 and compound 33, and revealed a bactericidal effect at 5 × MIC concentration 
after 7 h and 24 h, respectively, and a bacteriostatic effect at 1 × MIC or 
2 × MIC without any sign of bacterial membrane disruption at these lower 
concentrations. Finally, 20 and 33 were proved to target Ddl in bacterio via 
intracellular LC-MS dosage of d-Ala, l-Ala and d-Ala-d-Ala. Although, at this 
stage, our results indicate that other mechanisms might be involved to explain 
the antimicrobial potency of our compounds, their ability to inhibit the growth 
of strains resistant to usual antibiotics, as well as strains that express 
alternative ligases, sets the stage for the development of new antimicrobial 
agents potentially less sensitive to resistance mechanisms.

Copyright © 2020 Elsevier Masson SAS. All rights reserved.

DOI: 10.1016/j.ejmech.2020.112444
PMID: 32497961