New Chloramphenicol Derivatives with a Modified Dichloroacetyl Tail
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antibiotics Article New Chloramphenicol Derivatives with a Modified Dichloroacetyl Tail as Potential Antimicrobial Agents Artemis Tsirogianni 1, Georgia G. Kournoutou 2, Anthony Bougas 2 , Eleni Poulou-Sidiropoulou 2, George Dinos 2,* and Constantinos M. Athanassopoulos 1,* 1 Synthetic Organic Chemistry Laboratory, Department of Chemistry, University of Patras, 26504 Patras, Greece; [email protected] 2 Department of Biochemistry, School of Medicine, University of Patras, 26504 Patras, Greece; [email protected] (G.G.K.); [email protected] (A.B.); [email protected] (E.P.-S.) * Correspondence: [email protected] (G.D.); [email protected] (C.M.A.); Tel.: +30-2610-969-125 (G.D.); +30-2610-997-909 (C.M.A.) Abstract: To combat the dangerously increasing pathogenic resistance to antibiotics, we developed new pharmacophores by chemically modifying a known antibiotic, which remains to this day the most familiar and productive way for novel antibiotic development. We used as a starting material the chloramphenicol base, which is the free amine group counterpart of the known chloramphenicol molecule antibiotic upon removal of its dichloroacetyl tail. To this free amine group, we tethered alpha- and beta-amino acids, mainly glycine, lysine, histidine, ornithine and/or beta-alanine. Further- more, we introduced additional modifications to the newly incorporated amine groups either with protecting groups triphenylmethyl- (Trt) and tert-butoxycarbonyl- (Boc) or with the dichloroacetic group found also in the chloramphenicol molecule. The antimicrobial activity of all compounds Citation: Tsirogianni, A.; Kournoutou, G.G.; Bougas, A.; was tested both in vivo and in vitro, and according to the results, the bis-dichloroacetyl derivative Poulou-Sidiropoulou, E.; Dinos, G.; of ornithine displayed the highest antimicrobial activity both in vivo and in vitro and seems to be a Athanassopoulos, C.M. New dynamic new pharmacophore with room for further modification and development. Chloramphenicol Derivatives with a Modified Dichloroacetyl Tail as Keywords: antibiotics; chloramphenicol; ribosome; peptidyl transferase; antibiotic resistance Potential Antimicrobial Agents. Antibiotics 2021, 10, 394. https:// doi.org/10.3390/antibiotics10040394 1. Introduction Academic Editor: Carlos M. Franco Antibiotics changed the course of therapeutics and have saved countless lives world- wide since their first clinical introduction [1]. Infectious diseases that could not be previ- Received: 15 March 2021 ously treated and their catastrophic effects are now easily controlled. However, shortly Accepted: 3 April 2021 after their introduction, resistance began to emerge rapidly, and now multidrug-resistant Published: 6 April 2021 bacteria have become a major concern for these infections’ treatment [2]. It has become evident that novel anti-infective agents with better efficiency and/or alternative modes of Publisher’s Note: MDPI stays neutral action are urgently required to battle the ever-evolving multidrug-resistant bacteria. Many with regard to jurisdictional claims in published maps and institutional affil- research groups worldwide are now devoted to solving this crisis either looking for new iations. natural compounds or derivatizing known antibiotics [3,4]. Following the second strategy, we present here new derivatives of chloramphenicol (CAM) with important antimicrobial activity, which show great promise as new scaffolds for further development of superior antimicrobial agents. Discovered in 1947 [5], chloramphenicol (CAM) was the first ribosome-acting and Copyright: © 2021 by the authors. broad-spectrum antibiotic introduced, covering a wide range of gram-positive and gram- Licensee MDPI, Basel, Switzerland. negative pathogens. When issued for clinical use, the drug immediately became very This article is an open access article distributed under the terms and popular, not only for its low cost and effectiveness, but also for its few and mild side effects. conditions of the Creative Commons However, shortly after, its initial mild side effects became serious disorders, like bone Attribution (CC BY) license (https:// marrow depression and aplastic anemia [6,7]. As a result, the toxicity of the drug and the creativecommons.org/licenses/by/ development of safer alternative antibiotics narrowed the indications of chloramphenicol 4.0/). Antibiotics 2021, 10, 394. https://doi.org/10.3390/antibiotics10040394 https://www.mdpi.com/journal/antibiotics Antibiotics 2021, 10, 394 2 of 11 prescriptions and finally its use was declined. Although CAM was downgraded thera- peutically, it never ceased to be an attractive tool for protein synthesis studies, especially regarding the ribosome structure and function development. Following numerous func- tional and structural studies, it has been established that CAM binds in the large ribosomal subunit covering partially the binding surface of the A-site substrate and therefore inhibit- ing protein synthesis [8–11]. According to the previous references, the aromatic ring of the ribosome-bound CAM overlaps with the placement of side chains of the incoming aa-tRNAs, thus efficiently preventing the aminoacyl moiety of aa-tRNA from properly accommodating into the peptidyl-transferase center (PTC) active site. This model was recently revised based on the novel data which support the theory that CAM acts as a context-specific inhibitor of translation whose action depends on the nature of specific amino acids in the nascent chain and the identity of the residue entering the A site. More precisely, chloramphenicol-mediated inhibition is stimulated when a nascent peptide in the ribosome carries an alanine amino acid in the penultimate position and preferentially aspartic acid and lysine in the P and A sites, respectively. This ADK (alanine–aspartic acid–lysine) motif, is the most preferable for CAM action and is soundly characteristic of the functional interplay between the nascent chain and the ribosomal peptidyl transferase activity [12,13]. The emergence of undesirable side effects and increasing antimicrobial resistance led to an early onset of studies for chloramphenicol derivatization targeting both characteristics, improved antimicrobial activity and lowered toxicity side effects. Two of the most frequently and rapidly spreading resistance mechanisms operate through the enzymatic modification of either the CAM molecule or the ribosome-binding site. In the first case, CAM is modified via its acetylation with acetyltransferase enzymes (CATs) and in the second, via methylation of the A2503 base of 23S rRNA, with lost affinity in both cases of CAM for the ribosome [14]. To this day, numerous derivatives have been designed and synthesized, but none of them has been evaluated as superior to the parental chloram- phenicol molecule (reviewed in [15–20]). Although the derivatization approach has not been successful up till now, efforts have never ceased since it is one of the most productive methods for the development of new-generation antibiotics against pathogenic resistance. In this study, using the chloramphenicol base (CLB) as a starting material (Figure1), we tethered its amine group to the Boc- or Trt-protected amino acids glycine, histidine, lysine, ornithine or β-alanine through an amide bond. These compounds were then deprotected and the thus obtained free amine groups of the amino acid part were further modified, introducing either a dichloroacetic or a difluoroacetic group (Figure2). Figure 1. Structures of chloramphenicol (CAM) and chloramphenicol base (CLB). Antibiotics 2021, 10, 394 3 of 11 Figure 2. Structures of CLB derivatives encountered in this work. According to the in vivo and in vitro evaluation of all new compounds, the bis- dichloroacetyl derivative of ornithine (6) (Figure2) displayed the highest antimicrobial activity in vivo and in vitro and appears to be a dynamic new pharmacophore with room for further modification and development. Antibiotics 2021, 10, 394 4 of 11 2. Results and Discussion 2.1. Chemical Synthesis For the purpose of this work, we prepared 22 derivatives (33 compounds in total, including intermediates), starting from the commercially available CLB. Detailed synthetic schemes and procedures describing the preparation and the structural characterization by 1H, 13C NMR and ESI–MS spectra of each of the abovementioned compounds can be found in the Supplementary Materials section of this paper. 2.2. Antibacterial Activity All the synthesized compounds were first tested in vivo. Using an antibiotic-sensitive Escherichia coli DTolC strain, we monitored culture growth in the presence of increasing concentrations of all antibiotics. Among the compounds tested, only three of them dis- played significant inhibition (Figure3). Expressing the data as percent inhibition of the control grown in the absence of antibiotics, we calculated the EC50 concentrations which are presented in Table1 in both µM and µg/mL, ranging from 6.70 to 18.66 µg/mL (Table1) . Compound 6, which is the most effective, is a derivative of the CLB with the basic amino acid ornithine, where both of its amine groups bear a dichloroacetyl group in the form of an amide bond. The remaining two compounds, 21 and 14, with noticeable in vivo activity are both histidine derivatives, where the amine groups are protected either with Trt as in the case of (21) or with Boc in the case of (14). In reality, compound 21 is not a direct histidine derivative but rather a histamine one, which is tethered to the CLB through a succinic acid linker. Histidine–CLB is well-known