Design, synthesis and structure-based optimization of novel isoxazole-containing benzamide derivatives as FtsZ modulators

Highlights

• Two novel series of isoxazole-containing benzamide derivatives were designed and synthesized.

• They were evaluated for their biological activities as FtsZ-targeting antibacterial agents.

• Computational docking program was used to analyze for the structure-based rational optimization.

• This optimization strategy indeed led to a potent compound B14 with better activity.

• In-depth study on the mechanism confirmed this compound targeted FtsZ protein.

Abstract

Antibiotic resistance among clinically significant bacterial pathogens is becoming a prevalent threat to public health, and new antibacterial agents with novel mechanisms of action hence are in an urgent need. Utilizing computational docking method and structure-based optimization strategy, we rationally designed and synthesized two series of isoxazol-3-yl- and isoxazol-5-yl-containing benzamide derivatives that targeted the bacterial cell division protein FtsZ. Evaluation of their activity against a panel of Gram-positive and -negative pathogens revealed that compounds B14 and B16 that possessed the isoxazol-5-yl group showed strong antibacterial activity against various testing strains, including methicillin-resistant Staphylococcus aureus and penicillin-resistant S. aureus. Further molecular biological studies and docking analyses proved that the compound functioned as an effective inhibitor to alter the dynamics of FtsZ self-polymerization via a stimulatory mechanism, which finally terminated the cell division and caused cell death. Taken together, these results could suggest a promising chemotype for development of new FtsZ-targeting bactericidal agent.

Introduction

At present the widespread bacterial resistance to antibiotics is extremely severe throughout world in hospital and community settings, which has brought up an urgent need for the discovery and development of new generation of antibiotics [1,2]. With respect to the current research, the bacterial cell division process has been considered as a novel and attractive target for the discovery of new antibacterial drug [[2], [3], [4], [5]]. During this division process, FtsZ (Filamentous temperature-sensitive mutant Z) is considered as a fundamental protein forming the division machinery [[6], [7], [8]]. FtsZ is the first protein to be localized at the incipient division site, and assembles into a highly dynamic cytoskeleton scaffold, termed Z-ring, by undergoing GTP-dependent polymerization. Subsequently, this cytokinetic structure recruits other downstream division proteins and contracts enabling septum formation and cell separation, completing a successful bacterial cell division event [7,9,10]. Because of its high conservation and important function among drug-sensitive and -resistant bacteria, FtsZ has been confirmed as a promising target for the development of new antibacterial agents by inhibition of the cell division.

By the recent investigations, a number of small molecules have been identified as FtsZ inhibitors, a few of which just listed (Fig. 1). For example, PC190723 is a milestone potent FtsZ inhibitor from optimization of 3-methoxybenzamide (3-MBA). Despite its initial promise, the poor drug-like and pharmacokinetic properties have limited its further clinical development. To address this problem, strategy of prodrug was used and three prodrugs (e.g., TXY436, TXY541 and TXA709) were developed [[11], [12], [13]], especially TXA709, in which the Cl group on the pyridyl ring has been replaced with a CF₃ functionality resistant to metabolic attack. As a result, the product of TXA709 is associated with improved pharmacokinetic properties and superior in vivo antistaphylococcal efficacy relative to PC190723 [13].

The crystal structure of S. aureus FtsZ-PC190723 complex [14] and docking models [15] indicate that the amide group forms strong hydrogen bonds with surrounding amino acid residues in T7 loop of FtsZ, which provides the essential binding force between these benzamide compounds and FtsZ. Besides, the 3-Oside chain stretches into a hydrophobic cleft of FtsZ to form additional interaction. This explains why 3-MBA, a simple benzamide derivative, can possess FtsZ-targeting effect [16]. Further effective modification is concentrated on the 3-O-side chain. For instance, introduction of the lipophilic alkyl chain or aromatic ring at the 3-O-position (3 and 4 in Fig. 1) significantly enhance antibacterial activity but cannot compare favourably with PC190723. This gap indicates that heteroatom-containing side chain could be more suitable than the pure hydrocarbon side chain for the hydrophobic cleft of FtsZ. This is because heteroatoms of the 3-O-side chain such as N, O and S may bring wondrous interactions in this hydrophobic cleft, which differs from the hydrophobic interactions formed by C atoms of the 3-O-side chain.

In recent years, isoxazole scaffold has attracted more and more attention because of its wide spectrum of biological activities and therapeutic potential [17]. The structural features of the isoxazole make it possible for multiple non-covalent interactions, especially hydrogen bonds, pi-pi stack and hydrophobic interactions. The inclusion of isoxazole may contribute to the increased efficacy, decreased toxicity, and improved pharmacokinetics profiles [[18], [19], [20]]. Successful applications of developing isoxazole compounds have resulted in multiple corresponding drugs, including anticancer, antibacterial, antifungal, antiviral and anti-inflammatory in the market [21].

Based on the above considerations, we designed the series A of the isoxazol-3-yl-5-benzamide derivatives through introducing an isoxazol-3-yl group into the 3-O-side chain, and predicted their potential binding mode through computational docking method. And then, the docking mode was used to establish their key determinants for affinity to guide the structure-based design, which led to design of the series B of the isoxazol-5-yl-3-benzamide derivatives. Subsequently, five subordinate analogues were obtained by further elongating the methylene linkage or introducing Br, Cl or Me into the 3-O-side chain (Fig. 2). From this strategy, we did find some isoxazol-5-yl-3-benzamide derivatives possessing excellent antibacterial and FtsZ-inhibition activity. In particular, B14 with the isoxazol-5-yl group displayed about thirty-fold more potent activity against Bacillus subtilis and Bacillus pumilus (MIC, 0.015 μg/mL) than PC190723, and considerable activity against S. aureus including two drug-resistant strains (MIC, 2 μg/mL). Here we report the synthesis and antibacterial activity of series A and B. The cell morphology and the FtsZ polymerization are also performed to indicate their on-target activity. Their preliminary structure-activity relationships (SARs) and docking study are described.