DRUG DISCOVERY AND RESISTANCE New fluoroquinolones active against fluoroquinolones-resistant Mycobacterium tuberculosis strains Valentina Guerrini a, c , Maria De Rosa b, c, d , Serena Pasquini b , Claudia Mugnaini b , Antonella Brizzi b , Anna Maria Cuppone a , Gianni Pozzi a , Federico Corelli b, * a Dipartimento di Biotecnologie Mediche, Università di Siena, Policlinico Santa Maria alle Scotte, Viale Bracci 1, 53100 Siena, Italy b Dipartimento di Biotecnologie, Chimica e Farmacia, Università di Siena, Via A. De Gasperi 2, 53100 Siena, Italy article info Article history: Received 16 October 2012 Received in revised form 11 February 2013 Accepted 24 February 2013 Keywords: Fluoroquinolone Synthesis Antimycobacterial activity Fluoroquinolone-resistant strain Tuberculosis summary A set of 21 new fluoroquinolones bearing an aromatic or heteroaromatic moiety at C-7 and an alkyl group at N-1 were synthesized based on the lead structure of pirfloxacin and tested in vitro against Myco- bacterium tuberculosis (M. tuberculosis) H37Rv by MIC determination in liquid medium. Among the synthesized compounds, 1-(tert-butyl)-6-fluoro-7-(4-hydroxyphenyl)-4-oxo-1,4-dihydroquinoline-3- carboxylic acid (2o) and 1-(tert-butyl)-6-fluoro-7-(pyridin-3-yl)-4-oxo-1,4-dihydroquinoline-3- carboxylic acid (2n) were found to be the most active ones against M. tuberculosis H37Rv with the same MICs of reference compounds ciprofloxacin (CFX) and levofloxacin (LFX). MICs of 2o and 2n were determined for fluoroquinolone-sensitive and fluoroquinolone-resistant M. tuberculosis clinical isolates and 2o was the most active compound with up 4-fold difference of MIC with respect to CFX. The activity of 2o was also tested at the concentration of 16 mg/mL against M. tuberculosis H37Rv in infected murine macrophages. The results showed a 4-fold decrease in viable count of cell-associated mycobacteria with respect to untreated controls after 48 h of drug incubation. Ó 2013 Published by Elsevier Ltd. 1. Introduction The incidence of tuberculosis (TB) has risen in the last years. In particular the rates of new cases of multidrug-resistant tuberculosis (MDR-TB) and extensively drug-resistant tuberculosis (XDR-TB) continue to increase and this is now a major health concern. 1 The increase of the rate of TB worldwide, together with the emergence of Mycobacterium tuberculosis drug resistant strains, highlights the urgent need for new antitubercular drugs for TB treatment and control. Fluoroquinolones are important second line drugs for TB and one of the main tools to fight resistant TB strains. 2 Currently levofloxacin (LFX) and moxifloxacin (MFX) are recommended by WHO for treatment of MDR-TB. 3 The target of quinolones is the enzyme DNA gyrase which is involved in DNA supercoiling and bacterial replication. Quinolones exert their antibacterial activity by interfering with the enzymatic reaction cycle, binding to the enzyme-DNA binary complex, thereby stabilizing the covalent linkage enzyme-DNA. The resulting ternary complex blocks DNA replication and leads to cell death. 4 The increased use of quinolones in TB has led to decreased quinolone susceptibility in M. tuberculosis. Quinolone resistance is generally due to point mutations in gyrA and gyrB genes, in particular in quinolone resistance determining regions (QRDRs), which are involved in quinolone binding. 5e10 Piton and coworkers produced the first crystallographic model of the quinolone binding poket (QBP) of DNA gyrase, identifying original mechanistic properties of quino- lone binding and suggesting the relationships between amino acid mutations and resistance phenotype of M. tuberculosis. 11 Additional resistance mechanisms include mutations in regions of gyrA or gyrB outside of the QRDRs, decreased cell wall permeability to the drug, active drug efflux pump mechanism, sequestration or drug inacti- vation. 12e14 1.1. Structure-antimycobacterial activity of quinolones While structureeantibacterial activity relationships of fluo- roquinolones have been almost completely detailed in a number of review articles such as (but not limited to) those authored by * Corresponding author. Tel.: þ39 0577 234308; fax: þ39 0577 234333. E-mail addresses: guerrini49@unisi.it (V. Guerrini), derosa20@unisi.it (M. De Rosa), pasquini10@unisi.it (S. Pasquini), claudia.mugnaini@unisi.it (C. Mugnaini), brizzi3@unisi.it (A. Brizzi), annamaria.cuppone@unisi.it (A.M. Cuppone), gianni.pozzi@unisi.it (G. Pozzi), federico.corelli@unisi.it (F. Corelli). c These authors contributed equally to this work. d Present address: Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry, Uppsala University, P.O. Box 256, SE-75105 Uppsala, Sweden. Contents lists available at SciVerse ScienceDirect Tuberculosis journal homepage: http://intl.elsevierhealth.com/journals/tube 1472-9792/$ e see front matter Ó 2013 Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.tube.2013.02.017 Tuberculosis xxx (2013) 1e7 Please cite this article in press as: Guerrini V, et al., New fluoroquinolones active against fluoroquinolones-resistant Mycobacterium tuberculosis strains, Tuberculosis (2013), http://dx.doi.org/10.1016/j.tube.2013.02.017