Quinoxaline‐based efflux pump inhibitors restore drug susceptibility in drug‐resistant nontuberculous mycobacteria

Nontuberculous mycobacteria (NTM) comprise several ubiquitous, environmentally localized bacteria that may be responsible for serious human diseases. NTM‐associated pulmonary infections largely affect individuals with underlying respiratory disease or chronic disease and immunosuppressed patients. Mycobacterium simiae and M. abscessus are two NTMs responsible for lung disease in immunocompetent and immunocompromised individuals. In this study, two NTM strains were isolated from two patients admitted to an Italian hospital and were identified as M. simiae and M. abscessus. The two NTMs were tested for drug susceptibility against different antibiotics. To restore drug susceptibility, a new series of 2‐aryl‐3‐phenoxymethyl‐quinoxaline derivatives (QXs) was designed, synthesized, and investigated as efflux pump inhibitors (EPIs) against two clinical isolates of the above‐cited NTMs, evaluating how EPIs can influence the drug minimal inhibitory concentration values and, therefore, the activity. The different\ resistance levels tracked in the clinical strains were reduced by EPIs, and in several cases, the susceptibility was completely restored. QXs also resulted as potential chemical probes to be used in drug susceptibility tests to identify the resistance origin when detected.

nonpathogenic and only rarely associated with disease in humans.
More recently it has been associated with pulmonary disease in both immunocompromised and immunocompetent patients.
No treatment regimen was found to be effective against M. simiae caused pulmonary infections, not even multiple antimycobacterial associations, especially in subjects whose therapeutic treatment is difficult as this microorganism has intrinsic and acquired resistance to first-line drugs but also macrolides. [8] Mycobacterium abscessus is a fast-growing, ubiquitous mycobacterium in soil and water responsible for a wide variety of human infections; skin and respiratory tract are particularly affected. Infections caused by this NTM are difficult to treat due to the natural and acquired resistance to drugs and disinfectants. It is responsible for pneumonia mainly in hosts with underlying structural lung diseases, such as cystic fibrosis, bronchiectasis, and previous tuberculosis. [9] According to the 2007 American Thoracic Society/Infectious Disease Society of America guidelines, treatment regimens remain limited with current antimicrobial agents and, therefore, abscessus lung disease is considered a chronic incurable disease. [10] M. simiae and M.
abscessus infections have been revealed in patients in different states worldwide and drug susceptibility test to commonly used drugs has been performed on isolates, showing drug resistance for different tested drugs. [6,10,11] Intrinsic resistance can be due to a combination of different factors, such as the altered permeability of the cell envelope, low-affinity antibiotic target, drug efflux systems, and antibiotic neutralizing enzymes. Although acquired drug resistance is mainly caused by antibiotic target mutation or overexpression of efflux pumps and neutralizing enzymes, proved by upregulations or mutations detected in the bacterial genome. [12] The Mycobacterial membrane protein Large (MmpL) transporters belong to RND, Resistance-Nodulation Cell Division, an important family of multidrug resistance pumps. MmpLs are highly conserved between M. tuberculosis and NTMs. They are transporters that participate in the transport of the substrates through the periplasm to the extracellular environment and are involved in drug resistance mechanisms. [13] A high abundance of MmpLs in NTMs has been proved to be associated with drug resistance. [13][14][15][16] It was also found that efflux inhibitors such as verapamil, [17] reserpine, and carbonyl cyanide m-chlorophenylhydrazone (CCCP) [14] increased drug susceptibility. [18] Efflux pump inhibitors can also be used as chemical probes. As target mutation and efflux pump overexpression are the most detected causes connected with drug resistance, effective efflux pump inhibitors (EPIs) can also be used in drug susceptibility tests to elucidate the eventual drug resistance origin.
Clearly, there is an urgent need for the development of effective drugs or regimens against drug-resistant NTMs and fast enlightening of drug resistance origin. In the last decade, our research group has focused its attention on the synthesis and biological activity of compounds with antimycobacterial activity. [19][20][21] Furthermore, we synthesized effective EPIs active in enhancing chemotherapeutics in cancer cell lines, [22,23] M. tuberculosis, [24] and several bacterial, and fungal strains. [25] These latter compounds bear a quinoxaline (QX) functionalized aryl moiety in position 2. The high and interesting inhibition of different EP was paired with very low solubility, therefore, the QX compounds from the first generation were used as hit compounds to obtain more soluble but still active EP inhibitors. To increase solubility, methoxy groups from parental series were replaced by chlorine atoms and EPI activity was studied against several pathogen strains and cancer cell lines. We present here a new series of 2-aryl-3-phenoxymethyl-quinoxaline derivatives (QXs) that has been proved active as EPIs against two NTM clinical isolates.  [22,23,26] was then implemented in two final steps to get the desired compounds. The subsequent bromination and halogen substitution yielded designed compounds 8a and 8b.  Intermediates 7a,b were prepared by activating the proper salicylic acid derivative (11a,b) to a more reactive chloride derivative, and the following coupling with the desired 3,4-dichloroaniline (12) produced the designed salicylic amide intermediates, as shown in Scheme 3.

| Enhancing the effect of efflux pump inhibitors on antimicrobial activity
The enhancement of antimycobacterial-drugs activity exerted by the synthesized efflux pump inhibitors (8a,b and 10a,b) was evaluated by REMA assays. They were performed in parallel by administering the sole drug or the association of each drug with each synthesized EPI.
Minimum inhibitory concentrations (MICs) were measured for antimycobacterial drugs alone and when coadministered with EPIs. Table 1 shows the drug susceptibility of both NTM clinical isolated strains to six drugs commonly used in therapy: azithromycin, amikacin, ciprofloxacin, levofloxacin, moxifloxacin, and linezolid.  Table 2.
Derivative 10b reduced it only by two-old and it can be deemed as not considerable. While the antimycobacterial effect of amikacin was modulated by our QXs but drug susceptibility was not fully recovered, proving that the mechanism of amikacin resistance is not chargeable to the sole efflux pumps activity. Along the same line, ciprofloxacin, levofloxacin, moxifloxacin, and linezolid MIC values were not improved by the coadministration with our EPIs, proving that the drug resistance in this mycobacteria strain is not due to efflux pump overexpression.
Multidrug resistance of clinical M. simiae was reverted by the association of antimycobacterial drugs with our EPIs, as reported in Table 3. In the presence of all the tested compounds, the MICs of amikacin were reduced and compound 8a restored drug susceptibility with a fourfold enhancement. Levofloxacin activity was improved four-fold by all the QX derivatives while ciprofloxacin resistance was affected by the sole compound 10a with a MIC reduction of four times. Azithromycin, moxifloxacin, and linezolid resistance were not reverted by the association with our EPIs.
Compound 8a showed the widest activity improving the MIC values of all the reported antimycobacterial drugs. From a structure-activity relationship point of view, the slight structural differences among the four compounds resulted in a comparable activity of the QX derivatives when tested as efflux pump inhibitors in these NTM strains. We can highlight that the addition of a second, more hindered side-chain provided in compounds 10a and 10b did not result in a significant improvement of the activity. It also proved that the binding site is huge enough to accommodate huge molecules such as 10a and 10b, but it is occupied also by slightly smaller molecules such as 8a,b. These results proved quite clearly that EPIs can be used in coadministration to revert drug resistance in MDR NTM, as well as chemical probes, during in vitro assays, to identify the resistance origin.    (12) and inorganic reagents were commercially available and were purchased by Sigma Aldrich. Intermediates 1-6, and 9 were prepared following the procedure we previously described. [22,23,26] The known N-(3,4-dichlorophenyl)-2-hydroxybenzamide (7a) and 5-chloro-N-(3,4dichlorophenyl)-2-hydroxybenzamide (7b) intermediates were prepared as reported by Waisser et al. [27] and are here fully characterized.