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Please cite this article in press as: Vieira M, et al. Antimicrobial activity of quinoxaline 1,4-dioxide with 2- and 3-substituted derivatives. MicrobiolRes (2013), http://dx.doi.org/10.1016/j.micres.2013.06.015

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Microbiological Research xxx (2013) xxx– xxx

Contents lists available at SciVerse ScienceDirect

Microbiological Research

jo ur n al homepage: www.elsev ier .com/ locate /micres

Antimicrobial activity of quinoxaline 1,4-dioxide with 2- and3-substituted derivatives

1

2

Mónica Vieiraa,b,∗, Cátia Pinheirob, Rúben Fernandesb,c, João Paulo Noronhaa,Q1

Cristina Prudênciob,c3

4

a REQUIMTE/CQFB, Departamento de Química, FCT, Universidade Nova de Lisboa, 2829-516 Caparica, Portugal5b Ciências Químicas e das Biomoléculas, Centro de Investigac ão em Saúde e Ambiente, Escola Superior de Tecnologias da Saúde, Instituto Politécnico doPorto, Rua Valente Perfeito, 322, 4400-330 Vila Nova de Gaia, Portugal

6

7c Centro de Farmacologia e Biopatologia Química (U38-FCT), Faculdade de Medicina, Universidade do Porto, Alameda Prof. Hernâni Monteiro, 4200-319Porto, Portugal

8

9

10

a r t i c l e i n f o11

12

Article history:13

Received 9 April 201314

Received in revised form 11 June 201315

Accepted 12 June 201316

Available online xxx

17

Keywords:18

Antimicrobial activity19

Quinoxaline N,N-dioxide derivatives20

Minimum inhibitory concentration21

Cellular viability22

a b s t r a c t

Quinoxaline is a chemical compound that presents a structure that is similar toquinolone antibiotics. The present work reports the study of the antimicrobial activityof quinoxaline N,N-dioxide and some derivatives against bacterial and yeast strains. Thecompounds studied were quinoxaline-1,4-dioxide (QNX), 2-methylquinoxaline-1,4-dioxide(2MQNX), 2-methyl-3-benzoylquinoxaline-1,4-dioxide (2M3BenzoylQNX), 2-methyl-3-benzylquinoxaline-1,4-dioxide (2M3BQNX), 2-amino-3-cyanoquinoxaline-1,4-dioxide (2A3CQNX),3-methyl-2-quinoxalinecarboxamide-1,4-dioxide (3M2QNXC), 2-hydroxyphenazine-N,N-dioxide(2HF) and 3-methyl-N-(2-methylphenyl)quinoxalinecarboxamide-1,4-dioxide (3MN(2MF)QNXC). Theprokaryotic strains used were Staphylococcus aureus ATCC 6538, S. aureus ATCC 6538P, S. aureus ATCC29213, Escherichia coli ATCC 25922, E. coli S3R9, E. coli S3R22, E. coli TEM-1 CTX-M9, E. coli TEM-1, E. coliAmpC Mox-2, E. coli CTX-M2 e E. coli CTX-M9. The Candida albicans ATCC 10231 and Saccharomycescerevisiae PYCC 4072 were used as eukaryotic strains. For the compounds that presented activity usingthe disk diffusion method, the minimum inhibitory concentration (MIC) was determined. The alterationsof cellular viability were evaluated in a time-course assay. Death curves for bacteria and growth curvesfor S. cerevisiae PYCC 4072 were also accessed. The results obtained suggest potential new drugs forantimicrobial activity chemotherapy since the MIC’s determined present low values and cellular viabilitytests show the complete elimination of the bacterial strain. Also, the cellular viability tests for theeukaryotic model, S. cerevisiae, indicate low toxicity for the compounds tested.

© 2013 Published by Elsevier GmbH.

1. Introduction23

Antimicrobial agents are largely used in treatment and pre-24

vention of microorganism infections. Among others, the misuse25

and, especially, the abusive use of this kind of drugs, in human26

health, veterinary and animal production, led to the development27

of drug-resistant and multidrug-resistant (MDR) microorganismsQ228

(Roe 2008; Moreno et al. 2008). In addition, the permanent contact29

with some antimicrobial drugs, besides the resistance develop-30

ment, allows the increase of allergies and respiratory complications31

which are affecting the human population worldwide (Santos et al.32

2007; Vollaard and Clasener 1994; Butaye et al. 2001; Witte et al.33

2008). Resistant bacteria are increasing and the interval between34

∗ Corresponding author at: Rua Valente Perfeito, 322 Gab. 21, 4400-330 Vila Novade Gaia, Portugal. Tel.: +351 222061000; fax: +351 222061001.

E-mail address: mav@estsp.ipp.pt (M. Vieira).

the appearances of new and multi-drug resistant species is hap- 35

pening in short periods of time (Alanis 2005). These conditions 36

are becoming emergent public health issues in the sense that they 37

compromise pharmacological activity and the efficacy of these 38

antimicrobial agents (Fernandes et al. 2008, 2009) and thus the 39

heath of the population. 40

Because MDR bacteria are increasing worldwide human kind 41

deals with the urgent need of development of new drugs with 42

enhanced antimicrobial activity able to fight pathogens with no 43

adverse effects (Fernandes et al. 2013). It is also expected to develop 44

drugs that can reverse the resistance observed overturning the 45

actual bacterial profile. Some approaches have been developed in 46

order to evaluate the bioactivity of numerous compound families 47

against several strains of microorganisms (Gradelski et al. 2001; 48

Moellering 2011). 49

Quinoxaline is an organic heterocyclic compound that has been 50

used as base of synthesis of bioactive derivatives and several inves- 51

tigation groups have demonstrated their potential in medical and 52

0944-5013/$ – see front matter © 2013 Published by Elsevier GmbH.http://dx.doi.org/10.1016/j.micres.2013.06.015

Please cite this article in press as: Vieira M, et al. Antimicrobial activity of quinoxaline 1,4-dioxide with 2- and 3-substituted derivatives. MicrobiolRes (2013), http://dx.doi.org/10.1016/j.micres.2013.06.015

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pharmacological applications (Zanetti et al. 2005; Carta et al. 2002;53

Sanna et al. 1999). These studies point to chemotherapeutical54

interests regarding the anti-tumor, anti-bacterial, anti-fungal, and55

anti-viral including anti-HIV (De Clercq 1997; Waring et al. 2002;56

Haykal et al. 2008; Harakeh et al. 2004) applications of these com-57

pounds. Relevant bioactivity has been reported in Mycobacterium58

spp. strains (Zanetti et al. 2005). No studies were found reporting59

biological activity for the quinoxaline derivatives in the present60

study with the microbial strains used.61

The quinoxaline derivatives with N-oxide and N,N-dioxide have62

particular interest since they present relevant anti-oxidant activ-63

ity. Many compounds with nitrogen oxygen bonds play important64

biological roles by releasing NO groups or by cellular deoxygenating65

(Burguete et al. 2011; Hossain et al. 2012).66

The present study pretends to be a contribution to the67

characterization of antibacterial and antifungal activity some N,N-68

quinoxaline derivatives (Table 1).69

The activity of these compounds was tested against bacteria70

and yeast in order to understand the biological activity in both71

eukaryotic and prokaryotic microbial models. In the present work72

Saccharomyces cerevisiae and Candida albicans were used as repre-73

sentative models of eukaryotic microorganisms. Likewise, several74

strains of Staphylococcus aureus and Escherichia coli were used as75

prokaryotic representative models of Gram-positive and Gram-76

negative respectively.77

2. Material and methods78

2.1. Quinoxaline N,N-dioxide and quinoxaline derivatives79

The compounds used in the present study were previously used80

by some of our collaborators and were gently provided by the81

Center of Investigation in Chemistry of the University of Porto.82

Synthesis, spectra and thermochemical properties were already83

studied for the quinoxaline derivatives used in the present study84

(Table 1) (Acree et al. 1997; Ribeiro da Silva et al. 2004; Gomes et al.85

2005, 2007).86

Stock solutions of the compounds were prepared in a 500 mL87

volume at a 500 �g/L final concentration. Since the compounds are88

thermally stable, the solutions were sterilized in an autoclave (AJC89

Uniclave 88) for 20 min at 120 ◦C. From these solutions, standards90

were prepared at the final concentrations 500, 100, 50, 20, and91

5 �g/L.92

2.2. Bacterial strains93

The strains used in this study were stored deep frozen at −80 ◦C.94

The selected strains, in order to evaluate the susceptibility of a bac-95

terial cell model to the proposed compounds, included S. aureus96

ATCC 6538, S. aureus ATCC 6538P, S. aureus ATCC 29213, E. coli ATCC97

25922, E. coli S3R9 and E. coli S3R22 (a penicillin resistant strain and98

a multidrug resistant strain, respectively). It was included some99

E. coli strains harboring extended spectrum �-lactamases (ESBL)100

such as TEM-1, TEM-1 + CTX-M9, CTX-M2, CTX-M9 and the AmpC101

�-lactamase MOX-2.102

2.3. Yeast strains103

The strains used in this study were S. cerevisiae PYCC 4072 (UNL,104

Portugal) and C. albicans ATCC 10231 and were also stored deep-105

frozen at −80 ◦C.106

2.4. Microorganisms culture and zone inhibition107

In order to assess the potential microbial activity of the com-108

pounds presented, disk diffusion method was used with two109

purposes. The first one was to determine the sensibility of the 110

strains to known antibiotics, cefoxitin (FOX) and ciprofloxacin (CIP). 111

The second was to assess the inhibition zone for new compounds. 112

Bacteria and yeast cells were sub-cultured in broth agar (tryptic 113

soy broth – TSB) and incubated for 24 h at 37 ◦C. Freshly prepared 114

bacterial cells were transferred into a saline solution (NaCl 0.9%, 115

Carlo Erba Reactifs, France) and density was settled in the interval 116

0.09 and 0.10, corresponding to 0.5 McFarland (1–2 × 108 CFU/mL). 117

The density of the solutions was measured at 625 nm using a 118

spectrophotometer (Thermo Scientific Genesys 20). Solutions were 119

spread onto a Trypticase Soy Agar (TSA; Cultimed, Spain) nutrient 120

plate in a laminar flow cabinet. Yeast strains were spread onto Yeast 121

Extract Peptone Dextrose (YEPD; Oxoid, Basingstoke, UK) nutrient 122

plate, in laminar flow cabinet. Blank sterile disks were immersed 123

in the standard solutions, with the final concentration of 500, 100, 124

50, 20, and 5 �g/L for each compound. Plates were incubated for 125

24 h at 37 ◦C and zone inhibition diameters were measured in mil- 126

limeters. Each one of these bacteria was tested with cefoxitin disks 127

(FOX) 30 �g and ciprofloxacin (CIP) 5 �g. Cefoxitin is a �-lactam 128

and ciprofloxacin is a quinolone that has a similar structure of 129

quinoxaline. The compounds studied have no established reference 130

values regarding the sensitive/resistant behavior for the quinoxa- 131

line derivatives, so microdilution method was employed in order 132

to determine de minimum inhibitory concentration. Strains studied 133

were classified (Table 2) as susceptible (S) or resistant (R) accord- 134

ing to Clinical Laboratory Standard Institute (CLSI) guidelines, by 135

disk diffusion, considering the values of CLSI to the �-lactam and 136

quinolone used in the present study. All results were confirmed by 137

replica. 138

2.5. Minimum inhibitory assays 139

The minimum inhibitory concentration (MIC) for each quinox- 140

aline derivative was estimated using the microdilution method, 141

according to the CLSI (Rex, 2009; Wilder 2005, 2006). The MIC’s 142

were determined for each strain/compound pairs that presented 143

antimicrobial activity. 144

The microplates used consisted of 96 wells. The TSB was dis- 145

pensed into several wells, 8 for each chemical compound. One of 146

these 8 wells corresponded to the positive control (containing the 147

culture medium and bacterial suspension) and another to the neg- 148

ative control (containing only the culture medium). The remaining 149

wells were used to prepare volumetrically diluted in series from 150

stock solution (1:1, 1:2, 1:4, 1:8, 1:16 and 1:32) for each compound 151

and considering the concentrations that presented inhibition halo. 152

Fresh prepared cultures of the microorganisms were suspended in 153

a saline solution to a density of 0.100 at 625 nm. Each well was pre- 154

pared to a final volume of 200 �L. The microplates were closed and 155

incubated at 37 ◦C, for 16–20 h. The presence or absence of turbid- 156

ity was verified and rechecked by inoculating a fraction of the wells 157

in solid culture medium (Mueller-Hinton broth, Cultimed, Spain). 158

The plates were incubated for 24 h at 37 ◦C. The results obtained in 159

the wells and plates were compared. All results were confirmed by 160

replica. 161

2.6. Cellular viability of bacteria 162

In order to evaluate the growing or death performance cel- 163

lular viability was analyzed for all strain/quinoxaline derivative 164

pairs that presented growth inhibition and for which MIC’s were 165

determined. Microbial suspensions were prepared at 0.5 McFarland 166

density with TSB medium for bacterial strains. The solutions of 167

the studied compounds were colored and the optical density (OD) 168

measurements lack accuracy. In alternative to the construction of 169

cellular viability and death curves by platting was observed in a 170

time-course assay. For 24 h, the presence of viable cells was tested 171

Please cite this article in press as: Vieira M, et al. Antimicrobial activity of quinoxaline 1,4-dioxide with 2- and 3-substituted derivatives. MicrobiolRes (2013), http://dx.doi.org/10.1016/j.micres.2013.06.015

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Table 1Quinoxaline N,N-dioxide and quinoxaline derivatives.

Quinoxaline-1,4-dioxide (QNX) 2-Methylquinoxaline-1,4-dioxide(2MQNX)

2-Methyl-3-benzoylquinoxaline-1,4-dioxide(2M3BenzoilQNX)

2-Methyl-3-benzylquinoxaline-1,4-dioxide(2M3BQNX)

2-Amino-3-cyanoquinoxaline-1,4-dioxide(2A3CQNX)

3-Methyl-2-quinoxalinecarboxamide-1,4-dioxide(3M2QNXC)

2-Hidroxiphenazine-N-dioxide (2HF) 3-Methyl-N-(2-methylphenyl)quinoxalinecarboxamide-1,4-dioxide(3MN(2MF)QNXC)

Table 2Zone diameter interpretative Standards (Enterobacteriaceae) and equivalent minimal inhibitory concentration (MIC) according to CLSI document (Wilder 2005).

Zone diameter interpretative standards (mm) MIC (�g/mL)

R I S R S

Ciprofloxacin (CIP) (5 �g) ≤15 16–20 ≥21 ≥4 ≤1Cefoxitin (FOX) (30 �g) ≤14 15–17 ≥18 ≥32 ≤8

R – resistant; I – intermediate; S – susceptible.

and CFU/mL was determined. The time intervals were divided172

as presented in Table 3. A standard tube containing the studied173

strain and growing media was used as control and a test tube con-174

taining also the quinoxaline derivative in the MIC concentration175

determined previously was used as working solution. For CFU/mL176

counting, optimum dilution was determined by serial dilutions177

with 10−1 factor, in saline solution, adding 1/10 of strain media178

and 9/10 of saline solution (Table 3). All results were confirmed by179

replica.180

2.7. Cellular viability for eukaryotic model181

The aim of this method was to evaluate the cellular viabil-182

ity effects of compounds that presented antibacterial activity. The183

procedure was the same as described for bacteria, but the final184

concentration of compounds was 500 �g/L, corresponding to the185

highest MIC determined previously. Along 24 h, CFU/mL was deter- 186

mined if viable cells were present. The time intervals were divided 187

as presented in Table 3. All results were confirmed by replica. 188

3. Results and discussion 189

3.1. Disk diffusion method 190

The quinoxaline derivative compounds studied have no ref- 191

erence values for the disk diffusion method, according to CLSI. 192

Nevertheless, CLSI guidelines were used for known antibiotics. 193

For the results obtained by this method, the absence of inhibi- 194

tion zone for the compounds tested was considered an indicative 195

of no antimicrobial activity against that strain. Table 4 shows 196

antimicrobial activity results for each bacterial or yeast strain and 197

Table 3Dilution factor and time intervals for UFC/mL determinations.

Optimum dilution factor Time for counting (min)

Bacteria 10−5 0 30 60 90 120 180 1440Yeast 10−3 0 30 60 90 120 180 210 1440

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Table 4Disk diffusion diameters for the quinoxaline derivatives.

Strain Compound (500 �g/L)

QNX 2MQNX 2M3BenzoilQNX 2M3BQNX 2A3CQNX 3M2QNXC 2HF 3MN(2MF)QNXCInhibition halo/mm

S. aureus ATCC 6538S. aureus ATCC 6538PS. aureus ATCC 29213

0

E. coli ATCC 25922 0 0 0 0 11 24 0 0E. coli S3R9 0 0 0 0 16 26 0 0E. coli S3R22 0 0 0 0 24 26 0 0E. coli TEM-1 CTX M9 0 0 0 0 0 14 0 0E. coli TEM-1 24 16 0 0 16 26 14 0E. coli AmpC MOX-2 0 0 0 0 0 15 0 0E. coli CTX M2E. coli CTX M9

0

S. cerevisiae PYCC 4072C. albicans ATCC 10231

0

Table 5Classification of each strain as susceptible (S) or resistant (R) according to CLSI inpresence of cefoxitin disk (FOX) and ciprofloxacin disk (CIP) by disk diffusion method(Wilder 2005).

Strain Antibiotic

Cefoxitin (30 �g) Ciprofloxacin (5 �g)

S. aureus ATCC 6538 S SS. aureus ATCC 6538P R SS. aureus ATCC 29213 S S

E. coli ATCC 25922 S SE. coli S3R9 S SE. coli S3R22 S RE. coli TEM CTX-M9 S RE. coli TEM-1 S SE. coli AmpC MOX-2 R S

S. aureus: S – sensitive (CIP: zone inhibition ≥21 mm; FOX: zone inhibition ≥22 mm);R – resistant (CIP: zone inhibition ≤15 mm; FOX: zone inhibition <21 mm).E. coli: S – sensitive (CIP: zone inhibition ≥21 mm; FOX: zone inhibition ≥18 mm);R – resistant (CIP: zone inhibition <15 mm; FOX: zone inhibition <14 mm).

each compound tested. All the compounds studied showed no198

antimicrobial activity in the Gram-positive prokaryotic strains and199

eukaryotic strains since no inhibition zone was observed. On the200

other hand, the compounds only presented antimicrobial activity201

in Gram-negative prokaryotic strains, except for E. coli CTX-M2 and202

E. coli CTX-M9. The compound 2A3CQNX presented activity in four203

Gram-negative strains (E. coli ATCC 25922, E. coli S3R9, E. coli S3R22204

and E. coli TEM-1); whereas 3M2QNXC was the compound that205

presented activity in a greater number of strains studied, namely,206

E. coli ATCC 25922, E. coli S3R9, E. coli S3R22, E. coli TEM-1 and207

E. coli AmpC MOX-2. The other compounds, QNX, 2MQNX and208

2HF only presented activity in E. coli TEM-1. Several studies refer209

selective antibacterial activity. Meanwhile, others reveal activity210

in both Gram-positive and Gram-negative cells (Sykes et al. 1982;211

Blondeau et al. 2000; Neu and Labthavikul 1982). The results pre-212

sented in Table 5 seem to indicate that E. coli S3R22 and E. coli TEM213

CTX-M9 are resistant to ciprofloxacin (5 �g) and E. coli AmpC MOX-214

2 is resistant to cefoxitin (30 �g). The other strains, S. aureus and215

E. coli, seem to be sensitive to both antibiotics.216

3.2. Minimum inhibitory concentration217

For the compounds that presented activity using the disk218

diffusion method, minimum inhibitory concentration (MIC) was219

determined and presented in Table 6. These results can be com-220

pared with MIC values previously determined for cefoxitin and221

Table 6Minimum inhibitory concentration (�g/L) results for each group bacterialstrain/compound by microdilution method, CLSI (Wilder 2006).

Strain Compound Minimum inhibitoryconcentration (�g/L)

E. coli ATCC 259222A3CQNX 5003M2QNXC 350

E. coli S3R92A3CQNX 3203M2QNXC 125

E. coli S3R222A3CQNX 3203M2QNXC 200

E. coli TEM CTX-M9 3M2QNXC 125

E. coli TEM-1

QNX 2562MQNX 4002A3CQNX 1003M2QNXC 802HF 329

E. coli AmpC MOX-2 3M2QNXC 100

ciprofloxacin and presented in Table 2 (Rex 2009). The results are 222

presented for each well, bacteria, growth medium and different 223

concentrations of each compound. The results were validated with 224

both negative (compound and growth medium that presented no 225

turbidity) and positive controls (bacteria and growth medium that 226

presented turbidity). The MIC values presented are smaller than 227

those indicated by the CLSI to CIP and FOX, suggesting a more 228

effective activity of quinoxaline derivatives with respect to those 229

antibiotics. 230

3.3. Cellular viability and CFU variation for prokaryotic cells 231

Cellular viability was analyzed for all bacterial 232

strain/quinoxaline derivative pairs that presented growth inhibi- 233

tion by disk diffusion method and for which MIC’s were previously 234

determined. Fig. 1 shows an example of CFU/mL variation along 235

time for Gram-negative prokaryotic strain (E. coli TEM-1). The 236

results obtained show that every strain, in absence of any com- 237

pound, grows up in the first minutes (viable cells), as expected 238

(Chang-Li et al. 1988; Zwietering et al. 1990; Sezonov et al. 2007) 239

and then decreases the number of CFU/mL until 24 h. For each 240

bacterial strain/quinoxaline derivative pairs, the number of viable 241

cells decreases with time, as expected, and at 24 h there are no 242

viable cells. For E. coli TEM-1/2MQNX group the Fig. 1 shows that 243

there are no viable cells at 180 min. CFU/mL variation for other 244

bacterial strains/quinoxaline derivative pairs presented a similar 245

behavior to E. coli in presence and absence of compounds. 246

Please cite this article in press as: Vieira M, et al. Antimicrobial activity of quinoxaline 1,4-dioxide with 2- and 3-substituted derivatives. MicrobiolRes (2013), http://dx.doi.org/10.1016/j.micres.2013.06.015

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Fig. 1. Example of variation for Gram negative prokaryotic strains (E. coli TEM-1) in the presence of quinoxaline derivative at MIC determined.

Fig. 2. Variation along time for S. cerevisiae in absence/presence of the compounds tested.

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3.4. Cellular viability and CFU variation for eukaryotic cells247

Both eukaryotic strains, C. albicans and S. cerevisiae, grow up248

in the first minutes. This is followed by a decrease of cellular via-249

bility (Barford 1990; Bochu et al. 2003). By exposing S. cerevisiae to250

the compounds that presented antibacterial activity (QNX, 2MQNX,251

3M2QNXC, 2A3CQNX, 2HF), the number of viable cells calculated252

by CFU increases in the time-course. as observed in Fig. 2. These253

results seem to indicate that, in the presence of these quinoxaline254

derivatives, S. cerevisiae has a growth rate that increases. Surpris-255

ingly, all the quinoxaline derivatives studied showed no effect on256

growth or cell viability, when compared with the control. Actually,257

growth enhanced rather than declined. Naturally, we do not have258

at this point of the state of the art any explanation for these results,259

that we intend to clarify in further studies.260

Nevertheless, it seems promising that compounds that may be261

potential antimicrobial agents used in humans or veterinary, may262

not be toxic in eukaryotic cells.263

4. Conclusions264

Quinoxaline derivative compounds studied in the present work265

presented selective antimicrobial activity between both eukaryotic266

and prokaryotic strains. Antimicrobial activity was observed only267

in the Gram-negative strains studied, except in E. coli CTX-M2 and268

E. coli CTX-M9. We believe that these two strains may encode some269

kind of mechanism that confers resistance to this group of com-270

pound. No antimicrobial activity was observed for Gram-positive271

prokaryotic strains, possibly due to the presence of peptidoglycan272

in Gram-positive cell wall, permeability properties of the mem-273

branes, or even due to metabolic dissimilarities between these two274

phylogenetic groups, that enables the entrance/activity of the com-275

pounds. This fact may also point to the need of these compounds to276

cross cellular walls to act. At the present time, there are no clues that277

might explain why some quinoxaline derivatives have a distinctive278

behavior between G+ and G− inhibition.279

Moreover, we also have no data that supports why those quinox-280

aline derivatives which present antibacterial activity promote S.281

cerevisiae growth. The mode of action of these quinoxaline deriva-282

tives is not clear yet. We intend to develop further studies in order283

to contribute to this elucidation. The results showed in the present284

study, should guide these works. We now know that they seem285

not to be effective in yeast and in Gram positive strains. So, further286

studies on Gram negative response will be conducted. Since the287

mechanism of action of these compounds is not known it is diffi-288

cult at this point of the state of the art to discuss structure activity289

relationship based on cell viability studies.290

With exception to 2M3BenzoylQNX and 3M3BQNX, that pre-291

sented no activity against the strains studied, all the compounds292

presented activity against both quinolone and/or �-lactams Gram-293

negative resistant strains. The MIC values determined were294

between 80 and 500 �g/L, and are smaller than the admitted values295

for approved drugs with antibiotic activity (CIP and FOX) and with296

similar chemical constitution (CIP) (Table 2).297

The cellular viability for the eukaryotic strain and the death rate298

for the prokaryotic strains studied, at MIC concentrations, suggest299

these compounds as potential new drugs with selective antibacte-300

rial activity for Gram-negative bacteria.301

Funding302

The authors thank Institute Polytechnic of Porto and Santander-303

Totta bank for a scholarship to CV and FCT-MES for a PhD grant304

SFRH/BD/48116/2008 to MV.

Competing interests 305

None. 306

Ethical approval 307

Not required. 308

Acknowledgements 309

Thanks are due to Center of Health and Environmental Research 310

(CISA) to assignment of Integration into Research Fellowship for 311

performance this work, and Centre of Chemistry Investigation (CIQ) 312

of the Chemistry Department, University of Porto, Portugal, for the 313

supply of quinoxaline derivatives. 314

References 315

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