Insights into the Microbicidal, Antibiofilm, Antioxidant and Toxicity Profile of New O-Aryl-Carbamoyl-Oxymino-Fluorene Derivatives

The unprecedented increase in microbial resistance rates to all current drugs raises an acute need for the design of more effective antimicrobial strategies. Moreover, the importance of oxidative stress due to chronic inflammation in infections with resistant bacteria represents a key factor for the development of new antibacterial agents with potential antioxidant effects. Thus, the purpose of this study was to bioevaluate new O-aryl-carbamoyl-oxymino-fluorene derivatives for their potential use against infectious diseases. With this aim, their antimicrobial effect was evaluated using quantitative assays (minimum inhibitory/bactericidal/biofilms inhibitory concentrations) (MIC/MBC/MBIC), the obtained values being 0.156–10/0.312–10/0.009–1.25 mg/mL), while some of the involved mechanisms (i.e., membrane depolarization) were investigated by flow cytometry. The antioxidant activity was evaluated by studying the scavenger capacity of DPPH and ABTS•+ radicals and the toxicity was tested in vitro on three cell lines and in vivo on the crustacean Artemia franciscana Kellog. The four compounds derived from 9H-fluoren-9-one oxime proved to exhibit promising antimicrobial features and particularly, a significant antibiofilm activity. The presence of chlorine induced an electron-withdrawing effect, favoring the anti-Staphylococcus aureus and that of the methyl group exhibited a +I effect of enhancing the anti-Candida albicans activity. The IC50 values calculated in the two toxicity assays revealed similar values and the potential of these compounds to inhibit the proliferation of tumoral cells. Taken together, all these data demonstrate the potential of the tested compounds to be further used for the development of novel antimicrobial and anticancer agents.


Introduction
Antibiotics are one of the most important medical discoveries, leading to a remarkable decrease in the mortality and morbidity caused by infectious diseases, but also fostering the progress of modern medicine, by making possible procedures such as transplantation, cancer chemotherapy and surgery [1,2].
Unfortunately, numerous factors, such as the over-and inappropriate use of antimicrobials not only in human medicine but also in the agricultural and veterinary sectors have now led to the occurrence, enrichment and dissemination of antimicrobial resistance (AMR), mirrored in the high prevalence of antibiotic-resistant infections, accompanied by increased mortality rates (25,000 patients are killed each year in Europe by multi-drug resistant bacteria and the estimates of annual deaths will reach 10 million until 2050 if action is not taken) and a huge economic burden [3][4][5][6].
The problem of AMR is amplified by microbial biofilms, which are represented by microbial communities composed of cells adherent to a surface, protected by a matrix of extracellular polymeric substances, expressing a modified phenotype regarding the growth rate and gene transcription, and exhibiting increased tolerance (sometimes hundred up to thousand times higher than their planktonic counterparts) to antibiotics and other chemical inhibitors [12,13].
Antibiotic-resistant strains are currently isolated from hospital-and communityacquired infections and from the natural environment. A so-called "post-antibiotic era" is expected to appear, which would make it impossible to treat infections caused by multidrugresistant strains. Many international authorities thus advocate identifying incentives to encourage research in the field of antimicrobial drug discovery.
In this context, the aim of this paper was to evaluate the antimicrobial features of new O-aryl-carbamoyl-oxymino-fluorene derivatives, previously reported for their inhibitory activity against Gram-positive (e.g., Bacillus anthracis, S. aureus, including methicillin resistant strains) and Gram-negative (Escherichia coli, Proteus mirabilis, K. pneumoniae, P. aeruginosa, Burkholderiathailandensis and Francisella tularensis) bacterial strains, mycobacteria, yeasts and molds [14][15][16][17]. The antioxidant activity of these compounds has been also evaluated, as it could represent an advantage for novel antimicrobial leads by decreasing the intensity and duration of the inflammatory response often accompanying the infectious process, thus avoiding their deleterious effects on the host tissues [18]. It is well known that oxidative stress represents an imbalance between the generation of free radicals and a decrease in the concentration of endogenous antioxidants (such as glutathione, vitamin C, vitamin E and a series of enzymes-catalase, superoxide dismutase and peroxidases) [19]. Free radicals (ROS-reactive oxygen species or RNS-reactive nitrogen species) contain more than one unpaired electron, which is unstable and attacks proteins, nucleic acids and lipids [19]. Generally, ROS include superoxide anion (O 2 − ), hydrogen peroxide (H 2 O 2 ), hydroxyl radical (OH·), singlet oxygen or nitric oxide (NO) [19]. Endogenous sources of free radicals include the mitochondrial respiratory chain, mental stress, aging, inflammation, or ischemia/reperfusion [20,21]. Free radicals have a dual behavior, as at higher concentrations they have shown negative effects upon the biological system (being involved in autoimmune, cardiovascular, neurodegenerative, and metabolic diseases or cancer) [22], whilst low/moderate amounts have beneficial properties (modulation of different signaling pathways, phagocytosis, mitogenic response, etc.) [22]. However, infections with resistant bacteria (Staphylococcus aureus, Escherichia coli or Pseudomonas aeruginosa, Proteus sp.) [23][24][25][26], as previously mentioned, lead to chronic inflammation with the continuous generation of cytokines and chemokines by macrophages (such as interleukins IL-4, IL-5, IL-12 or tumor necrosis factor TNF-α). The activation of macrophages is further involved in the modulation of several pathways (mediated by nuclear factor kappa B, activator protein 1, nuclear factor of activated T cells, hypoxia-inducible factor 1-α), with the ongoing hyperproduction of ROS and RNS. An important consequence of chronic inflammation is tissue damage due to persistent oxidative stress and the excessive induction of tissue repair mechanisms [27].

Antimicrobial Activity against Planktonic and Biofilm Embedded Microbial Cells
The tested compounds showed an inhibitory effect on microbial growth at minimum inhibitory concentration (MIC) values of 0.156-10 mg/mL (Table 1) and minimum bactericidal concentration (MBC) values between 0.312 and 10 mg/mL ( Table 2). The four compounds inhibited the ability of bacterial and fungal strains to adhere and develop biofilms on the inert substratum, with a minimum biofilm inhibitory concentration (MBIC) of 0.009-1.25 mg/mL (Table 3).  The FCM assay results have shown that the green fluorescence of the potentialsensitive probe, DiBAC4(3), was enhanced at subinhibitory concentrations, supporting the hypothesis that the in vitro bactericidal activity of the tested compounds was the result of cytoplasmic membrane potential dissipation. However, it has been suggested that membrane depolarization is required to facilitate the entry of antibiotics into bacteria for the expression of activity. Additionally, the disruption of membrane function may actually have intracellular targets [57]. Thus, further studies are needed for the elucidation of the mechanisms of antimicrobial action.
The compound 1c did not cause changes in the membrane potential of the two Grampositive strains, i.e., E. faecalis ATCC 29212 and S. aureus ATCC 25923. Table 4 shows the membrane depolarization demonstrated by the increased values of the staining index (SI).

The Toxicity Profile of the Tested Compounds on the Artemia franciscana Kellog Model
Three of the four compounds (1a-c) were not toxic at the tested concentrations, all nauplii were alive and showing normal movements. Compound 1d manifested moderate toxicity, as evidenced by the lethality curve (concentration-response) ( Figure 1) and by the LC50 value (14.63 µg/mL, 95% CI 11.80-18.15 µg/mL).
Three of the four compounds (1a-c) were not toxic at the tested concentrations, all nauplii were alive and showing normal movements. Compound 1d manifested moderate toxicity, as evidenced by the lethality curve (concentration-response) ( Figure 1) and by the LC50 value (14.63 μg/ mL, 95% CI 11.80-18.15 μg/ mL).

Antioxidant Activity Evaluated by Scavenger Activity towards DPPH andABTS •+ Free Radicals
The antioxidant activity was tested only for the compounds 1a-c (tested in a two-fold concentration range from 25 to 1000 μM), which proved no cytotoxicity in the previous Artemia franciscana Kellog in vivo assay. The absorbance values for all analyzed compounds decreased with the increase in concentration. varying between 0.9310 nm (at 25 μM) and 0.8547 nm (at 1000 μM) for compound 1a; 0.9269 nm (at 25 μM) and 0.8923 nm (at 1000 μM) for compound 1b and 0.8770 nm (at 25 μM) and 0.8026 nm (at 1000 μM) for compound 1c. The results of the DPPH free radical scavenger activity are presented in Figure 2. DPPH free radical scavenger capacity varies between 12.66% (1a at the concentration of 25 μM) and 24.64 % (1c at the concentration of 1000 μM). Moreover, compound 1a inhibited with 19.81% free radical activity at the maximum concentration of 1000 μM. The lowest scavenger activity recorded at the highest tested concentration of 1000 µ M was observed for compound 1b (16.29%). However, since the scavenging activity was low (below 30%), all tested compounds have shown very high IC50 (μM) values beyond 1000 μM (Table 5), which is a strong indicator of the low antioxidant potential. Still, the lowest IC50 value, which indicates the best antioxidant activity, was obtained for compound 1c, followed by compounds 1a and 1b (Table 5). Significant differences have been found between IC50 values for all tested compounds (p < 0.0001) ( Table 6).

Antioxidant Activity Evaluated by Scavenger Activity towards DPPH and ABTS •+ Free Radicals
The antioxidant activity was tested only for the compounds 1a-c (tested in a two-fold concentration range from 25 to 1000 µM), which proved no cytotoxicity in the previous Artemia franciscana Kellog in vivo assay. The absorbance values for all analyzed compounds decreased with the increase in concentration. varying between 0.9310 nm (at 25 µM) and 0.8547 nm (at 1000 µM) for compound 1a; 0.9269 nm (at 25 µM) and 0.8923 nm (at 1000 µM) for compound 1b and 0.8770 nm (at 25 µM) and 0.8026 nm (at 1000 µM) for compound 1c. The results of the DPPH free radical scavenger activity are presented in Figure 2. DPPH free radical scavenger capacity varies between 12.66% (1a at the concentration of 25 µM) and 24.64 % (1c at the concentration of 1000 µM). Moreover, compound 1a inhibited with 19.81% free radical activity at the maximum concentration of 1000 µM. The lowest scavenger activity recorded at the highest tested concentration of 1000 µM was observed for compound 1b (16.29%). However, since the scavenging activity was low (below 30%), all tested compounds have shown very high IC50 (µM) values beyond 1000 µM (Table 5), which is a strong indicator of the low antioxidant potential. Still, the lowest IC50 value, which indicates the best antioxidant activity, was obtained for compound 1c, followed by compounds 1a and 1b (Table 5). Significant differences have been found between IC50 values for all tested compounds (p < 0.0001) ( Table 6).   In the second assay, the absorbance of ABTS •+ free radical solution in the presence tested compounds varied between 0.5802 (at 25 μM) and 0.5564 (at 1000 μM) for co pound 1a, 0.5332 (at 25μM) and 0.5109 (at 1000 μM) for compound 1b, 0.5848 (at 25 μ and 0.5658 (at 1000 μM) for compound 1c. The scavenger activity increases with conc tration, regardless of the analyzed compound ( Figure 3). Free radical scavenger (Table activity varied between 12.53% (1c at 25 μM) and 26.60% (1b at 1000 μM). For the tes concentration range, the highest scavenger activity was observed for compound 1b, a the lowest for compound 1c. In comparison with the DPPH assay, in the ABTS assay higher scavenging activity was observed for 1a, and b at all tested concentrations, wh for 1c, the inhibition was lower.  In the second assay, the absorbance of ABTS •+ free radical solution in the presence of tested compounds varied between 0.5802 (at 25 µM) and 0.5564 (at 1000 µM) for compound 1a, 0.5332 (at 25 µM) and 0.5109 (at 1000 µM) for compound 1b, 0.5848 (at 25 µM) and 0.5658 (at 1000 µM) for compound 1c. The scavenger activity increases with concentration, regardless of the analyzed compound ( Figure 3). Free radical scavenger (Table 7) activity varied between 12.53% (1c at 25 µM) and 26.60% (1b at 1000 µM). For the tested concentration range, the highest scavenger activity was observed for compound 1b, and the lowest for compound 1c. In comparison with the DPPH assay, in the ABTS assay, a higher scavenging activity was observed for 1a, and b at all tested concentrations, while for 1c, the inhibition was lower.  All tested compounds exhibited a low antioxidant effect, with high IC50 (μM) values beyond 1000 μM (Table 7). Analyzing Table 7, one can note that the best antioxidant activity was observed for 1b followed by 1a,c. These differences among antioxidant assays are probably the consequence of the compound lipophilicity and specific mechanism of action against free radicals. On the other hand, the higher antioxidant effect of compound 1b can be explained by the inductive effect of the methyl group that confers greater molecular stability. Significant differences have been found between IC50 values for all tested compounds (p < 0.0001) ( Table 8). CI-confidence interval; *** = p < 0.001

Cytotoxicity
The cytotoxicity of the investigated compounds was tested using theIncuCyte Basic Analysiskit, allowing to calculate the IC50, against three tumoral cell lines, i.e., HeLa (cervical cancer cells), HT29 (colon adenocarcinoma) and MG63 (osteosarcoma). The selected metric of the concentration-response module was set on phase and confluence (%). The All tested compounds exhibited a low antioxidant effect, with high IC50 (µM) values beyond 1000 µM (Table 7). Analyzing Table 7, one can note that the best antioxidant activity was observed for 1b followed by 1a,c. These differences among antioxidant assays are probably the consequence of the compound lipophilicity and specific mechanism of action against free radicals. On the other hand, the higher antioxidant effect of compound 1b can be explained by the inductive effect of the methyl group that confers greater molecular stability. Significant differences have been found between IC50 values for all tested compounds (p < 0.0001) ( Table 8).

Cytotoxicity
The cytotoxicity of the investigated compounds was tested using the IncuCyte Basic Analysiskit, allowing to calculate the IC50, against three tumoral cell lines, i.e., HeLa (cervical cancer cells), HT29 (colon adenocarcinoma) and MG63 (osteosarcoma). The selected metric of the concentration-response module was set on phase and confluence (%). The IC50 levels are presented in Table 9. The new derivatives exhibited cytotoxicity at concentrations lower than 100 µg/mL, from 6.33 ± 3.02 to 31.5 µg/mL, as observed from the IC50 calculation, the susceptibility of the tested cell lines increasing from MG63 cells to HeLa cells.

Discussion
Less than half a century after the discovery of antibiotics, we are now threatened to enter the post-antibiotic era, where the fatality rate due to infections will increase sharply, particularly in less developed countries and in the infant population. A lot of modern medical procedures, such as transplants, surgery or chemotherapy will no longer be possible because of infections with multidrug-resistant bacteria. Thus, there is an acute need to find antibiotics with original structures, having new microbial targets. Taking into account that fluorine is found in diverse pharmacologically active compounds with antimalarial (lumefantrine), antiarrhythmic (indecainide), muscle relaxant (hexafluronium bromide or antiviral (tilorone) activity as well as the pharmacological activities of carbamoyl and oximinic pharmacophore groups, we have combined these biologically active fragments into a single original molecule, to obtain new compounds of the class O-aryl-carbamoyloxymino-fluorene, previously characterized [17] (Figure 4) and to evaluate their potential bactericidal, fungicidal and antibiofilm effects.
IC50 levels are presented in Table 9. The new derivatives exhibited cytotoxicity at concentrations lower than 100 µ g/mL, from 6.33 ± 3.02 to 31.5 µ g/mL, as observed from the IC50 calculation, the susceptibility of the tested cell lines increasing from MG63 cells to HeLa cells.

Discussion
Less than half a century after the discovery of antibiotics, we are now threatened to enter the post-antibiotic era, where the fatality rate due to infections will increase sharply, particularly in less developed countries and in the infant population. A lot of modern medical procedures, such as transplants, surgery or chemotherapy will no longer be possible because of infections with multidrug-resistant bacteria. Thus, there is an acute need to find antibiotics with original structures, having new microbial targets. Taking into account that fluorine is found in diverse pharmacologically active compounds with antimalarial (lumefantrine), antiarrhythmic (indecainide), muscle relaxant (hexafluronium bromide or antiviral (tilorone) activity as well as the pharmacological activities of carbamoyl and oximinic pharmacophore groups, we have combined these biologically active fragments into a single original molecule, to obtain new compounds of the class O-aryl-carbamoyl-oxymino-fluorene, previously characterized [17] (Figure 4) and to evaluate their potential bactericidal, fungicidal and antibiofilm effects. The most susceptible strains in planktonic growth were S. aureus, followed by the P. aeruginosa strain. The most active compound against planktonic cells was 1d, which exhibited the lowest MIC value of 0.156 mg/mL against the S. aureus strain. The MBC values were similar or twice as high as the MIC ones, indicating that bactericidal activity and membrane depolarization were correlated for compound 1d-treated S. aureus cells, suggesting a dose-dependent bactericidal effect of the tested compounds on the membrane integrity, as revealed by the FCM analysis.
The biofilm formed by E. faecalis manifested an increased susceptibility to compound 1c, S. aureus to 1d, P. aeruginosa to 1a, E. coli to 1b and that of C. albicans to all tested compounds, and mainly to 1b. The MBEC values have been significantly lower (up to hundreds of times) than the corresponding MIC and MBC ones. The most susceptible strains in planktonic growth were S. aureus, followed by the P. aeruginosa strain. The most active compound against planktonic cells was 1d, which exhibited the lowest MIC value of 0.156 mg/mL against the S. aureus strain. The MBC values were similar or twice as high as the MIC ones, indicating that bactericidal activity and membrane depolarization were correlated for compound 1d-treated S. aureus cells, suggesting a dose-dependent bactericidal effect of the tested compounds on the membrane integrity, as revealed by the FCM analysis.
The biofilm formed by E. faecalis manifested an increased susceptibility to compound 1c, S. aureus to 1d, P. aeruginosa to 1a, E. coli to 1b and that of C. albicans to all tested compounds, and mainly to 1b. The MBEC values have been significantly lower (up to hundreds of times) than the corresponding MIC and MBC ones.
Taken together, the results of the antimicrobial activity assays suggest that the electronwithdrawing inductive effect of chlorine atoms enhanced the activity against planktonic and adhered S. aureus, while the +I effect of the methyl group enhanced the anti-fungal activity against C. albicans strain.
The evaluation of the undesired cytotoxic effects of new molecules aimed to be developed as a pharmaceutical is crucial to ensure drug safety and effectiveness. The toxicity on the Artemia franciscana Kellog crustacean species was assessed based on the method of B. N. Meyer et al. [58] and T.W. Sam [59], with slight adaptations suggested by more recent sources [60][61][62]. Robust methods were used to model the concentration-response relationship and to calculate the IC50 and IC 95% values. It must be underlined that an IC50 value of 10-30 µg/mL corresponds to moderate toxicity, as revealed in the case of compound 1d exhibiting an acute toxicity of 14.63 µg/ mL, which is very close to the one of cyclophosphamide, which has an IC50 value of 16.3 µg/mL [63,64]. The other evaluated compounds (1a, 1b and 1c) were not toxic at concentrations up to 100 µg/mL, therefore, at the solubility limit. The IC50 calculated for the four compounds using the in vitro cytotoxicity assay on three tumoral cell lines ranged between 6 and 32 µg/mL. These findings provide insights into the further investigation of these derivatives for their potential as a therapeutic targeting rapidly dividing cancer cells. Furthermore, in our future research, we will establish the cellular targets and pathways activated by these derivatives, which will provide insights into their mechanism of action.
The antioxidant activity was tested by two assays, i.e., the scavenger activity towards the DPPH and ABTS •+ free radicals. The DPPH (2,2-diphenyl-1-picrylhydrazyl) is a colored free radical that is reduced by antioxidants to pale-yellow hydrazine. The color change leads to a reduction in absorbance values [65][66][67]. Briefly, the method is based on electron transfer, although some authors consider that both electron and hydrogen atom transfer processes are involved. The method's advantages are that it is simple, and inexpensive and the provided results are well correlated with those obtained by other methods [3,68,69]. The ABTS •+ free radical is produced in the reaction of 2,2 -azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt with manganese (II) oxide [70], sodium/potassium persulfate [71], 2,2'-azobis(2-amidinopropane) dihydrochloride [72] or enzymes (peroxidase) [6]. The free radical has a blue color, with the ABTS •+ free radical being reduced in the presence of oxidants; the blue color loss is accompanied by a decrease in absorbance [3,67]. The method has several advantages: it is simple and reproducible, it does not depend on pH and can be used for the evaluation of both lipophilic and hydrophilic compounds. The method is based mainly on proton transfer [6]. The reaction time between ABTS and the substrate varies between 4-6 and 60 min [71,73].
Generally, the scavenger capacity towards both free radicals for the tested concentrations was below 30%. Significant differences have been found between IC50 (µM) values) of tested compounds by means of both antioxidant assays. The differences among DPPH and ABTS assays, between the analyzed compounds, are probably due to the lipophilicity of the compounds or to the specific mechanism of the antioxidant agent. Although the antioxidant activity is desirable for protecting the host cells from the toxic effects of an antibiotic, there are studies raising awareness that at least in anaerobic environments, the antioxidant activity could interfere with the ROS-mediated lethality of bactericidal antibiotics such as ampicillin, gentamicin or norfloxacin. The pretreatment with glutathione and ascorbic acid antioxidants decreased the lethality induced by ampicillin, gentamicin, and norfloxacin by at least 1-log at 4 h posttreatment [74].

Microbiological Assays
The inhibitory activity of the obtained compounds has been assessed on five microbial strains, respectively: E. coli ATCC 25922, P. aeruginosa ATCC 27853, S. aureus ATCC 25923, E. faecalis ATCC 29212 and C. albicans ATCC 90029, in a planktonic and adherent growth state.
The MIC assay was performed by the serial two-fold microdilutions in a liquid medium, using a concentration range from 5 to 0.009 mg/mL and microbial suspensions of 0.5 MacFarland density. The wells containing only the microbial culture served as positive control and those containing the sterile culture medium as the negative control. The MIC values were read after incubation at 37 • C for 24 h [75].
The MB assay was performed after reading the MIC values. For this purpose, 10 µL volumes of the liquid culture developed in the wells containing the highest concentration to the MIC value were plated on a solid culture medium to determine the MBC value (the concentration that has totally inhibited the microbial growth).
The anti-biofilm activity assay was performed by the purple violet microtiter method, as previously described, allowing us to determine the minimal biofilm inhibitory concentration (MBEC). The same range of concentrations, as in the MIC assay, i.e., from 5 to 0.009 mg/mL has been tested [76].
The investigation of potential mechanisms of antimicrobial action by flow cytometry (FCM) was performed on microbial cultures obtained after 18-24 h of microbial cell incubation in the presence of subinhibitory concentrations of the analyzed compounds. Thus, microbial suspensions with a density of approximately 10 6 CFU/mL were prepared in sterile saline phosphate buffer from exponentially growing microbial cultures, obtained on a solid culture medium. Subinhibitory concentrations of the compounds were prepared in Muller-Hinton liquid culture medium and inoculated with an equal amount of microbial suspension and incubated for 18-24 h at 37 • C.
Fluorescence intensity (FI) was measured with an Accuri C6 plus flow cytometer in the FITC fluorescence channel. Growth control was used to locate the microbial cell population for fluorescence measurements. DiBAC4 dye was used to detect changes in the microbial membrane potential. The fraction of microbial cells in the analyzed population that showed increased green fluorescence (corresponding to membrane depolarization) was calculated after the exclusion of untreated growth control fluorescence. A twofold increased fluorescence intensity (median fluorescence intensity = MFI) was considered to correspond to the depolarized microbial cells. For each subinhibitory concentration, a coloring index (CI) was calculated which represents the ratio of fluorescence intensity (FI) of treated versus untreated cells [77].

The Toxicity on the Artemia Franciscana Kellog Crustacean Species
The oocysts (Great Salt Lake, USA) provided by S.K. Trading were grown on artificial seawater medium (Coral Marine, Grotech), dissolved in distilled water with a few minutes of sonication, at 30 g/L concentration. The test was carried out in a 24-well plate, using three replicates. Because of their low solubility, the compounds 1a-d were suspended in the culture medium using sodium alginate (0.045%) (also used a negative control), the testing being carried out at the solubility limit. The concentrations 100, 50, 25, 12.5 and 6.2 µg/mL used for each substance were obtained by successive dilutions starting from the initial suspension. Between 10 and 15 nauplii per well were collected and placed in contact with the test suspensions (1.5 mL/well). All nauplii, dead or alive, were counted and recorded within 24 h of being placed in contact with the tested suspensions. The non-linear modeling of the concentration-lethality relationship was achieved through a four-parameter logistics model (4PL), implemented in several robust variants for estimating parameters in the R package "dr4pl" [78]. . For cytotoxicity kinetics, the IncuCyte ® S3 Live-Cell Analysis System (Sartorius, AG, Goettingen, Germany) was used. Briefly, 2 × 10 4 cells seeded in 96 well plates were treated with the new derivatives in a concentration ranging between 500 µg/mL and 3.9 µg/mL. The plates were placed in the IncuCyte ® S3 Live-Cell Analysis System, and five images per well were taken every six h over a 72-h period and then processed, according to kit recommendations.

Antioxidant Activity of Compounds
Scavenger Activity towards DPPH Free Radical The DPPH (Sigma-Aldrich, Darmstadt, Germany) solution of 0.1 mM concentration was obtained by dissolving 0.0039 g of free radical in 100 mL ethanol, in a volumetric flask. For all determinations, the DPPH solution was freshly prepared and kept in the dark.
The determination of the antioxidant activity was based on the Ohnishi M et al. method [79], quoted by Germano M.P. et al. [80].
For this purpose, 0.5 mL of 25-1000 µM tested solutions were treated with 3 mL DPPH solution (0.1 mM). The samples were kept at rest in the dark for 30 min [79,81]. Ethanol was used as a blank in order to measure the absorbance at 515 nm (Jasco V-530 spectrophotometer, Jasco, Japan).
The following formula was used to determine DPPH free radical scavenger activity (%) [5,82]: where, A = absorbance of the 0.1 mM DPPH solution in the absence (control)/presence (sample) of tested compounds after 30 min. Scavenger Activity towards ABTS •+ Free Radical The method according to Re R. et al. was used to evaluate the scavenger capacity of the free radical [71].
In order to obtain the ABTS •+ free radical reagent, equal volumes of solutions 1 and 2 were mixed for 16 h. The obtained reagent was kept in the dark; 1 mL of the obtained solution was brought to a 50 mL volumetric flask and diluted with ethanol so that the absorbance at λ = 734 nm would be 0.700 ± 0.02.
The tested compounds were dissolved and diluted similar to the previous antioxidant assay.
A volume of 0.5 mL of 25-1000 µM solutions was treated with 3 mL ABTS •+ solution, stirred and kept in the dark for 6 min and then, the absorbance was read at 734 nm (Jasco V-530 spectrophotometer) using ethanol as a blank.
The following formula was used for the calculation of the ABTS •+ free radical scavenger activity (%) where: Abst = ABTS •+ solution absorbance in the absence (0 min)/presence (6 min) of the tested compounds.
For both the above-mentioned methods, the antioxidant activity was expressed as IC 50 values (µM) which represent the concentration of each compound for which the scavenging activity of free radicals is 50%. The IC 50 values were calculated by linear regression plots, where the abscissa was represented by the concentration of the tested compound solution (25-1000 µM) and the ordinate the average percent of antioxidant capacity from three separate tests.

Statistical Analyses
For each tested concentration, all the determinations were carried out in triplicate; we established the mean ± standard deviation (SD) of three independent determinations. Microsoft Office (Excel 2007) and GraphPad Prism v.5 (GraphPad, SUA) were used to perform the statistical analysis. The antioxidant activity of the analyzed compounds was compared using the one-way ANOVA test followed by the Tukey post-test (p < 0.05 for statistical significance).

Conclusions
A series of four O-aryl-carbamoyl-oxymino-fluorene derivatives previously synthesized using the intermediate compound 9H-fluoren-9-one oxime and previously characterized have been bioevaluated in this paper, to formulate potential leads for their biomedical applications.
The four derivatives proved to inhibit the tested microbial strains' growth, in both planktonic and adherent states. The electron-withdrawing inductive effect of chlorine atoms enhanced the anti-staphylococcal activity, both against free-floating and adherent cells, while the +I effect of the methyl group favored the anti-fungal activity. Thus, they can be considered for further antimicrobial agent development.
The analysis of the effects on the membrane potential of the tested microbial strains showed that, at subinhibitory concentrations, they produce a depolarization of the plasma membrane, which is thus one of the targets of their antimicrobial activity.
The compounds were evaluated for their in vitro cytotoxicity on three cell lines and in vivo acute toxicity. All four tested compounds exhibited a similar profile of cytotoxicity on the three cellular lines, HeLa, HT29 and MG63, while three of them (1a-c) were non-toxic at the solubility limit on Artemia franciscana Kellog model.
The compounds have shown a modest scavenger capacity towards the DPPH free radical (<30%) in the tested concentration range, the most active being 1a,c. All tested compounds have shown scavenger activity towards the ABTS•+ free radical in the tested concentration range, with compound 1b exhibiting the best antioxidant activity, correlated with the +I effect and electron donating tendency of the methyl group. The antioxidant activity of these compounds could represent an advantage for novel antimicrobial leads, acting by decreasing the intensity and duration of the inflammatory response often accompanying the infectious process, thus avoiding their deleterious effects on the host tissues.
Taken together, all these data demonstrate the potential of the tested compounds to be further used for the development of novel antimicrobial and anticancer agents.