Christophe Ricco

Fatma Abdmouleh

Charlotte Riccobono

Léna Guenineche

Frédérique Martin

Elizabeth Goya-Jorge

Nathalie Lagarde

Mamdouh Bertrand Liagre

Mamdouh Ben Ali

Clotilde Ferroud

Mehdi El Arbi

Maité Sylla-Iyarreta Veitía

PEGylated triarylmethanes: Synthesis

Keywords: antimicrobial activity, anti-proliferative behavior and in silico studies triarylmethane, PEGylation, antimicrobial and anti-proliferative activity, antibiofilm activity, drug-likeness, Molecular modeling, DHFR and DNA gyrase B inhibitors aureus E. coli P. aeruginosa S. enterica C. albicans

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Triarylmethanes (TAMs) are molecules with three aryl groups (phenyls or heterocycles) bonded to a central carbon atom and they have numerous applications. They are essentially known as compounds derived from the dye industry and used for the dyeing of cotton, wool and silk [1,[START_REF] Rose | The condensed chemical dictionary[END_REF].

The various and very interesting properties of TAMs have attracted the attention of many scientists for several years [START_REF] Vijay Nair | Recent advances in the chemistry of triaryland triheteroarylmethanes[END_REF]. They are used in the food and textile industry, in the field of health, and as antifungal agents and antiparasitic agents in the fish farming industry or else as cell division inhibitors for plants [4,[START_REF] Nambo | Combination of Synthetic Chemistry and Live-Cell Imaging Identified a Rapid Cell Division Inhibitor in Tobacco and Arabidopsis thaliana[END_REF]. Recent studies show that TAMs and derivatives are interesting from the therapeutic point of view and in particular in the development of anti-cancer [START_REF] Palchaudhuri | The Complex Role of the Triphenylmethyl Motif in Anticancer Compounds[END_REF][START_REF] Arbiser | Triarylmethane analogs and their use in treating cancers[END_REF], anti-tuberculosis drugs [START_REF] Panda | Diaryloxy methano phenanthrenes: a new class of antituberculosis agents[END_REF][START_REF] Panda | Design, synthesis and antitubercular activity of compounds containing aryl and heteroaryl groups with alkylaminoethyl chains[END_REF], antivirals [START_REF] Douglas | Inhibition of respiratory syncytial virus fusion by the small molecule VP-14637 via specific interactions with F protein[END_REF][START_REF] Douglas | Small molecules VP-14637 and JNJ-2408068 inhibit respiratory syncytial virus fusion by similar mechanisms[END_REF] and anti-inflammatory drugs [START_REF] Sylla-Iyarreta | Fishing Anti-Inflammatories from Known Drugs: In Silico Repurposing, Design, Synthesis and Biological Evaluation of Bisacodyl Analogues[END_REF]. TAMs made up of heterocycles such as indoles, pyridines, pyrroles and aromatic rings such as naphthalenes, are often used in the pharmaceutical and cosmetic industries [13][START_REF] Wainwright | Uptake and cell-killing activities of a series of Victoria blue derivatives in a mouse mammary tumour cell line[END_REF][START_REF] Indig | Effect of molecular structure on the performance of triarylmethane dyes as therapeutic agents for photochemical purging of autologous bone marrow grafts from residual tumor cells[END_REF][START_REF] Guerin | Dyeing composition at acidic pH comprising a direct dye of triarylmethane structure[END_REF][START_REF] Seneca | Hair cosmetic water-in-oil emulsions comprising triarylmethane direct dyes[END_REF].

Concerned by the high interest in infectious disease research, we previously reported the antibacterial activity of bisacodyl (BSL), drug triarylmethane type, used in therapeutics as laxative and a series of analogues bearing the ferrocenyl moiety. Our study revealed the potentialities of bisacodyl and its derivatives as antibacterial agents against gram-positive pathogens showing an antimicrobial activity comparable to fusidic acid, a steroid antibiotic of narrow spectrum known as a protein synthesis inhibitor [START_REF] Görmen | Ferrocenyl analogues of bisacodyl: Synthesis and antimicrobial activity[END_REF]. However, some solubility problems were highlighted during the biological studies.

Small molecular drugs often suffer some drawbacks, such as low solubility, high toxicity, rapid excretion or untargeted biodistribution. To overcome these obstacles, one promising approach is to use a PEGylation strategy. Thanks to their favorable properties (nontoxic, non-immunogenic, nonantigenic and amphiphilic), PEG as modifying polymer plays an important role in drug discovery.

PEG-drug conjugates have several advantages: prolonged residence in body, decreased degradation by metabolic enzymes, reduction or elimination of protein immunogenicity, and so on [START_REF] Li | Current drug research on PEGylation with small molecular agents[END_REF][START_REF] Thakur | Synthesis of a PEGylated Dopamine Ester with Enhanced Antibacterial and Antifungal Activity[END_REF][START_REF] Mishra | PEGylation in anti-cancer therapy: An overview[END_REF][START_REF] Lopez | Antibacterial activity and cytotoxicity of PEGylated poly (amidoamine) dendrimers[END_REF].

As part of our ongoing research and considering the importance of triarylmethane scaffold as a privileged structure in drug discovery, the present work deals with the synthesis and the biological evaluation of novel PEGylated triarylmethanes 4a-e, 5, 7, 8a-f, 9, from commercial and simple raw materials. All the PEGylated compounds were evaluated against Gram-positive and Gram-negative pathogens, Candida albicans and by their antibiofilm activity. Furthermore, the antiproliferative activity of these derivatives was evaluated against two human colorectal cancer cell lines (HT-29 and HCT-116). Finally, docking studies were carried out to try to understand the mechanism of antimicrobial activity. In silico predictions were also used to analyze and comment ADME properties and drug-likeness of the novel PEGylated compounds.

Results and discussions

Chemistry

The synthesis described here involves the preparation of a novel series of PEGylated triarylmethanes to evaluate their biological potential. General synthetic methods to obtain the target compounds are outlined in Scheme 1 and 2. The non-symmetrical PEGylated derivatives were prepared following the synthetic pathways represented in Scheme 1. First the synthesis of the (4methoxyphenyl)(pyridin-2-yl)methanol 2 has been accomplished by a magnesium-bromine exchange following the procedure described by Park et al. 2013[23] , from 2-bromopyridine and panisaldehyde in anhydrous THF. Friedel-Crafts hydroxylalkylation of compound 2 with phenol using concentrated sulfuric acid as brønsted acid. The reaction was very simple, avoiding the use of costly catalyst systems. The PEGylated non-symmetrical triarylmethanes 4(a-e) were prepared from compound 3 and obtained in a range of 33% to 72% in high purity ( 1 H NMR ˃ 96%).

For the preparation of symmetrical PEGylated triarylmethanes 8(a-e), bisacodyl was previously saponified using standard conditions. After 72 h at room temperature compound 7 was obtained in 92% yield and was pure enough to be used in the next step without any purification as suggested by the 1 H NMR analysis. The preparation of symmetrical PEGylated triarylmethanes 8(a-e), was performed by reaction of deacetylated bisacodyl 7 with the corresponding glycol mono-tosylates in DMF. The reaction was conveniently carried out by mixing the reagents at 80 °C in an inert solvent and then allowing the mixture to stirring overnight. Under these conditions the PEGylated compounds 8(b-e) were obtained with yields ranging between 45% and 70% while compound 8a could not be obtained. Consequently, a different procedure has been employed to prepare compound 8a using 2-bromoethanol in DMF in the presence of K2CO3 and KI. After 96 hours the desired compound 8a was afforded in 15% yield mostly accompanied by the monosubstituted derivative 8f (50% yield) (Scheme 2). N-oxide 9 were isolated after purification by flash chromatography on silica gel with non-optimized yields (83% and 64% respectively). These results have not been optimized and could probably be improved by modifying the treatment conditions. All synthesized compounds were biologically evaluated.

Biological evaluation

Antimicrobial activity

All synthesized final molecules were screened for their antibacterial activity against Gram-positive and Gram-negative pathogens by the 96-well ELISA microdilution method. Gram-positive strains Micrococcus luteus (LB14110), Staphylococcus aureus (ATCC 9144), Gram-negative strains Escherichia coli (ATCC 8739), Salmonella enterica (NCTC 6017) and Pseudomonas aeruginosa (ATCC 9027), were used for inhibitory tests, using levofloxacin, a broad spectrum antibiotic and fusidic acid, a narrow antibiotic as controls.

The previous results obtained with bisacodyl concerning its potential as antimicrobial agent, encouraged us to develop a new series of PEGylated triarylmethanes with improved antimicrobial potency and good pharmacological profile. The Table 1 summarizes the minimum inhibitory concentration (MIC) values expressed in micrograms per milliliter (μg/mL) obtained for PEGylated derivatives (compounds 4a-e, 5, 7, 8a-f, 9), bisacodyl and controls. The MIC and minimum bactericidal concentration (MBC) values for levofloxacin were found to be < 1.52 μg/mL and 12.5-25 μg/mL for fusidic acid for all pathogens. The results revealed that the MBC of all derivatives was superior than 100 μg/mL which could mean that all evaluated compounds seem to be more bactericidal than bacteriostatic (MBC/MIC ratio is less than or equal to four) [START_REF] O'neill | Preclinical evaluation of novel antibacterial agents by microbiological and molecular techniques[END_REF] . All the synthesized compounds 4a-e, 5, 7, 8a-e, 9 had a moderate in vitro antimicrobial activity against all microorganisms tested.

Table 1

Antimicrobial activities of non-symmetrical and symmetrical PEGylated triarylmethanes. Minimum inhibitory concentration (MIC) in μg/mL.

3.125-6.25 or 6.25-12.5 μg/mL) as compared to fusidic acid MIC (12.5-25 μg/mL). Compounds 4b, 4c and 8f showed the best antibacterial activity against Micrococcus luteus with MIC values between 3.125-6.25 μg/mL. The influence of the size of the PEGylated chain on the antibacterial activity is not easily discernable. Compounds 4b and 4c bearing methoxy group and a PEGylated chain with n equal 2 or 3 carbon atoms showed a higher activity against Micrococcus luteus (MIC < 3.125-6.25 μg/mL). Nevertheless, the activity slightly declines when the length of the PEGylated chain increases (n= 4 or 5) (MIC < 6.25-12.5 μg/mL). Furthermore, when comparing the activities of compounds 4a (MIC < 6.25-12.5 μg/mL) and 8f (MIC < 3.125-6.25 μg/mL) against Micrococcus luteus, it could be assumed that methoxy group would be not critical for the activity. Compound 4e showed the best activity against Staphylococcus aureus, with MIC values between 12.5-25 μg/mL.

No difference for antimicrobial activity against both gram-negative pathogens was observed between non-symmetrical PEGylated triarylmethanes.

The symmetrical PEGylated derivatives (8a-e, 9) like their non-symmetrical analogues also exhibited the greatest antimicrobial activity against Micrococcus luteus (MIC values between 6,25-25 μg/mL). However, no significant improvement of the activity was observed compared with the non-symmetrical ones. A similar trend was noticed for the antibacterial activity against Staphylococcus aureus. These results could suggest that hindered compounds would have difficulties in crossing the thick peptidoglycan layer of Gram (+) pathogens.

In addition, compared with their non-symmetrical analogues, the symmetrical PEGylated compounds 8a-e showed better activity against Gram (-) pathogens. The best results were obtained against Escherichia coli with MIC values between 12.5-25 μg/mL for 8a-e versus 25-50 μg/mL for 4a-e. Even if no significant difference in activity against Candida albicans was observed for all evaluated compounds, symmetrical PEGylated derivatives 8a-e seem to be more active than the corresponding non-symmetrical ones. Compounds 8a-c displayed the greatest antimicrobial activity against Candida albicans (MIC values between 6.25-12.5 μg/mL). Almost all compounds displayed an antifungal activity comparable with that of fusidic acid with MIC values ranging from 6.25-12.5 or 12.5-25 μg/mL except for 4a.

Furthermore, N-oxide moiety was introduced (compound 5 and 9) in order to improve the antimicrobial activity. It has been noticed that the introduction of a pyridine N-oxide moiety could play a crucial role in the biological activity. This group could increase oxidation resistance improving the pharmacokinetic parameters [START_REF] Seto | Orally active CCR5 antagonists as anti-HIV-1 agents: synthesis and biological activity of 1-benzothiepine 1, 1-dioxide and 1-benzazepine derivatives containing a tertiary amine moiety[END_REF][START_REF] Alexander | CDP840. A prototype of a novel class of orally active anti-inflammatory phosphodiesterase 4 inhibitors[END_REF][START_REF] Guay | Discovery of L-791,943: a potent, selective, non emetic and orally active phosphodiesterase-4 inhibitor[END_REF][START_REF] Haginoya | Design, synthesis, and biological activity of non-amidine factor Xa inhibitors containing pyridine N-oxide and 2-carbamoylthiazole units[END_REF]. By matching the compounds 4a and 8a with their corresponding pyridine N-oxides 5 and 9 no significant differences in antimicrobial activity were observed for the different microorganisms tested.

Considering the antimicrobial activity of synthesized analogues, we estimate the antimicrobial effect of PEGylated compounds on Micrococcus luteus and Candida albicans strains by determining EC50 (effective concentration 50%), using levofloxacine and fusidic acid as controls.

The antibiotic potential is indicated by the fraction of bacteria killed by the drug and compounds were evaluated at concentrations between 200 and 1.152 µM. The EC50 was recorded as the concentration of drug required to produce 50% of the maximum biological response. The results obtained are outlined in Table 2.

Table 2

EC50 in µM of non-symmetrical and symmetrical PEGylated triarylmethanes for Micrococcus luteus.

BSL: bisacodyl; TAM OH deacetylated metabolite of bisacodyl, LvF: Levofloxacine, FA fusidic acid It may be noticed that compounds 4c and 9 are the more active derivatives against Micrococcus luteus with EC50 values of 62 and 63 µM respectively and comparable to that of bisacodyl (79 µM).

Biocidal activity

All synthesized compounds and bisacodyl were evaluated for their biocidal activity. The results are illustrated in Figure 1 and Table 3. Staphylococcus aureus was selected as a reference bacterium for biofilm production using Congo red agar (CRA) and 96 wells plate methods. The details of these procedures are given in the experimental section and supplementary data.

The evaluation of the ability of the synthetized compounds to inhibit the early biofilm formation was performed according to the minimum inhibitory concentration of each compound. The obtained results revealed that three symmetrical PEGylated compounds (8b, 8c and 8d), the bisacodyl and its desacetylated derivative 7, inhibited the biofilm formation with percentages superior to 45%.

Compounds from non-symmetrical series seemed to be less actives with a decrease of anti- adherence activity when the PEGylated chain increase. Results showed that the highest antiadhesive effect was observed for compound 8d with an inhibition percentage of 58% at 100 μg/mL.

The results displayed that all compounds induce efficient eradication of the biofilm formed with eradication activity percentages between 45% and 83% (Figure 1, Table 3). Compounds from nonsymmetrical series seemed to be better biofilm eradication agents than their symmetrical analogues.

These results could suggest that the introduction of a second PEGylated chain it is not necessary for the eradication of biofilm. Compounds 4d and deacetylated bisacodyl (compound 7) showed the best antibiofilm activities with eradication percentage superior to 80%. The antibiofilm activity of the compound with the best eradication percentage (compound 7, 83%) was confirmed by fluorescence microscopy. The images of the acridine orange staining treated slides with compound 7 displayed the reduction in the biofilm-coated surface compared to the control (Figure 2). The obtained results demonstrated the ability of compound 7 to modify the surface properties of bacterial cells and to reduce their adhesive properties. These results also confirm that compound 7 is more effective in eliminating the biofilm by eradication than by antiadhesion (83% and 48% respectively). The biofilm metabolic activity of compound 7 was measured using the MTT assay. The compound 7 reduced significantly the metabolic activity in the Staphylococcus aureus biofilm by anti-adhesion (78%) and by eradication (75%). These results suggest that the inhibition of the biofilm metabolism may be one of the mechanisms of action of antibiofilm activity.

Antiproliferative activity

Antiproliferative activity of derivatives 7, 8a-e, 9 was evaluated by MTT assay using human colorectal cancer cell lines (HT-29 and HCT-116). MTT assay were performed 24, 48 and 72 h after treatment and cell viability was expressed in percentage of each condition of treatment by normalizing to untreated cells. The results obtained for symmetrical triarylmethane series are outlined in Figure 3 and Table 4. Antiproliferative effects of evaluated PEGylated triarylmethanes were moderate. Comparing the cell viability percentages, HT-29 cells line seemed to be more sensitive than HCT-116. However, for HT-29 cells, some significant differences were observed for bisacodyl (BSL) and their PEGylated analogues at 24 h or 48 h after 100 µM treatment. At 72 h, BSL, its deacetylated derivative 7 and compound 8b displayed the best results with inhibition percentages of cell viability of 34.06%, 35.76% and 54.42% respectively. At this high in vitro concentration (100 µM) and due to a relatively long treatment time (72 h), it would therefore appear that these molecules are not only no-cytotoxic but also have no antiproliferative effect on HT-29 cancer cells.

Table 4

Cell viability of bisacodyl (BSL) TAM OH and their PEGylated analogues 8a-e and 9. Following the biological evaluation, docking studies were conducted in order to predict potential anti-bacterial targets for the synthesized molecules. Three well-known antibacterial compounds targets were selected according to the scientific literature: dihydrofolate reductase (DHFR) [START_REF] Nagendra Prasad | A novel copper (II) PAmPiCaT complex (cPAmPiCaTc) as a biologically potent candidate: A contraption evidence against methicillin-resistant Staphylococcus aureus (MRSA) and a molecular docking proof[END_REF],

Cpd. Num

DNA gyrase B [START_REF] Durcik | Synthesis and Evaluation of N-Phenylpyrrolamides as DNA Gyrase B Inhibitors[END_REF] and heptaprenyl diphosphate synthase (HepPPS) [START_REF] Desai | Structure, Function, and Inhibition of Staphylococcus aureus Heptaprenyl Diphosphate Synthase[END_REF]. For each of these targets, molecules BSL, 4a, 4d, 7, 8a and 8d were docked into the defined binding sites. For each target, the pose associated with the best docking score for each conformation of each molecule was conserved and submitted to a re-scoring procedure using the scoring function HYDE. For each pose, we thus obtained a range of estimated affinity with a lower limit and an upper limit (Figure 4). Two anti-bacterial targets, DHFR, a key enzyme controlling purine and thymidine nucleotide biosynthesis [START_REF] Schweitzer | Dihydrofolate reductase as a therapeutic target[END_REF] and DNA gyrase B that is able to modulate DNA topology [START_REF] Tihomir | Prospects for Developing New Antibacterials Targeting Bacterial Type IIA Topoisomerases[END_REF] constitute potential targets for the synthesized compounds herein presented.

ADME and drug-likeness profiles

In view of describing the potential for good bioavailability and to evaluate the drug-likeness of the synthesized PEGylated compounds, in silico predictions of ADME (absorption, distribution, metabolism and excretion)-related pharmacokinetic and physicochemical properties were performed. The results obtained are shown in Table 5 and the recommended values for each parameter are listed below [34,[START_REF] Leung | Simple predictive models of passive membrane permeability incorporating size-dependent membrane-water partition[END_REF]. e Estimated number of hydrogen bonds that would be donated by the solute to water molecules in an aqueous solution (recommended value: 0-6).

f Predicted octanol/water partition coefficient (recommended value: -2.0 to 6.5).

g Predicted aqueous solubility, log S. S in mol dm-3 is the concentration of the solute in a saturated solution that is in equilibrium with the crystalline solid (recommended value: -6.5 to 1.5).

h Predicted apparent Caco-2 cell permeability in nm/sec. Caco-2 cells are a model for the gut-blood barrier (< 25 is poor and > 500 is great).

i Predicted apparent MDCK cell permeability in nm/sec. MDCK cells are considered to be a good mimic for the blood-brain barrier (< 25 is poor and > 500 is great). j Predicted skin permeability, log Kp (recommended value: -8.0 to -1.0).

k Number of violations of Lipinski's rule of five (max is 4). Coloured cells correspond to excellent (green), medium (yellow) or poor (grey) compliance of the rule.

The molecular weight (MW), van der Waals surface area of polar nitrogen and oxygen atoms (PSA)

as well as the estimated number of hydrogen bonds donors (HBD) available to establish hydrogen bonds in an aqueous solution are consistent with the recommended values for drug-like compounds.

Meanwhile, the number of rotatable bonds exceeded the recommended maximum of 15 in the cases of 4d, 4e and from 8b-8e.

The 95% range of similarity respect to known drugs (#stars) reached the recommended values for all except for compounds 8d and 8e. The aqueous solubility (QPlogS) of these last two triarylmethanes was predicted as insufficient as well. These results could suggest that the introduction of bulky PEGylated chains on the triarylmethane skeleton is not favourable to develop drug-like compounds. It should be noticed that three of the predicted ADME properties satisfied the recommended values for all the compounds analysed: the octanol/water partition coefficient, the apparent Caco-2 cell permeability, and the MDCK cell permeability. Such predictions suggested a good bioavailability of the triarylmethanes synthetized. However, the skin permeability reached the recommended values only for the compounds 7, 8a, 8f and 9.

Overall, all the synthetized triarylmethanes are compliant with the Lipinski's rule of five, which demonstrates an adequate drug-likeness profile of the set. In fact, more than 60% of the compounds comply with the five rules, as recommended.

Conclusions

Experimental section

Chemicals

All reagents were obtained from commercial sources unless otherwise noted, and used as received.

Heated experiments were conducted using thermostatically controlled oil baths and were performed under an atmosphere oxygen-free in oven-dried glassware. All reactions were monitored by analytical thin layer chromatography (TLC). TLC was performed on aluminium sheets precoated silica gel plates (60 F254, Merck). TLC plates were visualized using irradiation with light at 254 nm or in an iodine chamber as appropriate. Flash column chromatography was carried out when necessary using silica gel 60 (particle size 0.040-0.063 mm, Merck).

Physical measurements

The structure of the products prepared by different methods was checked by comparison of their NMR, IR and MS data and by the TLC behavior. 1 H and 13 To a solution of compound 3 (1 equiv) in anhydrous DMF (8 mL) was added K2CO3 (4 equiv). The mixture was stirred at 80 °C for 1 h, under argon. Then a solution of the corresponding glycol mono-tosylate (1 equiv, except for 4a, 3 equiv) in dry DMF (2 mL) was added dropwise and the mixture was stirred at 80 °C (monitoring by TLC). After reaction completion, the reaction mixture was diluted with water (80 mL) and extracted with CH2Cl2 (5 x 80 mL). The organic layer was washed with a saturated solution of NaHCO3 (3 x 80 mL), brine (3 x 80 mL) and water (3 x 80 mL), and then dried over MgSO4. The solvent was removed in vacuo and then the product was purified by flash column chromatography on silica gel to afford the corresponding PEGylated nonsymmetrical triarylmethanes 4a-e Yields: 4a, 72% ; 4b, 40% ; 4c, 40% ; 4d, 33% ; 4e, 69%. To a solution of compound 7 (1.08 mmol ; 300 mg) in anhydrous DMF (10 mL) was added K2CO3 (6.49 mmol ; 896 mg). The mixture was stirred at 80°C for 30 min, under argon. Then a solution of 2-bromoethanol (4.32 mmol ; 310 µL) and KI (5.41 mmol ; 898 mg) was added and the mixture was stirred for 4 days at 80°C. The mixture was diluted with water (50 mL), and extracted with CH2Cl2 (3 x 30 mL). The organic layer was washed with saturated solution of NaCl (50 mL) then dried over MgSO4. The solvent was removed in vacuo and then the product was purified by flash column chromatography on silica gel (EtOAc/Cy 50:50) to afford the desired compound 8a (15% ;

59 mg) as a yellow oil and a second compound 8f (50% ; 176 mg) as a yellow oil.

General Procedure for the preparation of PEGylated symmetrical triarylmethanes

To a solution of compound 7 (1.08 mmol , 300 mg , 1 equiv) in anhydrous DMF (10 mL) was added K2CO3 (10 equiv). The mixture was stirred at 80 °C for 1 h, under argon. Then a solution of corresponding glycol mono-tosylate (4 equiv) in dry DMF (5 mL) was added dropwise and the mixture was stirred at 80 °C (monitoring by TLC). The mixture was diluted with water (50 mL), and extracted with EtOAc (3 x 50 mL). The organic layer was washed three times with water (25 mL). Then dried over MgSO4, filtered and the solvent was removed in vacuo and then the product was purified by flash column chromatography on silica gel to afford the corresponding PEGylated symmetrical triarylmethanes 8b-f Yields: 8b, 60% 8c,70%, 8d, 50% 8f, 45%. To a solution of the corresponding TAMs (1 equiv) in anhydrous CH2Cl2 under argon was added quickly m-CPBA (77%) (3 equiv). The reaction mixture was stirred at room temperature under argon. Then, the reaction mixture was quenched by the addition of a solution of KOH (40%) until pH 8-9. The medium was diluted with water (5 mL) and then extracted three times with CH2Cl2 (10 mL). The combined organic layers were dried over MgSO4, filtered and concentrated to dryness.

The crude product was purified by flash column chromatography on silica gel to afford the corresponding PEGylated symmetrical triarylmethanes N-oxides. Yields: 5, 83% ; 9,64%.

Minimal Inhibitory Concentration (MIC) and Minimal Bactericidal Concentration (MBC)

The synthesized compounds were tested in triplicate, using microplate dilution method. Minimal inhibitory concentrations (MICs) of compounds were determined according the National Committee for Clinical Laboratory Standard (NCCLS, 2002). The test was performed in sterile 96-well microplates.

The compounds were dissolved in dimethylsulfoxide (DMSO). Serial two fold dilutions of each sample to be evaluated were made to yield volumes of 100 μL per well with final concentrations ranging from 200 to 1.152 μg/mL. 100 μL of bacteria suspension with a concentration of 10 7 CFU/mL were added to each well. Negative control wells contained bacteria only in LB broth medium. After incubation at 37 °C for 20 h, the minimal inhibitory concentrations (MICs) were recorded as the lowest concentration of compound in the medium that showed no microbial growth.

Then, 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) was added to the wells to facilitate reading of the plates. If there is microbial growth, MTT turns to blue if not, the medium remains yellow. Solvent medium and positive growth controls were also run simultaneously. Then from each tube, one loopful was cultured on plate count agar and incubated for 24 h at 30 °C. The lowest concentration of the compound supporting no colony formation was defined as the MBC. The levofloxacin, broad spectrum antibiotic and fusidic acid, a narrow spectrum antibiotic.

The inhibitory activity of the tested compounds was calculated according to the formula: IA (%) = 100-100 (OD 600 (x) / OD 600 (i))

where (x) is the microbial culture containing the inhibitor and (i) is the microbial culture without inhibitor.

Antibiofilm activity

Biofilm production:

Congo Red Agar Method (CRA) Screening of biofilm production using Congo Red agar method was performed as described. The CRA medium was composed of Brain Heart Infusion (BHI), 37 (gL -1 ); sucrose, 50 (gL -1 ); agar, 10 (gL -1 ) and Congo red indicator (Sigma) 0.8 (gL -1 ). The plates were inoculated with different microorganisms tested, a non-biofilm producing E. coli DH5-α bacterium serving as a negative control. Plates were incubated at 28 °C for 24 h. Black colonies with dry crystalline consistency indicate a high production of biofilm.

well plate method

The ability to form a biofilm was determined using the crystal violet test. The isolated strains were inoculated into 10 mL of LB broth containing 1% glucose and incubated at 28 °C for 24 h. The culture strains were diluted to obtain OD 600 ~ 0. The 96-well flat bottom plates were used for biofilm cultures. S.aureus, a biofilm-forming strain, was grown overnight in LB medium at 37 °C and diluted with fresh medium supplemented with 1% (w/v) glucose. One hundred microliters of the bacterial culture dilution were added into each well to obtain a final OD at 600 nm of 0.1. Then, each compound was added at a concentration according to theirs MIC values against S. aureus. The final concentrations into the wells were ranged between 100 µg/mL and 25 µg/mL. Wells containing only LB medium supplemented with, 1% glucose and bacterial suspension were used as controls. Plates were incubated for 24 h at 37 °C. After incubation, the wells were emptied into a container by inverting the plates. Each well was gently washed twice with 250 µL of sterile PBS in order to remove the floating bacteria. After washing, plates were dried at 60 °C for 45 min. Then, wells were stained with 150 µL of crystal violet (0.2%) prepared in 20% ethanol for 15 min at room temperature. After staining incubation, crystal violet was removed and excess dye was washed three times with sterile water. Finally, 200 µL of glacial acetic acid was added to each well and plates were incubated for 1 h at room temperature. The optical density (OD) was measured at 570 nm using a Varioskan microplate reader (Thermofisher).

The percentage of the adhesion inhibition was determined by the following formula:

MTT assay of metabolic activity

The MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay is a colorimetric assay that measures the enzymatic reduction of MTT, a yellow tetrazole, to formazan. After biofilm formation, 150 µL of MTT dye (0.5 mg/mL MTT in PBS) was added to the microplate containing biofilm treated with compound 7 and incubated at 37°C for 1 h and bacterial suspension were served as controls. During this process, metabolically active bacteria reduced the MTT to purple formazan. After 1 h, the disks were transferred to a new 24-well plate, 1 mL of dimethyl sulfoxide (DMSO) was added to solubilize the formazan crystals, and the plate was incubated for 20 min with gentle mixing at room temperature, in the dark. After mixing via pipetting, 200 µL of the DMSO solution from each well was transferred to a 96-well plate, and the absorbance at 540 nm (optical density OD 540) was measured via a microplate reader (Varioskan, Thermofisher). A higher absorbance is related to a higher formazan concentration, which indicates a higher metabolic activity in the biofilm on the disk [START_REF] Cheng | Effect of amorphous calcium phosphate and silver nanocomposites on dental plaque microcosm biofilms[END_REF].

Antiproliferative activity

Cell culture

Scheme 1 .

 1 Scheme 1. Synthesis of non symmetrical PEGylated derivatives (a): (i) i-PrMgCl, THF, r.t., 2 h, Ar (ii) p-anisaldehyde, r.t., 2 h, Ar (b): phenol, H2SO4, PhNO2, 0 °C, 5 min (c): corresponding glycol mono-tosylate, K2CO3, DMF anh, 80 °C, 4-7 days, Ar; (d): m-CPBA, dichloromethane, r.t., 2 h.

Scheme 2 .

 2 Scheme 2. Synthesis of symmetrical PEGylated derivatives (a): KOH, H2O/EtOH, r.t., 72 h (b): corresponding glycol mono-tosylate, K2CO3, DMF anh, 80 °C, 24 h (c): 2-bromoethanol, KI, K2CO3, DMF anh, 80 °C, Ar, 96 h, (d): m-CPBA, dichloromethane, r.t., 3 h

Fig. 1

 1 Fig.1 Evaluation of anti-adhesive activity (pre-treatment) of PEGylated compounds, using Staphylococcus aureus as reference bacterium. The final concentrations into the wells were ranged between 100 µg/mL and 25 µg/mL according with MIC values against Staphylococcus aureus. The percentage of the adhesion inhibition was determined by the following formula: % adhesion inhibition (at 570 nm) = [(% OD (control) -OD (treated bacterium)) / OD (control)] * 100 Evaluation of antibiofilm activity (eradication) of PEGylated compounds at concentration of 200 μg/mL after 24 h of incubation at 37 °C. The percentage inhibition of the biofilm was determined at 570 nm by the following formula: % inhibition of biofilm = [(% OD (control) -OD (treated bacterium)) / OD (control)] * 100. Data are shown as mean ± SEM of antibiofilm activity in three independent experiments (n=3).

Fig. 2

 2 Fig.2 Fluorescence microscopy images (9 × 40) of anti-adherence activity of non-treated biofilm (A) compared with biofilm treated by the compound 7 against Staphylococcus aureus by antiadhesion (treatment at 50 µg/mL) (B) and eradication (treatment at 200 µg/mL) (C) The formation of biofilm (untreated biofilm) is represented by the green areas and the necrosis zones of the biofilm are represented by the black ones.

Fig. 4

 4 Fig. 4 Boxplots representing the estimated affinity values (in M) as predicted by HYDE for the docking poses obtained with the DHFR structure (2W9H, salmon), the DNA gyrase B structure (4DUH, green) and the HepPPS structure (5H9D, blue). The plain lines and dashed lines represent respectively the lower and the upper limits of estimated affinity

Fig. 5

 5 Fig. 5 Binding mode prediction of (A) compound 7 (represented in stick and colored in wheat) into the DHFR binding site and (B) compound 8a (represented in stick and colored in blue) into the DNA gyrase B binding site. The co-crystallized ligands are also represented in stick in their respective binding site (in pink for DHFR and in green for DNA gyrase B) and used as the reference.

  In summary two series of PEGylated triarylmethanes symmetrical and non-symmetrical were prepared, characterized and biologically evaluated. Twelve new compounds were straightforwardly synthesized by Friedel-Crafts hydroxylalkylation reaction or by nucleophilic substitution from deacetylated bisacodyl and then functionalized with PEG moieties. All compounds were tested on Gram-positive and Gram-negative foodborne pathogens and against Candida albicans. The biological results revealed that these compounds are potentially more effective against Gramnegative bacteria in particularly against Micrococcus luteus. Almost all PEGylated molecules displayed an antifungal activity comparable to that of fusidic acid with MIC values ranging from 6.25 to 50 μg/mL. They also showed a promising antibiofilm activity with inhibition percentages superior to 80% for the best molecules (compounds 4d and 7). The MTT assay suggested that the inhibition of the biofilm metabolism could be one of the mechanisms of action of antibiofilm activity. Nevertheless, those compounds have shown a modest anti-proliferative activity against colorectal cancer cell lines HT-29. Finally, in silico molecular docking studies proposed that DHFR and DNA gyrase B could be a potential antibacterial targets and in silico ADME predictions suggested that synthesized compounds have good drug-likeness and ADME profiles, except for 8d and 8e. Thus, the introduction of long PEGylated chains in triarylmethane skeleton seems to disfavor the drug-likeness properties of the studied compounds.The importance of anti-infective research field encourages us to continue the study of based triarylmethane skeleton compounds in order to find new potential antimicrobial molecules. The development of new series of triarylmethane bearing various aminoalkyl chains is the subject of current investigations. Moreover, additional studies focus on the biochemical mechanism of action of antibiofilm activity are now underway in our laboratory and results will be published in due course.

  DEPT experiments were used to establish the structures and to assign the signals. For the assignments of the NMR signals, we use the convention presented in Figure3. Low resolution mass spectra (ESI-LRMS) were performed from ionization by electrospray on a Waters Micromass ZQ2000. Infrared spectra were recorded over the 400-4000 cm -1 range with an Agilent Technologies Cary 630 FTIR/ ATR/ ZnSe spectrometer. High-resolution mass spectra (HRMS) analyses were acquired on a Thermo Scientific LTQ Orbitrap mass spectrometer.

Fig. 3

 3 Fig. 3 Convention adopted to assign signals of 1 H and 13 C-NMR spectra 4.2.3. General Procedure for the preparation of PEGylated non-symmetrical triarylmethanes

  (M-Na + ). 1 H NMR (DMSO-d6, 400 MHz) δ (ppm) = 3.41 (m, 2H, H26) ; 3.47 (m, 2H, H27) ; 3.50 (m, 4H, H22 or H23 or H24 or H25) ; 3.52 (m, 2H, H22 or H23 or H24 or H25) ; 3.57 (m, 2H, H22 or H23 or H24 or H25) ; 3.71 (m, 5H, H13 + H21) ; 4.03 (m, 2H, H20) ; 4.58 (t, 1H, HOH, J = 5.4 Hz) ; 5.54 (s, 1H, H6) ; 6.85 (m, 4H, H9 + H11 + H16 + H18) ; 7.09 (m, 4H, H8 + H12 + H15 + H19) ; 7.21 (m, 2H, H2 + H4) ; 7.71 (ddd, 1H, H3, J = 1.9, 7.6 and 9.6 Hz) ; 8.54 (ddd, 1H, H1, J = 1.0, 1.9 and 4.7 Hz). 13 C NMR (DMSO-d6, 100 MHz) δ (ppm) = 55.0 (C13) ; 56.5 (C6) ; 60.2 (C27) ; 67.0 (C20) ; 68.9 (C21) ; 69.7 (C22 or C23 or C24 or C25) ; 69.8 (C22 or C23 or C24 or C25) ; 69.9 (C22 or C23 or C24 or C25) 70.0 (C22 or C23 or C24 or C25) ; 72.

1 H 6 (

 16 NMR (DMSO-d6, 400 MHz) δ (ppm) = 3.68 (m, 4H, H20) ; 3.93 (t, 4H, H19, J = 5.0 Hz) ; 4.83 (t, 2H, HOH, J = 5.5 Hz) ; 5.53 (s, 1H, H6) ; 6.84 (m, 4H, H9 + H11 + H15 + H17) ; 7.08 (m, 4H, H8 + H12 + H14 + H18) ; 7.21 (m, 2H, H2 + H4) ; 7.71 (ddd, 1H, H3, J = 1.9, 7.7 and 9.6 Hz) ; 8.51 (m, 1H, H1). 13 C NMR (DMSO-d6, 100 MHz) δ (ppm) = 56.5 (C6) ; 59.6 (C20) ; 69.4 (C19) ; 114.2 (C9 + C11 + C15 + C17) ; 121.5 (C2 or C4) ; 123.4 (C2 or C4) ; 129.9 (C8 + C12 + C14 + C18) ; 135.4 (C7 + C13) ; 136.7 (C3) ; 149.1 (C1) ; 157.1 (C10 + C16) ; 163.1 (C5). HRMS: calcd. for C22H23NO4H (366.1700); found (366.1700). 4.2.37. 4-((4-(2-hydroxyethoxy)phenyl)(pyridin-2-yl)methyl)phenol 8f 1 HNMR (DMSO-d6, 400 MHz) δ (ppm) = 3.69 (m, 2H, H20) ; 3.93 (t, 2H, H19, J = 5.0 Hz) ; 4.83 (t, 1H, HOH aliphatique, J = 5.5 Hz) ; 5.47 (s, 1H, H6) ; 6.67 (d, 2H, H9 + H11, J = 8.1 Hz) ; 6.84 (d, 2H, H15 + H17, J = 8.1 Hz) ; 6.95 (d, 2H, H8 + H12, J = 8.1 Hz) ; 7.06 (d, 2H, H14 + H18, J = 8.3 Hz) ; 7.23 (m, 2H, H2 + H4) ; 7.71 (ddd, 1H, H3, J = 1.9, 7.7 and 9.6 Hz) ; 8.51 (m, 1H, H1). 13 C NMR (DMSO-d6, 100 MHz) δ (ppm) = 56.7 (C6) ; 59.6 (C20) ; 69.4 (C19) ; 114.1 (C15 + C17) ; 115.0 (C9 + C11) ; 121.4 (C2 or C4) ; 123.4 (C2 or C4) ; 129.8 (C8 + C12) ; 129.9 (C14 + C18) ; 133.6 (C13); 135.C7) ; 136.6 (C3) ; 149.1 (C1) ; 155.7 (C10) ; 157.0 (C16) ; 163.4 (C5). HRMS: calcd. for C20H19NO3H (322.1438); found (322.1439).

% 4 . 2 . 2 . 4 .

 4224 adhesion inhibition (at 570 nm) = [(% OD (control) -OD (treated bacterium)) / OD (control)] * 100 4.2.2.3. Biofilm inhibition (post-treatment)The stain of S. aureus is diluted to obtain OD 600 ~ 0.1. An individual well of sterile 96-well flatbottomed polystyrene plate containing 150 μL of LB medium was inoculated with 50 μL of glucose (1%) served as a negative control. In the remainder of the plate, 150 μL of LB medium with 50 μL of glucose (1%) and 50 μL of the bacterial culture were incubated. The plate was incubated for 24 h at 37 °C. The wells were washed twice with 250 μL of PBS (pH 7.2) to eliminate the floating bacteria. Then 150 μL of the culture medium (LB) with 50 μL of each sample were added to obtain a concentration of 200 μg/mL. The plate is again incubated at 37 °C for 24 h. Then culture medium of each well was removed by tapping the lower plates. The wells were washed twice with 250 μL of PBS (pH 7.2). Adherent bacteria were fixed at 60 °C for 1 hour and stained with 150 μL of 0.2% crystalline violet prepared in 20% (v / v) ethanol for 45 minutes. To remove excess water, the dye wells were washed three times with sterile water. The task-related bacterium was released by adding 200 μL of glacial acetic acid. After 1 hour at room temperature, the absorbance at 570 nm was determined using the microplate reader. The experiments were performed in triplicate. The percentage inhibition of the biofilm was determined by the following formula: % inhibition of biofilm (at 570 nm) = [(% OD (control) -OD (treated bacterium)) / OD (control)] * 100 Microscopic observation of antibiofilm activityThe antibiofilm activity of the compound 7 against Staphylococcus aureus was confirmed by microscopic observation using the OLYMPUS fluorescent microscope BX50 equipped with a digital camera OLYMPUS DP70. The biofilms were grown on glass pieces (Ø 10 mm) placed in 24-well polystyrene plates treated with the compound 7. Non-treated wells, containing LB supplemented with 10% (v/v) of DMSO, served as controls[START_REF] Padmavathi | Antibiofilm activity of biosurfactant producing coral associated bacteria isolated from gulf of mannar[END_REF][START_REF] Abdelli | Antibacterial, antiadherent and cytotoxic activities of surfactin(s) from a lipolytic strain Bacillus safensis F4[END_REF]. The compound 7 was added at according to MIC values against Staphylococcus aureus (50 µg/mL) for the test of antiadhesion and at 200 µg/mL for eradication in LB with 10% (v/v) of DMSO. The bacterial inoculation was adjusted to an OD 600nm of 0.1. Plates were incubated at 37 °C for 24 h. The wells were then carefully emptied with pipetting and glass slides were washed with sterile PBS (1X) before the treatment with 500 µL of acridine orange (0.1%, w/v, dissolved in PBS1X). Visualization was performed through a 40 × objective using U-MWB2 filter with excitation at 460-490 nm and emission at 520 nm.

Table 3

 3 Evaluation of adhesion inhibition* and eradication of biofilm**

	Cpd. Num	R, n, X	% of adhesion inhibition	% of eradication	Cpd. Num	R, n, X	% of adhesion inhibition	% of eradicati on

* Each compound was evaluated at a concentrations according to MIC values against S. aureus: At 100 µg/mL for compounds 4a, 4b, 4d, 8f, 5, 8d, 9; at 50 µg/mL for compounds 4c, 7, 8b, 8c, 8e; at 25 µg/mL for compounds 4e and 8a. ** Compounds were evaluated at concentration of 200 μg/mL

  treated at 100 µM and relative cell viability was assessed by MTT dye exclusion method for 24, 48 and 72 h. Data are shown as mean ± SEM of cell viability compared to untreated cells as control (100%) in three independent experiments (n=3). *p < 0.05.

	8d	n= 4	92.68	80.83	87.47	87.35	69.57	72.95
	8e	n= 5	100	88.94	87.31	84.04	67.10	79.84
	9	n= 1, X= O -	81.50	92.70	86.96	92.90	74.02	83.60
	BSL		94.35	82.44	65.94	99.97	77.01	73.28
	Control		100	100	100	100	100	100
	TAMOH: deacetylated metabolite of bisacodyl
	2.2.4. In silico studies
	A 2.2.4.1 Docking studies
	B
					% cell viability
				HT-29			HCT-116
			24 h	48 h	72 h	24 h	48 h	72 h
	7	TAMOH	99.72	87.65	64.24	98.84	77.79	81.51
	8a	n= 1	95.04	100	96.54	99.06	73.76	75.27
	8b	n= 2	90.33	71	45.58	100	91.28	93.23
	8c	n= 3	92.97	99.25	91.60	89.65	77.58	87.36
				12

* * * Fig. 3 Effect of compounds 7, 8a-e, 9 and bisacodyl (BSL) on cell proliferation. HT-29 and HCT116 cells were

Table 5

 5 Predictions of ADME-related parameters and drug-likeness of compounds 4a-

e, 5, 7, 8a-e, 9.

	Compound	MW a	#stars b	#rotor c	PSA d	HBD e	QPlogPo/W f	QPlogS g	QPPCaco h	QPPMDCK i	QPPKp j	Lipinski's
												Rule of five k
	4a	335.402	0	8	48.623	1	4.676	-5.343	2543.778	1357.141	-0.5	0
	4b	379.455	0	11	59.757	1	4.682	-5.245	2156.846	1135.453	-0.407	0
	4c	423.508	0	14	68.373	1	4.931	-5.749	2156.855	1135.458	-0.12	0
	4d	467.561	3	17	76.83	1	5.159	-6.192	2149.919	1131.511	0.166	1
	4e	511.614	4	20	85.409	1	5.215	-6.05	2151.926	1132.653	0.446	2
	8f	321.375	0	8	62.896	2	3.785	-4.67	774.734	375.437	-1.475	0
	5	351.401	0	8	57.487	1	4.627	-5.456	1784.746	925.283	-0.836	0
	7	277.322	0	5	54.363	2	3.536	-4.163	828.409	403.629	-1.65	0
	8a	365.428	0	11	73.898	2	3.779	-5.215	581.905	275.539	-1.507	0
	8b	453.534	3	17	91.238	2	4.285	-5.763	582.486	275.836	-0.932	0
	8c	541.64	5	23	106.507	2	4.625	-6.197	584.107	276.666	-0.353	1
	8d	629.746	8	29	121.093	2	5.147	-7.016	721.751	347.763	0.441	3
	8e	717.852	11	35	138.463	2	5.398	-7.359	734.68	354.502	1.004	3
	9	381.427	0	11	80.292	2	3.394	-5.338	508.191	238.013	-1.611	0
	a Molecular weight of the molecule (recommended value: 130-725).

b Number of property or descriptor values that fall outside the 95% range of similar values for known drugs (recommended value: 0-5). c Number of non-trivial (not CX3), non-hindered (not alkene, amide, small ring) rotatable bonds (recommended value: 0-15). d Van der Waals surface area of polar nitrogen and oxygen atoms (recommended value: 7-200).

  1. Individual wells of sterile 96-well flat-bottomed polystyrene plate (orange scientific) containing 200 μL of fresh medium were inoculated with 50 μL of the bacterial culture tested and a sterile broth was used as a control. The same procedure was repeated for E. coli DH5α and S. aureus which are negative and positive biofilm respectively. After incubation of the plate at 37 °C for 24 h, the culture medium of each well was removed by tapping To remove excess water, the dye wells were washed three times with sterile water. The task-related bacterium was released by adding 200 μL of glacial acetic acid. After 1 hour at room temperature, the absorbance at 570 nm was determined using the microplate reader.4.2.2.2. Biofilm formation inhibition (pre-treatment)

the lower plates. The wells were washed twice with 250 μL of Phosphate Buffer Saline, PBS (pH 7.2) to remove floating bacteria. Adherent bacteria were fixed at 60 °C for 1 hour and stained with 150 μL of 0.2% crystalline violet prepared in 20% (v/v) ethanol for 45 minutes.

Acknowledgements

Authors thank CAMPUS France (PHC Program, UTIQUE 2017 37094W) and the Ministère de l'Enseignement Supérieur et de la Recherche Scientifique of Tunisia for financial support. FA thanks Miss Mouna JLIDI for her help during the evaluation of antibiofilm activity. The authors also acknowledge the website SMART (Servier Medical Art) https://smart.servier.com/ for image supply used in the Graphical Abstract. LRMS: Low-resolution mass spectra LvF: Levofloxacin MBC: Minimal Bactericidal Concentration MIC: Minimal Inhibitory Concentration MGL: Molecular Graphics Laboratory MTT: 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide PBS: Phosphate Buffer Saline TAMs: Triarymethanes RT: room temperature SMART: Servier Medical Art

Human colorectal cancer (CRC) cell lines (HT-29 and HCT116) were purchased from the American Type Culture Collection (ATCC -LGC Standards, Molsheim, France). Cells were grown in DMEM medium for HT-29 cells and RPMI 1640 medium for HCT116 cells. Cells were supplemented with 10% FBS, 1% L-glutamine and 100 U/mL penicillin and 100 μg/mL streptomycin. Cultures were maintained in a humidified atmosphere containing 5% CO2 at 37 °C. Stock solutions of molecules were diluted in culture medium to obtain the appropriate final concentrations (10 to 100 µM). The same amount of vehicle (percentage of DMSO did not exceed 0.6%) was added to control cells.

In vitro antiproliferative effect of molecules

Antiproliferative effect of molecules was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) assays as previously described by Bretin et al. [START_REF] Bretin | Photodynamic Therapy Activity of New Porphyrin-Xylan-Coated Silica Nanoparticles in Human Colorectal Cancer[END_REF]. Human CRC cells were seeded at 4x10 3 cells/well and 7x10 3 cells/well for HCT116 and HT-29 cells, respectively, in 96-well culture plates and grown for 24 h in culture medium prior to exposure or not to molecules. MTT assay were performed 24, 48 and 72 h after treatment and cell viability was expressed in percentage of each condition of treatment by normalizing to untreated cells.

Experiments were performed in triplicate.

Statistical analysis

All quantitative results are expressed as the mean ± standard error of the mean (SEM) of separate experiments. Statistical significance was evaluated by the two-tailed unpaired Student's t-test and expressed as: *p < 0.05.

Docking studies

The 3D structures of Staphylococcus aureus Dihydrofolate Reductase (DHFR), Escherichia coli DNA gyrase B and Staphylococcus aureus heptaprenyl diphosphate synthase (HepPPS) were extracted from the PDB database [START_REF] Berman | The Protein Data Bank[END_REF] (PDB IDs : 2W9H, 4DUH and 5H9D respectively). Cocristallized ligands were removed from 2W9H and 4DUH structures (no ligand was co-cristallized in the 5H9D structure). The 3 structures (2W9H chain A ; 4DUH chain ; 4DUH chains A and C) were then prepared using Chimera [START_REF] Pettersen | UCSF Chimera-A visualization system for exploratory research and analysis[END_REF] and Molecular Graphics Laboratory (MGL) tools [START_REF] Morris | AutoDock4 and AutoDockTools4: Automated docking with selective receptor flexibility[END_REF] .

Three dimensional structures of compounds BSL, 4a, 4d, 7, 8a and 8d were generated using iCon, the LigandScout v.4.3. conformer generator [START_REF] Wolber | LigandScout: 3-D Pharmacophores Derived from Protein-Bound Ligands and Their Use as Virtual Screening Filters[END_REF] (defaults settings of the BEST option were used, except for the maximum number of conformations that was setted to 2000). The number of conformations obtained for each ligand was 19 for BSL, 12 for compound 4a, 354 for compound 4d, 7 for compound 7, 13 for compound 8a and 1624 for compound 8d. Protein-ligand docking of all these conformations of the 6 compounds was performed using AutoDock Vina v.1.1.2 [START_REF] Trott | AutoDock Vina: Improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading[END_REF]. The x, y, z AutoDock Vina grid center coordinates used are 8.960, -5.922, 16.425 for 2W9H ; 2.002, 29 2.167, 37.295 for 4DUH ; 27.226, 24.204, 24.816 for 5H9D. The size of the search space was set to 20 Å x 20 Å x 20 Å in the 3 cases. The pose associated with the best score was considered for each conformation and was re-scored using Hydrogen bonds and Dehydration (HYDE) scoring function [START_REF] Schneider | A consistent description of HYdrogen bond and DEhydration energies in protein-ligand complexes: methods behind the HYDE scoring function[END_REF] as implemented in SeeSAR v.9.2 [START_REF]SeeSAR version 9.2[END_REF].

ADME and drug-likeness profiles

The physicochemical and pharmacokinetic properties of the PEGylated triarylmethanes were predicted using the QikProp Panel of Schrodinger software v11.9 with a prior force field energy minimization of the structures.

Conflict of interest

All authors contributed to the drafting and revision of the article and approved the final version. The authors declare no conflict of interest Supplementary material Supplementary data associated with this article is available free of charge at http://www........

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