Prenylated Flavonoids with Potential Antimicrobial Activity: Synthesis, Biological Activity, and In Silico Study

Prenylated flavonoids are an important class of naturally occurring flavonoids with important biological activity, but their low abundance in nature limits their application in medicines. Here, we showed the hemisynthesis and the determination of various biological activities of seven prenylated flavonoids, named 7–13, with an emphasis on antimicrobial ones. Compounds 9, 11, and 12 showed inhibitory activity against human pathogenic fungi. Compounds 11, 12 (flavanones) and 13 (isoflavone) were the most active against clinical isolated Staphylococcus aureus MRSA, showing that structural requirements as prenylation at position C-6 or C-8 and OH at positions C-5, 7, and 4′ are key to the antibacterial activity. The combination of 11 or 12 with commercial antibiotics synergistically enhanced the antibacterial activity of vancomycin, ciprofloxacin, and methicillin in a factor of 10 to 100 times against drug-resistant bacteria. Compound 11 combined with ciprofloxacin was able to decrease the levels of ROS generated by ciprofloxacin. According to docking results of S enantiomer of 11 with ATP-binding cassette transporter showed the most favorable binding energy; however, more studies are needed to support this result.


Introduction
Prenylated flavonoids are a subclass of natural flavonoids characterized by the presence of a prenylated side chain (i.e., prenyl, geranyl and lavandulyl) attached to the flavonoid skeleton. Since prenylated flavonoids are generally more bioactive than their non-prenylated precursors, they are of interest as lead compounds for producing new drugs [1][2][3]. However, since prenylated flavonoids often exist at trace levels in their natural sources that make further biological studies difficult, it may be useful to synthesize prenylated flavonoids in appreciable quantities and at low costs to allow future biological studies [4]. Compounds of this type exhibit significant effects in the insect-plant interaction [5] as well as antifungal [6], antimicrobial [7], antiviral [8,9], anti-inflammatory [10], and anticancer [11] activities and anti-lipid properties, both in vitro and in vivo [12]. The Cprenylation of flavonoids seems to be crucial to the biological activities of these compounds,

Structure Determination
The place of C-alkylation was determined by 2D 1 H- 13 C HMBC NMR spectra. The main J 3 and J 2 interactions between the methylene proton CH 2 of the prenyl chain and aromatic peaks are schematically shown in Figure 1. Only the interaction between the methylene protons with the quaternary 8a carbon in the structure occurs when the Cprenylation is produced in C-8. Correlations J 3 and J 2 between the methylene protons CH 2 and quaternary 8a or 4a carbon are impossible when the prenylation is bounded at Position 6. For the rest of the molecules, the same reasoning was used to determine the place of C-alkylation.
The presence of the carbonyl and hydroxyl groups was confirmed by observing the typical signals in IR spectra. Stretching absorption values between 3550 and 3200 cm −1 as a broad signal indicated the presence of phenol groups and stretching absorption values between 1750 and 1680 cm −1 as one of the strongest singlet IR absorptions confirmed the presence of carbonyl groups.
The hydrogen bond to carbons was determined by J 1 interactions in the 2D 1 H-13 C HSQC NMR spectra. 2D NMR spectra (HSQC and HMBC) for all prenylated compounds were included in Supplementary Materials. main J3 and J2 interactions between the methylene proton CH2 of the prenyl chain and aromatic peaks are schematically shown in Figure 1. Only the interaction between the methylene protons with the quaternary 8a carbon in the structure occurs when the Cprenylation is produced in C-8. Correlations J3 and J2 between the methylene protons CH2 and quaternary 8a or 4a carbon are impossible when the prenylation is bounded at Position 6. For the rest of the molecules, the same reasoning was used to determine the place of C-alkylation.

In Vitro Antibacterial Effects on Human Pathogen Bacteria
From the results of bacterial growth inhibitory activity on sensitive and resistant S. aureus (Table 1), we can observe that not all prenylated flavonoids showed a good antibacterial effect, being prenylated flavanones 7 to 10, weak to moderate inhibitors. In Table 2, compounds 11 and 12, corresponding to prenylated naringenin, together with compound 13 (prenylated genistein), showed strong activity both on the sensitive strain and on the resistant strains, with MIC values ranging from 5 to 50 µg/mL. Compound 11 is the one that shows the best antibacterial action, especially on the resistant strain MRSA 97-7 with an MIC lower than that of the commercial agents tested. The results show that the saturation of the double bond at the C2-C3 position appears to be important for the antibacterial activity of flavonoids (compounds 11 and 12). This coincides with those reported in the literature, where prenylated flavanones show better activity than flavones [28,29]. The position of the hydroxyl substituents, as well as that of the prenyl group, appears to be important in the inhibitory activity of bacterial growth, observing that derivatives with OH at positions C-5, 7, and 4 show better inhibitory activity, as well as the presence of prenyl groups in the C6 and C8 positions of Ring A, as previously reported [30][31][32]. Araya-Coultier et al. (2018) showed that the position of the prenyl group affects the antimicrobial activity of flavonoids, when comparing the effect of lupiwighteone (5,7,4 -trihydroxy-8-prenylisoflavone) and wighteone (5,7,4 -trihydroxy-6-prenylisoflavone), on the Gram-positive strain L. monocytogenes. Lupiwighteone, chain prenylated at C-8, showed no inhibition, whereas wighteone, chain prenylated at position C-6, had high antibacterial activity [33].
When comparing compound 4 with compounds 11 and 12, where the base structure is maintained and only one prenyl substituent has been added in different positions, we can observe that the introduction of a prenyl group has an important effect on the inhibitory activity, reducing by up to at least one order of magnitude the MIC in MRSA 97-7 (Table 2). It has been reported that prenylated flavonoids are more hydrophobic than common flavonoids, facilitating this characteristic the ability to penetrate the cell membrane, thus improving their action at the active site [29]. Mun et al. (2014) proposed that prenylated flavonoids act through interaction with the bacterial membrane, binding directly with peptidoglycan, observing morphological changes when MRSA strains were treated with the prenylated flavonoid sophora-flavanone B [34]. Tables 3 and 4 show that a synergistic effect is produced between compounds 11, 12, and 13 when acting in conjunction with commercial antibiotics, showing that the molecules 11 and 12 reduce the MIC of ciprofloxacin over MRSA 97-7 by 10 times (FICI values 0.6 and 0.16, respectively) and 11, 12, and 13 reduce 100 times its MIC over MRSA 622-4 with FICI values from 0.1 to 0.2 (results shown in Table 4). They also acted synergistically with Vancomycin (FICI values from 0.15 to 0.43), decreasing its MIC 10 times against MRSA 97-7. Compound 11 also acted synergistically with Methicillin with an FICI value of 0.01, decreasing its MIC 100 times against MRSA 97-7. This potentiating effect of the antimicrobial agent action exerted by prenylated molecules is not observed when combining them with each other, as observed in Table 5, where, when combining compounds 11 and 12, an FICI value between 1.1 and 2 was achieved, given an account of an indifferent effect. This difference in the behavior of prenylated molecules, when compared to the action they exert when combined with antimicrobial agents, may be due to the fact that prenylated flavonoids act by a different mechanism than the commercial antibiotics evaluated, e.g., through the inhibition of pumps of expulsion such as ABC transporters, thus being able to enhance the action of the commercial antibiotic, as proposed by Wagner et al. (2009), who pointed out that the synergistic effect of phytodrugs in conjunction with antimicrobial agents can contribute to effects on the permeability of the cell wall of bacteria, the inhibition of the efflux pump, and the inhibition of bacterial enzymes, and that all these mechanisms would help the antimicrobial agent to reach its site of action [35].  To corroborate the synergistic action of compound 11 with vancomycin, a time kill assay experiment was carried out, the result of which is shown in Figure 2. We can see that compound 11 alone manages to slightly lower the bacterial growth curve, decreasing by less than 2 log the CFU/mL, showing a moderate bacteriostatic action. Vancomycin achieved a greater effect, lowering CFU/mL by more than 3 log10, but this effect is enhanced by acting in conjunction with compound 11, lowering CFU/mL by more than 5 log10, thus verifying the synergistic effect that compound 11 would exert on ciprofloxacin on MRSA 97-7. It has been described in the literature that prenylated flavonoids isolated from Morus alba L. show a broad spectrum of synergistic action towards Staphylococcus aureus, acting in conjunction with antibacterial agents such as aminoglycosides, β-lactams, glycopeptides, and fluoroquinolones [28]. prenylated molecules, when compared to the action they exert when combined with a microbial agents, may be due to the fact that prenylated flavonoids act by a diffe mechanism than the commercial antibiotics evaluated, e.g., through the inhibition pumps of expulsion such as ABC transporters, thus being able to enhance the actio the commercial antibiotic, as proposed by Wagner et al. (2009), who pointed out that synergistic effect of phytodrugs in conjunction with antimicrobial agents can contrib to effects on the permeability of the cell wall of bacteria, the inhibition of the efflux pu and the inhibition of bacterial enzymes, and that all these mechanisms would help antimicrobial agent to reach its site of action [35]. To corroborate the synergistic action of compound 11 with vancomycin, a time assay experiment was carried out, the result of which is shown in Figure 2. We can that compound 11 alone manages to slightly lower the bacterial growth curve, decreas by less than 2 log the CFU/mL, showing a moderate bacteriostatic action. Vancomy achieved a greater effect, lowering CFU/mL by more than 3 log10, but this effect is hanced by acting in conjunction with compound 11, lowering CFU/mL by more tha log10, thus verifying the synergistic effect that compound 11 would exert on ciproflox on MRSA 97-7. It has been described in the literature that prenylated flavonoids isola from Morus alba L. show a broad spectrum of synergistic action towards Staphyloco aureus, acting in conjunction with antibacterial agents such as aminoglycosides, β-lacta glycopeptides, and fluoroquinolones [28].  The results of the bacterial ROS level are shown in Figure 3. We can see that there is a significant decrease in ROS levels inside the bacteria as compounds 11, 12, and 13 are present, showing a protective effect by reducing ROS, with an effect as important as that shown by the agent Trolox, a known ROS species trapper. It has been described in the literature that polyhydroxylated flavonoids can act as antioxidant agents, trapping ROS [36], and this antioxidant property can be related to the ROS-reducing effect observed in this assay. The results of the bacterial ROS level are shown in Figure 3. We can see that there is a significant decrease in ROS levels inside the bacteria as compounds 11, 12, and 13 are present, showing a protective effect by reducing ROS, with an effect as important as that shown by the agent Trolox, a known ROS species trapper. It has been described in the literature that polyhydroxylated flavonoids can act as antioxidant agents, trapping ROS [36], and this antioxidant property can be related to the ROS-reducing effect observed in this assay. It has been previously reported that ciprofloxacin generates oxidative stress in S. aureus, generating an increase in ROS levels within the bacteria [37]. This result was corroborated in this study, where the levels of ROS generated in the bacteria in the presence of ciprofloxacin were statistically higher than those of the control. As a way to explain the synergistic action observed in the previous experiments, the effect on intrabacterial ROS levels was evaluated by combining ciprofloxacin with compound 11. As we can see in Figure 4, this combination markedly decreases the levels of ROS when compared to those generated by ciprofloxacin alone. From these results, it can be deduced that the prenylated compounds may be acting as trappers for the ROS generated by ciprofloxacin.  It has been previously reported that ciprofloxacin generates oxidative stress in S. aureus, generating an increase in ROS levels within the bacteria [37]. This result was corroborated in this study, where the levels of ROS generated in the bacteria in the presence of ciprofloxacin were statistically higher than those of the control. As a way to explain the synergistic action observed in the previous experiments, the effect on intrabacterial ROS levels was evaluated by combining ciprofloxacin with compound 11. As we can see in Figure 4, this combination markedly decreases the levels of ROS when compared to those generated by ciprofloxacin alone. From these results, it can be deduced that the prenylated compounds may be acting as trappers for the ROS generated by ciprofloxacin. The results of the bacterial ROS level are shown in Figure 3. We can see that there is a significant decrease in ROS levels inside the bacteria as compounds 11, 12, and 13 are present, showing a protective effect by reducing ROS, with an effect as important as that shown by the agent Trolox, a known ROS species trapper. It has been described in the literature that polyhydroxylated flavonoids can act as antioxidant agents, trapping ROS [36], and this antioxidant property can be related to the ROS-reducing effect observed in this assay. It has been previously reported that ciprofloxacin generates oxidative stress in S. aureus, generating an increase in ROS levels within the bacteria [37]. This result was corroborated in this study, where the levels of ROS generated in the bacteria in the presence of ciprofloxacin were statistically higher than those of the control. As a way to explain the synergistic action observed in the previous experiments, the effect on intrabacterial ROS levels was evaluated by combining ciprofloxacin with compound 11. As we can see in Figure 4, this combination markedly decreases the levels of ROS when compared to those generated by ciprofloxacin alone. From these results, it can be deduced that the prenylated compounds may be acting as trappers for the ROS generated by ciprofloxacin.  We used a fast and predictive tool as the molecular docking, which allowed us to obtain by the genetic algorithm the most suitable location of ligand on macromolecule without using molecular dynamic simulations. Thus, we carried out the analysis according to the most favorable binding energy, by grouping the results in a cluster. To analyze the good result of 11 and their correlation with the antibacterial activity, we used the in silico tool with a known and essential pharmacological target linked to this property. For the above, the ABC transporter was selected, which is crucial for balancing nutrients in bacteria and humans. This transporter is also responsible for several processes, such as drug-resistant bacteria [38]. Based on these reasons, we used docking tools to observe which interactions were relevant, especially with the most promising molecule 11, although it is worth taking into account that the biological tests were carried out with a racemic mixture. In this study, we decided to study both enantiomers in order to be able to discriminate them and determine whether the binding was stereoselective. The results showed a priority for the S enantiomer (−5.06 Kcal/mol ± 0.012) versus the R enantiomer (−3.37 Kcal/mol ± 0.016) (a difference of approximately 2 Kcal/mol between them) both binding in the similar place of the transporter. These analyses of results effectively indicated a different binding mode: The S enantiomer showed two hydrogen bonds with Glu320 and Asp321 of the ABC transporter; on the other hand, the R enantiomer showed two hydrogen bonds but with Glu353 and Gly378 ( Figure 5), and in both molecules, the hydrophobic interaction is important to the binding. We carried out a similar analysis by docking studies with 8 which has a bad antibacterial activity. The binding energy (-2.39 Kcal/mol) obtained was worse than the 11 enantiomers and the molecule only showed one hydrogen bond with Asp220 for stabilization in the binding area. Finally, we observed that the molecule is located in the same area as the isomers as we observed in Figure 5.
Docking of 11 (S and R enantiomers) and 8 with ATP-binding cassette (multi-drug ABC transporter Sav1866 from S. aureus in complex with AMP-PNP) We used a fast and predictive tool as the molecular docking, which allowed us to obtain by the genetic algorithm the most suitable location of ligand on macromolecule without using molecular dynamic simulations. Thus, we carried out the analysis according to the most favorable binding energy, by grouping the results in a cluster. To analyze the good result of 11 and their correlation with the antibacterial activity, we used the in silico tool with a known and essential pharmacological target linked to this property. For the above, the ABC transporter was selected, which is crucial for balancing nutrients in bacteria and humans. This transporter is also responsible for several processes, such as drug-resistant bacteria [38]. Based on these reasons, we used docking tools to observe which interactions were relevant, especially with the most promising molecule 11, although it is worth taking into account that the biological tests were carried out with a racemic mixture. In this study, we decided to study both enantiomers in order to be able to discriminate them and determine whether the binding was stereoselective. The results showed a priority for the S enantiomer (-5.06 Kcal/mol ± 0.012) versus the R enantiomer (-3.37 Kcal/mol ± 0.016) (a difference of approximately 2 Kcal/mol between them) both binding in the similar place of the transporter. These analyses of results effectively indicated a different binding mode: The S enantiomer showed two hydrogen bonds with Glu320 and Asp321 of the ABC transporter; on the other hand, the R enantiomer showed two hydrogen bonds but with Glu353 and Gly378 ( Figure 5), and in both molecules, the hydrophobic interaction is important to the binding. We carried out a similar analysis by docking studies with 8 which has a bad antibacterial activity. The binding energy (-2.39 Kcal/mol) obtained was worse than the 11 enantiomers and the molecule only showed one hydrogen bond with Asp220 for stabilization in the binding area. Finally, we observed that the molecule is located in the same area as the isomers as we observed in Figure 5.

In Vitro Antifungal Effects on Human Pathogenic fungi
In order to have a look into the potential usefulness of these compounds as candidates for the development of new antifungal drugs, we investigated the antifungal properties of compounds 1-5 and 7-13 against a panel of clinically important fungal species

In Vitro Antifungal Effects on Human Pathogenic fungi
In order to have a look into the potential usefulness of these compounds as candidates for the development of new antifungal drugs, we investigated the antifungal properties of compounds 1-5 and 7-13 against a panel of clinically important fungal species including two yeasts (Candida albicans and Cryptococcus neoformans), three Aspergillus spp. (A. niger, A. fumigatus, and A. flavus), and three dermatophytes (Trichophyton rubrum, T. mentagrophytes, and Microsporum gypseum) (voucher numbers are included in Table 6). The minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) of all compounds were determined with the microbroth dilution methods M27-A3 and M38-A2 of the Clinical and Laboratory Standards Institute (CLSI) [39,40]. The evaluation of the minimum fungicidal concentration (MFC) of the prenylated flavonoids showed whether the compounds kill fungi in addition to inhibiting them. This characteristic is highly appreciated in an antifungal drug since the recurrence of fungistatic drugs is mostly avoided because long treatments are no longer necessary.  Compounds with MICs of >250 µg/mL were considered inactive; those between 250 and 125 µg/mL were considered lowly active; those in the range 62.5-31.25 µg/mL were considered moderately active; and MICs of <31.25 µg/mL were considered highly active.
The results showed ( Table 6) that the non-prenylated structures (the flavone 1, the flavonols 2 and 3, the flavanone 4, and the isoflavone 5) were all inactive.
When these flavonoids were prenylated, the following results were obtained: (i) The prenylation of the flavone 1 or the isoflavone 5 did not improve the antifungal activity, since 7, 8, and 13 were inactive (MIC ≥ 250 µg/mL). (ii) The prenylation of the flavonols 2 and 3 led to 9 (which showed activity against the yeasts C. albicans and C. neoformans and dermatophytes at 62.5 µg/mL) and 10, which was inactive. Both compounds possessed the prenyl substituent at position 5 but differed in that 9 possesses a hydroxyl group in position 5. This structural difference could play a role in the antifungal activity of 9. (iii) The prenylation of the flavanone 4 led to both 11 (MIC = 125 µg/mL against C. neoformans and 62.5 µg/mL against dermatophytes) and the most active compound 12, which displayed MIC = 125 µg/mL against C. albicans, MIC = 62.5 µg/mL against C. neoformans, MIC = 15.6 µg/mL against the dermatophytes T. rubrum and T. mentagrophytes, and 31.2 µg/mL against M. gypseum). Probably the prenyl substituent at position 8 of the trihydroxyflavanone naringenin confers high activity to the whole flavanone molecule.
Considering that C. neoformans is the main cause of cryptococcal meningoencephalitis among HIV patients, which many times has not responded to any antifungal and has led to death [41], we decided to test compounds 9, 11, and 12 (which showed some activity against the ATCC strain) against a panel of five clinical C. neoformans strains. The results showed ( Table 7) that the behavior of the three compounds against clinical isolates is similar to that against the standard ATCC strain. In turn, since C. albicans is the fourth leading cause of nosocomial bloodstream infection (BSI) in intensive care units, causing fatal invasive candidiasis in a high percentage of patients [42], we decided to test 9 and 12 against five clinical isolates of C. albicans, in order to determine whether these two compounds have the potential to be further developed. The results showed ( Table 8) that both compounds behave against C. albicans clinical isolates similarly to how they behave against the standard ATCC strain.

In Vitro Antibacterial Effects on Plant Pathogen bacteria
The results of the antibacterial activity against phytopathogenic bacteria showed that all the prenylated compounds were able to inhibit the in vitro growth of the three bacteria tested. Interestingly, most of the compounds significantly inhibited the growth of the three bacterial species in high percentages, obtaining IC 50 values of less than 3.9 µM for several of them. Despite the above considerations, the 3, 4, and 5 molecules showed the highest IC 50 values in the series (Table 9). Statistical analysis revealed that the values obtained from inhibition are significant (p < 0.05) compared to the negative control. Antibacterial activities are shown as IC 50 and MIC values in µM concentrations; IC 50 values were calculated from the first kinetic assay. MIC: minimum inhibitory concentration.
IC 50 values for each compound indicated that the bacteria tested had different levels of sensitivity to the compounds tested. The most sensitive bacteria were P. syringae, and the most resistant were A. tumefaciens. This behavior could be due to the different mechanisms of action of the compounds on each tested Gram-negative pathogen.
After the first kinetic test, the bactericidal capacity of each compound was evaluated in a second growth kinetics test, using an inoculum of the first kinetic plate [43]. This new culture was incubated for 7 h, and the absorbance was measured. It was observed that the bacterial culture was able to recover its growth rate (with respect to the culture without compound) by 50-85%. This means that the compounds were bacteriostatic but not bactericide at the concentrations tested. Compound 13 showed selectivity against P. syringae and P. carotovorum and to a lesser extent against A. tumefaciens (>250, MIC).

In Vitro 5-hLOX Enzyme Inhibition Assay
The results showed that, of all the evaluated molecules (Table 10), only two (compounds 9 and 10) had a relevant inhibitory effect. The structural characteristic that differentiates these compounds from the rest of the structures is the catechol group (1,2dihydroxybenzene) in Ring B. Many known inhibitors of 5-LOX possess the catechol group that is a relevant characteristic for their inhibitory properties [44].

DPPH Radical Scavenging Activity
The results obtained from antioxidant activity assays are shown in Table 11. All compounds were compared with Trolox. All compounds possessing the catechol moiety were active. It is known that the flavonoids quercetin (2) and fisetin (3) have better antioxidant activity than Trolox [45]; however, their prenylated derivatives (9 and 10) show an even greater degree of this activity. This behavior follows the same trend found in some of our previous work [18]. Antioxidant activity is shown as IC 50 values in µM concentrations; NA = no activity. All compounds were analyzed in triplicate, and the results expressed as average ± standard deviation.

Cytotoxic Activity of 11 and 12
Anticancer activity for naringenin in diverse tumor cell lines has been previously reported [46][47][48]. However, in vitro activities were commonly observed using high flavonoid concentrations often exceeding 100 µM, which have limited physiological significance and must be interpreted with caution. Given this anticancer activity and that prenylated flavonoids have enhanced biological activities, we measured cytotoxic activity prenylated naringenin at positions 6 and 8 (11 and 12) against MDA-MB-231 (the human breast adenocarcinoma cell line), B16-F10 (mouse melanoma cells), and MEF (primary mouse embryonic fibroblasts). Our results in Table 12 show that the cytotoxic effect of both prenylated naringenins were low for both cancer cell lines and did not show any selectivity compared to the effect on non-cancerous cells (MEFs). When the cytotoxic effect of 11 and 12 against cell lines were compared with commonly used chemotherapeutic drugs, it could be verified that both prenylated flavonoids showed from 100-to 250-fold lower activity than Taxol and from 30-to 50-fold lower activity than etoposide. It is possible that the anticancer activity described for naringenin in vivo could be related to antiangiogenic and immunostimulating effects [49,50]. On the other hand, compounds 11 and 12 exhibited the most effective antibacterial activity, which had a synergistic effect with commercial antibiotics. A significant synergic activity was observed using approximately 7 µM for 11 and 15 µM for 12 (Table 4), which did not generate a cytotoxic effect in MEF cells. This clearly suggests that 11 and 12 could be useful for in vivo treatment against pathogenic resistant bacteria, although further studies are needed to confirm this possibility.

General Data
All chemical reagents obtained were purchased from Merck (Darmstadt, Germany), Sigma-Aldrich (St. Louis, MO, USA), or Alfa Aesar (Kandel, Germany), were of the highest commercially available purity, and were used without previous purification. Melting points (mp: • C) were measured on a melting point apparatus Stuart-Scientific SMP3 and are uncorrected. IR spectra were recorded as a KBr disk in a Thermo Scientific Nicolet 6700 FT-IR spectrometer (Massachusetts, USA), and frequencies are reported in cm −1 . High-resolution mass spectra were recorded on an Exactive TM Plus Orbitrap spectrometer (Thermo Scientific, Bremen, Germany) by infusion, applying a voltage of 5 or 8 kV in the positive ionization mode and 4 or 7 kV in the negative ionization mode. The spectra were recorded using full-scan mode, covering a mass range from m/z 214 to 470. The resolution was set to 140,000, and the maximum loading time for the ion cyclotron resonance (ICR) cell was set to 200 ms. 1 H-, 13

General Experimental Procedure for the Prenylation of Flavonoids
A solution of flavonoid (1 mol equiv) and dry ZnCl 2 (4 mol equiv) was placed in a round bottom flask and dissolved in ethyl acetate (100 mL). Under vigorous stirring, a solution of 3-methyl-2-buten-1-ol (6) (4 mol equiv) in ethyl acetate (10 mL) was added dropwise for 1 h at 40 • C. The reaction mixture was then allowed to warm up to reflux temperature and the stirring continued. After 4 h, water at pH 1 (100 mL) was added to the reaction mixture to decompose the ZnCl 2 . The organic layer was then separated, and a new extraction with ethyl acetate was made. The organic solutions obtained after extractions were mixed and dried over anhydrous sodium sulphate and filtered, and the solvent was evaporated under reduced pressure. The mixture was then subjected to silica gel flash column chromatography (ethyl acetate/hexane or dichloromethane/methanol or acetone/hexane mixtures were used as mobile phases) to obtain pure products.

Checkerboard Dilution Test
The assessments of synergy of prenylated flavonoids 11 and 12 combined with methicillin were investigated with the checkerboard method [57]. Briefly, the uppermost row (A) of a 96-well microtiter plate contained substance X in a concentration of about four times the expected MIC of the microorganism examined. Each following row (B-H) contained half the concentration of the previous one. The same procedure was carried out along the columns (1-12) with substance Y, but not necessarily with the same starting concentration. Therefore, each well contained a unique combination of the two substances (X and Y).

Time Kill Assay
Time kill curves were conducted with a final concentration of antimicrobial agent at four times the MIC [59]. Flasks containing 50 mL of MHB with the appropriate antimicrobial agent were inoculated with 50 mL of the test organism in a logarithmic growth phase adjusted to the appropriate density. Aliquots were removed, diluted, and plated at the 0, 2, 4, 6, and 24 h time points. The plates were incubated for 24 h, and the viable counts were determined. Bactericidal activity was defined as a reduction of 99.9% (≥3 log10) of the total count of CFU/mL in the original inoculum [56]. Bacteriostatic activity was defined as the maintenance of or a reduction of less than 99.9% (3 log10) of the total count of CFU/mL in the original inoculum.

Determination of Reactive Oxygen Species (ROS) Intrabacterial Levels
The production of ROS by S. aureus ATCC 6538 after treatment with the prenylated compounds was evaluated using the peroxynitrite indicator 2 ,7 -dichlorodihydrofluorescein diacetate (DCFH-DA) (Sigma-Aldrich), which can detect a broad range of ROS including nitric oxide and hydrogen peroxide. The adjusted bacterial culture (0.5 McFarland exponential phase bacteria culture) was treated with a concentration of each compound of 2 µM, using ciprofloxacin as an antibiotic control, Trolox as a ROS scavenger control, 1,4-naphtoquinone as a ROS generation control, and the presence of DCFH-DA at a final concentration of 5 mM in 0.85% saline and incubated at 37 • C aerobically at 200 rpm for 24 h. Untreated bacterial culture was served as the negative control. The fluorescence emission of DCFH-DA was measured at 525 nm using a Tecan microtiter plate reader with an excitation wavelength of 485 nm. The background fluorescence of 0.85% saline and autofluorescence of the bacterial cells incubated without the probe was measured to calculate the net fluorescence emitted from the assay itself. The experiment was conducted in triplicate [60]. Clinical isolates of C. neoformans (n = 5) were provided by IM. They included five strains whose voucher specimens are presented in Table 7. Clinical isolates of C. albicans (n = 5) were provided by CCC; their voucher specimens are presented in Table 8.

In Vitro Antifungal Activity Assays against Human Pathogens Microorganisms and Media
Strains were grown on Sabouraud-chloramphenicol agar slants for 48 h at 30 • C, maintained on slopes of Sabouraud-dextrose agar (SDA, Oxoid) and sub-cultured every 15 days to prevent pleomorphic transformations. Inocula were obtained in accordance with reported procedures [39,40] and adjusted to 1-5 × 10 3 cells with colony-forming units (CFUs)/mL.

Antifungal Susceptibility Testing
The minimum inhibitory concentration (MIC) of each compound was determined by using broth microdilution techniques according to the guidelines of the CLSI for yeasts (M27-A3) [39] and for filamentous fungi (including dermatophytes) M38-A2 [40]. MIC values were determined in RPMI-1640 (Sigma-Aldrich) and buffered to pH 7.0 with MOPS. Microtiter trays were incubated at 35 • C for yeasts and Aspergillus spp. and at 28-30 • C for dermatophyte strains in a moist, dark chamber. MICs were visually recorded at 48 h for yeasts, and at a time according to the control fungus growth, for the rest of the fungi. For the assay, stock solutions of pure compounds were two-fold diluted with RPMI-1640 from 250 to 0.98 µg/mL (final volume = 100 µL) and a final DMSO concentration of ≤1%. A volume of 100 µL of inoculum suspension was added to each well with the exception of the sterility control, where sterile water was added to the well instead. Terbinafine (Novartis Co, Basel, Switzerland) and amphotericin B (Sigma-Aldrich) were used as positive controls.
Endpoints were defined as the lowest concentration of drug resulting in the total inhibition of visual growth compared to the growth in the control wells containing no antifungal drug.
The MFC of each compound against an isolate was determined as follows: After determining the MIC, an aliquot of 5 µL was withdrawn from each clear well of the microtiter tray and plated onto a 150 mm RPMI-1640 agar plate buffered with MOPS (Remel Inc., Lenexa, KS, USA). Inoculated plates were incubated at 30 • C, and the MFC was recorded after 48 h. The MFC was defined as the lowest concentration of each compound that resulted in the total inhibition of visible growth.

In Vitro Antibacterial Activity against Plant Pathogens
Broth microdilution methods were used to evaluate the effects of the compounds on the growth of P. carotovorum (NCPPB 312), A. tumefaciens (strain C58C1), and P. syringae (NCPPB 281). Bacteria were grown in sterile tubes with 10 mL of Mueller-Hinton (MH) medium and incubated at 27 • C for 12 h with shaking to produce an initial culture. The antimicrobial activity was evaluated by observing the growth response of both microorganisms in samples with different concentrations of the compounds [61][62][63]. All assays were performed on sterile 96-well microplates with a final volume of 200 µL containing Mueller-Hinton broth (MH) inoculated with 1 µL of bacterial suspension (10 5 -10 6 UFC/mL, initial culture) in the presence of different concentrations of test compounds (3.9, 7.8, 15.6, 31.3, 62.5, 125, and 250 µM). MH was used as the negative control [C(−)], and MH with streptomycin [64] was used as the positive control [C(+)]. The plates were incubated for 7 h at 27 • C. Bacterial growth was monitored by measuring the optical density at 595 nm every hour with a microplate reader. All tests were performed in 10 repetitions for each microorganism evaluated. Bacterial growth was shown as the arithmetic mean expressed in terms of the negative control (100% growth). The lowest concentration of the compound preventing the appearance of turbidity was considered to be the minimal inhibitory concentration (MIC).
The first experiment (first kinetic assay) in which the compound was exposed to the bacterial cultures in MH was carried out over a period of 6 h. Subsequently, to determine the minimal bactericidal concentration (MBC), a second experiment (second kinetic assay) was conducted; this experiment involved taking inoculum from the first kinetic assay and adding it to MH in a new 96-well microplate containing culture medium, which was then cultured for 7 h. The aim of this second culture (second kinetic assay) was to determine whether the compounds have bactericidal or bacteriostatic properties [65].

Statistics
The Mann-Whitney U test (*) with p < 0.05 was performed to identify significant differences among the treatment and control groups and between the two treatments.

In Vitro 5-LOX Enzyme Inhibition Assay
The commercially available enzyme by Cayman Chemicals Inc., Ann Arbor, MI, USA, was diluted (1:500) in the assay buffer (HEPES 50 mM, EDTA 2 mM, ATP 10 µM, and CaCl 2 10 µM at pH 7.5) and mixed with 10 µM H 2 DCFDA dye in the reaction mixture and incubated for 15 min in the assay plate. Subsequently, 280 µL of buffer was added, and 10 µL of inhibitor with a final concentration of 10 µM was placed per well in the reaction mixture and incubated for 30 min. The reaction was started by the addition of a suitable concentration of arachidonic acid (0.5 µM). The fluorescence was read in a multimode detector Synergy™ HT Multi-Mode Microplate Reader (Biotek) at 480 nm excitation/520 nm emission after 1 h of incubation at room temperature. The inhibition percentage was obtained for analysis of oxidation of H 2 DCFDA dye to the highly fluorescent 2 ,7 -dichloro-fluorescein (DCF) product.

Docking of 11 (S and R Enantiomers) and with ABC Transporter
The prenylated flavonoids structures were built with the Molecular Operating Environment software [66]. ChelpG charges were obtained at the B3LYP/6-31G** level theory, employing the Gaussian 09 package [67]. Docking was done with the crystal structure of the ABC transporter (PDB code: 2ONJ, 3.40 Å resolution) using the AutoDock4 package [68] and a Lamarckian algorithm, assuming total flexibility of the inhibitors. The grid maps were made up first to 126 × 126 × 126 points and later to 60 × 60 × 60, with a grid-point spacing of 0.375 Å on the grid map. The AutoTors option was used to define the ligand torsions, and the docking results were then analyzed by a ranked cluster analysis, resulting in conformations with the highest overall binding energy (the most negative Gibbs free energy binding value, -∆G).

General Procedure to Determine the DPPH Radical Scavenging Activity
The radical scavenging activity of the prenylated compounds and starting materials towards the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical was measured as described [18], adapted to a screen to 96-well plates. Briefly, stock solutions of each compound were prepared in methanol at a 1-mM concentration (10 mL). Dilutions (1-200 µM) were prepared from the stock solutions. Methanol (90 µL), dilutions (150 µL), and DPPH (60 µL, Sigma-Aldrich) in methanol (0.5 mM), resulting in a final concentration of 0.1 mM of DPPH were added in a 96-well plate. Methanol was used as the blank sample. The mixtures were left for 30 min at room temperature, and the absorbances were then measured at 517 nm. Trolox was used as the standard antioxidant. The radical scavenging activity was calculated as follows: % Inhibition = [(blank absorbance − sample absorbance)/blank absorbance] × 100. The mean of three IC 50 (concentration causing 50% inhibition) values for each compound was determined graphically.

In Vitro Anticancer Activity of 11 and 12
The cell lines used in this work included MDA-MB-231 human breast adenocarcinoma cells, B16-F10 mouse metastatic melanoma cells, and MEF primary mouse embryonic fibroblasts. Cells were maintained in a DMEM high glucose medium (Mediatech, Manassas, VA, USA) supplemented with 10% (MDA-MB-231 and B16-F10) or 15% (MEF) heat-inactivated fetal bovine serum (HyClone Laboratories), 100 IU/mL penicillin, and 100 µg/mL streptomycin and maintained at 37 • C in a 5% CO 2 humidified atmosphere. Cell viability was measured using CyQuant®Direct Cell Proliferation Assay Kit (Life Technologies) following the manufacturer's instruction. Briefly, 5.000 cells/well were seeded onto a flat-bottomed 96-well plate in a 200 µL final volume. Six hours after seeding, the culture medium was replaced with the medium containing the tested compounds at concentrations ranging from 0 to 100 µM dissolved in DMSO (a 0.1% final concentration) for 72 h. The concentrations used to calculate the IC 50 values were 100, 30, 10, 3, 1, 0.3, 0.1, 0.01, and 0 µM. Untreated cells (medium containing 0.1% DMSO) were used as controls. At the end of the incubation, 100 µL of culture medium was removed from each experimental well and replaced by 2× detection reagent. Cells were incubated for 1 h, and fluorescence emission was measured at 535 nm with excitation at 480 nm in a microplate reader (Infinite 200 PRO, Tecan). At least four independent experiments were performed for each concentration. The results from each experiment were transformed to a percentage of controls, and the IC 50 values were graphically obtained from the dose-response curves. The IC 50 value was obtained adjusting the dose-response curve to sigmoidal curves (variable slope), generated using GraphPad Prisma 6.0 software [69].

Conclusions
Our results demonstrated that the 6-prenylated-(±)-naringenin (11), 8-prenylated-(±)naringenin (12) and the 8-prenylated genistein (13) are active molecules against Staphylococcus aureus-resistant bacteria with a strong synergistically effect in combination with commercial antibiotics vancomycin, ciprofloxacin, and methicillin, enhancing their effects in a factor of 10-100 times against drug-resistant bacteria. Compound 12 is the most active against the dermatophytes T. rubrum, T. mentagrophytes, and M. gypseum fungus. Prenylation substitution was shown to be fundamental for these biological activities, specifically at positions 6 and 8 of the (±)-naringenin (11 and 12, respectively); however, others structural requirements are important as well, such as hydroxylation at positions 5, 7 of ring A, and 4 of ring B. According to the docking results of 11 with ATP-binding cassette transporter, the S isomer showed better interaction energy than the R isomer. It is relevant to continue this study with enantiomerically pure isomers of prenylated flavanones 11 and 12.