And Dehydro-Ε-Viniferin for Antimicrobial Activity Against the Foodborne Pathogen Listeria Monocytogenes

International Journal of Molecular Sciences

Article Structural Requirements of Benzofuran Derivatives Dehydro-δ- and Dehydro-ε-Viniferin for Antimicrobial Activity Against the Foodborne Pathogen Listeria monocytogenes

Giorgia Catinella 1, Luce M. Mattio 1 , Loana Musso 1, Stefania Arioli 1 , Diego Mora 1 , Giovanni Luca Beretta 2 , Nadia Zaﬀaroni 2 , Andrea Pinto 1,* and Sabrina Dallavalle 1

1 Department of Food, Environmental and Nutritional Sciences (DeFENS), University of Milan, Via Celoria 2, 20133 Milan, Italy; [email protected] (G.C.); [email protected] (L.M.M.); [email protected] (L.M.); [email protected] (S.A.); [email protected] (D.M.); [email protected] (S.D.) 2 Molecular Pharmacology Unit, Department of Applied Research and Technological Development, Fondazione IRCCS Istituto Nazionale Tumori, Via Amadeo 42, 20133 Milan, Italy; [email protected] (G.L.B.); nadia.zaﬀ[email protected] (N.Z.) * Correspondence: [email protected]

Received: 15 February 2020; Accepted: 19 March 2020; Published: 21 March 2020

Abstract: In a recent study,we investigated the antimicrobial activity of a collection of resveratrol-derived monomers and dimers against a series of foodborne pathogens. Out of the tested molecules, dehydro-δ-viniferin and dehydro-ε-viniferin emerged as the most promising derivatives. To define the structural elements essential to the antimicrobial activity against the foodborne pathogen L. monocytogenes Scott A as a model Gram-positive microorganism, the synthesis of a series of simplified benzofuran-containing derivatives was carried out. The systematic removal of the aromatic moieties of the parent molecules allowed a deeper insight into the most relevant structural features affecting the activity. While the overall structure of compound 1 could not be altered without a substantial loss of antimicrobial activity, the structural simplification of compound 2 (minimal inhibitory concentration (MIC) 16 µg/mL, minimal bactericidal concentration (MBC) >512 µg/mL) led to the analogue 7 with increased activity (MIC 8 µg/mL, MBC 64 µg/mL).

Keywords: viniferin derivatives; benzofuran nucleus; antimicrobials; Listeria monocytogenes

1. Introduction Benzofuran derivatives are an important class of heterocyclic compounds, occupying a place in numerous bioactive natural products. Over time, they have attracted the attention of many researchers due to the broad scope of their activity, which includes anticancer, antimicrobial, immunomodulatory, antioxidant and anti-inflammatory properties [1,2]. In recent years, the benzofuran scaffold has emerged as a pharmacophore of choice for designing antimicrobial agents, and numerous achievements have revealed that benzofuran-based compounds have an extensive potential in this field [3]. As a part of our efforts in the search for new antimicrobial agents, we have recently evaluated the antimicrobial activity of a collection of resveratrol-derived monomers (e.g., resveratrol, pterostilbene) and dimers (e.g., trans-δ-viniferin, trans-ε-viniferin, dehydro-δ-viniferin 1, viniferifuran 2), which were screened as single molecules against a panel of foodborne pathogens (Figure1)[4].

Int. J. Mol. Sci. 2020, 21, 2168; doi:10.3390/ijms21062168 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2020, 21, 2168 2 of 10 Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 2 of 10

FigureFigure 1. Structures 1. Structures of resveratrol of resveratrol an andd representative representative resveratrol- resveratrol-derivedderived monomers monomers and and dimers. dimers. The most promising dimeric compound, dehydro-δ-viniferin 1, was endowed with significant Theantibacterial most promising activity against dimeric Gram-positive compound, bacteria.dehydro- Itδ-viniferin was verified 1, was that theendowed activity with of 1 againstsignificant antibacterialL. monocytogenes activitywas against achieved Gram-positive by damaging bact theeria. cytoplasmic It was verified membrane that with the significant activity of membrane 1 against L. monocytogenesdepolarization, was aachieved loss of membrane by damaging integrity, the and cytoplas severe morphologicalmic membrane changes. with The si dimericgnificant compound membrane depolarization,2 showed significant a loss of antimicrobial membrane activity integrity, as well and [4]. severe morphological changes. The dimeric compoundThese 2 showed promising significant results antimicrobial prompted us to activity explore as1 welland 2[4].as platforms in the search for new Theseantimicrobial promising scaffolds. results To provideprompted a deeper us to insight explore into 1 the and activity 2 as ofplatforms these compounds, in the search we planned for new antimicrobialto perform scaffolds. preliminary To structure–activityprovide a deeper relationship insight into (SAR) the studies.activity Theof these unique compounds, structural features we planned of to performcompounds preliminary1 and 2, bothstructure–activity containing a versatile relationship benzofuran (SAR) core studies. structure, The make unique them amenablestructural for features the generation of differently substituted analogues. To shed light on the minimal structural features which of compounds 1 and 2, both containing a versatile benzofuran core structure, make them amenable retain antimicrobial activity, we designed a small collection of simplified derivatives of compounds for the generation of differently substituted analogues. To shed light on the minimal structural 1 and 2 by a systematic removal of the moieties linked to the benzofuran rings (groups A, B, C) features(Figure which2), and retain we testedantimicrobial all the derivatives activity, against we designedListeria monocytogenesa small collectionas a representative of simplified foodborne derivatives of compoundsGram-positive 1 and bacterium, 2 by a systematic which was removal found to of be the sensitive moieties to compounds linked to the1 and benzofuran2 [4]. rings (groups A, B, C) (FigureTherefore, 2), and we designed we tested the all three the possiblederivatives simplified against analogues Listeria ofmonocytogenes compound 1 (compounds as a representative3–5) foodborneand the Gram-positive three possible simplifiedbacterium, analogues which was of compound found to be2 (compounds sensitive to6 –compounds8) (Figure2). 1 In and addition, 2 [4]. we designed three representative analogues (9–11) containing the -OMe groups in place of the phenolic -OH on the aromatic rings. The evaluation of the antimicrobial activity of the compounds provided an integrated overview of the structural determinants for the activity against the foodborne pathogenic species L. monocytogenes Scott A.

Int. J. Mol. Sci. 2020, 21, 2168 3 of 10 Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 3 of 10

Figure 2. Structures of compounds 1 and 2 and of the simplified analogues 3–8 and 9–11. Figure 2. Structures of compounds 1 and 2 and of the simplified analogues 3–8 and 9–11. 2. Results Therefore, we designed the three possible simplified analogues of compound 1 (compounds 3– In the present SAR study, different synthetic approaches were set up depending on the nature of 5) and the three possible simplified analogues of compound 2 (compounds 6–8) (Figure 2). In the benzofuran substitution pattern. addition, we designed three representative analogues (9–11) containing the -OMe groups in place of Initially, we focused on the synthesis of the simplified analogues of compound 1. Compound the phenolic -OH on the aromatic rings. 14 was obtained by a Cu-catalyzed tandem Sonogashira coupling–cyclization reaction starting with The evaluation of the antimicrobial activity of the compounds provided an integrated overview 12 13 14 of2-iodophenol the structuraland determinants 1-ethynyl-4-methoxybenzene for the activity against[5,6 ].the The foodborne iodination pathogenic of with species NIS gave L. 15 15 monocytogenescompound Scottin a 74%A. yield [7]. Suzuki coupling of with (3,5-dimethoxyphenyl)boronic acid gave the permethylated intermediate 16, which, after the deprotection of the phenolic -OH with BBr3, Int.2.aff Results ordedJ. Mol. Sci. 2,3-disubstituted 2020, 21, x FOR PEER benzofuran REVIEW 3 (Scheme1). 4 of 10 In the present SAR study, different synthetic approaches were set up depending on the nature of the benzofuran substitution pattern. Initially, we focused on the synthesis of the simplified analogues of compound 1. Compound 14 was obtained by a Cu-catalyzed tandem Sonogashira coupling–cyclization reaction starting with 2- iodophenol 12 and 1-ethynyl-4-methoxybenzene 13 [5,6]. The iodination of 14 with NIS gave compound 15 in a 74% yield [7]. Suzuki coupling of 15 with (3,5-dimethoxyphenyl)boronic acid gave the permethylated intermediate 16, which, after the deprotection of the phenolic -OH with BBr3, afforded 2,3-disubstituted benzofuran 3 (Scheme 1).

Scheme 1. Reagents and conditions: (a) (i) PdCl2(PPh3)2 (5% mol), CuI (3% mol), TEA:THF 1:1, Scheme 1. Reagents and conditions: a) i) PdCl2(PPh3)2 (5% mol), CuI (3% mol), TEA:THF 1:1, N2, 40 N2, 40 ◦C, 40 min; (ii) ACN, 100 ◦C, 90 min, 50%; (b) NIS, p-TsOH, ACN, N2, overnight, 74%; (c) °C, 40 min; ii) ACN, 100 °C, 90 min, 50%; b) NIS, p-TsOH, ACN, N2, overnight, 74%; c) (3,5- (3,5-dimethoxyphenyl)boronic acid, K2CO3, PdCl2(dppf) CH2Cl2, THF:H2O 1:1, MW, 70 ◦C, 30 min, dimethoxyphenyl)boronic acid, K2CO3, PdCl2(dppf)·CH2Cl· 2, THF:H2O 1:1, MW, 70 °C, 30 min, 83%; 83%; (d) BBr3, DCM, 0 ◦C to rt, overnight, 61%. d) BBr3, DCM, 0 °C to rt, overnight, 61%.

The synthesis of compound 4 was based on the same key Cu-catalyzed tandem Sonogashira coupling-cyclization described above for the construction of the 2-aryl-substituted benzofuran ring (Scheme 2). However, in this case the iodophenol should have a suitable moiety in position 4 to install the styryl functionality. Thus, compound 17 was reacted with 13 to obtain the ester 18, which was then converted into the corresponding aldehyde 19 by an LiAlH4 reduction, followed by a Dess– Martin periodinane oxidation. A Wittig–Horner olefination with diethyl (3,5- dimethoxyphenyl)phosphonate under microwave irradiation afforded 10. The final deprotection step of all the phenol groups was quite troublesome. Previous works reported that the demethylation of the stilbenoid derivatives was achieved using BBr3 in CH2Cl2 [8]. However, when applying these conditions to compound 10, only the partially demethylated compound 9 was obtained. We, therefore, decided to explore the alternative route reported by Vo et al. [9], based on the use of boron trichloride/tetra-n-butylammonium iodide (BCl3/TBAI). Actually, the reagent was found to be more effective than boron tribromide and allowed us to obtain the demethylated compound 4. A semi- preparative HPLC purification was required to obtain the pure compound in a 35% yield.

Scheme 2. Reagents and conditions: a) i) PdCl2(PPh3)2 (5% mol), CuI (3% mol), TEA:THF 1:1, N2, 40

°C, 40 min; ii) ACN, 100 °C, 90 min, 62%; b) i) LiAlH4, THF, N2, 0 °C, 10 min; ii) DMP, DCM, 0 °C 15 min, rt 90 min, 85%; c) diethyl (3,5-dimethoxyphenyl)phosphonate, NaH, THF, MW, 120 °C, 30 min,

76%; d) BBr3, DCM, 0 °C to rt, overnight, 60%; e) BCl3, TBAI, DCM, N2, 0 °C to rt, 6h, 35%.

The functionalization of positions 3 and 5 of the benzofuran ring required a different synthetic approach (Scheme 3). The heterocyclic core was synthesized efficiently from 20 by the sequence of alkylation with bromoacetaldehyde dimethylacetal and cyclodehydration using Amberlyst-15, following a procedure described by Liu et al. [10]. The introduction of a bromine in position 3 was obtained by the bromination of 22 followed by a treatment with KOH in MeOH [11]. The Suzuki coupling of compound 24 with (3,5 dimethoxyphenyl) boronic acid afforded compound 25. LiAlH4 reduction followed by a Dess–Martin periodinane oxidation gave the aldehyde 26, which underwent a Wittig–Horner olefination with diethyl(3,5-dimethoxyphenyl)phosphonate under microwave irradiation at 120 °C to give compound 27. Again, the critical step was the final deprotection of the

Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 4 of 10

Scheme 1. Reagents and conditions: a) i) PdCl2(PPh3)2 (5% mol), CuI (3% mol), TEA:THF 1:1, N2, 40

°C, 40 min; ii) ACN, 100 °C, 90 min, 50%; b) NIS, p-TsOH, ACN, N2, overnight, 74%; c) (3,5-

dimethoxyphenyl)boronic acid, K2CO3, PdCl2(dppf)·CH2Cl2, THF:H2O 1:1, MW, 70 °C, 30 min, 83%;

d) BBr3, DCM, 0 °C to rt, overnight, 61%.

TheInt. synthesis J. Mol. Sci. 2020 of, 21compound, 2168 4 was based on the same key Cu-catalyzed tandem 4Sonogashira of 10 coupling-cyclization described above for the construction of the 2-aryl-substituted benzofuran ring (Scheme 2). However,The synthesis in this of compound case the 4iodophenolwas based on shou theld same have key aCu-catalyzed suitable moiety tandem in position Sonogashira 4 to install the styrylcoupling-cyclization functionality. Thus, described compound above for 17 the was construction reacted ofwith the2-aryl-substituted 13 to obtain the benzofuran ester 18, ringwhich was (Scheme2). However, in this case the iodophenol should have a suitable moiety in position 4 to install then convertedthe styryl into functionality. the corresponding Thus, compound aldehyde17 was reacted 19 by with an13 LiAlHto obtain4 reduction, the ester 18, whichfollowed was thenby a Dess– Martin convertedperiodinane into the correspondingoxidation. aldehydeA Wittig–Horner19 by an LiAlH4 reduction,olefination followed bywith a Dess–Martin diethyl (3,5- dimethoxyphenyl)phosphonateperiodinane oxidation. A Wittig–Horner under microwave olefination irradiation with diethyl afforded (3,5-dimethoxyphenyl)phosphonate 10. The final deprotection step of all theunder phenol microwave groups irradiationwas quite a fftroublesome.orded 10. The finalPrevious deprotection works step reported of all the that phenol the groups demethylation was of quite troublesome. Previous works reported that the demethylation of the stilbenoid derivatives was the stilbenoid derivatives was achieved using BBr3 in CH2Cl2 [8]. However, when applying these achieved using BBr3 in CH2Cl2 [8]. However, when applying these conditions to compound 10, only conditionsthe partiallyto compound demethylated 10, compoundonly the9 waspartially obtained. demethylated We, therefore, decidedcompound to explore 9 thewas alternative obtained. We, therefore,route decided reported to byexplore Vo et al. the [9 ],alternative based on the route use of repo boronrted trichloride by Vo/ tetra-et al.n [9],-butylammonium based on the iodide use of boron trichloride/tetra-(BCl3/TBAI).n-butylammonium Actually, the reagent iodide was found (BCl to3 be/TBAI). more e ffActually,ective than the boron reagent tribromide was and found allowed to be more effective usthan to obtain boron the tribromide demethylated and compound allowed4. Aus semi-preparative to obtain the HPLCdemethylated purification compound was required 4 to. A semi- preparativeobtain HPLC the pure purification compound inwas a 35% required yield. to obtain the pure compound in a 35% yield.

Scheme 2. Reagents and conditions: (a) (i) PdCl2(PPh3)2 (5% mol), CuI (3% mol), TEA:THF 1:1, N2, Scheme 402. ◦ReagentsC, 40 min; (ii)and ACN, conditions: 100 ◦C, 90 min,a) i) 62%;PdCl (b)2(PPh (i) LiAlH3)2 (5%4, THF, mol), N2, 0CuI◦C, 10(3% min; mol), (ii) DMP, TEA:THF DCM, 0 ◦1:1,C N2, 40 °C, 40 min;15 min, ii) ACN, rt 90 min, 100 85%; °C, (c) 90 diethyl min, (3,5-dimethoxyphenyl)phosphonate,62%; b) i) LiAlH4, THF, N2, 0 °C, NaH, 10 THF, min; MW, ii) 120DMP,◦C, 30DCM, min, 0 °C 15 min, rt 9076%; min, (d) 85%; BBr3, DCM,c) diethyl 0 ◦C to(3,5-dimethoxyphe rt, overnight, 60%; (e)nyl)phosphonate, BCl3, TBAI, DCM, N 2NaH,, 0 ◦C toTHF, rt, 6 h,MW, 35%. 120 °C, 30 min, 76%; d) BBrThe3, functionalization DCM, 0 °C to rt, of overnight, positions 360%; and 5e) of BCl the3, benzofuranTBAI, DCM, ring N2 required, 0 °C to art, di 6h,fferent 35%. synthetic approach (Scheme3). The heterocyclic core was synthesized e fficiently from 20 by the sequence Theof functionalization alkylation with bromoacetaldehyde of positions 3 and dimethylacetal 5 of the benzofuran and cyclodehydration ring required using a Amberlyst-15, different synthetic approachfollowing (Scheme a procedure3). The heterocyclic described by core Liu etwas al. synthesized [10]. The introduction efficiently of from a bromine 20 by in the position sequence of 3 was obtained by the bromination of 22 followed by a treatment with KOH in MeOH [11]. alkylation with bromoacetaldehyde dimethylacetal and cyclodehydration using Amberlyst-15, The Suzuki coupling of compound 24 with (3,5 dimethoxyphenyl) boronic acid afforded compound following25 .a procedure LiAlH4 reduction described followed by byLiu a et Dess–Martin al. [10]. The periodinane introduction oxidation of a gavebromine the aldehyde in position26, 3 was obtainedwhich by the underwent bromination a Wittig–Horner of 22 followed olefination by with a treatment diethyl(3,5-dimethoxyphenyl)phosphonate with KOH in MeOH [11]. under The Suzuki couplingmicrowave of compound irradiation 24 with at 120 ◦(3,5C to dimethoxyphenyl) give compound 27. Again, boronic the critical acid step afforded was the finalcompound deprotection 25. LiAlH4 reductionof followed the phenolic by groups. a Dess–Martin After several periodinane attempts, compound oxidation5 was gave obtained the inaldehyde a 14% yield 26 by, which a treatment underwent with BBr and a troublesome purification by flash chromatography. a Wittig–Horner3 olefination with diethyl(3,5-dimethoxyphenyl)phosphonate under microwave Successively, we focused on the analogues of compound 2. Kim and Choi [12] employed a versatile irradiationand at mild 120 procedure °C to give to construct compound a 3-arylbenzofuran 27. Again, by the cyclization critical step of the was corresponding the finalβ-aryloxyketone deprotection of the using Bi(OTf)3. Following this protocol, compound 30 was prepared by the reaction of m-methoxyphenol with the α-bromoketone 29, on its turn obtained by treatment of 3,5-dimethoxyacetophenone 28 with CuBr2. The β-aryloxyketone 30 was subjected to a cyclodehydration reaction with Bi(OTf)3, involving an intramolecular Friedel–Craft acylation followed by dehydration to give the desired benzofuran 31. The same synthetic protocol was applied to the ketone 32 to obtain the benzofuran 33. In both cases, the intramolecular cyclization took advantage of the presence of the electron-donating OMe group. Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 5 of 10 Int. J. Mol. Sci. 2020, 21, 2168 5 of 10 phenolic groups. After several attempts, compound 5 was obtained in a 14% yield by a treatment with BBr3 and a troublesome purification by flash chromatography.

Scheme 3. Reagents and conditions: (a) 2-bromo-1,1-dimethoxyethane, Cs CO , MeCN, reﬂux, 3 days, Scheme 3. Reagents and conditions: a) 2-bromo-1,1-dimethoxyethane, Cs2CO23, MeCN,3 reflux, 3 days,

61%;61%; (b) Amberlyst-15, b) Amberlyst-15, toluene, toluene, reﬂux, reflux, 6 6h, h, 51%;51%; (c)c) Br Br2,2 DCM,, DCM, 0 °C 0 ◦ toC rt, to 75 rt, min, 75 min, 82%; 82%; d) KOH, (d) KOH, MeOH, MeOH, THF,THF, 0 ◦C, 0 20°C, min,20 min, 82%; 82%; (e) e) (3,5-dimethoxyphenyl)boronic (3,5-dimethoxyphenyl)boronic acid, acid, Pd(PPh Pd(PPh3)4, Na3)42CO, Na3, 2DME:HCO3, DME:H2O 5:1, 802O 5:1, 80 ◦C,°C, overnight, overnight, 74%; f) (f) LiAlH LiAlH4, THF,4, THF, 0 °C, 0 10◦C, min, 10 97%; min, g) 97%; DMP, g) DCM, DMP, 0 DCM,°C to rt,0 90◦ Cmin, to rt,78%; 90 h) min, diethyl 78%; (h) diethyl(3,5-dimethoxyphenyl)phosphonate, (3,5-dimethoxyphenyl)phosphonate, NaH, THF, NaH, MW, THF, 120 MW, °C, 12030 min,◦C, 3080%; min, i) BBr 80%;3, DCM, (i) BBr 0 3°C, DCM, to rt, 0 ◦C to rt,overnight, overnight, 14%. 14%.

TheSuccessively, installation ofwe afocused further on aromatic the analogues ring on of thecompound C-2 of compound2. Kim and 31Choiwas [12] obtained employed by a direct versatile and mild procedure to construct a 3-arylbenzofuran by the cyclization of the corresponding arylation by using Pd(OAc)2 and P(Cy)3 HBF4 [13]. The deprotection of the derivative 11 with BBr3 β-aryloxyketone using Bi(OTf)3. Following· this protocol, compound 30 was prepared by the reaction afforded compound 6 in a 52% yield (Scheme4). ofInt. J.m Mol.-methoxyphenol Sci. 2020, 21, x FOR with PEER theREVIEW α-bromoketone 29, on its turn obtained by treatment of6 of3,5- 10 dimethoxyacetophenone 28 with CuBr2. The β-aryloxyketone 30 was subjected to a cyclodehydration reaction with Bi(OTf)3, involving an intramolecular Friedel–Craft acylation followed by dehydration to give the desired benzofuran 31. The same synthetic protocol was applied to the ketone 32 to obtain the benzofuran 33. In both cases, the intramolecular cyclization took advantage of the presence of the electron-donating OMe group. The installation of a further aromatic ring on the C-2 of compound 31 was obtained by direct arylation by using Pd(OAc)2 and P(Cy)3·HBF4 [13]. The deprotection of the derivative 11 with BBr3 afforded compound 6 in a 52% yield (Scheme 4). Conversely, to obtain compound 7, it was necessary to introduce the styryl moiety in position 4. To avoid the formation of side products due to the reactivity of the styryl double-bond with the deprotecting reagent, we first treated compound 33 with BBr3 to afford compound 34 in a 91% yield. Finally,SchemeScheme a 4. HeckReagents 4. couplingReagents and conditions: withand pconditions:OH (a)styrene CuBr a) 2gave , EtOAc:CHClCuBr the2, EtOAc:CHCldesired3, reflux, scaffold3, overnight,reflux, 7. overnight, 67%; (b) m-methoxyphenol,67%; b) m- K2COmethoxyphenol,3, acetone, N2, reflux, K2CO 23, h,acetone, 90%; (c) N Bi(OTf)2, reflux,3, DCM,2h, 90%; N2 c), reflux, Bi(OTf) overnight,3, DCM, N 43%;2, reflux, (d) p-methoxybromobenzene, overnight, 43%; d) p-methoxybromobenzene, Pd(OAc)2, PCy3HBF4, K2CO3, pivalic acid, DMA, N2, 100 °C, 20h, 80%; e) Pd(OAc)2, PCy3HBF4,K2CO3, pivalic acid, DMA, N2, 100 ◦C, 20 h, 80%; (e) BBr3, DCM, 0 ◦C to rt, overnight, BBr3, DCM, 0 °C to rt, overnight, 52%; f) 3-bromo-5-methoxyphenol, K2CO3, acetone, N2, reflux, 2h, 52%; (f) 3-bromo-5-methoxyphenol, K2CO3, acetone, N2, reflux, 2 h, 89%; g) Bi(OTf)3, DCM, N2, reflux, overnight, 89%; g) Bi(OTf)3, DCM, N2, reflux, overnight, 83%; h) BBr3, DCM, 0 °C to rt, overnight, 91%; i) p- 83%; (h) BBr3, DCM, 0 ◦C to rt, overnight, 91%; (i) p-hydroxystyrene, Pd(OAc)2, TEA, dppp, DMF, N2, 120 ◦C, hydroxystyrene, Pd(OAc)2, TEA, dppp, DMF, N2, 120 °C, 20h, 80%. 20 h, 80%. The last simplified analogue was obtained as reported in Scheme 5. Compound 35 was Conversely, to obtain compound 7, it was necessary to introduce the styryl moiety in position methylated to give compound 36, following the procedure described by Iino [14]. However, in our 4. Tostudy, avoid the the selective formation methylation of side reaction products also due gave to 40% the of reactivity a dialkoxy of compound, the styryl which double-bond needed to with be the deprotectingseparated reagent,by column we chromatograp first treated compoundhy. The benzofuran33 with BBrderivative3 to aff ord38 was compound then obtained34 in aby 91% the yield. Finally, sequence a Heck of coupling alkylation with withpOH bromoacetaldehyde styrene gave the desireddimethylacetal scaffold and7. cyclodehydration using Amberlyst-15,The last simplified as described analogue before. was obtainedA treatment as reportedwith NBS in afforded Scheme 2-bromobenzofuran5. Compound 35 was 39, methylatedwhich to givewas compound isolated in36 a high, following yield as the the procedure only isomer described when we byused Iino dichloroethane [14]. However, as the in our solvent study, and the DMF selective methylationas the catalyst, reaction as alsodescribed gave 40%by Liu of [10]. a dialkoxy A Suzuki compound, coupling followed which needed by the conversion to be separated of theby ester column chromatography.group into aldehydes, The benzofuran as previously derivative reported,38 ledwas to thencompound obtained 41, bywhich the was sequence then subjected of alkylation to an with olefination reaction to give compound 42. Finally, the deprotection of the -OMe groups gave the simplified compound 8.

Scheme 5. Reagents and conditions: a) K2CO3, CH3I, DMF, rt, 45h, 35%; b) 2-bromo-1,1-

dimethoxyethane, Cs2CO3, CH3CN, reflux, 72h, 67%; c) Amberlyst-15, C6H5Cl, 120 °C, 3h, 63%; d)

NBS, DMF cat., ClCH2CH2Cl, 75 °C, 3h, 80%; e) (4-methoxyphenyl)boronic acid, Pd(PPh3)4, K2CO3,

DMF, 70 °C, overnight, 91%; f) LiAlH4, DCM, 0 °C, 10 min., 89%; g) DMP, DCM, 0 °C to rt, 90 min, 97%; h) diethyl (4-methoxybenzyl)phosphonate, NaH, dry THF, 120 °C - MW, 30 min, 54%; i) BBr3, 0 °C to rt, 6h, 24%.

The synthesized molecules 1–11 were tested against the foodborne pathogen L. monocytogenes Scott A, after being recognized as one of the most sensitive species as a result of our previous screening [4]. The results are reported in Table 1. The minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) values confirmed the higher sensitivity of L. monocytogenes to compound 1 (MIC 2 µg/mL, MBC 16 µg/mL) rather than to compound 2 (MIC 16 µg/mL, MBC >512 µg/mL). In general, the structural modifications of compounds 1 and 2 negatively interfered

Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 6 of 10

Scheme 4. Reagents and conditions: a) CuBr2, EtOAc:CHCl3, reflux, overnight, 67%; b) m-

methoxyphenol, K2CO3, acetone, N2, reflux, 2h, 90%; c) Bi(OTf)3, DCM, N2, reflux, overnight, 43%; d)

p-methoxybromobenzene, Pd(OAc)2, PCy3HBF4, K2CO3, pivalic acid, DMA, N2, 100 °C, 20h, 80%; e) BBr3, DCM, 0 °C to rt, overnight, 52%; f) 3-bromo-5-methoxyphenol, K2CO3, acetone, N2, reflux, 2h,

89%; g) Bi(OTf)3, DCM, N2, reflux, overnight, 83%; h) BBr3, DCM, 0 °C to rt, overnight, 91%; i) p-

hydroxystyrene, Pd(OAc)2, TEA, dppp, DMF, N2, 120 °C, 20h, 80%.

The last simplified analogue was obtained as reported in Scheme 5. Compound 35 was Int. J.methylated Mol. Sci. 2020 to, 21 give, 2168 compound 36, following the procedure described by Iino [14]. However, in our 6 of 10 study, the selective methylation reaction also gave 40% of a dialkoxy compound, which needed to be separated by column chromatography. The benzofuran derivative 38 was then obtained by the bromoacetaldehydesequence of alkylation dimethylacetal with bromoacetaldehyde and cyclodehydration dimethylacetal using Amberlyst-15, and cyclodehydration as described using before. A treatmentAmberlyst-15, with NBSas described afforded before. 2-bromobenzofuran A treatment with39 NBS, which afforded was isolated2-bromobenzofuran in a high yield 39, which as the only isomerwas when isolated we in used a high dichloroethane yield as the only as isomer the solvent when we and used DMF dichloroethane as the catalyst, as the as solvent described and by DMF Liu [10]. A Suzukias the coupling catalyst, as followed described by by the Liu conversion [10]. A Suzuki of the coupling ester group followed into aldehydes,by the conversion as previously of the ester reported, led togroup compound into aldehydes,41, which as waspreviously then subjected reported, toled an to olefinationcompound 41 reaction, which to was give then compound subjected 42to .an Finally, olefination reaction to give compound 42. Finally, the deprotection of the -OMe groups gave the the deprotection of the -OMe groups gave the simplified compound 8. simplified compound 8.

SchemeScheme 5. Reagents 5. Reagents and conditions: and conditions: (a) K2CO a)3 ,K CH2CO3I,3, DMF,CH3I, rt, DMF, 45h, 35%; rt, (b)45h, 2-bromo-1,1-dimethoxyethane, 35%; b) 2-bromo-1,1- Cs2COdimethoxyethane,3, CH3CN, reflux, Cs2CO 723, h,CH 67%;3CN, (c)reflux, Amberlyst-15, 72h, 67%; c) C Amberlyst-15,6H5Cl, 120 ◦ C,C6H 35 h,Cl, 63%; 120 °C, (d) 3h, NBS, 63%; DMF d) cat., ClCHNBS,2CH DMF2Cl, 75cat.,◦ C,ClCH 3 h,2CH 80%;2Cl, (e)75 °C, (4-methoxyphenyl)boronic 3h, 80%; e) (4-methoxyphenyl)boronic acid, Pd(PPh acid,3)4 ,KPd(PPh2CO33)4,, DMF,K2CO3, 70 ◦C, DMF, 70 °C, overnight, 91%; f) LiAlH4, DCM, 0 °C, 10 min., 89%; g) DMP, DCM, 0 °C to rt, 90 min, overnight, 91%; (f) LiAlH4, DCM, 0 ◦C, 10 min, 89%; (g) DMP, DCM, 0 ◦C to rt, 90 min, 97%; (h) diethyl 97%; h) diethyl (4-methoxybenzyl)phosphonate, NaH, dry THF, 120 °C - MW, 30 min, 54%; i) BBr3, 0 (4-methoxybenzyl)phosphonate, NaH, dry THF, 120 ◦C - MW, 30 min, 54%; (i) BBr3, 0 ◦C to rt, 6 h, 24%. °C to rt, 6h, 24%. The synthesized molecules 1–11 were tested against the foodborne pathogen L. monocytogenes The synthesized molecules 1–11 were tested against the foodborne pathogen L. monocytogenes Scott A, after being recognized as one of the most sensitive species as a result of our previous Scott A, after being recognized as one of the most sensitive species as a result of our previous screeningscreening [4]. [4]. The The results results are are reported reported inin TableTable1 1.. TheThe minimal minimal inhibitory inhibitory concentration concentration (MIC) (MIC) and and minimalminimal bactericidal bactericidal concentration concentration (MBC) (MBC) valuesvalues confirmed confirmed the the higher higher sensitivity sensitivity of L. of monocytogenesL. monocytogenes to compoundto compound1 (MIC 1 (MIC 2 µ2 gµg/mL,/mL, MBC MBC 1616 µµg/mL)g/mL) rather rather than than toto compound compound 2 (MIC2 (MIC 16 µg/mL, 16 µg /MBCmL, MBC >512>512µg/mL). µg/mL). In general, In general, the the structural structural modifications modifications of of compounds compounds1 1and and2 2negatively negatively interferedinterfered with their antimicrobial activity (Table1). However, compound 7, a simplified analogue of compound 2, showed an evident decrease in its MIC (8 µg/mL) and MBC (64 µg/mL) values, reflecting an improved antimicrobial activity.

Table 1. Antimicrobial activity of synthesized compounds against L. monocytogenes Scott A a and cytotoxic activity on WS1 b.

L. monocytogenes L. monocytogenes Compound WS1 Scott A Scott A a a b MIC (MBC) µg/mL MIC (µM) IC50 (µM) resveratrol 200 (-) c 876 >200 pterostilbene 64 (128) 250 57 10 ± 1 dehydro-δ-viniferin 2 (16) 4.42 37.0 1.4 ± 2 viniferifuran 16 (>512) 35.4 33.0 1.4 ± 3 16 (64) 50.3 98.7 1.8 ± 4 256 (>512) 743 97.8 3.0 ± 5 16 (64) 44.4 96.8 4.5 ± 6 64 (>512) 191 98.5 2.0 ± 7 8 (64) 23.2 45.0 1.2 ± 8 64 (>512) 178 85.0 4.6 ± 9 >512 (>512) 1370 95.0 2.3 ± 10 >256 (>256) 662 >100 11 >256 (>256) 656 >100 Chlorhexidine 8 (32) 15.8 - a MIC is the minimal inhibitory concentration inhibiting the growth; MBC (in brackets) is the minimal bactericidal b concentration. IC50 is deﬁned as the concentration of compound causing 50% cell growth inhibition. Twenty-four hours after seeding, cells were exposed for 48 h to the compounds and cytotoxicity was measured using MTS assay. Data represent mean values SD of three independent experiments. c L. monocytogenes LMG 16,779 [15]. ± Int. J. Mol. Sci. 2020, 21, 2168 7 of 10

3. Discussion The rapid development of the resistance of bacterial pathogens against antimicrobial agents still represents an important problem [16,17]. To overcome this drawback, the identification and development of novel scaffolds is imperative. Stilbenoids are an intriguing structural class of natural polyphenols characterized by a diverse array of biological properties [18]. Among stilbenoids, resveratrol and the dimethyl derivative pterostilbene have shown promising antimicrobial activity [19,20]. Recently, we investigated a focused collection of resveratrol-derived compounds, including resveratrol-derived monomers and dimers, which were screened against a series of foodborne pathogens [4]. The collected evidence indicated that most of the compounds were endowed with a significant antimicrobial activity against Gram-positive bacteria, with 1 and 2 being the most potent molecules of the series. To shed light on the SAR of these compounds, and to understand whether structural modifications could be exploited in the development of more potent congeners, we synthesized the simplified derivatives of 1 and 2. Importantly, the selective removal of the aromatic rings from compound 10s skeleton significantly reduced antimicrobial activity. In fact, compared to the parent compound 1, the derivatives 3, 4 and 5, obtained from the removal of the moieties in positions 5, 3 and 2, respectively, had significantly lower activity. In particular, the removal of the ring B (derivative 4), resulted in a completely inactive compound (MIC 256 µg/mL, MBC >512 µg/mL). This finding suggests that the three phenolic portions A, B, and C present in the parent compound 1 are all important and synergic for antimicrobial activity. Conversely, SAR studies on the analogues of compound 2 showed that the removal of the ring B increased the activity of the derivative. Indeed, compound 7 was more potent than 2 (MIC 8 µg/mL, MBC 64 µg/mL vs. MIC 16 µg/mL, MBC >512 µg/mL), whereas compounds 6 and 8, obtained by the selective removal of the moieties C and A, respectively, showed four-fold lower activity, only in terms of MIC, compared to the parent compound 2 (MIC 64 µg/mL vs. 16 µg/mL). It is interesting to notice that the most active compound of this series, compound 7, has three phenolic portions with a spatial orientation similar to that of compound 1. Interestingly, the introduction of a hydroxy group on the benzofuran skeleton of compound 3 (6 vs. 3), caused a four-fold reduction in activity (MIC 64 µg/mL, MBC >512 µg/mL vs. MIC 16 µg/mL, MBC 64 µg/mL). The replacement of the phenolic hydroxyl groups with the methoxy groups was, in any case, deleterious, giving inactive compounds (compounds 9, 10, 11). Notably, not only the spatial orientation but also the distance between the substituents of the rings seemed to play a role. In fact, compound 9 differed from pterostilbene only in a longer spacer connecting the two substituted rings (the benzofuran core). This difference results in a drop in compound activity (MIC and MBC >512 µg/mL vs. MIC 64 µg/mL, MBC 128 µg/mL). Overall, the results confirm that the shape and geometry of the molecules play a key role in the antimicrobial activity. Indeed, it is well documented that the hydroxy groups in polyphenolic compounds can interact with the bacterial cell membrane [21,22], and that the relative position of the -OH groups on the phenolic nucleus strongly influences the antibacterial efficacy [23,24]. To evaluate the potential toxicity of the compounds on healthy human cells, the antiproliferative activity of the synthesized molecules, together with the natural compounds resveratrol and pterostilbene, was evaluated on normal skin fibroblast WS1 cells (Table1). The concentration of compound causing 50% cell growth inhibition (IC50) of resveratrol was > 200 µM, whereas the IC50 of all the other compounds ranged from 33 to >100 µM. A comparison with the MIC of the compounds against L. monocytogenes Scott A (µM concentration, Table1) showed that 1 has a very good profile of selectivity. Indeed, cytotoxic effects were observed at a concentration ~10-fold higher than the lowest concentration of the compound that prevents the visible growth of bacteria. Moreover, the simplified analogues 3, 5, 7 displayed cytotoxic activity in a range of concentration two-fold higher than the MIC values. Overall, the collected data identified the scaffolds of compounds 1 and 7 as the most promising for further developments. Int. J. Mol. Sci. 2020, 21, 2168 8 of 10

4. Materials and Methods

4.1. Chemical Synthesis The procedures for the synthesis and characterization data for the various derivatives and intermediates are detailed in the Supplementary Materials.

4.2. Evaluation of the Minimal Inhibitory Concentration (MIC) and Minimal Bactericidal Concentration (MBC) All synthesized derivatives were stored in dry form at 20 C and dissolved in DMSO at a − ◦ ﬁnal concentration of 4.096 mg/mL prior to their use in the MIC and MBC determination. Brieﬂy, L. monocytogenes was cultivated in Brain Hearth Infusion (BHI) broth (Sigma, Italy) at 37 ◦C for 24 h under a shaking condition (200 rpm). After the growth, the cells were diluted in BHI to 0.2 OD 600 nm and then used for the 96-well plate inoculum. The concentrations tested ranged from 1 up to 512 µg/mL. The assay was carried out in triplicate in a 96-well plate, according to Mattio et al., 2019 [4].

4.3. Cytotoxicity on Human Skin Normal WS1 Fibroblast Cells The normal human skin WS1 fibroblast cells (ATCC CRL-1502) were cultured in Eagle’s Minimum Essential Medium plus 10% fetal bovine serum at 37 ◦C and 5% CO2. Cytotoxic potency was assessed by a growth inhibition assay (CellTiter 96® AQueous One Solution Cell Proliferation Assay MTS, Promega). The cells were seeded in a 96-well plate, and 24 h later were exposed to the compounds (concentration range 1 200 µM). After 48 h of exposure, 20 µL − of 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium salt was added to each well. The absorbance was measured using a FLUOstar OPTIMA plate reader (BMG Labtech GmbH, Offenburg, Germany) at 492 nm after 4 h of incubation at 37 ◦C in 5% CO2. The IC50 was defined as the drug concentration causing 50% cell growth inhibition, determined by the dose response curves. Experiments were performed in triplicate. − 5. Conclusions In the present study, we gained further insight into the properties of the viniferin analogues and reported the structural requirements which are fundamental in the antimicrobial activity of the derivatives 1 and 2. Moreover, we showed that, although less active than the parent compound 1, the simplified derivatives 3 and 5 retained antimicrobial activity. In addition, we were pleased to find that the simplification strategy adopted for compound 2 was successful. Indeed, compound 7, which showed two-fold higher potency compared with 2, represents an intriguing new versatile scaffold for further derivatization. The cytotoxic profile observed on normal human cells was interesting. Indeed, on the WS1 fibroblasts, compounds 1, 3, 5, 7 showed cytotoxic effects at concentrations at least two-times higher than that observed on Listeria monocytogenes. Among these, compound 1 resulted as the most selective of the series. In conclusion, this study identified dehydro-δ-viniferin 1 and compound 7 (the simplified derivative of viniferifuran 2) as promising scaffolds for the development of nature-inspired antimicrobials active against foodborne pathogenic species.

Supplementary Materials: Supplementary Materials can be found at http://www.mdpi.com/1422-0067/21/6/2168/s1. Author Contributions: Conceptualization, A.P., S.D. and D.M.; methodology, G.C., L.M.M., S.A., L.M. and G.L.B.; investigation, G.C., L.M.M., S.A., L.M. and G.L.B.; resources, A.P., S.D., L.M., N.Z. and D.M.; data curation, G.C., L.M.M., S.A. and G.L.B.; writing—original draft preparation, A.P., S.D.; writing—review and editing, all authors. G.C. and L.M.M. contributed equally to this work. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Int. J. Mol. Sci. 2020, 21, 2168 9 of 10

Acknowledgments: This work was financially supported by “Piano di Sostegno alla Ricerca 2015/2017-Linea 2_Azione A” of the University of Milan (DeFENS) and “Transition Grant 2015-2017 - Linea 1A” of the University of Milan. The work of Giorgia Catinella has been partially funded by Fondazione F.lli Confalonieri (PhD Scholarship). Conflicts of Interest: The authors declare no conflict of interest.

References

1. Khanam, H. Bioactive benzofuran derivatives: A review. Eur. J. Med. Chem. 2015, 97, 483–504. [CrossRef] [PubMed] 2. Naik, R.; Harmalkar, D.S.; Xu, X.; Jang, K.; Lee, K. Bioactive benzofuran derivatives: Moracins A-Z in medicinal chemistry. Eur. J. Med. Chem. 2015, 90, 379–393. [CrossRef][PubMed] 3. Hiremathad, A.; Patil, M.R.; Chethana, K.R.; Chand, K.; Amelia Santos, M.; Keri, R.S. Benzofuran: An emerging scaffold for antimicrobial agents. RSC Adv. 2015, 5, 96809–96828. [CrossRef] 4. Mattio, L.M.; Dallavalle, S.; Musso, L.; Filardi, R.; Franzetti, L.; Pellegrino, L.; D’Incecco, P.; Mora, D.; Pinto, A.; Arioli, S. Antimicrobial activity of resveratrol-derived monomers and dimers against foodborne pathogens. Sci. Rep. 2019, 9, 19525. [CrossRef] 5. Markina, N.A.; Chen, Y.; Larock, R.C. Efficient microwave-assisted one-pot three-component synthesis of 2,3-disubstituted benzofurans under Sonogashira conditions. Tetrahedron 2013, 69, 2701–2713. [CrossRef] 6. Jaseer, E.A.; Prasad, D.J.C.; Sekar, G. Domino synthesis of 2-arylbenzo[b]furans by copper (II)-catalyzed coupling of o-iodophenols and aryl acetylenes. Tetrahedron 2010, 66, 2077–2082. [CrossRef] 7. Yao, T.; Yue, D.; Larock, R.C. Synthesis of 2,3-disubstituted benzofurans by the palladium-catalyzed coupling of 2-iodoanisoles and terminal alkynes. Org. Synth. 2014, 91, 283–292. [CrossRef] 8. Kraus, G.A.; Gupta, V. A new synthetic strategy for the synthesis of bioactive stilbene dimers. A direct synthesis of amurensin H. Tetrahedron Lett. 2009, 50, 7180–7183. [CrossRef] 9. Vo, D.D.; Elofsson, M. Total Synthesis of Viniferifuran, Resveratrol-Piceatannol Hybrid, Anigopreissin A and Analogues—Investigation of Demethylation Strategies. Adv. Synth. Catal. 2016, 358, 4085–4092. [CrossRef] 10. Liu, J.; Do, T.J.; Simmons, C.J.; Lynch, J.C.; Gu, W.; Ma, Z.; Xuc, W.; Tang, W. Total synthesis of diptoindonesin G and its analogues as selective modulators of estrogen receptors. Org. Biomol. Chem. 2016, 14, 8927–8930. [CrossRef] 11. Saitoh, M.; Kunitomo, J.; Kimura, E.; Iwashita, H.; Uno, Y.; Onishi, T.; Uchiyama, N.; Kawamoto, T.; Tanaka, T.; Mol, C.D.; et al. (2-{3-[4-(Alkylsulfinyl)phenyl]-1-benzofuran-5-yl}-5-methyl-1,3,4-oxadiazole Derivatives as Novel Inhibitors of Glycogen Synthase Kinase-3ß with Good Brain Permeability. J. Med. Chem. 2009, 52, 6270–6286. [CrossRef] 12. Kim, I.; Choi, J. A versatile approach to oligostilbenoid natural products—Synthesis of permethylated analogues of viniferifuran, malibatol A., and shoreaphenol. Org. Biomol. Chem. 2009, 7, 2788–2795. [CrossRef][PubMed] 13. Lindgren, A.E.G.; Öberg, C.T.; Hillgren, J.M.; Elofsson, M. Total Synthesis of the Resveratrol Oligomers ( )-Ampelopsin B and ( )--Viniferin. Eur. J. Org. Chem. 2016, 3, 426–429. [CrossRef] ± ± 14. Iino, T.; Tsukahara, D.; Kamata, K.; Sasaki, K.; Ohyama, S.; Hosaka, H.; Hasegawa, T.; Chiba, M.; Nagata, Y.; Eiki, J.; et al. Discovery of potent and orally active 3-alkoxy-5-phenoxy-N-thiazolyl benzamides as novel allosteric glucokinase activators. Bioorg. Med. Chem. 2009, 17, 2733–2743. [CrossRef][PubMed] 15. Ferreira, S.; Domingues, F. The antimicrobial action of resveratrol against Listeria monocytogenes in food-based models and its antibiofilm properties. J. Sci. Food Agric. 2016, 96, 4531–4535. [CrossRef][PubMed] 16. Hammerum, A.M.; Heuer, O.E. Human health hazards from antimicrobial-resistant Escherichia coli of animal origin. Clin. Infect. Dis. 2009, 48, 916–921. [CrossRef][PubMed] 17. Allen, H.K.; Donato, J.; Wang, H.H.; Cloud-Hansen, K.A.; Davies, J.; Handelsman, J. Call of wild: Antibiotic resistance genes in natural environments. Nat. Rev. Microbiol. 2010, 8, 251–259. [CrossRef][PubMed] 18. Lee, H.H.; Molla, M.N.; Cantor, C.R.; Collins, J.J. Bacterial charity work leads to population wide-resistance. Nature 2010, 467, 82–85. [CrossRef] 19. Akinwumi, B.C.; Bordun, K.A.M.; Anderson, H.D. Biological activities of stilbenoids. Int. J. Mol. Sci. 2018, 19, 792. [CrossRef] 20. Ma, D.S.L.; Tan, L.T.; Chan, K.; Yap, W.; Pusparajah, P.; Chuah, L.; Ming, L.C.; Khan, T.M.; Lee, L.; Goh, B. Resveratrol-Potential antibacterial agent against foodborne pathogens. Front. Pharmacol. 2018, 9, 102. [CrossRef] Int. J. Mol. Sci. 2020, 21, 2168 10 of 10

21. Alvarez-Martinez, F.J.; Barrajon-Catalan, E.; Encinar, J.A.; Rodriguez-Diaz, J.C.; Micol, V. Antimicrobial Capacity of Plant Polyphenols against Gram-positive Bacteria: A Comprehensive Review. Curr. Med. Chem. 2019, 26, 1–29. [CrossRef][PubMed] 22. Chibane, L.B.; Degraeve, P.; Ferhout, H.; Bouajila, J.; Oulahal, N. Plant antimicrobial polyphenols as potential natural food preservatives. J. Sci. Food Agric. 2019, 99, 1457–1474. [CrossRef][PubMed] 23. Wu, Y.; Bai, J.; Zhong, K.; Huang, Y.; Qi, H.; Jiang, Y.; Gao, H. Antibacterial Activity and Membrane-Disruptive Mechanism of 3-p-trans-Coumaroyl-2-hydroxyquinic Acid, a Novel Phenolic Compound from Pine Needles of Cedrus deodara, against Staphylococcus aureus. Molecules 2016, 21, 1084. [CrossRef][PubMed] 24. Gyawali, R.; Ibrahim, S.A. Natural products as antimicrobial agents. Food Control 2014, 46, 412–429. [CrossRef]

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).