Antibacterial Evaluation and Virtual Screening of New Thiazolyl-Triazole Schiff Bases as Potential

18 Pages·2017·10.2 MB·English

International Journal o f
Molecular Sciences
Article
Antibacterial Evaluation and Virtual Screening of
New Thiazolyl-Triazole Schiff Bases as Potential
DNA-Gyrase Inhibitors
CristinaNastasă1,* ID,DanC.Vodnar2,*,IoanaIonu¸t1,AncaStana1 ID,DanielaBenedec3,
RaduTamaian4,5,* ID,OvidiuOniga1andBrîndus¸aTiperciuc1 ID
1 DepartmentofPharmaceuticalChemistry,“IuliuHa¸tieganu”UniversityofMedicineandPharmacy,
41VictorBabes¸Street,RO-400012Cluj-Napoca,Romania;[email protected](I.I.);
[email protected](A.S.);[email protected](O.O.);[email protected](B.T.)
2 DepartmentofFoodScienceandTechnology,UniversityofAgriculturalSciencesandVeterinaryMedicine,
3-5Mănăs¸turStreet,RO-400372Cluj-Napoca,Romania
3 DepartmentofPharmacognosy,“IuliuHa¸tieganu”UniversityofMedicineandPharmacy,
12IonCreangăStreet,RO-400010Cluj-Napoca,Romania;[email protected]
4 NationalInstituteforResearchandDevelopmentforCryogenicandIsotopicTechnologies,
4thUzineiStreet,RO-240050RâmnicuVâlcea,Romania
5 SCBiotechCorpSRL,4thUzineiStreet,RO-240050RâmnicuVâlcea,Romania
* Correspondence:[email protected](C.N.);[email protected](D.C.V.);
[email protected](R.T.);Tel.:[phone](C.N.);[phone](D.C.V.);[phone](R.T.)
Received:23November2017;Accepted:9January2018;Published:11January2018
Abstract: The global spread of bacterial resistance to drugs used in therapy requires new potent
and safe antimicrobial agents. DNA gyrases represent important targets in drug discovery.
Schiffbases,thiazole,andtriazolederivativesareconsideredkeyscaffoldsinmedicinalchemistry.
Fifteenthiazolyl-triazoleSchiffbaseswereevaluatedfortheirantibacterialactivity,measuringthe
growthinhibitionzonediameter,theminimuminhibitoryconcentration(MIC),andtheminimum
bactericidalconcentration(MBC),againstGram-positive(Staphylococcusaureus,Listeriamonocytogenes)
and Gram-negative (Escherichia coli, Salmonellatyphimurium, Pseudomonas aeruginosa) bacteria.
The inhibition of S. aureus and S. typhimurium was modest. Compounds B1, B2, and B9 showed
asimilareffectasciprofloxacin,theantimicrobialreference,againstL.monocytogenes. B10displayed
a better effect. Derivatives B1, B5–7, B9, and B11–15 expressed MIC values lower than the
reference,againstL.monocytogenes. B5,B6,andB11–15stronglyinhibitedthegrowthofP.aeruginosa.
All compounds were subjected to an in silico screening of the ADMET (absorption, distribution,
metabolism,elimination,toxicity)properties. MoleculardockingwasperformedonthegyrAand
gyrBfromL.monocytogenes. Thevirtualscreeningconcludedthatthiazolyl-triazoleSchiffbaseB8is
thebestdrug-likecandidate,satisfyingrequirementsforbothsafetyandefficacy,beingmorepotent
againstthebacterialgyrAthanciprofloxacin.
Keywords:Schiffbase;thiazole;triazole;antibacterialactivity;ADMET;moleculardocking;DNA-gyrase
1. Introduction
The alarming worldwide spread of the bacterial resistance, to most of the drugs available
nowadaysintherapy[1–3],urgentlyrequiresthedevelopmentofneweffectiveantibacterialagents.
TheDNAtopoisomerasesmanagethetopologicalstateoftheDNAinthecell,beinginvolved
inreplication, transcription, recombination, andchromatinremodeling. Inthebacterialproteome,
there are various types of topoisomerases, the most common being type I topoisomerases, type II
topoisomerases(DNAgyrases),andtypeIVtopoisomerases[4].
Int.J.Mol.Sci. 2018,19,222;doi:10.3390/ijms19010222 www.mdpi.com/journal/ijms
Int.J.Mol.Sci. 2018,19,222 2of18
One of the major targets of antibacterial compounds is represented by DNA gyrase (Type II
topoisomerase),anenzymeplayinganessentialroleinbacterialreplication[5]. Structurally,theDNA
gyraseisbuiltoftwoAsubunits(gyrA)andtwoBsubunits(gyrB)thatformanA2B2heterotetramer.
TopoisomeraseIVconsistsoftwoconstitutivesubunits: parE(homologoustoDNAgyrasesubunit
B–gyrB)andparC(homologoustoDNAgyrasesubunitA–gyrA)[6]. DNAgyrasehasfourfunctional
domains(GO(GeneOntology)terms)[7,8]. Domain1,theN-terminalofdomaingyrB,harborsthe
ATPase activity. The type II topoisomerases use the hydrolysis of ATP, in the presence of Mg2+,
tosimultaneouslycutbothstrandsofthedouble-helix,inordertomanageDNAtanglesandsupercoils.
Domain 2, the C-terminal domain of gyrB, which consists of a Toprim structural motif and a tail
region,contributestothebindingofDNA,viatheinteractionwiththegyrAsubunit[9,10]. Domain3,
theN-terminaldomainofgyrA,isresponsibleforthebreaking-rejoiningfunction,throughitscapacity
to form protein-DNA bridges; meanwhile, Domain 4, the C-terminal domain of gyrA (TOP4c),
is able to non-specifically bind DNA [11]. The Toprim domain (also known as the Rao-Rossmann
fold)isastructuralmotiffoundinDNAprimases,topoisomerases,andsomeenzymesinvolvedin
phosphotransfersorabletohydrolyzephosphodiesterbonds[10]. ThecentralDNA-bindingcoreof
gyrAcontainstheactivesitetyrosineresidueslocatedinthecatabolite-activator-protein-like(CAP-like)
domainwhichincludestheDNAbindinghelix-turn-helix(HTH)motif.TheCAP-liketyrosineresidues
arecrucialforthebreakageandreligationoftheDNA,byforminganesterwiththe5(cid:48)phosphateofthe
DNA[12]. TheToprimdomainofsubunitBisadjacenttothecatalytictyrosineintheCAPdomainof
subunitAandbothformanactivesite,essentialfortheDNA-cleavage[10].
QuinolonesaretheonlyclassofDNAgyraseinhibitorsthatareclinicallyused.Theireffectisbased
ontheinhibitionofthegyrAsubunit,thereforeperturbingtheDNAcleavageandtheintroductionof
negative supercoils into the bacterial DNA [13]. Cyclothialidines [14] and aminocoumarins [15]
are studied for their inhibition of the ATP-binding site of the gyrB subunit. Because of the
bacterialresistancetofluoroquinolonesusedintherapyandalsooftheirsideeffectsandlimitations,
therearegyraseinhibitorssearched,fromdifferentchemicalclasses: benzimidazoles,benzoxazole,
benzothiazole, oxazolopyridines [16], aminopyrazinamides [17], thiazole derivatives [18–20],
andtriazolederivatives[21,22],whichdifferentlybindthebiologicaltarget.
Schiff bases are a group of compounds which have gained popularity as biologically active
scaffoldsduetotheireaseofsynthesis,theirversatility,andtheirlargespectraofactivities,suchastheir
antimicrobial[23,24],anti-inflammatory,anticonvulsant,andantioxidant[25]properties. Schiffbases
havesufficientwatersolubilityandarestableinvitroandinvivo[26]. TheiminebondinSchiffbases
providesbindingopportunitieswithdifferentnucleophilesandelectrophiles,inhibitingenzymesor
DNAreplication. Theisostericreplacementofthequinolones’3-carboxylgroup,essentialforgyrase
binding,withanamino-thiazolicfragment[27]orotherazoles(thiazoles,triazoles),ledtomolecules
withimprovedantimicrobialeffectsandawiderspectrumofactivity. Thesecompoundsexpressfewer
sideeffectsandhaveabettercapacityinovercomingbacterialresistance[28,29].
Evenifresearcheffortsarecontinuouslymade,therestillremainsthechallengetodiscovernew
drugsofahighpotential,largespectrumofactivity,andwithagoodsafetyprofile. Asacontinuation
ofoureffortsindiscoveringnewheterocyclicSchiffbaseswithpotentantimicrobialproperties[30],
wesynthesizedaseriesofnewSchiffbasesofthiazolyl-triazole[31].Promptedbytheaspectsdescribed
above,theworkpresentedherefocusesontheinvestigationoftheantibacterialpotentialofthenew
azolyl-SchiffbasesagainstGram-positiveandGram-negativebacteria. Amoleculardockingstudy
wasrealizedonDNAgyrasesofListeriamonocytogenes. ADMETprofilingforrisksandsafetyriskswas
alsoconductedforthenewseriesofcompounds.
Int. J. Mol. Sci. 2018, 19, 222 3 of 19
Int.J.Mol.Sci. 2018,19,222 3of18
2. Result and Discussion
2. ResultandDiscussion
2.1. Antibacterial Activity
2.1. AntibacterialActivity
22..11..11.. DDeetteerrmmiinnaattiioonn ooff tthhee IInnhhiibbiittiioonn ZZoonnee DDiiaammeetteerrss
TThhee aannttiimmiiccrroobbiiaall aaccttiivviittyy wwaass tteesstteedd iinn vviittrroo uussiinngg tthhee ddiisskk ddiiffffuussiioonn mmeetthhoodd,, bbyy mmeeaassuurriinngg tthhee
ddiiaammeetteerrss ooff tthhee iinnhhiibbiittiioonn zzoonneess.. TThhee ssyynntthheessiizzeedd ccoommppoouunnddss BB11––1155 ((FFiigguurree 11)) [[3311]] wweerree ssccrreeeenneedd
aaggaaiinnsstt ttwwoo GGrraamm-p-poossitiitvivee( L(Lisitsetreiraiam monooncoyctyotgoegneenseAs TACTCC3C5 135521,5S2t,a Sphtayplohcyolcoccuosccauusr eauusreAuTs CACT2C5C92 235)9a2n3d)
tahnrde etGhrreaem G-nreagma-tniveeg(aEtisvceh er(EicshcihaecroiclihAiaT cColCi 2A5T9C22C, S2a5lm92o2n,e lSlaaltmypohniemllau rtiyupmhiAmTuCriCum1 3A31T1C,PC. a1e3ru3g1i1n, oPsa.
AaeTruCgCin2o7s8a 5A3)TbCaCc t2e7ri8a5l3s)t rbaaicntser(iTaalb sltera1i)n.s (Table 1).

FFiigguurree 11.. SSttrruuccttuurreess ooff tthhee SScchhiiffff bbaasseess BB11––1155..
TTaabbllee 11.. TThhee aannttiibbaacctteerriiaall aaccttiivviittyy ooff ccoommppoouunnddss BB11––1155..
Gram-Positive Bacteria Gram-Negative Bacteria
Gram-PositiveBacteria Gram-NegativeBacteria
Staphylococcus aureus Listeria monocytogenes Escherichia coli Salmonella typhimurium P. aeruginosa
Cp. SAtaTpChyClo 2c5o9c2c3u saureus LisAteTriCaCm 3o5n1o5c2y togenes AETscChCer i2c5h9i2a2c oli SalmAonTeCllCa t1y3p3h1i1m urium PA.aTeCruCg i2n7o8s5a3
Cp. DiameAteTrC C25923 DiamAetTeCr C35152 DiamAeTteCrC 25922 DiamAeTteCrC 13311 DiaAmTCetCer2 7853
%AI %AI %AI %AI %AI
D(miamm)e ter D(miamm)e ter D(imamme)t er D(imamme)t er Di(ammmet)e r
%AI %AI %AI %AI %AI
B1 1(m4 m) 50 (1m8 m) 100 (m14m ) 51.8 (m16m ) 72.7 (m1m9) 73
B2 B1 141 4 505 0 181 8 110000 1144 5511..88 1186 7821..78 1199 7373
B3 B2 141 4 505 0 161 8 8180.80 1144 5511..88 1188 8811..88 1196 7631.5
B4 B3 141 4 505 0 141 6 7878.7.8 1144 5511..88 1188 8811..88 1168 616.59.2
B5 B4 141 4 505 0 141 4 7777.7.7 1144 5511..88 1188 8811..88 1281 698.20.7
B5 14 50 14 77.7 14 51.8 18 81.8 21 80.7
B6 14 50 14 77.7 14 51.8 16 72.7 21 80.7
B6 14 50 14 77.7 14 51.8 16 72.7 21 80.7
B7 B7 161 6 575.71. 1 121 2 6666.6.6 1144 5511..88 1166 7722..77 1188 696.29.2
B8 B8 121 2 424.28. 8 121 2 6666.6.6 1144 5511..88 1166 7722..77 1188 696.29.2
B9 B9 141 4 505 0 181 8 110000 1166 5599..22 1166 7722..77 2200 767.96.9
B10B 10 181 8 646.42.2 202 0 111111.1.1 1166 5599..22 1188 8811..88 1188 696.29.2
B11B 11 121 2 424.28. 8 8 8 4444.4.4 1166 5599..22 1188 8811..88 2211 808.70.7
B12B 12 121 2 424.28. 8 141 4 7777.7.7 1144 5511..88 1188 8811..88 2211 808.70.7
B13 12 42.8 12 66.6 14 51.8 18 81.8 21 80.7
B13 12 42.8 12 66.6 14 51.8 18 81.8 21 80.7
B14 12 42.8 16 88.8 16 59.2 18 81.8 21 80.7
B14B 15 121 6 425.78. 1 161 0 8585.8.5 1164 5519..82 1188 8811..88 2211 808.70.7
B1C5 IP 162 8 571.10 0 101 8 5150.50 1247 5110.08 1282 81100.8 2261 10800.7
CIP 28 100 18 100 27 100 22 100 26 100
Thevaluesobtainedforthemostactivecompoundsaremarkedinbold. Cp.: Compounds;CIP:ciprofloxacin;
%ThAeI =vpaleurceesn toabgteaaicnteivdi tyfoinr dtehxe (m(Zoosnte aocftivthee cionmhibpiotiuonndosf asryen tmheatrickecdo minp obuonldd/. ZCopne.: oCfotmhepoinuhnidbist;i oCnIPof:
rceipferroenflcoexdarcuign); ×%A10I0 )=. percentage activity index ((Zone of the inhibition of synthetic compound/Zone
of the inhibition of reference drug) × 100).
Forevaluatingtheantimicrobialactivity,100µg/diskofthesynthesizedcompoundsandalso
oftheForer feevreanlucaetsinugb stthaen acne,ticmipircoroflboixaal caicnt,ivwiteyr,e 1u0s0e µd.gT/dhiesks oolfv tehnet sfyonrtthheesipzreedp acoramtipoonuonfdths eansodl ualtsioon osf,
dthiem reetfheyrlesnuclefo sxuidbseta(DncMe,S cOip),roefxlhoixbaicteind, nwoerine huisbeitdo.r Tyhaec tsiovlivtyenotn fothr ethbea cptreerpiaalrasttrioanin sofc othnes isdoelruetdiofnosr,
tdhiimsestthuydlys.ulfoxide (DMSO), exhibited no inhibitory activity on the bacterial strains considered for
this sRtuedgayr. dingtheactivityagainsttheGram-positivebacteria,thestrainofListeriamonocytogeneswas
moresensibletothetestedcompounds, threeofthem(B1, B2, andB9)showingasimilareffectto
Int.J.Mol.Sci. 2018,19,222 4of18
ciprofloxacin,withan18mminhibitionzoneandanAIvalueof100%. B10(3-nitro-phenyl)proved
tobemoreactivethantheantibacterialreference,displayinga20mmdiameterandanAIof111.1%.
The second Gram-positive bacterium, Staphylococcus aureus, was moderately inhibited by the new
molecules,withinhibitionzonesrangingfrom12to18mm,respectively,andAIbetween42.8and
64.2%. Inthecaseofbothstrains,compoundB10exhibitedthemostpronouncedeffect.
TheinhibitoryactivityagainstEscherichiacoli,Salmonellatyphimurium,andPseudomonasaeruginosa
wasmodestlyrelatedtociprofloxacin. Thecompoundsdeterminedzonesof14–16mm(AI51.8–59.2%)
againstEscherichiacoli. ThevaluesregisteredonSalmonellatyphimuriumrangedbetween16–18mm
(AI72.7–81.8%). ThepercentageactivityindexagainstPseudomonasaeruginosawasbetween61.5%,
corresponding to a 16 mm inhibitory zone diameter, and 80.7%, corresponding to a 21 mm zone
diameter. ThemostpotentderivativeswereB5,B6,andB11–15.
Fromtheresultsobtained,itcanbeobservedthatSchiffbaseB10expressedthemostpronounced
antibacterialeffect.
2.1.2. DeterminationofMICandMBCValues
The broth microdilution method was employed for the minimum inhibitory concentration
test. All synthesized compounds were tested against two Gram-positive bacterial strains
(Staphylococcus aureus ATCC 49444, Listeria monocytogenes ATCC 19115) and two Gram-negative
bacterial strains (Pseudomonas aeruginosa ATCC 27853, Salmonella typhimurium ATCC 14028).
Stock solutions (1 mg/mL) were prepared by dissolving the test compounds and the reference
antimicrobial,ciprofloxacin,insterileDMSO.TheresultsarepresentedinTable2(MICandMBC).
Table2.MinimumInhibitoryConcentration(MIC)andMinimumBactericidalConcentration(MBC)
(inµg/mL)ofcompoundsB1–15.
S.aureus L.monocytogenes P.aeruginosa S.typhimurium
Cp.
ATCC49444 ATCC19115 ATCC27853 ATCC14028
MIC MBC MIC MBC MIC MBC MIC MBC
B1 31.25 31.25 1.95 3.9 7.81 15.62 62.5 125
B2 31.25 31.25 3.9 7.8 7.81 15.62 62.5 62.5
B3 62.5 62.5 3.9 7.8 15.62 31.25 62.5 62.5
B4 31.25 62.5 3.9 7.8 7.81 15.62 62.5 62.5
B5 31.25 31.25 1.95 3.9 1.95 3.9 62.5 62.5
B6 31.25 62.5 1.95 3.9 1.95 3.9 62.5 62.5
B7 31.25 31.25 1.95 3.9 7.81 15.62 62.5 125
B8 62.5 62.5 3.9 3.9 7.81 15.62 62.5 125
B9 31.25 31.25 1.95 3.9 3.9 7.8 62.5 125
B10 31.25 31.25 3.9 7.8 7.81 15.62 62.5 62.5
B11 62.5 62.5 1.95 3.9 1.95 3.9 62.5 62.5
B12 31.25 62.5 1.95 3.9 1.95 1.95 62.5 125
B13 31.25 62.5 1.95 3.9 1.95 3.9 31.25 62.5
B14 15.62 31.25 1.95 3.9 1.95 3.9 62.5 125
B15 31.25 62.5 1.95 3.9 1.95 3.9 62.5 62.5
Ciprofloxacin 1.95 3.9 3.9 7.8 3.9 7.8 0.97 1.95
InoculumControl +++ +++ +++ +++
No No No No
Brothcontrol
growth growth growth growth
Cp.:Compounds;+++Indicatesgrowthinallconcentrations.Thevaluesobtainedforthemostactivecompounds
aremarkedinbold.
Analyzing the results obtained, it can be seen that the growth inhibitory activity against the
Gram-positivebacteriawasmorepronouncedagainstthestrainofListeriamonocytogenes,where10
of the compounds (B1, B5–7, B9, B11–15) expressed MIC values lower than that of ciprofloxacin.
The others showed the same effect as the reference. The strain of Staphylococcus aureus was less
sensitivetotheactivityofthenewmolecules.
Int.J.Mol.Sci. 2018,19,222 5of18
GrowthofPseudomonasaeruginosawasstronglyinhibitedbymostofthecompounds. Itcanbe
observed that B5, B6, and B11–15 had MICs lower than the antibacterial used as the reference,
whileB9hadthe same active concentration, in agreement with the inhibitory zone diameters.
TheinhibitionofSalmonellatyphimuriumwasmodestforallthetestedderivatives.
DeterminationofMBCconfirmedtheresultspreviouslyobtained,whenMICwasinvestigated.
TheMBCvalueofSchiffbaseB8(meta-hydroxy)againstL.monocytogenesATCC19115wasinferior
to that of ciprofloxacin. For this compound, MIC was equal to the antibacterial reference. Also,
theMBCof1.95µg/mLshouldbenoted,registeredforB12(ortho-methoxy-phenyl),againstthestrain
ofP.aeruginosa,whichismuchsmallerthanthatforciprofloxacin. TheMBC/MICratio(TableS1)is
oneortwoforallthetestedcompounds,suggestingthattheymayexertbactericidalactivity[32,33].
2.2. VirtualScreening
Virtualscreening(VS)emergedasanadaptiveresponseofcheminformaticstoorganicchemistry
requirements, in order to prioritize the synthesis of the most promising drug candidates [34–37].
Inthisrespect,variouscheminformaticstoolscanbeusedtofilterthecandidatecompounds,basedon
their absorption, distribution, metabolism, excretion, toxicity (ADMET) [38–40], and their spatial
interactionwiththetargetsandtheirbindingaffinity(BA)viamoleculardocking[40,41]. Development
ofnovelantimicrobialsasDNAgyraseinhibitors(validateddrugstargets)hasbeenexploitedinboth
computationalandwet-labstrategies[5,20,42].
In our VS setup, we assessed the activity and potency of the newly synthesized Schiff
bases, via molecular docking, on the two subunits of topoisomerases II (gyrA and gyrB) from
Listeriamonocytogenes,comparingtheresultswithciprofloxacin,chosenasthereferencedrug. Wealso
evaluated the safety-related concerns by the means of ADMET profiling. An academic license of
MarvinSketch was used for the drawing and displaying of 2D structures, 3D optimization of all
ligands,andalsoforgeneratingtheinputSDFfilesforADMETprofilingandTriposMOL2filesfor
docking,MarvinSketch17.6.0,2017,ChemAxon,Budapest,Hungary[43].
2.2.1. ADMETProfiling
ADMET profiling provides helpful guidance on the absorption, plasma clearance,
tissue distribution, metabolic effects, and both acute and later toxicity. The results of the
ADME study previously conducted and presented [31] showed that the thiazolyl-triazole Schiff
bases B1–15 displayed good pharmakocinetic properties and that all new molecules passed the
drug-likenesscriteria.
Toxicitystudiesaremandatoryforanewproduct,duetothefactthatadrughastonotonlymanifest
itsefficacy, butalsohavegoodtolerabilityandalowtoxicityrate. Consideringtheaboveaspectsas
agoodstartingpoint,wecontinuedtheanalysisofthenewlysynthesizedcompounds(Table3).
Table3.ADMETprofiling–risksandsafetyconcerns.
PAINSFilters
Cp. MW(Da) logP tPSA(Å2) PPIs UMSs CIs PPDI MedChem GSK4/400 Pfizer3/75
A B C
thiol
B1 446.38 5.69 104.1 Yes thiol ND ND ND NI hydr. bad warn.
hal.
thiol
B2 446.38 5.69 104.1 Yes thiol ND ND ND NI hydr. bad warn.
hal.
thiol
B3 446.38 5.69 104.10 Yes thiol ND ND ND NI hydr. bad warn.
hal.
thiol
B4 411.93 5.06 123.00 Yes thiol ND ND ND NI hydr. bad warn.
hal.
B5 456.38 5.13 123.00 Yes hal. thiol ND ND ND NI hydr. bad warn.
thiol
B6 395.48 4.53 123.00 Yes thiol ND ND ND NI hydr. good warn.
hal.F
thiol I479
B7 393.49 4.08 143.23 Yes phenol thiol I479b ND ND NI hydr. good warn.
Int.J.Mol.Sci. 2018,19,222 6of18
Table3.Cont.
PAINSFilters
Cp. MW(Da) logP tPSA(Å2) PPIs UMSs CIs PPDI MedChem GSK4/400 Pfizer3/75
A B C
thiol
B8 393.49 4.08 143.23 Yes thiol ND ND ND NI hydr. good warn.
phenol
B9 393.49 4.08 143.23 Yes thiol thiol I215 ND ND NI hydr. good warn.
phenol
thiol
B10 422.48 4.26 149.92 Yes thiol ND ND ND NI hydr. bad warn.
nitro
thiol
B11 422.48 4.26 149.92 Yes thiol ND ND ND NI hydr. bad warn.
nitro
B12 407.51 4.41 132.23 Yes thiol thiol ND ND ND NI hydr. bad warn.
B13 407.51 4.41 132.23 Yes thiol thiol ND ND ND NI hydr. bad warn.
thiol
B14 383.51 4.45 151.24 Yes thiol ND ND ND NI hydr. good warn.
thp.
B15 420.55 4.56 126.24 Yes thiol thiol ND ND ND NI hydr. bad warn.
CIP 331.34 0.28 81.98 Not hal.F ND I215 ND ND NI ND good bad
Cp.:Compounds;MW:molecularweight;logP:logarithmofcompoundpartitioncoefficientbetweenn-octanol
andwater;tPSA:topologicalpolarsurfacearea;PPIs:protein-proteininteractions;UMSs:undesirablemoieties
andsubstructures;hal.: halogenure;hal. F:halogenurewithFluorine;thp.: thiophene;CIs: covalentinhibitors;
PAINS:Pan-AssayInterferenceCompounds;ND:nonedetected(compoundisfreeofproblematicsub-structures
forthecorrespondingriskcriteria). I479: intermediatecompoundwhichembedsalow-riskstructuralPAINS
alertwithanumberofoccurrencesbelowthethreshold,accordingtothePAINSfiltermore150_hzone_phenol_A.
I479b:intermediatecompoundwhichembedsalow-riskstructuralPAINSalertwithanumberofoccurrencesbelow
thethreshold,accordingtothePAINSfiltermore150_hzone_phenol_A_bis.I215:intermediatecompoundwhich
embedsalow-riskstructuralPAINSalertwithanumberofoccurrencesbelowthethreshold,accordingtothe
PAINSfiltermore150_hzone_phenol_B.PPDI:phospholipidosisinduction;NI:non-inducerofphospholipidosis;
hydr.:hydrazine;warn.:warning(havetobeusedwithcautionasblindlyapplyingsuchrecipescandiscardfrom
developmentmanyinterestingmolecules)
Therisksandsafetyprofilingoftheinvestigatedcompounds(Table3)indicatesthatallcompounds
arenon-inducersofphospholipidosis. Ontheotherhand,ciprofloxacin(CIP)wasclassifiedasnotPPI
friendly,despiteitsapproveddrugstatus;meanwhile,alltheSchiffbasessuccessfullycompliedwith
thissafetycriterion.Referringtotheproblematicmoieties,CIPisfreeofcovalentinhibitors;meanwhile,
allSchiffbaseshaveintheirframeworkstructurethethiol(–SH)group,whichisconsideredresponsible
forcovalentbinding[44,45]. Moreover,allcompoundscontainatleastonelowriskUMSssubstructure:
ahalogenureinthecaseofCIP,respectivelytheaforementioned–SHgroupinthecaseofSchiffbases.
CompoundsB1–6alsohaveasecondarylowriskhalogenure. Meanwhile,otherlowriskgroupsare
presentasfollows: phenol(B7–9),nitro(B10–11),andthiophene(B14). PAINSgroupsweredetected
onlyinthestructureofB7,B9,andCIP(Table3).
ItcanbeobservedthatonlyfiveSchiffbases(B6–9andB14)andCIPhavegoodpredictionsfrom
GSK4/400rules, whichsaythatsubstanceswithclogP<4andamolecularweight<400Dahave
betterdrug-likeproperties[46]. Schiffbases,duetotheirhydrazonemoiety,mayhave,accordingto
theMedChemrules[47],potentiallyreactiveorpromiscuousbehaviorandalsoreceivedawarning
fromthePfizer3/75rule(substanceswithclogP>3andtPSA<75Å2aremorelikelytohaveinvivo
toxicity),duetothelikelihoodofpromiscuousbinding[48].
2.2.2. MolecularDocking
TheDNAgyraseisbuiltoftwoAsubunits(gyrA)andtwoBsubunits(gyrB)thatformanA2B2
heterotetramer. TopoisomeraseIVconsistsoftwoconstitutivesubunits: parE(homologoustoDNA
gyrasesubunitB–gyrB)andparC(homologoustoDNAgyrasesubunitA–gyrA)[6].
FluoroquinolonesactbyinhibitingthegyrAsubunitandalsoascompetitiveinhibitorsofthe
ATP-bindingsiteonthegyrBsubunit[5].
The best antibacterial activity for our Schiff bases was displayed against the Gram-positive
Listeriamonocytogenes. Inthisview,weselectedasdockingtargetsthetwoDNAgyrases(DNAgyrase
Int. J. Mol. Sci. 2018, 19, 222 7 of 19
from the Pfizer 3/75 rule (substances with clogP > 3 and tPSA < 75 Å2 are more likely to have in vivo
Int.J.Mol.Sci. 2018,19,222 toxicity), due to the likelihood of promiscuous binding [48]. 7of18
2.2.2. Molecular Docking
The DNA gyrase is built of two A subunits (gyrA) and two B subunits (gyrB) that form an A2B2
subunitA–gyrAandDNhetAerotgetyrarmaesr.e TospuoibsoumneriatseB IV– cgonysrisBts )off trwoom conLsti.tumtivoen suobcuyntitos:g peanrEe (sh,omsoinlogcoeust tho eDsNeA arevalidated
gyrase subunit B–gyrB) and parC (homologous to DNA gyrase subunit A–gyrA) [6].
drugtargets,currentlyusedindrugdesign[5,21,42].
Fluoroquinolones act by inhibiting the gyrA subunit and also as competitive inhibitors of the
TheresultsofthetAwTPo-bmindoinlge scitue olna rthed goyrcBk suinbugnitr [u5].n sarepresentedbrieflyinTables4and5,interms
The best antibacterial activity for our Schiff bases was displayed against the Gram-positive
of binding affinity (BAL)istfeoriar mtohnoecytbogeensets. Idn othcisk viinewg, wpeo sseleecst–edu ass idnogckinags tasrgceotsr tihne gtwco rDiNteAr igayraasesr (oDoNtA- mean-square
deviationequaltozero.gAyrasge rsaubpuhniit cAa–lgydrAe panidc tDiNoAn goyrfasteh seubdunoitc Bk–ignyrgB) rfreosmu Ll.t msoinsociyltolguensets,r saintceed thiense Farieg ures2and3.
validated drug targets, currently used in drug design [5,21,42].
DetailedbindingpatternsaTnhed retshultes otfo thtae tlweon meorlegcuelatri cdoicnkitnegr rauncst aioren prsesaenrteeds bhrioefwly inn TianbleTs a4 banlde 5S, i2n .terms
of binding affinity (BA) for the best docking poses–using as scoring criteria a root-mean-square
deviation equal to zero. A graphical depiction of the docking results is illustrated in Figures 2 and 3.
Table4. PredictedbiDnedtaiinlegd bainffidinngi tpya,ttienrntse arnadc tthieo tnotadl eonmergaeitinc ,inatenradctiponosl aarre sihnotwenr ianc Ttaibolne Ss2.b etweencompounds
B1–15andtheDNAgyraseAfromListeriamonocytogenes.
Table 4. Predicted binding affinity, interaction domain, and polar interactions between compounds
B1–15 and the DNA gyrase A from Listeria monocytogenes.
Backboneofthe Backbone of the
Compounds
CompoundsB1–15
B1–15

Compound gyrA BA (kcal/mol) Atom ID of Ligand Interacting AA Residue
Compound gyrABA(kcal/mol) AtomNI D(2) ofLigand ISnert1e12r actingAAResidue
B1 −8.9 N (4) Val113
N(2) Ser112
N (21) Ser98
B1 −8.9 N N(2) (4) Ser112, Val113 Val113
NN (4)( 21) Val113 Ser98
B2 −7.6
N (21) Ser98
S (N32) (2) Val268 Ser112,Val113
N N(2) (4) Val113 Val113
B2 −7.6 N (4) Val113, Val268
B3 −8.1 N(21) Ser98
N (21) Ser98
S (S32() 32) Val268 Val268
N (2) Val113
N(2) Val113
B4 −8.5 N (4) Val113, Val268
N (N21)( 4) Ser98 Val113,Val268
B3 −8.1
NN (2)( 21) Val113 Ser98
N (4) Val113, Val268
B5 −8.8 S(32) Val268
N (21) Ser98
S (N32) (2) Val268 Val113
N (2) Val113
B4 B6 −8.5 −8.8 N N(4) (4) Val113, ValV26a8l 113,Val268
N N(21() 21) Ser98 Ser98
N (2) Val113
B7 −8.1 N N(4) (2) Val113, Val268 Val113
N(4) Val113,Val268
B5 −8.8
N(21) Ser98
S(32) Val268
N(2) Val113
B6 −8.8 N(4) Val113,Val268
N(21) Ser98
N(2) Val113
N(4) Val113,Val268
B7 −8.1
N(21) Ser98
PhenolicO Gln95,Ser98
N(2) Val113
N(4) Val113,Val268
B8 −8.7 N(21) Ser98
PhenolicO Tyr266
S(32) Val268
N(2) Val113
N(4) Val113,Val268
B9 −8.5
N(21) Ser98
S(32) Val268
N(2) Val113
N(4) Val113,Val268
B10 −9.1 N(21) Ser98
NitroN Tyr266
NitroO Gln267
Int. J. Mol. Sci. 2018, 19, 222 7 of 19
from the Pfizer 3/75 rule (substances with clogP > 3 and tPSA < 75 Å2 are more likely to have in vivo
toxicity), due to the likelihood of promiscuous binding [48].
2.2.2. Molecular Docking
The DNA gyrase is built of two A subunits (gyrA) and two B subunits (gyrB) that form an A2B2
heterotetramer. Topoisomerase IV consists of two constitutive subunits: parE (homologous to DNA
gyrase subunit B–gyrB) and parC (homologous to DNA gyrase subunit A–gyrA) [6].
Fluoroquinolones act by inhibiting the gyrA subunit and also as competitive inhibitors of the
ATP-binding site on the gyrB subunit [5].
The best antibacterial activity for our Schiff bases was displayed against the Gram-positive
Listeria monocytogenes. In this view, we selected as docking targets the two DNA gyrases (DNA
gyrase subunit A–gyrA and DNA gyrase subunit B–gyrB) from L. monocytogenes, since these are
validated drug targets, currently used in drug design [5,21,42].
The results of the two molecular docking runs are presented briefly in Tables 4 and 5, in terms
Int.J.Mol.Sci. 2018,19,222 8of18
of binding affinity (BA) for the best docking poses–using as scoring criteria a root-mean-square
deviation equal to zero. A graphical depiction of the docking results is illustrated in Figures 2 and 3.
Detailed binding patterns and the total energetic interactions are shown in Table S2.
Table4.Cont.
Table 4. Predicted binding affinity, interaction domain, and polar interactions between compounds
B1–15 and the DNA gyrase A from Listeria monocytogenes.
Backboneofthe Backbone of the
Compounds
CompoundsB1–15
B1–15

Compound gyrA BA (kcal/mol) Atom ID of Ligand Interacting AA Residue
Compound gyrABA(kcal/mol) AtomNI D(2) ofLigand ISnert1e12r actingAAResidue
B1 −8.9 N (4) Val113
N(2) Val113
N (21) Ser98
B11 −8.8 N N(2) (4) Ser112, ValV11a3l 113,Val268
NN (4)( 21) Val113 Ser98
B2 −7.6
N S(21()3 2) Ser98 Val268
S (32) Val268
N N(2) (4) Val113 Val113,Val268
N (4) Val113, Val268
B3 −8.1 N(21) Ser98
B12 −8 N (21) Ser98
MSe (3th2)o xyO Val268 Ser98
NS (2() 32) Val113 Val268
B4 −8.5 N (4) Val113, Val268
N (N21)( 2) Ser98 Val113
N N(2) (4) Val113 Val113,Val268
B13 −8.8 N (4) Val113, Val268
B5 −8.8 N(21) Ser98
N (21) Ser98
MSe (3th2)o xyO Val268 Tyr266
N (2) Val113
N(2) Val113
B6 −8.8 N (4) Val113, Val268
N (N21)( 4) Ser98 Val113,Val268
B14 −7.9
B7 −8.1 NN (2)( 21) Val113 Ser98
N (4) Val113, Val268
S(32) Val268

N(2) Val113
B15 −8.8 N(4) Val113,Val268
N(21) Ser98
O(24) Ser172
CIP −7.1
O(25) Gly171
Int. J. Mol. Sci. 2018, 19, 222 9 of 19
Table5. Predictedbindingaffinity,interactiondomain,andpolarinteractionsbetweencompounds
B1–15andtheDNAgyraseBfromListeriamonocytogenes.
Table 5. Predicted binding affinity, interaction domain, and polar interactions between compounds
B1–15 and the DNA gyrase B from Listeria monocytogenes.
Backboneofthe Backbone of the
compoundsB1–15compounds B1–15

Compound gyrB BA (kcal/mol) Atom ID of Ligand Interacting AA Residue
Compound gyrBBA(kcal/mol) AtomN (4I)D ofLigand InAtsep6r1a1c tingAAResidue
B1 −6.3
S (32) Asp614
N(4) Asp611
B1 B2 −6.3−6.7 NA NA
B3 −6.6 NA S(32) NA Asp614
B4 −7.3 NA NA
B2 −6.7 NA NA
B5 −6.3 N (2) Asp614, Thr618
B3 B6 −6.6−7.2 NA NA NA NA
N (4) Asp611
B4 B7 −7.3−6.3 Phenolic ON (30A) Thr618 NA
Thiazole S (16) Asp614
B5 B8 −6.3−7.3 Phenolic ON (3(02) ) Lys610, AAsps6p161 14,Thr618
N (21) Asn608
B6 B9 −7.2−7.2 Phenolic ON (30A) Asp611, Ala615 NA
B10 −6.8 Nitro O (31,32) Arg518
N(4) Asp611
Nitro N (30) Arg518
B7 B11 −6.3−6.8 NPithroe On o(3l2i)c O(30) Arg518 Thr618
TSh (i3a3z) oleS(16) Gln542 Asp614
B12 −6.4 NA NA
B8 −7.3 PNh e(2n1o) licO(30) Gln5L42y s610,Asp611
B13 −6.6
Methoxy O (30) Arg518
N(21) Asn608
B9 B14 −7.2−6.5 NA NA
B15 −6.2 PhNe (n4)o licO(30) Asp6A11s p611,Ala615
CIP −6.5 O (25) Ala510
Int.J.Mol.Sci. 2018,19,222 9of18
Int. J. Mol. Sci. 2018, 19, 222 9 of 19
Table5.Cont.
Table 5. Predicted binding affinity, interaction domain, and polar interactions between compounds
B1–15 and the DNA gyrase B from Listeria monocytogenes.
Backboneofthe Backbone of the
compoundsB1–15compounds B1–15

Compound gyrB BA (kcal/mol) Atom ID of Ligand Interacting AA Residue
Compound gyrBBA(kcal/mol) AtomN (4I)D ofLigand InAtsep6r1a1c tingAAResidue
B1 −6.3
B10 −6.8 NSi t(3r2o) O(31,32) Asp614 Arg518
B2 −6.7 NA NA
B3 −6.6 NNiAt roN(30) NA Arg518
B4 −7.3 NA NA
B11 −6.8 NitroO(32) Arg518
B5 −6.3 N (2) Asp614, Thr618
B6 −7.2 NA S(33) NA Gln542
N (4) Asp611
B12 −6.4 NA NA
B7 −6.3 Phenolic O (30) Thr618
Thiazole NS (1(62) 1) Asp614 Gln542
B13 B8 −6.6−7.3 Phenolic O (30) Lys610, Asp611
MethoxyO(30) Arg518
N (21) Asn608
B9 −7.2
B14 −6.5 Phenolic ON (30A) Asp611, Ala615 NA
B10 −6.8 Nitro O (31,32) Arg518
B15 −6.2 Nitro N N(30)( 4) Arg518 Asp611
B11 −6.8 Nitro O (32) Arg518
CIP −6.5 S (33O) (25) Gln542 Ala510
Int. J. Mol. Sci. 2018, 19, 222 B12 −6.4 NA NA 10 of 19
N (21) Gln542
B13 −6.6
Methoxy O (30) Arg518
B14 −6.5 NA NA
B15 −6.2 N (4) Asp611
CIP −6.5 O (25) Ala510

(A)
(B)
Figure 2. General view (A) and detail (B) of the best docking poses of ligands against gyrA. Target is
Figure 2. General view (A) and detail (B) of the best docking poses of ligands against gyrA.
depicted as thin sticks with a secondary structure drawn as a cartoon backbone and semi-transparent
Target is depicted as thin sticks with a secondary structure drawn as a cartoon backbone and
electrostatic molecular surface (cropped in the detailed view), where ligands are figured as
semi-btarlaln-asnpda-srteinckt (eSlcehcitfrfo bsatsaetsi carme oClPeKcu cloalrorseudr, fmaceean(wcrhoiplep CeIdP iins ptihnek-dmeatgaeilnetda).v iew),whereligandsare
figuredasball-and-stick(SchiffbasesareCPKcolored,meanwhileCIPispink-magenta).

(A)
Int. J. Mol. Sci. 2018, 19, 222 10 of 19
(A)
(B)
Figure 2. General view (A) and detail (B) of the best docking poses of ligands against gyrA. Target is
depicted as thin sticks with a secondary structure drawn as a cartoon backbone and semi-transparent
Int.J.Mol.eSlceic.tr2o0s1t8a,t1ic9 ,m22o2lecular surface (cropped in the detailed view), where ligands are figured as 10of18
ball-and-stick (Schiff bases are CPK colored, meanwhile CIP is pink-magenta).

Int. J. Mol. Sci. 2018, 19, 222 11 of 19
(A)

(B)
(C)
Figure 3. General view (A) and details (B,C) of the best docking poses of ligands against gyrB. Target
Figure 3. General view (A) and details (B,C) of the best docking poses of ligands against gyrB.
is depicted as thin sticks with a secondary structure drawn as a cartoon backbone and
Target is depicted as thin sticks with a secondary structure drawn as a cartoon backbone and
semi-transparent electrostatic molecular surface (cropped in the detailed view), where ligands are
semi-transparentelectrostaticmolecularsurface(croppedinthedetailedview),whereligandsare
figured as ball-and-stick (CIP is pink-magenta, B8 is green, meanwhile the rest of the Schiff bases are
figuredasball-and-stick(CIPispink-magenta, B8isgreen,meanwhiletherestoftheSchiffbases
CPK colored). In the general view (A), the right group is made from CIP, B10, B11, and B13;
are CPK colored). In the general view (A), the right group is made from CIP, B10, B11, and B13;
meanwhile, the left group is made of the rest of the Schiff bases (including here B8); Detail (B) shows
meanwhile,theleftgroupismadeoftherestoftheSchiffbases(includinghereB8);Detail(B)shows
the left group (B8 group), image being focused on B8 (green) binding mode, emphasizing the three
thelHef-tbognrodus pes(tBab8ligshroeud pw),itihm Laygse61b0e ainngd fAoscpu6s1e1d (2o nHB-b8on(gdrse) e(ncr)obpipneddi nvgiewm oshdoew,einmgp ohnalys itzhien gnetahreestth ree
H-boAnAdss reessitdaubeliss–huendtilw ai t7h.5L Åy sd6i1st0ananced frAosmp 6B181); (D2eHta-ibl o(Cn)d ssh)o(wcrso pthpee rdigvhite gwrosuhpo (wCiInPg goronulyp)t,h imenageea rest
AAsbreeinsigd fuoecus–seudn toinl ath7e. 5CÅIP d(pisintakn-mceagfreonmta)B b8in);dDinegt amilo(dCe), esmhopwhasstizhiengr itghhet Hg-rboounpd (eCstIaPblgisrhoeudp )w,iitmh age
beinAglfao5c1u0 s(ecrdopopnetdh veieCwIP sh(powinikn-gm oanglye nthtea )nbeainredsitn AgAms oredseid,uemes–puhnatsili zai n7.g5 Åth ediHsta-bnocen fdroemst aCbIPli)s.h edwith
Ala510(croppedviewshowingonlythenearestAAsresidues–untila7.5ÅdistancefromCIP).
From Table 4, it can be easily observed that all Schiff bases are stronger binders than CIP (used
as the control inhibitor) to gyrA; meanwhile, on gyrB, all compounds are considerably weaker
FromTable4,itcanbeeasilyobservedthatallSchiffbasesarestrongerbindersthanCIP(usedas
binders. For CIP, the docking results are consistent with the data included in DrugBank [49,50].
thecontrolinhibitor)togyrA;meanwhile,ongyrB,allcompoundsareconsiderablyweakerbinders.
From the supplementary data (Table S2), it can be observed that Schiff bases have a common binding
For CIP, the docking results are consistent with the data included in DrugBank [49,50]. From the
pattern (also illustrated in Figure 2) against gyrA within the TOP4c domain (located between
suppploesmitieonntsa 1r2y–d46a5t ain( TLaisbteleriaS 2m)o,nitoccyatnogbeneeso,b ascecrovrdedingth watitShc thhief fUbnaiPserosthKaBv IeDa: Qco8mYAmVo6n), binin thdein rgegpioantt ern
(alsodielslucrsitbreadte bdetiwneFeing uPrhee288) aagnadi nLsyts2g7y0r;A mweainthwinhitleh,e CTIOP Pbi4ncdds osmligahitnly( ldoicffaetreedntblyet, wbeetewnepeno sGitliyo4n1s a1n2d– 465
Gln267. The TOP4c domain or DNA topoisomerase, type IIA, subunit A/C-terminal domain, has
been described as having DNA-binding activity (according to Simple Modular Architecture
Research Tool–SMART accession number: SM00434) [51,52]. In medallions, the canonical sequence
of gyrA with the TOP4c domain (positions 12–465) (Figure 2A) and the canonical sequence of gyrB
with the Toprim domain (positions 430–544) (Figure 3A), marked with yellow, are depicted.
CIP formed two H-bonds, between the carboxyl group from position 3 (O24, O25) with Gly171
and Ser172 of the TOP4c domain (Figure 4A). All Schiff bases formed at least three H-bonds between
the azomethine nitrogen (N21) and serine (Ser98) and between the triazole nitrogens (N2 and N4)
and the valine residues (Val113, Val268). Additional H-bonds are formed by B7 (with Gln95), B8
(with Tyr266–Figure 4B, Figure 5), B10 (with Tyr266 and Gln267), and B13 (with Tyr266). Even
though the Schiff bases and ciprofloxacin bind differently to the target, but in the same subunit A
(C-terminal domain of DNA topoisomerase IIA, (TOP4c)), both binding patterns competitively block
the access of O-(5′-phospho-DNA)-tyrosine intermediate at its binding site, located in position 123
(according UniProtKB ID: Q7BSI9), in Listeria monocytogenes. This confirms the importance of the
imine functional group for the binding mode of the Schiff bases.