Marine Alkaloid 2,2-Bis(6-bromo-3-indolyl) Ethylamine and Its Synthetic Derivatives Inhibit

14 Pages·2017·1.43 MB·English

microorganisms
Article
Marine Alkaloid 2,2-Bis(6-bromo-3-indolyl)
Ethylamine and Its Synthetic Derivatives Inhibit
Microbial Biofilms Formation and Disaggregate
Developed Biofilms
RaffaellaCampana1,* ,GianfrancoFavi2 ,WallyBaffone1andSimoneLucarini3,*
1 DepartmentofBiomolecularScience,DivisionofToxicological,HygieneandEnvironmentalScience,
ViaS.Chiara27,UniversityofUrbinoCarloBo,61029Urbino,Italy;[email protected]
2 DepartmentofBiomolecularScience,SectionofOrganicChemistryandOrganicNaturalCompounds,
UniversityofUrbinoCarloBo,ViaIMaggetti24,61029Urbino,Italy;[email protected]
3 DepartmentofBiomolecularScience,DivisionofChemistry,PiazzadelRinascimento6,
UniversityofUrbinoCarloBo,61029Urbino,Italy
* Correspondence:[email protected](R.C.);[email protected](S.L.);
Tel.:[phone](R.C.);[phone](S.L.)
(cid:1)(cid:2)(cid:3)(cid:1)(cid:4)(cid:5)(cid:6)(cid:7)(cid:8)(cid:1)
(cid:1)(cid:2)(cid:3)(cid:4)(cid:5)(cid:6)(cid:7)
Received:20December2018;Accepted:20January2019;Published:23January2019
Abstract: The antimicrobial activity of the marine bisindole alkaloid 2,2-bis(6-bromo-3-indolyl)
ethylamine(1)andrelatedsyntheticanalogues(compounds2–8)againsttargetmicroorganismswas
investigatedbyMinimumInhibitoryConcentration(MIC)determination. Compound1showedthe
greatestantimicrobialactivitywiththelowestMIC(8mg/L)againstEscherichiacoli,Staphylococcus
aureus, and Klebsiella pneumoniae, while the derivatives exhibited higher MICs values (from 16 to
128mg/L).Compounds1,3,4,and8,themostactiveones,werethentestedagainstE.coli,S.aureus,
K.pneumoniae,andCandidaalbicansduringbiofilmsformationaswellason24hdevelopedbiofilms.
Thenaturalalkaloid1inhibitedthebiofilmformationofallthetestedmicroorganismsupto82.2%
anddisaggregatedbiofilmsofE.coli,S.aureus,K.pneumoniae,andC.albicansafter30minofcontact,as
assessedbyviableplatecountandcrystalviolet(CV)staining(opticaldensityat570nm). Synthetic
derivatives3,4,and8displayedanti-biofilmactivitytowardindividualbacterialpopulations. This
studyhighlightsthepotentialofmarinebisindolealkaloid1asanti-biofilmagentandshows,through
apreliminarystructureactivityrelationship(SAR),theimportanceofhalogensandethylamineside
chainfortheantimicrobialandantibiofilmactivitiesofthisbisindoleseries.
Keywords: marinebisindolealkaloids;bisindolederivatives;anti-biofilmactivity
1. Introduction
Biofilms represent the predominant phenotype of most bacteria in their natural habitat.
The biofilm formation requires a first phase of adhesion to the surface, after the cells start to
replicateintomicro-coloniesandtoproduceanextracellularpolymericsubstance(EPS),composed
of polysaccharides and other macromolecules [1,2]. The EPS plays an important role in biofilm
becauseithelpsthebondbetweenthebacteriaandthesubstratumanditprotectsthecoloniesfrom
any environmental stress, including antimicrobial treatment [3]. Food surfaces as well as medical
environmentsaresuitablesurfacesformicrobialcolonizationandsubsequentlybiofilmformation[4],
thusrepresentingapotentialrisktotransmitpathogenstohumansbycross-contaminations. Moreover,
biofilmsthatweredevelopedonmedicaldevicesaredifficulttoeradicateduetothesignificantdecrease
inthesusceptibilitytoantimicrobialsofbacteriaorganizedinbiofilm,ascomparedtotheirplanktonic
form[5].
Microorganisms2019,7,28;doi:10.3390/microorganisms7020028 www.mdpi.com/journal/microorganisms
Microorganisms 2018, 6, x FOR PEER REVIEW 2 of 15
Microorganisms2019,7,28 2of14
significant decrease in the susceptibility to antimicrobials of bacteria organized in biofilm, as
compared to their planktonic form [5].
MMaarriinnee nnaattuurraall pprroodduuccttss sshhooww ppootteenntt aaccttiivviittyy aanndd sseelleeccttiivviittyy aaggaaiinnsstt aa wwiiddee ssppeeccttrruumm ooff
pphhaarrmmaaccoollooggiiccaall ttaarrggeettss aanndd tthheeiirr uunnpprreecceeddeenntteedd ssttrruuccttuurreess aarree oofftteenn aann iimmppoorrttaanntt ssoouurrccee ooff lleeaadd
ccoommppoouunnddss iinn ddrruugg ddiissccoovveerryy aanndd ddeevveellooppmmeenntt [[66]].. AAmmoonngg tthhee vvaarriioouuss ssttrruuccttuurraall ccllaasssseess,, tthhee mmaarriinnee
bbiissiinnddoollee aallkkaallooiiddss,, ddiimmeerrss ooff iinnddoollee ttyyppiiccaallllyy pprroodduucceedd bbyy mmaarriinnee ssppoonnggeess [[77,,88]],, hhaavvee rreecceeiivveedd mmuucchh
aatttteennttiioonn dduuee ttoo tthheeiirr ssiiggnniiffiiccaanntt ccyyttoottooxxiicciittyy,, aannttiinneeooppllaassttiicc aaccttiivviittyy,, aass wweellll aass aannttiimmiiccrroobbiiaall aanndd
aannttiibbiiooffiillmm aaccttiivviittyy [[99––1111]].. NNoottaabbllyy,, 22,,22--bbiiss((66--bbrroommoo--33--iinnddoollyyll))eetthhyyllaammiinnee ((ccoommppoouunndd 11)) ((FFiigguurree 11)),,
iissoollaatteedd ffrroomm tthhee CCaalliiffoorrnniiaann ttuunniiccaattee DDiiddeemmnnuumm ccaannddiidduumm aanndd tthhee NNeeww CCaalleeddoonniiaann ssppoonnggee OOrriinnaa
sspppp.. [[1122,,1133]],, hhaas sshsohwown nanatinptliapslmasomdoiadli aalctaivcittiyv i[t1y4,[1154],,1 c5y],toctyotxoictoitxyi caigtyainasgta sinevstersaelv teurmalortu cmelol rlinceesll,
sliuncehs ,assu Uch93a7s hUum93a7nh leuumkaenmlieau [1k6e,m17i]a, M[16C,1F7-7], hMumCFa-n7 bhrueamsta, nanbdre Caastc,oa-2n dhuCmaacno- e2phituhmelaianl ceopliothreecltiaall
[c1o8lo].r Secetvaelr[a1l 8n].atSueravle raanldn saytunrthaletainc dprsoydnuthctest iwcipthro adnutcibtsacwteitrhiala nantidb aacntetirbiaiolfailnmd aacnttivibitiyo fi(ltmuloancgtiivciinty,
(tuturlboonmgiyccinin, tBu,r beotcm.)y c(FinigBu,reet c1.)) (sFhiagruer ew1i)thsh caoremwpoituhncdo m1,p oa ucnodm1m,aonco 3m,3m’-odniin3d,3o’-ldyliminedtohlaynlme e(tDhIaMne)
(mDoIlMec)umlaorl eucnuitla [r19u–n2i1t][.1 9–21].

FFiigguurree 11.. ThTeh emmarainrein aelkaalkloaildo i1d, 1so,msoem neatunraatul raanlda nsydntshyenttich eatnictibaancttiebraicatle arigaelnatsg ehnatvsinhga vthineg 3t,3h’e-
d3,i3in’-ddoiilnydlmoleytlhmaenteh (aDnIeM(D) ImMo)lemcuollaerc uulnairt.u nit.
Forexample,tulongicin,derivedfromaTopsentiasp. marinesponge,isabisindolemethanelinked
For example, tulongicin, derived from a Topsentia sp. marine sponge, is a bisindolemethane
toanimidazolegroupthatinhibitedthegrowthofS.aureus[22]. Compound11,whichwasdeveloped
linked to an imidazole group that inhibited the growth of S. aureus [22]. Compound 11, which was
byDongandco-workers[23],showedtobeverypotentagainstseveralmethicillin-resistantS.aureus
developed by Dong and co-workers [23], showed to be very potent against several methicillin-
clinical strains both in the planktonic and biofilm forms. However, to the best of our knowledge,
resistant S. aureus clinical strains both in the planktonic and biofilm forms. However, to the best of
nostudiesweredevotedtotheantimicrobialandantibiofilmabilitiesof1andanalogues. Moreover,
our knowledge, no studies were devoted to the antimicrobial and antibiofilm abilities of 1 and
thepresenceofthealkylaminosidechaininthemarinealkaloid1couldimproveitssolubilityinwater,
analogues. Moreover, the presence of the alkylamino side chain in the marine alkaloid 1 could
possiblyenhancingtheantimicrobialand/orantibiofilmactivityandallowingthepossibilityoffurther
improve its solubility in water, possibly enhancing the antimicrobial and/or antibiofilm activity and
synthetictransformationsbyaminoderivatizationand/orconjugation.
allowing the possibility of further synthetic transformations by amino derivatization and/or
For these reasons, in the present study, the marine bisindole alkaloid 1 and some related
conjugation.
derivativesweresynthesized(Figure2)andtestedagainstseveraltargetmicroorganisms,including
For these reasons, in the present study, the marine bisindole alkaloid 1 and some related
Gram-postitive, Gram-negative, and mycetes, by Minimum Inhibitory Concentration (MIC)
derivatives were synthesized (Figure 2) and tested against several target microorganisms, including
determination. Themostactivecompoundswerethentestedfortheiranti-biofilmactivityduring
Gram-postitive, Gram-negative, and mycetes, by Minimum Inhibitory Concentration (MIC)
biofilmsformationaswellasondevelopedbiofilms.
determination. The most active compounds were then tested for their anti-biofilm activity during
biofilms formation as well as on developed biofilms.
MMiiccrroooorrggaanniissmmss 22001198,, 76,, 2x8 FOR PEER REVIEW 33 ooff 1145
Microorganisms 2018, 6, x FOR PEER REVIEW 3 of 15

R1 R2 R3 Compound
R1 R2 R3 Compound
Br H CH2NH2 1
Br H CH2NH2 1
H H CH2NH2 2
H H CH2NH2 2
F H CH2NH2 3
F H CH2NH2 3
Br Me CH2NH2 4
Br Me CH2NH2 4
H H H 5
H H H 5
Br H 6
Br H 6

Br H 7
Br H 7
H H 8
H H 8
Figure 2. Marine bisindole alkaloid 1 and its synthetic derivatives 2–8.
FFiigguurree 22.. MMaarriinnee bbiissiinnddoollee aallkkaallooiidd 11 aanndd iittss ssyynntthheettiicc ddeerriivvaattiivveess 22––88..
2. Materials and Methods
22.. MMaatteerriiaallss aanndd MMeetthhooddss
22..11.. CChheemmiiccaallss
2.1. Chemicals
BBiiss((11HH--iinnddooll--33--yyll))mmeetthhaannee ((ccoommppoouunndd 55)) aanndd aallll oorrggaanniicc ssoollvveennttss tthhaatt wweerree uusseedd iinn tthhiiss ssttuuddyy
Bis(1H-indol-3-yl)methane (compound 5) and all organic solvents that were used in this study
wweerree ppuurrcchhaasseedd ffrroomm SSiiggmmaa ((MMiillaann,, IIttaallyy)).. PPrriioorr ttoo uussee,, aacceettoonniittrriillee wwaass ddrriieedd wwiitthh mmoolleeccuullaarr ssiieevveess
were purchased from Sigma (Milan, Italy). Prior to use, acetonitrile was dried with molecular sieves
wwiitthh aann eeffffeeccttiivvee ppoorree ddiiaammeetteerr ooff 44 ÅÅ.. CCoolluummnn cchhrroommaattooggrraapphhyy ppuurriiffiiccaattiioonnss wweerree ppeerrffoorrmmeedd uunnddeerr
with an effective pore diameter of 4 Å. Column chromatography purifications were performed under
“‘‘flflaasshh””c coonnddiittiioonnss uussiinngg MMeerrcckk 223300––440000 mmeesshh ssiilliiccaa ggeell.. AAnnaallyyttiiccaall tthhiinn--llaayyeerr cchhrroommaattooggrraapphhyy ((TTLLCC))
‘‘flash” conditions using Merck 230–400 mesh silica gel. Analytical thin-layer chromatography (TLC)
wwaass ccaarrrriieedd oouutt oonn MMeerrcckk ssiilliiccaa ggeell ppllaatteess ((ssiilliiccaa ggeell 6600 FF225544)),, wwhhiicchh wweerree vviissuuaalliizzeedd bbyy eexxppoossuurree ttoo
was carried out on Merck silica gel plates (silica gel 60 F254), which were visualized by exposure to
uullttrraavviioolleett lliigghhtt aanndd aann aaqquueeoouuss ssoolluuttiioonn ooff cceerriiuumm aammmmoonniiuumm mmoollyybbddaattee ((CCAAMM)).. EESSII--MMSS ssppeeccttrraa
ultraviolet light and an aqueous solution of cerium ammonium molybdate (CAM). ESI-MS spectra
wweerree rreeccoorrddeedd wwiitthh aa WWaatteerrss MMiiccrroommaassss ZZQQ ssppeeccttrroommeetteerr)).. EEII--MMSS ssppeeccttrraa wweerree rreeccoorrddeedd wwiitthh aa
were recorded with a Waters Micromass ZQ spectrometer). EI-MS spectra were recorded with a
SShhiimmaaddzzuu QQPP--55000000 MMaassss ssppeeccttrroommeetteerr..1 1HH NNMMRR aanndd 1133CC NNMMRR ssppeeccttrraa wweerree rreeccoorrddeedd oonn aa BBrruukkeerr AACC
Shimadzu QP-5000 Mass spectrometer. 1H NMR and 13C NMR spectra were recorded on a Bruker AC
440000 oorr 110000,, rreessppeeccttiivveellyy,, ssppeeccttrroommeetteerr aanndd aannaallyyzzeedd uussiinngg tthhee TTooppSSppiinn ssooffttwwaarree ppaacckkaaggee.. CChheemmiiccaall
400 or 100, respectively, spectrometer and analyzed using the TopSpin software package. Chemical
sshhiiffttss wweerree mmeeaassuurreedd bbyy uussiinngg tthhee cceennttrraall ppeeaakk ooff tthhee ssoollvveenntt..
shifts were measured by using the central peak of the solvent.
22..22.. CChheemmiissttrryy
2.2. Chemistry
MMaarriinnee bbiissiinnddoollee aallkkaallooiidd 11 aanndd ccoommppoouunnddss 22––44 wweerree pprreeppaarreedd,, aass ddeessccrriibbeedd iinn FFiigguurree3 3..
Marine bisindole alkaloid 1 and compounds 2–4 were prepared, as described in Figure 3.

FFiigguurree 33.. RReeaaccttiioonn ccoonnddiittiioonnss:: ((aa)) ddiipphheennyyll pphhoosspphhaattee,, aacceettoonniittrriillee,, 8800 ◦°CC,, 2244 hh;; ((bb)) KK22CCOO33,, mmeetthhaannooll,,
Figure 3. Reaction conditions: (a) diphenyl phosphate, acetonitrile, 80 °C, 24 h; (b) K2CO3, methanol,
rreeflfluuxx,, 22 hh..
reflux, 2 h.
2.2.1. GeneralProcedurefortheSynthesisofDerivatives1–4

Diphenylphosphate(0.02mmol)wasaddedtoasolutionoftheappropriateindolederivative
(0.4 mmol) and (trifluoroacetylamino)acetaldehyde dimethyl acetal (0.2 mmol) in anhydrous
Microorganisms2019,7,28 4of14
acetonitrile(0.2mL),andtheresultingmixturewasstirredat80◦Cfor24hinasealedtube,monitoring
the progress of the reaction by TLC and HPLC-MS. After cooling to room temperature, saturated
aqueousNaHCO (30mL)anddichloromethane(30mL)wereaddedandthetwophaseswerethen
3
separated. The aqueous solution was extracted with dichloromethane (3 × 20 mL). After drying
over dry Na SO , the combined organic phases were concentrated in vacuum and the resulting
2 4
crudeproductwasutilizedwithoutfurtherpurification. Amixtureofthatcrudetrifluoroacetamide
derivative and potassium carbonate (1 mmol) in MeOH (1.87 mL) and H O (0.13 mL) was stirred
2
andheatedatrefluxfor2h. TheMeOHwasremovedunderreducedpressureandwaterwasadded
(30mL).Theaqueoussolutionwasextractedwithdichloromethane(3 × 30mL)andtheresulting
solutionwasdriedwithNa SO andthenconcentratedinvacuum. Thecrudematerialwaspurified
2 4
byflashchromatographyonneutralalumina.
2.2.2. 2,2-Bis(6-Bromo-1H-Indol-3-Yl)Ethylamine(1)and2,2-Bis(1H-Indol-3-Yl)Ethylamine(2)
The physico-chemical data of compounds 1 and 2 are in agreement with those that were
reported[17].
2.2.3. 2,2-Bis(6-Fluoro-1H-Indol-3-Yl)Ethylamine(3)
Compound 3 was prepared employing 6-fluoro-1H-indole and was isolated by column
chromatography (dichloromethane/methanol/triethylamine, 90:9:1) as a white solid in 70% yield
(twosteps). TLC:Rf=0.18(silicagel;dichloromethane/methanol/triethylamine,90:9:1;UV,CAM).
MS(ESI):m/z310[M-H]−. 1HNMR(400MHz,CD OD,293K):δ=3.37-3.41(m,2H,CHCH NH ),
3 2 2
4.52(dd,1H,J =J =7.5Hz,CHCH NH ),6.72(ddd,2H,J =2.0Hz,J =9.0Hz,J =9.5Hz,H5),
1 2 2 2 5-7 5-4 5-F
7.04(dd,2H,J =2.0Hz,J =9.5Hz,H7),7.14(d,2H,J=3.0Hz,H2),7.44(dd,2H,J =5.0Hz,
7-5 7-F 4-F
J =9.0Hz,H4)ppm. 13CNMR(100MHz,CD OD,293K):δ=37.2,45.6,96.7(d,2C,J=26Hz,C5),
4-5 3
106.5(d,2C,J=24Hz,C7),116.4(2C,C3),119.5(d,2C,J=10Hz,C4),122.3(d,2C,J=3Hz,C9),123.6
(2C,C2),137.0(d,2C,J=12Hz,C8),159.7(d,2C,J=233Hz,C6)ppm.
2.2.4. 2,2-Bis(6-Bromo-1-Methyl-1H-Indol-3-Yl)Ethylamine(4)
Compound4waspreparedemploying6-bromo-1-methylindoleanditwasisolatedbycolumn
chromatography(dichloromethane/methanol/triethylamine, 90:9:1)asyellowishsolid(foam)in
77%yield(twosteps). TLC:Rf=0.22(silicagel;dichloromethane/methanol/triethylamine,90:9:1;
UV,CAM).MS(ESI):m/z460(50),462(100),464(50)[M+H]+. 1HNMR(400MHz,CD OD,293K):
3
δ=3.32(d,2H,J=7.5Hz,CHCH NH ),3.72(s,6H,NCH ),4.49(dd,1H,J =J =7.5Hz,CHCH NH ),
2 2 3 1 2 2 2
7.06(s,2H,ArH),7.06(dd,2H,J =1.5Hz,J =8.5Hz,ArH),7.40(d,2H,J=8.5Hz,ArH),7.51(d,
1 2
2H,J=1.5Hz,ArH)ppm. 13CNMR(100MHz,CD OD,293K):δ=31.4,36.8,45.8,112.0,114.7,115.9,
3
120.2,121.4,126.3,127.2,138.2ppm.
Compounds6and7weresynthesized,asreportedinFigure4.
Microorganisms 2018, 6, x FOR PEER REVIEW 5 of 15
Microorganisms2019,7,28 5of14
Compounds 6 and 7 were synthesized, as reported in Figure 4.

FFiigguurree 44.. RReeaaccttiioonn ccoonnddiittiioonnss: :(a(a) )eetthhyyl lpprrooppiioollaattee, ,ddiicchhlolorroommeeththaannee, ,aat trroooomm tteemmppeerraattuurree ((RRTT)),,
oovveerrnniigghhtt ((OONN));; ((bb)) 99,, ttoolluueennee,, rreefflluuxx,, 22 hh;; ((cc)) ttrriiflfluuoorrooaacceettiicc aacciidd,, rreeflfluuxx,, 44hh.. ((dd)) 99,, aacceettoonniittrriillee,, RRTT,, 11hh;;
aanndd,, ((ee)) ppaarraaffoorrmmaallddeehhyyddee,, rreefflluuxx,, 44hh. .
2.2.5. 4-Ethyl3-Methyl1-(2,2-Bis(6-Bromo-1H-Indol-3-Yl)Ethyl)-2-Methyl-1H-Pyrrole-3,4-
2.2.5. 4-Ethyl 3-Methyl 1-(2,2-bis(6-Bromo-1H-Indol-3-Yl)Ethyl)-2-Methyl-1H-Pyrrole-3,4-Dicarboxylate
Dicarboxylate(6)
(6)
Amixtureof1(0.4mmol)andethylpropiolate(0.44mmol)wasstirredindichloromethane(1mL)
A mixture of 1 (0.4 mmol) and ethyl propiolate (0.44 mmol) was stirred in dichloromethane (1
overnight at room temperature. A solution of 9 (0.6 mmol) in toluene (4 mL) was added and the
mL) overnight at room temperature. A solution of 9 (0.6 mmol) in toluene (4 mL) was added and the
reactionwasrefluxedfor2h. Catalyticamountoftrifluoroaceticacid(twodrops)wasaddedandthe
reaction was refluxed for 2 h. Catalytic amount of trifluoroacetic acid (two drops) was added and the
reactionwasrefluxedforadditional4h. Afterremovalofthesolvent,thecrudemixturewaspurified
reaction was refluxed for additional 4 h. After removal of the solvent, the crude mixture was purified
bycolumnchromatographyonsilicagel(ethylacetate/cyclohexane25:75)toaffordproduct6in70%
byyie lcdolausmany eclhlorowmsaotloidg.raMpShy(E oIn) msi/lzic(a% g)e=l 6(e2t7h(yMl a+c)e(t3a)t,e4/c0y5c(l3o1h),e4x0a3ne(1 2050:)7,54)0 t1o (a3f8f)o.r1dH pNroMduRc(t4 60 0inM 7H0%z,
yield as a yellow solid. MS (EI) m/z (%) = 627 (M+) (3), 405 (31), 403 (100), 401 (38). 1H NMR (400 MHz,
DMSO-d6,293K):δ=1.17(t,3H,J=7.2Hz,OCH CH ),2.11(s,3H,CH ),3.64(s,3H,OCH ),4.06
2 3 3 3
D(qM,JS=O7-d.26H, 2z9,32 KH):, δO =C 1H.17C (Ht, 3)H,4,. 6J 3= (7d.2, 2HHz,, JO=C8H.02CHHz3,),C 2H.1C1 H(s, N3H),,4 C.8H73()t,, 31.H64, (Js=, 38H.0, HOCz,HC3H), C4.H06 N(q),,
2 3 2 2
J6 .=9 97.(2d Hd,z2, H2 ,HJ, O=C1H.62CHHz,3)J, 4=.683. 8(dH, 2zH,A, Jr H= )8,.07 .1H6z(, sC,H1HC,HA2NrH),) ,47.8.73 7(−t, 71.H42, J( m= ,84.0H H,Az,r CHH),C7H.429N()d, ,62.9H9,
1 2
(Jd=d1, .26HH, zJ1, A= r1-.H6 )H,1z1, .J026 =( b8r.8s, H2Hz,, ANrHH)).,1 73C.16N (Ms, R1H(1,0 A0rMHH), z7,.3D7M−7S.O42- d(m6,,2 49H3K, A):rδH=),1 70..479, 1(d4.,6 2,H35,. 2J ,=5 01..96,
Hz, Ar-H), 11.06 (brs, 2H, NH). 13C NMR (100 MHz, DMSO-d6, 293 K): δ = 10.7, 14.6, 35.2, 50.9, 51.4,
51.4,59.7,100.0,114.0,114.2,114.4,115.4,121.0,121.6,124.4,125.8,127.5,135.3,137.6,163.7,165.6.
59.7, 100.0, 114.0, 114.2, 114.4, 115.4, 121.0, 121.6, 124.4, 125.8, 127.5, 135.3, 137.6, 163.7, 165.6.
2.2.6. Methyl1-(2,2-Bis(6-Bromo-1H-Indol-3-Yl)Ethyl)-4-Ethyl-1H-Imidazole-5-Carboxylate(7)
2.2.6. Methyl 1-(2,2-bis(6-Bromo-1H-Indol-3-Yl)Ethyl)-4-Ethyl-1H-Imidazole-5-Carboxylate (7)
To a stirred solution of 1 (0.4 mmol) in acetonitrile (2 mL), 9 (0.4 mmol) was added at room
tempTeora atu srtei.rrAedft esrotlhuetidonis aopf p1e a(0ra.4n cmemofotlh) einr eaacgeetnotnsi,trpialer a(f2o rmmLa)l,d 9e h(y0.d4e m(0m.8oml)m woals) wadadseadd daet dr,oaonmd
ttehmenptehreatruesreu.l tAinfgtemr tihxetu dreiswapapseraerflaunxceed offo trh4eh re(TagLeCnctsh,e pcakr).afTohremsaolldveehnytdwea (s0e.8v ampmoroatl)e dwuans daedrdreedd,u acnedd
tphreenss uthree arensdutlhtiengcr umdixetruersei dwueasw raesflpuuxreidfi efdorb 4y cho l(uTmLCn cchhreocmk)a. tToghrea psohlyve(netth wylaasc eetvaatpe/orcaytceldoh uenxadneer
r8e0d:2u0c)etdo gpivrees7suarsea barnodw nthsoe lidcriund7e2 %reysiiedlude. MwSa(sE I)pmu/rzif(i%ed) =b5y5 6c(oMlu+m)(n1 00c)h,r4o0m1(a3t2o)g,r4a0p3h(y1 00(e),th40y5l
a(3c9e)t.a1teH/cNycMloRhe(4x0a0neM 8H0:z2,0D) MtoS gOiv-de6 7, 2a9s3 aK b)r:oδw=n2 s.2o6li(ds ,i3nH 7,2C%H yi)e,l3d..8 0M(Ss, (3EHI), mO/Cz H(%)), =4. 9515–64 (.M96+)(m (1,030H),,
3 3
4C0H1 C(3H22),N 4)0,37 .(0110(0d),d 4,025H (,3J91).= 1H1.6 NHMz,RJ 2(4=008 .4MHHzz,, ADrMHS),O7-.2d26,( s2,913H K,)A: rδH =) ,27.2.360 (s(d, 3,H2H, ,CJH=3)2, .03.H80z (,sA, r3HH),,
O7.C40H(3d),, 42.H91,-J4=.968 .(4mH, z3,HA, rCHH)C,7H.52N0()d, 7,.20H1 ,(dJd=, 12.H6,H J1z =, A1.r6H H),z1, 1J2.0 =4 8(.b4r Hs,z2,H A,rNHH), )7..1232C (sN, 1MHR, A(1r0H0)M, 7H.3z0,
(DdM, 2SHO, -Jd =6 ,22.09 3HKz),: Aδr=H1),6 .71.,430 5(.d4,, 521H.0, ,J 5=1 .88.,41 H14z.,2 A,1r1H4).,5 ,71.5105 .(4d,, 121H7.,9 J, 1= 210..68 ,H1z2,1 A.6r,H12),4 .141,.0142 5(.b9r,s1, 327H.6,,
N14H2.)2. ,113C47 N.4M,1R61 (.150.0 MHz, DMSO-d6, 293 K): δ = 16.1, 35.4, 51.0, 51.8, 114.2, 114.5, 115.4, 117.9, 120.8,
121.6C, 1o2m4.p4o, u12n5d.98, 1w3a7s.6p, r1e4p2a.2re, d14a7s.4r,e 1p6o1r.t5e.d inFigure5.
Microorganisms 2018, 6, x FOR PEER REVIEW 6 of 15
Microorganisms2019,7,28 6of14
Compound 8 was prepared as reported in Figure 5.

FFiigguurree 55.. RReeaaccttiioonn ccoonnddiittiioonnss:: ((aa)) NN,,NN--ddiiiissoopprrooppyylleetthhyylalammininee, ,eetthhaannool,l ,00 °◦CC, ,1100 mmiinn;; aanndd,, ((bb)) 1100,,
aacceettoonniittrriillee,, 00 °◦CC,, RRTT,, 22 hh..
2.2.7. 3,3(cid:48)-(2-(4-Methyl-1H-1,2,3-Triazol-1-Yl)Ethane-1,1-Diyl)Bis(1H-Indole)(8)
2.2.7. 3,3'-(2-(4-Methyl-1H-1,2,3-Triazol-1-Yl)Ethane-1,1-Diyl)Bis(1H-Indole) (8)
To a cooled solution (0 ◦C) of 2 (0.4 mmol) in ethanol (5 mL) was added
To a cooled solution (0 °C) of 2 (0.4 mmol) in ethanol (5 mL) was added N,N-
N,N-diisopropylethylamine (2.4 mmol, 6 eq). The solution was stirred for 10 min. after which
diisopropylethylamine (2.4 mmol, 6 eq). The solution was stirred for 10 min. after which hydrazone
hydrazone10(0.52mmol,1.3eq.) dissolvedinacetonitrile(4mL)wasaddeddropwisetothecooled
10 (0.52 mmol, 1.3 eq.) dissolved in acetonitrile (4 mL) was added dropwise to the cooled solution
solutionandstirringwascontinuedatroomtemperatureuntilcompletionofthereaction(TLCcheck).
and stirring was continued at room temperature until completion of the reaction (TLC check). After
Aftercompletionofthereaction,allvolatileswereremovedunderreducedpressureandtheresidue
completion of the reaction, all volatiles were removed under reduced pressure and the residue was
waspurifiedbycolumnchromatography(ethylacetate/cyclohexane60:40)togive8aspaleyellow
purified by column chromatography (ethyl acetate/cyclohexane 60:40) to give 8 as pale yellow solid
solidinquantitativeyield.
in quantitative yield.
MS(EI)m/z(%)=341(M+)(4),109(100). 1HNMR(400MHz,DMSO-d6,293K):δ=2.12(s,3H,
MS (EI) m/z (%) = 341 (M+) (4), 109 (100). 1H NMR (400 MHz, DMSO-d6, 293 K): δ = 2.12 (s, 3H,
CH ),5.04−5.16(m,3H,CH CHN),6.91(t,2H,J=7.6Hz,ArH),7.03(t,2H,J=7.6Hz,ArH),7.31−7.35
(CmH,334)H, 5,.0A4r−H5).1,67 .(5m4,( d3,H2,H C,HJ2=2C8H.0NH),z 6,.A91r H(t,) ,27H.6, 8J (=s ,71.6H H,Az,r AHr)H,1)0, .78.603(b (rts, ,22HH, ,JN = H7.)6; 1H3Cz, NAMrHR),( 170.301M−7H.3z5,
(m, 4H, ArH), 7.54 (d, 2H, J = 8.0 Hz, ArH), 7.68 (s, 1H, ArH), 10.86 (brs, 2H, NH); 13C NMR (100 MHz,
DMSO-d6,293K):δ=10.9,35.5,53.9,111.9,115.3,118.7,119.2,121.4,122.8,123.2,126.8,136.8,141.7.
DMSO-d6, 293 K): δ = 10.9, 35.5, 53.9, 111.9, 115.3, 118.7, 119.2, 121.4, 122.8, 123.2, 126.8, 136.8, 141.7.
2.3. BacterialStrains
2.3. Bacterial Strains
Sixreferencehumanpathogenswereusedinthisstudy,EscherichiacoliATCC35218,Pseudomonas
Six reference human pathogens were used in this study, Escherichia coli ATCC 35218,
aeruginosaATCC9027,StaphylococcusaureusATCC43387,EnterococcusfaecalisATCC29212,andCandida
Pseudomonas aeruginosa ATCC 9027, Staphylococcus aureus ATCC 43387, Enterococcus faecalis ATCC
albicansATCC14053. TheclinicalhumanstrainKlebsiellapneumoniae6/4,isolatedfrompatientwith
29212, and Candida albicans ATCC 14053. The clinical human strain Klebsiella pneumoniae 6/4, isolated
urinaryinfectionandkindlyprovidedbyGammaLaboratory(Fano,Pesaro,Italy),wasalsoincluded.
fromA plaltoiefntth wesitthra uinrisnwareyr einmfeacitniotani naendd ikninTdrylyp tpicroSvoiydeAdg bary (GTaSmA,mOax Loaidb,oMraitloarny, I(tFaalnyo),a Pte3s7a◦rCo,, Iwtahlyil)e,
was also included.
C.albicansATCC14053wasgrowninSabouraudDextroseAgar(Oxoid,Milan,Italy). Allthestock
cultuArelsl owf etrheek setrpatinats −w8e0re◦ CmaininNtauintreiden itnb Trroythpt(iOc Sxooiyd A,Mgailra (nT,SIAta,l yO)xwoiidth, M15i%lano,f Igtalylyc)e raot l3.7 °C, while
C. albicans ATCC 14053 was grown in Sabouraud Dextrose Agar (Oxoid, Milan, Italy). All the stock
c2u.4l.tuDreetse rwmeirnea tkioenpto faMt −i8n0im °Cum inI nNhuibtirtioernyt Cbornocthen (tOraxtoioind,( MMIiCla)n, Italy) with 15% of glycerol.
MICsweredeterminedbystandardmicro-dilutionmethod. First,eachcompoundwasdissolved
2.4. Determination of Minimum Inhibitory Concentration (MIC)
in dimethyl sulfoxide (DMSO) (Sigma, Milan, Italy) of biological grade. Several colonies of each
bacteMriaIClsst rwaienrew deerteeirnmoicnueldat bedy sinta1n0dmarLd omfisctreori-ldeilMutuioelnle mr-eHthinotdon. Fbirrsott,h e(aMchH cBo)m(Opoxuoindd) wanads idnicsusoblavteedd
iant d37im◦eCthfyolr su18lf–o2x4idhe. (DEMacShOb) a(cStiegrmiaal, sMusiplaenn, sIiotanlyw) oasf baidojluosgtiecdal tgoraadbeo.u Ste1v0e6raclf uco/lmonLie(sO oDf each
610nm
b0.a1c3t–e0ri.1a5l )satrnadin1 0w0eµrLe winaoscaudladteedd ininw 1e0ll smofLt hoef 9s6t-ewrielell cMelulecluleltru-Hreinptloante (bCroeltlhs ta(Mr®H,GBr)e i(nOexroBidio)- Oannde,
iFnrcicukbeantehda uast e3n7, °GCe rfomr a1n8y–)2,4t ohg. eEtahcehr bwaicthterthiael asupsppreonpsriioante wvaoslu amdjeusstoefdt htoe atebsotusto 1lu06t icofnus/m(fLro (mOD0.6510ntom
102.183–µ0g./15m) Lan).dT 1w0o0 rµoLw wsaws eardeduesde din fworelplso soift itvhee (9b6a-wcteerlli acealllo cnuelt)uarned plnaeteg a(Ctivelelsctoarn®t,r oGlrse(iMneHr BBioa-lOonnee),,
Frersicpkeecntihvaeulys.eGn,e Gntearmmiacniny)(,f rtoomge0th.1e2r5 wtoith1 2t8heµ ga/pmprLo)parniadtefl vuoclounmazeos loef( Sthigem teas,tM soillaunti,oIntasl y(f)rwomer e0.a5l stoo
1a2d8d eµdg/amsLin).t eTrwnaol rcoowntsr owlse.rPe ruesliemd ifnoarr ypoassistaivyes (wbiatchteDriMa SaOlonwe)e raencda rnreiegdatoivuet ctoonetxrcollusd (eMiHtsBp oaslosnibel)e,
rbeascpteercitoivstealtyic. Ganedn/taomrbicaicnt e(rfricoimda l0.a1c2t5iv tioty 1;2th8e µvgo/lmumL)e aonfdD fMluScOonaadzdoeled (iSnigeamcah, wMeilllanne, vIetarleyx) cweeedreed al5s%o
(avd/vd)eodf aths einfitnearnltaolt caolnvotrloulms. eP.rTehliemoipntaicryal adsesnasyisty w(6it0h0 DnmM)SoOf ewacehrew cealrlrwieads oausste tsos eedxculsuidnge aitsM puoltsissicbalne
bEaxcMteriicorsotpaltaict eanRde/aodre bra(cTthereircmidoalS accietinvtiitfiyc; ,thItea lvyo)l.uMmeIC ofw DaMs dSeOfi nadeddeads itnh eealcohw wesetllc onnevceenr terxacteioendeodf
5co%m (pv/ovu) nodf itnhhe ifibintainl gtotthael bvaoclutemriea.l Tghroew otphtiacfatle rde2n4shityo f(6in0c0u nbmat)i oonf .eAacllhd wateall wwearse aesxspersessesde dusaisngth ae
Mmeualtnisocfanth rEeex inMdiecpreonpdlaetnet eRxepaedreimr e(nTthsetrhmatow Secrieepnetirffioc,r mIteadlyi)n. dMupICli cawtea.s defined as the lowest
concentration of compound inhibiting the bacterial growth after 24 h of incubation. All data were
expressed as the mean of three independent experiments that were performed in duplicate.
2.5. Crystal Violet Biofilm Assay
Microorganisms2019,7,28 7of14
2.5. CrystalVioletBiofilmAssay
A static biofilm formation assay was performed in 24-wells polystyrene plates (VWR, Milan,
Italy) and biomass production was assessed after Cristal Violet (CV) staining, as described in
Campanaetal.[24]. All of the strains were grown in Tryptic Soy Broth (TSB, Oxoid, Milan, Italy)
at37◦Cfor24htoobtainabacterialsuspensionattheendofthelogarithmicphase. Subsequently,
theopticaldensityofeachsuspension(OD )wasadjustedtoabout0.13–0.15(correspondentto
610nm
106–107 cfu/mL);1mLofeachsuspension,diluted1:10inTSB,wasseededin24-wellpolystyrene
plates (Cellstar®, Greiner Bio-One, Frickenhausen, Germany), and incubated at 37 ◦C for 24 h to
allowbiofilmformation. Attheendofincubation,thewellswereoncewashedwithPBStoeliminate
unattachedcellsandcoveredwithcrystalviolet(CV)0.1%(v/v)for15min. Thesampleswereonce
washedagainwithPBSandair-dried. TheremainingCVwasdissolvedin85%ethanol(15minat
roomtemperature)and,finally,200µLfromeachwellwastransferredtoa96-wellcellcultureplate
(Cellstar®, Greiner Bio-One, Frickenhausen, Germany) for spectrophotometry at 570 nm, using a
MultiscanExMicroplateReader(ThermoScientific). Eachdatapointwasaveragedfromatleasteight
replicatewells. Theexperimentswereperformedtwiceusingindependentcultures.
2.6. BiofilmFormationInhibition
Theanti-biofilmactivityofeachcompoundagainsteachpathogenwasdeterminedintermsof
biofilm formation inhibition. Briefly, 200 µL of each bacterial suspension (about 106 bacteria/mL)
wereinoculatedin24-wellpolystyreneplates(Cellstar®,GreinerBio-One,Frickenhausen,Germany)
withthecorrespondingamountofeachselectedcompoundattheirrelativeMICand2×MICvalues.
TwowellsforeachpathogenwerealsoinoculatedwithbacteriainTrypticSoyBroth(TSB)(Oxoid,
MilanItaly)andincludedascontrols. Theplateswerethenincubatedfor24hat37◦Ctoallowbiofilm
development; at the end of the incubation, each sample was gently once washed by PBS and the
biofilmformationinhibitionforeachpathogenwasassessedbyCVstaining. Alldatawereexpressed
asthemeanofthreeindependentexperimentsperformedinduplicate.
2.7. EradicationofBiofilmswithSelectedChemicalCompounds
Intheselastexperiments,theactivityofselectedcompounds(1,3,4,and8)ondevelopedbiofilms
of E. coli ATCC 35218, S. aureus ATCC 43387, K. pneumoniae 6/4, and C. albicans ATCC 10231 was
determined. Briefly,biofilmsofeachpathogenwerepreparedwiththeproceduredescribedabove
(Crystalvioletbiofilmassaysection). After24hofincubationat37◦C,allofthebiofilmsweregently
oncewashedwithPBSandcoveredfor30minwiththerightamountofeachcompoundcorresponding
totheMIC.Foreachplate,twowellsweretreatedwithphysiologicalsaline(negativecontrols). After
antimicrobial treatment, all of the biofilms were once washed by PBS and adherent bacteria were
harvested bysterile scrapersin 1mL ofsterile physiologicalsaline solution, subsequentlyserially
dilutedinthesamemediumandplatedintriplicateonTSA(Oxoid,Milan,Italy). Theplateswere
thenincubatedat37◦Cfor24hattheendofwhichthecolonyformingunits(cfu)wereenumerated.
Toverifytheeventualreductionofbiomassproductionafterantimicrobialtreatment, CVstaining
wascarriedoutasdescribedabove. Allthedatawereexpressedasthemeanofthreeindependent
experimentsperformedinduplicate.
2.8. StatisticalAnalysis
StatisticalanalysiswasperformedusingPrismversion5.0(GraphPadInc.,LaJolla,CA,USA).
Theassumptionsforparametrictestwerecheekedpriortocarryoutthestatisticalanalysisbyt-Student
test. pvalues<0.05wereconsideredtobestatisticallysignificant.
Microorganisms2019,7,28 8of14
3. Results
3.1. DeterminationofMinimumInhibitoryConcentration
Thecompound1showedthegreatestantimicrobialactivityagainstallthepathogensincludedin
thisstudy. Indetail,thelowestMICvalueof8µg/mLwasobservedforE.coliATCC35218,S.aureus
ATCC43387,andK.pneumoniae6/4,whilehighervaluesweredeterminedforE.faecalisATCC29212
(32µg/mL),P.aeruginosaATCC9027,andC.albicansATCC10231(64µg/mL)(Table1). Asregards
compound3,thelowestMICvalue(64µg/mL)wasevidencedforE.coliATCC35218and128µg/mL
forall othersspecies. Compound 4 showed16 µg/mLMIC valuetoward E.coli ATCC 35218and
32µg/mLagainstK.pneumoniae6/4andC.albicansATCC10231. MICvaluesof128µg/mLwere
registeredforcompound8,whilealltheothercompoundsshowedMICs>128µg/mLforallthetested
microorganisms. With regard to the internal control, gentamicin inhibited microbial growth with
thelowestMICvalueof8µg/mLforK.pneumoniae6/4andthehighestMICvalueof64µg/mLfor
E.faecalisATCC29212(Table1). C.albicansATCC10231resultedinbeingsensitivetofluconazolewith
MICof1µg/mL.
Table1.MinimumInhibitoryConcentration(MIC)values(µg/mL)ofthetestedcompoundsagainst
selected microbial strains, assessed by broth microdilution method according to the Performance
StandardsforAntimicrobialSusceptibilityTesting(CLSIDocumentM100-S2,2013).Gentamicinand
fluconazolewereusedasinternalcontrols.
MicrobialStrains 1 2 3 4 5 6 7 8 Gentamicin
E.coliATCC35218 8 >128 64 16 >128 >128 >128 128 16
S.aureusATCC43387 8 >128 128 128 >128 >128 >128 128 16
E.faecalisATCC29212 32 >128 128 128 >128 >128 >128 128 64
P.aeruginosaATCC9027 64 >128 128 128 >128 >128 >128 128 16
K.pneumoniae6/4 8 >128 128 32 >128 >128 >128 128 8
C.albicansATCC10231 64 >128 128 32 >128 >128 >128 128 1a
aFluconazolewasusedascontrolforC.albicansATCC10231.
3.2. BiofilmFormationInhibition
The compounds 1, 3, 4, and 8 were tested at the relative MIC and 2× MIC values during the
biofilmformationoftheexaminedpathogens(Table2). Inmanycases,thecompoundsattheirrelative
MICand2×MICvaluesdeterminedthebactericidaleffectafter24hofincubation(novisiblegrowth
inthewells)and, inthesecasesbiofilmformationafterCVstainingwerenotassessed(FigureS1).
E.faecalisATCC29212andP.aeruginosaATCC9027werenotincludedonthebasisoftheresultsthat
wereobtainedduringthebiofilmformation,whereE.faecalisresultedinbeingcompletelyinhibitedby
allthetestedcompounds(bactericidaleffect)whileP.aeruginosawasresistant.
As general trend, compound 1 was able to reduce biofilm formation of most the examined
bacteriaattheirrelativeMICvalues,withtheonlyexceptionofP.aeruginosaATCC9027. Thelowest
concentration (8 µg/mL), tested against E. coli ATCC 35218 and S. aureus ATCC 43387, provoked
68.3 and 82.2% of biofilm formation inhibition, respectively; the same concentration induced only
40.0%ofK.pneumoniae6/4biofilmformationinhibition,reaching83.7%ofinhibitionwith16µg/mL
(2×MICvalue). Thehighestconcentration(64µg/mL)wasunabletoinhibitthebiofilmformation
of P. aeruginosa ATCC 9027, while it exhibited bactericidal action against C. albicans ATCC 10231.
Thecompound3(MIC64µg/mL)wasabletostronglyinhibitthebiofilmformationofE.coliATCC
35218 (97.6%). The highest MIC value (128 µg/mL) determined only 34.6% of biofilm formation
inhibitioninC.albicansATCC10231anditwasunabletocontrastthebiofilmformationofP.aeruginosa
ATCC 9027 (7.8%). On the other hand, 128 µg/mL resulted in being bactericidal against S. aureus
ATCC 43387, E. faecalis ATCC 29212, and K. pneumoniae 6/4. The compound 4 was tested at MIC
valuesrangingfrom16to128µg/mL.ThelowestconcentrationwasbactericidalagainstE.faecalis
Microorganisms2019,7,28 9of14
ATCC29212,whilethebiofilmformationofK.pneumoniae6/4wasremarkablyinhibited(64.7%)by
MICvalueof32µg/mL,reaching96.6%ofinhibitionwith64µg/mL(2×MICvalue). Lessbiofilm
inhibition(14.6%)wasobservedforC.albicansATCC10231,with32µg/mL,andalsowith2×MIC
value(40.9%). Thehighestconcentrationofcompound4(128µg/mL)caused56.6%ofS.aureusATCC
43387biofilmformationinhibitionandresultedinbeingbactericidalagainstE.faecalisATCC29212.
Asregardscompound8(MIC128µg/mL),remarkablebiofilmformationinhibitionwasevidenced
forK.pneumoniae6/4(65.8%),whilelowerpercentageswereobservedforE.coliATCC35218(54.1%)
and S. aureus ATCC 43387 (34.9%); also, in this case, the biofilm formation of P. aeruginosa ATCC
9027wasnotreduced,whileabactericidaleffectwasobservedagainstE.faecalisATCC29212andC.
albicansATCC10231. Increasedpercentagesofbiofilmformationinhibitionwerealwaysobserved
withcompound8at2×MICvalue.
Table2.Biofilmformationinhibitionexertedbycompounds1,3,4,and8.Eachmoleculewasaddedat
itsrelativeMICand2×MIC(µg/mL)valuesduringthebiofilmformationofthedifferentpathogens;
theinhibitionpercentagesweredeterminedafterspectrophotometerreaderat570nm.
1 3 4 8
MicrobialStrains Biofilm Biofilm Biofilm Biofilm
Inhibition Inhibition Inhibition Inhibition
MIC MIC 2×MIC MIC MIC 2×MIC MIC MIC 2×MIC MIC MIC 2×MIC
E.coliATCC35218 8 68.3% BE 64 97.6% BE 16 1.7% 48.1% 128 54.1% 92.0%
S.aureusATCC43387 8 82.2% BE 128 BE ND 128 56.6% BE 128 34.9% 62.9%
E.faecalisATCC29212 32 BE ND 128 BE ND 128 BE ND 128 BE ND
P.aeruginosaATCC9027 64 0.5% 0.9% 128 7.8% BE 128 4.8% BE 128 7.1% BE
K.pneumoniae6/4 8 40.0% 83.7% 128 BE ND 32 64.7% 96.6% 128 65.8% 98.7%
C.albicansATCC10231 64 BE ND 128 34.6% BE 32 14.6% 40.9% 128 BE ND
BE,bactericidaleffect:biomasswasnotdeterminedbyspectrophotometerreader(570nm)becausenobacterial
growthwasvisible.inthewells(seealsoFigureS1);ND:notdeterminedbecausealreadybactericidalatMICvalue.
3.3. EradicationofBiofilmsafterTreatmentwithCompounds1,3,4and8
InthecaseofE.coliATCC35218,thetreatmentofbiofilmswithcompound1(MIC8µg/mL)
reducedbacterialviabilityto2.0×107 cfu/mLincomparisonto5.28×108 cfu/mLoftherelative
control biofilm (p < 0.05), while the other compounds caused no significant reduction of cfu/mL
(Figure 6). For S. aureus ATCC 43387, compound 1 (MIC 8 µg/mL) reduced viable bacteria to
2.27×106cfu/mL in comparison to 3.85 × 108 cfu/mL of the untreated control (p < 0.001) and a
statisticalsignificantreductionincfu/mLvalueswasalsoobservedaftertreatmentwithcompounds4
(MIC128µg/mL)(p<0.01). AsregardsK.pneumoniae6/4,thetreatmentwithcompounds1(MIC
8µg/mL)and4(MIC32µg/mL)determinedvaluesof1.0×107and1.37×106cfu/mL,respectively,
withasignificantdecreaseincomparisontotherelativecontrolbiofilm(3.2×107cfu/mL)(p<0.01;
p<0.05). NoremarkablereductionofK.pneumoniae6/4growthwasobservedwithcompounds3and
8(MIC128µg/mL).InthecaseofC.albicansATCC10231,themostremarkabledecreaseofcfu/mL
wasobservedafterthetreatmentwithcompound1(MIC64µg/mL),showing2.0×104cfu/mLin
comparisonto2.6×107cfu/mLofthecontrolone(p<0.001).Similarly,thetreatmentwithcompounds
4(MIC32µg/mL)evidenced3.0×104cfu/mL(p<0.01),whilecompounds3and8(MICs128µg/mL)
inducedonlyaslightreduction.
ThebiomassanalysisrevealeddifferentdecreasesofOD valuesinbiofilmsaftertreatment
570nm
withthedifferentcompounds(Figure7). AsregardsE.coliATCC35218,afterexposuretocompound1
(MIC8µg/mL),anODof0.320wasobservedincomparisonto0.641oftherelativeuntreatedbiofilm
(p<0.01),whilethetreatmentwithcompounds3(MIC64µg/mL),4(MIC16µg/mL),and8(MIC
128 µg/mL) showed OD values that were quite similar (from 0.541 to 0.622) to that of the control.
TheexposureofS.aureusATCC43387biofilmstocompound1(MIC8µg/mL)determinedanOD
of0.250incomparisonto0.573oftherelativeuntreatedbiofilm(p<0.01),whileaftertreatmentwith
compounds3and4(MICs128µg/mL),ODvaluesof0.350and0.264,respectively(p<0.05)were
Microorganisms2019,7,28 10of14
registered. Onthecontrary,compound8(MIC128µg/mL)determinedanODvaluequitesimilarto
tMhiacrtooorfgatnhiesmrse 2la01ti8v, 6e, cxo FnOtRr oPlE.ER REVIEW 10 of 15

FFiigguurree 66.. AAsssseessssmmeenntt ooff vviiaabbllee cceellllss hhaarrvveesstteedd ffrroomm bbiiooffiillmmss ooff EE.. ccoollii AATTCCCC 3355221188,, SS.. aauurreeuuss AATTCCCC
4433338877,, KK.. ppnneeuummoonniiaaee 66//44 aanndd CC.. aallbbiiccaannss AATTCCCC1 100223311a aftfeterr3 300m minino offt rteraetamtmenetnwt withithco cmompopuonudnsd1s, 13,, 34,,
a4n, adn8d a8t atht ethireirre lraetliavteivMe MICICva vlualeuse.sD. aDtaatrae rperperseesnetntth tehme meaenan± ±S SDDo offc cfufu//mmLL vvaalluueess oobbttaaiinneedd iinn tthhrreeee
iinnddeeppeennddeenntt eexxppeerriimmeennttss ppeerrffoorrmmeedd iinn dduupplliiccaattee.. AAsstteerriisskkss rreepprreesseenntt vvaalluueess ssttaattiissttiiccaallllyy ssiiggnniifificcaanntt
Microo((r**g ppan <<is 00m..s00 552;0; 1***8* ,pp 6 ,<< x 0 0F..0O011R;; *P***E** EppR << R 0E0..0V00I0E11W)) aa s s ccoommppaarreedd ttoo tthhee rreellaatteedd ccoonnttrroollss.. 11 of 15
The biomass analysis revealed different decreases of OD 570 nm values in biofilms after treatment
with the different compounds (Figure 7). As regards E. coli ATCC 35218, after exposure to compound
1 (MIC 8 µg/mL), an OD of 0.320 was observed in comparison to 0.641 of the relative untreated biofilm
(p < 0.01), while the treatment with compounds 3 (MIC 64 µg/mL), 4 (MIC 16 µg/mL), and 8 (MIC 128
µg/mL) showed OD values that were quite similar (from 0.541 to 0.622) to that of the control. The
exposure of S. aureus ATCC 43387 biofilms to compound 1 (MIC 8 µg/mL) determined an OD of 0.250
in comparison to 0.573 of the relative untreated biofilm (p < 0.01), while after treatment with
compounds 3 and 4 (MICs 128 µg/mL), OD values of 0.350 and 0.264, respectively (p < 0.05) were
registered. On the contrary, compound 8 (MIC 128 µg/mL) determined an OD value quite similar to
that of the relative control.
For K. pneumoniae 6/4, compound 1 (MIC 8 µg/mL) determined an OD of 0.163 in comparison to
0.365 of the relative untreated biofilm (p < 0.01), while the treatment with compounds 3 (MIC 128
µg/mL), 4 (MIC 32 µg/mL), and 8 (MIC 128 µg/mL) induced 0.252, 0.214 (p > 0.05), and 0.201 (p < 0.01)
OD values, respectively. In the case of C. albicans ATCC 10231, the treatment with compound 1 (MIC
64 µg/mL) reduced the biomass to OD 0.191 in comparison to 0.290 of the control one (p < 0.01);
similarly, after exposure to compounds 3 (MIC 128 µg/mL), 4 (MIC 32 µg/mL), and 8 (MIC 128
µg/mL), OD values of 0.148 (p < 0.01), 0.178 (p < 0.05) and 0.168 (p < 0.01), respectively, were registered.
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bbiioofifillmmss ooff EE.. ccoollii AATTCCCC 3355221188,, SS.. aauurreeuuss AATTCCCC 4433338877,, KK.. ppnneeuummoonniiaaee 66//44 aanndd CC.. aallbbiiccaannss AATTCCCC 1100223311,,
aass aasssseesssseedd bbyy ssppeeccttrroopphhoottoommeetteerr rreeaaddeerr ((OODD5 57700nnmm)) aafftteerr 3300 mmiinn ooff ccoonnttaacctt.. DDaattaa rreepprreesseenntt tthhee mmeeaann
±± SSDD oobbttaaiinneedd iinn tthhrreeee iinnddeeppeennddeenntt eexxppeerriimmeennttss ppeerrffoorrmmeedd iinn dduupplliiccaattee.. AAsstteerriisskkss rreepprreesseenntt vvaalluueess
ssttaattiissttiiccaallllyy ssiiggnniifificcaanntt ((** pp << 00..0055;; **** pp << 00..0011)) aass ccoommppaarreedd ttoo tthhee rreellaatteedd ccoonnttrroollss..
FIno rTKab.lpen 3eu amreo nsiuame6m/a4r,iczoemd pthoeu npder1ce(nMtaIgCe8s µofg /bimomL)adsse treerdmuicntieodna, ninOdeDx ooff 0e.r1a6d3icinatcioonm apcatrivisiotyn,
tiond0u.3c6e5d obfy tehaechre cloamtivpeouunndtr eaagtaeidnsbt itohfiel mtes(tped< d0e.v0e1l)o,pwehdi lbeiotfhielmtrse. aAtms sehnotwwni,t haftceorm 30p omuinnd osf 3co(nMtaIcCt
with compound 1, reductions greater than 50% were observed for most of the examined strains, with
the exception of C. albicans ATCC 10231 (only 34.2%). On the contrary, all the derivatives (3, 4, and 8)
showed low activity against E. coli ATCC 35218 biofilms, in some cases, also the derivatives reached
remarkable percentages of biomass reduction. Interestingly, compound 3 reduced biofilm of C.
albicans ATCC 10231 (48.8%), resulting the most active compound against mycete biofilms.
Compound 4 showed strong anti-biofilm activity against S. aureus ATCC 43387 with percentage
comparable to compound 1 (53.9% vs 56.3%), while compound 8 acted as good disaggregating agent
on K. pneumoniae 6/4 (44.9%) and C. albicans ATCC 10231 (41.9%) biofilms.
Table 3 Percentages of biofilm eradication activity assessed for E. coli ATCC 35218, S. aureus ATCC
43387, K. pneumoniae 6/4 and C. albicans ATCC 10231 after 30 min of contact with compounds 1, 3, 4,
and 8 at their relative MIC (µg/mL) values. Percentages were determined after spectrophotometer
reader (OD 570nm).
Preformed Biofilms 1 3 4 8
E. coli ATCC 35218 50.0% 4.7% 15.7% 3.1%
S. aureus ATCC 43387 56.3% 38.9% 53.9% 14.2%
K. pneumoniae 6/4 55.3% 30.9% 41.3% 44.9%
C. albicans ATCC 10231 34.2% 48.8% 38.6% 41.9%
4. Discussion
Currently, the research is being conducted to discover novel compounds that are able to inhibit
microbial biofilms, without allowing bacteria to develop drug resistance. Marine invertebrates have
been proved to be a rich source of bioactive compounds with different ecological functions [6,25].
Among these, sponges are marine sessile filters exposed to large amounts of bacteria in the
surrounding seawater and their potential antimicrobial activity attracted the attention of many
researchers [10,26,27].