Bromopyrrole Alkaloids with the InhibitoryEffects against the Biofilm Formation of Gram Negative ...

15 Pages·2017·2.15 MB·English

marine drugs
Article
Bromopyrrole Alkaloids with the Inhibitory
Effects against the Biofilm Formation of Gram
Negative Bacteria
JingyuanSun1,JiruWu1,BangAn1,NicoleJ.deVoogd2,WeiCheng1,*andWenhanLin1,* ID
1 StateKeyLaboratoryofNaturalandBiomimeticDrugs,PekingUniversity,Beijing100191,China;
[email protected](J.S.);[email protected](J.W.);[email protected](B.A.)
2 TheNetherlandsCentreforBiodiversityNaturalis,P.O.Box9517,2300RALeiden,TheNetherlands;
[email protected]
* Correspondence:[email protected](W.C.);[email protected](W.L);Tel.:[phone](W.L.)
Received:27September2017;Accepted:11December2017;Published:2January2018
Abstract: Anti-biofilmassayguidedfractionationofthemarinespongeStylissamassarevealedthe
butanolsolublefractionthatwaspossessingtheinhibitoryactivitytowardthebiofilmformationof
bacteriumE.coli. Chromatographicseparationofthebioactivefractionresultedintheisolationof
32bromopyrrolealkaloids,includingsixnewalkaloids,namedstylisinesA–F(1–6). Thestructures
ofnewalkaloidswereestablishedbycomprehensiveanalysesofthetwo-dimensional(2D)NMR
(COSY, HMQC, and HMBC) and the high resolution electrospray ionization mass spectroscopy
(HRESIMS)data,whiletheabsoluteconfigurationsweredeterminedbytheX-raydiffractionandthe
electroniccirculardichroism(ECD)data. Bioassayresultsindicatedthatphakellin-basedalkaloids,
includingdibromoisophakellinanddibromophakellin,significantlyreducedthebiofilmformation
ofthebacteriumE.coli. Presentworkprovidedagroupofnewnaturalscaffoldsfortheinhibitory
effectsagainstthebiofilmformationofE.coli.
Keywords: marinesponge;Stylissamassa;antimicrobialactivity;antibiofilm;structureelucidation
1. Introduction
Planktonicbacteriaattachedontosolidsurfaces,creatingacomplexcommunityofbacteriato
formabiofilm, whichprotectsbacteriaagainstantibioticsandcausesmanyrecalcitrantinfections,
aswellasresistancetoantibiotics[1–4]. Biofilmsaccountfor>80%ofhumanbacterialinfections,anda
biofilmmatrixresistsantibioticsatconcentrationsover1000timeshigherthanconventionallyused
doses [5–7]. In nature, most bacteria are likely to form surface-attached biofilm communities as a
survivalstrategy. Pathogenicbiofilmsposeachallengebecausetheyenhanceresistancenotonlyto
conventionalantibiotics, butalsotohostdefensesandexternalstresses. Thus,theyaredifficultto
controlinmedicalandindustrialsettings. Sofar,nospecificbiofilminhibitoriscommerciallyavailable,
while bacterial biofilms remain a frontier of microbiology as they exhibit important tolerance and
resistanceinmanyaspects. Currently,anexplosiveamountofbiofilmresearchisbeingconducted
to discover novel compounds that are capable of inhibiting biofilms, without allowing bacteria to
developdrugresistance. Marinebenthicinvertebrateshavebeenprovedarichsourceofbioactive
compoundswithmultipleecologicalfunctions. Amongthesessileorganisms,spongesarethemarine
sessilefilterfeedersthatareexposedtolargeamountsofbacteriaandalsovirusesinthesurrounding
seawater. Becausebacteriaplayakeyroleinmicrofilmformation[8],itbecomesvitalforspongesto
regulatebacterialepibiosisontheirsurfaces. Oneepibacterialdefensestrategyforsponge-symbiont
communityistoproducebioactivesecondarymetabolitesinordertooutcompeteotherbacteriain
the water column from causing infection or biofouling [9–14]. Among the sponge-derived active
Mar.Drugs 2018,16,9;doi:10.3390/md16010009 www.mdpi.com/journal/marinedrugs
Mar. Drugs 2017, 15, 9 2 of 15
Mar.Drugs 2018,16,9 2of15
other bacteria in the water column from causing infection or biofouling [9–14]. Among the sponge-
derived active metabolites, pyrrole-imidazole alkaloids are a class of structurally unique natural
pmreotdaubcotlsit ews,itphy rar orlaen-igmei doafz oelceolaolgkiaclaoli dasnadr esaigcnliafsicsaonft sptrhuacrtmuraaclolylougniciaqlu ebinoaatcutirvailtiperso d[1u5c–t1s7w]. itFhoar
irnasntgaencoef, eocrooliodginic aanldan sdtesviegnnsiifincea wntepreh daremmaocnosltorgaitceadl tboi opaocstsievsisti easnt[i1f5e–ed17a]n.tF porroipnesrttaiensc,e w,ohriolei doirnoiadnidn
astnedv eintssi naenwaloergeudees mhoanvset rbateeedn toexptoesnsseisvselayn tsifteueddiaendt apsr obpieorftiilems, winhhiilbeiotororsid [i1n8a–n2d1]i.t sInan oaulorg ureessehaarcvhe
pberoengreaxmte nfosriv tehlye sdtiusdcoievderays boifo bfiilomacitnivheib intaotrusr[a1l8 –p2r1o]d.uInctso uthraret saeraer cdheprirvoegdr afmromfo rmthaeridnies csopvoenrygeosf,
HbiPoaLcCti-vDeAnDat-uMraSl dparotad uanctds tbhioaatsasraeyd geruiivdeeddf reoxmamminaartinioens pofo nthgee ss,pHonPgLeC -eDxtAraDc-tMs rSevdeaataleadn tdhabti otahses any-
BguuiOdHed eexxtraamctisn aotfi othne osfptohnegsep Sotnygliesseax mtraascstas croenvteaailne da tphraotfitlhee onf -bBruoOmHinaetxetdra acltksaolofitdhse, sapnodn pgoesSsetyslsi sasna
imnahsisbaitcoornyt eafifnecat pargoafiinlesto tfhber boimofiinlmat efdoramlkaatiloonid osf, aEn. dcopli.o Csshersosmanationghriabpithoicry seepffaercattaiogna ionfs tthteh ealbkiaolfioildm-
cfoornmtaaintiionng oefxEt.racocltis. Crehsruolmteadt oignr atphhe icissoelpaatiroanti oonf o3f2t hberaolmkaolpoyidrr-coolen taaliknainlogidesx,t riancctlsurdeisnugl tesdixi nntehwe
cisoomlaptioounnodfs,3 n2abmroemlyo sptyylrirsoinleesa lAka–lFo i(d1–s,6)in (cFliugduirneg 1s).i xTnhee wanctoibmapctoeurinadl sa,nnda amnetil-ybsiotyfillimsin eefsfeAct–sF o(f1 t–h6e)
(isFoiglautreed1 a)l.kTahloeidans twibearcet eerviaalluaantdeda.n ti-biofilmeffectsoftheisolatedalkaloidswereevaluated.

FFiigguurree 11.. SSttrruuccttuurreess ooff ssttyylliissiinneess AA––FF ((11––66))..
2. Results
2. Results
The sponge tissue was extracted with 95% alcohol, and the crude extract was partitioned
Thespongetissuewasextractedwith95%alcohol,andthecrudeextractwaspartitionedbetween
b40e%tweMeneO 4H0%-H MOeaOnHd-HC2HO Calndt oCrHem2Colv2 etoli priedms.ovTeh elipwidaste. rTshoelu wblaeteerx trsaoclutbwlea setxhtreanctp awrtaisti otnheedn
2 2 2
pbeatrwtiteieonneHd Obeatwndeenn- BHu2OOH an.Tdh ne-B1HuONHM. TRhsep 1eHct rNuMmRin spasescotrcuiamti oinn awssitohcitahteioLnC w-EiSthIM thSe dLaCta-EoSfInM-BSu dOatHa
2
of n-BuOH extract characterized a profile of brominated alkaloids. Chromatographic separation,
extractcharacterizedaprofileofbrominatedalkaloids. Chromatographicseparation,includingthe
including the semi-preparative HPLC purification, conduced the isolation of 32 bromopyrrole
semi-preparativeHPLCpurification,conducedtheisolationof32bromopyrrolederivatives,including
derivatives, including six new alkaloids (Figure 1).
sixnewalkaloids(Figure1).
Stylisine A (1) was obtained as a light yellow amorphous solid. Its molecular formula was
Stylisine A (1) was obtained as a light yellow amorphous solid. Its molecular formula was
ddeetteerrmmiinneedd aass CC11HH13NN5OOBBr roonn ththee bbaassisis ooff tthhee hhiigghh rreessoolluuttiioonn eelleeccttrroosspprraayy iioonniizzaattiioonn mmaassss
11 13 5
spectroscopy (HRESIMS) and NMR data. The pseudomolecular ion peaks at m/z 310 and 312, with a
spectroscopy(HRESIMS)andNMRdata. Thepseudomolecularionpeaksatm/z310and312,witha
rraattiioo ooff 11::11 iinn tthhee EESSIIMMSS ssppeeccttrruumm aallssoo ssuuppppoorrtteedd aa bbrroommiinnee aattoomm iinn tthhee mmoolleeccuullee.. TThhee 1133CC NNMMRR aanndd
distortionless enhancement by polarization transfer (DEPT) spectra exhibited 11 carbon resonances,
distortionlessenhancementbypolarizationtransfer(DEPT)spectraexhibited11carbonresonances,
which were classified into three methylenes, one methine, and seven quaternary carbons. The COSY
whichwereclassifiedintothreemethylenes,onemethine,andsevenquaternarycarbons. TheCOSY
in association with the HMBC correlations established a tetrahydropyrroloindolizinone nucleus, as
in association with the HMBC correlations established a tetrahydropyrroloindolizinone nucleus,
eavsiedveindte nfrtofmro mtheth CeOCSOYS Yrerlealtaiotinosnhsihpisp sfofro raann aalklkyyl lssppinin ssyysstteemm ffrroomm HH2--66 ((δδH 22..8877,, 22..9999)) ttoo HH2--88 ((δδH
2 H 2 H
33..9988,, 44..1100)),, iinn aaddddiittiioonn ttoo tthhee HHMMBBCC ccoorrrreellaattiioonnss ffrroomm tthhee ppyyrrrroollee pprroottoonnH H--11( (δδH 77..4466,, bbrrss)) ttoo CC--33 ((δδC
H C
112233..11)) aanndd CC--1100 ((δδC 112266..44)),, NNHH ((δδH 1122..5544,, bbrrss,, ppyyrrrroollee)) ttoo CC--22 ((δδC 8866..88)),, CC--33,, aanndd CC--99 ((δδC 115522..66)),, aanndd
C H C C
bbeettwweeeenn HH2--88//CC-9- 9anadn dHH2-6-/C6/-4C (-δ4C( 1δ021.80)2. .T8h).e Trehmeareinminagin minogiemtyo iientvyoilnvevdo lav eqduaateqrunaatreyr ncaarrbyocna rabt oδnC
2 2 C
157.5 (C-11) and three nitrogen atoms, which was characteristic of a guanidine unit that was
at δ 157.5(C-11)andthreenitrogenatoms,whichwascharacteristicofaguanidineunitthatwas
C
ssuubbssttiittuutteedd aatt CC--44 dduuee ttoo tthhee HHMMBBCC ccoorrrreellaattiioonnss ffrroomm aa NNHH ((99..1166,, ss)) ttoo CC--33,, CC--44,, CC--55 ((δδC 114422..22)),, aanndd
C
CC--1111 ((FFiigguurree 22)).. IInn aaddddiittiioonn,, tthhee ssiiggnniifificcaannttllyy sshhiieellddeedd ccaarrbboonn CC--22 ((δδC 8866..88)) wwaass aattttrriibbuutteedd ttoo aa bbrroommiinnee
C
atom substitution.
atomsubstitution.
Mar.Drugs 2018,16,9 3of15
Mar. Drugs 2017, 15, 9 3 of 15
Mar. Drugs 2017, 15, 9 3 of 15

FFiigguurree 22.. KKeeyy CCOOSSYY aanndd HHMMBBCC ccoorrrreellaattiioonnss ooff 11,, 22,, 44 aanndd 66..
Stylisine B (2) was obtained as a yellow amorphous solid. Its ESIMS data presented the ion peaks
Stylisine B (2) was obtained as a yellow amorphous solid. Its ESIMS data presented the ion
at m/z 401.9, 403.9, and 405.9 [M + H]+ with the ratio of 1:2:1, indicating two bromine atoms in the
peaksatm/z401.9,403.9,and405.9[M+H]+withtheratioof1:2:1,indicatingtwobromineatomsin
mthoelmecoulleec, uwleh,iwleh itlheet hHeRHERSEIMSISM aSnadn dNNMMR Rddataat apprroovvidideedd aa mmoolleeccuullaarr ffoorrmmuullaao offC C1H1 H10NN5OO2BBrr2..
11 10 5 2 2
Analyses of the two-dimensional (2D) NMR data revealed that compound 2 consists of two units of
Analysesofthetwo-dimensiFoignuarel 2(.2 KDey) CNOMSYR andd aHtMaBrCe vcoerarelleatdiontsh oaft 1,c 2o, m4 apndo 6u. nd2consistsoftwounitsof
substructures. The first unit was determined to be a pyrrole-2-carboxamide, based on the HMBC
substructures. The first unit was determined to be a pyrrole-2-carboxamide, based on the HMBC
correlations frSotmyli sHin-e3 B ( (δ2H) w 6a.9s 5ob, tsa)i nteod C as- 1a y(eδlClo 1w0 a5m.2o)r pahnodu sa s oclaidrb. Iotsn EySlI McaSr dbaotan p Cre-s5e n(tδeCd 1th5e9 i.o5n) ,p aeankds from NH
correlationsfromH-3(δ 6.95, s)toC-1(δ 105.2)andacarbonylcarbonC-5(δ 159.5), andfrom
(δH 12.68, ast, mp/zy r4r0o1.l9e, )4 0t3o.9 H,C a-n2d (4δ0C5 .99 8[M.4 )+, HC]-+ 3w iC(tδh Ct h1e1 r3a.t5io) ,o fa 1n:2d:1 ,C in-5d.i cTathineg stwecoo bnrodm uinnei at toCwmass i na tshsei gned to a
NH(δH 12m.6o8le,cus,lep, ywrhriolel et)het oHCRE-2SI(MδSC a9n8d. 4N),MCR- 3da(tδaC p1ro1v3i.d5e)d, aan mdoCle-c5u.laTr hfoermseuclao nofd Cu11nHi1t0Nw5Oa2sBra2.s signedto
faufruoriomimidiadAzaonzlaeoly lnseeusn coulfe ctuhlees u,t wsa,oc-cadocicmrdoerinndsgiion ntgaol t(ao2dDad) diNtdiMoitRnio adlna tHaal MrHevBMeaClBe cdCo thrcraoet rlcaroetmiloapntoiuso nnfdrs o2fm croo nHmsi-s7tHs (o-δ7f Htw( δ4o. 3u7n4,i. t3ms7 o),f mto )Cto-9C (δ-9C
H
128.6) ands uCbs-t1ru0c t(uδrCe s1. 5T3h.e5 f)i,r sat nudni tH w-a8s d(δeHte r5m.7in6e,d st)o tboe aC p-7y rr(oδlCe -27-4ca.0rb) oaxanmdi dCe,- 1b0as.e dA onn athme iHneM BgCro up to be
(δ 128.6) and C-10 (δ 153.5), and H-8 (δ 5.76, s) to C-7 (δ 74.0) and C-10. An amine group to
C C H C
positionedc oartr eCla-t1io1n s( δfrCo m15 H9.-53 )( δwH 6a.s9 5a, ss)c troi bCe-1d ( δbCy 1 0th5.e2) HanMd aB cCar cboonryrel claartbioonn Cb-e5 t(wδCe 1e5n9. 5N), Han2d (fδroHm 9 .N2H3, s) and C-
be positioned at C-11 (δ 159.5) was ascribed by the HMBC correlation between NH (δ 9.23, s)
11. The con(δnH e1c2t.i6o8n, s o, fp ytwrrooCl eu) ntoit sC -a2c (rδoCs 9s8 a.4 m), Ce-t3h y(δlCe n11e3 g.5r),o aunpd tCo- 5f.o Trmhe asenc oanmd iudnei tw waass calsasrigifnieedd t 2bo ya tHhe COSY
and C-11.fuTrohiemicdoanzonlee cntuicolenuso, factcworodinugn tiot sadadcirtioosnsal aHmMBeCth cyolrerenlaetiognrso furopmt oH-f7o (rδmH 4.a37n, mam) toid Ce-9w (δaCs clarified
rbeylatthioenCsOhi1Sp2Y8s. 6fr)r eoalnamdti oCHn-12s-06h ( iδ(pδC sH1 5f33r.o.45m)3, ,a Hmnd2) -Ht6o-8 ( aδ(nHδH a35m..476i3d, ,sem) Nt)o HtCo- 7(aδ n(Hδ Ca8 7m.14.5i0d,) eta,n NJd H=C -61(.0δ0.H HA8nz. 1)a m5a,nintd,e JgHr=o-7u6p,. 0 atolHo bnzeg) awnidthH th-7e,
HaloMnBgCw citohprrotehsiletaiotHinoeMnd sBa tCf Cro-c1mo1r ( rδCeC l-1a55t 9it.o5on) wtshafers oaamsmcriCibde-ed5 btpoyr otthhteoe Hna MmaBniCdd e cHoprr2re-o6lat.to iTonnh abene tsdwigeHenni f-Ni6c.HaT2n (htδleHy 9s .si2gh3n,i esi)fil dacneaddn C tql-yusahteiernldaerdy
2
carbons C-111 .a Tnhde c Con-n2e wctieorne o ifn twdoic uantiitvs eac orof stsh ae m seuthbyslteinteu gtiroounp o tfo bforromm anin aem aidtoe mwass aclta Crif-i1ed a bnyd th Ce -C2O, SrYes pectively
quaternarycarbonsC-1andC-2wereindicativeofthesubstitutionofbromineatomsatC-1andC-2,
[22]. The raeblastioolnustheip sc ofrnomfig Hu2r-6a t(δioH n3. 4a3t, mC) -t7o awn aams iddee NteHrm (δiHn 8e.d15 , ot,n J =t h6.e0 Hbza)s ains do Hf -7t,h aelo ncgo mwiptha trhies on of the
respectiveHlyM[B2C2 ]c.oTrrheleataiobnsso flruomte Cc-o5 ntofi gthue raamtiioden partotCon-7 anwda Hs2d-6e. tTehrem siignneifdicaonntlyt hsehiebladsedis qoufattehrneacryo mparison
experimental and calculated electronic circular dichroism (ECD) data. The experimental ECD
oftheexpecarribmonesn Cta-1l aanndd C-c2a wlceurel ainteddicaetilveec otrf othnei scucbisrtcituutliaorn dofi cbhrormoiinsem at(oEmCs aDt )Cd-1a atnad. CT-h2,e reesxppecetirviemlye ntalECD
spectrum [o2f2 ]2. Tehxeh iabbistoeludt et hcoen pfiogusriatitvioen Cato Ctt-o7n w eafsf edcettse r(mCinEe)d a ot n2 4th0e abnadsis 2 o8f0 t hnem c,o manpdar insoeng aotf ivthee CE at 230
spectrum of 2 exhibited the positive Cotton effects (CE) at 240 and 280 nm, and negative CE at
nm, whiche xwpeerrime einnt aal cacnodr dcaalncuclea twedi tehle tchtreo nciac lcciurclualtaerd d EicChrDoi sdma t(aE CfDor) tdhaeta .m Tohde eelx cpoermimpeonutanl dE CoDf 2 with 7S
230nm,whichwereinaccordancewiththecalculatedECDdataforthemodelcompoundof2with7S
configuratsipoenct,r uums ionfg 2 etxhhei bittiemd eth-ed peopseitnivdee Cnott todne enfsfeictyts (fCuEn) catti 2o4n0a aln dth 2e8o0 rnym ,( aTnDd DneFgTat)i vme CeEth aot d23 0( Figure 3)
configuratniomn, ,wuhsiicnhg wtehree itni maceco-drdeapnecen wdeitnh tthdee cnaslciutylatfeudn EcCtiDo ndaaltat hfoero trhye m(ToDdeDl cFoTm)pmouentdh oofd 2( wFiigthu 7rSe 3)[23,24].
[23,24]. Thus, C-7 of 2 was in agreement with S configuration.
Thus,C-7coofn2figwuraastioinn, augsrinege mtheen ttimwei-tdhepSencdoennfit gduenrsaittyio fnu.nctional theory (TDDFT) method (Figure 3)
[23,24]. Thus, C-7 of 2 was in agreement with S configuration.

Figure3.Experimentalandcalculatedelectroniccirculardichroism(ECD)datafor2and3inMeOH.
Figure 3. Experimental and calculated electronic circular dichroism (ECD) data for 2 and 3 in MeOH.

Figure 3. Experimental and calculated electronic circular dichroism (ECD) data for 2 and 3 in MeOH.
Mar.Drugs 2018,16,9 4of15
TheplanerstructureofstylisineC(3)wasthesameasthatof2,asdeterminedbytheduplicated
NMR(Table1)andMSdata. However, thespecificrotationof2([α]25 +30.8, MeOH)showedthe
D
oppositephasetothatof3([α]25−31.2,MeOH),suggesting3tobeastereoisomerof2.Thisassumption
D
wasconfirmedbytheoppositeCottoneffectsof3incomparisonwiththoseof2intheECDspectra
(Figure3). ThecomparableCottoneffectsoftheexperimentalECDof3andthecalculatedECDforthe
7Risomerof2,indicatedtheabsoluteconfigurationof3tobe7R.
Table1.1Hand13CNMRdataof1–6indimethylsulfoxide(DMSO-d ).
6
1 2/3 4/5 6
7.46,d 127.38, 121.52,
1 105.20,C 105.67,C 6.98,dd(2.4,2.8)
(2.8) CH CH
109.32,
2 86.79,C 98.36,C 99.24,C 6.52,dd(2.0,2.4)
CH
113.86,
3 123.14,C 6.95,s 113.46,CH 6.84,s 119.98,C
CH
4 102.79,C 128.36,C 125.81,C 126.22,C
5 142.18,C 159.51,C 157.56,C 163.29,C
2.99,m; 28.42, 42.36, 3.77,dd(2.0,13.20) 42.06, 37.60,
6 3.43,m 3.48,dd(4.8,7.2)
2.87,m CH2 CH2 3.34,dd(5.2,13.2) CH2 CH2
21.56, 74.01, 50.59, 132.54,
7 2.15,m 4.37,m 4.64,m 6.84,t(7.2)
CH2 CH CH CH
4.10,m; 48.27, 115.41, 2.63,dd(11.2,12.8) 36.16,
8 5.76,brs 131.59,C
3.98,m CH2 CH 2.26,dd(1.5,12.8) CH2
9 152.56,C 128.58,C 170.25,C 165.98,C
60.56,
10 126.39,C 153.49,C 4.21,q(7.0)
CH2
14.08,
11 157.51,C 159.50,C 1.26,t(7.0)
CH3
12.54,d
NH 12.68,s 7.87,brd(5.2) 11.85,dd(2.0,2.8)
(2.8)
8.15,t
NH 7.73,t(4.8)
(5.0)
NH
9.16,s
(CN3H4)
NH2 7.48,br 9.23,brs
(CN3H4)
NH
7.24,br
(CN3H4)
7.13,brs;
CONH2 7.54,brs
StylisineD(4)wasobtainedasyellowamorphoussolid. ItsHRESIMSandNMRdataestablished
amolecularformulaofC H N O Br ,containingtwobromineatoms. AcomparisonoftheNMR
9 10 3 2 2
data(Table1)indicatedthat4sharedthepartialstructure(1,2-dibromopyrrole-4-carboxamide)of2.
TheCOSYrelationshipsestablishedanalkylspinsystemfromH -6(δ 3.34,3.77)toH -8(δ 2.26,
2 H 2 H
2.63),whileH -6extendedtheCOSYcorrelationtoanNHproton(δ 7.87,t). TheHMBCcorrelations
2 H
fromacarbonylcarbonC-9(δ 170.3)toH -8,H-7(δ 4.64),andNH (δ 7.13,7.54)allowedforthe
C 2 H 2 H
linkageofaterminalamidetoC-8(δ 36.2). TheconnectionofC-7(δ 50.6)tothenitrogenatomof
C C
thepyrroleringwasdeducedbytheHMBCcorrelationsfromH-7toC-1(δ 105.7)andC-4(δ 125.8).
C C
Inaddition,theHMBCcorrelationsfromNH(δ 7.87)toC-4andthecarbonylcarbonC-5(δ 157.6)
H C
andbetweenH-3(δ 6.84,s)andC-5,resultedintheformationofaδ-lactam. TheexperimentalECD
H
datashowedpositiveCEat228nmandnegativeCEat260nm,whichwereinaccordancewiththe
calculatedECDdataforamodelcompoundof4with7Sconfiguration(Figure4).
Mar.Drugs 2018,16,9 5of15
Mar. Drugs 2017, 15, 9 5 of 15
35
30
25
20
15
10
eg) 5
d
m 0
D ( -5
C
-10 Exp. ECD for 4
-15 Exp. ECD for 5
Calc. ECD for 5
-20
Calc. ECD for 4
-25
F1
-30
-35
200 225 250 275 300 325 350 375 400
Wavelength (nm)
Figure 4. Experimental and calculated ECD data for 4 and 5 in MeOH.
Figure4.ExperimentalandcalculatedECDdatafor4and5inMeOH.
The identical NMR data of stylisine E (5) and 4 (Table 1), in association with the NMR and the
TheidenticalNMRdataofstylisineE(5)and4(Table1),inassociationwiththeNMRandthe
HRESIMS data, assigned the same planar structure of both 5 and 4. The distinction was attributed to
HRESIMSdata,assignedthesameplanarstructureofboth5and4. Thedistinctionwasattributed
the absolute configuration at C-7, which was determined to be 7R, according to the Cotton effects of
totheabsoluteconfigurationatC-7,whichwasdeterminedtobe7R,accordingtotheCottoneffects
5 showing opposite phases to those of 4, and to be similar with those calculated for 7R isomer of 4
of5showingoppositephasestothoseof4,andtobesimilarwiththosecalculatedfor7Risomerof4
(Figure 4).
(Figure4).
Stylisine F (6) was obtained as a yellow amorphous solid. Its molecular formula was established
StylisineF(6)wasobtainedasayellowamorphoussolid. Itsmolecularformulawasestablished
as C11H12N2O3 on the basis of the HRESIMS and NMR data, requiring seven degrees of unsaturation.
asC H N O onthebasisoftheHRESIMSandNMRdata,requiringsevendegreesofunsaturation.
The 1131C N12M2R a3nd DEPT spectra exhibited 11 carbon resonances, of which, eight sp2 carbons and three
The13CNMRandDEPTspectraexhibited11carbonresonances,ofwhich,eightsp2carbonsandthree
sp3 carbons were defined. The COSY correlations from H-1 (δH 6.98) to H-2 (δH 6.52) and NH (δH
sp3 carbonsweredefined. TheCOSYcorrelationsfromH-1(δ 6.98)toH-2(δ 6.52)andNH(δ
11.85), in association with the HMBC correlations from H-1 anHd H-2 to the quaHternary carbons C-H3
11.85),inassociationwiththeHMBCcorrelationsfromH-1andH-2tothequaternarycarbonsC-3(δ
(δC 120.0) and C-4 (δC 126.2), disclosed a 3,4-disubstituted pyrrole ring. The HMBC correlations fromC
120.0)andC-4(δ 126.2),discloseda3,4-disubstitutedpyrrolering. TheHMBCcorrelationsfromNH
NH to the carboCns in the pyrrole ring and a carbonyl carbon C-5 (δC 163.3) ascertained a carbonyl
tothecarbonsinthepyrroleringandacarbonylcarbonC-5(δ 163.3)ascertainedacarbonylgroupat
group at C-4. A spin system coupling H2-6 (δH 3.48, dd) with NCH (δH 7.73, t) and H-7 (δH 6.84, t) in the
C-4. AspinsystemcouplingH -6(δ 3.48,dd)withNH(δ 7.73,t)andH-7(δ 6.84,t)intheCOSY
COSY spectrum, in addition to2 the HHMBC correlations fromH H2-6 to C-5, and thHe olefinic carbons C-
spectrum,inadditiontotheHMBCcorrelationsfromH -6toC-5,andtheolefiniccarbonsC-7(δ
7 (δC 132.5) and C-8 (δC 131.6) and between H-7 and C2-3, established a pyrroloazepine nucleusC.
132.5)andC-8(δ 131.6)andbetweenH-7andC-3,establishedapyrroloazepinenucleus. Additional
Additional HMBCC correlations from H-7 and the ethoxy methylene protons (δH 4.21, t) to the carbonyl
HMBCcorrelationsfromH-7andtheethoxymethyleneprotons(δ 4.21,t)tothecarbonylcarbonC-9
carbon C-9 (δC 166.0) confirmed an ethoxycarbonyl group at C-8.H Compound 6 was suggested to be
(δ 166.0)confirmedanethoxycarbonylgroupatC-8. Compound6wassuggestedtobeanartifact
anC artifact derived during the process of the EtOH extraction, while 8-carboxylic analogue was
derivedduringtheprocessoftheEtOHextraction,while8-carboxylicanaloguewasconsideredtobe
considered to be the natural origin of 6.
thenaturaloriginof6.
In addition, 26 known alkaloids including pyrrole-2-carbamide type derivatives (1,2-
In addition, 26 known alkaloids including pyrrole-2-carbamide type
dibromopyrrole-4-carbamide (7) [25], 2-bromopyrrole-4-carbamide (8), 1,2-dibromopyrrole-4-
derivatives (1,2-dibromopyrrole-4-carbamide (7) [25], 2-bromopyrrole-4-carbamide (8),
ethylate (9) [26], oroidin (10) [27], hymenidin (11) [28], dispacamide 1 (12) [29], keramadine (13) [30],
1,2-dibromopyrrole-4-ethylate(9)[26],oroidin(10)[27],hymenidin(11)[28],dispacamide1(12)[29],
laughine (14) [31], taurodispacamide A (15) [32]); aldizine type analogues (aldizine (16), 1-
keramadine(13)[30],laughine(14)[31],taurodispacamideA(15)[32]);aldizinetypeanalogues(aldizine
bromoaldizine (17) [33], 10Z-hymenialdisine (18), 10E-hymenialdisine (19) [34], hymenin (20) [35],
(16), 1-bromoaldizine (17) [33], 10Z-hymenialdisine (18), 10E-hymenialdisine (19) [34], hymenin
stevensine (21) [36], (10E)-3-bromohymenialdisine (22), (10Z)-3-bromohymenialdisine (23) [37] and
(20) [35], stevensine (21) [36], (10E)-3-bromohymenialdisine (22), (10Z)-3-bromohymenialdisine
(10Z)-debromohymenialdisine (24)); phakellin type analogues (dibromoisophakellin (25),
(23)[37]and(10Z)-debromohymenialdisine(24));phakellintypeanalogues(dibromoisophakellin(25),
dibromophakellin (26) [38], monobromophakellin (27), monobromoisophakellin (28)); mamzacidin
dibromophakellin(26)[38],monobromophakellin(27),monobromoisophakellin(28));mamzacidintype
type analogues (manzacidins B-C (29, 30) [39], N-methylmanzacidin C (31) [40]); in addition to
analogues(manzacidinsB-C(29,30)[39],N-methylmanzacidinC(31)[40]);inadditiontolongamideB
longamide B (32) (Supporting Information, Figure S41) [41], were identified by the comprehensive
(32)(Supporting Information, FigureS41) [41], wereidentified bythe comprehensiveanalyses and
analyses and the comparison of one-dimensional (1D) and 2D NMR, and HRESIMS data, in
thecomparisonofone-dimensional(1D)and2DNMR,andHRESIMSdata,inassociationwiththe
association with the specific rotations. The absolute configurations of N-methylmanzacidin C were
specificrotations. TheabsoluteconfigurationsofN-methylmanzacidinCwereconfirmedbytheX-ray
confirmed by the X-ray diffraction of a single crystal using the Flack parameter for the first time
diffractionofasinglecrystalusingtheFlackparameterforthefirsttime(Figure5).
(Figure 5).
Mar.Drugs 2018,16,9 6of15
Mar. Drugs 2017, 15, 9 6 of 15
Mar. Drugs 2017, 15, 9 6 of 15

Figure 5. Plot of the X-ray diffraction of N-methylmanzacidin C.
Figure5.PlotoftheX-raydiffractionofN-methylmanzacidinC.
Figure 5. Plot of the X-ray diffraction of N-methylmanzacidin C.
Biogenetically, pyrrole-2-carbamide and brominated pyrrole-2-carbamides, such as 7 and 8,
Biogenetically, pyrrole-2-carbamide and brominated pyrrole-2-carbamides, such as 7 and 8,
were postulated to be the precursors to derive the isolated alkaloids [42–44]. Condensation of 8 with
Biogenetically, pyrrole-2-carbamide and brominated pyrrole-2-carbamides, such as 7 and 8,
were postulated to be the precursors to derive the isolated alkaloids [42–44]. Condensation of 8
howmeoraer pgoinstiunlea tyeide ltdoe bde ltahueg phrienceu r(s1o4r)s, two hdieleri vthe et hlea titseorl actoedm aplokualnodid us n[4d2e–r4w4]e.n Ct odnedheyndsarotigoenn oaft i8o wn iathn d
withhomoarginineyieldedlaughine(14),whilethelattercompoundunderwentdehydrogenation
cyhcloizmaotiaorng intion e gyeienledreadte l a1u.g hCinoen (d1e4n),s awtihoinle tohfe l7a ttweri tcho m3p-aomunindo u-ln-(d2e-rawmeinnto dimehidyadzrooglyeln)-aptiroonp -aln-edn e
and cyclization to generate 1. Condensation of 7 with 3-amino-l-(2-aminoimidazolyl)-prop-l-ene
gecnyecrlaizteadti oonr oitdoi ng (e1n0e)r, awteh i1ch. fCoollnodweendsa otixoyng eonfa t7io nw ainthd o3l-eafminiinco m-l-i(g2r-aamtioinno tiom aidffaozrodl ydl)i-sppraocpa-ml-eidnee 1
generatedoroidin(10),whichfollowedoxygenationandolefinicmigrationtoafforddispacamide1
(12g)e. nHereateterdo coyrcoliidzianti (o1n0 )o, fw 1h2i cthh rfooullgohw ethde o fxoyrgmenataitoionn o af nadn oeltehfienri cb rmidiggrea ttioo nd etori vaeff othrde dstiespreaociasmomideer 1s 2
(12). Heterocyclizationof12throughtheformationofanetherbridgetoderivethestereoisomers2
an(d1 23).. CHoemteproocuyncdlizs a4t iaonnd o 5f 1w2e trher cooungshi dtheree fdo rtmo batei ogne noef raante edt hfreor mbr 2id agned t o3 dbeyr irvine gt hree asrtrearenogiesmomenerts a 2n d
and3. Compounds4and5wereconsideredtobegeneratedfrom2and3byringrearrangementand
oxayngde n3a. tCioonm (pSocuhnedmse 4 1 a)n. d 5 were considered to be generated from 2 and 3 by ring rearrangement and
oxoyxgyegneantaiotinon(S (cShcehmemee1 )1.).

Scheme 1. Biogenetic postulation to derive 1–5.
Scheme 1. Biogenetic postulation to derive 1–5.
Scheme1.Biogeneticpostulationtoderive1–5.
Hymenidin, taurodiscapamide A, and oroidin have been reported to display significant growth
Hymenidin, taurodiscapamide A, and oroidin have been reported to display significant growth
inhibHitiyomn eangiadinins,t ttahuer Godraismca ppoasmitiidvee Aba,catnedriao rSotiadpihnylhoacovcecubse eanurreeupso arntedd Btaocidlliussp lsauybtsiilgisn, iafincda nthteg rGorwamth
inhibition against the Gram positive bacteria Staphylococcus aureus and Bacillus subtilis, and the Gram
ninehngeiabgtiiatviteoiv nbe aabcgataecitrneisarti aEt hsEceshcGehreriracihmcihaip ac oocsloiil tiai vanneddb PaPscseteueurddioaommSootnanpaashs yaaleeorrcuuoggciicnnuoosssaaa,u, araessu wswaeenlllld aasBs a tthchielel uf ufsunsngugubutsi slCi sC., .aa lanblidbciatcnhasne s[G2 [2r2]a2. m].
OnerOogiradotiiivnde iwnb awasca atsel asroilas orEe rpsecophroetrretidechd ti oato ic nionhlihibaibintid tt hthPees g egurrodowowmtthohn ooaffs RRahheroouddgoossinppiiorrsiilalll,uuamms sswaalleeexlxliiggaeesnnsts,h ,a ea m fmuanarirgniunes eb Cbaca.tcaetlrebiruicimuanm tsh t[ah2ta2 t] .
iOs rkiosn ikdonwinonww tnao st foao flrsomorm rbe ibpoiofoirfltmielmds st[ o1[91i9]n.] h.1 i1,b2,2i-tD-DtihbiebrrogomrmoowopptyhyrrorrofollReeh--44o--dccoaasrrpbbiraaimmlluiidmdee s pparlreooxmmigoeotnteesd,d al alamrrvavarali lnm meebteatamacmtoerorpiruhpmohsoitssh iaost fo ifs
tkhnetoh wea nsactisodciifdaonira mnC biCiooinofianl am ssasva[i1vg9ing]ny.yi1 i, 2[-2[D255]i.b] .r oIInmn oaapddyddriritotiioloenn-,4, -cooarrroobiiaddmiinni--dbbeaasspeerddo mddoeetreriivdvaaltaitvrivveeass l mininectocaormprpoororaprtahintoigns gis ao af2 t-h2e-
aamsaciimndoiinaimnoimCidiiaodnzaoazlsoeal vem igmontoyiftii fs[ 2es5rev]r.veIdend aa asd sdm mitoioloelnecc,uuolleresos i wdwiinitth-hb aaa s uuendniiqqduuereei vsscacaatiffvffooelslddi nttoco o iinrnphhoibirbiatit tia nangndda d 2dis-iapsmpereisrneso ebi mabcaitdcetareizaroila lel
bmioobfitioilfmfislesm r[vs4 e[54d,45a6,4s]6. m]. oleculeswithauniquescaffoldtoinhibitanddispersebacterialbiofilms[45,46].
TThhTeeh ellii ttleeitrreaarttauutrureer edd adattaaat ass ttsiitmmimuuullaalatteteeddd uuusss tttooo eeexxxaaammmiiinnneee ttthhheee iiisssooolllaaattteeeddd aaalllkkkaaallolooiididdsss iniinndddiviivvidiidduuaulaallylll yyf offroo rrg rggorrwoowwthtt hh
iinnhihniibhbiiitbtiiiootnino anag gaagaiiannisnstts ptp apatahtthohogogegenenniciic cb bbaaacctcetteerriraiiaa, ,i, niinncclcluuludddiinnigng g tthhteeh eGGrGraarmmam ppoopssioittsiivivteei v bbeaacbcttaeercritaiea rS iS.a .a aSuur.eraueuusr sAe uATsTCACCTC 2C 52C95292325,3 S9,2 .S 3.,
hSa.ehhmaaeeommlyootlliyyctutiicscu uAss ATACTTCCCC C2 922999799077,0 0a, ,naandnd dB B.B .s .ususbubtbitlitilisils i AsAATTCTCCCCC 11111515665226,,2 aa,nnanddd tthhtehe eGGGrraarmamm nnenegegagataitvitvieve be babacatcectrteeirari aiEa E. Ec. .oclcoio lAlii ATACTTCCC CC
22552995229222,, 2XX, aaXnnattnhhtoohmmomaannaeensse vvs eevsseiiscciaacttaootrroiiraai aAA ATTTCCCCCC 11 1111666333333,,, P PP..l. allacachchhrryryymmmaaannnsssA AATTTCCCCCC1 1111919292121,1,A ,A Aggrgrorobobabatateetrreiiruuimumm ttu utmummeeffeaafccaiiceeninessn NNs Noo..o 8.
8A AT8C TACCTC1C1 C111 511815,18R5, 8aR,l saRtloasntlsoitnaoinsaoi asla osnloaalnacneaacarecuaearmurumAm TA ACTTCCC1CC1 161191666.996U6.. n UUennxepexxeppcteeeccdtteelydd,llymy,, o mmsotoscstot c mcoompmopuponoudunsnddssh ss ohswhowoewdedew dwe waekaekao korr no or
naocnt iaovc iattiycvtiiatvgyiat yaing asagtianthisnets ttph taehn epe lpaonafnelbe lao cof tfbe braiacactetwerriiaitah wwMiitIthCh MM≥IIC1C2 ≥8≥ 1µ122g88/ mμμggL//mm(SLLu p((SSpuuopprptpionorgrttiIninngfg oI nrInmfofaormtrimoantai,toiTnoa,nb T,l eaTbaSlb1e)l e,S w1S)1i,t )h,
wthiwethei txthhc etehp eetx ieocxencpeotpifotoinor onoi fod ofi nroortiohdiaditnins th htohawat tse shdhooMwweIeCdd v MMaIlICuCe vvsaarllauuneegss i rrnaagnnfggriionnmgg ff3rro2o–mm1 2 33822–µ–11g22/88 mμ μgLg/.m/mLL. .
However, the initial bioassay indicated that the n-BuOH extract (an extract of total alkaloids)
However, the initial bioassay indicated that the n-BuOH extract (an extract of total alkaloids)
exerted inhibitory effects against the biofilm formation of E. coli. The bioassay guided fractionation
exerted inhibitory effects against the biofilm formation of E. coli. The bioassay guided fractionation
of n-BuOH extract revealed three fractions (F2 to F4) to be active (Table 2), while the most active
of n-BuOH extract revealed three fractions (F2 to F4) to be active (Table 2), while the most active
Mar.Drugs 2018,16,9 7of15
However, the initial bioassay indicated that the n-BuOH extract (an extract of total alkaloids)
exertedinhibitoryeffectsagainstthebiofilmformationofE.coli. Thebioassayguidedfractionation
of n-BuOH extract revealed three fractions (F2 to F4) to be active (Table 2), while the most active
fraction was attributed to F2, in which dibromoisophakellin and dibromophakellin were isolated
asthemaincomponents. Bothofthecompoundsexertedsignificantinhibitionagainstthebiofilm
formation of E. coli with IC value of 50.9 and 31.3 µg/mL, respectively. In order to understand
50
thebioactivitiesoftherelativeanalogues,weextendedtheanti-biofilmassayofremainingalkaloids.
However, those alkaloid derivatives showed weak or no inhibition toward the biofilm formation
(Table 3). Preliminary analyses of the structure-activity relationship indicated that phakellin-type
scaffoldwithanaminoimidazoleringisessentialtoinducetheinhibitoryactivity,whileanalogues
withdibromosubstitutionshowedmoreactivethanthosewithmonobromosubstitution. Inparallel,
confocal laser scanning microscopy (CLSM) was used to detect the ability of dibromophakellin
and dibromoisophakellin to interfere with the biofilm formation of E. coli. In this experiment,
thebiofilm-embeddedcellswereLIVE/DEADstained. Theviablebacteriawerestainedwithintact
membranesfluorescinggreen(SYTO9)todistinguishthedeadbacteriathatwerestainedwithdamaged
membrane fluorescing red (propidium iodine) [47,48]. As shown in Figure 6, the number of the
viablebacteriaofE.coliinthebiofilmandthetotalbiofilmthicknessweregreatlyreducedafterthe
treatmentwithdibromoisophakellinanddibromophakellinindividuallyatadoseof100µg/mLin
comparison with the compact and condensed biofilm control. These images provided additional
evidencetosupporttheinhibitoryeffectsofbothcompoundsagainstthebiofilmformation. Inparallel
experiment,theuntreatedbiofilmsofE.coliinthevehiclegroup(DMSO)showedaninhomogeneous
distribution with visible cell clusters and clearly rod shaped, whereas the E. coli cells treated by
dibromoisophakellinanddibromophakellinchangedtheircellmorphologytoamoresphericalshape,
andtendedtobeaggregated.
Table2.InhibitoryeffectsoffractionsagainstE.colibiofilmformation.
Sample E.coliBiofilmFormationInhibitionRate(%)a
Positivecontrolb 98.4
n-BuOHfraction 7.7
F1 na
F2 84.4
F3 26.4
F4 18.2
F5 na
aMeasuredinadoseof200µg/mL.bPositivecontrol:chloramphenicol.Measuredinadoseof100µg/mL.
Table3.InhibitoryeffectsofalkaloidsagainstE.colibiofilmformation.
Sample E.coliBiofilmFormationInhibitionRate(%)a IC (µg/mL)
50
Positivecontrol 98.4±0.8
12 10.4±1.7 >100
25 72.1±2.8 50.9
26 76.5±1.1 31.3
27 25.9±2.4 >100
aMeasuredinadoseof100µg/mL.Positivecontrol:chloramphenicol.
Mar. Drugs 2017, 15, 9 7 of 15
fraction was attributed to F2, in which dibromoisophakellin and dibromophakellin were isolated as
the main components. Both of the compounds exerted significant inhibition against the biofilm
formation of E. coli with IC50 value of 50.9 and 31.3 μg/mL, respectively. In order to understand the
bioactivities of the relative analogues, we extended the anti-biofilm assay of remaining alkaloids.
However, those alkaloid derivatives showed weak or no inhibition toward the biofilm formation
(Table 3). Preliminary analyses of the structure-activity relationship indicated that phakellin-type
scaffold with an aminoimidazole ring is essential to induce the inhibitory activity, while analogues
with dibromo substitution showed more active than those with monobromo substitution. In parallel,
confocal laser scanning microscopy (CLSM) was used to detect the ability of dibromophakellin and
dibromoisophakellin to interfere with the biofilm formation of E. coli. In this experiment, the biofilm-
embedded cells were LIVE/DEAD stained. The viable bacteria were stained with intact membranes
fluorescing green (SYTO9) to distinguish the dead bacteria that were stained with damaged
membrane fluorescing red (propidium iodine) [47,48]. As shown in Figure 6, the number of the viable
bacteria of E. coli in the biofilm and the total biofilm thickness were greatly reduced after the
treatment with dibromoisophakellin and dibromophakellin individually at a dose of 100 μg/mL in
comparison with the compact and condensed biofilm control. These images provided additional
evidence to support the inhibitory effects of both compounds against the biofilm formation. In
parallel experiment, the untreated biofilms of E. coli in the vehicle group (DMSO) showed an
inhomogeneous distribution with visible cell clusters and clearly rod shaped, whereas the E. coli cells
treated by dibromoisophakellin and dibromophakellin changed their cell morphology to a more
spherical shape, and tended to be aggregated.
Table 2. Inhibitory effects of fractions against E. coli biofilm formation.
Sample E. coliBiofilm Formation Inhibition Rate (%) a
Positive control b 98.4
n-BuOH fraction 7.7
F1 na
F2 84.4
F3 26.4
F4 18.2
F5 na
a Measured in a dose of 200 μg/mL. b Positive control: chloramphenicol. Measured in a dose of 100 μg/mL.
Table 3. Inhibitory effects of alkaloids against E. coli biofilm formation.
Sample E. coli Biofilm Formation Inhibition Rate (%)a IC50 (μg/mL)
Positive control 98.4 ± 0.8
12 10.4 ± 1.7 >100
25 72.1 ± 2.8 50.9
26 76.5 ± 1.1 31.3
27 25.9 ± 2.4 >100
Mar.Drugs 2018,16,9 8of15
a Measured in a dose of 100 μg/mL. Positive control: chloramphenicol.
Total mass Live bacteria Dead bacteria
control
Mar. Drugs 2017, 15, 9 8 of 15

dibromoisop
hakellin
dibromopha
kellin
control
Figure 6. Microscopy images of dibromoisophakellin and dibromophakellin showing inhibition
Figure6.Microscopyimagesofdibromoisophakellinanddibromophakellinshowinginhibitiontoward
toward Escherichia coli biofilms by LIVE/DEAD-stained after 24 h.
EscherichiacolibiofilmsbyLIVE/DEAD-stainedafter24h.
A–C = DMSO control, D–F = treated with dibromoisophakellin (100 μg/mL), G–I = treated with
dibromA–oCph=akDeMlliSnO (1c0o0n tμrgol/,mDL–)F, a=ntdr eJa–tLe d= wtreitahteddib wroitmh ocihsoloprhaamkpelhlienni(c1o0l0 (µ20g /μmg/Lm),LG).– VI=iabtrleea bteadctweriitah
adreib vroismibolpe hinak gerlleienn( 1a0n0dµ dge/amd Lb)a,catnedriaJ– iLn =retrde.a Lteedft wpiatnheclhs l(oAra, mDp, hGe,n Ji)c ofolr( 2i0ntµegg/ramteLd) .rVeida balnedb agcrteeernia
faloruerveissciebnlet cinhagnrneeenls,a mndidddelea dpabnaecltse r(Bia, iEn, Hre,d K. )L feofrt gpraeneenl s(l(iAve, Dba,cGte,rJi)a)f oarndin rteigghrat tpeadnreelsd (aCn, dF,g Ir,e Le)n
ffloro urerdes (cdeneatdch baancnteerlsia,)m. iddlepanels(B,E,H,K)forgreen(livebacteria)andrightpanels(C,F,I,L)for
red(deadbacteria).
3. Discussion
To the best of our knowledge, this study uncovered the largest numbers of brominated alkaloids
with diverse scaffolds that were derived from marine sponge S. massa, implying more complexity of
ecological environments, such as high biodiversity of predators, competitors, and overgrowths, as
well as natural sponge-microbial associations in tropical sea water [49]. In addition to the postulation
of the biogenetic relationships for new compounds in Scheme 1, complex hetercycles of pyrrole-
imidazole alkaloids, such as aldizine-type and phakellin-type alkaloids, were supposed to be
generated biogenetically from oroidin as a key intermediate through different manners of
rearrangement and cyclization [44,50]. Although sponges have large symbiotic bacterial populations,
oroidin and its analogues have been proved to be derived from sponge origin producing in its
spherulous cells [51]. The structure variation is suggested to be an active enzymatic process involving
the induction by symbiotic microorganisms. The prominent alkaloids, including oroidin (10),
hymenidin (11), 10Z or 10E hymenialdisine (18 or 19), and debromohymenialdisine (24) from the
sponge distributed at various biogeographic locations across Indo-Pacific reefs, suggesting that these
typical alkaloids could be employed as chemical marks for taxonomy.
Mar.Drugs 2018,16,9 9of15
3. Discussion
Tothebestofourknowledge,thisstudyuncoveredthelargestnumbersofbrominatedalkaloids
withdiversescaffoldsthatwerederivedfrommarinespongeS.massa,implyingmorecomplexityof
ecologicalenvironments,suchashighbiodiversityofpredators,competitors,andovergrowths,aswell
asnaturalsponge-microbialassociationsintropicalseawater[49]. Inadditiontothepostulationofthe
biogeneticrelationshipsfornewcompoundsinScheme1,complexhetercyclesofpyrrole-imidazole
alkaloids, such as aldizine-type and phakellin-type alkaloids, were supposed to be generated
biogeneticallyfromoroidinasakeyintermediatethroughdifferentmannersofrearrangementand
cyclization [44,50]. Although sponges have large symbiotic bacterial populations, oroidin and its
analogueshavebeenprovedtobederivedfromspongeoriginproducinginitsspherulouscells[51].
The structure variation is suggested to be an active enzymatic process involving the induction by
symbioticmicroorganisms. Theprominentalkaloids,includingoroidin(10),hymenidin(11),10Zor
10Ehymenialdisine(18or19),anddebromohymenialdisine(24)fromthespongedistributedatvarious
biogeographic locations across Indo-Pacific reefs, suggesting that these typical alkaloids could be
employedaschemicalmarksfortaxonomy.
Inthenaturallyoccurringpyrrole-imidazolealkaloids,asderivedfrommarinesponges,very
fewhavebeenshowntomodulatebiofilmformationwithoutkillingthebacteriaordisruptingtheir
growth [8]. Oroidin and bromoageliferin were able to interfere with the bacterial attachment of
RhodospirillumsalexigensandVibriovulnificus[52,53],whereR.salexigensisamarinebacteriumthat
isknowntoformbiofilms. Oroidinexhibitedmodestanti-biofilmactivityagainsttwostrains(PAO1
and PA14) of Pseudomonas aeruginosa, with IC values of 190 and 166 µM, whereas its analogue,
50
dihydrosventrin, showed stronger inhibition against PAO1 and PA14 with IC values of 52 and
50
111 µM, respectively [54]. In our work, oroidin was inactive toward the anti-biofilm formation
of E. coli, but dispacamide 1 (12) showed weak activity, with 10.4% inhibition rate at 100 µg/mL
without microbicidal effect. These findings suggested that different pyrrole-imidazole alkaloids
couldplayseparaterolesinecologicaldefense. Phakellin-basedalkaloidsdibromoisophakellinand
dibromophakellin exerted significant reduction against the biofilm formation of E. coli with weak
inhibitionagainstthegrowthinhibitionofplanktonicbacteria,indicatingtheinhibitoryeffectsvia
non-microbicidalmechanism. Themolecularmechanismoftheactivecompoundstoeliminatebiofilm
formationremainstobeassessed.
4. MaterialsandMethods
4.1. GeneralExperimentalProcedures
OpticalrotationsweremeasuredonaRudolphIVAutopolautomaticpolarimeter. IRspectra
were determined on a Thermo Nicolet Nexus 470 FT-IR spectrometer. 1D and 2D NMR spectra
wererecordedonaBrukerAvance400NMRspectrometer(400MHzfor1Hand100MHzfor13C,
respectively). Chemicalshiftswerereferencedtothesolventpeaksatδ 2.50andδ 39.8fordimethyl
H C
sulfoxide(DMSO-d ),respectively,andcouplingconstantswereinHz. ESIMSandHRESIMSspectra
6
were obtained from a Thermo Scientific LTQ Orbitrap XL instrument. Column chromatography
was carried out with silica gel (160–200 mesh, 200–300 mesh), and HF silica gel for TLC was
254
obtainedfromQingdaoMarineChemistryCo. Ltd. (Qingdao,China),ODSgel(50µm),andSephadex
LH-20 (18–110 µm) were obtained from YMC (Kyoto, Japan) and Amersham Pharmacia Biotech
AB,Uppsala,Sweden. HPLCchromatographywasperformedonanAlltechinstrument(426-HPLC
pump),equippedwithanAlltechUVIS-200detectorandsemipreparativereversed-phasecolumns
(GracePrevailC ,5µm,250mm×10mm). AnalyticalHPLCchromatographywasperformedon
18
ProminenceLC-20AD(Shimadzu,Kyoto,Japan)equippedwithpumpLC-20AD,detectorProminence
SPD-M20A PDA and Shimadzu VP-ODS column (150 mm × 4.6 mm), Thermo BDS Hypersil C
18
column(150mm×4.6mm,5µm).
Mar.Drugs 2018,16,9 10of15
4.2. AnimalMaterial
SpongeStylissamassawascollectedfromtheinnercoralreefatadepthofaround8minHainan
IslandofChina,inMay2013,andthesampleswerefrozenimmediatelyaftercollection. Thespecimen
wasidentifiedbyDr. NicoleJ.deVoogd(NaturalisBiodiversityCenter(Leiden,TheNetherlands)).
Thevoucherspecimens(XSA-24)aredepositedattheStateKeyLaboratoryofNaturalandBiomimetic
Drugs,PekingUniversity,China.
4.3. IsolationandPurification
Sponge tissue (3.5 kg, wet weight) was extracted with 95% EtOH for three times, and the
crude extract was partitioned between MeOH and n-hexane to remove lipids. The MeOH layer
was further partitioned between H O and n-BuOH to afford n-BuOH soluble extract (11.4 g).
2
The DAD-HPLC-ESIMS and the NMR data revealed that n-BuOH fraction contains a profile of
brominated alkaloids. The silica gel chromatography of n-BuOH-fraction (4.9 g) was eluted with
CH Cl -MeOH (a gradient from 6:1 to 2:1) to afford five fractions (F1 to F5). F1 (336 mg) was
2 2
chromatographedonasilicagelcolumnelutingwithpetroleumether(PE)-acetone(from3:1to1:1,
gradient), to yield 7 (6.0 mg), 17 (11.3 mg), 13 (8.6 mg), and 16 (29.3 mg). Part of F2 (140 mg) was
subjected to a silica gel column using CH Cl -MeOH (13:1) as a mobile phase followed by HPLC
2 2
purificationusingamobilephaseofCH CN-H O(28:72,1 CF COOH)toafford25(11.7mg)and
3 2 3
26(117.5mg). F3(795mg)wasseparatedonODScolumn(cid:104)elutingwithMeOH-H O(1:5)toafford
2
sixsubfractions(SF31–SF36). SF31(146mg)waspurifiedbyasemi-preparativeHPLCelutingwith
CH CN-H O (14:86, 3 CF COOH) to afford 1 (21.1 mg) and 27 (30.8 mg). Following the same
3 2 3
separationprotocolby(cid:104)thesemi-preparativeHPLCelutingwithMeOH-H O(3:1),28(1.2mg)was
2
purifiedfromSF32(82.8mg). SF33(248mg)wasseparatedonSephadexLH-20columnelutingwith
MeOHtoafford11(26mg). SF34(28mg)waspurifiedbythesemi-preparativeHPLCelutingwith
CH CN-H O(1:4,1 CF COOH)toafford18(3.3mg),12(7.2mg),and10(99.2mg). F4(515mg)
3 2 3
was separated on O(cid:104)DS column and eluted with a gradient of MeOH-H O (25–50%) to afford five
2
subfractions(SF41–SF45). Followingthesemi-preparativeHPLCseparation,19(6.7mg)wasobtained
fromSF42(177mg)(CH CN-H O12:88,1 CF COOH),15(17.5mg),20(13.5mg),8(3.1mg),and
3 2 3
21 (7.6 mg) were obtained from SF43 (120(cid:104)mg). SF44 (337 mg) was separated by semi-preparative
HPLCelutingwith32%CH CN-H O(1 CF COOH)toyield22(2.9mg),23(17.5mg),and32(3.6
3 2 3
mg). F5(2.9g)wassubsequentlysubject(cid:104)edtoMPLC,andthenpurifiedbysemi-preparativeHPLC
eluting with 36% MeOH-H O to afford 24 (9.9 mg), 31 (2.0 mg), 30 (39.7 mg), 29 (11.9 mg), 9 (39.1
2
mg),15(9.6mg),2/3mixture(5.2mg),4/5(4.1mg)mixture,and6(4.7mg). Theseparationof2/3(a
ratioof1:1)and4/5(aratioof5:4)wasachievedbythechiralHPLCcolumn(DacielCHIRALPAKIC
column)elutingwithn-hexane-isopropanol(78:22)toobtain2(2.4mg),3(2.5mg),4(2.0mg),and5
(1.8mg).
Stylisine A (1): light yellow solid, [α]25 − 9.4 (c 0.14, MeOH), IR (KBr film) ν 3322, 3149, 3079,
D max
2925,1679,1657,1585,1205,1136cm−1,1Hand13CNMR(DMSO-d ),seeTable1,ESIMSm/z310.03
6
[M+H]+andHRTOFMSm/z310.0311[M+H]+(calcd. forC H N OBr,310.0303).
11 13 5
StylisineB(2): lightyellowamorphous,[α]25+30.8(c0.05,MeOH),IR(KBrfilm)ν 3424,1677,1205,
D max
1138cm−1;1Hand13CNMR(DMSO-d ),seeTable1,ESIMSm/z401.92[M+H]+andHRTOFMSm/z
6
401.9199[M+H]+(calcd. forC H N O Br ,401.9201).
11 10 5 2 2
StylisineC(3): lightyellowamorphous,[α]25 −31.2(c0.05,MeOH),IR(KBrfilm)ν 3424,2853,1677,
D max
1439,1206,1138cm−1,1Hand13CNMR(DMSO-d ),seeTable1,ESIMSm/z401.92[M+H]+ and
6
HRTOFMSm/z401.9199[M+H]+(calcd. forC H N O Br ,401.9201).
11 10 5 2 2
StylisineD(4): lightyellowamorphous,[α]25 −4.0(c0.05,MeOH),IR(KBrfilm)ν 3452,1735,1719,
D max
1674,1653,1381,1367,1205,1139cm−1;1Hand13CNMR(DMSO-d ),seeTable1,ESIMSm/z349.91
6
[M+H]+andHRTOFMSm/z349.9146[M+H]+(calcd. forC H N O Br ,349.9140).
9 10 3 2 2