Bioactive bromotyrosine-derived alkaloids from the Polynesian sponge Suberea ianthelliformis

16 Pages·2017·1.21 MB·English

marine drugs
Article
Bioactive Bromotyrosine-Derived Alkaloids from the
Polynesian Sponge Suberea ianthelliformis
AmrEl-Demerdash1,2 ID,CélineMoriou1,JordanToullec3,MarcBesson4,5 ID,
StéphanieSoulet6,NellySchmitt6,SylvainPetek3 ID,DavidLecchini4,CécileDebitus3,*and
AliAl-Mourabit1,*
1 InstitutdeChimiedesSubstancesNaturelles,CNRSUPR2301,UniversityParis-Sud,
UniversityofParis-Saclay,1,AvenuedelaTerrasse,91198Gif-Sur-Yvette,France;
[email protected](A.E.-D.);[email protected](C.M.)
2 OrganicChemistryDivision,ChemistryDepartment,FacultyofScience,MansouraUniversity,
Mansoura35516,Egypt
3 LEMAR,IRD,UBO,CNRS,IFREMER,IUEM,29280Plouzané,France;
[email protected](J.T.);[email protected](S.P.)
4 CRIOBE,CNRS,EPHE,UPVD,PSLResearchUniversity,98729Moorea,FrenchPolynesia;
[email protected](M.B.);[email protected](D.L.)
5 ObservatoireOcéanologiquedeBanyuls-sur-Mer,UniversitéPierreetMarieCurieParis,
66650Banyuls-sur-Mer,France
6 EIO,UPF,ILM,IFREMER,IRD,Faa’a,98702Tahiti,FrenchPolynesia;
[email protected](S.S.);[email protected](N.S.)
* Correspondance:[email protected](C.D.);[email protected](A.A.-M.);
Tel.:[phone](C.D.);[phone](A.A.-M.)
(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:29March2018;Accepted:24April2018;Published:27April2018
Abstract:Herein,wedescribetheisolationandspectroscopicidentificationofeightnewtetrabrominated
tyrosine alkaloids 2–9 from the Polynesian sponge Suberea ianthelliformis, along with known major
compound psammaplysene D (1), N,N-dimethyldibromotyramine, 5-hydroxy xanthenuric acid,
andxanthenuricacid. Cytotoxicityandacetylcholinesteraseinhibitionactivitieswereevaluatedfor
someoftheisolatedmetabolites.TheyexhibitedmoderateantiproliferativeactivityagainstKBcancer
celllines,butpsammaplyseneD(1)displayedsubstantialcytotoxicityaswellasacetylcholinesterase
inhibitionwithIC valuesof0.7µMand1.3µM,respectively.
50
Keywords: brominatedtyrosinealkaloids;Subereaianthelliformis;cytotoxicity;acetylcholinesterase
inhibition
1. Introduction
Sponges of the marine genus Suberea (order: Verongiida) are known to produce a large array
of structurally diverse brominated tyrosine alkaloids, which are considered as chemotaxonomical
markers[1–3]. Nevertheless,bromotyrosine-derivedmetaboliteshavebeenisolatedfromsponges
belongingtodistinctorderssuchasAgelasida[4]andothers. Inaddition,aninterestingrecentstudy
showedthatcultureofthemarinesponge-derivedbacteriumPseudovibriodenitrificansAb134isolated
from the Arenosclera brasiliensis sponge (order Haplosclerida) can produce bromotyrosine-derived
alkaloidsaswell[5]. Althoughmisidentificationsandcontaminationsduetomisplacedspecimens
arepossible,thesereportscompromisethisfamilyofcompoundsasaVerongiidamarkerandsuggest
stayingcautiousaboutsecondarymetabolitesmarkersforspongephylogeny[6]. Thebromotyrosine
derivatives’architecturesvaryfromthesimplemonomericmoleculessuchassubereaphenolK[7],
orthedimericsuberedamineA[8,9],tomorecomplexderivativeslikethewell-knownfistularin-3[10].
Mar.Drugs2018,16,146;doi:10.3390/md16050146 www.mdpi.com/journal/marinedrugs
Mar.Drugs2018,16,146 2of16
Many compounds of this class of bromotyrosine alkaloids display a wide scope of bioactivities
includinMgarc. yDtrougtso 2x01ic8,i 1t6y, xa FgOaRi nPEsEtRm RuEVriIEnWe l eukemiaL1201cellsandKBcells[8,9],DNAdemeth2y olfa 1t6i ng[11],
antiproliferative [12], antimicrobial [13–16], and antiplasmodial activities [17]. In continuation of
wide scope of bioactivities including cytotoxicity against murine leukemia L1201 cells and KB cells
our sea[r8c,h9],f oDrNbAio daecmtievtehymlatainrign e[11n]a, taunrtaiplrpolrifoedrauticvtes [f1r2o]m, anStoimuitchroPbiaacl ifi[1c3–s1p6]o, nagneds a[n1t8ip],laisnmobdoitahl health
andcheamctiivcaitlieesc [o17lo].g Iny cfioenltdinsu,awtioenr oefp oourrt sheaerrceh tfhore biisooalcatitvioe nm,asrtirnue cntauturaralld perotedrumctsin fraotmio nSo,uatnhd Paccyiftioc toxicity
evaluatisopnonogfese i[g1h8]t, nine wbotthe trhaebalrtoh manodt ycrhoesminiceala lekcoalloogiyd sfi(eFldigs,u wree 1re),paolrot nhgerew tihthe ifsooulartikonn,o swtrnucctuormalp ounds
determination, and cytotoxicity evaluation of eight new tetrabromotyrosine alkaloids (Figure 1),
fromthespongeSubereaianthelliformis. TheknownmajorcompoundpsammaplyseneD(1)wasalso
along with four known compounds from the sponge Suberea ianthelliformis. The known major
evaluated for its acetylcholinesterase inhibition activity. Although acetylcholinesterase inhibition
compound psammaplysene D (1) was also evaluated for its acetylcholinesterase inhibition activity.
hasbeenmostlyexploredastreatmentforAlzheimer’spathology, ourattentionhasbeenbrought
Although acetylcholinesterase inhibition has been mostly explored as treatment for Alzheimer’s
to the cphaethmoliocgayl, eocuorl oatgteyntiimonp hoarst abneceen borfousughcth toe nthzey mcheemiinchali beictoolorsgyi nimtphoeritranecne voifr osuncmh eennztyomre against
predatioinnhi[b1i9to]r.sO inn tlhyeoirn eenvpiurobnlmiceantti oonr argealaintsets pfiresdhattiooxni c[1it9y]. oOnnlGy aomneb puusibaliacaffitinonis r(ewlaetesst efirsnh mtoxoicsiqtyu itofish)
and butoynl cGhamolbiunseias ateffrinaiss e(wfersotmerna mLoasqtruuitnocfiushli)a anmda bguntiyfilccahotloinxeisnter[a2s0e] f.roWm ea Ldaetsrucnrcibuleia omuagrnoifbicsae trovxiant ions of
[20]. We describe our observations of the inhibitory effect of psammaplysene D on
theinhibitoryeffectofpsammaplyseneDonacetylcholinesterase,usingbalneationexperimentson
acetylcholinesterase, using balneation experiments on two fish species, Poecilia reticulata (guppy) and
twofishspecies,Poeciliareticulata(guppy)andAcanthurustriostegus(reeffish).
Acanthurus triostegus (reef fish).

Figure 1. Structures of isolated compounds 1–12.
Figure1.Structuresofisolatedcompounds1–12.
2. Results and Discussion
2. ResultsandDiscussion
2.1. Isolation and Structure Elucidation
2.1. IsolationandStructureElucidation
Each crude extract obtained by CH2Cl2 (10.5 g) and n-BuOH, (13 g) of Suberea ianthelliformis was
Eacshubcjercutdede teox tnroarcmtaol bpthaainsee dMbPyLCC HandC ellu(te1d0 .5wigth) agnraddine-nBtsu OofH C,H(12C3l2g/M)oeOfSHu. bTehreea siuabn-tfrhaecltliiofonrsm iswas
2 2
were purified by preparative, semi-preparative, and analytical reversed-phase HPLC and led to the
subjected to normal phase MPLC and eluted with gradients of CH Cl /MeOH. The sub-fractions
isolation of eight new compounds 2–9, along with the known derivat2ives2 psammaplysene D (1),
werepurifiedbypreparative,semi-preparative,andanalyticalreversed-phaseHPLCandledtothe
N,N-dimethyldibromotyramine (10), 5-hydroxy xanthurenic acid (11) and xanthurenic acid acid (12)
isolation(Fiogfureei g1)h. tThnee wknocwonm npaotuurnald psro2d–u9c,tsa plosanmgmwapitlhysethnee Dk n(1o) w[2n1],d Ne,rNiv-daimtivetehsylpdsibarmommoatyprlaymsienne e D (1),
N,N-dim(1e0t) h[y22ld], ib5-rhoymdrootxyyr axmanitnheen(u1r0i)c, 5ac-ihdy d(4r,5o,x8-ytrxihayndtrhouxyrqeuniincoalicnied-2(-1ca1r)baonxidlicx aancitdh) u(r1e1n) i[c23a]c iadnda cid(12)
(Figurex1a)n.tThuhreenkinc oawcidn (n12a)t u[2r4a] lwperroe diduecnttsifipesda bmy mcoampplayrsiseonne oDf t(h1e)ir[ 21H1] N,NM,RN a-nddim MeSt hdyatlad wibirtho mthoostye ramine
(10) [22], 5-hydroxy xanthenuric acid (4,5,8-trihydroxyquinoline-2-carboxilic acid) (11) [23] and

xanthurenicacid(12)[24]wereidentifiedbycomparisonoftheir1HNMRandMSdatawiththose
reportedintheliterature. ItisworthtopointoutthatpsammaplyseneD(1)wasisolatedbynormal
phaseMPLCasthemajorcompound(6.7g)representingover50%oftheentiren-BuOHextract.
Mar. Drugs 2018, 16, x FOR PEER REVIEW 3 of 16
Mar.Drugs2018,16,146 3of16
reported in the literature. It is worth to point out that psammaplysene D (1) was isolated by normal
phase MPLC as the major compound (6.7 g) representing over 50% of the entire n-BuOH extract.
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a7n.3d9–δ7H.376./175.3–87–.171.3/57 .(0J 2=– 165.9.59 H(Jz=) a1n5.d5 δHHz 7).,1w5–e7re.1a1s/7si.0g2n–e6d.9t9o (tJh =e 1E5.o5l eHfizn),– wCeHre-7 a=sCsiHgn-8e–d, tsoa ttihsefy Ein ogletfhine
t–eCnHth-7d=eCgHre-e8–o,f suantissaftyuirnagt iothne. TtehnethH MdeBgCredea toaf aunnaslyastuisrasthioown. edThceo rHreMlatBioCn sdbateat waeneanlyHsi-s7 sahnodwthede
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cnoonnfi-prmroitnogntahteedc ocnanrbeoctnio Cn-o4f (tδhCe 1t3r0an.9s8-c) icnonnafmirmoyilnmg othtief tcoonthnee2ct,6io-dni borfo tmhei ntraatends-pcihnennaomliocyriln mgo(dtief dtou ctehde
w2,6it-hditbhreocmhienmatiecdal pshheifntsoloicf Cri-n2g/ (6daetdδuCce1d1 2w.8i0tha tnhde Cch-1ematicδaCl 1s5h4if.t1s0 o).f TCh-e2/s6e caot nδdC 1o1l2e.fi8n0 iacnpdr oCto-1n aHt δ-8C
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(32.7H4, (22mH), t2ot, tJh e= t7h.2r eHezp)r, o2p.2y2li cancdar 2b.o1n6 s(2eHqu, e2nmce) tNo –tCheH t2h-1re0e– CpHro2p-1y1li–cC cHar2b-1o2n– Ose,qwuehniclee tNho–CseHa2t-1δ0H–
3C.H302-(121H–C, mH2)-1a2n–dO3, .w00hi(l2eH t,hmos)e wate rδeH a3t.t3r0ib (u2tHed, mto) tahnede t3h.0y0l ic(2cHh,a min) CwHe2r-e1 9a–ttCriHbu2-te2d0. toT htheep ertohtoynlisc
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C(FHig2u-1r0e w2)a. sTchoen mneecttheydletoneth geraomupid CicHN2--1M0e w21absa csoendnoenctietsdc thoe mthiec aalmshidifitc (NδC-M47e.2114 b/a4s8e.2d) oann dittsh cehHemMiBcaCl
csohrifrte (laδtCi 4o7n.1t4o/4C8-.92)( aδnCd1 t6h8e.9 H6/M1B68C. 8c0o)rraenldatiCo-n2 1to( δCC-93 (4δ.7C 41/683.69.65/51)6.8T.8h0e) achndem Ci-c2a1l (sδhCi f3t4s.7o4f/3C6H.525-)1. 2Thaet
(cδhHem4.i1c1a/l 4s.h05if)tsa nodf δCCH(27-11.29 4a/t 7(2δ.H6 44).1sh1/o4w.0e5d) iatnsdli nδkC t(o71th.9e4e/7th2e.6r4o)x syhgoewn.eFdu irttsh leirnmk otroe ,ththee ectohrerre loaxtiyogneonf.
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mthoe leccounlenetoctitohne oothf etrh1is,2 ,p4,a6r-tt eotrfa stuhbes tmituotleedcu-pleh etnoy lthrien go.tFhuerr th1e,2r,m4,o6r-ete,tCraHsu2-b1s9ti(tδuHte3d.-0p0h)edniyspl laryinegd.
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(c1a3r4b.o6n3 /C13-146.5 6(δ),C c1o3n6fi.8r1m/1i3n6g.9t9h)e asnudb sCtitHu-t1io5n/1o7 f(t1h3i4s.6a3ro/1m34a.t5i6c)r, incognbfiyrmthiengN ,tNhe-d siumbestthityultaimoni noofe tthhyisl
cahroamina.tCico rminpgo ubyn dth2e wNa,Nsn-daimmeedthpyslaammminaopeltyhsyeln cehaF.in. Compound 2 was named psammaplysene F.
CCoommppoouunndd 33 ddiissppllaayyeedd tthhee ssaammee mmoolleeccuullaarr ffoorrmmuullaa tthhaann ccoommppoouunndd 22 oobbttaaiinneedd bbyy HHRREESSIIMMSS
((mm//zz 669988..88667755 [[MM ++ HH]]++)).. AA pprreelilmimininaaryry 1H1H NNMMRR ininssppeecctitoionn ((TTaabbllee 11)) sshhoowweedd ssiimmiillaarriittyy bbeettwweeeenn
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soyf sttwemo odfotwwnofiedlodw dnofiuebldletdso oufb ldeotsuobfledtso uabt leδtHs 6a.t49δH–6.65.24/96–.65.65–26/.568.5 6(J– 6=. 5182(.5J =H1z2, .5cisH) za,ncdis )δaHn 6d.1δ3H–
66..1136–/66..0196–/66.1.029 –(J6 .1=2 1(2J.5= H12z.)5 wHezr)ew aesrseigansesdig ntoed –tCoH–-C7=HC-H7=-C8 Ho-f8 tohfe tchienncainmnoayml omylomietoyi etwyitwh itah Za
Zcocnofingfiugruatriaotnio. nF.urFthuertrhmeormreo, trhe,e tChOeSCYO, SHYS,QHCS,Q aCnd, aHnMd BHCM aBnCalyasniasl dysaitsa d(Faitgau(rFei g2u) sreho2w) sehdo twhee dsatmhee
scaomrreelacotirornesla taiso npsraesviporuesvlyio ufosluynfdo ufnodr f2o. rT2h.uTsh, utsh,et hteettreatbrarobmroimnainteadte dtytryorsoinsien e3 3wwaass aassssiiggnneedd ttoo
ppssaammmmaappllyysseennee GG..

FFiigguurree 22.. CCOOSSYY aanndd kkeeyy HHMMBBCC ccoorrrreellaattiioonnss ffoorr ppssaammmmaappllyysseenneess FF ((22)) aanndd GG ((33))..
Mar.Drugs2018,16,146 4of16
Table1. 1HNMR(500MHz)and13CNMR(125MHz)dataforPsammaplysenesF(2)andG(3)in
CD OD.
3
Position PsammaplyseneF(2) PsammaplyseneG(3)
No. δC,Typea δHmult,(JinHz)a HMBC δC,Typea δHmult,(JinHz)a HMBC
1 154.10,C - - 153.29/153.04,C - -
2 112.80,C - - 112.49,C - -
3 133.09/133.01,CH 7.78/7.70,s 1,2,5,6,7 133.70/133.42,CH 7.53/7.54,s 1,2,5,6,7
4 130.98,C - - 130.70/131.16,C - -
5 133.09/133.01,CH 7.78/7.70,s 1,2,3,6,7 133.70/133.42,CH 7.53/7.54,s 1,2,3,6,7
6 112.80,C - - 112.49,C - -
7.36–7.39/7.35–7.38, 6.49–6.52/6.56–6.58,
7 140.89/141.19,CH 3,4,5,9 132.24/132.49,CH 3,4,5,9
d(15.5) d(12.5)
7.11–7.15/6.99–7.02, 6.13–6.16/6.09–6.12,
8 118.90/118.72,CH 4,7,9 123.90/124.59,CH 4,7,9
d(15.5) d(12.5)
9 168.96/168.80,C - - 171.13/171.01,C - -
10 48.20/47.14,CH2 3.89/3.74,t(7.2) 9,11,12,21 48.97/45.75,CH2 3.65/3.73,t(7.5) 9,11,12,21
11 30.86/29.16,CH2 2.22/2.16,m 10,12 29.32/28.68,CH2 2.02/2.18,m 10,12
12 71.94/72.64,CH2 4.11/4.05,t(6.4) 10,11,13 72.03/72.32,CH2 3.78/4.04,t(6.5) 10,11,13
13 153.88/153.94,C - - 153.55/153.86,C - -
14 119.57,C - - 119.36/119.59,C - -
15 134.63/134.56,CH 7.57,brs 13,14,17,18,19 134.51/134.57,CH 7.51/7.56,s 13,14,17,18,19
16 136.81/136.99,C - - 136.85,C - -
17 134.63/134.56,CH 7.57,brs 13,14,15,18,19 134.51/134.57,CH 7.51/7.56,s 13,14,15,18,19
18 119.57,C - - 119.36/119.59,C - -
19 30.45,CH2 3.00,m 15,16,17,20,22,23 30.51/30.56,CH2 3.00,m 15,16,17,20,22,23
20 59.24,CH2 3.30,m 16,19,22,23 59.32/59.24,CH2 3.34,m 16,19,22,23
21 34.74/36.55,CH3 3.09/3.28,s 9,10 32.59/36.67,CH3 3.06/3.03,s 9,10
22 43.62,CH3 2.91,s 20,23 43.70/43.73,CH3 2.91,s 20,23
23 43.62,CH3 2.91,s 20,22 43.70/43.73,CH3 2.91,s 20,22
aChemicalshiftsaregivenforbothrotamerswhendistinguishablesignalswereidentified.
Compounds 4 and 5 were obtained in a minute quantity (0.7 mg) as a 1:1 mixture that could
notbeseparated. Duetotheminutequantityofthemixture,wedecidedtodeterminethestructures
ofthelattertwometabolitesfromthemixture. HRESIMSanalysisrevealedthemolecularformulas
C H 79Br 81Br N O (m/z769.9521[M+H]+)andC H 79Br 81Br N O (m/z755.9309[M+H]+)
27 36 2 2 3 3 26 34 2 2 3 3
for4and5,withsimilarisotopicpatternsindicatingthepresenceoffourbromines,but14and28a.m.u.
(atomicmassunit)lessthanpsammaplyseneD(1),respectively. Directcomparisonofthe1HNMR
spectraofthismixture(Table2)andtheco-isolatedmajorcompoundpsammaplyseneD(1)showed
goodsuperimposition,includingtherotamericforms. Themaindifferencesconcernedthesignalsof
CH -3(cid:48),CH -19andCH -20(Figure3)indicatingmodificationsofthenumberoftheN-methylgroups
2 2 2
linkedtothem. The2DNMRexperiments(COSY,HSQC,andHMBC)ofbothcompoundsshowed
thesamecorrelationsastheco-isolatedpsammaplyseneD(1)(Figure2). 1HNMR(Table2)revealed
foursignalsofN-methylgroupsatδ 3.10and3.29(2s, 6H,s-trans-CH -21, s-cis-CH -21), δ 2.70
H 3 3 H
(s,CH -22,3H)andδ 2.74–2.79(CH -4(cid:48) andCH -5(cid:48),6H).TheHMBCanalysisshowedcorrelation
3 H 3 3
fromN-CH -22(δ 2.70,δ 34.0)toC-20(δ 51.1)forcompound4. Thechemicalshiftsdifferences
3 H C C
observedforH-19(δ 2.93),H-20(δ 3.21)andCH -22(δ 2.70)suggestedamodificationonthispart
H H 3 H
ofthemolecule. ThechemicalshiftsofC-20(δ 51.1)andCH -22(δ 34.0)suggestedtheexistence
C 3 C
ofonlyonemethylgrouponthissideofthechain. Attheotherendofthemolecule,correlationsof
N-CH -4(cid:48) and5(cid:48) (δ 2.74–2.79,δ 44.7)toC-3(cid:48) (δ 57.2)alongwiththechemicalshiftofC-3(cid:48),showed
3 H C C
the presence of two methyl groups on the nitrogen N-C-3(cid:48). The chemical shift of C-20 and C-22
were the same for compound 5, but the chemical shift of C-2(cid:48) (δ 27.9), C-3(cid:48) (δ 48.7), and CH -4(cid:48)
C C 3
(δ 34.0), aswellastheHMBCcorrelationsbetweenC-3(cid:48) andCH -4(cid:48), suggestedthepresenceofa
C 3
NH-MeconnectedtothecarbonC-3(cid:48). Therefore,ourmixturewascomposedoftwopsammaplyseneD
derivativeslackingoneandtwoN-methylsandnamedpsammaplyseneH(4)andpsammaplysene
I(5),respectively.
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FFiigguurree 33.. CCOOSSYY aanndd kkeeyy HHMMBBCC ccoorrrreellaattiioonnss ffoorr ppssaammmmaappllyysseenneess HH ((44)) aanndd II ((55))..
TTaabbllee 22.. 11HH NNMMRR ((550000 MMHHzz)) aanndd 131C3C NNMMR R(1(2152 5MMHHz)z d)adtaa tfaofro PrsPasmammampalypslyenseens eHs H(4)( 4a)ndan Id (5I)( 5in)
iCnDC3DO3DO. D.
Position Psammaplysene H (4) Psammaplysene I (5)
Position PsammaplyseneH(4) PsammaplyseneI(5)
No δC, Type a δH mult, (J in Hz) a HMBC δC, Type a δH mult, (J in Hz) a HMBC
No1 δC,1T5y4p.e7,a C δHmult,(Ji-n Hz)a HMBC- δC1,5T4y.p7,e Ca δHmult,(J- inHz)a HMB- C
12 1514.179,.C6, C - - - - 115149.7.,6C, C -- --
23 13131.95./61,3C3.4, CH 7.9-2/7.85, d 1, -2, 5, 6, 7 1331.51/91.63,3C.4, CH 7.92/-7.85, d 1, 2,- 5, 6, 7
3 133.5/133.4,CH 7.92/7.85,d 1,2,5,6,7 133.5/133.4,CH 7.92/7.85,d 1,2,5,6,7
4 136.2, C - - 136.2, C - -
4 136.2,C - - 136.2,C - -
55 13133.53/.153/133.43,.C4,H CH 7.972./972./875.,8d5, d 1,21,, 32,,6 3,,7 6, 7 113333.5.5//113333.4.4,C, CHH 77.9.92/2/77.8.855,d, d 11,,2 2,,3 ,36, ,67, 7
66 1119.169,.C6, C - - - - 111199.6.,6C, C -- --
77 14104.20/.124/104.00,.C0,H CH 7.470.–470.–474,.4m4, m 3,43,, 54,,9 5, 9 114400.2.2//114400.0.0,C, CHH 77.4.400––77.4.44,4m, m 33,,4 4,5, ,59, 9
8 121.6/121.7,CH 7.14–7.27,dd(15.0) 4,7,9 121.6/121.7,CH 7.14–7.27,dd(15.0) 4,7,9
8 121.6/121.7, CH 7.14–7.27, dd (15.0) 4, 7, 9 121.6/121.7, CH 7.14–7.27, dd (15.0) 4, 7, 9
9 168.5,Ca - - 168.5,Ca - -
109 48.4/14678.3.,5C, HC 2a 3.91/3.76,t- (7.0) 9,11,12,-2 1 48.41/6487..53,, CCH a 2 3.91/3.76-, t(7.0) 9,11,1-2 ,21
1110 30.468/.249/4.17,.C3,H C2H2 23.2.931//23..1776,,m t (7.0) 91,0 1,112, 12, 21 3408.6./4/2497.1.3,C, CHH22 32.9.213//3.27.167, ,tm (7.0) 9, 1101,,1 122, 21
1211 72.360/.761/2.99,.C1,H C2H2 4.122./243./027.,1m7, m 10,1110,1, 312 7320.6./6/7219.9.1,C, CHH22 42.1.22/3/42.0.177,m, m 10,1101,, 1123
1312 7125.64/.77,1C.9, CH2 4.12-/4.07, m 10-, 11, 13 72.165/47.17.,9C, CH2 4.12/-4.07, m 10, -11, 13
14 119.6,C - - 119.6,C - -
13 154.7, C - - 154.7, C - -
15 134.5,CH 7.56/7.53,m 13,14,17,18,19 134.5,CH 7.56/7.53,m 13,14,17,18,19
1614 1371.81,9C.6b, C - - - - 13171.89,.6C, bC -- --
1715 13143.54,.C5,H CH 7.576./576./573.,5m3, m 131,314, ,1145,, 1178,, 1198, 19 113344.5.5,C, CHH 77.5.56/6/77.5.533,m, m 1133,,1 144,,1 51,71, 81,81,9 19
1816 11193.67,.8C, C b - - - - 111397..68,,C C b -- --
1917 321.23,4C.5H, 2CH 2.973.5,6t/(78..05)3, m 1316, ,1240,, 1252, 18, 19 3123.24,.5C,H C2H 72..9536,/t7(.58.30,) m 13,1 164,,2 01,52, 218, 19
2018 51.11,1C9H.62, C 3.21,m- 16,19,2-2 511.11,9C.6H, 2C 3.21,-m 16,19-, 22
21 34.7/36.6,CH3 3.10/3.29,s 9,10 34.7/36.6,CH3 3.10/3.29,s 9,10
2219 34.302,.C2H, C3H2 22..7903,, st (8.0) 1260, 20, 22 3342.0.,2C, CHH32 2.29.37,0 t, s(8.0) 16,2 020, 22
1(cid:48)20 57.521,.C1H, C2H2 3.237.,2m1, m 1,126(cid:48),, 31(cid:48)9, 22 4581.7.,1C, CHH22 33.3.251,m, m 116,,2 (cid:48)1,93,(cid:48) 22
234(cid:48)(cid:48)(cid:48)2212 32744714....369374,,,/.3CCC06HHH, .C6223,H C3H 3 2.72438..21./138220,,../77mm340.,,2 ss9 , s 1(cid:48),321(cid:48)(cid:48)(cid:48),,,9345(cid:48)(cid:48)(cid:48),2, 1050 (cid:48) 34733.314704.../909.3,,06CCC, .HC6HH,2H 23C3H 3 23.7.2417..221/0.38/273,,.07mm.,26 s9, s, s 1(cid:48)1,9(cid:48)32,,2(cid:48)(cid:48)3 ,10(cid:48)40 (cid:48)
5(cid:48)1′ 44.537,.C2H, C3H2 2.79/32..2774,, ms 3(cid:48)1,4, (cid:48)2′, 3′ 48.7-, CH2 3.3-5, m 1, -2′, 3′
a C2′h emical s2h7i.f6t,s CHar2e given for2.b23o,t mh rotamers wh1e′,n 3′ distinguish3a0b.9l eCHsi2 gnals were2.i2d3e, nmt ified; b Dete1c′t,e 3d′ by
HM3′B Ccorrel7a1ti.9o,n CsH.2 4.18, m 1′, 2′, 4′, 5′ 71.9, CH2 4.18, m 1′, 2′, 4′
4′ 44.3, CH3 2.78/2.74, s 3′, 5′ 34.0, CH3 2.77/2.76, s 3′
5′ 44.3, CH3 2.79/2.74, s 3′, 4′ - - -
Ca Cohmempoicuanl sdhsift6s aarne dgiv7ene fxohr ibboitthe drottahmeerms owlheecnu ldairstifnogrumisuhlaablew seigrenadlse wteerrme iidneendtifibeyd; Hb DReEteScIteMd S as
C Hby7 H9BMrB8C1B crorNrelaOtioants.m /z743.9386[M+H]+,andC H 79Br 81Br N O atm/z729.9190[M+H]+
25 33 2 2 3 3 24 31 2 2 3 3
respectively, indicating nine degrees of unsaturation with an isotopic pattern of four bromines.
Compounds 6 and 7 exhibited the molecular formula were determined by HRESIMS as
These NMR and mass data showed that compound 7 has one methyl group less than compound
6C.2E5Hxa33m79Binr2a8t1iBorn2No3fOth3 ea1t HmN/z M74R3a.9n3d86H [SMQ C+ sHpe]+c,t raan(dT aCb2l4eH33)17d9Bisrp28l1aByr2eNd3sOig3 naatl smw/zi t7h2s9i.m91i9la0r [cMhe m+ iHca]l+
respectively, indicating nine degrees of unsaturation with an isotopic pattern of four bromines.
shiftstothosereportedforthetetrabromotyrosinecompoundsanomoiansAandB,previouslyisolated
These NMR and mass data showed that compound 7 has one methyl group less than compound 6.
fromAnomoianthellapopeae[26](order:Verongiida)andfromanunknownspeciesofthegenusHexadella
Examination of the 1H NMR and HSQC spectra (Table 3) displayed signals with similar chemical
(order: Verongiida)collectedinIndonesia[27]. 2DNMRanalysisincludingCOSY,HSQC,andHMBC
shifts to those reported for the tetrabromotyrosine compounds anomoians A and B, previously
(Table3,Figure4),allowedidentificationofthestructuresof6and7astheO-demethylanomoianB
isolated from Anomoianthella popeae [26] (order: Verongiida) and from an unknown species of the
andO-demethylanomoianA,respectively. Thus,thenewcompoundswerenamedanomoianC(6)
agnednuasn oHmexoaiadnellDa ((o7r).dTerh:e V1eHroNnMgiiRdasi)g cnoalllsecotfetdh eina rIonmdoantiecspiar o[t2o7n]s. 2CDH -N3/M5Ra nadnaClyHs-i1s 5i/n1c7luodfitnhge sCeOnSeYw,
HSQC, and HMBC (Table 3, Figure 4), allowed identification of the structures of 6 and 7 as the
compoundsindicateda6:4rotamericproportionality.
O-demethyl anomoian B and O-demethyl anomoian A, respectively. Thus, the new compounds
were named anomoian C (6) and anomoian D (7). The 1H NMR signals of the aromatic protons
CH-3/5 and CH-15/17 of these new compounds indicated a 6:4 rotameric proportionality.
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FFiigguurree 44.. CCOOSSYY aanndd kkeeyy HHMMBBCC ccoorrrreellaattiioonnss ffoorr aannoommooiiaannss CC ((66)) aanndd DD ((77))..
TTaabbllee 33.. 11HH NNMMRR ((550000 MMHHzz)) aanndd 1133CC NNMMRR ((112255 MMHHzz)) ddaattaa ffoorr AAnnoommooiiaannss CC ((66)) aanndd DD ((77)) iinn CCDD33OODD..
Position Anomoian C (6) Anomoian D (7)
Position AnomoianC(6) AnomoianD(7)
No δC, Type a δH mult, (J in Hz) a HMBC δC, Type a δH mult, (J in Hz) a HMBC
1N o 15δ1.C6,5T/1y5p1e.4a7, C δHmult,(Jin- Hz)a HMBC- δC,1T5y2p.7e, Ca δHmult,(JinH- z)a HMBC -
21 151.6151/21.3561,. 4C7 ,C - - - - 115122.7.,6C9, C - - - -
32 134.41172/1.3364,.4C1, CH 7.3-4/7.32, s 1, 2-, 4, 5, 6, 7 111324.6.490,,C CH -7.37/7.35, s 1-, 2, 4, 5, 6, 7
3 134.47/134.41,CH 7.34/7.32,s 1,2,4,5,6,7 134.40,CH 7.37/7.35,s 1,2,4,5,6,7
4 132.77/133.49, C - - 130.53/129.90, C - -
4 132.77/133.49,C - - 130.53/129.90,C - -
55 113344.4.477//113344..4411,, CCHH 7.347/.374.3/27,.3s2, s 1,21,,3 2,,4 3,,6 4,,7 6, 7 13143.44.04,0C, HCH 7.37/77..3357,/7s.35, s 1,2,31,, 42,,6 3,,7 4, 6, 7
66 111122.3.366,,C C - - - - 11121.269.6,9C, C - - - -
77 3333.1.144//3322..1144,,C CHH22 2.902/.920.8/32–.823.9–92,.9m9, m 3,43,,5 4,,8 5,,9 8, 9 373.470.4/03/83.80.20,2C, HCH22 2.83–32..0843/–23..9024,/2m.92, m 3,4,53,, 84,,9 5, 8, 9
8 66.86,CH 3.82,m 7,9,24,25 60.79/61.29,CH 4.26/4.17,m 7,9,24
8 66.86, CH 3.82, m 7, 9, 24, 25 60.79/61.29, CH 4.26/4.17, m 7, 9, 24
9 172.36/172.63,C - - 171.40/172.00,C - -
9 172.36/172.63, C - - 171.40/172.00, C 3.48–3.80/3.26–-3 .51, -
1100 4477.1.144//4488..2244,,C CHH22 3.403–.430.7–23,.m72, m 9,191,, 1211, 21 474.677.6/74/74.77.87,8C, HCH22 3.48–m3.80/3.26–3.51, m 9,11,92, 111, 21
1111112312 2727192925..03..30391.91//6//13737/01011....4754752352,,.,,3CC CC0HH,HH C2222 13.91.82.95–32–2.–3.802.590.–m03/30/1./3.917.3-.83 7/8–33,1.–8m.9182.,9 ,m2, m 1101,,11110123,,- 1123 279227..139255..13//155375//01373..01.567..01562,,01,CC ,,C HHCC HH2222 1.935.–9217./.093535./.–991297..,/903m55-., 9/1m9.,9 m5, m 1101,,11231101,,- 1123
13 153.61/153.30,C - - 153.72,C - -
1144 111199.4.433,,C C - - - - 11191.947.4,7C, C - - - -
15 134.47/134.50,CH 7.52/7.53,s 13,14,1136,, 1147,, 1168,, 1179, 134.51/134.53,CH 7.54/7.55,s 13,14,161,31, 71,41, 81,61, 917,
1156 113347..4977//113348..5204,, CCH 7.5-2/7.53, s -18, 19 1341.5317/.7123,4C.53, CH -7.54/7.55, s - 18, 19
17 134.47/134.50,CH 7.52/7.53,s 13,14,15,16,18,19 134.51/134.53,CH 7.54/7.55,s 13,14,15,16,18,19
16 137.97/138.24, C - - 137.72, C - -
18 119.43,C - - 119.47,C - -
1179 1313.44.04/7/3113.446.5,0C, HC2H 2.79.25,2m/7.53, s 15,1136,, 1147,, 1250, 16, 13341.5.212/1,3C4H.523, CH 2.937,.5m4/7.55, s 15,161,31, 71,42, 015, 16,
20 60.12/60.14,CH2 3.08,m 16,19,1282,, 1293 59.96,CH2 3.21,m 16,19,221,82,3 19
1281 36.641/1394.4.236,, CC H3 2.92/2.93-, s 9,10- 36.25/13149..2497,,C CH 3 2.50/2.47,s- 9,10 -
1222290342 4443642410...333..4101002/////4443641410.....3134149464,,,,,CCC CCHHHHH33322 222...747252///22223.....7479012128,,,,, sssmm 112285600,,,,, 2112256932,, 1272,, 2203 344344...21168835///19344..34429...26622,,811 CC,,,CCCHHHHH22 333 222...877244///322...07723044..92,,,31sss,, mm 2200118,,5622,, 321169,, 2127,, 2230
2215 4326.3.614//4314..1296,,C CHH33 2.452/.922.4/22,.9s3, s 8,294, 10 36.25/3-4.29, CH3 -2.50/2.47, s - 9, 10
22 44.a30C/4h4e.3m4,i CcaHl3s hiftsare2g.7iv2/e2n.71f,o sr bothrotam2e0r, s23w hendis4t4i.n1g8/u44is.2h1a, CblHe3 signalswe2r.7e4i/d2.e74n, tsi fied. 20, 23
23 44.30/44.34, CH3 2.72/2.71, s 20, 22 44.18/44.21, CH3 2.74/2.74, s 20, 22
24 42.31/41.19, CH3 2.45/2.42, s 8, 25 33.26/33.68, CH3 2.82/3.00, s 8
C25 ompo4u2.n31d/418.19h, CaHd3 aproto2n.45a/2t.e42d, s molecular8, i2o4 natm/z81-5 .0068[M+H]+-c orresponding- tothe
molecularaCfohremmiucalal sChiftHs are7 9gBivren81 fBorr bNothO ro,tadmeteerrsm wihneend dbisytiHngRuEisShIaMbleS .sTighnealms wasesres ipdeecnttrifuiemd. indicated
29 43 2 2 4 3
an isotopic matrix of a tetrabrominated compound. Quick examination of the 1H NMR spectrum
Compound 8 had a protonated molecular ion at m/z 815.0068 [M + H]+ corresponding to the
(Table4)showedsignalswithidenticalchemicalshiftstothosereportedforanomoianC(6)andD(7).
Fmuorltehceurlmaro froe,rmiturelav eCa2l9eHd4t3h79eBrp2r81eBsre2nNce4Oo3f, adnetaedrmdiitnioenda blyd iHmRetEhSyIlMamS.i nTohper ompayslsi cscpheacitnru,mas ionbdsiecravteedd faonr
isotopic matrix of a tetrabrominated compound. Quick examination of the 1H NMR spectrum (Table
psammaplyseneD(1). Thiswasconfirmedbythe2DNMRspectraanalysis,includingCOSY,HSQC,
4) showed signals with identical chemical shifts to those reported for anomoian C (6) and D (7).
andHMBC,enablingustosetthecorrelationsshowninFigure5. Thenewcompound8wasnamed
Furthermore, it revealed the presence of an additional dimethylaminopropylic chain, as observed for
anomoianE.
psammaplysene D (1). This was confirmed by the 2D NMR spectra analysis, including COSY,
TheHRESIMSdataof9revealedthemolecularformulaC H 79Br 81Br N O (m/z829.0259
30 45 2 2 4 3
HSQC, and HMBC, enabling us to set the correlations shown in Figure 5. The new compound 8 was
[M+H]+). Its 1H NMR spectrum was very similar to anomoian E (8) spectrum, except for the
named anomoian E.
two additional methyl groups at δ 2.51/2.54 integrating for six protons for 9. HSQC, COSY,
H
andHTMheB HCRsEpeScIMtraSa dnaatlay soifs 9le rdevtoeathleeds athmee mcoorlreeclualtaiorn fsoramsiunlac oCm30pHo4u57n9Bdr8281aBnrd2Nen4Oab3 (lemd/zt h8e29a.s0s2ig5n9 m[Men +t
H]+). Its 1H NMR spectrum was very similar to anomoian E (8) spectrum, except for the two
oftheN-8-methylgroupsthroughanadditionalcorrelationfromtheN–CH -24/25(δ 2.51/2.54,6H)
3 H
taoddthiteiomneatlh mineethgyrol ugproCuHps- 8at( δδH 32..9571//23..5849 ,inmte;gδrat6i6n.g7 9f/or6 6s.i5x8 p).roTthoenrse ffoorre 9,.s tHruScQtuCr,e C9OwSaYs, danetde rHmMinBeCd
H C
spectra analysis led to the same correlations as in compound 8 and enabled the assignment of the
asanomoianF,existingas6:4rotamers(Table4,Figure5).
N-8-methyl groups through an additional correlation from the N–CH3-24/25 (δH 2.51/2.54, 6H) to the
methine group CH-8 (δH 3.97/3.89, m; δC 66.79/66.58). Therefore, structure 9 was determined as
anomoian F, existing as 6:4 rotamers (Table 4, Figure 5).
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FigurFei5gu.rCeO 5.S CYOaSnYd ankde ykeHy HMMBBCCc coorrrreellaattiioonns sfofro arnaonmoomiaonsia En (s8)E a(n8d) Fa n(9d). F(9).
Table 4. 1H NMR (500 MHz) and 13C NMR (125 MHz) data for Anomoians E (8) and F (9) in CD3OD.
Table4.1HNMR(500MHz)and13CNMR(125MHz)dataforAnomoiansE(8)andF(9)inCD OD.
3
Position Anomoian E (8) Anomoian F (9)
No δC, Type a δH mult, (J in Hz) a HMBC δC, Type a δH mult, (J in Hz) a HMBC
Position1 153.29/153.15, C AnomoianE- (8) - 152.76/152.57, C Anom- oianF(9) -
No 2 δC,1T19y.p08e.1a19.14, C δHmult,(Jin- Hz)a HMB-C 1δ18C.,9T2,y Cp ea δHmu-l t,(JinHz)a - HMBC
3 135.35/135.19, CH 7.51/7.48, s 1, 2, 5, 6, 7 135.32/135.22, CH 7.51/7.48, s 1, 2, 5, 6, 7
1 153.29/153.15,C - - 152.76/152.57,C - -
4 137.14/138.15, C - - 138.66/137.72, C - -
2 119.08.119.14,C - - 118.92,C - -
5 135.35/135.19, CH 7.51/7.48, s 1, 2, 3, 6, 7 135.32/135.22, CH 7.51/7.48, s 1, 2, 3, 6, 7
3 135.35/135.19,CH 7.51/7.48,s 1,2,5,6,7 135.32/135.22,CH 7.51/7.48,s 1,2,5,6,7
6 119.08.119.14, C - - 118.92, C - -
4 137.14/138.15,C - - 138.66/137.72,C - -
5 7 135.35/3183.053./1398,.6C5H, CH2 7.52.18/47–.34.083,/s2.93, m 1,23,, 43,, 56,, 87, 9 3133.505.3/231/.19315, .C2H2,2C H 3.00/27.9.521–/3.70.74,8 m,s 3, 4, 15,, 28,, 39, 6,7
6 8 119.086.01.1792./1641,.2C3, CH 4.-15/4.06, m 4, -7, 9, 24 66.581/6168.7.992, ,CCH 3.97/3.89-, m 4, 7, 9, 24, -25
7 9 38.03/13782.6.753,/C17H3.246, C 2.84–3.03/2.9- 3,m 3,4,5,-8 ,9 13732..0105//13711..9615,, CC H2 3.00/2- .92–3.07,m 3-, 4,5,8,9
8 10 60.72/4671.5.72/34,7C.8H2, CH2 34..5115–/34.7.066/3,.m26–3.60, m 4,9,7 1,19,, 1224, 21 486.626.5/847/.6366.,7 C9H,C2 H 3.41–3.734./937.5/13–.38.96,4m, m 9, 141,, 172,,9 2,12 4,25
9 11 172.732/91.1763/.3406.,6C4, CH2 1.95-–2.03/1.95, m 1-0, 12 301.7721./1209/.0187, 1C.6H52, C 1.69–1.98/1.90-–1.98, m 10, 12 -
12 72.36/71.71, CH2 3.51–3.76/33..2996,– m3.6 0, 10, 11, 13 71.82/72.40, CH2 3.34.19–4/33.7.945/, 3m.5 1–3.64, 10, 11, 13
10 13 47.57/47.81253,.C54H, 2C m - 9,11,12-, 21 14583..2776//14573..3561,, CC H2 - m 9,- 11,12,21
14 119.42, C - - 119.52, C 1.69–1.-9 8/1.90–1.98, -
11 15 29.16/13340..5644/,1C34H.522, CH 1.95–2.073./551/.97.55,6m, s 13,1 104,, 1127, 18, 19 13340.5.771/1/3249.6.008, ,CCHH 2 7.57/7.5m8, s 13, 14, 17,1 108,,1 129
12 16 72.36/17317.7.714,/C13H8.252, C 3.99,m- 10,11,-1 3 71.18327/.2782,. 4C0 ,CH2 3.94- /3.95,m 1- 0,11,13
13 17 115334.5.544,/C134.52, CH 7-.55/7.56, s 13, 14-, 15, 18, 19 1314.5537./7173/41.6503, .C51H, C 7.57/7.58-, s 13, 14, 15, 18-, 19
14 18 119.421,1C9.42, C - - - - 11191.592.5, 2C, C - - - -
15 19 134.54/3113.341./5321,.4C1H, CH2 7.55/72.5.963,,s m 13,1145,, 1176,, 1187,, 1290 3103.467.5/730/.15394, .C6H0,2C H 27.9.597, /m7 .58,s 151, 316,,1 147,,1 270, 18,19
16 20 137.746/01.0318/.6502.,0C8, CH2 -3.09, m 16, 1-9, 22, 23 59.14337, .C2H8,2C 3.26, m- 16, 19, 22, -23
17 21 134.54/3163.244./5324,.3C3H, CH3 7.55/27.8.586/2,.s99, s 13,14,195,, 1108 ,19 3143.431.5/736/.16344, .C6H0,3C H 2.972./527.9/47, .s5 8,s 139,, 1140, 15,18,19
18 22 11494.4.129,/C44.24, CH3 2-.73/2.71, s 2-0, 23 43.17159, .C5H2,3C 2.85/2.85-, s 20, 23 -
19 23 31.31/4341.1.491/4,4C.2H42, CH3 2.923.,7m3/2.71, s 15,162,01,7 2,22 0 304.63.77/53, 0C.H593, CH2 2.85/22..8959,, sm 2105, ,2126 ,17,20
20 24 60.01/3630.7.018/3,4C.1H12, CH3 3.029.,4m2/2.39, s 16,19,42,2 8, 23 42.235/94.24.307,,C CHH23 2.51/23..4256,, sm 41, 86,, 2159 ,22,23
21 25 36.24/34.33,C- H3 2.88/2.99,- s 9,10- 4324.2.33/14/23.067.6, 4C,HC3H 3 2.521./922.4/52, .s9 4,s 4, 8, 294, 10
2222122345(cid:48)(cid:48) 12345′′′′′ 44374431....1176299166////.794437154441.-,6....25442216C667444412/....H91007,,,,55551CCCC2,,,,. 6HHHHCCCC22333HHHH, C2233 H2 222...77442332..02///95-222,,423...22773mm.....02311988956,,,55,,,sss,, mmmss 1221,1004′2(cid:48)1, ,,,,-(cid:48),1332 2283,′′′2′,,,,323(cid:48) ′,3454(cid:48) 3′′′′ , ′ 5 ′ 442272544..22167337244.....33163346077//....42799467744,,,,, 425522CCCCC,,,,..HHHHH00CCCC7722233HHHH,, CC2233HH33 422...0992322221224..24....,//.5885 075222t1551,,..1 . 99(2////mm,51172222t.,,5 , ....(ss4884)m5 5555.5,,,,)ssss 1′1, ,1332 ′′′2′,,,, ′1,345444 223′′′′1,,, ,00 ′2(cid:48)88 5,,,(cid:48),, ′,322 22332(cid:48)45(cid:48)
3(cid:48) 57a.1 C5,hCeHm2ical shifts ar3e.3 g6i,vmen for both ro1(cid:48)t,a2m(cid:48),4e(cid:48)r,s5 (cid:48)when disti5n7g.0u7i,sChHa2ble signals w3e.4r5e, midentified. 1(cid:48),2(cid:48),4(cid:48),5(cid:48)
4(cid:48) 44.05,CH3 2.85,s 3(cid:48),5(cid:48) 43.79,CH3 2.92/2.91,s 3(cid:48),5(cid:48)
5(cid:48) The44 a.0b5,sCoHlu3te configur2a.8t5io,sn of the stereo3c(cid:48),e4n(cid:48) ter C-8 for 4a3n.7o9,mCHo3ian C-F (62–.992)/ 2c.a91n, sbe propose3d(cid:48), 4b(cid:48)y
aChemicalshiftsaregivenforbothrotamerswhendistinguishablesignalswereidentified.
comparison of the values and signs of their optical rotations in MeOH ([α]25 +9°, +11°, +7° and +6°,
D
respectively), with the closely related congeners, anomoian A ([α]D +5.1°) [26], anomoian B ([α]D
Th+e1a0b.5s°o) l[u27t]e, cisoo-nAfingoumroaitaino nA o([fαt]h20 e+s4.t5e°r)e [o2c8e],n stuebreCre-d8amfoinreasn Ao m([αo]i2a5 n+2C1°-)F a(n6d– 9B) (c[αa]n25 b+e16p°)r o[9p],o sedby
D D D
compariinsdoincaotifntgh tehavta alull easrea nbdearsiinggn sL-otyfrothsienier roepsitdicuael. rTohtea tsiyonnthseitnic Misoe-OAnHom([oαia]n25 A+ 9([◦α,]+20 1+14◦.5,°+) 7a◦nda nd+6◦,
D D
respectisvuebleyr)e,dwamitinhest hAe ([cαlo]2Ds0 e+l1y9.5re°)l aaltleodwecdo nthgee dneeterrsm, iannaotimono oifa tnheAir a(b[αso]lDut+e 5co.1n◦fi)gu[2ra6t]i,onasn [o2m8].o ian B ([α]D
+10.5◦)[27],iso-AnomoianA([α]20 +4.5◦)[28],suberedaminesA([α]25 +21◦)andB([α]25 +16◦)[9],
2.2. Biological Activities D D D
indicating that all are bearing L-tyrosine residue. The synthetic iso-Anomoian A ([α]20 +4.5◦) and
D
suberedamiTnhees iAso(la[αte]d2 0co+m1p9o.5u◦n)dasl l1o–w3 aenddt h6–e1d1 ewteerrem tienstaetdio fnoro tfhtehire cirytaobtosxoilcu atceticvoitny fiagguairnastti ohnums[a2n8 ].
epidermoid carcDinoma cells (KB) cancer cell lines (Table 5) and acetylcholinesterase. The results
showed of the compounds exhibited moderate cytotoxicity. The major compound psammaplysene D
2.2. BiologicalActivities
(1) showed the most interesting cytotoxicity with an IC50 = 0.7 μM. Anomoian E (8) and F (9),
Thceoinstoailnaitnegd thceo mN,pNo-duinmdetshy1l–a3mainnodpr6o–p1yl1 swidee raes tpessatmedmafpolrystehneei rDc, ybtuot tloaxckicinag ctthiev idtoyuabglea binonstd h uman
epiderm–CoiHd-7c=aCrHci-n8,o mdiaspclaeylelsd (Ka Bl)owcaenr caecrticveitlyl. liMneosre(oTvaerb, leps5a)mamnadplaycseentye lcFh o(l2i)n, eswtheircahs eb.eaTrh ea results
showedofthecompoundsexhibitedmoderatecytotoxicity. Themajorcompoundpsammaplysene

D (1) showed the most interesting cytotoxicity with an IC = 0.7 µM. Anomoian E (8) and F (9),
50
containingtheN,N-dimethylaminopropylsideaspsammaplyseneD,butlackingthedoublebond
–CH-7=CH-8,displayedaloweractivity.Moreover,psammaplyseneF(2),whichbearatrans-cinnamoyl
part,wasfoundtoexhibitaloweractivitythanpsammaplyseneD(1). Therefore,fromastructural
Mar.Drugs2018,16,146 8of16
point of view, both motifs (N,N-dimethylaminopropyl and trans-cinnamoyl) seem to be together
correlatedtotheactivityofpsammaplyseneD(1).
Table5.Cytotoxicactivitiesinvitro(KBCellLine)forcompounds1–3,6–11.
Compounds 10µMa 1µMa
PsammaplyseneD(1) 100±0.2 95±0.5
PsammaplyseneF(2) 73±2 20±0.2
PsammaplyseneG(3) 75±0.4 17±1
AnomoianC(6) 28±5 15±3
AnomoianD(7) 29±2 17±2
AnomoianE(8) 82±1 6±2
AnomoianF(9) 100±0.5 20±0.6
N,N-dimethyldibromotyramine(10) 100±1 89±1
5-hydroxyxanthenuricacid(11) 25±1 6±0.5
a CellproliferationwasmeasuredwithCelltiter96AqueousOnesolutionreagent(Promega),andresultsare
expressedasthepercentageofinhibitionofcellularproliferationofKBcellstreatedfor72hwithcompounds
comparedtocellstreatedwithDMSOonly(mean±SEoftriplicate).
The screening of our French Polynesian sponges extract library was performed on
acetylcholinesteraseinhibitor(AChE)intheaimtoinvestigateapossiblechemicaldefenseofsessile
organismsonthereefagainstfishes,theirmainpredators.Thechemicalstudyofthebioactiveextractof
SubereaianthelliformisusingAChEledtotheidentificationofpsammaplyseneD(1)asitsuniqueinvitro
AChEinhibitorwithanIC =1.3µM.Noneoftheothercloselyrelatedcompoundsdisplayedany
50
activityonthistargetenhancinghereagaintheimportanceofthephenoxy-1-N,N-dimethylpropane
chainandthetrans-cinnamoylmotifs(Figure1). FurtherkineticsinvestigationofthemodeofAChE
inhibitionofpsammaplyseneD(convergenceofthelinesontheLineweaverBurkplot,decreasing
V values(from0.0036to0.0007µmol·min−1)andincreasingK values(from0.14to0.23µM))
max M
suggestedamixedcompetitive/non-competitiveinhibitionsuggestingthatpsammaplyseneD(1)not
onlybindstothefreeenzyme,butalsototheenzyme-substratecomplex. Inordertotrytoevaluate
theeffectofpsammaplyseneDintheenvironment,itwasfurthertestedonfreshwaterfish(guppy,
Poeciliareticulata)andreeffish(Acanthurustriostegus)feedingexperimentusingbathingwithorwithout
thebioactivecompound. Guppiesarefedwithcommercialflakes,butthewildreeffishfeedonnatural
coralheads,andtheirappetiteevaluatedbycountingthebitesonthecorals. Twoexperimentswere
runonguppiesusingSubereaianthelliformiscrudeextractandpurifiedpsammaplyseneD(1): anacute
toxicitybioassay(100µg/mLofcrudeextractorpurifiedpsammaplyseneD(1),1h)andachronic
toxicity(5µg/mLofcrudeextractor1µg/mLofpurifiedpsammalyseneD(1),72h).
Duringtheacuteexposure,fishweretotallyconfusedandhaduncontrolledmobility,displaying
suddenjumpsandbalancecontrolloss,assoonas20minaftertheadditionofpsammaplyseneDin
thetank. ThisresultcanbecomparedtotheobservationreportedbyNèemanetal. withLatrunculia
speciestoxins[20]. Exposureofguppiesduring24hofatasub-lethaldose(5µg/mLofcrudeextract
or1µg/mLofpurifiedpsammalyseneD(1)ofthecompoundcausedpassivityofthefishtowardsthe
foodflakesoffereddaily,whereasthoseofthecontrolsatenormally. Theseobservationsindicatea
clearlinkbetweenthelossofappetiteandpsammaplyseneD(1)toxicity. Similaractivitywasobserved
onreeffishrecruits(fishlarvaeundergoingmetamorphosis)ofAcanthurustriostegus: thepresence
psammaplyseneD(1)(1µg/mL)intankssignificantlyandreadilydecreasedthenumberofbitesof
recruits,suggestingarapidsaturationoftheenvironmentperceptionsensorsofthefishlarvaeduring
the72hofexperiment(Figure6).
Mar.DruMgasr.2 D01ru8g,s1 260,1184, 616, x FOR PEER REVIEW 9 of 196o f16
Mar. Drugs 2018, 16, x FOR PEER REVIEW 9 of 16
C = Control
S= Solvent
P = PsammaplyseneD
C = Control
S= Solvent
P = PsammaplyseneD
1
-h
1 .
-1-1 .hh-his .Fs
se
iFtiB
.s
e
tiB
C S P C S P C S P

Figure6.NumberoCf b i t e s So n c o r a PlpiecesperCA . t r i o s tSe g u s r e Pcr uitandpeCr h o u r . ES r r o r b Parsrepresent
Figure 6. Number of bites on coral pieces per A. triostegus recruit and per hour. Error bars represent
standarddeviationofthemean(N=6).**p<0.01.
standard deviation of the mean (N = 6). ** p < 0.01.
Figure 6. Number of bites on coral pieces per A. triostegus recruit and per hour. Error bars represent
PsamstmPansaadpmarlmyd sdaepenvlyieastDeionn(e 1 oD)f td(h1ies) mpdleiasapynl e(aNdy e=cd l6e )c.al *er*al ypr l<yb 0eb.h0e1ha. avvioiorraall eeffffeeccttss oonn bboothth gugpuppipesi easnadn rdeerf efeisfhfi lsahrvlaaer: vae:
they showed a loss of appetite from the first day of experiment. Reef fish juveniles’ behavior did not
theyshowedalossofappetitefromthefirstdayofexperiment. Reeffishjuveniles’behaviordidnot
seemPs taom bme aimplpyascetneed Dby ( 1p)s daimspmlaaypeldys celneea rDly. Ibte ihs ainvitoerreasl teinffge cttos noont eb oththa tg ounplpy iAes. tarniods treegeufs f liashrv laaer vwaeer: e
seemtobeimpactedbypsammaplyseneD.ItisinterestingtonotethatonlyA.triosteguslarvaewere
thimeyp sahcotewde bdy a t hloes sp uofr ea pppsaemtitme farpolmys ethnee fDir,s ts udgagye osft ienxgp ae rcihmaenngte. Roef esfe nfissihb ijluitvye ntoilwesa’r bdesh tahvei ocor mdipdo nuontd
impactedbythepurepsammaplyseneD,suggestingachangeofsensibilitytowardsthecompound
sedeumri ntog bteh eim mpeatcatmedo rbpyh possaism. mThaep ltyosxeicniety D t.e Istt ias ti nat ehriegsht idnogs teo (n1o0t0e μthga/mt oLn loyf Acr. utrdioes etexgturasc lta rovra peu wriefrieed
durinigmpsptahamectmemdae pbtlyay mstheoner epp uDhr)oe o spinss .athmTehm geauptpolpyxsiieecsni telye Ddt e, tssout tgahgteeaisr thdinieggah tah d cahofatsenerg (eo1 n0oe0f shµeongus/ri bmbilaLitthyoi ntfogcw rwuaridtdhes eethixteht recaroc mtthpoeo rcuprnuuddr eifi ed
psamdmeuxartiprnalgcy tts heoenr metheDet a)pmouonrirfptihehdeo sgpissu.a pTmphmiee atspoxlleyicdsietnytoe t etDsht e faritra cadt iheoiangt hh( adacouftsteeer (t1oo0xn0iec μithgyo/ meuxrLp beorafi tmchreuinndtge). weBxrittarhaincets iotarhn epdru grtiihlfliese dco rfu de
extracpttshaoemrsemt hgauepplpypsuieernsi efiw eDedr)e op ensx attrmhaecm tgeaudpp aplnyidess et hnleeed A DtCo hftrEhae caitcri todivneiaty(tha m cauefttaeesru toroenxdei cohintoy uthree bx eapxtehtrrianimcgts ew (nFitti)hg. uerBietrh 7aeairn, btsh) ea[2 nc9rd]u. dTgehil el s of
theseegxreutrspaucpltti seo wsr wethreee r cepleuearxrietfirrea wdc tiptehsd atmhanem dpautprhliyefiseAednC ep hsDaEm farmcatcaitpvioliytnys e(mnaceeu Dates, usthorexodiwcioitnyng e tahx pmeeierlixdmt breaunctt t)ss.i gB(Fnriiafgiincuasrn eatn 7ddae ,cbgr)ielal[s2s e9o ]of. f The
resulttshthewsee e argceutipcvlpietiyae rsoe wrf ewtrheie t ehexnttrhzayecmtpeedu rianinfi dteh dteh epg sAilalCsm,h mEsu aagcpgtielvysitstieynn gme aeDnas ,uascrhteiodow no nion tngh etah eemx torilaldfcatcbst uo(Frtiygs iuogrrneg ia7finacs,ba ()np [t2edr9i]ep. chTrehereaa sl eof
theacrtenisveuirtlvtyso uowsfe tsrheye sctleeenmazr)ey rtmh waeti tihcno tuthhlede p gnuiorltli sfbi,eesd uf pugsrgatehmsetmri niangpvlaeynssteiangcaet tiDeodn, stohhnoewrtehi.n eAgo algf rmaecaitltod rn byuumot rsbgiegarnn oisffi (cApaneCrth idpEeh cienrrehaailsbeni toeofrr vs ous
thtaer gaecttiivnigt y Aolfz htheiem eenrz’sy mdeis einas eth eh agviell s,b eseung giessotliantge da nf raocmti onna otunr atlh es ooulfraccetso r[y3 0o]r. gAanms o(npge ritphheemra, l 6
system)thatcouldnotbefurtherinvestigatedthere. AgreatnumberofAChEinhibitorstargeting
Alzhenicemorvmeorpu’ossu dsnyidsseste awmsei)t hthh aaavctt eicvobiuteileedsn nriaosnto glbaient gefud frrftohrmoerm IiCnn5v0a et1su ttiroga al1t0esd0o uμthrMceer ews. [eA3r0e g]i.rseAoalamt tneodun mfgrbotemhre omVfe ,Ar6oCnchgoEimi dinpa hosipubointnodgrsse sw ith
ta(rTgaebtlien g6 )A. lIzth ewimouerld’s bdei seinasteer ehsativneg baelesno itsoo lafuterdth efrr oimnv ensattiugraatel stohuorscee sc o[m30p].o uAnmdso nags tchheemm, ic6a l
activitiesrangingfromIC 1to100µMwereisolatedfromVerongiidasponges(Table6). Itwouldbe
coamntpi-opurenddast owriyt hd eafcetnivs5ie0tsie tsh rroaunggihn fgi sfhr oamnt iIfCee50d a1n tto a 1ct0i0v iμtyM, s iwnecere t hiseo fliasthe dm fordoeml iVs eearosinegr itiod as estp uopn gtheasn
intere(sTrteailbnalgtee ad6l bs).oi oItato sswfauyorsut hilndev robilenv ivniengst etsirpgeosanttigenegt h poarelssdeoa ctootorm s,f psuuorctuhhne adrs s innauvsdecisbhtiregamantciehc ast lhoaor nscetr iu-cspotramecdepaaontuson.r dys daesf enchseemsitcharlo ugh
fishaanntitfi-eperdeadnattoarcyt idveifteyn,sseins cthertohuegfihs fhishm aondtiefleeisdeanast iaecrtitvoitsye, tsuinpcet hthaen frisehla mteoddebli oisa esassaiyesr tion vseotl vuipn tghaspn onge
predarteolartse,ds ubciohasassaynsu idnivborlavnincgh sspoorncgreu psrteadceataonrss., such as nudibranchs or crustaceans.
Brain Gill
0.12 0.12
Brain Gill
0.10 0.10
10.12 0.12
-g0.08 0.08
m0.10 0.10
1. U0.06 0.06
-g0.08 0.08
m 0.04 0.04
. U0.00.602 0.00.602
0.04 0.04
0 0
0.02 0.02
Control 100 µg. mL-1 Control 100 µg. mL-1
0 0
(a)
Control 100 µg. mL-1 Control 100 µg. mL-1

(a)
Figure7.Cont.
Mar.Drugs2018,16,146 10of16
Mar. Drugs 2018, 16, x FOR PEER REVIEW 10 of 16
Brain Gill
0.10 0.10
0.08 0.08
1
-g
m0.06 0.06
.U
0.04 0.04
0.02 0.02
0 0
Control 5 µg. mL-1 Control 5 µg. mL-1

(b)
FigureFi7g.u(rae) 7A. (Ca)h AECahcEti vaicttyiv,iUty·,m Ug·m−1g,−1a, fatfetrer1 1h he exxppoossuurree ttoo 110000 µµgg/m/mL Lofo PfsPamsammamlyaselynsee Dne (1D). (B1o)x.pBlootx plot
distribduisttiroinbuwtioans mwaasd meawdiet hw5ithfi s5h fi(sNh (=N5 =) .5*). p* p< <0 0.0.055;;( (bb)) AACChhEE aaccttiivvitiyty, ,UU·m·mg−g1,− a1f,tearf t7e2r h7 2exhpoesxuproe stuor eto
5 µg/mL of crude extract. Boxplot distribution was made with 5 fish (N = 5).
5µg/mLofcrudeextract.Boxplotdistributionwasmadewith5fish(N=5).
Table 6. AChE inhibition of bromotyrosine compounds isolated from Verongiida sponges.
Table6.AChEinhibitionofbromotyrosinecompoundsisolatedfromVerongiidasponges.
Sponge/Compounds AChE Origin Activities Ref.
Pseudoceratina purpurea Eel
SpoPnugreea/Clidominpe oQu nds AChEOriginAIcCti5v0 =it i1e.s2 µM (NCI a) ORlaetfuenrejin ecte al. 2014 [31]
PseIusdooacneoramtioniaapnu Arp urea Eel IC50 = 70 µM (NCI)
PurAepalliydsiannezQine A IC50=1.2µM(NICC5I0 =a) 104 µM (NCI) Olatunjietal.2014[31]
IsoAancaonmthooiadnenAdrilla sp IC50=70µM(NHCuI)man recombinant AChE
ApHlyosamnozaineeroAthionin IC50=104µM(INCC50I =) 4.5 µM (CI b) Sirimangkalakitti et al. 2015 [32]
AcaFnisthtuodlaernidnr i1l lasp. HumanrecombIinCa50n =t A47c.h5 EµM (ND c)
Homoaerothionin IC50=4.5µM(CEIebl ) Insect recombinant Sirimangkalakittietal.2015[32]
FisUtunlaidrienn1tified Verongida AIpCly50sa=m4i7n.e5-µ4 M(KNI D= 1c6) µM (NCI) KI = 2 µM (NCI) Sepcic et al. 2001 [33]
UnidentifiedVa eNroCngI:i dnaon coEmelpetitive inhibition; b CI: coInmsepcettrieticvoem ibninhaibntition; c NSDep: nciocnet dael.s2c0r0ib1e[d33. ]
Aplysamine-4 KI=16µM(NCI) KI=2µM(NCI)
A commaNonC Is:indoen ecfofmecpt eotift iAveCihnhEi binithioinb;itbioCnI: ctroemaptmeteitnivtes iinsh aibniotiroenx;icaN [3D4:,n3o5n], dweshcircibhe dst.rengthens the
importance of natural AChE inhibitors as potential deterrents of predators such as fishes.
AFucrothmemrmoonres, idthee eofflfeaccttoorfyA dCyhsfEunincthioibn itiino nAltzrheeaitmmeern’st sdiisseaanseo rheaxsi aal[s3o4 ,b3e5e]n, wdehmicohnssttrraetnedg th[3e6n].s the
imporPtasanmcemoafpnlyasteunrea lDA Cish tEhein mhiabjoitro rcsomasppooutnedn toiaf ltdhee tesprroenngtes o(0f.p66r%ed awt/owr)s. sIut chhaas sbfieeshn essh.oFwunrt htheramt ore,
theolftaycrotosirnyed dyesrfiuvendc tcioomnpinouAnldzsh aeriem leorc’asteddi siena sspehhaaersuasl scoellbse oefn thdee smpoonngster, aatlelodw[3in6g] .thPesiarm remleaaspel yins etnhee Dis
aquifer channels and further in the close environment of the sponge [37]. It would be thus interesting
themajorcompoundofthesponge(0.66%w/w). Ithasbeenshownthattyrosinederivedcompounds
to track and quantify the concentration of this compound in the close reef environmental water of
arelocatedinsphaeruscellsofthesponge,allowingtheirreleaseintheaquiferchannelsandfurther
the sponges, using for example, solid phase adsorption toxin tracking (SPATT) [38], to allow the on
inthecloseenvironmentofthesponge[37]. Itwouldbethusinterestingtotrackandquantifythe
field deterrent or reef protective activity of this compound acting softly on olfactory sense of
concentrationofthiscompoundintheclosereefenvironmentalwaterofthesponges,usingforexample,
predators in the marine environment. The importance of AChE inhibitors in the marine environment
solidpcahna bsee aindcsluodrpedti oinn nteouxrionetcroalocgkyin ogf (cShPeAmTicTal) d[3e8fe],ntsoesa, lal omwertghiengo nfiefilde lodf dcheetemrirceanl teocorloregeyf, pwrhoitcehc tive
actividtyisoplfatyhsi sthceo mimppoourntadncaec toinf gthseo fltilnykos nbeotlwfaecetno reycosleongsye aonfdp reevdoalutotirosni, nsethcoenmdaarryi nmeeetnabvoirliotensm, ent.
Theimbephoarvtiaonr,c aenodf nAeuCrhosEciiennhceibs i[t3o9r]s. inthemarineenvironmentcanbeincludedinneuroecologyof
chemicaldefenses,amergingfieldofchemicalecology,whichdisplaystheimportanceofthelinks
3. Material and Methods
betweenecologyandevolution,secondarymetabolites,behavior,andneurosciences[39].
3.1. General Procedures
3. MaterialandMethods
Optical rotations were measured using MCP-300 polarimeter (Anton Paar, Les Ulis, France). IR
3.1. GsepneecrtarlaP wroecreed ruerceosrded on a BX FT-IR spectrometer (Perkin Elmer, Courtaboeuf, France). 1D and 2D
NMR spectra were recorded on a Avance 500 and 600 MHz (CNRS-ICSN, Bruker, Wissembourg,
Optical rotations were measured using MCP-300 polarimeter (Anton Paar, Les Ulis, France).
France). The chemical shifts are relative to the residual signal solvent (CD3OD: δH 3.31; δC 49.20;
IRspectrawererecordedonaBXFT-IRspectrometer(PerkinElmer,Courtaboeuf,France). 1Dand
DMF-d7: δH 8.03, 2.92, 2.75; δC 163.2, 34.9, 29.8). High-resolution mass spectra were obtained on a LCT
2DNMRspectrawererecordedonaAvance500and600MHz(CNRS-ICSN,Bruker,Wissembourg,
Premier XE spectrometer (Waters, Guyancourt, France) in electrospray ionization mode by direct
France). The chemical shifts are relative to the residual signal solvent (CD OD: δ 3.31; δ 49.20;
3 H C
DMF- d7: δH 8.03, 2.92, 2.75; δC 163.2, 34.9, 29.8). High-resolution mass spectra were obtained on
a LCT Premier XE spectrometer (Waters, Guyancourt, France) in electrospray ionization mode by
directinfusionofthepurifiedcompounds. PreparativeHPLCwasperformedusingAutoPrepsystem