Bioactive Pyridone Alkaloids from a Deep-Sea-Derived Fungus Arthrinium sp. UJNMF0008

14 Pages·2017·2.77 MB·English

marine drugs
Article
Bioactive Pyridone Alkaloids from a
Deep-Sea-Derived Fungus Arthrinium sp. UJNMF0008
JieBao1,HuijuanZhai1,KongkaiZhu1,Jin-HaiYu1,YuyingZhang1,YinyinWang1,
Cheng-ShiJiang1 ID,XiaoyongZhang2,YunZhang3andHuaZhang1,*
1 SchoolofBiologicalScienceandTechnology,UniversityofJinan,336WestRoadofNanXinzhuang,
Jinan250022,China;[email protected](J.B.);[email protected](H.Z.);[email protected](K.Z.);
[email protected](J.-H.Y.);[email protected](Y.Z.);[email protected](Y.W.);
[email protected](C.-S.J.)
2 CollegeofMarineSciences,SouthChinaAgriculturalUniversity,483WushanRoad,Guangzhou510642,
China;[email protected]
3 KeyLaboratoryofTropicalMarineBio-ResourcesandEcology,SouthChinaSeaInstituteofOceanology,
ChineseAcademyofSciences,164WestXingangRoad,Guangzhou510301,China;[email protected]
* Correspondence:[email protected];Tel.:[phone]
(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:29April2018;Accepted:18May2018;Published:22May2018
Abstract: Eightnew4-hydroxy-2-pyridonealkaloidsarthpyronesD–K(1–8),alongwithtwoknown
analoguesapiosporamide(9)andarthpyroneB(10),wereisolatedfromadeep-sea-derivedfungus
Arthriniumsp. UJNMF0008. Thestructuresoftheisolatedcompoundswereelucidatedonthebasis
of spectroscopic methods with that of 1 being established by chemical transformation and X-ray
diffractionanalysis. Compounds1and2boreanesterfunctionalitylinkingthepyridoneanddecalin
moietiesfirstreportedinthisclassofmetabolites,while3and4incorporatedararenaturalhexa-or
tetrahydrobenzofuro[3,2-c]pyridin-3(2H)-onemotif. Compounds3–6and9exhibitedmoderateto
significantantibacterialactivityagainstMycobacteriumsmegmatisandStaphylococcusaureuswithIC
50
valuesrangingfrom1.66–42.8µM,while9displayedcytotoxicityagainsttwohumanosteosarcoma
celllines(U2OSandMG63)withIC valuesof19.3and11.7µM,respectively.
50
Keywords: Arthrinium;pyridonealkaloid;antibacterialactivity;cytotoxicity
1. Introduction
Theincreasingantibioticresistanceandhighspreadingrateofpathogenicbacteriahavebecome
severe public healthcare threats globally, and the efforts towards new antibacterial drugs remain
a pressing task [1]. Marine fungi have been considered to be an invaluable resource for bioactive
compoundsandplayanimportantroleinthesearchfornovelantimicrobialcompounds[1–3].Fungiof
thegenusArthriniumarewidelydistributedthroughouttheworldandhaveproventoproducediverse
secondarymetaboliteswithavarietyofbioactivities,includingcytotoxicity[4–7],antimicrobial[8],
anti-HSV[9],AChEinhibitory[4],COX-2inhibitory[10],syncytiumformationinhibitory[11]and
Maxi-Kchannelmodulatory[12]activities,aswellaslethalityagainstbrineshrimp[13].
Duringthecourseofourongoingpursuitforantimicrobialcompoundsfrommarine-derived
fungi,anArthriniumsp.UJNMF0008fromadeep-seasedimentsample,collectedintheSouthChinaSea
(17◦55(cid:48)00(cid:48)(cid:48)N,115◦55(cid:48)31(cid:48)(cid:48)E;3858mdepth),cametoourattentionowingtoitsstronginhibitoryactivity
againstStaphylococcusaureus. Furtherchemicalinvestigationonalarge-scalecultureofthisfungal
strainledtotheisolationofeightpreviouslyunreported4-hydroxy-2-pyridonealkaloids. arthpyrones
D–K (1–8), together with two previously reported co-metabolites, apiosporamide (9) [14,15] and
arthpyroneB(10)[4]. Thestructures(Figure1)oftheisolatedcompoundswerecharacterizedonthe
Mar.Drugs2018,16,174;doi:10.3390/md16050174 www.mdpi.com/journal/marinedrugs
Mar. Drugs 2018, 16, x 2 of 14
fungal strain led to the isolation of eight previously unreported 4-hydroxy-2-pyridone alkaloids.
Mar.Drugs2018,16,174 2of14
arthpyrones D–K (1–8), together with two previously reported co-metabolites, apiosporamide (9)
[14,15] and arthpyrone B (10) [4]. The structures (Figure 1) of the isolated compounds were
bchaasirsacotfecroizmedp reohne nstihvee sbpaescitsr osocfo pciocmanparleyhseenss,iavned tshpeeicrtraobsscoolpuitce coannafilgyuseras,t ioannsdw etrheeairs siagbnseodlubtye
dcoifnfefirgeunrtamtioeanns siwncelrued inagssEigCnDedc ombyp ardisioffne,recnhte mmicaealntrsa nsinfocrlumdaitniogn aEnCdDX -rcaoymcpryasrtiasollno,g racphhemy.iAcalll
tthraenissfoolramteastwioenr eatnedst eXd-rfaoyr thcreyirstaanlltoimgriacprohbyi.a lA,clly ttohteo xiiscoalantdesa cweteyrlec htoelsitneeds teforar seth(eAirC hanEt)iminihcirboibtoiaryl,
acycttiovtiotxieics. aTnhde aacesstyalychreosliunletssteesrtaasbel i(sAhCedhEth) ianth3i–b6itaonryd a9ctwiveirtieems. iTldhet oasssiagyn irfiescuanltts aensttaibbalicstheerdia lthaagte 3n–t6s
aagnadi n9s twMeryec ombailcdte rtoiu msigsnmifeigcmanatt iasnatnibdacSt.earuiarle uasg,ewnthsi laeg9aisnhsot wMeydcombaocdteerriautme csmyteogtmoxaitcisi taynadg aSi.n asutrtewuso,
hwuhmilea n9 oshstoewoseadr cmomodaecrealtlel icnyetsotUox2OiciStya nadgaMinGst6 t3w. o human osteosarcoma cell lines U2OS and MG63.
24 23OHH22 O 1 3 11 HO 22OH OH O HO OHH
21 O H OH H 21 O O
OH 13 H
19 O 9 10 5 24 20 19 O 20 19
HO 20 H H H H
14 HO HO H H
17 N15 O 12 7 17 N O N O
H H H
1 2 3
HO OH OR2 R1
O
HO OH HO
O O OH O OH O
H H H
R2
H H R1O H H HO H H
N O N O N O
H H H
4 5 R1= H, R2= Me 7 R1= OH, R2= H
6 R1= Me, R2= H 8 R1= H, R2= OH
OH
O
HO
OH O O O
H OH H
HO H H HO H H
N O N O
H H
9 10
FFiigguurree 11.. CChheemmiiccaall ssttrruuccttuurreess ooff 11––1100..
2. Results and discussion
2. Resultsanddiscussion
22..11.. SSttrruuccttuurree EElluucciiddaattiioonn
CCoommppoouunndd 11 wwaass oobbttaaiinneedda assw whhitietep poowwddeer.r.T ThheeH HRR-E-ESISMIMSSio inonfo fro1r 1a tamt m/z/z4 44444.2.0230232( [(M[M− −H H]]−−,,
ccaallccdd.. 444444..22002288)) ssuuggggeesstteedda am moolelceuculalrarfo fromrmulualao foCf 2C4H24H313N1NOO7,7,w whhicihchw wasas1 61a6m amumu omreorteh atnhatnh atthoaft iotsf
citos- mcoe-tmabeotalibteolaitreth aprythropnyeroBn(e1 0B) ([140],)s [u4p],p sourtpivpeorotfivaen oofx yange onxaytegdenaantaeldo gaunea.lDogeutaei.l eDdeatanialelyds iasnoaflythseis1 oHf
athned 11H3C aNndM 1R3Cd NatMa(RTa dbaletas 1(Taanbdle2s) 1in adnicda t2e)d inthdaitca1tiendc othrpaot r1a itnedcoarpskoerlaetteodn as ismkielalerttoont hsiamtoilfaarr ttoh pthyarot noef
Bar(t1h0p)y[r4o],nwe iBth (1a0l)i k[e4l]y, waditdhi taio lnikaleleys taedrdgirtoiounpaal sessuteprp gorroteudp bays tshuepNpoMrtRedd ibffye rtehnec eNsMofRt hdeifrfeemreanrckeasb olyf
uthpefi reelmdashrkifatbedly Cu-p1f3ierleds osnhaifntecde (Cfr-o1m3 r2e0so8n.4ainnce1 0(ftroom17 22.098i.n4 i1n) a1n0 dtoo f17a2d.j9a cinen 1t)C a-n9d, Co-f1 a4d,jCa-c1e5n,t CC--197,
aCn-1d4C, C-1-915s,i gCn-a1l7s .aTnhdi sCd-1e9d suicgtnioanls.w Tahsisfu drethdeurctcioonnfi wrmase dfubrtyhienrs cpoencftiiromneodf 2bDy iNnsMpeRctdioatna .oTf h2De CNOMSYR
cdoartrae. laTthioen CsO(FSiYgu croer2re)loafti1onrse v(eFaigleudret w2)o osfp i1n rceovuepalleindg tswyost esmpins fcrooumplHin2g-1 s–yHs-t1em0(si nfrcolumd iHng2-1b–rHan-c1h0
f(rinacglmudeinntgs frbormancHh- 3f–rHag3m-1e1natns dfrHo-m8– HH3--31–2H)3o-1f1a daencda liHn-m8–oHie3t-y12a)n doff rao mdeHca-2li1n– Hm2o-2ie5t.yS uabnsde qfuroenmt
eHx-a2m1–iHna2t-i2o5n. oSfuHbsMeqBuCendta teax(aFmiginuaretio2n) reovf eHalMedBkCe ydcaotarr e(lFaitgiuonres f2r)o mrevHe-a1l7edto kCe-y1 4c,oCrr-1e5la,tCio-n18s ,fCro-1m9
aHn-d17C t-o2 0C,-H14-,2 C1-t1o5C, C-1-81,8C, C-2-019a nanddC C-2-250,,H H-2-221t otoC C-1-918a, nCd-2H0 -a9ntdo CC--2153,, Hle-a2d2i ntog Cto-1th9 eancodn Hst-r9u tcoti oCn-1o3f,
Fleraadgimnge nttos Athea ncdonBsttrhuacttiwoner eofi dFernatgicmalentotst hAo saenidn 1B0 .thTaht ewtweroe firdaegnmtiecnatls two etrheolsien kined 1v0i.a Tahne etswteor
bfroangdmaesndtse mwoernes tlriantkeeddb yvitah eanM eSstdeirf fberoenndc ea(sa mdeum1o6n)satnradte1d3C bNy MthRe cMheSm diicfafelrsehnifctev (aarmiauti o1n6s) faonrdC -113C3
(N∆Mδ R− c3h5e.m5)icaanld shCi-f1t 4v(a∆riδati1o0n.s9 )fobre tCw-1ee3n (Δ1δaCn −d3150.5.)T ahnedp Cla-n1a4r (ΔstδruC c1t0u.r9e) obfet1wweaesnt 1h uasndes 1ta0b. lTishhee pdlwanitahr
C C
astnruecsttuerreb roifd 1g ewbaest wtheuesn etshtaebpliysrhieddo nweitahn dand eesctaelrin bmridogieet ibeest.ween the pyridone and decalin moieties.
Therelativestereochemistryof1wasassignedtobeidenticaltothatofarthpyroneB(10)based
o nanalysesofNOESYdataand1H–1Hcouplings. Morespecifically,thekeyNOESYcorrelationsfrom
Mar.Drugs2018,16,174 3of14
H-10(δ 1.39)tobothH-4b(δ 0.78)andH -12(δ 1.11)(1,3-diaxialrelationship)suggestedthatH-4b,
H H 3 H
H-10andCH -12wereaxiallylocatedonthesameside. Meanwhile,thecorrelationsfromH-5(δ 1.80)
3 H
tobothH-3(δ 1.52)andH-9(δ 2.90)alsoindicatedtheiraxialandco-facialpositions. Therelative
H H
configuration of Fragment B was thus defined with a trans-conjunction. In addition, the relative
configuration of Fragment A was supported by the NOESY correlations from H-25a (δ 1.86) to
H
bothH-21(δ 3.74)andH-23(δ 3.84), aswellasJ (2.4Hz)andJ (2.4Hz). Unfortunately,
H H 21,22 22,23
the stereochemical relationship between Fragments A and B could not be determined due to the
interruption of the achiral pyridone unit. However, a further transformation of 10 to 1 (see the
ExperimentalSection)viaBaeyer–Villigeroxidationconnectedthechemicalrelationshipbetweenthe
twoco-metabolitesandalsoconfirmedthestructuralassignment. Finally,asuitablesinglecrystalof
1wasobtainedfromtheMeOH-H ObinarysystemandsubjecttoX-raydiffractionanalysis,which
2
notonlyverifiedthestructureof1,butalsoestablisheditsabsolutestereochemistry(Figure3)as3R,
5S,8R,9R,10R,20R,21S,22S,23S(Flackparameter,0.0(3)). Compound1wasthusunambiguously
characterizedandwasnamedarthpyroneD,followingarthpyronesA−C,isolatedfromanotherfungus
ofthesamegenus[4]. ItisinterestingtopointoutthattheassignedC-20configurationfor1isopposite
thatdescribedforarthpyronesAandB[4]. BycarefulexaminationofthereportedORTEPviewof
arthpyroneA,itisclearthatbotharthpyronesAandBalsobeara20Rabsoluteconfiguration,whilethe
authorsinvertedtheC-20stereochemistrybymistakewhendrawingthestructuresintwodimensions.
Table1.1HNMR(600MHz)datafor1–4(CD OD).
3
1 2 3 4
Positon
δH,mult.(JinHz) δH,mult.(JinHz) δH,mult.(JinHz) δH,mult.(JinHz)
1a 2.11,m 2.07,m 1.93,m 1.98,m
1b 1.08,m 1.07,m 0.90,m 0.96,m
2a 1.78,m 1.79,m 1.76,m 1.75,m
2b 1.01,m 1.01,m 1.05,m 1.03,m
3 1.52,m 1.54,m 1.52,m 1.49,m
4a 1.74,m 1.77,m 1.76,m 1.73,m
4b 0.78,q(12.2) 0.78,q(12.4) 0.81,q(12.3) 0.79,q(12.4)
5 1.80,m 1.82,m 1.83,m 1.84,m
6 5.41,brd(9.9) 5.41,brd(9.9) 5.41,brd(9.8) 5.38,brd(9.8)
7 5.61,ddd(9.9,4.5,2.8) 5.62,ddd(9.9,4.4,2.8) 5.61,m 5.57,ddd(9.8,4.5,2.7)
8 2.76,m 2.79,m 2.84,m 2.73,m
9 2.90,dd(11.7,5.9) 2.88,dd(11.6,5.9) 4.44,dd(11.3,5.8) 4.07,dd(11.3,5.7)
10 1.39,qd(11.3,2.5) 1.38,qd(11.5,2.3) 1.57,qd(11.2,2.0) 1.54,qd(11.3,2.6)
11 0.93,d(6.6) 0.93,d(6.6) 0.94,d(6.5) 0.92,d(6.5)
12 1.11,d(7.0) 1.08,d(7.1) 0.83,d(7.2) 0.76,d(7.1)
17 7.38,s 7.31,s 7.69,s 7.96,s
21 3.74,d(2.4) 3.71,d(7.0) 5.15,d(10.1)
22 4.60,brs 3.62,dd(7.0,6.2) 3.91,dd(10.1,2.9) 4.68,d(3.8)
23 3.84,ddd(11.6,5.1,2.4) 3.69,m 4.09,m 3.95,ddd(10.8,3.8,3.2)
24a 1.86,m 2.12,m 2.07,m 2.03,m
24b 1.34,m 1.54,m 1.72,m 1.89,m
25a 1.86,m 2.43,ddd(14.1,7.9,3.7) 2.88,m 2.69,m
25b 1.75,m 1.67,ddd(14.1,9.3,3.8) 1.51,m 2.51,m
Mar.Drugs2018,16,174 4of14
Mar. Drugs 2018, 16, x 4 of 14
Mar. Drugs 2018, 16, x 4 of 14

FFiigguurree 22.. KKeeyy 22DD NNMMRR ccoorrrreellaattiioonnss ffoorr 11..
Figure 2. Key 2D NMR correlations for 1.

Figure 3. ORTEP view of 1.
FFiigguurree3 3.. OORRTTEEPPv viieewwo off1 1..
Compound 2 was isolated as a pale yellow powder, and its molecular formula C24H33NO8 was
estabClCisoohmmepdpo ofuruonnmdd 2t2hw we aHassRi si-soEolSalatIeMteddSa aisosan a p apat almele/y zye l4ell6ol2ow.w2p1 4po3ow w(d[Mdere, r−a, nHadn]d−i,t sictamsl cmodlo.e lc4eu6c2lua.l2ra1fr3o f3rom),r mubeluainlaCg C 12H84H m3N3aNsOsO u8 wnwiatasss
24 33 8
meessottarabeb llitishshahened dt hffrraootm mof tt hh1e.e THHhRRe-- EENSSMIIMMRSS d iiaootnna aaottf m m2/ /zr ze4s64e26m.22b.12l41e34d 3 ([tMh([oM s−e −H o]fH− ,1 ]c −a(Tl,cacdba.l le4csd6 2.1. 24a16n32d3.2) ,21 b)3,e3 ai)n,ngbd e 1it8nh gem 1ao8sbsvm uioanusitsss
duminfofitersreem nthocaerens wtthheaarnte tothhfa e1t d.o oTfwh1.en TfNiheeMldNR sM hdiRfatteadd a ostfai g2on far2elssr eoemfs eCbml-e2bd0l e(tdδhCot h7s8eo .s5oe fio n1f 2 1( Ta(nTadbabl e7ls0e .s611 ianan n1dd) a22n)),,d aa Cnndd-2 t1thh (eeδ Coo b7bv7v.i3ioo uiunss
2dd iaifffnfeedrre eδnnCc ce7es0s. w0w eienrre e1 tt)hh, eea lddooonwwg nnwfifiieethlldd t hsshheii ffuttepeddfi essliidgg nnsaahllissf tooeffd CC r--e22s00o (n(δδaCn 7c78e8. 5.o5 fin iCn 2-2 2a2an nd(δd 7C 7070.65. 6.i3ni ni 1n)1 2)a naadnnd dC C-δ2C-1 28 1(5δ(.C2δ 7i7n7. 371 .)i3n.
C C
Fi2nu ar2tnhaden rdδ Ceδ x7a0m7.00 i.ni0na it1nio),1n a) ,looafnl ogCn OwgSiwtYhi ttdhhaetth aue p(uFfiipegfiluder lesd h4si)fh teirfedtve rdeeasrloeednsoa ntnwacneo c oesft roCuf-cC2tu2-2 r(a2δlC ( δm75o.7t3i5 fi.s3n oi2nf at2nhaden dδdCe δc8a5l.i82n5 i.an2n i1dn) .
C C C
C1F)H.urF-2tuh1re–trCh eHerx2ae-2mx5ai mnpaaitnritoa, tnwi oohnfi cohCf OCwSOeYrSe Y dthadteaa t sa(aFm(iFgeiug auresr et4h4)o )rsreeev vienea al1el.ed dT httwew opo isvsttorrutuacclt tuHurraMall BmmCoo cttioiffsrsr eoolffa ttthihoeen dsd ee(cFcaiaglliiunnr aea nn4d)d
fCCroHHm--2 21H1––-C9C HH(δ2H-- 22255. 8pp8aa)r rttto,, wwChh-1iic3chh, Hww-ee1rr7ee (ttδhhHee 7 ss.a3am1m)e et oaa ssC tt-hh1oo4s,s eeC ii-nn15 11,. .C TT-hh1e8e ,p pCiivv-o1o9tta aalln HHdMM CBB-2CC0 ccaoonrrdrree lHlaat-ti2ioo1nn (ssδ (H(FF i3igg.7uu1rr)ee t 4o4))
2
Cffrr-o1om8m, H CH--9-290( δ (aδnH2d .2 8C.88)-82t)5o t oCw -Ce1r3-e1, 3Ha,l -sH1o7- 1c(o7δ n(sδ7iHs. 3t7e1.n)3tt1 ow) Ctiot-h 1C 4t,-h1Co4-s,1 eC5 ,o-C1b5s-1,e 8rCv,-Ce1d8-1 ,f 9oCar- n11d9. TCanh-2de0r Ceafn-o2dr0e H,a tn-h2d1e H(aδf-o2r13e .m(7δ1eH)n t3toi.o7C1n-)e1 dt8o,
H H H
MCS-1 a8n, dC -N20M aRn dd iCff-e2r5e nwceerse, tahles oa bcosennsciset eonf tH wMitBh Cth coosrer eolbastieornv efdro fmor H1.- 2T2h teor eCfo-1r9e, atnhde athfoer memoeren tpioonlaerd
C-20andC-25werealsoconsistentwiththoseobservedfor1. Therefore,theaforementionedMSand
nMatSu raen odf N2 MallR s udpifpfeorretnecde tsh, eth pel aanbasre nstcreu octfu HreM oBf C2 wcoitrhreoluatt itohne ferpoomx yH b-2ri2d tgoe Cbe-1tw9 eaennd Cth-1e9 m anorde Cp-o2l2a.r
NMRdifferences,theabsenceofHMBCcorrelationfromH-22toC-19andthemorepolarnatureof2
Tnhaet urreel oatfi v2 ea llc osunpfipgourrtaetdio tnh eo pf la2n awr astsr uacstsuirgen oedf 2 owni ththoeu t bthaesi se poofx yN bOriEdSgYe bdetawtae e(nF iCg-u1r9e an4)d. CT-h2e2 .
allsupportedtheplanarstructureof2withouttheepoxybridgebetweenC-19andC-22. Therelative
cTohnefi gruerlaattiiovne ocfo tnhfei gduercaatliionn m oofi et2y wwaass idaesnsitgicnaeld to othna tt hofe 1 baass iinsd iocfa tNedO bEyS NY OdEaStYa c(oFrirgeulareti o4n)s. aTnhde
configuration of 2 was assigned on the basis of NOESY data (Figure 4). The configuration of the
sdciemocnialflaiigrnu NmraMotiiRoent dy oawft ata.h sTei hddeee ncNatOilcinaE lSmtYoo citehotrayrt ewolafat1sio iandssei nnfrtdoicimacal Htteo-d 2th5babyt (NoδfHO 1 1E a.S6s7Y i)n ctdooir cbraeotlteahdt i Hobny-2 sN1a O(nδdEH Ss3Yi.m7 c1io)l ararrneNdla MtHioR-n23sd aa(δntaHd.
3Ts.hi6me9)iN lsauOr gNEgSMeYsRtce oddr arttehale.a iTtri hoaenx siNaflOr oaEnmSdYH cco-o2-rp5rbleal(naδtairo n1n.sa6 tf7ur)ortemo, wbHoh-t2ihl5ebH t (h-δ2oH1s 1e(δ .6fr7o)3 mt.7o 1 Hb)o-a2tnh2d H(δH-H2- 123 3.(6δ(2Hδ) 3t.o73 1.b6)o9 at)nhsd uH gH-g1-e27s3 t( e(δδdHH
H H H
7t3h.3.e61i9)r) a asnxudiga lgHae-ns2td4ebdc o(tδ-hHpe li1ar.n 5aa4xr)i asnula patunprdoe r,ctewod-hp tilhlaeantta hHro n-s2ea2tf,u rHroem-,2 w4Hbh- ia2ln2e d(tδ htohse3e .p 6fy2rro)imdtoo Hnbeo- 2tuh2n H(iδt -Hw1 73e.r(6eδ2 a) x7tio.a3 lb1ly)o talhon cdHatH-e1d-72 o4(δnbH
H H
t(7hδ.e3 1o1)t. h5a4en)rd ss uiHdp-ep2 o4obfr t te(hδdeH t hh1e.a5xt4aH)n se-u2 r2pin,pHgo.r- T2te4hdbe tashtnardut cHtthu-e2r2ep ,yo Hrfi 2-d2 ow4nbae sau nhndein ttchwee e eprleuycraiixddioaatnlleeyd ul aonsci ats thweodewroenn aitnxhi eFaliolgytuh lreoerc 4as.ti dede oofn
H
the other side of the hexane ring. The structure of 2 was hence elucidated as shown in Figure 4.
thehexanering. Thestructureof2washenceelucidatedasshowninFigure4.
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Mar. Drugs 2018, 16, x 5 of 14
Mar. Drugs 2018, 16, x 5 of 14
COSY HMBC
COSY HMBC
NOESY
OH NOESY
OH 1
HO 22 OH O 25 1 33
HO 22 OHOH O 25 13
2244 HHOO2200 1199OH 111544OO 1133 99 2233 2222442211 2200 11771199 13112288 99 1100 55
17 15 22
17 N O
Fragment A HN O Fragment B
Fragment A H Fragment B

Figure 4. Key 2D NMR correlations for 2.
FFiigguurree 44.. KKeeyy 22DD NNMMRR ccoorrrreellaattiioonnss ffoorr 22..
CCoommppoouunndd 33 wwaass aassssiiggnneedd aa mmoolleeccuullaarr ffoorrmmuullaa ooff CC2244HH3311NNOO66 bbyy HHRR--EESSIIMMSS mm//zz 442288..22007744 (([[MM −−
H]−, cCalocmd.p 4o2u8n.2d0739)w, sausgagsessitginvee dofa anm iosloemcuelra orff aoprmiousplaoroafmCid24eH (931) N[1O4,615b]y. AHnRa-lEysSiIsM oSf tmhe/ Nz M42R8. 2d0a7ta4
H]−, calcd. 428.2079), suggestive of an isomer of apiosporamide (9) [14,15]. Analysis of the NMR data
((T[Mab−lesH 1] −an,dca 2lc) dfo.r4 32 8r.e2v0e7a9l)e,ds uthgeg epsrteivseenocfe aonf ais odmecearlionf mapoiioetsyp oasra imndidiceat(e9d) [b1y4 ,a1 5c]l.oAsen raelsyesmisbolafnthcee
(Tables 1 and 2) for 3 revealed the presence of a decalin moiety as indicated by a close resemblance
bNeMtwReedna taits( TaNbMlesR 1daantda 2a)nfdor t3hroesvee aolfe d9t,h eanpdr estehnec emoafina dNecMalRin dmifofieerteyncaessi nbdeitcwateeedn bythae ctlowsoe
between its NMR data and those of 9, and the main NMR differences between the two
croes-memetbalbaonlcietebs eotwbseeernveitds fNoMr tRhed aletfat-asniddet hForasgemofen9,t aAn.d Ftuhrethmear ininNspMecRtiodnif foefr eCnOceSsYb edtawtae e(nFitghueretw 5o)
co-metabolites observed for the left-side Fragment A. Further inspection of COSY data (Figure 5)
ccoon-mfiremtaebdo ltihtees eoxbissteernvceed offo rthteh edleecfatl-isnid meoFtriaf gamnden atlsAo. eFsutratbhleisrhiends pthecet isoanmoef sCpOinS Ycoduaptlain(gF isgyusrteem5)
confirmed the existence of the decalin motif and also established the same spin coupling system
fcfrrooonmmfi r HHm--e22d11––thHHe22--e22x55i s aatess n ttchheaatot fiinnth 99e.. dIInenc aaadldidndiimttiiooonnti,,f ttahhneed HHaMlMsoBBeCCs tccaoobrrlrriseehllaaettdiiootnnhsse ((sFFaiimgguuerrseep 55in)) fcfrroooummp l HiHn-g-99 s (y(δδsHHt e 44m..444f4r))o tmtoo
CHC---112331 –aaHnndd2- C2C5--11a44s vtvheearritiffiiineedd9 .tthhInee accodondnnniteeiocctntii,ootnnh ebbeeHttwwMeeBeeCnn FcForrararggemmlaeetinnottnss s AA(F aaingnuddr BeB,,5 ww)fhhrioillmee tthHhoo-s9see( δffrHroom4m.4 HH4)--1t1o77 C((δδ-H1H 3 77a..66n99d))
tCtoo- 1CC4--11v44e,,r iCCfi--e11d55,,t hCCe--11c8o8,n, CCne--11c9t9i oaannnddb e CCtw--22e00e,, n aalFloornnaggg m wweiintthhts ttAhhooassneed ffrrBoo,mmw hHHil--e2255thbbo ((sδδeHHf r11o..55m11))H ttoo-1 CC7--(22δ0H0 aa7nn.6dd9 )CCt--o221C1,,- e1en4n,aaCbbl-le1ed5d,
tCh-e1 8a,ssCe-m19balyn dofC th-2e0 ,paylroindgonwe irthintgh oansedf trhoem cHyc-l2o5hbex(δaHne1 .m51o)ietotyC, -a2s0 waenldl aCs- 2th1e,iern lainbkleadgeth feroamss eCm-1b8l–y
the assembly of the pyridone ring and the cyclohexane moiety, as well as their linkage from C-18–
Cof-2th0.e pMyroirdeoonveerr,i ngfoaunrd tehxecchyacnlgoehaebxlaen epmrootioentys, aswwereel laosbtsheerivreldin kaing eftrhoem 1CH-1 8N–CM-2R0 . Mspoercetoruvmer,
C-20. Moreover, four exchangeable protons were observed in the 1H NMR spectrum
(f(SoSuuupprppellxeecmmheaennntgtaaerrayyb IIlnneffoporrrmmotaaottniioosnnw TTeaarbbelleoe bSSs11e)),r, vmmeeedaassinuurrteehdde ii1nnH DDMNMMSSOOR--dds66p,, ewwchthriiucchhm ww(Seerureep aapsslsseiimggnneeenddta ffrooyrr NINnHHfo,,r 2m200a--OOtiHoHn,,
2T2a-bOleHS 1a)n,md e2a3s-uOrHed, irnesDpMecStiOve-dly6,. wThhiisc hwwaesr esuapsspiogrnteedd fboyr NHHM,B2C0- OaHnd, 2C2-OOSHY acnodrr2e3la-OtioHn,sr e(sFpigecutrive e5ly) .
22-OH and 23-OH, respectively. This was supported by HMBC and COSY correlations (Figure 5)
fTrhoims wNaHs tsou pHp-1o7rt, e2d0-bOyHH tMo CB-C20a,n Hd-2C2O tSoY 22c-oOrrHe laantido nHs-(2F3i gtou r2e3-5O)Hfr.o TmheN aHbotoveH-m-1e7n,t2io0n-OedH atnoalCy-s2e0s,
from NH to H-17, 20-OH to C-20, H-22 to 22-OH and H-23 to 23-OH. The above-mentioned analyses
aHc-c2o2utnote2d2 -fOoHr aalnl dbuHt -o23neto o2x3y-gOeHn .aTtohmea, bwohviec-hm wenatsi oansecdribanedal ytos etshaec cfoorumntaetdiofno rofa lalnb uetpoonxey obxryidggene
accounted for all but one oxygen atom, which was ascribed to the formation of an epoxy bridge
babeteottwwmee,eewnn h CCic--h1199w aaannsdda CsCc--r22i1b1,,e adasst owwtaahsse aafllossorom ssuautppipopnoorrotteefdda nbbyye p ttohhxee y ddboorwwidnngffieieelbldde tsswhhieiffetteneddC cc-h1he9emmaniiccdaallC ss-hh2ii1ff,tt aoosff wCCa--11s99a (l(δsδoCC
1s1u777p7..p22)o) raatnenddd buuypptffhiieeellddd osswhhiinffttfieeeddl d cchhsheemimftiieccdaallc hsshheimifftti cooaffl CCsh--2i2f11t o((δfδCCC 66-1779..77())δ ccCoo1mm7p7p.aa2rr)eeaddn dwwuiittphhfi ttehhlodossseeh iiinnft e22d ((cTThaaebbmlleei c22a))l.. sTThhhifeet
roeflaCt-i2v1e (cδoCn6f7ig.7u)rcaotimonp aorfe d3 wwitahs thesotsaebliinsh2e(dT avbilae 2a)n.aTlhyseerse loaft ivNeOcEonSYfig duaratati o(nFiogfu3rew 5a)s aesntda b1lHis–h1eHd
relative configuration of 3 was established via analyses of NOESY data (Figure 5) and 1H–1H
cvoiaupalninalgyss e(TsaobfleN 1O)E. STYhed saitmai(lFairgituyr ebe5t)waenedn 1tHhe– 11HH cNouMpRli nsgpsec(tTraab olef 1th).e Tdheecasliimn ilmaroiiteytyb eotfw 3e eanndth 9e
couplings (Table 1). The similarity between the 1H NMR spectra of the decalin moiety of 3 and 9
s1uHpNpoMrtReds ptheec tcroamomftohne rdeelactailvine smteorieeotcyhoefm3isatnryd, a9ss wupaps oarlsteod cothnefircmomedm boyn NreOlaEtSivYe csotrerreeloactihoenms.i sTthrye,
supported the common relative stereochemistry, as was also confirmed by NOESY correlations. The
carsuwciaasl aNlsOoEcSoYn ficromrreedlabtiyonNsO frEoSmY Hco-r2r1e–laHti-o2n5sa. aTnhde Hcr-u2c2i–aHlN-2O4aE SinYdcicoarrteedla ttihoen 1s,f3r-odmiaxHia-2l 1r–eHla-t2io5nasahnipd
crucial NOESY correlations from H-21–H-25a and H-22–H-24a indicated the 1,3-diaxial relationship
oHoff - t2thh2ee– Httww-2oo4 paprrionotdtooinnca ppteaadiirrstsh,, eww1hh,i3icc-hhd iiiannx iccaoolmmrebblaiinntiaaottniioosnhn i wpwioitthfh t ththheeet wpprroootptoornno tccooonuupppalliiinnrsgg, wccoohnnissctthaanninttssc ooofmf JJb2211i,,n2222a ((t11io00n..11 w HHiztzh))
atahnnedd p JJ2r22o2,,2t233o (n(22.c.99o HuHpzzl))i nddgeeffciionnneedsdt atthhneets rroeelflaaJtt2ii1vv,e2e2 cc(oo1nn0ff.i1iggHuurrzaa)ttiaioonnnd ooJf2f 2tt,hh23ee (pp2oo.9llyyHooxzxy)ygdgeeenfinaantteeeddd tcchyyecclrlooehlhaeetxxivaaennece o mmnofioiigeeuttyyr.a. tTTihohnee
sotfruthcetuproe lyoxyogfe nated3 cyclowheaxs anemthoeierteyb.yT hestcrhuacrtuacreteorifz3edw asthteor ebycfheaartaucrtee rizedat ofeartuarree
structure of 3 was thereby characterized to feature a rare
haerxaarheyhderxoabheyndzroofbuernoz[3o,f2u-rco]p[3y,r2i-dc]ipn-y3r(i2dHin)--3o(n2eH )f-roangemferangt mweintthw vitehryv efreywfe wexeaxmamplpesle srereppoortretedd ffrroomm
hexahydrobenzofuro[3,2-c]pyridin-3(2H)-one fragment with very few examples reported from
nnaattuurree [[1166––1188]]..
nature [16–18].

FFFiiiggguuurrreee 555... KKKeeeyyy 222DDD NNNMMMRRR cccooorrrrrreeelllaaatttiiiooonnnsss fffooorrr 333 (((pppiiinnnkkk cccooorrrrrreeelllaaatttiiiooonnnsss ooobbbssseeerrrvvveeeddd iiinnn DDDMMMSSSOOO---ddd666)))...
Mar.Drugs2018,16,174 6of14
Table2.13CNMR(150MHz)datafor1–8(CD OD).
3
1 2 3 4 5 6 7 8
Position
δc,Type δc,Type δc,Type δc,Type δc,Type δc,Type δc,Type δc,Type
1 30.7,CH2 30.8,CH2 31.0,CH2 30.8,CH2 31.0,CH2 31.0,CH2 30.4,CH2 30.4,CH2
2 36.5,CH2 36.5,CH2 36.6,CH2 36.7,CH2 36.6,CH2 36.6,CH2 30.8,CH2 34.6,CH2
3 34.3,CH 34.3,CH 34.4,CH 34.4,CH 34.4,CH 34.4,CH 42.0,CH 41.7,CH
4 43.0,CH2 43.0,CH2 43.2,CH2 43.2,CH2 43.2,CH2 43.2,CH2 37.4,CH2 79.6,CH
5 43.2,CH 43.2,CH 43.2,CH 43.4,CH 43.2,CH 43.2,CH 42.7,CH 49.8,CH
6 131.6,CH 131.6,CH 131.7,CH 131.7,CH 131.7,CH 131.7,CH 131.6,CH 127.1,CH
7 132.2,CH 132.2,CH 132.7,CH 132.7,CH 132.7,CH 132.6,CH 132.8,CH 133.3,CH
8 33.8,CH 33.7,CH 32.4,CH 32.9,CH 32.5,CH 32.5,CH 32.4,CH 32.0,CH
9 50.3,CH 50.3,CH 54.2,CH 55.5,CH 54.3,CH 54.3,CH 54.2,CH 54.1,CH
10 37.7,CH 37.7,CH 37.6,CH 38.0,CH 37.6,CH 37.6,CH 37.9,CH 35.9,CH
11 22.9,CH3 22.9,CH3 23.0,CH3 23.0,CH3 23.0,CH3 23.0,CH3 68.6,CH2 19.3,CH3
12 18.3,CH3 18.2,CH3 18.3,CH3 18.3,CH3 18.4,CH3 18.4,CH3 18.4,CH3 18.3,CH3
13 172.9,C 173.2,C 212.1,C 203.8,C 211.9,C 211.9,C 211.8,C 211.6,C
14 123.8,C 126.4,C 108.1,C 110.7,C 108.9,C 108.5,C 108.7,C 108.8,C
15 158.2,C 160.7,C 164.3,C 163.4,C 163.7,C 164.0,C 163.9,C 164.0,C
17 129.8,CH 132.2,CH 141.0,CH 133.3,CH 143.2,CH 144.2,CH 139.9,CH 139.9,CH
18 115.6,C 115.7,C 118.3,C 115.4,C 114.3,C 111.4,C 116.6,C 116.7,C
19 160.8,C 161.9,C 177.2,C 166.4,C 179.8,C 179.5,C 179.3,C 179.4,C
20 70.6,C 78.5,C 76.9,C 116.6,C 76.3,C 82.4,C 70.4,C 70.4,C
21 70.0,CH 77.3,CH 67.7,CH 156.2,C 78.7,CH 75.2,CH 60.5,CH 60.5,CH
22 85.2,CH 75.3,CH 74.1,CH 65.3,CH 86.6,CH 75.2,CH 57.6,CH 57.6,CH
23 71.6,CH 72.6,CH 71.3,CH 70.8,CH 72.4,CH 72.4,CH 67.1,CH 67.1,CH
24 27.7,CH2 27.6,CH2 27.1,CH2 26.7,CH2 28.2,CH2 26.1,CH2 25.8,CH2 25.8,CH2
25 37.6,CH2 31.1,CH2 31.2,CH2 18.4,CH2 30.4,CH2 23.7,CH2 31.6,CH2 31.6,CH2
OCH3 60.5,CH3 51.0,CH3
Themolecularformulaof4wasdeterminedasC H NO byHR-ESIMSm/z410.1974([M−H]−,
24 29 5
calcd.410.1973),indicativeofadehydratedanalogueof3.AnalysisoftheNMRdata(Tables1and2)for
4confirmedthishypothesis,withdiagnosticsignalsfortwoquaternarysp2carbons(δ 116.6and156.2)
C
in4insteadoftheoxyquaternarysp3carbonsatδ 76.9(C-20)andδ 67.7(C-21)in3. Thisobservation
C C
ledtothesuggestionthatthefuranringisformedbetweenthecyclohexadioland2-piperidonerings
andextendedtheconjugationsystemofthepyridonechromophore,whichwasfurtherconfirmedby
theUVdataof4withanabsorptionpeakred-shiftedto367nm. FurtherexaminationofCOSYand
HMBCcorrelations(SupplementaryInformationFiguresS42andS43)corroboratedtheaforementioned
structuralassignment. ThesimilarityoftheNMRdataofthedecalinmoietiesof4and3suggestedthe
commonrelativeconfigurationsasalsoconfirmedbyNOESYcorrelations(SupplementaryInformation
FigureS44). ThecrucialNOESYcorrelationfromH-23–H-25bandprotoncouplingconstantsofJ ,
2223
(3.8Hz)andJ , (10.8Hz)definedtherelativeconfigurationsatC-22andC-23asshowninFigure1.
2324a
Compounds5and6wereassignedthesamemolecularformulaofC H NO bytheirHR-ESIMS
25 35 7
m/z 460.2349 and 460.2337 ([M − H]−, calcd. 460.2341), respectively, suggestive of a pair of
isomers. The NMR data (Tables 2 and 3) for 5 revealed high similarity to those of apiosporamide
(9)[14,15], and the major differences were attributable to the shielded signals for the oxirane ring
in9insteadoftwooxymethines(δ 3.87and3.35;δ 78.7and86.6)andamethoxygroup(δ 3.63;
H C H
δ 60.5) in 5. Subsequent analysis of COSY and HMBC correlations (Supplementary Information
C
FiguresS50andS51) corroborated the aforementioned observations with a key HMBC correlation
fromthemethoxyprotonstoC-22(δ 86.6)andestablishedtheplanarstructureof5. Therelative
C
stereochemistryof5wasdeterminedtobeidenticaltothatof2basedonthecomparisonoftheirNMR
spectra, includingcrucial1H–1Hcouplingsbetweenthecorrespondingchiralcenters, whichwere
furtherconfirmedbyexaminationoftheNOESYspectrum(SupplementaryInformationFigureS52).
AnalysisoftheNMRdata(Tables2and3)for6suggestedthatitwasamethoxyregio-isomerof5,
andthemethoxygroupwasdeterminedtolocateatC-20(δ 82.4)viatheHMBCcorrelationfromthe
C
methoxyprotons(δ 3.18)toC-20. DetailedinspectionofCOSYandHMBCspectra(Supplementary
H
InformationFiguresS57andS58)verifiedtheplanarstructureof6,andtherelativeconfigurationwas
Mar.Drugs2018,16,174 7of14
alsodeterminedtobethesameasthatof5basedonanalysesofkey1H–1HcouplingsandNOESY
correlations(SupplementaryInformationFigureS59).
Compounds 7 and 8 were determined to possess the same molecular formula C H NO by
24 31 7
their HR-ESIMS m/z 444.2025 and 444.2030 ([M − H]−, calcd. 444.2028), respectively, indicative
of oxygenated analogues of apiosporamide (9) [14,15]. Analysis of the NMR data (Tables 2 and 3)
for7confirmedthishypothesiswithdiagnosticsignalsforanoxymethylene(δ 3.39, 2H; δ 68.6)
H C
in 7 instead of the CH -11 group in 9. Good resemblance between the remaining NMR data for 7
3
and9suggestedtheassignmentofcommonstructuralfeaturesbetweenthetwoco-metabolitesand
indicatedtheirsamerelativestereochemistry. Thestructurewiththerelativeconfigurationof7was
finally confirmed by further examination of 2D NMR data, especially COSY, HMBC and NOESY
spectra(SupplementaryInformationFiguresS64–S66). Similarly,theoxidationof8occurredatC-4as
supportedbytheCOSYcorrelationsfromH-4(δ 2.73)toH-3(δ 1.39)andH-5(δ 1.77),aswellasthe
H H H
HMBCcorrelationfromH -11(δ 1.03)toC-4(δ 79.6). ThecouplingconstantsofH-4(dd,J=9.9Hz)
3 H C
indicatedthatitwasaxiallylocated,andthus,thehydroxylgroupwasequatoriallyoriented. Thelow
chemicalshiftofH-4(<3.0ppm)wasduetoitsaxialposition,whichlocateditintheshieldingzone
ofC2-C3andC5-C6bonds. ThesimilaritybetweentheremainingNMRdatafor8and7,especially
key1H–1Hcouplingconstants,supportedcommonstructuralfeatures,includingconfigurationsofthe
stereocenters,betweenthetwoco-metabolites. Thestructureof8withtherelativeconfigurationwas
furtherconfirmedbyanalysisof2DNMRdata(SupplementaryInformationFiguresS71–S73).
Table3.1HNMR(600MHz)datafor5–8(CD OD).
3
5 6 7 8
Positon
δH,mult.(JinHz) δH,mult.(JinHz) δH,mult.(JinHz) δH,mult.(JinHz)
1a 1.91,m 1.91,m 1.98,m 1.86,m
1b 0.87,m 0.88,m 0.90,m 0.90,m
2a 1.74,m 1.74,m 1.81,m 1.74,m
2b 1.03,m 1.03,m 1.05,m 1.14,dq(13.1,3.5)
3 1.51,m 1.50,m 1.60,m 1.39,m
4a 1.74,m 1.74,m 1.84,m 2.73,dd(9.9,9.9)
4b 0.79,q(12.3) 0.79,q(12.4) 0.82,m
5 1.81,m 1.81,m 1.82,m 1.77,m
6 5.39,brd(9.8) 5.40,brd(9.8) 5.42,brd(9.8) 6.00,brd(10.1)
7 5.59,ddd(9.8,4.3,2.7) 5.59,ddd(9.8,4.4,2.7) 5.60,ddd(9.8,4.4,2.5) 5.68,ddd(10.1,4.4,2.6)
8 2.82,m 2.81,m 2.83,m 2.81,m
9 4.41,dd(11.3,5.8) 4.41,dd(11.1,5.6) 4.43,dd(11.3,5.7) 4.46,dd(11.2,5.8)
10 1.55,m 1.55,m 1.58,m 1.71,m
11 0.92,d(6.5) 0.92,d(6.5) 3.39,dd(6.0,1.2) 1.03,d(6.4)
12 0.81,d(7.2) 0.81,d(7.2) 0.82,d(7.2) 0.82,d(7.2)
17 8.00,s 7.77,s 7.57,s 7.56,s
21 3.87,d(7.9) 4.20,s 3.63,d(3.6) 3.63,d(3.8)
22 3.35,dd(9.8,7.9) 3.65.m 3.41,t(3.6) 3.41,dd,(3.8,2.8)
23 3.69,m 3.73,m 4.12,m 4.12,m
24a 1.91,m 1.98,m 1.81,m 1.81,m
24b 1.31,m 1.55,m 1.34,m 1.33,m
25a 2.82,m 2.59,m 2.20,m 2.20,m
1.69,ddd(14.2,10.2,
25b 1.48,m 1.76,m 1.69,m
2.8)
OCH3 3.63,s 3.18,s
Compounds9and10wereidentifiedtobethepreviouslyreportedapiosporamide[14,15]and
arthpyroneB[4],respectively,basedoncomprehensivespectroscopicanalysesincludingMS,NMR,
[α] andECD.Whiletheabsoluteconfigurationof1wasestablishedbyX-rayanalysisandchemical
D
transformation, those of the other analogues were assigned mainly by analysis of their ECD data.
Theabsoluteconfigurationsof3and5–8,whichhavethesame3-acylpyridonechromophore,were
characterizedbytheirECDspectra,whicharesimilartothatof9(Figure6),whoseabsolutestructure
Mar. Drugs 2018, 16, x 8 of 14
Mar. Drugs 2018, 16, x 8 of 14
Mar.Drugs2018,16,174 8of14
characterized by their ECD spectra, which are similar to that of 9 (Figure 6), whose absolute
characterized by their ECD spectra, which are similar to that of 9 (Figure 6), whose absolute
structure had been confirmed by total synthesis of its enantiomer [14,15]. The absolute
structure had been confirmed by total synthesis of its enantiomer [14,15]. The absolute
choandfibgeuernatcioonnsfi romf tehde bsytertoeotaclesnytenrtsh oesf i2s aonfdit s4 ewnearnet ipormopeors[e1d4, 1o5n] .bTiohgeenaebtsioc lgurtoeucnodnsfi (gSucrhaetmioen s1)o. fthe
configurations of the stereocenters of 2 and 4 were proposed on biogenetic grounds (Scheme 1).
stereocentersof2and4wereproposedonbiogeneticgrounds(Scheme1).

Figure 6. The ECD spectra of 3 and 5–9.
FFigiguurere6 6..T ThheeE ECCDDs sppeecctrtarao off3 3a anndd5 5––99..
WWiitthh aallll tthheessee mmeetatabbooliltietessi ninh hanandd,w, weew wereeraeb albelteo tpor opproopseosaep al apulsaiubsleibbleio bsyionstyhnetthiceptiact hpwatahywfaoyr
With all these metabolites in hand, we were able to propose a plausible biosynthetic pathway
ftohre ntheew naenwal oagnuaelso.gCuoesm. pCooumndpo1ucnodu l1d bceouolbdt abinee dobftraoimne1d0 fvrioamo n1e0-s tveipa Boaneey-esrt–epV ilBliageeyreorx–iVdialltiigoenr,
for the new analogues. Compound 1 could be obtained from 10 via one-step Baeyer–Villiger
woxhiidleatsieolne,c twivheiolex isdealeticotnivoef o9xaitdCat-i1o1na nodf 9C -a4t wCo-u11ld aanfdfo rCd-47 awnodu8l,dr easfpfoercdti v7e layn.dT h8e, lrikeesplyebctiiovseylnyt.h Tehtiec
oxidation, while selective oxidation of 9 at C-11 and C-4 would afford 7 and 8, respectively. The
loirkiegliyn boifo2s–y6ntisheotuictl ionreigdinin oSfc 2h–e6m ies 1o.uPtlrionteodn ainti oSnchoefmthee 1e.p Porxoytognraotuiopno fof9 twheo uelpdogxiyv egrtohuepin otef r9m wedoiualtde
likely biosynthetic origin of 2–6 is outlined in Scheme 1. Protonation of the epoxy group of 9 would
(gii)vteh atthceo iunltderumndederiagtoe in(it)r atmhaotl eccouulladr nuuncdleeorgpoh iilnictrsaumbsotlietucutiloanr bnyuc1l9e-oOpHhi,lfico llsouwbestditbuytiodne pbryo to1n9a-OtioHn,
give the intermediate (i) that could undergo intramolecular nucleophilic substitution by 19-OH,
ftoolyloiewlded3 .bSyu bsdeeqpureontotndaethioynd ratoti oynieolfd3 w3. ouSuldbsfueqrnuiesnht 4.dTehhyedinratetiromne doifa t3e (iw)ocouuldld faulsroniesxhp e4r. ieTnhcee
followed by deprotonation to yield 3. Subsequent dehydration of 3 would furnish 4. The
irnintegrmopeedniianteg b(iy) hcoyudlrdo lyaslsiso feoxllpoewrieedncbey rdinepgr ootpoennaitniogn btyo ahfyfodrrdoltyhseisi nftoelrlomweeddia tbey( idi)e,pwrhoitcohnawtiaosnn toot
intermediate (i) could also experience ring opening by hydrolysis followed by deprotonation to
aofbftoaridn ethdei ninttheermcuerdrieantet s(itui)d, wy.hTihche winatesr nmoetd oibattaei(niei)dc ionu tlhdep cruordruencet s2tuvdiayB. Taehyee irn–tVerilmligederiaotxe i(diai)t icoonuoldr
afford the intermediate (ii), which was not obtained in the current study. The intermediate (ii) could
purnoddeurgceo 2se vleiac tBivaeeymeer–thVyilllaitgioern otxoiydiaetlidon5 oorr u6.ndergo selective methylation to yield 5 or 6.
produce 2 via Baeyer–Villiger oxidation or undergo selective methylation to yield 5 or 6.

SScchheemmee 11.. PPrrooppoosseedd bbiioossyynntthheettiicc ppaatthhwwaayy ffoorr 22––66..
Scheme 1. Proposed biosynthetic pathway for 2–6.
2.2. Biological Activity
2.2. BiologicalActivity
2.2. Biological Activity
Compounds 1–10 were tested against two Gram-positive strains Mycobacterium smegmatis
Compounds1–10weretestedagainsttwoGram-positivestrainsMycobacteriumsmegmatisATCC
Compounds 1–10 were tested against two Gram-positive strains Mycobacterium smegmatis
ATCC 607 and Staphylococcus aureus ATCC 25923, two Gram-negative strains Escherichia coli ATCC
607andStaphylococcusaureusATCC25923,twoGram-negativestrainsEscherichiacoliATCC8739and
ATCC 607 and Staphylococcus aureus ATCC 25923, two Gram-negative strains Escherichia coli ATCC
8739 and Pseudomonas aeruginosa ATCC 9027 and a yeast Candida albicans ATCC10231. Compounds
PseudomonasaeruginosaATCC9027andayeastCandidaalbicansATCC10231. Compounds3–6and
8739 and Pseudomonas aeruginosa ATCC 9027 and a yeast Candida albicans ATCC10231. Compounds
39–e6x ahnibdi t9e dexahnibtiibteadct aenritaiblaacctteivriiatyl aacgtiavinitsyt aMga.isnmste gMm.a stmiseagnmdatSis. aaunrde uSs. awuirtehusI Cwithv aIClu50e svaralunegsi nragnfgrionmg
1fr3.6o–6m6– a 41n2..6d86 9µ– 4Me2x.h8(Ti bμaibMtlee d(4 Ta)a,nbwtilbeh ai4lce)t,e nwroiahnlie laeoc nftiotvhnietey t oeafsg ttaehidne scttoe Mmstep. dsom ucenogmdmspasothiuso nawdnesdd sShs.i ogawunriefieduc sas nwi5gt0inatihnf itIciCman50it cv raaonlbutiieamsl iarccartnoivbgiiitanylg
from 1.66–42.8 μM (Table 4), while none of the tested compounds showed significant antimicrobial
Mar.Drugs2018,16,174 9of14
againstE.coli,P.aeruginosaandC.albicansat50µM.Expandingourbiologicalactivityevaluations,
these isolates were also screened for AChE inhibitory and cytotoxic activities against two human
osteosarcomacelllinesU2OSandMG63. Only9displayedmildcytotoxicityagainstthetwocelllines
withIC valuesof19.3and11.7µM.
50
Table4.Antibacterialactivityof3–6and9(IC ,µM)a.
50
Compounds MS SA
3 11.4 8.97
4 >50 42.8
5 19.4 8.37
6 35.3 14.1
9 2.20 1.66
a Ceftriaxonesodiumwasusedasapositivecontrol(IC50 <1.0µM);MS:MycobacteriumsmegmatisATCC607;
SA:StaphylococcusaureusATCC25923.
3. ExperimentalSection
3.1. GeneralExperimentalProcedures
NMRspectrawererecordedonaBrukerAvanceDRX600NMRspectrometerwithresidualsolvent
peaksasreferences(CD OD:δ 3.31,δ 49.00;DMSO-d : δ 2.50,δ 39.52). Opticalrotationswere
3 H C 6 H C
measured on a Rudolph VI polarimeter. UV spectra were obtained using a Shimadzu UV-2600
spectrophotometer. The X-ray diffraction analysis was performed on an Oxford Gemini E Eos
CCD detector with graphite monochromated Cu Kα radiation (λ = 1.54178 Å). ECD spectra were
acquiredonaChirascancirculardichroismspectrometer(AppliedPhotophysics). ESIMSanalyses
wereconductedonanAgilent1260–6460TripleQuadLC-MSspectrometer. HR-ESIMSspectrawere
obtained on an Agilent 6545 Q-TOF mass spectrometer. Semipreparative HPLC separations were
carried out on an Agilent 1260 series using a YMC-Pack ODS-A column (250 × 10 mm, 5 µm).
Columnchromatography(CC)wasperformedonsilicagel(200–300mesh,YantaiJiangyouSilicaGel
DevelopmentCo.,Yantai,China),reversedphaseC silicagel(MerckKGaA,Darmstadt,Germany)
18
andSephadexLH-20gel(GEHealthcareBio-SciencesAB,Uppsala,Sweden).
3.2. FungalMaterial
ThefungusstrainUJNMF0008wasisolatedfromamarinesedimentsamplecollectedintheSouth
China Sea (17◦55(cid:48)00(cid:48)(cid:48) N, 115◦55(cid:48)31(cid:48)(cid:48) E; 3858 m depth). This strain was identified as an Arthrinium
sp. based on morphological traits and a molecular biological protocol by DNA amplification and
comparisonofitsITSregionsequencewiththeGenBankdatabase(100%similaritywithArthrinium
sp. zzz1842(HQ696050.1)). TheBLASTsequenceddataweredepositedatGenBank(No. MG010382).
ThestrainwasdepositedatChinaGeneralMicrobiologicalCultureCollectionCenter(CGMCCC),
InstituteofMicrobiology,ChineseAcademyofSciences.
3.3. FermentationandExtraction
Arthriniumsp. UJNMF0008fromaPDAcultureplatewasinoculatedin500-mLErlenmeyerflasks
containing150mLsolublestarchmedium(1%glucose,0.1%solublestarch,1%MgSO ,0.1%KH PO ,
4 2 4
0.1%peptoneand3%seasalt)at28◦Conarotaryshakerat180rpmfor3daysasseedcultures. Then,
eachoftheseedcultures(20mL)wastransferredintoautoclaved1-LErlenmeyerflaskswithsolidrice
medium(eachflaskscontained80gcommerciallyavailablerice,0.4gyeastextract,0.4gglucose,and
120mLwaterwith3%seasalt). Afterthat,thestrainwasincubatedstaticallyfor30daysat28◦C.
Afterfermentation,thetotal4.8kgriceculturewascrushedandextractedwith15.0L95%EtOH
threetimes. TheEtOHextractwasevaporatedunderreducedpressuretoaffordanaqueoussolution
andthenextractedwith2.0Lethylacetatethreetimestogive80gcrudegum.
Mar.Drugs2018,16,174 10of14
3.4. IsolationandPurification
TheextractwasfractionatedbyasilicagelcolumnelutingwithstepgradientCH Cl -MeOH
2 2
(v/v100:0,98:2,95:5,90:10,80:20,70:30,50:50and0:100,each8.0L)togivetenfractions(Fr.1–Fr.10)
basedonTLCandHPLCanalysis. Fr.7(12.2g)wasrepeatedlyappliedtothesilicagelcolumnwith
stepgradientCH Cl -(CH ) CO(v/v100:0–0:100)anddividedintothreesubfractions(Fr.7-1–Fr.7-3).
2 2 3 2
Fr.7-2(5.8g)wasfractionatedbytheMCIgelcolumnelutedwithgradientMeOH-H Otogivefour
2
subfractions(Fr.7-2-1–Fr.7-2-4). Fr.7-2-4-1(68.5mg)obtainedfromFr.7-2-4(0.9g)viaSephadexLH-20
CC(inMeOH)wasfurtherpurifiedbyHPLCelutingwithMeOH-H O(v/v73:27,2.5mLmin−1)to
2
give9(t =34.6min,45.2mg),whileFr.7-2-4-2(40.2mg)wasfurtherseparatedbyHPLCelutingwith
R
MeOH-H O(v/v71:29,2.5mLmin−1)toafford4(t =22.2min,2.8mg),3(t =32.3min,1.6mg)and
2 R R
5(t =48.3min,2.5mg). Fr.8(15.9g)wassubjecttosilicagelCCwithstepgradientCH Cl -(CH ) CO
R 2 2 3 2
(v/v100:0–0:100)anddividedintoeightsubfractions(Fr.8-1–Fr.8-8). Fr.8-2(3.2g)wasappliedtothe
MCIcolumnelutedwithgradientMeOH-H Otogivefoursubfractions(Fr.8-2-1–Fr.8-2-4). Fr.8-2-3
2
(0.9g)wasfurtherdividedbySephadexLH-20CCelutingwithMeOH-CH Cl (v/v1:1)andthen
2 2
purifiedbyHPLCelutingwithMeOH-H O-CH CO H(v/v/v50:50:10−4,2.5mLmin−1)toafford7
2 3 2
(t =21.1min,48.2mg)and8(t =23.9min,54.6mg). Fr.8-2-4(432.4mg)wasalsofractionatedby
R R
SephadexLH-20CCelutingwithMeOH-CH Cl (v/v1:1)andthenpurifiedbyHPLCelutingwith
2 2
MeOH-H O-CH CO H(v/v/v75:25:10−4, 2.5mLmin−1)toyield6(t =33.1min, 1.6mg). Fr.8-3
2 3 2 R
(6.4g)wasappliedtoMPLCwithanODScolumnelutingwithgradientMeOH-H O(v/v50:50–70:30)
2
toobtain1(3.3g). Fr.8-8(190.0mg)wasseparatedbySephadexLH-20CCelutingwithMeOH-CH Cl
2 2
(v/v1:1)toobtaintwosubfractions(Fr.8-8-1–Fr.8-8-2). Then,Fr.8-8-1(122.6mg)waspurifiedbyHPLC
eluting with MeOH-H O-CH CO H (v/v/v 64:36:10−4, 2.5 mL min−1) to yield 10 (t = 33.2 min,
2 3 2 R
52.6mg),andFr.8-8-2(35.6mg)wasisolatedbyHPLCelutingwithMeOH-H O-CH CO H(v/v/v
2 3 2
68:32:10−4,2.5mLmin−1)toyield2(t =13.5min,4.6mg).
R
3.4.1. ArthpyroneD(1)
Colorlesscrystals;mp204–207◦C;[α]26 −23.8(c1.0,MeOH);UV(MeOH)λ (logε)213(3.96),
D max
289(3.04)nm;ECD(0.05mgmL−1,MeOH)λ(∆ε)214(46.2),235(3.4),283(1.6)nm;1Hand13CNMR
data,Tables1and3;(+)-ESIMSm/z446.1[M+H]+;(−)-HR-ESIMSm/z444.2032[M−H]− (calcd. for
C H NO ,444.2028).
24 30 7
3.4.2. ArthpyroneE(2)
Paleyellowpowder;[α]26 −28.9(c0.5,MeOH);UV(MeOH)λ (logε)211(3.96),279(3.02)nm;
D max
ECD(0.05mgmL−1, MeOH) λ(∆ε)212(27.0), 285(−1.1)nm; 1Hand13CNMRdata, Tables1and3;
(−)-ESIMSm/z462.0[M−H]−;(−)-HR-ESIMSm/z462.2143[M−H]−(calcd.forC H NO ,462.2133).
24 32 8
3.4.3. ArthpyroneF(3)
Paleyellowpowder;[α]26 −108.7(c0.2,MeOH);UV(MeOH)λ (logε)230(2.87),279(2.35),
D max
332(2.56)nm;ECD(0.20mgmL−1,MeOH)λ(∆ε)258(0.6),316(0.8),341(−0.7)nm;1Hand13CNMR
data,Tables1and3;(−)-ESIMSm/z428.0[M−H]−;(−)-HR-ESIMSm/z428.2074[M−H]− (calcd.
forC H NO ,428.2079).
24 30 6
3.4.4. ArthpyroneG(4)
Paleyellowpowder;[α]26 −76.2(c0.2,MeOH);UV(MeOH)λ (logε)228(3.65),262(3.01),
D max
367 (2.67) nm; ECD (0.10 mg mL−1, MeOH) λ (∆ε) 238 (2.5); 319 (0.7) nm; 1H and 13C NMR data,
Tables1and3;(−)-ESIMSm/z410.0[M−H]−;(−)-HR-ESIMSm/z410.1974[M−H]− (calcd. for
C H NO ,410.1973).
24 28 5