全 文 ：天然产物研究与开发 NatProdResDev2008, 20:395-396
文章编号:1001-6880(2008)03-0395-02
　
　
　ReceivedApril10, 2007;AcceptedJuly17, 2007
　FoundationItem:ThisworkwassupportedinpartbytheNationalSci-
enceFund forDistinguished YoungScholarsto Y.-M.Shen
(30325044)andtheNationalNaturalScienceFoundationofChina
(30070007, 30430020).
＊CorrespondingauthorTel:86-871-5223111;E-mail:yshen@xmu.edu.cn
滑桃树内生链霉菌 5B发酵产物中的一个新吡唑生物碱
珠　娜 1, 2 ,赵沛基 1, 2 ,康前进 1, 2 ,沈月毛 1＊
1中国科学院昆明植物研究所 植物化学与西部植物资源持续利用国家重点实验室 ,昆明 650204;
2中国科学院研究生院 ,北京 100049
摘　要:从表面消毒的滑桃树(TrewianudifloraLinn.)根部分离得到一株内生链霉菌 5B, 经 10升固体 YMG培养
3周后 ,从发酵产物中分离得到一个新吡唑类生物碱。该生物碱通过理化性质 、NMR及 HRMS等波谱数据鉴定
为 5, 6-dihydro-2-isopropyl-4H-pyrrolo[ 1, 2-b] pyrazole。
关键词:滑桃树;内生放线菌;吡唑类生物碱
中图分类号:R915;Q939.9 文献标识码:A
ANewPyrazoleAlkaloidfromtheEndophyticStrain
Streptomycessp.5BofTrewianudifloraLinn.
ZHUNa1, 2 , ZHAOPei-ji1, 2 , KANGQian-jin1, 2 , SHENYue-mao1＊
1StateKeyLaboratoryofPhytochemistryandPlantResourcesinWestChina,
KunmingInstituteofBotany, ChineseAcademyofSciences, Kunming650204 , China;
2GraduateUniversityofChineseAcademyofSciences, Beijing100049 , China
Abstract:Anewcompound, 5, 6-dihydro-2-isopropyl-4H-pyrrolo[ 1, 2-b] pyrazole, wasisolatedfrom 10 LYMGagar
fermentationproductoftheendophyticstrainStreptomycessp.5B, whichwasisolatedfromthesurfacesterilizedrootof
TrewianudifloraLinn..Thestructurewaselucidatedbyphysico-chemicalandspectralevidence.
Keywords:Trewianudiflora;endophyticactinomycetes;pyrazole
Introduction
Endophytesaremicroorganisms(fungi, bacteriaand
actinomycetes)livingtheirwholeorpartiallifecycles
insidehealthyplanttissues, andcausenoapparentdis-
easesymptomstothehosts[ 1] .Endophyticmicrobes,
widelyresideinmostofplants, playanimportantrole
intheirhostplantsandaresourceoffunctionalmetabo-
liteswithchemicalandbioactivediversity[ 1-3] .Inthis
study, anewsubstitutedpyrazolealkaloidwasisolated
fromthefermentationproductoftheendophyticstrain
Streptomycessp.5Bthatwasobtainedfromsurfacester-
ilizedrootofTrewianudifloraLinn..Itsstructurewas
determinedtobe5, 6-dihydro-2-isopropyl-4H-pyrolo
[ 1, 2-b] pyrazole(1)basedonthespectraldata.
MaterialsandMethods
Generalexperimentalprocedure
MassspectrawereobtainedwithaVG-Auto-Spec-3000
andaThermoFinniganLCQAdvantagespectrometer.
NMRspectrawererecordedonaBrukerAM-400spec-
trometer(Bruker, Switzerland)withTMSasinternal
standards(δinppm, JinHz).HPLCwereperformed
onaWaters600 seriespumpingsystemequippedwith
photodiodearraydetector(Waters996)togetherwitha
symmetryC18 analyticalcolumn(5 μm, 3.9 ×150
mm, Waters).TLCplatesandsilicagel(200-300
mesh)werepurchasedfromQingdaoHaiyangChemical
Factory, Qingdao, P.R.China.SephadexLH-20andre-
versedphaseC18 silicagel(RP-18)wereboughtfrom
AmershanBioscienceandMerk, respectively.
Materials
AnendophyticStreptomycesstrainnamed5Bwasisola-
DOI :10.16333/j.1001-6880.2008.03.004
tedonHV(Humicacid-vitamin)agar[ 4] fromthesur-
facesterilizedrootofT.nudiflora, whichwascultivatedin
greenhouseofKunmingInstituteofBotany, ChineseAcad-
emyofSciences, China.10Lfermentationof5Bwasper-
formedonsolidYMGmedia(4.0 gglucose, 10.0gmalt
extract, 4.0gyeastextractand15.0 gagarin1 Ldis-
tiledwater, pH7.2)at24℃for3weeks.
Extractionandisolation
Thefermentationproductwasextracted3 timeswith
EtOAc/MeOH/AcOH(80:15:5, v/v/v)[ 5] toaford
11 gMeOHsolublecrudeextract.Theextractwassub-
jectedtoMPLC(mediumpressureliquidchromatogra-
phy)overRP-18 Sigel(130 g)elutedwith30%,
50%, 70%, 100% MeOH/H2O(800 mLforeach)to
givefourfractionsA-D.FractionBwassubjectedto
SephadexLH-20(130 g)columnelutedwithMeOHto
afordafraction(210 mg), whichcontainedcompo-
nentsgivingpositivereactionwithDragendrof srea-
gent.Thisfractionwasfurtherseparatedoversilicagel
(4 g)columnelutedwithCHCl3 /Acetone(100:0,
100:10, 100:20, 0:100 , 30 mLeach)togivefractions
I-IV.FractionIwassubjectedtoHPLC(symmetryC18
analyticalcolumn, 5 μm, 3.9×150 mm, Waters)eluted
with16% MeOH/H2Otogive1(5mg, tR=28.9min).
Compound1 wasisolatedaspaleyelowsolid.Themo-
lecularformulawasdeterminedtobeC9H15N2 byHR-
ESI-MSanalysis(m/z151.1238 ([ M +H] +);
calcd.151.1235).The13CNMR(DEPT)spectradis-
playedsignalsindicatingtwomethyls, threemethyl-
enes, twomethinesandtwoquaternarycarbons.The
skeletonof1 wasestablishedbycomparingitsNMR
datawiththosereportedforwithasomnine[ 6] , whichwas
furtherconfirmedby1H, 1H-COSYandHMBCexperi-
ments(Table1).Isopropylwasdeterminedandlocated
atC-2byHMBC(Fig.1):corelationsδH 1.20 (H-8
or/andH-9)/δC 20.6 (C-8 or9), 31.0 (C-7)and
160.8 (C-2).Therefore, compound1 wasdetermined
tobe5, 6-dihydro-2-isopropyl-4H-pyrrolo[ 1, 2-b]
pyrazole.Thisisthefirstreportofisolationofpyrazole
alkaloidfromStreptomyces.
Fig.1　Structureofcompound1
Table1　NMRdataofcompound1 inCD3OD
Position δC(DEPT) δH(multi., JHz) HMBC COSY
2 160.8s - - -
3 120.1 d 7.34(1H, s) 160.8 -
3a 143.2s - - -
4 30.2t 3.15(2H, t, 6.3)
22.4, 50.2,
120.1, 160.8w 2.26
5 22.4t 2.26(2H, m)
30.2,
50.2, 143.2 3.15, 4.13
6 50.2t 4.13(2H, t, 7.3)
22.4,
30.2, 143.2 2.26
7 31.0d 3.41(1H, m) 20.6, 160.8 1.20
8 20.6q 1.20(3H, d, 7.2)
20.6,
31.0, 160.8 3.41
9 20.6q 1.20(3H, d, 7.2)
20.6,
31.0, 160.8 3.41
5, 6-Dihydro-2-isopropyl-4H-pyrrolo [ 1, 2-b]
pyrazole(1)　Paleyelowsolid.EI-MSm/z(rel.
int):150 (M+, 19), 149 (57).HR-ESI-MSm/z:
151.1238 ([ M+H] + , C9H15 N2;calcd.for151.
1235).NMRdataseeTable1.
Acknowledgements　Thisworkwassupportedinpart
bytheNationalScienceFundforDistinguishedYoung
ScholarstoY.-M.Shen(30325044), theNationalNatural
ScienceFoundationofChina(30070007, 30430020).The
authorsaregratefultoMr.Y.-N.HeandMs.H.-L.Liang
intheStateKeyLaboratoryofPhytochemistryandPlant
ResourcesinWestChina, KunmingInstituteofBotanyof
theChineseAcademyofSciencesforrecordingNMRand
MSspectra, respectively.
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396 NatProdResDev　　　　　　　　　　　　　　　　　　　　　 　Vol.20