Phytochemistry\ Vol[ 38\ No[ 1\ pp[ 598Ð501\ 0887Þ 0887 Elsevier Science Ltd[ All rights reserved\ Pergamon Printed in Great Britain

9920Ð8311:87:,Ðsee front matterPII] S9920Ð8311"87#99030Ð0

LINGULATUSIN\ TWO EPIMERS OF AN UNUSUAL LINEARDITERPENE FROM ASTER LINGULATUS

IN HONOUR OF PROFESSOR G[ H[ NEIL TOWERS 64TH BIRTHDAY

YU SHAO\$ MING!FU WANG\$ CHI!TANG HO\$ CHEE!KOK CHIN\$ SHU!WEI YANG\%GEOFFREY A[ CORDELL\%� HERMANN LOTTER& and HILDEBERT WAGNER&

$Departments of Plant Science and Food Science\ Cook College\ Rutgers\ The State University of New Jersey\ NewBrunswick\ NJ 97892\ U[S[A[

%Program for Collaborative Research in the Pharmaceutical Sciences\ Department of Medicinal Chemistry andPharmacognosy\ College of Pharmacy\ University of Illinois at Chicago\ Chicago\ IL 59501\ U[S[A[

&Institut fu�r Pharmazeutische Biologie\ Universita�t\ der Mu�nchen 79222\ Mu�nchen\ Germany

"Received 10 October 0886^ received in revised form 11 January 0887^ accepted 11 January 0887#

Key Word Index*Aster lin`ulatus^ compositae^ lingulatusin^ diterpene^ X!ray crystallography

Abstract*A 0]0 mixture of two epimers of an unusual linear diterpene\ lingulatusin\ was isolated from thewhole plant of Aster lin`ulatus[ The structure was determined on the basis of IR\ MS and extensive NMRspectral studies\ and X!ray crystallographic data[ Þ 0887 Elsevier Science Ltd[ All rights reserved

INTRODUCTION

In previous papers we reported the isolation and struc!ture elucidation of four cytotoxic triterpenoid sap!onins from the n!BuOH soluble fraction of a 69)ethanol extract of Aster lin`ulatus "Compositae# ð0\1Ł[ In a continuing search for new bioactive naturalsubstances in this plant\ we undertook an examinationof the EtOAc soluble fraction[ Chromatography yiel!ded the isolate 0 which was identi_ed as a 0]0 mixtureof two epimers of an unusual linear diterpene with ag!hydroxy butenolide and two tetrahydropyran moiet!ies[ We herein describe the isolation and the structureelucidation of 0\ to which we have given the namelingulatusin\ through extensive NMR spectroscopictechniques and X!ray crystallography data[

RESULTS AND DISCUSSION

Lingulatusin "0# was obtained as colorless needles\mp 034Ð036>C\ UV"MeOH# lmax 196 nm "o 00\169#[

� Author to whom correspondence should be sent[

598

The molecular formula\ C19H21O5\ was derived frompositive FAB MS "ðM¦KŁ¦ at m:z 396 and ðM¦NaŁ¦

at m:z 280#\ and 0H and 02C NMR data\ and indicated_ve degrees of unsaturation[ The IR absorptions at2312 and 0634 cm−0 indicated the presence of alcoholand ester functions\ and the DEPT and APT 02CNMR spectra "Table 0# showed the signals for a totalof twenty carbons\ including two tertiary methyls\nine methylenes\ six methines\ and three quaternarycarbons\ suggesting a diterpenoid structure[

The 02C resonances of two ole_nic carbons at d

061[1 "s\ C!2# and 006[6 "d\ C!1#\ a carbonyl at d 063[9"s\ C!0# and a hemiacetal at d 099[8 "d\ C!06# led us topropose the presence of a hydroxy butenolide moiety"partial structure A# ð2Ł[ In the 0H NMR spectrum of0\ two broad singlets at d 4[77 and 4[88\ assigned toH!1 and H!06\ respectively\ showed very small long!range coupling\ and thus the butenolide moiety has ab!substitutent[ This unit accounted for three of the_ve unsaturations and three oxygen atoms of the sixoxygen atoms in the molecular formula[ Therefore\ 0

must contain two additional ring structures and threeadditional oxygen atoms[

The 0H!0H COSY\ with aid of TOCSY\ establishedthe major proton system from H!3 to H!03[ The iso!lated 0H NMR signal "H!3# resonating at d 1[27 waschosen as the starting point for the analysis of COSYand TOCSY spectra[ The correlations between H!3 "d1[27# and H!4 "d 0[50#\ H!4 and H!5 "d 0[07#\ H!5 andH!6 "d 0[40#\ H!6 and H!7 "d 0[82 and 0[00#\ H!6 andH!07 "d 1[86 and 2[77#\ H!7 and H!8 "d 0[13 and 0[60#\

YU SHAO et al[509

Table 0[ NMR spectral data of lingulatusin "0# in CD2OD

Position dC position dH HMBC

0 063[9 "s# 01 006[6 "d# 1 4[77 "s# C!0\ C!3\ C!062 061[1 "s# 23 17[6 "t# 3 1[27 "m# C!2\ C!44 13[7 "t# 4 0[50 "m# C!3\ C!55 22[0 "t# 5 0[07 "m# C!4\ C!66 26[9 "d# 6 0[40 "m# C!4\ C!5\ C!8\ C!077 20[1 "t# 7a 0[82 "m# C!09\ C!07

7b 0[00 "m# C!098 29[0 "t# 8a 0[13 "m# C!6\ C!7\ C!00

8b 0[60 "m# C!6\ C!709 70[0 "d# 09 1[85 "m# C!7\ C!00\ C!01\ C!07\ C!0800 31[4 "d# 00 0[42 "m# C!0901 16[2 "t# 01a 0[72 "m# C!02\ C!03\ C!08

01b 0[12 "m# C!0902 15[3 "t# 02a 0[21 "m# C!00\ C!01\ C!03\ C!04

02b 0[60 "m# C!00\ C!0103 74[5 "d# 03 1[87 "dd\ 00[1\ 3[9# C!01\ C!04\ C!05\ 08\ C!1904 61[7 "s# 0405 14[5 "q#� 05 0[01 "s#� C!03\ C!04\ C!1906 099[8 "d# 06 4[88 "s# C!0\ C!107 63[3 "t# 07a 2[77 "dd\ 00[1\ 3[9# C!6\ C!7\ C!09

07b 1[86 "dd\ 00[1\ 00[1# C!5\ C!7\ C!0908 61[2 "t# 08a 3[19 "dd\ 00[1\ 2[8# C!00\ C!01\ C!03

08b 2[02 "dd\ 00[1\ 00[1# C!09\ C!00\ C!01\ C!0319 14[6 "q#� 19 0[03 "s#� C!03\ C!04\ C!05

�Values may be interchanged in the same column[ Coupling constants "parentheses# are in Hz[

H!8 and H!09 "d 1[85#\ H!09 and H!00 "d 0[42#\ H!00and H!01 "d 0[12 and 0[72#\ H!00 and H!08 "d 2[02and 3[19#\ H!01 and H!02 "d 0[21 and 0[60#\ and H!02and H!03 "d 1[87# led to the assignment of the protonscontinuing from H!3 to H!03[ The 02C NMR datawhich were assigned by the HMQC experimentshowed that C!09 "d 70[0#\ C!03 "d 74[5#\ C!07 "d 63[3#and C!08 "d 61[2# were attached to oxygen atoms[ Inthe HMBC spectrum\ H!03 "d 1[87# was correlatedwith C!08 "d 61[2#\ and H!08 "d 3[19 and 2[02# wascorrelated with C!03 "d 74[5#[ The above correlationssuggested that C!03 and C!08 shared an oxygen atomto form a tetrahydropyran ring[ Similarly\ the long!range correlations between H!09 "d 1[85# and C!07 "d63[3#\ and H!07 "d 1[86 and 2[77# and C!09 "d 70[0#indicated that C!09 and C!07 shared another oxygenatom to form a second tetrahydropyran ring[ Thesedata taken together are in accordance with the partialstructure B[

Taking into account the molecular formula and thetwo partial structures A and B\ two methyls and ahydroxy!bearing quaternary carbon "d 61[5# remainedto be assigned[ The two methyls "Me!05 and Me!19#\appearing as singlets "d 0[01 and 0[03#\ were correlatedwith C!04\ thus\ establishing the partial structure C[

HMBC correlations were used to assemble the threepartial structures[ The correlation between C!04 "d61[7# and H!03 "d 1[87# established the linkage of thepartial structures B and C from C!03 to C!04\ and thecorrelation between H!3 "d 1[27# and C!2 "d 061[1#established the linkage of the partial structures A andB from C!2 to C!3[ These considerations yielded thegross structure of 0\ without stereochemical impli!cations[

The relative stereochemistry of 0 was deduced usinga combination of the 0H!0H coupling constants andNOE interactions from a NOESY experiment[ Thelarge couplings observed between H!03 and axial H!

Lingulatusin\ two epimers of an unusual linear diterpene from Aster lin`ulatus 500

Fig[ 0[ ORTEP drawing of the crystal structure of lingulatusin "0#[

02 "J03\02ax�00[1 Hz#\ and H!00 and axial H!08"J00\08ax�00[1 Hz# required that H!03 and H!00 werelocated in trans!diaxial positions "b and a\ respec!tively#[ It could be determined that the tetrah!ydropyran ring from C!03 to C!08 adopted a regularchair conformation with the aid of Dreiding stereomodels[ The observed NOEs from H!03 to Hax!01\ H!03 to Hax!08\ Hax!01 to H!08\ H!00 to Hax!02\ H!00 toequatorial H!08 favored the above deductions[ Like!wise\ the assignment of a b!con_guration for H!09and an a!con_guration for H!07 was based on thecoupling constant "J07ax\6�00[1 Hz# and observedNOEs between H!09 and Hax!7\ H!09 and Hax!07\ H!6 and Heq!07[ The tetrahydropyran ring from C!09 toC!07 was also present in a chair conformation[

The relative stereochemistry at C!06 could not bedetermined by NMR data[ Thus\ in order to _rmlyestablish the structure and stereochemistry\ 0 was sub!jected to single crystal crystallography[� The absolutecon_guration of 0 was _nally established and lin!gulatusin "0# was found to be a C!06 epimeric "06a or06b!hydroxy# mixture in the ratio 0]0[ The isolatecrystallized in a triclinic space group P0 with z�1molecules in the unit cell[ The statistic of =E�E−0=pointed clearly to a centrosymmetric space group\ butall attempts to solve the structure in P!0 failed[ A trialin the most common space group P0 revealed twoseparate epimeric molecules in the asymmetric unit\i[e[ the unit cell was as shown in Fig[ 0[ The moleculesdi}ered only in the con_guration of the hydroxy sub!stituent at the C!06\ one being up and the other downof the ring[ This explained the unusual statistical valueand the crystallization behavior[ The re_nement con!verged anisotropically with hydrogens indicated atR�4[0) for all 1749 measured data[ It is noteworthy

0 All X!ray structure calculations were performed withSHELXTL[ The crystal data are deposited at the CambridgeCrystallographic Data centre[

that the NMR data of 0 did not show two sets ofsignals for the two epimers[ Hence\ it was proposedthat the conversion of two epimers in solution wasvery fast[

Lingulatusin represents a new class of linear diter!penoid[ Even though several natural products bearingthe g!hydroxy butenolide moiety have been isolatedfrom marine sources ð3Ð01Ł and some of them haveexhibited potent anti!in~ammatory ð00\ 01Ł and anti!tumor activities ð7Ł\ to our knowledge\ no naturallyoccurring compound of this type has been reportedfrom higher plants[

EXPERIMENTAL

General

Mps were determined on a Ko~er apparatus and areuncorrected[ IR spectrum was obtained on a MattsonCYGNUS 099 Fourier!transform infrared spec!trometer[ FABMS spectrum was recorded on a Fin!nigan MAT!89 instrument[ 0H!NMR and 02C!NMRspectra were measured on a Nicolet NT!259 "259 MHzfor 0H\ 89 MHz for 02C# spectrometer in CD2OD withTMS as int[ standard[ The two!dimensional NMRspectra "0H!0H COSY\ HMQC\ TOCSY and HMBC#were recorded on a General Electric GN Omega499 MHz spectrometer operating at 499 MHz for 0HNMR and at 014 MHz for 02C NMR[ 02C!NMR mul!tiplicity was determined using DEPT and APT experi!ments[

Plant material

The plant material of Aster lin`ulatus was collectedin August 0881\ from Li!Jiang County\ Yunnan prov!ince\ China[ A voucher specimen was identi_ed byProfessor Z[ W[ Lu and is deposited in the Herbariumof Kunming Institute of Botany\ Academia Sinica\Kunming\ China[

YU SHAO et al[501

Extraction and isolation

The dried whole plants of A[ lin`ulatus "6 kg# werepercolated _ve times with 69) EtOH "each 0 week#at room temperature[ The combined extracts wereevaporated to dryness in vacuo to a}ord a residue"0 kg# that was suspended in H1O and then partitionedsuccessively with petroleum ether\ EtOAc\ and H1O!saturated n!BuOH to yield three corresponding frac!tions "034\ 179\ and 369 g\ respectively#[ The EtOAcfraction "179 g# was subjected to column chro!matography on silica gel eluting with chloroformÐacetone "09]0Ð0]0# gradient[ The chloroformÐacetone"5]0# eluent "419 mg# was further separated bySephadex LH!19 "MeOH# followed by crystallizationfrom ethanol to a}ord 0 "124 mg\ 9[9923)\ dry wt[#[

Lin`ulatusin "0#

Colorless needles^ mp 034Ð036>C^ UV "MeOH# lmax

196 nm "o 00169#^ C19H21O5^ positive!ion FABMS m:z]396 ðM¦KŁ¦\ 280 ðM¦NaŁ¦\ 258 ðM¦HŁ¦\ 240ðM−H1O¦0Ł¦\ 298 ðM−C2H6OŁ¦\ and 114ðM−C7H04O1Ł¦^ IR "KBr# gmax 2312 "OH#\ 0634"COOR#\ 0539 and 0434 "C1C#\ 0999Ð0099 "CÐO#cm−0^ 0H and 02C NMR] see Table 0[

Acknowled`ements*The authors thank ProfessorZhenwei Lu of Kunming Institute of Botany\ Aca!demia Sinica\ for the botanical identi_cation\ and theNuclear Magnetic Resonance and Mass SpectrometryLaboratories of the Research Resources Center\ Uni!

versity of Illinois at Chicago\ for providing assistanceand the spectroscopic instrument used in this study[

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