Tricyclic sesquiterpenes from rudbeckia laciniata in honour of professor G. H. Neil Towers 75th birthday

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Pergamon PII] S9920Ð8311"87#99140Ð8

Phytochemistry\ Vol[ 38\ No[ 1\ pp[ 482Ð599\ 0887 Þ 0887 Elsevier Science Ltd[ All rights reserved Printed in Great Britain 9920Ð8311:87:,Ðsee front matter

TRICYCLIC SESQUITERPENES FROM RUDBECKIA LACINIATA IN HONOUR OF PROFESSOR G[ H[ NEIL TOWERS 64TH BIRTHDAY YUKIHARU FUKUSHI$%\ CHIE YAJIMA$\ JUNYA MIZUTANI$& and SATOSHI TAHARA$% $Department of Applied Bioscience\ Faculty of Agriculture\ Hokkaido University\ Kita!ku\ Sapporo 959\ Japan %CREST\ Japan Science and Technology Corporation\ Kawaguchi\ Saitama 221\ Japan &Present address] Plant Ecochemicals Research Centre\ Megumino Kita\ Eniwa\ Hokkaido 950!02\ Japan "Received 4 December 0886^ received in revised form 14 February 0887#

Key Word Index*Rudbeckia laciniata^ Asteraceae^ phytotoxic^ tricyclic sesquiterpene^ ses! quiterpene lactone

Abstract*Three phytotoxic sesquiterpenes were isolated from the roots of Rudbeckia laciniata\ of which one of these was designated as lacinan!7!ol[ Its absolute con_guration was established by the MNCB ð1!"1?! methoxy!0?!naphthyl#!2\4!dichlorobenzoic acidŁ method[ It was chemically transformed into the corresponding secondary allylic alcohol\ whose absolute con_guration was also determined by MNCB and CD "circular dichroism# allylic benzoate methods[ Þ 0887 Elsevier Science Ltd[ All rights reserved

INTRODUCTION

The terpenoids and polyacetylenic compounds of Rudbeckia laciniata "Asteraceae# have previously been studied in detail ð0Ð2Ł[ This plant is naturalized in Japan and grows along roadsides and _elds of Hok! kaido island[ Our allelopathic interest in this plant resulted in the isolation of three phytotoxic ses! quiterpenes "0Ð2#[ Compounds 0 and 2 have been iso! lated from the same plant previously\ and the chemical characterization of 0 has been presented elsewhere ð3Ł[ In this paper\ the absolute con_guration of a new sesquiterpene "1# was determined by 0H NMR spec! troscopy using an axially chiral reagent "MNCB# ð3Ð 6Ł[ The chemical transformation of 1 into the cor! responding secondary allylic alcohol\ and application of the MNCB method and CD allylic benzoate method ð7Ł to the resulting product are also described[ RESULTS AND DISCUSSION

During screening of higher plants for phytotoxic secondary metabolites\ the EtOAc!soluble extract of R[ laciniata roots was found to be inhibitory against germination and growth of some plants[ Through the bioassay!guided fractionation method of Stevens and Merrill ð8Ł\ three phytotoxic sesquiterpenes "0Ð2# were obtained from the extract[ Prelacinan!6!ol "0# ð3Ł and igalan "2# ð2Ł have been reported as constituents of

 Author to whom correspondence should be addressed[

this plant[ The structure of 2 was determined by 0H and 02C NMR spectral data comparison to that pub! lished ð09Ð03Ł[ Compound 1 was new and named as lacinan!7!ol[ Lacinan!7!ol "1\ C04H15O# exhibited an IR absorp! tion at 2279 cm−0[ Its mass spectrum gave a dehy! dration ion at m:z 193\ and the corresponding tertiary carbinyl carbon appeared at d 63[19 "s# in pyridine!d4 indicated 1 to be a tertiary alcohol[ The DEPT spectra showed the presence of _fteen aliphatic carbons con! sisting of four methyls\ _ve methylenes\ three methines and three quaternary carbons[ All the carbon signals had chemical shifts less than 79 ppm\ indicating no double bond and therefore a tricyclic structure[ The 0 HÐ0H COSY spectrum established the correlations of i# H!01 to H!4 and ii# H!6 to H!09[ The HMBC spectrum showed correlations of i# H!04 and Cs!6\ 7\ 8^ ii# H!03 and Cs!4\ 5\ 6\ 02^ iii# H!02 and Cs!4\ 5\ 6\ 03^ iv# H!01 and Cs!0\ 1\ 2^ v# H!2 and Cs!0\ 1\ 3\ 4\ 01 and vi# C!0 and Hs! 1\ 2b\ 3ab\ 4\ 8ab\ 09ab\ 01 "Table 0#[ Taken together\ the above data led to the assignment of 1 as 1\ 5\ 5\ 7!tetramethyltricy! cloð4[1[4[90\4Łundecan!7!ol and the other HMBC and 0 H!0H COSY spectral correlations for 1 supported this structure[ The relative stereochemistry of 1 was concluded from several lines of data] i# W!type long!range coup! lings between H!8b and H!09a "J2 Hz#\ and H!09b and H!4 "J1 Hz#^ ii# the arrangement of the large pyridine!induced solvent shifts ð3\ 04Ł of signals around the hydroxyl group in the 0H NMR spectra "Figure 0A#^ and iii# the observation of NOEs detected

482

483

Table 0[ NMR spectral data "CDCl2 and C4D4N# for lacinan!7!ol "1# and its MNCB esters "3a and 3b# MNCB esters

1 C

d 02C

0 1 2

34[98 31[50 21[20

11[97

4

41[16

5 6

23[55 38[40

0

H!0H COSY

0[49 m a 0[12 dddd "6\7[4\00\02# b 0[70 dddd "5[4\8\8\02# a 0[48 m b 0[46 m 0[86 ddd "1\09\09[4#

2ab\01 1\2b\3ab

2ab\3b\4 2ab\3a\4 3ab\09b

2b\3ab\6\8a\09b02\03

2a\09a\03 2b\4 3b\02

0[15 dd "1\3#

00ab

3ab\4\6\00b\02\03 00ab\02\03\04

03

HMBC 1\2b\3ab\4\8ab\09ab\01 2ab\3a\8b\01 1\3ab\01

1\2a\3ab

NOE

d 02C

8b 3a\01

33[58 31[94 20[89

3b 2ab\4

10[65 41[17

d 0H

d 0H 3a "aS#

3b "aR#

0[49 0[12

9[65 0[90

0[14 0[03

0[71

0[48

0[66

ca 0[47 ca 0[47 0[69

ca 0[17 ca 0[17 9[45

0[31 0[34 0[09

0[73

0[61

0[54 0[02

9[68 −9[26

9[86 9[20

0[02

9[78

9[86

9[81

9[52

9[69

0[62

0[44

0[44

0[24

0[09

0[98

9[66 0[17 9[81 0[26 0[97

9[44 9[72 9[66 0[06

9[56 9[76 9[68 9[76

23[12 38[20 0[09

7 8

63[19 40[14

09

07[31

00

01 02 03 04 OH

19[65

03[69 23[19 17[03 21[59

a 0[61 d "02# b 0[34 dd "2\02# a 0[97 dddd "2\5\00\02# b 9[78 dddd "1\1[4\3\02# a 0[69 dddd "1[4\3\00\03# b 0[28 ddd "1\1[4\00\03# 9[79 d "6# 0[51 s 9[85 s 0[42 s 4[04 s

8b 8a\09a 8b\09b\00ab

6\8ab\00ab\04 1\4\09a\04

1\4\6\8ab\00b

0\09a\00ab 6\09ab\00b

3a

64[69 49[16

06[75

8a\01 6\09ab

6\09ab\00a 1

09b\01\04 1

03

19[45

04 1\2a 4\6\03 4\02 6\8b

2a\8a\09b 4\6\03 3a\00a\02 8a\00b

Assignments were based on DEPT\ 0H!0H COSY\ HMQC\ HMBC\ NOESY and di}erence NOE experiments[

03[26 23[12 16[61 21[42

Y[ FUKUSHI et al[

3

d 0H "J Hz#

Tricyclic sesquiterpenes from Rudbeckia laciniata

484

Fig[ 0[ "A# Pyridine!induced solvent shifts] Dd "dCDCl2!dC4D4N# in 0H NMR obtained for prelacinan!7!ol "1#[ "B# Key NOEs observed in 1[

by NOESY and NOE di}erence spectra in Fig[ 0B and Table 0[ To establish the absolute con_guration of 1\ it was esteri_ed with "aS#! and "aR#! ð4Ð6\ 05Ł MNCB in methylene chloride containing DCC and 3!pyr! rolidinopyridine ð06Ł to yield 3a and 3b\ respectively[ The chemical shift "d −9[26# of H!8b in the "aS#! MNCB ester "3a# indicates that H!8b faces to the centre of the naphthalene ring[ The "aS#! and "aR#! MNCB shifts ð3\ 07Ł dD "d1Ðd3a and d3b# in the 0H NMR spectra were positive values except that of H!6 "Figure 1A and Fig[ 1B#[ The negative Dd values for H!6 indicate that H!6 locates close to the carbonyl group and is anisotropically a}ected more intensively by the carbonyl group than by the naphthalene ring

in both esters[ The presumable conformations of the esters are depicted in Fig[ 1A and Fig[ 1B[ The values of the MNCB shifts were arranged according to the conformation in each ester[ NOE correlations in the "aR#!MNCB ester "3b# also supported the con! formations mentioned above and revealed the absol! ute con_guration of 1 "Figure 1B#[ The Dd "d3aÐ d3b# values were systematically arranged "Figure 1C#[ It was concluded that the MNCB method is applicable to tertiary alcohols\ when the conformation of esters are reasonably determined[ As an additional line of proof\ 1 was transformed to a secondary allylic alcohol "Scheme 0#[ Dehy! dration of 1 with thionyl chloride in pyridine ð08Ł gave a mixture of two alkenes 4 and 5 "Table 1#[ Oxidation

485

Y[ FUKUSHI et al[

Fig[ 1[ Shift e}ects in 0H NMR "CDCl2# caused by MNCB esteri_cation[ "A# "aS#!MNCB shifts] Dd  d1!d3a[ "B# "aR#! MNCB shifts] Dd  d1!d3b[ NOE correlations are also indicated[ "C# Chemical shift di}erences for the MNCB esters of 1] Dd  d3a!d3b[

Scheme 0[ Chemical conversion of lacinan!7!ol "1# to an allylic alcohol "6# and its esters "7a\ 7b\ 8#[

of 4 with SeO1 on silica gel and t!butylhydroperoxide ð19Ł stereoselectively a}orded an allylic alcohol 6[ Esteri_cation of compound 6 with "aS#! and "aR#! MNCB\ and benzoyl chloride yielded 7a\ 7b and 8\ respectively[ The relative con_guration at C!8 in 6 was deter! mined by NOESY and NOE di}erence spectra "Figure 2A#[ The R!con_guration at C!8 in 6 was determined by the application of MNCB method for 7a and 7b "Figure 2B and Table 2#\ and CD allylic benzoate method ð7Ł for 8 "117 nm\ De ¦1[39\ MeOH] Fig[

2C#\ respectively[ Hence\ the absolute con_guration of lacinan!7!ol was established as depicted in formula 1[ This _nding is in accordance with the conclusion of the 0H NMR MNCB method applied to the tertiary alcohol "1#[ Four sesquiterpenes with the same skeleton of pre! lacinan!6!ol "0# have been found\ such as 09 and 00 in Eremophila `eor`ei ð10\ 11Ł\ 09 in E[ metallicorum ð12Ł "Myoporaceae#\ 01 in Juniperus thurifera "Cup! ressaceae# ð13Ł\ 09 in Fabiana imbricata "Solanaceae# ð14Ł and 02 in Acorus calamus "Araceae# ð15Ł[ Three

Tricyclic sesquiterpenes from Rudbeckia laciniata

486

Table 1[ NMR spectral data "CDCl2# for compounds 4 and 5[ 4 C

d 02C

0 1 2

33[39 30[59 20[64

3

11[09

4 5 6

43[45 21[70 37[77

7 8

040[75 39[52

09

07[04

00

11[06

01 02 03 04

03[32 21[97 13[93 095[68

5 d 0H "J Hz#

d 02C 38[95 27[27 20[64

0[36 m a 0[13 m b 0[71 m a ca 0[43 m b ca 0[43 m 0[14 m

11[41 45[90 21[52 38[34

0[54 dd "1\2[4# a 1[23 ddd "1[4\1[4\03# b 0[61 dddd "1[4\1[4\00[4\03# a 0[12 m b 0[00 dddd "1[4\1[4\00[4\02[4# a 0[77 dddd "1[4\2[4\7\01# b 0[23 ddd "1\6\01\02[4# 9[68 d "6# 9[80 s 9[80 s E 3[57 ddd "1\1[4\1[4# Z 3[60 ddd "1\1[4\1[4#

032[36 017[55

d 0H

0[75 0[16 0[78 0[40 0[34 0[06 0[67

4[60

05[79

0[05 9[74

11[55

0[68 0[92

04[02 22[92 13[44 10[89

9[78 9[70 9[77 0[79 1

Assignments were based on DEPT\ 0H!0H COSY\ HMQC\ HMBC\ NOESY and di}erence NOE experiments[

Fig[ 2[ "A# Key NOEs detected for compound 6[ "B# Dd "d7a!d7b# values in 0H NMR "CDcl2#[ Protons encircled by dotted line are under the border place ð4Ł[ "C# Newman projection across the C!8!C!7 bond in compound 8[

sesquiterpenes "03!05# with the same skeleton of 1 have also been known like 03 in Cupressus dupreziana ð16Ł\ 03 and 04 in Juniperus thurifera ð13Ł\ and 05 in J[ chinensis ð17Ł "Cupressaceae#[ Among the compounds\ only 02 possesses a methyl group of a!con_guration at C!1 position[ The occurrence of 0 and 1 in the

same plant is interesting in the light of their biogenesis "Scheme 1# ð13\ 16\ 18Ł[ Compounds 0 and 1 showed inhibition in growth of seedlings of Nasturtium of_cinale and Phleum pra! tense at 49 ppm\ while compound 2 showed inhibition in seed germination and seedling growth at 49 ppm

487

Y[ FUKUSHI et al[ Table 2[ NMR spectral data "CDCl2# for compound 6 and its MNCB esters "7a and 7b#[ MNCB esters

6 C

d 02C

0 1 2

37[90 30[22 21[52

3

11[17

4

41[36

5 6

21[16 37[61

7 8 09

045[65 68[51 01[42

00

01 02 03 04

OH

11[09

05[46 20[84 13[97 009[56

d 0H "J Hz#

d 0H 7a "aS#

7b "aR#

0[65 m a 0[13 dddd "4[4\8\09\02# b 0[76 dddd "6\8[4\01\02# a 0[48 m b 0[59 m 0[00 ddd "1\8\00#

0[48 0[94

0[47 0[09

0[62

0[69

ca 0[34 ca 0[34 0[95

0[37 0[36 0[96

0[64 dd "1\2[4#

0[39

0[34

2[65 br s a 0[05 dddd "0[4\6\00[4\02# b 0[18 dd "00\02# a 0[89 dddd "1[4\2\00\02[4# b 0[36 dddd "1\6\00[4\02[4# 9[87 d "5[4# 9[74 s 9[83 s Z 4[97 dd "0[4\2# E 3[81 dd "0[4\2# 0[28 br

4[03 9[60

4[09 9[88

9[28

9[80

0[30

0[55

9[28

9[80

9[44 9[60 9[66 3[45

9[48 9[60 9[72 3[39

3[44

3[15

Assignments were based on DEPT\ 0H!0H COSY\ HMQC\ HMBC\ NOESY and di}erence NOE experiments[

Scheme 1[ Hypothetical biogenesis scheme for sesquiterpenes 0 and 1 in R[ laciniata[ Fpp  farnesyl pyrophosphate[

and 09!19 ppm\ respectively "data not shown#[ Hence it was found that igalan "2# is major phytotoxic con! stituent of this plant[ Moreover igalan "2# exhibited clear inhibitory zone on the TLC bioautogram using Cladosporium herbarum as the test fungus "data not shown#[

EXPERIMENTAL

General Mps are uncorr[ EIMS] direct inlet\ 69 eV[ IR] KBr pellet or NaCl plate[ 0H NMR "499 MHz# and 02C NMR "56[7 and 014 MHz# spectra were taken in

Tricyclic sesquiterpenes from Rudbeckia laciniata

CDCl2 or C4D4N at room temp[\ with TMS as int[ standard[ Silica gel "Wakogel C!199# was used for CC and silica gel 59 F143 "Merck# for TLC[ Compounds on TLC plates were detected under UV light "143 nm# or by colour produced using phosphomolybdic acid reagent[ Growth!re`ulation assay for N[ o.cinale and P[ pra! tense The assay method employed was originally described by Stevens and Merrill ð18Ł using N[ of_c! inale and P[ pratense seeds purchased from a local market[ To 0 ml of molten agar "9[4)# in a test tube "01 mm i[d[×094 mm# was added 09 ml of a DMSO soln containing an appropriate amount of the test compound[ The contents in the tube were mixed tho! roughly\ and the _nal concentration of each test com! pound was adjusted to 4\ 09\ 19\ 49 and 099 ppm[ For the control experiments\ 09 ml of DMSO was added to the agar medium[ Ten to _fteen seeds of each plant were sown on the medium and cultivated at 14> with a day length of 03 hr under ~uorescent light[ The per! centage seed germination of and root length of the hypocoyl "N[ of_cinale# or coleoptyl "P[ pratense# were measured 6 days after sowing\ with the mean of dupli! cate experimental results being calculated thereafter[ Extraction and isolation Roots of R[ laciniata were collected in Sapporo in September in 0880[ The fresh roots were sliced into pieces and extracted twice with EtOAc at room temp[ This EtOAc extract showed phytotoxic activity\ and was subjected on prep[ TLC plates developed with hexane] EtOAc "3]0#[ This separation gave active bands at Rf 9[11Ð9[32[ Rechromatography of these bands on silica gel plates\ eluted with CH1Cl1 also gave active bands at Rf 9[14Ð9[39[ From these\ three active compounds "0Ð2# were obtained in small amount[ This was then repeated at a larger scale as follows[ Thus the EtOAc extract "6[1 g# from roots "fr[ wt 1[9 kg# was fractionated into 05 sub!fractions by CC on 099 g of silica gel to give frs 0Ð4 "hexane#\ frs 5Ð7 "hexane]EtOAc\ 8]0#\ frs 8Ð00 "hexane]EtOAc\ 3]0#\ frs 01Ð03 "hexane]EtOAc\ 1]0# each 099 ml\ fr[ 04 "EtOAc\ 299 ml# and fr[ 05 "MeOH\ 199 ml#[ Frs containing 0\ 1 and 2 were further puri_ed to give 0 "59 mg#\ 1 "049 mg# and 2 "329 mg#\ respectively[ Lacinan!7!ol "1#[ Colourless plates from hexane^ mp 64[9Ð65[9>^ ðaŁ14 D ¦87[6> "CHCl2^ c 9[59#[ EIHR! MS] m:z 111[0878 ðMŁ¦\ C04H15O requires 111[0874[ EIMS\ m:z "rel[ int#] 111 ðMŁ¦ "09#\ 196 ðM!MeŁ¦ "07#\ 193 ðM!H1OŁ¦ "10#\ 078 "06#\ 050 "33#\ 038 "45#\ 026 KBr "16#\ 008 "83#\ 32 "099#[ IR nmax cm−0] 2279 "OH#\ 1869\ 1829\ 1769\ 0334\ 0259\ 0059\ 0039\ 0099[ 0H and 02C NMR] Table 0[ Igalan "2#[ Colourless prisms from hexane!Et1O^ mp 68[9Ð79[9>\ lit[ ð09Ł\ 68[9Ð68[4>^ ðaŁ14D ¦091[2> "CHCl2^ c 9[06\ lit[ ð09Ł\ ¦094>\ lit[ ð00Ł\ ¦33[8>#[

488

EIHR!MS] m:z 121[0346 ðMŁ¦\ C04H19O1 requires 121[0353[ The physicochemical properties of 2 coincided well with those of igalan\ secoeudesmanolid or elemasteiractinolide ð09Ð03Ł[

Esteri_cation of compound 1 with "aS#! and "aR#! MNCB To a soln of 1 "10[9 mg\ 84 mmol#\ "aS#!MNCB "08[9 mg\ 44 mmol# and 3!pyrrolidinopyridine "1[9 mg\ 02 mmol# in CH1Cl1 "299 ml# was added DCC "04[9 mg\ 62 mmol#\ and the solution was stirred at room temp[ for 4 days[ The reaction mixt was directly applied to prep[ TLC "hexane]EtOAc\ 09]0# to give the "aS#! ester "3a# "3[7 mg\ 05)#[ Under essentially the same conditions\ 3b was obtained in 5) yield[ Compounds 3a and 3b^ 0H NMR] for spectral analysis\ see Table 0[ 02C NMR "56[7 Hz\ CDCl2# of 3a] d 03[91 "C!01#\ 06[25 "C!09#\ 08[24 "C!00#\ 10[51 "C!3#\ 15[83 "C!04#\ 16[10 "C!03#\ 20[30 "C!2#\ 21[41 "C!02#\ 22[59 "C!5#\ 39[70 "C!0#\ 32[14 "C!1#\ 33[27 "C!6#\ 36[42 "C!8#\ 40[70 "C!4#\ 76[62 "C!7#\ "ds of carbons in MNCB moiety 45[30\ 002[09\ 012[45\ 013[59\ 015[57\ 017[92\ 017[78\ 017[82\ 018[70\ 021[95\ 022[95\ 022[83\ 025[71\ 026[49\ 042[31\ 054[24#[ EIMS of 3a\ m:z "rel[ int#] 449 ðMŁ¦ "9[8#\ 237 "53#\ 235 ðM!C04H13Ł¦ "099#\ 218 "7#\ 141 "6#\ 58 "6#[

Dehydration of compound 1 to alkene 4 and 5 To a stirred soln of 1 "012[1 mg\ 443 mmol# in CH1Cl1!pyridine "5 ml\ 4]0# was added dropwise a soln SOCl1 "199 ml# in CH1Cl1 "0 ml# at 9>[ The reaction soln was stirred for 0 hr at 9> followed by dilution with hexane "4 ml#\ and directly chromatographed on 09 g of silica gel with hexane to a}ord a mixt of alkenes "82[0 mg#[ These were separated by CC on 04 g of AgNO2!silica gel "0]3#\ eluted with hexane\ to give 4 "16[7 mg# and 5 "23[6 mg#\ respectively[ Compound 4[ Colourless oil^ ðaŁ14 D ¦35[8> "CHCl2^ c 0[09#[ EIMS\ m:z "rel[ int#] 193 ðMŁ¦ "20#\ 050 "13#\ 038 "47#\ 027 "64#\ 008 "099#\ 60 "41#\ 46 "68#\ 32 "58#[ −0 IR nneat ] 2954 "C1CH1#\ 1729\ 1614\ 0259\ 0390\ maxcm 0219\ 0179\ 679[ 0H and 02C NMR see Table 1[ Compound 5[ Colourless oil^ ðaŁ14 D ¦53[1> "CHCl2^ c 9[79#[ EIMS\ m:z "rel[ int#] 193 ðMŁ¦ "49#\ 050 "16#\ 022 "14#\ 008 "099#\ 094 "25#\ 82 "22#\ 80 "16#\ 66 "05#\ −0 44 "05#\ 30 "18#[ IR nneat ] 2999\ 1839\ 1759\ 0324\ maxcm 0254\ 0244\ 674 for 0H and 02C NMR spectral analyses] Table 1[

Oxidation of compound 4 to allylic alcohol 6 Selenium dioxide supported on silica gel "4)\ 29 mg# and 79) t!butylhydroperoxide "19 mg\ 066 mmol# were mixed in CH1Cl1 "299 ml# and stirred for 04 min at room temp[ To this soln was added dropwise a soln of compound 4 "00[6 mg\ 46 mmol# in CH1Cl1 "49 ml# with stirring[ After 0 hr\ the reaction

599

Y[ FUKUSHI et al[

mixt was directly applied to prep[ silica gel TLC "H! EA 09]0# to yield the allylic alcohol "6# "7[7 mg\ 69)#[ Compound 6[ Colourless prisms from hexane! EtOAc^ mp 62[4Ð64[9>^ ðaŁ14 D ¦60[5> "CHCl2^ c 9[63#[ EIMS\ m:z "rel[ int#] 119 ðMŁ¦ "29#\ 191 ðM!H1OŁ¦ "38#\ 076 "51#\ 048 "78#\ 034 "66#\ 58 "81#\ 30 "099#[ For 0 H and 02C NMR spectral analysis\ see] Table 2[ Esteri_cation of compound 6 with "aS#!and "aR#! MNCB To a soln of compound 6 "1[1 mg\ 09 mmol#\ "aS#! MNCB "4[9 mg\ 44 mmol# and 3!pyrrolidinopyridine "0[9 mg\ 6 mmol# in CH1Cl1 "299 ml# was added DCC "4[9 mg\ 13 mmol#\ and the solution was stirred at room temp[ for 7 hr[ The reaction mixt was directly applied to prep[ TLC "hexane]EtOAc\ 4]0# to give the "aS#! ester "7a# "2[6 mg\ 53)#[ Under essentially the same conditions\ 7b was obtained in 76) yield[ Compounds 7a and 7b[ For 0H NMR] spectral analysis\ see] Table 2[ EIMS of 7a\ m:z "rel[ int#] 449 ðM¦1Ł¦ "09#\ 437 ðMŁ¦ "03#\ 237 "57#\ 235 ðM!C04H11Ł¦ "099#\ 141 "7#\ 80 "6#[ Esteri_cation of compound 6 with benzoyl chloride To a soln of 6 "0[6 mg\ 7 mmol#\ Et2N "0[4 mg\ 04 mmol# and 3!dimethylaminopyridine "0[9 mg\ 7 mmol# in CH1Cl1 "199 ml# was added benzoyl chloride "1[0 mg\ 04 mmol#\ and the solution was stirred at room temp for 2 hr[ The reaction mixt was directly applied to prep[ TLC "hexane]CH1Cl1\ 0]4# to give the ben! zoate 8 "9[8 mg\ 49)#as a colourless semi!solid[ UV lmax "MeOH# nm "log e#] 118[3 "2[82#[ CD] De117[9 ¦1[39 "MeOH\ c 2[83×09−3M\ 14>#[ EIMS\ m:z "rel[ int#] 213 ðMŁ¦ "9[4#\ 191 "15#\ 076 "15#\ 048 "23#\ 034 "21#\ 011 "32#\ 094 "099#\ 80 "13#\ 66 "40#\ 58 "29#\ 32 "57#\ 0H NMR "CDCl2#] 9[67 "2H\ d\ J5[3 Hz#\ 9[81 "2H\ s#\ 9[87 "2H\ s#\ 0[0Ð1[0 "00H#\ 3[84 "0H\ d\ J9[8 Hz#\ 4[07 "0H\ d\ J9[8 Hz#\ 4[36 "0H\ s#\ 6[33 "1H\ br dd\ J6[1\ 7[1 Hz#\ 6[44 "0H\ t\ J6[1 Hz#\ 7[94 "1H\ d\ J7[1 Hz#[ Acknowled`ements*The authors are grateful to Dr[ Jun Kawabata\ Mr[ Kenji Watanabe and Dr[ Eri Fukushi "GC!MS + NMR Laboratory\ Faculty of Agriculture\ Hokkaido University# for measuring the 1D NMR and mass spectra[ The authors wish to thank for M[S[ M[ Tofazzal Islam for linguistic improvement of the manuscript[

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