Megastigmane glycosides from seeds of Trifolium alexandrinum

Article abstract of DOI:10.1016/S0031-9422(98)00603-7

Five megastigmane glycosides have been isolated from the seeds of Trifolium alexandrinum L., of which two are known compounds, while three are new compounds showing the presence of apiofuranosyl-(1→2)-glucopyranosyl residue as a sugar moiety. The structures of the isolated compounds were established on the basis of NMR and mass spectral data.

Full text of DOI:10.1016/S0031-9422(98)00603-7

\
PERGAMON
Phytochemistry 49 "0888# 748Ð751
Megastigmane glycosides from seeds of Trifolium alexandrinum
Khaled M[ Mohameda\ꢀ\ Mahmoud H[ Mohamedb\ Kazuhiro Ohtanic\ Ryoji Kasaic\
Kazuo Yamasakic
aDepartment of Pharmacognosy\ Faculty of Pharmacy\ Assiut University\ Assiut 60415\ Egypt
bDepartment of Pharmacognosy\ Faculty of Pharmacy\ Al!Azhar University\ Assiut\ Egypt
cInstitute of Pharmaceutical Sciences\ Hiroshima University School of Medicine\ 0!1!2 Kasumi\ Minami!Ku\ Hiroshima 623\ Japan
Received 13 July 0887
Abstract
Five megastigmane glycosides have been isolated from the seeds of Trifolium alexandrinum L[\ of which two are known compounds\
while three are new compounds showing the presence of apiofuranosyl!"0:1#!glucopyranosyl residue as a sugar moiety[ The
structures of the isolated compounds were established on the basis of NMR and mass spectral data[ Þ 0888 Elsevier Science Ltd[
All rights reserved[
Keywords] Trifolium alexandrinum^ Leguminosae^ Megastigmane glycosides
0[ Introduction
The 49) methanolic eluate was subjected to repeated
silica gel\ RP!07 column chromatography and pre!
parative HPLC using ODS column to give _ve com!
pounds "0Ð4#[
The seeds of Trifolium alexandrinum L[ is popularly
used in Egypt as an antidiabetic agent "Salah + El!
Awady\ 0850^ Helmi\ El!Mahdy\ Ali\ + Khayyal\ 0858#[
Previously\ oleanene!type triterpenoidal saponins
"Mohamed\ Ohtani\ Kasai\ + Yamasaki\ 0884#^ quer!
cetin\ quercetrin and a new ~avanonol derivative named
trifolexin have been isolated from the seeds of this plant
"Maatooq\ 0886#[
This paper deals with the isolation and structure eluci!
dation of _ve megastigmane glycosides from the seeds of
Trifolium alexandrinum\ two of which are known glu!
coside derivatives "0 and 1# and they are reported for the
_rst time from the family Leguminosae\ while compounds
2Ð4 are new megastigmane glycosides displaying an api!
ofuranosyl!"0:1#!glucopyranosyl moiety[
1[0[ Structural formulae
Compound 0 had a molecular formula of C08H29O7 as
determined from the negative HR!FAB!mass spectrum
and02 C NMR spectral data[ The02 C and0H NMR spectra
of 0 "Tables 0 and 1# together with DEPT mode measure!
ment showed the presence of a P!D!glucopyranosyl moi!
ety from the signals at dC 091[6 and dH 3[22 "0H\ d\ Jꢁ6[7
Hz#[ They showed also the existence of an aglycone with
02 carbon atoms which had a trans!disubstituted double
bond\ an a\P!unsaturated ketone\ an oxygenated quat!
ernary carbon\ three tertiary methyls and a secondary
methyl\ suggesting a megastigmane "a!ionol# skeleton\
which was con_rmed by measurement of 1!D NMR
including HÐH COSY and HSQC spectral analyses[
Enzymatic hydrolysis of 0 using P!glucosidase yielded D!
glucose and an aglycone "0a# having identical physical
data "m[p[ and ðaŁD# as those previously reported for
vomifoliol "Pousset + Poisson\ 0858^ Fukui\ Koshimizu\
Usuda\ + Yamazaki\ 0866^ Okamura\ Yagi\ + Nishioka\
0870#[ From the aforementioned data compound 0 could
be identi_ed as "5S\6E\8R#!5\8!dihydroxymegastigma!
3\6!dien!2!one!8!O!P!D!glucopyranoside or roseoside\
which has previously been found in Zizyphus jujuba "Oka!
1[ Results and discussion
The seeds of T[ alexandrinum were _rst defatted with
hexane followed by extraction with methanol[ The con!
centrated extract was diluted with water and partitioned
between ethylacetate and water[ The aqueous fraction
was then applied on a column of Diaion HP 19 and eluted
with water\ 49) methanol and _nally with methanol[
ꢀ Corresponding author[
9920!8311:88:, ! see front matter Þ 0888 Elsevier Science Ltd[ All rights reserved[
PII] S9920!8311 "87#99592!6

759
K[M[ Mohamed et al[:Phytochemistry 49 "0888# 748Ð751
Table 0
The molecular formula of compound 1 was assigned
from 02 C\ DEPT 02 C NMR and negative HR!FAB!mass
spectral data as C08H29O6[ The02 C and0H NMR "Tables
0Ð1# of 1 showed a close similarity to 0 except for the
signals at dC 45[7 with dH 1[55\ 0H\ d\ Jꢁ8[9 Hz assigned
for the methine carbon C!5 and other e}ects on C!0\ C!
4\ C!6\ C!01 and C!02 "see Table 0# due to the absence
of a C!5 axial hydroxyl group compared to 0[ The results
of 1!D HÐH COSY and HSQC together with comparison
the 02 C and 0H NMR data of compound 1 with those
reported before "Pabst\ Barron\ Semon\ + Schrier\ 0881#
proved its structure as "5R\6E\8R#!8!hydroxymega!
stigma!3\6!dien!2!one!8!O!P!D!glucopyranoside which
was isolated from raspberry fruit along with its
"5R\6E\8S#!diastereomer "Pabst et al[\ 0881^ Ito\ Yasu!
moto\ Kasai\ + Yamasaki\ 0883#\ and this is the _rst
report on its isolation from the family Leguminosae[ For
further con_rmation\ compound 1 was subjected to enzy!
matic hydrolysis using P!glucosidase to a}ord D!glucose
and the aglycone "5R\6E\8R#!8!hydroxymegastigma!3\6!
dien!2!one "1a# with ðaŁD ¦081> "Ref[ Aasen\ Kimland\
+ Enzell\ 0862^ Behr\ Wahlberg\ Nishida\ + Enzell\ 0867^
¦066>\ for the naturally occurring "5R\6E\8R#!derivative
and ¦58> for the synthetic "5R\6E\8S#!derivative "Behr
et al[\ 0867#[
The molecular formula of compound 2 was calculated
as C13H27O01 from 02 C NMR\ DEPT 02 C NMR and
negative HR!FAB!mass spectral data[ Acid hydrolysis of
2 using 0 M HCl a}orded 0a\ D!glucose and D!apiose
identi_ed by direct comparison with authentic samples[
The negative FAB!mass spectral data of 2 supported the
presence of an extra apiosyl moiety as compared with 0
by showing a quasi molecular anion at m:z 406 ðMÐHŁ−
and a fragment peak at m:z 274 ðMÐHÐApiŁ−\ indicating
that apiose is the terminal sugar[ Comparison the 02 C
and 0H NMR spectral data of 2 "Tables 0Ð1# with those
of 0 revealed that 2 is an apiofuranosyl!derivative of
02C NMR data of compounds 0Ð4 "099 MHz\ CD2OD#
C
0
1
2
3
4
4a
4aa
0
1
2
3
4
5
6
7
31[3
49[6
26[0
37[2
31[3
49[6
26[0
37[3
26[4
37[3
25[2
36[6
25[1
36[4
190[0
016[1
056[1
79[9
020[4
024[2
66[2
10[1
12[3
13[6
08[4
191[9 190[1 191[9 191[5 087[3 087[5
015[1 016[1 015[0 014[5 014[1 014[1
054[7 056[2 054[8 069[2 054[2 054[1
45[7
79[9
45[7
41[5
15[8
27[0
67[1
14[1
16[7
18[2
08[7
40[0
14[8
26[1
64[6
13[3
16[0
17[6
08[4
40[9
15[2
27[8
56[5
14[6
16[0
17[5
08[4
017[7 020[4 017[7
027[1 024[2 027[1
8
66[9
10[9
16[5
17[0
12[7
65[8
10[1
12[3
13[6
08[4
65[5
10[9
16[5
17[0
12[7
09
00
01
02
Glc
0?
1?
2?
3?
091[6
64[1
67[9
60[5
67[0
51[7
091[4 090[2 099[8 099[6 099[2
64[2
67[9
60[4
67[0
51[6
68[2
66[8
60[6
67[9
51[7
68[2
66[7
60[5
66[8
51[6
68[9
67[9
61[1
67[9
52[0
68[9
66[6
61[0
66[8
51[8
4?
5?
Api
0ý
1ý
2ý
3ý
009[7 009[7 009[6 009[3
67[5
79[5
55[0
64[3
67[5
79[5
55[0
64[2
67[7
79[8
55[5
64[6
67[1
79[8
55[6
64[6
4ý
a
These data were recorded in pyridine!d4[
mura et al[\ 0870#\ Vinca rosea "Bhakuni\ Joshi\ Uprety\
+ Kapil\ 0863#\ Cydonia oblonga\ Betula alba "Tschesche\
Ciper\ + Harz\ 0865#\ Nicotiana tabacum "Kodama\ Fuji!
mori\ + Kato\ 0870# and Pinus sylvestris "Andersson +
Lundgren\ 0877# and this is the _rst report on its occur!
rence in family Leguminosae[
Table 1
0H NMR data of compounds 0Ð4 "399 MHz\ CD2OD#
H
0
1
2
3
4
4a
1a
1b
3
1[49\ 0H\ d"05[4#
1[03\ 0H\ d"05[4#
4[70\ 0H\ s
1[31\ 0H\ d"05[3#
1[92\ 0H\ d"05[3#
4[75\ 0H\ s
1[41\ 0H\ d"05[4#
1[05\ 0H\ d"05[4#
4[71\ 0H\ s
1[35\ 0H\ d"05[7#
1[97\ 0H\ d"05[7#
4[80\ 0H\ s
1[38\ 0H\ d"06[0#
1[99\ 0H\ d"06[0#
4[73\ 0H\ s
1[32\ 0H\ d"05[7#
1[91\ 0H\ d"05[7#
5[32\ 0H\ s
5
1[55\ 0H\ d"8[9#
1[60\ 0H\ d"8[9#
0[21\ 0H\ m
0[34\ 0H\ m
6
4[75\ 0H\ d"04[6#
4[54\ 0H\ dd"04[3\ 8[9# 4[76\ 0H\ d"04[5#
4[56\ 0H\ dd"04[3\ 8[9# 0[37\ 1H\ m
0[51\ 1H\ m
7
4[73\ 0H\ dd"04[6\ 5[2# 4[65\ 0H\ dd"04[3\ 5[3# 4[74\ 0H\ dd"04[5\ 5[3# 4[79\ 0H\ dd"04[3\ 5[2# 0[55\ 1H\ m
0[66\ 1H\ m
8
3[30\ 0H\ quint[ "5[2#
0[16\ 2H\ d"5[2#
0[91\ 2H\ s
0[92\ 2H\ s
0[80\ 2H\ s
3[28\ 0H\ m
0[17\ 2H\ d"5[3#
9[88\ 2H\ s
0[91\ 2H\ s
0[81\ 2H\ s
3[31\ 0H\ m
0[16\ 2H\ d"5[3#
0[90\ 2H\ s
0[91\ 2H\ s
0[81\ 2H\ s
3[31\ 0H\ m
0[20\ 2H\ d"5[2#
0[93\ 2H\ s
2[15\ 0H\ m
0[10\ 2H\ d"5[0#
0[93\ 2H\ s
2[74\ 0H\ m
0[18\ 2H\ d"5[0#
9[81\ 2H\ s
0[93\ 2H\ s
0[74\ 2H\ s
09
00
01
02
0?
0ý
0[95\ 2H\ s
0[02\ 2H\ s
0[86\ 2H\ d"0[1#
3[34\ 0H\ d"6[7#
4[39\ 0H\ d"0[4#
1[98\ 2H\ d"0[2#
3[32\ 0H\ d"6[7#
4[39\ 0H\ d"0[1#
3[22\ 0H\ d"6[7#
3[23\ 0H\ d"6[8#
3[39\ 0H\ d"6[6#
4[28\ 0H\ d"0[3#
3[74\ 0H\ d"6[5#
4[77\ 0H\ br[s
a
Data was recorded in pyridine!d4^ J values in parentheses are recorded in Hz[

K[M[ Mohamed et al[:Phytochemistry 49 "0888# 748Ð751
750
roseoside "0# as indicated from the signals at dC 009[7 "C!
0ý# with dH 4[28 "0H\ d\ Jꢁ0[3 Hz\ H!0ý# of the P!D!
apiofuranosyl moiety "Otsuka et al[\ 0883# and the signals
at dC 090[2 "C!0?# with dH 3[39 "0H\ d\ Jꢁ6[6 Hz\ H!0?#
of the P!D!glucopyranosyl unit[ Their attachment tog!
ether by a "0:1# interglycosidic linkage was deduced
from the down_eld shift of C!1? of glucose to dC 68[2 with
up_eld shift of C!0? to dC 090[2 when compared with the
corresponding shifts of 0 at dC 64[1 and 091[6\ respec!
tively[ Therefore\ the new compound 2 was identi_ed
as "5S\6E\8R#!5\8!dihydroxymegastigma!3\6!dien!2!one!
8!O!P!D!apiofuranosyl!"0:1#!P!D!glucopyranoside and
named as trifostigmanoside I[
"Otsuka et al[\ 0883#[ In the NOESY experiment of 4\
correlation peaks between H!5 and H!01 and H!02 were
observed indicating a!axial orientation of H!5[ The
chirality at C!5 was established to be R!con_guration
as shown by molecular models which was also in ac!
cordance with the naturally occurring blumenol C
"Galbraith + Horn\ 0861#[ From the above mentioned
data\ the new compound 4 could be characterized
as "5R\8R#!8!hydroxymegastigma!3!en!2!one!8!O!P!D!
apiofuranosyl!"0:1#!P!D!glucopyranoside and named as
trifostigmanoside III[
2[ Experimental
The elemental composition of compound 3 was deter!
mined as C13H27O00 by negative HR!FAB!MS and 02
C
Mp[ uncorr[^0H and02 C NMR "TMS as int[ standard#]
399 and 099 MHz\ respectively[ FAB!MS] direct inlet
method at an ionizing voltage of 69 eV[ HPLC] D!ODS!
4 column "19 mm I[D[×14 cm# with di}erential refrac!
tometer as detector^ ~ow rate of mobile phase 5 ml:min\
injection volume 9[7Ð0[9 ml[ CC] Kieselgel 59 "69Ð129
mesh\ Merck# and Diaion HP 19 "Mitsubishi#[ TLC] silica
gel 59 precoated plates\ F!133 and HPTLC using RP!07
precoated plates\ F!133 s "Merck#[
NMR spectral analysis[ The 02 C and 0H NMR spectral
data of 3 showed a close similarity to that of 1\ and
inspection of the data "Tables 0Ð1# veri_ed that the
aglycone part was 1a and the sugar part could be assigned
as P!D!apiofuranosyl!"0:1#!P!D!glucopyranose\ when
compared with that of 2[ These assignments were
achieved from the signals at dC 009[7 "C!0ý# with dH 4[39
"0H\ d\ Jꢁ0[4 Hz\ H!0ý# and dC 099[8 "C!0?# with dH 3[34
"0H\ d\ Jꢁ6[7 Hz\ H!0?# with down_eld shift of C!1? of
glucose to dC 68[2 when compared with the correspond!
ing shift of 1 at dC 64[2[ For further con_rmation\ com!
pound 3 was hydrolysed using 0 M HCl and the obtained
products were identi_ed as 1a\ D!glucose and D!apiose
by direct comparison with authentic materials[
Consequently\ the new compound 3 was identi_ed
as "5R\6E\8R#!8!hydroxymegastigma!3\6!dien!2!one!8!
O!P!D!apiofuranosyl!"0:1#!P!D!glucopyranoside and
named as trifostigmanoside II[
2[0[ Plant material
Seeds of T[ alexandrinum L[ "local name] Bersim# was
collected from Assiut\ Egypt\ in August 0885[ The plant
was authenticated by Professor A[ Fayed\ from the
Department of Botany\ Faculty of Science\ Assiut Uni!
versity\ Assiut\ Egypt[ A voucher specimen is deposited
at the Herbarium of the Department of Pharmacognosy\
Faculty of Pharmacy\ Assiut University\ Assiut\ Egypt[
Compound 4 was analyzed for C13H39O00 "negative
0
HR!FAB!MS and DEPT 02 C NMR[ The 02 C and H
2[1[ Extraction and isolation of compounds "0Ð4#
NMR "CD2OD\ Tabs[ 0Ð1# of 4 exhibited signals cor!
responding to P!D!apiofuranosyl!"0:1#!P!D!gluco!
pyranosyl moiety when compared with those of 2 and 3[
The remaining signals of the aglycone part indicated the
presence of megastigmane skeleton with close similarity
to 2 and 3 except for saturation of the double bond at C!
6 to give two methylenes at dC 15[8 and 27[0 cor!
responding to C!6 and C!7\ respectively with dH 0[37
and 0[55 "each 1H\ m# for H!6 and H!7\ respectively[
Comparing the NMR data of 4 in pyridine!d4 "Tables 0Ð
1# with those reported earlier "Galbraith + Horn\ 0861^
Miyase\ Ueno\ Takizawa\ Kobayashi\ + Oguchi\ 0877#
identi_ed the aglycone part as blumenol C[ The acid
hydrolysis of 4 using 0 M HCl a}orded D!glucose and D!
apiose in addition to blumenol C "4a# with ðaŁD ¦009[2>\
which was in a good agreement with the reported
value "Ref[ Galbraith + Horn\ 0861^ ¦001[4>#[ The
NMR spectrum of 4a in pyridine!d4 "Table 0# showed
that C!8 was shifted up_eld to dC 56[5 in comparison
with that of 4 at dC 64[6 "glycosylation shift valueꢁ
−7[1# assuming the chirality at C!8 as R!con_guration
Air dried and powdered seeds "0 kg# were defatted
"×2# by maceration with hexane\ followed by extraction
with MeOH under re~ux conditions[ The combined
methanolic extracts were _ltered\ evapd[ under red[ pres[
The residue suspended in H1O and partitioned with
EtOAc[ The aq[ fr[ was applied to a column of Diaion
HP 19 and eluted with H1O\ 49) MeOH and _nally with
MeOH[ The 49) MeOH eluate "5[9 g# was chro!
matographed by silica gel CC using EtOAcÐMeOHÐH1O
"7]1]0 and 5]1]0 successively# to give 5 frs[ Fr[ 0 "419 mg#
was sepd[ by a column of reversed phase RP!07 using
49) MeOH to a}ord 0 "003 mg# and 1 "00 mg#[ Fr[ 2
"139 mg# was chromatographed on a column of RP!07
using 39) MeOH followed by prep[ ODS HPLC using
31[4) MeOH to give 2 "8 mg#\ 3 "02 mg# and 4 "14 mg#[
2[2[ Compound 0
Amorphous powder\ ðaŁ11 ¦000[1> "MeOH^ c 0[22#[
D
HR!FAB!MS "negative# m:z] 274[0773 ðMÐHŁ−

751
K[M[ Mohamed et al[:Phytochemistry 49 "0888# 748Ð751
0
C08H18O7\ req[ 274[0751\ 112 ðMÐHÐglcŁ−[ 02 C and H
2[8[ Acid hydrolysis of compounds 2Ð4
NMR "CD2OD\ Tables 0Ð1#[
Compound 2 "4 mg# in 0 M HCl "09 ml# was re~uxed
at 79>C for 2 h[ The reaction mixture was diluted with
H1O and then extracted with Et1O\ from the ethereal
layer the aglycone 0a "1[1 mg# was obtained[ In the aq[
phase D!apiose and D!glucose were identi_ed by TLC
comparison with authentic substances[ By the same pro!
cedure\ 5 mg of 3 was hydrolyzed to give D!apiose\ D!
glucose and the aglycone 1a "2 mg#[ Compund 4 "01 mg#
was also hydrolyzed by the same way to a}ord D!apiose\
D!glucose and 5[3 mg of the aglycone blumenol C "4a# as
an oily liquid\ ðaŁ11D ¦009[2> "CHCl2^ c 9[31#\ 02 C NMR
Table 0[
2[3[ Compound 1
Amorphous powder\ ðaŁ11 ¦84[1> "MeOH^ c 0[22#[
D
HR!FAB!MS "negative# m:z] 258[0818 ðMÐHŁ−
C08H18O6\ req[ 258[0802\ 196 ðMÐHÐglcŁ−[ 02 C and H
0
NMR "CD2OD\ Tables 0Ð1#[
2[4[ Enzymatic hydrolysis of 0 and 1
To a solution of 0 "39 mg# in 4 ml acetate bu}er "pH
4[9# was added 49 mg of P!glucosidase\ and the mixture
was incubated with stirring at 26>C for 37 h and the
mixture was extracted with Et1O "2×09 ml#[ The com!
bined ethereal layers were evapd[ and the residue was
puri_ed by repeated crystallization from C5H5ÐEtOAc
mixture to a}ord 00 mg of the aglycone vomifoliol "0a#\
mp 000Ð002>C\ ðaŁ11D ¦120[6> "CHCl2^ c 9[62#[ In the aq[
phase D!glucose was identi_ed by TLC comparison with
an authentic sample[ By the same procedure\ 5 mg of 1
was hydrolyzed to give D!glucose and the aglycone 1a
"2[1 mg# as an oily liquid\ ðaŁ11D ¦081[3> "CHCl2^ c 9[10#[
Acknowledgements
The authors are grateful to The Research Center of
Molecular Medicine of the Hiroshima University School
of Medicine\ Japan\ for NMR measurements[
References
Aasen\ A[ J[\ Kimland\ B[\ + Enzell\ C[ R["0862#[ Acta Chem[ Scand[\
16\ 1096[
Andersson\ R[\ + Lundgren\ L[ N["0877#[ Phytochemistry\ 16\ 448[
Behr\ D[\ Wahlberg\ I[\ Nishida\ T[\ + Enzell\ C[ R["0867#[ Acta Chem[
Scand[ B\ 21\ 280[
Bhakuni\ D[ S[\ Joshi\ P[ P[\ Uprety\ H[\ + Kapil\ R[ S["0863#[ Phy!
tochemistry\ 02\ 1430[
2[5[ Compound 2
Fukui\ H[\ Koshimizu\ K[\ Usuda\ S[\ + Yamazaki\ Y["0866#[ Agric[
Biol[ Chem[\ 30\ 064[
Galbraith\ M[ N[\ + Horn\ D[ H[ S["0861#[ J[C[S[ Chem[ Comm[\ 2\
002[
Helmi\ R[\ El!Mahdy\ S[ A[\ Ali\ H[\ + Khayyal\ M[ A[ H["0858#[ J[
Egypt[ Med[ Assoc[\ 41\ 427[
Amorphous powder\ ðaŁ11 ¦41[8> "MeOH^ c 9[51#[
D
HR!FAB!MS "negative# m:z] 406[1180 ðMÐHŁ−
C13H26O01\ req[ 406[1173\ 274 ðMÐHÐApiŁ−\ 112 ðMÐHÐ
ApiÐglcŁ−[ 02 C and 0H NMR "CD2OD\ Tables 0Ð1#[
Ito\ A[\ Yasumoto\ K[\ Kasai\ R[\ + Yamasaki\ K["0883#[ Phyto!
chemistry\ 25\ 0354[
Kodama\ H[\ Fujimori\ T[\ + Kato\ K["0870#[ Biol[ Chem[\ 34\ 830[
Maatooq\ G[ T["0886#[ Mans[ J[ Pharm[ Sci[\ 02\ 69[
Miyase\ T[\ Ueno\ A[\ Takizawa\ N[\ Kobayashi\ H[\ + Oguchi\
H["0877#[ Chem[ Pharm[ Bull[\ 25\ 1364[
Mohamed\ K[ M[\ Ohtani\ K[\ Kasai\ R[\ + Yamasaki\ K["0884#[ Phy!
tochemistry\ 39\ 0126[
Okamura\ N[\ Yagi\ A[\ + Nishioka\ I["0870#[ Chem[ Pharm[ Bull[\ 18\
2496[
2[6[ Compound 3
Amorphous powder\ ðaŁ11 ¦05[2> "MeOH^ c 9[79#[
D
HR!FAB!MS "negative# m:z
]
490[1240 ðMÐHŁ−
C13H26O00\ req[ 406[1225\ 258 ðMÐHÐApiŁ−\ 196 ðMÐHÐ
ApiÐglcŁ−[ 02 C and 0H NMR "CD2OD\ Tables 0Ð1#[
Otsuka\ H[\ Kamada\ K[\ Ogimi\ C[\ Hirata\ E[\ Takushi\ A[\ + Takeda\
Y["0883#[ Phytochemistry\ 24\ 0220[
Pabst\ A[\ Barron\ D[\ Semon\ E[\ + Schrier\ P["0881#[ Phytochemistry\
20\ 0538[
Pousset\ J[ L[\ + Poisson\ J["0858#[ Tetrahedron Lett[\ 04\ 0062[
Salah\ M[ K[\ + El!Awady\ M[ M["0850#[ J[ Pharmaceut[ Sci[ U[A[R[\
1\ 006[
2[7[ Compound 4
Amorphous powder\ ðaŁ11 ¦66[0> "MeOH^ c 9[76#[
D
HR!FAB!MS "negative# m:z] 492[1388 ðMÐHŁ−
C13H28O00\ req[ 492[1381\ 260 ðMÐHÐApiŁ−\ 198 ðMÐHÐ
ApiÐglcŁ−[ 02 C and 0H NMR "CD2OD\ Tables 0Ð1#[
Tschesche\ R[\ Ciper\ F[\ + Harz\ A["0865#[ Phytochemistry\ 04\ 0889[