Camptothecin-related alkaloids from hairy roots of Ophiorrhiza pumila

Article abstract of DOI:10.1016/S0040-4020(02)01196-1

Hairy roots of Ophiorrhiza pumila, a rubiaceous camptothecin-producing plant, were obtained. From an investigation of the secondary metabolites, it was found that the hairy roots produced camptothecin and its related alkaloids, i.e. (3S)-pumiloside, (3S)- and (3R)-deoxypumilosides and strictosamide. We also isolated two new camptothecinoids, OPHR-23 and OPHR-17, the structures of which including the absolute configurations, were determined by spectroscopic analyses and chemical conversion from tryptamine and secologanin.

Full text of DOI:10.1016/S0040-4020(02)01196-1

Tetrahedron 58 (2002) 9169–9178
Camptothecin-related alkaloids from hairy roots
of Ophiorrhiza pumila
a
Mariko Kitajima, Satoshi Yoshida, Kyoko Yamagata, Mio Nakamura, Hiromitsu Takayama,
a
a
a
a
a
Kazuki Saito, Hiroko Seki and Norio Aimi
b
a,
*
a
Graduate School of Pharmaceutical Sciences, Chiba University, 1-33 Yayoi-cho, Inage-ku, Chiba 263-8522, Japan
Chemical Analysis Center, Chiba University, 1-33 Yayoi-cho, Inage-ku, Chiba 263-8522, Japan
b
Received 19 August 2002; accepted 20 September 2002
Abstract—Hairy roots of Ophiorrhiza pumila, a rubiaceous camptothecin-producing plant, were obtained. From an investigation of the
secondary metabolites, it was found that the hairy roots produced camptothecin and its related alkaloids, i.e. (3S)-pumiloside, (3S)- and (3R)-
deoxypumilosides and strictosamide. We also isolated two new camptothecinoids, OPHR-23 and OPHR-17, the structures of which
including the absolute configurations, were determined by spectroscopic analyses and chemical conversion from tryptamine and
secologanin. q 2002 Elsevier Science Ltd. All rights reserved.
1. Introduction
2. Results and discussion
1
Camptothecin (1) is a well-known anti-tumor alkaloid,
which was first isolated from Camptotheca acuminata
belonging to Nyssaceae. This compound possesses inhibi-
tory activity against DNA topoisomerase I and also activity
against HIV-1. Recently, semi-synthetic camptothecins
such as irinotecanw and topotecan have been used as
clinical antitumor agents. In the course of our chemical
studies on indole alkaloids, we found that Ophiorrhiza
pumila (rubiaceae) produces camptothecin (1) and its
related alkaloids such as (3S)-pumiloside (2), (3S)- and
We succeeded in obtaining hairy roots of O. pumila by the
direct inoculation of Agrobacterium rhizogenes (pRi15834;
pGSGluc1) to the regenerated plant stems. Agrobacterium-
inoculated stems were maintained on the hormone-free half-
strength Murashige and Skoog medium and after about 80
days hairy roots appeared. The hairy roots obtained were
cultivated on the same medium under light at 258C and
frequently subcultivated on fresh medium at intervals of two
weeks.
5
w
(
3R)-deoxypumilosides (3 and 4) which might be plausible
Hairy roots (190 g of wet weight) cultivated on the solid
medium were extracted with hot MeOH to give the extract
(2.3 g). The MeOH extract was then partitioned between
H O and CHCl and the aqueous layer was extracted with
2
biogenetic precursors of camptothecin (1). For the purpose
of establishing an efficient production method of campto-
thecin (1) as well as finding novel secondary metabolites
and a clue to clarification of the camptothecin biosynthetic
pathway, we have also investigated the constituents of
callus cultures and regenerated plants of O. pumila. Callus
cultures produce anthraquinones that could not be detected
in the wild plants, and the regenerated plants produce almost
the same alkaloids as the wild plants. In continuation of our
2
3
n-BuOH to give the CHCl extract (263 mg), the n-BuOH
3
extract (210 mg) and the water-soluble portion (1.83 g),
respectively. From the extract, camptothecin-related alka-
loids such as (3S)-pumiloside (2) (33.7 mg), (3S)- and (3R)-
deoxypumilosides (3, 9.9 mg and 4, 9.4 mg) and strictosa-
mide (5, 9.4 mg) were isolated together with one new
alkaloid, OPHR-23 (6, 0.4 mg). This result revealed that
hairy roots of O. pumila maintained the alkaloid production
ability. Although a small amount of (3S)-deoxypumiloside
(3) was already isolated as a tetraacetate derivative from the
3
4
5
study, we obtained hairy roots of O. pumila and
investigated secondary metabolites of the hairy roots,
resulting in the isolation and structure elucidation of
alkaloids, including new camptothecinoids, which will be
described here in detail.
2
d
wild plants, at this time, we obtained (3S)-deoxypumilo-
side (3) as a genuine form and in comparable amount with
the 3-epimer (4).
The new alkaloid, OPHR-23 (6, 0.4 mg), having blue
fluorescence under UV lamp at the wavelength of 365 nm,
showed the characteristic UV spectrum of camptothecin at
Keywords: camptothecin; Ophiorrhiza pumila; alkaloids.
*
Corresponding author. Tel./fax: þ81-43-290-2901;
e-mail: aimi@p.chiba-u.ac.jp
0040–4020/02/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved.
PII: S0040-4020(02)01196-1

9170
M. Kitajima et al. / Tetrahedron 58 (2002) 9169–9178
Figure 1.
362, 290, 254, 219 nm. From the HR-FAB-MS, the
molecular formula was decided to be C H N O . The
weight) cultivated in the liquid medium were extracted with
hot MeOH to give the extract (10.6 g). The MeOH extract
was then partitioned between H O, CHCl and n-BuOH to
2
7 28 2 9
1
H NMR spectrum revealed five aromatic protons due to
the 2,3-disubstituted quinoline system [d 8.67 (s, H-7), 8.16
d, H-12), 8.11 (d, H-9), 7.85 (dd-like, H-11), 7.70 (dd-like,
2
3
give the CHCl3 extract (790 mg), the n-BuOH extract
(1666 mg) and the water-soluble portion (6.03 g), respec-
tively. Two new camptothecinoids (6, 0.3 mg and 7,
0.4 mg), one of which was the same as the new campto-
thecinoid isolated from the hairy roots cultivated on the
solid medium, were isolated together with camptothecin
(1, 17.7 mg), (3S)-pumiloside (2, 9.9 mg), (3R)-deoxy-
pumiloside (4, 1.3 mg) and strictosamide (5, 9.9 mg).
(
H-10)], one singlet aromatic proton due to H-14 (d 7.02) and
one methylene proton due to H -5 (d 5.24), which are
2
characteristic protons of camptothecin (1). A set of three
protons on a vinyl group [d 5.81 (ddd, J¼17.1, 10.1, 9.1 Hz,
H-19), 5.51 (br-d, J¼17.1 Hz, H-18), and 5.44 (dd, J¼10.1,
2
.1 Hz, H-18)], an acetal proton at d 5.46 (d, J¼7.0 Hz,
H-21), and one sugar unit containing an anomeric proton at
d 4.64 (d, J¼7.9 Hz, H-1 ) were also observed, which are
0
very similar to those of strictosamide (5), (3S)-pumiloside
The new alkaloid, OPHR-17 (7), having blue fluorescence
under UV lamp (l 365 nm), showed the UV spectrum (363,
280 sh, 254, 219 nm) and the molecular formula
C H N O ) which were the same as those of OPHR-23
(
acetal proton at d 5.65 (s, H-17) and methoxy protons at d
2), and deoxypumilosides (3 and 4), together with another
2
7 28 2 9
1
3
2
1
3
.47 (s). The C NMR showed 27 carbons including 16 sp
(6). The H NMR spectrum revealed five aromatic protons
due to the 2,3-disubstituted quinoline system [d 8.68 (s,
H-7), 8.15 (d, H-12), 8.12 (d, H-9), 7.85 (dd-like, H-11),
7.70 (dd-like, H-10)], one singlet aromatic proton due to
carbons, three acetal carbons (d 97.9, 95.2 and 91.7), a
methoxy carbon (d 55.2) and carbons due to one glucose
unit. In the HMBC spectrum, crosspeaks between the acetal
proton at d 5.65 and both carbons due to the methoxy group
and the acetal carbon at d 91.7 (C-21) and a crosspeak
H-14 (d 7.14) and one methylene proton due to H -5 (d 5.25)
2
as camptothecin, suggesting that the D-ring was oxidized to
the pyridone ring. Furthermore, a set of three protons on a
vinyl group at d 5.82 (ddd, J¼17.0, 10.2, 7.0 Hz, H-19),
5.37 (br-d, J¼17.0 Hz, H-18), and 5.23 (br-d, J¼10.2 Hz,
H-18), two acetal protons at d 5.53 (s, H-17), 5.45
0
between the anomeric proton (d 4.64, H-1 ) and the same
acetal carbon (C-21) were observed, suggesting that the
methoxy group was located on C-17 (Figs. 1 and 2). From
these data, the structure of this alkaloid was deduced to be 6.
(
d, J¼1.7 Hz, H-21), one anomeric proton at d 4.65
0
As the new compound (6) possesses a unique structure, we
tried again to get this alkaloid from the hairy roots cultivated
in the liquid medium. On the basis of the fundamental study
on the culture condition, we cultivated the hairy roots in
Gamborg’s B5 medium containing sucrose (1%) under light
(d, J¼7.8 Hz, H-1 ) and methoxy protons at d 3.57 (s)
1
3
were observed. The C NMR showed 27 carbons including
three acetal carbons (d 97.6, 94.5, 94.3) and a methoxy
carbon (d 57.1) which are the same as those in 6. From the
above spectral data, the structure of OPHR-17 was
presumed to be 7 and the new alkaloids 6 and 7 were
considered to be a pair of diastereomers at the C-17 position.
(
750 lx) at 258C at 66 rpm. Hairy roots (about 375 g of wet
We next planned the synthesis of two new camptothecinoids
starting from tryptamine (8) and secologanin (9) to establish
the structures, including the absolute configurations
(Scheme 1). Condensation of 8 and 9 by the Pictet–
Spenglar reaction under acidic condition followed by D-ring
closure by treatment with alkaline gave strictosamide (5, y.
2
3
6%) having 3S and vincoside lactam (10, y. 33%) having
6
R stereochemistry, respectively. (3S)-Pumiloside tetra-
acetate (11) was prepared from 5 by a three-step procedure
that included acetylation of the hydroxy groups in the
glucose unit, oxidative bond cleavage at C2–C7 by using
Figure 2.
excess NaIO , and ring closure between C2 and C6 to give a
4

M. Kitajima et al. / Tetrahedron 58 (2002) 9169–9178
9171
Scheme 1.
7
quinolone ring. Removal of the protecting group of 11 gave
3S)-pumiloside (2). 11 was treated with lithium diisopro-
pylamide (LDA) and then with N-phenyltrifluoromethane-
could be removed under acidic condition. Compound 16
having the Troc group on the hydroxy function was prepared
from (3R)-deoxypumiloside (4) and then treated with DDQ
in toluene–MeOH at 508C to afford 18 and 19 in 52 and
15% yield, respectively. These compounds showed the
characteristic UV absorptions of camptothecin (18: 358,
291, 253, 215 nm, 19: 359, 288, 253, 218 nm), suggesting
(
8
sulfonimide in THF–HMPA. The enol triflate thus
obtained was treated with Pd(OAc) , 1,1 -bis(diphenyl-
0
phosphino)-ferrocene (DPPF), Et N and HCOOH in
2
3
9
dioxane to afford the deoxygenated compound (14).
Deacetylation of 14 by NaOMe gave (3S)-deoxypumiloside
1
the aromatization of the D-ring. In the H NMR spectrum,
(
3), whose spectroscopic data, including the CD spectrum,
an aromatic proton at H-14 (18: d 7.20, 19: d 7.16), an acetal
proton at H-17 (18: d 5.78, 19: d 5.64) along with two acetal
protons at H-21 and H-1 , and 17-OMe were observed.
were identical with those of natural 3 that was obtained from
the hairy roots of O. pumila. The same procedure was
applied to vincoside lactam (10) to give (3R)-deoxypumilo-
side (4).
0
Furthermore, three acetal carbons, one methoxy carbon, and
aromatic carbons due to C-3, C-14–C-16 were observed in
1
3
the C NMR spectrum. Removal of the Troc group in each
compound by treatment with Zn in AcOH gave OPHR-23
(6) and OPHR-17 (7), respectively. Each product was
identified with the natural compound isolated from the hairy
roots of O. pumila by comparison of the spectroscopic data,
including CD spectra.
Next, (3R)-deoxypumiloside tetraacetate (15) was treated
1
0
with DDQ in toluene–MeOH at 508C to give a mixture of
7-epimers of 17 in 91% yield in the ratio of 4:1 (major/
1
minor). A mixture of these compounds showed the
characteristic UV absorptions of camptothecin and a
characteristic proton shifted to low field due to H-14 was
1
observed in the H NMR spectrum, suggesting the
The configuration of the C-17 position in OPHR-23 (6) and
OPHR-17 (7), a pair of diastereomers at C-17, was
determined by using the NMR techniques (Fig. 3). The
coupling constant between H-20 and H-21 in OPHR-23 (6)
and OPHR-17 (7) showed different values, suggesting that
each compound exists in a different conformation of the
E-ring. Therefore, four possible stereostructures could be
aromatization of the D-ring. As these compounds could
not be separated, the mixture was treated with NaOMe in
MeOH to give OPHR-23 (6) and OPHR-17 (7) along with
some undesired compounds such as 20 and 21 (main
product, y. 35%). To prevent elimination of the sugar unit
and migration of the double bond at C18–C19 to C19–C20
during deprotection of the acetyl group in 17, the protective
group on the hydroxy functions in the sugar moiety was
changed to a trichloroethoxycarbonyl (Troc) group which
considered for OPHR-23 (6) and OPHR-17 (7). OPHR-23
3
(6) has a large coupling constant (7.0 Hz) of J
1
in the
H NMR spectrum. In the differential NOE experiments of
H20,H21

9172
M. Kitajima et al. / Tetrahedron 58 (2002) 9169–9178
Scheme 2.
6
, irradiation of H-17 led to enhancement of H-21. These
at the C-17 position of 6 and 7 were determined to be 17S
and 17R, respectively.
observations revealed that 6 takes conformer I and has 17S
configuration. Furthermore, the observed C–H long-range
coupling constants between H-17 and C-15 (3.0 Hz),
between H-17 and C-21 (4.9 Hz) and between H-17 and
Camptothecin having a quinoline nucleus is biogenetically
derived from strictosidine via strictosamide (5), that was
1
1
1a,b
C-22 (1.5 Hz) obtained by PFG J-HMBC 2D spectroscopy
fitted well with the expected values from the torsion angles
Dreiding model), supporting the 17S configuration and
conformation I in 6 (Fig. 3). On the other hand, OPHR-17
actually proved by Hutchinson
(Scheme 2). However,
the biosynthetic pathway from strictosamide (5) to 1,
comprising (i) transformation of an indole 6–5–6 mem-
bered ring system of the ABC-ring to a quinoline 6–6–5
ring system, (ii) oxidation of the D-ring to a pyridone and
(iii) removal of a glucose unit and structural conversions in
the E-ring, is not clear yet. Existence of (3S)-pumiloside (2)
and (3S)-deoxypumiloside (3) suggests a possibility that
conversion of the ABC-ring of strictosamide (5) would be
the next step after the formation of strictosamide. Although
OPHR-23 (6) and OPHR-17 (7) seemed to be compounds
(
3
(7) has a small coupling constant of J
suggesting that it takes conformation II. The coupling
(1.7 Hz),
H20,H21
3
3
constants of JH17,C15 (2.5 Hz), JH17,C21 (6.2 Hz) and
3
JH17,C22 (1@ Hz) were in good agreement with those of 7
having 17R. From these results, the absolute configurations
1
2
similar to the presumed biosynthetic intermediate from 3
to 1, OPHR-23 (6) and OPHR-17 (7) might be artifacts
during the extraction or isolation process. A similar
observation was reported in the case of tetrahydroisoquino-
1
0
line-type glucosides isolated from Alangium lamarckii.
3. Conclusion
In conclusion, we have succeeded in obtaining the hairy
roots of a camptothecin-producing plant, O. pumila, in
which camptothecin and its related alkaloids, i.e. (3S)-
pumiloside, (3S)- and (3R)-deoxypumilosides and strictosa-
mide, are produced as in wild plants. The structures,
including absolute configurations of the two new alkaloids,
OPHR-23 and OPHR-17, isolated from the hairy roots were
determined by spectroscopic analyses including J-HMBC
Figure 3.

M. Kitajima et al. / Tetrahedron 58 (2002) 9169–9178
9173
2D method and chemical synthesis from tryptamine and
secologanin via pumilosides and deoxypumilosides.
CHCl and n-BuOH to give the CHCl extract (790 mg), the
3 3
n-BuOH extract (1666 mg) and the water-soluble portion
6.03 g), respectively. Camptothecin (1, 17.7 mg) was
obtained from the 50% AcOEt–MeOH—MeOH eluate of
SiO open column chromatography of the CHCl extract.
The n-BuOH extract (1666 mg) was separated by SiO2
open column chromatography eluted with MeOH–CHCl
gradient to give 10 fractions. The 20 and 25% MeOH–
CHCl eluates were purified by using a combination of
HPLC (SiO , 10% MeOH–CHCl ) and MPLC (SiO , 10%
MeOH–CHCl ) to give OPHR-17 (7, 0.4 mg) and OPHR-
23 (6, 0.3 mg), respectively. The 25% MeOH–CHCl eluate
(
4
. Experimental
2
3
Melting points were determined on a Yamato MP-21
1
3
13
apparatus and are uncorrected. H and C NMR spectra
were recorded on a JEOL JNM A-500, a JEOL JNM A-400
3
1
or a JEOL JNM FX270. H NMR spectra were measured at
1
2
3
2
3
5
1
00 or 400 MHz. C NMR spectra were measured at
25.65, 100 or 67.8 MHz. MS spectra were obtained by use
3
3
of a JEOL JMS-AM20 (EI-MS), a Hitachi RMU-7M
HR-EI-MS) or a JEOL JMS-HX110 (FAB-MS and
gave strictosamide (5, 9.9 mg). (3S)-Pumiloside (2, 9.9 mg)
and (3R)-deoxypumiloside (4, 1.3 mg) were obtained from
(
HR-FAB-MS). UV spectra were recorded on a JASCO
V-560. CD spectra were measured by a JASCO J-720WI
instrument. For TLC, precoated Silica gel 60 F254 plates
the CHCl
3
–MeOH–H
2
O¼10:5:1 eluate.
4.1.1. (3S)-Deoxypumiloside (3). HR-FAB-MS (positive,
þ
(
Merck, 0.25 mm thick) were used. For column chroma-
NBA) m/z: 497.1905 (MH ) (Calcd for C26
H
29
þ
N
2
O
8
,
1
tography, Silica gel 60 [Merck, 70–230 mesh (for open
chromatography) and 230–400 mesh (for flash chroma-
tography)], Aluminium oxide 90 [Merck] and DIAION
HP20 [Mitsubishikasei] were used. For medium pressure
liquid chromatography (MPLC), a C. I. G. prepacked
497.1924); FAB-MS (positive, NBA) m/z: 497 (MH ); H
NMR (500 MHz, CD OD) d: 8.24 (1H, s, H-7), 7.99 (1H, d,
3
J¼8.2 Hz, H-12), 7.89 (1H, d, J¼8.2 Hz, H-9), 7.71 (1H,
br-dd, J¼8.2, 8.2 Hz, H-11), 7.55 (1H, dd, J¼8.2, 8.2 Hz,
H-10), 7.16 (1H, d, J¼2.4 Hz, H-17), 5.83 (1H, ddd, J¼
17.1, 10.2, 10.2 Hz, H-19), 5.48 (1H, dd, J¼17.1, 1.8 Hz,
H-18), 5.47 (1H, d, J¼1.5 Hz, H-21), 5.34 (1H, dd, J¼10.2,
1.8 Hz, H-18), 4.94 (1H, d, J¼16.5 Hz, H-5), 4.8 (over-
lapped, H-3), 4.74 (1H, d, J¼16.5 Hz, H-5), 4.69 (1H, d,
column CPS-HS-221-05 (SiO ) and CPO-HS-221-20
2
(
ODS) [Kusano Kagakukikai] were used. For HPLC,
Senshu Pak AQUASIL SS-752N (10f£250 mm) [Senshu
Scientific Co., Ltd] was used.
0
0
J¼7.9 Hz, H-1 ), 3.86 (1H, dd, J¼12.0, 2.0 Hz, H-6 ), 3.64
0
H-3 ), 3.35 (overlapped, H-15), 3.30 (overlapped, H-5 ),
4.1. Extraction and isolation of alkaloids from hairy
roots of O. pumila
(1H, dd, J¼12.0, 5.8 Hz, H-6 ), 3.35 (1H, dd, J¼8.8, 8.8 Hz,
0
0
0
0
2.68 (1H, m, H-20), 2.65 (1H, m, H-14), 2.02 (1H, br-ddd,
3.25 (overlapped, H-4 ), 3.16 (1H, dd, J¼8.8, 7.9 Hz, H-2 ),
From hairy roots cultivated on the solid medium. Hairy
roots (190 g of wet weight), cultivated on the hormone-free
half-length Murashige and Skoog (MS) solid medium
containing sucrose (1%) under light at 258C, were extracted
with hot MeOH to give the extract (2.3 g). The MeOH
extract was then partitioned between H O and CHCl . The
1
3
J¼12.0, 12.0, 12.0 Hz, H-14);
C NMR (125 MHz,
CD OD) d: 168.2 (C-22), 163.2 (C-2), 149.0 (C-13),
3
147.7 (C-17), 133.6 (C-19), 132.6 (C-7), 131.0 (C-11),
129.7 (C-6), 129.3 (C-8, 9), 129.0 (C-12), 128.0 (C-10),
0
121.3 (C-18), 110.6 (C-16), 99.8 (C-1 ), 97.5 (C-21), 78.4
2
3
0
0
0
0
0
organic layer was washed with H O, dried over MgSO and
2
(C-5 ), 78.0 (C-3 ), 74.8 (C-2 ), 71.6 (C-4 ), 62.7 (C-6 ),
61.6 (C-3), 49.1 (C-5), 46.0 (C-20), 30.2 (C-14), 25.1
(C-15); UV lmax (MeOH) nm: 321, 313, 307, 300, 236,
206; CD (c¼0.532 mmol/L, MeOH, 248C) De (l nm):
0 (330), 21.58 (322), 25.03 (272), 23.79 (262),
223.61 (240), 0 (229), þ5.86 (223), þ4.12 (213), þ12.93
(205).
4
then evaporated to give the CHCl extract (263 mg). The
3
aqueous layer was extracted with n-BuOH and the organic
layer was evaporated to give the n-BuOH extract (210 mg).
The aqueous layer was freeze-dried to give the water-
soluble portion (1.83 g). The n-BuOH extract (210 mg) was
separated by SiO open column chromatography eluted with
2
CHCl —MeOH–CHCl —CHCl –MeOH–H O—MeOH
2
3
3
3
gradient to give the 12 fractions. The 20% MeOH–CHCl3
eluate was subjected to MPLC (SiO ) using 10% MeOH–
4.1.2. (3R)-Deoxypumiloside (4). FAB-MS (positive,
þ
NBA) m/z: 497 (MH ); H NMR (500 MHz, CD OD) d:
3
1
2
CHCl to give (3S)- and (3R)-deoxypumilosides (3, 9.9 mg
3
8.29 (1H, s, H-7), 8.04 (1H, d, J¼8.4 Hz, H-12), 7.94 (1H, d,
J¼8.4 Hz, H-9), 7.75 (1H, dd-like, J¼8.4, 8.4 Hz, H-11),
7.59 (1H, dd-like, J¼8.4, 8.4 Hz, H-10), 7.51 (1H, d, J¼
2.5 Hz, H-17), 5.56 (1H, d, J¼1.8 Hz, H-21), 5.52 (1H, ddd,
J¼17.1, 10.2, 10.2 Hz, H-19), 5.32 (1H, dd, J¼17.1, 1.8 Hz,
H-18), 5.23 (1H, d, J¼17.1 Hz, H-5), 5.19 (1H, dd, J¼10.2,
1.8 Hz, H-18), 5.07 (1H, dd, J¼12.5, 3.8 Hz, H-3), 4.72
and 4, 9.4 mg), respectively. The 10% MeOH–CHCl3
eluate of MPLC was purified by MPLC (SiO ) using 20%
2
MeOH–AcOEt to give the new compound, OPHR-23 (6,
.4 mg). (3S)-Pumiloside (2, 24.1 mg) and strictosamide (5,
0
9
eluate of SiO open column chromatography of the n-BuOH
.4 mg) were obtained from the 30–40% MeOH–CHCl3
2
0
extract. (3S)-Pumiloside (2, 9.6 mg) was also obtained
from the 50% MeOH–H O eluate of DIAION HP20 of the
water-soluble portion.
(1H, d, J¼7.9 Hz, H-1 ), 4.70 (1H, d, J¼17.1 Hz, H-5), 3.91
0
(1H, dd, J¼12.1, 1.9 Hz, H-6 ), 3.69 (1H, dd, J¼12.1,
2
0
overlapped, H-15), 3.33 (overlapped, H-4 , 5 ), 3.24 (1H,
0
5.6 Hz, H-6 ), 3.40 (1H, dd, J¼8.9, 8.9 Hz, H-3 ), 3.38
0
0
(
0
From hairy roots cultivated in the liquid medium. Hairy
roots (about 375 g of wet weight), cultivated in the
Gamborg’s B5 liquid medium containing sucrose (1%),
were extracted with hot MeOH to give the extract (10.6 g).
dd, J¼8.9, 7.9 Hz, H-2 ), 2.82 (1H, m, H-20), 2.67 (1H, ddd,
J¼12.5, 3.8, 3.8 Hz, H-14), 1.53 (1H, ddd, J¼12.5, 12.5,
1
3
12.5 Hz, H-14); C NMR (125 MHz, CD OD) d: 165.7
3
(C-22), 163.2 (C-2), 149.2 (C-17), 148.9 (C-13), 133.7
(C-19), 132.3 (C-7), 131.0 (C-11), 129.7 (C-6), 129.4 (C-8,
The MeOH extract was then partitioned between H O,
2

9174
M. Kitajima et al. / Tetrahedron 58 (2002) 9169–9178
9
9
), 129.1 (C-12), 128.0 (C-10), 120.6 (C-18), 108.8 (C-16),
0
4.2. Syntheses of OPHR-23 (6) and OPHR-17 (7)
0
0
9.7 (C-1 ), 97.5 (C-21), 78.4 (C-5 ), 78.0 (C-3 ), 74.8
0
0
0
(
C-2 ), 71.6 (C-4 ), 62.7 (C-3, 6 ), 49.6 (C-5), 44.8 (C-20),
4.2.1. Preparation of (3S)-pumiloside tetraacetate (11)
from strictosamide tetraacetate. To a MeOH solution
(60 mL) of strictosamide tetraacetate (806 mg, 1.210 mmol)
3
3
1.1 (C-14), 29.2 (C-15); UV lmax (MeOH) nm: 321, 313,
07, 300, 236, 206; CD (c¼0.151 mmol/L, MeOH, 268C)
De (l nm): 0 (330), þ1.82 (322), þ6.76 (278), 0 (268),
was added an aqueous solution (30 mL) of NaIO (6.0 g,
4
2
25.28 (240), 0 (229), þ1.50 (227), 0 (221), 20.38 (218), 0
0.023 mol) at room temperature under Ar. The mixture was
stirred for 22 h in dark. After concentration of MeOH, the
residue was extracted with CHCl . The organic layer was
(
215), þ3.70 (209).
3
4.1.3. OPHR-23 (6). Amorphous; HR-FAB-MS (positive,
NBA) m/z: 547.1699 (MNa ) (Calcd for C H N O Na,
washed with brine, dried over MgSO and evaporated. To a
4
þ
47.1693); FAB-MS (positive, NBA) m/z: 525 (MH ), 547
dry EtOH solution (50 mL) of the resulting dicarbonyl
derivative was added dry Et N (2.0 mL, 0.014 mol) and the
2
7
28
2
9
þ
MNa ); H NMR (500 MHz, DMSO-d ) d: 8.67 (1H, s,
5
(
3
þ
1
mixture was stirred for 16.5 h at room temperature under Ar.
Water was added to the reaction mixture and the whole was
extracted with CHCl . The organic layer was washed with
6
H-7), 8.16 (1H, d, J¼7.6 Hz, H-12), 8.11 (1H, d, J¼7.6 Hz,
H-9), 7.85 (1H, dd-like, J¼7.6, 7.6 Hz, H-11), 7.70 (1H,
dd-like, J¼7.6, 7.6 Hz, H-10), 7.02 (1H, s, H-14), 5.81 (1H,
ddd, J¼17.1, 10.1, 9.1 Hz, H-19), 5.65 (1H, s, H-17), 5.51
3
brine, dried over MgSO and evaporated. The residue was
4
purified by SiO open column chromatography (MeOH–
2
(
1H, br-d, J¼17.1 Hz, H-18), 5.46 (1H, d, J¼7.0 Hz, H-21),
CHCl3 gradient) to afford 11 (606 mg, y. 74%). (3S)-
1
5
5
4
4
.44 (1H, dd, J¼10.1, 2.1 Hz, H-18), 5.24 (2H, s, H -5),
Pumiloside tetraacetate (11): Mp 2188C (dec., EtOH); H
NMR (500 MHz, CDCl ) d: 11.60 (1H, br-s, Na-H), 8.36
2
.01 (1H, d, J¼5.5 Hz, OH), 4.96 (1H, d, J¼4.9 Hz, OH),
3
0
.92 (1H, d, J¼5.2 Hz, OH), 4.64 (1H, d, J¼7.9 Hz, H-1 ),
(1H, d, J¼7.6 Hz, H-9), 7.63 (1H, dd, J¼7.6, 7.6 Hz, H-11),
7.55 (1H, d, J¼7.6 Hz, H-12), 7.38 (1H, dd, J¼7.6, 7.6 Hz,
H-10), 7.10 (1H, d, J¼1.9 Hz, H-17), 5.69 (1H, m, H-19),
0
.51 (1H, dd, J¼6.0, 6.0 Hz, OH), 3.68 (1H, m, H-6 ), 3.53
(
H-6 ), 3.2–3.1 (2H, overlapped), 3.08 (1H, m), 2.99 (1H, m)
1H, m, H-20), 3.47 (3H, s, 17-OMe), 3.45 (overlapped,
0
H-2 , 3 , 4 , 5 ); C NMR (125 MHz, DMSO-d ) d: 157.8
C-22), 152.5 (C-2), 147.9 (C-13), 147.4 (C-15), 145.5
C-3), 134.6 (C-19), 131.5 (C-7), 130.3 (C-11), 129.9 (C-6),
0
5.25 (1H, dd, J¼9.5, 9.5 Hz, H-3 ), 5.21–5.15 (2H, m,
0
0
0
0
13
(
(
(
H -18), 5.18 (1H, m, H-21), 5.06 (1H, dd, J¼9.6, 9.6 Hz,
6
2
0
0
H-4 ), 5.01 (1H, dd, J¼8.8, 8.8 Hz, H-2 ), 4.95 (1H, d,
0
J¼8.3 Hz, H-1 ), 4.75 (2H, m, H-3, H-5), 4.57 (1H, br-d,
0
1
29.0 (C-12), 128.5 (C-9), 128.0 (C-8), 127.6 (C-10), 122.3
J¼12.7 Hz, H-5), 4.28 (1H, dd, J¼12.2, 4.3 Hz, H-6 ), 4.13
0
1.7 (C-21), 77.5, 77.0, 73.1 and 70.0 (C-2 , 3 , 4 , 5 ), 61.0
0
0
(
9
C-16), 121.0 (C-18), 99.2 (C-14), 97.9 (C-1 ), 95.2 (C-17),
0
(1H, d, J¼12.2 Hz, H-6 ), 3.75 (1H, br-d, J¼8.5 Hz, H-5 ),
3.12 (1H, m, H-15), 2.56 (2H, m, H-14, 20), 2.08 (s, 6H,
OCOCH £2), 2.03 and 2.01 (each 3H, s, OCOCH £2), 2.1–
0
0
0
0
(
C-6 ), 55.2 (OMe), 50.0 (C-5), 47.3 (C-20); UV lmax
MeOH) nm: 362, 290, 254, 219; CD (c¼0.382 mmol/L,
3
3
(
MeOH, 248C) De (l nm): 0 (390), 20.59 (364), 0 (334),
2.0 (1H, m, H-14); UV l
(MeOH) nm: 328, 315, 303
max
(sh), 289 (sh), 245, 239 (sh), 213.
2
0.89 (307), 0 (280), þ3.14 (260), 0 (254), 26.11 (239), 0
(
230), þ6.99 (223), 0 (216), 227.12 (205).
4.2.2. Preparation of (3R)-pumiloside tetraacetate (12)
from vincoside lactam tetraacetate. To a MeOH solution
(60 mL) of vincoside lactam tetraacetate (1161 mg,
1.743 mmol) was added an aqueous solution (30 mL) of
4.1.4. OPHR-17 (7). Amorphous; HR-FAB-MS (positive,
NBA) m/z: 547.1715 (MNa ) (Calcd for C H N O Na,
þ
47.1693); FAB-MS (positive, NBA) m/z: 525 (MH ), 547
2
7
28
2
9
þ
MNa ); H NMR (400 MHz, DMSO-d ) d: 8.68 (1H, s,
5
(
NaIO (6.0 g, 0.023 mol) at room temperature under Ar.
4
þ
1
The mixture was stirred for 41 h in dark. The residue
obtained by the same work up procedure as above was
dissolved in dry EtOH (50 mL) and then dry Et N (2.5 mL,
6
H-7), 8.15 (1H, d, J¼7.8 Hz, H-12), 8.12 (1H, d, J¼7.8 Hz,
H-9), 7.85 (1H, dd-like, J¼7.8, 7.8 Hz, H-11), 7.70 (1H,
dd-like, J¼7.8, 7.8 Hz, H-10), 7.14 (1H, s, H-14), 5.82 (1H,
ddd, J¼17.0, 10.2, 7.0 Hz, H-19), 5.53 (1H, s, H-17), 5.45
3
0.018 mol) was added to the solution. The mixture was
stirred for 54 h at room temperature under Ar. (3R)-
Pumiloside tetraacetate (12, 890 mg, y. 75%) was obtained
via the same work up manner and purification as described
above. (3R)-Pumiloside tetraacetate (12): Mp 213–2178C
(
5
1H, d, J¼1.7 Hz, H-21), 5.37 (1H, br-d, J¼ 17.0 Hz, H-18),
.25 (2H, s, H -5), 5.23 (1H, br-d, J¼ 10.2 Hz, H-18), 5.02
2
(
(
1H, d, J¼4.9 Hz, OH), 4.96 (1H, d, J¼4.9 Hz, OH), 4.65
0
þ
1
1H, d, J¼7.8 Hz, H-1 ), 4.59 (1H, dd, J¼6.0, 6.0 Hz, OH),
(EtOH); EI-MS m/z (%): 680 (M , 1), 43 (100); H NMR
(400 MHz, CDCl ) d: 11.39 (1H, s, Na-H), 8.36 (1H, d,
0
4
m, H-20), 3.57 (3H, s, 17-OMe), 3.45 (1H, m, H-6 ), 3.2
.12 (1H, d, J¼2.2 Hz, OH), 3.69 (1H, m, H-6 ), 3.68 (1H,
3
0
2H, overlapped), 3.03 (1H, m) (H-3 , 4 , 5 ), 2.96 (1H, m,
J¼7.8 Hz, H-9), 7.65 (1H, dd-like, J¼7.8, 7.8 Hz, H-11),
7.58 (1H, d, J¼7.8 Hz, H-12), 7.49 (1H, d, J¼2.2 Hz,
H-17), 7.40 (1H, dd-like, J¼7.8, 7.8 Hz, H-10), 5.46 (1H,
ddd, J¼17.1, 9.7, 9.7 Hz, H-19), 5.28 (1H, d, J¼1.7 Hz,
H-21), 5.24 (1H, d, J¼17.1 Hz, H-18), 5.23 (1H, d, J¼
0
H-2 ); C NMR (125 MHz, DMSO-d ) d: 157.7 (C-22),
0
0
(
0
52.6 (C-2), 147.9 (C-13), 145.7 (C-15), 145.5 (C-3), 135.8
13
6
1
(
(
(
9
6
C-19), 131.5 (C-7), 130.3 (C-11), 129.9 (C-6), 128.9
C-12), 128.5 (C-9), 128.0 (C-8), 127.6 (C-10), 121.2
0
9.7 Hz, H-18), 5.22 (1H, dd, J¼9.5, 9.5 Hz, H-3 ), 5.08 (1H,
0
0
C-16), 118.9 (C-18), 99.9 (C-14), 97.6 (C-1 ), 94.5 (C-17),
dd, J¼9.5, 9.5 Hz, H-4 ), 5.10–5.05 (2H, m, H-3, 5), 5.00
0
0
1.0 (C-6 ), 57.1 (OMe), 50.1 (C-5), 45.8 (C-20); UV lmax
0
0
0
0
4.3 (C-21), 77.4, 76.7 and 69.9 (C-3 , 4 , 5 ), 73.4 (C-2 ),
0
(1H, dd, J¼9.5, 8.2 Hz, H-2 ), 4.92 (1H, d, J¼8.2 Hz, H-1 ),
4.55 (1H, d, J¼13.9 Hz, H-5), 4.30 (1H, dd, J¼12.3, 4.7 Hz,
0
0
0
H-5 ), 2.90 (1H, m, H-15), 2.58 (1H, m, H-20), 2.47 (1H, m,
(
0
MeOH) nm: 363, 280 (sh), 254, 219; CD (c¼
H-6 ), 4.14 (1H, dd, J¼12.3, 2.2 Hz, H-6 ), 3.76 (1H, m,
.891 mmol/L, MeOH, 248C) De (l nm): 0 (400), þ0.36
(373), 0 (337), þ0.51 (308), 0 (285), 20.41 (261), 0
(248), 21.59 (237), 0 (231), þ4.17 (220), 0 (213), 210.30
(204).
H-14), 2.09, 2.04, 2.01 and 1.98 (each 3H, s, OCOCH £4),
3
1
3
1.61 (1H, ddd, J¼12.4, 12.4, 12.4 Hz, H-14); C NMR
(100 MHz, CDCl ) d: 174.5 (C-7), 170.6, 170.0, 169.47 and
3

M. Kitajima et al. / Tetrahedron 58 (2002) 9169–9178
9175
0
1
69.46 (OCOCH £4), 162.4 (C-22), 150.6 (C-2), 147.0
4.25 (1H, dd, J¼14.7, 1.5 Hz, H-5), 3.69 (1H, m, H-6 ),
3
0
0
0
2.72 (1H, m, H-20), 2.53 (1H, m, H-14), 1.37 (1H, ddd,
0
(
(
C-17), 140.9 (C-13), 132.3 (C-11), 131.5 (C-19), 125.8
C-8), 125.3 (C-9), 124.4 (C-10), 120.9 (C-18), 118.8
3.39–3.16 (3H, m, H-15, 3 , 5 ), 3.07–2.98 (2H, m, H-2 , 4 ),
0
2.29 and 72.26 (C-3 , 5 ), 70.5 (C-2 ), 68.2 (C-4 ), 61.8
0
signal); C NMR (67.8 MHz, DMSO-d ) d: 172.7 (C-7),
(
7
(
(
(
2
(
(
C-12), 114.3 (C-6), 107.7 (C-16), 96.2 (C-21), 96.0 (C-1 ),
J¼12.3, 12.3, 12.3 Hz, H-14) (H-6 was buried under H O
2
0
C-6 ), 61.5 (C-3), 49.0 (C-5), 42.8 (C-20), 29.5 (C-14), 28.4
0
0
0
13
6
0
161.6 (C-22), 149.8 (C-2), 146.4 (C-17), 140.2 (C-13),
132.6 (C-19), 131.5 (C-11), 125.3 (C-8), 124.7 (C-9), 123.2
(C-10), 120.0 (C-18), 118.3 (C-12), 112.9 (C-6), 107.1
0 0 0
(C-16), 97.9 (C-1 ), 94.9 (C-21), 77.2 and 76.4 (C-3 , 5 ),
73.1 (C-2 ), 70.0 (C-4 ), 61.0 (C-6 ), 60.4 (C-3), 48.1
C-15), 20.73, 20.65 and 20.56 (OCOCH £4); UV l
3
max
MeOH) nm: 328, 315, 303 (sh), 289 (sh), 245, 239 (sh),
13; CD (c¼0.171 mmol/L, MeOH, 238C) De (l nm): 0
0
0
0
340), 22.49 (327), 21.42 (315), 0 (295), 234.46 (245), 0
230), þ13.50 (215), 0 (205).
(C-5), 42.4 (C-20), 28.8 (C-14), 27.5 (C-15); CD (c¼
0
.117 mmol/L, MeOH, 248C) De (l nm): 0 (334), 21.88
4
(
.2.3. Preparation of (3S)-pumiloside (2). To a solution of
3S)-pumiloside tetraacetate (11, 10 mg, 0.015 mmol) in dry
MeOH (2 mL) was added 1N NaOMe in MeOH (50 mL,
.050 mmol) at room temperature under Ar. The mixture
(329), 0 (295), 234.52 (247), 0 (233), þ14.98 (218).
4.2.5. Preparation of (3S)-deoxypumiloside tetraacetate
(14). To a solution of (3S)-pumiloside tetraacetate (11,
606 mg, 0.891 mmol) in dry THF (30 mL) and dry HMPA
(310 mL, 1.782 mmol), was added a THF solution (4 mL) of
lithium diisopropylamide (LDA, 2 equiv.) at 2788C for 1 h
under Ar. A THF solution (3 mL) of N-phenyltrifluoro-
methanesulfonimide (637 mg, 1.783 mmol) was added
dropwise to the mixture and the reaction mixture was
stirred at 2788C for 30 min. Then the temperature was
gradually raised to room temperature over 1 h. Cold water
was added to the reaction mixture. After the solvent was
removed under reduced pressure, the whole was extracted
0
was stirred at the same temperature for 2 h. Water was
added to the reaction mixture and the whole was extracted
with n-BuOH. The organic layer was washed with water and
evaporated. The residue was purified by SiO open column
2
chromatography (MeOH–CHCl ¼1:5) to give (3S)-pumi-
3
loside (2, 6.9 mg, y. 92%). The synthetic compound was
identical with the natural (3S)-pumiloside (TLC, UV, H
NMR, CD, and MS). (3S)-Pumiloside (2): Mp .2958C
2
a
1
1
(
MeOH); H NMR (500 MHz, DMSO-d ) d: 12.08 (1H, s,
6
Na–H), 8.12 (1H, dd, J¼8.2, 1.2 Hz, H-9), 7.66 (1H,
dd-like, J¼8.2, 8.2 Hz, H-11), 7.60 (1H, d, J¼8.2 Hz,
H-12), 7.34 (1H, dd-like, J¼8.2, 8.2 Hz, H-10), 7.04 (1H, d,
J¼2.8 Hz, H-17), 5.80 (1H, ddd, J¼16.8, 10.1, 10.1 Hz, H-
with CHCl . The organic layer was washed with brine, dried
3
over MgSO4 and evaporated. The residue was purified
by SiO2 open column chromatography (CHCl –MeOH
3
1
9), 5.47 (1H, dd, J¼16.8, 1.8 Hz, H-18), 5.38 (1H, d,
gradient) to afford (3S)-7-triflate (590 mg, y. 82%). (3S)-7-
1
J¼1.5 Hz, H-21), 5.34 (1H, dd, J¼10.1, 1.8 Hz, H-18), 4.76
Triflate: H NMR (400 MHz, CDCl , selected data) d: 8.17
3
0
(
1H, br-d, J¼11.9 Hz, H-3), 4.54 (1H, d, J¼7.9 Hz, H-1 ),
(1H, br-d, J¼8.3 Hz, H-9), 8.09 (1H, br-d, J¼8.3 Hz, H-12),
7.84 (1H, dd-like, J¼8.3, 8.3 Hz, H-10), 7.72 (1H, dd-like,
J¼8.3, 8.3 Hz, H-11), 7.18 (1H, d, J¼2.6 Hz, H-17), 5.77
(1H, ddd, J¼17.1, 10.2, 10.2 Hz, H-19), 5.52 (1H, dd,
J¼17.1, 1.6 Hz, H-18), 5.41 (1H, dd, J¼10.2, 1.6 Hz,
4
H-5), 3.69 and 3.43 (each 1H, m, H -6 ), 3.3 (overlapped,
.47 (1H, br-d, J¼13.7 Hz, H-5), 4.32 (1H, d, J¼13.7 Hz,
0
H-15), 3.16 (2H, m, H-3 , 5 ), 3.03 (1H, m, H-4 ), 2.98 (1H,
2
0
0
m, H-2 ), 2.65 (1H, m, H-20), 2.5 (overlapped, H-14), 2.01
0
0
1
3
(
(
(
(
(
9
1H, ddd, J¼11.6, 11.6, 11.6 Hz, H-14);
C NMR
H-18), 2.11, 2.09, 2.04 and 2.01 (each 3H, s, OCOCH £4);
3
1
3
125 MHz, DMSO-d ) d: 172.9 (C-7), 163.9 (C-22), 149.7
C NMR (100 MHz, CDCl ) d: 170.6, 170.1, 169.8, 169.4,
3
6
C-2), 145.1 (C-17), 140.4 (C-13), 132.5 (C-19), 131.6
C-11), 125.3 (C-8), 124.7 (C-9), 123.2 (C-10), 120.5
C-18), 118.3 (C-12), 112.9 (C-6), 108.9 (C-16), 97.8 (C-1 ),
165.0, 163.8, 150.8, 146.9, 145.6, 131.2, 131.0, 129.4,
128.4, 123.6, 121.7, 121.3, 121.0, 120.4, 110.0, 96.2, 96.1,
72.34, 72.28, 70.4, 68.2, 61.7, 60.5, 46.6, 43.7, 29.7, 28.8,
23.7, 20.7, 20.63, 20.57; UV lmax (MeOH) nm: 321, 308,
236, 205. To a solution of (3S)-7-triflate (5.9 mg,
0.007 mmol) in dry 1,4-dioxane (0.6 mL), were added a
0
4.8 (C-21), 77.3 (C-5 ), 76.5 (C-3 ), 73.1 (C-2 ), 70.1
0
0
0
0
0
(
(
C-4 ), 61.0 (C-6 ), 59.4 (C-3), 47.4 (C-5), 43.6 (C-20), 28.2
C-14), 23.7 (C-15); CD (c¼0.127 mmol/L, MeOH, 248C)
De (l nm): 0 (334), þ1.27 (324), þ0.51 (319), þ0.99 (315),
solution of DPPF (1.5 mg, 0.003 mmol), Pd(OAc) (0.4 mg,
2
0.002 mmol) and dry Et N (5.6 mL, 0.040 mmol) in dry
3
0
(302), 219.52 (240), 0 (219), þ11.48 (211).
1,4-dioxane (0.2 mL) and a solution of HCOOH (1.1 mL,
4.2.4. Preparation of (3R)-pumiloside (13). To a solution
of (3R)-pumiloside tetraacetate (12, 17 mg, 0.025 mmol) in
0.029 mmol) in dry 1,4-dioxane (0.2 mL) successively at
room temperature under Ar. The reaction mixture was
heated at 608C for 30 min. A cold water was added to the
dry MeOH (2.5 mL) was added 1N NaOMe in MeOH
50 mL, 0.050 mmol) at 08C under Ar. The mixture was
(
mixture and the whole was extracted with CHCl . The
3
stirred at room temperature for 1 h. The reaction mixture
was directly subjected to SiO2 open column chroma-
tography (MeOH–CHCl gradient) to give (3R)-pumiloside
organic layer was washed with 5% Na CO aq. and then
2
water, dried over MgSO and evaporated. The residue was
4
3
purified by preparative TLC (SiO , 5% MeOH–CHCl ) to
3
3
2
(
(
13, 12.2 mg, y. 95%). (3R)-Pumiloside (13): Mp .3008C
1
afford (3S)-deoxypumiloside tetraacetate (14, 3.8 mg, y.
79%). The synthetic compound was identical with the
MeOH); H NMR (500 MHz, DMSO-d ) d: 8.12 (1H, dd,
6
2
d
J¼8.0, 1.1 Hz, H-9), 7.65 (1H, dd-like, J¼8.0, 8.0 Hz,
H-11), 7.60 (1H, d, J¼8.0 Hz, H-12), 7.34 (1H, overlapped,
H-10), 7.34 (1H, d, J¼2.4 Hz, H-17), 5.45 (1H, ddd,
J¼17.1, 10.0, 10.0 Hz, H-19), 5.42 (1H, d, J¼1.5 Hz,
H-21), 5.31 (1H, dd, J¼17.0, 2.1 Hz, H-18), 5.19 (1H, dd,
tetraacetate derived from natural (3S)-deoxypumiloside
1
(TLC, UV, H and C NMR, CD, and MS).
13
4.2.6. Preparation of (3R)-deoxypumiloside tetraacetate
(15). To a solution of (3R)-pumiloside tetraacetate (12,
10.5 mg, 0.015 mmol) in dry THF (0.5 mL) and dry HMPA
(0.1 mL, 0.575 mmol), was added a THF solution (0.2 mL)
J¼10.0, 2.1 Hz, H-18), 5.11 (1H, br-d, J¼9.4 Hz, H-3), 4.74
0
(
1H, dd, J¼14.7, 2.1 Hz, H-5), 4.53 (1H, d, J¼7.9 Hz, H-1 ),

9176
M. Kitajima et al. / Tetrahedron 58 (2002) 9169–9178
of LDA (2 equiv.) at 2788C for 1 h under Ar. A THF
solution (0.2 mL) of N-phenyltrifluoromethanesulfonimide
toluene (0.5 mL) was stirred at 508C for 3 h. The reaction
mixture was directly subjected to Al O column chroma-
2
3
(
29.1 mg, 0.082 mmol) was added dropwise to the mixture
tography (5% MeOH–CHCl ) and the residue was purified
3
and the reaction mixture was stirred at 2788C for 30 min.
Then the temperature was gradually raised to 2158C and the
mixture was stirred for 30 min. (3R)-7-triflate (11.1 mg, y.
by MPLC (2% MeOH–CHCl ) to afford 17 (26.2 mg, y.
3
91%, a mixture of 17-epimers¼4:1). 17: FAB-MS (positive,
þ
253, 218 nm; Main product: H NMR (500 MHz, CDCl ,
NBA) m/z: 693 (MH ); UV l
(MeOH) nm: 361, 287,
max
1
8
9%) was obtained via the same work up manner and
purification as described above. (3R)-7-Triflate: Mp 185–
878C (dec., MeOH); FAB-MS (positive, NBA) m/z: 813
3
selected data) d: 8.37 (s, 1H, H-7), 8.21 (1H, d, J¼8.5 Hz,
H-12), 7.92 (1H, d, J¼8.5 Hz, H-9), 7.81 (1H, dd-like,
J¼8.5, 8.5 Hz, H-11), 7.65 (1H, dd-like, J¼8.5, 8.5 Hz,
H-10), 7.22 (1H, s, H-14), 5.79 (1H, s, H-17), 3.70 (3H, s,
1
þ
1
(
MH ); H NMR (400 MHz, CDCl , selected data) d: 8.16
3
(
7
1H, br-d, J¼8.0 Hz, H-9), 8.08 (1H, br-d, J¼8.0 Hz, H-12),
.84 (1H, dd-like, J¼8.0, 8.0 Hz, H-10), 7.71 (1H, dd-like,
17-OMe), 2.08, 2.05, 2.03 and 2.01 (each 3H, s, OCOCH £
3
1
3
J¼8.0, 8.0 Hz, H-11), 5.50 (1H, d, J¼17.2 Hz, H-5), 5.46
4); C NMR (125 MHz, CDCl ) d: 170.6, 170.3, 169.4,
3
(
1H, ddd, J¼16.9, 9.8, 9.8 Hz, H-19), 2.11, 2.04, 2.02 and
158.8, 152.6, 148.9, 148.4, 145.7, 132.9, 131.0, 130.5,
129.7, 128.7, 128.1, 127.9, 122.6, 122.1, 100.9, 96.4, 96.1,
93.6, 72.9, 72.2, 71.0, 68.5, 62.1, 56.5, 49.8, 47.9, 20.7,
20.6.
1
3
2
CDCl ) d: 170.6, 170.0, 169.4, 163.9, 162.6, 150.7, 147.2,
.01 (each 3H, s, OCOCH £4); C NMR (100 MHz,
3
3
1
1
6
46.8, 131.4, 131.0, 129.4, 128.4, 121.3, 120.95, 120.93,
20.4, 120.1, 116.9, 108.0, 96.3, 96.0, 72.3, 70.5, 68.2, 61.8,
1.7, 47.1, 42.9, 29.8, 28.0, 20.7, 20.64, 20.56; UV lmax
4.2.10. Treatment of 17 with NaOMe. To a suspension
mixture of 17 (a mixture of 17-epimers, 22.6 mg,
0.326 mmol) in dry MeOH (3 mL) was added 1N NaOMe
in MeOH (16.3 mL, 0.016 mmol) at 08C under Ar.
The mixture was stirred at room temperature for 5.5 h.
(
MeOH) nm: 321, 308, 236, 205. To a solution of (3R)-7-
triflate (224 mg, 0.275 mmol) in dry 1,4-dioxane (10 mL),
were added a solution of DPPF (76.6 mg, 0.138 mmol),
Pd(OAc) (18.6 mg, 0.083 mmol) and dry Et N (250 mL,
2
3
1
.764 mmol) in dry 1,4-dioxane (3 mL) and a solution of
The reaction mixture was directly subjected to SiO open
2
HCOOH (47 mL, 1.247 mmol) in dry 1,4-dioxane (2 mL)
successively at room temperature under Ar. The reaction
mixture was heated at 608C for 1 h. The residue, which was
obtained by the same work up procedure as described above,
was purified by SiO2 open column chromatography
column chromatography (MeOH–CHCl3 gradient). The
residue was purified by MPLC (15% MeOH–CHCl ) to
3
give 21 (3.9 mg, y. 35%) together with OPHR-23 (6,
0.3 mg) and a mixture (3.0 mg) of OPHR-17 (7) and 20. 21:
þ
for C H N O , 345.1239); FAB-MS (positive, NBA) m/z:
HR-FAB-MS (positive, NBA) m/z: 345.1224 (MH ) (Calcd
(
tetraacetate (15, 182 mg, y. quant.). The synthetic com-
CHCl –MeOH gradient) to afford (3R)-deoxypumiloside
3
21 17 2 3
þ
1
345 (MH ); H NMR (500 MHz, CDCl ) d: 8.37 (1H, s,
3
2
d
pound was identical with the tetraacetate derived from
1
H-7), 8.23 (1H, d, J¼7.9 Hz, H-12), 7.93 (1H, d, J¼7.9 Hz,
H-9), 7.82 (1H, dd, J¼7.9, 7.9 Hz, H-11), 7.66 (1H, dd,
J¼7.9, 7.9 Hz, H-10), 7.44 (1H, s, H-14), 7.08 (1H, s, H-21),
13
natural (3R)-deoxypumiloside (TLC, UV, H and C NMR,
CD, and MS).
6
.67 (1H, dd, J¼17.1, 10.7 Hz, H-19), 6.35 (1H, s, H-17),
4
.2.7. Preparation of (3S)-deoxypumiloside (3). To a
suspension mixture of (3S)-deoxypumiloside tetraacetate
14, 20 mg, 0.030 mmol) in dry MeOH (1 mL) was added
N NaOMe in MeOH (30 mL, 0.030 mmol) at 08C under Ar.
5.53 (1H, dd, J¼17.1, 1.2 Hz, H-18), 5.33 (1H, d, J¼
19.2 Hz, H-5), 5.27 (1H, dd, J¼10.7, 1.2 Hz, H-18), 5.27
13
(
1
(1H, overlapped, H-5), 3.66 (3H, s, 17-OMe); C NMR
(125 MHz, CDCl ) d: 159.1, 152.9, 148.9, 145.9, 145.1,
3
The mixture was stirred at room temperature for 3 h. The
reaction mixture was directly subjected to SiO2 open
column chromatography (20% MeOH–CHCl ) and MPLC
140.8, 131.0, 130.5, 129.7, 129.1, 128.9, 128.1, 127.8,
115.5, 114.3, 113.1, 95.8, 95.7, 56.4, 49.9; UV lmax
(MeOH) nm: 355, 254, 219.
3
(
10% MeOH–CHCl ) to give (3S)-deoxypumiloside (3,
3
6
with the natural (3S)-deoxypumiloside (TLC, UV, H and
C NMR, CD, and MS).
.6 mg, y. 44%). The synthetic compound was identical
1
4.2.11. Preparation of 16 from (3R)-deoxypumiloside (4).
To a suspension mixture of (3R)-deoxypumiloside (4,
133 mg, 0.269 mmol) and DMAP (14 mg, 0.115 mmol) in
dry CH Cl (10.5 mL) were added dry pyridine (3.5 mL,
1
3
2
2
4
.2.8. Preparation of (3R)-deoxypumiloside (4). To a
43.27 mmol) and 2,2,2-trichloroethyl chloroformate
(0.44 mL, 3.196 mmol) at 08C under Ar. The mixture was
stirred at room temperature for 16.5 h. A cold water was
added to the reaction mixture and the whole was extracted
suspension mixture of (3R)-deoxypumiloside tetraacetate
15, 200 mg, 0.301 mmol) in dry MeOH (6 mL) was added
N NaOMe in MeOH (301 mL, 0.301 mmol) at 08C under
(
1
Ar. The mixture was stirred at room temperature for 5 h.
The reaction mixture was directly subjected to SiO open
with CHCl . The organic layer was washed with water,
3
dried over MgSO and evaporated. The residue was purified
4
2
column chromatography (30% MeOH–CHCl ) and MPLC
3
by SiO open column chromatography (CHCl ) to afford 16
2
(323 mg, y. quant.). 16: FAB-MS (NBA) m/z: 1192 (M ),
1
3
þ
1194, 1196, 1198, 1200, 1202, 1204; H NMR (400 MHz,
(
10% MeOH–CHCl ) to give (3R)-deoxypumiloside (4,
3
1
13 mg, y. 76%). The synthetic compound was identical
1
13
with the natural one (TLC, UV, H and C NMR, CD, and
MS).
CDCl , selected data) d: 8.08 (1H, d, J¼8.1 Hz, H-12), 8.08
3
(1H, s, H-7), 7.83 (1H, d, J¼8.1 Hz, H-9), 7.72 (1H, dd-like,
J¼8.1, 8.1 Hz, H-11), 7.56 (1H, dd-like, J¼8.1, 8.1 Hz,
H-10), 7.53 (1H, d, J¼2.4 Hz, H-17), 5.47 (1H, ddd,
J¼17.1, 9.9, 9.9 Hz, H-19), 5.20 (1H, dd, J¼9.9, 2.0 Hz,
H-18), 4.86–4.65 (8H, m, COOCH CCl £4), 4.51 (1H, dd,
4
.2.9. DDQ oxidation of (3R)-deoxypumiloside tetra-
acetate (15). A mixture of (3R)-deoxypumiloside tetra-
acetate (15, 27.7 mg, 0.417 mmol) and a solution of DDQ
2
3
0
0
(
60 mg, 0.264 mmol) in dry MeOH (0.5 mL) and dry
J¼12.2, 5.1 Hz, 6 -H), 4.43 (1H, dd, J¼12.2, 2.7 Hz, 6 -H);

M. Kitajima et al. / Tetrahedron 58 (2002) 9169–9178
9177
1
3
C NMR (100 MHz, CDCl ) d: 163.1, 162.0, 153.9, 153.5,
3
(c¼0.228 mmol/L, MeOH, 238C) De (l nm): 0 (388),
20.79 (333), 0 (321), þ1.76 (307), 0 (250), 27.19 (237),
0 (230), þ22.88 (220), 0 (212), 236.00 (204).
1
1
7
53.3, 153.2, 148.4, 147.2, 131.6, 130.5, 129.9, 129.3,
28.3, 128.1, 127.1, 121.3, 108.5, 97.0, 96.1, 94.6, 94.2,
6.7, 75.4, 73.1, 71.7, 66.2, 61.9, 48.9, 43.3, 30.3, 28.5.
4.3. Preparation of OPHR-23 (6) from 18
4
.2.12. DDQ oxidation of 16. A mixture of 16 (109 mg,
0
in dry MeOH (2.5 mL) and dry toluene (2.5 mL) was stirred
.091 mmol) and a solution of DDQ (114 mg, 0.502 mmol)
To a suspension mixture of 18 (18.0 mg, 0.147 mmol) in dry
MeOH (1 mL) was added zinc dust (50 mg) at room
temperature under Ar. The mixture was stirred at the same
temperature for 19 h and under reflux for 2 h. The reaction
mixture was filtrated and the filtrate was evaporated. The
at 508C for 6 h. The reaction mixture was directly subjected
to Al O
column chromatography (MeOH–CHCl3
gradient). The residue was purified by MPLC (35%
2
3
n-hexane–AcOEt) to afford 18 (58 mg, y. 52%) and 19
residue was purified by MPLC (10% MeOH–CHCl ) to
3
(
(
17 mg, y. 15%). 18: FAB-MS (positive, NBA) m/z: 1221
þ
afford OPHR-23 (6, 3.0 mg, y. 39%). The synthetic
1
1
MH ), 1223, 1225, 1227, 1229, 1231; H NMR (500 MHz,
compound was identical with the natural one (UV, H and
3
C NMR, CD, and MS).
1
CDCl ) d: 8.37 (s, 1H, H-7), 8.21 (1H, d, J¼8.2 Hz, H-12),
3
7
8
(
(
.92 (1H, d, J¼8.2 Hz, H-9), 7.81 (1H, dd-like, J¼8.2,
.2 Hz, H-11), 7.65 (1H, dd-like, J¼8.2, 8.2 Hz, H-10), 7.20
4.4. Preparation of OPHR-17 (7) from 19
1H, s, H-14), 5.80 (1H, m, H-19), 5.78 (1H, s, H-17), 5.52
1H, br-d, J¼10.1 Hz, H-18), 5.49 (1H, br-d, J¼17.1 Hz,
To a suspension mixture of 19 (29.4 mg, 0.024 mmol) in dry
MeOH (1.5 mL) was added zinc dust (60 mg) at room
temperature under Ar. The mixture was stirred at 608C for
7 h. The reaction mixture was filtrated and the filtrate was
evaporated. The residue was purified by MPLC (10%
H-18), 5.43 (1H, d, J¼7.3 Hz, H-21), 5.37 (1H, dd, J¼9.2,
0
9
.2 Hz, H-3 ), 5.28 (1H, d, J¼19.2 Hz, H-5), 5.23 (1H, d,
0
J¼19.2 Hz, H-5), 5.23 (1H, d, J¼7.6 Hz, H-1 ), 5.15 (1H,
0
0
dd, J¼9.2, 9.2 Hz, H-4 ), 5.02 (1H, dd, J¼9.2, 7.3 Hz, H-2 ),
.81–4.71 (8H, m, COOCH CCl £4), 4.44 (2H, m,
4
H -6 ), 3.99 (1H, m, H-5 ), 3.70 (3H, s, 17-OMe), 3.45
MeOH–CHCl ) to afford OPHR-17 (7, 0.6 mg, y. 5%). The
3
2
3
0
0
synthetic compound was identical with the natural one (UV,
1
H and C NMR, CD, and MS).
2
1
3
13
(
CDCl ) d: 158.8 (C-22), 153.7, 153.2, 153.0 and 152.8
1H, br-dd, J¼8.1, 8.1 Hz, H-20); C NMR (125 MHz,
3
(
(
(
COOCH CCl £4), 152.6 (C-2), 148.9 (C-13), 148.1
2
3
C-15), 145.8 (C-3), 132.7 (C-19), 131.0 (C-7), 130.5
C-11), 129.7 (C-12), 128.8 (C-6), 128.1 (C-8, 9), 127.9 (C-
References
1
9
9
0), 122.8 and 122.6 (C-16, 18), 100.7 (C-14), 96.3 (C-17),
5.7 (C-1 ), 94.2, 94.1, 94.03 and 93.95 (COOCH CCl £4),
0
3.5 (C-21), 75.4 (C-2 ), 73.1 (C-4 ), 71.4 (C-5 ), 66.1
2
3
1. (a) Hutchinson, C. R. Tetrahedron 1981, 37, 1047. (b) Cai,
J.-C.; Hutchinson, C. R. The Alkaloids; Brossi, A., Ed.;
Academic: New York, 1983; Vol. 21, p 101. (c) Wall, M. E.;
Wani, M. C. In The Monoterpenoid Indole Alkaloids; Saxton,
J. E., Ed.; Wiley: London, 1994; p 689 Chapter 13.
0
0
0
0
0
(
C-6 ), 56.6 (17-OMe), 49.8 (C-5), 48.1 (C-20) (C-3 and
four Troc methylene carbons were buried under CDCl3
signals.); UV lmax (MeOH) nm: 358, 291, 253, 215; CD
(
c¼0.253 mmol/L, MeOH, 238C) De (l nm): 0 (395),
(
d) Takayama, H.; Kitajima, M.; Aimi, N. J. Synth. Org.
2
2
1.32 (355), 20.99 (308), 0 (284), þ3.20 (260), 0 (254),
Chem. Jpn 1999, 57, 181.
8.02 (238), 0 (230), þ9.57 (221), 0 (214), 236.64 (205).
2
. (a) Aimi, N.; Nishimura, M.; Miwa, A.; Hoshino, H.; Sakai, S.;
Haginiwa, J. Tetrahedron Lett. 1989, 30, 4991. (b) Aimi, N.;
Hoshino, H.; Nishimura, M.; Sakai, S.; Haginiwa, J.
Tetrahedron Lett. 1990, 31, 5169. (c) Aimi, N.; Ueno, M.;
Hoshino, H.; Sakai, S. Tetrahedron Lett. 1992, 33, 5403.
þ
19: FAB-MS (positive, NBA) m/z: 1221 (MH ), 1223,
1225, 1227, 1229, 1231; H NMR (500 MHz, CDCl ) d:
3
8
1
.37 (1H, s, H-7), 8.22 (1H, d, J¼8.2 Hz, H-12), 7.93 (1H, d,
J¼8.2 Hz, H-9), 7.82 (1H, dd, J¼8.2, 8.2 Hz, H-11), 7.65
(
1H, dd, J¼8.2, 8.2 Hz, H-10), 7.16 (1H, s, H-14), 5.81 (1H,
(
d) Kitajima, M.; Masumoto, S.; Takayama, H.; Aimi, N.
m, H-19), 5.64 (1H, s, H-17), 5.44 (1H, d, J¼0.9 Hz, H-21),
Tetrahedron Lett. 1997, 38, 4255.
0
5
.34–5.19 (5H, overlapped, H -5, H -18, H-3 ), 5.15 (1H, d,
2
2
0
0
3. Kitajima, M.; Fischer, U.; Nakamura, M.; Ohsawa, M.; Ueno,
M.; Takayama, H.; Unger, M.; St o¨ ckigt, J.; Aimi, N.
Phytochemistry 1998, 48, 107.
J¼7.6 Hz, H-1 ), 5.10 (1H, dd, J¼9.5, 9.5 Hz, H-4 ), 4.92
0
(
4
1H, dd, J¼8.5, 7.6 Hz, H-2 ), 4.82 (br-d, J¼11.8 Hz),
.81–4.75 (m), 4.71 (br-d, J¼11.9 Hz), 4.58 (d, J¼
4
. (a) Kitajima, M.; Nakamura, M.; Takayama, H.; Saito, K.;
St o¨ ckigt, J.; Aimi, N. Tetrahedron Lett. 1997, 38, 8997.
(b) Kitajima, M.; Nakamura, M.; Watanabe, A.; Takayama,
H.; Aimi, N. J. Chem. Soc., Perkin Trans. 1 1998, 389.
1
1.6 Hz), 4.47 (d, J¼11.9 Hz, 8H, COOCH CCl £4), 4.51
2
3
0
(
2
3
1H, dd, J¼11.9, 4.9 Hz, H-6 ), 4.43 (1H, dd, J¼11.9,
0
0
.4 Hz, H-6 ), 4.02 (1H, m, H-5 ), 3.70 (3H, s, 17-OMe),
1
3
.59 (1H, br-d, J¼8.2 Hz, H-20); C NMR (125 MHz,
5
. (a) Saito, K.; Sudo, H.; Yamazaki, M.; Koseki-Nakamura, M.;
Kitajima, M.; Takayama, H.; Aimi, N. Plant Cell Rep. 2001,
CDCl ) d: 158.6 (C-22), 153.7, 153.3 and 153.0 (COOCH -
3
2
CCl £3), 152.8 (C-2), 152.4 (COOCH CCl ), 148.9 (C-13),
3
2
3
2
0, 267. (b) Sudo, H.; Yamakawa, T.; Yamazaki, M.; Aimi,
1
45.8 (C-15), 145.4 (C-3), 134.8 (C-19), 131.0 (C-7), 130.5
N.; Saito, K. Biotechnol. Lett. 2002, 24, 359. (c) Yamazaki, Y.;
Urano, A.; Sudo, H.; Kitajima, M.; Takayama, H.; Yamazaki,
M.; Aimi, N.; Saito, K., submitted.
(
(
(
C-11), 129.7 (C-12), 128.7 (C-6), 128.1 (C-8, 9), 127.8
C-10), 122.4 (C-16), 119.7 (C-18), 101.3 (C-14), 95.3
0
C-1 ), 94.9 (C-17), 94.8 (C-21), 94.2, 94.1, 94.01 and 93.97
0
6.1 (C-6 ), 57.3 (17-OMe), 49.8 (C-5), 46.7 (C-20) (C-3
0
0
6
7
. Blackstock, W. P.; Brown, R. T.; Chapple, C. L.; Fraser, S. B.
J. Chem. Soc., Chem. Commun. 1972, 1006.
(
6
COOCH CCl £4), 75.0 (C-2 ), 73.1 (C-4 ), 71.0 (C-5 ),
2
3
0
and four Troc methylene carbons were buried under CDCl3
0
. (a) Battersby, A. R.; Burnett, A. R.; Parson, P. G. J. Chem. Soc.
(C) 1969, 1193. (b) Hutchinson, C. R.; O’Loughlin, G. J.;
signals.); UV lmax (MeOH) nm: 359, 288, 253, 218; CD

9178
M. Kitajima et al. / Tetrahedron 58 (2002) 9169–9178
Brown, R. T.; Fraser, S. B. J. Chem. Soc., Chem. Commun.
974, 928.
. Zheng, Q.; Yang, Y.; Martin, A. R. Heterocycles 1994, 37,
761.
632. (b) Takayama, H.; Ichikawa, T.; Kuwajima, T.; Kitajima,
1
M.; Seki, H.; Aimi, N.; Nonato, M. G. J. Am. Chem. Soc. 2000,
122, 8635. (c) Hansen, P. E. Prog. NMR Spectrosc. 1981, 14,
175. (d) Sasaki, M.; Matsumori, N.; Maruyama, T.;
Nonomura, T.; Murata, M.; Tachibana, K.; Yasumoto, T.
Angew. Chem., Int. Ed. Engl. 1996, 35, 1672.
8
9
1
. Cacchi, C.; Gattini, P. G.; Morea, E.; Orter, G. Tetrahedron
Lett. 1986, 27, 5541.
1
0. Itoh, A.; Tanahashi, T.; Nagakura, N. Heterocycles 1998, 48,
99.
1. (a) Willker, W.; Leibfritz, D. Magn. Reson. Chem. 1995, 33,
1
2. Hutchinson, C. R.; Heckendorf, A. H.; Daddona, P. E.;
Hagaman, E.; Wenkert, E. J. Am. Chem. Soc. 1974, 96, 5609.
4
1