Camptothecins and two new monoterpene glucosides from Ophiorrhiza liukiuensis

Article abstract of DOI:10.1248/cpb.53.1355

Two new monoterpene glucosides, demethylsecologanol and 3?-O-glucosylsenburiside II, were isolated from Ophiorrhiza liukiuensis (Rubiaceae) together with 23 known compounds, including camptothecins and β-carboline-type alkaloids. Their structures were determined by spectroscopic analysis.

Full text of DOI:10.1248/cpb.53.1355

October 2005
Notes
Chem. Pharm. Bull. 53(10) 1355—1358 (2005)
1355
Camptothecins and Two New Monoterpene Glucosides from Ophiorrhiza
liukiuensis
Mariko KITAJIMA,* Norie FUJII, Fumie YOSHINO, Hiroshi SUDO, Kazuki SAITO, Norio AIMI, and
Hiromitsu TAKAYAMA*
Graduate School of Pharmaceutical Sciences, Chiba University; 1–33 Yayoi-cho, Inage-ku, Chiba 263–8522, Japan.
Received June 3, 2005; accepted June 30, 2005; published online July 4, 2005
Two new monoterpene glucosides, demethylsecologanol and 3ꢀ-O-glucosylsenburiside II, were isolated from
Ophiorrhiza liukiuensis (Rubiaceae) together with 23 known compounds, including camptothecins and b-carbo-
line-type alkaloids. Their structures were determined by spectroscopic analysis.
Key words Ophiorrhiza liukiuensis; alkaloid; iridoid; secoiridoid; camptothecin; b-carboline
Camptothecin (1),1—6) a well-known monoterpenoid indole
18.3 mg), 10-methoxycamptothecin (5, 4.3 mg), pumiloside
(6, 21.0 mg), (3R)-deoxypumiloside (7, 26.3 mg) and stric-
tosamide (8, 3.7 mg); b-carboline-type alkaloids, such as
lyalosidic acid (9, 339.4 mg), ophiorine A (10, 5.6 mg),
ophiorine B (11, 6.4 mg) and harman (12, 25.2 mg); and 13
known compounds (13—25). The structures of the known
compounds were deduced from spectroscopic data and con-
firmed by comparison with those of authentic samples or re-
ported data. From the results, it was proved that O. liukiuen-
sis produced both camptothecins and b-carboline-type alka-
loids.
alkaloid possessing remarkable anti-tumor activity, was iso-
lated for the first time from Camptotheca acuminata7) (Nys-
saceae). At present, two semi-synthetic camptothecins,
topotecan and irinotecan, are used clinically as anti-tumor
agents. To date, several camptothecin-producing plants have
been reported: Nothapodytes foetida,8) Merrilliodendron
megacarpum,9) Pyrenacantha klaineana10) (Icacinaceae), Er-
vatamia heyneana11) (Apocynaceae), Mostuea brunonis12)
(Loganiaceae), Ophiorrhiza mungos,13) and O. filistipula14)
(Rubiaceae). In our previous study of the chemical con-
stituents of Ophiorrhiza plants distributed in Japan, we found
that O. pumila15—18) produced camptothecin (1) and its re-
lated alkaloids (4, 6—8), whereas O. kuroiwai,5,19) which was
recently shown to be an interspecies hybrid of O. pumila and
O. liukiuensis by us, produced not only camptothecins (1,
4, 5) but also b-carboline-type alkaloids (9—12). On the
basis of these findings, we obtained callus20) and tissue cul-
tures21—23) of O. pumila and examined their constituents to
obtain camptothecin. As a continuation of our studies, we in-
vestigated the secondary metabolites of O. liukiuensis, which
is distributed in Okinawa, Japan, Taiwan, and the Philip-
pines, and isolated two new monoterpenoid glucosides (2, 3)
together with 23 known compounds, including camp-
tothecins and b-carboline-type alkaloids, which will be de-
scribed herein.
The HR-FAB-MS spectrum of new secoiridoid 2 gave a
protonated molecular ion peak at m/z 377.1426 ([MꢀH]ꢀ)
that corresponded to the molecular formula C16H25O10 (m/z
377.1447). The UV spectrum showed the characteristic ab-
sorption of b-alkoxyacrylic acid or its ester at 233.0 nm. The
1H-NMR spectrum showed signals that indicated the pres-
ence of the olefinic proton of b-alkoxyacrylic acid at d 7.35
(s, H-3), a set of three protons on the vinyl group at d 5.73
(ddd, Jꢁ17.4, 9.7, 9.7 Hz, H-8), 5.28 (d, Jꢁ17.4 Hz, H-10),
and 5.24 (d, Jꢁ9.7 Hz, H-10), an acetal proton at d 5.45
(d, Jꢁ6.3 Hz, H-1), and one sugar unit containing a b-linked
anomeric proton at d 4.76 (d, Jꢁ8.0 Hz, H-1ꢂ), which were
characteristic of secoiridoid-type monoterpene glycosides.
Furthermore, signals assigned to the protons of methylene
bearing a hydroxy group were observed at d 3.57—3.48 (2H,
overlapped, H2-7). The 13C-NMR spectrum revealed 16 car-
bons, including one carboxyl carbon (d 172.3, C-11), four
alkenyl carbons [d 151.4 (C-3) and d 112.0 (C-4) due to the
b-alkoxyacrylic acid group, d 134.0 (C-8) and d 119.6
(C-10) due to the vinyl group], one anomeric carbon (d 98.9,
C-1ꢂ), one acetal carbon (d 97.5, C-1), and two hydroxy-
methyl carbons (d 59.8, C-7 and d 60.8, C-6ꢂ). The above
data and 2D-NMR analysis indicated that 2 was a lactone
open form of sweroside (13). To confirm the structure and
the relative and absolute configurations, chemical correla-
tions of 2 with known compounds were performed. Swero-
side (13) was treated with 1 N LiOH–THF (1 : 2) to give a
mixture of 2 and the starting material that was regenerated
from 2 during workup with Amberlite IR-120B. Then, we
attempted to convert 2 into its methyl ester derivative known
as secologanol,24) by treatment with CH2N2. This, however,
gave sweroside (13). All the spectroscopic data of semi-syn-
thetic 13 (1H- and 13C-NMR, MS, [a]D) were identical with
those of natural 13. Therefore, the structure of 2 was deduced
Whole plants of O. liukiuensis (145.0 g) were extracted
with hot MeOH to give the extract (16.0 g). Partitioning the
MeOH extract between H2O and CHCl3 and extracting the
aqueous layer with n-BuOH gave the CHCl3 extract (2.7 g),
the n-BuOH extract (2.9 g) and the water-soluble portion
(10.3 g). Each extract was purified by repeated chromatogra-
phy to afford two new monoterpene glucosides (2, 6.5 mg,
3, 282.7 mg) as well as camptothecin (1, 18.4 mg) and
its related alkaloids, such as 9-methoxycamptothecin (4,
Fig. 1. Structures of New Compounds
∗ To whom correspondence should be addressed. e-mail: htakayam@p.chiba-u.ac.jp
© 2005 Pharmaceutical Society of Japan

1356
Vol. 53, No. 10
Fig. 2. Structures of Known Compounds Isolated from Ophiorrhiza liukiuensis
through an ether linkage. The HMBC correlations between
the oxymethine proton at d 4.97 (H-7) and the carbonyl car-
bon at d 166.8 and between the proton at d 7.86 (H-6ꢅ) and
the carbonyl carbon at d 166.1 were observed. Therefore, the
structure of 3 was deduced to be 3ꢅ-O-glucosylsenburiside
II.
Experimental
General Experimental Procedures Optical rotation: JASCO P-1020.
UV: JASCO V-560. IR: JASCO FT/IR-230. 1H- and 13C-NMR spectra:
JEOL JNM-ECP600 at 600 (1H) and 150 (13C) MHz, JEOL JNM A-500 at
500 (1H) and 125.65 (13C) MHz, or JEOL JNM-ECP400 at 400 (1H) and 100
(13C) MHz, respectively. FAB-MS and HR-FAB-MS: JEOL JMS-HX110.
CD: JASCO J-720WI. TLC: Precoated silica gel 60 F254 plates (Merck,
0.25 mm thick), RP-18 F254S plates (Merck, 0.25 mm thick). Column chro-
matography: Silica gel 60 (Merck, 230—400 mesh for flash column chro-
matography), Cosmosil 75 C18-OPN (ODS, Nacalai Tesque), DIAION HP20
(Mitsubishi Chemical). MPLC: C. I. G. prepacked column CPS-HS-221-05
(SiO2) and CPO-HS-221-20 (ODS) (Kusano Kagakukikai). HPLC: CAP-
CELL PAK NH2 UG80 (Shiseido Fine Chemicals).
Plant Material Ophiorrhiza liukiuensis HAYATA was collected from Ishi-
gaki Island, Okinawa, Japan, in July 2002. Taxonomic identification of the
collected plant was based on the specimen kept in Koishikawa Botanical
Gardens, Graduate School of Science, The University of Tokyo. A voucher
specimen was deposited at the Graduate School of Pharmaceutical Sciences,
Chiba University.
Extraction and Isolation Whole plants of O. liukiuensis (145.0 g, wet
weight) were extracted with hot MeOH five times to give the extract (16.0 g).
The MeOH extract was then partitioned between H2O and CHCl3. The or-
ganic layer was washed with H2O, dried over MgSO4 and evaporated to give
the CHCl3 extract (2.7 g). The aqueous layer was extracted with n-BuOH
and the organic layer was evaporated to give the n-BuOH extract (2.9 g). The
aqueous layer was freeze-dried to give the water-soluble portion (10.3 g).
The water-soluble portion was subjected to column chromatography on DI-
AION HP20 to give six fractions: fr. A, H2O (500 ml) 8.58 g; fr. B, H2O
(500 ml) 365 mg; fr. C, 30% MeOH/H2O (500 ml) 363 mg; fr. D, 50%
MeOH/H2O (500 ml) 389 mg; fr. E, MeOH (1000 ml) 572 mg; and fr. F, ace-
tone (500 ml) 10 mg. Fr. B was purified by using a combination of MPLC
(SiO2, CHCl3/MeOH/H2Oꢁ7 : 4 : 1), silica gel flash column chromatography
(CHCl3/MeOH/H2Oꢁ7 : 4 : 1), and MPLC (SiO2, CHCl3/MeOH/H2Oꢁ
Fig. 3. Selected HMBC Correlations of 3
to be demethylsecologanol.
The HR-FAB-MS spectrum of new iridoid glucoside 3
gave a protonated molecular ion peak at m/z 779.2371
([MꢀH]ꢀ, D ꢃ2.7 mmu) and established the molecular for-
mula as C36H43O19. Acid hydrolysis of 3 was performed to
1
give D-glucose. The H-NMR spectrum was very similar to
that of swertiaside A (17)25) except for the aromatic and
sugar regions. It showed signals indicative of an olefinic pro-
ton at d 7.46 (d, Jꢁ1.5 Hz, H-3), an acetal proton at d 5.51
(d, Jꢁ3.7 Hz, H-1), and a b-linked anomeric proton at d 4.66
(d, Jꢁ7.8 Hz, H-1ꢂ), as well as a doublet assignable to the
methyl group at d 1.24 (d, Jꢁ7.0 Hz, H3-10), all of which
were characteristic of iridoid glycosides. Furthermore, sig-
nals assignable to two sets of aromatic protons due to 1,3-
disubstituted benzenes and one extra glucose unit were ob-
served. The 13C-NMR spectrum revealed one carboxyl car-
bon (d 170.7), two ester carbons [d 166.8 (C-7ꢄ), d 166.1 (C-
7ꢅ)] and two anomeric carbons [d 100.2 (C-1ꢂ), d 102.4 (C-
1ꢄꢄ)]. The above data suggested that 3 was a glucosyl deriva-
tive of senburiside II.26) The HMBC spectrum of 3 showed
correlation between the anomeric proton at d 5.00 (H-1ꢄꢄ)
and an aromatic carbon at d 159.3, indicating that one extra
glucose was attached to C-3ꢅ of the terminal benzene ring

October 2005
1357
give sweroside {4.2 mg yield quant., [a]D22 ꢃ137.0° (cꢁ0.07, MeOH)}, as
identified from 1H- and 13C-NMR spectra.
10 : 5 : 1) to give new secoiridoid 2 (6.5 mg). Fr. E was separated by silica gel
flash column chromatography (CHCl3/MeOH/H2Oꢁ10 : 5 : 1—7 : 4 : 1—
7 : 5 : 1.5). The CHCl3/MeOH/H2Oꢁ7 : 4 : 1 eluate was purified by a combi-
nation of silica gel flash column chromatography (CHCl3/MeOH/H2O gradi-
ent), ODS open column chromatography (5% CH3CN/H2O), and MPLC
(SiO2, 15% MeOH/CHCl3 or ODS, 20% CH3CN/H2O) to give new iridoid
glucoside 3 (282.7 mg). From the water-soluble portion, lyalosidic acid (9,
145.0 mg), ophiorine A (10, 5.6 mg), ophiorine B (11, 6.4 mg), sweroside
Acid Hydrolysis of 3 A solution of 3 (14.8 mg) in 1 N HCl aq. (2.0 ml)
and 1,4-dioxane (0.25 ml) was heated at 130 °C for 2 h under Ar. The reac-
tion mixture was extracted with AcOEt. The organic layer was washed with
water, dried over MgSO4 and evaporated. The aqueous layer was neutralized
by passage through Amberlite IRA-93 and elution with H2O. This was fol-
lowed by evaporation in vacuo to give a sugar fraction. The identity and con-
figuration of the sugars were determined by comparison with authentic D-
(ꢀ)-glucose (tR, 20.0 min) on HPLC. HPLC conditions: column, Shiseido
Fine Chemicals CAPCELL PAK NH2 UG80 (4.6ꢆ250 mm i.d.); solvent,
85% CH3CN–H2O; flow rate, 0.5 ml/min; temperature, 40 °C; RI detection,
Shodex RI-72 and chiral detection, JASCO OR-1590. The sugar fraction
gave peak that corresponded to D-(ꢀ)-glucose (tR, 20.2 min).
(13, 20.8 mg),27,28) loganic acid (15, 5.6 mg),29) swertiaside
A (17,
45.0 mg),25) and chlorogenic acid (22, 111.8 mg)30,31) were isolated. From the
CHCl3 extract, camptothecin (1, 18.4 mg), 9-methoxycamptothecin (4,
18.3 mg), 10-methoxycamptothecin (5, 4.3 mg), harman (12, 12.5 mg),
scopoletin (18, 4.0 mg),32) ursolic acid (23, 38.4 mg),33,34) and daucosterol
(25, 4.2 mg)35) were isolated. From the n-BuOH extract, pumiloside (6,
21.0 mg), (3R)-deoxypumiloside (7, 26.3 mg), strictosamide (8, 3.7 mg),
lyalosidic acid (9, 194.4 mg), harman (12, 12.7 mg), sweroside (13,
15.4 mg), epi-vogeloside (14, 1.7 mg),36,37) loganin (16, 3.0 mg),36,38) hyperin
(19, 94.1 mg),39,40) (6S,9R)-roseoside (20, 2.2 mg),41,42) (6R,7E,9R)-9-hydro-
xymegastigma-4,7-dien-3-one-9-O-b-D-glucoside (21, 2.9 mg),43) and b-sito-
sterol (24, 24.2 mg)44) were obtained. The detailed NMR data of 10-
methoxycamptothecin (5) have not been reported elsewhere. 1H-NMR
(400 MHz, DMSO-d6) d: 8.55 (1H, s, H-7), 8.07 (1H, d, Jꢁ9.2 Hz, H-12),
7.52 (1H, s, H-9), 7.50 (1H, overlapped, H-11), 7.28 (1H, s, H-14), 6.51 (1H,
br-s, OH), 5.41 (2H, s, H2-17), 5.26 (2H, s, H2-5), 3.93 (3H, s, 10-OMe),
1.85 (2H, m, H2-19), 0.87 (3H, dd, Jꢁ7.2, 7.2 Hz, H3-18). 13C-NMR
(100 MHz, DMSO-d6) d: 172.7 (C-21), 158.4 (C-10), 157.1 (C-16a), 150.4
and 150.3 (C-2, 15), 146.0 (C-3), 144.2 (C-13), 130.7 (C-12), 130.5 (C-6),
130.2 (C-7), 129.7 (C-8), 123.2 (C-11), 118.6 (C-16), 106.5 (C-9), 96.3 (C-
14), 72.6 (C-20), 65.4 (C-17), 56.0 (OMe), 50.4 (C-5), 30.4 (C-19), 8.0 (C-18).
Demethylsecologanol (2) Amorphous. [a]D23 ꢃ108.3° (cꢁ0.06,
MeOH). UV lmax (MeOH) nm: 233.0. FAB-MS (Glycerol, positive) m/z:
377 [MꢀH]ꢀ. HR-FAB-MS (GlycerolꢀH2O/PEG) m/z: 377.1426 [MꢀH]ꢀ
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(Calcd for C16H25O10: 377.1447). H-NMR (600 MHz, D2O) d: 7.35 (1H, s,
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3ꢅ-O-Glucosylsenburiside II (3) Amorphous. [a]D26 ꢃ83.4° (cꢁ0.69,
MeOH). UV lmax (MeOH) nm: 232.5, 286.0. IR nmax (KBr) cmꢃ1: 3447,
1716, 1261, 1075. FAB-MS (Glycerol, positive) m/z: 779 [MꢀH]ꢀ. HR-
FAB-MS (GlycerolꢀH2O/PEG) m/z: 779.2371 [MꢀH]ꢀ (Calcd for
C36H43O19: 779.2398). 1H-NMR (500 MHz, CD3OD) d: 7.91 (1H, ddd,
Jꢁ7.9, 1.3, 1.3 Hz, H-6ꢄ), 7.90 (1H, overlapped, H-1ꢅ), 7.86 (1H, ddd,
Jꢁ7.9, 1.3, 1.3 Hz, H-6ꢅ), 7.82 (1H, br-dd, Jꢁ1.8, 1.8 Hz, H-2ꢄ), 7.55 (1H,
dd, Jꢁ7.9, 7.9 Hz, H-5ꢄ), 7.49 (1H, dd, Jꢁ7.9, 7.9 Hz, H-5ꢅ), 7.47 (1H, ddd,
Jꢁ7.9, 2.3, 1.3 Hz, H-4ꢄ), 7.46 (1H, d, Jꢁ1.5 Hz, H-3), 7.43 (1H, ddd,
Jꢁ7.9, 2.6, 1.3 Hz, H-4ꢅ), 5.51 (1H, d, Jꢁ3.7 Hz, H-1), 5.00 (1H, br-d,
Jꢁ7.6 Hz, H-1ꢄꢄ), 4.97 (1H, ddd, Jꢁ7.1, 4.4, 4.4 Hz, H-7), 4.66 (1H, d,
Jꢁ7.8 Hz, H-1ꢂ), 3.90 (1H, dd, Jꢁ12.2, 2.1 Hz, H-6ꢄꢄ), 3.83 (1H, br-dd,
Jꢁ11.9, 1.5 Hz, H-6ꢂ), 3.71 (1H, dd, Jꢁ12.2, 5.6 Hz, H-6ꢄꢄ), 3.64 (1H, br-
dd, Jꢁ11.9, 5.2 Hz, H-6ꢂ), 3.51—3.47 (1H, overlapped, H-2ꢄꢄ, 3ꢄꢄ, 5ꢄꢄ), 3.41
(1H, m, H-3ꢂ), 3.36 (1H, br-dd, Jꢁ9.0, 9.0 Hz, H-4ꢄꢄ), 3.31—3.27 (2H, over-
lapped, H-4ꢂ, 5ꢂ), 3.18 (1H, dd, Jꢁ9.2, 7.8 Hz, H-2ꢂ), 3.05 (1H, m, H-5),
2.53 (1H, ddd, Jꢁ14.3, 7.1, 7.1 Hz, H-6), 2.12 (1H, m, H-8), 2.03 (1H, ddd,
Jꢁ14.3, 4.4, 4.4 Hz, H-6), 2.00 (1H, ddd, Jꢁ8.2, 8.2, 3.7 Hz, H-9), 1.24
(3H, d, Jꢁ7.0 Hz, H3-10). 13C-NMR (125 MHz, CD3OD) d: 170.7 (C-11),
166.8 (C-7ꢄ), 166.1 (C-7ꢅ), 159.3 (C-3ꢅ), 152.4 (C-3, C-3ꢄ), 133.2 (C-1ꢄ),
131.8 (C-1ꢅ), 131.0 (C-5ꢅ), 130.8 (C-5ꢄ), 128.1 (C-6ꢄ), 127.7 (C-4ꢄ), 125.1
(C-6ꢅ), 124.0 (C-2ꢄ), 123.5 (C-4ꢅ), 119.3 (C-2ꢅ), 112.6 (C-4), 102.4 (C-1ꢄꢄ),
100.2 (C-1ꢂ), 96.2 (C-1), 83.8 (C-7), 78.3 (C-5ꢂ, 5ꢄꢄ), 77.9 (C-3ꢂ, 3ꢄꢄ), 74.9
(C-2ꢄꢄ), 74.7 (C-2ꢂ), 71.5 (C-4ꢂ), 71.3 (C-4ꢄꢄ), 62.7 (C-6ꢂ), 62.4 (C-6ꢄꢄ), 48.7
(C-9), 43.1 (C-8), 37.9 (C-6), 32.6 (C-5), 18.4 (C-10).
Reaction of Natural 2 with CH2N2 An ether solution of freshly pre-
pared CH2N2 was added to a solution of 2 (3.6 mg) in MeOH (1.0 ml). After
5 min, the solvent was removed under reduced pressure. The residue was pu-
rified by silica gel open column chromatography (20% MeOH/CHCl3) to

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