Cytotoxic calanquinone A from Calanthe arisanensis and its first total synthesis

Article abstract of DOI:10.1016/j.bmcl.2008.06.099

Calanquinone A (1) was isolated from an EtOAc-soluble extract of Calanthe arisanensis through bioassay-guided fractionation. Its structure was identified by spectroscopic methods. Compound 1 showed potent cytotoxicity (EC₅₀ < 0.5 μg/mL) against

Full text of DOI:10.1016/j.bmcl.2008.06.099

Bioorganic & Medicinal Chemistry Letters 18 (2008) 4275–4277
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Cytotoxic calanquinone A from Calanthe arisanensis and its first total synthesis
Chia-Lin Lee a,b, Kyoko Nakagawa-Goto a, Donglei Yu a, Yi-Nan Liu a, Kenneth F. Bastow c,
a,
Susan L. Morris-Natschke a, Fang-Rong Chang b, Yang-Chang Wu b,d, , Kuo-Hsiung Lee
*
*
a Natural Products Research Laboratories, School of Pharmacy, University of North Carolina at Chapel Hill, 315 Beard Hall, CB# 7360, NC 27599-7360, USA
b Graduate Institute of Natural Products, Kaohsiung Medical University, Kaohsiung 807, Taiwan
c Division of Medicinal Chemistry and Natural Products, School of Pharmacy, University of North Carolina, Chapel Hill, NC 27599, USA
d Center of Excellence for Environmental Medicine, Kaohsiung Medical University, Kaohsiung 807, Taiwan
a r t i c l e i n f o
a b s t r a c t
Article history:
Calanquinone A (1) was isolated from an EtOAc-soluble extract of Calanthe arisanensis through bioassay-
guided fractionation. Its structure was identified by spectroscopic methods. Compound 1 showed potent
cytotoxicity (EC50 < 0.5 lg/mL) against lung (A549), prostate (PC-3 and DU145), colon (HCT-8), breast
(MCF7), nasopharyngeal (KB), and vincristine-resistant nasopharyngeal (KB-VIN) cancer cell lines, and
interestingly, showed an improved drug resistance profile compared to paclitaxel. The total synthesis
of 1 was also achieved and is reported herein.
Received 27 March 2008
Revised 26 June 2008
Accepted 30 June 2008
Available online 3 July 2008
Keywords:
Ó 2008 Elsevier Ltd. All rights reserved.
Calanquinone A
Calanthe arisanensis
Cytotoxicity
Total synthesis
The Calanthe genus in the Orchidaceae family contains terres-
trial perennial herbs that are widely distributed from tropical
Africa and Madagascar to tropical and subtropical Asia, China,
Japan, southward through Malaysia and Indonesia to the Pacific
islands and Australia. This genus includes more than 150 species,
but only nineteen are found in Taiwan. Among them Calanthe
arisanensis Hayata is endemic to Taiwan and grows in forests from
1000 to 2000 m throughout the island.1
OCH3
OCH3
8a
9
8
7
10
B
C
5
6
10a
O
4b
O
1
4a
A
2
4
OH
3
A phytochemical study of this plant has not been reported to
date. In cytotoxicity screening of extracts of Formosan plants, an
EtOAc extract of C. arisanensis was found to be active against vari-
OCH3
ous human cancer cell lines with IC50 < 20 lg/mL. Bioassay-direc-
ted chromatographic fractionation of this extract produced a new
phenanthraquinone calanquinone A (1). Compound 1 showed sig-
nificant in vitro cytotoxic activity against seven human cancer cell
lines, as described below (see Fig. 1).
The active MeOH extract (225 g) of dry roots of C. arisanensis
(5.42 kg) was partitioned between EtOAc and water (1:1, v/v). Fur-
ther fractionation of the active EtOAc extract (32.7 g) by repeated
liquid chromatography on silica gel gave calanquinone A (1). HRE-
SIMS of 1 showed a [MÀH]À ion at m/z 313.0705 (C17H14O6–H),
indicating 11 degrees of unsaturation. The IR spectrum showed
absorptions for hydroxyl (3348 cmÀ1), carbonyl (1642 cmÀ1), and
aromatic ring (1626, 1514, 1460, 1411, and 844 cmÀ1) functional
Figure 1. Structure of calanquinone A (1).
groups. UV absorptions at 242, 308, and 426 nm also indicated
an aromatic system. Seventeen carbon signals, including three
methoxy, four methine, and ten quaternary carbons, were ob-
served in the NMR spectra of 1 (Table 1). Among the ten quaternary
carbons, two were identified as carbonyl carbons on the basis of
chemical shifts dC 184.7 and 186.2. Therefore, the data supported
the presence of two carbonyls, six olefins, and three ring moieties
to fulfill the 11 degrees of unsaturation, and 1 was postulated to be
a phenanthrenedione or anthrenedione.2 1D NMR and HSQC data
indicated the presence of three methoxy groups at dH 3.96, 4.01,
and 4.02 (dC 57.1, 56.2, and 61.0), one pair of ortho-coupled aro-
matic protons at dH 8.05 (d, J = 8.7 Hz, dC 137.1) and 8.10 (d,
J = 8.7 Hz, dC 122.0), and two olefinic protons at dH 6.15 (s, dC
* Corresponding authors. Tel.: +886 7 312 1101x2197; fax: +886 7 311 4773
(Y.C.W.); Tel.: +1 919 962 0066; fax: +1 919 966 3893 (K.H.L.).
E-mail addresses: yachwu@kmu.edu.tw (Y.-C. Wu), khlee@unc.edu (K.-H. Lee).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.06.099

4276
C.-L. Lee et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4275–4277
Table 1
we prepared 2-methoxy-5-carboxylic acid methyl ester-1,4-qui-
NMR data of calanquinone A (1)a
none (4)9 by AgO oxidation of the methyl ester (3) of commercially
available 2,4,5-trimethoxybenzoic acid (2). Compound 4 was cou-
pled with 3,4,5-trimethoxytoluene in the presence of 1 equiv. of
trifluoroacetic acid to produce quinone 5, although Kraus reported
the production of hydroquinone 10 under these reaction condi-
tions (Scheme 1). The quinone skeleton of 5 was confirmed from
13C NMR data, which showed two carbonyl groups at 180.6 and
183.6 ppm. In addition, reaction of 5 with Me2SO4 did not give a
methoxylated product (i.e., 11 in Scheme 1). We attempted to re-
duce 5 with Na2S2O4 to obtain the corresponding hydroquinone,
which could be transformed to the Kraus type of intermediate 11
(R = OMe) after methylation. Despite extending the reaction time
and adding more reducing agent, only starting material was
recovered.
Proton
13C (dC)
1H (dH)
HMBC (13C no)
3, 4, 10a
NOESY (1H no)
C3–OCH3
1
2
3
4
4a
4b
5
6
7
8
8a
9
10
10a
–OCH3
184.7
107.4
6.15 s
161.7
186.2
128.3
118.7
148.3
140.4
155.2
101.4
6.86 s
4b, 6, 7, 9
9, C7–OCH3
135.1
137.1
122.0
8.05 d (8.7)
8.10 d (8.7)
4b, 8, 10a
1, 4a, 8a
8, 10
9
133.0
C3–OCH357.1
C6–OCH361.0
C7–OCH356.2
C3–OCH33.96 s
C6–OCH34.02 s
C7–OCH34.02 s
10.73
3
6
7
2
Therefore, 5 was reduced with LAH (THF, reflux, 1 h) to alcohol,
which was oxidized selectively to aldehyde 6 with activated MnO2
(toluene, 110 °C, overnight) (Scheme 2). After an unsuccessful at-
tempt to obtain the phenanthraquinone 9 directly from 6 using
P4-tBu, quinone 6 was first reduced to the hydroquinone using
aq. Na2S2O4 (CH2Cl2, rt., overnight),10 and then methylated with
Me2SO4 in the presence of K2CO3(acetone, 60 °C, 1.5 h) to give
the desired compound 7 (Scheme 2). Cyclization of 7 with P4-tBu
(benzene, 100 °C, 63 h) gave 8, which was oxidized with AgO
(6 N HNO3, acetone, 50 °C, 2–3 min) to phenanthraquinone 9. Com-
pound 9 was converted to calanquinone A (1) by selective demeth-
ylation with TMSI in CH2Cl2 at rt (Scheme 2).
Synthesized 1 and intermediates 5–9 were screened in an in vi-
tro cytotoxicity assay (data shown in Table 3). Compound 1 exhib-
ited the highest potency (EC50 0.15–0.75 lg/mL) against all seven
tested cancer cell lines. The remaining compounds showed no
(6–8) or only weak (5 and 9) activity. Clearly, the potency of 1 mer-
C7–OCH3
8, C6–OCH3
C5–OH
a
Measured in CDCl3 (300 and 500 MHz, d in ppm, J in Hz).
107.4) and 6.86 (s, dC 101.4). In the HMBC spectra, the olefinic pro-
ton at dH 6.86 exhibited 2J interactions with a carbon at dC 155.2 (C-
7), as well as 3J interactions with carbons at dC 118.7 (C-4b), 140.4
(C-6), and 137.1 (C-9). The other olefinic proton at dH 6.15 exhib-
ited 2J interactions with a carbon at dC 161.7 (C-3), as well as 3J
interactions with carbons at dC 186.2 (C-4) and 133.0 (C-10a). Loca-
tions of methoxy groups at C-3, C-6, and C-7 were confirmed by the
following NOESY correlations: dH 6.15 (H-2)/3.96 (3-OMe), dH 6.86
(H-8)/4.01 (7-OMe) and 8.05 (H-9), dH 4.01 (7-OMe)/4.02 (6-OMe),
and dH 8.05 (H-9)/8.10 (H-10). Thus, compound 1 was identified as
5-hydroxy-3,6,7-trimethoxy-1,4-phenanthrenequinone and has
been named as calanquinone A (1).
Table 2
Compound 1 is related in structure to other naturally occurring
phenanthrenequinones, including the des-oxy analog sphenone
(lacking the C-5 OH group),3 cymbinodin A (lacking the two meth-
oxy groups at C-6 and C-7),4 and annoquinone A (lacking any sub-
stituents on ring C).5,6 In prior studies, sphenone and annoquinone
A showed cytotoxic activity against the KB cell line (reported EC50
Cytotoxicity of calanquinone A (1) isolated from C. arisanensis
Compound
EC50
DU145 HCT-8 MCF-7 KB
0.34 0.20 0.03 0.32
(lg/mL)/cell line
A549
0.19
PC-3
0.16
KBVIN
0.45
Calanquinone A (1)
Paclitaxela
<0.005 0.0097 <0.005 0.21
0.0072 <0.005 2.16
2.73 and 1.65
Compound 1 exhibited potent cytotoxicity (EC50 0.03–0.45
lg/mL, respectively).
a
Positive control.
lg/
mL) against human lung (A549), prostate (PC-3 and DU145), colon
(HCT-8), breast (MCF7), nasopharyngeal (KB), and vincristine-
resistant nasopharyngeal (KB-VIN) cancer cell lines. Paclitaxel
was used as a positive control (data shown in Table 2). Interest-
ingly, 1 exhibited comparable potency against both KB and its
drug-resistant KB-VIN subline, and thus showed an improved drug
resistance profile compared to paclitaxel. The cytotoxic values
demonstrate the strong potential of 1 as a promising lead com-
pound and C. arisanensis as a promising plant source of new agents
for cancer chemotherapy.
Table 3
Cytotoxicity of synthesized 1 and related intermediates
Compound
EC50 (lg/mL)/cell line
A549
PC3
DU145
HCT8
MCF7
KB
KBVIN
1
5
6
7
8
9
0.31
6.12
NA
NA
NA
0.75
6.09
NA
NA
NA
0.48
4.74
NA
0.29
5.85
NA
0.15
6.40
NA
0.30
4.02
NA
NA
NA
0.24
5.48
NA
NA
NA
NA
NA
NA
NA
NA
NA
In order to make sufficient quantities of 1 for extensive biolog-
ical evaluation, we modified the synthetic procedure of Kraus and
co-workers7,8 (Scheme 1) to synthesize 1. As shown in Scheme 2,
7.06
7.81
4.40
15.25
5.33
8.08
9.14
NA: no activity up to 20
l
g/mL.
O
OR
O
CO2Me
1) LAH
CO2Me
TFA
2) MnO4
MeO
MeO
3) P4-tBu
4) AgO
MeO
OR
MeO
O
MeO
O
OMe
10 (R=H)
11 (R=Me)
OMe
MeO
OMe
4
Me2SO4
Scheme 1. Synthetic procedure of Kraus.

C.-L. Lee et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4275–4277
4277
OMe
OMe
O
O
MeO
OMe
OMe
CO2R
CO2Me
OMe
CO2Me
AgO
TFA, ether
20%
(2 steps overall yield from 3)
6N HNO
3 MeO
MeO
MeO
O
O
MeO
OMe
K2CO3
Me2SO4
96%
2:
3:
R = H
R = Me
4
5
OMe
O
CHO
CHO
1. Na2S2O4/H2O,
CH2Cl2, 43%
1. LAH,THF, 69%
2. MnO2, toluene, 84%
MeO
MeO
OMe
2. Me2SO4, K2CO3,
acetone, 100%
OMe
MeO
O
OMe
MeO
OMe
OMe
OMe
7
6
O
AgO, 6N HNO3
acetone, reflux
P4-tBu, PhH
100 °C,44%
TMSI, CH2Cl2
30%
1
MeO
MeO
46%
O
MeO
OMe
MeO
OMe
OMe
OMe
OMe
9
8
Scheme 2. Our total synthesis of calanquinone A (1).
its further study and our synthetic route can efficiently produce
sufficient quantities of 1 for future extensive biological evaluation
and SAR investigation.
References and notes
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of Taiwan: Taipei, Taiwan, 2000; Vol. 5, pp. 776–777, 782–783.
2. Fan, C.; Wang, W.; Wang, Y.; Qin, G.; Zhao, W. Phytochemistry 2001, 57,
1255.
Acknowledgments
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Phytochemistry 1987, 26, 1623.
6. Krohn, K.; Loock, U.; Paavilainen, K.; Hausen, B.; Schmalle, H. W.; Kiesele, H.
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This work was supported by grants from National Cancer Insti-
tute, NIH, USA (CA 17625, K.H. Lee), National Science Council,
Taiwan (Y.-C. Wu) and KMU-EM-97-2.1.b (Y.-C. Wu).
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2008.06.099.