Cytotoxic pheophorbide-related compounds from Clerodendrum calamitosum and C. cyrtophyllum

Article abstract of DOI:10.1021/np000595b

Three pheophorbide-related compounds (1-3) were isolated from the leaves and stems of Clerodendrum calamitosum. The methyl ester of 3 (6) and the known (10S)-hydroxypheophytin a (7) also were isolated from leaves of the related plant Clerodendrum cyrtophyllum. Compounds 1 and 6 were isolated for the first time as naturally occurring products from a plant source. All structures were elucidated by detailed spectroscopic analysis. Biological evaluation showed that 1 and 2 exhibited strong cytotoxicity against human lung carcinoma (A549), ileocecal carcinoma (HCT-8), kidney carcinoma (CAKI-1), breast adenocarcinoma (MCF-7), malignant melanoma (SK-MEL-2), ovarian carcinoma (1A9), and epidermoid carcinoma of the nasopharynx (KB), and its etoposide- (KB-7d), vincristine- (KB-VCR), and camptothecin-resistant (KB-CPT) subclones. Compound 3 was less cytotoxic than 1 and 2. Compounds 4-6, the methyl esters of 1-3, showed strongly increased cytotoxicity compared with the parent acids. Interestingly, 6 was the most active derivative among these compounds. Compound 7 was inactive.

Full text of DOI:10.1021/np000595b

J . Nat. Prod. 2001, 64, 915-919
915
Cytotoxic P h eop h or bid e-Rela ted Com p ou n d s fr om Cler od en d r u m ca la m itosu m
a n d C. cyr toph yllu m §
†
†
†
†
†
†
Huey-Hwa Cheng, Hui-Kang Wang, J unko Ito, Kenneth F. Bastow, Yoko Tachibana, Yuka Nakanishi,
†
‡
,†
Zhihong Xu, Tsui-Yun Luo, and Kuo-Hsiung Lee*
Natural Products Laboratory, Division of Medicinal Chemistry and Natural Products, School of Pharmacy,
University of North Carolina, Chapel Hill, North Carolina 27599, and Department of Biochemistry,
Taipei Medical College, Taipei, Taiwan, Republic of China
Received December 8, 2000
Three pheophorbide-related compounds (1-3) were isolated from the leaves and stems of Clerodendrum
calamitosum. The methyl ester of 3 (6) and the known (10S)-hydroxypheophytin a (7) also were isolated
from leaves of the related plant Clerodendrum cyrtophyllum. Compounds 1 and 6 were isolated for the
first time as naturally occurring products from a plant source. All structures were elucidated by detailed
spectroscopic analysis. Biological evaluation showed that 1 and 2 exhibited strong cytotoxicity against
human lung carcinoma (A549), ileocecal carcinoma (HCT-8), kidney carcinoma (CAKI-1), breast
adenocarcinoma (MCF-7), malignant melanoma (SK-MEL-2), ovarian carcinoma (1A9), and epidermoid
carcinoma of the nasopharynx (KB), and its etoposide- (KB-7d), vincristine- (KB-VCR), and camptothecin-
resistant (KB-CPT) subclones. Compound 3 was less cytotoxic than 1 and 2. Compounds 4-6, the methyl
esters of 1-3, showed strongly increased cytotoxicity compared with the parent acids. Interestingly, 6
was the most active derivative among these compounds. Compound 7 was inactive.
Clerodendrum calamitosum L. (Verbenaceae) is a small
cytotoxic or antitumor activities of chlorophyll-related
1
6
shrub originally found in J ava, but now widely cultivated
compounds are very limited. Nakatani et al.
and
2
17
in Taiwan. The leaves and stems of this plant, known as
Nakamura et al. reported that pheophorbides showed
photocytotoxic effects toward the tumor cells upon direct
photoirradiation.
“
Hwa-Shih Tsao”, have been used in folk medicine to treat
2
bladder, kidney, and gall stones and as a diuretic. Previous
3
studies by Chen et al. reported that the crude extract of
We report herein on the isolation and characterization
of pheophorbide-type constituents of C. calamtosum and
C. cyrtophyllum and the cytotoxicity of these compounds
against a panel of human tumor cell lines, including A549,
HCT-8, CAKI-1, MCF-7, SK-MEL-2, 1A9, and KB and
its drug-resistant KB-7d, KB-VCR, and KB-CPT subclones,
compared with their structurally related compounds,
chlorophyll and chlorophyllin, under ambient light treat-
ment.
this plant exhibited antihypertensive, sedative, and anti-
bacterial effects, but no literature reports describing the
cytotoxic effects or the active chemical constituents of this
plant have yet appeared. The related Clerodendrum
cyrtophyllum Turcz (Verbenaceae) is a perennial shrub,
indigenous to central and northern Taiwan. Its stems
and roots are used in traditional Chinese medicine to
treat beriberi, fever, jaundice, leukorrhea, syphilis, and
typhoid.4
,5
Resu lts a n d Discu ssion
In the course of screening medicinal plants for potential
antitumor agents, we found that a chloroform-soluble
extract of the leaves of C. calamitosum showed significant
in vitro cytotoxicity against tissue-cultured cells of human
epidermoid carcinoma of the nasopharynx (KB). Subse-
quent bioassay-directed fractionation of this extract led to
the isolation of three pheophorbide-type compounds, 1-3.
Their structures were established from spectral data,
Ground, air-dried leaves and stems of C. calamitosum
were extracted with methanol, and the methanol extract
was further fractionated successively with n-hexane,
chloroform, and ethyl acetate. Repeated chromatography
of the chloroform fraction led to the isolation of 1-3. The
methyl ester derivatives, 4-6, were obtained from 1-3,
respectively, by diazomethane methylation. Similarly, the
dried leaves of C. cyrtophyllum were extracted with metha-
nol, and the methanolic extract was further fractionated
successively with n-hexane and chloroform. Repeated chro-
matography of the chloroform fraction led to the isolation
of 6 [methyl (10S)-hydroxypheophorbide a] and 7 [(10S)-
hydroxypheophytin a].
1
13
including UV, MS, H and C NMR, DEPT, COSY,
HETCOR, HMBC, HMQC, and NOESY, and by chemical
interconversions between these compounds. Among them,
1
has been isolated as a new naturally occurring compound
from this plant, although it has been prepared previously
6
semisynthetically.
7
8
Compound 1, a dark brown amorphous solid, had a
molecular weight of 638 by FABMS. It was identified as
Lai et al. and Terwel and van der Hoeven have shown
that vegetable extracts containing chlorophylls are anti-
mutagenic in a bacterial assay system, and chlorophyllin
1
13
purpurin 7 dimethyl ester by comparison of its H and
C
6
,18
9
-11
NMR data with those in the literature
and of similar
In addition, the struc-
has been shown to be a potent antimutagenic
and
compounds, including 2 and 3.1
9-21
anticarcinogenic agent.1
2-15
However, data demonstrating
ture was supported by NOE correlations of the 9a-methyl
protons with both the 5a- and 10b-methyl signals.
Compound 2, dark green crystals, had a molecular
weight of 592 by FABMS. It was identified as pheophorbide
*
To whom correspondence should be addressed. Tel: 919-962-0066.
Fax: 919-966-3893. E-mail: khlee@unc.edu.
§
Paper number 208 in the series Antitumor Agents. For Part 207, see
ref 1.
1
13
†
a by comparison of H and C NMR spectral data with
University of North Carolina.
Taipei Medical College.
‡
19-21
those reported in the literature.
1
0.1021/np000595b CCC: $20.00
© 2001 American Chemical Society and American Society of Pharmacognosy
Published on Web 07/04/2001

9
16 J ournal of Natural Products, 2001, Vol. 64, No. 7
Cheng et al.
Ta ble 1. 1H NMR Data of 1-7 (δ, in CDCl3)a-c
position
1
2
3
4
5
6
7d
1
2
2
2
a
a
3.26 (s)
7.80 (dd, 18, 12)
3.32 (s)
3.42 (s)
3.34 (s)
3.37 (s)
3.43 (s)
3.37 (s)
7.85 (dd, 18, 12) 8.01 (dd, 18, 12)
7.92 (dd, 18, 12) 7.91 (dd, 18, 12) 8.02 (dd, 18, 12) 7.89 (dd, 18, 12)
b (E) 6.21 (d, 18)
b (Z)
6.19 (d, 18)
6.09 (d, 12)
9.21 (s)
6.30 (d, 18)
6.19 (d, 12)
9.48 (s)
6.30 (d, 18)
6.15 (d, 12)
9.32 (s)
6.24 (d, 18)
6.13 (d, 12)
9.27 (s)
6.31 (d, 18)
6.21 (d, 12)
9.47 (s)
6.20 (dd, 18, 1)
6.11 (dd, 12, 1)
9.29 (s)
6.06 (d, 12)
9.21 (s)
3.10 (s)
3.58 (q, 8)
1.63 (t, 8)
9.54 (s)
R
3
4
4
â
5
7
7
7
7
7
7
8
8
δ
9
1
1
a
a
b
3.09 (s)
3.26 (s)
3.19 (s)
3.14 (s)
3.27 (s)
3.09 (s)
3.53 (q, 8)
1.61 (t, 8)
9.37 (s)
3.72 (q, 8)
1.70 (t, 8)
9.62 (s)
3.59 (q, 8)
1.70 (t, 8)
9.60 (s)
3.53 (s)
4.68 (br d, 9)
3.58 (q, 8)
1.65 (t, 8)
9.43 (s)
3.73 (q, 8)
1.71 (t, 8)
9.61 (s)
3.53 (q, 8)
1.592 (t, 8)
9.47 (s)
3.70 (s)
4.17 (dd, 9, 3)
2.95 (m)
2.34 (m)
2.57 (m)
2.27 (m)
a
3.58 (s)
3.61(s)
3.73(s)
3.60 (s)
3.73 (s)
4.68 (br dd, 9, 2) 4.18 (br dt, 9, 2) 4.18 (dd, 9, 3)
2.38 (m)
2.10 (m)
2.09 (dd, 3, 9)
1.76 (m)
4.21 (br. d, 8)
2.65 (m)
2.30 (m)
2.52 (m)
2.25 (m)
3.67 (s)
4.46 (m)
1.80 (d, 7)
8.55 (s)
4.15 (dd, 9, 3)
2.98 (m)
2.35 (m)
2.66 (m)
2.27 (m)
3.62 (s)
4.48 (q, 7)
1.61 (d, 7)
8.61(s)
a
a′
b
b′
d
2.61 (m)
2.28 (m)
2.55 (m)
2.21 (m)
2.98 (ddt, 14, 9, 3) 2.37 (m)
2.37 (ddd, 14, 9, 6) 2.10 (m)
2.64 (dt, 9, 9)
2.25 (dt, 14, 9, 4) 1.76 (m)
3.60 (s)
4.49 (q, 7)
1.61 (d, 7)
8.64 (s)
2.08 (dd, 3, 9)
4.28 (q, 7)
1.77 (d, 7)
8.45 (s)
4.42 (dq, 2, 7)
1.80 (d, 7)
8.51 (s)
4.30 (m)
1.79 (d, 7)
8.49 (s)
4.50 (q, 7)
1.586 (d, 7)
8.61(s)
a
a
0
0b
3.84 (s)
3.88 (s)
6.22 (s)
3.86 (s)
-1.69
6.27 (s)
3.90 (s)
-1.67
4.12 (s)
3.62 (s)
-1.81
5.60 (br. s)
4.14 (s)
3.56 (s)
5.31(s)
3.62 (s)
5.50 (s)
NH
OH-10
a
b
All assignments were based on COSY, NOESY, and TOCSY experiments. s ) singlet, d ) doublet, t ) triplet, m ) multiplet, br )
c
d
broad. 500 Hz, J values in parentheses in Hz. Phytyl proton assignments given in Experimental Section.
F igu r e 1. Structures and reactions of pheophorbide a related compounds.
Compound 3, dark green crystals, had a molecular
weight of 608 by FABMS and was identified as (10S)-
hydroxypheophorbide a by comparison of H and C NMR
spectral data with literature values.19
The detailed proton signal assignments of 1-3 are listed
in Table 1. All three compounds were converted to the
corresponding methyl esters (4-6) by treatment with
diazomethane. Furthermore, 6 underwent ring opening and
esterification to give the â-keto ester 4 using methyl iodide
in the presence of potassium carbonate.
Compound 7, a dark green amorphous solid, showed a
+
protonated molecular ion peak at m/z 886 [M ] in the
1
13
FABMS. Its 1H and C NMR spectra closely resembled
those of 6 except for the presence of a phytyl group and
the absence of one methyl ester. Compound 7 was identified
13
1
as (10S)-hydroxypheophytin a by comparison of its H NMR
data with those reported in the literature.16
All compounds isolated and prepared in this study were
assayed for in vitro cytotoxicity against a panel of human
tumor cell lines. The results are shown in Tables 2 and 3.
Compounds 1-3 showed similar trends of tumor specificity,
being most cytotoxic against the KB and 1A9 cell lines, less
cytotoxic against the CAKI-1 and SK-MEL-2 cell lines, and
the least cytotoxic against the HCT-8, A-549, and MCF-7
cell lines. Among these compounds, 2 displayed both strong
and broad activity for the KB, 1A9, CAKI-1, SK-MEL-2,
HCT-8, A549, and MCF-7 tumor cells, with ED50 values of
Compound 6, a dark green amorphous solid, showed a
+
protonated molecular ion peak at m/z 622 [M ] in the
FABMS. Its 1H NMR spectrum and TLC R
value were
f
identical to those of the methyl ester synthesized from 3
obtained from C. calamitosum above. This report is the first
description of its isolation as a naturally occurring com-
pound.

Cytotoxic Pheophorbide-Related Compounds
J ournal of Natural Products, 2001, Vol. 64, No. 7 917
Ta ble 2. Cytotoxicity (ED50) of Pheophorbide a Compounds and Pheophytins against Human Tumor Cell Linesb
a
compound
KB
A-549
HCT-8
CAKI-1
1A9 (OVCAR)
MCF-7
SK-MEL
1
2
3
4
5
6
7
0.70
0.46
1.3
0.20
0.23
0.30
12
63
38
1.8
1.5
>4
1.6
1.5
>4
0.79
1.0
1.3
0.88
3.0
0.55
0.62
1.9
0.64
0.48
1.2
0.20
0.26
0.27
22
1.6
1.6
>4
1.1
0.91
4.0
1.1
1.2
0.49
0.83
0.47
15
0.73
0.95
0.45
8.8
0.40
14
0.84
14
19
NT
NT
c,d
c,d
c,d
c,d
c,d
chlorophyll
copper sodium chlorophyllin
NT
NT
64
31
NT
NT
NT
d
d
39
>40
>40
a
Cytotoxicity as ED50 in µg/mL for each cell line is the concentration of compound that causes a 50% reduction in absorbance at 562
b
c
nm relative to untreated cells using SRB assay. Cell lines cultured in RPMI-1640, 10% (v/v) FBS, 100 µg/mL kanamycin. ED50 values
d
of chlorophyll against KB and 1A9 cell growth were >6 µg/mLsnot considered to be significant and not further evaluated. NT ) not
tested.
Ta ble 3. Cytotoxicity of Pheophorbide a Compounds against
Drug-Resistant Cell Lines
affected adversely by MRP (KB-7d) or P-glycoprotein
MDR) overexpression (KB-VCR). The cytotoxicity of 6 was
(
compound
KB-7da
KB-VCRa
KB-CPTa
not adversely affected in the KB-7d and KB-CPT cell lines;
however, cytotoxicity decreased significantly in the KB-
VCR cell line. Interestingly, compared with the parental
cell line, the growth of the two multidrug-resistant cell lines
was more susceptible to 1 (about 2-fold for the MDR
derivative).
1
2
6
0.7
1.0
0.8
119
1.0
0.5
0.6
>57
153
15
1.0
1.1
1.1
1.0
1.0
VP-16
Taxol
a
Values are fold-resistance relative to activity in KB cells.
Compounds 1-3 are structurally closely related. Com-
pounds 2 and 3 both contain the same cyclopentanone ring
and differ only in the substitution at the C-10 position (H
and OH, respectively), while 1 is a ring-opened analogue
with a ketone group at C-10. Both 1 and 2 showed
comparable cytotoxicity, while 3 was less cytotoxic than 1
and 2. Methylation of the COOH-7c groups in 1 and 2 led
to derivatives (4, 5) with equal or slightly higher potency
than the parent compounds. However, methylation of the
COOH group in 3 gave 6, which showed strongly increased
cytotoxicity compared with 3. Except in the CAKI-1 cell
line, 6 was as potent or more potent than 4 and 5. These
findings may indicate that, in the parent acid 3, intra-
molecular hydrogen bonding occurs between the OH-10 and
COOH-7c groups, and this interaction is subsequently
prevented by methylation of 3 at the 7c-position, allowing
the C-10 hydroxy group to interact more freely with a
target receptor. On the other hand, we found that chloro-
phyll, chlorophyllin, and the pheophytins showed no sig-
nificant cytotoxicity. This lack of activity might be due to
the long-chain phytyl group at the 7c-COOH, which could
limit the penetration through the tumor cell membrane and
thus decrease drug action. Detailed structure-cytotoxicity
relationships remain to be explored.
0
.46, 0.48, 0.88, 0.91, 1.5, 1.5, and 1.6 µg/mL, respectively.
Compound 1 showed similar but slightly lower cytotoxicity,
while 3 was less potent. This latter compound exerted
significant activity against the KB and 1A9 cell lines with
ED50 values of 1.3 and 1.2 µg/mL, respectively, moderate
activity against the CAKI-1 cell line with an ED50 value of
3
.0 µg/mL, borderline activity against the SK-MEL-2 cell
line, and insignificant activity (ED50 > 4 µg/mL) against
the HCT-8, A549, and MCF-7 cell lines. The cytotoxicities
of chlorophyll (commercial mixture of a and b) and chloro-
phyllin were also evaluated for comparison purposes. The
ED50 values of chlorophyllin against the KB, 1A9, and A549
cell lines were not significant. Chlorophyll was also inactive
against the KB and 1A9 cell lines, with ED50 values
significantly greater than 4 µg/mL. Therefore, chlorophyllin
and chlorophyll were not evaluated further. In addition,
(10S)-hydroxypheophytin a (7) was not active, as shown
in Table 2.
The methyl esters 4-6 showed increased cytotoxicity
against the cell lines tested except for the SK-MEL-2 cell
line, when compared with the corresponding acid. For
example, 4-6 were in turn 4-fold, 2-fold, and 4-fold more
cytotoxic against KB cells in comparison with 1-3, respec-
tively. In addition, all three methylated derivatives showed
parallel trends of cell line specificity to those of the parent
compounds and had similar cytotoxicity against KB and
Although some reports regarding antimutagenic and
anticarcinogenic effects of chlorophyll and chlorophyllin
have been published, data demonstrating antitumor activ-
ity of chlorophyll-related compounds have rarely been
1
A9 cells, with ED50 values ranging from 0.2 to 0.30 µg/
2
reported. In 1981, Nakatani et al. reported that 13 -
mL; however, cytotoxicity in other cell lines varied widely.
For example, 4-6 showed varying cytotoxicity against
HCT-8 cells, with ED50 values of 0.79, 1.0, and 0.40 µg/
mL, respectively. Methylation did not affect the cytotoxicity
of 1 and 2 against SK-MEL-2 cells, whereas methylation
markedly changed the cytotoxicity of 3 against SK-MEL-2
2
hydroxy-(13 -R,S)-pheophytin a and -pheophytin b, isolated
from silkworm excreta,16 have in vitro cytostatic activity
against hepatoma tissue culture (HTC) cells at a dose of
3
3 µg/mL in culture medium, and six methyl ester deriva-
tives of pheophorbides showed cytotoxic effects toward HTC
cells at 33 or 66 µg/mL under direct photoirradiation. In
the dark, all these substances are only weak inhibitors of
cell multiplication. The authors explained the observed
phenomena as photodynamic destruction of HTC cells by
5
cells (ED for 3, 4 µg/mL; for 6, 0.84 µg/mL). In general, 6
showed potent and broad spectrum activity against all
seven cell lines tested, with ED50 values ranging from 0.27
to 1.9 µg/mL, while the parent acid 3 was not significantly
cytotoxic against the A549, HCT-8, and MCF-7 cell lines.
Compounds 1, 2, and 6 were also evaluated against drug-
resistant cell lines (Table 3). Cross-resistance usually is
seen against natural products in at least one of two drug-
resistant cell lines, as shown for taxol and etoposide (VP-
2
2,23
24
singlet oxygen
and superoxide radical. Also, Nakamura
et al.17 reported that pheophorbides (2 and 3) from leaves
of Neptunia oleracea exhibited photocytotoxicity against
Raji cells with photoirradiation.
In our study, 1 and 2, extracted from the herb C.
calamitosum, were identified as potent cytotoxic agents
1
6) (Table 3). However, the cytotoxicity of 1 and 2 was not

9
18 J ournal of Natural Products, 2001, Vol. 64, No. 7
Cheng et al.
c
against seven solid tumor cell lines without direct il-
lumination. We demonstrated that methyl ester derivatives
(s, C-18 ), 100.7 (d, C-R), 106.3 (d, C-â), 106.1 (s, C-γ), 93.7 (d,
a,b
C-δ) ( assignments may be interchangeable); FABMS m/z 639
+
1
6
[M + H] .
5
and 6, which were also studied by Nakatani et al.,
1
P h eop h or bid e a (2): dark green amorphous solid; H NMR
displayed much stronger cytotoxicity against tumor cell
lines tested, without direct photoirradiation. Previous
papers2 postulating the mechanism of action for cytotoxic
pheophorbides focus on photodynamic cytotoxicity involving
several steps: penetration and probable fixation of the
compounds in cellular membranes, photosensitized forma-
tion of highly reactive singlet oxygen and superoxide
radical, and damage to essential cellular components
including nucleic acids, proteins, and lipids. Our recent
findings suggest that the cytotoxic effect of chlorophyll-
related compounds can occur through mechanisms other
than phytodynamic action. Mechanism of action studies are
currently in progress.
+
data, see Table 1; FABMS m/z 593 [M + 1] .
(
10S)-Hyd r oxyp h eop h or bid e a (3): dark green amor-
5,26
1
+
phous solid; H NMR data, see Table 1; FABMS m/z 608 [M] .
Meth yla tion of 1-3. To ethyl ether solutions of 1 (20 mg),
(10 mg), or 3 (10 mg) was added excessive fresh diazo-
methane ethyl solution. Each solution was stood overnight,
and the solvent was removed in vacuo. The residue was
purified by a cellulose microcrystalline column eluting with
2
3
CHCl to give 4 (16 mg), 5 (8 mg), or 6 (8 mg), respectively.
The structures of all methyl esters were confirmed from the
presence of one extra methoxy group signal in the proton NMR
spectra (Table 1).
Meth yla tion of 6. To a dichloromethane solution of 6 (10
2 3
mg) were added 0.5 mL of methyl iodide and 10 mg of K CO ,
and the mixture was stirred at room temperature overnight.
After filtration and evaporation, the residue was purified by
Exp er im en ta l Section
Gen er a l Exp er im en ta l P r oced u r es. Melting points were
determined on a Fisher-J ohns melting apparatus and are
uncorrected. The optical rotation was measured on a J ASCO
DIP-1000 digital polarimeter. The UV-vis spectrum was
recorded on a Shimadzu UV-2101PC UV-vis scanning spec-
trophotometer. NMR spectra were measured on a Varian Inova
a microcrystalline cellulose column eluting with CHCl
(8 mg).
Extr a ction a n d Isola tion of 6 a n d 7. The dried leaves
15 kg) of C. cyrtophyllum were extracted with MeOH. The
MeOH extract was treated as described above to give an
n-hexane extract (IM1) and three CHCl extracts (IM2-1, -2,
and -3). Bioassay-directed fractionation was used to isolate the
cytotoxic principles. The first CHCl fraction (IM2-1) was
subjected to Si gel column chromatography using CHCl
3
to give
4
(
3
5
00 MHz spectrometer with TMS as internal standard.
3
Chemical shifts are reported in δ (ppm). FABMS were mea-
sured on a VG analytical VG-70E mass spectrometer. Pre-
coated Si gel plates (Kieselgel 60 F254 0.25 mm, Merck) were
used in TLC analysis for monitoring isolation and separation.
Chlorophyll was obtained from Carl Roth GmbH, Germany,
and chlorophyllin from Spectrum, Inc., Gardena, CA.
P la n t Ma ter ia l. In September, 1997, leaves and stems of
Clerodendrum calamitosum were collected in Taipei, Taiwan,
and leaves of Clerodendrum cyrtophyllum were collected in
Cha-Yee, Taiwan. Dr. Zuei-Ching Chen, Department and
Graduate Institute of Botany, National Taiwan University,
Taipei, Taiwan, identified both species. Voucher specimens are
deposited at the Department of Biochemistry, Taipei Medical
College.
Extr a ction a n d Isola tion of 1-3. The freshly collected
leaves and stems (4 kg) of C. calamitosum were air-dried,
ground, and extracted repeatedly with MeOH. The combined
MeOH extracts were evaporated in vacuo to yield a dark
greenish syrup. The concentrated syrup was mixed with Celite,
dried in a vacuum oven, and packed on a Si gel column, then
3
-
MeOH (100:1 f 1:1) and finally MeOH as eluent. The eluted
fractions were monitored by TLC and combined into 10
fractions. The active fractions were chromatographed repeat-
edly over Si gel eluting with CHCl -MeOH (100:1 f 1:1),
3
MeOH, benzene-EtOAc (10:1 f 3:1), and acetone to give 6
(107 mg) and 7 (57 mg).
Meth yl (10S)-h yd r oxyp h eop h or bid e a (6): dark green
1
13
amorphous solid; H NMR data, see Table 1; C NMR δ 131.7
(s, C-1), 12.0 (q, C-1a), 136.3 (s, C-2), 128.9 (d, C-2a), 122.7 (t,
C-2b), 136.08 (s, C-3 ), 11.0 (q, C-3a), 145.0 (s, C-4), 19.3 (t,
C-4a), 17.3 (q, C-4b), 129.3 (s, C-5 ), 12.2 (q, C-5a), 126.9 (s,
C-6 ), 51.8 (d, C-7), 31.4 (t, C-7a), 31.1 (t, C-7b), 174.0 (s, C-7c),
a
b
b
51.7 (q, C-7d), 50.3 (d, C-8), 22.6 (q, C-8a), 191.9 (s, C-9), 89.0
c
(s, C-10), 172.3 (s, C-10a ), 53.4 (q, C-10b), 142.0 (s, C-11),
a
136.11 (s, C-12 ), 155.2 (s, C-13), 150.9 (s, C-14), 137.7 (s, C-15),
c
149.8 (s, C-16), 162.4 (s, C-17), 172.8 (s, C-18 ), 97.8 (d, C-R),
a-c
104.0 (d, C-â), 107.6 (s, C-γ), 93.6 (d, C-δ) ( assignments may
+
be interchangeable); FABMS m/z 622 [M ].
(10S)-Hyd r oxyp h eop h ytin a (7): dark green amorphous
1
eluted successively with n-hexane, CHCl
Isolation of cytotoxic principles from the MeOH extract was
guided by bioassay-directed fractionation. Because the CHCl
3
, EtOAc, and MeOH.
solid; H NMR data, see Table 1 for pheophorbide skeleton;
for phytyl protons, δ 4.57 (2H, dd, J ) 2, 7 Hz, H-P1), 5.23
3
(1H, dt, J ) 2, 7 Hz, H-P2), 1.59 (3H, s, H-P3a), 0.86 (3H, s,
a
a
fraction showed more potent cytotoxicity than the others in
the KB cell assay, this fraction was subjected to Si gel column
H-P7a ), 0.84 (3H, s, H-P11a ), 1.50 (1H, sept, J ) 7 Hz,
b
H-P15), 0.79 (1H, d, J ) 7 Hz, H-P15a ), 0.81 (1H, dd, J ) 7
b
13
chromatography using CHCl
3
-MeOH (100:1f1:1) and MeOH
Hz, H-P16 ); C NMR δ 131.7 (s, C-1), 12.0 (q, C-1a), 136.3
c
as eluents. The eluted fractions were monitored by TLC and
combined into 13 pooled fractions. The active fractions were
chromatographed repeatedly over Si gel, eluting with EtOAc,
(s, C-2), 129.0 (d, C-2a), 122.7 (t, C-2b), 136.2 (s, C-3 ), 11.1 (q,
C-3a), 145.0 (s, C-4), 19.3 (t, C-4a), 17.3 (q, C-4b), 127.0 (s,
d
d
C-5 ), 12.2 (q, C-5a), 129.3 (s, C-6 ), 51.8 (d, C-7), 31.7 (t, C-7a),
e
EtOAc-MeOH (10:1f1:1), CHCl
then MeOH, and over microcrystalline cellulose eluting with
CHCl to give active principles 1 (45 mg) and 2 (140 mg).
3
, CHCl
3
-MeOH (10:1f1:1),
31.2 (t, C-7b), 173.5 (s, C-7c), 50.4 (d, C-8), 22.7 (q, C-8a ), 191.9
(s, C-9), 89.0 (s, C-10), 172.3 (s, C-10a), 53.4 (q, C-10b), 142.0
f
c
3
(s, C-11 ), 136.1 (s, C-12 ), 155.2 (s, C-13), 150.9 (s, C-14), 137.8
(s, C-15), 149.9 (s, C-16), 162.5 (s, C-17), 172.8 (s, C-18), 97.8
(d, C-R), 104.1 (d, C-â), 107.7 (s, C-γ), 93.6 (d, C-δ), for phytyl
Compound 3 (265 mg) was obtained from the EtOAc fraction
by repeated cellulose microcrystalline chromatography eluting
f
with CHCl
3
.
carbons, 61.5 (t, C-P1), 118.0 (d, C-P2), 142.7 (s, C-P3 ), 16.3
P u r p u r in 7 d im eth yl ester (1): dark brown amorphous
(q, C-P3a), 39.8 (t, C-P4), 25.0 (t, C-P5), 36.7 (t, C-P6), 32.6
2
4
g
h
solid; mp 196-198 °C (softened); [R]
UV-vis (CHCl ) λmax (log ꢀ) 681 (4.25), 548 (3.91), 506 (3.81),
30-330 (flat top, 4.35), 279 (4.04) nm; H NMR data, see
D
+76.7° (c 0.07, CHCl
3
);
(d, C-P7), 19.71 (q, C-P7a ), 37.4 (t, C-P8 ), 24.4 (t, C-P9), 37.33
h
g
3
(t, C-P10 ), 32.8 (d, C-P11), 19.65 (q, C-P11a ), 37.27 (t,
C-P12 ), 24.8 (t, C-P13), 39.4 (t, C-P14), 27.9 (d, C-P15), 22.60
(q, C-P15a ), 22.66 (q, C-P16 ) ( assignments may be inter-
changeable); FABMS m/z 886 [M ].
1
h
4
1
3
e
e
a-h
Table 1; C NMR δ 131.4 (s, C-1), 11.9 (q, C-1a), 136.3 (s, C-2),
28.7 (d, C-2a), 122.6 (t, C-2b), 135.6 (s, C-3 ), 11.0 (q, C-3a),
45.7 (s, C-4), 19.4 (t, C-4a), 17.5 (q, C-4b), 129.6 (s, C-5), 12.9
a
+
1
1
Gr ow th In h ibition Assa y. The SRB assay previously
described by Skehan et al.2
7,28
was adapted to the measure-
(
q, C-5a), 119.4 (s, C-6), 52.5 (d, C-7), 31.3 (t, C-7a), 30.8 (t,
b
C-7b), 178.0 (s, C-7c ), 49.6 (d, C-8), 23.0 (q, C-8a), 167.7 (s,
C-9), 53.2 (s, C-9a), 186.4 (s, C-10), 166.5 (s, C-10a), 51.9 (q,
C-10b), 142.7 (s, C-11), 135.8 (s, C-12 ), 156.1 (s, C-13), 149.0
ment of cellular viability, but with some modification. This
assay is based on the spectrophotometric determination of
sulforhodamine B (SRB), a pink aminoxanthine dye, bound to
cellular protein.29 Drug stock solutions were prepared in
a
(s, C-14), 138.6 (s, C-15), 136.6 (s, C-16), 164.0 (s, C-17), 174.3

Cytotoxic Pheophorbide-Related Compounds
J ournal of Natural Products, 2001, Vol. 64, No. 7 919
DMSO and stored at -70 °C. Upon dilution into culture
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), renal cancer (CAKI-1), breast adenocarcinoma (MCF-7),
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HEPES, 0.2% (w/v) sodium bicarbonate, 10% (v/v) fetal bovine
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serum, and 100 µg/mL kanamycin at 37 °C in a humidified
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atmosphere containing 5% CO
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suspensions were seeded in 96-well mictrotiter plates at
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ties varied among the cell lines to ensure a final absorbance
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units. Tumor cells were incubated at 37 °C for 72 h in the
presence of various concentrations of drugs from DMSO-
diluted stock. Cultures were monitored briefly at daily inter-
vals by microscopic examination. Cytotoxic action generally
developed after 1 day exposure and resulted in crenulation and
shrinkage of cells. After 3 days, attached cells were fixed with
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247.
(
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(
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1982, 46, 2183-2193.
MO). The absorbance at 562 nm was measured using an
automated microculture plate reader (Molecular Devices,
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values, the drug concentration resulting in 50% growth inhibi-
tion, were interpolated from dose-response data. Each test
was performed in triplicate, and absorbance readings varied
no more than 5%. ED50 values determined in independent tests
varied no more than 30%.
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Ack n ow led gm en t. We thank Mr. and Mrs. Fong-Lan Lu
for kindly providing plant material cultivated in their yard.
We also thank Professor Zuei-Ching Chen for his kind iden-
tification of the plant material. This study was supported by
grant CA-17625 from the National Cancer Institute awarded
to K.H.L.
(
26) Yamada, K.; Nakamura, N. Eiyo to Shokuryo 1977, 30, 307-312.
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(
Refer en ces a n d Notes
(
1) For Part 207, see: VanVliet, D. S.; Tachibana, Y.; Bastow, K. F.;
Huang, E. S.; Lee, K. H. J . Med. Chem. 2001, 44, 1422-1428.
NP000595B