Antiproliferative Compounds from Cleistanthus boivinianus from the Madagascar Dry Forest

Article abstract of DOI:10.1021/np501020m

The two new lignans 3α-O-(β-d-glucopyranosyl)desoxypodophyllotoxin (1) and 4-O-(β-d-glucopyranosyl)dehydropodophyllotoxin (2) were isolated from Cleistanthus boivinianus, together with the known lignans deoxypicropodophyllotoxin (3), (±)-β-apopicropodophy

Full text of DOI:10.1021/np501020m

Article
pubs.acs.org/jnp
Antiproliferative Compounds from Cleistanthus boivinianus from the
Madagascar Dry Forest1
Yixi Liu,† Kelly Young,† L. Harinantenaina Rakotondraibe,†,# Peggy J. Brodie,† Jessica D. Wiley,‡
Maria B. Cassera,‡ Martin W. Callmander∥,§ R. Rakotondrajaona,⊥ Etienne Rakotobe,⊥
∥
Vincent E. Rasamison,⊥ Karen TenDyke, Yongchun Shen, and David G. I. Kingston*,†
‡
†Department of Chemistry and the Virginia Tech Center for Drug Discovery and Department of Biochemistry and Virginia Tech
Center for Drug Discovery, Virginia Tech, Blacksburg, Virginia 24061, United States
§Missouri Botanical Garden, P.O. Box 299, St. Louis, Missouri 63166, United States
⊥Centre National d’Application des Recherches Pharmaceutiques, B.P. 702, Antananarivo 101, Madagascar
∥Eisai Inc., 4 Corporate Drive, Andover, Massachusetts 01810, United States
S
* Supporting Information
ABSTRACT: The two new lignans 3α-O-(β-D -
glucopyranosyl)desoxypodophyllotoxin (1) and 4-O-(β-D-
glucopyranosyl)dehydropodophyllotoxin (2) were isolated
from Cleistanthus boivinianus, together with the known lignans
deoxypicropodophyllotoxin (3), ( )-β-apopicropodophyllin
(4), (−)-desoxypodophyllotoxin (5), (−)-yatein (6), and β-
peltatin-5-O-β-D-glucopyranoside (7). The structures of all
compounds were characterized by spectroscopic techniques.
Compounds 1, 4, and 5 showed potent antiproliferative
activities against the A2780 ovarian cancer cell line, with IC50
values of 33.0 3.6, 63.1 6.7, and 230 1 nM, respectively. Compounds 2 and 7 showed only modest A2780 activities, with
IC50 values of 2.1 0.3 and 4.9 0.1 μM, respectively, while compounds 3 and 6 had IC50 values of >10 μM. Compound 1 also
had potent antiproliferative activity against the HCT-116 human colon carcinoma cell line, with an IC50 value of 20.5 nM, and
compound 4 exhibited modest antiproliferative activity against the A2058 human caucasian metastatic melanoma and MES-SA
human uterine sarcoma cell lines, with IC50 values of 4.6 and 4.0 μM, respectively.
n the course of work as part of the Madagascar International
Cooperative Biodiversity Group (ICBG) program,2,3 an
ethanol extract of the stems of Cleistanthus boivinianus (Baill.)
chromatography on Sephadex LH-20 followed by reversed-
phase solid-phase extraction (SPE). The most active fractions
from the C18 SPE were subjected to C18 HPLC to yield
compounds 3, 5, and 6 and three semipure active fractions.
Further purification of these fractions by silica gel or diol HPLC
furnished compounds 1, 2, 4, and 7.
I
Mull. Arg. (Phyllanthaceae) was found to have moderate
̈
antiproliferative activity against the A2780 ovarian cancer cell
line (IC50 4.5 μg/mL) and was thus selected for evaluation of
the presence of novel antiproliferative agents. The genus
Cleistanthus is native to Africa, India, and Australia4 and
comprises approximately 140 species, but only five of these
have been investigated chemically: C. collinus, C. patulus, C.
schlechteri var. schlechteri, C. gracilis,4 and C. indochinensis.5
Arylnaphthalide lignans are major constituents of the genus,
while arytetralin, furofuranoid, and dibenzylbutane lignans and
terpenoids have also been isolated from them and are known
for their cytotoxic properties.4−12
Compound 1 was isolated as a white solid with the molecular
1
formula C28H32O13 based its HRESIMS data. Its H NMR
spectrum contained three singlet aromatic signals together with
signals for a methylenedioxy group and three aromatic methoxy
groups. Signals suggestive of a sugar unit were also present.
Analysis of COSY, HSQC, and HMBC data assigned the sugar
as a β-glucopyranosyl unit. The remaining aliphatic signals were
assigned by HSQC and COSY analysis to two isolated
methylene groups, one of which was oxygenated, and to two
adjacent methines. The above data indicated that 1 has an
aryltetralin lignan skeleton. This was supported by the HMBC
data (Figure 1), with cross-peaks from H-1 to C-8 and to C-2′/
C-6′, supporting the linkage of C-1 to both the A- and B-rings.
HMBC cross-peaks between H-5 and C-4 and between H2-4
RESULTS AND DISCUSSION
■
Dereplication of an active EtOAc-soluble fraction obtained
from liquid−liquid partition of the extract (100 mg) as
previously described13 indicated that it contained at least one
new bioactive compound, so a larger sample was investigated.
Fractionation of this extract yielded an antiproliferative EtOAc
fraction, which was further subjected to size-exclusion column
Received: December 17, 2014
© XXXX American Chemical Society and
American Society of Pharmacognosy
A
DOI: 10.1021/np501020m
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The 2.3 Hz J-value between H-1 and H-2 was suggestive of a
cis-relationship, which was confirmed by the NOESY
interaction of H-1 and H-2. The trans-configuration of the D-
ring was determined by the NOESY correlation between H-2
and H2-3a at δH 4.58. The 1R absolute configuration was
established by the electronic circular dichroism (ECD)
spectrum of 1, which gave a positive Cotton effect at 288 nm
(Δε +1.02), consistent with the positive effect for other 1R
lignans.14−19 These data combined with the relative config-
uration determined above allowed the R and S configurations to
be assigned for the C-2 and C-3 chiral centers, respectively.
Mild acid hydrolysis of 1 gave (+)-β-apopicropodophyllin
(4a) and a sugar that was identified as D-glucose by TLC and
optical rotation comparison with a standard sample. Placement
of the glucose unit at C-3 was evident from the HMBC
correlation between the anomeric proton at δH 4.18 and the
oxygenated quaternary carbon at δC 83.6 (C-3). The complete
assignment of all protons and carbons of 1 (Table 1) was
accomplished by analysis of the COSY, HSQC, HMBC, and
NOESY spectra. Thus, compound 1 was assigned as 3α-O-(β-D-
glucopyranosyl)desoxypodophyllotoxin.
As indicated by the experiment described above, compound
1 is prone to conversion to its α,β-unsaturated derivative 4a,
and a sample of 1 left in MeOH−CHCl3 for a few days
underwent decomposition, most likely caused by traces of HCl
in the CHCl3. Attempted enzymatic hydrolysis of 1 did not
proceed under normal conditions, so the aglycone could not be
isolated.
Compound 2 had the molecular formula of C28H29O13, based
1
on its HRESIMS data. Its H NMR spectrum was similar to
that obtained for 1, and its UV absorption maxima at 260, 315,
and 351 nm indicated the presence of a naphthalene nucleus,20
1
suggesting that 2 is an arylnaphthalide lignan. The H NMR
spectrum of 2 exhibited signals for four aromatic protons at δH
8.04 and 6.95 (s, each 1H, H-5 and H-8) and 6.59 (s, 2H, H-2′
and H-5′) and for an oxymethylene group at δH 5.75 and 5.53
(J = 15.4 Hz, each 1H, H2-3a), but lacked the signals for the
two methine protons at C-1 and C-2 and the methylene
protons at C-4 observed in 1. These facts confirmed the
presence of a naphthalene unit in 2. In the HMBC spectrum,
the correlation from H-5 (δH 8.04) and H2-3a (δH 5.75 and
5.53) to δC 144.3 (C-4) confirmed the linkage of C-4 to both
the B- and D-rings, while the correlations of δH 6.59 (s, 1H, H-
2′ and/or H-6′) and δH 6.95 (H-8) to δC 128.6 (C-1)
connected the A- and B-rings to C-1. A comparison of the
NMR spectra of 2 with those of dehydropodophyllotoxin (2a)
suggested that compound 2 is a glycosylated derivative of 2a.21
The HMBC correlation between the anomeric proton signal at
δH 4.89 (H-1″) and δC 144.3 (C-4) confirmed that the sugar is
located at C-4 of the aglycone. Acid hydrolysis of 2 gave
dehydropodophyllotoxin (2a) as the aglycon and a sugar that
Figure 1. Key HMBC and NOESY correlations of 1 and key HMBC
correlations of 2.
1
was identified as D-glucose by H NMR, TLC, and optical
rotation comparison with a standard sample. The complete
assignments of all protons and carbons of 2 (Table 1) were
accomplished by analysis of the HSQC and HMBC spectra.
Compound 2 was thus assigned as 4-O-(β-D-glucopyranosyl)-
dehydropodophyllotoxin.
and C-8a established the C-4a/C-4 connectivity, and the
presence of the lactone ring was confirmed by cross-peaks
between the methylene protons H2-3a and the ester carbonyl
carbon C-2a. Correlations of the methylenedioxy protons with
C-6 and C-7 indicated the position of the methylenedioxy
group. Finally, the positions of the methoxy groups were
assigned based on cross-peaks from the two methoxy signals at
δH 3.75 to C-3′ and C-5′ and for the other methoxy signal to C-
4′.
The five known aryltetralin lignans deoxypicropodophyllo-
toxin (3),22 ( )-β-apopicropodophyllin (4),20,23,24 (−)-des-
oxypodophyllotoxin (5),25 (−)-yatein (6),26 and β-peltatin-5-
O-β-D-glucopyranoside (7)27 were also isolated. Their
1
structures were determined by comparison of their H NMR
spectroscopic, mass spectrometric, and optical rotation values
B
DOI: 10.1021/np501020m
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1
Table 1. H NMR (500 MHz, CD3OD) and 13C NMR (125
and some were evaluated in other cell lines and for antimalarial
activity (Table 2). Compound 1 showed the highest
MHz, CD3OD) Chemical Shift Data (δ, ppm) for
Compounds 1 and 2
antiproliferative activity (IC50 33.0
A2780 cell line, followed by 4 (63.1 6.7 nM) and 5 (230
3.6 nM) against the
1
1
2
nM). The potency of 1 is similar to that of the anticancer drug
paclitaxel, which has an IC50 value of 73 nM in this assay.
Compounds 2 and 7 showed only modest antiproliferative
position
δH (J in Hz)
δC (DEPT)
δH (J in Hz)
δC (DEPT)
1
4.47 d (2.3)
3.76 d (2.3)
47.1 (CH)
52.8 (CH)
178.4 (C)
128.6 (C)
119.7 (C)
170.4 (C)
67.3 (CH2)
activities, with IC50 values of 2.1
0.3 and 4.9
0.1 μM,
2
respectively. The glycosylated compound 1 had slightly
improved activity compared with the racemic compound 4.
The unsaturated D-ring of 4 increased the activity about 4-fold
compared with the trans dihydro D-ring analogue 5, while the
cis D-ring analogue 3 was much less potent than 4, consistent
with previous studies indicating the significance of the trans-
fused lactone for activity.28,29 A glucose moiety at C-5 or C-4
and the aromatization of the C-ring reduced activity, as shown
by the fact that compounds 2 and 7 were about 10- and 20-fold
less potent than 5, respectively. Furthermore, although good
antiproliferative activity has been observed in other cell lines for
(−)-yatein (6),20 it was only weakly active against the A2780
cell line. Compound 1 also displayed potent antiproliferative
activity against the HCT-116 human colon carcinoma cell line,
with an IC50 value of 20.5 nM, and weak antimalarial activity
against Plasmodium falciparum with an IC50 value of 12.6 3.2
μM. Compound 4 displayed moderate antiproliferative activity
against A2058 human caucasian metastatic melanoma and
MES-SA human uterine sarcoma cells, with IC50 values of 4.6
and 4.0 μM, respectively.
2a
3a
4.58 d (10.2)
3.98 d (10.2)
76.9 (CH2) 5.75 d
(15.3)
5.53 d
(15.3)
3
4
83.6 (C)
130.4 (C)
144.3 (C)
3.35 d (15.1)
2.91 d (15.1)
37.0 (CH2)
4a
5
127.3 (C)
108.9 (CH)
147.3 (C)
147.3 (C)
105.7 (CH)
130.5 (C)
130.5 (C)
104.9 (CH)
154.4 (C)
136.2 (C)
154.4 (C)
104.9 (CH)
136.5 (C)
98.6 (CH)
149.6 (C)
149.6 (C)
102.8 (CH)
131.7 (C)
131.1 (C)
107.6 (CH)
152.9 (C)
137.2 (C)
152.9 (C)
107.6 (CH)
101.6 (CH2)
55.4
6.78 s
6.72 s
6.55 s
8.04 s
6.95 s
6.59 s
6.59 s
6
7
8
8a
1′
2′
3′
4′
5′
6′
6.55 s
OCH2O 5.94 s
101.1 (CH2) 6.11 s
In summary, compound 1 is a new lignan with potent
antiproliferative activity against the A2780 cell line. It is also the
first reported C-3 substituted podophyllotoxin analogue. It
would be an attractive substrate for further studies to explore its
mechanism of action were it not for its lability under acidic
conditions, which suggests that it would not be stable enough
for drug use.
OMe-3′
OMe-4′
OMe-5′
3.79 s
3.75 s
3.79 s
59.8
55.3
59.8
3.82 s
3.88 s
3.82 s
59.9
55.4
c
Glc
a
1″
2″
4.18 d (7.7)
99.2 (CH)
73.6 (CH)
4.89
104.8 (CH)
74.2 (CH)
3.14−3.09
3.63 dd (9.0,
7.8)
3″
4″
5″
3.24−3.17
3.24−3.17
3.02 ddd (9.2,
8.5, 2.4)
76.4 (CH)
69.8 (CH)
76.7 (CH)
3.48 t (9.0)
3.44 m
76.7 (CH)
70.1 (CH)
77.0 (CH)
EXPERIMENTAL SECTION
■
a
3.33
General Experimental Procedures. IR and UV spectra were
measured on MIDAC M-series FTIR and Shimadzu UV-1201
spectrophotometers, respectively. 1H and 13C NMR spectra were
recorded on a Bruker Avance 500 spectrometer in CD3OD (with
CD3OD as reference) and CDCl3 (with CDCl3 as reference). Mass
spectra were obtained on an Agilent 6220 mass spectrometer. Open
column chromatography was performed using Sephadex LH-20, and
solid-phase extraction was performed using C18 cartridges. Semi-
preparative HPLC was performed using Shimadzu LC-10AT pumps
coupled with a semipreparative Phenomenex C18 column (5 μm, 250
× 10 mm), a Shimadzu SPD M10A diode array detector, and a SCL-
10A system controller. All isolated compounds were purified to 95%
purity or better, as judged by HPLC (both UV and ELSD detection)
before determining bioactivity.
b
6″
3.79 dd (11.8,
2.4)
61.3 (CH2) 3.90
61.3 (CH2)
3.59 dd (11.8,
2.4)
3.71 dd
(11.9, 6.0)
a
Peaks covered by the CH3OH peak; chemical shift assigned from
HSQC and HMBC spectra. Peak covered by an OMe peak. Glc: β-
D-glucopyranosyl.
b
c
with the data reported in the literature, except for the case of β-
apopicropodophyllin (4), which was optically inactive. The
isolation of racemic 4 is noteworthy, since all previous isolates
have been of the dextrorotatory isomer, and indicates that it is
not formed by elimination of glucose from 1.
All isolated compounds were evaluated for their antiprolifer-
ative activity against the A2780 human ovarian cancer cell line,
Plant Material. Leaves of Cleistanthus boivinianus (collection:
Stephan Rakotonandrasana et al. 1036) were obtained at an elevation
́
of 51 m from a 3 m tall tree with yellow flowers. Collection was made
5 km northeast of the village of Marivorahona, PK 123,
Table 2. Antiproliferative and Antimalarial Activities of the Isolated Compounds (IC50 Values, μM)
cell line
A2780
paclitaxel
1
2
3
4
5
6
7
0.073 0.015
0.033 0.0036
0.0205
2.1 0.3
NT
>10
NT
NT
NT
NT
0.063 0.0067
0.23 0.001
>10
NT
NT
NT
NT
4.9 0.1
NT
HCT-116
A2058
NT
NT
NT
NT
NT
4.6
NT
NT
NT
NT
NT
NT
NT
MES-SA
NT
NT
4.0
NT
P. falciparum Dd2
12.6 3.2
NT
NT
NT
C
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Journal of Natural Products
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Andohanantsohihy, in a mosaic of dry forest and savanna trees;
coordinates 13°06′37″ S 049°09′39″ E. Collection was made by
́
Stephan Rakotonandrasana with assistance from R. Randrianaivo, R.
( )-β-Apopicropodophyllin (4): amorphous powder; [α]25 0 (c
D
0.5, CHCl3). The 1H and 13C NMR spectra were identical with
literature data.20,23,24
Acid Hydrolysis of Compounds 1 and 2. Compound 1 (2 mg)
was dissolved in dilute citrate−phosphate buffer (pH 7.0, 4 mL), and
β-D-glucosidase (4 mg) was added. The mixture was incubated for 2
weeks at 37 °C; no reaction was observed. The reaction mixture was
then adjusted to pH 5.0 with dilute HCl and stirred at 37 °C
overnight. The mixture was then briefly heated to boiling and extracted
with EtOAc (3 × 10 mL), and both the organic and the water layers
were evaporated to dryness under reduced pressure. The structure of
the white powder (1.2 mg) derived from the organic layer was
Rakotonandrasana, C. Claude, V. Benjara, and M. Modeste. Duplicate
voucher specimens are deposited at the Centre National d’Application
des Recherches Pharmaceutiques (CNARP), the Herbarium of the
Parc Botanique et Zoologique de Tsimbazaza, Antananarivo,
Madagascar (TAN), the Missouri Botanical Garden, St. Louis,
Missouri (MO), and the Museum National d’Histoire Naturelle in
Paris, France (P).
Extraction and Isolation. A ground sample of C. boivinianus
leaves (250 g) was extracted with EtOH at room temperature to yield
33.4 g of crude EtOH extract designated MG4031. A total of 5.88 g of
this extract was made available to Virginia Tech. An active EtOAc-
soluble fraction obtained from liquid−liquid partition of the extract
(100 mg) was subjected to dereplication studies using size-exclusion
chromatography, reversed-phase HPLC coupled with bioassay, high-
resolution ESIMS, 1H NMR spectroscopy, and a database search using
the online Dictionary of Natural Products. The results indicated the
extract to contain at least one new bioactive compound, and so a 3.0 g
sample was investigated. The crude EtOH extract was dissolved in 90%
MeOH(aq) (300 mL) and extracted with hexanes (3 × 200 mL).
Evaporation of the hexane-soluble fraction afforded 271 mg of residue.
The 90% MeOH(aq) layer was then evaporated, suspended in H2O
(300 mL), and extracted with EtOAc (3 × 200 mL) to yield 640 mg of
an EtOAc-soluble fraction with an IC50 value of 0.19 μg/mL. The
EtOAc fraction was subjected to Sephadex LH-20 open column
chromatography (CH2Cl2−MeOH, 1:1) to give six fractions. The
most active fraction, Fr 3 (IC50 0.11 μg/mL), was then divided into
three subfractions by C18 solid-phase extraction by using 40%
MeOH(aq) (Fr 3-1), 70% MeOH(aq) (Fr 3-2), and 100% MeOH
(Fr 3-3). Further purification of the most active subfraction, Fr 3-3
(IC50 0.11 μg/mL), by HPLC on a C18 column with a solvent gradient
of H2O−CH3CN, from 60:40 to 50:50 from 0 to 17 min, to 40:60
from 17 to 22 min, to 40:60 from 22 to 27 min, and ending with 100%
CH3CN from 27 to 35 min, yielded compounds 3 (1.2 mg, tR 23.5
min), 5 (1.5 mg, tR 24.5 min), and 6 (2.1 mg, tR 26.5 min). Purification
of the subfraction Fr 3-2 (IC50 0.1 μg/mL) by C18 HPLC furnished
eight fractions (solvent gradient of H2O−CH3CN, from 70:30 to
62:38 from 0 to 15 min, and ending with 100% CH3CN from 15 to 24
min), among which the third (tR 12 min), sixth (tR 14.5 min), and
seventh (tR 15.5 min) fractions were active, with IC50 values of 1.5, 0.1,
and 1.2 μg/mL, respectively. Further purification of the third fraction
by HPLC on a silica gel column with a solvent gradient of CHCl3−
MeOH, from 95:5 to 90:10 from 0 to 5 min, to 88:12 from 5 to 12
min, and ending with a 100% MeOH wash from 13 to 20 min, yielded
compound 7 (2.8 mg, tR 13.5 min). Further purification of the sixth
fraction by HPLC on a silica gel column with a solvent gradient of
CHCl3−MeOH, from 95:5 to 90:10 from 0 to 5 min, to 80:20 from 5
to 10 min, to 70:30 from 10 to 15 min, and ending with a 100%
1
determined to be 4a by its H NMR spectrum and optical rotation
([α]25 +86, c 1.2, CHCl3); lit.20 +96.8). The semisolid carbohydrate
D
from the water layer (0.5 mg) was dissolved in 2 mL of water and kept
overnight before determination of its optical rotation. Compound 2 (1
mg) was dissolved in dilute HCl (pH 5.0, 2 mL) and stirred at 37 °C
overnight. The reaction mixture was extracted with EtOAc (3 × 10
mL), and both the organic and the water layers were evaporated to
dryness under reduced pressure. The structure of the dried powder
from the organic layer was determined to be 2a by its 1H NMR
spectrum. TLC of the sugar from both 1 and 2 on a silica gel plate with
CHCl3−MeOH−H2O (15:6:1) showed that it had an identical Rf
value (0.23) to glucose. The isolated glucose had [α]25 (c 0.5, H2O)
D
values of +41.9 and +43.2, respectively.
Antiproliferative Bioassay. The A2780 ovarian cancer cell line
antiproliferative bioassay was performed at Virginia Tech as previously
reported.30,31 The A2780 cell line is a drug-sensitive ovarian cancer cell
line.32 Paclitaxel was used as the positive control and gave an IC50
value of 0.073 0.015 μM.
Antimalarial Bioassay. The effect of each compound on parasite
growth of the Dd2 strain of P. falciparum was measured after 72 h
treatment using the malaria SYBR green I-based fluorescence assay as
described previously.2,33,34 Artemisinin was used as the positive
control, and it had an IC50 value of 8 1 nM.
ASSOCIATED CONTENT
■
S
* Supporting Information
1H NMR spectra of compounds 1−7. The Supporting
Information is available free of charge on the ACS Publications
website at DOI: 10.1021/np501020m.
AUTHOR INFORMATION
Corresponding Author
*Tel: +1-540-231-6570. Fax: +1-540-231-3255. E-mail:
dkingston@vt.edu (D. G. I. Kingston).
■
Present Address
#College of Pharmacy, Division of Medicinal Chemistry and
Pharmacognosy, The Ohio State University, Columbus, Ohio
43210, United States.
MeOH wash from 16 to 25 min, yielded compounds 4 (5 mg, tR
6
Notes
min) and 1 (19 mg, tR 14 min). Further purification of the seventh
fraction by HPLC on a diol column with a solvent gradient of CHCl3−
MeOH, from 100:0 to 95:5 from 0 to 5 min, to 90:10 from 5 to 15
min, to 87.5:12.5 from 15 to 20 min, and ending with a 100% MeOH
wash from 20 to 25 min, yielded compound 2 (3 mg, tR 22.5 min).
3α-O-(β-D-Glucopyranosyl)desoxypodophyllotoxin (1): amor-
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This project was supported by the Fogarty International
Center, the National Cancer Institute, the National Institute of
Allergy and Infectious Diseases, the National Institute of
Mental Health, the National Institute on Drug Abuse, the
National Heart Lung and Blood Institute, the National Center
for Complementary and Alternative Medicine, the Office of
Dietary Supplements, the National Institute of General Medical
Sciences, the Biological Sciences Directorate of the National
Science Foundation, and the Office of Biological and
Environmental Research of the U.S. Department of Energy
under Cooperative Agreement U01 TW00313 with the
International Cooperative Biodiversity Groups. This project
phous powder; [α]25 +120 (c 0.1, MeOH); CD (c 0.031, MeOH)
D
λmax (Δε) 210 (+22.59), 234 (+5.98), 248 (−1.33), 288 (+1.02) nm;
UV (MeOH) λmax(log ε) 291 (3.19), 245 (3.7), 208 (4.4) nm; IR
(film) νmax 3402, 2912, 1760, 1590, 1053 cm−1; 1H and 13C NMR data,
see Table 1; HRESIMS m/z [M + Na]+ 599.1741 (calcd for
+
C28H32NaO13 599.1735).
,
4-O-(β-D-Glucopyranosyl)dehydropodophyllotoxin (2): amor-
phous powder; [α]25 −14 (c 0.22, MeOH); UV (MeOH) λmax (log
D
ε) 351 (3.69), 315 (4.0), 260 (4.7) nm; IR (film) νmax 3400, 2920,
1734, 1645, 1070 cm−1; 1H and 13C NMR data, see Table 1;
HRESIMS m/z [M + H]+ 573.1635 (calcd for C28H29O13+, 599.1603).
D
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Journal of Natural Products
Article
(20) Novelo, M.; Cruz, J. G.; Hernandez, L.; Pereda-Miranda, R.;
Chai, H.; Mar, W.; Pezzuto, J. M. J. Nat. Prod. 1993, 56, 1728−1736.
(21) Atta-ur-Rahman; Ashraf, M.; Choudhary, M. I.; Habib-ur-
Rehman; Kazmi, M. H. Phytochemistry 1995, 40, 427−431.
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was also supported by the National Research Initiative of the
Cooperative State Research, Education and Extension Service,
USDA, Grant 2008-35621-04732. J.D.W. was supported by
NSF S-STEM award DUE-0850198. These supports are
gratefully acknowledged. Work at Virginia Tech was supported
by the National Science Foundation under Grant CHE-
0722638 for the purchase of the Agilent 6220 mass
spectrometer. We thank Mr. B. Bebout for obtaining the
mass spectra. Fieldwork essential for this project was conducted
under a collaborative agreement between the Missouri
Botanical Garden and the Parc Botanique et Zoologique de
Tsimbazaza and a multilateral agreement between the ICBG
partners, including the Centre National d’Application des
Recherches Pharmaceutiques. We thank R. Randrianaivo, R.
Rakotonandrasana, C. Claude, V. Benjara, and M. Modeste for
assistance with plant collection, and we gratefully acknowledge
courtesies extended by the Government of Madagascar
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DOI: 10.1021/np501020m
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