Bioactive flavaglines and other constituents isolated from Aglaia perviridis

Article abstract of DOI:10.1021/np3007588

Eight new compounds, including two cyclopenta[b]benzopyran derivatives (1, 2), two cyclopenta[b]benzofuran derivatives (3, 4), three cycloartane triterpenoids (5-7), and an apocarotenoid (8), together with 16 known compounds, were isolated from the chloroform-soluble partitions of separate methanol extracts of a combination of the fruits, leaves, and twigs and of the roots of Aglaia perviridis collected in Vietnam. Isolation work was monitored using human colon cancer cells (HT-29) and facilitated with an LC/MS dereplication procedure. The structures of the new compounds (1-8) were determined on the basis of spectroscopic data interpretation. The Mosher ester method was employed to determine the absolute configurations of 5-7, and the absolute configuration of the 9,10-diol unit of compound 8 was established by a dimolybdenum tetraacetate [Mo₂(AcO)₄] induced circular dichroism procedure. Seven known rocaglate derivatives (9-15) exhibited significant cytotoxicity against the HT-29 cell line, with rocaglaol (9) being the most potent (ED₅₀ 0.0007 μM). The new compounds 2-4 were also active against this cell line, with ED₅₀ values ranging from 0.46 to 4.7 μM. The cytotoxic compounds were evaluated against a normal colon cell line, CCD-112CoN. In addition, the new compound perviridicin B (2), three known rocaglate derivatives (9, 11, 12), and a known sesquiterpene, 2-oxaisodauc-5-en-12-al (17), showed significant NF-κB (p65) inhibitory activity in an ELISA assay.

Full text of DOI:10.1021/np3007588

Article
pubs.acs.org/jnp
Bioactive Flavaglines and Other Constituents Isolated from Aglaia
perviridis
Li Pan,† Ulyana Munoz Acuna,‡ Jie Li,† Nivedita Jena,† Tran Ngoc Ninh,§ Caroline M. Pannell,⊥
̃
̃
Heebyung Chai,† James R. Fuchs,† Esperanza J. Carcache de Blanco,†,‡ Djaja D. Soejarto,∥,#
and A. Douglas Kinghorn*,†
†Division of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, The Ohio State University, Columbus, Ohio 43210,
United States
‡Division of Pharmacy Practice and Administration, College of Pharmacy, The Ohio State University, Columbus, Ohio 43210, United
States
§Institute of Ecology and Biological Resources, Vietnamese Academy of Science and Technology, Hoang Quoc Viet, Cau Giay, Hanoi,
Vietnam
⊥Department of Plant Sciences, University of Oxford, South Parks Road, Oxford OX1 3RB, U.K.
∥Program for Collaborative Research in the Pharmaceutical Sciences and Department of Medicinal Chemistry and Pharmacognosy,
College of Pharmacy, University of Illinois at Chicago, Chicago, Illinois 60612, United States
#Department of Botany, Field Museum of Natural History, 1400 S. Lake Shore Drive, Chicago, Illinois 60605, United States
S
* Supporting Information
ABSTRACT: Eight new compounds, including two
cyclopenta[b]benzopyran derivatives (1, 2), two cyclopenta-
[b]benzofuran derivatives (3, 4), three cycloartane triterpe-
noids (5−7), and an apocarotenoid (8), together with 16
known compounds, were isolated from the chloroform-soluble
partitions of separate methanol extracts of a combination of
the fruits, leaves, and twigs and of the roots of Aglaia perviridis
collected in Vietnam. Isolation work was monitored using
human colon cancer cells (HT-29) and facilitated with an LC/
MS dereplication procedure. The structures of the new compounds (1−8) were determined on the basis of spectroscopic data
interpretation. The Mosher ester method was employed to determine the absolute configurations of 5−7, and the absolute
configuration of the 9,10-diol unit of compound 8 was established by a dimolybdenum tetraacetate [Mo2(AcO)4] induced
circular dichroism procedure. Seven known rocaglate derivatives (9−15) exhibited significant cytotoxicity against the HT-29 cell
line, with rocaglaol (9) being the most potent (ED50 0.0007 μM). The new compounds 2−4 were also active against this cell line,
with ED50 values ranging from 0.46 to 4.7 μM. The cytotoxic compounds were evaluated against a normal colon cell line, CCD-
112CoN. In addition, the new compound perviridicin B (2), three known rocaglate derivatives (9, 11, 12), and a known
sesquiterpene, 2-oxaisodauc-5-en-12-al (17), showed significant NF-κB (p65) inhibitory activity in an ELISA assay.
Aglaia Lour., containing more than 120 species, is the largest
genus of the plant family Meliaceae.1 Aglaia species have
attracted considerable interest in the area of natural products
research, since they are a rich source of the “flavagline” class of
bioactive agents. Flavagline derivatives have been proposed to
be biogenetically derived from the coupling of a flavonoid unit
and a cinnamic acid amide moiety and can be divided into three
subtypes, namely, the cyclopenta[b]benzofurans, the
cyclopenta[b]benzopyrans, and the benzo[b]oxepines.2−5
More than 100 flavaglines have been isolated from over 30
Aglaia species to date.2−5
In a search for new anticancer agents from tropical plants,
several Aglaia species, including Aglaia crassinervia,6 A. edulis,7
A. elliptica,8 A. foveolata,9−11A. ponapensis,12 and A. rubiginosa,13
mainly collected from Indonesia, have been investigated
previously as promising candidate plants in our laboratories
for phytochemical investigation. Cyclopenta[b]benzopyran
derivatives, with most of them being rocaglaol and related
analogues, and triterpenoids, mainly of the glabretal, bacchar-
ane, and dammarane types, were isolated as the major
constituents from the above-mentioned taxa.6−13 Among the
flavaglines from Aglaia species, two cyclopenta[b]benzofurans
have been reported to exhibit inhibitory activity in vivo in
tumor-bearing experimental animals,2−5 namely, rocaglamide14
and silvestrol.9,15−18 Silvestrol was isolated and fully structurally
characterized from A. foveolata Pannell,9 obtained from
Kalimantan, Indonesia, and also obtained from A. stellatopilosa
Special Issue: Special Issue in Honor of Lester A. Mitscher
Received: October 29, 2012
© XXXX American Chemical Society and
American Society of Pharmacognosy
A
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Pannell,15,19 collected in Sarawak, Malaysia. It may be noted
that the taxonomic resolution of the complex species A.
leptantha in Borneo resulted in the recognition of three separate
species, namely, A. leptantha Miq., A. glabriflora Hiern, and the
new A. stellatopilosa Pannell, with the latter endemic to
Borneo.19 Silvestrol has been synthesized by the Porco20 and
Rizzacasa21,22 groups and found to be a translation inhibitor.23
Silvestrol has been accepted for preclinical development
through the NeXT program of the U.S. National Cancer
Institute as a result of its potential use in treating B-cell
malignancies.17,18,24
Aglaia perviridis Hiern (Meliaceae), a tree up to 15 m tall, is
distributed in the forest regions of southern mainland China,
Bangladesh, Bhutan, India, the Indian Ocean islands, Laos,
Malaysia, Thailand, and Vietnam.25 Previous phytochemical
studies on this plant have led to the isolation of bisamides,
lignans, sterols, sesquiterpenes, and triterpenes.26−29 Among
these compounds, two bisamides were reported as being
biologically active for inhibition of their NO production.28
Thus far, no antiproliferative agents have been isolated from
this plant. In the present investigation, a CHCl3 extract of a
combination of leaves, twigs, and fruits of A. perviridis collected
in Vietnam was found to exhibit cytotoxic activity (IC50 3.0 μg/
mL) against human colon cancer (HT-29) cells. Subsequent
bioassay-guided fractionation conducted using the same cell
line led to the isolation of two new cyclopenta[b]benzopyrans,
perviridisins A and B (1 and 2), three new cycloartane
triterpenoids, perviridisinols A−C (5−7), and a new apocar-
otenoid, (6R,9S)-9,10-dihydroxy-4-megastigmen-3-one (8),
together with 12 known compounds, including five rocaglate
derivatives, rocaglaol (9),30 methyl rocaglate (10),30 4′-
demethoxy-3′,4′-methylenedioxyrocaglaol (11),7 methyl 4′-
demethoxy-3′,4′-methylenedioxyrocaglate (12),7 and didesme-
RESULTS AND DISCUSSION
■
The molecular formula of compound 1 was determined as
C36H42N2O10 based on the [M + Na]+ ion peak at m/z
1
669.2809 (calcd 669.2788) in the HRESIMS. In the H NMR
spectrum, the 11 protons in the low-field region could be
attributed to three aromatic rings, including a monosubstituted
benzene ring at δH 7.29−7.40 (5H, m, H-2″-6″), a 1,4-
disubstituted benzene ring at δH 7.06 (2H, d, J = 8.9 Hz, H-3′,
5′) and 7.70 (2H, d, J = 8.9 Hz, H-2′, 6′), and a 1,2,3,5-
tetrasubstituted benzene ring at δH 6.16 (1H, d, J = 2.2 Hz, H-
7) and 6.19 (1H, d, J = 2.3 Hz, H-9). Also observed were an
oxygenated methine singlet at δH 4.67 (1H, s, H-10), two
methine protons at δH 3.50 (1H, d, J = 5.2 Hz, H-3) and 4.30
(1H, d, J = 5.4 Hz, H-4), which coupled to each other in the
1H−1H COSY spectrum, and three methoxy groups at δH 3.88
(3H, s, OCH3-6), 3.87 (3H, s, OCH3-4′), and 3.76 (3H, s,
OCH3-8) (Table 1). These proton signals suggested that 1 is
based on a cyclopenta[b]benzopyran skeleton typical of those
found previously in several other Aglaia species.3,10,12,41,42
Besides the signals ascribed to the general cyclopenta[b]-
benzopyran skeletal feature, a putrescinyl 4-hydroxytiglate
moiety was recognized. The latter was based on the carbon
signals of four CH2 groups at δC 38.3 (C-13), 27.9 (C-14), 26.2
(C-15), and 39.8 (C-16), a carbonyl group at δC 169.6 (C-18),
a trisubstituted double bond at δC 133.3 (C-19) and 133.7 (C-
20), an oxygenated methylene at δC 59.8 (C-21), and a methyl
group at δC 13.4 (C-22). These signals were consistent with the
proton resonances of two alkyl vicinal methylenes at δH 1.35
(2H, m, H-14) and 1.44 (2H, m, H-15), two N-vicinal
methylenes at δH 2.87 and 3.30 (each 1H, m, H-13) and 3.23
(2H, m, H-16), an olefinic proton at δH 6.27 (1H, brt, J = 5.9
Hz, H-20), an oxygenated methylene at δH 4.27 (2H, brd, J =
5.8 Hz, H-21), and a methyl group located on the double bond
at δH 1.82 (3H, s, H-22) (Table 1). The location of this
thylrocaglamide (13),31 a bisamide, gigantamide A,32
a
sesquiterpene, 2-oxaisodauc-5-en-12-al (17),33,34 scopoletin,35
5,7,4′-tri-O-methylkaempferol,36 and three triterpenes, cabra-
leahydroxylactone,6 24-methylenecycloartan-3β,21-diol,37 and
argenteanol.38
The CHCl3 extract of the roots of A. perviridis was also found
to be very active when evaluated for cytotoxicity against HT-29
cells (ED50 0.2 μg/mL). To avoid the reisolation of only the
same cytotoxic agents as from the plant parts investigated
above, the CHCl3-soluble extract of A. perviridis roots was
subjected to an LC-MS dereplication procedure, which revealed
the presence of four rocaglaol derivatives (9−12). In addition,
the occurrence of unknown cytotoxic compounds was
suggested with possible molecular formulas of C29H28O9 and
C27H26O7. Further fractionation on this extract led to the
purification of six additional rocaglaol derivatives, including two
new cyclopenta[b]benzofurans, 8b-O-methyl-4′-demethoxy-
3′,4′-methylenedioxyrocaglaol (3) and methyl 8b-O-methyl-
4′-demethoxy-3′,4′-methylenedioxyrocaglate (4), along with
four rare known rocaglates, methyl 1-formyloxy-4′-deme-
thoxy-3′,4′-methylenedioxyrocaglate (14),7 methyl 1-formylox-
yrocaglate (15),39 8b-O-methylrocaglaol,40 and methyl 8b-O-
methylrocaglate (16).40 The isolates were tested for their
cytotoxicity against the HT-29 cancer cell line as well as the
normal colon cell line, CCD-112CoN. Finally, the NF-κB
(p65) inhibitory effects of these compounds were also
evaluated using an ELISA assay.
B
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1
Table 1. H and 13C NMR Spectroscopic Data of
a
Compounds 1 and 2
1
2
position
δH, mult (J in Hz)
δC
δH, mult (J in Hz)
δC
2
84.7
60.0
54.7
80.0
87.1
60.3
58.1
79.9
3
3.50, d (5.2)
4.30, d (5.4)
4.28, d (6.7)
4.40, d (6.5)
4
5
Figure 1. Selected key HMBC (→) and NOESY (↔) correlations
observed for perviridisin A (1).
5-OH
5.38, s
5.23, s
5a
108.4
158.5
93.4
112.0
156.2
92.6
6
7
6.16, d (2.2)
6.19, d (2.3)
6.14, d (2.1)
6.16, d (2.2)
3.88, which implied the absence of any shielding effect from the
benzene ring and confirmed the β-orientation of the C-4 phenyl
group. The observed NOESY cross-peaks of H-10/H-3, H-
2′(6′), and H-2″(6″), as well as H-3/H-2′(6′) and H-2″(6″),
further supported the 3-βH and 4-αH orientation and
established the endo relationship between H-10 and H-3
(Figure 1). Thus, the structure of compound 1 was elucidated
as shown, and this substance has been accorded the trivial name
perviridisin A.
8
160.8
93.9
160.4
94.0
9
9a
153.8
76.2
153.0
82.4
10
4.67, s
nd
4.20, s
10-OH
11
2.97, brs
168.7
38.3
169.5
38.5
NH-12
13
5.04, brt (6.2)
2.87, m
5.21, brt (6.4)
2.86, m
3.30, m
3.17, m
The same molecular formula as that of 1, C36H42N2O10, was
assigned to compound 2 based on the [M + Na]+ ion peak at
m/z 669.2763 (calcd 669.2788) in the HRESIMS. The 1H and
13C NMR spectroscopic data of these two compounds were
found to be quite comparable. Thus, it was evident that no
rearrangements had occurred in the cyclopenta[b]benzopyran
system, and the phenyl ring and the amidic putrescinyl 4-
hydroxytiglate moiety at C-4 and C-3, respectively, were both
14
1.35, m
27.9
26.2
39.8
1.33, m
27.3
25.6
39.4
15
1.44, m
1.60, m
16
3.23, m
3.26, m
NH-17
18
6.15, brt (6.0)
6.10, brt (5.2)
169.6
133.3
133.7
59.8
169.1
133.0
133.1
59.4
19
20
6.27, brt (5.9)
4.27, brd (5.8)
1.82, s
6.27, brt (6.0)
b
21
4.27
1
identical to those of 1 (Table 1). In the H NMR spectrum of
22
13.4
1.82, s
13.0
2, a singlet appearing at δH 2.97 was found to show HMBC
correlations with C-2, C-10, and C-5 and was assigned
subsequently as a hydroxy group at C-10. In the NOESY
spectrum, no NOE effect between H-3 and H-10 was observed,
while the 10-H proton correlated with H-3, H-2′(6′), and H-
2″(6″). This analysis suggested OH-10 and H-3 to be spatially
close, consistent with a downfield shift of 0.78 ppm observed
for H-3 caused by the deshielding effect from OH-10. Thus, the
structure of compound 2 (perviridisin B) was deduced as the
C-10 epimer of 1, as shown.
1′
131.3
127.2
115.0
160.0
141.6
129.4
129.0
127.4
56.4
129.6
128.3
114.0
159.6
140.7
129.9
128.3
126.8
56.2
2′,6′
3′,5′
4′
7.70, d (8.9)
7.06, d (8.9)
7.84, d (8.9)
7.05, d (8.9)
1″
b
2″,6″
3″,5″
4″
OCH3-6
OCH3-8
OCH3-4′
7.36
7.52, d (7.3)
7.36, t (7.4)
b
7.40
b
7.29, m
3.88, s
3.76, s
3.87, s
7.29
3.89, s
3.77, s
3.87, s
55.8
55.4
The HRESIMS of compound 3 gave a sodiated molecular
ion peak at m/z 485.1582 [M + Na]+, consistent with a
molecular formula of C27H26O7. The NMR data of 3 proved to
be similar to values published for 4′-demethoxy-3′,4′-
methylenedioxyrocaglaol (11),7 a known compound isolated
55.9
55.5
a
1H NMR spectra measured at 400 MHz, 13C NMR spectra measured
at 100 MHz; obtained in CDCl3 with TMS as internal standard.
Assignments supported with 2D NMR spectra. Overlapping signals.
b
1
in this investigation. In the H NMR spectrum, signals for a
putrescinyl 4-hydroxytiglate group on C-3 through an amide
linkage was deduced by the HMBC correlation between H-3
and the carbonyl group at C-11. Only two cyclopenta[b]-
benzopyran derivatives isolated from A. dasyclada have been
reported to possess the same amidic putrescinyl 4-hydrox-
ytiglate group.43 The connection of the aromatic ring with C-4
was confirmed by HMBC correlations of H-2″ and H-6″ with
C-4. Further key HMBC correlations of H-4 with C-3, C-5, C-
5a, and C-10, H-3 with C-4 and C-1′, H-10 with C-5 and C-5a,
H-2′ and -6′ with C-2, and H-9 with C-9a supported the
proposed cyclopenta[b]benzopyran skeleton of 1 (Figure 1).
According to the literature, H-3β and H-4α substituents could
be suggested based on the coupling constant of 5.4 Hz between
H-3 and H-4.10,44 In addition, if there were a C-4 α-oriented
phenyl ring, the 6-OCH3 protons would be shielded from
monosubstituted benzene ring at δH 6.82 (2H, m, H-2″ and H-
6″) and 7.09 (3H, m, H-3″, H-4″, and H-5″), a 1,3,4-
trisubstituted benzene ring at δH 6.77 (1H, brs, H-2′), 6.61
(1H, d, J = 8.2 Hz, H-5′), and 6.74 (1H, brd, J = 8.2 Hz, H-6′),
and a 1,2,3,5-tetrasubstituted benzene ring at δH 6.31 (1H, d, J
= 1.9 Hz, H-5) and 6.19 (1H, d, J = 1.9 Hz, H-7) were
observed. An (−OCH−CH2−CH−) spin system was evident
based on the coupling patterns of the methylene protons at δH
1.97 (1H, dd, J = 13.8, 6.7 Hz, H-2α) and 2.67 (1H, ddd, J =
14.2, 14.2, 6.5 Hz, H-2β), as well as two methine protons at δH
4.90 (1H, d, J = 7.1 Hz, H-1) and 3.79 (1H, dd, J = 14.3, 6.7
1
Hz, H-3), which was confirmed by the analysis of the H−1H
COSY spectrum. In addition, two aromatic methoxy groups at
δH 3.86 (3H, OCH3-6) and 3.93 (3H, OCH3-8) and
methylenedioxy protons at δH 5.87 and 5.88 (each 1H, d, J =
1.4 Hz, OCH2O) (Table 2) were observed. Besides the above
characteristic protons assigned to a 4′-demethoxy-3′,4′-
around δH 3.88 to approximately 3.11.10,43 In the H NMR
1
spectrum of compound 1, the 6-OCH3 protons appeared at δH
C
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1
Table 2. H and 13C NMR Spectroscopic Data of
Compound 4 gave a molecular formula of C29H28O9, as
determined by a sodiated molecular ion peak at m/z 543.1638
[M + Na]+ in the HRESIMS. The NMR spectra of 3 and 4
were closely comparable, with the major differences focused on
signals of the cyclopentane ring. In the 1H NMR spectrum of 4,
instead of two geminal protons ascribed to H-2 of compound 3,
a methine signal appeared at δH 3.79 (dd, J = 14.8 and 7.1 Hz),
which showed COSY correlations with two methines at δH 5.09
(1H, d, J = 7.1 Hz) and 4.11 (1H, d, J = 14.4 Hz), respectively.
In addition, a methyl ester group was recognized at δH 3.60
(3H, s, COOCH3). Correspondingly, in the 13C NMR
spectrum, a methine group at δC 49.7 (C-2) and a methyl
group at δC 52.0 (COOCH3), as well as a carbonyl group at δC
170.2 (COOCH3), were evident (Table 2). These observations
suggested that a methoxycarbonyl group is located at C-2 in
compound 4, which was confirmed by a key HMBC correlation
between H-2 and the carbonyl carbon at δC 170.2. Thus, the
structure of 4 was elucidated as methyl 8b-O-methyl-4′-
demethoxy-3′,4′-methylenedioxyrocaglate.
a
Compounds 3 and 4
3
4
δH, mult. (J in
position
δH, mult. (J in Hz)
δC
Hz)
δC
1
4.90, d (7.1)
80.1 5.09, d (7.1)
80.3
49.7
2
1.97, α, dd, (13.8, 6.7)
35.5 3.79, dd (14.8,
7.1)
2.67, β, ddd (14.2, 14.2,
6.5)
3
3.79, dd (14.3, 6.7)
53.9 4.11, d (14.4)
55.2
99.4
3a
101.1
4a
161.2
161.4
89.9
5
6.31, d (1.9)
6.19, d (1.9)
89.7 6.31, d (1.9)
6
164.0
164.4
92.8
7
92.4 6.17, d (1.8)
8
157.4
104.0
100.6
128.9
157.6
104.0
99.8
8a
8b
Compound 5 was obtained as a white powder. Its molecular
formula was assigned as C30H48O3 based on the [M + Na]+ ion
peak at m/z 479.3502 (calcd 479.3501) in the HRESIMS. In
1′
128.8
108.0
147.4
146.7
107.4
120.6
136.5
128.0
127.8
127.0
101.0
b
2′
6.77, brs
107.8 6.75
3′
146.7
1
the high-field region of the H NMR spectrum, besides proton
4′
146.3
signals for four tertiary methyl groups at δH 0.81 (3H, s, H-29),
0.88 (3H, s, H-30), 0.97 (3H, s, H-28), and 0.98 (3H, s, H-18),
a secondary methyl group at δH 0.94 (3H, d, J = 6.0 Hz, H-21),
and two methyl groups located at a vinylic carbon at δH 1.97
(3H, s, H-26) and 2.19 (3H, s, H-27), two protons attributed to
a typical cyclopropyl methylene group were recognized at δH
0.33 (1H, d, J = 4.0 Hz, H-19α) and 0.55 (1H, d, J = 4.0 Hz, H-
19β). In the low-field region of the 1H NMR spectrum, proton
signals for two oxygenated methines were evident, at δH 3.28
(1H, dd, J = 11.0, 4.4 Hz, H-3) and 4.10 (1H, brs, H-22), and
an olefinic proton at δH 6.13 (1H, s, H-24). The 13C NMR
spectrum of 5 showed 30 carbon signals, which were classified
from the DEPT and HSQC spectra into seven methyls, nine
methylenes, four alkyl methines, five alkyl quaternary carbons,
two oxygenated methines (δC 78.8, C-3 and 81.0, C-22), a
trisubstituted double bond (δC 120.7, C-24 and 158.0, C-25),
and a carbonyl group (δC 201.5, C-23) (Table 3). These
characteristic NMR data suggested that 5 possesses a
cycloartane skeleton, which has been reported as one of the
major classes of triterpenes isolated from Aglaia species.38,44−48
In the HMBC spectrum, observed correlations from H-2, H3-
28, and H3-29 to C-3, as well as from H-22 to C-21 and C-17
and from H-21 to C-22 supported the location of hydroxy
groups at C-3 and C-22, respectively. The protons of two
geminal methyls, H3-26 and H3-27, showed correlations with
the Δ24 double-bond carbons, respectively. In addition, the
olefinic H-24 correlated with the C-23 carbonyl group, as well
as C-26 and C-27. These observations confirmed the presence
of a terminal dimethylvinyl moiety conjugated with a carbonyl
functionality in the side chain.
5′
6′
6.61, d (8.2)
107.0 6.62, brs
b
6.74, brd (8.2)
120.5 6.75
1″
137.8
b
2″,6″
3″,5″
4″
6.82, m
128.2 6.76
b
b
7.09
127.5 7.06
b
7.09
126.6 7.08, m
100.7 5.87, brs
5.88, brs
OCH2O
5.88, d (1.4)
5.87, d (1.4)
3.86, s
OCH3-6
55.7 3.86, s
55.9 3.90, s
51.7 2.47, s
55.9
56.0
OCH3-8
3.93, s
OCH3-8b
COOCH3
COOCH3
2.46, s
52.1
170.2
3.60, s
52.0
a
1H NMR spectra measured at 400 MHz, 13C NMR spectra measured
at 100 MHz; obtained in CDCl3 with TMS as internal standard.
Assignments supported with 2D NMR spectra. Overlapping signals.
b
methylenedioxyrocaglaol moiety, an extra methoxy group signal
appeared at δH 2.46 (3H, OCH3-8b), which suggested the
hydroxy group at C-8b of 4′-demethoxy-3′,4′-methylenediox-
yrocaglaol (11) to be methylated in 3. In comparison with the
13C NMR spectrum of 11, a downfield shift of 5 ppm for C-8b
and upfield shifts of ca. 2 and 3 ppm for C-3a and C-8a,
respectively, were observed for compound 3 due to this
substitution. In the HMBC spectrum, a key correlation between
the methoxy group at δH 2.46 and C-8b was observed. The β-
orientation of this methoxy group was supported by the NOE
cross-peak of OCH3-8b/H-2′ and H-6′. According to a
previous study, only two naturally occurring rocaglaol
analogues from A. duppereana, which were also isolated from
A. perviridis in the present investigation as the compounds 8b-
O-methylrocaglaol and methyl 8b-O-methylrocaglate (16), have
been reported to have the OH group at C-8b substituted by a
methoxy group.40 Furthermore, analysis of the additional NOE
effects gave supporting evidence that the relative configuration
of compound 3 is identical with those of previously reported
rocaglaol derivatives.7,40 Thus, the structure of 3 was
determined as 8b-O-methyl-4′-demethoxy-3′,4′-methylenediox-
yrocaglaol.
The absolute configurations of C-3 and C-22 in compound 5
were determined by the Mosher ester method. Treatment of 5
with (R)- and (S)-MTPA chloride gave the C-3 and C-22 (S)-
1
and (R)-MTPA ester derivatives. Analysis of the H NMR
chemical shift differences (ΔδS−R) between the (S)- and (R)-
MTPA ester derivatives led to the assignment of the S-
configuration at both C-3 and C-22 (Figure 2). Furthermore, in
the NOESY spectrum, cross-peaks of H3-28/H-19α and H-5,
H-8/H3-18 and H-19β, and H-17/H3-30 provided evidence
that the relative configurations of the remaining stereocenters
D
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Journal of Natural Products
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a
Table 3. 13C NMR Chemical Shifts of Compounds 5−8
5
6
7
8
position
1
δH, mult. (J in Hz)
δC
δH, mult. (J in Hz)
δC
δH, mult. (J in Hz)
δC
δH, mult. (J in Hz)
δC
b
b
b
1.55,
1.22,
1.75,
1.57,
α
β
α
β
31.9
1.55,
1.22,
1.75,
1.57,
α
β
α
β
31.9
1.54,
α
β
α
β
30.7
36.3
b
b
b
b
b
b
b
b
b
1.26,
1.99,
1.32,
2
30.3
30.3
34.8
2.00, d (17.3), α
2.48, d (17.3), β
47.0
3
4
5
6
3.28,dd (11.0, 4.4)
78.8
40.5
47.0
21.1
3.28,dd (10.8, 4.4)
78.8
40.5
46.9
21.0
3.22,ddd (10.4, 9.0, 4.5)
76.5
44.6
43.2
24.6
201.3
124.4
168.7
51.3
b
1.18
5.83, brs
b
b
b
1.30
1.28
1.19
b
b
b
b
1.59,
0.77,
α
β
1.60,
0.79,
α
β
1.68,
α
2.03
b
b
0.58, m, β
b
b
b
b
7
1.10 , α
26.0
1.11 , α
26.0
1.08 , α
25.1
1.48 , a
26.1
b
b
b
b
1.31 , β
1.32 , β
1.38 , β
2.02 , b
b
b
8
1.49, dd (12.0, 4.4)
47.9
19.9
26.0
26.3
1.50, dd (12.4, 5.0)
47.8
19.9
26.3
26.4
1.58
46.7
23.4
29.6
26.9
1.48, a; 1.68, m, b
33.0
72.2
66.2
28.0
9
3.56, dt (10.4, 5.4)
3.47, d (5.4)
1.04, s
10
11
b
b
b
1.96,
1.11,
α
β
1.98,
1.14,
1.55,
1.34,
α
β
α
β
1.99,
1.20,
1.68,
1.61,
α
β
α
β
b
b
b
b
b
b
b
b
12
1.59
1.59
32.7
31.6
32.0
1.12, s
26.5
23.8
b
13
14
15
16
45.8
48.5
35.9
27.8
45.9
47.9
35.9
26.2
45.2
48.9
35.2
27.5
2.07, d (0.9)
b
b
b
b
b
b
1.30
1.83
1.23
2.00
1.40
1.95
1.70
1.31
1.95
1.32
b
b
b
b
17
18
19
47.6
17.9
29.8
1.93, m
50.1
18.9
29.9
2.00, m
46.1
18.0
27.1
0.98, s
0.97, s
0.99, s
0.33, d (4.0), α
0.55, d (4.0), β
2.02, m
0.32, d (4.0), α
0.56, d (4.0), β
2.18, m
0.15, d (4.0), α
0.40, d (4.0), β
b
20
21
42.1
16.4
49.4
70.3
1.51
42.6
62.4
0.94, d (6.0)
3.61, t (8.6), α
4.07, t (8.6), β
3.69, t (7.0)
3.64, dd (11.0, 4.0), a
3.74, dd (11.0, 2.0), b
b
22
23
4.10, brs
81.1
83.1
80.5
1.98
31.7
28.2
b
2.14
b
201.5
4.28, dd (8.8, 7.0)
5.18, d (8.8)
1.58
b
1.50
24
25
26
27
28
29
30
31
6.13, s
120.7
158.0
28.0
21.4
25.4
14.0
19.3
123.5
139.0
26.1
18.5
25.4
14.0
19.3
156.6
33.8
22.0
21.9
14.4
2.25, quin (6.8)
1.04, d (6.8)
1.03, d (6.8)
0.98, d (6.4)
1.97, s
2.19, s
0.97, s
0.81, s
0.88, s
1.77, s
1.75, s
0.97, s
0.80, s
0.90, s
0.91, s
19.2
4.70, brs
4.74, brs
106.3
a
1H NMR spectra measured at 400 MHz, 13C NMR spectra measured at 100 MHz; spectra of compounds 5−7 were obtained in CDCl3 with TMS
b
as internal standard; spectrum of 8 was obtained in methanol-d4. Assignments supported with 2D NMR spectra. Overlapping signals.
5 was determined to be (3S,22S)-dihydroxycycloart-24-en-23-
one, and this substance has been accorded the trivial name
perviridisinol A.
Compound 6 gave the same molecular formula as that of 5,
C30H48O3, based on the [M + Na]+ ion peak at m/z 479.3496
1
in the HRESIMS. The H and 13C NMR spectra of 6 were
closely comparable to those of compound 5, with the major
differences occurring for signals in the side chain. On
1
comparison of the H NMR data of these two compounds,
Figure 2. ΔδS−R values of MTPA esters of 5.
the doublet of the secondary methyl group H3-21 at δH 0.94 in
compound 5 was absent, while resonances of an oxygenated
methylene appeared at δH 3.61 (1H, d, J = 8.6 Hz, H-21α) and
4.07 (1H, d, J = 8.6 Hz, H-21β). In addition, an extra
of compound 5 are identical with those of previously reported
related compounds.38,44−48 Hence, the structure of compound
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oxygenated methine resonance appeared at δH 4.28 (1H, dd, J =
7.0 and 8.8 Hz, H-23) and showed COSY correlations with an
olefinic signal at δH 5.18 (1H, d, J = 8.8 Hz, H-24) and an
oxygenated methine at δH 3.69 (1H, t, J = 7.0 Hz, H-22),
respectively. Correspondingly, in the 13C NMR spectrum of 6,
instead of the carbon signals of the C-21 methyl group and the
C-23 carbonyl group in 5, resonances for an oxygenated
methylene at δC 70.3 (C-21) and an oxygenated methine at δC
80.5 (C-23) were observed (Table 3). From the NMR data, in
combination with the molecular formula, an extra ring was
required in addition to the tetracyclic ring system of a
cycloartane triterpene skeleton. This analysis suggested that
C-21 is connected with C-23 through an oxygen bridge to form
a tetrahydrofuran ring in the side chain. This deduction was
supported by HMBC correlations of H-21 with C-23, H-20
with H-21 and H-22, and H-23 and H-21β with C-22. The
relative configurations of the stereocenters of the tetrahy-
drofuran ring were assigned as shown based on observed NOE
effects between H-20/H-21β and H-23, H-17/H-21α and H-
22, and H-24/H-22. The absolute configurations at both C-3
and C-22 were established as S by the Mosher ester procedure
(Figure 3). Thus, the structure of compound 6 (perviridisinol
B) was elucidated as 21,23R-epoxy-(3S,22S)-dihydroxycycloart-
24-ene.
methylenecycloartan-3β,21-diol, is absent in compound 7. This
observation explained the different coupling pattern observed
for H-3 (δH 3.22, 1H, ddd, J = 10.4, 9.0, and 4.5 Hz) when
compared with H-3 (δH 3.28, 1H, dd, J = 10.8 and 4.4 Hz) of
3β-hydroxy cycloartane derivatives.38,44−48 The absolute
configuration of C-3 was also determined as S by the Mosher
ester procedure (Figure 4). A NOESY experiment revealed the
Figure 4. ΔδS−R values of MTPA esters of 7.
consistent relative configuration of 7 with other cycloartane
analogues isolated in this investigation. Thus, the structure of
compound 7 (perviridisinol C) was determined as 3S,21-
dihydroxy-24-methylene-29-norcycloartane.
The molecular formula of compound 8 was determined as
C13H22O3 from the sodiated molecular ion peak at m/z
249.1459 [M + Na]+ (calcd 249.1467) in the HRESIMS. In the
1H NMR spectrum, proton signals of two tertiary alkyl methyls
at δH 1.04 (3H, s, H-11) and 1.12 (3H, s, H-12), a methyl
group located on a double bond at δH 2.07 (3H, d, J = 0.9 Hz,
H-13), a methine group at δH 3.56 (1H, d, J = 10.4 and 5.4 Hz),
and an oxygenated methylene group at δH 3.47 (2H, d, J = 5.4
Hz) were recognized (Table 3). Altogether, 13 carbon signals
in the 13C NMR spectrum were sorted by their DEPT and
HSQC data into three methyls, three alkyl methylenes, one
alkyl methine, one quaternary carbon, an oxygenated methine,
an oxygenated methylene, a trisubstituted double bond, and a
carbonyl group. An α,β-unsaturated carbonyl moiety could be
recognized based on the carbon signals of the carbonyl group at
δC 201.3 (C-3) and of the double bond at δC 124.4 (C-4) and
168.7 (C-5). In the HMBC spectrum, proton signals of two
geminal methyl goups, H3-11 and H3-12, showed strong
correlations with a quaternary carbon at δC 36.3 (C-1), a
methylene carbon adjacent to a carbonyl group at δC 47.0 (C-
2), and a methine carbon at δC 51.3 (C-6), which was further
found to correlate with H3-13, the proton signal of the methyl
group on the double bond. In addition, the proton signal of the
oxygenated methylene (H-10) correlated with the oxygenated
methine (C-9) and an alkyl methylene (C-8). In the COSY
spectrum, the alkyl methylenes, H-7 and H-8, correlated with
one another, with H-7 also correlating with H-6. Based on the
1D- and 2D-NMR data analysis, the planar structure of
compound 8 was elucidated as a C13 apocarotenoid
derivative,49 9,10-dihydroxy-4-megastigmen-3-one.
Figure 3. ΔδS−R values of MTPA esters of 6.
The HRESIMS of compound 7 gave a sodiated molecular
ion peak at m/z 465.3715 [M + Na]+, consistent with a
molecular formula of C30H50O2. The NMR data of 7 were
similar to those of 24-methylenecycloartan-3β,21-diol, a known
triterpene also isolated in the present study. In the low-field
1
region of the H NMR spectrum, characteristic proton signals
occurring as two singlets at δH 4.70 and 4.74 (each 1H, H-31),
attributed to a terminal vinyl group, as well as two geminal
protons at δH 3.64 (1H, dd, J = 11.0, 4.0 Hz, H-21a) and 3.74
(1H, dd, J = 11.0, 2.0 Hz, H-21b), corresponding to an
oxygenated methylene, were recognized. In the high-field
region of the 1H NMR spectrum, only five methyl groups were
observed (Table 3). Besides signals for two methyl groups
belonging to an isobutane moiety in the side chain (δH 1.04,
3H, d, J = 6.8 Hz, H-26; δH 1.03, 3H, d, J = 6.8 Hz, H-27) and
two tertiary methyls (δH 0.91, 3H, s, H-30; δH 0.99, 3H, s, H-
18) at the C/D ring junction, a secondary methyl group was
apparent at δH 0.98 (3H, d, J = 6.4 Hz, H3-28). This methyl
group signal displayed an HMBC correlation with the
oxygenated methine carbon at δC 76.5 (C-3) and exhibited
NOE associations with H-3 and H-19α. In the 13C NMR
spectrum, instead of the quaternary carbon around δC 40.0
corresponding to C-4 found in 24-methylenecycloartan-3β,21-
diol,37 a methine carbon appeared at δC 44.6 and showed an
HMBC correlation with H3-28. All these observations
suggested that C-29, the tertiary methyl group at C-4 in 24-
The absolute configuration of C-6 was determined as R based
on the positive absorptions around 245 and 335 nm in the
ECD spectrum (c 1.26 × 10−4 M, MeOH) of 8.49,50 As far as
the acyclic 9,10-diol moiety is concerned, neither the Mosher
ester procedure nor a regular electronic circular dichroism
(ECD) measurement could be used for the assignment of the
absolute configuration of C-9. In this case, a practical and
reliable method developed by Snatzke and Frelek was
employed to solve the problem.51,52 After mixing compound
8 and dimolybdenum tetraacetate [Mo2(AcO)4] in DMSO, a
ligand−metal complex possessing a suitable chromophoric
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Figure 5. ECD spectrum of compound 8 in a DMSO solution (blue). ECD spectrum of compound 8 in a DMSO solution of Mo2(OAc)4 (red).
(The inherent ECD was subtracted.)
group was formed, for which the induced circular dichroism
(ICD) spectrum was recorded and analyzed. According to
Snatzke’s theory, the absorption band around 310 nm (band
IV) is one of these most reliably related to the absolute
configuration of a diol derivative in the [Mo2(AcO)4]-induced
CD spectrum, which possesses the same sign of torsional angle
of the O−C−C−O unit in the favored conformation. In the
ICD spectrum of compound 8, the diagnostic Cotton effect
around 310 nm was positive (Figure 5), which corresponds to a
positive dihedral angle of the O−C−C−O moiety (Figure 6).
Thus, the absolute configuration of C-9 of the 9,10-diol moiety
in compound 8 was assigned as S.
Table 4. Bioactivity Evaluation of Compounds Isolated from
A. perviridis
a
b
c
d
compound
HT-29
CCD112CoNl
NF-κB (p65)
2
0.46
0.96
4.7
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
2.40
>20
3
4
>20
9
0.0007
0.012
0.0067
0.002
0.021
0.056
0.019
4.9
0.005
>20
10
11
12
13
14
15
16
17
0.33
3.54
>20
e
>20
>20
>20
0.005
f
paclitaxel
rocaglamide
0.001
0.005
23.0
g
>50
0.08
a
b
Figure 6. The O−C−C−O dihedral in the compound 8−[Mo2]4+
Results are expressed as ED50 values (μM). Compounds 1, 5−8, 17,
argenteanol, cabraleahydroxylactone, gigantamide A, 24-methylenecy-
cloartan-3β,21-diol, 5,7,4′-tri-O-methylkaempferol, 8b-O-methylroca-
glaol, and scopoletin were inactive against HT-29 cells (ED50 > 10
μM). Compounds with ED50 > 50 μM were considered inactive
against CCD112CoNl cells. Compounds 1, 3−8, 10, 13, 15, 16,
argenteanol, cabraleahydroxylactone, gigantamide A, 24-methylenecy-
cloartan-3β,21-diol, 5,7,4′-tri-O-methylkaempferol, 8b-O-methylroca-
glaol, and scopoletin were inactive in the NF-κB (p65) assay (ED50
20 μM). The NF-κB inhibitory activity for 14 was not tested due to
the limited quantity obtained. Used as a positive control substance for
the cytotoxicity assay. Used as a positive control substance for the
NF-κB (p65) assay.
complex.
c
All isolates were evaluated for their cytotoxic activity against
HT-29 human colon cancer cells. As shown in Table 4,
cyclopenta[b]benzofuran derivatives were demonstrated as
being the major cytotoxic substances from A. perviridis. Seven
known rocaglaol derivatives (9−15) exhibited pronounced
cytotoxicity against the HT-29 cell line, showing ED50 values
ranging from 0.0007 to 0.056 μM, with rocaglaol (9) as the
most potent compound. Four rocaglaol analogues, with the C-
8b hydroxy group replaced by a methoxy group, including the
new compounds 3 and 4, as well as two known compounds, 8b-
O-methylrocaglaol and methyl 8b-O-methylrocaglate (16),
were found to be much less potently cytotoxic. This
observation is consistent with the reported negative result
observed for 8b-O-methylrocaglaol and methyl 8b-O-methyl-
rocaglate (16) in the human monocytic leukemia cell lines
MONO-MAC-1 and MONO-MAC-639 and supported the
conclusion that a free hydroxy group at C-8b is an essential
feature of rocaglaol derivatives for the resulting cytotoxicity.
The new cyclopenta[b]benzopyran derivative, perviridisin B
(2), exhibited significant cytotoxicity (ED50 0.46 μM) against
HT-29 cells, while its C-10 epimer, perviridisin A (1), was
inactive in the same assay. In order to evaluate the selectivity of
d
>
e
f
g
these potent cytotoxic agents isolated from A. perviridis for a
tumorigenic cell line, compounds with ED50 values of less than
10 μM against HT-29 cells were further tested against the
CCD-112CoN normal colon cell line. None of the compounds
tested were found to show inhibitory activity against the
employed normal cells at the relatively high concentration of 50
μM. This preliminary selectivity testing result provided a
favorable in vitro selectivity profile for any further development
of these active flavaglines as oncology leads. In a previous
mechanistic study, rocaglaol (9) was shown to cause G2/M-
phase cell cycle arrest and to induce apoptosis of LNCaP
human prostate cancer cells.53
G
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LC-MS Conditions. HPLC conditions: mobile phase: a gradient
elution of MeOH−H2O (0−10 min, from 62:38 to 70:30; 11−30 min,
100% MeOH); injection volume: 45 μL (10 mg/mL). The mobile
phase flow rate was maintained at 0.75 mL/min and was split post-
column using a microsplitter valve (Upchurch Scientific, Oak Harbor,
WA, USA) to ca. 20 μL/min for introduction to the ESI source.
Optimal ESI conditions: capillary voltage, 3000 V; source temperature,
110 °C; cone voltage, 55 V. Q1 was set to optimally pass ions from m/
z 100−2000, and all ions transmitted into the pusher region of the
TOF analyzer were scanned over the m/z 100−1000 range with a 1 s
integration time. Data were acquired in a continuum mode during the
LC run. The result showed that the active fractions with cytotoxicity at
the concentration of 20 μg/mL mainly included 11 m/z values: 507.2,
529.2, 493.2, 515.2, 471.2, 457.2, 521.2, 543.2, 535.2, 557.2, and 485.2,
respectively.
All the isolates were also evaluated for their NF-κB (p65)
inhibitory activity in an enzyme-based ELISA assay. Rocaglaol
(9) and the known sesquiterpene 2-oxaisodauc-5-en-12-al (17)
were extremely active, both with an ED50 value of 0.005 μM,
more than 10 times more potent than the control compound,
rocaglamide. For other rocaglate derivatives (10−16), a notable
decrease of the NF-κB (p65) inhibitory activity was observed
due to the substitution of a methoxycarbonyl or carboxamide at
C-2 or/and the 4′-methoxy group being substituted by a 3′,4′-
methylenedioxy in their structures, when compared with
rocaglaol (9). Perviridisin B (2), a new compound with
significant cytotoxicity against HT-29 cells, was also found to
show moderate NF-κB inhibitory activity (ED50 = 2.4 μM) in
the present investigation.
Data Analysis. A NAPRALERT database (http://www.napralert.
org) search reported 656 compounds from the genus Aglaia,
comprising flavaglines, terpenoids, flavonoids, and bisamides. The
flavagline group is the major compound class of Aglaia species showing
EXPERIMENTAL SECTION
■
cytotoxic activity from the previous investigations.7,8,54−57
A
General Experimental Procedures. Melting points were
measured using a Fisher Scientific melting point apparatus and are
uncorrected. Optical rotations were measured on a Perkin-Elmer 343
automatic polarimeter (PerkinElmer, Waltham, MA, USA). UV spectra
were run on a Hitachi U-2910 spectrophotometer (Hitachi, Tokyo,
Japan). ECD spectra were recorded on a JASCO J-810 spectropo-
larimeter (JASCO Inc., Easton, MD, USA). IR spectra were obtained
on a Thermo Scientific Nicolet 6700 FT-IR spectrometer (Thermo
Scientific, Waltham, MA, USA). NMR spectroscopic data were
recorded at room temperature on Bruker Avance DRX-400
spectrometers (Bruker, Billerica, MA, USA), using standard Bruker
pulse sequences, and the data were processed using MestReNova 6.0
software (Mestrelab Research SL, Santiago de Compostela, Spain).
High-resolution electrospray ionization mass spectra (HRESIMS)
were performed on a Micromass Q-Tof II (Micromass, Wythenshawe,
UK) mass spectrometer operated in the positive-ion mode, with NaI
being used for mass calibration for a calibration range of m/z 100−
2000. LC-MS experiments were performed on a liquid chromato-
graphic/autosampler system that consisted of a Waters Alliance 2690
separations module (Waters, Milford, MA, USA) and a Micromass
LC-TOF II mass spectrometer (Micromass) equipped with an
orthogonal electrospray source (Z-spray). Column chromatography
was carried out with silica gel (230−400 mesh; Sorbent Technologies,
Atlanta, GA, USA). Analytical TLC was conducted on precoated 250
μm thick silica gel UV254 aluminum-backed plates (Sorbent
Technologies). Waters Xbridge (4.6 × 150 mm), semipreparative
(10 × 150 mm), and preparative (19 × 150 mm) C18 (5 μm) columns
were used for analytical, semipreparative, and preparative HPLC,
respectively, as conducted on a Waters system comprising a 600
controller, a 717 Plus autosampler, and a 2487 dual-wavelength
absorbance detector.
Plant Material. The roots and the combination of the fruits, leaves,
and twigs of Aglaia perviridis were collected in Nui Chua National Park
(11°43′ N; 109°08′ E; 730 m alt.), Ninh Thuan Province, Vietnam, in
January 2010, by D.D.S., T.N.N., and Vuong Tan Tu, who also
identified this plant, in cooperation with C.M.P. A voucher specimen
(original collection Soejarto et al.14595) has been deposited in the
John G. Searle Herbarium of the Field Museum of Natural History
(under accession number FM 2287877), Chicago, IL, USA.
LC-MS Dereplication Procedure. LC-UV Conditions. Sample
concentration: 10 mg/mL MeOH solution; mobile phase: gradient
elution of MeOH−H2O (0−10 min, from 62:38 to 70:30; 11−30 min,
100% MeOH); UV detection wavelength: 210 nm; flow rate: 0.75
mL/min; injection volume: 45 μL for the 96-well plate with sample
concentration of ca. 20 μg/mL, and 11.3 μL for the 96-well plate with
sample concentration of ca. 5 μg/mL.
combination analysis of the search results using each possible
molecular formula with the substance role “occurrence” and the key
word “Aglaia” in the SciFinder database (Chemical Abstracts Service)
was carried out to check if the molecular weights from the active peaks
match any known flavaglines isolated from the genus Aglaia.
Extraction and Isolation. The air-dried and finely ground
combination of the leaves, twigs, and fruits of A. perviridis (880 g)
was extracted by maceration in MeOH−H2O (95:5; 3 × 2 L) at room
temperature for two days each. The solvent was evaporated under
reduced pressure to yield 223 g of a crude extract, which was
suspended in a MeOH−H2O (9:1) mixture and then extracted with
hexanes (3 × 1 L) and CHCl3 (3 × 1 L), sequentially, to afford a
CHCl3-soluble extract (17 g). The CHCl3-soluble extract, with an IC50
value of 3.0 μg/mL against HT-29 human colon cancer cells, was
separated by column chromatography over Si gel using a CH2Cl2−
acetone gradient solvent system (30:1 to pure acetone). Of seven
subfractions obtained, F1 and F5 were found to be the most active,
with ED50 values of 0.3 and 0.8 μg/mL, respectively. Fraction F01 (1.1
g) was chromatographed over an open C18 column (2.2 × 20 cm),
using MeOH−H2O mixtures (50:50 to 100% MeOH) for elution, to
give 24 subfractions (F101−F124). Recrystallization of the yellow
precipitates from F101 and F108 in MeOH afforded scopoletin (3.0
mg) and 5,7,4′-tri-O-methylkaempferol (5.0 mg), respectively.
Compound 17 (2.0 mg) was obtained from subfraction F103 by
column chromatography over silica gel with acetone−n-hexane (5:1 to
2:1). Subfraction F105 was fractionated over an open C18 column,
eluted with MeOH−H2O (60:40 to 100% MeOH), to afford five
subfractions (F1051−F1055). F1051 was purified by HPLC on a
semipreparative RP-18 column, using MeOH−H2O (0.1% formic
acid) (55:45) as solvent system, to afford, in turn, 12 (3.0 mg), 10
(12.0 mg), 11 (5.0 mg), and 9 (4.0 mg). Subfraction F1052 was
chromatographed by HPLC on a semipreparative RP-18 column
(CH3CN−H2O, 30:70) to give perviridisin A (1, 4.0 mg) and
gigantamide (2.0 mg). Subfraction F1054 was passed over a
semipreparative RP-18 column (CH3CN−H2O, 30:70) by HPLC to
yield (6R,9S)-9,10-dihydroxy-4-megastigmen-3-one (8, 2 mg). Sub-
fraction F1055 was subjected to separation on a semipreparative RP-18
column by HPLC, using CH3CN−H2O (30:70) as solvent system, to
give a mixture of perviridisin B (2, 2.0 mg) and compound 13 (6.0
mg), which was resolved on the same HPLC column, using MeOH−
H2O (50:50) for elution. Cabraleahydroxylactone (15 mg) was
purified from subfraction F113 by chromatography over a silica gel
column and eluted with CH2Cl2−acetone mixtures (20:1 to 2:1). F115
was chromatographed on a semipreparative RP-18 column with a
CH3CN−H2O (80:20) solvent system to yield perviridisinol A (5, 7.0
mg). 24-Methylenecycloartan-3β,21-diol (12.0 mg) was recrystallized
from subfraction F119 using methanol as solvent. Perviridisinol C (7,
3.5 mg) was purified from subfraction F120 over a semipreparative
RP-18 column by HPLC, using CH3CN−H2O (50:50) as solvent
system. Subfraction F122 was purified by HPLC, using a semi-
Cytotoxicity Assay Screening. Fractions were collected into two
96-well plates (250 μL/well × 90 and negative control/well × 6) with
sample concentrations of 20 and 5 μg/mL, respectively, and were
tested for HT-29 cell growth inhibition activity, according to an
established protocol.11
H
dx.doi.org/10.1021/np3007588 | J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
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1
preparative RP-18 column (MeOH−H2O, 50:50), to afford
argenteanol (23.0 mg) and perviridisinol B (6, 7.0 mg), respectively.
The roots of A. perviridis (370 g) were extracted and partitioned
using the same procedure as described above to yield a CHCl3-soluble
extract (3.0 g), which exhibited potent cytotoxicity against HT-29 cells
(ED50 0.2 μg/mL). In order to decide whether or not to further pursue
this lead, and thereby make the isolation procedure on this plant
material more efficient, the CHCl3-soluble extract of the roots of A.
perviridis was subjected to an LC-MS dereplication analysis. During
this procedure, the effluent from the HPLC chromatography was split,
with the two effluents analyzed by MS and screened using HT-29
cancer cells cultured in a 96-well plate, respectively. The results
indicated the possible presence of four known cytotoxic rocaglate
derivatives (9−12), which also occurred in a sample of a combination
of the leaves, twigs, and fruits of A. perviridis, based on peaks at m/z
457, 515, 529, and 471, respectively. In addition, the putative
elemental formula, C29H38O9, was consistent with the presence of a
rare known rocaglate analogue that has not been found from A.
perviridis previously, methyl 1-formyloxyrocaglate (15).39 Besides
these known compounds, an unknown compound in a cytotoxic
well corresponding to a possible molecular formula of C27H26O7 was
evident from a sodiated ion peak at 485 amu. Accordingly, bioassay-
guided fractionation was used to facilitate the isolation process from A.
perviridis roots. This extract was fractionated over a Sephadex LH-20
column using MeOH to yield four fractions (F1′−F4′). The most
active subfraction, F4′ (700 mg, ED50 < 0.16 μg/mL), was subjected to
separation over a preparative RP-18 HPLC column using a MeOH−
H2O gradient solvent system (0−50 min 57:43; 50−80 min 70:30) for
elution, to afford a mixture of 14 and 15, 8b-O-methyl-4′-demethoxy-
3′,4′-methylenedioxyrocaglaol (3, 4.0 mg), 16 (4.5 mg), methyl 8b-O-
methyl-4′-demethoxy-3′,4′-methylenedioxyrocaglate (4, 2.0 mg), and
8b-O-methyl rocaglaol (4.0 mg). The mixture of compounds 14 (0.8
mg) and 15 (1.5 mg) was further separated by HPLC on a
semipreparative RP-18 column (CH3CN−H2O, 50:50).
1100, 1025, 755 cm−1; H NMR (400 MHz, CDCl3) and 13C NMR
(100 MHz, CDCl3) data, see Table 3; HRESIMS m/z 479.3502 [M +
Na]+ (calcd for C30H48O3Na, 479.3501).
Perviridisinol B (6): white powder; mp 198−200 °C; [α]20D +2.0 (c
0.1, MeOH); UV (MeOH) λmax (log ε) 204 (3.78, end absorption)
nm; IR (film) νmax 3395, 2930, 2866, 1699, 1457, 1378, 1215, 1097,
1
1023, 1006, 754 cm−1; H NMR (400 MHz, CDCl3) and 13C NMR
(100 MHz, CDCl3) data, see Table 3; HRESIMS m/z 479.3496 [M +
Na]+ (calcd for C30H48O3Na, 479.3501).
Perviridisinol C (7): white powder; mp 164−165 °C; [α]20 +29.0
D
(c 0.1, MeOH); UV (MeOH) λmax (log ε) 204 (3.40, end absorption)
nm; IR (film) νmax 3285, 2961, 2923, 2868, 1453, 1377, 1041, 1006,
882, 757, 669 cm−1; 1H NMR (400 MHz, CDCl3) and 13C NMR (100
MHz, CDCl3) data, see Table 3; HRESIMS m/z 465.3715 [M + Na]+
(calcd for C30H50O2Na, 465.3709).
(6R,9S)-9,10-Dihydroxy-4-megastigmen-3-one (8): colorless gum;
[α]20 +59.0 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 240 (3.81)
D
nm; ECD (c 1.26 × 10−4 M, MeOH) λmax (Δε) 245 (+1.13), 335
(+0.35) nm; ECD (c 1.14 × 10−3 M, DMSO) λmax (Δε) 274 (+0.81),
350 (+0.21) nm; IR (film) νmax 3400, 2956, 2872, 1650, 1440, 1379,
1304, 1257, 1101, 1043, 871 cm−1; 1H NMR (400 MHz, methanol-d4)
and 13C NMR (100 MHz, methanol-d4) data, see Table 3; HRESIMS
m/z 249.1459 [M + Na]+ (calcd for C13H22O3Na, 249.1467).
Preparation of the (R)- and (S)-MTPA Ester Derivatives of
Compounds 5−7. The (R)- and (S)-MTPA ester derivatives of
compounds 5−7 were prepared in a manner described previously.58,59
In brief, two portions of each compound (1 mg) were added into two
NMR tubes and dried completely. Pyridine-d5 was added to both tubes
(each 0.5 mL). Then, (S)-(+)-α-methoxy-α-(trifluoromethyl)-
phenylacetyl (MTPA) chloride (10 μL) or (R)-MTPA chloride (10
μL) was injected into the NMR tubes separately under N2 gas
protection and quickly mixed with the dissolved sample. The 1H NMR
chemical shifts of the (R)- and (S)-MTPA ester of 5−7 were recorded
after the reactions were complete. COSY and NOESY experiments
were used to establish the 1H NMR assignment, and only fully
assigned signals used for the ΔδS−R calculation.
Perviridisin A (1): colorless resin; [α]20 −22.0 (c 0.09, MeOH);
D
UV (MeOH) λmax (log ε) 218 (4.34), 272 (3.32) nm; ECD (c 4.64 ×
10−5 M, MeOH) λmax (Δε) 230 (+6.72), 280 (+1.94) nm; IR (film)
νmax 3420, 2937, 1662, 1618, 1592, 1516, 1457, 1438, 1252, 1216,
1201, 1149, 1098, 1031, 832, 752 cm−1; 1H NMR (600 MHz, CDCl3)
and 13C NMR (150 MHz, CDCl3) data, see Table 1; HRESIMS m/z
669.2809 [M + Na]+ (calcd for C30H48O3Na, 669.2788).
3,22-Di-(R)-MTPA ester of perviridisinol A (5): 1H NMR data (400
MHz, pyridine-d5) δ 6.402 (1H, s, H-24), 5.638 (1H, d, J = 2.6 Hz, H-
22), 4.991 (1H, dd, J = 11.7, 4.5 Hz, H-3), 2.430 (1H, m, H-20), 2.228
(3H, s, H-27), 2.206 (1H, m, H-17), 1.807 (3H, s, H-26), 1.209 (3H,
d, J = 6.4 Hz, H-21), 1.016 (3H, s, H-18), 0.919 (3H, s, H-30), 0.890
(3H, s, H-28), 0.878 (3H, s, H-29), 0.621 (1H, m, H-6β), 0.450 (1H,
d, J = 3.9 Hz, H-19β), 0.298 (1H, d, J = 3.9 Hz, H-19α).
Perviridisin B (2): colorless resin; mp 270−272 °C; [α]20D +21.0 (c
0.1, MeOH); UV (MeOH) λmax (log ε) 215 (4.32), 273 (3.18) nm;
ECD (c 4.64 × 10−5 M, MeOH) λmax (Δε) 222 (−6.03), 280 (−3.51)
nm; IR (film) νmax 3413, 2936, 1661, 1618, 1591, 1515, 1460, 1439,
3,22-Di-(S)-MTPA ester of perviridisinol A (5): 1H NMR data (400
MHz, pyridine-d5) δ 6.508 (1H, s, H-24), 5.627 (1H, d, J = 2.4 Hz, H-
22), 4.979 (1H, dd, J = 11.8, 4.3 Hz, H-3), 2.366 (1H, m, H-20), 2.237
(3H, s, H-27), 2.162 (1H, m, H-17), 1.848 (3H, s, H-26), 1.148 (3H,
d, J = 6.4 Hz, H-21), 1.004 (3H, s, H-28), 0.959 (3H, s, H-18), 0.908
(3H, s, H-29), 0.891 (3H, s, H-30), 0.630 (1H, m, H-6β), 0.420 (1H,
d, J = 3.6 Hz, H-19β), 0.246 (1H, d, J = 3.6 Hz, H-19α).
1
1253, 1215, 1201, 1150, 1098, 1033, 833, 753 cm−1; H NMR (400
MHz, CDCl3) and 13C NMR (100 MHz, CDCl3) data, see Table 1;
HRESIMS m/z 669.2763 [M + Na]+ (calcd for C36H42N2O10Na,
669.2788).
8b-O-Methyl-4′-demethoxy-3′,4′-methylenedioxyrocaglaol (3):
3,22-Di-(R)-MTPA ester of perviridisinol B (6): 1H NMR data (400
MHz, pyridine-d5) δ 5.647 (1H, d, J = 10.0 Hz, H-24), 5.524 (1H, t, J
= 4.8 Hz, H-22), 5.000 (1H, dd, J = 11.1, 4.4 Hz, H-3), 4.758 (1H, dd,
J = 8.7, 4.1 Hz, H-23), 4.206 (1H, t, J = 8.0 Hz, H-21β), 3.899 (1H, m,
H-21α), 2.613 (1H, m, H-20), 2.179 (1H, m, H-17), 1.741 (3H, s, H-
27), 1.667 (3H, s, H-26), 0.970 (3H, s, H-18), 0.910 (3H, s, H-30),
0.891 (3H, s, H-28), 0.881 (3H, s, H-29), 0.641 (1H, m, H-6β), 0.476
(1H, d, J = 3.6 Hz, H-19β), 0.290 (1H, d, J = 3.7 Hz, H-19α).
pale yellow, amorphous powder; [α]20 −32.0 (c 0.09, MeOH); UV
D
(MeOH) λmax (log ε) 216 (4.26), 233 (3.97), 280 (3.53) nm; ECD (c
5.41 × 10−5 M, MeOH) λmax (Δε) 219 (−15.10), 280 (−2.16) nm; IR
(film) νmax 3524, 2934, 1623, 1597, 1497, 1491, 1456, 1436, 1247,
1216, 1201, 1148, 1125, 1107, 1067, 1041, 1007, 936, 811, 756, 699
cm−1; 1H NMR (400 MHz, CDCl3) and 13C NMR (100 MHz,
CDCl3) data, see Table 2; HRESIMS m/z 485.1582 [M + Na]+ (calcd
for C27H26O7Na,485.1576).
1
3,22-Di-(S)-MTPA ester of perviridisinol B (6): H NMR data (400
Methyl 8b-O-methyl-4′-demethoxy-3′,4′-methylenedioxyroca-
glate (4): pale yellow, amorphous powder; [α]20 −7.5 (c 0.1,
MHz, pyridine-d5) δ 5.671 (1H, d, J = 8.7 Hz, H-24), 5.492 (1H, t, J =
5.0 Hz, H-22), 4.990 (1H, dd, J = 11.7, 4.8 Hz, H-3), 4.902 (1H, dd, J
= 8.8, 4.5 Hz, H-23), 4.186 (1H, t, J = 8.0 Hz, H-21β), 3.861 (1H, m,
H-21α), 2.480 (1H, m, H-20), 2.105 (1H, m, H-17), 1.774 (3H, s, H-
27), 1.746 (3H, s, H-26), 1.011 (3H, s, H-28), 0.914 (3H, s, H-29),
0.899 (3H, s, H-30), 0.861 (3H, s, H-18), 0.667 (1H, m, H-6β), 0.445
(1H, d, J = 3.7 Hz, H-19β), 0.236 (1H, d, J = 4.0 Hz, H-19α).
3,21-Di-(R)-MTPA ester of perviridisinol C (7): 1H NMR data (400
MHz, pyridine-d5) δ 4.949 (1H, td, J = 10.6, 4.5 Hz, H-3), 4.883 (1H,
s, H-31b), 4.839 (1H, s, H-31a), 4.822 (1H, m, H-21β), 4.405 (1H, dd,
J = 11.3, 5.1 Hz, H-21α), 2.238 (1H, m, H-25), 1.895 (1H, m, H-17),
D
MeOH); UV (MeOH) λmax (log ε) 216 (4.27), 233 (4.01), 280 (3.61)
nm; nm; ECD (c 4.80 × 10−5 M, MeOH) λmax (Δε) 218 (−12.41),
239 (+1.04), 277 (−1.35) nm; IR (film) νmax 3502, 2932, 1746, 1624,
1598, 1500, 1456, 1437, 1242, 1203, 1149, 1124, 1107, 1067, 1041,
1
1007, 936, 813, 699 cm−1; H NMR (400 MHz, CDCl3) and 13C
NMR (100 MHz, CDCl3) data, see Table 2; HRESIMS m/z 543.1638
[M + Na]+ (calcd for C29H28O9Na, 543.1631).
Perviridisinol A (5): white powder; mp 160−162 °C; [α]20 +76.0
D
(c 0.1, MeOH); UV (MeOH) λmax (log ε) 205 (3.54), 241 (3.85) nm;
IR (film) νmax 3446, 2936, 2869, 1699, 1676, 1618, 1457, 1379, 1217,
I
dx.doi.org/10.1021/np3007588 | J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
1.830 (1H, m, H-20), 1.062 (3H, d, J = 6.7 Hz, H-26), 1.056 (3H, d, J
= 6.9 Hz, H-27), 1.209 (3H, d, J = 6.4 Hz, H-21), 1.028 (3H, s, H-18),
0.899 (3H, s, H-30), 0.788 (3H, d, J = 6.3 Hz, H-28), 0.472 (1H, m,
H-6β), 0.379 (1H, d, J = 3.4 Hz, H-19β), 0.162 (1H, d, J = 3.8 Hz, H-
19α).
DEDICATION
■
Dedicated to Dr. Lester A. Mitscher, of the University of
Kansas, for his pioneering work on the discovery of bioactive
natural products and their derivatives.
1
3,21-Di-(S)-MTPA ester of perviridisinol C (7): H NMR data (400
MHz, pyridine-d5) δ 4.940 (1H, m, H-3), 4.900 (1H, s, H-31b), 4.846
(1H, s, H-31a), 4.692 (1H, dd, J = 11.3, 1.6 Hz, H-21β), 4.477 (1H,
dd, J = 11.6, 3.9 Hz, H-21α), 2.259 (1H, m, H-25), 1.877 (1H, m, H-
17), 1.803 (1H, m, H-20), 1.070 (3H, d, J = 6.7 Hz, H-26), 1.062 (3H,
d, J = 7.0 Hz, H-27), 1.209 (3H, d, J = 6.4 Hz, H-21), 0.982 (3H, s, H-
18), 0.975 (3H, d, J = 6.0 Hz, H-28), 0.823 (3H, s, H-30), 0.499 (1H,
m, H-6β), 0.344 (1H, d, J = 3.7 Hz, H-19β), 0.080 (1H, d, J = 3.8 Hz,
H-19α).
Determination of the Absolute Configuration of the Diol
Moiety in Compound 8 by Snatzke’s Method. According to a
published procedure,51,52 0.3 mg of compound 8 and 0.75 mg of
Mo2(OAc)4 were dissolved in 1.0 mL of dry DMSO to give a solution,
with the ligand to metal molar ratio being around 1.0:1.2. The
electronic transitions of the metal complex in DMSO were monitored
by CD measurement immediately in the UV/vis region of 200 to 450
nm after mixing (recording a spectrum every 10 min), until a
stationary ICD spectrum was observed around 30 min later. The
inherent ECD of compound 8 was subtracted to give a corrected ICD
spectrum, and the characteristic absorption around 310 nm of the
metal complex was used as key diagnostic information to analyze the
absolute configuration of C-9 in compound 8.
Cytotoxicity Assays. All compounds isolated were evaluated
against the HT-29 human colon cancer cell line, according to a
previously described protocol.11 Compounds with a ED50 value less
than 10 μM were further tested against the CCD-112CoN normal
colon cell line, according to a published protocol.60
Enzyme-Based ELISA NF-κB Assay. The enzyme-based ELISA
NF-κB assay was carried out according to a published protocol.61,62
Rocaglamide was used as a positive control, with an ED50 value of 0.08
μM in this assay.
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ASSOCIATED CONTENT
■
S
* Supporting Information
1H, 13C, and 2D-NMR spectra of compounds 1−8, 1H NMR of
(R)- and (S)-MTPA esters of 5−7. This material is available
free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Tel: +1-614-247-8094. Fax: +1-614-247-8119. E-mail:
kinghorn@pharmacy.ohio-state.edu.
■
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Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This study was supported by grant P01 CA125066 (awarded to
A.D.K.) from NCI, NIH. We are grateful to Mr. J. Fowble,
College of Pharmacy, The Ohio State University (OSU), and
Dr. C.-H. Yuan, Campus Chemical Instrument Center, OSU,
for facilitating the acquisition of the NMR spectra. We
acknowledge Ms. N. Kleinholz, Mr. M. Apsega, and Dr. K.
Green-Church, Campus Chemical Instrument Center, OSU, for
the mass spectrometric measurements. The plant material was
collected under the terms and conditions of a Memorandum of
Agreement between the University of Illinois at Chicago and
the Institute of Ecology and Biological Resources (IEBR) of the
Vietnam Academy of Science and Technology, Hanoi, Vietnam.
Thanks are expressed to the Director of Nui Chua National
Park for permission and to the Director of IEBR for overseeing
the field operation in the collection of the plant.
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