Alkaloids from microcos paniculata with cytotoxic and nicotinic receptor antagonistic activities

Article abstract of DOI:10.1021/np3007414

Microcos paniculata is a large shrub or small tree that grows in several countries in South and Southeast Asia. In the present study, three new piperidine alkaloids, microgrewiapines A-C (1-3), as well as three known compounds, inclusive of microcosamine A (4), 7′-(3′,4′- dihydroxyphenyl)- N-[4-methoxyphenyl)ethyl]propenamide (5), and liriodenine (6), were isolated from cytotoxic fractions of the separate chloroform-soluble extracts of the stem bark, branches, and leaves of M. paniculata. Compounds 1-6 and 1a (microgrewiapine A 3-acetate) showed a range of cytotoxicity values against the HT-29 human colon cancer cell line. When evaluated for their effects on human α3β4 or α4β2 nicotinic acetylcholine receptors (nAChRs), several of these compounds were shown to be active as nAChR antagonists. As a result of this study, microgrewiapine A (1) was found to be a selective cytotoxic agent for colon cancer cells over normal colon cells and to exhibit nicotinic receptor antagonistic activity for both the hα3β4 and hα4β2 receptor subtypes.

Full text of DOI:10.1021/np3007414

Article
pubs.acs.org/jnp
Alkaloids from Microcos paniculata with Cytotoxic and Nicotinic
Receptor Antagonistic Activities
Patrick C. Still,† Bitna Yi,‡ Tatiana F. Gonzalez-Cestari,‡ Li Pan,† Ryan E. Pavlovicz,†,§ Hee-Byung Chai,†
́
Tran Ngoc Ninh,⊥ Chenglong Li,† Djaja Djendoel Soejarto,∥,∇ Dennis B. McKay,‡
and A. Douglas Kinghorn*,†
†Division of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, The Ohio State University, Columbus, Ohio 43210,
United States
‡Division of Pharmacology, College of Pharmacy, The Ohio State University, Columbus, Ohio 43210, United States
§Biophysics Graduate Program, The Ohio State University, Columbus, Ohio 43210, United States
⊥Institute of Ecology and Biological Resources, Vietnam Academy of Science and Technology, Hoang Quoc Viet, Cau Giay, Hanoi,
Vietnam
∥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: Microcos paniculata is a large shrub or small tree
that grows in several countries in South and Southeast Asia. In
the present study, three new piperidine alkaloids, micro-
grewiapines A−C (1−3), as well as three known compounds,
inclusive of microcosamine A (4), 7′-(3′,4′-dihydroxyphenyl)-
N-[4-methoxyphenyl)ethyl]propenamide (5), and liriodenine
(6), were isolated from cytotoxic fractions of the separate
chloroform-soluble extracts of the stem bark, branches, and
leaves of M. paniculata. Compounds 1−6 and 1a (microgrewiapine A 3-acetate) showed a range of cytotoxicity values against the
HT-29 human colon cancer cell line. When evaluated for their effects on human α3β4 or α4β2 nicotinic acetylcholine receptors
(nAChRs), several of these compounds were shown to be active as nAChR antagonists. As a result of this study, microgrewiapine
A (1) was found to be a selective cytotoxic agent for colon cancer cells over normal colon cells and to exhibit nicotinic receptor
antagonistic activity for both the hα3β4 and hα4β2 receptor subtypes.
Microcos paniculata L. (Malvaceae) is a member of a genus
having about 60 species distributed in Asia, Africa, South
America, and Australia, with these plants being shrubs or small
trees.1 The dried bark, roots, fruits, and leaves of Microcos
species have been used ethnomedicially to treat diarrhea and
fever, as general tonics, and as insecticides.2−4 Previous
phytochemical work on the genus Microcos has led to reports
of 2,3,6-trisubstituted piperidine alkaloids, with several
substitution patterns and stereochemical configurations in the
piperidine ring possible for such alkaloids.2−4 M. paniculata
produces these alkaloids to defend against herbivory, with
Bandara and associates having reported the insecticidal activity
of a stem bark extract against the second instar larvae of the
mosquito Aedes aegypti.3 Piperidine alkaloids of this type have
also been found outside the plant kingdom, particularly
corydendramines A and B from the marine hydroid
Corydendrium parasiticum and the solenopsins from certain
fire ants (Solenopsis spp.).5−8
out the central nervous system, peripheral nervous system, and
a number of peripheral organs, nAChRs are ligand-gated ion
channels that mediate acetylcholine-mediated signaling, and
thus contribute to a wide range of physiological processes such
as pain, cognition, fast synaptic transmission, and inflamma-
tion.9,10,12 Associated with various diseases and disorders (e.g.,
depression, anxiety, autism, ADHD, schizophrenia, and cancer),
nAChRs have been a focus of drug discovery efforts as an
attractive therapeutic target.9−11,13
Reported herein is the purification of three new piperidine
alkaloids, microgrewiapines A−C (1-3), and three known
compounds, microcosamine A (4), 7′-(3′,4′-dihydroxyphenyl)-
N-[4-methoxyphenyl)ethyl]propenamide (5), and liriodenine
(6), from the separate stem bark, branches, and leaves of M.
paniculata. All plant parts were collected in Vietnam as part of
an ongoing investigation on the discovery of natural anticancer
agents from tropical plants. Chloroform extracts were purified,
Identification of natural products with activities on neuronal
nicotinic acetylcholine receptors (nAChRs) has substantial
implications for drug discovery.9−11 Widely expressed through-
Received: October 24, 2012
Published: January 17, 2013
© 2013 American Chemical Society and
American Society of Pharmacognosy
243
dx.doi.org/10.1021/np3007414 | J. Nat. Prod. 2013, 76, 243−249

Journal of Natural Products
Article
and their anticancer potential was evaluated by testing cytotoxic
effects against the HT-29 human colon cancer cell line.14 The
structures of the three new compounds were established by
spectroscopic data interpretation. Esterification at the C-3
hydroxy group using the Mosher ester method allowed
unambiguous assignment of the absolute configuration of
compound 1. The three known compounds 4−6 were
identified by spectroscopic analysis and comparison of the
data obtained with literature values.4,15,16 Structural similarity of
compounds 1−4 to the previously identified nAChR antagonist
1-(3-phenylpropyl)piperidin-3-yl)methyl [1,1′-biphenyl]-2-car-
boxylate (KAB-18), along with growing evidence to support the
involvement of nAChRs in various aspects of cancer, provided a
rationale to examine the effects of 1−6 on nAChRs.17−19
As a result of this study, microgrewiapine A (1) was found to
be a cytotoxic agent against HT-29 human colon cancer cells
and to exhibit nicotinic receptor antagonistic activity for both
the hα3β4 and hα4β2 receptor subtypes. This is the first report
of cytotoxicity and nAChR modulatory activity of piperidine
alkaloids from a plant in the genus Microcos.
molecular formula of C17H29NO was determined from the
molecular ion peak at m/z 264.2329 [M + H]+ (calcd for
C17H30NO, 264.2327) in the HRESIMS, which supported the
presence of a single nitrogen atom in the molecule. The IR
spectrum showed bands at 3402, 2929, and 1629 cm−1,
indicative of hydroxy group absorption and olefinic C−H
stretching, respectively. The UV spectrum exhibited a
maximum at 270 nm, suggesting conjugation in the molecule.
The 1H NMR and 13C NMR spectra (600 MHz, CDCl3, Table
1) showed signals for six methine groups in the region δH
5.53−6.16, along with resonances at δH 2.09 (2H, dt, J = 7.2,
6.9 Hz, H-7′), 1.29−1.38 (4H, H-8′, H-9′), and 0.89 (3H, t, J =
7.2 Hz, H-10′), which correlated in the 1H-13C HSQC
spectrum to six olefinic methine carbons [δC 130.1−136.7],
three methylene carbons [δC 32.5 (C-7′); 31.4 (C-8′); 22.0 (C-
9′)], and a methyl carbon [δC 14.1 (C-10′)], respectively.
These NMR signals indicated the presence of a deca-trienyl
group in the molecule of 1. Double-bond geometry at Δ1′,2′
and Δ5′,6′ was determined to be trans on the basis of the large
coupling constants J1′2′ = 14.6 Hz and J5′6′ = 14.5 Hz. For Δ3′,4′,
the configuration could not be determined by coupling
1
constants due to signal overlap. Differences in H and 13C
chemical shifts between (1′E,3′E,5′E) and (1′E,3′Z,5′E)
geometries, however, can be used to elucidate the Δ3′,4′
configuration. If the conjugated triene system adopts a
(1′E,3′E,5′E) geometry, the NMR chemical shifts of H-2′/H-
5′ and C-2′/C-5′ are typically around δH 6.0 ppm and δC 130
ppm, respectively.20 If the conjugated triene instead adopts a
(1′E,3′Z,5′E) geometry, δH H-2′/H-5′ shifts ∼0.4 ppm
downfield and δC C-2′/C-5′ shifts ∼5 ppm upfield.20 The
triene system of 1 shows chemical shifts of δH 6.03−6.16 (H-2′,
H-5′) and δC 130.1−132.6 (C-2′, C-5′), which support a trans
configuration for Δ3′,4′. The remaining 1H and 13C NMR
signals were attributed to a methyl group [δH 1.26 (3H, d, J =
6.1 Hz, CH3-2); δC 16.4], two methylene groups [δH 2.03 (1H,
m, H-4α), 1.29−1.38 (1H, H-4β), 1.49 (1H, ddd, J = 10.1, 3.0,
3.0 Hz, H-5α), 1.63 (1H, m, H-5β); δC 33.5 (C-4), 31.3 (C-5)],
an oxygen-bearing methine group [δH 3.27 (1H, ddd, J = 10.8,
8.9, 4.5, H-3); δC 72.6 (C-3)], two nitrogen-bearing methine
RESULTS AND DISCUSSION
■
Microgrewiapine A (1) was obtained as colorless needle
crystals, mp 127−128 °C, [α]15 +15.4 (c 0.1, MeOH). A
D
a
1
Table 1. H NMR and 13C NMR Data for 1−3
1
2
3
no.
δC
66.1
δH (J in Hz)
δC
76.5
δH (J in Hz)
δC
68.6
δH (J in Hz)
2
3
1.83, dq (8.9, 6.1)
3.27, ddd (10.8, 8.9, 4.5)
2.03, m
2.86, dq (10.7, 6.1)
3.90, ddd (11.2, 10.5, 4.8)
1.46, m
3.05, dq (12.7, 6.5)
3.82, brs
72.6
33.5
67.4
33.0
71.1
32.1
4α
1.57, m
b
4β
1.29−1.38
2.13, m
2.01, m
5α
31.3
1.49, ddd (10.1, 3.0, 3.0)
26.6
2.33, m
23.8
1.56, m
5β
1.63, m
1.62, m
2.67, ddd (12.0, 4.1, 3.6)
3.53, ddd (11.4, 9.4, 1.9)
5.98, dt (15.1, 7.0)
6
67.6
2.48, ddd (11.2, 8.7, 3.0)
5.53, dt (14.6, 8.8)
78.8
3.44, m
79.5
1′
2′-5′
6′
136.7
127.7
5.94, dt (15.3, 8.9)
127.9
b
b
b
130.1−132.6
135.7
6.03−6.16
5.71, dd (14.5, 7.4)
128.8−135.8
137.6
6.05−6.26
5.74, dd (14.2, 7.2)
129.2−136.1
137.8
5.96−6.25
5.72, dd (15.2, 9.0)
7′
32.5
2.09, dt (7.2, 6.9)
32.9
2.09, dt (7.2, 6.9)
32.7
2.09, dt (7.0, 6.2)
b
b
b
8′, 9′
10′
CH3-2
N-CH3
31.4; 22.0
14.1
1.29−1.38
30.9; 21.8
14.3
1.26−1.41
31.5; 22.5
14.1
1.24−1.39
0.89, t (7.2)
1.26, d (6.1)
2.21, s
0.89, t (7.0)
1.57, d (6.0)
2.95
0.89, t (7.1)
1.66, d (6.5)
2.86, s
16.4
10.6
13.5
40.4
51.1
54.1
a
Compound 1 measured at 600 MHz, compounds 2 and 3 measured at 400 MHz. All compound NMR spectra obtained in CDCl3 with TMS as
b
internal standard; J values (Hz) are given in parentheses. Assignments supported with H−1H COSY, HSQC, and HMBC spectra. Multiplicity
patterns unclear due to signal overlapping.
1
244
dx.doi.org/10.1021/np3007414 | J. Nat. Prod. 2013, 76, 243−249

Journal of Natural Products
Article
groups [δH 2.48 (1H, ddd, J = 11.2, 8.7, 3.0 Hz, H-6), 1.83 (1H,
dq, J = 8.9, 6.1 Hz, H-2); δC 67.6 (C-6), 66.1 (C-2)], and a N-
CH3 group [δH 2.21 (3H, s, N-CH3); δC 40.4]. The oxygen-
1
bearing methine group at δH 3.27 (H-3) showed a H−1H
COSY correlation with the nitrogen-bearing methine group at
δH 1.83 (H-2). The methylene signals δH 1.49 and 1.63,
1
corresponding to the C-5 protons, exhibited a H−1H COSY
correlation with the nitrogen-bearing methine group at δH 2.48
(C-6). A heteronuclear 1H−13C HMBC three-bond correlation
between CH3-2 (δH 1.26) and C-3 (δC 72.6), a four-bond
correlation between the N-CH3 (δH 2.21) group and CH3-2,
and a two-bond correlation between both C-5 protons and the
nitrogen-bearing methine group at δH 2.48 (C-6) were
observed. The chemical shift values and connectivity patterns
Figure 2. Key COSY, HMBC, and NOESY correlations of 1.
N, and aliphatic amine oxide functionalities, respectively. The
1H NMR and 13C NMR spectra (400 MHz, CDCl3, Table 1)
showed that signals in the piperidine ring were shifted
downfield, compared to 1, due to the presence of an N-oxide
moiety. Downfield shifts were pronounced in both the
nitrogen-bearing methine signals at δH 2.86 (1H, dq, J =
10.7, 6.12 Hz, H-2) and 3.44 (1H, m, H-6) and the oxygen-
bearing methine signal at δH 3.90 (1H, ddd, J = 11.2, 10.5, 4.8
Hz, H-3). Despite the downfield shifts of these signals, the
splitting patterns were comparable to the analogous signals in
compound 1 (Table 1). The coupling constant between the
oxygenated methine at δH 3.90 (H-3) and the signal at δH 2.86
(H-2) was 10.7 Hz, indicating a trans configuration between H-
3 and H-2.
1
1
from the H−1H COSY and H−13C HMBC spectra indicated
the presence of an N-methyl-2-methyl-3-piperidinol ring.2−4
The 1H NMR signal at δH 5.53 (1H, dt, J = 14.6, 8.8 Hz, H-1′)
was coupled to the nitrogen-bearing methine signal at δH 2.48
1
(C-6) with a magnitude of 8.7 Hz and also showed a H−1H
COSY correlation to the same methine proton, indicating that
the deca-1E,3E,5E-trienyl group is attached to the N-methyl-2-
methyl-3-piperidinol ring at C-6. The absolute configuration at
the C-3 hydroxy group position was accomplished using a
Mosher esterification procedure.21,22 Two portions of 1 (each
1.0 mg) were treated with (S)-(+)-α- and (R)-(−)-α-methoxy-
α-(trifluoromethyl)phenylacetyl chloride (14 μL) in deuterated
pyridine (0.5 mL) directly in separate NMR tubes at room
temperature, affording the (R)- and (S)-MTPA esters,
Two configurations at the nitrogen atom of 2 are possible.
1
For a 1R configuration, a H−1H NOESY correlation between
N-CH3 and H-3 would not be expected. Indeed, no NOE
correlation was observed between the signals at δH 2.95 (N-
1
CH3) and 3.90 (H-3) (Figure 3). Strong H−1H NOESY
respectively.21,22 Observed chemical shift differences (ΔδS−R
)
indicated the absolute configuration at the OH-3 group of 1 to
be R (Figure 1).
Figure 3. Key NOESY correlations and energy-minimized 3D
structure of 2.23
correlations were seen between the N-methyl (δH 2.95) and H-
2 (δH 2.86) signals, due to the proximity within 4 Å of these
functionalities (Figure 3). In addition, 1H−1H NOESY
correlations were observed between the signals at δH 2.86
(H-2) and 3.44 (1H, m, H-6), indicating these protons to be
located on the same face of the piperidinol ring. The 1H NMR
Figure 1. Chemical shift difference values, δ(S−R), for protons on the
esterified piperidinol ring of 1, as established by the Mosher ester
method.
1
coupling patterns and H−1H NOESY correlations observed
With the absolute configuration at the OH-3 position
determined, analysis of coupling constants and 1H−1H
NOESY correlations established the relative conformation of
the remaining substituents of the piperidinol ring. The coupling
constant between H-3α and the signal at δH 1.83 (H-2) was 8.9
Hz, indicating the substituents were trans configured. Strong
1H−1H NOESY correlations between H-2 and the signal at δH
2.48 (H-6) suggested that the latter proton is oriented on the
β-face of the piperidinol ring with H-2 (Figure 2). Accordingly,
microgrewiapine A (1) was assigned structurally as (2S,3R,6S)-
6-((1E,3E,5E)-deca-1,3,5-trien-1-yl)-1,2-dimethylpiperidin-3-ol.
Microgrewiapine B (2) was obtained as a clear, amorphous
for 2 were supported by in silico MP2 energy minimization
studies using the 6-311G(d,p) basis set, implemented in the
Guassian 09 program.23 Relative energies of the 1R and 1S
enantiomers of 2 in the gas phase, ethanol, and water were
computed, and the 1R enantiomer was found to be the more
energetically favorable. Energy difference values between the 1R
and 1S enantiomers of 2 were calculated to be 2.76, 1.08, and
0.94 kcal/mol for the gas phase, ethanol, and water,
respectively. The solvent effects were calculated with a
polarized continuum model that implicitly calculates the effects
the solvent has on the molecule. Microgrewiapine B (2) was
assigned structurally, therefore, as (2S,3R,6S)-6-(1E,3E,5E)-
deca-1,3,5-trien-1-yl)-3-hydroxy-1,2-dimethylpiperidine 1-
oxide.
solid, mp 134−136 °C, [α]25 +4.0 (c 0.1, MeOH). A
D
molecular formula of C17H29NO2 was determined from the
molecular ion peak at m/z 280.2269 [M + H]+ (calcd for
C17H30NO2, 280.2276) in the HRESIMS. The IR spectrum
showed absorption bands at 3400, 2967, 1670, 1204, and 1000
cm−1, indicative of the presence of hydroxy, olefinic C−H, C−
Microgrewiapine C (3) was obtained as colorless needle
crystals, mp 130−131 °C, [α]25 +77.8 (c 0.1, MeOH). A
D
molecular formula of C17H29NO2 was determined from the
molecular ion peak at m/z 280.2281 [M + H]+ (calcd for
245
dx.doi.org/10.1021/np3007414 | J. Nat. Prod. 2013, 76, 243−249

Journal of Natural Products
Article
C17H30NO2, 280.2277) in the HRESIMS. The UV and IR
spectra were closely comparable to those of the 3R enantiomer,
2. The 1H NMR signal at δH 3.82 (1H, brs, H-3) appeared as a
broad singlet when compared to the ddd pattern as observed
for 2. The methine signal at δH 3.05 (1H, dq, J = 12.7, 6.5 Hz,
H-2) would be expected to cause a split pattern for H-3;
however no peak multiplicity was observed for this proton. The
Table 2. Percent Inhibition of nAChR Activity by a Single
Concentration of Compounds 1−6 and 1a
hα3β4 nAChR
hα4β2 nAChR
ab
ab
,
,
compound
% inhibition
% inhibition
1
58.2 9.2
82.7 3.3
74.0 14.2
79.3 6.0
67.3 28.0
59.0 7.8
21.8 6.2
13.3 24.5
44.1 22.3
61.0 27.9
91.8 5.6
39.0 11.7
2
1
3S configuration was supported by a strong H−1H NOESY
3
82.8 11.5
53.0 9.5
14.0 6.6
19.3 24.4
4
correlation between the signals at δH 3.05 (1H, dq, J = 12.7, 6.5
Hz, H-2), 3.82 (1H, brs, H-3), and 3.53 (1H, ddd, J = 11.4, 9.4,
1.9 Hz, H-6), indicating that these substituents have the same
5
6
c
1
orientation. In addition, a strong H−1H NOESY correlation
1a
NT
d
d
D-tubocurarine
54.0 23.5
88.3 13.3
between δH 2.86 (3H, s, N-CH3) and 3.82 (1H, brs, H-3) was
observed, confirming the opposite configuration from that of 2,
for which this correlation was absent. Microgrewiapine C (3)
was therefore assigned as (2S,3S,6S)-6-((1E,3E,5E)-deca-1,3,5-
trien-1-yl)-3-hydroxy-1,2-dimethylpiperidine 1-oxide.
mecamylamine
d
e
KAB-18
NA
a
b
Percent inhibition was determined at 10 μM. Values represent
c
d
means SD, n = 2−5. NT = not tested. Known nicotinic receptor
antagonist. NA = no activity at 10 μM.
e
As part of an ongoing investigation on the discovery of
natural anticancer agents from tropical plants, compounds 1−6
were evaluated for their cytotoxic activity against HT-29 human
colon cancer cells. Compound 1 was found to be the most
active in inhibiting the proliferation of HT-29 cancer cells, with
an IC50 value of 6.8 μM. Screening of 1 against CCD-112CoN
normal colon cells resulted in an IC50 value of 30.4 μM.
Therefore, the selectivity ratio of compound 1 for cancerous
colon cells versus normal colon cells was found to be
approximately 4. Microgrewiapine 3-acetate (1a) and com-
pounds 2, 3, 5, and 6 showed IC50 values greater than 10 μM
when evaluated against the HT-29 cancer cell line and were
therefore deemed inactive.
Structural similarities between 1−4 and 1-(3-phenylpropyl)-
piperidin-3-yl)methyl [1,1′-biphenyl]-2-carboxylate, which was
previously identified as a novel ligand of neuronal nicotinic
receptors, prompted an examination of the effects of these
compounds on nAChRs.17 In particular, the phenyl rings, the
three-carbon aliphatic chain, and the piperidine ring in KAB-18
are comparable to the triene side-chain, aliphatic tail, and
piperidinol ring found in 1−4. The effects of 1−6 and 1a on
human nAChR activity were tested using a functional calcium
accumulation assay with HEKtsA201 cells stably expressing
either human hα4β2 or hα3β4 nAChRs. When assayed at a
single concentration (10 μM), all of the natural products (1−6)
and the semisynthetic acetate analogue (1a) inhibited the
epibatidine-stimulated calcium accumulation on hα4β2 or
hα3β4 nAChRs (Figure 4, Table 2). This supports their
classification as antagonists of human nAChRs. Specifically,
compound 1 exhibited approximately 60% and 70% inhibition
of hα4β2 and hα3β4 nAChR activity, respectively. In
comparison, the acetate 1a inhibited approximately 40% of
the hα4β2 nAChR response (compound 1a was not tested
against hα3β4). Analogues incorporating an N-oxide moiety (2
and 3) also caused ∼80% inhibition for both the hα4β2 and
hα3β4 nAChRs. Microcosamine A (4) inhibited 53.7% and
59.0% of the hα3β4 and hα4β2 activity, respectively.
Compounds 5 and 6 produced only weak inhibition of activity
for both the hα3β4 and hα4β2 nAChRs (less than 20%
inhibition). The acetate analogue 1a was synthesized from 1
and shows that compounds with a free hydroxy group at C-3
have more potent activity as nAChR antagonists.
It is also worth noting the mechanisms by which KAB-18 exerts
antagonistic effects on nAChRs. Functional studies with KAB-
18 have shown its insurmountable antagonism, suggesting that
its activity on nAChRs involves a noncompetitive and allosteric
mechanism.17 That is, KAB-18 does not directly compete with
the endogenous ligand acetylcholine for its binding site, but
instead acts at a distinct (allosteric) site. Therefore, it has been
defined as a negative allosteric modulator of nAChRs. As
allosteric modulators can offer significant therapeutic advan-
tages over competitive ligands in clinical practice,13 additional
attention is warranted on the discovery of natural products,
such as compounds 1−4, which show activity on nAChRs, with
a focus on the structural similarities to known allosteric
modulators.
Given the evidence implicating nAChRs in the development
and progression of cancer, the cytotoxicity of 1−3 against HT-
Figure 4. Effects of compounds 1−6 on recombinant nAChRs. An
inhibition assay for each compound was performed using HEK tsA201
cells stably expressing hα3β4 and hα4β2 nAChRs. Cells were loaded
with Calcium 5 NW dye and stimulated with 1 μM epibatidine in the
presence of a single concentration of each test compound (10 μM), as
described in the Experimental Section. Results are expressed as the
percentage of the control (epibatidine)-stimulated peak fluorescence
level. Values represent the means SD of three to five experiments
performed in triplicate. Percentage inhibitions achieved by the test
compounds are shown in Table 2.
The extent of inhibition shown by these natural products is
similar to those of other established nAChR antagonists
including bupropion, dihydro-β-erythroidine, d-tubocurarine,
mecamylamine, tetracaine, and KAB-18-like molecules.17,24−26
246
dx.doi.org/10.1021/np3007414 | J. Nat. Prod. 2013, 76, 243−249

Journal of Natural Products
Article
hexanes, to EtOAc, to MeOH, producing 11 pooled fractions (F1−
F11). Fraction F6 (eluted using EtOAc/10% MeOH) had the most
pronounced cytotoxicity (IC50 = 6.0 μg/mL) and was visualized with
Dragendorff’s reagent, indicating the presence of alkaloids. Isocratic
column chromatographic purification of F6 using silica gel (43−60
mesh; CHCl3/MeOH, 15:1) as adsorbent provided 18 further
fractions (R1−R18). Fraction R7 was subjected to Sephadex LH-20
column chromatography with elution by 100% MeOH, to afford
colorless needles of compound 1 (7 mg).
29 human colon cancer cells may be influenced by nAChR
modulation. Indeed, there is growing evidence implicating
nAChRs in various aspects of cancer development and
progression. nAChRs can influence the function, growth, and
survival of cancer cells by regulation of a number of
neurotransmitters and growth, angiogenic, and neurotrophic
factors (e.g., dopamine, glutamate, γ-aminobutyric acid
(GABA), serotonin, BDNF, VEGF, HGF, TGF-α, TGF-β,
and PDGF).18,27 In addition, nAChR activation and the
resulting increases in intracellular Ca2+ concentrations lead to
stimulation of a number of signaling pathways mediating cell
proliferation (e.g., Src kinase cascade, PI3-Akt pathway, ERK/
MAP kinase cascade, NF-κB pathway, and cyclic AMP signaling
cascade).28 Through activation of antiapoptotic proteins and
induction of NF-κB, nAChRs also directly influence cell
survival. Although the in vitro cytotoxicity of 1−3 may be
influenced by nAChR modulation, exploration of this
mechanism is outside the scope of this investigation, but
presents a worthy future direction.
Owing to the implication of nAChRs in various diseases and
disorders, the discovery of natural compounds with antagonistic
activity on nAChRs is of great relevance. In particular, the
involvement of nAChRs in various aspects of cancer growth
and progression suggests that the cytotoxic effects of
compound 1 on HT-29 human colon cancer cells, combined
with the nAChR modulation by compounds 1−3, warrant
further study. The role of nAChRs in physiological and
pathophysiological processes in the central nervous system
makes these types of molecules an interesting starting point for
drug discovery in this area.
From the branches of M. paniculata, the MeOH extract (39 g) was
concentrated in vacuo and partitioned to afford a hexanes extract (7 g)
and a CHCl3 extract (6 g). Bioactivity-guided fractionation of the
CHCl3-soluble extract (D3) was carried out using the HT-29 human
colon cancer cell line and showed an IC50 value of 13.1 μg/mL. D3
was chromatographed over silica gel (230−400 mesh), with a step
gradient from hexanes, to EtOAc, to MeOH, producing nine fractions
(F1−F9). Fractions F7 and F8 (eluted with EtOAc/10% MeOH) had
the most pronounced cytotoxicity, with IC50 values of 11.6 and 14.6
μg/mL, respectively. The use of Dragendorff’s reagent suggested that a
group of alkaloids in F7 and F8 could be contributing to the observed
cytotoxicity. Purification of F7 employed a reversed-phase (C18)
HPLC gradient separation (30:70 CH3CN/H2O to 60:40 CH3CN/
H2O) over 40 min, affording compound 5 (tR = 22 min; 3 mg) as the
most highly absorbing analyte in the mixture at 205 nm. Passage of F8
over silica gel (43−60 mesh) (eluted with CHCl3/MeOH, 15:1)
produced 34 fractions, which were combined on the basis of TLC
analysis to afford two fractions (R1 and R2). Fraction R1 yielded a
yellow, crystalline material, which was purified by reversed-phase
HPLC (C18) (5:95 MeOH/H2O to 95:5 MeOH/H2O) over 60 min,
affording compound 6 (tR = 30 min; 1.3 mg). Fraction R2 was purified
by passage over a silica gel (40−63 μm mesh) column (CHCl3/
MeOH, 10:1 to 5:1), affording four fractions (T1−T4). T2 was
purified by reversed-phase HPLC (C18) (48:52 MeOH/H2O with
0.01% NH4OH in H2O mobile phase), to afford microgrewiapine B (2,
tR = 87 min; 2 mg) as the most highly UV absorbing analyte in the
mixture at 270 nm. T3 was purified by reversed-phase HPLC (C18)
(30:70 CH3CN/H2O with 0.01% NH4OH in H2O mobile phase) to
afford microgrewiapine C (3, tR = 79 min; 1.5 mg).
EXPERIMENTAL SECTION
■
General Experimental Procedures. Melting points were
determined on a Fisher Scientific melting point apparatus and are
uncorrected. Optical rotations were determined on a PerkinElmer 343
automatic polarimeter (sodium lamp). UV spectra were obtained with
a PerkinElmer Lambda 10 UV/vis spectrometer. IR spectra were
measured on a Thermo Scientific Nicolet 6700 FT-IR spectrometer.
NMR spectroscopic data were run on Bruker Avance-300, 400, or 600
MHz spectrometers, and the data were processed using MestReNova
6.0 software. Accurate mass values were obtained on a Micromass LCT
ESI spectrometer. Sodium iodide was used for mass calibration for a
calibration range of m/z 100−2000. Silica gel (43−60 mesh; Sorbent
Technologies, Atlanta, GA, USA) was used for column chromatog-
raphy. Analytical TLC was performed on precoated 250 μm thickness
silica gel plates (UV254, glass backed; Sorbent Technologies). HPLC
was conducted using a Hitachi LaChrom Elite system composed of a
L-2450 diode array detector, a L-2200 autosampler, and a L-2130
pump. Waters X-Bridge C18 5 μm (4.6 × 150 mm) analytical and 5 μm
(10 × 150 mm) semipreparative HPLC columns were used.
From the leaves of M. paniculata, the MeOH extract (200 g) was
concentrated in vacuo and partitioned to afford a hexanes extract (21
g) and a CHCl3 extract (12 g). Bioactivity-guided fractionation of the
CHCl3-soluble extract (D3) was carried out using an HT-29 human
colon cancer cell line and showed an IC50 value of 2.9 μg/mL. D3 was
chromatographed over silica gel (230−400 mesh), with a step gradient
from hexanes, to EtOAc, to MeOH, producing 11 major fractions
(F1−F11). F11 (eluted with EtOAc/40% MeOH) had the most
pronounced cytotoxicity, with an IC50 value of 1.7 μg/mL. F11 was
loaded onto a Diaion HP-20 column and eluted with 50:50 MeOH/
H2O to obtain a Dragendorff-positive alkaloidal fraction. The retained
chlorophyll portion was eluted with pure acetone and stored. F11 was
then chromatographed over a silica gel column (43−63 mesh) with a
gradient elution (EtOAc/CH3CN/H2O/MeOH, 70:10:5:5, increasing
to 60:20:20:20 of this same solvent mixture), affording four fractions
(R1−R4). R1 was further separated by reversed-phase HPLC (C18)
(56:44 CH3CN/H2O with 0.01% NH4OH in H2O mobile phase), to
afford compound 4 (tR = 46 min; 4.5 mg) along with a minor impurity.
Crude 4 was subjected to silica gel (43−63 mesh) chromatography
using CHCl3/MeOH, 8:1, to afford 4 (0.6 mg) as an amorphous solid.
Microgrewiapine A (1): colorless needle crystals; mp 127−128 °C;
Plant Material. Separate samples of the stem bark, branches and
leaves of Microcos paniculata were collected in Kego Nature Reserve
(18°08.087′ N; 105°56.020′ E; 40 m alt.), Hatinh Province, Vietnam,
in December 2008, by T.N.N., Vuong Tan Tu, and D.D.S., who also
identified this plant. A voucher specimen (collection number DDS
14261) has been deposited in the John G. Searle Hebarium of the
Field Museum of Natural History, Chicago, Illinois.
Extraction and Isolation. The dried and milled stem bark (646
g), branches (1.1 kg), and leaves (770 g) of M. paniculata were
extracted separately with methanol. For the stem bark, the MeOH
extract (179 g) was concentrated in vacuo and partitioned to afford a
hexanes extract (2 g) and a CHCl3 extract (8 g). Bioactivity-guided
fractionation of the CHCl3-soluble extract (D3) was carried out using
an HT-29 human colon cancer cell line and showed an IC50 value of
9.9 μg/mL. D3 was chromatographed using vacuum-liquid chroma-
tography over silica gel (230−400 mesh) with a step gradient from
[α]25 +15.4 (c 1.0, MeOH); UV (MeOH) λmax (log ε) 270 (3.87)
D
nm; IR (film) νmax 3402, 2956, 2923, 2864 cm−1; 1H NMR (600 MHz,
CDCl3) and 13C NMR (150 MHz, CDCl3) data, see Table 1;
HRESIMS m/z 264.2329 [M + H]+ (calcd for C17H30NO, 264.2327).
Preparation of Microgrewiapine A 3-Acetate (1a). A catalytic
amount of N,N-dimethylaminopyridine (0.1 equiv) and 5 mg of 1 were
placed in a round-bottomed flask under argon. Anhydrous dichloro-
methane (500 μL) was added, and the mixture was cooled to 0 °C.
Triethylamine (2 equiv) was added, followed by dropwise addition of
acetic anhydride (3 equiv), and the reaction was warmed to room
temperature and stirred for 3 h. The reaction was quenched with a
247
dx.doi.org/10.1021/np3007414 | J. Nat. Prod. 2013, 76, 243−249

Journal of Natural Products
Article
saturated aqueous NaHCO3 solution at 0 °C. The mixture was
extracted two times with ethyl acetate (50 mL), and the combined
organic layers were washed with brine, dried over Na2SO4, filtered, and
evaporated in vacuo. The crude product was chromatographed over
silica gel (CHCl3/MeOH/AcOH, 10:1:0.1) to yield 1a as a white,
amorphous solid (3 mg, 60%).
plates by the addition of 100 μL of cold 20% trichloroacetic acid and
incubated at 4 °C for 30 min. Next, the plates were washed three times
with tap water and dried overnight. The fixed cells were stained for 30
min at room temperature with 0.4% (w/v) sulforhodamine B, an
anionic protein stain in 1% acetic acid, and rinsed three times with 1%
acetic acid to remove unbound dye and allowed to dry. The bound dye
was then solubilized with 10 mM unbuffered Tris base (pH 10, 200
μL/well) for 5 min on a shaker. Absorbance at 515 nm was measured
with a Bio-Tek μQuant microplate reader. The IC50 values of test
samples in serial dilutions were calculated using nonlinear regression
analysis (TableCurve 2D v4; AISN Software, Inc., Mapleton OR,
USA). Paclitaxel exhibited an IC50 value of 23.0 μM against CCD-
112CoN normal colon cells.
Microgrewiapine A 3-acetate (1a): amorphous solid; [α]25D +19.5
1
(c 1.0, MeOH); UV (MeOH) λmax (log ε) 259 (4.29) nm; H NMR
(400 MHz, CDCl3) δH 0.87 (3H, t, J = 7.0 Hz, H-10′), 1.11 (3H, d, J =
5.8, CH3-2α), 1.30 (1H, m, H-2β), 1.31−1.42 (1H, m, H-4α), 1.31−
1.42 (2H, m, H-8′, 9′), 1.35 (1H, m, H-5α), 1.55 (1H, ddd, J = 13.2,
3.3, 2.0 Hz, H-5β), 1.69 (1H, m, H-4β), 2.05 (3H, s, OCOCH3), 2.12
(1H, m, H-7′), 2.21 (3H, s, N-CH3), 2.54 (1H, ddd, J = 11.2, 8.6, 3.4
Hz, H-6β), 4.48 (1H, ddd, J = 10.6, 7.3, 2.6 Hz, H-3α), 5.55 (1H, dt, J
= 14.4, 8.8, H-1′), 5.72 (1H, dd, J = 7.1, 7.0, H-6′), 6.04−6.18 (4H, m,
H-2′−5′); 13C NMR (100 MHz, CDCl3) δC 170.7 (OCOCH3), 137.4
(C-1′), 136.7 (C-6′), 130.8−133.6 (C-2′−C-5′), 75.1 (C-3), 67.2 (C-
6), 33.7 (C-7′), 32.3 (C-4), 31.4 (C-8′), 30.3 (C-5), 22.4 (C-9′), 21.8
(C-2), 14.7 (C-10′); HRESIMS m/z 306.2419 [M + H]+ (calcd for
C19H31NO2, 306.2433).
Calcium Accumulation Assays. Calcium accumulation assays
were performed with HEK ts201 cells stably expressing either human
α3β4 nAChRs (hα3β4 nAChRs) or human α4β2 nAChRs (hαβ2
nAChRs) (obtained from Professor Jon Lindstrom, University of
Pennsylvania, Philadelphia, PA, USA), using a previously reported
procedure.17,24,26 Cells were plated at a density of 2.0 to 2.3 × 105 cells
per well in poly-L-ornithine-coated 96-well culture plates 24 h prior to
the assay. On the day of experiments, cells were washed with 100 μL of
HEPES-buffered Krebs (HBK) and incubated with 40 μL of HBK and
40 μL of Calcium 5 NW dye (Molecular Devices, Sunnyvale, CA,
USA) (1 h, room temperature). Changes in intracellular calcium levels
resulting from nAChR activation were then measured simultaneously
during and after the course of drug treatment period using a fluid
handling integrated fluorescence plate reader (FlexStationII, Molecular
Devices, Sunnyvale, CA, USA). In order to evaluate the antagonistic
effects of compounds, cells were treated with the compounds at the
first addition (20 s) and with 1 μM of the nAChR agonist epibatidine
(Thermo Fisher Scientific, Pittsburgh, PA, USA) at the second
addition (60 s) in the continued presence of the compounds. For the
quantification of compound antagonistic effects, a control-agonist-
treated group and a control-sham group were included. For the
control-agonist group, HBK (40 μL) and 1 μM epibatidine solution
(40 μL) were added at the first and second treatment period,
respectively. The control-sham group was treated with HBK (40 μL)
at the first and second treatment period, respectively. The changes in
fluorescence were measured continuously for 120 s from the bottom of
the well at an excitation of 485 nm and emission of 525 nm at ca. 1.5 s
intervals. Functional responses were quantified by first calculating the
net fluorescence (the difference between control-agonist-treated group
and control-sham group), and results were expressed as a percentage
of control-agonist group (1 μM epibatidine). All functional data were
calculated from the number of observations performed in triplicate.
For pharmacological evaluation, compounds were initially prepared
with 100% DMSO (0.01 M stocks). Further dilutions of compounds
were made in HBK buffer (≤100 μM).
Preparation of MTPA Esters of Microgrewiapine A (1). A
modified Mosher ester procedure carried out directly in NMR tubes
was used as previously reported.21,22After drying compound 1 (2 mg,
0.0078 mmol) in the NMR tube overnight in a desiccator,
approximately 400 μL of pyridine-d5 were added under argon.
Separate 2 mg portions of 1 were treated with (S)-α-methoxy-α-
(trifluoromethyl)phenylacetyl chloride and (R)-MTPA-Cl (0.075
mmol, 10 equiv) at room temperature. After 6 h, the reaction was
1
1
complete and the H NMR (400 MHz, CDCl3) and H−1H COSY
spectra were recorded. Ambiguous and overlapping signals were not
used for the ΔδS−R calculations.21,22 1H NMR data of (R)-MTPA ester
of 1 (400 MHz, pyridine-d5): δ 2.60 (3H, s, N-CH3), 2.37 (1H, m, H-
5), 1.89 (1H, m, H-4a), 1.50 (2H, m, H-4b), 1.25 (3H, m, CH3-2). 1H
NMR data of (S)-MTPA ester of 1 (400 MHz, pyridine-d5): δ 2.56
(3H, s, N-CH3), 2.42 (1H, m, H-5), 1.91 (1H, m, H-4a), 1.64 (2H, m,
H-4b), 1.12 (3H, m, CH3-2).
Microgrewiapine B (2): amorphous solid; mp 134−136 °C; [α]25
D
+4.0 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 272 (3.97) nm; IR
1
(film) νmax 3400, 2967, 1670, 1204, 1000 cm−1; H NMR (400 MHz,
CDCl3) and 13C NMR (100 MHz, CDCl3) data, see Table 1;
HRESIMS m/z 280.2269 [M + H]+ (calcd for C17H30NO2, 280.2276).
Microgrewiapine C (3): colorless needle crystals; mp 130−131 °C;
[α]25 +77.8 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 274 (3.92)
D
nm; IR (film) νmax 3400, 2967, 1670, 1204, 1000 cm−1; 1H NMR (400
MHz, CDCl3) and 13C NMR (100 MHz, CDCl3) data, see Table 1;
HRESIMS m/z 280.2281 [M + H]+ (calcd for C17H30NO2, 280.2277).
Cytotoxicity Assays. Isolation was directed using a gatekeeper
assay that tests cytotoxicity toward HT-29 human colon cancer cells.
Compounds 1−6 were evaluated in this assay and showed a range of
activities according to a previously described protocol.14 Paclitaxel was
used as a positive control and exhibited an IC50 value of 0.001 μM.
Compound 1 and paclitaxel were screened also against a normal
colon cell line (CCD-112CoN). Compounds with an IC50 value less
than 10 μM against HT-29 cells were further tested against the CCD-
112CoN normal colon cell line. For this assay, CCD-112CoN
noncancerous human colon cells (ATCC, CRL-1541) were cultured in
MEME medium (Hyclone, Logan, UT, USA) supplemented with 10%
FBS, PSF, 1.0 mM sodium pyruvate, 0.1 mM nonessential amino acid,
and 1.5 g/L sodium bicarbonate and placed in a humidified incubator
with an atmosphere of 95% air and 5% CO2 at 37 °C. Cells were
trypsinized and split for subculture when they reached near-confluent
state (5 days or later). Upon reaching about 70% confluence, the
medium was changed and the cells were used for the test procedure
one day later. The harvested cells, after appropriate dilution, were
seeded per well in 96-well CCD-112CoN normal colon cells (7600
cells/190 μL) plates using complete medium and were treated with the
test samples (10 μL/well in triplicate) at various concentrations. Test
samples were performed using 10% DMSO as the solvent. Then, 10
μL of 10% DMSO was also added to the control wells. After
incubation for 3 days at 37 °C in 5% CO2, the cells were fixed to the
ASSOCIATED CONTENT
■
S
* Supporting Information
1
NMR spectroscopic data for compounds 1−3 including H,
13C, and 2D NMR spectra and ESIMS/MS data of the new
compounds 1−3. 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: +1-614-247-8081. E-mail:
kinghorn.4@osu.edu.
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This study was supported, in part, by grant P01 CA125066
(awarded to A.D.K.) from NCI, NIH. Microcos paniculata
samples were collected under the terms of an agreement
248
dx.doi.org/10.1021/np3007414 | J. Nat. Prod. 2013, 76, 243−249

Journal of Natural Products
Article
(21) Reiser, M. J.; Hui, Y. H.; Rupprecht, J. K.; Kozlowski, J. F.;
Wood, K. V.; McLaughlin, J. L.; Hanson, P. R.; Zhuang, Z.; Hoye, T.
R. J. Am. Chem. Soc. 1992, 114, 10203−10213.
(22) Su, B.-N.; Park, E. J.; Mbwambo, Z. H.; Santarsiero, B. D.;
Mesecar, A. D.; Fong, H. H. S.; Pezzuto, J. M.; Kinghorn, A. D. J. Nat.
Prod. 2002, 65, 1278−1282.
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. We
thank the Director of Kego Nature Reserve for the permission,
and Dr. L. X. Canh, Director of IEBR, for the support and
facilitation in the collection of the plant material. Prof. J.
Lindstrom, University of Pennsylvania, Philadelphia, PA,
provided HEK tsA201 cells stably expressing either hα4β2 or
hα3β4 nAChRs. We acknowledge Mr. J. Fowble, College of
Pharmacy, The Ohio State University, and Dr. C.-H. Yuan,
OSU Campus Chemical Instrument Center, for facilitating the
acquisition of the 400 and 600 MHz NMR spectra. We
acknowledge Ms. N. Kleinholz, Mr. M. Apsega, and Dr. K.
Green-Church, Campus Chemical Instrument Center, The
Ohio State University, for the mass spectrometric measure-
ments. We acknowledge Dr. P. N. Patil, Professor Emeritus,
College of Pharmacy, The Ohio State University, for facilitating
this collaborative investigation and for many helpful sugges-
tions.
(23) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.;
Robb, M. A.; Cheeseman, J. R.; Scalmani, G.; Barone, V.; Mennucci,
B.; Petersson, G. A.; Nakatsuji, H.; Caricato, M.; Li, X.; Hratchian, H.
P.; Izmaylov, A. F.; Bloino, J.; Zheng, G.; Sonnenberg, J. L.; Hada, M.;
Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.; Nakajima,
T.; Honda, Y.; Kitao, O.; Nakai, H.; Vreven, T.; Montgomery, J. A., Jr.;
Peralta, J. E.; Ogliaro, F.; Bearpark, M.; Heyd, J. J.; Brothers, E.; Kudin,
K. N.; Staroverov, V. N.; Kobayashi, R.; Normand, J.; Raghavachari, K.;
Rendell, A.; Burant, J. C.; Iyengar, S. S.; Tomasi, J.; Cossi, M.; Rega,
N.; Millam, J. M.; Klene, M.; Knox, J. E.; Cross, J. B.; Bakken, V.;
Adamo, C.; Jaramillo, J.; Gomperts, R.; Stratmann, R. E.; Yazyev, O.;
Austin, A. J.; Cammi, R.; Pomelli, C.; Ochterski, J. W.; Martin, R. L.;
Morokuma, K.; Zakrzewski, V. G.; Voth, G. A.; Salvador, P.;
̈
Dannenberg, J. J.; Dapprich, S.; Daniels, A. D.; Farkas, O.;
Foresman, J. B.; Ortiz, J. V.; Cioslowski, J.; Fox, D. J. Gaussian 09,
Revision A.01; Gaussian, Inc.: Wallingford, CT, 2009.
́
(24) Gonzalez-Cestari, T. F.; Henderson, B. J.; Pavlovicz, R. E.;
REFERENCES
McKay, S. B.; El-Hajj, R. A.; Pulipaka, A. B.; Orac, C. M; Reed, D. D.;
Boyd, R. T.; Zhu, M. X.; Li, C.; Bergmeier, S. C.; McKay, D. B. J.
Pharmacol. Exp. Ther. 2009, 328, 504−515.
■
(1) Dorr, L. J. In Flora of China, Vol. 12; Wu, Z. Y.; Raven, P. H.;
Hong, D. Y., Eds.; Science Press, Beijing, and Missouri Botanical
Garden Press: St. Louis, MO, 2007; pp 251−258, 1753.
(2) Luo, J. Acta Pharm. Sin. 2009, 44, 150−153.
(3) Bandara, K. A. N. Phytochemistry 2000, 54, 29−32.
(4) Feng, S.-X.; Lin, L.-D.; Xu, H.-H.; Wei, X.-Y. J. Asian Nat. Prod.
Res. 2008, 10, 1155−1158.
́
(25) McKay, D. B.; Chang, C.; Gonzalez-Cestari, T. F.; McKay, S. B.;
El-Hajj, R.; Bryant, D. L.; Zhu, M. X.; Swann, P. W.; Arason, K. M.;
Pulipaka, A. B.; Orac, C. M.; Bergmeier, S. C. Mol. Pharmacol. 2007,
71, 1288−1297.
́
(26) Henderson, B. J.; Carper, D. J.; Gonzalez-Cestari, T. F.; Yi, B.;
Dalefield, M. L.; Coleman, R. S.; McKay, D. B. J. Med. Chem. 2011, 54,
8681−8692.
(5) Linquist, N.; Shigematsu, N.; Pannell, L. J. Nat. Prod. 2000, 63,
1290−1291.
(27) Paleari, L.; Grozio, A.; Cesario, A.; Russo, P. Sem. Cancer Biol.
2008, 18, 211−217.
(6) MacConnell, J. G.; Blum, M. S. Science 1970, 168, 840−841.
(7) Yi, G. B.; McClendon, D.; Desaiah, D.; Goddard, J.; Lister, A.;
Moffitt, J.; Vander Meer, R. K.; deShazo, R.; Lee, K. S.; Rockhold, R.
W. Int. J. Toxicol. 2003, 22, 81−86.
(28) Dasgupta, P.; Kinkade, R.; Joshi, B.; Decook, C.; Haura, E.;
Chellappan, S. Proc. Natl. Acad. Sci. 2006, 103, 6332−6337.
(8) Chen, L.; Sharma, K. R.; Fadamiro, H. Y. Naturwissenschaften
2009, 96, 1421−1429.
(9) Gotti, C.; Riganti, L.; Vailati, S.; Lementi, F. Curr. Pharm. Des.
2006, 12, 407−428.
̀
(10) Lukas, R. J.; Changeux, J.-P.; Le Novere, N.; Albuquerque, E. X.;
Balfour, D. J. K.; Berg, D. K.; Bertrand, D.; Chiappinelli, V. A.; Clarke,
P. B. S.; Collins, A. C.; Dani, J. A.; Grady, S. R.; Kellar, K. J.;
Lindstrom, J. M.; Marks, M. J.; Quik, M.; Taylor, P. W.; Wonnacott, S.
Pharmacol. Rev. 1999, 51, 397−401.
(11) Gotti, C.; Riganti, L.; Vailati, S.; Lementi, F. Curr. Pharm. Des.
2006, 12, 407−428.
(12) Dani, J. A.; Bertrand, D. Annu. Rev. Pharmacol. Toxicol. 2007, 47,
699−729.
(13) Taly, A.; Corringer, P.-J.; Guedin, D.; Lestage, P.; Changeux, J.-
P. Nat. Rev. Drug Discovery 2009, 8, 733−750.
(14) Pan, L.; Kardono, L. B. S.; Riswan, S.; Chai, H.; Carcache de
Blanco, E. J.; Pannell, C. M.; Soejarto, D. D.; McCloud, T. G.;
Newman, D. J.; Kinghorn, A. D. J. Nat. Prod. 2010, 73, 1873−1878.
(15) Anis, E.; Anis, I.; Ahmed, S.; Mustafa, G.; Malik, A.; Afza, N.;
Hai, S. M. A.; Shahzad-ul-Hussan, S.; Choudhary, M. I. Chem. Pharm.
Bull. 2002, 50, 112−114.
(16) Harrigan, G. G.; Gunatilaka, A. A. L.; Kingston, D. G. I. J. Nat.
Prod. 1994, 57, 68−73.
(17) Henderson, B. J.; Pavlovicz, R. E.; Allen, J. D.; Gonzalez-Cestari,
́
T. F.; Orac, C. M.; Bonnell, A. B.; Zhu, M. X.; Boyd, R. T.; Li, C.;
Bergmeier, S. C.; McKay, D. B. J. Pharmacol. Exp. Ther. 2010, 334,
761−774.
(18) Schuller, H. M. Nat. Rev. Cancer 2009, 9, 195−205.
(19) Egleton, R. D.; Brown, K. C.; Dasgupta, P. Trends Pharmacol.
Sci. 2008, 29, 151−158.
(20) Ando, T.; Ogura, Y.; Koyama, M.; Kurane, M.; Uchiyama, M.;
Seol, K. Y. Agric. Biol. Chem. 1988, 52, 2459−2468.
249
dx.doi.org/10.1021/np3007414 | J. Nat. Prod. 2013, 76, 243−249