Bioactive terpenoids from the fruits of Aphanamixis grandifolia

Article abstract of DOI:10.1021/np400126q

From the fruits of the tropical tree Aphanamixis grandifolia, five new evodulone-type limonoids, aphanalides I-M (1-5), one new apo-tirucallane-type triterpenoid, polystanin E (6), and three new chain-like diterpenoids, nemoralisins A-C (7-9), along with 12 known compounds were identified. The absolute configurations were determined by a combination of single-crystal X-ray diffraction studies, Mo₂(OAc)₄-induced electronic circular dichroism (ECD) data, the Mosher ester method, and calculated ECD data. The cytotoxicities of all the isolates and the insecticidal activities of the limonoids were evaluated.

Full text of DOI:10.1021/np400126q

Note
pubs.acs.org/jnp
Bioactive Terpenoids from the Fruits of Aphanamixis grandifolia
Yao Zhang,† Jun-Song Wang,*,† Dan-Dan Wei,† Yu-Cheng Gu,‡ Xiao-Bing Wang,† and Ling-Yi Kong*,†
†State Key Laboratory of Natural Medicines, Department of Natural Medicinal Chemistry, China Pharmaceutical University, 24 Tong
Jia Xiang, Nanjing 210009, People’s Republic of China
‡Syngenta, Jealott’s Hill International Research Centre, Bracknell, Berkshire RG42 6EY, United Kingdom
S
* Supporting Information
ABSTRACT: From the fruits of the tropical tree Aphanamixis
grandifolia, five new evodulone-type limonoids, aphanalides I−M
(1−5), one new apo-tirucallane-type triterpenoid, polystanin E
(6), and three new chain-like diterpenoids, nemoralisins A−C
(7−9), along with 12 known compounds were identified. The
absolute configurations were determined by a combination of
single-crystal X-ray diffraction studies, Mo2(OAc)4-induced
electronic circular dichroism (ECD) data, the Mosher ester
method, and calculated ECD data. The cytotoxicities of all the
isolates and the insecticidal activities of the limonoids were
evaluated.
eliaceous plants have attracted a broad range of interests
C26H33O7, 457.2232), indicative of 10 indices of hydrogen
deficiency. The strong IR absorptions implied the presence of
hydroxy (3466 cm−1), carbonyl (1729 cm−1), and olefinic
(1683 cm−1) functionalities. The UV absorption at 230 nm
featured an α,β-unsaturated ester carbonyl moiety. In
accordance with its molecular formula, all 26 carbons were
observed in the 13C NMR spectrum (Supporting Information,
Table S1), which were further classified by DEPT and HSQC
experiments as five methyl, two methylene, 12 methine (three
oxygenated and five olefinic), and seven quaternary carbons
(two carbonyls, one oxygenated, and one olefinic). These
functionalities took five out of the 10 indices of hydrogen
deficiency, and the remaining five indices suggested compound
1 to be pentacyclic. Its 1H NMR spectrum displayed signals for
five tertiary methyl groups (δH 1.55, 1.37, 1.30, 1.23, and 0.66,
each 3H, s), a β-substituted furan ring (δH 7.58, 7.58, and 6.58,
each 1H, s), and an α,β-unsaturated ester carbonyl moiety [δH
6.75 (1H, d, J = 12.0 Hz) and 5.79 (1H, d, J = 12.0 Hz)]
(Supporting Information, Table S2). The aforementioned data
indicated that 1 was an evodulone-type limonoid,20 structurally
similar to aphanagranin A.12
M
from researchers in the fields of both organic chemistry1
and agrochemistry.2 Limonoids are characteristic components
of the Meliaceae family and are well known for their insecticidal
activity. The commercial application of limonoids in agriculture
has experienced significant growth in recent decades.3
Aphanamixis grandifolia Bl. is a wild timber tree distributed
mainly in the tropical and subtropical areas of South and
Southeast Asia. The roots and leaves of this plant are utilized to
relieve rheumatoid joint pain and numbness of limbs in some
regions of China. Triterpenoids,4−6 limonoids,7−9 and other
kinds of secondary metabolites10,11 have been isolated from its
stem bark and seeds. Noteworthily, amoorastatin and
aphanastatin, two limonoids possessing a 14,15-oxirane moiety,
isolated from the seeds of the title plant, showed strong growth
inhibitory effects against murine P388 cells with an ED50 value
less than 0.065 μg/mL.7,8 Therefore, a study of the bioactive
components from the fruits of A. grandifolia was undertaken. As
a result, five new evodulone-type limonoids, aphanalides I−M
(1−5), one new apo-tirucallane-type triterpenoid, polystanin E
(6), and three new chain-like diterpenoids, nemoralisins A−C
(7−9), along with 12 known compounds, aphanagranin A
(10),12 aphanalide C (11),13 polystanin A (12),14 meliasenins S
(13) and T (14),15 agladupols E (15) and D (16),16
polystanins C (17) and D (18),14 21α-methoxy-21,23-epoxy-
24α-hydroxytirucall-7-en-3-one (19),17 23,26-dihydroxytirucall-
7,24-dien-3-one (20),18 and nemoralisin (21),19 were isolated
from the fruits of A. grandifolia. Herein, the details of the
structural elucidation of these isolates and their selective
bioactivities are presented.
The key HMBC correlations from H-1 to C-3, C-5, and C-
10, from H-2 to C-3, from gem-dimethyl Me-28 and Me-29 to
C-4, and from Me-19 to C-1 established an α,β-unsaturated
seven-membered lactone in the A-ring. Three one-proton
doublets at δH 5.22, 4.88, and 4.72 were ascribed to three
hydroxy groups since no cross-peaks to any carbons were
observed in the HSQC spectrum. Their positions at rings B and
2
C were determined by the J HMBC correlations from the
Aphanalide I (1) was obtained as colorless crystals (MeOH),
and its molecular formula of C26H34O7 was established by the
negative HRESIMS ion at m/z 457.2234 [M − H]− (calcd for
Received: February 20, 2013
© XXXX American Chemical Society and
American Society of Pharmacognosy
A
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Figure 1. X-ray crystal structure of aphanalide I (1).
Aphanalide J (2), a white, amorphous powder, showed an ion
at m/z 493.3 [M + Cl]− in the negative ESIMS mode. The
molecular formula was established as C26H34O7 by the
HRESIMS ion at m/z 457.2229 [M − H]− (calcd for
C26H33O7, 457.2232), the same molecular formula as that of
1. The 1H and 13C NMR spectra of 2 were similar to those of 1,
with the exception of the D-ring. The one-proton doublet at δH
4.80 (J = 3.5 Hz) was assigned to 15-OH based on the same
coupling constant as that of H-15 and its HMBC correlation
with the oxymethine at δC 75.4 (C-15). The quaternary
oxygenated carbon was assigned to C-14 by its HMBC
correlations with Me-18 and Me-30. The remaining index of
hydrogen deficiency required an additional ring in 2. The
HMBC correlations between H-7 and C-14 established an
oxetane ring moiety. The relative configurations in 2 were
similar to those of 1 according to its ROESY spectrum. The
cross-peaks of H-7/Me-30 and Me-30/15-OH determined the
oxetane ring moiety to be α-oriented, consistent with those for
aphanalides A and C,13 determined by single-crystal X-ray
diffraction analyses.
The threo configuration of the vicinal diol (11-OH and 12-
OH) moiety was established by the key ROESY correlations of
11-OH/Me-19β and 12-OH/Me-18α. The absolute config-
uration of the vic-diol was assigned by using the in situ
Mo2(OAc)4-induced electronic circular dichroism (ECD)
method, developed by Frelek.22 According to the empirical
rule proposed by Snatzke,23,24 the induced ECD curve at
around 310 nm (band IV) showing the same sign with the O−
C−C−O torsion angle in the favored conformation allowed the
assignment of the absolute configuration. The metal complexes
of compound 2 in DMSO gave a significant induced ECD
spectrum (Supporting Information, Figure S2), in which the
negative Cotton effect (CE) at 308 nm permitted the
assignment of 11R and 12R for 2. The structure of compound
2 was thus established as shown.
protons at δH 5.22, 4.88, and 4.72 to C-11, C-7, and C-12,
respectively. The HMBC correlations from H-14 and H2-16 to
a downfield ketocarbonyl at C-15 revealed the cyclopentenone
nature of the D-ring. The C-17 and C-20 linkage between the
terminal furan ring and the core skeleton was established by the
key HMBC correlations from H-17 to C-20, C-21, and C-22
and from H2-16 to C-20.
1
In the H NMR spectrum of 1, a large coupling constant
between H-5 and one of the C-6 methylene protons at δH 1.93
(JH5−H6ax = 13.0 Hz) indicated that the B-ring was in a chair
conformation and that this proton was in an axial position and
β-oriented. The double triplet of the other methylene proton at
δH 1.45 (dt, J = 13.0, 3.5 Hz) and the singlet of H-7 suggested
that the two protons were nearly orthogonal and that H-7 was
in an equatorial position and β-oriented. The strong ROESY
cross-peaks (Supporting Information, Figure S1) of H-6β/Me-
19, Me-19/11-OH, Me-19/Me-29, Me-19/Me-30, Me-30/H-7,
Me-30/H-12, and H-12/H-17 indicated that H-7, 11-OH, H-
12, H-17, Me-19, Me-29, and Me-30 were cofacial and β-
oriented. Thus, the ROESY correlations of H-6α/H-5, H-5/
Me-28, H-5/H-9, H-9/H-14, H-14/7-OH, H-14/Me-18, and
Me-18/12-OH revealed that they were α-oriented. To resolve
any ambiguity in structure and also its absolute configuration,
compound 1 was crystallized from MeOH to afford crystals for
an X-ray crystallography study at low temperature (150 K). On
the basis of seven oxygen atoms in the molecule, the final
refinement on the Cu Kα data allowed unambiguous
assignment of the absolute configuration with a Flack
parameter value x = −0.02(17).21 The configurations of the
10 stereogenic centers were thus established as 5R, 7R, 8R, 9R,
10R, 11R, 12R, 13S, 14S, and 17S. In its crystal structure
(Figure 1), the A-ring adopted a boat conformation, the B- and
C-rings chair conformations, and the D-ring an envelope
conformation. The absolute configuration of such an
evodulone-type limonoid containing a cyclopentenone D-ring
was determined for the first time via the single-crystal X-ray
diffraction method.
Aphanalide K (3) was also obtained as crystals (MeOH).
The HRESIMS data of 3 exhibited an ion at m/z 517.2437 [M
− H]− (calcd for C28H37O9 517.2443), suggesting a molecular
formula of C28H38O9, 60 mass units more than that of 1. Its
NMR spectra were in general similar to those of 1, especially in
rings B−E. The α,β-unsaturated ester carbonyl moiety in the A-
ring of 1 was absent in 3, as evidenced by the lack of the pair of
cis-olefinic doublets and the downfield-shifted ester carbonyl
carbon resonance at δC 170.1 (Δδ +3.1, C-3). Moreover, signals
B
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for an O-acetyl group [δH 1.90 (3H, s); δC 169.7 (C), 20.9
(CH3)] were observed. The HMBC correlations from H-1 to
C-2 and the two carbonyl carbons at δC 169.7 (OAc) and 170.1
(C-3) located the O-acetyl group at C-1. The relative
configurations of 3 were determined by the ROESY experiment
in a similar way to that of 1, in which the cross-peak between
H-1 and Me-19 determined the O-acetyl group to be α-
oriented. The absolute configuration of 3 was confirmed by
single-crystal X-ray diffraction analysis (Supporting Informa-
tion, Figure S4), which finally established the absolute
configuration of 3 as 1S, 5R, 7R, 8R, 9S, 10R, 11R, 12R, 13S,
14S, and 17S by the anomalous dispersion method with a Flack
parameter of −0.10(14)21 using Cu Kα radiation.
methyl, seven methylene (one oxygenated), eight methine
(three oxygenated and one olefinic), and eight quaternary
carbons (one hemiacetal and two oxygenated). Such data
indicated that 6 was an apo-tirucallane-type protolimonoid27
with a modified side chain, which was established mainly by the
HMBC experiment. The two O-acetyl groups were located at
C-1 and C-7 according to the HMBC correlations from H-1
and H-7 to two ester carbonyl signals at δC 171.5 and 172.0,
respectively. The HMBC correlations from H-1 and H2-2 to an
ester carbonyl at δC 173.6 indicated that the A-ring comprised a
seven-membered lactone moiety as in 3. Furthermore, the
HMBC correlations from Me-19 to C-5 and C-9, from Me-30
to C-7, C-9, and C-14, from Me-18 to C-12, C-14, and C-17,
and from an olefinic proton H-15 to C-8, C-13, and C-17
established the rings B−D. The HMBC correlations from the
C-21 oxymethylene protons to the C-24 hemiacetal carbon (δC
96.7) revealed the existence of an oxygen bridge between C-21
and C-24 in the side chain. The cross-peaks from Me-26 and
Me-27 protons to the hemiacetal carbon positioned a 2-
hydroxyisopropyl group at C-24. The HMBC correlations from
H-23 to C-22 and C-24 located a hydroxy group at C-23.
The relative configuration of 6 was established mainly by the
ROESY experiment. ROESY correlations of H-1/Me-19, Me-
19/Me-29, Me-19/Me-30, Me-30/H-7, and Me-30/H-17
indicated that H-1, H-7, Me-19, Me-29, and Me-30 were
cofacial and were randomly assigned as β-oriented. The ROESY
cross-peaks of Me-18/H-9, H-9/H-5, and H-5/Me-28 thus
indicated that they were α-oriented. The NOE associations via
a ROESY spectrum from ring to side chain protons could lead
to erroneous configurational conclusions. The relative config-
urations of C-20, C-23, and C-24 were determined by analysis
of the preferred conformation of 6 and by comparison of
carbon resonances. The small coupling constant between H-23
and H2-22 (JH23−H22ax = JH23−H22eq = 3.0 Hz) indicated that the
tetrahydropyran ring at C-17 was in a chair conformation and
that H-23 was in an equatorial position and β-oriented. The
chemical shifts of C-20 (δC 31.1, Δδ 0.0), C-23 (δC 69.2, Δδ
+0.1), and C-24 (δC 96.7, Δδ +0.2) of 6 were similar to those
of chisopanin A, which were confirmed by the single-crystal X-
ray diffraction analysis,28 suggesting that H-20 and 23-OH were
α-oriented and 24-OH was β-oriented. Therefore, the structure
of 6 was assigned as depicted.
Aphanalide L (4) was obtained as a white powder. Its
HRESIMS showed a negative ion at m/z 595.2307 [M + Cl]−
(calcd for C30H40ClO10 595.2315), corresponding to a
molecular formula of C30H40O10, indicating 11 indices of
hydrogen deficiency. Its 1D NMR spectra resembled those of 3.
The major differences between them were in the D-ring and the
presence of an additional O-acetyl group [δH 2.10 (s); δC 169.5
(C), 21.1 (CH3)] in 4. This additional O-acetyl group was
located at C-7 by the HMBC cross-peak between its ester
carbonyl carbon at δC 169.5 and H-7 (δH 4.42). The remaining
index of hydrogen deficiency and one unused oxygen atom
necessitated the formation of a 14,15-oxirane moiety [δH 3.31
(H-15); δC 72.4 (C-14), 56.1 (C-15)], which was confirmed by
the HMBC correlations from H-15, Me-18, and Me-30 to C-14.
The β-orientation of H-7 was determined by ROESY
correlation of H-7/Me-30. Due to the absence of ROESY
cross-peaks from H-15 to neighboring proton signals, it was
difficult to define the configuration of the 14,15-oxirane moiety.
However, limonoids incorporating a 14,15-oxirane moiety have
been structurally established by the X-ray crystallographic
analysis.25 The similar chemical shifts of C-14 and C-15 of 5 to
those of analogous limonoids favored the β-orientation of the
14,15-oxirane moiety. The significant negative CE observed at
308 nm in the induced ECD spectrum allowed the assignment
of the 11R and 12R for 4.
Aphanalide M (5) was obtained as a white powder with [α]D25
+43.6. The molecular formula of C34H41NO10, with 15 indices
of hydrogen deficiency, was determined from the positive
HRESIMS ion at m/z 646.2617 [M + Na]+ (calcd for
1
C34H41NO10Na, 646.2623). The H NMR spectrum showed
Nemoralisin A (7) was obtained as an oil, and its molecular
formula was determined to be C20H28O5 by the positive
HRESIMS ion at m/z 371.1835 [M + Na]+ (calcd for
C20H28O5Na, 371.1829), suggesting seven indices of hydrogen
the characteristic signals of a nicotinoyl moiety [δH 8.77 (1H, d,
J = 2.0 Hz, H-3′), 8.79 (1H, dd, J = 5.0, 2.0 Hz, H-5′), 7.54
(1H, ddd, J = 8.0, 5.0, 2.0 Hz, H-6′), and 8.00 (1H, dt, J = 8.0,
2.0 Hz, H-7′)]26 and an O-acetyl group [δH 1.83 (3H, s)].
Except for signals belonging to these substituents, the 1D NMR
data resembled those of 4. The nicotinoyl group was located at
C-12 according to the HMBC correlation between H-12 (δH
5.29) and C-1′ (δC 164.5). The relative configuration of 5 was
analyzed via a ROESY spectrum in the same procedure as for 1.
On the basis of these results and biosynthetic considerations,13
5 was assigned the same configurations as 1−4.
1
deficiency. The H NMR spectrum displayed signals for three
typical olefinic protons (δH 5.78, 5.55, and 5.31) and five
methyl groups (δH 1.96, 1.66, 1.32, 1.32, and 1.19), similar to
that of nemoralisin, isolated first from Polyalthia nemoralis
(Annonaceae).19 The observed 20 carbon resonances were well
resolved in the 13C NMR spectrum, which, aided by HSQC,
revealed the presence of one α,β-unsaturated ketone, one α,β-
unsaturated δ-lactone, three trisubstituted double bonds, and
12 sp3 carbon resonances. Deducting five indices of hydrogen
deficiency accounted for two carbonyls and three double bonds,
indicating a bicyclic system in 7. The α,β-unsaturated δ-lactone
in the A-ring, the 3-oxofurano B-ring, and the aliphatic chain
between rings A and B were established by HMBC correlations.
The ROESY correlations of H-5/Me-19, H-6/H-4a, and H-
6/H-4b indicated an E-geometry of the Δ6(7) double bond. The
ROESY cross-peak between H-13 and Me-18 inferred the
spatial proximity of these protons. The configurations at C-5,
Polystanin E (6) was isolated as a white powder. Its
molecular formula of C34H52O10 was determined by the positive
ion at m/z 643.3448 [M + Na]+ (calcd for C34H52O10Na,
1
643.3453) in the HRESIMS data. The H NMR spectrum
showed the presence of nine tertiary methyl groups (δH 2.08,
2.01, 1.55, 1.37, 1.32, 1.22, 1.21, 1.21, and 1.09, each 3H, s),
one olefinic proton (δH 5.27), and five oxygenated carbon
protons (δH 5.13, 4.83, 3.87, 3.80, and 3.56). In addition to two
O-acetyl groups, the 13C NMR spectrum exhibited seven
C
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C-8, and C-11 could not be assigned from the ROESY
spectrum because of the flexibility of the aliphatic chain.
Therefore, the Mosher ester method29 combined with
calculated ECD data established the absolute configuration of
7. Treatment of 7 with (R)-(−)- and (S)-(+)-α-methoxy-α-
(trifluoromethyl)phenylacetyl chloride (MTPA-Cl) gave the
7721, compared to the positive control doxorubicin. The
primary structure−activity relationships of these terpenoids
indicated that the presence of the multiple double bonds in
these isolates was important for their activities.
Limonoids 1−5, 10, and 11 were further screened for their
insecticidal activities against a panel of insects. As shown in
Table S5 (Supporting Information), compounds 4 and 5
showed strong insecticidal activities against Diabrotica balteata,
and 5 also showed strong activity against Sitobion avenae
compared to the positive controls, while 1 and 2 exhibited weak
to moderate activities against the four insects. The initial
structure−activity relationships of limonoids 1−5, 10, and 11
indicated that the presence of the 14,15-oxirane moiety was
important for their insecticidal activities and that the nitrogen
atom enhanced such activity.
1
(S)- and (R)-MTPA esters 7a and 7b, respectively. The H
NMR signals of the two MTPA esters were assigned
unambiguously based on 1H−1H COSY spectra, and the
ΔδH(S−R) values were then calculated (Figure 2). The results
EXPERIMENTAL SECTION
■
General Experimental Procedures. Melting points were
measured on an X-4 instrument and are uncorrected. Optical rotations
were measured with a JASCO P-1020 polarimeter. ECD spectra were
recorded on a JASCO J-810 spectrometer. UV spectra were recorded
on a UV-2450 UV/vis spectrophotometer. IR spectra (KBr disks, in
cm−1) were recorded on a Bruker Tensor 27 spectrometer. NMR
spectra were recorded on a Bruker Avance III NMR instrument using
standard pulse sequences (1H: 500 MHz, 13C: 125 MHz) with TMS as
internal standard. Mass spectra were obtained on an Agilent 1100
Series LC/MSD ion-trap mass spectrometer (ESIMS) and an Agilent
6520B UPLC-Q-TOF spectrometer (HRESIMS), respectively. Prep-
arative HPLC was carried out using a Shimadzu LC-8A equipped with
a Shim-pack RP-C18 column (20 × 200 mm, i.d.) with a flow rate of
10.0 mL/min, detected by a binary channel UV detector. Silica gel
(100−200 and 200−300 mesh) and YMC RP-C18 (50 μm) were used
for column chromatography (CC). Fractions obtained from CC were
monitored by TLC with precoated silica gel GF254 plates. Spots were
visualized by heating silica gel plates sprayed with vanillin−sulfuric
acid. All solvents used were of analytical grade.
Figure 2. Values of ΔδH(S−R) (measured in CDCl3) for the MTPA
esters of 7.
indicated the 8S absolute configuration. All four possible
isomers (5S,8S,11S)-7, (5S,8S,11R)-7, (5R,8S,11S)-7, and
(5R,8S,11R)-7 were then concluded and ECD spectra
calculated (Supporting Information, Figures S5 and S6). The
experimental ECD spectrum of 7 was in accordance with the
calculated ECD spectrum for (5S,8S,11R)-7, thus establishing
the assignment of the absolute configuration of 7 as depicted.
Nemoralisin B (8) was obtained in a minute amount as a
colorless oil. The positive ion at m/z 349.2008 [M + H]+
indicated a molecular formula of C20H28O5, the same as 7.
Their NMR data (Supporting Information, Table S3) were
similar, which combined with their HMBC spectra revealed
that they shared the same carbon skeleton and functionalities.
The major difference existed in the A-ring. Compound 8 might
be a C-5 epimer of 7 according to the large chemical shift
discrepancy (ca. Δδ +10.2) at C-5, due to the anisotropic
effects of the Δ6(7) double bond, which was also supported by
the absence of the ROESY cross-peak between H-5 and Me-19.
The calculated ECD spectrum of (5R,8S,11R)-8 matched well
with the experimental ECD spectrum of 8, thus establishing the
absolute configuration of 8 as depicted (Supporting Informa-
tion, Figure S7).
Nemoralisin C (9) had the molecular formula C20H28O5, as
determined by the HRESIMS. Its NMR data suggested that 9
was structurally related to 7. The major differences were the
disappearance of a methyl doublet in 9 and its replacement by a
downfield-shifted methyl singlet at δH 1.45, correlating with a
quaternary oxygenated carbon signal at δC 72.8 in the HMBC
spectrum. The experimental ECD spectrum of 9 was almost the
same as that of 7, thus defining the absolute configuration of 9
as shown.
Plant Material. The fruits of Aphanamixis gradifolia Bl. were
collected from Xishuangbanna, Yunnan Province, China, in April 2010,
and authenticated by Prof. Jing-Yun Cui of Xishuangbanna Tropical
Garden, Chinese Academy of Sciences. A voucher specimen (No. AG-
Fruits-2010-04-ZY) has been deposited in the Department of Natural
Medicinal Chemistry, China Pharmaceutical University.
Extraction and Isolation. The air-dried and powdered fruits of A.
grandifolia (2.8 kg) were extracted with 95% aqueous EtOH (3 × 10
L) under reflux. After removal of the solvent under reduced pressure,
the crude extract (405.3 g) was suspended in H2O (1 L) and
successively partitioned with petroleum ether (3 × 1 L) and EtOAc (3
× 1 L). The EtOAc extract (110.2 g) was subjected to a silica gel
column, eluted with a gradient of CH2Cl2−acetone (20:1 to 0:1), to
give seven fractions (A−G). Fraction B (12.8 g) was chromatographed
over a silica gel column, eluted with a gradient of petroleum ether−
acetone (6:1 to 2:1), to give 6 (4.3 mg), 13 (13.5 mg), 14 (5.4 mg),
15 (41.8 mg), 16 (20.0 mg), 17 (58.3 mg), 18 (15.1 mg), and 19
(17.8 mg). Fraction C (9.6 g) was subjected to a silica gel column,
eluted with a gradient of CHCl3−MeOH (100:1 to 10:1), to yield 7
(30.6 mg), 8 (2.3 mg), 9 (5.3 mg), 12 (200.6 mg), 20 (18.3 mg), and
21 (120.1 mg). Fraction D (10.0 g) was chromatographed over a silica
gel column, eluted with a gradient of petroleum ether−EtOAc (2:1 to
1:2), to give 4 (13.1 mg), 5 (2.8 mg), 10 (30.5 mg), and 11 (6.3 mg).
Fraction F (12.5 g) was subjected to a silica gel column, eluted with a
gradient of petroleum ether−EtOAc (1:1 to 0:1), to yield 1 (24.2 mg),
2 (10.6 mg), and 3 (20.5 mg) (detailed procedure for extraction and
isolation, see Supporting Information).
Compounds 1−21 were tested in vitro for their cytotoxicities
against the human tumor cell lines HL-60, Bel-7402, HepG2,
SMMC-7721, A549, and MCF-7. Compounds with IC50 values
over 10 μM were deemed inactive (Supporting Information,
Table S4). The ranges of IC50 values were 1.80−8.72 μM for 8,
12, 13, 15, and 17−21 against HL-60, 1.53−8.50 μM for 6, 12,
13, and 21 against Bel7402, 3.62−9.31 μM for 7, 8, 12, 20, and
21 against HepG2, and 0.67−7.84 μM for 6−8, 12−16, and
19−21 against SMMC-7721. Compounds 20 and 21 showed
the most potent cytotoxicities against HepG2 and SMMC-
D
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Note
(17) Mitsui, K.; Saito, H.; Yamamura, R.; Fukaya, H.; Hitotsuyanagi,
Y.; Takeya, K. Chem. Pharm. Bull. 2007, 55, 1442−1447.
(18) Luo, X. D.; Wu, S. H.; Ma, Y. B.; Wu, D. G. Phytochemistry
2001, 57, 131−134.
ASSOCIATED CONTENT
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S
* Supporting Information
Details for extraction and isolation, X-ray crystallographic
analyses of aphanalide I (1) and aphanalide K (3), absolute
configuration determination (Snatzke’s method, Mosher ester
method, and quantum chemical ECD calculation), and
bioassays (cytotoxicity and insecticidal activity assays). Copies
of HRESIMS, 1D and 2D NMR, ECD, and IR spectra of 1−9
and induced ECD spectra of 2 and 4. CIF files of 1 and 3.
These materials are available free of charge via the Internet at
http://pubs.acs.org.
(19) He, X. F.; Wang, X. N.; Fan, C. Q.; Gan, L. S.; Yin, S.; Yue, J. M.
Helv. Chim. Acta 2007, 90, 783−791.
(20) Sondengam, B. L.; Kamga, C. S.; Connolly, J. D. Tetrahedron
Lett. 1979, 20, 1357−1358.
(21) Flack, H. D. Acta Crystallogr. 1983, A39, 876−881.
(22) Gorecki, M.; Jablonska, E.; Kruszewska, A.; Suszczynska, A.;
Urbanczyk-Lipkowska, Z.; Gerards, M.; Morzycki, J. W.; Szczepek, W.
J.; Frelek, J. J. Org. Chem. 2007, 72, 2906−2916.
(23) Snatzke, G. Angew. Chem., Int. Ed. Engl. 1979, 18, 363−366.
(24) Di Bari, L.; Pescitelli, G.; Pratelli, C.; Pini, D.; Salvadori, P. J.
Org. Chem. 2001, 66, 4819−4825.
AUTHOR INFORMATION
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(25) Ndung’u, M.; Hassanali, A.; Hooper, A. M.; Chhabra, S.; Miller,
T. A.; Paul, R. L.; Torto, B. Phytochemistry 2003, 64, 817−823.
Corresponding Author
*Tel/Fax: +86-25-8327-1405. E-mail: cpu_lykong@126.com
(L.-Y.K.) or wang.junsong@gmail.com (J.-S.W.).
́
(26) Gonzalez, A. G.; Rodríguez, F. M.; Bazzocchi, I. L.; Ravelo, A. G.
J. Nat. Prod. 2000, 63, 48−51.
(27) Luo, X. D.; Wu, S. H.; Wu, D. G.; Ma, Y. B.; Qi, S. H.
Tetrahedron 2002, 58, 6691−6695.
Notes
The authors declare no competing financial interest.
(28) Yang, M. H.; Wang, J. S.; Luo, J. G.; Wang, X. B.; Kong, L. Y.
Bioorg. Med. Chem. 2011, 19, 1409−1417.
ACKNOWLEDGMENTS
(29) Huang, H. L.; Wang, C. M.; Wang, Z. H.; Yao, M. J.; Han, G. T.;
Yuan, J. C.; Gao, K.; Yuan, C. S. J. Nat. Prod. 2011, 74, 2235−2242.
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This research work was financially supported by the National
Natural Science Foundation of China (21272275), the Priority
Academic Program Development of Jiangsu Higher Education
Institutions (PAPD), the Program for Changjiang Scholars and
Innovative Research Team in University (IRT1193), and the
Fundamental Research Funds for the Central Universities
(JKY2011011, 3092013112014). The Syngenta Postgraduate
Fellowship (SPF-048) awarded to Y.Z. is also appreciated.
REFERENCES
■
(1) Yuan, T.; Zhu, R. X.; Zhang, H.; Odeku, O. A.; Yang, S. P.; Liao,
S. G.; Yue, J. M. Org. Lett. 2010, 12, 252−255.
(2) Ge, Y. H.; Liu, K. X.; Zhang, J. X.; Mu, S. Z.; Hao, X. J. J. Agric.
Food Chem. 2012, 60, 4289−4295.
(3) Ntalli, N. G.; Caboni, P. J. Agric. Food Chem. 2012, 60, 9929−
9940.
(4) Zeng, Q.; Guan, B.; Qin, J. J.; Wang, C. H.; Cheng, X. R.; Ren, J.;
Yan, S. K.; Jin, H. Z.; Zhang, W. D. Phytochemistry 2012, 80, 148−155.
(5) Zhang, Y.; Wang, J. S.; Wei, D. D.; Wang, X. B.; Luo, J.; Luo, J.
G.; Kong, L. Y. Phytochemistry 2010, 71, 2199−2204.
(6) Wang, J. S.; Zhang, Y.; Wei, D. D.; Wang, X. B.; Luo, J. G.; Luo,
J.; Yang, M. H.; Yao, H. Q.; Sun, H. B.; Kong, L. Y. Tetrahedron Lett.
2012, 53, 1705−1709.
(7) Polonsky, J.; Varon, Z.; Arnoux, B.; Pascard, C.; Pettit, G. R.;
Schmidt, J. H.; Lange, L. M. J. Am. Chem. Soc. 1978, 100, 2575−2576.
(8) Polonsky, J.; Varon, Z.; Arnoux, B.; Pascard, C.; Pettit, G. R.;
Schmidt, J. M.. J. Am. Chem. Soc. 1978, 100, 7731−7733.
(9) Polonsky, J.; Varon, Z.; Marazano, C.; Arnoux, B.; Pettit, G. R.;
Schmid, J. M.; Ochi, M.; Kotsuki, H. Experientia 1979, 35, 987−989.
(10) Nishizawa, M.; Inoue, A.; Hayashi, Y.; Sastrapradja, S.; Kosela,
S.; Iwashita, T. J. Org. Chem. 1984, 49, 3660−3662.
(11) Astulla, A.; Hirasawa, Y.; Rahman, A.; Kusumawati, I.; Ekasari,
W.; Widyawaruyanti, A.; Zaini, N. C.; Morita, H. Nat. Prod. Commun.
2011, 6, 323−326.
(12) Tong, L.; Zhang, Y.; He, H. P.; Hao, X. J. Chin. J. Chem. 2012,
30, 1261−1264.
(13) Wang, J. S.; Zhang, Y.; Wang, X. B.; Kong, L. Y. Tetrahedron
2012, 68, 3963−3971.
(14) Zhang, Y.; Wang, J. S.; Wang, X, B; Gu, Y. C.; Kong, L. Y. Chem.
Pharm. Bull. 2013, 61, 75−81.
(15) Hu, J. F.; Fan, H.; Wang, L. J.; Wu, S. B.; Zhao, Y. Phytochem.
Lett. 2011, 4, 292−297.
(16) Xie, B. J.; Yang, S. P.; Chen, H. D.; Yue, J. M. J. Nat. Prod. 2007,
70, 1532−1535.
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