Aphadilactones a-d, four diterpenoid dimers with dgat inhibitory and antimalarial activities from a meliaceae plant

Article abstract of DOI:10.1021/jo402340h

Aphadilactones A-D (1-4), four diastereoisomers possessing an unprecedented carbon skeleton, were isolated from the Meliaceae plant Aphanamixis grandifolia. Their challenging structures and absolute configurations were determined by a combination of spectroscopic data, chemical degradation, fragment synthesis, experimental CD spectra, and ECD calculations. Aphadilactone C (3) with the 5S,11S,5'S,11'S configuration showed potent and selective inhibition against the diacylglycerol O-acyltransferase-1 (DGAT- 1) enzyme (IC50 = 0.46 ± 0.09 μM, selectivity index > 217) and is the strongest natural DGAT-1 inhibitor discovered to date. In addition, compounds 1-4 showed significant antimalarial activities with IC50 values of 190 ± 60, 1350 ± 150, 170 ± 10, and 120 ± 50 nM, respectively.

Full text of DOI:10.1021/jo402340h

Article
pubs.acs.org/joc
Aphadilactones A−D, Four Diterpenoid Dimers with DGAT Inhibitory
and Antimalarial Activities from a Meliaceae Plant
Jia Liu,† Xiu-Feng He,† Gai-Hong Wang,† Emilio F. Merino,‡ Sheng-Ping Yang,† Rong-Xiu Zhu,∥
Li-She Gan,⊥ Hua Zhang,† Maria B. Cassera,‡ He-Yao Wang,† David G. I. Kingston,§ and Jian-Min Yue*,†
†State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zu Chong Zhi
Road, Shanghai 201203, P. R. China
‡Department of Biochemistry and the Virginia Tech Center for Drug Discovery, MC 0308, Virginia Polytechnic Institute and State
University, Blacksburg, Virginia 24061, United States
§Department of Chemistry and the Virginia Tech Center for Drug Discovery, MC 0212, Virginia Polytechnic Institute and State
University, Blacksburg, Virginia 24061, United States
∥School of Chemistry and Chemical Engineering, Shandong University, Jinan 250100, P. R. China
⊥College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, P. R. China
S
* Supporting Information
ABSTRACT: Aphadilactones A−D (1−4), four diaster-
eoisomers possessing an unprecedented carbon skeleton,
were isolated from the Meliaceae plant Aphanamixis
grandifolia. Their challenging structures and absolute config-
urations were determined by a combination of spectroscopic
data, chemical degradation, fragment synthesis, experimental
CD spectra, and ECD calculations. Aphadilactone C (3) with
the 5S,11S,5′S,11′S configuration showed potent and selective inhibition against the diacylglycerol O-acyltransferase-1 (DGAT-
1) enzyme (IC50 = 0.46 0.09 μM, selectivity index > 217) and is the strongest natural DGAT-1 inhibitor discovered to date. In
addition, compounds 1−4 showed significant antimalarial activities with IC50 values of 190 60, 1350 150, 170 10, and 120
50 nM, respectively.
them were reported to have activity against DGAT or DGAT-
associated diseases. The DGAT-1 inhibitory fraction was thus
subjected to extensive fractionation and purification to obtain
four major stereoisomers, aphadilactones A−D (1−4) (Figure
1), whose carbon skeleton represents a new structure class.
Further biological tests of the pure compounds 1−4 verified
INTRODUCTION
■
Excessive triglyceride accumulation in adipocytes is associated
with a number of human diseases such as obesity, diabetes, and
steatohepatitis (fatty liver disease). Two diacylglycerol O-
acyltransferase (DGAT) isozymes, DGAT-1 and DGAT-2, have
been reported to play important roles in triglyceride synthesis
and metabolism.1 DGAT-1-deficient mice are resistant to diet-
induced obesity and have decreased adiposity and improved
insulin and leptin sensitivity. In contrast, newborn DGAT-2-
deficient mice are lipopenic and die soon after birth.2 Selective
inhibition of DGAT-1 therefore represents a novel and
potential approach for the treatment of obesity, dyslipidemia,
and metabolic syndrome.2 A number of natural DGAT-1
inhibitors have been discovered in the past decade from both
plants and microbes, and the five most potent natural inhibitors
show IC50 values ranging from 2.5 to 9.8 μM.2,3
In our search for new classes of potent and selective DGAT
inhibitors, the plant Aphanamixis grandifolia (Meliaceae) came
to our attention, as one of the fractions from its ethanolic
extract exhibited significant inhibition against DGAT-1. A.
grandifolia, an arbor tree, grows mainly in the tropical and
subtropical areas of Asia,4 and its leaves and roots have been
used as folk medicine in China to treat rheumatism and
alleviate pain.5 Previous chemical studies on this plant afforded
sesquiterpenoids,6 triterpenoids,7 and limonoids,8 but none of
Figure 1. Structures of compounds 1−4.
Received: October 23, 2013
© XXXX American Chemical Society
A
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1
Table 1. H NMR Data [δH (mult, J in Hz)] for 1−8 in CD3OD
a
b
b
b
b
b
proton
1
2
3
4
5 (6)
7 (8)
2
5.79 (m)
5.79 (br s)
2.38 (m)
5.78 (br s)
5.78 (br s)
2.37 (m)
4a
2.35 (dd, 18.0, 4.3)
2.43 (dd, 18.0, 10.9)
5.21 (m)
2.35 (dd, 18.2, 4.7)
2.44 (m)
4b
2.45 (dd, 18.1, 10.2)
5.22 (m)
2.44 (m)
5
5.23 (m)
5.23 (m)
6
5.35 (br d, 8.5)
2.09 (m, 2H)
1.53 (m, 2H)
1.54 (m)
5.36 (br d, 8.1)
2.10 (m, 2H)
1.53 (m, 2H)
1.53 (m)
5.39 (br d, 8.6)
2.14 (br t, 7.1, 2H)
1.63 (m, 2H)
1.56 (m)
5.39 (br d, 8.6)
2.14 (m, 2H)
1.63 (m, 2H)
1.60 (m)
8
2.52 (m, 2H)
1.61 (m, 2H)
1.47 (m)
2.56 (m, 2H)
9
1.70 (m, 2H)
1.53 (m)
10a
10b
11
1.76 (m)
1.79 (m)
1.78 (m)
1.76 (m)
1.77 (m)
1.76 (m)
2.74 (m)
2.75 (m)
2.64 (m)
2.64 (m)
2.70 (m)
2.61 (m)
c
c
c
c
c
16
1.38 (s, 3H)
1.38 (s, 3H)
1.37 (s, 3H)
1.37 (s, 3H)
1.92 (m)
1.37 (s, 3H)
1.37 (s, 3H)
1.36 (s, 3H)
c
c
c
c
c
17
1.39 (s, 3H)
1.39 (s, 3H)
1.38 (s, 3H)
1.38 (s, 3H)
1.37 (s, 3H)
e
e
18α
18β
19
2.03 (m)
1.48 (m)
1.82 (m)
2.04 (m)
1.92 (m)
e
e
1.48 (m)
2.05 (m)
1.82 (m)
1.92 (m)
1.50 (m)
1.82 (m)
1.73 (d, 0.8, 3H)
2.02 (br s, 3H)
5.79 (br s)
1.73 (br s, 3H)
2.02 (br s, 3H)
5.79 (br s)
1.76 (d, 1.3, 3H)
2.02 (br s, 3H)
5.78 (br s)
1.76 (d, 1.2, 3H)
2.02 (br s, 3H)
5.78 (br s)
2.34 (dd, 18.1, 4.5)
2.44 (m)
2.11 (s, 3H)
2.15 (s, 3H)
20
2′
4′a
4′b
5′
2.34 (dd, 18.0, 4.2)
2.43 (dd, 18.0, 10.9)
5.21 (m)
2.34 (dd, 18.1, 4.4)
2.45 (dd, 18.1, 10.2)
5.20 (m)
2.34 (dd, 18.2, 4.7)
2.42 (m)
5.21 (m)
5.21 (m)
6′
8′
5.33 (br d, 8.5)
2.06 (m, 2H)
1.29 (m)
5.34 (br d, 8.2)
2.06 (m, 2H)
1.24 (m)
5.32 (br d, 8.6)
2.07 (br t, 7.4, 2H)
1.35 (m)
5.35 (br d, 8.6)
2.09 (m, 2H)
1.33 (m)
2.48 (m, 2H)
1.34 (m)
1.59 (m)
1.89 (m)
2.07 (m)
5.31 (s)
2.50 (m, 2H)
1.43 (m)
9′a
9′b
10′a
10′b
13′
16′
17′
18′α
18′β
19′
20′
1.50 (m)
1.50 (m)
1.47 (m)
1.46 (m)
1.56 (m)
1.89 (ddd, 13.0, 13.0, 4.4)
2.09 (m)
1.82 (m)
1.87 (m)
1.84 (m)
1.86 (m)
2.16 (m)
2.03 (m)
2.05 (m)
1.99 (ddd, 13.0, 13.0, 4.5)
5.41 (s)
5.29 (s)
5.27 (s)
5.34 (s)
5.33 (s)
d
d
d
d
d
d
1.33 (s, 3H)
1.33 (s, 3H)
1.31 (s, 3H)
1.31 (s, 3H)
1.32 (s, 3H)
1.30 (s, 3H)
d
d
d
d
d
d
1.36 (s, 3H)
1.36 (s, 3H)
1.35 (s, 3H)
1.35 (s, 3H)
1.35 (s, 3H)
1.34 (s, 3H)
e
1.82 (ddd, 13.4, 13.4, 2.7)
2.11 (m)
2.07 (m)
1.92 (m)
1.92 (m)
1.84 (m)
1.92 (m)
e
1.86 (m)
1.92 (m)
1.92 (m)
2.09 (m)
1.92 (m)
1.71 (d, 0.7, 3H)
1.69 (br s, 3H)
1.71 (d, 1.2, 3H)
1.68 (d, 1.2, 3H)
2.13 (s, 3H)
2.11 (s, 3H)
2.02 (br s, 3H)
2.01 (br s, 3H)
2.02 (br s, 3H)
2.02 (br s, 3H)
a
b
e
Recorded at 700 MHz. Recorded at 400 MHz. c,dMay be exchanged in the same column. The relative configurations of H-18 and H-18′ assigned
for 5 and 7 were reversed in 6 and 8 vertically.
that aphadilactone C (3) is the primary active component, with
potent inhibition against DGAT-1 (IC50 = 0.46 0.09 μM) but
only marginal activity against DGAT-2 (IC50 > 100 μM),
indicating that it is a highly selective DGAT-1 inhibitor with a
selectivity index of >217. Most excitingly, aphadilactone C is
the strongest natural DGAT-1 inhibitor discovered to date,
being >5-fold more active than the best previously reported
one, erysenegalensein O (IC50 = 2.5 μM).3b
Another pharmacological activity screening for 1−4 showed
that they are also active against Plasmodium falciparum in vitro
culture, with IC50 values of 190 60, 1350 150, 170 10,
and 120 50 nM, respectively. Malaria is caused by protozoan
parasites of the Plasmodium genus, of which P. falciparum is
responsible for most of the fatal cases. The parasite is
transmitted between human hosts by Anopheles mosquitoes.
It is widespread in tropical and subtropical regions, including
parts of America, Asia, and Africa. Malaria has infected humans
for over 50 000 years, and it continues to cause about 200−300
million cases and kills nearly a million people annually in Africa
alone.9 Historically, plants have been a prominent source of
antimalarial drugs such as quinine and artemisinin.10
Artemisinin combination therapies are recommended by the
World Health Organization as the first-line treatment for
malaria and are extremely safe and effective after 3 days of
dosing.11 However, given the emergence of resistance to
artemisinin and other antimalarials, the need for new medicines
is ever present.12 P. falciparum encodes only one DGAT
enzyme, which is essential for intraerythrocytic proliferation.13
Infected erythrocytes display a dramatic increase in triglyceride
content; however, P. falciparum lacks the capability to degrade
triglycerides to produce energy, and instead, triglycerides are
part of the lipid bodies in the food vacuole.14
We present herein the isolation, structural elucidation, and
biological activities of 1−4 along with a brief structure−activity
relationship (SAR) discussion of this compound class.
RESULTS AND DISCUSSION
■
Aphadilactone A (1) was obtained as a pale gum with [α]D22
−8.3 (c 0.145, MeOH). The molecular formula, C40H52O8 with
15 degrees of unsaturation, was determined by HR-ESI-MS(+),
which showed a peak at m/z 683.3569 [M + Na]+ (calcd
683.3560). Its IR spectrum displayed absorptions of carbonyl
(1699 cm−1) and double bond (1606 cm−1) functionalities. In
accord with the molecular formula, 40 carbon signals were
resolved in its 13C NMR spectrum (Table 2), including eight
methyls, 10 sp3 methylenes, eight methines (two oxygenated
and five olefinic), and 14 quaternary carbons (two oxygenated,
five olefinic, and six carbonyl), as distinguished by DEPT
B
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experiments in combination with HSQC and HMBC spectra
[S9 and S10 in the Supporting Information (SI)]. Overall
analysis of the NMR data (Tables 1 and 2) further indicated the
presence of furan-3(2H)-one and α,β-unsaturated δ-lactone
moieties,15 which was supported by the UV absorption at λmax
=
265 nm.
Table 2. 13C NMR Data for 1−8 in CD3OD
b
a
a
a
b
b
carbon
1
2
3
4
5 (6)
7 (8)
Figure 2. 1H−1H COSY (bold bonds) and H → C HMBC
correlations (arrows) in 1.
1
168.1
116.4
161.2
35.8
168.0
116.4
161.2
35.7
167.9
116.5
161.1
35.8
167.9
116.4
161.1
35.8
2
3
C-20 to C-3. The deshielded resonance of H-5 (δH 5.21)
suggested the formation of a six-membered lactone between C-
1 and C-5 to furnish a 4,6-dimethyl-5,6-dihydro-2H-pyran-2-
one motif (component B1). In the same way, an identical
component B2 (C-1′ to C-6′ and C-20′) was readily
established. The chemical shifts of C-12 (δC 193.1), C-13 (δC
112.4), and C-14 (δC 206.9) were suggestive of an α,β-
unsaturated ketone moiety, and its C-14 end was shown to be
connected to C-15 (δC 89.4) bearing two methyls by the
HMBCs of H-16 and H-17 with C-14 and C-15. This analysis
together with the deshielded resonances of C-12 and C-15
indicated the presence of a 2,2-dimethylfuran-3(2H)-one
subunit (C-12 to C-17).15 Similarly, another 2,2-dimethylfur-
an-3(2H)-one subunit (C-12′ to C-17′) was constructed on the
basis of chemical shifts and HMBCs (Figure 2). C-12 was
shown to be linked to C-11 by the HMBCs of H-10 and H-18
with C-12. The HMBCs of H-18 with C-11′, H-18′ with C-13
and C-12′, and H-10′ with C-13, C-11′, and C-12′ showed that
C-13, C-10′, and C-12′ are attached to C-11′ to fix a
cyclohexene ring. The HMBCs of H-19 with C-6, C-7, and
C-8 and H-19′ with C-6′, C-7′, and C-8′ revealed the linkages
of C-8 and C-19 to C-7 and C-8′ and C-19′ to C-7′,
respectively, to construct component A and also enabled us to
fix the Δ-6 and Δ-6′ double bonds, which were confirmed by
the HMBCs of H-5 with C-6 and C-7 and H-5′ with C-6′ and
C-7′, respectively. The planar structure of 1 was thus
established, and it was secured by spectral analysis of its
ozonized products,16 compounds 5 and 9 (Figure 3 and Figure
S1 in the SI).
4
5
76.0
75.9
76.0
76.0
6
123.6
143.6
40.2
123.7
143.5
40.2
123.9
143.4
40.0
123.8
143.5
40.1
7
211.3
43.9
21.4
31.3
37.7
193.0
112.2
206.9
89.5
23.0
23.3
25.8
29.8
211.4
43.8
22.2
32.2
36.8
193.1
112.0
206.8
89.3
23.1
23.3
24.7
29.8
8
9
25.0
24.9
25.9
25.9
10
11
12
13
14
15
16
17
18
19
20
1′
2′
3′
4′
5′
6′
7′
8′
9′
10′
11′
12′
13′
14′
15′
16′
17′
18′
19′
20′
31.2
31.1
32.1
32.1
37.7
37.6
36.5
36.6
193.1
112.4
206.9
89.4
193.2
112.3
206.8
89.4
193.1
112.1
206.6
89.2
193.1
112.0
206.6
89.2
c
c
c
c
c
c
c
c
c
c
c
23.1
23.1
23.1
23.1
c
23.4
23.4
23.5
23.4
25.9
25.9
24.9
24.8
16.7
16.6
16.6
16.6
23.0
23.0
23.0
23.0
168.1
116.4
161.2
35.8
168.0
116.4
161.2
35.8
167.9
116.5
161.1
35.8
167.9
116.5
161.2
35.8
76.0
76.0
76.0
76.0
123.6
143.5
40.4
123.9
143.4
40.5
123.6
143.4
40.4
124.0
143.3
40.5
211.3
44.2
19.6
35.4
43.4
197.7
102.2
209.8
90.5
22.9
22.9
31.4
29.9
211.3
44.1
20.0
35.7
43.1
197.4
102.0
210.1
90.3
22.9
22.9
30.4
29.9
23.4
23.2
23.7
23.6
35.6
35.2
35.8
35.6
43.5
43.4
43.1
43.1
197.9
102.2
209.8
90.6
198.0
102.2
209.7
90.5
197.5
101.9
209.8
90.3
197.6
101.9
209.9
90.5
23.0
23.0
23.0
23.0
23.0
23.0
23.0
23.0
31.6
31.5
30.4
30.5
16.6
16.5
16.8
16.5
23.0
23.0
23.0
23.0
a
b
c
Recorded at 100 MHz. Recorded at 125 MHz. May be
interchangeable in the same column.
1
The H−1H COSY and HSQC spectra of 1 revealed four
proton-bearing structural fragments: C-4 to C-6; C-8 to C-11,
C-18, and C-18′; C-4′ to C-6′; and C-8′ to C-10′ (shown as
bold bonds in Figure 2). The connections of these fragments
with the other functional groups of 1 were mainly determined
by analysis of the HMBC spectrum (Figure 2). The carbon
chemical shifts and the HMBCs of H-20 with C-2 (δC 116.4),
C-3 (δC 161.2), and C-4 and of H-2 with C-1 (δC 168.1)
indicated the presence of an α,β-unsaturated carbonyl group
linked to C-4 via the C-3/C-4 bond as well as the attachment of
Figure 3. Ozonolysis of compounds 1−4.
The ESI-MS, UV, IR, and 1H and 13C NMR (Tables 1 and 2)
data for compounds 2−4 showed high similarities to those of 1,
and the minor variations were the proton and carbon
resonances around the cyclohexene ring. It was also obvious
that the NMR data for 1 and 2 were very similar, as were those
for 3 and 4. These data suggested that these compounds were
C
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four stereoisomers with different configurations at C-11 and C-
11′. Comprehensive spectral analysis (Tables 1 and 2 and SI
S12−S41), especially of the 2D NMR spectra, verified that 2−4
possess the same planar structure as 1 (Figure 1).
The NOESY correlations of H-6 with H-8 and H-6′ with H-
8′ revealed that both the Δ-6 and Δ-6′ double bonds of 1−4
have the E geometry. In the NOESY spectrum of 1 (Figure 4
Figure 5. Experimental ECD spectra of 5−8, 5a, and 6a.
nm, negative CE at 238 nm) of 8 (ozonized from 4) indicated
that 4 and 8 have an 11R,11′R configuration. While 5
(ozonized from 1) exhibited an obvious positive CE at 239
nm arising from exciton coupling of the two chromophores, a
negative CE at ca. 274 nm expected by comparison to those of
7 and 8 was eclipsed for unspecified reasons. To remove the
ambiguity and the interactions among the multiple chromo-
phores, 5 was reduced with NaBH4 in CH3OH to afford its diol
derivative 5a (Figure 6), which showed a first negative CE
Figure 4. Key NOESY correlations (blue arrows) in 1−4.
and SI S11), the strong correlation of H-11 with H-18′α
indicated that they adopt 1,3-diaxial positions in the half-chair
cyclohexene ring, and they were arbitrarily fixed as α-oriented,
which was supported by the coupling constants of H-18′α at δH
1.82 [ddd, 13.4 (geminal), 13.4 (ax−ax), and 2.7 (ax−eq) Hz].
Consequently, the long chain at C-11′ of 1 was assigned as α-
directed by the key correlation of H-9′b with H-18′α (SI S11;
see the expanded parts). Thus, the two wings incorporating
lactone rings at C-11 and C-11′ of the cyclohexene ring in 1
were definitely assigned as trans-oriented, and this was
confirmed by the strong NOESY correlations of H-11 and H-
9′b with H-18′α observed for its ozonized product 5 (SI S51).
Similarly, the relative configurations at C-11 and C-11′ of 2
were assigned as depicted by the key NOESY correlations of H-
11 and H-9′b with H-18′β (Figure 4 and SI S21). In a similar
fashion, the two wings at C-11 and C-11′ in 3 and 4 were
determined to be cis-oriented in the half-chair cyclohexene ring
by the key NOESY correlations (Figure 4) observed for 3 (H-
11 with H-18′β and H-18α with H-9b′; SI S31) and 4 (H-11
with H-18′α and H-18β with H-9b′; SI S41). The mirror-image
CD spectra of the ozonized products (5/6 and 7/8) (Figure 5)
rigorously support the assignments of the relative config-
urations of C-11 and C-11′ in 1−4.
Figure 6. Reduction of compounds 5 and 6.
(weak, λ = 270 nm) and a second positive CE (λ = 240 nm),
suggestive of a negative helicity between the two chromo-
phores, indicating that 1, 5, and 5a have the 11R,11′S
configuration. The opposite CD curves of 6 (ozonized from
2) and 6a versus those of 5 and 5a thus revealed the reversed
configuration (11S,11′R) for 2, 6, and 6a. ECD calculations for
compounds 5−8 matched the experimental data well (Figure
7), which further confirmed the above assignments.
The CD spectra of 1−4 (Figure S2 in the SI) result from the
interactions of the multiple chromophores and did not provide
useful Cotton effects (CEs) for the assignment of absolute
stereochemistry. Fortunately, the typical CD curves of their
correspondingly ozonized products 5−8 representing two pairs
of enantiomers (5/6 and 7/8) allowed the absolute
configurations at C-11 and C-11′ of the molecules to be
established (Figure 5).17 The CD exciton couplets of 5−8
associated with the two chromophores of the 2,2-dimethylfur-
an-3(2H)-one moieties15 (centered at ca. λmax = 264 nm) were
observed. The clear first negative CE (λ = 274 nm) and the
second positive CE (λ = 239 nm) of 7 (ozonized from 3)
showed a negative helicity between two chromophores,
establishing an 11S,11′S configuration for compounds 3 and
7. Consequently, the opposite CD curves (positive CE at 274
Figure 7. Calculated ECD spectra of compounds 5−8 vs the
experimental ECD curves: (a) 5 and 6; (b) 8 and 7.
D
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The configurational assignment of the lactone rings of 1−4
was much more challenging because of their remote locations
from the central core of the molecules. Ozonolysis of 1 (or 2−
4) afforded 9 in low yields as an inseparable mixture of two
epimers instead of the expected aldehyde (9a), likely as a result
of the unstable nature of 9a in the presence of H2O or the
excess of ozone that may have further decomposed 9 or 9a
(Figure 3). In order to define the absolute configurations at C-5
and C-5′ in 1−4, two model compounds 13 and 14 with S and
R configurations, respectively, were synthesized from the
starting materials (R)- and (S)-oxiran-2-ylmethanol, respec-
tively (Figure 8).18 With 13 and 14 in hand, an alternative
ylation, oxidation, and reduction in a one-pot reaction, and each
reaction mixture was analyzed by chiral HPLC (Figure S4 in
the SI), which revealed that C-5 and C-5′ in 2−4 are also S-
configured. The structures of 1−4 were thus fully determined
as shown in Figure 1.
Aphadilactones A−D are proposed to be formed from two
molecules of nemoralisin-type diterpenoid i, enzyme-catalyzed
Diels−Alder reaction of which would give the key intermediate
ii. Compounds 1−4 would finally be produced from ii via a
typical 1,3-hydrogen migration (Figure 10). This hypothesis
Figure 8. Syntheses of 13 and 14. Reagents and conditions: (a)
TBDPSiCl, imidazole, DMF; (b) CuI, CH3CH(CH2)MgBr, THF,
−30 to 0 °C; (c) CH2CHCOCl, Et3N, DMAP, CH2Cl2, 0 °C; (d)
Grubbs’ catalyst II, CH2Cl2, 50 °C; (e) Bu4NF, THF.
Figure 10. Plausible biosynthetic pathway for 1−4.
strategy aiming to obtain 13 and/or 14 from 1−4 under mild
conditions was thus designed (Figure 9); we expected that if C-
5 and C-5′ bore different absolute configurations, they could be
distinguished by oxidative degradation of the partially
dihydroxylated products 10a and 11a. Fortunately, after
dihydroxylation with potassium osmate,19 compound 1 gave
the three products 10a−12a in isolated yields of 13−15%, as
was confirmed to be plausible by the simultaneous isolation of
nemoralisin22 in this investigation; its absolute structure was
established in this study by single-crystal X-ray diffraction
analysis of its derivative 15 (Figure 11 and Table S3 in the SI).
Compound 15 was obtained by dihydroxylation of nemoralisin
with the AD-mix-α reagent.
A review of the literature showed that the structures of 1−4
are biosynthetically related to those of aphanamenes A and B
from the same species, which were reported by Kong and co-
workers in a very recent paper.23 Although 1−4 could originate
from a similar or the same monomer as aphanamenes A and B,
their carbon framework is different from that in the latter two
compounds. Moreover, peak splitting was observed for the
20
expected. Each of these was further oxidized with Pb(OAc)4
and then reduced with NaBH4,21 and the resulting reaction
mixtures were directly subjected to chiral HPLC analysis. One
HPLC peak at ca. tR = 13.9 min in the degraded products of
10a−12a matched that of the authentic sample 13 (Figure S3
in the SI), and no peak corresponding to 14 was observed,
indicating that both C-5 and C-5′ of 1 have the S configuration.
Similarly, 2−4 separately underwent the cascade of dihydrox-
Figure 9. Oxidative degradation of 1. Reagents and conditions: (a) 5 mol % K2OsO4·2H2O, 5 equiv of MeSO2NH2, 15 equiv of K3Fe(CN)6, 15
t
equiv of K2CO3, BuOH/H2O (1:1), r.t.; (b) Pb(OAc)4, DCM, 0 °C; (c) NaBH4, MeOH.
E
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Article
μM, selectivity index > 217) whereas its stereoisomers 1, 2, and
4 showed only marginal inhibitions, suggesting that stereo-
specific binding between DGAT-1 enzyme and its inhibitors is
required. Interestingly, compound 2 showed the weakest
activity against P. falciparum, while 1, 3, and 4 were more
potent. Our study also revealed a gross SAR of DGAT-1
inhibition for this compound class, which will be important for
the structural optimization. To the best of our knowledge,
aphadilactone C (3) is the strongest natural DGAT-1 inhibitor
discovered to date,2,3 and it represents a new structural scaffold
for DGAT inhibitors and highlights the potential for future
structural optimization and/or total synthesis. We view
aphadilactone C as a promising new class of DGAT-1 inhibitor
that deserves further investigation. The strong antimalarial
activities of 1, 3, and 4 also make them promising candidates
for antimalarial drugs. This finding once again demonstrates the
robust capability of nature to produce stereochemically diverse
molecules that are a precious treasure for drug discovery.
Figure 11. Single-crystal X-ray structure of compound 15.
NMR data of aphanamene B, which was assumed by the
authors to be caused by cyclohexene conformation isomer-
ization.23 However, on the basis of our findings that
compounds 1 and 2 and compounds 3 and 4 are not separable
on normal achiral HPLC columns and that the NMR “peak
splitting” before further purification under chiral conditions was
due to mixtures of diastereoisomers, the structure character-
ization of aphanamene B might need to be re-examined.
Compounds 1−4 and their respective ozonized products 5−
8 were evaluated for inhibition of hDGAT enzymes.
Interestingly, aphadilactone C (3) exhibited significant
inhibitory activity against DGAT-1 with an IC50 of 0.46
0.09 μM (SI page 14) and very weak activity against DGAT-2
(IC50 > 100 μM), indicating that it is a highly selective DGAT-1
inhibitor (selectivity index > 217). In this test, the natural
DGAT-1 inhibitory agent betulinic acid (IC50 = 17.2 μM) was
used as the positive control.2 Inspection of the structures and
activities of 1−8 against DGAT-1 at 10 μM (Table S4 in the
SI) outlined a gross SAR. Compounds 1 and 3 exhibited 25.5%
and 85.9% inhibitions on DGAT-1 at 10 μM, respectively, while
the other analogues only showed marginal inhibitions. These
results primarily indicate the following: (1) an 11′S
configuration (as in 1 and 3) is crucial for the activity, and
the 11S configuration as in 3 that keeps the two lactone-
containing wings away from the two furan-3(2H)-one moieties
enhances the activity; (2) the length of the two wings, the
double bonds, and the lactone rings are also important for the
activity. This gross SAR analysis of these diastereoisomers
indicates that the active binding site of the DGAT-1 enzyme is
highly selective to the stereochemistry of the substrate.
EXPERIMENTAL SECTION
■
General Experimental Procedures. IR spectra were recorded
with KBr disks. Optical rotations were measured at room temperature.
NMR spectra were mainly measured on a 400 or 500 MHz
spectrometer, except for the NOESY spectra, which were measured
on a 700 MHz spectrometer. HR-ESI-MS was carried out on a TOF
mass spectrometer. EI-MS spectra were measured on a spectrometer
by direct inlet at 70 eV. An ODS-A column (10 mm × 250 mm, S-5
μm, 12 nm) and an AD-H column (10 mm × 250 mm, 5 μm) were
used for the semipreparative HPLC analysis. Data collections for
crystal structure analysis were performed at room temperature (293 K)
employing graphite-monochromatized Cu Kα radiation (λ = 1.54178
Å).
Plant Material. The leaves of A. grandifolia BL were collected from
Sanya of Hainan Island, People’s Republic of China, in May 2010 and
were identified by Prof. Shi-Man Huang (Department of Biology,
Hainan University, P. R. China). A voucher specimen has been
deposited with the Shanghai Institute of Materia Medica, Chinese
Academy of Sciences, P. R. China (accession number AP-2010-2Y).
Extraction and Isolation. The dried powder of the leaves of A.
grandifolia (5 kg) was extracted three times with 95% ethanol at room
temperature to give a crude extract (350 g), which was then
partitioned between EtOAc and water to yield an EtOAc-soluble
fraction E (150 g). Fraction E was subjected to a column of MCI gel
(MeOH/H2O, 50:50−90:10 v/v) to give four fractions E1−E4.
Fraction E3 (13.3 g) showed significant inhibition (ca. 41%, 10 μg/
mL) on DGAT-1 enzyme and was subjected to column chromatog-
raphy using reversed-phase C18 silica gel (MeOH/H2O, 65:35−85:15
v/v) to obtain the major fraction E3a, which mainly contained
aphadilactones A−D (553 mg). Fraction E3a was further separated by
semipreparative HPLC equipped with a C18 silica gel column (10 mm
× 250 mm, YMC Co., Ltd.) and eluted with the mobile phase of
CH3CN/H2O (68:32 v/v) to afford two fractions, E3a1 (260 mg) and
E3a2 (228 mg). Each of the fractions E3a1 and E3a2 showed a
homogeneous single peak in HPLC analysis (C18 silica gel column,
even run under a number of optimized conditions) but displayed very
complicated NMR characteristics suggestive of a mixture. Fraction
E3a1 was thus subjected to a further purification by semipreparative
HPLC using a chiral AD-H column (10 mm × 250 mm, Daicel
Chemical Industries, Ltd.) with n-hexane/isopropanol/ethanol
(70:10:20 v/v) as the mobile phase to yield compounds 1 (126 mg)
and 2 (100 mg). In the same way, fraction E3a2 afforded 3 (120 mg)
and 4 (55 mg).
Compounds 1−4 were also tested for in vitro antimalarial
activity, and they exhibited significant activities with IC50 values
of 190
60, 1350
150, 170
10, and 120
50 nM,
respectively. Artemisinin was used as the positive control, and
its IC50 value was 7.3 0.3 nM (Table 3). Further studies are
required in order to establish whether their mechanism of
action involves inhibition of PfDGAT enzyme.
CONCLUSION
■
Our chemical investigation into the Chinese herb A. grandifolia
has led to the isolation of a new class of diterpenoid dimers,
aphadilactones A−D (1−4). Compound 3 showed very potent
and selective inhibition against DGAT-1 (IC50 = 0.46 0.09
Table 3. Antimalarial (P. falciparum) Activities of Compounds 1−4 in Vitro
compound
IC50 (nM)
1
2
3
4
artemisinin
7.3 0.3
190 60
1350 150
170 10
120 50
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The Journal of Organic Chemistry
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Compounds 1−4 were confirmed to exist in the ethanolic crude
extract of the leaves by both TLC (four compounds showed the same
Rf = 0.6 in chloroform/MeOH, 20:1) and HPLC (tR = 14.8 min for 1
and 2, tR = 13.2 min for 3 and 4; C-18 column with a mobile phase of
CH3CN/H2O, 70:30 v/v).
Compound 8. Pale gum; [α]2D2 −42.5 (c 0.12, MeOH); 1H and 13
C
spectroscopic data, see Tables 1 and 2; IR (KBr) νmax 2931, 1701,
1612, 1577, 1458, 1415, 1377, 1362, 1261, 1221, 1174, 958, 939 cm−1;
UV/vis (MeOH) λmax/nm (log ε) 265.2 (4.35); CD (MeOH) λmax
/
nm (Δε) 274 (1.81), 238 (−0.92); ESI-MS(+) m/z 445.4 [M + H]+,
467.3 [M + Na]+, 911.6 [2M + Na]+; ESI-MS(−) m/z 443.5 [M −
H]−; HR-ESI-MS(+) m/z 445.2593 [M + H]+ (calcd for C26H37O6,
445.2590).
Aphadilactone A (1). Pale gum; [α]2D2 −8.3 (c 0.145, MeOH); H
1
and 13C spectroscopic data, see Tables 1 and 2; IR (KBr) νmax 2931,
1699, 1606, 1574, 1415, 1381, 1246, 1174, 1041, 850 cm−1; UV/vis
λmax/nm (log ε) 265.4 (4.21); CD (MeOH) λmax/nm (Δε) 283 (2.31),
253 (−5.22), 230 (1.03); ESI-MS(+) m/z 661.5 [M + H]+, 683.4 [M
+ Na]+; ESI-MS(−) m/z 659.9 [M − H]−; HR-ESI-MS(+) m/z
683.3569 [M + Na]+ (calcd for C40H52O8Na, 683.3560).
1
Compound 9. Light-yellow oil; H and 13C spectroscopic data, see
Table S1 in the SI; EI-MS (70 eV) m/z (%) 141 (1), 111 (100), 83
(16), 55 (36).
Reduction of Compounds 5 and 6. To a solution of 5 (1.5 mg,
0.0034 mmol) in MeOH (1 mL), excess NaBH4 (1.0 mg, 0.026 mmol)
was added at 0 °C, and the mixture was kept shaking for 10 min. The
solvent was removed under reduced pressure, and the residue was
treated with 0.1 mL of saturated NH4Cl solution, after which 2 mL of
water was added. The aqueous solution was extracted with ethyl ether
(3 × 2 mL), and the combined organic layers were dried over
anhydrous Na2SO4. After filtration and removal of the solvent,
compound 5a (1.2 mg) was obtained. In a similar procedure,
compound 6 (1.7 mg, 0.0038 mmol) was transformed to 6a (1.4 mg).
Compound 5a. CD (MeOH) λmax/nm (Δε) 286 (0.23), 270
(−0.21), 240 (1.42); ESI-MS(+) m/z 449.4 [M + H]+, 920.5 [2M +
Na]+; ESI-MS(−) m/z 447.4 [M − H]−; HR-ESI-MS(+) m/z
449.2901 [M + H]+ (calcd for C26H41O6, 449.2903).
Aphadilactone B (2). Pale gum; [α]2D2 −40.7 (c 0.15, MeOH); H
1
and 13C spectroscopic data, see Tables 1 and 2; IR (KBr) νmax 2931,
1697, 1606, 1574, 1439, 1415, 1381, 1246, 1221, 1174, 1041, 850
cm−1; UV/vis (MeOH) λmax/nm (log ε) 265.2 (4.36); CD (MeOH)
λmax/nm (Δε) 243 (−8.66), 224 (4.24); ESI-MS(+) m/z 661.5 [M +
H]+, 683.4 [M + Na]+; ESI-MS(−) m/z 659.7 [M − H]−; HR-ESI-
MS(+) m/z 661.3727 [M + H]+ (calcd for C40H53O8, 661.3740).
Aphadilactone C (3). Pale gum; [α]2D0 4.0 (c 0.125, MeOH); H
1
and 13C spectroscopic data, see Tables 1 and 2; IR (KBr) νmax 2931,
1699, 1608, 1576, 1458, 1381, 1362, 1246, 1176, 1072, 1041, 850
cm−1; UV/vis (MeOH) λmax/nm (log ε) 266.0 (4.08); CD (MeOH)
λmax/nm (Δε) 308 (1.02), 267 (−2.28), 252 (−1.87), 230 (0.15); ESI-
MS(+) m/z 661.6 [M + H]+, 683.5 [M + Na]+; ESI-MS(−) m/z 659.9
[M − H]−; HR-ESI-MS(+) m/z 683.3561 [M + Na]+ (calcd for
C40H52O8Na, 683.3560).
Compound 6a. CD (MeOH) λmax/nm (Δε) 277 (−0.61), 268
(−0.56), 238 (2.30); ESI-MS(+) m/z 449.4 [M + H]+, 919.6 [2M +
Na]+; ESI-MS(−) m/z 447.4 [M − H]−; HR-ESI-MS(+) m/z
449.2901 [M + H]+ (calcd for C26H41O6, 449.2903).
Aphadilactone D (4). Pale gum; [α]2D0 −15.1 (c 0.185, MeOH); 1H
and 13C spectroscopic data, see Tables 1 and 2; IR (KBr) νmax 2931,
1697, 1610, 1576, 1458, 1381, 1246, 1174, 1072, 1041, 850 cm−1; UV/
vis (MeOH) λmax/nm (log ε) 266.0 (4.01); CD (MeOH) λmax/nm
(Δε) 275 (1.49), 250 (−3.15), 224 (−1.39); ESI-MS(+) m/z 661.5
[M + H]+, 683.4 [M + Na]+; ESI-MS(−) m/z 659.9 [M − H]−; HR-
ESI-MS(+) m/z 683.3575 [M + Na]+ (calcd for C40H52O8Na,
683.3560).
Dihydroxylation of Aphadilactone A (1). To a solution of
compound 1 (15 mg, 0.023 mmol) in tert-butyl alcohol/water (1:1, 2
mL) were added K2OsO4 (0.42 mg, 0.0012 mmol), K3Fe(CN)6 (112
mg, 0.34 mmol), K2CO3 (47 mg, 0.34 mmol), and MeSO2NH2 (11
mg, 0.12 mmol) at room temperature.19 After about 10 h of reaction
(monitored by TLC), the reaction was quenched with 5 mL of
saturated Na2S2O3. The resulting aqueous mixture was then extracted
with EtOAc (3 × 10 mL). The organic phase was washed with brine
(30 mL) and dried over anhydrous MgSO4. After filtration, the solvent
was removed in vacuo, and the residue was purified by semipreparative
HPLC (CH3CN/H2O, 55:45 v/v) to afford 10a (2.0 mg, tR = 13.2
min), 11a (2.1 mg, tR = 14.6 min), and 12a (2.5 mg, tR = 7.3 min).
Ozonolysis of Aphadilactones A−D (1−4). Compound 1 (10.0
mg, 0.015 mmol) in CH2Cl2/MeOH (10 mL/2 mL) was ozonized at
−78 °C and then treated with dimethyl sulfide (0.2 mL).16 The
reaction mixture was allowed to warm to room temperature and stirred
overnight. After workup, the resulting residue was purified by
preparative TLC (developed by CH3Cl/MeOH, 30:1 v/v) to afford
6.0 mg of compound 5 (Rf = 0.5) and 1.5 mg of compound 9 (Rf =
0.3); the latter was an inseparable mixture of two epimers.
Aphadilactones B−D (2−4) (each 10.0 mg) were subjected to the
same ozonolysis procedure to produce compounds 6−8 (6, 5, and 6
mg, respectively) compound 9 (1.0 mg) was obtained only in the
ozonolysis of compound 4.
Compound 10a. Pale gum; [α]2D2 −4.6 (c 0.065, MeOH); H and
1
13C spectroscopic data, see Table S2 in the SI; UV/vis (MeOH) λmax
/
nm (log ε) 265.4 (4.06); ESI-MS(+) m/z 695.4 [M + H]+, 717.3 [M +
Na]+; ESI-MS(−) m/z 739.8 [M + HCOO]−; HR-ESI-MS(+) m/z
717.3613 [M + Na]+ (calcd for C40H54O10Na, 717.3615).
Compound 11a. Pale gum; [α]2D2 −6.0 (c 0.05, MeOH); H and
1
13C spectroscopic data, see Table S2 in the SI; UV/vis (MeOH) λmax
/
Compound 5. Pale gum; [α]2D2 25.7 (c 0.175, MeOH); 1H and 13
C
nm (log ε) 264.4 (3.83); ESI-MS(+) m/z 695.3 [M + H]+, 717.3 [M +
Na]+; HR-ESI-MS(+) m/z 717.3617 [M + Na]+ (calcd for
C40H54O10Na, 717.3615).
spectroscopic data, see Tables 1 and 2; IR (KBr) νmax 2931, 1699,
1608, 1576, 1456, 1414, 1377, 1362, 1221, 1174, 937 cm−1; UV/vis
(MeOH) λmax/nm (log ε) 264.8 (4.16); CD (MeOH) λmax/nm (Δε)
274 (1.95), 239 (3.12); ESI-MS(+) m/z 445.4 [M + H]+, 467.3 [M +
Na]+, 911.6 [2M + Na]+; ESI-MS(−) m/z 443.4 [M − H]−; HR-ESI-
MS(+) m/z 445.2591 [M + H]+ (calcd for C26H37O6, 445.2590).
Compound 12a. Pale gum; [α]2D2 −21.7 (c 0.06, MeOH); H and
1
13C spectroscopic data, see Table S2 in the SI; UV/vis (MeOH) λmax
/
nm (log ε) 214.2 (4.05), 265.2 (4.06); ESI-MS(+) m/z 729.4 [M +
H]+, 751.3 [M + Na]+; ESI-MS(−) m/z 773.6 [M + HCOO]−; HR-
ESI-MS(+) m/z 751.3676 [M + Na]+ (calcd for C40H56O12Na,
751.3669).
Compound 6. Pale gum; [α]2D2 −27.8 (c 0.115, MeOH); H and
1
13C spectroscopic data, see Tables 1 and 2; IR (KBr) νmax 2931, 1699,
1606, 1576, 1458, 1414, 1377, 1362, 1223, 1174, 937 cm−1; UV/vis
(MeOH) λmax/nm (log ε) 265.4 (4.29); CD (MeOH) λmax/nm (Δε)
273 (−1.83), 237 (−2.42); ESI-MS(+) m/z 445.3 [M + H]+, 467.2 [M
+ Na]+, 911.6 [2M + Na]+; ESI-MS(−) m/z 443.1 [M − H]−; HR-
ESI-MS(+) m/z 445.2588 [M + H]+ (calcd for C26H37O6, 445.2590).
Oxidative Cleavage of Compounds 10a−12a. Compound 10a
(1.0 mg) was dissolved in dried CH2Cl2 (1 mL), and a fresh batch of
lead tetraacetate (1.0 mg) was slowly added at 0 °C.20 After the raw
material was completely converted (as monitored by TLC), an excess
of newly prepared methanolic solution of NaBH4 was added, and the
reaction was continued for 10 min.21 After workup, the resulting
product was subsequently subjected to chiral HPLC analysis (chiral
AD-H column, 10 mm × 250 mm, Daicel Chemical Industries, Ltd.)
with n-hexane/isopropanol (2.7:0.3) as the mobile phase. In the same
fashion, compounds 11a (1.0 mg) and 12a (1.2 mg) were treated, and
the products were subjected to chiral HPLC analysis. As our aim was
to determine the chirality of 13 by comparing its chiral HPLC
retention time with those of authentic samples, the expected products
Compound 7. Pale gum; [α]2D2 40.7 (c 0.135, MeOH); 1H and 13
C
spectroscopic data, see Tables 1 and 2; IR (KBr) νmax 2931, 1697,
1612, 1577, 1458, 1417, 1377, 1362, 1223, 1174, 939, 804 cm−1; UV/
vis (MeOH) λmax/nm (log ε) 264.4 (4.17); CD (MeOH) λmax/nm
(Δε) 274 (−2.12), 239 (0.86); ESI-MS(+) m/z 445.4 [M + H]+,
467.4 [M + Na]+, 911.6 [2M + Na]+; ESI-MS(−) m/z 443.4 [M −
H]−; HR-ESI-MS(+) m/z 445.2590 [M + H]+ (calcd for C26H37O6,
445.2590).
G
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10b, 11b, 12b (5a), and 13 were not deliberately purified in the final
step.
Antimalarial Assay. Dose-dependent growth inhibition was
assessed against P. falciparum strain Dd2 (chloroquine-resistant)
using SYBR Green as described previously.27 Briefly, ring-stage
parasite cultures (200 μL/well, 1% hematocrit and 1% parasitemia)
were grown for 72 h in the presence of increasing concentrations of
drug. Ten-point dilutions were used to test the dose response at
concentrations ranging from 5 to 0.1 μM for compound 3 and from 1
to 0.01 μM for compounds 1, 2, and 4. Artemisinin was used as a
positive control. Parasite growth was normalized to untreated controls.
The IC50 calculations were performed with GraFit 5 (Erithacus
Software Ltd.) using nonlinear regression curve fitting, and the
reported values represent averages of two independent experiments
with standard deviations.
Compounds 2−4 (each 5 mg) were treated with a similar
dihydroxylation/oxidative cleavage cascade as for 1 in a one-pot
reaction to afford three final products for chiral HPLC analysis.
Synthesis of (S)-6-(Hydroxymethyl)-4-methyl-5,6-dihydro-
2H-pyran-2-one (13).18 To a solution of TBDPSiCl (9.2 g) and
imidazole (2.25 g) in DMF (100 mL) was added dropwise a solution
of (R)-oxiran-2-ylmethanol (13a, 2.02 g) in CH2Cl2 (10 mL) at 0 °C.
The solution was allowed to warm to room temperature and stirred for
5 h. After workup in the normal way, the product was purified on a
silica gel column (hexane/EtOAc, 50:1) to afford 13b (8.09 g, 95%).
To a slurry of CuI (95 mg) in THF (5 mL) at −30 °C was added
dropwise isopropenylmagnesium bromide (0.5 M in THF, 10 mL).
The thick orange mixture was stirred for 5 min, and 13b (1.02 g) in
THF (10 mL) was added dropwise. The reaction mixture was warmed
to 0 °C and kept stirring for 15 min. The reaction was quenched by
addition of a saturated aqueous solution of NH4Cl (10 mL), and the
mixture was worked up in the usual manner. The products were
purified by column chromatography (hexane/acetone, 50:1) to yield
13c as a colorless oil (0.98 g, 85%). To a solution of 13c (0.92 g) in
dried CH2Cl2 (5 mL) were added Et3N (550 mg) and DMAP (50 mg)
at 0 °C. A solution of acryloyl acid chloride (250 mg) in CH2Cl2 (10
mL) was then added dropwise to the reaction mixture, and stirring was
continued for a further 1 h. After workup, the reaction residue was
purified by silica gel column chromatography (hexane/EtOAc, 50:1)
to afford 13d (0.95 g, 90%). A solution of 13d (250 mg) in 150 mL of
dried CH2Cl2 was purged with dried nitrogen for 15 min, and a
solution of Grubbs’ catalyst II (44 mg) in 20 mL of dried CH2Cl2 was
added dropwise under a nitrogen atmosphere. Then the reaction
mixture was refluxed under a nitrogen atmosphere for 8 h. After
removal of the solvent under reduced pressure, the residue was
purified on a silica gel column (hexane/acetone, 20:1) to obtain 13e
(185 mg, 80%). To a solution of 13e (52 mg) in 2 mL of THF was
added TBAF (0.3 mL, 1 M in THF) at 0 °C, and stirring was
continued for 0.5 h. The mixture was warmed to room temperature
and kept stirring for another 2 h. After removal of the solvent in vacuo,
the residue was purified by preparative TLC (chloroform/MeOH,
20:1) to afford 13 (16 mg, 80%). [α]2D2 −74.0 (c 0.1, MeOH); EI-MS
(70 eV) m/z (%) 112 (8), 111 (100), 83 (9); HR-EI-MS m/z
111.0456 [M − CH2OH]+ (calcd for C6H7O2, 111.0446). For 1H and
13C spectroscopic data, see Table S1 in the SI.
ASSOCIATED CONTENT
* Supporting Information
■
S
Structural determination; tabulated NMR data; 1H, 13C, and 2D
NMR spectra, mass spectra, and IR spectra of 1−14; DGAT
inhibitions of 1−8; and ECD calculation methods. This
material is available free of charge via the Internet at http://
pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
■
*Tel: +86-21-50806718. Fax: +86-21-50806718. E-mail:
jmyue@mail.shcnc.ac.cn.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
Financial support for this work was provided by the National
Natural Science Foundation of China (81021062, 20902095),
the Foundation from the Ministry of Science and Technology
of the People’s Republic of China (2012CB721105), the
Jeffress Memorial Trust of Virginia (J-1058), and the Virginia
Commonwealth Health Research Board (208-03-12) (to
M.B.C.). We thank Professor S.-M. Huang (Department of
Biology, Hainan University) for the collection and identification
of the plant material.
Synthesis of (R)-6-(Hydroxymethyl)-4-methyl-5,6-dihydro-
2H-pyran-2-one (14). Except for the starting material (14a), the
same synthetic approach and experimental conditions as used for the
synthesis of 13 were applied to prepare 14. [α]2D2 80.0 (c 0.07,
MeOH); EI-MS (70 eV) m/z (%) 111 (100), 94 (6), 83 (18), 82 (6),
55 (24); HR-EI-MS m/z 111.0428 [M − CH2OH]+ (calcd for
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