Chemical constituents of Heteroplexis micocephala

Article abstract of DOI:10.1021/np900213w

Eleven new compounds including two sesquiterpenes with an unusual 2,2,5,9-tetramethylbicyclo[6.3.0]undecane carbon skeleton (1 and 2), five phytane-type diterpene dilactones (3-7), an ent-clerodane diterpene dilactone (8), and three phenylpropenol esters

Full text of DOI:10.1021/np900213w

1184
J. Nat. Prod. 2009, 72, 1184–1190
Chemical Constituents of Heteroplexis micocephala
Xiaona Fan, Jiachen Zi, Chenggen Zhu, Wendong Xu, Wei Cheng, Sen Yang, Ying Guo, and Jiangong Shi*
Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College (Key Laboratory of BioactiVe
Substances and Resources Utilization of Chinese Herbal Medicine, Ministry of Education), Beijing 100050, People’s Republic of China
ReceiVed April 5, 2009
Eleven new compounds including two sesquiterpenes with an unusual 2,2,5,9-tetramethylbicyclo[6.3.0]undecane carbon
skeleton (1 and 2), five phytane-type diterpene dilactones (3-7), an ent-clerodane diterpene dilactone (8), and three
phenylpropenol esters (9-11), together with a diacylphenol (12) and 38 known compounds, have been isolated from an
ethanolic extract of Heteroplexis micocephala. Their structures including absolute configurations were elucidated by
spectroscopic and chemical analyses. In the in Vitro assays, compound 6 showed a selective cytotoxic activity against
A2780 with an IC50 value of 4.37 µM, while sinapyl diangelate (13) showed a potent activity inhibiting HIV-1 replication
with an IC50 value of 4.04 µM.
Heteroplexis (Compositae) is a unique genus consisting of five
species found in the limestone terrains of Longzhou and Yangshuo,
Guangxi Province, mainland China. H. micocephala Y. L. Chen is
planted and used as a folk medicine for treatment of indigestion
and dropsy.1 However, nothing is known about the chemical
constituents of the genus. As part of a program to assess the
chemical and biological diversity of several traditional Chinese
medicines,2 an ethanolic extract of H. micocephala has been
investigated. We report herein the isolation, structural elucidation,
and in Vitro bioassays of 11 new compounds including two
sesquiterpenes with an unusual 2,2,5,9-tetramethylbicyclo[6.3.0]-
undecane carbon skeleton (1 and 2), five phytane-type diterpene
dilactones (3-7), an ent-clerodane diterpene dilactone (8), and three
phenylpropenol esters (9-11), together with a diacylphenol (12)
and 38 known compounds. Although derivatives with the 2,2,5,9-
tetramethylbicyclo[6.3.0]undecane carbon skeleton were semisyn-
thesized by the acid-catalyzed transannular cyclization of zerumbol
and zerumbone epoxide,3 compounds 1 and 2 are the first examples
of natural products with this skeleton.
(calcd for C15H22O2, 234.1620) with five degrees of unsaturation
1
was indicated by HREIMS at m/z 234.1623 [M]+•. The H NMR
spectrum of 1 in acetone-d6 showed signals attributed to three
tertiary methyls at δ 0.91 (s, H3-12), 1.07 (s, H3-13), and 1.31 (s,
H3-15) and an olefinic methyl at δ 1.84 (d, J ) 1.5 Hz, H3-14).
Signals attributed to olefinic protons at δ 6.21 (ddq, J ) 12.0, 12.0,
1.5 Hz, H-4) and 6.39 (d, J ) 2.0 Hz, H-7) indicated the presence
of two trisubstituted double bonds. An exchangeable singlet at δ
4.00 (OH-9) suggested the presence of a hydroxy group attached
at a quaternary carbon. A double doublet at δ 2.92 (dd, J ) 6.5
and 2.0 Hz, H-1) and a broadened triplet at δ 2.34 (brt, J ) 12.0
Hz, H-3a), together with partially overlapped multiplets integrating
for five protons between δ 1.63 and 1.96 (see Table 1), suggested
the presence of a methine and three methylenes in 1. The 13C NMR
and DEPT spectra showed carbon signals corresponding to the
above protonated units and five quaternary carbons (Table 2). On
the basis of chemical shifts, the quaternary carbons were differenti-
ated to be a carbonyl carbon, two olefinic carbons, an oxygen-
bearing carbon, and an aliphatic carbon. These spectroscopic data
suggested that 1 was an unusual bicyclic sesquiterpene containing
crossed conjugated dienone and hydroxy functional groups.
The structure was further elucidated by the 2D NMR spectro-
1
scopic data of 1. Analysis of H-1H COSY and HSQC spectra
provided unambiguous assignments of proton and carbon signals.
In the HMBC spectrum, long-range correlations from H-1 to C-8,
C-10, and C-11, from both HO-9 and H3-15 to C-8, C-9, and C-10,
and from HO-9 to C-15, in combination with chemical shifts and
coupling patterns of these protons and carbons, indicated the
presence of a 9-hydroxy-9-methylcyclopentane moiety. HMBC
correlations from H-4 to C-6 and C-14, from H-7 to C-1, C-5, and
C-8, from both H3-12 and H3-13 to C-1, C-2, and C-3, and from
H3-14 to C-4, C-5, and C-6, together with chemical shifts and
coupling patterns of these protons and carbons, indicated unequivo-
cally that there was an unusual 2,2,5-trimethylcycloocta-4,7-dien-
6-one moiety in 1. Accordingly, the planar structure of 1 was
assigned as 9-hydroxy-2,2,5,9-tetramethylbicyclo[6.3.0]undeca-4,7-
dien-6-one. The configuration of 1 was elucidated by an analysis
of the NOE difference experiment and CD data. Irradiation of H-1
enhanced H3-13 and H3-15, and in turn irradiation of H3-15
enhanced H-1 and H-7. This indicated that H-1, H3-13, and H3-15
were cofacially oriented. The CD spectrum of 1 showed negative
Cotton effects at 354 (∆ε -1.1) and 257 nm (∆ε -5.88). Molecular
modeling using the MM2 program indicated that the cross-
conjugated octadienone ring of 1S,9S- or 1R,9R-9-hydroxy-2,2,5,9-
tetramethylbicyclo[6.3.0]undeca-4,7-dien-6-one possessed a slightly
twisted boat conformation for the lowest energy conformation
(Figure S1, Supporting Information). On the basis of the octant
Results and Discussion
Compound 1 was obtained as a yellowish oil, [R]20 -84.0 (c
D
0.16, MeOH), and the presence of hydroxy (3424 cm-1) and
conjugated ketone (1730, 1714, 1650, 1623, and 1601 cm-1) groups
was indicated by its IR spectrum. The EIMS of 1 gave a molecular
ion peak at m/z 234 [M]+•, and the molecular formula C15H22O2
* To whom correspondence should be addressed. Tel: 86-10-83154789.
Fax: 86-10-63017757. E-mail: shijg@imm.ac.cn.
10.1021/np900213w CCC: $40.75  2009 American Chemical Society and American Society of Pharmacognosy
Published on Web 05/19/2009

Chemical Constituents of Heteroplexis micocephala
Journal of Natural Products, 2009, Vol. 72, No. 6 1185
Table 1. 1H NMR Spectroscopic Data (δ) of Compounds 1-8a
position
1
2
3
4
5
6
7
8
1
2.92 dd (6.5, 2.0) 3.04 td (6.0, 2.5)
2
5.84 s
5.84 t (2.0)
5.84 s
5.85 s
6.14 dd (10.0, 3.0)
6.58 dd (10.0, 3.0)
3a
3b
4
2.34 t (12.0)
1.72 t (12.0)
6.21 ddq
(12.0, 12.0, 1.5)
2.27 t (12.0)
1.75 dd (12.0, 8.5)
6.19 ddq
(12.0, 8.5, 1.0)
2.45 t (7.0)
2.55 m
2.53 m
2.36 m
2.53 m
2.35 m
3.37 t (3.0)
5a
5b
2.31 dt (14.0, 7.0) 1.91 m
1.82 m
6a
6b
7a
7b
5.13 t (7.0)
4.17 dt (7.0, 5.0)
5.26 t (6.5)
5.26 td (7.0, 1.0)
2.03 m
1.75 m
2.10 m
1.75 m
6.39 d (2.0)
6.40 d (2.5)
8a
8b
9
10a
10b
11a
11b
12a
2.16 t (7.0)
2.33 dt (15.0, 7.5) 2.22 m
2.22 dt (15.0, 7.5)
2.26 dt (14.0, 7.0) 2.42 m
6.66 m 6.57 m
2.75 d (7.0)
2.70 t (7.2) 2.54 dd (11.5, 3.0)
2.53 m
5.70 dt (15.0, 7.0)
6.71 td (7.5, 1.0) 5.57 dd (15.0, 8.5)
2.37 q (7.2)
1.89 m
1.66 m
1.93 m
1.65 m
0.91 s
1.85 m
1.75 m
1.90 m
1.70 m
0.81 s
6.49 m
3.01 s
3.49 dd (8.5, 4.5)
3.00 dd (12.0, 5.0)
1.95 t (12.0)
5.60 dd (12.0, 5.0)
3.03 dd (17.0, 5.0) 3.15 dd (16.5, 3.5) 4.93 dd (6.0, 5.0) 4.41 ddd (4.5, 4.5, 2.0) 3.18 dd
(18.4, 7.2)
2.55 m
12b
13
14
15
16
2.48 m
2.52 m
5.26 m
5.26 m
1.07 s
1.84 d (1.5)
1.31 s
1.05 s
1.84 brs
1.38 s
5.21 dt (7.0, 4.0)
5.22 d (4.0)
5.10 dd (9.0, 5.0) 5.15 dd (9.0, 4.5)
5.47 d (9.0)
5.25 m
5.25 m
5.47 d (9.0)
6.56 brs
7.64 brs
7.54 brs
1.75 s
1.77 s
1.63 s
1.75 s
1.75 s
5.12 brs
4.88 brs
4.83 d (2.0)
4.05 d (5.0)
1.75 s
1.79 s
1.67 s
1.75 s
1.76 s
1.64 s
1.75 s
1.75 s
2.11 s
17
19a
19b
20
4.60 d (9.5)
4.11 d (9.5)
1.16 s
4.73 s
4.81 s
4.40 d (6.0)
4.81 d (2.0)
4.44 d (2.0)
OH
4.00 s
3.87 s
a Data (δ) were measured in CDCl3 for 3 and in acetone-d6 for 1, 2, and 4-8 at 500 MHz. Proton coupling constants (J) in Hz are given in
1
parentheses. The assignments were based on DEPT, H-1H COSY, HSQC, and HMBC experiments.
Table 2. 13C NMR Spectroscopic Data (δ) of Compounds 1-8a
position
1
2
3
4
5
6
7
8
1
2
3
4
5
6
7
8
49.8
44.5
41.1
133.7
140.8
195.9
130.4
169.4
80.9
41.1
24.6
25.6
26.0
49.9
43.9
41.3
133.2
141.1
196.1
130.1
170.2
80.8
41.2
24.3
25.7
25.9
174.0
115.7
169.9
28.5
174.3
115.2
172.5
25.4
33.8
74.2
151.9
30.4
29.1
130.2
128.5
32.9
174.3
115.5
172.0
29.0
173.5
115.6
172.0
29.1
197.0
132.6
139.4
53.2
43.7
35.0
19.1
50.9
36.1
56.7
25.7
26.3
26.4
123.2
135.8
37.8
124.5
136.3
38.8
124.5
136.2
43.5
134.9
124.2
51.5
74.2
80.1
119.4
140.3
18.6
206.8
41.6
24.6
138.4
128.8
32.8
9
28.4
28.5
10
11
12
13
14
15
16
17
18
19
20
139.3
126.9
32.4
144.6
132.7
68.1
43.0
72.1
74.3
74.6
79.7
74.7
127.2
109.7
144.6
140.9
171.3
173.6
72.4
20.1
30.4
20.2
26.6
123.6
139.6
18.4
25.6
170.8
16.0
125.2
130.2
18.3
119.2
140.4
18.6
26.1
170.3
16.0
125.2
139.2
18.2
25.7
170.8
30.2
25.7
26.0
176.7
16.2
170.8
110.3
73.7
73.1
73.7
73.7
14.6
a Data (δ) were measured in CDCl3 for 3 at 125 MHz and in acetone-d6 for 1, 2, and 4-8 at 125 or 150 MHz. The assignments were based on
1
DEPT, H-1H COSY, HSQC, and HMBC experiments.
rule for cross-conjugated dienones,4 negative Cotton effects were
predicted by the octant projection diagram of the 1S,9S-enantiomer,
whereas positive Cotton effects were predicted for the 1R,9R-
enantiomer. Therefore, the structure of 1 was determined as (-)-
1S,9S-9-hydroxy-2,2,5,9-tetramethylbicyclo[6.3.0]undeca-4,7-dien-
6-one.
H3-12, and C-15 of 2 were shielded by ∆δH -0.07, -0.10 and
∆δC -3.8 ppm, respectively. These differences suggested that 2
was a C-1 or C-9 epimer of 1. The C-9 epimeric relationship was
confirmed by the NOE difference experiment of 2 that showed
enhancements of HO-9 and H3-13 by irradiation of H-1 and an
enhancement of H3-12 by irradiation of H3-15. This was supported
by the molecular modeling and the octant projection diagrams of
the C-1 and C-9 epimers of 1 (Figure S2, Supporting Information).
Therefore, the structure of 2 was determined as (-)-1S,9R-9-
hydroxy-2,2,5,9-tetramethylbicyclo[6.3.0]undeca-4,7-dien-6-one.
Compound 2 was obtained as a yellowish gum, [R]20D -55.3 (c
0.53, MeOH). Its IR, EIMS, and NMR spectroscopic features were
similar to those of 1 (see Tables 1 and 2 and Experimental Section),
indicating that 2 is an isomer of 1. Analysis of the 2D NMR
spectroscopic data of 2 further confirmed that it had a planar
structure identical to that of 1. However, the NMR resonances
assignable to H-1, H3-15, and C-8 of 2 (Tables 1 and 2) were
deshielded by ∆δH +0.12, +0.07 and ∆δC +0.8 ppm, respectively,
compared to those of 1. Meanwhile, the resonances due to H-3a,
Compound 3, a yellowish oil, [R]20 +31.3 (c 1.38, MeOH),
D
showed IR absorptions (1778, 1748, 1678, and 1637 cm-1
)
consistent with the presence of both an exo- and an endocyclic
unsaturated γ-lactone functionality. The EIMS of 3 gave a molecular
ion peak at m/z 330 [M]+•, and the HREIMS at m/z 330.1859 [M]+•

1186 Journal of Natural Products, 2009, Vol. 72, No. 6
Fan et al.
1
indicated that the molecular formula of 3 was C20H26O4. The H
NMR spectrum of 3 displayed signals attributed to three olefinic
tertiary methyls at δ 1.63 (s, H3-19), 1.75 (s, H3-16), and 1.77 (s,
H3-17) and signals assignable to five olefinic and/or oxygen-bearing
methines at δ 6.66 (m, H-10), 5.84 (s, H-2), 5.22 (d, J ) 4.0 Hz,
H-14), 5.21 (dt, J ) 7.0, 4.0 Hz, H-13), and 5.13 (t, J ) 7.0 Hz,
H-6). In addition, it showed signals due to a deshielded oxymeth-
ylene and five aliphatic methylenes (Table 1). The 13C NMR
spectrum of 3 showed 20 carbon resonances corresponding to the
above protonated units and six quaternary carbons including two
ester carbonyls and four olefinic carbons (Table 2). The above
spectroscopic data indicated that 3 is a dilactonized acyclic
diterpene. The protonated carbons and their bonded protons (see
Tables 1 and 2) were assigned unambiguously by the HSQC data.
In the 1H-1H COSY spectrum of 3, homonuclear vicinal coupling
correlation between H2-5 and both H2-4 and H-6, and between H2-9
and both H2-8 and H-10, in combination with the chemical shifts
and coupling patterns of these protons, indicated unambiguously
the presence of the two structural fragments from C-4 to C-6 and
from C-8 to C-10 in 3. Vicinal coupling correlations of H-13 with
both H2-12 and H-14, together with the chemical shifts and coupling
patterns of these protons, established the fragment from C-12 to
C-14 in 3. In the HMBC spectrum of 3, two- and three-bond
heteronuclear correlations from H-6 to C-4, C-5, C-8, and C-19,
from H2-8 to C-6, C-7, C-9, C-10, and C-19, and from H3-19 to
C-6, C-7, and C-8 indicated unequivocally that the two former
fragments were connected through C-7 to form a linear central
moiety and that one of the methyls was located at C-7 in 3. HMBC
correlations from H-2 to C-1, C-3, C-4, and C-20, from H2-4 to
C-2, C-3, C-5, C-6, and C-20, and from H2-20 to C-1, C-2, and
C-3, in combination with chemical shifts of these protons and
carbons, indicated the presence of the eastern R,ꢀ-unsaturated
γ-lactone ring fragment connecting to the central moiety through
a single bond between C-3 and C-4. HMBC correlations from H2-9
to C-11, from H-10 to C-12 and C-18, and from H-12a to C-11,
C-13, C-14, and C-18, along with the chemical shifts of these
protons and carbons, indicated the presence of the western γ-lactone
ring fragment connecting to the central moiety through a double
bond between C-10 and C-11. In addition, HMBC correlations from
H-14 to C-16 and C-17 and from both H3-16 and H3-17 to C-14
and C-15, together with chemical shifts of these protons and
carbons, established the connection of C-15 with C-14, C-16, and
C-17. In the NOE difference experiment of 3, irradiation of H-6
caused an enhancement of H2-8, while H2-9 was enhanced by
irradiation of H-12a. These NOE data indicated an E geometric
configuration for the double bonds between C-6 and C-7 and
between C-10 and C-11. On the basis of the specific rotations
reported for γ-substituted R-methylene-γ-lactones,5 the positive
specific rotation of 3 indicated that it had 13S-configuration. Thus,
the structure of 3 was determined and assigned the trivial name
heteroplexisolide A.
This suggestion was supported by changes of the chemical shifts
and coupling patterns of the central moiety of 4 as compared with
those of 3 (Tables 1 and 2). It was further confirmed by the 2D
NMR spectroscopic data of 4, which permitted unambiguous
assignment of the NMR data (Tables 1 and 2). In particular, in the
HMBC spectrum of 4, long-range heteronuclear correlations from
H2-19 to C-6, C-7, and C-8 and from HO-6 to C-5 and C-6, together
with chemical shifts and coupling patterns of these protons and
carbons, proved the location of the double bond between C-7 and
C-19 and the hydroxy group at C-6 in 4. The CD data of 4
(Experimental Section) were similar to those of 3. This suggested
that 4 also possessed S absolute configuration at C-13. Therefore,
the structure of 4 was proposed for heteroplexisolide B. The
determination of the absolute configuration at C-6 of 4 by using
the Mosher’s method failed due to a limitation of the sample
amount.
Compound 5 was obtained as a yellowish oil, [R]20D +67.0 (c 2.08,
MeOH). The IR and HRESIMS data of 5 (Experimental Section)
indicated that it is an isomer of 4. However, the NMR data of 5 (Tables
1 and 2) indicated that it possessed a central linear moiety and an
eastern R,ꢀ-unsaturated γ-lactone ring identical to those of 3. A further
analysis of the 1H NMR spectrum of 5 demonstrated that the chemical
shifts and coupling patterns assignable to the western γ-lactone moiety
[δH 4.40 (d, J ) 6.0 Hz, exchangeable, OH-12), 4.93 (dd, J ) 6.0 and
5.0 Hz, H-12), 5.10 (dd, J ) 9.0 and 5.0 Hz, H-13), and 5.47 (d, J )
9.0 Hz, H-14)] were significantly different from those of 3 and 4. These
data suggested that the hydroxy group is located at C-12 in 5 instead
of C-6 in 4. This suggestion was supported by the 13C NMR (Table
2) and 2D NMR data of 5. In particular, it was proved by two- and
three-bond correlations from HO-12 to C-11, C-12, and C-13 and from
H-12 to C-10, C-11, C-13, and C-18 in the HMBC spectrum. In the
NOE difference spectrum of 5, irradiation of HO-12 enhanced H-14,
while irradiation of H-12 caused enhancements of H2-9 and H-13. This
indicated a cis-orientation of H-12 and H-13 and an E geometric
configuration of the double bond between C-10 and C-11 for 5. On
the basis of comparison of the specific rotation of 5 with those of the
closely related analogues, such as 3-epilitsenolides D1 and D2,6 the
absolute configuration of 5 was proposed to be 12S,13S. Therefore,
the structure of 5 was determined for heteroplexisolide C.
The spectroscopic data of 6 (Tables 1 and 2 and Experimental
Section) indicated that it is also an isomer of 4. Comparison of the
NMR data of 5 and 6 indicated that they had the same eastern R,ꢀ-
unsaturated γ-lactone ring. However, the signals assigned to H2-9 and
H-10 of 5 were replaced by resonances attributed to an E-double bond
[δ 5.70 (dt, J ) 15.0, 7.0 Hz, H-9) and 5.57 (dd, J ) 15.0, 8.5 Hz,
H-10)] and a deshielded methine [3.49 (dd, J ) 8.5, 4.5 Hz, H-11)]
for 6. This suggested that the double bond between C-10 and C-11 in
5 shifted to between C-9 and C-10 in 6. The suggestion was supported
by a shielded shift of H-12 (∆δ -0.52 ppm) for 6 compared with 5.
The elucidation was further verified by the 13C NMR (Table 2) and
2D NMR data of 6. Although the C-1 resonance was not clearly
observable in the 13C NMR spectrum of 6, the HMBC spectrum
(Supporting Information) indicated that there was a carbon resonance
at δC 173.5 correlating with H2-20. HMBC correlations from H-9 to
C-8, C-10, and C-11, from H-10 to C-8, C-9, and C-11, from H-11 to
C-9, C-10, and C-18, and from H-13 to C-15, together with the
chemical shifts and coupling constants of these protons and carbons,
confirmed the location of the double bond between C-9 and C-10 and
the hydroxy group at C-12 in 6. In the NOE spectrum, irradiation of
H-11 enhanced H-12 and H-13; in turn irradiation of H-13 enhanced
H-11 and H-12. These data indicated that H-11, H-12, and H-13 are
oriented on the same side of the western lactone ring in 6. The positive
specific rotation suggested that 6 had an 11R,12S,13S configuration.6
Therefore, the structure of 6 was determined for heteroplexisolide D.
Compound 4, a yellowish oil, [R]20 +31.4 (c 0.50, MeOH),
D
exhibited a quasimolecular ion peak at m/z 369 [M + Na]+ in its
positive ESIMS. The molecular formula C20H26O5 (calcd for
C20H26O5Na, 369.1678) of 4, with one oxygen more than that of 3,
was indicated from the HRESIMS at m/z 369.1688 [M + Na]+.
The IR and NMR spectroscopic features of 4 showed some
similarities to those of 3 (see Tables 1 and 2 and Experimental
Section). However, the IR spectrum of 4 showed the presence of
hydroxy group(s) (3463 cm-1). The 1H NMR spectrum of 4 showed
that the signals of the olefinic methyl (CH3-19) and methine (CH-
6) of 3 were replaced respectively by signals attributable to an
olefinic methylene [δH 5.12 and 4.88 (brs each, H-19a and H-19b)
and δC 110.3] and an oxymethine [δH 4.17 (dt, J ) 7.0 and 5.0
Hz, H-6) and 4.05 (d, J ) 5.0 Hz, exchangeable, HO-6) and δC
74.2]. This suggested that 4 was a derivative of 3 containing a
double bond between C-7 and C-19 and a hydroxy group at C-6.
Compound 7 was obtained as a yellowish oil, [R]20 +15.6 (c
D
0.27, MeOH). Its IR spectrum showed the presence of conjugated
lactone (1752 and 1679 cm-1) and carbonyl (1715 cm-1) groups.

Chemical Constituents of Heteroplexis micocephala
Journal of Natural Products, 2009, Vol. 72, No. 6 1187
Table 3. NMR Spectroscopic Data (δ) for Compounds 9-11a
9
10
11
position
δH
δC
δH
δC
δH
δC
1
2
3
4
5
6
7
8
133.0
105.0
154.5
139.4
154.5
105.0
134.6
123.9
65.1
135.7
104.2
153.4
129.7
153.4
104.2
134.2
125.1
65.0
135.6
104.3
153.4
129.6
153.4
104.3
134.2
125.0
65.0
6.79 s
6.79 s
6.65 d (16.0)
6.36 dt (16.0, 6.5)
4.77 dd (6.5, 1.0)
6.85 s
6.85 s
6.69 d (15.5)
6.45 dt (15.5, 6.0)
4.79 dd (6.0, 1.0)
6.85 s
6.85 s
6.70 d (15.5)
6.44 dt (15.5, 6.0)
4.79 dd (6.0, 1.0)
9
1′
167.8
133.0
138.2
15.9
167.7
128.7
138.3
15.9
20.7
170.5
43.3
167.8
128.7
138.3
15.9
20.7
174.2
41.6
2′
3′
6.10 qd (7.0, 1.0)
1.96 dd (7.0, 1.0)
1.88 brs
6.11 qd (7.0, 1.5)
1.96 dd (7.0, 1.5)
1.88 t (1.5)
6.11 qd (7.0, 1.5)
1.96 dd (7.0, 1.5)
1.88 t (1.5)
4′
5′
20.7
1′′
2′′
2.40 d (7.0)
2.18 m
2.62 q (7.0)
1.77 m
1.59 m
1.01 t (7.0)
1.23 d (7.0)
3.81 s
3′′a
3′′b
4′′
26.7
27.6
1.04 d (6.5)
1.04 d (6.5)
3.81 s
22.6
22.6
56.4
11.7
17.2
56.6
5′′
OMe-3
OMe-4
OMe-5
3.83 s
3.71 s
3.83 s
56.4
60.5
56.4
3.81 s
56.4
3.81 s
56.6
a
1
Data (δ) were measured in acetone-d6 for 9, 10, and 11 at 500 MHz for H NMR and at 125 MHz for 13C NMR. Proton coupling constants (J) in
Hz are given in parentheses. The assignments were based on DEPT and HMBC experiments.
The EIMS gave a molecular ion peak at m/z 222 [M]+•, and the
HREIMS at m/z 222.1249 [M]+• indicated the molecular formula
C13H18O3 (calcd for C13H18O3, 222.1256). The NMR spectra
resembled those of 3 except for the absence of resonances attributed
to the eastern side moiety at C-7 of 3 (Tables 1 and 2). In addition,
in the NMR spectra of 7, the resonance corresponding to C-7 of 3
appeared to be a carbonyl at δC 206.8, while resonances corre-
sponding to H2-8 and H3-19 and C-8 and C-19 of 7 were deshielded,
respectiveyly, by ∆δH +0.54 and +0.48 ppm and ∆δC +3.8 and
+14.2 ppm, as compared with those of 3. The above data indicated
that compound 7 was a 7-oxo analogue of 3 eliminating the eastern
side moiety on C-7. This was confirmed by the HMBC experiment
of 7 that showed correlations from H2-8, H2-9, and H3-19 to C-7.
The positive specific rotation of 7 indicated that it had a 13S
configuration.5 Thus, the structure of 7 was determined for
heteroplexisolide E.
analysis of the 2D NMR data of 8 confirmed that it is a stereoisomer
of rhynchosperin A. In the NOE spectrum of 8, H-10 was enhanced
by irradiation of H-4 or H-8, while H-12 and H2-19 were enhanced
by irradiation of H3-20. These NOE enhancements indicated
unambiguously that 8 was a C-8 epimer of rhynchosperin A.8
Therefore, the structure of 8 was determined and given the trivial
name 8-epirhynchosperin A.
Compound 9 had the molecular formula C17H22O5 (cacld for
C17H22O5, m/z 306.1467) indicated from the HREIMS at m/z
306.1455 [M]+•. The IR spectrum of 9 displayed absorption bands
for a conjugated ester carbonyl (1670 cm-1) and an aromatic ring
1
(1582 and 1508 cm-1). The H NMR spectrum of 9 (Table 3)
showed characteristic signals of an (E)-3,4,5-trimethoxyphenyl-
propenyloxy moiety at δ 6.79 (s, H-2 and H-6), 6.65 (d, J ) 16.0
Hz, H-7), 6.36 (dt, J ) 16.0, 6.5 Hz, H-8), 4.77 (dd, J ) 6.5, 1.0
Hz, H2-9), 3.83 (s, OMe-3 and OMe-5), and 3.71 (s, OMe-4). In
addition, it showed characteristic signals for an angeloyl unit at δ
6.10 (qd, J ) 7.0, 1.0 Hz, H-3′), 1.96 (dd, J ) 7.0, 1.0 Hz, H3-4′),
and 1.88 (brs, H3-5′).9 On the basis of the above spectroscopic data,
the structure of 9 was determined as (E)-3,4,5-trimethoxyphenyl-
propenyl angelate, which was confirmed by the 13C NMR and
HMBC data (Table 3 and Supporting Information).
The molecular formula of compound 10, C21H28O6, was indicated
from the HREIMS at m/z 376.1885 [M]+•. The UV, IR, and NMR
spectroscopic data of 10 were similar to those of 9 (Table 3 and
Experimental Section) except that the C-4 O-methyl resonance in
9 was replaced by a group of signals reminiscent of an isopentanoyl
unit in 10. In addition, the chemical shift of C-4 was shielded by
∆δC -9.7 ppm compared with that of 9. These data suggested that
the O-methyl group at C-4 of 9 was replaced by an isopentanoyloxy
in 10. This was confirmed by the HMBC data of 10 (Supporting
Information). Thus, 10 was determined to be (E)-3,5-dimethoxy-
4-isopentanoyloxyphenylpropenyl angelate.
Compound 8, a white amorphous powder, [R]20D -22.8 (c 0.26,
MeOH), showed IR absorption bands for lactone (1783 and 1670
cm-1) and conjugated carbonyl (1668 cm-1) functional groups. Its
EIMS exhibited a molecular ion peak at m/z 356 [M]+•, and the
HREIMS at m/z 356.1241 [M]+• suggested a molecular formula of
C20H20O6 (calcd for C20H20O6, 356.1260). The NMR data of 8
displayed characteristic signals of a ꢀ-substituted furan ring at δ
7.64 (brs, H-15), 7.54 (brs, H-16) and 6.56 (brs, H-14),7 which
were in accordance with the IR absorption for a furan ring (νmax
3154, 1509, and 875 cm-1). A group of signals at δ 6.14 (dd, J )
10.0, 3.0 Hz, H-2), 6.58 (dd, J ) 10.0, 3.0 Hz, H-3), and 3.37 (t,
J ) 3.0 Hz, H-4) were assignable to a cis -CHdCH-CH- unit.
An ABX spin system at δ 3.00 (dd, J ) 12.0, 5.0 Hz, H-11a), 1.95
(t, J ) 12.0 Hz, H-11b), and 5.60 (dd, J ) 12.0, 5.0 Hz, H-12)
revealed the presence of a -CH2CH-O- unit. An AB spin system
at δ 4.60 and 4.11 (d each, J ) 9.5 Hz, H-19a and H-19b) indicated
the presence of an isolated oxymethylene. In addition, the NMR
spectrum showed signals due to an isolated methine at δ 3.01 (s,
H-10), a tertiary methyl at δ 1.16 (s, H3-20), and signals due to
two vicinal coupled methylenes attached to another methine (Table
1). The 13C NMR and DEPT spectra showed 20 carbon resonances
corresponding to the above structural units, two ester carbonyls
(C-17 and C-18), and two quaternary carbons (C-5 and C-9) (Table
2). These spectroscopic data are similar to those of rhynchosperin
A reported from Rhynchospermum Verticullatum.8 A comprehensive
The spectroscopic data of 11 (Table 3 and Experimental Section)
indicated that it was an isomer of 10. Comparison of the NMR
data of 10 and 11 indicated that the resonances due to the
isopentanoyl group of 10 were replaced by those attributed to a
2-methylbutyryl moiety in 11. Therefore, 11 was determined as
(E)-3,5-dimethoxy-4-(2-methylbutyryloxy)phenylpropenyl angelate.
The S-configuration at the stereogenic center of 11 was determined
by synthesis of 11 (Scheme 1). The synthetic product gave

1188 Journal of Natural Products, 2009, Vol. 72, No. 6
Scheme 1. Synthesis of 11
Fan et al.
Qingdao Marine Chemical Inc. Qingdao, People’s Republic of China)
and Pharmadex LH-20 (Amersham Biosciences Inc. Shanghai, People’s
Republic of China). Preparative TLC separation was preformed with
high-performance silica gel preparative TLC plates (HSGF254, glass
precoated, Yantai Jiangyou Silica Gel Development Co. Ltd. Yantai,
People’s Republic of China). HPLC separation was performed on an
instrument consisting of a Waters 600 controller, a Waters 600 pump,
and a Waters 2487 dual λ absorbance detector, with a Prevail (250 ×
10 mm i.d.) column packed with C18 (5 µm). TLC was carried out
with glass precoated silica gel GF254 plates. Spots were visualized under
UV light or by spraying with 7% H2SO4 in 95% EtOH followed by
heating. Unless otherwise noted, all chemicals were obtained from
commercially available sources and were used without further purification.
Plant Material. The herbs of H. micocephala were collected at
Dayao mountain, Guangxi Province, People’s Republic of China, in
2003. The plant was identified by Mr. Guang-Ri Long (Guangxi Forest
Administration, Guangxi 545005, China). A voucher specimen (no. YG
01025) was deposited at the Herbarium of the Department of Medicinal
Plants, Institute of Materia Medica.
Extraction and Isolation. The air-dried plant material of H.
micocephala (2.56 kg) was powdered and extracted with 95% EtOH
at room temperature. The EtOH extract was evaporated under reduced
pressure below 40 °C to yield a dark green residue (270.0 g). The
residue was suspended in H2O (1500 mL) and then partitioned with
EtOAc (4 × 1500 mL). After removal of solvent, the EtOAc extract
(125.0 g) was applied to a normal-phase silica gel column. Successive
elution of the column with a gradient of increasing EtOAc (2-100%)
in petroleum ether afforded 12 fractions (A1-A12) on the basis of
TLC analysis. Fraction A5 (15.3 g), eluted by 20% EtOAc in petroleum
ether, was chromatographed over Pharmadex LH-20 with CHCl3-
MeOH (2:1) as mobile phase to give four subfractions (A5-1-A5-4).
Fraction A5-3 (2.3 g) was purified by reversed-phase preparative HPLC,
using a mobile phase of 40% CH3CN in H2O, to yield 1 (2.0 mg) and
2 (6.4 mg). Fraction A5-4 (5.4 g) was separated by reverse-phase
preparative HPLC using 46% MeOH in H2O as a mobile phase to yield
9 (23.3 mg), 10 (6.1 mg), 11 (7.3 mg), 12 (4.5 mg), and 12a (2.3 mg).
Fraction A7 (30.2 g), eluted by 60% EtOAc in petroleum ether, was
subjected to CC over normal-phase silica gel with a gradient of
increasing MeOH (0-100%) in CHCl3, to yield eight subfractions (A7-
1-A7-8). A7-4 (12.5 g) was separated by reversed-phase preparative
HPLC, using a mobile phase of 45% MeOH in H2O, to yield 3 (1237.7
mg), 4 (2.1 mg), 5 (2683.5 mg), 6 (7.5 mg), 7 (5.4 mg), and 8
(13.4 mg).
spectroscopic data and specific rotation identical to those of the
natural product (Experimental Section).
Compound 12 was determined as 1-(5′-acetyl-2′-methoxyphenyl)-
3-hydroxy-3-methylbutan-1-one by its spectroscopic data (Experi-
mental Section). Although 12 was synthesized as an intermediate
in a preparation of 1-(2′-alkoxy-5′-carboxyphenyl)-R,ꢀ-unsaturated
ketones,10 it has not yet been obtained as natural product before.
In addition, the ethyl ether of 12 was also obtained in the isolation
procedure (12a); however, it is considered an artifact because of a
production of 12a by keeping 12 in ethanol at 40 °C for 24 h. The
spectroscopic data of 12 and 12a were included in the Experimental
Section due to their absence in the literature.
The known compounds were identified by comparison of
spectroscopic data with those reported in the literature as (-)-bornyl
ferulate,11 1ꢀ-hydroxy-R-cyperone,12 R-rotunol,13 10R-hydroxyca-
din-4-en-15-al,14 10R-hydroxyisodauc-3-en-15-al,15 germacrene
B,16 mandassidione,17 loliolide,18 12-epi-bacchotricuneatin A,7
cleroinermin,19 desoxyarticulin,20 andydroolearin,21 friedelin,22
ursolic acid,23 obtusalin,24 R-spinasterol,25 6-hydroxystigmasta-
4,22-dien-6ꢀ-ol-3-one,26 3-O-ꢀ-D-glucopyranosyl spinasterol,27 sco-
poletin,28 umbelliferone,29 ayapin,30 3,3′-dimethylquercetin,31
3-methoxy-5,7,3′,4′-tetrahydroxyflavone,32 5-hydroxy-7,4′-dimeth-
oxyflavone,33 isosakuranetin,34 7-methoxy-4′,5,6-trihydroxyfla-
vone,35 acacetin,36 morinin B,9a sinapyl diangelate (13),9b visci-
done,37 espeleton,38 6-methoxy-4-methyl-1-tetralone,39 4-hydroxy-
2,3-dimethyl-2-nonen-4-olide,40 1-eicosanyl 3,4-dihydroxycinnamate,
2,4-diacetylanisole, isovanillin, salicylic acid, and ferulic acid.
In the in Vitro bioassays, 6 showed a selective cytotoxic activity
against A2780 with an IC50 value of 4.37 µM [the positive control
camptothecin (CPT), IC50 0.28 µM], and 13 showed activity against
HIV-1 replication with an IC50 value of 4.04 µM [the positive
control zidovudine (AZT), IC50 0.048 µM]. However other com-
pounds were inactive. In addition, the isolates were also assessed
for their activities against the release of ꢀ-glucuronidase in rat
polymorphonuclear leukocytes (PMNs) induced by platelet-activat-
ing factor (PAF),41 neuroprotective activity against glutamate-
induced neurotoxicity in cultures of PC12 cells,2 the antioxidant
activity in Fe2+-cystine-induced rat liver microsomal lipid peroxi-
dation,42 and the inhibitory activity against protein tyrosine
phosphatase 1B (PTP1B),43 but were inactive at concentrations of
10-5 M.
(-)-1S,9S-9-Hydroxy-2,2,5,9-tetramethylbicyclo[6.3.0]undeca-4,7-
dien-6-one (1): yellowish oil; [R]20 -84.0 (c 0.16, MeOH); UV
D
(MeOH) λmax (log ε) 202 (3.62), 262 (3.76) nm; CD (MeOH) 354 (∆ε
-1.1), 257 (∆ε -5.88) nm; IR νmax 3424, 2961, 2925, 1730, 1714,
1650, 1623, 1601 cm-1; 1H NMR (acetone-d6, 500 MHz) data, see Table
1; 13C NMR (acetone-d6, 125 MHz) data, see Table 2; EIMS m/z 234
[M]+•; HREIMS m/z 234.1623 [M]+• (calcd for C15H22O2, 234.1620).
(-)-1S,9R-9-Hydroxy-2,2,5,9-tetramethylbicyclo[6.3.0]undeca-
4,7-dien-6-one (2): yellowish gum; [R]20D -55.3 (c 0.53, MeOH); UV
(MeOH) λmax (log ε) 202 (3.52), 259 (3.69) nm; CD (MeOH) 358 (∆ε
-1.23), 248 (∆ε -3.87) nm; IR νmax 3420, 2960, 2925, 1732, 1715,
1648, 1621, 1601 cm-1; 1H NMR (acetone-d6, 500 MHz) data, see Table
1; 13C NMR (acetone-d6, 125 MHz) data, see Table 2; EIMS m/z 234
[M]+•; HREIMS m/z 234.1613 [M]+• (calcd for C15H22O2, 234.1620).
Heteroplexisolide A (3): yellowish oil; [R]20 +31.3 (c 1.38,
D
MeOH); UV (MeOH) λmax (log ε) 206 (4.30) nm; CD (MeOH) 210
(∆ε +1.67) nm; IR νmax 2933, 1778, 1748, 1678, 1637, 1444, 1322,
1199, 1181, 1022, 971, 888 cm-1; H NMR (CDCl3, 500 MHz) data,
1
Experimental Section
see Table 1; 13C NMR (CDCl3, 125 MHz) data, see Table 2; EIMS
m/z 330 [M]+•; HREIMS m/z 330.1859 [M]+• (calcd for C20H26O4,
330.1831).
General Experimental Procedures. Optical rotations were measured
on a PE model 343 polarimeter. UV spectra were measured on a Cary
300 spectrometer. CD spectra were recorded on a JASCO-815 CD
spectrometer. IR spectra were recorded on a Nicolet 5700 FT-IR
microscope spectrometer (FT-IR microscope transmission). 1D- and
2D-NMR spectra were obtained at 500 or 600 MHz for 1H, and 125 or
150 MHz for 13C, respectively, on INOVA 500 MHz or SYS 600 MHz
spectrometers in acetone-d6 or CDCl3, with solvent peaks as references.
EIMS and HREIMS data were measured with a Micromass Autospec-
Ultima ETOF spectrometer. ESIMS data were measured with a Q-Trap
LC/MS/MS (Turbo Ionspray source) spectrometer. HRESIMS data were
measured using a JMS-T100CS AccuToF CS spectrometer. Column
chromatography was performed with silica gel (200-300 mesh,
Heteroplexisolide B (4): yellowish oil; [R]20 +31.4 (c 0.50,
D
MeOH); UV (MeOH) λmax (log ε) 208 (4.52) nm; CD (MeOH) 219
(∆ε +1.44) nm; IR νmax 3463, 2931, 1778, 1746, 1678, 1637, 1443,
1
1322, 1205, 1180, 1020, 970, 893 cm-1; H NMR (acetone-d6, 500
MHz) data, see Table 1; 13C NMR (acetone-d6, 125 MHz) data, see
Table 2; ESIMS m/z 369 [M + Na]+ and 385 [M + K]+; HRESIMS
m/z 369.1688 [M + Na]+ (calcd for C20H26O5Na, 369.1678).
Heteroplexisolide C (5): yellowish oil; [R]20 +67.0 (c 2.08,
D
MeOH); UV (MeOH) λmax (log ε) 208 (4.35) nm; IR νmax 3431, 2927,
1
1747, 1679, 1636, 1446, 1322, 1181, 1037, 965, 889, 846 cm-1; H

Chemical Constituents of Heteroplexis micocephala
Journal of Natural Products, 2009, Vol. 72, No. 6 1189
NMR (acetone-d6, 500 MHz) data, see Table 1; 13C NMR (acetone-d6,
125 MHz) data, see Table 2; ESIMS m/z 369 [M + Na]+ and 385 [M
+ K]+; HRESIMS m/z 369.1680 [M + Na]+ (calcd for C20H26O5Na,
369.1678).
stirred for 12 h at 0 °C and then extracted with H2O (3 × 15 mL).
After the organic layer was concentrated, the residue was separated by
preparative TLC (petroleum ether-acetone, 2:1) to give 11 (55.2 mg,
73.4%) as a yellowish gum: [R]20D +11.4 (c 1.26, MeOH). Its 1H NMR
(acetone-d6, 500 MHz), 13C NMR (acetone-d6, 125 MHz), and EIMS
data were identical to those of the natural product.
Heteroplexisolide D (6): yellowish gum; [R]20 +4.9 (c 0.24,
D
MeOH); UV (MeOH) λmax (log ε) 205 (4.28) nm; IR νmax 3429, 2933,
1
1743, 1637, 1445, 1379, 1177, 1032, 977, 896, 861 cm-1; H NMR
1-(5′-Acetyl-2′-methoxyphenyl)-3-hydroxy-3-methylbutan-1-
one (12): yellowish, amorphous powder; UV (MeOH) λmax (log ε) 208
(4.02), 240 (4.27), 268 (4.09) nm; IR νmax 3493, 2973, 2937, 1680,
(acetone-d6, 500 MHz) data, see Table 1; 13C NMR (acetone-d6, 150
MHz) data, see Table 2; ESIMS m/z 369 [M + Na]+ and 385 [M +
K]+; HRESIMS m/z 369.1687 [M + Na]+ (calcd for C20H26O5Na,
369.1678).
1
1597 cm-1; H NMR (acetone-d6, 500 MHz) δ 8.15 (1H, d, J ) 2.0
Hz, H-6′), 8.13 (1H, dd, J ) 8.5, 2.0 Hz, H-4′), 7.25 (1H, d, J ) 8.5
Hz, H-3′), 4.03 (3H, s, OMe-2′), 3.84 (1H, s, OH-3), 3.18 (2H, s, H2-
2), 2.54 (3H, s, Me-Ac), 1.25 (6H, s, H3-4 and H3-5); 13C NMR
(acetone-d6, 125 MHz) 203.4 (C-1), 196.1 (CO-Ac), 162.4 (C-2′), 134.2
(C-4′), 131.0 (C-1′), 130.8 (C-6′), 130.5 (C-5′), 112.8 (C-3′), 70.3 (C-
3), 56.6 (OMe-2′), 55.5 (C-2), 30.3 (C-4 and C-5), 26.4 (Me-Ac); EIMS
m/z 250 [M]+•; HREIMS m/z 250.1200 [M]+• (calcd for C14H18O4,
250.1205).
Heteroplexisolide E (7): yellowish oil; [R]20 +15.6 (c 0.27,
D
MeOH); IR νmax 2970, 2920, 1752, 1715, 1679 cm-1; 1H NMR (acetone-
d6, 500 MHz) data, see Table 1; 13C NMR (acetone-d6, 150 MHz) data,
see Table 2; EIMS m/z 222 [M]+•; HREIMS m/z 222.1249 [M]+• (calcd
for C13H18O3, 222.1256).
8-Epirhynchosperin A (8): white, amorphous powder; [R]20D -22.8
(c 0.26, MeOH); IR νmax 3154, 1783, 1730, 1670, 1668, 1509, 1380,
1147, 1022, 875 cm-1; 1H NMR (acetone-d6, 500 MHz) data, see Table
1; 13C NMR (acetone-d6, 125 MHz) data, see Table 2; EIMS m/z 356
[M]+•; HREIMS m/z 356.1241 [M]+• (calcd for C20H20O6, 356.1260).
(E)-3,4,5-Trimethoxyphenylpropenyl angelate (9): yellowish oil;
12a: yellowish oil; UV (MeOH) λmax (log ε) 209 (3.86), 240 (4.01),
268 (3.90) nm; IR νmax 2974, 2933, 1681, 1597, 1262 cm-1; 1H NMR
(acetone-d6, 500 MHz) δ 8.10 (1H, dd, J ) 8.5, 2.0 Hz, H-4′), 8.03
(1H, d, J ) 2.0 Hz, H-6′), 7.22 (1H, d, J ) 8.5 Hz, H-3′), 4.02 (3H,
s, OMe-2′), 3.34 (2H, q, J ) 7.0 Hz, CH2-OEt), 3.18 (2H, s, H2-2),
2.53 (3H, s, Me-Ac), 1.23 (6H, s, H3-4 and H3-5), 0.91 (3H, t, J ) 7.0
Hz, Me-OEt); 13C NMR (acetone-d6, 125 MHz) δ 201.8 (C-1), 196.1
(CO-Ac), 161.9 (C-2′), 133.6 (C-4′), 131.9 (C-1′), 131.0 (C-5′), 130.6
(C-6′), 112.5 (C-3′), 75.0 (C-3), 57.0 (CH2-OEt), 56.5 (OMe-2′), 53.3
(C-2), 26.4 (Me-Ac), 26.3 (C-4 and C-5), 16.2 (Me-OEt); EIMS m/z
278 [M]+•; HREIMS m/z 278.1501 [M]+• (calcd for C16H22O4,
278.1518).
1
IR νmax 2941, 1700, 1670, 1582, 1508, 1420, 1246, 1127 cm-1; H
NMR (acetone-d6, 500 MHz) and 13C NMR (acetone-d6, 125 MHz)
data, see Table 3; EIMS m/z 306 [M]+•; HREIMS m/z 306.1455 [M]+•
(calcd for C17H22O5, 306.1467).
(E)-3,5-Dimethoxy-4-isopentanoyloxyphenylpropenyl angelate (10).
yellowish oil; IR νmax 2961, 1760, 1715, 1598, 1462, 1338, 1233, 1134
cm-1; 1H NMR (acetone-d6, 500 MHz) and 13C NMR (acetone-d6, 125
MHz) data, see Table 3; EIMS m/z 376 [M]+•; HREIMS m/z 376.1885
[M]+• (calcd for C21H28O6, 376.1886).
Cells, Culture Conditions, and Cell Proliferation Assay. See ref
44.
(E)-3,5-Dimethoxy-4-(S-2-methylbutyryloxy)phenylpropenyl an-
Anti-HIV Activity Assay. A cell-based VSVG/HIV-1 pseudotyping
system was used for evaluating a compound’s anti-HIV replication
activity as described previously.45 Briefly, vesicular stomatitis virus
glycoprotein (VSV-G) plasmid was cotransfected with env-deficient
HIV-1 vector, pNL4-3.luc.R-E-,46 into 293T cells by using a modified
Ca3(PO4)2 method.47 Sixteen hours post-transfection, plates were washed
by PBS, and fresh media DMEM with 10% FBS was added into the
plates. Forty eight hours post-transfection, supernatant, which contained
VSVG/HIV-1 virions, was harvested and filtered through a 0.45 µm
filter. VSVG/HIV-1 pseudotyped virions were quantified by p24
concentrations, which were detected by ELISA (ZeptoMetrix, Cat.:
0801111), then diluted to 0.2 ng p24/mL, which can be used directly
or stored at -80 °C.
For the infection assay, 293T cells were plated on 24-well plates at
the density of 6 × 104 cells per well one day prior to infection.
Compounds were incubated with target cells for 15 min prior to adding
VSVG/HIV-1. The same amount of solvent alone was used as blank
control. After postinfection for 48 h, cells were lysed in 50 µL Cell
Lysis Reagent (Promega). Luciferase activity of the cell lysate was
measured by a FB15 luminometer (Berthold Detection System)
according to the manufacture’s instructions.
gelate (11): yellowish gum; [R]20D +6.5 (c 0.63, MeOH); UV (MeOH)
λ
max (log ε) 196 (4.35), 220 (4.69), 265 (4.29) nm; IR νmax 2969, 2939,
1
2878, 1758, 1715, 1597, 1460, 1231, 1133 cm-1; H NMR (acetone-
d6, 500 MHz) and 13C NMR (acetone-d6, 125 MHz) data, see Table 3;
EIMS m/z 376 [M]+•; HREIMS m/z 376.1868 [M]+• (calcd for C21H28O6,
376.1886).
Synthesis of 11. S-2-Methylbutyric acid (220 µL) was added into
SOCl2 (5 mL). The mixture was refluxed for 2 h, and then the excess
SOCl2 was removed by evaporation under reduced pressure. The residue
was dissolved in dried acetone (10 mL) and cooled in an ice bath.
(E)-3,5-Dimethoxy-4-hydroxyphenylpropenaldehyde (104.0 mg) and
triethylamine (TEA, 0.50 µL) were added, and the mixture was stirred
for 0.5 h at 0 °C and 8 h at room temperature. After the solvent was
removed under reduced pressure the residue was dissolved in H2O (15
mL) and extracted with EtOAc (3 × 15 mL). The organic phase was
dried over anhydrous MgSO4, filtered, and concentrated. The residue
was separated by preparative TLC (petroleum ether-acetone, 2:1) to
give (E)-3,5-dimethoxy-4-(S-2-methylbutyryloxy)phenylpropenaldehyde
(116.4 mg, 79.7%) as a yellowish gum: [R]20D +25.3 (c 0.13, MeOH);
1H NMR (acetone-d6, 600 MHz) δ 9.70 (1H, d, J ) 7.8 Hz, H-9), 7.63
(1H, d, J ) 15.6 Hz, H-7), 7.17 (2H, s, H-2 and H-6), 6.81 (1H, dd, J
) 15.6, 7.8 Hz, H-8), 3.87 (6H, s, OMe-3 and OMe-5), 2.65 (1H, m,
H-2′), 1.79 (1H, m, H-3′a), 1.61 (1H, m, H-3′b), 1.26 (3H, d, J ) 7.2
Hz, H3-5′), 1.02 (3H, t, J ) 7.2 Hz, H3-4′); EIMS m/z 292 [M]+•.
(E)-3,5-Dimethoxy-4-(S-2-methylbutyryloxy)phenylprope-
naldehyde (102.2 mg) was reduced by NaBH4 (20.0 mg) in EtOH (5
mL) for 30 min at room temperature. After the solution was acidified
by 1 M HCl to pH 2.0, the solvent was removed under reduced pressure.
Water (15 mL) was added to the residue, and the mixture was extracted
with EtOAc (3 × 15 mL). The organic layer was dried over MgSO4
and evaporated to yield (E)-3,5-dimethoxy-4-(S-2-methylbutyryloxy)-
Acknowledgment. Financial support from the National Natural
Sciences Foundation of China (NNSFC; grant nos. 30825044 and
20432030), the Program for Changjiang Scholars and Innovative
Research Team in University (PCSIRT, grant no. IRT0514), and the
national “973” program of China (grant nos. 2004CB13518906 and
2006CB504701) is acknowledged.
Supporting Information Available: . MS, 1H and 13C NMR,
HMBC, and NOE spectra of compounds 1-12a. This material is
available free of charge via the Internet at http://pubs.acs.org.
phenylpropenol (101.0 mg, 98.2%) as a yellowish gum: [R]20 +18.0
D
References and Notes
1
(c 0.14, MeOH); H NMR (acetone-d6, 600 MHz) δ 6.78 (2H, s, H-2
and H-6), 6.57 (1H, d, J ) 15.6 Hz, H-7), 6.40 (1H, dt, J ) 15.6, 4.8
Hz, H-8), 4.23 (2H, m, H2-9), 3.84 (1H, m, OH-9), 3.81 (6H, s, OMe-3
and OMe-5), 2.61 (1H, m, H-2′), 1.77 (1H, m, H-3′a), 1.59 (1H, m,
H-3′b), 1.24 (3H, d, J ) 7.2 Hz, H3-5′), 1.01 (3H, t, J ) 7.2 Hz, H3-
4′); EIMS m/z 294 [M]+•.
Angelic acid (80.0 mg), 1-(3-dimethylaminopropyl)-3-ethylcarbo-
diimide hydrochloride (EDCI, 176.2 mg), and DMAP (277.2 mg) were
added to a solution of (E)-3,5-dimethoxy-4-(S-2-methylbutyryloxy)phe-
nylpropenol (58.8 mg) in dry CH2Cl2 (5 mL) at 0 °C. The mixture was
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