(±)-torreyunlignans A-D, Rare 8-9′ linked neolignan enantiomers as phosphodiesterase-9A inhibitors from torreya yunnanensis

Article abstract of DOI:10.1021/np500528u

(±)-Torreyunlignans A-D (1a/1b-4a/4b), four pairs of new 8-9′ linked neolignan enantiomers featuring a rare (E)-2-styryl-1,3-dioxane moiety, were isolated from the trunk of Torreya yunnanensis. The structures were determined by combined spectroscopic and chemical methods, and the absolute configurations were elucidated by ECD calculations. The compounds were screened by using tritium-labeled adenosine 3′,5′-cyclic monophosphate ([³H]-cGMP) as a substrate for inhibitory affinities against phosphodiesterase-9A (PDE9A), which is a potential target for the treatment of diabetes and Alzheimer's disease. All of the enantiomers exhibited inhibition against PDE9A with IC₅₀ values ranging from 5.6 to 15.0 μM. This is the first report of PDE9A inhibitors from nature.

Full text of DOI:10.1021/np500528u

Article
pubs.acs.org/jnp
(
± )-Torreyunlignans A−D, Rare 8−9′ Linked Neolignan Enantiomers
as Phosphodiesterase-9A Inhibitors from Torreya yunnanensis
†
,§
†,§
†
†
†
†
Zhong-Bin Cheng, Xiao Lu, Jing-Mei Bao, Qing-Hua Han, Zhen Dong, Gui-Hua Tang,
Li-She Gan,* Hai-Bin Luo,* and Sheng Yin*
,‡
,†
,†
†School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou, Guangdong 510006, People’s Republic of China
‡College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, Zhejiang 310058, People’s Republic of China
*
S Supporting Information
ABSTRACT: (±)-Torreyunlignans A−D (1a/1b−4a/4b), four pairs of new 8−9′ linked neolignan enantiomers featuring a rare
E)-2-styryl-1,3-dioxane moiety, were isolated from the trunk of Torreya yunnanensis. The structures were determined by
(
combined spectroscopic and chemical methods, and the absolute configurations were elucidated by ECD calculations. The
3
compounds were screened by using tritium-labeled adenosine 3′,5′-cyclic monophosphate ([ H]-cGMP) as a substrate for
inhibitory affinities against phosphodiesterase-9A (PDE9A), which is a potential target for the treatment of diabetes and
Alzheimer’s disease. All of the enantiomers exhibited inhibition against PDE9A with IC50 values ranging from 5.6 to 15.0 μM.
This is the first report of PDE9A inhibitors from nature.
he second messengers adenosine and guanosine 3′,5′-
cyclic monophosphate (cAMP and cGMP) modulate
screening program aimed at the discovery of novel PDE
8
−11
T
inhibitors from natural resources,
a fraction of the ethanolic
many physiological processes such as steroid hormone function,
extract of T. yunnanensis showed inhibitory activity of 43%
toward PDE9 and no obvious activity against PDE4 (<5%) and
PDE5 (<5%) at a concentration of 10 μg/mL. Subsequent
chemical investigation led to the isolation of four pairs of new
neolignan enantiomers featuring a rare 8−9′ linkage skeleton
and a rare (E)-2-styryl-1,3-dioxane moiety. The compounds
were screened for inhibitory activity against PDE9A and
exhibited IC values ranging from 5.6 to 15.0 μM. Herein, the
platelet aggregation, apoptosis, leukocyte migration, inflamma-
1
tion, memory, and circadian regulation. Phosphodiesterases
(
PDEs) catalyze the hydrolysis of intracellular cAMP and
cGMP and thus play key roles in the cellular process. Among
the 11-membered PDE isoforms, PDE9 has the lowest Km
among cGMP-specific PDE families. Thus, selective PDE9A
inhibitors have been studied as potential therapeutics for the
treatment of types I and II diabetes as well as Alzheimer’s
disease. Although a number of chemically diverse molecules
have been developed as PDE9A inhibitors over the past decade,
PF-04447943 was the only compound reported to have
completed Phase II clinical trials for the treatment of
50
details of isolation, structure elucidation, and the inhibitory
activity of these enantiomers against PDE9A are described.
1
,2
RESULTS AND DISCUSSION
■
1
The air-dried powder of the trunk of T. yunnanensis was
extracted with 95% EtOH to give a crude extract, which was
Alzheimer’s disease. Therefore, the search for novel PDE9
inhibitors has continuously attracted interest.
Torreya yunnanensis (Taxaceae), a woody climber endemic to
suspended in H O and successively partitioned with petroleum
2
ether, EtOAc, and n-BuOH. Various column chromatographic
separations of the EtOAc extract afforded four pairs of
enantiomers 1a/1b−4a/4b.
the Yunnan Province of China, is often used in house and3
bridge building, furniture manufacture, and as a farm tool.
Previous chemical investigation of this plant led to the isolation
of biflavones, diterpenes, and lignans, some of which showed
significant inhibitory activity against β-secretase, dengue virus,
and neurofibromatosis type 1-deficient cancer cells. In the
4
5
6
Received: June 30, 2014
7
©
XXXX American Chemical Society and
American Society of Pharmacognosy
A
dx.doi.org/10.1021/np500528u | J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
of the three phenylpropanoid units were established by HMBC
correlations of H-8/C-9′, H-9′/C-8, and H-9″/C-7 and C-9,
which necessitated the presence of a 1,3-dioxane ring. The
locations of the methoxy groups were defined by detailed
HMBC and NOE analyses (Figures 1 and 2). Compound 1 was
Compound 1, a light yellow oil, had the molecular formula
1
3
C H O , as established by C NMR data and HRESIMS,
32
36
7
indicating 15 indices of hydrogen deficiency. The IR spectrum
exhibited absorption bands for aromatic functionalities (1592
−1
1
and 1467 cm ). The H NMR spectrum revealed the presence
of five methoxy groups [δ 3.80 (3H, s), 3.81 (3H × 2, s), 3.82
Figure 2. Key NOE correlations (←···→) of 1.
H
(
(
3H, s), and 3.83 (3H, s)], an oxygenated methylene [δ 4.31
1H, dd, J = 11.3, 4.4 Hz) and 3.71 (1H, dd, J = 11.3, 11.3
H
unstable upon standing in CDCl . The decomposed products
3
Hz)], two oxymethines [δ 5.32 (1H, dd, J = 4.6, 0.9 Hz) and
H
were isolated and identified to be the 1,3-diol 1c and 2-
4.46 (1H, d, J = 10.0 Hz)], two disubstituted E double bonds
12
methoxycinnamaldehyde (1d) (Scheme 1), which further
[δH 6.56 (1H, d, J = 16.0 Hz), 6.41 (1H, ddd, J = 16.0, 8.4, 6.2
confirmed the gross structure of 1.
The relative configuration of 1 was established on the basis of
Hz), 7.11 (1H, d, J = 16.4 Hz), and 6.30 (1H, dd, J = 16.4, 4.6
Hz)], a 1,2,4-trisubstituted benzene ring (ring A) [δ 7.08 (1H,
1
1
H
the interpretation of the NOESY data and H− H coupling
constants. The strong NOE interactions of H-9″/H-7 and H-9β
indicated that H-9″, H-7, and H-9β occupied the axial bonds of
the 1,3-dioxane-ring in a chair conformation and were
arbitrarily assigned β-orientations (Figure 2). Accordingly, the
coupling constant of 10.0 Hz between H-7 and H-8 indicated
d, J = 1.9 Hz), 6.93 (1H, d, J = 8.1 Hz), and 7.00 (1H, dd, J =
8.1, 1.9 Hz)], a symmetrically 1,2,3-trisubstituted benzene ring
(
ring B) [δ 6.61 (2H, d, J = 8.4 Hz) and 7.13 (1H, t, J = 8.4
H
Hz)], and a 1,2-disubstituted benzene ring (ring C) [δ 6.96
H
(
(
1H, d, J = 8.2 Hz), 7.25 (1H, ddd, J = 8.2, 7.8, 1.6 Hz), 6.92
1H, t, J = 7.8 Hz), and 7.50 (1H, dd, J = 7.8, 1.5 Hz)]. The C
13
13,14
that H-8 adopted an α-axial orientation.
NMR spectrum, in combination with DEPT experiments,
showed 32 carbon resonances attributable to three benzene
rings, two double bonds, an acetal, two methylenes (one
oxygenated), two methines (one oxygenated), and five methoxy
groups. As 14 of the 15 indices of hydrogen deficiency were
accounted for by three benzene rings and two double bonds,
the remaining degree of unsaturation required the presence of
an additional ring in 1. The gross structure of 1 was further
established by analyses of its 2D NMR data (Figure 1). The
COSY spectrum established three C spin systems from C-7 to
3
C-9, C-7′ to C-9′, and C-7″ to C-9″. The HMBC correlations
from H-7 to C-1, C-2, and C-6; from H-7′ to C-1′, C-2′, and C-
6
′; and from H-7″ to C-1″, C-2″, and C-6″ connected the three
3
C units to three benzene rings, respectively, thus indicating the
presence of three phenylpropanoid moieties. The connectivities
of (7R, 8R, 9″R)-1 was obtained by inversion of the calculated
ECD spectrum of (7S, 8S, 9″S)-1, which allowed the
assignment of the absolute configuration of 1b as (RRR).
Compounds 1a and 1b were given the trivial names
(
+)-torreyunlignan A and (−)-torreyunlignan A, respectively.
Compound 2 had a molecular formula of C H O , as
3
1
34
6
established by 13C NMR data and HRESIMS. The NMR data
of 2 were similar to those of 1 except for the absence of a
methoxy group and the presence of a 1,2-disubstituted ring A in
1
1
Figure 1. Selected H− H COSY () and HMBC (→) correlations
of 1.
2 [δ 6.99 (1H, d, J = 8.4 Hz), 7.03 (1H, td, J = 7.5, 0.8 Hz),
7.57 (1H, dd, J = 7.5, 1.6 Hz), and 7.30 (1H, ddd, J = 8.4, 7.5,
H
B
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Journal of Natural Products
Article
Scheme 1. Decomposition of 1 in CDCl3
Figure 3. (a) Experimental ECD spectra of (±)-torreyunlignans A (1a/1b) in MeCN and calculated ECD spectra of (SSS)-1 and (RRR)-1. (b)
Experimental ECD spectra of (±)-torreyunlignans B (2b/2a) in MeCN and calculated ECD spectra of (SSS)-2 and (RRR)-2. (c) Experimental ECD
spectra of (±)-torreyunlignans C (3b/3a) in MeCN and calculated ECD spectra of (SSS)-3 and (RRR)-3. (d) Experimental ECD spectra of
(±)-torreyunlignans D (4b/4a) in MeCN and calculated ECD spectra of (SSS)-4 and (RRR)-4. The calculated ECD spectra were computed at the
B3LYP/6-311++G(2d,2p) level.
1
.6 Hz)]. This indicated that compound 2 was a ring-A
given the trivial names (−)-torreyunlignan B and (+)-torreyun-
lignan B, respectively.
demethoxy derivative of 1. The methoxy group on ring A was
located at C-2 by the HMBC correlation from the methoxy
protons (δH 3.85) to C-2 (δC 157.7) and by the NOE
Compound 3, obtained as a light yellow oil, had the
13
molecular formula C H O , as established by C NMR data
32 36
7
and HRESIMS. The NMR data of 3 showed similarity to those
of 2 with major differences being attributable to the presence of
an additional methoxy group in the A-ring of 3. The aromatic
interaction between the methoxy protons and H-3 (δ 6.99).
H
The gross structure was further established by analyses of its 2D
NMR spectra. The relative configuration of 2 was assigned to
be the same as that of 1 based on the interpretation of the NOE
correlations (H-9″/H-9β and H-7/H-9β) and H− H coupling
constants (J7 = 10.0 Hz and J = 4.4 Hz). Compound 2 also
signals of the A-ring at δ 7.24 (1H, t, J = 8.1 Hz) and 6.66
H
(
2H, d, J = 8.1 Hz) implied a symmetrically 1,2,3-trisubstituted
1
1
benzene ring, requiring that the additional methoxy group in 3
should be located at C-6. This was confirmed by the HMBC
correlation from the methoxy protons (δ 3.83) to C-6 (δ
,8
8,9α
has negligible optical activity and an ECD spectrum devoid of
Cotton effects. Subsequent chiral resolution of 2 afforded the
H
C
1
61.4).
The relative configuration of 3 was assigned to be the same
2
0
20
enantiomers 2a ([α] D: −13.2) and 2b ([α] D: +13.7). By
comparison of the calculated ECD spectra of (7S, 8S, 9″S)-2
and (7R, 8R, 9″R)-2 with the experimental data (Figure 3b),
the absolute configurations of 2a and 2b were assigned as (SSS)
and (RRR), respectively. Compounds 2a and 2b were thus
as that of 2 based on their similar 1D NMR data and NOESY
correlations. However, in 3, the two A-ring O-methyl groups
relative to the C-1−C-7 bond slightly hinder the free rotation
about this bond and resulted in different chemical shifts of C-2
C
dx.doi.org/10.1021/np500528u | J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
δ 159.7) and C-6 (δ 161.4). The optical activity and ECD
Article
(
EXPERIMENTAL SECTION
C
C
■
spectrum of 3 were similar to those of 1 and 2, and subsequent
General Experimental Procedures. Optical rotations
20
chiral resolution of 3 afforded the enantiomers 3a ([α] D:
were measured on a Rudolph Autopol I automatic polarimeter,
ECD spectra were obtained on an Applied Photophysics
Chirascan spectrometer. UV spectra were recorded on a
Shimadzu UV-2450 spectrophotometer. IR spectra were
determined on a Bruker Tensor 37 infrared spectrophotometer.
NMR spectra were measured on a Bruker AM-400
spectrometer at 25 °C. ESIMS spectra were obtained on a
Finnigan LCQ Deca instrument, and HRESIMS was performed
on a Waters Micromass Q-TOF spectrometer. A Shimadzu LC-
0 AT equipped with a SPD-M20A PDA detector was used for
HPLC. A chiral-phase column (Phenomenex Lux, cellulose-2,
50 × 10 mm, 5 μm) was used for semipreparative HPLC
separation. Silica gel (300−400 mesh, Qingdao Haiyang
Chemical Co., Ltd.), C18 reversed-phase silica gel (12 nm, S-
2
0
−
19.4) and 3b ([α] D: +20.3). The absolute configurations of
3
a and 3b were defined as (SSS) and (RRR), respectively, by
using the same methods as described above (Figure 3c).
Compounds 3a and 3b were given the trivial names
(
−)-torreyunlignan C and (+)-torreyunlignan C, respectively.
Compound 4, a light yellow oil, had the molecular formula
1
3
C H O , as determined by C NMR data and HRESIMS.
30
32
5
The NMR data of 4 resembled those of 2 except for the
presence of a 1,2-disubstituted B-ring in 4 instead of the
symmetrically 1,2,3-trisubstituted ring in 2, indicating that it
was a 6′-demethoxy derivative of 2. Analyses of its 2D NMR
data confirmed the gross structure of 4. The relative
configuration of 4 was assigned to be the same as that of 2
based on their similar 1D NMR data and the NOESY
correlations. Chiral resolution of 4 afforded a pair of
2
2
5
0 μm, YMC Co., Ltd.), and MCI gel (CHP20P, 75−150 μm,
Mitsubishi Chemical Industries Ltd.) were used for column
chromatography (CC). All solvents were of analytical grade
2
0
20
enantiomers 4a ([α] : −10.0) and 4b ([α] D: +12.4). The
D
absolute configurations of 4a and 4b were defined as (SSS) and
(
Guangzhou Chemical Reagents Company, Ltd.). Expression
(RRR), respectively, by using the same methods as described
and purification of PDE9A2, PDE4D2, and PED5A1 were
carried out by using a Hielscher UP200S ultrasonic cell
disruption processor, a Sigma 6K15 centrifugal machine, an
Eppendorf BioPhotomer spectrophotometer and a Qiagen
nickel-nitriloacetic acid (Ni-NTA) column. The radioactivity of
the samples was measured on a PerkinElmer Tricarb 2910
liquid scintillation counter. The yeast extract and tryptone
prepared for the LB medium were purchased from Oxoid Ltd.
above (Figure 3d). Compounds 4a and 4b were given the trivial
names (−)-torreyunlignan D and (+)-torreyunlignan D,
respectively.
Torreyunlignans A−D represent a group of 8−9′ linked
neolignans featuring a rare (E)-2-styryl-1,3-dioxane moiety. In
particular, lignans featuring a C-8−C-9′ linked skeleton are
rarely found in nature, as only 18 examples of this chemotype
13−15
have been reported,
all from the genera Alpinia and
3
and the substrate [ H]-cGMP was from Waukesha GE
Morina.
Healthcare. BAY73-6691 was purchased from Sigma.
Plant Material. The trunk of T. yunnanensis was collected in
October 2012 in the Yunnan Province, P. R. China, and was
authenticated by Prof. You-Kai Xu of Xishuangbanna Tropical
Botanical Garden, Chinese Academy of Sciences. A voucher
specimen (accession number: YNFS201210) has been
deposited at the School of Pharmaceutical Sciences, Sun Yat-
sen University.
The enantiomers were screened for inhibitory activity toward
PDE9A at an initial concentration of 10 μM by using reported
1
6
methods. The compounds showed inhibition greater than
0% toward PDE9A. To further evaluate the potency of the
9
compounds, BAY73-6691, a well-known PDE9A inhibitor, was
used as the reference compound (IC 49.7 nM), comparable
5
0
17
to the reported value of 55 nM. The bioassay results showed
that the enantiomers had moderate activity with IC50 values
ranging from 5.6 to 15.0 μM (Table 1).
Extraction and Isolation. The air-dried powder of the
trunk of T. yunnanensis (900 g) was extracted with 95% EtOH
(
3 × 5 L) at room temperature (rt) to give 91 g of crude
Table 1. IC50 Values of the Compounds (1a/1b-4a/4b)
Against PDE9A2
extract. The extract was suspended in H O (0.5 L) and
2
successively partitioned with petroleum ether (PE, 3 × 1 L),
EtOAc (3 × 1 L), and n-BuOH (3 × 1 L). The EtOAc extract
(22 g) was subjected to MCI gel (CC) eluted with a MeOH/
H O gradient (3:7 → 10:0) to afford five fractions (I−V).
Fraction V (8.5 g) was chromatographed over C reversed-
phase (RP-18) silica gel CC eluted with MeOH/H O (5:5 →
10:0) to afford five fractions (Va−Ve). Fraction Vb was
subjected to RP-18 silica gel CC (MeOH/H O, 6:4 → 10:0) to
compound
IC50 (μM)
compound
IC50 (μM)
1
2
3
4
a
a
a
a
15.0 ± 0.9
10.1 ± 0.6
5.6 ± 0.2
6.4 ± 0.8
49.7 nM
1b
2b
3b
4b
8.8 ± 0.3
7.7 ± 0.7
7.8 ± 0.9
6.7 ± 0.6
2
1
8
2
a
BAY73-6691
a
Positive control.
2
afford fractions (Vb1−Vb4). Fraction Vc was subjected to silica
gel CC (PE/CH Cl , 6:1 → 1:1) to give four fractions (Vc1−
PDE9A has drawn considerable attention and is a potential
2
2
target for the treatment of diabetes and CNS diseases. The
bioassay results in the current study showed that all of the
enantiomers showed similar activity toward PDE9A, implying
that the chiral 1,3-dioxane moiety may have similar orientations
when interacting with the active site of PDE9A and the
different locations of the methoxy groups would not affect the
potency. The present findings represent the first examples of
natural products as PDE9A inhibitors, which may enable the
future design of novel PDE9 inhibitors. However, the
mechanism of inhibition on PDE9A and the selectivity against
other PDE families require further investigation.
Vc4). Fraction Vc4 was subjected to silica gel CC (PE/CH Cl ,
2 2
5:1 → 1:2) to afford (Vc4a−Vc4e), Vc4a and Vc4b were
further purified by HPLC system equipped with a chiral-phase
column (MeOH/H
17.3 min) and 4b (4.4 mg, t
min) and 2b (18.4 mg, t 18.6 min), respectively. Fraction Vb3
O, 93:7, 3 mL/min) to give 4a (4.6 mg, t
2
R
19.4 min), 2a (18.8 mg, t 17.6
R
R
R
and Vb4 were further purified by HPLC equipped with a chiral-
phase column (MeOH/H O, 90:10, 3 mL/min; MeOH/H O,
2
2
93:7, 3 mL/min) to give 1a (15.3 mg, t 22.2 min) and 1b
R
(15.4 mg, t 30.2 min), 3a (14.6 mg, t 22.5 min) and 3b (14.8
R
R
mg, t 23.8 min), respectively.
R
D
dx.doi.org/10.1021/np500528u | J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
1
Table 2. H NMR Data of Torreyunlignans A−D (1−4) in Acetone-d (J in Hz, δ in ppm)
6
a
b
b
b
no.
1
2
3
4
2
3
4
5
6
7
8
9
9
3
4
5
6
7
8
9
3
4
5
6
7
8
9
2
3
4
6
2
6
2
7.08, d (1.9)
6.99, d (8.4)
6.66, d (8.1)
7.24, t (8.1)
6.66, d (8.1)
7.01, m
7.29, m
7.02, m
7.30, ddd (8.4, 7.5, 1.6)
7.03, td (7.5, 0.8)
7.57, dd (7.5, 1.6)
5.14, d (10.0)
6.93, d (8.1)
7.00, dd (8.1, 1.9)
4.46, d (10.0)
2.15, m
7.53, dd (7.8, 1.7)
5.11, d (10.1)
2.14, m
5.24, d (10.2)
3.02, m
2.16, m
α
β
′
′
′
′
′
′
′
″
″
″
″
″
″
″
4.31, dd (11.3, 4.4)
3.71, dd (11.3, 11.3)
6.61, d (8.4)
4.34, dd (11.3, 4.4)
3.74, dd (11.3, 11.3)
6.60, d (8.4)
4.28, dd (11.2, 4.5)
3.62, dd (11.2, 11.2)
6.60, d (8.4)
4.27, dd (11.3, 4.5)
3.73, dd (11.3, 11.3)
6.92, d (8.1)
7.13, t (8.4)
7.12, t (8.4)
7.10, d (8.4)
7.18, ddd (8.1, 7.7, 1.7)
6.85, td (7.7, 0.7)
7.32, dd (7.7, 1.7)
6.59, d (15.9)
6.61, d (8.4)
6.60, d (8.4)
6.60, d (8.4)
6.56, d (16.0)
6.59, d (16.0)
6.51, d (16.0)
6.41, ddd (16.0, 8.4, 6.2)
1.87, m 2.06, m
6.96, d (8.2)
6.43, ddd (16.0, 7.9, 6.4)
1.94, m 2.05, m
6.95, d (8.4)
6.41, ddd (16.0, 7.6, 6.4)
1.83, m 1.98, m
6.96, dd (8.4, 0.7)
7.23, ddd (8.4, 7.5, 1.6)
6.90, t (7.5)
6.03, ddd (15.9, 7.8, 6.8)
1.95, m 2.01, m
6.98, dd (8.2, 0.7)
7.25, ddd (8.2, 7.7, 1.6)
6.92, t (7.7)
7.25, ddd (8.2, 7.8, 1.6)
6.92, t (7.8)
7.25, ddd (8.4, 7.7, 1.6)
6.93, t (7.7)
7.50, dd (7.8, 1.6)
7.11, d (16.4)
7.50, dd (7.7, 1.6)
7.13, d (16.3)
7.48, dd (7.5, 1.6)
7.07, d (16.3)
7.49, dd (7.7, 1.6)
7.07, d (16.3)
6.30, dd (16.4, 4.6)
5.32, dd (4.6, 0.9)
6.33, dd (16.3, 4.6)
5.34, dd (4.6, 1.0)
3.85, s
6.25, dd (16.3, 4.6)
5.26, dd (4.6, 1.1)
3.83, s
6.26, d (16.3, 4.7)
5.33, dd (4.7, 1.0)
3.87, s
-OCH3
-OCH3
-OCH3
-OCH3
′-OCH3
′-OCH3
″-OCH3
3.83, s
3.80, s
3.83, s
3.80, s
3.80, s
3.82, s
3.81, s
3.81, s
3.82, s
3.79, s
3.79, s
3.81, s
3.81, s
3.83, s
a
b
at 500 MHz. at 400 MHz.
(
+)-Torreyunlignan A (1a). Light yellow oil; [α]20 +4.2 (c
2927, 2836, 1593, 1472, 1434, 1372, 1247, 1124, 1104, 1022,
D
−
1
1
13
0
2
2
(
1
.3, CH Cl ); UV (MeOH) λ (log ε) 215 (4.72), 257 (4.55),
972, 752, 726 cm ; H and C NMR data, see Tables 2 and 3;
2
2
max
−4
+
99 (3.98) nm; ECD (c 1.09 × 10 M, CH CN) λ (Δε)
ESIMS m/z 533.3 [M + H] ; HRESIMS m/z 555.2365 [M +
3
max
+
51 (+1.89), 268 (−0.33), 283 (+0.26), 308 (−0.18) nm; IR
Na] (calcd for C H O Na, 555.2359).
32
36
7
2
0
KBr) ν 2931, 2837, 1592, 1516, 1467, 1434, 1245, 1139,
(+)-Torreyunlignan C (3b). Light yellow oil; [α] +20.3 (c
max
D
−1
1
13
−4
112, 1026, 972, 753, 723 cm ; H and C NMR data, see
Tables 2 and 3; ESIMS m/z 533.2 [M + H] ; HRESIMS m/z
55.2334 [M + Na] (calcd for C H O Na, 555.2353).
(
0.6, CH Cl ); ECD (c 1.05 × 10 M, CH CN) λ (Δε) 229
2
2
3
max
+
(+1.30), 253 (+1.79), 270 (−1.72) nm; UV, IR, NMR, MS, and
HRESIMS are the same as those of 3a.
(−)-Torreyunlignan D (4a). Light yellow oil; [α] −10 (c
0.7, CH Cl ); UV (MeOH) λ (log ε) 299 (3.99), 254 (4.43),
+
5
3
2
36
7
20
20
−)-Torreyunlignan A (1b). Light yellow oil; [α] −4.2 (c
D
D
−4
0
(
.3, CH Cl ); ECD (c 1.09 × 10 M, CH CN) λ (Δε) 253
2
2
3
max
2
2
max
−4
−2.41), 268 (+1.10), 284 (−0.67), 307 (+0.37) nm; UV, IR,
214 (4.54) nm; ECD (c 1.26 × 10 M, CH CN) λ (Δε)
3
max
NMR, MS, and HRESIMS are the same as those of 1a.
245 (−3.94), 276 (+1.14) nm; IR (KBr) ν 2957, 2927, 2854,
max
2
0
(
−)-Torreyunlignan B (2a). Light yellow oil; [α] −13.2 (c
2836, 1597, 1490,1460,1436,1288, 1242, 1138, 1026, 972, 889,
D
−1
1
13
0
2
2
2
.7, CH Cl ); UV (MeOH) λ (log ε) 216 (4.59), 258 (4.40),
839, 751 cm ; H and C NMR data, see Tables 2 and 3;
ESIMS m/z 473.1 [M + H] ; HRESIMS m/z 495.2140 [M +
Na] (calcd for C H O Na, 495.2142).
2
2
max
−4
+
99 (3.85) nm; ECD (c 1.19 × 10 M, CH CN) λ (Δε)
3
max
+
50 (−3.95), 281 (+3.59), 304 (0.67) nm; IR (KBr) νmax 2925,
30
32
5
2
0
D
854, 1585, 1465, 1377, 1287, 1244, 1112, 1024, 972, 750, 722
(+)-Torreyunlignan D (4b). Light yellow oil; [α] +12.4 (c
−
1
1
13
−4
cm ; H and C NMR data, see Tables 2 and 3; ESIMS m/z
0.7, CH Cl ); ECD (c 1.23 × 10 M, CH CN) λ (Δε) 243
2
2
3
max
+
+
5
03.2 [M + H] ; HRESIMS m/z 541.1993 [M + K] (calcd for
(+4.02), 275 (−1.38) nm; UV, IR, NMR, MS, and HRESIMS
are the same as those of 4a.
C H O K, 541.1992).
3
1
34
6
2
0
20
1
(
+)-Torreyunlignan B (2b). Light yellow oil; [α] +13.7 (c
Compound 1c. [α] 0 (c 0.1, CH Cl ); H NMR (CDCl ,
D
D 2 2 3
−4
0
(
.7, CH Cl ); ECD (c 1.18 × 10 M, CH CN) λ (Δε) 251
400 MHz) UV (MeOH) λmax (log ε) 357 (2.47), 207 (3.22)
2
2
3
max
−1
+3.48), 281 (−3.78), 298 (−0.51) nm; UV, IR, NMR, MS,
and HRESIMS are the same as those of 2a.
−)-Torreyunlignan C (3a). Light yellow oil; [α] −19.4 (c
nm; IR (KBr) νmax 3431, 2922, 2852, 1464, 1385, 1108 cm ;
δ 7.11 (1H, d, J = 8.2 Hz, H-4′), 6.95 (1H, d, J = 1.7 Hz, H-2),
H
2
0
(
6.91 (1H, dd, J = 8.1, 1.7 Hz, H-5), 6.84 (1H, d, J = 8.1 Hz, H-
5), 6.56 (1H, J = 16.1 Hz, H-7′), 6.54 (2H, d, J = 8.2 Hz, H-3′
and H-5′), 6.45 (1H, ddd, J = 16.1, 7.5, 6.5 Hz, H-8′), 4.75
(1H, d, J = 7.2 Hz, H-7), 3.93 (1H, m, H-9b), 3.89 (3H, s, 3-
D
0
2
2
.5, CH Cl ); UV (MeOH) λ (log ε) 299 (3.66), 256 (4.22),
11 (4.50) nm; ECD (c 1.04 × 10 M, CH CN) λ (Δε)
27 (−1.31), 251 (−1.55), 270 (+1.58) nm; IR (KBr) νmax
2 2 max
−4
3
max
E
dx.doi.org/10.1021/np500528u | J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
Table 3. 13C NMR Data of Torreyunlignans A−D (1−4) in
the absorbance at 280 nm (calculated by ProtParam software).
A typical batch of purification yielded 30−60 mg of PDE9A2
protein from a 0.5 L cell culture. The PDE9A2 proteins had
purity greater than 90% as shown by sodium dodecyl sulfate
polyacrylamide gel electrophoresis (SDS-PAGE).
Acetone-d (100 MHz, δ in ppm)
6
no.
1
2
3
4
1
2
3
4
5
6
7
8
9
1
2
3
4
5
6
7
8
9
1
2
3
4
5
6
7
8
9
2
3
4
6
2
6
2
133.6, C
129.6, C
116.5, C
129.7, C
112.4, CH
150.3, C
157.7, C
159.7, C
157.8, C
The catalytic domains of PDE4D2 (86−413) and PDE5A1
8
−11,16
111.3, CH
129.7, CH
121.5, CH
128.9, CH
77.3, CH
42.3, CH
106.9, CH
130.5, CH
104.9, CH
161.4, C
111.4, CH
129.8, CH
121.6, CH
129.1, CH
77.3, CH
42.3, CH
(
535−860) were purified by published protocols.
150.3, C
Enzymatic Assays. The enzymatic activities of the catalytic
112.3, CH
121.2, CH
85.2, CH
41.6, CH
domains of PDE9A2 and other PDEs were measured by using
cGMP or cAMP as substrates. The assay buffer contains 20−50
77.1, CH
37.1, CH
3
mM Tris-HCl (pH 8.0), 10 mM MgCl , and 1 mM DTT. [ H]-
2
cGMP was diluted with the assay buffer to 20000−30000 cpm/
assay. The reaction was carried out at rt (25 °C) for 15 min and
72.0, CH2
72.1, CH2
72.8, CH2
72.1, CH2
′
′
′
′
′
′
′
′
115.3, C
115.3, C
115.4, C
127.2, C
terminated by addition of 0.2 M ZnSO . The reaction product
4
159.1, C
159.1, C
159.1, C
157.3, C
3
was precipitated by 0.2 N Ba(OH) , whereas unreacted [ H]-
2
104.9, CH
128.5, CH
104.9, CH
159.1, C
104.8, CH
128.4, CH
104.8, CH
159.1, C
104.9, CH
128.4, CH
104.9, CH
159.1, C
111.8, CH
129.0, CH
121.3, CH
127.1, CH
127.0, CH
128.4, CH
32.5, CH2
126.0, C
cGMP remained in the supernatant. The radioactivity in the
supernatant was measured in 2.5 mL of Ultima Gold liquid
scintillation cocktails (PerkinElmer) by a PerkinElmer 2910
liquid scintillation counter. At least eight concentrations of
inhibitors were used for the measurement of IC of inhibitors.
123.1, CH
131.4, CH
34.0, CH2
125.9, C
122.8, CH
131.7, CH
33.7, CH2
125.9, C
122.6, CH
132.0, CH
34.5, CH2
126.1, C
50
Each measurement was repeated at least three times, and IC50
values were calculated by nonlinear regression.
′
″
″
″
″
″
″
″
″
″
158.0, C
158.0, C
158.0, C
158.1, C
ASSOCIATED CONTENT
Supporting Information
■
111.9, CH
130.1, CH
121.4, CH
127.8, CH
128.2, CH
127.8, CH
101.9, CH
111.8, CH
130.0, CH
121.3, CH
127.8, CH
128.1, CH
127.9, CH
102.1, CH
55.9
111.9, CH
129.9, CH
121.4, CH
127.7, CH
127.9, CH
128.3, CH
102.4, CH
56.7
112.0, CH
130.1, CH
121.4, CH
127.8, CH
128.3, CH
127.9, CH
102.2, CH
55.9
*
S
1
1
D and 2D NMR spectra of 1−4, 1c, H NMR spectrum of 1d,
and the theoretical calculation details of ECD spectra. This
material is available free of charge via the Internet at http://
pubs.acs.org.
AUTHOR INFORMATION
Corresponding Authors
-OCH3
-OCH3
-OCH3
-OCH3
′-OCH3
■
56.1
56.1
*E-mail: (L.-S.G.) lsgan@zju.edu.cn. Tel: +86-0571-88208457.
*E-mail: (H.-B.L.) luohb77@mail.sysu.edu.cn. Tel: +86-20-
39943031. Fax: +86-20-39943000.
56.5
56.0
56.0
55.7
56.0
56.0
55.8
56.0
56.0
55.7
55.8
55.8
′-OCH3
*E-mail: (S.Y.) yinsh2@mail.sysu.edu.cn. Tel: +86-20-
39943090. Fax: +86-20-39943090.
″-OCH3
Author Contributions
§
Z.-B.C. and X.L. contributed equally.
OCH ), 3.87 (3H, s, 4-OCH ), 3.83 (1H, m, H-9a), 3.81 (3H
3
3
×
2, s, 2′, 6′-OCH ), 2.27 (1H, m, H-9′a), 2.13 (1H, m, H-9′b),
Notes
3
1
3
2
.04 (1H, m, H-8); C NMR (CDCl , 100 MHz) δ 158.1 (C,
The authors declare no competing financial interest.
3
C
C-2′, 6′), 149.1 (C, C-3), 148.5 (C, C-4), 136.1 (C, C-1), 132.5
CH, C-8′), 127.5 (CH, C-4′), 122.3 (CH, C-7′), 118.9 (C, C-
), 114.8 (C, C-1′), 110.9 (C, C-5), 109.6 (CH, C-2), 104.0
CH, C-3′, 5′), 78.1 (CH, C-7), 64.7 (CH , C-9), 55.91 (4-
(
6
(
ACKNOWLEDGMENTS
■
The authors thank the National Natural Science Foundation of
China (Nos. 81102339, 21103234, and 81373258) and the
Opening Project of Guangdong Provincial Key Laboratory of
New Drug Design and Evaluation (2011A060901014-006) for
providing financial support to this work. We thank Prof. H. Ke
in the Department of Biochemistry and Biophysics at
University of North Carolina, Chapel Hill, for his help with
the expression, purification, and enzymatic assay of PDE9A,
PDE5A, and PDE4D.
2
OCH ), 55.87 (3-OCH ), 55.7 (2′, 6′-OCH ), 47.2 (CH, C-8),
3
3
3
+
3
4.2 (CH , C-9′); ESIMS m/z 353.2 [M − 2H O + H] , 799.3
2
2
+ − −
[
2 M + Na] , 775.3 [2 M − H] , 387.1 [M − H] .
Protein Expression and Purification. The recombinant
pET-PDE9A2 plasmid (catalytic domain, 181−506), which was
subcloned and purified by the same protocols as those in our
16
previously reported studies, was transferred into Escherichia
coli strain BL21 (Codonplus, Stratagene). The E. coli cells
carrying recombinant plasmid grew in LB medium (containing
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dx.doi.org/10.1021/np500528u | J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
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dx.doi.org/10.1021/np500528u | J. Nat. Prod. XXXX, XXX, XXX−XXX