Potential antiviral lignans from the roots of saururus chinensis with activity against epstein-barr virus lytic replication

Article abstract of DOI:10.1021/np400757k

Epstein-Barr virus (EBV) is a member of the γ-herpes virus subfamily and has been implicated in the pathogenesis of several human malignancies. Bioassay-guided fractionation was conducted on an EtOAc-soluble extract of the roots of Saururus chinensis and monitored using an EBV lytic replication assay. This led to the isolation of 19 new (1-19) and nine known (20-28) lignans. The absolute configurations of the new lignans were established by Mosher's ester, ECD, and computational methods. Eight lignans, including three sesquineolignans (19, 23, and 24) and five dineolignans (3, 4, 26, 27, and 28), exhibited inhibitory effects toward EBV lytic replication with EC₅₀ values from 1.09 to 7.55 μM and SI values from 3.3 to 116.4. In particular, manassantin B (27) exhibited the most promising inhibition, with an EC₅₀ of 1.72 μM, low cytotoxicity, CC₅₀ > 200 μM, and SI > 116.4. This is the first study demonstrating that lignans possess anti-EBV lytic replication activity.

Full text of DOI:10.1021/np400757k

Article
pubs.acs.org/jnp
Potential Antiviral Lignans from the Roots of Saururus chinensis with
Activity against Epstein−Barr Virus Lytic Replication
Hui Cui,† Bo Xu,‡ Taizong Wu,† Jun Xu,† Yan Yuan,*,‡,§ and Qiong Gu*,†
†Research Center for Drug Discovery, School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006,
People’s Republic of China
‡The Institute of Human Virology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, Guangdong 510080,
People’s Republic of China
§Department of Microbiology, School of Dental Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104,
United States
S
* Supporting Information
ABSTRACT: Epstein−Barr virus (EBV) is a member of the
γ-herpes virus subfamily and has been implicated in the
pathogenesis of several human malignancies. Bioassay-guided
fractionation was conducted on an EtOAc-soluble extract of the
roots of Saururus chinensis and monitored using an EBV lytic
replication assay. This led to the isolation of 19 new (1−19)
and nine known (20−28) lignans. The absolute configurations
of the new lignans were established by Mosher’s ester, ECD,
and computational methods. Eight lignans, including three sesquineolignans (19, 23, and 24) and five dineolignans (3, 4, 26, 27,
and 28), exhibited inhibitory effects toward EBV lytic replication with EC50 values from 1.09 to 7.55 μM and SI values from 3.3
to 116.4. In particular, manassantin B (27) exhibited the most promising inhibition, with an EC50 of 1.72 μM, low cytotoxicity,
CC50 > 200 μM, and SI > 116.4. This is the first study demonstrating that lignans possess anti-EBV lytic replication activity.
Saururus chinensis Baill (Saururaceae) is a perennial herbaceous
plant distributed in China and Korea. It has been used in
traditional Chinese medicine as an antipyretic, diuretic, and anti-
inflammatory agent to treat diseases such as edema, jaundice, and
gonorrhea.1 Previous photochemical investigations on species of
this genus led to the isolation of different types of compounds,
including lignans, aristolactams, flavonoids, anthraquinones, and
furanoditerpenes.1 Lignans are the main active constituents of
S. chinensis. Some have exhibited a broad spectrum of biological
activities including NF-κB,1 HIV-1 protease,2 arginase II,3 and
HIF-1 inhibitory activities,4 cardiovascular effects,5 and cytotoxic
activity.6
Epstein−Barr virus (EBV) is a γ-herpes virus associated with
several human diseases, including infectious mononucleosis,
Burkitt lymphoma, T-cell lymphoma, Hodgkin’s disease, naso-
pharyngeal carcinoma, and gastric carcinoma.7 EBV has two
modes of infection, latent and lytic viral replication.8 When EBV
enters the lytic cycle, the viral genome is amplified and virus
particles are produced.9 Therefore, reactivation of the lytic cycle
of EBV may play a role in the pathogenesis of malignancies.10−12
Two nucleotide analogues, acylovir and ganciclovir, are used
to treat herpesvirus infections in clinical practice.13,14 Some
natural products, including 4-O-acetyl-7-acetoxychavicol,15 trite-
rpenoids,16 sesquiterpenoids,17 and isoflavonoids,18 have been
demonstrated to inhibit EBV lytic replication.
an EC50 value of 34.38 μg/mL in P3HR-1 cell lines. Bioactivity-
guided fractionation of the EtOH extract using an EBV lytic
replication assay led to the isolation of 19 new (1−19) and nine
known lignans (20−28). Eight lignans, including sesquine-
olignans 19, 23, and 24 and dineolignans 3, 4, 26, 27, and 28,
demonstrated significant inhibitory effects toward EBV lytic
replication. Herein, the isolation and structure elucidation of
the new compounds (1−19) and the anti-EBV lytic replication
activities of all the isolates are reported.
RESULTS AND DISCUSSION
■
The 95% EtOH extract was separated into four portions ac-
cording to polarity by dissolving it in petroleum ether, EtOAc,
n-BuOH, and H2O, and each fraction was assayed for antiviral
activity. The EtOAc-soluble fraction exhibited the most potent
anti-EBV activity with an EC50 of 21.31 μg/mL. Bioactivity-
guided fractionation of the EtOAc extract using the EBV lytic
DNA replication assay led to the isolation of 19 new (1−19) and
nine known lignans (20−28).
The known compounds were identified as schinlignin A (20),21
epigrandisin (21),22 di-O-methyltetrahydrofuriguaiacin B (22),23
saucerneol methyl ether (23),24 saucerneol D (24),24 (7S,8S)-
polysphorin (25),1 manassantin A (26),25 manassantin B (27),25
and 4-O-demethylmanassantin B (28)4 by spectroscopic analysis
and comparison with literature data.
As part of continuing studies to identify antivirals from
medicinal plants,19,20 the 95% EtOH extract from the roots of
S. chinensis demonstrated potent inhibition ofEBV replication with
Received: September 16, 2013
Published: December 20, 2013
© 2013 American Chemical Society and
American Society of Pharmacognosy
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a
Table 1. 1H and 13C NMR Chemical Shifts of Compounds 1−4 and 19
1
2
3
4
19
δH (J in Hz)
position
δH (J in Hz)
δC
δH (J in Hz)
δC
δH (J in Hz)
δC
δH (J in Hz)
δC
δC
1
130.7
111.7
150.6
152.0
116.0
123.0
203.1
43.6
132.0
111.1
149.2
146.9
118.7
120.1
106.8
82.3
136.6
118.9
150.8
146.8
111.1
118.9
83.5
136.8
110.3
150.8
146.5
118.8
118.8
83.5
136.4
111.2
150.6
145.6
118.6
119.7
83.0
2
7.54, d (1.9)
6.92, m
6.93, m
6.92, m
6.92, d (1.7)
3
4
5
7.01, m
7.02, d (8.1)
6.92, m
6.78, m
6.97, d (8.1)
6.99, d (8.1)
5.46, d (5.8)
2.30, m
6.92, m
6
7.67, d (6.3)
6.93, m
6.86, m
7
5.46, d (5.8)
2.30, m
5.12, d (8.6)
1.79, m
8
3.91, m
44.4
44.3
46.1
9
1.31, d (6.0)
16.1
0.75, s
19.6
0.72, d (6.1)
15.0
0.72, d (6.1)
15.0
0.65, d (6.9)
15.1
1′
2′
3′
4′
5′
6′
7′
8′
9′
1″
2″
3″
4″
5″
6″
7″
8″
9″
130.7
111.7
150.6
152.1
116.1
123.0
203.0
43.5
134.8
111.1
149.2
147.9
110.7
119.4
79.9
136.6
110.4
149.1
146.8
110.4
118.9
83.5
136.6
110.3
150.8
146.3
119.1
118.8
83.5
132.1
109.6
146.7
146.7
114.3
119.4
87.5
7.54, d (1.9)
6.92, m
6.94, m
6.92, m
7.01, d (1.7)
7.01, m
6.92, m
6.85, m
6.84, m
6.86, m
7.67, d (6.3)
6.92, m
6.92, m
6.99, d (8.1)
5.46, d (5.9)
2.30, m
6.97, m
5.33, d (7.8)
2.87, m
5.46, d (5.8)
2.30, m
4.39, d (9.3)
2.25, m
3.91, m
46.2
44.4
44.3
47.8
1.31, d (6.0)
16.1
0.65, d (5.6)
11.4
0.72, d (6.1)
15.0
0.72, d (6.1)
15.0
1.05, d (6.5)
15.1
133.7
110.1
149.2
149.3
111.1
120.0
78.2
132.6
110.3
150.7
149.1
111.1
120.1
78.5
132.2
109.7
146.7
145.7
114.5
120.1
78.6
133.6
113.7
145.7
146.6
108.2
119.1
77.4
132.1
109.6
146.7
145.4
114.4
120.8
78.5
6.84, m
6.86, m
6.92, m
6.92, m
6.85, m
6.84, d (8.0)
6.91, m
6.80, m
6.78, m
6.93, m
6.94, m
6.87, m
6.87, m
6.99, m
4.70, d (9.0)
4.32, m
4.65, d (7.1)
4.15, m
4.63, d (8.3)
4.13, m
4.61, d (8.2)
4.13, m
4.60, d (8.3)
4.10, m
82.6
83.8
84.2
84.1
84.0
1.20, d (6.9)
16.7
1.21, d (6.1)
17.2
1.17, d (6.1)
17.2
1.16, d (6.1)
17.0
1.13, d (6.2)
17.1
1‴
2‴
3‴
4‴
5‴
6‴
7‴
8‴
132.3
107.6
148.0
147.7
108.3
121.1
78.2
132.5
110.3
150.7
149.1
111.1
120.1
78.5
132.8
110.4
149.2
150.8
110.4
120.8
78.6
134.1
107.7
147.9
147.5
110.6
121.2
78.6
6.92, brs
6.84, m
6.83, m
6.78, m
6.79, d (7.9)
6.85, m
6.84, d (8.0)
6.91, m
6.83, m
6.76, m
6.80, m
6.78, m
4.68, d (9.3)
4.32, m
4.65, d (7.1)
4.22, m
4.63, d (8.3)
4.13, m
4.62, d (8.2)
4.13, m
82.8
84.0
84.4
84.2
9‴
1.20, d (6.9)
3.89, s
16.8
1.18, d (6.2)
3.89, s
17.1
1.17, d (6.1)
3.92, s
17.2
1.16, d (6.1)
3.92, s
17.1
OMe-3/3′
OMe-3″/4″
OMe-3‴/4‴
CH2O−
−OH
56.1
56.1
56.1
56.0
3.84, s
3.88, s
56.1
56.0
3.89, s
56.1
3.89, s
56.1
3.89, s
56.1
3.89, s
56.1
3.87, s
56.1
3.87, s
56.1
5.96, s
101.2
5.94, s
101.2
5.63, brs
5.61, brs
5.72, brs
a
1H NMR measured at 400 MHz, 13C NMR measured at 100 MHz, obtained in CDCl3 with TMS as internal standard. Assignments were supported
with HSQC and HMBC NMR spectra.
Compound 1 was obtained as a white powder. Its HRESIMS
showed a sodiated molecular ion at m/z 753.3044, correspond-
ing to the molecular formula C41H46O12Na. Its IR spectrum
exhibited absorptions for hydroxy (3449 and 3367 cm−1) and
conjugated carbonyl (1658 cm−1) groups, as well as an aromatic
ring (1511 cm−1). The NMR spectra of 1 (Table 1) closely
resembled those of manassantin B (27).25 Comparison of the 13C
NMR spectra of 1 and manassantin B (27) revealed two carbon-
yls (δC 203.1, 203.0) in compound 1 instead of two oxymethines.
These data indicated a seco-tetrahydrofuran moiety with the two
carbonyl groups located at C-7 and C-7′. The HMBC corre-
lations of H-9/9′ (δH 1.31, d, J = 6.0 Hz) and H-8/8′ (δH 3.91, m)
with C-7/7′ (δC 203.1, 203.0), H-2 and H-6 with C-7, and H-2′
and H-6′ with C-7′ supported the above functional group
assignments (Supporting Information, Figure S7). Thus, the
molecular structure of 1 was elucidated as shown.
The coupling constants for H-7″ (J7″,8″ = 9.0 Hz) and H-7‴
(J7‴,8‴ = 9.3 Hz) revealed threo configurations for C-7″/8″ and
C-7‴/8‴.25 The absolute configurations of C-7″ and C-7‴ were
determined using the Mosher’s ester method.26 Treatment of
compound 1 with (R)- or (S)-MTPA chloride in anhydrous
pyridine yielded the (S)- and (R)-MTPA ester derivatives,
1
respectively. The H NMR chemical shift differences (ΔδS−R
)
between the 1R and 1S esters are shown in Figure 1. The negative
Δδ for H-8″, H-9″, H-8‴, and H-9‴ and the positive values for
3″-OMe, 4″-OMe, and the methylenedioxy indicated the R
configurations for both C-7″ and C-7‴. Therefore, C-8″ and
C-8‴ were assigned R configurations.
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Figure 1. ΔδS−R values of MTPA esters of 1.
Figure 2. Experimental and calculated ECD spectra of 1 (left) and 2 (right) in MeOH.
Electronic circular dichroism (ECD) methods were used to
determine the configuration of C-8/8′ in compound 1. ECD
spectra were calculated using the Gaussian 09 program at the
TD-DFT-B3LYP/6-31(d,p) level in MeOH. This calculation
for 8S,8′R configurations agreed with the experimental ECD
data (Figure 2; Supporting Information, Figure S145). Thus, the
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structure of compound 1 was unambiguously defined as
(8S,8′R)-7,7′-dioxo-seco-manassantin B and was given the trivial
name saurucinol A.
Compound 4 and 4-O-demethylmanassantin B had similar NMR
and ECD data (Supporting Information, Figure S140) and
negative optical rotations, suggesting that they have the same
absolute configurations.4 Thus, the structure of compound 4 was
delineated as 3″-O-demethylmanassantin B.
Compound 2 was obtained as a white, amorphous solid. Its
molecular formula was determined to be C42H51O12 from an ion
at m/z 747.3362 in the HRESIMS. The NMR data of 2 (Table 1)
were typical for a tetrahydrofuran-type dineolignan23 and
showed two oxygenated quaternary carbons at δC 106.8 and
82.3. These data indicated that the tetrahydrofuran ring is
oxygenated at C-7 and C-8. Comparison of the NMR data of
2 and manassantin A (26)25 revealed that compound 2 was a
7,8-epoxy derivative of 26. HMBC correlations of CH3-9 with the
two oxygenated carbons C-7 and C-8 and C-8′ and of CH3-9′
with C-8′, C-7′, and C-8 confirmed that the oxirane ring is
located at C-7 and C-8 (Supporting Information, Figure S15).
Because of the limited quantities of compound 2, we failed to
establish the absolute configurations of C-7″/8″ and C-7‴/8‴
using the Mosher’s method. The relative configurations of C-7″/7‴
and C-8″/8‴ in 2 were assumed to be R by comparison of their
spectroscopic data (Table 1) with those of manassantin A.25
The relative configuration of the tetrahydrofuran ring in
compound 2 was determined via a NOESY experiment. NOESY
cross-peaks of H-7′/H-8′ and CH3-9 and of H-2/H-6 and CH3-9
indicated that the tetrahydrofuran ring assumed a syn−anti−syn
conformation in compound 2, similar to manassantin A
analogues (Supporting Information, Figure S16).25 The ECD
data of compound 2 were also used to determine the absolute
configuration of the tetrahydrofuran ring. The calculated ECD
spectrum of the (7S,8S,7′S,8′S)-stereoisomer moderately agreed
with the experimental ECD spectrum (Figure 2; Supporting
Information, Figure S146). Thus, the structure of compound 2
was tentatively defined as rel-(7S,8S,7′S,8′S)-7,8-epoxymanas-
santin A and was assigned the trivial name saurucinol B.
Compound 5, a white powder, had the molecular formula
C23H28O8, as established by HRESIMS. The NMR data were
similar to those of (2,6-dimethoxy-4-propenylphenoxy)-1-
(3,4,5-trimethoxyphenyl)propan-1-one (12) (Tables 2 and 3)
except for the absence of one allyl group and the presence of an
E-propenyl moiety.28 The HMBC correlations of H-7′ (δH 6.52,
d, 15.8) and H-8′ (δH 6.26, dt, 15.8, 5.6) with C-1′ (δC 132.9),
H-7′ with C-2′/6′ (δC 103.7), and H-2′/6′ (δH 6.57, s) with C-7′
(δC 131.0) indicated that the E-propenyl group is located at C-1′
(Supporting Information, Figure S39). Therefore, 5 was iden-
tified as (+)-(8R)-3,4,5,3′,5′-pentamethoxy-Δ7′,8′-9′-hydroxy-7-
oxo-8-O-4′-neolignan, based on the specific rotation of +44 and
ECD data, which showed a positive Cotton effect at 358 nm.
Compound 5 was given the trivial name saurucinol C.
Compound 6 had the molecular formula C25H32O9. The NMR
data of 6 (Table 2) closely resembled those of erythro-2-(4″-allyl-
2″,6″-dimethoxyphenoxy)-(3′,4′,5′-trimethoxyphenyl)propan-
1,3-diol29 except for the presence of an O-acetyl group. The
O-acetyl group was connected to C-9 based on the HMBC
(Supporting Information, Figure S46) correlations of a methyl
(δH 2.05) with a hydroxymethyl (C-9, δC 64.1) and an acetoxy
carbonyl carbon (δC 170.7). The coupling constant of H-7 (J7,8
=
8.0 Hz) suggested that 6 possesses a 7,8-threo-configuration.23
Moreover, in the ECD spectrum, the negative Cotton effects at
209 and 280 nm indicated the 8R configuration for compound
630,31 (Supporting Information, Figure S141). Thus, the struc-
ture of 6 was defined as (7R,8R)-8-O-4′-7-(3,4,5-trimethoxy-
phenyl)-8-(1′-allyl-3′,5′-dimethoxy)-9-O-acetylpropanetriol and
was assigned the trivial name saurucinol D.
Compound 3 had a molecular formula of C41H50O11, as
established by HRESIMS. The NMR data (Table 1) resembled
those of manassantin A (26), except for the absence of one
methoxy group in compound 3. The 1H NMR spectrum showed
Compound 7, a white powder, had a molecular formula of
C25H32O9 by HRESIMS. The NMR data (Table 2) were similar
to those of 5, except for the absence of a carbonyl (δC 197.4)
resonance and additional resonances for an oxymethine (δC 79.9;
δH 5.85, d, 7.1) and an acetoxy group (δC 170.0, 21.2; δH 1.90, s).
HMBC (Supporting Information, Figure S53) correlation from
H-7 (δH 5.85) to the acetoxy carbonyl carbon (δC 170.0) indi-
cated that the acetoxy group was located at C-7. The absolute
configuration of compound 7 was determined to be 7R,8R based
on the ECD data showing negative Cotton effects at 209 and
294 nm (Supporting Information, Figure S141).30,31 Thus, based
on its specific rotation of −34, the structure of compound 7
was defined as (−)-(7R,8R)-3,4,5,3′,5′-pentamethoxy-Δ7′,8′-9′-
hydroxy-7-acetoxy- 8-O-4′-neolignan and given the trivial name
saurucinol E.
The NMR data of compound 8 (Table 2) was typical of an
8-O-4′-neolignan. One E-propenyl unit was also indicated by the
NMR data. The HMBC correlations of H-2′/6′ with C-7′ and
H-7′/H-8′ with C-1′ indicated that the E-propenyl moiety was
located at C-1′ (Supporting Information, Figure S60). Further-
more, the HMBC correlations of the methylenedioxy hydrogens
(δH 5.94, s) with C-4/C-5 indicated the C-4/C-5 location of
the presence of a phenolic hydroxy group (δH 5.63). In the 13
C
NMR spectrum of manassantin A (26), C-3″ and C-4″ resonated
at δC 149.1 and 148.9, respectively. However, these carbons in 3
resonated at δC 146.7 and 145.7, respectively. Furthermore, the
chemical shift of C-5″ (δC 111.0) in manassantin A was shifted
downfield to δC 114.5 in compound 3. These characteristic 13
C
NMR chemical shifts indicated a neolignan with 3-OMe and
4-OH substitutions.23 The HMBC spectrum (Supporting
Information, Figure S23) displayed correlations of the phenolic
proton (δH 5.63) with the carbons corresponding to resonances
at δC 145.7 and 114.5. These correlations implied that the
phenolic hydroxy group is located at C-4″ rather than C-3″.
Compound 3 and manassantin A have similar NMR spectra and
negative optical rotations, suggesting that they have the same
absolute configurations. The configuration of manassantin A was
determined to be 7″R,7‴R,8″R,8‴R,7S,7′S,8R,8′R through
synthetic methods.27 Thus, the structure of compound 3
(4″-O-demethylmanassantin A) was defined as (7″R,7‴R,8″R,8‴R)-
(7S,7′S,8R,8′R)-4″,7″,7‴-trihydroxy-3,3′,3″,3‴,4‴-pentamethoxy-
7,7′-epoxy-4,8‴:4′,8″-dioxy-8,8′-dineolignan.
the methylenedioxy group. The coupling constant of H-7 (J7,8
=
The NMR data of 4 (Table 1) were similar to those of 4-O-
demethylmanassantin B (28).4 Analysis of the NMR data of 4
and 28 revealed the presence of one hydroxy (δH 5.61) group in
both 4 and 28. HMBC correlations from H-7″ to C-1″ (δC 133.6),
C-2″ (δC 113.7), and C-6″ (δC 119.1) indicated the hydroxy
substitution at C-3 in 4 (Supporting Information, Figure S31).
8.4 Hz) indicated a 7,8-threo configuration. On the basis of
the ECD (Supporting Information, Figure S141) and NMR
data,30,31 the structure of compound 8 was delineated as (−)-
(7R,8R,7′E)-4,5-methylenedioxy-3,3′,5′-trimethoxy-7-hydroxy-
1′-propenyl-8-O-4′-neolignan and was given the trivial name
saurucinol F.
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a
Table 2. 1H and 13C NMR Data (δ) for Compounds 5−9
5
6
7
8
9
position
δH (J in Hz)
δC
δH (J in Hz)
δC
δH (J in Hz)
δC
δH (J in Hz)
δC
δH (J in Hz)
δC
1
2
3
4
5
6
7
8
9
130.5
107.3
152.9
142.7
152.9
107.3
197.4
80.8
135.5
104.2
153.3
137.9
153.3
104.2
74.6
133.8
104.8
153.2
138.1
153.3
104.8
79.9
135.6
101.7
143.4
134.9
148.9
107.0
79.3
134.9
7.48, d (4.6)
6.58, s
6.60, d (4.6)
6.55, s
6.55, s
101.7
143.6
136.1
148.8
107.2
79.3
7.48, d (4.6)
6.58, s
6.60, d (4.6)
5.85, d (7.1)
4.46, m
6.55, s
6.55, s
4.98, d (8.0)
4.57, d (8.4)
3.91, m
4.57, d (8.2)
3.92, m
5.30, q (6.7)
1.55, d (6.6)
4.02, dd (8.0, 3.4)
4.54, dd (12.0, 3.4)
3.94 (12.0, 2.6)
86.6
80.3
86.6
86.5
18.0
64.1
1.13, d (6.6)
17.3
1.19, d (6.3)
17.7
1.19, d (6.2)
17.7
1′
2′
3′
4′
5′
6′
7′
8′
132.9
103.7
153.3
135.5
153.3
103.7
131.0
128.4
63.6
135.0
105.5
152.6
136.7
152.6
105.5
40.6
132.1
104.0
153.3
137.4
153.3
104.0
131.3
128.0
63.8
134.1
103.0
152.9
136.1
152.9
103.0
130.9
125.7
18.5
135.3
105.6
152.8
135.6
152.8
105.6
40.6
6.57, s
6.42, s
6.61, d (4.7)
6.58, s
6.44, s
6.57, s
6.42, s
6.61, d (4.7)
6.54, d (15.8)
6.28, m
6.58, s
6.44, s
6.52, d (15.8)
6.26, dt (15.8, 5.6)
4.30, brs
3.34, d (6.6)
5.95, m
5.12, m
3.85, s
6.34, d (15.7)
6.17, qd (15.4, 6.6)
1.89, d (6.5)
3.89, s
3.35, d (6.6)
5.96, m
5.11, m
3.86, s
137.0
116.4
56.2
137.2
116.3
56.7
9′
4.32, d (4.3)
3.84
3/5-OMe
3′/5′-OMe
4-OMe
CH2O−
OAc
3.87, s
56.3
56.2
56.7
3.74, s
56.0
3.82, s
56.1
3.84
56.3
3.89, s
56.1
3.86, s
56.1
3.90, s
61.0
3.81, s
60.9
3.82
60.9
5.94, s
101.5
5.96, s
101.5
170.7
20.9
170.0
21.2
OAc
2.05, s
1.90, s
a
1H NMR measured at 400 MHz, 13C NMR measured at 100 MHz, obtained in CDCl3 with TMS as internal standard. Assignments were supported
with HSQC and HMBC NMR spectra.
a
Table 3. 1H and 13C NMR Data (δ) for Compounds 10−13
10
δH (J in Hz)
11
δH (J in Hz)
12
δH (J in Hz)
13
δH (J in Hz)
position
δC
δC
δC
δC
1
2
3
4
5
6
7
8
9
133.9
104.6
153.2
137.8
153.2
104.6
79.9
133.9
104.7
153.2
137.9
153.2
104.7
79.9
130.7
107.3
153.0
142.4
153.0
107.3
197.9
80.9
132.1
109.4
146.9
145.9
114.2
120.9
78.6
6.61, s
6.60, s
7.50, s
6.85−6.95
6.85−6.95
6.85−6.95
4.61, d (8.4)
4.08, m
6.61, s
6.60, s
7.50, s
5.85, d (8.8)
4.42, m
5.85, d (7.2)
4.45, m
80.2
80.3
5.25, m
84.4
1.12, d (6.6)
17.4
1.13, d (6.4)
17.4
1.54, d (6.7)
18.0
1.16, d (6.2)
17.2
1′
2′
3′
4′
5′
6′
7′
8′
135.4
105.7
153.1
135.6
153.1
105.7
40.6
133.5
103.2
153.2
136.6
153.2
103.2
131.1
125.2
18.5
133.8
105.6
153.3
136.2
153.3
105.6
40.7
133.6
109.5
151.2
146.9
118.9
119.2
130.6
125.0
18.5
6.57, s
6.56, s
6.38, s
6.85−6.95
6.85−6.95
6.85−6.95
6.35, d (15.6)
6.57, s
6.56, s
6.38, s
3.33, d (6.6)
6.26, m
5.09, m
3.84, s
6.33, d (15.7)
6.15, m
3.31, d (6.2)
5.94, m
5.09, m
3.87, s
137.4
116.1
56.3
137.3
116.2
56.0
6.14, dt (15.6, 6.5)
1.87, d (6.5)
3. 91, s
9′
1.88, d (6.5)
3.85, s
3/5-OMe
3′/5′-OMe
4-OMe
OAc
56.3
56.0
3.80, s
56.2
3.82, s
56.2
3.72, s
56.4
3.90, s
56.0
3.82, s
60.9
3.82, s
60.9
3.90, s
61.1
170.1
21.2
170.1
21.2
OAc
1.92, s
1.92 (s)
a
1H NMR measured at 400 MHz, 13C NMR measured at 100 MHz, obtained in CDCl3 with TMS as internal standard. Assignments were supported
with HSQC and HMBC NMR spectra.
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Journal of Natural Products
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a
Table 4. 1H and 13C NMR Data (δ) for Compounds 14−18
14
δH (J in Hz)
15
δH (J in Hz)
16
δH (J in Hz)
17
δH (J in Hz)
18
δH (J in Hz)
position
δC
δC
δC
δC
δC
1
134.9
110.1
149.1
148.7
111.2
118.8
87.7
134.8
110.2
149.3
148.6
111.3
118.9
87.7
136.8
103.3
153.3
137.4
153.3
103.3
87.5
133.3
103.3
147.1
134.3
147.1
103.3
87.4
133.5
109.0
148.8
146.3
110.9
118.5
83.6
2
6.99, s
6.97, s
6.65, s
6.64, s
6.80, s
3
4
5
6.86, d (7.9)
6.96, d (8.2)
4.52, d (6.4)
2.32, m
6.85, d (7.9)
6.96, d (7.9)
4.53, m
6.78, d (8.1)
6.84, s
6
6.65, s
6.64, s
7
4.48, d (6.4)
2.31, m
4.48, t (5.4)
2.30, m
5.42, m
8
44.6
2.33, m
44.8
44.5
44.3
2.26, m
44.2
9
1.05, t (6.1)
13.2
1.07, d (6.4)
13.5
1.05, d (6.5)
13.2
1.02, d (6.4)
13.0
0.69, d (5.8)
14.9
1′
2′
3′
4′
5′
6′
7′
8′
133.6
103.3
147.1
134.3
147.1
103.3
87.3
138.4
103.6
153.4
138.4
153.4
103.6
87.4
134.6
106.4
143.4
138.0
149.0
100.5
87.4
138.1
103.4
153.3
137.4
153.3
103.4
87.5
134.1
109.8
148.1
144.8
114.1
119.3
83.7
6.66, s
6.66, s
6.62, s
6.64, s
6.89, d (8.0)
6.84, s
6.66, s
6.66, s
6.62, s
6.64, s
4.50, d (6.2)
2.32, m
4.52, m
2.33, m
1.03, d (6.4)
3.88, s
4.46, d (6.6)
2.30, m
4.48, t (5.4)
2.30, m
5.42, m
44.2
44.2
44.4
44.4
2.26, m
44.2
9′
1.05, d (6.1)
3.88, s
13.1
13.0
1.02, d (6.5)
3.86, s
12.9
1.05, d (6.4)
3. 84, s
13.3
0.69, d (5.8)
3.89, s
14.9
3-OMe
4-OMe
5-OMe
3′-OMe
4′-OMe
5′-OMe
CH2O−
56.1
56.2
56.2
56.1
56.0
3.87, s
56.0
3.87, s
56.0
3.88, s
60.9
3.89, s
56.0
3.86, s
56.2
3. 84, s
3.81, s
3.80, s
3.81, s
56.1
56.3
60.9
56.3
3.88, s
3.88, s
56.4
56.4
3.85, s
3.84, s
3.85, s
56.3
61.0
56.3
3.88, s
56.1
3.83, s
5.95, s
56.7
101.5
a
1H NMR measured at 400 MHz, 13C NMR measured at 100 MHz, obtained in CDCl3 with TMS as internal standard. Assignments were supported
with HSQC and HMBC NMR spectra.
Compound 9 had a molecular formula of C22H26O7, the same
as that of compound 8. Comparison of the spectroscopic data of
compounds 9 and 8 (Table 2 and Experimental Section) indi-
cated the presence of an allyl group in 9 instead of an E-propenyl.
The structure of 9 was confirmed by 2D NMR (COSY, HMQC,
and HMBC). The coupling constant of H-7 (J7,8 = 8.2 Hz) indi-
cated a 7,8-threo configuration. Its ECD spectrum did not exhibit
clear Cotton effects (Supporting Information, Figure S142); thus,
the Mosher’s method (Supporting Information, Figure S148)
was utilized to determine its absolute configuration. Thus, com-
pound 9 was identified as (−)-(7R,8R)-4,5-methylenedioxy-
3,3′,5′-trimethoxy-7-hydroxy-1′-allyl-8-O-4′-neolignan and was
assigned the trivial name saurucinol G.
Compound 10 had the molecular formula C25H32O8. Its NMR
data (Table 3) were similar to those of (7R,8R)-raphidecursinol
B28 except for the presence of an acetoxy group (δH 1.92; δC
170.1, 21.2). The acetoxy group was located at C-7 based on the
HMBC correlations of H-7 (δH 5.85) and a CH3 (δH 1.92) with
the carbonyl carbon at δC 170.1. Thus, compound 10 was
identified as (−)-(7R,8R)-7-O-acetylraphidecursinol B based on
comparison of its specific rotation of −18 and spectroscopic data
with those of (7R,8R)-raphidecursinol B.28 This is the first time
that compound 10 has been isolated from nature; thus it is a new
natural product.
polysphorin, it was identified as a natural product for the first
time.
The NMR data of compound 12 (Table 3) were essentially
identical to those of (2,6-dimethoxy-4-propenylphenoxy)-1-
(3,4,5-trimethoxyphenyl)propan-1-one, which was synthesized
to determine the configuration of polysphorin.28 The 2D NMR
spectra confirmed that the molecular structure of 12 is as shown.
On the basis of its ECD spectrum (Supporting Information,
Figure S142) and its specific rotation of +48 (c 0.03, CHCl3),28
compound 12 was identified as (+)-(8R)-(2,6-dimethoxy-4-
propenylphenoxy)-1-(3,4,5-trimethoxyphenyl)propan-1-one.
Compound 12 has been reported as a synthetic compound;
however, this is the first report of its natural occurrence.
The NMR data of compound 13 (Table 3) were similar to
machilin D.23 The HMBC spectrum revealed that 13 had the
same molecular structure as machilin D (Supporting Informa-
tion, Figure S95).23 However, the configuration of machilin D
has not yet been determined. Mosher’s method was employed to
1
determine the configuration of 13. The negative Δδ H NMR
values for H-8, H-9, 3′-OMe, H-7′, and H-8′ and the positive
values for 3-OMe (Supporting Information, Figure S148) indi-
cated a 7R configuration. The coupling constant of J7,8 (8.4 Hz)
indicated a 7,8-threo configuration. Thus, on the basis of the
above mentioned data and a specific rotation of −88 (c 0.02,
CHCl3), compound 13 was defined as (−)-(7R,8R)-machilin D.
Compounds 14−18 are tetrahydrofuran-type lignans accord-
ing to their NMR data (Table 4). Tetrahydrofuran-type lignans
are the main components in plants of the Saururus genus.23 The
absolute configuration of the tetrahydrofuran ring in these
compounds was determined from the ECD spectra. In addition,
the configuration of the tetrahydrofuran ring was also identified
Compound 11 had the molecular formula C25H32O8. The
NMR data (Table 3) revealed the presence of an E-propenyl
group in 11. Compound 11 was synthesized to determine the
absolute configuration of polysphorin.28 Thus, on the basis of
the specific rotation of −24, the structure of compound 11
was defined as (−)-(7R,8R)-7-O-acetylpolysphorin. Although
compound 11 was reported as a semisynthetic derivative of
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Journal of Natural Products
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by comparing the chemical shifts of C-7/7′, C-8/8′, and C-9/9′
with reported data. The chemical shifts for the different
configurations of the tetrahydrofuran-type lignans are summar-
ized in Figure 3.23,29,32−37
tetrahydrofuran lignan with five methoxy groups. The IR and 1H
NMR data indicated the presence of a hydroxy group. The
molecular structure of 17 was confirmed to be 4-hydroxy-
3,5,3′,4′,5′-pentamethoxy-7,7′-epoxylignan by the 2D NMR data
(HMQC, HMBC, and NOESY). The ECD spectrum of 17
showed negative Cotton effects at 223 and 277 nm, which was
similar to compound 14. Thus, the absolute configuration of 17
was defined as (−)-(7S,8S,7′R,8′R)-4-hydroxy-3,5,3′,4′,5′-pentam-
ethoxy-7,7′-epoxylignan (Supporting Information, Figures S143,
S144) and was assigned the trivial name saurucinol J.
Compound 18 had the molecular formula C21H26O5. Through
comparison of the NMR data of compounds 14−18, the tetra-
hydrofuran ring of compound 18 had a different configuration
based on the 13C NMR chemical shifts of 9/9′-CH3 (δC 14.9).
The NMR data indicated the all-cis configuration of the tetra-
hydrofuran ring. On the basis of the 1H NMR, COSY, HMBC,
NOESY, and ECD spectra,23 the structure of compound 18 was
established as (7R,8S,7′S,8′R)-1-O-methyltetrahydrofuriguaiacin
B and was given the trivial name saurucinol K.
Compound 19 was obtained as a white powder. Its spec-
troscopic data (UV, IR, and NMR) were essentially identical to
those of saucerneol J, which was isolated from the roots of S.
chinensis.23 The HMBC (Supporting Information, Figure S138)
and NOESY (Supporting Information, Figure S139) spectra
confirmed that its molecular structure was identical to that of
saucerneol J. The relative configuration of the tetrahydrofuran
moiety in saucerneol J was identified as 7,8-cis-8,8′-trans-7′,8′-
trans. Mosher’s method was used to determine the absolute
Figure 3. 13C NMR chemical shifts for different configurations of the
tetrahydrofuran ring.
The spectroscopic data of 14 (Table 4 and Experimental
Section) were similar to those of (−)-(7S,7′R,8S,8′R)-4,4′-
dihydroxy-3,3′,5′-trimethoxy-7,7′-epoxylignan37 except for the
presence of an additional methoxy group. This methoxy group
was located at C-4 based on the 2D NMR data (HMQC, HMBC,
and NOESY). The relative configuration of 14 was determined
to be 7,8-trans-8,8′-cis-7′,8′-trans based on the chemical shifts,
as shown in Figure 3. The NOESY spectrum of 14 (Supporting
Information, Figure S103) confirmed its relative configuration.
The absolute configuration of 14 was established as 7S,8S,-
7′R,8′R based on comparison of its ECD data with that of
(−)-(7S,7′R,8S,8′R)-4,4′-dihydroxy-3,3′,5′-trimethoxy-7,7′-epoxy-
lignan37 as well as with the calculated ECD spectrum (Supporting
Information, Figure S147). Thus, the structure of compound 14
was defined as (−)-(7S,8S,7′R,8′R)-4′-dihydroxy-3,4,3′,5′-tetra-
methoxy-7,7′-epoxylignan and given the trivial name saurucinol H.
Compound 15 had the molecular formula C23H30O6 by
HRESIMS. The NMR data of compounds 15 and 14 (Table 4)
were similar except for an additional methoxy group (δH 3.84).
The methoxy group was located at C-4′ in compound 15 based
on comparison of their NMR data (Table 4). The relative con-
figuration of 15 was determined to be identical to 14 as shown.
The absolute configuration of 15 was also established as
7S,8S,7′R,8′R by comparison of its ECD spectrum with that of
14 (Supporting Information, Figure S143). Accordingly, com-
pound 15 was identified as 4-O-methylsaurucinol H.
1
configurations of C-7″ and C-8″. The negative Δδ H NMR
values for H-8″, H-9″, H-7′, H-8′, H-9′, and 3′-OMe and the
positive value for 3″-OMe indicated the 7″R absolute con-
figuration (Supporting Information, Figure S148). Because of the
C-7″/8″ threo configuration (J7″,8″ = 8.6 Hz), C-8″ was assigned
the R configuration. However, the absolute configurations of C-7,
C-8, C-7′, and C-8′ could not be determined. Thus, compound
19 was defined as (−)-(7″R,8″R)-saucerneol J, based on its
specific rotation of −86 (c 0.02, CHCl3) and its ECD spectrum
(Supporting Information, Figure S144).
The isolates were tested for their abilities to inhibit EBV lytic
DNA replication in P3HR-1 cells. As shown in Table 5, all com-
pounds showed significant to marginal inhibitory activity against
EBV DNA replication, with EC50 values from 1.09 to 80.1 μM.
Using the structural and biological data of the 28 lignans isolated
in this study, the structure−activity relationships were assessed.
Three sesquineolignans (19, 23, and 24) displayed significant
inhibition of EBV replication with EC50 values of 6.95, 1.70, and
1.09 μM, respectively. Five of the seven dineolignans (3, 4, 26,
27, and 28) showed relatively high antiviral activities, with EC50
values from 1.72 to 7.55 μM. The 8-O-4′ neolignans (5−13 and
25) and the tetrahydrofuran-type lignans (14−18 and 20−22)
showed moderate to weak activities, with EC50 values from 11.6
to 80.1 μM. These data indicated that the dineolignan or sesqui-
neolignan scaffold is required for significant inhibition. Com-
parison of the structures of these five significant dineolignans
(3, 4, 26, 27, and 28) with those of the less potent dineolignans
(1 and 2) revealed the importance of the tetrahydrofuran ring for
antiviral activity. The seco-tetrahydrofuran moiety of compound
1 and the oxirane positioned on the tetrahydrofuran ring of
compound 2 led to a sharp decrease in activities (1, 16.2 μM; 2,
14.5 μM), indicating that the modification of the tetrahydrofuran
ring is not tolerated. The configuration of the tetrahydrofuran
ring seems to be unimportant for activity, given that com-
pounds 19, 23, and 24 have tetrahydrofuran rings with different
The NMR data (Table 4) of compound 16 showed the
presence of a methylenedioxy group. The 2D NMR (Supporting
Information, Figures S115−S117) confirmed its molecular
structure to be as shown. The ECD spectrum of 16 (negative
Cotton effect at 227 nm) was the same as that of 14 (Supporting
Information, Figure S143). Therefore, the structure of com-
pound 16 was defined as (−)-(7S,8S,7′R,8′R)-3′,4′-methylene-
dioxy-3,4,5,5′-tetramethoxy-7,7′-epoxylignan and was assigned
the trivial name saurucinol I.
Compound 17 had the molecular formula C23H30O7 based on
the HRESIMS data. Comparison of the NMR data of com-
pounds 17 and 16 (Table 4) indicated that 17 was a symmetric
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Table 5. Anti-EBV Lytic Replication Activities of Compounds Isolated from S. chinensis
a
d
b
d
c
a
d
b
d
c
no.
EC50
R2
CC50
>200
R2
SI
no.
EC50
R2
CC50
R2
SI
1
16.2
14.5
0.6309
0.9132
0.9637
0.9491
0.9136
0.7378
0.9545
0.8543
0.9340
0.9636
0.9551
0.8343
0.9424
0.9624
12.4
4.4
9.2
20.2
2.5
3.7
4.2
4.2
4.0
4.0
3.0
9.1
2.6
8.6
15
16
17
18
19
20
21
22
23
24
25
26
27
28
67.9
27.3
29.5
26.9
0.6741
0.7378
0.8462
0.9347
0.9574
0.7967
0.9669
0.7598
0.9377
0.8748
0.9713
0.9619
0.9852
0.9703
172.2
55.0
65.8
62.3
23.2
32.0
0.6141
0.9765
0.9195
0.8294
0.9613
0.9725
2.5
2.0
2
64.8
69.5
26.5
0.8692
0.9756
0.9038
3
7.55
2.69
2.2
4
2.3
5
80.1
>200
194.5
>200
6.95
16.7
3.3
6
53.3
44.3
21.7
12.8
21.5
25.4
21.9
23.8
11.6
0.6301
1.9
7
25.0
49.9
>200
8.0
8
90.3
50.6
85.0
76.8
0.7986
0.9027
0.8292
0.8448
59.8
110.2
45.0
0.9140
0.7076
0.9263
0.8532
1.2
9
1.70
1.09
24.0
65.0
41.1
3.2
10
11
12
13
14
77.3
>200
3.42
1.72
3.52
>200
>200
70.8
58.5
116.4
20.2
61.3
0.7890
0.8751
100.5
0.6523
a
b
The inhibitory effects of the compounds against EBV lytic replication were tested and expressed as EC50 values (μM). Cytotoxicities were
c
d
measured after 2 days of compound treatments and expressed as CC50 values (μM). Selective Index (SI) = CC50/EC50. The regression coefficients
of the dose−response curves.
Han (Kunming Institute of Botany, CAS, China). A voucher specimen
(No. 20120301) was deposited in the School of Pharmaceutical
Sciences, Sun Yat-sen University, Guangzhou, China.
configurations and all showed good activities. Furthermore, in
the dineolignan series, compounds 3, 4, and 28, which have a free
phenolic hydroxy group, exhibited rather good inhibitory
activities with EC50 values of 7.55, 2.69, and 3.52 μM, re-
spectively; however, they also exhibited greater cytotoxicities
with CC50 values of 69.5, 26.5, and 70.8 μM. Compound 27,
which has a methoxy instead of a phenolic hydroxy group,
exhibited significant activity and low cytotoxicity, with EC50,
CC50, and SI values of 1.72 μM, >200 μM, and 116.4, re-
spectively. Similarly, when the phenolic hydroxy group of com-
pound 3 was O-methylated as in compound 26, the activity
increased by 2-fold and the cytotoxicity decreased by 3-fold. The
same trend was also observed in the sesquineolignan series.
Compounds 23 and 24, which do not possess a phenolic hydroxy
moiety, exhibited significant inhibition with EC50 (SI) values of
1.70 (65.0) and 1.09 (41.1) μM, respectively. Compound 19,
which has two phenolic hydroxy substituents, had an EC50 (SI)
value of 6.95 μM (3.3). These data strongly suggested that the
phenolic hydroxy group in the structures of the sesquineolignans
and dineolignans increases cytotoxicity and decreases antiviral
activity.
Extraction and Isolation. The air-dried and milled roots of
S. chinensis (5 kg) were extracted by maceration in 95% EtOH (3 × 10 L)
at room temperature for two days. The solvent was evaporated under
reduced pressure to yield a crude extract (296 g), which was suspended
in H2O and extracted with petroleum ether (3 × 3 L), EtOAc (3 × 3 L),
and n-BuOH (3 × 3 L). The EtOAc-soluble extract exhibited significant
activity against EBV lytic replication, with an EC50 value of 21.31 μg/mL
in HR-1 cell lines. Bioactivity-guided fractionation of the EtOAc-soluble
(Fr. EA) extract was carried out using an anti-EBV assay in the P3HR-1
cell line. Fr. EA (96 g) was chromatographed on a silica gel column
(800 g, 100−200 mesh, 10 × 70 cm) eluting with a step gradient of
petroleum ether−EtOAc to give nine fractions (F1−F9). F2, F3, and F4
were the most active, and F2 (6.8 g) was further separated on ano-
ther silica gel column using petroleum ether−EtOAc (90:10) as the
mobile phase to yield five subfractions (F201 to F205). Compounds 17
(120.0 mg) and 22 (230.0 mg) were obtained from F203 by column
chromatography on silica gel eluting with petroleum ether−EtOAc
(90:10). F204 was chromatographed over an open C18 column (2.6 ×
40 cm) eluting with a MeOH−H2O mixture (80:20) to yield four
subfractions (F2041−F2044). F2041 and F2044 were purified using
HPLC on a semipreparative RP-18 column, using MeOH−H2O (55:45)
as the solvent system, to afford 8 (5.0 mg), 10 (130.0 mg), 13 (8.0 mg),
15 (5.0 mg), 18 (35.0 mg), 19 (8.0 mg), and 23 (40.0 mg). F3 was
chromatographed on Sephadex LH-20 eluting with CHCl3−MeOH
(50:50) to give four subfractions (F301−F304). F301 was purified by
RP-HPLC using MeOH−H2O (55:45) as the solvent system to obtain 2
(9.0 mg), 9 (5.0 mg), 11 (15.0 mg), and 25 (14.0 mg). F302 was
subjected to silica gel column chromatography eluting with CH2Cl2−
MeOH (100:1) to obtain 26 (600.0 mg) and 27 (600.0 mg). F303 was
purified by HPLC on a semipreparative RP-18 column using MeOH−
H2O (55:45) as the solvent to afford 3 (10.0 mg), 4 (40.0 mg), 14
(15.0 mg), and 28 (30.0 mg). F304 was subjected to CC on Sephadex
LH-20, eluting with MeOH, to afford compounds 1 (12.0 mg) and 24
(800.0 mg). F4 was purified using MPLC, eluting with CHCl3−MeOH
(100:1), to give three subfractions (F401−F403). F402 was chromato-
graphed over MCI gel eluting with an increasing gradient of MeOH
(30−100%) in H2O to give six subfractions (F4021−F4026). F4021 was
subjected to a silica gel column eluting with a gradient of CH2Cl2−
EtOAc to yield 5 (80.0 mg), 6 (13.0 mg), and 7 (15.0 mg). F4022 was
purified by RP-HPLC using a mobile phase of MeOH−H2O (55:45) to
afford 12 (8.0 mg), 16 (6.0 mg), 20 (15.0 mg), and 21 (10.0 mg).
Saurucinol A (1): white powder; [α]20D −288 (c 0.01, CHCl3); UV
(MeOH) λmax (log ε) 212 (4.37), 230 (4.44), 280 (4.35) nm; ECD
(MeOH) λmax (Δε) 217 (−0.07), 348 (−0.20), 375 (+0.01) nm; IR νmax
EXPERIMENTAL SECTION
■
General Experimental Procedures. Optical rotations were
measured on a Perkin-Elmer 341 polarimeter. UV spectra were recorded
using a Shimadzu UV2457 spectrophotometer. ECD spectra were ob-
tained on an Applied Photophysics Chirascan spectrometer. IR spectra
were recorded on a Bruker Tensor 37 infrared spectrophotometer. The
1H (400 MHz), 13C (100 MHz), and 2D NMR spectra were obtained on
a Bruker AVANCE-400 using TMS as an internal reference. HRESIMS
were acquired on a Shimadzu LCMS-IT-TOF, and the ESIMS data were
measured on an Agilent 1200 series LC-MS/MS system. TLC analysis
was carried out on silica gel plates (Marine Chemical Ltd., Qingdao,
China). RP-C18 silica gel (Fuji, 40−75 μm), MCI gel (CHP20P, 75−
150 μm, Mitsubishi Chemical Corporation, Tokyo, Japan), silica gel
(200−300 Mesh Marine Chemical Ltd., Qingdao, China), and Sephadex
LH-20 (GE Healthcare Bio-Sciences AB, Sweden) were used for column
chromatography (CC). Analytical and semipreparative HPLC separa-
tion were carried out on an LC-20AT Shimadzu liquid chromatography
system with a ZORBA SB-C18 column (250 × 9.4 mm, 5 μm) or an
Agilent SB-C18 column connected with an SPD-M20A diode array
detector.
Plant Material. The roots of S. chinensis were collected in the Yulin
District, Guangxi Province, China, and authenticated by Chunyan
107
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Journal of Natural Products
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1
3449, 3367, 2924, 2853, 1658, 1511, 1261, 1219, 771 cm−1; H NMR
(CDCl3, 400 MHz) and 13C NMR (CDCl3, 100 MHz) data, see
Table 1; HRESIMS m/z 753.2892 [M+Na]+ (calcd for C41H46O12Na,
753.2882).
100 MHz) data, see Table 3; HRESIMS m/z 483.2019 [M + Na]+ (calcd
for C25H32O8Na, 483.1989).
(+)-(8R)-(2,6-Dimethoxy-4-propenylphenoxy)-1-(3,4,5-
trimethoxyphenyl)propan-1-one (12): white powder; [α]20 +48
D
Saurucinol B (2): white, amorphous solid; [α]20 −80 (c 0.01,
(c 0.03, CHCl3); UV (MeOH) λmax (log ε) 222 (4.11), 281 (4.01) nm;
ECD (MeOH) λmax (Δε) 365 (+0.12) nm; IR (KBr) νmax 2932, 2839,
D
CHCl3); UV (MeOH) λmax (log ε) 214 (4.28), 233 (4.37), 279 (4.00)
nm; ECD (MeOH) λmax (Δε) 207 (−0.10), 283 (−0.10) nm; IR (KBr)
1
1680, 1645, 1500, 1459, 1330, 1233, 1126 cm−1; H NMR (CDCl3,
400 MHz) and 13C NMR (CDCl3, 100 MHz) data, see Table 3;
νmax 3477, 2925, 2853, 1660, 1512, 1264, 1219, 1035, 771 cm−1; H
1
HRESIMS m/z 439.1737 [M + Na]+ (calcd for C23H28O7Na, 439.1727).
NMR (CDCl3, 400 MHz) and 13C NMR (CDCl3, 100 MHz) data, see
Table 1; positive mode HRESIMS m/z 747.3362 [M + H]+ (calcd for
C42H51O12, 747.3375).
(−)-(7R,8R)-Machilin D (13): colorless oil; [α]20 −88 (c 0.02,
D
CHCl3); UV (MeOH) λmax (log ε) 211 (4.41), 267 (4.07) nm; ECD
(MeOH) λmax (Δε) 220 (−0.02) nm; IR (KBr) νmax 3499, 2929, 1510,
1458, 1264, 1223, 1130, 1034, 769 cm−1; 1H NMR (CDCl3, 400 MHz)
and 13C NMR (CDCl3, 100 MHz) data, see Table 3; HRESIMS m/z
367.1532 [M + Na]+ (calcd for C20H24O5Na, 367.1516).
4″-O-Demethylmanassantin A (3): white, amorphous solid; [α]20
D
−35 (c 0.03, CHCl3); UV (MeOH) λmax (log ε) 210 (4.49), 231 (4.37),
280 (3.88) nm; ECD (MeOH) λmax (Δε) 220 (−0.17), 278 (−0.05)
nm; IR (KBr) νmax 3483, 2963, 2930, 1512, 1263, 1219, 1031, 771 cm−1;
1H NMR (CDCl3, 400 MHz) and 13C NMR (CDCl3, 100 MHz)
data, see Table 1; HRESIMS m/z 741.3245 [M + Na]+ (calcd for
C41H50O11Na, 741.3294).
Saurucinol H (14): colorless oil; [α]20 −13 (c 0.02, CHCl3); UV
D
(MeOH) λmax (log ε) 237 (4.05), 277 (3.68) nm; ECD (MeOH) λmax
(Δε) 217 (−0.03), 289 (−0.03) nm; IR (KBr) νmax 3361, 2925, 2852, 1608,
1513, 1460, 1216, 1115, 1028, 759 cm−1; 1H NMR (CDCl3, 400 MHz) and
13C NMR (CDCl3, 100 MHz) data, see Table 4; HRESIMS m/z 411.1778
[M + Na]+ (calcd for C22H28O6Na, 411.1755).
3″-O-Demethylmanassantin B (4): white, amorphous solid; [α]20
D
−112 (c 0.02, CHCl3); UV (MeOH) λmax (log ε) 208 (4.44), 231 (4.03),
286 (3.61) nm; ECD (MeOH) λmax (Δε) 220 (−0.07), 290 (−0.03)
nm; IR (KBr) νmax 3499, 2963, 2926, 1596, 1506, 1452, 1262, 1035, 755
cm−1; 1H NMR (CDCl3, 400 MHz) and 13C NMR (CDCl3, 100 MHz)
data, see Table 1; HRESIMS m/z 725.2950 [M + Na]+ (calcd for
C40H46O11Na, 725.2932).
4-O-Methylsaurucinol H (15): white, amorphous solid; [α]20D −8.9
(c 0.05, CHCl3); UV (MeOH) λmax (log ε) 215(4.32), 227 (4.27), 277
(3.68) nm; ECD (MeOH) λmax (Δε) 226 (−0.15), 277 (−0.09) nm; IR
(KBr) νmax 2925, 2850, 1591, 1509, 1459, 1262, 1229, 1127, 1019,
769 cm−1; 1H NMR (CDCl3, 400 MHz) and 13C NMR (CDCl3,
100 MHz) data, see Table 4; HRESIMS m/z 425.1954 [M + Na]+ (calcd
for C23H30O6Na, 425.1935).
Saurucinol C (5): white powder; [α]20 +44 (c 0.01, CHCl3); UV
D
(MeOH) λmax (log ε) 228 (4.36), 271 (4.34) nm; ECD (MeOH) λmax
(Δε) 217 (−0.07), 358 (+0.24) nm; IR (KBr) νmax 3743, 3361, 3193,
2923, 2853, 1658, 1583, 1462, 1416, 1330, 1236, 1126 cm−1; 1H NMR
(CDCl3, 400 MHz) and 13C NMR (CDCl3, 100 MHz) data, see Table 2;
HRESIMS m/z 455.1651 [M + Na]+ (calcd for C23H28O8Na,
455.1676).
Saurucinol I (16): colorless oil; [α]20 −1.8 (c 0.06, CHCl3); UV
D
(MeOH) λmax (log ε) 217 (4.42), 273 (3.31) nm; ECD (MeOH) λmax
(Δε) 226 (−0.05), 277 (−0.03) nm; IR (KBr) νmax 2961, 2840, 1633,
1591, 1507, 1456, 1220, 1128, 772 cm−1; 1H NMR (CDCl3, 400 MHz)
and 13C NMR (CDCl3, 100 MHz) data, see Table 4; HRESIMS m/z
439.1740 [M + Na]+ (calcd for C23H28O7Na, 439.1727).
Saurucinol D (6): white powder; [α]20 −40 (c 0.02, CHCl3); UV
D
(MeOH) λmax (log ε) 228 (4.10), 270 (3.33) nm; ECD λmax (Δε)
(MeOH) 209 (−0.06), 282 (−0.05) nm; IR (KBr) νmax 3744, 2923,
2852, 1741, 1512, 1462, 1366, 1220, 771 cm−1; 1H NMR (CDCl3, 400
MHz) and 13C NMR (CDCl3, 100 MHz) data, see Table 2; HRESIMS
m/z 499.1904 [M + Na]+ (calcd for C25H32O9Na, 499.1939).
Saurucinol J (17): white, amorphous solid; [α]20 −10 (c 0.05,
D
CHCl3); UV (MeOH) λmax (log ε) 220 (4.09), 271 (3.29) nm; ECD
(MeOH) λmax (Δε) 223 (−0.03), 277 (−0.02) nm; IR (KBr) νmax 3449,
1
2960, 2937, 2840, 1591, 1514, 1462, 1219, 1121, 1011, 772 cm−1; H
NMR (CDCl3, 400 MHz) and 13C NMR (CDCl3, 100 MHz) data, see
Table 4; HRESIMS m/z 441.1909 [M + Na]+ (calcd for C23H30O7Na,
441.1884).
Saurucinol E (7): white powder; [α]20 −34 (c 0.02, CHCl3); UV
D
(MeOH) λmax (log ε) 228 (4.15), 268 (4.15) nm; ECD λmax (Δε)
(MeOH) 212 (−0.04), 298 (−0.03) nm; IR (KBr) νmax 3744, 3360,
Saurucinol K (18): white, amorphous solid; [α]20 +63 (c 0.01,
D
2923, 2854, 1734, 1652, 1585, 1506, 1460, 1228, 1123, 771 cm−1; H
1
CHCl3); UV (MeOH) λmax (logε) 210 (4.31), 230 (4.18), 277 (3.71)
nm; ECD (MeOH) λmax (Δε) 223 (−0.16), 275 (−0.08) nm; IR (KBr)
νmax 3499, 2961, 2929, 1608, 1514, 1458, 1262, 1221, 1030, 772 cm−1;
1H NMR (CDCl3, 400 MHz) and 13C NMR (CDCl3, 100 MHz)
data, see Table 4; HRESIMS m/z 381.1686 [M + Na]+ (calcd for
C21H26O5Na, 381.1672).
NMR (CDCl3, 400 MHz) and 13C NMR (CDCl3, 100 MHz) data, see
Table 2; HRESIMS m/z 499.1969 [M + Na]+ (calcd for C25H32O9Na,
499.1939).
Saurucinol F (8): colorless oil; [α]20 −102 (c 0.01, CHCl3); UV
D
(MeOH) λmax (logε) 225 (4.30), 266 (4.12) nm; ECD (MeOH) λmax
(Δε) 216 (−0.03), 288 (−0.03), 348 (+0.01) nm; IR (KBr) νmax 3465,
3362, 2922, 2851, 1633, 1219, 1126, 772 cm−1; 1H NMR (CDCl3, 400
MHz) and 13C NMR (CDCl3, 100 MHz) data, see Table 2; HRESIMS
m/z 425.1571 [M + Na]+ (calcd for C22H26O7Na, 425.1571).
(-)-(7″R,8″R)-Saucerneol J (19): white powder; [α]20D −86 (c 0.02,
CHCl3); UV (MeOH) λmax (log ε) 212 (4.27), 231 (4.28), 280 (3.89) nm;
ECD (MeOH) λmax (Δε) 220 (−0.02), 278 (−0.01) nm; IR (KBr) νmax
3499, 2961, 1607, 1513, 1459, 1376, 1269, 1224, 1132, 1032,
764 cm−1; 1H NMR (CDCl3, 400 MHz) and 13C NMR (CDCl3,
100 MHz) data, see Table 1; HRESIMS m/z 547.2334 [M + Na]+ (calcd
for C30H36O8Na, 547.2302).
Saurucinol G (9): colorless oil; [α]20 −111 (c 0.02, CHCl3); UV
D
(MeOH) λmax (log ε) 214 (4.54), 271 (2.97) nm; ECD (MeOH) λmax
(Δε) 283 (−0.01) nm; IR (KBr) νmax 3449, 2932, 1632, 1590, 1501,
1425, 1225, 1124, 1039, 770 cm−1; 1H NMR (CDCl3, 400 MHz) and
13C NMR (CDCl3, 100 MHz) data, see Table 2; HRESIMS m/z
425.1585 [M + Na]+ (calcd for C22H26O7Na, 425.1571).
Preparation of (R)- and (S)-MTPA Esters of 1, 9, 13, and 19.
Compound (1 mg) was dissolved in 1 mL of pyridine and stirred at rt for
10 min. An excess of (R)- or (S)-MTPA chloride (10 μL) was added, and
the reaction was stirred overnight at rt. The solvent was removed in
vacuo, and the crude reaction product was purified by preparative silica
gel TLC using petroleum ether−EtOAc (5:1) as developing solvent.
1H NMR data of (R)-MTPA ester of 1 (400 MHz, CDCl3): δH 7.61
(2H, s, H-6, H-6′), 7.49 (2H, s, H-2, H-2′), 6.90−6.70 (8H, m, Ar-H),
6.06 (1H, d, J = 8.6 Hz, H-7″), 5.99 (1H, d, J = 8.5 Hz, H-7‴), 5.96
(2H, s, OCH2O), 4.73 (1H, m, H-8″), 4.72 (1H, m, H-8‴), 3.87 (3H,
4″-OCH3), 3.70 (3H, 3″-OCH3), 3.80 (6H, 3-OCH3, 3′-OCH3), 1.23
(−)-(7R,8R)-7-O-Acetylraphidecursinol B (10): white, amorphous
solid; [α]20D −18 (c 0.04, CHCl3); UV (MeOH) λmax (log ε) 217 (4.37),
270 (3.40) nm; ECD (MeOH) λmax (Δε) 230 (+0.04) nm; IR (KBr)
νmax 2961, 2839, 1589, 1502, 1459, 1422, 1234, 1125, 1026 cm−1; H
1
NMR (CDCl3, 400 MHz) and 13C NMR (CDCl3, 100 MHz) data, see
Table 3; HRESIMS m/z 483.1988 [M + Na]+ (calcd for C25H32O8Na,
483.1989).
(−)-(7R,8R)-7-O-Acetylpolysphorin (11): white powder; [α]20D −24
(c 0.02, CHCl3); UV (MeOH) λmax (log ε) 222 (4.15), 268 (4.15) nm;
ECD (MeOH) λmax (Δε) 239 (+0.01), 296 (−0.01) nm; IR (KBr)
νmax 2934, 2841, 1739, 1586, 1502, 1461, 1419, 1372, 1334, 1237, 1126,
761 cm−1; 1H NMR (CDCl3, 400 MHz) and 13C NMR (CDCl3,
1
(6H, s, H-9, H-9′), 1.12 (6H, s, H-9″, H-9‴). H NMR data of (S)-
MTPA ester of 1 (400 MHz, CDCl3): δH 7.57 (2H, t, J = 8.8 Hz, H-6,
H-6′), 7.47 (2H, d, J = 4.3 Hz, H-2, H-2′), 6.97−6.77 (8H, m, Ar-H),
6.17 (1H, d, J = 7.2 Hz, H-7″), 6.12 (1H, d, J = 7.4 Hz, H-7‴), 5.97
108
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Article
(2H, d, J = 4.1 Hz, OCH2O), 4.71 (1H, m, H-8″), 4.70 (1H, m, H-8‴),
3.87 (3H, 4″-OCH3), 3.83 (3H, 3″-OCH3), 3.79 (6H, 3-OCH3,
3′-OCH3), 1.28 (6H, d, J = 6.1 Hz, H-9, H-9′), 1.10 (6H, d, J = 5.8 Hz,
H-9″, H-9‴).
Cytotoxicity Assays. The viabilities of P3HR-1 cells after being
treated or untreated with compounds were assessed by counting Trypan
blue-stained cells two days post-treatment using a light microscope. Cell
viabilities were defined relative to the control cells (nondrug treated).
The half-maximal cytotoxic concentration (CC50) was calculated from
the dose−response curves with GraphPad Prism software.
1H NMR data of (R)-MTPA ester of 9 (400 MHz, CDCl3): δH 6.46
(2H, s, OCH2O), 6.37 (2H, s, H-3′, H-5′), 6.04 (1H, d, J = 7.5 Hz, H-7),
5.94 (2H, s, H-2, H-6), 5.11 (1H, m, H-8′), 5.08 (1H, d, J = 8.5 Hz, H-
9′), 4.51 (1H, m, H-8), 3.77 (3H, 3-OCH3), 3.73 (6H, 2′-OCH3, 6′-
OCH3), 3.32 (1H, d, J = 6.5 Hz, H-7′), 0.95 (1H, d, J = 6.4 Hz, H-9). 1H
NMR data of (S)-MTPA ester of 9 (400 MHz, CDCl3): δH 6.68 (2H, d,
J = 8.6 Hz, OCH2O), 6.35 (2H, s, H-3′, H-5′), 6.21 (1H, d, J = 5.8 Hz,
H-7), 5.94 (2H, s, H-2, H-6), 5.10 (1H, m, H-8′), 5.07 (1H, d, J = 8.2 Hz,
H-9′), 4.43 (1H, m, H-8), 3.84 (3H, 3-OCH3), 3.73 (6H, 2′-OCH3,
6′-OCH3), 3.30 (1H, d, J = 6.6 Hz, H-7′), 0.97 (1H, d, J = 6.1 Hz, H-9).
1H NMR data of (R)-MTPA ester of 13 (400 MHz, CDCl3): δH
6.98−6.82 (6H, m, Ar-H), 6.35 (1H, d, J = 14.9 Hz, H-7′), 6.15 (1H, m,
H-8′), 6.11 (1H, d, J = 7.6 Hz, H-7), 4.62 (1H, m, H-8), 3.80 (3H,
3′-OCH3), 3.67 (3H, 3-OCH3), 1.88 (1H, d, J = 6.6Hz, H-9′), 1.09 (1H, d,
ASSOCIATED CONTENT
* Supporting Information
■
S
The IR, MS, 1D and 2D NMR, and CD spectra for compounds
1−19. This material is available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Authors
■
*E-mail: (Y. Yuan) yuan2@pobox.upenn.edu.
*E-mail: (Q. Gu) guqiong@mail.sysu.edu.cn. Tel: 86-20-
39943077. Fax: 86-20-39943077.
1
J = 6.2 Hz, H-9). H NMR data of (S)-MTPA ester of 13 (400 MHz,
CDCl3): δH 7.06−6.77 (6H, m, Ar-H), 6.34 (1H, d, J = 15.9 Hz, H-7′),
6.24 (1H, d, J = 6.5 Hz, H-7), 6.14 (1H, m, H-8′), 4.60 (1H, m, H-8), 3.79
(3H, 3′-OCH3), 3.72 (3H, 3-OCH3), 1.87 (1H, d, J = 6.5 Hz, H-9′), 1.07
(1H, d, J = 6.4 Hz, H-9).
Author Contributions
Hui Cui and Bo Xu contributed equally.
Notes
1H NMR data of (R)-MTPA ester of 19 (400 MHz, CDCl3): δH
7.38−6.79 (9H, m, Ar-H), 6.11 (1H, d, J = 7.4 Hz, H-7″), 5.13 (1H, d, J =
8.2 Hz, H-7), 4.61 (1H, m, H-8″), 4.46 (1H, d, J = 9.3 Hz, H-7′), 3.82
(3H, 3″-OCH3), 3.74 (3H, 3′-OCH3), 3.71 (3H, 3-OCH3), 2.32
(1H, m, H-8), 1.75 (1H, m, H-8′), 1.10 (1H, m, H-9′), 1.08 (1H, m, H-
9″), 0.65 (1H, d, J = 7.0 Hz, H-9). 1H NMR data of (S)-MTPA ester of
19 (400 MHz, CDCl3): δH 7.29−6.79 (9H, m, Ar-H), 6.23 (1H, d, J =
6.3 Hz, H-7″), 5.12 (1H, d, J = 8.4 Hz, H-7), 4.59 (1H, m, H-8″), 4.45
(1H, d, J = 8.9 Hz, H-7′), 3.82 (3H, 3″-OCH3), 3.72 (3H, 3′-OCH3), 3.70
(3H, 3-OCH3), 2.23 (1H, m, H-8), 1.74 (1H, m, H-8′), 1.08 (1H, d, J =
6.5 Hz, H-9′), 1.07(1H, d, J = 6.2 Hz, H-9″), 0.65 (1H, d, J = 6.9 Hz, H-9).
Computational Methods. The calculated ECD spectra were
obtained by density functional theory (DFT) and time-dependent DFT
(TD-DFT) using Gaussian 09 and analyzed using GUIs GaussView
(version 5.0). The calculated ECD spectra calculations were first per-
formed at the HF/6-31G level in the gas phase for initial optimization.
The minimum geometries were fully optimized at the B3LYP/6-31G(d)
level in the gas phase to yield more accurate conformers. The ECD
spectra were simulated at the B3LYP/6-31G(d,p) level in MeOH. The
calculated ECD curve was generated using SpecDis with σ = 0.2 eV.
Analysis of Intracellular EBV Genomic DNA Content and
Inhibition of EBV DNA Replication by Extracts and Compounds.
P3HR-1 cells, a Burkitt lymphoma cell line latently infected with EBV,
were cultured in RPMI 1640 medium (Gibco-BRL, Gaithersburg, MD,
USA) supplemented with 10% fetal bovine serum (Gibco-BRL), strep-
tomycin (100 μg/mL), and penicillin (100 units/mL). For viral lytic
replication, P3HR-1 cells were induced with TPA (20 ng/mL) and
sodium butyrate (0.3 mM). After three hours, the cells were treated with
the extracts or compounds over a wide range of concentrations. Forty-
eight hours postinduction, the total DNA in the cells was purified using
the DNeasy tissue kit according to the manufacturer’s protocol (Takara
Bio. Inc., Japan). The EBV genomic copy number was quantified by real-
time PCR on a Roche 480 LightCycler instrument using the LightCycler
FastStart DNA Masterplus SYBR green kit with primers for the detection
of EBNA1 (sense: 5′-CATTGAGTCGTCTCCCCTTTGGAAT-3′;
antisense: 5′-TCATAACAAGGTCCTTAATCGCATC-3′). The half-
maximal antiviral effective concentration (EC50) value of each com-
pound was determined from a dose−response curve of EBV DNA
content values from TPA/butyrate-induced and compound-treated
cells. The viral DNA contents were reduced by the contents of
noninduced cells, divided by that from the control cells without drug
treatment and then represented on the Y axes of the dose−response
curves. Y axis value = (TPAx − no TPAx)/(TPA0 − no TPA0), where x is
any concentration of the drug, and 0 represents no-drug treatment. The
EC50 for viral DNA synthesis for each compound was calculated with the
aid of GraphPad Prism software.
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This study was supported in part by the National High-Tech
R&D Program of China (863 Program) (No. 2012AA020307),
the R&D team program of Guangdong Province (No.
2009010058), the National Natural Science Foundation of
China (No. 81001372, 81173470, 81271805, 81171575), the
National Science and Technology Major Project of China (No.
2010ZX09102-305), and the External Cooperation Program of
the Chinese Academy of Sciences (No. P2010-KF08).
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