Four new cyclohexylideneacetonitrile derivatives from the hypocotyl of mangrove (bruguiera gymnorrhiza)

Article abstract of DOI:10.3390/molecules200814565

Four new cyclohexylideneacetonitrile derivatives 1-4, named menisdaurins B-E, as well as three known cyclohexylideneacetonitrile derivatives-menisdaurin (5), coclauril (6), and menisdaurilide (7)-were isolated from the hypocotyl of a mangrove (Bruguiera gymnorrhiza). The structures of the isolates were elucidated on the basis of extensive spectroscopic analysis. Compounds 1-7 showed anti-Hepatitis B virus (HBV) activities, with EC50 values ranging from 5.1 ± 0.2 μg/mL to 87.7 ± 5.8 μg/mL.

Full text of DOI:10.3390/molecules200814565

Molecules 2015, 20, 14565-14575; doi:10.3390/molecules200814565
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molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Four New Cyclohexylideneacetonitrile Derivatives from the
Hypocotyl of Mangrove (Bruguiera gymnorrhiza)
1
,†
1,†
2
1
2
1
Xiang-Xi Yi , Jia-Gang Deng , Cheng-Hai Gao , Xiao-Tao Hou , Fei Li , Zhi-Ping Wang ,
Er-Wei Hao , Yan Xie , Zheng-Cai Du , Hui-Xue Huang and Ri-Ming Huang 3,4,*
1
1
1
1
1
School of Pharmaceutical Sciences, Guangxi University of Chinese Medicine, Nanning 530001,
China; E-Mails: xiangxiyi81@aliyun.com (X.-X.Y.); dengjg53@126.com (J.-G.D.);
xthou@126.com (X.-T.H.); wangzhiping@163.com (Z.-P.W.); ewhao@163.com (E.-W.H.);
xiey2009@126.com (Y.X.); duzhengcai8@163.com (Z.-C.D.); hhx123@hotmail.com (H.-X.H.)
2
Guangxi Key Laboratory of Marine Environmental Science, Guangxi Academy of Sciences,
Nanning 530007, China; E-Mails: gaochenghai@gxas.cn (C.-H.G.); finylee@yeah.net (F.L.)
3
Key Laboratory of Plant Resources Conservation and Sustainable Utilization,
South China Botanical Garden, Chinese Academy of Sciences, Guangzhou 510650, China
4
Guangdong Provincial Key Laboratory of Applied Botany, South China Botanical Garden,
Chinese Academy of Sciences, Guangzhou 510650, China
†
These authors contributed equally to this work.
*
Author to whom correspondence should be addressed; E-Mail: huangriming@scib.ac.cn;
Tel./Fax: +86-20-3725-2958.
Academic Editor: Derek J. McPhee
Received: 03 July 2015 / Accepted: 31 July 2015 / Published: 12 August 2015
Abstract: Four new cyclohexylideneacetonitrile derivatives 1–4, named menisdaurins
B–E, as well as three known cyclohexylideneacetonitrile derivatives—menisdaurin (5),
coclauril (6), and menisdaurilide (7)—were isolated from the hypocotyl of a mangrove
(
Bruguiera gymnorrhiza). The structures of the isolates were elucidated on the basis of
extensive spectroscopic analysis. Compounds 1–7 showed anti-Hepatitis B virus (HBV)
activities, with EC50 values ranging from 5.1 ± 0.2 μg/mL to 87.7 ± 5.8 μg/mL.
Keywords: antiviral; Bruguiera gymnorrhiza; cyclohexylideneacetonitrile derivative

Molecules 2015, 20
. Introduction
Bruguiera gymnorrhiza (L.) Savigny (Rhizophoraceae) is a common buttressed tree found in the
14566
1
mangrove forests [1] which are native to many countries of southern and eastern Africa, Asia, and
northern Australia [2]. Different parts of these mangrove plants have traditionally been used as herbal
medicines in Thailand and China [2]. Previous phytochemical investigations on B. gymnorrhiza have
shown the presence of diterpenes, triterpenes, flavonoids, aromatic compounds, and sulfur-containing
compounds [1–6]. As part of our continuing program aimed at exploring the bioactive natural
products from mangrove plants collected on the coast of South China Sea [7,8], four new
cyclohexylideneacetonitrile derivatives 1–4 (Figure 1), which have been named menisdaurins B–E, as
well as three known cyclohexylideneacetonitrile derivatives—menisdaurin (5) [9], coclauril (6) [10], and
menisdaurilide (7) [11]—were isolated from the hypocotyl of a mangrove B. gymnorrhiza. Herein, we
will discuss the isolation, structural elucidation, and antiviral activity of these secondary metabolites.
OH
OH
3
3
HO
HO
OH
OH
OH
OH
R
8 O
O
8
R
8 O
O
HO
OH
6
5
6
5
1
6
1
3
3
3
CN
CN
CN
1
1
2
1
2
4
2
4
4
H
H
H
2
3
R = OMe
R = OH
1
5
R = OMe
R = OH
4
HO
OH
HO
O
H
O
CN
6
7
Figure 1. Secondary metabolites 1–7.
2
. Results and Discussion
The molecular formula of menisdaurin B (1), a yellow powder, was established as C15
H
21NO
7
based
−
on the NMR and HRESIMS data ([M − H] , m/z: 326.1238; calcd. for C15
H
20NO
7
m/z: 326.1240). A
−1
13
characteristic, sharp band at 2225 cm in the IR spectrum and a signal at δ
C
117.9 (C-1) in the C-NMR
1
13
showed the presence of an α,β-unsaturated nitrile [9]. The H- and C-NMR (Table 1) data suggested
the presence of a sugar joined to an aglycone by an α,β-glycosidic linkage (anomeric proton and carbon;
δ
H
4.38 (1H, d, J = 7.8 Hz, H-1′) and δ
C
102.3 (C-1′), respectively). The acid hydrolysis of 1 with HCl
1
gave a D-glucose, which was confirmed by TLC. In the H-NMR spectrum of the aglycone in 1 (Table 1),
two olefinic protons at δ
methine protons at δ 4.72 (1H, dd, J = 10.2, 3.5 Hz, H-8) and 4.45 (1H, ddd, J = 9.0, 5.5 and 2.5 Hz,
H-6), and one methene proton at δ 2.39 (1H, ddd, J = 12.5, 5.5 and 3.5 Hz, H-7a) and 1.62 (1H, ddd,
J = 12.5, 10.2 and 9.0 Hz, H-7b), and also from the COSY and HMQC, the sequence from C-4 to C-8 was
established as –CH–CH–CH(O)–CH –CH(O)–. The remaining methine signal (δ 97.2 (C-2) and δ
H
6.20 (1H, d, J = 10.0 Hz, H-4) and 6.18 (1H, dd, J = 10.0, 2.5 Hz, H-5), two
H
H
2
C
H

Molecules 2015, 20
.66 (1H, d, J = 1.8 Hz, H-1)) and a quaternary carbon (δ
14567
5
C
155.2 (C-3)) resembled the cyanomethylene
group of menisdaurin (1) [9] also isolated from the same plant B. gymnorrhiza, whose structure was
confirmed by comparison the [α] , IR spectrum and detailed NMR data with those in the literature [9,12,13].
In contrast to the low optical rotation of a [α]
D
2
2
D
(−145°, in MeOH, c 0.5) reported for menisdaurin in [12], we
(−78.2°, in MeOH, c 0.2) for our isolated menisdaurin (5). Although this differs from
value given for menisdaurin in [12], it is acceptable considering the compound 5 was the same
2
D
0
measured an [α]
2
2
the [α]
D
−1
substance, namely menisdaurin. This is underlined by a characteristic sharp band at 2223 cm in the IR
1
3
spectrum and a signal at δ 118.5 (C-1) in the C-NMR showed the presence of an α,β-unsaturated
C
nitrile, which are in good agreement with those of menisdaurin in [9,13]. Finally the detailed NMR data
given for 5 fit the reported data of menisdaurin in [9,12,13].
The spin systems of H-1′/H-2′/H-3′/H-4′/H-5′/H
2
-6′ and H-4/H-5/H-6/H -7/H-8 present in 1, as the
2
1
1
analysis of the H- H COSY correlations revealed, were assembled with the assistance of the HMBC
correlations (Figure 2). The structure of 1 was further confirmed by the HMBC spectrum (Figure 2). There
were significant long range couplings between H-2 and C-1, C-4 and C-8, which further established the
position of the α,β-unsaturated nitrile. The anomeric proton H-1′ has a long range correlation with C-8
which showed that the sugar moiety is attached to C-8 of the aglycone. Moreover, the methoxyl attached
to C-6 was secured by the HMBC correlation of OCH
3
to C-6.
1
The stereochemistry of 1 was established by a comprehensive analysis of the H-NMR coupling
1
3
constants, NOESY (Figure 3), C-NMR and CD data. The large coupling characteristics showed the
pseudodiaxial relationships of H-7b (ddd, J7b,8 = 10.2 Hz, J7b,6 = 9.0 Hz, and J7a,7b = 12.5 Hz) with both
H-6 and H-8, together with a NOE correlation between H-6 and H-8 in the NOESY spectrum (Figure 3),
and demonstrated that H-6, H-7b and H-8 occupied the pseudodiaxial positions and that H-6 and H-8
2
D
0
were oriented on the same side of the ring system in 1. Compound 1 showed a [α]
in MeOH of −44.7°.
−145°) [12], whose stereochemistry of the
aglycone and β-glucosyl residue have been established by the X-ray single crystallographic analysis [14]
and enzymatic hydrolysis [15], respectively. Moreover, the NMR data of in C-8 (δ 73.2) in 1 is identical
to that of C-8 (δ 73.2) observed in menisdaurin [13], which indicated that C-8 in 1 has the same absolute
2
2
The reported value for menisdaurin was negative ([α]
D
C
C
stereostructure as C-8 in menisdaurin. Finally, the absolute configurations of C-6 and C-8 were further
confirmed by its CD spectrum, in which a negative Cotton effect by the cyclohexene group was show at
2
15 nm (Δε215nm −25.5). The appplicaton of the octant rule [16] to the compound depicted in the formula
of 1 found that the expected sign of the Cotton effect should be negative. Accordingly, the absolute
configurations at the chiral centres C-6 and C-8 of the aglycone in 1 were assigned as S and R,
respectively. The structure of 1 is thus predicted to be as shown in Figure 1.
Menisdaurin C (2), was obtained as a yellow powder, and had the molecular formula C15
H
23NO as
7
−
deduced from the HR-ESI-MS ([M − H] , m/z: 328.1394; calcd for C15
H
22NO m/z: 328.1396) and NMR
7
−1
data (Table 1). Furthermore, its NMR spectra and the sharp band at 2222 cm in the IR spectrum
established that 1 possessed an α,β-unsaturated nitrile [9] and a β-glucosyl moiety, which were greatly
similar to those of 1 and menisdaurin (5), indicating that 2, 1, and 5 are structurally related. A careful
1
13
comparison of the H- and C-NMR spectra of 2 with those of 1 revealed that the C-4 and C-5 are double
bonds in 1, while C-4 and C-5 in 2 are methylene groups.
It was possible to differentiate between the separate spin systems of H-1′/H-2′/H-3′/H-4′/H-5′/H
2
-6′
1
1
and H
2
2 2
-4/H -5/H-6/H -7/H-8 from the H- H COSY spectrum of 2 (Figure 2). These data, together with

Molecules 2015, 20
14568
the key HMBC correlations between H-1′/C-8; H-2/C-1, C-4, and C-8; and MeO-6/C-6 (Figure 2),
permitted the elucidation of the carbon skeleton of 2.
The relative configuration of the aglycone in 2 was determined by the NOESY experiment (Figure 3)
1
1
and H-NMR J values. In the H-NMR, a large coupling constant (J7b,8 = 9.7 Hz) was observed for H-8,
which required a trans-diaxial relationship between H-8 and H-7b. In addition, a NOESY correlation
between H-8 and H-6 indicated that these two protons were in the axial orientations and cis to one another.
2
0
Compound 2 showed a [α]
D
in MeOH of −56.2°. The corresponding reported value for menisdaurin
−145°) [12], and the observed value for 1 was −44.7°. The NMR data of C-8 (δ 73.9)
in 2 was very similar to that observed at C-8 (δ 73.2) in compound 1 and C-8 (δ 73.2) in menisdaurin [13],
22
was negative ([α]
D
C
C
C
which suggested that the absolute configuration of C-8 was the same as those of C-8 in compound 1 and
menisdaurin. Furthermore, the absolute configurations of C-6 and C-8 were confirmed by its CD
spectrum, in which a negative Cotton effect caused by the nitrylidenecyclohexane group appeared at 215 nm
(
Δε215nm −22.7), the applicaton of the octant rule to the compound depicted as compound 2 found that
the expected sign of the Cotton effect should be negative [17]. Therefore, the absolute configuration of
the aglycone in 2 was assigned as S and R, respectively. Compound 2 is thus determined as shown in
Figure 1 on the basis of the above spectroscopic evidence.
Menisdaurin D (3), was obtained as a yellow powder, and the molecular formula C14
H
21NO was
7
−
obtained from the HR-ESI-MS ([M − H] , m/z: 314.1238; calcd for C14
H
20NO m/z: 314.1240) and NMR
7
spectral data (Table 1). The molecular formula indicated that 3 was a derivative of 2. Comparison of the
1
13
H- and C-NMR spectra of 3 with those of 2 confirmed the overall similarity between their structures.
1
13
However, a careful comparison of the H- and C-NMR spectra of 3 with those of 2 revealed that the
major differences between 2 and 3 were the 6-OMe in 2 and an especially downfield signal of the
hydroxyl group 6-OH at δ
H
4.79 in 3, and the relative up shift of C-6 at δ
C
67.2 in 3.
1
1
The molecular framework was established by the H- H COSY and HMBC correlations (Figure 2).
1
1
The comprehensive analysis of the H- H COSY correlations of 3 established the spin systems of
H-1′/H-2′/H-3′/H-4′/H-5′/H -6′ and H -4/H -5/H-6/H -7/H-8. The MeO-6 group attached to C-6 was
2
2
2
2
confirmed by the HMBC correlations from MeO-6 to C-6. The planar structure of 3 was further
confirmed by the HMBC correlations between H-2 and C-1, C-4, and C-8, which established the position
of the α,β-unsaturated nitrile, and the correlations between the anomeric proton H-1′ and C-8, which
showed that the sugar moiety is attached to C-8 of the aglycone.
The NOESY correlation of H-8 and H-6 (Figure 3) and the a large coupling constant (J7b,8 = 9.4 Hz)
confirmed that these two protons were cofacial and were at the axial positions. Compound 3 showed a
2
D
0
22
D
[α]
in MeOH of −76.2°. The reported value for menisdaurin was negative ([α]
−145°) [12], and the
observed values for 1 and 2 were −44.7° and −56.2°, respectively. Combined with the NMR data of C-8
74.8) in 3 is greatly similar with that of C-8 (δ 73.9) observed in 2 and the key NOESY correlation
(
δ
C
C
of H-6 and H-8, the configurations of C-6 and C-8 in compound 3 should be the same as those of C-6
and C-8 in 2, namely S and R, respectively. Moreover, the Cotton effect values of both 2 and 3 showed
negative Cotton effects at 215 nm (Δε215nm −22.7) and (Δε215nm −42.6), respectively. Compound 3 is
identified as a derivative of 2 on the basis of the above spectroscopic evidence.
Menisdaurin E (4) was obtained as colorless plates, and the molecular formular C
8
H
11NO was
2
+
obtained from the HR-ESI-MS data ([M + Na] , m/z: 176.0679; calcd for C
8
H
11NO Na m/z: 176.0687)
2
−
1
and NMR spectral data (Table 1). A characteristic, sharp band at 2219 cm in the IR spectrum and a

Molecules 2015, 20
14569
1
3
signal at δ
C
116.8 (C-1) in the C-NMR showed the presence of an α,β-unsaturated nitrile [9]. Analyses
1
3
of the C-NMR data indicated that 4 is the aglycone of compound 3.
1
1
The molecular framework was established by the H- H COSY and HMBC correlations (Figure 2).
1
1
The comprehensive analysis of the H- H COSY correlations of 4 established the spin system of
-4/H -5/H-6/H -7/H-8. The planar structure of 4 was further confirmed by the HMBC correlations
between H-2 and C-1, C-4, and C-8, which established the position of the α,β-unsaturated nitrile.
H
2
2
2
1
1
Figure 2. Selected H- H COSY and HMBC correlations of 1–4.
Figure 3. Selected NOESY correlations of 1–4.
The NOESY correlation of H-8 and H-6 (Figure 3) and the large coupling constant (J7b,8 = 9.3 Hz)
confirmed that these two protons were cofacial and were at the axial positions. Compound 4 showed a
2
D
0
[
α] in MeOH of −47.3°. The observed value for 3 was −76.2°. Moreover, the Cotton effect values of
both 3 and 4 showed negative Cotton effects at 215 nm (Δε215nm −42.6) and (Δε215nm −33.4), respectively.
Compound 4 is identified as the aglycone of 3 on the basis of the above spectroscopic evidence.
A comparison of the anti-HBV activities for selected sets of 1–7 are displayed in Table 2. The
standard deviations listed in Table 2 for the EC50 and CC50 values were calculated using the coefficients
of variance produced by each regression analysis. A selectivity index (SI) was calculated for each
compound and is expressed as the ratio of CC50 to EC50 (Table 2).

Molecules 2015, 20
14570
1
13
Table 1. H- and C-NMR data of 1–4.
1 a
2 a
3 a
(J in Hz)
4 b
No.
C
δ , Mult
δ
H
(J in Hz)
δ
C
, Mult
δ
H
(J in Hz)
δ
C
, Mult
δ
H
δ
C
, Mult
δ
H
(J in Hz)
1
2
3
117.9, C
97.2, CH
152.2, C
125.4, CH
117.1, C
94.7, CH
166.0, C
118.7, C
94.3, CH
166.5, C
116.8, C
89.4, CH
170.5, C
5.66 (d, 1.8)
5.53 (d, 1.5)
5.51 (d, 1.8)
5.6 (s)
4
a
6.20 (d, 10.0)
29.7, CH
2
2.46 (d, 11.9)
2.25 (dd, 11.9, 7.8)
1.86–1.92 (m)
29.6, CH
2
2.45 (d, 11.0)
2.11 (dd, 11.0, 7.8)
1.69–1.74 (m)
1.36–1.42 (m)
25.2, CH
2
2.79 (d, 11.0)
4
b
2.59 (dd, 11.0, 7.8)
1.87–1.93 (m)
5
a
144.3, CH
62.8, CH
6.18 (dd, 10, 2.5)
36.4, CH
2
36.3, CH
2
33.5, CH
2
5
b
1.19–1.24 (m)
1.61–1.67 (m)
6
4.45 (ddd, 9.0, 5.5, 2.5)
2.39 (ddd, 12.5, 5.5, 3.5)
1.62 (ddd, 12.5, 10.2, 9.0)
4.72 (dd, 10.2, 3.5)
63.9, CH
3.92–3.87 (m)
67.2, CH
3.75–3.70 (m)
67.7, CH
4.48–4.54 (m)
7
a
37.1, CH
2
36.1, CH
2
1.87 (ddd, 11.5, 5.2, 3.5)
1.17 (ddd, 11.5, 9.7, 8.8)
3.08 (dd, 9.7, 3.5)
41.1, CH
2
2.09 (ddd, 11.3, 5.0, 3.8) 42.9, CH
2
2.18 (ddd, 10.1, 5.0, 3.3)
1.56 (ddd, 10.1, 9.3, 8.5)
4.17 (dd, 9.3, 3.3)
7
b
1.55 (ddd, 11.3, 9.4, 8.5)
8
73.2, CH
102.3, CH
73.8, CH
77.4, CH
70.6, CH
77.3, CH
73.9, CH
102.8, CH
75.9, CH
77.6, CH
70.4, CH
77.5, CH
74.8, CH
102.6, CH
75.8, CH
78.8, CH
71.9, CH
78.6, CH
3.10 (dd, 9.4, 3.8)
4.27 (d, 7.6)
65.7, CH
1
′
′
′
′
′
4.38 (d, 7.8)
4.15 (d, 7.3)
2
3
4
5
3.16 (ddd, 8.7, 7.8, 4.5)
3.39 (ddd, 8.7, 8.7 4.2)
3.01 (ddd, 8.7, 8.7, 5.4)
3.11 (ddd, 8.7, 6.8, 6.2)
3.45 (ddd, 11.8, 6.2, 5.2)
3.98 (ddd, 11.8, 6.8, 2.3)
2.95 (ddd, 8.5, 7.3, 4.3)
3.10 (ddd, 8.7, 8.5, 4.5)
3.04 (ddd, 8.7, 8.7, 5.2)
2.94 (ddd, 8.7, 6.4, 2.4)
3.39 (ddd, 11.3, 6.2, 5.1)
3.65 (ddd, 11.3, 6.8, 2.2)
3.09 (ddd, 8.4, 7.6, 4.3)
3.11 (ddd, 8.4, 8.3, 4.5)
3.01 (ddd, 8.3, 7.8, 5.2)
3.05 (ddd, 7.8, 6.4, 2.2)
3.39 (ddd, 11.0, 6.2, 5.1)
3.65 (ddd, 11.0, 6.5, 2.2)
6
′a
′b
-OMe
61.8, CH
2
61.4, CH
2
63.0, CH
2
6
6
49.2, CH
3
49.2, CH
3
6
-OH
′-OH
′-OH
′-OH
′-OH
4.79 (d, 3.5)
5.02 (d, 4.3)
5.01 (d, 4.5)
4.99 (d, 5.2)
4.13 (d, 5.1)
2
4.97 (d, 4.5)
4.94 (d, 4.2)
4.93 (d, 5.4)
5.09 (d, 4.3)
5.04 (d, 4.5)
5.02 (d, 5.2)
4.15 (d, 5.1)
3
4
6
4.17 (d, 5.2)
a
1
13
b
1
13
In DMSO-d
6
, 600 MHz for H- and 150 MHz for C-NMR; In CD
3
OD, 600 MHz for H- and 150 MHz for C-NMR.

Molecules 2015, 20
14571
Table 2. Anti-HBV activity of 1–7.
Compounds EC50 (μg/mL) CC50 (μg/mL) SI (CC50/EC50
)
1
23.8 ± 0.5
30.7 ± 1.0
19.4 ± 0.3
8.7 ± 1.1
5.1 ± 0.2
7.6 ± 0.5
80.7 ± 5.8
0.1 ± 0.03
178.2 ± 13.3
164.9 ± 10.4
182.0 ± 6.8
72.3 ± 4.1
57.8 ± 3.9
83.9 ± 2.3
214.8 ± 14.7
/
7.5
5.4
9.4
8.3
11.3
11.0
2.7
/
2
3
4
5
6
7
Lamivudine
According to the results described in Table 2, it is easy to understand that most of the tested
compounds 1–7 showed moderate antiviral properties. In general, this study revealed the activity order
as 5 > 6 > 4 > 3 > 1 > 2 > 7. Compound 5 had the most effective activity against HBV replication in the
human hepatoblastoma cell line. Compound 7, by comparison, demonstrated a lesser degree of specific
activity against HBV replication.
3. Experimental Section
3.1. General Procedures
UV spectra were recorded in MeOH on a Lambda 35 UV-Vis spectrophotometer (Perkin-Elmer,
Wellesley, MA, USA). The IR spectra were measured in KBr on a WQF-410 FT-IR spectrophotometer
Beifen-Ruili, Beijing, China). NMR spectra were recorded on an AV 600 MHz NMR spectrometer with
(
TMS as an internal standard (Bruker, Bremen, Germany). HRESIMS data were obtained from a Bruker
Maxis mass spectrometer (Bruker). A Waters-2695 HPLC system, using a Sunfire™ C18 column (150 mm ×
10 mm i.d., 10 μm, Waters, Milford, MA, USA) coupled to a Waters 2998 photodiode array detector
was used. Optical rotation data were measured by a Perkin-Elmer Model 341 polarimeter. CD spectra
were recorded on a MODEL J-810-150S spectropolarimeter (MODEL J-810-150S, Tokyo, Japan). The
silica gel GF254 used for TLC was supplied by the Qingdao Marine Chemical Factory (Qingdao, China).
Spots were detected on TLC under UV light or by heating after spraying with 5% H
2
SO in EtOH. All
4
solvent ratios are measured v/v.
3
.2. Plant Material
The hypocotyl of B. gymmorrhiza was collected from Beilun, Guangxi Province, China, in May 2012.
The specimen was identified by Professor Hangqing Fan from the Guangxi Mangrove Research Center,
Guangxi Academy of Sciences. A voucher specimen (2012-GXAS-001) was deposited in the Guangxi
Key Laboratory of Marine Environmental Science, Guangxi Academy of Sciences, China.
3.3. Extraction and Isolation
The hypocotyls of B. gymmorrhiza (25.4 kg) were exhaustively extracted with EtOH–CH
2
Cl (2:1, 30 L)
2
at 25 °C for 4 days three times in a large metal bowl (diameter 80 cm, volume 50 L). The solvent was
evaporated in vacuo (0.09 MPa, 40 °C, rotary evaporator) to afford a syrupy residue (435 g) that was

Molecules 2015, 20
14572
suspended in distilled water (1.5 L) and extracted successively with petroleum ether (3 × 2 L), ethyl acetate
3 × 2 L) and n-butanol (3 × 2 L). The ethyl acetate portion (52.3 g) was subjected to column
chromatography on silica gel, using CHCl /Me CO (from 10:0 to 5:4) and CHCl /MeOH (from 10:1
(
3
2
3
to 0:10) as gradient eluents, giving twelve fractions (A−L). Fraction E was subjected to column
chromatography on silica gel, using petroleum/ethyl acetate (from 10:0 to 0:10) as an eluent, giving six
sub-fractions (E1−E6), then the sub-fraction E6 was separated by HPLC, using mixtures of MeOH/H
2
O
O
(
5:95) to yield 7 (4.1 mg, R
10:90) to yield 6 (1.6 mg, R
O (5:95) to yield 4 (1.2 mg, R
subjected to column chromatography on silica gel, using CHCl
giving fourteen fractions (A −N ). Fraction N was subjected to a Sephadex LH-20 column chromatography
with MeOH, then separated by HPLC, using mixtures of MeOH/H O (5:95) to yield 5 (1.6 mg, R
5.2 min), 1 (3.4 mg, R = 27.2 min), 2 (1.9 mg, R = 33.2 min) and 3 (3.7 mg, R = 48.3 min), respectively.
t
= 27.0 min). Fraction F was separated by HPLC, using mixtures of MeOH/H
2
(
t
= 18.7 min). Fraction G was separated by HPLC, using mixtures of
= 19.0 min). The n-butanol soluble portion (171.4 g) was
/MeOH (from 10:0 to 0:10) as an eluent,
MeOH/H
2
t
3
n
n
n
2
t
=
1
t
t
t
2
0
Menisdaurin B (1): Yellow powder; [α]
MeOH) λmax (log ε) nm: 271 (4.12); IR (KBr) νmax 3381 (OH), 2225 (CN), and 1621 (C=C) cm ;
1H- (DMSO-d ) and 13C-NMR (DMSO-d
), see Table 1; HRESIMS m/z 326.1238 (calcd for
− H, 326.1240).
D
−44.7° (c 0.54, MeOH); CD (MeOH) Δε215nm −25.5; UV
−1
(
6
6
C
15
H21NO
7
2
0
Menisdaurin C (2): Yellow powder; [α]
MeOH) λmax (log ε) nm: 267 (3.72); IR (KBr) νmax 3398 (OH), 2222 (CN), and 1620 (C=C) cm ;
1H- (DMSO-d ) and 13C-NMR (DMSO-d
), see Table 1; HRESIMS m/z 328.1394 (calcd for
− H, 328.1396).
D
−56.2° (c 0.47, MeOH); CD (MeOH) Δε215nm −22.7; UV
−1
(
6
6
C
15
H23NO
7
2
0
Menisdaurin D (3): Yellow powder; [α]
MeOH) λmax (log ε) nm: 270 (4.53); IR (KBr) νmax 3428 (OH), 2220 (CN), and 1623 (C=C) cm ;
1H- (DMSO-d ) and 13C-NMR (DMSO-d
), see Table 1; HRESIMS m/z 314.1238 (calcd for
− H, 314.1240).
D
−76.2° (c 0.63, MeOH); CD (MeOH) Δε215nm −42.6; UV
−1
(
6
6
C
14
H21NO
7
2
0
Menisdaurin E (4): Colorless plates; [α]
MeOH) λmax (log ε) nm: 269 (4.51); IR (KBr) νmax 3425 (OH) and 2219 (CN) cm ; H (CD
13C-NMR (CD
OD), see Table 1; HRESIMS m/z 176.0679 (calcd for C + Na, 176.0687).
D
−47.3° (c 0.51, MeOH); CD (MeOH) Δε215nm −33.4; UV
−1 1
(
3
OD) and
3
8
H
11NO
2
3
.4. Acid Hydrolysis of 1–3
Acid hydrolysis of 1−3 afforded glucose, which was identified in comparison with standard sugars as
described in the literature [15,18,19]. Compounds 1−3 (1 mg each) were hydrolysed with 2 mol/L of
HCl in H O for 6 h at 85 °C. The reaction mixtures were concentrated. The reaction mixtures of 1−3 and
the authentic D-glucose were subjected to TLC, using Me CO/CH CH(OH)CH /H O (26:14:7) as an
eluent. The R values for the reaction mixtures of 1−3 (R for compounds 1−3 were 0.382, 0.383, and 0.382,
respectively) were greatly similar to this of the authentic D-glucose (R = 0.384) under the same conditions.
2
2
3
3
2
f
f
f

Molecules 2015, 20
.5. Antiviral Assay
Antiviral activity and toxicity of 1−7 were assessed using a standardized culture assay [20], which
14573
3
uses cultures of the HBV-producing, human hepatoblastoma cell line [21]. This cell line, which
chronically produces infectious HBV [22], has been shown to be an accurate and predictive model for
all measured aspects of cellular HBV replication [23].
4
. Conclusions
To our best knowledge, cyclohexylideneacetonitrile derivatives have been found in many
sources [10,13,24–28], and some of molecules possessed prominent biological properties, such as
anti-HBV [14], antioxidant [29], insecticidal [30], antifeedant [30], and antifungal [30] activities. In our
work, four new cyclohexylideneacetonitrile derivatives, menisdaurins B–E (compounds 1–4), as well as
three known cyclohexylideneacetonitrile derivatives, menisdaurin (5), coclauril (6), and menisdaurilide
(7), were isolated from the hypocotyl of the mangrove B. gymnorrhiza. Compounds 1–7 showed moderate
anti-HBV activities. Compounds 1–7 were isolated for the first time from B. gymnorrhiza, which
enriches the class of cyclohexylideneacetonitrile molecules originated from marine resources.
Acknowledgments
This study was supported by grants from the National Natural Science Foundation of China (No.
3
1100260, 81260480), Guangxi Laboratory on Applied Techniques and Product Development of Beibu
Gulf Marine Chinese Materia Medica (2013-8-2-16), the Project of Development of TCM Ph.D Degree,
Guangxi University of Chinese Medicine (201410-02), Guangxi Key Laboratory on Generic Technology
Research and Development of TCM Preparation (14-6-70), Guangxi Key Laboratory of Marine
Environmental Science, Guangxi Academy of Sciences (GXKLHY13-06), the Foundation of Key
Laboratory of Plant Resources Conservation and Sustainable Utilization, South China Botanical Garden,
Chinese Academy of Sciences (No. 201210ZS), Guangxi Key Laboratory of Efficacy Study on Chinese
Materia Medica (10-046-04), the Key Project of Science and Technology Department of Guangxi
(No. 15104001-11).
Author Contributions
X.-X.Y. and J.-G.D. contributed in writing the manuscript. R.-M.H. conceived and designed the
format of the manuscript. X.-T.H. and Y.X. were responsible for structure identification. H.-X.H. and
Z.-C.D. were responsible for the isolation of the compounds. F.L. and C.-H.G. were in charge of the
biological activity. E.-W.H. and Z.-P.W. were responsible for the analysis of the data of the biological activity.
Conflicts of Interest
The authors declare no conflict of interest.

Molecules 2015, 20
14574
References
1
.
Homhual, S.; Zhang, H.J.; Bunyapraphatsara, N.; Kondratyuk, T.P.; Santarsiero, B.D.; Mesecar, A.D.;
Herunsalee, A.; Chaukul, W.; Pezzuto, J.M.; Fong, H.H. Bruguiesulfurol, a new sulfur compound
from Bruguiera gymnorrhiza. Planta Med. 2006, 72, 255–260.
2
.
Homhual, S.; Bunyapraphatsara, N.; Kondratyuk, T.; Herunsalee, A.; Chaukul, W.; Pezzuto, J.M.;
Fong, H.H.S.; Zhang, H.J. Bioactive dammarane triterpenes from the mangrove plant
Bruguiera gymnorrhiza. J. Nat. Prod. 2006, 69, 421–424.
3.
4.
5.
6.
7.
Han, L.; Huang, X.S.; Sattler, I.; Dahse, H.M.; Fu, H.Z.; Lin, W.H.; Grabley, S. New diterpenoids
from the marine mangrove Bruguiera gymnorrhiza. J. Nat. Prod. 2004, 67, 1620–1623.
Han, L.; Huang, X.S.; Sattler, I.; Moellmann, U.; Fu, H.Z.; Lin, W.H.; Grabley, S. New aromatic
compounds from the marine mangrove Bruguiera gymnorrhiza. Planta Med. 2005, 71, 160–164.
Sun, Y.Q.; Guo, Y.W. Gymnorrhizol, an unusual macrocyclic polydisulfide from the Chinese
mangrove Bruguiera gymnorrhiza. Tetrahedron Lett. 2004, 45, 5533–5535.
Panyadee, A.; Sahakitpichan, P.; Ruchirawat, S.; Kanchanapoom, T. 5-Methyl ether flavone
glucosides from the leaves of Bruguiera gymnorrhiza. Phytochem. Lett. 2015, 11, 215–219.
Gao, C.H.; Yi, X.X.; Xie, W.P.; Chen, Y.N.; Xu, M.B.; Su, Z.W.; Yu, L.; Huang, R.M. New
antioxidative secondary metabolites from the fruits of a Beibu Gulf mangrove, Avicennia marina.
Mar. Drugs 2014, 12, 4353–4360.
8
9
1
1
1
1
.
Yi, X.X.; Chen, Y.; Xie, W.P.; Xu, M.B.; Chen, Y.N.; Gao, C.H.; Huang, R.M. Four new jacaranone
analogs from the fruits of a Beibu Gulf mangrove Avicennia marina. Mar. Drugs 2014, 12, 2515–2525.
Ueda, K.; Yasutomi, K.; Mori, I. Structure of a new cyanoglucoside from Ilex warburgii Loesn.
Chem. Lett. 1983, 1, 149–150.
.
0. Yogo, M.; Ishiguro, S.; Murata, H.; Furukawa, H. Coclauril, a nonglucosidic 2-cyclohexen-1-
ylideneacetonitrile, from Cocculus lauriforius DC. Chem. Pharm. Bull. 1990, 38, 225–226.
1. Takahashi, K.; Matsuzawa, S.; Takani, M. Studies on constituents of medicinal plants: 20. The
constituent of vines of Menispermum dauricum DC. Chem. Pharm. Bull. 1978, 26, 1677–1681.
2. Nahrstedt, A.; Wray, V. Structural revision of a putative cyanogenic glucoside from Ilex aquifolium.
Phytochemistry 1990, 29, 3934–3936.
3. Nakanishi, T.; Nishi, M.; Somekawa, M.; Murata, H.; Mizuno, M.; Iinuma, M.; Tanaka, T.; Murata, J.;
Lang, F.A.; Inada, A. Structures of new and known cyanoglucosides from a North-American plant,
Purshia tridentata DC. Chem. Pharm. Bull. 1994, 42, 2251–2255.
1
1
4. Geng, C.A.; Huang, X.Y.; Lei, L.G.; Zhang, X.M.; Chen, J.J. Chemical constituents of
Saniculiphyllum guangxiense. Chem. Biodivers. 2012, 9, 1508–1516.
5. Tanaka, T.; Ikeda, T.; Kaku, N.; Zhu, X.H.; Okawa, M.; Yokomizo, K.; Uyeda, M.; Nohara, T.
A new lignan glycoside and phenylethanoid glycosides from Strobilanthes cusia BREMEK.
Chem. Pharm. Bull. 2004, 52, 1242–1245.
1
6. Kirk, D.N. The chiroptical properties of carbonyl-compounds. Tetrahedron 1986, 42, 777–818.
7. Burnett, R.D.; Kirk, D.N. Chiroptical studies. 101. An empirical-analysis of circular-dichroism data
for steroidal and related transoid α,β-unsaturated ketones. J. Chem. Soc. Perkin Trans. 1 1981,
1
1
460–1468, doi:10.1039/p19810001460.

Molecules 2015, 20
14575
1
1
8. Prakash, I.; Chaturvedula, V.S.P. Structures of some novel α-glucosyl diterpene glycosides from
the glycosylation of steviol glycosides. Molecules 2014, 19, 20280–20294.
9. Wang, X.Y.; Wang, M.C.; Xu, M.; Wang, Y.; Tang, H.F.; Sun, X.L. Cytotoxic oleanane-type
triterpenoid saponins from the rhizomes of Anemone rivularis var. flore-minore. Molecules 2014,
1
9, 2121–2134.
2
2
2
0. Korba, B.E.; Gerin, J.L. Use of a standardized cell-culture assay to assess activities of nucleoside
analogs against Hepatitis B virus replication. Antivir. Res. 1992, 19, 55–70.
1. Sells, M.A.; Zelent, A.Z.; Shvartsman, M.; Acs, G. Replicative intermediates of Hepatitis B virus
in HepG2 cells that produce infectious virions. J. Virol. 1988, 62, 2836–2844.
2. Acs, G.; Sells, M.A.; Purcell, R.H.; Price, P.; Engle, R.; Shapiro, M.; Popper, H. Hepatitis B virus
produced by transfected HepG2 cells causes hepatitis in chimpanzees. Proc. Natl. Acad. Sci. USA
1
987, 84, 4641–4644.
2
3. Korba, B.E.; Gerin, J.L. Antisense oligonucleotides are effective inhibitors of Hepatitis B virus
replication in vitro. Antivir. Res. 1995, 28, 225–242.
2
4. Seigler, D.S.; Pauli, G.F.; Frohlich, R.; Wegelius, E.; Nahrstedt, A.; Glander, K.E.;
Ebinger, J.E. Cyanogenic glycosides and menisdaurin from Guazuma ulmifolia, Ostrya virginiana,
Tiquilia plicata, and Tiquilia canescens. Phytochemistry 2005, 66, 1567–1580.
2
2
2
2
2
5. Damtoft, S.; Jensen, S.R. Cyclohexyl butenolide glucosides from Epacridaceae. Phytochemistry
1
995, 40, 157–159.
6. Manga, S.S.E.; Messanga, B.B.; Sondengam, B.L. 7,8-Dihydrobenzofuranones from Ouratea reticulata.
Fitoterapia 2001, 72, 706–708.
7. Sosa, A.; Winternitz, F.; Wylde, R.; Pavia, A.A. Structure of a cyanoglucoside of
Lithospermum purpureo-caeruleum. Phytochemistry 1977, 16, 707–709.
8. Murakami, A.; Ohigashi, H.; Tanaka, S.; Hirota, M.; Irie, R.; Takeda, N.; Tatematsu, A.;
Koshimizu, K. Bitter cyanoglucosides from Lophira alata. Phytochemistry 1993, 32, 1461–1466.
9. Silva, T.M.S.; Lins, A.C.D.; Sarmento-Filha, M.J.; Ramos, C.S.; Agra, M.D.; Camara, C.A. Riachin,
a new cyanoglucoside from Bauhinia pentandra and its antioxidant activity. Chem. Nat. Compd.
2
013, 49, 685–690.
3
0. Abbassy, M.A.; Abdelgaleil, S.A.M.; Belal, A.S.H.; Rasoul, M.A.A.A. Insecticidal, antifeedant
and antifungal activities of two glucosides isolated from the seeds of Simmondsia chinensis.
Ind. Crop. Prod. 2007, 26, 345–350.
Sample Availability: Samples of the compounds are not available from the authors.
©
2015 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution license
http://creativecommons.org/licenses/by/4.0/).
(