Four New Cyclohexylideneacetonitrile Derivatives from the Hypocotyl of Mangrove (Bruguiera gymnorrhiza)

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.


Introduction
Bruguiera gymnorrhiza (L.) Savigny (Rhizophoraceae) is a common buttressed tree found in the 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][2][3][4][5][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.

Results and Discussion
The molecular formula of menisdaurin B (1), a yellow powder, was established as C15H21NO7 based on the NMR and HRESIMS data ([M − H] − , m/z: 326.1238; calcd. for C15H20NO7 m/z: 326.1240). A characteristic, sharp band at 2225 cm −1 in the IR spectrum and a signal at δC 117.9 (C-1) in the 13 C-NMR showed the presence of an α,β-unsaturated nitrile [9]. The 1 H-and 13 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; δH 4.38 (1H, d, J = 7.8 Hz, H-1′) and δC 102.3 (C-1′), respectively). The acid hydrolysis of 1 with HCl gave a D-glucose, which was confirmed by TLC. In the 1 H-NMR spectrum of the aglycone in 1 (Table 1)  ) and a quaternary carbon (δC 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]D, IR spectrum and detailed NMR data with those in the literature [9,12,13]. In contrast to the low optical rotation of a [α] 22 D (−145°, in MeOH, c 0.5) reported for menisdaurin in [12], we measured an [α] 20 D (−78.2°, in MeOH, c 0.2) for our isolated menisdaurin (5). Although this differs from the [α] 22 D value given for menisdaurin in [12], it is acceptable considering the compound 5 was the same substance, namely menisdaurin. This is underlined by a characteristic sharp band at 2223 cm −1 in the IR spectrum and a signal at δC 118.5 (C-1) in the 13 C-NMR showed the presence of an α,β-unsaturated 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′/H2-6′ and H-4/H-5/H-6/H2-7/H-8 present in 1, as the analysis of the 1 H-1 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 OCH3 to C-6.
The stereochemistry of 1 was established by a comprehensive analysis of the 1 H-NMR coupling constants, NOESY (Figure 3), 13 [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 (δC 73.2) in 1 is identical to that of C-8 (δC 73.2) observed in menisdaurin [13], which indicated that C-8 in 1 has the same absolute 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 215 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 C15H23NO7 as deduced from the HR-ESI-MS ([M − H] − , m/z: 328.1394; calcd for C15H22NO7 m/z: 328.1396) and NMR data (Table 1). Furthermore, its NMR spectra and the sharp band at 2222 cm −1 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 comparison of the 1 H-and 13 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.
The relative configuration of the aglycone in 2 was determined by the NOESY experiment ( Figure 3) and 1 H-NMR J values. In the 1 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.
Compound 2 showed a [α] 20 D in MeOH of −56.2°. The corresponding reported value for menisdaurin was negative ([α] 22 D −145°) [12], and the observed value for 1 was −44.7°. The NMR data of C-8 (δC 73.9) in 2 was very similar to that observed at C-8 (δC 73.2) in compound 1 and C-8 (δC 73.2) in menisdaurin [13], 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 C14H21NO7 was obtained from the HR-ESI-MS ([M − H] − , m/z: 314.1238; calcd for C14H20NO7 m/z: 314.1240) and NMR spectral data ( Table 1). The molecular formula indicated that 3 was a derivative of 2. Comparison of the 1 H-and 13 C-NMR spectra of 3 with those of 2 confirmed the overall similarity between their structures. However, a careful comparison of the 1 H-and 13 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 δH 4.79 in 3, and the relative up shift of C-6 at δC 67.2 in 3.
The Menisdaurin E (4) was obtained as colorless plates, and the molecular formular C8H11NO2 was obtained from the HR-ESI-MS data ([M + Na] + , m/z: 176.0679; calcd for C8H11NO2Na m/z: 176.0687) and NMR spectral data (Table 1). A characteristic, sharp band at 2219 cm −1 in the IR spectrum and a signal at δC 116.8 (C-1) in the 13 C-NMR showed the presence of an α,β-unsaturated nitrile [9]. Analyses of the 13 C-NMR data indicated that 4 is the aglycone of compound 3.
The molecular framework was established by the 1 H-1 H COSY and HMBC correlations ( Figure 2). The comprehensive analysis of the 1 H-1 H COSY correlations of 4 established the spin system of H2-4/H2-5/H-6/H2-7/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.  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).  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.

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% H2SO4 in EtOH. All solvent ratios are measured v/v.

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.

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 H2O 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 Me2CO/CH3CH(OH)CH3/H2O (26:14:7) as an eluent. The Rf values for the reaction mixtures of 1−3 (Rf for compounds 1−3 were 0.382, 0.383, and 0.382, respectively) were greatly similar to this of the authentic D-glucose (Rf = 0.384) under the same conditions.

Antiviral Assay
Antiviral activity and toxicity of 1−7 were assessed using a standardized culture assay [20], which 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].