Three New Sesquiterpenoids and One New Sesquiterpenoid Derivative from Chinese Eaglewood

Three new sesquiterpenoids (1–3) and one new sesquiterpenoid derivative (4), along with three known sesquiterpenoids (5–7), were isolated from the 95% ethanolic extract of Chinese eaglewood [Aquilaria sinensis (Lour.) Gilg]. The structures of these compounds were elucidated through extensive analysis of spectroscopic data including IR, NMR, HRESIMS, and X-ray diffraction experiments. In addition, the above new compounds were detected for their bioactivities against LPS-induced NO production in RAW 264.7 cells. Among them, compound 2 exhibited obvious anti-inflammatory activity with an IC50 value of 8.1 μM.


Introduction
Chinese eaglewood, the resinous wood of Aquilaria sinensis (Lour.) Gilg (Thymelaeaceae), commonly known in different countries as Chenxiang, agarwood, agalloch, jinkoh, or aloeswood, is widely distributed in Southern China in such provinces as Hainan, Fujian, Yunnan and Guangxi [1]. The resinous part of eaglewood can be formed by any of many possible natural or artificial factors, such as lightning strikes, bacteria invasion, insects, burning, holing, physical cutting, and artificial chemical stimulation. Traditionally, eaglewood has been used as analgesic, sedative, and digestive medicine in many Asian countries. Moreover, some rare and precious eaglewoods also serve as incense, items for collection, and decorations all over the world. Research on the chemical components of the eaglewood began half a century ago. So far, a large number of compounds have been reported from this resin wood, including aromatics [2,3], sesquiterpenes [4], chromone derivatives [5][6][7][8], triterpenes, and diterpenes [9]. Some of them showed certain anti-microbial, anti-inflammation, neuroprotective, and anti-depressant activities. Previous phytochemical investigations of Chinese eaglewood have resulted in the isolation and identification of some chromone derivatives and sesquiterpenes [5,10]. As a continuation of studies on the bioactive metabolites from this plant, three new sesquiterpenoids (1-3) and one new sesquiterpenoid derivative (4), together with three known sesquiterpenoids (5-7) (Figure 1), were isolated from the 95% ethanolic extract of Chinese eaglewood. Their structures were elucidated mainly via IR, NMR, HRESIMS, and X-ray diffraction experiments. Herein, the isolation and structural identification of the compounds obtained from Chinese eaglewood and the inhibitory effects of the new compounds on LPS-induced NO production in RAW 264.7 cells in vitro are reported.

Structure Elucidation of Compounds
Compound 1 was obtained as colorless needles via crystallization from acetone. Its molecular formula was established as C 15 3 -15) and one oxymethine (δ H 3.86, td, J = 9.6 Hz, 3.6 Hz) protons. The 13 C-NMR data ( Table 2) displayed 15 carbon signals, sorted into three methyls, three methylenes, five methines, and four quaternary carbons according to DEPT spectrum, respectively. Double-bond carbon (δ C 138.5, C-1; δ C 150.3, C-10), carbonyl (δ C 209.4, C-2), and acetoxyl (δ C 180.7, C-12) signals could also be observed in the 13 C NMR spectrum. In addition, there were no exchanging hydrogen atoms in compound 1, based on the molecular formula by combination with the 13 C-NMR and DEPT spectroscopic data. Therefore, the presence of a tricyclic unit was determined by the distinctive signals at δ C 180.7 and δ C 81.4.
Compound 3 was obtained as a colorless oil. Its molecular formula was assigned as C15H24O4 by HRESIMS at m/z 291.1594 (calcd for C15H24O4Na, 291.1572), accounting for four degrees of unsaturation. In the 1 H-NMR data (Table 1)  compound 1, the Cu Kα X-ray diffraction experiment was conducted, which confirmed the above conclusion ( Figure 4). Therefore, the structure of compound 1 was identified to be 7βH-guaia-1(10)en-12,8β-olide (Figure 1).  Compound 2 had the molecular formula C15H20O4, as determined by HRESIMS at m/z 287.1251 (calcd for C15H20O3Na, 287.1259). The 1 H-NMR data of compound 2 (Table 1)  . The 13 C-NMR and DEPT spectra of compound 2 exhibited 15 carbon signals, which was similar to the known compound postiaseco-guaianolide [11,12], except for the differences in the numbers and chemical shifts of double bonds. Detailed analysis of 2D NMR data, the terminal double bond (-11, -13) in postiaseco-guaianolide was substituted by a methyl in compound 2 on the basis of the HMBC correlation of δH 1.30 to δC 178.1. Furthermore, the position of the double bond in compound 2 was determined by the key HMBC correlations of H-14 to δC 168.2 (C-3), and H-2, H-3 to δC 210.8 (C-1). The NOESY correlation of H-2/H-3 illustrated the geometric configuration of C=C double bond between C-2 and C-3, which belonged to Z-type. The relative configuration of compound 2 was also disclosed through the NOESY spectrum by comparison with postiaseco-guaianolide. On the assumption that H-5 and H-7 were assigned as α-oriented and β-oriented, respectively, the correlations of CH3-13/H-7, H-4/H-5, and H-8/H-11 allowed the assignments of H-11, H-4, and H-8 as α-orientation ( Figure 3). Consequently, the structure of compound 2 was tentatively identified to be 1,10-dioxo-4αH-5αH-7βH-11αH-1,10-secoguaia-2(3)-en-12,8β-olide (Figure 1).
Compound 3 was obtained as a colorless oil. Its molecular formula was assigned as C15H24O4 by HRESIMS at m/z 291.1594 (calcd for C15H24O4Na, 291.1572), accounting for four degrees of unsaturation. In the 1 H-NMR data (Table 1) , H-3). The 13 C-NMR and DEPT spectra of compound 2 exhibited 15 carbon signals, which was similar to the known compound postiaseco-guaianolide [11,12], except for the differences in the numbers and chemical shifts of double bonds. Detailed analysis of 2D NMR data, the terminal double bond (-11, -13) in postiaseco-guaianolide was substituted by a methyl in compound 2 on the basis of the HMBC correlation of δ H 1.30 to δ C 178.1. Furthermore, the position of the double bond in compound 2 was determined by the key HMBC correlations of H-14 to δ C 168.2 (C-3), and H-2, H-3 to δ C 210.8 (C-1). The NOESY correlation of H-2/H-3 illustrated the geometric configuration of C=C double bond between C-2 and C-3, which belonged to Z-type. The relative configuration of compound 2 was also disclosed through the NOESY spectrum by comparison with postiaseco-guaianolide.
Compound 3 was obtained as a colorless oil. Its molecular formula was assigned as C 15 3 -15), and two olefinic protons at δ H (4.90 s, 5.07 s) were observed. The 13 C-NMR and DEPT spectra showed 15 carbon signals including two methyls, six methylenes (an oxygenated and one olefinic carbons), four methines, and three quaternary carbons (one carbonyl, an oxygenated, and one olefinic carbons). The 1 H-1 H COSY, HSQC, and HMBC spectra disclosed that compound 3 was an 8, 9 secoguaiane-type sesquiterpenoid (Figure 2). The 1 H-1 H COSY correlations of H-7/H-11, H-11/H-12 and the HMBC correlation of H-12 to δ C 210.8 (C-8) attested to the presence of a five-numbered lactone ring. The HMBC correlations of H-15 and H-9 to δ C 85.8 (C-1) determined that the isopropenyl moiety was connected to C-1. Combined with the 1 H-1 H COSY correlations of H-2 through H-3, H-4, H-5, H-6 to H-7, HMBC correlations of Me-14 to C-3, C-4, and C-5, and HMBC correlations of H-13 to C-11 and C-12, the planar structure of compound 3 was deduced as shown in Figure 1. The relative configuration was inferred from the NOESY spectrum in reference to compound 1. If H-5 and H-7 were both assigned to be β-oriented, H-11 and Me-14 were situated at the opposite side of the molecule with α-orientation by NOESY correlations of H-13/H-7 and H-4/H-5. The 1-OH group was inferred to be α-oriented according to the NOESY signal between H-4 and Me-15. Therefore, the structure of compound 3 was tentatively established to be 1α-hydroxy-4βH-5βH-7βH-11αH-8,9-secoguaia-9(10)-en-8,12-olide (Figure 1).
Compound 4 was isolated as a colorless oil. Its molecular formula was assigned as C 12 H 20 O 2 on the basis of the [M + Na] + ion peak at m/z 219.1387 (calcd for C 12 H 20 O 2 Na, 219.1361) in the positive HRESIMS. The IR spectrum of compound 4 showed absorptions for hydroxyl (3447 cm´1) and carbonyl (1698 cm´1) groups. The 1 H-NMR data (Table 1) displayed two methyl groups at δ H (0.98 d, J = 7.2 Hz, H 3 -11 and 1.08 d, J = 6.8 Hz, CH 3 -12). The 13 C-NMR and DEPT spectra ( Table 2) exhibited 12 carbon signals including two methyls, five methylenes, three methines, and two quaternary carbons (one carbonyl and an oxygenated carbons). The above information indicated that compound 4 was identified as a natural sesquiterpenoid derivative. The interpretation of 1D and 2D NMR data disclosed that compound 4 originated from guaianesesquiterpenoid by degradation of an isopropyl fragment (C-11/12/13). The positions of the hydroxyl group (δ C 86.9, qC) and the carbonyl group (δ C 213.9) were deduced via HMBC correlations ( Figure 2). The key NOESY correlations of H-4/H-5, H-10/H-5 were observed, which disclosed that Me-11 and Me-12 were α-oriented while H-5 was β-oriented. The relative configuration of OH-1 was determined by the NOESY correlation of between OH-1 (δ H 4.13, in DMSO-d 6 ) and Me-12 ( Figure 3). Thus, the structure of compound 4 was elucidated to be 1α-hydroxy-4α,10α-dimethyl-5βH-octahydro-azulen-8-one (Figure 1).

Evalution of Anti-Inflammatory Activity
Compounds 1-4 were tested for their inhibitory effects against LPS induced NO production in RAW 264.7 cells. Aminoguanidine hydrochloride acted as the positive control. The results showed that compound 2 exhibited significant inhibitory activity with an IC 50 value of 8.1 µM, compared to the positive control Aminoguanidine hydrochloride with an IC 50 value of 11.6 µM, while compounds 1, 3, and 4 did not exhibit obvious inhibitory activities with IC 50 values higher than 100 µM. In addition, none of the compounds showed apparent cytotoxicities (see Supplementary Materials).

Anti-Inflammatory Assay
RAW 264.7 cells were maintained in Dulbecco's Modified Eagles Medium containing 10% fetal bovine serum in a humid atmosphere of 5% CO 2 at 37˝C and plated into 96-well plates at a density of approximately 1ˆ10 5 cells per well. The cells were pretreated with different concentrations of the detected compounds for 2 h and then incubated for 12 h with or without LPS (2 µg/mL). The nitrite content in the culture supernatant was measured by the Griess reaction. The IC 50 value was calculated by GraphPad Prism software with the inhibition rates of different concentrations. Moreover, the formula of inhibition rate is (OD Model´O D Compound )/(OD Model´O D Control )ˆ100%. Cell viability was measured by Cell Counting Kit-8 (CCK-8, Dojindo, Kumamoto, Japan) method.

Conclusions
A systematic chemical search was performed and resulted in the separations of three new sesquiterpenoids and one sesquiterpenoid derivative, together with three known sesquiterpenoids from Chinese eaglewood. The structures of the new compounds were identified based on detailed spectroscopic analysis and published analogues. In addition, the stereo configuration of compound 1 was confirmed by Cu Kα X-ray crystallographic experiment. Besides, the new compound 2 showed significant inhibitory activity against LPS induced NO production in RAW 264.7 cells. Author Contributions: Huan Zhao conceived and designed the main ideas of this paper, carried out the experiments, analyzed the experimental results, and wrote the paper. Qinghua Peng participated in the experiments. Zhuzhen Han and Li Yang revised this paper and guided the writing of this paper. Zhuzhen Han and Li Yang put forward the main ideas of this paper and guided the experiments. Zhengtao Wang put forward the main ideas of this paper. The authors read and approved the final manuscript.

Conflicts of Interest:
The authors declare no conflict of interest.

Abbreviations
The following abbreviations are used in this manuscript: