**1. Introduction**

Series of cembranoidal secondary metabolites from soft corals have been shown to exhibit attractive biological activities including cytotoxicity [1–14] and anti-inflammatory ability [6,7,9,11, 13–18]. From previous investigations of exploring bioactive natural products from soft corals, many cembranoids were discovered from organisms of the genera *Sarcophyton*, [1–8,16], *Sinularia* [9–12,17,18], and *Lobophyton* [13–15]. In some cases, two cembranoid units could be linked to produce biscembranoids via various reactions [18–24], marking the high diversity and complexity in chemical structures of cembrane-related soft coral natural products.

Many studies have revealed that soft corals of the genus *Sarcophyton* are important sources of various types of natural products, some of them with notable bioactivies [25–28]. Our previous

chemical study on *Sarcophyton cherbonnieri* led to the isolation of six new cembranoids cherbonolides A–E and one biscembranoid bischerbolide peroxide, along with a known compound, isosarcophine [24]. In continuation of our effort on discovery of new and bioactive compounds from marine animals, we further explored the chemical constituents of *S. cherbonnieri*. This investigation again led to the discovery of new cembranoids, cherbonolides F–L (**1–7**). The structures of **1**–**7** (Figure 1) were determined by spectroscopic analysis, including two-dimensional (2D) NMR experiments and a chemical reaction. Compounds **2** and **4** were elucidated as cembranoids possessing an allylic peroxy group. Cembranoids of isosarcophine-type have been reported frequently [24,27–30].

The screening of the in vitro anti-inflammatory activities through the inhibition of superoxide anion generation and elastase release in *<sup>N</sup>*-formyl-methionyl-leucyl-phenylalanine/cytochalasin B (fMLF/CB)-induced primary human neutrophils was also performed in order to unveil the anti-inflammatory ability of these compounds. We report herein the isolation, structure determination, and bioactivity of the new metabolites **1**–**7**.

**Figure 1.** New cembranoids isolated from *Sarcophyton cherbonnieri*.

### **2. Results and Discussion**

Solvent-free residue of the ethyl acetate extract of the soft coral *S. cherbonnieri* was separated and further purified by chromatographic methods to yield metabolites **1**–**7**. The structures were established by extensive analyses of MS and NMR spectra (Figures S1–S49, Supplementary Materials). 13C- and 1H-NMR data which were essential for structure determination of **1**–**7** are listed in Tables 1–3.

Cherbonolide F (**1**) was obtained as a colorless oil. The molecular formula of **1**, C20H28O4, was established by high-resolution electrospray ionization mass spectrometry (HR-ESI-MS) (*m*/*z* calculated 355.1880; found 355.1879, [M + Na]+), implying seven degrees of unsaturation. The IR spectrum of **1** revealed the absorptions of a hydroxy (<sup>ν</sup>max 3460 cm<sup>−</sup>1) and a lactonic carbonyl group (<sup>ν</sup>max 1748 cm<sup>−</sup>1). The 13C-NMR spectrum of **1** showed 20 signals which were assigned to four methyls, five *sp<sup>3</sup>* methylenes, two *sp<sup>3</sup>* oxygenated methines, three *sp<sup>2</sup>* methines, and two *sp<sup>3</sup>* and four *sp<sup>2</sup>* nonprotonated carbon atoms (Table 1) with the assistance of distortionless enhancement by polarization transfer (DEPT) spectra. Carbon signals resonating at δC 173.9 (C), 160.5 (C), 123.6 (C), 78.6 (CH), and 9.0 (CH3) and proton signals resonating at δH 4.95 (1H, dd, *J* = 10.0, 1.6 Hz) and δH 1.66 (3H, s) were attributed to signals of an <sup>α</sup>-methyl-<sup>α</sup>,β-unsaturated-γ-lactone ring by comparing the NMR data of the γ-lactone ring of the known compound isosarcophine (**7**). Signals at δC 61.2

(CH), 60.2 (C), and δH 2.54 (1H, dd, *J* = 6.0, 6.0 Hz) showed the appearance of a trisubstituted epoxide. One trisubstituted and one disubstituted double bond were identified by NMR signals resonating at δC 120.9 (CH), 142.4 (C) and δH 4.54 (1H, dd, *J* = 10.0, 0.8 Hz), and at δC 140.3 (CH), 124.5 (CH) and δH 5.32 (1H, d, *J* = 16.0 Hz) and 5.38 (1H, ddd, *J* = 16.0, 6.8, 6.8 Hz), respectively. 1H–1H correlation spectroscopy (COSY) correlations established four separate proton sequences, which were connected by heteronuclear multiple bond correlation (HMBC) correlations (Figure 2). Essential HMBC correlations from H-2 to C-1 and C-4, H2-14 to C-1 and C-2, H3-17 to C-1, C-15, and C-16, H3-18 to C-3, C-4, and C-5, H3-19 to C-7, C-8, and C-9, and H3-20 to C-11, C-12, and C-13 established the 14-membered ring carbon skeleton of **1**, which also indicated the presence of a hydroxyl at C-8.

Furthermore, analysis of nuclear Overhauser effect (NOE) correlations was applied to establish the relative configuration of **1**, as shown in Figure 3. It was revealed that H-2 showed NOE correlation with H3-18, but not with H-3; therefore, assuming the β-orientation of H-2, H3-18 should be located on the β face. Moreover, H3-18 exhibited NOE correlation with H-7, but not with H-6, revealing the β-orientation of H-7 and the α-orientation of H-6. Both H-6 and H-7 exhibited NOE interactions with H3-19, thus established the β-orientation of H3-19 as shown in Figure 3. One methylene proton at C-13 exhibited NOE correlation with H-2 and was characterized as H-13β (δH 0.99, m), while the other proton was assigned as H-13α (δH 1.49, m). NOE correlations of H-13β with H-11 and H-13α with H3-20 reflected the β-orientation of H-11 and the α-orientation of H3-20. The *E* geometries of the trisubstituted C-3/C-4 and C-6/C-7 double bonds were also assigned from the NOE correlations of H3-18 (δH 1.35, s) with H-2, but not with H-3, as well as the large coupling constant *J* = 16.0 Hz between H-6 and H-7, and the observed more shielded signal of C-18 (δC 16.7). According to the above observations, the relative configuration of this compound was established. As **1** was isolated together with the previous reported compounds isosarcophine and cherbonolides A−E [24] from the same organism, it should possess the same (2*S*,8*S*,11*R*,12*R*)-configuration from the shared biosynthetic pathway.

**Figure 2.** The selected COSY and HMBC correlations of **1**–**4**.


**Table 1.** 13C-NMR spectroscopic data of compounds **1**–**7.**

a Spectra recorded in C6D6 at 100 MHz at 25 ◦C. b Attached protons were deduced by distortionless enhancement by polarization transfer (DEPT) experiments. c Spectra recorded in C6D6 at 125 MHz. d Spectra recorded in CDCl3 at 100 MHz.

**Table 2.** 1H-NMR spectral data for compounds **1**−**4**.


a Spectra recorded in C6D6 at 400 MHz at 25 ◦C. b Spectra recorded in C6D6 at 500 MHz at 25 ◦C. c Coupling constants (*J* values) in Hz are shown in parentheses.


**Table 3.** 1H-NMR spectral data for compounds **5**−**7**.

a Spectra recorded in C6D6 at 400 MHz at 25 ◦C. b Spectra recorded in CDCl3 at 400 MHz at 25 ◦C. c Coupling constants (*J* values) in Hz are shown in parentheses.

The molecular formula of cherbonolide G (**2**) was found to be C20H28O5 by analysis of HR-ESI-MS (*m*/*z* calculated 371.1829; found 371.1830, [M + Na]+), revealing that **2** possesses an additional oxygen atom to that of **1**. Moreover, both **1** and **2** showed the very similar 1H- and 13C-NMR data (Table 1), except that the chemical shift of C-8 was shifted from δC 71.7 of **1** to δC 83.7 of **2**. The very similar COSY, HMBC (Figure 2), and NOE (Figure 3) correlations of **1** and **2** also revealed the very close structures for both compounds. However, the hydroxy group of **1** at C-8 was replaced by a hydroperoxy group in **2**, with a broad singlet appearing at δH 6.72 and the downfield shift of C-8. Accordingly, the molecular skeleton and the (2*S*,8*S*,11*R*,12*R*)-configuration of **2** were determined.

Cherbonolide H (**3**) has the same molecular formula as that of **1**, as determined by HR-ESI-MS experiment. Moreover, most of the 1H–1H COSY and HMBC correlations (Figure 2) of **3** were similar to those of isosarcophine except for the presence of a hydroxyl at C-9 leading to the shift of CH-9 to lower field (δC 76.2; δH 3.68), and the shift of C-6/C-7 double bond of **1** to C-7/C-8 double bond of **3**. Analysis of NOE correlations (Figure 3) showed that the β-oriented H-2 exhibited NOE interactions with both H3-18 and H-13β, but not with H-3, assigning the *E*-geometry of the trisubstituted C-3/C-4 double bond. These results, along with the found NOE correlations (Figure 3) of H-13α/ H3-20, H3-20/H-9, led to the assignment of the α-orientation of H-9.

Cherbonolide I (**4**) was found to contain one additional oxygen atom than **3**, according to HR-ESI-MS experiment. These two compounds also showed very similar 1H–1H COSY and HMBC correlations, revealing the identical molecular framework of both compounds. NMR data of **3** and **4** were similar (Table 1), except for those of CH-9, suggesting that **4** is possibly the C-9 hydroperoxy derivative of **3**. By analysis of NOE correlations (Figure 3), the *E* geometries of both C-3/C-4 and C-7/C-8 double bonds of **4** and the (2*S*,9*R*,11*R*,12*R*)-configuration were also established. Reduction of **4** by triphenylphosphine yielded **3**, further confirming the structure of **4**.

Cherbonolide J (**5**) was given as a colorless oil with a molecular formula C20H30O5 on the basis of HR-ESI-MS data (*m*/*z* calculated for C20H30O5Na 373.1986; found 373.1984), revealing six degrees of unsaturation. The IR absorptions at 3443 and 1748 cm<sup>−</sup><sup>1</sup> were due to hydroxy and ester carbonyl groups, respectively. The 13C- and 1H-NMR spectroscopic data (Tables 1 and 3) of **5** measured at C6D6 were very close to a known compound sarcophyolide E [29], and the 2D NMR (COSY, HSQC, and HMBC) correlation analysis revealed that both compounds had the same molecular framework (Figure 4). Detailed analysis of the NOE correlations (Figure 5) showed that both compounds possessed the same relative configuration. However, the [α]D25 values in CHCl3 (−6 for **5** and +4.4 for sarcophyolide E) were close but with different signs, suggesting that **5** is the enantiomer of this known compound. The absolute configurations of **5** and sarcophyolide E were deduced by comparison of the circular dichroism (CD) spectroscopic data. As shown in Figure 6, the negative Cotton effect at 247 nm and positive effect at 228 nm for **5** in comparison with the positive and negative Cotton effects at 252 and 226 nm for sarcophyolide E [29], respectively, confirmed that **5** is the newly found enantiomer of sarcophyolide E.

**Figure 4.** Selected COSY and HMBC correlations of **5**–**7**.

**Figure 5.** Selected NOE correlations for **5**–**7**.

**Figure 6.** the CD spectrum of **5** (1.2 × 10−<sup>4</sup> M, MeOH).

Cherbonolide K (**6**) is a colorless oil which was shown to have the molecular formula C20H28O4 by HR-ESI-MS experiment, appropriate for seven degrees of unsaturation. The infrared (IR) spectrum of **6** showed peaks of hydroxy and estercarbonyl groups at 3444 and 1763 cm<sup>−</sup>1, respectively. 13C-NMR data (Table 1) with signals at δC 151.2 (C), 147.2 (C), 116.2 (CH), 72.7 (C), 123.6 (C), 169.5 (C), 9.1 (CH3), and 29.9 (CH3) and 1H NMR data (Table 3) with signals at δH 5.50 (s, 1H), 1.95 (s, 3H), and 1.41 (s, 3H) were attributed to the cembranoidal <sup>α</sup>-methyl-<sup>α</sup>,β-unsaturated-<sup>γ</sup> lactone ring with a conjugated 2,3-double bond that further connected with the methyl and hydroxyl substituted C-4. The above results were supported by HMBC correlations of **6** (Figure 4) from H-3 (δH 5.50) to C-1 (δC 151.2), C-2 (δC 147.2), and C-4 (δC 72.7), H3-17 (δH 1.95) to C-1, C-15 (δC 123.6), and C-16 (δC 169.5), and H3-18 (δH 1.41) to C-3 (δC 116.2) and C-4 (δC 72.6). The remainder of the structure from C-5 to C-14 was found to be identical to isosarcophine [24]. Thus, the planar structure of **6** was established. Furthermore, the NOE correlation analysis shown in Figure 5 revealed the α-orientations of 4-OH and 12-CH3, β-orientation of H-11, (*Z*)-2,3-double bond, and (*E*)-7,8-double bond. An isomer of **6**, cherbonolide L (**7**), was also subsequently isolated. The metabolite **7** had nearly the same NMR data as **6** except for CH2-5 and CH2-6. Thus, it can be assumed that **7** is the C-4 epimer of **6**. Analysis of the 2D NMR correlations of **7** (Figures 4 and 6) further supported this assumption.

For the screening of bioactivities, the anti-inflammation activities of **1**–**7** toward inhibition of *<sup>N</sup>*-formyl-methionyl-leucyl-phenylalanine/cytochalasin B (fMLF/CB)-induced generation of superoxide anion (O2•–) and release of elastase in primary human neutrophils were measured. The results (Table 4) showed that, although none of the isolates exhibited strong inhibitory activities in the assay, **2** and **3** were found to display notable ability to inhibit the elastase release (48.2% ± 6.2%) and superoxide anion generation (44.5% ± 4.6%) at 30 μM, respectively. In comparison with (+)-isosarcophine, cherbonolides A–E, and bischerbonolide peroide discovered previously from *S. cherbonnieri* [24], it was found that, although **2** and **3** exhibited weaker activities than bischerbonolide peroxide, they displayed comparable activities to those of cherbonolides A and C. In general, allylic oxidation at the 7,8-double bond of (+)-isosarcophine might be able to produce derivatives with stronger bioactivities.


**Table 4.** Inhibitory effects of metabolites **1**–**7** against elastase release and superoxide anion generation in *<sup>N</sup>*-formyl-methionyl-leucyl-phenylalanine/cytochalasin B (fMLF/CB)-induced primary human neutrophils. IC50, half maximal inhibitory concentration.

a Concentration necessary for 50% inhibition (IC50). b Percentage of inhibition (Inh %) at 30 μM. Results presented as mean ± S.D. The anti-inflammatory assays were performed with eight biological replicates. \* *p* < 0.05, \*\* *p* < 0.01, and \*\*\* *p* < 0.001 compared with the control.

### **3. Materials and Methods**

### *3.1. General Experimental Procedures*

Values of the specific optical rotation of the isolates were measured with a JASCO P-1020 polarimeter (JASCO Corporation, Tokyo, Japan). Infrared spectra were recorded using a JASCO FT/IR-4100 infrared spectrophotometer (JASCO Corporation, Tokyo, Japan). The CD spectrum was recorded on a Jasco J-815 circular dichroism (CD) spectropolarimeter (JASCO, Tokyo, Japan) in MeOH. 1H- and 13C-NMR spectra were acquired on a Varian 400MR FT-NMR (or Varian Unity INOVA500 FT-NMR) instrument (Varian Inc., Palo Alto, CA, USA) at 400 MHz (or 500 MHz) and 100 MHz (or 125 MHz), respectively, in CDCl3 or C6D6. LR-ESI-MS and HR-ESI-MS experiments were carried out using a Bruker APEX II (Bruker, Bremen, Germany) mass spectrometer. Silica gel (230–400 mesh) was used as the adsorbent for normal-phase column chromatography. Thin-layer chromatography (TLC) analyses were performed with precoated silica gel plates (Kieselgel 60 F-254, 0.2 mm, Merck, Darmstadt, Germany). Further purification of impure fractions or compounds was further achieved by high-performance liquid chromatography on a Hitachi L-7100 HPLC instrument (Hitachi Ltd., Tokyo, Japan) with a Merck Hibar Si-60 column (250 mm × 21 mm, 7 μm; Merck, Darmstadt, Germany) and on a Hitachi L-2455 HPLC apparatus (Hitachi, Tokyo, Japan) with a Supelco C18 column (250 mm × 21.2 mm, 5 μm; Supelco, Bellefonte, PA, USA).
