New Cembranoids and a Biscembranoid Peroxide from the Soft Coral Sarcophyton cherbonnieri

Six new cembranoids, cherbonolides A−E (1–5) and bischerbolide peroxide (6), along with one known cembranoid, isosarcophine (7), were isolated from the Formosan soft coral Sarcophyton cherbonnieri. The structures of these compounds were elucidated by detailed spectroscopic analysis and chemical methods. Compound 6 was discovered to be the first example of a molecular skeleton formed from two cembranoids connected by a peroxide group. Compounds 1–7 were shown to have the ability of inhibiting the production of superoxide anions and elastase release in N-formyl-methionyl-leucyl-phenylalanine/cytochalasin B (fMLF/CB)-induced human neutrophils.


Results and Discussion
The soft coral S. cherbonnieri (1.2 kg, wet weight) was collected using SCUBA diving from Jihui Port of Taitung, Taiwan, in March 2013, and stored in a freezer before extraction. The freeze-dried organisms (207 g) were sliced into small pieces, followed by exhaustive extraction with ethyl acetate (EtOAc). The EtOAc extract was dried with anhydrous sodium sulfate (Na2SO4). After removal of EtOAc under reduced pressure, the residue yielded was separated by silica gel column chromatography and the resolved fractions were further purified by reverse-phase C18 highperformance liquid chromatography (HPLC) to afford compounds 1-7 (Figure 1), the structures of which were elucidated on the basis of spectroscopic analyses (Supplementary Materials, Figures S1-S46).
Cherbonolide A (1) was isolated as a colorless oil. The molecular formula C20H28O4 of 1 was determined by the high-resolution electrospray ionization mass spectrometry (HRESIMS) (m/z calcd 355.1880; found 355.1879, [M + Na] + ), which required seven degrees of unsaturation. The IR spectrum of 1 showed the presence of a hydroxyl group (νmax 3457 cm −1 ) and a lactonic carbonyl group (νmax 1746 cm −1 ). The presence of 20 carbons in the structure of 1, including four methyls, five sp 3 methylenes, three sp 3 oxygenated methines, two sp 2 methines, one sp 3 and five sp 2 nonprotoned carbon

Results and Discussion
The soft coral S. cherbonnieri (1.2 kg, wet weight) was collected using SCUBA diving from Jihui Port of Taitung, Taiwan, in March 2013, and stored in a freezer before extraction. The freeze-dried organisms (207 g) were sliced into small pieces, followed by exhaustive extraction with ethyl acetate (EtOAc). The EtOAc extract was dried with anhydrous sodium sulfate (Na 2 SO 4 ). After removal of EtOAc under reduced pressure, the residue yielded was separated by silica gel column chromatography and the resolved fractions were further purified by reverse-phase C 18 high-performance liquid chromatography (HPLC) to afford compounds 1-7 (Figure 1), the structures of which were elucidated on the basis of spectroscopic analyses (Supplementary Materials, Figures S1-S46).
(m/z calcd 371.1830; found 371.1829, [M + Na] + ), having one more oxygen than 1. Moreover, both 1 and 2 had almost identical 1 H and 13 C NMR data (Table 1), except for those of C-6. The allylic hydroxy group of 1 at C-6 was substituted by a hydroperoxyl in 2, with the characteristic signal of a broad singlet in the downfield region, δH 7.99 [26,33,34]. Obvious downfield shifts of C-6 (δC 65.2 in 1, 78.3 in 2) and H-6 (δH 4.70 in 1, 4.97 in 2) were also observed, indicating that 2 possesses the hydroperoxy group at C-6. Furthermore, reduction of 2 by reaction with triphenylphosphine afforded 1. On the basis of the above analyses, the planar structure and the (2S,6R,11R,12R)-configuration of 2 were determined.  Cherbonolide C (3) should have the same molecular formula as 1, according to HRESIMS data.
Also, the 1 H-1 H COSY and HMBC correlations ( Figure 2) of 3 are similar to those of 1, suggesting that these compounds possess almost the same molecular skeleton. Analysis of NOE correlations ( Figure  5) showed that the relative configurations at C-2, C-11 and C-12 in 1 and 3 are the same. Assuming the β-orientation of H-2, as H3-18 showed NOE interaction with H-2 but not with H-3, the E geometry was assigned to the trisubstituted C-3/C-4 double bond. One of the methylene protons at C-  The molecular formula of cherbonolide B (2) was determined to be C20H28O5 by the HRESIMS (m/z calcd 371.1830; found 371.1829, [M + Na] + ), having one more oxygen than 1. Moreover, both 1 and 2 had almost identical 1 H and 13 C NMR data (Table 1), except for those of C-6. The allylic hydroxy group of 1 at C-6 was substituted by a hydroperoxyl in 2, with the characteristic signal of a broad singlet in the downfield region, δH 7.99 [26,33,34]. Obvious downfield shifts of C-6 (δC 65.2 in 1, 78.3 in 2) and H-6 (δH 4.70 in 1, 4.97 in 2) were also observed, indicating that 2 possesses the hydroperoxy group at C-6. Furthermore, reduction of 2 by reaction with triphenylphosphine afforded 1. On the basis of the above analyses, the planar structure and the (2S,6R,11R,12R)-configuration of 2 were determined.   The molecular formula of cherbonolide B (2) was determined to be C 20 H 28 O 5 by the HRESIMS (m/z calcd 371.1830; found 371.1829, [M + Na] + ), having one more oxygen than 1. Moreover, both 1 and 2 had almost identical 1 H and 13 C NMR data (Table 1), except for those of C-6. The allylic hydroxy group of 1 at C-6 was substituted by a hydroperoxyl in 2, with the characteristic signal of a broad singlet in the downfield region, δ H 7.99 [26,33,34]. Obvious downfield shifts of C-6 (δ C 65.2 in 1, 78.3 in 2) and H-6 (δ H 4.70 in 1, 4.97 in 2) were also observed, indicating that 2 possesses the hydroperoxy group at C-6. Furthermore, reduction of 2 by reaction with triphenylphosphine afforded 1. On the basis of the above analyses, the planar structure and the (2S,6R,11R,12R)-configuration of 2 were determined.  Cherbonolide C (3) should have the same molecular formula as 1, according to HRESIMS data. Also, the 1 H-1 H COSY and HMBC correlations ( Figure 2) of 3 are similar to those of 1, suggesting that these compounds possess almost the same molecular skeleton. Analysis of NOE correlations ( Figure 5) showed that the relative configurations at C-2, C-11 and C-12 in 1 and 3 are the same. Assuming the β-orientation of H-2, as H 3 -18 showed NOE interaction with H-2 but not with H-3, the E geometry was assigned to the trisubstituted C-3/C-4 double bond. One of the methylene protons at C-5 (δ H 2.42, dd, J = 12.0, 3.2 Hz) displayed NOE interaction with H-3, but not with H 3 -18, and was hence determined as H-5α. Further, H-6 (δ H 3.84, dd, J = 9.2, 9.2 Hz) showed NOE correlations with H-5α and H-9α, but not with H-9β and H 3 -19. These observations, together with NOE correlations of H-9β/H 3 -19, H 3 -19/H-7 and H-7/H 3 -18, enabled deduction of the α-orientation of H-6 and led to the assignment of a 6S* configuration and a Z geometry of the trisubstituted C-7/C-8 double bond in 3. The olefinic methyl group attaching at C-8 showed carbon signal at δ C 22.2 ppm further confirmed the Z geometry of C-7/C-8 double bond [30]. The absolute configuration of 3 at C-6 was also verified by using the modified Mosher's method. Determination of the ∆δ values (δ S − δ R , shown in Figure 4) for protons neighboring C-6 further confirmed the S configuration at C-6 in 3 ( Figure 4). The absolute configuration of 3 was thus assigned as 2S,6S,11R and 12R. Thus, cherbonolide C (3) is the 7Z isomer of cherbonolide A (1).
Cherbonolide D (4) was found to be an isomer of 3 according to HRESIMS. Both compounds have almost the same 1 H-1 H COSY and HMBC correlations, indicating they have the same molecular skeleton. NMR data of 3 and 4 are nearly the same (Table 1), except for those of CH-6, suggesting that 4 could be the C-6 epimer of 3. The (2S,6R,11R,12R)-configuration and the E and Z geometries of C-3/C-4 and C-7/C-8 double bonds of 4, respectively, were also established by analysis of NOE correlations to be as those of 3 ( Figure 5).
Cherbonolide E (5) was determined to have a molecular formula C 20 H 28 O 5 from its HRESIMS data (m/z calcd 371.1830; found 371.1829, [M + Na] + ), with one more oxygen than in 4. Compounds 4 and 5 displayed almost identical 1 H and 13 C NMR data (Table 2), except for those of CH-6. It was found that the hydroxy substituent of 4 at C-6 was replaced by a hydroperoxy group in 5, with the characteristic signal of a broad singlet at δ H 7.25 [26,33,34]. The obvious downfield shifts of C-6 (δ C 64.8 in 4, 78.9 in 5) and H-6 (δ H 4.21 in 4, 4.58 in 5) also confirmed the substitution of a hydroperoxy group at C-6 of 5. Furthermore, reduction of 5 with triphenylphosphine could afford 4. Therefore, the structure of 5, with the (2S,6R,11R,12R)-configuration, was determined.
Mar. Drugs 2018, 16, x 6 of 16 the Z geometry of C-7/C-8 double bond [30]. The absolute configuration of 3 at C-6 was also verified by using the modified Mosher's method. Determination of the Δδ values (δS − δR, shown in Figure 4) for protons neighboring C-6 further confirmed the S configuration at C-6 in 3 ( Figure 4). The absolute configuration of 3 was thus assigned as 2S,6S,11R and 12R. Thus, cherbonolide C (3) is the 7Z isomer of cherbonolide A (1). Cherbonolide D (4) was found to be an isomer of 3 according to HRESIMS. Both compounds have almost the same 1 H-1 H COSY and HMBC correlations, indicating they have the same molecular skeleton. NMR data of 3 and 4 are nearly the same (Table 1), except for those of CH-6, suggesting that 4 could be the C-6 epimer of 3. The (2S,6R,11R,12R)-configuration and the E and Z geometries of C-3/C-4 and C-7/C-8 double bonds of 4, respectively, were also established by analysis of NOE correlations to be as those of 3 ( Figure 5). Cherbonolide E (5) was determined to have a molecular formula C20H28O5 from its HRESIMS data (m/z calcd 371.1830; found 371.1829, [M + Na] + ), with one more oxygen than in 4. Compounds 4 and 5 displayed almost identical 1 H and 13 C NMR data (Table 2), except for those of CH-6. It was found that the hydroxy substituent of 4 at C-6 was replaced by a hydroperoxy group in 5, with the characteristic signal of a broad singlet at δH 7.25 [26,33,34]. The obvious downfield shifts of C-6 (δC 64.8 in 4, 78.9 in 5) and H-6 (δH 4.21 in 4, 4.58 in 5) also confirmed the substitution of a hydroperoxy group at C-6 of 5. Furthermore, reduction of 5 with triphenylphosphine could afford 4. Therefore, the structure of 5, with the (2S,6R,11R,12R)-configuration, was determined. Bischerbolide peroxide (6) Table 2). The DEPT spectra of 6 showed the presence of eight methyls, twelve sp 3 methylenes, six sp 3 oxygenated methines, four sp 2 methines, two sp 3 and eight sp 2 nonprotoned carbons (including two ester carbonyls). As the 13 C NMR spectrum of 6 was constituted by twenty sets of signals with each set containing two peaks of very similar chemical shifts, 6 was thus identified as a compound formed from the connection of two quite similar diterpenoid subunits. The entire planar structure was established by examination of 1 H and 13 C NMR data and 1 H-1 H COSY and HMBC correlations ( Figure 6). Two methines resonating at δ C 114.3 and δ C 114.4 were considered to be the positions at which the two cembranoidal units were connected by insertion of a peroxyl group. Based on the above analyses, the molecular skeleton of 6 was elucidated as the biscembranoid formed by the connection of two molecules of isosarcophytoxide [36] via a peroxyl group at C-16 and C-16 . The fragmentation pattern of ESIMS (Figure 7) could further prove the dimeric nature of 6 and the peroxyl linkage at C-16/C-16 . One ion peak displayed at m/z 339 can be explained by the cleavage of O-O bond and the following elimination of H-16 from a monocembranoidal unit in 6 to form a sodiated cembranoid lactone molecular ion A (pathway a). The other ion peaks can be interpreted by the cleavage of the single bond between C-16 and peroxyl oxygen to afford ion B (m/z 301), and a peroxycembranoidal radical which could further abstract an hydrogen atom and form the sodium adduct C (m/z 357) (pathway b). Moreover, compound 6 was found to be the first example of a biscembranoid with a molecular skeleton formed by two cembranoid units connected by a peroxyl group.   As the 13 C NMR spectrum of 6 was constituted by twenty sets of signals with each set containing two peaks of very similar chemical shifts, 6 was thus identified as a compound formed from the connection of two quite similar diterpenoid subunits. The entire planar structure was established by examination of 1 H and 13 C NMR data and 1 H-1 H COSY and HMBC correlations ( Figure 6). Two methines resonating at δC 114.3 and δC 114.4 were considered to be the positions at which the two cembranoidal units were connected by insertion of a peroxyl group. Based on the above analyses, the molecular skeleton of 6 was elucidated as the biscembranoid formed by the connection of two lactone molecular ion A (pathway a). The other ion peaks can be interpreted by the cleavage of the single bond between C-16 and peroxyl oxygen to afford ion B (m/z 301), and a peroxycembranoidal radical which could further abstract an hydrogen atom and form the sodium adduct C (m/z 357) (pathway b). Moreover, compound 6 was found to be the first example of a biscembranoid with a molecular skeleton formed by two cembranoid units connected by a peroxyl group. The relative configuration of 6 was determined from a literature survey [36,37] and NOE correlations ( Figure 8). The 13 C NMR spectrum of 6 displayed 40 signals of two sets signals with nearly identical chemical shifts, representing the very similar stereochemical environments of the two structural units. In addition, compound 6 was found to have nearly identical chemical shifts for H-11 (11′), H3-18 (18′), H3-20 (20′) and C-20 (20′) to those of (2S,11R,12R)-isosarcophytoxide (8), and were in turn found to exhibit distinguishable differences to the corresponding chemical shifts of (2R,11R,12R)-isosarcophytoxide (9) (Table 3 and Figure 9). Thus, 6 possessed the cembranoidal structural unit derived from 8, as also proven by observed NOE correlations (Figure 8). Different proton values were observed for H-2 (δH 5.28) and H-2′ (δH 5.50), indicating that H-2′ was on the same planar face as the peroxide group and was deshielded, and H-2 was on the same planar face as H-16 and was shielded. As compounds 1−7 were isolated from the same organism in this study, they are likely to possess the same absolute S,R,R-configurations at the chiral centers C-2, C-11 and C-12, respectively, as those of 1 and 3. A previous report also showed that different absolute configurations at C-2 of the related diasteromeric dihydrofuran ring-containing cembranoids could significantly influence the sign of the specific optical rotation [36,38]. For cembranoids with 2S configuration a significant positive and for those with 2R configuration a negative optical rotation were found. The  The relative configuration of 6 was determined from a literature survey [36,37] and NOE correlations (Figure 8). The 13 C NMR spectrum of 6 displayed 40 signals of two sets signals with nearly identical chemical shifts, representing the very similar stereochemical environments of the two structural units. In addition, compound 6 was found to have nearly identical chemical shifts for H-11 (11 ), H 3 -18 (18 ), H 3 -20 (20 ) and C-20 (20 ) to those of (2S,11R,12R)-isosarcophytoxide (8), and were in turn found to exhibit distinguishable differences to the corresponding chemical shifts of (2R,11R,12R)-isosarcophytoxide (9) (Table 3 and Figure 9). Thus, 6 possessed the cembranoidal structural unit derived from 8, as also proven by observed NOE correlations (Figure 8). Different proton values were observed for H-2 (δ H 5.28) and H-2 (δ H 5.50), indicating that H-2 was on the same planar face as the peroxide group and was deshielded, and H-2 was on the same planar face as H-16 and was shielded. As compounds 1−7 were isolated from the same organism in this study, they are likely to possess the same absolute S,R,R-configurations at the chiral centers C-2, C-11 and C-12, respectively, as those of 1 and 3. A previous report also showed that different absolute configurations at C-2 of the related diasteromeric dihydrofuran ring-containing cembranoids could significantly influence the sign of the specific optical rotation [36,38]. For cembranoids with 2S configuration a significant positive and for those with 2R configuration a negative optical rotation were found. The [α] 25 D of 6 was +41; thus, the absolute configuration of 6 was deduced to be 2S,11R,12R,16R, 2 S,11 R,12 R,16 S.   (8) and (2R,11R,12R)-isosarcophytoxide (9). The plausible biosynthesis of 6 might arise from the proton abstraction at C-16 of 8 by hydrogen peroxide radical HOO  to form a radical intermediate 10, which could react with O2 from one plane side of radical center C-16 to afford cembranoidal peroxide radical 11. Further reaction of 11 with 10 from another side could lead to the formation of 6 (Scheme 1). However, the possibility that 6 might be generated by autooxidation of 8 could not be neglected.   Figure 8. Selected NOESY correlations for 6. Table 3. Selected 1 H and 13 C NMR data comparison with 6, (2S,11R,12R)-isosarcophytoxide (8) and (2R,11R,12R)-isosarcophytoxide (9). H and 13 C data were cited from ref. [36,37].
The plausible biosynthesis of 6 might arise from the proton abstraction at C-16 of 8 by hydrogen peroxide radical HOO  to form a radical intermediate 10, which could react with O2 from one plane side of radical center C-16 to afford cembranoidal peroxide radical 11. Further reaction of 11 with 10 from another side could lead to the formation of 6 (Scheme 1). However, the possibility that 6 might be generated by autooxidation of 8 could not be neglected.  The plausible biosynthesis of 6 might arise from the proton abstraction at C-16 of 8 by hydrogen peroxide radical HOO• to form a radical intermediate 10, which could react with O 2 from one plane side of radical center C-16 to afford cembranoidal peroxide radical 11. Further reaction of 11 with 10 from another side could lead to the formation of 6 (Scheme 1). However, the possibility that 6 might be generated by autooxidation of 8 could not be neglected. It is known that the proteolytic enzymes and toxic reactive oxygen species produced by stimulated neutrophils might play a critical role in the pathogenesis of many inflammatory diseases [39,40]. By measuring the capability to inhibit N-formyl-methionyl-leucyl-phenylalanine/ cytochalasin B (fMLF/CB)-induced superoxide anion generation and elastase release in human neutrophils, the in vitro anti-inflammatory effects for metabolites 1-7 were evaluated [41,42]. According to the results (shown in Table 4), compound 6 had a significant inhibitory effect (64.6 ± 0.8%), with an IC50 value of 26.2 ± 1.0 μM, on the generation of superoxide anions, and compounds 1 and 3 had moderate inhibitory effects (32.1 ± 4.3% and 44.5 ± 4.6%, respectively) at 30 μM. Compounds 1, 3 and 6 revealed moderate inhibitory effects (37.6 ± 5.0%, 35.6 ± 6.2% and 42.4 ± 5.1%, respectively) on elastase release at the same concentration. These results, obtained after stimualting the neutrophils with fMLF/CB, may suggest that 1, 3 and 6 have potential merits against inflammatory disorders.
In summary, examination of the chemical constituents of the soft coral Sarcophyton cherbonnieri led to the discovery of six new compounds 1-6, along with one known compound 7. Although a number of natural compounds possessing a peroxyl group, such as artemisinin [43], neovibsanin C [44], cardamom peroxide [45], plakortin [46] and chondrillin [47], have been discovered, compound 6 was discovered to be the first compound with a molecular skeleton consisting of two cembranoidal units connected by a peroxide group. Similar to the results of previous studies indicating that natural peroxides could possess promising biological activity [48], compound 6 was found to possess antiinflammatory activity by exhibiting stronger ability on inhibition on the generation of superoxide anions and release of elastase in fMLF/CB-induced human neutrophils.

General Procedures
The values of optical rotation of the metabolites were determined with a JASCO P-1020 polarimeter (JASCO Corporation, Tokyo, Japan). Infrared absorptions were recorded using a JASCO Scheme 1. Proposed biosynthetic pathway for 6.
It is known that the proteolytic enzymes and toxic reactive oxygen species produced by stimulated neutrophils might play a critical role in the pathogenesis of many inflammatory diseases [39,40]. By measuring the capability to inhibit N-formyl-methionyl-leucyl-phenylalanine/cytochalasin B (fMLF/CB)-induced superoxide anion generation and elastase release in human neutrophils, the in vitro anti-inflammatory effects for metabolites 1-7 were evaluated [41,42]. According to the results (shown in Table 4), compound 6 had a significant inhibitory effect (64.6 ± 0.8%), with an IC 50 value of 26.2 ± 1.0 µM, on the generation of superoxide anions, and compounds 1 and 3 had moderate inhibitory effects (32.1 ± 4.3% and 44.5 ± 4.6%, respectively) at 30 µM. Compounds 1, 3 and 6 revealed moderate inhibitory effects (37.6 ± 5.0%, 35.6 ± 6.2% and 42.4 ± 5.1%, respectively) on elastase release at the same concentration. These results, obtained after stimualting the neutrophils with fMLF/CB, may suggest that 1, 3 and 6 have potential merits against inflammatory disorders. In summary, examination of the chemical constituents of the soft coral Sarcophyton cherbonnieri led to the discovery of six new compounds 1-6, along with one known compound 7. Although a number of natural compounds possessing a peroxyl group, such as artemisinin [43], neovibsanin C [44], cardamom peroxide [45], plakortin [46] and chondrillin [47], have been discovered, compound 6 was discovered to be the first compound with a molecular skeleton consisting of two cembranoidal units connected by a peroxide group. Similar to the results of previous studies indicating that natural peroxides could possess promising biological activity [48], compound 6 was found to possess anti-inflammatory activity by exhibiting stronger ability on inhibition on the generation of superoxide anions and release of elastase in fMLF/CB-induced human neutrophils.

Animal Material
The soft coral S. cherbonnieri was collected by hand using scuba diving from Jihui Fish Port, Taiwan, in March 2013, at a depth of 10-15 m. Organisms of the marine animal were stored in a freezer until extraction.

Reduction of Cherbonolides B and E (2 and 5)
In diethyl ether (5.0 mL), compound 2 (3.2 mg) was added followed by addition of excess amount of triphenylphosphine (2.9 mg) and the mixture was stirred at room temperature for 4 h. The solvent of the solution was evaporated under reduced pressure to afford a residue, which was purified by silica gel column chromatography using n-hexane-acetone (3:1) as an eluent to yield 1 (2.9 mg, 95%). Similarly, 5 (2.1 mg) was converted to 4 (1.7 mg) in 85% yield.

Preparation of (S)-and (R)-MTPA Esters of 1 and 3
Compound 1 (1.3 mg) was dissolved in pyridine 100 µL and added (R)-(−)-α-methoxy-α-(trifluoromethyl) phenylacetyl chloride (MTPA chloride) 10 µL. The mixture was permitted to stand at room temperature overnight and the reaction was found to complete by monitoring with normal-phase TLC plate. The solution was dried completely under the vacuum of an oil pump and the residue was purified by a short silica gel column using acetone to n-hexane (1:3) to yield the (S)-MTPA ester 1a (0.9 mg, 62.9%). The same procedure was applied to obtain the (R)-MTPA ester 1b (1.0 mg, 69.9%) from the reaction of (S)-(+)-α-methoxy-α-(trifluoromethyl) phenylacetyl chloride with 1 in pyridine.

Conclusions
Six new cembranoids, cherbonolides A−E (1-5) and a cembrane dimer (bischerbolide peroxide, 6), along with isosarcophine (7) were isolated from the Formosan soft coral Sarcophyton cherbonnieri. Bischerbolide peroxide (6) was discovered as the first example of cembranoid dimers possessing a peroxide group as a linking group. Compounds 1, 3 and 6 showed an anti-inflammatory activity through their inhibitory effects on the generation of superoxide anion in fMLF/CB-induced human neutrophils. Moreover, peroxide 6 was also shown to exhibit stronger activity in inhibiting the elastase release which supported its anti-inflammatory activity.