marine drugs Article
New Cembranoids and a Biscembranoid Peroxide from the Soft Coral Sarcophyton cherbonnieri Chia-Chi Peng 1 , Chiung-Yao Huang 1 , Atallah F. Ahmed 2,3 Chang-Feng Dai 7 and Jyh-Horng Sheu 1,8,9, * 1 2 3 4 5 6 7 8 9
*
ID
, Tsong-Long Hwang 4,5,6
ID
,
Department of Marine Biotechnology and Resources, National Sun Yat-sen University, Kaohsiung 804, Taiwan;
[email protected] (C.-C.P.);
[email protected] (C.-Y.H.) Department of Pharmacognosy, College of Pharmacy, King Saud University, Riyadh 11451, Saudi Arabia;
[email protected] Department of Pharmacognosy, Faculty of Pharmacy, Mansoura University, Mansoura 35516, Egypt Graduate Institute of Natural Products, College of Medicine, Chang Gung University, Taoyuan 333, Taiwan;
[email protected] Research Center for Industry of Human Ecology and Graduate Institute of Health Industry Technology, Chang Gung University of Science and Technology, Taoyuan 333, Taiwan Department of Anesthesiology, Chang Gung Memorial Hospital, Taoyuan 333, Taiwan Institute of Oceanography, National Taiwan University, Taipei 112, Taiwan;
[email protected] Graduate Institute of Natural Products, Kaohsiung Medical University, Kaohsiung 807, Taiwan Frontier Center for Ocean Science and Technology, National Sun Yat-sen University, Kaohsiung 804, Taiwan Correspondence:
[email protected]; Tel.: +886-7-525-2000 (ext. 5030); Fax: +886-7-525-5020
Received: 27 June 2018; Accepted: 31 July 2018; Published: 6 August 2018
Abstract: 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. Keywords: Sarcophyton cherbonnieri; cembranoid; biscembranoid peroxide; anti-inflammatory activity; elastase release inhibition
1. Introduction Many cembrane-based natural products have been shown to exhibit significant activities such as cytotoxicity [1–14] and anti-inflammatory activity [9,11,13–18]. From the experience of searching bioactive metabolites from soft corals, series of cembranoids have been unveiled from octocorals (Alcyonaceae) belonging to the genera Sarcophyton, [1–8,16], Sinularia [9–12,17,18] and Lobophyton [13–15]. Also, previous studies showed that two cembranoid units could form biscembranoid-type compounds by Diels-Alder reaction [19–21], radical dimerization [22,23], or connection with a sulfur atom [18], making the chemistry of cembranes more complex and interesting than monomeric form. Our current chemical investigation on Sarcophyton cherbonnieri led to the discovery of six new cembranoids, cherbonolides A−E (1–5) and bischerbolide peroxide (6), and one known compound, isosarcophine (7) [24]. The structures of 1–7 (Figure 1) were elucidated by extensive spectroscopic analysis, including detailed 2D nuclear magnetic resonance (NMR) experiments and chemical methods. Compounds 2, 5, and 6 were characterized as cembranoids bearing an allylic peroxyl group as those Mar. Drugs 2018, 16, 276; doi:10.3390/md16080276
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as those previously discovered cembranoidal peroxides [25–29]. Furthermore, compound is previously discovered marine marine cembranoidal peroxides [25–29]. Furthermore, compound 6 is 6the the first example a biscembranoid with twocembranoidal cembranoidalunits unitsinterconnected interconnectedby by aa peroxyl first example of aofbiscembranoid with two peroxyl group. group. The absolute configurations of 1 and 3 were further established using a modified Mosher’s reaction. The absolute configurations of 1 and 3 were further established using a modified Mosher’s reaction. Also, evaluation evaluationofofthe theinin vitro anti-inflammatory activities through the inhibition of superoxide Also, vitro anti-inflammatory activities through the inhibition of superoxide anion −• ) generation anion (O2−•) generation release in N-formyl-methionyl-leucyl-phenyl(O and elastase and releaseelastase in N-formyl-methionyl-leucyl-phenylalanine/cytochalasin B 2 alanine/cytochalasin human B (fMLF/CB)-induced human was carried out. (fMLF/CB)-induced neutrophils was carriedneutrophils out.
Figure 1. Cembranoid isolated from Sarcophyton cherbonnieri.
2. Results and Discussion
soft coral coralS.S.cherbonnieri cherbonnieri(1.2 (1.2kg, kg, wet weight) was collected using SCUBA diving The soft wet weight) was collected using SCUBA diving fromfrom JihuiJihui Port Port of Taitung, Taiwan, in March 2013, andinstored in abefore freezer before extraction. The freeze-dried of Taitung, Taiwan, in March 2013, and stored a freezer extraction. The freeze-dried organisms organisms (207 g) were small pieces,by followed by exhaustive ethyl(EtOAc). acetate (207 g) were sliced into sliced small into pieces, followed exhaustive extractionextraction with ethylwith acetate (EtOAc). The EtOAc was anhydrous dried with sodium anhydrous sodium sulfate (Naremoval 2SO4). After removal of The EtOAc extract wasextract dried with sulfate (Na2 SO of EtOAc under 4 ). After EtOAc under reduced pressure, thewas residue yielded wasgelseparated by silica gel and column reduced pressure, the residue yielded separated by silica column chromatography the chromatography and further the resolved were further purified by reverse-phase C18 highresolved fractions were purifiedfractions by reverse-phase C18 high-performance liquid chromatography performance liquid chromatography (HPLC) to structures afford compounds (Figure 1), theonstructures (HPLC) to afford compounds 1–7 (Figure 1), the of which1–7 were elucidated the basis of which were elucidated on the basis of spectroscopic analyses (Supplementary Materials, Figures S1– spectroscopic analyses (Supplementary Materials, Figures S1–S46). S46).Cherbonolide A (1) was isolated as a colorless oil. The molecular formula C20 H28 O4 of 1 was Cherbonolide A (1) was isolated as a colorless oil. The formula C20H28O(m/z 4 of 1calcd was determined by the high-resolution electrospray ionization massmolecular spectrometry (HRESIMS) + 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 −1 ) and a lactonic carbonyl group (ν of 1 showed the 355.1879, presence [M of a+hydroxyl group (νmax 3457 355.1880; found Na]+), which required sevencm degrees of unsaturation. The IR spectrum max −1 ). The presence of 20 carbons in the structure of 1, including 3 methylenes, −1 1746 cm four methyls, five sp of 1 showed the presence of a hydroxyl group (νmax 3457 cm ) and a lactonic carbonyl group (νmax 3 1 2 nonprotoned carbon atoms were3 three methines, twocarbons sp2 methines, sp3 and of five1,spincluding 1746 sp cm−oxygenated ). The presence of 20 in theone structure four methyls, five sp 3 2 3 methylenes, three sp oxygenated methines, two sp methines, one sp and five sp2 nonprotoned carbon
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atoms were the aid ofenhancement distortionless by polarization transfer (DEPT) assigned withassigned the aid ofwith distortionless byenhancement polarization transfer (DEPT) spectra. The NMR spectra. The NMRatpeaks resonating at δ(C), C 174.4 (C), 160.7 123.8 and 8.8 (CH 3), and peaks resonating δC 174.4 (C), 160.7 123.8 (C), 77.8(C), (CH) and(C), 8.877.8 (CH(CH) ), and δ 5.44 (1H, dd, 3 H δ H 5.44 (1H, dd, J = 10.0, 1.6 Hz) and δ H 1.86 (3H, s), are characteristic signals of an α-methyl-α,βJ = 10.0, 1.6 Hz) and δH 1.86 (3H, s), are characteristic signals of an α-methyl-α,β-unsaturated-γ-lactone unsaturated-γ-lactone comparison of the NMR data γ-lactone ring of known compound ring by comparison ofring the by NMR data of the γ-lactone ringofofthe known compound isosarcophine (7). isosarcophine (7). Signals δC 60.8 (C), (1H, 10.8, 2.8the Hz) showedof the Signals at δC 60.8 (C), 61.4at(CH) and δH61.4 2.42(CH) (1H,and dd,δJH =2.42 10.8, 2.8dd, Hz)J =showed presence a presence of a trisubstituted epoxide. Two trisubstituted double bonds could also be identified by trisubstituted epoxide. Two trisubstituted double bonds could also be identified by NMR signals at δCδ122.2 (CH), δC 141.6 (C) and δH 4.90 (1H, d, J = 10.0 Hz), and at δC 128.1 NMR signals resonating at resonating δC 122.2 (CH), C 141.6 (C) and δH 4.90 (1H, d, J = 10.0 Hz), and at δC 128.1 (CH), δC 1 the 1 (CH), (C) δC 139.8 and δH 5.20 (1H, d, J = 10.4 Hz), respectively. The correlations identified from 139.8 and δ(C) H 5.20 (1H, d, J = 10.4 Hz), respectively. The correlations identified from the H- H 1 1 H-1H correlation spectroscopy H-1H COSY) spectrum revealed four separate sequences, as correlation spectroscopy (1 H-1 H( COSY) spectrum revealed four separate proton proton sequences, as shown shown in2,Figure which were assembled by heteronuclear multiple bond (HMBC) correlation (HMBC) in Figure which 2, were assembled by heteronuclear multiple bond correlation correlations correlations (Figure 2). Key HMBC correlations of H-2 (δHdd, 5.44, dd, 10.0, Hz) C-1; 2-14 (Figure 2). Key HMBC correlations of H-2 (δH 5.44, 1H, J =1H, 10.0, 1.6J =Hz) to1.6 C-1; H2to -14 (δHH2.01, (δH 2.01, H m)/C-1; H31.86, -17 (δ 1.86, to C-1, C-16; 3-18 (δ to C-3, C-4 and C-5; 3-19 m)/C-1; s)Hto C-1,s)C-15 andC-15 C-16;and H3 -18 (δHH1.70, s)Hto1.70, C-3,s)C-4 and C-5; H3 -19 (δHH1.86, 3 -17 (δH (δHto1.86, to C-7, and C-9; HH 3-20 (δH s) 1.33, s) to C-11, C-13, established connection s) C-7,s)C-8 andC-8 C-9; and H3and -20 (δ 1.33, to C-11, C-12C-12 and and C-13, established thethe connection of of the four proton sequences, and thusconstructed constructedthe the14-membered 14-memberedring ringcarbon carbonskeleton, skeleton, which which also the four proton sequences, and thus hydroxyl at at C-6. C-6. Thus, the planar structure structure of of 11 was was established. established. indicated the presence of a hydroxyl
OR
18
6
4
1
19
8
O
2
16
O
O
15
O
OR 17
14
10
O
12
O
20
1: R=H 2: R=OH
3. 4: R=H 5: R=OH COSY correlations HMBC correlations 1
1
Figure 2. 2. Selected Selected1 HH-1 H COSY and HMBC correlations correlations of of 1, 1, 22 and and 33−5. Figure −5.
Further, careful analysis of nuclear Overhauser enhancement (NOE) correlations was applied to Further, careful analysis of nuclear Overhauser enhancement (NOE) correlations was applied to establish the relative stereochemistry of 1, as shown in Figure 3. The NOE spectrum revealed that Hestablish the relative stereochemistry of 1, as shown in Figure 3. The NOE spectrum revealed that H-2 2 (δH 5.44, dd, J = 10.0, 1.6 Hz) showed NOE correction with H3-18 (δH 1.70, s); therefore, assuming a (δH 5.44, dd, J = 10.0, 1.6 Hz) showed NOE correction with H3 -18 (δH 1.70, s); therefore, assuming a β-orientation of H-2, H3-18 should be β-oriented, too. Moreover, H3-18 exhibited NOE correlation β-orientation of H-2, H3 -18 should be β-oriented, too. Moreover, H3 -18 exhibited NOE correlation with with H-6 (δH 4.70, ddd, J = 10.4, 10.4, 5.2 Hz), revealing the β-orientation of H-6 and the R* H-6 (δH 4.70, ddd, J = 10.4, 10.4, 5.2 Hz), revealing the β-orientation of H-6 and the R* configuration of configuration of C-6. One methylene proton at C-13 exhibited NOE correlation with H-2 and was C-6. One methylene proton at C-13 exhibited NOE correlation with H-2 and was characterized as H-13β characterized as H-13β (δH 1.06, m), while the other proton was assigned as H-13α (δH 2.03, m). NOE (δH 1.06, m), while the other proton was assigned as H-13α (δH 2.03, m). NOE correlations of H-13β correlations of H-13β with H-11 (δH 2.42, dd, J = 10.8, 2.8 Hz) and H-13α with H3-20 (δH 1.33, s) with H-11 (δH 2.42, dd, J = 10.8, 2.8 Hz) and H-13α with H3 -20 (δH 1.33, s) reflected the β-orientation of reflected the β-orientation of H-11 and the α-orientation of H3-20, and hence the R* configurations of H-11 and the α-orientation of H3 -20, and hence the R* configurations of both C-11 and C-12. The E both C-11 and C-12. The E geometries of the trisubstituted C-3/C-4 and C-7/C-8 double bonds were geometries of the trisubstituted C-3/C-4 and C-7/C-8 double bonds were also assigned from the NOE also assigned from the NOE correlations of H3-18 (δH 1.70, s) with H-2, but not with H-3 (δH 4.90, d, J correlations of H3 -18 (δH 1.70, s) with H-2, but not with H-3 (δH 4.90, d, J = 10.0 Hz), as well as H3 -19 = 10.0 Hz), as well as H3-19 (δH 1.86, s) with H-6, but not with H-7 (δH 5.20, d, J = 10.4 Hz), and were (δH 1.86, s) with H-6, but not with H-7 (δH 5.20, d, J = 10.4 Hz), and were also confirmed by the upfield also confirmed by the upfield chemical shifts (δC < 20 ppm) observed for both C-18 (δC 15.9) and C-19 chemical shifts (δC < 20 ppm) observed for both C-18 (δC 15.9) and C-19 (δC 14.9) [30]. Based on the (δC 14.9) [30]. Based on the above observations and the detailed analysis of other NOE correlations, above observations and the detailed analysis of other NOE correlations, the relative configuration of the relative configuration of this compound was established. Furthermore, the absolute configuration this compound was established. Furthermore, the absolute configuration of 1 at C-6 was determined of 1 at C-6 was determined by the modified Mosher’s esterification method [31,32]. The (S)- and (R)by the modified Mosher’s esterification method [31,32]. The (S)- and (R)-MTPA esters of 1 (1a and 1b, MTPA esters of 1 (1a and 1b, respectively, as shown in Figure 4) were afforded by the reaction of 1 respectively, as shown in Figure 4) were afforded by the reaction of 1 with (R)-(-) and (S)-(+)-MTPA with (R)-(-) and (S)-(+)-MTPA chloride, respectively. Determination of the Δδ values (δS–δR) for chloride, respectively. Determination of the ∆δ values (δS –δR ) for protons nearing C-6 resulted in protons nearing C-6 resulted in the establishment of the R configuration at C-6 in 1 (Figure 4). The the establishment of the R configuration at C-6 in 1 (Figure 4). The absolute configuration of 1 was absolute configuration of 1 was thus assigned as 2S,6R,11R,12R, mostly the same as that of isosarcophine (7) [24], except that of C-6. Therefore, cherbonolide A (1) was identified as 6-αhydroxyisosarcophine.
The molecular formula of cherbonolide B (2) was determined to be C20H28O5 by the HRESIMS ++ (m/z calcd ),having havingone onemore moreoxygen oxygenthan than1.1.Moreover, Moreover,both both11 (m/z calcd371.1830; 371.1830;found found371.1829, 371.1829,[M [M++Na] Na]), 11 1313 and and22had hadalmost almostidentical identical H Hand and CCNMR NMRdata data(Table (Table1), 1),except exceptfor forthose thoseof ofC-6. C-6.The Theallylic allylichydroxy hydroxy group groupof of11at atC-6 C-6was wassubstituted substitutedby byaahydroperoxyl hydroperoxylin in2,2,with withthe thecharacteristic characteristicsignal signalof ofaabroad broad singlet in the downfield region, δδHH7.99 [26,33,34]. Obvious downfield shifts of C-6 (δ CC65.2 in 1,1,78.3 singlet in the downfield region, 7.99 [26,33,34]. Obvious downfield shifts of C-6 (δ 65.2 in 78.3 Mar. Drugs 2018, 16, 276 4 of 17 in 2) and H-6 (δ H 4.70 in 1, 4.97 in 2) were also observed, indicating that 2 possesses the hydroperoxy in 2) and H-6 (δH 4.70 in 1, 4.97 in 2) were also observed, indicating that 2 possesses the hydroperoxy group groupat atC-6. C-6.Furthermore, Furthermore,reduction reductionof of22by byreaction reactionwith withtriphenylphosphine triphenylphosphineafforded afforded1.1.On Onthe the thus as 2S,6R,11R,12R, same as that of(2S,6R,11R,12R)-configuration isosarcophine (7) [24], except that C-6. basis of analyses, planar structure and the of basisassigned of the the above above analyses, the themostly planarthe structure and the (2S,6R,11R,12R)-configuration of 22ofwere were Therefore, cherbonolide A (1) was identified as 6-α-hydroxyisosarcophine. determined. determined.
Figure 3. Key NOESY correlations of 1. Figure Figure3. 3.Key KeyNOESY NOESYcorrelations correlationsof of1. 1.
OR OR
+0.006 +0.006
-0.001 -0.001
O O
-0.110 -0.110
O O +0.057 +0.057
+0.109 +0.109 +0.082 +0.082
+0.056 +0.056
HH -0.009 -0.009 -0.016 -0.016
O O
-0.052 -0.052 -0.052 -0.052
HH
1a: 1a:R= R=(S)-MTPA (S)-MTPA 1b: 1b:R= R=(R)-MTPA (R)-MTPA
O O
O O
OR OR
O O
3a: 3a:R= R=(S)-MTPA (S)-MTPA 3b: 3b:R= R=(R)-MTPA (R)-MTPA
1HNMR Figure 4.4.11H Figure4. NMRchemical chemicalshift shiftdifferences differencesΔδ Δδ(δ (δSSS−− −δδRRR)))inin ppm for the MTPA estersof of11and and3.3. Figure H NMR chemical shift differences ∆δ (δ inppm ppmfor forthe theMTPA MTPAesters
Cherbonolide CherbonolideCC(3) (3)should shouldhave havethe thesame samemolecular molecularformula formulaas as1,1,according accordingto toHRESIMS HRESIMSdata. data. The 1molecular formula of cherbonolide B (2) was determined to be C20 H28 O5 by the HRESIMS 1 11 Also, H and correlations 2) 33are similar to suggesting that + ), having Also,the the HHHCOSY COSYfound andHMBC HMBC correlations (Figure 2)of of are similar tothose those of suggesting that (m/z calcd 371.1830; 371.1829, [M + Na](Figure one more oxygen thanof 1.1,1, Moreover, both 1 these compounds possess almost the same molecular skeleton. Analysis of NOE correlations (Figure 1 13 these compounds possess almost the same molecular skeleton. Analysis of NOE correlations (Figure and 2 had almost identical H and C NMR data (Table 1), except for those of C-6. The allylic hydroxy 5) the relative configurations at and and same. 5)showed showed that thewas relative configurations atC-2, C-2,C-11 C-11in and C-12in in11characteristic and33are arethe thesignal same.Assuming Assuming group of 1 that at C-6 substituted by a hydroperoxyl 2, C-12 with the of a broad the β-orientation of H-2, as H 3-18 NOE interaction with H-2 but not with H-3, the E the β-orientation of H-2, as H 3-18showed showed NOE interaction with H-2 but not with H-3, the E geometry singlet in the downfield region, δH 7.99 [26,33,34]. Obvious downfield shifts of C-6 (δC 65.2geometry in 1, 78.3 was the trisubstituted bond. of protons at C-5 was assigned toH the trisubstituted C-3/C-4 double bond.One One ofthe themethylene methylene protons athydroperoxy C-5(δ (δHH2.42, 2.42, in 2)assigned and H-6 to (δ 4.70 in 1, 4.97 inC-3/C-4 2) were double also observed, indicating that 2 possesses the dd, 3.2 NOE was dd, JJ =at = 12.0, 12.0,Furthermore, 3.2 Hz) Hz) displayed displayed NOE interaction with H-3, but but not not with with H H3-18, 3-18, and and was hence group C-6. reduction of 2interaction by reactionwith with H-3, triphenylphosphine afforded 1. On thehence basis determined as H-5α. Further, H-6 (δ HH3.84, dd, J = 9.2, 9.2 Hz) showed NOE correlations with H-5α determined as H-5α. Further, H-6 (δ 3.84, dd, J = 9.2, 9.2 Hz) showed NOE correlations with H-5α of the above analyses, the planar structure and the (2S,6R,11R,12R)-configuration of 2 were determined. and H-9α, but not with H-9β and H 3 -19. These observations, together with NOE correlations of and H-9α, but not with H-9β and H3-19. These observations, together with NOE correlations ofHH9β/H 9β/H3-19, 3-19, H H3-19/H-7 3-19/H-7 and and H-7/H H-7/H3-18, 3-18, enabled enabled deduction deduction of of the the α-orientation α-orientation of of H-6 H-6 and and led led to to the the assignment assignmentof ofaa6S* 6S*configuration configurationand andaaZZgeometry geometryof ofthe thetrisubstituted trisubstitutedC-7/C-8 C-7/C-8double doublebond bondin in3.3. The olefinic methyl group attaching at C-8 showed carbon signal at δ C 22.2 ppm further confirmed The olefinic methyl group attaching at C-8 showed carbon signal at δC 22.2 ppm further confirmed
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Table 1. 1 H and 13 C NMR chemical shifts for compounds 1–4.
Position
1 δH , m (J in Hz)
1 2 3
5β 6 7 8
a
2.20, m 2.76, dd (12.8, 5.2) 4.70, ddd (10.4, 10.4, 5.2) 5.20, d (10.4) 2.03, m
9β
2.38, m
10α 10β
1.29, m 2.51, m 2.42, dd (10.8, 2.8)
12 13α 13β 14α 14β 15 16 17 18 19 20 6-OOH
δC
type
δH , m (J in Hz)
77.8, CH 122.2, CH
2.03, m 1.06, m 2.49, m 2.01, m
1.86, s 1.70, s 1.86, s 1.33, s
47.9, CH2
65.2, CH 128.1, CH 139.8, C 36.8, CH2
a
3 δC
b,
type
77.8, CH 122.7, CH
2.07, m
42.6, CH2
78.3, CH 123.1, CH 144.2, C 36.9, CH2
23.6, CH2
61.4, CH
2.43, m
61.4, CH
123.8, C 174.4, C 8.8, CH3 15.9, CH3 14.9, CH3 15.8, CH3
1.86, s 1.72, s 1.89, s 1.33, s 7.99, br s
δC
type
78.4, CH 123.9, CH
60.8, C 37.0, CH2 23.7, CH2 123.8, C 174.4, C 8.7, CH3 15.9, CH3 15.3, CH3 15.8, CH3
δH , m (J in Hz) c
49.1, CH2
3.84, dd (9.2, 9.2) 5.09, d (9.2) 2.21, ddd (13.6, 13.6, 2.4)
69.6, CH 131.6, CH 138.4, C 28.2, CH2
1.16, m 1.84, m 2.24, dd (10.4, 2.4) 1.49, m 0.98, m 1.58, m 1.58, m
1.64, s 1.31, s 1.45, s 1.00, s
23.9, CH2 58.9, CH 59.7, CH 35.5, CH2 22.2, CH2 123.8, C 173.9, C 8.8, CH3 18.1, CH3 22.2, CH3 17.1, CH3
δC d , type 159.8, C
4.98, d (10.4) 4.45, d (10.4)
140.2, C 2.42, dd (12.0, 3.2)
1.63, m
1.35, m 2.17, m
23.7, CH2
4 d,
189, m
23.5, CH2
2.02, m 1.07, m 2.52, m 2.03, m
c
160.6, C 4.91, dd (10.0, 1.6) 4.55, d (10.0)
140.8, C 2.18, dd (12.4, 10.8) 2.87, dd (12.4, 4.4) 4.97, ddd (10.8, 9.2, 4.4) 5.05, d (9.2)
2.42, m
60.8, C 36.9, CH2
δH , m (J in Hz)
160.6, C 5.43, dd (10.0, 1.6) 4.91, d (10.0)
141.6, C
9α
11
2 b,
160.7, C 5.44, dd (10.0, 1.6) 4.90, d (10.0)
4 5α
a
77.8, CH 123.5, CH 139.4, C
1.98, m 2.25, dd (12.8, 3.6) 4.21, ddd (11.2, 9.2, 3.6) 4.84, d (9.2) 1.58, m 2.30, dd (13.2, 4.8) 1.60, m 1.28, m 1.98, m 1.61, m 0.65, m 2.08, m 1.65, m
1.63, s 1.28, s 1.32, s 0.99, s
49.3, CH2
64.8, CH 131.2, CH 139.4, C 28.5, CH2
22.7, CH2 62.6, CH 60.9, C 37.1, CH2 23.1, CH2 123.7, C 174.3, C 8.8, CH3 16.9, CH3 21.8, CH3 16.4, CH3
Spectrum recorded at 400 MHz in CDCl3 . b Spectrum recorded at 100 MHz in CDCl3 . c Spectrum recorded at 400 MHz in C6 D6 . d Spectrum recorded at 100 MHz in C6 D6 .
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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-5 (δH 2.42, dd, Mar. Drugs 2018, 16, x 6 of 16 J = 12.0, 3.2 Hz) displayed NOE interaction with H-3, but not with H3 -18, and was hence determined as H-5α. Further, H-6 (δH 3.84, dd, J = 9.2, 9.2 Hz) showed NOE correlations with H-5α and H-9α, the Z geometry of C-7/C-8 [30]. The absolute of 3 at of C-6 was also verified but not with H-9β anddouble H3 -19. bond These observations, togetherconfiguration with NOE correlations H-9β/H 3 -19, and H-7/H deduction of the α-orientation H-6 and(δ led assignment by usingHthe modified Mosher’s method. Determination of the Δδofvalues S −toδthe R, shown in Figure 4) 3 -19/H-7 3 -18, enabled of a neighboring 6S* configuration a Z geometry of thethe trisubstituted C-7/C-8at double bond in 3. The4). olefinic for protons C-6and further confirmed S configuration C-6 in 3 (Figure The absolute methyl group attaching at C-8 showed carbon signal at δC 22.2 ppm further confirmed the Z geometry configuration of 3 was thus assigned as 2S,6S,11R and 12R. Thus, cherbonolide C (3) is the 7Z isomer of C-7/C-8 double bond [30]. The absolute configuration of 3 at C-6 was also verified by using the of cherbonolide A (1). modified Mosher’s method. Determination of the ∆δ values (δS − δR , shown in Figure 4) for protons Cherbonolide D (4) wasconfirmed found to an isomer atofC-6 3 in according HRESIMS. Both compounds neighboring C-6 further thebe S configuration 3 (Figure 4).toThe absolute configuration 1 1 of 3 was assigned 2S,6S,11R and 12R. Thus, cherbonolide C (3) is the they 7Z isomer cherbonolide have almost thethus same H- HasCOSY and HMBC correlations, indicating haveofthe same molecular A (1). skeleton. NMR data of 3 and 4 are nearly the same (Table 1), except for those of CH-6, suggesting that Cherbonolide D (4) was found to be an isomer of 3 according to HRESIMS. Both compounds 4 could be the C-6 epimer of 3. The (2S,6R,11R,12R)-configuration and the E and Z geometries of Chave almost the same 1 H-1 H COSY and HMBC correlations, indicating they have the same molecular 3/C-4 and C-7/C-8 double of nearly 4, respectively, were also established by suggesting analysis of NOE skeleton. NMR data of bonds 3 and 4 are the same (Table 1), except for those of CH-6, correlations be asbethose ofepimer 3 (Figure that 4tocould the C-6 of 3. 5). The (2S,6R,11R,12R)-configuration and the E and Z geometries of C-3/C-4 and C-7/C-8 bonds of respectively, were also established by28analysis NOE Cherbonolide E (5) was double determined to4,have a molecular formula C20H O5 fromofits HRESIMS correlations to be as those of 3 (Figure 5). data (m/z calcd 371.1830; found 371.1829, [M + Na]+), with one more oxygen than in 4. Compounds 4 Cherbonolide E (5) was determined to have a molecular formula C20 H28 O5 from its HRESIMS 1 and 5 displayed identical and 13C 2),oxygen exceptthan forinthose of CH-6. It was data (m/z almost calcd 371.1830; foundH 371.1829, [MNMR + Na]+data ), with(Table one more 4. Compounds 1 13 4 and 5 displayed almost identical H and 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 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 characteristic signal of a broad singlet at δH 7.25 [26,33,34]. The obvious downfield shifts of C-6 (δC 64.8 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 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 group atC-6 C-6ofof5. 5. Furthermore, reduction oftriphenylphosphine 5 with triphenylphosphine afford Therefore, the Furthermore, reduction of 5 with could afford 4.could Therefore, the4. structure structureofof5, 5, with (2S,6R,11R,12R)-configuration, was determined. with the the (2S,6R,11R,12R)-configuration, was determined.
Figure 5. Key NOESY correlations correlations forfor 3 and 4. 4. Figure 5. Key NOESY 3 and
Bischerbolide peroxide (6) was afforded as a white powder with the molecular formula C40H58O6 from HRESIMS (m/z calcd 657.4124; found 657.4125, [M + Na]+), appropriate for twelve degrees of unsaturation. The 13C NMR spectroscopic data of 6 revealed the presence of 40 carbons (Table 2). The DEPT spectra of 6 showed the presence of eight methyls, twelve sp3 methylenes, six sp3 oxygenated methines, four sp2 methines, two sp3 and eight sp2 nonprotoned carbons (including two ester carbonyls). NMR signals resonating at δC 114.3 (CH), 141.4 (C), 124.9 (C), 82.7 (CH) and 10.2 (CH3), and δH 5.28 (1H, d, J = 10.0 Hz) and δH 1.72 (3H, s), and another group of signals observed at δC 114.4
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Table 2. 1 H and 13 C NMR chemical shifts for compounds 5 and 6.
Position 1 2 3 4 5α 5β 6 6α 6β 7 8 9α 9β 10α 10β 11 12 13α 13β 14α 14β 15 16 17 18 19 20 6-OOH a
5 δH , m (J in Hz)
a
4.95, d (10.0) 4.42, d (10.0) 1.95, m 2.47, br d (11.0) 4.58, ddd (11.0, 9.5, 2.5)
4.78, d (9.5) 1.65, m 2.52, dd (14.0, 4.5) 1.28, m 1.62, m 1.97, m 1.59, m 0.64, m 2.07, m 1.61, m
1.63, s 1.29, s 1.34, s 0.98, s 7.25, br s
6 δC
b,
type
160.4, C 78.4, CH 124.6, CH 139.2, C 44.6, CH2
δH , m (J in Hz)
c
5.28, d (10.0) 5.06, d (10.0) 2.21, m 2.32, m
78.9, CH
126.6, CH 143.8, C 29.8, CH2 23.4, CH2 63.3, CH 61.5, C 37.6, CH2 23.7, CH2 124.3, C 174.3, C 9.4, CH3 17.3, CH3 22.5, CH3 16.9, CH3
δC
d,
141.4, C 82.7, CH 126.4, CH 140.2, C 38.5, CH2 24.2, CH2
2.07, m 2.42, m 4.98, dd (9.2, 9.2) 1.96, m 2.27, m 1.22, m 2.04, m 2.51, m 1.83, m 0.95, m 2.33, m 1.81, m 6.13, br s 1.72, s 1.58, s 1.65, s 1.27, s
δH , m (J in Hz) c
type
125.6, CH 133.1, C 36.6, CH2 23.6, CH2 62.1, CH 61.2, CH 37.3, CH2 22.6, CH2 124.9, C 114.3, C 10.2, CH3 14.6, CH3 14.7, CH3 15.7, CH3
10 20 30 40 50 α 50 β 60 60 α 60 β 70 80 90 α 90 β 100 α 100 β 110 120 130 α 130 β 140 α 140 β 150 160 170 180 190 200
5.50, d (10.0) 4.92, d (10.0) 2.21, m 2.31, m
δC d , type 141.5, C 81.9, CH 125.1, CH 141.0, C 38.8, CH2 24.2, CH2
2.07, m 2.42, m 4.95, dd (9.2, 9.2) 1.96, m 2.27, m 1.22, m 2.04, m 2.51, m 1.83, m 0.95, m 2.33, m 1.81, m 6.17, d (3.6) 1.73, s 1.59, s 1.65, s 1.27, s
125.5, CH 133.3, C 36.6, CH2 23.7, CH2 62.2, CH 61.3, C 37.4, CH2 22.7, CH2 124.9, C 114.4, CH 10.2, CH3 14.6, CH3 14.7, CH3 15.7, CH3
Spectrum recorded at 500 MHz in C6 D6 . b Spectrum recorded at 125 MHz in C6 D6 . c Spectrum recorded at 400 MHz in CDCl3 . d Spectrum recorded at 100 MHz in CDCl3 .
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Mar. Drugs 2018, 16, 276 Table
1 13 2. H and C NMR chemical shifts for compounds 5 and 6.
8 of 17
5 6 a b, type c d was afforded δBischerbolide H, m (J in Hz)peroxide δC(6) δH, mas (J ainwhite Hz) cpowder δC d,with typethe molecular δHformula , m (J in C Hz) 40 H58 O6δC , type + from HRESIMS (m/z calcd 160.4, 657.4124; appropriate for twelve degrees of 141.5, C 1 C found 657.4125, [M + Na] 141.4,), C 1′ 13 unsaturation. The C NMR spectroscopic of 6 revealed carbons (Table 2).81.9, CH 2 4.95, d (10.0) 78.4, CH 5.28,data d (10.0) 82.7,the CHpresence 2′ of 405.50, d (10.0) The DEPT theCH presence of eight methyls,126.4, twelveCH sp3 methylenes, six spd3 (10.0) oxygenated125.1, CH 3 4.42,spectra d (10.0)of 6 showed 124.6, 5.06, d (10.0) 3′ 4.92, methines, four sp2 methines,139.2, two spC3 and eight sp2 nonprotoned carbons two ester carbonyls).141.0, C 4 140.2, C (including 4′ NMR signals at44.6, δC 114.3 82.7 and 2.21, (C), m 124.9 (C), 38.5, CH(CH) 2 5′α 10.2 (CH 2.21, m δH 5.2838.8, CH2 5α 1.95, resonating m CH2 (CH), 141.4 3 ), and 5β d (11.0) m group of signals observed 5′β at δC 114.4 2.31, (CH), m (1H, 2.47, d, J =br 10.0 Hz) and δH 1.72 (3H, s), and 2.32, another 141.5 6 4.58,124.9 ddd(C), (11.0, 9.5,(CH) 2.5) and 78.9, CH2Hz) and 6′ δH 1.73 (3H, s), revealed24.2, CH2 (C), 81.9 10.2CH (CH3 ), and δH 5.50 (1H, d,24.2, J = 10.0 6α 2.07, m 6′α group2.07, m the presence of two slightly different 2,5-dihydrofuran rings with a peroxyl by comparison 6β min the literature [35]. Also, 6′β two groups 2.42, mof signals of the similar NMR data of five-membered2.42, rings 7 4.78,atd δ(9.5) 126.6, CHand4.98, dd (1H, (9.2, m), 9.2) and125.6, CH 7′ (CH) 4.95, resonating (CH) δH 2.51 δC 61.3 (C), 62.2 anddd δH(9.2, 2.519.2) (1H, m)125.5, CH C 61.2 (C), 62.1 8 C 133.1, C 8′ showed the presence of two143.8, trisubstituted epoxides. Four trisubstituted olefinic bonds were revealed 133.3, C 2 1.96, 36.6,δHCH 2 (1H, 9′αd, J = 10.01.96, 9α 1.65, m appearing 29.8, from NMR signals at CH δC 126.4 (CH), δCm 140.2 (C) and 5.06 Hz);m at δC 125.636.6, CH2 9β 2.52, (14.0, 2.27, (CH), δC dd 133.1 (C)4.5) and δH 4.98 (1H, dd, J = 9.2, 9.2mHz); at δC 125.1 (CH), δ9′β and δm C 141.0 (C) 2.27, H 4.92 (1H, 2 1.22,(C) m and δH 4.95 23.6,(1H, CH2dd, J10′α 1.22, m 23.7, CH2 10α 1.28, m and at δC 23.4, dd, J = 10.0 Hz); 125.5CH (CH), δC 133.3 = 9.2, 9.2 Hz), respectively. 13 10β 1.62, m 2.04, m 10′β 2.04, m As the C NMR spectrum of 6 was constituted by twenty sets of signals with each set containing 11 1.97, CH shifts, 2.51, m thus identified 62.1, CH 11′ 2.51, m from the62.2, CH two peaks of m very similar63.3, chemical 6 was as a compound formed 12 61.5, C CHplanar12′ connection of two quite similar diterpenoid subunits. The61.2, entire structure was established 61.3, C 13 CCH 13α 1.59, m of 1 H and 37.6, 2 37.3, CH 13′α correlations 1.83, m by examination NMR data1.83, andm1 H-1 H COSY and2 HMBC (Figure 6).37.4, CH2 13β 0.64, mresonating at δC 114.3 and 0.95, m were considered to13′β 0.95, m at which Two methines δC 114.4 be the positions 14α 2.07, m 23.7, CH 2 2.33, m 22.6, CH 2 14′α 2.33, the two cembranoidal units were connected by insertion of a peroxyl group. Based onmthe above22.7, CH2 14β 1.61, m 1.81, m 14′β 1.81, m analyses, the molecular skeleton of 6 was elucidated as the biscembranoid formed by the connection of 0 15 124.3, C [36] via a peroxyl group 124.9, C and 15′C-16 . The fragmentation 124.9, C two molecules of isosarcophytoxide at C-16 16 C further 6.13, brthe s dimeric 114.3, C of 616′ 6.17, d (3.6) pattern of ESIMS (Figure 7)174.3, could prove nature and the peroxyl linkage at114.4, CH 0 3 1.72, s 10.2, CH 3 17′ 1.73, s 17 1.63, s 9.4, CH C-16/C-16 . One ion peak displayed at m/z 339 can be explained by the cleavage of O–O bond and10.2, CH3 3 1.58, s 14.6, 18′ a sodiated 1.59,cembranoid s 14.6, CH3 18 1.29, elimination s 17.3, CHfrom the following of H-16 a monocembranoidal unitCH in36 to form 19 1.34, s 22.5, CH 3 1.65, s 14.7, CH 3 19′ 1.65, s lactone molecular ion A (pathway a). The other ion peaks can be interpreted by the cleavage of the14.7, CH3 3 1.27,tos afford ion 15.7, CH3301), 20′ 1.27, s 15.7, CH3 20 0.98, s single bond between C-1616.9, and CH peroxyl oxygen B (m/z and a peroxycembranoidal 6-OOH radical which 7.25, brcould s further abstract an hydrogen atom and form the sodium adduct C (m/z 357)
Position
a Spectrum recorded at 500 MHz in C6D6. b Spectrum recorded at 125 MHz in C6D6. c Spectrum (pathway b). Moreover, compound 6 was found to be the first example of a biscembranoid with a recorded at 400 MHz in CDCl . d Spectrum recorded 100 MHzby in aCDCl 3. molecular skeleton formed by 3two cembranoid units at connected peroxyl group.
20' 18 6
4
O 8
2
19
16
O
1 15 10
12
O 20
14
19'
16'
O
8'
2' 4'
17
10'
1'
15'
O
O
12'
14'
17'
6'
18'
6
COSY correlations HMBC correlations
1 H-11 H COSY and HMBC correlations of 6. Figure Selected1HFigure 6.6.Selected H COSY and HMBC correlations of 6.
As the 13C 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 1H and 13C NMR data and 1H-1H 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 Mar. Drugs 2018, 16, 276 9 of 17 molecular skeleton formed by two cembranoid units connected by a peroxyl group.
Figure Figure7.7.ESIMS ESIMS fragmentation fragmentation ofof6.6.
The The relative configuration from aaliterature literaturesurvey survey [36,37] relative configurationofof6 6was was determined determined from [36,37] andand NOENOE 13 13 correlations (Figure 8). 8). TheTheC NMR 40signals signals two signals correlations (Figure C NMRspectrum spectrumof of 66 displayed displayed 40 of of two setssets signals withwith nearly identical chemical shifts, representing the very similar stereochemical environments of nearly identical chemical shifts, representing the very similar stereochemical environments the two two structural In addition, compound was found to have nearly identical chemical shifts structural units. Inunits. addition, compound 6 was 6found to have nearly identical chemical shifts for H0 ), H -18 (180 ), H -20 (200 ) and C-20 (200 ) to those of (2S,11R,12R)-isosarcophytoxide (8), for H-11 (11 3 3-20 (20′) and 3 11 (11′), H3-18 (18′), H C-20 (20′) to those of (2S,11R,12R)-isosarcophytoxide (8), and were and were in turn found to exhibit distinguishable differences to the corresponding chemical shifts in turn found to exhibit distinguishable differences to the corresponding chemical shifts of of (2R,11R,12R)-isosarcophytoxide (9) (Table 3 and Figure 9). Thus, 6 possessed the cembranoidal (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 structural unit derived from 8, as also proven0 by observed NOE correlations (Figure 8). Different values were observed for H-2 (δH 5.28) and H-2 (δH 5.50), indicating that H-20 was on the same planar proton observed for was H-2 deshielded, (δH 5.28) and H 5.50), indicating that H-2′ was on the same facevalues as the were peroxide group and andH-2′ H-2(δ was on the same planar face as H-16 and was planar face asAs thecompounds peroxide group andisolated was deshielded, andorganism H-2 was in onthis thestudy, samethey planar shielded. 1−7 were from the same are face likelyastoH-16 and was shielded. As compounds 1−7 were isolated from the same organism in this study, they are possess the same absolute S,R,R-configurations at the chiral centers C-2, C-11 and C-12, respectively, likelyasto possess the3.same absolute S,R,R-configurations at the chiral configurations centers C-2, at C-11 those of 1 and A previous report also showed that different absolute C-2 and of theC-12, related diasteromeric dihydrofuran ring-containing cembranoids could significantly influence the sign respectively, as those of 1 and 3. A previous report also showed that different absolute configurations of the specific optical rotation [36,38]. For cembranoids with 2S configuration a significant positive at C-2 of the related diasteromeric dihydrofuran ring-containing cembranoids could significantly 25 and for those with 2R configuration a negative optical rotation were found. The [ α ] of 6 was +41; a D configuration influence the sign of the specific optical rotation [36,38]. For cembranoids with 2S 0 0 0 0 thus, the absoluteand configuration of 6 was to be 2S,11R,12R,16R, 2 S,11rotation R,12 R,16 S. found. The significant positive for those with 2R deduced configuration a negative optical were [α] 25D 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.
Mar. Drugs 2018, 16, x
9 of 16
20'
13'
Drugs Mar. DrugsMar. 2018, 16,2018, x 16, 276
10 of 17
9 of 16
13'
18
11'
2
16'
16
20'
13'
2'
13
13'
18
11
18'
11'
13
20
2
16'
16
2'
13
Figure 8. Selected NOESY correlations for 6.
11
18' 1 13 13C NMR data comparison with 6, (2S,11R,12R)-isosarcophytoxide (8) and Table 3. Selected H and (2R,11R,12R)-isosarcophytoxide (9).
20
9a 6 8a δH 2.51 (H-11, H-11′) δH 2.50 δH 2.75 C 62.1NOESY (C-11) δ Figure 8. Selected correlations for 6. for 6. δC 61.2 δC 62.3 C-11 Figure 8. Selected NOESY correlations δC 62.2 (C-11′) 1 H and 13 C NMR dataδcomparison C 61.2 (C-12)with 6, (2S,11R,12R)-isosarcophytoxide (8) and Table 3. Selected 1 13 δC 61.4 δC 60.7 data comparison Table 3.(2R,11R,12R)-isosarcophytoxide Selected H andC-12C NMR(9). δC 61.3 (C-12′) with 6, (2S,11R,12R)-isosarcophytoxide (8) and δC 37.3 (C-13) (2R,11R,12R)-isosarcophytoxide (9). δC 37.4 δC 35.4 C-13 δ C 37.4 (C-13′) Position 6 8a 9a δC 22.66(C-14) 9a Position 8a δC 22.5 δH δ2.75 C-14 C 20.4 H-11 δH 2.51 (H-11, H-110 ) δH 2.50 δC 22.7 (C-14′) H-11 δH δ2.51 (H-11, H-11′) δH 2.50 δH 2.75 C 62.1 (C-11) δC 62.3 δC 61.2 δH 1.58 (H3-18) C-11 0) δδ 62.2 (C-11 δ H3-18 H 1.58 δ H 1.70 C 62.1 (C-11) C 1.59(C-12) (H3-18′) δC 62.3 C-11 δC 61.2 δCδH61.2 δ C 62.2 (C-11′) δC 61.4 δC δ60.7 C-12 H3-20 δHδ 1.27 (H 3-20, 0H3-20′) δ H 1.28 H 1.18 C 61.3 (C-12 ) C37.3 61.2 (C-12) (C-20, C-20′) δC 15.7 δC 17.7 C-20 δδCδC15.7 (C-13) C-12 C-13 δC 37.4 δC 61.4 δC 35.4 δC 60.7 13 (C-130 ) a The selected 1Hδδ 37.4 C 61.3 (C-12′) C and C data were cited from ref. [36,37]. δC 22.6 (C-14) δC 37.3 (C-13) δC 22.5 δ 20.4 C-14 δC 22.7 (C-140 ) δC 37.4C at C-16 δC of 35.4 The plausibleC-13 biosynthesis of 6 might arise from the proton abstraction 8 by hydrogen δC1.58 37.4 δH (H(C-13′) 3 -18) δH 1.58 δH 1.70 3 -18 0 peroxide radical HOOHto form a radical intermediate 10, which could react with O 2 from one plane δHδC 1.59 (H3(C-14) -18 ) 22.6 0 δC 22.5 C-14C-16 δC 20.4of 11 with 10 side of radical center to affordδH cembranoidal radical Further H3 -20 1.27 (H -20, Hperoxide δH 1.28 11. δH 1.18reaction 3 3 -20 ) δC 22.7 (C-14′) 0 δC 15.7 (C-20, ) δC However, 15.7 δthe C 17.7 from another side couldC-20 lead to the formation of C-20 6 (Scheme 1). possibility that 6 might Position H-11
δH 1.58 (H3-18) δH 1.59 (H3-18′) δH 1.27 (H3-20, H3-20′) δC 15.7 O (C-20, C-20′)
a The selected 1 H and 13 C data were cited from ref. [36,37]. δH 1.58 H3-18 be generated by autooxidation of 8 could not be neglected.
H3-20 C-20 7 a
18
δH 1.28 δC 15.7 O
16
δH 1.70 δH 1.18 δC 17.7
1 The selected H and 13C data were cited from ref. [36,37]. 5 3
19
2S
1
H
2R
15
17
H
The plausible biosynthesis of 6 might arise from the proton abstraction at C-16 of 8 by hydrogen 11 9 13 peroxide radical HOO to form could react with O2 from one plane O a radical intermediate 10, which O 20 cembranoidal peroxide radical 11. Further reaction of 11 with 10 side of radical center C-16 to afford 9 from another side could lead to the the possibility that 6 might 8 formation of 6 (Scheme 1). However, be generatedFigure by autooxidation of 8 could not be neglected. 9. (8) (8) andand (2R,11R,12R)-isosarcophytoxide (9) [36]. 9. Structures Structuresofof(2S,11R,12R)-isosarcophytoxide (2S,11R,12R)-isosarcophytoxide (2R,11R,12R)-isosarcophytoxide (9) [36].
18
The plausible biosynthesis of 6 might arise from the proton abstraction at C-16 of 8 by hydrogen O intermediate 10, which could react withO peroxide radical HOO• to form a radical O2 from one plane 16 side of radical 7 center C-16 to afford cembranoidal peroxide radical 11. Further reaction of 11 with 10 5 3 2S 2R from another side could of 6 (Scheme 1). However, the possibility that 6 might be 19 lead to the formation 15 1 generated by autooxidation H of 8 could not be neglected. H 9
17
11
13
O
O 20
8
9
Mar. Drugs 2018, 16, 276 Mar. Drugs 2018, 16, x
11 of 17 10 of 16
O 8
HOO
O
H O2
H
O
O 10
6
O 10
11
Scheme 1. Proposed biosynthetic pathway for 6.
It isisknown known that proteolytic enzymes andreactive toxic reactive oxygenproduced species by produced by It that the the proteolytic enzymes and toxic oxygen species stimulated stimulated neutrophils playrole a critical in the pathogenesis many inflammatory neutrophils might playmight a critical in therole pathogenesis of many of inflammatory diseasesdiseases [39,40]. [39,40]. By measuring the capability to inhibit N-formyl-methionyl-leucyl-phenylalanine/ By measuring the capability to inhibit N-formyl-methionyl-leucyl-phenylalanine/cytochalasin B cytochalasin B (fMLF/CB)-induced superoxide anion generation and elastase release in human (fMLF/CB)-induced superoxide anion generation and elastase release in human neutrophils, neutrophils, the in vitro anti-inflammatory effects for metabolites 1–7 were evaluated [41,42]. the in vitro anti-inflammatory effects for metabolites 1–7 were evaluated [41,42]. According to the According to the results (shown in Table 4), compound 6 had a significant inhibitory effect (64.6 ± results (shown in Table 4), compound 6 had a significant inhibitory effect (64.6 ± 0.8%), with an IC50 0.8%), with an IC50 value of 26.2 ± 1.0 μM, on the generation of superoxide anions, and compounds 1 value of 26.2 ± 1.0 µM, on the generation of superoxide anions, and compounds 1 and 3 had moderate and 3 had moderate inhibitory effects (32.1 ± 4.3% and 44.5 ± 4.6%, respectively) at 30 μM. inhibitory effects (32.1 ± 4.3% and 44.5 ± 4.6%, respectively) at 30 µM. Compounds 1, 3 and 6 revealed Compounds 1, 3 and 6 revealed moderate inhibitory effects (37.6 ± 5.0%, 35.6 ± 6.2% and 42.4 ± 5.1%, moderate inhibitory effects (37.6 ± 5.0%, 35.6 ± 6.2% and 42.4 ± 5.1%, respectively) on elastase release respectively) on elastase release at the same concentration. These results, obtained after stimualting at the same concentration. These results, obtained after stimualting the neutrophils with fMLF/CB, the neutrophils with fMLF/CB, may suggest that 1, 3 and 6 have potential merits against may suggest that 1, 3 and 6 have potential merits against inflammatory disorders. inflammatory disorders. In summary, examination of the chemical of the soft coraland Sarcophyton cherbonnieri Table 4. Inhibitory effects of compounds 1–7 onconstituents superoxide anion generation elastase release in led to the discovery of six new compounds 1–6, along with one known compound 7. Although a fMLF/CB-induced human neutrophils. number of natural compounds possessing a peroxyl group, such as artemisinin [43], neovibsanin C [44], cardamom peroxide [45], plakortinSuperoxide [46] and chondrillin [47], have beenRelease discovered, compound Anion Elastase Compounds a 6 was discovered to be the first compound with a molecular skeleton consisting b b IC50 (µM) Inh % Inh % of two cembranoidal units connected by a peroxide group. Similar to the results of previous studies indicating that natural 1 >30 32.1 ± 4.3 ** 37.6 ± 5.0 ** peroxides could possess activity 6 was found to possess anti2 promising biological >30 4.0 [48], ± 6.7compound23.5 ± 6.6 * inflammatory activity3 by exhibiting >30 stronger ability inhibition on35.6 the ±generation of superoxide 44.5on ± 4.6 *** 6.2 ** 4 >30 6.4 ± 4.2 27.6 ± 6.4 ** anions and release of elastase in fMLF/CB-induced human neutrophils. 5 >30 2.6 ± 6.2 30.5 ± 4.6 ** 6 26.2 ± 1.0 64.6 ± 0.8 *** 42.4 ± 5.1 ** Table 4. Inhibitory effects of compounds 1–7 on superoxide anion generation and elastase release in 7 >30 3.5 ± 5.3 20.7 ± 4.1 ** fMLF/CB-induced human neutrophils. Idelalisib 0.07 ± 0.01 102.8 ± 2.2 *** 99.6 ± 4.2
a
Concentration necessary for 50% inhibitionSuperoxide (IC50 ). b Percentage (Inh %) at 30 µM. Data are presented Anionof inhibition Elastase Release as mean ± S.E.M. (n =Compounds 3–4); * p < 0.05, ** p < 0.01, ***a p < 0.001 compared with the control value. b b
IC50 (μM) Inh % Inh % 1 >30 32.1 ± 4.3 ** 37.6 ± 5.0 ** In summary, examination constituents cherbonnieri led 2 of the chemical >30 4.0 ± 6.7 of the soft 23.5coral ± 6.6Sarcophyton * to the discovery of six new compounds 1–6, known compound 3 >30 along with 44.5 ±one 4.6 *** 35.6 ± 6.2 ** 7. Although a number of 4 a peroxyl >30 4.2 27.6 6.4 ** natural compounds possessing group, such6.4 as ±artemisinin [43],± neovibsanin C [44], cardamom 5 >30 2.6 ± 6.2 30.5 ± 4.6 ** peroxide [45], plakortin [46] and chondrillin [47], have been discovered, compound 6 was discovered 6 a molecular 26.2 ± 1.0 64.6consisting ± 0.8 *** of 42.4 5.1 ** to be the first compound with skeleton two ±cembranoidal units connected 7 >30 3.5 ± 5.3 20.7 ± 4.1 ** by a peroxide group. Similar to the results of previous studies indicating that natural peroxides Idelalisib 0.07 ± 0.01 102.8 ± 2.2 *** 99.6 ± 4.2
could possess promising biological activity [48], compound 6 was found to possess anti-inflammatory a Concentration necessary for 50% inhibition (IC50). b Percentage of inhibition (Inh %) at 30 μM. Data activity by exhibiting stronger ability on inhibition on the generation of superoxide anions and release are presented as mean ± S.E.M. (n = 3–4); * p < 0.05, ** p < 0.01, *** p < 0.001 compared with the control of elastase in fMLF/CB-induced human neutrophils. value.
3. Materials and Methods 3.1. 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
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3. Materials and Methods 3.1. 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 FT/IR-4100 infrared spectrophotometer (JASCO Corporation, Tokyo, Japan). 1 H and 13 C NMR spectra were obtained 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 C6 D6 . The data of LRESIMS and HRESIMS were measured using a Bruker APEX II (Bruker, Bremen, Germany) mass spectrometer. Silica gel (230–400 mesh) was used as adsorbent for column chromatography. TLC analyses were performed using precoated silica gel plates (Kieselgel 60 F-254, 0.2 mm) (Merck, Darmstadt, Germany). Further purification of impure fractions or compounds were performed 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). 3.2. 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. 3.3. Extraction and Isolation The frozen marine organisms, S. cherbonnieri (1.2 kg, wet wt), were freeze-dried (yield: 207 g), minced to small pieces and then extracted thoroughly with EtOAc (1 L × 5). The combined EtOAc extract (10.2 g) was concentrated under reduced pressure to yield a residue, which was chromatographed over a silica gel column by eluting with acetone in n-hexane (0–100%, stepwise), and then with MeOH in acetone (0–100%, stepwise) to yield 19 fractions. Fraction 9, eluting with n-hexane–acetone (6:1), was repeatedly purified by column chromatography over silica gel to yield a solid which was immersed in cold MeOH (0 ◦ C) to afford a white powder 6 (24.3 mg). Fraction 10, eluting with n-hexane–acetone (4:1), was further purified over silica gel using n-hexane–acetone (6:1) to afford seven subfractions (A1–A7) and afford 7 (320.4 mg). Subfraction A2 was further separated by reverse-phase HPLC using acetonitrile–H2 O (1:1.3) to afford 1 (11.0 mg). Subfraction A3 was purified by reverse-phase HPLC using acetonitrile–H2 O (1:1.1) to afford 2 (13.3 mg) and 5 (10.1 mg). Fraction 13, eluting with n-hexane–acetone (1:1), eluting with acetone by sephadex LH-20 to afford five subfractions (B1–B5). Subfraction B3 was purified by reverse-phase HPLC using acetonitrile–H2 O (1:1.4) to afford 3 (10.6 mg) and 4 (9.4 mg). 3.3.1. Cherbonnolide A (1) −1 13 1 Colorless oil; [α]25 D +43 (c 1.00, CHCl3 ); IR (neat) νmax 3444, 1746, and 1003 cm ; C and H NMR + data see Table 1; HRMS (ESI-TOF) m/z: [M + Na] Calcd for C20 H28 O4 Na 355.1880; Found 355.1879.
3.3.2. Cherbonnolide B (2) −1 13 C Colorless oil; [α]25 D +59 (c 1.00, CHCl3 ); IR (neat) νmax 3363, 1741, 1678, and 1093 cm ; + and NMR data see Table 1; HRMS (ESI-TOF) m/z: [M + Na] Calcd for C20 H28 O5 Na 371.1829; Found 371.1830. 1H
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3.3.3. Cherbonnolide C (3) −1 13 C Colorless oil; [α]25 D +26 (c 1.00, CHCl3 ); IR (neat) νmax 3445, 1749, 1678, and 1094 cm ; + and NMR data see Table 1; HRMS (ESI-TOF) m/z: [M + Na] Calcd for C20 H28 O4 Na 355.1880; Found 355.1882. 1H
3.3.4. Cherbonnolide D (4) −1 13 1 White powder; [α]25 D +3 (c 1.00, CHCl3 ); IR (neat) νmax 3445, 1748, and 1096 cm ; C and H NMR data see Table 1; HRMS (ESI-TOF) m/z: [M + Na]+ Calcd for C20 H28 O4 Na 355.1880; Found 355.1883.
3.3.5. Cherbonnolide E (5) −1 13 C Colorless oil; [α]25 D +8 (c 1.00, CHCl3 ); IR (neat) νmax 3389, 1748, 1678 and 1096 cm ; and 1 H NMR data see Table 2; HRMS (ESI-TOF) m/z: [M + Na]+ Calcd for C20 H28 O5 Na 371.1829; Found 371.1830.
3.3.6. Bischerbolide Peroxide (6) −1 White powder; [α]25 D +41 (c 1.00, CHCl3 ); IR (neat) νmax 3420, 1733, 1232, 1166 and 1040 cm ; 1 + and H NMR data see Table 2; HRMS (ESI-TOF) m/z: [M + Na] Calcd for C40 H58 O6 Na 657.4125; Found 657.4124.
13 C
3.3.7. 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. 3.3.8. 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. Selective 1 H NMR (CDCl3 , 400 MHz) data of 1a: δH 4.925 (1H, d, J = 10.0 Hz, H-3), 1.769 (3H, s, H3 -18), 2.821 (1H, dd, J = 12.8, 4.8 Hz, H-5a), 2.376 (1H, m, H-5b), 5.107 (1H, d, J = 9.6 Hz, H-7), 1.880 (3H, s, H3 -19), 2.460 (1H, m, H-9a), 1.989 (1H, m, H-9b); selective 1 H NMR (CDCl , 400 MHz) data of 1b: δ 4.905 (1H, d, J = 10.0 Hz, H-3), 1.763 (3H, s, H -18), 2.739 3 H 3 (1H, dd, J = 12.8, 5.6 Hz, H-5a), 2.267 (1H, m, H-5b), 5.217 (1H, d, J = 9.6 Hz, H-7), 1.905 (3H, s, H3 -19), 2.476 (1H, m, H-9a), 1.998 (1H, m, H-9b). Preparation of (S)- and (R)- MTPA esters of 3 used the same reaction and purification procedures as the reduction of 1, the solution of 3 (1.1 mg) was converted to the (S)-MTPA ester 3a (0.8 mg) in 74% yield and (R)-MTPA ester 3b (0.9 mg) in 80% yield, respectively. Selective 1 HNMR (C6 D6 , 400 MHz) data of 3a: δH 1.243 (3H, s, H3 -18), 2.311 (1H, dd, J = 11.2, 2.4 Hz, H-5a), 1.930 (1H, dd, J = 11.2, 11.2 Hz, H-5b), 5.065 (1H, d, J = 9.2 Hz, H-7), 1.416 (3H, s, H3 -19); selective 1 H NMR (C6 D6 , 400 MHz) data of 3b: δH 1.244 (3H, s, H3 -18), 2.363 (1H, dd, J = 12.0, 3.2 Hz, H-5a), 1.982 (1H, dd, J = 12.0, 11.6 Hz, H-5b), 4.9515 (1H, d, J = 10.0 Hz, H-7), 1.360 (3H, s, H3 -19).
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3.4. In Vitro Anti-Inflammatory Testing 3.4.1. Human Neutrophils Blood was obtained from elbow vein of healthy adult volunteers (years 20–30). Neutrophils were enriched by dextran sedimentation, Ficoll-Hypaque centrifugation, and hypotonic lysis. Neutrophils were incubated in an ice-cold Ca2+ -free HBSS buffer (pH 7.4) [42]. 3.4.2. Superoxide Anion Generation Neutrophils (6 × 105 cells mL−1 ) incubated in HBSS with ferricytochrome c (0.5 mg mL−1 ) and Ca2+ (1 mM) at 37 ◦ C were treated with DMSO (as control) or compound for 5 min. Neutrophils were primed by cytochalasin B (CB, 1 µg mL−1 ) for 3 min before activating fMLF (100 nM) for 10 min (fMLF/CB) [40,49]. 3.4.3. Elastase Release Neutrophils (6 × 105 cells mL−1 ) incubated in HBSS with MeO-Suc-Ala-Ala-Pro-Val-p-nitroanilide (100 µM) and Ca2+ (1 mM) at 37 ◦ C were treated with DMSO or compound for 5 min. Neutrophils were activated with fMLF (100 nM)/CB (0.5 µg mL−1 ) for 10 min [40]. 3.4.4. Statistical Analysis Data are displayed as the mean ± SEM and comparisons were performed by Student’s t-test. A probability value of 0.05 or less was considered to be significant. The software Sigma Plot (version 8.0, Systat Software, San Jose, CA, USA) was used for the statistical analysis. 4. 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. Supplementary Materials: HRESIMS, 1 H, 13 C, DEPT, HMQC, COSY, HMBC and NOESY spectra of new compounds 1–6, and 1 H NMR spectra of (+)-sarcophytoxide and sarcophytonin after different treatments are available online at http://www.mdpi.com/1660-3397/16/8/276/s1. Figure S1: HRESIMS spectrum of 1, Figure S2: 1 H NMR spectrum of 1 in CDCl3 , Figure S3: 13 C NMR spectrum of 1 in CDCl3 , Figure S4: HSQC spectrum of 1 in CDCl3 , Figure S5: 1 H-1 HCOSY spectrum of 1 in CDCl3 , Figure S6: HMBC spectrum of 1 in CDCl3 , Figure S7: NOESY spectrum of 1 in CDCl3 , Figure S8: HRESIMS spectrum of 2, Figure S9: 1 H NMR spectrum of 2 in CDCl3 , Figure S10: 13 C NMR spectrum of 2 in CDCl3 , Figure S11: HSQC spectrum of 2 in CDCl3 , Figure S12: 1 H-1 HCOSY spectrum of 2 in CDCl , Figure S13: HMBC spectrum of 2 in CDCl , Figure S14: NOESY spectrum of 3 3 2 in CDCl3 , Figure S15: HRESIMS spectrum of 3, Figure S16: 1 H NMR spectrum of 3 in C6 D6 , Figure S17: 13 C NMR spectrum of 1 in C6 D6 , Figure S18: HSQC spectrum of 1 in C6 D6 , Figure S19: 1 H-1 HCOSY spectrum of 3 in C6 D6 , Figure S20: HMBC spectrum of 3 in C6 D6 , Figure S21: NOESY spectrum of 3 in C6 D6 , Figure S22: HRESIMS spectrum of 4, Figure S23: 1 H NMR spectrum of 4 in C6 D6 , Figure S24: 13 C NMR spectrum of 4 in C6 D6 , Figure S25: HSQC spectrum of 4 in C6 D6 , Figure S26: 1 H-1 HCOSY spectrum of 4 in C6 D6 , Figure S27: HMBC spectrum of 4 in C6 D6 , Figure S28: NOESY spectrum of 4 in C6 D6 , Figure S29: HRESIMS spectrum of 5, Figure S30: 1 H NMR spectrum of 5 in C6 D6 , Figure S31: 13 C NMR spectrum of 5 in C6 D6 , Figure S32: HSQC spectrum of 5 in C6 D6 , Figure S33: 1 H-1 HCOSY spectrum of 5 in C6 D6 , Figure S34: HMBC spectrum of 5 in C6 D6 , Figure S35: NOESY spectrum of 5 in C6 D6 , Figure S36: HRESIMS spectrum of 6, Figure S37: ESIMS spectrum of 6, S38: 1 H NMR spectrum of 6 in CDCl3 , Figure S39: 13 C NMR spectrum of 6 in CDCl3 , Figure S40: HSQC spectrum of 6 in CDCl3 , Figure S41: 1 H-1 HCOSY spectrum of 6 in CDCl3 , Figure S42: HMBC spectrum of 6 in CDCl3 , Figure S43: NOESY spectrum of 6 in CDCl3 , Figure S44: 1 H NMR spectrum of (+)-sarcophytoxide in CDCl3 before treatment with acetone and silica gel under air, Figure S45. 1 H NMR spectrum of (+)-sarcophytoxide
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in CDCl3 after treatment with acetone and silica gel under air, Figure S46. 1 H NMR spectrum of sarcophytonin A in CDCl3 before treatment with acetone and silica gel under air, Figure S47. 1 H NMR spectrum of sarcophytonin A in CDCl3 after treatment with acetone and silica gel under air. Author Contributions: J.-H.S. conceived and guided the whole experiment. C.-C.P. isolated the compounds, and performed spectroscopic data measurement and analysis, and structure interpretation. C.-Y.H. and A.F.A. performed spectroscopic data analysis, confirmation of structures and preparation of the manuscript. T.-L.H. performed the anti-inflammatory assay. C.-F.D. contributed to species identification of the soft coral. Funding: This research was funded by Ministry of Science and Technology of Taiwan (MOST102-2113-M-110-001-MY2, 104-2113-M-110-006, and 104-2811-M-110-026) and International Scientific Partnership Program (ISPP) at King Saud University, Saudi Arabia (ISPP-116). Acknowledgments: Financial supported was mainly provided by the Ministry of Science and Technology (MOST102-2113-M-110-001-MY2, 104-2113-M-110-006, and 104-2811-M-110-026) to J.-H.S. The authors extend their appreciation to the International Scientific Partnership Program ISPP at King Saud University for funding this research work through ISPP-116. Conflicts of Interest: The authors declare no conflict of interest.
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