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Article Casbane Diterpenes from Red Sea Coral Sinularia polydactyla

Mohamed-Elamir F. Hegazy 1, Tarik A. Mohamed 1, Abdelsamed I. Elshamy 2, Montaser A. Al-Hammady 3, Shinji Ohta 4 and Paul W. Paré 5,*

1 Department of Phytochemistry, National Research Centre, El-Tahrir Street, Dokki, Giza 12622, Egypt; [email protected] (M.-E.F.H.); [email protected] (T.A.M.) 2 Department of Natural Compound Chemistry, National Research Centre, El-Tahrir Street, Dokki, Giza 12622, Egypt; [email protected] 3 National Institute of Oceanography and Fisheries, Red Sea Branch, Hurghada 84511, Egypt; [email protected] 4 Graduate School of Biosphere Science, Hiroshima University, 1-7-1 Kagamiyama, Higashi-Hiroshima 739-8521, Japan; [email protected] 5 Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, TX 79409, USA * Correspondence: [email protected]; Tel.: +1-806-834-0461; Fax: +1-806-742-1289

Academic Editor: Derek J. McPhee Received: 11 February 2016 ; Accepted: 29 February 2016 ; Published: 3 March 2016

Abstract: The soft coral genus Sinularia is a rich source of bioactive metabolites containing a diverse array of chemical structures. A solvent extract of Sinularia polydactyla resulted in the isolation of three new casbane diterpenes: sinularcasbane M (1), sinularcasbane N (2) and sinularcasbane O (3); in addition, known metabolites (4–5) were isolated. Compounds were elucidated on the basis of spectroscopic analyses; the absolute configuration was confirmed by X-ray analysis.

Keywords: soft coral; alcyoniidae; Sinularia polydactyla; diterpenes

1. Introduction In Alcyonacean soft coral, the genus Sinularia is a rich source of diverse natural products with over 500 metabolites including sesquiterpenes, diterpenes, polyhydroxylated steroids, alkaloids and polyamines already having been chemically characterized [1–5]. Sinularia consists of almost 90 species of which approximately 50 have been profiled for biological activity; such studies have established that the genus is a rich source of secondary metabolites with biological properties including cytotoxic, anti-inflammatory and antimicrobial activities [5–10]. While Sinularia species occur in marine waters around the world, S. polydactylais (Figure1) is endemic to the Red Sea. Although the marine environment contains extensive reef formations, this ecosystem is not as well characterized in terms of chemical studies of marine organisms compared to other large coral reef systems [11]. As a part of a comprehensive chemical inventory of marine natural products from Red Sea soft coral [12–14], herein, we reported the chemical characterization of solvent-extracted casbane diterpenes from S. polydactyla (Figure2). Casbane-type diterpene structures are related to the 14-membered cembrane ring system except for the dimethyl-cyclopropyl moiety instead of an isopropyl residue fused to the ring. These extremely-rare natural products are predominantly isolated from select coral species [15]. Several soft coral casbanes have been isolated from Sinularia depressa with biologically active 10-hydroxydepressin exhibiting cytotoxicity against several tumor cell lines with IC50 values near 50 µM[16].

Molecules 2016, 21, 308; doi:10.3390/molecules21030308 www.mdpi.com/journal/molecules Molecules 2016, 21, 308 2 of 8 Molecules 2016, 21, 308 2 of 8 Molecules 2016, 21, 308 2 of 8

Figure 1. Soft coral Sinularia polydactyla. FigureFigure 1. 1.Soft Soft coralcoral Sinularia polydactyla polydactyla. .

18 18 18 18 16 O OH OH H 16 O H 4 OH4 OH H H 4 4 16 3 16 6 3 3 6 3 7 7 6 6 4 6 6 3 H H 7 4 7 7 3 HOHO 5 5 2 2 H 7 5 2 5 5 2 H 5 2 15 2 15 15 1717 O 15 1 1 O 1 1 8 8 19 19 11 88 1818 OO 14 14 15 15 8 8 13 1616 HH 11 11 13 17 17 9 9 99 1111 1313 10 10 13 13 1010 12 1414 9 9 14 14 11 11 12 12 1010 12 12 HH 17 17 OH HH O O 19 19 20 O 20 20 O 1 2 3 1 2 O 3 O 18 O O 6 6 18 O HH O HH 5 7 8 4 5 7 8 H 3 4 H 2 3 O 17 2 H O O 17 13 H 11 O 9 O 14 13 12 11 O 9 1 14 12 15 1 H 15 H H 10 HHH 10 16 HH19 H O 16 19 O H O O O O O O 4 5 4 5 FigureFigure 2. 2.Structures Structures of metabolites metabolites 1–15–. 5. Figure 2. Structures of metabolites 1–5. 2. Results2. Results and and Discussion Discussion 2. Results and Discussion 25 Compound 1 was obtained as a colorless oil with a negative optical rotation ([α]D −25 9.6 in MeOH). 25 Compound 1 was obtained as a colorless oil with a negative optical rotation+ (rαsD ´ 9.6 in MeOH). HR-ESI-FTMSCompound analysis1 was obtained exhibited as aa molecularcolorless oil ion with peak a negativeat m/z 327.2293 optical [M rotation + Na] ([ correspondingα]D − 9.6 in MeOH). to a + HR-ESI-FTMS analysis exhibited a molecular ion peak at m/z 327.2293 [M + Na]+ corresponding to a HR-ESI-FTMSmolecular formula analysis of Cexhibited20H32O2 (calcd. a molecular for C20H 32ionO2 Na,peak 327.2300) at m/z 327.2293with five [Mdegrees + Na] of unsaturation.corresponding The to a molecularmolecularIR spectrum formula formula exhibited of of C C2020 bandsHH32OO2 for2 (calcd.(calcd. hydroxyl for for C and20 CH20 32olefinicHO322Na,O2 Na,substituents327.2300) 327.2300) with at 3409, withfive degreesand five 1650 degrees of cm unsaturation.−l, respectively. of unsaturation. The ´1 TheIR IRThespectrum spectrum 1 H-NMR exhibited exhibitedspectrum bands showed bands for hydroxyl four for hydroxylmethyl and olefinic signals, and substituents including olefinic substituents two at 3409,olefinic and methyls at 1650 3409, cm at− andlδ, Hrespectively. (1.56 1650 brs cm , 1 respectively.Theand 1 H-NMR 1.74 Thebrs, spectrumeachH-NMR 3H) and showed spectrum two tertiary four showed methyl methyls four signals, at methyl δH (1.00 including signals, s and 1.06 two including s, olefinic each 3H). two methyls Olefinic olefinic at protons δ methylsH (1.56 at brs at δ H (1.56andδ brsH 1.74 5.02 and brs, (brd, 1.74 each J = brs, 8.3 3H) Hz) each and and 3H)two 5.19 andtertiary (brd, two J methyls= tertiary 8.3 Hz) at were methyls δH (1.00attributed ats andδH to (1.001.06 two s, strisubstituted each and 1.063H). s,Olefinic eachdouble 3H). protons bonds; Olefinic at exomethylene singlets were observed at δH 4.89 and 5.04. In addition, two secondary oxygenated protonsδH 5.02 at (brd,δH 5.02J = 8.3 (brd, Hz) Jand= 8.3 5.19 Hz) (brd, and J =5.19 8.3 Hz) (brd, wereJ = attributed 8.3 Hz) were to two attributed trisubstituted to two double trisubstituted bonds; proton signals appeared at δH 3.93 (brd, J = 11.1 Hz) and 4.39 (dd, J = 10.0, 4.3 Hz). The 13C-NMR doubleexomethylene bonds; exomethylene singlets were singletsobserved were at δ observedH 4.89 and at 5.04.δH 4.89In addition, and 5.04. two In addition,secondary two oxygenated secondary spectrum displayed 20 carbon resonances which were classified by DEPT spectra as four methyls,13 six oxygenatedproton signals proton appeared signals at appeared δH 3.93 (brd, at δH J 3.93= 11.1 (brd, Hz)J and= 11.1 4.39 Hz) (dd, and J = 4.39 10.0, (dd, 4.3 JHz).= 10.0, The 4.3 C-NMR Hz). The methylenes, six methines and four quaternary carbons. In addition, two oxygenated carbons at δC 13C-NMRspectrum spectrum displayed displayed 20 carbon 20resonances carbon resonances which werewhich classified were by classifiedDEPT spectra byDEPT as four spectra methyls, as six four 71.2 and 77.0 and six olefinic carbons at δC 125.4, 137.9, 124.4, 136.9, 154.0, and 109.8 were observed. methylenes, six methines and four quaternary carbons. In addition, two oxygenated carbons at δC methyls,A casbane-type six methylenes, diterpene six skeleton methines was and predicted four quaternary based on NMR carbons. data Incomparisons addition, of two analogous oxygenated 71.2 and 77.0δ and six olefinic carbons at δC 125.4, 137.9, δ124.4, 136.9, 154.0, and 109.8 were observed. carbonsstructures at C 71.2previously and 77.0 isolated and from six olefinicthe same carbonsgenus [4,15,16]. at C 125.4, Characteristic 137.9, 124.4,signals 136.9,at δC 25.0, 154.0, 31.5, and and109.8 A casbane-type diterpene skeleton was predicted based on NMR data comparisons of analogous were20.1 observed. were proposed A casbane-type to constitute diterpene a cyclopropane skeleton moiety was assigned predicted to C-1, based C-14 onand NMR C-15, respectively data comparisons by structures previously isolated from the same genus [4,15,16]. Characteristic signals at δC 25.0, 31.5, and of analogousDQF-COSY structures and HMBC previously analyses. The isolated DQF-COSY from showed the same correlations genus [4 ,between15,16]. Characteristic signals at δH 0.69 signals (td, at 20.1 were proposed to constitute a cyclopropane moiety assigned to C-1, C-14 and C-15, respectively by δC 25.0,J = 10.9, 31.5, 2.6 and Hz) 20.1 and were 0.67 (td, proposed J = 10.9, to 4.0 constitute Hz) with methylene a cyclopropane mutiplet moiety signals assigned δH 1.26 and to C-1,1.80/2.19, C-14 and DQF-COSY and HMBC analyses. The DQF-COSY showed correlations between signals at δH 0.69 (td, C-15,respectively. respectively Additionally, by DQF-COSY HMBC and data HMBC confirmed analyses. these The carbon DQF-COSY assignments showed with correlationscorrelations with between J = signals10.9, 2.6 at δHz)H 0.69 and (td, 0.67 J = 10.9 (td, and J = 10.9,2.6, H-1), 4.0 Hz)0.67 (td,with J = methylene 10.9 and 4.0 mutiplet Hz, H-14), signals 1.26 (m, δ H 1.262-2) and and 1.80/2.19 1.80/2.19, signals at δH 0.69 (td, J = 10.9, 2.6 Hz) and 0.67 (td, J = 10.9, 4.0 Hz) with methylene mutiplet signals respectively.(m, H2-13). Additionally,Two singlet signals HMBC at δdataC 15.8 confirmed and 28.7 were these assigned carbon toassignments methyl groups with at correlations C-16 and C-17 with δH 1.26 and 1.80/2.19, respectively. Additionally, HMBC data confirmed these carbon assignments signalsrespectively at δH 0.69 and (td, attached J = 10.9 andto C-15 2.6, H-1),based 0.67 on HMBC(td, J = 10.9correlations and 4.0 Hz,(Figure H-14), 3). 1.26The (m,olefinic H2-2) signal and 1.80/2.19 at δH with correlations with signals at δH 0.69 (td, J = 10.9 and 2.6 Hz, H-1), 0.67 (td, J = 10.9 and 4.0 Hz, (m, H2-13). Two singlet signals at δC 15.8 and 28.7 were assigned to methyl groups at C-16 and C-17 H-14), 1.26 (m, H -2) and 1.80/2.19 (m, H -13). Two singlet signals at δ 15.8 and 28.7 were assigned respectively and2 attached to C-15 based 2on HMBC correlations (FigureC 3). The olefinic signal at δH to methyl groups at C-16 and C-17 respectively and attached to C-15 based on HMBC correlations Molecules 2016, 21, 308 3 of 8

Molecules 2016, 21, 308 3 of 8 (Figure3). The olefinic signal at δH 5.19 (d, J = 8.3 Hz) was assigned to H-3 based on an observed DQF-COSY5.19 (d, Jcorrelation = 8.3 Hz) was with assigned H-2 andto H-3 HMBC based correlationon an observed between DQF-COSY H-3 andcorrelation an olefinic with H-2 methyl and and oxygenatedHMBC correlation methine atbetweenδC 10.5 H-3 (C-18) and an and olefinic 77.0 (C-5),methyl respectively. and oxygenated Additionally, methine at δC HMBC 10.5 (C-18) correlations and allowed77.0 for(C-5), the respectively. assignment Additionally, of H-3 (δH 5.19),HMBC H corr3-18elations (δH 1.74), allowed and for H-5 the (δ Hassignment4.39), (Figure of H-33). (δ DQF-COSYH 5.19), H3-18 (δH 1.74), and H-5 (δH 4.39), (Figure 3). DQF-COSY correlations with H-5 allowed for a methylene correlations with H-5 allowed for a methylene signal assignment at δH 1.60 (ddd, J = 13.8, 10.0, 2.2 Hz, signal assignment at δH 1.60 (ddd, J = 13.8, 10.0, 2.2 Hz, H-6)/1.94 (ddd, J = 13.8, 10.0, 2.2 Hz, H-6) as H-6)/1.94 (ddd, J = 13.8, 10.0, 2.2 Hz, H-6) as well as the oxygenated signal at δH 3.93 (brd, J = 11.1 Hz, well as the oxygenated signal at δH 3.93 (brd, J = 11.1 Hz, H-7). C-7 (δC 71.2) exhibited a HMBC H-7). C-7 (δC 71.2) exhibited a HMBC correlation with exocyclic broad singlets at δH 4.89 (H-19) and correlation with exocyclic broad singlets at δH 4.89 (H-19) and 5.04 (H-19). HMBC correlations were 5.04 (H-19). HMBC correlations were observed between C-19 and δH 2.07 (m, H-9)/2.19 (m, H-9) as observed between C-19 and δH 2.07 (m, H-9)/2.19 (m, H-9) as well as between H-9 and δC 154 (C-8). well as between H-9 and δ 154 (C-8). DQF-COSY correlation between H-9 and δ 2.07/2.19 (m) and DQF-COSY correlation Cbetween H-9 and δH 2.07/2.19 (m) and 5.02 (brd, J = 8.3 Hz) Hallowed for the 5.02 (brd,assignmentsJ = 8.3 of Hz) H-10 allowed and H-11, for respectively the assignments and the ofendocyclic H-10 and proton H-11, H-11 respectively coupled with and δC 40.7 the endocyclic(C-13) protonin the H-11 HMBC coupled spectrum. with Finally,δC 40.7 HMBC (C-13) corre in thelations HMBC were spectrum. observed between Finally, H-11 HMBC and correlationsδC 15.8 (C-20) were observedas well between as H-20 H-11and endocyclic and δC 15.8 signal (C-20) δC 136.0 as well (C-12). as H-20 and endocyclic signal δC 136.0 (C-12).

18 OH OH 4 6 3 HO 7 5 2 1 8 19

15 9 16 10 13 14 11 12 17 OH 20 1 O 2

O O

O O 3 Figure 3. Selected 1H-1H COSY (__) and HMBC (→) correlations of 1–3. Figure 3. Selected 1H-1H COSY (—) and HMBC (Ñ) correlations of 1–3. NOESY correlations observed between H-1, H-14 and H-16, indicated protons oriented on the NOESYsame side correlations consistent with observed a cis ring between junction. H-1,The spectrum H-14 and showed H-16, indicatedtwo proton protonssignals attributed oriented to on the same1,2-cyclopropane side consistent moiety with a atcis δHring 0.69 junction. (td, J = 10.9, The 2.6 spectrum Hz, H-1) and showed 0.67 (td, two J = proton 10.9, 4.0 signals Hz, H-14). attributed Due to to the flexible nature of the 14-membered macrocyclic ring, the relative configuration of the hydroxyl 1,2-cyclopropane moiety at δH 0.69 (td, J = 10.9, 2.6 Hz, H-1) and 0.67 (td, J = 10.9, 4.0 Hz, H-14). Due to thegroups flexible at C-5 nature and ofC-7 the could 14-membered not be determined macrocyclic by NOESY; ring, however the relative coupling configuration constants for of H-5/H-6a the hydroxyl and H-6b/H-7 of 4.3 and 11.1 Hz respectively, indicated an cis-configuration for the hydroxyls. H-1α, groups at C-5 and C-7 could not be determined by NOESY; however coupling constants for H-5/H-6a H-3 and H-5 NOE correlations led to an assignment of H-5α and H-7α (Figure 4) which was confirmed and H-6b/H-7 of 4.3 and 11.1 Hz respectively, indicated an cis-configuration for the hydroxyls. H-1α, by chemical shifts of δC 15.6 and 28.7 for C-16 and C-17 methyl groups respectively, and consistent H-3 andwith H-5 previously NOE correlations reported cis led-fused to ancasbane assignment derivatives of H-5 [4,15].α and Based H-7 onα (Figure this spectral4) which analysis, was confirmed1 was by chemicalidentified shifts as 5β,7 ofβδ-dihydroxy-1C 15.6 andα 28.7,14α for-casba-3,8(19),11-triene, C-16 and C-17 methyl named groups here as respectively, a sinularcasbane and M. consistent 25 with previouslyCompound reported 2 was cisobtained-fused as casbane colorless derivatives crystals with [4 a,15 negative]. Based optical on this rotation spectral ([α] analysis,D − 15.5 in1 was identifiedMeOH). as HR-ESI-FTMS 5β,7β-dihydroxy-1 analysisα showed,14α-casba-3,8(19),11-triene, a molecular ion peak at namedm/z 327.2295 here [M as + a Na] sinularcasbane+ corresponding M. 25 to a molecular formula of C20H32O2 (calcd. for C20H32O2Na, 327.2300) with five degrees of unsaturation. Compound 2 was obtained as colorless crystals with a negative optical rotation (rαsD ´ 15.5 in MeOH).The HR-ESI-FTMSIR spectrum exhibited analysis bands showed for hydroxyl a molecular and olefinic ion peak groups at m at/z 3409,327.2295 and 1650 [M +cm Na]−l, respectively.+ corresponding Spectroscopic data was similar to 1 except for oxygenated and cyclopropane proton signals. The to a molecular formula of C20H32O2 (calcd. for C20H32O2Na, 327.2300) with five degrees of DQF-COSY spectrum showed a correlation between one of the cyclopropane signal δH 1.28 (dd, J = 10.1, unsaturation. The IR spectrum exhibited bands for hydroxyl and olefinic groups at 3409, and 1650 cm´1, 8.9 Hz) and the olefinic signal at δH 5.09 (dd, J = 10.1, 1.2 Hz) introducing the possibility of the respectively. Spectroscopic data was similar to 1 except for oxygenated and cyclopropane proton cyclopropane ring or double bond shifting. The second cyclopropane signal at δH 0.77 (ddd, J = 11.9, signals. The DQF-COSY spectrum showed a correlation between one of the cyclopropane signal 8.9, 2.7 Hz) correlates with the methylene protons at δH 1.13 (1H, dddd, J = 13.4, 12.8, 11.9, 3.4 Hz) and δH 1.281.55 (dd,(1H, Jdddd,= 10.1, J = 8.914.0, Hz) 13.4, and 4.9, the2.7 Hz) olefinic which signal is consistent at δH 5.09 with (dd, the cyclopropaneJ = 10.1, 1.2 moiety Hz) introducing being the possibilityshifted to C-1 of and the C-2 cyclopropane and the olefinic ring bond or doubleremaining bond at C-3 shifting. and C-4. The Also second consistent cyclopropane with this shift, signal at δHthe0.77 olefinic (ddd, protonJ = 11.9, H-3 8.9, correlates 2.7 Hz) with correlates an oxygenated with the signal methylene at δC 79.5 protons in the atHMBCδH 1.13 spectrum (1H, dddd, J = 13.4,allowing 12.8, for 11.9, a hydroxyl 3.4 Hz) group and 1.55to be (1H, located dddd, at C-5J =(δ 14.0,H 3.97, 13.4, dd, J 4.9,= 10.7, 2.7 4.6 Hz) Hz). which H-5 correlated is consistent with with the cyclopropane moiety being shifted to C-1 and C-2 and the olefinic bond remaining at C-3 and C-4. Also consistent with this shift, the olefinic proton H-3 correlates with an oxygenated signal Molecules 2016, 21, 308 4 of 8

at δC 79.5 in the HMBC spectrum allowing for a hydroxyl group to be located at C-5 (δH 3.97, dd, Molecules 2016, 21, 308 4 of 8 J = 10.7, 4.6 Hz). H-5 correlated with the methylene signals at δH 2.31 (ddd, J = 13.4, 10.7, 7.9 Hz)/2.38 (brdd,MoleculesJ = 13.4,2016, 21 4.6, 308 Hz) in DQF-COSY, allowing for the assignment of H-6. H-6 showed a correlation4 of 8 the methylene signals at δH 2.31 (ddd, J = 13.4, 10.7, 7.9 Hz)/2.38 (brdd, J = 13.4, 4.6 Hz) in DQF-COSY, with the olefinic methine signal at δH 4.86 (brdd, J = 7.9, 1.2 Hz, H-7) as well as the broad methyl allowingthe methylene for the signals assignment at δH 2.31of H-6. (ddd, H-6 J showed= 13.4, 10.7, a correlation 7.9 Hz)/2.38 with (brdd, the olefinic J = 13.4, methine 4.6 Hz) signal in DQF-COSY, at δH 4.86 singlet at δH 1.63 (H3-19) in DQF-COSY and HMBC, respectively. Similar spectra for 1 and 2 allowed (brdd,allowing J = 7.9,for the1.2 Hz,assignment H-7) as well of H-6. as the H-6 broad showed methyl a correlation singlet at withδH 1.63 the (H olefinic3-19) in methineDQF-COSY signal and at HMBC, δH 4.86 for the exomethylene to be assigned to C-12/C-20. HMBC correlation between C-12 and δ 3.90 respectively.(brdd, J = 7.9, 1.2Similar Hz, H-7) spectra as well for as1 andthe broad 2 allowed methyl for singlet the exomethylene at δH 1.63 (H3-19) to bein DQF-COSYassigned to and C-12/C-20. HMBC,H (dd, 7.6, 5.2 Hz, H-11) as well DQF-COSY correlations between H-11 and δH 1.52/1.66 (m, H-10) and HMBCrespectively. correlation Similar between spectra C-12 for 1 and and δ H2 3.90allowed (dd, for7.6, the 5.2 exomethyleneHz, H-11) as well to be DQF-COSY assigned to correlations C-12/C-20. H-10 with δ 1.98 (dd, 15.6, 7.6, H-9)/2.09 (ddd, 15.6, 13.1, 3.1, H-9) established methylene groups betweenHMBC correlation H-11H and δbetweenH 1.52/1.66 C-12 (m, and H-10) δH 3.90 and (dd, H-10 7.6, with 5.2 δHz,H 1.98 H-11) (dd, as 15.6, well 7.6, DQF-COSY H-9)/2.09 correlations (ddd, 15.6, δ at C-9-C-1013.1,between 3.1, H-9)H-11 and established aand hydroxyl δH 1.52/1.66 methylene at C-11. (m, AH-10) groups HMBC and at correlationC-9-C-10H-10 with and δ betweenH a1.98 hydroxyl (dd, C-20 15.6, at andC-11. 7.6, AHH-9)/2.09 HMBC1.66 (m, correlation(ddd, H-13)/2.14 15.6, δ (ddd,between13.1, 12.8, 3.1, 12.8,C-20H-9) andestablished 4.9, δ H-13)H 1.66 along (m,methylene H-13)/2.14 with DQF-COSYgroups (ddd, at 12.8, C-9-C-10 correlations 12.8, 4.9, and H-13) a hydroxyl between along withat H-13 C-11. DQF-COSY and A HMBCH 1.13 correlations correlation (dddd, 13.4, 12.8,between 11.9, 3.4, C-20H-13 H-14)/1.55 andand δ δHH 1.66 1.13 (dddd,(m, (dddd, H-13)/2.14 14.0, 13.4,13.4, 12.8, (ddd, 4.9,11.9 12.8, 2.7,, 3.4, 12.8, H-14), H-14)/1.55 4.9, H-13)δH 0.77 (dddd, along (ddd, with 14.0 11.9, ,DQF-COSY 13.4, 8.9, 4.9, 2.7, 2.7, correlations H-1) H-14), and δHδ H 1.280.77between (dd, (ddd, 10.1, H-13 11.9, 8.9, and 8.9, H-2) δ2.7,H allowed1.13 H-1) (dddd, and for δ 13.4, theH 1.28 assignment 12.8, (dd, 11.9 10.1,, 3.4, 8.9, of theH-14)/1.55 H-2) other allow NMR (dddd,ed for signals the14.0 assignment, 13.4, (Figure 4.9,3 2.7,). of The theH-14), relativeother δH configurationNMR0.77 (ddd, signals 11.9, of (Figure hydroxyl 8.9, 2.7, 3). The groupH-1) relative and at C-11δH configuration1.28 was (dd, based 10.1, onof 8.9, hydroxyl biogenetic H-2) allow group considerationsed atfor C-11 the wasassignment of based casbane on of biogenetic derivativesthe other publishedconsiderationsNMR signals by Yin, (Figure ofet casbane al. 3).in The 2013, derivatives relative Yang, configuration etpublished al., 2015, by and ofYin, hydroxyl Li, etet al. al. inin group2013, 2010 Yang, at [4 C-11,15 et,16 wasal.],, which2015, based and wason biogeneticLi, supported et al. in by2010considerations coupling [4,15,16], constant whichof casbane and was NOESY derivativessupported experiments publishedby coupling by to constantYin, be in et theal. and inβ 2013, position.NOESY Yang, experiments Consequently,et al., 2015, andto be Li, the in et relativethe al. inβ configurationsposition.2010 [4,15,16], Consequently, of which H-1 andwas the H-2supported relative showed configurations by interaction coupling constantof with H-1 H-16and and H-2 inNOESY NOESYshowed experiments interaction experiments to with be to inH-16 be the in inβ the α positionNOESYposition. experiments (FigureConsequently,3). Absoluteto be the in relativethe configuration α position configurations (Figure was 3). establishedof Absolute H-1 and confH-2 byiguration X-rayshowed crystallography wasinteraction established with (Figureby H-16 X-ray in5 ). Therefore,crystallographyNOESY experiments2 was assigned(Figure to be5). as inTherefore, 5 Sthe,11 αR position-dihydroxy-1 2 was (Figure assignedS ,23).R Absolute-casba-3,7,12(20)-triene, as 5S,11 confR-dihydroxy-1iguration wasS a,2 established newR-casba-3,7,12(20)- natural by product X-ray crystallography (Figure 5). Therefore, 2 was assigned as 5S,11R-dihydroxy-1S,2R-casba-3,7,12(20)- namedtriene, here a new as sinularcasbanenatural product N.named here as sinularcasbane N. triene, a new natural product named here as sinularcasbane N.

FigureFigure 4. 4.Energy-minimized Energy-minimized 3D3D structurestructure and NOESY correlations correlations (→ (Ñ) for) for 1–13–. 3. Figure 4. Energy-minimized 3D structure and NOESY correlations (→) for 1–3.

Figure 5. ORTEP depictions of 2 with oxygens (O1–O5) labeled in red. FigureFigure 5. 5.ORTEP ORTEP depictions depictions ofof 22 withwith oxygens (O1–O5) labeled labeled in in red. red. 25 Compound 3 was obtained as a colorless oil with a positive optical rotation ([α]D + 10.2 in 25 MeOH).Compound HR-ESI-FTMS 3 was analysisobtained showed as a colorless a molecular oil with ion apeak positive at m /zoptical 355.1516 rotation [M + Na]([α]+D (calcd. + 10.2 for in MeOH). HR-ESI-FTMS analysis showed a molecular ion peak at m/z 355.1516 [M + Na]+ (calcd. for Molecules 2016, 21, 308 5 of 8

25 Compound 3 was obtained as a colorless oil with a positive optical rotation (rαsD + 10.2 in MeOH). HR-ESI-FTMS analysis showed a molecular ion peak at m/z 355.1516 [M + Na]+ (calcd. for C19H24O5Na: 355.1521), consistent with a molecular formula C19H24O5, indicating eight degrees of unsaturation. Spectroscopic data was similar to previously reported 4 by Saitman, et al. in 2011, except around C-5, raising the possibility that 3 was an epimer of 4. Spectral shift differences included a downfield shift of H-5 (δH 4.42 d, J = 10.3 Hz) and an upfield shift of H-4 (2.61 and 2.49); downfield shifts for C-5, C-7, and C-4 in comparison with 4 were also observed [17–19]. The relative configuration of the four chiral centers at C-1, C-5, C-8, and C-10 were also determined by 13C signal comparisons as well as observed NOESY correlations (Figure4). Assignment of H-1 to a β-orientation was based on Alcyonacea biogenesis of cembrane-type compounds [5,19]. In the NOESY spectrum, H-1 correlates with H-14β (δH 1.75 m) and one proton of C-2 methylene (δH 2.24, H-2β). NOESY correlations between H-5 (δH 4.42, d) with H-11α at (δH 7.20, s), H-4α at (δH 2.57, m), and H-10α at (δH 5.15 s) confirmed an α-orientation for H-5, indicating that epimerization occurs at C-5. Therefore, the norcembranoid was assigned to be the epimer of scabrolide F (sinularcasbane O). In addition, previously reported metabolites scabrolide F (4)[18] and ineleganolide (5)[20] were identified by NMR and MS comparisons with literature values.

3. Materials and Methods

3.1. General Information Specific rotation was measured with a Horiba SEPA-300 digital polarimeter (Kyoto, Japan, l = 5 cm) and IR spectra were collected on a Shimadzu FTIR-8100 spectrometer (Kyoto, Japan). For X-ray, a Bruker SMART-APEX II ULTRA (Billerica, MA, USA) was used. ESI-MS and HR-ESI-MS were carried out using a Thermo Fisher Scientific LTQ Orbitrap XL mass spectrometer (Waltham, MA, USA), and 1H (600 MHz) and 13C (150 MHz) NMR spectra were recorded on a JEOL JNM-ECA 600 spectrometer (Tokyo, Japan) with tetramethylsilane as an internal standard. Purification was run on a Shimadzu HPLC system equipped with a RID-10A refractive index detector and compound separation was performed on YMC-Pack ODS-A (Tokyo, Japan, 250 ˆ 4.6 mm i.d.) and (250 ˆ 20 mm i.d.) columns for analytical and preparative separation, respectively. Chromatography separation included normal-phase silica BW-200 (Fuji Silysia Chemical, Ltd., Kasugai, Japan, 150–350 mesh) and ODS reverse phase Chromatorex DM1020T (Fuji Silysia Chemical, Ltd., 100–200 mesh) columns as well as silica gel 60F254 (Merck, Darmstadt, Germany, 0.25 mm) and RP-18 WF254S (Merck, 0.25 mm) TLC plates with spots developed with heating of H2SO4-MeOH (1:9) sprayed plates.

3.2. Animal Material Soft coral S. polydactyla was collected from the Egyptian Red Sea off the coast of Hurghada in March 2013. The soft coral was identified by Montaser A. Al-Hammady with a voucher specimen (03RS100) deposited in the National Institute of Oceanography and Fisheries, marine biological station, Hurghada, Egypt.

3.3. Extraction and Separation Frozen soft coral (6.5 kg, total wet weight) was chopped into small pieces and extracted with methylene chloride/methanol (1:1) at room temperature (4 L ˆ 5 times). The combined extracts were concentrated in vacuo to a brown gum. The dried material (243 g) was subjected to gravity chromatography in a silica gel column (6 cm ˆ 120 cm) eluting with n-hexane (3000 mL) followed by a gradient of n-hexane-CH2Cl2 up to 100% CH2Cl2 and CH2Cl2–MeOH up to 50% MeOH (3000 mL each of the solvent mixture). The n-hexane/CH2Cl2 (1:2) fraction (2.2 g) eluted with n-hexane/EtOAc (6:1) was subjected to silica gel column separation. Fractions were obtained and combined into two main sub-fractions, A and B, according to a TLC profile. Sub-fraction A was re-purified by reversed-phase HPLC using MeOH/H2O (70%:30%) to afford 3 (11 mg), 4 (18 mg) and 5 (22 mg). Molecules 2016, 21, 308 6 of 8

Sub-fraction B was re-purified by reversed-phase HPLC (Shimadzu HPLC system equipped with a RID-10A refractive index detector and compound separation was performed on YMC-Pack ODS-A (250 ˆ 10 mm i.d.) column for separation) using MeOH/H2O (65%:35%) to afford 6 (10 mg). Sub-fraction C was re-purified by reversed-phase HPLC using MeOH/H2O (60%:30%) to afford 1 (11 mg) and 2 (18 mg).

25 1 13 Sinularcasbane M (1): colorless oil; rαsD = ´9.6 (c 0.01, CHCl3); H-NMR and C-NMR data, see Table1; + HR-ESI-FTMS [M + Na] m/z 327.2293 (calc. 327.2300, C20H32O2Na); also see Figure S1. 25 1 13 Sinularcasbane N (2): colorless crystal; rαsD = ´15.5 (c 0.01, CHCl3); H-NMR and C-NMR data, see + Table1; HR-ESI-FTMS [M + Na] m/z 327.2295 (calc. 327.2300, C20H32O2Na); also see Figure S1.

1 13 Table 1. H- and C-NMR spectral data recorded in CDCl3, at 600 and 150 MHz, respectively.

1 2 3 4 Position δH (J in Hz) δC δH (J in Hz) δC δH (J in Hz) δC δC 1 0.69 (td, 10.9, 2.6) 25.0 0.77 (ddd, 11.9, 8.9, 2.7) 31.1 2.58 m 40.8 38.8 2.94 d (3.42), 2 1.26 (m) 29.6 1.28 (dd, 10.1, 8.9) 25.2 50.2 50.2 2.96 d (2.04) 3 5.19 (brd, 8.3) 125.4 5.09 (dd, 10.1, 1.2) 125.6 208.4 208.4 4 137.9 136.8 2.47 m, 2.57 m 44.0 44.0 4.12 d (3.42), 5 4.39 ( dd, 10.0, 4.3) 77.0 3.97 (dd, 10.7, 4.6) 79.5 74.8 77.8 4.14 (2.76) 1.60 (ddd, 13.8, 11.1, 4.3) 40.2 2.31 (ddd, 13.4, 10.7, 7.9) 33.2 212.7 212.5 6 1.94 (ddd, 13.8, 10.0, 2.2) 2.38 (brdd, 13.4, 4.6) 7 3.93 (brd, 11.1) 71.2 4.86 (brdd, 7.9, 1.2) 119.5 2.21 m, 2.39 m 48.2 51.1 8 154.0 136.0 78.7 79.4 2.07 m 33.4 1.98 (dd, 15.6, 7.6) 34.3 2.11 m, 2.35 m 43.1 41.7 9 2.19 m 2.09 (ddd, 15.6, 13.1, 3.1) 2.07 m 24.8 1.52 (m) 34.2 5.11 s 78.3 79.0 10 2.19 m 1.66 (m) 11 5.02 (brd, 8.3) 124.4 3.90 (dd, 7.6, 5.2) 71.2 7.20 s 150.7 151.6 12 136.9 154.5 131.2 131.1 1.80 m 40.7 1.66 (m) 34.1 1.92 m, 2.59 m 20.8 20.1 13 2.19 m 2.14 (ddd, 12.8, 12.8, 4.9) 0.67 (td, 10.9, 4.0) 31.5 1.13 (dddd, 13.4, 12.8, 11.9, 3.4) 26.1 1.29 m, 1.75 m 27.6 29.2 14 1.55 (dddd, 14.0, 13.4, 4.9, 2.7) 15 20.1 20.1 145.8 145.4 16 1.00 s 15.6 1.02 s 15.5 4.67 s, 4.79 s 113.1 113.0 17 1.06 s 28.7 1.06 s 29.1 1.64 s 25.3 27.8 18 1.74 brs 10.5 1.71 (d, 1.2) 10.2 1.30 s 18.1 18.4 4.89 brs 109.8 1.63 (brs) 17.1 174.0 174.4 19 5.04 brs 1.56 brs 15.8 4.85 brs, 108.6 20 5.02 brs

X-ray Crystallography Data Single crystal X-ray analysis established the complete structure and absolute configuration of 2 and the crystal data are summarized as follows: C40H61O3, formula wt. 589.88, triclinic, space group, a = 9.437(4) Å, b = 9.499(4) Å, c = 10.543(4) Å, α = 98.763(6)˝, β = 95.390(5)˝, γ = 99.422(5)˝, volume = 914.6(6) Å, are based upon the refinement of the XYZ-centroids of 5460 reflections above 20 σ(I) with 5.389˝ < 2θ < 41.63˝. Data were corrected for absorption effects using the multi-scan method (SADABS). The ratio of minimum to maximum apparent transmission was 0.750. 3 3 3 3 Dcacld = 1.071 g/cm , crystal size 0.369 mm ˆ 0.344 mm ˆ 0.267 mm . A total of 720 frames were collected, exposure time was 0.40 h and the frames were integrated with the Bruker SAINT Software package (V8.34A; Bruker AXS Inc., Madison, Wisconsin, USA, 2013) using a narrow-frame algorithm. The integration of the data using a triclinic unit cell yielded a total of 5460 reflections to a maximum θ angle of 27.85˝ (0.76 Å resolution), of which 4717 were independent (average redundancy 1.158, completeness = 95.8%, Rint = 2.69%, Rsig = 6.64%) and 3571 (75.70%) were greater than 2σ (F2). CCDC 1407158 contains the supplementary crystallographic data for this paper. These data can be obtained Molecules 2016, 21, 308 7 of 8 free of charge via http://www.ccdc.cam.ac.uk/conts/retrieving.html (or from the CCDC, 12 Union Road, Cambridge CB2 1EZ, UK; Fax: +44 1223 336033; E-mail: [email protected].).

25 1 13 Sinularcasbane O (3): colorless oil; rαsD = +10.2 (c 0.01, CHCl3); H-NMR and C-NMR data, see + Table1; HR-ESI-FTMS [M + Na] m/z 355.1517 (calc. 355.1521 C19H24O5Na); also see Figure S1. 25 + Scabrolide F (4): rαsD = ´12.3 (c 0.01, CHCl3), HR-ESI-FTMS [M + Na] m/z 355.1516 (calc. 355.1521 C19H24O5Na). 25 + Ineleganolide (5): colorless oil; rαsD = ´35.0 (c 0.01, CHCl3), HR-ESI-FTMS [M + Na] m/z 353.1370 (calc. 353.1365 C19H22O5Na).

4. Conclusions The methylene chloride/methanol (1:1) extract from the Red Sea coral S. polydactyla afforded three new metabolites, sinularcasbane M (1), sinularcasbane N (2) and sinularcasbane O (3). The structures were elucidated by spectroscopic analyses; the absolute configuration of 2 was confirmed by X-ray analysis.

Supplementary Materials: Supplementary materials can be accessed at: http://www.mdpi.com/1420-3049/21/ 3/308/s1. Acknowledgments: This project was supported financially by the Science and Technology Development Fund (STDF), Egypt, Grant No. 1102. Author Contributions: Tarik A. Mohamed, Abdelsamed I. Elshamy contributed to extraction, isolation, identification and manuscript preparation. Montaser A. Al-Hammady contributed to soft coral collection and identification, Shinji Ohta and Paul W. Paré contributed to structure elucidation, guiding the experiments, analyses and manuscript writing. Mohamed-Elamir F. Hegazy is the project leader organizing and guiding the experiments, structure elucidation, and manuscript writing. Conflicts of Interest: The authors declare no conflict of interest.

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