marine drugs

Article New Furanocembranoids from violaceum

Pin-Chang Huang 1,2,†, Wen-Sou Lin 3,4,†, Bo-Rong Peng 2, Yu-Chia Chang 5, Lee-Shing Fang 6,7, Guo-Qiang Li 8,9 , Tsong-Long Hwang 5,10,11,12 , Zhi-Hong Wen 4,* and Ping-Jyun Sung 1,2,4,13,14,15,*

1 Graduate Institute of Marine Biology, National Dong Hwa University, Pingtung 94450, Taiwan; [email protected] 2 Department of Planning and Research, National Museum of Marine Biology and Aquarium, Pingtung 94450, Taiwan; [email protected] 3 Department of Neurology, Kaohsiung Armed Forces General Hospital, Kaohsiung 80284, Taiwan; [email protected] 4 Department of Marine Biotechnology and Resources, National Sun Yat-sen University, Kaohsiung 80424, Taiwan 5 Research Center for Chinese Herbal Medicine, Research Center for Food and Cosmetic Safety, Graduate Institute of Healthy Industry Technology, College of Human Ecology, Chang Gung University of Science and Technology, Taoyuan 33303, Taiwan; [email protected] (Y.-C.C.); [email protected] (T.-L.H.) 6 Center for Environmental Toxin and Emerging-Contaminant Research, Cheng Shiu University, Kaohsiung 83347, Taiwan; [email protected] 7 Super Micro Mass Research and Technology Center, Cheng Shiu University, Kaohsiung 83347, Taiwan 8 Key Laboratory of Marine Drugs, Chinese Ministry of Education, School of Medicine and Pharmacy, Ocean University of China, Qingdao 266033, China; [email protected]. 9 Laboratory of Marine Drugs and Biological Products, National Laboratory for Marine Science and Technology, Qingdao 266235, China 10 Graduate Institute of Natural Products, College of Medicine, Chang Gung University, Taoyuan 33302, Taiwan 11 Chinese Herbal Medicine Research Team, Healthy Aging Research Center, Chang Gung University, Taoyuan 33302, Taiwan 12 Department of Anaesthesiology, Chang Gung Memorial Hospital, Taoyuan 33305, Taiwan 13 Graduate Institute of Natural Products, Kaohsiung Medical University, Kaohsiung 80708, Taiwan 14 Research Center for Natural Products and Drug Development, Kaohsiung Medical University, Kaohsiung 80708, Taiwan 15 Chinese Medicine Research and Development Center, China Medical University Hospital, Taichung 40447, Taiwan * Correspondence: [email protected] (Z.-H.W.); [email protected] (P.-J.S.); Tel.: +886-7-525-2000 (ext. 5038) (Z.-H.W.); +886-8-882-5037 (P.-J.S.); Fax: +886-7-525-5021 (Z.-H.W.); +886-8-882-5087 (P.-J.S.) † These authors contributed equally to this work.

 Received: 17 February 2019; Accepted: 3 April 2019; Published: 5 April 2019 

Abstract: Three new furanocembranoids—briaviodiol F (1) and briaviotriols A (2) and B (3)—along with a known analogue, briaviodiol A (4), were obtained from a cultured-type octocoral Briareum violaceum. The structures of cembranoids 1–3 were elucidated by using spectroscopic methods. In vitro study demonstrated that compounds 2 and 4 exerted inhibition effects on inducible nitric oxide synthase (iNOS) release from RAW 264.7, a macrophage cell line that originated from a mouse monocyte macrophage, stimulated with lipopolysaccharides.

Keywords: Briareum violaceum; briaviodiol; briaviotriol; anti-inflammatory; iNOS

Mar. Drugs 2019, 17, 214; doi:10.3390/md17040214 www.mdpi.com/journal/marinedrugs Mar. Drugs 2019, 17, 214 2 of 9

1. Introduction

BriareumMar. Drugs violaceum 2019, 17, x (Quoy and Gaimard, 1883) is a soft of the family Briareidae2 of 9 [1,2], which has been found to contain cembrane-type diterpenoids in abundance [3–10]. Diterpenoids of this type have[3–10]. been Recently, reported in toour have research complicated into the structureschemical constituents and possess and a properties variety of of bioactivities a cultured [3–10]. Recently,octocoral in our B. research violaceum into, we thehave chemical isolated constituentsthree previously and unreported properties furanocembranoids— of a cultured octocoral briaviodiol F (1), and briaviotriols A (2) and B (3)—along with a known analogue, briaviodiol A (4) B. violaceum 1 [9] (Figure, we have1). A pro-inflammatory isolated three previously suppression unreportedassay was employed furanocembranoids— to assess the activities briaviodiol of these F ( ), and briaviotriolsisolated compounds A (2) and against B ( 3the)—along release of with inducibl a knowne nitric analogue,oxide synthase briaviodiol (iNOS) from A macrophage (4)[9] (Figure 1). A pro-inflammatorycells. suppression assay was employed to assess the activities of these isolated compounds against the release of inducible nitric oxide synthase (iNOS) from macrophage cells. 2. Results and Discussion

O O O

17 16 O 15 O O

2 1 3 14 OH OH OH R OCH3 OCH 18 OH OH 3 OH 4 H H HH HH 13 HO H 5 12 20 O O O 6 11 H H 9 H H 7 8 10 HO H H H 19 1: R = α-OCH3, 4: R = β-OCH3 2 3 B. violaceum

Figure 1. Structures of briaviodiol F (1), briaviotriols A (2) and B (3), and briaviodiol A (4), and a Figure 1. Structures of briaviodiol F (1), briaviotriols A (2) and B (3), and briaviodiol A (4), and a picture of the octocoral B. violaceum. picture of the octocoral B. violaceum. Briaviodiol F (1) was isolated as a colorless oil. Compound 1 displayed a pseudomolecular ion 2. Results and Discussion at m/z 403.20886 in the (+)-HRESIMS, which indicated its molecular formula was C21H32O6 (calcd. for BriaviodiolC21H32O6 + F Na, (1) 403.20911), was isolated suggesting as a colorless six degrees oil. of Compound unsaturation.1 displayed Additionally, a pseudomolecular IR absorptions at ion at 3497 and 1754 cm–1 indicated that 1 contained hydroxy and ester groups. As shown in Table 1, DEPT m/z 403.20886 in the (+)-HRESIMS, which indicated its molecular formula was C21H32O6 (calcd. for and 13C NMR spectra indicated that a suite of 13C resonances at δC 172.1 (C-17), 154.3 (C-1), 127.2 C H O + Na, 403.20911), suggesting six degrees of unsaturation. Additionally, IR absorptions at 21 32 (C-15),6 109.5 (C-2), and 9.2 (CH3-16) were due to an α-methyl-γ-butenolide moiety by comparison –1 3497 andwith 1754 the cmdata ofindicated known cembranoids that 1 contained briaviodiol hydroxy A (4) [9] and and ester pachyclavulariolide groups. As shown F [6]. inMoreover, Table1, DEPT 13 δ 13 δ and Cresonances NMR spectra at Cindicated 127.1 (C-4) that and a135.1 suite (CH-5), of C and resonances the olefinic at δprotonC 172.1 at (C-17),H 5.30 (1H, 154.3 dd, (C-1), J = 8.0, 127.2 5.6 (C-15), 109.5 (C-2),Hz, H-5) and (Table 9.2 (CH 1), 3indicated-16) were an dueadditional to an αunsatu-methyl-ratedγ functionality,-butenolide suggesting moiety by the comparison presence of a with the 2 δ data of knowntrisubstituted cembranoids olefin. In briaviodiolthe HSQC spectrum, A (4)[9 ]an and sp pachyclavulariolidecarbon ( C 135.1) correlated F [6]. with Moreover, the methine resonances proton (δH 5.30). This proton had 3J-correlations with H2-6 (δH 1.93–1.99, 2H, m) in the 1H–1H COSY at δ 127.1 (C-4) and 135.1 (CH-5), and the olefinic proton at δ 5.30 (1H, dd, J = 8.0, 5.6 Hz, H-5) C spectrum, and had 3J-correlations with C-3 and C-18 in the HMBCH spectrum (Table 1), further (Table1),confirming indicated the an existence additional of a unsaturated trisubstituted functionality, olefin. In light suggesting of the 1H and the 13 presenceC NMR data, of a together trisubstituted 2 olefin. Inwith the the HSQC degrees spectrum, of unsaturation, an sp 1 wascarbon determined (δC 135.1) as a correlatedtricyclic cembrane with thediterpene. methine proton (δH 5.30). 13 1 1 This protonThe had HJ -correlationsNMR coupling withinformation H2-6 ( δinH the1.93–1.99, COSY spectrum 2H, m) of in 1 theenabledH– theH determination COSY spectrum, of the and had 3J-correlationsproton sequences with C-3 between and C-18 H-5/H in the2-6/H HMBC2-7/H-8/H-9/H spectrum2-10/H (Table2-11 and1), furtherH-8/H3-19 confirming (Table 1). The the carbon existence of a trisubstitutedskeleton of olefin. 1 was In elucidated light of thebased1H on and the13 keyC NMRHMBC data, fromtogether H-3β, H-13, with H-14, the H degrees3-16 to C-1; of unsaturation, H2-3, H-14 to C-2; H2-3, H3-18 to C-4; H-10β, H-11α, H-13, H-14, H3-20 to C-12; H-14, H3-16 to C-15; and 1 was determined as a tricyclic cembrane diterpene. H3-16 to C-17. The presence of a vinyl methyl group on C-4 was supported by HMBC from H3-18 to 1 TheC-3,H C-4, NMR C-5; couplingH-3α (δH 2.78) information to C-18 and in H-5 the to COSY C-18. spectrumFurthermore, of HMBC1 enabled from theOH-13 determination to C-13, of the protonC-14 sequencesand OH-14 to between C-1, C-13, H-5/H C-14 suggested2-6/H2-7/H-8/H-9/H the existence of 2hydroxy-10/H2 -11groups and at H-8/HC-13 and3-19 C-14, (Table 1). The carbonrespectively. skeleton Therefore, of 1 was the elucidated methoxy group based was on on the C-2, key since HMBC the fromHMBC H-3 spectrumβ, H-13, exhibited H-14, Ha 3-16 to correlation between the singlet at δH 3.39 (OMe) and C-2 (δC 109.5). Taking into account the C-1; H2-3, H-14 to C-2; H2-3, H3-18 to C-4; H-10β, H-11α, H-13, H-14, H3-20 to C-12; H-14, H3-16 to molecular formula, the remaining oxygen atom must be part of the tetrahydrofuran ring located C-15; and H -16 to C-17. The presence of a vinyl methyl group on C-4 was supported by HMBC from between3 C-9 and C-12. α δ H3-18 to C-3,Based C-4, on C-5; NOESY H-3 (correlationsH 2.78) to and C-18 further and H-5information to C-18. provided Furthermore, by MM2 HMBC forcefield from OH-13 to C-13,calculations C-14 and [11], OH-14 the torelative C-1, stereochemistry C-13, C-14 suggested of 1 with the the stable existence conformation of hydroxy is shown groups in Figure at C-13 2 and C-14, respectively.(Supplementary Therefore, Figures S1–10). the methoxy When H-9 group was α-oriented was on C-2,in 1, a since correlation the HMBC between spectrum H-9 and H exhibited3-19 a was observed, suggesting that these protons were on the α-face, and H-8 was β-oriented. H-8 correlation between the singlet at δH 3.39 (OMe) and C-2 (δC 109.5). Taking into account the molecular formula, the remaining oxygen atom must be part of the tetrahydrofuran ring located between C-9 and C-12. Mar. Drugs 2019, 17, 214 3 of 9

Based on NOESY correlations and further information provided by MM2 forcefield calculations [11], the relative stereochemistry of 1 with the stable conformation is shown in Figure2 (Supplementary Figures S1–10). When H-9 was α-oriented in 1, a correlation between H-9 and H3-19 was observed, suggesting that these protons were on the α-face, and H-8 was β-oriented. H-8 correlated Mar. Drugs 2019, 17, x 3 of 9 with H-13, and the hydroxy proton OH-13 correlated with H3-20, suggesting that the hydroxy group at C-13 and the Me-20 at C-12 were α-oriented. H-14 exhibited a NOESY correlation with H-13, and no correlated with H-13, and the hydroxy proton OH-13 correlated with H3-20, suggesting that the 1 couplinghydroxy constant group was at C-13 detected and the between Me-20 at C-12 H-13 were and α H-14-oriented. in theH-14 Hexhibited NMR a spectrum, NOESY correlation implying that ◦ the dihedralwith H-13, angle and located no coupling between constant H-13 was anddetected H-14 between was about H-13 and 90 H-14, and in thethe 1 14-hydroxyH NMR spectrum, group was β-oriented.implying Correlations that the dihedral between angle H-5 located and H-3,between and H-13 H-14 and and H-14 H-3 was (δ aboutH 2.78) 90°, suggested and the 14-hydroxy that this proton δ is α, andgroup the protonwas β-oriented. at δ 3.04 Correlations is 3β. Additionally, between H-5 and the H-3, proton and signalH-14 and of aH-3 methoxy ( H 2.78) groupsuggested displayed H δ NOESYthat correlations this proton with is α, both and H-3the protonα/β, whichat H 3.04 indicated is 3β. Additionally, that the methoxy the proton group signal at C-2of a wasmethoxyα-oriented. group displayed NOESY correlations with both H-3α/β, which indicated that the methoxy group at H -18 was found to show a NOESY correlation with H-3β, but not with H-5, and H-5 was shown to 3 C-2 was α-oriented. H3-18 was found to show a NOESY correlation with H-3β, but not with H-5, be correlatedand H-5 with was H-3shownα andto be H-14, correlated which with suggested H-3α and H-14, an E -configurationwhich suggested ofan theE-configuration C-4/5 double of bond. The aforementionedthe C-4/5 double results bond. enabled The aforementioned establishment ofresults the relativeenabled configurationestablishment of 1the, and relative therefore its stereogenicconfiguration carbons of were 1, and assigned therefore as its 2R stereogenic*,8R*,9S*,12 carbonsR*,13S were*, 14 Rassigned*. as 2R*,8R*,9S*,12R*,13S*, 14R*. 1 13 Table 1.TableH (4001. 1H MHz,(400 MHz, CDCl CDCl3) and3) and 13CC (100(100 MHz, CDCl CDCl3) NMR,3) NMR, COSY, COSY, HMBC HMBC data for data 1. for 1.

PositionPosition δHδ (HJ (inJ inHz) Hz) δC, typeδC , type COSY COSY HMBC HMBC

11 154.3, 154.3,C C

22 109.5, 109.5,C C

3α3α//ββ 2.782.78 d (14.0); d (14.0); 3.04 3.04 d (14.0) d (14.0) 44.8, CH 44.8,2 CH2 C-1, C-2,C-1, C-4, C-2, C-5, C-4, C-18 C-5, C-18 44 127.1, 127.1,C C 55 5.30 5.30 dd dd (8.0, (8.0, 5.6) 5.6) 135.1, 135.1,CH CH H2-6 H2-6C-3, C-18 C-3, C-18 66 1.93–1.99 1.93–1.99 m m 26.3, CH 26.3,2 CH2 H-5, H2-7 H-5, H2-7 C-4, C-5, C-4, C-7, C-5, C-8 C-7, C-8 α β 77α//β 1.801.80 m; m; 1.26 1.26 m m 34.9, CH 34.9,2 CH2 H2-6, H-8 H2-6, H-8 C-5, C-6,C-5, C-8, C-6, C-9, C-8, C-19 C-9, C-19 8 0.68 m 41.4, CH H -7, H-9, H -19 C-9 8 0.68 m 41.4, CH H2-7, H-9, H23-19 3 C-9 9 3.68 ddd (9.6, 9.6, 6.0) 85.2, CH H-8, H2-10 C-7 9 3.68 ddd (9.6, 9.6, 6.0) 85.2, CH H-8, H2-10 C-7 10α/β 1.51 m; 1.98 m 30.3, CH2 H-9, H2-11 C-11, C-12 10α/β 1.51 m; 1.98 m 30.3, CH2 H-9, H2-11 C-11, C-12 C-9, C-10, C-12, C-13, 11α/β 1.56 m; 2.38 dd (12.0, 6.4) 36.9, CH H -10 11α/β 1.56 m; 2.38 dd (12.0, 6.4) 36.9, CH2 2 H2-10 2 C-9, C-10, C-12, C-13,C-20 C-20

1212 84.1, C 84.1, C 1313 3.60 3.60 d (10.0) d (10.0) 70.7, CH 70.7, CH OH-13 OH-13 C-1, C-11, C-1, C-12, C-11, C-20 C-12, C-20 1414 5.06 5.06 s s 63.9, CH 63.9, CH - - C-1, C-2, C-1, C-12, C-2, C-15 C-12, C-15 1515 127.2, 127.2,C C

1616 2.11 2.11 s s 9.2, CH 9.2,3 CH3 C-1, C-15,C-1, C-17 C-15, C-17 1717 172.1, 172.1,C C 18 1.44 s 15.3, CH C-3, C-4, C-5 18 1.44 s 15.3, CH3 3 C-3, C-4, C-5 19 0.78 d (6.4) 17.3, CH3 H-8 C-7, C-8, C-9 19 0.78 d (6.4) 17.3, CH3 H-8 C-7, C-8, C-9 20 1.30 s 20.9, CH3 C-11, C-12, C-13 20 1.30 s 20.9, CH3 C-11, C-12, C-13 OMe-2 3.39 s 51.0, CH3 C-2 OH-13OMe-2 3.14 3.39 d s (10.0) 51.0, CH3 H-13C-2 C-13, C-14

OH-14OH-13 3.14 d 3.34 (10.0) s H-13 -C-13, C-1,C-14 C-13, C-14 OH-14 3.34 s - C-1, C-13, C-14

17

2.543 2.667 2R* 18 3 15 16 2.298 1 4

2.437 14R* 5 2.253 2.354 6 2.734

2.574 13S*

2.363 2.475 2.346 7 12R* 2.055 20 8R* 2.262 11 9S* 19

10 2.173 2.706 : NOESY Correlation Figure 2. Computer-depicted model drawing of 1 and calculated distances (unit = Å) between Figure 2. Computer-depicted model drawing of 1 and calculated distances (unit = Å) between protons protons with main NOESY correlations. with main NOESY correlations.

Mar. Drugs 2019, 17, 214 4 of 9

Briaviotriol A (2) was found to have the molecular formula C21H32O7, as established by 1 13 (+)-HRESIMS at m/z 419.20377 (calcd. for C21H32O7 + Na, 419.20402). The H and C NMR spectra of 2 were very similar to those of 1. Comparison between the 1H and 13C NMR data of 2 (Table2) and those of 1 suggested that the double bond is located between C-4 and C-18 in 2 instead of C-4 and C-15 in 1. HMBC from H2-18 to C-3, C-4, C-5; and from H2-3 and H-5 to C-18, corroborated the existence of an exocyclic double bond at C-4. In the HSQC spectrum, an oxymethine carbon (δC 69.1) correlated 3 with the methine proton (δH 4.57), and this proton had J-correlations with H2-6 (δH 1.81, 1H, m and 1.93, 1H, m) in the 1H–1H COSY spectrum, demonstrating that a hydroxy group was attached to C-5.

1 13 Table 2. H (400 MHz, CDCl3) and C (100 MHz, CDCl3) NMR, COSY, HMBC data for 2.

Position δH (J in Hz) δC, type COSY HMBC 1 157.9, C 2 107.8, C 3α/β 2.67 d (14.4); 3.15 d (14.4) 42.2, CH2 C-1, C-2, C-4, C-5, C-18 4 146.4, C 5 4.57 dd (7.2, 6.0) 69.1, CH H2-6 C-4, C-6, C-18 0 6/6 1.81m; 1.93 m 32.2, CH2 H-5, H2-7 - 0 7/7 1.33 m; 1.89 ddd (14.4, 4.8, 4.4) 30.6, CH2 H2-6, H-8 C-5 8 1.53 m 37.4, CH H2-7, H-9, H3-19 - 9 3.65 ddd (8.4, 8.4, 6.0) 85.4, CH H-8, H2-10 - 0 10/10 1.52 m; 2.10 m 31.5, CH2 H-9, H2-11 - 0 11/11 1.65 m; 2.16 m 37.2, CH2 H2-10 C-9, C-10, C-12, C-13, C-20 12 84.4, C 13 3.50 d (5.6) 75.2, CH OH-13 - 14 5.18 br s 67.7, CH OH-14 - 15 130.0, C 16 2.11 s 10.1, CH3 C-1, C-15, C-17 17 171.1, C 18a/b 5.13 s; 5.30 s 115.6, CH2 C-3, C-4, C-5 19 0.85 d (6.4) 16.7, CH3 H-8 C-7, C-8, C-9 20 1.29 s 21.8, CH3 C-11, C-12, C-13 OMe-2 3.26 s 50.7, CH3 C-2 OH-13 2.42 br d (5.6) H-13 - OH-14 2.29 br d (4.4) H-14 -

The stereochemistry of 2 was established from the correlations observed in the NOESY spectrum (Figure3 and Supplementary Figures S2–10). In addition, in the NOESY spectrum H-9 was correlated with H3-19, which suggested that these protons were positioned on the same face and were assigned as α protons, as H-8 was β-oriented. H-13 correlated with H-8 and H-14, but no coupling between H-13 and H-14 was observed, demonstrating that the hydroxy groups at C-13 and C-14 were α- and β-oriented, respectively. Correlations between an oxygen-bearing methyl (δH 3.26) and H-13 suggested that the C-2 methoxy group was situated on the β face. Additionally, correlation between H-5 and H-8 supported a β-orientation of H-5. Based on the aforementioned results, the relative configurations of the stereogenic carbons of 2 were determined as 2S*,5S*,8R*, 9S*,12R*,13S*,14R*. Mar. Drugs 2019, 17, x 4 of 9

Briaviotriol A (2) was found to have the molecular formula C21H32O7, as established by (+)-HRESIMS at m/z 419.20377 (calcd. for C21H32O7 + Na, 419.20402). The 1H and 13C NMR spectra of 2 were very similar to those of 1. Comparison between the 1H and 13C NMR data of 2 (Table 2) and those of 1 suggested that the double bond is located between C-4 and C-18 in 2 instead of C-4 and C-15 in 1. HMBC from H2-18 to C-3, C-4, C-5; and from H2-3 and H-5 to C-18, corroborated the existence of an exocyclic double bond at C-4. In the HSQC spectrum, an oxymethine carbon (δC 69.1) correlated with the methine proton (δH 4.57), and this proton had 3J-correlations with H2-6 (δH 1.81, 1H, m and 1.93, 1H, m) in the 1H–1H COSY spectrum, demonstrating that a hydroxy group was attached to C-5.

Table 2. 1H (400 MHz, CDCl3) and 13C (100 MHz, CDCl3) NMR, COSY, HMBC data for 2.

Position δH (J in Hz) δC, type COSY HMBC 1 157.9, C 2 107.8, C

3α/β 2.67 d (14.4); 3.15 d (14.4) 42.2, CH2 C-1, C-2, C-4, C-5, C-18 4 146.4, C 5 4.57 dd (7.2, 6.0) 69.1, CH H2-6 C-4, C-6, C-18 6/6′ 1.81m; 1.93 m 32.2, CH2 H-5, H2-7 - 7/7′ 1.33 m; 1.89 ddd (14.4, 4.8, 4.4) 30.6, CH2 H2-6, H-8 C-5 8 1.53 m 37.4, CH H2-7, H-9, H3-19 - 9 3.65 ddd (8.4, 8.4, 6.0) 85.4, CH H-8, H2-10 - 10/10′ 1.52 m; 2.10 m 31.5, CH2 H-9, H2-11 - 11/11′ 1.65 m; 2.16 m 37.2, CH2 H2-10 C-9, C-10, C-12, C-13, C-20 12 84.4, C 13 3.50 d (5.6) 75.2, CH OH-13 - 14 5.18 br s 67.7, CH OH-14 - 15 130.0, C

16 2.11 s 10.1, CH3 C-1, C-15, C-17 17 171.1, C 18a/b 5.13 s; 5.30 s 115.6, CH2 C-3, C-4, C-5 19 0.85 d (6.4) 16.7, CH3 H-8 C-7, C-8, C-9

20 1.29 s 21.8, CH3 C-11, C-12, C-13

OMe-2 3.26 s 50.7, CH3 C-2 Mar. Drugs 2019, 17, 214 5 of 9 OH-13 2.42 br d (5.6) H-13 - OH-14 2.29 br d (4.4) H-14 -

17 16 18 15

2S* 4 3 1

2.579

5S* 14R* 2.205 2.356 6 2.074 2.760 2.081 7 2.760 13S*

12R* 20 8R*

9S* 19 11

10

2.585 : NOESY Correlation FigureFigure 3. Computer-depicted 3. Computer-depicted model model drawing drawing of of2 and2 and calculated calculated distances distances (unit (unit = = Å) Å) between between protons withprotons main NOESYwith main correlations. NOESY correlations.

Compound 3 has a molecular formula C21H32O7 according to its (+)-HRESIMS m/z 419.20399 1 13 (calcd. for C21H32O8 + Na, 419.20402). The H and C NMR features of 3 resemble those of 1; comparison of the 1H and 13C NMR chemical shifts of the sp2 methine proton and its respective 2 carbon (δH 5.29, 1H, d, J = 8.4 Hz; δC 133.5, CH-5), and the sp quaternary carbon (δC 131.0, C-4) of 3 (Table3) with those of 1 (δH 5.30, 1H, dd, J = 8.0, 5.6 Hz; δC 135.1, CH-5; δC 127.1, C-4) (Table1), as well as a NOESY correlation between H-5 and H3-18, indicated the Z-configuration of the C-4/5 double bond (Figure4 and Supplementary Figure S3–10). Furthermore, the HSQC spectrum showed that an oxymethine carbon (δC 68.7) was correlated with a methine proton (δH 4.86; H-6), and this 3 proton exhibited J-correlations with the olefinic proton H-5 (δH 5.29) and H2-7 (δH 1.34, 1H, m; 1.80, 1H, m) in the COSY spectrum, which confirmed a hydroxy group at C-6. As H-6 showed a NOESY correlation with H-3β, this suggested that the C-6 hydroxy group was α-oriented. Based on a NOESY experiment (Figure4 and Supplementary Figures S3–10), 3 was identified to have the stereogenic centers 2R*,6S*,8R*,9S*,12R*,13S*,14R*. Since 3 has never been previously reported, it was named briaviotriol B.

1 13 Table 3. H (400 MHz, CDCl3) and C (100 MHz, CDCl3) NMR, and COSY, HMBC data for 3.

Position δH (J in Hz) δC, type COSY HMBC 1 159.8, C 2 107.7, C 3α/β 1.76 d (14.4); 3.56 d (14.4) 39.6, CH2 C-1, C-2, C-4, C-5, C-18 4 131.0, C 5 5.29 d (8.4) 133.5, CH H-6 C-3, C-18 6 4.86 ddd (12.0, 8.4, 3.2) 68.7, CH H-5, H2-7 - 0 7/7 1.34 m; 1.80 m 44.9, CH2 H-6, H-8 C-5, C-6, C-8, C-9, C-19 8 1.21 m 35.3, CH H2-7, H-9, H3-19 - 9 3.65 ddd (10.0, 10.0, 4.4) 87.3, CH H-8, H2-10 - 0 10/10 1.38 m; 2.05 m 32.8, CH2 H-9, H2-11 C-9, C-11, C-12 0 11/11 1.61 m; 2.22 dd (13.2, 8.0) 35.5, CH2 H2-10 C-9, C-12, C-13, C-20 12 85.1, C 13 3.40 d (8.4) 76.7, CH OH-13 C-1, C-12, C-20 14 4.75 d (5.2) 63.5, CH OH-14 C-1, C-2, C-12, C-13, C-15 15 128.5, C 16 2.08 s 9.6, CH3 C-1, C-15, C-17 17 171.9, C 18 1.83 s 24.0, CH3 C-3, C-4, C-5 19 0.84 d (6.8) 20.1, CH3 H-8 C-7, C-8, C-9 20 1.28 s 21.1, CH3 C-11, C-12, C-13 OMe-2 3.20 s 51.1, CH3 C-2 OH-13 3.05 d (8.4) H-13 C-13, C-14 OH-14 2.70 d (5.2) H-14 C-13, C-14 Mar. Drugs 2019, 17, 214 6 of 9 Mar. Drugs 2019, 17, x 6 of 9

19 17

16

15 18

2R* 3 1 4 2.303 2.148 14R* 5 2.837 2.083 2.827 13S* 2.919 6S* 2.642 12R*

11 7 20 10 8R* 9S*

2.350 19 2.329 : NOESY Correlation Figure 4.4.Computer-depicted Computer-depicted model model drawing drawing of 3ofand 3 calculatedand calculated distances distances (unit= (unit Å) between = Å) between protons withprotons main with NOESY main correlations.NOESY correlations.

CompoundUsing an in 4vitrowas pro-inflammatory identified as briaviodiol suppression A (Figure assay,1), the by comparisoneffects of 1–4 of on its the1H release and 13 ofC NMRiNOS dataprotein with from those lipopolysaccharide in the literature [ 9(L].PS)-stimulated RAW 264.7 macrophage cells were assessed. First, alamarUsing blue an cellin vitroviabilitypro-inflammatory assessment revealed suppression that 1– assay,4 did thenot effectshave significant of 1–4 on thecytotoxic release effects of iNOS in proteinRAW 264.7 from cells. lipopolysaccharide The results of the (LPS)-stimulated in vitro pro-inflammatory RAW 264.7 macrophagesuppression cellsassay were showed assessed. that 2 First, and alamar4 at 10 blueμM suppressed cell viability the assessment release of revealediNOS to that67.7 1an–4ddid 61.9%, not haverespectively, significant when cytotoxic compared effects with in RAWresults 264.7 of the cells. cells The stimulated results of with the inonly vitro LPSpro-inflammatory (Table 4). Compound suppression 1 showed assay no showedsuppression that 2effectand 4onat iNOS 10 µ Mrelease. suppressed the release of iNOS to 67.7 and 61.9%, respectively, when compared with results of the cells stimulated with only LPS (Table4). Compound 1 showed no suppression effect on iNOSTable release. 4. Effects of 1–4 on LPS-induced pro-inflammatory iNOS release in RAW 264.7 cells at a concentration of 10 μM. The data presented are the relative intensity normalized to the LPS- Tablestimulated 4. Effects group. of Compounds1–4 on LPS-induced 2 and 4 were pro-inflammatory found to have iNOS the higher release inhibition in RAW 264.7effects cells on LPS- at a concentrationinduced iNOS of expression 10 µM. The in data macrophages presented expression. are the relative intensity normalized to the LPS- stimulated group. Compounds 2 and 4 were found to have the higher inhibition effects on LPS- induced iNOS expression in macrophages expression. iNOS Expression (% of LPS) LPS 100.0 ± 7.0iNOS ± 1 109.0Expression 19.2 (% of LPS) 2 67.7 ± 2.4 LPS 100.0 ± 7.0 3 79.5 ± 9.4 1 109.0 ± 19.2 4 61.9 ± 7.3 2 67.7 ± 2.4 3 79.5 ± 9.4 3. Experimental Section 4 61.9 ± 7.3

3.3.1. Experimental General Experimental Section Procedures The JEOL NMR spectrometer (model ECZ400S, Tokyo, Japan) was used to record the spectra 3.1. General Experimental Procedures with the solvent peak of CHCl3 (δH 7.26 ppm) and CDCl3 (δC 77.1 ppm) as internal references for 1H NMRThe and JEOL 13C NMRNMR, spectrometer respectively. (model ESIMS ECZ400S, and HRESIMS Tokyo, Japan) were was obtained used to from record the the Bruker spectra mass with 1 thespectrometer solvent peak with of CHCl7 Tesla3 ( δmagnetsH 7.26 ppm) (model: and CDClSolariX3 (δ CFTMS77.1 ppm)system) asinternal (Bremen, references Germany). for ColumnH NMR andchromatography,13C NMR, respectively. IR spectra ESIMS and optical and HRESIMS rotation we werere obtainedperformed from according the Bruker to our mass earlier spectrometer research with[10]. 7 Tesla magnets (model: SolariX FTMS system) (Bremen, Germany). Column chromatography, IR spectra and optical rotation were performed according to our earlier research [10]. 3.2. Material Specimens of B. violaceum used for this study were collected in December 2016 from the cultivation tank (capacity = 270 tons) at the National Museum of Marine Biology and Aquarium

Mar. Drugs 2019, 17, 214 7 of 9

3.2. Animal Material Specimens of B. violaceum used for this study were collected in December 2016 from the cultivation tank (capacity = 270 tons) at the National Museum of Marine Biology and Aquarium (NMMBA) in Southern Taiwan. For its identification, this coral species was compared to reliable sources published earlier [1,2]. A voucher specimen was deposited in the NMMBA (voucher no.: NMMBA-CSC-005).

3.3. Extraction and Isolation Sliced bodies (wet/dry weight = 358.7/144.5 g) of the coral specimen were prepared and extracted with a 1:1 mixture of MeOH and CH2Cl2 to give 17.2 g of crude extract which was partitioned between EtOAc and H2O to obtain 6.3 g of the EtOAc extract. The EtOAc extract was then applied onto a silica gel column and eluted with gradients of n-hexane/EtOAc (100% n-hexane−100% EtOAc, stepwise), to furnish 14 fractions (fractions: A−N). Fraction G was further chromatographed on a silica gel column and eluted with gradients of n-hexane/Me2CO (20:1−100% Me2CO, stepwise) to afford 11 subfractions (fractions: G1−G11). Fraction G4 was applied onto a silica gel column and eluted with gradients of n-hexane and Me2CO (20:1−100% Me2CO, stepwise) to give 12 subfractions (fractions: G4A−G4L). Afterwards, fraction G4E was then separated by normal-phase HPLC (NP-HPLC) using a mixture of n-hexane and Me2CO (5:1) as solvent to obtain 5 subfractions (fractions: G4E1−G4E5). Then, fraction G4E1 was separated by NP-HPLC using a mixture of CH2Cl2 and Me2CO (with volume: volume = 80:1; at a flow rate = 3.0 mL/min) to afford 1 (62.7 mg). Fraction G4H was separated by NP-HPLC using a mixture of n-hexane and Me2CO (with volume: volume = 4:1; at a flow rate = 2.0 mL/min) to afford 4 (17.0 mg). Fraction G4J was repurified by NP-HPLC using a mixture of n-hexane and Me2CO (with volume: volume = 3:1; at a flow rate = 2.0 mL/min) to afford 3 (0.9 mg). Fraction G8 was separated by NP-HPLC using a mixture of n-hexane and Me2CO (3:1) to obtain 6 subfractions G8A−G8F. Fraction G8F was repurified by reverse-phase HPLC (RP-HPLC) using a mixture of MeCN and H2O (with volume: volume = 1:1; at a flow rate = 1.0 mL/min) to yield 2 (1.2 mg). 21 −1 1 13 Briaviodiol F (1): Colorless oil; [α]D +223 (c 1.48, CHCl3); IR (neat) νmax 3497, 1754 cm ; H and C + NMR data (see Table1); ESIMS: m/z 403 [M + Na] ; HRESIMS: m/z 403.20886 (calcd. for C21H32O6 + Na, 403.20911).

22 −1 1 13 Briaviotriol A (2): Colorless oil; [α]D −68 (c 0.06, CHCl3); IR (neat) νmax 3424, 1749 cm ; H and C + NMR data (see Table2); ESIMS: m/z 419 [M + Na] ; HRESIMS: m/z 419.20377 (calcd. for C21H32O7 + Na, 419.20402).

23 −1 1 13 Briaviotriol B (3): Colorless oil; [α]D −39 (c 0.04, CHCl3); IR (neat) νmax 3424, 1749 cm ; H and C + NMR data (see Table3); ESIMS: m/z 419 [M + Na] ; HRESIMS: m/z 419.20399 (calcd. for C21H32O7 + Na, 419.20402). 21 23 Briaviodiol A (4): Colorless crystal; [α]D −52 (c 0.85, CHCl3) (Reference [9] [α]D −31 (c 0.14, CHCl3)); −1 1 13 IR (neat) νmax 3467, 1747 cm ; H and C NMR data were found to be in absolute agreement with previous study [9]; ESIMS: m/z 403 [M + Na]+.

3.4. Molecular Mechanics Calculations The molecular models were generated by implementing the MM2 force field [11] in ChemBio 3D Ultra software (ver. 12.0) which was created by CambridgeSoft (PerkinElmer, Cambridge, MA, USA).

3.5. In Vitro Anti-Inflammatory Assay The pro-inflammatory suppression assay was performed using a murine macrophage cell line, RAW 264.7, which was purchased from the American Type Culture Collection (ATCC cell line no. TIB-71; Manassas, VA, USA). Untreated or LPS-induced RAW 264.7 cells were used to determine the anti-inflammatory activities of cembranoids 1–4 by assessing the inhibition of pro-inflammatory iNOS release from macrophage cells. The iNOS protein levels were measured by using western blotting Mar. Drugs 2019, 17, 214 8 of 9 analysis [12–14]. Briefly, in the control group, macrophages were incubated in compound-free medium with LPS (10 µM) alone for 16 h; and in the cembranoid-treated groups, the cells were pre-treated with cembranoids 1–4 (10 µM) for 10 min followed by an LPS challenge for 16 h. After the incubation, cell lysates were collected, and equal amounts of the total protein samples were subjected to western blot analysis. The immunoreactivities were caculated based on the optical densities of the corresponding iNOS bands of each group on the membrane, and the cells with LPS treatment alone were set to be 100%. Viability of macrophage cells of different groups was determined after treatment with alamar blue (Invitrogen, Carlsbad, CA, USA), a chemical of tetrazolium dye that is reduced by living cells to a fluorescent substance. The assay has been shown to have accurate measurement in determining the survival of RAW 264.7 cells [15,16], which is based on a mechanism similar to that of an assay using 3-(4,5-dimethyldiazol-2-yl)-2,5- diphenyltetrazolium bromide. Data analyses were firstly performed using one-way analysis of variance (ANOVA), and further analyzed by the Student-Newman-Keuls post hoc test for multiple comparison. All the data with a p-value of < 0.05 were considered as a significant difference.

4. Conclusions B. violaceum has been demonstrated to have a wide structural diversity of interesting diterpenoids that possess various pharmacological properties [17]. This specimen was encrusted on different species of scleractinian hard in the Indo-Pacific coral reef system [18]. In our continued study of B. violaceum, three previously unreported furanocembranoids 1–3 were isolated, together with the previously described briaviodiol A (4). In the present study, the anti- inflammatory activities of 1–4 were assessed using inhibition of pro-inflammatory iNOS release from macrophages. The results indicated that briaviotriol A (2) and briaviodiol A (4) showed the most potent suppressive effects on iNOS release.

Supplementary Materials: The Supplementary Materials are available online at http://www.mdpi.com/1660- 3397/17/4/214/s1. ESIMS, HRESIMS, IR, 1D (1H NMR, 13C NMR, and DEPT spectra), and 2D (COSY, HSQC, HMBC, and NOESY) spectra of new compounds 1–3 and 1H and 13C NMR spectra of 4. Author Contributions: P.-C.H., W.-S.L., Z.-H.W., and P.-J.S. designed the whole experiment and contributed to manuscript preparation. B.-R.P., Y.-C.C., L.-S.F., G.-Q.L., and T.-L.H. analyzed the data and performed data acquisition. Funding: This research was supported by grants from the National Museum of Marine Biology and Aquarium; the National Dong Hwa University; and the Ministry of Science and Technology, Taiwan (Grant Nos: MOST 104-2320-B-291-001-MY3 and 107-2320-B-291-001-MY3) awarded to Ping-Jyun Sung. Conflicts of Interest: The authors declare no conflicts of interest.

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