marine drugs
Article Briarenones A-C, New Briarellin Diterpenoids from the Gorgonian Briareum violaceum
Yang Cheng 1, Atallah F. Ahmed 2,3 , Raha S. Orfali 2, Chang-Feng Dai 4 and Jyh-Horng Sheu 1,5,6,7,* 1 Department of Marine Biotechnology and Resources, National Sun Yat-sen University, Kaohsiung 804, Taiwan; [email protected] 2 Department of Pharmacognosy, College of Pharmacy, King Saud University, Riyadh 11451, Saudi Arabia; [email protected] (A.F.A.); [email protected] (R.S.O.) 3 Department of Pharmacognosy, Faculty of Pharmacy, Mansoura University, Mansoura 35516, Egypt 4 Institute of Oceanography, National Taiwan University, Taipei 112, Taiwan; [email protected] 5 Graduate Institute of Natural Products, Kaohsiung Medical University, Kaohsiung 807, Taiwan 6 Frontier Center for Ocean Science and Technology, National Sun Yat-sen University, Kaohsiung 804, Taiwan 7 Department of Medical Research, China Medical University Hospital, China Medical University, Taichung 404, Taiwan * Correspondence: [email protected]; Tel.: +886-7-525-2000 (ext. 5030); Fax: +886-7-525-5020
Received: 9 January 2019; Accepted: 12 February 2019; Published: 17 February 2019
Abstract: Three new eunicellin-derived diterpenoids of briarellin type, briarenones A-C (1-3), were isolated from a Formosan gorgonian Briareum violaceum. The chemical structures of the compounds were elucidated on the basis of extensive spectroscopic analyses, including two-dimensional (2D) NMR. The absolute configuration of 1 was further confirmed by a single crystal X-ray diffraction analysis. The in vitro cytotoxic and anti-inflammatory potentialities of the isolated metabolites were tested against the growth of a limited panel of cancer cell lines and against the production of superoxide anions and elastase release in N-formyl-methionyl-leucyl-phenyl-alanine and cytochalasin B (fMLF/CB)-stimulated human neutrophils, respectively.
Keywords: Briareum violaceum; briarenones; briarellin; gorgonian
1. Introduction Gorgonians belonging to genus Briareum (phylum Cnidaria, family Briareidae) are considered to be a rich source of highly oxygenated diterpenoids, particularly those derived from briarein (briarane) [1–11], eunicellin (cladiellin) [1,12–16], asbestinin [11,15,17–19], and, to a lesser extent, cembrane [20]. These metabolites were shown to exhibit various bioactivities such as anti-inflammatory [2,4,5,13,21], analgesic [22], cytotoxic [7–10,14,15,23], antimalarial [12,15], antiviral [10,15], and antimicrobial [15] activities. Briarellins constitute a class of tetracyclic oxygenated diterpenoids in which an additional seven-membered ether ring (oxepane) is formed between C-3 and C-16 of the eunicellin skeleton. These metabolites were originally discovered from Caribbean gorgonians of genus Briareum, e.g., B. asbestinum [13,14,16] and B. polyanthes [12,15], and also showed interesting bioactivities. Yet, briarellins remain to be isolated from the Pacific Briareum genus, although briarellins with the α,β-conjugated enone in the six-membered ring analogs were previously reported from one species of a taxonomically linked Pachyclavularia genus [24–26]. This prompted us to extensively investigate the chemical constituents of a gorgonian Briareum violaceum growing wildly in the Taiwanese waters and to screen their cytotoxic and anti-inflammatory activities. The current study led to the discovery of a group of three new briarellin diterpenoids, briarenones A-C (1-3), characterized by having cis fused cyclohexane, cyclodecane, and oxepane rings. The chemical structures of these molecules were resolved by various spectroscopic
Mar. Drugs 2019, 17, 120; doi:10.3390/md17020120 www.mdpi.com/journal/marinedrugs Mar. Drugs 2019, 17, 120 2 of 10 Mar. Drugs 2018 , 16 , x 2 of 11 analyses,including including two-dimensional two-dimensional (2D) NMR (2D) correlation NMR correlation analysis, analysis, while their while absolute their absolute configurations configurations were wereassigned assigned by single-crystal by single-crystal X-ray X-ray diffraction diffraction analys analysisis for for1. 1.
2. Results and Discussion The lyophilized specimen specimen of of the the gorgonian gorgonian was was extracted extracted with with ethyl ethyl acetate acetate (EtOAc). (EtOAc). The chromatographic fractionation fractionation of of the the solvent-free solvent-free extract extract on silica on silicagel and gel reversed-phase and reversed-phase C18 columns C18 andcolumns the final and separation the final using separation reversed-phase using reversed-pha high-performancese high-performance chromatography chromatography (RP-HPLC) afforded (RP- diterpenoidsHPLC) afforded1-3 (Figurediterpenoids1; Figures 1‒3 (Figure S1–S26, 1; Supplementary Figures S1 –S26, Materials). Supplementary Materials).
Figure 1. 1. StructuresStructures of of new new briarellin briarellin diterpenoids diterpenoids ( (11‒-3) isolated from Briareum violaceum and pachyclavulariaenone FF (( 44).).
25 Briarenone A (1) was obtained as a needle crystal, [α] 25 +224.4 (CHCl ). Its molecular formula Briarenone A ( 1) was obtained as a needle crystal, [α]D +224.4 (CHCl33). Its molecular formula D + C22H32O6 was determined by the sodium adduct peak [M + Na]+ at m/z 415.2089 in the high-resolution C22 H32 O6 was determined by the sodium adduct peak [M + Na] at m/z 415.2089 in the high-resolution electrospray ionization mass spectrometry (HRESIMS), inferring seven degrees of unsaturation. The electrospray ionization mass spectrometry (HRESIMS), inferring−1 seven degrees of unsaturation. The infrared (IR) absorption bands at νmax 3477, 1728, and 1670 cm −1 indicated the presence of hydroxyl, infrared (IR) absorption bands at νmax 3477, 1728, and 1670 cm indicated the presence of hydroxyl, ester carbonyl, and α,β-unsaturated carbonyl functionalities, respectively. As the NMR spectra of ester carbonyl, and α,β-unsaturated carbonyl functionalities, respectively. As the NMR spectra of 1 1 showed numerous broad and very weak signals due to the likely presence of a mixture of slowly showed numerous broad and very weak signals due to the likely presence of a mixture of slowly interconverting conformers on the NMR time scale, remeasuring the spectra at −10 ◦C allowed us to interconverting conformers on the NMR time scale, remeasuring the spectra at −10 °C allowed us to have better resolution for the signals. The 1H NMR spectrum displayed the signals of four methyls, have better resolution for the signals. The 1H NMR spectrum displayed the signals of four methyls, including one olefinic (δH 1.99 ppm, s), two methyls attached to oxygen-bearing carbons (δH 1.27 ppm, including one olefinic (δ H 1.99 ppm, s), two methyls attached to oxygen-bearing carbons (δ H 1.27 ppm, s and 1.28 ppm, s), and one secondary methyl (δH 1.09 ppm, d, J = 7.2 Hz), signifying the terpenoid s and 1.28 ppm, s), and one secondary methyl (δ H 1.09 ppm, d, J = 7.2 Hz), signifying the terpenoid nature of 1. Furthermore, the NMR data (Table1) indicated the existence of an acetyl ( δC 171.3 ppm, nature of 1. Furthermore, the NMR data (Table 1) indicated the existence of an acetyl (δ C 171.3 ppm, δC/δH 21.5/2.08 ppm) and an α,β-unsaturated ketone (δC 198.0, 157.0, and δC/δH 128.0/5.95 ppm). TakingδC/δ H 21.5/2.08 into account ppm) the and unsaturation an α,β-unsaturated degrees mentionedketone (δC 198.0,above, 157.0, the 22 and carbon δ C/δ H signals 128.0/5.95 in the ppm).13C 13 NMRTaking spectrum into account of 1 the(Table unsaturation1) are, thus, degrees ascribable mentio to thened presence above, the of a22 tetracyclic carbon signals diterpenoid in the acetate.C NMR Threespectrum ring-juncture of 1 (Table methines 1) are, thus, (δC ascribable/δH 51.1/2.71, to the 48.3/2.5, presence and of a 36.8/3.13 tetracyclic ppm), diterpenoid two tetrahydrofuran acetate. Three (THF)-oxymethinesring-juncture methines (δC / (δδHC/δ83.4/3.72H 51.1/2.71, and 48.3/2.5, 77.9/4.79 and ppm), 36.8/3.13 and one ppm), oxymethylene two tetrahydrofuran (δC/δH 63.7/3.62 (THF)- andoxymethines 3.34 ppm) (δ suggestedC/δ H 83.4/3.72 a briarellin-related and 77.9/4.79 ppm), [13,16 ,24and,25 one] or anoxymethylene asbestinin-related (δ C/δ H [1563.7/3.62,17,18] structureand 3.34 forppm)1. suggested However, a since briarellin-related the protons of [13,16,24,25] two methyl o groupsr an asbestinin-related (δH 1.99 ppm, s,[15,17,18] H3-20 and structure 1.09 ppm, for d,1. 3 JHowever,= 7.2 Hz, since H3-17), the but protons not one of methyltwo methyl group, groups showed (δ H 1.99JCH correlationsppm, s, H 3-20 in and the 1.09 heteronuclear ppm, d, J = multiple 7.2 Hz, 3 bondH3-17), correlation but not one (HMBC) methyl spectrum group, with showed two ofJCH the correlations angular methine in the carbons heteronuclear (δC 51.1 multiple and 48.3 bond ppm forcorrelation C-10 and (HMBC) C-14, respectively), spectrum with the two briarellin-type of the angular structure methine was carbons designated (δ C 51.1 for and1 (Figure 48.3 ppm2). Threefor C- partial10 and structuresC-14, respectively), were assigned the briarellin-type by the analysis structure of proton was correlationdesignated spectroscopyfor 1 (Figure 2). (1H- Three1H COSY), partial 1 1 includingstructures thatwere assigned assigned by by the the allylic analysis correlation of proton of correlation the H3-20 (spectroscopyδH 1.99 ppm) ( withH- H the COSY), olefinic including proton H-12that assigned (δH 5.95 by ppm, the s)allylic (Figure correlation2). The connectivityof the H 3-20 of(δ H these 1.99 partialppm) with structures, the olefinic the positionsproton H-12 of the (δ H hydroxyl,5.95 ppm, acetoxyl,s) (Figure and 2). ketoneThe connectivity carbonyl, and of these the ring-fusion partial structures, carbons of the THF positions and oxepane of the ringshydroxyl, were deducedacetoxyl, fromand ketone the complete carbonyl, correlation and the ring-fusion analyses of carbons the HMBC of THF spectrum and oxepane as illustrated rings were in Figurededuced2. Furthermore,from the complete a comparison correlationof analyses the 13C of NMR the HMBC data ofspectrum1 with as those illustrated of pachyclavulariaenone in Figure 2. Furthermore, F (4), isolateda comparison from Pachyclavularia of the 13 C NMR violacea data [ of25 ],1 verified with those the replacement of pachyclavulariaenone of the C-4 hydroxymethine F ( 4), isolated group from (PachyclavulariaδC 69.7 ppm, CH) violacea in 4 [25],by a verified methylene the replacement group (δC 33.6 of the ppm, C-4 CH hydroxym2) in 1. Thisethine was group associated (δ C 69.7 with ppm, a significantCH) in 4 by upfield a methylene chemical group shift (δ C at 33.6 C-5 ppm, (∆δC CH-8.52) ppm) in 1. This in 1 relativewas associated to that inwith4. The a significant planar structure upfield chemical shift at C-5 (∆δC ‒8.5 ppm) in 1 relative to that in 4. The planar structure of 1 was accordingly
Mar. Drugs 2019, 17, 120 3 of 10 of 1 was accordingly established (Figure2). The relative configuration of 1 at C-1, C-2, C-3, C-6, C-7, C-9, C-10, C-14, and C-15, was assigned by the analysis of nuclear rotating-frame Overhauser effect spectroscopy (ROESY) correlations (Figure2), which were found to be consistent with that of 4 as illustrated in Figure2. Consequently, two opposite sets of nuclear Overhauser effect (NOE) interactions were observed for 1. One set displayed NOEs for H-1/H-10, H-1/H-14, H-1/H3-17, H-14/H3-17, H-1/H-8β (δH 2.03 ppm, d, J = 15.0, 12.0 Hz), and H-8β/H3-19, and another set exhibited NOEs for H-2/H3-18 and H3-18/H-6, while H-9 did not show any NOE correlation with H-10. Thus, H-1, H-10, H-14, H3-17, and H3-19 should be positioned in the β-face of the molecule, whereas H-2, H-9, H3-18, and H-6 should be positioned in its α-face. In order to confirm the molecular structure of 1, including the absolute configuration, a single-crystal X-ray structure analysis was further performed (Figure3). The compound was slowly crystallized from MeOH as a monohydrate C22H32O6·H2O, with H-bond holding the H2O molecule in a hydrophilic pocket. From a dataset collected using copper radiation, the X-ray crystallographic analysis (Tables S1–S3, Supplementary Materials) of 1 determined the absolute configurations of briarenone A (1) as 1S,2R,3R,6S,7S,9R,10R,14S,15S, which forms a hydrogen bond from 7-OH with a molecule of water.
Table 1. The 1H and 13C NMR chemical shifts for 1-3.
1 2 3 a b c d c d # δH, m (J in Hz) δC , type δH, m (J in Hz) δC , type δH, m (J in Hz) δC , type 1 3.13 ddd (9.6, 5.4, 4.8) 36.8, CH 3.12 ddd (10.0, 7.0, 4.5) 37.7, CH 2.89 m 40.4, CH 2 3.72 d (9.6) 83.4, CH 3.76 d (10.0) 86.4, CH 3.62 d (10.5) 85.8, CH 3 76.5, C 75.4, C 75.5, C 4α 1.94 br d (10.8) 33.6, CH2 1.58 m 31.6, CH2 1.72 d (15.0) 46.4, CH2 4β 1.49 m 1.58 m 2.36 dd (14.5, 8.5) 5α 1.94 br d (10.8) 27.2, CH2 2.04 m 28.7, CH2 4.84 dd (8.5, 8.5) 65.0, CH 1.58 ddd (11.4, 10.8, 5β 1.79 m 3.6) 6 5.77 dd (11.4, 3.6) 78.0, CH 4.07 dd (11.0, 4.5) 78.3, CH 5.52 d (8.5) 135.4, CH 7 74.6, C 148.3, C 128.8, C 8α 1.81 dd (15.0, 3.6) 45.5, CH2 2.77 dd (14.0,4.5) 37.0, CH2 2.74 d (14.5) 38.6, CH2 8β 2.03 dd (15.0, 12.0) 2.14 dd (14.0,5.0) 2.01 dd (14.5, 3.5) 9 4.79 dd (12.0, 3.6) 77.9, CH 4.45 dd (5.0, 4.0) 82.2, CH 4.29 dd (6.5, 3.0) 81.2, CH 10 2.71 br d (6.0) 51.1, CH 2.97 br d (7.0) 50.3, CH 2.82 br d (8.5) 48.3, CH 11 157.0, C 156.6, C 156.4, C 12 5.95 s 128.0, CH 5.93 s 127.4, CH 5.91 s 128.6, CH 13 198.1, C 198.1, C 198.1, C 14 2.50 br d (4.8) 48.3, CH 2.42 br d (4.5) 48.5, CH 2.31 dd (4.5, 5.5) 48.4, CH 15 2.67 m 30.0, CH 2.59 m 30.4, CH 2.58 m 32.3, CH 16α 3.62 d (13.8) 63.8, CH2 3.35 dd (13.5,3.5) 63.9, CH2 3.52 m 65.2, CH2 16β 3.34 br d (13.8) 3.54 d (13.0) 3.52 m 17 1.09 d (7.2) 17.2, CH3 1.04 d (7.5) 17.2, CH3 1.02 d (7.5) 18.5, CH3 18 1.27 s 22.2, CH3 1.25 s 23.8, CH3 1.41 s 27.5, CH3 19 1.28 s 23.7, CH3 5.35 s; 5.18 s 117.7, CH2 1.93 s 29.2, CH3 20 1.99 s 21.9, CH3 1.98 s 21.8, CH3 1.91 s 21.9, CH3 Ac 2.08 s 21.5, CH3 171.3, C a ◦ b ◦ c Spectra recorded at 600 MHz in CDCl3 at −10 C; spectra recorded at 150 MHz in CDCl3 at −10 C; spectra d recorded at 500 MHz in CDCl3; spectra recorded at 125 MHz in CDCl3.
Briarenone B (2) was obtained as a white powder. It possessed a molecular formula of C20H28O4, as established from the sodiated ion peak in the HREIMS (m/z 355.1880 [M + Na]+) of 2, accounting for −1 seven degrees of unsaturations. The IR absorptions at νmax 3420 and 1670 cm indicated the presence of hydroxyl and conjugated carbonyl functionalities, respectively. The NMR data of 2, measured in ◦ CDCl3 at −10 C, were comparable to those of the framework of 1 except in the replacement of an 3 sp oxycarbon (δC 74.6 ppm, C, C-7) and a methyl group (δC/δH 23.7/1.28 ppm, CH3, CH3-19) in 1 by an exomethylene group (δC/δH 148.3 ppm, C and 117.7/5.35 and 5.18 ppm, CH2, respectively) in 2. Analysis of NMR data of 2 (Table1) and correlations found in the 1H-1H COSY and HMBC spectra (Figure4) enabled the establishment of the gross structure of 2. Furthermore, the observed NOE correlations for 2 (Figure5) assigned similar β-positioning for H-1, H-10, H-14, and H3-17, and Mar. Drugs 2019, 17, 120 4 of 10
α-orientation for H-2, H-9, and H3-18 as found in 1. Moreover, one of the exomethylene protons (δH 5.18 ppm, s) exhibited NOE interaction with H-10 (δH 2.97 ppm, br d, J = 7.0 Hz), while the other one (δH 5.35, s) displayed NOE with the hydroxymethine proton at C-6 (δH 4.07 ppm, dd, J = 11.0, 4.5 Hz). A molecular modeling investigation revealed that H-6 did not exhibit NOE interaction in the nuclear Overhauser effect spectroscopy (NOESY) with the methylene protons at C-8 (distances >3.7 Å). This designated the α-orientation of 6-OH and, hence, the 6R configuration as shown in Figure6. Moreover, it was found that eunicellins with similar substitutions in the ten-membered ring, but with an 6β-OH (Figure6), displayed distinctive chemical shifts at C-6 ( δC < 74.0 ppm), C-7 (δC ≥ 150.0 ppm), and H-6 (δH > 4.25 ppm) [27–30] than those assigned for 2 (δ 78.3, 148.3, and 4.07 ppm, respectively). This observation also suggests the α-orientation of 6-OH in 2. On the basis of the Mar.above Drugs findings, 2018 , 16 , the x absolute configuration of 1, and the biogenetic consideration, the configuration4 of 1 11S, Mar.2R Drugs,3R,6 2018R,9R, 16,10, xR ,14S,15S was, thus, established for 2. 4 of 11
Figure 2. Key 1H-1H COSY, HMBC, and NOE correlations for 1. 1 1 FigureFigure 2. 2.KeyKey 1H-H-1H COSY,H COSY, HMBC, HMBC, and and NOE NOE correlations correlations for for 1. 1.
FigureFigure 3. 3. OakOak RidgeRidge ThermalThermal Ellipsoid Plot (ORTEP) (ORTEP) diagram diagram of of the the molecular molecular structure structure of of 1 1asas determineddetermined by by X-ray X-ray analysis. analysis. Figure 3. Oak Ridge Thermal Ellipsoid Plot (ORTEP) diagram of the molecular structure of 1 as determined by X-ray analysis. + BriarenoneThe HREIMS B ( (2m) /wasz 355.1880 obtained [M as + a Na] white) and powder. NMR It data possessed (Table1 a) indicatedmoleculara formula molecular of C formula20 H28 O4, asC20 establishedH28O4 for briarenonefrom the sodiated C (3), inferringion peak thatin the3 HRis anEIMS isomer ( m/z of355.18802. Therefore, [M + Na] it+ was) of 2 found, accounting that 3 Briarenone B ( 2) was obtained as a white powder. It possessed−1 a molecular formula of C 20 H28 O4, possessed an α,β-unsaturated carbonyl (IR: νmax 1668 cm δ 198.1 ppm, C, 156.4 ppm,−1 C, and δ /δ asfor established seven degrees from the of sodiated unsaturations. ion peak The in IR the absorpti HREIMSons ( m/z atC ν355.1880max 3420 [M and + 1670Na]+) cmof 2, indicatedaccountingC the H 128.6/5.91 ppm, CH) and a hydroxy methine group (IR: ν 3299 cm−1; δ /δ 65.0/4.84 ppm, CH). presence of hydroxyl and conjugated carbonyl functionalities,max respectively.C H The −1 NMR data of 2, for seven degrees of unsaturations. The IR absorptions at νmax 3420 and 1670 cm indicated the However, compound 3 differs in the presence of a trisubstituted double bond (δC/δH 135.4/5.52 ppm, presencemeasured of hydroxylin CDCl 3 and at −10 conjugated °C, were carbonyl comparable functi toonalities, those of respectively. the framewo Therk of NMR 1 except data of in 2 the, CH and δ 128.8 ppm, C), instead of a 1,1-disubstituted double bond in 2, with the appearance of replacementC of an sp 3 oxycarbon (δ C 74.6 ppm, C, C-7) and a methyl group (δ C/δ H 23.7/1.28 ppm, CH 3, measured in CDCl 3 at −10 °C, were comparable to those of the framework of 1 except in the an olefinic methyl (δ /δ 29.2/1.93 ppm). The hydroxyl group was found to be linked to the allylic CH 3-19) in 1 by an C exomethyleneH group (δ C/δ H 148.3 ppm, C and 117.7/5.35 and 5.18 ppm, CH 2, replacement of an sp 3 oxycarbon (δ C 74.6 ppm, C, C-7) and a methyl group (δ C/δ H 23.7/1.28 ppm, CH 3, carbon (C-5) due to the HMBC correlation observed from the olefinic methyl (δH 1.931 1 ppm, H3-19) CHrespectively)3-19) in 1 by in an2. Analysis exomethylene of NMR group data of (δ C 2/δ (TableH 148.3 1) ppm,and correlations C and 117.7/5.35 found in and the 5.18 H- H ppm, COSY CH and2, respectively)HMBC spectra in 2 . (FigureAnalysis 4) of enabled NMR data the of establishment 2 (Table 1) and of correlations the gross structure found in ofthe 2 .1 H- Furthermore,1H COSY and the HMBCobserved spectra NOE (Figure correlations 4) enabled for 2 (Figure the establishment 5) assigned o similarf the gross β-positioning structure for of 2H-1,. Furthermore, H-10, H-14, the and observedH3-17, and NOE α-orientation correlations for for H-2, 2 (Figure H-9, and 5) assigned H 3-18 as similar found inβ-positioning 1. Moreover, for one H-1, of H-10,the exomethylene H-14, and protons (δ H 5.18 ppm, s) exhibited NOE interaction with H-10 (δ H 2.97 ppm, br d, J = 7.0 Hz), while H3-17, and α-orientation for H-2, H-9, and H 3-18 as found in 1. Moreover, one of the exomethylene the other one (δ H 5.35, s) displayed NOE with the hydroxymethine proton at C-6 (δ H 4.07 ppm, dd, J protons (δ H 5.18 ppm, s) exhibited NOE interaction with H-10 (δ H 2.97 ppm, br d, J = 7.0 Hz), while = 11.0, 4.5 Hz). A molecular modeling investigation revealed that H-6 did not exhibit NOE interaction the other one (δ H 5.35, s) displayed NOE with the hydroxymethine proton at C-6 (δ H 4.07 ppm, dd, J = 11.0,in the 4.5 nuclear Hz). A Overhauser molecular modeling effect spectroscopy investigation (NOES revealedY) with that the H-6 methylene did not exhibit protons NOE at C-8 interaction (distances in>3.7 the nuclearÅ). This Overhauser designated effect the α-orientationspectroscopy of(NOES 6-OHY) and, with hence,the methylene the 6 R configurationprotons at C-8 as(distances shown in >3.7Figure Å). This6. Moreover, designated it was the found α-orientation that eunicellins of 6-OH with and, similar hence, substitutions the 6 R configuration in the ten-membered as shown ring, in Figurebut with 6. Moreover, an 6β-OH it (Figurewas found 6), displayedthat eunicellins distinctive with similar chemical substitutions shifts at C-6 in (δtheC
Mar. Drugs 2018 , 16 , x 5 of 11
findings, the absolute configuration of 1, and the biogenetic consideration, the configuration 1 S, 2R,3 Mar.R,6 RDrugs,9 R ,102018 R, 16,14, xS ,15 S was, thus, established for 2. 5 of 11 + findings,The HREIMS the absolute ( m/z 355.1880 configuration [M + Na] of 1,) andand theNMR biogenetic data (Table consideration, 1) indicated the a configuration molecular form 1 S, ula C20 H228RO,3 R4 ,6 forR,9 briarenoneR,10 R,14 S,15 CS was, ( 3), thus, inferring established that 3 for is 2an. isomer of 2. Therefore, it was found that 3 −1 possessedThe an HREIMS α,β-unsaturated ( m/z 355.1880 carbonyl [M + Na] (IR:+) νandmax 1668NMR cmdata δ(TableC 198.1 1) ppm,indicated C, 156.4 a molecular ppm, C, form andula δ C/δ H −1 128.6/5.91C20 H28 O ppm,4 for briarenoneCH) and a C hydroxy ( 3), inferring methine that group3 is an (IRisomer: νmax of 3299 2. Therefore, cm ; δC/δ itH was65.0/4.84 found ppm, that 3CH). −1 However,possessed compound an α,β-unsaturated 3 differs in carbonyl the presence (IR: νmax of 1668a trisubstituted cm δC 198.1 double ppm, C, bond 156.4 (δ ppm,C/δ H 135.4/5.52C, and δC/δ ppm,H −1 CH 128.6/5.91and δC 128.8 ppm, ppm, CH) C), and instead a hydroxy of a 1,1-disubstitutedmethine group (IR doub: νmax le3299 bond cm in; δ2C, /δwithH 65.0/4.84 the appearance ppm, CH). of an Mar. Drugs 2019, 17, 120 5 of 10 olefinicHowever, methyl compound (δ C/δ H 29.2/1.93 3 differs ppm). in the Thepresence hydroxyl of a trisubstituted group was found double to bond be linked (δ C/δ H to 135.4/5.52 the allylic ppm, carbon C 2 (C-5)CH due and to δ the 128.8 HMBC ppm, correlation C), instead ofobserved a 1,1-disubstituted from the olefinic double bondmethyl in (δ, Hwith 1.93 the ppm, appearance H 3-19) toof thean sp 2 olefinic methyl (δ C/δ H 29.2/1.93 ppm). The hydroxyl group was found1 1 to be linked to the allylic carbon tomethine the sp 2carbonmethine (δ C carbon135.4 ppm, (δ 135.4C-6), ppm,which C-6),in turn which exhibited in turn H- exhibitedH COSY1 correlationH-1H COSY with correlation H-5 (δ H (C-5) due to the HMBC correlationC observed from the olefinic methyl (δ H 1.93 ppm, H 3-19) to the sp 2 with4.84 H-5 ppm, (δ H dd,4.84 J ppm,= 8.5, dd, 8.5J = Hz). 8.5, 8.5 Moreover, Hz). Moreover, comparison comparison 1 of1 NMR of NMR data data of of3 3 withwith those those of methine carbon (δ C 135.4 ppm, C-6), which in turn exhibited H- H COSY correlation with H-5 (δ H pachyclavulariaenone D pointed out that 3 is the 5-hydroxy isomer of pachyclavulariaenone D [25]. pachyclavulariaenone4.84 ppm, dd, J = D 8.5, pointed 8.5 Hz). out that Moreover,3 is the comparison 5-hydroxy isomer of NMR of pachyclavulariaenone data of 3 with those D of [25]. 3 The pachyclavulariaenone gross gross structure structure of of3 was D was pointed further further out elucidated that elucidated 3 isfrom the 5-hydroxy from the full the 2D isomer full NMR 2D of spectroscopic NMRpachyclavulariaenone spectr correlationoscopic correlationD analyses[25]. 3 H ofanalyses3The(Figure gross of 43). (Figure structure The NOE 4). of The interaction 3 was NOE further interaction of H elucidated3-19 of with H from-19 the with olefinic the fullthe methineolefinic 2D NMR methine protons spectroscopic H-6protons (δ H correlation 5.52H-6 ppm,(δ 5.52 d, Jppm,= 8.5analyses d, Hz) J = (Figure 8.5 of 3Hz) (Figure5), (Figure in addition4). The 5), NOEin to addition the interaction downfield to the of H shiftdownfield3-19 ofwith C-19 the shift ( δolefinicC of29.2 C-19 ppm),methine (δ C indicated29.2 protons ppm), H-6 the indicated Z(δ Hgeometry 5.52 the ofZ geometry theppm, 6,7-double d, J of= 8.5 the bondHz) 6,7-double (Figure [31]. Moreover,5), bond in addition [31]. the Moreover, to NOE the downfield correlations the NOE shift forcorrelations of H-2/HC-19 (δ 3C-18, 29.2 for Hppm),H-2/H3-18/H-5, indicated3-18, H H-5/H-83-18/H-5, the α (H-5/H-8αδH Z2.74 geometry ppm, (δ H d,2.74 ofJ the= ppm, 14.5 6,7-double Hz),d, J and= 14.5 bond H-8 Hz), α[31]./H-9 and Moreover, disclosed H-8α/H-9 the the NOEdisclosed 5S configuration.correlations the 5 S forconfiguration. TheseH-2/H NOEs3-18, H These were3-18/H-5, further NOEs validatedwereH-5/H-8α further by a(δ validated molecularH 2.74 ppm, by modeling d, a J molecular= 14.5 study Hz), modelingand (Figure H-8α/H-96). stud Finally, disclosedy (Figure full NOEthe 6). 5 S correlation Finally,configuration. full analysis NOE These correlation (FigureNOEs 5) coupledanalysiswere (Figure with further the 5) validated previous coupled by with identification a molecular the previous modelingof the ident absolute ification study (Figureconfiguration of the 6). absolute Finally, of 1configuration , full which NOE was correlation co-isolatedof 1, which analysis (Figure 5) coupled with the previous identification of the absolute configuration of 1, which alongwas co-isolated with 3, designated along with the 3, 1 designatedS,2R,3R,5S,9 theR,10 1SR,2,14R,3S,15R,5 SS,9configurationR,10 R,14 S,15 forS configuration3. for 3. was co-isolated along with 3, designated the 1S,2 R,3 R,5 S,9 R,10 R,14 S,15 S configuration for 3.
Figure 4. Key1 1H-11H COSY and HMBC correlations for 2 and 3. FigureFigure 4.4. KeyKey 1H-1HH COSY COSY and and HMBC HMBC correlations correlations for 2 and 3..
Mar. Drugs 2018 , 16 , x Figure 5. Key NOE correlations for 2 and 3. 6 of 11 Figure 5. Key NOE correlations for 2 and 3.
Figure 5. Key NOE correlations for 2 and 3.
FigureFigure 6. A 6. partialA partialstructure structure of eunicellin-derived of eunicellin-derived diterpenoids: diterpenoids: cladiellisin [27], cladiellisin 3-acetyl [cladiellisin27], 3-acetyl [28],cladiellisin pachycladin [28], B pachycladin [29], and klysimplexins B [29], and klysimplexins C and E [30]. C and E [30].
Although briarellin-type diterpenoids (e.g., briarellins A–S) were discovered initially from Caribbean gorgonians of genus Briareum [12–16], since 1995, numerous related analogs were successfully isolated and identified from Pachyclavularia violacea inhabiting Taiwanese [24,25] and Indonesian [26] waters. Unlike the previously identified briarellins, the structures of briarenones A– C ( 1–3), isolated in this study from B. violaceum , were found to be similar to those of pachyclavulariaenones [24,25] in possessing the three ring-juncture protons (H-1, H-10, and H-14) cis to each other and forming the cis fusion of the cyclohexane, cyclodecane, and oxepane rings. Also, the determination of the absolute configurations of 1–3 by NOE correlation analysis coupled with X- ray crystallographic analysis of 1 using copper radiation, could imply the absolute configurations for similar briarellins isolated from the genera of Briareum and Pachyclavularia , such as pachyclavulariaenones A–G [24,25], to be similar to that of 1. Compounds of this kind possessing a six-membered carbocyclic ring cis -fused to both of the ten-membered carbocyclic and seven- membered ether rings, making three ring-junction protons (H-1, H-10, and H-14) cis to each other, including those reported from Pachyclavularia violacea [24–26], can be considered as a useful chemotaxonomic marker in classification of gorgonians and in approaching a resolution of the argument for considering the gorgonian species of Pachyclavularia to be the same as those of genus Briareum [26,32]. The isolated compounds were evaluated against the growth of DLD-1 (human colon adenocarcinoma), HT-29 (human colon carcinoma), and HuCC-T1 (human colon cholangiocellular carcinoma) cell lines. The in vitro anti-inflammatory activities of compounds 1‒3 were also measured against the release of elastase and the production of superoxide anions in N-formyl-methionyl-leucyl- phenyl-alanine and cytochalasin B (fMLF/CB)-activated neutrophils. However, the compounds did not show either cytotoxic or anti-inflammatory activities in the tested in vitro models.
3. Materials and Methods
3.1. General Experimental Procedures Optical rotations and IR spectra were measured on a JASCO P-1020 polarimeter and a JASCO FT/IR-4100 (JASCO Corporation, Tokyo, Japan) spectrophotometer, respectively. HRESIMS spectra were measured on a Bruker APEX II mass spectrometer (Bruker, Bremen, Germany). 1H and 13 C NMR spectra were obtained on a Varian Unity INOVA 600 FT-NMR (or Varian Unity INOVA500 FT-NMR) instruments (Varian Inc., Palo Alto, CA, USA) at 600 MHz (or 500 MHz) for 1H, and 150 MHz (or 125 13 MHz) for C in CDCl 3. Thin-layer chromatography (TLC) analyses were performed on precoated silica (Si) gel plates (Kieselgel 60 F-254, 0.2 mm), and Si gel (230–400 mesh) (Merck, Darmstadt, Germany) and C18-reversed phase Si gel (RP-18; 40–63 µM) (Parc-Technologique BLVD, Quebec, Canada) were used for column chromatography. Further purification and the isolation of compounds were performed by reversed-phase high-performance liquid chromatography (RP-HPLC) on a Hitachi L-2455 HPLC apparatus with a Supelco C18 column (250 × 21.2 mm, 5 µm).
Mar. Drugs 2019, 17, 120 6 of 10
Although briarellin-type diterpenoids (e.g., briarellins A–S) were discovered initially from Caribbean gorgonians of genus Briareum [12–16], since 1995, numerous related analogs were successfully isolated and identified from Pachyclavularia violacea inhabiting Taiwanese [24,25] and Indonesian [26] waters. Unlike the previously identified briarellins, the structures of briarenones A–C (1–3), isolated in this study from B. violaceum, were found to be similar to those of pachyclavulariaenones [24,25] in possessing the three ring-juncture protons (H-1, H-10, and H-14) cis to each other and forming the cis fusion of the cyclohexane, cyclodecane, and oxepane rings. Also, the determination of the absolute configurations of 1–3 by NOE correlation analysis coupled with X-ray crystallographic analysis of 1 using copper radiation, could imply the absolute configurations for similar briarellins isolated from the genera of Briareum and Pachyclavularia, such as pachyclavulariaenones A–G [24,25], to be similar to that of 1. Compounds of this kind possessing a six-membered carbocyclic ring cis-fused to both of the ten-membered carbocyclic and seven-membered ether rings, making three ring-junction protons (H-1, H-10, and H-14) cis to each other, including those reported from Pachyclavularia violacea [24–26], can be considered as a useful chemotaxonomic marker in classification of gorgonians and in approaching a resolution of the argument for considering the gorgonian species of Pachyclavularia to be the same as those of genus Briareum [26,32]. The isolated compounds were evaluated against the growth of DLD-1 (human colon adenocarcinoma), HT-29 (human colon carcinoma), and HuCC-T1 (human colon cholangiocellular carcinoma) cell lines. The in vitro anti-inflammatory activities of compounds 1-3 were also measured against the release of elastase and the production of superoxide anions in N-formyl-methionyl-leucyl-phenyl-alanine and cytochalasin B (fMLF/CB)-activated neutrophils. However, the compounds did not show either cytotoxic or anti-inflammatory activities in the tested in vitro models.
3. Materials and Methods
3.1. General Experimental Procedures Optical rotations and IR spectra were measured on a JASCO P-1020 polarimeter and a JASCO FT/IR-4100 (JASCO Corporation, Tokyo, Japan) spectrophotometer, respectively. HRESIMS spectra were measured on a Bruker APEX II mass spectrometer (Bruker, Bremen, Germany). 1H and 13C NMR spectra were obtained on a Varian Unity INOVA 600 FT-NMR (or Varian Unity INOVA500 FT-NMR) instruments (Varian Inc., Palo Alto, CA, USA) at 600 MHz (or 500 MHz) for 1H, and 150 13 MHz (or 125 MHz) for C in CDCl3. Thin-layer chromatography (TLC) analyses were performed on precoated silica (Si) gel plates (Kieselgel 60 F-254, 0.2 mm), and Si gel (230–400 mesh) (Merck, Darmstadt, Germany) and C18-reversed phase Si gel (RP-18; 40–63 µM) (Parc-Technologique BLVD, Quebec, Canada) were used for column chromatography. Further purification and the isolation of compounds were performed by reversed-phase high-performance liquid chromatography (RP-HPLC) on a Hitachi L-2455 HPLC apparatus with a Supelco C18 column (250 × 21.2 mm, 5 µm).
3.2. Animal Material The soft coral B. violaceum was collected from Jihui Fish Port (Taitung, Taiwan) by scuba divers at a depth of 10–15 m on March 2013 and stored in a freezer at −20 ◦C until extraction. Moreover, the soft coral was taxonomically identified by Prof. Chang-Feng Dai, National Taiwan University, Taipei. A voucher specimen was deposited in the Department of Marine Biotechnology and Resources, National Sun Yat-sen University, Kaohsiung.
3.3. Extraction and Isolation The freeze-dried soft coral (0.5 kg) was extracted with EtOAc (3 × 3 L) and filtered. The filtrate was evaporated in vacuo to yield the EtOAc extract (3.90 g). The extract was fractionated by Si gel column chromatography using EtOAc in n-hexane (6.25% to 100%) as a gradient elution system to Mar. Drugs 2019, 17, 120 7 of 10 afford 21 fractions (A01–A21). Fraction A04 was further fractionated on an RP-18 Si gel column, using MeOH-H2O (2.5:1) as a mobile phase, into eight subfractions (A0401–A0408). Compound 1 (7.4 mg) was obtained from A405 after two-step purification on RP-HPLC using MeOH-H2O (3:2) and acetyl nitrite (CH3CN)-H2O (1:2). Subfractions A402 and A403 were combined together on the basis of their similar TLC chromatogram and were further separated on RP-HPLC using CH3CN-H2O (1:2) to yield compounds 2 (4.5 mg) and 3 (2.2 mg).
3.3.1. Briarenone A (1) 25 Colorless needles; [α]D +224.4 (c 7.4, CHCl3); IR (neat) νmax 3477, 2964, 2929, 1728, 1670, and 756 −1 1 13 + cm ; H and C NMR data (600/150 MHz; CDCl3), Table1; ESIMS m/z 415 [M + Na] ; HRESIMS m/z + 415.2089 [M + Na] (calculated for C22H32O6Na, m/z 415.2091).
3.3.2. Briarenone B (2)
25 −1 Colorless powder; [α]D −10.7 (c 4.5, CHCl3); IR (neat) νmax 3420, 2912, 2851, 1670, and 772 cm ; 1 13 + H and C NMR data (500/125 MHz; CDCl3), Table1; ESIMS m/z 355 [M + Na] ; HRESIMS m/z + 355.1880 [M + Na] (calculated for C20H28O4Na, m/z 355.1880).
3.3.3. Briarenone C (3)
25 −1 1 Colorless powder; [α]D −14.8 (c 2.2, CHCl3); IR (neat) νmax 3299, 2921, 2851, and 1668 cm ; H 13 + and C NMR data (500/125 MHz; CDCl3), Table1; ESIMS m/z 355 [M + Na] ; HRESIMS m/z 355.1880 + [M + Na] (calculated for C20H28O4Na, m/z 355.1880).
3.3.4. Single-Crystal X-Ray Crystallography of 1 A suitable colorless prism of compound 1 was obtained from a solution in MeOH by slow evaporation for a month at 4 ◦C. The crystal (0.20 × 0.18 × 0.17 mm3) was analyzed at 100(2) K, space 3 group P212121 (# 19). Cell: a = 8.3456(3) Å, b = 10.6813(3) Å, c = 23.4466(8) Å, V = 2090.07(12) Å , Z = 4, 3 −1 Dcalcd = 1.305 mg/m , and µ(CuKα) = 0.790 mm . Intensity data of single-crystal X-ray diffraction were measured on a Bruker APEX DUO diffractometer. Of the 13326 reflections collected, only 3658 independent reflections [R(int) = 0.0331] with I > 2σ(I) were used for the analysis. The structure was solved by direct method and refined by a full-matrix least squares on F2 method. The refinement converged to a final R1 = 0.0401, wR2 = 0.1064, with goodness-of-fit = 1.091. For coordinates corresponding to the absolute stereochemistry represented, absolute structure parameter 0.04(6) was obtained [33].
3.4. Bioassays
3.4.1. Cytotoxicity Assay Cancer cell (DLD-1, HT-29, and HuCC-T1) lines were purchased from the American Type Culture Collection (ATCC). Compounds 1-3 were evaluated for cytotoxic activity using an Alamar blue assay. Alamar Blue (resazurin) is an important non-toxic redox indicator that is utilized to assess metabolic function and cell health. A complete description of this test was previously described [34,35]. The absorbance was measured at 570 nm using an ELISA plate reader (Thermo Fisher Scientific Instruments Co., Ltd., Vantaa, Finland).
3.4.2. In Vitro Anti-Inflammatory Assays Human neutrophils were obtained from blood by dextran sedimentation, Ficoll-Hypaque centrifugation, and hypotonic lysis and then incubated as previously described [36]. Neutrophils (6 × 105 cells·mL−1) incubated at 37 ◦C in Hank’s balanced salt solution (HBSS) with MeO-Suc-Ala-Ala-Pro-Val-p-nitroanilide (100 µM) and Ca2+ (1 mM) were treated with dimethyl sulfoxide (DMSO) or the tested compound for 5 min. The activation of neutrophils was challenged Mar. Drugs 2019, 17, 120 8 of 10
for 10 min with fMLF (100 nM)/CB (0.6 and 0.5 µg·mL−1 for superoxide anion generation and elastase release, respectively). The anti-inflammatory activities of compounds 1-3 were measured with examining the inhibition of FMLP/CB-induced human neutrophils producing superoxide anion and elastase, using UV spectrometer detection at wavelengths of 550 nm and 405 nm, respectively [36,37].
4. Conclusions Three new briarellin type diterpenoids, briarenones A-C (1-3), were identified from Briareum violaceum inhabiting Taiwanese waters. The compounds have three cis ring-juncture protons (H-1, H-10, and H-14) due to the cis fusion of the cyclohexane, cyclodecane, and oxepane rings. The molecular structures of this type may be considered as a useful chemotaxonomic marker in the identification of some species of genus Briareum. The absolute configurations of the compounds were assigned on the basis NOE correlation analysis coupled with a single-crystal X-ray diffraction analysis for briarenone A. The isolated metabolites showed no in vitro cytotoxicity against DLD-1, HT-29, and HuCC-T1 cells and did not inhibit the superoxide anion generation or elastase release in fMLF/CB-stimulated neutrophils. Compounds 1-3 did not exhibit cytotoxic and anti-inflammatory activities in this study; however, more biological activity screening should be carried out to discover their pharmaceutical potential. Moreover, the absolute configuration of 1 analyzed by X-ray diffraction would be useful for elucidation of the structurally similar metabolites isolated from the genera Briareum and Pachyclavularia.
Supplementary Materials: HRESIMS, 1H, 13C, HMQC, COSY, HMBC, and NOESY spectra of new compounds 1–3; and crystal data, atomic coordinate bond lengths [Å] and angles [◦] for 1 are available online at http: //www.mdpi.com/1660-3397/17/2/120/s1. Figure S1: HRESIMS spectrum of 1; Figure S2: 1H NMR spectrum 1 13 of 1 in CDCl3; Figure S3: H NMR spectrum of 1 in CDCl3 (1.08–2.82 and 3.10–5.92 ppm); Figure S4: C NMR 1 1 spectrum of 1 in CDCl3 and DEPT spectra; Figure S5: HSQC spectrum of 1 in CDCl3; Figure S6: H- H COSY spectrum of 1 in CDCl3; Figure S7: HMBC spectrum of 1 in CDCl3; Figure S8: ROESY spectrum of 1 in CDCl3; 1 1 Figure S9: HRESIMS spectrum of 2; Figure S10: H NMR spectrum of 2 in CDCl3; Figure S11: H NMR spectrum 13 of 2 in CDCl3 (0.96–3.38 and 3.24–6.08ppm); Figure S12: C NMR spectrum of 2 in CDCl3; Figure S13: DEPT 1 1 spectra of 2 in CDCl3; Figure S14: HSQC spectrum of 2 in CDCl3; Figure S15: H- H COSY spectrum of 2 in CDCl3; Figure S16: HMBC spectrum of 2 in CDCl3; Figure S17: NOESY spectrum of 2 in CDCl3; Figure S18: 1 1 HRESIMS spectrum of 3; Figure S19: H NMR spectrum of 3 in CDCl3; Figure S20: H NMR spectrum of 3 in 13 CDCl3 (0.82–3.04 and 3.38–6.03 ppm); Figure S21: C NMR spectrum of 1 in CDCl3; Figure S22: DEPT spectra of 1 1 3 in CDCl3; Figure S23: HSQC spectrum of 1 in CDCl3; Figure S24: H- HCOSY spectrum of 3 in CDCl3; Figure S25: HMBC spectrum of 3 in CDCl3; Figure S26: NOESY spectrum of 3 in CDCl3; Table S1: Crystal data and structure refinement for 1; Table S2: Atomic coordinates (× 104) and equivalent isotropic displacement parameters (Å2 × 103) for 1; Table S3: Bond lengths and bond angles for 1. Author Contributions: J.-H.S. designed the experiment. Y.C. isolated the compounds and performed spectroscopic data measurement and structure interpretation. A.F.A. performed the spectroscopic data analysis, final structure determination, and preparation of the manuscript. R.S.O. contributed to data analysis and editing of the manuscript. C.-F.D. contributed to species identification of the soft coral. Funding: The research was funded by the Ministry of Science and Technology of Taiwan (MOST104-2113-M-110-006 and 104-2811-M-110-026) and the 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 (MOST104-2113-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 conflicts of interest.
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