marine drugs

Article New Cytotoxic Secondary Metabolites from Marine Bryozoan

Xiang-Rong Tian 1,†, Yan-Qing Gao 1,†, Xiao-Lin Tian 1, Jiao Li 2, Hai-Feng Tang 3,*, Yu-Shan Li 4, Hou-Wen Lin 5 and Zhi-Qing Ma 1,* 1 Research & Development Center of Biorational Pesticide, Northwest A&F University, Yangling 712100, China; [email protected] (X.-R.T.); [email protected] (Y.-Q.G.); [email protected] (X.-L.T.) 2 State Key Laboratory of Elemento-Organic Chemistry, College of Chemistry, Nankai University, Tianjin 300071, China; [email protected] 3 Institute of Materia Medica, School of Pharmacy, Fourth Military Medical University, Xi’an 710032, China 4 School of Traditional Chinese Medicines, Shenyang Pharmaceutical University, Shenyang 110016, China; [email protected] 5 Department of Pharmacy, Renji Hospital, Affiliated to School of Medicine, Shanghai Jiao-Tong University, Shanghai 200127, China; [email protected] * Correspondence: [email protected] (H.-F.T.); [email protected] (Z.-Q.M.); Tel.: +86-29-8477-4748 (H.-F.T.); +86-29-8709-2122 (Z.-Q.M.); Fax: +86-29-8477-4748 (H.-F.T.); +86-29-8709-3344 (Z.-Q.M.) † These authors contributed equally to this work.

Academic Editor: Anake Kijjoa Received: 24 January 2017; Accepted: 11 April 2017; Published: 13 April 2017

Abstract: A new sterol, (23R)-methoxycholest-5,24-dien-3β-ol (1), two new ceramides, (2S,3R,4E,8E)- 2-(tetradecanoylamino)-4,8-octadecadien-l,3-diol (6) and (2S,3R,20R,4E,8E)-2-(tetradecanoylamino)- 4,8-octadecadien-l,3,20-triol (7), together with three known sterols (2–4), a lactone (5) and two ceramides (8,9), were isolated from the marine bryozoan Cryptosula pallasiana, collected at Huang Island of China. The structures of the new compounds were elucidated by extensive spectroscopic analyses, chemical methods and quantum electronic circular dichroism (ECD) calculations. Among the isolated compounds, sterol 1 possessed a rare side chain with a methoxy group at C-23, and a double bond between C-24 and C-25. Ceramides 6 and 7 possessed 14 carbons in their long-chain fatty acid base (FAB), which were different from the normal ceramides with 16 carbons in the FAB. Moreover, compounds 5 and 8 were isolated for the first time from marine bryozoans. Compounds 1–9 were evaluated for their cytotoxicity against human tumor cell lines HL-60, Hep-G2 and SGC-7901. The results showed that lactone 5 appears to have strong cytotoxicity against the test tumor cell lines, with IC50 values from 4.12 µM to 7.32 µM, and sterol 1 displayed moderate cytotoxicity with IC50 values between 12.34 µM and 18.37 µM, while ceramides 6–9 showed weak cytotoxicity with IC50 ranging from 21.13 µM to 58.15 µM.

Keywords: marine bryozoan; Cryptosula pallasiana; sterol; ceramides; cytotoxicity

1. Introduction Marine bryozoans are important sources for the discovery of new bioactive secondary metabolites. There are three reports containing 19 new metabolites isolated from this understudied phylum in the last year alone [1]. Modern phytochemical and pharmacological investigations of marine bryozoans demonstrated that secondary metabolites, including macrolides, alkaloids, sterols, as well as halogen-containing compounds, etc., exhibited remarkable cytotoxic activities on tumor cell lines, such as human myeloid leukemia HL-60, human leukemia U937, murine lymphocytic leukemia P388,

Mar. Drugs 2017, 15, 120; doi:10.3390/md15040120 www.mdpi.com/journal/marinedrugs Mar. Drugs 2017, 15, 120 2 of 10

Mar. Drugs 2017, 15, 120 2 of 10 and so on [2,3]. Cryptosula pallasiana is an encrusting colonial marine bryozoan, belonging to the class ,leukemia P388, order and Cheilostomata so on [2,3]. Cryptosula and family pallasiana . is an encrusting In our colonial previous marine investigation bryozoan, on the bioactivebelonging compounds to the fromclass thisGymnolaemata, marine bryozoan, order Cheilostomata 16 alkaloids, 13and sterols, family three Cryptosulidae. aromatic compoundsIn our and twoprevious glycerol investigation derivatives on the have bioactive been identified compounds [4 –from6]. In this the marine course bryozoan, of our ongoing 16 alkaloids, discovery 13 of new biologicallysterols, three active aromatic secondary compounds metabolites and two fromglycerol marine derivatives bryozoans, have beenC. pallasiana, identifiedcollected [4–6]. In fromthe the coastscourse of Huang of our Island ongoing in Qingdaodiscovery City,of new Shandong biologically Province active ofsecondary China, wasmetabolites investigated. from marine We report bryozoans, C. pallasiana, collected from the coasts of Huang Island in Qingdao City, Shandong herein the isolation and structure elucidation of three new compounds including one sterol (1) and Province of China, was investigated. We report herein the isolation and structure elucidation of three two ceramidesnew compounds (6 and including7), along one with sterol six known (1) and secondary two ceramides metabolites (6 and 7 including), along with three six sterols known ( 2–4), one lactonesecondary (5) metabolites and two ceramides including ( 8three,9). Thesterols structures (2–4), one of lactone the known (5) and compounds two ceramides were (8 identified,9). The by comparisonstructures of theirof the physical known andcompounds spectroscopic were dataidentified with thoseby comparison reported inof thetheir literature physical (Figure and 1). In addition,spectroscopic the cytotoxicity data with those of the reported isolated in compounds the literature against (Figure human1). In addition, tumor the cell cytotoxicity lines HL-60, of Hep-G2the and SGC-7901isolated compounds are also described. against human tumor cell lines HL‐60, Hep‐G2 and SGC‐7901 are also described.

Figure 1. Structures of compounds 1–9 from marine bryozoan C. pallasiana. Figure 1. Structures of compounds 1–9 from marine bryozoan C. pallasiana. 2. Results 2. Results The 95% EtOH extract of C. pallasiana was concentrated and partitioned with petroleum ether, TheCCl4 95%and EtOH n‐BuOH, extract respectively. of C. pallasiana The wasresulting concentrated CCl4 portion and partitioned was separated with petroleumby column ether, CCl4 chromatographyand n-BuOH, respectively. (CC) and high The‐performance resulting CCl liquid4 portion chromatography was separated (HPLC) by to column afford chromatographya new sterol (CC)( and1) and high-performance two new ceramides liquid(6 and 7 chromatography), along with six known (HPLC) compounds to afford (2–5, a 8 newand 9) sterol (Figure (1 1).) andThe two newknown ceramides compounds (6 and were7), alongidentified with as cholest six known‐5‐en‐3β‐ compoundsol (2) [7], (22 (E2)–‐5cholesta, 8 and‐7,229)‐dien (Figure‐3β,51α).,6β‐ The knowntriol compounds (3) [8], (24S,22 wereE)‐methylcholesta identified as‐ cholest-5-en-37,22‐dien‐3β,5αβ,6-olβ‐triol (2)[ (74)], [8], (22 Eloliolide)-cholesta-7,22-dien-3 (5) [9], (2S,3R,4Eβ)‐,52‐α,6β- triol ((tetradecanoylamino)3)[8], (24S,22E)-methylcholesta-7,22-dien-3‐4‐octadecen‐l,3‐diol (8) [10]β ,5andα,6 β(2-triolS,3R,4 (4E)[,8E8)],‐2 loliolide‐(hexadecanoylamino) (5)[9], (2S,3‐4,8R,4‐ E)-2- octadecadien‐l,3‐diol (9) [11], respectively, by comparison of their spectroscopic data (see (tetradecanoylamino)-4-octadecen-l,3-diol (8)[10] and (2S,3R,4E,8E)-2-(hexadecanoylamino)-4,8- supplementary information) with those reported in the literature. octadecadien-l,3-diol (9)[11], respectively, by comparison of their spectroscopic data (see Compound 1 was obtained as a white amorphous powder. Its molecular formula was supplementary information) with those reported in the literature. determined as C28H46O2 on the basis of the positive high‐resolution electrospray ionization mass Compoundspectroscopy1 (HRESIMS)was obtained m/z as 437.3395 a white [M amorphous + Na]+ (C28 powder.H46O2Na, Its calcd. molecular for 437.3396). formula The was 1H determined‐NMR as C28spectrumH46O2 on of the1 showed basis two of the tertiary positive methyl high-resolution resonance signals electrospray at δH 0.73, s ionization (H3‐18) and mass 1.00, s spectroscopy (H3‐19), + 1 (HRESIMS) m/z 437.3395 [M + Na] (C28H46O2Na, calcd. for 437.3396). The H-NMR spectrum of 1 showed two tertiary methyl resonance signals at δH 0.73, s (H3-18) and 1.00, s (H3-19), three secondary methyl groups at δH 0.95, d, J = 5.9 Hz (H3-21), δH 1.67, d, J = 1.0 Hz (H3-26) and 1.74, d, J = 1.1 Hz Mar. Drugs 2017, 15, 120 3 of 10 Mar. Drugs 2017, 15, 120 3 of 10 three secondary methyl groups at δH 0.95, d, J = 5.9 Hz (H3‐21), δH 1.67, d, J = 1.0 Hz (H3‐26) and 1.74, d, J = 1.1 Hz (H3‐27), one methoxy group at δH 3.21 s (OMe‐23), two oxymethine protons at δH 3.52, m, (H(H3‐-27),3) and one 3.94, methoxy dt, J = group9.3, 3.1 at HzδH (H3.21‐23), s (OMe-23), and two tri two‐substituted oxymethine olefinic protons protons at δH at3.52, δH m,5.35, (H-3) t, J = and 2.5 3.94,Hz (H dt,‐6)J and= 9.3, 5.03, 3.1 td, Hz J (H-23),= 9.0, 2.6, and 1.3 two Hz (H tri-substituted‐24). The 13C‐NMR olefinic spectrum protons showed at δH 5.35, 28 carbon t, J = 2.5 resonances, Hz (H-6) 13 andwhich 5.03, were td, J =assigned 9.0, 2.6, 1.3by Hz distortionless (H-24). The C-NMRenhancement spectrum by showedpolarization 28 carbon transfer resonances, (DEPT) which and wereheteronuclear assigned bysingle distortionless quantum coherence enhancement (HSQC) by polarization spectra to one transfer methoxy (DEPT) carbon and heteronuclear at δC 55.7 (OMe single‐23), quantumfive methyl coherence (C‐18, C (HSQC)‐19, C‐21, spectra C‐26 and to one C‐27), methoxy nine methylene carbon at spδC3 55.7(C‐1, (OMe-23), C‐2, C‐4, C five‐7, C methyl‐11, C‐ (C-18,12, C‐ 3 C-19,15, C C-21,‐16 and C-26 C‐ and22), C-27),seven ninemethine methylene sp3 (C‐ sp3, C(C-1,‐8, C C-2,‐9, C C-4,‐14, C-7,C‐17, C-11, C‐20 C-12, and C-15,C‐23) C-16 including and C-22), two 3 sevenhydroxymethines methine sp C(C-3,‐3 (δC C-8, 71.8) C-9, and C-14, C‐23 C-17, (δC 74.8), C-20 two and quaternary C-23) including sp3 carbons two hydroxymethines (C‐10 and C‐13), C-3two 3 2 (methineδC 71.8) andsp2 carbons C-23 (δ C ‐74.8),6 (δC 121.7) two quaternary and C‐24 ( spδC 127.0),carbons and (C-10 two andquaternary C-13), twosp2 carbons methine C sp‐5 (δcarbonsC 140.7) 2 C-6and ( δCC‐25121.7) (δC and134.5). C-24 These (δC 127.0),data suggested and two quaternary that 1 possessed sp carbons a 3‐hydroxy C-5 (δC ∆140.7)5‐steroid and C-25nucleus (δC with134.5). a 5 Thesemethoxy data group suggested and double that 1 possessedbond in its a side 3-hydroxy chain. Further∆ -steroid structural nucleus information with a methoxy for compound group and 1 doublewas obtained bond in itsby side 2D chain. NMR Further analysis structural including information 1H‐1H forcorrelation compound spectroscopy1 was obtained (COSY) by 2D NMR and 1 1 analysisheteronuclear including multipleH- H bond correlation correlation spectroscopy (HMBC) (COSY) spectra and (Figure heteronuclear 2). The observation multiple bond of correlations correlation 1 1 (HMBC)from H‐23 spectra to H2‐ (Figure22 and2 H).‐ The24 in observation the 1H‐1H COSY of correlations spectrum, from and H-23 correlations to H 2-22 from and methoxy H-24 in the protonH- Hto COSYC‐23, from spectrum, H2‐22 andto C‐ correlations23, from H3‐26 from to C methoxy‐24 and C proton‐25, and to from C-23, H from3‐27 to H C2-22‐24 toand C-23, C‐25 from in the H 3HMBC-26 to C-24spectrum and C-25, indicated and fromthat the H3 methoxy-27 to C-24 group and is C-25 on C in‐23, the and HMBC the tri spectrum‐substituted indicated double that bond the is methoxy between groupC‐24 and is on C‐ C-23,25. Based and theon the tri-substituted above evidences, double the bond planar is between structure C-24 of sterol and C-25. 1 could Based be established on the above as evidences,23‐methoxycholest the planar‐5,24 structure‐dien‐3‐ ofol. sterol 1 could be established as 23-methoxycholest-5,24-dien-3-ol.

FigureFigure 2. 2.Key Key1 1H-H‐11H COSY and HMBC correlations of 1..

The stereochemistry of 1 was established by extensive analysis of nuclear overhauser effect The stereochemistry of 1 was established by extensive analysis of nuclear overhauser effect spectroscopy (NOESY) spectrum and quantum electronic circular dichroism (ECD) calculations. The spectroscopy (NOESY) spectrum and quantum electronic circular dichroism (ECD) calculations. 1H‐NMR assignment of α and β protons for compound 1 (Table 1) was based on careful analysis of The 1H-NMR assignment of α and β protons for compound 1 (Table1) was based on careful analysis of the NOESY spectrum as well as comparison with the reference [5] (Figure 3). The NOESY correlations the NOESY spectrum as well as comparison with the reference [5] (Figure3). The NOESY correlations from H3‐19 to H‐1α and H‐1β, from H‐3 to H‐4α and H‐2α, and from H‐4α to H‐6, suggested that the from H -19 to H-1α and H-1β, from H-3 to H-4α and H-2α, and from H-4α to H-6, suggested that the A‐ring 3exists in a chair conformation. The 3‐hydroxyl group was oriented in an equatorial position A-ring exists in a chair conformation. The 3-hydroxyl group was oriented in an equatorial position and therefore in a β‐orientation, which was confirmed by the chemical shift of C‐3 (δC 71.8 > 70.0) and therefore in a β-orientation, which was confirmed by the chemical shift of C-3 (δ 71.8 > 70.0) [12]. [12]. Similarly, the presence of a cross‐peak between H‐14 and H‐17 implied that theC C‐17 side chain Similarly, the presence of a cross-peak between H-14 and H-17 implied that the C-17 side chain was was in a β‐position. The correlations from H3‐21 to H‐12β and H‐17, and from H‐20 to H‐16β and H3‐ in a β-position. The correlations from H -21 to H-12β and H-17, and from H-20 to H-16β and H -18 18 indicating the α‐orientation of the methyl3 group at C‐20, and determined the C‐20S configuration3 indicating the α-orientation of the methyl group at C-20, and determined the C-20S configuration for 1. The C‐23R configuration was determined by ECD calculations of 23R and 23S for 1 using the for 1. The C-23R configuration was determined by ECD calculations of 23R and 23S for 1 using the time‐dependent density functional theory (TD‐DFT) method at the B3LYP/6‐31G (d) level [13–16]. time-dependent density functional theory (TD-DFT) method at the B3LYP/6-31G (d) level [13–16]. The preliminary conformational distribution search was performed by HyperChem 7.5 software The preliminary conformational distribution search was performed by HyperChem 7.5 software (Hypercube, Inc., Gainesville, FL, USA). The corresponding minimum geometries were fully (Hypercube, Inc., Gainesville, FL, USA). The corresponding minimum geometries were fully optimized optimized by using DFT at the B3LYP/6‐31G (d) levels implemented in the Gaussian 09 program by using DFT at the B3LYP/6-31G (d) levels implemented in the Gaussian 09 program package. The package. The ECD calculations were performed after optimization of the selected conformers at the ECD calculations were performed after optimization of the selected conformers at the B3LYP/6-31G B3LYP/6‐31G (d) [17,18]. The result showed that the experimental circular dichroism (CD) curve of 1 (d) [17,18]. The result showed that the experimental circular dichroism (CD) curve of 1 was consistent was consistent with the calculated ECD curve of 23R, while opposite to that of 23S (Figure 4). The with the calculated ECD curve of 23R, while opposite to that of 23S (Figure4). The NOESY correlations NOESY correlations from H‐23 to H‐20, H‐22a and H3‐26, and from H‐24 to H‐22α, H‐22β and H3‐27, from H-23 to H-20, H-22α and H -26, and from H-24 to H-22α, H-22β and H -27, indicated the indicated the α‐orientation of the methoxy3 group at C‐23, which also confirmed the3 23R configuration. α-orientation of the methoxy group at C-23, which also confirmed the 23R configuration. Thus, sterol 1 Thus, sterol 1 was unambiguously elucidated as (23R)‐methoxycholest‐5,24‐dien‐3β‐ol. was unambiguously elucidated as (23R)-methoxycholest-5,24-dien-3β-ol.

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Table 1. 1H‐ and 13C‐NMR (500 and 125 MHz, CDCl3) data of 1 a. 1 13 a Mar. Drugs 2017,, 15,, 120Table 1. H- and C-NMR (500 and 125 MHz, CDCl3) data of 1 . 4 of 10 No. δC δH mult., J in Hz No. δC δH mult., J in Hz

δ 1 δ 13 δ δ a 1 No. 37.3 αTableC 1. 1H‐ andH mult.,1.86, 13C‐ NMRmJ in Hz (500 and No.12513 MHz, CDClC42.53) data of 1H a.mult., J in Hz– 1 37.3 β 1.08,α 1.86, m m 1314 42.556.9 – 1.04, m No. δC δH mult., J in Hz No. δC δH mult., J in Hz 2 31.7 α 1.84,β 1.08, m m 1415 56.924.3 α 1.04, m 1.57, m 1 37.3 α 1.86, m 13 42.5 – 2 31.7 β 1.50,α 1.84, m m 15 24.3 α 1.57, β m 1.07, m ββ 1.08,1.50, m 14 56.9 β 1.07,1.04, m m 3 2 71.831.7 α3.52, 1.84, m m 1516 24.328.2 α α 1.57, 1.83, m m 3 71.8 3.52, m 16 28.2 α 1.83, m 4 42.3 α β2.28, 1.50, m m β 1.07, β 1.30, m m 3 471.8 42.3 β 2.23,α3.52,2.28, mm m 1617 28.256.8 α β 1.30, 1.83, m1.01, m m β 2.23, m 17 56.8 1.01, m 5 4 140.742.3 α 2.28,– m 18 12.0 β 1.30,0.73, m s 6 5121.7 140.75.35, β 2.23, t –(2.5) m 181719 12.056.819.4 0.73,1.01, s 1.00, m s 7 5 631.9140.7 α 121.7 5.35,1.50,– t (2.5)m 191820 19.412.032.5 1.00,0.73, s1.66, s m 6 7121.7 31.9 β5.35, 1.97,α 1.50, t (2.5)m m 201921 32.519.418.7 1.66,1.00,0.95, m s d (5.9) 7 31.9 αβ 1.50,1.97, m 2120 18.732.5 0.95,1.66, d (5.9) m 8 31.9 1.50, m 22 42.5 α 0.92, m β 1.97, m 21 18.7 α0.95, d (5.9) 8 31.9 1.50, m 22 β 42.5 0.92, m 1.68, m 8 31.9 1.50, m 22 42.5 α β 1.68,0.92, m m 9 50.1 0.93, m 23 74.8 3.94, dt (9.3, 3.1) 9 50.1 0.93, m 23 β 74.8 3.94, dt (9.3,1.68, 3.1) m 10 9 36.550.1 0.93,– m 2324 74.8127.0 3.94,5.03, dt td (9.3, (9.0, 3.1) 2.6, 1.3) 10 36.5 – 24 127.0 5.03, td (9.0, 2.6, 1.3) 11 10 21.136.5 α 1.43,– m 2425 127.0134.5 5.03, td (9.0, 2.6,– 1.3) 11 21.1 α 1.43, m 25 134.5 – 11 21.1 α β 1.49, 1.43, m m 2526 134.518.2 1.67,– d (1.0) β 1.49, m 26 18.2 1.67, d (1.0) 12 39.8 α β1.16, 1.49, m m 2627 18.225.9 1.67,1.74, d (1.0) d (1.1) 12 39.8 α 1.16, m 27 25.9 1.74, d (1.1) 12 39.8 α β 2.03, 1.16, m m 23‐27OCH 3 25.955.7 1.74, d 3.21,(1.1) s β 2.03, m 23-OCH3 55.7 3.21, s 3 a β 2.03, m 23‐OCH3 55.7 3.21, s Assignmentsa Assignments based based on DEPTsDEPTs and and HSQC. HSQC. a Assignments based on DEPTs and HSQC.

Figure 3. Key NOESY correlations ( ) for 1. FigureFigure 3. 3. KeyKey NOESY NOESY correlations correlations ( ( )) forfor 11..

FigureFigure 4. 4.Experimental Experimental andand calculated ECD ECD spectra spectra for for 1. 1.

Compound 6 was obtained as a white amorphous powder. The (+)‐HRESIMS showed a Compound 6 wasFigure obtained 4. Experimental as a white and amorphous calculated ECD powder. spectra The for 1 (+)-HRESIMS. showed a + pseudomolecular ion peak at m/z 530.4545 [M + Na]+ (calcd. for C32H61NO3Na, 530.4549), which, pseudomolecular ion peak at m/z 530.4545 [M + Na] (calcd.− for C32H61NO3Na, 530.4549), which, together with the pseudomolecular ion peak at m/z 506 [M − H] in− the (‒)‐ESIMS spectrum, enabled togetherCompound with the 6 pseudomolecular was obtained as ion a peak white at m/zamorphous506 [M − powder.H] in the The (-)-ESIMS (+)‐HRESIMS spectrum,1 showed13 enabled a the determination of the molecular formula for 6 as C32H61NO3. A careful analysis of the 1H‐ and 13C‐ + 1 thepseudomolecular determination ion of thepeak molecular at m/z 530.4545 formula [M for 6+ asNa] C32 (calcd.H61NO for3. A C careful32H61NO analysis3Na, 530.4549), of the H-which, and 13togetherC-NMR with spectra the pseudomolecular of 6 (Table2) by DEPT ion peak and HSQCat m/z 506 experiments [M − H]− in revealed the (‒)‐ESIMS the presence spectrum, of an enabled amide the determination of the molecular formula for 6 as C32H61NO3. A careful analysis of the 1H‐ and 13C‐

Mar. Drugs 2017, 15, 120 5 of 10

0 group δC 174.0 (C-1 ), δH 6.25, d, J = 7.5 Hz (NH), a nitrogen- or oxygen-bearing methine δC 54.5 (C-2), 3 δH 3.95, dd, J = 11.3, 3.5 Hz (H-2), an oxymethylene sp δC 62.5 (C-1), δH 3.70, dd, J = 11.3, 3.3 Hz 3 2 and 3.90, m (H2-1), an oxymethine sp δC 74.7 (C-3), δH 4.32, m (H-3), and four methine sp δC 129.2 (C-4), δH 5.54, dd, J = 15.4, 6.4 Hz (H-4); δC 133.6 (C-5), δH 5.78, dt, J = 15.4, 6.6 Hz (H-5); δC 131.4 (C-8), δH 5.42, dt, J = 15.2, 6.3 Hz (H-8) and δC 129.0 (C-9), δH 5.37, dt, J = 15.2, 6.4 Hz (H-9), which together with a series of alkyl proton signals (δC 29.2–29.7, δH 1.26, br s), indicated an existence of a 1 1 sphinga-4,8-diene skeleton for 6 [19]. The H- H COSY correlations from H-2 to NH, H2-1 and H-3, 0 0 0 0 and the HMBC correlations from NH to C-1 , from H2-2 to C-1 and C-3 , and from H-3 to C-1 and C-2 confirmed the sphingosine skeleton. That the two double bonds were placed on C-4 and C-8 1 1 was supported by H- H COSY correlations of H-3/H-4, H-4/H-5, H-5/H2-6, H2-6/H2-7, H2-7/H-8, H-8/H-9 and H-9/H2-10, as well as by HMBC correlations from H-3 to C-4 and C-5, from H-8 to C-6 and C-7, and from H2-10 to C-8 and C-9 (Figure5A). That the sphingoid long chain base (LCB) and the long chain fatty acid base (FAB) of the amide portion contained 18 and 14 carbons, respectively, was evidenced by the electrospray ionization mass spectroscopy (ESI-MS) fragments at m/z 219, 228, 262, 283, 394 (Figure5B) [ 20]. The number of the carbon for the FAB was further validated through electron impact mass spectrometry (EI-MS) molecular ion at m/z 242 [M]+ for the methyl tetradecanoate obtained after methanolysis of 6 with 5% HCl-MeOH. Therefore, the planar structure and the key connectivities of 6 were established.

1 13 a Table 2. H- and C-NMR (500 and 125 MHz, CDCl3) of 6 and 7 .

6 7 No. δC δH mult., J in Hz δC δH mult., J in Hz 1 62.5 3.70, dd (11.3, 3.3) 62.2 3.72, dd (11.0, 3.0) 3.90, m 3.89, m 2 54.5 3.95, dd (11.3, 3.5) 54.4 3.92, br d (11.0) 3 74.7 4.32, m 74.7 4.30, m 4 129.2 5.54, dd (15.4, 6.4) 129.2 5.52, dd (15.5, 6.0) 5 133.6 5.78, dt (15.4, 6.6) 133.5 5.75, dt (15.5, 6.1) 6 32.3 1.97, q (6.7) 32.4 1.96, q (6.5) 7 32.1 2.12, m 32.2 2.11, m 8 131.4 5.42, dt (15.2, 6.3) 131.3 5.41, m 9 129.0 5.37, dt (15.2, 6.4) 129.0 5.38, m 10 32.6 2.08, m 32.2 2.07, m 11~15 29.2–29.7 1.26, br s 29.2–29.7 1.26, br s 16 31.9 1.26, br s 32.0 1.26, br s 17 22.7 1.26, br s 22.7 1.26, br s 18 14.1 0.88, t (6.8) 14.1 0.87, t (7.0) 10 174.0 – 175.0 – 20 36.9 2.23, t (7.5) 72.4 4.12, dd (7.8, 3.4) 30 25.8 1.64, m 34.8 2.16, t (3.3) 40 29.2–29.7 1.26, br s 31.8 1.26, br s 50 29.2–29.7 1.26, br s 25.1 1.63, m 60~110 29.2–29.7 1.26, br s 29.2–29.7 1.26, br s 120 31.9 1.26, br s 31.9 1.26, br s 130 22.7 1.26, br s 22.7 1.26, br s 140 14.1 0.88, t (6.8) 14.1 0.87, t (7.0) NH – 6.25, d (7.5) – 6.20, d (7.8) a Assignments based on DEPTs and HSQC. Mar. Drugs 2017, 15, 120 6 of 10 Mar. Drugs 2017, 15, 120 6 of 10 Mar. Drugs 2017, 15, 120 6 of 10

Figure 5. Key 1H‐11H COSY and HMBC correlations (A), and main ESI‐MS fragment ions (B) of 6. Figure 5. Key 1HH-‐1HH COSY COSY and and HMBC HMBC correlations correlations ( (AA),), and and main main ESI ESI-MS‐MS fragment fragment ions ions ( (BB)) of of 66.. (aa positive fragment ions; b negative fragment ions). (a positive fragment ions; b negativenegative fragment fragment ions). ions). That the geometry of C‐4/C‐5 and C‐8/C‐9 double bonds are trans was based on the vicinal That the geometrygeometry ofof C-4/C-5C‐4/C‐5 and C C-8/C-9‐8/C‐9 double double bonds bonds are are transtrans waswas based on the vicinal coupling constants J4,5 = 15.4 Hz and J8,9 = 15.2 Hz, as well as the chemical shifts of C‐6 (δC 32.3) and coupling constants JJ4,54,5 == 15.4 15.4 Hz Hz and and J8,9J 8,9= 15.2= 15.2 Hz, Hz, as well as well as the as thechemical chemical shifts shifts of C of‐6 C-6(δC 32.3) (δC 32.3) and C‐7 (δC 32.1) [21]. The absolute stereochemistry of 6 was determined as D‐(−)‐erythro‐2S,3R andC‐7 C-7(δC (32.1)δC 32.1) [21]. [21 The]. The absolute absolute stereochemistry stereochemistry of of 66 waswas determineddetermined asas DD-(‐−(−)-)‐erythroerythro-2‐2S,3,3R configuration based on the chemical shifts of C‐2 (δC 54.5) and C‐3 (δC 74.7), which were in good configurationconfiguration based on the chemical shifts of C-2C‐2 ((δδCC 54.5) and C-3C‐3 (δCC 74.7), which were in good agreement with those reported for N‐palmitoyl‐D‐erythro‐(2S,3R)‐octadecasphinga‐4(E)‐en (C‐2 for agreement with thosethose reportedreported forfor NN-palmitoyl-‐palmitoyl‐DD-‐erythroerythro-(2‐(2S,3,3R)-octadecasphinga-4()‐octadecasphinga‐4(E))-en‐en (C (C-2‐2 for 54.5 and C‐3 for 74.7) [22,23]. Based on the above analyses, the structure of 6 was determined as 54.5 and C-3C‐3 for 74.7)74.7) [[22,23].22,23]. Based on the above analyses, the structure of 6 was determined as (2S,3R,4E,8E)‐2‐(tetradecanoylamino)‐4,8‐octadecadien‐l,3‐diol. (2S,3,3R,4,4E,8,8E)-2-(tetradecanoylamino)-4,8-octadecadien-l,3-diol.)‐2‐(tetradecanoylamino)‐4,8‐octadecadien‐l,3‐diol. The molecular formula of compound 7 was assigned as C32H61NO4 on the basis of (+)‐HRESIMS The molecular formulaformula ofof compoundcompound7 7was was assigned assigned as as C C3232HH6161NO4 on the basis of (+)-HRESIMS(+)‐HRESIMS + 32 61 4 1 pseudomolecular ion peak at m/z 546.4496 [M]+ (calcd.+ for C H NO Na, 546.4498) and the 1H‐ and pseudomolecular ion ion peak peak at at m/zm/z 546.4496546.4496 [M] [M] (calcd.(calcd. for C for32H61 CNOH4Na,NO 546.4498)Na, 546.4498) and the andH‐ and the 13C‐NMR spectra (Table 2). The 13C‐NMR spectrum of 7 showed 32a close61 resemblance4 to that of 6, 113H-C‐NMR and 13 spectraC-NMR (Table spectra 2). (Table The 132C).‐NMR The 13spectrumC-NMR spectrumof 7 showed of 7a showedclose resemblance a close resemblance to that of to6, except for the additional hydroxyl group on C‐2′ in the FAB, since 7 showed different chemical shifts thatexcept of for6, exceptthe additional for the hydroxyl additional group hydroxyl on C‐ group2′ in the on FAB, C-2 since0 in the 7 showed FAB, since different7 showed chemical different shifts of C‐1′ (δC 175.0), C‐2′ (0δC 72.4) and C‐30′ (δC 34.8) in its FAB.0 HMBC correlations of H‐2′ (δH 4.12) to C‐ chemicalof C‐1′ (δC shifts 175.0), of C C-1‐2′ (δ(Cδ 72.4)175.0), and C-2C‐3′ ((δδC 34.8)72.4) in and its FAB. C-3 (HMBCδ 34.8) correlations in its FAB. of HMBC H‐2′ (δ correlationsH 4.12) to C‐ 1′ and C‐3′ confirmed thatC the hydroxyl groupC was on C‐2′ (FigureC 6A). A series of fragment ions at of1′ and H-2 C0 (‐δ3′ confirmed4.12) to C-1 that0 andthe hydroxyl C-30 confirmed group was that on the C‐ hydroxyl2′ (Figure group6A). A wasseries on of C-2 fragment0 (Figure ions6A). at m/z 227, 242,H 283 and 296 in the (‒)‐ESIMS spectrum (Figure 6B), deduced the lengths of LCB and FAB Am/z series 227, 242, of fragment283 and 296 ions in the at m/z(‒)‐ESIMS227, 242, spectrum 283 and (Figure 296 6B), in the deduced (-)-ESIMS the lengths spectrum of LCB (Figure and 6FABB), to be 18 and 14 carbons, respectively. Additionally, the length of FAB was further validated through to be 18 and 14 carbons, respectively. Additionally, the length of FAB was further validated through deduced the lengths of LCB and FAB to+ be 18 and 14 carbons, respectively. Additionally, the (+)‐EIMS molecular ion at m/z 258 [M]+ of (R)‐methyl 2‐hydroxytetradecanoate +obtained from length(+)‐EIMS of FABmolecular was further ion at validated m/z 258 through[M] of (+)-EIMS(R)‐methyl molecular 2‐hydroxytetradecanoate22 ion at m/z 258 [M] obtainedof (R)-methyl from methanolysis of 7. Comparisons of the optical rotation of 7 ([α]22D +9.5°) with (2S,3R,2′R,4E)‐2′‐hydroxy‐ 2-hydroxytetradecanoatemethanolysis of 7. Comparisons obtained of the from optical methanolysis rotation of 7 of ([α7.]D Comparisons+9.5°) with (2S,3 ofR the,2′R,4 opticalE)‐2′‐hydroxy rotation‐ C16‐ceramide22 ([◦α]D +5.2°) and its0 2′S‐isomer0 ([α]D −16.1°), suggested that 7 ◦and (2S,3R,20 ′R,4E)‐2′‐ ofC167‐ceramide([α]D +9.5 ([α)]D with +5.2°) (2 Sand,3R ,2itsR 2,4′SE‐)-2isomer-hydroxy-C16-ceramide ([α]D −16.1°), suggested ([α] Dthat+5.2 7 and) and (2S its,3R 2,2S′R-isomer,4E)‐2′‐ hydroxy‐C16◦ ‐ceramide have the same absolute0 configurations0 for core chiral centers C‐2, C‐3 and C‐ ([hydroxyα]D −16.1‐C16),‐ceramide suggested have that the7 and same (2S absolute,3R,2 R,4 configurationsE)-2 -hydroxy-C16-ceramide for core chiral havecenters the C same‐2, C‐ absolute3 and C‐ 2′ [24]. Thus, compound 7 was determined as 0(2S,3R,2′R,4E,8E)‐2‐(tetradecanoylamino)‐4,8‐ configurations2′ [24]. Thus, forcompound core chiral 7 centers was determined C-2, C-3 and as C-2 (2[S24,3].R,2 Thus,′R,4E compound,8E)‐2‐(tetradecanoylamino)7 was determined‐4,8 as‐ octadecadien0 ‐l,3,2′‐triol. 0 (2octadecadienS,3R,2 R,4E,8‐l,3,2E)-2-(tetradecanoylamino)-4,8-octadecadien-l,3,2′‐triol. -triol.

Figure 6. Key 1H‐1H COSY and HMBC correlations (A), and main ESI‐MS fragments (B) of 7. FigureFigure 6.6. Key 11HH-‐1HH COSY COSY and and HMBC HMBC correlations correlations ( A),), and and main ESI ESI-MS‐MS fragments fragments ( B) of 7.. Compounds 1–9 were evaluated for their cytotoxicity against human myeloid leukemia cell line Compounds 1–9 were evaluated for their cytotoxicity against human myeloid leukemia cell line HL‐60,Compounds human hepatocellular1–9 were evaluated carcinoma for theirHepG cytotoxicity‐2, and human against gastric human carcinoma myeloid SGC leukemia‐7901 using cell line an HL‐60, human hepatocellular carcinoma HepG‐2, and human gastric carcinoma SGC‐7901 using an HL-60,MTT assay, human and hepatocellular results are shown carcinoma in Table HepG-2, 3. Compound and human 1 displayed gastric carcinomaa moderate SGC-7901 cytotoxicity using to the an MTT assay, and results are shown in Table 3. Compound 1 displayed a moderate cytotoxicity to the MTTtest cell assay, lines and with results IC are50 values shown between in Table3 .12.34 Compound μM and1 displayed 18.37 μM, a moderate while 2– cytotoxicity4 did not show to the any test test cell lines with IC50 values between 12.34 μM and 18.37 μM, while 2–4 did not show any cellcytotoxicity. lines with Since IC50 values5 exhibited between strong 12.34 cytotoxicityµM and 18.37 againstµM, the while test2 tumor–4 did notcell showlines with any cytotoxicity. IC50 values cytotoxicity. Since 5 exhibited strong cytotoxicity against the test tumor cell lines with IC50 values Sincebetween5 exhibited 4.12 μM strong and 7.32 cytotoxicity μM, it can against be considered the test tumor as a potential cell lines for with the IC furthervalues development between 4.12 ofµ anM between 4.12 μM and 7.32 μM, it can be considered as a potential for the further50 development of an andanticancer 7.32 µM, agent. it can Compounds be considered 6–9 asshowed a potential weak forcytotoxicity the further against development all the test of tumor an anticancer cell lines agent. with anticancer agent. Compounds 6–9 showed weak cytotoxicity against all the test tumor cell lines with CompoundsIC50 values ranging6–9 showed from weak 21.13 cytotoxicity μM to 58.15 against μM; however, all the test they tumor showed cell lines stronger with cytotoxicity IC50 values ranging against IC50 values ranging from 21.13 μM to 58.15 μM; however, they showed stronger cytotoxicity against fromHepG 21.13‐2 andµM SGC to 58.15‐7901µ thanM; however, HL‐60, suggesting they showed a selectivity stronger cytotoxicity of these ceramides against HepG-2 against and HepG SGC-7901‐2 and HepG‐2 and SGC‐7901 than HL‐60, suggesting a selectivity of these ceramides against HepG‐2 and thanSGC‐ HL-60,7901. Furthermore, suggesting a selectivityceramide of7, thesehaving ceramides the 2′‐OH against group HepG-2 in its and FAB, SGC-7901. displayed Furthermore, stronger SGC‐7901. Furthermore, ceramide 7, having the 2′‐OH group in its FAB, displayed stronger cytotoxicity than its dehydroxy counterparts, indicating that the 2′‐OH group in the FAB may play cytotoxicity than its dehydroxy counterparts, indicating that the 2′‐OH group in the FAB may play an important role in the expression of cytotoxicity for ceramides [24,25]. an important role in the expression of cytotoxicity for ceramides [24,25].

Mar. Drugs 2017, 15, 120 7 of 10 ceramide 7, having the 20-OH group in its FAB, displayed stronger cytotoxicity than its dehydroxy counterparts, indicating that the 20-OH group in the FAB may play an important role in the expression of cytotoxicity for ceramides [24,25].

Table 3. Cytotoxicity of compounds 1–9 against HL-60, HepG2 and SGC7901 tumor cell lines in vitro a (IC50, µM) .

Compound HL-60 HepG-2 SGC-7901 1 17.64 ± 0.32 12.34 ± 0.12 18.37 ± 0.17 2 >100 >100 >100 3 >100 >100 >100 4 >100 >100 >100 5 6.29 ± 0.11 4.12 ± 0.15 7.32 ± 0.26 6 32.26 ± 0.23 26.69 ± 0.21 27.14 ± 0.30 7 25.32 ± 0.17 21.13 ± 0.13 22.74 ± 0.16 8 35.72 ± 0.36 28.53 ± 0.24 30.31 ± 0.14 9 58.15 ± 0.28 46.21 ± 0.17 45.79 ± 0.12 Adriamycin b 2.51 ± 0.14 2.73 ± 0.23 2.65 ± 0.17 a IC50 values are means from three independent experiments in which each compound concentration was tested in three replicate wells; b Adriamycin as a positive control.

3. Experimental Section

3.1. General Experimental Procedures Optical rotations were measured on a Perkin-Elmer 343 polarimeter. 1D and 2D NMR spectra were obtained in CDCl3 or CD3OD on a Bruker AVANCE-500 spectrometer, with tetramethylsilane (TMS) as an internal standard. Chemical shifts (δ) were expressed in ppm and coupling constants in Hz. EI-MS spectrum was obtained on a Finnigan MAT 212 mass spectrometer. ESI-MS and HR-ESI-MS spectra were taken on a Micromass Quattro mass spectrometer (Micromass, Manchester, UK). CD spectrum was recorded on JASCOJ-720 spectrophotometer (JASCO Corporation, Tokyo, Japan). Separation and purification were performed by CC on silica gel H (10–40 µm, Qingdao Marine Chemical Inc., Qingdao, China), Sephadex LH-20 (Pharmacia Inc., New Jersey, NJ, USA) and reversed-phase Si gel (Lichroprep RP-18, 40–63 µm, Merck Inc., Darmstadt, Germany). Semi-preparative HPLC was carried out on a Dionex P680 liquid chromatograph equipped with a UV 170 UV/Vis detector at 206 nm using a YMC-Pack R&D ODS-A column (250 mm × 20 mm i.d., 5 µm, YMC, Kyoto, Japan). Compounds detection in the thin-layer chromatography (TLC) plate was achieved by spraying the silica gel plates (Qingdao Marine Chemical Inc., Qingdao, China) with 15% H2SO4 in ethanol followed by heating.

3.2. Material The marine bryozoan Cryptosula pallasiana was collected in March 2009 from Huang Island, Qingdao City, Shandong Province of China, was identified by one of us (H.-W. Lin). A voucher specimen (No: QD-0903-1) was deposited in Marine Laboratory, Changzheng Hospital, Second Military Medical University.

3.3. Extraction and Isolation The fresh animal C. pallasiana (about 20 kg) was exhaustively extracted with 95% EtOH at room temperature. Followed the extraction procedure we described previously, CCl4 fraction (about 12.9 g) was further separated by CC on Sephadex LH-20 with CHCl3/MeOH (1:1) as an eluting solvent to afford three fractions (Frs. A–C) [5]. Fr. A (5.56 g) was subjected to a reversed-phase silica gel column eluting with a mixture of MeOH/H2O (4:1) and MeOH to afford Fr. A1 and Fr. A2, respectively. Fr. A2 was submitted to a silica gel column and eluted with petroleum ether/EtOAc (15:1, 10:1, 5:1, 1:1) to give 12 fractions (Frs. A2-1–A2-12). The isolation of 16 alkaloids, 13 sterols, three aromatic compounds and Mar. Drugs 2017, 15, 120 8 of 10

two glycerol derivatives from Fr. B, Fr. C, and Fr. A2-4 to Fr. A2-9 have been reported previously [4–6]. Fr. A2-3 (183.0 mg) was purified by semi-preparative HPLC to give 1 (2.5 mg, tR = 67.2 min) and 2 (20.9 mg, tR = 73.4 min), using MeOH/H2O (19:1) as mobile phase at a flow rate of 8.0 mL/min. Fr. A2-11 (114.7 mg) was purified by semi-preparative HPLC to obtain 3 (6.2 mg, tR = 47.7 min) and 4 (20.9 mg, tR = 54.4 min), using MeOH/H2O (9:1) as mobile phase at a flow rate of 8.0 mL/min. Fr. A1 (2.4 g) was eluted with CHCl3/MeOH (1:1) on Sephadex LH-20, and then further purified by semi-preparative HPLC to give 5 (4.0 mg, tR = 39.6 min), using MeOH/H2O (1:3) as mobile phase at a flow rate of 8.0 mL/min. Fr. A2-10 (150.0 mg) was eluted with CHCl3/MeOH (1:1) on Sephadex LH-20, and then further purified by semi-preparative HPLC to afford 6 (9.3 mg, tR = 49.5 min), 7 (7.4 mg, tR = 46.3 min), 8 (8.5 mg, tR = 51.4 min) and 9 (7.1 mg, tR = 54.7 min), using MeOH/H2O (19:1) as mobile phase at a flow rate of 8.0 mL/min.

3.3.1. (23R)-Methoxycholest-5,24-dien-3β-ol (1)

22 ◦ 1 13 White amorphous powder, [α]D −22.0 (c 0.05, CHCl3); H- and C-NMR data see Table1; (+)-ESI-MS m/z: 437 [M + Na]+, 415 [M + H]+, 385, 349, 318, 274, 256; (+)-HRESIMS m/z: 437.3395 + [M + Na] (calcd. for C28H46O2Na, 437.3396).

3.3.2. (2S,3R,4E,8E)-2-(Tetradecanoylamino)-4,8-octadecadien-l,3-diol (6)

22 ◦ 1 13 White amorphous powder, [α]D −5.7 (c 0.10, CHCl3); H- and C-NMR data see Table2; (+)-ESIMS m/z: 530 [M + Na]+, 437, 330, 302, 274, 262, 256, 228, 219; (-)-ESIMS m/z: 552 [M + COOH]−, 542 [M + Cl]−, 506 [M − H]−, 447, 394, 341, 283, 255, 218, 186, 143, 126; (+)-HRESIMS m/z: 530.4545 + [M + Na] (calcd. for C32H61NO3Na, 530.4549).

3.3.3. (2S,3R,20R,4E,8E)-2-(Tetradecanoylamino)-4,8-octadecadien-l,3,20-triol (7)

22 ◦ 1 13 White amorphous powder, [α]D +9.5 (c 0.10, CHCl3); H- and C-NMR data see Table2; (+)-ESIMS m/z: 546 [M + Na]+, 437, 330, 274, 262, 256; (-)-ESIMS m/z: 558 [M + Cl]−, 522 [M − H]−, 340, + 296, 283, 242, 227; (+)-HRESIMS m/z: 546.4496 [M + Na] (calcd. for C32H61NO4Na, 546.4498).

3.4. Methanolysis of Compounds 6 and 7 Compounds 6 and 7 (each about 3 mg) were dissolved in 5% HCl-MeOH (3 mL), respectively, and then refluxed for 12 h at 80 ◦C. The reaction mixture was extracted with n-hexane (3 × 4 mL). The n-hexane portion was washed with H2O and concentrated in vacuo to yield methyl tetradecanoate and (R)-methyl 2-hydroxytetradecanoate, respectively. The H2O portion was neutralized with NH4OH, 22 and then further concentrated in vacuo to yield the corresponding LCB with specific rotation [α]D at ◦ −3.4 (c 0.05, CHCl3). + + + Methyl tetradecanoate: EI-MS m/z 242 [M] (25), 211 [M − CH3] (11), 199 [M − COCH3] (22), 185 (8), 171 (4), 157 (7), 143 (22), 129 (9), 115 (4), 101 (9), 87 (61), 74 (100), 55 (24). 22 ◦ + (R)-Methyl 2-hydroxytetradecanoate: [α]D −6.5 (c 0.05, CHCl3); EI-MS m/z 258 [M] (7), + 199 [M − COCH3] (65), 125 (30), 111 (56), 90 (47), 83 (85), 69 (100), 55 (92).

3.5. MTT Cytotoxicity Assays Compounds 1–9 were evaluated for their cytotoxic activity against human tumor cell lines HL-60, HepG-2 and SGC-7901 using MTT assay. The cell lines were obtained from American type culture collection (ATCC), and were seeded to RPMI-1640 medium with 10% fetal bovine serum and 100 U/mL ◦ benzyl penicillin-streptomycin solutions, at 37 C in a humidified atmosphere with 5% CO2/air for 24 h. Then the test samples were added and incubated at 37 ◦C for another 72 h. The detailed procedure for MTT assay can be found in our previous published literature [5]. The cytotoxic activity was expressed as IC50 value using adriamycin as a positive control. Mar. Drugs 2017, 15, 120 9 of 10

4. Conclusions A new sterol (1), two new ceramides (6,7) and six known compounds (2–5,8,9) were identified from the marine bryozoan C. pallasiana. Among the isolated compounds, sterol 1 showed novelty in the C-23R methoxy group and double bond between C-24 and C-25 in its side chain. Since this is the first report of ceramides in this , it can be considered important for its chemotaxonomic significance. Furthermore, ceramides with 14 carbons in the FAB were different from those we previously obtained from Bugula neritina [25,26]. All compounds were evaluated for their cytotoxicity, and lactone (5) was found to exhibit stronger cytotoxicity than sterols and ceramides, suggesting that it could be responsible for the cytotoxicity of C. pallasiana.

Supplementary Materials: The following are available online at www.mdpi.com/1660-3397/15/4/120/s1, Figures S1–S9: HR-ESI-MS, ESI-MS, 1H-NMR, 13C-NMR, DEPT135, 1H-1H COSY, HSQC, HMBC and NOESY spectra for new compound 1, Figures S10, S12 and S13: HR-ESI-MS, 1H-NMR and 13C-NMR spectra for new compound 6, Figures S14, S16 and S17: HR-ESI-MS, 1H-NMR and 13C-NMR spectra for new compound 7, Figures S11 and S15: EI-MS spectra of methyl tetradecanoate and (R)-methyl 2-hydroxytetradecanoate obtained from methanolysis of ceramides 6 and 7, respectively, Data S18–S22: 1H-NMR and 13C-NMR data for the known compounds 2–5, Data S23 and S24: 1H-NMR, 13C-NMR and ESI-MS data for the known compounds 8 and 9. Acknowledgments: This work was financially supported by National Natural Science Foundation of China (No. 31201552 and No. 81473132), and National High-Tech Research and Development Project (863 Project, No. 2007AA09Z401). Author Contributions: Xiang-Rong Tian, Yan-Qing Gao and Xiao-Lin Tian carried out the experiment, including isolation and bioactive evaluation of the related materials. First author Xiang-Rong Tian also wrote the manuscript. Jiao Li tested the CD spectrum and calculated quantum ECD. Yu-Shan Li participated in the structural elucidation. Hou-Wen Lin collected and identified the samples of Cryptosula pallasiana. As corresponding authors, Hai-Feng Tang and Zhi-Qing Ma organized and designed the experiments. Conflicts of Interest: The authors declare no conflict of interest.

Abbreviations The following abbreviations are used in this manuscript:

CC Column Chromatography HPLC High-Performance Liquid Chromatography MTT Microculture Tetrazolium ATCC American Type Culture Collection RPMI Roswell Park Memorial Institute LCB Long Chain Base FAB Fatty Acid Base TD-DFT Time-Dependent Density Functional Theory

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