Steroids 78 (2013) 866–873

Contents lists available at SciVerse ScienceDirect

Steroids

journal homepage: www.elsevier .com/locate /s teroids

Hurgadacin: A new steroid from Sinularia polydactyla

0039-128X/$ - see front matter � 2013 Elsevier Inc. All rights reserved.http://dx.doi.org/10.1016/j.steroids.2013.05.006

⇑ Corresponding author at: Chemistry of Natural Compounds Department,Division of Pharmaceutical Industries, National Research Centre, El-Behoos St. 33,Dokki-Cairo 12622, Egypt. Tel.: +20 1282851960.

E-mail address: mshaaba_99@yahoo.com (M. Shaaban).1 These authors contributed equally to the work.

Mohamed Shaaban a,b,⇑,1, Khaled A. Shaaban b,1, Mohamed A. Ghani c

a Chemistry of Natural Compounds Department, Division of Pharmaceutical Industries, National Research Centre, El-Behoos St. 33, Dokki-Cairo 12622, Egyptb Institute of Organic and Biomolecular Chemistry, University of Göttingen, Tammannstrasse 2, D-37077 Göttingen, Germanyc Red Sea Marine Parks, P.O. Box 363, Hurghada, Red Sea, Egypt

a r t i c l e i n f o a b s t r a c t

Article history:Received 20 September 2012Received in revised form 6 May 2013Accepted 8 May 2013Available online 17 May 2013

Keywords:HurgadacinSinularia polydactylaBiological activity

Hurgadacin (1), a 24,25-bishomo-26-methylenecholesterol was isolated from the soft coral Sinularia poly-dactyla, collected from the Red Sea, near Hurghada at the Egyptian coast. The new steroid 1 was isolatedtogether with the closely related polyhydroxy steroids 24-methylenecholestane-3b,5a,6b-triol (2) and24-methylenecholestane-1a,3b,5a,6b,11a-pentol (3), in addition to the sesquiterpene lactiflorenol (4)and the trinorcarotenolide acetate peridinin (5), The structures of the isolated compounds were con-firmed by intensive studies of their 1D and 2D NMR spectra and mass data. The antimicrobial and cyto-toxic activities of the soft coral extract and the corresponding constituents were evaluated against diversepathogenic microorganisms and brine shrimps, respectively.

� 2013 Elsevier Inc. All rights reserved.

1. Introduction

The Red Sea represents one of the most promising areas as asource of medicinal and nutritional natural products [1]. Many col-lections made on the Red Sea reefs led to numerous comprehensivestudies on the taxonomy of Red Sea soft corals [2–4]. Few studies,however, focused on marine organisms from the Red Sea as asource for drug leads. Soft corals (Octocorallia, Alcyonacea) are ahighly diverse group of marine organisms, which are known tocontain a rich variety of secondary metabolites. These substancesfrom corals show not only great significance in chemical ecology,but also various biological activities such as antitumor, antibacte-rial, antiviral and antifungal properties [1,5,6]. Soft corals belong-ing to genus Sinularia (phylum Cnidaria, class Alcyonaria, andfamily Alcyoniidae) are most widely distributed and constitute adominant portion of the biomass in the tropical reef environment.The taxonomic identification is largely based on the examination ofthe spicules, and a fairly reliable taxonomic key is available toguide species identification [7]. The genus Sinularia consists of al-most 90 species of which more than 50 have been chemicallyexamined: About 500 metabolites that have been reported fromthe genus Sinularia, including sesquiterpenes, diterpenes, poly-hydroxylated steroids, and polyamines [8,9]. Soft corals (Coelen-terate) contain highly diverse mono- and polyhydroxysterols,most of which are derivatives of (24S)-ergostane-type C28 sterols

[10–12]. The predominant component of this 3b-monohydroxys-terol mixture is, in most cases, (24S)-ergost-5-en-3b-ol (22,23-dihydrobrassicasterol), in contrast to sponges, which often containthe compounds as mixture of C-24 epimers [13,14]. Many of thesemetabolites were shown to possess interesting antimicrobial, anti-inflammatory, and cytotoxic activities [15–17].

The present work is focused on the isolation, purification andidentification of secondary metabolites from the Red Sea soft coralSinularia polydactyla and evaluation of their cytotoxic and antimi-crobial activities. The structures of the isolated compounds wereelucidated on the basis of spectral analysis by MS, 1H and 13CNMR, and 2D NMR techniques. The extract of S. polydactyla, col-lected from the Red Sea at the Hurghada coasts in Egypt, showedfour interesting TLC bands in the chemical screening: three of themwere not UV absorbing but were detected as violet and blue zoneson spraying with anisaldehyde/sulphuric acid reagent, as indica-tion of terpenes and/or steroids. The fourth band appeared as redin colour during TLC, which turned blue on treatment with conc.sulphuric acid, pointing to a carotene system. Working up of theextract by chromatographic techniques delivered five compounds,belonging to three structure classes: Three steroids, two of thembeing poly-hydroxylated: 24-methylenecholestane-3b,5a,6b-triol(2), and 24-methylenecholestane-1a,3b,5a,6b,11a-pentol (3),while the third was assigned as the new 24,25-bishomo-26-methylenecholesterol (1) and named hurgadacin, along with thesesquiterpene lactiflorenol (4) and the trinorcarotenolide acetateperidinin (5). The structures of the isolated compounds 1–5(Fig. 1) were intensively studied and elucidated by NMR (1 and2D) and MS data. The biological activities of the soft coral extractalong with isolated compounds were evaluated against diversepathogenic microorganisms and brine shrimps for cytotoxicity.

1

3 5 7

1113

15

17

18

19

20

2122

26

27

28

29

30H

H

HOH

CH2

CH3

H3C9

1

3 5 7

1113

15

17

18

19

20

2122

24

25

26

27

28

H

H

HOH

CH2

CH3

H3C9

RR

HO OH

12

1 2: R1 = R2 = H3: R1 = R2 = OH

135 7

9

11

12

13

14OHH3C

H H

H

H3CCH3H

1'3'

5'

19'

16'

17'

18'

CH37'

CH3

CH3

H3C OH

O

O

H3C

11' 15'1

35

7

91113

15

1920 16 17

18

CH3 OO

H3C CH3

OHH3CO

4 5

Fig. 1. Structural formula of the obtained bioactive compounds (1–5) from Sinularia polydactyla.

M. Shaaban et al. / Steroids 78 (2013) 866–873 867

2. Experimental section

Optical rotations: Polarimeter (Perkin-Elmer, model 343). UV/vis spectra were recorded on a Perkin-Elmer Lambda 15 UV/VISspectrometer. The NMR spectra were measured on Varian Unity300 (300.145 MHz) and Varian Inova 600 (150.820 MHz) spec-trometers. ESI MS was recorded on a Finnigan LCQ with a quater-nary pump Rheos 4000 (Flux Instrument). EI-MS spectra wererecorded on a Finnigan MAT 95 spectrometer (70 eV) with perflu-orokerosene as reference substance for EI HRMS. HRMS wererecorded by ESI MS on an Apex IV 7 Tesla Fourier-Transform IonCyclotron Resonance Mass Spectrometer (Bruker Daltonics, Bille-rica, MA, USA). GC–MS was carried out using a Trace GC–MS Ther-mo Finnigan, EI ionization mode at 70 eV, instrument equippedwith a capillary column CP-Sil 8 CB for amines (length: 30 m;inside diameter: 0.25 mm; outside diameter: 0.35 mm; film thick-ness: 0.25 lm). The analysis was carried out at a programmedtemperature: initial temperature 40 �C (kept for 1 min), thenincreasing at a rate of 10 �C/min until the final temperature of280 �C (kept for 10 min); injector temp was 250 �C and detectortemp. was set to 250 �C; He was used as carrier gas at a flow rate1 mL/min; total run time 27 min, injection volume 0.2 lL. Flashchromatography was carried out on silica gel (230–400 mesh).Rf-values were measured on Polygram SIL G/UV254 (Macherey–Na-gel & Co.). Size exclusion chromatography was done on SephadexLH-20 (Lipophilic Sephadex; Amersham Biosciences, Ltd., pur-chased from Sigma–Aldrich Chemie, Steinheim, Germany).

2.1. Animal material

The Soft coral S. polydactyla (1.2 kg) was collected in January2008 near Mahmieat of the Read Sea at Hurghada coast, east Egyptat a depth �10 m. The organism was taxonomically identified by

Table 1Physico-chemical properties of Hurgadacin (1).

Appearance Colourless solid

Rf 0.51 (CH2Cl2/5% MeOH)Molecular formula C30H50OEI-MS: m/z (%) 426 ([M]+, 28), 398 ([M�CO]+, 30), 383 (11), 314 (100), 271 (64),HREI-MS (m/z):Found 426.3859Calcd. 426.3862

½a�20D

�25 (c 0.1, MeOH)

UV/vis:kmax (log e) MeOH: 229.6 (3.33); MeOH/HCl: 230.8 (3.47); MeOH/NaOH: 23

MSc. Mohamed Abdel-Ghani, Institute of Environmental Protec-tion, Hurghada, Egypt.

2.2. Extraction and isolation

The raw organism (1.2 kg), was crushed mechanically, treatedwith CH2Cl2–MeOH (2:1) (6 L) and kept at �5 �C for 8 days. Organicsolvent and water from the liquid phase were collected by filtra-tion and separated. The dichloromethane extract was concentratedin vacuum at 40 �C to afford 2.40 g of a reddish brown crudeextract.

2.3. Isolation and purification

The obtained extract was separated by column chromatography(2 � 100 cm, 170 g silica gel, eluted with a cyclohexane–CH2Cl2–MeOH gradient (cyclohexane 0.25 L followed by CH2Cl2/0–100%MeOH) under TLC control into five fractions: I (550 mg), II(720 mg), III (70 mg), IV (170 mg) and V (320 mg). Fractions I andII were combined as their chromatograms showed similarity fol-lowed by GC–MS analysis which were out of our interest. Purifica-tion of fraction III using silica gel column (eluted with CH2Cl2)followed by Sephadex LH-20 (CH2Cl2/40% MeOH) afforded lactif-lorenol (4) as colourless oil (15.2 mg). Fraction IV showed two ma-jor bands on TLC: one of them was colourless and not UVabsorbing, but turned blue after spraying with anisaldehyde/sul-phuric acid. The other one was orange red and changed to blueby moistening with conc. sulphuric acid. Fractionation on silicagel (CH2Cl2/MeOH) followed by Sephadex LH-20 (CH2Cl2/40%MeOH) afforded hurgadacin (1) as colourless solid (46.1 mg). Thered component was further purified by Sephadex LH-20 (MeOH)to deliver an orange red solid of peridinin (5) (65.2 mg). The polarfraction V was applied to silica gel (column 40 � 3 cm, CH2Cl2/

255 (24), 229 (18), 161 (13), 159 (18), 145 (22), 107 (22), 95 (17), 81 (16), 55 (8)

1.6 (3.50) nm

Table 213C and 1H NMR data of steroids 1–3 (J in [Hz]).

No. Hurgadacin (1) (CDCl3) 2 (DMSO-d6) 3 (DMSO-d6)

dCa dH

b dCa dH

b dCa dH

c

1 37.2 1.05 (m), 1.80 (m) 31.9 1.50 (m), 1.13 (m) 76.2 3.96 (brm)1-OH 5.22 (d, 4.1)2 31.6 1.80 (m), 1.48 (m) 31.0 1.60 (m) 37.5 1.68 (m), 1.86 (m)3 71.7 3.49 (m) 65.7 3.81 (m) 62.1 3.75 (m)3-OH – – 4.10 (d, 5.4) 4.32 (d, 5.4)4 42.3 2.22 (m) 40.8 1.37 (m), 1.86 (m) 41.7 1.46 (m), 1.94 (m)5 140.7 – 74.1 – 77.1 –5-OH 3.56 (s) 5.06 (s)6 121.6 5.32 (brm) 74.3 3.30 (brm) 73.4 3.18 (brm)6-OH 4.33 (d, 3.9) 4.51 (d, 4.1)7 31.9 1.94 (m), 1.43 (m) 34.4 1.32 (m), 1.58 (m) 35.0 1.64 (m), 1.38 (m)8 31.9 1.42 (m) 29.9 1.60 (m) 29.1 1.74 (m)9 50.1 0.92 (m) 44.5 1.35 (m) 46.4 1.82 (m)10 36.5 – 37.7 – 41.7 –11 21.1 1.46 (m), 1.98 (m) 20.7 1.29 (m) 65.8 3.75 (brm)11-OH 5.02 (brs)12 39.8 1.14 (m), 1.98 (m) 39.7 1.92 (m), 1.10 (m) 50.3 1.14 (m), 2.26 (m)13 42.3 – 42.2 – 42.5 –14 56.7 0.98 (m) 55.7 1.00 (m) 54.6 1.08 (m)15 24.3 1.56 (m) 23.8 1.52 (m), 0.98 (m) 23.9 1.00 (m), 1.52 (m)16 28.2 1.24 (m), 1.83 (m) 27.7 1.21 (m), 1.79 (m) 27.8 1.84 (m), 1.24 (m)17 56.0 1.10 (m) 55.6 1.12 (m) 55.3 1.14 (m)18 11.2 0.65 (s) 11.8 0.63 (s) 12.5 0.61 (s)19 19.4 0.98 (s) 16.2 1.03 (s) 15.9 1.07 (s)20 35.7 1.40 (m) 35.1 1.37 (m) 35.0 1.64 (m)21 18.7 0.92 (d, 6.5) 18.4 0.91 (d, 6.5) 18.4 0.94 (d, 6.6)22 34.7 1.12 (m), 1.52 (m) 34.2 1.10 (m), 1.50 (m) 34.1 1.08 (m), 1.52 (m)23 28.2 1.48 (m) 30.5 1.86 (m), 2.06 (m) 30.4 1.88 (m), 2.08 (m)24 31.6 1.48 (m) 155.7 – 155.7 –25 31.0 1.86 (m), 2.08 (m) 106.3 4.70 (s), 4.63 (s) 106.4 4.70 (s), 4.64 (s)26 156.8 – 33.0 2.20 (m) 33.0 2.20 (m)27 105.9 4.68 (s), 4.63 (d, 1.3) 21.7 0.99 (d, 6.8) 21.7 0.99 (d, 6.8)28 33.8 2.20 (m) 21.5 0.98 (d, 6.8) 21.6 0.98 (d, 6.8)29 22.0 1.00 (d, 6.8) – – – –30 21.8 0.99 (d, 6.8) – – – –

a 125 MHz.b 600 MHz.c 300 MHz.

868 M. Shaaban et al. / Steroids 78 (2013) 866–873

MeOH) followed by Sephadex LH-20 (MeOH) to afford 24-methyl-enecholestane-3b,5a,6b-triol (2, 60.2 mg) and 24-methylenecho-lestane-1a,3b,5a,6b,11a-pentol (3, 15.1 mg) as colourless solids.

24-Methylenecholestane-3b,5a,6b-triol (2): Rf = 0.66 (CH2Cl2/10% MeOH); mp 240–242 �C, lit (mp 241–242 �C [11]); ½a�20

D = �2�(c 0.1, MeOH), lit ½a�20

D = �4� (c 0.26, pyridine) [11]; 1H NMR(CDl3, 600 MHz): see Table 2; 13C NMR (CDCl3, 125 MHz): see Ta-ble 2; EI-MS: m/z (%) 432 [M]+, (12), 398 (12), 381 (13), 348 (33),335 (78), 305 (100), 269 (62), 202 (24), 159 (16), 123 (24), 109(30), 107 (32), 95 (49), 81 (56), 55 (56), 43 (100); DCI MS: m/z(%) 451 ([M+NH4+H]+, 100), 433 ([M+H]+, 100); (+)-ESIHR-MS: m/z 455.34961 [M+Na]+ ([calcd 455.34957 for C28H48O3Na]).

24-Methylenecholestane-1a,3b,5a,6b,11a-pentol (3): Rf = 0.55(CH2Cl2/10% CH3OH); mp 197–199 �C, lit (196–198 �C [18]);½a�20

D = �7� (c 0.1, MeOH), lit ½a�20D = �4.0� (c 1.60, C5H5N) [12], lit

Table 313C and 1H NMR data of Lactiflorenol (4) in CDCl3, (J in [Hz]).

No. dCa dH

b No. dCa dH

b

1 47.2 2.24 (m) 9 37.0 2.46 (dd, 13.4, 9.9)2 24.7 1.86 (m), 1.69 (m) 10 153.8 –3 40.1 1.69 (m) 11 106.4 4.69 (d, 32.7)4 80.5 – 12 24.0 1.19 (s)5 54.8 2.24 (m) 13 37.3 2.21 (m)6 121.3 5.51 (d, 2.9) 14 21.4 0.95 (d, 6.8)7 149.5 – 15 21.2 0.94 (d, 6.9)8 29.9 2.16 (m), 1.96 (m)

a 125 MHz.b 300 MHz.

½a�20D = �20.0� (c 0.5, CHCl3) [18]; 1HNMR (CDl3, 300 MHz): see Ta-

ble 2; 13C NMR (CDCl3, 125 MHz): see Table 2; EI MS: m/z (%) 464([M]+, 3), 446 ([M�H2O]+, 11), 428 ([M�2H2O]+, 25), 410 ([M�3H2-

O]+, 32), 392 ([M�4H2O]+, 8), 359 (12), 283 (9), 187 (8), 174 (10),143 (18), 121 (20), 109 (22), 95 (25), 81 (30), 58 (36), 43 (100);(+)-ESI-MS: m/z (%) 951 ([2 M+Na]+, 100), 465 ([M+H]+, 18); (�)-ESI-MS: m/z (%) 927 [2 M�H]�, (60), 463 [M�H]�, (100); (+)-ESI-HR-MS: m/z 465.35750 [M+H]+ ([calcd 465.3574511 forC28H49O5) and m/z 487.33935 [M+Na]+ ([calcd 487.3393957 forC28H48O5Na).

Lactiflorenol; 6,10-Guaiadien-4-ol (4): (220); Rf = 0.56 (CH2Cl2);½a�20

D = 0� (c 0.1, CHCl3); 1HNMR (CDl3, 300 MHz): see Table 3; 13CNMR (CDCl3, 125 MHz): see Table 3; (�)-EI-MS: m/z (%): 220([M]+., 48, C15H24), 202 ([M�H2O]+, 28), 177 (36), 162 (76), 147(42), 119 (100), 93 (58), 69 (46), 55 (51), 43 (96).

Peridinin (5): Rf = 0.31 (CH2Cl2/15% MeOH); mp 108–110 �C, litmp 107–109 �C [35]; ½a�20

D = �36� (c 0.1, CHCl3); 1H NMR (CDl3,600 MHz): see Table 4; 13C NMR (CDCl3, 125 MHz): see Table 4;(+)-ESI-MS: m/z (%): 1283 ([2 M+Na]+, 65), 653 ([2 M+Na]+, 100);(�)-ESI-MS: m/z (%) 1259 ([2 M�H]+, 25), 629 ([M�H]�, 90); (+)-ESIHR-MS: m/z 631.3628300 [M+H]+ ([calcd 631.36291 forC39H51O7]).

2.4. Biological activities

2.4.1. Antimicrobial activityAntimicrobial assays were conducted utilizing the disc-agar

method [19] against diverse sets of microorganisms, listed in the

Table 413C and 1H NMR data of peridinin (5) in CDCl3, (J in [Hz]).

No. Peridinin (5) (Experimental) No. Peridinin (5) (Literature) [38] (CDCl3)

dCa dH

b dC dH

1 35.3 – 1 35.3 –2 47.1 1.60 (m), 1.22 (m) 2 47.1 1.62 (m), 1.26 (dd, 10.8, 12.8)3 64.2 3.88 (m) 3 64.2 3.91 (m)4 40.9 2.37 (dd, 14.4, 4.0), 1.60 (m) 4 40.9 2.40 (br dd, 14.4, 4.9), 1.63 (m)5 67.5 – 5 67.5 –6 70.4 – 6 70.5 –7 133.6 7.14 (d, 15.6) 7 133.6 7.17 (d, 15.6)8 121.8 6.35 (d, 15.5) 8 121.8 6.37 (d, 15.6)9 124.9 – 9 124.8 –10 136.3 7.00 (s) 10 136.3 7.02 (s)11 146.8 – 11 146.8 –12 119.2 5.71 (s) 12 119.2 5.73 (s)13 133.9 – 13 134.0 –14 133.0 6.42 (d, 11.7) 14 133.0 6.45 (brd, 10.8)15 137.2 6.50 (dd, 14.2, 11.3) 15 137.2 6.51 (dd, 14.3, 10.8)16 29.5 1.18 (s) 16 29.5 1.21 (s)17 24.9 0.95 (s) 17 24.9 0.97 (s)18 19.9 1.17 (s) 18 19.9 1.20 (s)19 168.7 – 19 168.7 –20 15.4 2.20 (s) 20 15.4 2.23 (s)10 35.8 – 10 35.8 –20 45.4 1.96 (m), 1.39 (m) 20 45.4 1.99 (brd, 12.8), 1.40 (m)30 68.0 5.35 (m) 30 67.9 5.38 (m)

30-COCH321.4 2.01 (s) 30-COCH3

21.4 2.04 (s)

30-COCH3170.4 – 30-COCH3

170.4 –

40 45.8 1.48 (m), 2.26 (m) 40 45.2 1.50 (m), 2.28 (brd, 12.8)50 72.6 – 50 70.7 –60 117.6 – 60 117.6 –70 202.6 – 70 202.6 –80 103.3 6.03 (s) 80 103.3 6.05 (s)90 133.0 – 90 133.9 –100 128.1 6.08 (d, 11.7) 100 128.1 6.11 (d, 11.5)110 131.5 6.58 (dd, 14.6, 11.8) 110 131.5 6.58 (dd, 14.6, 11.8)140 133.0 6.37 (dd, 14.5, 11.0) 140 133.0 6.38 (dd, 14.3, 11.5)150 128.9 6.61 (dd, 14.3, 11.5) 150 128.9 6.61 (dd, 14.3, 11.5)160 32.0 1.04 (s) 160 32.1 1.07 (s)170 29.1 1.36 (s) 170 29.2 1.38 (s)180 31.2 1.32 (s) 180 31.3 1.35 (s)190 14.0 1.78 (s) 190 14.0 1.80 (s)

a 125 MHz.b 600 MHz.

M. Shaaban et al. / Steroids 78 (2013) 866–873 869

theoretical part. The extract of the soft coral S. polydactyla was dis-solved in CH2Cl2/10% MeOH at a concentration of 1 mg/mL. Ali-quots of 40 lL were soaked on filter paper discs (9 mm diameter,No. 2668, Schleicher & Schüll, Germany) and dried for 1 h at roomtemperature under sterile conditions. The paper discs were placedon inoculated agar plats and incubated for 24 h at 38 �C for bacteriaand 48 h (30 �C) for fungal isolates, while the algal test strains wereincubated at �22 �C in day light for 8–10 days. The soft coral ex-tract and the corresponding compounds 1–5 were examinedagainst Bacillus subtilis, Staphylococcus aureus, Streptomyces virido-chromogenes (Tü 57), Escherichia coli, Candida albicans, Mucor miehi,Chlorella vulgaris, Chlorella sorokiniana, Scenedesmus subspicatus,Rhizoctonia solani and Pythium ultimum.

2.4.2. Brine shrimp microwell cytotoxic assayThe cytotoxic assay was performed according to the literature

methods of Takahashi et al. [20] and Sajid et al. [21].

3. Results and discussion

The Soft coral Sinularia polydactyla (1.2 kg) was collected fromRed Sea, Hurghada, at Egyptian cost; mechanically crushed andexhaustively extracted using dichloromethane/methanol. Theorganic phase was consequently, separated and concentrated invacuo yielding 2.40 g of a reddish brown crude extract. The latter

was applied to fractionation by silica gel column chromatographyunder TLC control using cyclohexane/dichloromethane/methanolas eluent system to afford five fractions. Purification of the middlepolar fraction III delivered lactiflorenol (4), while hurgadacin (1)and peridinin (5) were obtained from fraction IV. Finally, the polarfraction V afforded 24-methylenecholestane-3b,5a,6b-triol (2) and24-methylenecholestane-1a,3b,5a,6b,11a-pentol (3) (see experi-mental part).

3.1. Hurgadacin (24,25-bishomo-26-methylenecholesterol)

Compound 1 was obtained from fraction IV as moderately polarcolourless solid, which did not show UV absorption on TLC, butgave a blue colour on spraying with anisaldehyde/sulphuric acid,pointing to a terpenoid or steroid nature (Table 1). The molecularweight of 1 was deduced by EI MS as 426 Da, which exhibited asubsequent expulsion of carbonyl group, giving a further fragmention at m/z 398. HREI MS of 1 delivered the molecular formulaC30H50O, corresponding to six double bond equivalents. In the UVspectrum, compound 1 exhibited a maximum at kmax = 230 nm,which showed nearly no shift in acidic or alkaline solution; theseobservations pointed to a compound bearing one or more non-con-jugated double bonds.

In the 1H NMR and HMQC/HSQC spectra (Table 2), compound 1exhibited three olefinic 1H singlets at d 5.32, 4.68 and 4.63. Two ofthem (d 4.68 and 4.63) represented an exo-methylene group (C-27,

870 M. Shaaban et al. / Steroids 78 (2013) 866–873

dC: 105.9), while the third one corresponding to H-6 (dC: 121.6)was due to a –CH@Cq-fragment. An oxymethine proton (H-3, dC

71.7) appeared as multiplet at d 3.49; two further multiplets at d2.22 (2H) and 2.20 (1H), indicated sp2-attached methylene (C-4,dC: 42.3) and methine (C-28, dC: 33.8) groups, respectively. Twofurther sp2-attached methylene groups were found at d 2.08/1.86(H2-25, dC: 31.0) and 1.94/1.43 (H2-7, dC: 31.9), and two methylsinglets at d 0.98 (H3-19) and 0.68 (H3-18), and three methyl dou-blets at d 1.00 (H3-29, J � 6.8 Hz), 0.99 (H3-30, J � 6.8 Hz) and 0.92(H3-21, J � 6.5 Hz) were seen. The first two methyl doublets (1.00and 0.99) represented presumably the gem-dimethyls of an isopro-pyl group, with the respective methine at d 2.20 (H-28) accordingto the H,H COSY data.

In the region of d 1.98–0.92, multiplets integrating for 23 pro-tons were visible, representing nine methylenes (dC 39.8–21.1)and five methines (dC: 56.7, 56.0, 50.1, 31.9 and 35.7). Accordingto the 13C NMR/HMQC spectra, four quaternary carbons were pres-ent; two of them at d 156.8 (C-26) and 140.7 (C-5) were olefinic,while the others were aliphatic (d 42.3, C-13 and 36.5, C-10). Basedon these features, compound 1 bears eight methines (including anoxymethine and an olefinic one), thirteen methylenes (amongthem one sp2 exo-methylene), 5 methyls and four quaternary car-bons (two of them with sp2 hybridisation), giving 30 carbons, as ex-pected from the aforementioned molecular formula.

According to the studied spectroscopic data and searches in dat-abases (Dictionary of Natural Products [DNP] and SciFinder), com-pound 1 was confirmed as a new steroid, and detailed 2D NMRexperiments were performed therefore (Fig. 2). The methyl singletat d 0.99 (CH3-19) displayed four HMBC correlations to C-1 (37.2,3J), C-9 (50.1, 3J), C-5 (140.7, 3J) and C-10 (36.5, 2J), interpreted asdirect attachment to the quaternary carbon C-10. The oxymethineproton H-3 (d 3.49) showed a COSY correlation with H2-2 (d 1.80,1.48) and H2-4 (d 2.22); H2-2 showed additional cross signals withH2-1 (1.80, 1.03), confirming the structure of ring A. The methylenecarbon C-4 (d 42.3) showed an HMBC coupling with the olefinicmethine H-6 (d 5.32), while the latter one exhibited further corre-lations with d 31.9 (C-7, 2J) and C-8 (3J). The connection between H-6 and H2-7 (1.94, 1.43) was further established by H,H COSY sig-nals. The H,H COSY coupling between the methine protons H-8(1.42) and H-9 (0.92) was weak, but completed the fused rings Aand B.

The second methyl singlet CH3-18 (d 0.65) displayed four cru-cial correlations to CH2-12 (39.8, 3J), Cq-13 (d 42.3, 2J), CH-14 (d56.7, 3J), and CH-17 (d 56.0, 3J), confirming its attachment to C-13. In the H,H COSY spectrum, the protons H2-12 (1.98, 1.14)showed a correlation with H2-11 (1.46, 1.98), and the latter exhib-ited a weak coupling with the methine proton H-9 (d 0.92). In addi-tion, a COSY correlation between H-8 (1.42) and H-14 (d 0.98, dC

56.7) was found, closing ring C. The latter proton showed H ? Ccorrelations to C-13 (d 42.3, 2J) and C-17 (d 56.0, 3J). The two meth-ylenes H2-15 (1.56) and H2-16 (1.83, 1.24) were assigned accordingto H,H COSY data, completing the five-membered ring D.

These data confirmed a 3-hydroxy-D5-steroid with the partialformula C19H30O, so that the residual atoms C11H20 and one doublebond equivalent must account for the side chain. Based on the

1

3 5 7

1113

15

17

18

19

20

2122

26

27

28

29

30

HO

CH3

H3C9

COSY

HMBC

3

HO

Fig. 2. Selected HMBC (?), H,H COSY (—) and N

NMR data, two of the three methyl doublets belonged to an isopro-pyl group, and the third one was assigned to CH3-21 (dH 0.92, dC

18.7) which is attached to CH-20 (dH: 1.40, dC: 35.7). Respectively,CH3-21 showed HMBC correlations to C-17 (d 56.0), CH-20 (35.7),and CH2-22 (34.7). On the other hand, the sp2 exo-methylene pro-tons (4.68/4.63) showed correlations to the methine carbon C-28 (d33.8) of the terminal isopropyl group and to the methylene carbonC-25 (d 31.0). The C-26–C-30 fragment was further confirmed by3J-couplings of the two methyl doublets CH3-29 (1.00) and CH3-30 (0.99) with the quaternary sp2-carbon C-26 (dC 156.8). Theremaining ethanediyl group (–CH2-23–CH2-24) was placed be-tween CH2-25 (d 31.0) and CH2-22 (d 34.7) according to the H,HCOSY experiment. Consequently, structure of 1 was assigned as24,25-bishomo-26-methylenecholesterol, which we named ashurgadacin.

In the NOE experiment (Fig. 2), as its planarity, the olefinicmethine H-6 showed NOE correlations to CH3-19, H-8, H-3, H2-4,H2-7 and H-9. Moreover, the methyl singlet CH3-18 showed NOEcorrelation to H-8 and CH3-19 suggesting a b-orientation of H-8.In contrast, H3-18, did not exhibit an NOE correlation to H-14and H-9, thereby suggesting a-orientation of these protons. Hence,the stereochemistry of 1 at C-13, C-19- and C-20 was deduced to beas the same in most reported steroids [22] and to those of the clo-sely related steroids 2 and 3 with a ß-orientation for the side chainas well. Finally, the exo-methylene protons @CH2-27 showed anNOE correlations to CH2-24 and CH2-25 and CH-28 in the fragment[–CH2(25)–C(26)@CH2(27)@CH2(28)–] as shown in figure 2 (Sup-porting information, NOE Figs. S8–11). Finally, the stereochemistryof compound 1 at the chiral centers C-3, C-8, C-9, C-10, C-13, C-14,C-17 and C-20 were confirmed to have the same configurations asin compounds 2 and 3, displaying as well the same sign of the opti-cal activity.

It is worth mentioning that soft corals (coelenterate) are charac-terized by the presence of diverse mono- and poly-hydroxy sterolsmost of which are derivatives of (24S)-ergostane-type C28 sterols[10,14,23–25]. The predominant component of the 3b-monohydr-oxysterol mixture of soft corals, in most cases, (24S)-ergost-5-ene-3b-ol (22,23-dihydrobrassicasterol). Therefore, hurgadacin(1), is the first reported C30 sterol from soft corals, containingtwo extra vicinal methylene carbons (C24 and C25) in the sidechain, although (24Z)-stigmast-24(28)-ene-3b,5a,6b-triol, C29 ste-rol was reported from Royal Jelly of Honeybees (Apis mellifera)[26]. In contrast, sterols of C22: C24 (e.g. Bendigoles A–C, D–F)were reported from marine derived Actinomycetes [22,27].

To the best of our knowledge, there is no report on such steroidswith extended side chain with two methylene units. However, ahypothetical pathway that leads to this unusual side chain is pro-posed in the Scheme 1. The introduction of methyl and methylenegroups onto phytosterol side chains can occur by transfer of amethyl group from S-adenosylmethionine (SAM), catalyzed by ste-rol methyltransferase (SMT), to a C@C double (e.g. 24,25 C@C) withformation of a carbocation. The methyl transfer reaction catalyzedby SMT is proposed to proceed through a nucleophilic attack by thep electrons of the D24 double bond on the S-methyl group of Ado-Met [28,29]. Subsequently, a 24 ? 25-hydride shift and proton

1

5 7

11 13

15

17

18

19 20

21

22

26

27

28

29

30

9

Selected NOE

CH2

CH3

CH3

CH3CH3

H

H

HH

H

CH3

H H

OE ( ) correlations in hurgadacin (1).

22 24

25

CH322

2425

22

24 25-H

22

24 25

26

22

2426

24,25-methano steroid

homo steroid

repeat identical stepsCH3

22

2426

CH322

2426

SAM

SAM-DependentMethylation

-H22

2426

bishomo steroid

SAM

SAM-DependentMethylation

-H

CH222

24 26

28

29

27

30Desired side chain or hurgadacin

+H

Scheme 1. Proposed biosynthetic pathway for the unusual terminal side chain of Hurgadacin (1).

M. Shaaban et al. / Steroids 78 (2013) 866–873 871

elimination occur, thus generating a 24 methylene sterol bearing aterminal isopropyl group. Alternatively, elimination may occurwithout hydride shift and with elimination into a terminal methylgroup, thus generating a 24-methyl, 25-methylene sterol. Elimina-tion of a proton from the 24-methyl group (i.e. C28) with formationof a C28–C25 bond would generate a cyclopropane ring (24,25-methano sterol). Many such methano sterols are known especiallyin marine algae [30]. Thus, a logical route to homosterols such ashurgadacin would be through the ring opening of such a 24,25-methano sterol, protonation at C24 and then ring cleavage leadingto the insertion of CH2 between C24 and C25. Termination by pro-ton elimination from C26 would generate a C26–C25 C@C. Repeti-tion of this process would insert another carbon into the side chain.Finally, SAM-dependent methylation of the C26–C27 C@C followedby a hydride shift and elimination of proton would lead to the pro-duction of hurgadacin side chain [31].

Structures of the known compounds; 24-methylenecholestane-3b,5a,6b-triol (2) [11,26], 24-methylenecholestane-1a,3b,5a,6b,11a-pentol (3) [12] (Table 2), lactiflorenol [32–34] (4, Table 3)and peridinin [35–38] (5, Table 4), were fully assigned using inten-sive studies of 2D NMR (HMBC & HH COSY) spectroscopic data asshown below.

3.2. 24-methylenecholestane-3b,5a,6b-triol (2) and 24-methylenecholestane-1a,3b,5a,6b,11a-pentol (3)

As two colourless solids with closely related structures andpolarity, steroids 2 and 3 were isolated from the polar fraction V,showing high similarity in their chromatographic properties. Themolecular weights of 2 and 3 were deduced as 432 and 464 Da,with corresponding molecular formulas C28H48O3 and C28H48O5,respectively. Their structures were consequently established onthe bases of intensive studies of 1D (1H and 13C NMR) and 2D(HMQC, MHSQC and HMBC) (Fig. 3) and optical activity, and com-parison with literature as 24-methylenecholestane-3b,5a,6b-triol;ergost-24(28)-ene-3b,5a,6b-triol (2) [11] and 24-methylenecholes-tane-1a,3b,5a,6b,11a-pentol; ergost-24(28)-ene-1a,3b,5a,6b,11a-triol (3) [12], respectively (Table 2, Fig. 2). Structures of 2 and 3

CH3

H3C

H

H

HOHO

OH

H

1

3 5 7

1113

15

17

18

19

20

2122

9

24

25

2627

28

COSYHMBC

H

2

Fig. 3. Selected HMBC (?) and COSY (bold lines) correlations in 24-methylenecholes

were assigned here to first time by the 2D NMR (HMQC, H,H COSYand HMBC).

3.3. Lactiflorenol

From fraction III, compound 4 was isolated as low polar colour-less oil, showing the molecular weight 220 Da and the correspond-ing molecular formula C15H24O, pointing to its sesquiterpenenature. Based on an intensive 1D NMR (1H NMR and 13C NMR)and 2D NMR (H,H COSY, HMQC and HMBC) spectroscopy, com-pound 4 was confirmed as Lactiflorenol [32–34] (Table 3, Fig. 4).A full assignment of compound 4 was reported here for the firsttime on the bases of 2D NMR.

3.4. Peridinin

As middle polar red solid, compound 5 was isolated from frac-tion IV, showing a colour changing to blue on treatment with sul-phuric acid, pointing to its polyene nature. The molecular weight of5 was deduced as 630 Da and the corresponding molecular formulaas C39H50O7. Subsequently, the structure of compound 5 was fullyassigned using intensive elucidation of its 1D (1H NMR, 13C NMR)and 2D (H,H COSY, HMQC, HMBC) NMR spectroscopic data asperidinin [35–38] (Table 4, Fig. 5). Peridinin was first isolatedand named by Schütt in 1890 [39]. Peridinin (5), as a homogenouspigment, has since been isolated from several different dinoflagel-late sources, principally Ceratium tripos, and its name was derivedfrom Peridineen (peridnians) [37]. Peridinin and related com-pounds were known to inhibit the growth of human cancer cellsas evaluated in the Japanese Foundation for Cancer Research 39cell line assay [40]. In accordance, peridinin is reported to exhibitmoderate activities against HCT-116 (colon cancer), MKN7 (stom-ach cancer) and other cells [37,38].

3.5. Biological activities

The soft coral extract and the isolated metabolites 1–5 wereantibiotically inactive against Bacillus subtilis, Staphylococcus

CH3

H3C

H

H

HOO

OH

H

1

3 5 7

1113

15

17

18

19

2021

22

9

24

25

2627

28

COSYHMBC

HOOH

3

tane-3b,5a,6b-triol (2) and 24-methylenecholestane-1a,3b,5a,6b,11a-pentol (3).

1

35 7

9

11

12

13

14OHH3C

H H

H

H3CCH3H

COSY

Selected HMBC

Fig. 4. Selected HMBC (?) and H,H COSY (bold lines) couplings in Lactiflorenol (4).

9'

1'3'

5'

19'

16'

17'

18'

CH37'

CH3

CH3

H3C OH

O

O

H3C

11' 15'1

35

7

91113

15

1920 16 17

18

CH3 OO

H3C CH3

OHH3CO

COSY

Selected HMBC

Fig. 5. Selected HMBC (?) and H,H COSY (bold lines) couplings in peridinin (5).

872 M. Shaaban et al. / Steroids 78 (2013) 866–873

aureus, Streptomyces viridochromogenes (Tü 57), Escherichia coli,Candida albicans, Mucor miehi, Chlorella vulgaris, Chlorella soroki-niana, Scenedesmus subspicatus, Rhizoctonia solani and Pythiumultimum, at 40 lg/disk, although the crude coral extract displayeda high activity (27 mm inhibition zone diameter) against the phy-topathogenic fungus R. solani. Moreover, the extract and most ofthe isolated compounds displayed only marginal cytotoxicityagainst brine shrimps (4–7% mortality at 10 lg/mL), except 24-methylenecholestane-1a,3b,5a,6b,11a-pentol (3), which showeda mortality rate of 24%.

Acknowledgements

The authors are greatly thankful to Prof. H. Laatsch, Institute ofOrganic and Biomolecular Chemistry, University of Göttingen, Ger-many, for his lab facilities and support. We thank Dr. H. Frauendorfand Mr. R. Machinek for MS and NMR measurements. Mrs. F. Lissyand Mr. A. Kohl are deeply appreciated for biological activity test-ing and technical assistance, respectively. We thank Dr. Madan K.Kharel (College of Pharmacy, University of Kentucky, USA) for theinsightful discussion regarding the biosynthesis of the Hurgadacin.M. S. thanks the German Academic Exchange Service (DAAD) for ashort-term grant.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.steroids.2013.05.006.

References

[1] Mohammed R, Seliem MAE, Mohammed TAA, Abed-ElFatah A, Abo-YoussefAM, Thabet MM. Bioactive secondary metabolites from the Red Sea soft coralHeteroxenia fuscescens. Int J Appl Res Nat Prod 2012;4:15–27.

[2] Benayahu Y. Xeniidae (Cnidaria: Octocorallia) from the Red Sea withdescription of a new species. Zool Med Leiden 1990;64:113–20.

[3] Verseveldt J, Benayahu Y. On two old and fourteen new species of Alcyonacea(Coelentrata, Octocorallia) from the Red Sea. Zool Verh Leiden 1983;208:1–33.

[4] Benayahu Y, Loya Y. Long-term recruitment of soft-corals (Octocorallia:Alcyonacea) on artificial substrata at Eilat (Red Sea). Mar Ecol Prog Ser1987;38:161–7.

[5] Coll JC. The chemistry and chemical ecology of octocorals (Coelenterata,Anthozoa, Octocorallia). Chem Rev 1992;92:613–31.

[6] Shaaban M, Shaaban KA, Abd-Alla HI, Hanna AG, Laatsch H. Dendrophen, anovel glycyrrhetyl amino acid from Dendronephthya hemprichi. Z Naturforsch2011;66b:1199–207.

[7] Verseveldt J. A revision of the genus Sinularia (Octocorallia,Alcyonacea). Leiden, The Netherlands: Brill Leiden; 1980. p. 1–128.

[8] Kamel HN, Slattery M. Terpenoids of Sinularia: chemistry and biomedicalapplications. Pharm Biol 2005;43:253–69.

[9] Anjaneyulu ASR, Venkateswarlu R. The chemical constituents of soft coral-species of Sinularia genus: a review. J Sci Ind Res 1995;54:637–49.

[10] Kobayashi M, Krishna MM, Anjaneyulu V. Isolation of 24-Methylenecholestane-1a,3b,5a,6b,16b-pentol from. Sinularia sp. of soft coral.Chem Pharm Bull 1992;40:2845–6.

[11] Kobayashi M, Krishna MM, Haribabu B, Anjaneyulu V. Isolation of 23-demethylgorgost-7-ene-3b,5a,6b-triol and (24S)-ergostane-3b,5a,6b,7b,15b-pentol from soft corals of the Andaman and Nicobar Coasts. Chem PharmBull 1993;41:87–9.

[12] Kobayashi M, Appa Rao KMCh, Krishna MM, Anjaneyulu V. Marine Sterols. Part30. Isolation of 24-methylene-cholestane-1á,3â,5á,6â,11á-pentol and its 11-monoacetate from the Andaman Sea Soft Coral Sinularia dissecta. J Chem Res1994:180–1.

[13] Schmitz FJ. Marine natural products. In: Scheuer PJ, editor, vol. 1. NewYork: Academic Press, New Press; 1978. p. 241.

[14] (a) Faulkner DJ. Marine natural products: metabolites of marine invertebrates.Literature from 1977–07/84. Nat Prod Rep 1984;1:251. ibidem 1984; 1: 551;ibidem 1986; 3: 1; ibidem 1987; 4: 539;(b) Crebs HC. Progress in the chemistry of organic natural products. In: HerzW, Griesebach H, Kirby GW, Tamm C, editors, vol. 49. Vienna: Springer-Verlag;1986. p. 151.

[15] Venkateswarlu V, Reddy SN, Venkatesham U. Recent advances in marinebiotechnology, 6: Bio-organic compounds and biomedicalapplications. NH: Science Publisher, Inc.; 2001. p. 101–129.

[16] Alejandro MSM, Gustafson KR. Marine pharmacology in 2000: antitumor andcytotoxic compounds. Int J Cancer 2003;105:291–9.

[17] Aceret TL, Coll JC, Uchio Y, Sammarco PW. Antimicrobial activity of thediterpenes flexibilide and sinulariolide derived from Sinularia flexibilis Quoyand Gaimard 1833 (Coelenterata: Alcyonacea, Octocorallia). Comp BiochemPhysiol (C) 1998;120:121–6.

[18] Ramesh P, Venkateswarlu Y. Novel steroid constituents of the soft coralSinularia dissecta. Steroids 1999;64:785–9.

[19] Burkholder PR, Burkholder LM, Almodovar LR. Antibiotic activity of somemarine algae of Puerto Rico. Bot Mar 1960;2:149–56.

[20] Takahashi A, Kurasawa S, Ikeda D, Okami Y, Takeuchi T. Altemicidin, a newacaricidal and antitumor substance. I. Taxonomy, fermentation, isolation andphysico-chemical and biological properties. J Antibiot 1989;32:1556–61.

[21] Sajid I, Yao CBF, Shaaban KA, Hasnain S, Laatsch H. Antifungal and antibacterialactivities of indigenous Streptomyces isolates from saline farmlands:prescreening, ribotyping and metabolic diversity. World J MicrobiolBiotechnol 2009;25:601–10.

[22] Schneider K, Graf E, Irran E, Nicholson G, Stainsby FM, Goodfellow M, BordenSA, Keller S, Süssmuth RD, Fiedler H-P. Bendigoles A–C, new steroids fromGordonia australis Acta 2299. J Antibiot 2008;61:356–64.

[23] Note that introduction of a double bond at C-22 of ergostane (24b/S) causes achange of the (R/S) notation at C-20 and C-24, e.g. (20R,24S)-ergostane (24b) to(20S, 24R)-ergost-22-ene (24b).

[24] Schmitz FJ. Marine natural products. In: Scheuer PJ, editor, vol. 1. NewYork: Academic Press; 1978. p. 241.

[25] (a) Kobayashi M, Hayashi T, Nakajima F, Mitsuhashi H. Marine sterols. IX.Occurrence of 24 xi-methylcholestane-1 beta, 3 beta, 5 alpha, 6 beta, 25-pentol 25-monoacetate in the soft coral, Sarcophyton glaucum. Steroids1979;34:285–93;(b) Kobayashi M, Hayashi T, Hayashi K, Tanabe M, Nakagawa T, Mitsuhashi H.Marine sterols. XI. Polyhydroxsterols of the soft coral sarcophyton glaucum:Isolation and synthesis of 5-cholestane-1a,3b,5,6b-terol. Chem Pharm Bull1983;31:1848–55. 4127–4129.

[26] Kodai T, Umebayashi K, Nakatani T, Ishiyama K, Noda N. Compositions of royaljelly II. Organic acid glycosides and sterols of the royal jelly of honeybees (Apismellifera). Chem Pharm Bull 2007;55:1528–31.

[27] Simmons L, Kaufmann K, Garcia R, Schwär G, Huch V, Bendigoles D-F MüllerR.Bioactive sterols from the marine sponge-derived Actinomadura sp., SBMs009.Bioorg Med Chem 2011;19:6570–5.

[28] Nes WD, Song Z, Dennis AL, Zhou W, Nam J, Miller MB. Biosynthesis ofphytosterols kinetic mechanism for the enzymatic C-methylation of sterols. JBiol Chem 2003;278:34505–16.

[29] Moore JT, Gaylor JL. Investigation of an S-adenosylmethionine:D24-sterolmethyltransferase in ergosterol biosynthesis in yeast specificity of sterolsubstrates and inhibitors. J Biol Chem 1970;245:4684–8.

[30] Giner J-L, Djerassi C. Biosynthetic studies of marine Lipids. 40. Generation ofthe cyclopropane ring of Sormosterol. Acta Chem Scand 1992;46:678–9;Giner J-L, Djerassi C. Biosynthetic studies of marine lipids. 31. Evidence for aprotonated cyclopropyl intermediate in the biosynthesis of 24-propylidenecholesterol. J Am Chem Soc 1991;113:1386–93.

[31] Paul M. Dewick, Medicinal natural products. A biosynthetic approach, 2nd Ed.,vol. 535, John Wiley & Sons, Ltd.; 2002. ISBN: 9780471496403.

[32] Bowden BF, Coll JC, Mitchell SJ. Studies of Australian soft corals. XXI. A newses-quiterpene from Nephthea chabrolii and an investigation of common clamTridacna maxima. Aust J Chem 1980;33:1833–9.

[33] Jurek J, Scheuer PJ. Sesquiterpenoids and norsesquiterpenoids from the softcoral Lemnalia africana. J Nat Prod 1993;56:508–13.

M. Shaaban et al. / Steroids 78 (2013) 866–873 873

[34] Lange GL, Gottardo C, Merica A. Synthesis of terpenoids using a free radicalfragmentation/elimination sequence. J Org Chem 1999;64:6738–44.

[35] Tsutsui M, Ostfeld D. The structure of peridinin, the characteristicDinoflagellate Carotenoid. J Am Chem Soc 1971;93:1823–5.

[36] Mclean S, Reynold WF. Total assignment of the 1H and 13C spectra of peridinin.Magn Reson Chem 1992;30:362–3.

[37] Strain HH, Svec WA, Wegfahrt P, Hapoport H, Taxos FT, Norgard S, Kjosen H,Liaaen-Jensen S. Algal carotenoids 14, structural studies on peridinin, Part 1.Structure elucidation. Acta Chim Scand B 1976;30:109–20.

[38] Suzuki M, Watanabe K, Fujiwara S, Kurasawa T, Wakabayashi T, Tsuzuki M,Iguchi K, Yamori T. Isolation of peridinin-related norcarotenoids with cell

growth-inhibitory activity from the cultured dinoflagellate of Symbiodiniumsp., a Symbiont of the Okinawan soft coral Clavularia viridis, and analysis offatty Acids of the dinoflagellate. Chem Pharm Bull 2003;51:724–7.

[39] Schütt F. Ueber Peridineenfarbstoffe. Ber Deut Bot Ges 1890;8:9–32.[40] Yamori T, Matsunaga A, Sato S, Yamazaki K, Komi A, Ishizu K, Mita I, Edatsugi

H, Matsuba Y, Takezawa K, Nakanishi O, Kohno H, Nakajima Y, Komatsu H,Andoh T, Tsuruo T. Potent activity of MS-247, novel DNA minor groove binder,evaluated by an in vitro and in vivo human cancer cell line panel. Cancer Res1999;59:4042–9.