9,11-Secosterols with antiproliferative activity from the gorgonian Eunicella cavolini

Bioorganic & Medicinal Chemistry 17 (2009) 4537–4541

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9,11-Secosterols with antiproliferative activity from the gorgonian Eunicella cavolini Efstathia Ioannou a, Ayman F. Abdel-Razik a,b, Xanthippi Alexi c, Constantinos Vagias a, Michael N. Alexis c, Vassilios Roussis a,* a

Department of Pharmacognosy and Chemistry of Natural Products, School of Pharmacy, University of Athens, Panepistimiopolis Zografou, Athens 15771, Greece Natural Products Chemistry Department, National Research Center, Dokki 12622, Cairo, Egypt c Institute of Biological Research and Biotechnology, National Hellenic Research Foundation, Vas. Constantinou 48, Athens 11635, Greece b

a r t i c l e

i n f o

Article history: Received 19 January 2009 Revised 28 April 2009 Accepted 4 May 2009 Available online 8 May 2009 Keywords: Eunicella cavolini 9,11-Secosterols Structure elucidation Antiproliferative activity

a b s t r a c t Four new 9,11-secosterols (2, 4–6), along with two previously reported ones (1, 3) were isolated from the organic extract of the gorgonian Eunicella cavolini. The structures and relative configurations of the isolated natural products were established on the basis of detailed NMR spectroscopic analysis. Metabolites 1 and 2 were found to strongly inhibit the growth of LNCaP human prostate adenocarcinoma cells and the estrogen-dependent growth of MCF-7 human breast adenocarcinoma cells. Ó 2009 Elsevier Ltd. All rights reserved.

AcO

1. Introduction

11

18 12

Secosteroids are highly oxidized metabolites with bond cleavage in the rings of the steroid tetracyclic nucleus that have exhibited a diverse array of pharmacological activities, such as antimicrobial, antiviral, anti-inflammatory, antiproliferative, antifouling, cytotoxic, and ichthyotoxic effects. 9,11-Secosteroids have been isolated almost exclusively from sponges and coelenterates. Among them, members of the orders Alcyonacea, Gorgonacea, and Stolonifera (Cnidaria, Anthozoa) produce biologically active 9,11-secosteroids.1 Even though species of the genus Eunicella (Gorgoniidae) have been the subject of numerous chemical investigations in the past, the presence of 9,11-secosteroid metabolites has not been reported so far.2,3 In the course of our ongoing efforts to isolate bioactive natural products from marine organisms found along the coastlines of Greece, we decided to carry out a chemical analysis of Eunicella cavolini, one of the most abundant gorgonian species in the Mediterranean Sea.4 Recently, we reported the isolation and structural characterization of four new pregnanes and three new 5a,8a-epidioxysterols from E. cavolini.5,6 Herein, we describe the isolation and structure elucidation of four new (2, 4–6) and two previously reported (1, 3) 9,11-secosterols and the effects of 1, 2, 4, and 5 on the growth of MCF-7 human breast adenocarcinoma cells and LNCaP human prostate adenocarcinoma cells. * Corresponding author. Tel./fax: +30 210 7274592. E-mail address: [email protected] (V. Roussis). 0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.bmc.2009.05.004

19 2 3

HO

1

4

10 5

H

O

13

9 6

8 7

R

17 16 14 15

H

OH 28

26

1 R=

21

22 20

25

4 R=

27

23

22

21 20

23 24

26

28

2 R=

21

22 20

23

24

27 25

26 25

26

5 R=

22

21

23

20

24

25 27

28

3 R=

21

22 20

23

27

24

25 26

6 R=

22

21 20

23

24

27 25 26

2. Results and discussion Colonies of the gorgonian E. cavolini, collected from the Lichadonissia Isles, Greece, were exhaustively extracted with a mixture of CH2Cl2/MeOH and the organic extract was subsequently subjected to a series of chromatographic separations to allow for the isolation of compounds 1–6.

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The spectroscopic characteristics of metabolites 1–6 (Tables 1 and 2) were strikingly similar. Specifically, the common and highly conserved signals observed in the 1H and 13C NMR spectra of 1–6 included an ester carbonyl (dC 171.2), a ketone functionality (dC 203.6), two oxygenated methines (dH/C 3.57/70.0; 4.24/69.4), one oxygenated methylene (dH/C 4.15/61.5), a trisubstituted double bond (dH/C 6.49/146.1; 136.9), two methyl groups on quaternary carbons (dH/C 0.65/17.3; 1.09/16.1), and an acetate methyl group (dH/C 1.99/21.1). Furthermore, the mass spectra of 1–6 exhibited common fragment ions at m/z 303 [M–OAc–H–side chain]+, 287 [M–OAc–OH–side chain]+, and 269 [M–OAc–H2O–OH–side chain]+. The above information, in combination with the correlations provided by HSQC, HMBC, and COSY experiments (Fig. 1), indicated that all isolated compounds possess an 11-acetoxy-3,6-dihydroxy-9,11-seco-5-steroidal nucleus and differ only in the side chains. The relative configuration of the steroidal nucleus for 1– 6, established to be the same for all isolated compounds, was assigned on the basis of key NOE correlations and coupling constants. Enhancements observed in the NOESY spectrum for H-3/H-4a, H3/H-5, and H-4a/H-5, as well as for H-4b/H-6, H-4b/H3-19, and H-6/H3-19 provided evidence that H-3, H-5, and H3-19 were axial, thus revealing the trans fusion of rings A and B and the b- and aorientation of the hydroxy groups at C-3 and C-6, respectively. The latter was also confirmed by comparison of the chemical shift and multiplicity of H-3 (d 3.57 m) with those of other known 3bsterols and the large value of the coupling constant between H-5 and H-6 (9.9 Hz), reflecting a diaxial orientation for these two protons. The stereochemistry of ring D was deduced by the crosspeaks detected between H-11 and H-14, H-14 and H-17, and H318 and H-20, suggesting a trans relationship between H3-18 and both H-14 and H-17. Therefore, the structure elucidation of 1–6 focused in the establishment of their side chains. Compound 2, isolated as yellow oil, displayed an ion peak at m/z 457.3314 (HRFABMS), corresponding to C29H45O4 and consistent for [MOH]+. The mass spectrum showed diagnostic fragment ions at m/z 303 [M–OAc–H–C8H15]+, 287 [M–OAc–OH–C8H15]+, and 269 [M–OAc–H2O–OH–C8H15]+, indicating the presence of a C8H15 unsaturated side chain. The structural features present in the 1H and 13C NMR spectra of 2 corresponding to its side chain included

Table 2 13 C NMR data (50.3 MHz, CDCl3) of compounds 2 and 4–6 Position

2

4

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 OAc OAc

31.8 30.5 70.0 32.8 48.7 69.4 146.1 136.9 203.6 44.7 61.5 37.1 45.9 42.8 26.9 25.8 50.8 17.3 16.1 38.6 21.6 133.6 134.8 38.5 29.9 11.9

CH2 CH2 CH CH2 CH CH CH C C C CH2 CH2 C CH CH2 CH2 CH CH3 CH3 CH CH3 CH CH CH CH2 CH3

20.6 171.2 21.1

CH3 C CH3

5

31.7 30.5 70.0 32.8 48.7 69.5 146.1 136.9 203.5 44.7 61.4 37.1 46.0 42.6 26.9 26.1 50.5 17.0 16.1 34.6 19.0 34.0 31.8 156.6 33.8 21.8 22.0 106.2 171.1 21.1

CH2 CH2 CH CH2 CH CH CH C C C CH2 CH2 C CH CH2 CH2 CH CH3 CH3 CH CH3 CH2 CH2 C CH CH3 CH3 CH2 C CH3

O O

O

31.8 30.5 70.0 32.8 48.7 69.4 146.1 137.0 203.6 44.7 61.5 37.1 46.0 42.7 26.9 26.0 50.8 17.3 16.1 38.7 21.7 134.4 133.2 43.1 33.2 20.1 19.7 17.8 171.1 21.1

6 CH2 CH2 CH CH2 CH CH CH C C C CH2 CH2 C CH CH2 CH2 CH CH3 CH3 CH CH3 CH CH CH CH CH3 CH3 CH3 C CH3

31.8 30.5 70.0 32.8 48.7 69.4 146.1 136.9 203.6 44.7 61.5 37.1 45.9 42.8 26.9 26.2 50.5 17.2 16.1 34.7 19.0 35.4 24.5 39.5 28.0 22.8 22.6

CH2 CH2 CH CH2 CH CH CH C C C CH2 CH2 C CH CH2 CH2 CH CH3 CH3 CH CH3 CH2 CH2 CH2 CH CH3 CH3

171.1 21.1

C CH3

R COSY HMBC

HO OH Figure 1. COSY and important HMBC correlations for the steroidal nucleus of compounds 1–6.

Table 1 1 H NMR data (400 MHz, CDCl3) of compounds 2 and 4–6 Position

2

1 2 3 4 5 6 7 11 12 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 OAc

1.86 1.91 3.57 2.28 1.76 4.24 6.49 4.15 1.60 3.22 1.57 1.70 1.69 0.65 1.09 2.15 1.01 5.21 5.16 1.91 1.24 0.81

4 m, 1.46 m m, 1.44 m m m, 1.43 m m dd (9.9, 1.7) d (1.7) m m, 1.23 m t (9.8) m m, 1.44 m m s s m d (6.8) dd (15.1, 8.0) dd (15.1, 7.4) m m t (6.7)

0.90 d (6.7) 1.99 s

5

1.88 1.90 3.58 2.28 1.76 4.25 6.51 4.15 1.63 3.24 1.59 1.85 1.67 0.66 1.10 1.44 0.99 1.54 2.09

m, 1.45 m m, 1.46 m m m, 1.42 m m dd (9.9, 1.6) d (1.6) m m, 1.21 m t (10.0) m m, 1.43 m m s s m d (6.8) m, 1.13 m m, 1.86 m

2.20 1.00 1.01 4.70 1.99

m d (6.7) d (6.7) br s, 4.63 br s s

1.87 1.91 3.58 2.28 1.74 4.25 6.50 4.15 1.62 3.23 1.58 1.70 1.67 0.66 1.10 2.15 1.02 5.21 5.20 1.86 1.45 0.81 0.80 0.89 1.99

6 m, 1.46 m m, 1.44 m m m, 1.41 m m dd (9.9, 1.9) d (1.9) m m, 1.21 m t (10.0) m m, 1.42 m m s s m d (6.8) m m m m d (6.9) d (6.9) d (6.8) s

1.86 1.93 3.58 2.27 1.75 4.25 6.51 4.16 1.63 3.23 1.57 1.80 1.63 0.66 1.10 1.39 0.96 1.33 1.34 1.11 1.49 0.85 0.84

m, 1.46 m m, 1.45 m m m, 1.42 m m dd (9.8, 2.0) d (2.0) m m, 1.24 m t (9.8) m m, 1.43 m m s s m d (6.6) m, 0.98 m m, 1.13 m m m d (6.6) d (6.6)

1.99 s

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sibility of the E geometry for the D22 double bond. Moreover, the strong absorption band at 753 cm1 observed in the IR spectrum indicated the Z orientation of the disubstituted double bond, since in the opposite case an absorption around 970 cm1 would be expected.8–10 The configuration at C-24 could not be defined through spectroscopic analysis. On the basis of the above mentioned data, 5 was identified as (22Z)-11-acetoxy-3b,6a-dihydroxy-24n-methyl9,11-seco-5a-cholesta-7,22-dien-9-one. Compound 6, isolated as a colorless oil, displayed an ion peak at m/z 477.3623 (HRFABMS), corresponding to C29H49O5 and consistent for [M+H]+. The mass spectrum contained fragment ions at m/z 303 [M–OAc–H–C8H17]+, 287 [M–OAc–OH–C8H17]+, and 269 [M–OAc–H2O–OH–C8H17]+, indicating the presence of a C8H17 saturated side chain. The structural features present in the 1H and 13C NMR spectra of 6 corresponding to its side chain included three methyl groups on tertiary carbons (dH/C 0.84/22.6; 0.85/22.8; 0.96/19.0). The correlations between all geminal and vicinal protons observed in the COSY spectrum, as well as the HMBC correlations of C-20 with H3-21 and H-22, C-24 with H-23 and H-25, and C-25 with H-24, H3-26, and H3-27 were used to establish the side chain of 6 unambiguously. Thus, metabolite 6 was identified as 11acetoxy-3b,6a-dihydroxy-9,11-seco-5a-cholest-7-en-9-one. Compounds 1 and 3 were identified by comparison of their spectroscopic and physical characteristics with those reported in the literature as (22E)-11-acetoxy-3b,6a-dihydroxy-24-nor-9,11seco-5a-cholesta-7,22-dien-9-one (1) and (22E)-11-acetoxy3b,6a-dihydroxy-9,11-seco-5a-cholesta-7,22-dien-9-one (3), previously isolated from the soft coral Gersemia fruticosa.11,12 It has been reported that 9,11-secosterols, in the low lmol/L range of concentrations, exert growth inhibitory effects against human cancer cells of different origin, including leukemic, hepatic, cervical, breast, prostate, colon, skin, and lung cancer cells,11,13–16 as well as against a variety of rat and murine cancer cells.17,18 In these reports, compound effects on cell growth were assessed mainly using conversion of yellow MTT [3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide] to a purple formazan as a means to indirectly assess the number of viable cells.14–18 We sought to similarly determine the effect of 1, 2, 4, and 5 on the growth of MCF-7 human breast adenocarcinoma cells and LNCaP human prostate adenocarcinoma cells using the MTT assay (Table 3). We found that the numbers of viable LNCaP cells growing in the presence of 10% fetal bovine serum (FBS) and 10 lM of 1, 2 and 5 were 76 ± 4, 83 ± 2 and 45 ± 12% lower (p <0.05 vs vehicle; ANOVA; n = 4), respectively, compared to cells exposed to vehicle alone (0.1% DMSO). The IC50 values for 1, 2, and 5 on LNCaP cells were found to be 7.0 ± 0.6, 5.4 ± 0.7, and 6.7 ± 0.8 lM, respectively. In contrast, compound 4 failed to significantly inhibit the growth of LNCaP cells. Similarly, the numbers of MCF-7 cells growing in the presence of 10% FBS and 10 lM of 1, 2, and 5 were observed to be 34 ± 5, 49 ± 6, and 27 ± 3% lower, respectively, compared to in the presence of vehicle alone, while 4 was ineffective in inhibiting

one 1,2-disubstituted double bond (dH/C 5.16/134.8; 5.21/133.6), two methyl groups on tertiary carbons (dH/C 0.90/20.6; 1.01/ 21.6), and a methyl group on a secondary carbon (dH/C 0.81/11.9). The correlations between all geminal and vicinal protons observed in the COSY spectrum, as well as the HMBC correlations of C-20 with H3-21 and H-22, C-23 with H-22, H-25, and H3-28, and C25 with H-24, H3-26, and H3-28 were used to establish the side chain of 2 unambiguously. The geometry of the D22 double bond was assigned as E on the basis of the coupling constant between protons H-22 and H-23 (J = 15.1 Hz). The configuration at C-24 could not be determined, since only one of the two diastereomers was isolated and the observed chemical shift differences for other pairs of sterols with the same side chain are marginal and can only be used to determine between the two isomers comparatively.7 Thus, metabolite 2 was identified as (22E)-11-acetoxy-3b,6a-dihydroxy-24n-methyl-27-nor-9,11-seco-5a-cholesta-7,22-dien-9-one. Compound 4, obtained as yellow oil, displayed an ion peak at m/ z 471.3470 (HRFABMS), corresponding to C30H47O4 and consistent for [MOH]+. In the mass spectrum the fragment ions at m/z 303 [M–OAc–H–C9H17]+, 287 [M–OAc–OH–C9H17]+, and 269 [M–OAc– H2O–OH–C9H17]+ suggested the presence of a C9H17 unsaturated side chain. The signals corresponding to the side chain of 4 in the 1 H and 13C NMR spectra included three methyl groups on tertiary carbons (dH/C 0.99/19.0; 1.00/21.8; 1.01/22.0) and a 1,1-disubstituted double bond (dH/C 4.63, 4.70/106.2; 156.6). The cross peaks between all geminal and vicinal protons observed in the COSY spectrum, in conjunction with the correlations of C-24 and C-28 with H-23 and H-25 displayed in the HMBC spectrum clearly indicated the nature of the side chain of 4. Therefore, metabolite 4 was identified as 11-acetoxy-3b,6a-dihydroxy-24-methyl-9,11-seco5a-cholesta-7,24(28)-dien-9-one. Compound 5 was isolated as a colorless oil. The ion peak at m/z 489.3541 observed in the HRFABMS was consistent for [M+H]+ and corresponded to C30H49O5. The characteristic fragment ions at m/z 303 [M–OAc–H–C9H17]+, 287 [M–OAc–OH–C9H17]+, and 269 [M– OAc–H2O–OH–C9H17]+ displayed in the mass spectrum indicated the presence of a C9H17 unsaturated side chain. In the 1H and 13C NMR spectra of 5, the signals of the side chain were evident, including four methyl groups on tertiary carbons (dH/C 0.80/19.7; 0.81/20.1; 0.89/17.8; 1.02/21.7) and a 1,2-disubstituted double bond (dH/C 5.20/133.2; 5.21/134.4). Cross peaks between all geminal and vicinal protons evident in the COSY spectrum allowed the determination of the side chain of 5, while HMBC correlations of C20 and C-22 with H3-21, C-24 with H-23, H-25, and H3-28, and C25 with H-24, H3-26, and H3-27 further verified the side chain structure. The geometry of the D22 double bond was difficult to assign, due to the fact that both olefinic protons overlapped, thus not allowing a reliable measurement of their coupling constant. The use of various NMR solvents (C3D6O, C6D6, C5D5N) did not help in resolving the overlapping of H-22 and H-23, but the small width measured at the base of their multiplet (9.6 Hz) excluded the pos-

Table 3 Effects of compounds 1, 2, 4, and 5 on the growth of LNCaP and MCF-7 cells Compoundsa

LNCaP cells in MEM+10% FBS

MCF-7 cells in MEM+10% FBS

b

b

Growth inhibition d

1 2 4 5

76 ± 4 83 ± 2d 610 45 ± 12d a b c d e

IC50 (lM) 7.0 ± 0.6 5.4 ± 0.7 n.d.e 6.7 ± 0.8

Growth inhibition d

34 ± 5 49 ± 6d 610 27 ± 3

All compounds were tested at a concentration of 10 lM. Effects were calculated by [(ODvehicleODtest compound) * 100]/ODvehicle. Effects were calculated by [(ODvehicleODtest compound) * 100]/(ODvehicleODICI p <0.05 versus vehicle; ANOVA; n = 4. Not determined.

182,780).

MCF-7 cells in MEM+5% DCC-FBS+1 nM 17b-estradiol

IC50 (lM)

Growth inhibitionc

IC50 (lM)

7.6 ± 0.4 7.0 ± 0.5 n.d.e n.d.e

86 ± 9d 90 ± 13d 50 ± 5d 62 ± 12d

7.4 ± 0.2 5.4 ± 0.2 3.8 ± 0.7 6.0 ± 0.4

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the growth of these cells. Under these conditions only 1 and 2 could significantly (p <0.05 vs vehicle) inhibit the growth of MCF7 cells. Metabolites 1 and 2 exhibited IC50 values of 7.6 ± 0.4 and 7.0 ± 0.5 lM, respectively, against MCF-7 cells. It has been reported that 1 can inhibit the growth of human K562 leukemic cells and HeLa cervical carcinoma cells at IC50 values of 6.5 ± 1.1 and 7.0 ± 0.6 lM, respectively.13 Since it was found that 1 was considerably more effective against LNCaP cells compared to MCF-7 cells, the presumption is that the activity of secosterols may vary considerably depending on the cell line used, as previously reported.18 Estrogen and growth factors are known to jointly promote proliferation and survival of MCF-7 cells through complex interactions of the estrogen receptor (ER) with transmembrane growth factor receptors (GFR) at the plasma membrane, which result in activation of downstream kinases in a manner that is fully inhibited by ER antagonists, like tamoxifen and ICI 182,780.19,20 ICI 182,780, in particular, is an estrogen receptor (ER)-destabilizer capable of strongly down-regulating ER expression, thereby fully inhibiting the estrogen/ER-dependent growth of these cells.21,22 In line with these notions, we have established that the rate of growth of MCF-7 cells in the presence of 5% DCC-FBS (heat-inactivated FBS treated with dextran-coated charcoal; the treatment removes labile growth factors, as well as endogenous steroids) and low physiological levels of 17b-estradiol (1 nM; this concentration promotes cell proliferation in the presence of DCC-FBS to its maximum) is lower than that in the presence of 10% FBS (contains enough endogenous estrogen for maximal cell proliferation) and that in either case, cell proliferation is abolished in the presence of 1 lM ICI 182,780. We sought to determine the growth inhibitory effects of 1, 2, 4, and 5 on the estrogen-dependent growth of MCF-7 cells and to compare them to that of ICI 182,780. Therefore, we exposed MCF-7 cells growing in the presence of 5% DCC-FBS and 1 nM 17b-estradiol to 1, 2, 4, and 5 (10 lM), to ICI 182,780 (1 lM) and to vehicle only (Table 3). We found that compounds 1, 2, 4, and 5 exerted significant (p <0.05 vs vehicle; ANOVA; n = 4) growth inhibitory effects that were equal to 86 ± 9, 90 ± 13, 50 ± 5 and 62 ± 12%, respectively, of the effect of ICI 182,780 (100% inhibition of growth). Metabolites 1, 2, 4, and 5 exhibited IC50 values of 7.4 ± 0.2, 5.4 ± 0.2, 3.8 ± 0.7, and 6.0 ± 0.4 lM, respectively, against MCF-7 cells. The interference of the secosterols assayed with estrogen-dependent growth seems to be favored by the presence of a double bond at C-22, especially with E geometry, as in 1 and 2. Comparison of the growth inhibitory effects of 1, 2, 4, and 5 in FBS and in 17b-estradiol-supplemented DCC-FBS suggests, however, that growth factors presumably present in FBS but not (as much) in DCC-FBS, stimulate GFR/ER cross-talk to partially inhibit the effect of 1 and 2, render the effect of 5 non-significant and abolish the effect of 4. Thus, it appears that the antiproliferative activity of the tested secosterols can vary considerably depending on cell growth conditions.

3. Experimental 3.1. General experimental procedures Optical rotations were measured on a Perkin–Elmer model 341 polarimeter with a 1 dm cell. UV spectra were obtained on a Shimadzu UV-160A spectrophotometer. IR spectra were obtained on a Paragon 500 Perkin–Elmer spectrometer. NMR spectra were recorded on Bruker AC 200 and Bruker DRX 400 spectrometers. Chemical shifts are given on the d (ppm) scale using TMS as internal standard. The 2D experiments (HMQC, HMBC, COSY, NOESY) were performed using standard Bruker pulse sequenses. High-resolution mass spectra were provided by the University of Notre Dame, Department of Chemistry and Biochemistry, Notre Dame,

IN, USA. Low-resolution CI mass spectra were recorded in the positive mode on a Thermo Electron Corporation DSQ mass spectrometer using a Direct-Exposure Probe and methane as the CI reagent gas. Column chromatography was performed using Kieselgel 60 (Merck). HPLC separations were conducted using a CECIL 1100 Series liquid chromatography pump equipped with a GBC LC-1240 refractive index detector, using a Kromasil 100 C18 (MZ-Analysentechnik, 25 cm  8 mm) column. TLC was performed using Kieselgel 60 F254 (Merck aluminum support plates) and spots were detected after spraying with 15% H2SO4 in MeOH reagent and heating at 100 °C for 1 min. 3.2. Animal material Colonies of E. cavolini were collected from the Lichadonissia Isles in Maliakos Gulf, Greece, at a depth of 15–20 m in October, 2004. A voucher specimen of the soft coral has been deposited at the Herbarium of the Department of Pharmacognosy and Chemistry of Natural Products, University of Athens (ATPH/MO/164). 3.3. Extraction and isolation Specimens of the gorgonian (1.7 kg) were exhaustively extracted with CH2Cl2/MeOH (3:1) at room temperature. Evaporation of the solvent in vacuo afforded a dark orange oily residue (48 g) which was subjected to gravity column chromatography on silica gel, using cyclohexane with increasing amounts of EtOAc, followed by EtOAc with increasing amounts of MeOH as the mobile phase, to afford eighteen fractions (I–XVIII). Fraction XIV (85% EtOAc, 530 mg) was further fractionated by gravity column chromatography on silica gel, using cyclohexane/EtOAc (35:65) as the mobile phase, to yield fifteen fractions (XIV1–XIV15). Fractions XIV12 (32.1 mg), XIV13 (38.0 mg), XIV14 (26.4 mg), and XIV15 (27.5 mg) were subjected repeatedly to reversed-phase HPLC, using MeOH–H2O (88:12) as eluant, to yield in order of elution compounds 1 (2.5 mg), 2 (3.0 mg), 3 (0.3 mg), 4 (4.0 mg), 5 (2.0 mg), and 6 (0.4 mg). 3.3.1. (22E)-11-Acetoxy-3b,6a-dihydroxy-24n-methyl-27-nor9,11-seco-5a-cholesta-7,22-dien-9-one (2) Yellow oil; ½a20 D þ 27:0 (c 0.20, CHCl3); UV (CHCl3) kmax (log e) 248.4 (3.57) nm; IR (thin film) mmax 3408, 2958, 2864, 1734, 1673, 1463, 1370, 1258, 1043, 973 cm1; 1H NMR data, see Table 1; 13C NMR data, see Table 2; PCIMS (CH4) m/z 474 (21), 457 (26), 439 (17), 415 (63), 397 (93), 379 (54), 347 (17), 303 (20), 287 (50), 269 (30), 217 (19), 111 (100), 81 (39), 69 (84); HRFABMS m/z 457.3314 [MOH]+ (calcd for C29H45O4, 457.3318). 3.3.2. 11-Acetoxy-3b,6a-dihydroxy-24-methyl-9,11-seco-5acholesta-7,24(28)-dien-9-one (4) Yellow oil; ½a20 D þ 22:0 (c 0.10, CHCl3); UV (CHCl3) kmax (log e) 248.6 (3.60) nm; IR (thin film) mmax 3408, 2958, 2873, 1739, 1673, 1463, 1379, 1253, 1043 cm1; 1H NMR data, see Table 1; 13 C NMR data, see Table 2; PCIMS (CH4) m/z 488 (9), 471 (21), 453 (27), 429 (37), 411 (79), 393 (99), 303 (9), 287 (17), 269 (23), 125 (22), 109 (66), 95 (48), 69 (84), 61 (100); HRFABMS m/z 471.3470 [MOH]+ (calcd for C30H47O4, 471.3474). 3.3.3. (22Z)-11-Acetoxy-3b,6a-dihydroxy-24n-methyl-9,11seco-5a-cholesta-7,22-dien-9-one (5) Colorless oil; ½a20 D þ 18:0 (c 0.13, CHCl3); UV (CHCl3) kmax (log e) 247.4 (3.39) nm; IR (thin film) mmax 3437, 2958, 2864, 1734, 1669, 1450, 1370, 1258, 1043, 753 cm1; 1H NMR data, see Table 1; 13C NMR data, see Table 2; PCIMS (CH4) m/z 488 (9), 471 (36), 453 (26), 429 (81), 411 (100), 393 (64), 303 (32), 287 (63), 269 (42),

E. Ioannou et al. / Bioorg. Med. Chem. 17 (2009) 4537–4541

4541

125 (38), 109 (40), 69 (77); HRFABMS m/z 489.3541 [M+H]+ (calcd for C30H49O5, 489.3580).

material. Financial support from the University of Athens, in the form of a ‘Kapodistrias’ grant, is gratefully acknowledged.

3.3.4. 11-Acetoxy-3b,6a-dihydroxy-9,11-seco-5a-cholest-7-en9-one (6) Colorless oil; ½a20 D þ 7:5 (c 0.03, CHCl3); UV (CHCl3) kmax (log e) 247.6 (3.29) nm; IR (thin film) mmax 3446, 2939, 2857, 1737, 1671 cm1; 1H NMR data, see Table 1; 13C NMR data, see Table 2; PCIMS (CH4) m/z 476 (5), 459 (25), 441 (20), 417 (55), 399 (100), 381 (59), 303 (20), 287 (40), 269 (27), 113 (7), 69 (48); HRFABMS m/z 477.3623 [M+H]+ (calcd for C29H49O5, 477.3580).

References and notes

3.4. Assessment of antiproliferative activity Stock solutions of test compounds (0.01 mM) were prepared in DMSO (vehicle). Compound effects on the growth of MCF-7 and LNCaP cells were assessed in 96-well flat-bottomed microculture plates using MTT, as previously reported.23,24 MCF-7 cells cultured in minimal essential medium in the presence of 10% FBS or 5% DCC-FBS and 1 nM 17b-estradiol were exposed to the tested secosterols, 17b-estradiol or the ER-destabilizer ICI 182,780 for four days and assayed spectrophotometrically (absorbance at 550 nm) using MTT conversion to colored formazan as a means to indirectly assess the number of viable cells. Absorbance following exposure to test compounds (10 lM), 17b-estradiol (1 nM) and ICI 182,780 (1 lM) was expressed as the percentage of that of vehicle, set equal to 100. Growth inhibitory effects (mean ± SEM of at least three independent assays) were calculated as [(ODvehicleODtest compound) * 100]/ODvehicle. Alternatively, they could be expressed as % of that of ICI 182,780 by [(ODvehicle ODtest compound) * 100]/(ODvehicleODICI 182,780). ICI 182,780 (FaslodexÒ, Fulvestrant) was used as positive control, inhibiting the cell growth by 100% and exhibiting an IC50 value of 18 nM against both MCF-7 and LNCaP cells.22,25 Acknowledgments The authors thank the Greek State Scholarships Foundation (I.K.Y.) for a postdoctoral research studies scholarship awarded to A.A.R. and Mr. Dimitrios Christofidis for the collection of the animal

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