Arch. Pharm. Res. (2015) 38:18–25 DOI 10.1007/s12272-014-0480-8

RESEARCH ARTICLE

Cytotoxic 5a,8a-epidioxy sterols from the marine sponge Monanchora sp. Bora Mun • Weihong Wang • Hiyoung Kim • Dongyup Hahn • Inho Yang • Dong Hwan Won • Eun-hee Kim • Jihye Lee • Chulkyeong Han • Hyunji Kim Merrick Ekins • Sang-Jip Nam • Hyukjae Choi • Heonjoong Kang



Received: 16 August 2014 / Accepted: 3 September 2014 / Published online: 19 September 2014 Ó The Pharmaceutical Society of Korea 2014

Abstract Three new sterols, 5a,8a-epidioxy-24-norcholesta-6,9(11),22-trien-3b-ol (1), 5a,8a-epidioxy-cholesta6,9(11),24-trien-3b-ol (2), and 5a,8a-epidioxy-cholesta6,23-dien-3b,25-diol (3), with four known sterols (4–7) were isolated from a marine sponge Monanchora sp. Their chemical structures were elucidated by extensive spectroscopic analysis. Compounds 1 and 3–7 showed moderate cytotoxicity against several human carcinoma cell lines including renal (A-498), pancreatic (PANC-1 and MIA PaCa-2), and colorectal (HCT 116) cancer cell lines.

Electronic supplementary material The online version of this article (doi:10.1007/s12272-014-0480-8) contains supplementary material, which is available to authorized users. B. Mun  W. Wang  H. Kim  D. Hahn  I. Yang  D. H. Won  E. Kim  J. Lee  C. Han  H. Kim  H. Kang Center for Marine Natural Products and Drug Discovery, School of Earth and Environmental Sciences, Seoul National University, NS-80, Seoul 151-747, Republic of Korea M. Ekins Queensland Museum, South Brisbane BC, QLD 4101, Australia S.-J. Nam Department of Chemistry and Nano Science, Ewha Womans University, Seoul 120-750, Republic of Korea H. Choi (&) College of Pharmacy, Yeungnam University, 280, Daehak-ro, Gyeongsan 712-749, Republic of Korea e-mail: [email protected] H. Kang (&) Research Institute of Oceanography, Seoul National University, NS-80, Seoul 151-747, Republic of Korea e-mail: [email protected]

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Keywords 5a,8a-epidioxy sterol  Monanchora sp.  Cytotoxicity  Pancreatic cancer  Renal cancer  Colorectal cancer

Introduction Marine sponges (phylum Porifera) are broadly recognized as a highly prolific source of structurally unique and biologically active natural products (Seo et al. 1997; Sarma et al. 2005). Steroids and terpenoids are two abundant classes of compounds found in marine sponges (Gross and Ko¨nig 2006; Sheikh and Djerassi 1974). In particular, sterols, also known as steroid alcohols, are a subgroup of steroids, which are ubiquitously found in marine invertebrates, plants, and fungi. Sterols play a pivotal role in cell signaling and serve as biosynthetic precursors of vitamins and steroidal hormones (Sarma et al. 2009). Sterols from marine sources have been reported to possess diverse bioactivities, including antiproliferative, cytotoxic, antifouling, and farnesoid X-activated receptor antagonistic activities (Ioannou et al. 2009; Luo et al. 2006; Liu et al. 2011; Im et al. 2000; Sera et al. 1999; Shin et al. 2012). During the course of our search for cytotoxic secondary metabolites against cancer cells, we studied a marine sponge of the genus Monanchora (class: Demospongiae, order: Poecilosclerida, family: Crambeidae). In the literature, most of the natural products isolated from the genus Monanchora have been guanidine alkaloids which exhibited a wide range of biological activities, including ichthyotoxic, antibacterial, antifungal, antiviral, cytotoxic, apoptosis-inducing, and Ca2? channel-blocking activities (Berlinck et al. 1992; Takishima et al. 2009; Gallimore et al. 2005; Chang et al. 2003; Makarieva et al. 2011; Guzii et al. 2010; Berlinck et al. 1993). In addition, marine

Cytotoxic 5a,8a-epidioxy sterols

sponges of the genus Monanchora have also yielded sterols in previous studies (Kapustina et al. 2012; Santalova et al. 2007).

Materials and methods General experimental procedures Optical rotations were measured in methanol (MeOH) using a 1.0 cm cell on a Rudolph Research Autopol III. UV spectra were obtained using a Hitachi JP/U-3010 UV spectrophotometer. CD spectra were taken in MeOH using a Chirascan plus Circular Dichroism Detector. IR spectra were acquired on a JASCO FT/IR 4200 spectrophotometer. NMR spectra were recorded on a Bruker Avance DPX-500 or DPX-600 or an AscendTM 700 spectrometer using methanol-d4 as solvent. Chemical shifts were referenced to the respective solvent peaks [dH 3.31 and dC 49.0 for methanol-d4]. Electrospray ionization source (ESI) low resolution mass spectra were recorded on an Agilent Technologies 6120 Quadrupole mass spectrometer coupled to an Agilent Technologies 1260 series HPLC. High resolution mass data were collected on a Jeol JMS-700 double-focusing (B/E configuration) instrument. Animal material The specimen, Monanchora sp. (QM G331990), was collected by SCUBA at a depth of 15–20 m near Ga-geo Island in the South Sea of Korea, and immediately frozen in dry ice. The sponge was 120 9 100 9 80 mm sized and had many oscular bearing papillae (0.5–4 mm in diameter). In life it was reddish brown, but this turned to a dark brown in alcohol. The tissue was soft and compressible. The specimen was taxonomically identified as Monanchora sp. 4,831 (class Demospongiae, order Poecilosclerida, family Crambeidae). A voucher specimen (QM G331990) was deposited at the Queensland Museum, Australia, and at the Center for Marine Natural Products and Drug Discovery, Seoul National University, Korea (CMDD09A0401). Extraction and isolation Lyophilized specimens (dry weight 400 g) were extracted three times with MeOH/dichloromethane (CH2Cl2) (1:1 v/v) at room temperature. Extracts were combined and partitioned three times between water and CH2Cl2. The CH2Cl2soluble layer was further partitioned between hexanes and 90 % aqueous MeOH to afford a hexanes-soluble fraction (15.8 g) and an aqueous MeOH-soluble fraction (17 g). The aqueous MeOH fraction was then subjected to reversed-phase silica gel flash column chromatography

19

˚ , 40–60 lm) using a stepwise (YMC Gel ODS-A, 120 A solvent gradient of 50 % aqueous MeOH to 100 % MeOH and washing with acetone to afford 14 fractions. Fraction 8 was further separated by normal-phase MPLC, eluting with a solvent of 3–100 % ethyl acetate (EtOAc) in hexane to afford 11 subfractions (8-1 through 8-11). Subfraction 8-5 was subjected to reversed-phase preparative HPLC (Phe˚ 21.2 9 250 mm, nomenex Luna 10 l C18 (2) 100 A 8.0 mL/min, UV = 210 nm), eluting with 75 % aqueous acetonitrile to afford two pure compounds, 2 (6.0 mg, retention time 95 min) and 4 (7.4 mg, retention time 105 min), and subfractions 8-5-1–8-5-31. Compound 1 (2.5 mg) was obtained from the subfraction 8-5-23 by reversed-phase HPLC (Waters Xterra RP18 5 lm 4.6 9 250 mm, 0.7 mL/min, UV = 200 nm) using 70 % aqueous acetonitrile as eluent (retention time for 1: 23 min). To isolate compound 3, fraction 8-6 was further separated by reversed-phase preparative HPLC (Phenomenex ˚ 21.2 9 250 mm, 8.0 mL/min, Luna 10 l C18 (2) 100 A UV = 200 nm) using 75 % aqueous acetonitrile as eluent to afford 16 fractions. Compound 3 (0.5 mg) was obtained from subfraction 8-6-5 by reversed-phase HPLC (Waters Xterra RP18 5 lm 4.6 9 250 mm, 0.7 mL/min, UV = 200 nm), eluting with 50 % aqueous acetonitrile (retention time for 3: 21 min). Compounds 5 (3.0 mg) and 6 (2.0 mg) were isolated from fraction 9 along with compounds (1–4). Compound 7 (2.5 mg) was also isolated from fraction 8-5-28. 5a,8a-epidioxy-24-norcholesta-6,9(11),22-trien-3b-ol (1) White amorphous powder; ½a25 D ? 51° (c 0.003, MeOH); UV (MeOH) kmax (log e) 205 (3.42) nm; IR(thin film) mmax: 3418, 2930, 2871, 1737, 1650, 1462, 1377, 1241, 1075, 1034, 973, 756 cm-1; 1H, 13C, and 2D NMR data, see Table 1; HRFABMS, m/z 399.2859 (calcd for C26H39O3, 399.2899). 5a,8a-epidioxy-cholesta-6,9(11),24-trien-3b-ol (2) Yellow amorphous solid; ½a25 D ? 52° (c 0.01, MeOH); UV (MeOH) kmax (log e): 203 (3.99) nm; IR(thin film) mmax: 3393, 2929, 2870, 1456, 1375, 1338, 1236, 1076, 1031, 969, 932 cm-1; 1H, 13C, and 2D NMR data, see Table 2; HRFABMS, m/z 413.3050 (calcd for C27H41O3, 413.3056). 5a,8a-epidioxy-cholesta-6,23-dien-3b,25-diol (3) White amorphous powder; ½a25 D - 24° (c 0.001, MeOH); UV (MeOH) kmax (log e): 209 (3.51) nm; IR(thin film) mmax: 3417, 2925, 1666, 1461, 1378, 1260, 1075, 1043,

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Table 1 NMR data of 1 (methanol-d4) Number

dC, ma

dH, m, J (Hz)

COSY

HMBC

1

32.3, t

2.01 (m) a, 1.70 (m) b

2

C-6

2

29.9, t

1.88 (m) a, 1.57 (m) b

1, 3

C-3, 4

3

65.4, d

3.83 (m)

2, 4

C-2, 4

4

35.4, t

2.01 (m) a, 1.96 (m) b

3

C-2, 3, 5, 6

5

82.6, s

6

135.5, d

6.34 (d, 8.6)

7

C-4, 5,10

7

130.3, d

6.67 (d, 8.6)

6

C-5, 8, 9, 14, 15

8

78.3, s

9

142.7, s

10

37.8, s

11 12

119.3, d 41.0, t

5.50 (dd, 5.8, 1.5) 2.30 (m) a, 2.09 (m) b

12 11

C-8, 10, 12, 13 C-9, 11, 13, 14, 18

13

43.3, s

14

48.2, d

1.81 (m)

15

C-7, 12, 18

15

20.3, t

1.62 (m)

14, 16

C-7, 16

16

28.2, t

1.80 (m) a, 1.40 (m) b

15, 17

C-15

17

55.7, d

1.38 (m)

16, 20

18

11.9, q

0.79 (s)

C-12,13,14, 17

19

24.5, q

1.13 (s)

C-1, 5, 9, 10

20

39.6, d

2.01 (m)

21, 22

C-21, 22, 23

21

19.7, q

1.03 (d, 6.6)

20

C-17, 20, 22

22

133.0, d

5.22 (dd, 15.2, 8.6)

20, 23

C-17, 20, 21, 23, 24

23

135.2, d

5.33 (dd, 15.2, 6.8)

22, 24

C-20, 22, 24, 25, 26

24

30.8, d

2.21 (dqq, 6.7, 6.7, 6.7)

23, 25, 26

C-22, 23, 25, 26

25

21.8, q

0.98 (d, 6.7)

24

C-23, 24, 25

0.97 (d, 6.7)

24

C-23, 24, 26

21.8, q

26 1

700 MHz for H NMR and 175 MHz for a

C-18, 21, 22

13

C NMR

Multiplicity was determined by analysis of 1H NMR and HSQC data

971 cm-1; 1H, 13C, and 2D NMR data, see Table 2; HREIMS, m/z 430.3086 (calcd for C27H42O4, 430.3083). Cytotoxicity assay One human renal cancer cell line (A498), two human pancreatic cancer cell lines (MIA-paca and PANC-1), and a human colorectal cancer cell line (HCT 116) were seeded separately in 96-well plates in DMEM supplemented with 10 % fetal bovine serum (FBS) in humidified air containing 5 % CO2 at 37 °C. After incubation for 24 h, the indicated compounds were dissolved in DMSO and administered to cells at different concentrations. After incubation for 72 h, cells were harvested and incubated with 5 mg/mL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra-zolium bromide (MTT) solution for 4 h at 37 °C. DMSO was added to each well to dissolve the resulting purple formazan crystals, and the absorbance of each well was measured at 450 nm using an ELISA reader. The assay was carried out in triplicate.

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Results and discussion Solvent partitioning followed by open column chromatography, normal-phase MPLC and reversed-phase HPLC resulted in the isolation of three new and four known epidioxy sterols that have not been isolated previously from the genus Monanchora. Epidioxy sterols have been reported to exhibit cytotoxicity against a series of human cell lines including A549, SK-OV-3, SK-MEL-2, XF498, HCT 15, P-388, SGC-7901, HepG2, HeLa, KB, HT-29, HTLV-1, and MCF-7 (Ioannou et al. 2009; Luo et al. 2006; Liu et al. 2011; Im et al. 2000; Sheu et al. 2000; Gauvin et al. 2000). However, their cytotoxicity against human renal and pancreatic cancer cells has not been previously studied, and renal and pancreatic cancers account for nearly 5 % of all cancer cases globally (Ferlay et al. 2010). Furthermore, the 5-year survival rates of renal and pancreatic cancer patients in the malignant stage are less than 12 and 2 %, respectively (Howlader et al. 1975–2009). Notably, the 5-year survival rate of pancreatic cancer

Cytotoxic 5a,8a-epidioxy sterols

21

Table 2 NMR data of 2 and 3 (methanol-d4) 2a

Number

3a

dC, ma

dC, ma

dH, m, J (Hz)

dH, m, J (Hz)

1

33.8, t

2.00 (m) a, 1.69 (m) b

34.4, t

1.89 (m) a, 1.73 (m), b

2

31.4, t

1.86 (m) a, 1.54 (m) b

29.3, t

1.80 (m) a, 1.55 (m) b

3

66.9, d

3.82 (m)

65.6, d

3.80 (m)

4

37.0, t

1.99 (m) a, 1.93 (m) b

36.2, t

1.98 (m)

5

84.1, s

6

137.0, d

6.30 (d, 8.5)

135.5, d

6.31 (d, 8.4)

7 8

131.9, d 79.8, s

6.64 (d, 8.5)

130.3, d 79.6, s

6.56 (d, 8.4)

9

144.3, s

51.2, d

1.46 (m)

10

39.3, s

36.7, s

82.3, s

11

120.8, d

5.47 (dd, 6, 1.8)

39.2, t

2.01 (m) a, 1.24 (m) b

12

42.7, t

2.31 (dd, 17, 6) a 2.06 (d, 7) b

27.8, t

1.98 (m) a, 1.46 (m) b

13

45.1, s

14

49.6, d

1.77 (dd, 11.7, 8.7)

51.5, d

15

21.9, t

1.65 (m)

20.2, t

1.53 (m)

16

29.2, t

1.97 (m) a, 1.45 (m) b

22.9, t

1.58 (m) a, 1.25 (m) b

17

57.5, d

1.35 (m)

55.8, d

1.24 (m)

18

13.3, q

0.76 (s)

11.7, q

0.87 (s)

19

26.0, q

1.11 (s)

17.2, q

0.92 (s)

20

36.4, d

1.45 (m)

35.4, d

1.51 (m)

21

19.0, d

0.95 (d, 6.5)

17.6, q

0.96 (d, 6.44)

22 23

37.1, t 25.8, t

1.46 (m), 1.08 (m) 2.05 (m), 1.91 (m)

38.4, t 124.4, d

2.17 (m), 1.80(m) 5.60 (br s)

24

126.1, d

5.09 (br t, 6.3)

139.4, d

5.60 (br s)

25

132.0, s

26

26.0, q

1.67 (s)

28.6, q

1.28 (s)

27

17.8, q

1.61 (s)

28.6, q

1.28 (s)

1

700 MHz for H NMR and 175 MHz for a

44.5, s 1.53 (m)

69.6, s

13

C NMR

Assignments and multiplicity were aided by analysis of COSY, HSQC and HMBC spectroscopic data analysis

patients in any stage is only 6 % (Howlader et al. 1975–2009). Only a small number of chemotherapeutic agents are effective for treating patients with either of these two cancers. Temsirolimus and 5-fluorouracil (5-FU) have been used clinically as chemotherapeutics to treat renal and pancreatic cancers, respectively. Nevertheless, there is a huge demand for new chemotherapeutic agents with the potential to treat renal and pancreatic cancers. To discover agents with biomedical potentials, we evaluated the cytotoxicities of the purified compounds against four human cancer cell lines, including renal cancer, pancreatic cancer and colorectal cancer cell lines. Compounds 1, 3–5 and 7 were observed to display moderate cytotoxicity against the human pancreatic cancer cell line PANC-1, and compound 6 was found to possess cytotoxicity against the human colorectal cancer cell line HCT 116 (Fig. 1).

The molecular formula of compound 1 was established as C26H38O3 based on its HRFABMS ion peak at m/ z 399.2859 [M?H]? (D -4.0 mmu), which requires 8° of unsaturation. The IR absorption band observed at 3,418 cm-1 suggested the presence of a hydroxy functionality. Two methyl singlets (dH 0.79 and 1.13) and three methyl doublets (dH 0.97, 0.98, and 1.03) in the 1H NMR spectrum of 1 (Table 1) were reminiscent of a steroidal skeleton, which was confirmed by further analyses of COSY, HSQC and HMBC spectra (Fig. 2). Four isolated spin systems based on the series of COSY cross peaks between H-1 and H-4, H-6 and H-7, H-11 and H-12, and between H-14 and H-20 of 1 depicted the typical spectroscopic features of the steroidal tetracyclic ring structure. All vicinal proton correlations for the side chain were also observed in the COSY spectra and confirmed by HMBC correlations from H3-21 to C-17, C-20, and C-22 as well as

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Fig. 1 Chemical structures of 1–7

from H3-25 and H3-26 to C-23 and C-24 (Fig. 2). Five olefinic protons, observed at dH 6.34, 6.67, 5.50, 5.22, and 5.33, were associated with the olefinic carbons at dC 135.5, 130.3, 119.3, 133.0, and 135.2, respectively, in the HSQC spectra. This accounts for 3° of unsaturation, indicating that 1 contains five rings including the steroidal tetracyclic rings. The large 1H–1H coupling constant (J = 15.2 Hz) between H-22 and H-23 suggested a trans configuration. Two oxygenated quaternary carbons at dC 78.3 (C-8) and 82.6 (C-5) in the 13C NMR spectrum indicated the presence of an endocyclic double bond between C-6 and C-7 and a peroxy group between C-5 and C-8 in 1. This resolved the remaining unsaturation. The relative configurations of 1 were established by ROESY data analysis. The ROESY correlations between

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H-3/H-1a, H-12a/H-21, H-18/H-20b, H-18/H-12b, H-19/ H-1b, H-19/H-4b, H-14a/H-17a, and H-17a/H-21 were observed, and the relative configuration of the hydroxy group at C-3 was established as b. The chemical shift of H-6 (dH 6.34) and H-7 (dH 6.67) indicated a 5a,8a-epidioxy system, not a 5b,8b-epidioxy system (Luo et al. 2006; Liu et al. 2011; Im et al. 2000). Thus, the structure of 1 was established as a norcholesterol, 5a,8a-epidioxy-24-norcholesta-6,9(11),22-trien-3b-ol (1). The molecular formula of compound 2 was also determined to be C27H40O3 based on the presence of a HRFABMS ion peak at m/z 413.3050 [M?H]? (D 0.6 mmu), which indicated 8° of unsaturation, and the IR absorption band at 3,393 cm-1 indicated the presence of a hydroxy group.

Cytotoxic 5a,8a-epidioxy sterols

Fig. 2 Key COSY and HMBC correlations of 1

Four methyl singlets (dH 0.76, 1.11, 1.67, and 1.61) and one methyl doublet (dH 0.95) in the 1H NMR spectrum of 2 suggested that it also possess a steroidal skeleton like 1. Six olefinic carbons (dC 137.0, 131.9, 144.3, 120.8, 126.1, and 132.0) observed in the 13C NMR spectrum of 2 indicated the presence of three double bonds, which suggested the presence of five rings as was found in 1. However, four olefinic protons were observed at dH 6.30, 6.64, 5.47, and 5.09 in the 1H NMR spectrum of 2, while five olefinic protons were observed in that of 1. Furthermore, both singlet methyls H3-26 (dH 1.67) and H3-27 (dH 1.61) showed HMBC correlations to an olefinic methine carbon C-24 (dC 126.1) and an olefinic quaternary carbon C-25 (dC 132.0). Thus, the double bond, found at C-22 of 1 was placed at C-24 in the side chain of 2. ROESY data revealed that the absolute configuration of each ring junction and chiral center in 2 was the same as observed in 1 (supplementary data). Accordingly, the structure of compound 2 was determined to be 5a,8aepidioxy-cholesta-6,9(11),24-trien-3b-ol. The molecular formula of compound 3 was determined to be C27H42O4 based on a HREIMS ion peak at m/z 430.3086 [M] ? (D ?0.3 mmu), indicating 7° of unsaturation, and the IR absorption band at 3,417 cm-1 indicated the presence of a hydroxy group. Four methyl singlets (dH 0.87, 0.92, 1.28, and 1.28) and one methyl doublet (dH 0.96) in the 1H NMR spectrum of 3 suggested it possessed a steroidal skeleton (Table 2). Four olefinic carbons observed at dC 135.5 (C-6), 130.3 (C-7), d 124.4 (C-23) and d 139.4 (C-24) and four associated olefinic protons at dH 6.31 (1H, d, J = 8.4 Hz, H-6), d 6.56 (1H, d, J = 8.4 Hz, H-7), and dH 5.60 (2H, br s, H-23 and H-24) indicated the presence of two 1,2-disubstituted double bonds. One of the double bonds in 3 was found to be an endocyclic double bond between C-6 and C-7 with a peroxy group between C-5 and C-8, as was found in 1 and 2. The position of the second double bond between C-23 and C-24 was established by HMBC correlations from H-26 (dH 1.28) and H-27 (dH 1.28) to C-24 and from a methylene H-22 (dH 2.17, m, 1H; 1.80, m, 1H) to C-23 and

23

C-24. The chemical shifts of olefinic protons H-23 and H-24 at dH 5.60-5.61 suggested they were in a trans configuration (Takahashi et al. 2007). In addition, H-23, H-24, H-26 and H-27 showed HMBC correlations to an oxygenated quaternary carbon observed at dC 69.6, and the position of quaternary carbon was established at C-25. Thus, the structure of compound 3 was determined to be 5a,8a-epidioxy-cholesta-6,23-dien-3b,25-diol. Four previously reported sterols, 5a,8a-epidioxy24-norcholesta-6,22-dien-3b-ol (4), 5a,8a-epidioxy-24methylcholesta-6,24(28)-dien-3b-ol (5), 5a,8a-epidioxycholesta-6,24-dien-3b-ol (6), and 5a,8a-epidioxy-24methylcholesta-6,9(11),24(28)-trien-3b-ol (7) were also isolated. Compounds 4, 5, and 7, were previously isolated from the marine organisms Ascidia nigra (a tunicate), Dendrogyra cylindrus (a coral), Thalysias juniperina (a sponge), Phallusia mammillata, and Ciona intestinalis (tunicates). Compound 6 was previously isolated from the marine tunicates Aplysia punctate and Dendrodoa grossularia (Gunatilaka et al. 1981; Guyot and Durgeat 1981; Jimenez et al. 1986). 5a,8a-Epidioxysterols have been reported to possess growth inhibiting activity against human cancer cells from lung adenocarcinoma, gastric carcinoma, promyelocytic leukemia, colon adenocarcinoma, myelogenous leukemia, nasopharyngeal carcinoma, breast adenocarcinoma, liver adenocarcinoma, and acute monocytic leukemia (Sheu et al. 2000; Gauvin et al. 2000; Kahlos et al. 1989; Takei et al. 2005; Pan et al. 2006). However, the cytotoxicities of 5a,8a-epidioxysterols against renal and pancreatic cancer cells have not been previously studied. Therefore, in the present study, we evaluated the cytotoxicities of compounds 1–7 against four human cancer cell lines, including renal cancer cells, pancreatic cancer cells, and colorectal cancer cells using the colorimetric methyl thiazolyl tetrazolium bromide (MTT) assay (Table 3). Compound 6 had a potent cytotoxic effect against the human colorectal cancer cell line (HCT 116) with an IC50 value of 2.5 lM, compounds 1 and 3 exhibited moderate cytotoxic effects against PANC-1 cancer cells with IC50 values of 33.5 and 35.0 lM, respectively. In addition, compounds 5 and 7 were found to be slightly more cytotoxic than clinically used positive controls against A-498 (a renal cancer cell line; positive control, temsirolimus), and MIA PaCa-2 and PANC-1 (both pancreatic cancer cell lines; positive control, 5-fluorouracil) cells. Interestingly, cytotoxicity against the two pancreatic cancer cell lines appeared to be dependent on the position of a double bond in the side chain of 5a,8a-epidioxysterols, whereas cytotoxicity against the renal cancer cell appeared to be independent of this double bond. Two compounds (5 and 7) with a terminal vinyl group were more cytotoxic than compounds (2 and 6) with a double bond between C-24 and C-25 to

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B. Mun et al.

Table 3 Cytotoxicity of compounds 1–7 Compound

Cancer cell lines (IC50, lM) A-498a

MIA PaCa-2b

PANC-1b

HCT 116c

1

41.6

53.0

33.5

12.0

2

45.1

69.0

62.0

27.0

3

46.4

62.0

35.0

62.4

4

30.1

42.0

39.0

[100

5

23.0

39.0

31.7

[100

6

31.8

54.5

53.0

2.5

7 Temd

24.9 33.5

20.0 –

28.5 –

12.4 –

5-FUd



41.6

40.0



DOXd







2.9

a

A-498: human renal cancer cell line

b

MIA PaCa-2, PANC-1: human pancreatic cancer cell line

c

HCT 116: human colorectal cancer cell line

d

Tem (temsirolimus), 5-FU (5-fluorouracil) and DOX (doxorubicin) were used as positive controls

renal and pancreatic cancer cells. This is the first report to be issued on the cytotoxicities of epidioxysterols against renal and pancreatic cancer cells, and thus, expands the biomedical potential of 5a,8a-epidioxysteroids. Our observations indicate that 5a,8a-epidioxysteroids with a double bond between C-24 and C-28 (a terminal vinyl group) offer potential leads for the discovery of new antipancreatic cancer agents. Acknowledgments This work was supported by the Marine Biotechnology Program, Ministry Oceans and Fisheries and by the 2012 Yeungnam University Research Grant. B. Mun, H. Kim, D. Hahn, J. Lee were in part supported by the BK21 program, Ministry of Education, Korea. Conflict of interest All the authors have no conflict of interest to declare.

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Cytotoxic 5α,8α-epidioxy sterols from the marine sponge Monanchora sp.

Three new sterols, 5α,8α-epidioxy-24-norcholesta-6,9(11),22-trien-3β-ol (1), 5α,8α-epidioxy-cholesta-6,9(11),24-trien-3β-ol (2), and 5α,8α-epidioxy-ch...
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