New marine sterols from an algal-bearing gorgonian coral Pinnigorgia sp

Steroids 115 (2016) 123–129

Contents lists available at ScienceDirect

Steroids journal homepage: www.elsevier.com/locate/steroids

New marine sterols from an algal-bearing gorgonian coral Pinnigorgia sp Yu-Chia Chang a,b,1, Nan-Fu Chen c,1, Tsong-Long Hwang d,e, Chung-Chih Tseng f,g, Tung-Ying Wu h, Bo-Rong Peng b,i, Zhi-Hong Wen a,f, Lee-Shing Fang j, Yang-Chang Wu h,k,l,m,⇑, Jyh-Horng Sheu a,f,⇑, Ping-Jyun Sung b,f,h,i,m,⇑ a

Doctoral Degree Program in Marine Biotechnology, National Sun Yat-sen University and Academia Sinica, Kaohsiung 804, Taiwan National Museum of Marine Biology and Aquarium, Pingtung 944, Taiwan Division of Neurosurgery, Department of Surgery, Kaohsiung Armed Forces General Hospital, Kaohsiung 802, Taiwan d Graduate Institute of Natural Products, School of Traditional Chinese Medicine, College of Medicine and Chinese Herbal Medicine Research Team, Healthy Aging Research Center, Chang Gung University, Taoyuan 333, Taiwan e Research Center for Industry of Human Ecology and Graduate Institute of Health Industry Technology, Chang Gung University of Science and Technology, Taoyuan 333, Taiwan f Department of Marine Biotechnology and Resources, National Sun Yat-sen University, Kaohsiung 804, Taiwan g Division of Dentistry, Zuoying Branch of Kaohsiung Armed Forces General Hospital, Kaohsiung 813, Taiwan h Chinese Medicine Research and Development Center, China Medical University Hospital, Taichung 404, Taiwan i Graduate Institute of Marine Biology, National Dong Hwa University, Pingtung 944, Taiwan j Department of Sport, Health, and Leisure, Cheng Shiu University, Kaohsiung 833, Taiwan k School of Pharmacy, College of Pharmacy, China Medical University, Taichung 404, Taiwan l Center for Molecular Medicine, China Medical University Hospital, Taichung 404, Taiwan m Graduate Institute of Natural Products, Kaohsiung Medical University, Kaohsiung 807, Taiwan b c

a r t i c l e

i n f o

Article history: Received 2 March 2016 Received in revised form 20 July 2016 Accepted 14 August 2016 Available online 21 August 2016 Keywords: Gorgonian Marine sterol Pinnigorgia HSCs

a b s t r a c t Four new marine sterols, (22E,24R)-ergosta-5,22-diene-3b,11a-diol (1), (24S)-ergosta-5-ene-3b,11a-diol (2), 5a,6a-epoxy-23-demethylgorgost-8-ene-3b,7a-diol (3), and 5a,6a-epoxy-23-demethylgorgost-8 (14)-ene-3b,7a-diol (4), along with a known metabolite, 23-demethylgorgost-7-ene-3b,5a,6b-triol (5), were isolated from an algal-bearing gorgonian coral Pinnigorgia sp., collected off the waters of Taiwan. The structures of these sterols were elucidated on the basis of spectroscopic methods. Sterols 1–5 were tested for in vitro cytotoxicity in hepatic stellate cells (HSCs). Proliferation of HSCs plays a key role in the pathogenesis of liver fibrosis. Ó 2016 Elsevier Inc. All rights reserved.

1. Introduction Gorgonian corals have been well-recognized as marine organisms containing various steroid analogues [1] and the ergosteroland gorgosterol-type metabolites isolated from algal-bearing gorgonian corals were suggested be originally synthesized by the symbiotic zooxanthellae and not by the host corals [2,3]. In continuation of our research into new substances from marine

⇑ Corresponding authors at: Chinese Medicine Research and Development Center, China Medical University Hospital, Taichung 404, Taiwan. Fax: +886 4 220 60248 (Y.-C.Wu). Department of Marine Biotechnology and Resources, National Sun Yatsen University, Kaohsiung 804, Taiwan. Fax: +886 7 525 5020 (J.-H.Sheu). National Museum of Marine Biology and Aquarium, Pingtung 944, Taiwan. Fax: +886 8 882 5087 (P.-J.Sung). E-mail addresses: [email protected] (Y.-C. Wu), [email protected]. tw (J.-H. Sheu), [email protected] (P.-J. Sung). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.steroids.2016.08.018 0039-128X/Ó 2016 Elsevier Inc. All rights reserved.

invertebrates collected off the waters of Taiwan, a series of novel 24-methyl sterols derivatives, including pinnigorgiols A–C [4], pinnisterols A–C [5], 11-acetoxy-24S-methyl-3b,5a,6a-trihydroxy9,11-secocholest-7-en-9-one, and 5b,6b-epoxy-(22E,24R)-ergosta8,22-diene-3b,7b-diol [6], along with two plant-orignated metabolites, pubinernoid and apo-90 -fucoxanthinone [7], have been isolated from an algal-bearing gorgonian coral identified as Pinnigorgia sp. (phylum Cinidaria, class Anthozoa, subclass Octocorallia, order Alcyonacea, family Gorgoniidae). Recently, chemical examination of this interesting organism resulted in the isolation of five marine sterols, including two new ergosterols, (22E,24R)-ergosta5,22-diene-3b,11a-diol (1), (24S)-ergosta-5-ene-3b,11a-diol (2), two new 23-demethylgorgosterols, 5a,6a-epoxy-23-demethylgorgost-8-ene-3b,7a-diol (3), 5a,6a-epoxy-23-demethylgorgost-8 (14)-ene-3b,7a-diol (4), and a known metabolite, 23-demethylgorgost-7-ene-3b,5a,6b-triol (5) [8] (Fig. 1). The structures of new sterols 1–4 were elucidated by spectroscopic methods and by comparison of their NMR features with those of related sterol

124

Y.-C. Chang et al. / Steroids 115 (2016) 123–129

2 3

HO

12

19

11 9 H

1 5

4

R

18

HO

10 H 8 7

17

R H

H

15

1: R =

20

23

HO

OH

O H

24

H

H HO

28 22

H

13 16 14

6

21

R H

OH

O H

29

27 3: R =

25

4: R =

H

26

2: R =

H

7: R =

9: R =

H

H 6: R =

H

H

H

HO

HO OH

OH

H

H

5

H

H O O

8

Fig. 1. The structures of (22E,24R)-ergosta-5,22-diene-3b,11a-diol (1), (24S)-ergosta-5-ene-3b,11a-diol (2), 5a,6a-epoxy-23-demethylgorgost-8-ene-3b,7a-diol (3), 5a,6aepoxy-23-demethylgorgost-8(14)-ene-3b,7a-diol (4), 23-demethylgorgost-7-ene-3b,5a,6b-triol (5), stigmasta-5,22E-diene-3b,11a-diol (6), 5a,6a-epoxy-24R-ethylcholest8-ene-3b,7a-diol (7), (22R,23R,24R)-5a,8a-epidioxy-22,23-methylene-24-methyl-6,9(11)-dien-3b-ol (8), and 5a,6a-epoxy-24R-ethylcholest-8(14)-ene-3b,7a-diol (9).

analogues. We report herein the isolation, structure determination, and bioactivity of sterols 1–5.

used in this study (NMMBA-TW-GC-2012-130) and the specimen that was used in a previous study cited in Ref. [6] (NMMBA-TWGC-2010-099) are identical.

2. Experimental 2.3. Extraction and isolation 2.1. General procedures Optical rotations were measured on a Jasco P-1010 digital polarimeter. Infrared spectra were recorded on a Jasco FT/IR4100 spectrometer; peaks are reported in cm1. The NMR spectra were recorded on a Varian Mercury Plus 400 spectrometer, using the residual CHCl3 signal (dH 7.26 ppm) as an internal standard for 1H NMR and CDCl3 (dC 77.1 ppm) for 13C NMR; coupling constants (J) are given in Hz. ESIMS and HRESIMS were recorded using a Bruker 7 Tesla solariX FTMS system. Column chromatography was performed on silica gel (230–400 mesh, Merck). TLC was carried out on precoated Kieselgel 60 F254 (0.25 mm, Merck); Spots were visualized by spraying with 10% H2SO4 solution followed by heating. Normal-phase HPLC (NP-HPLC) was performed using a system comprised of a Hitachi L-7110 pump and a Rheodyne 7725 injection port. A normal-phase column (Supelco Ascentis Si Cat #:581515-U, 25 cm  21.2 mm, 5 lm, Sigma-Aldrich) was used for NP-HPLC. Reversed-phase HPLC (RP-HPLC) was performed using a system comprised of a Hitachi L-2130 pump, a Hitachi L2455 photodiode array detector, and a Rheodyne 7725 injection port. A reverse phase column (LunaÒ 5 lm C18(2) 100 Å, AXIA Packed, 25 cm  21.2 mm) was used for RP-HPLC. 2.2. Animal material Specimen of the gorgonian corals Pinnigorgia sp. was collected by hand using scuba diving off the coast of Green Island, Taiwan in August 2012 and stored in a freezer until extraction. A voucher specimen (NMMBA-TW-GC-2012-130) was deposited in the National Museum of Marine Biology and Aquarium, Taiwan. This organism was identified by comparison with previous descriptions [9]. Since collected from the same colony, the specimen that was

Sliced bodies of Pinnigorgia sp. (wet weight 1.98 kg; dry weight 0.86 kg) were extracted with EtOAc (1 L  6) at room temperature. The EtOAc extract (84.9 g) was partitioned between MeOH and nhexane (500 mL/500 mL  4). The MeOH layer (12.6 g) was separated on Sephadex LH-20 and eluted using a mixture of CH2Cl2 and MeOH (1:1) to yield seven subfractions A–G. Fraction F was separated by silica gel and eluted using n-hexane/acetone (stepwise, 1:1–pure acetone) to afford eight subfractions F1–F8. Fraction F2 was purified by silica gel and eluted using n-hexane/ acetone (stepwise, 9:1–pure acetone) to yield ten subfractions F2A–F2J. Fraction F2D was purified by NP-HPLC using a mixture of n-hexane/EtOAc (3:1) to yield seventeen subfractions F2D1– F2D17. Fractions F2D14 and F2D15 were purified by RP-HPLC, using a mixture of MeOH/H2O (95:5) to afford 2 (2.7 mg) and 1 (8.9 mg), respectively. Fraction F2E was purified by NP-HPLC using a mixture of n-hexane/EtOAc (3:1) to yield eight subfractions F2E1–F2E8. Fraction F2E8 was repurified by NP-HPLC using a mixture of n-hexane/EtOAc (1:1), followed by RP-HPLC using a mixture of MeOH/H2O (85:15) to afford 4 (1.8 mg) and 3 (1.5 mg), respectively. Fraction F4 was purified by NP-HPLC using a mixture of nhexane/acetone (2:1) to yield nine subfractions F4A–F4I. Then, fraction F4I was purified by NP-HPLC using a mixture of n-hexane/acetone (2:1) to afford nine subfractions F4I1–F4I9. Fraction F4I6 was purified by NP-HPLC, using a mixture of n-hexane/acetone (2:1) to yield 5 (1.8 mg). 2.3.1. (22E,24R)-Ergosta-5,22-diene-3b,11a-diol (1) White amorphous powder: mp 153–155 °C; ½a27 D 252 (c 0.9, CHCl3); IR (neat) mmax 3391 cm1; 1H and 13C NMR data, see Table 1; ESIMS, m/z 437 [M + Na]+; HRESIMS, m/z 437.33888 (calcd for C28H46O2Na, 437.33900).

125

Y.-C. Chang et al. / Steroids 115 (2016) 123–129 Table 1 H and 13C NMR data, 1H–1H COSY, and HMBC correlations for sterol 1.

1

C/H 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 a b c d e

dHa

d Cb c

2.56 1.79 3.53 2.28

ddd (14.0, 4.0, 4.0) ; 1.16 m m; 1.53 m m m

5.40 1.95 1.39 0.99

d (4.0) m; 1.58 m dd (10.4, 4.4) d (10.4)

1 d

4.05 m 2.24 m; 1.23 m

39.1 (CH2) 31.8 (CH2) 71.8 (CH) 42.8 (CH2) 141.1 (C) 121.5 (CH) 31.9 (CH2) 31.7 (CH) 56.9 (CH) 38.1 (C) 69.2 (CH) 51.3 (CH2)

1.19 1.56 1.69 1.09 0.71 1.16 2.02 1.03 5.14 5.21 1.83 1.47 0.83 0.81 0.90

42.9 (C) 55.8 (CH) 24.1 (CH2) 28.7 (CH2) 56.0 (CH) 13.0 (CH3) 19.1 (CH3) 40.1 (CH) 20.9 (CH3) 135.4 (CH) 132.0 (CH) 42.8 (CH) 33.1 (CH) 19.9 (CH3) 19.6 (CH3) 17.6 (CH3)

m m; 1.06 m m; 1.29 m m s s m d (6.4) dd (15.2, 7.6) dd (15.2, 7.2) m m d (6.8) d (6.4) d (6.8)

H–1H COSY

HMBC (H ? C)

H2-2 H2-1, H-3 H2-2, H2-4 H-3

C-2, -3, -5, -10, -19 C-1, -10 n.o.e C-2, -3, -5, -6, -10

H2-7 H-6, H-8 H2-7, H-9, H-14 H-8, H-11

C-4, C-5, C-7, C-1,

H-9, H2-12 H-11

n.o. C-7, -9, -11, -13, -14, -17, -18, -20

H-8, H2-15 H-14, H2-16 H2-15, H-17 H2-16, H-20

C-9, -16 C-8, -13, -14, -16, -17 C-13, -14, -15, -17 C-13, -15, -18, -20 C-12, -13, -14, -17 C-1, -5, -9, -10 C-17, -22, -23 C-17, -20, -22 C-17, -20, -21, -23 C-20, -22, -25, -28 C-22, -23, -25, -26, -27, -28 C-23, -24, -26, -27, -28 C-24, -25, -27 C-24, -25, -26 C-23, -24, -25

H-17, H-20 H-20, H-22, H-23, H-24, H-25 H-25 H-24

H3-21, H-22 H-23 H-24 H-25, H3-28 H3-26, H3-27

-7, -6, -9, -7,

-10 -8, -9, -14 -10, -14 -8, -10, -11, -19

Spectra recorded at 400 MHz in CDCl3 at 25 °C. Spectra recorded at 100 MHz in CDCl3 at 25 °C. J values (in Hz) in parentheses. The values are downfield in ppm from TMS. Multiplicity deduced from DEPT and HMQC spectra and indicated by the usual symbols. n.o. = not observed.

Table 2 H and 13C NMR data, 1H–1H COSY, and HMBC correlations for sterol 2.

1

a b c d e

H–1H COSY

C/H

dH a

d Cb

1

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

2.56 ddd (14.0, 3.2, 3.2)c; 1.13 m 1.81 m; 1.50 m 3.53 m 2.31–2.23 m

39.1 (CH2)d 31.8 (CH2) 71.8 (CH) 42.7 (CH2) 141.1 (C) 121.5 (CH) 31.9 (CH2) 31.7 (CH) 56.9 (CH) 38.1 (C) 69.2 (CH) 51.4 (CH2) 43.0 (C) 55.8 (CH) 24.2 (CH2) 28.3 (CH2) 55.9 (CH) 12.8 (CH3) 19.0 (CH3) 36.1 (CH) 18.8 (CH3) 33.5 (CH2) 30.5 (CH2) 39.0 (CH) 31.4 (CH) 17.6 (CH3) 20.5 (CH3) 15.4 (CH3)

H2-2 H2-1, H-3 H2-2, H2-4 H-3

C-19 C-1 C-2 C-3, -5, -6, -10

H2-7 H-6, H-8 H2-7, H-9, H-14 H-8, H-11

C-4, -7, -10 C-5, -6, -8, -9 C-14 C-1, -7, -8, -10, -11, -19

H-9, H2-12 H-11

n.o.e C-9, -11, -13, -14, -18

H-8, H2-15 H-14, H2-16 H2-15, H-17 H2-16, H-20

C-7, -8, -13, -16, -18 C-14 n.o. C-15 C-12, -13, -14, -17 C-1, -5, -9, -10 C-22 C-17, -20, -22 C-17, -20, -24 C-20, -22, -24 C-22 C-24, -26, -27, -28 C-24, -25, -27 C-24, -25, -26 C-23, -24

5.41 1.97 1.43 1.00

d (6.4) dddd (18.4, 6.4, 6.4, 2.0); 1.56 m m br d (10.4)

4.04 m 2.30 m; 1.21 m 1.16 1.60 1.88 1.10 0.70 1.16 1.34 0.94 1.39 1.37 1.21 1.60 0.78 0.85 0.77

m m; 1.07 m; 1.30 m s s m d (6.4) m; 0.94 m; 0.94 m m d (6.8) d (7.2) d (6.8)

m m

m m

H-17, H3-21, H2-22 H-20 H-20, H2-23 H2-22, H-24 H-23, H-25, H3-28 H-24, H3-26, H3-27 H-25 H-25 H-24

Spectra recorded at 400 MHz in CDCl3 at 25 °C. Spectra recorded at 100 MHz in CDCl3 at 25 °C. J values (in Hz) in parentheses. The values are downfield in ppm from TMS. Multiplicity deduced from DEPT and HMQC spectra and indicated by the usual symbols. n.o. = not observed.

HMBC (H ? C)

126

Y.-C. Chang et al. / Steroids 115 (2016) 123–129

2.3.2. (24S)-Ergosta-5-ene-3b,11a-diol (2)

3. Results and discussion

White amorphous powder: mp 193–195 °C; ½a25 D 28 (c 0.1, CHCl3); IR (neat) mmax 3386 cm1; 1H and 13C NMR data, see Table 2; ESIMS, m/z 439 [M + Na]+; HRESIMS, m/z 439.35459 (calcd for C28H48O2Na, 439.35465).

The new metabolite (22E,24R)-ergosta-5,22-diene-3b,11a-diol (1) was isolated as a white amorphous powder, and its molecular formula was established as C28H46O2 (6° of unsaturation) from a sodium adduct at m/z 437 in the ESIMS and further supported by the HRESIMS at m/z 437.33888 (calcd for C28H46O2Na, 437.33900). The 13C and DEPT spectroscopic data of 1 showed that this compound has 28 carbons (Table 1), including six methyls, seven sp3 methylenes, nine sp3 methines (including two oxymethines), two sp3 quaternary carbons, three sp2 methines, and one sp2 quaternary carbon. The IR spectrum of 1 revealed the presence of hydroxy (mmax 3391 cm1) groups. The 1H NMR spectrum (Table 1) showed signals due to two tertiary methyl groups (dH 0.71, 3H, s, H3-18; 1.16, 3H, s, H3-19), a 24methyl-D22-sterol side chain (dH 0.81, 3H, d, J = 6.4 Hz, H3-27; 0.83, 3H, d, J = 6.8 Hz, H3-26; 0.90, 3H, d, J = 6.8 Hz, H3-28; 1.03, 3H, d, J = 6.4 Hz, H3-21; 5.14, 1H, dd, J = 15.2, 7.6 Hz, H-22; 5.21, 1H, dd, J = 15.2, 7.2 Hz, H-23), a trisubstituted carbon–carbon double bond (dH 5.40, 1H, d, J = 4.0 Hz, H-6), and two methine protons on carbons bearing a hydroxy group (dH 3.53, 1H, m, H-3; 4.05, 1H, m, H-11). 1H NMR coupling information in the 1H–1H COSY spectrum of 1 enabled identification of H2-1/H2-2/H-3/H2-4, H-6/H2-7/H-8/ H-9/H-11/H2-12, H-8/H-14/H2-15/H2-16/H-17/H-20/H-22/H-23/ H-24/H-25/H3-26, H-20/H3-21, H-25/H3-27, and H-24/H3-28 (Table 1). These data, together with the key HMBC correlations between protons and quaternary carbons, such as H2-1, H2-4, H27, H3-19/C-5; H2-1, H2-2, H2-4, H-6, H-8, H-9, H3-19/C-10; and H2-12, H2-15, H2-16, H-17, H3-18/C-13 (Table 1), permitted the elucidation of the main carbon skeleton of 1. The relative configuration of 1 was elucidated by comparison of 13 C data with those of a known, stigmasta-5,22E-diene-3b,11a-diol

2.3.3. 5a,6a-Epoxy-23-demethylgorgost-8-ene-3b,7a-diol (3) White amorphous powder: mp 178–180 °C; ½a25 8 (c 0.5, D CHCl3); IR (neat) mmax 3426 cm1; 1H and 13C NMR data, see Table 3; ESIMS, m/z 465 [M + Na]+; HRESIMS, m/z 465.33400 (calcd for C29H46O3Na, 465.33392). 2.3.4. 5a,6a-Epoxy-23-demethylgorgost-8(14)-ene-3b,7a-diol (4) White amorphous powder: mp 167–169 °C; ½a25 3 (c 0.6, D CHCl3); IR (neat) mmax 3358 cm1; 1H and 13C NMR data, see Table 4; ESIMS, m/z 465 [M + Na]+; HRESIMS, m/z 465.33399 (calcd for C29H46O3Na, 465.33392). 2.3.5. 23-Demethylgorgost-7-ene-3b,5a,6b-triol (5) White amorphous powder: mp 261–263 °C (Ref. [8], mp 229– 232 °C, colorless needles); ½a25 D 238 (c 0.4, CHCl3) (Ref. [8], [a]D 45 (c 2.18, pyridine)); IR (neat) mmax 3442 cm1; 1H (400 MHz, CDCl3) and 13C (100 MHz, CDCl3) NMR data were found to be in full agreement with those reported previously [8]; ESIMS, m/z 467 [M + Na]+. 2.4. Anti-hepatofibric assay The anti-hepatofibric effects of tested sterols 1–5 were assayed using a WST-1 assay method. Anti-hepatofibric assays were carried out according to the procedures described previously [10].

Table 3 H and 13C NMR data, 1H–1H COSY, and HMBC correlations for sterol 3.

1

C/H 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 29 7-OH a b c d e

dHa 1.78 1.99 3.96 2.18

d Cb m; 1.62 m m; 1.62 m m m; 1.47 m

3.32 d (2.8)c 4.23 dd (10.0, 2.8)

2.16 m; 1.30 m 1.99 m; 1.40 m 2.18 2.05 2.18 1.32 0.54 1.14 0.79 0.93 0.56 0.30 0.52 1.65 0.88 0.86 0.91 0.12 1.77

m m; 1.32 m m; 1.47 m m s s m d (6.8) m m m m d (6.0) d (6.8) d (6.8) m d (10.0)

1

HMBC (H ? C)

H2-2 H2-1, H-3 H2-2, H2-4 H-3

C-3, -5, -10, -19 C-1, -4, -10 n.o.e C-2, -3, -5, -6, -10

H-7 H-6, 7-OH

C-7, -8 n.o.

H2-12 H2-11

n.o. C-9, -11, -14

H2-15 H-14, H2-16 H2-15, H-17 H2-16, H-20

C-13, -15 C-14 C-13, -15 C-12, -13 C-12, -13, -14, -17 C-1, -5, -9, -10 n.o. C-17, -20, -22 C-17 C-24 n.o. C-24 C-24, -25, -27 C-24, -25, -26 C-23, -24 C-20, -22, -24 C-7

H–1H COSY

d

30.2 (CH2) 30.9 (CH2) 68.6 (CH) 39.2 (CH2) 65.6 (C) 62.6 (CH) 67.1 (CH) 126.9 (C) 134.6 (C) 38.0 (C) 23.7 (CH2) 35.7 (CH2) 42.5 (C) 49.3 (CH) 23.9 (CH2) 29.1 (CH2) 55.1 (CH) 10.9 (CH3) 22.8 (CH3) 40.5 (CH) 19.2 (CH3) 24.0 (CH) 25.2 (CH) 45.0 (CH) 32.8 (CH) 20.7 (CH3) 18.6 (CH3) 15.8 (CH3) 10.4 (CH2)

H-17, H-20 H-20, H-22, H-23, H-24, H-25, H-25, H-24 H-22, H-7

H3-21, H-22 H-23, H2-29 H-24, H2-29 H-25, H3-28 H3-26, H3-27 H3-27 H3-26 H-23

Spectra recorded at 400 MHz in CDCl3 at 25 °C. Spectra recorded at 100 MHz in CDCl3 at 25 °C. J values (in Hz) in parentheses. The values are downfield in ppm from TMS. Multiplicity deduced from DEPT and HMQC spectra and indicated by the usual symbols. n.o. = not observed.

127

Y.-C. Chang et al. / Steroids 115 (2016) 123–129 Table 4 H (400 MHz, CDCl3) and

1

C/H 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 29 7-OH a b c d e

13

C (100 MHz, CDCl3) NMR data and 1H–1H COSY and HMBC correlations for sterol 4.

dHa 1.67 1.94 3.92 2.13

dCb m; 1.43 m m; 1.57 m m dd (11.6, 8.8)c; 1.40 m

3.15 d (3.6) 4.44 dd (9.6, 3.6) 2.34 m 1.51–1.37 m 1.93 m; 1.18 dd (13.6, 4.0)

2.64 2.20 1.33 0.82 0.86 0.92 0.92 0.57 0.34 0.54 1.65 0.89 0.86 0.91 0.13 1.78

m; 2.31 m m; 1.47 m m s s m d (6.4) m m m m d (6.8) d (6.8) d (7.6) m d (9.6)

1 d

32.2 (CH2) 31.1 (CH2) 68.7 (CH) 39.6 (CH2) 67.8 (C) 61.3 (CH 65.1 (CH) 125.1 (C) 38.7 (CH) 35.8 (C) 19.0 (CH2) 36.6 (CH2) 43.3 (C) 152.6 (C) 25.3 (CH2) 27.3 (CH2) 58.2 (CH) 17.7 (CH3) 16.5 (CH3) 39.2 (CH) 19.2 (CH3) 24.0 (CH) 25.1 (CH) 44.8 (CH) 32.8 (CH) 20.7 (CH3) 18.5 CH3) 15.7 (CH3) 10.6 (CH2)

H–1H COSY

HMBC (H ? C)

H2-2 H2-1, H-3 H2-2, H2-4 H-3

C-2, -3, -5, -10 C-1, -3 n.o.e C-2, -3, -5, -6

H-7 H-6, 7-OH

C-7, -8 n.o.

H2-11

C-8

H-9, H2-12 H2-11

C-10 C-11

H2-16 H2-15, H-17 H2-16, H-20

C-8, -14 C-13, -15, -17 n.o. C-12, -13, -14, -17 C-1, -5, -9, -10 C-13, -17, -21, -22, -23 C-17, -20, -22 n.o. n.o. C-22 C-24 C-24, -25, -27 C-24, -25, -26 C-23, -24, -25 C-24 C-7

H-17, H-20 H-20, H-22, H-23, H-24, H-25, H-25, H-24 H-22, H-7

H3-21, H-22 H-23, H2-29 H-24, H2-29 H-25, H3-28 H3-26, H3-27 H3-27 H3-26 H-23

Spectra recorded at 400 MHz in CDCl3 at 25 °C. Spectra recorded at 100 MHz in CDCl3 at 25 °C. J values (in Hz) in parentheses. The values are downfield in ppm from TMS. Multiplicity deduced from DEPT and HMQC spectra and indicated by the usual symbols. n.o. = not observed.

(6) (Fig. 1), isolated from the resinous exudates of Commiphora mukul [11]. The relative stereochemistry at C-3 (dC 71.8), C-8 (dC 31.7), C-9 (dC 56.9), C-10 (dC 38.1), C-11 (dC 69.2), C-13 (dC 42.9), C-14 (dC 55.8), C-17 (dC 56.0), and C-20 (dC 40.1) in 1 were found to be the same as those of 6 (C-3, dC 72.0; C-8, dC 31.9; C-9, dC 57.0; C-10, dC 38.3; C-11, dC 69.4; C-13, dC 43.0; C-14, dC 56.1; C17, dC 55.9; and C-20, dC 40.6). A large coupling constant observed between H-22 and H-23 (J = 15.2 Hz) supported a trans relationship between H-22 and H-23. A stereogenic center (C-24) was identified in the side chain. The configuration at C-24 was suggested to be R on the basis of the 13C NMR chemical shift of C-28 (dC 17.6). It was reported that the 13C NMR value of C-28 resonates at dC 17.68 ppm in the 24R epimer of a known sterol, (22E,24R)-24methylcholesta-5,22-dien-3b-ol, with the same chain, and the 24S epimer, (22E,24S)-24-methylcholesta-5,22-dien-3b-ol, has a relative 0.4 ppm downfield chemical shift (Fig. 2) [12]. (24S)-Ergosta-5-ene-3b,11a-diol (2) was isolated as a white amorphous powder, and its molecular formula was established as C28H48O2 (6° of unsaturation) by HRESIMS at m/z 439.35459 (calcd for C28H48O2Na, 439.35465). The IR spectrum of 2 indicated the presence of hydroxy (3386 cm1) groups. The whole series of spectroscopic data obtained from 1D and 2D NMR experiments (Table 2) clearly indicated that sterol 2 had the same core structure as that of sterol 1, the differences being limited to the absence in 2 of the carbon–carbon double bond between C-22/23. The 1H and 13 C NMR data assignments of 2 were compared with the values of 1. The HMBC correlations observed fully supported the locations of the functional groups, and hence sterol 2 was assigned as structure 2, with the same configurations as sterol 1 in the core rings AD; the chiral carbons C-3, C-8, C-9, C-10, C-11, C-13, C-14, and

C-17 of 2 were identical to those of 1, and the 1H and 13C NMR chemical shifts and proton coupling constants were also in accord. The configuration of stereogenic center at C-24 was assigned as S on the basis of the 13C NMR chemical shifts of C-25 (dC 31.4), C-26 (dC 17.6), and C-27 (dC 20.5). It was reported that the 13C NMR values of C-25, C-26, and C-27 resonates at dC 31.54, 17.68, and 20.56 ppm, in a 24S epimer of a known sterol, (24S)-24-methylcholest-5-en-3b-ol, with the same side chain, and the 13C NMR values of C-25, C-26, and C-27 in a 24R epimer, (24R)-24methylcholest-5-en-3b-ol, were appeared at dC 32.49, 20.26, and 18.32 ppm (Fig. 3) [12]. Sterol 3 (5a,6a-epoxy-23-demethylgorgost-8-ene-3b,7a-diol) was isolated as a white amorphous powder. Its HRESIMS displayed a signal at m/z 465.33400 [M + Na]+ corresponding to the molecular formula C29H46O3 (calcd for C29H46O3Na, 465.33392), with seven degrees of unsaturation. The IR spectrum of 3 showed a broad band for OH group at 3426 cm1. The 1H NMR spectrum (Table 3) exhibited the presence of two methyl singlets (dH 0.54, H3-18; 1.14, H3-19), four methyl doublets (dH 0.86, J = 6.8 Hz, H327; 0.88, J = 6.0 Hz, H3-26; 0.91, J = 6.8 Hz, H3-28; 0.93, J = 6.8 Hz, H3-21), and three oxymethine protons (dH 3.32, d, J = 2.8 Hz, H-6; 3.96, m, H-3; 4.23, dd, J = 10.0, 2.8 Hz, H-7). Four protons at dH 0.12 (2H, m, H2-29), 0.30 (1H, m, H-23), and 0.56 (1H, m, H-22), indicating the presence of a cyclopropyl moiety in 3. The 13C NMR spectrum resolved 29 C-atom signals (Table 3), which were assigned by HMQC correlation as six methyls, eight sp3 methylenes, ten sp3 methines (three oxygenated at dC 62.6, 67.1, and 68.6), three sp3 quaternary carbons (one oxygenated at dC 65.6), as well as two quaternary sp2 carbons.

128

Y.-C. Chang et al. / Steroids 115 (2016) 123–129

1

28 (δ C 17.6)

28 (δ C 17.68)

28 ( δ C 18.08)

24 (δ C 42.8) 27 (δ C 19.6) 25 ( δ C 33.1)

24 (δ C 42.90) 27 (δ C 19.69) 25 ( δ C 33.16)

24 ( δ C 43.12) 27 ( δ C 20.19) 25 (δ C 33.28)

26 ( δ C 19.9)

A

B

26 ( δ C 20.02)

26 ( δ C 19.69)

Fig. 2. The 13C NMR chemical shifts of the side-chain of (22E,24R)-ergosta-5,22-diene-3b,11a-diol (1), (22E,24R)-24-methylcholesta-5,22-dien-3b-ol (A) and (22E,24S)-24methylcholesta-5,22-dien-3b-ol (B) [12].

2 Fig. 3. The (B) [12].

13

28 (δ C 15.4)

28 (δ C 15.51)

28 ( δ C 15.44)

24 (δ C 39.0) 27 (δ C 20.5) 25 ( δ C 31.4)

24 (δ C 39.17) 27 (δ C 20.56) 25 ( δ C 31.54)

24 ( δ C 38.92) 27 ( δ C 18.32) 25 (δ C 32.49)

26 ( δ C 17.6)

A

B

26 ( δ C 17.68)

26 ( δ C 20.26)

C NMR chemical shifts of the side-chain of (24S)-ergost-5-ene-3b,11a-diol (2), (24S)-24-methylcholest-5-en-3b-ol (A) and (24R)-24-methylcholest-5-en-3b-ol

Compound Basal 1 2 3 4 5

HSC-T6 cell viability (% of basal) 100 % 101.19 % 93.85 % 96.20 % 107.99 % 57.59 %

Fig. 4. Sterols 1–5 decreased viability of HSC-T6 in 10 lM for 24 h. Cells were treated with DMSO (control). Cytotoxicity assay was monitored spectrophotometrically at 450 nm. Quantitative data are expressed as the mean ± S.E.M. (n = 3–4). ***p < 0.001 compared to basal.

From the 1H–1H COSY spectrum of 3 (Table 3), it was possible to establish the separate system that maps out the proton sequences from H2-1/H2-2/H-3/H2-4, H-6/H-7, H2-11/H2-12, H-14/H2-15/H216/H-17/H-20/H-22/H-23/H-24/H-25/H3-26, H-20/H3-21, H-22/ H2-29/H-23, H-25/H3-27, and H-24/H3-28 (Table 3). These data, together with the key HMBC correlations between protons and quaternary carbons, such as H2-1, H2-4, H3-19/C-5; H-6/C-8; H212, H3-19/C-9; H2-1, H2-2, H2-4, H3-19/C-10, and H-14, H2-16, H17, H3-18/C-13, permitted the elucidation of the main carbon skeleton of 3 (Table 3). The ring junction C-18 and C-19 methyls were positioned at C-13 and C-10 from the HMBC correlations between H3-18/C-12, -13, -14, -17 and H3-19/C-1, -5, -9, -10. The oxymethine units at dC 68.6 and 67.1 correlated to the methine protons at dH 3.96 and 4.23 in the HMQC spectrum, proving the attachments of hydroxy group at C-3 and C-7, respectively. The presence of a trisubstituted epoxy group was established from the signals of an oxymethine at dC 62.6 (CH-6) and 65.6 (C-5) and further confirmed by the proton signal at dH 3.32 (1H, d, J = 2.8 Hz, H-6). By comparison of the 1H and 13C NMR chemical shifts of 3 with those of a known sterol, 5a,6a-epoxy-24R-ethylcholest-8-ene3b,7a-diol (7) (Fig. 1) from the Colombian marine sponge Polymastia tenax [13], sterol 3 possessed the same configuration as sterol 7 in the core rings AD; the chiral carbons C-3, C-5, C-6, C-7, C-10, C13, C-14, and C-17 of 3 were identical to those of 7, and the 1H and 13 C NMR chemical shifts and proton coupling constants were also

in accord. In addition, by comparison of the 1H and 13C NMR chemical shifts of Me-21, Me-26, Me-27, and Me-28 with those of a known 23-demethylgorgosterol derivative, (22R,23R,24R)-5a,8aepidioxy-22,23-methylene-24-methylcholest-6,9(11)-dien-3b-ol (8) (Fig. 1) from the soft coral Sinularia gaweli [14], it was suggested that the stereochemistry of 3 at the side chain should be assigned as 22R, 23R, and 24R [15]. 5a,6a-Epoxy-23-demethylgorgost-8(14)-ene-3b,7a-diol (4) was isolated as a white amorphous powder. The molecular formula of 4 was established as C29H46O3 by HRESIMS at m/z 465.33399 (calcd for C29H46O3Na, 465.33392) and indicated that sterol 4 must be an isomer of sterol 3. Comparison of the NMR data of 4 with those of 3 (see Tables 4 and 3) showed that both compounds possessed the same side chain and a similar sterol skeleton nucleus, but differed in the location of the fully substituted carbon–carbon double bond. The whole series of spectroscopic data obtained from 1D and 2D NMR experiments (Table 4) clearly indicated that sterol 4 had the same core structure as a known sterol, 5a,6a-epoxy-24Rdemethylgorgost-8(14)-ene-3b,7a-diol (9) [13]. Consequently, the structure of 4 was determined as 5a,6a-epoxy-23-demethylgorgost-8(14)-ene-3b, 7a-diol. Sterol 5 was identified as 23-demethylgorgost-7-ene-3b,5a,6btriol, which has been previously isoalted from an Andaman Sea soft coral Sinularia sp [8]. To the best of our knowledge, it is the first time to obtain this natural compound with a pure form.

Y.-C. Chang et al. / Steroids 115 (2016) 123–129

In the cytotoxicity testing, sterol 5 was found to show low cytotxicity towards the HSC-T6 cells at a concentration at 10 lM (inhibition rate 57.59%) after 24 h testing and sterols 1–4 were not active (Fig. 4). Acknowledgments This research was supported by grants from the National Museum of Marine Biology and Aquarium; the National Sun Yatsen University; the National Dong Hwa University; the Ministry of Science and Technology (Grant Nos. NSC 103-2911-I-002-303; MOST 104-2911-I-002-302, 103-2325-B-039-008, 103-2325-B039-007-CC1, 103-2325-B-291-001, 104-2325-B-291-001, 1042320 -B-291-001-MY3, and NSC 101-2320-B-291-001-MY3); the National Health Research Institutes (NHRI-EX103-10241BI); and in part by a grant from the Chinese Medicine Research Center, China Medical University (Ministry of Education, Aim for the Top University Plan), Taiwan, awarded to Yang-Chang Wu, Jyh-Horng Sheu, and Ping-Jyun Sung. References [1] J.W. Blunt, B.R. Copp, R.A. Keyzers, M.H.G. Munro, M.R. Prinsep, Marine natural products, Nat. Prod. Rep. 32 (2015) 116–211. and literature cited in previous reviews. [2] N.W. Withers, W.C.M.C. Kokke, W. Fenical, C. Djerassi, Sterol patterns of cultured zooxanthellae isolated from marine invertebrates: synthesis of gorgosterol and 23-desmethylgorgosterol by aposymbiotic algae, Proc. Natl. Acad. Sci. USA 79 (1982) 3764–3768. [3] W.C.M.C. Kokke, S. Epstein, S.A. Look, G.H. Rau, W. Fenical, C. Djerassi, On the origin of terpenes in symbiotic associations between marine invertebrates and algae (zooxanthellae), J. Biol. Chem. 259 (1984) 8168–8173. [4] Y.C. Chang, L.M. Kuo, J.H. Su, T.L. Hwang, Y.H. Kuo, C.S. Lin, Y.C. Wu, J.H. Sheu, P. J. Sung, Pinnigorgiols A-C, 9,11-secosterols with a rare ring arrangement from a gorgonian coral Pinnigorgia sp, Tetrahedron 72 (2016) 999–1004.

129

[5] Y.C. Chang, L.M. Kuo, T.L. Hwang, J. Yeh, Z.H. Wen, L.S. Fang, Y.C. Wu, C.S. Lin, J. H. Sheu, P.J. Sung, Pinnisterols A-C, new 9,11-secosterols from a gorgonian Pinnigorgia sp, Mar. Drugs 14 (2016) 12. [6] Y.D. Su, C.H. Cheng, Z.H. Wen, Y.C. Wu, P.J. Sung, New anti-inflammatory sterols from a gorgonian Pinnigorgia sp, Bioorg. Med. Chem. Lett. 26 (2016) 3060–3063. [7] H.H. Chang, Y.C. Chang, W.F. Chen, T.L. Hwang, L.S. Fang, Z.H. Wen, Y.H. Chen, Y. C. Wu, P.J. Sung, Pubinernoid A and apo-90 -fucoxanthinone, secondary metabolites from a gorgonian coral Pinnigorgia sp, Nat. Prod. Commun. 11 (2016) 707–708. [8] M. Kobayashi, M.M. Krishna, B. Haribabu, V. Anjaneyulu, Marine sterols. XXV. Isolation of 23-demethylgorgost-7-ene-3b,5a,6b-triol and (24S)-ergostane3b,5a,6b,7b,15b-pentol from soft corals of the Andaman and Nicobar coasts, Chem. Pharm. Bull. 41 (1993) 87–89. [9] K. Fabricius, P. Alderslade, Soft Corals and Sea Fans–A Comprehensive Guide to the Tropical Shallow-Water Genera of the Central-West Pacific, the Indian Ocean and the Red Sea, first ed., Australian Institute of Marine Science, Queensland, Australia, 2001, pp. 218–219. [10] L.M. Kuo, C.Y. Kuo, C.Y. Lin, M.F. Hung, J.J. Shen, T.L. Hwang, Intracellular glutathione depletion by oridonin leads to apoptosis in hepatic stellate cells, Molecules 19 (2014) 3327–3344. [11] T. Shen, L. Zhang, Y.Y. Wang, P.H. Fan, X.N. Wang, Z.M. Lin, H.X. Lou, Steroids from Commiphora mukul display antiproliferative effect against human prostate cancer PC3 cells via induction of apoptosis, Bioorg. Med. Chem. Lett. 22 (2012) 4801–4806. [12] J.L.C. Wright, A.G. McInnes, S. Shimizu, D.G. Smith, J.A. Walter, D. Idler, W. Khalil, Identification of C-24 alkyl epimers of marine sterols by 13C nuclear magnetic resonance spectroscopy, Can. J. Chem. 56 (1978) 1898–1903. [13] G. Santafé, V. Paz, J. Rodríguez, C. Jiménez, Novel cytotoxic oxygenated C29 sterols from the Colombian marine sponge Polymastia tenax, J. Nat. Prod. 65 (2002) 1161–1164. [14] W.H. Yen, W.F. Chen, C.H. Cheng, C.F. Dai, M.C. Lu, J.H. Su, Y.D. Su, Y.H. Chen, Y. C. Chang, Y.H. Chen, J.H. Sheu, C.S. Lin, Z.H. Wen, P.J. Sung, A novel 5a,8aepidioxysterol from the soft coral Sinularia gaweli, Molecules 18 (2013) 2895– 2903. [15] P.A. Blanc, C. Djerassi, Isolation and strucrure elucidation of 22(S),23(S)methylenecholesterol. Evidence for direct bioalkylation of 22dehydrocholesterol, J. Am. Chem. Soc. 102 (1980) 7113–7114.