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Columnaristerol A, a novel 19-norsterol from the Formosan octocoral Nephthea columnaris
Bioorganic & Medicinal Chemistry Letters 26 (2016) 4966–4969
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl
Columnaristerol A, a novel 19-norsterol from the Formosan octocoral Nephthea columnaris Ta-Yuan Whuang a,b,y, Wen-Cheng Tsai c,y, Nan-Fu Chen d,e,y, Zhi-Cheng Chen f, Kuan-Hao Tsui g,h,i, Zhi-Hong Wen f,j, Yin-Di Su b, Yu-Chia Chang b,j, Yu-Hsin Chen b,k, Mei-Chin Lu a,b, Lee-Shing Fang l, Jih-Jung Chen i, Tung-Ying Wu m, Yang-Chang Wu m,n,o,p,⇑, Ping-Jyun Sung a,b,f,m,p,⇑ a
Graduate Institute of Marine Biology, National Dong Hwa University, Pingtung 944, Taiwan National Museum of Marine Biology and Aquarium, Pingtung 944, Taiwan Medicine Department, Zuoying Branch of Kaohsiung Armed Forces General Hospital, Kaohsiung 833, Taiwan d Division of Neurosurgery, Department of Surgery, Kaohsiung Armed Forces General Hospital, Kaohsiung 802, Taiwan e Department of Neurological Surgery, Tri-Service General Hospital, National Defense Medical Center, Taipei 114, Taiwan f Department of Marine Biotechnology and Resources, National Sun Yat-sen University, Kaohsiung 804, Taiwan g Department of Obstetrics and Gynecology, Kaohsiung Veterans General Hospital, Kaohsiung 813, Taiwan h Department of Obstetrics and Gynecology and Institute of Clinical Medicine, National Yang-Ming University, Taipei 112, Taiwan i Department of Pharmacy and Graduate Institute of Pharmaceutical Technology, Tajen University, Pingtung 907, Taiwan j Doctoral Degree Program of Marine Biotechnology, National Sun Yat-sen University and Academia Sinica, Kaohsiung 804, Taiwan k Department of Life Science and Institute of Biotechnology, National Dong Hwa University, Hualien 974, Taiwan l Department of Sport, Health and Leisure, Cheng Shiu University, Kaohsiung 833, Taiwan m Chinese Medicine Research and Development Center, China Medical University Hospital, Taichung 404, Taiwan n School of Pharmacy, College of Pharmacy, China Medical University, Taichung 404, Taiwan o Center for Molecular Medicine, China Medical University Hospital, Taichung 404, Taiwan p 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 16 July 2016 Revised 26 August 2016 Accepted 2 September 2016 Available online 4 September 2016
a b s t r a c t Columnaristerol A (1), a rare natural 19-norsterol possessing a 10b-hydroxy group was isolated from the Formosan octocoral Nephthea columnaris, and its structure was elucidated by spectroscopic methods. Sterol 1 was found to be a cytotoxic agent that exhibited in vitro moderate cytotoxic activity against MOLT-4 and SUP-T1 human leukemia–lymphoma cell lines. Ó 2016 Elsevier Ltd. All rights reserved.
Keywords: Nephthea columnaris 19-Norsterol Columnaristerol Cytotoxicity
Sterols are a group of organic molecules that are highly diverse and metabolically active. They have been found to be key metabolites in extracts of Nephthea spp. octocorals distributed in the waters of Taiwan,1–12 which are an important source of natural sterol products. In our continuing studies on the chemical constituents of Nephthea columnaris,13,14 a novel 19-norsterol, columnaristerol A (1) (Fig. 1), was isolated. In this paper, we report the separation procedure and further structure determination of the newly-isolated compound 1, in addition to the results of investiga⇑ Corresponding authors. Tel.: +886 4 220 57513; fax: +886 4 220 60248 (Y.-C.W.); tel.: +886 8 882 5037; fax: +886 8 882 5087 (P.-J.S.). E-mail addresses: [email protected] (Y.-C. Wu), [email protected] (P.-J. Sung). y These authors contributed equally to this work. http://dx.doi.org/10.1016/j.bmcl.2016.09.007 0960-894X/Ó 2016 Elsevier Ltd. All rights reserved.
tion into its cytotoxic activity against different human leukemia– lymphoma cell lines. Columnaristerol A (1), [a]25 D 37 (c 0.02, CHCl3), was isolated as an amorphous powder, and the HRESIMS of 1 exhibited a pseudo molecular peak at m/z 423.32338 [M+Na]+ (calcd for C27H44O2+Na, 423.32335) and established a molecular formula of C27H44O2, indicating six degrees of unsaturation. Using data from the 1H NMR and DEPT spectra and comparing with the molecular formula of the compound, the results suggested that there were two exchangeable protons that required the presence of two hydroxy groups. This deduction was confirmed by the IR spectrum, which exhibited a broad absorption at 3419 cm–1. The 13C NMR and DEPT data of 1 revealed the presence of 27 carbons in the compound (Table 1), including four methyls, ten sp3 methylenes, seven sp3
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28 22
21 18
R 1 10
2 3
HO
4
5
11 9
H 6
12
H 8
20
H
27 23
24
26
17
13
21
HO
16
14
15
1
H
1: R = OH 2: R = CH3
7
10
3
9
8
HO
25
20
26
17
14
5 6
Figure 1. The structures of columnaristerol A (1) and 24-methylenecholesterol (2).
methines (including one oxymethine, dC 71.0), two sp3 quaternary carbons (including one oxygenated quaternary carbon, dC 69.1), one trisubstituted double bond (dC 137.6 and 125.2), and one exocyclic double bond (dC 156.9 and 105.9). In addition, the 1H NMR spectrum (Table 1) exhibited four methyl signals at dH 1.03 (3H, d, J = 6.8 Hz), 1.02 (3H, d, J = 6.8 Hz), 0.95 (3H, d, J = 6.4 Hz), and 0.70 (3H, s). Therefore, based on the NMR data, two degrees of unsaturation were accounted for, which implied that 1 was a tetracyclic compound. From the 1H–1H COSY spectrum of 1, the proton sequences from H2-1/H2-2/H-3/H2-4, H-6/H2-7/H-8/H-9/H2-11/H2-12, H-8/H-14/ H2-15/H2-16/H-17/H-20/H2-22/H2-23, H-20/H3-21, H-25/H3-26, and H-25/H3-27 were established (Table 1 and Fig. 2). These data, together with the key HMBC correlations between H2-4/C-5, -6, -10; H-6/C-4, -10; H2-7/C-5; H-9/C-1; H-17/C-13; H3-18/C-12, -13, -14, -17; H2-23/C-24; H-25/C-24; and H2-28/C-23, -25, enabled elucidation of the carbon skeleton of 1 (Table 1 and Fig. 2). By comparison of the 13C NMR data with those of a known typical phytosterol, 24-methylenecholesterol (2) (Fig. 1),15 the planar structure of 1 was established. The stereochemistry of 1 was determined based on the results of a NOESY experiment, and by comparison of NMR data with those
13
11
27
24
18
25
: 1H-1H COSY : HMBC
Figure 2. The 1H–1H COSY and selective key HMBC correlations of 1.
of sterol 2. The configurations at C-3 (dC 71.0), C-8 (dC 32.2), C-9 (dC 49.0), C-13 (dC 42.3), C-14 (dC 55.8), C-17 (dC 56.0), and C-20 (dC 35.7) in 1 were found to be similar as those of 2 (C-3, dC 71.84; C-8, dC 31.94; C-9, dC 50.17; C-13, dC 42.31; C-14, dC 56.80; C-17, dC 56.04; C-20, dC 35.79).15 Key NOESY correlations for 1 showed interactions between H-3/H-2a (dH 1.86), H-3/H-1a (dH 1.17), and H3-18/H-8 (dH 1.42) (Fig. 3). Thus, the hydroxy groups at C-3 and C-10 should be positioned on the b-face by modeling analysis. According to the 13C NMR chemical shifts of C-10 (dC 69.1), the configuration of the chiral carbon C-10 was assigned as S form. It was reported that the 13C NMR value of C-10 resonates at dC 68.91 ppm in a known synthetic sterol, 3b,10b-dihydroxy-19norandrost-5-en-17-one, with the same A–C rings as those of 1.16 Naturally-occurring 19-norsterols are rarely found in marine organisms8,11,12,17 and columnaristerol A (1) was proven to be a rare natural 19-norsterol possessing a 10b-hydroxy group. A possible biosynthetic pathway of 1 was proposed as illustrated in Figure 4. Columnaristerol A (1) might be derived from 24-methylenecholesterol (2) through the intermediates A and B. This pathway involves oxidation and decarboxylation to produce
Table 1 H and 13C NMR data, 1H–1H COSY, and HMBC correlations for sterol 1
1
a b c
Position
dH (J in Hz)a
dC, Mult.b
1
HMBC (H?C)
1a/b 2a/b 3 4 5 6 7/70 8 9 10 11 12/120 13 14 15/150 16/160 17 18 20 21 22/220 23/230 24 25 26 27 28a/b
1.17 1.86 3.58 2.36
m; 2.11 ddd (14.4, 3.6, 3.2) m; 1.71 m m br d (15.6) (2H)
H2-2 H2-1, H-3 H2-2, H2-4 H-3
n.o.c n.o. n.o. C-2, -3, -5, -6, -10
5.54 2.00 1.42 0.99
br d (5.2) m; 1.50 m m m
35.0, CH2 31.0, CH2 71.0, CH 41.6, CH2 137.6, C 125.2, CH 31.6, CH2 32.2, CH 49.0, CH 69.1, C 20.2, CH2 39.3, CH2 42.3, C 55.8, CH 24.3, CH2 28.2, CH2 56.0, CH 11.7, CH3 35.7, CH 18.7, CH3 34.7, CH2 30.9, CH2 156.9, C 33.8, CH 21.8, CH3 22.0, CH3 105.9, CH2
H2-7 H-6, H-8 H2-7, H-9, H-14 H-8, H2-11
C-4, -8, -10 C-5, -6, -8, -9 C-14 C-1, -8, -11
H-9, H2-12 H2-11
n.o. n.o.
H-8, H2-15 H-14, H2-16 H2-15, H-17 H2-16, H-20
C-15 C-14 n.o. C-13, C-12, n.o. C-17, n.o. C-24,
-28
C-24, C-24, C-24, C-23,
-26, -27, -28 -25, -27 -25, -26 -25
1.61 m (2H) 2.04 m; 1.22 m 1.07 1.61 1.86 1.17 0.70 1.43 0.95 1.56 2.07
m m; 1.07 m; 1.30 m s m d (6.4) m; 1.17 m; 1.90
2.23 1.02 1.03 4.71
m d (6.8) d (6.8) s; 4.66 s
m m
m m
Spectra recorded at 400 MHz in CDCl3 at 25 °C. Spectra recorded at 100 MHz in CDCl3 at 25 °C. n.o. = not observed.
H–1H COSY
H-17, H3-21, H2-22 H-20 H-20, H2-23 H2-22 H3-26, H3-27 H-25 H-25
-18 -13, -14, -17 -20, -22
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21 28 20R
18
24 17R 13R 14S
9S
1 10S
8S 5
3S
6
Figure 3. Selected protons with key NOESY correlations of 1.
H H
H
H
H
H
H
2
HO
H
HO
[O]
A
HO
[O]
H HO
H (1)
H
CO2
1
HO
H
(2) OH H
H
HOOC
H
H
B
HO
Figure 4. Proposed biosynthetic pathway of columnaristerol A (1).
Table 2 Cytotoxic data of sterol 1 Cell lines IC50 (lM)
Compounds
1 Doxorubicina a b
MOLT-4
K-562
SUP-T1
U-937
18.3 0.02
NAb 0.28
25.5 0.04
NA 0.42
Doxorubicin was used as a positive control. NA = not active at 20 lg/mL for 72 h.
1. Based on the biosynthetic derivation, the absolute configurations for the chiral carbons of 1 were assigned as 3S, 8S, 9S, 10S, 13R, 14S, 17R, and 20R. The cytotoxic effects of columnaristerol A (1) against the cell proliferation of a panel of human leukemia–lymphoma cell lines, including K-562 (chronic myelogenous leukemia), MOLT-4 (acute lymphoblastic leukemia), SUP-T1 (T-cell lymphoblastic lymphoma), and U-937 (histiocytic lymphoma), were tested. The results were as shown in Table 2, which demonstrated that sterol 1 possessed moderate cytotoxic effects towards MOLT-4 and SUP-T1 cells.
Acknowledgments This research was supported by grants from the National Museum of Marine Biology and Aquarium; the National Dong Hwa University; the National Sun Yat-sen University; the Ministry of Science and Technology (Grant Nos. MOST 105-2325-B-291-001 and 104-2320-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 Y.-C.W. and P.-J.S.
T.-Y. Whuang et al. / Bioorg. Med. Chem. Lett. 26 (2016) 4966–4969
Supplementary data Supplementary data (experimental details, HRESIMS, and NMR spectra of 1) associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bmcl.2016.09.007. References and notes 1. Duh, C.-Y.; Wang, S.-K.; Chu, M.-J.; Sheu, J.-H. J. Nat. Prod. 1998, 61, 1022. 2. El-Gamal, A. A. H.; Wang, S.-K.; Dai, C.-F.; Duh, C.-Y. J. Nat. Prod. 2004, 67, 1455. 3. Su, J.-H.; Lin, F.-Y.; Huang, H.-C.; Dai, C.-F.; Wu, Y.-C.; Hu, W.-P.; Hsu, C.-H.; Sheu, J.-H. Tetrahedron 2007, 63, 703. 4. Cheng, S.-Y.; Dai, C.-F.; Duh, C.-Y. Steroids 2007, 72, 653. 5. Cheng, S.-Y.; Dai, C.-F.; Duh, C.-Y. J. Nat. Prod. 2007, 70, 1449. 6. Huang, Y.-C.; Wen, Z.-H.; Wang, S.-K.; Hsu, C.-H.; Duh, C.-Y. Steroids 2008, 73, 1181.
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7. Cheng, S.-Y.; Wen, Z.-H.; Wang, S.-K.; Chiang, M. Y.; El-Gamal, A. A. H.; Dai, C.F.; Duh, C.-Y. Chem. Biodivers. 2009, 6, 86. 8. Cheng, S.-Y.; Huang, Y.-C.; Wen, Z.-H.; Chiou, S.-F.; Wang, S.-K.; Hsu, C.-H.; Dai, C.-F.; Duh, C.-Y. Tetrahedron Lett. 2009, 50, 802. 9. Cheng, S.-Y.; Huang, Y.-C.; Wen, Z.-H.; Hsu, C.-H.; Wang, S.-K.; Dai, C.-F.; Duh, C.-Y. Steroids 2009, 74, 543. 10. Wang, S.-K.; Puu, S.-Y.; Duh, C.-Y. Mar. Drugs 2012, 10, 1288. 11. Wang, S.-K.; Puu, S.-Y.; Duh, C.-Y. Mar. Drugs 2013, 11, 571. 12. Tsai, T.-C.; Huang, Y.-T.; Chou, S.-K.; Shih, M.-C.; Chiang, C.-Y.; Su, J.-H. Chem. Pharm. Bull. 2016, 64. in press. 13. Hsiao, T.-H.; Sung, C.-S.; Lan, Y.-H.; Wang, Y.-C.; Lu, M.-C.; Wen, Z.-H.; Wu, Y.C.; Sung, P.-J. Mar. Drugs 2015, 13, 3443. 14. Hsiao, T.-H.; Cheng, C.-H.; Wu, T.-Y.; Lu, M.-C.; Chen, W.-F.; Wen, Z.-H.; Dai, C.F.; Wu, Y.-C.; Sung, P.-J. Molecules 2015, 20, 13205. 15. McInnes, A. G.; Walter, J. A.; Wright, J. L. C. Org. Magn. Reson. 1980, 13, 302. 16. Kocˇovsky´, P.; Baines, R. S. J. Org. Chem. 1994, 59, 5439. 17. Fang, H.-Y.; Liaw, C.-C.; Chao, C.-H.; Wen, Z.-H.; Wu, Y.-C.; Hsu, C.-H.; Dai, C.-F.; Sheu, J.-H. Tetrahedron 2012, 68, 9694.
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl
Columnaristerol A, a novel 19-norsterol from the Formosan octocoral Nephthea columnaris Ta-Yuan Whuang a,b,y, Wen-Cheng Tsai c,y, Nan-Fu Chen d,e,y, Zhi-Cheng Chen f, Kuan-Hao Tsui g,h,i, Zhi-Hong Wen f,j, Yin-Di Su b, Yu-Chia Chang b,j, Yu-Hsin Chen b,k, Mei-Chin Lu a,b, Lee-Shing Fang l, Jih-Jung Chen i, Tung-Ying Wu m, Yang-Chang Wu m,n,o,p,⇑, Ping-Jyun Sung a,b,f,m,p,⇑ a
Graduate Institute of Marine Biology, National Dong Hwa University, Pingtung 944, Taiwan National Museum of Marine Biology and Aquarium, Pingtung 944, Taiwan Medicine Department, Zuoying Branch of Kaohsiung Armed Forces General Hospital, Kaohsiung 833, Taiwan d Division of Neurosurgery, Department of Surgery, Kaohsiung Armed Forces General Hospital, Kaohsiung 802, Taiwan e Department of Neurological Surgery, Tri-Service General Hospital, National Defense Medical Center, Taipei 114, Taiwan f Department of Marine Biotechnology and Resources, National Sun Yat-sen University, Kaohsiung 804, Taiwan g Department of Obstetrics and Gynecology, Kaohsiung Veterans General Hospital, Kaohsiung 813, Taiwan h Department of Obstetrics and Gynecology and Institute of Clinical Medicine, National Yang-Ming University, Taipei 112, Taiwan i Department of Pharmacy and Graduate Institute of Pharmaceutical Technology, Tajen University, Pingtung 907, Taiwan j Doctoral Degree Program of Marine Biotechnology, National Sun Yat-sen University and Academia Sinica, Kaohsiung 804, Taiwan k Department of Life Science and Institute of Biotechnology, National Dong Hwa University, Hualien 974, Taiwan l Department of Sport, Health and Leisure, Cheng Shiu University, Kaohsiung 833, Taiwan m Chinese Medicine Research and Development Center, China Medical University Hospital, Taichung 404, Taiwan n School of Pharmacy, College of Pharmacy, China Medical University, Taichung 404, Taiwan o Center for Molecular Medicine, China Medical University Hospital, Taichung 404, Taiwan p 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 16 July 2016 Revised 26 August 2016 Accepted 2 September 2016 Available online 4 September 2016
a b s t r a c t Columnaristerol A (1), a rare natural 19-norsterol possessing a 10b-hydroxy group was isolated from the Formosan octocoral Nephthea columnaris, and its structure was elucidated by spectroscopic methods. Sterol 1 was found to be a cytotoxic agent that exhibited in vitro moderate cytotoxic activity against MOLT-4 and SUP-T1 human leukemia–lymphoma cell lines. Ó 2016 Elsevier Ltd. All rights reserved.
Keywords: Nephthea columnaris 19-Norsterol Columnaristerol Cytotoxicity
Sterols are a group of organic molecules that are highly diverse and metabolically active. They have been found to be key metabolites in extracts of Nephthea spp. octocorals distributed in the waters of Taiwan,1–12 which are an important source of natural sterol products. In our continuing studies on the chemical constituents of Nephthea columnaris,13,14 a novel 19-norsterol, columnaristerol A (1) (Fig. 1), was isolated. In this paper, we report the separation procedure and further structure determination of the newly-isolated compound 1, in addition to the results of investiga⇑ Corresponding authors. Tel.: +886 4 220 57513; fax: +886 4 220 60248 (Y.-C.W.); tel.: +886 8 882 5037; fax: +886 8 882 5087 (P.-J.S.). E-mail addresses: [email protected] (Y.-C. Wu), [email protected] (P.-J. Sung). y These authors contributed equally to this work. http://dx.doi.org/10.1016/j.bmcl.2016.09.007 0960-894X/Ó 2016 Elsevier Ltd. All rights reserved.
tion into its cytotoxic activity against different human leukemia– lymphoma cell lines. Columnaristerol A (1), [a]25 D 37 (c 0.02, CHCl3), was isolated as an amorphous powder, and the HRESIMS of 1 exhibited a pseudo molecular peak at m/z 423.32338 [M+Na]+ (calcd for C27H44O2+Na, 423.32335) and established a molecular formula of C27H44O2, indicating six degrees of unsaturation. Using data from the 1H NMR and DEPT spectra and comparing with the molecular formula of the compound, the results suggested that there were two exchangeable protons that required the presence of two hydroxy groups. This deduction was confirmed by the IR spectrum, which exhibited a broad absorption at 3419 cm–1. The 13C NMR and DEPT data of 1 revealed the presence of 27 carbons in the compound (Table 1), including four methyls, ten sp3 methylenes, seven sp3
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T.-Y. Whuang et al. / Bioorg. Med. Chem. Lett. 26 (2016) 4966–4969 28
28 22
21 18
R 1 10
2 3
HO
4
5
11 9
H 6
12
H 8
20
H
27 23
24
26
17
13
21
HO
16
14
15
1
H
1: R = OH 2: R = CH3
7
10
3
9
8
HO
25
20
26
17
14
5 6
Figure 1. The structures of columnaristerol A (1) and 24-methylenecholesterol (2).
methines (including one oxymethine, dC 71.0), two sp3 quaternary carbons (including one oxygenated quaternary carbon, dC 69.1), one trisubstituted double bond (dC 137.6 and 125.2), and one exocyclic double bond (dC 156.9 and 105.9). In addition, the 1H NMR spectrum (Table 1) exhibited four methyl signals at dH 1.03 (3H, d, J = 6.8 Hz), 1.02 (3H, d, J = 6.8 Hz), 0.95 (3H, d, J = 6.4 Hz), and 0.70 (3H, s). Therefore, based on the NMR data, two degrees of unsaturation were accounted for, which implied that 1 was a tetracyclic compound. From the 1H–1H COSY spectrum of 1, the proton sequences from H2-1/H2-2/H-3/H2-4, H-6/H2-7/H-8/H-9/H2-11/H2-12, H-8/H-14/ H2-15/H2-16/H-17/H-20/H2-22/H2-23, H-20/H3-21, H-25/H3-26, and H-25/H3-27 were established (Table 1 and Fig. 2). These data, together with the key HMBC correlations between H2-4/C-5, -6, -10; H-6/C-4, -10; H2-7/C-5; H-9/C-1; H-17/C-13; H3-18/C-12, -13, -14, -17; H2-23/C-24; H-25/C-24; and H2-28/C-23, -25, enabled elucidation of the carbon skeleton of 1 (Table 1 and Fig. 2). By comparison of the 13C NMR data with those of a known typical phytosterol, 24-methylenecholesterol (2) (Fig. 1),15 the planar structure of 1 was established. The stereochemistry of 1 was determined based on the results of a NOESY experiment, and by comparison of NMR data with those
13
11
27
24
18
25
: 1H-1H COSY : HMBC
Figure 2. The 1H–1H COSY and selective key HMBC correlations of 1.
of sterol 2. The configurations at C-3 (dC 71.0), C-8 (dC 32.2), C-9 (dC 49.0), C-13 (dC 42.3), C-14 (dC 55.8), C-17 (dC 56.0), and C-20 (dC 35.7) in 1 were found to be similar as those of 2 (C-3, dC 71.84; C-8, dC 31.94; C-9, dC 50.17; C-13, dC 42.31; C-14, dC 56.80; C-17, dC 56.04; C-20, dC 35.79).15 Key NOESY correlations for 1 showed interactions between H-3/H-2a (dH 1.86), H-3/H-1a (dH 1.17), and H3-18/H-8 (dH 1.42) (Fig. 3). Thus, the hydroxy groups at C-3 and C-10 should be positioned on the b-face by modeling analysis. According to the 13C NMR chemical shifts of C-10 (dC 69.1), the configuration of the chiral carbon C-10 was assigned as S form. It was reported that the 13C NMR value of C-10 resonates at dC 68.91 ppm in a known synthetic sterol, 3b,10b-dihydroxy-19norandrost-5-en-17-one, with the same A–C rings as those of 1.16 Naturally-occurring 19-norsterols are rarely found in marine organisms8,11,12,17 and columnaristerol A (1) was proven to be a rare natural 19-norsterol possessing a 10b-hydroxy group. A possible biosynthetic pathway of 1 was proposed as illustrated in Figure 4. Columnaristerol A (1) might be derived from 24-methylenecholesterol (2) through the intermediates A and B. This pathway involves oxidation and decarboxylation to produce
Table 1 H and 13C NMR data, 1H–1H COSY, and HMBC correlations for sterol 1
1
a b c
Position
dH (J in Hz)a
dC, Mult.b
1
HMBC (H?C)
1a/b 2a/b 3 4 5 6 7/70 8 9 10 11 12/120 13 14 15/150 16/160 17 18 20 21 22/220 23/230 24 25 26 27 28a/b
1.17 1.86 3.58 2.36
m; 2.11 ddd (14.4, 3.6, 3.2) m; 1.71 m m br d (15.6) (2H)
H2-2 H2-1, H-3 H2-2, H2-4 H-3
n.o.c n.o. n.o. C-2, -3, -5, -6, -10
5.54 2.00 1.42 0.99
br d (5.2) m; 1.50 m m m
35.0, CH2 31.0, CH2 71.0, CH 41.6, CH2 137.6, C 125.2, CH 31.6, CH2 32.2, CH 49.0, CH 69.1, C 20.2, CH2 39.3, CH2 42.3, C 55.8, CH 24.3, CH2 28.2, CH2 56.0, CH 11.7, CH3 35.7, CH 18.7, CH3 34.7, CH2 30.9, CH2 156.9, C 33.8, CH 21.8, CH3 22.0, CH3 105.9, CH2
H2-7 H-6, H-8 H2-7, H-9, H-14 H-8, H2-11
C-4, -8, -10 C-5, -6, -8, -9 C-14 C-1, -8, -11
H-9, H2-12 H2-11
n.o. n.o.
H-8, H2-15 H-14, H2-16 H2-15, H-17 H2-16, H-20
C-15 C-14 n.o. C-13, C-12, n.o. C-17, n.o. C-24,
-28
C-24, C-24, C-24, C-23,
-26, -27, -28 -25, -27 -25, -26 -25
1.61 m (2H) 2.04 m; 1.22 m 1.07 1.61 1.86 1.17 0.70 1.43 0.95 1.56 2.07
m m; 1.07 m; 1.30 m s m d (6.4) m; 1.17 m; 1.90
2.23 1.02 1.03 4.71
m d (6.8) d (6.8) s; 4.66 s
m m
m m
Spectra recorded at 400 MHz in CDCl3 at 25 °C. Spectra recorded at 100 MHz in CDCl3 at 25 °C. n.o. = not observed.
H–1H COSY
H-17, H3-21, H2-22 H-20 H-20, H2-23 H2-22 H3-26, H3-27 H-25 H-25
-18 -13, -14, -17 -20, -22
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21 28 20R
18
24 17R 13R 14S
9S
1 10S
8S 5
3S
6
Figure 3. Selected protons with key NOESY correlations of 1.
H H
H
H
H
H
H
2
HO
H
HO
[O]
A
HO
[O]
H HO
H (1)
H
CO2
1
HO
H
(2) OH H
H
HOOC
H
H
B
HO
Figure 4. Proposed biosynthetic pathway of columnaristerol A (1).
Table 2 Cytotoxic data of sterol 1 Cell lines IC50 (lM)
Compounds
1 Doxorubicina a b
MOLT-4
K-562
SUP-T1
U-937
18.3 0.02
NAb 0.28
25.5 0.04
NA 0.42
Doxorubicin was used as a positive control. NA = not active at 20 lg/mL for 72 h.
1. Based on the biosynthetic derivation, the absolute configurations for the chiral carbons of 1 were assigned as 3S, 8S, 9S, 10S, 13R, 14S, 17R, and 20R. The cytotoxic effects of columnaristerol A (1) against the cell proliferation of a panel of human leukemia–lymphoma cell lines, including K-562 (chronic myelogenous leukemia), MOLT-4 (acute lymphoblastic leukemia), SUP-T1 (T-cell lymphoblastic lymphoma), and U-937 (histiocytic lymphoma), were tested. The results were as shown in Table 2, which demonstrated that sterol 1 possessed moderate cytotoxic effects towards MOLT-4 and SUP-T1 cells.
Acknowledgments This research was supported by grants from the National Museum of Marine Biology and Aquarium; the National Dong Hwa University; the National Sun Yat-sen University; the Ministry of Science and Technology (Grant Nos. MOST 105-2325-B-291-001 and 104-2320-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 Y.-C.W. and P.-J.S.
T.-Y. Whuang et al. / Bioorg. Med. Chem. Lett. 26 (2016) 4966–4969
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