Accepted Manuscript Five new quassinoids and cytotoxic constituents from the roots of Eurycoma longifolia SeonJu Park, Nguyen Xuan Nhiem, Phan Van Kiem, Chau Van Minh, Bui Huu Tai, Nanyoung Kim, Jae-Hyoung Song, Hyun-Jeong Ko, Seung Hyun Kim PII: DOI: Reference:
S0960-894X(14)00686-6 http://dx.doi.org/10.1016/j.bmcl.2014.06.058 BMCL 21777
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date: Revised Date: Accepted Date:
18 March 2014 11 June 2014 20 June 2014
Please cite this article as: Park, S., Nhiem, N.X., Kiem, P.V., Minh, C.V., Tai, B.H., Kim, N., Song, J-H., Ko, H-J., Kim, S.H., Five new quassinoids and cytotoxic constituents from the roots of Eurycoma longifolia, Bioorganic & Medicinal Chemistry Letters (2014), doi: http://dx.doi.org/10.1016/j.bmcl.2014.06.058
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Five new quassinoids and cytotoxic constituents from the roots of Eurycoma longifolia
SeonJu Parka, Nguyen Xuan Nhiema,b, Phan Van Kiem b, Chau Van Minhb, Bui Huu Taib, Nanyoung Kima, Jae-Hyoung Songc, Hyun-Jeong Koc, and Seung Hyun Kima,∗
a
College of Pharmacy, Yonsei Institute of Pharmaceutical Science, Yonsei University, Incheon 406-840, Korea b Institute of Marine Biochemistry, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet, Caugiay, Hanoi, Vietnam c Laboratory of Microbiology and Immunology, College of Pharmacy, Kangwon National University, Chuncheon 200-701, Korea
∗ Corresponding author. Tel.: +82-32-749-4514; fax: +82-32-749-4105; e-mail: [email protected]
1
ABSTRACT Eurycoma longifolia has been widely used for various traditional medicinal purposes in South-East Asia. In this study, five new quassinoids, eurylactone E (1), eurylactone F (2), eurylactone G (3), eurycomalide D (4), and eurycomalide E (5), along with ten known quassinoids (6 − 15) were isolated from the roots of E. longifolia. Their structures were determined by extensive spectroscopic methods, including 1D and 2D NMR, and MS spectra data. Among the isolated compounds, 13β-methyl,21-dihydroeurycomanone (6) has been reported as a synthetic derivative. However, it was isolated from the natural product for the first time in this study. The cytotoxic activities of fifteen compounds were evaluated against human lung cancer cell line, A549 and human cervical cancer cell line, HeLa.
Keywords Eurycoma longifolia; quassinoid; cytotoxicity; A549 (human lung cancer cell); HeLa (human cervical cancer cell).
2
Eurycoma longifolia (Simaroubaceae), commonly known as Tongkat Ali, is an herbal medicinal plant popularly used in South-East Asian countries. The plant extract has been used in local traditional medicines for antimalarial, anti-pyretic, and antiulcer as well as for enhancing testosterone levels in men.1, 2 Tongkat Ali can be consumed as a tonic drink or available in the health-food market either in the form of raw crude powder, or capsule mixed with other aphrodisiac herbs. It is also available as an additive mixed with coffee or in certain health products as a replacement for ginseng.1,
3
Since many recent studies proved its efficacy as a general health and
testosterone booster, and for its known aphrodisiac properties, both popularity and its demand increased.1-3 Although quassinoid, alkaloid, and squalene derivatives are reported as major chemical components of Tongkat Ali,4-9 studies of its chemical constituent have been not carried out extensively but rather recent studies were mainly focused on its extract or fraction-based bioactivities of anti-angiogenic, antiosteoporotic, reproductive, and genocytotoxic activities.10-13 Studying chemical constituent is important for consuming product concerning on the quality of the food supply, mainly food adulteration and contamination issues. Therefore, it necessitates explorative study of chemical constituents of Tongkat Ali, universally consuming natural product for one’s interest. In the present study, along with 5 newly isolated compounds, total 15 quassinoids of four different types: eurycomalactone, laurycolactone, klaineanone, and longilactone were isolated from E. longifolia. The structures of new compounds (1 – 5) were elucidated on the basis of spectroscopic analysis and comparison with literature data. Among 10 known compounds, 13β-methyl,21-dihydroeurycomanone (6) was isolated from a natural source for the first time. These compounds were screened for 3
in-vitro cytotoxicity against A549 and HeLa tumor cell lines. The roots of E. longifolia were collected in Dak Lak province, Vietnam, in March 2013, and authenticated by Dr. Bui Van Thanh in Institute of Ecology and Biological Resources, Vietnamese Academy of Science and Technology, Vietnam. A voucher specimen was deposited at the Herbarium of Pharmacy, Yonsei Institute of Pharmaceutical Sciences, Yonsei University, Incheon, Korea. The dried roots of E. longifolia (18.0 kg) were extracted with MeOH (3 × 10 L, 50℃) under sonication for 4 h to yield 400.0 g extract after evaporation of the solvent. This extract was suspended in H2O and successively partitioned with CHCl3 and n-BuOH to obtain the CHCl3 (EL1, 105.0 g), n-BuOH (EL2, 234.0 g), and H2O (EL3, 60.0 g) extracts after removal of the solvents in vacuo. Using various chromatographic resin and isolation techniques, fifteen quassinoids were isolated.14 Compound 1 was obtained as a white amorphous powder and its molecular formula was determined as C19H22O9, by the HR-ESI-MS [M + H]+ ion at m/z 395.1316 (calcd for C19H23O9, 395.1342). The 1H NMR spectrum showed three methyl resonances at δH 1.36 (3H, d, J = 6.0 Hz), 1.60 (3H, s) and 2.21 (3H, s) and the methylene protons at δH 1.86 (d, J = 16.0 Hz) and 2.24 (dd, J = 6.0, 16.0 Hz). One olefinic proton at δH 5.98, and five oxymethine protons at δH 3.79 (d, J = 11.8 Hz), 4.05 (d, J = 11.8 Hz), 4.64, 4.80 and 5.82 were also observed (Table 1). The
13
C-NMR and DEPT spectra of 1
revealed 19 carbons signals, including nine quaternary (δC 49.80, 57.20, 80.81, 137.80, 145.66, 170.54, 173.99, 174.35, and 197.08), five methine (δC 49.50, 71.13, 85.06, 88.48, and 120.69), two methylene (δC 40.60, and 64.37), and three methyl carbons (δC 10.20, 16.67, and 23.75). The NMR data of 1 were similar to those of eurylactone 4
A6 except for the addition of one carbonyl and one hydroxymethyl group and the presence of double bond. The position of three carbonyls were verified by HMBC correlations between H-5 (δH 5.82) and C-2 (δC 173.99), between H-21 (δH 1.36) and C-12 (δC 197.08), and between H-7 (δH 4.80) and C-16 (δC 174.35), leading three carbonyls were located at C-2, C-12, and C-16. The HMBC correlations between H20 (δH 3.79 and 4.05) and C-7 (δC 85.05), C-9 (δC 137.79), and C-14 (δC 80.80) suggested the presence of one hydroxymethyl group at C-8. The presence of double bond between C-9 and C-11 was confirmed by HMBC correlations between H-19 (δH 1.60) and quaternary C-9 (δC 137.79) and between H-13 (δH 3.48) and quaternary C11 (δC 145.65) (Figure 2). Since compound 1 is a biogenetic derivative of quassinoids from E. longifolia, it was supposed to have the same configurations at C-8 and C-10 of eurylactone A. ROESY correlations between H-19 (δH 1.60) and H-18 (δH 2.21); H-7 (δH 4.80) and H-20 (δH 3.79 and 4.05) suggested the configuration of both H-5 and H-7 to be β. Furthermore, ROESY correlation between H-15 (δH 4.64) and H-21 (δH 1.36) as well as no ROESY correlations between H-15 (δH 4.64) and H-20 (δH 3.79 and 4.05) confirmed the configurations of H-15 and methyl group at C-13 to be α. Based on the evidence above, compound 1 was established and named eurylactone E.15 Compound 2 was isolated as a white amorphous powder. HR-ESI-MS spectra resulted as the same molecular formula as that of 1. The 1H and
13
C NMR, HSQC,
HMBC, and COSY analyses of 2 indicated the same constitution as those of 1. The chemical shifts of C-13 (δC 45.74) and C-21 (δC 14.06) in 2 were different from the chemical shifts of C-13 (δC 49.50) and C-21 (δC 10.19) in 1, suggesting the possibility of different configurations at the chiral carbon C-13 (Table 1). Moreover, the ROESY 5
correlation between H-20 (δH 3.75 and 3.82) and H-21 (δH 1.44); H-7 (δH 4.97) and H20; H-13 (δH 3.28) and H-15 (δH 4.57) suggested that the configuration of the methyl group at C-13 to be β. Thus, the structure of 2 was elucidated and named eurylactone F.16 Compound 3 was obtained as a white amorphous powder and its molecular formula was deduced as C19H20O9, by the HR-ESI-MS ion [M*]- at m/z 392.1080 (calcd for C19H20O9, 392.1107). The 1H NMR spectrum showed two methyl resonances: a singlet at δH 1.64 (3H, s) and 2.23 (3H, s). Three olefinic protons at δH 5.90, 6.01 and 6.47, and five oxymethine protons at δH 3.61 (d, J = 11.6 Hz), 3.81 (d, J = 11.6 Hz), 4.65, 4.96 (d, J = 6.1 Hz) and 5.82 were also observed (Table 1). The 13C-NMR and DEPT spectra of 3 revealed 19 carbons signals including ten quaternary (δC 50.48, 57.36, 81.43, 139.62, 144.57, 146.48, 170.54, 173.54, 174.00, and 184.41), four methine (δC 70.79, 85.57, 88.69, and 120.99), three methylene (δC 41.04, 66.75, and 125.53), and two methyl carbons (δC 16.84, and 23.86). One additional double bond at C-13/C-21 was observed compared to compounds 1 and 2. This was confirmed by HMBC correlations between H-21 (δH 5.90 and 6.47) and C-12 (δC 184.41), C-13 (δC 144.54), and C-14 (δC 81.43). Compound 3 was supposed to be a biogenetic derivative resulted from the dehydrogenation of 1 and 2. The important NOESY correlations shown in Figure 2 proved the configurations of chiral carbons. Based on these, the compound 3 was named eurylactone G.17 Compound 4 was obtained as a white amorphous powder. HR-ESI-MS exhibited [M + H]+ signal at m/z 367.1755 (calcd for C19H27O7, 367.1757), suggesting molecular formula of C19H26O7. The
1
H NMR spectrum showed four methyl
resonances at δH 1.11 (3H, d, J = 6.8 Hz), 1.39 (3H, s), 1.49 (3H, s) and 1.73 (3H, s), 6
one methylene proton at δH 1.46 (d, J = 13.4 Hz) and 2.09 (dd, J = 4.8, 13.4 Hz), and three methine protons at δH 1.84 (d, 4.0), 2.84 (s), and 2.98 (m). One olefinic proton at δH 5.96 (s), and four oxymethine protons at δH 3.19 (d, J = 8.8 Hz), 4.09, 4.28 (d, J = 5.2 Hz), and 4.83 were also detected (Table 2). The 13C-NMR and DEPT spectra of 4 revealed 19 carbons signals, including six quaternary (δC 47.29, 48.81, 73.04, 170.85, 179.06, and 202.80), eight methine (δC 33.02, 49.82, 54.37, 68.44, 70.09, 83.83, 85.72 and 122.91), one methylene (δC 45.84), and four methyl carbons (δC 16.89, 19.90, 23.16, and 29.18). The NMR data of 4 were similar to those of eurycomalide B18 except for the appearance of hydroxyl group at C-4. This location was determined by the HMBC correlation between H-16 (δH 1.39) and C-3 (δC 45.84), C-4 (δC 73.04), and C-5 (δC 170.85) (Figure 2). The NOESY correlations between H-17 and H-2; H18 and H-13 suggested that the configurations of hydroxyl group at C-2 and methyl group at C-8 are α and β, respectively. Moreover, all β-configurations of three hydroxyl groups at C-1, C-4, and C-11 were proved by the NOESY correlations between H-1/H-9, H-1/H-11, and H-1/H-16. Based on the evidence above, the structure of eurycomalide D19 was established. Compound 5 was isolated as white amorphous powder and its molecular formula was determined as C19H22O9, by the HR-ESI-MS [M + H]+ ion at m/z 411.1638 (calcd for C20H27O9, 411.1655). The 1H NMR spectrum showed three methyl resonances at δH 1.18 (3H, s), 1.20 (3H, d, J = 6.0 Hz), and 1.70 (3H, s), three methylene proton at δH 2.12 and 2.34, δH 2.58 (d, J = 15.6 Hz) and δH 2.96 (d, J = 15.6 Hz), and 4.36 (d, J = 12.4 Hz) and 5.02 (dd, J = 12.4 Hz), and two methine protons at δH 2.22 (m), and 2.26 (s). Four oxymethine protons at δH 3.28 (d, J = 10.2 Hz), 3.72 (m), 4.29 (s), and 4.52 (s) were also observed (Table 2). The 13C-NMR and DEPT spectra of 5 revealed 7
20 carbons signals, including eight quaternary (δC 46.93, 47.89, 75.79, 79.70, 128.82, 129.98, 175.54 and 177.87), six methine (δC 53.91, 54.36, 66.87, 68.85, 76.69, and 82.03), three methylene (δC 29.16, 41.75 and 69.62), and three methyl carbons (δC 11.44, 16.04, and 19.98). The NMR data of 5 were similar to those of shinjudilactone20 except for the removal of one carbonyl group and the addition of two oxymethines. The HMBC correlation between H-1 (δH 3.38 (d, J = 10.2 Hz)) and C-2 (δC 66.87) suggested that instead of carbonyl group at C-2 as in shinjudilactone,20 hydroxyl group is attached. In addition, attachment of hydroxyl group at C-14 and C15 was determined by the HMBC correlations between H-21 (δH 1.20 (d, J = 6.0 Hz)), and H-20 (δH 4.36 (d, J = 12.4 Hz) and 5.02 (dd, J = 12.4 Hz)) and C-14 (δC 79.70) and between H-15 (δH 4.52) and C-14 (δC 79.70), and C-16 (δC 175.54) (Figure 2). The NOESY correlations between H-19 (δH 1.18) and H-2 (δH 3.74) and between H21 (δH 1.20) and H-15 (δH 4.52) determined the configurations of C-2 and C-15 as S and R, respectively (Figure 3). Thus, the structure of 5 was determined and named eurycomalide E.21 13β-methyl,21-dihydroeurycomanone (6) was isolated as a white amorphous powder and was isolated from the natural product for the first time in the present investigation. Previously reported 13β-methyl,21-dihydroeurycomanone was obtained by hydrogenating eurycomanone catalyzed by (Ph3P)3RhCl.22 HR-ESI-MS spectra showed [M + H]+ signal at m/z 411.1643 (calcd for C20H27O9, 411.1655), implying the molecular formula of C20H26O9. The 1H NMR spectrum showed three methyl resonances at δH 1.08 (3H, d, J = 6.0 Hz), 1.13 (3H, s), and 2.03 (3H, s), two methylene protons at δH 2.16 and 2.29, and δH 3.65 and 4.27, and one methine proton at δH 1.08 (d, J = 7.0 Hz). One olefinic proton at δH 6.05 and four oxymethine protons 8
at δH 3.51 (d, J = 3.9 Hz), 4.21 (s), 4.22 (s), and 4.71 (d, J = 4.1 Hz) were also observed (Table 2). The 13C-NMR and DEPT spectra of 6 revealed 20 carbons signals, including seven quaternary (δC 46.08, 51.29, 78.27, 110.36, 164.10, 173.61 and 198.77), eight methine (δC 33.13, 43.45, 46.55, 73.95, 74.02, 81.41 and 84.37), two methylene (δC 25.62, and 71.85), and three methyl carbons (δC 9.12, 9.80, and 22.99). 1
H and 13C NMR data of 6 were very similar to those of 13,21-dihydroeurycomanone4
except for C-13 and C-21 chemical shifts, suggesting the possibility of different configurations at the chiral carbon C-13 (Table 2), deducing the structure identification as 13β-methyl,21-dihydroeurycomanone.23 Comparison of the NMR and MS data with reported values in the literature led to identification of the structures of the known compounds to be eurylactone A (7),6 eurycomalactone (8),5 13,21-dihydroeurycomanone (9),4 eurycomanone (10),4 13α(21)-epoxyeurycomanone
(11),8
longilactone
(12),4
2-dihydro-18-
dedihydrolongilactone (13),7 14,15β-dihydroxyklaineanone (14),4 and 5α,14β,15βtrihydroxyklaineanone (15)18 (Figure 1). All compounds were tested for cytotoxicity against A549 and HeLa cell lines. Kuo et al. previously reported cytotoxic constituents from the E. longifolia against human lung cancer (A549) and human breast cancer (MCF-7).18 In this study, the cytotoxicity was evaluated by modified the SRB assay previously described.24 In our experimental condition, compounds 8, 12, and 14 showed significant cytotoxicity in both A549 and MCF-7. Compound 9 was more selective against A549 and compound 10 showed cytotoxic effect only in MCF-7. The cytotoxic effects of the tested compounds in A549 were almost same as the previous report18 (Table 3). In HeLa cell line, compounds 8 – 12 and 14 displayed significant cytotoxicity showing the relative 9
cell viability ranging from 21.01 ± 2.46% to 66.9 ± 6.67% at the concentration of 100 µM (Table 3). Among them, compound 14 showed the most potent cytotoxicity in HeLa cell line with IC50 value of 48.18 µM.
Acknowledgements This research was supported by Ministry of Food and Drug Safety (13182MFDS725), Republic of Korea.
10
References and notes 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.
11. 12. 13. 14.
Abdullah, M. Z.; Rahman, A. S. A.; Shakaff, A. Y. M.; Noor, A. M. T. I. Meas. Control. 2004, 26, 19. Ang, H. H.; Cheang, H. S.; Yusof, A. P. M. Exp. Anim. 2000, 49, 35. Tambi, M. I. B. M.; Imran, M. K.; Henkel, R. R. Andrologia. 2012, 44, 226. Morita, H.; Kishi, E.; Takeya, K.; Itokawa, H.; Tanaka, O. Chem. Lett. 1990, 19, 749. Chan, K. L.; Iitaka, Y.; Noguchi, H.; Sugiyama, H.; Saito, I.; Sankawa, U. Phytochem. 1992, 31, 4295. Itokawa, H.; Qin, X.-R.; Morita, H.; Takeya, K. J. Nat. Prod. 1993, 56, 1766. Itokawa, H.; Qin, X.-R.; Morita, H.; Takeya, K.; Iitaka, Y. Chem. Pharm. Bull. 1993, 41, 403. Morita, H.; Kishi, E.; Takeya, K.; Itokawa, H.; Iitaka, Y. Phytochem. 1993, 33, 691. Chua, L. S.; Amin, N. A. M.; Neo, J. C. H.; Lee, T. H.; Lee, C. T.; Sarmidi, M. R.; Aziz, R. A. J. Chromatogr. B. 2011, 879, 3909. Al-Salahi, O. S. A.; Kit-Lam, C.; Majid, A. M. S. A.; Al-Suede, F. S. R.; Mohammed Saghir, S. A.; Abdullah, W. Z.; Ahamed, M. B. K.; Yusoff, N. M. Microvasc. Res. 2013. Ahmad, N. S.; Mohd, F. A. B.; Tajul, A. A. S.; Norliza, M.; Norazlina, M.; Ima, N. S. Aging Male. 2011, 14, 150. Solomon, M. C.; Erasmus, N.; Henkel, R. R. Andrologia. 2013, n/a. Li, C.-H.; Liao, J.-W.; Liao, P.-L.; Huang, W.-K.; Tse, L.-S.; Lin, C.-H.; Kang, J.-J.; Cheng, Y.-W. Evid. Based Complement. Alternat. Med. 2013, 2013, 11. The EL1 fraction (105.0 g) was chromatographed on a silica gel column and eluting with a gradient of n-hexane – acetone (40 : 1 → 0 : 1, v/v) to obtain six sub-fractions, EL1A (14.2 g), EL1B (11.3 g), EL1C (17.2 g), EL1D (21.6 g), EL1E (25.2 g), and EL1F (7.3 g). The EL1D fraction was chromatographed on silica gel column eluting with CHCl3 – acetone (6 : 1, v/v) to yield 8 (407.0 mg). The EL2 fraction was chromatographed on a Diaion HP-20P column eluting with H2O containing increasing concentrations of MeOH (0, 25, 50, 75, and 100%) to obtain five sub-fractions EL2A (82.0 g), EL2B (26.3 g), EL2C (32.8 g), EL2D (12.4 g), and EL2E (72.5 g). The EL2B fraction was chromatographed on a silica gel column eluting with CHCl3 – MeOH (8 : 1, v/v) to yield 9 (109.0 mg), 10 (875.0 mg), 11 (10.0 mg), 12 (110.0 mg), and 13 (58.0 mg). The EL2C fraction was chromatographed on HPLC using J’sphere ODS H-80 250 mm × 20 mm column, 11% aq. MeCN, and a flow rate of 5 mL/min to yield 1 (1.3 mg), 2 (12.1 mg), 3 (7.4 mg) and 15 (1.2 mg). The EL2D fraction was chromatographed on HPLC using J’sphere ODS H-80 250 mm × 20 mm column, 15% aq. MeCN, and a flow rate of 5 mL/min to yield 4 (1.0 mg), 5 (1.0 mg), and 6 (10.3 mg). The EL2E fraction was 11
15.
16.
17.
18. 19.
20. 21.
22. 23.
24.
chromatographed on a silica gel column eluting with CHCl3 – acetone (1 : 1, v/v) to yield 7 (5.0 mg), and 14 (45.0 mg). Eurylactone E: white amorphous powder; [α]20 : +114.4 (c=0.1 in MeOH); IR D -1 (film) νmax 3415, 1748, 1655, 1116, 1042 cm ; HRESIMS m/z 393.1183 [M – H]- (calcd for C20H21O9, 393.1186). 1H (methanol-d4, 400 MHz) and 13C NMR data (methanol-d4, 100 MHz), see Table 1. Eurylactone F: white amorphous powder; [α]20 : +81.1 (c=0.1 in MeOH); IR D -1 (film) νmax 3355, 1660, 1049, 1032, 1016 cm ; HRESIMS m/z 395.1325 [M + H]+ (calcd for C19H23O9, 395.1337). 1H (methanol-d4, 400 MHz) and 13C NMR data (methanol-d4, 100 MHz), see Table 1. Eurylactone G: white amorphous powder; [α]20 : +66.9 (c=0.1 in MeOH); IR D -1 (film) νmax 3363, 1748, 1053, 1032, 1014 cm ; HRESIMS m/z 392.1094 [M*](calcd for C19H20O9, 392.1107). 1H (methanol-d 4, 400 MHz) and 13C NMR data (methanol-d4, 100 MHz), see Table 1. Kuo, P.-C.; Damu, A. G.; Lee, K.-H.; Wu, T.-S. Bioorg. Med. Chem. 2004, 12, 537. Eurycomalide D: white amorphous powder; [α]20 : +205.8 (c=0.1 in MeOH); D -1 IR (film) νmax 3398, 1650, 1016, 707 cm ; HRESIMS m/z 367.1755 [M + H]+ (calcd for C19H27O7, 367.1757). 1H (methanol-d 4, 400 MHz) and 13C NMR data (methanol-d4, 100 MHz), see Table 2. Kubota, K.; Fukamiya, N.; Hamada, T.; Okano, M.; Tagahara, K.; Lee, K.-H. J. Nat. Prod. 1996, 59, 683. Eurycomalide E: white amorphous powder; [α]20 : +19.2 (c=0.1 in MeOH); IR D (film) νmax 3465, 1614, 1070, 616 cm-1; HRESIMS m/z 411.1638 [M + H]+ (calcd for C20H27O9, 411.1655), 409.1494 [M – H]- (calcd for C20H25O9, 409.1499). 1H (methanol-d 4, 400 MHz) and 13C NMR data (methanol-d 4, 100 MHz), see Table 2. Tada, H.; Yasuda, F.; Otani, K.; Doteuchi, M.; Ishihara, Y.; Shiro, M. Eur. J. Med. Chem. 1991, 26, 345. 13β-methyl,21-dihydroeurycomanone: white amorphous powder; [α]20 : +51.4 D -1 (c=0.1 in MeOH); IR (film) νmax 3398, 1647, 1016, 698 cm ; HRESIMS m/z 411.1643 [M + H]+ (calcd for C20H27O9, 411.1655), 409.1628 [M – H]- (calcd for C20H25O9, 409.1499). 1H (methanol-d4, 400 MHz) and 13C NMR data (methanol-d 4, 100 MHz), see Table 2. Lin, Z. X.; Hoult, J. R. S.; Raman, A. J. Ethnopharmacol. 1999, 66, 141.
12
Table 1. NMR Spectroscopic Data for Compounds 1 – 3 1 δHa, c (J in Hz) 5.98 (s) 5.82 (s) 1.86 (d, 16.0), 2.24 (dd, 6.0, 16.0) 7 85.05 4.80 8 57.20 9 137.79 10 49.80 11 145.65 12 197.08 13 49.50 3.48 14 80.80 15 71.12 4.64 (s) 16 174.36 18 16.67 2.21 (s) 19 23.74 1.60 (s) 20 64.37 3.79 (d, 11.8), 4.05 (d, 11.8) 21 10.19 1.36 (d, 6.0) a Measured in methanol-d4 Pos. 2 3 4 5 6
δCa, b 173.99 120.69 170.53 88.47 40.60
b
100 MHz
c
400 MHz
δCa, b 173.89 120.82 170.47 88.53 40.58 85.11 56.56 139.28 49.98 145.54 198.21 45.74 78.39 71.87 173.85 16.69 23.74 67.88 14.06
2 δHa, c (J in Hz) 6.00 (s) 5.76 (s) 1.88 (d, 16.0), 2.28 (dd, 6.0, 16.0) 4.97 (d, 6.0) 3.28 4.57 (s) 2.21 (s) 1.60 (s) 3.75 (d, 11.6), 3.82 (d, 11.6) 1.44 (d, 7.6)
Assignments were done by HSQC, HMBC, COSY and ROESY experiments.
13
3 δCa, b 174.00 120.99 170.54 88.69 41.04 85.57 57.36 139.62 50.48 146.48 184.41 144.54 81.43 70.79 173.54 16.84 23.86 66.76 125.53
δHa, c (J in Hz) 6.01 (s) 5.82 (s) 1.94 (d, 16.0), 2.25 (d, 6.0) 4.96 (d, 6.0) 4.65 (s) 2.23 (s) 1.64 (s) 3.61 (d, 11.6), 3.81 (d, 11.6) 5.90 (s), 6.47 (s)
Table 2. NMR Spectroscopic Data for Compounds 4 – 6 Pos. 1 2 3
83.83 68.44 45.84
4 5
73.04 170.85
4 (J in Hz) 3.19 (d, 8.8) 4.09 1.46 (d, 13.4) 2.09 (dd, 4.8, 13.4) -
6
122.91
7 8 9 10 11 12 13 14 15 16 17 18 19
202.80 48.81 49.82 47.29 70.09 85.72 33.02 54.38 179.06 29.17 19.90 23.16 16.89
δCa, b
δHa, c
5 δCa, b
Pos. 1 2 3
82.02 66.87 41.75
4 5
129.98 128.82
5.96 (s)
6
29.15
1.84 (d, 3.6) 4.83 4.28 (d, 5.2) 2.98 (m) 2.84 (s) 1.39 (s) 1.73 (s) 1.49 (s) 1.11 (d, 6.8)
7 8 9 10 11 12 13 14 15 16 18 19 20
76.68 46.93 54.35 47.78 75.79 177.87 53.91 79.70 68.84 175.54 19.98 16.04 69.61
21
11.44
a
Measured in methanol-d4
b
100 MHz
c
400 MHz
δHa, c
(J in Hz) 3.38 (d, 10.2) 3.74 (m) 2.12, 2.34
84.37 198.77 126.03
δHa, c (J in Hz) 4.21 (s) 6.05 (s)
-
164.10 43.45
2.77 (d, 13.0)
2.58 (d, 15.6) 2.96 (d, 15.6) 4.29 (s) 2.26 (s) 2.22 (m) 4.52 (s) 1.70 (s) 1.18 (s) 4.36 (d, 12.4) 5.02 (d, 12.4) 1.20 (d, 6.0)
Assignments were done by HSQC, HMBC, COSY and ROESY experiments.
14
6 δCa, b
25.62 73.95 51.29 46.55 46.08 110.36 81.41 33.13 78.27 74.02 173.61 22.99 9.80 71.85 9.12
2.16, 2.29 4.71 (d, 4.1) 3.17 (s) 3.51 (d, 3.9) 2.51 (dt) 4.22 (s) 2.03 (s) 1.13 (s) 3.65 (d, 9.9) 4.27 (d, 3.8) 1.08 (d, 7.0)
Table 3. Cytotoxicity of Compounds 1 – 15 in A549 and HeLa Cells A549 1 uM
10 uM
100 uM
1 uM
10 uM
100 uM
1
101.8 ± 0.39
104.1 ± 0.45
102.7 ± 0.60
102.2 ± 0.42
103.2 ± 0.76
105.5 ± 1.57
2
101.1 ± 0.65
104.4 ± 0.11
103.9 ± 1.81
102.5 ± 0.21
104.0 ± 0.25
105.6 ± 0.04
3
100.4 ± 0.13
101.7 ± 0.62
100.1 ± 0.15
101.6 ± 0.51
103.5 ± 0.47
101.0 ± 0.64
4
96.64 ± 0.43
101.5 ± 0.77
101.3 ±0.30
101.2 ± 0.40
102.3 ± 0.17
101.9 ± 0.42
5
103.0 ± 0.73
103.0 ± 0.86
103.0 ± 0.82
105.5 ± 0.51
105.4 ± 0.28
103.2 ± 0.04
6
99.17 ± 0.43
101.0 ± 0.43
99.82 ± 1.01
99.77 ± 0.25
102.0 ± 0.32
101.0 ± 0.64
7
102.0 ± 0.26
102.0 ± 0.26
100.0 ± 1.25
104.4 ± 1.10
103.5 ± 0.74
103.1 ± 0.66
8
101.0 ± 0.04
71.54 ± 0.95
41.50 ±1.64
105.0 ± 1.13
77.65 ± 1.87
44.46 ± 1.06
9
100.5 ± 0.41
91.77 ± 0.84
67.35 ± 1.70
100.4 ± 0.08
75.55 ± 4.08
50.81 ± 1.25
10
96.78 ± 1.77
101.4 ± 0.17
87.22 ± 0.41
100.9 ± 0.47
97.47 ± 3.08
47.03 ± 5.65
11
96.21 ± 0.32
102.0 ± 0.69
99.10 ± 0.34
101.0 ± 0.02
101.0 ± 0.17
61.66 ± 2.27
12
99.66 ± 0.86
99.79 ± 0.58
66.06 ± 3.66
101.0 ± 0.13
102.0 ± 0.13
66.9 ± 6.67
13
96.80 ± 0.50
101.0 ± 0.22
101.0 ± 0.60
99.71 ± 0.87
101.0 ± 0.47
98.74 ± 2.59
14
99.47 ± 0.04
86.42 ± 0.22
43.30 ± 1.27
100.6 ± 0.93
71.58 ± 4.76
21.01 ± 2.46
99.80 ± 0.26
101.0 ± 0.45
99.32 ± 0.56
101.0 ± 0.13
103.0 ± 0.36
100.0 ± 0.62
15 a
epirubicin a
HeLa
0.61 ± 0.04
24.30 ± 9.49
Epirubicin used as a positive control.
15
Figure Legends Figure 1. Chemical structures of compounds 1 – 15. Figure 2. The key HMBC and COSY correlations of compounds 1 – 6. Figure 3. The key NOESY/ROESY correlations of compounds 1 – 5.
16
Figure 1. O
O 18
HO
13
11
19
OH
20
4 2 10
O H
HO OH
OH O
6
H
OH
11 18 15
O
HO OH
12
OH OH
1 10
OH
8
10
O
O
O
O
O
OH
16
4
5
18
6 O
OH
OH
HO
HO
HO OH
OH O
OH OH O O
O
14
8
4
O
OH
HO
1
Me
21
20
11
19
O
O
3 OH
O OH
O
19
O
O
2
O
HO
O
H O
1
17
OH O
O
16
HO
OH
OH
8
O
OH
OH
OH
OH
14
O
O
21
HO
O OH OH
O
O H O
O
O
7
OH
OH
HO OH
9
8 OH
OH
OH
O
O
O
OH
O OH
OH
O
HO
HO OH
O
O
O
O
O
O
11
10
12
OH
OH
OH
OH
HO
HO OH
HO
OH
OH OH
HO
O
O
O
OH OH
O
OH
O
O O
O
O OH
OH
13
15
14
17
O
Figure 2.
18
Figure 3.
19
Graphical Abstract
Five new quassinoids and cytotoxic constituents from the roots of Eurycoma longifolia SeonJu Park, Nguyen Xuan Nhiem, Phan Van Kiem, Chau Van Minh, Bui Huu Tai, Nanyoung Kim, Jae-Hyoung Song, Hyun-Jeong Ko and Seung Hyun Kim
20
S0960-894X(14)00686-6 http://dx.doi.org/10.1016/j.bmcl.2014.06.058 BMCL 21777
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date: Revised Date: Accepted Date:
18 March 2014 11 June 2014 20 June 2014
Please cite this article as: Park, S., Nhiem, N.X., Kiem, P.V., Minh, C.V., Tai, B.H., Kim, N., Song, J-H., Ko, H-J., Kim, S.H., Five new quassinoids and cytotoxic constituents from the roots of Eurycoma longifolia, Bioorganic & Medicinal Chemistry Letters (2014), doi: http://dx.doi.org/10.1016/j.bmcl.2014.06.058
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Five new quassinoids and cytotoxic constituents from the roots of Eurycoma longifolia
SeonJu Parka, Nguyen Xuan Nhiema,b, Phan Van Kiem b, Chau Van Minhb, Bui Huu Taib, Nanyoung Kima, Jae-Hyoung Songc, Hyun-Jeong Koc, and Seung Hyun Kima,∗
a
College of Pharmacy, Yonsei Institute of Pharmaceutical Science, Yonsei University, Incheon 406-840, Korea b Institute of Marine Biochemistry, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet, Caugiay, Hanoi, Vietnam c Laboratory of Microbiology and Immunology, College of Pharmacy, Kangwon National University, Chuncheon 200-701, Korea
∗ Corresponding author. Tel.: +82-32-749-4514; fax: +82-32-749-4105; e-mail: [email protected]
1
ABSTRACT Eurycoma longifolia has been widely used for various traditional medicinal purposes in South-East Asia. In this study, five new quassinoids, eurylactone E (1), eurylactone F (2), eurylactone G (3), eurycomalide D (4), and eurycomalide E (5), along with ten known quassinoids (6 − 15) were isolated from the roots of E. longifolia. Their structures were determined by extensive spectroscopic methods, including 1D and 2D NMR, and MS spectra data. Among the isolated compounds, 13β-methyl,21-dihydroeurycomanone (6) has been reported as a synthetic derivative. However, it was isolated from the natural product for the first time in this study. The cytotoxic activities of fifteen compounds were evaluated against human lung cancer cell line, A549 and human cervical cancer cell line, HeLa.
Keywords Eurycoma longifolia; quassinoid; cytotoxicity; A549 (human lung cancer cell); HeLa (human cervical cancer cell).
2
Eurycoma longifolia (Simaroubaceae), commonly known as Tongkat Ali, is an herbal medicinal plant popularly used in South-East Asian countries. The plant extract has been used in local traditional medicines for antimalarial, anti-pyretic, and antiulcer as well as for enhancing testosterone levels in men.1, 2 Tongkat Ali can be consumed as a tonic drink or available in the health-food market either in the form of raw crude powder, or capsule mixed with other aphrodisiac herbs. It is also available as an additive mixed with coffee or in certain health products as a replacement for ginseng.1,
3
Since many recent studies proved its efficacy as a general health and
testosterone booster, and for its known aphrodisiac properties, both popularity and its demand increased.1-3 Although quassinoid, alkaloid, and squalene derivatives are reported as major chemical components of Tongkat Ali,4-9 studies of its chemical constituent have been not carried out extensively but rather recent studies were mainly focused on its extract or fraction-based bioactivities of anti-angiogenic, antiosteoporotic, reproductive, and genocytotoxic activities.10-13 Studying chemical constituent is important for consuming product concerning on the quality of the food supply, mainly food adulteration and contamination issues. Therefore, it necessitates explorative study of chemical constituents of Tongkat Ali, universally consuming natural product for one’s interest. In the present study, along with 5 newly isolated compounds, total 15 quassinoids of four different types: eurycomalactone, laurycolactone, klaineanone, and longilactone were isolated from E. longifolia. The structures of new compounds (1 – 5) were elucidated on the basis of spectroscopic analysis and comparison with literature data. Among 10 known compounds, 13β-methyl,21-dihydroeurycomanone (6) was isolated from a natural source for the first time. These compounds were screened for 3
in-vitro cytotoxicity against A549 and HeLa tumor cell lines. The roots of E. longifolia were collected in Dak Lak province, Vietnam, in March 2013, and authenticated by Dr. Bui Van Thanh in Institute of Ecology and Biological Resources, Vietnamese Academy of Science and Technology, Vietnam. A voucher specimen was deposited at the Herbarium of Pharmacy, Yonsei Institute of Pharmaceutical Sciences, Yonsei University, Incheon, Korea. The dried roots of E. longifolia (18.0 kg) were extracted with MeOH (3 × 10 L, 50℃) under sonication for 4 h to yield 400.0 g extract after evaporation of the solvent. This extract was suspended in H2O and successively partitioned with CHCl3 and n-BuOH to obtain the CHCl3 (EL1, 105.0 g), n-BuOH (EL2, 234.0 g), and H2O (EL3, 60.0 g) extracts after removal of the solvents in vacuo. Using various chromatographic resin and isolation techniques, fifteen quassinoids were isolated.14 Compound 1 was obtained as a white amorphous powder and its molecular formula was determined as C19H22O9, by the HR-ESI-MS [M + H]+ ion at m/z 395.1316 (calcd for C19H23O9, 395.1342). The 1H NMR spectrum showed three methyl resonances at δH 1.36 (3H, d, J = 6.0 Hz), 1.60 (3H, s) and 2.21 (3H, s) and the methylene protons at δH 1.86 (d, J = 16.0 Hz) and 2.24 (dd, J = 6.0, 16.0 Hz). One olefinic proton at δH 5.98, and five oxymethine protons at δH 3.79 (d, J = 11.8 Hz), 4.05 (d, J = 11.8 Hz), 4.64, 4.80 and 5.82 were also observed (Table 1). The
13
C-NMR and DEPT spectra of 1
revealed 19 carbons signals, including nine quaternary (δC 49.80, 57.20, 80.81, 137.80, 145.66, 170.54, 173.99, 174.35, and 197.08), five methine (δC 49.50, 71.13, 85.06, 88.48, and 120.69), two methylene (δC 40.60, and 64.37), and three methyl carbons (δC 10.20, 16.67, and 23.75). The NMR data of 1 were similar to those of eurylactone 4
A6 except for the addition of one carbonyl and one hydroxymethyl group and the presence of double bond. The position of three carbonyls were verified by HMBC correlations between H-5 (δH 5.82) and C-2 (δC 173.99), between H-21 (δH 1.36) and C-12 (δC 197.08), and between H-7 (δH 4.80) and C-16 (δC 174.35), leading three carbonyls were located at C-2, C-12, and C-16. The HMBC correlations between H20 (δH 3.79 and 4.05) and C-7 (δC 85.05), C-9 (δC 137.79), and C-14 (δC 80.80) suggested the presence of one hydroxymethyl group at C-8. The presence of double bond between C-9 and C-11 was confirmed by HMBC correlations between H-19 (δH 1.60) and quaternary C-9 (δC 137.79) and between H-13 (δH 3.48) and quaternary C11 (δC 145.65) (Figure 2). Since compound 1 is a biogenetic derivative of quassinoids from E. longifolia, it was supposed to have the same configurations at C-8 and C-10 of eurylactone A. ROESY correlations between H-19 (δH 1.60) and H-18 (δH 2.21); H-7 (δH 4.80) and H-20 (δH 3.79 and 4.05) suggested the configuration of both H-5 and H-7 to be β. Furthermore, ROESY correlation between H-15 (δH 4.64) and H-21 (δH 1.36) as well as no ROESY correlations between H-15 (δH 4.64) and H-20 (δH 3.79 and 4.05) confirmed the configurations of H-15 and methyl group at C-13 to be α. Based on the evidence above, compound 1 was established and named eurylactone E.15 Compound 2 was isolated as a white amorphous powder. HR-ESI-MS spectra resulted as the same molecular formula as that of 1. The 1H and
13
C NMR, HSQC,
HMBC, and COSY analyses of 2 indicated the same constitution as those of 1. The chemical shifts of C-13 (δC 45.74) and C-21 (δC 14.06) in 2 were different from the chemical shifts of C-13 (δC 49.50) and C-21 (δC 10.19) in 1, suggesting the possibility of different configurations at the chiral carbon C-13 (Table 1). Moreover, the ROESY 5
correlation between H-20 (δH 3.75 and 3.82) and H-21 (δH 1.44); H-7 (δH 4.97) and H20; H-13 (δH 3.28) and H-15 (δH 4.57) suggested that the configuration of the methyl group at C-13 to be β. Thus, the structure of 2 was elucidated and named eurylactone F.16 Compound 3 was obtained as a white amorphous powder and its molecular formula was deduced as C19H20O9, by the HR-ESI-MS ion [M*]- at m/z 392.1080 (calcd for C19H20O9, 392.1107). The 1H NMR spectrum showed two methyl resonances: a singlet at δH 1.64 (3H, s) and 2.23 (3H, s). Three olefinic protons at δH 5.90, 6.01 and 6.47, and five oxymethine protons at δH 3.61 (d, J = 11.6 Hz), 3.81 (d, J = 11.6 Hz), 4.65, 4.96 (d, J = 6.1 Hz) and 5.82 were also observed (Table 1). The 13C-NMR and DEPT spectra of 3 revealed 19 carbons signals including ten quaternary (δC 50.48, 57.36, 81.43, 139.62, 144.57, 146.48, 170.54, 173.54, 174.00, and 184.41), four methine (δC 70.79, 85.57, 88.69, and 120.99), three methylene (δC 41.04, 66.75, and 125.53), and two methyl carbons (δC 16.84, and 23.86). One additional double bond at C-13/C-21 was observed compared to compounds 1 and 2. This was confirmed by HMBC correlations between H-21 (δH 5.90 and 6.47) and C-12 (δC 184.41), C-13 (δC 144.54), and C-14 (δC 81.43). Compound 3 was supposed to be a biogenetic derivative resulted from the dehydrogenation of 1 and 2. The important NOESY correlations shown in Figure 2 proved the configurations of chiral carbons. Based on these, the compound 3 was named eurylactone G.17 Compound 4 was obtained as a white amorphous powder. HR-ESI-MS exhibited [M + H]+ signal at m/z 367.1755 (calcd for C19H27O7, 367.1757), suggesting molecular formula of C19H26O7. The
1
H NMR spectrum showed four methyl
resonances at δH 1.11 (3H, d, J = 6.8 Hz), 1.39 (3H, s), 1.49 (3H, s) and 1.73 (3H, s), 6
one methylene proton at δH 1.46 (d, J = 13.4 Hz) and 2.09 (dd, J = 4.8, 13.4 Hz), and three methine protons at δH 1.84 (d, 4.0), 2.84 (s), and 2.98 (m). One olefinic proton at δH 5.96 (s), and four oxymethine protons at δH 3.19 (d, J = 8.8 Hz), 4.09, 4.28 (d, J = 5.2 Hz), and 4.83 were also detected (Table 2). The 13C-NMR and DEPT spectra of 4 revealed 19 carbons signals, including six quaternary (δC 47.29, 48.81, 73.04, 170.85, 179.06, and 202.80), eight methine (δC 33.02, 49.82, 54.37, 68.44, 70.09, 83.83, 85.72 and 122.91), one methylene (δC 45.84), and four methyl carbons (δC 16.89, 19.90, 23.16, and 29.18). The NMR data of 4 were similar to those of eurycomalide B18 except for the appearance of hydroxyl group at C-4. This location was determined by the HMBC correlation between H-16 (δH 1.39) and C-3 (δC 45.84), C-4 (δC 73.04), and C-5 (δC 170.85) (Figure 2). The NOESY correlations between H-17 and H-2; H18 and H-13 suggested that the configurations of hydroxyl group at C-2 and methyl group at C-8 are α and β, respectively. Moreover, all β-configurations of three hydroxyl groups at C-1, C-4, and C-11 were proved by the NOESY correlations between H-1/H-9, H-1/H-11, and H-1/H-16. Based on the evidence above, the structure of eurycomalide D19 was established. Compound 5 was isolated as white amorphous powder and its molecular formula was determined as C19H22O9, by the HR-ESI-MS [M + H]+ ion at m/z 411.1638 (calcd for C20H27O9, 411.1655). The 1H NMR spectrum showed three methyl resonances at δH 1.18 (3H, s), 1.20 (3H, d, J = 6.0 Hz), and 1.70 (3H, s), three methylene proton at δH 2.12 and 2.34, δH 2.58 (d, J = 15.6 Hz) and δH 2.96 (d, J = 15.6 Hz), and 4.36 (d, J = 12.4 Hz) and 5.02 (dd, J = 12.4 Hz), and two methine protons at δH 2.22 (m), and 2.26 (s). Four oxymethine protons at δH 3.28 (d, J = 10.2 Hz), 3.72 (m), 4.29 (s), and 4.52 (s) were also observed (Table 2). The 13C-NMR and DEPT spectra of 5 revealed 7
20 carbons signals, including eight quaternary (δC 46.93, 47.89, 75.79, 79.70, 128.82, 129.98, 175.54 and 177.87), six methine (δC 53.91, 54.36, 66.87, 68.85, 76.69, and 82.03), three methylene (δC 29.16, 41.75 and 69.62), and three methyl carbons (δC 11.44, 16.04, and 19.98). The NMR data of 5 were similar to those of shinjudilactone20 except for the removal of one carbonyl group and the addition of two oxymethines. The HMBC correlation between H-1 (δH 3.38 (d, J = 10.2 Hz)) and C-2 (δC 66.87) suggested that instead of carbonyl group at C-2 as in shinjudilactone,20 hydroxyl group is attached. In addition, attachment of hydroxyl group at C-14 and C15 was determined by the HMBC correlations between H-21 (δH 1.20 (d, J = 6.0 Hz)), and H-20 (δH 4.36 (d, J = 12.4 Hz) and 5.02 (dd, J = 12.4 Hz)) and C-14 (δC 79.70) and between H-15 (δH 4.52) and C-14 (δC 79.70), and C-16 (δC 175.54) (Figure 2). The NOESY correlations between H-19 (δH 1.18) and H-2 (δH 3.74) and between H21 (δH 1.20) and H-15 (δH 4.52) determined the configurations of C-2 and C-15 as S and R, respectively (Figure 3). Thus, the structure of 5 was determined and named eurycomalide E.21 13β-methyl,21-dihydroeurycomanone (6) was isolated as a white amorphous powder and was isolated from the natural product for the first time in the present investigation. Previously reported 13β-methyl,21-dihydroeurycomanone was obtained by hydrogenating eurycomanone catalyzed by (Ph3P)3RhCl.22 HR-ESI-MS spectra showed [M + H]+ signal at m/z 411.1643 (calcd for C20H27O9, 411.1655), implying the molecular formula of C20H26O9. The 1H NMR spectrum showed three methyl resonances at δH 1.08 (3H, d, J = 6.0 Hz), 1.13 (3H, s), and 2.03 (3H, s), two methylene protons at δH 2.16 and 2.29, and δH 3.65 and 4.27, and one methine proton at δH 1.08 (d, J = 7.0 Hz). One olefinic proton at δH 6.05 and four oxymethine protons 8
at δH 3.51 (d, J = 3.9 Hz), 4.21 (s), 4.22 (s), and 4.71 (d, J = 4.1 Hz) were also observed (Table 2). The 13C-NMR and DEPT spectra of 6 revealed 20 carbons signals, including seven quaternary (δC 46.08, 51.29, 78.27, 110.36, 164.10, 173.61 and 198.77), eight methine (δC 33.13, 43.45, 46.55, 73.95, 74.02, 81.41 and 84.37), two methylene (δC 25.62, and 71.85), and three methyl carbons (δC 9.12, 9.80, and 22.99). 1
H and 13C NMR data of 6 were very similar to those of 13,21-dihydroeurycomanone4
except for C-13 and C-21 chemical shifts, suggesting the possibility of different configurations at the chiral carbon C-13 (Table 2), deducing the structure identification as 13β-methyl,21-dihydroeurycomanone.23 Comparison of the NMR and MS data with reported values in the literature led to identification of the structures of the known compounds to be eurylactone A (7),6 eurycomalactone (8),5 13,21-dihydroeurycomanone (9),4 eurycomanone (10),4 13α(21)-epoxyeurycomanone
(11),8
longilactone
(12),4
2-dihydro-18-
dedihydrolongilactone (13),7 14,15β-dihydroxyklaineanone (14),4 and 5α,14β,15βtrihydroxyklaineanone (15)18 (Figure 1). All compounds were tested for cytotoxicity against A549 and HeLa cell lines. Kuo et al. previously reported cytotoxic constituents from the E. longifolia against human lung cancer (A549) and human breast cancer (MCF-7).18 In this study, the cytotoxicity was evaluated by modified the SRB assay previously described.24 In our experimental condition, compounds 8, 12, and 14 showed significant cytotoxicity in both A549 and MCF-7. Compound 9 was more selective against A549 and compound 10 showed cytotoxic effect only in MCF-7. The cytotoxic effects of the tested compounds in A549 were almost same as the previous report18 (Table 3). In HeLa cell line, compounds 8 – 12 and 14 displayed significant cytotoxicity showing the relative 9
cell viability ranging from 21.01 ± 2.46% to 66.9 ± 6.67% at the concentration of 100 µM (Table 3). Among them, compound 14 showed the most potent cytotoxicity in HeLa cell line with IC50 value of 48.18 µM.
Acknowledgements This research was supported by Ministry of Food and Drug Safety (13182MFDS725), Republic of Korea.
10
References and notes 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.
11. 12. 13. 14.
Abdullah, M. Z.; Rahman, A. S. A.; Shakaff, A. Y. M.; Noor, A. M. T. I. Meas. Control. 2004, 26, 19. Ang, H. H.; Cheang, H. S.; Yusof, A. P. M. Exp. Anim. 2000, 49, 35. Tambi, M. I. B. M.; Imran, M. K.; Henkel, R. R. Andrologia. 2012, 44, 226. Morita, H.; Kishi, E.; Takeya, K.; Itokawa, H.; Tanaka, O. Chem. Lett. 1990, 19, 749. Chan, K. L.; Iitaka, Y.; Noguchi, H.; Sugiyama, H.; Saito, I.; Sankawa, U. Phytochem. 1992, 31, 4295. Itokawa, H.; Qin, X.-R.; Morita, H.; Takeya, K. J. Nat. Prod. 1993, 56, 1766. Itokawa, H.; Qin, X.-R.; Morita, H.; Takeya, K.; Iitaka, Y. Chem. Pharm. Bull. 1993, 41, 403. Morita, H.; Kishi, E.; Takeya, K.; Itokawa, H.; Iitaka, Y. Phytochem. 1993, 33, 691. Chua, L. S.; Amin, N. A. M.; Neo, J. C. H.; Lee, T. H.; Lee, C. T.; Sarmidi, M. R.; Aziz, R. A. J. Chromatogr. B. 2011, 879, 3909. Al-Salahi, O. S. A.; Kit-Lam, C.; Majid, A. M. S. A.; Al-Suede, F. S. R.; Mohammed Saghir, S. A.; Abdullah, W. Z.; Ahamed, M. B. K.; Yusoff, N. M. Microvasc. Res. 2013. Ahmad, N. S.; Mohd, F. A. B.; Tajul, A. A. S.; Norliza, M.; Norazlina, M.; Ima, N. S. Aging Male. 2011, 14, 150. Solomon, M. C.; Erasmus, N.; Henkel, R. R. Andrologia. 2013, n/a. Li, C.-H.; Liao, J.-W.; Liao, P.-L.; Huang, W.-K.; Tse, L.-S.; Lin, C.-H.; Kang, J.-J.; Cheng, Y.-W. Evid. Based Complement. Alternat. Med. 2013, 2013, 11. The EL1 fraction (105.0 g) was chromatographed on a silica gel column and eluting with a gradient of n-hexane – acetone (40 : 1 → 0 : 1, v/v) to obtain six sub-fractions, EL1A (14.2 g), EL1B (11.3 g), EL1C (17.2 g), EL1D (21.6 g), EL1E (25.2 g), and EL1F (7.3 g). The EL1D fraction was chromatographed on silica gel column eluting with CHCl3 – acetone (6 : 1, v/v) to yield 8 (407.0 mg). The EL2 fraction was chromatographed on a Diaion HP-20P column eluting with H2O containing increasing concentrations of MeOH (0, 25, 50, 75, and 100%) to obtain five sub-fractions EL2A (82.0 g), EL2B (26.3 g), EL2C (32.8 g), EL2D (12.4 g), and EL2E (72.5 g). The EL2B fraction was chromatographed on a silica gel column eluting with CHCl3 – MeOH (8 : 1, v/v) to yield 9 (109.0 mg), 10 (875.0 mg), 11 (10.0 mg), 12 (110.0 mg), and 13 (58.0 mg). The EL2C fraction was chromatographed on HPLC using J’sphere ODS H-80 250 mm × 20 mm column, 11% aq. MeCN, and a flow rate of 5 mL/min to yield 1 (1.3 mg), 2 (12.1 mg), 3 (7.4 mg) and 15 (1.2 mg). The EL2D fraction was chromatographed on HPLC using J’sphere ODS H-80 250 mm × 20 mm column, 15% aq. MeCN, and a flow rate of 5 mL/min to yield 4 (1.0 mg), 5 (1.0 mg), and 6 (10.3 mg). The EL2E fraction was 11
15.
16.
17.
18. 19.
20. 21.
22. 23.
24.
chromatographed on a silica gel column eluting with CHCl3 – acetone (1 : 1, v/v) to yield 7 (5.0 mg), and 14 (45.0 mg). Eurylactone E: white amorphous powder; [α]20 : +114.4 (c=0.1 in MeOH); IR D -1 (film) νmax 3415, 1748, 1655, 1116, 1042 cm ; HRESIMS m/z 393.1183 [M – H]- (calcd for C20H21O9, 393.1186). 1H (methanol-d4, 400 MHz) and 13C NMR data (methanol-d4, 100 MHz), see Table 1. Eurylactone F: white amorphous powder; [α]20 : +81.1 (c=0.1 in MeOH); IR D -1 (film) νmax 3355, 1660, 1049, 1032, 1016 cm ; HRESIMS m/z 395.1325 [M + H]+ (calcd for C19H23O9, 395.1337). 1H (methanol-d4, 400 MHz) and 13C NMR data (methanol-d4, 100 MHz), see Table 1. Eurylactone G: white amorphous powder; [α]20 : +66.9 (c=0.1 in MeOH); IR D -1 (film) νmax 3363, 1748, 1053, 1032, 1014 cm ; HRESIMS m/z 392.1094 [M*](calcd for C19H20O9, 392.1107). 1H (methanol-d 4, 400 MHz) and 13C NMR data (methanol-d4, 100 MHz), see Table 1. Kuo, P.-C.; Damu, A. G.; Lee, K.-H.; Wu, T.-S. Bioorg. Med. Chem. 2004, 12, 537. Eurycomalide D: white amorphous powder; [α]20 : +205.8 (c=0.1 in MeOH); D -1 IR (film) νmax 3398, 1650, 1016, 707 cm ; HRESIMS m/z 367.1755 [M + H]+ (calcd for C19H27O7, 367.1757). 1H (methanol-d 4, 400 MHz) and 13C NMR data (methanol-d4, 100 MHz), see Table 2. Kubota, K.; Fukamiya, N.; Hamada, T.; Okano, M.; Tagahara, K.; Lee, K.-H. J. Nat. Prod. 1996, 59, 683. Eurycomalide E: white amorphous powder; [α]20 : +19.2 (c=0.1 in MeOH); IR D (film) νmax 3465, 1614, 1070, 616 cm-1; HRESIMS m/z 411.1638 [M + H]+ (calcd for C20H27O9, 411.1655), 409.1494 [M – H]- (calcd for C20H25O9, 409.1499). 1H (methanol-d 4, 400 MHz) and 13C NMR data (methanol-d 4, 100 MHz), see Table 2. Tada, H.; Yasuda, F.; Otani, K.; Doteuchi, M.; Ishihara, Y.; Shiro, M. Eur. J. Med. Chem. 1991, 26, 345. 13β-methyl,21-dihydroeurycomanone: white amorphous powder; [α]20 : +51.4 D -1 (c=0.1 in MeOH); IR (film) νmax 3398, 1647, 1016, 698 cm ; HRESIMS m/z 411.1643 [M + H]+ (calcd for C20H27O9, 411.1655), 409.1628 [M – H]- (calcd for C20H25O9, 409.1499). 1H (methanol-d4, 400 MHz) and 13C NMR data (methanol-d 4, 100 MHz), see Table 2. Lin, Z. X.; Hoult, J. R. S.; Raman, A. J. Ethnopharmacol. 1999, 66, 141.
12
Table 1. NMR Spectroscopic Data for Compounds 1 – 3 1 δHa, c (J in Hz) 5.98 (s) 5.82 (s) 1.86 (d, 16.0), 2.24 (dd, 6.0, 16.0) 7 85.05 4.80 8 57.20 9 137.79 10 49.80 11 145.65 12 197.08 13 49.50 3.48 14 80.80 15 71.12 4.64 (s) 16 174.36 18 16.67 2.21 (s) 19 23.74 1.60 (s) 20 64.37 3.79 (d, 11.8), 4.05 (d, 11.8) 21 10.19 1.36 (d, 6.0) a Measured in methanol-d4 Pos. 2 3 4 5 6
δCa, b 173.99 120.69 170.53 88.47 40.60
b
100 MHz
c
400 MHz
δCa, b 173.89 120.82 170.47 88.53 40.58 85.11 56.56 139.28 49.98 145.54 198.21 45.74 78.39 71.87 173.85 16.69 23.74 67.88 14.06
2 δHa, c (J in Hz) 6.00 (s) 5.76 (s) 1.88 (d, 16.0), 2.28 (dd, 6.0, 16.0) 4.97 (d, 6.0) 3.28 4.57 (s) 2.21 (s) 1.60 (s) 3.75 (d, 11.6), 3.82 (d, 11.6) 1.44 (d, 7.6)
Assignments were done by HSQC, HMBC, COSY and ROESY experiments.
13
3 δCa, b 174.00 120.99 170.54 88.69 41.04 85.57 57.36 139.62 50.48 146.48 184.41 144.54 81.43 70.79 173.54 16.84 23.86 66.76 125.53
δHa, c (J in Hz) 6.01 (s) 5.82 (s) 1.94 (d, 16.0), 2.25 (d, 6.0) 4.96 (d, 6.0) 4.65 (s) 2.23 (s) 1.64 (s) 3.61 (d, 11.6), 3.81 (d, 11.6) 5.90 (s), 6.47 (s)
Table 2. NMR Spectroscopic Data for Compounds 4 – 6 Pos. 1 2 3
83.83 68.44 45.84
4 5
73.04 170.85
4 (J in Hz) 3.19 (d, 8.8) 4.09 1.46 (d, 13.4) 2.09 (dd, 4.8, 13.4) -
6
122.91
7 8 9 10 11 12 13 14 15 16 17 18 19
202.80 48.81 49.82 47.29 70.09 85.72 33.02 54.38 179.06 29.17 19.90 23.16 16.89
δCa, b
δHa, c
5 δCa, b
Pos. 1 2 3
82.02 66.87 41.75
4 5
129.98 128.82
5.96 (s)
6
29.15
1.84 (d, 3.6) 4.83 4.28 (d, 5.2) 2.98 (m) 2.84 (s) 1.39 (s) 1.73 (s) 1.49 (s) 1.11 (d, 6.8)
7 8 9 10 11 12 13 14 15 16 18 19 20
76.68 46.93 54.35 47.78 75.79 177.87 53.91 79.70 68.84 175.54 19.98 16.04 69.61
21
11.44
a
Measured in methanol-d4
b
100 MHz
c
400 MHz
δHa, c
(J in Hz) 3.38 (d, 10.2) 3.74 (m) 2.12, 2.34
84.37 198.77 126.03
δHa, c (J in Hz) 4.21 (s) 6.05 (s)
-
164.10 43.45
2.77 (d, 13.0)
2.58 (d, 15.6) 2.96 (d, 15.6) 4.29 (s) 2.26 (s) 2.22 (m) 4.52 (s) 1.70 (s) 1.18 (s) 4.36 (d, 12.4) 5.02 (d, 12.4) 1.20 (d, 6.0)
Assignments were done by HSQC, HMBC, COSY and ROESY experiments.
14
6 δCa, b
25.62 73.95 51.29 46.55 46.08 110.36 81.41 33.13 78.27 74.02 173.61 22.99 9.80 71.85 9.12
2.16, 2.29 4.71 (d, 4.1) 3.17 (s) 3.51 (d, 3.9) 2.51 (dt) 4.22 (s) 2.03 (s) 1.13 (s) 3.65 (d, 9.9) 4.27 (d, 3.8) 1.08 (d, 7.0)
Table 3. Cytotoxicity of Compounds 1 – 15 in A549 and HeLa Cells A549 1 uM
10 uM
100 uM
1 uM
10 uM
100 uM
1
101.8 ± 0.39
104.1 ± 0.45
102.7 ± 0.60
102.2 ± 0.42
103.2 ± 0.76
105.5 ± 1.57
2
101.1 ± 0.65
104.4 ± 0.11
103.9 ± 1.81
102.5 ± 0.21
104.0 ± 0.25
105.6 ± 0.04
3
100.4 ± 0.13
101.7 ± 0.62
100.1 ± 0.15
101.6 ± 0.51
103.5 ± 0.47
101.0 ± 0.64
4
96.64 ± 0.43
101.5 ± 0.77
101.3 ±0.30
101.2 ± 0.40
102.3 ± 0.17
101.9 ± 0.42
5
103.0 ± 0.73
103.0 ± 0.86
103.0 ± 0.82
105.5 ± 0.51
105.4 ± 0.28
103.2 ± 0.04
6
99.17 ± 0.43
101.0 ± 0.43
99.82 ± 1.01
99.77 ± 0.25
102.0 ± 0.32
101.0 ± 0.64
7
102.0 ± 0.26
102.0 ± 0.26
100.0 ± 1.25
104.4 ± 1.10
103.5 ± 0.74
103.1 ± 0.66
8
101.0 ± 0.04
71.54 ± 0.95
41.50 ±1.64
105.0 ± 1.13
77.65 ± 1.87
44.46 ± 1.06
9
100.5 ± 0.41
91.77 ± 0.84
67.35 ± 1.70
100.4 ± 0.08
75.55 ± 4.08
50.81 ± 1.25
10
96.78 ± 1.77
101.4 ± 0.17
87.22 ± 0.41
100.9 ± 0.47
97.47 ± 3.08
47.03 ± 5.65
11
96.21 ± 0.32
102.0 ± 0.69
99.10 ± 0.34
101.0 ± 0.02
101.0 ± 0.17
61.66 ± 2.27
12
99.66 ± 0.86
99.79 ± 0.58
66.06 ± 3.66
101.0 ± 0.13
102.0 ± 0.13
66.9 ± 6.67
13
96.80 ± 0.50
101.0 ± 0.22
101.0 ± 0.60
99.71 ± 0.87
101.0 ± 0.47
98.74 ± 2.59
14
99.47 ± 0.04
86.42 ± 0.22
43.30 ± 1.27
100.6 ± 0.93
71.58 ± 4.76
21.01 ± 2.46
99.80 ± 0.26
101.0 ± 0.45
99.32 ± 0.56
101.0 ± 0.13
103.0 ± 0.36
100.0 ± 0.62
15 a
epirubicin a
HeLa
0.61 ± 0.04
24.30 ± 9.49
Epirubicin used as a positive control.
15
Figure Legends Figure 1. Chemical structures of compounds 1 – 15. Figure 2. The key HMBC and COSY correlations of compounds 1 – 6. Figure 3. The key NOESY/ROESY correlations of compounds 1 – 5.
16
Figure 1. O
O 18
HO
13
11
19
OH
20
4 2 10
O H
HO OH
OH O
6
H
OH
11 18 15
O
HO OH
12
OH OH
1 10
OH
8
10
O
O
O
O
O
OH
16
4
5
18
6 O
OH
OH
HO
HO
HO OH
OH O
OH OH O O
O
14
8
4
O
OH
HO
1
Me
21
20
11
19
O
O
3 OH
O OH
O
19
O
O
2
O
HO
O
H O
1
17
OH O
O
16
HO
OH
OH
8
O
OH
OH
OH
OH
14
O
O
21
HO
O OH OH
O
O H O
O
O
7
OH
OH
HO OH
9
8 OH
OH
OH
O
O
O
OH
O OH
OH
O
HO
HO OH
O
O
O
O
O
O
11
10
12
OH
OH
OH
OH
HO
HO OH
HO
OH
OH OH
HO
O
O
O
OH OH
O
OH
O
O O
O
O OH
OH
13
15
14
17
O
Figure 2.
18
Figure 3.
19
Graphical Abstract
Five new quassinoids and cytotoxic constituents from the roots of Eurycoma longifolia SeonJu Park, Nguyen Xuan Nhiem, Phan Van Kiem, Chau Van Minh, Bui Huu Tai, Nanyoung Kim, Jae-Hyoung Song, Hyun-Jeong Ko and Seung Hyun Kim
20
