Letter ‐ spectral assignment Received: 13 October 2014
Revised: 30 December 2014
Accepted: 31 December 2014
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI 10.1002/mrc.4214
1H and 13C NMR assignments of new ecdysteroids
from Callisia fragrans
Dan Thi Thuy Hang, Nguyen Thi Minh Hang, Hoang Le Tuan Anh, Nguyen Xuan Nhiem, Cao Thi Hue, Pham Thanh Binh, Nguyen Tien Dat, Nguyen Hoai Nam, Pham Hai Yen, Chau Van Minh, Nguyen Van Hung* and Phan Van Kiem* Introduction Ecdysteroids are a class of insect hormones with a characteristic 7en-6-one moiety in the B-ring of polyhydroxysteroids. They were discovered as insect molting hormones. These compounds display important physiological effects on insects and play defensive role.[1] Ecdysteroids also exert beneficial pharmacological properties such as decreasing the blood cholesterol and glucose level in experimental animals, anticancer, and wound healing activities.[2] The increasing numbers of patents have been deposited concerning various beneficial effects of ecdysteroids in many medical or cosmetic domains, which make ecdysteroids very attractive candidates for several practical uses. Callisia fragrans (Lindl.) belongs to Commelinaceae family and is native in South America. Chemical studies of this plant showed the presence of phenolic compounds[3] and amino acids.[4] In addition, the methanol extract of C. fragrans showed antiherpetic activity.[5] In this paper, we report the isolation and structure determination of three new and two known ecdysteroids from the stems of C. fragrans.
Results and Discussion The stems of C. fragrans were extracted with hot methanol to give methanol extract. This extract was suspended in water and then partitioned with n-hexane and chloroform to give the n-hexane, chloroform, and water layers. Three new and two known ecdysteroids were isolated from the water layer using combined chromatographic separations (refer to Fig. 1). Known ecdysteroids were elucidated to be 2β,3β,11α,14α-tetrahydroxy-5α-androst-7(8)en-6,17-dione (4)[6] and 2β,3β,14α,17β-tetrahydroxy-5α-androst-7 (8)-ene-6-one (5)[6,7] on the basic of spectral data, which were in good agreement with those reported in the literature.[8] These compounds were isolated from Callisia genus for the first time. Compound 1 was obtained as a white amorphous powder, and its molecular formula was determined to be C19H28O6 by HR-ESIMS at m/z 387.1589 [M + Cl] (Calcd. C19H28O6Cl for 387.1580). The 1H NMR spectrum of 1 showed the presence of one olefinic proton at δH 5.79 (d, J = 2.5 Hz), four oxymethine protons at δH 3.97 (ddd, J = 2.5, 2.5, 3.5 Hz), 4.03 (ddd, J = 3.5, 3.5, 12.5 Hz), 4.11 (m), and 4.36 (dd, J = 6.5, 9.0 Hz), two tertiary methyl groups at δH 0.72 (s) and 1.09 (s). The 13C NMR and DEPT spectra showed the signals for 19 carbons including one carbonyl at δC 206.7, two olefinic at δC 122.2 and 165.0, three non-protonated at δC 40.0,
Magn. Reson. Chem. (2015)
48.2, and 83.0, six methine at δC 43.3, 52.9, 68.6, 68.9, 69.4, and 78.9, five methylene at δC 29.8, 31.5, 33.3, 39.1, and 40.3, and two methyl carbons at δC 16.5 and 24.7. The analytical 1H and 13C NMR data (Table 1) suggested that the structure of 1 was similar to 2β,3β,11α,14α-tetrahydroxy-5α-androst-7(8)-en-6,17-dione (4)[6] except for the hydrogenation of carbonyl group at C-17. The HMBC correlations from H-2 (δH 4.03) to C-1 (δC 39.1)/C-3 (δC 68.6)/C-4 (δC 33.3)/C-10 (δC 40.0); from H-3 (δH 3.97) to C-1 (δC 39.1)/C-2 (δC 68.9)/C-4 (δC 33.3)/C-5 (δC 52.9) suggested two hydroxyl groups at C-2 and C-3. The HMBC correlations between H-5 (δH 2.36) and C6 (δC 206.7)/C-7 (δC 165.0); H-7 (δH 5.79) and C-5 (δC 52.9)/C-6 (δC 206.7)/C-8 (δC 165.0)/C-9 (δC 43.3)/C-14 (δC 83.0) suggested the position of carbonyl group at C-6 and the double bond at C-7/C-8. The HMBC correlations from H-18 (δH 0.72) to C-12 (δC 40.3)/C-13 (δC 48.2)/C-14 (δC 83.0)/C-17 (δC 78.9), from H-9 (δH 3.16)/H-12 (δH 1.90 and 2.18) to C-11 (δC 69.4) suggested three hydroxyl groups at C-11, C-14, and C-17. The constitution of 1 was confirmed again by COSY analysis (Fig. 2). Compound 1 was supposed to have the same configurations as 4, a biogenetic derivative of 1, at C-5, C-9, C-18, and C-19. The α or β position of hydroxyl groups were established by a careful analysis of coupling constant of protons as well as by NOESY analysis. The large coupling constant between Hβ-1 and H-2, J = 12.5 Hz; small coupling constant of H-2/Hα-3, J = 2.5 Hz; and NOESY correlations of H-2 (δH 4.03)/Hα-9 (δH 3.16). These suggested the configurations of both hydroxyl groups at C2 and C-3 to be β. Furthermore, the NOESY correlations between H-11 (δH 4.11) and H-18 (δH 0.72)/H-19 (δH 1.09); large coupling constant between H-9 and H-11, J = 9.0 Hz proved the configuration of hydroxyl group at C-11 to be α. Moreover, the NOESY correlations between H-17 (δH 4.36) and Hα-12 (δH 2.18)/Hα-16 (δH 2.30); H-18 (δH 0.72) and Hβ-15 (δH 2.09)/Hβ-16 (δH 1.61) confirmed the α and β configurations of hydroxyl groups at C-14 and C-17, respectively. Based on the previous evidence, the structure of 1 was established to be 2β,3β,11α,14α,17β-pentahydroxy-5α-androst-7(8)-en-6-one, a new compound named callecdysterol A.
* Correspondence to: Phan Van Kiem and Nguyen Van Hung, Institute of Marine Biochemistry, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet, Caugiay, Hanoi, Vietnam. E-mail: [email protected]; [email protected] Institute of Marine Biochemistry, Vietnam Academy of Science and Technology (VAST), 18 Hoang Quoc Viet, Caugiay, Hanoi, Vietnam
Copyright © 2015 John Wiley & Sons, Ltd.
D. T. T. Hang et al.
Figure 1. Structures of 1–5 from C. fragrans.
Table 1. The NMR spectral data of compounds 1–3 in CD3OD Pos.
1 δC
1
39.1
2 3 4
68.9 68.6 33.3
5 6 7 8 9 10 11 12
52.9 206.7 122.2 165.0 43.3 40.0 69.4 40.3
13 14 15
48.2 83.0 31.5
16
29.8
17 18 19
78.9 16.5 24.7
δH (mult., J in Hz) 1.40 (β, dd, 12.5, 12.5) 2.61 (α, dd, 3.5; 12.5) 4.03 (ddd, 3.5, 3.5, 12.5) 3.97 (ddd, 2.5, 2.5, 3.5) 1.70 (β, ddd, 3.5, 3.5, 13.5) 1.79 (α, ddd, 2.5, 13.5, 13.5) 2.36 (β, dd, 3.5, 13.5) — 5.79 (d, 2.5) — 3.16 (α, dd, 2.5, 9.0) — 4.11 (m) 1.90 (β, dd, 6.0, 11.5) 2.18 (α, t, 11.5) — — 1.60 (α, m)2.09 (β, dd, 7.0, 12.0) 1.61 (β, m) 2.30 (α, m) 4.36 (dd, 6.5, 9.0) 0.72 (s) 1.09 (s)
2 δC 35.3 68.7 70.3 36.6 80.6 202.6 120.6 164.4 46.8 46.0 70.2 40.3 48.2 83.0 31.4 29.8 78.8 16.5 17.3
δH (mult., J in Hz) 1.74 (β, dd, 13.5, 13.5) 2.60 (α, dd, 4.5, 13.5) 4.12a 4.00 (ddd, 2.5, 3.0, 3.0) 1.79 (β, dd, 3.0, 15.0) 2.10 (α, dd, 2.5, 15.0) — — 5.85 (d, 3.0) — 3.22 (α, dd, 3.0, 9.0) — 4.13a 1.89 (β, dd, 5.5, 12.0) 2.18 (α, t, 12.0) — — 1.60 (α, m) 2.09 (β, m) 1.60 (β, m) 2.30 (α, m) 4.35 (dd, 6.5, 9.0) 0.72 (s) 1.04 (s)
3 δC 37.4 68.7 68.3 35.8 51.6 207.0 118.8 155.7 136.5 41.0 134.0 35.3 47.9 82.6 31.1 30.1 78.9 15.9 31.6
δH (mult., J in Hz) 1.73 (β, dd, 12.5, 12.5) 2.11 (α, dd, 3.0, 12.5) 3.74 (ddd, 3.0, 3.0, 12.5) 3.87 (ddd, 3.0, 3.0, 3.5) 1.59 (β, ddd, 3.0, 3.5, 13.5) 1.78 (α, ddd, 3.0, 12.5, 13.5) 2.47 (β, dd, 3.5, 12.5) — 5.75 (s) — — — 6.39 (br d, 6.5) 2.21 (β, dd, 6.5, 17.5) 2.66 (α, d, 17.5) — — 1.85 (α, m) 2.12 (β, m) 1.61 (β, m) 2.33 (α, m) 4.44 (dd, 7.0, 9.0) 0.76 (s) 1.13 (s)
a
overlapped signals.
Compound 2 was obtained as a white amorphous powder, and its molecular formula was deduced to be C19H28O7, by the HR-ESIMS ion [M + Cl] at m/z 403.1537 (Calcd. C19H28O7Cl for 403.1530). The 1H and 13C NMR analyses indicated that the structure of 2 was similar to 1 except for the additional hydroxyl group at C-5. The hydroxyl group at C-5 was further confirmed by the HMBC correlations between H-19 (δH 1.04) and C-1 (δC 35.3)/C-5 (δC 80.6)/C-9
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(δC 46.8). In addition, the β configuration of this hydroxyl group was confirmed by detecting 7.4 ppm diamagnetic shift (γ-gauche effect) on the C-19 signal. The positions of the remaining hydroxyl groups were determined by HSQC, HMBC, and COSY spectra. The observation of NOESY correlations between H-2 (δH 4.12) and H-9 (δH 4.13); H-2 (δH 4.12) and H-3 (δH 4.00) confirmed β configuration of both 2,3-hydroxyl groups. The α-orientation of hydroxyl group at C-11
Copyright © 2015 John Wiley & Sons, Ltd.
Magn. Reson. Chem. (2015)
Ecdysteroids from Callisia fragrans
Figure 2. Key HMBC, COSY, and NOESY correlations of 1–3.
was based on the NOESY correlation between H-11 (δH 4.13) and H18 (δH 0.72). Consequently, compound 2 was elucidated to be 2β,3β,5β,11α,14α,17β-hexahydroxy-5α-androst-7(8)-en-6-one, a new compound named callecdysterol B. The molecular formula of 3 was also determined to be C19H26O5, by the HR-ESI-MS ion at m/z: 369.1478 [M + Cl] in the HR-ESI-MS (Calcd. C19H26O5Cl for 369.1470). The 1H-NMR of 3 showed the signals of two olefinic protons at δH 5.75 (s) and 6.39 (br d, J = 6.5 Hz), three oxygenated methine protons at δH 3.74 (dt, J = 3.0, 3.0, 12.5 Hz), 3.87 (ddd, J = 3.0, 3.0, 3.5 Hz), and 4.44 (dd, J = 7.0, 9.0 Hz), two tertiary methyl groups at δH 0.76 (s) and 1.13 (s). The 13C NMR and DEPT spectra of 3 exhibited the presence of 19 carbons including one carbonyl, four olefinic, three non-protonated, four methine, five methylene, and two methyl carbons. The 1H and 13C NMR data of 3 were similar to those of callecdysterol A (1) except for the additional double bond at C-9/C-11. The HMBC correlations between H-7 (δH 5.75) and C-5 (δC 51.6)/C-6 (δC 207.0)/ C-9 (δC 136.5)/C-14 (δC 82.6), between H-11 (δH 6.39) and C-8 (δC 155.7)/C-9 (δC 136.5)/ C-10 (δC 41.0)/C-12 (δC 35.3)/C-13 (δC 47.9) suggested the carbonyl group at C-6 and two double bonds at C-7/C-8 and C-9/C-11. The positions of two hydroxyl groups at C-14 and C-17 were confirmed by HMBC correlations from H-18 (δH 0.76) to C-12 (δC 35.3)/C-13 (δC 47.9)/C-14 (δC 82.6)/C-17 (δC 78.9). In addition, the NOESY correlations between H-18 (δH 0.76) and Hβ-12 (δH 2.21)/Hβ-15 (δH 2.12); Hα-12 (δH 2.66) and H-17 (δH 4.44) suggested the configurations of hydroxyl groups at C-14 and C-17 to be α and β, respectively. Thus, the structure of 3 was elucidated to be 2β,3β,14α,17βtetrahydroxy-5α-androst-7(8),9(11)-dien-6-one, a new compound named callecdysterol C.
Experimental General experimental procedures The HR-ESI-MS was obtained on an AGILENT 6550 iFunnel Q-TOF LC/MS spectrometer. Optical rotations were determined on a Jasco DIP-370 automatic polarimeter. Column chromatography was performed using a silica-gel (Kieselgel 60, 70–230 mesh and 230–400 mesh, Merck) or YMC RP-18 resins (30–50 μm, Fujisilisa Chemical Ltd.), thin layer chromatography using a pre-
Magn. Reson. Chem. (2015)
coated silica gel 60 F254 (0.25 mm, Merck), and RP-18 F254S plates (0.25 mm, Merck). NMR measurements All NMR spectra were recorded on a Bruker AM500 FT-NMR spectrometer operated at 500 and 125 MHz for hydrogen and carbon, respectively. The NMR measurements, including 1H, 13C, DEPT, HSQC, HMBC, COSY, and NOESY experiments, were carried out using 5-mm probe tubes at temperature of 304.5 K in CD3OD solutions, with TMS as the internal standard.[9] The pulse conditions were as follows: for the 1H spectrum, spectrometer frequency (SF) = 500.13 MHz, acquisition time (AQ) = 3.2769 s, relaxation delay (RD) = 1.0 s, pulse width = 10.5, flip angle = 30.0o, spectral width (SW) = 10 000 Hz, and digital resolution (DR) = 0.15 Hz; for the 13C spectrum, SF = 125.76 MHz, AQ = 1.0 s, RD = 2 s, pulse width = 6.4, SW = 31446.5 Hz, and DR = 0.48 Hz; the mixing time for the NOESY spectrum was 0.3 s. Chemical shifts are reported in parts per million from TMS. Plant material The stems of C. fragrans were collected in Thuynguyen, Haiphong, Vietnam during September, 2012, and identified by Prof. Dr. Tran Huy Thai, Institute of Ecology and Biological Resources, VAST, Vietnam. A voucher specimen (CF1209) was deposited at the Institute of Marine Biochemistry, VAST, Vietnam Extraction and isolation The dried stems of C. fragrans (5.0 kg) were extracted with MeOH three times by sonicator for 2 h to yield 180.0 g of a dark solid extract, which was then suspended in water (2.0 l) and successively partitioned with n-hexane and chloroform (CHCl3) to obtain nhexane (CF1, 70.0 g), CHCl3 (CF2, 60.0 g), and water (CF3, 50.0 g) layers after removal of the solvents in vacuo. The water soluble fraction (CF3, 50.0 g) was chromatographed on a diaion HP-20P column eluting with water containing increasing concentrations of MeOH (0, 25, 50, 75, and 100%) to obtain five sub-fractions, CF3A (10.0 g), CF3B (14.0 g), CF3C (10.0 g), CF3D (7.0 g), and CF3E (9.0 g). The CF3E fraction was chromatographed on a silica gel column
Copyright © 2015 John Wiley & Sons, Ltd.
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D. T. T. Hang et al. eluting with gradient of CHCl3–MeOH (20 : 1 → 5 : 1, v/v) to give three smaller fractions, CF3E1 (2.2 g), CF3E2 (2.1 g), and CF3E3 (1.8 g). The CF3E1 fraction was chromatographed on a silica gel column eluting with chloroform–acetone (3 : 1, v/v) to obtain 1 (10.0 mg) and 4 (7.0 mg). The CF3E2 fraction was chromatographed on a silica gel column eluting with chloroform–ethyl acetate (1 : 1, v/v) to obtain 2 (8.0 mg). The CF3E3 fraction was chromatographed on an YMC column eluting with acetone–water (1 : 1, v/v) to obtain 3 (8.0 mg) and 5 (8.0 mg). Callecdysterol A (1): A white amorphous powder; ½α25 D : 20.9 (c = 0.1 in MeOH); IR (KBr) νmax 3376, 1690, 1250 cm1; C19H28O6, HR-ESI-MS found m/z: 387.1589 [M + Cl] (Calcd. [C19H28O6Cl] for 387.1580); 1H and 13C NMR: refer to Table 1. Callecdysterol B (2): A white amorphous powder; ½α25 D : +90.4 (c = 0.1 in MeOH); IR (KBr) νmax 3380, 1683, 1230 cm1; C19H28O7, HR-ESI-MS found m/z: 403.1537 [M + Cl] (Calcd. [C19H28O7Cl] for 403.1530); 1H and 13C NMR: refer to Table 1. Callecdysterol C (3): A white amorphous powder; ½α25 D : +35.6 (c = 0.1 in MeOH); IR (KBr) νmax 3340, 1650, 1601 cm1; C19H26O5, HR-ESI-MS found m/z: 369.1478 [M + Cl] (Calcd. C19H26O5Cl for 369.1470); 1H and 13C NMR: refer to Table 1.
Acknowledgements This research project is proposition of Academician Nguyen Van Hieu. We would like to express our thanks to him and Vietnam Academy of Science and Technology for the support.
References [1] L. Dinan. Arch. Insect Biochem. Physiol. 2009, 72, 126. [2] R. Lafont, L. Dinan. Journal Insect Science 2003, 3, 1. [3] D. N. Olennikov, T. A. Ibragimov, I. N. Zilfikarov, V. A. Chelombit’ko. Chemisrty Natural Compounds 2008, 44, 776. [4] I. G. Nikolaeva, G. G. Nikolaeva. Chemistry Natural Compounds 2009, 45, 939. [5] L. Yarmolinsky, M. Zaccai, S. Ben-Shabat, M. Huleihel. Open Virology Journal 2010, 4, 57. [6] C. Y. Tan, J. H. Wang, X. Li, J. Asian. Nat. Prod. Res. 2003, 5, 237. [7] M. Báthori, J. P. Girault, I. Máthé, R. Lafont. Biomed. Chromatogr. 2000, 14, 464. [8] K. Yuasa, T. Ide, H. Otsuka, C. Ogimi, E. Hirata, A. Takushi, Y. Takeda. Phytochemistry 1997, 45, 611. [9] Avance 1D and 2D Course. version 010220, Bruker AG, Fällanden, Switzerland, 2001.
Supporting information Declaration of interest
Additional supporting information may be found in the online version of this article at the publisher’s web site.
The authors report no conflicts of interest.
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Copyright © 2015 John Wiley & Sons, Ltd.
Magn. Reson. Chem. (2015)
Revised: 30 December 2014
Accepted: 31 December 2014
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI 10.1002/mrc.4214
1H and 13C NMR assignments of new ecdysteroids
from Callisia fragrans
Dan Thi Thuy Hang, Nguyen Thi Minh Hang, Hoang Le Tuan Anh, Nguyen Xuan Nhiem, Cao Thi Hue, Pham Thanh Binh, Nguyen Tien Dat, Nguyen Hoai Nam, Pham Hai Yen, Chau Van Minh, Nguyen Van Hung* and Phan Van Kiem* Introduction Ecdysteroids are a class of insect hormones with a characteristic 7en-6-one moiety in the B-ring of polyhydroxysteroids. They were discovered as insect molting hormones. These compounds display important physiological effects on insects and play defensive role.[1] Ecdysteroids also exert beneficial pharmacological properties such as decreasing the blood cholesterol and glucose level in experimental animals, anticancer, and wound healing activities.[2] The increasing numbers of patents have been deposited concerning various beneficial effects of ecdysteroids in many medical or cosmetic domains, which make ecdysteroids very attractive candidates for several practical uses. Callisia fragrans (Lindl.) belongs to Commelinaceae family and is native in South America. Chemical studies of this plant showed the presence of phenolic compounds[3] and amino acids.[4] In addition, the methanol extract of C. fragrans showed antiherpetic activity.[5] In this paper, we report the isolation and structure determination of three new and two known ecdysteroids from the stems of C. fragrans.
Results and Discussion The stems of C. fragrans were extracted with hot methanol to give methanol extract. This extract was suspended in water and then partitioned with n-hexane and chloroform to give the n-hexane, chloroform, and water layers. Three new and two known ecdysteroids were isolated from the water layer using combined chromatographic separations (refer to Fig. 1). Known ecdysteroids were elucidated to be 2β,3β,11α,14α-tetrahydroxy-5α-androst-7(8)en-6,17-dione (4)[6] and 2β,3β,14α,17β-tetrahydroxy-5α-androst-7 (8)-ene-6-one (5)[6,7] on the basic of spectral data, which were in good agreement with those reported in the literature.[8] These compounds were isolated from Callisia genus for the first time. Compound 1 was obtained as a white amorphous powder, and its molecular formula was determined to be C19H28O6 by HR-ESIMS at m/z 387.1589 [M + Cl] (Calcd. C19H28O6Cl for 387.1580). The 1H NMR spectrum of 1 showed the presence of one olefinic proton at δH 5.79 (d, J = 2.5 Hz), four oxymethine protons at δH 3.97 (ddd, J = 2.5, 2.5, 3.5 Hz), 4.03 (ddd, J = 3.5, 3.5, 12.5 Hz), 4.11 (m), and 4.36 (dd, J = 6.5, 9.0 Hz), two tertiary methyl groups at δH 0.72 (s) and 1.09 (s). The 13C NMR and DEPT spectra showed the signals for 19 carbons including one carbonyl at δC 206.7, two olefinic at δC 122.2 and 165.0, three non-protonated at δC 40.0,
Magn. Reson. Chem. (2015)
48.2, and 83.0, six methine at δC 43.3, 52.9, 68.6, 68.9, 69.4, and 78.9, five methylene at δC 29.8, 31.5, 33.3, 39.1, and 40.3, and two methyl carbons at δC 16.5 and 24.7. The analytical 1H and 13C NMR data (Table 1) suggested that the structure of 1 was similar to 2β,3β,11α,14α-tetrahydroxy-5α-androst-7(8)-en-6,17-dione (4)[6] except for the hydrogenation of carbonyl group at C-17. The HMBC correlations from H-2 (δH 4.03) to C-1 (δC 39.1)/C-3 (δC 68.6)/C-4 (δC 33.3)/C-10 (δC 40.0); from H-3 (δH 3.97) to C-1 (δC 39.1)/C-2 (δC 68.9)/C-4 (δC 33.3)/C-5 (δC 52.9) suggested two hydroxyl groups at C-2 and C-3. The HMBC correlations between H-5 (δH 2.36) and C6 (δC 206.7)/C-7 (δC 165.0); H-7 (δH 5.79) and C-5 (δC 52.9)/C-6 (δC 206.7)/C-8 (δC 165.0)/C-9 (δC 43.3)/C-14 (δC 83.0) suggested the position of carbonyl group at C-6 and the double bond at C-7/C-8. The HMBC correlations from H-18 (δH 0.72) to C-12 (δC 40.3)/C-13 (δC 48.2)/C-14 (δC 83.0)/C-17 (δC 78.9), from H-9 (δH 3.16)/H-12 (δH 1.90 and 2.18) to C-11 (δC 69.4) suggested three hydroxyl groups at C-11, C-14, and C-17. The constitution of 1 was confirmed again by COSY analysis (Fig. 2). Compound 1 was supposed to have the same configurations as 4, a biogenetic derivative of 1, at C-5, C-9, C-18, and C-19. The α or β position of hydroxyl groups were established by a careful analysis of coupling constant of protons as well as by NOESY analysis. The large coupling constant between Hβ-1 and H-2, J = 12.5 Hz; small coupling constant of H-2/Hα-3, J = 2.5 Hz; and NOESY correlations of H-2 (δH 4.03)/Hα-9 (δH 3.16). These suggested the configurations of both hydroxyl groups at C2 and C-3 to be β. Furthermore, the NOESY correlations between H-11 (δH 4.11) and H-18 (δH 0.72)/H-19 (δH 1.09); large coupling constant between H-9 and H-11, J = 9.0 Hz proved the configuration of hydroxyl group at C-11 to be α. Moreover, the NOESY correlations between H-17 (δH 4.36) and Hα-12 (δH 2.18)/Hα-16 (δH 2.30); H-18 (δH 0.72) and Hβ-15 (δH 2.09)/Hβ-16 (δH 1.61) confirmed the α and β configurations of hydroxyl groups at C-14 and C-17, respectively. Based on the previous evidence, the structure of 1 was established to be 2β,3β,11α,14α,17β-pentahydroxy-5α-androst-7(8)-en-6-one, a new compound named callecdysterol A.
* Correspondence to: Phan Van Kiem and Nguyen Van Hung, Institute of Marine Biochemistry, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet, Caugiay, Hanoi, Vietnam. E-mail: [email protected]; [email protected] Institute of Marine Biochemistry, Vietnam Academy of Science and Technology (VAST), 18 Hoang Quoc Viet, Caugiay, Hanoi, Vietnam
Copyright © 2015 John Wiley & Sons, Ltd.
D. T. T. Hang et al.
Figure 1. Structures of 1–5 from C. fragrans.
Table 1. The NMR spectral data of compounds 1–3 in CD3OD Pos.
1 δC
1
39.1
2 3 4
68.9 68.6 33.3
5 6 7 8 9 10 11 12
52.9 206.7 122.2 165.0 43.3 40.0 69.4 40.3
13 14 15
48.2 83.0 31.5
16
29.8
17 18 19
78.9 16.5 24.7
δH (mult., J in Hz) 1.40 (β, dd, 12.5, 12.5) 2.61 (α, dd, 3.5; 12.5) 4.03 (ddd, 3.5, 3.5, 12.5) 3.97 (ddd, 2.5, 2.5, 3.5) 1.70 (β, ddd, 3.5, 3.5, 13.5) 1.79 (α, ddd, 2.5, 13.5, 13.5) 2.36 (β, dd, 3.5, 13.5) — 5.79 (d, 2.5) — 3.16 (α, dd, 2.5, 9.0) — 4.11 (m) 1.90 (β, dd, 6.0, 11.5) 2.18 (α, t, 11.5) — — 1.60 (α, m)2.09 (β, dd, 7.0, 12.0) 1.61 (β, m) 2.30 (α, m) 4.36 (dd, 6.5, 9.0) 0.72 (s) 1.09 (s)
2 δC 35.3 68.7 70.3 36.6 80.6 202.6 120.6 164.4 46.8 46.0 70.2 40.3 48.2 83.0 31.4 29.8 78.8 16.5 17.3
δH (mult., J in Hz) 1.74 (β, dd, 13.5, 13.5) 2.60 (α, dd, 4.5, 13.5) 4.12a 4.00 (ddd, 2.5, 3.0, 3.0) 1.79 (β, dd, 3.0, 15.0) 2.10 (α, dd, 2.5, 15.0) — — 5.85 (d, 3.0) — 3.22 (α, dd, 3.0, 9.0) — 4.13a 1.89 (β, dd, 5.5, 12.0) 2.18 (α, t, 12.0) — — 1.60 (α, m) 2.09 (β, m) 1.60 (β, m) 2.30 (α, m) 4.35 (dd, 6.5, 9.0) 0.72 (s) 1.04 (s)
3 δC 37.4 68.7 68.3 35.8 51.6 207.0 118.8 155.7 136.5 41.0 134.0 35.3 47.9 82.6 31.1 30.1 78.9 15.9 31.6
δH (mult., J in Hz) 1.73 (β, dd, 12.5, 12.5) 2.11 (α, dd, 3.0, 12.5) 3.74 (ddd, 3.0, 3.0, 12.5) 3.87 (ddd, 3.0, 3.0, 3.5) 1.59 (β, ddd, 3.0, 3.5, 13.5) 1.78 (α, ddd, 3.0, 12.5, 13.5) 2.47 (β, dd, 3.5, 12.5) — 5.75 (s) — — — 6.39 (br d, 6.5) 2.21 (β, dd, 6.5, 17.5) 2.66 (α, d, 17.5) — — 1.85 (α, m) 2.12 (β, m) 1.61 (β, m) 2.33 (α, m) 4.44 (dd, 7.0, 9.0) 0.76 (s) 1.13 (s)
a
overlapped signals.
Compound 2 was obtained as a white amorphous powder, and its molecular formula was deduced to be C19H28O7, by the HR-ESIMS ion [M + Cl] at m/z 403.1537 (Calcd. C19H28O7Cl for 403.1530). The 1H and 13C NMR analyses indicated that the structure of 2 was similar to 1 except for the additional hydroxyl group at C-5. The hydroxyl group at C-5 was further confirmed by the HMBC correlations between H-19 (δH 1.04) and C-1 (δC 35.3)/C-5 (δC 80.6)/C-9
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(δC 46.8). In addition, the β configuration of this hydroxyl group was confirmed by detecting 7.4 ppm diamagnetic shift (γ-gauche effect) on the C-19 signal. The positions of the remaining hydroxyl groups were determined by HSQC, HMBC, and COSY spectra. The observation of NOESY correlations between H-2 (δH 4.12) and H-9 (δH 4.13); H-2 (δH 4.12) and H-3 (δH 4.00) confirmed β configuration of both 2,3-hydroxyl groups. The α-orientation of hydroxyl group at C-11
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Ecdysteroids from Callisia fragrans
Figure 2. Key HMBC, COSY, and NOESY correlations of 1–3.
was based on the NOESY correlation between H-11 (δH 4.13) and H18 (δH 0.72). Consequently, compound 2 was elucidated to be 2β,3β,5β,11α,14α,17β-hexahydroxy-5α-androst-7(8)-en-6-one, a new compound named callecdysterol B. The molecular formula of 3 was also determined to be C19H26O5, by the HR-ESI-MS ion at m/z: 369.1478 [M + Cl] in the HR-ESI-MS (Calcd. C19H26O5Cl for 369.1470). The 1H-NMR of 3 showed the signals of two olefinic protons at δH 5.75 (s) and 6.39 (br d, J = 6.5 Hz), three oxygenated methine protons at δH 3.74 (dt, J = 3.0, 3.0, 12.5 Hz), 3.87 (ddd, J = 3.0, 3.0, 3.5 Hz), and 4.44 (dd, J = 7.0, 9.0 Hz), two tertiary methyl groups at δH 0.76 (s) and 1.13 (s). The 13C NMR and DEPT spectra of 3 exhibited the presence of 19 carbons including one carbonyl, four olefinic, three non-protonated, four methine, five methylene, and two methyl carbons. The 1H and 13C NMR data of 3 were similar to those of callecdysterol A (1) except for the additional double bond at C-9/C-11. The HMBC correlations between H-7 (δH 5.75) and C-5 (δC 51.6)/C-6 (δC 207.0)/ C-9 (δC 136.5)/C-14 (δC 82.6), between H-11 (δH 6.39) and C-8 (δC 155.7)/C-9 (δC 136.5)/ C-10 (δC 41.0)/C-12 (δC 35.3)/C-13 (δC 47.9) suggested the carbonyl group at C-6 and two double bonds at C-7/C-8 and C-9/C-11. The positions of two hydroxyl groups at C-14 and C-17 were confirmed by HMBC correlations from H-18 (δH 0.76) to C-12 (δC 35.3)/C-13 (δC 47.9)/C-14 (δC 82.6)/C-17 (δC 78.9). In addition, the NOESY correlations between H-18 (δH 0.76) and Hβ-12 (δH 2.21)/Hβ-15 (δH 2.12); Hα-12 (δH 2.66) and H-17 (δH 4.44) suggested the configurations of hydroxyl groups at C-14 and C-17 to be α and β, respectively. Thus, the structure of 3 was elucidated to be 2β,3β,14α,17βtetrahydroxy-5α-androst-7(8),9(11)-dien-6-one, a new compound named callecdysterol C.
Experimental General experimental procedures The HR-ESI-MS was obtained on an AGILENT 6550 iFunnel Q-TOF LC/MS spectrometer. Optical rotations were determined on a Jasco DIP-370 automatic polarimeter. Column chromatography was performed using a silica-gel (Kieselgel 60, 70–230 mesh and 230–400 mesh, Merck) or YMC RP-18 resins (30–50 μm, Fujisilisa Chemical Ltd.), thin layer chromatography using a pre-
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coated silica gel 60 F254 (0.25 mm, Merck), and RP-18 F254S plates (0.25 mm, Merck). NMR measurements All NMR spectra were recorded on a Bruker AM500 FT-NMR spectrometer operated at 500 and 125 MHz for hydrogen and carbon, respectively. The NMR measurements, including 1H, 13C, DEPT, HSQC, HMBC, COSY, and NOESY experiments, were carried out using 5-mm probe tubes at temperature of 304.5 K in CD3OD solutions, with TMS as the internal standard.[9] The pulse conditions were as follows: for the 1H spectrum, spectrometer frequency (SF) = 500.13 MHz, acquisition time (AQ) = 3.2769 s, relaxation delay (RD) = 1.0 s, pulse width = 10.5, flip angle = 30.0o, spectral width (SW) = 10 000 Hz, and digital resolution (DR) = 0.15 Hz; for the 13C spectrum, SF = 125.76 MHz, AQ = 1.0 s, RD = 2 s, pulse width = 6.4, SW = 31446.5 Hz, and DR = 0.48 Hz; the mixing time for the NOESY spectrum was 0.3 s. Chemical shifts are reported in parts per million from TMS. Plant material The stems of C. fragrans were collected in Thuynguyen, Haiphong, Vietnam during September, 2012, and identified by Prof. Dr. Tran Huy Thai, Institute of Ecology and Biological Resources, VAST, Vietnam. A voucher specimen (CF1209) was deposited at the Institute of Marine Biochemistry, VAST, Vietnam Extraction and isolation The dried stems of C. fragrans (5.0 kg) were extracted with MeOH three times by sonicator for 2 h to yield 180.0 g of a dark solid extract, which was then suspended in water (2.0 l) and successively partitioned with n-hexane and chloroform (CHCl3) to obtain nhexane (CF1, 70.0 g), CHCl3 (CF2, 60.0 g), and water (CF3, 50.0 g) layers after removal of the solvents in vacuo. The water soluble fraction (CF3, 50.0 g) was chromatographed on a diaion HP-20P column eluting with water containing increasing concentrations of MeOH (0, 25, 50, 75, and 100%) to obtain five sub-fractions, CF3A (10.0 g), CF3B (14.0 g), CF3C (10.0 g), CF3D (7.0 g), and CF3E (9.0 g). The CF3E fraction was chromatographed on a silica gel column
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D. T. T. Hang et al. eluting with gradient of CHCl3–MeOH (20 : 1 → 5 : 1, v/v) to give three smaller fractions, CF3E1 (2.2 g), CF3E2 (2.1 g), and CF3E3 (1.8 g). The CF3E1 fraction was chromatographed on a silica gel column eluting with chloroform–acetone (3 : 1, v/v) to obtain 1 (10.0 mg) and 4 (7.0 mg). The CF3E2 fraction was chromatographed on a silica gel column eluting with chloroform–ethyl acetate (1 : 1, v/v) to obtain 2 (8.0 mg). The CF3E3 fraction was chromatographed on an YMC column eluting with acetone–water (1 : 1, v/v) to obtain 3 (8.0 mg) and 5 (8.0 mg). Callecdysterol A (1): A white amorphous powder; ½α25 D : 20.9 (c = 0.1 in MeOH); IR (KBr) νmax 3376, 1690, 1250 cm1; C19H28O6, HR-ESI-MS found m/z: 387.1589 [M + Cl] (Calcd. [C19H28O6Cl] for 387.1580); 1H and 13C NMR: refer to Table 1. Callecdysterol B (2): A white amorphous powder; ½α25 D : +90.4 (c = 0.1 in MeOH); IR (KBr) νmax 3380, 1683, 1230 cm1; C19H28O7, HR-ESI-MS found m/z: 403.1537 [M + Cl] (Calcd. [C19H28O7Cl] for 403.1530); 1H and 13C NMR: refer to Table 1. Callecdysterol C (3): A white amorphous powder; ½α25 D : +35.6 (c = 0.1 in MeOH); IR (KBr) νmax 3340, 1650, 1601 cm1; C19H26O5, HR-ESI-MS found m/z: 369.1478 [M + Cl] (Calcd. C19H26O5Cl for 369.1470); 1H and 13C NMR: refer to Table 1.
Acknowledgements This research project is proposition of Academician Nguyen Van Hieu. We would like to express our thanks to him and Vietnam Academy of Science and Technology for the support.
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Supporting information Declaration of interest
Additional supporting information may be found in the online version of this article at the publisher’s web site.
The authors report no conflicts of interest.
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