Fitoterapia 98 (2014) 22–26
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Fitoterapia journal homepage: www.elsevier.com/locate/fitote
Norcassane- and cassane-type furanoditerpenoids from the seeds of Caesalpinia sappan Hai-Feng Wu a, Yin-Di Zhu a, Zhong-Hao Sun a, Jing-Quan Yuan b, Hua Wei c, Xiao-Po Zhang a, Yu Tian a, Jun-Shan Yang a, Guo-Xu Ma a,⁎, Xu-Dong Xu a,⁎ a Key Laboratory of Bioactive Substances and Resources Utilization of Chinese Herbal Medicine, Ministry of Education, Institute of Medicinal Plant Development, Peking Union Medical College, Chinese Academy of Medical Sciences, Beijing 100193, PR China b National Engineering Laboratory of Southwest Endangered Medicinal Resources Development, National Development and Reform Commission, Guangxi Botanical Garden of Medicinal Plant, Nanning 530023, PR China c Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing 100700, PR China
a r t i c l e
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Article history: Received 4 May 2014 Accepted in revised form 20 June 2014 Available online 10 July 2014 Keywords: Caesalpinia sappan Norcassane diterpene Cytotoxicity
a b s t r a c t Three novel furanoditerpenoids, norcaesalpinin J (1) featuring an unusual 20-norcassane hydroperoxide and phangininoxys B (2) and C (3) possessing cassane hemiketal skeletons, were isolated from the seeds of Caesalpinia sappan. Their structures were elucidated by extensive spectroscopic methods. All isolates were evaluated for the cytotoxic activities on three human cancer cell lines. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Plants belonging to the genus Caesalpinia (Fabaceae) are known to be a rich source of norcassane and cassane diterpenoids [1], most of which have been shown to possess antitumor activities [2–11]. Caesalpinia sappan, also known as Sappanwood and Brazilwood, is a thorny tree native to Southeast Asia, possessing many medicinal properties such as antibacterial and analgesic activities [12,13]. Our previous phytochemical investigation of C. sappan resulted in the isolation of three new cassane-type diterpenes [14]. Continuing with our earlier studies on the species, another three novel furanoditerpenoids, norcaesalpinin J (1) featuring an
⁎ Corresponding authors at: Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical College, No. 151, Malianwa North Road, Haidian District, Beijing, PR China. Tel./ fax: + 86 10 5783 3296. E-mail addresses: [email protected] (G.-X. Ma), [email protected] (X.-D. Xu).
http://dx.doi.org/10.1016/j.fitote.2014.07.001 0367-326X/© 2014 Elsevier B.V. All rights reserved.
unusual 20-norcassane hydroperoxide and phangininoxys B (2) and C (3) possessing cassane hemiketal skeletons were isolated from the seeds of C. sappan (Fig. 1). Herein, we report the isolation and structure elucidation of these compounds as well as their cytotoxic activity evaluation. 2. Experimental section 2.1. General Optical rotations were obtained on a Perkin-Elmer 341 digital polarimeter. UV and IR spectra were recorded on Shimadzu UV2550 and FTIR-8400S spectrometers, respectively. NMR spectra were obtained with a Bruker AV III 600 NMR spectrometer (chemical shift values are presented as δ values with TMS as the internal standard). HRESIMS spectra were performed on a LTQ-Obitrap XL spectrometer. Preparative HPLC was performed on a Lumtech K1001 analytic LC equipped with two pumps of K-501, a UV detector of K-2600, and a Kromasil (250 mm × 10 mm) semi-preparative column packed with C18 (5 μm). C18 reversed-phase silica gel
H.-F. Wu et al. / Fitoterapia 98 (2014) 22–26
23
Fig. 1. Chemical structures of compounds 1–4.
(40–63 μm, Merck, Darmstadt, Germany), Sephadex LH-20 (Pharmacia, Uppsala, Sweden), and silica gel columns (100– 200 and 300–400 mesh, Qingdao Marine Chemical Plant, Qingdao, People's Republic of China) were used for column chromatography, and precoated silica gel GF254 plates (Zhi Fu Huang Wu Pilot Plant of Silica Gel Development, Yantai, People's Republic of China) were used for TLC. All solvents used were of analytical grade (Beijing Chemical Works).
Table 1; HRESIMS m/z: 371.1833 [M + Na]+ (calcd for C20H28O5Na, 371.1834). 2.3.2. Phangininoxy B (2) C21H26O5, white amorphous powder; IR (KBr) νmax: 1720, 1637, and 1459 cm−1; UV (MeOH) λmax (log ε): 218 (5.24) nm. For 1H NMR (600 MHz, CDCl3) and 13C APT (150 MHz, CDCl3) spectroscopic data see Table 1; HRESIMS m/z: 381.1674 [M + Na]+ (calcd for C21H26O5Na, 381.1678).
2.2. Plant material The seeds of C. sappan were collected in April 2013 from Nanning, Guangxi Province, China and identified by Prof. Jing-Quan Yuan, Department of Pharmaceutical Chemistry, Guangxi Botanical Garden of Medical Plant. A voucher specimen (No. 13418) was deposited at the Guangxi Botanical Garden of Medical Plant.
2.3.3. Phangininoxy C (3) C23H30O6, white amorphous powder; [α]25 D − 20 (c = 0.50, MeOH); IR (KBr) νmax: 1726, 1638, and 1452 cm−1; UV (MeOH) λmax (log ε): 218 (4.33) nm. For 1H NMR (600 MHz, CDCl3) and 13C APT (150 MHz, CDCl3) spectroscopic data see Table 1; HRESIMS m/z: 425.1950 [M + Na]+ (calcd for C23H30O6Na, 425.1940).
2.3. Extraction and isolation
2.4. Cytotoxic assay
Air-dried and powered seeds of C. sappan (0.8 kg) were extracted three times with methanol. Removal of the methanol under reduced pressure yielded the extract (40 g). The residue was suspended in water and extracted with hexane, chloroform, and n-butanol, respectively. The CHCl3-soluble fraction (4.7 g) was subjected to silica gel column chromatography using a petroleum ether–EtOAc gradient (from 20:1 to 0:1) as eluent, to yield five fractions (Frs. A–E). Fr. C (750 mg) was applied to a silica gel (300– 400 mesh) column eluting with petroleum ether–EtOAc gradient (50:1; 30:1; 10:1; 8:1; 6:1; 4:1; 2:1; 0:1) to afford 1 (3.4 mg). Fr. D (550 mg) was subjected to silica gel column chromatography (300–400 mesh) eluting with petroleum ether–CHCl3 gradient (1:1, 1:2, 1:2.5, 1:3, 1:4, 1:5, 0:1) to yield 2 (3.6 mg). Fr. E (620 mg) was subjected to C18 reversed-phase silica gel and sephadex LH-20 column, and four fractions (Frs. E1–E4) were collected. Fr. E3 (170 mg) was separated by semi-preparative liquid chromatography using a MeOH–H2O (62:38) system to yield 3 (3.2 mg).
The cytotoxicity of compounds 1–3 were assessed against HepG-2, MCF-7, and HCT-8 human cancer cell lines by the MTT method. Cells were grown in DMEM medium supplied with 10% fetal bovine serum and cultured at a density of 6 × 104 cells/ml per well in a 96-well microtiter plate. Then five different concentrations of each compound dissolved in dimethyl sulfoxide (DMSO) were added to each well. Each concentration was tested in triplicate. After incubation at 37 °C in 5% CO2 for 48 h, 10 μl of MTT (4 mg/ml) was added to each well and incubated for another 4 h. Then the liquid in the well was removed and DMSO was added (200 μl) to each well. The absorbance was recorded on a microplate reader at a wavelength of 570 nm.
2.3.1. Norcaesalpinin J (1) C20H28O5, white amorphous powder; [α]25 D + 132 (c = 0.50, MeOH); IR (KBr) νmax: 3400 and 1726 cm−1; UV (MeOH) λmax (log ε): 204 (3.96) nm. For 1H NMR (600 MHz, CDCl3) and 13C APT (150 MHz, CDCl3) spectroscopic data see
3. Results and discussion Compound 1 was obtained as a white amorphous powder with [α]25 D + 132 (c = 0.50, MeOH) and showed a dark bluish spot on the paper strip with hydrogen peroxide [15]. The IR spectrum showed the presence of hydroperoxyl (3400 cm−1) and carbonyl (1726 cm−1) groups. The HRESIMS spectrum showed a quasi-molecular ion at m/z 371.1833 [M + Na]+ (calcd for C20H28O5Na, 371.1834), from which in conjunction with NMR data the molecular formula was established as C20H28O5, representing seven indices of
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H.-F. Wu et al. / Fitoterapia 98 (2014) 22–26
Table 1 1 H and 13C NMR spectroscopic data of 1–3 (600 and 150 MHz, CDCl3). Position
1 δC
1
29.8 t
2
18.7 t
3
29.0 t
4 5 6
45.0 s 43.8 d 23.1 t
7
24.0 t
8 9 10 11
37.4 d 36.8 d 86.9 s 21.6 t
12 13 14 15 16 17 18 19
149.8 s 122.0 s 31.6 d 109.6 d 140.6 d 17.7 q 26.8 q 182.1 s
2 δH (J in Hz) 1.26, m 1.71, m 1.41, m 1.64, m 1.30, m 2.00, m 2.42, m 1.38, m 1.85, m 1.58, m – 1.72, m 2.58, m 2.45, m 2.72, m
2.67, m 6.18, d (1.8) 7.22, d (1.8) 1.02, d (7.2) 1.11, s
20 18-OCH3 19-OCH2CH3 19-OCH2CH3 19-OCH3 OOH
3
δC 35.0 t 18.0 t 35.7 t 47.5 s 46.2 d 27.8 t 28.1 t 38.1 d 45.1 d 43.4 s 70.0 d 146.8 s 129.5 s 32.6 d 109.5 d 143.2 d 14.3 q 176.1 s 67.1 t 106.2 d 52.2 q
δH (J in Hz) 1.46, m 1.80, m 1.82, m 2.03, m 1.59, m 1.98, m 1.85, m 1.17, m 1.52, m 1.57, m 2.18, m 1.78, m 4.73, d (3.0)
2.65, qd (7.2, 1.2) 6.23, d (1.2) 7.32, d (1.2) 0.98, d (7.2) 3.50, d (12.0) 4.89, d (12.0) 4.98, s 3.72, s
δC 35.2 t 19.7 t 30.6 t 52.0 s 46.7 d 27.5 t 27.8 t 38.0 d 45.0 d 44.0 s 70.3 d 146.8 s 129.3 s 32.6 d 109.4 d 143.1 d 14.3 q 175.6 s 98.7 d 101.3 d 52.2 q 63.9 t 15.2 q
53.0 q
δH (J in Hz) 1.47, m 1.80, m 1.70, m 1.99, m 1.75, m 2.09, m 1.98, m 1.17, m 1.96, m 1.46, m 1.69, m 2.11, m 1.77, m 4.73, d (3.6)
2.65, qd (7.2, 2.4) 6.22, d (1.2) 7.30, d (1.2) 0.97, d (7.2) 5.64, s 5.34, s 3.71, s 3.86, m 3.62, m 1.21, t (7.2)
3.78, s 8.64, s
Underline refers to corresponding methyl or methylene in oxyethyl group.
hydrogen deficiency. The 1H NMR spectrum (Table 1) indicated the presence of two methyl signals at δH 1.02 (d, J = 7.2 Hz, H-17) and δH 1.11 (s, H-18), one methoxyl at δH 3.78 (s), a low-field exchangeable hydroperoxy proton at δH 8.64, and two mutually coupled olefinic protons at δH 6.18 (d, J = 1.8 Hz, H-15) and 7.22 (d, J = 1.8 Hz, H-16) suggesting the presence of a fused furan ring. Except for one methoxy (δC 53.0), the 13C APT spectrum (Table 1) showed 19 carbon resonances for an ester carbonyl (δC 182.1), a 1,2-disubstituted furan ring (δC 109.6, 122.0, 140.6, 149.8), two methyls (δC 17.7, 26.8), six methylenes (δC 18.7, 21.6, 23.1, 24.0, 29.0, 29.8), four methines (δC 31.6, 37.4, 36.8, 43.8), and two quaternary carbons (δC 45.0, 86.9), which clearly indicated a diterpenoid skeleton. The carbon framework of 1 had 19 carbon signals, suggesting 1 to be a norditerpene. The partial structures deduced by the COSY (bold line) and HSQC spectra were connected based on the long-range correlations (arrows) observed in the HMBC spectrum (Fig. 2). The methyl protons at δH 1.11 (H3-18) showed the long-range correlations with the carbons at δC 29.0 (C-3), 45.0 (C-4), 43.8 (C-5), and 182.1 (C-19). Thus, C-3, C-5, C-18 and C-19 were connected with the quaternary carbon C-4. Moreover, the connectivity of C-11 (δC 21.6) and C-14 (δC 31.6) with olefinic carbon C-13 (δC 122.0) was established on the basis of HMBC correlation between the methylene protons (H2-11) and the carbon C-13. Likewise, the connectivity between C-12 (δC 149.8)
and C-14 was established based on the HMBC correlation between C-12 and H-14. The methyl group (C-17) was located at C-14 by the HMBC correlations of H3-17 (δH 1.02) with C-8 (δC 37.4), C-13, and C-14 (δC 31.6). The ether linkage between C-16 (δC 140.6) and C-12 (δC 149.8), i.e. presence of furan ring, was confirmed by the long-range correlation observed between H-16 and C-12. Thus, all the nineteen carbons in the framework of 1 have been accounted, which have one carbon (C-20 attached at C-10) less than that of deoxycaesaldekarin C (4) [16]. Finally, the correlations observed in the HMBC experiment between C-10 (δC 86.9) and H-6 (δH 1.85), H-9 (δH 2.60), and H-11 (δH 2.49) confirmed that the hydroperoxy group was assigned at C-10, which also explained the downfield chemical shift of C-10 (δC 86.9) compared with that of other cassane diterpenoids.
Fig. 2. Key 1H–1H COSY (▬), HMBC (→) and NOESY (↔) correlations of 1.
H.-F. Wu et al. / Fitoterapia 98 (2014) 22–26
Accordingly, 1 could be deduced as a 10-hydroperoxy-20norcassane diterpene. All cassane diterpenes isolated so far from the genus Caesalpinia share the same carbon skeleton with the trans/ anti/trans system of the three six-membered rings A, B, and C, and the β-oriented proton at C-8 and the α-oriented protons at C-5/C-9 are well established [17]. Considering the biogenetic relationship in the cassane and norcassane diterpenoids, 1 can be inferred to have a similar absolute configuration to 4 as shown in Fig. 1. The relative stereochemistry of 1 was determined on the basis of NOESY experiments (Fig. 2). The NOEs from OOH-10 to H-8 (δH 1.72, m) and OCH3-19 (δH 3.78) indicated that rings A and B are in chair conformations with a trans-fused ring junction, thus, confirming the relative configurations at C-10. The interaction of H3-17 with H-9 and H3-18 with H-5 suggested that they were α-oriented. The present compound is a novel norcassane furanoditerpenoid hydroperoxide. The biogenetic origin of the tertiary hydroperoxyl is not obvious, and might be related to the oxidative removal of the C-10 methyl [18,19]. Thus, the structure of 1 was established and named norcaesalpinin J. Although a number of naturally occurring peroxy abietane diterpenoids have already been reported [20,21], norcaesalpinin J represents the first example of hydroperoxyl-containing 20-norcassane diterpenoid. Compound 2 had the molecular formula C21H26O5 ([M + Na]+ m/z: 383.1813) based on HRESIMS. The 1H NMR spectrum showed one methyl signal at δH 0.98 (d, J = 7.2 Hz, H-17), one methoxyl at δH 3.72 (s), and two signals of a furan ring at δH 6.23 (d, J = 1.2 Hz, H-15) and 7.32 (d, J = 1.2 Hz, H-16). The lower field signal at δH 4.98 (s) was deduced to be a hemiacetal proton H-20 whose HMBC spectrum showed correlations with carbons at δC 35.0 (C-1), 45.1 (C-9), 43.4 (C-10), 70.0 (C-11) and 67.1 (C-19). The 13C APT spectrum displayed 21 carbon signals ascribed to an ester carbonyl (δC 176.1), a 1,2-disubstituted furan ring (δC 109.5, 129.5, 143.2, 146.8), a methyl, a methoxy, six methylenes, six methines, and two quaternary carbons. All carbon-bound protons were assigned by HSQC and 1H–1H COSY spectra (Fig. 3). The NMR spectra of 2 (Table 1) were quite similar to those of phangininoxy A [22], indicating that 2 was an epimer in hemiacetal carbon (C-20) of the latter. In the HMBC spectrum (Fig. 3), the correlations of H2-19 (δH 3.50, 4.98, d, J = 12.0 Hz) with C-3 (δC 35.7), C-4 (δC 47.5), C-5 (δC 46.2), C-18 (δC 176.1), and C-20 (δC 106.2) implied that C-19 was oxygenated methylene. The HMBC correlation of methoxyl (δH 3.72) with C-18 suggested that the methoxy was located at C-18. In the NOESY spectrum (Fig. 3), the key
Fig. 3. Key 1H–1H COSY (▬), HMBC (→) and NOESY (↔) correlations of 2.
25
correlations of H-8 (δH 2.18)/H-20 (δH 4.98) and H-11 (δH 4.73)/H-9 (δH 1.78) indicated the stereochemistry of H-11 (α) and H-20 (β), respectively. Thus, the structure of 2 was identified and named phangininoxy B. Compound 3 had the same carbon skeleton as 2, and its molecular formula was assigned to be C23H30O6 based on its positive HRESIMS (mnz [M + Na]+: 425.1950; calcd for 425.1940). Comparison of the NMR spectroscopic data of 3 with those of 2 showed that compound 3 is a 19-O-ethyl derivative of the latter, which was supported on the basis of the HMBC correlations of H-19 (δH 5.64) with C-3 (δC 30.6), C-4 (δC 52.0), C-18 (δC 175.6), and C-20 (δC 101.3), and the methyl signal (δH 1.21, t, J = 7.2 Hz) with oxygenated methylene carbon (δC 63.9) and the methylene signal (δH 3.62, m; 3.86, m) with methyl carbon (δC 15.2) and C-19 (δC 98.7) (Fig. 4). In the 1H–1H COSY spectrum, the correlation between methyl protons and oxymethylene also confirmed the presence of oxygenated ethyl side-chain. The HMBC correlation of methoxyl (δH 3.71) with C-18 (δC 175.7) suggested that the methoxy was attached to C-18. The relative configurations of compound 3 were established to be identical to those of 2 on the basis of analysis of the NOESY spectrum (Fig. 4). The NOESY correlations between the dioxymethine proton at δH 5.64 (H-19) displayed a cross-peak with protons at δH 1.98 (H-5) and 1.77 (H-9), indicating that this dioxymethine proton was α-oriented. Accordingly, compound 3 was named phangininoxy C. All isolates were tested for their cytotoxicity against three human cancer cell lines (HepG-2, MCF-7, and HCT-8) using the MTT method with cisplatin as the positive control. Compound 1 showed moderate cytotoxicity against all the tested cell lines, while 2 and 3 displayed weak activity with IC50 values of 22.3–28.1 μM (Table 2). Conflict of interest We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work, and there is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the position presented in, or the review of, the manuscript entitled. Acknowledgments The work was financially supported by the technological large platform for comprehensive research and development
Fig. 4. Key 1H–1H COSY (▬), HMBC (→) and NOESY (↔) correlations of 3.
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H.-F. Wu et al. / Fitoterapia 98 (2014) 22–26
Table 2 In-vitro cytotoxic activity of compounds 1–3. Compounds
IC50 (μM) HepG-2
1 2 3 Cisplatina
8.9 22.3 25.5 4.7
± ± ± ±
MCF-7 1.1 0.98 2.4 0.53
13.7 23.2 38.1 12.7
± ± ± ±
HCT-8 1.3 2.3 0.73 0.95
10.2 36.7 43.5 2.5
± ± ± ±
1.9 0.86 4.1 0.28
Values present mean ± SD of triplicate experiments. a Positive control substance.
of new drugs in the Twelfth Five-Year “Significant New Drugs Created” Science and Technology Major Projects (No. 2012ZX09301-002-001-026), the National Science and Technology Support Program (No. 2012BA127B06), and the Innovation Capacity building in Guangxi Science and Technology Agency (No. 10100027-3). References [1] Maurya R, Ravi M, Singh S, Yadav PP. A review on cassane and norcassane diterpenes and their pharmacological studies. Fitoterapia 2012;83:272–80. [2] Yodsaoue O, Cheenpracha S, Karalai C, Ponglimanont C, Chantrapromma S, Fun HK, et al. Phanginin A–K, diterpenoids from the seeds of Caesalpinia sappan Linn. Phytochemistry 2008;69:1242–9. [3] Yadav PP, Maurya R, Sarkar J, Arora A, Kanojiya S, Sinha S, et al. Cassane diterpenes from Caesalpinia bonduc. Phytochemistry 2009;70:256–61. [4] Cota BB, de Oliveira DM, de Siqueira EP, Souza-Fagundes EM, Pimenta AM, Santos DM, et al. New cassane diterpenes from Caesalpinia echinata. Fitoterapia 2011;82:969–75. [5] Ma GX, Xu XD, Cao L, Yuan JQ, Yang JS, Ma LY. Cassane-type diterpenes from the seeds of Caesalpinia minax with their antineoplastic activity. Planta Med 2012;78:1363–9. [6] Zhang JY, Abdel-Mageed WM, Liu MM, Huang P, He WN, Li L, et al. Caesanines A–D, new cassane diterpenes with unprecedented N bridge from Caesalpinia sappan. Org Lett 2013;15:4726–9.
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Fitoterapia journal homepage: www.elsevier.com/locate/fitote
Norcassane- and cassane-type furanoditerpenoids from the seeds of Caesalpinia sappan Hai-Feng Wu a, Yin-Di Zhu a, Zhong-Hao Sun a, Jing-Quan Yuan b, Hua Wei c, Xiao-Po Zhang a, Yu Tian a, Jun-Shan Yang a, Guo-Xu Ma a,⁎, Xu-Dong Xu a,⁎ a Key Laboratory of Bioactive Substances and Resources Utilization of Chinese Herbal Medicine, Ministry of Education, Institute of Medicinal Plant Development, Peking Union Medical College, Chinese Academy of Medical Sciences, Beijing 100193, PR China b National Engineering Laboratory of Southwest Endangered Medicinal Resources Development, National Development and Reform Commission, Guangxi Botanical Garden of Medicinal Plant, Nanning 530023, PR China c Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing 100700, PR China
a r t i c l e
i n f o
Article history: Received 4 May 2014 Accepted in revised form 20 June 2014 Available online 10 July 2014 Keywords: Caesalpinia sappan Norcassane diterpene Cytotoxicity
a b s t r a c t Three novel furanoditerpenoids, norcaesalpinin J (1) featuring an unusual 20-norcassane hydroperoxide and phangininoxys B (2) and C (3) possessing cassane hemiketal skeletons, were isolated from the seeds of Caesalpinia sappan. Their structures were elucidated by extensive spectroscopic methods. All isolates were evaluated for the cytotoxic activities on three human cancer cell lines. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Plants belonging to the genus Caesalpinia (Fabaceae) are known to be a rich source of norcassane and cassane diterpenoids [1], most of which have been shown to possess antitumor activities [2–11]. Caesalpinia sappan, also known as Sappanwood and Brazilwood, is a thorny tree native to Southeast Asia, possessing many medicinal properties such as antibacterial and analgesic activities [12,13]. Our previous phytochemical investigation of C. sappan resulted in the isolation of three new cassane-type diterpenes [14]. Continuing with our earlier studies on the species, another three novel furanoditerpenoids, norcaesalpinin J (1) featuring an
⁎ Corresponding authors at: Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical College, No. 151, Malianwa North Road, Haidian District, Beijing, PR China. Tel./ fax: + 86 10 5783 3296. E-mail addresses: [email protected] (G.-X. Ma), [email protected] (X.-D. Xu).
http://dx.doi.org/10.1016/j.fitote.2014.07.001 0367-326X/© 2014 Elsevier B.V. All rights reserved.
unusual 20-norcassane hydroperoxide and phangininoxys B (2) and C (3) possessing cassane hemiketal skeletons were isolated from the seeds of C. sappan (Fig. 1). Herein, we report the isolation and structure elucidation of these compounds as well as their cytotoxic activity evaluation. 2. Experimental section 2.1. General Optical rotations were obtained on a Perkin-Elmer 341 digital polarimeter. UV and IR spectra were recorded on Shimadzu UV2550 and FTIR-8400S spectrometers, respectively. NMR spectra were obtained with a Bruker AV III 600 NMR spectrometer (chemical shift values are presented as δ values with TMS as the internal standard). HRESIMS spectra were performed on a LTQ-Obitrap XL spectrometer. Preparative HPLC was performed on a Lumtech K1001 analytic LC equipped with two pumps of K-501, a UV detector of K-2600, and a Kromasil (250 mm × 10 mm) semi-preparative column packed with C18 (5 μm). C18 reversed-phase silica gel
H.-F. Wu et al. / Fitoterapia 98 (2014) 22–26
23
Fig. 1. Chemical structures of compounds 1–4.
(40–63 μm, Merck, Darmstadt, Germany), Sephadex LH-20 (Pharmacia, Uppsala, Sweden), and silica gel columns (100– 200 and 300–400 mesh, Qingdao Marine Chemical Plant, Qingdao, People's Republic of China) were used for column chromatography, and precoated silica gel GF254 plates (Zhi Fu Huang Wu Pilot Plant of Silica Gel Development, Yantai, People's Republic of China) were used for TLC. All solvents used were of analytical grade (Beijing Chemical Works).
Table 1; HRESIMS m/z: 371.1833 [M + Na]+ (calcd for C20H28O5Na, 371.1834). 2.3.2. Phangininoxy B (2) C21H26O5, white amorphous powder; IR (KBr) νmax: 1720, 1637, and 1459 cm−1; UV (MeOH) λmax (log ε): 218 (5.24) nm. For 1H NMR (600 MHz, CDCl3) and 13C APT (150 MHz, CDCl3) spectroscopic data see Table 1; HRESIMS m/z: 381.1674 [M + Na]+ (calcd for C21H26O5Na, 381.1678).
2.2. Plant material The seeds of C. sappan were collected in April 2013 from Nanning, Guangxi Province, China and identified by Prof. Jing-Quan Yuan, Department of Pharmaceutical Chemistry, Guangxi Botanical Garden of Medical Plant. A voucher specimen (No. 13418) was deposited at the Guangxi Botanical Garden of Medical Plant.
2.3.3. Phangininoxy C (3) C23H30O6, white amorphous powder; [α]25 D − 20 (c = 0.50, MeOH); IR (KBr) νmax: 1726, 1638, and 1452 cm−1; UV (MeOH) λmax (log ε): 218 (4.33) nm. For 1H NMR (600 MHz, CDCl3) and 13C APT (150 MHz, CDCl3) spectroscopic data see Table 1; HRESIMS m/z: 425.1950 [M + Na]+ (calcd for C23H30O6Na, 425.1940).
2.3. Extraction and isolation
2.4. Cytotoxic assay
Air-dried and powered seeds of C. sappan (0.8 kg) were extracted three times with methanol. Removal of the methanol under reduced pressure yielded the extract (40 g). The residue was suspended in water and extracted with hexane, chloroform, and n-butanol, respectively. The CHCl3-soluble fraction (4.7 g) was subjected to silica gel column chromatography using a petroleum ether–EtOAc gradient (from 20:1 to 0:1) as eluent, to yield five fractions (Frs. A–E). Fr. C (750 mg) was applied to a silica gel (300– 400 mesh) column eluting with petroleum ether–EtOAc gradient (50:1; 30:1; 10:1; 8:1; 6:1; 4:1; 2:1; 0:1) to afford 1 (3.4 mg). Fr. D (550 mg) was subjected to silica gel column chromatography (300–400 mesh) eluting with petroleum ether–CHCl3 gradient (1:1, 1:2, 1:2.5, 1:3, 1:4, 1:5, 0:1) to yield 2 (3.6 mg). Fr. E (620 mg) was subjected to C18 reversed-phase silica gel and sephadex LH-20 column, and four fractions (Frs. E1–E4) were collected. Fr. E3 (170 mg) was separated by semi-preparative liquid chromatography using a MeOH–H2O (62:38) system to yield 3 (3.2 mg).
The cytotoxicity of compounds 1–3 were assessed against HepG-2, MCF-7, and HCT-8 human cancer cell lines by the MTT method. Cells were grown in DMEM medium supplied with 10% fetal bovine serum and cultured at a density of 6 × 104 cells/ml per well in a 96-well microtiter plate. Then five different concentrations of each compound dissolved in dimethyl sulfoxide (DMSO) were added to each well. Each concentration was tested in triplicate. After incubation at 37 °C in 5% CO2 for 48 h, 10 μl of MTT (4 mg/ml) was added to each well and incubated for another 4 h. Then the liquid in the well was removed and DMSO was added (200 μl) to each well. The absorbance was recorded on a microplate reader at a wavelength of 570 nm.
2.3.1. Norcaesalpinin J (1) C20H28O5, white amorphous powder; [α]25 D + 132 (c = 0.50, MeOH); IR (KBr) νmax: 3400 and 1726 cm−1; UV (MeOH) λmax (log ε): 204 (3.96) nm. For 1H NMR (600 MHz, CDCl3) and 13C APT (150 MHz, CDCl3) spectroscopic data see
3. Results and discussion Compound 1 was obtained as a white amorphous powder with [α]25 D + 132 (c = 0.50, MeOH) and showed a dark bluish spot on the paper strip with hydrogen peroxide [15]. The IR spectrum showed the presence of hydroperoxyl (3400 cm−1) and carbonyl (1726 cm−1) groups. The HRESIMS spectrum showed a quasi-molecular ion at m/z 371.1833 [M + Na]+ (calcd for C20H28O5Na, 371.1834), from which in conjunction with NMR data the molecular formula was established as C20H28O5, representing seven indices of
24
H.-F. Wu et al. / Fitoterapia 98 (2014) 22–26
Table 1 1 H and 13C NMR spectroscopic data of 1–3 (600 and 150 MHz, CDCl3). Position
1 δC
1
29.8 t
2
18.7 t
3
29.0 t
4 5 6
45.0 s 43.8 d 23.1 t
7
24.0 t
8 9 10 11
37.4 d 36.8 d 86.9 s 21.6 t
12 13 14 15 16 17 18 19
149.8 s 122.0 s 31.6 d 109.6 d 140.6 d 17.7 q 26.8 q 182.1 s
2 δH (J in Hz) 1.26, m 1.71, m 1.41, m 1.64, m 1.30, m 2.00, m 2.42, m 1.38, m 1.85, m 1.58, m – 1.72, m 2.58, m 2.45, m 2.72, m
2.67, m 6.18, d (1.8) 7.22, d (1.8) 1.02, d (7.2) 1.11, s
20 18-OCH3 19-OCH2CH3 19-OCH2CH3 19-OCH3 OOH
3
δC 35.0 t 18.0 t 35.7 t 47.5 s 46.2 d 27.8 t 28.1 t 38.1 d 45.1 d 43.4 s 70.0 d 146.8 s 129.5 s 32.6 d 109.5 d 143.2 d 14.3 q 176.1 s 67.1 t 106.2 d 52.2 q
δH (J in Hz) 1.46, m 1.80, m 1.82, m 2.03, m 1.59, m 1.98, m 1.85, m 1.17, m 1.52, m 1.57, m 2.18, m 1.78, m 4.73, d (3.0)
2.65, qd (7.2, 1.2) 6.23, d (1.2) 7.32, d (1.2) 0.98, d (7.2) 3.50, d (12.0) 4.89, d (12.0) 4.98, s 3.72, s
δC 35.2 t 19.7 t 30.6 t 52.0 s 46.7 d 27.5 t 27.8 t 38.0 d 45.0 d 44.0 s 70.3 d 146.8 s 129.3 s 32.6 d 109.4 d 143.1 d 14.3 q 175.6 s 98.7 d 101.3 d 52.2 q 63.9 t 15.2 q
53.0 q
δH (J in Hz) 1.47, m 1.80, m 1.70, m 1.99, m 1.75, m 2.09, m 1.98, m 1.17, m 1.96, m 1.46, m 1.69, m 2.11, m 1.77, m 4.73, d (3.6)
2.65, qd (7.2, 2.4) 6.22, d (1.2) 7.30, d (1.2) 0.97, d (7.2) 5.64, s 5.34, s 3.71, s 3.86, m 3.62, m 1.21, t (7.2)
3.78, s 8.64, s
Underline refers to corresponding methyl or methylene in oxyethyl group.
hydrogen deficiency. The 1H NMR spectrum (Table 1) indicated the presence of two methyl signals at δH 1.02 (d, J = 7.2 Hz, H-17) and δH 1.11 (s, H-18), one methoxyl at δH 3.78 (s), a low-field exchangeable hydroperoxy proton at δH 8.64, and two mutually coupled olefinic protons at δH 6.18 (d, J = 1.8 Hz, H-15) and 7.22 (d, J = 1.8 Hz, H-16) suggesting the presence of a fused furan ring. Except for one methoxy (δC 53.0), the 13C APT spectrum (Table 1) showed 19 carbon resonances for an ester carbonyl (δC 182.1), a 1,2-disubstituted furan ring (δC 109.6, 122.0, 140.6, 149.8), two methyls (δC 17.7, 26.8), six methylenes (δC 18.7, 21.6, 23.1, 24.0, 29.0, 29.8), four methines (δC 31.6, 37.4, 36.8, 43.8), and two quaternary carbons (δC 45.0, 86.9), which clearly indicated a diterpenoid skeleton. The carbon framework of 1 had 19 carbon signals, suggesting 1 to be a norditerpene. The partial structures deduced by the COSY (bold line) and HSQC spectra were connected based on the long-range correlations (arrows) observed in the HMBC spectrum (Fig. 2). The methyl protons at δH 1.11 (H3-18) showed the long-range correlations with the carbons at δC 29.0 (C-3), 45.0 (C-4), 43.8 (C-5), and 182.1 (C-19). Thus, C-3, C-5, C-18 and C-19 were connected with the quaternary carbon C-4. Moreover, the connectivity of C-11 (δC 21.6) and C-14 (δC 31.6) with olefinic carbon C-13 (δC 122.0) was established on the basis of HMBC correlation between the methylene protons (H2-11) and the carbon C-13. Likewise, the connectivity between C-12 (δC 149.8)
and C-14 was established based on the HMBC correlation between C-12 and H-14. The methyl group (C-17) was located at C-14 by the HMBC correlations of H3-17 (δH 1.02) with C-8 (δC 37.4), C-13, and C-14 (δC 31.6). The ether linkage between C-16 (δC 140.6) and C-12 (δC 149.8), i.e. presence of furan ring, was confirmed by the long-range correlation observed between H-16 and C-12. Thus, all the nineteen carbons in the framework of 1 have been accounted, which have one carbon (C-20 attached at C-10) less than that of deoxycaesaldekarin C (4) [16]. Finally, the correlations observed in the HMBC experiment between C-10 (δC 86.9) and H-6 (δH 1.85), H-9 (δH 2.60), and H-11 (δH 2.49) confirmed that the hydroperoxy group was assigned at C-10, which also explained the downfield chemical shift of C-10 (δC 86.9) compared with that of other cassane diterpenoids.
Fig. 2. Key 1H–1H COSY (▬), HMBC (→) and NOESY (↔) correlations of 1.
H.-F. Wu et al. / Fitoterapia 98 (2014) 22–26
Accordingly, 1 could be deduced as a 10-hydroperoxy-20norcassane diterpene. All cassane diterpenes isolated so far from the genus Caesalpinia share the same carbon skeleton with the trans/ anti/trans system of the three six-membered rings A, B, and C, and the β-oriented proton at C-8 and the α-oriented protons at C-5/C-9 are well established [17]. Considering the biogenetic relationship in the cassane and norcassane diterpenoids, 1 can be inferred to have a similar absolute configuration to 4 as shown in Fig. 1. The relative stereochemistry of 1 was determined on the basis of NOESY experiments (Fig. 2). The NOEs from OOH-10 to H-8 (δH 1.72, m) and OCH3-19 (δH 3.78) indicated that rings A and B are in chair conformations with a trans-fused ring junction, thus, confirming the relative configurations at C-10. The interaction of H3-17 with H-9 and H3-18 with H-5 suggested that they were α-oriented. The present compound is a novel norcassane furanoditerpenoid hydroperoxide. The biogenetic origin of the tertiary hydroperoxyl is not obvious, and might be related to the oxidative removal of the C-10 methyl [18,19]. Thus, the structure of 1 was established and named norcaesalpinin J. Although a number of naturally occurring peroxy abietane diterpenoids have already been reported [20,21], norcaesalpinin J represents the first example of hydroperoxyl-containing 20-norcassane diterpenoid. Compound 2 had the molecular formula C21H26O5 ([M + Na]+ m/z: 383.1813) based on HRESIMS. The 1H NMR spectrum showed one methyl signal at δH 0.98 (d, J = 7.2 Hz, H-17), one methoxyl at δH 3.72 (s), and two signals of a furan ring at δH 6.23 (d, J = 1.2 Hz, H-15) and 7.32 (d, J = 1.2 Hz, H-16). The lower field signal at δH 4.98 (s) was deduced to be a hemiacetal proton H-20 whose HMBC spectrum showed correlations with carbons at δC 35.0 (C-1), 45.1 (C-9), 43.4 (C-10), 70.0 (C-11) and 67.1 (C-19). The 13C APT spectrum displayed 21 carbon signals ascribed to an ester carbonyl (δC 176.1), a 1,2-disubstituted furan ring (δC 109.5, 129.5, 143.2, 146.8), a methyl, a methoxy, six methylenes, six methines, and two quaternary carbons. All carbon-bound protons were assigned by HSQC and 1H–1H COSY spectra (Fig. 3). The NMR spectra of 2 (Table 1) were quite similar to those of phangininoxy A [22], indicating that 2 was an epimer in hemiacetal carbon (C-20) of the latter. In the HMBC spectrum (Fig. 3), the correlations of H2-19 (δH 3.50, 4.98, d, J = 12.0 Hz) with C-3 (δC 35.7), C-4 (δC 47.5), C-5 (δC 46.2), C-18 (δC 176.1), and C-20 (δC 106.2) implied that C-19 was oxygenated methylene. The HMBC correlation of methoxyl (δH 3.72) with C-18 suggested that the methoxy was located at C-18. In the NOESY spectrum (Fig. 3), the key
Fig. 3. Key 1H–1H COSY (▬), HMBC (→) and NOESY (↔) correlations of 2.
25
correlations of H-8 (δH 2.18)/H-20 (δH 4.98) and H-11 (δH 4.73)/H-9 (δH 1.78) indicated the stereochemistry of H-11 (α) and H-20 (β), respectively. Thus, the structure of 2 was identified and named phangininoxy B. Compound 3 had the same carbon skeleton as 2, and its molecular formula was assigned to be C23H30O6 based on its positive HRESIMS (mnz [M + Na]+: 425.1950; calcd for 425.1940). Comparison of the NMR spectroscopic data of 3 with those of 2 showed that compound 3 is a 19-O-ethyl derivative of the latter, which was supported on the basis of the HMBC correlations of H-19 (δH 5.64) with C-3 (δC 30.6), C-4 (δC 52.0), C-18 (δC 175.6), and C-20 (δC 101.3), and the methyl signal (δH 1.21, t, J = 7.2 Hz) with oxygenated methylene carbon (δC 63.9) and the methylene signal (δH 3.62, m; 3.86, m) with methyl carbon (δC 15.2) and C-19 (δC 98.7) (Fig. 4). In the 1H–1H COSY spectrum, the correlation between methyl protons and oxymethylene also confirmed the presence of oxygenated ethyl side-chain. The HMBC correlation of methoxyl (δH 3.71) with C-18 (δC 175.7) suggested that the methoxy was attached to C-18. The relative configurations of compound 3 were established to be identical to those of 2 on the basis of analysis of the NOESY spectrum (Fig. 4). The NOESY correlations between the dioxymethine proton at δH 5.64 (H-19) displayed a cross-peak with protons at δH 1.98 (H-5) and 1.77 (H-9), indicating that this dioxymethine proton was α-oriented. Accordingly, compound 3 was named phangininoxy C. All isolates were tested for their cytotoxicity against three human cancer cell lines (HepG-2, MCF-7, and HCT-8) using the MTT method with cisplatin as the positive control. Compound 1 showed moderate cytotoxicity against all the tested cell lines, while 2 and 3 displayed weak activity with IC50 values of 22.3–28.1 μM (Table 2). Conflict of interest We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work, and there is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the position presented in, or the review of, the manuscript entitled. Acknowledgments The work was financially supported by the technological large platform for comprehensive research and development
Fig. 4. Key 1H–1H COSY (▬), HMBC (→) and NOESY (↔) correlations of 3.
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H.-F. Wu et al. / Fitoterapia 98 (2014) 22–26
Table 2 In-vitro cytotoxic activity of compounds 1–3. Compounds
IC50 (μM) HepG-2
1 2 3 Cisplatina
8.9 22.3 25.5 4.7
± ± ± ±
MCF-7 1.1 0.98 2.4 0.53
13.7 23.2 38.1 12.7
± ± ± ±
HCT-8 1.3 2.3 0.73 0.95
10.2 36.7 43.5 2.5
± ± ± ±
1.9 0.86 4.1 0.28
Values present mean ± SD of triplicate experiments. a Positive control substance.
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