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Ivorenoids A–F: limonoids from Khaya ivorensis
Tetrahedron 70 (2014) 3570e3575
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Ivorenoids AeF: limonoids from Khaya ivorensis Wen-Bin Wu, Hua Zhang, Hong-Chun Liu, Shi-Hui Dong, Yan Wu, Jian Ding, Jian-Min Yue * State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi Road, Shanghai 201203, People’s Republic of China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 25 January 2014 Received in revised form 27 March 2014 Accepted 3 April 2014 Available online 12 April 2014
Six new limonoids, ivorenoids AeF (1e6), along with ten known analogues, were isolated from an ethanolic extract of the stems of Khaya ivorensis. Their structures were elucidated on the basis of spectroscopic analyses. Compounds 1 and 2 possessed a rare rearranged skeleton of khayanolides and a unique g-lactone (C-16/C-8) replacing the common and characteristic d-lactone D-ring (C-16/C-17) of limonoids. A mechanism of the interesting deuteration of H-2 and H-15b of 1, and H-15b of the solvent CD3OD due to the ketoeenol tautomerism was demonstrated. Compounds 3 and 6 showed moderate activity against HL-60 cell line with IC50 values of 15.3 and 17.5 mM, respectively. Ó 2014 Elsevier Ltd. All rights reserved.
Keywords: Khaya ivorensis Limonoid Ivorenoid Cytotoxicity
1. Introduction Khaya ivorensis A. Chev. (Meliaceae) grows widely in Angola, Cameroon, and Nigeria.1 It is also cultivated in southern China. This plant has been demonstrated to exhibit a wide range of biological properties, such as anti-inflammatory, anti-malarial, anti-fungal and cytotoxic activities.2,3 In ourprevious work, four limonoids and two macrolides were isolated from the stems of K. ivorensis.4e6 As a continuation of our chemical investigation on this plant species, six new limonoids, ivorenoids AeF (1e6), including two D-ring demolished khayanolides (1 and 2), three mexicanolides (3e5), and one andirobin type limonoid (6), were isolated and characterized (Fig. 1). Khayanolides, a very rare class of limonoids biosynthetically produced from phragmalins by the cleavage of the C-1eC-2 bond and formation of a C-1eC-30 bond, were first isolated from Khaya senegalensis.7 Compounds 1 and 2 possessed a rare rearranged skeleton of khayanolides and a unique g-lactone (C-16/C-8) replacing the common and characteristic d-lactone (C-16/C-17) of limonoids. It is very interesting that the phenomenon of deuteration of H-2 and H-15b in ivorenoid A (1), and H-15b in ivorenoid B (2) were observed 1H NMR acquirement, which was rationalized by involving the enolization procedures in the solution. We present herein the isolation and structure elucidation of these new compounds, the mechanism of deuteration of H-2 and H-15b in
Fig. 1. Limonoids 1e6 from K. ivorensis.
ivorenoid A (1), and H-15b in ivorenoid B (2), and the biological evaluation on these limonoids. 2. Results and discussion
* Corresponding author. Tel./fax: þ86 21 50806718; e-mail addresses: jmyue@ mail.shcnc.ac.cn, [email protected] (J.-M. Yue). 0040-4020/$ e see front matter Ó 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tet.2014.04.007
Ivorenoid A (1) displayed a sodiated ion at m/z 581.2002 [MþNa]þ in the HR-ESI(þ)MS analysis, consistent with an
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elemental composition of C29H34O11 requiring 13 degrees of unsaturation. The 1H NMR data (Table 1) of 1 revealed the presence of three tertiary methyls (dH 1.02, 1.03, 1.46, each 3H, s), an acetyl methyl (dH 2.09, 3H, s), and a methoxyl (dH 3.76, 3H, s), as well as a typical b-substituted furan ring (dH 7.38, br s; 6.42, d, J¼1.2 Hz; 7.44, t, J¼1.7 Hz). The 13C NMR and HSQC spectrum showed 29 resonances in agreement with the HR-ESI(þ)MS analysis, and the DEPT and 2D NMR experiments indicated that 32 of the 34 hydrogen atoms were directly attached to carbons (CH35, CH24, and CH9), and the remaining two protons were assignable to the presence of two hydroxy groups. One proton signal at dH 1.74, which showed no correlation with any carbons in the HSQC spectrum, was assigned as H-11b by its correlations with H-9 and H-11a in the HeH COSY, and this was confirmed by its correlation to C-12 in the HMBC spectrum. The furan ring and four carbonyl groups as distinguished by NMR analysis accounted for seven out of the 13 degrees of unsaturation, which required compound 1 being hexacyclic in the limonoid core. The aforementioned facts and biogenetic reasoning suggested that 1 was a member of the limonoid family. Fig. 2. Selected HMBC (H/C) and ROESY correlations (H4H) of 1. Table 1 1 H and 13C NMR data of compounds 1e3 Position 1a
dH
2a
dC
(mult, J in Hz) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 28 29
4.69 (d, 11.1)
3.36 (d, 9.9) 4.27 (d, 9.9)
2.56 (d, 8.0)
a 2.17 (m) b 1.74 (m) a 1.80 (m) b 0.81 (m)
a 3.55 (d, 18.5) b 3.01 (d, 18.5) 4.95 (s) 1.02 (s) 1.46 (s)
30
7.38 (br s) 6.42 (d, 1.2) 7.44 (t, 1.7) 1.03 (s) a 2.75 (d, 13.0) b 2.14 (d, 13.0) 3.67 (d, 11.1)
OAc
2.09 (s)
7-OMe
3.76 (s)
3b
dH
dC
(mult, J in Hz) 91.4 78.1 206.7 51.9 41.6 71.5 175.4 101.3 48.9 62.6 18.0
3.31 (d, 10.2) 4.27 (d, 10.2)
2.54 (d, 7.9)
a 2.17 (m) b 1.71 (m) 30.0 a 1.80 (m) b 0.80 (m)
42.9 90.7 39.3 a 3.60 (d, 18.7) b 3.69 (d, 18.7) 177.9 73.1 4.90 (s) 15.0 1.02 (s) 17.6 1.47 (s) 128.0 142.3 7.37 (br s) 112.2 6.42 (d, 1.3) 144.0 7.44 (t, 1.6) 16.0 1.03 (s) 41.6 a 2.78 (d, 13.1) b 2.15 (d, 13.0) 60.2 3.54 (s) 22.1 2.10 (s) 172.5 53.2 3.76 (s)
Recorded at 400 or 100 MHz for 1H and a Data measured in CD3OD. b Data measured in CDCl3.
13
dH
dC
(mult, J in Hz) 91.0 102.3 204.7 50.5 41.3 71.4 175.3 101.7 49.5 62.5 17.7
2.73 (m) 4.93 (d, 8.8) 2.52 (d, 3.8) 4.42 (br s)
1.61 (m)
a 1.79 (m) b 2.15 (m) 30.2 a 1.36 (m) b 1.84 (m)
42.8 90.6 2.12 (m) 42.4 2.82 (d, 4.8) 178.2 73.0 15.2 17.4 127.9 142.3 112.1 144.0 15.8 41.2
5.36 (s) 0.98 (s) 1.32 (s) 7.48 6.40 7.43 1.25 0.99
(br s) (d, 1.1) (t, 1.7) (s) (s)
68.4 a 1.75 (m) b 2.17 (m) 22.1 2.08 (s) 172.5 53.2 3.82 (s)
107.8 44.7 76.3 37.4 49.2 71.3 175.8 80.2 63.6 44.6 20.2 35.5 35.3 45.1 27.7 170.2 78.0 22.5 22.9 121.2 140.7 109.9 143.0 24.4 25.2 28.6 21.1 170.4 52.6
C, respectively.
Analysis of its HMBC spectrum (Fig. 2) outlined a carbon skeleton of khayanolide for 1, which was structurally related to a known limonoid, khayanolide E.8 However, both the proton and carbon resonances of CH-17 (dH 4.95, s; dC 73.1) in compound 1 were obviously upfield shifted as compared to those of khayanolides bearing a D-ring of d-lactone (C-16/C-17), suggesting that the characteristic D-ring of d-lactone was demolished in 1, which was
supported by the absence of the HMBC correlation from H-17 to C16. The presence of a hydroxy group at C-17 was revealed by the chemical shifts of H-17 and C-17.9 The HMBC correlations of H3-18/ C-12, C-13, C-14 and C-17; H-30/C-8, C-9 and C-14; and H-11a/C-9 and C-13 furnished the C-ring embodying two oxygenated quaternary carbons C-8 (dC 101.3) and C-14 (dC 90.7). The ether linkage between C-2 and C-14 was demonstrated by the key HMBC correlation from H-2 and C-14. Besides A1, A2, B1, B2 and C rings, the presence of an additional ring in the limonoid core was required to meet the degrees of unsaturation in 1. The severely downfield shifted signal of the quaternary carbon C-8 (dC 101.3) caused by the deshielding and electron-withdrawing effects of the g-lactone group indicated the formation of a lactone ring between C-8 and C16 in 1, which was very rare in the limonoid family. The relative configuration of compound 1 was mainly determined by the ROESY experiment (Fig. 2). The correlations of H-5/ H-11b, and H-5/H-12b revealed that H-5, H-11b, and H-12b were co-facial, and assigned in a b-orientation randomly. Consequently, the correlation of H-11b/H-17 along with the chemical shifts of H17 (dH 4.95, s) and C-17 (dC 73.1) tentatively fixed OH-17 to be adirected,9,10 which is consistent with the biosynthetic reasoning, and the fact that all the limonoids isolated from this plant shared the same C-17 configuration.4 The ROESY correlations of H-9/H-11a, H-11a/H3-18, and H3-18/H-15a showed that H-9, H-11a, CH3-18 and the newly formed five-membered g-lactone ring were co-facial and a-configured. The relative configuration of C-6 could not be assigned via the available NMR data. The structure of ivorenoid A was thus assigned as depicted. It was interesting that the integration values of the proton signals H-2 at dH 4.69 (d, J¼11.1 Hz) and H-15b at dH 3.01 (d, J¼18.5 Hz) of 1 shrunk severely while measuring 2D NMR data in CD3OD, and in concomitant the doublet proton signals H-15a at dH 3.55 (d, J¼18.5 Hz) and H-30 at dH 3.67 (d, J¼11.1 Hz) became into singlets, which were sharply contrary to the 1H NMR data acquired ten days before (Fig. 3). The exchanges between the hydrogen atoms of compound and the deuterium of solvent CD3OD were supposed to happen in the NMR tube. To prove this assumption, the sample was treated with CH3OH after removal of CD3OD. When the 1H NMR spectrum of the recovered sample was acquired in CD3OD again, the H-2 and H-15b signals of 1 were observed normally. The deuteration mechanism by involving a keto-enol tautomerism as the driving force was proposed (Fig. 4). Compound 1 coexisted with its enolized intermediate 1a in an equilibrium. When it was dissolved
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Fig. 3. Deuteration observed in the 1H NMR of 1 in CD3OD.
Fig. 4. Deuteration mechanism proposed for compound 1.
in CD3OD for several days, the intermediate 1a was mainly transformed into the stereospecific deuterated product 1b due to steric hindrance of CH3-18, which made the deuterium atom only accessible to attack the double bond from the upside of the fivemembered enol ring. This explanation was finally confirmed by keeping 1b in CH3OH to yield 1 again, where a similar equilibrium between 1b and 1c existed. Ivorenoid B (2) had the molecular formula C29H34O12. Analysis of its 1H and 13C NMR data (Table 1) indicated that 2 was a structural analogue of 1, and the only difference lain in that the H-2 in 1 was replaced by an OH group in 2. This was verified by the downfield shifted carbon signal of C-2 at dC 102.3, which showed an HMBC correlation with H-30. The relative configuration of 2 was assigned by comparing the NMR data with those of 1. A similar deuteration on H-15b was only observed in 2 (Supplementary data), which supported the mechanism proposed for the deuteration of H-2 and H-15b in 1 (Fig. 4). Ivorenoid C (3) showed a HR-EIMS ion at m/z 546.2457 [M]þ, corresponding to the molecular formula C29H38O10 with 11 degrees of unsaturation. The proton signals of four tertiary methyls (dH 0.98, 0.99, 1.25, 1.32, each 3H, s), one acetyl (dH 2.08, 3H, s), and a methoxy (3.82, 3H, s) were observed in the 1H NMR spectrum (Table 2). The 13C NMR with DEPT and HSQC experiments revealed 29 carbon resonances including 6CH3, 4CH2, 10CH (three oxygenated and three olefinic), and 9C (three carbonyl, one olefinic and two oxygenated) groups. Comprehensive analysis of the NMR
data suggested that compound 3 was a mexicanolide-type limonoid, whose structure was closely related to that of grandifolide A.11 The only difference was the presence of H-2 in 3 replacing the 2-OH of grandifolide A, which was confirmed by the HMBC correlations from H-2 to C-1, C-3, C-4, C-10 and C-30 (Supplementary data). Ivorenoid D (4) was assigned a molecular formula of C31H40O12 by HR-ESI(þ)MS. Analysis of its 1H and 13C NMR data (Table 1) suggested that its structure was closely related to tabulvelutin B,12 the only difference was the presence of one more hydroxyl in 4. The hydroxy group was then located at C-11 (dC 64.2) based on the chemical shifts of the related protons and carbons, and the 2D NMR spectra of 4 (Supplementary data). The b-orientation for H-11 was assigned on the ground of ROESY correlation between H-11 and H-5 (Supplementary data). The structure of 4 was thus elucidated as shown. Ivorenoid E (5) showed similar spectroscopic characteristics to those of compound 4. By comparing its NMR data (Table 2) with those of 4, the C-2 signal of 5 was upfield shifted to dC 79.7, indicating that a hydroxy group was located at this position instead of the acetoxyl group of 4; the H-11 resonance was obviously downfield shifted, revealing the presence of an acetoxyl group at C-11, which was confirmed by the HMBC correlation from H-11 to the acetyl carbonyl. Furthermore, 1H and 13C NMR analysis showed the presence of an isobutyryloxy moiety (dH 1.14, 3H, d, J¼7.0 Hz; 1.15, 3H, d, J¼7.1 Hz; 2.65, 1H, m; dC 19.0, 19.0, 34.0, 178.0), which was then located at C-3 by the key HMBC correlation from H-3 at dH 4.80 to C-10 at dC 178.0 (Supplementary data). The stereochemistry of H3 was assigned to be a-oriented by the ROESY correlation between H-3 and the proton of 1-OH (Supplementary data). Ivorenoid F (6) gave a molecular formula of C27H34O9 with eleven degrees of unsaturation as determined by HR-EIMS at m/z 502.2198 [M]þ. The 1H NMR data of 6 (Table 2) revealed the existence of four tertiary methyls (dH 1.04, 1.05, 1.45 and 1.47, each 3H, s), a methoxy group (dH 3.85, 3H, s), and a b-substituted furan ring. The 13C NMR and DEPT spectra of 6 showed 27 carbon resonances comprising five methyls, four methylenes (one olefinic), nine methines (three olefinic), and nine quaternary carbons (three carbonyl and two olefinic). These data suggested that compound 6 was likely an andirobin-type limonoid. Extensive analysis of the 1D and 2D NMR data (Fig. 5) further revealed that the structure of 6 was closely related to that of 17-epi-methyl-6-hydroxyangolensate,13 and only differed in the presence of an additional hydroxy group, which was attached at C-11 by the chemical shift of C-11 at dC 67.8. The b-orientation of H-11 in 6 was fixed by the key ROESY correlation (Fig. 5) between H-11 and H-5. The H-17 was assigned in a borientation by the ROESY correlations of H-5/H-12b and H-12b/H17. The ten known compounds were characterized as khayanone,14 6R,8a-dihydroxycarapin,15 khayalenoid K,9 1-O-acetylkhayanolide B,16 khayanolide D,9 khayalactone,17 seneganolide,18 khayalactol,15 senegalension B and ivorenolide A5,19 by comparing their spectroscopic data with those reported in the literature. All the isolates were tested for cytotoxicities against HL-60 (human leukaemia) and P388 (murine leukaemia) cell lines using the MTT method.20 Ivorenoids C (3) and F (6) exhibited selective activities against HL-60 with IC50 values of 15.3 and 17.5 mM, respectively. Doxorubicin (Melone Pharmaceutical Co. Ltd., purity: 98.48%) was used as the positive control (IC50: 0.19 mM for HL-60 and 0.63 mM for P388). 3. Conclusions In conclusion, six new limonoids, including compounds 1 and 2 possessed a rare rearranged skeleton of khayanolides and a unique g-lactone (C-16/C-8) ring, were obtained and characterized from K. ivorensis. These findings are important addition to the structurally
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Table 2 1 H and 13C NMR data of compounds 4e6 in CDCl3 Position
4
5
dH (mult, J in Hz)
dC
1 2
6
dH (mult, J in Hz)
108.0 93.4
3 4 5 6
5.50 (s)
76.0 39.6 40.4 32.1
2.73 (m) a 3.02 (d, 16.1) b 2.33 (m),
7 8 9 10 11 12
1.55 (d, 11.0) 4.22 (ddd, 11.0, 11.0, 4.0) a 1.36 (m) b 2.00 (dd, 14.2, 4.1)
13 14 15
36.6 43.6 27.3
2.17 (m) 2.82 (m)
16 17 18 19 20 21 22 23 28 29 30
169.6 78.0 22.3 22.7 120.9 140.9 109.8 143.2 22.1 24.2 39.5
5.22 (s) 1.05 (s) 1.17 (s) 7.54 (s) 6.44 (t, 0.8) 7.45 (t, 1.5) 0.74 (s) 1.35 (s) a 1.94 (d, 14.9) b 2.67 (d, 14.9) 7.17 (s)
1-OH 2-OH 2-OAc
2.08 (s)
3-OAc
2.12 (s)
7-OMe 11-OAc
3.71 (s)
177.3 78.7 67.5 47.5 64.2 45.2
22.1 174.6 21.0 170.4 52.2
10 20 30 40
4.80 (s) 2.65 (m) a 2.68 (m) b 2.33 (dd, 17.1, 11.2)
1.82 (d, 11.2) 5.01 (ddd, 11.1, 11.1, 3.7) a 1.17 (m) b 2.49 (dd, 13.8, 3.7) 2.17 (d, 7.3) a 2.81 (dd, 19.6, 7.9) b 2.95 (d, 19.6) 5.31 (s) 0.92 (s) 1.18 (s) 7.72 (s) 6.66 (d, 0.8) 7.40 (t, 1.6) 0.70 (s) 1.27 (s) a 1.59 (d, 13.9) b 2.60 (m) 5.22 (br s) 4.31 (br s)
3.65 (s) 1.90 (s)
2.65 (m) 1.15 (d, 7.1) 1.14 (d, 7.0)
Recorded at 400 or 100 MHz for 1H and
13
dC
dH (mult, J in Hz)
dC
108.4 79.7
3.64 (dd, 5.6, 2.6) a 2.37 (d, 2.4) b 3.10 (d, 5.8)
78.2 39.0
82.0 38.5 39.2 32.0 174.3 78.4 65.8 44.5 68.7 40.7 36.3 44.1 27.1 170.3 78.4 22.1 22.0 120.7 141.2 109.8 142.8 22.0 24.3 40.8
51.9 20.9 169.7 178.0 34.0 19.0 19.0
2.55 (s) 4.46 (d, 1.5)
2.33 (br s) 4.44 (br s) a 1.36 (d, 14.2) b 1.89 (dd, 14.6, 4.0)
a 2.94 (d, 18.0) b 2.64 (d, 17.9) 5.60 (s) 1.04 (s) 1.45 (s) 7.44 (br s) 6.37 (d, 1.0) 7.40 (t, 1.6) 1.47 (s) 1.05 (s) a 5.15 (s) b 5.36 (s)
3.85 (s)
211.6 48.8 47.7 72.2 176.1 142.2 59.0 45.3 67.8 36.3 40.7 80.8 33.4 169.6 79.1 16.9 23.3 120.5 140.8 109.8 142.9 23.7 24.7 115.7
53.6
C, respectively.
diverse and complex limonoid family. In particular, the observation on the exchange of the hydrogen atoms in compounds 1 and 2 with the deuterium of the solvent CD3OD due to the ketoeenol tautomerism is a good scientific input for understanding the involved chemistry. 4. Experimental section 4.1. General experimental procedures
Fig. 5. Selected HMBC (H/C) and ROESY correlations (H4H) of 6.
Specific rotations were acquired on a PerkineElmer 341 polarimeter. UV spectra were measured on a Shimadzu UV-2550 UVevisible spectrophotometer. IR spectra were recorded on a PerkineElmer 577 spectrometer. NMR spectra were measured on a Bruker AM-400 spectrometer and were referenced to the solvent peaks (dH at 3.34 and dC at 49.86 in CD3OD; dH at 7.26 and dC at 77.16 in CDCl3). EIMS (70 eV) and HR-EIMS were carried out on a Finnigan MAT 95 mass spectrometer. ESIMS and HR-ESIMS were carried out on a Bruker Daltonics Esquire 3000plus instrument and a Waters QTOF Ultima mass spectrometer, respectively. Silica gel (300e400 mesh), C18 reverse-phased silica gel (150e200 mesh, Merck), and MCI gel (CHP20P, 75e150 mM, Mitsubishi Chemical
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Industries, Ltd.) were used for column chromatography, and precoated silica gel GF254 plates (Qingdao Marine Chemical Plant, Qingdao, People’s Republic of China) were used for TLC. Semipreparative HPLC was performed on a Waters 515 pump equipped with a Waters 2487 UV detector (254 nm) and a YMC-Pack ODS-A column (25010 mm, S-5 mm, 12 nm). All solvents used were of analytical grade (Shanghai Chemical Plant, Shanghai, People’s Republic of China), and solvents used for HPLC were of HPLC grade (J&K Scientific Ltd.). 4.2. Plant material Plant material of K. ivorensis was collected from Yunnan Province of China in July 2009 and identified by Professor Y.-K. Xu of Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences. A voucher specimen has been deposited in the Shanghai Institute of Materia Medica, Chinese Academy of Sciences (accession number: Khivo-2009-1Y). 4.3. Extraction and isolation The air-dried powder of K. ivorensis plant material (10.0 kg) was extracted with 95% EtOH three times at room temperature (each 10 L), and the crude was partitioned between H2O and ethyl acetate. The EtOAc-soluble fraction (260 g) was subjected to a column of MCI gel eluted with methanol in water (MeOH/H2O, 30:70 to 90:10) to give four fractions, A-D. Fraction B was separated over a column of silica gel and eluted with CHCl3/MeOH (100:1 to 5:1) to obtain fraction B1eB5. Fraction B3 was chromatographed over silica gel column eluted with petroleum ether/ ethyl acetate (10:1 to 1:1) to give four subfractions, B3aeB3d. Fraction B3b was purified over a column of reverse-phased silica gel (MeOH/H2O, 65:35) to afford 1 (3 mg) and 2 (10 mg). Fraction B3c was successfully separated by semi-preparative HPLC (MeOH/ H2O, 65/35, 3 mL/min) to afford khayalactone (5 mg). Fraction B3d was applied to a column eluted with petroleum ether/acetone (10:1 to 2:1) to yield 1-O-acetylkhayanolide B (9 mg). Fraction B4 was separated by silica gel (petroleum ether/ethyl acetate, 10:1 to 1:5), and each major components was purified by semipreparative HPLC (MeOH/H2O, 65/35, 3 mL/min) to afford 3 (6 mg), khayanone (12 mg) and khayalactol (8 mg), sequentially. Part B5 was applied to a silica gel column (petroleum ether/ethyl acetate) to give two fractions B5a and B5b. Fraction B5a was separated by semi-preparative HPLC (MeOH/H2O, 60/40, 3 mL/ min) to yield 4 (10 mg) and 8a-dihydroxycarapin (9 mg). Fraction B5b was subjected to silica gel column (petroleum ether/acetone, 5:1 to 1:5) to afford khayanolide D (12 mg). Fraction C was chromatographed over silica gel column eluted with CHCl3/MeOH (100:1 to 5:1) to give three subfractions, C1eC3. Fraction C1 was separated over a column of silica gel (petroleum ether/ethyl acetate, 20/1 to 3/1) to obtain three major parts, each of which was purified by semi-preparative HPLC (MeOH/H2O, 55/45, 3 mL/min) to afford 5 (12 mg), senegalension B (6 mg) and khayalenoid K (3 mg), respectively. Fraction C2 was separated by semipreparative HPLC (MeOH/H2O, 55/45, 3 mL/min) to yield 6 (9 mg), seneganolide (8 mg) and ivorenolide A (5 mg), successively.
LR-ESI()MS m/z 603.8 [MþHCOO]; HR-ESI(þ)MS m/z 581.2002 [MþNa]þ (calcd for C29H34O11Na 581.1999). 4.4.2. Ivorenoid B (2). White amorphous powder; [a]20 D þ3 (c 0.1, MeOH); UV (MeOH) lmax (log ε) 271 (4.79) nm; IR (KBr) nmax 3431, 2925, 1736, 1630, 1385, 1250, 1052, 602 cm1; 1H and 13C NMR data: see Table 1; LR-ESI(þ)MS m/z 597.2 [MþNa]þ; LR-ESI()MS m/z 573.6 [MH], 1147.3 [2MH]; HR-ESI(þ)MS m/z 597.1952 [MþNa]þ (calcd for C29H34O12Na 597.1948). 4.4.3. Ivorenoid C (3). White amorphous powder; [a]20 D 59 (c 0.1, MeOH); UV (MeOH) lmax (log ε) 265 (4.13) nm; IR (film) nmax 3438, 2929, 1720, 1651, 1456, 1373, 1240, 1026, 756 cm1; 1H and 13C NMR data: see Table 1; LR-ESI(þ)MS m/z 569.3 [MþNa]þ, 1115.3 [2MþNa]þ; LR-ESI()MS m/z 591.5 [MþHCOO]; EIMS m/z 546 [M]þ (4), 486 (7), 390 (13), 343 (68), 304 (42), 218 (75), 159 (66), 121 (58), 95 (100); HR-EIMS m/z 546.2457 [M]þ (calcd for C29H38O10 546.2465). 4.4.4. Ivorenoid D (4). White amorphous powder; [a]20 D 31 (c 0.1, MeOH); UV (MeOH) lmax (log ε) 265 (4.24) nm; IR (KBr) nmax 3466, 2952, 1739, 1637, 1437, 1373, 1306, 1232, 1159, 1107, 1058, 1027, 968, 930, 876, 806, 602 cm1; 1H and 13C NMR data: see Table 2; LRESI(þ)MS m/z 627.3 [MþNa]þ, 1231.5 [2MþNa]þ; LR-ESI()MS m/ z 649.6 [MþHCOO]; HR-ESI(þ)MS m/z 627.2410 [MþNa]þ (calcd for C31H40O12Na 627.2417). 4.4.5. Ivorenoid E (5). White amorphous powder; [a]20 D 57 (c 0.1, MeOH); UV (MeOH) lmax (log ε) 266 (4.14) nm; IR (KBr) nmax 3440, 2975, 2937, 2879, 1747, 1651, 1603, 1470, 1381, 1317, 1234, 1219, 1144, 1061, 1026, 901, 604 cm1; 1H and 13C NMR data: see Table 2; LRESI(þ)MS m/z 655.3 [MþNa]þ, 1287.5 [2MþNa]þ; LR-ESI()MS m/ z 631.7 [MH], 1263.3[2MH]; HR-ESI(þ)MS m/z 655.2722 [MþNa]þ (calcd for C33H44O12Na 655.2730). 4.4.6. Ivorenoid F (6). White amorphous powder; [a]20 D 54 (c 0.05, MeOH); UV (MeOH) lmax (log ε) 268 (4.27) nm; IR (KBr) nmax 3450, 2927, 1724, 1462, 1435, 1387, 1246, 1111, 1045, 876, 756 cm1; 1H and 13 C NMR data: see Table 2; LR-ESI(þ)MS m/z 525.3 [MþNa]þ, 1027.4 [2MþNa]þ; EIMS m/z 502 [M]þ (88), 470 (21), 374 (38), 359 (34), 332 (36), 149 (77), 137 (100), 121 (79), 95 (90); HR-EIMS m/z 502.2198 [M]þ (calcd for C27H34O9 502.2203).
4.5. Cytotoxic assay The cytotoxic activities of all the isolated compounds were evaluated by using the MTT method. Briefly, the tested cells in culture medium 100 mL were plated in the wells of 96-well plates (Falcon, CA). The cells were treated in triplicate with graded concentrations of the compounds at 37 C for 72 h. A 20 mL aliquot of MTT solution (5 mg/mL) was put directly in the wells. The cultures were incubated for 4 h, and 100 mL of ‘triplex solution’ (10% SDS/5% i BuOH/12 mM HCl) was added. The plates were incubated at 37 C overnight and the measured using a plate reader at 570 nm (VERSA Max, Molecular Devices). The results were expressed as IC50 as calculated by the Logit method.
Acknowledgements 4.4. Characterization of new limonoids 4.4.1. Ivorenoid A (1). White amorphous powder; [a]20 D þ26 (c 0.05, MeOH); UV (MeOH) lmax (log ε) 266 (4.68) nm; IR (film) nmax 3438, 2924, 2852, 1782, 1730, 1462, 1369, 1246, 1147, 1020, 758, 602 cm1; 1 H and 13C NMR data: see Table 1; LR-ESI(þ)MS m/z 581.1 [MþNa]þ;
The financial support (No. 81273398) of the National Natural Science Foundation, and the foundation (2012CB721105) from the Ministry of Science and Technology of the People’s Republic of China are gratefully acknowledged. We thank Professor You-Kai Xu for the identification of the plant material.
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Supplementary data The NMR, ESIMS, EIMS, IR, 2D NMR spectra of compounds 1e6. This material is available free of charge via the Internet. Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.tet.2014.04.007.
References and notes 1. Adesida, G. A.; Adesogan, E. K.; Okorie, D. A.; Taylor, D. A. H.; Styles, B. T. Phytochemistry 1971, 10, 1845e1853. 2. Abdelgaleil, S. A. M.; Hashinaga, F.; Nakatani, M. Pest Manag. Sci. 2005, 61, 186e190. 3. Agbedahunsi, J. M.; Fakoya, F. A.; Adesanya, S. A. Phytomedicine 2004, 11, 504e508. 4. Zhang, B.; Yang, S. P.; Yin, S.; Zhang, C. R.; Wu, Y.; Yue, J. M. Phytochemistry 2009, 70, 1305e1308. 5. Zhang, B.; Wang, Y.; Yang, S. P.; Zhou, Y.; Wu, W. B.; Tang, W.; Zuo, J. P.; Li, Y.; Yue, J. M. J. Am. Chem. Soc. 2012, 134, 20605e20608. 6. Wang, Y.; Liu, Q. F.; Xue, J. J.; Zhou, Y.; Yu, H. C.; Yang, S. P.; Zhang, B.; Zuo, J. P.; Li, Y.; Yue, J. M. Org. Lett. 2014, 16, 2062e2065. 7. Olmo, L. R. V.; Silva, M. F.; Das, G. F. da; Fo, E. R.; Vieira, P. C.; Fernandes, J. B.; Marsaioli, A. J.; Pinheiro, A. L.; Vilela, E. F. Phytochemistry 1996, 42, 831e837.
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8. Nakatani, M.; Abdelgaleil, S. A. M.; Kassem, S. M. I.; Takezaki, K.; Okamura, H.; Iwagawa, T.; Doe, M. J. Nat. Prod. 2002, 65, 1219e1221. 9. Wu, J.; Yang, S. X.; Li, M. Y.; Feng, G.; Pan, J. Y.; Xiao, Q.; Sinkkonen, J.; Satyanandamurty, T. J. Nat. Prod. 2010, 73, 644e649. 10. Chen, Y. Y.; Wang, X. N.; Fan, C. Q.; Yin, S.; Yue, J. M. Tetrahedron Lett. 2007, 48, 7480e7484. 11. Zhang, H.; Odeku, O. A.; Wang, X. N.; Yue, J. M. Phytochemistry 2008, 69, 271e275. 12. Yin, J. L.; Di, Y. T.; Fang, X.; Liu, E. D.; Liu, H. Y.; He, H. P.; Li, S. L.; Li, S. F.; Hao, X. J. Tetrahedron Lett. 2011, 52, 3083e3085. 13. Rahman, A.; Zareen, S.; Choudhary, M. I.; Akhtar, M. N.; Khan, S. N. J. Nat. Prod. 2008, 71, 910e913. 14. Khalid, S. A.; Friedrichsen, G. M.; Kharazmi, A.; Theander, T. G.; Olsen, C. E.; Christensen, S. B. Phytochemistry 1998, 49, 1769e1772. 15. Tchimene, M. K.; Tane, P.; Ngamga, D.; Connolly, J. D.; Farrugia, L. J. Phytochemistry 2005, 66, 1088e1093. 16. Abdelgaleil, S. A. M.; Okamura, H.; Iwagawa, T.; Doe, M.; Nakatani, M. Heterocycles 2000, 53, 2233e2240. 17. Tchuendem, M. H. K.; Ayafor, J. F.; Connolly, J. D.; Sterner, O. Tetrahedron Lett. 1998, 39, 719e722. 18. Nakatani, M.; Abdelgaleil, S. A. M.; Okamura, H.; Iwagawa, T.; Doe, M. Chem. Lett. 2000, 29, 876e877. 19. Yuan, C. M.; Zhang, Y.; Tang, G. H.; Li, S. L.; Di, Y. T.; Hou, L.; Cai, J. Y.; Hua, H. M.; He, H. P.; Hao, X. J. Chem. Asian J. 2012, 7, 2024e2027. 20. Tao, Z.; Zhou, Y.; Lu, J.; Duan, W.; Qin, Y.; He, X.; Lin, L.; Ding, J. Cancer Biol. Ther. 2007, 6, 691e696.
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Ivorenoids AeF: limonoids from Khaya ivorensis Wen-Bin Wu, Hua Zhang, Hong-Chun Liu, Shi-Hui Dong, Yan Wu, Jian Ding, Jian-Min Yue * State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi Road, Shanghai 201203, People’s Republic of China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 25 January 2014 Received in revised form 27 March 2014 Accepted 3 April 2014 Available online 12 April 2014
Six new limonoids, ivorenoids AeF (1e6), along with ten known analogues, were isolated from an ethanolic extract of the stems of Khaya ivorensis. Their structures were elucidated on the basis of spectroscopic analyses. Compounds 1 and 2 possessed a rare rearranged skeleton of khayanolides and a unique g-lactone (C-16/C-8) replacing the common and characteristic d-lactone D-ring (C-16/C-17) of limonoids. A mechanism of the interesting deuteration of H-2 and H-15b of 1, and H-15b of the solvent CD3OD due to the ketoeenol tautomerism was demonstrated. Compounds 3 and 6 showed moderate activity against HL-60 cell line with IC50 values of 15.3 and 17.5 mM, respectively. Ó 2014 Elsevier Ltd. All rights reserved.
Keywords: Khaya ivorensis Limonoid Ivorenoid Cytotoxicity
1. Introduction Khaya ivorensis A. Chev. (Meliaceae) grows widely in Angola, Cameroon, and Nigeria.1 It is also cultivated in southern China. This plant has been demonstrated to exhibit a wide range of biological properties, such as anti-inflammatory, anti-malarial, anti-fungal and cytotoxic activities.2,3 In ourprevious work, four limonoids and two macrolides were isolated from the stems of K. ivorensis.4e6 As a continuation of our chemical investigation on this plant species, six new limonoids, ivorenoids AeF (1e6), including two D-ring demolished khayanolides (1 and 2), three mexicanolides (3e5), and one andirobin type limonoid (6), were isolated and characterized (Fig. 1). Khayanolides, a very rare class of limonoids biosynthetically produced from phragmalins by the cleavage of the C-1eC-2 bond and formation of a C-1eC-30 bond, were first isolated from Khaya senegalensis.7 Compounds 1 and 2 possessed a rare rearranged skeleton of khayanolides and a unique g-lactone (C-16/C-8) replacing the common and characteristic d-lactone (C-16/C-17) of limonoids. It is very interesting that the phenomenon of deuteration of H-2 and H-15b in ivorenoid A (1), and H-15b in ivorenoid B (2) were observed 1H NMR acquirement, which was rationalized by involving the enolization procedures in the solution. We present herein the isolation and structure elucidation of these new compounds, the mechanism of deuteration of H-2 and H-15b in
Fig. 1. Limonoids 1e6 from K. ivorensis.
ivorenoid A (1), and H-15b in ivorenoid B (2), and the biological evaluation on these limonoids. 2. Results and discussion
* Corresponding author. Tel./fax: þ86 21 50806718; e-mail addresses: jmyue@ mail.shcnc.ac.cn, [email protected] (J.-M. Yue). 0040-4020/$ e see front matter Ó 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tet.2014.04.007
Ivorenoid A (1) displayed a sodiated ion at m/z 581.2002 [MþNa]þ in the HR-ESI(þ)MS analysis, consistent with an
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elemental composition of C29H34O11 requiring 13 degrees of unsaturation. The 1H NMR data (Table 1) of 1 revealed the presence of three tertiary methyls (dH 1.02, 1.03, 1.46, each 3H, s), an acetyl methyl (dH 2.09, 3H, s), and a methoxyl (dH 3.76, 3H, s), as well as a typical b-substituted furan ring (dH 7.38, br s; 6.42, d, J¼1.2 Hz; 7.44, t, J¼1.7 Hz). The 13C NMR and HSQC spectrum showed 29 resonances in agreement with the HR-ESI(þ)MS analysis, and the DEPT and 2D NMR experiments indicated that 32 of the 34 hydrogen atoms were directly attached to carbons (CH35, CH24, and CH9), and the remaining two protons were assignable to the presence of two hydroxy groups. One proton signal at dH 1.74, which showed no correlation with any carbons in the HSQC spectrum, was assigned as H-11b by its correlations with H-9 and H-11a in the HeH COSY, and this was confirmed by its correlation to C-12 in the HMBC spectrum. The furan ring and four carbonyl groups as distinguished by NMR analysis accounted for seven out of the 13 degrees of unsaturation, which required compound 1 being hexacyclic in the limonoid core. The aforementioned facts and biogenetic reasoning suggested that 1 was a member of the limonoid family. Fig. 2. Selected HMBC (H/C) and ROESY correlations (H4H) of 1. Table 1 1 H and 13C NMR data of compounds 1e3 Position 1a
dH
2a
dC
(mult, J in Hz) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 28 29
4.69 (d, 11.1)
3.36 (d, 9.9) 4.27 (d, 9.9)
2.56 (d, 8.0)
a 2.17 (m) b 1.74 (m) a 1.80 (m) b 0.81 (m)
a 3.55 (d, 18.5) b 3.01 (d, 18.5) 4.95 (s) 1.02 (s) 1.46 (s)
30
7.38 (br s) 6.42 (d, 1.2) 7.44 (t, 1.7) 1.03 (s) a 2.75 (d, 13.0) b 2.14 (d, 13.0) 3.67 (d, 11.1)
OAc
2.09 (s)
7-OMe
3.76 (s)
3b
dH
dC
(mult, J in Hz) 91.4 78.1 206.7 51.9 41.6 71.5 175.4 101.3 48.9 62.6 18.0
3.31 (d, 10.2) 4.27 (d, 10.2)
2.54 (d, 7.9)
a 2.17 (m) b 1.71 (m) 30.0 a 1.80 (m) b 0.80 (m)
42.9 90.7 39.3 a 3.60 (d, 18.7) b 3.69 (d, 18.7) 177.9 73.1 4.90 (s) 15.0 1.02 (s) 17.6 1.47 (s) 128.0 142.3 7.37 (br s) 112.2 6.42 (d, 1.3) 144.0 7.44 (t, 1.6) 16.0 1.03 (s) 41.6 a 2.78 (d, 13.1) b 2.15 (d, 13.0) 60.2 3.54 (s) 22.1 2.10 (s) 172.5 53.2 3.76 (s)
Recorded at 400 or 100 MHz for 1H and a Data measured in CD3OD. b Data measured in CDCl3.
13
dH
dC
(mult, J in Hz) 91.0 102.3 204.7 50.5 41.3 71.4 175.3 101.7 49.5 62.5 17.7
2.73 (m) 4.93 (d, 8.8) 2.52 (d, 3.8) 4.42 (br s)
1.61 (m)
a 1.79 (m) b 2.15 (m) 30.2 a 1.36 (m) b 1.84 (m)
42.8 90.6 2.12 (m) 42.4 2.82 (d, 4.8) 178.2 73.0 15.2 17.4 127.9 142.3 112.1 144.0 15.8 41.2
5.36 (s) 0.98 (s) 1.32 (s) 7.48 6.40 7.43 1.25 0.99
(br s) (d, 1.1) (t, 1.7) (s) (s)
68.4 a 1.75 (m) b 2.17 (m) 22.1 2.08 (s) 172.5 53.2 3.82 (s)
107.8 44.7 76.3 37.4 49.2 71.3 175.8 80.2 63.6 44.6 20.2 35.5 35.3 45.1 27.7 170.2 78.0 22.5 22.9 121.2 140.7 109.9 143.0 24.4 25.2 28.6 21.1 170.4 52.6
C, respectively.
Analysis of its HMBC spectrum (Fig. 2) outlined a carbon skeleton of khayanolide for 1, which was structurally related to a known limonoid, khayanolide E.8 However, both the proton and carbon resonances of CH-17 (dH 4.95, s; dC 73.1) in compound 1 were obviously upfield shifted as compared to those of khayanolides bearing a D-ring of d-lactone (C-16/C-17), suggesting that the characteristic D-ring of d-lactone was demolished in 1, which was
supported by the absence of the HMBC correlation from H-17 to C16. The presence of a hydroxy group at C-17 was revealed by the chemical shifts of H-17 and C-17.9 The HMBC correlations of H3-18/ C-12, C-13, C-14 and C-17; H-30/C-8, C-9 and C-14; and H-11a/C-9 and C-13 furnished the C-ring embodying two oxygenated quaternary carbons C-8 (dC 101.3) and C-14 (dC 90.7). The ether linkage between C-2 and C-14 was demonstrated by the key HMBC correlation from H-2 and C-14. Besides A1, A2, B1, B2 and C rings, the presence of an additional ring in the limonoid core was required to meet the degrees of unsaturation in 1. The severely downfield shifted signal of the quaternary carbon C-8 (dC 101.3) caused by the deshielding and electron-withdrawing effects of the g-lactone group indicated the formation of a lactone ring between C-8 and C16 in 1, which was very rare in the limonoid family. The relative configuration of compound 1 was mainly determined by the ROESY experiment (Fig. 2). The correlations of H-5/ H-11b, and H-5/H-12b revealed that H-5, H-11b, and H-12b were co-facial, and assigned in a b-orientation randomly. Consequently, the correlation of H-11b/H-17 along with the chemical shifts of H17 (dH 4.95, s) and C-17 (dC 73.1) tentatively fixed OH-17 to be adirected,9,10 which is consistent with the biosynthetic reasoning, and the fact that all the limonoids isolated from this plant shared the same C-17 configuration.4 The ROESY correlations of H-9/H-11a, H-11a/H3-18, and H3-18/H-15a showed that H-9, H-11a, CH3-18 and the newly formed five-membered g-lactone ring were co-facial and a-configured. The relative configuration of C-6 could not be assigned via the available NMR data. The structure of ivorenoid A was thus assigned as depicted. It was interesting that the integration values of the proton signals H-2 at dH 4.69 (d, J¼11.1 Hz) and H-15b at dH 3.01 (d, J¼18.5 Hz) of 1 shrunk severely while measuring 2D NMR data in CD3OD, and in concomitant the doublet proton signals H-15a at dH 3.55 (d, J¼18.5 Hz) and H-30 at dH 3.67 (d, J¼11.1 Hz) became into singlets, which were sharply contrary to the 1H NMR data acquired ten days before (Fig. 3). The exchanges between the hydrogen atoms of compound and the deuterium of solvent CD3OD were supposed to happen in the NMR tube. To prove this assumption, the sample was treated with CH3OH after removal of CD3OD. When the 1H NMR spectrum of the recovered sample was acquired in CD3OD again, the H-2 and H-15b signals of 1 were observed normally. The deuteration mechanism by involving a keto-enol tautomerism as the driving force was proposed (Fig. 4). Compound 1 coexisted with its enolized intermediate 1a in an equilibrium. When it was dissolved
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Fig. 3. Deuteration observed in the 1H NMR of 1 in CD3OD.
Fig. 4. Deuteration mechanism proposed for compound 1.
in CD3OD for several days, the intermediate 1a was mainly transformed into the stereospecific deuterated product 1b due to steric hindrance of CH3-18, which made the deuterium atom only accessible to attack the double bond from the upside of the fivemembered enol ring. This explanation was finally confirmed by keeping 1b in CH3OH to yield 1 again, where a similar equilibrium between 1b and 1c existed. Ivorenoid B (2) had the molecular formula C29H34O12. Analysis of its 1H and 13C NMR data (Table 1) indicated that 2 was a structural analogue of 1, and the only difference lain in that the H-2 in 1 was replaced by an OH group in 2. This was verified by the downfield shifted carbon signal of C-2 at dC 102.3, which showed an HMBC correlation with H-30. The relative configuration of 2 was assigned by comparing the NMR data with those of 1. A similar deuteration on H-15b was only observed in 2 (Supplementary data), which supported the mechanism proposed for the deuteration of H-2 and H-15b in 1 (Fig. 4). Ivorenoid C (3) showed a HR-EIMS ion at m/z 546.2457 [M]þ, corresponding to the molecular formula C29H38O10 with 11 degrees of unsaturation. The proton signals of four tertiary methyls (dH 0.98, 0.99, 1.25, 1.32, each 3H, s), one acetyl (dH 2.08, 3H, s), and a methoxy (3.82, 3H, s) were observed in the 1H NMR spectrum (Table 2). The 13C NMR with DEPT and HSQC experiments revealed 29 carbon resonances including 6CH3, 4CH2, 10CH (three oxygenated and three olefinic), and 9C (three carbonyl, one olefinic and two oxygenated) groups. Comprehensive analysis of the NMR
data suggested that compound 3 was a mexicanolide-type limonoid, whose structure was closely related to that of grandifolide A.11 The only difference was the presence of H-2 in 3 replacing the 2-OH of grandifolide A, which was confirmed by the HMBC correlations from H-2 to C-1, C-3, C-4, C-10 and C-30 (Supplementary data). Ivorenoid D (4) was assigned a molecular formula of C31H40O12 by HR-ESI(þ)MS. Analysis of its 1H and 13C NMR data (Table 1) suggested that its structure was closely related to tabulvelutin B,12 the only difference was the presence of one more hydroxyl in 4. The hydroxy group was then located at C-11 (dC 64.2) based on the chemical shifts of the related protons and carbons, and the 2D NMR spectra of 4 (Supplementary data). The b-orientation for H-11 was assigned on the ground of ROESY correlation between H-11 and H-5 (Supplementary data). The structure of 4 was thus elucidated as shown. Ivorenoid E (5) showed similar spectroscopic characteristics to those of compound 4. By comparing its NMR data (Table 2) with those of 4, the C-2 signal of 5 was upfield shifted to dC 79.7, indicating that a hydroxy group was located at this position instead of the acetoxyl group of 4; the H-11 resonance was obviously downfield shifted, revealing the presence of an acetoxyl group at C-11, which was confirmed by the HMBC correlation from H-11 to the acetyl carbonyl. Furthermore, 1H and 13C NMR analysis showed the presence of an isobutyryloxy moiety (dH 1.14, 3H, d, J¼7.0 Hz; 1.15, 3H, d, J¼7.1 Hz; 2.65, 1H, m; dC 19.0, 19.0, 34.0, 178.0), which was then located at C-3 by the key HMBC correlation from H-3 at dH 4.80 to C-10 at dC 178.0 (Supplementary data). The stereochemistry of H3 was assigned to be a-oriented by the ROESY correlation between H-3 and the proton of 1-OH (Supplementary data). Ivorenoid F (6) gave a molecular formula of C27H34O9 with eleven degrees of unsaturation as determined by HR-EIMS at m/z 502.2198 [M]þ. The 1H NMR data of 6 (Table 2) revealed the existence of four tertiary methyls (dH 1.04, 1.05, 1.45 and 1.47, each 3H, s), a methoxy group (dH 3.85, 3H, s), and a b-substituted furan ring. The 13C NMR and DEPT spectra of 6 showed 27 carbon resonances comprising five methyls, four methylenes (one olefinic), nine methines (three olefinic), and nine quaternary carbons (three carbonyl and two olefinic). These data suggested that compound 6 was likely an andirobin-type limonoid. Extensive analysis of the 1D and 2D NMR data (Fig. 5) further revealed that the structure of 6 was closely related to that of 17-epi-methyl-6-hydroxyangolensate,13 and only differed in the presence of an additional hydroxy group, which was attached at C-11 by the chemical shift of C-11 at dC 67.8. The b-orientation of H-11 in 6 was fixed by the key ROESY correlation (Fig. 5) between H-11 and H-5. The H-17 was assigned in a borientation by the ROESY correlations of H-5/H-12b and H-12b/H17. The ten known compounds were characterized as khayanone,14 6R,8a-dihydroxycarapin,15 khayalenoid K,9 1-O-acetylkhayanolide B,16 khayanolide D,9 khayalactone,17 seneganolide,18 khayalactol,15 senegalension B and ivorenolide A5,19 by comparing their spectroscopic data with those reported in the literature. All the isolates were tested for cytotoxicities against HL-60 (human leukaemia) and P388 (murine leukaemia) cell lines using the MTT method.20 Ivorenoids C (3) and F (6) exhibited selective activities against HL-60 with IC50 values of 15.3 and 17.5 mM, respectively. Doxorubicin (Melone Pharmaceutical Co. Ltd., purity: 98.48%) was used as the positive control (IC50: 0.19 mM for HL-60 and 0.63 mM for P388). 3. Conclusions In conclusion, six new limonoids, including compounds 1 and 2 possessed a rare rearranged skeleton of khayanolides and a unique g-lactone (C-16/C-8) ring, were obtained and characterized from K. ivorensis. These findings are important addition to the structurally
W.-B. Wu et al. / Tetrahedron 70 (2014) 3570e3575
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Table 2 1 H and 13C NMR data of compounds 4e6 in CDCl3 Position
4
5
dH (mult, J in Hz)
dC
1 2
6
dH (mult, J in Hz)
108.0 93.4
3 4 5 6
5.50 (s)
76.0 39.6 40.4 32.1
2.73 (m) a 3.02 (d, 16.1) b 2.33 (m),
7 8 9 10 11 12
1.55 (d, 11.0) 4.22 (ddd, 11.0, 11.0, 4.0) a 1.36 (m) b 2.00 (dd, 14.2, 4.1)
13 14 15
36.6 43.6 27.3
2.17 (m) 2.82 (m)
16 17 18 19 20 21 22 23 28 29 30
169.6 78.0 22.3 22.7 120.9 140.9 109.8 143.2 22.1 24.2 39.5
5.22 (s) 1.05 (s) 1.17 (s) 7.54 (s) 6.44 (t, 0.8) 7.45 (t, 1.5) 0.74 (s) 1.35 (s) a 1.94 (d, 14.9) b 2.67 (d, 14.9) 7.17 (s)
1-OH 2-OH 2-OAc
2.08 (s)
3-OAc
2.12 (s)
7-OMe 11-OAc
3.71 (s)
177.3 78.7 67.5 47.5 64.2 45.2
22.1 174.6 21.0 170.4 52.2
10 20 30 40
4.80 (s) 2.65 (m) a 2.68 (m) b 2.33 (dd, 17.1, 11.2)
1.82 (d, 11.2) 5.01 (ddd, 11.1, 11.1, 3.7) a 1.17 (m) b 2.49 (dd, 13.8, 3.7) 2.17 (d, 7.3) a 2.81 (dd, 19.6, 7.9) b 2.95 (d, 19.6) 5.31 (s) 0.92 (s) 1.18 (s) 7.72 (s) 6.66 (d, 0.8) 7.40 (t, 1.6) 0.70 (s) 1.27 (s) a 1.59 (d, 13.9) b 2.60 (m) 5.22 (br s) 4.31 (br s)
3.65 (s) 1.90 (s)
2.65 (m) 1.15 (d, 7.1) 1.14 (d, 7.0)
Recorded at 400 or 100 MHz for 1H and
13
dC
dH (mult, J in Hz)
dC
108.4 79.7
3.64 (dd, 5.6, 2.6) a 2.37 (d, 2.4) b 3.10 (d, 5.8)
78.2 39.0
82.0 38.5 39.2 32.0 174.3 78.4 65.8 44.5 68.7 40.7 36.3 44.1 27.1 170.3 78.4 22.1 22.0 120.7 141.2 109.8 142.8 22.0 24.3 40.8
51.9 20.9 169.7 178.0 34.0 19.0 19.0
2.55 (s) 4.46 (d, 1.5)
2.33 (br s) 4.44 (br s) a 1.36 (d, 14.2) b 1.89 (dd, 14.6, 4.0)
a 2.94 (d, 18.0) b 2.64 (d, 17.9) 5.60 (s) 1.04 (s) 1.45 (s) 7.44 (br s) 6.37 (d, 1.0) 7.40 (t, 1.6) 1.47 (s) 1.05 (s) a 5.15 (s) b 5.36 (s)
3.85 (s)
211.6 48.8 47.7 72.2 176.1 142.2 59.0 45.3 67.8 36.3 40.7 80.8 33.4 169.6 79.1 16.9 23.3 120.5 140.8 109.8 142.9 23.7 24.7 115.7
53.6
C, respectively.
diverse and complex limonoid family. In particular, the observation on the exchange of the hydrogen atoms in compounds 1 and 2 with the deuterium of the solvent CD3OD due to the ketoeenol tautomerism is a good scientific input for understanding the involved chemistry. 4. Experimental section 4.1. General experimental procedures
Fig. 5. Selected HMBC (H/C) and ROESY correlations (H4H) of 6.
Specific rotations were acquired on a PerkineElmer 341 polarimeter. UV spectra were measured on a Shimadzu UV-2550 UVevisible spectrophotometer. IR spectra were recorded on a PerkineElmer 577 spectrometer. NMR spectra were measured on a Bruker AM-400 spectrometer and were referenced to the solvent peaks (dH at 3.34 and dC at 49.86 in CD3OD; dH at 7.26 and dC at 77.16 in CDCl3). EIMS (70 eV) and HR-EIMS were carried out on a Finnigan MAT 95 mass spectrometer. ESIMS and HR-ESIMS were carried out on a Bruker Daltonics Esquire 3000plus instrument and a Waters QTOF Ultima mass spectrometer, respectively. Silica gel (300e400 mesh), C18 reverse-phased silica gel (150e200 mesh, Merck), and MCI gel (CHP20P, 75e150 mM, Mitsubishi Chemical
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Industries, Ltd.) were used for column chromatography, and precoated silica gel GF254 plates (Qingdao Marine Chemical Plant, Qingdao, People’s Republic of China) were used for TLC. Semipreparative HPLC was performed on a Waters 515 pump equipped with a Waters 2487 UV detector (254 nm) and a YMC-Pack ODS-A column (25010 mm, S-5 mm, 12 nm). All solvents used were of analytical grade (Shanghai Chemical Plant, Shanghai, People’s Republic of China), and solvents used for HPLC were of HPLC grade (J&K Scientific Ltd.). 4.2. Plant material Plant material of K. ivorensis was collected from Yunnan Province of China in July 2009 and identified by Professor Y.-K. Xu of Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences. A voucher specimen has been deposited in the Shanghai Institute of Materia Medica, Chinese Academy of Sciences (accession number: Khivo-2009-1Y). 4.3. Extraction and isolation The air-dried powder of K. ivorensis plant material (10.0 kg) was extracted with 95% EtOH three times at room temperature (each 10 L), and the crude was partitioned between H2O and ethyl acetate. The EtOAc-soluble fraction (260 g) was subjected to a column of MCI gel eluted with methanol in water (MeOH/H2O, 30:70 to 90:10) to give four fractions, A-D. Fraction B was separated over a column of silica gel and eluted with CHCl3/MeOH (100:1 to 5:1) to obtain fraction B1eB5. Fraction B3 was chromatographed over silica gel column eluted with petroleum ether/ ethyl acetate (10:1 to 1:1) to give four subfractions, B3aeB3d. Fraction B3b was purified over a column of reverse-phased silica gel (MeOH/H2O, 65:35) to afford 1 (3 mg) and 2 (10 mg). Fraction B3c was successfully separated by semi-preparative HPLC (MeOH/ H2O, 65/35, 3 mL/min) to afford khayalactone (5 mg). Fraction B3d was applied to a column eluted with petroleum ether/acetone (10:1 to 2:1) to yield 1-O-acetylkhayanolide B (9 mg). Fraction B4 was separated by silica gel (petroleum ether/ethyl acetate, 10:1 to 1:5), and each major components was purified by semipreparative HPLC (MeOH/H2O, 65/35, 3 mL/min) to afford 3 (6 mg), khayanone (12 mg) and khayalactol (8 mg), sequentially. Part B5 was applied to a silica gel column (petroleum ether/ethyl acetate) to give two fractions B5a and B5b. Fraction B5a was separated by semi-preparative HPLC (MeOH/H2O, 60/40, 3 mL/ min) to yield 4 (10 mg) and 8a-dihydroxycarapin (9 mg). Fraction B5b was subjected to silica gel column (petroleum ether/acetone, 5:1 to 1:5) to afford khayanolide D (12 mg). Fraction C was chromatographed over silica gel column eluted with CHCl3/MeOH (100:1 to 5:1) to give three subfractions, C1eC3. Fraction C1 was separated over a column of silica gel (petroleum ether/ethyl acetate, 20/1 to 3/1) to obtain three major parts, each of which was purified by semi-preparative HPLC (MeOH/H2O, 55/45, 3 mL/min) to afford 5 (12 mg), senegalension B (6 mg) and khayalenoid K (3 mg), respectively. Fraction C2 was separated by semipreparative HPLC (MeOH/H2O, 55/45, 3 mL/min) to yield 6 (9 mg), seneganolide (8 mg) and ivorenolide A (5 mg), successively.
LR-ESI()MS m/z 603.8 [MþHCOO]; HR-ESI(þ)MS m/z 581.2002 [MþNa]þ (calcd for C29H34O11Na 581.1999). 4.4.2. Ivorenoid B (2). White amorphous powder; [a]20 D þ3 (c 0.1, MeOH); UV (MeOH) lmax (log ε) 271 (4.79) nm; IR (KBr) nmax 3431, 2925, 1736, 1630, 1385, 1250, 1052, 602 cm1; 1H and 13C NMR data: see Table 1; LR-ESI(þ)MS m/z 597.2 [MþNa]þ; LR-ESI()MS m/z 573.6 [MH], 1147.3 [2MH]; HR-ESI(þ)MS m/z 597.1952 [MþNa]þ (calcd for C29H34O12Na 597.1948). 4.4.3. Ivorenoid C (3). White amorphous powder; [a]20 D 59 (c 0.1, MeOH); UV (MeOH) lmax (log ε) 265 (4.13) nm; IR (film) nmax 3438, 2929, 1720, 1651, 1456, 1373, 1240, 1026, 756 cm1; 1H and 13C NMR data: see Table 1; LR-ESI(þ)MS m/z 569.3 [MþNa]þ, 1115.3 [2MþNa]þ; LR-ESI()MS m/z 591.5 [MþHCOO]; EIMS m/z 546 [M]þ (4), 486 (7), 390 (13), 343 (68), 304 (42), 218 (75), 159 (66), 121 (58), 95 (100); HR-EIMS m/z 546.2457 [M]þ (calcd for C29H38O10 546.2465). 4.4.4. Ivorenoid D (4). White amorphous powder; [a]20 D 31 (c 0.1, MeOH); UV (MeOH) lmax (log ε) 265 (4.24) nm; IR (KBr) nmax 3466, 2952, 1739, 1637, 1437, 1373, 1306, 1232, 1159, 1107, 1058, 1027, 968, 930, 876, 806, 602 cm1; 1H and 13C NMR data: see Table 2; LRESI(þ)MS m/z 627.3 [MþNa]þ, 1231.5 [2MþNa]þ; LR-ESI()MS m/ z 649.6 [MþHCOO]; HR-ESI(þ)MS m/z 627.2410 [MþNa]þ (calcd for C31H40O12Na 627.2417). 4.4.5. Ivorenoid E (5). White amorphous powder; [a]20 D 57 (c 0.1, MeOH); UV (MeOH) lmax (log ε) 266 (4.14) nm; IR (KBr) nmax 3440, 2975, 2937, 2879, 1747, 1651, 1603, 1470, 1381, 1317, 1234, 1219, 1144, 1061, 1026, 901, 604 cm1; 1H and 13C NMR data: see Table 2; LRESI(þ)MS m/z 655.3 [MþNa]þ, 1287.5 [2MþNa]þ; LR-ESI()MS m/ z 631.7 [MH], 1263.3[2MH]; HR-ESI(þ)MS m/z 655.2722 [MþNa]þ (calcd for C33H44O12Na 655.2730). 4.4.6. Ivorenoid F (6). White amorphous powder; [a]20 D 54 (c 0.05, MeOH); UV (MeOH) lmax (log ε) 268 (4.27) nm; IR (KBr) nmax 3450, 2927, 1724, 1462, 1435, 1387, 1246, 1111, 1045, 876, 756 cm1; 1H and 13 C NMR data: see Table 2; LR-ESI(þ)MS m/z 525.3 [MþNa]þ, 1027.4 [2MþNa]þ; EIMS m/z 502 [M]þ (88), 470 (21), 374 (38), 359 (34), 332 (36), 149 (77), 137 (100), 121 (79), 95 (90); HR-EIMS m/z 502.2198 [M]þ (calcd for C27H34O9 502.2203).
4.5. Cytotoxic assay The cytotoxic activities of all the isolated compounds were evaluated by using the MTT method. Briefly, the tested cells in culture medium 100 mL were plated in the wells of 96-well plates (Falcon, CA). The cells were treated in triplicate with graded concentrations of the compounds at 37 C for 72 h. A 20 mL aliquot of MTT solution (5 mg/mL) was put directly in the wells. The cultures were incubated for 4 h, and 100 mL of ‘triplex solution’ (10% SDS/5% i BuOH/12 mM HCl) was added. The plates were incubated at 37 C overnight and the measured using a plate reader at 570 nm (VERSA Max, Molecular Devices). The results were expressed as IC50 as calculated by the Logit method.
Acknowledgements 4.4. Characterization of new limonoids 4.4.1. Ivorenoid A (1). White amorphous powder; [a]20 D þ26 (c 0.05, MeOH); UV (MeOH) lmax (log ε) 266 (4.68) nm; IR (film) nmax 3438, 2924, 2852, 1782, 1730, 1462, 1369, 1246, 1147, 1020, 758, 602 cm1; 1 H and 13C NMR data: see Table 1; LR-ESI(þ)MS m/z 581.1 [MþNa]þ;
The financial support (No. 81273398) of the National Natural Science Foundation, and the foundation (2012CB721105) from the Ministry of Science and Technology of the People’s Republic of China are gratefully acknowledged. We thank Professor You-Kai Xu for the identification of the plant material.
W.-B. Wu et al. / Tetrahedron 70 (2014) 3570e3575
Supplementary data The NMR, ESIMS, EIMS, IR, 2D NMR spectra of compounds 1e6. This material is available free of charge via the Internet. Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.tet.2014.04.007.
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