Original Papers
1767
Authors
Hong-Ying Wang, Jun-Song Wang, Si-Ming Shan, Xiao-Bing Wang, Jun Luo, Ming-Hua Yang, Ling-Yi Kong
Affiliation
State Key Laboratory of Natural Medicines, Department of Natural Medicinal Chemistry, China Pharmaceutical University, Nanjing, P. R. China
Key words " Trichilia connaroides l " Meliaceae l " nortriterpenoids l " pregnanes l " lignans l " NO production inhibition l " α‑glucosidase inhibition l
Abstract !
Phytochemical investigation of the stem and bark of Trichilia connaroides led to the isolation of eight new nortriterpenoids (1–8), along with fifteen known compounds (9–23). Their structures were established based on extensive spectroscopic analysis. The absolute configuration of 2 was confirmed by X‑ray crystallographic study. The nitric oxide production and α-glucosidase inhibitory ef-
Introduction !
received revised accepted
April 10, 2013 August 5, 2013 October 13, 2013
Bibliography DOI http://dx.doi.org/ 10.1055/s-0033-1351045 Planta Med 2013; 79: 1767–1774 © Georg Thieme Verlag KG Stuttgart · New York · ISSN 0032‑0943 Correspondence Prof. Dr. Ling-Yi Kong State Key Laboratory of Natural Medicines Department of Natural Medicinal Chemistry China Pharmaceutical University 24 Tong Jia Xiang Nanjing 210009 People′s Republic of China Phone: + 86 25 83 27 14 05 Fax: + 86 25 83 27 14 05 [email protected]
Trichilia connaroides (Wight & Arn.) Bentv. (Meliaceae) is widely distributed throughout the southeast of Asia. It has been traditionally used in China for the treatment of arthritis, pharyngitis, tonsillitis, and other ailments [1]. The crude extracts obtained from T. connaroides have exhibited antihyperlipidemic, hepatoprotective, analgesic, and anti-inflammatory effects [2–4]. Its twigs, leaves, roots, and fruits afforded a series of highly rearranged limonoids with a complex ring system and steroids [5–13]. To seek bioactive and novel components from this plant, we studied the dichloromethane extract of the stem and bark of T. " Fig. 1) connaroides. As a result, 23 components (l were isolated including one new 1,2-seco phragmalin-type limonoid with a rare 9-oxa-tricyclo [3.3.2.17,10]undecane-2-ene moiety (secotrichagmalin A, 1), five new mexicanolides (trichanolides A–E, 2–6), two new highly rearranged 30-nortrijugins (trijugins I–J, 7–8), and five known limonoids (9–13), five pregnanes (14–18), and five lignans (19–23). Their structures were elucidated on the basis of spectroscopic methods, or by comparison with the reported data in the literature. In addition, the absolute configuration of 2 was determined by means of single-crystal X‑ray diffraction analysis. All these isolates were evaluated for their inhibitory effects on lipopolysaccharide-in-
fects for these isolates were evaluated: moderate to strong nitric oxide production inhibitory activities were found for 5, 6, and 11–15, with IC50 values ranging from 7.5 to 26.3 µM; marked α-glucosidase inhibitory effects were observed for 22 and 23, with IC50 values of 2.3 and 0.4 µM, respectively. Supporting information available online at http://www.thieme-connect.de/ejournals/toc/ plantamedica
duced nitric oxide production in RAW264.7 cells and on α-glucosidase. Herein, we describe the isolation, structural elucidation, and biological evaluation of these isolates.
Results and Discussion !
Secotrichagmalin A (1), white amorphous powder, has a molecular formula of C32H38O9 as established by the pseudomolecular ion at m/z 565.2451 [M – H]−, (calcd. for C32H37O9, 565.2443, errors in 1.49 ppm) in the negative HRESIMS, indicating 14 degrees of unsaturation. The IR spectrum implied the presence of hydroxyl (3442 cm−1), carbonyl (1713 cm−1), and olefinic groups (1631 cm−1). Except for the easily distinguishable signals for three angular methyls (δH 0.85, 1.00, and 1.08; δC 19.4, 22.0, and 22.7) and a β-substituted furanyl moiety (δH 6.58, 7.54, and 7.67; δC 111.4, 121.7, 143.4, and 144.5), the NMR " Tables 1 and 2) also presented a spectrum of 1 (l methoxycarbonyl moiety (δH 3.61; δC 52.2 and 175.5), a tigloyl group (δH 1.92, 1.95, 7.11; δC 12.4, 14.5, 129.5, 139.7, and 168.4), and a hemiacetal group (δC 98.7). The typical 4,29,1-ring bridge was characterized by a pair of geminal doublets at δH 1.63 (1H, d, J = 13.0 Hz) and 1.97 (1H, d, J = 13.0 Hz), as evidenced by the HMBC cor" Fig. 2) of H-1/C-29, H -29/C-1, and relations (l 2
Wang H-Y et al. Chemical Constituents from …
Planta Med 2013; 79: 1767–1774
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Chemical Constituents from Trichilia connaroides and Their Nitric Oxide Production and α-Glucosidase Inhibitory Activities
Original Papers
Fig. 1
Chemical structures of compounds 1–23. (Color figure available online only.)
H2-29/C‑4. The 13C NMR data of 1 resembled those of andhraxylocarpin A [14], except for the great shift discrepancy in one oxygenated methine carbon (δC 87.5, Δ+5.9 ppm) and the hemiacetal carbon (δC 98.7, Δ-7.7 ppm). In the HMBC spectrum, this methine carbon signal (δC 87.5) corresponded to an oxygenated methine proton signal at δH 3.67 in the HSQC spectrum and had an HMBC correlation with H2-29, thus assigning itself to C-1. The position of the hemiacetal carbon at C-2 was determined by its HMBC correlations with H-1 and H-3. Thus, the gross structure of 1 was established as a hydroxyl positional isomer of andhraxylocarpin A. The relative configuration of 1 was deduced mainly from the " Fig. 2). The ROESY correlations from H-1 ROESY experiment (l to H-9, Me-19 and H2-29, from H-9 to H-11α, Me-18 and Me-19, and from H-3 to H-29b, indicated that H-1, H-3, H-9, Me-18, Me19, and H2-29 were cofacial and were arbitrarily assigned with an α-orientation. The correlations of H-17/H-12β, H-17/H‑21, H-5/ Me-28, and H-5/H-15 revealed H-5, H-17, and Me-28 to be β-oriented. Therefore, the structure of 1 was elucidated as depicted, and the compound was named secotrichagmalin A. To the best of our knowledge, secotrichagmalin A is the second report of a 1,2-seco phragmalin-type limonoid with a rare 9-oxa-tricyclo [3.3.2.17,10]undecane-2-ene moiety found in nature [14]. Trichanolide A (2), colorless crystals (MeOH/H2O), has a molecular formula of C32H38O11 as deduced from the pseudomolecular ion peak at m/z 621.2309 [M + Na]+ in the positive-ion mode HRESIMS. The 1D NMR data combined with subsequent 2D NMR " Tables 1 and 2, Fig. 3) presented signals for a β-substianalysis (l tuted furan ring, a methyl esterified C-6–C-7 appendage, a C-16/ C-17 δ-lactone ring D, four angular methyls, and a ketone carbonWang H-Y et al. Chemical Constituents from …
Planta Med 2013; 79: 1767–1774
yl, which suggested 2 was a B,D-seco mexicanolide-type limonoid [11, 15, 16]. The NMR spectroscopic data of 2 were similar to those of quivisianolide A [17], except for the downfield shifted signal of H-3′ to δH 7.14 (Δ+0.90), and upfield shifted signals of C4′ to δC 14.8 (Δ-1.5) and of C-5′ to δC 12.9 (Δ-8.3), suggesting the presence of the tigloyl group in 2 instead of an angeloyl group. Thus, the planar structure of 2 was established. The relative configuration of 2 was established mainly by the ROESY experiment " Fig. 3). The observed cross-peaks of H-14/Me-18, H-14/H-15α, (l H-3/Me-29, Me-18/H-12α and Me-19/Me-29 indicated that H-3, H-14, Me-18, Me-19, and Me-29 were co-facial and were arbitrarily assigned with an α-orientation. Likewise, the ROESY correlations from Me-28 to H-5 and H-3′ suggested that H-5 and Me-28 were β-oriented. The furan ring in the α-orientation was determined according to the ROESY correlation of H-14/H‑22, accordingly, H-17 was thus β-oriented. The 9,11-epoxide ring was assigned to be α-oriented on the basis of the J11,12 coupling constant [17]. To further clarify the structure, compound 2 was subjected to an X‑ray diffraction study using a mirror Cu Kα radiation " Fig. 4, the structure [Flack parameter 0.00(17)]. As shown in l was confirmed, and the absolute configuration of 2 was finally determined to be 2S,3S,5S,8R,9R,10S,11R,13R,14R,17R,30R. Compounds 3-6 were determined as analogues of 2 according to " Tables 1 and 2). Compound 3 was similar to 2, their NMR data (l with the exception of the presence of an additional double bond and the absence of one epoxy ring. In the HMBC spectrum, both of the two carbons of this double bond at δC 137.4 and 127.1 correlated with the proton signal at δH 2.14 ascribed to one proton of H2-12; the carbon at δC 137.4 showed a cross-peak with Me-19
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1768
Original Papers
Pos.
1 2 3 5 6a 6b 9 11α 11β 12α 12β 14 15α 15β 17 18 19 21 22 23 28 29a 29b 30 MeO-7 3′ 4′ 5′ a
1
H (500 MHz) data of compounds 1–6. 1a
2a
3b
4b
5b
6b
δH (J in Hz)
δH (J in Hz)
δH (J in Hz)
δH (J in Hz)
δH (J in Hz)
δH (J in Hz)
5.14, s 3.37, dd (7.0, 6.0) 2.56, dd (17.0, 6.0) 2.52, dd (17.0, 7.0)
5.10, s 3.04* 2.34, d (6.5)
3.75, m 5.04, d (9.5) 3.38, dd (7.5, 6.0) 2.48, dd (16.5, 6.0) 2.39*
4.90/4.95, br s 3.22/3.17, d (10.0) 2.46/2.37, m 2.38/2.35, br s 2.25 *
4.91, br s 3.42, br d (10.0) 2.45/2.48, d (6.0) 2.38/2.33, dd (10.0, 6.0) 2.28 *
3.55, br s
5.85, t (4.0)
3.23, br s
2.07, d (15.5) 2.35, d (15.5) 1.84, dd (12.5, 7.0) 2.98, dd (18.5, 7.0) 3.57* 5.15, s 1.01, s 0.89, s 7.53, s 6.41, s 7.53, s 0.86, s 0.85, s
2.14, dd (18.5, 4.0) 2.58, dd (18.5, 4.0) 1.82, dd (12.5, 7.0) 2.73, dd (18.5, 7.0) 3.05* 5.14, s 1.04, s 1.29, s 7.35, s 6.26, s 7.42, s 0.83, s 0.81, s
1.99, d (16.0) 2.40* 1.60, dd (13.0, 7.0) 2.67, dd (18.5, 7.0) 3.63, dd (18.5, 12.5) 4.98, s 1.11, s 0.83, s 7.30, s 6.17, s 7.39, br s 0.95, s 0.83, s
1.78, m 2.06, m 1.66/1.78, m 2.18, m 2.25 * 2.81/2.86* 2.86/2.81* 5.46/5.55, s 1.13/1.25, s 1.25, s 6.09/6.31, s 6.27/6.22, s
1.71, m 2.28 * 1.45, td (14.5, 4.5) 1.93, m 2.28 * 2.83* 2.84* 5.57/5.54, s 1.02/1.04, s 1.24, s
3.61, s 3.77, s 7.14, q (7.0) 1.98, d (7.0) 2.01, s
3.44, s 3.71, s 7.05, q (7.0) 1.95, d (7.0) 1.97, s
3.17, d (2.5) 3.75, s 7.07, q (7.0) 1.94, d (7.0) 1.95, s
3.67, d (2.5) 5.00, s 2.96, t (7.5) 2.28, m 2.84, dt (13.0, 3.5) 1.72, m 1.12* 1.13* 1.98, d (13.0) 6.09, s 5.33, s 1.08, s 1.00, s 7.67, s 6.58, br s 7.54, s 0.85, s 1.97, d (13.0) 1.63, d (13.0) 6.07, d (2.5) 3.61, s 7.11, q (7.0) 1.92, q (7.0) 1.95, s
0.79/0.74, s 0.85/0.83, s
7.37/7.34, s 6.20, s 0.76, s 0.82, s
5.32/5.39, s 3.75/3.70, s 6.99, q (7.0) 1.81/1.90, d (7.0) 1.85/1.88, s
5.38, br s 3.67/3.65, s 6.94, q (7.0) 1.83/1.82, d (7.0) 1.85, s
Measured in MeOH-d4; b Measured in CDCl3; * Overlapped with other signals
(δH 1.29), which assigned the carbon signals at δC 137.4 and 127.1 to C-9 and C-11, respectively. The main difference of 4 was in the presence of an additional methine carbon and the absence of an oxygenated quaternary carbon. This methine proton (δH 3.75) correlated with C-1, C-8, and C-30 in the HMBC spectrum, and corresponded to a methine carbon at δC 49.1 in the HSQC spectrum, which in turn had an HMBC correlation with H-3, thus assigning itself to H-2. A comprehensive analysis of the HSQC and HMBC spectra allowed the establishment of 3 and 4 as shown, and they were named trichanolide B and C, respectively. Detailed analysis of the 1H and 13C NMR spectroscopic data of 5 and 6 indicated that they were mexicanolide-type limonoids as in 2, but were lacking the characteristic signals for the furan ring which was replaced by the rare γ-hydroxybutenolide moiety [18, 19]. The NMR data of 5 were similar to those of granatumin E [20], except for the presence of one additional hydroxyl. Compared with granatumin E, both the C-3 and C-30 signals were shifted downfield by 8.4 and 6.2 ppm, respectively, and the H-3 signal of 5 appeared as a singlet instead of a doublet. In addition, the C-2 methine signal in granatumin E was absent, an oxygenated quaternary carbon resonance at δC 77.3 was observed, which correlated with H-3 in the HMBC spectrum, thus assigning the additional hydroxyl to C-2. Consequently, the structure of 5 was elucidated and the compound named trichanolide D. Compounds 6 and 5 were superimposable except for those of the ring E. The γhydroxybutenolide ring was also present in 6 as characterized by two broad singlet proton at δH 7.37/7.34 (H-22) and 6.20 (H-23), and resonances at δC 135.7 (C-20), 168.3/168.4 (C-21), 149.5/
149.9 (C-22), and 97.0/96.6 (C-23). The large difference in the γhydroxybutenolide ring was due to a different tautomeric position, confirmed by the HMBC correlation between the singlet proton signal at δH 5.57/5.54 (H-17) and the α,β-unsaturated γlactone carbonyl (C-21), thus establishing a 23-hydroxybutenolide ring in 6 [18, 21]. Compound 6 was established as trichanolide E. Trijugin I (7) had the molecular formula C28H32O13, suggesting 13 " Table 3) exdegrees of unsaturation. The 13C NMR spectrum (l hibited 28 carbon signals. After deduction of those belonging to one methoxyl and one acetyl group, the remaining 25 carbons suggested a pentanortriterpenoid nature for 7. Four singlet methyls at δH 0.99/1.10, 1.57, 1.40, and 1.07 were attributable to Me18, Me-19, Me-28, and Me-29. The characteristic lactone ring D was confirmed by the HMBC correlations from the H2-15 protons to C-14, C-13, and C-16, from H-17 to C-13, and from Me-18 to C13 and C-14, and signals of a carbomethoxy group at C-7 was also observed. The only unassigned acetyl group was located at C-8 according to the downfield shifted signal of C-8 at δC 89.4. Its NMR data suggested that it was structurally related to trijugin D [8], but differing in the presence of the 21-hydroxybutenolide ring instead of a β-furanyl ring in trijugin D. The relative configu" Fig. 5). Hence, ration of 7 was determined by ROESY spectrum (l the structure of 7 was elucidated as shown. Trijugin J (8) had a molecular formula of C29H34O13. Its NMR data " Table 3) resembled those of 7, especially those associated with (l rings A–D, but differing in ring E. The characteristic carbon signals at δC 104.2, 135.7, 148.6, 169.5, and 57.5 could be attributed Wang H-Y et al. Chemical Constituents from …
Planta Med 2013; 79: 1767–1774
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Table 1
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Original Papers
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 30 MeO-7 1′ 2′ 3′ 4′ 5′ a
1a
2a
3b
4b
5b
6b
δC
δC
δC
δC
δC
δC
87.5 98.7 83.1 49.4 38.8 34.9 175.5 141.3 46.3 54.4 23.9 34.0 39.6 169.0 115.0 167.0 80.7 22.0 22.7 121.7 143.4 111.4 144.5 19.4 40.4 134.8 52.2 168.4 129.5 139.7 14.5 12.4
210.2 80.3 86.8 41.2 44.7 32.8 175.7 63.0 63.6 52.4 56.8 29.0 35.1 40.1 30.7 172.0 84.3 24.1 11.2 123.5 142.9 110.4 145.2 23.8 21.4 65.6 53.0 168.5 129.4 140.6 14.8 12.9
210.2 79.3 85.4 39.8 47.7 31.4 173.2 61.2 137.4 51.7 127.1 32.1 35.6 39.6 29.7 169.5 82.2 23.5 14.7 122.2 140.9 109.7 143.9 24.0 20.4 64.5 52.5 167.0 128.3 139.6 14.9 12.8
209.1 49.1 77.9 39.2 43.6 32.4 173.9 59.6 62.9 50.2 56.3 28.0 34.4 39.0 29.3 169.3 83.1 23.7 10.7 122.3 140.9 109.7 143.9 24.9 21.7 60.8 52.8 167.0 128.0 140.1 15.0 12.7
214.3 77.3 85.4/84.9 39.8/39.7 42.3/42.6 33.4/33.2 176.3/176.4 136.3/136.1 56.6/56.3 49.2/50.0 21.1/20.6 34.7/34.9 37.8/37.1 45.7/46.4 29.6/29.8 167.2/166.8 75.8/76.0 21.4/21.2 16.4/15.8 163.3/160.6 97.7/99.4 122.4/121.1 169.5 22.8/22.1 19.8/20.3 130.3/130.5 53.30/53.33 167.6/167.7 127.5/127.3 140.6/140.8 15.00/14.97 12.2/12.3
215.4/215.3 77.6 85.1/85.2 39.9 40.8 33.0 173.7/173.3 136.5 56.6/56.7 50.4/50.3 21.2/21.1 34.8 37.0/36.9 45.5 29.4 167.7 76.7 22.8 15.70/15.73 135.7 168.3/168.4 149.5/149.9 97.0/96.6 22.2 20.6 129.4/129.3 52.5/52.4 167.8 127.9 139.9/139.8 14.9 12.2
Table 2 13C NMR (125 MHz) data of compounds 1–6.
Measured in MeOH-d4; b Measured in CDCl3
Fig. 2 1H-1H COSY, Key HMBC, and ROESY correlations of 1. (Color figure available online only.)
to C-23, C-20, C-22, C-21, and OMe-23 comprising a rare 23-methoxy-α,β-unsaturated γ-lactone moiety of ring E with the corresponding proton resonances at δH 5.88 (1H, br s) and 7.21 (1H, br s), assigned to H-23 and H-22, respectively. The three-proton singlet at δH 3.42 was determined to be at C-23 according to its HMBC correlation with C-23 [22–24]. Interestingly, the NMR signals due to the γ-methoxybutenolide ring in 8 were not observed in pairs compared with those possessing a γ-hydroxybutenolide Wang H-Y et al. Chemical Constituents from …
Planta Med 2013; 79: 1767–1774
ring, which could be explained by the blockade effect of the methoxyl to the tautomerization. The similar ROESY correlations of 7 and 8 suggested the same relative configurations. However, the available evidences were insufficient to determine the configuration at C-23. The structure of 8 was thus established as shown. The known isolates were identified as trijugins D (9) and F (10) [8], methyl-2-hydroxy-3β-tigloyloxy-1-oxomeliac-8(30) enate (11) [25], febrifugin A (12) [26], and granatumin E (13) [20], (E)-
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Position
Original Papers
1771
Fig. 4 Perspective drawing of the X‑ray structure of 2. (Color figure available online only.)
to 26.3 µM, and compounds 2–4 displayed weak activities with IC50 values ranging from 48.7 to 57.5 µM. These results revealed the potential of mexicanolide-type limonoids in this regard. Interestingly, 14 and 15 possessing a Δ17,20 double bond showed striking activities with IC50 values lower than 10 µM, as compared with compound 18 which has the same skeleton with an identical Δ5,6 double bond and two β-OHs substituted at C-3 and C-4, suggesting the important role of the Δ17,20 double bond in the inhibitory effect on nitric oxide production in LPS-activated macrophages. The α-glucosidase inhibitory activities of these isolates were also " Table 4). Of them, compounds 9, 10, and 14 disevaluated (l played moderate activities in inhibition of α-glucosidase with IC50 values ranging from 30 to 50 µM. Amazingly, 22 and 23, with an extremely simple skeleton, were the most active, with IC50 values of 2.3 and 0.4 µM, respectively. Compounds 22 and 23 have been isolated from many plants [36], hence, since easily obtained, they may be considered as lead compounds providing a new thread for synthesis and modification of hypoglycemic agents.
Materials and Methods volkendousin (14), (Z)-volkendousin (15) [27], 3β,4α-dihydroxypregnan-20-one (16) [8], 3β,4α-dihydroxypregnan-16-one (17) [7], ekeberin B (18) [28], episyringaresinol (19) [29], (+)-syringaresinol (20) [30], (+)-medioresinol (21) [30], honokiol (22), and magnolol (23) [31] by comparison of their spectroscopic data with those previously reported. Limonoids containing γ-hydroxybutenolide were isolated in this study. The γ-hydroxybutenolide could be transformed from the common furan via singlet oxygen (1O2) oxidation. Singlet oxygen is produced mainly in plant leaves by light through chlorophylls which act as photosensitizers [32]. The [4 + 2] cycloaddition of singlet oxygen to β-substituted furanyl moiety in compounds, e.g., 9 and 11, can readily afford unstable furan endoperoxides. The subsequent breakdown of these endoperoxides affords diverse products including hydroxybutenolides 5–7 and the solvent addition product 8 [33–35]. Twenty-three isolates were evaluated for their inhibitory effects on the release of NO in lipopolysaccharide (LPS)-induced RAW264.7 cells using N-monomethyl-L-arginine as the reference " Table 4. compound (IC50 = 37.7 µM). Results are summarized in l Compounds 5, 6, 11, 12, and 13, exhibited moderate to strong NO production inhibitory activity with IC50 values ranging from 7.5
!
Plant material The air-dried stem and bark of T. connaroides were collected in November 2011 from Xishuangbanna, Yunnan Province, People′ s Republic of China, and authenticated by Prof. Shun-Cheng Zhang, Xishuangbanna Botanical Garden, Chinese Academy of Sciences, People′s Republic of China. A voucher specimen (No. ZGH-201111) is deposited in the Department of Natural Medicinal Chemistry, China Pharmaceutical University.
Extraction and isolation The air-dried powder of the plant material (10.0 kg) was extracted with 95% EtOH under reflux and then evaporated in vacuo to remove the solvents so as to give a crude extract (1280.0 g). The extract was suspended in H2O (5 L) and successively partitioned with petroleum ether and CH2Cl2 to give two parts. The CH2Cl2 extract (64.0 g) was fractionated by silica gel column eluted with petroleum ether-EtOAc in a gradient (20 : 1 to 0 : 1) to give seven major fractions (I–VII). Fraction III (2.7 g) was chromatographed on MCI, RP‑C18, and successive preparative HPLC to afford 19 (5.0 mg), 20 (9.0 mg), 21 (6.0 mg), 22 (3.0 mg), and 23. Fraction IV (12.4 g) was subjected to a column of MCI, RP‑C18, and successive Wang H-Y et al. Chemical Constituents from …
Planta Med 2013; 79: 1767–1774
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Fig. 3 Key HMBC and ROESY correlations of 2. (Color figure available online only.)
1772
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1 2 3 4 5 6 7 8 9 10 11α 11β 12α 12β 13 14 15α 15β 16 17 18 19 20 21 22 23 28 29 MeO-7 MeO-23 8-OAc
a
8b
δC
δH (J in Hz)
δC
δH (J in Hz)
80.5 51.8 207.8 48.0 45.6 74.8 170.1 89.4 175.9 43.4 42.2
4.43/4.41, d (4.5) 3.62/3.61, d (4.5)
81.8 53.0 209.6 48.9 46.6 75.6 172.0 90.8 177.0 44.4 42.8
4.46, d (4.5) 3.66, d (4.5)
33.7 47.4 93.3/93.5 36.9 167.2 81.2 16.7 18.9 163.5 97.1/97.8 121.0/121.9 169.2 26.4 22.1 53.1 170.6 21.8
3.33/3.34, d (8.5) 5.06, d (8.5)
2.50/2.46, m 1.71, m 2.03, m
2.95/2.97, d (18.0) 2.25/2.24, d (18.0) 5.59/5.53, br s 0.99/1.10, s 1.57, s 6.00/6.24, s 6.26/6.15, br s 1.40, s 1.07, s 3.85, s
2.20, s
35.2 48.3 94.5 37.8 168.0 80.7 16.8 19.4 135.7 169.5 148.6 104.2 26.7 22.7 53.6 57.5 170.8 22.1
Table 3 1H (500 MHz) and 13C (125 MHz) data of compounds 7– 8.
3.42, d (8.5) 5.17, d (8.5)
2.35, m 2.41, m 1.43, dd (13.0, 8.0) 1.91*
2.88, d (18.0) 2.10, d (18.0) 5.29, br s 0.90, s 1.50, s
7.21, br s 5.88, br s 1.30, s 0.98, s 3.74, s 3.42, s 2.00, s
b
Measured in CDCl3; Measured in Acetone-d6; * Overlapped with solvent signal
Fig. 5 Key HMBC and ROESY correlations of 7. (Color figure available online only.)
preparative HPLC to give 14 (8.0 mg), 15 (2.0 mg), 16 (10.0 mg), 17 (20.0 mg), and 18 (10.0 mg). Fraction V (10.5 g) was chromatographed on MCI, silica gel, RP‑C18, Sephadex LH-20, and preparative HPLC to yield 1 (2.5 mg), 2 (5.5 mg), 3 (4.3 mg), 4 (5.0 mg), 5 (2.5 mg), 6 (3.2 mg), 7 (7.8 mg), 8 (1.5 mg), 9 (90.0 mg), 10 (2.4 mg), 11 (4.5 mg), 12 (3.0 mg), and 13 (2.0 mg) (for detailed procedure for extraction and isolation, see Supporting Information).
Wang H-Y et al. Chemical Constituents from …
Planta Med 2013; 79: 1767–1774
New isolates Secotrichagmalin A (1): white, amorphous powder; [α]25 D + 232.2 (c 0.07, MeCN); UV (MeCN) λmax (log ε) 202 (4.19), 267 (3.45) nm; IR (KBr) νmax 3442, 2971, 2923, 1713, 1631, 1454, 1396, " Tables 1 and 1253, 1162, 1049 cm−1; 13C and 1H NMR data, see l 2; HRESIMS m/z 565.2451 [M – H]− (calcd. for C32H37O9, 565.2443).
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7a
Position
Table 4 Nitric oxide production in LPS-stimulated RAW264.7 and α-glucosidase inhibitory activities of the isolates from Trichilia connaroides a,b. Compounds 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 Genisteinc N-monomethyl-L-arginine c a
α-Glucosidase
Effects of inhibiting
inhibitory activities
NO production
> 100 > 100 > 100 64.7 ± 3.9 > 100 > 100 > 100 > 100 32.5 ± 2.1 49.0 ± 4.5 > 100 > 100 > 100 30.7 ± 1.9 > 100 > 100 > 100 > 100 > 100 83.5 ± 10.9 > 100 2.3 ± 0.2 0.4 ± 0.1 10.6 ± 0.6
49.2 ± 2.9 57.5 ± 4.5 48.7 ± 3.4 54.3 ± 3.7 16.6 ± 1.6 20.3 ± 1.9 > 100 > 100 > 100 > 100 26.3 ± 1.8 12.8 ± 2.3 7.5 ± 1.1 7.6 ± 1.3 8.2 ± 0.4 > 100 > 100 > 100 > 100 > 100 > 100 > 100 46.7 ± 5.8 37.7 ± 2.7
Results are expressed as IC50 values in µM; b Compounds with IC50 > 100 µM are not
Trijugin I (7): white, amorphous powder; [α]25 D − 10.5 (c 0.06, MeCN); UV (MeCN) λmax (log ε) 195 (3.93), 205 (3.84) nm; IR (KBr) νmax 3442, 2919, 2850, 1745, 1633, 1399, 1236, 1129, " Table 3; HRESIMS m/z 1050 cm−1; 1H and 13C NMR data, see l 575.1778 [M – H]− (calcd. for C28H31O13, 575.1770). Trijugin J (8): white, amorphous powder; [α]25 D + 7.1 (c 0.09, MeCN); UV (MeCN) λmax (log ε) 192 (3.59), 205 (3.47), 260 (2.18) nm; IR (KBr) νmax 3447, 2917, 2850, 1741, 1680, 1468, 1384, 1204, 1130, 1050, 1021 cm−1; 1H and 13C NMR data, see " Table 3; HRESIMS m/z 589.1928 [M – H]− (calcd. for C l 29H33O13, 589.1927). X‑ray crystallographic study of 2: C32H38O11, M = 598.62: orthorhombic, space group P 21 21 21, unit cell dimensions a = 8.0814 (10) Å, b = 18.8427 (2) Å, c = 19.7979 (3) Å, V = 3014.73 (7) Å3, Z = 4, T = 290 (2) K, µ (CuKα) = 0.829 mm−1, Dcalc = 1.319 g/mm3, Flack = 0.01(17). Colorless crystals of 2 were obtained in a mixed solvent of methanol and water. A suitable crystal was selected, investigated on a diffractometer (λ = 1.54 184 Å) and kept at 290 (2) K during data collection. The structure was solved with the SHELXS-97 [37] structure solution program using direct methods and refined with the SHELXL-97 [38] refinement package using least squares minimization. Data collection yielded a total of 18 863 integrated intensities, and resulted in 5597 unique and averaged observations with Rint = 0.0266. Full matrix least-squares refinement on F2 led to a final R indices [I > 2 σ(I)]: R1 = 0.0366, wR2 = 0.0882, R indices (all data) [I > 2σ(I)]: R1 = 0.0449, wR2 = 0.0946 and GOOF of 1.044. Crystallographic data for compound 1 have been deposited at the Cambridge Crystallographic Data Centre (deposition No. CCDC 911539).
shown; c Positive controls
NO production bioassay
Trichanolide A (2): colorless crystals (MeOH/H2O); mp 251– 252 °C; [α]25 D − 55.0 (c 0.10, MeCN); UV (MeCN) λmax (log ε) 212 (4.16) nm; IR (KBr) νmax 3449, 2919, 2850, 1735, 1640, 1400, 1261, 1204, 1123, 1068, 1025 cm−1; 1H and 13C NMR data, see " Tables 1 and 2; HRESIMS m/z 621.2309 [M + Na]+ (calcd. for l C32H38O11Na, 621.2306). Trichanolide B (3): white, amorphous powder; [α]25 D + 34.8 (c 0.05, MeCN); UV (MeCN) λmax (log ε) 194 (4.24), 209 (4.21), 269 (3.41) nm; IR (KBr) νmax 3425, 2921, 2851, 1731, 1622, 1400, 1259, " Tables 1 and 2; 1163, 1063 cm−1; 1H and 13C NMR data, see l HRESIMS m/z 605.2349 [M + Na]+ (calcd. for C32H38O10Na, 605.2357). Trichanolide C (4): white, amorphous powder; [α]25 D − 79.0 (c 0.09, MeCN); UV (MeCN) λmax (log ε) 213 (4.14) nm; IR (KBr) νmax 3423, 1631, 1400, 1146, 1058 cm−1; 1H and 13C NMR data, see " Tables 1 and 2; HRESIMS m/z 605.2378 [M + Na]+ (calcd. for l C32H38O10Na, 605.2357). Trichanolide D (5): white, amorphous powder; [α]25 D − 31.6 (c 0.05, CHCl3); UV (MeCN) λmax (log ε) 196 (5.00), 206 (5.01), 287 (3.66) nm; IR (KBr) νmax 3415, 1725, 1638, 1618, 1400, 1131 cm−1; 1H " Tables 1 and 2; HRESIMS m/z 623.2461 and 13C NMR data, see l + [M + Na] (calcd. for C32H40O11Na, 623.2463). Trichanolide E (6): white, amorphous powder; [α]25 D 0 (c 0.08, CHCl3); UV (MeCN) λmax (log ε) 195 (5.04), 205 (4.98), 283 (3.66) nm; IR (KBr) νmax 3414, 1638, 1618, 1400, 1385, 1090 cm−1; 1H " Tables 1 and 2; HRESIMS m/z 623.2465 and 13C NMR data, see l + [M + Na] (calcd. for C32H40O11Na, 623.2463).
The details of the NO production bioassays were provided in a previously published paper [39]. N-monomethyl-L-arginine (Sigma, purity ≥ 99.9 %) was used as a positive control, and all experiments were performed in three independent replicates.
α-Glucosidase inhibitory assay The details of the α-glucosidase inhibitory assay were according to the method previously reported [40]. Genistein (G6649, Sigma-Aldrich Co., purity ≥ 99.9 %) was used as a positive control and averages of 3 replicates were adopted.
Supporting information The general experimental procedures and details of extraction and isolation, as well as 1D and 2D NMR spectra for new compounds 1-8 are available as Supporting Information.
Acknowledgements !
The research was financially supported by the Project of National Natural Science Foundation of China (21272275 and 81202899), the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD), and the Program for Changjiang Scholars and Innovative Research Team in University (IRT1193).
Conflict of Interest !
The authors declare no conflict of interest.
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Original Papers
Original Papers
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Authors
Hong-Ying Wang, Jun-Song Wang, Si-Ming Shan, Xiao-Bing Wang, Jun Luo, Ming-Hua Yang, Ling-Yi Kong
Affiliation
State Key Laboratory of Natural Medicines, Department of Natural Medicinal Chemistry, China Pharmaceutical University, Nanjing, P. R. China
Key words " Trichilia connaroides l " Meliaceae l " nortriterpenoids l " pregnanes l " lignans l " NO production inhibition l " α‑glucosidase inhibition l
Abstract !
Phytochemical investigation of the stem and bark of Trichilia connaroides led to the isolation of eight new nortriterpenoids (1–8), along with fifteen known compounds (9–23). Their structures were established based on extensive spectroscopic analysis. The absolute configuration of 2 was confirmed by X‑ray crystallographic study. The nitric oxide production and α-glucosidase inhibitory ef-
Introduction !
received revised accepted
April 10, 2013 August 5, 2013 October 13, 2013
Bibliography DOI http://dx.doi.org/ 10.1055/s-0033-1351045 Planta Med 2013; 79: 1767–1774 © Georg Thieme Verlag KG Stuttgart · New York · ISSN 0032‑0943 Correspondence Prof. Dr. Ling-Yi Kong State Key Laboratory of Natural Medicines Department of Natural Medicinal Chemistry China Pharmaceutical University 24 Tong Jia Xiang Nanjing 210009 People′s Republic of China Phone: + 86 25 83 27 14 05 Fax: + 86 25 83 27 14 05 [email protected]
Trichilia connaroides (Wight & Arn.) Bentv. (Meliaceae) is widely distributed throughout the southeast of Asia. It has been traditionally used in China for the treatment of arthritis, pharyngitis, tonsillitis, and other ailments [1]. The crude extracts obtained from T. connaroides have exhibited antihyperlipidemic, hepatoprotective, analgesic, and anti-inflammatory effects [2–4]. Its twigs, leaves, roots, and fruits afforded a series of highly rearranged limonoids with a complex ring system and steroids [5–13]. To seek bioactive and novel components from this plant, we studied the dichloromethane extract of the stem and bark of T. " Fig. 1) connaroides. As a result, 23 components (l were isolated including one new 1,2-seco phragmalin-type limonoid with a rare 9-oxa-tricyclo [3.3.2.17,10]undecane-2-ene moiety (secotrichagmalin A, 1), five new mexicanolides (trichanolides A–E, 2–6), two new highly rearranged 30-nortrijugins (trijugins I–J, 7–8), and five known limonoids (9–13), five pregnanes (14–18), and five lignans (19–23). Their structures were elucidated on the basis of spectroscopic methods, or by comparison with the reported data in the literature. In addition, the absolute configuration of 2 was determined by means of single-crystal X‑ray diffraction analysis. All these isolates were evaluated for their inhibitory effects on lipopolysaccharide-in-
fects for these isolates were evaluated: moderate to strong nitric oxide production inhibitory activities were found for 5, 6, and 11–15, with IC50 values ranging from 7.5 to 26.3 µM; marked α-glucosidase inhibitory effects were observed for 22 and 23, with IC50 values of 2.3 and 0.4 µM, respectively. Supporting information available online at http://www.thieme-connect.de/ejournals/toc/ plantamedica
duced nitric oxide production in RAW264.7 cells and on α-glucosidase. Herein, we describe the isolation, structural elucidation, and biological evaluation of these isolates.
Results and Discussion !
Secotrichagmalin A (1), white amorphous powder, has a molecular formula of C32H38O9 as established by the pseudomolecular ion at m/z 565.2451 [M – H]−, (calcd. for C32H37O9, 565.2443, errors in 1.49 ppm) in the negative HRESIMS, indicating 14 degrees of unsaturation. The IR spectrum implied the presence of hydroxyl (3442 cm−1), carbonyl (1713 cm−1), and olefinic groups (1631 cm−1). Except for the easily distinguishable signals for three angular methyls (δH 0.85, 1.00, and 1.08; δC 19.4, 22.0, and 22.7) and a β-substituted furanyl moiety (δH 6.58, 7.54, and 7.67; δC 111.4, 121.7, 143.4, and 144.5), the NMR " Tables 1 and 2) also presented a spectrum of 1 (l methoxycarbonyl moiety (δH 3.61; δC 52.2 and 175.5), a tigloyl group (δH 1.92, 1.95, 7.11; δC 12.4, 14.5, 129.5, 139.7, and 168.4), and a hemiacetal group (δC 98.7). The typical 4,29,1-ring bridge was characterized by a pair of geminal doublets at δH 1.63 (1H, d, J = 13.0 Hz) and 1.97 (1H, d, J = 13.0 Hz), as evidenced by the HMBC cor" Fig. 2) of H-1/C-29, H -29/C-1, and relations (l 2
Wang H-Y et al. Chemical Constituents from …
Planta Med 2013; 79: 1767–1774
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Chemical Constituents from Trichilia connaroides and Their Nitric Oxide Production and α-Glucosidase Inhibitory Activities
Original Papers
Fig. 1
Chemical structures of compounds 1–23. (Color figure available online only.)
H2-29/C‑4. The 13C NMR data of 1 resembled those of andhraxylocarpin A [14], except for the great shift discrepancy in one oxygenated methine carbon (δC 87.5, Δ+5.9 ppm) and the hemiacetal carbon (δC 98.7, Δ-7.7 ppm). In the HMBC spectrum, this methine carbon signal (δC 87.5) corresponded to an oxygenated methine proton signal at δH 3.67 in the HSQC spectrum and had an HMBC correlation with H2-29, thus assigning itself to C-1. The position of the hemiacetal carbon at C-2 was determined by its HMBC correlations with H-1 and H-3. Thus, the gross structure of 1 was established as a hydroxyl positional isomer of andhraxylocarpin A. The relative configuration of 1 was deduced mainly from the " Fig. 2). The ROESY correlations from H-1 ROESY experiment (l to H-9, Me-19 and H2-29, from H-9 to H-11α, Me-18 and Me-19, and from H-3 to H-29b, indicated that H-1, H-3, H-9, Me-18, Me19, and H2-29 were cofacial and were arbitrarily assigned with an α-orientation. The correlations of H-17/H-12β, H-17/H‑21, H-5/ Me-28, and H-5/H-15 revealed H-5, H-17, and Me-28 to be β-oriented. Therefore, the structure of 1 was elucidated as depicted, and the compound was named secotrichagmalin A. To the best of our knowledge, secotrichagmalin A is the second report of a 1,2-seco phragmalin-type limonoid with a rare 9-oxa-tricyclo [3.3.2.17,10]undecane-2-ene moiety found in nature [14]. Trichanolide A (2), colorless crystals (MeOH/H2O), has a molecular formula of C32H38O11 as deduced from the pseudomolecular ion peak at m/z 621.2309 [M + Na]+ in the positive-ion mode HRESIMS. The 1D NMR data combined with subsequent 2D NMR " Tables 1 and 2, Fig. 3) presented signals for a β-substianalysis (l tuted furan ring, a methyl esterified C-6–C-7 appendage, a C-16/ C-17 δ-lactone ring D, four angular methyls, and a ketone carbonWang H-Y et al. Chemical Constituents from …
Planta Med 2013; 79: 1767–1774
yl, which suggested 2 was a B,D-seco mexicanolide-type limonoid [11, 15, 16]. The NMR spectroscopic data of 2 were similar to those of quivisianolide A [17], except for the downfield shifted signal of H-3′ to δH 7.14 (Δ+0.90), and upfield shifted signals of C4′ to δC 14.8 (Δ-1.5) and of C-5′ to δC 12.9 (Δ-8.3), suggesting the presence of the tigloyl group in 2 instead of an angeloyl group. Thus, the planar structure of 2 was established. The relative configuration of 2 was established mainly by the ROESY experiment " Fig. 3). The observed cross-peaks of H-14/Me-18, H-14/H-15α, (l H-3/Me-29, Me-18/H-12α and Me-19/Me-29 indicated that H-3, H-14, Me-18, Me-19, and Me-29 were co-facial and were arbitrarily assigned with an α-orientation. Likewise, the ROESY correlations from Me-28 to H-5 and H-3′ suggested that H-5 and Me-28 were β-oriented. The furan ring in the α-orientation was determined according to the ROESY correlation of H-14/H‑22, accordingly, H-17 was thus β-oriented. The 9,11-epoxide ring was assigned to be α-oriented on the basis of the J11,12 coupling constant [17]. To further clarify the structure, compound 2 was subjected to an X‑ray diffraction study using a mirror Cu Kα radiation " Fig. 4, the structure [Flack parameter 0.00(17)]. As shown in l was confirmed, and the absolute configuration of 2 was finally determined to be 2S,3S,5S,8R,9R,10S,11R,13R,14R,17R,30R. Compounds 3-6 were determined as analogues of 2 according to " Tables 1 and 2). Compound 3 was similar to 2, their NMR data (l with the exception of the presence of an additional double bond and the absence of one epoxy ring. In the HMBC spectrum, both of the two carbons of this double bond at δC 137.4 and 127.1 correlated with the proton signal at δH 2.14 ascribed to one proton of H2-12; the carbon at δC 137.4 showed a cross-peak with Me-19
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Original Papers
Pos.
1 2 3 5 6a 6b 9 11α 11β 12α 12β 14 15α 15β 17 18 19 21 22 23 28 29a 29b 30 MeO-7 3′ 4′ 5′ a
1
H (500 MHz) data of compounds 1–6. 1a
2a
3b
4b
5b
6b
δH (J in Hz)
δH (J in Hz)
δH (J in Hz)
δH (J in Hz)
δH (J in Hz)
δH (J in Hz)
5.14, s 3.37, dd (7.0, 6.0) 2.56, dd (17.0, 6.0) 2.52, dd (17.0, 7.0)
5.10, s 3.04* 2.34, d (6.5)
3.75, m 5.04, d (9.5) 3.38, dd (7.5, 6.0) 2.48, dd (16.5, 6.0) 2.39*
4.90/4.95, br s 3.22/3.17, d (10.0) 2.46/2.37, m 2.38/2.35, br s 2.25 *
4.91, br s 3.42, br d (10.0) 2.45/2.48, d (6.0) 2.38/2.33, dd (10.0, 6.0) 2.28 *
3.55, br s
5.85, t (4.0)
3.23, br s
2.07, d (15.5) 2.35, d (15.5) 1.84, dd (12.5, 7.0) 2.98, dd (18.5, 7.0) 3.57* 5.15, s 1.01, s 0.89, s 7.53, s 6.41, s 7.53, s 0.86, s 0.85, s
2.14, dd (18.5, 4.0) 2.58, dd (18.5, 4.0) 1.82, dd (12.5, 7.0) 2.73, dd (18.5, 7.0) 3.05* 5.14, s 1.04, s 1.29, s 7.35, s 6.26, s 7.42, s 0.83, s 0.81, s
1.99, d (16.0) 2.40* 1.60, dd (13.0, 7.0) 2.67, dd (18.5, 7.0) 3.63, dd (18.5, 12.5) 4.98, s 1.11, s 0.83, s 7.30, s 6.17, s 7.39, br s 0.95, s 0.83, s
1.78, m 2.06, m 1.66/1.78, m 2.18, m 2.25 * 2.81/2.86* 2.86/2.81* 5.46/5.55, s 1.13/1.25, s 1.25, s 6.09/6.31, s 6.27/6.22, s
1.71, m 2.28 * 1.45, td (14.5, 4.5) 1.93, m 2.28 * 2.83* 2.84* 5.57/5.54, s 1.02/1.04, s 1.24, s
3.61, s 3.77, s 7.14, q (7.0) 1.98, d (7.0) 2.01, s
3.44, s 3.71, s 7.05, q (7.0) 1.95, d (7.0) 1.97, s
3.17, d (2.5) 3.75, s 7.07, q (7.0) 1.94, d (7.0) 1.95, s
3.67, d (2.5) 5.00, s 2.96, t (7.5) 2.28, m 2.84, dt (13.0, 3.5) 1.72, m 1.12* 1.13* 1.98, d (13.0) 6.09, s 5.33, s 1.08, s 1.00, s 7.67, s 6.58, br s 7.54, s 0.85, s 1.97, d (13.0) 1.63, d (13.0) 6.07, d (2.5) 3.61, s 7.11, q (7.0) 1.92, q (7.0) 1.95, s
0.79/0.74, s 0.85/0.83, s
7.37/7.34, s 6.20, s 0.76, s 0.82, s
5.32/5.39, s 3.75/3.70, s 6.99, q (7.0) 1.81/1.90, d (7.0) 1.85/1.88, s
5.38, br s 3.67/3.65, s 6.94, q (7.0) 1.83/1.82, d (7.0) 1.85, s
Measured in MeOH-d4; b Measured in CDCl3; * Overlapped with other signals
(δH 1.29), which assigned the carbon signals at δC 137.4 and 127.1 to C-9 and C-11, respectively. The main difference of 4 was in the presence of an additional methine carbon and the absence of an oxygenated quaternary carbon. This methine proton (δH 3.75) correlated with C-1, C-8, and C-30 in the HMBC spectrum, and corresponded to a methine carbon at δC 49.1 in the HSQC spectrum, which in turn had an HMBC correlation with H-3, thus assigning itself to H-2. A comprehensive analysis of the HSQC and HMBC spectra allowed the establishment of 3 and 4 as shown, and they were named trichanolide B and C, respectively. Detailed analysis of the 1H and 13C NMR spectroscopic data of 5 and 6 indicated that they were mexicanolide-type limonoids as in 2, but were lacking the characteristic signals for the furan ring which was replaced by the rare γ-hydroxybutenolide moiety [18, 19]. The NMR data of 5 were similar to those of granatumin E [20], except for the presence of one additional hydroxyl. Compared with granatumin E, both the C-3 and C-30 signals were shifted downfield by 8.4 and 6.2 ppm, respectively, and the H-3 signal of 5 appeared as a singlet instead of a doublet. In addition, the C-2 methine signal in granatumin E was absent, an oxygenated quaternary carbon resonance at δC 77.3 was observed, which correlated with H-3 in the HMBC spectrum, thus assigning the additional hydroxyl to C-2. Consequently, the structure of 5 was elucidated and the compound named trichanolide D. Compounds 6 and 5 were superimposable except for those of the ring E. The γhydroxybutenolide ring was also present in 6 as characterized by two broad singlet proton at δH 7.37/7.34 (H-22) and 6.20 (H-23), and resonances at δC 135.7 (C-20), 168.3/168.4 (C-21), 149.5/
149.9 (C-22), and 97.0/96.6 (C-23). The large difference in the γhydroxybutenolide ring was due to a different tautomeric position, confirmed by the HMBC correlation between the singlet proton signal at δH 5.57/5.54 (H-17) and the α,β-unsaturated γlactone carbonyl (C-21), thus establishing a 23-hydroxybutenolide ring in 6 [18, 21]. Compound 6 was established as trichanolide E. Trijugin I (7) had the molecular formula C28H32O13, suggesting 13 " Table 3) exdegrees of unsaturation. The 13C NMR spectrum (l hibited 28 carbon signals. After deduction of those belonging to one methoxyl and one acetyl group, the remaining 25 carbons suggested a pentanortriterpenoid nature for 7. Four singlet methyls at δH 0.99/1.10, 1.57, 1.40, and 1.07 were attributable to Me18, Me-19, Me-28, and Me-29. The characteristic lactone ring D was confirmed by the HMBC correlations from the H2-15 protons to C-14, C-13, and C-16, from H-17 to C-13, and from Me-18 to C13 and C-14, and signals of a carbomethoxy group at C-7 was also observed. The only unassigned acetyl group was located at C-8 according to the downfield shifted signal of C-8 at δC 89.4. Its NMR data suggested that it was structurally related to trijugin D [8], but differing in the presence of the 21-hydroxybutenolide ring instead of a β-furanyl ring in trijugin D. The relative configu" Fig. 5). Hence, ration of 7 was determined by ROESY spectrum (l the structure of 7 was elucidated as shown. Trijugin J (8) had a molecular formula of C29H34O13. Its NMR data " Table 3) resembled those of 7, especially those associated with (l rings A–D, but differing in ring E. The characteristic carbon signals at δC 104.2, 135.7, 148.6, 169.5, and 57.5 could be attributed Wang H-Y et al. Chemical Constituents from …
Planta Med 2013; 79: 1767–1774
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Table 1
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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 30 MeO-7 1′ 2′ 3′ 4′ 5′ a
1a
2a
3b
4b
5b
6b
δC
δC
δC
δC
δC
δC
87.5 98.7 83.1 49.4 38.8 34.9 175.5 141.3 46.3 54.4 23.9 34.0 39.6 169.0 115.0 167.0 80.7 22.0 22.7 121.7 143.4 111.4 144.5 19.4 40.4 134.8 52.2 168.4 129.5 139.7 14.5 12.4
210.2 80.3 86.8 41.2 44.7 32.8 175.7 63.0 63.6 52.4 56.8 29.0 35.1 40.1 30.7 172.0 84.3 24.1 11.2 123.5 142.9 110.4 145.2 23.8 21.4 65.6 53.0 168.5 129.4 140.6 14.8 12.9
210.2 79.3 85.4 39.8 47.7 31.4 173.2 61.2 137.4 51.7 127.1 32.1 35.6 39.6 29.7 169.5 82.2 23.5 14.7 122.2 140.9 109.7 143.9 24.0 20.4 64.5 52.5 167.0 128.3 139.6 14.9 12.8
209.1 49.1 77.9 39.2 43.6 32.4 173.9 59.6 62.9 50.2 56.3 28.0 34.4 39.0 29.3 169.3 83.1 23.7 10.7 122.3 140.9 109.7 143.9 24.9 21.7 60.8 52.8 167.0 128.0 140.1 15.0 12.7
214.3 77.3 85.4/84.9 39.8/39.7 42.3/42.6 33.4/33.2 176.3/176.4 136.3/136.1 56.6/56.3 49.2/50.0 21.1/20.6 34.7/34.9 37.8/37.1 45.7/46.4 29.6/29.8 167.2/166.8 75.8/76.0 21.4/21.2 16.4/15.8 163.3/160.6 97.7/99.4 122.4/121.1 169.5 22.8/22.1 19.8/20.3 130.3/130.5 53.30/53.33 167.6/167.7 127.5/127.3 140.6/140.8 15.00/14.97 12.2/12.3
215.4/215.3 77.6 85.1/85.2 39.9 40.8 33.0 173.7/173.3 136.5 56.6/56.7 50.4/50.3 21.2/21.1 34.8 37.0/36.9 45.5 29.4 167.7 76.7 22.8 15.70/15.73 135.7 168.3/168.4 149.5/149.9 97.0/96.6 22.2 20.6 129.4/129.3 52.5/52.4 167.8 127.9 139.9/139.8 14.9 12.2
Table 2 13C NMR (125 MHz) data of compounds 1–6.
Measured in MeOH-d4; b Measured in CDCl3
Fig. 2 1H-1H COSY, Key HMBC, and ROESY correlations of 1. (Color figure available online only.)
to C-23, C-20, C-22, C-21, and OMe-23 comprising a rare 23-methoxy-α,β-unsaturated γ-lactone moiety of ring E with the corresponding proton resonances at δH 5.88 (1H, br s) and 7.21 (1H, br s), assigned to H-23 and H-22, respectively. The three-proton singlet at δH 3.42 was determined to be at C-23 according to its HMBC correlation with C-23 [22–24]. Interestingly, the NMR signals due to the γ-methoxybutenolide ring in 8 were not observed in pairs compared with those possessing a γ-hydroxybutenolide Wang H-Y et al. Chemical Constituents from …
Planta Med 2013; 79: 1767–1774
ring, which could be explained by the blockade effect of the methoxyl to the tautomerization. The similar ROESY correlations of 7 and 8 suggested the same relative configurations. However, the available evidences were insufficient to determine the configuration at C-23. The structure of 8 was thus established as shown. The known isolates were identified as trijugins D (9) and F (10) [8], methyl-2-hydroxy-3β-tigloyloxy-1-oxomeliac-8(30) enate (11) [25], febrifugin A (12) [26], and granatumin E (13) [20], (E)-
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Position
Original Papers
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Fig. 4 Perspective drawing of the X‑ray structure of 2. (Color figure available online only.)
to 26.3 µM, and compounds 2–4 displayed weak activities with IC50 values ranging from 48.7 to 57.5 µM. These results revealed the potential of mexicanolide-type limonoids in this regard. Interestingly, 14 and 15 possessing a Δ17,20 double bond showed striking activities with IC50 values lower than 10 µM, as compared with compound 18 which has the same skeleton with an identical Δ5,6 double bond and two β-OHs substituted at C-3 and C-4, suggesting the important role of the Δ17,20 double bond in the inhibitory effect on nitric oxide production in LPS-activated macrophages. The α-glucosidase inhibitory activities of these isolates were also " Table 4). Of them, compounds 9, 10, and 14 disevaluated (l played moderate activities in inhibition of α-glucosidase with IC50 values ranging from 30 to 50 µM. Amazingly, 22 and 23, with an extremely simple skeleton, were the most active, with IC50 values of 2.3 and 0.4 µM, respectively. Compounds 22 and 23 have been isolated from many plants [36], hence, since easily obtained, they may be considered as lead compounds providing a new thread for synthesis and modification of hypoglycemic agents.
Materials and Methods volkendousin (14), (Z)-volkendousin (15) [27], 3β,4α-dihydroxypregnan-20-one (16) [8], 3β,4α-dihydroxypregnan-16-one (17) [7], ekeberin B (18) [28], episyringaresinol (19) [29], (+)-syringaresinol (20) [30], (+)-medioresinol (21) [30], honokiol (22), and magnolol (23) [31] by comparison of their spectroscopic data with those previously reported. Limonoids containing γ-hydroxybutenolide were isolated in this study. The γ-hydroxybutenolide could be transformed from the common furan via singlet oxygen (1O2) oxidation. Singlet oxygen is produced mainly in plant leaves by light through chlorophylls which act as photosensitizers [32]. The [4 + 2] cycloaddition of singlet oxygen to β-substituted furanyl moiety in compounds, e.g., 9 and 11, can readily afford unstable furan endoperoxides. The subsequent breakdown of these endoperoxides affords diverse products including hydroxybutenolides 5–7 and the solvent addition product 8 [33–35]. Twenty-three isolates were evaluated for their inhibitory effects on the release of NO in lipopolysaccharide (LPS)-induced RAW264.7 cells using N-monomethyl-L-arginine as the reference " Table 4. compound (IC50 = 37.7 µM). Results are summarized in l Compounds 5, 6, 11, 12, and 13, exhibited moderate to strong NO production inhibitory activity with IC50 values ranging from 7.5
!
Plant material The air-dried stem and bark of T. connaroides were collected in November 2011 from Xishuangbanna, Yunnan Province, People′ s Republic of China, and authenticated by Prof. Shun-Cheng Zhang, Xishuangbanna Botanical Garden, Chinese Academy of Sciences, People′s Republic of China. A voucher specimen (No. ZGH-201111) is deposited in the Department of Natural Medicinal Chemistry, China Pharmaceutical University.
Extraction and isolation The air-dried powder of the plant material (10.0 kg) was extracted with 95% EtOH under reflux and then evaporated in vacuo to remove the solvents so as to give a crude extract (1280.0 g). The extract was suspended in H2O (5 L) and successively partitioned with petroleum ether and CH2Cl2 to give two parts. The CH2Cl2 extract (64.0 g) was fractionated by silica gel column eluted with petroleum ether-EtOAc in a gradient (20 : 1 to 0 : 1) to give seven major fractions (I–VII). Fraction III (2.7 g) was chromatographed on MCI, RP‑C18, and successive preparative HPLC to afford 19 (5.0 mg), 20 (9.0 mg), 21 (6.0 mg), 22 (3.0 mg), and 23. Fraction IV (12.4 g) was subjected to a column of MCI, RP‑C18, and successive Wang H-Y et al. Chemical Constituents from …
Planta Med 2013; 79: 1767–1774
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Fig. 3 Key HMBC and ROESY correlations of 2. (Color figure available online only.)
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1 2 3 4 5 6 7 8 9 10 11α 11β 12α 12β 13 14 15α 15β 16 17 18 19 20 21 22 23 28 29 MeO-7 MeO-23 8-OAc
a
8b
δC
δH (J in Hz)
δC
δH (J in Hz)
80.5 51.8 207.8 48.0 45.6 74.8 170.1 89.4 175.9 43.4 42.2
4.43/4.41, d (4.5) 3.62/3.61, d (4.5)
81.8 53.0 209.6 48.9 46.6 75.6 172.0 90.8 177.0 44.4 42.8
4.46, d (4.5) 3.66, d (4.5)
33.7 47.4 93.3/93.5 36.9 167.2 81.2 16.7 18.9 163.5 97.1/97.8 121.0/121.9 169.2 26.4 22.1 53.1 170.6 21.8
3.33/3.34, d (8.5) 5.06, d (8.5)
2.50/2.46, m 1.71, m 2.03, m
2.95/2.97, d (18.0) 2.25/2.24, d (18.0) 5.59/5.53, br s 0.99/1.10, s 1.57, s 6.00/6.24, s 6.26/6.15, br s 1.40, s 1.07, s 3.85, s
2.20, s
35.2 48.3 94.5 37.8 168.0 80.7 16.8 19.4 135.7 169.5 148.6 104.2 26.7 22.7 53.6 57.5 170.8 22.1
Table 3 1H (500 MHz) and 13C (125 MHz) data of compounds 7– 8.
3.42, d (8.5) 5.17, d (8.5)
2.35, m 2.41, m 1.43, dd (13.0, 8.0) 1.91*
2.88, d (18.0) 2.10, d (18.0) 5.29, br s 0.90, s 1.50, s
7.21, br s 5.88, br s 1.30, s 0.98, s 3.74, s 3.42, s 2.00, s
b
Measured in CDCl3; Measured in Acetone-d6; * Overlapped with solvent signal
Fig. 5 Key HMBC and ROESY correlations of 7. (Color figure available online only.)
preparative HPLC to give 14 (8.0 mg), 15 (2.0 mg), 16 (10.0 mg), 17 (20.0 mg), and 18 (10.0 mg). Fraction V (10.5 g) was chromatographed on MCI, silica gel, RP‑C18, Sephadex LH-20, and preparative HPLC to yield 1 (2.5 mg), 2 (5.5 mg), 3 (4.3 mg), 4 (5.0 mg), 5 (2.5 mg), 6 (3.2 mg), 7 (7.8 mg), 8 (1.5 mg), 9 (90.0 mg), 10 (2.4 mg), 11 (4.5 mg), 12 (3.0 mg), and 13 (2.0 mg) (for detailed procedure for extraction and isolation, see Supporting Information).
Wang H-Y et al. Chemical Constituents from …
Planta Med 2013; 79: 1767–1774
New isolates Secotrichagmalin A (1): white, amorphous powder; [α]25 D + 232.2 (c 0.07, MeCN); UV (MeCN) λmax (log ε) 202 (4.19), 267 (3.45) nm; IR (KBr) νmax 3442, 2971, 2923, 1713, 1631, 1454, 1396, " Tables 1 and 1253, 1162, 1049 cm−1; 13C and 1H NMR data, see l 2; HRESIMS m/z 565.2451 [M – H]− (calcd. for C32H37O9, 565.2443).
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7a
Position
Table 4 Nitric oxide production in LPS-stimulated RAW264.7 and α-glucosidase inhibitory activities of the isolates from Trichilia connaroides a,b. Compounds 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 Genisteinc N-monomethyl-L-arginine c a
α-Glucosidase
Effects of inhibiting
inhibitory activities
NO production
> 100 > 100 > 100 64.7 ± 3.9 > 100 > 100 > 100 > 100 32.5 ± 2.1 49.0 ± 4.5 > 100 > 100 > 100 30.7 ± 1.9 > 100 > 100 > 100 > 100 > 100 83.5 ± 10.9 > 100 2.3 ± 0.2 0.4 ± 0.1 10.6 ± 0.6
49.2 ± 2.9 57.5 ± 4.5 48.7 ± 3.4 54.3 ± 3.7 16.6 ± 1.6 20.3 ± 1.9 > 100 > 100 > 100 > 100 26.3 ± 1.8 12.8 ± 2.3 7.5 ± 1.1 7.6 ± 1.3 8.2 ± 0.4 > 100 > 100 > 100 > 100 > 100 > 100 > 100 46.7 ± 5.8 37.7 ± 2.7
Results are expressed as IC50 values in µM; b Compounds with IC50 > 100 µM are not
Trijugin I (7): white, amorphous powder; [α]25 D − 10.5 (c 0.06, MeCN); UV (MeCN) λmax (log ε) 195 (3.93), 205 (3.84) nm; IR (KBr) νmax 3442, 2919, 2850, 1745, 1633, 1399, 1236, 1129, " Table 3; HRESIMS m/z 1050 cm−1; 1H and 13C NMR data, see l 575.1778 [M – H]− (calcd. for C28H31O13, 575.1770). Trijugin J (8): white, amorphous powder; [α]25 D + 7.1 (c 0.09, MeCN); UV (MeCN) λmax (log ε) 192 (3.59), 205 (3.47), 260 (2.18) nm; IR (KBr) νmax 3447, 2917, 2850, 1741, 1680, 1468, 1384, 1204, 1130, 1050, 1021 cm−1; 1H and 13C NMR data, see " Table 3; HRESIMS m/z 589.1928 [M – H]− (calcd. for C l 29H33O13, 589.1927). X‑ray crystallographic study of 2: C32H38O11, M = 598.62: orthorhombic, space group P 21 21 21, unit cell dimensions a = 8.0814 (10) Å, b = 18.8427 (2) Å, c = 19.7979 (3) Å, V = 3014.73 (7) Å3, Z = 4, T = 290 (2) K, µ (CuKα) = 0.829 mm−1, Dcalc = 1.319 g/mm3, Flack = 0.01(17). Colorless crystals of 2 were obtained in a mixed solvent of methanol and water. A suitable crystal was selected, investigated on a diffractometer (λ = 1.54 184 Å) and kept at 290 (2) K during data collection. The structure was solved with the SHELXS-97 [37] structure solution program using direct methods and refined with the SHELXL-97 [38] refinement package using least squares minimization. Data collection yielded a total of 18 863 integrated intensities, and resulted in 5597 unique and averaged observations with Rint = 0.0266. Full matrix least-squares refinement on F2 led to a final R indices [I > 2 σ(I)]: R1 = 0.0366, wR2 = 0.0882, R indices (all data) [I > 2σ(I)]: R1 = 0.0449, wR2 = 0.0946 and GOOF of 1.044. Crystallographic data for compound 1 have been deposited at the Cambridge Crystallographic Data Centre (deposition No. CCDC 911539).
shown; c Positive controls
NO production bioassay
Trichanolide A (2): colorless crystals (MeOH/H2O); mp 251– 252 °C; [α]25 D − 55.0 (c 0.10, MeCN); UV (MeCN) λmax (log ε) 212 (4.16) nm; IR (KBr) νmax 3449, 2919, 2850, 1735, 1640, 1400, 1261, 1204, 1123, 1068, 1025 cm−1; 1H and 13C NMR data, see " Tables 1 and 2; HRESIMS m/z 621.2309 [M + Na]+ (calcd. for l C32H38O11Na, 621.2306). Trichanolide B (3): white, amorphous powder; [α]25 D + 34.8 (c 0.05, MeCN); UV (MeCN) λmax (log ε) 194 (4.24), 209 (4.21), 269 (3.41) nm; IR (KBr) νmax 3425, 2921, 2851, 1731, 1622, 1400, 1259, " Tables 1 and 2; 1163, 1063 cm−1; 1H and 13C NMR data, see l HRESIMS m/z 605.2349 [M + Na]+ (calcd. for C32H38O10Na, 605.2357). Trichanolide C (4): white, amorphous powder; [α]25 D − 79.0 (c 0.09, MeCN); UV (MeCN) λmax (log ε) 213 (4.14) nm; IR (KBr) νmax 3423, 1631, 1400, 1146, 1058 cm−1; 1H and 13C NMR data, see " Tables 1 and 2; HRESIMS m/z 605.2378 [M + Na]+ (calcd. for l C32H38O10Na, 605.2357). Trichanolide D (5): white, amorphous powder; [α]25 D − 31.6 (c 0.05, CHCl3); UV (MeCN) λmax (log ε) 196 (5.00), 206 (5.01), 287 (3.66) nm; IR (KBr) νmax 3415, 1725, 1638, 1618, 1400, 1131 cm−1; 1H " Tables 1 and 2; HRESIMS m/z 623.2461 and 13C NMR data, see l + [M + Na] (calcd. for C32H40O11Na, 623.2463). Trichanolide E (6): white, amorphous powder; [α]25 D 0 (c 0.08, CHCl3); UV (MeCN) λmax (log ε) 195 (5.04), 205 (4.98), 283 (3.66) nm; IR (KBr) νmax 3414, 1638, 1618, 1400, 1385, 1090 cm−1; 1H " Tables 1 and 2; HRESIMS m/z 623.2465 and 13C NMR data, see l + [M + Na] (calcd. for C32H40O11Na, 623.2463).
The details of the NO production bioassays were provided in a previously published paper [39]. N-monomethyl-L-arginine (Sigma, purity ≥ 99.9 %) was used as a positive control, and all experiments were performed in three independent replicates.
α-Glucosidase inhibitory assay The details of the α-glucosidase inhibitory assay were according to the method previously reported [40]. Genistein (G6649, Sigma-Aldrich Co., purity ≥ 99.9 %) was used as a positive control and averages of 3 replicates were adopted.
Supporting information The general experimental procedures and details of extraction and isolation, as well as 1D and 2D NMR spectra for new compounds 1-8 are available as Supporting Information.
Acknowledgements !
The research was financially supported by the Project of National Natural Science Foundation of China (21272275 and 81202899), the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD), and the Program for Changjiang Scholars and Innovative Research Team in University (IRT1193).
Conflict of Interest !
The authors declare no conflict of interest.
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Original Papers
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