Letter - spectral assignment Received: 25 June 2014
Revised: 26 July 2014
Accepted: 30 July 2014
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI 10.1002/mrc.4132
Structure elucidation of four new megastigmanes from Sonneratia ovata Backer Thi Hoai Thu Nguyen,a Nguyen Kim Tuyen Pham,b Khanitha Pudhom,c Poul Erik Hansend and Kim Phi Phung Nguyene* Introduction For a long time, fruits, bark, and leaves of some Sonneratia species have been used in folk medicine for the treatment of different diseases such as asthma, febrifuge, ulcers, hepatitis, piles, sprain, and hemorrhages.[1] Moreover, these species of the Sonneratia genus growing widely in the mangrove forests in tropical areas possess a huge biomass. Therefore, this genus is attractive for pharmacological and chemical studies. Previously, pharmaceutical investigations on Sonneratia genus showed antidiabetic,[2] antioxidant, antibacterial, [3] and anti-tumor properties.[4] Chemical studies are reported on S. alba,[5] S. caseolaris,[6,7] S. apeltala,[8] S. hainanensis,[4,9] S. paracaseolaris [10] to obtain diverse types of compounds. In previous chemical studies on Sonneratia ovata, a number of compounds were isolated.[6,11] The chemical investigation on the leaves of S. ovata using efficient separation techniques leads to the isolation of four new megastigmanes together with six known megastigmane ones. Enantiomers cannot be directly distinguished by nuclear magnetic resonance (NMR). However, the presence of a glucose moiety may establish diastereomers. The use of an intrinsic glucose molecule to determine the absolute configuration based on NMR parameters and density functional theory (DFT) calculations is investigated.
Results and Discussion The methanolic crude extract from the dried leaves of S. ovata was partitioned with petroleum ether and subsequently with ethyl acetate to give petroleum ether and ethyl acetate extracts. The ethyl acetate extract was applied to efficient separation and purification techniques to afford four new megastigmane compounds, sonnerstigmane A (1a), sonnerstigmane B (1b), sonnerstigmane C (5), and sonnerstigmane D (6), together with six known megastigmanes (as shown in Fig. 1). The structures of these known compounds are determined by analyses of their NMR spectral data, as well as comparing their data with those in the literature. They are (6S,9R)-roseoside (2),[12] (6S,9R)-ionone 9-O-(6-O-galloyl)-β-D-glucopyranoside (3),[13] (S)-dehydrovomifoliol (4),[14] 8,9-dihydromegastigmane-4,6diene-3-one (7),[15] 5,8-epoxymegastigmane-6-ene-3-one 9-O-β-Dglucopyranoside (8),[16] and dehydrololiolide (9).[17] Norisoprenoids or megastigmanes are known in many kinds of species; however, these are now reported in Sonneratia genus for the first time. They are considered to be very important flavor and fragrance precursors in tea, tobacco, fruits, essential oils,
Magn. Reson. Chem. (2014)
vegetables, spices, and wine.[18] The isolated megastigmanes constitute an interesting case as structure with 6 is a glycosylated derivative of 7. Compound 1–4 possess the 6,9-dihydroxymegastigmane-4,7diene-3-one skeleton. This moiety can be recognized basically by the presence of two singlet gem-methyl signals at around 1.0 ppm of (H3C)2C < moiety (H-11 and H-12), a doublet methyl signal at about 1.2 ppm of H3C-CH-O (H-10), a singlet proton signal at around 1.9 ppm of a methyl group (H-13) attached to a double bond and two doublet gem-proton signals with a large coupling constant of 17.0 Hz of H-2a and H-2b at δH from 2.1 to 2.5 ppm. At the high frequency range, the proton spectrum shows three olefinic proton signals at δH from 5.7 to 5.9 ppm of two double bonds C4 = C5 and C7 = C8, in which C-5 is a quaternary carbon. Additionally, the 13C spectrum shows carbon signals of a conjugated carbonyl group at round δC 197 (>C¼O, C-3), 162 (>C¼, C-5), and 127 (–CH¼, C-4) in CDCl3. These two first signals are shifted to lower field at 201 (C-3) and 167 (C-5) by the nucleophilic effect of methanol on a carbonyl carbon of an enone system when recording NMR spectra in CD3OD. Moreover, the 13C spectrum shows two olefinic carbon signals of a remaining isolated double bond at around δC 135 and 131. An oxygenated quaternary carbon and an oxygenated methine carbon, a methylene carbon, a quaternary carbon, and four methyl carbons are also observed at round δC 80 (C-6), 77 (C-9), 50 (C-2), 42 (C-1), 24 and 23 (C-11 and C-12), 21 (C-10), and 19 (C-13), respectively (Table 2). Compound 1 is obtained as a pale yellow oil. The analysis of the 1H-NMR and 13C-NMR data of 1 shows that it may possess a 6,9-dihydroxymegastigmane-4,7-diene-3-one skeleton as mentioned earlier (Tables 1 and 2). The skeleton of 1 is confirmed
* Correspondence to: Kim Phi Phung Nguyen, Department of Organic Chemistry, VNUHCM – University of Science, 227 Nguyen Van Cu Str., Dist. 5, Ho Chi Minh City, Vietnam. E-mail: [email protected] a Department of Basic Science, University of Medicine and Pharmacy, Ho Chi Minh City, Vietnam b Department of Environmental Science, Saigon University, Ho Chi Minh City, Vietnam c Department of Chemistry, Faculty of Science, Chulalongkorn University, Bangkok, Thailand d Department of Science, Systems and Models, Roskilde University, Roskilde, Denmark e Department of Organic Chemistry, VNUHCM – University of Science, Ho Chi Minh City, Vietnam
Copyright © 2014 John Wiley & Sons, Ltd.
T. H. T. Nguyen et al.
Figure 1. Structures of compounds isolated from the leaves of Sonneratia ovata.
Table 1.
1
H-NMR data of 1a, 1b, 2, 3, and 4, δH (ppm), J (Hz)
N°
1aa
1ba
2m
3m
4a
2
2.26 (d, 16.8) 2.41 (d, 16.8) 5.89 (s) 5.73 (d, 15.6) 5.72 (dd, 15.6, 5.2) 3.80 (quint-like, 6.0) 1.25 (d, 6.0) 1.07 (s) 0.99 (s) 1.90 (s)
2.26 (d, 16.8) 2.41 (d, 16.8) 5.89 (s) 5.73 (d, 15.6) 5.70 (dd, 15.6, 5.2) 3.80 (quint-like, 6.0) 1.23 (d, 6.0) 1.07 (s) 1.01 (s) 1.88 (s)
2.17 (d, 17.0) 2.49 (d, 17.0) 5.87 (m) 5.87 (m) 5.87 (m) 4.42 (m) 1.30 (d, 6.5) 1.03 (s) 1.03 (s) 1.92 (d, 1.5) 4.35 (d, 8.0) 3.17 (dd, 9.0, 8.0) 3.34 (m) 3.25 (m) 3.23 (m) 3.63 (dd, 12.0, 5.5) 3.83 (dd, 11.5, 1.5)
2.11 (d, 17.0) 2.40 (d, 17.0) 5.81 (d, 2.5) 5.80 (d, 15.0) 5.81 (dd, 15.0, 5.5) 4.38 (m) 1.29 (d, 6.0) 0.99 (s) 0.95 (s) 1.84 (d, 1.0) 4.40 (d, 7.5) 3.22 (dd, 9.0, 8.0) 3.40 (dd, 9.0, 9.0) 3.44 (dd, 9.0, 9.0) 3.51 (ddd, 9.0, 5.0, 2.0) 4.37 (dd, 12.0, 5.0) 4.47 (dd, 12.0, 2.0) 7.08 (s)
2.45 (d, 17.2) 2.34 (d, 17.2) 5.93 (s) 6.84 (d, 15.6) 6.47 (d, 15.6
3.24 (1H, s)
3.26 (1H, s)
4 7 8 9 10 11 12 13 1′ 2′ 3′ 4′ 5′ 6′ 2″, 6″ -OCH3
2.28 (s) 1.09 (s) 1.01 (s) 1.87 (s)
a
Chloroform-d Methanol-d4
m
by the COSY and Heteronuclear Multiple-Bond Correlation (HMBC) correlations (Fig. 2). The E configuration of the double bond C7 = C8 is determined by the large three-bond H–H coupling of 15.6 Hz. Besides that, 1 has a methoxy group’s signals at δH 3.24 (3H, s, –OCH3) and δC 56.3 (–OCH3). The methoxy group attached to the megastigmane skeleton at C-9 is determined via the HMBC correlation of the methoxy proton with carbon C-9. The 1H-NMR and 13C-NMR spectra of 1 display pairs of signals with a ratio of about 1.2:1.0 corresponding to two stereoisomers of 9-methoxyvomifoliol. According to Yamano and Ito,[12] specific rotations of (6S,9R) and (6S,9S) isomers of vomifoliol are
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dextrorotatory, and of (6R,9S) and (6R,9R), isomers are levorotatory. Compound 1 differs from vomifoliol only by a methoxy group at C-9 and possesses a positive optical rotation. Therefore, 1 is suggested to be a mixture of (6S,9R) and (6S,9S) isomers with a ratio of about 1.2:1.0. The high resolution electrospray ionisation mass spectroscopy (HR-ESI-MS) showed a pseudo molecular ion peak at m/z 261.1479, calcd for [C14H22O3 + Na]+, 261.1467. All assignments suggest that the new compound 1 is a mixture with the ratio (1.2:1.0) of sonnerstigmane A [(6S,7E,9R)-6-hydroxy-9-methoxymegastigmane4,7-diene-3-one (1a)] and sonnerstigmane B [(6S,7E,9S)-6-hydroxy9-methoxymegastigmane-4,7-diene-3-one (1b)].
Copyright © 2014 John Wiley & Sons, Ltd.
Magn. Reson. Chem. (2014)
Structure elucidation of four new megastigmanes from Sonneratia ovata Backer Table 2.
13
C-NMR data of 1a, 1b, 2, 3, and 4 1aa
1ba
2m
3m
4a
41.3 49.9 198.0 127.0 162.8 79.2 131.3 133.8 77.5 21.5 23.0 24.2 19.0
41.2 49.9 198.0 127.1 162.7 79.3 131.2 133.9 77.4 21.5 23.0 24.3 19.0
42.4 50.7 201.2 127.1 167.2 80.0 131.5 135.2 77.3 21.2 23.4 24.7 19.5 102.7 75.2 78.1 71.6 78.0 62.8
42.4 50.7 201.4 127.2 167.1 80.0 131.6 134.9 77.1 21.2 23.4 24.6 19.7 103.1 75.2 78.0 71.5 75.4 64.7 121.4 110.3 146.5 139.9 168.4
41.6 49.7 197.3 127.8 160.9 79.3 145.4 130.5 197.7 28.4 23.1 24.5 18.8
56.3
56.3
N° 1 2 3 4 5 6 7 8 9 10 11 12 13 1′ 2′ 3′ 4′ 5′ 6′ 1″ 2″, 6″ 3″, 5″ 4″ –COO– –OCH3 a
Chloroform-d Methanol-d4
m
Compound 6 is obtained as a colorless oil. 1H-NMR and 13C-NMR data of 6 prove that it possesses the same megastigmane skeleton as 1 (Table 3). However compound 6 differs from 1 by the position of the double bond which is at C6 = C7 and the presence of one more hydroxyl group at C-8 at δ 4.75 (1H, dd, 10.0, 5.0 Hz, H-8) and one less olefinic proton signal. Besides that, the 13C spectrum of 6 displays an oxygenated methine carbon at higher magnetic field at δ 72.3 (C-8), instead of an oxygenated quaternary carbon signal at around 80 ppm of C-6 in 1. These are confirmed by the COSY correlations of the olefinic proton H-7 at δ 6.09 (1H, d, 10.0 Hz) with the carbinol proton H-8 at δ 4.75 (1H, dd, 10.0, 5.0 Hz); of H-8 with the carbinol proton H-9 at δ 3.83 (1H, m); and of H-9 with the methyl protons at δ 1.30 (3H, d, 6.5 Hz, H-10) (Fig. 2). Additionally, the HMBC spectrum reveals the correlations of H-10 with two oxygenated carbons at δ 72.3 (C-8) and 82.1 (C-9), of H-13 at δ 2.15 (3H, s) with olefinic carbons at δ 127.3 (C-4), 158.9 (C-5), and 145.2 (C-6) (Fig. 2). The 1H-NMR spectrum also displays an anomeric proton signal at δ 4.46 (1H, d, 7.5 Hz, H-1′) and other oxygenated protons from
3.26–3.86 ppm of one β-glucose unit, which are matched with the observation of an anomeric carbon at δ 105.7 and five oxygenated carbons at δ from 62.7 to 78.0. The attachment of the glucose moiety to the aglycone at C-9 is confirmed via the HMBC correlation between the anomeric proton H-1′ and C-9. The NOESY spectrum reveals the proximity in space between the olefinic proton H-7 and methyl protons H-13; therefore, the configuration of the double bond C6 = C7 is E. The following analysis is based on H,H coupling constants, NOESY cross peak intensities and calculated 13C nuclear shieldings as well as rotamers’ calculated energies. The aim is to determine the absolute configurations for oxygenated carbons 8 and 9 by using the glucose moiety as an asymmetric discriminator. The glycosidic linkage is β orientation as judged from 3JH1′,H2′ value of 7.5 Hz. In the HMBC spectrum, 3J (H-1′, C-9) and 3J (H-9, C-1′) show intense cross peaks and in NOE experiment, H-9 and H-1′ also show strong interaction. These findings show that these two protons must be proximate in space. Additionally, the strong NOESY cross peaks between H-8 and the methyl protons H-11 and H-12 show that H-8 is primarily directed toward these methyl groups. Therefore, the rotamers of 6 are suggested as shown in Fig. 4. In order to assign the configuration of chiral carbons of these rotamers, the following criteria are used: A strong NOESY cross between H-8 and CH3-10 shows that the gauche rotamers A, D, I and L are not suitable. A second very important finding is the absence of a NOESY cross peak between H-8 and H-9 showing that these two protons are not in the gauche conformations and therefore rotamers B, H, E, and K are excluded. This leaves only C, F, G, and J as the possibilities. J and G can be excluded because of the small value of correlation coefficient (R2) between the observed 13C chemical shifts and the calculated nuclear shieldings (Fig. 4) (In these rotamers, carbons C-7–C-10, C-1′, CH3-11, and CH3-12 are put in the consideration because they are most likely influenced by the conformations and configurations). Results from weak to medium of NOESY cross peaks between H-7 and H-9 as well as weak cross peaks in the HMBC spectrum between H-9 and C-7 and H-8 and C-10 show that both rotamers C and F are good candidates. The distinction between these two rotamers lies in their energies. The calculated energy of F is 23 KJ higher than that of E-rotamer therefore F-rotamer is not highly populated and is excluded. In contrast, C is favored by 21 KJ over B. These are also suitable with the two bulky substituents of C, glucose and quinone moieties, are far apart in space. Up to this point, rotamer C is the most suitable. All data in the preceding text suggest that 6 is (7E,8R,9R)-8-hydroxymegastigmane-4,6-diene-3one 9-O-β-D-glucopyranoside and is named sonnerstigmane D. Compound 7 has been reported earlier with a gross structure without stereochemical assignments.[15] The NOESY cross peaks and the H,H coupling constants combined with DFT calculations can shed some light on this problem in analogy with what is described above for compound 6. The NOESY correlations (Fig. 3)
Figure 2. Heteronuclear Multiple-Bond Correlation (arrows point from H → C) and COSY correlations of 1a, 1b, 5, and 6.
Magn. Reson. Chem. (2014)
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Copyright © 2014 John Wiley & Sons, Ltd.
m
6.09 (1H, d, 10.0) 4.75 (1H, dd, 10.0, 5.0) 3.83 (1H, m) 1.30 (3H, d, 6.5) 1.39 (3H, s) 1.33 (3H, s) 2.15 (3H, s) 4.46 (1H, d, 7.5) 3.26 (1H, m) 3.37 (1H, m) 3.29 (1H, m) 3.27 (1H, m) 3.68 (1H, dd, 12.0, 5.0) 3.88 (1H, m)
5.98 (1H, s)
2.37 (1H, d, 16.0) 2.40 (1H, d, 16.0)
δH (ppm), J (Hz)
13
39.9 54.3
δC
202.0 127.3 158.9 145.2 134.7 72.3 82.1 19.3 29.8 29.9 22.8 105.7 75.5 78.0 71.5 77.9 62.7
H-NMR and C-NMR data of 6 and 7
1
Chloroform-d Methanol-d4
a
8-OH 9-OH
3 4 5 6 7 8 9 10 11 12 13 1′ 2′ 3′ 4′ 5′ 6′
1 2
N°
Table 3.
4′, 5′ 4′
5′
2′
1, 5, 6, 8, 9, 11, 12 6, 7, 9, 10 7, 10, 1′ 8, 9 1, 2, 6, 12, 3 1, 2, 6, 11, 3 4, 5, 6 9, 1′
2, 6, 13
1, 3, 4, 6, 11, 12
HMBC
8 7, 9 8, 10
13
COSY
6m
9, 13 10, 11, 12 7, 10, 1′ 8, 9 8 8 4, 7 9
13
11, 12
NOESY
5.87 (1H, d, 10.0) 4.67 (1H, dd, 10.0, 6.3) 3.71 (1H, m) 1.24 (3H, d, 6.0) 1.38 (3H, s) 1.29 (3H, s) 2.09 (3H, s) 4.43 (1H, d, 8.0) 3.24–3.39 (m) 3.24–3.39 (m) 3.24–3.39 (m) 3.24–3.39 (m) 3.73 (1H, dd, 12.0, 4.5) 3.80 (1H, dd, 12.0, 2.5)
5.95 (1H, s)
2.31 (1H, d, 16.0 2.38 (1H, d, 16.0)
δH (ppm)
m+a
6
201.1 127.2 157.7 145.2 133.3 71.9 82.3 19.2 29.7 29.9 22.8 105.2 74.7 77.2 70.8 77.0 62.2
39.5 53.9
δC
3.03 (1H,brs) 2.96 (1H, brs)
5.83 (1H, d, 9.6) 4.57 (1H, t-like, 7.6) 3.69 (1H, quint-like, 6.4) 1.18 (3H, d, 6.4) 1.38 (3H, s) 1.29 (3H, s) 2.06 (3H, s)
5.95 (1H, s)
2.32 (1H, d, 16.0) 2.38 (1H, d, 16.0)
δH (ppm), J (Hz)
7a
199.3 127.4 155.5 145.7 132.2 72.1 71.8 19.6 30.1 29.7 22.7
39.1 55.7
δC
T. H. T. Nguyen et al.
Magn. Reson. Chem. (2014)
Structure elucidation of four new megastigmanes from Sonneratia ovata Backer confirm the basic structure as seen in Fig. 1. The NOESY cross peak between H-9 and H-10 serves as an internal standard. Cross peaks between H-9 and H-7 and between H-10 and H-8 are clearly observed, whereas the one between H-8 and H-9 is very weak. This is similar to the observations for compound 6. The fitting of the 13C chemical shifts and calculated 13C nuclear shieldings (R2 of rotamers: B′ = 0.9992, C′ = 0.9994, E′ = 0.9987, and F′ 0.9907, carbons included in correlation C-7–C-13) shows that either rotamer C′ or its enantiomer G′ are good candidates. As presented in the preceding text, 6 is a glycosylated derivative of 7, and the rotamer C is attributed for 6; therefore, C′ is proposed as the preferred conformation for compound 7. Compound 5 is a colorless oil. 1H-NMR and 13C-NMR spectra of 5 show signals of a megastigmane skeleton (Table 4). 5 differs from 1 by the presence of two hydroxyl groups at C-7 and C-8 instead of a double bond because the proton NMR spectrum reveals two hydroxyl protons with one at δ 4.19 (1H, d, 5.0 Hz, 7-OH), which has the coupling with proton H-7 and the second at δ 3.10 (1H, brs, 8-OH), which has the COSY correlation with proton H-8 (Fig. 2). Moreover, the proton NMR spectrum displays three oxygenated methine proton signals at δ 4.14 (1H, dd, 7.6, 5.0 Hz, H-7), 4.66 (1H, brdd, 7.6, 7.6 Hz, H-8), and 4.45 (1H, quint-like, 7.2 Hz, H-9), and only one olefinic proton signal at δ 5.85 (1H, s, H-4). These correspond to the presence of three oxygenated methine carbons at δ 84.6 (C-7), 77.0 (C-8), and 75.2 (C-9), an oxygenated quaternary carbon at δ 85.5 (C-6) and two olefinic carbons at δ 126.8 (–CH¼, C-4) and 166.8 (>C¼, C-5). The COSY spectrum shows the cross peaks of H-7/H-8/H-9/H-10 indicating the structure of a –CH(OH)–CH(OH)– CH(O)–CH3 moiety. The chemical shifts of C-6 and C-9 resonate at the higher frequency range at δ 85.5 (C-6) and 75.2 (C-9), compared
with the normal open chain ones at δ 68.8 and 69.2, respectively.[19] Therefore, 5 is suggested to have an ether linkage between C-6 and C-9. This is proved by a quasi molecular ion peak at m/z 239.1284 [M-H], (calcd for C13H10O4 -H, 239.1283). According to Takahashi and Nakagawa,[20] the stereochemistry of C-8, C-9 is determined by the analysis of the coupling constant of H-8/H-9. If J is smaller than 5.5 Hz, two protons H-8 and H-9 are in a trans configuration, otherwise, if J is bigger than 6.0 Hz, they are in a cis configuration. In compound 5, the cis configuration between H-8 and H-9 is assigned by their coupling constant of 7.2 Hz. Moreover, the NOESY spectrum (Fig. 3) reveals the cross peaks of H-8 with H-9, and of H-12 with both H-8 and H-9, which confirm the cis configuration of H-8 and H-9. A very weak NOE cross peak between H-7 and H-8 shows that these two protons are not on the same side. Additionally, NOESY correlations of H-7 with either H-13 or H-10 assign the trans configuration of H-7 and H-8. Based on all the aforementioned analysis, the structure of compound 5 is identified as 6,9-epoxy-7,8-dihydroxymegastigmane-4-ene-3one and is named sonnerstigmane C.
Experimental General experimental procedures The NMR spectra were measured on a Bruker Avance spectrometer, at 500 MHz for 1H-NMR and 125 MHz for 13C-NMR, or on a Bruker Avance spectrometer, at 400 MHz for 1H-NMR and 100 MHz for 13 C-NMR, or on a Varian spectrometer, at 300 MHz for 1H-NMR and 75 MHz for 13C-NMR. The HR-ESI-MS war recorded on a HR-ESI-MS MicroOTOF-Q mass spectrometer. Thin layer
Figure 3. NOESY correlations of 5, 6, and 7.
Table 4. Nuclear magnetic resonance spectral data of compound (5) (CDCl3) N° 1 2 3 4 5 6 7 7-OH 8 8-OH 9 10 11 12 13
δC (ppm) 41.2 49.5 200.5 126.8 166.8 85.5 84.6 77.0 75.2 17.7 26.3 26.3 19.8
Magn. Reson. Chem. (2014)
δH (ppm), J (Hz)
COSY
3.18 (1H, d, 18.0) 2.05 (1H, d, 18.0)
HMBC
NOESY
1, 3, 11, 12 1, 3, 4, 6, 11, 12
5.85 (1H, s)
13
2, 6, 13
4.14 (1H, dd, 7.6, 5.0) 4.19 (1H, d, 5.0) 4.66 (1H, br-dd, 7.6, 7.6) 3.10 (1H, brs) 4.45 (1H, quint-like, 7.2) 1.24 (3H, d, 6.8) 1.00 (3H, s) 1.19 (3H, s) 1.97 (3H, s)
8
1, 5, 6, 8
13 10
7, 9, 8-OH 8 8, 10
7, 10
7 (w), 9, 12
7 8, 9 1, 2, 6, 12 1, 2, 3, 6, 11 4, 5, 6
8, 10, 12
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8, 9 7
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T. H. T. Nguyen et al. chromatography was carried out on silica gel 60 F254 or silica gel 60 RP-18 F254S (Merck), and spots were visualized by spraying with 20% H2SO4 solution followed by heating. Gravity column chromatography was performed with silica gel 60 (0.040–0.063 mm, Himedia), or with silica gel 60 RP-18 (0.040–0.063 mm, Himedia). All NMR experiments were acquired at ambient temperature. Chemical shifts are expressed in ppm with reference to the residual protonated solvent signals (chloroform-d with δH 7.260 and δC 77.160 and methanol-d4 with δH 3.310 and δC 49.000) or the internal TMS (0.000). On a 400 Bruker Avance spectrometer, 1H spectra spectrometer spectral width (SW) 6393 Hz, number of data points (NP) 32 K, acquisition time (AQ) 2.5 s, pulse width (PW) 10.9 μs, relaxation delay (RD) 2 s, number of scans (NS) 1, line broadening (LB) 0.3 Hz. 13 C spectra: SW 26785 Hz, NP 64 K, AQ 1.2 s, PW 9 μs, RD 1.5 s, NS 1024, LB 1.0 Hz. HSQC spectra were carried out using the Bruker pulse sequence hsqcedetgp: AQ 0.17 s, RD 1.4 s, PW 13C 9.0 μs, PW 1 H 10.9 μs, SW 1H 3067 Hz; SW 13C 180 ppm, NP 1024, NI 256, FT size 1024 × 1024. The experiment was optimized for a 1JCH of 145 Hz and run in the echo-antiecho mode. HMBC spectrum was carried out using the Bruker pulse sequence hmbcgplpndqf: AQ 0.17 s, RD 1.1 s, PW 13C 9.0 μs, PW 1H 10.9 μs, SW 1H 3067 Hz; SW 13C 250 ppm, NP 1024, NI 256, FT size 2048 × 1024, nJCH optimized for 8.0 Hz. COSY: AQ 0.33 s, NS 6, PW 10.9 μs, SW 3067 Hz, NI 256, NP 2048, FT size 1024 × 1024. On a 500 Bruker Avance spectrometer, 1H spectra: SW 10330 Hz, NP 128 K, AQ 6.3 s, PW 11.5 μs, RD 3 s, NS 1, LB 0.3 Hz. 13C spectra: SW 32894 Hz, NP 64 K, AQ 0.99 s, PW 9 μs, RD 1.5 s, NS 509, LB 1.5 Hz. HSQC spectra were carried out using the Bruker pulse sequence hsqcedetgp: AQ 0.057 s, RD 1.5 s, PW 13C 9.7 μs, PW 1H 11.6 μs, SW 1H 9014 Hz; SW 13C 260 ppm, NP 1024, NI 256, FT size 1024 × 1024. The experiment was optimized for a 1JCH of 145 Hz and run in the echo-antiecho mode. HMBC spectrum was carried out using the Bruker pulse sequence hmbcgplpndqf: AQ 0.23 s, RD 1.0 s, PW 13C 9.9 μs, PW 1H 11.6 μs, SW 1H 9014 Hz; SW 13C 260 ppm, NP 4096, NI 128, FT size 2048 × 1024, nJCH optimized for 8.0 Hz. COSY: AQ 0.15 s, PW 11.6 μs, SW 6684 Hz, NI 128, NP 2048, FT size 1024 × 1024. NOESY: AQ 0.17 s, PW 11.6 μs, SW 6000.6 Hz, NI 512, NP 2048, FT size 1024 × 1024.
(column: 50 × 6 cm) eluting with chloroform – methanol – water (20:1:0, 9:1:0, 20:6:1, 14:6:1) to afford ten sub-fractions (A8.1 to A8.10). Sub-fraction A8.4 (1.3 g) was applied on RP-18 silica gel CC and eluted with water-methanol (8:2, 7:3, 1:1, 0:1) to obtain 6 (25 mg). The fraction A7 (24 g) was chromatographed on silica gel (column: 50 × 6 cm) eluting with chloroform – methanol – water (20:1:0, 9:1:0, 20:6:1, 14:6:1) to afford ten sub-fractions (A7.1 to A7.10). Sub-fraction A7.8 (2.2 g) was applied on RP-18 silica gel CC and eluted with water-methanol (9:2, 8:2, 1:1) to obtain 3 (16 mg). Sub-fraction A7.7 (2.8 g) was subjected to reverse-phase RP-18 silica gel CC, eluting with water-methanol (20:1, 9:1, 4:1, 1:1) to afford 2 (340 mg). The same manner applied on sub-fraction A7.6 (1.8 g) to yield 8 (6 mg). The fraction A4 (8.4 g) was subjected to silica gel CC, eluting with dichloromethane-methanol (95:5, 85:15) and then purified by RP-18 silica gel CC, eluted with water:methanol (6:4) to afford 5 (8.6 mg) and 7 (32.8 mg). The fraction A3 (2.9 g) was applied on RP-18 silica gel CC and eluted with water-methanol (6:4, 5:5, 2:8) to obtain 4 (25 mg) and 9 (5 mg). The fraction A2 (1.2 g) was applied on RP-18 silica gel CC, eluted with water:methanol (5:5) to obtain a mixture of 1a and 1b (23 mg).
Plant material
Sonnerstigmane C (5)
Leaves of S. ovata Backer were collected at Can Gio mangrove forest in Ho Chi Minh city, Vietnam in March 2010. The scientific name of the plant was authenticated by pharmacist Phan Duc Binh. A voucher specimen (No US-B006) was deposited in the herbarium of the Department of Organic Chemistry, VNUHCM – University of Science.
Colorless oil. ½α25 D 41.1 (c 0.35 MeOH). HR-ESI-MS m/z 239.1284 [M-H]-, (Calcd for C13H10O4 –H, 239.1283). 1H-NMR, 13C-NMR (CDCl3, 400 MHz for 1H and 100 MHz for 13C) data (Table 4). COSY and HMBC data (Fig. 2). NOESY data (Fig. 3).
Extraction and isolation
Colorless oil. ½α25 D + 2.3 (c 0.48 MeOH). HR-ESI-MS m/z 409.1858 [M + Na]+, (Calcd for C19H30O8Na, 409.1838). 1H-NMR, 13C-NMR (CD3OD, 500 MHz for 1H and 125 MHz for 13C) data (Table 3). COSY and HMBC data (Fig. 2). NOESY data (Fig. 3).
The air-dried powder of leaves (10.9 kg) was macerated with methanol (50 L × 3) at room temperature, and after filtration, the methanolic solution was concentrated at reduced pressure to yield a residue of 1.5 kg. This crude extract was suspended in water with 10% of methanol and was partitioned first with petroleum ether (60–80 °C) and then with ethyl acetate. After evaporation at the reduced pressure, three extracts were obtained: petroleum ether (245 g), ethyl acetate (390 g), and aqueous layer (865 g). The ethyl acetate extract was chromatographed on the silica gel CC (column: 120 × 6 cm) eluting with petroleum ether – ethyl acetate (1:4, 0:1) and then ethyl acetate – methanol (9:1, 4:1, 1:1, 0:1) to give fractions A1 to A9. The fraction A8 (30 g) was chromatographed on silica gel
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Mixture of sonnerstigmane A (1a) and sonnerstigmane B (1a)
Pale yellow oil. ½α25 D + 158.3 (c 1.2 MeOH). HR-ESI-MS m/z 261.1479 [M + Na]+ (Calcd for C14H22O3Na, 261.1467). 1H-NMR, 13C-NMR (CDCl3, 400 MHz for 1H and 100 MHz for 13C) data (Tables 1 and 2). COSY and HMBC data (Fig. 2). (6S,9R)-Roseoside (2)
Pale yellow oily wax. 1H-NMR, 13C-NMR (CDCl3, 300 MHz for 1H and 75 MHz for 13C) data (Tables 1 and 2). (6S,9R)-Ionone 9-O-(6-O-galloyl)-β-D-glucopyranoside (3) 1 13 Yellowish oil. ½α25 D + 340.0 (c 0.47 MeOH). H-NMR, C-NMR (CDCl3, 1 13 300 MHz for H and 75 MHz for C) data (Tables 1 and 2).
(S)-Dehydrovomifoliol (4) 1 13 Colorless oil. ½α25 C-NMR (CDCl3, D + 209.0 (c 1.3 MeOH). H-NMR, 400 MHz for 1H and 100 MHz for 13C) data (Tables 1 and 2).
Sonnerstigmane D (6)
8,9-Dihydromegastigmane-4,6-diene-3-one (7) 1 13 Pale yellow oil. ½α25 D + 54.0 (c 1.52 MeOH). H-NMR, C-NMR (CDCl3, 1 13 400 MHz for H and 100 MHz for C) data (Table 3). NOESY data (Fig. 3).
5,8-Epoxymegastigmane-6-ene-3-one 9-O-β-D-glucopyranoside (8)
Yellowish oil. 1H-NMR, 75 MHz for 13C).
Copyright © 2014 John Wiley & Sons, Ltd.
13
C-NMR (CD3OD, 300 MHz for 1H and
Magn. Reson. Chem. (2014)
Structure elucidation of four new megastigmanes from Sonneratia ovata Backer
Figure 4. Rotamers of compound 6 (The replacement of O-glucopyranosyl group by hydroxyl groups gives the rotamers A′, B′, C′, D′, E′, F′, G′, H′, I′, J′, and K′, respectively, of compound 7). Dehydrololiolide (9) 1 Colorless oil. ½α25 D + 3.6 (c 0.25 MeOH). H-NMR, 300 MHz for 1H and 75 MHz for 13C).
13
C-NMR (CDCl3,
Theoretical calculations The DFT calculations were performed with the Gaussian 09 package,[21] and the molecular geometries were fully optimized using the B3LYP variant of the DFT [22,23] with the 6-31G (d) basis set. The NMR nuclear shieldings were calculated with the same level of theory and basis set using the GIAO method.[24,25] Correlation coefficients between observed 13C chemical shifts and calculated 13 C nuclear shielding are shown in Fig. 4.
(S)-dehydrovomifoliol (4), 8,9-dihydromegastigmane-4,6-diene-3-one (7), 5,8-epoxymegastigmane-6-ene-3-one 9-O-β-D-glucopyranoside (8), and dehydrololiolide (9). Their chemical structures are established primarily by NMR and MS spectroscopy. 1a, 1b, 5, and 6 are new compounds and 2, 3, 4, 7, 8, and 9 have not been isolated from Sonneratia genus before. The presence of a glucose molecule in 6 allows a discrimination based in the present case on the proximity between H-1′and H-9. The DFT calculations not only provide 13C nuclear shieldings but also rotamers’ energies. Furthermore, calculated energies for rotamers are so different that also a qualified suggestion can be made regarding the populations. Furthermore, the comparison of correlation coefficients for least square between observed 13C chemical shifts and calculated 13C nuclear shielding may also be used to assign or leave out rotamers.
Conclusions From the leaves of S. ovata, ten compounds are isolated, including a mixture of sonnerstigmane A (1a), sonnerstigmane B (1a), sonnerstigmane C (5), sonnerstigmane D (6), (6S,9R)-roseoside (2), (6S,9R)-ionone 9-O-(6-O-galloyl)-β-D-glucopyranoside (3),
Magn. Reson. Chem. (2014)
Acknowledgements This research was supported by the Department of Science and Technology – HCM city, grant #1058/QĐ-SKHCN. N.T.H.T would like to thank Vietnamese government for a scholarship at Roskilde
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T. H. T. Nguyen et al. University under the 322 projects, to thank Chulalongkorn University for a scholarship at Chulalongkorn University under the One Semester Scholarships Program for ASEAN Countries.
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[16] H. Tazaki, N. Ohta, K. Nabeta, H. Okuyama, M. Okumura. Phytochemistry 1993, 34, 1067–1070. [17] Q. Zeng, J. Ye, J. Ren, X. R. Cheng, J. J. Qin, H. Z. Jin, W. D. Zhang. Chem. Nat. Compd. 2013, 49, 486–492. [18] P. Winterhalter, R. L. Rouseff. In carotenoid-derived aroma compounds; ACS Symposium Series; American Chemical Society: Washington, DC, 2001, 1–17. [19] L. Luo, R. Wang, X. Wang, Z. Ma, N. Li. Food Chem. 2012, 131, 992–998. [20] K. Takahashi, T. Nakagawa. Chem. Pharm. Bull. 1966, 14, 641–647. [21] M. J. Frisch, G. W. Trucks, H. B. Schlegel, G. E. Scuseria, M. A. Robb, J. R. Cheeseman, G. Scalmani, V. Barone, B. Mennucci, G. A. Petersson, H. Nakatsuji, M. Caricato, X. Li, H. P. Hratchian, A. F. Izmaylov, J. Bloino, G. Zheng, J. L. Sonnenberg, M. Hada, M. Ehara, K. Toyota, R. Fukuda, J. Hasegawa, M. Ishida, T. Nakajima, Y. Honda, O. Kitao, H. Nakai, T. Vreven, J. A. Montgomery Jr., J. E. Peralta, F. Ogliaro, M. Bearpark, J. J. Heyd, E. Brothers, K. N. Kudin, V. N. Staroverov, R. Kobayashi, J. Normand, K. Raghavachari, A. Rendell, J. C. Burant, S. S. Iyengar, J. Tomasi, M. Cossi, N. Rega, J. M. Millam, M. Klene, J. E. Knox, J. B. Cross, V. Bakken, C. Adamo, J. Jaramillo, R. Gomperts, R. E. Stratmann, O. Yazyev, A. J. Austin, R. Cammi, C. Pomelli, J. W. Ochterski, R. L. Martin, K. Morokuma, V. G. Zakrzewski, G. A. Voth, P. Salvador, J. J. Dannenberg, S. Dapprich, A. D. Daniels, Ö. Farkas, J. B. Foresman, J. V. Ortiz, J. Cioslowski, D. J. Fox, Gaussian 09, Revision A.1, Gaussian, Inc., Wallingford CT, 2009. [22] A. D. Becke. J. Chem. Phys. 1993, 98, 5648–5652. [23] C. Lee, W. Yang, R. G. Parr. Phys. Rev. B. 1988, 37, 785–789. [24] R. Ditchfield. Mol. Phys. 1974, 27, 789–807. [25] K. Wolinski, J. F. Hinton, P. Pulay. J. Am. Chem. Soc. 1990, 112, 8251–8260.
Supporting Information Additional supporting information may be found in the online version of this article at the publisher’s website.
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Magn. Reson. Chem. (2014)
Revised: 26 July 2014
Accepted: 30 July 2014
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI 10.1002/mrc.4132
Structure elucidation of four new megastigmanes from Sonneratia ovata Backer Thi Hoai Thu Nguyen,a Nguyen Kim Tuyen Pham,b Khanitha Pudhom,c Poul Erik Hansend and Kim Phi Phung Nguyene* Introduction For a long time, fruits, bark, and leaves of some Sonneratia species have been used in folk medicine for the treatment of different diseases such as asthma, febrifuge, ulcers, hepatitis, piles, sprain, and hemorrhages.[1] Moreover, these species of the Sonneratia genus growing widely in the mangrove forests in tropical areas possess a huge biomass. Therefore, this genus is attractive for pharmacological and chemical studies. Previously, pharmaceutical investigations on Sonneratia genus showed antidiabetic,[2] antioxidant, antibacterial, [3] and anti-tumor properties.[4] Chemical studies are reported on S. alba,[5] S. caseolaris,[6,7] S. apeltala,[8] S. hainanensis,[4,9] S. paracaseolaris [10] to obtain diverse types of compounds. In previous chemical studies on Sonneratia ovata, a number of compounds were isolated.[6,11] The chemical investigation on the leaves of S. ovata using efficient separation techniques leads to the isolation of four new megastigmanes together with six known megastigmane ones. Enantiomers cannot be directly distinguished by nuclear magnetic resonance (NMR). However, the presence of a glucose moiety may establish diastereomers. The use of an intrinsic glucose molecule to determine the absolute configuration based on NMR parameters and density functional theory (DFT) calculations is investigated.
Results and Discussion The methanolic crude extract from the dried leaves of S. ovata was partitioned with petroleum ether and subsequently with ethyl acetate to give petroleum ether and ethyl acetate extracts. The ethyl acetate extract was applied to efficient separation and purification techniques to afford four new megastigmane compounds, sonnerstigmane A (1a), sonnerstigmane B (1b), sonnerstigmane C (5), and sonnerstigmane D (6), together with six known megastigmanes (as shown in Fig. 1). The structures of these known compounds are determined by analyses of their NMR spectral data, as well as comparing their data with those in the literature. They are (6S,9R)-roseoside (2),[12] (6S,9R)-ionone 9-O-(6-O-galloyl)-β-D-glucopyranoside (3),[13] (S)-dehydrovomifoliol (4),[14] 8,9-dihydromegastigmane-4,6diene-3-one (7),[15] 5,8-epoxymegastigmane-6-ene-3-one 9-O-β-Dglucopyranoside (8),[16] and dehydrololiolide (9).[17] Norisoprenoids or megastigmanes are known in many kinds of species; however, these are now reported in Sonneratia genus for the first time. They are considered to be very important flavor and fragrance precursors in tea, tobacco, fruits, essential oils,
Magn. Reson. Chem. (2014)
vegetables, spices, and wine.[18] The isolated megastigmanes constitute an interesting case as structure with 6 is a glycosylated derivative of 7. Compound 1–4 possess the 6,9-dihydroxymegastigmane-4,7diene-3-one skeleton. This moiety can be recognized basically by the presence of two singlet gem-methyl signals at around 1.0 ppm of (H3C)2C < moiety (H-11 and H-12), a doublet methyl signal at about 1.2 ppm of H3C-CH-O (H-10), a singlet proton signal at around 1.9 ppm of a methyl group (H-13) attached to a double bond and two doublet gem-proton signals with a large coupling constant of 17.0 Hz of H-2a and H-2b at δH from 2.1 to 2.5 ppm. At the high frequency range, the proton spectrum shows three olefinic proton signals at δH from 5.7 to 5.9 ppm of two double bonds C4 = C5 and C7 = C8, in which C-5 is a quaternary carbon. Additionally, the 13C spectrum shows carbon signals of a conjugated carbonyl group at round δC 197 (>C¼O, C-3), 162 (>C¼, C-5), and 127 (–CH¼, C-4) in CDCl3. These two first signals are shifted to lower field at 201 (C-3) and 167 (C-5) by the nucleophilic effect of methanol on a carbonyl carbon of an enone system when recording NMR spectra in CD3OD. Moreover, the 13C spectrum shows two olefinic carbon signals of a remaining isolated double bond at around δC 135 and 131. An oxygenated quaternary carbon and an oxygenated methine carbon, a methylene carbon, a quaternary carbon, and four methyl carbons are also observed at round δC 80 (C-6), 77 (C-9), 50 (C-2), 42 (C-1), 24 and 23 (C-11 and C-12), 21 (C-10), and 19 (C-13), respectively (Table 2). Compound 1 is obtained as a pale yellow oil. The analysis of the 1H-NMR and 13C-NMR data of 1 shows that it may possess a 6,9-dihydroxymegastigmane-4,7-diene-3-one skeleton as mentioned earlier (Tables 1 and 2). The skeleton of 1 is confirmed
* Correspondence to: Kim Phi Phung Nguyen, Department of Organic Chemistry, VNUHCM – University of Science, 227 Nguyen Van Cu Str., Dist. 5, Ho Chi Minh City, Vietnam. E-mail: [email protected] a Department of Basic Science, University of Medicine and Pharmacy, Ho Chi Minh City, Vietnam b Department of Environmental Science, Saigon University, Ho Chi Minh City, Vietnam c Department of Chemistry, Faculty of Science, Chulalongkorn University, Bangkok, Thailand d Department of Science, Systems and Models, Roskilde University, Roskilde, Denmark e Department of Organic Chemistry, VNUHCM – University of Science, Ho Chi Minh City, Vietnam
Copyright © 2014 John Wiley & Sons, Ltd.
T. H. T. Nguyen et al.
Figure 1. Structures of compounds isolated from the leaves of Sonneratia ovata.
Table 1.
1
H-NMR data of 1a, 1b, 2, 3, and 4, δH (ppm), J (Hz)
N°
1aa
1ba
2m
3m
4a
2
2.26 (d, 16.8) 2.41 (d, 16.8) 5.89 (s) 5.73 (d, 15.6) 5.72 (dd, 15.6, 5.2) 3.80 (quint-like, 6.0) 1.25 (d, 6.0) 1.07 (s) 0.99 (s) 1.90 (s)
2.26 (d, 16.8) 2.41 (d, 16.8) 5.89 (s) 5.73 (d, 15.6) 5.70 (dd, 15.6, 5.2) 3.80 (quint-like, 6.0) 1.23 (d, 6.0) 1.07 (s) 1.01 (s) 1.88 (s)
2.17 (d, 17.0) 2.49 (d, 17.0) 5.87 (m) 5.87 (m) 5.87 (m) 4.42 (m) 1.30 (d, 6.5) 1.03 (s) 1.03 (s) 1.92 (d, 1.5) 4.35 (d, 8.0) 3.17 (dd, 9.0, 8.0) 3.34 (m) 3.25 (m) 3.23 (m) 3.63 (dd, 12.0, 5.5) 3.83 (dd, 11.5, 1.5)
2.11 (d, 17.0) 2.40 (d, 17.0) 5.81 (d, 2.5) 5.80 (d, 15.0) 5.81 (dd, 15.0, 5.5) 4.38 (m) 1.29 (d, 6.0) 0.99 (s) 0.95 (s) 1.84 (d, 1.0) 4.40 (d, 7.5) 3.22 (dd, 9.0, 8.0) 3.40 (dd, 9.0, 9.0) 3.44 (dd, 9.0, 9.0) 3.51 (ddd, 9.0, 5.0, 2.0) 4.37 (dd, 12.0, 5.0) 4.47 (dd, 12.0, 2.0) 7.08 (s)
2.45 (d, 17.2) 2.34 (d, 17.2) 5.93 (s) 6.84 (d, 15.6) 6.47 (d, 15.6
3.24 (1H, s)
3.26 (1H, s)
4 7 8 9 10 11 12 13 1′ 2′ 3′ 4′ 5′ 6′ 2″, 6″ -OCH3
2.28 (s) 1.09 (s) 1.01 (s) 1.87 (s)
a
Chloroform-d Methanol-d4
m
by the COSY and Heteronuclear Multiple-Bond Correlation (HMBC) correlations (Fig. 2). The E configuration of the double bond C7 = C8 is determined by the large three-bond H–H coupling of 15.6 Hz. Besides that, 1 has a methoxy group’s signals at δH 3.24 (3H, s, –OCH3) and δC 56.3 (–OCH3). The methoxy group attached to the megastigmane skeleton at C-9 is determined via the HMBC correlation of the methoxy proton with carbon C-9. The 1H-NMR and 13C-NMR spectra of 1 display pairs of signals with a ratio of about 1.2:1.0 corresponding to two stereoisomers of 9-methoxyvomifoliol. According to Yamano and Ito,[12] specific rotations of (6S,9R) and (6S,9S) isomers of vomifoliol are
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dextrorotatory, and of (6R,9S) and (6R,9R), isomers are levorotatory. Compound 1 differs from vomifoliol only by a methoxy group at C-9 and possesses a positive optical rotation. Therefore, 1 is suggested to be a mixture of (6S,9R) and (6S,9S) isomers with a ratio of about 1.2:1.0. The high resolution electrospray ionisation mass spectroscopy (HR-ESI-MS) showed a pseudo molecular ion peak at m/z 261.1479, calcd for [C14H22O3 + Na]+, 261.1467. All assignments suggest that the new compound 1 is a mixture with the ratio (1.2:1.0) of sonnerstigmane A [(6S,7E,9R)-6-hydroxy-9-methoxymegastigmane4,7-diene-3-one (1a)] and sonnerstigmane B [(6S,7E,9S)-6-hydroxy9-methoxymegastigmane-4,7-diene-3-one (1b)].
Copyright © 2014 John Wiley & Sons, Ltd.
Magn. Reson. Chem. (2014)
Structure elucidation of four new megastigmanes from Sonneratia ovata Backer Table 2.
13
C-NMR data of 1a, 1b, 2, 3, and 4 1aa
1ba
2m
3m
4a
41.3 49.9 198.0 127.0 162.8 79.2 131.3 133.8 77.5 21.5 23.0 24.2 19.0
41.2 49.9 198.0 127.1 162.7 79.3 131.2 133.9 77.4 21.5 23.0 24.3 19.0
42.4 50.7 201.2 127.1 167.2 80.0 131.5 135.2 77.3 21.2 23.4 24.7 19.5 102.7 75.2 78.1 71.6 78.0 62.8
42.4 50.7 201.4 127.2 167.1 80.0 131.6 134.9 77.1 21.2 23.4 24.6 19.7 103.1 75.2 78.0 71.5 75.4 64.7 121.4 110.3 146.5 139.9 168.4
41.6 49.7 197.3 127.8 160.9 79.3 145.4 130.5 197.7 28.4 23.1 24.5 18.8
56.3
56.3
N° 1 2 3 4 5 6 7 8 9 10 11 12 13 1′ 2′ 3′ 4′ 5′ 6′ 1″ 2″, 6″ 3″, 5″ 4″ –COO– –OCH3 a
Chloroform-d Methanol-d4
m
Compound 6 is obtained as a colorless oil. 1H-NMR and 13C-NMR data of 6 prove that it possesses the same megastigmane skeleton as 1 (Table 3). However compound 6 differs from 1 by the position of the double bond which is at C6 = C7 and the presence of one more hydroxyl group at C-8 at δ 4.75 (1H, dd, 10.0, 5.0 Hz, H-8) and one less olefinic proton signal. Besides that, the 13C spectrum of 6 displays an oxygenated methine carbon at higher magnetic field at δ 72.3 (C-8), instead of an oxygenated quaternary carbon signal at around 80 ppm of C-6 in 1. These are confirmed by the COSY correlations of the olefinic proton H-7 at δ 6.09 (1H, d, 10.0 Hz) with the carbinol proton H-8 at δ 4.75 (1H, dd, 10.0, 5.0 Hz); of H-8 with the carbinol proton H-9 at δ 3.83 (1H, m); and of H-9 with the methyl protons at δ 1.30 (3H, d, 6.5 Hz, H-10) (Fig. 2). Additionally, the HMBC spectrum reveals the correlations of H-10 with two oxygenated carbons at δ 72.3 (C-8) and 82.1 (C-9), of H-13 at δ 2.15 (3H, s) with olefinic carbons at δ 127.3 (C-4), 158.9 (C-5), and 145.2 (C-6) (Fig. 2). The 1H-NMR spectrum also displays an anomeric proton signal at δ 4.46 (1H, d, 7.5 Hz, H-1′) and other oxygenated protons from
3.26–3.86 ppm of one β-glucose unit, which are matched with the observation of an anomeric carbon at δ 105.7 and five oxygenated carbons at δ from 62.7 to 78.0. The attachment of the glucose moiety to the aglycone at C-9 is confirmed via the HMBC correlation between the anomeric proton H-1′ and C-9. The NOESY spectrum reveals the proximity in space between the olefinic proton H-7 and methyl protons H-13; therefore, the configuration of the double bond C6 = C7 is E. The following analysis is based on H,H coupling constants, NOESY cross peak intensities and calculated 13C nuclear shieldings as well as rotamers’ calculated energies. The aim is to determine the absolute configurations for oxygenated carbons 8 and 9 by using the glucose moiety as an asymmetric discriminator. The glycosidic linkage is β orientation as judged from 3JH1′,H2′ value of 7.5 Hz. In the HMBC spectrum, 3J (H-1′, C-9) and 3J (H-9, C-1′) show intense cross peaks and in NOE experiment, H-9 and H-1′ also show strong interaction. These findings show that these two protons must be proximate in space. Additionally, the strong NOESY cross peaks between H-8 and the methyl protons H-11 and H-12 show that H-8 is primarily directed toward these methyl groups. Therefore, the rotamers of 6 are suggested as shown in Fig. 4. In order to assign the configuration of chiral carbons of these rotamers, the following criteria are used: A strong NOESY cross between H-8 and CH3-10 shows that the gauche rotamers A, D, I and L are not suitable. A second very important finding is the absence of a NOESY cross peak between H-8 and H-9 showing that these two protons are not in the gauche conformations and therefore rotamers B, H, E, and K are excluded. This leaves only C, F, G, and J as the possibilities. J and G can be excluded because of the small value of correlation coefficient (R2) between the observed 13C chemical shifts and the calculated nuclear shieldings (Fig. 4) (In these rotamers, carbons C-7–C-10, C-1′, CH3-11, and CH3-12 are put in the consideration because they are most likely influenced by the conformations and configurations). Results from weak to medium of NOESY cross peaks between H-7 and H-9 as well as weak cross peaks in the HMBC spectrum between H-9 and C-7 and H-8 and C-10 show that both rotamers C and F are good candidates. The distinction between these two rotamers lies in their energies. The calculated energy of F is 23 KJ higher than that of E-rotamer therefore F-rotamer is not highly populated and is excluded. In contrast, C is favored by 21 KJ over B. These are also suitable with the two bulky substituents of C, glucose and quinone moieties, are far apart in space. Up to this point, rotamer C is the most suitable. All data in the preceding text suggest that 6 is (7E,8R,9R)-8-hydroxymegastigmane-4,6-diene-3one 9-O-β-D-glucopyranoside and is named sonnerstigmane D. Compound 7 has been reported earlier with a gross structure without stereochemical assignments.[15] The NOESY cross peaks and the H,H coupling constants combined with DFT calculations can shed some light on this problem in analogy with what is described above for compound 6. The NOESY correlations (Fig. 3)
Figure 2. Heteronuclear Multiple-Bond Correlation (arrows point from H → C) and COSY correlations of 1a, 1b, 5, and 6.
Magn. Reson. Chem. (2014)
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Copyright © 2014 John Wiley & Sons, Ltd.
m
6.09 (1H, d, 10.0) 4.75 (1H, dd, 10.0, 5.0) 3.83 (1H, m) 1.30 (3H, d, 6.5) 1.39 (3H, s) 1.33 (3H, s) 2.15 (3H, s) 4.46 (1H, d, 7.5) 3.26 (1H, m) 3.37 (1H, m) 3.29 (1H, m) 3.27 (1H, m) 3.68 (1H, dd, 12.0, 5.0) 3.88 (1H, m)
5.98 (1H, s)
2.37 (1H, d, 16.0) 2.40 (1H, d, 16.0)
δH (ppm), J (Hz)
13
39.9 54.3
δC
202.0 127.3 158.9 145.2 134.7 72.3 82.1 19.3 29.8 29.9 22.8 105.7 75.5 78.0 71.5 77.9 62.7
H-NMR and C-NMR data of 6 and 7
1
Chloroform-d Methanol-d4
a
8-OH 9-OH
3 4 5 6 7 8 9 10 11 12 13 1′ 2′ 3′ 4′ 5′ 6′
1 2
N°
Table 3.
4′, 5′ 4′
5′
2′
1, 5, 6, 8, 9, 11, 12 6, 7, 9, 10 7, 10, 1′ 8, 9 1, 2, 6, 12, 3 1, 2, 6, 11, 3 4, 5, 6 9, 1′
2, 6, 13
1, 3, 4, 6, 11, 12
HMBC
8 7, 9 8, 10
13
COSY
6m
9, 13 10, 11, 12 7, 10, 1′ 8, 9 8 8 4, 7 9
13
11, 12
NOESY
5.87 (1H, d, 10.0) 4.67 (1H, dd, 10.0, 6.3) 3.71 (1H, m) 1.24 (3H, d, 6.0) 1.38 (3H, s) 1.29 (3H, s) 2.09 (3H, s) 4.43 (1H, d, 8.0) 3.24–3.39 (m) 3.24–3.39 (m) 3.24–3.39 (m) 3.24–3.39 (m) 3.73 (1H, dd, 12.0, 4.5) 3.80 (1H, dd, 12.0, 2.5)
5.95 (1H, s)
2.31 (1H, d, 16.0 2.38 (1H, d, 16.0)
δH (ppm)
m+a
6
201.1 127.2 157.7 145.2 133.3 71.9 82.3 19.2 29.7 29.9 22.8 105.2 74.7 77.2 70.8 77.0 62.2
39.5 53.9
δC
3.03 (1H,brs) 2.96 (1H, brs)
5.83 (1H, d, 9.6) 4.57 (1H, t-like, 7.6) 3.69 (1H, quint-like, 6.4) 1.18 (3H, d, 6.4) 1.38 (3H, s) 1.29 (3H, s) 2.06 (3H, s)
5.95 (1H, s)
2.32 (1H, d, 16.0) 2.38 (1H, d, 16.0)
δH (ppm), J (Hz)
7a
199.3 127.4 155.5 145.7 132.2 72.1 71.8 19.6 30.1 29.7 22.7
39.1 55.7
δC
T. H. T. Nguyen et al.
Magn. Reson. Chem. (2014)
Structure elucidation of four new megastigmanes from Sonneratia ovata Backer confirm the basic structure as seen in Fig. 1. The NOESY cross peak between H-9 and H-10 serves as an internal standard. Cross peaks between H-9 and H-7 and between H-10 and H-8 are clearly observed, whereas the one between H-8 and H-9 is very weak. This is similar to the observations for compound 6. The fitting of the 13C chemical shifts and calculated 13C nuclear shieldings (R2 of rotamers: B′ = 0.9992, C′ = 0.9994, E′ = 0.9987, and F′ 0.9907, carbons included in correlation C-7–C-13) shows that either rotamer C′ or its enantiomer G′ are good candidates. As presented in the preceding text, 6 is a glycosylated derivative of 7, and the rotamer C is attributed for 6; therefore, C′ is proposed as the preferred conformation for compound 7. Compound 5 is a colorless oil. 1H-NMR and 13C-NMR spectra of 5 show signals of a megastigmane skeleton (Table 4). 5 differs from 1 by the presence of two hydroxyl groups at C-7 and C-8 instead of a double bond because the proton NMR spectrum reveals two hydroxyl protons with one at δ 4.19 (1H, d, 5.0 Hz, 7-OH), which has the coupling with proton H-7 and the second at δ 3.10 (1H, brs, 8-OH), which has the COSY correlation with proton H-8 (Fig. 2). Moreover, the proton NMR spectrum displays three oxygenated methine proton signals at δ 4.14 (1H, dd, 7.6, 5.0 Hz, H-7), 4.66 (1H, brdd, 7.6, 7.6 Hz, H-8), and 4.45 (1H, quint-like, 7.2 Hz, H-9), and only one olefinic proton signal at δ 5.85 (1H, s, H-4). These correspond to the presence of three oxygenated methine carbons at δ 84.6 (C-7), 77.0 (C-8), and 75.2 (C-9), an oxygenated quaternary carbon at δ 85.5 (C-6) and two olefinic carbons at δ 126.8 (–CH¼, C-4) and 166.8 (>C¼, C-5). The COSY spectrum shows the cross peaks of H-7/H-8/H-9/H-10 indicating the structure of a –CH(OH)–CH(OH)– CH(O)–CH3 moiety. The chemical shifts of C-6 and C-9 resonate at the higher frequency range at δ 85.5 (C-6) and 75.2 (C-9), compared
with the normal open chain ones at δ 68.8 and 69.2, respectively.[19] Therefore, 5 is suggested to have an ether linkage between C-6 and C-9. This is proved by a quasi molecular ion peak at m/z 239.1284 [M-H], (calcd for C13H10O4 -H, 239.1283). According to Takahashi and Nakagawa,[20] the stereochemistry of C-8, C-9 is determined by the analysis of the coupling constant of H-8/H-9. If J is smaller than 5.5 Hz, two protons H-8 and H-9 are in a trans configuration, otherwise, if J is bigger than 6.0 Hz, they are in a cis configuration. In compound 5, the cis configuration between H-8 and H-9 is assigned by their coupling constant of 7.2 Hz. Moreover, the NOESY spectrum (Fig. 3) reveals the cross peaks of H-8 with H-9, and of H-12 with both H-8 and H-9, which confirm the cis configuration of H-8 and H-9. A very weak NOE cross peak between H-7 and H-8 shows that these two protons are not on the same side. Additionally, NOESY correlations of H-7 with either H-13 or H-10 assign the trans configuration of H-7 and H-8. Based on all the aforementioned analysis, the structure of compound 5 is identified as 6,9-epoxy-7,8-dihydroxymegastigmane-4-ene-3one and is named sonnerstigmane C.
Experimental General experimental procedures The NMR spectra were measured on a Bruker Avance spectrometer, at 500 MHz for 1H-NMR and 125 MHz for 13C-NMR, or on a Bruker Avance spectrometer, at 400 MHz for 1H-NMR and 100 MHz for 13 C-NMR, or on a Varian spectrometer, at 300 MHz for 1H-NMR and 75 MHz for 13C-NMR. The HR-ESI-MS war recorded on a HR-ESI-MS MicroOTOF-Q mass spectrometer. Thin layer
Figure 3. NOESY correlations of 5, 6, and 7.
Table 4. Nuclear magnetic resonance spectral data of compound (5) (CDCl3) N° 1 2 3 4 5 6 7 7-OH 8 8-OH 9 10 11 12 13
δC (ppm) 41.2 49.5 200.5 126.8 166.8 85.5 84.6 77.0 75.2 17.7 26.3 26.3 19.8
Magn. Reson. Chem. (2014)
δH (ppm), J (Hz)
COSY
3.18 (1H, d, 18.0) 2.05 (1H, d, 18.0)
HMBC
NOESY
1, 3, 11, 12 1, 3, 4, 6, 11, 12
5.85 (1H, s)
13
2, 6, 13
4.14 (1H, dd, 7.6, 5.0) 4.19 (1H, d, 5.0) 4.66 (1H, br-dd, 7.6, 7.6) 3.10 (1H, brs) 4.45 (1H, quint-like, 7.2) 1.24 (3H, d, 6.8) 1.00 (3H, s) 1.19 (3H, s) 1.97 (3H, s)
8
1, 5, 6, 8
13 10
7, 9, 8-OH 8 8, 10
7, 10
7 (w), 9, 12
7 8, 9 1, 2, 6, 12 1, 2, 3, 6, 11 4, 5, 6
8, 10, 12
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8, 9 7
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T. H. T. Nguyen et al. chromatography was carried out on silica gel 60 F254 or silica gel 60 RP-18 F254S (Merck), and spots were visualized by spraying with 20% H2SO4 solution followed by heating. Gravity column chromatography was performed with silica gel 60 (0.040–0.063 mm, Himedia), or with silica gel 60 RP-18 (0.040–0.063 mm, Himedia). All NMR experiments were acquired at ambient temperature. Chemical shifts are expressed in ppm with reference to the residual protonated solvent signals (chloroform-d with δH 7.260 and δC 77.160 and methanol-d4 with δH 3.310 and δC 49.000) or the internal TMS (0.000). On a 400 Bruker Avance spectrometer, 1H spectra spectrometer spectral width (SW) 6393 Hz, number of data points (NP) 32 K, acquisition time (AQ) 2.5 s, pulse width (PW) 10.9 μs, relaxation delay (RD) 2 s, number of scans (NS) 1, line broadening (LB) 0.3 Hz. 13 C spectra: SW 26785 Hz, NP 64 K, AQ 1.2 s, PW 9 μs, RD 1.5 s, NS 1024, LB 1.0 Hz. HSQC spectra were carried out using the Bruker pulse sequence hsqcedetgp: AQ 0.17 s, RD 1.4 s, PW 13C 9.0 μs, PW 1 H 10.9 μs, SW 1H 3067 Hz; SW 13C 180 ppm, NP 1024, NI 256, FT size 1024 × 1024. The experiment was optimized for a 1JCH of 145 Hz and run in the echo-antiecho mode. HMBC spectrum was carried out using the Bruker pulse sequence hmbcgplpndqf: AQ 0.17 s, RD 1.1 s, PW 13C 9.0 μs, PW 1H 10.9 μs, SW 1H 3067 Hz; SW 13C 250 ppm, NP 1024, NI 256, FT size 2048 × 1024, nJCH optimized for 8.0 Hz. COSY: AQ 0.33 s, NS 6, PW 10.9 μs, SW 3067 Hz, NI 256, NP 2048, FT size 1024 × 1024. On a 500 Bruker Avance spectrometer, 1H spectra: SW 10330 Hz, NP 128 K, AQ 6.3 s, PW 11.5 μs, RD 3 s, NS 1, LB 0.3 Hz. 13C spectra: SW 32894 Hz, NP 64 K, AQ 0.99 s, PW 9 μs, RD 1.5 s, NS 509, LB 1.5 Hz. HSQC spectra were carried out using the Bruker pulse sequence hsqcedetgp: AQ 0.057 s, RD 1.5 s, PW 13C 9.7 μs, PW 1H 11.6 μs, SW 1H 9014 Hz; SW 13C 260 ppm, NP 1024, NI 256, FT size 1024 × 1024. The experiment was optimized for a 1JCH of 145 Hz and run in the echo-antiecho mode. HMBC spectrum was carried out using the Bruker pulse sequence hmbcgplpndqf: AQ 0.23 s, RD 1.0 s, PW 13C 9.9 μs, PW 1H 11.6 μs, SW 1H 9014 Hz; SW 13C 260 ppm, NP 4096, NI 128, FT size 2048 × 1024, nJCH optimized for 8.0 Hz. COSY: AQ 0.15 s, PW 11.6 μs, SW 6684 Hz, NI 128, NP 2048, FT size 1024 × 1024. NOESY: AQ 0.17 s, PW 11.6 μs, SW 6000.6 Hz, NI 512, NP 2048, FT size 1024 × 1024.
(column: 50 × 6 cm) eluting with chloroform – methanol – water (20:1:0, 9:1:0, 20:6:1, 14:6:1) to afford ten sub-fractions (A8.1 to A8.10). Sub-fraction A8.4 (1.3 g) was applied on RP-18 silica gel CC and eluted with water-methanol (8:2, 7:3, 1:1, 0:1) to obtain 6 (25 mg). The fraction A7 (24 g) was chromatographed on silica gel (column: 50 × 6 cm) eluting with chloroform – methanol – water (20:1:0, 9:1:0, 20:6:1, 14:6:1) to afford ten sub-fractions (A7.1 to A7.10). Sub-fraction A7.8 (2.2 g) was applied on RP-18 silica gel CC and eluted with water-methanol (9:2, 8:2, 1:1) to obtain 3 (16 mg). Sub-fraction A7.7 (2.8 g) was subjected to reverse-phase RP-18 silica gel CC, eluting with water-methanol (20:1, 9:1, 4:1, 1:1) to afford 2 (340 mg). The same manner applied on sub-fraction A7.6 (1.8 g) to yield 8 (6 mg). The fraction A4 (8.4 g) was subjected to silica gel CC, eluting with dichloromethane-methanol (95:5, 85:15) and then purified by RP-18 silica gel CC, eluted with water:methanol (6:4) to afford 5 (8.6 mg) and 7 (32.8 mg). The fraction A3 (2.9 g) was applied on RP-18 silica gel CC and eluted with water-methanol (6:4, 5:5, 2:8) to obtain 4 (25 mg) and 9 (5 mg). The fraction A2 (1.2 g) was applied on RP-18 silica gel CC, eluted with water:methanol (5:5) to obtain a mixture of 1a and 1b (23 mg).
Plant material
Sonnerstigmane C (5)
Leaves of S. ovata Backer were collected at Can Gio mangrove forest in Ho Chi Minh city, Vietnam in March 2010. The scientific name of the plant was authenticated by pharmacist Phan Duc Binh. A voucher specimen (No US-B006) was deposited in the herbarium of the Department of Organic Chemistry, VNUHCM – University of Science.
Colorless oil. ½α25 D 41.1 (c 0.35 MeOH). HR-ESI-MS m/z 239.1284 [M-H]-, (Calcd for C13H10O4 –H, 239.1283). 1H-NMR, 13C-NMR (CDCl3, 400 MHz for 1H and 100 MHz for 13C) data (Table 4). COSY and HMBC data (Fig. 2). NOESY data (Fig. 3).
Extraction and isolation
Colorless oil. ½α25 D + 2.3 (c 0.48 MeOH). HR-ESI-MS m/z 409.1858 [M + Na]+, (Calcd for C19H30O8Na, 409.1838). 1H-NMR, 13C-NMR (CD3OD, 500 MHz for 1H and 125 MHz for 13C) data (Table 3). COSY and HMBC data (Fig. 2). NOESY data (Fig. 3).
The air-dried powder of leaves (10.9 kg) was macerated with methanol (50 L × 3) at room temperature, and after filtration, the methanolic solution was concentrated at reduced pressure to yield a residue of 1.5 kg. This crude extract was suspended in water with 10% of methanol and was partitioned first with petroleum ether (60–80 °C) and then with ethyl acetate. After evaporation at the reduced pressure, three extracts were obtained: petroleum ether (245 g), ethyl acetate (390 g), and aqueous layer (865 g). The ethyl acetate extract was chromatographed on the silica gel CC (column: 120 × 6 cm) eluting with petroleum ether – ethyl acetate (1:4, 0:1) and then ethyl acetate – methanol (9:1, 4:1, 1:1, 0:1) to give fractions A1 to A9. The fraction A8 (30 g) was chromatographed on silica gel
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Mixture of sonnerstigmane A (1a) and sonnerstigmane B (1a)
Pale yellow oil. ½α25 D + 158.3 (c 1.2 MeOH). HR-ESI-MS m/z 261.1479 [M + Na]+ (Calcd for C14H22O3Na, 261.1467). 1H-NMR, 13C-NMR (CDCl3, 400 MHz for 1H and 100 MHz for 13C) data (Tables 1 and 2). COSY and HMBC data (Fig. 2). (6S,9R)-Roseoside (2)
Pale yellow oily wax. 1H-NMR, 13C-NMR (CDCl3, 300 MHz for 1H and 75 MHz for 13C) data (Tables 1 and 2). (6S,9R)-Ionone 9-O-(6-O-galloyl)-β-D-glucopyranoside (3) 1 13 Yellowish oil. ½α25 D + 340.0 (c 0.47 MeOH). H-NMR, C-NMR (CDCl3, 1 13 300 MHz for H and 75 MHz for C) data (Tables 1 and 2).
(S)-Dehydrovomifoliol (4) 1 13 Colorless oil. ½α25 C-NMR (CDCl3, D + 209.0 (c 1.3 MeOH). H-NMR, 400 MHz for 1H and 100 MHz for 13C) data (Tables 1 and 2).
Sonnerstigmane D (6)
8,9-Dihydromegastigmane-4,6-diene-3-one (7) 1 13 Pale yellow oil. ½α25 D + 54.0 (c 1.52 MeOH). H-NMR, C-NMR (CDCl3, 1 13 400 MHz for H and 100 MHz for C) data (Table 3). NOESY data (Fig. 3).
5,8-Epoxymegastigmane-6-ene-3-one 9-O-β-D-glucopyranoside (8)
Yellowish oil. 1H-NMR, 75 MHz for 13C).
Copyright © 2014 John Wiley & Sons, Ltd.
13
C-NMR (CD3OD, 300 MHz for 1H and
Magn. Reson. Chem. (2014)
Structure elucidation of four new megastigmanes from Sonneratia ovata Backer
Figure 4. Rotamers of compound 6 (The replacement of O-glucopyranosyl group by hydroxyl groups gives the rotamers A′, B′, C′, D′, E′, F′, G′, H′, I′, J′, and K′, respectively, of compound 7). Dehydrololiolide (9) 1 Colorless oil. ½α25 D + 3.6 (c 0.25 MeOH). H-NMR, 300 MHz for 1H and 75 MHz for 13C).
13
C-NMR (CDCl3,
Theoretical calculations The DFT calculations were performed with the Gaussian 09 package,[21] and the molecular geometries were fully optimized using the B3LYP variant of the DFT [22,23] with the 6-31G (d) basis set. The NMR nuclear shieldings were calculated with the same level of theory and basis set using the GIAO method.[24,25] Correlation coefficients between observed 13C chemical shifts and calculated 13 C nuclear shielding are shown in Fig. 4.
(S)-dehydrovomifoliol (4), 8,9-dihydromegastigmane-4,6-diene-3-one (7), 5,8-epoxymegastigmane-6-ene-3-one 9-O-β-D-glucopyranoside (8), and dehydrololiolide (9). Their chemical structures are established primarily by NMR and MS spectroscopy. 1a, 1b, 5, and 6 are new compounds and 2, 3, 4, 7, 8, and 9 have not been isolated from Sonneratia genus before. The presence of a glucose molecule in 6 allows a discrimination based in the present case on the proximity between H-1′and H-9. The DFT calculations not only provide 13C nuclear shieldings but also rotamers’ energies. Furthermore, calculated energies for rotamers are so different that also a qualified suggestion can be made regarding the populations. Furthermore, the comparison of correlation coefficients for least square between observed 13C chemical shifts and calculated 13C nuclear shielding may also be used to assign or leave out rotamers.
Conclusions From the leaves of S. ovata, ten compounds are isolated, including a mixture of sonnerstigmane A (1a), sonnerstigmane B (1a), sonnerstigmane C (5), sonnerstigmane D (6), (6S,9R)-roseoside (2), (6S,9R)-ionone 9-O-(6-O-galloyl)-β-D-glucopyranoside (3),
Magn. Reson. Chem. (2014)
Acknowledgements This research was supported by the Department of Science and Technology – HCM city, grant #1058/QĐ-SKHCN. N.T.H.T would like to thank Vietnamese government for a scholarship at Roskilde
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T. H. T. Nguyen et al. University under the 322 projects, to thank Chulalongkorn University for a scholarship at Chulalongkorn University under the One Semester Scholarships Program for ASEAN Countries.
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