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Lignans and a neolignan from Virola oleifera leaves
Phytochmistry, Vol. 32, No. 6, pp. 1567.1572,
Pnntedin GreatBritain.
1993
0031-9422/93 $6.00+0.00 PergamonPressLtd
LIGNANS AND A NEOLIGNAN
FROM
VIROLA OLEIFERA
ANNA MARIA A. P. FERNANDES, LAURO E. S. BARATA
LEAVES
and PEDRO H. FERRI*
Instituto de Quimica, Universidade Estadual de Campinas, C. P. 6154 (13 081-970)Campinas, SP, Brazil; *Institute de Quimica e Geociencias, Universidade Federal de Go&, (74 001-970)Goilnia, GO, Brazil (Received 4 August 1992)
Key Word Index-Virola oleifera; Myristicaceae; leaves; 4’,7-epoxy-8,3’-neolign-7’-ene; nans; lignan-7-01s; 2,7’-cyclolignan-7-01; lignans; neolignan; oleiferins A-E.
7,7’-epoxylig-
Abstract-The chlorophyll-free dichloromethane fraction of the ethanolic extract from leaves of Virola olefera yielded four new natural lignans, including a known lignan-7-01, and galbacin, eupomatenoid-8 and (+)-aristolignin. Structural elucidations were made by spectroscopic methods.
INTRODUCTION
Although numerous Virola species from the Amazonian forest have been submitted to phytochemical studies [ 11, few chemical analyses have been reported from specimens growing in the Atlantic forest in the southeastern region of Brazil. Virola oleifera is widely distributed in this region and has been used in folk medicine as a cicatrizant, anti-inflammatory, antirheumatic, anti-asthmatic, etc. [2]. However, no study of the chemical constituents from this myristicaceous tree has been carried out so far. This paper reports, besides the known compounds galbacin (1) [3], eupomatenoid-8 (2) [4] and (+)-aristolignin (3) [S], the isolation and spectroscopic identification of four new natural lignans, oleiferin-A (4), its known substitutional variant 5 [6], and oleiferins C (6), D (7) and E (8). In order to facilitate comparisons among different lignans and the neolignan, the nomenclature of these compounds follows the IUPAC-IUB Joint Commission on Biochemical Nomenclature recommendations outlined in ref. [7]. RESULTS AND DISCUSSlON
The chlorophyll-free dichloromethane fraction of the 95% ethanol extract from the leaves of V. oleijkra was separated by vacuum liquid chromatography [S] and continuous preparative TLC. These procedures led to the isolation of minor components, 1-8, which were identified by spectroscopic methods as described below. Compound 1, C2,,H,,0,, showed in its ‘HNMR spectrum a characteristic signal pattern of a structurally symmetrical 7,7’-epoxylignan with two 3&methylenedioxyphenyl groups. From a comparison of the spectroscopic data with reported values, 1 was identified as galbacin, which had been isolated previously from Himantandru baccata [3]. Compound 2, CzoHzoO, ([Ml’ nr/z 324), [a];’ +42.1” (CHCl,; c 0.202), was obtained as crystals, mp PHYTO 32:6-O
90-91” (MeOH). From its ‘H NMR spectrum [S 1.35 (d, J =6.8 Hz, Me-8), 63.31 (dq, J=6.8,8.8 Hz, H-8), 64.98 (d, J = 8.8 Hz, H-7)] the (7c(,8/?)-4’,7-epoxy-8,3’-neolignan system could be readily recognized, as well as a (E)propenyl group [S 1.86 (dd, J= 1.5, 6.6 Hz, Me-g’), 66.10 (dq, J=6.6,16 Hz, H-8’), 66.35 (d, J= 16 Hz, H-7’)], a 3,4methylenedioxy group (65.95, s) and a methoxyl signal (6 3.89, s). Recently published 13C NMR values [9] helped us in the assignment of the i3C signals (Table 1). Spectral data of 2 also closely resembled those reported for eupomatenoid-8 isolated from the bark of Eupomatia laurina [4]. Although eupomatenoid-8 was hrst isolated and synthesized as a gum [lo] in our hands the compound was isolated as a crystalline material. Compound 3, C2iHZ605 ([Ml’ m/z 358), was isolated as a crystalline solid, mp 82-83”, [a]h3 + 27.5” (CHCl,; c 0.309). The ‘H NMR spectrum showed signals of two secmethyls c60.63 (J = 7.0 Hz, Me-8’), 6 1.06 (.I = 6.6 Hz, Me8)], two methines (H-8 and H-8’) at 6 1.69 and 2.15 as two multiplets, and two benzylic methines C64.27 (d, J =9.2 Hz, H-7), 64.98 (d, J = 8.4 Hz, H-7’)] which were essentially similar to those of a veraguensin-type 7.7’epoxylignan [ll]. Furthermore, the ‘H and i3C NMR (Table 1) spectra revealed the presence of 3,4-dimethoxyphenyl and 4-hydroxy-3-methoxyphenyl groups. The differential NOE correlation spectrum showed appreciable NOE between H-7 and H-2/H-6/H-g/Me-8, and between H-7’ and H-2/H&‘/H-8, indicating that a 4-hydroxy-3methoxyphenyl group was attached to C-7 of the tetrahydrofuran ring and a 3,4_dimethoxyphenyl group to C7’. Comparison of these spectral data led to the conclusion that 3 is (+)-aristolignin which has been isolated previously as an oil from roots of Aristolochiu chilensis c51. Compound 4, an oil, [a];’ +41.4” (CHCl,; c 0.828), displayed a [M]’ at m/z 358 for CZ1HZ605. The ‘H NMR spectrum revealed the presence of two methyl
1567
A. M. A. P. FERNANDES et al.
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doublets C60.77 (Me-8’) and 60.92 (Me-8); 5=6.8 Hz], two methine multiplets (61.53, H-8’ and 61.60, H-8), benzylic methylene signals at 62.32 (dd, J = 6.8, 13.7 Hz, H-7’) and 62.42 (dd, J= 8.4, 13.7 Hz, H-7’) and a benzylic methine substituted by oxygen (64.28, d, J = 8.8 Hz, H-7). The homonuclear COSY spectrum indicated coupling between the methyl signal at 60.92 and the methine signal at 61.60, which was also coupled to the oxygenated methine at 64.28. On the other hand, the methine signal at 6 1.53 was coupled to the methyl signal at 60.77 and the benzylic methylene signals at 62.32 and 62.42, indicating that the isolated compound has the lignan-7-olic skeleton. The aromatic proton at 66.55 (d, J= 1.8 Hz, H-2) was coupled with the signal at 66.54 (dd, J = 1.8, 8.1 Hz, H-6) which also coupled with the signal at 66.62 (d, 3=8.1 Hz, H-5). As expected, the signals at 66.32, (d, J = 1.8 Hz, H-2’), 66.43 (dd, J= 1.8.8.1 Hz, H-6’), and S6.60 (d, J= 8.1 Hz, H-5’) were found to be coupled. The 13C NMR spectrum revealed two sets of aromatic nuclei
and showed the presence of 3,4-dimethoxyphenyl and 3,4methylenedioxyphenyl moieties. Carbon-13 proton-shift correlation (13C-‘H HETCOR) and long-range COLOC spectra confirmed the structure of 4 and enabled us to assign all the proton and carbon signals (Table 1). The long-range 13C-rH correlation spectrum (in CDCl,) indicated that appreciable long-range spin coupling was present between C-7’ and H-2’ (66.47) or between H-7 and C-l’(6 133.6), C-2’ (S 111.8) and C-6 (S 120.9), indicating that a secondary carbinol is attached to the benzylic carbon of the 3,4_methylenedioxyphenyl group (Table 1). The EI mass spectrum of 4 was in agreement with the structure proposed from the ‘H and 13C NMR analyses. It exhibited a [M]’ at m/z 358, accompanied by characteristic peaks at m/z 340 [M -H,O] +, a base peak at m/z 151 [CH(OH)C,H,(OCH,O)]+, besides a peak at miz 149. Since this should represent [C6H3(OCH20)(C0)]+, it is the piperonyl rather than the veratryl group which is connected to the benzyl alcohol unit. Based on this
A. M. A. P. FERNAWESet al.
1570
0
1'
MC Lafferty Rearrangement m/z
m/z
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m/z
163
180
d-cleavage
m/z
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Scheme 1. Proposed mass spectral fragmentation of compound 7.
(dq, J = 4.0, 6.2 Hz, H-8)] and benzylic methylene signals at 62.lO(dd, Jz9.0, 14.7Hz, H-7’)and 62.78(dd, J=6.0, 14.7 Hz, H-7’). On irradiation at 6 ca 2.08-2.10, the doublet at 60.82 (Me-8’) and the double doublet at 62.78 (H-7’) became two singlets. The spectrum showed further signals for methoxyl(64.0), methylenedioxy (6 5.87, s), one phenolic hydroxyl (65.70, sl) and six aromatic protons. The latter were shown clearly in the form of two sets cS7.46 (d, 5=1.6Hz, H-2), 66.85 (d, 5=8.3 Hz, H-5), 67.40(dd,J=1.6,8.3 Hz.H-6)and66.50(d,J=1.5 Hz,H2’), 66.59 (d, J= 7.9 Hz, H-5’) 66.45 (dd, J= 1.5, 7.9 Hz, H6’11. Additionally, the 13C NMR spectrum (Table 1) showed two separate sets of aromatic carbon atoms, one due to a 4-hydroxy-3-methoxy moiety, the other due to a 3,4-methylenedioxyphenyl unit. The EI mass fragmentation showed, besides intense peaks at m/z 162, 151 and 135, a base peak at m/z 180 which arises from a McLafferty rearrangement (Scheme 1). On the basis of these data, the structure of 7 was concluded to be (8S*,8’R*)-4hydroxy-3-methoxy-3’.4’-methyIenedioxylignan-7-one, a new natural product. Compound 8 (named oleiferin E), C,,H,,O, ([Ml’ m/z 340), was isolated as needles, mp 217--219’, [a]i4 - 6.4” (CHCI,; c 0.230). The ‘H NMR spectrum showed doublets at SO.98 (d, J = 6.2 Hz, Me-8’) and 6 1.18 (d, J =6.2 Hz, Me-8) with their related methine protons (6 1.52, m, H-8’; 6 1.43, m, H-8), and an oxygenated benzylic methine at 64.32 (d, J=9.5 Hz, H-7), similar to those of compounds 4-6, but with signal at 63.50 (d, J = 10.2 Hz, H-7’) ascribed to the presence of a doubly benzylic methine proton in place of a common benzylic methylene present in the lignan-7-ol-type series. Interproton couplings were established though a homonuclear COSY spectrum. The methyl signal at 60.98 was coupled with the methine signal at 6 1.52, which itself was coupled with the benzylic proton at 63,50. On the other hand, the methyl signal at 6 1.18 was coupled with the methine signal at 61.43, which was itself coupled with the oxygenated methine signal at 64.32. Furthermore, the aromatic proton at 66.53 (d, J= 1.7 Hz, H-2’) was coupled
with the signal at S6.60 (dd, J= 1.7, 8.0 Hz, H-6’), which also coupled with the signal at 66.70 (d, J= 8.0 HZ, H-5’). The aromatic region displayed again a set of signals at (56.74 (d, 5=8.1 Hz, H-5) and 67.16 (d, 5=8.1 Hz, H-6). As expected, the latter set was found to be coupled. These observations are consistent with the 2,7’-cyclolignanic skeleton with two methylenedioxyl groups, one at 65.92 (s) and one at 65.63 (d, J= 1.1 Hz, 1H) and 65.71 (d, J = 1.1 Hz, 1H). The assignment of these methylenedioxyls was based on the known chemical shift difference between ring A or C substitution [16-J. The observed coupling constant of the H-7 and H-8 signals (J = 9.5 Hz), H-7’ and H-8’ (J= 10.2 Hz), and a relatively large coupling constant of the H-8 and H-8’ signals rather than 3 Hz [17], confirm the all tran.s stereochemistry with the hydroxyl, methyl groups and the pendant phenyl substituent all being pseudo-equatorial. The 13C NMR spectrum (Table 1) and the mass fragmentation pattern (m/z) 322 [M-H,O]‘, 284 [M-C,Hs]+ 162 [CH,CH: CH(C,H,)(OCH,O)]+, 151 (base peak) [CH(OH)C,H,(OCH,O)] +,149 [C,H,(OCH,O)CO] +, 135 [CH&H&OCH,O)] ‘) supported the structure of 8, On the basis of these data, the structure of 8 was concluded to be (7~,7’8,8/3,8’n)-3,4: 3’,4’-bis(methy1 lenedioxy)-2,7’-cyclolignan-7-01. Although 8 has been synthesized [18], this is the first time that it has been isolated from a natural source. While 2,7’-cyclolignans and 2,7’-cyclolignan-7-one are well known [15], compound 8 is distinguished by a hydroxyl substitution pattern which has been reported only recently from a natural source [ 191. EXPERIMENTAL General. Mp: uncorr. ‘H and 13CNMR spectra were recorded at 300 and 75 MHz, respectively, using TMS as int. ref. EIMS were measured at 70eV. Plant material. Leaves of V. oleifera (Schott) A. C. Smith were collected by Dr Gentil Godoy (Atlantic Forestal Reserve, Ubatuba, Sao Paulo State, Brazil) in May 1990 and authenticated by Dr Jorge Tamashiro
Lignans from h-da (Department of Botany, Unicamp). A voucher specimen is deposited in the Herbarium of Unicamp, Sgo Paulo, Brazil. Isolation. In order to eliminate chlorophylls and other colouring matter, air-dried powdered leaves (0.8 kg) were extracted with EtOH at room temp. The EtOH extract (24 g) was dild with MeOH-H,O (4: 1), filtered over Celite and extracted first with hexane and then with CH,C12. Both chlorophyll-free extracts (2.7 g from hexane, 2.6 g from CH,Cl,) were fractionated by VLC [8] over silica gel H (40 pm) using hexane and hexane-EtOAc mixts of increasing polarity. Thus, from the chlorophyllfree hexane extract were isolated l(5 mg) and 2 (2 mg); the chlorophyll-free CH,C12 extract gave 3 (14 mg), 4 (20 mg), 5 (15 mg), 6 (3.5 mg), 7 (4 mg) and 8 (1.5 mg). Galbacin (1). Oil, the spectral properties of which were identical with those described in ref. [I3], Eupomatenoid-8 (2). Crystals, mp 40-91” (MeOH). [~]g +42.1” (CHCl,; c 0.202). UV v”,EF” nm (log E): 215 (4.4), 272 (4.1). ‘H NMR (300 MHz, CDCl,) 6: 1.35 (d, J =6.8 Hz, Me-8), 1.86 (dd, J= 1.5,6.6 Hz, Me-8’), 3.31 (dq, J=6.8, 8.8 Hz, H-8), 3.89 (s, OMe), 4.98 (d, J=8.8 Hz, H7), 5.95 (s, OCH,O), 6.10 (dq, J =6.6, 16 Hz, H-8’), 6.35 (d, J=l6 Hz, H-7’), 6.75 (sI, lH), 6.77 (sl, lH), 6.78 (d, J = 8.1 Hz, H-5), 6.87 (dd, J = 1.5, 8.1 Hz, H-6), 6.93 (d, J = 1.5 Hz, H-2). 13C NMR (75.5 MHz, CDCl,) in Table 1. EIMS m/z (rel. int.): 324 [M]+ (lOO), 309 (6), 203 (4), 162 (lo), 149 (16). Aristolignin (3). Crystals, mp 82-83”. [ali +27.5” (CHCl,; c 0.309). UV v:;;” nm (log E): 230 (4.0), 278 (3,6); +NaOH: 234 (3.9), 250 (3,9), 282 (3.6), 294 (sh, 3.5). ‘H NMR (300 MHz, CCl,/D,O): 60.63 (d, J =7.0 Hz, Me-8’), 1.06 (d, J = 6.6 Hz, Me-8), 1.69 (m, H-8), 2.15 (m, HS’), 3.76 (s, OMe), 3.79 (s, OMe), 3.92 (s, OMe), 4.27 (d, J =9.2 Hz, H-7),4.98 (d,J=8.4 Hz, H-7’), 5.31 (s, OH), 6.71 (d, J= 1.2 Hz, H-2’), 6.73 (dd, J= l-2,9.3 Hz, H-6’), 6.74 (d, J=9.3 Hz,H-5’),6.79(d,J=8.2 Hz,H-5), 6.85(dd, J= 1.7, 8.2 Hz, H-6), 6.98 (d, J = 1.7 Hz, H-2). ’ 3C NMR (75.5 MHz, Ccl,) in Table 1. EIMS m/z (rel. int.): 358 CM]’ (48), 206 (46), 192 (lOO), 191 (18), 177 (20), 151 (27). Ole$erin A (4). Oil. [a]kl +41.4” (CHCl,; c 0.828). UV vzFH nm (log&): 229 (3.8), 280 (3.6). ‘H NMR (300 MHz, CCldD,O) 6: 0.77 (d, J=6.8 Hz, Me-8’), 0.92 (d, J =6.8 Hz, Me-8), 1.53 (m, H-8’), 1.58 (~1,OH), 1.60(m, H-8), 2.32 (dcd,J=6.8, 13.7 Hz, H-7’), 2.42 (dd, J=8.4, 13.7 Hz, H-7’), 3.68 (s, OMe-3’), 3.74 (s, OMe-4’), 4.28 (d, J =8.8 Hz, H-7), 5.95 (dd, J= 1.4,3.8 Hz, OCH,O), 6.32 (d, J= 1.8 Hz, H-2’), 6.43 (dd, J = 1.8,8.1 Hz, H-6’), 6.54 (dd, J = 1.8, 8.1 Hz, H-6), 6.55 (d, J = 1.8 Hz, H-2), 6.60 (d, J =8.1 Hz, H-5’), 6.62 (d, J=8.1 Hz, H-5). ‘H NMR (300 MHz, CDCl,): 60.79 (d, J = 6.8 Hz, Me-8’), 1.01 (d, J = 6.8 Hz, Me-8), 1.60(m, H-8), 1.70 (m, H-8’), 1.70 (sl, OH), 2.41 (dd, J=6.8, 13.7 Hz, H-7’), 2.45 (dd, J=8.4, 13.7 Hz, H-7’), 3.80 (s, OMe-3’), 3.86 (s, OMe-4’), 4.37 (d, J =8.8 Hz, H-7), 5.94 (d, J= 1.4 Hz, OCH,O), 6.47 (d, J = 1.7 Hz, H-2’), 6.56 (dd, J= 1.8, 8.1 Hz, H-6’), 6.64 (dd, J = 1.7 Hz, 8.1 Hz, H-6), 6.65 (d, J= 1.7 Hz, H-2), 6.73 (d, J =8.1 Hz, H-5’), 6.74 (d, J =8.1 Hz, H-5). 13C NMR (75.5 MHz, CDCl,) in Table 1. EIMS m/z (rel. int.): 358 [M]’ (32), 340 (2), 151 (lOO), 149 (11).
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ole~eera
Ole$erin B (5). Oil. [z];’ + 54.4” (CHCl,; c 0.469). UV vii:” nm (loge): 221 (3.8), 274 (3.5). ‘H NMR (300 MHz, CCi,,/D,O): 60.77 (d, J =6.7 Hz, Me-8’), 0.93 (d, J = 6.7 Hz, Me-8), 1.40 (sl, OH), 1.52 (m, H-8’), 1.62 (m, H-8), 2.30 (dd, J=6.8, 13.8 Hz, H-7’), 2.38 (dd, J=9.1, 13.8 Hz, H-7’), 3.70 (s, OMe), 3.78 (s, OMe), 4.27 (d, J=8.5 Hz, H7), 5.86 (s, OCH,O), 6.35 (d, lH), 6.37 (dd, J =8.0 Hz, lH), 6.55 (d, J=9.0 Hz, lH), 6.55 (dd, J=9_0Hz, lH), 6.59 (d, lH),664(d, J=8.1 Hz, IH). 13CNMR(75.5MHz,CC1,) in Table 1. EIMS m/z (rel. int.): 348 [MI+ (25), 340 (2), 167 (lOO), 165 (2), 135 (14). OEeiferin C (6). Oil. [a];’ +44.0” (CHCI,; c 0.772). UV ~~~~~”nm (log E): 228 (3.7), 279 (3.7). ‘H NMR (300 MHz, CCl,/D,O): SO.76 (d, J=6.9 Hz, Me-8’), 0.92 (d, J = 6.9 Hz, Me-8), 1.29 (sl, OH), 1.54 (m, H-8’), 1.62 (m, H-8), 2.30 (dd, J=6.9, 13.5 Hz, H-7’), 2.45 (dd, J=8.1, 13.5 Hz, H-7’), 4.33 (d, J=8.1 Hz, H-7), 5.87 (dd, J=O.8, 1.5Hz, OCH,O-3,4), 5.92 (s, OCH,O-3’,4’), 6.37-6.39 (m, 2H), 6.57-6.59(m, 2H), 6.59 (d, Jz8.1 Hz, lH), 6.65 (d,8.4 Hz, 1H). 13C NMR (75.5 MHz, Ccl,) in Table 1. EIMS m/z (rel. int.): 342 [M]’ (181, 324 (3), 151 (lOO), 149 (5), 135
t 16). OEeiferinD (7). Oil. [a]:’ - 52.5” (CHCl,; c 0.162). UV vixen nm: (log&): 228 (3.8), 280 (3.7), 304 (sh, 3.5), 320 (sh, 3.2); +NaOH: 245 (3.6), 290 (3.4), 342 (3.9). ‘H NMR (300 MHz, CCl,/D,O): 60.82 (d, J = 6.0 Hz, Me-8’), 1.18 (d, J=6.6 Hz, Me-8), 2.08 (m, H-8’), 2.10 (dd, J=9.0, 14.7 Hz, H-7’), 2.78 (dd, J=6.0, 14.7 Hz, H-7’), 3.29 (dq, J =4.0, 6.2 Hz, H-8), 4.0 (s, OMe), 5.70 (sl, OH), 5.87 (s, OCH,O), 6.45 (dd, J= 1.5, 7.9 Hz, H-6’), 6.50 (d, J =1.5 Hz, H-2’), 6.59 (d, J=7.9 Hz, H-S), 6.85 (d, J = 8.3 Hz, H-5), 7.40 (dd, J= 1.6, 8.3 Hz, H-6), 7.46 (d, J = 1.6 Hz, H-2). 13C NMR (75.5 MHz, Ccl,) in Table 1. EIMS m/z (rel. int.): 342 [M]’ (S), 180 (lOO), 162 (60), 151 (30), 135 (21). Oleiferin E (8). Needles, mp. 217-219”. [a]? -6.4” (CHCl,; c 0.230). UV 1;:;” nm (loge): 232 (3.9), 285 (3.7). ‘H NMR (300 MHz, CDCl,): 50.98 (d, J = 6.2 Hz, Me-8’), l.l8(d,J=6.2Hz,Me-8),1.43(m,H-8),1.52(m,H-8’),3.50 (d, J=10.2 Hz, H-7’), 4.32 (d, J=9.5 Hz, H-7), 4.36 (sl, OH), 5.63 (d, J= 1.1 Hz, lH, OCH,O-3,4), 5.71 (d, J = 1.1 Hz, lH, OCH,-3,4), 5.92 (s, OCH,O-3’,4’), 6.53 (d, J= 1.7 Hz, H-2’), 6.60 (dd, J= 1.7, 8.0 Hz, H-6’), 6.70 (d, J =8.0 Hz, H-5’), 6.74 (d, J= 8.1 Hz, H-5), 7.16 (d, J = 8.1 Hz, H-6). 13C NMR (75.5 MHz, CDCl,) in Table 1. EIMS m/z (rel. int.): 340 [M]’ (6), 322 (50), 284 (4), 162 (16) 151 (lOO), 249 (36), 135 (27). Acknowledgements-We thank FAPESP and CNPq fur a fellowship to AMAPF and PHF. Thanks are also due to Dr Gentil Godoy (Instituto Agron8mico de CampinasIAC), Dr Jorge Tamashiro (Department of Botany, Unicamp) for the botanical identification, Mrs Paula Pilli and Mrs S6nia Crisostomo for the measurement of ‘H and 13C NMR.
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Pnntedin GreatBritain.
1993
0031-9422/93 $6.00+0.00 PergamonPressLtd
LIGNANS AND A NEOLIGNAN
FROM
VIROLA OLEIFERA
ANNA MARIA A. P. FERNANDES, LAURO E. S. BARATA
LEAVES
and PEDRO H. FERRI*
Instituto de Quimica, Universidade Estadual de Campinas, C. P. 6154 (13 081-970)Campinas, SP, Brazil; *Institute de Quimica e Geociencias, Universidade Federal de Go&, (74 001-970)Goilnia, GO, Brazil (Received 4 August 1992)
Key Word Index-Virola oleifera; Myristicaceae; leaves; 4’,7-epoxy-8,3’-neolign-7’-ene; nans; lignan-7-01s; 2,7’-cyclolignan-7-01; lignans; neolignan; oleiferins A-E.
7,7’-epoxylig-
Abstract-The chlorophyll-free dichloromethane fraction of the ethanolic extract from leaves of Virola olefera yielded four new natural lignans, including a known lignan-7-01, and galbacin, eupomatenoid-8 and (+)-aristolignin. Structural elucidations were made by spectroscopic methods.
INTRODUCTION
Although numerous Virola species from the Amazonian forest have been submitted to phytochemical studies [ 11, few chemical analyses have been reported from specimens growing in the Atlantic forest in the southeastern region of Brazil. Virola oleifera is widely distributed in this region and has been used in folk medicine as a cicatrizant, anti-inflammatory, antirheumatic, anti-asthmatic, etc. [2]. However, no study of the chemical constituents from this myristicaceous tree has been carried out so far. This paper reports, besides the known compounds galbacin (1) [3], eupomatenoid-8 (2) [4] and (+)-aristolignin (3) [S], the isolation and spectroscopic identification of four new natural lignans, oleiferin-A (4), its known substitutional variant 5 [6], and oleiferins C (6), D (7) and E (8). In order to facilitate comparisons among different lignans and the neolignan, the nomenclature of these compounds follows the IUPAC-IUB Joint Commission on Biochemical Nomenclature recommendations outlined in ref. [7]. RESULTS AND DISCUSSlON
The chlorophyll-free dichloromethane fraction of the 95% ethanol extract from the leaves of V. oleijkra was separated by vacuum liquid chromatography [S] and continuous preparative TLC. These procedures led to the isolation of minor components, 1-8, which were identified by spectroscopic methods as described below. Compound 1, C2,,H,,0,, showed in its ‘HNMR spectrum a characteristic signal pattern of a structurally symmetrical 7,7’-epoxylignan with two 3&methylenedioxyphenyl groups. From a comparison of the spectroscopic data with reported values, 1 was identified as galbacin, which had been isolated previously from Himantandru baccata [3]. Compound 2, CzoHzoO, ([Ml’ nr/z 324), [a];’ +42.1” (CHCl,; c 0.202), was obtained as crystals, mp PHYTO 32:6-O
90-91” (MeOH). From its ‘H NMR spectrum [S 1.35 (d, J =6.8 Hz, Me-8), 63.31 (dq, J=6.8,8.8 Hz, H-8), 64.98 (d, J = 8.8 Hz, H-7)] the (7c(,8/?)-4’,7-epoxy-8,3’-neolignan system could be readily recognized, as well as a (E)propenyl group [S 1.86 (dd, J= 1.5, 6.6 Hz, Me-g’), 66.10 (dq, J=6.6,16 Hz, H-8’), 66.35 (d, J= 16 Hz, H-7’)], a 3,4methylenedioxy group (65.95, s) and a methoxyl signal (6 3.89, s). Recently published 13C NMR values [9] helped us in the assignment of the i3C signals (Table 1). Spectral data of 2 also closely resembled those reported for eupomatenoid-8 isolated from the bark of Eupomatia laurina [4]. Although eupomatenoid-8 was hrst isolated and synthesized as a gum [lo] in our hands the compound was isolated as a crystalline material. Compound 3, C2iHZ605 ([Ml’ m/z 358), was isolated as a crystalline solid, mp 82-83”, [a]h3 + 27.5” (CHCl,; c 0.309). The ‘H NMR spectrum showed signals of two secmethyls c60.63 (J = 7.0 Hz, Me-8’), 6 1.06 (.I = 6.6 Hz, Me8)], two methines (H-8 and H-8’) at 6 1.69 and 2.15 as two multiplets, and two benzylic methines C64.27 (d, J =9.2 Hz, H-7), 64.98 (d, J = 8.4 Hz, H-7’)] which were essentially similar to those of a veraguensin-type 7.7’epoxylignan [ll]. Furthermore, the ‘H and i3C NMR (Table 1) spectra revealed the presence of 3,4-dimethoxyphenyl and 4-hydroxy-3-methoxyphenyl groups. The differential NOE correlation spectrum showed appreciable NOE between H-7 and H-2/H-6/H-g/Me-8, and between H-7’ and H-2/H&‘/H-8, indicating that a 4-hydroxy-3methoxyphenyl group was attached to C-7 of the tetrahydrofuran ring and a 3,4_dimethoxyphenyl group to C7’. Comparison of these spectral data led to the conclusion that 3 is (+)-aristolignin which has been isolated previously as an oil from roots of Aristolochiu chilensis c51. Compound 4, an oil, [a];’ +41.4” (CHCl,; c 0.828), displayed a [M]’ at m/z 358 for CZ1HZ605. The ‘H NMR spectrum revealed the presence of two methyl
1567
A. M. A. P. FERNANDES et al.
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doublets C60.77 (Me-8’) and 60.92 (Me-8); 5=6.8 Hz], two methine multiplets (61.53, H-8’ and 61.60, H-8), benzylic methylene signals at 62.32 (dd, J = 6.8, 13.7 Hz, H-7’) and 62.42 (dd, J= 8.4, 13.7 Hz, H-7’) and a benzylic methine substituted by oxygen (64.28, d, J = 8.8 Hz, H-7). The homonuclear COSY spectrum indicated coupling between the methyl signal at 60.92 and the methine signal at 61.60, which was also coupled to the oxygenated methine at 64.28. On the other hand, the methine signal at 6 1.53 was coupled to the methyl signal at 60.77 and the benzylic methylene signals at 62.32 and 62.42, indicating that the isolated compound has the lignan-7-olic skeleton. The aromatic proton at 66.55 (d, J= 1.8 Hz, H-2) was coupled with the signal at 66.54 (dd, J = 1.8, 8.1 Hz, H-6) which also coupled with the signal at 66.62 (d, 3=8.1 Hz, H-5). As expected, the signals at 66.32, (d, J = 1.8 Hz, H-2’), 66.43 (dd, J= 1.8.8.1 Hz, H-6’), and S6.60 (d, J= 8.1 Hz, H-5’) were found to be coupled. The 13C NMR spectrum revealed two sets of aromatic nuclei
and showed the presence of 3,4-dimethoxyphenyl and 3,4methylenedioxyphenyl moieties. Carbon-13 proton-shift correlation (13C-‘H HETCOR) and long-range COLOC spectra confirmed the structure of 4 and enabled us to assign all the proton and carbon signals (Table 1). The long-range 13C-rH correlation spectrum (in CDCl,) indicated that appreciable long-range spin coupling was present between C-7’ and H-2’ (66.47) or between H-7 and C-l’(6 133.6), C-2’ (S 111.8) and C-6 (S 120.9), indicating that a secondary carbinol is attached to the benzylic carbon of the 3,4_methylenedioxyphenyl group (Table 1). The EI mass spectrum of 4 was in agreement with the structure proposed from the ‘H and 13C NMR analyses. It exhibited a [M]’ at m/z 358, accompanied by characteristic peaks at m/z 340 [M -H,O] +, a base peak at m/z 151 [CH(OH)C,H,(OCH,O)]+, besides a peak at miz 149. Since this should represent [C6H3(OCH20)(C0)]+, it is the piperonyl rather than the veratryl group which is connected to the benzyl alcohol unit. Based on this
A. M. A. P. FERNAWESet al.
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0
1'
MC Lafferty Rearrangement m/z
m/z
162
m/z
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180
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Scheme 1. Proposed mass spectral fragmentation of compound 7.
(dq, J = 4.0, 6.2 Hz, H-8)] and benzylic methylene signals at 62.lO(dd, Jz9.0, 14.7Hz, H-7’)and 62.78(dd, J=6.0, 14.7 Hz, H-7’). On irradiation at 6 ca 2.08-2.10, the doublet at 60.82 (Me-8’) and the double doublet at 62.78 (H-7’) became two singlets. The spectrum showed further signals for methoxyl(64.0), methylenedioxy (6 5.87, s), one phenolic hydroxyl (65.70, sl) and six aromatic protons. The latter were shown clearly in the form of two sets cS7.46 (d, 5=1.6Hz, H-2), 66.85 (d, 5=8.3 Hz, H-5), 67.40(dd,J=1.6,8.3 Hz.H-6)and66.50(d,J=1.5 Hz,H2’), 66.59 (d, J= 7.9 Hz, H-5’) 66.45 (dd, J= 1.5, 7.9 Hz, H6’11. Additionally, the 13C NMR spectrum (Table 1) showed two separate sets of aromatic carbon atoms, one due to a 4-hydroxy-3-methoxy moiety, the other due to a 3,4-methylenedioxyphenyl unit. The EI mass fragmentation showed, besides intense peaks at m/z 162, 151 and 135, a base peak at m/z 180 which arises from a McLafferty rearrangement (Scheme 1). On the basis of these data, the structure of 7 was concluded to be (8S*,8’R*)-4hydroxy-3-methoxy-3’.4’-methyIenedioxylignan-7-one, a new natural product. Compound 8 (named oleiferin E), C,,H,,O, ([Ml’ m/z 340), was isolated as needles, mp 217--219’, [a]i4 - 6.4” (CHCI,; c 0.230). The ‘H NMR spectrum showed doublets at SO.98 (d, J = 6.2 Hz, Me-8’) and 6 1.18 (d, J =6.2 Hz, Me-8) with their related methine protons (6 1.52, m, H-8’; 6 1.43, m, H-8), and an oxygenated benzylic methine at 64.32 (d, J=9.5 Hz, H-7), similar to those of compounds 4-6, but with signal at 63.50 (d, J = 10.2 Hz, H-7’) ascribed to the presence of a doubly benzylic methine proton in place of a common benzylic methylene present in the lignan-7-ol-type series. Interproton couplings were established though a homonuclear COSY spectrum. The methyl signal at 60.98 was coupled with the methine signal at 6 1.52, which itself was coupled with the benzylic proton at 63,50. On the other hand, the methyl signal at 6 1.18 was coupled with the methine signal at 61.43, which was itself coupled with the oxygenated methine signal at 64.32. Furthermore, the aromatic proton at 66.53 (d, J= 1.7 Hz, H-2’) was coupled
with the signal at S6.60 (dd, J= 1.7, 8.0 Hz, H-6’), which also coupled with the signal at 66.70 (d, J= 8.0 HZ, H-5’). The aromatic region displayed again a set of signals at (56.74 (d, 5=8.1 Hz, H-5) and 67.16 (d, 5=8.1 Hz, H-6). As expected, the latter set was found to be coupled. These observations are consistent with the 2,7’-cyclolignanic skeleton with two methylenedioxyl groups, one at 65.92 (s) and one at 65.63 (d, J= 1.1 Hz, 1H) and 65.71 (d, J = 1.1 Hz, 1H). The assignment of these methylenedioxyls was based on the known chemical shift difference between ring A or C substitution [16-J. The observed coupling constant of the H-7 and H-8 signals (J = 9.5 Hz), H-7’ and H-8’ (J= 10.2 Hz), and a relatively large coupling constant of the H-8 and H-8’ signals rather than 3 Hz [17], confirm the all tran.s stereochemistry with the hydroxyl, methyl groups and the pendant phenyl substituent all being pseudo-equatorial. The 13C NMR spectrum (Table 1) and the mass fragmentation pattern (m/z) 322 [M-H,O]‘, 284 [M-C,Hs]+ 162 [CH,CH: CH(C,H,)(OCH,O)]+, 151 (base peak) [CH(OH)C,H,(OCH,O)] +,149 [C,H,(OCH,O)CO] +, 135 [CH&H&OCH,O)] ‘) supported the structure of 8, On the basis of these data, the structure of 8 was concluded to be (7~,7’8,8/3,8’n)-3,4: 3’,4’-bis(methy1 lenedioxy)-2,7’-cyclolignan-7-01. Although 8 has been synthesized [18], this is the first time that it has been isolated from a natural source. While 2,7’-cyclolignans and 2,7’-cyclolignan-7-one are well known [15], compound 8 is distinguished by a hydroxyl substitution pattern which has been reported only recently from a natural source [ 191. EXPERIMENTAL General. Mp: uncorr. ‘H and 13CNMR spectra were recorded at 300 and 75 MHz, respectively, using TMS as int. ref. EIMS were measured at 70eV. Plant material. Leaves of V. oleifera (Schott) A. C. Smith were collected by Dr Gentil Godoy (Atlantic Forestal Reserve, Ubatuba, Sao Paulo State, Brazil) in May 1990 and authenticated by Dr Jorge Tamashiro
Lignans from h-da (Department of Botany, Unicamp). A voucher specimen is deposited in the Herbarium of Unicamp, Sgo Paulo, Brazil. Isolation. In order to eliminate chlorophylls and other colouring matter, air-dried powdered leaves (0.8 kg) were extracted with EtOH at room temp. The EtOH extract (24 g) was dild with MeOH-H,O (4: 1), filtered over Celite and extracted first with hexane and then with CH,C12. Both chlorophyll-free extracts (2.7 g from hexane, 2.6 g from CH,Cl,) were fractionated by VLC [8] over silica gel H (40 pm) using hexane and hexane-EtOAc mixts of increasing polarity. Thus, from the chlorophyllfree hexane extract were isolated l(5 mg) and 2 (2 mg); the chlorophyll-free CH,C12 extract gave 3 (14 mg), 4 (20 mg), 5 (15 mg), 6 (3.5 mg), 7 (4 mg) and 8 (1.5 mg). Galbacin (1). Oil, the spectral properties of which were identical with those described in ref. [I3], Eupomatenoid-8 (2). Crystals, mp 40-91” (MeOH). [~]g +42.1” (CHCl,; c 0.202). UV v”,EF” nm (log E): 215 (4.4), 272 (4.1). ‘H NMR (300 MHz, CDCl,) 6: 1.35 (d, J =6.8 Hz, Me-8), 1.86 (dd, J= 1.5,6.6 Hz, Me-8’), 3.31 (dq, J=6.8, 8.8 Hz, H-8), 3.89 (s, OMe), 4.98 (d, J=8.8 Hz, H7), 5.95 (s, OCH,O), 6.10 (dq, J =6.6, 16 Hz, H-8’), 6.35 (d, J=l6 Hz, H-7’), 6.75 (sI, lH), 6.77 (sl, lH), 6.78 (d, J = 8.1 Hz, H-5), 6.87 (dd, J = 1.5, 8.1 Hz, H-6), 6.93 (d, J = 1.5 Hz, H-2). 13C NMR (75.5 MHz, CDCl,) in Table 1. EIMS m/z (rel. int.): 324 [M]+ (lOO), 309 (6), 203 (4), 162 (lo), 149 (16). Aristolignin (3). Crystals, mp 82-83”. [ali +27.5” (CHCl,; c 0.309). UV v:;;” nm (log E): 230 (4.0), 278 (3,6); +NaOH: 234 (3.9), 250 (3,9), 282 (3.6), 294 (sh, 3.5). ‘H NMR (300 MHz, CCl,/D,O): 60.63 (d, J =7.0 Hz, Me-8’), 1.06 (d, J = 6.6 Hz, Me-8), 1.69 (m, H-8), 2.15 (m, HS’), 3.76 (s, OMe), 3.79 (s, OMe), 3.92 (s, OMe), 4.27 (d, J =9.2 Hz, H-7),4.98 (d,J=8.4 Hz, H-7’), 5.31 (s, OH), 6.71 (d, J= 1.2 Hz, H-2’), 6.73 (dd, J= l-2,9.3 Hz, H-6’), 6.74 (d, J=9.3 Hz,H-5’),6.79(d,J=8.2 Hz,H-5), 6.85(dd, J= 1.7, 8.2 Hz, H-6), 6.98 (d, J = 1.7 Hz, H-2). ’ 3C NMR (75.5 MHz, Ccl,) in Table 1. EIMS m/z (rel. int.): 358 CM]’ (48), 206 (46), 192 (lOO), 191 (18), 177 (20), 151 (27). Ole$erin A (4). Oil. [a]kl +41.4” (CHCl,; c 0.828). UV vzFH nm (log&): 229 (3.8), 280 (3.6). ‘H NMR (300 MHz, CCldD,O) 6: 0.77 (d, J=6.8 Hz, Me-8’), 0.92 (d, J =6.8 Hz, Me-8), 1.53 (m, H-8’), 1.58 (~1,OH), 1.60(m, H-8), 2.32 (dcd,J=6.8, 13.7 Hz, H-7’), 2.42 (dd, J=8.4, 13.7 Hz, H-7’), 3.68 (s, OMe-3’), 3.74 (s, OMe-4’), 4.28 (d, J =8.8 Hz, H-7), 5.95 (dd, J= 1.4,3.8 Hz, OCH,O), 6.32 (d, J= 1.8 Hz, H-2’), 6.43 (dd, J = 1.8,8.1 Hz, H-6’), 6.54 (dd, J = 1.8, 8.1 Hz, H-6), 6.55 (d, J = 1.8 Hz, H-2), 6.60 (d, J =8.1 Hz, H-5’), 6.62 (d, J=8.1 Hz, H-5). ‘H NMR (300 MHz, CDCl,): 60.79 (d, J = 6.8 Hz, Me-8’), 1.01 (d, J = 6.8 Hz, Me-8), 1.60(m, H-8), 1.70 (m, H-8’), 1.70 (sl, OH), 2.41 (dd, J=6.8, 13.7 Hz, H-7’), 2.45 (dd, J=8.4, 13.7 Hz, H-7’), 3.80 (s, OMe-3’), 3.86 (s, OMe-4’), 4.37 (d, J =8.8 Hz, H-7), 5.94 (d, J= 1.4 Hz, OCH,O), 6.47 (d, J = 1.7 Hz, H-2’), 6.56 (dd, J= 1.8, 8.1 Hz, H-6’), 6.64 (dd, J = 1.7 Hz, 8.1 Hz, H-6), 6.65 (d, J= 1.7 Hz, H-2), 6.73 (d, J =8.1 Hz, H-5’), 6.74 (d, J =8.1 Hz, H-5). 13C NMR (75.5 MHz, CDCl,) in Table 1. EIMS m/z (rel. int.): 358 [M]’ (32), 340 (2), 151 (lOO), 149 (11).
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Ole$erin B (5). Oil. [z];’ + 54.4” (CHCl,; c 0.469). UV vii:” nm (loge): 221 (3.8), 274 (3.5). ‘H NMR (300 MHz, CCi,,/D,O): 60.77 (d, J =6.7 Hz, Me-8’), 0.93 (d, J = 6.7 Hz, Me-8), 1.40 (sl, OH), 1.52 (m, H-8’), 1.62 (m, H-8), 2.30 (dd, J=6.8, 13.8 Hz, H-7’), 2.38 (dd, J=9.1, 13.8 Hz, H-7’), 3.70 (s, OMe), 3.78 (s, OMe), 4.27 (d, J=8.5 Hz, H7), 5.86 (s, OCH,O), 6.35 (d, lH), 6.37 (dd, J =8.0 Hz, lH), 6.55 (d, J=9.0 Hz, lH), 6.55 (dd, J=9_0Hz, lH), 6.59 (d, lH),664(d, J=8.1 Hz, IH). 13CNMR(75.5MHz,CC1,) in Table 1. EIMS m/z (rel. int.): 348 [MI+ (25), 340 (2), 167 (lOO), 165 (2), 135 (14). OEeiferin C (6). Oil. [a];’ +44.0” (CHCI,; c 0.772). UV ~~~~~”nm (log E): 228 (3.7), 279 (3.7). ‘H NMR (300 MHz, CCl,/D,O): SO.76 (d, J=6.9 Hz, Me-8’), 0.92 (d, J = 6.9 Hz, Me-8), 1.29 (sl, OH), 1.54 (m, H-8’), 1.62 (m, H-8), 2.30 (dd, J=6.9, 13.5 Hz, H-7’), 2.45 (dd, J=8.1, 13.5 Hz, H-7’), 4.33 (d, J=8.1 Hz, H-7), 5.87 (dd, J=O.8, 1.5Hz, OCH,O-3,4), 5.92 (s, OCH,O-3’,4’), 6.37-6.39 (m, 2H), 6.57-6.59(m, 2H), 6.59 (d, Jz8.1 Hz, lH), 6.65 (d,8.4 Hz, 1H). 13C NMR (75.5 MHz, Ccl,) in Table 1. EIMS m/z (rel. int.): 342 [M]’ (181, 324 (3), 151 (lOO), 149 (5), 135
t 16). OEeiferinD (7). Oil. [a]:’ - 52.5” (CHCl,; c 0.162). UV vixen nm: (log&): 228 (3.8), 280 (3.7), 304 (sh, 3.5), 320 (sh, 3.2); +NaOH: 245 (3.6), 290 (3.4), 342 (3.9). ‘H NMR (300 MHz, CCl,/D,O): 60.82 (d, J = 6.0 Hz, Me-8’), 1.18 (d, J=6.6 Hz, Me-8), 2.08 (m, H-8’), 2.10 (dd, J=9.0, 14.7 Hz, H-7’), 2.78 (dd, J=6.0, 14.7 Hz, H-7’), 3.29 (dq, J =4.0, 6.2 Hz, H-8), 4.0 (s, OMe), 5.70 (sl, OH), 5.87 (s, OCH,O), 6.45 (dd, J= 1.5, 7.9 Hz, H-6’), 6.50 (d, J =1.5 Hz, H-2’), 6.59 (d, J=7.9 Hz, H-S), 6.85 (d, J = 8.3 Hz, H-5), 7.40 (dd, J= 1.6, 8.3 Hz, H-6), 7.46 (d, J = 1.6 Hz, H-2). 13C NMR (75.5 MHz, Ccl,) in Table 1. EIMS m/z (rel. int.): 342 [M]’ (S), 180 (lOO), 162 (60), 151 (30), 135 (21). Oleiferin E (8). Needles, mp. 217-219”. [a]? -6.4” (CHCl,; c 0.230). UV 1;:;” nm (loge): 232 (3.9), 285 (3.7). ‘H NMR (300 MHz, CDCl,): 50.98 (d, J = 6.2 Hz, Me-8’), l.l8(d,J=6.2Hz,Me-8),1.43(m,H-8),1.52(m,H-8’),3.50 (d, J=10.2 Hz, H-7’), 4.32 (d, J=9.5 Hz, H-7), 4.36 (sl, OH), 5.63 (d, J= 1.1 Hz, lH, OCH,O-3,4), 5.71 (d, J = 1.1 Hz, lH, OCH,-3,4), 5.92 (s, OCH,O-3’,4’), 6.53 (d, J= 1.7 Hz, H-2’), 6.60 (dd, J= 1.7, 8.0 Hz, H-6’), 6.70 (d, J =8.0 Hz, H-5’), 6.74 (d, J= 8.1 Hz, H-5), 7.16 (d, J = 8.1 Hz, H-6). 13C NMR (75.5 MHz, CDCl,) in Table 1. EIMS m/z (rel. int.): 340 [M]’ (6), 322 (50), 284 (4), 162 (16) 151 (lOO), 249 (36), 135 (27). Acknowledgements-We thank FAPESP and CNPq fur a fellowship to AMAPF and PHF. Thanks are also due to Dr Gentil Godoy (Instituto Agron8mico de CampinasIAC), Dr Jorge Tamashiro (Department of Botany, Unicamp) for the botanical identification, Mrs Paula Pilli and Mrs S6nia Crisostomo for the measurement of ‘H and 13C NMR.
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