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Secoiridoid glucosides fromChelonanthus chelonoides
Pergamon
SECOIRIDOID
0031-9422(94)00!534-6
GLUCOSIDES
Phytochemistry, Vol. 37, No. 6, pp. 1649 1652, 1994 Copyright Q 1994 Elsevkr Science Ltd Printed in Great Bntain. All nghts reserved 0031-9422/94 S7.00 + 0.00
FROM CHELONANTHUS
CHELONOIDES*
YOSHINORI SHIOBARA, KEIKO KATO, YUKARI UEDA, KAZUKO TANIUE, EMI SYOHA, NOBUSHIGE NISHIMOTO,~ FERNANDO DE OLIVEIRA,~ GOKITHI AKISUE,~ MARIA KUBOTA AKISUE$ and GORO HASHIMOTO$ Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Yamashiro-cho, Tokushima 770, Japan; IDepartment de Farmacia da Faculdade de Cii%tciasFarma&uticas da Universidade de SHo Paula, SBo Paula 30786, Brazil; $Centro de Peaquisas Historia Natural, Vila Leopoldina, SHo Paula 05318, Brazil (Received in revisedform 6 June 1994) Key Word Index-Chelonanthus chelonoides; Gentianaceae; dihydrochelonanthoside; sweroside.
sccoiridoid glucoside; chelonanthoside;
Abstract-Two new sccoiridoid glucosideq chelonanthoside and dihydrochelonanthoside, have been isolated from whole plants of Chelonanthus chelonoides along with sweroside. Their structures were elucidated on the basis of chemical and extensive spectral analyses.
INTRODIJCTION
Chelonanthus chelonoides Gilg. is commonly known as Tabacarana’ in Amazonas State, Brazil, and the whole plant has been used as a tonic, a carminative and a stomachic [l-3]. In the course of our investigation on Brazilian medicinal plants [4], our interest in C. chelonoides was initiated because the plant has a bitter taste. The present work describes the isolation and characterization of two new secoiridoid glucosides from C. chelonoides.
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\
RFSULTS AND DISCUSSION
Fractionation of the methanol extract of the whole plants of C. chelonoides as described in the Experimental section resulted in the isolation of two new glucosides, chelonanthoside (1) and dihydrochelonanthoside (2) along with the known secoiridoid glucoside, sweroside (3)
0 Tw
ca
Chelonanthoside (l), C,,H,,O,,, was obtained as a powder. Its UV absorptions, 224 and 244 nm, indicated the presence of two conjugated systems, the latter of which was characteristic of an iridoid enolether system conjugated with a carbonyl group. A comparison of the “C and ‘HNMR spectra of 1 with those of sweroside (3) [6] showed, in the former, the presence of an additional trisubstituted double bond (6c128.8, 141.1; 6,6.95), a conjugated ester carbonyl (6c166.9) and two vinylic methyl groups (6,182, 1.83), and the replacement of the C-7 methylene carbon signal by an acetal carbon signal at
R’ 1. Tgo
R2
2. 3. 4. 5. 6. 7.
:: H AC
(S )-Mho H Tgo (S )-Mho H
AC AC
OH
H
0 (S )-Mbo
*Part of this work was presented at the 114th Annual Meetitq of the Pharmaceutical Society of Japan, Tokyo, March 1994, Abstract Papers(2), p. 196. tAuthor to whom correspondence should he addreaaed.
6, 93.5. Moreover, the CI-mass spectrum of 1 exhibited ions at m/z 457 [M+H]+, 357, 195 and 101. The fragmentations could be explained by the elimination of a tigloyloxy moiety (m/z 357 [(M + H)- lOO]‘) and subsequent loss ofglucose(m/z 195 [(M +H)-(lOO+ 162)]+, in which the fragment ions at m/z 357 and 101 would correspond to the pseudomolecular ions of a dehydro derivative of sweroside [7] and C,H,CO,H, respectively. Dihydrochelonanthoside (2), C,,H,,O,, was obtained as needles. A comparison of the ‘HNMR spectrum of 2 with that of 1 showed that the two vinylic
1649
Y.SHIOBARA et al.
1650
methyl signals in 1 were replaced by a primary and a secondary methyl signal at 6nO.91 and 1.14, respectively. In addition the 13C NMR spectrum indicated the presence of a saturated ester carbonyl group (6cl75.9). The CI-mass spectrum of 2 exhibited ions at m/z 459 [M ions at +Hl+, 357, 195 and 103. The fragmentation m/z 357 [(M+H)-102]+ and at 195 [(M+H)-(102 + 162)]+ suggested that 2 was a dihydro derivative of 1. These results showed that 1 and 2 are 7-acylated derivatives of secologanic acid lactol from (7) [S]. On acetylation 1 and 2 gave tetraacetates 4 and 5, respectively. The compounds foliamenthin, dihydrofoliamenthin and menthiafolin from Menyanthes trifoliata [9, lo] are other examples of esters of 7. The mixture of 1 and 2 was chemically degraded by Battersby’s procedure, i.e. methanolic alkaline hydrolysis, followed by reduction with NaBH, and subsequent acetylation to give sweroside tetraacetate (6) [ll]. On alkaline hydrolysis 1 and 2 furnished (E)-2-methyl-2-butenoic acid (tiglic acid) and 2methylbutanoic acid, respectively. The absolute configuration of 2-methylbutanoic acid was determined as (S) from its positive optical rotation [12]. Assignments of the ‘HNMR spectra of 1, 2, 4-6 (Experimental) were performed by extensive decoupling experiments. More recently, Junior reported the stereochemistries of 7-acyloxyswerosides, menthiafolin [13] and exaltoside [14], in which the orientation of the acyl moiety was assigned by means of the coupling constants of H-7. The ‘HNMR spectra of 1 and 2 indicated that the chemical shifts and coupling constants of H-7 at 6,6.63 (t, J = 2.1 Hz) and at 6n6.61 (t, J = 2.1 Hz), respectively, are in agreement with those of the compounds described above. Furthermore, NOE experiments were carried out using chelonanthoside tetraacetate (4), the ‘H NMR spectrum of which showed an angular methine proton signal (H-5) at 6,3.24 (ddt, J,_,=2.3 Hz; J,_6.=13.6Hz; J5-68 = 5.6 Hz; J, _ 9 = 5.6 Hz) as a well separated signal. When H-5 was irradiated, a methine proton at Su2.70 (H-9) and a vinyl proton of the tigloyloxy moiety at 6n7.08 (H-3”) were enhanced in the NOE difference specta. The above results clearly demonstrated that the lactone ring of 1 and 2 exists in a chair conformation and the acyloxy moieties must be p-oriented. The i3C NMR spectra (Experimental) of 1 and 4 were assigned by the INEPT spectra and comparison with the reported values of exaltoside [14], 6 [15] and tiglic acid [16]. The assignment of the i3C NMR spectrum of 5 was performed by means of r3C-‘H COSY spectrum, based on the assignation of 2. Compounds 1 and 2 were thus deduced as tigloyl and (S)-2-methylbutanoyl esters of the /I-lactol form of secologanic acid (7), respectively, Foliamenthin-type lactol ester derivatives of secoiridoid/open chain monoterpene carboxylic acid have not been encountered outside the Menyanthaceae family [14]. Two compounds from the Gentianaceae plant described above are novel examples of the esters of a secoiridoid/hemiterpene carboxylic acid. EXPERIMENTAL
Mps: uncorr; ‘HNMR (400 MHz) and r3CNMR (100 MHz): TMS as int. standard; TLC: Kiesel gel 60FZS,
precoated silica gel plates (Merck). Spots were visualized by UV light for glucosides and tiglic acid or by spraying with 0.04% soln of Bromocresol Green in 20% EtOH and heating for 2-methylbutanoic acid; HPLC: 25 cm x 10 mm i.d. column of Cosmosil 5C,, (Nacalai tesque) using 30% MeCN, flow rate: 2 ml min- ’ UV: 240 nm; LC: 50 cm x 22 mm i.d. column of silica gel (TLC, Kieselgel 60 H, 15 pm, Merck). Plant muterial. Whole plants of C. chelonoides collected in Manaus, Amazonas State, Brazil and identified by G. H. Samples were deposited in the Herbarium of the Institute of Pharmacognosy, Tokushima Bunri University (voucher specimen No. G-023). Extraction and separation. The air-dried plants (650 g) were extracted with MeOH (1.5 1 x 3). A suspension of the resulting MeOH extract in water was treated with nBuOH (500 ml x 3). The BuOH layer was coned in uacuo to give a brown syrup (94 g), 10 g of which was chromatographed on silica gel (100 g) using CHCl,-MeOH (19: 1) and 20 ml fractions were collected. Fractions 72-84 were collected and coned to give a mixture of chelonanthoside (1) and dihydrochelonanthoside (2) (950 mg) as an amorphous powder, 200 mg of which was subjected to prep. HPLC. The components corresponding to R, 14 and 16 min were collected and lyophilized to afford 1 (I35 mg) and 2 (60 mg), respectively. From fractions 176-214, sweroside (3) was obtained as a powder (70 mg). On acetylation, 3 afforded sweroside tetraacetate (6) as needles, mp 169 -171’ (lit. mp 167- 168” [5]), which was identified by comparison with an authentic sample (‘H, i3CNMR spectra and mmp). Chelonanthoside (1). Powder; [a];‘-52.6” (MeOH; c 1). CI-MS (CH,, pos. ion mode) m/z (rel. int.): 457 [M + H]+(1),439(4),357(9),245(27),195(73),177(13),151 (18), 125 (74), 101 (100) 83 (58); HRCI-MS m/z: 457.1707 (talc for C,,H,,O,,: 457.1710); UV j.EgH mn (logs): 224 (4.02), 244 (3.94); IR ~5:; cm-‘: 3350, 1710, 1620; ‘H NMR (CD,OD): 6 1.82 (3H, br d, J=7.0 Hz, H-4”), 1.83(3H, brs, H-5”), 1.89(1H,dt, J=13.6,2.1 Hz,H-6a), 1.97(1H,ddd,J=13.6,5.3,2.1 Hz,H-68),2.74(1H,ddd,J =9.6,5.3, 1.5 Hz, H-9), 3.21 (lH,dd, J=9.2,7.8 Hz,H-2’) 3.28 (lH, t, J=9.2 Hz, H-4’) 3.33 (lH, ddd, J=9.2, 5.6, 1.8 Hz, H-5’), 3.37 (lH, t, J = 9.2 Hz, H-3’), 3.43 (lH, ddt, J = 13.6,5.3,2.3 Hz, H-5), 3.67 (lH, dd, J= 11.7,5.6 Hz, H6’a), 3.89 (lH, dd, J= 11.7, 1.8 Hz, H-6’b), 4.71 (lH, d, J =7.8 Hz, H-l’), 5.30(1H, dd, J=9.6, 1.8 Hz, H-lOa), 5.33 (lH, dd, J=17.0, 1.8 Hz, H-lob), 5.54 (lH, dt, J=17.0, 9.6 Hz, H-8), 5.59 (lH, d, J= 1.5 Hz, H-l), 6.63 (lH, t, J =2.1 Hz, H-7), 6.95(1H, br q, J=7.0 Hz, H-3”), 7.66(1H, d, J=2.3 Hz, H-3); i3CNMR (CD,OD): 6 12.0 (C-S’), 14.7 (C-4”), 23.0 (C-5), 29.0 (C-6), 43.4 (C-9), 62.6 (C-6’), 71.4 (C-4’), 74.7 (C-2’), 78.1 (C-3’), 78.3 (C-5’), 93.5 (C-7), 98.7(C-1), 100.3 (C-l’), 104.5 (C-4), 121.4(C-lo), 128.8 (C2”), 133.0 (C-8), 141.1 (C-3”), 155.3 (C-3), 166.0 (C-11) 166.9 (C-l”). Dihydrochelonanthoside (2). Needles, mp 159-161”; CUIF - 63.5” (MeOH; c 0.75). CI-MS (CH,, pos. ion mode) m/z (rel. int.): 459 [M + H]+ (3), 441 (5), 357 (lo), 247(17),195(100),177(11),151(15),125(18),103(22),85 (21); HRCI-MS m/z: 459.1872 (talc for C,,H,,O,,: 459.1866); W E.EgH nm (log&): 244 (3.98); IR vi:: cm-i:
Secoiridoid ghcosides from Chelo~~th~ 3350,1730,1710,1620; ‘HNMR (CD,OD): SO.91 (3H, t, J=7.0Hz,H-4”), 1.14(3H,d,J=7.0Hz,H-S’), 1.51 and 1.67 (each lH, septet, J=7.0Hz, H-3”), 1.87 (lH, dt, J =14.2,2.1 Hz, H-&Y), 1.92 (lH, ddd, J=14.2, 5.6, 2.1 Hz, H-6@),2.45 (lH, sextet, J= 7.0 Hz, H-2”), 2.72 (lH, ddd, J =9.6,5.4,1.5 Hz, H-9), 3.21 (lH, dd,J=9.2,7.8 Hz, H-2’), 3.28 (lH, t, J=9.2 Hz, H-4’), 3.30-3.42 (3H, m, H-5, H-3’ and H-S), 3.66 (lH, dd, J= 12.45.4 Hz, H-6’a), 3.89 (lH, dd, J= 12.0, 1.8 Hz, H-6’b), 4.70(1H, d, J=7.8 Hz, H-l’), 5.3O(lH,dd,J=9.6,1.8 Hz,H-lOa),5.33(1H,dd,J=17.1, 1.8 Hz, H-lob), 5.54 (IH, dt, J= 17.1, 9.6 Hz, H-8), 5.59 (lH, d, J--1.5 Hz, H-l), 6.61 (lH, t, J=2.1 Hz, H-7), 7.67 (lH, 4 J=2.3Hz, H-3); 13CNMR (CD,OD): 611.8 (C-4”), 16.8 (C-5”), 22.9 (C-5), 27.6 (C-3”), 28.9 (C-6), 42.0 (C-2”), 43.4 (C-9), 62.6 (C-6’), 71.4 (C-4’), 74.7 (C-2’), 78.1 (C-3’), 78.3 (C-5’), 93.1 (C-7), 98.7(C-1), 100.4 (C-l’), 104.4 (C-4), 121.5 (C-lo), 133.0 (C-S), 155.3 (C-3), 165.9 (C-11), 175.9 (C-l”). Sweroside tetruucetute (6). Needles, mp 169-171”; [u]:: - 170” (CHCI,; c 1). FAB-MS m/z: 527 EM +HJ+; UV l!$TH nm (loge); 242 (3.98); IR vE:13 cm-‘: 1750, 1710, 1620; ‘HNMR (CDCI,): 61.68-1.75 (2H, m, H-6), 1.97, 2.01, 2.04, 2.10 (each 3H, s, OAc), 2.69 (lH, ddd, J=9.5, 6.1, 1.8 Hz, H-9), 2.87 (lH, ddt, J= 11.4,6.1,2.5 Hz, H-5), 3.79 (lH, ddd, J = 9.6,4.6, 2.2 Hz, H-S}, 4.15 (lH, dd, J = 12.4, 2.2 Hz, H-6’a), 4.28-4.35 (2H, m, H-7j?, H-6’b), 4.46 (fH, dt, J=ll.O, 2.8 Hz, H-7a), 4.93 (lH, d, J =8.1 Hz, H-l’), 5.0 (lH, dd, J=9.6, 8.1 Hz, H-2’), 5.10 (lH, t, J=9.6 Hz, H-4’), 5.22-5.32 (4H, m, H-l, H-10, H3’), 5.46 (LH, dt, J=17.2, 9.5 Hz, H-8), 7.56 (lH, d, J =2.5 Hz, H-3); 13C NMR (CDCI,): 620.4 (3 x OMe), 20.6fCOMe), 24.5 (C-6), 27.3 (C-5), 41.9 (C-9), 61.6(C-6’), 68.0 (C-4’), 68.1 (C-7), 70.4 (C-2’), 72.1 (C-3’, S), 95.9 (Cl’), 96.4 (C-l), 105.3 (C-4), 120.9 (C-lo), 131.0 (C-8), 151.3 (C-3), 164.8 (C-11), 169.2, 169.3, 169.8, 170.4 (COMe). Acetylation of the mixture of compounds 1 and 2. The mixture of 1 and 2 (200mg) was acetylated with Ac,O-pyridine in the usual manner. The reaction mixture was subjected to LC using hexane-EtOAc (7: 3) and 20 ml fractions were collected. Fractions 31-39 and 45-60 afforded 5 (65 mg) and 4 (110 mg), respectively. Chelonanthoside detraacetate (4). Powder; [u]f: -47.1” (CHCl,; c 1). FAB-MS: m/z 625 [M +H]+; UV A::” nm (log E): 226 (4.10), 242 (3.98); IR ~2:‘s cm- I: 1750,1710,1620; ‘HNMR (CDCI,): 61.78-1.87 (2H, m, H-6, overlapped with H-4” and H-5”), 1.85 (3H, br d, J = 7.0 Hz, H-4”), 1.87 (3H, br s, H-5”), 1.92,2.01,2.04,2.10 (each 3H, s, OAc), 2.70 (lH, ddd, J=9.5, 5.6 and 1.5 Hz, H-9), 3.24 (lH, ddr, J= 13.6, 5.6 and 2.3 Hz, H-5), 3.78 (lH, ddd, J=9.6, 5.0 and 2.4Hz, H-S), 4.15 (IH, dd, J =12.0 and 2.4 Hz H-6’a), 4.32 (lH, dd, J=12.0 and 5.0 Hz., H&‘b), 4.96 (IH, d, J = 8.0 Hz, H-l’), 5.04 (lH, dd, J=9.6 and 8.0Hz, H-2’), 5.10 (lH, t, J=9.6Hz, H-4’), 5.26 (lH, t, J=9.6Hz, H-3’), 5.32 (lH, dd, J=17.6 and 1.8 Hz, H-lOa), 5.33 (lH, dd, J=9.5, 1.8 Hz, H-lob), 5.38 (IH, 4 J= 1.5 Hz, H-l), 5.47 (lH, dt, J= 17.6 and 9.5 Hz, H-8), 6.69 (lH, t, J=2.2Hz, H-7), 7.08 (lH, br q, J =7.0 Hz, H-3”), 7.61 (lH, d, J=2.3 Hz, H-3); 13C NMR (CDCI,): 6 11.8 (C-S’), 14.5 (C-4”), 20.4 (COMe), 20.5 (2 x COMe), 20.7 (COMe), 21.7 (C-S), 27.9 (C-6), 41.4 (C-9), 61.5 (C-6’), 68.0 (C-4’), 70.0 (C-2’), 72.1 (C-3’ and C-S), PHYTO 37-6-L
chelonoides
1651
91.4(C-7),96.O(C-1’),96.4(C-l), 104.2(C-4), 121.5(C-IO), 127.1 (C-2”), 130.6 (C-8), 140.5 (C-3”), 152.1 (C-3), 162.9 (C-11), 165.3 (C-l”), 168.8, 169.3, 169.8, 170.4 (each COMe). ~ihydroche~o~un~bos~e terracetate (5). Powder; [a$’ - 56.5” (CHC&; c 1). FAB-MS: m/z 627 [M + H]‘; UV kgzH nm (logs): 242 (3.96); IR vZ;~~ cm-? 1750, 1730, 1710, 1620; ‘HNMR (CDCI,): 60.94 (3H, t, J=7.0Hz, H-4”), 1.19 (3H, d, J-:7.0 Hz, H-5”), 1.53, 1.72 (each lH, septet, J=7.0 Hz, H-3”), 1.82 (lH, dt, J= 13.6, 2.2 Hz, H6a), 1.83 (lH, ddd, J=13.6, 5.6, 2.2 Hz, H-6&, 1.95, 2.01, 2.04,2.10 (each 3H, s, OAc), 2.52 (lH, sextet, J=7.0 Hz, H-2”), 2.68 (lH, ddd, J=9.5, 5.6 and 1.5Hz, H-9), 3.22 (lH, ddt, J= 13.6, 5.6 and 2.3 Hz, H-5), 3.78 (lH, ddd, J =9.6, 4.6 and 2.2 Hz, H-5’), 4.16 (lH, dd, J=12.4 and 4.6 Hz, H-6’a), 4.30 (lH, dd, J = 12.4 and 2.2 Hz, H-6’b), 4.94 (lH, d, J = 8.0 Hz, H-l’), 5.04 (lH, dd, J = 9.6 and S.OHz, H-2’), 5.10 (lH, t, J = 9.6Hz, H-4’), 5.25 (lH, t, J = 9.6Hz,H-3’),5.31 (lH,dd,J = 17.6and 1.8Hz,H-lOa), 5.32 (lH, dd, J==9.5, 1.8H2, H-lob), 5.38 (lH, d, J = 1.5 Hz, H-l), 5.46 (lH, dt, J= 17.6 and 9.5 Hz, H-8), 6.65 (IH, t, J=2.2 Hz, H-7), 7.62 (lH, d, J=2.3 Hz, H-3k 13C NMR (CDCI,): 6 11.4 (C-4”), 16.2 (C-5”), 20.1 (COMe), 20.4 (2 x COMe), 20.6 (COMe), 21.8 (C-5), 26.4 (C3”), 27.9 (C-6), 40.7 (C-2”), 41.4 (C-9), 61.6 (C-6’), 68.0 (C4’),70.2 (C-2’), 72.2 (C-3’ and C-S), 91.3 (C-7), 96.0 (C-l’), 96.4 (C-l), 104.3 (C-4), 121.7 (C-lo), 130.6 (C-8), 152.2 (C3), 163.0 (C-11), 174.5 (C-l”), 168.8, 169.3, 169.9, 170.5 (each COMe). Conversion of the mixture of compounds 1 and 2 to sweroside te~ra~etate (6). To a soln of the mixture of 1
and 2 (200 mg) in 50% MeOH (20 ml) was added dropwise 0.1 M NaOH (10 ml). After stirring for 1 hr at room temp., NaBH, (50 mg) was added and the mixture was stirred for 12 hr at room temp. The reaction mixture was neutralized with 1 M NC1 under ice-cooling and extracted with n-BuOH. The residue obtained from the BuOH layer was acetylated with Ac,O-pyridine in the usual manner. The crude product was subjected to silica gel CC using hexane-EtOAc (1: 1) and the eluate was coned and recrystallized from EtOH to give needles (75 mg), mp 169-171”, identical with 6. Aniline hydrolysis o~~omFounds 1 and 2. Compound 1 (50 mg) was added to 0.1 M NaOH (2 ml) and stirred for 1 hr at room temp. The resulting soln was acidified to pH 4 with 1 M HCI and then extracted with CI-I,Cl,. The organic layer was washed with brine and coned. The residue was chromatographed on silica gel using CH,Cl,-MeOH (99: 1) to give tiglic acid (7 mg), plates, mp 63-W, TLC (~HCi3-MeOH, 19 : 1, R, 0.26). Compound 2 (40 mg) was hydrolysed in the same manner to afford (S)-2-methylbutanoic acid (6 mg), oil, [a]$’ +16.1” (MeOH; c 0.3) (lit. [a]:: f 16.3” [12]), TLC (CHCl,-MeOH, lP:l, R, 0.24). Each compound was identified with an authentic specimen by comparison of the “H NMR spectrum and TLC.
Acknowledgements-We are grateful to Prof. Emeritus H. Inouye, Kyoto University, and Prof. K. Inoue, Gifu Pharmaceutical University, for a generous supply of an
Y. SHIOBARAet ul.
1652
authentic sample of sweroside tetraacetate. We also thank Miss Y. Okamoto of this university for the measurement of high resolution mass spectra. REFERENCES
1. Pio Corria, M. (1952) Dictionirrio das Plantas cteis do Brasil, Vol. 3, p. 377. Imprensa National, Rio de Janeiro. 2. Amorozo, M. C. M. and GCly, A. (1988) Boletim Paraense Emilio Goeldi, Se?ie Botijnica 4, 87. 3. Amorozo, M. C. M. and Gily, A. (1988) Boletim Paraense Emilio Goeldi, Se’rie Bota^nica 4, 123. 4. Shiobara, Y., Inoue, S., Kato, K., Nishiguchi, Y., Oishi, Y., Nishimoto, N., Oliveira, F., Akisue, G., Akisue, M. K. and Hashimoto, G. (1993) Phytochemistry 32, 1527. 5. Inouye, H., Ueda, S. and Nakamura, Y. (1970) Chem. Pharm. Bull. 18, 1856. 6. van Beek, T. A., Lankhorst, P. P., Verpoorte, R. and Baerheim Svendsen, A. (1982) Planta Med. 44, 30.
7. Shaufelberger, D., Domon, B. and Hostettmann, K. (1984) Planta Med. 50, 398. 8. Guarnaccia, R., Botta, L. and Coscia, C. J. (1974) J. Am. Chem. Sot. 96, 7079. 9. Loew, P., Sczepanski, Ch. V., Coscia, C. J. and Arigoni, D. (1968) J. Chem. Sot., Chem. Commun. 1276. 10. Battersby, A. R., Burnett, A. R., Knowles, G. D. and Parsons, P. G. (1968) J. Chem. Sot., Chem. Commun. 1277. 11. Battersby, A. R., Burnett, A. R. and Parsons, P. G. (1969) J. Chem. Sot. (C) 1187. 12. Yoshikawa, K., Nakagawa, M., Yamamoto, R., Arihara, S. and Matsuura, K. (1992) Chem. Pharm. Bull. 40, 1779. 13. Junior, P. (1989) Planta Med. 55, 83. 14. Junior, P. (1991) Planta Med. 57, 181. 15. El-Naggar, L. J., Beal, J. L. and Doskotch, R. W. (1982) J. Nat. Prod. 45, 539. 16. Brouwer, H. and Stothers, J. B. (1972) Can. J. Chem. 50, 601.
SECOIRIDOID
0031-9422(94)00!534-6
GLUCOSIDES
Phytochemistry, Vol. 37, No. 6, pp. 1649 1652, 1994 Copyright Q 1994 Elsevkr Science Ltd Printed in Great Bntain. All nghts reserved 0031-9422/94 S7.00 + 0.00
FROM CHELONANTHUS
CHELONOIDES*
YOSHINORI SHIOBARA, KEIKO KATO, YUKARI UEDA, KAZUKO TANIUE, EMI SYOHA, NOBUSHIGE NISHIMOTO,~ FERNANDO DE OLIVEIRA,~ GOKITHI AKISUE,~ MARIA KUBOTA AKISUE$ and GORO HASHIMOTO$ Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Yamashiro-cho, Tokushima 770, Japan; IDepartment de Farmacia da Faculdade de Cii%tciasFarma&uticas da Universidade de SHo Paula, SBo Paula 30786, Brazil; $Centro de Peaquisas Historia Natural, Vila Leopoldina, SHo Paula 05318, Brazil (Received in revisedform 6 June 1994) Key Word Index-Chelonanthus chelonoides; Gentianaceae; dihydrochelonanthoside; sweroside.
sccoiridoid glucoside; chelonanthoside;
Abstract-Two new sccoiridoid glucosideq chelonanthoside and dihydrochelonanthoside, have been isolated from whole plants of Chelonanthus chelonoides along with sweroside. Their structures were elucidated on the basis of chemical and extensive spectral analyses.
INTRODIJCTION
Chelonanthus chelonoides Gilg. is commonly known as Tabacarana’ in Amazonas State, Brazil, and the whole plant has been used as a tonic, a carminative and a stomachic [l-3]. In the course of our investigation on Brazilian medicinal plants [4], our interest in C. chelonoides was initiated because the plant has a bitter taste. The present work describes the isolation and characterization of two new secoiridoid glucosides from C. chelonoides.
‘OR2
\
RFSULTS AND DISCUSSION
Fractionation of the methanol extract of the whole plants of C. chelonoides as described in the Experimental section resulted in the isolation of two new glucosides, chelonanthoside (1) and dihydrochelonanthoside (2) along with the known secoiridoid glucoside, sweroside (3)
0 Tw
ca
Chelonanthoside (l), C,,H,,O,,, was obtained as a powder. Its UV absorptions, 224 and 244 nm, indicated the presence of two conjugated systems, the latter of which was characteristic of an iridoid enolether system conjugated with a carbonyl group. A comparison of the “C and ‘HNMR spectra of 1 with those of sweroside (3) [6] showed, in the former, the presence of an additional trisubstituted double bond (6c128.8, 141.1; 6,6.95), a conjugated ester carbonyl (6c166.9) and two vinylic methyl groups (6,182, 1.83), and the replacement of the C-7 methylene carbon signal by an acetal carbon signal at
R’ 1. Tgo
R2
2. 3. 4. 5. 6. 7.
:: H AC
(S )-Mho H Tgo (S )-Mho H
AC AC
OH
H
0 (S )-Mbo
*Part of this work was presented at the 114th Annual Meetitq of the Pharmaceutical Society of Japan, Tokyo, March 1994, Abstract Papers(2), p. 196. tAuthor to whom correspondence should he addreaaed.
6, 93.5. Moreover, the CI-mass spectrum of 1 exhibited ions at m/z 457 [M+H]+, 357, 195 and 101. The fragmentations could be explained by the elimination of a tigloyloxy moiety (m/z 357 [(M + H)- lOO]‘) and subsequent loss ofglucose(m/z 195 [(M +H)-(lOO+ 162)]+, in which the fragment ions at m/z 357 and 101 would correspond to the pseudomolecular ions of a dehydro derivative of sweroside [7] and C,H,CO,H, respectively. Dihydrochelonanthoside (2), C,,H,,O,, was obtained as needles. A comparison of the ‘HNMR spectrum of 2 with that of 1 showed that the two vinylic
1649
Y.SHIOBARA et al.
1650
methyl signals in 1 were replaced by a primary and a secondary methyl signal at 6nO.91 and 1.14, respectively. In addition the 13C NMR spectrum indicated the presence of a saturated ester carbonyl group (6cl75.9). The CI-mass spectrum of 2 exhibited ions at m/z 459 [M ions at +Hl+, 357, 195 and 103. The fragmentation m/z 357 [(M+H)-102]+ and at 195 [(M+H)-(102 + 162)]+ suggested that 2 was a dihydro derivative of 1. These results showed that 1 and 2 are 7-acylated derivatives of secologanic acid lactol from (7) [S]. On acetylation 1 and 2 gave tetraacetates 4 and 5, respectively. The compounds foliamenthin, dihydrofoliamenthin and menthiafolin from Menyanthes trifoliata [9, lo] are other examples of esters of 7. The mixture of 1 and 2 was chemically degraded by Battersby’s procedure, i.e. methanolic alkaline hydrolysis, followed by reduction with NaBH, and subsequent acetylation to give sweroside tetraacetate (6) [ll]. On alkaline hydrolysis 1 and 2 furnished (E)-2-methyl-2-butenoic acid (tiglic acid) and 2methylbutanoic acid, respectively. The absolute configuration of 2-methylbutanoic acid was determined as (S) from its positive optical rotation [12]. Assignments of the ‘HNMR spectra of 1, 2, 4-6 (Experimental) were performed by extensive decoupling experiments. More recently, Junior reported the stereochemistries of 7-acyloxyswerosides, menthiafolin [13] and exaltoside [14], in which the orientation of the acyl moiety was assigned by means of the coupling constants of H-7. The ‘HNMR spectra of 1 and 2 indicated that the chemical shifts and coupling constants of H-7 at 6,6.63 (t, J = 2.1 Hz) and at 6n6.61 (t, J = 2.1 Hz), respectively, are in agreement with those of the compounds described above. Furthermore, NOE experiments were carried out using chelonanthoside tetraacetate (4), the ‘H NMR spectrum of which showed an angular methine proton signal (H-5) at 6,3.24 (ddt, J,_,=2.3 Hz; J,_6.=13.6Hz; J5-68 = 5.6 Hz; J, _ 9 = 5.6 Hz) as a well separated signal. When H-5 was irradiated, a methine proton at Su2.70 (H-9) and a vinyl proton of the tigloyloxy moiety at 6n7.08 (H-3”) were enhanced in the NOE difference specta. The above results clearly demonstrated that the lactone ring of 1 and 2 exists in a chair conformation and the acyloxy moieties must be p-oriented. The i3C NMR spectra (Experimental) of 1 and 4 were assigned by the INEPT spectra and comparison with the reported values of exaltoside [14], 6 [15] and tiglic acid [16]. The assignment of the i3C NMR spectrum of 5 was performed by means of r3C-‘H COSY spectrum, based on the assignation of 2. Compounds 1 and 2 were thus deduced as tigloyl and (S)-2-methylbutanoyl esters of the /I-lactol form of secologanic acid (7), respectively, Foliamenthin-type lactol ester derivatives of secoiridoid/open chain monoterpene carboxylic acid have not been encountered outside the Menyanthaceae family [14]. Two compounds from the Gentianaceae plant described above are novel examples of the esters of a secoiridoid/hemiterpene carboxylic acid. EXPERIMENTAL
Mps: uncorr; ‘HNMR (400 MHz) and r3CNMR (100 MHz): TMS as int. standard; TLC: Kiesel gel 60FZS,
precoated silica gel plates (Merck). Spots were visualized by UV light for glucosides and tiglic acid or by spraying with 0.04% soln of Bromocresol Green in 20% EtOH and heating for 2-methylbutanoic acid; HPLC: 25 cm x 10 mm i.d. column of Cosmosil 5C,, (Nacalai tesque) using 30% MeCN, flow rate: 2 ml min- ’ UV: 240 nm; LC: 50 cm x 22 mm i.d. column of silica gel (TLC, Kieselgel 60 H, 15 pm, Merck). Plant muterial. Whole plants of C. chelonoides collected in Manaus, Amazonas State, Brazil and identified by G. H. Samples were deposited in the Herbarium of the Institute of Pharmacognosy, Tokushima Bunri University (voucher specimen No. G-023). Extraction and separation. The air-dried plants (650 g) were extracted with MeOH (1.5 1 x 3). A suspension of the resulting MeOH extract in water was treated with nBuOH (500 ml x 3). The BuOH layer was coned in uacuo to give a brown syrup (94 g), 10 g of which was chromatographed on silica gel (100 g) using CHCl,-MeOH (19: 1) and 20 ml fractions were collected. Fractions 72-84 were collected and coned to give a mixture of chelonanthoside (1) and dihydrochelonanthoside (2) (950 mg) as an amorphous powder, 200 mg of which was subjected to prep. HPLC. The components corresponding to R, 14 and 16 min were collected and lyophilized to afford 1 (I35 mg) and 2 (60 mg), respectively. From fractions 176-214, sweroside (3) was obtained as a powder (70 mg). On acetylation, 3 afforded sweroside tetraacetate (6) as needles, mp 169 -171’ (lit. mp 167- 168” [5]), which was identified by comparison with an authentic sample (‘H, i3CNMR spectra and mmp). Chelonanthoside (1). Powder; [a];‘-52.6” (MeOH; c 1). CI-MS (CH,, pos. ion mode) m/z (rel. int.): 457 [M + H]+(1),439(4),357(9),245(27),195(73),177(13),151 (18), 125 (74), 101 (100) 83 (58); HRCI-MS m/z: 457.1707 (talc for C,,H,,O,,: 457.1710); UV j.EgH mn (logs): 224 (4.02), 244 (3.94); IR ~5:; cm-‘: 3350, 1710, 1620; ‘H NMR (CD,OD): 6 1.82 (3H, br d, J=7.0 Hz, H-4”), 1.83(3H, brs, H-5”), 1.89(1H,dt, J=13.6,2.1 Hz,H-6a), 1.97(1H,ddd,J=13.6,5.3,2.1 Hz,H-68),2.74(1H,ddd,J =9.6,5.3, 1.5 Hz, H-9), 3.21 (lH,dd, J=9.2,7.8 Hz,H-2’) 3.28 (lH, t, J=9.2 Hz, H-4’) 3.33 (lH, ddd, J=9.2, 5.6, 1.8 Hz, H-5’), 3.37 (lH, t, J = 9.2 Hz, H-3’), 3.43 (lH, ddt, J = 13.6,5.3,2.3 Hz, H-5), 3.67 (lH, dd, J= 11.7,5.6 Hz, H6’a), 3.89 (lH, dd, J= 11.7, 1.8 Hz, H-6’b), 4.71 (lH, d, J =7.8 Hz, H-l’), 5.30(1H, dd, J=9.6, 1.8 Hz, H-lOa), 5.33 (lH, dd, J=17.0, 1.8 Hz, H-lob), 5.54 (lH, dt, J=17.0, 9.6 Hz, H-8), 5.59 (lH, d, J= 1.5 Hz, H-l), 6.63 (lH, t, J =2.1 Hz, H-7), 6.95(1H, br q, J=7.0 Hz, H-3”), 7.66(1H, d, J=2.3 Hz, H-3); i3CNMR (CD,OD): 6 12.0 (C-S’), 14.7 (C-4”), 23.0 (C-5), 29.0 (C-6), 43.4 (C-9), 62.6 (C-6’), 71.4 (C-4’), 74.7 (C-2’), 78.1 (C-3’), 78.3 (C-5’), 93.5 (C-7), 98.7(C-1), 100.3 (C-l’), 104.5 (C-4), 121.4(C-lo), 128.8 (C2”), 133.0 (C-8), 141.1 (C-3”), 155.3 (C-3), 166.0 (C-11) 166.9 (C-l”). Dihydrochelonanthoside (2). Needles, mp 159-161”; CUIF - 63.5” (MeOH; c 0.75). CI-MS (CH,, pos. ion mode) m/z (rel. int.): 459 [M + H]+ (3), 441 (5), 357 (lo), 247(17),195(100),177(11),151(15),125(18),103(22),85 (21); HRCI-MS m/z: 459.1872 (talc for C,,H,,O,,: 459.1866); W E.EgH nm (log&): 244 (3.98); IR vi:: cm-i:
Secoiridoid ghcosides from Chelo~~th~ 3350,1730,1710,1620; ‘HNMR (CD,OD): SO.91 (3H, t, J=7.0Hz,H-4”), 1.14(3H,d,J=7.0Hz,H-S’), 1.51 and 1.67 (each lH, septet, J=7.0Hz, H-3”), 1.87 (lH, dt, J =14.2,2.1 Hz, H-&Y), 1.92 (lH, ddd, J=14.2, 5.6, 2.1 Hz, H-6@),2.45 (lH, sextet, J= 7.0 Hz, H-2”), 2.72 (lH, ddd, J =9.6,5.4,1.5 Hz, H-9), 3.21 (lH, dd,J=9.2,7.8 Hz, H-2’), 3.28 (lH, t, J=9.2 Hz, H-4’), 3.30-3.42 (3H, m, H-5, H-3’ and H-S), 3.66 (lH, dd, J= 12.45.4 Hz, H-6’a), 3.89 (lH, dd, J= 12.0, 1.8 Hz, H-6’b), 4.70(1H, d, J=7.8 Hz, H-l’), 5.3O(lH,dd,J=9.6,1.8 Hz,H-lOa),5.33(1H,dd,J=17.1, 1.8 Hz, H-lob), 5.54 (IH, dt, J= 17.1, 9.6 Hz, H-8), 5.59 (lH, d, J--1.5 Hz, H-l), 6.61 (lH, t, J=2.1 Hz, H-7), 7.67 (lH, 4 J=2.3Hz, H-3); 13CNMR (CD,OD): 611.8 (C-4”), 16.8 (C-5”), 22.9 (C-5), 27.6 (C-3”), 28.9 (C-6), 42.0 (C-2”), 43.4 (C-9), 62.6 (C-6’), 71.4 (C-4’), 74.7 (C-2’), 78.1 (C-3’), 78.3 (C-5’), 93.1 (C-7), 98.7(C-1), 100.4 (C-l’), 104.4 (C-4), 121.5 (C-lo), 133.0 (C-S), 155.3 (C-3), 165.9 (C-11), 175.9 (C-l”). Sweroside tetruucetute (6). Needles, mp 169-171”; [u]:: - 170” (CHCI,; c 1). FAB-MS m/z: 527 EM +HJ+; UV l!$TH nm (loge); 242 (3.98); IR vE:13 cm-‘: 1750, 1710, 1620; ‘HNMR (CDCI,): 61.68-1.75 (2H, m, H-6), 1.97, 2.01, 2.04, 2.10 (each 3H, s, OAc), 2.69 (lH, ddd, J=9.5, 6.1, 1.8 Hz, H-9), 2.87 (lH, ddt, J= 11.4,6.1,2.5 Hz, H-5), 3.79 (lH, ddd, J = 9.6,4.6, 2.2 Hz, H-S}, 4.15 (lH, dd, J = 12.4, 2.2 Hz, H-6’a), 4.28-4.35 (2H, m, H-7j?, H-6’b), 4.46 (fH, dt, J=ll.O, 2.8 Hz, H-7a), 4.93 (lH, d, J =8.1 Hz, H-l’), 5.0 (lH, dd, J=9.6, 8.1 Hz, H-2’), 5.10 (lH, t, J=9.6 Hz, H-4’), 5.22-5.32 (4H, m, H-l, H-10, H3’), 5.46 (LH, dt, J=17.2, 9.5 Hz, H-8), 7.56 (lH, d, J =2.5 Hz, H-3); 13C NMR (CDCI,): 620.4 (3 x OMe), 20.6fCOMe), 24.5 (C-6), 27.3 (C-5), 41.9 (C-9), 61.6(C-6’), 68.0 (C-4’), 68.1 (C-7), 70.4 (C-2’), 72.1 (C-3’, S), 95.9 (Cl’), 96.4 (C-l), 105.3 (C-4), 120.9 (C-lo), 131.0 (C-8), 151.3 (C-3), 164.8 (C-11), 169.2, 169.3, 169.8, 170.4 (COMe). Acetylation of the mixture of compounds 1 and 2. The mixture of 1 and 2 (200mg) was acetylated with Ac,O-pyridine in the usual manner. The reaction mixture was subjected to LC using hexane-EtOAc (7: 3) and 20 ml fractions were collected. Fractions 31-39 and 45-60 afforded 5 (65 mg) and 4 (110 mg), respectively. Chelonanthoside detraacetate (4). Powder; [u]f: -47.1” (CHCl,; c 1). FAB-MS: m/z 625 [M +H]+; UV A::” nm (log E): 226 (4.10), 242 (3.98); IR ~2:‘s cm- I: 1750,1710,1620; ‘HNMR (CDCI,): 61.78-1.87 (2H, m, H-6, overlapped with H-4” and H-5”), 1.85 (3H, br d, J = 7.0 Hz, H-4”), 1.87 (3H, br s, H-5”), 1.92,2.01,2.04,2.10 (each 3H, s, OAc), 2.70 (lH, ddd, J=9.5, 5.6 and 1.5 Hz, H-9), 3.24 (lH, ddr, J= 13.6, 5.6 and 2.3 Hz, H-5), 3.78 (lH, ddd, J=9.6, 5.0 and 2.4Hz, H-S), 4.15 (IH, dd, J =12.0 and 2.4 Hz H-6’a), 4.32 (lH, dd, J=12.0 and 5.0 Hz., H&‘b), 4.96 (IH, d, J = 8.0 Hz, H-l’), 5.04 (lH, dd, J=9.6 and 8.0Hz, H-2’), 5.10 (lH, t, J=9.6Hz, H-4’), 5.26 (lH, t, J=9.6Hz, H-3’), 5.32 (lH, dd, J=17.6 and 1.8 Hz, H-lOa), 5.33 (lH, dd, J=9.5, 1.8 Hz, H-lob), 5.38 (IH, 4 J= 1.5 Hz, H-l), 5.47 (lH, dt, J= 17.6 and 9.5 Hz, H-8), 6.69 (lH, t, J=2.2Hz, H-7), 7.08 (lH, br q, J =7.0 Hz, H-3”), 7.61 (lH, d, J=2.3 Hz, H-3); 13C NMR (CDCI,): 6 11.8 (C-S’), 14.5 (C-4”), 20.4 (COMe), 20.5 (2 x COMe), 20.7 (COMe), 21.7 (C-S), 27.9 (C-6), 41.4 (C-9), 61.5 (C-6’), 68.0 (C-4’), 70.0 (C-2’), 72.1 (C-3’ and C-S), PHYTO 37-6-L
chelonoides
1651
91.4(C-7),96.O(C-1’),96.4(C-l), 104.2(C-4), 121.5(C-IO), 127.1 (C-2”), 130.6 (C-8), 140.5 (C-3”), 152.1 (C-3), 162.9 (C-11), 165.3 (C-l”), 168.8, 169.3, 169.8, 170.4 (each COMe). ~ihydroche~o~un~bos~e terracetate (5). Powder; [a$’ - 56.5” (CHC&; c 1). FAB-MS: m/z 627 [M + H]‘; UV kgzH nm (logs): 242 (3.96); IR vZ;~~ cm-? 1750, 1730, 1710, 1620; ‘HNMR (CDCI,): 60.94 (3H, t, J=7.0Hz, H-4”), 1.19 (3H, d, J-:7.0 Hz, H-5”), 1.53, 1.72 (each lH, septet, J=7.0 Hz, H-3”), 1.82 (lH, dt, J= 13.6, 2.2 Hz, H6a), 1.83 (lH, ddd, J=13.6, 5.6, 2.2 Hz, H-6&, 1.95, 2.01, 2.04,2.10 (each 3H, s, OAc), 2.52 (lH, sextet, J=7.0 Hz, H-2”), 2.68 (lH, ddd, J=9.5, 5.6 and 1.5Hz, H-9), 3.22 (lH, ddt, J= 13.6, 5.6 and 2.3 Hz, H-5), 3.78 (lH, ddd, J =9.6, 4.6 and 2.2 Hz, H-5’), 4.16 (lH, dd, J=12.4 and 4.6 Hz, H-6’a), 4.30 (lH, dd, J = 12.4 and 2.2 Hz, H-6’b), 4.94 (lH, d, J = 8.0 Hz, H-l’), 5.04 (lH, dd, J = 9.6 and S.OHz, H-2’), 5.10 (lH, t, J = 9.6Hz, H-4’), 5.25 (lH, t, J = 9.6Hz,H-3’),5.31 (lH,dd,J = 17.6and 1.8Hz,H-lOa), 5.32 (lH, dd, J==9.5, 1.8H2, H-lob), 5.38 (lH, d, J = 1.5 Hz, H-l), 5.46 (lH, dt, J= 17.6 and 9.5 Hz, H-8), 6.65 (IH, t, J=2.2 Hz, H-7), 7.62 (lH, d, J=2.3 Hz, H-3k 13C NMR (CDCI,): 6 11.4 (C-4”), 16.2 (C-5”), 20.1 (COMe), 20.4 (2 x COMe), 20.6 (COMe), 21.8 (C-5), 26.4 (C3”), 27.9 (C-6), 40.7 (C-2”), 41.4 (C-9), 61.6 (C-6’), 68.0 (C4’),70.2 (C-2’), 72.2 (C-3’ and C-S), 91.3 (C-7), 96.0 (C-l’), 96.4 (C-l), 104.3 (C-4), 121.7 (C-lo), 130.6 (C-8), 152.2 (C3), 163.0 (C-11), 174.5 (C-l”), 168.8, 169.3, 169.9, 170.5 (each COMe). Conversion of the mixture of compounds 1 and 2 to sweroside te~ra~etate (6). To a soln of the mixture of 1
and 2 (200 mg) in 50% MeOH (20 ml) was added dropwise 0.1 M NaOH (10 ml). After stirring for 1 hr at room temp., NaBH, (50 mg) was added and the mixture was stirred for 12 hr at room temp. The reaction mixture was neutralized with 1 M NC1 under ice-cooling and extracted with n-BuOH. The residue obtained from the BuOH layer was acetylated with Ac,O-pyridine in the usual manner. The crude product was subjected to silica gel CC using hexane-EtOAc (1: 1) and the eluate was coned and recrystallized from EtOH to give needles (75 mg), mp 169-171”, identical with 6. Aniline hydrolysis o~~omFounds 1 and 2. Compound 1 (50 mg) was added to 0.1 M NaOH (2 ml) and stirred for 1 hr at room temp. The resulting soln was acidified to pH 4 with 1 M HCI and then extracted with CI-I,Cl,. The organic layer was washed with brine and coned. The residue was chromatographed on silica gel using CH,Cl,-MeOH (99: 1) to give tiglic acid (7 mg), plates, mp 63-W, TLC (~HCi3-MeOH, 19 : 1, R, 0.26). Compound 2 (40 mg) was hydrolysed in the same manner to afford (S)-2-methylbutanoic acid (6 mg), oil, [a]$’ +16.1” (MeOH; c 0.3) (lit. [a]:: f 16.3” [12]), TLC (CHCl,-MeOH, lP:l, R, 0.24). Each compound was identified with an authentic specimen by comparison of the “H NMR spectrum and TLC.
Acknowledgements-We are grateful to Prof. Emeritus H. Inouye, Kyoto University, and Prof. K. Inoue, Gifu Pharmaceutical University, for a generous supply of an
Y. SHIOBARAet ul.
1652
authentic sample of sweroside tetraacetate. We also thank Miss Y. Okamoto of this university for the measurement of high resolution mass spectra. REFERENCES
1. Pio Corria, M. (1952) Dictionirrio das Plantas cteis do Brasil, Vol. 3, p. 377. Imprensa National, Rio de Janeiro. 2. Amorozo, M. C. M. and GCly, A. (1988) Boletim Paraense Emilio Goeldi, Se?ie Botijnica 4, 87. 3. Amorozo, M. C. M. and Gily, A. (1988) Boletim Paraense Emilio Goeldi, Se’rie Bota^nica 4, 123. 4. Shiobara, Y., Inoue, S., Kato, K., Nishiguchi, Y., Oishi, Y., Nishimoto, N., Oliveira, F., Akisue, G., Akisue, M. K. and Hashimoto, G. (1993) Phytochemistry 32, 1527. 5. Inouye, H., Ueda, S. and Nakamura, Y. (1970) Chem. Pharm. Bull. 18, 1856. 6. van Beek, T. A., Lankhorst, P. P., Verpoorte, R. and Baerheim Svendsen, A. (1982) Planta Med. 44, 30.
7. Shaufelberger, D., Domon, B. and Hostettmann, K. (1984) Planta Med. 50, 398. 8. Guarnaccia, R., Botta, L. and Coscia, C. J. (1974) J. Am. Chem. Sot. 96, 7079. 9. Loew, P., Sczepanski, Ch. V., Coscia, C. J. and Arigoni, D. (1968) J. Chem. Sot., Chem. Commun. 1276. 10. Battersby, A. R., Burnett, A. R., Knowles, G. D. and Parsons, P. G. (1968) J. Chem. Sot., Chem. Commun. 1277. 11. Battersby, A. R., Burnett, A. R. and Parsons, P. G. (1969) J. Chem. Sot. (C) 1187. 12. Yoshikawa, K., Nakagawa, M., Yamamoto, R., Arihara, S. and Matsuura, K. (1992) Chem. Pharm. Bull. 40, 1779. 13. Junior, P. (1989) Planta Med. 55, 83. 14. Junior, P. (1991) Planta Med. 57, 181. 15. El-Naggar, L. J., Beal, J. L. and Doskotch, R. W. (1982) J. Nat. Prod. 45, 539. 16. Brouwer, H. and Stothers, J. B. (1972) Can. J. Chem. 50, 601.