Sesquiterpene pyridine alkaloids from Peritassa campestris

Phytochemistry 58 (2001) 1205–1207 www.elsevier.com/locate/phytochem Sesquiterpene pyridine alkaloids from Peritassa campestris Luciano M. Liãoa,*, Paulo C. Vieirab, Edson Rodrigues-Filhob, João B. Fernandesb, Maria F.G.F. da Silvab a Instituto de Quı´mica, Universidade Federal de Goiás, C.P. 131, 74001-970 Goiânia, GO, Brazil Departamento de Quı´mica, Universidade Federal de São Carlos, C.P. 676, 13565-905 São Carlos, SP, Brazil b Received 20 December 2000; received in revised form 14 May 2001 Abstract An investigation of the methanol and ethyl acetate extracts from the roots of Peritassa campestris (Hippocrateaceae) afforded the sesquiterpene pyridine alkaloid, 4-hydroxy-7-epi-chuchuhuanine E-V, and nine known alkaloids, forrestine, euonimine, ebenifoline E-I, wilforine, euojaponine F, euonine, wilforjine, neowilforine, and wilforzine. The structures of the isolates were elucidated on the basis of spectral data, particularly HMQC and HMBC experiments. # 2001 Published by Elsevier Science Ltd. Keywords: Peritassa campestris; Hippocrateaceae; Sesquiterpene pyridine alkaloids; 4-Hydroxy-7-epi-chuchuhuanine E-V 1. Introduction Sesquiterpene pyridine alkaloids, based on a highly oxygenated dihydro-b-agarofuran core on a sesquiterpene moiety, and evoninoate or wilfordate esters on their alkaloid moiety, constitute a relatively small group of natural products frequently found in plants of the Celastraceae family (Brüning and Wagner, 1978). Recently, these alkaloids have also been described in plants of the Hippocrateaceae (Mata et al., 1990; Lião et al., 1997). This chemical aspect reinforces the recent botanical classification in which the two families, Celastraceae and Hippocrateaceae, appear to be grouped in the Celastraceae (Brüning and Wagner, 1978; Heywood, 1993). These alkaloids have also been of interest due to their cytotoxicity against several human tumour cell lines (Kuo et al., 1994), and their insect antifeedant, and insecticidal activities (Shirota et al., 1994). Sesquiterpene pyridine alkaloids isolated from Tripterygium species have also shown immunosuppressive activity (Zheng et al., 1989). As part of our continuing search for bioactive compounds from plants, especially from the savanna, we investigated the MeOH and EtOAc soluble portions of the crude extract of the roots of Peritassa campestris Cambess. (Hippocrateaceae), a plant that occurs in the * Corresponding author. Tel.: +55-62-521-1008; fax: +55-62-5211008. E-mail address: [email protected] (L.M. Lião). Brazilian savanna (Cerrado), popularly known as ‘‘bacupari do campo’’, and which is used in folk medicine for wound healing in the form of a decoction (Septı́mio, 1994). In this paper we report the isolation and structure elucidation of a new sesquiterpene pyridine alkaloid, 4-hydroxy-7-epi-chuchuhuanine E-V (1), and the isolation of nine known alkaloids forrestine (Chunquan et al., 1992), euonimine, ebenifoline E-I, euojaponine F, euonine (Itokawa et al., 1993), wilforine (Beroza, 1951), wilforjine (Deng et al., 1992), neowilforine (He et al., 1989), and wilforzine (He et al., 1987). 2. Results and discussion 4-Hydroxy-7-epi-chuchuhuanine E-V (1) was isolated as a colorless amorphous solid, in low yield, and its IR spectrum displayed absorptions at 3614, 1740, and 1583 cm
1. The 1H and 13C NMR spectra suggested a dihydro-b-agarofuran sesquiterpene skeleton with an evoninic diester bridge. The sesquiterpene moiety was characterised in the 1H NMR spectrum by tertiary methyl groups at
1.67 (3H, s, Me-14) and
1.58 (3H, d, J=1.2 Hz, Me-12), coupling with one hydrogen-bonded hydroxyl group at
4.50 (q, J=1.2 Hz, OH-4), two sets of methylene protons at
4.62 (d, J=13.6 Hz, H-11a),
5.28 (d, J=13.6 Hz, H-11b),
3.67 (d, J=11.6 Hz, H15a) and
5.98 (d, J=11.6 Hz, H-15b), seven methine protons at
5.43 (d, J=4.0 Hz, H-1), 4.07 (dd, J=4.0 0031-9422/01/$ - see front matter # 2001 Published by Elsevier Science Ltd. PII: S0031-9422(01)00315-6 1206 L.M. Lião et al. / Phytochemistry 58 (2001) 1205–1207 and 2.4 Hz, H-2), 4.76 (d, J=2.4 Hz, H-3), 7.03 (s, H-5), 2.33 (d, J=4.0 Hz, H-6), 5.53 (dd, J=6.0 and 4.0 Hz, H7) and 5.35 (d, J=6.0 Hz, H-8), and five acetyl groups at
2.31, 2.21, 2.15, 2.01 and 1.95. A further oxygenated functional group was assumed to be a hydroxyl group, based on a broad singlet at
3.65 (exchanged with D2O). The evoninic acid fragment in 1 was assigned by the signals at
8.05 (dd, J=8.0 and 2.0 Hz, H-40 ), 7.26 (dd, J=8.0 and 4.8 Hz, H-50 ) and 8.69 (dd, J=4.8 and 2.0 Hz, H-60 ), corresponding to a 2,3-disubstituted pyridine unit. It also contains two secondary methyl groups at
1.38 (d, J=6.8 Hz, Me-90 ) and 1.15 (d, J=7.2 Hz, Me-100 ), coupled with two methine protons at
4.66 (dq, J=6.8 and 1.0 Hz, H-70 ) and 2.53 (dq, J=7.2 and 1.0 Hz, H-80 ). This sesquiterpene pyridine alkaloid includes one macrocyclic structure formed by two ester linkages between one sesquiterpene molecule and one evoninic acid, at positions 3 and 15. In order to determine the positions of the acetate groups and consequently the hydroxyl group position, 1H–1H COSY, HMQC and HMBC spectra were recorded. In the former the crosspeak of H-2 (
4.07) with H-1 (
5.43) and H-3 (
4.76), suggested the position of the hydroxyl group at C-2, which reinforced by the observation that H-2 was at higher field than the corresponding proton in alkaloids containing a benzoyl or acetyl group, attached to this position. This proposal was confirmed by the HMBC spectrum which located the acetate groups by crosspeaks between these groups and H-1, H-5, H-7, H-8, and H-11. The 13C NMR spectrum showed 36 signals, characteristic of evoninoate sesquiterpene pyridine alkaloids and showing five acetate groups. The carbon attached to the hydroxyl group appeared at
69.65, approximately
1.0 deshielded when compared with alkaloids with an acetate group at carbon 2. Additional support was obtained by a full scan ESI mass spectrum, where the molecular ion peak for this compound was detected at m/z 764 ([M+H]+), reinforcing this structural proposal. The relative stereochemistry around the dihydroagarofuran nucleus was determined from the coupling constants to be the same as other alkaloids isolated, including wilforine, wherein the stereochemistry was determined by X-ray analysis (Lião et al., 1997). The C7 epimeric relationship between this compound and chuchuhuanine E-V (1a), was defined by the coupling constant between H-7 and H-8. While in 1 this coupling constant was 6.0 Hz, in 1a it was 9.4 Hz (Shirota et al., 1994), indicating a cis and trans H-7/H-8 relationship for 1 and 1a, respectively. Alkaloid 1 was assayed for lethality in the brine shrimp toxicity (BST) test, and showed LC50 99.0 mg/ml. This test is proposed as a simple bioassay for natural product research, which active compounds could then be subjected to more elaborate bioassays for specific pharmacological activities. Sesquiterpene pyridine alkaloids was assayed against several tumor cell lines, and emarginatine F turned out to be active against some cell lines (Kuo et al., 1994). 3. Experimental 3.1. General experimental procedures NMR: Bruker DRX 400, in CDCl3 and TMS as int. standard; ESI-MSMS: low resolution on a triple quadrupole Micromass Quattro LC instrument, equipped with a ‘‘Z-spray’’ ion source; HRMS were obtained on a Fisons VG Autospec; [a]D: Perkin-Elmer 241 instrument; DCCC: Tokyo Rikakikai Co. with 300 columns of 40 cm2 mm i.d. 3.2. Plant material Roots of P. campestris were collected in a Savanna Reserve at Universidade Federal de São Carlos, in October 1992 and identified by Dr. Maria Helena de O. Antunes, Departamento de Botânica, Universidade Federal de São Carlos, São Paulo, Brazil, wherein a voucher specimen (no. 2845) is deposited. 3.3. Extraction and isolation The roots were dried, powdered (2.8 kg) and successively extracted with hexane, CH2Cl2 and MeOH at room temperature. The combined extracts were submitted to liquid-liquid partition, resulting in hexane (5.1 g), methanol (35.84 g), ethyl acetate (5.87 g), n-butanol (5.21 g), and aqueous (23.80 g) extracts. The methanolic extract (3.5 g) was submitted to droplet countercurrent chromatography (DCCC), using the aqueous layer of hexane:MeOH:H2O:EtOAc (5:4:1:2) as the stationary and the organic layer as the mobile phase, in a flow rate of 0.6 ml/min. The collected fractions were monitored by TLC and grouped in 22 fractions. Fractions 20–22 were separately filtered on Florisil (70–230 mesh) using hexane:CH2Cl2:MeOH (8:2:0.5). Fraction 20 was flash chromatographed on silica gel (230–400 mesh), eluting with hexane: CH2Cl2:MeOH (8:2:0.5), affording wilforine L.M. Lião et al. / Phytochemistry 58 (2001) 1205–1207 (330.0 mg, 9.428%) and a mixture of sesquiterpene pyridine alkaloids. Fraction 22 was submitted to recycling HPLC using a GS-310P column (MeOH; 50.0 2.15; 3 ml/min; UV 254 nm), affording euojaponine F (15.7 mg, 0.448%), wilforjine (5.9 mg, 0.168%), wilforzine (3.5 mg, 0.100%), and another mixture of alkaloids. The ethyl acetate extract (3.0 g) was also submitted to DCCC, using the aqueous layer of CHCl3: MeOH:n-BuOH:H2O (10:10:1:6) as the stationary and the organic layer as the mobile phase, in a flow rate of 0.6 ml/min, and after TLC was grouped in 12 fractions. Fractions 3, 4 and 5, after Florisil filtration, were individually submitted to recycling HPLC under the same conditions as the methanol extract, affording forrestine (186.8 mg, 6.226%), wilforine (50.1 mg, 1.670%), euojaponine F (0.4 mg, 0.013%), euonine (4.2 mg, 0.140%), neowilforine (4.3 mg, 0.143%), euonimine (6.1 mg, 0.203%), and a mixture of sesquiterpene pyridine alkaloids. The mixtures of alkaloids obtained were monitored by TLC using the Dragendorff reagent and grouped into two sets. The second set was rechromatographed on recycling HPLC using a Shim-pack column (silica 5 m; 2.025 cm; hexane: CH2Cl2:MeOH
(8:2:0.5); 3 ml/min; UV 254 nm), affording the alkaloid 1 (1.5 mg, 0.023%) and ebenifoline E-I (2.0 mg, 0.038%). 3.4. Biological assays The in vitro lethality assay against Artemia salina (BST) was carried out according to procedures described in Alkofahi et al. (1989). 3.4.1. 4-Hydroxy-7-epi-chuchuhuanine E-V (1) This was obtained as a colorless amorphous solid: MeOH  ½a20 nm (log "): 201 D
37.4 (CHCl3; 0.002,); UV lmax 3 (3.92), 223 (3.61), 265 (3.24) nm; IR CHCl cm
1: 3614, max
1 1 1740, 1583, 1566, 1231 cm ; H NMR (400 MHz, CDCl3): see text; 13C NMR (100 MHz, CDCl3):
9.3 (q, C-100 ), 11.8 (q, C-90 ), 18.5 (q, C-14), 20.5 (q, OCOCH3), 20.8 (q, OCOCH3), 21.0 (q, OCOCH3), 21.4 (q, OCOCH3), 21.6 (q, OCOCH3), 22.7 (q, C-12), 36.3 (d, C-70 ), 44.9 (d, C-80 ), 50.4 (d, C-6), 52.5 (s, C-9), 60.3 (t, C-11), 68.9 (d, C-7), 69.6 (d, C-2), 69.9 (t, C-15), 70.4 (s, C-4), 70.9 (d, C-8), 73.8 (d, C-5), 75.3 (d, C-1), 77.9 (d, C-3), 83.9 (s, C-13), 94.4 (s, C-10), 121.1 (d, C-50 ), 125.3 (s, C-30 ), 137.6 (d, C-40 ), 151.4 (d, C-60 ), 165.1 (s, C-20 ), 165.1 (s, OCOCH3), 168.5 (s, C-120 ), 169.1 (s, OCOCH3), 169.3 (s, OCOCH3), 169.9 (s, OCOCH3), 170.1 (s, OCOCH3), 174.4 (s, C-110 ); ESI-MSMS (probe) 30 eV, m/z (rel. int.): 764 [M+H]+ (5), 746 (21), 728 92), 686 (9), 206 (100), 188 (9), 178 (33), 160 (6); HR-MS m/z: 763.2672 (calcd for C36H45NO17, 763.2687). 1207 Acknowledgements The authors thank CNPq (Conselho Nacional de Desenvolvimento Cientı́fico e Tecnológico), FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo), FUNAPE/UFG (Fundação de Apoio à Pesquisa da UFG), and CAPES/PICDT (Fundação Coordenação de Aperfeiçoamento de Pessoal de Nı́vel Superior/Programa de Incentivo a Capacitação Docente e Técnica) for financial support. References Alkofahi, A., Rupprecht, J.K., Anderson, J.E., McLaughlin, J.L., Mikolajczak, K.L., Scott, B.A., 1989. Search for new pesticides from higher-plants. In: American Chemical Society Symposium Series, Vol. 387. Washington, pp. 25–43. Beroza, M., 1951. Alkaloids from Tripterygium wilfordii Hook, wilforine and wilfordine. J. Am. Chem. Soc. 73, 3656–3659. Brüning, R., Wagner, H., 1978. Übersicht über die Celastraceeninhaltsstoffe: Chemie, Chemotaxonomie, Biosynthese, Pharmakologie. Phytochemistry 17, 1821–1858. Chunquan, C., Jikai, L., Dagang, W., 1992. Forrestine, an alkaloid from Tripterygium forrestii. Phytochemistry 31, 4391–4392. Deng, F., Xia, Z., Xu, R., Chen, J., 1992. The structures of triptotetraolide and wilforjine. Zhiwu Xuebao 34, 618–621. He, Z.S., Li, Y., Fang, S.D., Hong, S.H., 1987. Structures of wilforgine, wilforzine and wilformine from Tripterygium wilfordii. Acta Chim. Sinica 45, 510–513. He, Z.S., Li, Y., Fang, S.D., Hong, S.H., 1989. Structures of wilfortrine and neowilforine from Tripterygium wilfordii Hook. F. Huaxue Xuebao 47, 178–181. Heywood, V.H., 1993. Flowering Plants of the World. Oxford University Press, New York. pp. 178–183. Itokawa, H., Shirota, O., Morita, H., Takeya, K., Litaka, Y., 1993. Isolation, structural elucidation and conformational analysis of sesquiterpene pyridine alkaloids from Maytenus ebenifolia Reiss. X-ray molecular structure of ebenifoline W-I. J. Chem. Soc. Perkin Trans. 1, 1247–1253. Kuo, Y.H., King, M.L., Chen, C.F., Chen, H.Y., Chen, C.H., Chen, K., Lee, K.H., 1994. Two new macrolide sesquiterpene pyridine alkaloids from Maytenus emarginata: emarginatine G and the cytotoxic emarginatine F. J. Nat. Prod. 57, 263–269. Lião, L.M., Caracelli, I., Vieira, P.C., Silva, M.F.G.F., Fernandes, J.B., Rodrigues-Filho, E., Zukerman-Schpector, J., 1997. Crystal structure of wilforine. A sesquiterpene pyridine alkaloid from Salacia campestris. Anais Assoc. Bras. Quı́m. 46, 184–188. Mata, R., Calzada, F., Dı́az, E., Toscano, R.A., 1990. Chemical studies on Mexican plants used in traditional medicine, XV. Sesquiterpene evoninoate alkaloids from Hippocratea excelsa. J. Nat. Prod. 53, 1212–1219. Septı́mio, L. R., 1994. A Fitoterapia Baseada em Ervas Medicinais do Cerrado. In: Ministério da Cultura, SIPE (Secretaria de Intercâmbio e Projetos Especiais), Brası́lia, p. 103. Shirota, O., Otsuka, A., Morita, H., Takeya, K., Itokawa, H., 1994. Sesquiterpene pyridine alkaloids from Maytenus chuchuhuasca. Heterocycles 38, 2219–2229. Zheng, Y.L., Xu, Y., Lin, J.F., 1989. Immunosuppressive effects of wilfortrine and euonine. Acta Pharm. Sinica 24, 568–572.