New Melampolides from Schkuhria s chkuhrioides

J. Nat. Prod. 1998, 61, 1082-1085 1082 New Melampolides from Schkuhria schkuhrioides Guillermo Delgado,*,† Verónica Tejeda,† América Salas,† Marı́a Isabel Chávez,† Salvador Guzmán,† Alberto Bolaños,† Marı́a Isabel Aguilar,‡ Vı́ctor Navarro,§ and Marı́a Luisa Villarreal§ Instituto de Quı́mica, Universidad Nacional Autónoma de México, Ciudad Universitaria, Circuito Exterior, Coyoacán 04510, Mexico, D. F., Facultad de Quı́mica, Departamento de Farmacia, Universidad Nacional Autónoma de México, Ciudad Universitaria, Circuito Exterior, Coyoacán 04510, Mexico, D. F., and Unidad de Investigación Biomédica del Sur, Instituto Mexicano del Seguro Social, Argentina 1, Xochitepec, Morelos, Mexico Received December 3, 1997 The novel melampolides (11R)-11,13-dihydro-schkuhriolide (7), (11S)-11,13-dihydro-schkuhriolide (8), and schkuhrioidiol (11), along with the known constituents, frutescin (1), schkuhriolide (2), frutescinic acid (4), allo-schkuhriolide (5), and epoxyschkuhriolide (6) were isolated from the aerial parts of Schkuhria schkuhrioides. The structures of the new compounds were determined by spectroscopic methods. Compounds 1, 2, 4, 5, and 6 displayed no significant cytotoxic or antimicrobial activities. Species belonging to the genus Schkuhria1 are known sources of sesquiterpene lactones,2-6 diterpenes,7 polyacetylenes, and other constituents.8,9 Some species and their varieties are used in traditional medicine,1,2 and different biological activities have been reported for some constituents.10,11 Previous papers have reported a series of melampolides,12,13 elemanolides,14,15 and flavonoids from the aerial parts of S. schkuhrioides (Link & Otto) Thellung (Compositae). We have now characterized additional sesquiterpene lactones from this source, and the antimicrobial and cytotoxic activities of some melampolides were evaluated. Aerial parts of S. schkuhrioides were extracted with n-hexane and then with acetone. This extract was chromatographed using vacuum liquid chromatography (VLC)16,17 to yield frutescin (1),18,19 schkuhriolide (2),12,13 frutescinic acid (4),20 allo-schkuhriolide (5),12,21,22 epoxyschkuhriolide (6),13,23 and the novel natural sesquiterpenes 7, 8, and 11. Spectroscopic data of 1, 2, 4, 5, and 6 were identical to those reported previously. Some fractions containing a complex mixture of minor constituents were acetylated and separated by repeated column chromatography and preparative TLC, to afford epimers 9 and 10. The structures were deduced from their 1H NMR data (Table 1), which were very closely related to those of acetyl schkuhriolide (3), previously characterized.13 The structures 9 and 10 were established as the 11,13-dihydroderivatives of acetyl schkuhriolide, in agreement with the molecular formula and expected changes in the NMR data. The configurations at C-11 in 9 and 10 were determined by observing the changes in the chemical shifts of H-11 and H-13 (in CHCl3 and C6D6).24,25 The major difference in the chemical shifts of H-13 (∆δ ) δCDCl3 - δC6H6), due to the shielding effect of the solvent, observed for 9 (∆δH(13) ) 0.3) with respect to that of 10 (∆δH(13) ) 0.17) indicated that the secondary methyl group in 9 is oriented to the R-(convex) face of the macrocycle. The same trend is observed for H-11 in 10 (∆δH(11) ) 0.84) when compared to 9 (∆δH(11) ) 0.17), corroborating the R-orientation of H-11 in 10. Therefore, 7 [(11R)-11,13-dihydro-schkuhriolide] and 8 [(11S)-11,13-dihydro-schkuhriolide] are natural constituents of S. schkuhrioides. The most polar compound, schkuhrioidiol (11), was also a sesquiterpene lactone as suggested by the EIMS and 13C NMR data. The 1H NMR data, which also closely resembled those of 2, indicated the presence of an hydroxymethylene at C(14). 1H COSY, HMBC, and HMQC experiments26 of 11 and 12 (obtained by acetylation of 11) allowed the assignment of all 1H and 13C signals (See Table 2), confirming the structures. The * To whom correspondence should be addressed. Tel.: (525) 6224446. Fax: (525) 616-2217. E-mail: [email protected]. † Instituto de Quı́mica, UNAM. ‡ Facultad de Quı́mica, UNAM. § Unidad de Investigación Biomédica del Sur, IMSS. S0163-3864(97)00548-X CCC: $15.00 © 1998 American Chemical Society and American Society of Pharmacognosy Published on Web 07/31/1998 Melampolides from Schkuhria schkuhrioides Table 1. a 1H Journal of Natural Products, 1998, Vol. 61, No. 9 1083 NMR (300 MHz, CDCl3, δ, Coupling Constants in Parentheses) Spectral Data for Melampolides 9 and 10 hydrogen 9 9a 10 10a H-1 H-2 H-3 H-5 H-6a H-6b H-7 H-8 H-9a H-9b H-11 H-13a H-13b H-14 H-15 H-16 H-17a H-17b OAc 6.62 ddd (9.4,7,2) b 5.57 ddd (8.1,7,2) b 6.63 ddd (9.6,7,2) b 5.57 ddd (9.4,7,3) b 4.69 br d (10.5) 4.50 br d (10.2) 4.65 br d (9.6) 4.30 br d (9.1) 5.14 t (10.6) b 5.77 dt (12,5.1) b 5.29 t (10.6) b 5.86 dt (11.4,5) b 5.29 t (10.6) b 5.53 dt (12,5.8) b 5.38 dd (10,10.6) b 5.43 dt (12,5.8) 2.62 q (7.8) 2.63 q (7.7) 2.96 m 2.12 m 1.36 d (7.8) 9.44 d (1.8) 1.94 br s 1.06 d (8.1) 9.02 d (1.8) 1.59 br s 1.20 d (6.8) 9.46 d (1.8) 1.92 br s 1.07 d (7.3) 9.06 d (1.9) 1.53 s 2.01 s 1.52 s 1.96 s 1.50 s Taken in C6D6. b Superimposed signals. Table 2. 1H (500 MHz) and 13C NMR (125 MHz) Spectral Data for Melampolides 6, 11, and 12 (δ Values in ppm from TMS)a 6 11 position δH, mult J (Hz) δC δH, mult 1 2a 2b 3a 3b 4 5 6 7 8 9a 9b 10 11 12 13a 13b 14a 14b 15 C(6)OAc C(14)OAc 6.82 ddd 2.50 br t 2.52-2.55 m 2.35 br t 2.32-2.34 m 8.5, 8.0, 2.5 4.1 155.86 24.43 5.53 br t 2.13 m 1.87 dd 2.20 m 1.75 m 2.80 d 3.31 dd 2.89 m 5.59 ddd 3.05 br dd 2.59 br dd 9 10, 9 a 6.33 d 5.84 d 9.46 153 s 4.5 10, 9, 2.5 14.5, 2.5 14.5, 10 2.0 2.0 2.0 35.82 62.86 63.87 65.02 48.67 75.35 140.17 136.89 169.24 126.05 196.60 17.55 4.95 d 4.03 t 2.88 dd 4.91 ddd 2.59 dd 2.29 m 5.82 t 6.30 t 4.13 d 4.07 d 1.77 s J (Hz) 8.1 12 δC 128.30 24.22 2.9, 12.3 38.09 10.2 10.2 10.2, 5.1 12.3, 5.1, 4.5 12.3, 4.5 1 1 12 12.0 135.89 125.37 65.74 49.90 79.36 29.77 136.48 138.48 169.91 124.76 67.58 16.90 δH, mult 5.64 br t 2.20 m 1.92 m 2.22 m 1.75 dd 4.83 d 5.25 t 3.02 br dd 4.74 ddd 2.60 m 2.32 br t 6.27 t 5.73 t 4.61 d 4.47 d 1.91 d 2.00 2.08 J (Hz) 7.5 δC 131.58 24.37 38.04 12, 10.5 10.5 10.5 10.5, 5.0 13, 5, 5 131.44 122.12 68.97 47.08 78.22 29.79 13 0.5 0.5 12.5 12.5 1.5 131.77 137.70 169.37 124.70 68.31 17.04 170.42, 20.86 168.82, 20.75 Assignments were made on the basis of HMBC, HMQC, and NOESY correlation methods. relative stereochemical assignments of 12 were accomplished by NOESY experiments. The observation of a strong NOE between H-14 methylene protons and H-1, and between H-7 and H-8 confirmed the cisconfiguration of the C(1)-C(10) double bond and the γ-lactone, respectively. The NOE observed between H-14 and H-7 and H-8, as well as the NOE between H-1 and H-5, allowed us to establish the [1D14 ; 15D5]27 conformation for 12, which is similar to that found for 2. NaBH4 reduction of 2 afforded 11, confirming the structure of the new melampolide. The acetone extract of S. schkuhrioides investigated was found not to be active against several microorganisms (Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Salmonella tiphi, Candida albicans, Tricophyton mentagrophytes, Microsporum gypseum; MIC > 400 µg/mL),28,29 tumor cancer cell lines (KB, nasopharingeal carcinoma, UISO, cervix carcinoma, COLON, colon carcinoma; DE50 > 20 µg/mL),30 or Artemia salina (LC50 > 800 ppm).31 Compounds 1, 2, 4, 5, and 6 displayed no significant activities in the above-mentioned bioassays. Compound 5 showed the best cytotoxicities with ED50 values of 5.7 × 10-4, 1.82, and 0.9 µg/mL against KB, UISO, and COLON, respectively. Experimental Section General Experimental Procedures. 1H and 13C NMR spectra were recorded on Varian VXR-300 and Varian Unity Plus-500 instruments, and the chemical shifts are expressed in parts per million (δ) relative to TMS. Samples for NOE experiments were degassed (freeze, pump, thaw, 3×) and sealed under argon. IR spectra were recorded with a Nicolet Magna IR TM 750 and Perkin-Elmer 283B instruments. MS data were recorded with a JEOL JMS-AX 505 HA mass spectrometer. EIMS were obtained at 70 eV ionization energy. Vacuum chromatography was performed on Merck Kieselgel 60 (0.040-0.863 mm).16,17 All separations were carried out using distilled solvents. TLC analyses were performed on Alugram Sil G/UV254 Si gel plates. 1084 Journal of Natural Products, 1998, Vol. 61, No. 9 Plant Material. Aerial parts of S. schkuhrioides were collected near Teoloyucan (State of Mexico), in September 1993. A voucher specimen (MEXU 636061) has been deposited at the National Herbarium, Instituto de Biologı́a de la Universidad Nacional Autónoma de México. Extraction and Isolation. The air-dried plant material (5 kg) was powdered and extracted with n-hexane (twice, 48 h) and then with Me2CO (twice, 48 h) at room temperature, to give 177 g of residue. This extract (170 g) was chromatographed using VLC over Si gel (670 g) with a n-hexane-EtOAc gradient to obtain 14 fractions. The residue obtained from the fraction 7 eluted with n-hexane-EtOAc (9:1) (15 g) was subjected to column rechromatography over Si gel to afford 85 mg of 1.18,19 Repeated rechromatography over Si gel of fraction 8 (12.5 g, eluted with n-hexane-EtOAc 7:3) with n-hexane-EtOAc gradient gave a residue that was further purified by column chromatography over Si gel using CH2Cl2-MeOH (4:1), to afford 240 mg of frutescinic acid (4).20 Fraction 9 (8.1 g, eluted with n-hexaneEtOAc 3:2) was rechromatographed using VLC over Si gel (n-hexane-EtOAc gradient), and some fractions were further purified by column chromatography using CH2Cl2-MeOH (81:19) as eluent to obtain 35 mg of alloschkuhriolide (5).12,21,22 Subsequent fractions of this rechromatography, which contained the mixture 7 + 8 (960 mg), were acetylated following the standard procedure to afford a residue that was chromatographed over Si gel using n-hexane-EtOAc mixtures of increasing polarity to obtain 9 (12 mg) and 10 (16 mg). Compound 212,13 (2g) crystallized from the eluates of the chromatography of fraction 10 (eluted with n-hexaneEtOAc, 1:1). Some polar fractions of the rechromatography of fraction 10 (7 g) were rechromatographed over Si gel using VLC with n-hexane-EtOAc gradient, and some fractions were further subjected to repeated column chromatography on Si gel (n-hexane-EtOAc and CH2Cl2-MeOH gradients) to obtain epoxyschkuhriolide (6)13,23 (52 mg). Schkuhrioidiol (11) (53 mg) was isolated from fraction 11 (eluted with n-hexane-EtOAc 2:3) after repeated column chromatography over Si gel followed by preparative TLC (CH2Cl2-MeOH (4:1). Standard acetylation of 11 afforded 12. (11R)-11,13-Dihydro-acetyl-schkuhriolide (9): colorless oil, [R]25D + 9.1 (c 0. 11, MeOH); UV (MeOH) λmax (log ǫ) 204 (3.89), 226 (3.80) nm; IR (CHCl3) νmax 2953, 2928, 2855, 1773, 1732, 1685, 1630, 1460, 1373, 1179, 1009, 945 cm-1; 1H NMR (300 MHz, CDCl3 and in C6D6), see Table 1;13C NMR (75 MHz, C6D6) δ 195.6 s (C-14), 178.1 (s, C-12), 154.8 (d, C-1), 141.3 (s, C-10), 137.3 (s, C-4), 123.7 (s, C-5), 70.4 (d, C-8), 68.9 (d, C-6), 41.2 (d, C-7), 38.9 (d, C-11), 37.3 (t, C-3), 29.7 (t, C-9), 27.6 (t, C-2), 17.1 (q, C-15), 14.8 (q, C-13); EIMS m/z 306 [M]+ (2), 277 (4), 262 (4), 246 (25), 240 (17), 217 (40), 203 (10), 173 (25), 143 (45), 131 (15), 105 (22), 83 (32), 69 (30), 43 (100). (11S)-11,13-Dihydro-acetyl-schkuhriolide (10): colorless oil, [R]25D + 2.63 (c 0. 19, MeOH); UV (MeOH) λmax (log ǫ) 204 (3.77), 227 (3.77) nm; IR (CHCl3) νmax 3686, 2937, 2857, 1769, 1730, 1687, 1522, 1429, 1011, 932 cm-1;1H NMR (300 MHz, CDCl3 and in C6D6), see Table 1; EIMS m/z 306 [M]+ (1), 217 (33), 143 (47), 43 (100), 41 (35). Delgado et al. (4R,5R)-4(5)-Epoxyschkuhriolide (6): colorless needles (Me2CO) mp 132-134 °C [lit:13 134-136 °C]; IR (CHCl3) νmax 3597, 2932, 2865, 1766, 1713, 1688, 1635, 1522, 1425, 1367, 1337, 997 cm-1;1H and 13C NMR (500 and 125 MHz, CDCl3), see Table 2; EIMS m/z 278 [M]+ (2), 263 (6), 250 (6), 240 (15), 217 (32), 198 (24), 171 (25), 138 (31), 135 (22), 105 (33), 95 (38), 83 (58), 55 (46), 43 (100), 41 (54). Schkuhrioidiol (11): pale yellow oil, [R]25D + 124.1 (c 0.2, MeOH); UV (MeOH) λmax (log ǫ) 204 (4.13) nm; IR (CHCl3) νmax 3600, 3027, 2938, 2875, 1765, 1672, 1456, 1384, 1296, 1046 cm-1;1H and 13C NMR (500 and 125 MHz, CDCl3), see Table 2; EIMS m/z 264 [M]+(1), 246 (3), 231 (2), 180 (20), 145 (29), 143 (28), 135 (34), 117 (48), 105 (67), 84 (89), 83 (100), 79 (65), 67 (52), 55 (50), 41 (76), 39 (58); anal. C 68.29%, H 7.88%, calcd for C15H20O4, C 68.16%, H 7.63%.. Compound 11 obtained via reduction of 2. To a stirred solution of NaBH4 (6 mg, 0.15 mmol) in MeOH (5 mL) was added dropwise a solution of 2 (80 mg, 0.30 mmol) and CeCl3‚8H2O (149 mg, 0.38 mmol) in MeOH (5 mL). The resultant mixture was stirred for 5 min at room temperature, decomposed with diluted 10% HCl (to pH 6), and extracted with EtOAc (× 3, 25 mL). The combined organic layer was washed with H2O and dried over Na2SO4. Solvent was removed under reduced pressure, and the resultant material was purified by column chromatography (n-hexane-EtOAc gradient) to give 11 (74 mg). Diacetylschkuhrioidiol (12): obtained by standard acetylation of 11; pale yellow oil, IR (CHCl3) νmax 2960, 1770, 1737, 1672, 1460, 1372, 1164, 1116, 1010, 957, 947 cm-1;1H and 13C NMR (500 and 125 MHz, CDCl3), see Table 2; EIMS m/z 318[M]+ (1), 290 (4), 303 (2), 258 (6), 162 (12), 143 (15), 105 (33), 84 (70), 83 (100), 55 (45). Bioassays. The antimicrobial studies,28,29 the cytotoxicity assays,30 and the brine shrimp (Artemia salina Leach) lethality tests31 for the Me2CO extract, fractions, and isolated compounds (1, 2, 4, 5, and 6) from the title plant were performed using standard protocols. Acknowledgment. Financial support for this work from Universidad Nacional Autónoma de México (grant DGAPA-IN212195) and from Consejo Nacional de Ciencia y Tecnologı́a, México (grant 940040), is acknowledged. We thank Rocı́o Patiño, Beatriz Quiroz, Anı́bal Julián, Luis Velasco, and Javier Pérez-Flores for technical assistance. References and Notes (1) Heiser, C. B. Ann. Mo. Bot. Gar. 1945, 32, 265-278. (2) Pacciaroni, A. del V.; Sosa, V. E.; Ariza-Espinar, L.; Oberti, J. C. Phytochemistry 1995, 39, 127-131. (3) Bohlmann, F.; Zdero, C. Phytochemistry 1981, 20, 2431-2432. (4) Pérez, A. L.; Mendoza, J. S.; Romo de Vivar, A. Phytochemistry 1984, 23, 2911-2913. (5) Ganzer, U.; Jakupovic, J. Phytochemistry 1990, 29, 535-539. (6) Stewart, E.; Mabry, T. J. Phytochemistry 1985, 24, 2731-2733. (7) Bohlmann, F.; Jakupovic, J.; Robinson, H.; King, R. M. Phytochemistry 1980, 19, 881-884. (8) Bohlmann, F.; Kleine, K.-M. Chem. Ber. 1965, 98, 3081-3083. (9) Bohlmann, F.; Zdero, C. Phytochemistry 1977, 16, 780-781. (10) Pettei, M. J.; Miura, I.; Kubo, I.; Nakanishi, K. Heterocycles 1978, 11, 471-480. (11) Herz, W.; Govindan, S. V. Phytochemistry 1980, 19, 1234-1236. (12) Samek, Z..; Holub, M.; Bloszyk, E.; Drozdz, B. Z. Chem. 1979, 19, 449. Melampolides from Schkuhria schkuhrioides (13) Romo de Vivar, A.; Pérez, A. L.; León, C.; Delgado, G. Phytochemistry 1982, 21, 2905. (14) Delgado, G.; Hernández, H.; Romo de Vivar, A. J. Org. Chem. 1984, 49, 2994-2996. (15) Delgado, G.; Guzmán, S.; Romo de Vivar, A. Phytochemistry 1987, 26, 755-759. (16) Pelletier, S. W.; Chokshi, H. P.; Desai, H. J. Nat. Prod. 1986, 49, 892-900. (17) Coll, J. C.; Bowden, B. F. J. Nat. Prod. 1986, 49, 934-936. (18) Herz, W.; Bhat, S. V.; Sudarsanam, V. Phytochemistry 1972, 11, 1829-1831. (19) Herz, W.; Prasad, J. S.; Blount, J. F. J. Org. Chem. 1982, 47, 2206-2208. (20) Tsichritzis, F.; Jakupovic, J.; Bohlmann, F. Phytochemistry 1990, 29, 195-203. (21) Stewart, E.; Mabry, T. J. Phytochemistry 1985, 24, 2733-2734. (22) Macı́as, F. A.; Torres, A.; Molinillo, J. M. G.; Varela, R. M.; Castellano, D. Phytochemistry 1996, 43, 1205-1215. Journal of Natural Products, 1998, Vol. 61, No. 9 1085 (23) Delgado, G.; Guzmán, S.; Toscano, R. A. An. Esc. Nac. Cienc. Biol. (Méx) 1993, 39, 109-118. (24) Ledaal, T. Tetrahedron Lett. 1966, 1653-1658. (25) Ledaal, T. Tetrahedron Lett. 1968, 1683-1688. (26) Bax, A. J. Magn. Res. 1984, 57, 314-318. (27) Samek, Z.; Harmatha, J. Collect. Czech. Chem. Comm. 1978, 43, 2779-2798. (28) Mitscher, L. A.; Drake, S.; Gollapudi, S. R.; Okwute, K. J. J. Nat. Prod. 1987, 50, 1025-1040. (29) Van den Berghe, D. A.; Vlietinck, A. J., in: Methods in Plant Biochemistry, Hostettmann, K., Ed.; Academic Press: London, 1991; Vol. 6, 47-70. (30) Geran, R. I.; Greenberg, N. H.; McDonald, M. M.; Schumacher, A. M.; Abbott, B. J. Cancer Chemother. Rep., Part 3, 1972, 1-17. (31) Meyer, B. N.; Ferrigni, N. R.; Putnam, J. E.; Jacobsen, L. B.; Nichols, D. E.; McLaughlin, J. L. Planta Med. 1982, 45, 31-36. NP970548S