A NEW EREMOPHILANOLIDE FROM SENECIO ATACAMENSIS

GLAUCO MORALES * , PATRICIA SIERRA, LUIS A. LOYOLA
and JORGE BORQUEZ

Laboratorio de Productos Naturales , Departamento de Química , Facultad de Ciencias
Básicas , Universidad de Antofagasta, Casilla 170 . Antofagasta , Chile.
gmorales@uantof.cl
(Received: June 14, 2000 - Accepted: August 14, 2000)

SUMMARY

The fractionation work of the aerial parts of Senecio atacamensis Phil. afforded, in addition to the already known 6b -hydroxyeremophilenolide, a new sesquiterpene lactone 6b - hydroxy - 8a - methoxyeremophil -1(10),7(11) - dien - 8b,12 olide.

The structures were established by spectral data, in particular 2D NMR (DEPT, DQF-COSY, HMQC, HMBC and ROESY).

Keyword Index: Senecio atacamensis Phil, Asteraceae; sesquiterpenoids; 6b - hydroxyeremophilenolide; 6b - hydroxy - 8a - methoxyeremophil - 1(10),7(11) - dien - 8b,12 olide

RESUMEN

Desde las partes aéreas de Senecio atacamensis Phil. se ha aislado una nueva lactona sesquiterpénica : 6b - hidroxi - 8a - metoxieremofil - 1(10),7(11) - dien - 8b,12 - olida, junto a la ya conocida 6b - hidroxieremofilanólida, Las estructuras fueron establecidas usando la información espectral , en particular, las técnicas de RMN en 2D (DEPT, DQF-COSY, HMQC, HMBC and ROESY).

Palabras claves: Senecio atacamensis Phil, Asteraceae; sesquiterpenoides; 6b -hidroxieremofilenolida; 6b - hidroxi - 8a - metoxieremofil - 1(10),7(11) - dien - 8b,12 olida

INTRODUCTION

Aerial parts of several endemic Senecio species of northern Chile are widely employed in folk medicinal as a remedy for the altitudinal sickness ("puna" or "soroche") ¹. In continuation of a phytochemical investigation of the Senecio which grows on the western flank of the Andes in northern Chile ( 3500 m) ² - 4 we wish to report the isolation and structural elucidation of a new sesquiterpene lactone of the eremophilanolide type 1, isolated together with the known 6b - hydroxyeremophilenolide 2 from Senecio atacamensis Phil.

EXPERIMENTAL

General.

Melting point was determined on a Kofler block and is uncorrected . The solvents used for spectral measurements were TMS- CDCl ₃ , ¹ H(300 MHz) and ¹³ CNMR (75 MHz), as well as ¹ H - ¹ H COSY, DEPT, HMQC, HMBC and ROESY data were obtained on a Brucker AMX 300 spectrometers. Optical rotations were determined on a Perkin - Elmer 241 polarimeter. IR (KBr) was measured on a Perkin -Elmer 1600 FTIR spectrometer. UV spectra were taken in MeOH on a Beckmann DU - 7. Mass spectra were determined on a Hewlett Packard 5995 and VG micromass ZAB - 2F mass spectrometers. The column chromatography was carried out on silica gel ( Merck 70- 230 mesh); TLC was perfomed on precoated Kiesegel 60 F -254 plates 0.5 mm thick ( Merck).

Plant material.

Flowers and leaves of Senecio atacamensis .Phil. were collected from Socaire in northern Chile ( 23º 36’ 40s S ; 67º50’33s W, 3500 m) by G.M. The material was authenticated by Professor Clodomiro Marticorena, Facultad de Ciencias Biológicas y de Recursos Naturales de la Universidad de Concepción, and voucher specimens were kept at the Herbarium of Universidad de Concepción, Chile.

Extraction and isolation.

The air-dried flowers and leaves (404 g) were ground and percolated with EME (petrol (60-80 ºC), MeOH and Et ₂ O, 1:1:1). The EME ext. (31 g) (F001) was partitioned between CH ₂ Cl ₂ and H ₂ O to give a H ₂ O layer and a CH ₂ Cl ₂ layer. The residue of the CH₂Cl₂ (23 g) (F003) was further partitioned between 90% aq. MeOH and hexane, giving a aq. MeOH layer and hexane layer. The aq.MeOH residue ( 15 g , F005) was chromatographed over open silica gel column over, using gradients of hexane - EtOAc, and 60 fractions were collected. Fractions eluted with hexane - EtOAc 20% (6.3 g ) were combined on the basis of TLC analysis, and further resolved on another Si gel column eluted with hexane and EtOAc (15 %). 20 fractions were collected and pooled according to their similar TLC patterns. The pool II (3.2 g) was subject to another Si gel column eluted with mixture of hexane and EtOAc to give atacamensine 1 (1.25 g ). The successive CC Si gel of the fractions from F005 eluted with hexane - EtOAc 40% (4.3 g ) were combined and further resolved on another Si gel column, eluted with hexane and EtOAc(25 - 35%), to give 6b - hydroxyeremophilenolide 2 (0.8 g).

Atacamensine , 6b - hydroxy - 8a - methoxyeremophil - 1(10),7(11) - dien - 8b,12 olide 1, was crystallized from Me₂CO as colourless needles , mp 161º , [a]_D²⁴ = - 244 º (CHCl₃; c 0.11). IR (KBr ) cm^-1: 3575, 2940 - 2880, 1752, 1690, 1320, 1230, 1020, 960, 780. UV l_max (MeOH) 218 nm, log e 3.5. ¹H NMR and ¹³C NMR spectral data: see Table 1.

EIMS m/z 278( M ⁺, 42%), 260 (35), 246(96), 233(15), 232(15), 231(50), 219(55), 218(72), 205(55), 203(50), 189(30), 176(95), 156(58), 149(25), 140(40), 124(68), 123(98), 107(100), 105(52), 93(88),91(80), 79(62), 77(48) 55(46).

HREIMS m/z 278.1514 for C₁₆H₂₂O₄ (calcd 278.1698), 189.1665 for C₁₄H₂₁, (calcd 189.1624), 71.0560 for C₄H₇O (calcd 71.0498).

6b - Hydroxyeremophilenolide : 6b - hydroxy - 10bH - eremophila - 7(11) -en - 8a, 12 - olide 2 . Colourless needles , mp 207 -208º . [a]_D²⁴ +205 º (CHCl₃ ;c 1.0) . UV 218 nm , log e 3.95. IR n _max (KBr ) cm^-1: 3448, 2980 - 2880, 1746, 1688, 1450, 1360, 1020, 910, 780. HREIMS: m/z ( composition): 250.1574 (C₁₅H₂₂O₃, M+, 73%), 235.1336 (C_14H19O₃, 0.2%), 232.1464(C₁₅H₂₀O₂, 9 %), 217.1228 (C₁₄H₁₇O₂, 3.5%), 206.1308 (C₁₃H₁₈O₂, 4%),141.0554 (C₇H₉O₃, 50%), 126.0321 (C₆H₆O₃, 100%), 123.1177(C₉H₁₅, 61%), 109.1023 (C₈H₁₃, 97.4%), 95.0863 (C₇H₁₁ ,18%), 81.0718(C₆H₉ ,21%).

¹H NMR ( 300 Mz, CDCl₃ ) d 0.78 ( 3H,d, J = 6.0 Hz, Me-15 ), 1.17(3H,s, Me-14), 1.85(3H,d,J = 1.6 Hz, Me -13), 1.30(1H,m, H - 4), 1.33 (1H,m, H -1a), 1.40 (2H,m, H-3), 1.43 (2H,m, H-2), 1.66 (1H,m, H -9a), 1.75(1H,m, H - 1b),2.10 (1H,m, H - 10 ), 2.17 (1H,m, H - 9 b) , 4.69 (1H,s,H - 6) , 5.12 (1H,dd,d J = 10.2, 8,5 ,1.5 Hz, H-8 ) . ¹³C NMR (75 Mz, CDCl3 ) d 8.69 ( Me - 13) , 16.26 (Me- 14) , 16.37 ( Me - 15) , 19.99 (C - 2) , 25.84 (C - 1) , 29.47 (C - 4) , 30.61 (C - 3) , 33.84(C -10 ) , 35.04 (C - 9) , 42.95 (C -5 ), 69.94 ( C - 6 ) ,78.44 (C - 8) , 121.67(C - 11 ) , 161.60 (C - 7) , 174.85 (C - 12)

RESULTS AND DISCUSSION

Flowers and leaves were extracted with EME ( petrol: MeOH : Et₂O 1:1:1). The EME residue ( FOO1) was partitioned between H₂O (FOO2) and CH₂Cl₂ (FOO3), and FOO3 was partitioned between MeOH aq (90%) (FOO5) and hexane(FOO6). FOO5 residue was repeatedly chromatographed on Si gel using as solvent mixture of hexane - EtOAc of increasing polarity to yield 6b-hydroxy-8a- methoxyeremophil - 1(10),7(11) - dien - 8b,12 olide 1 and 6b - hydroxyeremophilanolide 2.

Compound 1, named atacamensine, displayed spectral properties that were similar to those of eremophilenolides isolated from Petasites japonicus ^5,6 and Cacalia roborowskii ⁷. The elemental formula of atacamensine 1 was deduced as C₁₆H₂₂O₄ by HREIMS which showed a molecular peak at 278.1514 (calcd. 278.1698). Based on the spectral data, atacamensine 1, was deduced to be 6b - hydroxy - 8a - methoxyeremophil - 1(10), 7(11) - dien - 8b, 12 - olide. The IR and UV spectra supported the presence of the a, b - unsaturated - g - lactone (1753, 1690 cm -1, 218 nm, log e 1.8) and a hydroxyl group (3575 cm^-1). The ¹HNMR spectra of 1 exhibited signals for four CH₃ groups, one of them secondary (d 0.85), a tertiary (d 1.09), a vinylic (d 2.02) and a methoxyl group (d 3.09), for a methyne proton germinal to a OH group (d 4.37) and for a proton olefinic (d 5.62)(Table 1). ¹³CNMR spectra of 1 showed all - resolved resonances for 16 carbon atoms)(Table 1). The multiplicity of each carbon atom was determined using DEPT experiments which revealed the presence of four primary carbons (d 9.25, 12.93, 18.94 and 50.74), three secondary carbons (d 25.15, 29.00 and 43.84), three tertiary carbons (d 38.14, 79.04, and 129.48) and six carbon atoms nonhydrogenated (d 47.26, 105.62, 126.34, 134.87, 157.83 and 175.40). The strongly low field shifted signal of five quaternary carbons was consistent with the presence of a carbonyl group and two olefinic doubled bond. Deshielded quaternary carbons at d 126.34, 157.83 and 175.40 and a vinyl methyl group at d 9.25 suggested a methyl substituted a , b - unsaturated - g - lactone with an endo-double bond. The base peak in the HREIMS of 1 at mz 107.0865 [C₈H₁₁]⁺ indicated that the A-ring of the homobicylic system corresponds to a dimetylcyclohexen residue in accordance to a doubled bond between C -1 and C -10 (d 5.62 1H, br s). While the peak at mz 171.1171 [C₈H₁₁O₄]⁺ indicated that the four oxygen atoms of the molecule of 1 are neighbouring and located at B - ring and the g - lactone ring. In the ¹H NMR of 1 the downfield shifted signal attributable to H - 8 (ca d 4.2 - 5.6) was missing indicating that C - 8 was substituted. This was supported by a pair of signals at d 2.69 (1H, d, J = 14.0 Hz) and 2.33 (1H,dd,J=14.0, 2.5 Hz) assigned to the resonance of the methylene group at C - 9. The position of the OCH₃ group bonded at C - 8 (d 105.62 s), the OH group at C - 6 (d 79.04 and 4.34, 1H,d,J=1.5 Hz) and the ¹ H, ¹³ C NMR spectral chemical shifts of 1 were deduced through direct and long-range ¹H - ¹H and ¹H - ¹³C correlations using COSY, HMQC and HMBC techniques. Among the most relevant couplings observed in the HMQC spectrum of atacamensine 1 are: methyl protons signal at d 0.85 s with carbon atom at d 12.93 assigned to C -14; at d 1.09 d with carbon atom at d 18.94 assigned to C - 15; at d 2.02 d with carbon atom at d 9.25 assigned to C - 13; methine protons at d 5.62 br s with carbon atom at d 129.48 assigned to C - 1; at d 4.24 d with carbon atom at d 38.14 assigned to C - 4. The connectivities detected in the HMBC spectrum are shown in Figure 1. The relative stereochemistry of the chiral centres of the rings was determined by ROESY, which in the 2 - D - ROESY experiments showed NOE between H - 6 and H - 4 and OCH ₃. The same effects were observed between Me - 15 and Me - 14. From the above, it was evident that in the atacamensine, the OCH₃ at C - 8 was a - oriented while Me - 15, Me - 14 and OH at C - 6 were b - oriented and the structure of atacamensine 1 was established as 6 b - hydroxy - 8 - methoxyeremophil - 1(10),7(11) - dien - 8 b, 12 olide. On the other hand, the relative chemical shifts of the protons and carbons of the Me - 14 and Me - 15 groups and the coupling between H-6 and Me - 13, in the ¹H and ¹³CNMR spectrum of atacamensine 1, were in complete agreement with the generalization reported for the 8a - methoxyeremophilenolide derivatives. ^5,8 . Finally, atacamensine 1 may be an artifact by the methanol extraction procedure used to isolated the compound.

Compound 2, obtained as a colourless needles - mp 204º - was identified as 6 b - hydroxyeremophilenolide ^{5,9 - 11} on the basis of physical and spectral properties. The molecular formula was established as C₁₅H₂₂O₃ (M+250, 1574, calcd 250.1678) by HREIMS. The IR and UV spectrum of 2 showed the presence of an a, b - unsaturated - g - lactone and a hydroxyl group. A detailed analysis of the ¹H and ¹³CNMR spectrum, plus 2D - experiments allowed the unambiguous assignments of ¹H and ¹³C signals shown in Experimental. ROESY spectrum of 2 clearly indicated that the Me - 14, Me - 15, H - 8 and H - 10 were b - oriented, while H - 6 was a.

ACKNOWLEDGEMENTS

We are grateful to Prof. Clodomiro Marticorena, Universidad de Concepción (Chile), for the botanical classification of the plant material. This work was subsidised by Fondecyt( grant Nº 1980391) and DGI (Universidad de Antofagasta).

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