A Labdane Diterpene from the Aerial Parts of Egletes viscosa Less

Egletes viscosa Less (Asteraceae) is an annual herb mostly native to intertropical Americas. It is known in the Northeast of Brazil as “macela” or “macela-da-terra” and grows throughout the margins of river and lakes as soon as the raining season ends. The flower buds are largely sold in the herbal stores and used in the folk medicine as emmenagogue, diaphoretic,1 stomachic and antidiarrhoeal.2 Previous studies with E. viscosa have reported the isolation of biologically active flavonoids and diterpenoids.3-14 Recently, Lee et al reported the isolation of labdane diterpene glycosides and flavonoids from the entire plant native from Peru.15 In continuation of the work with this species, we report now the isolation of the new labdane glycoside 8αhydroxylabd-14(15)-ene-13(S)-O-β-D-ribopyranoside 1 in addition to 13-epi-sclareol,16 barbatol,17 tarapacol,18 spinasterol,19 ternatin,7, 20-23 and triacontane from the aerial parts of E. viscosa. The complete proton and carbon assignments of the known compounds and of the new diterpene 1, including the unpublished 13C and 1H NMR data of the new derivatives 2-4 were accomplished by the use of a series of 2D NMR experiments such as 1H-1H COSY, HMQC and HMBC.


Introduction
Egletes viscosa Less (Asteraceae) is an annual herb mostly native to intertropical Americas.It is known in the Northeast of Brazil as "macela" or "macela-da-terra" and grows throughout the margins of river and lakes as soon as the raining season ends.The flower buds are largely sold in the herbal stores and used in the folk medicine as emmenagogue, diaphoretic, 1 stomachic and antidiarrhoeal. 2][5][6][7][8][9][10][11][12][13][14] Recently, Lee et al reported the isolation of labdane diterpene glycosides and flavonoids from the entire plant native from Peru. 15 In continuation of the work with this species, we report now the isolation of the new labdane glycoside 8αhydroxylabd-14(15)-ene-13(S)-O-β-D-ribopyranoside 1 in addition to 13-epi-sclareol, 16 barbatol, 17 tarapacol, 18 spinasterol, 19 ternatin, 7,[20][21][22][23] and triacontane from the aerial parts of E. viscosa.The complete proton and carbon assignments of the known compounds and of the new diterpene 1, including the unpublished 13 C and 1 H NMR data of the new derivatives 2-4 were accomplished by the use of a series of 2D NMR experiments such as 1 H-1 H COSY, HMQC and HMBC.
The unequivocal assignment of NMR spectral data of compound 1 shown in Table 1 was established by data analysis from COSY, HMQC and HMBC experiments.The spectral data attributed to the aglycone unit showed a very similar feature to those described for sclareol. 25he HMBC experiment confirmed the position of glycosylation at C-13 by the correlations of the anomeric hydrogen at δ 4.32 (H-1´) and both methylidene hydrogens at δ 5.22 and 5.17 (H-15a and H-15b) with the oxygenated carbon at δ 80.6 (C-13).The decaline ring system was characterized by long-range correlations of both methyl groups at δ 0.88 (H-18) and 0.81(H- 19)  with the carbons at δ 42.0 (C-3) and 56.3 (C-5), as well as the methyl groups at δ 0.81 (H-20) with the carbons at δ 39.9 (C-1), 56.3 (C-5) and 61.7 (C-9).Further, pertinent correlations of the methyl group at d 1.11 (H-17) with the carbons at δ 43.7 (C-7), 61.7 (C-9) and 74.0 (C-8) allowed to confirm the allocation of the hydroxyl group at C-8.
On the basis of the above spectroscopic data, in addition to all optical rotation considerations, compound 1 was identified as the novel labdane diterpenoid 8αhydroxylabd-14(15)-ene-13(S)-O-β-D-ribopyranoside.Chemical derivatization by usual methods was used to confirm the proposed structure and yielded the new labdane derivatives 2-4, to which the 1 H and 13 C NMR data are presented in Table 1.

General procedures
Melting points were obtained on a Mettler FP82HT apparatus and are uncorrected.IR spectra were recorded using a Perkin Elmer 1000 FT-IR spectrophotometer.Optical rotations were measured on a Perkin Elmer 341 polarimeter.The mass spectra were obtained on a Hewlett-Packard 5971 mass spectrometer by electron impact ionization (70 eV). 1 H and 13 C NMR spectra were recorded on a Bruker Avance DRX-500 (500 MHz for 1 H and 125 MHz for 13 C); chemical shifts are given in ppm relative to residual CHCl 3 (7.24 and 77.0 ppm).Silica Gel 60 (Merck, 230-400 mesh) was used for analytical TLC.Silica gel 60 (Merck, 60 F 254 , 0.2 mm) was used for column chromatography.All compounds were visualized on TLC by spraying with vanillin/perchloric acid/EtOH followed by heating.

Plant material
Aerial parts of cultivated Egletes viscosa Less were harvested from garden beds at the Departamento de Fitotecnia of the Universidade Federal do Ceará, Fortaleza, Ceará State.Voucher specimens (36694) have been identified by Dr. Edson Paula Nunes and deposited at the Herbário Prisco Bezerra (EAC), Departamento de Biologia, Universidade Federal do Ceará, Fortaleza, CE, Brazil.

Extraction and isolation
Dried aerial parts of E. viscosa (1.35 kg) were pulverized and extracted with hexane at room temperature (3 × 8 L).The solvent was removed under reduced pressure to give the correspondent extract and the mark obtained after hexane extraction was reextracted with EtOH (3 × 8 L).

Hydrogenation of 1
Compound 1 (12.0 mg) dissolved in EtOAc (5.0 mL) was added to a suspension containing Pd/Rh (2.0 mg) in MeOH (10.0 mL), that was previously saturated with H 2 .The mixture was stirred at room temperature during 30 minutes.Usual work up and SiO 2 column chromatography using hexane/CHCl 3 2:1 yielded compound 2 (10.0 mg, 83.3 %) as a white solid.

Acetylation of 1
To a solution of compound 1 (5.0 mg) in pyridine (1.0 mL) were added Ac 2 O (1.0 mL) and a catalytic amount of DMAP.The mixture was stirred for 30 minutes at room temperature.Subsequent workup afforded a residue that was chromatographed using hexane/CHCl 3 (3:1) as eluent to yield compound 3 (6.0mg, 94.0 %) as a yellowish solid.

Hydrogenation of 2
Compound 2 (10.0 mg) dissolved in EtOAc (4.0 mL) was added to a suspension containing Pd/Rh (2.0 mg) in MeOH (80.0 mL) that was previously saturated with H 2 .The mixture was stirred at room temperature during 30 minutes.Usual work up and SiO 2 column chromatography using hexane/CHCl 3 2:1 yielded compound 4 (8.0 mg, 80.0 %) as a white solid.