HETEROCYCLES

Paper | Regular issue | Vol. 78, No. 5, 2009, pp. 1253-1263
Received, 12th December, 2008, Accepted, 19th January, 2009, Published online, 20th January, 2009.
DOI: 10.3987/COM-08-11625

■ Eremophilane Derivatives from Pittocaulon praecox

Amira Arciniegas,* Ana-L. Pérez-Castorena, Erick Gastélum, José Luis Villaseñor, and Alfonso Romo de Vivar

Instituto de Química, Universidad Nacional Autónoma de México, Circuito Exterior, Ciudad Universitaria, Coyoacán 04510, D.F., Mexico

Abstract

Five new compounds; two eremophilane glucosides (1, 2) and three eremophilanolides (3, 11, 12), together with several known compounds were isolated from Pittocaulon praecox. Their structures were elucidated by spectroscopic methods. The anti-inflammatory activity of extracts and main isolated metabolites was evaluated on 2-O-tetradecanoylphorbol-13-acetate (TPA) induced model of acute inflammation.

INTRODUCTION
Pittocaulon praecox is one of the five species of the genus Pittocaulon (Asteraceae, Senecioneae, Tussilagininae) recently segregated from the genus Senecio.1 P. praecox is used in folk medicine to cure rheumatism and injuries.2 Previous chemical studies showed the furanoeremophilanes as its main secondary metabolites.3,4 The presence of pyrrolizidine alkaloids has also been reported.5 Nevertheless as no one of the compounds already reported justified the anti-inflammatory activity attributed to P. praecox we undertook a new investigation in search of possible anti-inflammatory substances. Five new compounds, two glucosides of eremophilane (1, 2) and three eremophilanolides (3, 11, 12), the known compounds 4-15,4,6-14 and the pyrrolizidine alkaloids senecionine and integerrimine15,16 were obtained. The anti-inflammatory activity of extracts and compounds 1-7, 9-12 was evaluated.

RESULTS AND DISCUSSION
Compound 1 showed a molecular formula C21H32O8 by HRFABMS. In the IR displayed bands at 3359, 1653 and 1605 cm-1 and in the UV, absorptions at 204 and 248 nm suggesting the presence of hydroxyl and conjugated carbonyl groups. In the 13C NMR spectrum, the signal at δ 193.3 showed the presence of a carbonyl group. COSY and COLOC experiments allow to assign the signals at δ 103.2 (d), 78.3 (d), 77.9 (d), 75.0 (d), 71.7 (d), and 62.7 (t) to a sugar moiety, and those at δ 71.7 (d) and 69.9 (t) to C-3 and C-12, respectively. These experiments permitted to attribute the signal observed at δ 5.75 (d, J = 2.1 Hz), in the 1H NMR spectrum, to H-9, as well as those at δ 4.87 (dd, J = 13.5, 1.5 Hz) and δ 4.49 (dd, J = 13.5, 2.1 Hz) to CH2-12. Finally, the presence of three methyl group signals at δ 1.97 (d, J = 1.2 Hz), δ 1.11 (d, J = 6.6 Hz) and 1.05 (s) supported structure 1 (Figure 1). The sugar residue was identified as β-glucose by gas chromatography of the hydrolyzed product and by the coupling constant of its anomeric proton (δ 4.17, d, J = 7.6 Hz). Linkage of the glucose moiety to C-12 was determined by the COLOC interaction observed between this carbon atom and the anomeric proton. The Z configuration of the 7(11) double bond was determined by the cross peaks of CH3-13 with H-6β, observed in the NOESY experiment. In the same spectrum, the interaction between H-14 and H-3 which showed an axial-axial coupling allowed to propose the α-orientation of the hydroxyl group at C-3, since on biogenetic grounds, the CH3-14 is β-oriented.17

Compound 2 exhibited the same molecular formula and very similar spectroscopic data to those of compound 1. In the 1H NMR spectrum of 2, H-12 resonated at δ 6.30 (q, J = 1.5 Hz) and its signal integrated for one hydrogen, H-7, absent in compound 1, appeared at δ 3.03 (dd, J = 14.0, 5.0 Hz), and the signals of CH2-6 were localized at δ 1.97 (dd, J = 13.5, 5.0 Hz) and δ 1.90 (dd, J = 14.0, 13.5 Hz), in accordance with structure 2. NOESY correlations between H-7 and H-12 and CH3-14 suggested the E configuration of the C-11 double bond and the β orientation of H-7, the cross peak between H-3 and CH3-14, permitted to propose the α orientation of the hydroxyl group. Because, the circular dichroism curve of 2 showed the same pattern as that reported for steroidal 4-en-3-ones,18 its absolute stereochemistry should be 3R, 4R, 5R, 7S.
Eremophilanolide 3 presented a molecular formula C15H21O4 by HRFABMS. The IR bands at 3351 and 1755 cm-1 indicated the presence of hydroxyl and unsaturated γ lactone groups. In the 1H NMR spectrum, the signal of a proton at δ 3.46 (ddd, J = 10.5, 10.5, 4.5 Hz), geminal to OH, was attributed to H-3. Since this proton showed an axial-axial coupling and correlated with the CH3-14 in the NOESY experiment, the hydroxyl group should be α-equatorial. At higher field, the signals of CH3-13 (δ 1.78, d, J = 1.5 Hz), CH3-14 (δ 0.97 s) and CH3-15 (δ 1.10, d, J = 6.5 Hz) provided an eremophilanolide profile where C-8 (δ 100.9 s) was bearing a hydroxyl group. This hydroxyl group should be α-orientated by the homoallylic coupling between H-13 (δ 1.78 d, J = 1.5) and H-6α (δ 2.38, br d, J = 12.5 Hz).19 The orientation of H-6α was determined by its NOESY interaction with H-4, while H-6β interacted with CH3-14 and CH3-15, in the same experiment. The signal at δ 5.66 (d, J = 2.0 Hz) was assigned to the vinylic proton 9, according to structure 3.

Eremophilanolide 11 presented a molecular formula C21H28O8 by HRFABMS. The presence of hydroxyl and carbonyl groups were deduced by the absorption bands observed in the IR spectrum (3346, 1743, 1729, 1701 cm-1). In the 1H NMR spectrum, the signals at δ 5.88 (d, J = 1.5 Hz) and 4.86 (ddd, J = 11.5, 7.0, 4.0 Hz) were assigned to the hydrogen atoms, geminal to ester functions, at C-6 and C-3, respectively, based on COSY and HMBC experiments. The ester group at C-6 was identified as isobutyrate by the cross peaks observed, in the HMBC spectrum, between C-1' (δ 174.9) and H-6 (δ 5.88), while H-3 (δ 4.86) correlated with the carbonyl of the acetate group (δ 169.5). In the 13C NMR spectrum, 21 carbon atom signals were observed, six of them being attributed to ester functions, the remaining 15 signals corresponded to an eremophilanolide profile. The presence of an epoxide ring at C-1, C-10 (δ 59.4 d, 60.7 s) was indicated by bidimentional NMR experiments. The NOESY experiment provided cross peaks of the OH with H-6, H-1 and H-9α, of H-3 with H-6 and H-4 and between H-9β and H-14, suggesting that the OH, H-1, H-3, H-4, and H-6 should be α-oriented. Structure 11 was confirmed by an X-ray analysis (Figure 2).
Compound 12 showed a molecular formula C22H28O8 by HRFABMS. The spectroscopic data of 11 and 12 were very similar. The 1H and 13C NMR spectra together with the HMBC experiment of 12 showed the presence of an ageloyloxy group (δ 6.27, qq; 2.02, dq; 1.97, quint) bonded at C-6 instead of the isobutyroyloxy observed in 11. Structure an relative stereochemistry of compound 12 were confirmed by X-ray analysis (Figure 3).

Compounds 5 was previously reported in mixture,4 and compound 8 was obtained as intermediary of synthesis,10 therefore the spectroscopic data of both compounds are included in the experimental section. The structures of known compounds 4, 6, 7, 9-10, 12-14 were determined by comparison of their physical constants and spectroscopic features with those reported in literature. Senecionine and integerrimine were identified by comparison with authentic samples and spectroscopic data.
The anti-inflammatory activity was tested on 12-O-tetradecanoylphorbol-13-acetate (TPA) induced model of acute inflammation.20 The MeOH extracts of roots and stems and the hexanic extract of stems showed mild activity with 39.9, 30.4, 34.3 % of edema inhibition, respectively, while the EtOAc extract of stems exhibited the highest activity with 66.6 % of edema inhibition. Compounds 1-7, 9-12 were also tested and unfortunately none of them show any relevant activity. Since P. praecox is used to cure rheumatism in folk medicine, this effect is probably related with the presence of the flavonoids naringenine (13), aromadendrin (14), and taxifolin (15) whose anti-inflammatory activity has been widely reported.21.22

EXPERIMENTAL
General experimental procedures. Melting points were determined on a Fisher-Jones melting point apparatus and are uncorrected. Optical rotations were determined on a Perkin-Elmer 343 polarimeter. UV and IR spectra were recorded on a Shimadzu UV 160U and a Bruker Tensor 27 spectrometer, respectively. 1D and 2D NMR spectra were obtained on an Eclipse JEOL 300 MHz, Bruker Avance 300 MHz or a Varian-Unity Inova 500 MHz spectrometer with tetramethylsilane (TMS) as internal standard. EIMS data were determined on a Bruker Daltonics Analysis 3.2 mass spectrometer. HRFABMS were performed at 10.000 resolution using electric field scans and polyethylene glycol ions (Fluka 200 and 300) as reference material. Circular dichroism was obtained on a Jasco J-720 spectropolarimeter. Column chromatography was carried out under vacuum on silica gel G 60 (Merck, Darmstadt, Germany). Flash chromatography was performed on silica gel 60 (230-400 Macherey-Nagel). TLC was carried out on Si gel 60 and preparative TLC on Si gel GF254 (Merck), layer thickness 2.0 mm. GC analyses was performed on an Agilent 6890 GC system with AT5 30 m x 0.25 mm 0.1 μm film thickness column at 100˚C initial temperature raised in 10 minutes to 200˚C final temperature. X-ray crystallographic analyses were realized on a Bruker Smart Apex CCD diffractometer with graphite-monochromated Mo Kα radiation (λ = 0.71073 Å). The structures were solved by direct methods using the program SHELXS. Non-hydrogen atoms were refined with anisotropic displacement parameters. Hydrogen atoms were included at calculated positions and were not refined.
Plant material. Pittocaulon praecox from Pedregal de San Angel Mexico D.F. and grown in Ozumba, Mexico State, Mexico, was collected in march 2007. A voucher specimen (MEXU 1236997) was deposited at the Herbario del Instituto de Biología, Universidad Nacional Autónoma de México.
Extraction and isolation. Dried and ground root barks (1.8 kg) were extracted with MeOH at rt to obtain 250 g of extract. A portion (85 g) of this extract was submitted to reductive process23 to obtain the alkaloidal residue (3.1 g). Purification of this residue by VCC (CH2Cl2-MeOH gradient system) produced senecionine (5 mg, mp 227-230 °C, MeOH),15,16 and integerrimine (4.3 mg, mp 170-172 °C, MeOH).15,16 The remaining MeOH extract (165 g) was fractionated through a VCC (EtOAc-MeOH gradient system) to obtain fraction A (eluted with EtOAc), fraction B (eluted with EtOAc-MeOH 19:1, 9:1 and 4:1) and fraction C (eluted with EtOAc-MeOH 1:1 and MeOH). Fraction A (55 g) was purified by VCC eluted with hexane-EtOAc gradient system to obtain fraction D (eluted with hexane and hexane-EtOAc 19:1) and fraction E (eluted with hexane-EtOAc 9:1). Purification of fraction D (35 g) by VCC (hexane-EtOAc gradient system) afforded fractions F (eluted with hexane-EtOAc 49:1) and G (eluted with hexane-EtOAc 19:1). Fraction F, after two successive chromatographies (hexane-EtOAc 49:1), produced a mixture of compounds 4 and 5 (1.2 g), purification of 200 mg of this mixture by preparative TLC eluted with hexane-EtOAc 99:1 produced senemorin (4, 125 mg, [α]D25 – 18.4°, c 0.14, CHCl3)6 and compound 5 (18 mg, [α]D25 –34.6°, c 0.13, CHCl3).4 Fraction G (19.2 g), after purification by VCC eluted with hexane-EtOAc 49:1, produced compounds 6 (1.1 g, [α]D25 –54.5°, c 0.20, CHCl3),7 7 (0.7 g, [α]D25 –38.2°, c 0.22, CHCl3)8 and a mixture (3:2) of compounds 6 and 7 (10.8 g). Purification of fraction E (9.5 g) by flash column (hexane-EtOAc 19:1) afforded 8 (30 mg, mp 190-192 °C, [α]D25 –94°, c 0.25, MeOH),10 9 (75 mg, [α]D25 –37.2°, c 0.14, CHCl3),9 10 (60 mg, [α]D25 –16.0°, c 0.20, CHCl3),9 a mixture (3:2) of compound 9 and 10 (5.8 g) and compound 11 (17 mg). Fraction B produced by crystallization (EtOAc-MeOH) compound 1 (5.8 g). Further purification of mother liquors of 1 (70 g) by VCC (EtOAc-MeOH in increasing gradient) produced fraction H (eluted with EtOAc-MeOH 49:1) and 5.5 g of 1, from fractions eluted with EtOAc-MeOH 19:1, purification of this last mother liqueurs (8.5 g) by a new chromatography eluted with EtOAc-MeOH 49:1 afforded 2.8 g of 1 and 1.2 g of compound 2. Fraction H (10 g) by flash column (EtOAc-MeOH 49:1) afforded 2 (1.2 g) and 3 (75 mg). Purification of fraction C (31 g) by VCC (EtOAc-MeOH gradient system) produced compound 1 (105 mg) and sucrose (5.5 g).
Dry and ground stem bark (1.7 kg) was extracted successively with hexane, EtOAc and MeOH. The hexane extract (122 g) was fractionated by VCC (hexane-EtOAc gradient mixture) to obtain from fractions eluted with hexane, after two successive chromatographies (hexane-EtOAc 49:1), compounds 6 (1.4 g), 7 (0.9 g) and 15.5 g of a mixture (3:2) of both compounds. From fractions eluted with hexane-EtOAc 19:1 (30 g) after purification by VCC (hexane-EtOAc 49:1) 1.2 g of 6, 0.8 of 7 and 13.8 g of their mixture (3:2) were obtained. The EtOAc extract (76 g) was purified by VCC eluted with hexane-EtOAc in increasing gradient. Fractions eluted with hexane (8 g) were purified by flash chromatography (hexane-EtOAc 49:1) to produce compounds 6 (180 mg), 7 (140 mg) and 5.3 g of their mixture. From fractions eluted with hexane-EtOAc 17:3 (3 g) after two successive chromatographies eluted with hexane-Me2CO 19:1 and CH2Cl2, respectively, compounds 11 (75 mg) and 12 (80 mg) were obtained. Fractions eluted with hexane-EtOAc 4:1 (13 g) were purified by a new VCC (hexane-EtOAc 9:1) to obtain naringenin (13, 370 mg, mp 255-256 °C, [α]D25 – 21.5°, c 0.2.5, MeOH).11,12 Purification of fraction obtained with hexane-EtOAc 7:3 (20 g) by VCC eluted with hexane-EtOAc 4:1, afforded 13 (60 mg), aromadendrin (14, 320 mg, mp 238-240 °C, [α]D25 + 11.9°, c 0.13, EtOH),13 a mixture (1:1) of 13 and 14 (3.2 g) and taxifolin (15, 13 mg, mp 218-220 °C, [α]D25 + 18.5°, c 0.2, Me2CO).14 Fractions eluted with EtOAc (10.5 g) were purified using EtOAc-MeOH mixture in increasing gradient to obtain compound 1 (670 mg), compound 2 (250 mg) and a mixture (2:1) of 1 and 2 (4,5 g). The MeOH extract (220 g) was fractionated with EtOAc-MeOH mixture in increasing gradient. Fractions eluted with EtOAc (20 g) produced by purification by VCC (hexane-EtOAc gradient mixture) sitosterol β-glucoside (550 mg), 13 (135 mg) and 14 (120 mg). Fractions eluted with EtOAc-MeOH 9:1 (80 g) afforded after purification by VCC column (EtOAc-MeOH 98:2) 1 (5.3g), 2 (7.8 g) and 48 g of a mixture (2:1) of both compounds.
Compound 1: Colorless needles (EtOAc-MeOH); mp 204-206 °C; [α]D25 –25.4° (c 0.28, MeOH); UV (MeOH) λmax (log ε): 204 (4.1), 248 nm (4.2); IR (Nujol): ν 3359, 1653, 1605 cm-1; 1H NMR (CD3OD, 300 MHz): δ 5.75 (1H, d, J = 2.1 Hz, H-9), 4.87 (1H, dd, J = 13.5, 1.5 Hz, H-12a), 4.49 (1H, dd, J = 13.5, 2.1 Hz, H-12b), 4.17 (1H, d, J = 7.6 Hz, H-1'), 3.80 (1H, dd, J = 12.0, 2.4 Hz, H-6'a), 3.65 (1H, dd, J = 12.0, 5.4 Hz, H-6'b), 3.54 (1H, ddd, J = 11.4. 11.4, 4.2 Hz, H-3), 3.33 (2H, m, H-3', H-4'), 3.19 (1H, m, H-2'), 3.14 (1H, m, H-5'), 3.02 (1H, d, J = 13.5 Hz, H-6β), 2.50 (1H, dddd, J = 15.0, 15.0, 5.1, 2.1 Hz, H-1β), 2.36 (1H, ddd, J = 15.0, 4.5, 2.7 Hz, H-1α), 2.19 (1H, br d, J = 13.5 Hz, H-6α), 2.13 (1H, m, H-2β), 1.97 (3H, d, J = 1.2 Hz, H-13), 1.43 (1H, m, H-2α), 1.39 (1H, m, H-4), 1.11 (3H, d, J = 6.6 Hz, H-15), 1.05 (3H, s, H-14); 13C NMR (CD3OD, 75.4 MHz): δ 193.3 (C, C-8), 171.2 (C, C-10), 144.5 (C, C-11), 132.2 (C, C-7), 126.3 (CH, C-9), 103.2 (CH, C-1'), 78.3 (CH, C-4')a, 77.9 (CH, C-5'), 75.0 (CH, C-2'), 71.7 (CH, C-3')a, 71.7 (CH, C-3), 69.9 (CH2, C-12), 62.7 (CH2, C-6'), 50.6 (CH, C-4), 43.8 (C, C-5), 42.4 (CH2, C-6), 36.4 (CH2, C-2), 31.7 (CH2, C-1), 17.9 (CH3, C-14), 16.5 (CH3, C-13), 11.1 (CH3, C-15). aExchangeable signals. MS (EI, 70 eV) m/z (%): 412 (2) [M]+, 250 (100), 233 (30); HRMS (FAB+) m/z: 413.2175 (calcd. 413.2175 for C21H33O8, [M+H]+).
Compound 2: Colorless needles (EtOAc-MeOH); mp 133-135 °C; [α]D25 +32.6° (c 0.17, MeOH); UV (MeOH) λmax (log ε): 237 nm (4.1); IR (Nujol): ν 3387, 1656, 1618 cm-1; CD (MeOH) Δελmax: Δε 270 -1.39, Δε 236 +8.10, Δε 205 +13.75 (c 3.4 x 10-5 M); 1H NMR (CD3OD, 500 MHz): δ 6.30 (1H, q, J = 1.5 Hz, H-12), 5.73 (1H, d, J = 1.5 Hz, H-9), 4.49 (1H, d, J = 7.5 Hz, H-1'), 3.84 (1H, dd, J = 13.5, 1.5 Hz, H-6'a), 3.66 (1H, dd, J = 13.5, 4.5 Hz, H-6'b), 3.56 (1H, ddd, J = 11.0, 11.0, 4.5 Hz, H-3), 3.37 (1H, m, H-5'), 3.35 (1H, m, H-2'), 3.32 (2H, m, H-3', H-4'), 3.03 (1H, dd, J = 14.0, 5.0 Hz, H-7), 2.53 (1H, dddd, J = 14.5, 14.5, 5.0, 1.5 Hz, H-1β), 2.33 (1H, ddd, J = 14.5, 4.0, 2.5 Hz, H-1α), 2,12 (1H, m, H-2β), 1.97 (1H, dd, J = 13.5, 5.0 Hz, H-6β), 1.90 (1H, dd, J = 14.0, 13.5 Hz, H-6α), 1.56 (3H, d, J = 1.5 Hz, H-13), 1.40 (1H, m, H-2α), 1.29 (1H, m, H-4), 1.20 (3H, s, H-14), 1.05 (3H, d, J = 6.5 Hz, H-15); 13C NMR (CD3OD, 125 MHz): δ 201.9 (C, C-8), 171.7 (C, C-10), 142.9 (CH, C-12), 124.6 (CH, C-9), 114.5 (C, C-11), 103.7 (CH, C-1'), 78.1 (CH, C-4')a, 77.7 (CH, C-5'), 74.3 (CH, C-2'), 71.1 (CH, C-3')a, 71.1 (CH, C-3), 62.4 (CH2, C-6'), 51.3 (CH, C-4), 43.1 (CH2, C-6), 40.9 (C, C-5), 36.0 (CH2, C-2), 31.9 (CH2, C-1), 17.3 (CH3, C-14), 10.6 (CH3, C-13), 10.6 (CH3, C-15). aExchangeable signals. MS (EI, 70 eV) m/z (%): 250 (55), 233 (20), 125 (100); HRMS (FAB+) m/z: 413.2175 (calcd. 413.2175 for C21H33O8, [M+H]+).
Compound 3: Colorless prisms (hexane-EtOAc); mp 175-177 °C; [α]D25 -31.2° (c 0.25, Me2CO); UV (MeOH) λmax (log ε): 226 nm (3.9); IR (KBr): ν 3351, 1755, 1651 cm-1; 1H NMR, (Me2COd6, 500 MHz): δ 5.66 (1H, d, J = 2.0 Hz, H-9), 3.46 (1H, ddd, J = 10.5, 10.5, 4.5 Hz, H-3), 2.88 (1H, d, J = 12.5 Hz, H-6β), 2.38 (1H, br d, J = 12.5 Hz, H-6α), 2.28 (1H, dddd, H = 14.5, 14.5, 5.0, 2.0 Hz, H-1β), 2.13 (1H, ddd, J = 14.5, 7.5, 4.5 Hz, H-1α), 2.03 (1H, m, H-2β), 1.78 (3H, d, J = 1.5 Hz, H-13), 1.41 (1H, dq, J = 10.5, 6.5 Hz, H-4), 1.31 (1H, m, H-2α), 1.10 (3H, d, J = 6.5 Hz, H-15), 0.97 (3H, s, H-14); 13C NMR (Me2COd6, 125 MHz): δ 171.9 (C, C-12), 159.1 (C, C-7), 150.3 (C, C-10), 122.6 (C, C-11), 120.3 (CH, C-9), 100.9 (C, C-8), 71.1 (CH, C-3), 51.6 (CH, C-4), 46.3 (C, C-5), 37.1 (CH2, C-6), 36.9 (CH2, C-2), 31.0 (CH2, C-1), 19.1 (CH3, C-14), 11.2 (CH3, C-15), 8.2 (CH3, C-13), - MS (EI, 70 eV) m/z (%): 265 (3), 246 (35), 219 (100), 178 (50); HRMS (FAB+) m/z: 265.1440 (calcd. 265.1440 for C15H21O4, [M+H]+).
Compound 5: Yellow oil; [α]D25 –34.6° (c 0.13, CHCl3); IR (CHCl3): ν 1728, 1464, 1152, 964 cm-1; 1H NMR (CDCl3, 300 MHz): δ 7.07 (1H, br s, H-12), 6.35 (1H, q, J = 1.2 Hz, H-6), 3.23 (1H, dd, J = 16.8, 1.8 Hz, H-9a), 3.09 (1H, d, J = 5.1 Hz, H-1), 2.65 (1H, hept., J = 7.2 Hz, H-2'), 2.18 (1H, d, J = 16.8 Hz, H-9b), 2.05 (1H, m, H-3a), 1.95-1.83 (2H, m, H-2a, H-3b), 1.85 (3H, d, J = 1.2 Hz. H-13), 1.52 (1H, ddq, J = 14.4, 7.2, 3.6 Hz, H-4), 1.34 (1H, m, H-2b), 1.26 (3H, s, H-14), 1.24 (6H, d, J = 7.2 Hz, H-3' and H-4'), 1.07 (3H, d, J = 7.2 Hz, H-15); 13C NMR (CHCl3, 75 MHz): δ 177.0 (C. C-1'), 148.4 (C, C-8), 138.9 (CH, C-12), 119.7 (C, C-7), 116.9 (C, C-11), 69.3 (CH, C-6), 63.3 (C, C-10), 62.9 (CH, C-1), 41.0 (C, C-5), 34.4 (CH, C-4), 32.1 (CH, C-2'), 30.6 (CH2, C-9), 23.6 (CH2, C-3), 19.9 (CH2, C-2), 19.5 (CH3, C-3’), 18.7 (CH3, C-4’), 16.7 (CH3-C-14), 15.2 (CH3, C-15), 8.6 (CH3, C-13).
Compound 8: Yellow needles (EtOAc-MeOH); mp 190-192 °C; [α]D25 -94.0° (c 0.1.5, MeOH); IR (KBr): ν 3471, 1665 cm-1; 1H NMR (CD3OD, 500 MHz): δ 5.86 (1H, s, H-9), 5.67 (1H, dd, J = 6.0, 2.5 Hz, H-1), 3.70 (1H, ddd, J = 10.0, 10.0, 6.0 Hz, H-3), 2.90 (1H, d, J = 16.5 Hz, H-6β), 2.62 (1H, ddd, J = 19.0, 6.0, 6.0 Hz, H-2β), 2.22 (1H, dd, J = 16.5, 1.5 Hz, H-6α), 2.10 (1H, ddd, J = 19.0, 10.0, 2.5 Hz, H-2α), 1.83 (3H, d, J = 1.5 Hz, H-13), 1.67 (1H, dq, J = 10.0, 7.0 Hz, H-4), 1.11 (3H, d, J = 7.0 Hz, H-15), 0.97 (3H, s, H-14); 13C NMR (CD3OD, 125 MHz): δ 175.2 (C, C-12), 142.1 (C, C-10), 141.2 (C, C-7)a, 137.0 (C, C-8)a, 127.2 (CH, C-1), 126.4 (C, C-11), 111.8 (CH, C-9), 69.2 (CH, C-3), 47.3 (CH, C-4), 40.9 (C, C-5), 36.9 (CH2, C-2), 35.8 (CH2, C-6), 20.8 (CH3-C14), 10.7 (CH3, C-15), 7.9 (CH3, C-13). aExchangeable signals.
Compound 11: Colorless prisms (EtOAc-MeOH); mp 258-260 °C; [α]D25 -46.0° (c 0.1.5, DMSO); UV (MeOH) λmax (log ε): 214 nm (3.8); IR (KBr): ν 3346, 1743, 1729, 1701 cm-1; 1H NMR (DMSOd6, 500 MHz): δ7.78 (s, OH), 5.88 (1H, d, J = 1.5, H-6), 4.86 (1H, ddd, J = 11.5, 7.0, 4.0 Hz, H-3), 3.19 (1H, d, J = 5.5, Hz, H-1), 2.70 (1H, hept, J = 7.0 Hz, H- 2'), 2.32 (1H, d, J = 13.5 Hz, H-9β), 2.26 (1H, ddd, J = 14.0, 7.0, 6.0 Hz, H-2α), 1.98 (3H, s, OAc), 1.90 (1H, br d, J = 13.0 Hz, H-2β), 1.85 (1H, m, H-4), 1.74 (3H, d, J = 1.5 Hz, H-13), 1.67 (1H, d, J = 13.5 Hz, H-9α), 1.17 (3H, d, J = 7.0 Hz, H-3'), 1.14 (3H, d, J = 7.0 Hz, H-4'), 1.08 (3H, s, H-14), 0.91 (3H, d, J = 7.5 Hz, H-15); 13C NMR (DMSOd6, 125 MHz): δ 174.9 (C, C-1'), 170.6 (C, C-12), 169.5 (C, OAc), 154.4 (C, C-7), 123.3 (C, C-11), 101.1 (C, C-8), 71.2 (CH, C-6), 67.3 (CH. C-3), 60.7 (C, C-10), 59.4 (CH, C-1), 44.1 (C, C-5), 41.8 (CH2, C-9), 33.3 (CH, C-4), 33.1 (CH, C-2'), 24.4 (CH2, C-2), 20.8 (CH3, OAc), 18.9 (CH3, C-3'), 18.3 (CH3, C-4'), 15.0 (CH3, C-14), 9.1 (CH3, C-15), 8.2 (CH3, C-13); MS (EI, 70 eV) m/z (%): 408 (10) [M]+, 320 (30), 260 (30), 71 (100); HRMS (FAB+) m/z: 409.1865 (calcd. 409.1862 for C21H29O8, [M+H]+).
Compound 12: Colorless prisms (EtOAc); mp 210-212 °C; [α]D25 -111.1° (c 0.27, CHCl3); UV (MeOH) λmax (log ε): 213.5 nm (3.6); IR (KBr): ν 3364, 1732, 1709 cm-1; 1H NMR (Me2COd6, 500 MHz): δ 6.68 (s, OH), 6.27 (1H, qq J = 7.5, 1.5 Hz, H-3'), 6.11 (1H, q, J = 1.5 Hz, H-6), 5.04 (1H, ddd, J = 11.5, 7.0, 4.5 Hz, H-3), 3.24 (1H, d, J = 6.0 Hz, H-1), 2.39 (1H, d, J = 13.0 Hz, H-9β), 2.36 (1H, ddd, J = 12.5, 7.0, 6.0 Hz, H-2α), 2.02 (1H, dq, J = 7.5, 1.5 Hz, H-4'), 1.97 (3H, quint, J = 1.5 Hz, H-5'), 1.99-1.95 (1H, m, H-2β), 1.96 (3H, s, OAc), 1.97-1.95 (1H, m, H-4), 1.79 (1H, d, J = 19.0 Hz, H-9α), 1.75 (3H, d, J = 1.5 Hz, H-13), 1.23 (3H, s, H-14), 1.00 (3H, d, J = 7.5 Hz, H-15); 13C NMR (Me2COd6, 125 MHz): δ 171.3 (C, C-12), 170.3 (C, OAc), 166.6 (C, C-1') 155.1 (C, C-7), 141.2 (CH, C-3'), 127.7 (C, C-2'), 124.9 (C, C-11), 101.9 (C, C-8), 72.3 (CH, C-6), 68.4 (CH, C-3), 61.7 (C, C-10), 60.9 (CH, C-1), 45.4 (C, C-5), 43.2 (CH2, C-9), 35.0 (CH, C-4), 25.7 (CH2, C-2), 20.9 (CH3, OAc)a, 20.6 (CH3, C-5')a, 16.0 (CH3, C-4'), 16.0 (CH3, C-14), 9.5 (CH3, C-15), 8.0 (CH3, C-13). aExchangeable signals. MS (EI, 70 eV) m/z (%): 420 (2), 320 (10), 83 (100); HRMS (FAB+) m/z: 421.1869 (calcd. 421.1862 for C22H29O8, [M+H]+).
Acid Hydrolysis of Compounds 1 and 2: Compounds 1 and 2 (100 mg each) were separately heated at 70 ºC for 0.5 h in 10% HCl. The reaction mixtures were extracted with CH2Cl2 and the aqueous layers were evaporated, sililated with HMDS:TMCS:pyridine (3:1:9) and analyzed by GC to identify glucose as the sugar present in both compounds 1 and 2.
Crystal data of 11: C21H28O8, Mr 408.43, orthorhombic, space group P 21 21 21 , a = 8.4002 (6) Å, α = 90o, b = 11.0620 (8) Å, β = 90o, c = 22.1000 (16) Å; γ = 90o, V = 2053.6(3) Å3, Z = 4, Dc = 1.321 Mg/m3, F(000) = 872; crystal dimensions / shape / color 0.34 x 0.16 x 0.08 mm / prism / colorless. Reflections collected 22413, independent reflections 3770. Number of parameters refined 274; final R indices [I>2σ(I)] R1 = 0.0356, wR2 = 0.0783; R indices (all data) R = 0.0465, wR2 = 0.0815.
Crystal data of 12: C22H28O8, Mr 420.44, orthorhombic, space group P 21 21 21 , a = 9.664 (1) Å, α = 90o, b = 11.878 (2) Å, β = 90o, c = 18.889 (2) Å; γ = 90o, V = 2168.1(4) Å3, Z = 4, Dc = 1.288 Mg/m3, F(000) = 896; crystal dimensions / shape / color 0.484 x 0.382 x 0.298 mm / block / colorless. Reflections collected 21590, independent reflections 2917. Number of parameters refined 338; final R indices [I>2σ(I)] R1 = 0.0553, wR2 = 0.1302; R indices (all data) R = 0.0653, wR2 = 0.1371.
Crystallographic data for 11 (CCDC-715671) and 12 (CCDC-715672) are available free of charge via the Internet at http://www.ccdc.cam.ac.uk/conts/retrieving.html (or from the CCDC, 12 Union Road, Cambridge CB2 1EZ, U.K.; fax: + 441223336033; deposit@ccdc.cam.ac.uk).

ACKNOWLEDGEMENTS
We are indebted to Carmen Márquez, Eréndira Ríos, Rubén A. Toscano, Simón Hernández, Ma. Rocío Patiño, Ma. Teresa Ramírez-Apan, Antonio Nieto, Beatríz Quiróz, Ma. Isabel Chávez, Héctor Ríos, Angeles Peña, Elizabeth Huerta, Nieves Zavala, Luis Velasco, Javier Pérez for technical assistance.

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