Isoprenoid Compounds from Euphorbia portlandica . X-ray Structure of Lupeportlandol , a New Lupane Triterpene

O estudo fitoquímico dos extratos de Me 2 CO da planta inteira e seca de Euphorbia portlandica levou ao isolamento de um novo álcool triterpênico pentacíclico, com o esqueleto do lupano, designado por lupeportlandol. A sua estrutura foi estabelecida como 3α-hidroxi-19αH-lup-20(29)-eno. Foram também isolados os já conhecidos triterpeno pentacíclico glutinol e o esteróide β-sitostenona. A caracterização do novo composto e do seu derivado acetilado foi baseada em métodos espectroscópicos e numa análise de difração de raio-X. O acetato de lupeportlandol mostrou-se inativo em ensaios de citotoxicidade in vitro contra três linhagens de células tumorais humanas: MCF-7 (câncer da mama), NCI-H460 (câncer do pulmão) e SF-268 (câncer do SNC).


Introduction
Euphorbia portlandica L., from Euphorbiaceae family, is frequently found in the coast of Portugal, mainly in sand and rocks near the sea.][11] Previous studies on this species have afforded rearranged jatrophane-type diterpenes, which have been found to be effective modulators of multidrug resistance in tumor cells. 12The present paper reports the isolation and structure determination of a new pentacyclic triterpene alcohol with the lupane skeleton (1), as well as the isolation of the known compounds glutinol (2) and β-sitostenone (3) from the Me 2 CO extracts of the whole dried plant Euphorbia portlandica.The evaluation of the acetylated derivative of compound 1 (1a) for its in vitro effect on the growth of three human cancer cell lines is also reported.

General experimental procedures
Melting points were determined on a Kofler apparatus and are not corrected.Optical rotations were obtained using a Perkin-Elmer 241-MC polarimeter.IR spectra were determined on a Perkin-Elmer 1310 instrument.The NMR spectra were recorded on a Varian Unity-300 NMR spectrometer ( 1 H 300 MHz; 13 C 75.4 MHz), with TMS as internal standard and CDCl 3 as solvent.MS were taken on a Kratos MS25RF spectrometer (70 eV) and HRMS on a Finnigan FT/MS 2001-DT.Column chromatography was carried out on SiO 2 (Merck 9385).TLC was performed on precoated SiO 2 F 254 plates (Merck 5554 and 5744) and visualized under UV light and by spraying with sulphuric acid-acetic acid-water (1:20:4) followed by heating.HPLC was carried out on a Merck-Hitachi instrument, with UV detection, using a Merck LiChrospher 100 RP-18 (10 µm, 250 x 10 mm) column.The purity of the isolated compounds was monitored by means of analytical TLC and HPLC or GC being the latter analyses performed on a Hewlet Packard-5890 with a HP-17 column (10 m x 0.53 mm x 2.0 µm, 50 % PhMe silicone), isothermally, at 270 ºC, with He as carrier gas (20 mL min -1 ) and injection and detection temperature 300 ºC; cholesterol acetate was used as an internal standard.

Plant material
Euphorbia portlandica was collected in the west coast of Portugal near the beach of Vale Furado, Nazaré, in September 1997 and identified by Dr. Teresa Vasconcelos of Instituto Superior de Agronomia, University of Lisboa.A voucher specimen (nº 248) has been deposited at the herbarium (LISI) of Instituto Superior de Agronomia.

X-ray crystallographic analysis
Appropriate crystals of 1 for X-ray diffraction analysis were obtained by recrystallization from Me 2 CO.The crystal data were collected using a Turbo CAD4 diffractometer using graphite monochromated Cu radiation.A summary of the crystal data and refinement conditions is presented in Table 3.During data collection, the intensity of three standard reflections was monitored every hour of X-ray exposure time showing no significant decay.The structure was solved and refined using the WinGX package. 13The program used to solve the structure was SIR97, 14 and to refine was SHELXL 97. 15 All hydrogens were found except the O-H hydrogen that was placed in calculated position riding on a carrier atom with isotropic displacement parameter amounting 1.5 times the value of the equivalent isotropic displacement parameter of the carrier atom.Absolute structure configuration was confirmed by the Flack parameter (see Table 3).Graphics were done with ORTEP-3 for Windows version 1.076, 16 included in WinGX system.

Cancer cell growth assay
][19] Doxorubicin was used as the positive control.

Results and Discussion
The acetone extract of the whole dried plant Euphorbia portlandica was partitioned between a MeOH/H 2 O solution and hexane.Fractionation of the hexane extract yielded a new pentacyclic triterpene alcohol with a lupane skeleton, named lupeportlandol, which structure was established as 3α-hydroxy-19αH-lup-20(29)-ene (1).The known pentacyclic triterpene glutinol (2) and the steroid β-sitostenone (3) were also isolated and identified.
Compound 1 showed a molecular ion, in the HREIMS, at m/z 426.38552 indicating the molecular formula C 30 H 50 O (calc.426.38561).The IR spectrum of 1 showed absorption bands for a hydroxyl group (3405 cm -1 ) and an exocyclic methylene group (3104, 1640, 884 cm -1 ).The EI-MS spectrum showed the molecular ion peak at m/z 426, the common fragments of pentacyclic triterpenes and a base peak at m/z 189 (Figure 1).This fragment may arise from the cleavage between C-8/C-14 and C-12/C-13 bonds with H-transfer and suggests the lupane or hopane skeleton for 1. 20,21 The 1 H NMR spectrum of 1 (Table 1) showed the presence of six tertiary methyl groups (δ 0.83, 0.86, 0.91, 0.93, 0.94, and 1.06) and one methyl group attached to an sp 2 carbon at δ 1.69.The latter methyl group and the The proton at δ 3.39 was ascribed to H-3 and must be equatorially (3α-OH) oriented since it appears as a broad singlet (W 1/2 = 7 Hz), resulting from small couplings to the protons H-2 (J 3eq,2ax ≅ J 3eq, 2eq ) and is slightly downfield shifted (∆δ ≅ 0.20 ppm) relatively to the normal chemical shift of H-3, axially oriented in common triterpenes, that appears as a double doublet near δ 3.20. 22,23 he multiplet appearing at δ 2.54 was assigned to H-19.The 13 C NMR spectrum showed signals for 30 carbon atoms with multiplicities assigned by DEPT experiment The low field region of this spectrum shows the olefinic carbons at δ 108.9 (CH 2 ) and 150.6 (C) assigned to the terminal double bond of the isopropenyl group and one methine carbon at δ 76.3.In the high-field region seven CH 3, ten CH 2 , five methines, and five quaternary carbons were identified.Acetylation of 1, with Ac 2 O-pyridine, yielded a monoacetyl derivative 1a that showed essentially the same 1 H NMR spectrum, except for the signal due to H-3 shifted to lower field (δ 4.72 brs) and the acetyl methyl resonance (δ 2.08.) The above results agree with a lupane skeleton for 1 having an unusual configuration for the hydroxyl group at C-3 (3α-OH) that was further confirmed by the carbon resonances of ring A which were shifted upfield (except C-10), when compared to those reported for lupeol (4), whereas paramagnetic shielding is observed for Me-24. 23However, by comparing the NMR data of 1 with those reported for epilupeol (5) significant differences are observed for proton and carbon resonances of rings D and E.  23 The stereochemistry of 1 was partially solved by through space proton-proton correlations observed in a NOESY spectrum.Indeed, the correlations between Me-25/Me-26, Me-26/Me-28 and Me-28/ isopropenyl (Figure 2) and the assumption of the same β configuration for both methyl groups at C-10 and C-8, as found in lupeol, provided evidence for a β configuration for both the isopropenyl group and Me-28.
The less clear stereochemical aspects of 1, as the ambiguity in the stereochemistry of C-18, led to an X-ray diffraction analysis of compound 1. Figure 3 shows the ORTEP diagram of the molecule establishing that the structure of lupeportlandol is 3α-hydroxy-19αH-lup-20(29)ene, a new epimer of both compounds 5 (at C-19) and 8 (at C-3).9][30][31] This data also agree with the conformation found both in 3α,4α-epoxy-D:A-friedo-18β,19αH-lupane 30 and 3β-hydroxy-20-oxo-29(20→19)abeolupane 31 where no double bonds were found in the carbon skeleton of the molecule.
Compounds 2 and 3, respectively identified as glutinol and β-sitostenone, were also isolated.[34] The ability of compound 1a to inhibit the in vitro growth of MCF-7, NCI-H460 and SF-268 cell lines was evaluated.It showed to be inactive (GI 50 > 100 µM) against the three cell lines.

Table 3 .
Crystal data and structure refinement for lupeportlandol(1)