Rearranged Jatrophane-Type Diterpenes from Euphorbia Species. Evaluation of their Effects on the Reversal of Multidrug Resistance

• Abstract
• Introduction
• Materials and Methods
• General experimental procedures
• Plant material
• Extraction and isolation
• Assay for MDR reversal effect
• Results and Discussion
• Acknowledgements
• References

Abstract

The rearranged jatrophane-type diterpenes (1 - 3), isolated from the Me₂CO extracts of Euphorbia portlandica and Euphorbia segetalis, were examined for their effects on multidrug resistance (MDR) in mouse lymphoma cells. Compounds 2 and 3 revealed to be active with the latter being more active than the positive control verapamil, a known resistance modifier. The new compound 1, named portlandicine, was isolated from Euphorbia portlandica and its structure characterised by high-field NMR spectroscopic methods including 2D NMR techniques: COSY, HMQC, HMBC and NOESY. The known diterpene 2, together with aleuritolic acid (4), oleanolic acid (5), and betulin diacetate (6), were also isolated from the same species.

Introduction

In recent years, considerable attention has been devoted to the structure characterisation and biological evaluation of polyfunctional jatrophane diterpenes isolated from Euphorbia species (Euphorbiaceae) [1], [2], [3], [4]. Among these compounds, several jatrophane esters have been considered as potent modulators of multidrug resistance (MDR) [5].

The major difficulty for success in cancer chemotherapy is the resistance of cancer cells to multiple anticancer drugs. This resistance is mostly associated with the overexpression of P-glycoprotein, which functions as an ATP-dependent drug-efflux pump, reducing the intracellular accumulation of drugs. The use of MDR reversal agents is a promising approach to overcome the MDR phenotype and several attempts to develop effective modulators and find structure-reversal activity relationships have been reported [6], [7].

In a previous publication we have described the isolation and structure characterisation of compound 3 from E. segetalis L., a diterpene with a novel carbon skeleton [8]. In the present study, we are reporting the MDR-reversing effects of 3, as well as those of the new diterpene polyester 1 and the known diterpene 2, both isolated from Euphorbia portlandica L. (Euphorbiaceae), a herb widely distributed along the coastline of Portugal. The isolation of compounds 1, 2, 4 - 6 and the structure elucidation of 1 are also reported.

Materials and Methods

General experimental procedures

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 Bruker ARX-400 NMR spectrometer (¹H 400 MHz; ¹³C 100.61 MHz), with TMS as internal standard and CDCl₃ as solvent. MS were taken on a Kratos MS25RF spectrometer (70 eV) and EI-FTICR-MS (15 eV) and LDI-FTICR-HRMS on a Finnigan FT/MS 2001-DT. Column chromatography was carried out on SiO₂ (Merck 9385). TLC were performed on precoated SiO₂ F₂₅₄ 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 × 10 mm) column.

Plant material

Euphorbia portlandica was collected on 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 (n° 248) has been deposited at the herbarium (LISI) of Instituto Superior de Agronomia.

Extraction and isolation

The air-dried whole powdered plant (4.8 kg) was extracted (maceration) with acetone (7 × 8 L) at room temperature. Evaporation of the solvent (under vacuum, 40 °C) from the crude extract gave a residue of 367 g, which was suspended in an MeOH/H₂O mixture (1 : 1, 2 L) and extracted with n-hexane (3 × 1 L), for removal of the waxy material, and Et₂O (6 × 1.5 L). The ether extract was dried (Na₂SO₄) and evaporated (40 °C), yielding a residue (114 g) that was chromatographed on SiO₂ (1.5 kg), using mixtures of n-hexane/EtOAc (1 : 0, 3 L; 19 : 1, 2 L; 9 : 1, 2 L; 17 : 3, 1 L; 4 : 1, 1 L; 3 : 1, 1 L; 7 : 3, 5 L; 13 : 7 to 0 : 1, 5 % gradient, 1 L each eluent) and EtOAc/MeOH (3 : 1, 1 L; 1 : 1, 1 L) as eluting solvents.

The residue (10 g) of the crude Fr A (n-hexane/EtOAc; 7 : 3, 1 L) was subjected to CC on SiO₂ (700 g) using n-hexane/CH₂Cl₂ (1 : 0 to 3 : 7, 10 % gradient; 1 : 3 to 0 : 1, 5 % gradient; 1 L each eluent), CH₂Cl₂/EtOAc (19 : 1 to 7 : 3, 5 % gradient; 3 : 2 to 0 : 1, 10 % gradient; 1 L each eluent). The residue of Frs eluted with CH₂Cl₂/EtOAc (3 : 1, 0.5 L; Fr A1, 1.8 g) gave 3 mg of compound 4 (R_f: 0.50, CH₂Cl₂/MeOH, 19 : 1), after being subjected to CC (100 g) with n-hexane/CH₂Cl₂ (1 : 3 to 1 : 19; 10 % gradient; 0.5 L each eluent) and CH₂Cl₂/EtOAc (19 : 1 to 11 : 9; 5 % gradient; 0.5 L each eluent), and preparative TLC (2 plates, 4 × CH₂Cl₂/acetone, 49 : 1; 4 × n-hexane/EtOAc, 17 : 3). Frs eluted with CH₂Cl₂/EtOAc (2 : 3 to 1 : 9) were associated (Fr A2; 200 mg) and submitted to CC (20 g of SiO₂; CH₂Cl₂/EtOAc; 7 : 3 to 1 : 1; 5 % gradient; 0.2 L each eluent); the Frs eluted with CH₂Cl₂/EtOAc (181 mg, 7 : 3, 100 mL) afforded 2 mg of compound 5 (R_f: 0.60, CH₂Cl₂/MeOH, 19 : 1), after being rechromatographed by CC (CH₂Cl₂/EtOAc 1 : 0 to 3 : 1; 5 % gradient; 1 : 1; 0.25 L each eluent) and preparative TLC (3 plates, 3 × CH₂Cl₂/MeOH, 99 : 1; CH₂Cl₂/MeOH, 49 : 1). Fraction A3 (0.6 g), eluted with CH₂Cl₂/EtOAc (1 : 9, 1 L; 0 : 1, 2 L) and EtOAc/MeOH (3 : 1, 1 L), was chromatographed on SiO₂ (50 g; CH₂Cl₂/EtOAc, 1 : 1, 0.6 L) to afford two main Frs that were combined (0.2 L; 320 mg) and further purified by CC with the same solvents, yielding compound 6 (R_f: 0.40, CH₂Cl₂/MeOH, 19 : 1; 15 mg).

The crude Fr B (40 g; n-hexane/EtOAc 7 : 3, 2 L; 13 : 7; 1 L) was subjected to CC on SiO₂ (800 g) using a 5 % gradient of n-hexane/EtOAc (1 : 0 to 0 : 1; 1 L each eluent). The Frs eluted with n-hexane/EtOAc (1 : 1, 0.5 L; 9 : 11, 0.5 L) were associated (Fr B1, 5.5 g) and chromatographed on SiO₂ (550 g) with n-hexane/EtOAc (1 : 0, 9 : 1, 4 : 1, 7 : 3, 13 : 7 3 : 2, 11 : 9, 1 : 1, 2 : 3, 2 : 3, 0 : 1; 1 L each eluent). Combined Frs eluted with n-hexane/EtOAc (11 : 9, 0.9 L; 1 : 1, 0.2 L) yielded 3.3 g of a product (Fr B1a) that was rechromatographed with n-hexane/CHCl₃ (0 : 1 to 1 : 0; 25 % gradient; 0.5 L each eluent) and CHCl₃/MeOH (99 : 1, 2 L; 39 : 1, 19 : 1, 13 : 7, 4 : 1, 0 : 1; 0.5 L each eluent); Frs eluted with CHCl₃/MeOH (99 : 1, 0.35 L) afforded 400 mg of a mixture, which was submitted again to CC on SiO₂ (40 g) with n-hexane/CHCl₃ (1 : 1 to 0 : 1; 10 % gradient; 0.2 L each eluent) from which the Frs eluted with n-hexane/CHCl₃ (3 : 7, 0.2 L; 1 : 4, 0.2 L) were associated (252 mg) and submitted to preparative TLC (8 plates; 5 × CHCl₃), yielding two spots (R_f: 0.50 and 0.30). The spot with R_f: 0.50 was removed, submitted again to preparative TLC (4 plates, 4 × CHCl₃), and crystallized (n-hexane/CHCl₃), yielding 40 mg of 2 (Rf: 0.49, CH₂Cl₂/MeOH, 19 : 1). The mother liquor was associated to the product of spot with R_f: 0.30, and the residue (160 mg) was subjected to CC on SiO₂ (20 g) with CH₂Cl₂/acetone (1 : 0 to 19 : 1; 1 % gradient; 93 : 7, 9 : 1, 4 : 1, 1 : 1, 0 : 1; 30 mL each eluent), yielding two Frs [(35 mg; CH₂Cl₂/acetone, 99 : 1, 5 mL; 49 : 1, 10 mL) (32 mg; CH₂Cl₂/acetone, 49 : 1, 20 mL)]. The first one was subjected to successive reverse phase HPLC (MeOH/H₂O, 7 : 3; 2.5 mL/min) and preparative TLC (3 plates, 3 × CH₂Cl₂/MeOH; 99 : 1), yielding 1 (6 mg; t_R: 17 min; Rf: 0.37, CH₂Cl₂/MeOH, 19 : 1) and 2 (6 mg; t_R: 17 min; Rf: 0.49, CH₂Cl₂/MeOH, 19 : 1). Using the same procedure, 1 (8 mg) and 2 (2 mg) were obtained from the second Fr.

Portlandicine (2α,5α,14α,17α-tetraacetoxy-3β-benzoyloxy-15β-hydroxy-9-oxoparaliane 1): Amorphous solid; [α]_D ²⁵: -57.7° (CHCl₃, c 0.10); LDI-FTICR-HRMS: m/z (rel. int.) = 679.2722 [M + Na]⁺ (100) (C₃₅H₄₄O₁₂Na requires 679.2725); IR (film): ν_max = 3480, 2940, 2860, 1740, 1710, 1490, 1380, 1240, 715 cm^-1; ¹H- and ¹³C-NMR see Table [1]; EIMS: m/z (rel. int.) = 656 [M]⁺ (0.1), 638 (0.1), 596 (0.4), 581 (0.4), 536 (1), 518 (0.4), 414 (11), 396 (1), 354 (5), 294 (9), 105 (100), 77 (18), 43 (45); EI-FTICR-MS: m/z (rel. int.) = 657 [M + H]⁺ (3), 639 (13), 597 (46), 579 (10), 536 (39), 537 (35), 518 (8), 519 (6), 473 (77), 413 (71), 414 (11), 396 (7), 397 (10), 372 (52), 354 (78), 355 (48), 343 (55), 312 (59), 294 (91), 281 (100), 266 (34), 247 (82),188 (28), 105 (21).

1β,5α,14α,17α-Tetraacetoxy-3β-benzoyloxy-15β-hydroxy-9-oxo- paraliane (2): Amorphous solid (lit. not described); [α]_D ²⁵: -31.8° (CHCl₃, c 0.10) (lit. not described); IR (film): ν_max = 3480, 2940, 2860, 1740, 1710, 1490, 1380, 1240, 715 cm-1; EIMS: m/z (rel. int.) = 656 [M]⁺, 638 (0.1), 596 (1), 581 (0.1), 536 (4), 518 (1), 473 (6), 414 (13), 372 (10), 354 (9), 396 (1), (9), 294 (10), 105 (100), 77 (12), 43 (79). ¹H- and ¹³C-NMR as described [9].

Assay for MDR reversal effect

Cells: The L5178 Y mouse T-lymphoma parental cell line was transfected with the pHa MDR1/A retrovirus as previously described [10]. The L5178 MDR cell line and the L5178 Y parental cell line, (obtained from Prof. M. Gottesmann, NCI and FDA, USA), were grown in McCoy’s 5A medium with 10 % heat-inactivated horse serum, L-glutamine and antibiotics. MDR1 expressing cell lines were selected by culturing the infected cells with 60 ng/mL colchicine to maintain expression of the MDR phenotype. Cell viability was determined by trypan blue.

Rhodamine 123 (R123) uptake assay: The cells were adjusted to a density of 2 × 10⁶/mL, resuspended in serum-free McCoy’s 5A medium and distributed in 0.5 mL aliquots into Eppendorf centrifuge tubes. From 2.0 to 16.0 μL of the 2.0 mM stock solutions of the compounds in DMSO were then added and the samples were incubated for 10 min at room temperature. A total of 10 μL (5.2 μM final concentration) of Rhodamine 123 (R123; Sigma) was next added to the samples and the cells were incubated for further 20 min at 37 °C, washed twice and resuspended in 0.5 mL phosphate-buffered saline for analysis. The fluorescence of the cell population was measured by flow cytometry with a Beckton Dickinson FACScan instrument. Verapamil (5 μL of a 2.0 mM solution) was used as a positive control in the Rhodamine 123 exclusion experiments [11]. The mean fluorescence intensity was calculated as a percentage of the control for the parental and MDR cell lines as compared to untreated cells. An activity ratio (R) was calculated on the basis of the measured fluorescence values (FL-1) measured via the following equation: R = (FL-1_{MDR treated}/FL-1_{MDR control})/(FL-1_{parental treated}/FL-1_{parental control}) [11], [12].

Results and Discussion

The Et₂O soluble part of the acetone extract of the air-dried whole powdered plant of Euphorbia portlandica was submitted to successive chromatographic fractionation and purification, as described above, to afford the tetracyclic diterpenes 1 and 2, and the pentacyclic triterpenes aleuritolic acid (4), oleanolic acid (5), and betulin diacetate (6).

Compound 1, a new diterpene named portlandicine, was obtained as a white amorphous powder, whose molecular formula was determined as C₃₅H₄₄O₁₂ from its LDI-FTICR-HRMS, which showed a quasi-molecular ion at m/z = 679.2722 [M + Na]⁺, indicative of fourteen unsaturations. The IR spectrum of 1 exhibited the characteristic absorptions of a hydroxyl group, carbonyl groups and an aromatic ring. Its EI mass spectrum displayed a weak molecular ion peak at m/z = 656, a base peak at m/ z = 105 [C₆H₅CO]⁺, and ions at m/z = 77 [C₆H₅]⁺, 596 [M - AcOH]⁺, 536 [M - 2 × AcOH]⁺, 414 [M - 2 × AcOH - C₆H₅CO₂H]⁺, 354 [M - 3 × AcOH - C₆H₅CO₂H]⁺ and 294 [M - 4 × AcOH - C₆H₅CO₂H]⁺, which indicated the presence of a benzoyl moiety and four acetoxy groups in the molecule. These structural features were confirmed by the NMR spectral data of 1, which showed the presence of four acetyl methyl groups (δ = 2 × 2.09, 2.06, 1.99; δ_C = 22.2, 21.2, 2 × 20.9), one benzoyl group (δ = 7.48 - 7.96, 5 H; δ_C = 133.6, 2 × 129.6, 129.1, 2 × 128.7), and five acyl carbonyl resonances at δ_C = 170.7, 170.5, 170.0, 169.7 and 165.2 (Table [1]). Moreover, the ¹H-NMR spectrum of 1 showed signals for four tertiary methyl groups (δ = 1.57, 1.13, 1.06, 0.64), three methine protons (δ = 5.86, d; 5.66 d; 4.90, s), and one methylene (δ = 4.39 d; 4.30 d; J = 12.0 Hz) bounded to an ester function. The remaining ¹³C-NMR spectral data showed resonances of twenty carbons, which were grouped according to the DEPT spectrum into four CH₃, four CH₂ (one oxygenated carbon at δ_C = 63.5), six CH (three oxygenated carbons at δ_C = 78.2, 72.8 and 67.9), and six quaternary carbons (a carbonyl group at δ_C = 223.8, and two oxygen bearing C at δ_C = 89.8 and 83.8). The above MS and NMR data clearly resemble those reported in literature for the tetracyclic diterpenes 2 and 3 [8], [9], [13]. When comparing the NMR spectra, the Me-16, which appears as a doublet (δ = 0.85, J = 7.2 Hz) and has a ¹³C resonance at δ_C = 10.2 in compound 2, is displayed as a lower field singlet (δ = 1.57) and located at an oxygenated carbon (δ_C 89.8), in the spectrum of portlandicine. Besides, the H-1 AB-quartet in the spectrum of 1, at δ = 2.40 and 2.16, is absent in the ¹H-NMR spectrum of 2, and replaced by a low field methine proton (δ = 5.06 d; J = 10.2 Hz) attached to a carbon (δ_C = 74.4) bearing an acetoxy group. These features indicate that portlandicine and 2 are isomers with one acetoxy group permuted between C-1 and C-2. Accordingly to this change, other differences in proton and carbon chemical shits of ring A and B were also observed. The structure of 1 was confirmed by 2D NMR experiments including COSY, HMQC and HMBC, which allowed the unambiguous assignment of all of the proton and carbon signals of the skeleton (Table [1]).

The relative stereochemistry of 1 was deduced from a NOESY spectrum and the J _H-H values [8], [9], [13]. The NOE interactions of the α-oriented H-4, taken as a reference point on a biogenetic basis [14], with H-3 (very strong), H-1α and H-17, confirmed the α orientation for these three protons. Further cross peaks of the OH-15 with H-1β, Me-16, the o-aromatic protons, H-5, H-14, and H-12, established the trans A/B ring junction and the configuration at C-2, C-3, C-5, C-12, C-14 and C-15. The strong NOE enhancements of H-12 at H-8 indicated that they have the same β configuration and a cis C/D ring connection. In addition, the cis B/C ring fusion was deduced from the observed cross peaks between H-8/Me-19 and Me-18/Me-20. The absence of NOE correlations between H-12/H-17a,b and H-12/Me-20 and between H-8/H-17a,b and H-8/Me-20 confirmed this stereochemistry.

Compounds 1 - 3 were examined for their MDR-reversing activity on L5178 mouse lymphoma cells. The results are displayed in Table [2] . Compounds 2 and 3 were shown to be effective resistance modulators in Pgp expressing cells. These results also showed that this effect is dose-dependent. Compounds 3 exhibited the highest effect in reversing MDR (fluorescence activity ratio R = 15.59 at 16 μM concentration), and stronger than that of the positive control verapamil (R = 6.46 at 20 μM). The differences of activity observed for compounds 1 - 3 may be related with the substitution pattern of ring A that may induce small conformation changes in the molecule.

Jathrophane diterpenes have been considered, because of their unique structural character and their exclusive distribution in the family Euphorbiaceae, as a group of diterpenes with potential taxonomic importance within this plant family [1]. The carbon skeleton type of portlandicine and of compounds 2 and 3 has been previously found only in Euphorbia segetalis and in Euphorbia paralias, two species that are placed in the paralias section as is also the case for Euphorbia portlandica. Therefore, the occurrence of this skeleton in these three species seems to reinforce the taxonomic significance of jatrophane derivatives.

The diterpene 1β,5α,14α,17α-tetraacetoxy-3β-benzoyloxy-15β-hydroxy-9-oxoparaliane (2), and the pentacyclic triterpenes aleuritolic acid (4) {[α]_D ²⁵: + 14.0° (CHCl₃, c 0.10)}, oleanolic acid (5) {[α]_D ²⁵: + 60.0° (CHCl₃, c 0.15)}, and betulin diacetate (6) {[α]_D ²⁵: + 23.9° (CHCl₃, c 0.10)}, were identified by comparison of their spectral data with those reported in the literature [13], [15], [16], [17], [18].

Acknowledgements

The authors thank Dr. Teresa Vasconcelos (ISA, University of Lisbon) for identification of the plant and Mr. I. Marques (IST) for low resolution mass spectra.

References

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Prof. Maria José Umbelino Ferreira

Centro de Estudos de Ciências Farmacêuticas

Faculdade de Farmácia da Universidade de Lisboa

Av. das Forças Armadas

1600-083 Lisboa

Portugal

Fax: +351-21-7946470

Email: mjuferreira@ff.ul.pt

References

• 1 Hohmann J, Vasas A, Günther G, Máthé I, Evanics F, Dombi G, Jerkovitch G. Macrocyclic diterpene polyesters of the jatrophane type from Euphorbia esula .  J Nat Prod. 1997;  60 331-5 and related references cited therein
  CrossrefPubMedSearch in Google Scholar
• 2 Yamamura S, Kosemura S, Ohba S, Ito M, Saito Y. The isolation and structures of euphoscopins A and B.  Tetrahedron Lett. 1981;  22 5315-8
  CrossrefPubMedSearch in Google Scholar
• 3 Kupchan S M, Siegel C W, Matz M J, Gilmore C J, Bryan R F. Structure and stereochemistry of jatrophone, a novel macrocyclic diterpenoid tumor inhibitor.  J Am Chem Soc. 1976;  98 2295-300
  CrossrefPubMedSearch in Google Scholar
• 4 Zheng W F, Cui Z, Zhu Q. Cytotoxic and antiviral activity of the compounds from Euphorbia kansui .  Planta Med. 1998;  64 754-6
  Thieme ConnectPubMedSearch in Google Scholar
• 5 Hohmann J, Molnár J, Rédei D, Evanics F, Forgo P, Kálmán A, Argay G, Szabó P. Discovery and biological evaluation of a new family of potent modulators of multidrug resistance: reversal of multidrug resistance of mouse lymphoma cells by new natural jatrophane diterpenoids isolated from Euphorbia species.  J Med Chem. 2002;  45 2425-31
  CrossrefPubMedSearch in Google Scholar
• 6 Kim S E, Hong Y S, Kim Y C, Lee J J. Mode of action of torilim in multidrug-resistance cancer cell lines.  Planta Med. 1998;  64 335-8
  PubMedSearch in Google Scholar
• 7 Wiese M, Pajeva I K. Structure-activity relationships of multidrug resistance reversers.  Curr Med Chem. 2001;  8 685-713
  CrossrefPubMedSearch in Google Scholar
• 8 Ferreira M JU, Madureira A M, Ascenso J R. A tetracyclic diterpene and triterpenes from Euphorbia segetalis .  Phytochemistry. 1998;  49 79-83
  CrossrefPubMedSearch in Google Scholar
• 9 Jakupovic J, Jeske F, Morgenstern T, Tsichritzis F, Marco J A, Berendsohn W. Diterpenes from Euphorbia segetalis .  Phytochemistry. 1998;  47 583-1600
  CrossrefPubMedSearch in Google Scholar
• 10 Coenwell M M, Pastan I, Gottesmann M M. Certain calcium channel blockers bind specifically to multidrug-resistance human KB carcinoma membrane vesicles and inhibit drug binding to P-glycoprotein.  J Biol Chem. 1987;  262 2166-70
  PubMedSearch in Google Scholar
• 11 Weaver J L, Szabo G, Pine P S, Gottesman M M, Goldenberg S, Aszalos A. The effect of ion channel blockers, immunosuppressive agents, and other drugs on the activity of the multidrug transporter.  Int J Cancer. 1993;  54 456-61
  PubMedSearch in Google Scholar
• 12 Kessel D. Exploring multidrug resistance using Rhodamine 123.  Cancer Commun. 1989;  1 145-9
  PubMedSearch in Google Scholar
• 13 Jakupovic J, Morgenstern T, Marco J A, Berendsohn W. Diterpenes from Euphorbia paralias .  Phytochemistry. 1998;  47 1611-9
  CrossrefPubMedSearch in Google Scholar
• 14 Liu L G, Tan R X. New jatrophane diterpenoid esters from Euphorbia turczaninowii .  J Nat Prod. 2001;  64 1064-8 and related references cited therein
  CrossrefPubMedSearch in Google Scholar
• 15 Chaudhuri S K, Fullas D, Brown D M, Wani M C, Wall M E. Isolation and structural elucidation of pentacyclic triterpenoids from Maprounea africana .  J Nat Prod. 1995;  58 1-9
  CrossrefPubMedSearch in Google Scholar
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Prof. Maria José Umbelino Ferreira

Centro de Estudos de Ciências Farmacêuticas

Faculdade de Farmácia da Universidade de Lisboa

Av. das Forças Armadas

1600-083 Lisboa

Portugal

Fax: +351-21-7946470

Email: mjuferreira@ff.ul.pt