AZORELLANE DITERPENOIDS FROM LARETIA ACAULIS,
AND ITS TOXOPLASMACIDAL ACTIVITY

LUIS A. LOYOLA^1* , JORGE BORQUEZ¹, GLAUCO MORALES¹,
JORGE ARAYA², JORGE GONZALEZ², IVAN NEIRA², HERNAN SAGUA²
AND AURELIO SAN-MARTÍN³.

¹Laboratorio de Productos Naturales, Departamento de Química, Facultad de Ciencias
Básicas, Universidad de Antofagasta, Antofagasta, Chile. ² Unidad de Parasitología,
Facultad de Ciencias de la Salud, Universidad de Antofagasta. Chile.³Departamento de
Química, Facultad de Ciencias, Universidad de Chile, Chile.
(Received: December 2, 1999 - Accepted: September 7, 2000)

SUMMARY

In addition to azorellanol a new diterpenoid 7-deacetylazorellanol, has been isolated from the aerial parts of Laretia acaulis (Cav.) Gill et Hook (Umbelliferae). Its structure was based on spectroscopic comparison with azorellanol and chemical grounds. Azorellanol and 7-deacetylazorellanol showed marked inhibitor activity against trophozoites of protozoan parasite Toxoplasma gondii. The 50 % inhibitory dose for azorellanol was calculated in 54 mM and for 7-deacetylazorellanol was 42 mM.

KEY WORDS: Laretia acaulis, diterpenoids, azorellanol, 7-deacetylazorellanol, Toxoplasma gondii.

RESUMEN

Desde de las partes aéreas de Laretia acaulis (Cav.) Gill et Hook hemos aislado azorellanol y un nuevo diterpenoide 7-desacetilazorellanol. Su estructura esta basada en la comparación de sus datos espectroscópicos con los de azorellanol y transformaciones químicas. Azorellanol y 7-desacetilzorellanol muestran una marcada actividad inhibitoria frente a los trofozoitos del protozoo parásito Toxoplasma gondii. La dosis inhibitoria 50 (DI₅₀) de azorellanol es 54 mM y para 7-desacetylazorellanol 42 mM.

PALABRAS CLAVES: Laretia acaulis, diterpenoides, azorellanol, 7-desacetilazorellanol, Toxoplasma gondii.

INTRODUCTION

In a previous work on the petroleum ether extract of Laretia acaulis (Cav.) Gill et Hook (Umbelliferae)¹), we described the isolation of a new mulinane diterpenoid, 13-epimulinolic acid, and two known mulinane-type diterpenoids, mulinolic acid and mulin -11, 13 - dien -20-oic acid . Further investigation on the same plant material has revealed the presence of azorellane-type diterpenoids, azorellanol (1) previously isolated from Azorella compacta²) and a new diterpenoid, named 7-deacetylazorellanol (2). This paper describe the structural determination of the new diterpenoid 2 by detailed analysis of its¹H and ¹³CNMR spectra and the acetylation of 2. Looking to the biological activities of these compounds, we have examined the anti-Toxoplasma gondii effect of the diterpenoids 1 and 2 using trophozoites a strain of T. gondii isolated in Chile (Santiago strain)³). We have found that both compounds have strong toxoplasmacidal activity.

EXPERIMENTAL

General. Mp. uncorr. Plant material, collected in Los Morteros, Vallenar, Chile, in November 1997, was identified by Prof. Clodomiro Marticorena, Universidad de Concepción and a voucher specimens were deposited in the Herbarium of Universidad de Concepción, Concepción, Chile.

Extraction and isolation of diterpenoid. Dried and finely powdered whole plant of Laretia acaulis (1200 g) was extracted with petrol at room temp. After filtration, the solvent was evapd. to dryness under red. pres. and low temp. yielding a gum (187 g). The petrol ext. was adsorbed on silica gel (220 g) and slurred on top of a column containing silica gel (800 g) in petrol and eluted with petrol-EtOAc gradient with increasing amounts of EtOAc²).

Fraction eluted with petrol-EtOAc (20 %) (17.8 g) was rechromatographed on silica gel (350 g) eluted with 15 % petrol-EtOAc to yielded azorellanol 1 (4.6 g) and 7-deacetylazorellanol 2 (1.8g).

7-Deacetylazorellanol (2): Prisms (from Et₂O ). mp 167-169 ºC . (Et₂O); IR n_max^KBr cm^-1: 3.385 (OH). ¹H NMR (see Table 2). ¹³C NMR (see Table 1). EIMS (70 eV, direct inlet) m/z (rel. Int.): 306 (3), 288 [M-H₂O]⁺ (83), 273 (16), 255 (13), 245 (24), 227 (29), 216 (25), 203 (31), 191 (39), 181 (19), 177 (20), 122 ( 63), 95 (100), 81 (65).

Acetylation of 2. A mixt. of 2 (25 mg), Ac₂O (2 mL) and pyridine (3 mL) was stood at room temp. overnight. The reaction mixt. was evapd. in vacuo. The residue was chromatographed on a silica gel column [ EP-EtOAc (10:2) ] to yield the azorellanol 1 (23 mg).

Assay of toxoplasmacidal activity. Trophozoites from virulent Santiago strain³) of Toxoplasma gondii, maintained by intraperitoneal passages in Swiss mice, were harvested from peritoneal cavities with RMPI 1640 medium. In vitro toxoplasmicidal studies were carried out using a method described by Cover and Gutteridge⁴⁾ and modified by González⁵⁾ . Briefly, 45 mL of trophozoites suspension containing 5.10⁶ cells/mL were incubated, with 5mL of each biocompounds (dissolved in DMSO at final concentration of 1 and 2 , 0.5 and 0.1 mM) at 37º C, using 96 wells microplates. Cell vitality were determined in an inverted microscope with a trypan blue dye exclusion test⁶ after 24 hr. of incubation in 10 % CO₂ chamber. The results were expressed as % mortality. Clindamicyn ( Sigma, USA) a drug that has been used in human and animal anti-T. Gondii theraphy was used as positive control⁶⁾ . Data was evaluated by the probit analysis7) with a 95 % of confidence interval

RESULTS AND DISCUSION

Compound 1 was identified by spectral comparison with authentic sample of azorellanol²⁾ . The structure of 2 was determined as described below.

The molecular formula C₂₀H₃₄O₂of compound 2 was established based on ¹³C NMR, DEPT and EI mass spectrometry. The ¹H-decoupled ¹³C-NMR spectrum of 2 (Table 1) showed resonances of 20 carbons; DEPT analysis using a angle of 90º, indicated five saturated methines at d 76.5, 59.5, 47.3, 31.7 and 25.8. The DEPT 135º spectrum showed six methylenes and five methyl carbons indicating, after comparison with a decoupled spectrum (Table 1), that the carbons at d 70.0, 42.4, 35.1 and 26.4 were not attached to hydrogens. The IR, 1H-NMR (Table 2) and 13C-NMR data of 2 suggested the presence of a secondary hydroxyl group [ 3385 cm^-1, d 4.05 (1H, dd, J= 11.3 and 6.9 Hz), d 76.5 (d) ] and revealed that the remaining proton was part of a tertiary hydroxyl group (d 70.0) .

The lack of olefinic or carbonyl resonances in the ¹³C NMR spectrum of compound 2 (Table 1) indicated that rings had to account for remaining four sites of unsaturation in the molecule. The 1H and ¹³C-NMR spectra of 2 (Tables 1, 2), together with ¹H COSY and HMQC and HMBC experiments, revealed the presence of an isopropyl group [dC 31.7 (CH), 22.8 (CH₃) and 22.7 (CH₃); dH 1.47 overlapped signal, 0.84 d and 0.96 d (J= 6.5 Hz in both signals)], two tertiary methyl groups [ dC 17.6 (CH₃), 17.7 (CH₃); dH 0.96 s, 0.87 s ], another tertiary methyl group attached to hydroxyl carbon [dC 30.4 (CH₃), 70.0 (C); dH 1.28 s ] and three shielded proton resonances at dH 0.13 t (J= 4.6 Hz), 0.66 dd (J= 10.0 and 4.6 Hz) and 0.77 dd (J= 10.0 and 4.6 Hz), which were assigned to the protons of a cyclopropane ring (methylene carbon at dC 10.8, methine carbon at dC 25.8 and quaternary carbon at dC 26.4). Comparison of these data (Tables 1, 2) with those of 1, previously isolated of Azorella compacta²⁾ suggested a structure closely related to azorellanol (1). In fact, the only difference resided in the presence of a hydroxyl group in 2 instead of the acetoxyl group in 1. Finally, the structure depicted in formula 2 was confirmed by acetylation of OH-7, which led to azorellanol 1. Additionally 2 is identical in all respect to hydrolysis product of azorellanol²⁾ 1 . Thus, compound 2 is 7-deacetylazorellanol .

The two diterpenoids isolated from L. Acaulis were examined for their parasiticidal effect against trophozoites of Toxoplasma gondii, the aethiologic agent of human and animal toxoplasmosis. Azorellanol 1 and deacetylazorellanol 2 showed marked cytotocic effect against free trophozoites at concentration of 1 and 0.5 mM, with 91 % and 98 % of lysis, respectively. The 50 % inhibitory dose (ID50) for 1 was calculated in 54 mM and 42 mM for 2. Similar toxoplasmacidal activity was observed when clindamicyn was used as experimental control, with ID50 of 84 mM.

During the last decade, toxoplasmosis has reached special importance as an opportunistic agent of encephalopathies in patients with Acquired Immunodeficiency Syndrome (AIDS) or in congenital diseases. Some of these diseases could be avoided by use of chemoprophylactic drugs. However the drugs available are not entirely efficient, have some side-effects and are expensive if were used in all patients with risk of develop the disease that in AIDS patients is always lethal⁸⁾ . This paper describes the first example of in vitro activity of diterpenoids against T. gondii. The evidence presented in this communication supports the hypothesis that azorellanol, desacetylazorellanol, and other related compounds may prove to be good replacements for toxoplasmacidal drugs currently in use. Further studies are in progress in order to support the use in animal models, and identified the biochemical pathways involved in the parasiticidal effect of these natural compounds.

ACKNOWLEDGEMENTS

We thank Prof. C. Marticorena, Universidad de Concepción (Chile), for the botanical classification of the plant material and Dr. Jose Darias, Instituto de Productos Naturales y Agrobiologia de Canarias, CSIC (Tenerife-España) for the mass spectral measurements. This work was subsidised by the FONDECYT (Chile, Grant Nº 1980376) and DICYT (Universidad de Antofagasta)

*Author to whom correspondence should be addressed

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