Synthesis and pharmacological evaluation of pyrazolo[4,3-c]quinolinones as high affinity GABAA-R ligands and potential anxiolytics.

The synthesis, in vitro ligand binding study and in vivo Elevated Plus Maze test (EPM) of a series of pyrazolo[4,3-c]quinolin-3-ones (PQs) are reported. Multistep synthesis of PQs started from anilines and diethyl 2-(ethoxymethylene)malonate to give the quinolin-4-one nucleus, via the Gould-Jacobs reaction. These quinolinones were transformed to 4-chloroquinolines, which react with aryl-hydrazines affording the final compounds. PQs exhibited different potency in displacing specific [3H]Flunitrazepam binding from the benzodiazepine binding site at the γ-aminobutyric acid receptor (GABAA-R) depending on the substitution of the pyrazoloquinolone nucleus. PCA helped determine how different substituents contributed to the differential behavior of the PQs studied. Compounds with high affinity for the GABAA-R were tested regarding their anxiolytic properties in Wistar adult male rats using the Elevated Plus Maze (EPM). Thus, PQs with a p-methoxy phenyl group at N-1 (7b-ii and 7c-ii) displayed a remarkable anxiolytic activity at low doses (0.5-1.0 mg/kg). Meanwhile, PQs featuring an unsubstituted phenyl (7b-i) or p-fluoro phenyl group (7b-iii) at the N-1 showed anxiogenic effects in the EPM test.

Volatiles and Nonvolatiles in Flourensia campestris Griseb. (Asteraceae), How Much Do Capitate Glandular Trichomes Matter?

The distribution and ultrastructure of capitate glandular trichomes (GTs) in Flourensia species (Asteraceae) have been recently elucidated, but their metabolic activity and potential biological function remain unexplored. Selective nonvolatile metabolites from isolated GTs were strikingly similar to those found on leaf surfaces. The phytotoxic allelochemical sesquiterpene (-)-hamanasic acid A ((-)-HAA) was the major constituent (ca. 40%) in GTs. Although GTs are quaternary ammonium compounds (QACs)-accumulating species, glycine betaine was not found in GTs; it was only present in the leaf mesophyll. Two (-)-HAA accompanying surface secreted products: compounds 4-hydroxyacetophenone (piceol; 1) and 2-hydroxy-5-methoxyacetophenone (2), which were isolated and fully characterized (GC/MS, NMR), were present in the volatiles found in GTs. The essential oils of fresh leaves revealed ca. 33% monoterpenes, 26% hydrocarbon- and 30% oxygenated sesquiterpenes, most of them related to cadinene and bisabolene derivatives. Present results suggest a main role of GTs in determining the volatile and nonvolatile composition of F. campestris leaves. Based on the known activities of the compounds identified, it can be suggested that GTs in F. campestris would play key ecological functions in plant-pathogen and plant-plant interactions. In addition, the strikingly high contribution of compounds derived from cadinene and bisabolene pathways, highlights the potential of this species as a source of high-valued bioproducts.

Distribution of (-)-hamanasic acid A in South American species of Flourensia and phytotoxic effects of leaf aqueous extracts.

The presence of the phytotoxic sesquiterpene (-)-hamanasic acid A {(-)HAA; 7-carboxy-8-hydroxy-1(2), 12(13)-dien-bisabolene} isolated from Flourensia campestris (FC), was investigated in the South American species of the genus, together with the evaluation of the phytotoxic activity of their leaf aqueous extracts. (-)HAA was identified and isolated from F. fiebrigii (FF) and F. oolepis (FO), being chemically (GC-MS, NMR, [a]) and biologically (bioassayed on lettuce) indistinguishable from that of FC, while no (-)HAA was found in F. hirta (FH), F. riparia (FR) and F. niederleinii (FN). Its leaf content in FF was similar to that found in FC (ca. 15 mg g-1 WT) and significantly higher than in FO (0.8 mg g-1 WT). The screening for the presence of (-)HAA in other species of Flourensia communities showed that its natural occurrence is restricted only to Flourensia species. No (-)HAA could be detected in any of the 37 -most representative- species of these communities (26 natives, 11 exotics), despite many of them belong to the same family and tribe as Flourensia spp. Leaf aqueous extracts of all Flourensia species exhibited strong inhibitory effects on lettuce germination and on root and shoot growth, regardless of the presence and content of (-)HAA. These results strongly suggest the existence of other powerful phytotoxic compounds in those Flourensia spp lacking (-)HAA. Our results clearly show that (-)HAA only pertains to some species of the genus Flourensia. Relative to previous exomorphologic groupings of the genus, our chemotaxonomic data would give support to the close link described between FC and FF, but not with FR. In addition, the fact that (-)HAA was also found in FO, which belongs to a second different line, also points out that species position in this lineage would deserve to be revisited. The restricted production of (-)HAA by Flourensia in their communities suggests its special link with the genus, and sustains its putative allelochemical role.

Phytotoxic activity in Flourensia campestris and isolation of (--)-hamanasic acid A as its active principle compound.

An aqueous extract from Flourensia campestris (Asteraceae) dry aerial parts showed strong inhibition on the germination and growth of Lactuca sativa. Based on bio-guided chromatographic fractionation of aq. extracts from dry and fresh leaves and spectroscopic means, (-)-hamanasic acid A (7-carboxy-8-hydroxy-1(2), 12(13)-dien-bisabolene (1)) was isolated as the most inhibitory active principle on germination (ECg(50)=2.9 mM) and on root (ECr(50)=1.5 mM)/shoot (ECs(50)=2.0 mM) growth. As measured by GC, and correlated with a simple designed 2D-TLC, compound 1 was distributed throughout the plant, with a remarkably high concentration (1.6%) in the leaves and the inflorescences. At least a quarter of the amount of 1 was found in aqueous extracts suggesting that leaching would be a key route for its release into the environment. By contrast, leaf essential oils (HD) between 0.5 and 1.5 µl ml(-1) did not show herbicidal effects and 1 was not found in them (TLC) nor among volatiles (HS-SPME). Volatile compositions were assessed by GC-FID and GC-MS and led to the identification of 23 compounds (4 monoterpenes and 19 sesquiterpenes) with a wide seasonal (spring-summer%) variation, represented principally by bicyclo-germacrene (37-6%), spathulenol (4-32%), globulol (20-0%), beta-caryophyllene (15-6%), caryophyllene oxide (1-13%) and bicycloelemene (10-1%), respectively. The high amount of 1 in F. campestris together with its feasibility of being extracted with water suggest that (-)-hamanasic acid A is an allelochemical in this species. Species-specific studies must be carried out to evaluate the potential of 1 as a natural herbicidal compound.