Biological activity of amides and analogs from Piper species and synthetic analogs

Foram isoladas de Piper scutifolium e P. corcovadensis seis amidas, incluindo a piperovatina, piperlonguminina, isopiperlonguminina e corcovadina, duas inéditas (scutifoliamida A e scutifoliamida B), além de duas aristolactamas (piperolactama C e stigmalactama). A partir de P. tuberculatum foram isoladas as amidas piplartina e pelitorina e os ésteres (E)-3,4,5-trimetoxicinamato de etila (11) e 3,4,5- trimetoxidiidrocinamato de etila (12), esse último obtido pela primeira vez de origem natural. Em função da atividade biológica apresentada pelas amidas isoladas, foram sintetizadas 5-fenil-pentadienamidas e 3-cinamamidas com diferentes substituintes nos anéis aromáticos e na porção amídica. Os extratos, produtos naturais isolados e sintéticos foram submetidos a ensaios de atividade antifúngica frente Cladosporium cladosporioides e C. sphaerospermum, antimicrobiana frente à Candida albicans, C. krusei, C. parapsilosis e Cryptococcus neoformans, inibidora de acetilcolinesterase, carrapaticida frente à Boophilus microplus e citotóxica frente a linhagens tumorais humanas. Para a atividade antifúngica contra C. cladosporioides e C. sphaerospermum foi constatada maior atividade para as N-isobutil e N-pentilamidas sem substituintes no anel aromático. As N-isobutilamidas foram mais ativas como inibidores de acetilcolinesterase; contra B. microplus grande atividade foi apresentada por piperovatina, e os ensaios de citotoxicidade indicaram que o grupo 5,6-dihidropiridin- 2(1H)-ona é fundamental para a atividade. As plântulas de P. corcovadensis apresentaram em sua composição predominantemente piperovatina e assim foram utilizadas para estudos com incorporação de acetato de sódio-[1-14C] e L-fenilalanina - [U-14C] na mesma. As plântulas e calos obtidos para P. scutifolium não apresentaram produção significativa de metabólitos secundários. O estudo do metabolismo fenilpropanoídico de P. regnellii resultou no isolamento parcial da enzima responsável pela conversão de conocarpano em eupomatenóide-6.Six amides were isolated from Piper scutifolium and P. corcovadensis including piperovatine, piperlonguminine, isopiperlonguminine and corcovadine, the new scutifoliamide A and scutifoliamide B and also two aristolactams (piperolactam C and stigmalactam). From P. tuberculatum the amides piplartine and pellitorine and two esters, ethyl (E)-3,4,5-trimethoxycinnamate (11) and ethyl 3,4,5- trimethoxydihydrocinnamate (12) were isolated, this last one obtained for the first time from natural sources. Due to the biological activity observed for the natural amides, 5- phenyl-pentadienamides and 3-cinnamamides having different substituents in the aromatic ring and amide moietis were synthesized. Extracts, isolated and synthetic compounds were assayed against Cladosporium cladosporioides, C. sphaerospermum, Candida albicans, C. krusei, C. parapsilosis and Cryptococcus neoformans, inhibitory of acetylcholinesterase, against the tick Boophilus microplus and citotoxic against human cell lines. A higher activity was observed against C. cladosporioides and C. sphaerospermum for N-isobutylamides and N-pentylamides having no substituents in the aromatic ring. N-isobutylamides were very active inhibitors of acetylcholinesterase; piperovatin was the most active against B. microplus and 5,6-dihydropyridin-2(1H)-one moiety was essential as the citotoxicity is concerned. Plantlets of P. corcovadensis contained piperovatine as major compound and thus were used as model for incorporation of sodium acetate-[1-14C] and L-phenylalanine-[U-14C]. The production of secondary metabolites in plantlets and callus of P. scutifolium was not significant. The studies of phenylpropanoid metabolism in P. regnellii was addressed to partial purification and characterization of the enzyme involved in the conversion of conocarpan to eupomatenoid-6

Fragmentation pattern of amides by EI and HRESI: Study of protonation sites using DFT-3LYP data

Proc. 2005/51850-9 and 2014/50316-7. POCI-01-0145-FEDER-007265.Amides are important natural products which occur in a few plant families. Piplartine and piperine, major amides in Piper tuberculatum and P. nigrum, respectively, have shown a typical N-CO cleavage when analyzed by EI-MS or HRESI-MS. In this study several synthetic analogs of piplartine and piperine were subjected to both types of mass spectrometric analysis in order to identify structural features influencing fragmentation. Most of the amides showed an intense signal of the protonated molecule [M + H]+ when subjected to both HRESI-MS and EI-MS conditions, with a common outcome being the cleavage of the amide bond (N-CO). This results in the loss of the neutral amine or lactam and the formation of aryl acylium cations. The mechanism of N-CO bond cleavage persists in α,β-unsaturated amides because of the stability caused by extended conjugation. Computational methods determined that the protonation of the piperamides and their derivatives takes place preferentially at the amide nitrogen supporting the dominant the N-CO bond cleavage.publishe

Morphological changes in <i>D. rerio</i> during the 48 hours of exposure to piplartine.

<p><b>A</b>) Leakage of the ocular pigment caused by 1.8 ppm piplartine, <b>B</b>) tissue alterations on the head and mouth caused by 1.6 µg/ml piplartine, <b>C</b>) exophthalmia and hemorrhage caused by 1.4 µg/ml piplartine and D) control group.</p

Toxicity of piplartine and niclosamide to the freshwater microcrustacean <i>D. similis</i> and the fish <i>D. rerio</i>.

<p>Data are presented as EC₅₀ or LC₅₀ (µg/ml) with the respective 95% confidence limits in brackets.</p><p>Toxicity classification: Cat. 1 - acute toxicity ≤1.00 µg/ml; Cat. 2 - acute toxicity >1.00 but ≤10.0 µg/ml; Cat. 3 - acute toxicity >10.0 but <100 µg/ml <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002251#pntd.0002251-Abiquim1" target="_blank">[28]</a>.</p

Mortality of <i>B. glabrata</i> adults exposed to methanolic extracts of different organs of <i>Piper tuberculatum</i>.

<p>n = 30 adult snails.</p><p>Values were obtained at the end of the 7^th day of observation.</p

LC₅₀ and LC₉₀ for <i>B. glabrata</i> embryos and adults exposed to piplartine.

<p>[ ] 95% confidence interval.</p><p>nc – not calculated.</p

Mortality of <i>B. glabrata</i> adults and embryos exposed to amides (20 µg/ml).

<p>n = 10 snails for the adult stage; total embryo number for the other stages.</p><p>0^* = negative control (1% DMSO).</p><p>Values were obtained at the end of the 7^th day of observation.</p

Calculated (squares) and predicted (circles) PLS data.

<p>These data were generated from ESIMS data, versus measured and autoscaled LC₅₀ values of <i>P. tuberculatum</i> extracts for <i>B. glabrata</i>.</p

<i>B. glabrata</i> embryos exposed to piplartine at the blastula stage.

<p><b>A</b>) Immediately following exposure to 1.2 µg/ml piplartine, <b>B</b>) during the 7 day observation period after exposure to 1.0 µg/ml piplartine (1- dead, 2 - normal, 3 – malformed).</p