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Proactive response to tackle the threat of emerging drugs: synthesis and toxicity evaluation of new cathinones

Publication . Gaspar, Helena; Bronze, Soraia; Oliveira, Catarina; Victor, Bruno L.; Machuqueiro, Miguel; Pacheco, Rita; Caldeira, Maria João; Santos, Susana

The emergence of potentially dangerous new psychoactive substances (NPS) imposes enormous challenges on forensic laboratories regarding their rapid and unambiguous identification. Access to comprehensive databases is essential for a quick characterization of these substances, allowing them to be categorized according to national and international legislations. In this work, it is reported the synthesis and structural characterization by NMR and MS of a library encompassing 21 cathinones, 4 of which are not yet reported in the literature, but with structural characteristics that make them a target for clandestine laboratories. This in-house library will be an important tool accessible to forensic laboratories, for the quick identification of seized NPS. The in vitro cytotoxicity of all cathinones was assessed in HepG2 cells, to have a preliminary but effective indication of their human hepatotoxicity potential. The two new cathinones DMB (8) and DMP (9) were the more cytotoxic, followed by the already seized mephedrone (2), 3,4-DMMC (3), 4-MDMC (7), NEB (12) with EC50 values ranging from 0.81 mM for (3) to 1.28 mM for (2). Results suggest an increase of cytotoxicity with the increase of the chain length of the acyl moiety and with the substitution (with one or two methyl groups) in the aromatic ring. The nature of the amine moiety seems to play only a minor role in the cytotoxic effect. Molecular dynamics simulations were performed to evaluate the molecular details related with the observed cytotoxicities. Although these studies indicated that cathinones are able to cross lipid bilayers with relative ease, when in their neutral forms, it was observed only a partial correlation between lipophilicity and cytotoxicity, indicating that membrane trafficking may not be the only key factor influencing the bioactivity of these compounds. This work is a valuable contribution to the forensic science field since a quick identification of novel cathinones is urgent to match their rapid increase in the market.

2018Journal article

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Structure-cytotoxicity relationship profile of 13 synthetic cathinones in differentiated human SH-SY5Y neuronal cells

Publication . Soares, Jorge; Costa, Vera Marisa; Gaspar, Helena; Santos, Susana; Bastos, Maria de Lourdes; Carvalho, Félix; Capela, João Paulo

Synthetic cathinones also known as β-keto amphetamines are a new group of recreational designer drugs. We aimed to evaluate the cytotoxic potential of thirteen cathinones lacking the methylenedioxy ring and establish a putative structure-toxicity profile using differentiated SH-SY5Y cells, as well as to compare their toxicity to that of amphetamine (AMPH) and methamphetamine (METH). Cytotoxicity assays [mitochondrial 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) reduction and lysosomal neutral red (NR) uptake] performed after a 24-h or a 48-h exposure revealed for all tested drugs a concentration-dependent toxicity. The rank order regarding the concentration that promoted 50 % of toxicity, at 24 h exposure, by the MTT assay was: 3,4-dimethylmethcathinone (3,4-DMMC)>METH > mephedrone ≈ α-pyrrolidinopentiophenone > AMPH ≈ methedrone > pentedrone > buphedrone ≈ flephedrone>α-pyrrolidinobutiophenone > methcathinone ≈ N-ethylcathinone>α-pyrrolidinopropiophenone>N,N-dimethylcathinone ≈ amfepramone. Apoptotic cell death signs were seen for all studied cathinones. 3,4-DMMC, methcathinone and pentedrone triggered autophagy activation, as well as increased reactive oxygen species production, and N-acetyl-L-cysteine (NAC) totally prevented that rise. Importantly, NAC was also able to prevent the cytotoxicity promoted by 6 tested drugs, ruling for an involvement of oxidative stress in the toxic events observed. The increased lipophilic chain on the alpha carbon, the presence and the high steric volume occupied by the substituents on the aromatic ring, and the substitution of the pyrrolidine ring by its secondary amine analogue have proved to be key points for the cytotoxicity profile of these cathinones. The structure-toxicity relationship established herein may enlighten future human relevant mechanistic studies, and future clinical approaches on intoxications.

2019Journal article

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