Nuno Milhazes

Simple coumarins and analogues in medicinal chemistry: occurrence, synthesis and biological activity.

Coumarins, also known as benzopyrones, are present in remarkable amounts in plants, although their presence has also been detected in microorganisms and animal sources. The structural diversity found in this family of compounds led to the division into different categories, from simple coumarins to many other kinds of policyclic coumarins, such as furocoumarins and pyranocoumarins. Simple coumarins and analogues are a large class of compounds that have attracted their interest for a long time due to their biological activities: they have shown to be useful as antitumoural, anti-HIV agents and as CNS-active compounds. Furthermore, they have been reported to have multiple biological activities (anticoagulant, anti-inflammatory), although all these properties have not been evaluated systematically. In addition, their enzyme inhibition properties, antimicrobial and antioxidant activities are other foremost topics of this field of research. The present work is to survey the information published or abstracted from 1990 till 2003, which is mainly related to the occurrence, synthesis and biological importance of simple coumarins and some analogues, such as biscoumarins and triscoumarins. Data are also highlighted, concerning the development of new synthetic strategies that could help in drug design and in the work on SAR or QSAR.

Methamphetamine‐induced neuroinflammation and neuronal dysfunction in the mice hippocampus: preventive effect of indomethacin

Methamphetamine (METH) causes irreversible damage to brain cells leading to neurological and psychiatric abnormalities. However, the mechanisms underlying life‐threatening effects of acute METH intoxication remain unclear. Indeed, most of the hypotheses focused on intra‐neuronal events, such as dopamine oxidation, oxidative stress and excitotoxicity. Yet, recent reports suggested that glia may contribute to METH‐induced neuropathology. In the present study, we investigated the hippocampal dysfunction induced by an acute high dose of METH (30 mg/kg; intraperitoneal injection), focusing on the inflammatory process and changes in several neuronal structural proteins. For that, 3‐month‐old male wild‐type C57BL/6J mice were killed at different time‐points post‐METH. We observed that METH caused an inflammatory response characterized by astrocytic and microglia reactivity, and tumor necrosis factor (TNF) system alterations. Indeed, glial fibrillary acidic protein (GFAP) and CD11b immunoreactivity were upregulated, likewise TNF‐α and TNF receptor 1 protein levels. Furthermore, the effect of METH on hippocampal neurons was also investigated, and we observed a downregulation in beta III tubulin expression. To clarify the possible neuronal dysfunction induced by METH, several neuronal proteins were analysed. Syntaxin‐1, calbindin D28k and tau protein levels were downregulated, whereas synaptophysin was upregulated. We also evaluated whether an anti‐inflammatory drug could prevent or diminish METH‐induced neuroinflammation, and we concluded that indomethacin (10 mg/kg; i.p.) prevented METH‐induced glia activation and both TNF system and beta III tubulin alterations. In conclusion, we demonstrated that METH triggers an inflammatory process and leads to neuronal dysfunction in the hippocampus, which can be prevented by an anti‐inflammatory treatment.

Methamphetamine‐Induced Early Increase of IL‐6 and TNF‐α mRNA Expression in the Mouse Brain

The mechanisms by which methamphetamine (METH) causes neurotoxicity are not well understood. Recent studies have suggested that METH‐induced neuropathology may result from a multicellular response in which glial cells play a prominent role, and so it is plausible to suggest that cytokines may participate in the toxic effects of METH. Therefore, in the present work we evaluated the effect of an acute administration of METH (30 mg/kg in a single intraperitoneal injection) on the interleukin (IL)‐1β, IL‐6, and tumor necrosis factor (TNF)‐α mRNA expression levels in the hippocampus, frontal cortex, and striatum of mice. We observed that METH did not induce changes in the IL‐1β mRNA expression levels in both hippocampus and striatum, with immeasurable levels in the frontal cortex. Regarding IL‐6, METH induced an increase in the expression levels of this cytokine in the hippocampus and striatum, 1 h and 30 min post injection, respectively. In the frontal cortex, the increase in IL‐6 mRNA levels was more significant and remained high even after 2 h. Moreover, the expression levels of TNF‐α were increased in both hippocampus and frontal cortex 30 min post METH administration, with immeasurable levels in the striatum. We conclude that the pro‐inflammatory cytokines IL‐6 and TNF‐α rapidly increase after METH administration, providing a new insight for understanding the effect of this drug of abuse in the brain.

Lipophilic Caffeic and Ferulic Acid Derivatives Presenting Cytotoxicity against Human Breast Cancer Cells

In the present work, lipophilic caffeic and ferulic acid derivatives were synthesized, and their cytotoxicity on cultured breast cancer cells was compared. A total of six compounds were initially evaluated: caffeic acid (CA), hexyl caffeate (HC), caffeoylhexylamide (HCA), ferulic acid (FA), hexyl ferulate (HF), and feruloylhexylamide (HFA). Cell proliferation, cell cycle progression, and apoptotic signaling were investigated in three human breast cancer cell lines, including estrogen-sensitive (MCF-7) and insensitive (MDA-MB-231 and HS578T). Furthermore, direct mitochondrial effects of parent and modified compounds were investigated by using isolated liver mitochondria. The results indicated that although the parent compounds presented no cytotoxicity, the new compounds inhibited cell proliferation and induced cell cycle alterations and cell death, with a predominant effect on MCF-7 cells. Interestingly, cell cycle data indicates that effects on nontumor BJ fibroblasts were predominantly cytostatic and not cytotoxic. The parent compounds and derivatives also promoted direct alterations on hepatic mitochondrial bioenergetics, although the most unexpected and never before reported one was that FA induces the mitochondrial permeability transition. The results show that the new caffeic and ferulic acid lipophilic derivatives show increased cytotoxicity toward human breast cancer cell lines, although the magnitude and type of effects appear to be dependent on the cell type. Mitochondrial data had no direct correspondence with effects on intact cells suggesting that this organelle may not be a critical component of the cellular effects observed. The data provide a rational approach to the design of effective cytotoxic lipophilic hydroxycinnamic derivatives that in the future could be profitably applied for chemopreventive and/or chemotherapeutic purposes.

Methamphetamine transiently increases the blood–brain barrier permeability in the hippocampus: Role of tight junction proteins and matrix metalloproteinase-9

Methamphetamine (METH) is a powerful stimulant drug of abuse that has steadily gained popularity worldwide. It is known that METH is highly neurotoxic and causes irreversible damage of brain cells leading to neurological and psychiatric abnormalities. Recent studies suggested that METH-induced neurotoxicity might also result from its ability to compromise blood-brain barrier (BBB) function. Due to the crucial role of BBB in the maintenance of brain homeostasis and protection against toxic molecules and pathogenic organisms, its dysfunction could have severe consequences. In this study, we investigated the effect of an acute high dose of METH (30mg/kg) on BBB permeability after different time points and in different brain regions. For that, young adult mice were sacrificed 1h, 24h or 72h post-METH administration. METH increased BBB permeability, but this effect was detected only at 24h after administration, being therefore a transitory effect. Interestingly, we also found that the hippocampus was the most susceptible brain region to METH, comparing to frontal cortex and striatum. Moreover, in an attempt to identify the key players in METH-induced BBB dysfunction we further investigated potential alterations in tight junction (TJ) proteins and matrix metalloproteinase-9 (MMP-9). METH was able to decrease the protein levels of zonula occludens (ZO)-1, claudin-5 and occludin in the hippocampus 24h post-injection, and increased the activity and immunoreactivity of MMP-9. The pre-treatment with BB-94 (30mg/kg), a matrix metalloproteinase inhibitor, prevented the METH-induced increase in MMP-9 immunoreactivity in the hippocampus. Overall, the present data demonstrate that METH transiently increases the BBB permeability in the hippocampus, which can be explained by alterations on TJ proteins and MMP-9.

Lipophilic phenolic antioxidants: Correlation between antioxidant profile, partition coefficients and redox properties

Lipophilic compounds structurally based on caffeic, hydrocaffeic, ferulic and hydroferulic acids were synthesized. Subsequently, their antioxidant activity was evaluated as well as their partition coefficients and redox potentials. The structure-property-activity relationship (SPAR) results revealed the existence of a clear correlation between the redox potentials and the antioxidant activity. In addition, some compounds showed a proper lipophilicity to cross the blood-brain barrier. Their predicted ADME properties are also in accordance with the general requirements for potential CNS drugs. Accordingly, one can propose these phenolic compounds as potential antioxidants for tackling the oxidative status linked to the neurodegenerative processes.

Structure-property-activity relationship of phenolic acids and derivatives. Protocatechuic acid alkyl esters.

The esterification of hydrophilic phenolic antioxidants is an efficient approach to enhance their solubility in apolar media. Herein, structure-property studies on the antiradical activity of a series of protocatechuic acid alkyl esters have been accomplished. The increase of the lipophilicity was shown to significantly improve the antioxidant activity of protocatechuic esters. Their efficiency as radical scavengers was evaluated using distinctive analytical methods, namely, 2,2-diphenyl-1-picrylhydrazyl (DPPH) UV/visible method, electrochemistry, and differential scanning calorimetry. All the new alkyl protocatechuate antioxidants studied possessed better radical-scavenging capacity than the natural antioxidant protocatechuic acid. This work has shown that the alkyl ester side chain markedly influences the lipophilicity of this type of phenolic system without disturbing the core of the molecule responsible for antioxidant activity. The data on the antioxidant activity obtained using the different analytical methods correlated well with each other and have revealed the interesting antioxidant potential of alkyl esters of protocatechuic acid.

Chromone 3-phenylcarboxamides as potent and selective MAO-B inhibitors.

Monoamine oxidase (MAO) is an enzyme, present in mammals in two isoforms MAO-A and MAO-B. These isoforms have a crucial role in neurotransmitters metabolism, representing an attractive drug target in the therapy of neurodegenerative diseases (MAO-B) and depression (MAO-A). In this context, our work has been focused on the discovery of new chemical entities (NCEs) for MAO inhibition, based on the development of chromone carboxamides. Chromone derivatives with a carboxamide function located in position 2- and 3- of the benzo-γ-pyrone core, (compounds 2-6 and 8-12) were synthesized, with moderate/good yields, by a one-pot condensation reaction using phosphonium salts as coupling reagents. The synthetic compounds were screened towards human MAO isoforms (hMAO) to evaluate their potency and selectivity. The chromone-3-carboxamides show high selectivity to hMAO-B, with compounds 9 and 12 displaying IC(50) values at nanomolar range.

New insights into the antioxidant activity of hydroxycinnamic acids: Synthesis and physicochemical characterization of novel halogenated derivatives

An interdisciplinary research project was developed combining the synthesis of a series of hydroxycinnamic acid derivatives and the evaluation of their physicochemical parameters (namely redox potentials and partition coefficients), along with the corresponding antioxidant activity. A structure-property-activity relationship (SPAR) approach was then applied aiming at establishing a putative relation between the physicochemical parameters of the compounds under study and their antioxidant activity. The results gathered allow concluding that the redox potentials could contribute to the understanding of the antioxidant activity and that the presence of an electron withdrawing group (EWG) of halogen type, namely a bromo atom, in an ortho position to a phenolic group of the cinnamic scaffold does not influence the antioxidant activity. On the other hand after the introduction of this type of substituent a significant increase on the lipophilicity of cinnamic derivatives was observed, which is a feature of extreme importance in the development of novel lipophilic antioxidants. The SPAR results revealed a relation between the redox potentials and the antioxidant activity of hydroxycinnamic acids and derivatives. The data obtained operate as a positive reinforce of the tendency to use redox properties as a guideline of the rational design of this type of compounds.

Chromone-2- and -3-carboxylic acids inhibit differently monoamine oxidases A and B

Chromone carboxylic acids were evaluated as human monoamine oxidase A and B (hMAO-A and hMAO-B) inhibitors. The biological data indicated that only chromone-3-carboxylic acid is a potent hMAO-B inhibitor, with a high degree of selectivity for hMAO-B compared to hMAO-A. Conversely the chromone-2-carboxylic acid resulted almost inactive against both MAO isoforms. Docking experiments were performed to elucidate the reasons of the different MAO IC(50) data and to explain the absence of activity versus selectivity, respectively.