Application of Computational Methods for the Design of New Potential Therapeutic Agents

Computer-aided drug discovery (CADD) represents a very useful tool to search for potential drug candidates and plays a strategic role in the discovery of new potential therapeutic agents for both pharmaceutical companies and academic research groups. Nevertheless, the modelling of biological systems still represents a challenge for computational chemists, and, at present, a single computational method able to face such challenge is not available. Computational tools are therefore evolving in the direction of combining molecular-mechanic (MM), molecular dynamics (MD), and quantum-mechanical (QM) approaches in order to achieve an overall better simulation of the actual molecular behaviour. In addition, many sampling methods have been developed and applied for the characterisation and comparison of the collective motions of protein structures related to the dynamics of proteins, protein folding and ligand-protein docking simulations. This prompted us, as computational medicinal chemists, to develop various CADD approaches, depending on the specific case under study, integrating theoretical and experimental data. In particular, the research activity carried out during the three years of my PhD led to: i) the development of three-dimensional (3-D) pharmacophore models for the analysis of 3-D structure-activity relationships (SARs) of bioactive compounds, ii) the identification of new molecular targets, iii) the simulation of large-scale protein conformational changes, iv) the simulation of protein/protein and ligand/protein interactions, and v) the design of new bioactive compounds. Computational studies were always performed in the frame of multi-disciplinary projects guided by a unique research strategy, which involved several international and national research groups, and were carried out by integrating and validating our computational studies with the experimental data coming from the other researchers involved in the various projects. The results obtained enabled to: i) identify a new class of anticancer agents against paclitaxel resistant cancer cells, ii) provide important information on the mechanism of action of cationic porphyrins, a novel class of proteasome conformational regulators with great potentiality as “lead” pharmacophores, and iii) optimise the cellular pharmacokinetic and pharmacodynamic properties of a new series of antimalarial agents. In addition, I spent a training period abroad of eight-months at the Institute of Research in Biomedicine (IRB) in Barcelona, under the supervision of prof. Modesto Orozco, during which I have had the opportunity to extend my computational background by learning and, then, performing metadynamic and MD simulations, investigating the open/close conformational transition of 20S human proteasome by molecular dynamics simulations

Novel C11 amino derivatives of cryptolepine : synthesis and in vitro studies with DNA and haeme

Tese de doutoramento, Farmácia (Química Farmacêutica e Terapêutica), Universidade de Lisboa, Faculdade de Farmácia, 2010Malaria is one of the most widespread infectious diseases of our time. The global malaria map has been shrinking over the past 60 years, but today more people are at risk of suffering from malaria than any other time in history. In the past few years malaria has once again attracted more attention, partly because it is recognized that malaria spread in sub-Saharan Africa has increased in the recent years, mainly due to the development of drug resistances. Cryptolepine (1), is an indoloquinoline alkaloid, extracted from the West African climbing shrub Cryptolepis sanguinolenta (Lindl.) Schltr, a traditional herb used in folk medicine for the treatment of malaria. Several authors hypothesized that the mechanism of action of cryptolepine could be by inhibition of haemozoin formation in the digestive vacuole (DV) of the parasite, however in a microscopic fluorescence study, the indoloquinoline chromophore, was suggested to accumulate into specific parasite structures that could correspond to the parasite nuclei, and thus justifying its activity due to cytotoxic effects on DNA and topoisomerase II activity. Cryptolepine derivatives (3) have been synthesized through the incorporation of basic side-chains in the C-11 position of the 10H-indolo[3,2-b]quinoline scaffold and evaluated for their antiplasmodial and cytotoxicity properties. The derivative containing a conformationally restricted piperidine sidechain (3n) presented IC50 values of 23-44 nM against chloroquine resistant strains and a selectivity index value of ca 1400, i.e. a 1000-fold improvement in selectivity when compared with 1. The introduced side chains are weakly basic, with pKa values in the terminal amine functionality ranging from 5.2 to 12.5, and are predicted to promote accumulation inside the DV to an extent similar to that of chloroquine. All compounds within this series showed the ability to interact with monomeric haematin (FPIX-OH), with a stoichiometry of 1:1 (3:FPIX-OH) and with association constants (Kass) values between 0.062 and 0.41 x106 M-1, comparable to chloroquine (Kass = 0.085 x106 M-1). The complex stabilization is assured by π-π stacking interactions modulated by the aromatic core, and H-bond between the terminal amine side chain and haematin carboxylate anions, thus capable to inhibit haemozoin formation in DV. However, localization studies of compound 3n inside parasite blood stages suggest an additional mechanism of action, like interactions with DNA, besides inhibition of haemozoin crystal growth. Cryptolepine derivatives (3) bind strongly to double-stranded d(GATCCTAGGATC)2 oligonucleotide with association constants ranging from 105 M-1 to 107 M-1. Furthermore, molecular docking simulations showed that, in contrast with 1, compounds 3 are predicted to not intercalate into DNA double helix, binding essentially to single- and double-stranded DNA, with a stoichiometry of 2:1 (3:DNA), through electrostatic and H-bonding interaction involving charged nitrogens. In order to explore the indolo[3,2-b]quinolin-11-one (quindolone) scaffold (4), and get new antimalarial chemotypes, we decided to synthesize a series of quindolones derivatives (5), targeting malaria parasite digestive vacuole and haeme detoxification pathway, through the introduction of N,N diethylethanamine in the indolo[3,2-b]quinoline aromatic nucleus (N5,N10-alkylation). This reaction gave N,O- (94), N,N- (5) and O-(95) alkylated products containing two or one basic side-chains, which were evaluated for antiplasmodial activity against chloroquine-resistant P. falciparum W2 strain and cytotoxicity for HepG2 A16 hepatic cells. By incorporating alkylamine side chains and chlorine atoms in the quindolone nucleus we transformed the inactive tetracyclic parent quindolones (4, 91a and 91b) into moderate or highly active and selective compounds to the resistant P. falciparum W2 strain, with IC50 ranging from 51 to 2638 nM and with selectivity ratios up to 98. All the quindolone derivatives in the series showed the ability to complex FPIX-OH (1:1 stoichiometry) with associations constants (Kass) ranging from 0,074 to 0,14 x106 M-1, being the main intermolecular interactions due to π-π stacking interactions and H-bond between derivatives and haematin. Cryptolepine and the new antimalarial chemotype, quindolone, are suitable scaffolds for the design of active and selective compounds targeting parasite haemozoin detoxification pathway, with potential for development as antimalarial agents.A malária ou paludismo é uma doença infecciosa provocada por parasitas do género Plasmodium e transmitida pela picada do mosquito fêmea do género Anopheles. A malária é uma das infecções mais difundida por todo o mundo. Apesar propagação ter diminuído nos últimos 50 anos, nos dias de hoje há mais pessoas em risco de contaminação com malária do que em qualquer outra época da história. Em 2008, a malária era endémica em 108 países, contando com cerca de 247 milhões de casos reportados, 3,3 mil milhões de pessoas em risco. Anualmente, entre 1 a 3 milhões de casos culminam em morte, dos quais, muitos são crianças com idade inferior a 5 anos. A malária é a principal causa de morte infantil em África, sendo que 60 % dos novos casos registados todos os anos ocorrem na África sub-Sahariana, onde ocorrem 90 % dos casos fatais de malária. Para além de ser um grave problema de saúde pública, a malária é também um problema sócio-económico, não só devido ao elevado investimento efectuado na prevenção e tratamento, mas também devido a custos indirectos resultantes da perda de productividade que advêm da elevada morbilidade e mortalidade. No entanto, recentemente a malária voltou a chamar a atenção da comunidade, muito porque foi reconhecido que o número de casos reportados em África tem aumentado nos últimos anos devido ao aumento de fenómenos de resistência nos parasitas aos fármacos utilizados para tratamento da infecção. Apesar da enorme variedade de compostos com actividade antimalárica, a sua eficácia contínua no entanto a ser reduzida devido aos fenómenos de resistência associados. A cloroquina (2) é uma 4- amionoquinolina sintetizada em 1934 e tem sido um dos pilares do tratamento da malária nos últimos 60 anos, sendo de consenso geral, que a sua actividade antimalárica se deva à inibição da formação do cristal de hemozoína no vacúolo digestivo do parasita. No organismo humano, o parasita ingere hemoglobina e digere-a, libertando os amino ácidos necessários para o seu desenvolvimento, e o heme, tóxico para o parasita. Este heme é então biocristalizado pelo parasita a hemozoina, um cristal inerte e não tóxico. A cloroquina, devido às suas propriedades básicas, apresenta a capacidade de se acumular no interior do vacúolo digestivo acídico e formar complexos estáveis cloroquina:heme, através de interacções π-π entre os anéis aromáticos, impedindo assim a formação da hemozoina e originando a morte do parasita. Vários autores referem ainda que a cloroquina apresenta também a capacidade de complexar com as faces em crescimento do cristal de hemozoina, inibindo assim o processo de cristalização. Nos últimos 30 anos, extractos de uma enorme variedade de espécies de plantas, incluindo muitas utilizadas na medicinal tradicional, têm sido avaliadas in vitro quanto à sua actividade antimalárica. O alcalóide criptolepina (1), constituinte maioritário da raíz da Cryptolepis sanguinolenta, um arbusto trepador africano normalmente utilizado na medicina tradicional para tratamento da malária, demonstrou possuir propriedades antiplasmodicas equivalentes à cloroquina. A criptolepina parece exercer as propriedades antiplasmodicas devido à capacidade de inibir a formação da hemozoina, tal como a cloroquina, ligando-se ao heme e bloqueando assim o mecanismo de destoxificação do parasita. No entanto, a criptolepina é também um agente intercalante de cadeias de ADN ricas em guanina (G) e citosina (C), e tendo preferência por sequências CC não alternadas. Assim, a criptolepina apresenta propriedades citotóxicas devido à inibição da topoisomerase II e da síntese do ADN. Estas propriedades citotóxicas podem também estar na origem da actividade antiplasmódica uma vez que, um estudo de localização intracelular em eritrócitos infectados com P. falciparum, revelou que o alcalóide se acumula em estruturas no interior do parasita que poderão corresponder ao núcleo. Neste trabalho foram sintetizados 25 análogos da criptolepina (3) com cadeias laterais diaminoalquílicas, na posição C11 do núcleo aromático da indolo[3,2-b]quinolina e avaliados quanto as suas propriedades antiplasmodicas e citotóxicas em linhas celulares de mamífero. O análogo com uma cadeia lateral de piperidina (3n), apresentou uma actividade antiplasmódica (IC50) variando entre 23 e 44 nM, contra diferentes estripes resistentes à cloroquina, e um índice de selectividade de aproximadamente 1400, representando um aumento de cerca de 1000 vezes quando comparado com 1. Os nossos estudos sugerem que a introdução de cadeias laterais com aminas terminais basicas, apresentando valores de pKa variando entre 5,2 e 12,5, promove a acumulação dos compostos no interior do vacúolo digestivo do parasita, em níveis de concentração semelhantes aos da cloroquina. Todos os análogos da criptolepina sintetizados apresentam a capacidade de formar complexos com o monómero da hematina (FPIX-OH), com constantes de associação (Kass) variando entre 0,062 e 0,41 x106 M-1, semelhante à constante de associação determinada para a cloroquina (Kass = 0,085 x106 M-1). Os complexos são estabilizados maioritariamente através de interacções π-π entre o núcleo aromático da indolo[3,2-b]quinolina e o núcleo porfirínico da hematina. Estudos de modelação molecular revelaram também que os azotos protonados nas aminas terminais das cadeias laterais podem formar pontes de hidrogénio com os iões carboxilato da hematina. Estes resultados demonstraram que os novos análogos da criptolepina apresentam a capacidade de complexar com a FPIX-OH e inibir a formação da hemozoina. No entanto, o estudo de localização intracelular realizado por microscopia de fluorescência em eritrócitos infectados com P. falciparum, demonstrou que os análogos da criptolepina também apresentam a capacidade de se acumularem no núcleo do parasita e assim, potenciar a actividade antiplasmódica. De modo a avaliar a capacidade de 3 para interagir com estruturas de ADN, foram realizados estudos de interacção com um oligonucleótido de cadeia única d(5’-GCCAAACACAGAATCG-3’) e de cadeia dupla d(GATCCTAGGATC)2. Os compostos 3 apresentaram forte capacidade de complexação com ambas as estruturas de DNA e valores de constante de associação (Kass) variando entre 105 M-1 e 107 M-1. Estudos de modelação molecular com estruturas de ADN de hélice duplas semelhante à utilizada no ensaios in vitro, demonstraram que os compostos não são agentes intercalantes, tal como verificado para a criptolepina, mas ligam-se à fenda menor/maior, com uma estequiometria 2:1 (análogo da criptolepina:ADN) e interagem preferencialmente com a cadeia de fosfatos através de interacções electrostáticas e pontes de hidrogénio. Estes resultados demonstraram que a actividade antiplasmódica dos novos análogos da criptolepina parece ser justificada por efeitos sinérgicos ou aditivos à inibição da formação da hemozoina e citotoxicidade associada à interacção com estruturas de ADN. Com o objectivo de aumentar a diversidade de esqueletos químicos com actividade antimalárica, foram sintetizados novos análogos da indolo[3,2-b]quinolin-11-ona (11-quindolona), tendo como propósito aumentar a retenção destes compostos no interior do vacúolo digestivo do parasita. Para tal, foram introduzidas duas cadeias amino-alquílicas (N,N-dietiletanoamina) no núcleo aromático da quindolona (alquilação em N5 e N10). A reacção originou no entanto padrões de alquilação adicionais, N,O- (94) e O- (95), que foram também avaliados quanto ao seu potencial antiplasmódico e citotoxicidade em células hepáticas HepG2 A16. Com introdução de uma ou duas cadeias aminoalquílicas e átomos de cloro no núcleo aromático, as quindolonas (4, 91a e 91b), inicialmente inactivas, deram origem a compostos com actividade moderada a forte contra a estirpe W2 do P. falciparum resistente à cloroquina, apresentando valores de IC50 entre 51 e 2638 nM, e com maior selectividade para o parasita. Todos os análogos da quindolona sintetizados apresentam também a capacidade de formar complexos com a hematina, com uma estequiometria 1:1 (análogo:FPIX-OH) e constantes de associação (Kass) que variam entre 0,074 e 0,14 x106 M-1. A estabilidade do complexo é assegurada pela formação de interacções π-π entre o núcleo aromático e o anel de porfírina da hematina e estudos de modelação molecular confirmaram a possibilidade de formação de pontes de hidrogénio entre a amina terminal da cadeia lateral e os aniões carboxilato do dimero da hematina. Estes resultados demonstraram que a introdução de cadeias amino-alquílicas no núcleo da quindolona origina compostos com boa actividade antiplasmódica, com aparente capacidade de inibição da formação da hemozoína e possivelmente com maior capacidade de acumulação no vacúolo digestivo do parasita. As indolo[3,2-b]quinolinas demonstraram assim serem bons esqueletos para o desenho e desenvolvimento de compostos para tratamento da malária, obtendo-se compostos mais activos e selectivos para o parasita.Fundação para a Ciência e Tecnologia (SFRH/BD/29202/2006); Faculdade de Farmácia, Universidade de Lisboa, Portugal; Scholl of Pharmacy and Pharmaceutical Sciences, University of Manchester, UK(material and equipment necessary to the development of the study)

Application of Computational Methods for the Design of New Potential Therapeutic Agents

Computer-aided drug discovery (CADD) represents a very useful tool to search for potential drug candidates and plays a strategic role in the discovery of new potential therapeutic agents for both pharmaceutical companies and academic research groups. Nevertheless, the modelling of biological systems still represents a challenge for computational chemists, and, at present, a single computational method able to face such challenge is not available. Computational tools are therefore evolving in the direction of combining molecular-mechanic (MM), molecular dynamics (MD), and quantum-mechanical (QM) approaches in order to achieve an overall better simulation of the actual molecular behaviour. In addition, many sampling methods have been developed and applied for the characterisation and comparison of the collective motions of protein structures related to the dynamics of proteins, protein folding and ligand-protein docking simulations. This prompted us, as computational medicinal chemists, to develop various CADD approaches, depending on the specific case under study, integrating theoretical and experimental data. In particular, the research activity carried out during the three years of my PhD led to: i) the development of three-dimensional (3-D) pharmacophore models for the analysis of 3-D structure-activity relationships (SARs) of bioactive compounds, ii) the identification of new molecular targets, iii) the simulation of large-scale protein conformational changes, iv) the simulation of protein/protein and ligand/protein interactions, and v) the design of new bioactive compounds. Computational studies were always performed in the frame of multi-disciplinary projects guided by a unique research strategy, which involved several international and national research groups, and were carried out by integrating and validating our computational studies with the experimental data coming from the other researchers involved in the various projects. The results obtained enabled to: i) identify a new class of anticancer agents against paclitaxel resistant cancer cells, ii) provide important information on the mechanism of action of cationic porphyrins, a novel class of proteasome conformational regulators with great potentiality as “lead” pharmacophores, and iii) optimise the cellular pharmacokinetic and pharmacodynamic properties of a new series of antimalarial agents. In addition, I spent a training period abroad of eight-months at the Institute of Research in Biomedicine (IRB) in Barcelona, under the supervision of prof. Modesto Orozco, during which I have had the opportunity to extend my computational background by learning and, then, performing metadynamic and MD simulations, investigating the open/close conformational transition of 20S human proteasome by molecular dynamics simulations

Defining the chemical and molecular mechanisms of cytotoxicity Induced by the endoperoxide class of antimalarials

Artemisinin-derived endoperoxide drugs find widespread employment as frontline treatment against malaria. Although evidence of their potential to express toxicity within a clinical setting remains limited, outcomes derived from animal studies attest their ability to induce neurological and developmental toxicity in mammalian systems. Activity is further demonstrated in vitro within rapidly proliferating human cells – most notably those belonging to immortalised, cancer-derived lines – with significant cytotoxic effects being observed upon drug treatment across a range of settings. It is believed that these find their origin through a mechanism dependent upon Fe(II)-mediated reduction of the endoperoxide bridge functionality, culminating in molecular bioactivation and the subsequent formation of carbon-centred free radical species which in turn, owing to their great reactivity, impart deleterious effects upon cellular functioning. Evidence suggests that dysfunction of the mitochondrion and the formation of reactive oxygen species (ROS) are key stages in the route through which artemisinin derivatives are able to induce death. The characteristics of artesunate-stimulated impact upon mitochondrial functioning are examined. It is demonstrated that culturing of cells in the presence of galactose enhances cytotoxic potential within the HeLa line. The magnitude of this variation in sensitivity is indicative that targeting of the mitochondrion affords a route through which activity is mediated. Falls in cell viability are further preceded by declines in ATP production, providing evidence that disruption of oxidative phosphorylation occurs as an early event in the route towards death. Studies performed on mitochondrial bioenergetic function using the Seahorse XF analyser indicate that artesunate imparts dose-dependent and timedependent decreases in respiratory reserve capacity and oxidative phosphorylation coupling efficiency, whilst stimulating a switch towards glycolytic energy production. Attempts to delineate the root causes of these effects are focused upon examining the relationship between oxidative stress, Fe(II) content and mitochondrial performance. The mitochondrially-localised antioxidant tiron and the lysosomal Fe(II) chelator desferrioxamine are shown to induce substantial cytoprotective effects against artesunate within the HeLa line. Evidence derived from Seahorse XF analysis indicates strongly that these outcomes are related to the capacity of both compounds to abrogate drug impact upon the functions of the mitochondrion. It can thus be posited that mitochondrial damage has its origins in the emergence of oxidative stress, with Fe(II) content acting as key determinant in its progression. The outcomes of further examinations performed within the ρ0 HeLa line suggest an origin for ROS emergence independent of the respiratory chain. In order to test the hypothesis that artemisinin derivatives might induce direct peroxidation of the mitochondrial phospholipid cardiolipin, the impact of a cytochrome c peroxidase inhibitor TPP-IOA is examined on the response of HeLa and HL-60 cells towards artesunate treatment. Results indicate that the inhibitor has variable effects upon cardiolipin oxidation state and subsequent cell survival, leaving doubt as to the true validity of such a connection. As a further study, the cytotoxic capacities of a range of novel artemisininderived anticancer agents and wholly synthetic tetraoxane and trioxolane antimalarials are given assessment. In conclusion, it can be stated that the outcomes of the studies performed in this thesis emphasise the importance of mitochondrial liability towards the progression of artemisinininduced cell death. Further insights into the mechanistic routes through which drug administration contributes, via oxidative stress and free Fe(II) content, to the defective functioning of the organelle have been achieved

Molecular Design as a Tool to Understand and Manipulate Cell Death

Phenotypic screening is a powerful technique for discovering small organic molecules with the ability to effect a desired change in biological systems. Yet, because of the diversity of ways that a small molecule can alter a biological system, phenotypic screens provide little to no insight into how a hit molecule elicits such a change. Understanding a molecule’s mechanism of action—which proteins it engages, where it localizes, and its reactivity—is critical to fully developing a hit molecule into a research tool or a therapeutic. In this dissertation, strategic, hypothesis-driven molecular design is used as a cornerstone technique to understand the mechanism of action of molecules that regulate cell death. In the first part, we examine the structural requirements for ferroptotic death induction by the 1,2-dioxolane FINO2. Next, we create a panel of ferroptosis inhibiting molecules that are targeted to specific organelles and used as imaging agents in order to examine the contribution of different organelles to ferroptosis. I then apply molecular design in a target-based context to discover which molecular features of LOC14 promote association with its receptor protein. Finally, I discuss a computational approach to developing a ligand that inhibits protein-protein interactions mediated by the small GTPase Rheb

Physicochemical, biological and β-haematin inhibiting activity of pyrido-dibemequines, pyrido[1,2-α]benzimidazoles and their derivatives

There is an urgent need for new antimalarials following the emergence of Plasmodium falciparum strains with reduced sensitivity to the currently used artemisinin combination therapies. Classical aminoquinoline-based drugs inhibit the formation of haemozoin (HZ) thereby causing parasite death from the cellular accumulation of toxic 'free' haem. Coincidentally, this immutable pathway also exists in Schistosoma mansoni, and presents a vulnerable target for drug design in these haematophagus organisms. Therefore, it would be of interest to explore novel scaffolds that can inhibit HZ formation as well as exploit the merits of established drugs via structural modifications that would harness their pharmacological and pharmacokinetic advantages while circumventing their therapeutic shortcomings. This project investigated the physicochemical, biological and mechanistic profiles of pyrido-dibemequine (pDBQ) and pyrido[1,2-α]benzimidazole (PBI) derivatives whose structural motifs were informed by previously synthesised prototype molecules. Specifically, the aqueous solubility, membrane permeability, lipophilicity, metabolic stability and potential for cardiotoxicity of seven pDBQs, their metabolites and ten PBIs were tested through computational and experimental methods. In addition, their antiplasmodial and antischistosomal activities were determined and correlated with their respective physicochemical properties. As regards mechanistic evaluation, their ability to inhibit formation of abiotic HZ, β-haematin (βH), was assessed and intracellular inhibition of HZ formation probed. The pDBQs constitute reversed chloroquines with a 4-aminoquinoline nucleus hybridised to a dibenzylmethylamine side group that serves as a chemosensitising moiety. The pDBQ derivatives showed moderate to high solubility (52 - 197 μM) and permeability (LogPₐₚₚ: -4.6 - -3.6) at pH 6.5. Their lipophilicity, indexed by cLogP, ranged between 3.7 and 5.6 while the mean LogD at both cytosolic (7.4) and vacuolar (5.0) pH was 3.15 and 0.93, respectively. The compounds also showed low-nanomolar range antiplasmodial activity against both chloroquine (CQ)-sensitive (CQS) and resistant (CQR) strains (IC₅₀ range CQS: 14.4 - 126.6 nM, CQRᴰᵈ²: 44.5 - 162 nM and CQR⁷ᴳ⁸: 69.6 - 307.1 nM), with no discernible cross-resistance with CQ and the antiplasmodial activity directly correlated with lipophilicity. Mechanistically, all the pDBQs inhibited βH formation (IC₅₀: 13 - 25 μM) and haem-pyridine fractionation profiles revealed they produced a CQ-like dose-dependent increase in toxic 'free' haem with corresponding decrease in HZ levels. Predicted human-Ether-a-Go-Go-Related Gene (hERG) channel inhibition pIC₅₀ ranged between 6.2 and 6.6, and correlated strongly with the cLogP and molecular weight. The derivatives were also highly susceptibility to microsomal metabolism, with N-dealkylation identified as the main biotransformation route. The pDBQ metabolites exhibited solubility and membrane permeability profiles similar to the parent compounds at pH 6.5, albeit with reduced lipophilicity (cLogP range: 2.3 - 3.5). Their βH inhibition activity (IC₅₀: 15 - 24 μM) was also comparable to the parent compounds as were the haem-pyridine fractionation profiles. However, they showed greater antiplasmodial activity, with 4/7 derivatives exhibiting IC₅₀ < 80 nM against PƒDd2 (CQR strain). The metabolites had reduced hERG channel inhibition potential (pIC₅₀: 5.0 - 5.7) and significantly improved metabolic stability upon incubation with mouse and human liver microsomes. The PBIs comprise molecules with structural likeness to CQ, including a planar heterocyclic moiety and a basic amine side group. PBI analogues showed low to moderate solubility (<5 - 80 μM) and were moderately lipophilic (mean LogD7.4: 3.04). Although most of the derivatives were stable in liver microsomes, their predicted hERG channel inhibition potential was higher (pIC₅₀: 6.11 - 7.50), presumably due to their high molecular weights. All but one derivative had submicromolar activity against CQS and CQR strains, with analogues bearing halo-substituents on the left of the PBI core showing the best antiplasmodial activity (mean IC₅₀: CQS = 26.7 nM and CQR = 30.0 nM), highest selectivity (188 - 341) as well as complete cures in P. berghei-infected mice. The PBIs also inhibited βH formation (IC₅₀: 6.8 - 120 μM) but did not all display intracellular inhibition of HZ formation. All derivatives were active against juvenile (mean IC₅₀: 1.97 μM) and adult (mean IC₅₀: 4.38 μM) schistosomes, with the 3, 4-dichloro-substituted analogue exhibiting 48% reduction of worm burden in vivo. In summary, the pDBQs evaluated in this project constitute potent antiplasmodial inhibitors of HZ formation but whose activity is compromised by metabolic and hERG liability while their metabolites seem to possess improved biological and physicochemical features. The observed activity of the PBIs against P. falciparum and S. mansoni complements the already-established broad antimicrobial potency of this chemotype

Synthesis, characterisation and antitumour activities of lanthanide complexes with hydrazones and carboxylic acid ligands

The tridentate hydrazone ligands, (E)-N'-(2-hydroxybenzylidene)benzohydrazide (H2phen) and (E)-N'-(2-hydroxybenzylidene)nicotinohydrazide (H2Nic), were synthesised and complexed to Ln(III) acetates. The centrosymmetric, acetato-bridged dinuclear coordination compounds with the formulae, [La2(Hphen)2(OAc)4(H2O)2]·DMF·H2O (1), [Ln2(HNic)2(OAc)4(H2O)2]·DMF·H2O (Ln = La (2) and Nd (3)) and [Ln2(HNic)2(OAc)4(H2O)2]·DMF (Ln = Er (4) and Yb (5)) were isolated and characterised by elemental analyses, IR spectroscopy, UV-Vis spectroscopy, X-ray diffraction studies and SHAPE 2.1. The nine-coordinate complexes 1–3 crystallise in the triclinic space group P-1, with the metal centres having the distorted spherical capped square antiprism geometry (C4v), while the eight-coordinate Er(III) and Yb(III) complexes (monoclinic system, space group P21/c) display the geometry of distorted triangular dodecahedron (D2d). Geometry optimisation of the monoanionic forms of the hydrazone ligands (Hphen– and HNic– ) were performed using Density Functional Theory (DFT) with Becke’s three parameter hybrid method and correlation functional of Lee, Yang and Parr (B3LYP) with augcc-pVTZ basis set. Natural population analysis (NPA) and molecular electrostatic potential (MEP) maps indicated that the most preferred sites for electrophilic attack in the anionic ligands are the phenolate and carbonyl oxygens, and the azomethine nitrogens. The evaluation of the cytotoxic activity of the compounds on breast cancer (MCF-7), the endometrial carcinoma (HEC-1A) and the human monocytic (THP-1) cell lines using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl tetrazolium bromide (MTT) assay revealed that the hydrazone ligands and complexes 1–4 are partially cytotoxic against MCF-7 cells, while the Schiff bases and complexes 3–5 significantly inhibit cell growth in HEC-1A cells. The complexation reactions of Ce(III), Nd(III), Gd(III) and Er(III) with the chelating/ bridging monoanionic ligand N-(2,6-dimethylphenyl)oxamate (Hpma– ) in basic media were performed in view of the potential applications of oxamate derivatives as cytotoxic agents. The coordination compounds were characterised by different Abstract T. Madanhire Nelson Mandela University xxvi physico-chemical techniques: elemental analysis, conductivity measurements, IR, 1 H NMR and UV-Vis-NIR spectroscopy. The anionic Hpma– was obtained through conversion of the proligand ethyl (2,6-dimethylphenylcarbamoyl)formate (Hdmp). The reactions afforded lanthanide(III)–oxamate coordination polymers of formulae: {[Ln(Hpma)3(MeOH)(H2O)]∙2MeOH}n (Ln = Ce (1) and Nd (2)), {[Gd2(Hpma)6(MeOH)4]∙6MeOH}n (3), {[Er2(Hpma)6(MeOH)(H2O)3]∙2MeOH}n (4) and [Ln2Na2(Hpma)8(EtOH)(H2O)6]n (Ln = Nd (5) and Gd (6)). The polymeric complexes feature Ln-Hpma moieties bridged by μ2-η 1 :η 1 :η 1 Hpma– , giving onedimensional zig-zag chains of the –Ln–O–C–O–Ln– type. Atomic charge analysis and the MEP map of the Hpma– moiety done using the DFT/B3LYP method were found to be consistent with the chelating and bridging modes of the anionic ligand through all the oxygen atoms. The evaluation of the cytotoxic activities of the metal salts, the proligand and the novel lanthanide complexes on MCF-7, HEC-1A and THP-1 cell lines revealed that only the rare-earth metal salts [Ce(NO3)3∙6H2O] and [Nd(NO3)3∙6H2O] showed modest cytotoxicity against MCF-7 and HEC-1A cells, respectively

Non-covalent interactions in organotin(IV) derivatives of 5,7-ditertbutyl- and 5,7-diphenyl-1,2,4-triazolo[1,5-a]pyrimidine as recognition motifs in crystalline self- assembly and their in vitro antistaphylococcal activity

Non-covalent interactions are known to play a key role in biological compounds due to their stabilization of the tertiary and quaternary structure of proteins [1]. Ligands similar to purine rings, such as triazolo pyrimidine ones, are very versatile in their interactions with metals and can act as model systems for natural bio-inorganic compounds [2]. A considerable series (twelve novel compounds are reported) of 5,7-ditertbutyl-1,2,4-triazolo[1,5-a]pyrimidine (dbtp) and 5,7-diphenyl- 1,2,4-triazolo[1,5-a]pyrimidine (dptp) were synthesized and investigated by FT-IR and 119Sn M\uf6ssbauer in the solid state and by 1H and 13C NMR spectroscopy, in solution [3]. The X-ray crystal and molecular structures of Et2SnCl2(dbtp)2 and Ph2SnCl2(EtOH)2(dptp)2 were described, in this latter pyrimidine molecules are not directly bound to the metal center but strictly H-bonded, through N(3), to the -OH group of the ethanol moieties. The network of hydrogen bonding and aromatic interactions involving pyrimidine and phenyl rings in both complexes drives their self-assembly. Noncovalent interactions involving aromatic rings are key processes in both chemical and biological recognition, contributing to overall complex stability and forming recognition motifs. It is noteworthy that in Ph2SnCl2(EtOH)2(dptp)2 \u3c0\u2013\u3c0 stacking interactions between pairs of antiparallel triazolopyrimidine rings mimick basepair interactions physiologically occurring in DNA (Fig.1). M\uf6ssbauer spectra suggest for Et2SnCl2(dbtp)2 a distorted octahedral structure, with C-Sn-C bond angles lower than 180\ub0. The estimated angle for Et2SnCl2(dbtp)2 is virtually identical to that determined by X-ray diffraction. Ph2SnCl2(EtOH)2(dptp)2 is characterized by an essentially linear C-Sn-C fragment according to the X-ray all-trans structure. The compounds were screened for their in vitro antibacterial activity on a group of reference staphylococcal strains susceptible or resistant to methicillin and against two reference Gramnegative pathogens [4] . We tested the biological activity of all the specimen against a group of staphylococcal reference strains (S. aureus ATCC 25923, S. aureus ATCC 29213, methicillin resistant S. aureus 43866 and S. epidermidis RP62A) along with Gram-negative pathogens (P. aeruginosa ATCC9027 and E. coli ATCC25922). Ph2SnCl2(EtOH)2(dptp)2 showed good antibacterial activity with a MIC value of 5 \u3bcg mL-1 against S. aureus ATCC29213 and also resulted active against methicillin resistant S. epidermidis RP62A

Treatment of Depression in Rats Exposed To Chronic Unpredictable Mild Stress Using Synthetic Derivatives of Isatin

This study highlights the possible interaction between stress and immune-inflammatory pathways in the pathogenesis of depression and suggests an animal model that addresses these pathways. The molecular docking with AutoDock 4.2 using the enzymes monoamine oxidase and indoleamine 2,3-dioxygenase for the selected compounds of synthetic derivatives of isatin resulted in excellent inhibitory potential when compared to that of standard. Based on the results of docking studies synthetic derivatives of isatin 3 and 6 were selected and evaluated for in vitro studies and tested at two dose levels (100 and 200 mg/kg) on experimental rats induced with chronic unpredictable mild stress depression. The test compounds exerted good antidepressant effect in rats. Treatment with SDI 3 and SDI 6 augmented the endogenous neurotransmitters (serotonin and norepinephrine), significantly restoring most of the altered behavioural parameters and established protective effects against oxidative stress caused by free radicals. Chronic unpredictable mild stress stimulates hypothalamus-pituitary adrenal axis and enhances the release of corticosterone contributing to stress-induced depression in rats. Chronic unpredictable mild stress augmented the production of indoleamine 2,3-dioxygenase enzyme and resulted in increased production of kynurenine thereby stimulating the formation of free radicals with reduced synthesis of serotonin producing depression. Treatment with SDI 3 and SDI 6 decreased corticosterone level, blocked indoleamine 2,3-dioxygenase and effectively reduced kynurenine production and increase the formation of serotonin. Synthetic derivatives of isatin 3 and 6 inhibited formation of monoamine oxidase-A and decreased the metabolism of serotonin and increased the availability of serotonin in hippocampal and prefrontal regions in brain of rat and prevented depression. Since, the synthetic derivatives of isatin 3 and 6 inhibited monoamine oxidase-B it might also have the potential to treat neurodegeneration disease like Parkinson’s. In conclusion, the study suggested that synthetic derivatives of isatin 3 and 6 compounds possess antidepressant and antioxidant properties, which might be helpful in preventing stress induced depression and the related neurodegenerative diseases as a result of imbalance of monoamines. Molecular level studies and further in vitro and in vivo models are required to substantiate this antidepressant activity and to corroborate the results of synthetic derivatives of isatin 3 and 6 before these compounds can be taken for further studies in human volunteers

Plant Science

The book "Plant Science" consists of 12 chapters divided into three sections authored by many researchers from different parts of the Globe. Section-I: Plant and Environment, describes the relationship between plants and environment, particularly enumerating species-environment relationship and response of plants to different environmental stress conditions. Section-II: Plant-Microbe relation, embodies broadly on both positive and negative aspects of microbes on plants. Section-III: Plant Biotechnology, shed light on current biotechnological research to develop modern technology for producing biologicals and also increasing plant immunity in present environmental conditions. The book "Plant Science" will be helpful to a wide group peoples; readers, scientists, researchers and allied professionals. We recommend it to you; enjoy reading it, save the plant and save life