Christopher J. Parkinson

An assessment of theoretical procedures for the calculation of reliable radical stabilization energies

The performance of a variety of theoretical methods in computing stabilization energies of the substituted methyl and vinyl radicals ˙CH2F, ˙CH2CN, ˙CH2CHCH2, ˙CH2CHO, CH2C˙F and CH2C˙CN is examined. The influence of electron correlation (UHF, UMP2, PMP2, RMP2, UB3-LYP, UQCISD, UQCISD(T), UCCSD(T), URCCSD(T) and RRCCSD(T)) and basis set size (from 6-31G(d) to 6-311++G(3df,3pd)) on stabilization energies is evaluated, as well as the performance of compound methods such as G2, G3, CBS-Q and CBS-APNO and their variants. The results indicate that generally reliable radical stabilization energies can be obtained at modest cost using RMP2/6-311+G(2df,p)//RMP2/6-31G(d) energies. A slightly less accurate but more economical procedure is RMP2/6-311+G(d)//B3-LYP/6-31G(d). UMP2 and PMP2 are unsuitable for obtaining radical stabilization energies for spin-contaminated radicals, while UB3-LYP appears generally to overestimate stabilization energies.

In vitro anti-plasmodial activity of Dicoma anomala subsp. gerrardii (Asteraceae): identification of its main active constituent, structure-activity relationship studies and gene expression profiling.

BackgroundAnti-malarial drug resistance threatens to undermine efforts to eliminate this deadly disease. The resulting omnipresent requirement for drugs with novel modes of action prompted a national consortium initiative to discover new anti-plasmodial agents from South African medicinal plants. One of the plants selected for investigation was Dicoma anomala subsp. gerrardii, based on its ethnomedicinal profile.MethodsStandard phytochemical analysis techniques, including solvent-solvent extraction, thin-layer- and column chromatography, were used to isolate the main active constituent of Dicoma anomala subsp. gerrardii. The crystallized pure compound was identified using nuclear magnetic resonance spectroscopy, mass spectrometry and X-ray crystallography. The compound was tested in vitro on Plasmodium falciparum cultures using the parasite lactate dehydrogenase (pLDH) assay and was found to have anti-malarial activity. To determine the functional groups responsible for the activity, a small collection of synthetic analogues was generated - the aim being to vary features proposed as likely to be related to the anti-malarial activity and to quantify the effect of the modifications in vitro using the pLDH assay. The effects of the pure compound on the P. falciparum transcriptome were subsequently investigated by treating ring-stage parasites (alongside untreated controls), followed by oligonucleotide microarray- and data analysis.ResultsThe main active constituent was identified as dehydrobrachylaenolide, a eudesmanolide-type sesquiterpene lactone. The compound demonstrated an in vitro IC50 of 1.865 μM against a chloroquine-sensitive strain (D10) of P. falciparum. Synthetic analogues of the compound confirmed an absolute requirement that the α-methylene lactone be present in the eudesmanolide before significant anti-malarial activity was observed. This feature is absent in the artemisinins and suggests a different mode of action. Microarray data analysis identified 572 unique genes that were differentially expressed as a result of the treatment and gene ontology analysis identified various biological processes and molecular functions that were significantly affected. Comparison of the dehydrobrachylaenolide treatment transcriptional dataset with a published artesunate (also a sesquiterpene lactone) dataset revealed little overlap. These results strengthen the notion that the isolated compound and the artemisinins have differentiated modes of action.ConclusionsThe novel mode of action of dehydrobrachylaenolide, detected during these studies, will play an ongoing role in advancing anti-plasmodial drug discovery efforts.

Structure-Activity Relationship Study of Sesquiterpene Lactones and Their Semi-Synthetic Amino Derivatives as Potential Antitrypanosomal Products

Sesquiterpene lactones (STLs) are natural products that have potent antitrypanosomal activity in vitro and, in the case of cynaropicrin, also reduce parasitemia in the murine model of trypanosomiasis. To explore their structure-antitrypanosomal activity relationships, a set of 34 natural and semi-synthetic STLs and amino-STLs was tested in vitro against T. b. rhodesiense (which causes East African sleeping sickness) and mammalian cancer cells (rat bone myoblast L6 cells). It was found that the α-methylene-γ-lactone moiety is necessary for both antitrypanosomal effects and cytotoxicity. Antitrypanosomal selectivity is facilitated by 2-(hydroxymethyl)acrylate or 3,4-dihydroxy-2-methylenebutylate side chains, and by the presence of cyclopentenone rings. Semi-synthetic STL amines with morpholino and dimethylamino groups showed improved in vitro activity over the native STLs. The dimethylamino derivative of cynaropicrin was prepared and tested orally in the T. b. rhodesiense acute mouse model, where it showed reduced toxicity over cynaropicrin, but also lost antitrypanosomal activity.

Stereocontrolled synthesis of calyculin A: construction of the C(1)–C(14) tetraene nitrile unit

An enantioselective and geometrically selective synthesis of the C(1)–C(14) tetraene nitrile unit of calyculin A, using aldol chemistry with (+)-(E)-diisopinocampheylborane and Stille coupling, is described.

Stereocontrolled synthesis of calyculin A: construction of the C(26)–C(37) amide-oxazole unit

(–)-B-[3-(Diisopropylaminodimethylsilyl)allyl]diisopinocampheylborane and Cornforth–Meyers chemistry, and Evans alkylation were employed to construct the C(26)–C(37) amide–oxazole unit of calyculin A.

Stereocontrolled synthesis of calyculin A: Construction of the C(15)-C(25) spiroketal unit

Two concise enantioselective syntheses of the C(15)–C(25) spiroketal unit of calyculin A, using derivatives of allyldiisopinocampheylborane efficieintly to control 1,2- and 1,3-diol stereochemistries, are reported.

ATP and luciferase assays to determine the rate of drug action in in vitro cultures of Plasmodium falciparum

BackgroundKnowledge of the rate of action of compounds against cultured malaria parasites is required to determine the optimal time-points for drug mode of action studies, as well as to predict likely in vivo parasite clearance rates in order to select optimal hit compounds for further development. In this study, changes in parasite ATP levels and transgenic luciferase reporter activity were explored as means to detect drug-induced stress in cultured parasites.MethodsIn vitro cultures of Plasmodium falciparum 3D7 wild-type or firefly luciferase-expressing parasites were incubated with a panel of six anti-malarial compounds for 10 hours and parasite ATP levels or luciferase activity determined at two-hour intervals using luminescence-based reagents. For comparative purposes, parasite morphology changes were evaluated by light microscopy, as well as the extent to which parasites recover after 48 hours from a six-hour drug treatment using a parasite lactate dehydrogenase assay.ResultsChanges in parasite ATP levels displayed three phenotypes: mild or no change (chloroquine, DFMO); 2–4 fold increase (mefloquine, artemisinin); severe depletion (ritonavir, gramicidin). The respective phenotypes and the rate at which they manifested correlated closely with the extent to which parasites recovered from a six-hour drug treatment (with the exception of chloroquine) and the appearance and severity of morphological changes observed by light microscopy. Luciferase activity decreased profoundly in parasites treated with mefloquine, artemisinin and ritonavir (34-67% decrease in 2 hours), while chloroquine and DFMO produced only mild changes over 10 hours. Gramicidin yielded intermediate decreases in luciferase activity.ConclusionsATP levels and luciferase activity respond rapidly to incubation with anti-malarial drugs and provide quantitative read-outs to detect the appearance and magnitude of drug-induced stress in cultured parasites. The correlation between the observed changes and irreversible parasite toxicity is not yet sufficiently clear to predict clinical clearance rates, but may be useful for ranking compounds against each other and standard drugs vis-à-vis rate of action and for determining early time-points for drug mode of action studies.

Selective induction of oxidative stress in cancer cells via synergistic combinations of agents targeting redox homeostasis

Cancer cell resistance to chemotherapy is still a heavy burden that impairs the response of many cancer patients to conventional chemotherapy. Using drug combinations is one therapeutic approach to overcome the developing resistance to any one drug. Oxidative stress is now a generally regarded hallmark of cancer that can be one approach to selectively target cancer cells while sparing normal cells. With the aim of increasing oxidative stress in cancer cells to a lethal set point, we have generated and combined several series of redox active compounds that act at different points of the cellular oxidative cascade. The premise of such combinations is to deplete of endogenous antioxidant defence proteins (e.g., Glutathione) while concomitantly increasing the generation of ROS via metal redox recycling and Fenton chemistry which eventually leads to the disruption of cellular redox homeostasis and induction of cell death. Through this approach, we have identified highly synergistic combinations of two distinctive classes of compounds (Azines and Copper(II) complexes of 2-pyridyl ketone thiosemicarbazones) which are capable of eliminating cancer cells without concomitant increase in toxicity toward normal cells. In one of our most potent combinations, a combination index (CI) value of 0.056 was observed, representing a 17 fold enhancement in activity beyond additive effects. Such new combination regimen of redox active compounds can be one step closer to potentially safer low dose chemotherapy.

Cytotoxic activity of expanded coordination bis-thiosemicarbazones and copper complexes thereof

A series of bis-thiosemicarbazone agents with coordinating groups capable of multiple metal coordination modes has been generated and evaluated for potential cytotoxic effects against melanoma (MelRm) and breast adenocarcinoma (MCF-7) cell lines. The bis-thiosemicarbazones in this study generally demonstrated superior cytotoxic activity against MelRm than MCF-7 in the absence of metal ion supplementation, but in most cases could not be considered superior to the reference thiosemicarbazone Dp44mT. The key structural features for the cytotoxic activity were the central metal binding atom on the aromatic core, the thiocarbonyl residue and the nature of substitution on the N4-terminus in terms of size and lipophilicity. The cytotoxicity of bis-thiosemicarbazone ligands improved significantly with Cu(II) supplementation, particularly against MCF-7 cells. The mechanism of cytotoxicity of bis-thiosemicarbazones was proposed to be dependent on the combined effect of metal mobilisation and ROS generation which is so called a “double-punch effect”.

Use of the electrophilic arylation reaction of aryllead triacetates in syntheses of (±)-O-methyljoubertiamine and (±)-mesembrine

p-Methoxyphenyllead triacetate and 3,4-dimethoxyphenyllead triacetate are used as electrophilic arylating agents to generate the quaternary benzylic centres in formal syntheses of (±)-Omethyljoubertiamine and (±)-mesembrine respectively.