Lead Optimization of a Pyrrole-Based Dihydroorotate Dehydrogenase Inhibitor Series for the Treatment of Malaria.

Malaria puts at risk nearly half the world's population and causes high mortality in sub-Saharan Africa, while drug resistance threatens current therapies. The pyrimidine biosynthetic enzyme dihydroorotate dehydrogenase (DHODH) is a validated target for malaria treatment based on our finding that triazolopyrimidine DSM265 (1) showed efficacy in clinical studies. Herein, we describe optimization of a pyrrole-based series identified using a target-based DHODH screen. Compounds with nanomolar potency versus Plasmodium DHODH and Plasmodium parasites were identified with good pharmacological properties. X-ray studies showed that the pyrroles bind an alternative enzyme conformation from 1 leading to improved species selectivity versus mammalian enzymes and equivalent activity on Plasmodium falciparum and Plasmodium vivax DHODH. The best lead DSM502 (37) showed in vivo efficacy at similar levels of blood exposure to 1, although metabolic stability was reduced. Overall, the pyrrole-based DHODH inhibitors provide an attractive alternative scaffold for the development of new antimalarial compounds.

Structure-Activity Relationship of Antischistosomal Ozonide Carboxylic Acids.

Semisynthetic artemisinins and other bioactive peroxides are best known for their powerful antimalarial activities, and they also show substantial activity against schistosomes-another hemoglobin-degrading pathogen. Building on this discovery, we now describe the initial structure-activity relationship (SAR) of antischistosomal ozonide carboxylic acids OZ418 (2) and OZ165 (3). Irrespective of lipophilicity, these ozonide weak acids have relatively low aqueous solubilities and high protein binding values. Ozonides with para-substituted carboxymethoxy and N-benzylglycine substituents had high antischistosomal efficacies. It was possible to increase solubility, decrease protein binding, and maintain the high antischistosomal activity in mice infected with juvenile and adult Schistosoma mansoni by incorporating a weak base functional group in these compounds. In some cases, adding polar functional groups and heteroatoms to the spiroadamantane substructure increased the solubility and metabolic stability, but in all cases decreased the antischistosomal activity.

Using Human Plasma as an Assay Medium in Caco-2 Studies Improves Mass Balance for Lipophilic Compounds.

PURPOSE: To examine the utility of human plasma as an assay medium in Caco-2 permeability studies to overcome poor mass balance and inadequate sink conditions frequently encountered with lipophilic compounds. METHODS: Caco-2 permeability was assessed for reference compounds with known transport mechanisms using either pH 7.4 buffer or human plasma as the assay medium in both the apical and basolateral chambers. When using plasma, Papp values were corrected for the unbound fraction in the donor chamber. The utility of the approach was assessed by measuring the permeability of selected antimalarial compounds using the two assay media. RESULTS: Caco-2 cell monolayer integrity and P-gp transporter function were unaffected by the presence of human plasma in the donor and acceptor chambers. For many of the reference compounds having good mass balance with buffer as the medium, higher Papp values were observed with plasma, likely due to improved acceptor sink conditions. The lipophilic antimalarial compounds exhibited low mass balance with buffer, however the use of plasma markedly improved mass balance allowing the determination of more reliable Papp values. CONCLUSIONS: The results support the utility of human plasma as an alternate Caco-2 assay medium to improve mass balance and permeability measurements for lipophilic compounds.

Revisiting the SAR of the Antischistosomal Aryl Hydantoin (Ro 13-3978).

The aryl hydantoin 1 (Ro 13-3978) was identified in the early 1980s as a promising antischistosomal lead compound. However, this series of aryl hydantoins produced antiandrogenic side effects in the host, a not unexpected outcome given their close structural similarity to the antiandrogenic drug nilutamide. Building on the known SAR of this compound series, we now describe a number of analogs of 1 designed to maximize structural diversity guided by incorporation of substructures and functional groups known to diminish ligand-androgen receptor interactions. These analogs had calculated polar surface area (PSA), measured LogD7.4, aqueous kinetic solubility, and estimated plasma protein binding values in ranges predictive of good ADME profiles. The principal SAR insight was that the hydantoin core of 1 is required for high antischistosomal activity. We identified several compounds with high antischistosomal efficacy that were less antiandrogenic than 1. These data provide direction for the ongoing optimization of antischistosomal hydantoins.

Haemoglobin degradation underpins the sensitivity of early ring stage Plasmodium falciparum to artemisinins.

Current first-line artemisinin antimalarials are threatened by the emergence of resistant Plasmodium falciparum. Decreased sensitivity is evident in the initial (early ring) stage of intraerythrocytic development, meaning that it is crucial to understand the action of artemisinins at this stage. Here, we examined the roles of iron (Fe) ions and haem in artemisinin activation in early rings using Fe ion chelators and a specific haemoglobinase inhibitor (E64d). Quantitative modelling of the antagonism accounted for its complex dependence on the chemical features of the artemisinins and on the drug exposure time, and showed that almost all artemisinin activity in early rings (>80%) is due to haem-mediated activation. The surprising implication that haemoglobin uptake and digestion is active in early rings is supported by identification of active haemoglobinases (falcipains) at this stage. Genetic down-modulation of the expression of the two main cysteine protease haemoglobinases, falcipains 2 and 3, renders early ring stage parasites resistant to artemisinins. This confirms the important role of haemoglobin-degrading falcipains in artemisinin activation, and shows that changes in the rate of artemisinin activation could mediate high-level artemisinin resistance.

Evidence for the in vitro bioactivation of aminopyrazole derivatives: trapping reactive aminopyrazole intermediates using glutathione ethyl ester in human liver microsomes.

Drug-induced toxicity is a leading cause of drug withdrawal from clinical development and clinical use and represents a major impediment to the development of new drugs. The mechanisms underlying drug-induced toxicities are varied; however, metabolic bioactivation to form reactive metabolites has been identified as a major contributor.1,2 These electrophilic species can covalently modify important biological macromolecules and thereby increase the risk of adverse drug reactions or idiosyncratic toxicity. Consequently, screening compounds for their propensity to form reactive metabolites has become an integral part of drug discovery programs. This screening process typically involves identification of structural alerts as well as the generation of reactive metabolites in vitro in subcellular hepatic fractions, followed by trapping the reactive species with nucleophiles and characterization via LC-MS. This article presents evidence for the bioactivation of a series of aminopyrazole derivatives via LC-MS detection of glutathione ethyl ester-trapped reactive intermediates formed in human liver microsomal incubations. These results indicate that the aminopyrazole motif, within specific contexts, may be considered a new structural alert for the potential formation of reactive metabolites.

The hypoxia imaging agent CuII(atsm) is neuroprotective and improves motor and cognitive functions in multiple animal models of Parkinson's disease.

Parkinson's disease (PD) is a progressive, chronic disease characterized by dyskinesia, rigidity, instability, and tremors. The disease is defined by the presence of Lewy bodies, which primarily consist of aggregated α-synuclein protein, and is accompanied by the loss of monoaminergic neurons. Current therapeutic strategies only give symptomatic relief of motor impairment and do not address the underlying neurodegeneration. Hence, we have identified Cu(II)(atsm) as a potential therapeutic for PD. Drug administration to four different animal models of PD resulted in improved motor and cognition function, rescued nigral cell loss, and improved dopamine metabolism. In vitro, this compound is able to inhibit the effects of peroxynitrite-driven toxicity, including the formation of nitrated α-synuclein oligomers. Our results show that Cu(II)(atsm) is effective in reversing parkinsonian defects in animal models and has the potential to be a successful treatment of PD.

Synthesis and biological evaluation of polysulfated oligosaccharide glycosides as inhibitors of angiogenesis and tumor growth.

A series of polysulfated penta- and tetrasaccharide glycosides containing alpha(1-->3)/alpha(1-->2)-linked mannose residues were synthesized as heparan sulfate (HS) mimetics and evaluated for their ability to inhibit angiogenesis. The compounds bound tightly to angiogenic growth factors (FGF-1, FGF-2, and VEGF) and strongly inhibited heparanase activity. In addition, the compounds exhibited potent activity in cell-based and ex vivo assays indicative of angiogenesis, with tetrasaccharides exhibiting activity comparable to that of pentasaccharides. Selected compounds also showed good antitumor activity in vivo in a mouse melanoma (solid tumor) model resistant to the phase III HS mimetic 1 (muparfostat, formerly known as PI-88). The lipophilic modifications also resulted in reduced anticoagulant activity, a common side effect of HS mimetics, and conferred a reasonable pharmacokinetic profile in the rat, as exemplified by the sulfated octyl tetrasaccharide 5. The data support the further investigation of this class of compounds as potential antiangiogenic, anticancer therapeutics.

CpG methylation potentiates pixantrone and doxorubicin-induced DNA damage and is a marker of drug sensitivity.

DNA methylation is an epigenetic modification of the mammalian genome that occurs predominantly at cytosine residues of the CpG dinucleotide. Following formaldehyde activation, pixantrone alkylates DNA and particularly favours the CpG motif. Aberrations in CpG methylation patterns are a feature of most cancer types, a characteristic that may determine their susceptibility to specific drug treatments. Given their common target, DNA methylation may modulate the DNA damage induced by formaldehyde-activated pixantrone. In vitro transcription, mass spectrometry and oligonucleotide band shift assays were utilized to establish that pixantrone-DNA adduct formation was consistently enhanced 2-5-fold at discrete methylated CpG doublets. The methylation-mediated enhancement was exquisitely sensitive to the position of the methyl substituent since methylation at neighboring cytosine residues failed to confer an increase in pixantrone-DNA alkylation. Covalent modification of DNA by formaldehyde-activated doxorubicin, but not cisplatin, was augmented by neighbouring CpG methylation, indicating that modulation of binding by CpG methylation is not a general feature of all alkylators. HCT116 colon cancer cells vastly deficient in CpG methylation were 12- and 10-fold more resistant to pixantrone and doxorubicin relative to the wild-type line, suggesting that these drugs may selectively recognize the aberrant CpG methylation profiles characteristic of most tumour types.

Formaldehyde-activated Pixantrone is a monofunctional DNA alkylator that binds selectively to CpG and CpA doublets.

The topoisomerase II poison mitoxantrone is important in the clinical management of human malignancies. Pixantrone, a novel aza-anthracenedione developed to improve the therapeutic profile of mitoxantrone, can efficiently alkylate DNA after formaldehyde activation. In vitro transcriptional analysis has now established that formaldehyde-activated pixantrone generates covalent adducts selectively at discrete CpG or CpA dinucleotides, suggesting that the activated complex binds to guanine or cytosine (or both) bases. The stability of pixantrone adduct-induced transcriptional blockages varied considerably, reflecting a mixture of distinct pixantrone adduct types that may include relatively labile monoadducts and more stable interstrand cross-links. 6,9-Bis-[[2-(dimethylamino)ethyl]amino]benzo[g]isoquinoline-5,10-dione (BBR 2378), the dimethyl N-substituted analog of pixantrone, could not form adducts, suggesting that pixantrone alkylates DNA through the primary amino functions located in each side chain of the drug. Pixantrone generated DNA adducts only when guanine was present in substrates and exhibited a lack of adduct formation with inosine-containing polynucleotides, confirming that the N2 amino group of guanine is the site for covalent attachment of the drug. Mass spectrometric analysis of oligonucleotide-drug complexes confirmed that formation of covalent pixantrone-DNA adducts is mediated by a single methylene linkage provided by formaldehyde and that this occurs only with guanine-containing double stranded oligonucleotide substrates. CpG methylation, an epigenetic modification of the mammalian genome, significantly enhanced the generation of pixantrone-DNA adducts within a methylated DNA substrate, indicating that the methylated dinucleotide may be a favored target in a cellular environment.

Iron-mediated degradation kinetics of substituted dispiro-1,2,4-trioxolane antimalarials.

The iron-mediated reactivity of various dispiro-1,2,4-trioxolanes was determined by automated kinetic analysis under standard reaction conditions. The active antimalarial compounds underwent peroxide bond cleavage by Fe(II) resulting in products indicative of carbon-centered radical formation. The rate of reaction was heavily influenced by the presence of spiro-substituted adamantane and cyclohexane rings, and was also significantly affected by cyclohexane ring substitution. Steric hindrance around the peroxide oxygen atoms appeared to be the major determinant of reaction rate, however polar substituents also affected reactivity by an independent mechanism. A wide range of reaction rates was observed within this class of peroxide antimalarials, however iron-mediated reactivity did not directly correlate with in vitro antimalarial activity.

Spiro and dispiro-1,2,4-trioxolanes as antimalarial peroxides: charting a workable structure-activity relationship using simple prototypes.

This paper describes the discovery of synthetic 1,2,4-trioxolane antimalarials and how we established a workable structure-activity relationship in the context of physicochemical, biopharmaceutical, and toxicological profiling. An achiral dispiro-1,2,4-trioxolane (3) in which the trioxolane is flanked by a spiroadamantane and spirocyclohexane was rapidly identified as a lead compound. Nonperoxidic 1,3-dioxolane isosteres of 3 were inactive as were trioxolanes without the spiroadamantane. The trioxolanes were substantially less effective in a standard oral suspension formulation compared to a solubilizing formulation and were more active when administered subcutaneously than orally, both of which suggest substantial biopharmaceutical liabilities. Nonetheless, despite their limited oral bioavailability, the more lipophilic trioxolanes generally had better oral activity than their more polar counterparts. In pharmacokinetic experiments, four trioxolanes had high plasma clearance values, suggesting a potential metabolic instability. The toxicological profiles of two trioxolanes were comparable to that of artesunate.

Kinetics of iron-mediated artemisinin degradation: effect of solvent composition and iron salt.

The antimalarial endoperoxides, such as artemisinin, are postulated to exert their potent parasiticidal activity via the formation of reactive intermediates in the iron-rich infected erythrocyte. The in vitro chemical reaction profile of putative endoperoxide antimalarials and ferrous iron is often qualitatively used to assess their potential antimalarial activity and to develop a structure-reactivity relationship. This study utilized LCMS to monitor the kinetics of artemisinin degradation and product formation in the presence of iron. A second order degradation reaction (k = 18 M(-1) h(-1)) was observed from the reaction of artemisinin with ferrous sulphate in aqueous acetonitrile to produce a number of stable isomeric rearrangement products. A systematic study of the effect of a number of solvent systems and different iron salts showed pronounced changes in reaction rate and product distribution. The significant effects observed in the current study highlight the need to carefully control reaction conditions when studying peroxide antimalarial stability or attempting to develop in vitro/in vivo correlations of endoperoxide antimalarials and their reactivity with iron.