Unusual structure-genotoxicity relationship in mouse lymphoma cells observed with a series of kinase inhibitors.

During development of a novel kinase inhibitor for an anti-inflammatory therapy at AstraZeneca UK, the lead compound was found to be potently active in the mouse lymphoma assay (MLA). This was not believed to be due to primary pharmacology because structural alert relationships and a negative Ames test indicated that the compound was unlikely to form DNA adducts. A number of investigations were performed to assess whether mammalian cell genotoxicity was inherent to the chemical series. The in vitro micronucleus assay (MN(vit)) combined with a semi-automated analysis system, was used as a high-throughput screen. A number of additional compounds were selected for testing, all with different substituents around a core isoquinolinone. These modifications did not affect the kinase and non-kinase selectivity of the compounds. Several of these compounds were positive in the MN(vit), however, two compounds were found to be negative and these were also confirmed to be negative in the MLA. It was considered possible that topoisomerase II or off-target kinase inhibition may have been responsible for the observed mammalian cell genotoxicity. The present investigations show how an iterative chemical design, along with genotoxicity screening by use of a semi-automated MN(vit), can identify and remove the genotoxic hazard from pharmaceutical projects at an early stage of development, and produce high-quality molecules suitable for further progression.

Lipophilicity of acidic compounds: impact of ion pair partitioning on drug design.

In drug discovery projects the ability to show a relationship between a compound's molecular structure and its pharmacokinetic, in vivo efficacy, or toxicity profile is paramount for the design of better analogues. To aid this understanding the measurement of distribution coefficients at some physiologically relevant pH, for example, log D(7.4), is common practice as they are used as a key descriptor in mathematical models for predicting various biological parameters. Evidence is presented that under typical experimental conditions ion pair partitioning can contribute greatly to log D(7.4) results for acidic compounds; if this is ignored it may compromise data analysis within drug discovery projects where the modulation of lipophilicity is a primary design strategy. The work herein focuses on acidic compounds and reflects the experience of AstraZeneca R&D Charnwood (AZ) where ion pair partitioning contributions can be minimized by the routine measurement of log D(5.5) data. The magnitude of ion pair partitioning contributions to the log D(7.4) measurements of 24 acidic drugs are investigated, and the risks to drug discovery projects that ignore such contributions are discussed. The superiority of measured lipophilicity data over calculated data for a set of AZ proprietary acidic compounds is also presented.

Switching between agonists and antagonists at CRTh2 in a series of highly potent and selective biaryl phenoxyacetic acids.

A novel series of biaryl phenoxyacetic acids was discovered as potent, selective antagonists of the chemoattractant receptor-homologous expressed on Th2 lymphocytes receptor (CRTh2 or DP2). A hit compound 4 was discovered from high throughput screening. Modulation of multiple aryl substituents afforded both agonists and antagonists, with small changes often reversing the mode of action. Understanding the complex SAR allowed design of potent antagonists such as potential candidate 34.

Substituted indole-1-acetic acids as potent and selective CRTh2 antagonists-discovery of AZD1981.

Novel indole-3-thio-, 3-sulfonyl- and 3-oxy-aryl-1-acetic acids are reported which are potent, selective antagonists of the chemoattractant receptor-homologous expressed on Th2 lymphocytes receptor (CRTh2 or DP2). Optimization required maintenance of high CRTh2 potency whilst achieving a concomitant reduction in rates of metabolism, removal of cyp p450 inhibition and minimization of aldose reductase and aldehyde reductase activity. High quality compounds suitable for in vivo studies are highlighted, culminating in the discovery of AZD1981 (22).

Strategies to improve in vivo toxicology outcomes for basic candidate drug molecules.

A valid PLS-DA model to predict attrition in pre-clinical toxicology for basic oral candidate drugs was built. A combination of aromatic/aliphatic balance, flatness, charge distribution and size descriptors helped predict the successful progression of compounds through a wide range of toxicity testing. Eighty percent of an independent test set of marketed post-2000 basic drugs could be successfully classified using the model, indicating useful forward predictivity. The themes within this work provide additional guidance for medicinal design chemists and complement other literature property guidelines.

In silico prediction of acyl glucuronide reactivity.

Drugs and drug candidates containing a carboxylic acid moiety, including many widely used non-steroidal anti-inflammatory drugs (NSAIDs) are often metabolized to form acyl glucuronides (AGs). NSAIDs such as Ibuprofen are amongst the most widely used drugs on the market, whereas similar carboxylic acid drugs such as Suprofen have been withdrawn due to adverse events. Although the link between these AG metabolites and toxicity is not proven, there is circumstantial literature evidence to suggest that more reactive acyl glucuronides may, in some cases, present a greater risk of exhibiting toxic effects. We wished therefore to rank the reactivity of potential new carboxylate-containing drug candidates, and performed kinetic studies on synthetic acyl glucuronides to benchmark our key compounds. Driven by the desire to quickly rank the reactivity of compounds without the need for lengthy synthesis of the acyl glucuronide, a correlation was established between the degradation half-life of the acyl glucuronide and the half life for the hydrolysis of the more readily available methyl ester derivative. This finding enabled a considerable broadening of chemical property space to be investigated. The need for kinetic measurements was subsequently eliminated altogether by correlating the methyl ester hydrolysis half-life with the predicted (13)C NMR chemical shift of the carbonyl carbon together with readily available steric descriptors in a PLS model. This completely in silico prediction of acyl glucuronide reactivity is applicable within the earliest stages of drug design with low cost and acceptable accuracy to guide intelligent molecular design. This reactivity data will be useful alongside the more complex additional pharmacokinetic exposure and distribution data that is generated later in the drug discovery process for assessing the overall toxicological risk of acidic drugs.

Prediction of efficacious inhalation lung doses via the use of in silico lung retention quantitative structure-activity relationship models and in vitro potency screens.

Lung concentrations of a drug are expected to drive the pharmacodynamic response to local inflammation after inhalation delivery, and the only way of determining the efficacious dose has been to measure it directly in animal models. In this study, we present a method to predict efficacious lung doses after inhalation in a rat lipopolysaccharide challenge model from in silico predictions of lung concentration and in vitro measurements only. A quantitative structure-activity relationship (QSAR) model, based on calculated physical properties predicted the partitioning of 34 compounds between lung and plasma. Because it was observed that lung/plasma partitioning correlated with lung concentration, it was possible to use this relationship to predict lung concentration at a given dose and time point. Based on the pharmacokinetic-pharmacodynamic (PKPD) relationship observed, a minimal free lung concentration relative to potency to drive significant inhibition of neutrophilia was established. By using predicted lung concentrations, measured fraction unbound in plasma, and cellular potency, it was possible to estimate an inhaled lung dose that would be expected to achieve this target exposure. These predictions were made for 23 compounds, which were not part of the original QSAR training set, and all except one were predicted to within 3-fold of their measured values. This novel approach shows that by understanding PKPD relationships and drivers for lung affinity after inhalation dosing, it is possible to estimate in vivo lung doses required for efficacy. This methodology provides a useful screening tool to rank candidate compounds and minimizes the use of extensive animal testing.

Deferitazole, a new orally active iron chelator.

Following a systematic search of desferrithiocin analogs, a polyether derivative, deferitazole (formerly FBS0701), has entered into phase 1 and 2 clinical trials with promising biological properties. However, until now, detailed physicochemical properties of this chelator have not been reported. The compound displays a high affinity and selectivity for iron(III) as demonstrated by the log ß2 = 33.39 ± 0.03 and the pFe(3+) value of 22.3. Two equilibrating isomeric forms of the iron(III) complex exist under biological conditions. Deferitazole also binds the trivalent metals Al(III) and La(III) with high affinity; log ß2 values, 26.68 and 21.55 respectively. The affinity of deferitazole for divalent cations is somewhat lower, with the exception of Cu(II) which possesses a log ß2 value of 25.5; deferitazole scavenges iron from labile sources such as citrate and albumin with efficiencies comparable with those of other therapeutic iron chelators, including deferasirox, deferiprone and desferrioxamine. The Fe(III)(deferitazole)2 is stable under physiological conditions and does not redox cycle. The high affinity of deferitazole for iron(III) renders it unlikely that this chelator will lead to the redistribution of iron and consequently deferitazole shows considerable promise as a therapeutic iron(III) chelator.

Synthesis and Further Reactivity of Functionalized Lactam-Derived Enol Triflates.

Pyrrolidinone- and piperidinone-derived enol triflates 2 were prepared in high yield (60-97%) from the corresponding lactams 1 using KHMDS and N-(5-chloro-2-pyridyl)triflimide. A structure-stability study on the less stable pyrrolidinone-derived triflates revealed that an N-tosyl group is essential, and an alpha-ethoxy substituent enhances thermal stability. Substituents at the 3- and 4-position are tolerated. Substitution of the triflate moiety by a wide variety of functional groups was achieved under mild conditions via metal-mediated reactions. Although cuprate couplings proceeded in only moderate yields, several palladium-catalyzed reactions gave good yields of interesting molecules for further synthetic operations (for example, Stille coupling with vinylstannanes, cross-coupling with arylzincs, and carbonylation processes). Preparation of the first enantiopure lactam-derived enol triflate 15 (from (S)-pyroglutamic acid) is described. Enamide hydrogenation of derivative 17 allowed the synthesis of a proline analogue 18 in excellent yield and diastereoselectivity (86% de).

trans-2-Tritylcyclohexanol as a chiral auxiliary in permanganate-mediated oxidative cyclization of 2-methylenehept-5-enoates: application to the synthesis of trans-(+)-linalool oxide.

The permanganate-mediated oxidative cyclization of a series of 2-methylenehept-5-eneoates bearing different chiral auxiliaries was investigated, leading to the discovery of trans-2-tritylcyclohexanol (TTC) as a highly effective chiral controller for the formation of the 2,5-substituted THF diol product with high diastereoselectivity (dr ∼97:3). Chiral resolution of (±)-TTC, prepared in one step from cyclohexene oxide, afforded (-)-(1S,2R)-TTC (er >99:1), which was applied to the synthesis of (+)-trans-(2S,5S)-linalool oxide.

Zwitterionic CRTh2 antagonists.

A novel series of zwitterions is reported that contains potent, selective antagonists of the chemoattractant receptor-homologous expressed on Th2 lymphocytes receptor (CRTh2 or DP2). A high quality lead compound 2 was discovered from virtual screening based on the pharmacophore features present in a literature compound 1. Lead optimization through side chain modification and preliminary changes around the acid are disclosed. Optimization of physicochemical properties (log D, MWt, and HBA) allowed maintenance of high CRTh2 potency while achieving low rates of metabolism and minimization of other potential concerns such as hERG channel activity and permeability. A step-change increase in potency was achieved through addition of a single methyl group onto the piperazine ring, which gave high quality compounds suitable for progression into in vivo studies.

Molybdenum-mediated carbonylation of aryl halides with nucleophiles using microwave irradiation.

A new, efficient, and practical molybdenum-mediated carbonylation of aryl and heteroaryl halides with a variety of nucleophiles is described using microwave irradiation. A range of reactions illustrating the wide scope of this chemistry were carried out and proceeded in good to excellent yields.

Enantioselective formal synthesis of eurylene: synthesis of the cis- and trans-THF fragments using oxidative cyclization.

A formal synthesis of eurylene is described, where both cis- and trans-THF-containing fragments were prepared using diastereo- and chemoselective permanganate-mediated oxidative monocyclizations of trienes.

Permanganate oxidation of 1,5,9-trienes: stereoselective synthesis of tetrahydrofuran-containing fragments.

Permanganate oxidation of farnesoate esters 12a-d afforded perhydro-2,2'-bifuranyl compounds 16a-d, with control of relative stereochemistry at four new stereocenters. Subsequent oxidative cleavage of 16a-d then provided tetrahydrofuran-containing fragments 17a-d, one of them 17b possessing the same relative stereochemistry present in the C13-C21 portion of the polyether antibiotic semduramycin (1). Control of the absolute stereochemistry was achieved through the use of the Oppolzer sultam chiral auxiliary. The requisite starting trienes were prepared stereoselectively in just three steps from geranyl chloride or neryl chloride, providing a short and versatile route to polyether fragments.