Skin sensitization in silico protocol.

The assessment of skin sensitization has evolved over the past few years to include in vitro assessments of key events along the adverse outcome pathway and opportunistically capitalize on the strengths of in silico methods to support a weight of evidence assessment without conducting a test in animals. While in silico methods vary greatly in their purpose and format; there is a need to standardize the underlying principles on which such models are developed and to make transparent the implications for the uncertainty in the overall assessment. In this contribution, the relationship between skin sensitization relevant effects, mechanisms, and endpoints are built into a hazard assessment framework. Based on the relevance of the mechanisms and effects as well as the strengths and limitations of the experimental systems used to identify them, rules and principles are defined for deriving skin sensitization in silico assessments. Further, the assignments of reliability and confidence scores that reflect the overall strength of the assessment are discussed. This skin sensitization protocol supports the implementation and acceptance of in silico approaches for the prediction of skin sensitization.

Principles and procedures for handling out-of-domain and indeterminate results as part of ICH M7 recommended (Q)SAR analyses.

The International Council for Harmonization (ICH) M7 guideline describes a hazard assessment process for impurities that have the potential to be present in a drug substance or drug product. In the absence of adequate experimental bacterial mutagenicity data, (Q)SAR analysis may be used as a test to predict impurities' DNA reactive (mutagenic) potential. However, in certain situations, (Q)SAR software is unable to generate a positive or negative prediction either because of conflicting information or because the impurity is outside the applicability domain of the model. Such results present challenges in generating an overall mutagenicity prediction and highlight the importance of performing a thorough expert review. The following paper reviews pharmaceutical and regulatory experiences handling such situations. The paper also presents an analysis of proprietary data to help understand the likelihood of misclassifying a mutagenic impurity as non-mutagenic based on different combinations of (Q)SAR results. This information may be taken into consideration when supporting the (Q)SAR results with an expert review, especially when out-of-domain results are generated during a (Q)SAR evaluation.

It's difficult, but important, to make negative predictions.

At the confluence of predictive and regulatory toxicologies, negative predictions may be the thin green line that prevents populations from being exposed to harm. Here, two novel approaches to making confident and robust negative in silico predictions for mutagenicity (as defined by the Ames test) have been evaluated. Analyses of 12 data sets containing >13,000 compounds, showed that negative predictivity is high (∼90%) for the best approach and features that either reduce the accuracy or certainty of negative predictions are identified as misclassified or unclassified respectively. However, negative predictivity remains high (and in excess of the prevalence of non-mutagens) even in the presence of these features, indicating that they are not flags for mutagenicity.

Evaluation of a statistics-based Ames mutagenicity QSAR model and interpretation of the results obtained.

The relative wealth of bacterial mutagenicity data available in the public literature means that in silico quantitative/qualitative structure activity relationship (QSAR) systems can readily be built for this endpoint. A good means of evaluating the performance of such systems is to use private unpublished data sets, which generally represent a more distinct chemical space than publicly available test sets and, as a result, provide a greater challenge to the model. However, raw performance metrics should not be the only factor considered when judging this type of software since expert interpretation of the results obtained may allow for further improvements in predictivity. Enough information should be provided by a QSAR to allow the user to make general, scientifically-based arguments in order to assess and overrule predictions when necessary. With all this in mind, we sought to validate the performance of the statistics-based in vitro bacterial mutagenicity prediction system Sarah Nexus (version 1.1) against private test data sets supplied by nine different pharmaceutical companies. The results of these evaluations were then analysed in order to identify findings presented by the model which would be useful for the user to take into consideration when interpreting the results and making their final decision about the mutagenic potential of a given compound.

An expert system to predict the forced degradation of organic molecules.

In this paper we describe Zeneth, a new expert computational system for the prediction of forced degradation pathways of organic compounds. Intermolecular reactions such as dimerization, reactions between the query compound and its degradants, as well as interactions with excipients can be predicted. The program employs a knowledge base of patterns and reasoning rules to suggest the most likely transformations under various environmental conditions relevant to the pharmaceutical industry. Building the knowledge base is facilitated by data sharing between the users.

Fine tuning reactivity: synthesis and isolation of 1,2,3,12b-tetrahydroimidazo[1,2-f]phenanthridines.

A facile route for the synthesis and isolation of 1,2,3,12b-tetrahydroimidazo[1,2-f]phenanthridines (TIPs) has been developed. The heterocycle is a reactive intermediate in the three-step cascade synthesis of 2,3-dihydro-1H-imidazo[1,2-f]phenanthridinium cations (DIPs), a biologically active DNA intercalating framework; however, the intermediate has previously only been characterized in situ. Derivatization of the structure at the imidazo-N position controls the reactivity of the intermediate with respect to electronic potential and pK(a) allowing isolation of a selection of TIP structures. Correlations between these parameters and reaction outcome have been made, and other influences such as steric and solvent effects have also been investigated.

A new C-C bond forming annulation reaction leading to pH switchable heterocycles.

A C-C bond forming reaction resulting from the alpha-addition of carbon based nucleophiles to N-bromoethyl phenanthridinium leads to the formation of 2,3-dihydro-12H-pyrrolo[1,2-f]phenanthridine-based derivatives which undergo reversible ring-opening/closing under pH control.

Spontaneous assembly and real-time growth of micrometre-scale tubular structures from polyoxometalate-based inorganic solids.

We report the spontaneous and rapid growth of micrometre-scale tubes from crystals of a metal oxide-based inorganic solid when they are immersed in an aqueous solution containing a low concentration of an organic cation. A membrane immediately forms around the crystal, and this membrane then forms micrometre-scale tubes that grow with vast aspect ratios at controllable rates along the surface on which the crystal is placed. The tubes are composed of an amorphous mixture of polyoxometalate-based anions and organic cations. It is possible for liquid to flow through the tubes, and for the direction of growth and the overall tube diameter to be controlled. We demonstrate that tube growth is driven by osmotic pressure within the membrane sack around the crystal, which ruptures to release the pressure. These robust, self-growing, micrometre-scale tubes offer opportunities in many areas, including the growth of microfluidic devices and the self-assembly of metal oxide-based semipermeable membranes for diverse applications.

Realization of a "lockable" molecular switch via pH- and redox-modulated cyclization.

A switchable organic system involving four distinct states that can be interconverted by use of both pH and redox chemistry as control parameters has been developed. The key molecules involved in this system are the phenanthridine-based heterocycles 1-isobutyl-1,2,3,12b-tetrahydroimidazo[1,2-f]phenanthridine (TIP) and 5-[2-(isobutylamino)ethyl]phenanthridinium (AEP). These two states are interchangeable via pH control, and in addition they can also be further manipulated by oxidation or reduction to convert them to their "pH-inert" forms: 1-isobutyl-2,3-dihydro-1H-imidazo[1,2-f]phenanthridinium (DIP) and 5-[2-(isobutylamino)ethyl]-5,6-dihydrophenanthridine (AEDP), respectively. UV and (1)H NMR experiments carried out in a biphasic dichloromethane (DCM)/water solution were used for in situ structure determination. The results showed that the pH-modulated cyclization and phase-transfer process between the TIP and AEP states was essentially quantitative and repeatable without any significant loss in activity and that reduction or oxidation could be used to lock out these states against such acid-base-induced changes.

Metal-mediated transformation of a triazinephenanthridinium ligand leading to a {Pd5} coordination complex observed crystallographically and by cryospray mass spectrometry.

The formation of a pentanuclear palladium(II) complex with a phenanthridinonetriazine-based ligand system, which itself is formed by a metal-mediated rearrangement of a triazinephenanthridinium proligand, is described.

Incorporation of N-heterocyclic cations into proteins with a highly directed chemical modification.

N-Heterocyclic cations are incorporated into proteins using 5-(2-bromoethyl)phenanthridinium bromide, which selectively reacts with either cysteine or lysine residues, resulting in ethylphenanthridinium (Phen) or highly stable cyclised dihydro-imidazo-phenanthridinium (DIP) adducts respectively; these modifications have been found to manipulate the observed structure of lysozyme and bovine serum albumin by AFM.

A general and efficient five-step one-pot procedure leading to nitrogen-bridgehead heterocycles containing an imidazole ring.

A very simple annulation reaction was designed, allowing an imidazole moiety to be fused onto a range of pyridine-based derivatives. The methodology consists of an activation step via the formation of a pyridinium salt to increase the electrophilicity of the pyridine ring, followed by a cascade reaction triggered by a nucleophilic attack of the iminium moiety. Depending on the pyridinium salt, it is possible to obtain functionalized imidazole moieties.

Microcalorimetry of interaction of dihydro-imidazo-phenanthridinium (DIP)-based compounds with duplex DNA.

Isothermal titration (ITC) and differential scanning calorimetry (DSC) have been used to screen the binding thermodynamics of a family of DNA intercalators based on the dihydro-imidazo-phenanthridinium (DIP) framework. All members of this DIP-based ligand family bind to both genomic (calf thymus and/or salmon testes) and a synthetic dodecamer d(CGCGAATTCGCG) duplex DNA with broadly similar affinities regardless of side chain size or functionality. Viscosity measurements confirm that binding satisfies standard criteria for intercalation. Binding is exothermic but with an additional favourable positive entropy contribution in most cases at 25 degrees C, although a significant negative heat capacity effect (DeltaC(p)) means that both DeltaH(0) and DeltaS(0) decrease with increasing temperature. DIP-ligand binding to DNA also shows significant entropy-enthalpy compensation effects that are now almost standard in such situations, probably reflecting the conformational flexibility of macromolecular systems involving a multiplicity of weak non-covalent interactions. This ability to vary side chain functionality without compromising DNA binding suggests that the DIP framework should be a promising basis for more adventurous chemistry at the DNA level.

Dihydroimidazophenanthridinium (DIP)-based DNA binding agents with tuneable structures and biological activity.

We have synthesised a library of dihydroimidazophenanthridinium cations (DIPs) with large structural diversity (1-29) using a "one-pot" approach. The DNA binding constants of DIPs range from 2x10(4) to 1.3x10(5) M(-1), and the free energies for binding range from -5.9 to -6.40 kcal mol(-1). Viscosity measurements demonstrated that the binding of the compounds caused DNA lengthening, thus signifying binding by intercalation. The cytotoxicities of the compounds were determined by tetrazolium dye-based microtitration assays and showed a large range of values (0.09-11.7 microM). Preliminary molecular modelling studies of the DNA-DIP interactions suggested that the DIP moieties can interact with DNA by intercalation, and some R groups might facilitate binding by minor-groove binding. The results provide insight into how to design biologically active DNA binding agents that can interact in these ways.

Discovery of an imidazo-phenanthridine synthon produced in a 'five-step one-pot reaction' leading to a new family of heterocycles with novel physical properties.

A new class of heterocyclic aromatic cation with novel physical properties has been constructed by an unprecedented reaction pathway that proceeds via five spontaneous steps to yield a 'synthon' that can be further derivatised by a final nucleophilic substitution step.

Highly stable phenanthridinium frameworks as a new class of tunable DNA binding agents with cytotoxic properties.

A new class of cytotoxic heteroaromatic cations is presented, based on the dihydro-imidazo-phenanthridinium framework (DIP), that have affinity for DNA and cytotoxicity toward cancerous cells. The DIP framework is particularly tunable due to the flexible synthetic methodology. Furthermore, the central moiety has proved to be very stable to hydrolysis and reduction compared to other phenanthridinium-based agents.

General one-pot, three-step methodology leading to an extended class of N-heterocyclic cations: spontaneous nucleophilic addition, cyclization, and hydride loss.

A new class of phenanthridinium derivative has been isolated from the reaction of 2-bromoethyl-phenanthridinium bromide with a range of primary amines in excellent yields. The reaction pathway is unprecedented and proceeds via three cascade steps: nucleophilic attack of a primary amine on the iminium moiety of a heteroaromatic ring system and cyclization to form a five-membered ring, followed by hydride loss to yield a rearomatized dihydro-1H-imidazo[1,2-f]phenanthridinium derivative. A range of NMR phase transfer experiments were carried out to elucidate the mechanistic pathway, and the methodology has been further developed by means of a biphasic system using N-bromosuccinimide as a co-oxidizing agent. The method has also been extended to other N-heterocyclic cation derivatives such as quinolinium and quinazolinium.