An underlying goal of drug discovery is to develop safe and stable substances that specifically target essential elements that cause disease. Molecular chirality adds an additional level of specificity and complexity in achieving this objective, as mirror image molecules are distinct substances and must be treated as such. Classical chiral-center enantiomers ( Figure 1A) have been shown to differ significantly in biological activity, pharmacodynamics, pharmacokinetics, and toxicity. 1 The cases of thalidomide 2 and perhexiline, 3 whose enantiomers differ dramatically with respect to toxicity and metabolic properties, emphasize the importance of addressing stereochemistry in drug development.In this Perspective, we address the pharmaceutical implications of a largely overlooked alternative source of drug chirality, atropisomerism, 4 which has the distinct feature of creating molecular chirality as a result of hindered rotation about a bond axis ( Figure 1B). Figure 1C shows space-filling models where it is evident that rotation about the vertical axis is hindered because of steric clashes between the bulky R1 and R2 groups with R3 and R4.Unlike compounds with classical chiral centers, which are often stable and which racemize via a bond breaking and making process, atropisomers racemize via an intramolecular dynamic process that only involves bond rotation. As bond rotation is time-dependent, racemization half-lives for atropisomers can vary dramatically between minutes to years, depending on the degree of steric hindrance, electronic influences, temperature, solvent, etc. Because of this time-dependent feature, drug discovery campaigns can become more complex, or may even be abandoned, when atropisomeric properties are observed. Atropisomerism frequently results as researchers strive to design more compact and conformationally constrained inhibitors. Even for courageous design and synthetic campaigns that attempt to develop atropisomeric compounds, important differences in properties have been reported for enantiomeric pairs, such as in vitro inhibition, crystallization, in vivo racemization rates, and absorption, distribution, metabolism, excretion, and toxicity (ADMET) properties. There are also examples of compounds that were unknowingly developed as a racemic mixture of atropisomers and required chiral detection experiments to finally reveal their existence. Overall, many view atropisomer chirality as a lurking menace with the potential to increase the cost of pharmaceutical research and development and to derail drug Figure 1. (A) Mirror-image enantiomers S and R arise from a classical chiral center (atom). (B) Other enantiomers S a and R a can arise from hindered rotation that creates a chiral axis. (C) Atropisomeric enantiomers S a and R a are shown as space-filling models. Reproduced with permission from ChemMedChem (LaPlante, S. R.; Edwards, P. J.; Fader, L. D.; Jakalian, A.; Hucke, O. Revealing atropisomer axial chirality in drug discovery. 2011, 6, 505À513,

An often overlooked source of chirality is atropisomerism, which results from slow rotation along a bond axis due to steric hindrance and/or electronic factors. If undetected or not managed properly, this time-dependent chirality has the potential to lead to serious consequences, because atropisomers can be present as distinct enantiomers or diastereoisomers with their attendant different properties. Herein we introduce a strategy to reveal and classify compounds that have atropisomeric chirality. Energy barriers to axial rotation were calculated using quantum mechanics, from which predicted high barriers could be experimentally validated. A calculated rotational energy barrier of 20 kcal mol(-1) was established as a suitable threshold to distinguish between atropisomers and non-atropisomers with a prediction accuracy of 86%. This methodology was applied to subsets of drug databases in the course of which atropisomeric drugs were identified. In addition, some drugs were exposed that were not yet known to have this chiral attribute. The most valuable utility of this tool will be to predict atropisomerism along the drug discovery pathway. When used in concert with our compound classification scheme, decisions can be made during early discovery stages such as "hit-to-lead" and "lead optimization," to foresee and validate the presence of atropisomers and to exercise options of removing, further stabilizing, or rendering the chiral axis of interest more freely rotatable via SAR design, thereby decreasing this potential liability within a compound series. The strategy can also improve drug development plans, such as determining whether a drug or series should be developed as a racemic mixture or as an isolated single compound. Moreover, the work described herein can be extended to other chemical fields that require the assessment of potential chiral axes.

An assay recapitulating the 3′ processing activity of HIV-1 integrase (IN) was used to screen the Boehringer Ingelheim compound collection. Hit-to-lead and lead optimization beginning with compound 1 established the importance of the C3 and C4 substituent to antiviral potency against viruses with different aa124/ aa125 variants of IN. The importance of the C7 position on the serum shifted potency was established. Introduction of a quinoline substituent at the C4 position provided a balance of potency and metabolic stability. Combination of these findings ultimately led to the discovery of compound 26 (BI 224436), the first NCINI to advance into a phase Ia clinical trial.

The emergence of resistance to existing classes of antiretroviral drugs necessitates finding new HIV-1 targets for drug discovery. The viral capsid (CA) protein represents one such potential new target. CA is sufficient to form mature HIV-1 capsids in vitro, and extensive structure-function and mutational analyses of CA have shown that the proper assembly, morphology, and stability of the mature capsid core are essential for the infectivity of HIV-1 virions. Here we describe the development of an in vitro capsid assembly assay based on the association of CA-NC subunits on immobilized oligonucleotides. This assay was used to screen a compound library, yielding several different families of compounds that inhibited capsid assembly. Optimization of two chemical series, termed the benzodiazepines (BD) and the benzimidazoles (BM), resulted in compounds with potent antiviral activity against wild-type and drug-resistant HIV-1. Nuclear magnetic resonance (NMR) spectroscopic and X-ray crystallographic analyses showed that both series of inhibitors bound to the N-terminal domain of CA. These inhibitors induce the formation of a pocket that overlaps with the binding site for the previously reported CAP inhibitors but is expanded significantly by these new, more potent CA inhibitors. Virus release and electron microscopic (EM) studies showed that the BD compounds prevented virion release, whereas the BM compounds inhibited the formation of the mature capsid. Passage of virus in the presence of the inhibitors selected for resistance mutations that mapped to highly conserved residues surrounding the inhibitor binding pocket, but also to the C-terminal domain of CA. The resistance mutations selected by the two series differed, consistent with differences in their interactions within the pocket, and most also impaired virus replicative capacity. Resistance mutations had two modes of action, either directly impacting inhibitor binding affinity or apparently increasing the overall stability of the viral capsid without affecting inhibitor binding. These studies demonstrate that CA is a viable antiviral target and demonstrate that inhibitors that bind within the same site on CA can have distinct binding modes and mechanisms of action.

BI 224436 is an HIV-1 integrase inhibitor with effective antiviral activity that acts through a mechanism that is distinct from that of integrase strand transfer inhibitors (INSTIs). This 3-quinolineacetic acid derivative series was identified using an enzymatic integrase long terminal repeat (LTR) DNA 3=-processing assay. A combination of medicinal chemistry, parallel synthesis, and structure-guided drug design led to the identification of BI 224436 as a candidate for preclinical profiling. It has antiviral 50% effective concentrations (

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Ataxia telangiectasia and Rad3-related (ATR) kinase protects genome integrity during DNA replication. RP-3500 is a novel, orally bioavailable clinical-stage ATR kinase inhibitor (NCT04497116). RP-3500 is highly potent with IC 50 values of 1.0 and 0.33 nM in biochemical and cell-based assays, respectively. RP-3500 is highly selective for ATR with 30-fold selectivity over mTOR and >2,000-fold selectivity over ATM, DNA-PK, and PI3Kkinases. In vivo, RP-3500 treatment results in potent single-agent efficacy and/or tumor regression in multiple xenograft models at minimum effective doses (MED) of 5-7 mg/kg once daily. Pharmacodynamic assessments validate target engagement, with dose-proportional tumor pCHK1 inhibition (IC 80 = 18.6 nM) and induction of -H2AX, pDNA-PKcs, and pKAP1. RP-3500 exposure at MED indicates that circulating free plasma levels above the in vivo tumor IC 80 for 10-12 hours are sufficient for efficacy on a continuous schedule. However, short-duration intermittent (weekly 3 days on/4 days off) dosing schedules as monotherapy or given concomitantly with reduced doses of olaparib or niraparib, maximize tumor growth inhibition while minimizing the impact on red blood cell depletion, emphasizing the reversible nature of erythroid toxicity with RP-3500 and demonstrating superior efficacy compared with sequential treatment. These results provide a strong preclinical rationale to support ongoing clinical investigation of the novel ATR inhibitor, RP-3500, on an intermittent schedule as a monotherapy and in combination with PARP inhibitors as a potential means of maximizing clinical benefit.