Design and Synthesis of 56 Shape-Diverse 3D Fragments.

Fragment-based drug discovery is now widely adopted for lead generation in the pharmaceutical industry. However, fragment screening collections are often predominantly populated with flat, 2D molecules. Herein, we describe a workflow for the design and synthesis of 56 3D disubstituted pyrrolidine and piperidine fragments that occupy under-represented areas of fragment space (as demonstrated by a principal moments of inertia (PMI) analysis). A key, and unique, underpinning design feature of this fragment collection is that assessment of fragment shape and conformational diversity (by considering conformations up to 1.5 kcal mol-1 above the energy of the global minimum energy conformer) is carried out prior to synthesis and is also used to select targets for synthesis. The 3D fragments were designed to contain suitable synthetic handles for future fragment elaboration. Finally, by comparing our 3D fragments with six commercial libraries, it is clear that our collection has high three-dimensionality and shape diversity.

A fragment-based approach leading to the discovery of a novel binding site and the selective CK2 inhibitor CAM4066.

Recently we reported the discovery of a potent and selective CK2α inhibitor CAM4066. This compound inhibits CK2 activity by exploiting a pocket located outside the ATP binding site (αD pocket). Here we describe in detail the journey that led to the discovery of CAM4066 using the challenging fragment linking strategy. Specifically, we aimed to develop inhibitors by linking a high-affinity fragment anchored in the αD site to a weakly binding warhead fragment occupying the ATP site. Moreover, we describe the remarkable impact that molecular modelling had on the development of this novel chemical tool. The work described herein shows potential for the development of a novel class of CK2 inhibitors.

Asymmetric catalysis on the nanoscale: the organocatalytic approach to helicenes.

The first asymmetric organocatalytic synthesis of helicenes is reported. A novel SPINOL-derived phosphoric acid, bearing extended π-substituents, catalyzes the asymmetric synthesis of helicenes through an enantioselective Fischer indole reaction. A variety of azahelicenes and diazahelicenes could be obtained with good to excellent yields and enantioselectivities.

Design of a Lead-Like Cysteine-Targeting Covalent Library and the Identification of Hits to Cys55 of Bfl-1.

Covalent hit identification is a viable approach to identify chemical starting points against difficult-to-drug targets. While most researchers screen libraries of <2k electrophilic fragments, focusing on lead-like compounds can be advantageous in terms of finding hits with improved affinity and with a better chance of identifying cryptic pockets. However, due to the increased molecular complexity, larger numbers of compounds (>10k) are desirable to ensure adequate coverage of chemical space. Herein, the approach taken to build a library of 12k covalent lead-like compounds is reported, utilizing legacy compounds, robust library chemistry, and acquisitions. The lead-like covalent library was screened against the antiapoptotic protein Bfl-1, and six promising hits that displaced the BIM peptide from the PPI interface were identified. Intriguingly, X-ray crystallography of lead-like compound 8 showed that it binds to a previously unobserved conformation of the Bfl-1 protein and is an ideal starting point for the optimization of Bfl-1 inhibitors.

Development of a Series of Pyrrolopyridone MAT2A Inhibitors.

The optimization of an allosteric fragment, discovered by differential scanning fluorimetry, to an in vivo MAT2a tool inhibitor is discussed. The structure-based drug discovery approach, aided by relative binding free energy calculations, resulted in AZ'9567 (21), a potent inhibitor in vitro with excellent preclinical pharmacokinetic properties. This tool showed a selective antiproliferative effect on methylthioadenosine phosphorylase (MTAP) KO cells, both in vitro and in vivo, providing further evidence to support the utility of MAT2a inhibitors as potential anticancer therapies for MTAP-deficient tumors.

Enantioselective α-hydroxylation of ß-ketoamides.

The first enantioselective α-hydroxylation reaction of α-substituted ß-ketoamides has been developed by using the commercially available hydroquinine/TBHP system. The tertiary alcohols are obtained in good to high yield and up to 83% ee, which can be improved by a single crystallization.

Trends in Molecular Properties, Bioavailability, and Permeability across the Bayer Compound Collection.

For oral drugs, medicinal chemists aim to design compounds with high oral bioavailability, of which permeability is a key determinant. Taking advantage of >2000 compounds tested in rat bioavailability studies and >20,000 compounds tested in Caco2 assays at Bayer, we have examined the molecular properties governing bioavailability and permeability. In addition to classical parameters such as logD and molecular weight, we also investigated the relationship between calculated pKa and permeability. We find that neutral compounds retain permeability up to a molecular weight limit of 700, while stronger acids and bases are restricted to weights of 400-500. We also investigate trends for common properties such as hydrogen bond donors and acceptors, polar surface area, aromatic ring count, and rotatable bonds, including compounds which exceed Lipinski's rule of five (Ro5). These property-structure relationships are combined to provide design guidelines for bioavailable drugs in both traditional and "beyond rule of 5" (bRo5) chemical space.

A fragment-based approach leading to the discovery of inhibitors of CK2α with a novel mechanism of action.

CK2 is a ubiquitous protein kinase with an anti-apoptotic role and is found to be overexpressed in multiple cancer types. To this end, the inhibition of CK2 is of great interest with regard to the development of novel anti-cancer therapeutics. ATP-site inhibition of CK2 is possible; however, this typically results in poor selectivity due to the highly conserved nature of the catalytic site amongst kinases. An alternative methodology for the modulation of CK2 activity is through allosteric inhibition. The recently identified αD site represents a promising binding site for allosteric inhibition of CK2α. The work presented herein describes the development of a series of CK2α allosteric inhibitors through iterative cycles of X-ray crystallography and enzymatic assays, in addition to both fragment growing and fragment merging design strategies. The lead fragment developed, fragment 8, exhibits a high ligand efficiency, displays no drop off in activity between enzymatic and cellular assays, and successfully engages CK2α in cells. Furthermore, X-ray crystallographic analysis provided indications towards a novel mechanism of allosteric inhibition through αD site binding. Fragments described in this paper therefore represent promising starting points for the development of highly selective allosteric CK2 inhibitors.

Exploration of piperidine 3D fragment chemical space: synthesis and 3D shape analysis of fragments derived from 20 regio- and diastereoisomers of methyl substituted pipecolinates.

Fragment-based drug discovery is now widely adopted for lead generation in the pharmaceutical industry. However, fragment screening collections are often predominantly populated with flat, 2D molecules. Herein, we report the synthesis of piperidine-based 3D fragment building blocks - 20 regio- and diastereoisomers of methyl substituted pipecolinates using simple and general synthetic methods. cis-Piperidines, accessed through a pyridine hydrogenation were transformed into their trans-diastereoisomers using conformational control and unified reaction conditions. Additionally, diastereoselective lithiation/trapping was utilised to access trans-piperidines. Analysis of a virtual library of fragments derived from the 20 cis- and trans-disubstituted piperidines showed that it consisted of 3D molecules with suitable molecular properties to be used in fragment-based drug discovery programs.

Organocatalytic stereoselective epoxidation of trisubstituted acrylonitriles.

The first diastereospecific and enantioselective epoxidation of trans-2-aroyl-3-arylacrylonitriles by means of the commercially available diaryl L-prolinol/tert-butyl hydroperoxide system has been developed. These diversely functionalized epoxides were obtained in excellent yield (up to 99%), complete diastereoselectivity for the trans-isomer, and good enantioselectivity (up to 84% ee). Highly enantioenriched epoxides can be easily obtained after a single crystallization (ee > 90%).

Fragment-Based Design of a Potent MAT2a Inhibitor and in Vivo Evaluation in an MTAP Null Xenograft Model.

MAT2a is a methionine adenosyltransferase that synthesizes the essential metabolite S-adenosylmethionine (SAM) from methionine and ATP. Tumors bearing the co-deletion of p16 and MTAP genes have been shown to be sensitive to MAT2a inhibition, making it an attractive target for treatment of MTAP-deleted cancers. A fragment-based lead generation campaign identified weak but efficient hits binding in a known allosteric site. By use of structure-guided design and systematic SAR exploration, the hits were elaborated through a merging and growing strategy into an arylquinazolinone series of potent MAT2a inhibitors. The selected in vivo tool compound 28 reduced SAM-dependent methylation events in cells and inhibited proliferation of MTAP-null cells in vitro. In vivo studies showed that 28 was able to induce antitumor response in an MTAP knockout HCT116 xenograft model.

Second-generation CK2α inhibitors targeting the αD pocket.

CK2 is a critical cell cycle regulator that also promotes various anti-apoptotic mechanisms. Development of ATP-non-competitive inhibitors of CK2 is a very attractive strategy considering that the ATP binding site is highly conserved among other kinases. We have previously utilised a pocket outside the active site to develop a novel CK2 inhibitor, CAM4066. Whilst CAM4066 bound to this new pocket it was also interacting with the ATP site: herein, we describe an example of a CK2α inhibitor that binds completely outside the active site. This second generation αD-site binding inhibitor, compound CAM4712 (IC50 = 7 µM, GI50 = 10.0 ± 3.6 µM), has numerous advantages over the previously reported CAM4066, including a reduction in the number of rotatable bonds, the absence of amide groups susceptible to the action of proteases and improved cellular permeability. Unlike with CAM4066, there was no need to facilitate cellular uptake by making a prodrug. Moreover, CAM4712 displayed no drop off between its ability to inhibit the kinase in vitro (IC50) and the ability to inhibit cell proliferation (GI50).

Specific inhibition of CK2α from an anchor outside the active site.

The development of selective inhibitors of protein kinases is challenging because of the significant conservation of the ATP binding site. Here, we describe a new mechanism by which the protein kinase CK2α can be selectively inhibited using features outside the ATP site. We have identified a new binding site for small molecules on CK2α adjacent to the ATP site and behind the αD loop, termed the αD pocket. An elaborated fragment anchored in this site has been linked with a low affinity fragment binding in the ATP site, creating a novel and selective inhibitor (CAM4066) that binds CK2α with a Kd of 320 nM and shows significantly improved selectivity compared to other CK2α inhibitors. CAM4066 shows target engagement in several cell lines and similar potency to clinical trial candidate CX4945. Our data demonstrate that targeting a poorly conserved, cryptic pocket allows inhibition of CK2α via a novel mechanism, enabling the development of a new generation of selective CK2α inhibitors.

Noncovalent organocatalytic synthesis of enantioenriched terminal aziridines with a quaternary stereogenic center.

A high-yielding and enantioselective access to novel N-Boc terminal aziridines, bearing a quaternary stereogenic center, has been developed via an aza-Michael initiated ring-closure (aza-MIRC) reaction of α-acyl acrylates with an N-tosyloxy tert-butyl carbamate catalyzed by a chiral amino thiourea. The feasibility of the aziridine regioselective ring-opening to valuable α,α-disubstituted α-amino acid esters has been demonstrated.

Hexafluorobenzene: a powerful solvent for a noncovalent stereoselective organocatalytic Michael addition reaction.

A dramatic enhancement of the diastereo- and enantioselectivity in the nitro-Michael addition reaction organocatalysed by a commercially available α,α-L-diaryl prolinol was disclosed when performing the reaction in unconventional hexafluorobenzene as a medium. DFT calculations were performed to clarify the origin of stereoselectivity and the role of C(6)F(6).