New bioactive small molecules are urgently required to seed both medicinal chemistry and chemical biology research. Within the two last decades, however, organic synthesis has been identified as a limiting factor in this regard owing to the historically uneven exploration of chemical space. Whilst fragment-based screening has proven a fruitful strategy for identifying novel bioactive small molecules, in a similar vein to high-throughput screening approaches, obstacles within this paradigm have been recognised. This relates to a lack of fragment novelty, the overrepresentation of sp2-rich compounds and the lack of readily accessible exit vectors currently demonstrated by commercial screening collections. Thus, the development of novel screening libraries that address these deficiencies is therefore of significant importance. Moreover, innovative strategies are required to aid the somewhat challenging hit validation phase. In this context, the development of high-throughput platforms to accelerate the synthesis of derivatives could provide the opportunity to lower the economic impact of this process.

This thesis seeks to explore how diversity-oriented synthesis can be leveraged to tackle these issues. To populate new areas of fragment space, in the first chapter we utilise an α,α-disubstituted amino alcohol building block to generate 12 novel three-dimensional fragments containing an underrepresented N-substituted quaternary carbon moiety. These molecules ultimately contributed to a screening library of 40 compounds featuring this key motif. Importantly, throughout the library medicinally relevant functionalities and modifiable substituents were installed to enable biological recognition and three-dimensional fragment growth. Finally, cheminformatic analysis demonstrated the broad molecular shape diversity of the library and adherence of the library to commonly adopted guidelines within the field. In addition, the library was also shown to compare favourably with existing commercial collections, exhibiting a higher number of chiral centres, a lower fraction aromatic and an improved molecular shape distribution.

In the second chapter, we demonstrate the ability of this library to deliver novel hits against challenging biological targets and facilitate multidirectional fragment growth. To achieve this, state-of-the-art high-throughput X-ray crystallographic fragment screening was conducted, leading to the identification of six hits against three novel biological targets (CFI25, Activin A and Penicillin-binding protein 3). Utilising the modular nature of the synthetic route developed, derivatives of each hit were readily generated interrogating several synthetic exit vectors, including the N-substituted quaternary centre. In turn, these analogues were successfully employed as a hit validation strategy, thus providing novel starting points for drug discovery or chemical probe development.

Finally, the third chapter describes investigations toward the coupling of rapid analogue construction with the XChem screening platform to provide a direct structural-binding readout via screening of crude reaction mixtures. In this section, we demonstrate the applicability of this library toward high-throughput derivative synthesis using solid-phase catalysis to generate 78 analogues in less than one week. In addition, these studies formed the basis of preliminary investigations toward the development of methodology to enable click chemistry to be implemented within the crude screening protocol.

Together, these findings demonstrate the effective nature of structurally diverse N-substituted quaternary small molecules within a fragment-based screening context. Herein, we demonstrate libraries of this nature can provide novel three-dimensional scaffolds to seed screening campaigns and ultimately deliver novel starting points for drug discovery.