The construction of small-molecule libraries with high skeletal diversity and biological relevancy is an invaluable resource to discover new therapeutics and chemical modulators (Dandapani and Marcaurelle, 2010; Galloway et al., 2010; O’ Connor et al., 2011). In this respect, we have developed a privileged substructure-based diversity-oriented synthesis (pDOS) strategy over the last decade. The pDOS strategy focuses on creatively reconstructing heterocyclic moieties around the biologically relevant privileged substructures through diversity-generating reactions efficiently (Oh and Park, 2011; Kim et al., 2014). Continuing our endeavor to develop novel pDOS pathways, we recently focused on pyrimidine as a valuable privileged substructure since pyrimidine and its analogs have been extensively used as nucleotide analogs. Although pyrimidine is a well-known molecular framework ensuring good bioactivity, previously reported pyrimidine-containing compounds are mostly monocycles or flat bicycles with limited structural diversity (Wakeling et al., 2002; Yaziji et al., 2011; Lawrence et al., 2012). To overcome this structural limitation, we have developed several pDOS pathways to construct unprecedented pyrimidine-embedded polyheterocycles (Kim et al., 2013; Kim et al., 2016; Choi et al., 2019) and identified a list of small-molecule modulators controlling the cellular contents of lipid droplets (Choi et al., 2015), inhibiting the protein-protein interaction (Kim et al., 2016), and regulating tau proteostasis (Shin et al., 2021).

This report proposed a new pDOS strategy for pyrimidine-containing polyheterocycles with conformational diversity. Either rigid or flexible conformation of small molecules can be critical for specific modulation of 3-dimensional ligand-protein interactions. As the overall orientation of crucial substituents can be pre-organized in the conformationally constrained molecular skeletons, they might exhibit specific interaction toward biological targets with high affinity and selectivity. On the other hand, flexible molecules of which different conformations exist in equilibrium enable distinct binding modes for different proteins, which rigid molecules cannot achieve (Becker et al., 2000; Rubin and Qvit, 2016). In this regard, the chemical library with diverse skeletal flexibility would be a valuable resource for chemical biology and drug discovery community to investigate the influences of skeletal flexibility on the biological system.

Although bioactive medium/macro- and bridged cyclic scaffolds are frequently found in many natural products with conformational diversity (Figure 1A), they are scarce in the reported chemical libraries due to the limited synthetic accessibility (Gradillas and Pérez-Castells, 2006; Shiina, 2007; Hussain et al., 2014; Cheng-Sánchez et al., 2018; Mu et al., 2020). To visualize the conformational diversity of core scaffolds, we conducted a conformational analysis of bioactive natural products shown in Figure 1A. Among the searched conformers of these natural products containing 8- to 15- member rings, we selected the conformers of which Boltzmann population is higher than 2.5% at room temperature and displayed the distribution of the chosen conformers on the bubble chart (Figure 1B, please see the Supplementary Material for detailed search method and raw data in a tabular form). The size of the circle represents the population of the corresponding conformer. As a result, the natural products containing medium-sized rings and macrocycles are relatively flexible with various low-energy conformations. On the other hand, the natural products with bridged cycles are more constrained with a lower number of possible conformers. Although these natural products show apparent conformational diversity, it is impossible to compare their biological activities in response to their skeletal flexibility because these molecules are not directly comparable with different structures.

To construct a set of small molecules that allows comparable skeletal flexibility, we paid attention to medium/macro- and bridged cyclic scaffolds sharing similar structural motifs. Therefore, we proposed an efficient skeletal transformation strategy encompassing medium/macro- and bridged rings (Figure 2A). The rigid polyheterocyclic starting scaffolds are composed of 6,5,6- and 6,5,7-membered tricyclic rings (1) that can be easily accessed from the reaction of functionalized 4-formyl pyrimidines with cyclic hydrazines. The cleavage of the central N–N bond in azatricycles (1) would afford relatively flexible 9- and 10-membered medium-sized rings (3). The subsequent intramolecular cyclization would convert them to conformationally restricted bridged cyclic skeletons (4). The resulting bridged cycles can be converted to conformationally labile 12- to 14-membered macrocycles (5) via selective C–C bond cleavage. The most significant advantage of this skeletal transformation strategy is that all respective products for each scaffold can be used as the starting materials for the following transformation. Therefore, we do not need to prepare the intermediate separately.

Before exploring each scaffold, we conducted a conformational analysis to investigate the skeletal flexibility of each scaffold as we did for the representative natural products in Figure 1B. As shown in Figure 2B, initial azatricycles (1) and bridged cyclic skeletons (4) were conformationally restricted in a single or a small number of possible conformations (see the Supplementary Material for the raw data in a tabular form). On the other hand, the medium-sized rings (3) and macrocycles (5) showed relative skeletal flexibility with multiple low-energy conformers depending on the ring size. The conformational flexibility of each skeleton was also visualized through the overlay of selected conformers aligned by the pyrimidine moiety (Figure 2C). From this chemoinformatic analysis, we envisioned that this set of polyheterocycles sharing pyrimidine moiety with conformational diversity could be a valuable resource to investigate how skeletal flexibility influences their biological functions.

In conclusion, we constructed a small collection of structurally diverse pyrimidine-embedded medium/macro- and bridged cyclic molecules with unique conformational flexibility. We envisioned that building a set of pyrimidine-containing polyheterocycles with skeletal diversity could be a valuable resource to investigate how differences in skeletal flexibility influence the function of the molecules. Using skeletal transformation strategy, we accessed conformationally flexible 9- to 14-member medium/macrocycles as well as rigid tricycles and bridged cycles sharing pyrimidine moiety. Conformational analysis showed that all synthesized molecules in this study have broad coverage of 3D molecular shapes in PMI, unlike the known bioactive pyrimidine molecules. Therefore, we are confident that this collection of pyrimidine-embedded polyheterocycles would serve as a valuable resource in exploring unique biological activities unattainable with previous pyrimidine compounds.