Structure-Based Approach for the Discovery of Pyrrolo[3,2‑<i>d</i>]pyrimidine-Based EGFR T790M/L858R Mutant Inhibitors

The epidermal growth factor receptor (EGFR) family plays a critical role in vital cellular processes and in various cancers. Known EGFR inhibitors exhibit distinct antitumor responses against the various EGFR mutants associated with nonsmall-cell lung cancer. The L858R mutation enhances clinical sensitivity to gefitinib and erlotinib as compared with wild type and reduces the relative sensitivity to lapatinib. In contrast, the T790M mutation confers drug resistance to gefitinib and erlotinib. We determined crystal structures of the wild-type and T790M/L858R double mutant EGFR kinases with reversible and irreversible pyrrolo­[3,2-<i>d</i>]­pyrimidine inhibitors based on analogues of TAK-285 and neratinib. In these structures, M790 adopts distinct conformations to accommodate different inhibitors, whereas R858 allows conformational variations of the activation loop. These results provide structural insights for understanding the structure–activity relationships that should contribute to the development of potent inhibitors against drug-sensitive or -resistant EGFR mutations

Synthetic Studies on Centromere-Associated Protein‑E (CENP-E) Inhibitors: 2. Application of Electrostatic Potential Map (EPM) and Structure-Based Modeling to Imidazo[1,2‑<i>a</i>]pyridine Derivatives as Anti-Tumor Agents

To develop centromere-associated protein-E (CENP-E) inhibitors for use as anticancer therapeutics, we designed novel imidazo­[1,2-<i>a</i>]­pyridines, utilizing previously discovered 5-bromo derivative <b>1a</b>. By site-directed mutagenesis analysis, we confirmed the ligand binding site. A docking model revealed the structurally important molecular features for effective interaction with CENP-E and could explain the superiority of the inhibitor (<i>S</i>)-isomer in CENP-E inhibition vs the (<i>R</i>)-isomer based on the ligand conformation in the L5 loop region. Additionally, electrostatic potential map (EPM) analysis was employed as a ligand-based approach to optimize functional groups on the imidazo­[1,2-<i>a</i>]­pyridine scaffold. These efforts led to the identification of the 5-methoxy imidazo­[1,2-<i>a</i>]­pyridine derivative (+)-(<i>S</i>)-<b>12</b>, which showed potent CENP-E inhibition (IC₅₀: 3.6 nM), cellular phosphorylated histone H3 (p-HH3) elevation (EC₅₀: 180 nM), and growth inhibition (GI₅₀: 130 nM) in HeLa cells. Furthermore, (+)-(<i>S</i>)-<b>12</b> demonstrated antitumor activity (<i>T</i>/<i>C</i>: 40%, at 75 mg/kg) in a human colorectal cancer Colo205 xenograft model in mice

Design and Synthesis of Pyrrolo[3,2-<i>d</i>]pyrimidine Human Epidermal Growth Factor Receptor 2 (HER2)/Epidermal Growth Factor Receptor (EGFR) Dual Inhibitors: Exploration of Novel Back-Pocket Binders

To develop novel human epidermal growth factor receptor 2 (HER2)/epidermal growth factor receptor (EGFR) kinase inhibitors, we explored pyrrolo­[3,2-<i>d</i>]­pyrimidine derivatives bearing bicyclic fused rings designed to fit the back pocket of the HER2/EGFR proteins. Among them, the 1,2-benzisothiazole (<b>42m</b>) ring was selected as a suitable back pocket binder because of its potent HER2/EGFR binding and cell growth inhibitory (GI) activities and pseudoirreversibility (PI) profile as well as good bioavailability (BA). Ultimately, we arrived at our preclinical candidate <b>51m</b> by optimization of the <i>N</i>-5 side chain to improve CYP inhibition and metabolic stability profiles without a loss of potency (HER2/EGFR inhibitory activity, IC₅₀, 0.98/2.5 nM; and GI activity BT-474 cells, GI₅₀, 2.0 nM). Reflecting the strong <i>in vitro</i> activities, <b>51m</b> exhibited potent tumor regressive efficacy against both HER2- and EGFR-overexpressing tumor (4–1ST and CAL27) xenograft models in mice at oral doses of 50 mg/kg and 100 mg/kg

Discovery of Potent Mcl-1/Bcl-xL Dual Inhibitors by Using a Hybridization Strategy Based on Structural Analysis of Target Proteins

Mcl-1 and Bcl-xL are crucial regulators of apoptosis, therefore dual inhibitors of both proteins could serve as promising new anticancer drugs. To design Mcl-1/Bcl-xL dual inhibitors, we performed structure-guided analyses of the corresponding selective Mcl-1 and Bcl-xL inhibitors. A cocrystal structure of a pyrazolo­[1,5-<i>a</i>]­pyridine derivative with Mcl-1 protein was successfully determined and revealed the protein–ligand binding mode. The key structure for Bcl-xL inhibition was further confirmed through the substructural analysis of ABT-263, a representative Bcl-xL/Bcl-2/Bcl-w inhibitor developed by Abbott Laboratories. On the basis of the structural data from this analysis, we designed hybrid compounds by tethering the Mcl-1 and Bcl-xL inhibitors together. The results of X-ray crystallographic analysis of hybrid compound <b>10</b> in complexes with both Mcl-1 and Bcl-xL demonstrated its binding mode with each protein. Following further optimization, compound <b>11</b> showed potent Mcl-1/Bcl-xL dual inhibitory activity (Mcl-1, IC₅₀ = 0.088 μM; and Bcl-xL, IC₅₀ = 0.0037 μM)

Discovery of a Selective Kinase Inhibitor (TAK-632) Targeting Pan-RAF Inhibition: Design, Synthesis, and Biological Evaluation of <i>C</i>‑7-Substituted 1,3-Benzothiazole Derivatives

With the aim of discovering a selective kinase inhibitor targeting pan-RAF kinase inhibition, we designed novel 1,3-benzothiazole derivatives based on our thiazolo­[5,4-<i>b</i>]­pyridine class RAF/VEGFR2 inhibitor <b>1</b> and developed a regioselective cyclization methodology for the <i>C</i>-7-substituted 1,3-benzothiazole scaffold utilizing meta-substituted anilines. Eventually, we selected 7-cyano derivative <b>8B</b> (TAK-632) as a development candidate and confirmed its binding mode by cocrystal structure with BRAF. Accommodation of the 7-cyano group into the BRAF-selectivity pocket and the 3-(trifluoromethyl)­phenyl acetamide moiety into the hydrophobic back pocket of BRAF in the DFG-out conformation contributed to enhanced RAF potency and selectivity vs VEGFR2. Reflecting its potent pan-RAF inhibition and slow off-rate profile, <b>8B</b> demonstrated significant cellular activity against mutated <i>BRAF</i> or mutated <i>NRAS</i> cancer cell lines. Furthermore, in both A375 (<i>BRAF</i>^V600E) and HMVII (<i>NRAS</i>^Q61K) xenograft models in rats, <b>8B</b> demonstrated regressive antitumor efficacy by twice daily, 14-day repetitive administration without significant body weight loss

Design and Synthesis of Novel DFG-Out RAF/Vascular Endothelial Growth Factor Receptor 2 (VEGFR2) Inhibitors. 1. Exploration of [5,6]-Fused Bicyclic Scaffolds

To develop RAF/VEGFR2 inhibitors that bind to the inactive DFG-out conformation, we conducted structure-based drug design using the X-ray cocrystal structures of BRAF, starting from an imidazo­[1,2-<i>b</i>]­pyridazine derivative. We designed various [5,6]-fused bicyclic scaffolds (ring A, <b>1</b>–<b>6</b>) possessing an anilide group that forms two hydrogen bond interactions with Cys532. Stabilizing the planarity of this anilide and the nitrogen atom on the six-membered ring of the scaffold was critical for enhancing BRAF inhibition. The selected [1,3]­thiazolo­[5,4-<i>b</i>]­pyridine derivative <b>6d</b> showed potent inhibitory activity in both BRAF and VEGFR2. Solid dispersion formulation of <b>6d</b> (<b>6d-SD</b>) maximized its oral absorption in rats and showed significant suppression of ERK1/2 phosphorylation in an A375 melanoma xenograft model in rats by single administration. Tumor regression (<i>T</i>/<i>C</i> = −7.0%) in twice-daily repetitive studies at a dose of 50 mg/kg in rats confirmed that <b>6d</b> is a promising RAF/VEGFR2 inhibitor showing potent anticancer activity

Design and Synthesis of Potent Inhibitor of Apoptosis (IAP) Proteins Antagonists Bearing an Octahydropyrrolo[1,2‑<i>a</i>]pyrazine Scaffold as a Novel Proline Mimetic

To develop novel inhibitor of apoptosis (IAP) proteins antagonists, we designed a bicyclic octahydropyrrolo­[1,2-<i>a</i>]­pyrazine scaffold as a novel proline bioisostere. This design was based on the X-ray co-crystal structure of four N-terminal amino acid residues (AVPI) of the second mitochondria-derived activator of caspase (Smac) with the X-chromosome-linked IAP (XIAP) protein. Lead optimization of this scaffold to improve oral absorption yielded compound <b>45</b>, which showed potent cellular IAP1 (cIAP1 IC₅₀: 1.3 nM) and XIAP (IC₅₀: 200 nM) inhibitory activity, in addition to potent tumor growth inhibitory activity (GI₅₀: 1.8 nM) in MDA-MB-231 breast cancer cells. X-ray crystallographic analysis of compound <b>45</b> bound to XIAP and to cIAP1 was achieved, revealing the various key interactions that contribute to the higher cIAPI affinity of compound <b>45</b> over XIAP. Because of its potent IAP inhibitory activities, compound <b>45</b> (T-3256336) caused tumor regression in a MDA-MB-231 tumor xenograft model (T/C: −53% at 30 mg/kg)