Synthesis and structure‑activity‑relationship of 3,4‑Diaryl‑1H‑pyrrolo[2,3‑b]pyridines as irreversible Inhibitors of mutant EGFR‑L858R/T790M

Abstract

The epidermal growth factor receptor (EGFR) is a well‑validated drug target for the treatment of non‑small cell lung cancer. Here we present an optimization approach and preliminary structure‑activity relationship for 1H‑pyrrolo[2,3‑b]pyridines as covalent irreversible mutant EGFR inhibitors. We synthesized a focused library to investigate the effect of different aromatic substituents in the 4‑position of this scaffold, interacting with the gatekeeper. We determined the activity of the synthesized compounds mutant EGFR enzyme assays and determined the selectivity over the wild type.

Introduction

The development of epidermal growth factor receptor (EGFR) inhibitors for the treatment of non‑small cell lung cancer (NSCLC) resulted in three generations of small molecular drugs in one decade (Fig. 1) (Russo et al., 2015). EGFR represents a well‑validated drug target for patients harboring so‑called activating mutations (Lynch et al., 2004; Gazdar, 2009). These mutations lead to a highly increased level of EGF signaling independent from extracellular stimuli. This results in an aberrant equilibrium between pro‑apoptotic and pro‑survival signals in malignant cells. Because the survival of these abnormal cells is highly dependent on the EGFR signaling pathway, EGFR inhibition leads to a rapid inhibition of tumor cell growth in vitro and tumor shrinkage in vivo (Pao et al., 2004; Takeda et al., 2014; Juchum et al., 2015). However, patients harboring wild type (wt) EGFR showed poor response rates under an anti EGFR therapy. The most frequently observed activating mutation in NSCLC patients is the single amino acid exchange L858R, located on exon 21 that codes for the tyrosine kinase domain of EGFR (Lynch et al., 2004; Kawahara et al., 2010). First generation EGFR inhibitors like gefitinib (N‑(3‑chloro‑4‑fluorophenyl)‑7‑methoxy‑6‑(3‑morpholinopropoxy)quinazolin‑4‑amine) are highly potent inhibitors of this particular kinase variant. However, during long‑term treatment, most of the initially responding patients develop secondary resistances to first generation EGFR inhibitors. The secondary point mutation T790 M is the leading cause for drug resistance in 60% of NSCLC patients (Pao et al., 2005; Yu et al., 2013). Second generation EGFR inhibitor afatinib ((S,E)‑N‑(4‑((3‑chloro‑4‑fluorophenyl)amino)‑7‑((tetrahydrofuran‑3‑yl)oxy)quinazolin‑6‑yl)‑4‑(dimethylamino)but‑2‑enamide) overcomes L858R/T790M resistance by a covalent irreversible interaction with the enzyme. Thus, this compound bears a reactive Michael acceptor group, an acrylamide, for the alkylation of a non‑catalytic cysteine (Cys797) (Kwak et al., 2005). Dose limiting toxicity compromised the clinical efficacy of afatinib. The potent inhibition of wt EGFR has been linked to the serious side effects of afatinib (Katakami et al., 2013; Miller et al., 2012). The recently FDA approved third generation EGFR inhibitor osimertinib (N‑(2‑((2‑(dimethylamino)ethyl)(methyl)amino)‑4‑methoxy‑5‑((4‑(1‑methyl‑1H‑indol‑3‑yl)pyrimidin‑2‑yl)amino)phenyl)acrylamide) was designed to covalently inhibit L858R and L858R/T790M mutant EGFR, while sparing the wild type (Cross et al., 2014). Osimertinib inhibits EGFR‑L858R/T790M with subnanomolar IC₅₀ values and the wt EGFR inhibition in the literature ranges from ~1 to 184 nM, depending on the assay format (Cross et al., 2014; Gunther et al., 2017; Finlay et al., 2014). Even though dose limiting toxicity could be reduced for osimertinib when compared with afatinib, this third generation EGFR inhibitor still shows the typical wt associated EGFR inhibitor side effects in up to 30% of treated patients, namely skin rash, neutropenia, leukopenia and diarrhea (Lamb and Scott, 2017). The active metabolite AZ5104 (N‑[2‑[[2‑(dimethylamino)ethyl]methylamino]‑5‑[[4‑(1H‑indol‑3‑yl)‑2‑pyrimidinyl]amino]‑4‑methoxyphenyl]‑2‑propenamide), which is the N‑indole‑demethylated derivative of osimertinib is less selective and displays a potent inhibitor of wt EGFR (15‑fold more potent than osimertinib), which partially explains the adverse effects of osimertinib, observed in clinical trials (Gao et al., 2016; Chu et al., 2018).

Here we present a rational design approach of novel covalent irreversible EGFR inhibitors towards improved activity and selectivity versus wild type, employing the 1H‑pyrrolo[2,3‑b]pyridine scaffold as a hinge binder. We previously described trisubstituted imidazoles bearing the 1H‑pyrrolo[2,3‑b]pyridine group to be a valuable scaffold to act as bidentate hinge binders in EGFR (e.g. MJ‑341 (N‑(3‑(4‑(4‑(4‑fluorophenyl)‑2‑(3‑hydroxypropyl)‑1H‑imidazol‑5‑yl)‑1H‑pyrrolo[2,3‑b]pyridin‑3‑yl)phenyl)acrylamide), Fig. 1) (Juchum et al., 2017). We found the 3‑position of the hinge binding heterocycle to be a suitable attachment point for a Michael acceptor linked via phenyl residues in order to covalently bind Cys797. MJ‑277 (N‑(3‑(4‑(4‑(4‑fluorophenyl)‑2‑(3‑hydroxypropyl)‑1H‑imidazol‑5‑yl)‑1H‑pyrrolo[2,3‑b]pyridin‑3‑yl)phenyl)acrylamide) served as a new hit compound for the development of EGFR inhibitors with decreased molecular weight when compared with the structure class of MJ‑341 and promising biological activity. We could develop highly potent compounds with this chemotype for EGFR‑L858R/T790M and for EGFR‑L858R/T790M/C797S in the past (Juchum et al., 2017). However, we could not improve the selectivity over EGFR wild type. In the present work, we further synthesized a focused library of closely related compounds decorated with different substituents in the 4‑position in order to improve the inhibitory activity and the selectivity over EGFR wild type. It has been recently shown that placing aromatic residues between the gatekeeper Met790 residue and Lys745 can be used to increase EGFR‑L858R/T790M activity while maintaining selectivity versus wild type (Tomassi et al., 2017). Docking predicted the 4‑position of the 1H‑pyrrolo[2,3‑b]pyridine scaffold to be the most promising attachment point for substituents towards this direction (Fig. 2). Covalent docking of 17 and 19 predicted a bidentate hinge‑binding motif and orientation of the aromatic residues between Met790 and Lys745 but avoiding to fully occupy hydrophobic region I, which might be detrimental for selectivity. For 19, docking predicted an additional hydrogen bond between the protonated amine of Lys745 and the sulfonamide group (Fig. 2B) that might be beneficial for inhibitory activity.