Discovery of N-((3R,4R)-4-Fluoro-1-(6-((3-methoxy-1-methyl-1H-pyrazol-4-yl)amino)-9-methyl-9H-purin-2-yl)pyrrolidine-3-yl)acrylamide (PF-06747775) through Structure-Based Drug Design: A High Affinity Irreversible Inhibitor Targeting Oncogenic EGFR Mutants with Selectivity over Wild-Type EGFR

Abstract

Mutant epidermal growth factor receptor (EGFR) is a major driver of non-small-cell lung cancer (NSCLC). Marketed first generation inhibitors, such as erlotinib, effect a transient beneficial response in EGFR mutant NSCLC patients before resistance mechanisms render these inhibitors ineffective. Secondary oncogenic EGFR mutations account for approximately 50% of relapses, the most common being the gatekeeper T790M substitution that renders existing therapies ineffective. The discovery of PF-06459988 (1), an irreversible pyrrolopyrimidine inhibitor of EGFR T790M mutants, was recently disclosed.1 Herein, we describe our continued efforts to achieve potency across EGFR oncogenic mutations and improved kinome selectivity, resulting in the discovery of clinical candidate PF-06747775 (21), which provides potent EGFR activity against the four common mutants (exon 19 deletion (Del), L858R, and double mutants T790M/L858R and T790M/Del), selectivity over wild-type EGFR, and desirable ADME properties. Compound 21 is currently being evaluated in phase-I clinical trials of mutant EGFR driven NSCLC.

Proteome-wide Map of Targets of T790M-EGFR-Directed Covalent Inhibitors

“…Each inhibitor and probe pair ( 1 / 4 , 2 / 5 , 3 / 6 ) displayed similar potency for inhibition of phosphorylated EGFR (pY1068) in H1975 cells, which express L858R/T790M-EGFR, and equivalent intrinsic reactivity in a conjugation assay with glutathione (GSH) (Table S1). Moreover, competitive ABPP assays with probes 4–6 provided estimates of inhibitor 1–3 cross-reactivity with WT-EGFR, as measured in A431 cells, that matched those determined using pY1068-EGFR measurements (Table S2 and Figure S2A) and were generally consistent with other literature reports, where inhibitors 1 and 2 have been found to cross-react to a greater extent with WT-EGFR than inhibitor 3 (Planken et al, 2017). Finally, probes 4–6 showed much weaker labeling of WT-EGFR in A431 cells compared to probe 7 (Figure S2B), which was based on a second-generation inhibitor with potent WT-EGFR activity (Lanning et al, 2014).…”

“…This limitation could explain, for instance, why some of the kinase off-targets reported previously for inhibitor 2 , such as FER and TNK2 (Planken et al, 2017) were not detected in our studies. Likewise, we only evaluated a single cell line in this study and additional off-targets reported will likely be uncovered by extending our chemical proteomic analysis to other cell/tissue types (e.g., some of the other reported kinase off-targets of inhibitor 2 – ERBB4, BMX, BTK, and TXK – show very low or negligible expression in H1975 cells (Barretina et al, 2012, Klijn et al, 2015)).…”

“…Here, we sought to extend these initial findings by determining the proteome-wide reactivity of third-generation inhibitors for EGFR designed to selectively and irreversibly target the T790M drug-resistant mutant form of this kinase. All three inhibitors investigated – the recently approved osimertinib (Tagrisso, AZD9291; 1 , Figure 1A), PF-06747775 ( 2 , Figure 1A) and rociletinib (CO-1686; 3 , Figure 1A) – were optimized for an EGFR inhibition profile that maintained good potency against the two original drug-sensitive, kinase-activating mutants (exon19Del and L858R) and the two major drug-resistant T790M mutants (exon19Del/T790M and L858R/T790M), while showing reduced activity against wild type (WT)-EGFR to minimize mechanism-based toxicity (Cross et al, 2014, Finlay et al, 2014, Tjin Tham Sjin et al, 2014, Walter et al, 2013, Planken et al, 2017). Our chemical proteomic studies reveal that, despite the highly engineered EGFR mutant inhibition profile achieved by all three third-generation inhibitors and their shared unsubstituted acrylamide reactive group, the inhibitors exhibited strikingly distinct proteome-wide reactivity profiles in human cancer cells.…”

Third generation EGFR TKIs: current data and future directions

“…It has potent pre-clinical EGFR activity against the four common mutants (exon 19 deletion (Del), L858R, and double mutants T790 M/L858R and T790 M/Del), selectivity over wild-type EGFR [27]. …”

The synthesis and the antimicrobial activity of N1-substituted 5-amino-4-arylsulfonyl-3-N-phenylaminopyrazoles

“…For example, Ceftolozane is a cephalosporin antibiotic of the 5th generation [1], Tozasertib is a pan-Aurora inhibitor [2]. Polysubstituted derivatives of pyrazole can act as antibacterial [3][4][5][6], anti-inflammatory [3,7,8], cytostatic [2,9,10], anesthetizing [11], anticancer [12][13][14][15], antiepileptic agents [16][17][18] and as insecticides [19,20].…”