First investigation of numerous membered ether linked macroc

First, investigation of numerous 12-14-membered ether-linked macrocycles revealed that smaller motifs were most lipophilic efficient. Compound (3) (12-membered macrocycle) exhibited good cellular potencies (ALK IC50 = 1.0 nM; ALK-L1196 M IC50 = 20 nM) and the highest LipE (4.4) with picomolar binding affinity. Remarkably, compared to acyclic analogs of (2), the macrocyclic ethers were generally too lipophilic and lack of desired efficiency for more facile overlap of potency, ADME, and CNS availability were questionable. Molecular modeling guidance was suggestive to incorporate single carbon Cy3 NHS ester (non-sulfonated) linking the amide with the proximal pyrazole moiety which led to development of 12-membered lactams with minimal binding strain and optimal log D space (2–3). Varieties of lactam linked templates were synthesized to harness enhanced LipE of the 12-membered ether macrocycles and to lower the lipophilicity with amide linkage. Compared to acylic analoges of (1), metabolic stability (low clearance), stereochemical sensitivity, imperious Pgp efflux potential, improved cellular potency (119-fold) and optimal log D of lactams (4) were among the compelling motives to investigate macrocyclic lactams as potential inhibitors capable of brain penetration. Overall, strategy was to construct lactams decorated with variety of substituents at C-ring. With the initial inspiring outcome of macrocyclic lactams and to further achieve the best balance of potency, CNS availability, ADME, and selectivity in a single macrocycle, lead optimization of 2-aminopyridine/pyrazine was surveyed. It has been reported that Trk kinase family (TrkA, TrkB, and TrkC) has shown close association with central and peripheral nervous system processes. If several Trk kinases are simultaneously targeted then selectivity might be an issue than for other kinases due to potential CNS side effects [80]. To gain selectivity over other kinases, residues different from ALK were targeted. Based on a set of residues in the active site, as a surrogate for other kinases comprising of Phe/Tyr residue at the position corresponding to ALK Leu1198 (an avenue to gain selectivity), the TrkB and ALK proteins were aligned by superposition. ALK co-crystal X-ray structures were investigated for numerous macrocyclic lactams closely related to (6) to address selectivity issue over other kinases. The pyrazole moiety (C-ring) of lactams (4) substituted at the ortho position (C3) posed more probability to approach the atoms of TrKB Tyr635 with contact distance (Me, 3.2–4.1 Å, cyclopropane, 3.2 to 3.7 Å, CN, 2.1–3.1 Å). These pyrazole based analogs exhibited picomolar enzymatic potencies against TrkB and unselective on the basis of the ALK-L1196 M Ki (approximately 2-fold) for several lactams analogous to (4). Significant attenuation in TrkB potency (Ki = 23 nM), robust selectivity ratio (∼38-fold) was demonstrated by cyanopyrazole lactam 6 (ALK Ki = 0.70 nM, ALK L1196 M Ki = 8.2 nM, pALK cell IC50 = 1.3 nM in 3T3-EML4-ALK engineered cell lines, pALK-L1196 M cell IC50 = 21 nM, logD = 2.3, MDR BA/AB = 1.5). The enhanced selectivity was attributed to an unfavorable approach of electron rich cyano (CN) to adjacent carbon atom of the Tyr635 in TrkB. Substantial improvement in cell potency against ALK wt and ALK clinical mutants compared with crizotinib (1) and PF-06439015 (2), lorlatinib (6) was a superior outcome of designing and development efforts. Comparison of cell-based pALK inhibition (IC50) in 3T3-EML4-ALK engineered cell lines shown in Table 2. Compound (6) (lorlatinib) demonstrated improved potency (from ALK IC50 = 80 nM for 1–1.3 nM for (6), almost 62-fold) and 40–825 fold improved potency to clinically known mutants in engineered NIH-3T3 ALK wild-type phosphorylation assay. In addition to high potency (ALK = 1.3 nM, ROS1 <0.02 nM), in biochemical assays compound (6) exhibited catalytic activity (wt ALK, Ki < 0.07 nM) and a range of mean inhibition constant (Ki < 0.1–0.9 nM) against previously reported crizotinib-resistant ALK mutants including L1196 M, G1269A, 1151Tins, F1174L, C1156Y, L1152R, and S1206Y [58]. Compound (6) demonstrated potent activity to both L1196 M (IC50 = 21 nM) and G1269A (IC50 = 15 nM) which are most reported crizotinib-resistant mutations in clinical settings. Besides this, compound (6) retained additional potency seriously confronted by all 2nd_generation ALK inhibitor specially 1151Tins (IC50 = 38 nM) and G1202R (IC50 = 77 nM) but ALK L1198F mutation conferred resistance to lorlatinib and resensitization to crizotinib to restore sensitivity has been reported recently [66]. Moreover, compound (6) was potent against all ALK mutants clinically available and showed 40−825-fold increment in potency compared to crizotinib.