5-Methoxybenzothiophene-2-Carboxamides as Inhibitors of Clk1/4: Optimization of Selectivity and Cellular Potency

Abstract

: Clks have been shown by recent studies to be promising targets for cancer therapy, as they are considered key regulators in the process of pre-mRNA splicing, which in turn affects every aspect of tumor biology. In particular, Clk1 and -4 are overexpressed in several human tumors. Most of the potent Clk1 inhibitors reported in the literature are non-selective, mainly showing off-target activity towards Clk2, Dyrk1A and Dyrk1B. Herein, we present new 5-methoxybenzothiophene-2-carboxamide derivatives with unprecedented selectivity. In particular, the introduction of a 3,5-difluoro benzyl extension to the methylated amide led to the discovery of compound 10b (cell-free IC50 = 12.7 nM), which was four times more selective for Clk1 over Clk2 than the previously published flagship compound 1b. Moreover, 10b showed an improved growth inhibitory activity with T24 cells (GI50 = 0.43 µM). Furthermore, a new binding model in the ATP pocket of Clk1 was developed based on the structure-activity relationships derived from new rigidified analogues.

An overview of cdc2‐like kinase 1 (Clk1) inhibitors and their therapeutic indications

“…Similar to ( 59 ), compound ( 60 ) showed a good selectivity profile, with no significant inhibition towards the most common off‐targets previously reported for Clk1 inhibitors. The selectivity factor for Clk1 inhibition over that of Dyrk1A was 28, whereas an equal potency against Clk4 was found 67 ; however, this was not unexpected as Clk1 and Clk4 share fully identical ATP binding pockets 11,112 …”

“…In an attempt to improve the potency, selectivity, and cellular activity of compound ( 59 ), other structural modifications were exploited at scaffold ( 58 ) by Elhady et al, which included modifications at the amide linker, as well as using disubstituted phenyl extensions at R 2 . Compound ( 60 ) gave the most interesting set of results: (i) an IC 50 of 12.7 nM against Clk1, (ii) a four times enhanced selectivity over Clk2 relative to ( 59 ), and (iii) a higher growth inhibitory activity against T24 cells than found with ( 59 ) (GI 50 = 0.43 µM) 67 …”

“…Clk1 inhibition has been established as a novel effective strategy in treating various types of tumors such as hypoxia‐induced prostate 63 and lung cancers, 58 gastrointestinal cancer, 64,65 colorectal cancer, 66 urinary bladder carcinoma, 18,67 and several hematological malignancies 68,69 . Clk1 was shown to phosphorylate and activate splicing factor 45 (SPF45) on eight serine residues leading to stabilization of SPF45 protein levels 20 .…”

“…Pan‐Clk inhibitory activity was suggested to be crucial for sufficient in cell and in vivo antitumor activity 65 . However, other studies proposed that application of selective Clk1 inhibitors in vivo would influence lower number of modulated pre‐mRNA splicings and result in presumably fewer adverse side effects while maintaining their aimed biological effectiveness 18,67 . Clk2 in particular has been identified as a key player in the hepatic response to refeeding, where insulin‐induced hepatic Clk2 phosphorylates PGC‐1α.…”

Discovery of Some Heterocyclic Molecules as Bone Morphogenetic Protein 2 (BMP-2)-Inducible Kinase Inhibitors: Virtual Screening, ADME Properties, and Molecular Docking Simulations

“…In particular, compounds I , II III , and IV ( Figure 1 ) enhanced BMP-2 expression in vitro and stimulated bone formation and trabecular connectivity restoration in vivo [ 18 ]. In contrast, benzothiophene and benzofuran derivatives can inhibit several protein kinases and act as anticancer agents such as compounds V – VIII ( Figure 2 ) [ 16 , 20 , 21 , 22 ].…”