Exploration of the imidazo[1,2-b]pyridazine scaffold as a protein kinase inhibitor

Highlights

• A large variety of imidazo[1,2-b]pyridazines were prepared.

• The prepared compounds were assayed against a panel of mammalian and protozoan kinases.

• Several molecules were found to be selective DYRK and CLK inhibitors.

• Despite their potencies against the kinases studied, the compounds were not cytotoxic.

Abstract

3,6-Disubstituted imidazo[1,2-b]pyridazine derivatives were synthesized to identify new inhibitors of various eukaryotic kinases, including mammalian and protozoan kinases. Among the imidazo[1,2-b]pyridazines tested as kinase inhibitors, several derivatives were selective for DYRKs and CLKs, with IC₅₀ < 100 nM. The characterization of the kinome of several parasites, such as Plasmodium and Leishmania, has pointed out profound divergences between protein kinases of the parasites and those of the host. This led us to investigate the activities of the prepared compounds against 11 parasitic kinases. 3,6-Disubstituted imidazo[1,2-b]pyridazines showed potent inhibition of Plasmodium falciparum CLK1 (PfCLK1). Compound 20a was found to be the most selective product against CLK1 (IC₅₀ = 82 nM), CLK4 (IC₅₀ = 44 nM), DYRK1A (IC₅₀ = 50 nM), and PfCLK1 (IC₅₀ = 32 nM). The compounds were also tested against Leishmania amazonensis. Several compounds showed anti-leishmanial activity at rather high (10 μM) concentration, but were not toxic at 1 μM or 10 μM, as judged by viability assays carried out using a neuroblastoma cell line.

Introduction

Reversible protein phosphorylation is the most common post-translational modification occurring in many fundamental cellular processes, such as differentiation, division, proliferation, apoptosis, and signal transduction mechanisms. Due to their strong involvement in essentially all physiological processes, dysfunctional phosphorylation can generate many human diseases, such as cancer [1], inflammation, auto-immune and neurodegenerative diseases, and parasite infections. Among the human kinome described by Manning et al. [2] in 2002, the CMGC group, which comprises nine kinase families, has been extremely well-studied as a group of therapeutic targets. Cdc2-like kinases (CLKs) and dual-specificity tyrosine-phosphorylation-regulated kinases (DYRKs) are families of conserved groups of dual-specificity kinases belonging to the CMGC group, including CDKs (cyclin-dependent kinases), MAP kinases (mitogen-activated protein kinases), GSK-3 (glycogen synthase kinase), and CLKs (Cdc2-like kinases). These two families of kinases auto-phosphorylate at tyrosine residues in their activation loop, but exclusively phosphorylate serine/threonine residues. The CLK family consists of four isoforms: CLK1, CLK2, CLK3, and CLK4. CLKs phosphorylate serine/arginine-rich splicing proteins (SRp), which are implicated in the regulation of alternative splicing [3]. CLK1 is involved in Alzheimer's disease (AD) [4] (splicing of microtubule-associated protein Tau pre-mRNA). The DYRK family comprises five isoforms, DYRK1A, DYRK1B, DYRK2, DYRK3, and DYRK4. The human DYRK1A gene is located on chromosome 21 in the “Down syndrome (DS) critical region,” and overexpression of DYRK1A in DS has been associated with neurodegenerative disease [5]. DYRK1A and DYRK1B are also implicated in diverse types of cancers [6], [7]. Orthologs of mammalian CLKs and DYRKs are also found in unicellular parasites, such as Leishmania, Trypanosoma, Cryptosporidium, Giardia, Toxoplasma, and Plasmodium. Deregulation and dysfunction of CLKs and DYRKs have been linked to several diseases, which make these kinases attractive potential therapeutic targets.

In the present paper, we describe potent and selective CLK and DYRK inhibitors. We optimized the C-3 position of disubstituted imidazo[1,2-b]pyridazines as CLK and DYRK kinase inhibitors. Our interest in this scaffold is based on the fact that imidazo[1,2-b]pyridazines are frequently described as kinase inhibitors. Imidazo[1,2-b]pyridazines have been reported to be inhibitors of CDK2 [8], Pim [9], IKKβ [10], [11], [12], VEGFR2 [13], and Syk [14] (Fig. 1). We have identified more than 20 patents issued since 2007 that describe imidazo[1,2-b]pyridazines as kinase inhibitors, the most advanced product being Ponatinib [15], which in 2012 was launched on the market as a BCR-ABL inhibitor in the treatment of chronic myelocytic leukemia (CML). The MRC (Medical Research Council) technology team has described PfPK7 [16] and PfCDPK1 [17], [18] inhibitors as being potential antimalarial agents. More recently, diarylimidazo [1,2-b]pyridazines have been identified and evaluated for antiplasmodial activity [19] (Fig. 1).

Alzheimer's disease (AD) is a neurodegenerative disease responsible for the most common form of dementia. It is characterized by extracellular accumulation of amyloid plaque (Aβ) and intracellular deposition of neurofibrillary tangles (NFTs). NFTs mainly result from the aggregation of hyperphosphorylated microtubule-associated Tau protein. Aberrant hyperphosphorylation of Tau induces decreased microtubule binding, causing loss of function and aggregation. CLK1 [4] and DYRK1A [20] have been shown to be involved in neurodegenerative disease, particularly AD. Many CLK and DYRK inhibitors have been described over the last few years, including harmine, TG003 [21] and Indy [22], leucettines [23], [24], quinazolines [25], [26], [27], KH-CB19 [3], meriolins [28], meridianins [29], imidazopyridazines [30], and most recently, furo- or thieno [3,2-d]pyrimidin-4-amines [31] derived from harmine. We describe the synthesis strategy and biological evaluation of a new series of disubstituted imidazo [1,2-b]pyridazine inhibitors of CLKs and DYRKs. The aim of this work is first to identify new mixed inhibitors of DYRK1A and CLKs, which could be further evaluated in cell and animal AD models. A second objective is to identify inhibitors of orthologous parasitic kinases which could be useful in the treatment of Leishmania and Plasmodium infections.