Research paper
New pyrido[3,4-g]quinazoline derivatives as CLK1 and DYRK1A inhibitors: synthesis, biological evaluation and binding mode analysis

https://doi.org/10.1016/j.ejmech.2019.01.052Get rights and content

Highlights

  • New pyrido[3,4-g]quinazolines were synthesized.

  • Nanomolar inhibitors of CLK1 and/or DYRK1A were identified.

  • Two alternative binding modes within CLK1 were identified by X-ray crystallography.

Abstract

Cdc2-like kinase 1 (CLK1) and dual specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) are involved in the regulation of alternative pre-mRNA splicing. Dysregulation of this process has been linked to cancer progression and neurodegenerative diseases, making CLK1 and DYRK1A important therapeutic targets. Here we describe the synthesis of new pyrido[3,4-g]quinazoline derivatives and the evaluation of the inhibitory potencies of these compounds toward CDK5, CK1, GSK3, CLK1 and DYRK1A. Introduction of aminoalkylamino groups at the 2-position resulted in several compounds with low nanomolar affinity and selective inhibition of CLK1 and/or DYRK1A. Their evaluation on several immortalized or cancerous cell lines showed varying degree of cell viability reduction. Co-crystal structures of CLK1 with two of the most potent compounds revealed two alternative binding modes of the pyrido[3,4-g]quinazoline scaffold that can be exploited for future inhibitor design.

Introduction

Protein kinases are important cellular targets for the development of novel chemical probes and drugs in many therapeutic areas, including cancer, neurodegenerative disorders, inflammation or pain therapy. To date, more than five hundred protein kinases have been identified in the human kinome. All of them share the same co-factor (ATP), and the ATP binding pocket shows high structural conservation in most of them. Developing kinase inhibitors that display selectivity within the human kinome, therefore, remains a major challenge [1]. However, as shown by FDA approved drugs, especially in oncology, absolute selectivity for a single kinase is not always needed [2]. As part of our ongoing efforts toward the development of potent and selective kinase inhibitors, we recently described pyrido[3,4-g]quinazolines as inhibitors of Cdc2-like kinases (CLK1) and dual specificity tyrosine phosphorylation-regulated kinases (DYRK1A) [3]. CLK1 and DYRK1A are involved in the regulation of alternative pre-mRNA splicing via SR-protein phosphorylation, and dysfunction of this tightly regulated process is linked to the progression of cancer, neurodegenerative diseases, and viral infections [4,5]. As reported in recent reviews, various chemical scaffolds could lead to potent DYRK1A and/or CLK1 inhibitors [[6], [7], [8], [9]]. We carried out a structure-activity relationship (SAR) study around the tricyclic pyridoquinazoline scaffold. Previous results demonstrated that the substitution by nitro/amino groups at the 10-position is essential to target CLK1 and/or DYRK1A kinases [3]. The introduction of alkyl/aryl substituents at the 5-position led to a change in the kinase inhibition profile, as 5-substituted derivatives exhibited improved potencies toward CDK5/GSK3, while CLK1/DYRK1A inhibition was impaired (Fig. 1A) [10]. Since modification of this heteroaromatic scaffold resulted in a major change in activity and selectivity, we decided to further extend the SAR studies on the pyrido[3,4-g]quinazoline scaffold by varying the substitution at the 2-position (Fig. 1A).

Section snippets

Synthesis of compound library

Analysis of the binding mode of the best CLK1/DYRK1A inhibitor of the pyrido[3,4-g]quinazolines series showed that the aminopyrimidine moiety forms two hydrogen-bonds with the backbone amine and carbonyl group of Leu244 upon binding to CLK1 (Fig. 1B). Additionally, the pyridine nitrogen atom is hydrogen-bonded to the Lys191 side chain [3]. Our aim was to extend this lead compound by adding a series of aminoalkylamino groups at the 2-position to figure out if this alters the potency or

Conclusion

A new series of pyrido[3,4-g]quinazolines with diverse substitutions at the 2-position and either an amino or a nitro group at the 10-position was synthesized and evaluated against five protein kinases (CDK5/p25, CLK1, DYRK1A, CK1δ/ε, and GSK-3α/β). The results demonstrated that the aminopyrimidine part is essential to the kinase inhibitory potency of this series (compounds 2 and 3). The most strongly inhibited kinases were CLK1 and DYRK1A, with a better selectivity toward CLK1 for 10-nitro

General

Starting materials were obtained from commercial suppliers and used without further purification. Solvents were distilled prior to use. IR spectra were recorded on a Shimadzu FTIR-8400S spectrometer (v¯ in cm−1). NMR spectra, performed on a Bruker AVANCE 400 (1H: 400 MHz, 13C: 100 MHz), are reported in ppm using the solvent residual peak as an internal standard; the following abbreviations are used: singlet (s), doublet (d), triplet (t), quadruplet (q), quintet (quint), doublet of doublet (dd),

Accession codes

The atomic coordinates and structure factors of the CLK1-pyrido[3,4-g]quinazoline complexes have been deposited in the Protein Data Bank (PDB), www.pdb.org. Accession codes: 6Q8K (CLK1-10i complex) and 6Q8P (CLK1-9m complex).

Acknowledgements

The authors thank Aurélie Job for HPLC analysis. The Auvergne Region (Jeune Chercheur Program) is acknowledged for funding of W. Z., F. A., F. G. and P. M. as well as the French Ministry of Higher Education and Research for Y. J. E. PhD fellowship. H. T. is gratefull to the Carthage University (Tunisia) for a doctoral mobility fellowship. KISSf facility is supported by Biogeneouest/Région Bretagne, Cancéropôle Grand Ouest and GIS IBiSA. A.C.J. is funded by German Research Foundation grant JO

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