Research paperStructure-based design and synthesis of imidazo[1,2-a]pyridine derivatives as novel and potent Nek2 inhibitors with in vitro and in vivo antitumor activities
Graphical abstract
Introduction
NIMA-related kinase 2 (Nek2) is a serine/threonine kinase that serves as a key regulator of mitotic processes such as centrosome duplication and spindle assembly [1], [2], [3]. Deregulation of mitotic processes can lead to genomic instability and aneuploidy, which is a characteristic of all tumors and is a hallmark of cancer. Nek2 expression is varied during the cell cycle and has maximal expression between the S and G2 phases. Nek2 binds to microtubules and is enriched in the centrosome, where it contributes to centrosome splitting during the G2/M phase of the cell cycle. This has been shown to occur through Nek2 phosphorylation of β-catenin [4], C-Nap1, rootletin, and the centrosomal protein Nlp (ninein-like protein) [5]. Furthermore, Nek2 actively participates in the spindle assembly checkpoint (SAC) process through interaction with kinetochore proteins Mad1/2 and by phosphorylating the kinetochore protein Hec1 [6]. Nek2 has been found essential for appropriate mitotic exit as cells with inactive, splice variants of Nek2 were unable to complete cell division resulting in polyploidy [7].
Nek2 overexpression is associated with aggressive cancer phenotypes and poor prognosis in many malignancies [8], [9], [10], [11], [12]. Nek2 expression in cancer cells is directly related to enhanced genomic instability [13], cell proliferation, and drug resistance [14], [15]. Targeting Nek2 with anti-Nek2 shRNA or siRNA constructs can overcome drug resistance and inhibit cancer growth both in vitro and in vivo [16], [17]. Therefore, Nek2 represents a novel biomarker to predict cancer prognosis and drug resistance and can also serve as an important therapeutic target to treat human cancers [8], [18].
To further investigate the role of Nek2 biology and to progress Nek2 as an oncology target, there is a need to develop novel and potent Nek2 inhibitors with adequate in vitro and in vivo antitumor activity. However, due to strong geometric constraints at the ATP binding site (MET86 and PHE148), the design of a Nek2 inhibitor with adequate drug-like properties has been challenging [19]. There are very few Nek2 inhibitors described in the literature and no inhibitor has entered clinical trials for the treatment of cancer [20]. Therefore, there is a pivotal knowledge gap in the understanding of Nek2 cancer biology and Nek2 as a therapeutic target due to a lack of adequate, drug-like inhibitors.
Recently, as described in Fig. 1, an irreversible inhibitor of Nek2 1, based on an indolone-core warhead, displayed a Nek2 IC50 of 770 nM [21]. Benzimidazole-based compound 2 (Nek2 IC50 = 360 nM) achieved good permeability and selectivity [19]. In addition, pyrazine-based compound 3 was identified to have an IC50 of 230 nM but did not attain cellular activity because of permeability issues [22]. Aminopyridine-based compound 4 (Nek2 IC50 = 22 nM) was discovered based on exploration of hybrids between benzimidazole-core (2) and pyrazine-core (3) warheads. Compound 4 was the most potent, reversible Nek2 inhibitor reported in the literature [23]. Although compound 4 exhibited strong activity on Nek2, the compound displayed only 3-fold selectivity against GSK-3β [23]. Further, compound 4 only demonstrated moderate cellular activity in cancer cell-lines and displayed characteristics of off-target inhibition [23]. Therefore, because of a lack of selectivity, potency, and/or pharmacokinetic properties current Nek2 inhibitors are not optimized to further elucidate Nek2 cancer biology and Nek2 as a therapeutic target.
A significant bioisostere of benzimidazole is imidazo[1,2-a]pyridine, which has been identified as a kinase-targeting scaffold that reversibly engages the ATP binding domain (hinge) of a protein kinase through hydrogen-bonding [24], [25], [26], [27], [28], [29], [30]. In order to identify highly potent and selective Nek2 inhibitors with adequate in vitro and in vivo antitumor activity, we further investigated the replacement of a benzimidazole-core with an imidazo[1,2-a]pyridine-core (MBM-1). To accomplish our study, we designed and synthesized a series of novel imidazo[1,2-a]pyridine derivatives based on molecular modeling with compound 2. The newly identified derivatives displayed high potency and selectivity, with drug-like pharmacokinetic and antitumor properties. Herein, we report the detailed synthesis and biological evaluation novel, imidazo[1,2-a]pyridine-based Nek2 inhibitors.
Section snippets
Chemistry
Cyclization of compound 5 with 2-chloroacetaldehyde provided imidazo[1,2-a]pyridine 6 [31], which was coupled with 1-Boc-4-hydroxypiperidin to give protected amine 7 via the Mitsunobu reaction [19]. 3-iodoimidazo[1,2-a]pyridine 8 was readily obtained through the iodination of compound 7 with NIS [31]. Then, free amine 9 was constructed through the treatment of compound 8 with TFA to remove the Boc-protecting group with subsequent reductive amination to generate intermediate 10 as depicted in
In vitro evaluation of antitumor activity
Despite a lack of cellular activity with compound 2, the compound still displayed decent activity and selectivity in Nek2 biochemical assays and therefore served as a validated starting point to generate a more potent, selective Nek2 inhibitor with enhanced in vitro and in vivo antitumor properties. Simultaneously, imidazo[1,2-a]pyridine, a bioisostere for benzimidazole, was investigated as a novel scaffold to help enhance hydrogen bonding at the ATP kinase domain [28], [29], [30]. Therefore,
Conclusion
A series of novel imidazo[1,2-a]pyridine Nek2 inhibitors were designed and synthesized via an innovative bioisostere approach. A nonlinear SAR was identified displaying that substitution at the 7-position of imidazo[1,2-a]pyridine and benzyl group substituents are critical for Nek2 potency and cancer cell activity. Furthermore, an unsubstituted primary amide is important to maintain Nek2 activity. Structure-based design led to compounds MBM-17 and MBM-55, which were potent and selective Nek2
Chemistry. General material and methods
Starting reagents, materials, and solvents for reactions were reagent grade and used as purchased. Chromatography solvents were HPLC grade and were used directly without further purification. Reaction mixtures were monitored via thin-layer chromatography (TLC) on silica gel F-254 TLC plates. Flash column chromatography was carried out using silica gel (200–300 mesh). Chemical shifts (δ) are reported in parts per million (ppm) and coupling constants (J) are reported in Hertz (Hz). NMR spectra
Acknowledgments
This work was supported by the National Natural Science Foundation of China (21572067), NSF of China (21332003), the The Science and Technology Commission of Shanghai Municipality (14431902700), and Open Funds of State Key Laboratory of Oncology in South China (HN2016-03).
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These authors contributed equally.