Elsevier

European Journal of Medicinal Chemistry

Volume 126, 27 January 2017, Pages 1083-1106
European Journal of Medicinal Chemistry

Research paper
Structure-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

https://doi.org/10.1016/j.ejmech.2016.12.026Get rights and content

  • A series of novel and potent Nek2 inhibitors based on a new imidazo[1,2-a]pyridine scaffold were designed and synthesized.

  • MBM-17 and MBM-55 effectively inhibited the proliferation of cancer cells by inducing cell cycle arrest and apoptosis.

  • MBM-17S and MBM-55S showed good selectivity and in vitro and in vivo antitumor activity without apparent toxicity.

Abstract

We present herein the discovery and development of novel and potent Nek2 inhibitors with distinctive in vitro and in vivo antitumor activity based on an imidazo[1,2-a]pyridine scaffold. Our studies identified a nonlinear SAR for activity against both Nek2 and cancer cells. Bioisostere and structure-based design techniques were employed to identify compounds 42c (MBM-17, IC50 = 3.0 nM) and 42g (MBM-55, IC50 = 1.0 nM), which displayed low nanomolar activity and excellent selectivity for Nek2. Both compounds effectively inhibited the proliferation of cancer cells by inducing cell cycle arrest and apoptosis. Importantly, the salts form of these two compounds (MBM-17S and MBM-55S) significantly suppressed tumor growth in vivo without apparent toxicity based on appearance and changes in body weight. In summary, MBM-17 and MBM-55 displayed the potential for substantial therapeutic application in cancer treatment.

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).

References (37)

  • A.M. Fry et al.

    Cell cycle regulation by the NEK family of protein kinases

    J. Cell Sci.

    (2012)
  • S. Bahmanyar et al.

    β-Catenin is a Nek2 substrate involved in centrosome separation

    Genes Dev.

    (2008)
  • J. Rapley et al.

    Coordinate regulation of the mother centriole Component Nlp by Nek2 and Plk1 protein kinases

    Mol. Cell. Biol.

    (2005)
  • J. Du et al.

    The mitotic checkpoint kinase NEK2A regulates kinetochore microtubule attachment stability

    Oncogene

    (2008)
  • Y. Fang et al.

    Targeting NEK2 as a promising therapeutic approach for cancer treatment

    Cell Cycle

    (2016)
  • Y.-R. Zeng et al.

    Overexpression of NIMA-related kinase 2 is associated with progression and poor prognosis of prostate cancer

    BMC Urol.

    (2015)
  • X. Zhong et al.

    Aberrant expression of NEK2 and its clinical significance in non-small cell lung cancer

    Oncol. Lett.

    (2014)
  • C.P. Neal et al.

    Overexpression of the Nek2 kinase in colorectal cancer correlates with beta-catenin relocalization and shortened cancer-specific survival

    J. Surg. Oncol.

    (2014)
  • Cited by (41)

    • Copper-imidazo[1,2-a]pyridines differentially modulate pro- and anti-apoptotic protein and gene expression in HL-60 and K562 leukaemic cells to cause apoptotic cell death

      2022, Biochimica et Biophysica Acta - Molecular Cell Research
      Citation Excerpt :

      Imidazo[1,2-a]pyridines are nitrogen containing heterocyclic molecules consisting of an imidazole moiety bound to a pyridine ring and have been ascribed a variety of pharmacological activities, including anti-cancer activity [21]. They are effective inhibitors of phosphatidyl inositol-3-kinase and the serine-threonine kinase, Nek2, both important kinases in cancer [22,23]. The imidazo[1,2-a]pyridine structure is a useful scaffold for the binding of metals such as copper, and in this context, we evaluated the potential anti-cancer activity of copper-imidazo[1,2-a]pyridine derivatives on breast and colorectal cancer cell lines as well as leukaemic cells, as reported by Dam et al, 2017 [17].

    • Discovery of imidazo[1,2-a]pyridine-thiophene derivatives as FLT3 and FLT3 mutants inhibitors for acute myeloid leukemia through structure-based optimization of an NEK2 inhibitor

      2021, European Journal of Medicinal Chemistry
      Citation Excerpt :

      CMP5 exhibited a further reduction in NEK2 potency without a significant change in FLT3 inhibition (Table 1). The amide portion of CMP3a engages in a hydrogen bond network in the back pocket of NEK2 and this moiety was subsequently removed in the final scaffold to further limit NEK2 activity (Fig. 1) [19,25]. Due to the mutant types of FLT3 are not commercially available, we started our studies with validating the inhibition profile of the scaffold on FLT3-wt in enzymatic level.

    • Design, synthesis, and structure activity relationship (SAR) studies of novel imidazo[1,2-a] pyridine derivatives as Nek2 inhibitors

      2020, Bioorganic and Medicinal Chemistry
      Citation Excerpt :

      Herein, we report a novel series of 3-(thiophen-2-yl)imidazo[1,2-a]pyridine-based compounds and investigate their potential antitumor properties on MGC-803 cells. Compound 2 was synthesized from 2-amino-4-bromopyridine 1 and 2-chloroacetaldehyde by a cyclization reaction in high yield.26 Compound 3 was synthesized by a Suzuki coupling reaction of compound 2 with 1-methyl-4-pyrazole boronic acid pinacol ester.

    • Pyrrolo[2,3-d]pyrimidine derivatives as inhibitors of RET: Design, synthesis and biological evaluation

      2020, European Journal of Medicinal Chemistry
      Citation Excerpt :

      We also explored the substitution pattern on the central phenyl ring. As part of a continuous effort targeting kinases for cancer therapy [33–39], we have reported inhibitors for several kinases. One of them, Pz-1, an RET inhibitor, has been widely studied in the scientific community [27].

    View all citing articles on Scopus
    1

    These authors contributed equally.

    View full text