Research paper
Structure-based design of novel quinoxaline-2-carboxylic acids and analogues as Pim-1 inhibitors

https://doi.org/10.1016/j.ejmech.2018.04.056Get rights and content

Abstract

We identified a new series of quinoxaline-2-carboxylic acid derivatives, targeting the human proviral integration site for Moloney murine leukemia virus-1 (HsPim-1) kinase. Seventeen analogues were synthesized providing useful insight into structure-activity relationships studied. Docking studies realized in the ATP pocket of HsPim-1 are consistent with an unclassical binding mode of these inhibitors. The lead compound 1 was able to block HsPim-1 enzymatic activity at nanomolar concentrations (IC50 of 74 nM), with a good selectivity profile against a panel of mammalian protein kinases. In vitro studies on the human chronic myeloid leukemia cell line KU812 showed an antitumor activity at micromolar concentrations. As a result, compound 1 represents a promising lead for the design of novel anticancer targeted therapies.

Introduction

Proviral integration site for Moloney murine leukemia virus (Pim) kinases belong to a small family of constitutively activated proto-oncogenic serine/threonine protein kinases, constituted of three isoforms: Pim-1, Pim-2 and Pim-3 [1]. These oncoproteins control many cellular functions like cell cycle regulation, apoptosis, cell survival, proliferation and differentiation [2,3], and are overexpressed in a large number of human cancer types, such as hematopoietic malignancies [4,5] and solid cancers (e. g. bladder [6], prostate [7], breast [8] or oral cancers [9]). These kinases are positive regulators of cell cycle progression at G1/S and G2/M checkpoints, and inhibit apoptosis, acting as oncogenic survival factors [10]. Interestingly, it has been demonstrated that Pim1−/−2−/−3−/− triple knockout mice were viable and fertile, which make these kinases very interesting for targeted cancer therapies [11].

Recently, Pim-1 has been shown to play a significant role in cancer stem cells growth, and in resistance to chemotherapy drugs, promoting multiple drug resistance [12,13]. This kinase is thus considered as a relevant target for cancer therapy and a large variety of small molecule inhibitors have been developed [[14], [15], [16], [17], [18]]. Many of these Pim-1 kinase inhibitors demonstrated significant in vitro activity in cancer cell lines and in different in vivo tumor xenograft models, and clinical trials are currently ongoing for the most promising candidates [14,18].

A remarkable characteristic of Pim-1 active site in comparison to other protein kinases is the presence of an original hinge region (region containing backbone peptide atoms that forms hydrogen bond interactions (H-bonds) with the adenine moiety of ATP). Indeed, this region contains a proline residue (Pro123), which has no H-bond donor property and precludes the formation of one of the conserved H-bond involving the hinge backbone and the ATP adenine ring, as it can be observed in other kinases. Thus, Pim-1 bounds ATP via only one hinge H-bond between the ATP adenine amino moiety and the backbone carbonyl of glutamate 121 (Glu121). Moreover, the insertion in the hinge of a valine (Val126), absent in other kinases, changes the hinge conformation, enlarging the catalytic pocket. This unique feature can be exploited for the design of selective inhibitors [19].

The vast majority of Pim-1 inhibitors mainly act as ATP competitive inhibitors, targeting the ATP-binding pocket. They can be classified into two categories: ATP-mimetics, which bind to the Glu121 residue of the hinge region, and non-ATP mimetics, which interact with the ATP binding cleft in a different manner from ATP [20].

In a continuing effort to develop new small molecule inhibitors with anticancer properties, our laboratory has been recently focusing on the study of new inhibitors of the signal transducer and activator of transcription 5 (STAT5) activation and expression and their interest in chronic myeloid leukemia (CML) [21]. Indeed, the STAT family transcription factors are commonly activated in cancer by upstream mutations or cell surface signaling molecules. It has been demonstrated that the Pim kinases are induced by the STAT family transcription factors (particularly STAT 3/5) [14]. Regarding the potential of Pim-1 as target in cancer therapy and particularly in leukemia [22,23], we decided to further explore the STAT signaling pathway, by developing new Pim-1 kinase specific inhibitors. In this purpose, we first performed a target-based approach, by realizing a focused in vitro screening of our chemical library on a limited panel of kinases, comprising Homo sapiens Pim-1 (HsPim-1), allowing the identification of the quinoxaline-2-carboxylic acid 1 as a new lead compound (Fig. 1). This molecule was able to inhibit the in vitro enzymatic activity of HsPim-1 with an IC50 of 74 nM.

Docking studies, using program GOLD (GOLD version 4.0; CCDC, Cambridge, UK), were performed to understand the binding interactions between the lead compound 1 and the ATP pocket of HsPim-1 (PDB ID 3A99) (Fig. 2). Data analysis suggests that the carboxylate group of this molecule can form a key salt bridge with the protonated amino group side chain of catalytic Lys67, as it has already been described in other Pim-1 inhibitors [24,25], and shares also a H-bond interaction with the backbone NH of Asp186 belonging to the DFG motif. Additionally, an H-bond interaction between the 3-hydroxyphenyl moiety and the carboxylate group of residue Asp186 can be observed. These studies suggested that compound 1 could act as an ATP competitive inhibitor, with a non-ATP mimetic binding mode.

Sixteen new analogues were then synthesized, exploiting the unique sequence of HsPim-1 ATP-binding cleft.

We report herein the design, synthesis, structure-activity relationships (SAR) and in vitro evaluations of this new class of Pim-1 inhibitors.

Section snippets

Chemistry

The preparation of quinoxaline-2-carboxylic acids 1, 5c-e, and 5h-i and potassium carboxylate salts 5b, and 5g was performed as shown in Scheme 1 by amination of the intermediate ethyl 3-chloroquinoxaline-2-carboxylate 3 with the appropriate amine derivatives.

The synthesis of ethyl 3-chloroquinoxaline-2-carboxylate 3 was achieved in two steps from commercial o-phenylenediamine according to literature procedures [26,27] (Scheme 1). First, the o-phenylenediamine was condensed with diethyl

Pim-1 enzymatic activity inhibition

Compounds were first evaluated for their efficacy to inhibit the in vitro enzymatic activity of HsPim-1, using a luminescence-based kinase assay [29]. Compounds that displayed HsPim-1 IC50 > 10 μM were considered inactive.

To get closer insight into the potential binding mode of our compounds within the ATP binding pocket of HsPim-1, we first decided to structurally vary the substitution patterns of the quinoxaline scaffold of 1 in position 3 (Table 1). Docking analysis revealed that the

Conclusion

In this study, we identified a new series of quinoxaline-2-carboxylic acids and analogues, exhibiting a potent activity against the HsPim-1 oncoprotein. Among the 17 compounds synthesized, 5 significantly blocked HsPim-1 with IC50 values in the submicromolar to low micromolar range. In particular, lead compound 1 showed the best inhibitory effect against HsPim-1, with an IC50 value of 74 nM. SAR in positions 2 and 3 of the quinoxaline scaffold confirmed the molecular modeling studies,

General remarks

All solvents were anhydrous reagents from commercial sources. Unless otherwise noted, all chemicals and reagents were obtained commercially and used without purification. Microwave heating was carried out with a single-mode Initiator Alstra (Biotage) unit. Melting points (Mp) were determined on a Stuart capillary apparatus and are uncorrected. High-resolution mass spectra (HRMS) were performed in positive mode with an ESI source on a Q-TOF mass spectrometer (Bruker maXis) with an accuracy

Acknowledgments

This work was supported by a grant from the « Association pour le Développement de la Recherche et de l'Innovation dans le NORD PAS DE CALAIS » (ADRINORD) (to B. O.). The authors thank the « Plateforme Scientifique et Technique Analyses des Systèmes Biologiques » (PST-ASB), Tours (France), for NMR spectrometry and the « Fédération de Recherche » ICOA/CBM (FR2708) platform, for HRMS analyses. The authors also thank the Cancéropôle Grand Ouest (axis: Natural sea products in cancer treatment), GIS

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