Synthesis and biological evaluation of selected 7H-pyrrolo[2,3-d]pyrimidine derivatives as novel CDK9/CyclinT and Haspin inhibitors
Graphical abstract
Introduction
In 1955 Fischer and Krebs reported evidence that the conversion of phosphorylase b to a, as observed in cell-free muscle extracts, is dependent on nucleotides with high energy phosphates in the presence of high energy divalent metal ions [1]. This opened a door into the world of protein kinases (PKs) as phosphorylase became the first known PK to be purified [2]. Since then, it has become apparent that the human kinome consists of 518 PKs (equating to 1.7% of all human genes) and is indispensable to normal cellular function [3]. PKs play a key part in regulating signal transduction by promoting the transfer of phosphoryl groups from phosphate donors, like adenosine triphosphate (ATP), to receptor substrates [4]. The associated signal transduction pathways significantly influence biological systems as they determine cellular responses to environmental stimuli and modify gene expression [5]. Erroneous signal transduction may lead to disruption of normal biological processes and could ultimately result in diseases such as, cancer, inflammation, diabetes and neurodegenerative disease [[6], [7], [8], [9]]. Thus, the appeal of PK inhibitors as drug targets is rooted in the expectation that erroneous signal transduction can be halted by inhibition of the appropriate PK. Nowadays, 52 PK inhibitor-based drugs are approved by the US food and drugs administration, where 46 of the 52 is used in the treatment of neoplastic diseases. Examples include abemaciclib, palbociclib and ricociclib (CDK4/6 inhibitors) [10].
Cyclin-dependant kinase CDK9/CyclinT complex is an example of such a therapeutic target. CDK9 is a member of CMGC family of kinases: for Cyclin-dependent kinase (CDK), Mitogen-activated protein kinase (MAPK), Glycogen synthase kinase (GSK) and CDC-like kinase (CLK). CMGC kinases are key regulators involved in the regulation of DNA repair and cell fate (cell proliferation, differentiation, apoptosis) [11]. CDK9 is indispensable in the governance of non-ribosomal transcription, which includes super-enhancers regulated gene expression. Super-enhancers are clusters of DNA regulatory components that guide transcription of genes of significance in cell-identity [12]. As abnormalities in the regulation of CDK9 activity have been described in acute myeloid leukaemia (AML), other haematological malignancies and solid tumours, the chemical inhibition of CDK9 is considered as a promising strategy in cancer therapy [11].
Another kinase of interest is the Haploid germ cell-specific nuclear protein kinase (Haspin). Haspin is one of the newer additions to the kinome and remains relatively underexplored as a drug target ([13]. It is present across a variety of eukaryotic species (animal, fungi and plants) and was first detected in male germ cells [14,15]. This Ser/Thr kinase is structurally unique as compared to other members of the PK family [16]. It displays essential function during cell division and is involved in histone code printing [13,17,18]. Haspin depletion by CRISPR/Cas9 in Breast cancer HeLa cells triggers severe mitotic defects and ultimately cell death by mitotic catastrophe [19].
In humans, Haspin mRNA expression has been marked in all proliferating cell-lines such as, thymus, bone marrow and foetal liver cell-lines. Haspin has also been found overexpressed in several highly proliferative malignant tumours such as melanomas, myelomas, pancreatic and bladder cancer [[20], [21], [22], [23]]. The degree of Haspin expression in tissue correlates with the degree of cellular proliferation and differentiation each tissue experiences and often correlates with the most aggressive stages of the cancers [18,[24], [25], [26], [27]]. Furthermore, Haspin inhibition or knockdown has been shown to slow down the development of several cancers in animal models [20]; [22,28,29]. Consequently, Haspin could prove to be a valuable target in mitosis and thus cancer therapy.
In a previous study [30], 7-azaindole derivatives were identified as dual-inhibitors of CDK9/CyclinT and Haspin (see 1 and 2, Fig. 1). This illustrates the ability of the 7-azaindole scaffold to produce potential novel CDK9/CyclinT and Haspin inhibitors. In the previous study, the substituents were selected based on their commercial availability, relative affordability and the fact that the scaffold was novel to most of the targets on the PK panel, thus allowing for simpler structures to be examined whilst retaining novelty [30]. Prompted by the success of the previous study, a series of selected 6-substituted 7-azaindole derivatives and a series of structurally related 7H-pyrrolo[2,3-d]pyrimidine (7-deazapurine) derivatives were synthesised. In the case of the current study, the substituents were chosen to match their counterparts in the previous study to determine if results would mirror that of the previous study. After the initial synthesis, it was decided to expand the series in order to examine a more diverse set of compounds. The additional substituents (7c–e, 7h–l) were selected to complement the existing substituents (7a, 7b, 7f, 7g, 7m–p).
These compounds were evaluated in vitro against CDK9/CyclinT and Haspin kinases (see Table 1, Table 2, Table 3). Additionally, these compounds were screened against other PKs: (i) CDK2/CyclinA, (ii) CDK5/p25, (iii) Pim-1, (iv) CK1ε, (v) GSK-3β, (vi) LmCK1 (from the parasite Leishmania major) and (vii) ABL1. The structures of the selected 7-azaindole and 7-deazapurine derivatives were subjected to a SciFinder search in order to establish the novelty of each compound. The structure search for compounds 5b, 5c, 5f, 5g–k, 7c–e, 7g, 7h, 7l–n, and 8a – c produced no former references and they were considered novel. Compounds 5a [31], 5d, 5e [32], 7a, 7b, 7f [33], 7i, 7j, 7k [34], 7o [35] and 7p [36] had been referenced in previous literature works. Nevertheless, none of the compounds had previously been examined as CDK9/CyclinT or Haspin inhibitors. In this study, we described the inhibitory activity in the low micromolar range of six compounds against CDK9/CyclinT and five compounds against Haspin. Among these molecular hits, four compounds are dual-target inhibitors.
Section snippets
Material and methods
The selected 7-azaindole and 7-deazapurine derivatives were synthesised as described in the experimental section. Materials for synthesis were sourced from AK Scientific (for 5a–k, 7a, 7b, 7e, 7f, 7g, 7l, 7m, 7n, 7o, 7p and 8a – c) and Ambeed (for 7c, 7d, 7h, 7j, and 7k), while CHR 6494 and flavopiridol were purchased from Sigma Aldrich. All materials were used without further purification, unless otherwise specified. Proton (1H) and carbon (13C) nuclear magnetic resonance (NMR) spectra were
Chemistry
6-Chloro-7-azaindole and 4-chloro-7H-pyrrolo[2,3-d]pyrimidine (6-Chloro-7-deazapurine) were reacted with a variety of commercially available boronic acids, by means of Suzuki-Miyaura coupling, to afford a series of C6-substituted 7-azaindole derivatives (5a–k) and a series of C4-substituted-7H-pyrrolo[2,3-d]pyrimidine derivatives (7a–p), respectively. Furthermore, the 4,7-bis-substituted-7H-pyrrolo[2,3-d]pyrimidine derivatives (8a–c) were derived from 4-chloro-7H-pyrrolo[2,3-d]pyrimidine
Results and discussion
Previously, it has been determined that derivatives of the 7-azaindole scaffold are likely to be PK inhibitors [30]. In this study, thirteen (13) 7-azaindole and twenty (20) 7-deazapurine derivatives were synthesised and screened against the CDK9/CyclinT and Haspin kinases. In addition, the compounds were also screened against HsCDK2/CyclinA, HsCDK5/p25, HsPim-1, HsCK1ε, HsGSK3β, LmCK1 and HsABL1. Screenings were performed at 10 and 1 μM concentrations (diluted in 1% DMSO v/v). The most
Conclusion
This study evaluated the inhibitory potential of two structurally related chemical series against selected disease-related protein kinases. The first series consisted of C6-substituted 7-azaindole derivatives while second series is constituted by C4-substitued 7H-pyrrolo[2,3-d]pyrimidine derivatives. The kinase-based screening assays performed against several PKs, revealed that compounds belonging to the 7-deazapurine scaffold and substituted on the C4-position, exhibited activity toward the
CRediT authorship contribution statement
Lianie Pieterse: Investigation, Formal analysis, Writing – original draft. Richard M. Beteck: Supervision, Reviewing and Editing. Blandine Baratte: Investigation. Omobolanle J. Jesumoroti: Reviewing and Editing. Thomas Robert: Writing – review & editing. Sandrine Ruchaud: Writing – review & editing. Stéphane Bach: Formal analysis, Writing – review & editing. Lesetja J. Legoabe: Conceptualization, Supervision, Reviewing and Editing.
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
The authors are grateful to Dr. Otto and Dr. Jordaan of the SASOL Centre for Chemistry, North-West University, for recording the NMR and MS spectra, respectively. The authors also thank the Cancéropôle Grand Ouest (3 MC network - Marine Molecules, Metabolism and Cancer), GIS IBiSA (Infrastructures en Biologie Santé et Agronomie) and Biogenouest (Western France life science and environment core facility network) for supporting KISSf screening facility.
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