Cancer is a massive health burden with almost two million new cases in the US in 2023 [1] and with expected thirteen million mortalities per year by 2030 according to the World Health Organization (WHO) [2]. One of the six hallmarks of cancer is the uncontrolled cell growth, and in oncology, sustained cell cycle suppression has been shown to be beneficial in cancer treatment [3]. Cyclins and their regulatory partners; cyclin-dependent kinases (CDKs), normally regulate the cell cycle. Its progression is maintained by important stages known as checkpoints that promote cell cycle cessation via CDKs [4], [5]. CDKs are a set of serine/threonine kinases involving 20 members, some of which are associated with the regulation of cell-cycle progression by phosphorylating proteins essential for cell division [6]. Abrogation of cell cycle control may occur due to deregulation of CDKs or cyclins, as well as the loss of endogenous inhibitory proteins leading to the growth of various tumors [7], [8]. Consequently, CDKs are regarded as crucial targets for anticancer medications.

CDK2 is a member of the CDK family that is considered pivotal for controlling cell cycle progress, specifically through G1 to S phase transition and G2 modulation [9]. Additionally, CDK2 is essential for cell differentiation, DNA repair and apoptosis [10], [11]. Despite the fact that normal tissues generally express trivial amounts of CDK2, the overexpression of CDK2 was typically detected in many tumors viz leukemia [12], melanoma [13], [14], breast [15], renal [16], CNS [17], [18], [19] and colon cancers [20], [21] leading to uncontrolled cancer cell proliferation. Consequently, CDK2 has attracted much attention recently as a druggable target for developing new chemotherapeutic approaches.

Up to date, several CDK2 inhibitors with different scaffolds have been developed as potential antiproliferative agents and some of them entered clinical trials such as milciclib [22], roniciclib [23], AZD5438 [24], roscovitine (seliciclib) [25], dinaciclib [26], BS-194 (4 K) [27] and fadraciclib (CYC065) [28] (Fig. 1). However, many of these agents revealed some adverse effects, limited efficacy and high toxicity [9], [29]. Thus, the innovation of novel CDK2 inhibitors still represents an interesting research field for medicinal chemists to develop new anticancer agents.

CDK2 inhibitors are classed as type I, type II and allosteric inhibitors [30]. The type I inhibitors bind to the ATP-binding site of the CDK2 enzyme in its active conformation. The vast majority of CDK2 inhibitors are type I inhibitors which are close to the ATP structure to mimic the ATP binding mode in the CDK2 active site and block its kinase activity [9]. The first generation CDK inhibitor roscovitine IV is a purine analogue and a CDK2 inhibitor that undergoes phase 2 clinical evaluation against breast and lung cancer [31].

Based on literature review [32], [33], the design of ATP-competitive CDK2 inhibitors generally depends on the ATP structure to mimic the ligand–protein interactions of ATP with CDK2 enzyme. An extensive analysis of type I inhibitors revealed that there are three main pharmacophoric features for their interactions with CDK2 ATP binding site; a) A planar ring such as the purine ring and its bioisosteres that occupies approximately the same conformational space as the adenine ring of ATP, b) H-bond donor and/or acceptor that interacts with the hinge backbone residues and c) Aromatic fragments that afford hydrophobic interactions with several amino acids lining both hydrophobic pocket 1 and/or hydrophobic pocket 2. It is worth mentioning that substitution by those hydrophobic fragments was reported to be essential for activity [34].

Herein, the design of the novel compounds relied on the purine derivative; roscovitine IV as a lead compound where the X-Ray crystal structure of CDK2 with bound roscovitine indicates that the purine ring as the core portion of roscovitine roughly overlaps with the adenine region of the ATP binding site and the two nitrogen atoms of the purine ring generate two cardinal hydrogen bonds with Leu83 (NH to Leu83 CO and N atom of imidazole ring to Leu83 NH) of the hinge region. In the same vein, the isopropyl group fills a narrow hydrophobic pocket formed by the hydrophobic amino acids, and the benzyl ring of roscovitine is directed towards the solvent-accessible region of the kinase, facing the outside of the ATP-binding pocket and forming hydrophobic interactions with several amino acids mainly Ile10, Phe82, Phe80, Val18, His84, Ala31 and Ala44 (Fig. 2) [34].

Besides, numerous heterocyclic scaffolds are well known for their different biological activities [35], [36], [37], [38], [39]. Curiously, pyrazolo[3,4-d]pyrimidine is a rigid hetero-bicyclic scaffold that is considered a purine analog and literature survey disclosed the biological importance of several pyrazolopyrimidine derivatives as CDK2 inhibitors with potential anticancer activity and as apoptotic inducers [40], [41], [42], [43], [44], [45]. For instance, the pyrazolo[3,4-d]pyrimidine derivatives VIII, IX and X exhibited CDK2 inhibitory activities with IC50 values of 0.5, 0.9, and 5 μM, sequentially [44], [46] (Fig. 3).

Provoked by these findings and in pursuance of our efforts to develop new CDK2 inhibitors [47], [48], [49], [50], new series of 4-substituted pyrazolo[3,4-d]pyrimidine derivatives were designed and featured the following in an attempt to mimic type I CDK2 inhibitors i) A pyrazolopyrimidine ring (by bioisosteric replacement of the purine ring of roscovitine to accommodate the adenine region of the ATP binding site in CDK2) ii) An amino linker appended at position 4 of the pyrazolopyrimidine scaffold which is responsible for the essential hydrogen bond formation with amino acid residue Leu83. iii) N-phenyl ring appended to N2 of the pyrazole ring to accommodate the narrow hydrophobic pocket 1 (by replacing the isopropyl moiety of roscovitine), iv) Different aromatic fragments attached to the amino group to occupy hydrophobic pocket 2 that is not occupied with any fragments of ATP such as phenyl, benzoic acid, benzophenone, pyridine, benzothiazole, antipyrine, phenyl thiazole, phenyl pyridazine and benzylidene thiazolinone rings (by replacing the benzyl group of roscovitine) to confer different polarities and electronic effects and might elicit additional hydrogen bindings and hydrophobic interactions with the hot spots in the ATP binding site of CDK2 thus augmenting the activity.

A molecular docking study was firstly performed for all the designed compounds in the CDK2 binding site to support the design hypothesis of the novel compounds and to ensure their ability to attain the essential binding interactions. The predesigned target compounds were then synthesized and subjected to various biological investigations to ensure their CDK2 inhibitory activity and anticancer achievability.