Pyridines, pyridazines and guanines as CDK2 inhibitors: a review

Cell cycle progresses by the activation of cyclin and cdk complexes. They act as check points and regulate the transition of cell cycle from one phase to another. Cdk2 inhibitors decrease the kinase activities by blocking the transition from G1 to S phases. In this article, we present a review on purine based cdk2 inhibitors. The review covers in great detail the different structures and the effects on cdk2 inhibition by various substitutions. The substitutions that most greatly influence the orientation and binding towards the ATP binding site are discussed and the effects of several substituents that explored the active site region of cdk2 with bound inhibitor has provided a rationale to review four relatively new families of purine based cdk2 inhibitors such as imidazo[1,2-a ]pyridines, imidazo[1,2-b ]pyridazines, 1 H -pyrazolo[3,4-b ]pyridines and O 6 - substituted guanines, respectively. The orientation and hydrogen bond interactions of analogues with Leu83, Asp86 and Lys33 residues influence the binding and the major features responsible for effective cdk2 inhibitor discovery and design are presented.


Introduction
Cell proliferation is a consequence of positive signals which promote cell division and negative signals which suppress the process.Key factors in this signaling cascade are a series of cyclin dependent kinases (cdks) 1 .It has been shown that they are also required for replication of viruses that replicate only in dividing cells, such as adeno-and papillomaviruses as well as in nondividing cells, such as HIV-1 and herpes simplex virus types 1 and 2 (HSV-1 and HSV-2) 2 .Cyclin-dependent kinases are a family of serine/threonine kinases which play a crucial role in cell cycle control 3 and are involved in diverse cellular processes, in regulation of cell division (cdks1, 2, 3, 4, 6 and 7), transcription (cdks7, 8 and 9) or maintenance of the structure of the cytoskeleton (cdk5) 2 .
Cyclin dependent kinases control the cell cycle progression operating at the transition from G 2 to M, G 1 to S phases, and progression through S phase, regulated by a complex set of mechanisms, including the presence of activating cyclins, regulatory phosphorylations, and endogenous cdk inhibitors at checkpoints 4 .Cell cycle progresses by the activation of Cyclin and cdk complexes 5 .These cyclins and cdks function as check points regulating the transition from one phase of cell cycle to another.Structural studies have explored the active and inactive states of cdk2.Monomeric form was inactive, while association of Cyclin A with cdk2 and Thr160 phosphorylation results active cdk2 6 .Active complex phosphorylate and inactivate members of the retinoblastoma protein (Rb) family that are negative regulators of G1 and S-phase progression, leading to induction of E2F-regulated gene expression and cell proliferation 7 .Cdk inhibitors decrease the kinase activities of the cyclin/cdk complexes, blocking the transition from G1 to S phases 8 .Activation of cdk2 results in rotation of N-and C-terminal domains leading to a slight widening of ATP cleft 9 .The movement of PSTAIRE helix and Glu51 and the subsequent reorganization leads to reshaping of the phosphate-binding site 10,11 .
Following the discovery of olomoucine and roscovitine (Figure 1) as selective ATP competitive cdk2 inhibitors, much effort has been devoted to find more specific and potent inhibitors because kinases within the cell share a high degree of sequence similarity at the active site.X-ray crystallographic analysis of a substantial number of cdk2/inhibitor complexes has elaborated the diverse binding modes of different inhibitors in atomic detail 3 .Several types of cdk inhibitors, shown in Figure 1, have so far been described: staurosporine, UCN-01 12 , flavopiridol (L86-8275) 13 , butyrolactone I 14 , other purine derivatives [15][16][17][18] , indirubin 19 , paullones 20 and others [21][22][23] .Based on the studies that explored the active site region of cdk2 with bound inhibitor, and the role of cdk2 in cell cycle progression and proliferation, cdk2 acts as a potential therapeutic target in several proliferative diseases, including cancer 11 .Development of successful small molecule cdk2 inhibitors as anticancer agents has provided a rationale to review four relatively new families of purine based cdk2 inhibitors with emphasis on substituted groups and their influence towards activity and selectivity at the ATP binding site.Therefore, analogues with sulfonamide and amino substituted imidazo[1,2-a]pyridines, imidazo [1,2-b]pyridazines, 4-, 5-or 6substituted 1H-Pyrazolo [3,4-b]pyridines and O 6 -substituted guanines are reviewed to explore the effect of substituents on cdk2 inhibition.

Effect of sulfonamide substitution
Structural studies showed that the aniline group substitution with sulfonamide was beneficial for cdk2 activity 37

Effect of 4-position substitution
Replacement of oxygen with either nitrogen or sulfur at position 4 (32, 33; IC 50 : 4.4µM, >25µM) resulted in a significant loss of cdk2 activity.Straight chain, branched and cyclic alkoxy groups are tolerated as they extend into the space occupied by the ribose of ATP without any specific contacts with the protein.Polar substituents like hydroxyl and dimethylamino resulted in loss of activity 41 .

O 6 -substituted guanines
ATP ribose binding domain was probed with ATP competitive O 6 -substituted guanine derivatives 44 .An increase in inhibition observed with increase in chain length (Figure 9) from O 6 -methylguanine to O 6 -pentylguanine and for O 6 -alkoxyalkyl purine derivatives 40 and 41 (IC 50 : 15 ± 2 µM, 16 ± 1 µM), respectively.Cyclohexylmethyloxypurine, NU2058 showed similar inhibition (42, IC 50 : 17 ± 2 µM) as observed with other 6-alkoxyalkylpurine derivatives.Replacement of the cyclohexyl group of NU2058 with a phenyl group in compound 43, (O 6benzylguanine) resulted in a 2-3 fold reduction in activity (IC 50 : 35 ± 6 µM), whereas substitution on the O 6 -benzyl group (44, 45 and 46) resulted in loss of potency (% inhibition: 52 ± 7, 52 ± 3 at 100 µM and 49 ± 14 at 10 µM).This might be due to the substituents unable to form favorable hydrogen bonds within the ribose binding domain.SAR studies suggest that a broad range of substituents are tolerated at the O 6 -position, but none resulted in appreciable activity or specificity as seen with NU2058, which suggests that the hydrogen bonds formed by the substituents within the ribose pocket contributed greatly towards potency and selectivity.
X-ray structure of NU2058 bound to monomeric cdk2 revealed that it forms a triplet of hydrogen bonds within the ATP binding site between residues in the hinge region of cdk2 and the NH 2 , N3, and N9 nitrogen atoms of the inhibitor 43 .Comparison of structures (Figure 9) of NU2058 (pdb code: 1e1v) and olomoucine (pdb code: 1w0x) bound to monomeric cdk2 revealed that NU2058 represents a different class of inhibitor to olomoucine [15] (IC 50 : 7 µM) as the two compounds bind in different orientations 45 .Extending this observation, SAR studies for a series of N 2 -substituted O 6 -cyclohexylmethylguanine derivatives 46,47 resulted in the discovery of potent inhibitor NU6102 (IC 50 : 0.0054 ± 0.001 µM) (Figure 10, pdb code: 1h1s).

Conclusions
A number of compounds with diverse substituents explored the potential active site region of cdk2 resulted in micromolar inhibition.The orientation and hydrogen bond interactions of analogues with Leu83, Asp86 and Lys33 greatly influence the binding for purine based cdk2 inhibitors and the major features responsible for effective cdk2 inhibitor discovery and design for four families are: -presence of hydrophobic phenylamino moiety on imidazo

Figure 2 .
Figure 2. Imidazo[1,2-a]pyridine class of inhibitors (values of IC 50 refer to the affinity of the compound with ATP).Binding of imidazo[1,2-a]pyridine analogues, compounds 5 and 6 bound with selected residues (labeled and drawn in stick representation) of pdb proteins 1oiq and 1oir.Compound 5 makes three hydrogen bond interactions with Leu83 and Lys33 residues and compound 6 displaying three hydrogen bond interactions with Leu83 and Lys 89 respectively.Hydrogen bonds are indicated by dotted lines.
[1,2-a]pyridines.-presence of 4-sulfonamide substituent on the phenylamino moiety of imidazo[1,2-b]pyridazines to reduce lipophilicity -4-, 5-and 6-position heterocycle substitutions are well tolerated on pyrazolo[3,4-b]pyridines.-increase in chain length from O 6 -methylguanine to O 6 -pentylguanine and presence of O 6cyclohexyl group.Artemisia annua.At present a few potential microorganisms that can carryout the same transformation reaction have been identified.Ten of her students have been awarded their Ph.D. degrees and more than six students are in different stages of completion.She has 26 years of research experience and her research interests include plant tissue culture of medicinal plants, environmental biotechnology and bioinformatics.In the area of plant tissue culture her group was successful in obtaining enhanced amounts of podophyllotoxin (anticancer compound) from transformed cultures of Podophyllum hexandrum.She published 35 papers in the fields of plant tissue culture, molecular biology and environmental biotechnology.Professor Srinivas was born in Andhra Pradesh, India in 1962.He obtained his doctorate from Berhampur University in 2002 for his work in the field of Pharmacognosy and Phytochemistry.The work dealt with the biological screening of some folklore medicinal plants used in Arthritis, Diabetes and Liver disorders.He was Principal Investigator for AICTE sponsored R&D project 'Bioassay guided principles of medicinal plants having different biological activities and attempt to made formulations' during 1998-2000.He worked as a Lecturer (1991-94), Sr. Lecturer (1997-02) and obtained a Reader (2002-04) position at Roland Institute of Pharmaceutical Sciences, Berhampur, Orissa.Since 2004, he accepted the position of Principal and Professor at Sri Venkateswara College of Pharmacy, Etcherla, Srikakulam.His research interests cover pharmacognosy, phytochemistry, medicinal chemistry, molecular modeling and drug design.Five PhDs have been awarded under his guidance and three students are at various stages of their research.He published 30 papers in the field of pharmacognosy, phytochemistry and medicinal plants.