Cyclin-dependent kinases (CDKs), belong to a group of serine/threonine kinases involved in the regulation of cell cycle progression, neuronal function, differentiation and apoptosis. Their activity is tightly regulated by multiple mechanisms including binding to corresponding cyclins, of which level of expression oscillates throughout the different phases of the cell cycle. Different CDK/cyclin complexes are activated during each cell cycle step through G1, S, G2, M phases. Sequential phosphorylation of the retinoblastoma protein (pRb) by CDK4/cyclin D, CDK6/cyclin D in early G1 phase and CDK2/cyclin E in late G1 phase causes the release of the E2F, proteins of transcription factor. In turn, E2F proteins lead to transcriptional activation of a set of genes required for entry into S-phase of the cell cycle. CDK2 is subsequently activated by cyclin A during the late stages of DNA replication, S-phase, and promotes appropriately timed deactivation of E2F to prevent apoptosis triggered by persistent E2F activity. Finally, CDK1 in complex with cyclin A or B is thought to be have roles in regulating the G2/M checkpoint and driving cells through mitosis.
In addition to the cell cycle control, other roles have been determined for CDK2, 7, 8 and 9. For example, CDK2/Cyclin E is important to the p53 mediated DNA damage response pathway and CDK7, 8 and 9 are involved in the regulation of transcription initiation and elongation through phosphorylation of RNA polymerase. Therefore, CDKs affect cell growth and survival through several different mechanisms and proper regulation of CDK activity is important to various cellular processes. It is now recognized that deregulation of CDKs by abnormal high expression of cyclin such as cyclin D and cyclin E or mutation occurs in many human tumors. For example, the expression and catalytic activity of CDK2/cyclin E complexes is increased in colorectal, ovarian, breast, and prostate cancers and elevated expression of cyclin E in primary tumors has correlation with poor survival rates for breast cancer patients. Abnormal expression of CDK1/cyclin B complexes has been also observed in some cases, prostate adenocarcinoma and lung cancer.
Although a report have shown that CDK2 may not be required for cell cycle progression and proliferation, recent reports suggested that melanocytes and hepatocytes may be dependent on CDK2 for proliferation and survival. Also, an investigation of simultaneous depletion of CDK1 and CDK2 was reported to provide increased efficacy in anti-proliferation of tumor cell lines, compared with targeting either CDK1 or CDK2 alone. In addition, emerging evidences indicate that certain tumor cells may require specific interphase CDKs for proliferation. Thus inhibition of CDKs may provide an effective strategy to control tumor growth as attractive targets for cancer therapy.
To date, a number of small-molecule CDK inhibitors are currently under clinical trials. These inhibitors are flat, small heterocycles which act by competition with ATP in the kinase ATP-binding site. Among them, flavopyridol, was the first CDK inhibitor to enter clinical evaluations. R-Roscovitine (trisubstituted purine analog) and BMS-387032 (aminothiazole) are selective for CDK2/cyclin E and PD-0332991 (pyridopyrimidine) is highly selective for CDK4/cyclin D and CDK6/cyclin D. Indirubin, a bis-indole scaffold and its derivatives have been investigated with considerable interests as potent inhibitors targeting important protein kinases such as CDK, GSK-3β, and aurora kinases.
In WO 2005/070416 A1, the inventors of present application has disclosed ‘indirubin derivatives having anticancer property against human cancer cell line’. In this disclosure, indirubin derivative having anticancer property by inhibiting cell proliferation as to human cancer cell line has been disclosed. Among disclosed indirubin derivatives, 5′-bromo-5-nitro-indirubin-3′-oxime showed the most potent anti-proliferative effects (IC50=0.79˜2.9 μM) against various cancer cell lines. However, the further substitution of radicals at the 5′ position in indirubin skeleton has not been made as well as anti-cancer activity regarding these compounds has not been measured. Further, there has been no report regarding the synthesis and biological evaluations exploring the effect of various substitutions at the 5′ position of indirubin skeleton.
On the other hand, the inventors of present application have found that the additional substitution at the 5′ position is clearly favorable compared with 5-nitro-indirubin-3′-oxime analog. Therefore, the inventors of present application have completed the invention by design, synthesis and biological evaluation including CDK inhibitory activity, anti-proliferative activity and in-vivo anti-cancer activity of novel 5′,5-substituted indirubin-3′-oxime analogs.