br Acknowledgements We are grateful to the National Natural
Acknowledgements We are grateful to the National Natural Science Foundation of China (Grant Nos. 21571033 and 81503099) and the New Drug Creation Project of the National Science and Technology Major Foundation of China (Grant No. 2015ZX09101032) for financial aids to this work. The research was also supported by Jiangsu Province Natural Science Foundation (Grant No. BK20150643). The authors would also like to thank the Fundamental Research Funds for the Central Universities (Project 2242013K30011) for supplying basic facilities to our key laboratory. We also want to express our gratitude to the Priority Academic Program Development of Jiangsu Higher Education Institutions for the construction of fundamental facilities (Project 1107047002). Jiangsu KeyGen Biotech Company was appreciated for completing the in vivo tests.
Introduction Protein kinase CK2 is a highly conserved serine/threonine protein kinase that is ubiquitously distributed in multiple cell types . It is a holoenzyme comprises a heterotetramer consisting of two catalytic (α and/or α′) and two regulatory (β) subunits. It is recognized as one of the most pleiotropic protein kinase with more than 300 substrates , many of which have been confirmed to play crucial roles in the dysfunction of programmed cell apoptosis and death, migration, differentiation and oncogenic transformation [, , , ]. Overexpression of CK2 has been observed in many cancers, including hematologic cancers such as acute myelogenous leukemia (AML), chronic lymphocytic leukemia (CLL), T-cell acute lymphoblastic leukemia (T-ALL) and multiple myeloma (MM) [, , , ]. Downregulation of CK2, either by transfection of specific siRNA or plasmid-based expression of kinase-inactive CK2, resulted in reduction of cancer cell viability and induction of apoptosis [, , ]. Thus, CK2 is becoming an important target for the treatment of cancer and other life-threatening diseases. Several compounds, belonging to different idasanutlin of chemical compounds like anthraquinones, xanthenones, coumarins, pyrazolo-triazynes, peptides, benzimidazoles and polyoxygenated benzotriazoles have been reported as CK2 inhibitors [, , , , , , , , , ]. However until now, only one of them, the pyridoquinoline CX-4945, has entered in Phase II clinical trials as potential anticancer drug [, , , ]. Herein, we report the structural optimization and docking studies of a new family of ATP-competitive CK2 inhibitors with the 2-amino-3-cyano-4H-pyranobenzoquinone core.
Results and discussion Most of the CK2 inhibitors known today are small and flat scaffolds having several rings, which fits into the active site of the CK2 and competes with ATP . One of the representative CK2 inhibitors is the anthraquinone emodin . Due to our interest in antitumoral quinones [, , , , ], a set of 60 structurally diverse natural and synthetic quinones  was screened on CK2 activity. From this screening compound 1 emerged with an IC50 < 1 μM. Compound 1 was obtained through a multicomponent reaction from the natural benzoquinone embelin (I) , 4-chlorobenzaldehyde (II) and malonotrile (III). Based on calculated values of the Fukui function (f−) as local density functional descriptor , the synthesis of this compound can be rationalized by the initial formation of a conjugated electron-deficient enone (A) through a Knoevenagel condensation of embeline (I) and 4-chlorobenzaldehyde (III). The next step of this mechanism could involve a Michael addition of malononitrile (II) to the reactive quinone methide intermediate to yield the intermediate (B), which experiments an intramolecular cyclization to yield the intermediate C that tautomerizes to compound 1 (Scheme 1). Compound 1 was tested for its inhibitory activity towards the human CK2 holoenzyme following the procedure described earlier [22,35]. The synthetic peptide RRRDDDSDDD was used as a substrate. For initial testing, inhibition was determined at inhibitor concentrations of 10 μM in DMSO as solvent. IC50 value was determined by measuring CK2 inhibition at eight different concentrations ranging from 0.001 to 100 μM in appropriate intervals and calculated from the resulting dose-response curve . Compound 1 was active toward the target enzyme with an IC50 value of 0.90 ± 0.05 μM. As a control the well-known CK2 inhibitor, emodin was subjected to the same test conditions which had an IC50 value of 0.58 ± 0.05 μM (Fig. 1).