The synthesis of 5-hetarylamino-3-aryl-1H-indazoles as inhibitors of protein kinase CK2

Aim. Basing on our earlier finding of inhibitory activity of 5-(4-quinazolylamino)-3-arylinda-zoles against human protein kinase CK2, the synthesis of new nitrogen containing heterocyclic derivatives was performed in order to find novel inhibitors of this kinase. Methods. Organic synthesis, NMR spectroscopy. Results. A series of 4-chloroquinazolines, 4-chloroquinolines, 4-chloropyrazolo[3,4-d]pyrimidines and 4-chlorothieno[2,3-d]pyrimidine was synthesized. Reaction of these intermediates with 5-amino-3-(3,4-dichlorophenyl)-indazole gave us a series of 14 novel heterоcyclic derivatives of 5-amino-3-arylindazole. Conclusions. Besides new quinazoline derivatives – the quinoline and thieno[2,3-d]pyrimidine derivatives of similar structure but different polarity were obtained. Also a series of 1-methylpyrazolo[3,4-d]py-rimidine derivatives with decreased lipophilicity was synthesized.


Introduction
regulating transcription, translation, cell proliferation, survival, and apoptosis [6]. Overexpression and overactivity of CK2 are associated with the development of neurodegenerative, inflammatory, cardio-vascular diseases, virus infections, and all the cancers that have been examined [7,8]. Therefore, smallmolecular inhibitors of CK2 would be important compounds for the development of clinical agents.
Indazole, also called benzopyrazole, is a heterocyclic molecule in which the pyrazole ring is fused with the benzene ring. Indazole derivatives have been reported as inhibitors of FGFR1 kinase, Aurora kinase, Pim kinase, Bcr-Abl tyrosine kinase [27]. Also, previously it has been described that 5-amino-3-arylindazole core (Fig. 1) could be a good starting point for the protein kinase inhibitors design [28].
Taking into account this fact, the main aim of this work was to find novel inhibitors of human protein kinase CK2 among the 5-hetarylamino-1H-indazole derivatives.

Materials and Methods
Starting materials and solvents were purchased from commercial suppliers and used without further purification. 1 H NMR spectra were recorded on a Varian Mercury 300 instrument at 302 MHz or Varian VXR 400 instrument at 400 MHz. 13 C NMR spectra were recorded on a Varian Mercury 300 instrument at 76 MHz or Varian VXR 400 instrument at 101 MHz. Chemical shifts were described as parts per million (δ) downfield from an internal standard of tetramethylsilane. All tested compounds had ≥ 95 % purity as determined by this method.
( 2 -C h l o r o -5 -n i t r o p h e n y l ) -3 , 4dichlorobenzophenone, 5. To a stirred suspension of 25 g (0.124 mol) of 2-chloro-5-nitrobenzoic acid 4 in 50 ml of 1,2-dichloroethane, 2-3 drops of DMF and 15 ml (0.205 mol) of thionyl chloride were added. The mixture was being refluxed until the complete dissolution of acid and cessation of release of gaseous SO 2 and hydrogen chloride in a bubble counter over the condenser. Then volatile components were evaporated under reduced pressure, 25 ml of 1,2-dichloroethane were added to the residue and evaporation of volatile components was repeated. To the crude 2-chloro-5-nitrobenzoyl chloride, 70 ml (0.62 mol) of 1,2-dichlorobenzene and 17 g (0.127 mol) of aluminium chloride were added. The mixture was being heated at 100 °C under stirring for 4-6 hours until cessation of release of gaseous hydrogen chloride. After cooling to room temperature, the viscous reaction mixture was diluted with 250 ml of cold water and 10 ml of conc. hydrochloric acid. Crude reaction product was extracted with 100 ml of CH 2 Cl 2 . The organic layer was evaporated at 100 °C under 20 mm Hg. The residue was recrystallized from isopropanol giving 35 g (85 %) of 5 as yellow crystals. 1  3-(3,4-Dichlorophenyl)-1H-indazol-5amine, 6. To a solution of 35 g (0.106 mol) of ketone 5 in 50 ml of dimethylformamide, 7 ml of hydrazine hydrate were added. The reaction mixture was being refluxed for 1 hour. After cooling to room temperature 5-nitro-3-(3,4dichlorophenyl)-1H-indazole hydrochloride appeared as a bright yellow precipitate. It was separated by filtration and further recrystallized from dimethylformamide to remove the impurity of (2,3-dichlorophenyl)-1H-indazole isomer. After recrystallization, 8.5 g (23 %) of pure 5-nitro-3-(3,4-dichlorophenyl)-1H-indazole hydrochloride were obtained as a yellow powder. 1  8.5 g of 5-nitro-3-(3,4-dichlorophenyl)-1Hindazole hydrochloride were dissolved in a mixture of 300 ml of isopropanol and 100 ml dimethylformamide, then 3 g of Raney nickel were added. Well stirred reaction mixture was heated under reflux while 10 ml of hydrazine hydrate were being added slowly dropwise. The reduction process took at least 8 hours and an additional amount of hydrazine hydrate was added if disappearance of deep yellow-orange coloration of reaction mixture did not take place. Hot reaction mixture was filtered from nickel and solvents were evaporated under reduced pressure. Solid residue was washed with water and dried giving 5.35 g (78 %) of 6 as red-brown solid. 1  General procedure of synthesis of heterocyclic chloro derivatives. To a suspension of 20 mmol of heterocyclic compound 8-21 in 20 ml of phosphorous oxychloride[,] 4.2 g (20 mmol) of phosphorous pentachloride were added. The mixture was being refluxed under stirring until complete dissolution of starting material. After cooling to room temperature the volatile material was evaporated under reduced pressure. Residue was dissolved in 50 ml of chloroform and carefully basified with NaHCO 3 solution under good stirring (caution! effervescence takes place). Organic layer was separated, dried with Na 2 SO 4 and evaporated, leaving chloro-derivative 22-35, that was immediately used in the next step without purification.
General procedure of arylation of 5-amino-3-(3,4-dichlorophenyl)-1H-indazol 6. A solution of 2.78 g (10 mmol) of 6 in 5 ml of dimethylformamide was added to the 10 mmol of corresponding chloro-derivative 22-35. The mixture was refluxed for 3-5 min to complete dissolution of all components and cooled to room temperature. Solid hydrochloride salts of products 36-49 were filtered off, washed with dimethylformamide and acetone. Soluble in DMF products were converted to the bases by adding 0.25 ml of triethylamine and 25 ml of water to the reaction mixture before filtration. Several products were additionally purified with column chromatography on silica gel, using the eluent system CH 2 Cl 2 :MeOH 95:5 ÷ 9:1.

Results and Discussion
Earlier we have found new promising CK2 inhibitors among the 4-quinazoline derivatives of 5-amino-3-arylindazoles 1-3 [29]. The inhibitory activity of these compounds against CK2 was tested in in vitro assay with radiolabeled ATP [30]. They have shown IC 50 values in a range 0.007-0.1 µM [29]. The structures and inhibitory activity of 1-3 are represented in Figure 2.
Besides the high IC 50 values all three compounds 1-3 also have a significant disadvantage. They have too high lipophilicity (LogP>5) and as a result -poor water solubility. We have tried to modify chemical structure to overcome this disadvantage. We have selected the 3,4-dichlorophenyl substituent in the 3-position as a substituent of the most active compound 3 and left it unchanged. Meanwhile, we have focused on varying of available heterocyclic residues attached to the 5-amino-group. The 5-amino-3-(3,4-dichlorophenyl)-1H-indazole 6 interme- diate has been synthesized following Scheme 1.
All novel synthesized compounds were tested for in vitro inhibitory activity towards CK2. Results of this testing as well as SAR and docking studies will be described in the separate article in the next issue of this journal.

Conclusions
Thus, following the previously used synthetic route, a series of 14 novel 5-hetarylamino-3-arylindazole derivatives was synthesized.