Identification of novel protein kinase CK2 inhibitors among indazole derivatives

Aim. To synthesize the novel purine bioisosteres — indazole derivatives and evaluate inhibitory activity of these compounds towards CK2 in the in vitro system. Methods. Chemical synthesis, 1 H and 13 C NMR spectroscopy, LC-MS method, determination of residual enzyme activity using ATP consumption tests with a luciferase Kinase-Glo® luminescent kinase assay. Results. Known synthetic methods of indazole chemistry were originally applied to the synthesis of 3-aryl-indazole-7-carboxylic acids. Conditions of cross-coupling of 3-bromo-indazole derivatives with arylboronic acids were substantially improved. 3-aryl-indazole 5- and 7-car-boxylic acids have shown IC 50 in a range 3.1–6.5 μM in luciferase luminescent kinase assay. Conclusions. The synthesis of 3-aryl-indazole-7-carboxylic acids has been developed. Novel inhibitors of the protein kinase CK2 among indazole derivatives have been identified among the 3-aryl-indazole 5- and 7-carboxylic acids. It has been shown the crucial impact of carboxyl group on the inhibitory activity


Introduction
Casein kinase 2 (CK2) is a serine/threonine protein kinase usually consisting of two catalytic subunits (α) and/or (α') and two regulatory (β) subunits. This enzyme stands out among the most protein kinases by its high pleiotropicity. CK2 is involved in various biological processes -it has hundreds of substrates for phosphorylation [1] and can use both ATP and GTP as a phosphate source [2]. For a long time, this ubiquity was considered as an obstacle to use CK2 in the drug development until discovering the fact that cancer cells are much more dependent on CK2 than normal cells [3]. It has turned CK2 into an important player in overcoming drug resistance, albeit with some limitations [4]. Abnormally high V. S. Vdovin, S. S. Lukashov, I. P. Borysenko Bioorganic Chemistry ISSN 1993-6842 (on- [5,6]. This increased CK2 level having no genetic background often reduces the treatment efficiency, so until the reasons are not clarified the only possible way of attenuating CK2 activity is therapeutic use of the potent and selective CK2 inhibitors [7]. A number of CK2 inhibitors belonging to different chemical classes have been published [8][9][10][11] and one of them -silmitasertib (CX-4945) -is undergoing clinical trials now supporting the notion that CK2 is recognized as a druggable protein kinase. Therefore, the discovery of small-molecular CK2 inhibitors is a significant area of medicinal chemistry research.
Earlier, we found a number of chemical classes of CK2 inhibitors using the computeraided drug design approach. Here we are representing the CK2 inhibitors identified among the indazole derivatives. Purine bioisosteres are a common target in search for new binders of the ATP-binding site as protein kinase inhibitors [7]. The indazole carboxylic acid derivatives drew our attention for two reasons: the first -an indazole ring is a bioisostere of an adenine ring, the second -a carboxyl group is an essential part of several indazolecontaining CK2 inhibitors, such as 4,5,6,7-tetrabromo-1H-indazole and 3,4,5,6,7-pentabromo-1H-indazole derivatives [8], benzo[g]indazole derivatives [9] and a series of 4,6-disubstituted pyrazine derivatives including inhibitor CC04820. The silmitasertib (CX-4945) molecule also contains a carboxyl group involved in electrostatic interaction with the Lys68 of CK2 ATP-binding site. Thus, this work aims at the synthesis of indazole-carboxylic acids and related derivatives and evaluation of their inhibitory activity against CK2 in the in vitro system.

Materials and Methods
Starting materials and solvents were purchased from commercial suppliers and used without further purification. NMR spectra were recorded on a Varian VXR 400 instrument at 400 MHz for 1 H NMR and 101 MHz for 13 C NMR spectra. Chemical shifts are described as parts per million (δ) downfield from an internal standard of tetramethylsilane, and spin multiplicities are given as s (singlet), d (doublet), dd (double doublet), td (triple doublet), t (triplet), q (quartet), quintet (quintet) or m (overlapped multiplets). HPLC-MS analysis was performed using the Agilent 1100 LC/MSD SL separations module and Mass Quad G1956B mass detector with electrospray ioni za tion (+ve or -ve ion mode as indicated), and HPLC was performed using a Zorbax SB-C18, Rapid Resolution HT cartridge, 4.6 mm × 30 mm, 1.8 µm i.d. column (Agilent P/N 823975-902) at a temperature of 40 °C with gradient elution of 0-100 % CH 3 CN (with 1 mL/L HCOOH)/ H 2 O (with 1 mL/L HCOOH) at a flow rate of 3 mL/min and a run time of 2.8 min. Compounds were detected at 215 nm using a diode array G1315B detector. All tested compounds gave ≥ 95 % purity as determined by these methods.
3-Phenyl-1H-indazole-7-carboxylic acid hydrochloride (8). Procedure A: The mixture of 1.00 g (2.71 mmol) of 1-tert-butyl-7-et hyl 3-bromoindazole-1,7-dicarboxylate 6, 0.35 g (2.88 mmol) of phenylboronic acid, 0.50 g (4.72 mmol) of sodium carbonate, 0.03 g (0.136 mmol) of palladium diacetate, 0.10 g (0.38 mmol) of triphenylphosphine, 15 ml of ethanol and 5 ml of water was refluxed under stirring in Ar atmosphere for 16 hours. After evaporation of solvents under reduced pressure the residue was diluted with 20 ml of dichloromethane and 10 ml of water, dark insoluble precipitate was filtered off and two layers were separated. Chromatographic purification of the residue after evaporation of the dichloromethane layer mixed with the insoluble precipitate Procedure B: The mixture of 1.00 g (2.71 mmol) of 1-tert-butyl-7-ethyl 3-bromoindazole-1,7-dicarboxylate 6, 0.35 g (2.88 mmol) of phenylboronic acid, 0.50 g (4.72 mmol) of sodium carbonate, 0.03 g (0.136 mmol) of palladium diacetate, 0.075 g (0.135 mmol) of dppf, 15 ml of ethanol and 5 ml of water was refluxed under stirring in Ar atmosphere for 16 hours. Then 0.45 g (8 mmol) of potassium hydroxide were added and the mixture was refluxed for one more hour. After evaporation of solvents under reduced pressure the residue was diluted with 20 ml of water, treated with charcoal and filtered. Acidification of the mother liquid gave 0.62 g (83 %) of 3-phenyl-1H-indazole-7-carboxylic acid hydrochloride 8 as a light grey precipitate.

3-(4-Fluoro-phenyl)-1H-indazol-5-ylamine
The reaction was stopped by adding 30 μl of luciferase mix (Kinase-Glo® Luminescent Kinase Assay, Promega) to each microplate well. Luciferase suppresses ATP consumption by kinase and starts the luciferase reaction. The luminescence was counted with Victor reader using Dual Luciferase assay protocol. The heating option on "Victor" was switched on 21 °C.

Results
In order to provide enough hydrophobic interactions to keep binding in the pocket of ATPbinding site we have focused on a synthesis of indazole-carboxylic acids carrying the arylmoiety at the 3-rd position of the indazole ring.
The synthesis of 3-aryl-indazole-5-carboxy lic acid derivatives as the inhibitors of various protein kinases was reported earlier [11][12][13][14]. At the same time only few instances of synthesis of indazole-7-carboxylic acids and no examples of synthesis of 3-aryl-substituted indazole-7-carboxylic acids have been reported. Here we are representing a method of synthesis of 3-phenyl-indazole-7-carboxylic acid that can be easily applied to the synthesis of any 3-aryl-substituted indazole-7-carboxylic acid. We have used synthetic methods known for indazole derivatives with other substitution. The indazole ring assembly starts from the 3-methyl-anthranilic acid 1 ester (Fig. 1). Bromination or iodination of the indazole-7-carboxylic acid ester 2 leads to the 3-halogeno-substituted intermediates 3 [15] that could possibly be converted into the 3-aryl-indazole-7-carboxylic acid 4 esters under the Suzuki-Miyaura cross-coupling conditions. This convenient synthetic route may involve a wide range of aryl-and heteroaryl-boronic acids, however, it requires additional steps of protection/deprotection of NH on the indazole ring. The halogen on the 3-rd position of the indazole ring shows no reactivity unless it's NH is covered with a protective group, moreover, NH-arylation with the arylboronic acids takes place under the same conditions as crosscoupling.
Moving along this synthetic route (Scheme 1) we have obtained the ethyl indazole-7-carboxylate 2 in moderate yield by the nitrosylation of the ethyl 3-methylanthranilate 1 with tret-butylnitrite in acetic acid. After the bromination and the N-Boc protection, this intermediate was used in the Suzuki-Miyaura cross-coupling with phenylboronic acid. Several attempts were unsuccessful. The crosscoupling product was not formed when BINAP-Pd(0), tBuOK in toluene were used. However, the ethyl 3-phenyl-indazole-7-carboxylate appeared in the reaction mixture when the reaction ran in aqueous ethanol media in presence of Pd(PPh 3 ) 4 and Na 2 CO 3 as a base. The replacement of Pd(PPh 3 ) 4 with Pd(OAc) 2 /dppf enhanced the yield of the process. Noteworthy, under the conditions of mentioned reaction the indazole moiety completely lost its N-Boc protection. Additionally, a significant amount -up to half the ethyl es- ter -underwent hydrolysis, so, it turned out reasonable to complete the hydrolysis by adding KOH and refluxing for one more hour before the isolation of the final product. The isolation of carboxylic acid also has an advantage -the rest of a catalyst and most impurities can be easily removed from the basic aqueous solution with further precipitation of the 3-phenyl-indazole-7-carboxylic acid after acidification (Fig. 2). 5-substituted 3-aryl-1H-indazoles 9-14 were synthesized following known procedures.

Discussion
We have tested the compounds 8-14 for inhibitory activity toward human CK2α catalytic subunit using Kinase-Glo® Luminescent Kinase Assay. The chemical structure of substituents and IC 50 values are presented in Table 1.
Several important structural features of indazole derivatives can be identified from a qualitative analysis of their activity toward CK2. As can be seen from with a carboxyl group at the 7-th position of the indazole ring shows the same level of inhibition of CK2 as compounds 10-12 with a carboxyl group at the 5-th position. In our opinion[, the] electrostatic interaction between negatively charged carboxylate and side chain residues of the ATP-binding pocket of CK2 plays an important role in the affinity of compounds 8 and 10-12. Compounds 9 and 14 having neutral nitrile group (which meanwhile can act as a hydrogen bond acceptor in the same way as carboxylate) demonstrate no inhibitory activity unlike the corresponding carboxylic acid derivatives. Compound 13 with the slightly basic hydrogen bond donor amino group also shows no inhibitory activity. The structure of the aromatic R 3 substituents in a range of the studied compounds seems not to be important. A slight advantage of the pyridine moiety over phenyl rings among other reasons may be a result of the contribution of higher polarity pyridine ring in the water solubi lity of the compound.

Conclusion
The synthesis of 3-aryl-indazole-7-carboxylic acid has been developed. Novel inhibitors of the protein kinase CK2 among indazole derivatives have been identified. The activity of the studied compounds strongly depends on the presence of carboxyl group as substituent R 5 or R 7 . The identified indazole derivatives have shown promising inhibitory activity toward CK2, although they require further chemical optimization and biological investigations.