Synthesis and cytotoxic activity of (2-arylquinazolin-4-yl)hydrazones of 2-hydroxybenzaldehydes as potential casein kinase 2 (CK2) inhibitors published by Ural Federal University eISSN 2411-1414 chimicatechnoacta.ru ARTICLE 2023, vol. 10(2), No. 202310211 DOI: 10.15826/chimtech.2023.10.2.11 1 of 8 Synthesis and cytotoxic activity of (2-arylquinazolin- 4-yl)hydrazones of 2-hydroxybenzaldehydes Emiliya V. Nosova ab* , Ilya I. Butorin a , Margarita D. Likhacheva a, Svetlana K. Kotovskaya a a: Institute of Chemical Engineering, Ural Federal University, Ekaterinburg 620009, Russia b: I. Postovsky Institute of Organic Synthesis, Ural Division of the Russian Academy of Sciences, Ekaterinburg 620219, Russia * Corresponding author: emilia.nosova@yandex.ru This paper belongs to a Regular Issue. Abstract 2-Phenyl-6,7-difluoro and 2-(4-fluorophenyl)quinazoline derivatives bear- ing salicylidenhydrazino fragments at position 4 were prepared based on 4,5-difluoroantranilic acid or anthranilamide. Molecular docking to casein kinase 2 was performed; compounds with high in silico activity to CK2 were revealed. Cytotoxic activity of the synthesized compounds was stud- ied on cancer cell line MDA-MB-231 and normal cell line WI26 VA4. Keywords 2-arylquinazolines salicylidenhydrazines in silico activity casein kinase 2 inhibitor cytotoxic activity Received: 28.03.23 Revised: 26.04.23 Accepted: 27.04.23 Available online: 04.05.23 Key findings ● N-Salicylidene-N’-(6,7-difluoro-2-phenylquinazolin-4-yl)-hydrazines were obtained based on 4,5-difluoroantranilic acid. ● N-Salicylidene-N’-(2-(4-fluorophenyl)quunazolin-4-yl)-hydrazines were synthesized based on anthranilamide. ● Molecular docking towards casein kinase 2 was performed, cytotoxic activity was studied. © 2023, the Authors. This article is published in open access under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). 1. Introduction Fluorine-containing quinazoline derivatives represent a class of anticancer agents. Phosphoinositide 3-kinase and Idelalisib (Zydelig) is used as a medication to treat certain blood cancers; the molecule acts as inhibitor of P110δ, the delta isoform of the enzyme phosphoinositide 3-kinase [1]. Gefitinib (Iressa) is a selective EGFR tyrosine kinase (EGFR-TK) inhibitor. This agent retards growth of various human tumor cell lines, metastasis, and angiogenesis, ac- celerates the apoptosis of tumor cells and enhances the ef- ficiency of chemotherapy, radiation, and hormone therapy [2]. Casein kinase 2 is a promising template for designing anticancer drugs. Protein kinase CK2 is a ubiquitous, highly conserved, and constitutively active serine/threonine pro- tein kinase; overexpression and hyperactivation of CK2 was observed in a wide variety of cancers, including breast, lung, prostate, colorectal, and renal [3–11]. For this reason, CK2 represents an attractive target for chemotherapy [12– 16]. Anilino-substituted 2,6-naphthyridines were found to act as potent CK2 protein kinase inhibitors [17]. The search of antitumor agents inhibiting kinases among bi- and tricy- clic derivatives of six-membered heterocycles with two ni- trogen atoms is promising [18–23]. (2-Phenylquinazolin-4-yl)hydrazones of 2-hydroxyben- zaldehydes 1a–c, 2a–c, 3a,b (Figure 1) were reported previ- ously [24]. The aim of the present article is to describe a series of new 2-arylquinazolines 3c,d, 9a,b, bearing salicylidenhy- drazino group at position 4, to estimate the interaction of quinazolines 1–3, 9 with important target of anticancer agents (CK2) by using molecular docking method, and to study cytotoxic and CK2 inhibition activity of some salicyl- idenhydrazino-substituted 2-arylquinazolines. 2. Experimental 2.1. General Unless otherwise indicated, all common reagents and solvents were used from Sigma Aldrich without further purification. http://chimicatechnoacta.ru/ https://doi.org/10.15826/chimtech.2023.10.2.11 mailto:emilia.nosova@yandex.ru http://creativecommons.org/licenses/by/4.0/ http://orcid.org/0000-0002-0177-1582 https://orcid.org/0000-0003-3403-5079 https://crossmark.crossref.org/dialog/?doi=https://doi.org/10.15826/chimtech.2023.10.2.11&domain=pdf&date_stamp=2023-05-04 https://journals.urfu.ru/index.php/chimtech/rt/suppFiles/6708/0 Chimica Techno Acta 2023, vol. 10(2), No. 202310211 ARTICLE 2 of 8 DOI: 10.15826/chimtech.2023.10.2.11 Figure 1 Structure of anticancer agents Idelalisib, Gefitinib and previously prepared salicilidenehydrazono substituted quinazolines (1a–c, 2a–c, 3a,b). 1, 2: R = H (a), 4-OH (b), 3,5-diBr (c); 3: R = H (a), 5-NO2 (b). The 1Н NMR, 13C NMR and 19F NMR spectra were obtained on a Bruker Avance II DMX400 spectrometer using DMSO- d6 as the solvent. The 1Н NMR experiments chemical shifts were referenced to the hydrogen resonances of the solvent (DMSO, δ = 2.50 ppm). Carbon chemical shifts were refer- enced to the carbon resonances of the solvent (CDCl3, δ = 77.16 ppm). 19F NMR spectra were recorded with CFCl3 (C6F6 was used as secondary reference, δF –162.9 ppm). Mass spectra were recorded on a SHIMADZU GCMS-QP2010 Ultra instrument with electron ionization (EI) of the sam- ple. Microanalyses (C, H, N) were performed using a Per- kin–Elmer 2400 elemental analyzer. Melting points were measured on the instrument Boetius. Microanalyses (C, H, N) were performed using the Perkin–Elmer 2400 elemental analyzer. 2.2. Preparation of intermediates 2.2.1. (E)-2-(4-Fluorophenylydeneamino)benzamide (6) 4-Fluorobenzaldehyde (0.67 g, 5.4 mmol) was added to a solution of 2-aminobenzamide 5 (0.74 g, 5.4 mmol) in eth- anol (9.62 mL). The mixture was stirred at room tempera- ture for 3 h, and the white precipitate was filtered off and recrystallized from acetonitrile. Yield 1.07 g (82 %), mp 194–196 °C (lit. 197–200 °C [25]). 1Н NMR, δ, ppm: 7.18 d (1H, Ar, 3J 8.1 Hz), 7.25–7.40 m (3H, Ar), 7.4 br. s (1H, NH), 7.5–7.6 m (2H, Ar), 7.95–8.05 m (2H, Ar), 8.2 br. s (1H, NH), 8.59 s (1H, CH=N). 19F{H} NMR, δ, ppm: –107.05 s. Found, %: C 69.32, Н 4.62, N 11.49. C14H11FN2O. Calculated, %: C 69.41, Н 4.58, N 11.56. 2.2.2. 2-(4-Fluorophenyl)quinazolin-4(3Н)-one (7) Copper(II) chloride (0.67 g, 4.9 mmol) was added to the suspension of 2-(4-fluorophenylydeneamino)benzamide 6 (0.90 g, 3.7 mmol) in ethanol (14 mL) and the mixture was refluxed for 5 h. After cooling the precipitate was filtered off and recrystallized from dimethyl sulfoxide. Yield 0.705 g (79%), mp 282–284 °C (lit. 284–286 °C [36]). 1Н NMR, δ, ppm: 7.26–7.31 m (2H, Ar), 7.46–7.49 m (1Н, Ar), 7.68– 7.70 m (1Н, Ar), 7.77–7.81 m (1Н, Ar), 8.13–8.15 m (1Н, Ar), 8.27–8.30 m (2Н, Ar), 12.4 br. s (1Н, NH). 19F{H} NMR, δ, ppm: –108.90 s. Found, %: C 70.11, Н 3.90, N 11.53. C14H9FN2O. Calculated, %: C 69.99, Н 3.78, N 11.66. 2.2.3. 2-(4-Fluorophenyl)-4-chloroquinazoline (8) Phosphorus oxychloride (3.2 mL) was added to quinazolin- 4-one 7 (0.65 g, 2.7 mmol), and the mixture was refluxed for 2 h. The reaction mixture was cooled to room tempera- ture and poured onto ice. The white precipitate was filtered off, washed with water, dried in air and used without addi- tional purification. Yield 0.52 g (75%), mp 148−150 °C. 1Н NMR, δ, ppm: 7.41–7.45 m (2H, Ar), 7.86–7.88 m (1Н, Ar), 8.14–8.15 m (2Н, Ar), 8.29–8.32 m (1Н, Ar), 8.53–8.57 m (2Н, Ar). 19F{H} NMR, δ, ppm: –109.38 s. Found (%): C 64.89, H 3.03, N 10.92. C14H8ClFN2. Calculated (%): C 65.00, H 3.12, N 10.83. 2.2.4. 2-(4-Fluorophenyl)-4-hydrazinoquinazoline (4b) Hydrazine monohydrate (0.72 mL, 9.7 mmol, 65% solution) was added to a solution of 4-chloroquinazoline 8 (0.5 g, 1.94 mmol) in ethanol (10 mL). The mixture was stirred at 70 °C for 3 h, then cooled. The bright yellow precipitate was filtered off and recrystallized from acetonitrile. Yield 0.39 g (80%), mp 174–176 °C. 1Н NMR, δ, ppm: 4.8 br. s (2H, NH2), 7.19–7.24 m (2H, Ar), 7.35–7.45 m (1Н, Ar), 7.65– 7.72 m (2Н, Ar), 8.15–8.20 m (1Н, Ar), 8.60–8.63 m (2Н, Ar), 9.6 br. s (1H, NH). 19F{H} NMR, δ, ppm: –111.81 s. Found (%): C 66.04, H 4.27, N 22.12. C14H11FN4. Calculated (%): C 66.13, H 4.36, N 22.03. 2.3. Preparation of target hydrazonoquinazolines 3c, d, 9a, b General method. The corresponding salicylic aldehyde (0.897 mmol) was added to a solution of 4-hydrazino- quinazoline 4a or 4b (0.753 mmol) in ethanol (7 mL). The reaction mixture was refluxed for 1.5 h, then cooled; the precipitate formed was filtered off and recrystallized from ethanol. 2.3.1. N-(3,5-Di(t-butyl)salicylidene)-N’-(6,7-difluoro-2- phenylquinazolin-4-yl)-hydrazine (3c) Yield 83%, mp 184−186 °C. NMR, δ, ppm: 1.37 s (9H, 3CH3), 1.55 s (9H, 3CH3), 7.25 s (1Н, Н4”), 7.33 s (1Н, Н6”), 7.49– 7.53 m (3Н, Н3’, Н4’, Н5’), 7.76 dd (1Н, Н5, 3JHF 9.2, 4JHF 4.9 Hz), 8.42 dd (1Н, Н8, 3JHF 9.2, 4JHF 4.9 Hz), 8.58 s (1H, CH=N), 8.68–8.72 m (2Н, Н2’, Н6’), 12.0 br. s (1Н, NH), 13.1 br. s (1H, OH). 19F{H} NMR, , ppm: –136.94 d (1F, 3JFF 24.3 Hz), –128.62 d (1F, 3JFF 24.3 Hz). 13C NMR: 29.32 (3CH3), 31.34 (3CH3), 33.89 (s, CMe3), 34.76 (s, CMe3), 108.87 (s, C-2’’), 110.04 (s, C-3’’, C-5’’), 114.90 (s), 117.40 (s), 125.20 (s), 125.38 (s), 128.05 (s), 128.22 (s), 130.80 (s), 135.76 (s), 137.51 (s), 140.10 (s), 148.01 (dd, C- 6 or C-7, 1JCF = 249.4, 2JCF = 12.6 Hz), 148.56 (s), 148.85 (d, C-5, C-8, 2JCF = 17.1 Hz), 153.60 (dd, C-7 or C-6, 1JCF = 254.9, 2JCF = 17.1 Hz), 155.11 (s), 155.61 (s), 159.85 (s). MS, m/z (Irel (%)): 488 [M]+ (99), 257 [M−di-tBu-isobenzoxazole]+ (100), 154 [M−di-tBu-isobenzoxazole-PhCN]+ (14). Found, %: C 71.41, Н 6.32, N 11.39. C29H30F2N4O. Calculated, %: C 71.29, Н 6.19, N 11.47. https://doi.org/10.15826/chimtech.2023.10.2.11 Chimica Techno Acta 2023, vol. 10(2), No. 202310211 ARTICLE 3 of 8 DOI: 10.15826/chimtech.2023.10.2.11 2.3.2. N-(5-Chlorosalicylidene)-N’-(6,7-difluoro-2-phe- nylquinazolin-4-yl)-hydrazine (3d) Yield 78%, mp 192–194 °C. 1Н NMR, δ, ppm: 7.03 d (1Н, Н3”, 3JHН 7.2 Hz), 7.28 d (1Н, Н4”, 3JHН 7.2 Hz), 7.50–7.54 m (3Н, Н3’, Н4’, Н5’), 7.63 s (1Н, Н6”), 7.76 dd (1Н, Н5, 3JHF 10.1, 4JHF 5.8 Hz), 8.50–8.52 m (1Н, Н8), 8.57 s (1H, CH=N), 8.58–8.62 m (2Н, Н2’, Н6’), 12.0–12.2 br. s (2Н, NH, OH). 19F{H} NMR, δ, ppm: –136.75 d (1F, 3JFF 24.0 Hz), –128.41 d (1F, 3JFF 24.0 Hz). 13C NMR: 108.92 (s, C-2’’), 114.72 (s), 114.85 (s), 118.41 (s), 120.40 (s), 122.87 (s), 127.86 (s), 128.44 (s), 130.54 (s), 130.74 (s), 137.52 (s), 143.57 (s), 146.97 (s), 149.08 (s), 149.23 (d, C-5, C-8, 2JCF = 17.1 Hz), 151.64 (dd, C-6 or C-7, 1JCF = 249.4, 2JCF = 12.6 Hz), 153.82 (dd, C-7 or C-6, 1JCF = 249.8, 2JCF = 16.5 Hz), 156.32 (s), 159.99 (s), 159.93 (s). MS, m/z (Irel (%)): 410 [M]+ (56), 257 [M−chloroisobenzoxazole]+ (100), 154 [M−chloroisobenzoxazole-PhCN]+ (29). Found, %: C 61.52, Н 3.30, N 13.59. C21H13ClF2N4O. Calculated, %: C 61.40, Н 3.19, N 13.64. 2.3.3. N-(3,5-Di(t-butyl)salicylidene)-N’-(2-(4-fluoro- phenyl)quinazolin-4-yl)-hydrazine (9а) Yield 76%, mp 152–154 °C. 1Н NMR, δ, ppm: 1.36 s (9H, 3CH3), 1.55 s (9H, 3CH3), 7.20–7.24 m (3Н, Н4”, H3’, H5’), 7.31 s (1Н, Н6”), 7.59 m (1Н, H7), 7.85–7.89 m (2Н, Н2’, Н6’), 8.34–8.36 m (1Н, H5), 8.62 s (1H, CH=N), 8.75–8.79 m (2Н, H6, H8), 12.1 br. s (1Н, NH), 13.3 br. s (1H, OH). 19F{H} NMR, δ, ppm: –111.28 s. 13C NMR: 29.33 (3CH3), 31.34 (3CH3), 33.89 (s, CMe3), 34.76 (s, CMe3), 112.14 (s, C-2’’), 114.93, 115.14 (both s, C-3’’, C-5’’), 117.53 (s), 122.60 (s), 125.20 (d, C-3’, C-5’, 2JCF = 21.6 Hz), 126.09 (s), 127.98 (s), 130.34 (d, C-2’, C-6’, 3JCF = 8.8 Hz), 133.50 (s), 134.57 (s), 135.76 (s), 140.08 (s), 148.45 (s), 150.51 (s), 155.10 (s), 156.08 (s), 158.18 (s), 163.85 (d, C-4’, 1JCF = 259.4 Hz). MS, m/z (Irel (%)): 470 [M]+ (67), 239 [M−di-tBu-isobenzoxa- zole]+ (100), 118 [M−di-tBu-isobenzoxazole-FC6H4CN]+ (12). Found, %: C 74.15, Н 6.80, N 11.79. C29H31FN4O. Cal- culated, %: C 74.02, Н 6.64, N 11.91. 2.3.4. N-(5-Chlorosalicylidene)-N’-(2-(4-fluoro- phenyl)quinazolin-4-yl)-hydrazine (9b) Yield 74%, mp 202–204 °C. 1Н NMR, δ, ppm: 7.02 d (1Н, Н3”, 3JHН 7.1 Hz), 7.25–7.35 m (3Н, Н4”, Н3’, Н5’), 7.58– 7.62 m (2Н, Н6’’, Н7), 7.85–7.89 m (2Н, Н2’, Н6’), 8.38– 8.42 m (1Н, H5), 8.60–8.70 m (3H, CH=N, H6, H8), 12.2 br. s (1Н, NH), 12.3 br. s (1H, OH). 19F{H} NMR, δ, ppm: –111.13 s. 13C NMR: 112.18 (s, C-2’’), 115.20 (s), 115.48 (s), 118.28 (s), 118.48 (s), 120.30 (s), 122.78 (s), 126.11 (s), 127.87 (d, C-3’, C-5’, 2JCF = 21.7 Hz), 128.77 (s), 130.09 (d, C-2’, C-6’,3JCF = 9.2 Hz), 130.39 (s), 133.64 (s), 134.47 (s), 145.43 (s), 150.51 (s), 156.21 (s), 156.45 (s), 157.24 (s), 158.21 (s), 163.84 (d, C-4’, 1JCF = 256.2 Hz). MS, m/z (Irel (%)): 392 [M]+ (47), 239 [M−chloroisobenzoxazole]+ (100), 118 [M−chloroisobenzoxazole-FC6H4CN]+ (27). Found, %: C 64.34, Н 3.70, N 14.19. C21H14ClFN4O. Calculated, %: C 64.21, Н 3.59, N 14.26. 2.4. Molecular docking studies Protein preparation. The ligand protein complex CK2 with the azolopyrimidine derivative CHEMBL2062585 (PDB ID: 3U4U) was uploaded from the Protein Data Bank database in .pdb format. Further, in the ArgusLab 4.0.1 software, third-party molecules (water, ions, etc.) were removed from the complex and then hydrogen atoms were added. Binding site was determined relative to the position of na- tive ligand with nanomolar activity (IC50 = 3 nM). Valida- tion of docking parameters was carried out by redocking the native ligand with the following parameters: GADock (elitism: 3), Scoring function: Ascore, Binding site box size: 18.6×17.5×16.5 Å, Grid resolution: 0.2 Å. A quantitative assessment of the docking protocol was carried out according to the RMSD (root-mean-square devi- ation). For the native structure RMSD < 2 Å, which indicates sufficient calculation accuracy. The parameters used for re- docking were then used for docking the studied structures without changes. Ligand preparation. Ligands were prepared in DataWar- rior software. 3D coordinates of the ligands (1 conformer per 1 structure) were generated using the Self-organized al- gorithm and the MMFF94s+ force field. Docking protocol. Molecular docking was performed in ArgusLab 4.0.1 software on a previously prepared protein with established parameters and binding site size. For docking, the Lamarckian genetic algorithm GADock and the empirical function AScore were used to calculate the free binding energy; the protein is taken as a rigid structure, and the ligands are flexible. A quantitative assessment of the af- finity of ligands to the protein was carried out by analyzing the calculated docking score. For the hit compound 9a with the best (lowest) docking score, refined docking (Grid resolution 0.1 Å) was carried out with the initial generation of structures in ArgusLab with geometry optimization on the UFF molecular force field. The evaluation of docking results, i.e., the calculation of the 3D position of the hit compound in the target protein and the 2D-map of non-covalent interactions, was carried out in ArgusLab and the PoseView module of the pro- teins.plus web service, respectively. 2.5. CK2 Assay Kinase activity was determined using the CK2a1 enzyme system (Promega V4482, Madison, WI, USA) and the ADP- GloTM kit (Promega V9101, Madison, USA) in white 96- well plates (Nunc U96 Microwell 267350, Denmark). Bo- vine casein was used as the peptide substrate. Staurospor- ine ATP-competitive inhibitor (STS, CAS 62996-74-1, Alfa Aesar J62837, 99 +%) was used as a positive control. The assay was carried out using 10 ng/well of N-GST labelled human recombinant CK2a1 expressed in Sf9 cells, 0.1 mg/mL casein, 10 mM ATP in a 40 mM Tris buffer (pH 7.50) containing 20 mM MgCl2, 0.1 mg/mL BSA and 50 mM DTT. Compounds were introduced in 1.25% DMSO and https://doi.org/10.15826/chimtech.2023.10.2.11 Chimica Techno Acta 2023, vol. 10(2), No. 202310211 ARTICLE 4 of 8 DOI: 10.15826/chimtech.2023.10.2.11 preincubated with kinase at 450 rpm for 10 min. The re- action was carried out for 60 min at 25 °C in PST-60HL shaker (Biosan, Latvia). ATP-dependent luminescence was measured at an integration time of 1000 ms using Infinite M200 PRO microplate reader (Tecan GmbH, Grödig, Aus- tria). The experiments were run in two replicates. The ac- tivity of CK2 in the sample wells was normalized against the control and enzyme-blank wells. 2.6. MTT assay and cell culture MDA-MB-231 breast cancer cells and WI-26 VA4 lung epi- thelial-like cells were purchased from the ATCC (Manas- sas, VA, USA). MDA-MB-231 cells were maintained in Dul- becco's modified Eagle's medium (DMEM), supplemented with 1× non-essential amino acids, 25 mM Hepes, 1× pen- icillin/streptomycin, and, where indicated, 10% (v/v) foe- tal bovine serum (FBS), all obtained through Gibco (Thermo Fisher Scientific, Inc., Waltham, MA, USA) [37]. WI-26 VA4 cells were maintained in Advanced MEM (Gibco, Loughborough, UK) supplemented with 5% fetal bovine serum (Fetal Bovine Serum, qualified, Australia, Gibco, UK), penicillin (100 UI mL–1), streptomycin (100 mg mL–1), and GlutaMax (1.87 mM, Gibco, Loughborough, UK). All all cell lines were cultivated under a humidified atmosphere of 95% air/5% CO2 at 37 °C. Subconfluent monolayers, in the log growth phase, were harvested by a brief treatment with TrypLE Express solution (Gibco, Loughborough, UK) in phosphate buffered saline (PBS, Capricorn Scientific, Germany) and washed three times in serum-free PBS. The number of viable cells was deter- mined by trypan blue exclusion. The effects of the synthesized compounds on cell via- bility were determined using the MTT colorimetric test [38]. All examined cells were diluted with the growth me- dium to 3.5·104 cells per mL, and the aliquots (7·103 cells per 200 mL) were placed in individual wells in 96-well plates (Eppendorf, Hamburg, Germany) and incubated for 24 h. The next day, the cells were treated with the synthe- sized compounds separately in 10 and 100 mM concentra- tions (or 200.0 mM concentration and diluted at various concentrations for determination of IC50) and incubated for 72 h at 37 °C in 5% CO2 atmosphere. Each compound was tested in triplicate. After incubation, the cells were treated with 40 mL MTT solution (3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyltetrazolium bromide, 5 mg mL–1 in PBS) and incubated for 4 h. After additional 4 h incubation, the medium with MTT was removed and DMSO (150 mL) was added to dissolve the formazan crystals. The plates were shaken for 10 min. The optical density of each well was determined at 560 nm using GloMax Multi+ (Promega, Madison, WI, USA) microplate reader. Each of the tested compounds was evaluated for cytotoxicity in three sepa- rate experiments. All stock solutions for biological evalu- ations were prepared via dissolving synthesized com- pounds in DMSO. 3. Results and Discussion 3.1. Synthesis Synthesis of hydrazones 3c,d was realized by the heating of 2-phenyl-4-hydrazino-6,7-difluoroquinazoline 4a [24], with 3,5-di(t-butyl)-2-hydroxybenzaldehyde and 5-chloro- 2-hydroxybenzaldehyde, respectively (Scheme 1). For obtaining 2-(4-fluorophenyl)-derivatives 9a,b we performed the synthesis of 4-hydrazinoquinazoline 4b. The key intermediate 7 was synthesized by condensation of 2- aminobenzamide 5 with 4-fluorobenzaldehyde (under stir- ring in ethanol at room temperature) and subsequent oxi- dation of azomethine 6 with copper(II) chloride (Scheme 2). Earlier, the synthesis of 2-(4-fluorophenyl)quinazolin- 4-one 7 from aminoamide 5 and 4-fluorobenzaldehyde was achieved in the presence of other oxidants, such as iodine in ionic liquid [bmim+][BF4–] [25], antimony chloride with- out solvent at microwave irradiation [26], or air in the pres- ence of vanadium bis(acetylacetonate) VO(acac)2 as a cata- lyst [27]. The formation of intermediate 6 in [bmim+][BF4–] at room temperature was mentioned [25]. Notably, that in- teraction between anthranilamide 5 and aryl carbaldehydes leads to the formation of Schiff bases or 2,3-dihydro- quinazolin-4(1H)-ones depending on the reaction conditions and the nature of aldehydes [28]. Therefore, it is not surpris- ing that at room temperature the interaction of 4-fluoroben- zaldehyde and aminoamide 5 resulted in the formation of az- omethine 6. The chloro-derivative 8 obtained by refluxing of quinazolinone 7 with phosphorus oxychloride was used for the introduction of the hydrazine group into position 4. The synthesis of hydrazones 9a,b was carried out by heating of 2-(4-fluorophenyl)-4-hydrazinoquinazoline 4b with corre- spondent aldehyde in ethanol (Scheme 2). The structure of target quinazolines 3c, d and 9a,b was determined based on their 1H NMR, 19F NMR and 13C NMR spectroscopy as well as mass spectrometry data (Figures S1-S18). Scheme 1 Synthesis of 2-phenyl-6,7-difluoro-4-salicylidenehydra- zino quinazolines 3c,d. Reaction conditions: (i) ethanol, reflux, 1.5 h. https://doi.org/10.15826/chimtech.2023.10.2.11 Chimica Techno Acta 2023, vol. 10(2), No. 202310211 ARTICLE 5 of 8 DOI: 10.15826/chimtech.2023.10.2.11 The 1H NMR spectra of salicylidenehydrazones 3c,d, 9a,b (Figures S1, S5, S11, S15) characteristically showed sig- nals of aryl fragment protons, benzene or difluorobenzene ring, singlets of –CН=N-groups at 8.57–8.70 ppm, broaden singlets of NH at 12.0–12.2 ppm and OН at 12.0–13.3 ppm. Two doublet signals present at 19F{H} NMR spectra of com- pounds 3c,d, and one singlet in the case of derivatives 9a,b. Structures 3c,d and 9a,b are also evidenced by the mass spectra data; the relative intensities of molecular ion peaks are 47–99%. The most abundant ions in the mass spectra of the correspondent 2-aryl-4-aminoquinazolines are m/z 257 for 2-phenyl-6,7-difluoroquinazolines 3c,d and m/z 239 for 2-(4-fluorophenyl)quinazolines 9a,b, which can form due to isobenzoxazole elimination. 3.2. Molecular docking Molecular docking was performed using ArgusLab 4.0.1 software [29]. Previously, a protein-ligand complex was taken from the Protein Data Bank database [30]: a CK2 complex with the azolopyrimidine derivative CHEMBL2062585 [31] (PDB ID: 3U4U) [32]. After prepara- tion (see Experimental) validation of the docking protocol was carried out for the protein-ligand complex by redock- ing the native ligand as a nanomolar inhibitor. According to the results of redocking, the standard deviation (RMSD) of the known position of CK2 protein inhibitor was 1.5 Å (Fig- ure S19). The results of molecular docking of several studied lig- ands prepared in DataWarrior [33] (see Experimental) with the calculated free binding energy (ΔG) as individual activ- ity indicator are given in Table 1. All docking compounds are more active than the comparison compound CHEMBL2062585 (IC50 (CK2) = 3 nM). To deepen the understanding of the action mechanism of probable CK2 inhibitors among the studied quinazoline derivatives, an updated docking refinement was carried out for the leader compound 9a based on in silico activity indi- cators. The position of the calculated ligand 9a in the active site of the protein with the recalculated values of ΔG is pre- sented in Figure S20A. The profile of non-covalent interac- tions is defined using the PoseView module of the pro- teins.plus service [34, 35]. Molecular docking towards CK2 shows higher in silico activity with partial coincidence of non-covalent interac- tions for all compounds compared to known nanomolar in- hibitor of this protein. In silico experiment revealed two hit compounds with the best affinity for casein kinase 2: the G value for hydrazone 3a is –13.91 kcal/mol, and for deriva- tive 9a G = –14.16 kcal/mol (Table 1). The leader compound 9a is characterized by another binding method to the CK2 active site compared to the na- tive ligand (Figure S20B). The main contribution to the binding of 9a with the protein is made by several hydropho- bic interactions (Figures 2, S20C), π-π stacking between the fluorophenyl moiety and the residue Phe113, and a hydro- gen bond between the hydroxy group of the ligand and the residue His160. At the same time, both native ligand and 9a are characterized by interactions only with residues of Asp175 and Met163. Thus, despite the significant in silico activity of 9a in terms of free binding energy relative to the nanomolar CK2 inhibitor, a different profile of non-covalent interactions can influence negatively the in vitro experiments. Scheme 2 Synthesis of 2-(4-fluorophenyl)-4-salicylidenehydrazino quinazolines 9a,b. Reagents and conditions: (i) ethanol, r.t., 3 h; (ii) CuCl2, ethanol, reflux, 5 h; (iii) POCl3, reflux, 2 h, (iv) H2N- NH2·H2O, ethanol, 70 °C, 3 h; (v) ethanol, reflux, 1.5 h. Figure 2 Non-covalent interactions of the docked ligand 9а. Table 1 Docking results and indicators of in vitro/in silico activity to casein kinase 2. Compound ΔG, kcal/mol Compound ΔG, kcal/mol 1a –12.33 3a –13.91 1b –13.16 3b –12.47 1c –12.20 3c –11.33 2a –11.84 3d –12.69 2b –12.77 9a –14.16 –13.86* 2c –13.10 9b –12.70 CHEMBL 2062585 (IC50 = 3 nM) –10.67 * The docking score obtained after docking refinement https://doi.org/10.15826/chimtech.2023.10.2.11 Chimica Techno Acta 2023, vol. 10(2), No. 202310211 ARTICLE 6 of 8 DOI: 10.15826/chimtech.2023.10.2.11 3.3. CK2 inhibition and cytotoxicity study The target compounds were evaluated against human re- combinant CK2 using the luminescent ADP-GloTM assay. Due to the limited solubility of the compounds, only four derivatives 1a, 3c, 9a,b was tested. The CK2 activity was determined after the casein phosphorylation reaction by the value of ATP-dependent luminescence. The data ob- tained and the CK2 activity in the presence of 50 μM of the tested compounds are shown in Table 2. Initial screening at 50 µM revealed that the compounds are not active towards CK2. All tested compounds showed no inhibitory activity at the level of the reference drug (Table 2). Even though the compounds 1, 3, 9 did not act as inhibitors of casein kinase 2, their cytostatic activity was investigated, since a differ- ent mechanism of cytotoxic action is possible. Some hydrazonoquinazolines 1, 3, 9 were evaluated for their anticancer properties against MDA-MB-231 cells. Addi- tionally, non-cancerous lung fibroblast cells WI-26 VA4 were used to control non-specific cytotoxicity (Table 3, Figure S21). As can be concluded from the Table 3, compounds 1b, 3b–d and 9b possess pronounced antiproliferative activities against MDA-MB-231 cells with IC50 values ranging between 4.91 and 65.2 M. Noteworthy, non-cancerous lung fibro- blast WI-26 VA4 cell line was less sensitive only to two tested molecules, including the most active nitrosalicyli- dene derivative 3b. 4. Limitations Structural modification is required to improve the solubil- ity and bioavailability of the compounds. Unfortunately, the possibilities of purchasing reagents for various modifica- tions are limited, and study of expanded range of com- pounds is hardly achievable. 5. Conclusions The novel fluorine-containing 2-aryl-4-salicilidenhydrazino quinazolines 3, 9 were synthesized and characterized by us- ing NMR and MS, and all the data are in accordance with the proposed structures. The results of molecular docking clearly indicate a high potential activity of this series of compounds in terms of free binding energy. On the other hand, other profiles of non-covalent interactions of the leader compounds compared to the nanomolar inhibitor can have various consequences. Therefore, an in vitro activity study with an in vitro-in silico correlation assay is expected to be the best direction for further targeted development of CK2 inhibitors for the considered class of compounds. N- (6,7-Difluoro-2-phenylquinazolin-4-yl)-N’-(5-nitrosalicyli- dene)hydrazine 3b exhibited enhanced activity against breast cancer (MDA-MB-231) cells in combination with moderate selectivity toward normal WI-26 VA4 cells. The rest of compounds do not show noticeable selectivity to nor- mal and tumour cells. ● Supplementary materials This manuscript contains supplementary materials, which are available on the corresponding online page. ● Funding This work was supported by the Ministry of Science and Higher Education of the Russian Federation, State Contract no FEUZ-2023-0021 (N687.42B.325/23). ● Acknowledgments Authors thank Academician Alexander A. Spasov (Depart- ment of Pharmacology & Bioinformatics, Scientific Center for Innovative Drugs, Volgograd State Medical University) for assistance with study of human recombinant CK2 inhi- bition and the Head of Medicinal Chemistry Center Alex- ander S. Bunev (Togliatti State University) for cytotoxicity data. ● Author contributions Conceptualization: E.V.N., S.K.K. Data curation: E.V.N. Investigation: M.D.L. Methodology: E.V.N, M.D.L., I.I.B. Software: I.I.B. Validation: I.I.B. Visualization: I.I.B. Writing – original draft: E.V.N. Writing – review & editing: S.K.K. Project administration: S.K.K. Table 2 Data of CK2 inhibition activity study for some hydrazono- quinazolines. Compound CK2 Activity (mSD), % CK2 Inhibition at 50 μM (mSD), % 1a 102.1418.22 –2.1418.22 3c 94.514.98 5.514.98 9a 96.0222.88 3.9822.88 9b 108.161.1 –8.161.1 Staurosporine 7.871.34* 92.131.34* Statistical significance relative to negative control, 1-factor ANOVA; *p <0.05 Table 3 MTT assay results for 2-aryl-4-salicylidenehydrazino quinazolines against breast cancer cell line (MDA-MB-231). Compound IC50, M MDA-MB-231 (Breast cancer) WI26 VA4 (Normal cell) 1a >100 46.612.57 1b 65.27.67 69.819.15 3b 4.910.66 6.320.47 3c 41.912.6 17.492.41 3d 7.430.87 4.020.38 9a n.d. 42.44.75 9b 14.51.80 11.271.02 n.d. – no data https://doi.org/10.15826/chimtech.2023.10.2.11 Chimica Techno Acta 2023, vol. 10(2), No. 202310211 ARTICLE 7 of 8 DOI: 10.15826/chimtech.2023.10.2.11 ● Conflict of interest The authors declare no conflict of interest. ● Additional information Author IDs: Emiliya V. Nosova, Scopus ID 35498195300; Margarita D. Likhacheva, Scopus ID 58084945000; Svetlana K. Kotovskaya, Scopus ID 6602187240; Ilya I. Butorin, Scopus ID 57202983112. Websites: Ural Federal University, https://urfu.ru/en; I. Postovsky Institute of Organic Synthesis, https://io- suran.ru. References 1. Furman RR, Sharman JP, Coutre SE, Cheson BD, Pagel JM, Hillmen P, Barrientos JC, Zelenetz AD, Kipps TJ, Flinn I, Ghia P, Eradat H. 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Methods Mol Biol. 2011;716:157–168. doi:10.1007/978-1-61779-012-6_9 https://doi.org/10.15826/chimtech.2023.10.2.11 https://doi.org/10.1080/00397911.2012.717669 https://doi.org/10.24820/ark.5550190.p009.894 https://doi.org/10.1093/nar/28.1.235 https://doi.org/10.1074/jbc.M114.634683 https://doi.org/10.1093/nar/gkw1074 https://doi.org/10.1021/ci500588j https://doi.org/10.1093/nar/gkaa235 https://doi.org/10.1093/bioinformatics/btl150 https://doi.org/10.1134/S1070428020080163 https://doi.org/10.1039/D0TB00620C https://doi.org/10.1007/978-1-61779-012-6_9 1. Introduction 2. Experimental 2.1. General 2.2. Preparation of intermediates 2.2.1. (E)-2-(4-Fluorophenylydeneamino)benzamide (6) 2.2.2. 2-(4-Fluorophenyl)quinazolin-4(3Н)-one (7) 2.2.3. 2-(4-Fluorophenyl)-4-chloroquinazoline (8) 2.2.4. 2-(4-Fluorophenyl)-4-hydrazinoquinazoline (4b) 2.3. Preparation of target hydrazonoquinazolines 3c, d, 9a, b 2.3.1. N-(3,5-Di(t-butyl)salicylidene)-N’-(6,7-difluoro-2-phenylquinazolin-4-yl)-hydrazine (3c) 2.3.2. N-(5-Chlorosalicylidene)-N’-(6,7-difluoro-2-phenylquinazolin-4-yl)-hydrazine (3d) 2.3.3. N-(3,5-Di(t-butyl)salicylidene)-N’-(2-(4-fluorophenyl)quinazolin-4-yl)-hydrazine (9а) 2.3.4. N-(5-Chlorosalicylidene)-N’-(2-(4-fluorophenyl)quinazolin-4-yl)-hydrazine (9b) 2.4. Molecular docking studies 2.5. CK2 Assay 2.6. MTT assay and cell culture MDA-MB-231 breast cancer cells and WI-26 VA4 lung epithelial-like cells were purchased from the ATCC (Manassas, VA, USA). MDA-MB-231 cells were maintained in Dulbecco's modified Eagle's medium (DMEM), supplemented with 1× non-essential amino acids, 25... 3. Results and Discussion 3.1. Synthesis 3.2. Molecular docking 3.3. CK2 inhibition and cytotoxicity study 4. Limitations 5. Conclusions ● Supplementary materials ● Funding ● Acknowledgments ● Author contributions ● Conflict of interest ● Additional information References