However, they suffered from generally poor central nervous system (CNS) permeability. Here, we describe the design, synthesis, and in vitro characterization of novel imidazopyridine-1-carboxamide (IMID 1) and imidazopyridine-3-carboxamide (IMID 2) compounds (7–18) to overcome such liability. In detail, structure-based approaches and fine-tuning of physicochemical properties guided the design of derivatives 7–18 resulting in ameliorated absorption, distribution, metabolism, and excretion (ADME) properties. A crystal structure of 16 in complex with GSK-3β enzyme (PDB entry 6Y9S) confirmed the in silico models. Despite the nanomolar inhibition activity, the new core compounds showed a reduction in potency with respect to INDZ derivatives 1–6. In this context, Molecular Dynamics (MD) and Quantum Mechanics (QM) based approaches along with NMR investigation helped to rationalize the observed structure activity relationship (SAR). With these findings, the key role of the acidic hydrogen of the central core for a tight interaction within the ATP pocket of the enzyme reflecting in good GSK-3β affinity was demonstrated.The interest of research groups and pharmaceutical companies to discover novel GSK-3β inhibitors has increased over the years considering the involvement of this enzyme in many pathophysiological processes and diseases. Along this line, we recently reported on 1H-indazole-3-carboxamide (INDZ) derivatives  Glycogen synthase kinase-3 (GSK-3) is a ubiquitous, multifunctional and constitutively active serine/threonine kinase, involved in numerous pathways and over one hundred different putative substrates .In humans, GSK-3 enzyme is encoded by two independent genes that regulate the expression of two diverse isoforms, namely GSK-3α and GSK-3β, with high sequence homology (~98%) in their kinase domain  and subsGSK-3β is a significantly networked enzyme, which regulates a plethora of physiological functions in peripheral tissues and the central nervous system (CNS), spanning from metabolism and cell cycle regulation to brain development. Accordingly, balanced GSK-3β regulation and signaling is key to human physiology but often compromised in diverse disease conditions such as diabetes, cancers, and brain disorders ,7. As foBased on this evidence, intensive efforts have been made in the search for novel GSK-3β inhibitors as innovative therapeutic agents, which, however, have not been translated into clinical practice yet. Therefore, GSK-3β inhibitors are still needed.1–6 pyridine-1-carboxamide (IMID 1) and imidazopyridine-3-carboxamide (IMID 2) derivatives 7–18  as putat1–6 were synthesized as previously described pyridine . Subsequent silver nitrate-mediated oxidation led to the carboxylic acid 20, which was then coupled to 1-[1-(2-methoxyethyl)piperidin-4-yl]methanamine and 1-(oxan-4-yl)methanamine under standard carboxylic acid-amine coupling reaction to afford amides 21 and 22, respectively. Final Suzuki coupling of 21 and 22 with the desired boronic acids gave the target compounds 7–10 and 12. The alkoxy-substituted pyridinyl derivative 11 was synthesized by the additional conversion of the 7-bromo-N-[(oxan-4-yl)methyl]imidazopyridine-1-carboxamide 22 to the corresponding boronic ester 23 by treatment with bis(pinacolato)diboron and subsequent Suzuki coupling with (3-bromopyridin-4-yl)methanol piperidin-4-yl]methanamine and 1-(oxan-4-yl)methanamine to provide the target compounds. Similarly to compound 11, synthesis of the alkoxy-substituted pyridinyl derivative 17 required the additional conversion of bromide 24 to the corresponding boronic ester 25, which then entered the same three-step synthetic route as described above . Interestingly, both compounds 2 and 4 carry an isopropoxypyridinyl group as R2 substituent, resulting from 3 to 5-fold more potent than the corresponding pyridinyl analogues 1 and 3, respectively. The high-resolution X-ray crystal structure of 2 in complex with GSK-3β  supported the expected binding mode pyridinyl (5) or a di-F-phenyl (6) also retained double-digit nanomolar inhibition activity. Finally, when comparing the enzymatic potency of the pyridinyl compound 1 against 3 or the isopropoxypyridinyl derivative 2 against 4, it appeared that the two matching pairs resulted almost equipotent, suggesting that the R1 substituents do not significantly impact on the GSK-3β inhibitory activity. This is in line with the observed INDZ binding mode, where the R1 group is solvent oriented.Compounds hibitors ,18,19, aing mode b. Here, 2, 3, 4, and 6 that, being the most potent derivatives of the dataset, were selected as INDZ representatives for experimental testing in an in vitro MDCK-MDR1 permeability assay (app) were calculated by measuring the transport of the compounds from the apical (A) to the basolateral (B) direction , whereas compounds 2 and 4 resulted only poorly permeable (Papp < 1.5 × 10−6 cm/s). Moreover, high ER values were obtained for derivatives 2 and 4 and lower ratios for compounds 3 and 6. The observed experimental data could be rationalized by analysis of the physicochemical profile. Indeed, several properties have consistently been found to be relevant for optimal brain exposure, such as lipophilicity, expressed by the calculated logarithm of the octanol/water partition coefficient (cLogP) or calculated logarithm of the octanol/water partition coefficient at physiological pH 7.4 (cLogD), topological polar surface area (TPSA), ionization state (pKa), number of H-bond donors (HBD) and acceptors (HBA), rotatable bond (RB) count, and molecular weight (MW). Up to now, many published works endeavor to provide guidance in terms of physicochemical property range for the design of CNS penetrant molecules pyridine-1-carbaldehyde (19). To a solution of 7-bromoimidazopyridine  in toluene (2.5 mL), DMF  and phosphorus oxychloride  were added. After stirring at 100 °C for 1.5 h, the reaction mixture was carefully quenched with ice and 1N aqueous NaOH solution. The aqueous phase was extracted with DCM (×3), the combined organic layers were dried over anhydrous MgSO4, filtered and evaporated under reduced pressure. The crude was purified by flash chromatography , affording 327 mg of the title compound. Yield: 57%. LC-MS (ESI) m/z: 224.9 [M + H]+.7-Bromoimidazopyridine-1-carboxylic acid (20). A mixture of 7-bromoimidazopyridine-1-carbaldehyde 19 , silver nitrate , and sodium hydroxide  in water (4 mL) was stirred at rt for 3 h. The reaction was then diluted with water and the black solid was filtered off and washed with water. The filtrate was made acidic with 1N aqueous HCl solution and the resulting precipitate was filtered and dried. The crude (375 mg) was used in the next step without further purification. Yield: 61%. LC-MS (ESI) m/z: 240.9 [M + H]+.7-Bromo-N-{[1-(2-methoxyethyl)piperidin-4-yl]methyl}imidazopyridine-1-carboxamide (21). The title compound was obtained according to general procedure (A) using 7-bromoimidazopyridine-1-carboxylic acid 20 (0.73 mmol) and 1-[1-(2-methoxyethyl)piperidin-4-yl]methanamine (0.73 mmol). Yield = 56% (200 mg). LC-MS (ESI) m/z: 395.1 [M + H]+. 1H-NMR  δ 8.35-8.32 , 8.30 , 8.28 , 6.94 , 3.72–3.68 , 3.57 , 3.40 , 3.38 , 3.30-3.26 , 3.06–2.93 , 2.07–1.90 , 1.66–1.53 ppm .7-Bromo-N-[(oxan-4-yl)methyl]imidazopyridine-1-carboxamide (22). The title compound was obtained according to general procedure (A) using 7-bromoimidazopyridine-1-carboxylic acid 20 (0.73 mmol) and 1-(oxan-4-yl)methanamine (0.73 mmol). Yield = 78% (192 mg). LC-MS (ESI) m/z: 338.0 [M + H]+. 1H-NMR  δ 8.33 , 8.27 , 8.25 , 6.91 , 3.95 , 3.40 , 3.30 , 1.94-1.82 , 1.74-1.65 , 1.42-1.29 ppm .N-((Tetrahydro-2H-pyran-4-yl)methyl)-7-imidazopyridine-1-carboxamide (23). The title compound was obtained according to general procedure (B) using 7-bromo-N-[(oxan-4-yl)methyl]imidazopyridine-1-carboxamide 22 (1.36 mmol). Yield = 76% (340 mg). LC-MS (ESI) m/z: 386.2 [M + H]+. 1H-NMR  δ 8.62 , 8.31 , 8.27 , 6.99 , 3.95 , 3.41 , 3.34-3.31 , 1.96–1.84 , 1.71 , 1.43–1.28 , 1.36 ppm .Ethyl 6-imidazopyridine-3-carboxylate (25). The title compound was obtained according to general procedure (B) using 6-bromoimidazopyridine-3-carboxylate 24 (11.0 mmol). Yield = 100% (4.08 g). LC-MS (ESI) m/z: 317.2 [M + H]+. 1H-NMR  δ 9.70 , 7.61–7.57 , 7.36–7.30 , 4.51 , 1.49 , 1.36 ppm .Ethyl 6-(pyridin-3-yl)imidazopyridine-3-carboxylate (26). The title compound was obtained according to general procedure (C) using 6-bromoimidazopyridine-3-carboxylate 24 (0.55 mmol) and pyridin-3-ylboronic acid (0.55 mmol). Yield = 64% (94 mg). LC-MS (ESI) m/z: 268.1 [M + H]+.Ethyl 6-{5-[(propan-2-yl)oxy]pyridin-3-yl}imidazopyridine-3-carboxylate (27). The title compound was obtained according to general procedure (C) using 6-bromoimidazopyridine-3-carboxylate 24 (0.45 mmol) and {5-[(propan-2-yl)oxy]pyridin-3-yl}boronic acid (0.45 mmol). Yield = 93% (140 mg). LC-MS (ESI) m/z: 326.1 [M + H]+.Ethyl 6-[4-(hydroxymethyl)pyridin-3-yl]imidazopyridine-3-carboxylate (28). The title compound was obtained according to general procedure (C) using ethyl 6-imidazopyridine-3-carboxylate 25 (0.68 mmol) and (3-bromopyridin-4-yl)methanol (0.68 mmol). Yield = 51% (105 mg). LC-MS (ESI) m/z: 298.1 [M + H]+.Ethyl 6-imidazopyridine-3-carboxylate (29). The title compound was obtained according to general procedure (C) using 6-bromoimidazopyridine-3-carboxylate 24 (1.16 mmol) and boronic acid (1.16 mmol). Yield = 90% (318 mg). LC-MS (ESI) m/z: 303.1 [M + H]+.Lithium 6-(pyridin-3-yl)imidazopyridine-3-carboxylate (30). The title compound was obtained according to general procedure (D) using ethyl 6-(pyridin-3-yl)imidazopyridine-3-carboxylate 26 (0.35 mmol). Yield = 100% (85 mg). LC-MS (ESI) m/z: 240.1 [M + H]+. 1H-NMR  δ 9.66 , 8.91 , 8.63 , 8.22 , 7.95 , 7.74 , 7.61 , 7.54 ppm .Lithium 6-{5-[(propan-2-yl)oxy]pyridin-3-yl}imidazopyridine-3-carboxylate (31). The title compound was obtained according to general procedure (D) using ethyl 6-{5-[(propan-2-yl)oxy]pyridin-3-yl}imidazopyridine-3-carboxylate 27 (0.43 mmol). Yield = 95% (123 mg). LC-MS (ESI) m/z: 298.1 [M + H]+. 1H-NMR  δ 9.63 , 8.44 , 8.28 , 7.93 , 7.74 , 7.71–7.69 , 7.52 , 4.87–4.78 , 1.40 ppm .Lithium 6-(4-(hydroxymethyl)pyridin-3-yl)imidazopyridine-3-carboxylate (32). The title compound was obtained according to general procedure (D) using ethyl 6-[4-(hydroxymethyl)pyridin-3-yl]imidazopyridine-3-carboxylate 28 (0.35 mmol). Yield = 100% (97 mg). LC-MS (ESI) m/z: 270.1 [M + H]+.Lithium 6-imidazopyridine-3-carboxylate (33). The title compound was obtained according to general procedure (D) using ethyl 6-imidazopyridine-3-carboxylate 29 (1.05 mmol). Yield = 93% (273 mg). LC-MS (ESI) m/z: 275.1 [M + H]+.N-((1-(2-Methoxyethyl)piperidin-4-yl)methyl)-7-(pyridin-3-yl)imidazopyridine-1-carboxamide (7). The title compound was obtained according to general procedure (C) using 7-bromo-N-{[1-(2-methoxyethyl)piperidin-4-yl]methyl}imidazopyridine-1-carboxamide 21 (0.25 mmol) and pyridin-3-ylboronic acid (0.25 mmol). Yield = 19% (20 mg). HRMS (ESI): calculated 394.2237 [M + H]+, found 394.2241 [M + H]+. 1H-NMR  δ 8.98 , 8.64–8.59 , 8.49 , 8.38 , 8.17 , 8.09 , 7.54 , 7.31 , 3.41–3.39 , 3.22 , 3.20–3.18 , 2.84 , 2.42 , 1.89 , 1.62 , 1.55 , 1.21–1.14 ppm . 13C-NMR  δ 163.2, 149.8, 147.8, 134.4, 133.8, 132.4, 131.3, 128.5, 125.4, 125.3, 124.5, 116.1, 113.2, 70.6, 58.4, 57.9, 54.0, 44.2, 36.5, 30.3 ppm.7-(5-Isopropoxypyridin-3-yl)-N-((1-(2-methoxyethyl)piperidin-4-yl)methyl)imidazopyridine-1-carboxamide (8). The title compound was obtained according to general procedure (C) using 7-bromo-N-{[1-(2-methoxyethyl)piperidin-4-yl]methyl}imidazopyridine-1-carboxamide 21 (0.25 mmol) and {5-[(propan-2-yl)oxy]pyridin-3-yl}boronic acid (0.25 mmol). Yield = 16% (19 mg). HRMS (ESI): calculated 452.2656 [M + H]+, found 452.2652 [M + H]+. 1H-NMR  δ 8.54 , 8.49 , 8.30 , 8.07 , 8.03 , 7.47 , 7.29 , 7.02 , 4.73–4.65 , 3.52 , 3.39 , 3.34 , 3.00 , 2.58 , 2.02 , 1.80 , 1.71–1.63 , 1.52-1.42 , 1.39 ppm . 13C-NMR  δ 163.4, 154.3, 139.8, 139.2, 134.4, 132.7, 131.9, 126.6, 125.7, 122.9, 120.2, 117.6, 113.8, 70.9, 70.3, 58.9, 58.2, 54.0, 44.4, 36.3, 29.8, 21.9 ppm.7-(Pyridin-3-yl)-N-((tetrahydro-2H-pyran-4-yl)methyl)imidazopyridine-1-carboxamide (9). The title compound was obtained according to general procedure (C) using 7-bromo-N-[(oxan-4-yl)methyl]imidazopyridine-1-carboxamide 22 (0.25 mmol) and pyridin-3-ylboronic acid (0.25 mmol). Yield = 34% (28 mg). HRMS (ESI): calculated 337.1659 [M + H]+, found 337.1662 [M + H]+. 1H-NMR  δ 8.95 , 8.58 , 8.48–8.46 , 8.34 , 8.23–8.15 , 7.57–7.50 , 7.23 , 3.96–3.88 , 3.42 , 3.36–3.34 , 1.98–1.85 , 1.77–1.68 , 1.44–1.28 ppm .7-(5-Isopropoxypyridin-3-yl)-N-((tetrahydro-2H-pyran-4-yl)methyl)imidazopyridine-1-carboxamide (10). The title compound was obtained according to general procedure (C) using 7-bromo-N-[(oxan-4-yl)methyl]imidazopyridine-1-carboxamide 22 (0.25 mmol) and {5-[(propan-2-yl)oxy]pyridin-3-yl}boronic acid (0.25 mmol). Yield = 28% (30 mg). HRMS (ESI): calculated 395.2078 [M + H]+, found 395.2077 [M + H]+. 1H-NMR  δ 8.49 , 8.46 – 8.43 , 8.34 , 8.23 , 7.75–7.73 , 7.24–7.20 , 4.83–4.81 , 3.96 , 3.42 , 3.35 , 1.91–2.11 , 1.72 , 1.40 , 1.42–1.31 ppm .7-(4-(Hydroxymethyl)pyridin-3-yl)-N-((tetrahydro-2H-pyran-4-yl)methyl)imidazo pyridine-1-carboxamide (11). The title compound was obtained according to general procedure (C) using N-((tetrahydro-2H-pyran-4-yl)methyl)-7-imidazopyridine-1-carboxamide 23 (0.35 mmol) and (3-bromopyridin-4-yl)methanol (0.35 mmol). Yield = 48% (12 mg). 1H-NMR  δ 8.90 , 8.79 , 8.74 , 8.67 , 8.44 , 8.04 , 7.23 , 4.99 , 4.29–4.24 , 3.72 , 3.65 , 2.28–2.15 , 2.05–1.98 , 1.72–1.62 ppm .7--N-((tetrahydro-2H-pyran-4-yl)methyl)imidazopyridine-1-carboxamide (12). The title compound was obtained according to general procedure (C) using 7-bromo-N-[(oxan-4-yl)methyl]imidazopyridine-1-carboxamide 22 (0.18 mmol) and boronic acid (0.18 mmol). Yield = 61% (42 mg). HRMS (ESI): calculated 372.1518 [M + H]+, found 372.1527 [M + H]+. 1H-NMR  δ 8.56 , 8.51 , 8.30 , 8.16 , 7.52–7.47 , 7.35–7.37 , 7.10 , 3.83 , 3.25 , 3.19 , 1.86–1.77 , 1.57 , 1.27–1.16 ppm . 13C-NMR  δ 163.2, 150.7, 147.8, 130.9, 129.2, 128.8, 128.7, 125.9, 125.8, 125.6, 124.9, 118.9, 117.8, 114.6, 67.2, 44.3, 35.6, 31.0 ppm.N-((1-(2-Methoxyethyl)piperidin-4-yl)methyl)-6-(pyridin-3-yl)imidazopyridine-3-carboxamide (13). The title compound was obtained according to general procedure (A) using lithium 6-(pyridin-3-yl)imidazopyridine-3-carboxylate 30 (0.40 mmol) and 1-[1-(2-methoxyethyl)piperidin-4-yl]methanamine (0.40 mmol). Yield = 39% (63 mg). HRMS (ESI): calculated 394.2237 [M + H]+, found 394.2229 [M + H]+. 1H-NMR  δ 9.70 , 8.92 , 8.66–8.62 , 8.12 , 7.91 , 7.64 , 7.55 , 7.45 , 3.40 , 3.22–3.19 , 2.83 , 2.42 , 1.89 , 1.63 , 1.59–1.56 , 1.22–1.14 ppm . 13C-NMR  δ 159.7, 149.7, 148.0, 134.8, 132.9, 132.5, 130.5, 125.2, 124.5, 122.6, 121.9, 121.0, 119.2, 70.6, 58.4, 57.9, 54.0, 44.4, 36.3, 30.3 ppm.6-(5-Isopropoxypyridin-3-yl)-N-((1-(2-methoxyethyl)piperidin-4-yl)methyl)imidazopyridine-3-carboxamide (14). The title compound was obtained according to general procedure (A) using lithium 6-{5-[(propan-2-yl)oxy]pyridin-3-yl}imidazopyridine-3-carboxylate 31 (0.30 mmol) and 1-[1-(2-methoxyethyl)piperidin-4-yl]methanamine (0.30 mmol). Yield = 46% (60 mg). HRMS (ESI): calculated 452.2656 [M + H]+, found 452.2647 [M + H]+. 1H-NMR  δ 9.75 , 8.45 , 8.31 , 7.66 , 7.53-7.50 , 7.49 , 7.42 , 7.21 , 4.72 – 4.67 , 3.51 , 3.38 , 3.34 , 2.99 , 2.57 , 2.01 , 1.79 , 1.69–1.63 , 1.48–1.42 , 1.40 ppm . 13C-NMR  δ 159.8, 154.2, 140.2, 138.5, 133.6, 132.7, 130.3, 125.6, 123.3, 121.8, 121.0, 120.6, 118.3, 70.8, 70.0, 58.9, 58.0, 53.8, 44.5, 36.1, 29.6, 21.9 ppm.6-(Pyridin-3-yl)-N-((tetrahydro-2H-pyran-4-yl)methyl)imidazopyridine-3-carboxamide (15). The title compound was obtained according to general procedure (A) using lithium 6-(pyridin-3-yl)imidazopyridine-3-carboxylate 30 (1.35 mmol) and 1-(oxan-4-yl)methanamine (1.35 mmol). Yield = 9% (41 mg). HRMS (ESI): calculated 337.1659 [M + H]+, found 337.1657 [M + H]+. 1H-NMR  δ 9.72 , 8.90–8.89 , 8.60 , 8.18 , 7.85 , 7.61–7.57 , 7.41 , 3.96 , 3.42 , 3.35 , 1.99–1.87 , 1.75–1.71 , 1.45–1.33 ppm .6-(5-Isopropoxypyridin-3-yl)-N-((tetrahydro-2H-pyran-4-yl)methyl)imidazopyridine-3-carboxamide (16). The title compound was obtained according to general procedure (A) using lithium 6-{5-[(propan-2-yl)oxy]pyridin-3-yl}imidazopyridine-3-carboxylate 31 (0.43 mmol) and 1-(oxan-4-yl)methanamine (0.43 mmol). Yield = 22% (37 mg). HRMS (ESI): calculated 395.2078 [M + H]+, found 395.2075 [M + H]+. 1H-NMR  δ 9.66 , 8.39 , 8.23 , 7.80 , 7.65–7.63 , 7.56 , 7.35 , 4.83–4.79 , 3.95 , 3.44–3.38 , 3.33 , 1.97–1.88 , 1.74–1.70 , 1.42–1.32 ppm . 6-(4-(Hydroxymethyl)pyridin-3-yl)-N-((tetrahydro-2H-pyran-4-yl)methyl)imidazopyridine-3-carboxamide (17). The title compound was obtained according to general procedure (A) using lithium 6-(4-(hydroxymethyl)pyridin-3-yl)imidazopyridine-3-carboxylate 32 (0.35 mmol) and 1-(oxan-4-yl)methanamine (0.42 mmol). Yield = 45% (34 mg). HRMS (ESI): calculated 367.1764 [M + H]+, found 367.1763 [M + H]+. 1H-NMR  δ 9.43 , 8.61 , 8.49 , 7.81 , 7.74 , 7.62 , 7.11 , 4.66 , 3.95 , 3.41 , 3.34 , 1.97–1.85 , 1.74–1.68 , 1.42–1.30 ppm .6--N-((tetrahydro-2H-pyran-4-yl)methyl)imidazopyridine-3-carboxamide (18). The title compound was obtained according to general procedure (A) using lithium 6-imidazopyridine-3-carboxylate 33 (1.05 mmol) and 1-(oxan-4-yl)methanamine (1.05 mmol). Yield = 15% (59 mg). HRMS (ESI): calculated 372.1518 [M + H]+, found 372.1520 [M + H]+. 1H-NMR  δ 9.66 , 8.69 , 7.90 , 7.66 , 7.56–7.50  7.49–7.46 , 7.39–7.35 , 7.30 , 3.84 , 3.25 , 3.21 , 1.89–1.81 , 1.58 , 1.25–1.17 ppm . 1H13C-NMR  δ 159.8, 150.7, 147.8, 132.3, 130.5, 127.3, 126.2, 125.9, 124.6, 123.0, 121.8, 121.1, 118.8, 117.9, 67.2, 44.5, 35.5, 31.0 ppm.2. Crystals of GSK-3β/Compound 2 were grown in the following conditions: 12–20% v/v PEG 8000, 0.13 M NaCl, 0.10 M TrisAcetate pH 7.5–8.0, 5 mM TCEP at 4 °C. Crystals appeared within 3 to 7 days in hanging drop vapor diffusion setups and were cryo-protected with glycerol, and vitrified in liquid nitrogen. Data collection and model statistics of the crystal structure of GSK-3β in complex with ligand 2 are shown in the 2 at the SWISS LIGHT SOURCE  using cryogenic conditions. The crystals belong to space group C 2 2 21. Data were processed to 2.08 Å resolution using the programs XDS and XSCALE pyridine-3-carboxamide inhibitor d XSCALE . The pharization . Subsequrization ,49. For rization ,49. The v/v). Analytical standards were prepared from test compound DMSO dilutions and transferred to buffer, maintaining a 1% v/v DMSO concentration. Assay buffer was composed of supplemented HBSS pH 7.4. For assessment of A–B permeability, HBSS was removed from the apical compartment and replaced with test compound dosing solution. The apical compartment insert was then placed into a companion plate containing fresh buffer (containing 1% v/v DMSO). For assessment of B–A permeability, HBSS was removed from the companion plate and replaced with test compound dosing solution. Fresh buffer (containing 1% v/v DMSO) was added to the apical compartment insert, which was then placed into the companion plate. At 60 min, the apical compartment inserts and the companion plates were separated and apical and basolateral samples diluted for analysis. Test compound permeability was assessed in duplicate. Two control compounds were screened alongside the test compounds, propranolol (highly permeable), and prazosin (a substrate for P-gp). Test and control compounds were quantified by LC-MS/MS cassette analysis using an 8-point calibration with appropriate dilution of the samples. Cyprotex generic analytical conditions were used. The starting concentration (C0) was determined from the dosing solution and the experimental recovery calculated from C0 and both apical and basolateral compartment concentrations. The permeability coefficient (Papp) for each compound was calculated from the following Equation (1):0 is the donor compartment concentration at time zero, and A is the area of the cell monolayer. C0 was obtained from analysis of the dosing solution. For bi-directional experiments, ER was calculated from mean A–B and B–A data. This was derived from Equation (2):MDCK-MDR1 cells were obtained from the NIH . Cells were seeded onto Millipore Multiscreen Transwell plates at 3.4 × 105 cells/cm2 and cultured in DMEM. Cell culture and assay incubations were carried out at 37 °C in an atmosphere of 5% CO2 with a relative humidity of 95%. The monolayers were prepared by rinsing both apical and basolateral surfaces twice with Hanks Balanced Salt Solution (HBSS) at the desired pH warmed to 37 °C. Cells were then incubated with HBSS at the desired pH in both apical and basolateral compartments for 40 min to stabilize physiological parameters. The dosing solutions were prepared by diluting test compound with assay buffer to give a final test compound concentration of 10 μM  were used. The protein was prepared using Maestro Protein Preparation Wizard [3 centered on the centroid of the co-crystallized inhibitor. In addition, we treated thiol and hydroxyl groups of Tyr134, Thr138, and Cys199 as rotatable (flexible) groups. Three H-bond constraints were defined being the carbonyl oxygen of the hinge residues Asp133 and Val135, and the hydrogen of the N-H group of Val135. In Glide SP docking, we required that at least one such H-bond crucial for compound activity was formed. We also used the enhanced sampling option to enhance the sampling of conformational space by adding variations on the input structure to the conformational search and included aromatic ligand hydrogen atoms as donors. Visual inspection of the docking results was carried out enabling the picking of one pose per ligand as a starting point for further studies. The Schrödinger suite was used for all calculations. Ligands were converted to 3D structures and prepared with LigPrep tool using default setting . Overalln Wizard . HydrogeA rigid-body superposition of INDZ vs. IMID 1 and IMID 2 analogues within each triplets was performed using Maestro software , and RMS16 in complex of GSK-3β (f GSK-3β  was prep1–18 within the ATP binding site of GSK-3β were processed in MD simulation using the graphics processing unit (GPU) tool of the Desmond MD package as supplied with the Schrödinger 2019-1 suite [+ ions in defined positions by using the ion placement option. Default parameters of OPLS-3e force field were used. The prepared systems included approximately 58,900 atoms. The MD simulation protocol followed an extended relaxation schedule consisting of the following steps: 1.2 restraints on the solute heavy atoms;100 ps, constant–volume ensemble (NVT) (T = 10 K); 50 kcal/mol/Å2.2 restraints on the solute heavy atoms;120 ps, NVT (T = 10 K); 50 kcal/mol/Å3.2 restraints on the solute heavy atoms;120 ps, constant–pressure ensemble (NPT) ; 50 kcal/mol/Å4.2 restraints on the solute heavy atoms;120 ps, NPT ; 50 kcal/mol/Å5.2400 ps, NPT ; unrestrained;6a.2, 8 and 14: 500 ns, NPT ; unrestrained;Derivatives 6b.Remaining compounds: 100 ns, NPT ; unrestrained.The selected binding poses of -1 suite ,56. The From 6a and 6b MD production step, 5000 and 1000 frames were respectively stored. The analysis of the MD trajectory was basically performed with the Schrödinger suite. RMSD and RMSF of both proteins and ligands, ligand–protein contacts along the simulations were calculated with the Simulation Interaction Diagram (SID) tool implemented in the Desmond MD package. MD simulations were visual inspected with VMD 1.9.4 . By defa2, 8, and 14, raw data from the SID output were processed using bash command lines and Knime workflows [For the comparison of 500 and 100 ns MD simulations of orkflows . In FiguThe percentages of interaction of the outer and central H-bond across each MD trajectory  and of oThe distances of the outer, central, and deep H-bonds along all the simulations were calculated with VMD 1.9.4, combined with Knime workflow and plotted in By way of illustration, snapshots of some MD trajectories were extracted using VMD 1.9.4 and reported in The average binding free energy (ΔG_bind) based on MM-GBSA was calculated using the thermal_mmgbsa.py script on 100 frames extracted from each production trajectory ,36 Tabl. The predictions of hydrogen bond acidity are based on the work of Kenny . The Vα using 2048 × 256 data points, 16 transients, with a relaxation delays of 2 s. NOESY experiments were acquired with a spectral window of 15 × 15 ppm (carrier frequency at 4.5 ppm) using 4096 × 256 data points, 32 transients, with a relaxation delay of 2 s and a mixing time of 600 ms. 1H-13C HSQC experiments were acquired with a spectral window of 15 ppm × 200 ppm (carrier frequencies at 6.5 and 100 ppm) using 2048 × 256 data points, 16 transients with relaxation delays of 2 s. 1H-13C HMBC experiments were acquired with a spectral window of 15 ppm × 220 ppm (carrier frequency at 6.5 and 110 ppm) using 4096 × 512 data points, 32 transients with relaxation delays of 2 s. All data were processed with TopSpin using a sine squared window function for each dimension of the 2D data. The complete NMR spectra assignment of the three compounds was performed in CDCl3, detected in the 1H-NMR spectra at 298 K.The variations of chemical shift (Δδ) for protons of interest were calculated from the difference of the chemical shift in DMSO-d6 and in CDClcoeff determination, 1H-NMR spectra of the three compounds were performed in deuterated DMSO at five different temperatures: 298 K, 303 K, 308 K, 313 K, and 318 K.For T1H-NMR spectra:The empirical estimation of the H-bonding strength was performed using the following equation, based on the Δδ values obtained from In this study, novel derivatives around the lead series core scaffold were investigated by means of ligand docking and MD simulations. The novel IMID derivatives turned out to be GSK-3β inhibitors in the nanomolar scale with a potency drop compared to the previous INDZ series but improved CNS permeability. Interestingly, the in silico-derived models of the binding mode generated during the design process well suited the crystallographic data (PDB entry 6Y9S). In order to rationalize SAR, computational tools were also employed retrospectively. In particular, MD and QM-based calculations shed light on the differences of the H-bond interactions between the core scaffolds and the hinge residues moving from INDZ to IMID 1 and IMID 2 derivatives. The pharmacological profile of IMID 1 and IMID 2 derivatives was associated to the lower acidity of the C-Ha versus N-Ha group of the core scaffolds. NMR-based studies were also carried out to substantiate this hypothesis. Overall, our findings provided adequate evidence that low C-Ha acidity and consequent unfavorable geometric rearrangement affected H-bond donating abilities so reducing the affinity for GSK-3β enzyme. This represented significant structural activity insight into our GSK-3β inhibitor design and demonstrated the pivotal role of computational approaches properly supported by experimental proofs in the different steps of the drug discovery program. In the future, prospective application of the aforementioned in silico tools may lead to further optimized classes of GSK-3β inhibitors.


During the past decades, several inhibitors were developed, but they lack specificity or efficacy. From the non-specific SOCE inhibitor 2-aminoethyl diphenylborinate (2-APB), we synthetized 16 new analogues by replacing/modifying the phenyl groups. Among them, our compound P11 showed the best inhibitory capacity with a Ki ≈ 75 nM. Furthermore, below 1 µM, P11 was devoid of any inhibitory activity on the two other main cellular targets of 2-APB, the IP3 receptors, and the SERCA pumps. Interestingly, Jurkat T cells secrete interleukin-2 under phytohemagglutinin stimulation but undergo cell death and stop IL-2 synthesis when stimulated in the presence of increasing P11 concentrations. Thus, P11 could represent the first member of a new and potent family of immunosuppressors.The store-operated calcium entry, better known as SOCE, forms the main Ca Indeed, many cellular functions, including contraction, differentiation, and proliferation, are regulated by an increase of the intracellular Ca2+ ion concentration cyt). To test 2-APB and its analogues, different concentrations of compounds were added 30 s prior to Ca2+ addition.To visualize SOCE, we performed a classical protocol: Jurkat cells in Ca2+]cyt increase) and an almost total inhibition at 50 µM. No significant differences were found between commercial and our 2-APB (not shown).As shown in 2+]cyt, confirming that enlarging the two phenyl groups impairs the potentiation process, resulting in inhibitors with a Ki ranging 1000-fold , Ki ≈ 0.5 µM (The addition of one methyl on both phenyl rings in para and meta positions respectively for compound P3 (and P5) allowed the creation of inhibitors with an inhibition constant of 2–3 µM B. Intere≈ 0.5 µM , and comKi = 75 ± 21 µM, We also created 2-APB analogs with one methoxy group on the two aromatic rings (compounds P2 and P4). Even if the presence of a methoxy group on the aromatic rings in para position had the same effect as a methyl group (compound P3 ≈ P2), the replacement in meta drastically impaired the inhibitory capacity of the compound P4  and compound P14 (F) vs. 3.5 µM for compound P3  or a linear alkyl chain . P8 and P9 are better inhibitors with Ki = 350 ± 40 nM and 641 ± 103 nM, respectively . Our coM n = 3, .Ki, 275 ± 17 nM  and the Na+/Ca2+ exchanger (“NCX”) counteracting the SOCE . To ensure that compound P11 acted on the SOCE and not on the Ca2+-efflux mechanisms, we next performed a Mn2+-quenching experiment with Indo-1. Mn2+ ions enter the cells through SOCC but cannot be pumped outside the cells by PMCA and NCX; once in the cells, Mn2+ bound to Indo-1 and quenched its fluorescence measured at 430 nm. Thus, an increase of the Mn2+ influx amplitude was associated with an increase of the Indo-1 quenching speed, and reversely cyt rise in LCL expressing Orai1 proteins, confirming the SOCE default of the SCID patient. Noteworthy, 1 µM P11 only induced an immediate decrease of [Ca2+]cyt in Orai1-expressing LCL and was inefficient in Orai1-deficient LCL. These results confirm that Orai1 is the target of P11 in these cells. We next tested P11 efficacy on two lymphoblastoid cell lines (“LCL”) established from an Orai1-deficient with a severe combined immunodeficiency (“LCL Orai1−”) and a healthy (“LCL Orai1+”) patients . Thus, Among our various molecules, we selected to perform experiments on P11 and two structurally close compounds, P8 (Ki ≈ 300 nM) and P9 (Ki ≈ 600 nM). 45Ca2+ store loading and IP3-dependent release from the ER Ca2+ store were performed on permeabilized cells in unidirectional conditions. As this approach required the use of cells forming strongly adherent monolayers, we used L15 fibroblasts stably overexpressing IP3R1, similarly to our previous work cyt increase for several minutes to allow the synthesis of interleukin-2 (“IL-2”) and the further activation of the immune functions. Therefore, inhibition of the [Ca2+]cyt rise due to SOCE impairs T cell activation and proliferation . Noteworthy, PHA was able to induce a weak Ca2+ release from internal stores (orange curve). One micromolar of P11 did not affect this Ca2+ release as the PHA-induced Ca2+ rise was very similar to the one recorded in absence of extracellular Ca2+ ions, showing that P11 only acted on the SOCE and not the ER Ca2+ release. However, after reaching a peak at 180 s, the [Ca2+]cyt started to decay and decreased to a value beneath the [Ca2+]cyt before cell stimulation. Thus, high [P11] concentrations not only blocked the SOCE of PHA-stimulated cells, but also emptied the cytosolic Ca2+ content. Having shown that compound P11 was able to block the Jurkat cell SOCE, we next performed experiments using phytohemagglutinin (“PHA”). PHA is commonly used to cross-link the T cell receptor allowing the synthesis of IL-2 and thereafter the T cell activation . As showWe next studied the effect of increasing [P11] on the neo-synthesized IL-2 concentration in the medium of PHA -stimulated Jurkat cells after 24 h.n = 3, n = 3, In the absence of PHA stimulation, Jurkat cells did not produce any IL-2 “UT”, B. On theAs this diminution in IL-2 synthesis can both be explained by the decrease of its synthesis by all cells, or by a decrease in the number of cells that can synthesize IL-2, we next evaluated cell death by trypan blue staining in the presence of PHA stimulation with and without the addition of compound P11.n = 5, n = 4). Furthermore, compound P11 by itself was not toxic as no significant increase in dead cells was noticed at a maximal concentration of the compound . Under resting culture conditions, the percentage of dead cells measured was lower than 5% . Interestingly, the additional presence of 10 nM compound P11 already induced a maximal activating effect on caspase-3 activity with a ≈ threefold increase . As shown in n = 3). PHA alone induced a threefold increase in the number of cells with fragmented DNA . At concentrations > 10 nM, compound P11 induced a ≈ 4.5-fold increase .Moreover, similar results were obtained when the percentage of dead cells was measured with the TUNEL assay . In restTaken together, these results demonstrate that compound P11 is not toxic for the cells when added alone but could profoundly impair Jurkat cell activation by inducing their apoptosis. Since the discovery of the molecular partners implied in SOCE in the middle of the 2000s, the characterization of their modulators has emerged as attracting enough for pharmaceutical companies like GSK and Hoffmann-La Roche or newcomers like Calcimedica. Indeed, it appeared that aberrant SOCE could be associated with autoimmune disorders, inflammation, or cancers . Inhibit2+ channels mainly consisting of Orai1 proteins  for 24 h to study the apoptosis induction and to measure IL-2 synthesis and caspase-3 activity. Remarkably, P11 was not by itself cytotoxic, and it exerted a cytotoxic effect only when the cells were stimulated and the [Ca2+]cyt rise impaired  was maintained in same conditions as the leukocyte cell lines except that DMEM  medium was used instead of RPMI1640. Epstein-Barr virus immortalized B cells from Orai1-deficient and healthy patients were a kind gift of Capucine Picard and Alain Fischer  supplemented with 10% heat-inactivated fetal calf serum, 3.8 mM L-glutamine, 1% non-essential amino acids, 85 IU mL−1 penicillin, 85 µg/mL streptomycin, 400 µg/mL geneticin, and 25 mM Na-Hepes (pH 7.4) at 37 °C and 10% CO2.Jurkat (acute T cell leukemia from ATCC), DG75 , and U937 (monocytic cell line from ATCC) cell lines were basically maintained in RPMI-1640 medium  supplemented with 10% heat-inactivated fetal calf serum and 2 mM L-glutamine, at 37 °C in a 5% COs France ) and maince [3R1  was a ge2+]cyt was recorded by a fluorimetric ratio technique as previously described cyt was calculated according to the equation [Ca2+]cyt = Kd (R-Rmin)/(Rmax-R), where Kd is the apparent dissociation constant of Indo-1-calcium complex (230 nM), and R is the ratio of fluorescence values recorded at 380 nm in absence and presence of 10 mM CaCl2 cyt subsequently to Ca2+ influx +.1.21 g of 2-((bis(4-methoxyphenyl)boryl)oxy)ethanamine were obtained following the general procedure H using 2.06 g of 3-bromoanisole as a white solid (81% yield). P3, 2-((di-p-tolylboryl)oxy)ethanamine (19565-45-8)  1H NMR : δ (ppm) 7.25 , 6.94 , 5.92 , 3.73 , 2.80 , 2.20 . 11B NMR : δ (ppm) 5.7 (s) 13C NMR : δ (ppm) 148.2, 133.3, 131.6, 127.3, 62.4, 41.3, 20.9. Melting point: 185.3 °C. LC-MS (ESI): C16H20BNO [2M+H]+: 506.80.1.04 g of 2-((di-p-tolylboryl)oxy)ethanamine were obtained following the general procedure using 1.88 g of 4-bromotoluene as a white solid (90% yield). P4, 2-((bis(3-methoxyphenyl)boryl)oxy)ethanamine1H NMR : δ (ppm) 7.06 , 6.96 , 6.60 , 6.05 , 3.76 , 3.68 , 2.81 . 11B NMR : δ (ppm) 5.3 (s). 13C NMR : δ (ppm) 158.2, 127.6, 123.9, 116.9, 110.1, 62.4, 54.5, 41.3. Melting point: 137.7 °C. LC-MS (ESI): C16H20BNO3 570.70 [2M+H]+.1.12 g of 2-((bis(3-methoxyphenyl)boryl)oxy)ethanamine were obtained following the general procedure using 2.06 g of 3-bromoanisole as a white solid (83% yield). P5, 2-((di-m-tolylboryl)oxy)ethanamine (97979-11-8)  1H NMR : δ (ppm) 7.26–7.10 , 7.01 , 6.84 , 6.00 , 3.74 , 2.80 , 2.22  11B NMR : δ (ppm) 4.8 (s). 13C NMR : δ (ppm) 135.2, 132.7, 129.1, 126.9, 125.9, 62.80, 41.8, 21.9. Melting point: 193.4 °C LC-MS (ESI): C16H20BNO [2M+H]+: 506.8.1.05 g of 2-((di-m-tolylboryl)oxy)ethanamine were obtained following the general procedure using 1.88 g of 3-bromotoluene as a white solid (82% yield). P6, 2-boryl)oxy)ethanamine (515157-62-7) 1H NMR : δ (ppm) 7.95 , 7.76 , 7.73–7.61 , 7.44–7.30 , 6.34 , 3.88 , 3.02–2.88 . 11B NMR : δ (ppm) 6.0 (s). 13C NMR : δ (ppm) 132.9, 131.9, 130.8, 129.7, 127.4, 127.2, 125.4, 124.9, 124.3, 62.6, 41.6. Melting point: 206.2 °C. LC-MS (ESI): C22H20BNO 650.80 [2M+H]. 1.50 g of 2-boryl)oxy)ethanamine were obtained following the general procedure using 2.28 g of 2-bromonaphtalene as a white solid (92% yield). P7, 2-boryl)oxy)ethanamine (99269-70-2)o-xylene as a white solid (79% yield). 1H NMR : δ (ppm) 7.12 , 7.06 , 6.87 , 5.87 , 3.71 , 2.78 , 2.13 , 2.12 . 11B NMR : δ (ppm) 5.9. 13C NMR : δ (ppm) 133.4, 133.1, 131.9, 129.2, 127.8, 62.30, 41.3, 19.7, 19.2. Melting point: 209.2 °C. LC-MS (ESI): C18H24BNO 562.75 [2M+H]+.1.11 g of 2-boryl)oxy)ethanamine were obtained following the general procedure using 2.04 g of 4-bromo-P8, 2-((bis(4-(tert-butyl)phenyl)boryl)oxy)ethanamine (1379680-33-7)1H NMR : δ (ppm) 7.32 , 7.15 , 5.98 , 3.75 , 2.80 , 1.23 . 11B NMR : δ (ppm) 6.1 (s). 13C NMR : δ (ppm) 146.7, 131.2, 123.3, 62.4, 41.4, 33.9, 31.4. Melting point: 255.5 °C. LC-MS (ESI): C22H32BNO 674.90 [2M+H]+.1.24 g of 2-((bis(4-(tert-butyl)phenyl)boryl)oxy)ethanamine were obtained following the general procedure using 2.34 g of 4-terbutyl-bromobenzene as a white solid (85% yield). P9, 2-(bis(4-(tert-butyl)phenyl)boryl)oxy)ethanaminen-butyl-bromobenzene as a white solid. (70% yield). 1H NMR : δ(ppm) 7.29 , 6.95 , 5.96 , 3.75 , 2.88–2.77 , 2.58–2.42 , 1.51 , 1.30 , 0.89 . 11B NMR : δ (ppm) 5.8 (s). 13C NMR : δ (ppm) 148.6, 138.4, 131.5, 126.6, 62.4, 41.3, 34.8, 33.5, 21.8, 13.8. Melting point: 99.0 °C. LC-MS (ESI): C22H32BNO 674.85 [2M+H]+.1.48 g of 2-(bis(4-(tert-butyl)phenyl)boryl)oxy)ethanamine were obtained following the general procedure using 2.34 g of 4-P10, 2-boryl)oxy)ethanaminem-xylene as a white solid (85% yield). 1H NMR : δ (ppm) 6.97 , 6.65 , 5.92 , 3.70 , 2.78 , 2.18 . 11B NMR : δ (ppm) 5.6 (s). 13C NMR : δ (ppm) 134.6, 129.4, 126.3, 62.3, 41.3, 21.3. Melting point: 294.5 °C. LC-MS (ESI): C18H24BNO 562.75 [2M+H]+.1.02 g of 2-boryl)oxy)ethanamine were obtained following the general procedure using 2.04 g of 4-bromo-P11, 2-(dibiphenyl-4-ylboryloxy)ethanamine (102032-41-7)1H NMR : δ (ppm) 7.60 , 7.55 , 7.46 , 7.41 , 7.30 , 6.19 , 3.83 , 2.95–2.83 . 11B NMR : δ (ppm) 7.8 (s). 13C NMR : δ (ppm) 151.0, 141.3, 137.0, 132.2, 128.8, 126.7, 126.4, 125.1, 62.6, 41.5. Melting point: 232.0 °C. LC-MS (ESI): C26H24BNO 754.80 for [2M+H]+.1.55 g of 2-(dibiphenyl-4-ylboryloxy)ethanamine were obtained following the general procedure using 2.56 g of 4-bromobiphenyl as a white solid (79 % yield). P12, 2-(dibenzo[b]thiophen-5-ylboryloxy)ethanamine1H NMR : δ (ppm) 7.92 , 7.75 , 7.55 , 7.47 , 7.33 , 6.21 , 3.85 , 2.98–2.85 . 11B NMR : δ (ppm) 6.7 (s) 13C NMR : δ (ppm) 147.3, 138.9, 136.4, 128.8, 126.4, 125.2, 124.0, 120.5, 62.6, 41.5. Melting point: 228.0 °C. LC-MS (ESI): C18H16BNOS2 674.55 [2M+H]+.1.34 g of 2-(dibenzo[b]thiophen-5-ylboryloxy)ethanamine were obtained following the general procedure using 2.34 g of 5-bromobenzo[b]thiophene as a white solid (68 % yield). P13, 2-(bis(4-chlorophenyl)boryl)oxy)ethanamine (61733-90-2)1H NMR : δ (ppm) 7.38–7.35 , 7.19–7.16 , 6.18 , 3.77–3.73 , 2.87–2.79 . 11B NMR : δ (ppm) 4.0. 13C NMR : δ (ppm) 133.8, 130.5, 129.1, 127.0, 62.9, 41.8.1.132g of 2-(bis(4-chlorophenyl)boryl)oxy)ethanamine were obtained as a white solid starting from 575 mg of diisopropylamine borane complex following the general procedure (80%). P14, 2-(bis(4-fluorophenyl)boryl)oxy)ethanamine (176913-70-5)1H NMR : δ (ppm) 7.40-7.36 , 6.98–6.92 t, J = 9Hz, 4H), 6.11 , 3.77 , 2.84 . 11B NMR : δ (ppm) 3.9. 13C NMR : δ (ppm) 163.0, 159.8, 133.6, 113.5, 62.9, 41.8.871 mg of 2-(bis(4-fluorophenyl)boryl)oxy)ethanamine were obtained as a pale yellow solid starting from 575 mg of diisopropylamine borane complex following the general procedure (80%). P16, 2-(((4-methoxyphenyl)(p-tolyl)boryl)oxy)ethanamine (7294-53-3)2SO4, filtered and concentrated under reduced pressure. The residue was dissolved in Et2O (25 mL) and 1.2 eq of ethanolamine was added. Depending on substituents, precipitation occurs spontaneously or requires addition of pentane. Crystals were filtered, washed with pentane, and dried under vacuum to yield 2-(((4-methoxyphenyl)(p-tolyl)boryl)oxy)ethanamine as a white solid in 90% yield). 1H NMR : δ (ppm) 7.26 , 6.94 , 6.71 , 5.89 , 3.74 , 3.67 , 2.80 , 2.21 . 11B NMR : δ (ppm) 5.19 (s). 13C NMR : δ (ppm) 157.3, 133.3, 132.6, 131.6, 127.3, 112.3, 62.4, 54.6, 41.3, 20.9. Melting point: 154.3°C LC-MS (ESI): C16H20BNO2 538.70 [2M+H]+: 539.32To a suspension of magnesium  in a solution of diisopropylaminoborane  in anhydrous THF (30 mL), at room temperature, was added 855 mg of 4-bromotoluene. The mixture was then heated at 70 °C under stirring during 20 h. After cooling at room temperature, all the volatiles were removed under vacuum and to the resulting oil were added anhydrous THF (40 mL) and 1.40 g of 4-bromoanisole (7.5 mmol) and the mixture was heated at 70 °C during 20 h. After cooling at −78 °C, the reaction was quenched with a 3 M aqueous HCl solution (25 mL) and was stirred for 45 min at room temperature. To this solution was added water (20 mL), diethyl ether (20 mL), and the organic phase was separated. The aqueous phase was then extracted with DCM (6 × 30 mL) and the combined organic phases were dried over anhydrous Nat-test < 0.05 is considered as significant.Given values are representative of at least three independent experiments and are given as mean ± SEM. When used, a 3R, absence of cytotoxicity on non-activated cells, but the ability to induce the caspase-3 activity and the apoptosis of PHA-activated cells. Thus, P11 is able to highjack the Ca2+ signaling of the T Jurkat cells from proliferation to death.This study confirms that from the non-specific SOCE inhibitor, 2-APB, it is possible to create new analogues with more efficient properties. Thus, we designed, synthetized, and characterized 16 new molecules. Among them, the molecule we called P11 unveils interesting properties: Full SOCE inhibition for concentration less than 1 µM with an inhibition constant between 30 and 75 nM according to the cell lines, absence of effect on the two other main targets of 2-APB, SERCA and IPThe 2-APB analogues are patented: FR1907184.


Correction to: Bioelectronic Med (2019) 5:18https://doi.org/10.1186/s42234-019-0035-xThe original version of this article  exhibited significant reductions in congestion symptoms, compared to pretreatment, at all time points measured (a). Mean difference in CQ7 score from before treatment peaked at -8.8 (− 45.0%) points at week four (b). Data represented as mean ± SEM. ****p < 0.0001, paired, two-sided t-testDaily microcurrent treatment reduces sinonasal congestion over 4 weeks. Sinonasal congestion was assessed weekly using the Congestion Quanitfier 7 (CQ7) instrument. Subjects that enrolled with moderate or worse congestion ; Week 2, -6.3 (CI95 -4.2 to -8.4); Week 3, -7.4 (CI95 -4.8 to -10.1); Week 4, -8.8 (CI95 -6.5 to -11.1)Percentage Decrease in CQ7 from Enrollment: Week 1, -23.0%; Week 2, -32.8%; Week 3, -38.1%; Week 4, -45.0%P < 0.0001 for all data points.Statistics remain unchanged at


Naphthalimides were more cytotoxic than naphthalic anhydrides, with the highest IC50 value determined for compound 9 (3.10 µM). These compounds were capable of inducing cell cycle arrest at G0/G1 or G2M phase and promoting apoptosis, autophagy or ferroptosis. The most promising conjugate 35 caused strong apoptosis and induced ROS production, which was proven by the increased level of 2′-deoxy-8-oxoguanosine in DNA. The tested conjugates were found to be weak topoisomerase II inhibitors and classical DNA intercalators. Compounds 33, 34, and 36 fluorescently stained lysosomes in HepG2 cells. Additionally, we performed a similarity-based assessment of the property profile of the conjugates using the principal component analysis. The creation of an inhibitory profile and descriptor-based plane allowed forming a structure–activity landscape. Finally, a ligand-based comparative molecular field analysis was carried out to specify the (un)favorable structural modifications (pharmacophoric pattern) that are potentially important for the quantitative structure–activity relationship modeling of the carborane–naphthalimide conjugates.We synthesized a series of novel 3-carboranyl-1,8-naphthalimide derivatives, mitonafide and pinafide analogs, using click chemistry, reductive amination and amidation reactions and investigated their in vitro effects on cytotoxicity, cell death, cell cycle, and the production of reactive oxygen species in a HepG2 cancer cell line. The analyses showed that modified naphthalic anhydrides and naphthalimides bearing  These compounds have been used in biological and nonbiological applications and have mainly been tested as DNA intercalators and anticancer as well as antibacterial, antiviral, and analgesic agents , receptoN-acetylamonafide, and neuromuscular dose-limiting toxicity, respectively amine (TBTA) as a ligand to complex and “protect” copper(I) -1,8-naphthalimide (21), or 3-amino-N-[2-(N-pyrrolidinyl)ethyl]-1,8-naphthalimide (22) -1,8-naphthalimide (21) or 3-amino-N-[2-(N-pyrrolidinyl)ethyl]-1,8-naphthalimide (22) with 3-propionic acid (37) -1,8-naphthalimide (21) and 3-amino-N-[2-(N-pyrrolidinyl)ethyl]-1,8-naphthalimide (22) were obtained in two steps: (1) synthesis of 3-aminonaphthalic anhydride (12) -1H-1,2,3-triazol-4-yl}-1,8-naphthalic anhydride (6): white solid, yield 12.2 mg (60%). TLC : Rf = 0.28; 1H-NMR : δ (ppm) = 8.98 , 8.96 , 8.90 , 8.58 , 8.50 , 7.93 , 5.19 , 4.47 , 2.40–2.37 , 2.13–2.08 , 3.0–1.5 ; 13C-NMR : δ (ppm) = 160.49 , 160.46 , 144.67 , 135.36–119.08 , 75.54 , 63.05 , 48.63 , 33.53 , 29.26 ; 11B-NMR {1H BB} : δ (ppm) = −3.19 , −6.19 , −9.77 , −11.64–−13.11 ; UV (99.8% EtOH): λmax (nm) = 260, 266, 335, λmin = 296, λsh = 320, 367; FT-IR: νmax (cm−1) = 2964 , 2575 (B-H), 1770 (C=O), 1733 (C=O), 725 (B-B); RP-HPLC (gradient A): tR = 18.23 min; APCI-MS: m/z: 450 [M + H]+, 492 [M + H + CH3CN]+, calcd for C19H23B10N3O3: 449.3-{1-[3-propyl]-1H-1,2,3-triazol-4-yl}-1,8-naphthalic anhydride (7): white solid, yield 38.5 mg, (48%). TLC : Rf = 0.43; 1H-NMR : δ (ppm) = 8.93 , 8.86 , 8.54 , 8.46 , 7.90 , 4.42 , 4.05 , 2.10–2.07 , 2.00–1.97 , 3.0–1.5 ; 13C-NMR : δ (ppm) = 160.41 , 160.40 , 144.57 , 135.32–118.98 , 75.73 , 56.30 , 48.75 , 32.83 , 29.92 ; 11B-NMR {1H BB} : δ (ppm) = −4.57 , −11.09 , −13.54 , −15.02 ; UV (99.8% EtOH): λmax (nm) = 261, 266, 334, λmin = 296, λsh = 234, 323, 365; FT-IR: νmax (cm−1) = 2956 , 2594 (B-H), 1769 (C=O), 1741 (C=O), 724 (B-B); RP-HPLC (gradient A): tR = 16.80 min; APCI-MS: m/z: 450 [M + H]+, 492 [M + H + CH3CN]+, calcd for C19H23B10N3O3: 449.3-{1-[3-propyl]-1H-1,2,3-triazol-4-yl}-1,8-naphthalic anhydride (6) or 3-{1-[3-propyl]-1H-1,2,3-triazol-4-yl}-1,8-naphthalic anhydride (7) (1 equiv.) was dissolved in absolute EtOH (10 mL per 0.45 mmoL) and N,N-dimethylethylenediamine (1.1 equiv.) (for compound 8) or N-(2-aminoethyl)pyrrolidine (1.1 equiv.) (for compound 9) was added. The reaction mixture was stirred for 1 h at 35 °C and then for 1 h at 45 °C under an inert (Ar) atmosphere. Subsequently, the solvent was evaporated to dryness under vacuum and crude product was purified by column chromatography on silica gel (230–400 mesh) with a gradient of MeOH (0–10%) in CH2Cl2. Additionally (only for compound 11), purified product was dissolved in CHCl3 (1 mL) and poured into hexane (5 mL). A precipitate was isolated by centrifugation.N-{2-(Dimethylamino)ethyl]-3-[1-propyl]-1H-1,2,3-triazol-4-yl}-1,8-naphthalimide (8): yellow solid, yield 9.5 mg (41%). TLC : Rf = 0.29; 1H-NMR : δ (ppm) = 8.86 , 8.76 , 8.69 , 8.45 , 8.37 , 7.83 , 4.73 , 4.61 , 4.28 2), 2.67 2), 2.58–2.55 , 2.33–2.30 2 overlapped with CH2-CH2-CH2), 3.0–1.5 ; 13C-NMR : δ (ppm) = 164.60 , 164.54 , 146.82 , 133.26–123.84 , 76.54 , 63.82 , 57.95 2), 50.13 , 46.14 , 38.92 2), 35.55 , 30.89 ; 11B-NMR {1H BB} : δ (ppm) = −2.80 , −5.95 , −9.59 , −11.53–−11.80 , −12.98 ; UV (99.8% EtOH): λmax (nm) = 257, 338, λmin = 295, λsh = 231, 321, 372; FT-IR: νmax (cm−1) = 2942 , 2574 (B-H), 1698 (C=O),1656 (C=O), 722 (B-B); RP-HPLC (gradient B): tR = 22.28 min; APCI-MS: m/z: 520 [M+H]+, calcd for C23H33B10N5O2 = 519; HRMS (ESI+) 520.3867 [M + H]+, calcd for C23H33B10N5O2 = 520.3711 [M + H]+.N-[2-(N-Pyrrolidinyl)ethyl]-3-[1-propyl]-1H-1,2,3-triazol-4-yl}-1,8-naphthalimide (9): yellow solid, yield 14.3 mg (59%). TLC : Rf = 0.27; 1H-NMR : δ (ppm) = 8.84 , 8.74 , 8.68 , 8.44 , 8.34 , 7.81 , 4.73 , 4.60 , 4.33 2), 2.95 , 2.79 , 2.58–2.56 , 2.34–2.28 , 1.80 , 3.0–1.5 ; 13C-NMR : δ (ppm) = 164.46 , 164.40 , 146.60 , 134.95–122.81 , 76.36 , 63.64 , 54.93 , 54.23 , 49.94 , 39.41 2), 35.36 , 30.68 , 24.29 ; 11B-NMR {1H BB} : δ (ppm) = −2.80 , −5.95 , −9.60 , −11.54 , −13.03 ; UV (99.8% EtOH): λmax = 257, 338 nm, λmin = 296 nm, λsh = 238, 323, 372 nm; FT-IR: νmax (cm−1) = 2960 , 2579 (B-H),1698 (C=O), 1659 (C=O), 723 (B-B), RP-HPLC (gradient B): tR = 21.84 min; APCI-MS: m/z: 547 [M + H]+, calcd for C25H35B10N5O2 = 546.N-{2-(Dimethylamino)ethyl]-3-[1-propyl]-1H-1,2,3-triazol-4-yl}-1,8-naphthalimide (10): yellow solid. Yield: 19.3 mg (84%). TLC : Rf = 0.33; 1H-NMR : δ (ppm) = 8.89 , 8.85–8.83 , 8.45–8.41 , 7.84 , 4.42 2), 4.16 , 4.05 , 2.53 2), 2.22 , 2.09–2.07 , 1.99–1.97 , 3.0–1.5 ; 13C-NMR : δ (ppm) = 163.17 , 163.11 , 144.98 , 134.25–121.97 , 75.74 , 56.42 2), 56.30 , 48.72 , 45.30 , 37.58 2), 32.84 , 29.95 ; 11B-NMR {1H BB} : δ (ppm) = −4.62 , −11.11 , −13.58 , −15.05 ; UV (99.8% EtOH): λmax = 257, 339 nm, λmin = 2978 nm, λsh = 234, 373 nm; FT-IR: νmax (cm−1) = 2953 , 2577 (B-H), 1698 (C=O), 1655 (C=O), 722 (B-B); RP-HPLC (gradient B): tR = 22.20 min; APCI-MS: m/z: 520 [M + H]+, calcd for C23H33B10N5O2 = 519.N-[2-(N-Pyrrolidinyl)ethyl]-3-[1-propyl]-1H-1,2,3-triazol-4-yl}-1,8-naphthalimide (11): white solid. Yield: 15 mg (55%). TLC : Rf = 0.34; 1H-NMR : δ (ppm) = 8.83 , 8.79 , 8.54 , 8.22 , 8.00 , 7.74 , 4.43 2), 4.40 , 2.99 , 2.93 , 2.88 , 2.10–2.07 , 1.88 , 3.0–1.5 ; 13C-NMR : δ (ppm) = 164.18 , 164.15 (1C C12), 146.58 , 134.45–120.71 , 74.72 , 55.21 , 54.47 , 53.61 , 49.80 , 38.70 2), 33.83 , 30.53 , 23.79 ; 11B-NMR {1H BB} : δ (ppm) = −4.18 , −9.62 , −10.59–−10.98 , −13.45 , −15.32 ; UV (99.8% EtOH): λmax = 257, 338 nm, λmin = 295 nm, λsh = 233, 373 nm; FT-IR: νmax (cm−1) = 2956 , 2593 (B-H), 1698 (C=O), 1660 (C=O), 730 (B-B); RP-HPLC (gradient B): tR = 22.58 min; APCI-MS: m/z: 547 [M + H]+, calcd for C25H35B10N5O2 = 546.ortho-carborane (4) or meta-carborane (5)) (1 equiv.) was dissolved in THF/H2O . 3-(Prop-2-yn-1-yloxy)-1,8-naphthalic anhydride (14) (1 equiv.), CuSO4 5H2O (0.06 equiv.) and sodium ascorbate (0.1 equiv.) were added. Reaction mixture was stirred for 3–4 h in 35 °C. For compound 15 after 1 h additional portion of CuSO4⋅5H2O (0.06 equiv.) and sodium ascorbate (0.1 equiv.) were added. The reaction was quenched by evaporation of the solvents. The crude compound was purified by column chromatography on silica gel (230–400 mesh) with a gradient of MeOH (0–5%) in CH2Cl2 as the eluent to afford product.1-(3-Azidopropyl)-carborane propyl)-1H-1,2,3-triazol-4-yl]methoxy}-1,8-naphthalic anhydride (15): white solid. Yield: 93.6 mg (74%). TLC : Rf = 0.23; 1H-NMR : δ (ppm) = 8.41 , 8.37 , 8.31 , 8.17 , 8.12 , 7.87 , 5.44 , 5.14 , 4.37 , 2.28–2.25 , 2.01–1.96 , 3.0–1.5 ; 13C-NMR : δ (ppm) = 160.48 (C11), 160.16 (C12), 156.37 (C3), 142.07 (Ctriazole), 134.05–115.85 (9Carom + CHtriazole), 75.42 (Ccarborane), 62.85 (CHcarborane), 61.97 (O-CH2-triazole), 48.25 (CH2-triazole), 33.49 (CH2-carborane), 29.34 (CH2-CH2-CH2); 11B-NMR {1H BB} : δ (ppm) = −3.25 , −6.23 , −9.85 , −11.76–−13.13 ; UV (99.8% EtOH): λmax = 236.5, 329.1, 371.7 nm, λmin = 227.5, 281.7, 344.9 nm, λsh = 245.9, 314.6 nm; FT-IR: νmax (cm−1) = 2924 , 2579 (B-H), 1770 (C=O), 1732 (C=O), 724 (B-B); RP-HPLC (gradient A): tR = 17.61 min; APCI-MS: m/z: 480 [M + H]+, 513 [M + H + MeOH]+, 522 [M + H + CH3CN]+, calcd for C20H25B10N3O4 = 479.3-{methoxy}-1,8-naphthalic anhydride (16): white solid. Yield: 39 mg (52%). TLC : Rf = 0.30; 1H-NMR : δ (ppm) = 8.41 , 8.37 , 8.30 , 8.17 , 8.12 , 7.87 , 5.44 , 4.33 , 4.01 , 1.92–1.85 , 3.0–1.5 ; 13C-NMR : δ (ppm) = 160.52 , 160.18 , 156.35 , 142.03 , 134.07–115.87 , 75.62 , 61.94 , 56.26 , 48.39 , 32.72 , 30.05 ; 11B-NMR {1H BB} : δ (ppm) = −4.56 , −11.16 , −13.61 , −15.15 ; UV (99.8% EtOH): λmax = 235, 328, 372 nm, λmin = 227, 282, 346 nm, λsh = 314, 356 nm; FT-IR: νmax (cm−1) = 2954 , 2596 (B-H), 1771 (C=O), 1733 (C=O), 725 (B-B); RP-HPLC (gradient A): tR = 17.42 min; APCI-MS: m/z: 480 [M + H]+, 513 [M + H + MeOH]+, 522 [M + H + CH3CN]+, calcd for C20H25B10N3O4 = 479.3-{methoxy}-1,8-naphthalic anhydride (15) or 3-{methoxy}-1,8-naphthalic anhydride (16) (1 equiv.) was dissolved in absolute EtOH (10 mL per 0.45 mmoL) and N,N-dimethylethylenediamine (1.1 equiv.) or N-(2-aminoethyl)pyrrolidine (1.1 equiv.) was added. The reaction mixture was stirred for 1 h at 35 °C, then for 1 h at 45 °C, under an inert (Ar) atmosphere. For compound 20 additional portion of N-(2-aminoethyl)pyrrolidine (0.6 equiv.) were added and reaction mixture was stirred for 2 h in 45 °C. Subsequently, the solvent was evaporated to dryness under vacuum and crude product was purified by column chromatography on silica gel (230–400 mesh) with a gradient of MeOH (0–15%) in CH2Cl2. Additionally (for compound 18 and 20), purified product was dissolved in CHCl3 (0.5–1 mL) and poured into hexane (5–8 mL). A precipitate was isolated by centrifugation.N-[2-(Dimethylamino)ethyl]-3-{metoxy}-1,8-naphthalimide (17): yellow solid. Yield: 14.4 mg (59%). TLC : Rf = 0.23; 1H-NMR : δ (ppm) = 8.32–8.30 , 8.07 , 8.05 , 7.81 , 5.41 , 5.13 , 4.37 , 4.15 2), 2.57 2), 2.26–2.23 2 overlapped with CH2-carborane), 2.00–1.95 , 3.0–1.5 ; 13C-NMR : δ (ppm) = 163.36 , 162.91 , 156.27 , 142.24 , 133.02–114.70 , 75.41 , 62.84 , 61.84 , 56.26 2), 48.25 , 45.10 , 37.42 2), 33.49 , 29.35 ; 11B-NMR {1H BB} : δ (ppm) = −3.20 , −6.15 , −9.82 , −11.72–−13.07 ; UV (99.8% EtOH): λmax = 238, 334, 375 nm, λmin = 226, 280, 351 nm, λsh = 320 nm; FT-IR: νmax (cm−1) = 2925 , 2594 (B-H), 1698 (C=O), 1658 (C=O), 723 (B-B); RP-HPLC (gradient B): tR = 21.64 min; APCI-MS: m/z: 550 [M + H]+, calcd for C24H35B10N5O3 = 549.N-[2-(N-Pyrrolidinyl)ethyl]-3-{metoxy}-1,8-naphthalimide (18): white solid. Yield: 22.7 mg (58%). TLC : Rf = 0.24; 1H-NMR : δ (ppm) = 8.40 , 8.22 , 8.05 , 7.69 , 7.67 , 7.62 , 5.39 , 4.38–4.35 2), 3.58 , 2.90 , 2.77 , 2.25–2.22 , 2.16–2.13 (CH2-CH2-CH2), 1.83 , 3.0–1.5 ; 13C-NMR : δ (ppm) = 164.22 , 163.85 , 156.76 , 143.91 , 133.23–114.58 , 73.77 , 62.68 , 61.78 , 54.50 , 53.71 , 49.28 , 38.99 2), 35.08 , 29.81 , 23.77 ; 11B-NMR {1H BB} : δ (ppm) = −2.15 , −5.45 , −9.17 , −11.86–−12.97 ; UV (99.8% EtOH): λmax = 239, 335, 374 nm, λmin = 225, 280, 352 nm, λsh = 318 nm; FT-IR: νmax (cm−1) = 2926 , 2578 (B-H), 1698 (C=O), 1659 (C=O), 723 (B-B); RP-HPLC (gradient B): tR = 21.87 min; APCI-MS: m/z: 577 [M + H]+, calcd for C26H37B10N5O3 = 576.N-[2-(Dimethylamino)ethyl]-3-{metoxy}-1,8-naphthalimide (19): yellow solid. Yield: 16.3 mg (71%). TLC : Rf = 0.39; 1H-NMR : δ (ppm) = 8.43 , 8.26 , 8.08 , 7.69–7.64 , 5.41 , 4.35 2), 4.30 , 2.90 , 2.75 2), 2.42 2), 1.98–1.97 , 3.0–1.5 ; 13C-NMR : δ (ppm) = 164.36 , 163.96 , 156.84 , 143.79 , 133.29–114.05 , 74.65 , 62.72 , 56.91 2), 55.19 , 49.64 , 45.56 , 37.95 2), 33.75 , 30.45 ; 11B-NMR {1H BB} : δ (ppm) = −4.26 , −9.68 , −10.68–−11.06 , −13.49 , −15.38 ; UV (99.8% EtOH): λmax = 238, 334, 375 nm, λmin = 225, 283, 351 nm, λsh = 319 nm; FT-IR: νmax (cm−1) = 2936 , 2595 (B-H), 1698 (C=O), 1655 (C=O), 731 (B-B); RP-HPLC (gradient B): tR = 21.23 min; APCI-MS: m/z: 550 [M + H]+, calcd for C24H35B10N5O3 = 549.N-[2-(N-Pyrrolidinyl)ethyl]-3-{metoxy}-1,8-naphthalimide (20): white solid. Yield: 28.3 mg (83%). TLC : Rf = 0.39; 1H-NMR : δ (ppm) = 8.40 , 8.23 , 8.05 , 7.66–7.63 , 5.40 , 4.34 2), 4.30 , 2.90 , 2.83 , 2.70 , 2.01–1.95 , 1.80 , 3.0–1.5 ; 13C-NMR : δ (ppm) = 164.22 , 163.80 , 156.81 , 143.76 , 133.24–114.33 , 74.64 , 62.67 , 55.18 , 54.47 , 53.75 , 49.61 , 39.25 2), 33.73 , 30.43 , 23.77 ; 11B-NMR {1H BB} : δ (ppm) = −4.24 , −9.68 , −10.64–−11.06 , −13.49 , −15.38 ; UV (99.8% EtOH): λmax = 238, 334, 375 nm, λmin = 226, 286, 351 nm, λsh = 319 nm; FT-IR: νmax (cm−1) = 2950 , 2595 (B-H), 1698 (C=O), 1658 (C=O), 731 (B-B); RP-HPLC (gradient B): tR = 21.32 min; APCI-MS: m/z: 577 [M+H]+, calcd for C26H37B10N5O3 = 576; HRMS (ESI+) 576.4156 [M + H]+, calcd for C26H37B10N5O3 = 576.3973 [M + H]+.3-Amino-1,8-naphthalic anhydride (12)  was dissolved in anhydrous THF (0.9 mL) and 2-ethanal (23) (1.3 equiv.) or 2-ethanal (24) (1.2 equiv.) was added. The reaction mixture was stirred for 24 h at reflux under an inert (Ar) atmosphere. Next, to the Schiff base 25, 26 NaBH3CN (3 equiv.) was added and reaction mixture was stirred for 24 h at RT under an inert (Ar) atmosphere. Subsequently, concentrated HCl (4–6 equiv.) was added and the reaction was stirred for additional 1 h. Then the reaction mixture was diluted with brine (2–4 mL) and crude product was extracted with CH2Cl2 (3 × 2–5 mL). The organic phase was separated, dried over MgSO4, filtered, and evaporated to dryness. The residue was purified by column chromatography on silica gel (230–400 mesh) using as an eluent CH2Cl2 to afford product.3-[ethylamino]-1,8-naphthalic anhydride (31): yellow solid. Yield: 16.3 mg (62%). TLC (2 × CH2Cl2): Rf = 0.33. 1H-NMR : δ (ppm) = 8.18 , 8.11 , 7.94 , 7.69 , 7.30 , 6.67 , 5.26 , 3.32 , 2.62 , 3.0–1.5 ; 13C-NMR : δ (ppm) = 160.88 , 160.79 , 146.75–109.25 , 74.28 , 62.94 , 41.79 , 34.94 ; 11B-NMR {1H BB} : δ (ppm) = −3.05 , −5.91 , −9.76 , −11.45–−13.06 ; UV (99.8% EtOH): λmax = 277, 336, 437 nm, λmin = 239, 305, 359 nm; FT-IR: νmax (cm−1) = 2926 , 2587 (B-H), 1763 (C=O), 1733 (C=O), 724 (B-B); RP-HPLC (gradient A): tR = 18.50 min; APCI-MS: m/z: 384 [M + H]+, 416 [M+MeOH+H]+calcd for C16H21B10NO3 = 383.3-[ethylamino]-1,8-naphthalic anhydride (32): yellow solid. Yield: 9.8 mg (55%). TLC (2 × CH2Cl2): Rf = 0.38. 1H-NMR : δ (ppm) = 8.19 , 8.11  7.93 , 7.69 , 7.27 , 6.61 , 4.08 , 3.21 , 2.31 , 3.0–1.5 ; 13C-NMR : δ (ppm) = 160.92 , 160.82 , 146.87–109.09 , 74.33 , 56.48 , 42.29 , 34.38 ; 11B-NMR {1H BB} : δ (ppm) = −4.59 , −11.11 , −13.61–−15.04 ; UV (99.8% EtOH): λmax = 279, 336, 439 nm, λmin = 239, 305, 360 nm; FT-IR: νmax (cm−1) = 2926 , 2596 (B-H), 1762 (C=O), 1732 (C=O), 724 (B-B); RP-HPLC (gradient A): tR = 19.17 min; APCI-MS: m/z: 384 [M + H]+, 416 [M+MeOH+H]+calcd for C16H21B10NO3 = 383.3-Amino-N-[2-(dimethylamino)ethyl]-1,8-naphthalimide (21)  or 3-amino-N-[2-(N-pyrrolidinyl)ethyl]-1,8-naphthalimide (22)  was dissolved in anhydrous THF (14.9 mL per 1 mmoL)  or in anhydrous MeOH (14 mL per 1 mmoL)  and 2-ethanal (23) (1.3 equiv.) or 2-ethanal (24) (1.3 equiv.) was added. The reaction mixture was stirred for 24 h at reflux under an inert (Ar) atmosphere. Next, to the Schiff base 27–30 NaBH3CN (3 equiv.) was added and reaction mixture was stirred for 24 h at RT under an inert (Ar) atmosphere. Subsequently, concentrated HCl (5–6 equv.) was added and the reaction was stirred for additional 1 h. Then, the reaction mixture was diluted with brine (57 mL per 1 mmoL) and crude product was extracted with CH2Cl2 (4 × 2–10 mL). The organic phase was separated, dried over MgSO4, filtered, and evaporated to dryness. Crude product was purified by column chromatography on silica gel (230–400 mesh) with a gradient of MeOH (3–14%) in CH2Cl2 as the eluent to afford product.N-[2-(Dimethylamino)ethyl]-3--1,8-naphthalimide (33): yellow solid. Yield: 15.7 mg (57%). TLC : Rf = 0.30. 1H-NMR : δ (ppm) = 8.11–8.08 , 7.95 , 7.65 , 7.23 , 6.58 , 5.27 , 4.12 2), 3.31 , 2.62 , 2.50 2 overlapped with DMSO), 2.21 2), 3.0–1.5 ; 13C-NMR : δ (ppm) = 163.68 , 163.47 , 146.55–108.34 , 74.33 , 62.94 , 56.45 2), 45.30 , 41.83 , 37.45, 2), 34.99 (CH2carborane); 11B-NMR {1H BB} : δ (ppm) = −3.17 , −6.02 , −9.82 , −11.62–−13.11 ; UV (99.8% EtOH): λmax = 256, 341, 438 nm, λmin = 236, 312, 366 nm, λsh = 280 nm; FT-IR: νmax (cm−1) = 2973 , 2579 (B-H), 1704 (C=O),1651 (C=O), 721 (B-B); RP-HPLC (gradient B): tR = 22.36 min; APCI-MS: m/z: 454 [M + H]+, calcd for C20H31B10N3O2 = 453.N-[2-(N-Pyrrolidinyl)ethyl]-3--1,8-naphthalimide (34): yellow solid. Yield: 23.5 mg (47%). TLC : Rf = 0.30. 1H-NMR : δ (ppm) = 8.12–8.09 , 7.96 , 7.65 , 7.24 , 6.59 , 5.26 , 4.17 2), 3.31 , 2.78 , 2.65–2.61 , 1.70 , 3.0–1.5 ; 13C-NMR : δ (ppm) = 163.77 , 163.55 , 146.57–108.37 , 74.35 , 62.95 , 53.75 , 52.92 , 41.83 , 38.21 2), 34.99 , 23.11 ; 11B-NMR {1H BB} : δ (ppm) = −3.14 , −6.10 , −9.82–−13.12 ; UV (99.8% EtOH): λmax = 25, 341, 439 nm, λmin = 236, 314.4, 366 nm, λsh = 280 nm; FT-IR: νmax (cm−1) = 2956 , 2576 (B-H), 1697 (C=O), 1656 (C=O), 722 (B-B), RP-HPLC (gradient B) tR = 22.83 min; APCI-MS: m/z: 480 [M + H]+, calcd for C22H33B10N3O2 = 479.N-[2-(Dimethylamino)ethyl]-3--1,8-naphthalimide (35): yellow solid. Yield: 11.7 mg (73%). TLC : Rf = 0.33. 1H-NMR : δ (ppm) = 8.28 , 7.92 , 7.88 , 7.60 , 6.98 , 4.35 2), 4.24 , 3.31 , 2.98 , 2.75 2), 2.43 2), 2.33 , 3.0–1.5 ; 13C-NMR : δ (ppm) = 164.65 , 164.41 , 145.79–109.83 , 73.48 , 57.02 2), 55.37 , 45.66 , 43.24 , 37.88, 2), 35.52 ; 11B-NMR {1H BB} : δ (ppm) = −4.14 , −9.43–−10.87 , −13.37 , −15.19 ; UV (99.8% EtOH): λmax = 257, 341, 440 nm, λmin = 236, 314, 367 nm, λsh = 280 nm; FT-IR: νmax (cm−1) = 2953 , 2598 (B-H), 1703 (C=O), 1655 (C=O), 729 (B-B); RP-HPLC O (gradient B): tR = 22.52 min; APCI-MS: m/z: 454 [M+H]+, calcd for C20H31B10N3O2 = 453; HRMS (ESI+) 454.3642 [M + H]+, calcd for C20H31B10N3O2 = 454.3493 [M + H]+.N-[2-(N-Pyrrolidinyl)ethyl]-3--1,8-naphthalimide (36): yellow solid. Yield: 12.5 mg (74%). TLC : Rf = 0.33. 1H-NMR : δ (ppm) = 8.27 , 7.91 , 7.87 , 7.59 , 6.95 , 4.36 2), 4.27 , 3.30 , 2.98 , 2.86 , 2.73 , 2.32 , 1.82 , 3.0–1.5 ; 13C-NMR : δ (ppm) = 164.58 , 164.32 , 145.78–109.69 , 73.47 , 55.36 , 54.50 , 53.87 , 43.23 , 39.11, 2), 35.48 , 23.79 ; 11B-NMR {1H BB} : δ (ppm) = −4.18 , −9.40 , −10.52–−10.92 , −13.41 , −15.22 ; UV (99.8% EtOH): λmax = 257, 341, 441 nm, λmin = 236, 313, 367 nm, λsh = 280 nm; FT-IR: νmax (cm−1) = 2961 , 2593 (B- H), 1697 (C=O),1657 (C=O), 728 (B-B); RP-HPLC (gradient B): tR = 22.95 min; APCI-MS: m/z: 480 [M + H]+, calcd for C22H33B10N3O2 = 479.3-Amino-N-[2-(dimethylamino)ethyl]-1,8-naphthalimide (21)  or 3-amino-N-[2-(N-pyrrolidinyl)ethyl]-1,8-naphthalimide (22)  and 3-propionic acid (37)  or 3-propionic acid (38)  were dissolved in anhydrous CH2Cl2 (0.6 mL), then anhydrous TEA  and PyBOP  were added. The reaction mixture was stirred for 2–3 h at RT under an inert (Ar) atmosphere. After that an additional portion of PyBOP  was added and mixture was stirred for 2–3 h at RT under an inert (Ar) atmosphere. The reaction mixture was diluted with CH2Cl2 (2 mL) and washed witH-NaHCO3  and water (2 × 1.5 mL). The organic layer was dried over MgSO4, then the drying agent was filtered off and washed with CH2Cl2. Filtrate and washings were combined, and evaporated to dryness under a vacuum. The crude product was purified twice by column chromatography on silica gel (230–400 mesh) using a gradient of MeOH (3–10%) in CH2Cl2 as an eluting solvent system.N-[2-(Dimethylamino)ethyl]-3-[propanamido]-1,8-naphthalimide (39): white solid. Yield: 18 mg (54%). TLC : Rf = 0.31. 1H-NMR : δ (ppm) = 9.85 , 8.69 , 8.56 , 8.38 , 8.24 , 7.77 , 4.75 , 4.24 2), 2.84 , 2.59 2), 2.27 2), 3.0–1.5 ; 13C-NMR : δ (ppm) = 170.29 , 164.50 , 164.22 , 138.74–121.91 , 76.53 , 63.88 , 57.74 2), 46.01 , 38.81 2), 36.72 . 33.37 ; 11B-NMR {1H BB} : δ (ppm) = −2.69 , −5.98 , −9.67 , −11.55–−12.92 ; UV (99.8% EtOH): λmax = 253, 337, 375 nm, λmin = 293, 357 nm, λsh = 228 nm; FT-IR: νmax (cm−1) = 2923 , 2581 (B-H), 1698 (C=O), 1656 (C=O), 722 (B-B); RP-HPLC (gradient B): tR = 21.35 min; APCI-MS: m/z: 482 [M+H]+, calcd for C21H31B10N3O3 = 481.N-[2-(N-Pyrrolidinyl)ethyl]-3-[propanamido]-1,8-naphthalimide (40): white solid. Yield: 23.2 mg (66%). TLC : Rf = 0.31. 1H-NMR : δ (ppm) = 10.64 , 8.67 , 8.60 , 8.35–8.34 , 7.79 , 5.24 , 4.18 2), 2.76 , 2.71–2.67 , 2.62 , 1.69 , 3.0–1.5 ; 13C-NMR : δ (ppm) = 169.32 , 163.32 , 163.10 , 137.65–120.79 , 75.66 , 63.47 , 53.69 , 52.85 , 38.42 2), 35.59 . 31.88 , 23.11 ; 11B-NMR {1H BB} : δ (ppm) = −3.10 , −6.05 , −9.75 , −11.69–−12.92 ; UV (99.8% EtOH): λmax = 254, 338, 375 nm, λmin = 293, 357 nm, λsh = 228 nm; FT-IR: νmax (cm−1) = 2922 , 2576 (B-H), 1704 (C=O), 1661 (C=O), 722 (B-B); RP-HPLC (gradient B): tR = 21.39 min; APCI-MS: m/z: 508 [M+H]+, calcd for C23H33B10N3O3 = 507; HRMS (ESI+) 508.3761 [M+H]+, calcd for C23H33B10N3O3 = 508.3598 [M + H]+.N-[2-(Dimethylamino)ethyl]-3-[propanamido]-1,8-naphthalimide (41): white solid. Yield: 18.2 mg, (55%). TLC : Rf = 0.34. 1H-NMR : δ (ppm) = 9.72 , 8.73 , 8.55 , 8.39 , 8.26 , 7.79 , 4.25 2), 3.71 , 2.67 , 2.61 2), 2.54 , 2.30 2), 3.0–1.5 ; 13C-NMR : δ (ppm) = 170.34 , 164.46 , 164.18 , 138.85–121.73 , 76.72 , 57.74 2), 56.92 , 46.03 , 38.79 2), 37.14 , 32.49 ; 11B-NMR {1H BB} : δ (ppm) = −4.33 , −9.87–−10.92 , −13.45 , −14.93 ; UV (99.8% EtOH): λmax = 258, 338, 375 nm, λmin = 293, 358 nm, λsh = 228 nm; FT-IR: νmax (cm−1) = 2922 , 2597 (B-H), 1697 (C=O), 1657 (C=O), 730 (B-B); RP-HPLC (gradient B): tR = 21.03 min; APCI-MS: m/z: 482 [M + H]+, calcd for C21H31B10N3O3 = 481.N-[2-(N-Pyrrolidinyl)ethyl]-3-[propanamido]-1,8-naphthalimide (42): white solid. Yield: 17.2 mg (49%). TLC : Rf = 0.34. 1H-NMR : δ (ppm) = 10.59 , 8.69 , 8.58 , 8.35–8.33 , 7.79 , 4.18 2), 4.09 , 2.72 , 2.59 , 2.54 , 2.40 , 1.69 , 3.0–1.5 ; 13C-NMR : δ (ppm) = 169.58 , 163.31 , 163.08 , 137.74–120.69 , 75.84 , 56.42 , 53.71 , 52.93 , 38.57 2), 36.16 , 31.14 , 23.16 ; 11B-NMR {1H BB} : δ (ppm) = −4.58 , −11.09 , −13.55 , −14.94 ; UV (99.8% EtOH): λmax = 254, 338, 375 nm, λmin = 293, 358 nm, λsh = 229 nm; FT-IR: νmax (cm−1) = 2921 , 2597 (B-H), 1696 (C=O), 1654 (C=O), 728 (B-B); RP-HPLC (gradient B): tR = 21.27 min; APCI-MS: m/z: 508 [M + H]+, calcd for C23H33B10N3O3 = 507.The cytotoxic properties of synthesized compounds were evaluated using human cancer cell line HepG2 established from hepatocellular carcinoma. Cell lines was purchased from Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures  and from ECACC .2.HepG2 cells were propagated in Dulbecco′s Modified Eagle Medium  supplemented with 10% heat-inactivated fetal bovine serum  and 100 units/mL penicillin G with 100 mg/mL streptomycin . The cells were incubated at 37 °C in a humidified atmosphere containing 5% CO2, the culture medium was removed and replaced with a freshly prepared solution of compounds in culture medium or medium itself as the control group.Upon reaching 80–90% confluency, cells were harvested with 0.25% trypsin in 1 mM EDTA  and transferred into 96-well microplates at 5000 cells/well (HepG2 cells). After overnight incubation of cells at 37 °C in a humidified atmosphere containing 5% CO2 for additional 24 h. Upon completion of the incubation, the medium was aspirated and replaced with 3--2,5-diphenyltetrazolium bromide (MTT) dye solution . After 3 h incubation, the resulting MTT formazan crystals were dissolved in DMSO (100 µL). To ensure the complete dissolution of formazan, the plates were shaken on an orbital microplate shaker at 1000 rpm for 15 min . The optical density of each well was then measured on a microplate spectrophotometer  at a wavelength of 570 nm. Each experiment consisted of 5 replications of each concentration, and each experiment was repeated three times independently. The results were calculated as percentage of control group viability. The IC50 values were determined using a nonlinear regression from the plot of % viability against log dose of compounds by using GraphPad Prism 6.0 software .Stock solution of each compound was prepared in DMSO at 50 mM. Stock solutions were diluted with the growth medium supplemented with 5% FBS to ensure drug dissolution for obtaining concentrations ranging from 0.1 to 200 μM. The cytotoxicity was evaluated by the MTT assay. The final content of DMSO in solutions did not exceed 0.2%, and an additional control group with 0.2% DMSO was included to rule out the effect of solvent. After treatment, cells were incubated at 37 °C in 5% CO5) cells were seeded onto 6-well cell culture plates and incubated for 24 h with the analyzed compounds at a concentration corresponding to whole IC50 values. DNA content was determined by flow cytometry with PI ) staining. After incubation, the cells were trypsinized and washed twice with PBS (1 mL). In the next step, the cells were fixed with an ice-cold 80% ethanol. After 1 h incubation in 4 °C, the cells were stored at −20 °C for further analysis. After double wash with PBS, the fixed cells were stained with PI (50 μg/mL) with the addition of RNAse A (100 μg/mL) for 30 min at 37 °C in the dark. The PI fluorescence was measured by FACSCalibur , and data were analyzed by FlowJo software.HepG2 (5 × 105) were seeded onto 6-well plates, cultured with EMEM media at 37 °C and 5% CO2 saturation, and incubated until 60–70% confluence. Subsequently, the cells were treated for 24 h with the tested compounds at a concentration that corresponds to a half of IC50 value. Next, the cells were detached with trypsin ) and washed twice with DPBS (1 mL) ), and the level of intracellular ROS generation was analyzed by dual staining with H2DCFDA/PI according to the manufacturer′s protocol ), in which fluorescence was triggered in the presence of ROS.HepG2 cells (3.5 × 106) were seeded onto flasks (25 cm2) and cultured in the supplemented growth medium for 16 h. Subsequently, the cells were treated with compounds 31 (53 μM), 33 (5 μM), 34 (8 μM), 35 (9 μM), and 36 (6 μM) for 24 h. After incubation, the cells were trypsinized, rinsed twice with PBS, and pelleted by centrifugation . Total DNA was isolated from the treated cells and untreated control cells by using Genomic mini DNA isolation kit  according to the manufacturer′s protocol. Quality of the total DNA was assessed spectrophotometrically.HepG2 cells (1 × 102 (0.1 mM) and digested witH-Nuclease P1 (3 μg). Samples were incubated for 3 h at 37 °C. Subsequently, Tris–HCl  and alkaline phosphatase (1 U) were added and incubated 2 h at 37 °C. All DNA hydrolysates were ultrafiltered using cut off 10,000 Da filter units.DNA (1 μg) was dissolved in sodium acetate buffer  containing ZnClv/v). Detection of dG was performed at 254 nm. 8-Oxo-dG was performed by the electrochemical detector: guard cell + 400 mV, detector 1: +130 mV (as a screening electrode), detector 2: +350 mV (as a measuring electrode set to sensitivity of 500 nA/V). All results are expressed as mean ± SD.8-Oxo-dG, and dG in hydrolysates were determined using HPLC-UV followed by electrochemical detection and ED . DNA hydrolysates were chromatographed isocratically by using ammonium acetate (50 mM pH 5.3)/methanol  were seeded onto 6-well plates. On the next day, the cells were treated for 24 h with the analyzed compounds at a concentration corresponding to whole IC50 values. Subsequently, the cells were detached with trypsin ), washed twice with DPBS (1 mL) ), and stained with YO-PRO-1 and PI according to the manufacturer′s protocol for 30 min at 37 °C in the dark. The cells were analyzed immediately after staining with 488 nm excitation by Accuri C6 flow cytometer .Apoptosis/necrosis assay was performed by double staining of cells with YO-PRO-1 ) and propidium iodide ) fluorescent dyes. Briefly, HepG2 cells (0.5 × 105) were seeded onto 6-well plates, cultured with EMEM media at 37 °C and 5% CO2, and incubated until 60–70% confluence. Subsequently, the cells were treated for 24 h with the tested compounds 31, 33–36 were added at concentration that corresponds to the whole IC50 value. After 24 h incubation, the cells were washed with ice-cold PBS buffer , harvest with 1 × trypsin/EDTA (Corning), and centrifuged . Next, the cells were washed in ice-cold PBS and recentrifuged. Supernatants were discarded, and the cells were suspended in annexin-binding buffer . The cells were stained by adding of the Annexin V Alexa Fluor 647 conjugate (5 µL) ). The cells were incubated at RT, for 15 min, in the dark. After incubation, annexin binding buffer (400 µL) was added to the Alexa Fluor 647-stained cells, mixed gently, and kept on ice until analysis. The rate of apoptosis was evaluated immediately after incubation by FACSCalibur flow cytometer with excitation at 635 nm.HepG2 cells (4 × 105) were seeded onto 6-well plates containing the growth medium (EMEM) and incubated until 70–80% confluency. On the next day, the cells were treated for 24 h with the analyzed compounds at a concentration corresponding to whole IC50 values. Rapamycin (autophagy inducer) (1 µM) was used to create a strong, single positive green fluorescence signal. Subsequently, the cells were detached with trypsin ), washed twice with DPBS (1 mL) ), and stained with Green Detection Reagent according to the manufacturer′s protocol for 30 min at 37 °C in the dark. The cells were analyzed immediately after staining with 488 nm excitation by Accuri C6 flow cytometer .Autophagy assay was performed in HepG2 cells by staining with Green Detection Reagent  fluorescent dye with excitation/emission at 463/534 nm. Briefly, the cells (3.85 × 105) were seeded onto 6-well plates, cultured with EMEM media, at 37 °C and 5% CO2 saturation, and incubated until 60–70% confluence. Subsequently, the cells were treated for 24 h with the tested compounds at a concentration that corresponds to whole IC50 values. Next, the cells were detached with trypsin ), washed twice with DPBS (1 mL) ) and intracellular oxidation of lipids was analyzed by staining with BODIPY® 581⁄591 C11 reagent according to the manufacturer’s protocol ). Upon oxidation in live cells, the reagent shifts fluorescence emission peak from 590 nm (red) to 510 nm (green). Cumene peroxide (10 µM) was used as a positive control to induce lipid peroxidation. Cells were analyzed immediately after staining, with 488 nm excitation by FACSCalibur flow cytometer  and data were analyzed by FlowJo software. The ratios of the signal from the 590 to 510 channels were used to quantify lipid peroxidation in cells.HepG2 cells ) for 15 min, and nuclei were stained with 3 μg/mL of Hoechst 33342 ) for 5 min. After incubation, the cells were gently rinsed in PBS to remove free dyes, placed in the FluoroBrite DMEM ). Live cell imaging was performed using a Leica TCS SP5 II confocal laser scanning microscope equipped with a White Light Laser (470–670 nm), a 405 laser and an environmental cell culture chamber that provided controlled conditions of temperature, CO2 saturation and humidity. Fluorescence images were collected using a Plan Apo 63x 1.4 NA oil-immersion objective at Ex/Em 488/500–600 nm for autofluorescence of tested compounds, 561/585–655 nm for lysosomes labeling and 405/430–480 nm for nuclei staining. LAS AF  and Leica LAS X software with a deconvolution module were used for image processing and fluorescence analysis, respectively.HepG2 wells were seeded on a glass-bottom 4-well CELLview cell culture dishes  at a density of 10 × 10w/v) sucrose, Tris-HCl , EDTA (1 mM), bromophenol blue (0.5 mg/mL)). The products were analyzed by electrophoresis using agarose gel (1%) in TEA buffer at 70 V for 2 h, followed by the ethidium bromide staining For the most active compounds 6 and 7 the reaction was repeated in a concentration range of 25–200 µM. The percentage of Topo-II alpha activity inhibition was calculated by densitometric quantification . The occurrence of a band representing supercoiled DNA on an agarose gel indicated the inhibition of enzyme activity.The Human Topoisomerase II-alpha Relaxation Assay Kit was purchased from Inspiralis . The Topo II-alpha inhibition assay was performed as described be the manufacturer. Briefly, the reaction mixture (30 μL) containing tested compound (100 µM) dissolved in DMSO , supercoiled pBR322 (0.5 μg) in 1X Assay Buffer and ATP (1 mM) was incubated at 37 °C for 15 min. Next, Topo-II alpha (1 U) was added and the reaction mixture was incubated at 37 °C for an additional 45 min. The reaction was terminated by addition of the STEB buffer  p > 0.05, (*) p < 0.05, (**) p < 0.01, (***) p < 0.001, and (****) p < 0.0001.Statistical analyzes were performed using GraphPad Prism version 6.0 for Windows, . Apoptosis and necrosis as well as cell cycle assay was analyzed by two-way analysis of variance (ANOVA), followed by Tukey′s multiple comparison test. ROS induction was analyzed by one-way ANOVA followed by Tukey′s multiple comparison test. Statistical significance for mitochondrial ROS production was determined by one-way ANOVA followed by Dunnett′s multiple comparison test. The results are presented as mean ± SEM from three independent experiments; Calf-thymus (ct-DNA) was purchased from Sigma  and used without purification. Sodium cacodylate (for the preparation of cacodylate buffer) was purchased from Acros Organics . Water was obtained from a Milli-Q purification system. All experiments were performed with freshly prepared solutions.2O, reconstituted overnight at 4 °C to dissolve all the material, and then filtered through a 0.45-μm filter. The molar concentration of ct-DNA was determined from UV-visible spectra by using molar absorption coefficient (ε) of 6600 M−1 cm−1at 260 nm [The ct-DNA was dissolved in Ht 260 nm . The purTm curves were collected at r = 0.3 (r = [compound]/[ct-DNA]) to assure the dominant binding mode. Thermal melting curves were determined by following the absorption change at 260 nm as a function of temperature by using a GBC Cintra10 UV-VIS spectrometer equipped with a GBC Thermocell Peltier Power Supply using a 1 cm path length cell. The absorbance of the samples was monitored at 260 nm from 30 °C to 90 °C with a heating rate of 1 °C/min. Tm values are the midpoints or the transition curves determined from the maximum of the first derivative. The ΔTm values were calculated by subtracting Tm of the free nucleic acid from Tm of the sample. Every ΔTm value reported in the study was the average of at least three measurements. The error in ΔTm was ±0.5 °C.The measurements were performed by adding aliquots of acetone stock solution of the tested compounds to the buffer solution . The The measurements were performed by adding aliquots of DMSO stock solution of the tested compounds to the buffer solution . Changes in the CD spectrum of ct-DNA upon the addition of compound were measured at different molar ratios r = [compound]/[ct-DNA]. Circular dichroism spectra were recorded on a JASCO J-815 CD spectrometer with a JASCO CDF-426S Peltier thermostated cell holder  by using a rectangular quartz cuvette of path length 0.5 cm (1 mL) in the 230–400 nm region. The reported CD profiles are an average of three successive scans with 200 nm per minute scan time and an appropriately corrected baseline. The temperature was maintained at 20 °C during the experiment.b is the binding constant, A0 and a are absorbance of the free tested compound and the apparent one, εG and εH−G are their coefficient respectively, [DNA] is the concentration of [DNA] in base pair. The slope to intercept ratio of the plot between A0/A−A0 versus 1/[DNA] yielded the binding constant.The measurements were performed by adding aliquots of acetone stock solution of the tested compounds to the buffer solution  to the final concentration 10 µM. The tested compounds were incubated 5 min, with increasing concentrations ranging from 0 to 15 µM of ct-DNA at 37°, then the UV-vis absorption spectra, between 315–455 nm, were recorded using a GBC Cintra10 UV-VIS spectrometer equipped with a GBC Thermocell Peltier Power Supply using a 1 cm path length cell. The binding constant was calculated according to the following equation :(1)A0A−A9 according to the active analogue approach (AAA) was used in FIT method to cover the entire bonding topology of naphtalimide ring (pharmacophore hypothesis). The steric and electrostatic potentials were calculated using sp3 carbon probe atom with a charge of +1. CoMFA grid spacing was 2.0 Å for all the dimensions within the defined region, which extended beyond the van der Waals envelopes of all molecules by at least 4.0 Å—for each molecule the energies with a total of 1680 grid points were calculated with 2 Å spacing in a lattice of 14 × 12 × 10, respectively. The potential energies at each lattice point can be plotted as three-dimensional color-coded contour maps indicating the regions where steric hindrance and/or charged substituents enhance or diminish the binding affinity.Sybyl-X 2.0/Certara and HyperChem 6.0 programs were engaged to conduct the molecular modeling simulations. OpenBabel (inter)change file format converter was employed for data conversion. The crystallographic geometry of carborane cluster was retrieved from Crystallographic Data Centre (deposition code: 1010195) and modified accordingly. The initial compound geometry optimization was performed using the optimized potentials for liquid simulations (OPLS) force field and Polak-Ribiere (conjugate gradient) method implemented in HyperChem 6.0 with a 0.1 kcal/A mol energy gradient convergence criterion. The specification of the electrostatic potential values based on the partial atomic charges was carried out with the Gasteiger–Hückel method implemented in Sybyl-X. CoMFA modeling efficiency of electronic and steric potentials in the molecular environment is directly controlled by the specification of the atomic superimposition. Hence, one 6-ordered atom trial alignment on the most active molecule x-th and y-th molecule and sim is the pair-wise similarity evaluation [x and y, x, y, respectively.The numerical sampling of similarity-related structure-activity landscape index (SALI) can be quantitatively expressed according to the following formula:aluation ,84. Tanif principal components for a data matrix X can be calculated as follows:X is a data matrix with m objects and n variables, T is the score matrix with dimensions (m × f), TP is a transposed matrix of loadings with dimensions (f × n) and E is a matrix of the residual variance (m × n) not explained by the first f principal components. On the whole, the first few principal components (PCs) frequently describe sufficiently data variance and reveal the groups of objects.The human-friendly 2D/3D plots of the compound distribution in the experimental-based (FCS) and virtual-derived (VCS) molecular space might be displayed using the principal component analysis (PCA) method. PCA is a linear projection method, that can be applied to model multivariate data with a relatively small number of so-called principal components (scores and loadings) generated to maximize the description of variance within the input data . The PCAX according to the following equation:b is a vector of the regression coefficients and e is a vector of the errors. The complexity of PLS models was estimated using the leave–one–out cross–validation procedure (LOO-CV) [obs is the assayed value; pred is the predicted value; mean is the mean value of obs, and i refers to the object index ranging from 1 to m. The cross-validated standard error of prediction s is estimated using the equation:m is the number of objects and k is the number of the PLS factors in the model. The quality of external predictions was validated by the standard deviation of error of prediction (SDEP), The partial least squares (PLS) approach generates a regression relation between variable Y and an ensemble of descriptors (LOO-CV) . The deportho- or meta-carborane clusters at position 3 of the heteroaromatic skeleton. The X-ray structure of the 1,8-naphthalimide–carborane conjugates 39 and 41 was established. In addition, we determined the cytotoxic activity of the modified conjugates against HepG2 cells and found that modified naphthalimides were significantly more active than modified anhydrides. Conjugates modified with ortho-carboranes were rather more active than those bearing meta-carboranes. The type of linker chain between the carborane cluster and the heterocyclic system influenced the antiproliferative activity of the naphthalimides, and the activity varied as follows: 8–11 (-triazole- linker) > 33–36 (-NH-CH- linker) > 39–42 (-NH-CO- linker) > 17–20 (-O-triazole- linker). Furthermore, our study showed that modified conjugates could effectively induce cell cycle arrest at G0/G1 or G2M phase and activate mainly apoptosis as well as autophagy and ferroptosis which was confirmed using the flow cytometry analysis. Among the studied compounds, conjugate 35 induced ROS production resulting in almost four times higher number of 8-oxo-dG in comparison to mitonafide, and strongly promoted apoptosis which might inhibit the growth of HepG2 cells. However, the presence of the carboranyl cluster at position 3 of these compounds did not promote them as effective Topo II inhibitors.In this paper, we have described convenient protocols for synthesizing 1,8-naphthalimide derivatives modified with an 33, 34, and 36 and their imaging capacity w studied by co-localization experiments, which revealed that these conjugates selectively localized in the lysosomes, which additionally enabled the localization of boron/carborane in the cells.The DNA binding properties of the synthesized compounds were also investigated by UV–vis, CD, and thermal denaturation experiments. It was found that these compounds were rather weak classical DNA intercalators, which indicated another type of interaction with DNA. The lysosome-targeting behavior of compounds 50 values on the PC1 vs. PC2 plane clearly indicated the diagonal separation of the active and nonactive conjugates. Moreover, the enhancement of the planar descriptor-driven projection with response data resulted in a structure–activity landscape that can be regarded as a subtle picture of (dis)allowed structural adjustment(s) potentially valid for molecular activities. Interestingly, the replacement of the anhydride-like fragment with the imide-based motif influenced the molecular potency, as shown by the comparison of the most active  and nonactive  compounds, respectively. Finally, we performed a quantitative CoMFA ligand-based study to specify the potentially valid steric and electrostatic features of the pharmacophore pattern. In this case, the bundle of steric bulk was indicated as the privileged feature contributing (un)favorably to the CoMFA model that was validated accordingly.We assessed the similarity-driven property space for the series of carborane-containing conjugates using PCA. The investigation of the spatial space defined by the first three orthogonal components (PC1–PC3) revealed that carborane-based derivatives are clustered into four subgroups. The projection of the pICThe quantitative comparison of the field-based descriptors can lead to a useful picture of the drug–receptor recognition pattern; however, it should be treated as a crude approximation of the underlying biological reality.The present work demonstrated that new organic and inorganic hybrids can be considered as a novel class of compounds with potential antitumor activities. Studies on the synthesis and biological properties of new naphthalimides bearing modified carborane at position 4 of the naphthalic ring are carried out in our laboratory and will be published soon.