KBJK557 was synthesized according to the procedure (Gunasekaran et al. 2020), and the experimental section is provided in Additional file 1. As described previously (Gunasekaran et al. 2017), all reactions were performed under argon atmosphere in flame-dried glassware using dry solvents, unless otherwise noted. Anhydrous organic solvents (99.9%) were purchased from Sigma-Aldrich and used in the reaction. Sigma- All reagents and few starting materials were purchased from Aldrich, TCI, and Across Organics and utilized in the reaction. Merck aluminum sheets with silica gel 60 F254 using 0.25 mm plates were used for thin-layer chromatography (TLC) and visualized by ultraviolet light, staining with phosphomolybdic acid (PMA), KMnO4, and ninhydrin. Merck silica gel 60 (70–230 mesh or 230–400 mesh) was used for column chromatography purification. Bruker DRX-400 and DRX-500 NMR spectrometers were used to record 1H and 13C NMR spectra. NMR chemical shifts (δ) are denoted in parts per million (ppm) and coupling constants (J) are given in hertz (Hz) (Additional file 1: Supplementary Information). MALDI-TOF mass was recorded using a Shimadzu mass spectrometer.
Tert-butyl (2-(4-formyl-3-phenyl-1H-pyrazol-1-yl)ethyl)carbamate (3)
3-phenyl-1H-pyrazole-4-carbaldehyde (1) (0.5 g, 2.906 mmol) in anhydrous DMF (5 mL) was added slowly to a stirred solution of tert-butyl (2-bromoethyl)carbamate (2) (0.604 g, 2.642 mmol) and potassium carbonate (1.47 g, 7.90 mmol) in anhydrous DMF (10 mL). After completion of the addition, the temperature was slowly raised to 60 °C and stirred for 16 h. The reaction mixture was quenched by the addition of water (20 mL) and extracted with ethyl acetate (20 mL × 3). The combined organic extracts were washed with brine (40 mL), dried over Na2SO4, and evaporated. The crude product was purified by trituration using DCM-hexane to yield 3 as a white solid (0.67 g, 73%). 1H NMR (500 MHz, CDCl3) δ 9.98 (s, 1H), 8.04 (s, 1H), 7.75 (d, J = 6.8 Hz, 2H), 7.56–7.41 (m, 3H), 4.87 (s, 1H), 4.34 (s, 2H), 3.75–3.58 (m, 2H), 1.47 (s, 9H). 13C NMR (101 MHz, CDCl3) δ 185.2, 154.6, 150.3, 138.4, 134.8, 131.4, 130.1, 129.1, 128.8, 120.9, 120.3, 80.2, 52.4, 40.4, 28.3.
1-(2-aminoethyl)-3-phenyl-1H-pyrazole-4-carbaldehyde hydrochloride (4)
2 M HCl (11 mL, 22 mmol) in diethyl ether was added to the stirred solution of 3 (700 mg, 2.22 mmol) in 10 mL dichloromethane. The resultant solution was stirred for 16 h at rt and the solid formed was filtered and washed with anhydrous diethyl ether to yield 4 as pale-yellow solid (537 mg, 96%). 1H NMR (400 MHz, DMSO-d6) δ 9.89 (s, 1H), 8.66 (s, 1H), 8.64–8.36 (m, 3H), 7.90–7.79 (m, 2H), 7.51–7.34 (m, 3H), 4.58 (d, J = 6.1 Hz, 2H), 3.34 (q, J = 5.8 Hz, 2H). 13C NMR (101 MHz, DMSO-d6) δ 185.0, 152.6, 138.7, 132.1, 129.3, 129.1, 128.9, 121.0, 49.5, 38.7. Maldi-tof m/z calcd for C12H13N3O: 215.10, found 215.44.
N-(2-(4-formyl-3-phenyl-1H-pyrazol-1-yl)ethyl)-4-(methylamino)benzamide (6)
To a stirred solution of EDCI (607 mg, 3.18 mmol), HOBt (428 mg, 3.18 mmol) in DMF (10 mL), 4-(methylamino)benzoic acid (5) (480 mg, 3.18 mmol) and DIEA (2.76 mL, 15.86 mmol) was added and stirred for 30 min. To the resultant solution, 4 (957 mg, 3.81 mmol) in DMF (5 mL) was added and allowed to stir at rt for 18 h. The reaction mixture was treated with 5% NaHCO3 solution (20 mL) and extracted with ethyl acetate (2 × 15 mL), the combined organic layer was washed with H2O and brine, dried over Na2SO4, and evaporated under vacuum. The crude residue was purified by flash column chromatography (CH2Cl2/ethyl acetate (3:1)) to provide 6 (0.834 g, 63%) as semi-solid. 1H NMR (400 MHz, CDCl3) δ 9.89 (s, 1H), 7.96 (s, 1H), 7.74 (dd, J = 7.6, 1.8 Hz, 2H), 7.60 (d, J = 8.7 Hz, 2H), 7.52–7.39 (m, 3H), 6.80 (t, J = 5.2 Hz, 1H), 6.52 (d, J = 8.7 Hz, 2H), 4.47–4.33 (m, 2H), 4.17 (s, 1H), 3.90 (q, J = 5.6 Hz, 2H), 2.85 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 184.8, 167.7, 154.3, 152.1, 135.2, 131.5, 129.1, 128.8, 128.7, 121.7, 120.88, 111.4, 52.1, 39.9, 30.2. (traces of dichloromethane are present). Maldi-tof m/z calcd for C20H20N4O2: 348.15, found 348.77, 360.76 (M+Na) +.
4-(methylamino)-N-(2-(3-phenyl-4-((2,4,6-trioxotetrahydropyrimidin-5(2H)-ylidene)methyl)-1H-pyrazol-1-yl)ethyl) benzamide (KBJK557)
Barbituric acid (7) (0.269 g, 2.1 mmol) was added to a stirred solution of 6 (800 mg, 2.3 mmol) in dry methanol (15 mL) and heated to reflux for 16 h. The resultant yellow solid was filtered and washed with cold methanol (3 × 5 mL) and dried to yield a pure compound, KBJK557 as a yellow solid (798 mg, 83%). 1H NMR (400 MHz, DMSO-d6) δ 11.19 (s, 2H), 9.32 (s, 1H), 8.23 (t, J = 5.4 Hz, 1H), 8.15 (s, 1H), 7.70–7.40 (m, 7H), 6.51 (d, J = 8.7 Hz, 2H), 6.19 (q, J = 4.7 Hz, 1H), 4.47 (t, J = 5.8 Hz, 2H), 3.70 (q, J = 5.8 Hz, 2H), 2.71 (d, J = 4.9 Hz, 3H). 13C NMR (101 MHz, DMSO-d6) δ 167.2, 164.3, 163.2, 157.8, 152.7, 150.7, 144.9, 139.0, 131.7, 130.0, 129.5, 129.2, 129.2, 121.1, 113.8, 112.9, 110.8, 52.2, 40.6, 40.4, 40.2, 40.1, 39.8, 39.6, 39.5, 29.8. Maldi-tof m/z calcd for C24H22N6O4: 458.17, found 458.74.
Cell viability assay
As reported previously (Gunasekaran et al. 2020), human lung cancer cells (A549, PC9, and H1975) were purchased from the Korean Cell Line Bank (Seoul, Korea), Sigma-Aldrich (Missouri, USA) and ATCC (Virginia, USA). Lung cancer cells were propagated in RPMI 1640 medium (Cytiva, USA), supplemented with 10% fetal bovine serum (FBS, Hyclone, USA), and 1% of penicillin–streptomycin (Sigma-Aldrich, USA) at 37 °C in a humidified incubator with 5% CO2. Lung cancer cells were seeded at a density of 1 × 103 cells per 96-well plates in 100 μL medium. After 24 h of incubation, Lung cancer cells were treated with KBJK557 at various concentrations (0, 12.5, 25, 50, 100, 200, 400, 500, and 600 μM) for 24 h, then added to the MTT assay (Enhanced Cell Viability Assay Kit, DoGenBio, Korea). Absorbance was measured at 450 nm using a microplate reader (Molecular Devices Corp, USA).
Cell cycle arrest study by fluorescence-activated cell sorter
As reported previously (Gunasekaran et al. 2020), lung cancer cells were seeded into 2 × 105 cells in 6-well plates and treated with KBJK557 (400 μM) for 24 h. Cells were washed with PBS three times and fixed using 70% cold ethanol during vortexing. The fixed cells were stained with propidium iodide (PI) (1 μg/mL, Sigma-Aldrich, USA) and treated RNase (1 μg/mL) (Thermo Scientific, USA) in PBS for 30 min at 37 °C in an incubator. Flow cytometric analysis was performed using a fluorescence-activated cell sorter (FACS; Beckman Coulter, CytoFLEX, USA).
Apoptosis effect of cancer cells
As reported previously (Gunasekaran et al. 2020), lung cancer cells (1 × 103 cells) were cultured in 96-well microplates for 24 h at 37 °C. Cells were treated with KBJK557 (400 μM) at 24 h. Then, 2.5 μL of mixtures of kit (LIVE/DEAD™ Viability/Cytotoxicity Kit, Invitrogen™, USA) were added to 1 mL of PBS, and 100 μL of this solution was added to each well. After 30 min of incubation at room temperature, images of cells were captured by fluorescence microscope (ZOE™ Fluorescent Cell Imager, USA). Cells count was measured using ImageJ software.
Animals
As reported previously (Gunasekaran et al. 2020), five-week-old male BALB/c nude mice were purchased from the Nara Bio animal center (NARA Biotech, Seoul, Korea), housed under specific pathogen-free conditions. The mice were housed in groups of four in transparent plastic cages bedded with aspen chip and were provided with standard mouse chow diet and tap water ad libitum when not being treated. The environment of the animal room was carefully controlled, with a 12 h dark–light cycle. And the temperature was maintained around 20–21 °C with relative humidity of 40–45%. The animal experiments were carried out according to a protocol approved by the KBSI Committee (KBSI-IACUC-22-6) and all experiments were performed in accordance with relevant guidelines and regulations.
The anticancer effect in tumor-bearing mice
As reported previously (Gunasekaran et al. 2020), we induced mouse xenograft tumor models by subcutaneously injecting 1 × 106 A549 cells in PBS into the right thigh region of six-week-old male BALB/c nu/nu mice. When the tumor volume (length × width × height × 0.52, mm3) reached approximately 50 mm3, a tail vein injection of vehicle solution, and KBJK557 (15 mg/kg) was carried out. KBJK557 was dissolved in Dimethyl sulfoxide (DMSO, Sigma-Aldrich, USA), Polyethylene glycol 300 (PEG300, Selleck Chemical, USA), Tween 80 (Fluka, Switzerland), and distilled water (D.W.) (5:30:5:60, v/v/v/v, 100 μL). We injected vehicle solution and KBJK557 at 3–4 day intervals for 18 days and the tumor size was measured using calipers.