Design, synthesis and pharmacological evaluation of 1,4-naphthoquinone- 1,2,3-triazole hybrids as new anticancer agents with multi-kinase inhibitory activity

Chemistry

All chemicals and materials were procured from Merck, Fluka, and Sigma-Aldrich commercial providers and were used without additional purification. Termo Scientifc Electrothermal digital equipment (Termo Fisher Scientifc Inc.) was used to determine the melting points of the final compounds using open capillary tubes. Thin layer chromatography (TLC) on aluminum plates coated with silica gel 60 GF254 was performed, with spots detected under UV light (254 nm). The IR spectra of synthetic compounds were acquired by a Perkin-Elmer Spectrum RXI FTIR spectrophotometer, employing KBr disks as the sample medium. ¹H NMR (400 MHz and 300 MHz) and ¹³C NMR (75 MHz) spectra were recorded on a Bruker Avance DPX250 spectrometer at constant temperature and the signals of the NMR spectrums are represented as δ value (ppm) relative to TMS in the downfield direction. Each of the compound’s coupling constants (J) are stated in hertz (Hz). Spectra characterization was performed utilizing MestReC 14.0.0–23239 (Mestrelab Research S.L, MestReNova) software. Mass spectra were analyzed utilizing Agilent Technology (HP) 7000 triple quadrupole mass spectrometer under an electron impact mode, employing an ionization voltage of 70 electron voltage (eV). CHNS elemental analysis was performed by FLASH EA 1112 SERIES thermo Finnigan equipment.

Synthesis of 1-Nitro-4-(prop-2-yn-1-yloxy)benzene compound (1)

4-Nitro phenol (1 mmol, 1 meq, 0.139 g) and potassium carbonate (3 mmol, 3 meq, 0.41 g) were added to a round bottle flask in 5 mL acetone as the solvent. The suspension was heated for 30 min, followed by the dropwise addition of propargyl bromide (1.2 mmol, 1.2 meq, 0.1 mL). The mixture was refluxed for 5 h. The reaction’s progress was tracked using thin-layer chromatography (TLC) until completion. Once cooled, the solid was filtered, rinsed with water, and then subjected to recrystallization from ethanol to yield pure compound 1.

1-Nitro-4-(prop-2-yn-1-yloxy)benzene (1)⁶⁰. Light yellow solid, mp: 101–103 °C, Yield: 95%; FT-IR (KBr) υ_max (cm⁻¹): 3253, 2119, 1595, 1498, 1331, 1249, 1015. ¹H NMR (400 MHz, DMSO-d₆) δ_H (ppm): 3.69 (t, H, J = 4 Hz, C ≡ CH), 4.98 (d, 2 H, J = 4 Hz, CH₂–C ≡ C), 7.16–7.22 (m, 2 H, Ar–H), 8.18–8.26 (m, 2 H, Ar–H). ¹³C NMR (100 MHz, DMSO-d₆) δ_C (ppm): 56.7, 78.6, 79.6, 115.9 (2 C), 126.2 (2 C), 141.8, 162.8.

Synthesis of substituted derivatives of 1-Benzyl-4-((4-nitrophenoxy)methyl)−1 H-1,2,3-triazole (2a-j)

Various derivatives of (2a-j) were synthesized by reacting the appropriate aryl halide (1.1 mmol, 1.1 meq) with sodium azide (1 mmol, 1 m eq, 0.065 g) in triethylamine (TEA) as a base, using t-BuOH/water as the solvent. Following approximately 30 min, 1-nitro-4-(prop-2-yn-1-yloxy) benzene compound (1) (1 mmol, 1 meq, 0.17 g) dissolved in absolute t-BuOH (5 ml) was added to the mixture alongside with CuSO₄·5H₂O (0.075 mmol, 0.075 meq, 0.015 g, 10 mol%) and Sodium ascorbate (0.75 mmol, 0.75 meq, 0.148 g, 25 mol%). The reaction was conducted at 40 °C and mixed continuously for 48 h. Following confirming the reaction’s completion, verified by TLC analysis, the solvent was removed under vacuum, washed with water and the resulting residue was purified by recrystallization from a mixture of ethyl acetate and n-hexane to isolate the desired compound ⁶¹.

Synthesis of 1-Benzyl-4-((4-nitrophenoxy)methyl)−1H-1,2,3-triazole (2a): Yellow solid, mp: 94–96 °C, Yield: 92%; FT-IR (KBr) υ_max (cm⁻¹): 1595, 1496, 1337, 1265, 1113. ¹H NMR (300 MHz, DMSO-d₆) δ_H (ppm): 5.32 (s, 2 H, N − CH₂ − Ph), 5.62 (s. 2 H, CH₂ − O−Ph), 7.20–7.33 (m, 4 H, Ar–H), 7.54–7.64 (m, 2 H, Ar–H), 8.17–8.28 (m, 2 H, Ar-H), 8.36 (s, H, triazole-H). ¹³C NMR (100 MHz, DMSO-d₆) δ_C (ppm): 53.3, 62.3, 115.8 (2 C), 125.5, 126.3 (2 C), 128.4 (2 C), 128.6. 129.2 (2 C), 136.4, 141.5, 142.5, 163.7.

Synthesis of substituted derivatives 4-((1-Benzyl-1 H-1,2,3-triazol-4-yl)methoxy)aniline (3a-3j)

Compound 2a-2j (1.1 mmol, 1.1 meq) dissolved in EtOH/H₂O (8:2) as a solvent at 60 ^⁰C and hydrazine hydrate (0.25 mmol, 0.12 meq, 0.08 mL) was added. Subsequently, a small quantity of Raney Ni (0.25 mL) was added. After allowing the solution to proceed (15 to 60 min), the hot solution was filtered to remove the Ni and the solvent was then evaporated to gain a light-yellow solid. This solid was washed with water and recrystallization in EtOH and H₂O (8:2) ⁶².

4-((1-Benzyl-1H-1,2,3-triazol-4-yl)methoxy)aniline (3a): Yellow solid, mp: 106–108 °C, Yield: 92%; FT-IR (KBr) υ_max (cm⁻¹): 3370, 3308, 1598, 1506, 1236, 1045. ¹H NMR (300 MHz, DMSO-d₆) δ_H (ppm): 4.71 (s, 2 H, NH₂), 5.01 (s. 2 H, CH₂ − O−Ph), 5.63 (s, 2 H, N − CH₂ − Ph), 6.46–6.59 (m, 2 H, Ar–H), 6.76 (d, J = 9 Hz, 2 H, Ar–H), 7.3 (d, J = 6 Hz, 2 H, Ar–H), 7.62 (d, J = 9 Hz, 2 H, Ar–H), 8.27(s, H, triazole-H). ¹³C NMR (100 MHz, DMSO-d₆) δ_C (ppm): 53.2, 62.2, 115.3 (2 C), 116.2 (2 C), 124.9, 128.3 (2 C), 128.6, 129.2 (2 C), 136.5, 143.2, 144.1, 149.8.

4-((1-(4-Bromobenzyl)−1H-1,2,3-triazol-4-yl)methoxy)aniline (3b): Light pink solid, mp: 112–114 °C, Yield: 86%; FT-IR (KBr) υ_max (cm⁻¹): 3458, 3369, 1607, 1507, 1225, 1006. ¹H NMR (400 MHz, DMSO-d₆) δ_H (ppm): 4.66 (s, 2 H, NH₂), 4.97 (s. 2 H, CH₂ − O−Ph), 5.60 (s, 2 H, N − CH₂ − Ph), 6.48–6.53 (m, 2 H, Ar–H), 6.69–6.74 (m, 2 H, Ar–H), 7.24–7.31 (m, 2 H, Ar–H), 7.56–7.61 (m, 2 H, Ar–H), 8.22 (s, H, triazole-H). ¹³C NMR (100 MHz, DMSO-d₆) δ_C (ppm): 52.5, 62.2, 115.3 (2 C), 116.2 (2 C), 121.8, 124.9, 130.6 (2 C), 132.1 (2 C), 135.9, 143.2, 144.1, 149.8.

4-((1-(4-Fluorobenzyl)−1H-1,2,3-triazol-4-yl)methoxy)aniline (3c): Cream solid, mp: 98–100 °C, Yield: 91%; FT-IR (KBr) υ_max (cm⁻¹): 3451, 3337, 1618, 1507, 1224, 1006. ¹H NMR (400 MHz, DMSO-d₆) δ_H (ppm): 4.66 (s, 2 H, NH₂), 4.97 (s. 2 H, CH₂ − O−Ph), 5.60 (s, 2 H, N − CH₂ − Ph), 6.48–6.53 (m, 2 H, Ar–H), 6.69–6.75 (m, 2 H, Ar–H), 7.18–7.25 (m, 2 H, Ar–H), 7.35–7.41 (m, 2 H, Ar–H), 8.22 (s, H, triazole-H). ¹³C NMR (100 MHz, DMSO-d₆) δ_C (ppm): 52.4, 62.2, 115.3 (2 C), 115.9, 116.2 (2 C), 124.8, 126.3, 130.7, 132.8, 132.8, 143.2, 144.1, 149.7, 161.1.

4-((1-(4-Chlorobenzyl)−1H-1,2,3-triazol-4-yl)methoxy)aniline (3d): Caramel solid, mp: 110–112 °C, Yield: 88%; FT-IR (KBr) υ_max (cm⁻¹): 3458, 3368, 1621, 1507, 1226, 1007. ¹H NMR (400 MHz, DMSO-d₆) δ_H (ppm): 4.68 (s, 2 H, NH₂), 4.97 (s. 2 H, CH₂ − O−Ph), 5.61 (s, 2 H, N − CH₂ − Ph), 6.47–6.54 (m, 2 H, Ar–H), 6.69–6.75 (m, 2 H, Ar–H), 7.29–7.37 (m, 2 H, Ar–H), 7.41–7.47 (m, 2 H, Ar–H), 8.22 (s, H, triazole-H). ¹³C NMR (100 MHz, DMSO-d₆) δ_C (ppm): 52.4, 62.2, 115.3 (2 C), 116.2 (2 C), 124.9, 129.2 (2 C), 130.3 (2 C), 133.3, 135.5, 143.2, 144.1, 149.8.

4-((1-(3,4-Dichlorobenzyl)−1H-1,2,3-triazol-4-yl)methoxy)aniline (3e): Yellow solid, mp: 116–118 °C, Yield: 84%; FT-IR (KBr) υ_max (cm⁻¹): 3450, 3397, 1626, 1508, 1226, 1019. ¹H NMR (400 MHz, DMSO-d₆) δ_H (ppm): 4.66 (s, 2 H, NH₂), 4.98 (s. 2 H, CH₂ − O−Ph), 5.63 (s, 2 H, N − CH₂ − Ph), 6.48–6.53 (m, 2 H, Ar–H), 6.69–6.74 (m, 2 H, Ar–H), 7.24–7.31 (m, H, Ar–H), 7.61–7.67 (m, 2 H, Ar–H), 8.27 (s, H, triazole-H). ¹³C NMR (100 MHz, DMSO-d₆) δ_C (ppm): 51.8, 62.2, 115.3 (2 C), 116.2 (2 C), 125.0, 126.3, 128.8, 130.5, 131.5, 131.7, 137.5, 143.3, 144.2, 149.7.

4-((1-(3-Chlorobenzyl)−1H-1,2,3-triazol-4-yl)methoxy)aniline (3f): light chocolate solid, mp: 85–87 °C, Yield: 87%; FT-IR (KBr) υ_max (cm⁻¹): 3385, 3315, 1637, 1508, 1227, 1050. ¹H NMR (400 MHz, DMSO-d₆) δ_H (ppm): 4.67 (s, 2 H, NH₂), 4.98 (s. 2 H, CH₂ − O−Ph), 5.63 (s, 2 H, N − CH₂ − Ph), 6.48–6.53 (m, 2 H, Ar–H), 6.68–6.76 (m, 2 H, Ar–H), 7.22–7.29 (m, H, Ar–H), 7.38–7.43 (m, 3 H, Ar–H), 8.26 (s, H, triazole-H). ¹³C NMR (100 MHz, DMSO-d₆) δ_C (ppm): 52.4, 62.2, 115.3 (2 C), 116.2 (2 C), 125.0, 127.1, 128.2, 128.6, 131.2, 133.7, 138.9, 143.2, 144.2, 149.8.

4-((1-(4-Isopropylbenzyl)−1H-1,2,3-triazol-4-yl)methoxy)aniline (3 g): Cream solid, mp: 102–104 °C, Yield: 88%; FT-IR (KBr) υ_max (cm⁻¹): 3383, 3321, 1643, 1510, 1235, 1050. ¹H NMR (400 MHz, DMSO-d₆) δ_H (ppm): 1.18 (d, 6 H, J = 8 Hz, CH₃ (Isopropyl)), 2.79–2.92 (m, 1H, CH₂ (Isopropyl)), 4.67 (s, 2 H, NH₂), 4.96 (s. 2 H, CH₂ − O−Ph), 5.55 (s, 2 H, N − CH₂ − Ph), 6.45–6.53 (m, 2 H, Ar–H), 6.69–6.74 (m, 2 H, Ar–H), 7.22–7.28 (m, 4 H, Ar–H), 8.20 (s, H, triazole-H). ¹³C NMR (100 MHz, DMSO-d₆) δ_C (ppm): 24.2 (2 C, CH₃), 33.6 (CH), 53.0, 62.2, 115.3 (2 C), 116.2 (2 C), 124.7, 127.1 (2 C), 128.5 (2 C), 133.9, 143.1, 144.0, 148.8, 149.8.

4-((1-(4-(ert-Butyl)benzyl)−1H-1,2,3-triazol-4-yl)methoxy)aniline (3 h): Light yellow solid, mp: 93–95 °C, Yield: 89%; FT-IR (KBr) υ_max (cm⁻¹): 3386, 3321, 1640, 1509, 1235, 1050. ¹H NMR (400 MHz, DMSO-d₆) δ_H (ppm): 1.26 (s, 9 H, −C (CH₃)₃), 4.66 (s, 2 H, NH₂), 4.96 (s. 2 H, CH₂ − O−Ph), 5.55 (s, 2 H, N − CH₂ − Ph), 6.48–6.54 (m, 2 H, Ar–H), 6.69–6.75 (m, 2 H, Ar–H), 7.22–7.28 (m, 2 H, Ar–H), 7.36–7.42 (m, 2 H, Ar–H), 8.20 (s, H, triazole-H). ¹³C NMR (100 MHz, DMSO-d₆) δ_C (ppm): 31.5 (3 C), 34.7, 52.9, 62.2, 115.3 (2 C), 116.1 (2 C), 124.7, 126.0 (2 C), 128.2 (2 C), 133.6, 143.2, 144.1, 149.8, 151.1.

4-((1-(4-Methylbenzyl)−1H-1,2,3-triazol-4-yl)methoxy)aniline (3i): Cream solid, mp: 98–100 °C, Yield: 83%; FT-IR (KBr) υ_max (cm⁻¹): 3429, 3324, 1629, 1508, 1223, 1043. ¹H NMR (400 MHz, DMSO-d₆) δ_H (ppm): 2.29 (s, 3 H, (CH₃)), 4.78 (s, 2 H, NH₂), 4.96 (s. 2 H, CH₂ − O−Ph), 5.54 (s, 2 H, N − CH₂ − Ph), 6.48–6.56 (m, 2 H, Ar–H), 6.69–6.76 (m, 2 H, Ar–H), 7.15–7.24 (m, 4 H, Ar–H), 8.17 (s, H, triazole-H). ¹³C NMR (100 MHz, DMSO-d₆) δ_C (ppm): 21.1, 53.0, 62.2, 115.4 (2 C), 116.2 (2 C), 124.7, 128.4 (2 C), 129.7 (2 C), 133.5, 137.9, 142.8, 144.7, 149.9.

2-(4-((4-((4-Aminophenoxy)methyl)−1H-1,2,3-triazol-1-yl)methyl)phenethyl) isoindoline-1,3-dione (3j): Brown solid, mp: 120–122 °C, Yield: 53%; FT-IR (KBr) υ_max (cm⁻¹): 3234, 3205, 1586, 1505, 1249, 1051. ¹H NMR (400 MHz, DMSO-d₆) δ_H (ppm): 3.99–4.05 (m, 2 H, CO–N–CH₂–CH₂), 4.64–4.70 (m, 2 H, N = N–N–CH₂–CH₂), 4.99 (s, 2 H, NH₂), 5.29 (s. 2 H, CH₂ − O−Ph), 5.39 (s, 2 H, N − CH₂ − Ph), 7.19–7.31 (m, 8 H, Ar–H), 7.83 (s, H, triazole-H), 8.21–8.26 (m, 4 H, Ar–H). ¹³C NMR (100 MHz, DMSO-d₆) δ_C (ppm): 56.7, 62.1, 67.1, 79.6, 109.6, 115.8 (2 C), 115.9, 123.6, 125.8, 126.2 (2 C), 126.3, 131.8, 134.9, 137.7, 141.4, 141.6, 141.8, 142.2, 142.9, 162.8, 163.2, 163.6, 167.8 (2 C).

Synthesis of substituted derivatives 2-((4-((1-Benzyl-1 H-1,2,3-triazol-4-yl)methoxy) phenyl) amino)naphthalene-1,4-dione (4a-4j)

Several derivatives of 4a-4j were produced by reacting 1,4-Naphthoquinones (1.2 mmol. 1.2 meq, 0.18 g) with 3a–3j derivatives in EtOH at room temperature. The reaction progressed until the color changed (1 to 4 h) evident by TLC analysis in EtOAc/ n-hexane (4:6), resulting in the formation of the final compound, which was then subjected to recrystallization in EtOH.

2-((4-((1-Benzyl-1H-1,2,3-triazol-4-yl)methoxy)phenyl)amino)naphthalene-1,4-dione (4a). Red solid, mp: 177–179 °C, Yield: 93%; FT-IR (KBr) υ_max (cm⁻¹): 3310, 1671, 1623, 1600, 1571, 1507, 1353, 1306, 1241. ¹H NMR (300 MHz, DMSO-d₆) δ_H (ppm): 5.17 (s, 2 H, CH₂ − O−Ph), 5.63 (s, 2 H, N − CH₂ − Ph), 5.94 (s, 1H, 1,4-Naphthoquinone (1,4-NQ)-H), 7.11 (d, J = 9 Hz, 2 H, Ar-H), 7.28–7.38 (m, 7 H, Ar-H), 7.77 (t, J = 6 Hz, 1H, 1,4-NQ-H), 7.85 (t, J = 6 Hz, 1H, 1,4-NQ-H), 7.94 (d, J = 6 Hz, 1H, 1,4-NQ-H), 8.05 (d, J = 6 Hz, 1H, 1,4-NQ-H), 8.33 (s, 1H, Triazole-H), 9.20 (s, 1H, NH). ¹³C NMR (75 MHz, DMSO-d₆) δ_C (ppm): 53.3, 61.7, 101.6, 115.8 (2 C), 125.2, 125.7, 126.0 (2 C), 126.5, 128.4 (2 C), 128.7, 129.3 (2 C), 130.9, 131.4, 133.0, 133.2, 135.4, 136.5, 143.4, 147.3, 156.1, 182.1 (C = O), 182.7 (C = O). MS (EI) m/z (%): 438 ([M + 2], 4), 436 (M⁺, 70), 265 (100), 173 (26), 91 (78), 144 (42). Anal. Calcd for C₂₆H₂₀N₄O₃: C 71.55; H 4.62; N 12.84%; found: C 70.49, H 4.58, N 13.24%.

2-((4-((1-(4-Bromobenzyl)−1H-1,2,3-triazol-4 yl)methoxy) phenyl) amino) naphthalene-1,4-dione (4b). Violet solid, mp: 172–174 °C, Yield: 96%; FT-IR (KBr) υ_max (cm⁻¹): 3231, 1675, 1596, 1568, 1507, 1355, 1292, 1241. ¹H NMR (300 MHz, CDCl₃) δ_H (ppm): 5.22 (s, 2 H, CH₂ − O−Ph), 5.53 (s, 2 H, N − CH₂ − Ph), 6.23 (s, 1H, 1,4-NQ-H), 7.02 (d, J = 9 Hz, 2 H, Ar-H), 7.19–7.23 (m, 4 H, Ar-H), 7.46 (s, 1H, NH), 7.53 (d, J = 9 Hz, 2 H, Ar-H), 7.58 (s, 1H, Triazole-H), 7.68 (t, J = 9 Hz, 1H, 1,4-NQ-H), 7.77 (t, J = 9 Hz, 1H, 1,4-NQ-H), 8.08–8.15 (m, 2 H, 1,4-NQ-H). ¹³C NMR (75 MHz, CDCl₃) δ_C (ppm): 53.6, 62.3, 102.6, 115.8 (2 C),122.7, 123.1, 124.9 (2 C), 126.2, 126.5, 129.8 (2 C), 130.4, 130.6, 132.3, 132.4 (2 C), 133.3, 133.4, 134.9, 144.5, 145.6, 156.1, 182.1, 183.8. MS (EI) m/z (%): 517 ([M + 2], 15), 515 (M⁺, 15), 265 (100), 264 (66), 169 (42), 171 (41), 90 (14). Anal. Calcd for C₂₆H₁₉BrN₄O₃: C 60.59; H 3.72; N 10.87%; found: C 59.95, H 3.69, N 11.20%.

2-((4-((1-(4-Fluorobenzyl)−1H-1,2,3-triazol-4-yl) methoxy) phenyl) amino) naphthalene-1,4-dione (4c). Dark violet solid, mp: 190–192 °C, Yield: 94%; FT-IR (KBr) υ_max (cm⁻¹):3307, 1667, 1601, 1570, 1510, 1354, 1298, 1221. ¹H NMR (300 MHz, DMSO-d₆) δ_H (ppm): 5.17 (s, 2 H, CH₂ − O−Ph), 5.63 (s, 2 H, N − CH₂ − Ph), 5.95 (s, 1H, 1,4-NQ-H), 7.11 (d, J = 9 Hz, 2 H, Ar-H), 7.24 (d, J = 9 Hz, 2 H, Ar-H), 7.30 (d, J = 9 Hz, 2 H, Ar-H), 7.38–7.48 (m, 2 H, Ar-H), 7.72–7.89 (m, 2 H, 1,4-NQ-H), 8.90–8.98 (m 1H, 1,4-NQ-H), 8.04 (d, J = 9 Hz, 1H, 1,4-NQ-H), 8.33 (s, 1H, Triazole-H), 9.20 (s, 1H, NH). ¹³C NMR (75 MHz, DMSO-d₆) δ_C (ppm): 52.5, 61.7, 101.6, 115.8 (2 C),116.0, 116, 3, 125.2, 125.7, 126.0 (2 C), 126.5, 130.8, 130.9 (2 C),131.4, 132.8, 132.8, 133.0, 133.2, 135.4, 143.4, 147.3, 156.1, 182.1 (C = O), 182.8 (C = O). MS (EI) m/z (%): 456 ([M + 2], 2), 454 (M⁺, 34), 265 (98), 264 (70), 191 (26), 162 (30), 109 (100). Anal. Calcd for C₂₆H₁₉FN₄O₃: C 68.72; H 4.21; N 12.33%; found: C 68.20, H 4.30, N 12.79%.

2-((4-((1-(4-Chlorobenzyl)−1H-1,2,3-triazol-4-yl)methoxy)phenyl)amino) naphthalene-1,4-dione (4d). Purple solid, mp: 164–166 °C, Yield: 92%; FT-IR (KBr) υ_max (cm⁻¹): 3234, 1672, 1616, 1598, 1589, 1508, 1351, 1291, 1238, 1216. ¹H NMR (300 MHz, CDCl₃) δ_H (ppm): 5.22 (s, 2 H, CH₂ − O−Ph), 5.54 (s, 2 H, N − CH₂ − Ph), 6.23 (s, 1H, 1,4-NQ-H), 7.02 (d, J = 9 Hz, 2 H, Ar-H), 7.18–7.26 (m, 4 H, Ar-H), 7.38 (d, 2 H, J = 6 Hz, Ar-H), 7.46 (s, 1H, NH), 7.58 (s, 1H, Triazole-H), 7.67 (t, J = 6 Hz, 1H, 1,4-NQ-H), 7.77 (t, J = 6 Hz, 1H, 1,4-NQ-H), 8.08–8.15 (m, 2 H, 1,4-NQ-H). ¹³C NMR (75 MHz, CDCl₃) δ_C (ppm): 53.6, 62.3, 102.6, 115.8 (2 C), 122.7, 124.9 (2 C), 126.2, 126.5, 129.4 (2 C), 129.5 (2 C), 130.4, 130.6, 132.3, 132.9, 133.3, 135.0 (2 C), 144.5, 145.6, 156.1, 182.1, 183.8. MS (EI) m/z (%): 472 ([M + 2], 14), 470 (M⁺, 44), 265 (100), 264 (71), 207 (17), 125 (59). Anal. Calcd for C₂₆H₁₉ClN₄O₃: C 66.32; H 4.07; N = 11.90%; found: C 66.19, H 3.94, N 11.18%.

2-((4-((1-(3,4-Dichlorobenzyl)−1H-1,2,3-triazol-4-yl)methoxy)phenyl)amino) naphthalene-1,4-dione (4e). Violet solid, mp: 179–181 °C, Yield: 82%; FT-IR (KBr) υ_max (cm⁻¹): 3444, 1678, 1623, 1596, 1575, 1520, 1353, 1247. ¹H NMR (300 MHz, CDCl₃) δ_H (ppm): 5.23 (s, 2 H, CH₂ − O−Ph), 5.52 (s, 2 H, N − CH₂ − Ph), 6.22 (s, 1H, 1,4-NQ-H), 6.96–7.07 (m, 2 H, Ar-H), 7.09–7.25 (m, 3 H, Ar-H), 7.34–7.52 (m, 2 H, Ar-H & s, 1H, NH), 7.59–7.72 (m, 2 H, 1,4-NQ-H), 7.76 (s, 1H, Triazole-H), 8.04–8.17 (m, 2 H, 1,4-NQ)-H. ¹³C NMR (75 MHz, CDCl₃) δ_C (ppm): 53.0, 62.3, 102.6, 115.8 (2 C),122.7, 124.9 (2 C), 126.2, 126.5, 127.3, 129.3, 123.0 (2 C), 130.4, 130.7, 131.2, 132.3, 133.3, 134.6, 134.9, 144.7, 145.6, 156.1, 182.1, 183.8. MS (EI) m/z (%): 506 ([M + 2], 17), 504 (M⁺, 25), 265 (100), 264 (74), 159 (45). Anal. Calcd for C₂₆H₁₈Cl₂N₄O₃: C 61.80; H 3.59; N 11.09%; found: C 61.84, H 3.59, N 11.05%.

2-((4-((1-(3-Chlorobenzyl)−1H-1,2,3-triazol-4-yl)methoxy)phenyl)amino) naphthalene-1,4-dione (4f). Light violet solid, mp: 168–170 °C, Yield: 87%; FT-IR (KBr) υ_max (cm⁻¹): 3244, 1674, 1613, 1601, 1572, 1353, 1295. ¹H NMR (300 MHz, CDCl₃) δ_H (ppm): 5.23 (s, 2 H, CH₂ − O−Ph), 5.55 (s, 2 H, N − CH₂ − Ph), 6.23 (s, 1H, 1,4-NQ-H), 7.02 (d, J = 9 Hz, 2 H, Ar-H), 7.14–7.23 (m, 4 H, Ar-H), 7.32–7.37 (m, 2 H, Ar-H), 7.47 (s, 1H, NH), 7.62 (s, 1H, Triazole-H), 7.66 (t, J = 9 Hz, 1,4-NQ-H), 7.76 (t, J = 9 Hz, 1H, 1,4-NQ-H), 8.06–8.14 (m, 2 H, 1,4-NQ-H). ¹³C NMR (75 MHz, CDCl₃) δ_C (ppm): 53.6, 62.3, 102.6, 115.8 (2 C), 122.8, 124.9 (2 C), 126.1, 126.2, 126.5,128.2, 129.1, 130.4, 130.5, 130.6, 132.9, 133.3, 135.0, 135.1, 136.4, 144.5, 145.6, 156.1, 182.1, 183.8. MS (EI) m/z (%): 472 ([M + 2], 10), 470 (M⁺, 26), 265 (100), 264 (67), 207 (22), 125 (54). Anal. Calcd for C₂₆H₁₉ClN₄O₃: C 66.32; H 4.07; N = 11.90%; found: C 66.32, H 3.97, N 11.92%.

2-((4-((1-(4-Isopropylbenzyl)−1H-1,2,3-triazol-4-yl)methoxy)phenyl)amino) naphthalene-1,4-dione (4 g). Dark red solid, mp: 182–185 °C, Yield: 78%; FT-IR (KBr) υ_max (cm⁻¹): 3343, 1667, 1616, 1605, 1520, 1303, 1244. ¹H NMR (300 MHz, CDCl₃) δ_H (ppm): 1.25 (d, 6 H, J = 9 Hz, CH₃ (Isopropyl)), 2.87–2.97 (m, 1H, CH₂ (Isopropyl)), 5.20 (s, 2 H, CH₂ − O−Ph), 5.53 (s, 2 H, N − CH₂ − Ph), 6.23 (s, 1H, 1,4-NQ-H), 7.02 (d, J = 9 Hz, 2 H, Ar-H), 7.17–7.26 (m, 6 H, Ar-H), 7.48 (s, 1H, NH), 7.58 (s, 1H, Triazole-H), 7.66 (t, J = 9 Hz, 1H, 1,4-NQ-H), 7.76 (t, J = 9 Hz, 1H, 1,4-NQ-H), 8.07–8.13 (m, 2 H, 1,4-NQ-H). ¹³C NMR (75 MHz, CDCl₃) δ_C (ppm): 23.9 (2 C, CH₃), 33.9 (CH), 54.1, 62.3, 102.6, 115.8 (2 C), 122.7, 124.8 (2 C), 126.2, 126.5, 127.3 (2 C), 128.3 (2 C), 130.4, 130.5, 131.7, 132.3, 133.3, 134.9, 144.1, 145.6, 149.8, 156.2, 182.1, 183.8. MS (EI) m/z (%): 480 ([M + 2], 2), 478 (M⁺, 36), 265 (76), 264 (41), 236 (10), 215 (23), 133 (100). Anal. Calcd for C₂₉H₂₆N₄O₃: C 72.79; H 5.48; N 11.71%; found: C 72.83, H 5.20, N 10.72%.

2-((4-((1-(4-(Tert-butyl)benzyl)−1H-1,2,3-triazol-4yl)methoxy)phenyl)amino) naphthalene-1,4-dione (4 h). Red solid, mp: 212–215 °C, Yield: 76%; FT-IR (KBr) υ_max (cm⁻¹): 3445, 1626, 1608, 1458, 1248.¹H NMR (300 MHz, CDCl₃) δ_H (ppm): 1.33 (s, 9 H, −C (CH₃)₃), 5.20 (s, 2 H, CH₂ − O−Ph), 5.54 (s, 2 H, N − CH₂ − Ph), 6.24 (s, 1H, 1,4-NQ -H), 7.02 (d, J = 9 Hz, 2 H, Ar-H), 7.22 (dd, J = 9 Hz, 4 H, Ar-H), 7.42 (d, J = 6 Hz, 2 H, Ar-H), 7.47 (s, 1H, NH), 7.58 (s, 1H, Triazole-H), 7.66 (t, J = 6 Hz, 1H, 1,4-NQ-H), 7.76 (t, J = 6 Hz,1H, 1,4-NQ-H), 8.07–8.14 (m, 2 H, 1,4-NQ-H). ¹³C NMR (75 MHz, CDCl₃) δ_C (ppm): 31.3 (3 C), 34.7, 54.0, 62.4, 102.6, 115.8 (2 C), 122.7, 124.8 (2 C), 126.1 (2 C), 126.5, 128.0 (2 C), 130.4, 130.6, 131.4, 132.3, 133.4, 134.9, 144.1, 145.6, 152.0, 156.2, 182.1, 183.8. MS (EI) m/z (%): 494 ([M + 2], 5), 492 (M⁺, 66), 265 (89), 264 (49), 229 (24), 147 (100). Anal. Calcd for C₂₉H₂₈N₄O₃: C 73.15; H 5.73; N 11.37%; found: C 73.13, H 5.57, N 11.15%.

2-((4-((1-(4-Methylbenzyl)−1H-1,2,3-triazol-4-yl)methoxy)phenyl)amino) naphthalene-1,4-dione (4i). Red solid, mp: 187–191 °C, Yield: 79%; FT-IR (KBr) υ_max (cm⁻¹): 3293, 1675, 1595, 1584, 1515, 1303, 1241. ¹H NMR (300 MHz, DMSO-d₆) δ_H (ppm): 2.38 (s, 3 H, (CH₃)), 5.21 (s, 2 H, CH₂ − O−Ph), 5.53 (s, 2 H, N − CH₂ − Ph), 6.24 (s, 1H, 1,4-NQ-H), 7.03 (d, J = 9 Hz, 2 H, Ar-H), 7.17–7.24 (m, 6 H, Ar-H), 7.45 (s, 1H, NH), 7.55 (s, 1H, Triazole-H), 7.64–7.72 (m, 1H, 1,4-NQ-H), 7.78 (t, J = 6 Hz, 1H, 1,4-NQ -H), 8.09–8.16 (m, 2 H, 1,4-NQ-H). ¹³C NMR (75 MHz, DMSO-d₆) δ_C (ppm): 21.2 (CH₃), 53.1, 61.7, 101.5, 115.8 (2 C), 125.1, 125.7, 126.0 (2 C), 126.5, 128.5 (2 C), 129.8 (2 C), 130.9, 131.4, 133.0, 133.2, 133.5, 135.4, 138.0, 143.3, 147.3, 156.1, 182.1, 182.8. MS (EI) m/z (%): 452 ([M + 2], 2), 450 (M⁺, 40), 265 (61), 264 (37), 236 (9), 187 (20), 158 (37), 105 (100). Anal. Calcd for C₂₇H₂₂N₄O₃: C 71.99; H 4.92; N 12.44%; found: C 70.39, H 4.58, N 10.69%.

2-(2-(4-((4-((1,4-Dioxo-1,4-dihydronaphthalen-2-yl)amino)phenoxy)methyl)−1H-1,2,3-triazol-1-yl)ethyl)isoindoline-1,3-dione (4j). Purple solid, mp: 254–257 °C, Yield: 62%; FT-IR (KBr) υ_max (cm⁻¹): 3447, 2960, 2922,1713, 1678, 1628, 1608, 1493, 1394, 1213. ¹H NMR (300 MHz, DMSO-d₆) δ_H (ppm): 3.95–4.06 (m, 2 H, CO–N–CH₂–CH₂), 4.61–4.72 (m, 2 H, N = N–N–CH₂–CH₂), 5.12 (s, 1H, 1,4-NQ-H), 5.28 (s, 2 H, CH₂ − O−Ph), 7.17–7.26 (m, 4 H, Ar-H), 7.79–7.87 (m, 4 H, Ar-H), 8.19 (d, J = 9 Hz, 2 H, 1,4-NQ-H), 8.31–8.36 (m 2 H, 1,4-NQ-H), 8.50 (s, 1H, Triazole-H), 9.20 (s, 1H, NH). ¹³C NMR (75 MHz, DMSO-d₆) δ_C (ppm): 14.0, 19.1, 30.4, 64.5, 115.8, 123.6, 124.5, 125.8, 126.0, 126.3, 129.1 (2 C), 131.5, 131.8, 132.0 (2 C), 132.2, 135.0, 137.6, 139.6, 141.0, 142.2, 152.6, 155.2, 163.7, 167.5, 168.9, 173.5. MS (EI) m/z (%): 521 ([M + 2], 2), 519 (M⁺, 10), 265 (69), 227 (51), 130 (34), 160 (100).

Biological study

Cell culture

Human PDAC cells, Mia-Paca-2, and human lung adenocarcinoma cells, EBC-1, were purchased from the Japanese Collection of Research Bio Resources Cell Bank (JCRB). The pancreatic ductal adenocarcinoma (PDAC) cells, AsPC-1, the colorectal adenocarcinoma cells, HT-29, and breast adenocarcinoma cells, MCF-7, all of human origin were provided from Iranian Biological Resource Center in Tehran, Iran. The cell culture media, including DMEM and RPMI 1640, as well as streptomycin as antibiotics, fetal bovine serum (FBS), and penicillin, were procured from Gibco (Thermo Fisher Scientific). The EBEWE Pharma, Unterach am Attersee, Austria provided cisplatin, doxorubicin, and 3-(4,5-Dimethylthiazol-2-yl)−2,5-diphenyltetrazolium bromide (MTT). Cabozantinib was obtained from LC Laboratories, Woburn, MA, USA. HT-29 cells were maintained in culture media consisting of high-glucose DMEM with 15% FBS, while Mia-Paca-2 cells were grown in a culture media composed of low-glucose DMEM containing 10% FBS. A 10% FBS supplement was included in the RPMI 1640 media for all other cell lines to sustain their optimal growth. Penicillin and streptomycin were added to all growth media. All cell lines were grown in monolayer cultures at a temperature of 37 °C within a humidified incubator, which was set to maintain a controlled atmosphere with 5% CO2.

MTT assay

The viability of cancer cells following treatment with synthetic compounds was evaluated using the MTT reduction assay, as reported previously ^63,64. In brief, the cells were cultured in 96-well plates at various densities for EBC-1 (100 × 10³), AsPC-1 (20 × 10³), Mia-Paca-2 (7.5 × 10³), HT-29 (30 × 10³), MCF-7 (30 × 10³), NIH3T3 (40 × 10³). All compounds, both synthetic and reference, were initially dissolved in dimethyl sulfoxide (DMSO) and then further diluted in the culture medium to a minimum of 400-fold to achieve the desired concentration. After 24 h, duplicate wells were treated with test compounds at different concentrations. Synthetic derivatives were tested at variant concentrations between 0.05 and 100 µM. Reference compounds included doxorubicin, cisplatin and cabozantinib. After an additional incubation of 72 h at 37 °C, the media in each well was replaced with an equal volume of fresh growth media containing 0.5 mg/mL MTT, followed by a 4-hour incubation at 37 °C to allow the formazan crystals to form. In each well, the DMSO was added to dissolve the formazan crystals after a one-hour incubation, followed by a 30-minute shaking period. Ultimately, a microplate reader was used to measure the absorbance of the resulting solutions at 570 nm. The IC₅₀ value of each compound was calculated by CurveExpert 1.34 software. Each experiment was repeated a minimum of 3 to 5 times to enhance the credibility and reliability of the results.

Kinase inhibition experiment

In order to assess the kinase inhibitory activity of the most promising compounds as antiproliferative agents including 4a, 4b, 4e, 4 g, and 4i against crucial oncogenic kinases, a radiometric assay was used. The compounds 4a and 4e were tested against a panel of 30 kinases at 10 µM, while the other 3 compounds were examined against some the most important ones.

Furthermore, the activities of compounds 4a and 4i against CDK2/cyclin A, VEGFR3, and PDGFRα were evaluated at five concentrations to generate dose-response curves, and IC₅₀ values were calculated through curve fitting. These assays were carried out by Eurofins Discovery (Eurofins Cerep, Celle-Lévescault, France).

Cell cycle test

The effect of select compounds (4a, 4e, and 4i) on the cell cycle distribution of AsPC-1 cells was examined by propidium iodide (PI)- RNase flow cytometric assay to acquire additional insight into the mechanism of action of these derivatives ⁶⁵. AsPC-1 cells were seeded in 12-well microplates at a density of 2 × 10⁵ cells/ml (1 mL per well) and incubated overnight. Later, different concentrations of 4a, 4e, and 4i derivatives at 3, 10, 30, and 50 µM were added to different wells. The cells were collected after trypsinization and collected into small tubes during 48 h of incubation period. After two PBS washes, the cells were fixed overnight at −20 ^◦C in 70% ethanol. Following fixation and washing AsPC-1 cells with PBS, they were stained for 30 min at room temperature, in the absence of light, using a DNA staining solution containing 20 µg/ml PI and 200 µg/ml RNase. Subsequence, analysis of a total of 20,000 events was performed using a FACS Calibur flow cytometer from BD Biosciences. CellQuest software (BD, USA) was used to determine the distribution of cells across sub-G1, G0/G1, S, and G2/M. Every experiment was repeated no fewer than three times.

Determination of apoptosis by Hoechst 33,258 staining

Hoechst 33,258 staining, employed as a DNA dye, was used to study apoptosis in AsPC-1 cells. Hoechst dyes attach to DNA molecules and produce fluorescence when exposed to ultraviolet (UV) light. AsPC-1 cells were initially seeded at a density of 15 × 10⁴ cells/mL (2 mL per well) and incubated for 24 h in 6-well plates. After removing the media, the wells were treated with different concentration of 4a, 4e, and 4i compounds, which were diluted in media, and then incubated for an additional 48 h. The media was removed, and one mL of the newly generated of 4% cold paraformaldehyde (PFA) was added, followed by a 20 min incubation. Afterward, the cells were washed twice with cold PBS and subsequently incubated with 1 mL of Hoechst 33,258 at a concentration of 2.5 µg/mL for 30 min in the dark. Finally, the morphological alterations characteristic of apoptosis was evaluated using a fluorescence microscope (Nikon model DS-Ri2) after a final wash with PBS.

Statistics

One-way analysis of variance (ANOVA) was used for multiple comparisons, followed by Fisher’s Least Significant Difference (LSD) test, performed with Statistical Package for the Social Sciences (SPSS) version 13 for Windows. Statistical significance was considered at P 

Computational study

Molecular modeling analysis

The smina docking program aimed to explore the binding affinity and crucial molecular interactions between the target binding sites and the synthetic compounds ⁶⁶. The crystal structures of CDK2/cyclin A, FLT4, and PDGFRA were obtained from RCSB-PDB website and EBI AlphaFold database and each protein was subsequently purified by removing any associated water molecules and co-crystal structures. AutoDock Tools 1.5.6 (ADT) ( were allocated to further preparation of the protein by adding the hydrogens and assigning Kollmann charges. The Marvin sketch (https://chemaxon.com/) software was utilized to draw all the compounds, and HyperChem software ⁶⁷ was employed to add hydrogen atoms. The synthetic compounds were optimized (gasteiger charge and energy) using the Open Babel ⁶⁸. Subsequently, the smina software was employed to determine the interactions of the binding modes and minimize affinity within the binding site of the enzymes. To explore binding interactions, the Discovery Studio 2021 client (https://discover.3ds.com/discovery-studio-visualizer) was used.

Molecular dynamics simulation

The Gromacs dynamics package 2019.1, operating on a Centos Linux server with GPUs, was employed to conduct molecular modeling and dynamics simulations of the target compound (4a) in complex with CDK2, FLT4, and PDGFRA kinase. The MD simulation parameters were obtained by the Amber99sb force field to define at 300 K and neutral pH (7.0). Force field parameters were generated by ACPYPE software ⁶⁹ and the AM1 partial charges were added by chimera software. A dodecahedral box surrounded the solute, which was then saturated by TIP3P water molecules with the addition of an adequate amount of Na⁺ ions to neutralize the system. The steepest descent method was employed twice, with constraints and then without it, to minimize the energy of the system in the explicit solvent. Subsequently, NVT heating with V-rescale thermostats was executed, imposing restraints on the positions of the macromolecule and ligand atoms for a duration of 500 ps. Using the particle-mesh Ewald (PME) approaches and the LINCS constraint, which is faster than the SHAKE technique the periodic boundary conditions were examined. After the 500 ps equilibration phase in the NPT ensemble, the system’s Pressure stabilized at 1 bar. The MD was run for 100 ns to achieve the best equilibration point. Following that, the trajectory was saved to account for any deviations in its path caused by the periodic boundary condition, and the complex was then repositioned at the center of the box. The root-mean-square deviation (RMSD) for protein backbone atoms, radius of gyration (Rg), and residue root-means-square fluctuation (RMSF) were analyzed in each snapshot against the primary frame. Also. the Gromos method was applied to cluster the trajectory in the equilibrium time range with cut-off values 0.14, 0.18, and 0.25 for CDK2/cyclin A, FLT4, and PDGFRA respectively. All binding interactions and generating images were analyzed by Discovery Studio Visualizer v20.1.0.19295.