*p* < 0.001 compared with control group, \* *p* < 0.05 \*\* *p* < 0.01 compared with LPS group.

#### Docking Analysis

Toll-like receptor 4 (TLR4) and its coreceptor—MD-2—are the target of LPS, which could influence innate immune response to release potentially cytotoxic molecules. MD-2 is linked with the extracellular domain of TLR4 and is indispensable for LPS recognition [13]. Therefore, this docking analysis was applied to explore whether or not the target compounds inhibit MD-2 to reduce the release of inflammatory mediators. Umeharu Ohto et al. [39] had revealed the crystal structure of mouse TLR4/MD-2/LPS complex, which was shown in Figure 4. In their study, MD-2 directly bound to LPS in its hydrophobic cavity with high affinity. With this background, compound **5n** was used as the ligand to explore the relationship between DCH and TLR4/MD-2/LPS complex. As shown in Figure 5, compound **5n** could integrate closely with the hydrophobic cavity of MD-2, which is the important TLR4/MD-2/LPS binding position. That means the DCH might disturb the interactions between LPS and TLR4/MD-2 complex. The minimum binding energy of (ΔG) is −10.08 kcal/mol. Given to this result, we supposed TLR4/MD-2 complex is likely to be one of the drug targets of DCH.

**Figure 4.** The surface representation of LPS (red) and Toll-like receptor 4 (TLR4) (green)/MD-2 (cyan) binding pocket (PDB: 3VQ2).

**Figure 5.** Interaction diagrams of the selected docked conformations of compound 5n to TLR4/MD-2 complex. (**A**) The surface representation of compound **5n** and TLR4 (limon)/MD-2 (yellow) binding pocket. (**B**) The magnified representation of compound **5n** (red) and TLR4 (limon)/MD-2 (cyan) binding pocket. (**C**) The interface of compound **5n** and TLR4/MD-2 complex; (**D**) 3D ligand interactions diagram. Dotted line; pink: alkyl hydrophobic; blue: pi hydrophobic; green: hydrogen bond.

#### **3. Conclusions**

Total-thirty new DCH were designed and synthesized. Most of the target compounds exhibited moderate activities against the tested cell lines and they showed a general promotion under hypoxia conditions. Meanwhile, the new potential activity of DCH was found that DCH could exhibit anti-neuroinflammatory activity. DCH could inhibit the release of NO, TNF-α, and IL-6, which were induced by LPS. The docking analysis was shown that MD-2, the coreceptor of TLR4, might be one of the targets of DCH. Additionally, DCH could also reduce NO levels by direct NO capture. In brief, DCH were the potential anti-neuroinflammatory agents which might reduce CRF of cancer patients.

#### **4. Experimental Section**

#### *4.1. Chemistry*

Unless otherwise specified, all solvents and reagents were commercially available and used without further purification. Analytical TLC was performed using silica gel precoated GF254 plates. The 1H-NMR and 13C-NMR spectra were recorded at 600 MHz and 150 MHZ on a Bruker AV-400 spectrometer (Bruker Bioscience, Billerica, MA, USA), using CDCl3 as solvent with tetramethylsilane as the internal standard. NMR spectra were analyzed using MestReNova. High-resolution mass spectra (HRMS) were measured using an Agilent Accurate-Mass Q-TOF 6530 (Agilent, Santa Clara, CA, USA) instrument in ESI mode. The 1H-NMR and 13C-NMR spectra of synthesized compounds, please see the supplementary materials.

#### 4.1.1. General Procedure for the Synthesis of Compounds **1a**–**1e** and **2a**–**2e**

Substituted coumarin (**a**–**e**) (10 mmol), substituted DHA (**III** or **IV**) (10 mmol), and triethylamine (10 mmol) were dissolved in 20 mL CH2Cl2. In addition, CuI (100 mg) was added and the reaction mixture was stirred at room temperature for 8 h under the protection of nitrogen. The mixture was filtered, washed with water, dried over Na2SO4 and evaporated to dryness. The crude product was purified through column chromatography.

*3-((4-((10S)-dihydroartemisininoxy)methyl-1H-1,2,3-triazol-1-yl)-methyl)-2H-1-chromen-2-one* (**1a**) White solid; m.p. 73–75 ◦C; yield 52%; 1H NMR (600 MHz, CDCl3) δ (ppm): 7.77 (s, 1H), 7.68 (s, 1H), 7.56 (t, *J* = 7.9 Hz, 1H), 7.47 (d, *J* = 7.7 Hz, 1H), 7.34 (d, *J* = 8.4 Hz, 1H), 7.30 (t, *J* = 7.6 Hz, 1H), 5.47 (s, 2H), 5.39 (s, 1H), 4.94 (d, *J* = 12.6 Hz, 1H), 4.92 (d, *J* = 3.5 Hz, 1H), 4.68 (d, *J* = 12.6 Hz, 1H), 2.67–2.60 (m, 1H), 2.38–2.31 (m, 1H), 2.01–1.97 (m, 1H), 1.85–1.81 (m, 1H), 1.77–1.68 (m, 3H), 1.59 (dd, *J* = 13.2, 3.4 Hz, 1H), 1.46–1.43 (m, 1H), 1.41 (s, 3H), 1.31–1.18 (m, 1H), 1.26–1.16 (m, 2H), 0.91 (d, *J* = 6.3 Hz, 3H), 0.88 (d, *J* = 7.3 Hz, 3H); 13C NMR (150 MHz, CDCl3) δ (ppm): 159.64, 152.65, 144.62, 141.31, 131.46, 127.30, 123.92, 122.67, 121.84, 117.53, 115.71, 103.10, 100.77, 86.93, 80.06, 76.22, 76.01, 75.80, 60.70, 51.47, 47.97, 43.33, 36.33, 35.36, 33.52, 29.78, 25.09, 23.59, 23.41, 19.30, 11.99; ESI-HRMS [M + Na]+: (*m*/*z*) Calcd. for C28H33N3O7Na: 546.2216. Found: 546.2181.

*6-bromo-3-((4-((10S)-dihydroartemisininoxy)methyl-1H-1,2,3-triazol-1-yl)-methyl)-2H-1-chromen-2-one* (**1b**) White solid; m.p. 78–80 ◦C; yield 48%; 1H NMR (600 MHz, CDCl3) δ (ppm): 7.75 (s, 1H), 7.64 (dd, *J* = 8.8, 2.3 Hz, 1H), 7.61 (d, *J* = 2.3 Hz, 1H), 7.55 (s, 1H), 7.24 (d, *J* = 8.8 Hz, 1H), 5.47 (s, 2H), 5.39 (s, 1H), 4.94 (d, *J* = 12.7 Hz, 1H), 4.93 (d, *J* = 3.5 Hz, 1H), 4.69 (d, *J* = 12.8 Hz, 1H), 2.67–2.63 (m, 1H), 2.38–2.33 (m, 1H), 2.03–1.99 (m, 1H), 1.86–1.82 (m, 1H), 1.75–1.70 (m, 2H), 1.62–1.58 (m, 2H), 1.42 (s, 3H), 1.31–1.19 (m, 4H), 0.92 (d, *J* = 6.3 Hz, 3H), 0.89 (d, *J* = 7.4 Hz, 3H); 13C NMR (150 MHz, CDCl3) δ (ppm): 158.90, 151.42, 144.74, 139.67, 134.18, 129.51, 123.17, 122.67, 119.00, 117.43, 116.53, 103.12, 100.87, 86.95, 80.06, 76.20, 75.99, 75.78, 60.76, 51.46, 47.82, 43.32, 36.36, 35.36, 33.52, 29.79, 25.10, 23.62, 23.42, 19.30, 12.00; ESI-HRMS [M + Na]+: (*m*/*z*) Calcd. for C28H32BrN3O7Na: 624.1321. Found: 624.1264.

*6-chloro-3-((4-((10S)-dihydroartemisininoxy)methyl-1H-1,2,3-triazol-1-yl)-methyl)-2H-1-chromen-2-one* (**1c**) White solid; m.p. 93–95 ◦C; yield 41%; 1H NMR (600 MHz, CDCl3) δ (ppm): 7.75 (s, 1H), 7.56 (s, 1H), 7.51 (dd, *J* = 8.8, 2.5 Hz, 1H), 7.45 (d, *J* = 2.4 Hz, 1H), 7.30 (d, *J* = 8.9 Hz, 1H), 5.47 (s, 2H), 5.39 (s, 1H), 4.95 (d, *J* = 12.7 Hz, 1H), 4.93 (d, *J* = 3.6 Hz, 1H), 4.69 (d, *J* = 12.6 Hz, 1H), 2.68–2.62 (m, 1H), 2.35 (td, *J* = 14.0, 4.0 Hz, 1H), 2.03–1.99 (m, 1H), 1.87–1.82 (m, 1H), 1.75–1.71 (m, 2H), 1.62–1.59 (m, 1H), 1.48–1.43 (m, 2H), 1.42 (s, 3H), 1.31–1.19 (m, 3H), 0.92 (d, *J* = 6.3 Hz, 3H), 0.89 (d, *J* = 7.4 Hz, 3H); 13C NMR (150 MHz, CDCl3) δ (ppm): 158.96, 150.95, 144.73, 139.79, 131.37, 129.27, 126.46, 123.19, 122.67, 118.51, 117.16, 103.12, 100.86, 86.95, 80.06, 76.21, 75.99, 75.78, 60.75, 51.47, 47.84, 43.33, 36.36, 35.36, 33.52, 29.79, 25.10, 23.62, 23.42, 19.30, 12.00; ESI-HRMS [M + Na]+: (*m*/*z*) Calcd. for C28H32ClN3O7Na: 580.1826. Found: 580.1788.

*6,8-dichloro-3-((4-((10S)-dihydroartemisininoxy)methyl-1H-1,2,3-triazol-1-yl)-methyl)-2H-1-chromen-2-one* (**1d**) White solid; m.p. 113–115 ◦C; yield 60%; 1H NMR (600 MHz, CDCl3) δ (ppm): 7.75 (s, 1H), 7.61 (d, *J* = 2.3 Hz, 1H), 7.55 (s, 1H), 7.37 (d, *J* = 2.4 Hz, 1H), 5.48 (s, 2H), 5.37 (s, 1H), 4.96–4.92 (m, 2H), 4.70 (d, *J* = 12.7 Hz, 1H), 2.69–2.62 (m, 1H), 2.38–2.33 (m, 1H), 2.04–1.99 (m, 1H), 1.87–1.82 (m, 1H), 1.76–1.72 (m, 3H), 1.62–1.59 (m, 1H), 1.42 (s, 3H), 1.32–1.18 (m, 4H), 0.92 (d, *J* = 6.3 Hz, 3H), 0.89 (d, *J* = 7.3 Hz, 3H); 13C NMR (150 MHz, CDCl3) δ (ppm): 157.92, 146.92, 144.86, 139.45, 131.36, 129.12, 125.03, 124.04, 122.68, 121.71, 119.30, 103.13, 100.95, 86.94, 80.04, 76.20, 75.99, 75.78, 60.80, 51.44, 47.63, 43.31, 36.37, 35.35, 33.51, 29.79, 25.09, 23.61, 23.42, 19.28, 12.00; ESI-HRMS [M + Na]+: (*m*/*z*) Calcd. for C28H31Cl2N3O7Na: 614.1437. Found: 614.1428.

*6,8-dibromo-3-((4-((10S)-dihydroartemisininoxy)methyl-1H-1,2,3-triazol-1-yl)-methyl)-2H-1-chromen-2-one* (**1e**) White solid; m.p. 103–105 ◦C; yield 61%; 1H NMR (600 MHz, CDCl3) δ (ppm): 7.90 (d, *J* = 2.2 Hz, 1H), 7.75 (s, 1H), 7.56 (d, *J* = 2.2 Hz, 1H), 7.52 (s, 1H), 5.48 (s, 2H), 5.37 (s, 1H), 4.96–4.92 (m, 2H), 4.70 (d, *J* = 12.7 Hz, 1H), 2.67–2.63 (m, 1H), 2.38–2.33 (m, 1H), 2.03–1.99 (m, 1H), 1.87–1.82 (m, 1H), 1.76–1.72 (m, 2H), 1.62–1.58 (m, 4H), 1.42 (s, 3H), 1.25–1.19 (m, 2H), 0.92 (d, *J* = 6.3 Hz, 3H), 0.90 (d, *J* = 7.4 Hz, 3H); 13C NMR (150 MHz, CDCl3) δ (ppm): 158.01, 148.45, 144.87, 139.36, 136.89, 128.75, 123.97, 122.67, 119.74, 116.48, 110.29, 103.13, 100.97, 86.95, 80.04, 76.20, 75.99, 75.78, 60.82, 51.45, 47.56, 43.31, 36.37, 35.35, 33.51, 29.79, 25.09, 23.62, 23.42, 19.30, 12.00; ESI-HRMS [M + Na]+: (*m*/*z*) Calcd. for C28H31Br2N3O7Na: 702.0426. Found: 702.0421.

*3-((4-(2-((10S)-dihydroartemisininoxy)ethyl)-1H-1,2,3-triazol-1-yl)-methyl)-2H-1-chromen-2-one* (**2a**) White solid; m.p. 143–145 ◦C; yield 66%; 1H NMR (600 MHz, CDCl3) δ (ppm): 7.65 (s, 1H), 7.63 (s, 1H), 7.56 (ddd, *J* = 8.7, 7.3, 1.6 Hz, 1H), 7.47 (dd, *J* = 7.8, 1.6 Hz, 1H), 7.35 (d, *J* = 8.4 Hz, 1H), 7.30 (td, *J* = 7.5, 1.1 Hz, 1H), 5.44 (s, 2H), 5.29 (d, *J* = 9.7 Hz, 1H), 4.78 (d, *J* = 3.6 Hz, 1H), 4.13–4.09 (m, 1H), 3.69–3.65 (m, 1H), 3.07–2.97 (m, 2H), 2.61–2.55 (m, 1H), 2.33 (td, *J* = 14.0, 4.0 Hz, 1H), 2.01–1.97 (m, 1H), 1.85–1.81 (m, 1H), 1.71–1.67 (m, 2H), 1.59–1.56 (m, 1H), 1.41 (s, 3H), 1.40–1.38 (m, 1H), 1.28–1.16 (m, 3H), 0.91 (d, *J* = 6.3 Hz, 3H), 0.89–0.83 (m, 1H), 0.80 (d, *J* = 7.3 Hz, 3H); 13C NMR (150 MHz, CDCl3) δ (ppm): 159.65, 152.64, 144.77, 141.15, 131.43, 127.30, 123.90, 121.98, 121.78, 117.52, 115.70, 103.03, 100.92, 86.84, 80.02, 76.21, 76.00, 75.78, 66.18, 51.47, 47.86, 43.28, 36.34, 35.36, 33.55, 29.79, 25.57, 25.14, 23.62, 23.34, 19.33, 11.93; ESI-HRMS [M + Na]+: (*m*/*z*) Calcd. for C29H35N3O7Na: 560.2373. Found: 560.2417.

*6-bromo-3-((4-(2-((10S)-dihydroartemisininoxy)ethyl)-1H-1,2,3-triazol-1-yl)-methyl)-2H-1-chromen-2-one* (**2b**) White solid; m.p. 138–140 ◦C; yield 52%; 1H NMR (600 MHz, CDCl3) δ (ppm): 7.64 (dd, *J* = 8.8, 2.3 Hz, 1H), 7.61 (d, *J* = 2.3 Hz, 1H), 7.60 (s, 1H), 7.52 (s, 1H), 7.23 (d, *J* = 8.8 Hz, 1H), 5.43 (s, 2H), 5.28 (s, 1H), 4.79 (d, *J* = 3.5 Hz, 1H), 4.13–4.09 (m, 1H), 3.70–3.66 (m, 1H), 3.07–2.98 (m, 2H), 2.62–2.57 (m, 1H), 2.34 (td, *J* = 14.0, 4.0 Hz, 1H), 2.01–1.98 (m, 1H), 1.86–1.81 (m, 1H), 1.71–1.67 (m, 2H), 1.60–1.56 (m, 1H), 1.42 (s, 3H), 1.40–1.37 (m, 1H), 1.27–1.17 (m, 3H), 0.92 (d, *J* = 6.2 Hz, 3H), 0.89–0.85 (m, 1H), 0.82 (d, *J* = 7.3 Hz, 3H); 13C NMR (150 MHz, CDCl3) δ (ppm): 158.90, 151.40, 144.86, 139.50, 134.15, 129.51, 123.31, 121.76, 118.99, 117.41, 116.51, 103.06, 100.92, 86.85, 80.00, 76.20, 75.99, 75.78, 66.18, 51.45, 47.72, 43.26, 36.37, 35.35, 33.55, 29.78, 25.56, 25.14, 23.63, 23.35, 19.32, 11.95; ESI-HRMS [M + Na]+: (*m*/*z*) Calcd. for C29H34BrN3O7Na: 638.1478. Found: 638.1508.

*6-chloro-3-((4-(2-((10S)-dihydroartemisininoxy)ethyl)-1H-1,2,3-triazol-1-yl)-methyl)-2H-1-chromen-2-one* (**2c**) White solid; m.p. 138–140 ◦C; yield 52%; 1H NMR (600 MHz, CDCl3) δ (ppm): 7.60 (s, 1H), 7.53 (s, 1H), 7.50 (dd, *J* = 8.8, 2.5 Hz, 1H), 7.45 (d, *J* = 2.4 Hz, 1H), 7.29 (d, *J* = 8.8 Hz, 1H), 5.43 (d, *J* = 1.1 Hz, 2H), 5.28 (s, 1H), 4.79 (d, *J* = 3.6 Hz, 1H), 4.13–4.09 (m, 1H), 3.70–3.66 (m, 1H), 3.05–2.99 (m, 2H), 2.61–2.58 (m, 1H), 2.37–2.31 (m, 1H), 2.01–1.98 (m, 1H), 1.86–1.81 (m, 1H), 1.70–1.68 (m, 2H), 1.60–1.56 (m, 1H), 1.42 (s, 3H), 1.41–1.36 (m, 1H), 1.26–1.17 (m, 3H), 0.92 (d, *J* = 6.2 Hz, 3H), 0.89–0.85 (m, 1H), 0.82 (d, *J* = 7.3 Hz, 3H); 13C NMR (150 MHz, CDCl3) δ (ppm): 158.97, 150.93, 144.87, 139.62, 131.34, 129.25, 126.47, 123.35, 121.76, 118.51, 117.14, 103.06, 100.93, 86.85, 80.00, 76.20, 75.99, 75.78, 66.19, 51.45, 47.72, 43.27, 36.37, 35.35, 33.56, 29.79, 25.57, 25.14, 23.63, 23.35, 19.32, 11.95; ESI-HRMS [M + Na]+: (*m*/*z*) Calcd. for C29H34ClN3O7Na: 594.1983. Found: 594.2002.

*6,8-dichloro-3-((4-(2-((10S)-dihydroartemisininoxy)ethyl)-1H-1,2,3-triazol-1-yl)-methyl)-2H-1-chromen-2-one* (**2d**) White solid; m.p. 101–103 ◦C; yield 50%; 1H NMR (600 MHz, CDCl3) δ (ppm): 7.60 (d, *J* = 2.2 Hz, 2H), 7.52 (s, 1H), 7.37 (d, *J* = 2.3 Hz, 1H), 5.45 (s, 2H), 5.27 (s, 1H), 4.79 (d, *J* = 3.5 Hz, 1H), 4.13–4.08 (m, 1H), 3.71–3.67 (m, 1H), 3.04–3.01 (m, 2H), 2.61–2.59 (m, 1H), 2.34 (td, *J* = 14.0, 4.0 Hz, 1H), 2.02–1.98 (m, 1H), 1.85–1.82 (m, 1H), 1.71–1.68 (m, 2H), 1.60–1.58 (m, 1H), 1.42 (s, 3H), 1.41–1.36 (m, 1H), 1.26–1.17 (m, 3H), 0.92 (d, *J* = 6.1 Hz, 3H), 0.90–0.85 (m, 1H), 0.82 (d, *J* = 7.3 Hz, 3H); 13C NMR (150 MHz, CDCl3) δ (ppm): 157.93, 146.90, 144.92, 139.29, 131.34, 129.12, 125.04, 124.16, 121.82, 121.70, 119.30, 103.07, 100.94, 86.86, 79.99, 76.20, 75.99, 75.78, 66.17, 51.44, 47.57, 43.26, 36.39, 35.35, 33.55, 29.78, 25.53, 25.13, 23.63, 23.35, 19.32, 11.95; ESI-HRMS [M + Na]+: (*m*/*z*) Calcd. for C29H33Cl2N3O7Na: 628.1593. Found: 628.1360.

*6,8-dibromo-3-((4-(2-((10S)-dihydroartemisininoxy)ethyl)-1H-1,2,3-triazol-1-yl)-methyl)-2H-1-chromen-2-one (***2e**) White solid; m.p. 93–95 ◦C; yield 44%; 1H NMR (600 MHz, CDCl3) δ (ppm): 7.83 (d, *J* = 2.1 Hz, 1H), 7.53 (s, 1H), 7.49 (d, *J* = 2.2 Hz, 1H), 7.42 (s, 1H), 5.38 (s, 2H), 5.21 (s, 1H), 4.73 (d, *J* = 3.4 Hz, 1H), 4.06–4.02 (m, 1H), 3.64–3.60 (m, 1H), 2.97–2.94 (m, 2H), 2.56–2.51 (m, 1H), 2.30–2.25 (m, 1H), 1.95–1.91 (m, 1H), 1.79–1.75 (m, 1H), 1.64–1.60 (m, 2H), 1.52 (dd, *J* = 13.2, 3.3 Hz, 1H), 1.35 (s, 3H), 1.34–1.29 (m, 1H), 1.19–1.11 (m, 3H), 0.85 (d, *J* = 6.1 Hz, 3H), 0.83–0.78 (m, 1H), 0.75 (d, *J* = 7.4 Hz, 3H); 13C NMR (150 MHz, CDCl3) δ (ppm): 158.01, 148.41, 144.96, 139.16, 136.85, 128.75, 124.12, 121.77, 119.74, 116.47, 110.26, 103.07, 100.93, 86.86, 80.00, 76.21, 76.00, 75.79, 66.19, 51.44, 47.48, 43.26, 36.39, 35.35, 33.55, 29.78, 25.56, 25.13, 23.63, 23.35, 19.33, 11.96; ESI-HRMS [M + Na]+: (*m*/*z*) Calcd. for C29H33Br2N3O7Na: 716.0583. Found: 716.0381.

#### 4.1.2. General Procedure for the Synthesis of Compounds **3f**–**3i** and **4f**–**4i**

Substituted coumarin (**f**–**i**) (10 mmol), substituted DHA (**III** or **IV**) (10 mmol) and triethylamine (10 mmol) were dissolved in 20 mL CH2Cl2. In addition, CuI (100 mg) was added and the reaction mixture was stirred at room temperature for 8 h under the protection of nitrogen. The mixture was filtered, washed with water, dried over Na2SO4 and evaporated to dryness. The crude product was purified through column chromatography.

*7-hydroxy-4-((4-((10S)-dihydroartemisininoxy)methyl-1H-1,2,3-triazol-1-yl)-methyl)-2H-1-chromen-2-one* (**3f**) White solid; m.p. 165–167 ◦C; yield 54%; 1H NMR (600 MHz, CDCl3) δ (ppm): 9.61 (s, 1H), 7.68 (d, *J* = 8.8 Hz, 1H), 7.67 (s, 1H), 6.99 (dd, *J* = 8.8, 2.4 Hz, 1H), 6.85 (d, *J* = 2.4 Hz, 1H), 6.15 (s, 1H), 5.76 (d, *J* = 15.4 Hz, 1H), 5.57 (d, *J* = 15.3 Hz, 1H), 5.20 (s, 1H), 4.87 (d, *J* = 1.9 Hz, 1H), 4.86 (d, *J* = 7.4 Hz, 1H), 4.68 (d, *J* = 12.9 Hz, 1H), 2.62–2.60 (m, 1H), 2.34–2.29 (m, 1H), 2.02−1.98 (m, 1H), 1.82–1.78 (m, 1H), 1.66−1.61 (m, 3H), 1.61−1.57 (m, 1H), 1.45–1.42 (m, 1H), 1.40 (s, 3H), 1.20−1.08 (m, 3H), 0.84 (d, *J* = 7.4 Hz, 3H), 0.82 (d, *J* = 6.1 Hz, 3H); 13C NMR (150 MHz, CDCl3) δ (ppm): 160.88, 159.75, 154.96, 146.45, 145.19, 124.19, 122.29, 112.78, 111.57, 108.80, 103.21, 103.04, 101.40, 86.91, 79.92, 76.20, 75.99, 75.78, 60.44, 51.32, 50.16, 43.18, 36.28, 35.26, 33.42, 29.74, 25.00, 23.53, 23.32, 19.26, 11.89; ESI-HRMS [M + Na]+: (*m*/*z*) Calcd. for C28H33N3O8Na: 562.2165. Found: 562.2172.

*5,7-dimethyl-4-((4-((10S)-dihydroartemisininoxy)methyl-1H-1,2,3-triazol-1-yl)-methyl)-2H-1-chromen-2-one* (**3g**) White solid; m.p. 133~135 ◦C; yield 57%; 1H NMR (600 MHz, CDCl3) δ (ppm): 7.56 (s, 1H), 7.07 (s, 1H), 6.95 (s, 1H), 5.90 (d, *J* = 17.0 Hz, 1H), 5.86 (d, *J* = 17.0 Hz, 1H), 5.51 (s, 1H), 5.41 (s, 1H), 4.98 (d, *J* = 12.8 Hz, 1H), 4.93 (d, *J* = 3.5 Hz, 1H), 4.73 (d, *J* = 12.8 Hz, 1H), 2.68 (s, 3H), 2.40 (s, 3H), 2.35 (dd, *J* = 14.0, 4.0 Hz, 1H), 2.05−2.01 (m, 1H), 1.88−1.85 (m, 1H), 1.75–1.70 (m, 2H), 1.60 (dd, *J* = 13.2, 3.4 Hz, 1H), 1.48–1.46 (m, 1H), 1.43 (s, 3H), 1.32−1.20 (m, 5H), 0.93 (d, *J* = 6.3 Hz, 3H), 0.89 (d, *J* = 7.4 Hz, 3H); 13C NMR (150 MHz, CDCl3) δ (ppm): 158.67, 154.26, 149.33, 145.35, 142.10, 133.93, 129.26, 122.10, 115.63, 113.36, 112.80, 103.16, 100.93, 86.98, 80.05, 76.21, 76.00, 75.79, 60.80, 52.05, 51.48, 43.31, 36.34, 35.36, 33.50, 29.78, 25.11, 23.61, 23.44, 23.28, 20.24, 19.31, 12.01; ESI-HRMS [M + Na]+: (*m*/*z*) Calcd. for C30H37N3O7Na: 574.2539. Found: 574.2553.

*7-methyl-4-((4-((10S)-dihydroartemisininoxy)methyl-1H-1,2,3-triazol-1-yl)-methyl)-2H-1-chromen-2-one* (**3h**) White solid; m.p. 107–109 ◦C; yield 50%; 1H NMR (600 MHz, CDCl3) δ (ppm): 7.58 (s, 1H), 7.50 (d, *J* = 8.1 Hz, 1H), 7.19 (s, 1H), 7.11 (d, *J* = 8.1 Hz, 1H), 6.01 (s, 1H), 5.74 (d, *J* = 16.3 Hz, 1H), 5.64 (d, *J* = 16.0 Hz, 1H), 5.30 (s, 1H), 4.92–4.88 (m, 2H), 4.72 (d, *J* = 12.9 Hz, 1H), 2.65–2.61 (m, 1H), 2.45 (s, 3H), 2.34 (td, *J* = 14.0, 4.0 Hz, 1H), 2.04–2.00 (m, 1H), 1.86–1.81 (m, 1H), 1.71–1.66 (m, 2H), 1.58–1.55 (m, 1H), 1.42 (s, 3H), 1.26–1.18 (m, 5H), 0.91 (d, *J* = 5.7 Hz, 3H), 0.86 (d, *J* = 7.3 Hz, 3H); 13C NMR (150 MHz, CDCl3) δ (ppm): 159.01, 152.86, 146.75, 145.42, 143.16, 125.03, 122.23, 121.95, 116.71, 113.56, 113.41, 103.15, 101.10, 86.93, 80.00, 76.21, 75.99, 75.78, 60.90, 51.41, 49.29, 43.27, 36.34, 35.31, 33.47, 29.77, 25.06, 23.58, 23.38, 20.70, 19.30, 11.96; ESI-HRMS [M + Na]+: (*m*/*z*) Calcd. for C29H35N3O7Na: 560.2373. Found: 560.2375.

*6-methyl-4-((4-((10S)-dihydroartemisininoxy)methyl-1H-1,2,3-triazol-1-yl)-methyl)-2H-1-chromen-2-one* (**3i**) White solid; m.p. 88–90◦C; yield 60%; 1H NMR (600 MHz, CDCl3) δ (ppm): 7.58 (s, 1H), 7.39 (d, *J* = 8.8 Hz, 2H), 7.28 (d, *J* = 8.3 Hz, 1H), 6.01 (s, 1H), 5.75 (d, *J* = 16.3 Hz, 1H), 5.66 (d, *J* = 16.3 Hz, 1H), 5.32 (s, 1H), 4.92 (d, *J* = 12.8 Hz, 1H), 4.90 (d, *J* = 3.5 Hz, 1H), 4.73 (d, *J* = 12.8 Hz, 1H), 2.67–2.60 (m, 1H), 2.41 (s, 3H), 2.34 (td, *J* = 14.0, 4.0 Hz, 1H), 2.04–1.99 (m, 1H), 1.86–1.81 (m, 1H), 1.72–1.68 (m, 2H), 1.57 (dd, *J* = 13.2, 3.1 Hz, 1H), 1.42 (s, 3H), 1.26–1.19 (m, 5H), 0.91 (d, *J* = 5.7 Hz, 3H), 0.86 (d, *J* = 7.3 Hz, 3H); 13C NMR (150 MHz, CDCl3) δ (ppm): 158.91, 150.87, 146.72, 145.41, 133.69, 132.71, 122.26, 121.95, 116.32, 115.68, 114.29, 103.15, 101.05, 86.94, 80.00, 76.21, 76.00, 75.78, 60.86, 51.42, 49.17, 43.27, 36.34, 35.31, 33.47, 29.76, 25.07, 23.59, 23.39, 20.01, 19.30, 11.96; ESI-HRMS [M + Na]+: (*m*/*z*) Calcd. for C29H35N3O7Na: 560.2373. Found: 560.2403.

*7-hydroxy-4-((4-(2-((10S)-dihydroartemisininoxy)ethyl)-1H-1,2,3-triazol-1-yl)-methyl)-2H-1-chromen-2-one* (**4f**) White solid; m.p. 121–123 ◦C; yield 54%; 1H NMR (600 MHz, CDCl3) δ (ppm): 9.98 (s, 1H), 7.68 (d, *J* = 8.8 Hz, 1H), 7.49 (s, 1H), 7.01 (dd, *J* = 8.8, 2.4 Hz, 1H), 6.85 (d, *J* = 2.4 Hz, 1H), 6.16 (s, 1H), 5.68–5.59 (m, 2H), 5.20 (s, 1H), 4.71 (d, *J* = 3.5 Hz, 1H), 4.08–4.04 (m, 1H), 3.67–3.63 (m, 1H), 3.03–2.94 (m, 2H), 2.56–2.53 (m, 1H), 2.33 (td, *J* = 14.1, 4.0 Hz, 1H), 2.03–1.99 (m, 1H), 1.86–1.82 (m, 1H), 1.58–1.54 (m, 2H), 1.51–1.48 (m, 1H), 1.40 (s, 3H), 1.40–1.35 (m, 2H), 1.27–1.25 (m, 1H), 1.20–1.16 (m, 2H), 0.88 (d, *J* = 5.6 Hz, 3H), 0.68 (d, *J* = 7.3 Hz, 3H); 13C NMR (150 MHz, CDCl3) δ (ppm): 161.02, 159.72, 155.05, 146.56, 145.44, 124.28, 121.23, 112.79, 111.51, 108.74, 103.16, 103.05, 100.88, 86.87, 79.91, 76.20, 75.99, 75.78, 65.75, 51.37, 50.15, 43.11, 36.37, 35.30, 33.45, 29.66, 25.24, 25.09, 23.62, 23.29, 19.29, 11.73; ESI-HRMS [M + Na]+: (*m*/*z*) Calcd. for C29H35N3O8Na: 576.2322. Found: 576.2352.

*5,7-dimethyl-4-((4-(2-((10S)-dihydroartemisininoxy)ethyl)-1H-1,2,3-triazol-1-yl)-methyl)-2H-1-chromen-2-one* (**4g**) White solid; m.p. 135–137 ◦C; yield 52%; 1H NMR (600 MHz, CDCl3) δ (ppm): 7.41 (s, 1H), 7.06 (s, 1H), 6.94 (s, 1H), 5.84 (s, 2H), 5.54 (s, 1H), 5.28 (s, 1H), 4.80 (d, *J* = 3.4 Hz, 1H), 4.15–4.10 (m, 1H), 3.73–3.69 (m, 1H), 3.11–3.00 (m, 2H), 2.66 (s, 3H), 2.61–2.58 (m, 1H), 2.40 (s, 3H), 2.35 (td, *J* = 14.1, 4.0 Hz, 1H), 2.03–1.99 (m, 1H), 1.89–1.84 (m, 1H), 1.70–1.63 (m, 2H), 1.59–1.55 (m, 1H), 1.49–1.43 (m, 1H), 1.42 (s, 3H), 1.29–1.18 (m, 3H), 0.93 (d, *J* = 6.3 Hz, 3H), 0.87 (dd, *J* = 12.4, 4.6 Hz, 1H), 0.80 (d, *J* = 7.3 Hz, 3H); 13C NMR (150 MHz, CDCl3) δ (ppm): 158.63, 154.30, 149.34, 145.42, 142.09, 133.98, 129.26, 121.15, 115.62, 113.33, 112.98, 103.07, 100.86, 86.87, 80.00, 76.21, 76.00, 75.79, 65.93, 52.03, 51.47, 43.25, 36.36, 35.37, 33.55, 29.76, 25.58, 25.15, 23.66, 23.32, 23.21, 20.24, 19.35, 11.98; ESI-HRMS [M + Na]+: (*m*/*z*) Calcd. for C31H39N3O7Na: 588.2686. Found: 588.2680.

*7-methyl-4-((4-(2-((10S)-dihydroartemisininoxy)ethyl)-1H-1,2,3-triazol-1-yl)-methyl)-2H-1-chromen-2-one* (**4h**) White solid; m.p. 100–102 ◦C; yield 53%; 1H NMR (600 MHz, CDCl3) δ (ppm): 7.48 (d, *J* = 8.1 Hz, 1H), 7.40 (s, 1H), 7.18 (s, 1H), 7.11 (d, *J* = 8.2 Hz, 1H), 6.00 (s, 1H), 5.68–5.62 (m, 2H), 5.29 (d, *J* = 12.6 Hz, 1H), 4.76 (d, *J* = 3.5 Hz, 1H), 4.11–4.08 (m, 1H), 3.69–3.65 (m, 1H), 3.06–2.96 (m, 2H), 2.60–2.54 (m, 1H), 2.45 (s, 3H), 2.34 (td, *J* = 14.0, 4.0 Hz, 1H), 2.05–1.99 (m, 1H), 1.88–1.83 (m, 1H), 1.66–1.63 (m, 2H), 1.60–1.58 (m, 1H), 1.56–1.53 (m, 1H), 1.41 (s, 3H), 1.27–1.17 (m, 3H), 0.93 (d, *J* = 6.1 Hz, 3H), 0.89–0.84 (m, 1H), 0.74 (d, *J* = 7.3 Hz, 3H); 13C NMR (150 MHz, CDCl3) δ (ppm): 158.98, 152.88, 146.91, 145.50, 143.18, 125.06, 122.27, 120.82, 116.69, 113.56, 113.35, 103.08, 100.86, 86.86, 79.97, 76.21, 76.00, 75.78, 65.94, 51.44, 49.21, 43.21, 36.36, 35.35, 33.54, 29.72, 25.55, 25.14, 23.65, 23.31, 20.69, 19.34, 11.86; ESI-HRMS [M + Na]+: (*m*/*z*) Calcd. for C30H37N3O7Na: 574.2529. Found: 574.2567.

*6-methyl-4-((4-(2-((10S)-dihydroartemisininoxy)ethyl)-1H-1,2,3-triazol-1-yl)-methyl)-2H-1-chromen-2-one* (**4i**) White solid; m.p. 100–102 ◦C; yield 43%; 1H NMR (600 MHz, CDCl3) δ (ppm): 7.40 (s, 1H), 7.40 (s, 1H), 7.38 (d, *J* = 1.9 Hz, 1H), 7.28 (d, *J* = 8.3 Hz, 1H), 6.00 (d, *J* = 1.2 Hz, 1H), 5.67 (s, 2H), 5.29 (s, 1H), 4.77 (d, *J* = 3.4 Hz, 1H), 4.13–4.09 (m, 1H), 3.71–3.67 (m, 1H), 3.05–2.99 (m, 2H), 2.61–2.56 (m, 1H), 2.41 (s, 3H), 2.36–2.32 (m, 1H), 2.03–2.00 (m, 1H), 1.88–1.84 (m, 1H), 1.66 (dd, *J* = 13.7, 3.5 Hz, 1H), 1.61 (d, *J* = 4.2 Hz, 1H), 1.58–1.53 (m, 2H), 1.42 (s, 3H), 1.24–1.16 (m, 3H), 0.93 (d, *J* = 6.2 Hz, 3H), 0.88–0.85 (m, 1H), 0.75 (d, *J* = 7.3 Hz, 3H); 13C NMR (150 MHz, CDCl3) δ (ppm): 158.88, 150.89, 146.89, 145.51, 133.70, 132.73, 122.29, 120.86, 116.31, 115.69, 114.22, 103.09, 100.87, 86.86, 79.98, 76.20, 75.99, 75.78, 65.94, 51.45, 49.06, 43.22, 36.37, 35.35, 33.54, 29.73, 25.57, 25.14, 23.65, 23.31, 20.03, 19.34, 11.86; ESI-HRMS [M + Na]+: (*m*/*z*) Calcd. for C30H37N3O7Na: 574.2529. Found: 574.2558.

#### 4.1.3. General Procedure for the Synthesis of Compounds **5j**,**5l**–**5o**, and **6j**–**6p**

Substituted coumarin (**j**–**p**) (10 mmol), substituted DHA (**I** or **II**) (10 mmol) and triethylamine (10 mmol) were dissolved in 20 mL CH2Cl2. In addition, CuI (100 mg) was added and the reaction mixture was stirred at room temperature for 8h under the protection of nitrogen. The mixture was filtered, washed with water, dried over Na2SO4 and evaporated to dryness. The crude product was purified through column chromatography.

*4-((4-(2-((10S)-dihydroartemisininoxy)ethyl)-1H-1,2,3-triazol-1-yl)-methoxy)-2H-1-chromen-2-one* (**5j**) White solid; m.p. 91–93 ◦C; yield 51%; 1H NMR (600 MHz, CDCl3) δ (ppm): 7.79–7.75 (m, 2H), 7.55 (ddd, *J* = 8.7, 7.3, 1.6 Hz, 1H), 7.33 (d, *J* = 8.3 Hz, 1H), 7.25–7.22 (m, 1H), 5.87 (s, 1H), 5.34 (d, *J* = 2.7 Hz, 2H), 5.16 (s, 1H), 4.77 (d, *J* = 3.6 Hz, 1H), 4.72–4.68 (m, 1H), 4.58–4.54 (m, 1H), 4.32–4.29 (m, 1H), 3.87–3.83 (m, 1H), 2.63–2.60 (m, 1H), 2.33 (td, *J* = 14.0, 4.0 Hz, 1H), 2.01–1.97 (m, 1H), 1.87–1.81 (m, 1H), 1.68–1.64 (m, 1H), 1.58–1.55 (m, 1H), 1.53–1.51 (m, 1H), 1.41 (s, 3H), 1.38 (d, *J* = 11.2 Hz, 1H), 1.26–1.18 (m, 3H), 0.91 (d, *J* = 5.9 Hz, 3H), 0.88–0.83 (m, 1H), 0.78 (d, *J* = 7.4 Hz, 3H); 13C NMR (150 MHz, CDCl3) δ (ppm): 163.90, 161.51, 152.34, 140.45, 131.58, 122.86, 122.85, 122.04, 115.80, 114.40, 103.21, 101.15, 90.15, 86.86, 79.73, 76.20, 75.99, 75.78, 65.36, 61.67, 51.32, 49.57, 42.97, 36.35, 35.25, 33.41, 29.53, 25.06, 23.58, 23.32, 19.29, 11.80; ESI-HRMS [M + Na]+: (*m*/*z*) Calcd. for C29H35N3O8Na: 576.2322. Found: 576.2336.

*5,7-dimethyl-4-((4-(2-((10S)-dihydroartemisininoxy)ethyl)-1H-1,2,3-triazol-1-yl)-methoxy)-2H-1-chromen-2-one* (**5l**) White solid; m.p. 111–113 ◦C; yield 49%; 1H NMR (600 MHz, CDCl3) δ (ppm): 7.74 (s, 1H), 6.98 (s, 1H), 6.83 (s, 1H), 5.75 (s, 1H), 5.28 (s, 2H), 5.14 (s, 1H), 4.76 (d, J = 3.6 Hz, 1H), 4.70–4.66 (m, 1H), 4.58–4.54 (m, 1H), 4.31–4.27 (m, 1H), 3.87–3.84 (m, 1H), 2.84 (s, 1H), 2.61–2.59 (m, 1H), 2.53 (s, 3H), 2.36 (s, 3H), 2.35–2.30 (m, 1H), 2.04–1.97 (m, 2H), 1.87–1.82 (m, 1H), 1.58–1.54 (m, 1H), 1.52–1.50 (m, 1H), 1.41 (s, 3H), 1.25–1.17 (m, 3H), 0.91 (d, *J* = 5.9 Hz, 3H), 0.88–0.85 (m, 1H), 0.76 (d, *J* = 7.4 Hz, 3H); 13C NMR (150 MHz, CDCl3) δ (ppm): 166.97, 161.69, 153.91, 141.76, 140.42, 135.57, 127.92, 122.68, 114.43, 110.62, 103.21, 101.15, 89.15, 86.85, 79.74, 76.21, 76.00, 75.79, 65.38, 61.48, 51.33, 49.59, 42.97, 36.36, 35.25, 33.42, 29.53, 25.05, 23.58, 23.30, 22.49, 20.35, 19.30, 11.80; ESI-HRMS [M + Na]+: (*m*/*z*) Calcd. for C31H39N3O8Na: 604.2635. Found: 604.2645.

*6,8-dimethyl-4-((4-(2-((10S)-dihydroartemisininoxy)ethyl)-1H-1,2,3-triazol-1-yl)-methoxy)-2H-1-chromen-2-one* (**5m**) White solid; m.p. 111–113 ◦C; yield 49%; 1H NMR (600 MHz, CDCl3) δ (ppm): 7.77 (s, 1H), 7.39 (s, 1H), 7.22 (s, 1H), 5.83 (s, 1H), 5.32 (d, *J* = 2.5 Hz, 2H), 5.17 (s, 1H), 4.78 (d, *J* = 3.0 Hz, 1H), 4.72–4.68 (m, 1H), 4.59–4.55 (m, 1H), 4.33–4.29 (m, 1H), 3.87–3.83 (m, 1H), 2.64–2.58 (m, 1H), 2.41 (s, 3H), 2.37–2.33 (m, 1H), 2.33 (s, 3H), 2.02–1.98 (m, 1H), 1.88–1.82 (m, 1H), 1.68–1.63 (m, 1H), 1.58–1.53 (m, 1H), 1.53–1.48 (m, 1H), 1.41 (s, 3H), 1.41–1.38 (m, 1H), 1.28–1.16 (m, 3H), 0.91 (d, *J* = 5.9 Hz, 3H), 0.88–0.84 (m, 1H), 0.78 (d, *J* = 7.4 Hz, 3H); 13C NMR (150 MHz, CDCl3) δ (ppm): 165.31, 162.94, 149.91, 141.55, 134.98, 133.03, 125.90, 123.94, 120.25, 114.82, 104.23, 102.18, 90.77, 87.87, 80.76, 77.24, 77.02, 76.81, 66.39, 62.54, 52.35, 50.58, 44.01, 37.37, 36.28, 34.44, 30.57, 26.07, 24.61, 24.35, 20.82, 20.31, 15.65, 12.84; ESI-HRMS [M + Na]+: (*m*/*z*) Calcd. for C31H39N3O8Na: 604.2635. Found: 604.2639.

*7,8-dimethyl-4-((4-(2-((10S)-dihydroartemisininoxy)ethyl)-1H-1,2,3-triazol-1-yl)-methoxy)-2H-1-chromen-2-one* (**5n**) White solid; m.p. 91–93 ◦C; yield 51%; 1H NMR (600 MHz, CDCl3) δ (ppm): 7.76 (s, 1H), 7.50 (d, *J* = 8.1 Hz, 1H), 7.03 (d, *J* = 8.1 Hz, 1H), 5.79 (s, 1H), 5.32 (d, *J* = 3.0 Hz, 2H), 5.15 (s, 1H), 4.77 (d, *J* = 3.5 Hz, 1H), 4.71–4.67 (m, 1H), 4.58–4.53 (m, 1H), 4.33–4.27 (m, 1H), 3.86–3.82 (m, 1H), 2.63–2.58 (m, 1H), 2.37 (s, 3H), 2.36 (s, 3H), 2.34–2.30 (m, 1H), 2.01–1.97 (m, 1H), 1.86–1.81 (m, 1H), 1.68–1.62 (m, 1H), 1.58–1.53 (m, 1H), 1.51 (dd, *J* = 13.4, 3.5 Hz, 1H), 1.41 (s, 3H), 1.39–1.36 (m, 1H), 1.27–1.15 (m, 3H), 0.91 (d, *J* = 5.8 Hz, 3H), 0.88–0.82 (m, 1H), 0.77 (d, *J* = 7.3 Hz, 3H); 13C NMR (150 MHz, CDCl3) δ (ppm): 165.56, 163.09, 151.58, 142.30, 141.69, 125.34, 124.53, 123.85, 119.71, 113.06, 104.24, 102.18, 89.91, 87.88, 80.78, 77.25, 77.04, 76.82, 66.39, 62.61, 52.37, 50.60, 44.02, 37.37, 36.29, 34.45, 30.58, 26.09, 24.61, 24.35, 20.46, 20.33, 12.85, 11.67; ESI-HRMS [M + Na]+: (*m*/*z*) Calcd. for C31H39N3O8Na: 604.2635. Found: 604.2646.

*8-methyl-4-((4-(2-((10S)-dihydroartemisininoxy)ethyl)-1H-1,2,3-triazol-1-yl)-methoxy)-2H-1-chromen- 2-one* (**5o**) White solid; m.p. 84–86 ◦C; yield 43%; 1H NMR (600 MHz, CDCl3) δ (ppm): 7.77 (s, 1H), 7.61 (d, *J* = 7.9 Hz, 1H), 7.39 (d, *J* = 7.4 Hz, 1H), 7.13 (t, *J* = 7.7 Hz, 1H), 5.85 (s, 1H), 5.33 (d, *J* = 2.8 Hz, 2H), 5.15 (s, 1H), 4.77 (d, *J* = 3.5 Hz, 1H), 4.71–4.67 (m, 1H), 4.58–4.54 (m, 1H), 4.33–4.27 (m, 1H), 3.86–3.82 (m, 1H), 2.62–2.59 (m, 1H), 2.45 (s, 3H), 2.33 (td, *J* = 14.1, 4.0 Hz, 1H), 2.01–1.97 (m, 1H), 1.86–1.81 (m, 1H), 1.65 (dd, *J* = 14.0, 3.6 Hz, 1H), 1.58–1.54 (m, 1H), 1.51 (dd, *J* = 13.4, 3.5 Hz, 1H), 1.41 (s, 3H), 1.40–1.35 (m, 1H), 1.27–1.17 (m, 3H), 0.90 (d, *J* = 5.8 Hz, 3H), 0.88–0.84 (m, 1H), 0.77 (d, *J* = 7.3 Hz, 3H); 13C NMR (150 MHz, CDCl3) δ (ppm): 165.30, 162.70, 151.74, 141.60, 133.84, 126.28, 123.84, 123.42, 120.66, 115.15, 104.24, 102.18, 90.88, 87.89, 80.77, 77.25, 77.03, 76.82, 66.39, 62.71, 52.36, 50.60, 44.01, 37.38, 36.28, 34.45, 30.57, 26.09, 24.61, 24.35, 20.33, 15.76, 12.84; ESI-HRMS [M + Na]+: (*m*/*z*) Calcd. for C30H37N3O8Na: 590.2478. Found: 590.2477.

*4-((4-(3-((10S)-dihydroartemisininoxy)propyl)-1H-1,2,3-triazol-1-yl)-methoxy)-2H-1-chromen-2-one* (**6j**) White solid; m.p. 74–76 ◦C; yield 47%; 1H NMR (600 MHz, CDCl3) δ (ppm): 7.79 (dd, *J* = 8.0, 1.6 Hz, 1H), 7.72 (s, 1H), 7.55 (ddd, *J* = 8.7, 7.4, 1.6 Hz, 1H), 7.32 (dd, *J* = 8.4, 1.1 Hz, 1H), 7.26–7.23 (m, 1H), 5.86 (s, 1H), 5.41 (s, 1H), 5.34 (s, 2H), 4.79 (d, *J* = 3.6 Hz, 1H), 4.56–4.46 (m, 2H), 3.93–3.89 (m, 1H), 3.44–3.41 (m, 1H), 2.69–2.63 (m, 1H), 2.39–2.34 (m, 1H), 2.27–2.20 (m, 2H), 2.05–2.01 (m, 1H), 1.91–1.86 (m, 1H), 1.79–1.75 (m, 1H), 1.67–1.64 (m, 1H), 1.51–1.46 (m, 2H), 1.42 (s, 3H), 1.39–1.33 (m, 1H), 1.28–1.22 (m, 2H), 0.96 (d, *J* = 6.4 Hz, 3H), 0.94 (d, *J* = 7.4 Hz, 3H); 13C NMR (150 MHz, CDCl3) δ (ppm): 163.96, 161.59, 152.33, 140.41, 131.55, 122.89, 122.40, 122.13, 115.78, 114.42, 103.18, 101.19, 90.16, 86.93, 79.93, 76.21, 76.00, 75.79, 63.62, 61.66, 51.47, 46.73, 43.23, 36.45, 35.33, 33.53, 29.78, 29.43, 25.12, 23.64, 23.54, 19.33, 12.07; ESI-HRMS [M + Na]+: (*m*/*z*) Calcd. for C30H37N3O8Na: 590.2478. Found: 590.2463.

*7-methyl-4-((4-(3-((10S)-dihydroartemisininoxy)propyl)-1H-1,2,3-triazol-1-yl)-methoxy)-2H-1-chromen-2-one* (**6k**) White solid; m.p. 80–82 ◦C; yield 51%; 1H NMR (600 MHz, CDCl3) δ (ppm): 7.71 (s, 1H), 7.65 (d, *J* = 8.1 Hz, 1H), 7.12 (s, 1H), 7.05 (d, *J* = 8.1 Hz, 1H), 5.80 (s, 1H), 5.41 (s, 1H), 5.32 (s, 2H), 4.79 (d, *J* = 3.6 Hz, 1H), 4.53–4.46 (m, 2H), 3.93–3.89 (m, 1H), 3.44–3.40 (m, 1H), 2.69–2.64 (m, 1H), 2.44 (s, 3H), 2.37 (td, *J* = 14.0, 4.0 Hz, 1H), 2.27–2.21 (m, 2H), 2.05–2.01 (m, 1H), 1.91–1.86 (m, 1H), 1.80–1.75 (m, 2H), 1.68–1.64 (m, 1H), 1.51–1.46 (m, 2H), 1.42 (s, 3H), 1.28–1.24 (m, 3H), 0.96 (d, *J* = 6.4 Hz, 3H), 0.94 (d, *J* = 7.3 Hz, 3H); 13C NMR (150 MHz, CDCl3) δ (ppm): δ 164.21, 161.95, 152.44, 142.81, 140.51, 124.11, 122.39, 121.82, 115.88, 111.91, 103.19, 101.18, 89.25, 86.93, 79.94, 76.20, 75.99, 75.78, 63.62, 61.55, 51.47, 46.73, 43.24, 36.44, 35.33, 33.53, 29.78, 29.42, 25.12, 23.63, 23.53, 20.72, 19.33, 12.07; ESI-HRMS [M + Na]+: (*m*/*z*) Calcd. for C31H39N3O8Na: 604.2635. Found: 604.2617.

*5,7-dimethyl-4-((4-(3-((10S)-dihydroartemisininoxy)propyl)-1H-1,2,3-triazol-1-yl)-methoxy)-2H-1-chromen-2-one* (**6l**) White solid; m.p. 86–88 ◦C; yield 42%; 1H NMR (600 MHz, CDCl3) δ (ppm): 7.68 (s, 1H), 6.98 (s, 1H), 6.83 (s, 1H), 5.74 (s, 1H), 5.40 (s, 1H), 5.28 (s, 2H), 4.78 (d, *J* = 3.7 Hz, 1H), 4.53–4.48 (m, 2H), 3.91–3.87 (m, 1H), 3.42–3.38 (m, 1H), 2.69–2.64 (m, 1H), 2.53 (s, 3H), 2.40–2.33 (m, 4H), 2.25–2.19 (m, 2H), 2.05–2.01 (m, 1H), 1.91–1.86 (m, 1H), 1.80–1.77 (m, 1H), 1.76–1.73 (m, 1H), 1.67–1.64 (m, 1H), 1.51–1.47 (m, 2H), 1.42 (s, 3H), 1.30–1.21 (m, 3H), 0.96 (d, *J* = 6.4 Hz, 3H), 0.94 (d, *J* = 7.4 Hz, 3H); 13C NMR (150 MHz, CDCl3) δ (ppm): 166.99, 161.73, 153.90, 141.72, 140.46, 135.57, 127.92, 122.26, 114.43, 110.63, 103.18, 101.15, 89.14, 86.91, 79.93, 76.21, 76.00, 75.78, 63.49, 61.58, 51.46, 46.66, 43.23, 36.46, 35.33, 33.53, 29.78, 29.42, 25.11, 23.64, 23.54, 22.46, 20.35, 19.33, 12.08; ESI-HRMS [M + Na]+: (*m*/*z*) Calcd. for C32H41N3O8Na: 618.2791. Found: 618.2827.

*6,8-dimethyl-4-((4-(3-((10S)-dihydroartemisininoxy)propyl)-1H-1,2,3-triazol-1-yl)-methoxy)-2H-1-chromen-2-one* (**6m**) White solid; m.p. 90–92 ◦C; yield 55%; 1H NMR (600 MHz, CDCl3) δ (ppm): 7.71 (s, 1H), 7.40 (s, 1H), 7.21 (s, 1H), 5.82 (s, 1H), 5.41 (s, 1H), 5.32 (s, 2H), 4.79 (d, *J* = 3.6 Hz, 1H), 4.55–4.46 (m, 2H), 3.94–3.90 (m, 1H), 3.44–3.40 (m, 1H), 2.69–2.63 (m, 1H), 2.40 (s, 3H), 2.39–2.34 (m, 1H), 2.33 (s, 3H), 2.27–2.21 (m, 2H), 2.06–2.01 (m, 1H), 1.91–1.86 (m, 1H), 1.79–1.77 (m, 1H), 1.76–1.73 (m, 1H), 1.67–1.63 (m, 1H), 1.51–1.45 (m, 2H), 1.42 (s, 3H), 1.37–1.35 (m, 1H), 1.28–1.22 (m, 2H), 0.95 (d, *J* = 6.4 Hz, 3H), 0.94 (d, *J* = 7.4 Hz, 3H); 13C NMR (150 MHz, CDCl3) δ (ppm): 165.37, 163.02, 149.90, 141.55,

134.97, 133.07, 125.88, 123.45, 120.35, 114.83, 104.21, 102.21, 90.79, 87.96, 80.97, 77.25, 77.04, 76.83, 64.68, 62.56, 52.51, 47.77, 44.27, 37.47, 36.37, 34.56, 30.82, 30.47, 26.15, 24.67, 24.57, 20.83, 20.36, 15.66, 13.11; ESI-HRMS [M + Na]+: (*m*/*z*) Calcd. for C32H41N3O8Na: 618.2791. Found: 618.2808.

*7,8-dimethyl-4-((4-(3-((10S)-dihydroartemisininoxy)propyl)-1H-1,2,3-triazol-1-yl)-methoxy)-2H-1-chromen-2-one* (**6n**) White solid; m.p. 115–117 ◦C; yield 44%; 1H NMR (600 MHz, CDCl3) δ (ppm): 7.71 (s, 1H), 7.52 (d, *J* = 8.1 Hz, 1H), 7.04 (d, *J* = 8.1 Hz, 1H), 5.80 (s, 1H), 5.40 (s, 1H), 5.32 (s, 2H), 4.79 (d, *J* = 3.5 Hz, 1H), 4.56–4.45 (m, 2H), 3.93–3.89 (m, 1H), 3.44–3.40 (m, 1H), 2.69–2.64 (m, 1H), 2.39–2.34 (m, 7H), 2.27–2.19 (m, 2H), 2.06–2.01 (m, 1H), 1.92–1.86 (m, 1H), 1.81–1.77 (m, 1H), 1.74 (dd, *J* = 13.4, 3.5 Hz, 1H), 1.67–1.63 (m, 1H), 1.52–1.45 (m, 2H), 1.42 (s, 3H), 1.39–1.34 (m, 1H), 1.27–1.24 (m, 2H), 0.96 (d, *J* = 6.4 Hz, 3H), 0.94 (d, *J* = 7.4 Hz, 3H); 13C NMR (150 MHz, CDCl3) δ (ppm): 165.61, 163.15, 151.58, 142.28, 141.66, 125.37, 124.51, 123.38, 119.78, 113.07, 104.21, 102.21, 89.92, 87.96, 80.97, 77.25, 77.04, 76.82, 64.66, 62.60, 52.51, 47.76, 44.27, 37.47, 36.37, 34.56, 30.82, 30.46, 26.15, 24.67, 24.57, 20.46, 20.37, 13.11, 11.67; ESI-HRMS [M + Na]+: (*m*/*z*) Calcd. for C32H41N3O8Na: 618.2791. Found: 618.2793.

*8-methyl-4-((4-(3-((10S)-dihydroartemisininoxy)propyl)-1H-1,2,3-triazol-1-yl)-methoxy)-2H-1-chromen-2-one* (**6o**) White solid; m.p. 143–145◦C; yield 38%; 1H NMR (600 MHz, CDCl3) δ (ppm): 7.71 (s, 1H), 7.63 (d, *J* = 8.0 Hz, 1H), 7.39 (d, *J* = 7.3 Hz, 1H), 7.14 (t, *J* = 7.7 Hz, 1H), 5.85 (s, 1H), 5.40 (s, 1H), 5.33 (s, 2H), 4.79 (d, *J* = 3.7 Hz, 1H), 4.55–4.46 (m, 2H), 3.93–3.89 (m, 1H), 3.44–3.40 (m, 1H), 2.70–2.63 (m, 1H), 2.45 (s, 3H), 2.37 (td, *J* = 14.0, 3.9 Hz, 1H), 2.28–2.19 (m, 2H), 2.05–2.01 (m, 1H), 1.92–1.86 (m, 1H), 1.81–1.72 (m, 2H), 1.67–1.64 (m, 1H), 1.53–1.46 (m, 2H), 1.42 (s, 3H), 1.39–1.33 (m, 1H), 1.30–1.22 (m, 2H), 0.96 (d, *J* = 6.4 Hz, 3H), 0.94 (d, *J* = 7.4 Hz, 3H); 13C NMR (150 MHz, CDCl3) δ (ppm): 165.35, 162.77, 151.73, 141.56, 133.82, 126.25, 123.45, 123.40, 120.73, 115.17, 104.22, 102.21, 90.89, 87.96, 80.97, 77.24, 77.03, 76.82, 64.65, 62.69, 52.51, 47.76, 44.27, 37.48, 36.37, 34.56, 30.82, 30.46, 26.15, 24.67, 24.57, 20.37, 15.76, 13.11; ESI-HRMS [M + Na]+: (*m*/*z*) Calcd. for C31H39N3O8Na: 604.2635. Found: 604.2646.

*6-methyl-4-((4-(3-((10S)-dihydroartemisininoxy)propyl)-1H-1,2,3-triazol-1-yl)-methoxy)-2H-1-chromen-2-one* (**6p**) White solid; m.p. 80–82 ◦C; yield 45%; 1H NMR (600 MHz, CDCl3) δ (ppm): 7.72 (s, 1H), 7.56 (s, 1H), 7.35 (dd, *J* = 8.5, 2.0 Hz, 1H), 7.21 (d, *J* = 8.4 Hz, 1H), 5.84 (s, 1H), 5.41 (s, 1H), 5.33 (s, 2H), 4.80 (d, *J* = 3.6 Hz, 1H), 4.55–4.47 (m, 2H), 3.94–3.90 (m, 1H), 3.45–3.41 (m, 1H), 2.70–2.64 (m, 1H), 2.40–2.33 (m, 4H), 2.27–2.21 (m, 2H), 2.06–2.01 (m, 1H), 1.91–1.87 (m, 1H), 1.80–1.73 (m, 2H), 1.65 (dd, *J* = 13.3, 3.4 Hz, 1H), 1.53–1.46 (m, 2H), 1.42 (s, 3H), 1.38–1.34 (m, 1H), 1.28–1.22 (m, 2H), 0.96 (d, *J* = 6.4 Hz, 3H), 0.94 (d, *J* = 7.3 Hz, 3H); 13C NMR (150 MHz, CDCl3) δ (ppm): 165.02, 162.88, 151.52, 141.46, 133.69, 133.59, 123.52, 122.80, 116.56, 115.08, 104.22, 102.22, 91.11, 87.97, 80.97, 77.24, 77.03, 76.82, 64.68, 62.60, 52.51, 47.80, 44.27, 37.48, 36.37, 34.57, 30.82, 30.48, 26.15, 24.67, 24.58, 20.90, 20.37, 13.12; ESI-HRMS [M + Na]+: (*m*/*z*) Calcd. for C31H39N3O8Na: 604.2635. Found: 604.2621.

#### *4.2. Biology*

#### 4.2.1. Storage and Preparation of Samples

All the target compounds were dissolved in dimethylsulfoxide and storage under −20 ◦C. They get to be diluted to different concentration when they will be used.

#### 4.2.2. Cell Culture

After recovery, the cells were cultured in 96-well plates for 24 h at 37 ◦C in a humidified atmosphere with 95% air and 5% CO2. After treated with different tested compounds, the hypoxia groups were placed in a sealed hypoxia incubator chamber (Stemcell Technologies, Inc., Vancouver, BC, Canada) filled with 5% CO2 and 95% N2 for 24 h, and then transferred to the incubator chamber filled with air for 72 h. On the contrary, the normoxia groups were cultured under air condition for 96h.

Cancer cells HT-29 (Human Colorectal Adenocarcinoma cell line), MDA-MB-231 (Human Breast Cancer cell line), HCT-116 (Human Colorectal Carcinoma cell line), and A549 (Human Lung Adenocarcinomic cell line) were from ATCC.

#### 4.2.3. MTT Assay

When the adherent cells reached 80% confluence, the culture medium was discarded. The cells were digested by trypsin and collected after centrifugation. The fresh culture medium was gently blown into it to form single-cell suspension. Cell suspension will be diluted to 1.5–3 <sup>×</sup> 104 cells/mL. Each hole of 96-well plates was added 100 μL cell suspension and incubated for 24 h (5% CO2; 37 ◦C). Then the holes were added different concentrations of tested compounds. The early screening concentration is set to 100 μM, 50 μM, 25 μM, 12.5 μM, and 6.25 μM. Three replicates were made for each concentration of the tested compounds. After the cells were grown for 96h, 20 μL MTT (5 mg/mL) was added to each well and incubated for 4 h. The medium was discarded and each well was added 100 μL DMSO to dissolve the formazan blue. Absorbance was measured at 570 nm with a microplate reader (Synergy-HT, BioTek Instruments, Winooski, VT, USA).

Hypoxia condition: The cells were treated with different concentration of tested compounds, placed in hypoxia chamber (Catalog Number 27310, Stemcell Technologies, Inc., Vancouver, BC, Canada), and incubated for 24 h (5% air; 95% N2; 37 ◦C). Then the cells were placed in normoxia condition (5% CO2; 37 ◦C) and incubated for 72 h.

#### 4.2.4. Preparation of N9 Cells

The murine microglial cell line N9 was a kind gift from Dr. P. Ricciardi-Castagnoli (Universita Degli Studi di Milano-Bicocca, Milan, Italy).

N9 cells, which were in the logarithmic phase, were incubated for 24 h by adherent culture. Subsequently, the medium was changed to new one without serum. The cells were treated with different concentration of tested compounds. After specific time in the LPS condition, the supernatants were collected and analyzed.

#### 4.2.5. Nitrite Measurement

The nitrite levels in medium were determined by Griess reaction. The absorbance was measured at 540 nm using a microplate reader.

#### 4.2.6. NO Capture Analysis

SNP was dissolved in PBS to prepare the 100 mM stock solution. In this experiment, SNP solution of 25 μL was added to 975 μL PBS solution which included different concentration of tested compounds. After 60 min under r.t. condition, the concentration of NO2 − was tested using Griess assay.

#### 4.2.7. Evaluation of Inflammatory Mediator

The secreted level of TNF-α and IL-6 was measured using mouse Th1/Th2/Th17 Cytokine Kit, purchased from BD Pharningen. The data was analyzed by FCAP Array v3.0. IL-2, IL-4, IL-10, IL-17A, and IFN-γ were not detected in the experiment.

#### 4.2.8. Molecular Docking Simulations

The crystallographic structure of TLR4/MD-2 complex was retrieved from the Protein DataBank (PDB ID: 3VQ2), which was optimized by removing water molecules, cofactors and heteroatoms. Receptor grid was generally around the MD-2 active site. Docking calculations were accomplished using AutoDock4. The docking results were further analyzed and visually optimized by Discovery Studio 4.0 and PyMOL.

**Supplementary Materials:** The H and 13 C NMR spectra of synthesized compounds are available online.

**Author Contributions:** H.Y. performed experiments, analyzed data, and drafted the manuscript. Z.H. performed experiments, analyzed data, and revised the manuscript. X.Y. performed experiments. Y.M. and C.G. conceived the work, gave critical comments, and revised the manuscript.

**Funding:** We greatly appreciate the funding support for this research provided by the National Natural Science Foundation of China (Grant No. 81573292).

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **Abbreviations**


#### **References**


**Sample Availability:** Not available.

© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

*Article*

## **A Comparative Study of the Anticancer Activity and PARP-1 Inhibiting E**ff**ect of Benzofuran–Pyrazole Sca**ff**old and Its Nano-Sized Particles in Human Breast Cancer Cells**

**Manal M. Anwar 1, Somaia S. Abd El-Karim 1, Ahlam H. Mahmoud 1, Abd El-Galil E. Amr 2,3,\* and Mohamed A. Al-Omar <sup>2</sup>**


Academic Editor: Qiao-Hong Chen Received: 26 May 2019; Accepted: 26 June 2019; Published: 29 June 2019

**Abstract:** Breast cancer is considered the most common and deadly cancer among women worldwide. Nanomedicine has become extremely attractive in the field of cancer treatment. Due to the high surface to volume ratio and other unique properties, nanomaterials can be specifically targeted to certain cells and tissues to interact with the living systems. The strategic planning of this study is based on using the nanoprecipitation method to prepare nanoparticles **BZP-NPs** (3.8–5.7 nm) of the previously prepared benzofuran–pyrazole compound (**IV**) **BZP** which showed promising cytotoxic activity. The capacity of **BZP** and **BZP-NPs** to suppress the growth of human breast tumor MCF-7 and MDA-MB-231 cells was evaluated using MTT assay. The IC50 doses of **BZP** and **BZP-NPs** targeting normal breast cells MCF-12A exceeded those targeting the cancer cells by >1000-fold, demonstrating their reasonable safety profiles in normal cells. Furthermore, cell cycle analysis, apoptosis induction detection, assessment of p53, Bcl-2, caspase-3, and PARP-1 levels of **BZP** and its nano-sized-**BZP-NPs** particles were also evaluated. Although the obtained results were in the favor of compound **IV** in its normal-sized particles, **BZP-NPs** appeared as a hit compound which showed improved cytotoxicity against the tested human breast cancer cells associated with the induction of pre-G1 apoptosis as well as cell cycle arrest at G2/M phase. The increase in caspase-3 level, upregulation of p53, and downregulation of Bcl-2 protein expression levels confirmed apoptosis. Furthermore, ELISA results exhibited that **BZP-NPs** produced a more favorable impact as a PARP-1 enzyme inhibitor than the parent **BZP**.

**Keywords:** breast cancer; benzofuran–pyrazole; nanoparticles; cytotoxic activity; apoptosis; PARP-1 inhibition

#### **1. Introduction**

Breast cancer is the most common malignancy in women, accounting for about 18% of female cancers and over half a million new cases are diagnosed worldwide each year. Its incidence increases with age and is currently rising. Although earlier diagnosis has improved the survival rates, saving lives and elevating treatment rates, metastatic breast cancer is still considered as the major factor of breast cancer-related mortality [1–4]. Despite the continuous development in the treatment of cancer disease, the strategy for cancer management has remained essentially unchanged: surgical resection of the malignant tumor followed by either chemotherapeutic administration, radiotherapy, or

a combination of the two. In fact, both of these therapies cause unselective and undesirable damage to the healthy tissues. In addition, a number of factors can lead to treatment failure, such as the remaining of some residual cells after the surgery, resistance to chemotherapies, physiological obstacles against the treatments, such as the blood–brain barrier and cellular barriers which hamper the access to drug targets, debilitating systemic toxicities, and poor pharmacokinetics of the chemotherapeutic [5–7].

Nanomedicine is defined as the use of nanotechnology for different medical purposes. Nanotechnology deals with research of materials of dimensions ranges between 1 to 100 nm (National Nanotechnology Initiative). This active protocol is applied in various science applications including cancer chemotherapeutics [8]. Deep studies have shed light on the combination of nanotechnology with cancer biology advances to gain novel techniques for cancer care. The concept that the strategy of nanomedicines is based on improving the therapeutic index of anticancer drugs by optimizing their pharmacokinetics and tissue distribution to facilitate and fasten delivery to the site of action is well known and has been investigated clinically [9–11]. Smaller (sub-100 nm) nanomedicine systems and lower molecular weight macromolecules have been shown to extravasate to a greater extent and/or penetrate farther from the vasculature than larger systems. This size effect has also been associated with improved efficacy [11,12]. Because of their sizes, the chemical properties and biodistribution of the nanomaterials are different from bulky materials. Thus, in recent years, nanomedicine has been considered as one of the most promising and important tools to defeat the problems obtained due to the administration of the traditional antitumor drugs. In addition, nanoparticle formulations can reduce or prevent systemic toxicities by specific delivery of the drugs to the cancer cells via size-mediated passive targeting and physiologically mediated active targeting. They can overcome drug resistance by the delivery of complimentary treatments and they can improve early detection of the disease using targeted delivery of molecular imaging agents to tumors in order to improve the diagnostic imaging of the tumor tissues, thus beginning the treatment before the onset of metastasis [13,14].

Inspired by the literature studies mentioned above and in continuation with our previous efforts in developing new effective agents of significant anticancer activity [15–17], this study deals with generating the previously prepared 1-(5-(3-(benzofuran-2-yl)-1-phenyl-1*H*-pyrazol-4-yl)-4,5 dihydro-3-(1*H*-pyrrol-2-yl)pyrazol-1-yl)ethanone (**IV**) **BZP** in nanoparticles **BZP-NPs** of sizes 3.8–5.7 nm (Figure 1).

**Figure 1.** The chemical structure of the benzofuran-pyrazole compound **IV** (**BZP**).

In our previous research, compound **IV** (**BZP**) was subjected, among other eight different benzofuran-pyrazole derivatives, to NCI for *in vitro* anticancer evaluation targeting full 60 human cancer cell lines using a single high dose concentration (10−<sup>5</sup> M) under the drug discovery program of the NCI [15]. The derivatives were chosen depending upon the degree of structural variations and computer modeling techniques in NCI. Fortunately, compound **IV** (**BZP)** exhibited promising cytotoxic potency against various cancer cell lines, so it was further evaluated by NCI team at five different minimal concentrations (0.01, 0.1, 1, 10, and 100 μM). It displayed cell growth inhibition of different breast cancer lines in the range of 45.95–55.44%. These data motivated the authors to convert compound **IV** (**BZP**) to nano-sized **BZP-NPs** to study the influence of the nanorange and whether nano-sized particles enhance the cytotoxic potency of the benzofuran compound.

The anticancer activity of **BZP** compound **IV** was assessed in comparison with its nano-sized **BZP-NPs** against MCF-7 and MDA-MB-231cancer cell lines. Various cellular mechanisms of action were also studied, such as apoptosis, cell cycle analysis, detection of caspase-3, p53, and Bcl-2 intensities, in addition to the efficiency of PARP-1 enzyme inhibition in the two types of the tested breast cancer cell lines

#### **2. Results and Discussion**

#### *2.1. Chemistry*

The preparation approach of the benzofuran–pyrazole derivative **IV** was outlined in Scheme 1 according to the reported method [15]. Using the Vilsmeier–Haach reaction, the key starting 1-(benzofuran-2-yl)ethanone (**I**) was converted to the intermediate pyrazole-4-carbaldehyde (**II**). The chalcone analogue **III** was obtained in a good yield by Claisen–Schmidt condensation of **II** with 2-acetylpyrrole in ethanolic sodium hydroxide solution. Cyclocondensation of **III** with hydrazine hydrate in acetic acid yielded the target compound **IV** in 85% yield (Scheme 1).

**Scheme 1.** Synthetic route of the benzofuran–pyrazole derivative (**IV**).

The nano-sized benzofuran–pyrazole **BZP-NPs** of different sizes (3.8–5.7 nm) were synthesized using the nanoprecipitation method [18]. The sizes and morphology of the nanobenzofuran–pyrazole hybrid **BZP-NPs** were examined by dynamic light scattering (DLS) and transmission electron microscopy (TEM). The results showed that nanoparticles were spherical in shape and their average size was 3.8–5.7 nm (Figure 2). The stability of the **BZP-NPs** was further investigated by X-ray diffraction (XRD) using a Pananalylical Empyrean X-ray Diffractometer and thermal analysis using a SDT Q600 V20.9 Build 20 thermal gravimetric instrument (Figures S1 and S2, Supplementary material).

Benzofuran–pyrazole **IV** (**BZP-NPs**)

**Figure 2.** Electron micrograph of the **BZP** and **BZP-NPs**. The bar marker represents 50 nm.

Surface charge and stability of the nanoparticles were analyzed using the Malvern Zetasizer nano Zs instrument (MAL1074157) and the zeta potential was −27.3 mV with a polydispersity index (PDI) of 0.77 (Figure 3).

**Figure 3.** Zeta potential distribution of **BZP-NPs**.

#### *2.2. Biological Analysis*

#### 2.2.1. In Vitro Anticancer Activity

The sensitivity of two human breast cancer cell lines, MCF-7 and MDA-MB-231, was evaluated against the benzofuran–pyrazole compound **BZP** and the target nano-sized benzofuran–pyrazole nanoparticles **BZP-NPs** using MTT assay. Doxorubicin served as a standard drug [17]. The resultant data were expressed as IC50 (nM) values which are the average of at least three independent experiments and are tabulated in Table 1. The obtained results showed that compound **IV** (**BZP**) produced significant cytotoxic activity against both breast cancer cell lines, with about 85- and 62-fold, respectively, greater potency than that of doxorubicin. Dramatic increase in the activity was observed by about 620 and 1000-fold for targeting both types of cancer cells by **BZP-NPs** compared to the reference drug doxorubicin. It could be detected that MDA-MB-231 cancer cells represented significant sensitivity against the nano-sized particles **BZP-NPs** more than their sensitivity against **BZP**.


**Table 1.** In vitro cytotoxic activity of compound **IV** (**BZP**) and **BZP-NPs**.

Despite the obvious benefits of chemotherapeutic drugs, there are several treatment-related damages to the normal cells that should be considered before finalizing the cancer treatment strategy. This study investigated the impact of compound **IV** (**BZP**) and **BZP-NPs** on normal breast cells (MRC-12A) using MTT assay [16]. Interestingly, a significant increase in the IC50 doses of **IV BZP** and **BZP-NPs** against the normal breast cells was detected when compared to their IC50 doses against both cancer cell lines (>1000-fold) (Table 1). This result confirmed the significant safety profile of the benzofuran-pyrazole compound **IV** either in the normal size particles or in its nano-sized particles **BZP-NPs**.

#### 2.2.2. Cell Cycle Analysis

Due to the antiproliferative efficacy of compound **IV** (**BZP**) and **BZP-NPs**, it was of interest to study its modes of action in both tested types of cancer cells, including cell cycle progression and apoptosis induction. Cell death occurs via different pathways, including apoptosis or type I cell-death, and autophagy or type II cell-death, which are both forms of programmed cell death, whereas necrosis is a nonphysiological process resulting from an infection or injury [19,20]. Apoptosis rate in MCF-7 and MDA-MB-231 cells was detected by flow cytometry, using propidium iodide (PI) and an annexin-V–FITC double staining assay [21]. After incubation of both types of the tested cancer cells for 24 h with BZP and BZP-NPs at their IC50 concentrations of 7 nM and 1 nM for MCF-7 cells and at 10 nM and 0.6 nM for MDA-MB-231 cells, they were labeled with the two dyes. The corresponding red (PI) and green (FITC) fluorescence was detected using flow cytometry and the results were compared to DMSO-treated cells which served as a negative control. Marked alterations in cell cycle phases have occurred. There was a great enhancement in the percentage of apoptotic cells at the pre-G phase. The tested particles (**BZP**) and (**BZP-NPs**) induced total apoptotic cells (annexin V+/PI<sup>−</sup> and annexin V+/PI+) of percentages 9.18%, 21.54%, respectively, in MCF-7 cells vs. 2.64% in the control MCF-7 cells. They also induced total apoptotic percentages of 11.09% and 23.17%, respectively, in MDA-MB-231 cells vs. 2.82% in the control MDA-MB-231cells. It is observable that the **BZP-NPs** produced a more favorable impact about two-fold more potent than **BZP** particles against both types of the tested cancer cells, especially MDA-MB-231 cells, which was consistent with the cytotoxicity assay results (Figures 4 and 5). Also, exposure of MCF-7 and MDA-MB-231 cells to **BZP** and **BZP-NPs** led to an interference with the cell cycle distribution, inducing a pronounced elevation in the percentage of cells at the G2/M phase, reaching 11.26% and 17.52%, respectively, in MCF-7 vs. 6.28% in the control cells and to 12.11% and 19.24% in MDA-MB-231 cells, respectively, vs. 4.92% in the negative control (Table 2). Accordingly, it can be concluded that the compound **IV** (**BZP)** inhibits the cancer cells' proliferation with a synchronous significant arrest at the G2/M phase. The inhibition potency was signified by the **BZP-NPs** (Figures 6 and 7). Accumulation of cells at G2/M phase is a remarkable hallmark of the apoptotic role of **BZP** and **BZP-NPs** in both tested cancer cell lines.

**Figure 4.** Representative dot plots of MCF-7 and MDA-MB-231 cells treated with compound **IV** (**BZP**) and **BZP-NPs** at their IC50 (μM) for 24 h, analyzed by flow cytometry after double staining of the cells with annexin-V FITC and PI.

**Figure 5.** Percentage of compound **IV** (**BZP**) and **BZP-NPs** in MCF-7 and MDA-MB-231 cancer cells.

(**A**): The effect of compound **IV** (**BZP**) on MCF-7 cancer cells

(**B**): The effect of compound **IV** (**BZP-NPs**) on MCF-7 cancer cells

**Figure 6.** *Cont*.

(**C**): The effect of compound **IV** (**BZP**) on MDA-MB-231cancer cells

(**D**): The effect of compound **IV** (**BZP-NPs**) on MDA-MB-231cancer cells

(**E**): The negative control of MCF-7 cancer cells

**Figure 6.** *Cont*.

(**F**): The negative control of MDA-MB-231 cancer cells

**Figure 6.** Flow cytometric analysis of compound **IV** (**BZP**) and **BZP-NPs** on MCF-7 and MDA-MB-231 cells. The orange color represents G1 phase percentage and red color represents G2/M phase percentage.

**Figure 7.** Cycle analysis of compound **IV** (**BZP**) and **BZP-NPs** in MCF-7 and MDA-MB-231 cancer cells.

**Table 2.** Determination of cell cycle inhibition of MCF-7 and MDA-MB-231 cancer cells by **BZP** and **BZP-NPs**.


#### 2.2.3. Effect Compound **IV** (**BZP**) and **BZP-NPs** on the Levels of Caspase-3/p53/Bax/Bcl-2

Caspases are cysteine protease enzymes in humans. Their presence is critical in starting the phase of programmed cell death (apoptosis). Some caspases initiate the intracellular cascade, whereas others (effector caspases) produce their activities downstream by controlling the cellular break via splitting of the structural proteins [19–21]. Caspase-3 displays an important role in the apoptotic process which includes cell shrinkage, chromatin condensation, and DNA fragmentation [22,23]. Enzyme Linked Immuno-Sorbent Assay (ELISA) was used to analyze the apoptotic events of both types of the tested breast cancer cells [16]. Table 3 shows that 24 h treatment of MCF-7 with compound **IV** (**BZP**) and **BZP-NPs** at concentrations of 7 nM and 1 nM and MDA-MB-231 cells at concentrations of 10 nM and 0.6 nM led to significant overexpression of caspase-3 compared to the doxorubicin-treated cells. **BZP** elevated the level of caspase-3 by six and five-fold compared to the untreated cells. A detectable enhancement in the level of caspase-3 occurred upon treatment of the tested cells with **BZP-NPs** by 14 and 17-fold compared to the untreated MCF-7 and MDA-MB-231 cells (Table 3).


**Table 3.** Determination of caspase-3, p53, Bax, and Bcl-2 levels in the tested cancer cells.

The tumor suppressor protein p53 serves as a transcription factor. It induces the expression of several downstream targets which are very important in regulation of the cell cycle, apoptosis, and DNA repair, among other mechanisms [24,25]. Cellular stress leads to the activation of the p53 pathway, compromising tumor development and preventing the proliferation of the damaged cells of oncogenic potential. p53 is considered as one of the most relevant tumor suppressor genes [26]. In addition, the B-cell lymphoma protein 2 (Bcl-2) plays a key role in tumor progression via inhibition of the intrinsic apoptotic pathway triggered by mitochondrial dysfunction. Cancer cells can resist apoptosis by modulating the expression of Bcl-2 family proteins which in turn regulate the mitochondrial apoptotic pathway via production Bcl-2 or downregulating pro-apoptotic proteins, such as Bax [26]. Accordingly, in this study, the impact **of IV** (**BZP**) and **BZP-NPs** was assessed on the intrinsic apoptotic pathway via measuring the levels of p53, Bax, and Bcl-2 after treatment of MCF-7 and MDA-MB-231 cells with **BZP** and **BZP-NPs** at their IC50 concentrations for 24 h. In comparison to the untreated control, p53 level increased seven-fold in both types of the tested cancer cells upon **BZP** treatment, while the level was doubled to 14-fold by the **BZP-NPs**. On the other hand, **BZP** and **BZP-NPs** elevated the level of the proapoptotic protein Bax by 5.7–13.1-fold with concurrent reduction in the expression levels of the antiapoptotic protein Bcl-2 by four to seven-fold in both tested cancer cell lines in comparison with the untreated control (Table 3).

#### 2.2.4. PARP-1 Cleavage Assay

Breast cancer is among the targets of a new class of drugs known as Poly (ADP-ribose) polymerase-1 (PARP-1) inhibitors. PARP-1 is a nuclear enzyme plays a role in the repair of single-stranded DNA (ss DNA) breaks [26]. The rationale for the therapeutic benefit for the pharmacological inhibition of PARP-1 in breast cancer comes from the following points: (a) sensitization of tumor cells to anticancer therapies such as radiation and cytotoxic agents [27]; (b) certain polymorphisms in PARP-1 can lead to breast cancer and negatively affect the efficacy of the hormone therapies; and (c) breast tumors with deficiencies in DNA-repair genes such as BRCA-1 or BRCA-2 represent acute sensitivity in

response to the inhibition of PARP-1. Interestingly, clinical evidence investigates that the usage of PARP-1-inhibiting candidates are not limited to BRCA-1 or BRCA-2 mutated cancers, but they also target non-BRCA mutated breast and ovarian cancers and produce a valuable impact in combination therapy [28,29]. Thus, it was of interest to study the inhibitory effects of **BZP** and **BZP-NPs** against PARP-1 enzyme in MCF-7 and MDA-MB-231 cancer cells using staurosporine as a standard drug. The resulting data were expressed as IC50 (nM) values and are summarized in (Table 4). It should be noted that **BZP** produced a slightly weaker inhibitory effect against PARP-than the reference drug, with an IC50 of 40 nM vs. 10 nM for staurosporine in MCF-7 cells. A dramatic decrease, about 13-fold, in the sensitivity was noticed in the case of MDA-MB-231 cancer cells against **BZP** which produced an IC50 of 60 nM vs. 8 nM for staurosporine. On the other hand, an interesting increase in the inhibitory potency was observed for the **BZP-NPs**, exhibiting an IC50 value of 10 nM, which is equal to that obtained by the standard drug in MCF-7 cells. The suppression activity was intensified against PARP-1 in MDA-MB-231 cells, producing an IC50 of 6 nM vs. 8 nM for staurosporine.



#### **3. Experimental**

*3.1. Synthesis of 1-(5-(3-(Benzofuran-2-yl)-1-phenyl-1H-pyrazol-4-yl)-4,5-dihydro-3-(1H-pyrrol-2-yl) pyrazol-1-yl)ethanone (***IV***).*

Compound **IV** was synthesized according to the previously reported procedure [15].

#### *3.2. Preparation of Nanobenzofuran–Pyrazole* **BZP-NPs**

The nanoparticles were prepared by the nanoprecipitation method [18]. The sizes and morphology of the nanoparticles **BZP-NPs** were examined by transmission electron microscopy (TEM) (H-7600; Hitachi Ltd., Tokyo, Japan). The results exhibited that the nanoparticles were spherical in shape and their average size was 3.8–5.7 nm (Figure 2).

#### *3.3. Physicochemical Characterization of the Nanobenzofuran–Pyrazole Compound* **BZP-NPs**

#### 3.3.1. Particle Size and Zeta Potential Using Photon Correlation Spectroscopy

Particle size was measured by dynamic light scattering (DLS) using a Zetasizer NANO-ZS (Ver. 7.04, Serial Number: MAL 1074157, Malvern Instruments Ltd., London, United Kingdom) at a wavelength of 633 nm with a 4.0 mW light source for collecting data at a fixed scattering angle of 173◦. The electrophoretic mobility (zeta potential) measurements were made using a Zetasizer NANO-ZS (Ver. 7.04, Serial Number: MAL 1074157, Malvern Instruments Ltd., United Kingdom) at 25 ◦C. **BZP-NPs** morphology was determined using JEOL Transmission Electron Microscope (JEM-1230, Tokyo, Japan) with 500,000 × magnification power, 100 kV acceleration voltage, and 0.5 nm resolving power.

#### 3.3.2. In Vitro Anticancer Activity

In vitro evaluation of the anticancer activity of the **BZP** compound and **BZP-NPs** targeting MCF-7 and MDA-MB-231 cells was performed using MTT assay according to a previously reported method [19]. Each experiment was performed at least three times.

#### *3.4. Cell Cycle Analysis and Apoptosis Detection*

Cell cycle analysis and apoptosis detection was carried out by flow cytometry (Beckman Coulter, Brea, CA, USA) [19–21]. Apoptosis detection was performed using a FITC Annexin-V/PI commercial kit (Becton Dickenson, Franklin Lakes, NJ, USA) following the manufacturer's protocol.

#### *3.5. Caspases-3 Assays*

Caspase-3 activity was measured using a Caspase-3 (Active) (human) ELISA kit, Catalog # KHO1091 (96 tests) (Invitrogen Corporation, Carlsbad, CA, USA) according to the manufacturer's instructions [16].

#### *3.6. In Vitro Determination of p53, Bax, and Bcl-2 Levels*

The levels of p53, Bax, and Bcl-2 markers were assessed using a BIORAD iScript TM One-Step RT-PCR kit with SYBR® Green according to the manufacturer's instructions [26,30].

#### *3.7. In Vitro PARP-1 Assay*

The procedure was done according to the supplied protocol of ab119690 Cleaved PARP Human ELISA (Enzyme-Linked Immunosorbent Assay) kit for the quantitative measurement of the 89 kDa fragment of Human PARP-1 in cell and tissue lysates [31].

#### **4. Conclusions**

This study demonstrates that the conversion of the benzofuran-pyrazole compound **IV** (**BZP**) to nanoparticles **BZP-NPs** greatly intensified its cytotoxic activity against two breast cancer cell lines, MCF-7 and MDA-MB-231, with respective IC50 values of 7 nM, 10 nM vs. nanoparticle IC50 values of 1 nM, 0.6 nM. The IC50 value of DOX was 620 nM. Furthermore, the IC50 doses of BZP and BZP-NPs against normal breast cells were >1000-fold greater than those against cancer cells, suggesting acceptable safety profiles in normal cells. The resultant data of cell cycle and apoptosis determination revealed that the tested derivative in both forms induced G2/M phase arrest, accompanied by an increase in apoptosis in the tested cancer cells. Further modes of action of the target compound were also predicted in both types of breast cancer cells. The biological results revealed that **BZP** significantly increased p53, caspase-3, and Bax levels and decreased Bcl-2 levels, and their levels were intensified upon treating the tested cancer cells with (**BZP-NPs**). The PARP1 enzyme assay showed that the efficiency of PARP-1 inhibition by (**BZP**) was slightly less than that of staurosporine, while **BZP-NPs** inhibited the enzyme efficiently as staurosporine in MCF-7 or to a greater extent in case of MDA-MB-231 cancer cells. It has been detected that the nanoparticles were more effective as an anticancer agent against MDA-MB-231 cells than against MCF-7 cancer cells.

**Supplementary Materials:** The following are available online, Figure S1: Diffraction (XRD) of BZP-NPs, Figure S2: Analysis of **BZP-NPs**.

**Author Contributions:** S.S.A.E.-K., M.M.A. performed most of the experiments; A.E.-G.E.A., M.A.A.-O., analyzed the data; A.H.M. the contributed to the anticancer activity assays; All authors read and approved the final manuscript.

**Funding:** This work was supported financially by National Research Centre, Dokki, Cairo, Egypt, under the project No. 11010317, entitled "Development of Novel Poly (ADP-ribose) Polymerase-1 Inhibitors as Anticancer and Chemo-sensitizers Targeting Breast Cancer Disease". Also, the authors extend their appreciation and thanking to Essam Rashwan, the head of confirmatory diagnostic unit, Vacsera-Egypt, for helping in performing the pharmacological screening.

**Acknowledgments:** The authors are grateful to the Deanship of Scientific Research, king Saud University for funding through Vice Deanship of Scientific Research Chairs.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


**Sample Availability:** Samples of the compounds are available from the authors.

© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

### *Article* **Synthesis of New Derivatives of Benzofuran as Potential Anticancer Agents**

### **Mariola Napiórkowska 1,\*, Marcin Cie´slak 2,\*, Julia Ka ´zmierczak-Bara ´nska 2, Karolina Królewska-Goli ´nska <sup>2</sup> and Barbara Nawrot <sup>2</sup>**


Academic Editor: Qiao-Hong Chen Received: 28 March 2019; Accepted: 16 April 2019; Published: 18 April 2019

**Abstract:** The results of our previous research indicated that some derivatives of benzofurans, particularly halogeno-derivatives, are selectively toxic towards human leukemia cells. Continuing our work with this group of compounds we here report new data on the synthesis as well as regarding the physico-chemical and biological characterization of fourteen new derivatives of benzofurans, including six brominated compounds. The structures of all new compounds were established by spectroscopic methods (1H- and, 13C-NMR, ESI MS), and elemental analyses. Their cytotoxicity was evaluated against K562 (leukemia), MOLT-4 (leukemia), HeLa (cervix carcinoma), and normal cells (HUVEC). Five compounds (**1c**, **1e**, **2d**, **3a**, **3d**) showed significant cytotoxic activity against all tested cell lines and selectivity for cancer cell lines. The SAR analysis (structure-activity relationship analysis) indicated that the presence of bromine introduced to a methyl or acetyl group that was attached to the benzofuran system increased their cytotoxicity both in normal and cancer cells.

**Keywords:** benzofurans; chemical synthesis; cytotoxic properties; HeLa; MOLT-4; K562

#### **1. Introduction**

Benzofuran skeleton holds an important position in organic chemistry and it is considered to be one of the most important heterocyclic systems because of its diverse profile of biological activity. This structural unit is a central part of a variety of biologically active compounds. Natural and synthetic benzofuran derivatives have been reported to possess wide therapeutic properties, including antiviral, immunosuppressive, antioxidant, antifungal, anti-inflammatory, antimicrobial, analgesic, antihyperglycemic, and antitumor activities [1–6]. *Cicerfuran, Conocarpan*, and *Ailanthoidol* are the best known biologically active natural benzofurans (Figure 1). Specifically, the *Cicerfuran* shows antifungal activity, *Conocarpan* has been reported as an antifungal and antitrypanosomal agent, and *Ailanthoidol* exhibits anticancer, antiviral, immunosuppressive, antioxidant, and antifungal activity [1,2,7]. The synthetic benzofuran derivatives are represented by *Amiodarone* (Figure 1), being used in the treatment of ventricular and supraventricular arrhythmias, and by *Bufuralol*, which is a non-specific β-adrenergic blocker with an affinity for β1 and β2-adrenergic receptors [3,4,7].

Nowadays, when cancer, after cardiovascular diseases, is the second most common cause of death and still constitutes an unresolved problem of clinical medicine and pharmacology, extensive research regarding new anticancer compounds is especially important. These new drugs should possess improved pharmacokinetics and specifically destroy cancer cells, without causing negative side effects. Research in the group of benzofuran derivatives is justified, especially by the fact that one can find many examples of data in the literature on benzofurans with anticancer activity. In many cases, the benzofuran skeleton is fused with other heterocyclic or aromatic moieties (Figure 2).

**Figure 1.** Structures of natural and synthetic benzofuran derivatives with biological activity.

**Figure 2.** Structures of synthetic benofuran derivatives **I**–**V** with anticancer activity [7].

There are several benzofuranyl imidazole derivatives among them (**I** and **II**), which were found to be cytotoxic towards an ovarian carcinoma cell line (Skov-3). The study of *N*-(5-(2-bromobenzyl) thiazole-2-yl) benzofuran-2-carboxamide (**III**) showed that this compound inhibited the growth of HCC (human hepatocellular carcinoma) cells and induced their apoptosis. The synthetic derivative **IV** was found to have antitumor activity and it was an effective chemopreventive and chemotherapeutic agent against malignant T cells. Moreover, a series of triazole derivatives **V** showed moderate antitumor activity. Recent data reported this type of benzofuran derivatives as potential therapeutic agents for breast cancer [7].

These are only a few examples from a large group of benzofurans with anticancer activity. Moreover, examples of compounds with cytotoxic activity were found among the simple derivatives of benzofuran like 2- and 3-benzofuranocarboxylic acid derivatives **VI**–**VII** (Figure 3). The above-mentioned compounds exhibit significant cytotoxic activity against human cancer cell lines [8].

**Figure 3.** Structures of 2- and 3-benzofurancarboxylic acid derivatives **VI–VII** with cytotoxic activity.

The literature survey shows that benzofurans containing halogens in their structure constitute an important group of compounds, with antitumor, cytotoxic, spasmolytic, antiarrhythmic, and antifungal activity [9–20]. Thus, this group of compounds is interesting for researchers, especially because the presence of halogen can increase the activity and selectivity of derivatives. This is probably related to the ability of halogens to create a "halogen bond", which results from the formation of the σ-hole. Although the halogen bonds are weaker than hydrogen bonds, they have specific effects and can lead to significant gains in binding affinity. These interactions can be found in protein-receptor complexes as well as in small molecules [21–24].

Furthermore, we have identified three bromo derivatives **VIII**–**X** (Figure 4) that showed selective toxicity towards human leukemia cells in our previous studies [25,26]. Compound **VIII** is especially cytotoxic towards K562 and HL-60 leukemic cell lines (IC50 5.0 and 0.1 μM, respectively), however it is not toxic towards HeLa cancer cells and healthy endothelial cells (HUVEC) (IC50 > 1 mM). Moreover, the observed remarkable cytotoxicity of **VIII** towards K562 cells resulted from cells apoptosis. Compounds **IX** and **X** proved to be highly toxic towards cancer cells (IC50 in a few μM range) and non-toxic towards endothelial cells (HUVEC) [25–27]. Unfortunately, these compounds are poorly soluble in water, which limits their use in the cell culture or animal studies.

**Figure 4.** Structures of the benzofuran derivatives **VIII**–**X**, lead compounds used in the present studies.

Using **VIII**–**X** as the lead compounds, we designed and synthesized fourteen new derivatives with hopes for their better solubility in aqueous solutions (of lower lipophilicity when compared to **VIII**–**X**). The biological activity (i.e., cytotoxicity, activation of apoptosis, interaction with DNA) of these new derivatives was also evaluated.

#### **2. Results**

#### *2.1. Synthesis*

Our goal was to obtain a small library of new, less lipophilic derivatives/analogs of lead compounds **VIII**–**X**. We designed the synthesis of a set of compounds containing a carboxyl (**1**), formamide (**1a**), and methoxycarbonyl groups (**1b**), instead of an acetyl group in the position 2 of the parent benzofuran ring to obtain new benzofuran **VIII** analogs.

Thus, the starting acid **1**, which was obtained by the multistep synthesis according to the previously reported procedures [28] was submitted either to oxalyl chloride and ammonium solution treatment or methylated with dimethyl sulphate, delivering the amide derivative **1a** and methyl ester **1b**, respectively (Scheme 1). In the next step, compounds **1** and **1b** were submitted to bromination. For this purpose, ester **1b** was reacted with molecular bromine in chloroform. Under these conditions, hydrogen in the methyl group at position 3 was substituted by a bromine atom to give compound **1c**, which only differed by the substituent in position 2 (methoxycarbonyl versus acetyl). During bromination of the acid **1** using bromine in chloroform or NBS in CCl4, a mixture of products was obtained, which was difficult to separate. Thus, the reaction conditions were changed and ethanol, instead of CCl4, was used as a solvent in bromination reaction that was carried out in the presence of the NBS, while acetic acid was used as a solvent in the respective reaction that was carried out in the presence of bromine. Under these conditions, we managed to isolate the bromo-derivative **1d**, with satisfactory yield. Moreover, bromo-derivative **1e** was also obtained, but only in the reaction that was facilitated by NBS. The analyses of nuclear magnetic resonance spectra (1H- and 13C-NMR), mass spectra, and elemental analysis showed that the structures of the received compounds were different from the assumed ones (bromination of the methyl group at position 3). Instead, the derivatives in which the carboxyl group was replaced by the bromine atom at the position 2 were isolated. Moreover, we confirmed the formation of **1e**, in which one bromine atom substituted the hydrogen atom in the acetyl group of the benzene moiety of benzofuran ring. The use of polar protic solvents (acetic acid, ethanol) could explain this substitution.

**Scheme 1.** Synthesis of analogues of compound **VIII**.

Analysis of the calculated log*P* values (Table 1) has shown that carboxyl and formamide analogs of lead compound **VIII** (**1** and **1a**, respectively) are much less lipophilic, while methoxycarbonyl analogs **1b** and **1c** exhibit similar properties as **VIII**. In contrast, an introduction of the bromine atom at position 2 of the furane ring resulted in significant increase of the benzofuran system hydrophobicity.


**Table 1.** Theoretical log*P* values (clog*P*) for obtained compounds calculated by the OSIRIS Property Explorer program.

Schemes 2 and 3 show the syntheses of analogs of compounds **IX** and **X**, respectively. The starting material in both cases was 6-acetyl-5-hydroxy-2-methylbenzofuran-3-carboxylic acid (**2**), which was subjected to multidirectional transformations (Scheme 2). In the first approach, the amide-derivative **2a** was obtained in the reaction of the acid **2** with oxalyl chloride and ammonium hydroxide. Next, the bromo-derivative **2b** was obtained by the bromination of **2a** with bromine in acetic acid as a solvent. The nuclear magnetic resonance (1H- and 13C-NMR), mass spectrometry and elemental analyses confirmed the substitution of hydrogen by bromine in aromatic ring in the position 4. We assume that the presence of the OH group in position 5 of the benzene ring assisted in the electrophilic substitution of bromide cation in its ortho position. In the second path, the acid **2** was brominated in the same conditions and the bromo-derivative **2e**, also with a bromine substitution to the benzene ring, was obtained. In the third approach, an ester-derivative **2c**, which was obtained in the reaction of the acid **2** with dimethyl sulphate, was brominated by using NBS in CCl4 to give the derivative **2d**, with a bromomethyl group in the position 2. Interestingly, all of the obtained derivatives **2** (**2a**–**2e**) exhibited lower clog*P* values, confirming the better water solubility of derivatives compared to lead compound **IX**, and the most pronounced occurred primary carboxamides **2a** and **2b**.

To obtain analogs of compound **X**, the starting acid **2** was reacted with an excess of dimethyl sulphate and the obtained derivative **3** was subjected to a multidirectional synthesis (Scheme 3). In the first case, bromine was introduced into the methyl group to give a compound **3d**, by reaction with NBS in CCl4. In the reaction of the compound **3** with a bromine in acetic acid, the lead compound **X** was obtained and then finally reduced to provide a hydroxyl-derivative **3a**. In the third path, ester **3** was hydrolyzed in alkaline conditions to acid **3b**, and finally this derivative was converted to an amide **3c** by reaction with oxalyl chloride and ammonium hydroxide. Importantly, all of the new benzofuran derivatives related to **X** are characterized by lower clog*P* values when compared to the lead compound, indicating their improved solubility in aqueous media (Table 1).

The introduced substituents significantly affected the lipophilicity of the obtained benzofurans. In most cases, the new derivatives had a lower clog*P* value in comparison with the lead compounds. The exception are three derivatives **1c**, **1d**, **1e**, where the substitution of bromine in the furan ring (compounds **1d** and **1e**) or in the methyl group caused the clog*P* to increase (Table 2).

**Scheme 2.** Synthesis of analogues of compound **IX**.

**Scheme 3.** Synthesis of analogues of compound **X**.

#### *2.2. MTT Cytotoxicity Studies*

Fourteen new benzofuran derivatives were tested for their cytotoxic properties in K562, MOLT-4 (leukemia, suspension cells), HeLa (cervix carcinoma, adherent cells), and normal endothelial cells (HUVEC). First, we measured the viability of cells after 48 h incubation with the given compound at the concentration of 100 μM. Next, for compounds that reduced cancer cells survival for more than 50%, we determined IC50 values. Cells that were exposed to 1% DMSO (a vehicle) served as the control with 100% survival. Cells treated with 1 μM staurosporine served as the internal control of the cytotoxicity experiments.

We have identified five compounds **1c**, **1e**, **2d**, **3a**, and **3d** in the initial screening, which, at the concentration of 100 μM, reduced the viability of all tested cancer cells K562, HeLa and MOLT-4 for more than 50% (data not shown). These compounds were also cytotoxic against human normal endothelial cells, so these compounds did not show any selectivity between the cancer and normal cells.

Next, for compounds **1c**, **1e**, **2d**, **3a**, and **3d**, the IC50 values were calculated (data given in Table 2). The test compounds were similarly toxic toward both cancer and normal cells, with IC50 values in the range of 20–85 μM. The exceptions were compounds **1c** and **3d**, which show IC50 out of this range (180 μM for MOLT-4 and 6 μM for HUVEC cells, respectively). We did not observe any significant differences in susceptibility between adherent and suspension cell lines.


**Table 2.** The IC50 values [μM] after 48 h incubation with cells.

For compounds **1c**, **1e**, **2d**, and **3d**, that exhibited the highest toxicity for K562 leukemia cells (IC50 below 50 μM), we investigated whether they induce apoptosis in these cells. We have measured the activity of caspases 3 and 7 (caspase 3/7), which are markers of programmed cell death. The K562 cells were treated with 1% DMSO (negative control), 1 μM staurosporine (positive control), or a test compound at the concentration of 5 × IC50 for 18 h. The activity of caspase 3/7 was measured using pro-fluorescent peptide substrate.

As shown in Figure 5, staurosporine, which is a strong inducer of apoptosis significantly increased the activity of caspase 3/7 in K562 cells. On the other hand, 1% DMSO had no effect on the activation of caspases. Interestingly, the incubation of cells with compound **1e** resulted in nearly five-fold increase in the activity of caspase 3/7, while compounds **1c** and **2d** activated caspase 3/7 to a lesser extent (about two-fold increase). In the presence of compound **3d**, the activation of caspases was minimal, if any. Altogether, this result suggests that the cytotoxic activity of test benzofurans **1c**, **1e**, and **2d** in K562 cells may be due to the induction of death by apoptosis.

**Figure 5.** Activity of caspase 3 and 7 in K562 cells treated with the test benzofurans **1c**, **1e**, **2d**, **3d**, or staurosporine for 18 h. Apoptosis was determined by Apo-ONE® Homogeneous Caspase-3/7 Assay (Promega, Madison, WI, USA). Abbreviations: DMSO—K562 cells treated with 1% DMSO. The caspase activation level in cells exposed to 1% DMSO was normalized to 1.0. Mean values +/− SD are shown.

#### *2.3. Interaction with DNA*

The results of MTT cytotoxicity experiments indicated that compounds **1c**, **1e**, **2d**, and **3d** were highly toxic towards the used cell lines. We hypothesized that a possible explanation of observed cytotoxicity might be due to an interaction of test benzofurans with genomic DNA (e.g., by intercalation). To verify this hypothesis, we investigated whether the test benzofurans have any effect on digestion of a plasmid DNA (pcDNA3.1 HisC) with endonuclease *BamH1*. pcDNA3.1 HisC contains a unique *BamH1* restriction site which allows for plasmid linearization. Plasmid DNA exists in linear, superhelical, and circular forms that differ in electrophoretic mobility (Figure 6 lane 1). Plasmid DNA was converted to a linear form upon digestion with *BamH1* (lane 2). Daunorubicin, which is a strong intercalator to double-stranded DNA, was used as a control in this experiment and it completely inhibited the digestion of plasmid DNA with *BamH1* (lane 3). In the presence of test compounds, pcDNA3.1 HisC was partially digested with *Bam H1* restriction enzyme (Figure 6 lanes 4–7). Most of the plasmid DNA was converted to a linear form, however there is a circular form still present. These results suggest that test benzofurans, to some extent, interact with DNA (especially compounds **1c**, **1e**, **2d**), and this interaction inhibits the digestion of double stranded DNA chain with restriction endonuclease.

**Figure 6.** Digestion of pcDNA3.1HisC (total length 5.5kbp) with *BamH1* endonuclease. M—marker DNA; 1—not digested plasmid DNA; 2—plasmid DNA digested with *BamH1* (DNA present in linear form); 3—plasmid DNA + daunorubicin + *BamH1*; 4—plasmid DNA + **1c** + *BamH1*; 5—plasmid DNA + **2d** + *BamH1*; 6—plasmid DNA + **1e** + *BamH1*; and, 7—plasmid DNA + **3d** + *BamH1.*

#### **3. Discussion**

We have previously identified benzofurans **VIII**, **IX**, and **X** (lead benzofurans), which efficiently killed cancer cells and were not toxic toward normal endothelial cells [25–27]. Moreover, lead compound **VIII** demonstrated selective toxicity toward leukemia cell lines. However, these compounds were poorly soluble in aqueous solutions. Based on their structure, we have synthesized 14 new derivatives with decreased lipophilicity. The polarity of new compounds was predicted based on the calculated log*P* values. We tested their cytotoxic properties in human cells of cancer and normal origin. Five compounds, **1c**, **1e**, **2d**, **3a**, and **3d**, displayed the highest cytotoxicity toward cancer cell lines. However, these compounds were less toxic than lead compounds **VIII**, **IX**, **X**, and did not demonstrate any selectivity toward leukemia cells (Table 2). Moreover, new derivatives exhibited significant toxicity in normal endothelial cells. Cells death is usually carried out in one of the two major mechanisms: apoptosis or necrosis. Apoptosis is a highly regulated and controlled process that does not elicit an inflammation response at the site of cell death. Necrosis leads to sudden and uncontrolled cell disintegration that is associated with release of the cellular content and massive inflammation [29]. Therefore, in the next experiments, we investigated whether the cellular toxicity of new benzofurans is the result of apoptosis or necrosis. Our data demonstrate that the activity of caspase 3/7 (an apoptosis marker) is significantly increased (1.5- to 5-fold) in the presence of benzofurans (Figure 5). It suggests that these derivatives induce apoptosis in cancer cells. In the search of cellular targets for testing benzofurans, we examined whether DNA may be such a target. Using biochemical assay, we found out that the incubation of test compounds with plasmid DNA inhibited its cleavage with selected endonuclease (*BamH1*). A similar result was obtained with daunorubicin, which is a strong DNA intercalating agent. The presence of undigested plasmid DNA suggests that benzofurans intercalate to DNA (or bind DNA in other way). However, a comparison of DNA digestion products clearly indicates that the binding of benzofurans to DNA is much weaker than daunorubicin (Figure 6).

The presence of a bromine substituent in the alkyl chain attached to the furan ring is most likely to be responsible for cytotoxic activity of compounds **1c**, **2d**, and **3d**. The activity of compound **1e** is probably related to the presence of a bromoacetyl substituent in a benzene ring. Whilst the presence of a halogen (bromine) directly substituted to the benzene ring or the furan skeleton does not seem to increase the cytotoxic activity of the tested compounds, for example, **2e**, **2b**, and **1d**. The amide derivatives of benzofurans (compounds **2a**, **3c**, **1a**) that lack halogen-containing alkyl substituents (a bromine atom) did not show the cytotoxic properties toward cancer cell lines. A similar effect is observed for derivatives with a free acidic group (compounds **1**, **3b**) and ester derivatives (**1b**).

We can observe a marked decrease in the activity and selectivity of these derivatives when comparing the activity of bromo-derivatives **1c** and **1e** with the activity of the lead compound **VIII**. This effect is probably due to the absence of the acetyl group at the 2-position of the furan ring. The derivative **1c** has an ester group and the compound **1e** a bromine atom in this position. Thus, it can be concluded that the arrangement of substituents: the acetyl group at the 2-position and the bromomethyl at the three-position determines the activity and selectivity of the lead compound.

Analysis of the results for the active **2d** derivative in comparison to its lead compound **IX** also indicates that the structural modifications of **2d** resulted in a loss of selectivity and decreased activity. In this case, the derivatives differ in the location of the halogen atom. The **2d** derivative contains the bromomethyl substituent in the two-position and the acetyl group in the six-position of the benzofuran system, while the **IX** contains the methyl group in the two-position and the bromoacetyl substituent in the six-position of the benzofuran system. It can again be assumed that the presence of a halogen atom substituted to an alkyl/acetyl moiety determines the activity of the derivatives, but the appropriate positioning of substituents is important in their selectivity. Finally, by comparing the active derivatives **3a** and **3d** with their lead compound **X**, we also observe a decrease in activity and selectivity. The **3a** compound differs from the leading compound by the presence of a hydroxyl group. It can be hypothesized that the reduction of the keto group and the possibility of creating additional hydrogen bonds, as well as an increase in the hydrophilicity could affect the activity of this derivative.

Compound **3d** contains a bromomethyl substituent at the two-position and an acetyl group at the six-position, while **X** contains a bromoacetyl group at the six-position and a methyl at the two-position. Both of the compounds exhibit cytotoxicity, but the absence of the bromoacetyl substituent in compound **3d** eliminated its selectivity and decreased cytotoxicity to the cancer cells.

#### **4. Materials and Methods**

#### *4.1. Chemistry*

All of the solvents, reagents, and chemicals used in these studies were purchased from Aldrich Chemical (Saint Louis, MO, USA) and Merck AG (Saint Louis, MO, USA). The melting points were determined with Electrothermal 9100 capillary apparatus and they are uncorrected. The nuclear magnetic resonance spectra (University of Warsaw, Warsaw, Poland) were recorded in DMSO-*d*<sup>6</sup> or CDCl3 on VMNRS300 operating at 300 MHz (1H-NMR) and 75 MHz (13C-NMR). Chemical shifts (δ) are expressed in parts per million relative to tetramethylsilane used as the internal reference. The coupling constants (*J*) values are given in hertz (Hz) and spin multiples are given as s (singlet), d (dublet), t (triplet), and m (multiplet). Mass spectral ESI (Electrospray Ionization) measurements

were carried out on a MicrOTOF II, Bruker instrument with a TOF detector (Jagiellonian Univeristy in Krakov, Poland). The spectra were obtained in the positive ion mode. Elemental analyses were recorded with CHNS micro analyzer elementary model Vario Micro Cube with electronic microbalance (Jagiellonian Univeristy in Krakow, Poland). Flash chromatography was performed on Merck Kieselgel 0.05–0.2 mm reinst (70–325 mesh ASTM, Saint Louis, MO, USA) silica gel using chloroform as eluent. TLC monitored progress of the reactions described in the experimental section on silica gel (plates with fluorescent indicator 254 nm, layer thickness 0.2 mm, Kieselgel G. Merck, Saint Louis, MO, USA), using chloroform-methanol as an eluent system at the *v*/*v* ratio of 9.8:0.2 or 9.5:0.5.

#### *4.2. General Synthetic Procedures*

Procedure 1. Procedure for Synthesis of Amides.

An appropriate carboxylic acid (0.004 mol) was suspended in anhydrous dichlorometane (DCM) (10 mL). Next, oxalyl chloride (0.43 mL, 0.005 mol) and the one drop of dimethylformamide (DMF) were added to the solution. The reaction mixture was stirred at room temperature for 24 h. Then, ammonium solution (aq. 30%, 5 mL) was added drop by drop and the mixture was stirred at room temperature for additional 12 h. When the reaction was complete (TLC control) the resulting mixture was diluted with water (50 mL) and extracted with DCM (3 × 50 mL). The organic extracts were dried with magnesium sulfate and concentrated under reduced pressure. The resulting solid was purified by a silica gel column chromatography (eluent: chloroform or chloroform:methanol; 50:0.2, *v*/*v*).

Procedure 2. Procedure for the Preparation of Methyl ester.

Procedure according to the method described earlier [11]. Thus, a mixture of appropriate carboxylic acid (0.02 mol), K2CO3 (0.1 mol) and (CH3O)2SO2 (0.02 mol) in acetone was refluxed for 48 h. When the reaction was complete, the mixture was filtered and the solvent was removed on rotary evaporator [11]. The residue was purified by a silica gel column chromatography (eluent: chloroform or chloroform:methanol; 50:0.2 *v*/*v*).

Procedure 3. Procedure for Bromination by Using N-Bromosuccinimide (NBS).

In this method, the procedure was used, as described earlier [11]. Briefly, *N*-bromosuccinimide (NBS) (0.02 mol) and the catalytic amount of benzoyl peroxide were added to a solution of the appropriate ester or acid (0.02 mol) in dry carbon tetrachloride or alternatively in ethanol (50 mL). The reaction mixture was refluxed for 24 h. When the reaction was complete (TLC monitoring), the mixture was filtered and the solvent was removed under reduced pressure. Silica gel column chromatography purified the residue (eluent: chloroform or chloroform:methanol; 50:0.2 *v*/*v*).

Procedure 4. Procedure for Bromination by Using Br2.

Method a. Procedure according to the method that was described earlier [11]. Thus, an appropriate ester, amide, or acid (0.02 mol) was dissolved in CHCl3 (20 mL), and then a solution of bromine in CHCl3 (0.02 mol in 10 mL) was added dropwise with stirring for 1 h. The obtained mixture was stirred at room temperature for 24 h. When the reaction was finished, the solvent was removed under reduced pressure. The residue was purified by a silica gel column chromatography (eluent: chloroform or chloroform:methanol; 50:0.2 *v*/*v*).

Method b. Procedure according to the method described earlier [11]. Thus, an appropriate ester, amide, or acid (0.02 mol) was dissolved in CH3COOH (80%, 20 mL), and then a solution of bromine in CH3COOH (0.02 mol in 10 mL) was added dropwise with stirring for 1 h. The obtained mixture was stirred at room temperature for 24 h. When the reaction was complete, the resulting mixture was diluted by Na2S2O3 solution (10 mL) and extracted with DCM (3 × 50 mL). The obtained organic extracts were dried with calcium chloride, filtered, and concentrated under reduced pressure. Silica gel column chromatography purified the residue (eluent: chloroform or chloroform:methanol; 50:0.2 *v*/*v*).

Procedure 5. Procedure for Reduction.

A starting ketone (0.0024 mol) was dissolved in the peroxides-free dioxane (20 mL), and then NABH(OAc)3 (0.0048 mol) was added. The mixture was stirred at room temperature for 24–48 h. When the reaction was complete, the solvent was removed under reduced pressure. The solid residue

was dissolved in CHCl3 (50 mL) and then washed with water (3 × 20 mL). The organic solution was dried with magnesium sulfate, filtered, and concentrated under reduced pressure. Finally, silica gel column chromatography purified the residue (eluent: chloroform or chloroform:methanol; 50:0.2 *v*/*v*).

#### 4.2.1. Synthesis of Analogues of Compound **VIII**

Synthesis of 7-Acetyl-5,6-Dimethoxy-3-Methylbenzofuran-2-Carboxylic Acid (**1**)

7-Acetyl-5,6-dimethoxy-3-methylbenzofuran-2-carboxylic acid was obtained in the multistep reaction according to the method described earlier [28].

M.W. = 278.2573; C14H14O6; Yield: 30%; white powder, m.p. 212–214 ◦C; 1H-NMR (300 MHz, DMSO, δ/ppm): 2.50 (3H, s, -CH3), 2.62 (3H, s, -COCH3), 3.84 (3H, s, -OCH3), 3.92 (3H, s, -OCH3), 7.47 (1H, s, Ar-H), 13.41 (1H, br.s, -COOH); 13C-NMR: δ 9.15, 32.13, 56.43, 61.755, 105.50, 119.64, 124.39, 124.68, 142.19, 144.27, 147.40, 150.07, 160.76, 197.33;.HRMS (*m*/*z*): calculated value for [M + Na] 100% = 301.0683; found 100% = 301.0681<sup>+</sup>; Anal. Calc. for C14H14O6: 60.43% C; 5.07% H, found 59.25% C; 4.92% H.

#### Synthesis of 7-Acetyl-5,6-Dimethoxy-3-Methylbenzofuran-2-Carboxamide (**1a**)

7-Acetyl-5,6-dimethoxy-3-methylbenzofuran-2-carboxamide was obtained according to Procedure 1. M.W. = 277.2726; C14H15NO5, Yield: 37%; white powder, m.p. 203–205 ◦C; 1H-NMR (300 MHz, CDCl3, δ/ppm): 2.50 (3H, s, -CH3), 2.68 (3H, s, -COCH3), 3.80 (3H, s, -OCH3), 3.90 (3H, s, -OCH3), 7.41 (1H, s, Ar-H), 7.70 (2H, br.m, -NH2); 13C-NMR: δ 8.73, 32.47, 56.04, 61.85, 104.99, 120.02, 121.07, 125.07, 143.14, 144.08, 146.34, 150.07, 160.93, 197.55; HRMS (*m*/*z*): calculated value for [M + Na] 100% = 300.0842, found 100% = 300.0842<sup>+</sup>. Anal. Calc. for C14H15NO5: 60.64% C; 5.45% H, 5.05% N, found 60.04% C; 4.537% H, 4.81% N.

#### Synthesis of Methyl 7-Acetyl-5,6-Dimethoxy-3-Methylbenzofuran-2-Carboxylate (**1b**)

Methyl 7-acetyl-5,6-dimethoxy-3-methyl-1-benzofuran-2-carboxylate was obtained according to Procedure 2. M.W. = 292.2839; C15H16O6; Yield: 60%; white powder, m.p. 98–100 ◦C; 1H-NMR (300 MHz, CDCl3, δ/ppm): 2.55 (3H, s, -CH3), 2.73 (3H, s, -COCH3), 3.94 (3H, s, -OCH3), 3.95 (3H, s, -OCH3), 3.95 (3H, s, -COOCH3), 7.10 (1H, s, Ar-H); 13C-NMR: δ 9.22, 32.34, 51.84, 56.38, 62.40, 104.42, 120.28, 124.84, 125.43, 141.68, 145.57, 148.67, 150.58, 160.41, 197.50; HRMS (*m*/*z*): calculated value for [M + Na] 100% = 315.0839, found 100% = 315.0839. Anal. Calc. for C15H16O6: 61.64% C; 5.52% H, found 61.35% C; 5.516% H.

#### Synthesis of Methyl 7-Acetyl-3-(Bromomethyl)-5,6-Dimethoxybenzofuran-2-Carboxylate (**1c**)

Methyl 7-acetyl-3-(bromomethyl)-5,6-dimethoxybenzofuran-2-carboxylate was obtained according to Procedure 4 (method a). M.W. = 371.1800; C15H15BrO6; Yield: 20%; white powder, m.p. 124–125 ◦C; 1H-NMR (300 MHz, CDCl3, δ/ppm): 2.72 (3H, s, -COCH3), 3.97 (9H, m, -OCH3, -OCH3, -COOCH3), 4.90 (2H, -CH2Br), 7.33 (1H, s, Ar-H); 13C-NMR: δ 20.46, 32.30, 52.35, 56.43, 62.41, 104.32, 120.53, 122.56, 124.99, 141.66, 145.71, 149.11, 151.01, 159.61, 197.15; HRMS (*m*/*z*): calculated value for [M + Na] 100% = 392.9944, 99% = 394.9927, found 100% = 392.9945, 99% = 394.9926. Anal. Calc. for C15H14 BrO6: 48.54% C; 4.07% H, found 48.82% C; 4.15% H.

#### Synthesis of 1-(2-Bromo-5,6-Dimethoxy-3-Methylbenzofuran-7-yl)ethanone (**1d**)

1-(2-Bromo-5,6-dimethoxy-3-methylbenzofuran-7-yl)ethanone was obtained according to Procedure 3 (in ethanol) as well as Procedure 4 (method b). Yield: M.W. = 313.1439; C13H13BrO4; Yield: 27%; white powder, m.p. 94–95 ◦C; 1H-NMR (300 MHz, CDCl3, δ/ppm): 2.16 (3H, s, -CH3), 2.69 (3H, s, -COCH3), 3.90 (3H, s, -OCH3), 3.93 (3H, s, -OCH3), 6.98 (1H, s, Ar-H); 13C-NMR: δ 8.69, 32.26, 56.47, 62.38, 103.39, 114.99, 119.83, 125.37, 126.39, 145.20, 1454.74, 150.22, 197.82; HRMS (*m*/*z*): calculated

value for [M + Na] 100% = 334.9889, 99% = 336.9870, found 100% = 334.9897, 99% = 336.9869. Anal. Calc. for C13H13BrO4: 49.86% C; 4.18% H, found 49.76% C; 4.159% H.

#### Synthesis of 2-Bromo-1-(2-Bromo-5,6-Dimethoxy-3-Methylbenzofuran-7-yl]ethanone (**1e**)

2-Bromo-1-(2-bromo-5,6-dimethoxy-3-methylbenzofuran-7-yl)ethanone was obtained according to Procedure 3 (in ethanol). M.W. = 392.0399; C13H12Br2O4; Yield: 27%; white powder, m.p. 128–129 ◦C; 1H-NMR (300 MHz, CDCl3, δ/ppm): 2.17 (3H, s, -CH3), 3.94 (6H, s, -OCH3, -OCH3), 4.58 (2H, s, -COCH2Br), 7.03 (1H, s, Ar-H); 13C-NMR: δ 8.69, 36.49, 56.53, 62.49, 104.60, 115.16, 116.09, 125.46, 126.66, 145.84, 145.95, 150.10, 190.29; HRMS (*m*/*z*): calculated value for [M + Na] 50% = 412.8995, 100% = 414.8975, 49% = 416.8956, found 50% = 412.8991, 100% = 414.8973, 49% = 416.8944. Anal. Calc. for C13H12 Br2O4: 39.83% C; 3.09% H, found 40.21% C; 3.066% H.

#### 4.2.2. Synthesis of Analogues of Compound **IX**

Synthesis of 6-Acetyl-5-Hydroxy-2-Methylbenzofuran-3-Carboxamide (**2a**)

6-Acetyl-5-hydroxy-2-methylbenzofuran-3-carboxamide was obtained according to Procedure 1. M.W. = 233.2200; C12H11NO4; Yield: 50%; white powder, m.p. 268–267 ◦C; 1H-NMR (300 MHz, DMSO, δ/ppm): 2.65 (3H, s, -COCH3), 2.68 (3H, s, -CH3), 7.22 (1H, s, Ar-H), 7.48 (2H, br.s, -NH2), 8.11 (1H, s, Ar-H), 11.97 (1H, s, -OH); 13C-NMR: δ 14.20, 27.52, 107.03, 112.69, 112.74, 116.38, 133.39, 146.00, 157.39, 163.86, 164.22, 204.26; HRMS (*m*/*z*): calculated value for [M + Na] 100% = 256.0580, found 100% = 256.0582. Anal. Calc. for C12H11NO4: 61.80% C; 4.75% H, 6.01% N, found 60.85% C; 4.766% H, 5.83% N.

#### Synthesis of 6-Acetyl-4-Bromo-5-Hydroxy-2-Methylbenzofuran-3-Carboxamide (**2b**)

6-Acetyl-4-bromo-5-hydroxy-2-methylbenzofuran-3-carboxamide was obtained according to Procedure 4 (method b). M.W. = 312.1161; C12H10BrNO4; Yield: 40%; white powder, m.p. 249–248 ◦C; 1H-NMR (300 MHz, DMSO, δ/ppm): 2.50 (3H, s, -COCH3), 2.68 (3H, s, -CH3), 7.69 (1H, br.s, -NH2), 7.94 (1H, br.s, -NH2), 8.23 (1H, s, Ar-H), 12.88 (1H, s, -OH); 13C-NMR: δ 13.20, 27.05, 99.21, 112.59, 115.82, 115.86, 133.19, 145.62, 154.37, 160.33, 163.79, 204.99; HRMS (*m*/*z*): calculated value for [M + Na] 100% = 333.9685, 98% = 335.9666, found 100% = 333.9685, 98% = 335.9669. Anal. Calc. for C12H10BrNO4: 46.18% C; 3.23% H, 4.49% N, found 46.33% C; 3.265% H, 4.35% N.

Synthesis of Methyl 6-Acetyl-5-Hydroxy-2-Methylbenzofuran-3-Carboxylate (**2c**)

Methyl 6-acetyl-5-hydroxy-2-methylbenzofuran-3-carboxylate (**2c**) was obtained according to the method described previously [11].

#### Synthesis of Methyl 6-Acetyl-2-(Bromomethyl)-5-Hydroxybenzofuran-3-Carboxylate (**2d**)

Methyl 6-acetyl-2-(bromomethyl)-5-hydroxybenzofuran-3-carboxylate was obtained according to Procedure 3 (in CCl4). M.W. = 327.1274; C13H11BrO5; Yield: 30%; white powder, m.p. 94–95 ◦C (chyba 138–140); 1H-NMR (300 MHz, CDCl3, δ/ppm): 2.70 (3H, s, -COCH3), 3.99 (3H, s, -COOCH3), 4.94 (2H, s, -CH2Br), 7.53 (1H, s, Ar-H), 7.88 (1H, s, Ar-H), 12.11 (1H, s, -OH); 13C-NMR: δ 14.19, 26.80, 52.08, 110.51, 112.63, 117.59, 132.64, 146.73, 155.99, 161.21, 162.63, 164.00, 203.35; HRMS (*m*/*z*): calculated value for [M + Na] 100% = 348.9682, 99% = 350.9663, found 100% = 348.9683, 99% = 350.9663. Anal. Calc. for C13H11BrO5: 47.73% C; 3.39% H, found 47.33% C; 3.265% H.

Synthesis of 6-Acetyl-4-Bromo-5-Hydroxy-2-Methylbenzofuran-3-Carboxylic Acid (**2e**)

6-Acetyl-4-bromo-5-hydroxy-2-methylbenzofuran-3-carboxylic acid was obtained according Procedure 4 (method b). M.W. = 313.1008; C12H9BrO5; Yield: 30%; white powder, m.p. 196–197 ◦C; 1H-NMR (300 MHz, CDCl3, δ/ppm): 2.48 (3H, s, -COCH3), 2.69 (3H, s, -CH3), 7.72 (1H, s, Ar-H), 12.89 (1H, s, -OH); 13C-NMR: δ 13.20, 26.70, 94.45, 100.68, 111.33, 116.11, 132.08, 145.99, 155.30, 159.72, 203.35; HRMS (*m*/*z*): calculated value for [M + Na] 100% = 334.9526, 99% = 336.9506, found 100%= 334.9525, 99% = 336.9505. Anal. Calc. for C12H9BrO5: 46.03% C; 2.90% H, found 46.33% C; 2.265% H.

4.2.3. Synthesis of Analogues of Compound **X**

Synthesis of Methyl 6-Acetyl-5-Methoxy-2-Methylbenzofuran-3-Carboxylate (**3**) and Methyl 6-(Dibromoacetyl)-5-Methoxy-2-Methyl-1-Benzofuran-3-Carboxylate (**X**)

Methyl 6-acetyl-5-methoxy-2-methylbenzofuran-3-carboxylate (**3**) and methyl 6-(dibromoacetyl)-5 methoxy-2-methylbenzofuran-3-carboxylate (**X**) were obtained according to the method described previously [11].

Synthesis of Methyl 6-(2,2-Dibromo-1-Hydroxyethyl)-5-Methoxy-2-Methylbenzofuran-3-Carboxylate (**3a**)

Methyl 6-(2,2-dibromo-1-hydroxyethyl)-5-methoxy-2-methylbenzofuran-3-carboxylate (**3a**) was obtained according to Procedure 5. M.W. = 422.0659; C14H14Br2O5; Yield: 70%; white powder, m.p. 170–172 ◦C; 1H-NMR (300 MHz, DMSO, δ/ppm): 2.74 (3H, s, -COOCH3), 3.18 (1H, br.s, -CH-), 3.93 (3H, s, -OCH3), 3.94 (3H, s, -CH3), 5.34 (1H, br.s, –OH), 6.14 (1H, d, -CH-, *J* = 3 Hz), 7.41 (1H, s, Ar-H), 7.59 (1H, s, Ar-H); 13C-NMR: δ 14.73, 51.46, 51.77, 55.93, 75.00, 102.26, 108.96, 110.78, 123.97, 126.92, 148.14, 153.17, 164.71, 164.75; HRMS (*m*/*z*): calculated value for [M + Na] 50% = 442.9100, 100% = 444.9081, 50% = 446.9063, found 50% = 442.9094, 100% = 444.9079, 50% = 446.9065. Anal. Calc. for C14H14Br2O6: 39.84% C; 3.34% H, found 40.20% C; 3.37% H.

Synthesis of 6-Acetyl-5-Methoxy-2-Methylbenzofuran-3-Carboxylic Acid (**3b**)

A mixture of methyl 6-acetyl-5-methoxy-2-methylbenzofuran-3-carboxylate (0.0008 mol) and 2 M NaOH (0.6 mL, 0.0012 mol) in ethanol (1.2 mL) was heated for 1 h. The bulk of the solvent was evaporated and the residue was acidified with 2 M HCl (1.2 mL) to give a fine precipitate. Next, the mixture was cooled to room temperature and then filtered to give the product. M.W. = 248.2313; C13H12O5; Yield: 30%; white powder, m.p. 249–250 ◦C; 1H-NMR (300 MHz, DMSO, δ/ppm): 2.55 (3H, s, -COCH3), 2.71 (3H, s, -CH3), 3.90 (3H, s, -OCH3), 7.45 (1H, s, Ar-H), 7.69 (1H, s, Ar-H); 13C-NMR: δ 14.41, 31.61, 56.03, 103.27, 109.23, 111.32, 124.90, 130.79, 147.06, 155.84, 164.66, 166.58, 197.99; HRMS (*m*/*z*): calculated value for [M + Na] 100% = 271.0577, found 100% = 270.0737. Anal. Calc. for C13H12O5\* <sup>1</sup> <sup>2</sup> H2O: 60.50% C; 5.07% H, found 60.05% C; 4.715% H.

Synthesis of 6-Acetyl-5-Methoxy-2-Methylbenzofuran-3-Carboxamide (**3c**)

6-Acetyl-5-methoxy-2-methylbenzofuran-3-carboxamide was obtained according to Procedure 1. M.W. = 247.2466; C13H13NO4; Yield: 30%; white powder, m.p. 223–224 ◦C; 1H-NMR (300 MHz, DMSO, δ/ppm): 2.55 (3H, s, -COCH3), 2.68 (3H, s, -CH3), 3.98 (3H, s, -OCH3), 7.22 (1H, s, Ar-H), 7.53 (2H, br.s, -NH2), 7.70 (1H, s, Ar-H); 13C-NMR: δ 14.11, 31.61, 56.17, 103.10, 111.14, 112.81, 124.65, 130.56, 146.94, 155.49, 162.02, 164.42, 198.08; HRMS (*m*/*z*): calculated value for [M + Na] 100% = 270.0737, found 100% = 270.0737. Anal. Calc. for C13H13NO4: 63.15% C; 5.30% H, 5.67% N, found 62.90% C; 5.245% H, 5.63% N.

Synthesis of Methyl 6-Acetyl-2-(Bromomethyl)-5-Methoxybenzofuran-3-Carboxylate (**3d**)

Methyl 6-acetyl-2-(bromomethyl)-5-methoxybenzofuran-3-carboxylate was obtained according to Procedure 3 (in CCl4). M.W. = 341.1540; C14H13BrO5; Yield: 30%; white powder, m.p. 148–150 ◦C; 1H-NMR (300 MHz, CDCl3, δ/ppm): 2.65 (3H, s, -COCH3), 3.98 (3H, s, -OCH3), 4.01 (3H, s, -COOCH3), 4.91 (2H, s, -CH2Br), 7.50 (1H, s, Ar-H), 7.86 7.70 (1H, s, Ar-H); 13C-NMR: δ 20.94, 31.08, 52.07, 56.03, 103.76, 110.48, 112.98, 127.44, 129.80, 148.49, 156.56, 161.91, 163.30, 199.04; HRMS (*m*/*z*): calculated value for [M + Na] 100% = 362.9839, 99% = 364.9820, found 100% = 362.9839, 99% = 364.9820. Anal. Calc. for C14H13BrO5: 49.29% C; 3.84% H, found 48.87% C; 3.755% H.

#### *4.3. Anticancer Activity*

#### 4.3.1. Cells and Cytotoxicity Assay

Human umbilical vein endothelial cells (Life Technologies, Waltham, MA, USA) were cultured (according to the manufacturer instructions) in Medium 200 supplemented with Low Serum Growth Supplement. 1 <sup>×</sup> 10<sup>4</sup> HUVEC cells were seeded on each well on a 96-well plate (Nunc). The HeLa (human cervix carcinoma) K562 and MOLT-4 (leukemia) cells were cultured in RPMI 1640 medium supplemented with antibiotics and 10% fetal calf serum (HeLa, K562) in a 5% CO2-95% air atmosphere. <sup>7</sup> <sup>×</sup> 103 HeLa, K562, or MOLT-4 cells were seeded on each well on 96-well plate (Nunc). 24 h later cells were treated with the test compounds and then incubated for an additional 48 hours. Stock solutions of test compounds were freshly prepared in DMSO (dimethylsulfoxide). The final concentrations of compounds that were tested in the cell cultures were: 2 <sup>×</sup> 10−1, 1 <sup>×</sup>10−1, 5 <sup>×</sup> 10−2, 1 <sup>×</sup> 10−2, 1 <sup>×</sup> 10−<sup>3</sup> and 1 <sup>×</sup> <sup>10</sup>−<sup>4</sup> mM. The concentration of DMSO in the cell culture medium was 1%.

The values of IC50 (the concentration of test compound that is required to reduce the cell survival fraction to 50% of the control) were calculated from dose-response curves and used as a measure of cellular sensitivity to a given treatment.

The MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Sigma, St. Louis, MO, USA] assay determined the cytotoxicity of all the compounds, as described previously [30]. Briefly, after 24 h or 48 h of incubation with the drug, the cells were treated with the MTT reagent, and incubation was continued for 2 h. MTT-formazan crystals were dissolved in 20% SDS and 50% DMF at pH 4.7 and absorbance was read at 570 and 650 nm on an ELISA-PLATE READER (FLUOstar Omega, BMG LABTECH GmbH, Ortenberg, Germany). As a control (100% viability), cells that were grown in the presence of medium vehicle only with 1% DMSO were used.

#### 4.3.2. Induction of Cell Apoptosis Analyzed by Caspase-3/7 Assay

<sup>20</sup> <sup>×</sup> <sup>10</sup><sup>3</sup> K562 cells were seeded on each well of 96-well plate in RPMI 1640 medium supplemented with 10% fetal calf serum and antibiotics. Cells were grown for 24 h at 37 ◦C and 5% CO2. The test compounds were dissolved in DMSO and added to the cell culture. The concentration of tested benzo[b]furans in cell culture was 5 × IC50.

Cells treated with 1% DMSO served as a negative control, while cells incubated with staurosporine (a strong inducer of apoptosis) were used as a positive control. Cells were exposed to test compounds for 18 h at 37 ◦C and 5% CO2. Subsequently, Apo-ONE® Homogeneous Caspase-3/7 Assay (Promega, Madison, WI, USA) measured the activity of caspase 3 and 7, according to the manufacturer's instructions. Briefly, the cells were lysed and incubated for 1.5 h with profluorescent substrate for caspase 3 and 7. Next, fluorescence was read at an excitation wavelength of 485 nm and emission of 520 nm with FLUOStar Omega plate reader (BMG-Labtech, Ortenberg, Germany).

#### 4.3.3. Digestion of Plasmid DNA with BamHI Restriction Nuclease

0.5 μg of plasmid DNA (pcDNAHisC, total length 5.5 kbp) containing a unique *BamHI* restriction site was dissolved in a 1× *BamHI* reaction buffer and then incubated overnight at 37 ◦C with the test compounds or daunorubicin, a strong intercalating agent, which was used as a positive control. The concentration of the test compounds and daunorubicin samples was 100 μM. In the next step, the reaction mixtures were digested with *BamHI* restriction endonuclease (2 U/μL) for 3 h at 37 ◦C. The total reaction volume was 10 μL. Products of the reaction were subjected to the 1% agarose gel electrophoresis in TBE buffer. The gel was stained with ethidium bromide and DNA fragments were visualized under a UV lamp (GBox, Syngene, Cambridge, UK).

#### **5. Conclusions**

We synthesized and tested a group of new benzofuran derivatives. The presence of bromine in the alkyl group in the furan ring is most likely responsible for the cytotoxic properties of the tested derivatives (compounds **1c**, **2d**, **3d**). Compound **1e** shows the cytotoxic property, and contains an acetyl halide substituent (bromine) in the benzene ring and a bromine atom that is directly attached to the furan ring. The most active compounds **2d** and **3d**, showed increased polarity when compared to the lead compounds **VIII**-**X**, but their cytotoxicity against human cancer cells decreased by 5–10 folds and the toxicity against normal cells increased. The formation of amide derivatives of benzofurans (compounds **1a**, **2a**, **2b**, **3c**) and the lack of a halogen-containing alkyl substituent in their structure resulted in better water solubility but loss of cytotoxic properties towards the cancer cells studied. A reduction of the bromoacetyl group in compound **X** increased its polarity but also eliminated the selectivity of the compound and diminished its toxicity towards tumor cells.

**Author Contributions:** Conceptualization, M.N.; Methodology, M.N. and M.C.; Performed the experiments M.N., M.C., J.K.-B., K.K.-G. Writing—Original Draft Preparation, M.N.; Writing—Review & Editing, M.N., M.C. and B.N.; Visualization, M.N. and M.C.; Supervision, B.N.; Project Administration, M.N. and M.C.; Funding Acquisition, M.N. and B.N.

**Funding:** This work was supported by the Polish Ministry of Science, project NCN OPUS UMO-2014/15/B/NZ7/00966.

**Acknowledgments:** The cytotoxicity, activation of caspases and DNA interaction studies were performed in the Screening Laboratory at the Division of Bioorganic Chemistry, Centre of Molecular and Macromolecular Studies of the Polish Academy of Sciences.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


**Sample Availability:** Not available.

© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

*Article*

## **In Vitro and In Vivo Anti-Breast Cancer Activities of Some Newly Synthesized 5-(thiophen-2-yl)thieno- [2,3-d]pyrimidin-4-one Candidates**

**Abd El-Galil E. Amr 1,2,\*, Alhussein A. Ibrahimd 2, Mohamed F. El-Shehry 3,4, Hanaa M. Hosni 3, Ahmed A. Fayed 2,5 and Elsayed A. Elsayed 6,7**


Academic Editor: Qiao-Hong Chen

Received: 29 May 2019; Accepted: 14 June 2019; Published: 17 June 2019

**Abstract:** In this study, some of new thiophenyl thienopyrimidinone derivatives **2**–**15** were prepared and tested as anti-cancer agents by using thiophenyl thieno[2,3-d]pyrimidinone derivative **2** as a starting material, which was prepared from cyclization of ethyl ester derivative **1** with formamide. Treatment of **2** with ethyl- chloroacetate gave thienopyrimidinone *N*-ethylacetate **3**, which was reacted with hydrazine hydrate or anthranilic acid to afford acetohydrazide **4** and benzo[d][1,3]oxazin-4-one **5,** respectively. Condensation of **4** with aromatic aldehydes or phenylisothiocyanate yielded Schiff base derivatives **6,7**, and thiosemicarbazise **10**, which were treated with 2-mercaptoacetic acid or chloroacetic acid to give the corresponding thiazolidinones **8**, **9,** and phenylimino-thiazolidinone **11**, respectively. Treatment of **4** with ethylacetoacetate or acetic acid/acetic anhydride gave pyrazole **12** and acetyl acetohydrazide **13** derivatives, respectively. The latter compound **13** was reacted with ethyl cycno-acetate or malononitrile to give **14** and **15**, respectively. In this work, we have studied the anti-cancer activity of the synthesized thienopyrimidinone derivatives against MCF-7 and MCF-10A cancer cells. Furthermore, in vivo experiments showed that the synthesized compounds significantly reduced tumor growth up to the 8th day of treatment in comparison to control animal models. Additionally, the synthesized derivatives showed potential inhibitory effects against pim-1 kinase activities.

**Keywords:** thiopene; thienopyrimidinone; thiazolidinone; anticancer activity

#### **1. Introduction**

Cancer is a major health problem acting as a global killer, so synthesizing new compounds, which may act as potent antitumor agents, is a great target for chemists working in this field. In this study, we are interested in synthesizing and studying biological activities of

thieno[2,3-d]pyrimidinone derivatives [1–11]. Thienopyrimidinones are very important moieties that act as keys for pharmacological and pharmaceutical properties. They are reported to cause antiviral [12], antimicrobial [13], antihypertensive [14], analgesic, and anti-inflammatory activities [15]. They also inhibit various protein kinase enzymes, such as CK2 involved in particular anticancer activity [16]. Additionally, the nitrogenous ring system was associated with some types of biological activities such as: anti-inflammatory [17], insecticidal [18], antimicrobial and antituberculosis [19,20] activities. On the other hand, thienopyrimidinones contain a thiophene ring fused with a pyrimidinone nucleus. In general, this system was thought to be interesting in development of pharmaceutical compounds [21,22], and was not only evaluated as cGMP phosphodiesterase inhibitors [23], anti-viral [24], anti-inflammatory [25], anti-microbial agents [26], but also as kinase inhibitors and potential anti-cancer agents [27,28]. In continuation to our previous work, and to extend our research [1–11], from the above points, we have studied the anticancer activity of the newly synthesized substituted thienopyrimidinone derivatives against MCF-7 and MCF-10A cancer cells. Furthermore, the work was extended to evaluate the effects of synthesized derivatives on the inhibition of tumor growth in an in vivo animal model. Finally, we evaluated the inhibitory effects of our synthesized compounds against pim-1 kinase activity as a possible mechanism of their action.

#### **2. Results and Discussion**

#### *2.1. Chemistry*

A series of thiophenyl thienopyrimidinone derivatives **2**–**15** were prepared and tested as anti-cancer agents. Cyclization of ethyl 5'-amino-[2,3'-bithiophene]-4'-carboxylate (**1**) with formamide gave the corresponding thiophenylthieno[2,3-d]pyrimidinone derivative (**2**), which was treated with ethylchloroacetate to give thienopyrimidinone N-ethylacetate **3**. Reaction of **3** with hydrazine hydrate or anthranilic acid afforded the corresponding hydrazide **4** and benzooxazinone **5** derivatives, respectively (Scheme 1).

**Scheme 1.** Synthetic route for compounds **2**–**5**.

Condensation of **4** with aromatic aldehydes, namely, 2,3-dimethoxybenzaldehyde or 4-chlorobenzaldehyde gave the corresponding Schiff base derivatives **6** and **7**, which were cyclized via reaction with 2-mercaptoacetic acid in dry benzene to give the corresponding thiazolidinone derivatives **8** and **9**, respectively. Treatment of **4** with phenylisothiocyanate gave thiosemicarbazide , which was condensed with chloroacetic acid to afford phenyliminothiazolidinone derivative **11** (Scheme 2).

**Scheme 2.** Synthetic route for compounds **6**–**11**.

Finally, treatment of **4** with ethylacetoacetate or acetic acid/acetic anhydride gave the corresponding pyrazolyl derivative **12** and N-acetyl hydrazide **13**, respectively. The latter compound **13** was reacted with ethylcycnoacetate or malononitrile to give pyridine derivatives **14** and **15**, respectively (Scheme 3).

**Scheme 3.** Synthetic route for compounds **12**–**15**.

#### *2.2. Biological Evaluation*

MCF-7 cells were used to investigate the potential in vitro anti-proliferative potential of the synthesized compounds. With the exception of Cpd. **2** (data not shown), we found that all compounds have promising activities when used in μM concentration. On the other hand, Cisplatin and Milaplatin showed higher IC50 values (13.34 ± 0.11 and 18.43 ± 0.13 μM, respectively). DMSO at concentrations of 0.1% and 0.5%, had little or no toxicity, whereas higher concentrations inhibited the growth of MCF-7cells. Therefore, it seems DMSO could be solvents of choice acceptable to be used at concentrations < 0.5% (*v*/*v*) towards the examined cells and possibly for other cell lines. Also, the effect on cell viability was proportional to the concentration applied. From Figure 1, we can see that Cpd. **15, 14** and **8** (IC50, 1.18 ± 0.032, 1.19 ± 0.042, 1.26 ± 0.052 μM, respectively) followed by **9** and **11** (IC50, 2.37 ± 0.053 and 2.48 ± 0.054 μM respectively) produced the highest effect on cell viability. Secondly, compounds **12**, **10** and **13**, showed moderate activities (IC50, 3.36 ± 0.063, 3.55 ± 0.065 and 3.64 ± 0.074 μM, respectively). Compounds **7, 6, 5, 4** and **3** were the least active ones (IC50, 4.33 ± 0.076, 4.52 ± 0.085, 4.76 ± 0.087, 4.87 ± 0.098 and 5.98 ± 0.099 μM, respectively). The order of activities can be arranged as **15** > **14** > **8** > **9** > **11** > **12** > **10** > **13** > **7** > **6** > **5** > **4** > **3**.

Results revealed that the substitution with pyridine moiety at the terminal NH improved the cytotoxic effect than the pyrimidone derivatives. In contrast, substitution with 5-membered di-heterocyclic ring system with aryl moiety decreased the obtained activities (methoxy phenyl > chlorophenyl). Attaching five membered pyrazolinone ring system bearing no aryl moiety at terminal NH (compound **12** decreased the activities than those containing aryl substitutions (compounds **9** and **11**). Compounds **10, 7** and **6** that contain aromatic *N*-substitution still have more potent activity. The increased effect of the aromatic ring may be attributed to ring aromaticity and electron resonance. On the other hand, aliphatic side chains (compounds **4** and **3**) or methylene bridges (Compound **5**) have less potent activities.

Additionally, results against non-tumorigenic MCF-10A proved that our derivatives have higher degrees of safety towards normal cells.

**Figure 1.** Obtained IC50 values for MCF-7 and MCF-10A cells.

In Vivo Xenograft Model

The in vivo anti-breast cancer activities of different synthesized derivatives were evaluated using a breast cancer mouse xenograft model. Figure 2 shows the increase in percentage of inhibition in tumor growth with treatment time when animals were exposed to different compounds. This was also compared with tumor development in control animals. It can be seen, that our derivatives reduced tumor growth starting day 2. The maximal effect was obtained after 8 days. Furthermore, the in vivo effect showed also the same inhibitory pattern obtained in the in vitro experiments. The average weight of each group of mice treated with drug and the control group summarized in Table 1.

**Figure 2.** Relative percentage of decrease in tumor volume in response to prepared compounds. **Table 1.** The average weight of each group of mice treated with drug and the control group.


The Provirus Integration in Maloney (Pim) kinases represents a family of constitutively active serine/threonine kinases and includes three subtypes (pim-1, pim-2 and pim-3). Pim kinases regulate many biological processes such as cell cycle, cell proliferation, apoptosis and drug resistance [29–32]. Being expressed in many types of solid and hematological cancers and almost absent in benign lesions, pim kinases proved to be a successful anti-cancer drug target of low toxicity [33–40]. Results obtained in Figure 3 showed that all synthesized compounds were showed potent inhibitory effects against pim-1 kinase.

**Figure 3.** IC50 of the tested compounds against pim-1 Kinase.

#### **3. Materials and Methods**

#### *3.1. Chemistry*

"Melting points were determined in open glass capillary tubes with an Electro Thermal Digital melting point apparatus (model: IA9100) and are uncorrected. Elemental microanalyses were carried out in the microanalysis unit of NRC and were found within the acceptable limits of the calculated values. Infrared spectra (KBr) were recorded on a Nexus 670 FTIR Nicolet, Fourier Transform infrared spectrometer. 1H- and 13C NMR spectra were run in (DMSO-d6) on Jeol 500 MHz instruments. Mass spectra were run on a MAT Finnigan SSQ 7000 spectrometer, using the electron impact technique (EI)."

*Synthesis of 5-(thiophen-2-yl)thieno[2,3-d]pyrimidin-4(3H)-one (2).* A mixture of compound **1** (1 mmol, 253 mg) and formamide (20 mL) was heated at 180 ◦C in oil bath for 2 h. The formed solid was collected by filtration, washed with cold methanol, dried and crystallized from EtOH to give compound **2**. Yield 80%, M.p 192–194 ◦C; IR (KBr, cm−1): υ¯ 3323 (NH), 1659 (C=O). 1H NMR (DMSO-d6) δH: 7.11–7.72 (m, 4H, thiophene-H), 8.50 (s, 1H, CH-pyrimidine), 13.30 (s, 1H, NH, disappeared with D2O). 13C NMR: 119.98, 122.01, 122.17, 126.84, 127.75, 128.74, 131.26, 136.42, (8C, thiophene-C), 157.05 (1C, pyrimidine-C), 165.56 (C=O). Mass spectrum, *m*/*z* (EI, %): 234 (M+, 100), 235 (M<sup>+</sup> + 1, 11), 236 (M<sup>+</sup> + 2, 9). Analysis for C10H6N2OS2 (234.29): Calculated: C, 51.27; H, 2.58; N, 11.96; S, 27.37. Found: C, 51.20; H, 2.50; N, 11.90; S, 27.30.

*Synthesis of ethyl 2-(4-oxo-5-(thiophen-2-yl)thieno[2,3-d]pyrimidin-3(4H)-yl)acetate (3).* A mixture of **2** (1 mmol, 234 mg), ethylchloroacetate (1 mmol, 122 mg) and anhydrous potassium carbonate (8 mmol) in dry acetone (30 mL) was heated under reflux for 4h. The obtained solid was removed by filtration, the filtrate was concentrated, the precipitate solid was filtered off, dried, and crystallized from EtOH to give the ester derivative **3**. Yield 70%, m.p 135–137 ◦C. IR (KBr, cm<sup>−</sup>1): ν 1753 (C=O, ester), 1655 (CO); 1H NMR (DMSO-d6), δ: 1.24 (t, 3H, CH3), 4.20 (s, 2H, CH2), 4.85 (q, 2H, CH2-ethyl), 7.10–7.72 (m, 4H, thiophene-H), 8.50 (s, 1H, pyrimidine-H). 13C-NMR (DMSO)-d6) δc: 14.5 (CH3), 40.2 (CH2), 47.8 (CH2), 119.9, 122.2, 126.9, 127.8, 128.7, 128.9, 131.3, 136.4 (8C, thiophene-C), 157.0 (1C, pyrimidine-C), 165.6, 168.3 (2C, 2CO). Mass spectrum, *m*/*z* (EI, %): 320 (M+, 100), 321 (M<sup>+</sup> + 1, 18). Analysis for C14H12N2O3S2 (320.38): Calculated: C, 52.49; H, 3.78; N, 8.74; S, 20.01. Found: C, 52.40; H, 3.70; N, 8.68; S, 19.86.

*Synthesis of 2-(4-oxo-5-(thiophen-2-yl)thieno[2,3-d]pyrimidin-3(4H)-yl)acetohydrazide (4).* To a solution of **3** (1 mmol, 320 mg) in ethanol (50 mL), hydrazine hydrate (4 mmol, 85%) was added and refluxed for 8 h. The precipitated solid was collected by filteration, dried and crystallized from EtOH to give compound **4**. Yield 75%, m.p. 205–207 ◦C, IR (KBr, cm−1): ν 3322 (NH), 3246 (NH2), 1659 (C=O). 1H-NMR (DMSO-d6) δ: 3.39 (s, 2H, CH2), 4.65 (s, 2H, NH2, disappeared with D2O), 7.10–7.65 (m, 4H, thiophene-H), 8.17 (s, 1H, pyrimidine-H), 12.58 (s, 1H, NH, disappeared with D2O). 13C-NMR (DMSOd6) δc: 40.1 (CH2), 120.5, 120.8, 126.6, 127.8, 128.7, 131.4, 133.5, 136.8 (8C, thiophene-C), 157.9 (1C, pyrimidine-C), 166.3, 169.4 (2C, 2CO). Mass spectrum, m/z (EI, %): 306 (M+, 100), 307 (M<sup>+</sup> + 1, 14). Analysis for C12H10N4O2S2 (306.36): Calculated: C, 47.05; H, 3.29; N, 18.29; S, 20.93. Found: C, 46.85; H, 3.20; N, 18.20; S, 20.85.

*Synthesis of 2-((4-oxo-5-(thiophen-2-yl)thieno[2,3-d]pyrimidin-3(4H)-yl)methyl)-4H-benzo[d]*-[1,3] *oxazin-4-one (5).* A mixture of **3** (1 mmol, 320 mg) and anthranilic acid (1 mmol, 137 mg) was fused together at 110 ◦C in an oil bath for 3hr. The residue was boiled with ethanol, the formed solid was removed by filtration, the solid formed was filtered off, and crystallized from EtOH to give **5**. Yield 60%, m.p. 225–227 ◦C. IR (KBr, Cm<sup>−</sup>1): ν 1750 (C=O), 1684 (C=O). 1H-NMR (DMSO d6) δH: 4.58 (s, 2H, CH2), 7.10–7.54 (m, 4H, thiophene-H), 7.68-8.16 (m, 4H, Ph-H), 8.64 (s, 1H, pyrimidine-H). 13C-MNR (DMSO-d6) δc: 43.0 (CH2), 120.0, 120.1, 121.3, 121.7, 126.7, 126.8, 127.7, 128.7, 131.3, 136.5, 136.6, 149.5, 149.7, 158.2 (14C, thiophene + Ph-C), 157.2 (1C, pyrimidine-C), 160.1, 165.6, 166.5 (3C, 3C=O). Mass spectrum, *m*/*z* (EI, %): 393 (M<sup>+</sup>, 100). Analysis for C19H11N3O3S2 (393.44): Calculated: C, 58.00; H, 2.82; N, 10.68; S, 16.30. Found: C, 57.90; H, 2.78; N, 10.60; S, 16.25.

*Synthesis of hydrazone derivatives 6 and 7.* To a mixture of **4** (1 mmol, 306 mg) and aromatic aldehydes, namely 3,4-dimethoxybenazaldehyed or 4-chlorobenzaldehyde (1 mmol) in ethanol (50 mL), few drops of piperidine were added and refluxed for 5 h, with stirring. After cooling, the formed solid was filtered off and recrystallized from dioxan to give the corresponding derivatives **6** and **7** respectively.

*N -(2,3-Dimethoxybenzylidene)-2-(4-oxo-5-(thiophen-2-yl)thieno[2,3-d]pyrimidin-3(4H)-yl)-acetohydrazide (6).* Yield 68%, m.p. 248–250 ◦C. IR (KBr, cm<sup>−</sup>1): ν 3388 (NH), 1660 (C=O). 1H-NMR (DMSO-d6) δH: 3.72, 3.86 (2s, 6H, 2OCH3), 4.11 (s, 2H, CH2), 6.98-7.61 (m, 7H, thiophene + Ph-H), 8.60 (s, 1H, CH=N), 9.05 (s, 1H, pyrimidine-H), 10.51 (s, 1H, NH, disappeared with D2O). 13C-NMR (MDSO-d6) δc: 44.1 (1C, CH2), 56.1, 60.1 (2C, OCH3), 114.3, 116.1, 119.5, 121.8, 124.0, 126.7, 127.7, 128.7, 129.3, 130.6, 131.3, 136.47, 149.07, 149.77 (14C, thiophene-C + Ph-C), 148.56 (1C, CH=N), 157.19 (1C, pyrimidine-C), 163.66, 169.74 (2C, 2C=O). Mass spectrum, *m*/*z* (EI, %): 454 (M<sup>+</sup>, 100). Analysis for C21H18N4O4S2 (454.52): Calculated: C, 55.49; H, 3.99; N, 12.33; S, 14.11. Found: C, 55.40; H, 3.90; N, 12.25; S, 13.96.

*N -(4-Chlorobenzylidene)-2-(4-oxo-5-(thiophen-2-yl)thieno[2,3-d]pyrimidin-3(4H)-yl)acetohydrazide (7).* Yield 65%, m.p. 250–252 ◦C. IR (KBr, Cm<sup>−</sup>1): ν 3408 (NH), 1670 (CO), 1659 (CO). 1H-NMR (DMSO-d6) δH: 4.10 (s, 2H, CH2), 7.10–7.80 (m, 9H, Ar-H + CH=N), 9.05 (s, 1H, pyrimidine-H), 10.56 (s, 1H, NH, disappeared with D2O). 13C-NMR (MDSO-d6) δc: 44.1 (1C, CH2), 145.2 (1C, CH=N), 119.5, 121.9, 124.0, 126.8, 127.8, 128.1, 129.3, 130.6, 131.3, 136.5, 149.1, 149.8 (14C, thiophene-C + Ph-C), 157.5 (1C, pyrimidine-C), 162.7, 169.9 (2C, 2C=O). Mass spectrum, *m*/*z* (EI, %): 428 (M+, 100), 430 (M<sup>+</sup> + 2, 40). Analysis for C19H13ClN4O2S2 (428.91): Calculated: C, 53.21; H, 3.06; N, 13.06; S, 14.95. Found: C, 53.12; H, 3.00; N, 13.00; S, 14.88.

*Synthesis of thiazolidinone derivatives 8 and 9.* To a stirred solution of **6** or **7** (1 mmol) in dry benzene (40 mL), thioglycollic acid (1 mmol, 92 mg) in dry benzene (10 mL) was added and refluxed for 12 h. The solvent was evaporated to dryness. The formed product was collected, and crystallized with dioxan to obtain the corresponding products **8** and **9**, respectively.

*N-(2-(2,3-Dimethoxyphenyl)-4-oxothiazolidin-3-yl)-2-(4-oxo-5-(thiophen-2-yl)thieno[2,3-d]pyrimidin-3(4H) yl)acetamide (8).* Yield 60%, m.p. 280–282 ◦C. IR (KBr, cm−1): ν 3417 (NH), 1670, 1680, 1630 (3 C=O). 1H-NMR (DMSO-d6) δH: 3.65, 3.90 (2s, 6H, 2OCH3), 4.66 (s, 2H, CH2), 4.77 (s, 2H, CH2), 5.86 (s, 1H, CH), 6.95-7.65 (m, 7H, thiophene + Ph-H), 8.49 (s, 1H, pyrimidine-H), 10.84 (s, 1H, NH, disappeared with D2O). 13C-NMR (DMSO-d6) δc: 35.9, 47.4 (2C, 2CH2), 56.5, 58.42 (2C, 2OCH3), 59.2 (1C, CH), 113.4, 117.9, 118.9, 120.7, 121.8, 126.8, 127.5, 128.7, 130.7, 131.0, 136.5, 145.3, 149.5, 149.9 (14C, thiophene-C + Ph-C), 156.9 (1C, pyrimidine-C), 162.5, 165.4, 169.2 (3C, 3C=O). Mass spectrum, *m*/*z* (EI, %): 528 (M+, 100), 529 (M<sup>+</sup> + 1, 30). Analysis for C23H20N4O5S3 (528): Calculated: C, 52.26; H, 3.81; N, 10.60; S, 18.19. Found: C, 52.18; H, 3.75; N, 10.52; S, 18.10.

*N-(2-(4-Chlorophenyl)-4-oxothiazolidin-3-yl)-2-(4-oxo-5-(thiophen-2-yl)thieno[2,3-d]-pyrimidin-3(4H)-yl) acetamide (9).* Yield 75%, m.p. 175-177 ◦C. IR (KBr, cm−1): ν 3417 (NH), 1720, 1630, 1660 (3C=O). 1H-NMR (DMSO-d6) δH: 3.81, 5.16 (2s, 4H, 2CH2), 5.90 (s, 1H, CH), 7.08–7.72 (m, 8H, thiophene-H + Ph-H), 8.50 (s, 1H, pyrimidine-H), 10.92 (s, 1H, NH, disappeared with D2O). 13CNMR (DMSO-d6) δc: 40.1, 48.2 (2C, 2CH2), 65.2 (1C, CH), 120.0, 121.8, 127.7, 128.7, 129.3, 129.4, 131.3, 133.3, 135.0, 136.5, 143.5, 149.7 (14C, thiophene-C + Ph-C), 157.2 (1C, pyrimidine-C), 163.8, 165.6, 169.9 (3C, 3C=O). Mass spectrum, *m*/*z* (EI, %): 503 (M+, 100), 505 (M<sup>+</sup> + 2, 34). Analysis for C21H15ClN4O3S3 (503.01): Calculated: C, 50.14; H, 3.01; N, 11.14; S, 19.12. Found: C, 50.02; H, 3.00; N, 11.04; S, 19.06.

*Synthesis of 2-(2-(4-oxo-5-(thiophen-2-yl)thieno[2,3-d]pyrimidin-3(4H)-yl)acetyl)-N-phenylhydrazine-1 carbothioamide (10).* A mixture of **4** (1mmol, 306 mg) and phenylisothiocynate (1 mmol, 135 mg) in dry dioxan (50 mL) was refluxed for 6 h. The obtained solid was filtered off, washed with ether, dried and recrystallized from ethanol to give thiosemicarbazide **10**. Yield 60%, m.p. 240–242 ◦C. IR (KBr, cm<sup>−</sup>1): ν 3414-3323 (NH), 1680, 1660 (2CO). 1H-NMR (DMSO-d6) δH: 4.66 (s, 2H, CH2), 6.95-7.58 (m, 9H, thiophene + Ph-H), 8.49 (s, 1H, pyrimidine-H), 8.70, 10.71, 12.78 (3s, 3H, 3NH, disappeared with D2O). 13CNMR (DMSO-d6) δc: 40.16 (CH2), 119.9, 121.8, 126.8, 127.9, 128.8, 129.1, 130.2, 131.3, 134.1, 136.5, 137.9, 149.9 (14C, thiophene + Ph-C), 156.5 (1C, pyrimimidine-C), 166.5, 169.1 (2C, 2C=O), 171.0 (1C, C=S). Mass spectrum, *m*/*z* (EI, %): 441 (M+, 100), 442 (M<sup>+</sup> + 1, 26). Analysis for C19H15N5O2S3 (441.54): Calculated: C, 51.68; H, 3.42; N, 15.86; S, 21.78. Found: C, 51.60; H, 3.40; N, 15.80; S, 21.70.

*Synthesis of N-(4-oxo-2-(phenylimino)thiazolidin-3-yl)-2-(4-oxo-5-(thiophen-2-yl)thieno[2,3-d]-pyrimidin-3(4H)-yl)acetamide (11).* A mixture of 10 (1 mmol, 441 mg) and chloroacetic acid (1 mmol, 94 mg) in absolute ethanol (30 mL) was heated under reflux for 8 h. The solid formed was filtered off and crystallized with dioxane to give thiazole derivative 11. Yield 60%, m.p. 255–257 ◦C. IR (KBr, cm<sup>−</sup>1): ν 3420 (NH), 1720, 1630 (2C=O). 1H-NMR (DMSO-d6) δH: 3.76 (s, 2H, CH2), 4.85 (s, 2H, CH2), 6.95-7.65 (m, 9H, thiophene-H + Ph-H), 8.50 (s, 1H, pyrimidine-H), 11.10 (s, 1H, NH, disappeared with D2O). 13CNMR (DMSO-d6) δc: 40.2 (CH2), 56.8 (CH2), 120.0, 122.0, 126.7, 128.7, 130.7, 131.3, 132.5, 136.4, (8C, thiophene-C), 145.2, 149.3, 150.8, 151.9 (6C, Ph-C), 156.6 (1C, pyrimidine-C), 158.1 (1C, C=N), 163.4, 165.7, 169.6 (3C, 3C=O). Mass spectrum, *m*/*z* (EI, %): 481 (M+. 100), 482 (M<sup>+</sup> + 1, 24). Analysis for C21H15N5O3S3 (481.56): Calculated: C, 52.38; H, 3.14; N, 14.54; S, 19.97. Found: C, 52.30; H, 3.10; N, 14.50; S, 19.90.

*Synthesis of 3-(2-(3-methyl-5-oxo-2,5-dihydro-1H-pyrazol-1-yl)-2-oxoethyl)-5-(thiophen-2-yl)thieno- [2,3-d]pyrimidin-4(3H)-one (12).* A mixture of compound 4 (1 mmol, 306 mg) and ethylacetoacetate (1 mmol, 130 mg) in ethanolic sodium hydroxide (0.5 mmol/50 mL) was refluxed with stirring for 6 h. The precipitate was collected by filtration and crystallized from dioxane to give pyrazole derivative 12. Yield 80%, m.p. 225–227 ◦C. IR (KBr, cm−1): ν 3417 (NH), 1650, 1630 (2C=O). 1H-NMR (DMSOd6) δH: 1.70 (s, 3H, CH3), 4.35 (s, 2H, CH2), 5.65 (s, 1H, pyrazole-CH), 7.51–7.69 (m, 4H, thiophene-H), 8.46 (s, 1H, pyrimidine-H), 12.93 (s, 1H, NH, disappeared with D2O). 13C-NMR (DMSO-d6) δc: 34.4 (CH3), 47.0 (CH2), 120.0, 123.8, 127.7, 128.7, 129.4, 130.9, 132.6, 136.5 (8C, thiophene), 98.3, 151.9 (2C, Pyrazole-C), 156.6 (1C, pyrimidine-C), 163.4, 166.5, 169.7 (3C, 3C=O). Mass spectrum, *m*/*z* (EI, %): 372

(M+, 100), 373 (M<sup>+</sup> + 1, 18). Analysis for C16H12N4O3S2 (372.42): Calculated: C, 51.60; H, 3.25; N, 15.04; S, 17.22. Found: C, 51.50; H, 3.20; N, 15.00; S, 17.16.

*Synthesis of N -acetyl-2-(4-oxo-5-(thiophen-2-yl)thieno[2,3-d]pyrimidin-3(4H)-yl)aceto-hydrazide (13).* A solution of **4** (1 mmol, 306 mg) in a mixture of AcOH acid and Ac2O (50 m, 1:1 *v*/*v*) was refluxed with stirring for 8 h. The reaction mixture was dropped onto iced-water. The obtained precipitate was filtered off, washed with water, and recrystallized from ethanol to give N-acetyl derivative **13**. Yield 70%, m.p. 235–237 ◦C. IR (KBr, cm−1): ν 3369-3232 (NH, NH), 1732 (C=O). 1H-NMR (DMSOd6) δH: 1.86 (s, 3H, CH3), 4.68 (s, 2H, CH2), 7.10–7.70 (m, 4H, thiophene-H), 8.44 (s, 1H, pyrimidine-H), 10.70, 10.82 (2s, 2NH, disappeared with D2O). 13C-NMR (DMSO-d6) δc: 20.1 (CH3), 50.1 (CH2), 119.9, 122.0, 122.2, 126.8, 127.8, 128.7, 131.3, 136.4 (8C, thiophene-C), 157.0 (1C, pyrimidine-C), 162.8, 165.6, 169.1 (3C, 3C=O). Mass spectrum, *m*/*z* (EI, %): 348 (M+, 100), 349 (M<sup>+</sup> + 1, 16). Analysis for C14H12N4O3S2 (348.40): Calculated: C, 48.27; H, 3.47; N, 16.08; S, 18.40. Found: C, 48.20; H, 3.40; N, 16.00; S, 18.32.

*Synthesis of compounds 14 and 15.* To a mixture of **13** (1 mmol, 348 mg) and ethylcyanoacetate or malononitrile (1 mmol) in EtOH (40 mL), a few drops of triethylamine were refluxed for 8 h, poured into iced-water. The precipitate was filtered off, and crystallized from EtOH to obtain compounds 14 and **15**, respectively.

*N-(6-Amino-4-hydroxy-2-oxopyridin-1(2H)-yl)-2-(4-oxo-5-(thiophen-2-yl)thieno[2,3-d]-pyrimidin-3(4H) yl)acetamide (14).* Yield 75%, m.p. 280–282 ◦C. IR (KBr, cm<sup>−</sup>1): ν 3492-3196 (OH, NH2, NH), 1420, 1680, 1653 (3 C=O). 1H-NMR (DMSO-d6) δH: 4.15 (s, 2H, CH2), 4.95, 5.70 (2s, 2H, 2CH), 6.50 (s, 2H, NH2, disappeared with D2O), 7.15–7.74 (m, 4H, thiophene-H), 8.50 (s, 1H, pyrimidine-H), 10.25 (s, 1H, OH, disappeared with D2O), 10.65 (s, 1H, NH, disappeared with D2O). 13C-NMR (DMSO-d6) δc: 49.00 (CH2), 116.1, 120.2, 121.8, 126.7, 127.9, 128.9, 131.3, 136.5 (8C, thiophene-C), 86.5, 100.2, 145.5, 158.7 (4C, pyridine-C), 156.2 (1C, pyrimidine-C), 164.1, 165.5, 169.5 (3C, 3CO). Mass spectrum, *m*/*z* (EI, %): 415 (M<sup>+</sup>, 75). Analysis for C17H13N5O4S2 (415.44): Calculated: C, 49.15; H, 3.15; N, 16.86; S, 15.43. Found: C, 49.05; H, 3.10; N, 16.80; S, 15.35.

*N-(2,4-Diaminopyridin-1(2H)-yl)-2-(4-oxo-5-(thiophen-2-yl)thieno[2,3-d]-pyrimidin-3(4H)-yl)acetamide (15).* Yield 75%, m.p. 290–292 ◦C, IR (KBr, cm−1). ν 3460-3345 (NH, NH2), 1680, 1653 (2C=O). 1H-NMR (DMSO-d6) δH: 4.13 (s, 2H, CH2), 4.60 (s, 2H, NH2, exchangeable with D2O), 5.60–6.10 (m, 4H, 4CH), 7.10–8.72 (m, 4H, thiophene-H), 8.47 (s, 1H, pyrimidine-H), 9.12 (s, 2H, NH2, disappeared with D2O), 10.32 (s, 1H, NH, disappeared with D2O). 13C-NMR (DMSO-d6) δc: 48.00 (CH2), 115.1, 120.0, 121.8, 126.8, 127.7, 128.7, 131.3, 136.5 (8C, thiophene-C), 78.5, 105.1, 118.5, 139.7, 150.0 (5C, pyridine-C), 157.0 (1C, pyrimidine-C), 165.6, 169.3 (2C, 2CO). Mass spectrum, *m*/*z* (EI, %): 400 (M+, 50). Analysis for C17H16N6O2S2 (400.48): Calculated: C, 50.99; H, 4.03; N, 20.99; S, 16.01. Found: C, 50.90; H, 4.00; N, 20.90; S, 15.95.

#### *3.2. Biological Evaluation*

#### 3.2.1. Cytotoxic Assay

"Human breast cancer cells (MCF-7) and normal non-tumorigenic MCF-10A cells were used throughout the work. Cells were obtained from ATCC, Gaithersburg, MD, USA. Standard MTT assay was used to explore the possible cytotoxic effects of the synthesized compounds [41,42]. Medium composition, cultivation conditions and assay performance were exactly the same as our previous work [43,44]. Cells were treated with varying concentrations (0–1 μM) of the compounds prepared in DMSO. After MTT addition, the absorbance of the dissolved formazan crystals was read at 570 nm [45]. The IC50 values were obtained with linear regression equations using Origin® 6.1 software (Origin Lab Corporation, Northampton, MA, USA)".

#### 3.2.2. Human Breast Cancer Xenograft Animal Model

"In this work, MCF-7 mouse xenograft model was used. The animal protocol was approved by the Institutional Animal Use Ethics and Care Committee of the University of Alabama at Birmingham (50-01-05-08B). Female athymic pathogen-free nude mice (nu/nu, 4–6 weeks) were purchased from Frederick Cancer Research and Development Center (Frederick, MD, USA). To establish MCF-7 human breast cancer xenografts, each of the female nude mice was first implanted with a 60-day (subcutaneously, s.c.) slow release estrogen pellet (SE-121, 1.7 mg 17α-estradiol/pellet; Innovative Research of America, Sarasota, FL, USA). After 24 h, grown cells were harvested, washed twice with serum-free medium, resuspended, and injected subcutaneously (5 million cells/0.2 mL) into the left inguinal area of the mice. During the experiment, animals were checked periodically and the percentages of tumor growth, as well as animal weights, were recorded. Every 48 h, the size of the tumor was recorded by measuring two perpendicular diameters of the tumor and tumor volume was calculated according to Wang et al. [46]".

"Treated animals and control groups (7–10 mice/group) received different compounds and vehicles, respectively. The tested compounds were dissolved in PEG400:ethanol:saline (57.1:14.3:28.6, *v*/*v*/*v*), and injected intraperitoneal (i.p.) at doses of 5 and 10 μM/kg/d, 3 d/wk for 3 weeks. The higher dose (10 μM/kg/d, 3 d/wk) inhibited MCF-7 xenograft tumor growth".

#### 3.2.3. Pim-1 Kinase Inhibitory Activity

#### Materials and Methods

"The kinase inhibitory activity of the synthesized compounds was determined using the Kinexus compound profiling service, Canada. Compounds were tested at 50 nM concentration. The kinase used was cloned, expressed and purified using proprietary methods. Quality control testing is routinely performed to ensure compliance to acceptable standards. 33P-ATP was purchased from PerkinElmer. All other materials were of standard laboratory grade".

#### Pim-1 Kinase Protein Assay

"The protein kinase target profiling was executed via employing a radioisotope assay format. All the assays were performed in a prepared radioactive working area. The protein kinase profiling assays were performed at room temperature for 20–30 min in a final volume of 25 μL according to the reported method [47]".

#### **4. Conclusions**

During the current work, different new **14** thiophenyl thienopyrimidinone derivatives were synthesized using variable cyclization and condensation routes. The synthesized derivatives showed promising potential biological potentials for their use in the pharmaceutical industry. They revealed higher in vitro cytotoxic activities against breast cancer cell line MCF-7 in comparison to known drugs, e.g., Cisplatin and Milaplatin. Furthermore, the prepared derivatives proved to be less toxic against the non-tumorigenic MCF-10A cell line. In vivo studies also showed potential reduction in tumor growth in animal models for all synthesized derivatives compared to control animals. Finally, mechanism of action studies showed that the newly synthesized derivatives exert their anticancer effects through the inhibition of pim-1 kinase enzymes.

**Author Contributions:** M.F.E.-S., A.A.I., A.A.F. and H.M.H. performed most of the experiments; A.E.-G.E.A. and M.A.A. analyzed the data; E.A.E. contributed to the anticancer activity assays; All authors read and approved the final manuscript.

**Funding:** The authors are grateful to the Deanship of Scientific Research, King Saud University for funding through Vice Deanship of Scientific Research Chairs.

**Acknowledgments:** The authors are grateful to the Deanship of Scientific Research, King Saud University for funding through Vice Deanship of Scientific Research Chairs.

**Conflicts of Interest:** The authors declare no conflict of interest.

### **References**


**Sample Availability:** Samples of the all compounds are available from the authors.

© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

*Article*

## **Cytotoxic E**ff**ects of Newly Synthesized Heterocyclic Candidates Containing Nicotinonitrile and Pyrazole Moieties on Hepatocellular and Cervical Carcinomas**

### **Amira A. El-Sayed 1,\*, Abd El-Galil E. Amr 2,3,\*, Ahmed K. EL-Ziaty <sup>1</sup> and Elsayed A. Elsayed 4,5**


Academic Editor: Qiao-Hong Chen Received: 28 April 2019; Accepted: 16 May 2019; Published: 22 May 2019

**Abstract:** In this study, a series of newly synthesized substituted pyridine **9, 11**–**18**, naphthpyridine derivative **10** and substituted pyrazolopyridines **19**–**23** by using cycnopyridone **8** as a starting material. Some of the synthesized candidates are evaluated as anticancer agents against different cancer cell lines. In vitro cytotoxic activities against hepatocellular and cervical carcinoma cell lines were evaluated using standard MTT assay. Different synthesized compounds exhibited potential in vitro cytotoxic activities against both HepG2 and HeLa cell lines. Furthermore, compared to standard positive control drugs, compounds **13** and **19** showed the most potent cytotoxic effect with IC50 values of 8.78 ± 0.7, 5.16 ± 0.4 μg/mL, and 15.32 ± 1.2 and 4.26 ± 0.3 μg/mL for HepG2 and HeLa cells, respectively.

**Keywords:** cyanopyridone; substituted pyridine; pyridotriazine; pyrazolopyridine; thioxotriazopyridine; anticancer activity; HepG2; HeLa

#### **1. Introduction**

Multicomponent reactions (MCR) "in which three or more starting materials react to form a product" play a significant role in the synthesis of heterocyclic compounds with pharmaceutical and chemical importance [1]. Several nicotinonitriles have been constructed via (MCR) and showed antitumor [2], antimicrobial [3], and antioxidant [4] activities. Also nicotinonitriles have been utilized as a scaffold for the synthesis of heterocyclic compounds containing a pyridine moiety with antimicrobial and antiviral activities [5]. A series of nicotinonitriles **1**–**3** (Figure 1) and have been synthesized and anti-proliferative [6], anti-Alzheimer's [7], and anti-inflammatory [8] activities.

**Figure 1.** Nicotinonitriles with anti-proliferative, anti-Alzheimer's anti-inflammatory activities.

The pyrazole moiety is both pharmacologically and medicinally significant [9]. A series of pyrazoles **4**–**7** (Figure 2) has been reported as anti-inflammatory activity by Bekhit et al. [10], they observed that the synthesized pyrazoles showed more anti-inflammatory activity than the standard indomethacin [11]. Trisubstituted pyrazoles have been constructed by Christodoulou et al. (2010) [11] and evaluated as anti-angiogenic agents; these derivatives showed a potent anti-angiogenic efficacy and moreover inhibited the growth of Mammary gland breast cancer (MCF-7) and cervical carcinoma (Hela) [12]. Recently novel derivatives of pyrazoles **5**,**6** have been prepared as antimicrobial [13] and anticonvulsant [14] agents. The pyrazole **7** has been prepared by Bonesi et al. (2010) [15] and showed effective Angiotensin -1-Converting Enzyme (ACE) inhibitor activity [15].

**Figure 2.** Pyrazoles as anti-inflammatory antimicrobial and anticonvulsant activities.

Based on the previous facts about the importance of pyrazoles and nicotinonitriles in medicinal chemistry, we have herein synthesized of some novel heterocyclic candidates containing nicotinonitrile and pyrazole moieties and tested their anticancer activity.

#### **2. Results**

#### *2.1. Chemistry*

The nicotinonitriles were obtained by two different ways, from the reaction of chalcone with ethylcyanoacetate, ammonium acetate and drops of piperidine as a base and from one pot four components reaction of methylketone, aldehyde, ethylcyanoacetate, ammonium acetate and drops of piperidine as a base [15]. In prolongation of our work in the synthesis of heterocyclic compounds and evaluation of their medicinal importance [16–27] and based on the literature survey about the pharmacological and medicinal importance of pyrazoles and nicotinonitriles, we have devoted our efforts to design and synthesize novel heterocyclic compounds containing pyrazol and nicotine-nitrile moieties, 4-(3-(4-fluorophenyl)-1-phenyl-1*H*-pyrazol-4-yl)-2-hydroxy-6-(naphthalen-1-yl)-nicotinenitrile **8** has been obtained by reacting of 1-acetylnaphthalene (**A**), 3-(4-fluorophenyl)-1-phenyl-1*H*pyrazole-4-carbaldehyde (**B**), ethyl 2-cyanoacetate, ammonium acetate and piperidine (Scheme 1).

**Scheme 1.** Synthesis of compound **8** as starting material.

The structure of the nicotinonitrile **8** has been confirmed from its spectral data. IR spectrum showing absorption frequencies at <sup>ν</sup> 3159 cm−1, 2220 cm−<sup>1</sup> and <sup>ν</sup> 1647 cm−<sup>1</sup> for OH, C≡N and C=N groups, respectively. Also, 1H-NMR spectrum of the assigned compound displayed signals at δ 12.89 ppm (disappeared with D2O) corresponding to acidic OH. A compelling evidence for the structure of **8** was provided by 13C-NMR spectrum that showed a singlet signal at δ 149.8, 139.3 and 139.3 ppm for C-OH, C=N and C≡N groups respectively. Mass spectra of **<sup>8</sup>** showed [M+] at *m*/*z* (%) 482 (22). Treatment of **8** with ethylchloroacetate afforded compound **9**, which was hydrazinolysis with NH2NH2 to give the corresponding cyclized product **10**.

Remediation of the nicotinonitrile derivative **8** with malononitrile in the presence of few drops of piperidine afforded 1,8-naphthyridine-3-carbonitrile derivative **11**. Chlorination of **8** by a mixture of (POCl3/PCl5) afforded 2-chloronicotinonitrile derivative **12**, which was reacted with malono nitrile as a carbon nucleophile gave the nicotinonitrile derivative **13**. Reaction of **12** with primary and secondary amines, namely, *o*-aminothiophenol, morpholine, 1-methylpiperazine and hydrazine hydrate gave novel nicotinonitriles **14**, **15a**, **b** and **16** (Scheme 2). The mechanism formation route of compound **11** has been shown in Figure 3.

**Figure 3.** The mechanism formation route of compound **11**.

**Scheme 2.** Synthetic route for compounds **9**–**16**.

Compound **16** was utilized as a building block for novel nicotinonitriles containing two pyrazole moieties. 2-Pyrazolyl nicotinonitrile derivatives **17** and **18** were prepared by treatment of **16** with acetyl acetone and 4,4,4,-trifluoro-1-(thiophen-2-yl)butane-1,3-dione, respectively. Treatment of **16** with acetic anhydride and acetic acid afforded pyrazolopyridine derivative **19**. The derivative **16** was treated with acetic anhydride to afford the *N*-acetyl pyrazolopyridine as a sole product **20**. The structure of compound **20** was confirmed chemically by acetylation of the amino pyrazopyridine **19** (Scheme 3).

Treatment of **16** with 4-chlorobenzaldehyde and/or tetrachlorophthalic anhydride in the presence of acetic acid afforded the cyclized **19** followed by condensation to give the Schiff's base **21** and tetra chloroisoindoline **22**, respectively. The structures of **21** and **22** were confirmed chemically by condensation of compound **19** with 4-chlorobenzaldehyde and/or tetrachlorophthalic anhydride to provide compounds **21** and **22**, respectively. Treatment of hydrazinyl derivative **16** with CS2 in the presence of alcoholic KOH provided thioxotriazolo pyridine derivative **23** (Scheme 3).

**Scheme 3.** Synthetic route for compounds **17**–**23**.

#### *2.2. Cytotoxic Activity*

The newly synthesized compounds were screened for their anticancer potentials against hepatocellular carcinoma HepG2 and cervical carcinoma HeLa. The cytotoxicity of the compounds was determined using MTT assay and DOX as a positive control [28–31].

The cytotoxic activities of the novel synthesized compounds **8**–**23** were estimated and the obtained results are presented in Figure 4. In general, it can be seen that all synthesized compounds exhibited cytotoxic activities against both tested cancer cell lines. Moreover, it can be seen that both cells reacted in a dose-dependent manner toward the applied concentrations. Additionally, both tested cell lines varied in their response toward different synthesized compounds. Furthermore, based on the IC50 values (Table 1) obtained for the tested compounds, it can be seen that cytotoxic activities ranged from very strong to non-cytotoxic. Compounds **13** and **19** exhibited the most potent cytotoxic effect (very strong activity) with IC50 8.78 ± 0.7, 5.16 ± 0.4 μg/mL, and 15.32 ± 1.2 and 4.26 ± 0.3 μg/mL for HepG2 and HeLa cells, respectively. Furthermore, it can be noticed that **Cpd. 19** exhibited more or less stronger activity similar to DOX towards HepG2 cells, (IC50 5.16 ± 0.4 and 4.50 ± 0.2 μg/mL, respectively). On the other hand, it was stronger by about 23.5% than DOX against HeLa cells (4.50 ±

0.2 and 5.57 ± 0.4 μg/mL, respectively). Additionally, **Cpd. 18** showed very strong activity towards HeLa cells with IC50value of 7.67 ± 0.6 μg/mL, while it exhibited strong activity towards HepG2 cells (IC50 16.70 ± 1.3 μg/mL). Moreover, **Cpd. 14** showed strong cytotoxic activities towards both tested cell lines (IC50values 12.20 ± 1.0 and 19.44 ± 1.4 μg/mL for HepG2 and HeLa cells, respectively). Meanwhile, **Cpds. 16** and **22** showed moderate and strong activities towards both cell lines. **Cpd. 16** showed IC50value of 33.45 ± 2.3 and 10.37 ± 0.9 μg/mL against HepG2 and HeLa cells, respectively. Also, **Cpd. 22** showed IC50 of 26.64 ± 1.9 and 9.33 ± 0.8 μg/mL for HepG2 and HeLa cells, respectively. On the other hand, **Cpd. 17** showed strong activity towards HepG2 cells (IC50 20.00 ± 1.7 μg/mL) and moderate activity towards HeLa cells (IC50 35.58 ± 2.6 μg/mL). Finally, **Cpds. 9**, **10**, **11**, **12**, **15a**, **b**, **17**, **20**, **21** and **23** showed activities ranging from moderate to non-cytotoxic, with IC50 values ranging from 24.83 ± 1.8 to >100 μg/mL.

**Figure 4.** Relative viabilities of HepG2 and HeLa cells as affected by different synthesized compounds.


**Table 1.** IC50 values obtained for the tested compounds against both HepG2 and HeLa cell lines.

\* IC50: 1–10 is (very strong), 11–20 is (strong), 21–50 is (moderate), 51–100 is (weak) and above is 100 (non-cytotoxic).

#### **3. Discussion**

During current work, multi-component reaction strategy was used to synthesize of compound **8**, which was used as a building block for preparing **16** new derivatives. The cytotoxic potential of the new prepared compounds has been evaluated against HepG2 and HeLa cells. Results obtained showed potential cytotoxic activities against both cell lines. Compounds **13** and **19** showed the most cytotoxic effects (IC50 8.78 ± 0.7 and 5.16 ± 0.4 μg/mL, for HepG2 cells, and 15.32 ± 1.2 and 4.26 ± 0.3 μg/mL for HeLa cells, respectively). Also, results showed that both tested cell lines varied in their response toward different synthesized compounds. This can be attributed to the inherent differences in both cell lines in terms of membrane structure and organization, hence different cell lines react differently towards different compounds [32–35].

Different activities of the prepared compounds may be attributed to the structure–activity relationship of these compounds. It can be seen that conversion of **Cpd. 12** to **13**, **14** and **16**, **18**, **19** and **22** altered the cytotoxicity from weak to moderate and strong activity towards two cell lines. This explained due to the introduction of two more nitrile groups, which significantly increased the activity. Compound **14** exhibited very strong activity due to the entity of the SH and NH groups, which may be added to any unsaturated group in DNA (thia or aza Michael addition) or the formation of hydrogen bonds with either one of the nucleo-bases of the DNA, thus causing DNA damage. Furthermore, the cytotoxicity of **Cpd. 16** may be due to the intermolecular hydrogen bonding of NH and NH2 groups with DNA moieties. Additionally, conversion of **Cpd. 16** to **18**, **19** and **22** increased their cytotoxic activities against both cell lines. Introducing thiophene ring increases the cytotoxic effect of **Cpd. 18** beside the effect of the pyrazole ring and the trifluoromethyl group. Additionally, introducing pyrazole ring bearing NH2 group to **Cpd. 16** increases the cytotoxic effect of **Cpd. 19** to very strong effect against both cell lines. The introduction of chloroiso- indoline-1,3-dione increases the cytotoxic effects of **Cpd. 22**. The chloro- group, with more electron withdrawing properties, may be the crucial for tumor cell inhibition beside the effect of the isoindoline-1,3-dioneas moderate cytokine inhibitor in cancer cells.

#### **4. Materials and Methods**

#### *4.1. Chemistry*

"Melting points reported are inaccurate. IR spectra were registered on Shimadzu FT-IR 8300 E (Shimadzu Corporation, Kyoto, Japan) spectrophotometer using the (KBr) disk technique. 1H-NMR spectra were determined on a Varian Spectrophotometer at 400 MHz using (TMS) as an internal reference and DMSO-d6 as solvent using (TMS) as internal standard. All chemical shifts (δ) are uttered in ppm. The mass spectra were determined using (MP) model MS-5988 and Shimadzu single focusing mass spectrophotometer (70 eV). Elemental analysis was investigated by Elemental analyzer Vario EL III".

4.1.1. Synthesis of 4-(3-(4-fluorophenyl)-1-phenyl-1*H*-pyrazol-4-yl)-2-hydroxy-6-(naphthalen-1-yl) nicotinenitrile (**8**)

A mixture of 1-acetyl naphthalene (**A**) (1.7 g, 0.01 mol), ethyl cyanoacetate (1.3 g, 0.01 mol), aldehyde (**B**) (3.6 g, 0.01 mol), ammonium acetate (5.40 g, 0.07 mol) and three drops of piperidine in ethanol (20 mL) was heated under reflux for 3 h. The obtained precipitate was filtered off, washed with cold water, dried and crystallized from ethanol/dioxane to give compound **8**. Yield 75%, yellow powder, m.p. > 300 ◦C; IR (KBr): <sup>ν</sup> (cm−1) 3159 (OH), 2220 (C≡N), 1647 (C=N); 1H-NMR (DMSO-d6): <sup>δ</sup> (ppm) 12.89 (s, 1H, OH, disappeared by D2O), 9.80 (s, 1H, pyrazole-H), 8.39–7.78 (m, 7H, Ar-H for naphthalene), 7.75–7.37 (m, 10H, Ar-H). 13C NMR (DMSO-d6): δ (ppm) 149.8 (C-OH), 139.3 (C=N), 119.3 (C≡N), 139.4, 134.3, 133.8, 133.5, 131.6, 131.2, 131.0, 130.9, 130.4, 130.3, 130.2, 129.9, 129.4, 129.3, 129.2, 129.1, 128.9, 128.2, 127.8, 127.6, 127.0, 125.6, 125.1, 117.4, 114.8 (Ar-CH), 40.6, 39.9 (aliph-C); MS *m*/*z* (ESI): 482 [M<sup>+</sup>] (22), 465 (21), 440 (12), 237 (100), 204; Anal. Calcd. for C31H19FN4O (482.50): C, 77.17; H, 3.97; N, 11.61. Found C, 76.98; H, 3.78; N, 11.52%.

4.1.2. Synthesis of ethyl 2-(3-cyano-4-(3-(4-fluorophenyl)-1-phenyl-1*H*-pyrazol-4-yl)-6-(naphthalene-1 yl)-2-oxopyridin-1(2*H*)-yl)acetate (**9**)

A mixture of **8** (4.84 g, 0.01 mol), ethylchloroacetate (1.22 g, 0.01 mol) and K2CO3 (2.2 g, 0.015 mol) in (CH3)2O (40 mL) was heated under reflux for 24 h, concentrated and poured on water; the obtained precipitate was collected by filteration off, dried and crystallized from EtOH/dioxane to give **9**. Yield 74%, m.p. 158–160 ◦C; IR (KBr): <sup>ν</sup> (cm<sup>−</sup>1) 2204 (C≡N), 1751 (C=O ester), 1651 (C=O pyridine); 1H-NMR (DMSO-d6): δ (ppm) 9.15 (s, 1H, pyrazole-5H), 8.10–7.49 (m, 7H, Ar-H for naphthalene), 7.48–7.33 (m, 10H, Ar-H), 4.16 (q, 2H, -CH2 ester), 3.40 (s, 2H, -CH2), 1.20 (t, 3H, -CH3, ester); MS *m*/*z* (ESI): 568 [M+] (2.5), 495 (65), 237 (80), 127 (100); Anal. Calcd. for C35H25FN4O3 (568.60): C, 73.93; H, 4.43, N, 9.85. Found C, 73.80; H, 4.21; N, 9.64%.

4.1.3. Synthesis of 8-(3-(4-fluorophenyl)-1-phenyl-1*H*-pyrazol-4-yl)-6-(naphthalen-1-yl)-3-oxo-3,4 dihydro-2*H*-pyrido[2,1-c][1,2,4]triazine-9-carbonitrile (**10**)

A mixture of **9** (5.7 g, 0.01 mol), NH2NH2ηH2O (2 mL, 0.04 mol) and EtOH (20 mL) was heated under reflux for 3 h. The outward appearance solid was filtered off, dried and crystallized from EtOH/dioxane to give **10**. Yield 71%, yellow powder, m.p. > 300 ◦C; IR (KBr): ν (cm−1) 3209 (NH), 2218 (C≡N), 1647 (C=O); 1H-NMR (DMSO-d6): <sup>δ</sup> (ppm) 12.38 (s, 1H, NH, disappeared in D2O), 9.13 (s, 1H, pyrazole-5H), 8.87–7.65 (m, 7H, Ar-H for naphthalene), 7.63–6.85 (m, 10H, Ar-H), 6.10 (s, 2H, CH2). 13C-NMR (DMSO-d6): δ (ppm) 165.8 (C=O), 139.7 (C=N), 136.1 (C=N), 133.8, 133.4, 131.7, 130.9, 130.8, 130.7, 130.6, 130.3, 130.2, 130.1, 129.9, 129.7, 129.2, 129.1, 128.8, 128.5, 128.1, 127.3, 126.8, 125.8, 125.6, 119.2, 119.1, 118.9, 118.5, 117.6 (Ar-CH), 119.3 (C≡N), 40.5, 39.9 (2CH), 17.6 (CH2); MS *m*/*z* (ESI): 519 [M<sup>+</sup> <sup>−</sup> OH] (82), 393 (64), 284 (100), 237 (68), 127 (56); Anal. Calcd. for C33H21FN6O (536.50): C, 73.87; H, 3.94; N, 15.66. Found C, 73.68; H, 3.24; N, 15.06%.

4.1.4. Synthesis of 5-(3-(4-fluorophenyl)-1-phenyl-1*H*-pyrazol-4-yl)-7-(naphthalen-1-yl)-2-oxo-1,2 dihydro-1,8-naphthyridine-3-carbonitrile (**11**)

Refluxing of compound **8** (4.84 g, 0.01 mol) with malononitrile (0.015 mol) in ethanol (20 mL) in the presence of drops of TEA for 5 h, then cooled, poured on ice/water, neutralized with drops of conc. HCl. The obtained solid was collected by filtration, crystallized from EtOH/dioxane to afford **11**. Yield 71%, pale brown powder, m.p. > 300 ◦C; IR (KBr): ν (cm−1) 3386, 3273 (NH2), 3158 (NH), 2218 (C≡N), 1646 (C=O), 1H-NMR (DMSO-d6): <sup>δ</sup> (ppm) 12.89 (s, 1H, NH, disappeared by D2O), 9.08 (s, 1H, pyrazole-5H), 8.07–7.61 (m, 7H, Ar-H for naphthalene), 7.60–7.37 (m, 10H, Ar-H), 6.22 (s, 2H, NH2, disappeared in D2O). 13C-NMR (DMSO-d6): δ (ppm) 149.9 (C=O), 139.3 (C=N), 133.8, 133.5, 131.2, 131.1, 131.00 (2), 130.9, 130.4, 130.3 (2), 130.2, 129.9 (2), 129.4, 129.1, 128.9 (2), 128.2, 127.8(2), 127.6, 127.1 (2), 125.6, 125.2 (2), 117.4, 116.8, 110.0 (Ar-CH), 119.3 (C≡N), 40.6, 39.9 (2CH); MS *<sup>m</sup>*/*<sup>z</sup>* (ESI): 532 [M<sup>+</sup> <sup>−</sup> NH3] (82), 516 (76), 440 (28), 310 (20), 237 (100); Anal. Calcd. for C34H21FN6O (548.50): C, 74.44; H, 3.89; N, 15.32. Found C, 74.24; H, 3.25; N, 14.98%.

4.1.5. Synthesis of 2-chloro-4-(3-(4-fluorophenyl)-1-phenyl-1*H*-pyrazol-4-yl)-6-(naphthalen-1-yl) nicotinenitrile (**12**)

A mixture of **8** (4.82 g, 0.01 mol), PCl5 (3 g, 0.03 mol) and POCl3 (5 mL, 0.03 mol) was heated under reflux for 8 h, then it was poured on crushed ice. The formed solid was filtered off, dried and crystallized from EtOH/dioxane to give **12**. Yield 61%, yellow powder, m.p. 164–166 ◦C; IR (KBr): ν (cm−1) 2227 (C≡N), 1628 (C=N); 1H-NMR (DMSO-d6): <sup>δ</sup> (ppm) 9.16 (s, 1H, pyrazole-5H), 8.35–7.63 (m, 7H, Ar-H for naphthalene), 7.61–7.39 (m, 10H, Ar-H). 13C-NMR (DMSO-d6): δ (ppm) 152.7, 150.0, 148.4, 139.2 (C=N), 135.3(C=N), 133.8, 131.5, 131.0, 130.4, 130.3, 130.2, 129.8, 129.5, 129.2 (2), 129.1, 127.9, 127.6, 126.9, 125.8 (2), 125.4, 125.1, 119.3 (C≡N), 116.6, 115.5, 107.8 (Ar-CH), 40.6, 39.9 (2CH); MS *m*/*z* (ESI): 503 [M<sup>+</sup> + 2] (6), 501 [M<sup>+</sup>] (50), 465 (100), 237 (82); Anal. Calcd. for C31H18ClFN4 (500.90): C, 74.32; H, 3.62; N, 11.84. Found C, 74.12; H, 3.26; N, 11.42%.

4.1.6. Synthesis of 2-[4-(3-(4-fluorophenyl)-1-phenyl-1*H*-pyrazol-4-yl)-6-(naphthalen-1-yl)-3-cyanopyridinyl]malononitrile (**13**)

To a solution of **12** (5.0 g, 0.01 mol) in EtOH (20 mL), malononitrile (0.01 mol) and TEA (1 mL) were added. The reaction mixture was heated under for 3 h. After cooling, it was poured on water and neutralized with diluted HCl. The obtained solid was separated by filtration, washed with water, dried and crystallized from EtOH/dioxane to yield **13**. Yield 76%, pale brown powder, m.p. 194–196 ◦C; IR (KBr): <sup>ν</sup> (cm−1) 2203 (C≡N), 1H-NMR (DMSO-d6): <sup>δ</sup> (ppm) 9.15 (s, 1H, pyrazole-5H), 8.11–7.66 (m, 7H, Ar-H for naphthalene), 7.65–7.36 (m, 10H, Ar-H), 7.07 (s, 1H, CH of CH(CN)2), MS *m*/*z* (ESI): 530 [M<sup>+</sup>] (12), 440 (100), 237 (76), 204 (31); Anal. Calcd. for C34H19FN6 (530.50): C, 76.97; H, 3.61; N, 15.84. Found C, 76.78; H, 3.42; N, 15.24%.

#### 4.1.7. Synthesis of **14** and **15a**,**b**

A mixture of 2-chloronicotinonitrile **12** (5.0 g, 0.01 mol) and the appropriate amine, namely, o-aminothiophenol, morpholine or 2-methylpiperidine (0.01 mol) in EtOH (20 mL) was heated under reflux for 3 h, then it was poured on cold water, filtered off and crystallized from EtOH/dioxane to afford **14** and **15a,b**, respectively.

4-(3-(4-Fluorophenyl)-1-phenyl-1*H*-pyrazol-4-yl)-2-(2-mercaptophenylamino)-6-(naphthalen-1-yl) nicotinonitrile (**14**). Yield 74%, brown powder, m.p. 108–110 ◦C; IR (KBr): ν (cm−1) 3330 (NH), 2208 (C≡N), 1H-NMR (DMSO-d6): <sup>δ</sup> (ppm) 9.29 (s, 1H, pyrazole-5H), 9.06–8.54 (m, 4H, Ar-H, thionyl-H), 8.26–7.66 (m, 7H, Ar-H for naphthalene), 7.60–6.66 (m, 10H, Ar-H), 3.34 (s, 1H, NH, disappeared in D2O), 1.20 (s, 1H, SH, disappeared in D2O). MS *m*/*z* (ESI): 589 [M+] (32), 465 (82), 441 (62), 237 (100), 127(12), 124 (20); Anal. Calcd. for C37H24FN5O (589.60): C, 75.36, H, 4.10; N, 11.88. Found C, 75.18; H, 4.05; N, 11.73%.

4-(3-(4-Fluorophenyl)-1-phenyl-1*H*-pyrazol-4-yl)-2-morpholino-6-(naphthalen-1-yl)nicotinonitrile (**15a**). Yield 65%, pale brown powder, m.p. 130–133 ◦C; IR (KBr): <sup>ν</sup> (cm<sup>−</sup>1) 2226 (C≡N), 1H-NMR (DMSO-d6): δ (ppm) 9.16 (s, 1H, pyrazole-5H), 8.71–7.56 (m, 7H, Ar-H for naphthalene), 7.55–7.15 (m, 10H, Ar-H), 3.76 (t, 4H, *J* = 8.8 Hz), 3.05 (t, 4H, *J* = 8.8 Hz), MS *m*/*z* (ESI): 552 [M+] (52), 465 (28), 237 (100), 230 (7), 127 (12), 87 (22); Anal. Calcd. for C35H26FN5O (551.60): C, 76.21; H, 4.75; N, 12.70. Found C, 75.98; H, 4.26; N, 12.31%.

4-(3-(4-Fluorophenyl)-1-phenyl-1*H*-pyrazol-4-yl)-2-(4-methylpiperazin-1-yl)-6-(naphthalen-1-yl) nicotinonitrile (**15b**). Yield 61%, brown powder, m.p. 156–158 ◦C; IR (KBr): ν (cm−1) 2918 (aliph-H), 2227 (C≡N), 1H-NMR (DMSO-d6): <sup>δ</sup> (ppm) 9.18 (s, 1H, pyrazole-5H), 8.71–7.65 (m, 7H, Ar-H for naphthalene), 7.64–7.12 (m, 10H, Ar-H), 3.30–3.25 (m, 4H, 2CH2), 2.43–2.23 (m, 4H, 2CH2), 2.24 (s, 3H, CH3), MS *m*/*z* (ESI): 564 [M<sup>+</sup>] (27), 538 (25), 439 (12), 237 (100), 100 (23); Anal. Calcd. for C35H29FN6 (564.60): C, 76.58, H, 5.18; N, 14.88. Found C, 75.98; H, 4.92; N, 14.72%.

4.1.8. Synthesis of 4-(3-(4-Fluorophenyl)-1-phenyl-1*H*-pyrazol-4-yl)-2-hydrazinyl-6-(naphthalen-1-yl) nicotinonitrile (**16**)

A mixture of the 2-chloronicotinonitrile **12** (5.0 g, 0.01 mol) and NH2NH2·H2O (0.04 mol) in EtOH (20 mL) was heated under reflux for 4h. The obtained solid was collected by filtration, dried and crystallized from EtOH/dioxane to yield **16**. Yield 86%, yellow powder, m.p. 164–168 ◦C; IR (KBr): <sup>ν</sup> (cm−1) 3417, 3310 (NH2), 3199 (NH), 2206 (C≡N), 1H-NMR (DMSO-d6): <sup>δ</sup> (ppm) 9.16 (s, 1H, pyrazole-5H), 8.35–7.97 (m, 7H, Ar-H for naphthalene), 7.96–6.88 (m, 10H, Ar-H), 4.82 (s, 1H, NH, disappeared in D2O), 3.43 (s, 2H, NH2, disappeared in D2O). 13C-NMR (DMSO-d6): δ (ppm) 149.3 (C-NHNH2), 148.3, 139.7 (C≡N), 139.2, 138.5, 136.1 (C=N), 135.3, 134.0, 133.8, 131.7, 131.5, 131.0, 130.9, 130.4, 130.2, 130.1, 129.5, 129.1, 128.1, 127.9, 127.6, 127.3, 126.9, 126.7, 126.4, 125.8, 125.4, 119.3 (C≡N), 118.2 (Ar-CH), 40.6, 40.0 (2CH); MS *<sup>m</sup>*/*<sup>z</sup>* (ESI): 496 [M+] (12), 465 (81), 440 (100), 237 (20), 204 (76); Anal. Calcd. for C31H21FN6 (496.55): C, 74.99; H, 4.26; N, 16.93. Found C, 74.86; H, 4.12; N, 16.78%.

#### 4.1.9. Synthesis of **17** and **18**

A mixture of **16** (4.9 g, 0.01 mol), acetylacetone or 4,4,4-trifluoro-1-(thiophen-2-yl)butane-1,3-dione (0.01 mol) in EtOH (10 mL) and AcOH (4 mL) was heated reflux for 3 h. After cooling, the solid obtained was filtered off, dried and crystallized from EtOH/dioxane to afford **17** and **18**, respectively. 2-(3,5-Dimethyl-1*H*-pyrazol-1-yl)-4-(3-(4-fluorophenyl)-1-phenyl-1*H*-pyrazol-4-yl)-6-

(naphthalen-1-yl)nicotinonitrile (**17**). Yield 85%, pale orange powder, m.p. 270–272 ◦C; IR (KBr): ν (cm<sup>−</sup>1) 2209 (C≡N), 1620 (C=N), 1H-NMR (DMSO-d6): <sup>δ</sup> (ppm) 9.24 (s, 1H, pyrazole-5H), 8.17–7.96 (m, 7H, Ar-H for naphthalene), 7.66–7.35 (m, 10H, Ar-H), 7.25 (s, 1H, pyrazole-4H), 2.48 (s, 6H, 2 CH3); MS *m*/*z* (ESI): 560 [M<sup>+</sup>] (13), 533 (26), 438 (62), 237 (15), 95 (100); Anal. Calcd. for C36H25FN6 (560.60): C, 77.13; H, 4.49; N, 14.99. Found C, 76.92; H, 4.32; N, 14.81%.

4-(3-(4-Fluorophenyl)-1-phenyl-1*H*-pyrazol-4-yl)-6-(naphthalen-1-yl)-2-(5-(thiophen-2-yl)-3-(trifluoromethyl)-1*H*-pyrazol-1-yl)nicotinonitrile (**18**). Yield 82%, dark yellow powder, m.p. 117–119 ◦C; IR (KBr): <sup>ν</sup> (cm−1) 2209 (C≡N), 1H-NMR (DMSO-d6): <sup>δ</sup> (ppm) 8.92 (s, 1H, pyrazole-5H), 8.03–7.89 (m, 7H, Ar-H for naphthalene), 7.59–7.54 (m, 3H, thionyl-H), 7.53–7.33 (m, 10H, Ar-H), 6.88 (s, 1H, pyrazole-4H); MS *m*/*z* (ESI): 583 [M+] (10), 465 (72), 237 (100), 299 (8), 217 (5); Anal. Calcd. for C39H22F4N6S (682.60): C, 68.61; H, 3.25; N, 12.31. Found C, 68.02; H, 3.12; N, 12.03%.

#### 4.1.10. Synthesis of **19** and **20**

A solution of **16** (4.9 g, 0.01 mol) in a mixture of AcOH/Ac2O (10 mL) or in glacial AcOH (10 mL) was refluxed for 2 h, poured on ice/water, filtered off and crystallized from EtOH/dioxane to give **19** and **20**, respectively. Also, refluxing of **19** (0.5 g, 0.01 mol) in acetic anhydride (7 mL) afforded compound **20**.

4-(3-(4-Flurophenyl)-1-phenyl-1*H*-pyrazol-4-yl)-6-(naphthalen-1-yl)-1*H*-pyrazolo[3,4-b]pyridin-3 amine (**19**). Yield 84%, pale yellow powder, m.p. 140–143 ◦C; IR (KBr): ν (cm−1) 3425–3354 (NH2),

3198 (NH), 1H-NMR (DMSO-d6): δ (ppm) 8.92 (s, 1H, pyrazole-5H), 8.22–7.90 (m, 7H, Ar-H for naphthalene), 7.66–7.34 (m, 10H, Ar–H), 5.02 (s, 2H, NH2, disappeared in D2O), 4.63 (s, 1H, NH, disappeared in D2O); MS *m*/*z* (ESI): 496 [M+] (28), 479 (76), 244 (50), 237 (100); Anal. Calcd. for C31H21FN6 (496.52): C, 74.99; H, 4.26; N, 16.93. Found C, 74.76; H, 4.15; N, 16.82%.

*N*-(4-(3-(4-Flurophenyl)-1-phenyl-1*H*-pyrazol-4-yl)-6-(naphthalen-1-yl)-1*H*-pyrazolo-[3,4-b] pyridin-3-yl)acetamide (**20**). Yield 78%, yellow powder, m.p. 138–140 ◦C; IR (KBr): ν (cm−1) 3196 (NH), 1690 (C=O), 1H-NMR (DMSO-d6): δ (ppm) 12.37 & 10.31 (s, NH, OH), 8.88 (s, 1H, pyrazole- 5H), 7.98–7.59 (m, 7H, Ar-H for naphthalene), 7.57–6.88 (m, 10H, Ar-H), 4.82 (s, 1H, NH, disappeared in D2O), 2.73 (s, 3H, acetyl); MS *m*/*z* (ESI): 538 [M+] (20), 479 (36), 244 (20), 237 (100); Anal. Calcd. for C33H23FN6O (538.59): C, 73.59; H, 4.30; N, 15.60. Found C, 73.28; H, 4.19; N, 15.32%.

4.1.11. Synthesis of *N*-(4-chlorobenzylidene)-4-(3-(4-fluorophenyl)-1-phenyl-1*H*-pyrazol-4-yl)-6- (naphthalen-1-yl)-1*H*-pyrazolo[3,4-b]pyridine-3-amine (**21**)

A solution of **16** or **19** (0.01 mol) in AcOH (10 mL) in the presence of 4-chlorobenzaldehyde (0.01 mol) was heated under reflux for 2 h, left to precipitate, filtered and crystallized from EtOH/ dioxane to afford **21**. Yield 58%, yellow powder, m.p. 158–160 ◦C; IR (KBr): ν (cm−1) 3192 (NH), 1H-NMR (DMSO-d6): δ (ppm) 9.89 (s, 1H, pyrazole-5H), 9.06 (s, 1H, N=C-H), 8.87–7.56 (m, 7H, Ar-H for naphthalene), 7.52–6.88 (m, 14H, Ar-H), 4.82 (s, 1H, NH, disappeared in D2O); MS *m*/*z* (ESI): 621 [M+] (15), 619 (48), 479 (20), 237 (80), 139 (35), 137 (100); Anal. Calcd. for C38H24ClFN6 (619.10): C, 73.72; H, 3.91; N, 13.57. Found C, 73.25; H, 3.82; N, 13.27%.

4.1.12. Synthesis of 2-(4-(3-(4-fluorophenyl)-1-phenyl-1*H*-pyrazol-4-yl)-6-(naphthalen-1-yl)-1*H*pyrazolo[3,4-b]-pyridin-3-yl)isoindoline-1,3-dione (**22**)

A mixture of **16** or **19** (0.01 mol) and tetrachlorophthalic anhydride (0.01 mol) in glacial acetic acid (10 mL) was refluxed for 1 h, poured on ice water, filtered off and crystallized from EtOH/dioxane to yield **22**. Yield 94%, yellow powder, m.p. 115–117 ◦C; IR (KBr): ν (cm<sup>−</sup>1) 3196 (NH), 1785, 1731 (C=O); 1H-NMR (DMSO-d6): δ (ppm) 8.87 (s, 1H, pyrazole-5H), 8.04–7.56 (m, 7H, Ar-H for naphthalene), 7.55–7.33 (m, 10H, Ar-H), 4.28 (s, 1H, NH, disappeared in D2O); Anal. Calcd. for C39H19Cl4FN6O2 (764.42): C, 61.28; H, 2.51; N, 10.99. Found C, 61.00; H, 2.42; N, 10.89%.

4.1.13. Synthesis of 7-(3-(4-fluorophenyl)-1-phenyl-1*H*-pyrazol-4-yl)-5-(naphthalen-1-yl)-3-thioxo-2,3-dihydro[1,2,4]triazolo[4,3-a]pyridine-8-carbonitrile (**23**)

Solution of hydrazinyl derivative **16** (4.9 g, 0.01 mol) in alcoholic KOH (10%, 20 mL) and CS2 (0.01 mol) was refluxed for 2 h, lift overnight, then poured on ice water, filtered off the solid obtained and crystallized from EtOH/dioxane to afford **23**. Yield 47% yellow powder, m.p. 288–290 ◦C; IR (KBr): <sup>ν</sup> (cm<sup>−</sup>1) 3192 (NH), 2218 (C≡N), 1240 (C=S); 1H-NMR (DMSO-d6): <sup>δ</sup> (ppm) 8.73 (s, 1H, pyrazole-5H), 7.97–7.63 (m, 7H, Ar-H for naphthalene), 7.53–6.77 (m, 10H, Ar-H), 3.76 (s, 1H, NH, disappeared in D2O). 13C-NMR (DMSO-d6): δ (ppm) 148.1 (C=S), 142.3, 138.7 (C=N), 133.8 (2), 133.4 (C=N), 131.7 (2), 131.2, 130.6 (2), 130.1, 129.9 (2), 129.4, 129.2 (2), 128.9, 128.4 (2), 126.9, 126.4 (2), 126.3 (2), 125.9 (2), 119.1 (C≡N), 110.0 (Ar-CH), 40.5, 39.9 (2CH); MS *m*/*z* (ESI): 538 [M+] (45), 494 (18), 479 (10), 453 (50), 237 (100); Anal. Calcd. for C32H19FN6S (538.60): C, 71.36; H, 3.56; N, 15.60. Found C, 71.31; H, 3.52; N, 15.58%.

#### *4.2. Cytotoxicity Assay*

#### 4.2.1. Materials and Cell Lines

Hepatocellular carcinoma (HepG2) and cervical Carcinoma (HeLa) cell lines, ATCC, VA, USA, were used throughout the work. All used chemicals and reagents were of high purity-cell culture grade.

#### 4.2.2. MTT Assay

Cytotoxic assay depends on the formation of purple formazan crystals by the action of dehydrogenase in living cells. Cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum, antibiotic solution (100 units/mL penicillin, 100 μg/mL streptomycin) at 37 ◦C in a 5% CO2 incubator. Cells were seeded in a 96-well plate (104 cells/well), and the plates were incubated for 48 h. Afterwards, cells were exposed to variable concentrations of prepared derivatives and incubation proceeded for further 24 h. After treatment, 20 μL of MTT solution (5 mg/mL) was added and incubated for 4 h. DMSO (100 μL/well) is added and the developed color density was measured at 570 nm using a plate reader (ELx 800, BioTek, Winuski, VT, USA). Relative cell viability was calculated as (Atreated/Auntreated) ×100 [36,37]. Results were compared with doxorubicin as a positive control.

### **5. Conclusions**

During the current investigation, we synthesized a new building block; namely 4-(3-(4-fluorophenyl)-1-phenyl-1*H*-pyrazol-4-yl)-2-hydroxy-6-(naphthalen-1-yl)nicotinonitril, with the help of multicomponent reaction systems. From that compound, a series of **16** different nicotinonitril derivatives were synthesized, and their structural and spectral data were elucidated. Furthermore, in vitro cytotoxic activities against hepatocellular and cervical carcinoma cell lines were investigated. Obtained results revealed that different synthesized compounds showed promising in vitro cytotoxic activities against both HepG2 and HeLa cell lines. Compounds **13** and **19** showed the most potent cytotoxic effect (IC50: 8.78 ± 0.7, 5.16 ± 0.4 μg/mL, and 15.32 ± 1.2 and 4.26 ± 0.3 μg/mL for HepG2 and HeLa cells, respectively.

**Author Contributions:** The listed authors contributed to this work as described in the following: A.A.E.-S. and A.K.E.-Z. synthesis, and interpreted the spectroscopic identification of the synthesized compounds, A.E.-G.E.A. and E.A.E. are interpreted the results, the experimental part and E.A.E. performed the revision before submission. All authors read and approved the final manuscript.

**Funding:** The authors are grateful to the Deanship of Scientific Research, king Saud University for funding through Vice Deanship of Scientific Research Chairs.

**Acknowledgments:** The authors are appreciative to Faculty of Science, Ain Shams University where the experimental part carried out in its laboratories and Faculty of Pharmaceutical, El-Masoura University to carry the anticancer activity in it.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


**Sample Availability:** Samples of the compounds are available from the authors.

© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

*Article*

## **Design, Synthesis and Biological Evaluation of a New Series of 1-Aryl-3-{4-[(pyridin-2 ylmethyl)thio]phenyl}urea Derivatives as Antiproliferative Agents**

**Chuanming Zhang 1, Xiaoyu Tan 1, Jian Feng 1, Ning Ding 1, Yongpeng Li 1, Zhe Jin 1, Qingguo Meng 2, Xiaoping Liu 1,\* and Chun Hu 1,\***


Academic Editor: Qiao-Hong Chen Received: 30 April 2019; Accepted: 30 May 2019; Published: 4 June 2019

**Abstract:** To discover new antiproliferative agents with high efficacy and selectivity, a new series of 1-aryl-3-{4-[(pyridin-2-ylmethyl)thio]phenyl}urea derivatives (**7a**–**7t**) were designed, synthesized and evaluated for their antiproliferative activity against A549, HCT-116 and PC-3 cancer cell lines in vitro. Most of the target compounds demonstrated significant antiproliferative effects on all the selective cancer cell lines. Among them, the target compound, 1-[4-chloro-3-(trifluoromethyl)phenyl]-3- {4-{{[3-methyl-4-(2,2,2-trifluoroethoxy)pyridin-2-yl]methyl}thio}phenyl}urea (**7i**) was identified to be the most active one against three cell lines, which was more potent than the positive control with an IC50 value of 1.53 ± 0.46, 1.11 ± 0.34 and 1.98 ± 1.27 μM, respectively. Further cellular mechanism studies confirmed that compound **7i** could induce the apoptosis of A549 cells in a concentration-dependent manner and elucidated compound **7i** arrests cell cycle at G1 phase by flow cytometry analysis. Herein, the studies suggested that the 1-aryl-3-{4-[(pyridin-2-ylmethyl)thio]phenyl}urea skeleton might be regarded as new chemotypes for designing effective antiproliferative agents.

**Keywords:** antiproliferative agent; urea; synthesis; antiproliferative activity; apoptosis

#### **1. Introduction**

Cancer is a major public health problem in developed countries and will become the most serious life-threatening disease worldwide in the near future [1]. Some advances in cancer treatment by molecule-targeted drugs, such as imatinib, gefitinib, and trastuzumab, were expected to improve cancer cure rates and also to reduce severe adverse reactions because of the high specificity of the targeted molecules, which are expressed and have critical roles in cancer cells, but not in normal cells. However, the clinical effect was found to be limited and did not last for a long time period because of the acquired resistance of the tumor cells. Furthermore, these molecules often cause on-target and/or off-target severe toxicity [2]. Therefore, the development of more target-specific therapy, with minimum toxicity, is warranted to extend disease-free survival and improve the quality of life of cancer patients.

In recent years, proton pump inhibitors (PPIs) as potential anticancer agents were intensively studied in cancer treatment. Lugini et al. compared the anti-tumor efficacy of different PPIs, including omeprazole, esomeprazole, lansoprazole, rabeprazole and pantoprazole in vitro and in vivo. The

result indicated that all the PPIs have shown different degrees of antitumor efficacy and lansoprazole showed a higher anti-tumor effect when compared to the other PPIs. [3]. Recently, the research by Zeng and Zheng et al. indicated that T-cell originated protein kinase (TOPK) activities were inhibited by pantoprazole and ilaprazole with high affinity and selectivity [4,5]. TOPK (also known as PBK or PDZ-binding kinase) was first reported by Abe et al. in 2000 [6], and it is a Ser/Thr protein kinase overexpressed in hematologic tumors, breast cancer, melanoma, colorectal cancer, prostate cancer, cervical cancer, bladder cancer and lung cancer [7–14]. The results of their studies demonstrated that pantoprazole can suppress the growth of colorectal cancer cells as a TOPK inhibitor both in vitro and in vivo, and also showed that the TOPK activities were inhibited by ilaprazole in HCT-116, ES-2, A549, SW1990 cancer cells in vitro [4,5]. As shown in Figure 1, all of the PPIs molecules contain thiomethylpyridine fragments. It can be predicted that these fragments should play an important role in the antiproliferative activity of proton pump inhibitors.

**Figure 1.** Chemical structures of proton pump inhibitors (PPIs).

As known, the diaryl urea is a fragment of great importance in medicinal chemistry and can be used for the synthesis of numerous heterocyclic compounds with diversified biological activities, including antithrombotic [15], antimalarial [16], antibacterial [17,18] and anti-inflammatory [19] properties, and it is characterized by its ability to form hydrogen bond interactions with drug targets [20–22]. The carbonyl oxygen atom acts as a proton acceptor while the two amide nitrogen atoms are proton donors (Figure 2). This unique type of structure endows urea derivatives with the ability to bind a variety of enzymes and receptors in the biological systems. Remarkably, the diaryl urea moiety is widely used in the design of anticancer drugs, such as sorafenib, regorafenib, linifanib and tivozanib (Figure 3).

**Figure 2.** H-bond acceptor and donors within the diaryl urea scaffold.

**Figure 3.** Some anticancer drugs of the diaryl urea moiety.

Molecular hybridization strategy is a useful concept in drug design and development based on the combination of pharmacophoric moieties of different bioactive substances to produce a new structure, the affinity and efficacy would be improved, when compared to the parent drugs [23]. These above interesting findings and our continuous quest to identify more potent antiproliferative agents led to the molecular hybridization of diaryl urea and thiomethylpyridine to integrate them in one molecular platform to generate a new hybrid, as shown in Figure 4, and expected that taking this way could get the antiproliferative agents with highly inhibitory activity.

**Figure 4.** Rational design of the target compounds based on molecular hybridization strategy.

#### **2. Results and Discussion**

#### *2.1. Chemistry*

The general synthetic route is illustrated in Scheme 1. The reaction of the commercially available 4-nitrobenzenethiol (**1**) with 2-(chloromethyl)pyridine derivatives (**2**) in ethanol at r.t. (room temperature) obtained compounds **3a**–**3d [24]**, which converted to key intermediates **4a**–**4d** via Pd-C catalytic hydrogenation reduction [25]. The aryl isocyanates **6a**–**6e** were prepared by reaction between aromatic amines and bis(trichloromethyl)carbonate (BTC) [26]. Finally, treatment of **4a**–**4d** with aryl isocyanates **6a**–**6e** in methylene dichloride yielded 1-aryl-3-{4-[(pyridin-2-ylmethyl)thio]phenyl}urea derivatives (**7a**–**7t**) as the target compounds [26]. The structures of the target compounds were characterized by infraredspectra (IR), proton nuclear magnetic resonance spectra (1H-NMR), carbon nuclear magnetic resonance spectra (13C-NMR), electrospray ionization mass spectra (ESI-MS) and high-resolution mass spectra (HRMS).

**Scheme 1.** Synthetic route of the target compounds **7a**–**7t.** Reagents and conditions: (a) NaOH (aq. 2M), EtOH, r.t.; (b) H2, 1 atm, 10% Pd-C, MeOH, r.t.; (c) BTC, Et3N, CH2Cl2, r.t.; (d) intermediate **4**, aryl isocyanates **6a**–**6e**, CH2Cl2, r.t.

#### *2.2. Biological Evaluation*

#### 2.2.1. Antiproliferative Activity

Using sorafenib as a positive control, all of the target compounds were evaluated for the antiproliferative activity in vitro against cancer cell lines, including A549 (lung cancer), HCT-116 (colorectal cancer), and PC-3 (prostate cancer) cell lines by MTT assay. The antiproliferative assay results evaluated as IC50 value (Table 1) and demonstrated that several target compounds have shown moderate to excellent potency against A549, HCT-116, and PC-3 cancer cell lines. Among the target compounds**7i** showed the more potent inhibitory effect against three cancer cell lines than positive control with IC50 values of 1.53 ± 0.46, 1.11 ± 0.34 and 1.98 ± 1.27 μM, respectively.

All the target compounds could be divided into four classes according to different substituents on the pyridine ring (Figure 5). The analyses of the structure-activity relationships (SARs) were summarized as follows: (1) The results of cytostatic activity assay showed that the substitutions of the 4 (R4) and 5 (R5) positions of the C ring had a weak effect on the inhibitory activity. However, if the 3-position (R3) hydrogen atom of the C ring was substituted by a methoxy group, the inhibitory activity was significantly decreased. At the same time, it could be seen that the inhibitory activity was better than other classes when the 4-position (R4) of the C ring was occupied by the trifluoroethoxy group. (2) The substituents on the A ring had a significant effect on the inhibitory activity of each class. When the substituents on C ring were the same, if there was no substituent on A ring, the inhibitory activity was worst in each class, such as compound **7a**, **7f**, **7k** and **7p**. Moreover, when the substituents in A ring were electron-withdrawing groups, such as 4-Cl or 3-CF3, the inhibitory activity was better than that substitution of the electron-donating groups, such as 4-OCH3 in each class. Furthermore, when the two electron-withdrawing groups coexist on the A ring, the target compounds displayed the strongest inhibitory activity, such as compound **7d**, **7i, 7n** and **7s**.

**Figure 5.** SARs summary for the target compounds.

**Table 1.** The chemical structures and inhibitory activities of the target compounds.


<sup>a</sup> Inhibitory activity was assayed by exposure for 72 h to substance and expressed as the concentration required to inhibit tumor cell proliferation by 50% (IC50). Data are presented as the means ± SEMs of three independent experiments.

#### 2.2.2. Cell Apoptosis Assay

The acceptable antiproliferative activity of compound **7i** promoted us to investigate its effect on cell apoptosis. To explore the effect of compound **7i** on cell apoptosis, the apoptotic analysis was performed with Annexin V-FITC/PI double staining and analyzed with flow-cytometry calculation. Treatment of A549 cells with compound **7i** resulted in a concentration-dependent apoptosis increase, as shown in Figure 6. Specifically, the percentage of early/primary apoptotic cells was about 4.33% for the low concentration (1 μM) of compound **7i**. When treated with high concentration (10 μM) of compound **7i**, around 29.47% of early/primary apoptosis rate was observed. While the late apoptosis rate of A549 cells was not changed significantly with increasing concentrations.

#### 2.2.3. Cell Cycle Analysis

The effect of compound **7i** on the cell cycle was also evaluated. After treatment of A549 cells with compound **7i** for 24 h at indicated concentrations (1, 5, 10 μM), the percentage of cells in G1 phase were 60.77%, 78.05% and 90.65%, respectively (Figure 7), suggesting that compound **7i** caused an obvious G1 arrest in a concentration-dependent manner with a concomitant decrease in terms of the number of cells in other phases of the cell cycle.

**Figure 6.** Compound **7i** induced apoptosis of A549 cells. (**A**) Apoptosis effect on A549 cell line induced by compound **7i** for 24 h using Annexin V-FITC/PI double staining and flow-cytometry calculation. The lower left quadrant represents live cells, the lower right is for early/primary apoptotic cells, upper right is for late/secondary apoptotic cells, and the upper left represents cells damaged during the procedure; (**B**) Quantitative analysis of apoptotic cells. The experiments were performed three times, and a representative experiment is shown.

**Figure 7.** Effects of compound **7i** on A549 cell cycle progress for 24 h. (**A**) Treatment of A549 cells with compound **7i** at different concentrations (1 μM, 5 μM, 10 μM) for 24 h. (**B**) Quantitative analysis of cell cycle. The experiments were performed three times, and a representative experiment is shown.

#### **3. Materials and Methods**

#### *3.1. Synthesis*

All reagents were obtained from commercial suppliers and used without further purification. Reaction progress was monitored by thin layer chromatography (TLC) on silica gel plates. The spots were visualized by ultraviolet (UV) light (254 nm). The column chromatography was performed using 200−300 mesh silica gel (Qingdao PUKE, Qingdao, China). Melting points were obtained by X-5 micro-melting point apparatus (Beijing Zhongyi Boteng Technology Co., Ltd., Beijing, China) and were uncorrected. 1H-NMR and 13C-NMR spectra were recorded on Bruker NMR spectrophotometers (Karlsruhe, Germany) using DMSO-*d*<sup>6</sup> as the solvent and TMS as the internal standard. Mass spectra were measured with an electrospray (ESI-MS) on a Waters spectrometer (Waters Corporation, Milford, MA, USA). High resolution mass spectrometry (HRMS) analyses were performed on an Agilent Technologies 6530 Accurate-Mass Q-TOF Mass Spectrometer (Santa Clara, CA, USA). The purities were determined by high-performance liquid chromatography (HPLC) using an Agilent 1100 series HPLC (Santa Clara, CA, USA).

The original figures of 1H-NMR, 13C-NMR, MS and HRMS of all the target compounds as the Supplementary Materials are available online.

3.1.1. General Procedure for the Preparation of 2-{[(4-nitrophenyl)thio]methyl}pyridine Derivatives (**3a**–**3d**)

4-Nitrobenzenethiol **1** (1.55 g, 0.01 mol), and 2-(chloromethyl)pyridine hydrochloride derivatives **2** (0.01 mol) were dissolved in EtOH (100 mL), then aqueous NaOH (2M) was added dropwise. After the addition completed, the solution was stirred for 8 h at room temperature. Upon completion, the excess ethanol was evaporated to give the residue. A large number of white solids have been precipitated when 200 mL of water was added. The Precipitate was filtered off and washed with water to obtain the intermediates (**3a**–**3d**), which was used for next step without further purification.

*4-(3-Methoxypropoxy)-3-methyl-2-{[(4-nitrophenyl)thio]methyl}pyridine* (**3a**) by using compound **1** (1.55 g, 0.01 mol) and 2-(chloromethyl)-4-(3-methoxypropoxy)-3-methylpyridine hydrochloride (2.66 g, 0.01 mol), obtained a yellow solid (3.12 g) in 89.7% yield. ESI-MS (*m*/*z*): 349.3 ([M + H]+).

*3-Methyl-2-{[(4-nitrophenyl)thio]methyl}-4-(2,2,2-trifluoroethoxy)pyridine* (**3b**) by using compound **1** (1.55 g, 0.01 mol) and 2-(chloromethyl)-3-methyl-4-(2,2,2-trifluoroethoxy)pyridine hydrochloride (2.76 g, 0.01 mol), obtained a yellow solid (3.26 g) in 91.2% yield. ESI-MS (*m*/*z*): 359.1 ([M + H]+).

*4-Methoxy-3,5-dimethyl-2-{[(4-nitrophenyl)thio]methyl}pyridine* (**3c**) by using compound **1** (1.55 g, 0.01 mol) and 2-(chloromethyl)-4-methoxy-3,5-dimethylpyridine hydrochloride (2.22 g, 0.01 mol), obtained a yellow solid (2.69 g) in 88.5% yield. ESI-MS (*m*/*z*): 305.0 ([M + H]+).

*3,4-Dimethoxy-2-{[(4-nitrophenyl)thio]methyl}pyridine* (**3d**) by using compound **1** (1.55 g, 0.01 mol) and 2-(chloromethyl)-3,4-dimethoxypyridine hydrochloride (2.24 g, 0.01 mol), obtained a yellow solid (2.86 g) in 93.5% yield. ESI-MS (*m*/*z*): 307.4 ([M + H]+).

3.1.2. General Procedure for the Preparation of 4-{[(pyridin-2-yl)methyl]thio}aniline Derivatives (**4a**–**4d**)

A mixture of **3a**–**3d** (5 mmol) and 0.1 g of preequilibrated 10% palladium/carbon in MeOH (50 mL) was hydrogenated at room temperature and atmospheric pressure. The reaction was completely monitored by TLC. When the reaction has completed, the mixture was filtered, and the filtrate was evaporated to yield intermediate (**4a**–**4d**) as yellowish oil, which was used for the next step without further purification.

*4-{{[4-(3-Methoxypropoxy)-3-methylpyridin-2-yl]methyl}thio}aniline* (**4a**) by using compound **3a** (1.74 g, 5 mmol), H2 and 10% Pd-C, obtained a yellowish oil (1.52 g) in 95.4% yield. ESI-MS (*m*/*z*): 319.3 ([M + H]+).

*4-{{[3-Methyl-4-(2,2,2-trifluoroethoxy)pyridin-2-yl]methyl}thio}aniline* (**4b**) by using compound **3b** (1.79 g, 5 mmol), H2 and 10% Pd-C, obtained a yellowish oil (1.58 g) in 96.1% yield. ESI-MS (*m*/*z*): 329.6 ([M + H]+).

*4-{[(4-Methoxy-3,5-dimethylpyridin-2-yl)methyl]thio}aniline* (**4c**) by using compound **3c** (1.52 g, 5 mmol), H2 and 10% Pd-C, obtained a yellowish oil (1.30 g) in 94.8% yield. ESI-MS (*m*/*z*): 275.2 ([M + H]+).

*4-{[(3,4-Dimethoxypyridin-2-yl)methyl]thio}aniline* (**4d**) by using compound **3d** (1.53 g, 5 mmol), H2 and 10% Pd-C, obtained a yellowish oil (1.30 g) in 94.2% yield. ESI-MS (*m*/*z*): 276.1 ([M + H]+).

3.1.3. General Procedure for the Preparation of the Target Compounds (**7a**–**7t**)

To a solution of BTC (1 mmol) in CH2Cl2 (20 mL) was added dropwise to primary aromatic amine **5** (1 mmol) in CH2Cl2 (20 mL) followed by the dropwise addition of triethylamine (1 mL) in CH2Cl2 (10 mL). The solvent was evaporated. The resulting residue was dissolved in CH2Cl2 (20 mL), and intermediates (**4a**–**4d**) (1 mmol) in CH2Cl2 (10 mL) was added dropwise. The mixture was stirred for about 3 h, monitored by TLC. After the reaction completed, the solvent was washed with water and brine, then dried over anhydrous magnesium sulfate. The mixture was filtered, the filtrate was evaporated and purified by silica gel chromatography (CH2Cl2/MeOH = 60/1, *v*/*v*) to obtain target compounds.

*1-{4-{{[4-(3-Methoxypropoxy)-3-methylpyridin-2-yl]methyl}thio}phenyl}-3-phenylurea* (**7a**)

Compound **7a** was prepared according to the general procedure by using compound **4a** (0.32 g, 1 mmol) and aniline (0.10 g, 1 mmol), obtained a white solid (0.20 g) in 45.1% yield. m.p. 105.2–106.8 ◦C. IR (KBr, cm<sup>−</sup>1): υ 3421.1, 2922.7, 2852.4, 1596.1, 1545.1, 1492.4, 1460.8, 1440.3, 1398.2, 1385.2, 1309.3, 1231.3, 1174.2, 1092.1, 1006.7, 894.6, 832.0, 799.2, 751.9, 693.4, 617.3, 507.5. 1H-NMR (400 MHz, DMSO-*d*6) δ 8.74 (s, 1H), 8.69 (s, 1H), 8.17 (d, *J* = 5.6 Hz, 1H), 7.46 (s, 1H), 7.44 (s, 1H), 7.41 (s, 1H), 7.39 (s, 1H), 7.32 (s, 1H), 7.31-7.29 (m, 1H), 7.28 (s, 1H), 7.26 (s, 1H), 6.97 (t, *J* = 7.3 Hz, 1H), 6.90 (d, *J* = 5.7 Hz, 1H), 4.22 (s, 2H), 4.09 (t, *J* = 6.2 Hz, 2H), 3.48 (t, *J* = 6.2 Hz, 2H), 3.25 (s, 3H), 2.12 (s, 3H), 2.02–1.94 (m, 2H). 13C-NMR (101 MHz, DMSO-*d*6) δ 163.17, 156.31, 152.87, 147.91, 140.07, 131.77, 129.23, 128.07, 122.34, 120.30, 119.09, 118.69, 106.49, 68.79, 65.48, 58.44, 31.14, 29.16, 10.88. ESI-MS (*m*/*z*): 438.4 ([M + H]+), 460.2 ([M + Na]+). HRMS (ESI) (*m*/*z*): Calcd. for C24H27N3O3S, 438.1846 ([M + H]+), found: 438.1856 ([M + H]+). Purity (HPLC): 99.27%.

*1-(4-Chlorophenyl)-3-{4-{{[4-(3-methoxypropoxy)-3-methylpyridin-2-yl]methyl}thio}phenyl}urea* (**7b**)

Compound **7b** was prepared according to the general procedure by using compound **4a** (0.32 g, 1 mmol) and 4-chloroaniline (0.13 g, 1 mmol), obtained a white solid (0.36 g) in 75.3% yield. m.p. 191.7–192.5 ◦C. IR (KBr, cm−1): υ 3428.1, 2923.1, 2852.9, 1631.8, 1490.8, 1398.9, 1384.8, 1298.9, 1273.5, 1237.0, 1174.2, 1121.4, 1086.2, 1008.0, 881.2, 832.1, 702.8, 619.7, 506.0. 1H-NMR (400 MHz, DMSO-*d*6) δ 8.83 (s, 1H), 8.77 (s, 1H), 8.17 (d, *J* = 5.6 Hz, 1H), 7.49 (d, *J* = 2.2 Hz, 1H), 7.47 (d, *J* = 2.1 Hz, 1H), 7.40 (d, *J* = 2.0 Hz, 1H), 7.39 (d, *J* = 2.1 Hz, 1H), 7.34 (s, 1H), 7.33 (s, 1H), 7.32 (d, *J* = 2.1 Hz, 1H), 7.31 (s, 1H), 6.90 (d, *J* = 5.7 Hz, 1H), 4.22 (s, 2H), 4.08 (t, *J* = 6.2 Hz, 2H), 3.48 (t, *J* = 6.2 Hz, 2H), 3.25 (s, 3H), 2.12 (s, 3H), 2.00–1.94 (m, 2H). 13C-NMR (101 MHz, DMSO-*d*6) δ 163.09, 156.35, 152.77, 147.99, 139.09, 138.88, 131.62, 129.07, 128.41, 125.86, 120.22, 119.24, 106.47, 68.80, 65.45, 58.43, 31.14, 29.16, 10.89. ESI-MS (*m*/*z*): 473.3 ([M + H]+), 495.2 ([M + Na]+). HRMS (ESI) (*m*/*z*): Calcd. for C24H26ClN3O3S, 472.1456 ([M + H]+), found: 472.1467 ([M + H]+). Purity (HPLC): 98.66%.

*1-(4-Methoxyphenyl)-3-{4-{{[4-(3-methoxypropoxy)-3-methylpyridin-2-yl]methyl}thio}phenyl}urea* (**7c**)

Compound **7c** was prepared according to the general procedure by using compound **4a** (0.32 g, 1 mmol) and 4-methoxyaniline (0.12 g, 1 mmol), obtained a white solid (0.22 g) in 46.3% yield. m.p. 144.8–146.6 ◦C. IR (KBr, cm−1): υ 3428.6, 2984.7, 2923.0, 2852.7, 1635.5, 1599.5, 1562.6, 1510.7, 1492.7, 1461.8, 1441.3, 1398.1, 1289.8, 1245.2, 1173.5, 1120.3, 1093.5, 1035.2, 1005.6, 800.0, 617.1, 548.4, 522.7. 1H-NMR (400 MHz, DMSO-*d*6) δ 8.64 (s, 1H), 8.47 (s, 1H), 8.16 (d, *J* = 5.7 Hz, 1H), 7.39 (d, *J* = 1.9 Hz, 1H), 7.37 (d, *J* = 2.2 Hz, 1H), 7.35 (d, *J* = 2.2 Hz, 1H), 7.33 (d, *J* = 2.3 Hz, 1H), 7.30 (d, *J* = 2.1 Hz, 1H), 7.29 (d, *J* = 2.0 Hz, 1H), 6.89 (d, *J* = 5.6 Hz, 1H), 6.87 (d, *J* = 2.2 Hz, 1H), 6.86 (d, *J* = 2.2 Hz, 1H), 4.21 (s, 2H), 4.08 (t, *J* = 6.2 Hz, 2H), 3.71 (s, 3H), 3.48 (t, *J* = 6.2 Hz, 2H), 3.25 (s, 3H), 2.12 (s, 3H), 2.00–1.94 (m, 2H). 13C-NMR (101 MHz, DMSO-*d*6) δ 163.06, 156.41, 154.97, 153.07, 148.01, 139.37, 133.09, 131.78, 127.82, 120.52, 120.22, 118.98, 114.44, 106.44, 68.79, 65.42, 58.43, 55.63, 31.14, 29.16, 10.88. ESI-MS (*m*/*z*): 468.4 ([M + H]+), 490.2 ([M + Na]+). HRMS (ESI) (*m*/*z*): Calcd. for C25H29N3O4S, 468.1952 ([M + H]+), found: 468.1959 ([M + H]+). Purity (HPLC): 98.91%.

*1-[4-Chloro-3-(trifluoromethyl)phenyl]-3-{4-{{[4-(3-methoxypropoxy)-3-methylpyridin-2-yl]methyl}thio}phe nyl}urea* (**7d**)

Compound **7d** was prepared according to the general procedure by using compound **4a** (0.32 g, 1 mmol) and 3-chloro-4-(trifluoromethyl)aniline (0.20 g, 1 mmol), obtained a white solid (0.24 g) in 43.8% yield. m.p. 136.0–137.2 ◦C. IR (KBr, cm−1): υ 3425.2, 2922.0, 2852.7, 1590.1, 1546.1, 1482.2, 1463.1, 1384.5, 1306.6, 1175.4, 1117.7, 1034.4, 820.6. 1H-NMR (400 MHz, DMSO-*d*6) δ 9.16 (s, 1H), 8.89 (s, 1H), 8.16 (d, *J* = 5.6 Hz, 1H), 8.10 (d, *J* = 2.2 Hz, 1H), 7.63 (d, *J* = 2.2 Hz, 1H), 7.62 (s, 1H), 7.42 (d, *J* = 2.0 Hz, 1H), 7.40 (d, *J* = 2.2 Hz, 1H), 7.33 (d, *J* = 2.1 Hz, 1H), 7.32 (d, *J* = 2.0 Hz, 1H), 6.89 (d, *J* = 5.7 Hz, 1H), 4.23 (s, 2H), 4.09 (t, *J* = 6.2 Hz, 2H), 3.48 (t, *J* = 6.2 Hz, 2H), 3.25 (s, 3H), 2.13 (s, 3H), 2.00–1.94 (m, 2H). 13C-NMR (101 MHz, DMSO-*d*6) δ 163.08, 156.34, 152.76, 148.01, 139.76, 138.48, 132.45, 131.46, 128.91, 123.55, 122.80, 120.24, 119.57, 117.23, 106.48, 68.80, 65.44, 58.43, 31.14, 29.16, 10.89. ESI-MS (*m*/*z*): 540.2 ([M + H]+), 562.0 ([M + Na]+). HRMS (ESI) (*m*/*z*): Calcd. for C25H25ClF3N3O3S, 540.1330 ([M + H]+), found: 540.1320 ([M + H]+). Purity (HPLC): 97.33%.

*1-{4-{{[4-(3-Methoxypropoxy)-3-methylpyridin-2-yl]methyl}thio}phenyl}-3-[3-(trifluoromethyl)phenyl]urea* (**7e**)

Compound **7e** was prepared according to the general procedure by using compound **4a** (0.32 g, 1 mmol) and 3-(trifluoromethyl)aniline (0.16 g, 1 mmol), obtained a white solid (0.27 g) in 53.1% yield. m.p. 136.1–137.9 ◦C. IR (KBr, cm−1): υ 3327.8, 2958.7, 2927.8, 2859.0, 2377.4, 2350.2, 2311.0, 1724.0, 1648.5, 1585.5, 1552.2, 1492.5, 1465.2, 1397.5, 1338.6, 1295.8, 1230.8, 1166.2, 1116.1, 1092.4, 1068.9, 1005.4, 890.0, 804.1, 732.9, 699.2, 602.3, 505.8. 1H-NMR (400 MHz, DMSO-*d*6) δ 9.05 (s, 1H), 8.84 (s, 1H), 8.17 (d, *J* = 5.6 Hz, 1H), 8.09 (s, 0H), 8.00 (d, *J* = 2.3 Hz, 1H), 7.57 (d, *J* = 8.4 Hz, 1H), 7.51 (t, *J* = 7.9 Hz, 1H), 7.42 (d, *J* = 2.0 Hz, 1H), 7.40 (s, 1H), 7.33 (d, *J* = 2.1 Hz, 1H), 7.32 (d, *J* = 2.1 Hz, 1H), 7.30 (s, 1H), 6.90 (d, *J* = 5.7 Hz, 1H), 4.23 (s, 2H), 4.09 (t, *J* = 6.2 Hz, 2H), 3.48 (t, *J* = 6.2 Hz, 2H), 3.25 (s, 3H), 2.13 (s, 3H), 2.01–1.94 (m, 3H). 13C-NMR (101 MHz, DMSO-*d*6) δ 163.11, 156.32, 152.85, 147.97, 140.98, 138.68, 131.55, 130.34, 129.10, 128.64, 122.30, 120.25, 119.40, 118.53, 106.46, 68.79, 65.44, 58.42, 31.13, 29.16, 10.88. ESI-MS (*m*/*z*): 506.3 ([M + H]+). HRMS (ESI) (*m*/*z*): Calcd. for C25H26F3N3O3S, 506.1720 ([M + H]+), found: 506.1728 ([M + H]+). Purity (HPLC): 97.09%.

*1-{4-{{[3-Methyl-4-(2,2,2-trifluoroethoxy)pyridin-2-yl]methyl}thio}phenyl}-3-phenylurea* (**7f**)

Compound **7f** was prepared according to the general procedure by using compound **4b** (0.33 g, 1 mmol) and aniline (0.10 g, 1 mmol), obtained a white solid (0.18 g) in 39.9% yield. m.p. 143.3–145.1 ◦C. IR (KBr, cm−1): υ 3424.1, 2923.9, 2852.6, 1687.8, 1639.9, 1600.0, 1548.5, 1495.9, 1441.2, 1399.4, 1384.7, 1307.8, 1284.4, 1266.4, 1232.1, 1176.6, 1112.2, 970.6, 915.9, 854.7, 836.1, 801.5, 783.1, 751.0, 696.2, 657.1, 618.8, 574.4. 1H-NMR (400 MHz, DMSO-*d*6) δ 8.73 (s, 1H), 8.67 (s, 1H), 8.24 (d, *J* = 5.7 Hz, 1H), 7.46 (s, 1H), 7.44 (s, 1H), 7.41 (s, 1H), 7.39 (s, 1H), 7.32 (s, 1H), 7.30 (s, 1H), 7.28 (s, 1H), 7.26 (s, 1H), 7.03 (d, *J* = 5.7 Hz, 1H), 6.97 (t, *J* = 7.3 Hz, 1H), 4.89 (q, *J* = 8.7 Hz, 2H), 4.25 (s, 2H), 2.16 (s, 3H). 13C-NMR (101 MHz, DMSO-*d*6) δ 161.62, 157.24, 152.87, 148.00, 140.05, 139.22, 131.90, 129.23, 127.85, 125.66, 122.90, 122.35, 120.44, 119.10, 118.71, 107.07, 64.92, 31.13, 10.74. ESI-MS (*m*/*z*): 448.4 ([M + H]+), 470.2 ([M + Na]+). HRMS (ESI) (*m*/*z*): Calcd. for C22H20F3N3O2S, 448.1301 ([M + H]+), found: 448.1295 ([M + H]+). Purity (HPLC): 97.04%.

*1-(4-Chlorophenyl)-3-{4-{{[3-methyl-4-(2,2,2-trifluoroethoxy)pyridin-2-yl]methyl}thio}phenyl}urea* (**7g**)

Compound **7g** was prepared according to the general procedure by using compound **4b** (0.33 g, 1 mmol) and 4-chloroaniline (0.13 g, 1 mmol), obtained a white solid (0.29 g) in 61.0% yield. m.p. 203.6–205.2 ◦C. IR (KBr, cm−1): υ 3424.1, 2984.9, 2923.1, 2851.9, 2350.0, 2311.0, 1611.4, 1548.6, 1491.9, 1440.3, 1399.6, 1384.9, 1370.1, 1311.1, 1268.3, 1172.6, 1111.7, 1051.4, 1004.4, 897.2, 798.3, 668.5, 615.4. 1H-NMR (400 MHz, DMSO-*d*6) δ 8.81 (s, 1H), 8.75 (s, 1H), 8.23 (d, *J* = 5.7 Hz, 1H), 7.48 (d, *J* = 2.1 Hz, 1H), 7.47 (d, *J* = 2.2 Hz, 1H), 7.40 (d, *J* = 2.0 Hz, 1H), 7.38 (s, 1H), 7.33 (d, *J* = 2.1 Hz, 1H), 7.32 (d, *J* = 2.2 Hz, 1H), 7.31 (s, 1H), 7.30 (d, *J* = 1.9 Hz, 1H), 7.03 (d, *J* = 5.7 Hz, 1H), 4.88 (q, *J* = 8.7 Hz, 2H), 4.25 (s, 2H), 2.16 (s, 3H). 13C-NMR (101 MHz, DMSO-*d*6) δ 161.62, 157.22, 152.77, 148.00, 139.08, 138.98, 131.80, 129.07, 128.11, 125.87, 122.90, 120.43, 120.23, 119.23, 107.08, 65.09, 31.14, 10.74. ESI-MS (*m*/*z*): 482.6 ([M + H]+), 504.3 ([M + Na]+). HRMS (ESI) (*m*/*z*): Calcd. for C22H19ClF3N3O2S, 482.0911 ([M + H]+), found: 482.0916 ([M + H]+). Purity (HPLC): 99.19%.

*1-(4-Methoxyphenyl)-3-{4-{{[3-methyl-4-(2,2,2-trifluoroethoxy)pyridin-2-yl]methyl}thio}phenyl}urea* (**7h**)

Compound **7h** was prepared according to the general procedure by using compound **4b** (0.33 g, 1 mmol) and 4-methoxyaniline (0.12 g, 1 mmol), obtained a white solid (0.22 g) in 45.9% yield. m.p. 171.3–172.1 ◦C. IR (KBr, cm−1): υ 3383.8, 2922.1, 2851.4, 2377.6, 2349.6, 1703.0, 1656.7, 1619.2, 1591.3, 1546.4, 1511.1, 1492.7, 1465.5, 1399.3, 1312.1, 1264.5, 1231.2, 1175.1, 1112.8, 1040.3, 1006.7, 970.2, 918.1, 897.2, 831.5, 799.7, 658.3. 1H-NMR (400 MHz, ) δ 8.64 (s, 1H), 8.47 (s, 1H), 8.16 (d, *J* = 5.7 Hz, 1H), 7.39 (d, *J* = 1.9 Hz, 1H), 7.37 (d, *J* = 2.2 Hz, 1H), 7.35 (d, *J* = 2.2 Hz, 1H), 7.33 (d, *J* = 2.3 Hz, 1H), 7.30 (d, *J* = 2.1 Hz, 1H), 7.29 (d, *J* = 2.0 Hz, 1H), 6.89 (d, *J* = 5.6 Hz, 1H), 6.87 (d, *J* = 2.2 Hz, 1H), 6.86 (d, *J* = 2.2 Hz, 1H), 4.91-4.85 (m, 2H), 4.21 (s, 2H), 3.71 (s, 3H), 2.12 (s, 3H). 13C-NMR (101 MHz, DMSO-*d*6) δ 161.84, 156.03, 154.81, 153.88, 153.39, 148.09, 133.72, 133.38, 125.65, 121.84, 120.37, 120.33, 115.40, 114.43, 108.04, 65.10, 55.63, 31.13, 10.44. ESI-MS (*m*/*z*): 478.3 ([M + H]+). HRMS (ESI) (m/z): Calcd. for C23H22F3N3O3S, 478.1407 ([M + H]+), found: 478.1408 ([M + H]+). Purity (HPLC): 99.66%.

*1-[4-Chloro-3-(trifluoromethyl)phenyl]-3-{4-{{[3-methyl-4-(2,2,2-trifluoroethoxy)pyridin-2-yl]methyl}thio}phe nyl}urea* (**7i**)

Compound **7i** was prepared according to the general procedure by using compound **4b** (0.33 g, 1 mmol) and 3-chloro-4-(trifluoromethyl)aniline (0.20 g, 1 mmol), obtained a white solid (0.27 g) in 49.5% yield. m.p. 142.0–143.0 ◦C. IR (KBr, cm−1): υ 3422.3, 2922.1, 2852.6, 1587.4, 1547.6, 1480.8, 1419.0, 1309.8, 1263.9, 1177.2, 1111.0, 1035.5, 974.7, 819.3, 618.3. 1H-NMR (400 MHz, DMSO-*d*6) δ 9.17 (s, 1H), 8.90 (s, 1H), 8.24 (d, *J* = 5.7 Hz, 1H), 8.10 (d, *J* = 2.2 Hz, 1H), 7.64 (dd, *J* = 8.9, 2.2 Hz, 1H), 7.61 (d, *J* = 8.7 Hz, 1H), 7.42 (d, *J* = 2.0 Hz, 1H), 7.40 (d, *J* = 2.2 Hz, 1H), 7.33 (d, *J* = 2.2 Hz, 1H), 7.32 (d, *J* = 2.1 Hz, 1H), 7.04 (d, *J* = 5.7 Hz, 1H), 4.89 (q, *J* = 8.7 Hz, 2H), 2.16 (s, 3H). 13C-NMR (101 MHz, DMSO-*d*6) δ 161.61, 157.18, 152.75, 148.00, 139.74, 138.60, 132.42, 131.64, 128.58, 127.33, 127.02, 125.65, 123.53, 122.80, 120.44, 119.55, 117.28, 117.22, 107.07, 64.79, 39.69, 10.72. ESI-MS (*m*/*z*): 550.1, 552.1, 553.1 ([M + H]+). HRMS (ESI) (*m*/*z*): Calcd. for C23H18ClF6N3O2S, 550.0785 ([M + H]+), found: 550.0769 ([M + H]+). Purity (HPLC): 99.97%.

*1-{4-{{[3-Methyl-4-(2,2,2-trifluoroethoxy)pyridin-2-yl]methyl}thio}phenyl}-3-[3-(trifluoromethyl)phenyl] urea* (**7j**)

Compound **7j** was prepared according to the general procedure C by using compound **4b** (0.33 g, 1 mmol) and 3-(trifluoromethyl)aniline (0.16 g, 1 mmol), obtained a white solid (0.34 g) in 66.1% yield. m.p. 174.7–176.4 ◦C. IR (KBr, cm−1): υ 3421.4, 2985.6, 2924.1, 2852.9, 2349.2, 2311.0, 1614.9, 1491.8, 1445.1, 1399.1, 1339.8, 1313.1, 1288.0, 1264.6, 1231.2, 1173.2, 1114.6, 1071.2, 1006.3, 976.0, 832.4, 798.2, 700.6, 616.4. 1H-NMR (400 MHz, DMSO-*d*6) δ 9.07 (s, 1H), 8.87 (s, 1H), 8.24 (d, *J* = 5.7 Hz, 1H), 8.01 (d, *J* = 2.0 Hz, 1H), 7.60–7.54 (m, 1H), 7.51 (t, *J* = 7.9 Hz, 1H), 7.43 (d, *J* = 2.0 Hz, 1H), 7.41 (d, *J* = 2.1 Hz, 1H), 7.33 (d, *J* = 2.1 Hz, 1H), 7.33–7.31 (m, 1H), 7.30 (d, *J* = 1.7 Hz, 1H), 7.04 (d, *J* = 5.7 Hz, 1H), 4.89 (q, *J* = 8.7 Hz, 2H), 4.26 (s, 2H), 2.16 (s, 3H). 13C-NMR (101 MHz, DMSO-*d*6) δ 161.66, 157.16, 152.86, 147.96, 140.97, 138.80, 131.76, 130.35, 130.15, 129.84, 128.31, 122.30, 120.47, 119.40, 118.54, 114.59, 107.09, 65.09, 49.05, 31.13, 10.73. ESI-MS (*m*/*z*): 516.2 ([M + H]+). HRMS (ESI) (*m*/*z*): Calcd. for C23H19F6N3O2S, 516.1175 ([M + H]+), found: 516.1174 ([M + H]+). Purity (HPLC): 97.29%.

*1-{4-{[(4-Methoxy-3,5-dimethylpyridin-2-yl)methyl]thio}phenyl}-3-phenylurea* (**7k**)

Compound **7k** was prepared according to the general procedure by using compound **4c** (0.27 g, 1 mmol) and aniline (0.10 g, 1 mmol), obtained a white solid (0.23 g) in 57.3% yield. m.p. 188.1–188.9 ◦C. IR (KBr, cm−1): υ 3422.4, 2923.8, 2852.4, 2351.0, 2321.9, 1644.0, 1597.4, 1553.7, 1494.2, 1441.5, 1398.0, 1312.9, 1270.7, 1237.5, 1173.3, 1127.7, 1073.5, 1002.5, 798.1, 755.7, 738.0, 694.0, 616.4. 1H-NMR (400 MHz, DMSO-*d*6) δ 8.72 (s, 1H), 8.67 (s, 1H), 8.12 (s, 1H), 7.45 (d, *J* = 1.3 Hz, 1H), 7.44-7.42 (m, 1H), 7.40 (d, *J* = 2.1 Hz, 1H), 7.39 (d, *J* = 2.1 Hz, 1H), 7.31 (d, *J* = 2.2 Hz, 1H), 7.29 (d, *J* = 2.1 Hz, 1H), 7.28 (s, 1H), 7.26 (d, *J* = 1.6 Hz, 1H), 6.97 (t, *J* = 7.4 Hz, 1H), 4.21 (s, 2H), 3.70 (s, 3H), 2.18 (s, 6H). 13C-NMR (101 MHz, DMSO-*d*6) δ 163.86, 155.80, 152.86, 148.97, 140.05, 139.16, 131.86, 129.24, 127.98, 125.19, 125.03, 122.35, 119.11, 118.70, 60.17, 31.15, 13.38, 11.34. ESI-MS (*m*/*z*): 394.6 ([M + H]+), 416.3 ([M + Na]+). HRMS (ESI) (*m*/*z*): Calcd. for C22H23N3O2S, 394.1584 ([M + H]+), found: 394.1586 ([M + H]+). Purity (HPLC): 99.89%.

*1-(4-Chlorophenyl)-3-{4-{[(4-methoxy-3,5-dimethylpyridin-2-yl)methyl]thio}phenyl}urea* (**7l**)

Compound **7l** was prepared according to the general procedure by using compound **4c** (0.27 g, 1 mmol) and 4-chloroaniline (0.13 g, 1 mmol), obtained a white solid (0.28 g) in 66.2% yield. m.p. 206.8–208.2 ◦C. IR (KBr, cm−1): υ 3422.5, 2923.0, 2852.0, 2377.1, 2349.6, 2310.8, 1630.4, 1547.7, 1491.7, 1439.7, 1399.2, 1385.1, 1309.9, 1270.9, 1235.5, 1173.0, 1124.1, 1051.3, 1004.6, 832.1, 798.2, 702.1, 668.3, 617.0. 1H-NMR (400 MHz, DMSO-*d*6) δ 8.81 (s, 1H), 8.75 (s, 1H), 8.11 (s, 1H), 7.48 (d, *J* = 2.1 Hz, 1H), 7.46 (d, *J* = 2.2 Hz, 1H), 7.40 (d, *J* = 2.0 Hz, 1H), 7.38 (d, *J* = 2.2 Hz, 1H), 7.33 (d, *J* = 2.1 Hz, 1H), 7.32 (s, 1H), 7.31 (s,

1H), 7.30 (d, *J* = 2.1 Hz, 1H), 4.21 (s, 2H), 3.70 (s, 3H), 2.18 (s, 3H), 2.18 (s, 3H). 13C-NMR (101 MHz, DMSO-*d*6) δ 163.86, 155.79, 152.77, 148.97, 139.08, 138.94, 131.76, 129.07, 127.98, 128.25, 125.87, 125.19, 125.03, 120.23, 119.25, 60.17, 31.14, 13.37, 11.33. ESI-MS (*m*/*z*): 428.7 ([M + H]+). HRMS (ESI) (*m*/*z*): Calcd. for C22H22ClN3O2S, 428.1194 ([M + H]+), found: 428.1199 ([M + H]+). Purity (HPLC): 99.53%.

*1-{4-{[(4-Methoxy-3,5-dimethylpyridin-2-yl)methyl)]thio}phenyl}-3-(4-methoxyphenyl)urea* (**7m**)

Compound **7m** was prepared according to the general procedure by using compound **4c** (0.27 g, 1 mmol) and 4-methoxyaniline (0.12 g, 1 mmol), obtained a white solid (0.21 g) in 49.2% yield. m.p. 171.4–172.6 ◦C. IR (KBr, cm−1): υ 3422.5, 2921.1, 2850.5, 1642.5, 1593.2, 1547.5, 1493.7, 1468.2, 1439.0, 1397.7, 1311.5, 1292.4, 1270.1, 1240.4, 1173.4, 1073.2, 1053.2, 1031.3, 1003.9, 828.2, 797.9, 616.3. 1H-NMR (400 MHz, DMSO-*d*6) δ 8.68 (s, 1H), 8.50 (s, 1H), 8.15 (s, 1H), 7.39 (d, *J* = 1.9 Hz, 1H), 7.37 (d, *J* = 2.1 Hz, 1H), 7.35 (d, *J* = 2.0 Hz, 1H), 7.33 (d, *J* = 2.2 Hz, 1H), 7.29 (d, *J* = 2.1 Hz, 1H), 7.27 (d, *J* = 1.9 Hz, 1H), 6.87 (d, *J* = 2.3 Hz, 1H), 6.86 (d, *J* = 2.2 Hz, 1H), 4.21 (s, 2H), 3.73 (s, 3H), 3.71 (s, 3H), 2.20 (s, 3H), 2.17 (s, 3H). 13C-NMR (101 MHz, DMSO-*d*6) δ 164.47, 155.40, 154.98, 153.08, 148.27, 139.64, 133.09, 132.28, 127.19, 125.70, 125.46, 125.41, 120.49, 118.95, 118.40, 114.46, 60.30, 55.65, 31.14, 13.45, 11.35. ESI-MS (*m*/*z*): 424.3 ([M + H]+). HRMS (ESI) (*m*/*z*): Calcd. for C23H25N3O3S, 424.1689 ([M + H]+), found: 424.1698 ([M + H]+). Purity (HPLC): 96.88%.

*1-[4-Chloro-3-(trifluoromethyl)phenyl]-3-{4-{[(4-methoxy-3,5-dimethylpyridin-2-yl)methyl]thio}phenyl}urea* (**7n**)

Compound **7n** was prepared according to the general procedure by using compound **4c** (0.27 g, 1 mmol) and 3-chloro-4-(trifluoromethyl)aniline (0.20 g, 1 mmol), obtained a white solid (0.35 g) in 70.1% yield. m.p. 152.3–153.1 ◦C. IR (KBr, cm−1): υ 3422.2, 2921.6, 2852.2, 1719.2, 1593.8, 1546.4, 1480.4, 1419.8, 1384.6, 1311.1, 1265.2, 1229.0, 1174.6, 1130.8, 1073.5, 1031.7, 823.3, 619.9. 1H-NMR (400 MHz, DMSO-*d*6) δ 9.18 (s, 1H), 8.91 (s, 1H), 8.12 (s, 1H), 8.10 (d, *J* = 2.0 Hz, 1H), 7.64 (d, *J* = 8.9 Hz, 1H), 7.62 (s, 1H), 7.42 (d, *J* = 2.1 Hz, 1H), 7.40 (d, *J* = 2.1 Hz, 1H), 7.33 (s, 1H), 7.31 (d, *J* = 1.9 Hz, 1H), 4.22 (s, 2H), 3.70 (s, 3H), 2.19 (s, 3H), 2.18 (s, 3H). 13C-NMR (101 MHz, DMSO-*d*6) δ 164.07, 154.69, 154.49, 153.03, 149.00, 140.01, 132.84, 132.38, 126.45, 126.36, 124.20, 123.34, 122.49, 121.03, 115.40, 60.18, 31.13, 13.43, 11.00. ESI-MS (*m*/*z*): 496.1; 497.1; 498.1; 499.1 ([M + H]+). HRMS (ESI) (*m*/*z*): Calcd. for C23H21ClF3N3O2S, 496.1068 ([M + H]+), found: 496.1066 ([M + H]+). Purity (HPLC): 98.56%.

*1-{4-{[(4-Methoxy-3,5-dimethylpyridin-2-yl)methyl]thio}phenyl}-3-[3-(trifluoromethyl)pHenyl]urea* (**7o**)

Compound **7o** was prepared according to the general procedure by using compound **4c** (0.27 g, 1 mmol) and 3-(trifluoromethyl)aniline (0.16 g, 1 mmol), obtained a white solid (0.30 g) in 64.7% yield. m.p. 156.9–158.1 ◦C. IR (KBr, cm−1): υ 3420.6, 2984.8, 2922.8, 2851.8, 2350.3, 2321.1, 1609.8, 1491.8, 1443.2, 1398.4, 1369.9, 1338.2, 1311.7, 1271.2, 1229.2, 1172.1, 1124.2, 1072.1, 1002.9, 797.9, 698.2, 616.0. 1H-NMR (400 MHz, DMSO-*d*6) δ 9.04 (s, 1H), 8.84 (s, 1H), 8.12 (s, 1H), 8.00 (d, *J* = 2.0 Hz, 1H), 7.57 (d, *J* = 8.8 Hz, 1H), 7.51 (t, *J* = 7.8 Hz, 1H), 7.42 (d, *J* = 1.9 Hz, 1H), 7.40 (d, *J* = 2.2 Hz, 1H), 7.33 (s, 1H), 7.32 (s, 1H), 7.30 (d, *J* = 2.6 Hz, 1H), 4.22 (s, 2H), 3.70 (s, 3H), 2.19 (s, 3H), 2.18 (s, 3H). 13C-NMR (101 MHz, DMSO-*d*6) δ 163.88, 155.76, 152.85, 148.95, 140.97, 138.73, 131.69, 130.35, 128.51, 125.21, 125.05, 123.31, 122.32, 119.43, 118.58, 114.64, 60.16, 31.13, 13.36, 11.33. ESI-MS (*m*/*z*): 462.3 ([M + H]+). HRMS (ESI) (*m*/*z*): Calcd. for C23H22F3N3O2S, 462.1458 ([M + H]+), found: 462.1469 ([M + H]+). Purity (HPLC): 99.79%.

*1-{4-{[(3,4-Dimethoxypyridin-2-yl)methyl]thio}phenyl}-3-phenylurea* (**7p**)

Compound **7p** was prepared according to the general procedure by using compound **4d** (0.28 g, 1 mmol) and aniline (0.10 g, 1 mmol), obtained a white solid (0.18 g) in 45.8% yield. m.p. 127.7–128.5 ◦C. IR (KBr, cm<sup>−</sup>1): υ 3287.2, 2937.3, 1654.0, 1593.7, 1548.0, 1487.0, 1442.7, 1421.2, 1379.4, 1297.9, 1270.4, 1231.6, 1175.0, 1071.7, 997.3, 932.8, 829.0, 782.9, 742.9, 692.4, 618.5, 516.5. 1H-NMR (400 MHz, DMSO-*d*6) δ 8.72 (s, 1H), 8.67 (s, 1H), 8.12 (d, *J* = 5.5 Hz, 1H), 7.49–7.45 (m, 1H), 7.44 (s, 1H), 7.41 (d, *J* = 2.0 Hz, 1H), 7.40 (d, *J* = 2.2 Hz, 1H), 7.33 (d, *J* = 2.0 Hz, 1H), 7.31 (d, *J* = 1.9 Hz, 1H), 7.29 (d, *J* = 7.7 Hz, 1H), 7.28–7.25 (m, 1H), 7.03 (d, *J* = 5.5 Hz, 1H), 6.97 (t, *J* = 7.3 Hz, 1H), 4.17 (s, 2H), 3.87 (s, 3H), 3.74 (s, 3H). 13C-NMR (101 MHz, DMSO-*d*6) δ 158.52, 152.88, 151.53, 145.72, 143.40, 138.98, 131.39, 129.24, 128.54, 122.33, 119.17, 118.69, 108.29, 60.99, 56.34, 36.37, 31.14. ESI-MS (*m*/*z*): 396.3 ([M + H]+), 418.2 ([M + Na]+). HRMS (ESI) (*m*/*z*): Calcd. for C21H21N3O3S, 396.1376 ([M + H]+), found: 396.1380 ([M + H]+). Purity (HPLC): 99.90%.

*1-(4-Chlorophenyl)-3-{4-{[(3,4-dimethoxypyridin-2-yl)methyl]thio}phenyl}urea* (**7q**)

Compound **7q** was prepared according to the general procedure by using compound **4d** (0.28 g, 1 mmol) and 4-chloroaniline (0.13 g, 1 mmol), obtained a white solid (0.21 g) in 49.1% yield. m.p. 141.7–142.9 ◦C. IR (KBr, cm−1): υ 3345.3, 3096.8, 2924.2, 2852.2, 1711.6, 1631.2, 1590.8, 1535.1, 1490.0, 1449.2, 1427.9, 1399.4, 1300.6, 1284.5, 1237.1, 1195.2, 1174.0, 1087.1, 1067.2, 996.9, 828.3, 703.0, 509.1. 1H-NMR (400 MHz, DMSO-*d*6) δ 8.83 (s, 1H), 8.76 (s, 1H), 8.11 (d, *J* = 5.5 Hz, 1H), 7.49 (s, 1H), 7.47 (s, 1H), 7.41 (s, 1H), 7.39 (s, 1H), 7.34-7.31 (m, 2H), 7.03 (d, *J* = 5.5 Hz, 1H), 4.17 (s, 2H), 3.87 (s, 3H), 3.74 (s, 3H). 13C-NMR (101 MHz, DMSO-*d*6) δ 158.52, 152.78, 151.50, 145.71, 143.40, 139.10, 138.75, 131.29, 129.07, 128.79, 125.85, 120.21, 119.30, 108.30, 60.99, 56.34, 36.29, 31.14. ESI-MS (*m*/*z*): 430.6 ([M + H]+), 452.1 ([M + Na]+). HRMS (ESI) (*m*/*z*): Calcd. for C21H20ClN3O3S, 430.0987 ([M + H]+), found: 430.0993 ([M + H]+). Purity (HPLC): 99.33%.

*1-{4-{[(3,4-Dimethoxypyridin-2-yl)methyl]thio}phenyl}-3-(4-methoxyphenyl)urea* (**7r**)

Compound **7r** was prepared according to the general procedure by using compound **4d** (0.28 g, 1 mmol) and 4-methoxyaniline (0.12 g, 1 mmol), obtained a white solid (0.20 g) in 47.9% yield. m.p. 179.0–180.6 ◦C. IR (KBr, cm−1): υ 3428.5, 2985.2, 2923.2, 2851.3, 1630.7, 1587.2, 1557.0, 1510.3, 1490.9, 1442.6, 1398.6, 1299.7, 1270.7, 1232.4, 1173.6, 1072.2, 1033.0, 1000.9, 934.0, 829.1, 799.5, 617.4, 549.0, 523.3. 1H-NMR (400 MHz, DMSO-*d*6) δ 8.62 (s, 1H), 8.46 (s, 1H), 8.11 (d, *J* = 5.5 Hz, 1H), 7.39 (d, *J* = 2.0 Hz, 1H), 7.38 (d, *J* = 2.2 Hz, 1H), 7.35 (d, *J* = 2.2 Hz, 1H), 7.33 (d, *J* = 2.2 Hz, 1H), 7.31 (d, *J* = 2.2 Hz, 1H), 7.30 (d, *J* = 2.0 Hz, 1H), 7.03 (d, *J* = 5.5 Hz, 1H), 6.87 (d, *J* = 2.2 Hz, 1H), 6.85 (d, *J* = 2.2 Hz, 1H), 4.15 (s, 2H), 3.87 (s, 3H), 3.74 (s, 3H), 3.71 (s, 3H). 13C-NMR (101 MHz, DMSO-*d*6) δ 158.51, 154.98, 153.07, 151.55, 145.73, 143.40, 139.22, 133.08, 131.45, 128.24, 120.54, 119.06, 114.46, 108.30, 60.99, 56.35, 55.65, 36.43, 31.15. ESI-MS (*m*/*z*): 426.3 ([M + H]+). HRMS (ESI) (*m*/*z*): Calcd. for C22H23N3O4S, 426.1482 ([M + H]+), found: 426.1489 ([M + H]+). Purity (HPLC): 98.84%.

*1-[4-Chloro-3-(trifluoromethyl)phenyl]-3-{4-{[(3,4-dimethoxypyridin-2-yl)methyl]thio}pHenyl}urea* (**7s**)

Compound **7s** was prepared according to the general procedure by using compound **4d** (0.28 g, 1 mmol) and 3-chloro-4-(trifluoromethyl)aniline (0.20 g, 1 mmol), obtained a white solid (0.32 g) in 64.5% yield. m.p. 188.1–189.2 ◦C. IR (KBr, cm−1): υ 3425.5, 2921.9, 2852.4, 1589.9, 1545.2, 1485.2, 1419.2, 1384.4, 1306.1, 1229.2, 1175.6, 1132.0, 1068.8, 1033.0, 824.9. 1H-NMR (400 MHz, DMSO-*d*6) δ 9.15 (s, 1H), 8.88 (s, 1H), 8.11 (d, *J* = 5.5 Hz, 1H), 8.10 (d, *J* = 2.2 Hz, 1H), 7.64 (d, *J* = 8.8 Hz, 1H), 7.62–7.58 (m, 1H), 7.42 (d, *J* = 2.0 Hz, 1H), 7.41 (d, *J* = 2.2 Hz, 1H), 7.34 (d, *J* = 2.2 Hz, 1H), 7.33 (d, *J* = 2.0 Hz, 1H), 7.03 (d, *J* = 5.5 Hz, 1H), 4.17 (s, 2H), 3.88 (s, 3H), 3.74 (s, 3H). 13C-NMR (101 MHz, DMSO-*d*6) δ 158.52, 152.76, 151.47, 145.73, 143.40, 139.76, 138.36, 132.44, 131.12, 129.28, 123.53, 122.78, 119.62, 117.24, 108.32, 61.00, 56.35, 36.17, 31.14. ESI-MS (*m*/*z*): 498.2 ([M + H]+). HRMS (ESI) (*m*/*z*): Calcd. for C22H19ClF3N3O3S, 498.0861 ([M + H]+), found: 498.0844 ([M + H]+). Purity (HPLC): 98.10%.

*1-{4-{[(3,4-Dimethoxypyridin-2-yl)methyl]thio}phenyl}-3-[3-(trifluoromethyl)phenyl]urea* (**7t**)

Compound **7t** was prepared according to the general procedure by using compound **4d** (0.28 g, 1 mmol) and 3-(trifluoromethyl)aniline (0.16 g, 1 mmol), obtained a white solid (0.21 g) in 44.3% yield. m.p. 198.4–199.8 ◦C. IR (KBr, cm−1): υ 3422.2, 2985.4, 2377.5, 2349.8, 2320.7, 2024.8, 1712.9, 1594.1, 1564.4, 1537.2, 1491.3, 1445.8, 1399.4, 1370.2, 1316.3, 1273.6, 1230.1, 1173.8, 1124.9, 1068.3, 1002.1, 932.4, 892.8, 828.2, 798.3, 743.3, 697.8, 615.6. 1H-NMR (400 MHz, DMSO-*d*6) δ 9.04 (s, 1H), 8.83 (s, 1H), 8.11 (d,

*J* = 5.5 Hz, 1H), 8.01 (d, *J* = 2.0 Hz, 1H), 7.60–7.54 (m, 1H), 7.51 (t, *J* = 7.9 Hz, 1H), 7.43 (d, *J* = 2.0 Hz, 1H), 7.41 (d, *J* = 2.2 Hz, 1H), 7.34 (d, *J* = 2.2 Hz, 1H), 7.32 (d, *J* = 2.5 Hz, 1H), 7.30 (s, 1H), 7.03 (d, *J* = 5.6 Hz, 1H), 4.17 (s, 2H), 3.88 (s, 3H), 3.75 (s, 3H). 13C-NMR (101 MHz, DMSO-*d*6) δ 158.52, 152.87, 151.50, 145.72, 143.41, 140.98, 138.55, 131.22, 130.34, 129.08, 122.31, 119.49, 118.56, 114.65, 108.31, 60.99, 56.34, 36.24, 31.12. ESI-MS (*m*/*z*): 464.2 ([M + H]+). Purity (HPLC): 98.85%.

#### *3.2. Biological Evaluation*

#### 3.2.1. Antiproliferative Activity Assays

The antiproliferative activities of target compounds were determined using a standard MTT assay [27–30]. Exponentially growing cells A549 (3 <sup>×</sup> <sup>10</sup><sup>3</sup> cells/well), HCT-116 (1 <sup>×</sup> <sup>10</sup><sup>4</sup> cells/well) and PC-3 (8 <sup>×</sup> 103 cells/well) were seeded into 96-well plates and incubated for 24 h to allow the cells to attach. After 24 h of incubation, the culture medium was removed and fresh medium containing various concentrations of the candidate compounds was added to each well. The cells were then incubated for 72 h, thereafter MTT assays were performed and cell viability was assessed at 570 nm by a microplate reader (ThermoFisher Scientific (Shanghai) Instrument Co., Ltd., Shanghai, China).

#### 3.2.2. Cell Apoptosis Assay

A549 cells were seeded into a 6-well plate (2 <sup>×</sup> 105/well) and incubated for 24 h. Then the cells were treated with different concentrations of the tested compound **7i** for 24 h. Thereafter, the cells were collected and the Annexin-V-FITC/PI apoptosis kit (Biovision, Milpitas, CA, USA) was used according to the manufacturer's protocol. The cells were analyzed by Accuri C6 flow cytometric (Becton Dickinson, Franklin Lakes, NJ, USA) [31].

#### 3.2.3. Cell Cycle Analysis

For flow cytometric analysis of DNA content, 5 <sup>×</sup> 105 A549 cells in exponential growth were treated with different concentrations of the compound **7i** for 24 or 48 h. After an incubation period, the cells were collected, centrifuged and fixed with ice cold ethanol (70%). The cells were then treated with buffer containing RNAse A and 0.1% Triton X-100 and then stained with the propidium iodide (PI). The samples were analyzed on Accuri C6 flow cytometer (Becton Dickinson). [32].

#### **4. Conclusions**

In summary, a new series of 1-aryl-3-{4-[(pyridin-2-ylmethyl)thio]phenyl}urea derivatives were designed and synthesized based on molecular hybridization strategy. Majority of target compounds showed moderate to good growth inhibition against the tested cancer cells. Particularly, compound **7i** exhibited more potent antiproliferative activity than well-known anticancer drug sorafenib against all three cancer cell lines (A549, HCT-116 and PC-3). The preliminary mechanism investigation showed that compound **7i** could induce A549 cells to apoptosis, and halted cell cycle progression at the G1 phase. The SARs illustrated that these target compounds in this work might serve as bioactive fragments, and compound **7i** could be used as a lead compound for the development of potent cancer chemotherapeutic agents in the drug discovery process.

**Supplementary Materials:** The following are available online. 1H-NMR, 13C-NMR, ESI-MS and HRMS of the target compounds, respectively.

**Author Contributions:** C.Z., X.T., J.F., Y.L., N.D. and Z.J. contributed to the synthetic work and the characterization of all target compounds. C.Z. and Z.J. the preparation of the manuscript. C.Z., X.T. and Q.M. performed the biological assays. X.L. and C.H. proposed the studies and contributed to their design, as well as to the writing of the manuscript. All authors have read and approved the final manuscript.

**Funding:** This work was supported by the National Science Foundation of China (Grant No. 21342006), the Program for Innovative Research Team of the Ministry of Education of China (Grant No. IRT\_14R36), the Natural Science Foundation of Liaoning Province, China (Grant No. 201602695), and the Scientific Research Foundation of Department of Education of Liaoning Province, China (Grant No. L2015517).

**Conflicts of Interest:** The authors confirm that this article content has no conflict of interest.

#### **References**


**Sample Availability:** Samples of all the target compounds are available from the authors.

© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

### *Article* **Potent Cytotoxicity of Novel L-Shaped Ortho-Quinone Analogs through Inducing Apoptosis**

**Sheng-You Li 1,2, Ze-Kun Sun 2,3, Xue-Yi Zeng 2,4, Yue Zhang 2,5, Meng-Ling Wang 2,4, Sheng-Cao Hu 4, Jun-Rong Song 2,4, Jun Luo 4, Chao Chen 2,4,\*, Heng Luo 2,4,\* and Wei-Dong Pan 1,2,4,\***


Academic Editor: Qiao-Hong Chen Received: 10 October 2019; Accepted: 11 November 2019; Published: 15 November 2019

**Abstract:** Twenty-seven L-shaped ortho-quinone analogs were designed and synthesized using a one pot double-radical synthetic strategy followed by removing methyl at C-3 of the furan ring and introducing a diverse side chain at C-2 of the furan ring. The synthetic derivatives were investigated for their cytotoxicity activities against human leukemia cells K562, prostate cancer cells PC3, and melanoma cells WM9. Compounds **TB1**, **TB3**, **TB4**, **TB6**, **TC1**, **TC3**, **TC5**, **TC9**, **TC11**, **TC12**, **TC14**, **TC15**, **TC16**, and **TC17** exhibited a better broad-spectrum cytotoxicity on three cancer cells. **TB7** and **TC7** selectively displayed potent inhibitory activities on leukemia cells K562 and prostate cancer cells PC3, respectively. Further studies indicated that **TB3**, **TC1**, **TC3**, **TC7**, and **TC17** could significantly induce the apoptosis of PC3 cells. **TC1** and **TC17** significantly induced apoptosis of K562 cells. **TC1**, **TC11**, and **TC14** induced significant apoptosis of WM9 cells. The structure-activity relationships evaluation showed that removing methyl at C-3 of the furan ring and introducing diverse side chains at C-2 of the furan ring is an effective strategy for improving the anticancer activity of L-shaped ortho-quinone analogs.

**Keywords:** ortho-quinones; antitumor activity; beta-lapachone; tanshione IIA

#### **1. Introduction**

Over several decades, cancer continues to be the most awful disease due to its uncontrolled cell growth and the fact that it is a dominate killer of human beings worldwide [1]. Especially in China, millions of deaths have been caused by tumor. The common cancer types in Chinese male, in 2018, were lung, stomach, colorectum, liver, and esophageal cancer. Additionally, breast, lung, colorectum, thyroid, and stomach cancer were the common types in Chinese female [2]. The incidence of colorectal cancer in males and females has increased, however, the incidence of esophageal, stomach, and liver cancer has decreased between 2000 and 2011 [3]. Meanwhile, the incidence and mortality of prostate cancer and bladder cancer in males, together with obesity and hormonal exposure-related cancers, namely thyroid, breast, and ovarian cancer in females have shown a rising trend [3]. However, the standardized treatments of cancer, including surgery, chemotherapy, and radiation therapy, show many limitations, such as severe adverse effects, recurrence, and increasing drug resistance [4].

Currently, phytochemicals have become a valuable source of anticancer drugs. Actually, over 75% of nonbiological anticancer drugs approved are either plant-derived natural products or developed based on these products [5]. Therefore, natural products have continued to be a hot research topic for the development of new antitumor drugs [6–9].

Tanshinone IIA is a natural ortho-quinone isolated from the rhizome of *Salvia miltiorrhiza Bunge* with antineoplastic activity, such as gastric cancer, breast cancer, osteosarcoma, etc. [10–13]. These various properties demonstrate that tanshinone IIA is a potential antitumor drug candidate. Furthermore, beta-lapachone is another natural ortho-quinone which has been reported to selectively kill many human cancer cells [14], however, the pyran ring of beta-lapachone has been found to be unstable during metabolism in the human body, and may led to side effects on normal tissues [15,16]. Recently, studies have revealed that some tanshinone analogs show similar or stronger antitumor activity when the ring-A is removed but the furan ring is retained [17,18]. You et al. [19,20] discovered that the binding site for quinone oxidoreductase-1 (NQO1) substrates was an L-shaped pocket (Figure 1B) which binds well with tanshinone analogs, and showed higher antitumor activities than the planar compound **1** and beta-lapachone. Therefore, we surmise that removing methyl at C-3 of the furan ring is more suitable for the binding site, and we anticipate that a novel L-shaped molecule without methyl at C-3 of the furan ring could provide better antitumor activities. Considering that some nitrogen, oxygen-substituted, and amino acid substrates can improve aqueous solubility and antitumor activities [21–25], we have attempted to introduce a great diversity of oxygen-substituted, nitrogen-containing groups and amino acids. Thus, in this work, we developed quinone-directed agents by removing methyl at C-3 of the furan ring and introducing a diverse side chain at C-2 of the furan ring, culminating in the discovery of a promising scaffold. The inhibitory activity was assessed in vitro using three cell lines including K562, PC3, and WM9.

**Figure 1.** (**A**) Structural design strategy and (**B**) l-shaped pocket. The figure is available in reference [19].

#### **2. Discussion and Results**

#### *2.1. Chemistry*

The synthesis of two substituted naphtho[1,2-b]furan-4,5-diones is outlined in Scheme 1. Briefly, treatment of lawsone **6** with allyl bromide followed by subsequent Claisen rearrangement afforded **7**, which was then cyclized to get the ortho-quinone **8** by using Lewis acid NbCl5 at room temperature [26].

**Scheme 1.** Synthesis of L-shaped ortho-quinone analogs.

Initially, dealing **8** with *N*-bromosuccinimide (NBS) and 2,2 -azobis(2-methylpropionitrile) (AIBN) afforded only trace amounts of **8a**. Another intermediate, **8b**, was obtained by Nelson's method [27] as shown in Scheme 1. Then, compound **9** was obtained from **8b** through a second radical reaction. Considering the same reaction condition, we successfully got **9** from **8** through a bis-radical reaction. The brominated intermediate **9** was reacted with substituted phenol or amine to provide ortho-quinone derivatives **TB1**–**TB9** and **TC1**–**TC18**, respectively. All the structures of ortho-quinone derivatives were identified through 1H, 13C, and HRMS.

In summary, we successfully established an effective synthetic strategy, which removed the methyl at C-3 of the furan ring and introduced diverse side chains at C-2 of the furan ring. In addition, we replaced the bromide of **9** with a variety of oxygen-substituted, nitrogen-containing group, and amino acid by a nucleophilic substitution.

#### *2.2. In Vitro Cytotoxicity Assay*

The cytotoxic activities of 5 μmol/L of the synthesized l-shaped ortho-quinone analogs were determined by using three cancer cell lines (Table 1). The results revealed that compounds **TB1**, **TB3**, **TB4**, **TB6**, **TC1**, **TC3**, **TC5**, **TC9**, **TC11**, **TC12**, **TC15**, **TC16**, and **TC17** showed a broad-spectrum potent inhibitory activity on the proliferation of the cancer cell lines, with a more than 70% inhibition rate, and **TB7** showed better inhibitory activity on K562 cells as compared with other cells. Moreover, we observed that **TC7** inhibited the growth of PC3 cells more efficiently than other cells.


**Table 1.** The structures and inhibitory rates after treating cancer cell lines with 5 μmol/L of target compounds, respectively. Data was presented as the mean ± SD of three independent experiments.


**Table 1.** *Cont*.

The concentration inhibition curves (Figure 2) were analyzed to calculate the IC50 values of the selected active compounds. The results indicated there was a dose-dependent trend of the inhibitory response of all active compounds on three cancer cells for treating 48 h. The IC50 values were summarized in Table 2 and show that the cytotoxicity of compounds **TB3**, **TC1**, **TC3**, **TC7**, **TC9**, and **TC17** on PC3 were better (*P* < 0.05) than that of the positive control (tanshinone IIA and paclitaxel), and another active compound exhibited similar activity to that of the positive control. The inhibition activity of **TC1** against the growth of K562 was better than that of the positive control, paclitaxel, and tanshinone IIA. Compounds **TB6**, **TC1**, **TC11**, **TC14**, and **TC15** inhibited the growth of WM9 better (*P* < 0.05) than that of tanshinone IIA and paclitaxel. In summary, most of novel L-shaped ortho-quinone analogs exhibited relatively better cytotoxicity activity as compared with the two positive controls, which indicated that the analogs containing L-shaped ortho-quinone as the core structure, possessed stronger anticancer activity. This result provided a preliminary biological activity basis for the investigation of anticancer candidate agents.

**Figure 2.** Growth inhibition induced by the active L-shaped ortho-quinone analogs on PC3, K562, and WM9 cells by MTT assay. The IC50 values (μM) of the compounds were determined according to these curves at different incubation times. The 100 μL tested compounds were added to 96-well microculture plates and 100 <sup>μ</sup>L cells (a final concentration of 5 <sup>×</sup> 104/well) were incubated for 48 h at 37 ◦C. Cell survival was evaluated by MTT assay. The inhibition ratio (%) was calculated as described in the Methods section. Data was presented as mean ± SD of three independent experiments.


**Table 2.** IC50 values of selected compounds in vitro.

Note: \* represents *p* < 0.05 and \*\* represents *p* < 0.01, vs. the inhibition of the positive control to the cancer cell lines. The data represented the average of three independent experiments.

#### *2.3. Structure-Activity Relationships Study*

To obtain two series of analogs, we successfully built an effective synthetic strategy by removing the methyl at C-3 of the furan ring and introducing diverse side chains at C-2 of the furan ring. On the basis of the cytotoxicity results (Tables 1 and 2), a preliminary structure-activity relationships could be established. The TB series molecules bearing electron-withdrawing groups or multi-substituted groups such as compounds **TB3**, **TB4**, and **TB6** showed a better inhibitory effect on PC3 cell lines, K562 cell lines, and WM9 cell lines, whereas the TB series molecules bearing alkane groups at the 2-position showed decreased cytotoxicity in PC3 cell lines, K562 cell lines, and WM9 cell lines, such as compounds **TB9**. The TC series molecules with electron-withdrawing groups, saturate six-membered rings, or multi-substituted groups emerged greater inhibitory effects on three cancer cell lines, such as **TC11**, **TC12**, **TC15**, and **TC16**, whereas the TC series molecules bearing donating groups or alkane groups at the 2-position showed reduced cytotoxicity in three cancer cell lines. The structure-activity relationships evaluation also showed that removing methyl at C-3 of the furan ring and introducing

diverse side chains at C-2 of the furan ring were good strategies for improving the anticancer activity of L-shaped ortho-quinone analogs.

#### *2.4. E*ff*ects of Active Compounds on Cell Apoptosis*

According to the above IC50 values of all active compounds, we selected six active compounds (**TB3**, **TC1**, **TC3**, **TC7**, **TC9**, and **TC17**) for PC3, three active compounds (**TB6**, **TC1**, and **TC17**) for K562, and four active compounds (**TB6**, **TC1**, **TC11**, and **TC14**) for WM9, based on their higher activities than that of the positive control and better selectivity and, then, studied their effects on cell apoptosis by microscope observation (Figure 3) and flow cytometry (Figure 4). The microscopic observations (Figure 3A) showed that the number of PC3 cells was significantly reduced by treatments with 2.5 μmol/L of **TB3**, **TC1**, **TC3**, and **TC7**; the apoptotic bodies and cell fragments were significantly observed as compared with the control group. The PC3 cells treated with **TC9** showed that the number of cells were significantly reduced, while fewer cells died and there were no significant apoptotic bodies. The PC3 cells treated with **TC7** showed a significant decrease in the number of cells, meanwhile, some cells died, apoptotic bodies appeared obviously, and the morphology of some cells became an irregular shape of spindle length. Above all, the inhibitory activity of **TB3**, **TC1**, **TC3**, and **TC7** may be through inducing apoptosis, another two compounds may be through different types.

**Figure 3.** *Cont*.

**Figure 3.** Effects of the active compounds on PC3 (**A**), K562 (**B**), and WM9 (**C**) cell growth and apoptosis. Cell number and morphological appearance of the two types of cell lines treated with 2.5 μmol/L of active compounds, then, it was observed by a fluorescent inverted microscope after 24 h. Scale bar = 100 μM in all images. All experiments were performed in triplicate.

**Figure 4.** *Cont*.

**Figure 4.** Effects of the active compounds on PC3 (**A**), K562 (**B**)**,** and WM9 (**C**) cell growth and apoptosis. Cell apoptosis induced by the compounds and tested by flow cytometry and the date was analyzed with Origin Pro 9.0 (**D**) and presented as means ± SEM from at least three independent experiments. \* *p* < 0.05, \*\* *p* < 0.01 (*n* = 3) as compared with the control.

The K562 cells treated with **TC1** were obviously dead and dispersed, with the appearance of apoptotic bodies as comparing with the control group (Figure 3B). The cells treated with **TB6** had a significantly reduced number of cells and most cells clumped growth similar to the control cells. For the cells treated with **TC17** we observed both dead cells and fewer clumps of cells. Furthermore, for the WM9 cell lines treated with **TB6**, **TC1**, **TC11**, and **TC14** (Figure 3C), we observed that the cells treated with **TC1** and **TC11** were obviously dead with a large number of apoptotic bodies and dispersed cells; the cells treated with **TB6** showed a significant decrease in the number of cells and fewer dead cells. Observation of the cells treated with **TC14** showed that the number of cells was significantly reduced, while some cells were obviously dead with apoptotic bodies appearing, and the morphology of some cells also became an irregular shape of spindle length. The above results indicated that **TC1** can induce apoptosis for K562 and WM9 cells to inhibit the growth; **TB6**, **TC11**, **TC14**, and **TC17** can jointly inhibit the proliferation of cell through a variety of mechanisms.

Flow cytometry analyzed results (Figure 4) confirmed that **TB3** (*p* < 0.01), **TC1** (*p* < 0.01), **TC3** (*p* < 0.01), **TC7** (*p* < 0.01), and **TC17** (*p* < 0.05) could significantly induce the apoptosis of PC3 cells (Figure 4A), while **TC9** did not. **TC1** (*p* < 0.01) and **TC17** (*p* < 0.05) significantly induced apoptosis of K562 cells (Figure 4B), while **TB6** had no significant effect on apoptosis of K562 and WM9 cells. **TC1** (*p* < 0.01), **TC11** (*p* < 0.05), and **TC14** (*p* < 0.01) could potently induce apoptosis for WM9 cells (Figure 4C).

#### **3. Materials and Methods**

#### *3.1. Instruments and Materials*

High-resolution mass spectra (HRMS) were obtained on an electrospray ionization (ESI) mode on a Bruker ESI-QTOF mass spectrometry. Nuclear magnetic resonance (NMR) spectra were recorded on a Bruker Avance NEO (1H NMR, 600 MHz; 13C NMR, 150 MHz, Bruker, Switzerland) with TMS as an internal standard. The IR spectra were recorded by using a FTIR Spectrometer (IR 200 Fourier Energy Spectrum Technology Co., Ltd., TianJin, China) and the KBr disk method was adopted. The melting points (mp) were determined on an WRX-4 microscope melting point apparatus. The column chromatography was performed on silica gel (Qingdao, 200–300 mesh) and the thin-layer (0.25 mm) chromatography (TLC) analysis was carried out on silica gel plates (Qingdao, China). Other reagents were analytical grade or guaranteed reagent commercial product and used without further purification, unless otherwise noted.

#### *3.2. Methods of Synthesis*

#### 3.2.1. Synthesis of 2-Allyl-3-hydroxy-1,4-naphthoquinone (**7**)

A mixture of lawsone **6** (10.0 g, 57.42 mmol) and anhydrous K2CO3 (7.94 g, 57.42 mmol) in anhydrous DMF (100 mL) were stirred for 15 min at room temperature. Allyl bromide (17.37 g, 143.55 mmol) in DMF (5 mL) was added dropwise and stirred for 15 min at 0 ◦C. The mixture was refluxed at 120 ◦C for 3 h and then cooled to room temperature before it was poured into water and extracted with EA. The organic phase was washed with brine, dried over Na2SO4, filtered, and concentrated in vacuo. The crude product was purified by column chromatography on silica gel (eluent: petroleum ether/EtOAc 15:1) to afford **7** (7.8 g, 63% yield) as a light yellow solid. Other data was found in reference [26].

#### 3.2.2. Synthesis of 2-Methyl-2,3-dihydrolnaphthol[1,2-*b*]furan-4,5-dione (**8**)

NbCl5 (18.92 g, 70.02 mmol) was added into **7** (3.0 g, 14.00 mmol) in anhydrous DCM (50 mL) at 0 ◦C. After stirring for 45 min at 30 ◦C, the mixture was poured into ice water and extracted with DCM. The organic phase was washed with brine, dried over Na2SO4, filtered, and concentrated in vacuo. The crude product was purified by column chromatography on silica gel (eluent: petroleum ether/EtOAc 4:1) to afford **8** (2.16 g, 72% yield) as red solid. Other data was found in reference [26].

#### 3.2.3. Synthesis of 2-Bromomethyl-naphtho[1-*b*]furan-4,5-dione (**9**)

A mixture of **8** (1.5 g, 7.00 mmol), anhydrous *N*-bromosuccinimide (2.49 g, 1.40mmol), and 2,2 -azobis(2-methylpropionitrile) (114.98 mg, 1.40 mmol) in anhydrous CCl4 (50 mL) was stirred under argon at 70 ◦C, until **8** were disappeared. Then, the mixture was cooled to room temperature, and anhydrous N-bromosuccinimide (2.49 g, 1.40 mmol) and 2,2 -azobis(2-methylpropionitrile) (114.98 mg, 1.40 mmol) were added and stirred at 70 ◦C for 2 h. The mixture was cooled to room temperature, and poured into water, extracted with EA, washed with brine, dried over Na2SO4, filtered, and concentrated in vacuo. The crude product was purified by column chromatography on silica gel (eluent: petroleum ether/EtOAc 12:1) to afford **9** (1.0 g, 67% yield) as red solid. Mp: 169–170 ◦C. 1H NMR (600 MHz, CDCl3) δ 8.09 (d, *J* = 7.8 Hz, 1H), 7.77 (d, *J* = 7.6 Hz, 1H), 7.69 (t, *J* = 7.6 Hz, 1H), 7.51 (t, *J* = 7.6 Hz, 1H), and 6.83 (s, 1H), 4.55 (s, 2H). 13C NMR (150 MHz, CDCl3) δ 180.06, 174.13, 160.94, 153.46, 135.56, 130.74, 130.69, 129.03, 127.98, 122.69, 122.34, 108.04, and 21.87. IR (ν, cm<sup>−</sup>1): 3118.33, 2920.28, 2339.32, 1670.84, 1551.16, and 691.42. HRMS (ESI) calcd. for [M + Na]<sup>+</sup> C13H7O3BrNa<sup>+</sup>: 312.9471, found 312.9460.

#### 3.2.4. Synthesis of **TB1**–**9** and **TC1**–**18**

A mixture of the corresponding amines or alcohols (0.52 mmol), K2CO3 (142 mg, 1.03 mmol), and **9** (100 mg, 0.34 mmol) in THF (5 mL) was stirred at 30 ◦C to 50 ◦C for 4 h. After cooling, the mixture was poured into water and extracted with EA. The combined organic layer was washed with brine and dried over anhydrous Na2SO4, filtered, and concentrated to afford a crude product which was purified through column chromatography on silica gel.

**TB1**: *2-(((4-methoxyphenyl)oxy)methyl)naphtho[1-b]furan-4,5-dione.* Red solid, yield: 34%. Mp: 112–113 ◦C. 1H NMR (600 MHz, CDCl3) δ 8.09 (d, *J* = 7.6 Hz, 1H), 7.75 (d, *J* = 7.6 Hz, 1H), 7.66 (t, *J* = 7.5 Hz, 1H), 7.48 (t, *J* = 7.6 Hz, 1H), 6.94–6.92 (m, 2H), 6.87–6.85 (m, 2H), 6.83 (s, 1H), 5.04 (s, 2H), and 3.78 (s, 3H). 13C NMR (150 MHz, CDCl3) δ 180.34, 174.38, 160.88, 154.72, 153.82, 151.95, 135.47, 130.66, 130.50, 128.98, 128.28, 122.59, 122.12, 116.34, 114.81, 108.37, 63.03, and 55.73. IR (ν, cm−1): 3445.32, 2358.40, 2339.32, 1671.47, 1598.48, 1253.20, 1219.81, 1161.58, and 1057.34. HRMS (ESI) calcd. for [M + Na]<sup>+</sup> C20H14O5Na<sup>+</sup>: 357.0733, found 357.0723.

**TB2**: *2-(((4-acetylphenyl)oxy)methyl)naphtho[1,2-b]furan-4,5-dione.* Light orange solid, yield: 40%. Mp: 190–191 ◦C. 1H NMR (600 MHz, CDCl3) δ 8.13 (d, *J* = 6.4 Hz, 1H), 8.00 (d, *J* = 8.9 Hz, 2H), 7.78 (d, *J* = 9.1 Hz, 1H), 7.69 (t, *J* = 7.6 Hz, 1H), 7.53 (t, *J* = 7.6 Hz, 1H), 7.06 (d, *J* = 8.9 Hz, 2H), 6.94 (s, 1H), 5.19 (s, 2H), and 2.60 (s, 3H). 13C NMR (150 MHz, CDCl3) δ 196.71, 180.20, 174.31, 161.58, 161.10, 152.60, 135.53, 131.28, 130.74, 130.70, 129.02, 128.11, 122.64, 122.08, 114.48, 108.98, 61.92, and 26.43. IR (ν, cm<sup>−</sup>1): 3445.93, 2358.51, 2341.16, 1671.86, 1599.84, 1249.41, 1217.01, 1178.57, and 1008.47. HRMS (ESI) calcd. for [M + Na]<sup>+</sup> C21H14O5Na<sup>+</sup>: 369.0733, found 369.0721.

**TB3**: *2-(((4-propiophenyl)oxy)methyl)naphtho[1,2-b]furan-4,5-dione.* Orange solid, yield: 37%. Mp: 179–180 ◦C. 1H NMR (600 MHz, CDCl3) δ 8.11 (d, *J* = 7.6 Hz, 1H), 8.00 (d, *J* = 8.9 Hz, 2H), 7.77 (d, *J* = 6.4 Hz, 1H), 7.71–7.68 (m, 1H), 7.53–7.50 (m, 1H), 7.06 (d, *J* = 8.9 Hz, 2H), 6.93 (s, 1H), 5.18 (s, 2H), 2.99 (q, *J* = 7.2 Hz, 2H), and 1.24 (t, *J* = 7.3 Hz, 3H). 13C NMR (150 MHz, CDCl3) δ 199.42, 180.20, 174.30, 161.40, 161.06, 152.68, 135.53, 130.98, 130.72, 130.68, 130.36, 129.01, 128.11, 122.63, 122.08, 114.47, 108.92, 61.90, 31.53, and 8.38. IR (ν, cm−1): 3447.03, 2358.62, 2341.16, 1669.65, 1601.07, 1222.31, 1180.35, and 1002.86. HRMS (ESI) calcd. for [M + Na]<sup>+</sup> C22H16O5Na<sup>+</sup>: 383.0890, found 383.0876.

**TB4**: *2-(((4-nitrophenyl)oxy)methyl)naphtho[1,2-b]furan-4,5-dione.* Red solid, yield: 48%. Mp: 205–206 ◦C. 1H NMR (600 MHz, DMSO-*d6*) δ 8.25 (d, *J* = 9.3 Hz, 2H), 7.96 (d, *J* = 7.6 Hz, 1H), 7.77–7.74 (m, 2H), 7.59–7.55 (m, 1H), 7.34 (d, *J* = 9.3 Hz, 2H), 7.18 (s, 1H), and 5.42 (s, 2H). 13C NMR (150 MHz, DMSO-*d6*) δ 179.58, 174.56, 163.29, 160.07, 152.42, 141.83, 135.43, 130.84, 130.17, 129.83, 127.89, 126.41, 122.50, 122.36, 115.94, 110.32, and 62.45. IR (ν, cm<sup>−</sup>1): 3445.75, 2358.70, 2341.16, 1681.37,1592.17, 1507.95, 1384.15, 1340.19, 1277.67, and 1110.13. HRMS (ESI) calcd. for [M + Na]<sup>+</sup> C19H11O6NNa<sup>+</sup>: 372.0479, found 372.0465.

**TB5**: *2-(((2-methoxyl-4-formyl)phenyl)oxy)methyl)naphtho[1,2-b]furan-4,5-dione.* Red solid, yield: 34%. Mp: 203–204 ◦C. 1H NMR (600 MHz, CDCl3) δ 9.91 (s, 1H), 8.13 (d, *J* = 6.4 Hz, 1H), 7.77 (d, *J* = 7.6 Hz, 1H), 7.72–7.68 (m, 1H), 7.54–7.48 (m, 3H), 7.15 (d, *J* = 8.0 Hz, 1H), 6.96 (s, 1H), 5.28 (s, 2H), and 3.97 (s, 3H). 13C NMR (150 MHz, CDCl3) δ 190.88, 180.19, 174.31, 161.15, 152.50, 152.42, 150.30, 135.52, 131.28, 130.75, 129.67, 129.03, 128.10, 122.68, 122.09, 115.30, 112.93, 109.87, 109.40, 62.85, and 56.09. IR (ν, cm−1): 3446.32, 2358.18, 2341.16, 1702.84, 1676.79, 1586.53, 1508.23, 1267.05, 1236.25, 1137.10, and 999.76. HRMS (ESI) calcd. for [M + Na]<sup>+</sup> C21H14O6Na<sup>+</sup>: 385.0683, found 385.0668.

**TB6**: *2-(((4-formylphenyl)oxy)methyl)naphtho[1,2-b]furan-4,5-dione.* Orange solid, yield: 30%. Mp: 212–213 ◦C. 1H NMR (600 MHz, CDCl3) δ 9.95 (s, 1H), 8.13 (d, *J* = 7.8 Hz, 1H), 7.92 (d, *J* = 8.9 Hz, 2H), 7.78 (d, *J* = 7.8 Hz, 1H), 7.73–7.69 (m, 1H), 7.56–7.51 (m, 1H), 7.14 (d, *J* = 8.7 Hz, 2H), 6.96 (s, 1H), and 5.21 (s, 2H). 13C NMR (150 MHz, CDCl3) δ 190.71, 180.17, 174.30, 162.67, 161.14, 152.36, 135.53, 132.10, 130.82, 130.77, 130.74, 129.05, 128.09, 122.64, 122.08, 115.06, 109.11, and 61.99. IR (ν, cm−1): 3446.21, 2358.24, 2337.30, 1687.41, 1671.45, 1598.48, 1253.19, 1219.95, 1161.49, and 1057.40. HRMS (ESI) calcd. for [M + Na]<sup>+</sup> C20H12O5Na<sup>+</sup>: 355.0577, found 355.0565.

**TB7**: *2-(((4-bromo-2-formyl)phenyl)oxy)methyl)naphtho[1,2-b]furan-4,5-dione.* Light orange solid, yield: 33%. Mp: 201–202 ◦C. 1H NMR (600 MHz, CDCl3) δ 10.43 (s, 1H), 8.14 (d, *J* = 7.6 Hz, 1H), 7.99 (d, *J* = 2.7 Hz, 1H), 7.76 (d, *J* = 7.6 Hz, 1H), 7.73–7.69 (m, 2H), 7.57–7.51 (m, 1H), 7.06 (d, *J* = 8.7 Hz, 1H), 6.96 (s, 1H), and 5.24 (s, 2H). 13C NMR (150 MHz, CDCl3) δ 187.86, 180.06, 174.25, 161.30, 158.94, 151.80, 138.27, 135.59, 131.52, 130.87, 130.82, 129.07, 127.95, 126.84, 122.64, 121.99, 115.01, 114.95, 109.43, and 62.80. IR (ν, cm<sup>−</sup>1): 3445.63, 2358.34, 2337.30, 1671.90, 1599.56, 1556.29, 1249.42, 1217.50, 1178.60, and 1008.51. HRMS (ESI) calcd. for [M + Na]<sup>+</sup> C20H11O5BrNa<sup>+</sup>: 432.9682, found 432.9673.

**TB8**: *2-(methoxymethyl)naphtho[1,2-b]furan-4,5-dione.* Red solid, yield: 12%. Mp: 53–54 ◦C. 1H NMR (600 MHz, CDCl3) δ 8.11 (dd, *J* = 7.8, 1.5 Hz, 1H), 7.78 (dd, *J* = 7.7, 1.4 Hz, 1H), 7.70–7.66 (m, 1H), 7.52–7.48 (m, 1H), 6.80 (s, 1H), 4.51 (s, 2H), and 3.46 (s, 3H). 13C NMR (150 MHz, CDCl3) δ 180.43, 174.46, 160.91, 154.77, 135.45, 130.64, 130.44, 128.96, 128.34, 122.59, 122.05, 107.97, 66.02, and 58.32. IR (ν, cm<sup>−</sup>1): 3446.10, 2358.55, 2337.96, 1677.60, 1276.65, 1216.21, 1153.22, and 1082.89. HRMS (ESI) calcd. for [M + Na]<sup>+</sup> C14H10O4Na<sup>+</sup>: 265.0471, found 265.0463.

**TB9**: *2-(ethoxymethyl)naphtho[1,2-b]furan-4,5-dione.* Red solid, yield: 11%. Mp: 60–61 ◦C. 1H NMR (600 MHz, CDCl3) δ 8.10 (dd, *J* = 8.2, 1.3 Hz, 1H), 7.77 (dd, *J* = 7.6, 1.2 Hz, 1H), 7.71–7.65 (m, 1H), 7.52–7.46 (m, 1H), 6.78 (s, 1H), 4.55 (s, 2H), 3.63 (q, *J* = 7.0 Hz, 2H), and 1.29 (t, *J* = 7.0 Hz, 3H). 13C NMR (150 MHz, CDCl3) δ 180.47, 174.45, 160.81, 155.23, 135.45, 130.61, 130.37, 128.90, 128.39, 122.59, 122.08, 107.66, 66.26, 64.25, and 15.10. IR (ν, cm<sup>−</sup>1): 3446.08, 2358.74, 2342.78, 1681.54, 1275.43, 1215.49, 1159.01, and 1083.47. HRMS (ESI) calcd. for [M + Na]<sup>+</sup> C15H12O4Na<sup>+</sup>: 279.0628, found 279.0621.

**TC1**: *2-(diethylaminomethyl)naphtho[1,2-b]furan-4,5-dione.* Red solid, yield: 51%. Mp: 78–79 ◦C. 1H NMR (600 MHz, CDCl3) δ 8.07 (d, *J* = 7.6 Hz, 1H), 7.72 (d, *J* = 7.6 Hz, 1H), 7.65 (t, *J* = 7.0 Hz, 1H), 7.46 (t, *J* = 7.6 Hz, 1H), 6.66 (s, 1H), 3.77 (s, 2H), 2.62 (q, *J* = 7.2 Hz, 4H), and 1.14 (t, *J* = 7.2 Hz, 6H). 13C NMR (150 MHz, CDCl3) δ 180.65, 174.55, 160.34, 156.44, 135.41, 130.55, 130.12, 128.74, 128.58, 122.45, 122.21, 107.11, 48.69, 47.10, and 12.01. IR (ν, cm<sup>−</sup>1): 3445.24, 2953.88, 2358.48, 2339.23, 1700.91, 1676.52, 1216.10, and 1111.39. HRMS (ESI) calcd. for [M + Na]<sup>+</sup> C17H18O3Na<sup>+</sup>: 284.1281, found 284.1271.

**TC2**: *2-(diisopropylaminomethyl)naphtho[1,2-b]furan-4,5-dione*. Red solid, yield: 18%. Mp: 69–70 ◦C. 1H NMR (600 MHz, CDCl3) δ 8.06 (d, *J* = 7.8 Hz, 1H), 7.68 (d, *J* = 6.2 Hz, 1H), 7.64 (t, *J* = 7.5 Hz, 1H), 7.44 (t, *J* = 6.7 Hz, 1H), 6.66 (s, 1H), 3.72 (s, 2H), 3.13 (p, *J* = 6.5 Hz, 2H), and 1.08 (d, *J* = 6.7 Hz, 12H). 13C NMR (150 MHz, CDCl3) δ 180.88, 174.66, 161.17, 159.76, 135.37, 130.50, 129.81, 128.91, 128.65, 122.50, 122.09, 105.12, 49.12, 42.36, and 20.81. IR (ν, cm<sup>−</sup>1): 3445.80, 2966.49, 2358.61, 2337.30, 1676.07, 1558.32, 1215.85, and 1149.51. HRMS (ESI) calcd. for [M + Na]<sup>+</sup> C19H22O3Na<sup>+</sup>: 312.1594, found 312.1583.

**TC3**: *2-((*l*-methionine methyl ester-1-yl)methyl)naphtho[1,2-b]furan-4,5-dione.* Red solid, yield: 12%. Mp: 52–53 ◦C. 1H NMR (600 MHz, CDCl3) δ 8.08 (d, *J* = 7.8 Hz, 1H), 7.72 (d, *J* = 7.6 Hz, 1H), 7.66 (t, *J* = 7.6 Hz, 1H), 7.47 (t, *J* = 7.6 Hz, 1H), 6.66 (s, 1H), 3.95 (d, *J* = 15.1 Hz, 1H), 3.79 (d, *J* = 15.1 Hz, 1H), 3.74 (s, 3H), 3.52 (dd, *J* = 8.4, 5.0 Hz, 1H), 2.64 (t, *J* = 7.2 Hz, 2H), 2.10 (s, 3H), 2.02–1.97 (m, 1H) and 1.89–1.82 (m, 1H). 13C NMR (150 MHz, CDCl3) δ 180.55, 175.18, 174.44, 160.36, 157.00, 135.44, 130.58, 130.19, 128.80, 128.47, 122.35, 122.17, 106.00, 59.14, 52.14, 44.65, 32.68, 30.48, and 15.38. IR (ν, cm−1): 3446.14, 2923.56, 2358.62, 2335.37, 1698.61, 1670.40, 1215.45, and 1147.65. HRMS (ESI) calcd. for [M + Na]<sup>+</sup> C19H19O5NSNa<sup>+</sup>: 396.0876, found 396.0865.

**TC4**: *2-((*l*-alanine methyl ester-1-yl)methyl)naphtho[1,2-b]furan-4,5-dione.* Red solid, yield: 27%. Mp: 83–84 ◦C. 1H NMR (600 MHz, CDCl3) δ 8.07 (d, *J* = 6.9 Hz, 1H), 7.73 (d, *J* = 4.9 Hz, 1H), 7.65 (d, *J* = 8.6 Hz, 1H), 7.46 (t, *J* = 6.6 Hz, 1H), 6.66 (s, 1H), 3.94 (d, *J* = 14.1 Hz, 1H), 3.81 (d, *J* = 14.9 Hz, 1H), 3.74 (s, 3H), 3.46 (q, *J* = 6.9 Hz, 1H), and 1.37 (d, *J* = 6.6 Hz, 3H). 13C NMR (150 MHz, CDCl3) δ 180.52, 175.63, 174.42, 160.36, 156.93, 135.40, 130.56, 130.18, 128.80, 128.46, 122.39, 122.18, 105.99, 55.69, 52.05, 44.26, and 19.13. IR (ν, cm<sup>−</sup>1): 3328.35, 2958.60, 2358.44, 2337.30, 1735.12, 1676.39, 1216.30, and 1139.72. HRMS (ESI) calcd. for [M + Na]<sup>+</sup> C17H15O5NNa<sup>+</sup>: 336.0842, found 336.0831.

**TC5**: *2-((*l*-isoleucinate methyl ester-1-yl)methyl)naphtho[1,2-b]furan-4,5-dione.* Red solid, yield: 39%. Mp: 112–113 ◦C. 1H NMR (600 MHz, CDCl3) δ 8.07 (d, *J* = 7.7 Hz, 1H), 7.71 (d, *J* = 7.7 Hz, 1H), 7.67–7.62

(m, 1H), 7.48–7.43 (m, 1H), 6.65 (s, 1H), 3.92 (d, *J* = 15.1 Hz, 1H), 3.72 (d, *J* = 16.4 Hz, 4H), 3.17 (d, *J* = 5.8 Hz, 1H), 1.57–1.50 (m, 1H), 1.26–1.16 (m, 2H), and 0.93–0.88 (m, 6H). 13C NMR (150 MHz, CDCl3) δ 180.60, 175.16, 174.46, 160.27, 157.37, 135.45, 130.55, 130.15, 128.78, 128.52, 122.32, 122.19, 105.82, 65.31, 51.64, 45.08, 38.47, 25.39, 15.66, and 11.49. IR (ν, cm−1): 3445.72, 2924.70, 2358.43, 2341.16,1732.42, 1682.86, 1209.15, and 1150.10. HRMS (ESI) calcd. for [M + Na]<sup>+</sup> C20H21O5NNa<sup>+</sup>: 378.1312, found 378.1299.

**TC6**: *2-((*l*-valine methyl ester-1-yl)methyl)naphtho[1,2-b]furan-4,5-dione*. Red solid, yield: 41%. Mp: 54–55 ◦C. 1H NMR (600 MHz, CDCl3) δ 8.09 (d, *J* = 6.2 Hz, 1H), 7.73 (d, *J* = 7.6 Hz, 1H), 7.70–7.64 (m, 1H), 7.50–7.45 (m, 1H), 6.67 (s, 1H), 3.95 (d, *J* = 15.3 Hz, 1H), 3.77–3.72 (m, 4H), 3.11 (d, *J* = 5.8 Hz, 1H), 2.02–1.95 (m, 1H), and 0.98 (t, *J* = 6.3 Hz, 7H). 13C NMR (150 MHz, CDCl3) δ 180.62, 175.22, 174.49, 160.28, 157.39, 135.43, 130.59, 130.14, 128.82, 128.56, 122.32, 122.22, 105.83, 66.36, 51.70, 45.16, 31.75, 19.28, and 18.35. IR (ν, cm<sup>−</sup>1): 3425.42, 2923,62, 2358.56, 2337.30, 1698.57, 1670.37, 1187.10, and 1118.00. HRMS (ESI) calcd. for [M + Na]<sup>+</sup> C19H19O5NNa<sup>+</sup>: 364.1155, found 364.1141.

**TC7**: *2-((*l*-glycine methyl ester-1-yl)methyl)naphtho[1,2-b]furan-4,5-dione*. Red solid, yield: 17%. Mp: 55–56 ◦C. 1H NMR (600 MHz, CDCl3) δ 8.06 (d, *J* = 7.6 Hz, 1H), 7.71 (d, *J* = 6.4 Hz, 1H), 7.68–7.62 (m, 1H), 7.49–7.43 (m, 1H), 6.67 (s, 1H), 3.93 (s, 2H), 3.75 (s, 3H), and 3.50 (s, 2H). 13C NMR (150 MHz, CDCl3) δ 180.49, 174.42, 172.45, 160.49, 156.62, 135.44, 130.57, 130.25, 128.79, 128.39, 122.41, 122.14, 106.25, 52.03, 49.50, and 45.38. IR (ν, cm<sup>−</sup>1): 3328.36, 2958.26, 2358.57, 2337.30, 1735.10, 1676.24, 1216.29, and 1180.25. HRMS (ESI) calcd. for [M + Na]<sup>+</sup> C16H13O5NNa<sup>+</sup>: 322.0686, found 322.0680.

**TC8**: *2-((*l*-leucinate methyl ester-1-yl)methyl)naphtho[1,2-b]furan-4,5-dione*. Red solid, yield: 19%. Mp: 54–55 ◦C. 1H NMR (600 MHz, CDCl3) δ 8.09 (d, *J* = 7.6 Hz, 1H), 7.73 (d, *J* = 7.6 Hz, 1H), 7.67 (t, *J* = 7.6 Hz, 1H), 7.47 (t, *J* = 7.6 Hz, 1H), 6.66 (s, 1H), 3.94 (d, *J* = 15.1 Hz, 1H), 3.77 (d, *J* = 15.1 Hz, 1H), 3.73 (s, 3H), 3.38 (t, *J* = 7.2 Hz, 1H), 1.82–1.77 (m, 1H), 1.52 (t, *J* = 7.4 Hz, 2H), 0.95 (d, *J* = 6.7 Hz, 3H), and 0.90 (d, *J* = 6.7 Hz, 3H). 13C NMR (150 MHz, CDCl3) δ 180.59, 176.01, 174.47, 160.32, 157.18, 135.42, 130.59, 130.17, 128.84, 128.51, 122.32, 122.20, 105.94, 59.08, 51.88, 44.63, 42.75, 24.89, 22.78, and 22.09. IR (ν, cm<sup>−</sup>1): 3434.68, 2958.49, 2358.68, 2339.23, 1670.06, 1518.72, 1211.01, and 1107.62. HRMS (ESI) calcd. for [M + Na]<sup>+</sup> C20H21O5NNa<sup>+</sup>: 378.1312, found 378.1302.

**TC9**: *2-((4-boc-piperazin-1-yl)methyl)naphtho[1,2-b]furan-4,5-dione*. Red solid, yield: 26%. Mp: 56–57 ◦C. 1H NMR (600 MHz, CDCl3) δ 8.10 (d, *J* = 7.4 Hz, 1H), 7.76 (d, *J* = 7.6 Hz, 1H), 7.67 (t, *J* = 7.6 Hz, 1H), 7.48 (t, *J* = 7.6 Hz, 1H), 6.70 (s, 1H), 3.68 (s, 2H), 3.49 (s, 4H), 2.52 (s, 4H), and 1.47 (s, 9H). 13C NMR (150 MHz, CDCl3) δ 180.53, 174.48, 160.57, 155.01, 154.68, 135.44, 130.64, 130.29, 128.81, 128.46, 122.53, 122.17, 107.66, 79.88, 54.60, 52.60, and 28.42. IR (ν, cm−1): 3434.73, 2358.58, 2339.23, 1669.96, 1518.77, 1211.18, and 1107.93. HRMS (ESI) calcd. for [M + H]<sup>+</sup> C22H25O5N2 <sup>+</sup>: 397.1758, found 397.1751.

**TC10**: *2-((pyrrolidin-1-yl)methyl)naphtho[1,2-b]furan-4,5-dione*. Red solid, yield: 24%. Mp: 62–63 ◦C. 1H NMR (600 MHz, CDCl3) δ 8.08 (d, *J* = 7.8 Hz, 1H), 7.76 (d, *J* = 7.6 Hz, 1H), 7.65 (t, *J* = 7.6 Hz, 1H), 7.46 (t, *J* = 7.6 Hz, 1H), 6.68 (s, 1H), 3.76 (s, 2H), 2.69–2.63 (m, 4H), and 1.89–1.83 (m, 4H). 13C NMR (150 MHz, CDCl3) δ 180.64, 174.54, 160.39, 156.44, 135.40, 130.56, 130.14, 128.80, 128.56, 122.55, 122.24, 106.71, 54.01, 51.84, and 23.53. IR (ν, cm<sup>−</sup>1): 3388.30, 3110.62, 2358.68, 2337.30, 1660.65, 1510.49, 1222.52, and 1091.51. HRMS (ESI) calcd. for [M + H]<sup>+</sup> C17H16O3N<sup>+</sup>: 282.1125, found 282.1115.

**TC11**: *2-(morpholinomethyl)naphtho[1,2-b]furan-4,5-dione*. Red solid, yield: 38%. Mp: 98–99 ◦C. 1H NMR (600 MHz, CDCl3) δ 8.09 (d, *J* = 7.6 Hz, 1H), 7.76 (d, *J* = 7.6 Hz, 1H), 7.69–7.64 (m, 1H), 7.51–7.45 (m, 1H), 6.70 (s, 1H), 3.78–3.74 (m, 4H), 3.66 (s, 2H), and 2.61–2.55 (m, 4H). 13C NMR (150 MHz, CDCl3) δ 180.50, 174.46, 160.50, 155.01, 135.36, 130.59, 130.23, 128.86, 128.47, 122.46, 122.21, 107.64, 66.79, 54.93, and 53.28. IR (ν, cm−1): 3438.84, 2807.27, 2358.54, 2342.76, 1676.47, 1557.77, 1215.99, 1111.32, and 1006.63. HRMS (ESI) calcd. for [M + Na]<sup>+</sup> C17H15O4NNa<sup>+</sup>: 320.0893, found 320.0886.

**TC12**: *2-(((4-fluorophenyl)amino)methyl)naphtho[1,2-b]furan-4,5-dione*. Dark red solid, yield: 51%. Mp: 174–175 ◦C. 1H NMR (600 MHz, DMSO-*d6*) δ 7.92 (d, *J* = 7.3 Hz, 1H), 7.73 (t, *J* = 7.5 Hz, 1H), 7.65 (d, *J* = 7.6 Hz, 1H), 7.52 (t, *J* = 7.5 Hz, 1H), 6.94 (t, *J* = 8.9 Hz, 2H), 6.74 (s, 1H), 6.73–6.68 (m, 2H), 6.20 (t, *J* = 6.4 Hz, 1H), and 4.37 (d, *J* = 6.0 Hz, 2H). 13C NMR (150 MHz, DMSO-*d6*) δ 179.86, 174.62, 159.03, 157.32, 155.16 (d, *J* = 231.0 Hz), 145.06, 135.47, 130.37, 129.85, 129.69, 128.27, 122.42, 122.06, 115.77 (d,

*J* = 21.0 Hz), 113.88 (d, *J* = 6.0 Hz), and 105.93. IR (ν, cm<sup>−</sup>1): 3378.05, 2923.56, 2358.52, 2335.37, 1662.70, 1514.18, 1215.45, and 1161.45. HRMS (ESI) calcd. for [M + Na]<sup>+</sup> C19H12O3NFNa<sup>+</sup>: 344.0693, found 344.0681.

**TC13**: *2-(((3-fluorophenyl)amino)methyl)naphtho[1,2-b]furan-4,5-dione*. Dark red solid, yield: 45%. Mp: 169–170 ◦C. 1H NMR (600 MHz, DMSO-*d6*) δ 7.92 (d, *J* = 7.6 Hz, 1H), 7.74 (t, *J* = 7.5 Hz, 1H), 7.65 (d, *J* = 7.6 Hz, 1H), 7.53 (t, *J* = 7.6 Hz, 1H), 7.10 (q, *J* = 8.0 Hz, 1H), 6.78 (s, 1H), 6.61 (t, *J* = 6.3 Hz, 1H), 6.54 (d, *J* = 8.2 Hz, 1H), 6.50 (d, *J* = 12.2 Hz, 1H), 6.35 (t, *J* = 8.4 Hz, 1H), and 4.41 (d, *J* = 6.2 Hz, 2H). 13C NMR (150 MHz, DMSO-*d6*) δ 179.83, 174.63, 163.90 (d, *J* = 238.5 Hz), 159.09, 156.86, 150.51 (d, *J* = 10.5 Hz), 135.46, 130.79 (d, *J* = 9.0 Hz), 130.40, 129.85, 129.73, 128.25, 122.42, 122.04, 109.34, 106.10, 102.99 (d, *J* = 21.0 Hz), and 99.24 (d, *J* = 27.0 Hz). IR (ν, cm<sup>−</sup>1): 3390.68, 3105.83, 2360.44, 2337.30, 1665.28, 1618.38, 1220.09, and 1152.73. HRMS (ESI) calcd. for [M + Na]<sup>+</sup> C19H12O3NFNa<sup>+</sup>: 344.0693, found 344.0688.

**TC14**: *2-(((2-fluorophenyl)amino)methyl)naphtho[1,2-b]furan-4,5-dione*. Dark red solid, yield: 46%. Mp: 170–171 ◦C. 1H NMR (600 MHz, CDCl3) δ 8.08 (d, *J* = 7.8 Hz, 1H), 7.69 (d, *J* = 7.4 Hz, 1H), 7.66 (t, *J* = 7.4 Hz, 1H), 7.47 (t, *J* = 7.5 Hz, 1H), 7.05–7.00 (m, 2H), 6.79 (t, *J* = 8.5 Hz, 1H), 6.75–6.72 (m, 1H), 6.71 (s, 1H), and 4.51 (d, *J* = 6.4 Hz, 2H). 13C NMR (150 MHz, CDCl3) δ 180.39, 174.38, 160.36, 155.98, 151.74 (d, *J* = 237.0 Hz), 135.45, 135.22 (d, *J* = 12.0 Hz), 130.63, 130.29, 128.79, 128.36, 124.67 (d, *J* = 4.5 Hz), 122.26 (d, *J* = 15.0 Hz), 118.16 (d, *J* = 6.0 Hz), 114 (d, *J* = 19.5 Hz), 112.52, 112.50, 106.03, and 40.74. IR (ν, cm<sup>−</sup>1): 3425.04, 2923.94, 2358.97, 2854,13, 1670.34, 1513.50, 1186.96, and 1117.57. HRMS (ESI) calcd. for [M + Na]<sup>+</sup> C19H12O3NFNa<sup>+</sup>: 344.0693, found 344.0684.

**TC15**: *2-(((2,4-difluorophenyl)amino)methyl)naphtho[1,2-b]furan-4,5-dione*. Red solid, yield: 33%. Mp: 154–155 ◦C. 1H NMR (600 MHz, CDCl3) δ 8.10 (d, *J* = 7.6 Hz, 1H), 7.71 (d, *J* = 7.6 Hz, 1H), 7.67 (t, *J* = 7.4 Hz, 1H), 7.49 (t, *J* = 7.4 Hz, 1H), 7.14 (t, *J* = 8.1 Hz, 1H), 6.77 (d, *J* = 8.0 Hz, 1H), 6.71 (s, 2H), 6.59 (d, *J* = 8.2 Hz, 1H), and 4.46 (d, *J* = 6.4 Hz, 2H). 13C NMR (150 MHz, CDCl3) δ 180.36, 174.38, 160.42, 155.83 (d, *J* = 9.0 Hz), 154.24 (d, *J* = 10.5 Hz), 151.95 (d, *J* = 12.0 Hz), 150.34 (d, *J* = 12.0 Hz), 135.49, 131.73 (d, *J* = 12.0 Hz), 130.52 (d, *J* = 46.5 Hz), 129.67, 128.55 (d, *J* = 73.5 Hz), 122.24 (d, *J* = 24 Hz), 115.33, 112.73 (dd, *J* = 12.0 Hz), 110.86 (dd, *J* = 25.5 Hz), 106.09, 103.90 (dd, *J* = 49.5 Hz), and 41.22. IR (ν, cm<sup>−</sup>1): 3434.72, 2358.46, 2337.30, 1689.86, 1518.53, 1210.93, 1107.68, and 957.02. HRMS (ESI) calcd. for [M + Na]<sup>+</sup> C19H11O3NF2Na<sup>+</sup>: 362.0599, found 362.0589.

**TC16**: *2-(((2,4,6-trimethylphenyl)amino)methyl)naphtho[1,2-b]furan-4,5-dione.* Red solid, yield: 33%. Mp: 173–174 ◦C. 1H NMR (600 MHz, CDCl3) δ 8.10 (d, *J* = 7.6 Hz, 1H), 7.67 (d, *J* = 3.8 Hz, 2H), 7.50–7.46 (m, 1H), 6.85 (s, 2H), 6.60 (s, 1H), 4.21 (s, 2H), 2.29 (s, 6H), and 2.25 (s, 3H). 13C NMR (150 MHz, CDCl3) δ 180.53, 174.47, 160.18, 157.50, 141.61, 135.47, 132.42, 130.65, 130.23, 130.13, 129.64, 128.84, 128.47, 122.23, 122.18, 105.72, 44.94, 20.62, and 18.23. IR (ν, cm<sup>−</sup>1): 3445.76, 2357.38, 2327.66, 1670.28, 1557.44, 1215.32, 1147.47, and 1025.94. HRMS (ESI) calcd. for [M + Na]<sup>+</sup> C22H19O3NNa<sup>+</sup>: 368.1257, found 368.1250.

**TC17**: *2-(((4-chlorophenyl)amino)methyl)naphtho[1,2-b]furan-4,5-dione.* Dark red solid, yield: 28%. Mp: 205–206 ◦C. 1H NMR (600 MHz, DMSO-*d6*) δ 7.92 (d, *J* = 6.8 Hz, 1H), 7.74 (t, *J* = 7.5 Hz, 1H), 7.65 (d, *J* = 7.6 Hz, 1H), 7.52 (t, *J* = 7.1 Hz, 1H), 7.12 (d, *J* = 8.9 Hz, 2H), 6.75 (s, 1H), 6.72 (d, *J* = 8.9 Hz, 2H), 6.49 (t, *J* = 6.3 Hz, 1H), and 4.40 (d, *J* = 6.2 Hz, 2H). 13C NMR (150 MHz, DMSO-*d*6) δ: 179.82, 174.61, 159.06, 156.95, 147.33, 135.46, 130.39, 129.84, 129.72, 129.09, 128.24, 122.41, 122.05, 120.26, 114.42, and 106.08. IR (ν, cm<sup>−</sup>1): 3388.22, 3110.62, 2358.84, 2342.68, 1660.69, 1510.19, 1222.68 and 1118.65. HRMS (ESI) calcd. for [M + Na]<sup>+</sup> C19H12O3NClNa<sup>+</sup>, 360.0398, found 360.0384.

**TC18**: *2-(((4-methoxyphenyl)amino)methyl)naphtho[1,2-b]furan-4,5-dione*. Dark solid, yield: 64%. Mp: 112–113 ◦C. 1H NMR (600 MHz, CDCl3) δ 8.08 (d, *J* = 6.4 Hz, 1H), 7.70 (d, *J* = 7.6 Hz, 1H), 7.69–7.63 (m, 1H), 7.50–7.44 (m, 1H), 6.82 (d, *J* = 8.9 Hz, 2H), 6.70–6.67 (m, 3H), 4.42 (s, 2H), and 3.77 (s, 3H). 13C NMR (150 MHz, CDCl3) δ 180.49, 174.43, 160.22, 156.83, 152.98, 140.82, 135.43, 130.62, 130.21, 128.77, 128.46, 122.27, 115.00, 114.74, 105.84, 55.77, and 42.13. IR (ν, cm<sup>−</sup>1): 3370.45, 3105.23, 2358.89, 2337.30, 1660.16, 1514.41, 1234.80, and 1040.25. HRMS (ESI) calcd. for [M + Na]<sup>+</sup> C20H15O4NNa<sup>+</sup>: 356.0893, found 356.0883.

#### *3.3. In Vitro Cytotoxicity Assay*

The *human cancer cell lines*, including prostate cancer cells PC3, leukemia cells K562, and melanoma cells WM9, were stored in the biology laboratory of the Key Laboratory of Chemistry for Natural Products of Guizhou Province and Chinese Academy of Sciences (Guiyang, China). All cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin (Sijiqing, Hangzhou, China) and incubated at 37 ◦C under 5% CO2, 95% air, and 95% humidity. Cytotoxicity was evaluated by performing the MTT assay [24]. Briefly, the cells were seeded in 96-well microculture plates at a density from 4 <sup>×</sup> 103 to 8 <sup>×</sup> 103 cells/well. Cells were then exposed to different concentrations of the assayed compounds for 48 h. Then, 20 μL of MTT solution (5 mg/mL) was added to each well and incubated at 37 ◦C for an additional 4 h. The medium was then removed and 200 μL Tris-DMSO solution was added. Plates were lightly shaking up to dissolve the dark blue formazan crystals and the absorbance was measured in an ELISA plate reader at 570 nm.

#### *3.4. Flow Cytometry Assay*

Cell apoptosis was determined by an inverted fluorescence microscope observation and flow cytometry as describe in our previous study [28]. Briefly, the cancer cells treated with compounds were harvested for centrifugation at 1000 rpm for 5 min at room temperature, washed twice with PBS and resuspended with binding buffer, and then PI (Sigma, St. Louis, MO, USA) was added to a final concentration of 20 mg/mL. The cell lines were analyzed by flow cytometry (Becton Dickinson, Franklin Lakes, NJ, USA).

#### *3.5. Statistical Analysis*

The IC50 values were calculated from the semilogarithmic dose-response curves. The data were analyzed using SPSS 18.0 and reported as mean ± SD of the number of experiments indicated. For all measurements, one-way ANOVA followed by Student's t-test was used to assess the statistical significance of the difference between each group. The LSD method was used to assess the statistical significance of the difference between the two groups. A statistically significant difference was considered at the level of *P* < 0.05. The data are presented as the mean ± SEM of three assays.

#### **4. Conclusions**

In this study, 27 novel L-shaped ortho-quinone analogs were synthesized and evaluated for their anti-cancer activities. Compounds **TB1**, **TB3**, **TB4**, **TB6**, **TC1**, **TC3**, **TC5**, **TC9**, **TC11**, **TC12**, **TC14**, **TC15**, **TC16**, and **TC17** possessed broad-spectrum potent cytotoxicity against PC3, K562, and WM9 cells. With more than a 70% inhibitory rate, **TB7** showed better inhibitory activity of K562 cells as compared with other cells. Moreover, we observed that **TC7** inhibited the growth of PC3 cells more efficiently than other cells. Some of the active compounds such as **TB3**, **TC1**, **TC3**, and **TC7** inhibited cell proliferation mainly through inducing apoptosis. The structure-activity relationships evaluation showed that removing methyl at C-3 of the furan ring and introducing diverse side chains at C-2 of the furan ring is an effective strategy for improving the anticancer activity of L-shaped ortho-quinone analogs.

**Author Contributions:** W.-D.P. and H.L. conceived and designed the experiments; S.-Y.L. and X.-Y.Z. performed part of chemical experiment; Z.-K.S., Y.Z., M.-L.W. performed the biology experiment; S.-C.H., J.L., J.-R.S. and C.C. contributed reagents and materials and revised the paper; S.-Y.L. and H.L. wrote the paper.

**Funding:** This work was supported by the Science and Technology Department of Guizhou Province (no. QKHJC (2017)1412, QKHRC (2016)4037, QKHZC (2019)2757, QKHJC (2016)1099, and QKHPTRC (2017) 5737), the National Science Foundation of China (NSFC no. 81660580 and 81702914), and Financial support from Guizhou Provincial Engineering Research Center for Natural Drugs.

**Conflicts of Interest:** The authors declare no conflict of interest.

### **References**


**Sample Availability:** Samples of the compounds are available from the authors.

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## *Article* **Synthesis, Antiproliferative, and Antioxidant Evaluation of 2-Pentylquinazolin-4(3***H***)-one(thione) Derivatives with DFT Study**

### **Amira A. El-Sayed 1, Mahmoud F. Ismail 1, Abd El-Galil E. Amr 2,3,\* and Ahmed M. Naglah 2,4**


Academic Editor: Qiao-Hong Chen

Received: 5 October 2019; Accepted: 20 October 2019; Published: 21 October 2019

**Abstract:** The current study was chiefly designed to examine the antiproliferative and antioxidant activities of some novel quinazolinone(thione) derivatives **6**–**14**. The present work focused on two main points; firstly, comparing between quinazolinone and quinazolinthione derivatives. Whereas, antiproliferative (against two cell lines namely, HepG2 and MCF-7) and antioxidant (by two methods; ABTS and DPPH) activities of the investigated compounds, the best quinazolinthione derivatives were **6** and **14,** which exhibited excellent potencies comparable to quinazolinone derivatives **5** and **9**, respectively. Secondly, we compared the activity of four series of Schiff bases which included the quinazolinone moiety (**11a**–**d**). In addition, the antiproliferative and antioxidant activities of the compounds with various aryl aldehyde hydrazone derivatives (**11a**–**d**) analogs were studied. The compounds exhibited potency that increased with increasing electron donating group in *p*-position (OH > OMe > Cl) due to extended conjugated systems. Noteworthy, most of antiproliferative and antioxidant activities results for the tested compounds are consistent with the DFT calculations.

**Keywords:** quinazolin-4(3*H*)-one; quinazolin-4(3*H*)-thione; Schiff base; antiproliferative activity; antioxidant activity; DFT study

### **1. Introduction**

Cancer is the second leading cause of death globally, and the contribution of cancer disease to the overall mortality rate is increasing. Economically, the total annual cost of cancer in 2010 was estimated at approximately US\$ 1.16 trillion [1]. So that, more rational design, synthesis, and evaluation of new compounds as anticancer, with higher efficiency is considered as urgent mission in the medicinal chemistry field.

Quinazoline and quinazolinone derivatives are considered as tremendous targets for the medicinal chemists, due to the fact that they are the scaffold of different potent anticancer drugs, such as Gefitinib (trade name Iressa®), Erlotinib (trade name Tarceva®) [2–4], Methaqualone [5], Afloqualone (as anticonvulsant activity) [6,7], Chloroqualone (as antitussive), and Diproqualone (as sedative-hypnotic agents) [8] (Figure 1).

**Figure 1.** Some structures of synthetic drugs scaffold quinazoline and quinazolinone derivatives.

In the last decades and until now, various compounds including quinazolinone moiety conspicuously exhibited broad spectrum in numerous pharmacological activities such as anticancer [9–14], anticonvulsants [15], antiproliferative [16], anti-inflammatory [17], antihypertensive [18], antifungal [19], antibacterial, antioxidant [20], antimicrobial [21], anti-allergic [22], antimalarial [23], antileishmanial [24], and treatment of Alzheimer's disease (AD) [25].

Generally, the natural products are considered as one of the most interesting sources of biologically active compounds. Among them, naturally occurring quinazolin-4(3*H*)-one derivatives, which can be isolated from various plants and microorganisms such as Luotonin A (sources; *Peganum nigellastrum*) [26], 2-(heptan-3-yl)quinazolinone (sources; *Bacillus cereus*) [27], Dictyoquinazol A (sources; *Dictyophora indusiata*) [28], and Echinozolinone (sources; *Echinops echinatus*) [29] (Figure 2).

**Figure 2.** Some structures of naturally occurring quinazolin-4(3*H*)-one derivatives.

Quinazolinones have been synthesized by different methodologies [28,30–37], in the present study, the conventional methodology to construct novel qunizolinone compounds has been adopted, followed by the study of the antiproliferative activity, antioxidant activity, and DFT calculations for the synthesized compounds.

#### **2. Results and Discussion**

#### *2.1. Chemistry*

In this interesting work, curing of anthranilic acid **1** with hexanoyl chloride **2** in dry pyridine afforded the corresponding *N*-hexanoyl derivative **3** [38], which was cyclized by heating in distilled acetic anhydride to give 2-pentyl-4*H*-benzo[*d*][1,3]oxazin-4-one **4** [39,40] (Scheme 1).

**Scheme 1.** The strategy for synthesis of compound **4**.

Benzoxazinone derivative **4** was utilized in situ as a precursor to construct new quinazolinone derivatives. For instance, reaction of benzoxazinone derivative **4** with formamide afforded 2-pentylquinazolin-4(3*H*)-one **5** [41] (Scheme 2). The 1H NMR spectrum of **5** exhibited a singlet peak at 12.13 ppm exchangeable with D2O corresponding to NH proton, two doublet and two triplet peaks in the aromatic region at 8.05–7.42 ppm corresponding to four aromatic protons, and four characteristic peaks upfield at 2.56–0.84 ppm for *n*-pentyl protons.

Afterwards, sulfuration of 2-pentylquinazolin-4(3*H*)-one **5** by utilizing of phosphorus pentasulfide in dry toluene afforded 2-pentylquinazoline-4(3*H*)-thione **6** (Scheme 2). The formation of compound **6** was unambiguously elaborated by the presence of intense band at 1236 cm−<sup>1</sup> corresponding to υc=s and the absence of the stretching band of υc=o in the IR spectrum. On the other hand, the incorporation of β-d-glucose pentaacetate with quinazolinone derivative **5** at the nitrogen atom of the later awarded *N*-(β-d-glucopyranosyl-2,3,4,6-tetraacetate)-2-pentyl quinazolin-4(3*H*)-one **7** (Scheme 2), via attacking of the lone pair of nitrogen atom of quinazolinone derivative **5** at the anomeric carbon (C1) of β-d-glucose pentaacetate, followed by ring opening and then ring closure with expulsion of acetate as a leaving group.

The chemical structure of compound **7** was explained by the IR spectrum, whereas it showed a band at 1746 cm−<sup>1</sup> compatible with υC=<sup>O</sup> of the acetate groups and lacked the absorption band for the NH group. Moreover, this structure was also interpreted by the 1H-NMR spectrum which revealed seven signals at 5.92–3.51 ppm and four singlet signals at 1.97–1.91 ppm all of them corresponding to the protons of β-d-glucopyranosyl-2,3,4,6-tetraacetate moiety.

Curing of the benzoxazinone derivative **4** with ethanolamine under reflux for 3 h afforded 3-(2-hydroxyethyl)-2-pentylquinazolin-4(3*H*)-one **8** as the sole product. The IR spectrum of compound **8** showed a broad band at 3395 cm−<sup>1</sup> corresponding to OH functionality. Furthermore, the 1H-NMR spectrum appreciably emerged a triplet peak at 4.95 ppm exchangeable with D2O corresponding to OH proton, triplet, and quartet peaks at 4.11 and 3.65 ppm, respectively, compatible with ethyl protons of 2-hydroxyethyl moiety. As well, the 13C-NMR spectrum exhibited two peaks at 58.8 and 46.1 ppm corresponding to the two carbons of 2-hydroxyethyl moiety.

3-Amino-2-pentylquinazolin-4(3*H*)-one **9** was commenced by refluxing of compound **4** with hydrazine monohydrate in absolute ethanol for 4 h (Scheme 2). The formation of compound **9** was confirmed by spectroscopic and elemental data. In particular, the 1H-NMR spectrum of compound **9** manifested a singlet signal commutable in D2O at 5.70 ppm corresponding to NH2 protons.

**Scheme 2.** Synthetic route to compounds **5**–**9**.

Reaction of 3-amino-2-pentylquinazolin-4(3*H*)-one **9** with various aldehydes **10a**–**d** gave Schiff bases **11a**–**d** as the sole product in each case (Scheme 3). The 1H-NMR spectra of compounds **11a**–**d** exhibited the appearance of a singlet signal in the region between 8.81–8.69 ppm compatible with methine proton of N=CH group.

The thiazolidin-4-one moiety **12** was constructed by the reaction of Schiff base **11a** with methyl thioglycolate in absolute ethanol including a small amount of piperidine as a catalyst for 3 h (Scheme 3). The prospective structure **12** is in keeping with its spectral and elemental analyses.

Additionally, the nucleophilicity of the amino group of compound **9** was also estimated by fusion of it with 4,5,6,7-tetrachloroisobenzofuran-1,3-dione in oil bath for an hour and that afforded phthalimido derivative **13** in an excellent yield (Scheme 3). The foreseeable structure of compound **13** was elucidated by their spectral data and elemental analysis. Obviously, its IR spectrum showed stretching absorption bands at 1788, and 1746 cm−<sup>1</sup> corresponding to the carbonyl groups of phthalimido moiety and at 1707 cm−<sup>1</sup> corresponding to carbonyl group of the quinazolinone moiety. The 1H-NMR spectrum exhibited four peaks for four aromatic protons and another four peaks for n-pentyl protons. Furthermore, its 13C-NMR spectrum emerged variant peaks, all of them fit with the proposed structure.

Eventually, the thione derivative **14** was obtained via sulfuration of compound **9** by utilizing phosphorus pentasulfide as the above pervious method (Scheme 3). The structure of **14** was unequivocally explained via the existence of a peak in the 13C NMR spectrum at 182.1 ppm compatible with the carbon of the thione functional group.

**Scheme 3.** Synthetic route to compounds **11**–**14**.

#### *2.2. Biological Evaluation*

#### 2.2.1. Antiproliferative Screening

Twelve compounds possessing quinazolinone(thione) moieties **5**–**14** along with compound **3** were screened against two cell lines, namely hepatocellular carcinoma (HepG2) and mammary gland (MCF-7) in vitro by utilizing MTT assay [42,43]. The latter assay is a colorimetric test based on the change of the yellow MTT (3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide) to a purple formazan derivative by mitochondrial succinate dehydrogenase in viable cells and the Doxorubicin (DOX) was used as a standard reference.

The results listed in Table 1 and illustrated in Figure 3, demonstrate that compounds **6** and **11d** have a very strong efficacy against HePG2 cell line with IC50 values at 5.20 ± 0.5 and 7.63 ± 0.6 μM, respectively. Meanwhile, compounds **6, 11d,** and **14** have a very strong efficacy against the MCF-7 cell line with IC50 values at 6.88 ± 0.4, 8.60 ± 0.7, and 10.78 ± 0.9 μM, respectively. Compounds **11b, 11c** have a strong efficacy against both cell lines with IC50 values in the range (12.54 ± 1.1–19.68 ± 1.6 μM). For the HePG2 cell line, compounds **5, 9, 11a,** and **14** have a moderate efficacy with IC50 values in the range (23.75 ± 1.9–41.92 ± 2.8 μM). Where, for the MCF-7 cell line, compounds **3, 5, 9,** and **11a** have a moderate efficacy with IC50 in the range (21.98 ± 1.8–47.53 ± 2.9 μM). Ultimately, the remaining compounds in both cases have weak efficacies with IC50 values > 50 μM.


**Table 1.** Cytotoxic efficacy of thirteen compounds against hepatocellular carcinoma (HePG2) and mammary gland (MCF-7) cell lines.

**Figure 3.** Cytotoxic efficacy of thirteen compounds against HePG2 and MCF-7 cell lines.

Through our screening of the antiproliferative efficacy of the synthesized compounds, it was determined that the average of relative viability of cells (%) with different concentrations such as 100, 50, 25, 12.5, 6.25, 3.125, and 1.56 μM against two cell lines (HePG2 and MCF-7) as shown in Figures 4 and 5.

**Figure 4.** Average of relative viability of HePG-2 cell line (%) with different concentrations.

**Figure 5.** Average of relative viability of MCF-7 cell line (%) with different concentrations.

Structure Activity Relationship (SAR)

By comparing the antiproliferative efficacy of the thirteen synthesized compounds in this study to their chemical structures, it was concluded that the following structure activity relationship's (SAR's) is hypothesized:

1. Conversion of quinazolin-4(3*H*)-one derivative **5** to quinazolin-4(3*H*)-thione derivative **6** enhanced the antiproliferative activity against both cell lines from moderate activity to very strong activity.

2. Similarly, conversion of 3-amino-2-pentylquinazolin-4(3*H*)-one **9** to 3-amino-2-pentylquinazoline-4(3*H*)-thione **14** enhanced the antiproliferative activity against both cell lines.

3. Reaction of **9** with various aryl aldehydes afforded hydrazone derivatives **(11a**–**d)** analoges with variable potencies according to the following sequence: 3,5-(OMe)2-4-OH-C6H2 **11d** > 3-OH-4-(OMe)-C6H3 **11c** > 4-(OMe)-C6H4 **11b** > 4-Cl-C6H4 **11a**, whereas, the OH group in *p*-position is more electron donating group than the OMe group and Cl atom (i.e., the delocalization of n-π electrons decreased in the above sequence).

4. Construction of the thiazolidinone ring in compound **12** decreased the antiproliferative activity comparable with the hydrazone derivative **11a**, due to decreasing of the delocalization of n-π electrons after replacement of the C=N group (electron attracting group) by the thiazolidinone ring.

#### 2.2.2. Antioxidant Activity Screening

One of the aims of this work is the screening of all synthesized compounds for antioxidant activity using two different methods, namely ABTS [2,2 -azino-bis(3-ethyl benzothiazoline-6-sulfonic acid)] and DPPH assays. DPPH (2,2-diphenyl-1-picryl-hydrazyl-hydrate) free radical assay based on electron-transfer that produces a violet solution in ethanol. This free radical is stable at ambient temperature and reduced in the presence of an antioxidant molecule, leading to colorless ethanol solution. After investigation of these results as listed in Table 2 and Figure 6, it was realized that, compounds **6** and **11d** have promising activity through using ABTS assay. Meanwhile, in the case of using DPPH assay, compounds **6**, **11d** and **14** have also very high activity. Ascorbic acid was used as a reference through the antioxidant activity screening.

The results depicted in Table 2 and Figure 6 demonstrated that, DPPH assay findings are very approximately related to those of ABTS assay with only one exception, compound **14** has an excellent antioxidant activity against DPPH (IC50 = 26.87 ± 0.23 μM) than that of the ABTS method (IC50 = 71.42 ± 0.52 μM). Noteworthy, all the screened compounds in the case of the DPPH method exhibited IC50

smaller than the corresponding ones of the same compounds in the case of the ABTS method, and it proposed that these compounds are more promising scavengers of the DPPH radical than those of the ABTS radical.

By comparing the antioxidant efficacy of the thirteen synthesized compounds in this study to their chemical structures, it was concluded that the following structure antioxidant activity relationship's (SAR's) is hypothesized:

1. The presence of C=S enhanced antioxidant activity than the presence of C=O, as shown in compounds **6** and **14** comparable with compounds **5** and **9**, respectively.

2. The hydrazone derivatives **(11a**–**d)** analogs have variable potencies according to the following sequence: 3,5-(OMe)2-4-OH-C6H2 **11d** > 3-OH-4-(OMe)-C6H3 **11c** > 4-(OMe)-C6H4 **11b** > 4-Cl-C6H4 **11a**, whereas, OH group in *p*-position is a more electron donating group (has more conjugated system) than OMe group and Cl atom.

3. In compound **12**, replacement of C=N group by the thiazolidinone ring decreased the antioxidant activity comparable with **11a**, because of the lack of the conjugated system.


**Table 2.** Antioxidant activities of all synthesized compounds by using 2,2 -azino-bis(3-ethyl benzothiazoline-6-sulfonic acid (ABTS) and 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH) methods.

**Figure 6.** Antioxidant activities of all synthesized compounds by using ABTS and DPPH methods.

Previous reports of structurally similar compounds (in quinazoline ring) but with different substituents have demonstrated different results in antiproliferative and antioxidant activities from our results [9,33,44].

#### *2.3. Density Functional Theory*

According to the frontier molecular orbital (FMO) theory, the highest occupied molecular orbital (HOMO) acts as an electron-donor and the lowest unoccupied molecular orbital (LUMO) acts as an electron-acceptor [45]. Meanwhile, both play remarkable roles in the electronic studies by using quantum chemical calculations and they are also of significant importance in modern biochemistry and molecular biology [46]. A molecule is considered as a softer and has an excellent chemical reactivity when it has a smaller energy gap. Meanwhile, a molecule is considered to have a higher chemical hardness and assumed to have good stability when it has a larger energy gap [47–51].

The quantum chemical calculations were implemented by the density functional theory (DFT) method by using the Gaussian(R) 09 program at the B3LYP level in conjunction with 6-31G(d,p) basis set and computed parameters are summarized in Table 3.

By computing and using the energy gap (Δ*E* = *E*LUMO − *E*HOMO) and dipole moment values beside another quantum chemical parameters such as ionization energy (*I* = −*E*HOMO), electron affinity (*A* = −*E*LUMO) [52], chemical hardness (η = (I − A)/2), chemical softness (S = 1/η) [53], and binding energy, we can rationally explicate the relation between the chemical structure and the antiproliferative activity (SAR's). Whereas, the energy gap of compound **6** (Δ*E* = 3.98 eV) is smaller than that corresponding of compound **5** (Δ*E* = 4.85 eV) and also compound **6** has a higher chemical softness value (*S* = 0.50 eV<sup>−</sup>1) than that corresponding of compound **5** (*S* = 0.41 eV<sup>−</sup>1). These results are matching with the results of the antiproliferative screening whereas, compound **6** has a higher potency comparable with compound **5** for both cell lines (HepG2 and MCF-7) as shown in Table 1 and Figure 3. Similarly, compound **14** has a smaller energy gap and a higher chemical softness than that corresponding to compound **9** as listed in Table 2. In addition, in vitro compound **14** showed a remarkable higher efficacy comparable with compound **9** as shown in Table 1 and Figure 3. Notably, the dipole moment values of compounds **6** (μ = 3.4641 D) and **14** (μ = 1.852 D) are higher than that of compounds **5** (μ = 3.2867 D) and **9** (μ = 1.764 D), respectively.

On the other hand, compounds **11a**–**d** possess antiproliferative activity in the following order **11d** > **11c** > **11b** > **11a**, meanwhile, the energy gaps of these compounds increase in the following order **11a** (Δ*E* = 2.99 eV) < **11d** (Δ*E* = 3.05 eV) < **11c** (Δ*E* = 3.10 eV) < **11b** (Δ*E* = 3.13 eV). The lower of the antiproliferative activity of compound **11a** may be explained by values of the dipole moment whereas; the dipole moment of compound **11a** is smaller than that of compounds **11b**–**d** as shown in Table 3.


**Table 3.** Quantum chemical parameters of the selected compounds with Density Functional Theory (DFT) at B3LYP/6-31G (d,p) basis set.

The distributions of the HOMO and LUMO orbitals of the selected compounds are computed at the same level of the DFT theory and are provided in Figures 7 and 8. The results manifested that possible reactive sites exist as shown below:

1. The HOMO of compounds **5** and **9** are nearly similar and the distribution of orbitals are mainly situated on the quinazolinone moiety, also, the LUMO of these compounds are situated on the same moiety.

2. The HOMO of compounds **6** and **14** are nearly similar and the distribution of orbitals are mainly situated on C=S, while, the LUMO of these compounds are mainly situated on the quinazolinthione moiety.

3. The HOMO of compounds **11a**–**d** are nearly similar and the distribution of orbitals are mainly situated on the quniazolinone moiety, meanwhile, the LUMO of these compounds are mainly situated on the aryl aldehyde hydrazone system.

**Figure 7.** Schematic representation of HOMO and LUMO coefficient distribution of compounds **5, 6, 9**, and **14**.

**Figure 8.** Schematic representation of highest occupied molecular orbital (HOMO) and loest unoccupied molecular orbital (LUMO) coefficient distribution of compounds **11a**–**d**.

#### **3. Materials and Methods**

#### *3.1. Chemistry*

The melting point is uncorrected and was measured on a Stuart SMP 30 advanced digital electric melting point apparatus (Cole-Parmer, Staffordshire, UK). All reactions were monitored by TLC (Kieselgel 60 F254, Merck, Munchen, Germany) and spots were visualized using UV (254 nm), In the region (400−4000 cm−1), the IR spectrum was measured in the KBr phase by using the Nicolet iS10 FT-IR spectrometer (Shimadzu Corporation, Kyoto, Japan). The 1H-NMR (at 400 MHz) and 13C-NMR (at 100 MHz) spectra were performed at chemical warfare labs, Egypt, with a Varian Gemini spectrometer (Metrohim, California, United States) in DMSO-*d*<sup>6</sup> as a solvent by using tetramethylsilane (TMS) as a reference. Perkin-Elmer 2400 CHN elemental analyzer (Waltham, MA, USA) was used to record CHN elemental analysis at the Faculty of Science, Cairo University, Egypt. The mass spectrum was measured on Shimadzu GC-MS QP1000EX apparatus (Shimadzu Corporation, Kyoto, Japan) at the central analytical lab, Ain Shams University, Cairo, Egypt.

#### 3.1.1. 2-Hexanamidobenzoic Acid **3**

Hexanoyl chloride **2** (1.39 mL, 0.01 mol) was added dropwise to anthranilic acid **1** (1.37 g, 0.01 mol) dissolved in dry pyridine (20 mL) at ambient temperature with stirring. The stirring was continued for an hour, and then the resulting emulsion was acidified with cold 10% HCl solution. The white solid which separated was collected by filtration and then recrystallized from benzene to give **3** [38] as white crystals; m.p.: 92–95 ◦C (Lit. m.p.: 93−95 ◦C) [38], yield: 92%. IR (KBr, cm−1): 3426−2463 (br) (OH), 3206 (NH), 2959, 2934, 2861 (CHaliph.), 1691, 1637 (C=O). 1H-NMR (400 MHz, DMSO-*d*6) δ (ppm): 13.51 (br.s, 1H, OH, exchangeable with D2O), 11.09 (s, 1H, NH, exchangeable with D2O), 8.48 (d, 1H, Ar-H, Ha, *J* = 8.8 Hz), 7.95 (d, 1H, Ar-H, Hd, *J* = 8.0 Hz), 7.55 (t, 1H, Ar-H, Hc, *J* = 7.8 Hz, *J* = 8.0 Hz), 7.11 (t, 1H, Ar-H, Hb, *J* = 7.4 Hz, *J* = 7.8 Hz), 2.35 (t, 2H, COCH2, *J* = 7.2 Hz, *J* = 7.6 Hz), 1.60 (quintet, 2H, COCH2CH2, *J* = 7.2 Hz, *J* = 7.6 Hz), 1.31−1.26 (m, 4H, CH3CH2CH2), 0.85 (t, 3H, CH3, *J* = 6.8 Hz, *J* = 7.2 Hz), MS *m*/*z* (%): 235 (M.<sup>+</sup>; 29.4). Anal. Calcd. for C13H17NO3 (235.28): C, 66.36; H, 7.28; N, 5.95. Found: C, 66.36; H, 7.28; N, 5.95.

#### 3.1.2. 2-Pentyl-4*H*-benzo[*d*][1,3]oxazin-4-one **4**

A suspension of 2-hexanamidobenzoic acid **3** (2.35 g, 0.01 mol) in freshly distilled acetic anhydride (10 mL) was heated in water bath for an hour followed by a concentration of the mixture in vacuo and used in situ [39,40].

#### 3.1.3. 2-Pentylquinazolin-4(3*H*)-one **5**

A solution of benzoxazinone **4** (2.17 g, 0.01 mol) in formamide (15 mL) was refluxed for 7 h. After cooling, the reaction mixture was poured onto ice cold water, the obtained solid was filtered off, dried, and recrystallized from petroleum ether 60–80 ◦C to give **5** [41] as white crystals; m.p.: 142–144 ◦C (Lit. m.p.: 153–154 ◦C) [41], yield: 92%. IR (KBr, cm−1): 3171 (NH), 2958, 2928, 2860 (CHaliph.), 1680 (C=O), 1614 (C=N or C=C). 1H-NMR (400 MHz, DMSO-*d*6) δ (ppm): 12.13 (s, 1H, NH, exchangeable with D2O), 8.05 (d, 1H, Ar-H, Ha, *J* = 8.0 Hz), 7.74 (t, 1H, Ar-H, Hc, *J* = 7.6 Hz, *J* = 7.8 Hz), 7.56 (d, 1H, Ar-H, Hd, *J* = 8 Hz), 7.42 (t, 1H, Ar-H, Hb, *J* = 7.6 Hz), 2.56 (t, 2H, N=CCH2, *J* = 7.6 Hz, *J* = 8.0 Hz), 1.70 (quintet, 2H, N=CCH2CH2, *J* = 7.6 Hz, *J* = 7.2 Hz), 1.30–1.26 (m, 4H, CH3CH2CH2), 0.84 (t, 3H, CH3, *J* = 6.8 Hz, *J* = 7.2 Hz). 13C-NMR (100 MHz, DMSO-*d*6) δ (ppm): 162.2, 157.9, 149.4, 134.7, 127.2, 126.3, 126.1, 121.2, 34.9, 31.1, 26.9, 22.2, 14.2. MS *m*/*z* (%): 216 (M.<sup>+</sup>; 26.3). Anal. Calcd. for C13H16N2O (216.28): C, 72.19; H, 7.46; N, 12.95. Found: C, 72.26; H, 7.49; N, 12.86.

#### 3.1.4. 2-Pentylquinazoline-4(3*H*)-thione **6**

To a solution of quinazolinone **5** (2.16 g, 0.01 mol) in dry toluene (30 mL), P2S5 (2.22 g, 0.01 mol) was added. The reaction mixture was refluxed for 1 h, and then filtered off. The obtained filtrate was evaporated under reduced pressure, the formed solid was collected by filtration, dried, and recrystallized from ethanol to give **6** as light brown crystals; m.p.: 101–103 ◦C, yield: 76%. IR (KBr, cm−1): 3184, 3141 (NH), 2967, 2935, 2852 (CHaliph.), 1618 (C=N), 1604 (C=C), 1236 (C=S). 1H-NMR (400 MHz, DMSO-*d*6) δ (ppm): 13.69 (s, 1H, NH, exchangeable with D2O), 8.52 (d, 1H, Ar-H, Hd, *J* = 8.4 Hz), 7.83 (t, 1H, Ar-H, Hc, *J* = 7.6 Hz), 7.63 (d, 1H, Ar-H, Ha, *J* = 8.4 Hz), 7.52 (t, 1H, Ar-H, Hb, *J* = 7.4 Hz, *J* = 7.8 Hz), 2.70 (t, 2H, N=CCH2, *J* = 7.6 Hz, *J* = 8.0 Hz), 1.72 (quintet, 2H, N=CCH2CH2, *J* = 7.6 Hz, *J* = 7.2 Hz), 1.31−1.26 (m, 4H, CH3CH2CH2), 0.85 (t, 3H, CH3, *J* = 6.8 Hz). MS *m*/*z* (%): 232 (M.<sup>+</sup>; 16.7). Anal. Calcd. for C13H16N2S (232.35): C, 67.20; H, 6.94; N, 12.06; S, 13.80. Found: C, 67.31; H, 7.03; N, 12.01; S, 13.85.

### 3.1.5. *N*-(β-d-Glucopyranosyl-2,3,4,6-tetraacetate)-2-pentylquinazolin-4(3H)-one **7**

Quinazolinone **5** (2.16 g, 0.01 mol) was refluxed with β-d-glucose pentaacetate (3.90 g, 0.01 mol) in absolute ethanol (50 mL) for 3 h. the solid obtained after slow evaporation of the resulting solution was collected and recrystallized from ethanol to give **7** as white crystals; m.p.: 135–137 ◦C, yield: 62%. IR (KBr, cm<sup>−</sup>1): 2955, 2924, 2854 (CHaliph.), 1746 (C=Oester), 1678 (C=Oquinazolinone), 1613 (C=N). 1H-NMR (400 MHz, DMSO-*d*6) δ (ppm): 8.05 (d, 1H, Ar-H, Ha, *J* = 8.0 Hz), 7.74 (t, 1H, Ar-H, Hc, *J* = 8.4 Hz, *J* = 6.8 Hz), 7.56 (d, 1H, Ar-H, Hd, *J* = 8.4 Hz), 7.43 (t, 1H, Ar-H, Hb, *J* = 8.0 Hz, *J* = 7.2 Hz), 5.92 (d, 1H, C1-H, *J* = 8.4 Hz), 5.39 (t, 1H, C2-H, *J* = 9.6 Hz), 4.93 (t, 1H, C3-H, *J* = 9.6 Hz), 4.90 (t, 1H, C4-H, *J* = 8.4 Hz, *J* = 10.0 Hz), 4.14, 412 (d,d, 1H, C6-H, *J* = 10.4 Hz, *J* = 5.6 Hz), 3.97 (d, 1H, C6-H, *J* = 10.4 Hz), 3.51 (m, 1H, C5-H), 2.56 (t, 2H, N=CCH2, *J* = 8.0 Hz, *J* = 7.6 Hz), 1.978 (s, 3H, CH3), 1.973 (s, 3H, CH3), 1.961 (s, 3H, CH3), 1.917 (s, 3H, CH3), 1.69 (quintet, 2H, N=CCH2CH2, *J* = 7.6 Hz, *J* = 6.8 Hz), 1.31−1.26 (m, 4H, CH3CH2CH2), 0.84 (t, 3H, CH3, *J* = 6.8 Hz, *J* = 7.2 Hz). MS *m*/*z* (%): 546 (M+; 32.4). Anal. Calcd. for C27H34N2O10 (546.57): C, 59.33; H, 6.27; N, 5.13. Found: C, 59.18; H, 6.21; N, 5.08.

#### 3.1.6. 3-(2-Hydroxyethyl)-2-pentylquinazolin-4(3*H*)-one **8**

A solution of benzoxazinone **4** (2.17 g, 0.01 mol) in ethanolamine (15 mL) was heated under reflux for 3 h. The reaction mixture was poured onto ice cold water, the obtained solid was filtered off, dried, and then recrystallized from ethanol to give **8** as white crystals; m.p.: 84–85 ◦C, yield: 47%. IR (KBr, cm<sup>−</sup>1): 3395 (OH), 2953, 2931, 2872 (CHaliph.), 1648 (C=O), 1611 (C=N). 1H-NMR (400 MHz, DMSO-*d*6) δ (ppm): 8.07 (d, 1H, Ar-H, Ha, *J* = 8.0 Hz), 7.75 (t, 1H, Ar-H, Hc, *J* = 7.8 Hz, *J* = 7.6 Hz), 7.57 (d, 1H, Ar-H, Hd, *J* = 8.0 Hz), 7.44 (t, 1H, Ar-H, Hb, *J* = 7.6 Hz, *J* = 7.2 Hz), 4.95 (t, 1H, OH, exchangeable with D2O, *J* = 5.6 Hz), 4.11 (t, 2H, CH2CH2OH, *J* = 5.6 Hz, *J* = 6.0 Hz), 3.65 (q, 2H, CH2OH, *J* = 6.0 Hz, *J* = 5.6 Hz), 2.93 (t, 2H, N=CCH2, *J* = 7.6 Hz, *J* = 8.0 Hz), 1.75 (quintet, 2H, N=CCH2CH2, *J* = 7.6 Hz, *J* = 7.6 Hz), 1.40−1.32 (m, 4H, CH3CH2CH2), 0.88 (t, 3H, CH3, *J* = 7.2 Hz). 13C-NMR (100 MHz, DMSO-*d*6) δ (ppm): 161.7, 158.4, 147.4, 134.6, 127.1, 126.5, 126.4, 120.4, 58.8, 46.1, 34.6, 31.3, 26.3, 22.4, 14.3. MS *m*/*z* (%): 260 (M<sup>+</sup>; 41.2). Anal. Calcd. for C15H20N2O2 (260.34): C, 69.20; H, 7.74; N, 10.76. Found: C, 69.17; H, 7.68; N, 10.81.

#### 3.1.7. 3-Amino-2-pentylquinazolin-4(3*H*)-one **9**

A mixture of benzoxazinone **4** (2.17 g, 0.01 mol) and hydrazine hydrate (1.5 mL) in absolute ethanol (20 mL) was refluxed for 3 h. The mixture was poured onto ice cold water, the formed solid was filtered off, and recrystallized from ethanol to give **9** as buff crystals; m.p.: 58–60 ◦C, yield: 43%. IR (KBr, cm<sup>−</sup>1): 3306, 3263 (NH2), 2954, 2931, 2910, 2856 (CHaliph.), 1673 (C=O), 1630 (C=N). 1H NMR (400 MHz, DMSO-*d*6) δ (ppm): 8.08 (d, 1H, Ar-H, Ha, *J* = 7.8 Hz), 7.75 (t, 1H, Ar-H, Hc, *J* = 7.4 Hz, *J* = 7.8 Hz), 7.59 (d, 1H, Ar-H, Hd, *J* = 7.6 Hz), 7.45 (t, 1H, Ar-H, Hb, *J* = 7.2 Hz, *J* = 7.6 Hz), 5.70 (s, 2H, NH2, exchangeable with D2O), 2.90 (t, 2H, N=CCH2, *J* = 7.2 Hz, *J* = 8.0 Hz), 1.74 (quintet, 2H, N=CCH2CH2, *J* = 7.6 Hz, *J* = 7.2 Hz), 1.37−1.30 (m, 4H, CH3CH2CH2), 0.87 (t, 3H, CH3, *J* = 6.4 Hz, *J* =7.2 Hz). 13C-NMR (400 MHz, DMSO-d6) δ (ppm): 160.9, 158.8, 147.0, 134.3, 127.2, 126.39, 126.31, 120.2, 34.0, 31.4, 26.0, 22.3, 14.3. MS *m*/*z* (%): 231 (M<sup>+</sup>; 41.1). Anal. Calcd. for C13H17N3O (231.30): C, 67.51; H, 7.41; N, 18.17. Found: C, 67.39; H, 7.34; N, 18.24.

#### 3.1.8. General Procedure for Synthesis of **11a**–**d**

A mixture of compound **9** (2.31 g, 0.01 mol) and the appropriate aldehydes **10a**–**d** (0.01 mol) in absolute ethanol (30 mL) was refluxed for 4−6 h. The reaction mixture was evaporated under reduced pressure; the obtained residue was collected and recrystallized from the proper solvent to give the corresponding benzylidene derivatives **11a**–**d**, respectively.

#### 3-((4-Chlorobenzylidene)amino)-2-pentylquinazolin-4(3*H*)-one **11a**

Yellow crystals; m.p.: 176–178 ◦C (ethanol), yield: 72%. IR (KBr, cm<sup>−</sup>1): 2943, 2866 (CHaliph.), 1667 (C=O), 1624 (C=N). 1H-NMR (400 MHz, DMSO-*d*6) δ (ppm): 8.69 (s, 1H, N=CH), 7.95 (d, 1H, Ar-H, Ha, *J* = 7.8 Hz), 7.88 (d, 2H, Ar-H, HE + HF, *J* = 8.8 Hz), 7.66 (t, 1H, Ar-H, Hc, *J* = 8.4 Hz), 7.57 (d, 3H, Ar-H, Hd + HX + HZ), 7.46 (t, 1H, Ar-H, Hb, *J* = 8.4 Hz), 2.34 (t, 2H, N=CCH2, *J* = 7.6 Hz), 1.60 (quintet, 2H, N=CCH2CH2, *J* = 7.2 Hz), 1.31−1.26 (m, 4H, CH3CH2CH2), 0.85 (t, 3H, CH3, *J* = 6.8 Hz). MS *m*/*z* (%): 353 (M<sup>+</sup>; 4.0). Anal. Calcd. for C20H20ClN3O (353.85): C, 67.89; H, 5.70; Cl, 10.02; N, 11.88. Found: C, 67.78; H, 5.62; Cl, 9.89; N, 11.79.

#### 3-((4-Methoxybenzylidene)amino)-2-pentylquinazolin-4(3*H*)-one **11b**

White crystals; m.p.: 83–84 ◦C (petroleum ether 60–80 ◦C), yield: 64%. IR (KBr, cm<sup>−</sup>1): 2946, 2912, 2882, 2843 (CHaliph.), 1669 (C=O), 1606 (C=N or C=C). 1H-NMR (400 MHz, DMSO-*d*6) δ (ppm): 8.81 (s, 1H, N=CH), 8.12 (d, 1H, Ar-H, Ha, *J* = 7.8 Hz), 7.89 (d, 2H, Ar-H, HE + HF, *J* = 8.8 Hz), 7.80 (t, 1H, Ar-H, Hc, *J* = 8.2, Hz, *J* = 7.4 Hz), 7.65 (d, 1H, Ar-H, Hd, *J* = 8.0 Hz), 7.50 (t, 1H, Ar-H, Hb, *J* = 7.6, Hz, *J* = 7.4 Hz), 7.12 (d, 2H, Ar-H, HX + HZ, *J* = 8.8 Hz), 3.85 (s, 3H, OCH3), 2.80 (t, 2H, N=CCH2, *J* = 7.6 Hz, *J* = 8.0 Hz), 1.71 (quintet, 2H, N=CCH2CH2, *J* = 7.6 Hz, *J* = 7.2 Hz), 1.33−1.26 (m, 4H, CH3CH2CH2), 0.81 (t, 3H, CH3, *J* = 7.2 Hz). MS *m*/*z* 7(%): 349 (M<sup>+</sup>; 11.). Anal. Calcd. for C21H23N3O2 (349.43): C, 72.18; H, 6.63; N, 12.03. Found: C, 72.29; H, 6.69; N, 11.88.

#### 3-((3-Hydroxy-4-methoxybenzylidene)amino)-2-pentylquinazolin-4(3*H*)-one **11c**

White crystals; m.p.: 150–152 ◦C (ethanol), yield: 57%. IR (KBr, cm−1): 3277 (OH), 2956, 2927, 2892, 2863, 2845 (CHaliph.), 1678 (C=O), 1603 (C=N or C=C). 1H-NMR (400 MHz, DMSO-*d*6) δ (ppm): 9.49 (s, 1H, OH, exchangeable with D2O), 8.70 (s, 1H, N=CH), 8.12 (d, 1H, Ar-H, Ha, *J* = 8.0 Hz), 7.79 (t, 1H, Ar-H, Hc, *J* = 8.0 Hz, *J* =8.4 Hz), 7.65 (d, 1H, Ar-H, Hd, *J* = 7.6 Hz), 7.49 (t, 1H, Ar-H, Hb, *J* = 8.0 Hz, *J* = 7.2 Hz), 7.42 (d, 1H, HF, *Jm* = 2 Hz), 7.30, 7.28 (d,d, 1H, Ar-H, HE, *Jo* = 8.4 Hz, *Jm* = 2 Hz), 7.07 (d, 1H, Ar-H, HX, *J* = 8.4 Hz), 3.85 (s, 3H, OCH3), 2.78 (t, 2H, N=CCH2, *J* = 7.6 Hz), 1.71 (quintet, 2H, N=CCH2CH2, *J* = 7.6 Hz, *J* = 7.2 Hz), 1.31−1.28 (m, 4H, CH3CH2CH2), 0.81 (t, 3H, CH3, *J* = 6.8 Hz, *J* = 7.2 Hz). MS *m*/*z* (%): 365 (M<sup>+</sup>; 23.4). Anal. Calcd. for C21H23N3O3 (365.43): C, 69.02; H, 6.34; N, 11.50. Found: C, 68.88; H, 6.28; N, 11.62.

#### 3-((4-Hydroxy-3,5-dimethoxybenzylidene)amino)-2-pentylquinazolin-4(3*H*)-one **11d**

White crystals; m.p.: 148–150 ◦C (benzene), yield: 61%. IR (KBr, cm−1): 3408 (OH), 2952, 2911, 2844 (CHaliph.), 1668 (C=O), 1591 (C=N or C=C). 1H-NMR (400 MHz, DMSO-d6) δ (ppm): 9.36 (br.s, 1H, OH, exchangeable with D2O), 8.72 (s, 1H, N=CH), 8.12 (d, 1H, Ar-H, Ha, *J* = 8.2 Hz), 7.79 (t, 1H, Ar-H, Hc, *J* = 8.0 Hz, *J* = 7.4 Hz), 7.65 (d, 1H, Ar-H, Hd, *J* = 7.6 Hz), 7.50 (t, 1H, Ar-H, Hb, *J* = 8.0 Hz, *J* = 7.2 Hz), 7.23 (s, 2H, HE + HF), 3.82 (s, 6H, 2OCH3), 2.81 (t, 2H, N=CCH2, *J* = 7.6 Hz, *J* = 8.0 Hz), 1.72 (quintet, 2H, N=CCH2CH2, *J* = 7.6 Hz, *J* = 7.2 Hz), 1.36−1.26 (m, 4H, CH3CH2CH2), 0.81 (t, 3H, CH3, *J* = 6.8 Hz, *J* =7.2 Hz). 13C-NMR (100 MHz, DMSO-*d*6) δ (ppm): 169.8, 158.0, 156.5, 148.6 (2), 146.7, 140.8, 134.6, 127.4, 127.0, 126.7, 122.8, 121.3, 106.8 (2), 56.5 (2), 34.5, 31.3, 26.0, 22.2, 14.2. MS *m*/*z* (%): 395 (M<sup>+</sup>; 62.1), Anal. Calcd. for C22H25N3O4 (395.46): C, 66.82; H, 6.37; N, 10.63. Found: C, 66.95; H, 6.41; N, 10.58.

#### 3.1.9. 2-(4-Chlorophenyl)-3-(4-oxo-2-pentylquinazolin-3(4*H*)-yl)thiazolidin-4-one **12**

A mixture of compound **11a** (3.53 g, 0.01 mol) and methyl thioglycolate (0.89 mL, 0.01 mol) in absolute ethanol (30 mL) containing piperidine (0.5 mL) was refluxed for 3 h. The obtained solid after evaporation of the solvent was collected and recrystallized from petroleum ether 60–80 ◦C to give **12** as pale yellow crystals; m.p.: 78–80 ◦C, yield: 47%. IR (KBr, cm<sup>−</sup>1): 2951, 2925, 2868 (CHaliph.), 1736 (C=Othiazolidinone), 1671 (C=Oquinazolinone), 1608 (C=N or C=C). 1H-NMR (100 MHz, DMSO-*d*6) δ (ppm): 8.13 (d, 1H, Ar-H, Ha, *J* = 8.2 Hz), 7.96 (d, 2H, Ar-H, HE + HF, *J* = 8.0 Hz), 7.80 (t, 1H, Ar-H, Hc, *J* = 7.6 Hz), 7.65 (d, 1H, Ar-H, Hd, *J* = 8.0 Hz), 7.64 (d, 2H, Ar-H, HX + HZ, *J* = 8.4 Hz), 7.51 (t, 1H, Ar-H, Hb, *J* = 7.6 Hz, *J* = 7.8 Hz), 5.70 (s, 1H, SCH), 3.75, 3.67 (d,d, 2H, CH2(thiazolidinone), *J* = 23.6 Hz, *J* = 23.2 Hz), 2.81 (t, 2H, N=CCH2, *J* = 8.0 Hz, *J* = 7.6 Hz), 1.71 (quintet, 2H, N=CCH2CH2, *J* = 7.6 Hz, *<sup>J</sup>* <sup>=</sup> 8.0 Hz), 1.35−1.17 (m, 4H, CH3CH2CH2), 0.80 (t, 3H, CH3, *<sup>J</sup>* <sup>=</sup> 7.2 Hz). MS *<sup>m</sup>*/*<sup>z</sup>* (%): 427 (M+; 11.8). Anal. Calcd. for C22H22ClN3O2S (427.95): C, 61.75; H, 5.18; Cl, 8.28; N, 9.82; S, 7.49. Found: C, 61.66; H, 5.12; Cl, 8.31; N, 9.75; S, 7.55.

#### 3.1.10. 4,5,6,7-Tetrachloro-2-(4-oxo-2-pentylquinazolin-3(4*H*)-yl)isoindoline-1,3-dione **13**

Compound **9** (2.31 g, 0.01 mol) was fused with 4,5,6,7-tetrachloroisobenzofuran-1,3-dione (2.85 g, 0.01 mol) in oil bath for an hour. The resulting solid was recrystallized from ethanol to give **13** as orange crystals; m.p.: 178–180 ◦C, yield: 86%. IR (KBr, cm−1): 2943, 2856 (CHaliph.), 1788, 1746 (C=Oimide), 1705 (C=Oquinazolinone), 1606 (C=N or C=C). 1H-NMR (400 MHz, DMSO-*d*6) δ (ppm): 8.08 (d, 1H, Ar-H, Ha, *J* = 8 Hz), 7.94 (t, 1H, Ar-H, Hc, *J* = 8.0 Hz, *J* = 7.2 Hz), 7.76 (d, 1H, Ar-H, Hd, *J* = 8.0 Hz), 7.60 (t, 1H, Ar-H, Hb, *J* = 7.6 Hz), 2.73 (t, 2H, N=CCH2, *J* = 6.8 Hz, *J* = 8.0 Hz), 1.70−1.60 (m, 2H, N=CCH2CH2), 1.29−1.27 (m, 4H, CH3CH2CH2), 0.81 (t, 3H, CH3, *J* = 6.8 Hz, *J* =7.2 Hz). 13C-NMR (100 MHz, DMSO-*d*6) δ (ppm): 161.5, 161.3, 157.9, 157.7, 147.4, 146.7, 140.0, 138.8, 136.6 (2), 128.2 (2), 128.0, 127.1, 126.8, 119.8, 32.6, 30.9, 26.0, 22.2, 14.2. MS *m*/*z* (%): 231 (M+; 41.1). Anal. Calcd. for C21H15Cl4N3O3 (499.17): C, 50.53; H, 3.03; Cl, 28.41; N, 8.42. Found: C, 50.61; H, 3.09; Cl, 28.37; N, 8.53.

#### 3.1.11. 3-Amino-2-pentylquinazoline-4(3*H*)-thione **14**

A mixture of compound **9** (2.31 g, 0.01 mol) and P2S5 (2.22 g, 0.01 mol) in dry toluene (15 mL) was heated under reflux for 4 h. The mixture was filtered off, the filtrate was evaporated under reduced pressure, the obtained solid was collected, dried, and recrystallized from ethanol to give **14** as yellow crystals; m.p.: 57–59 ◦C, yield: 53%. IR (KBr, cm−1): 3240, 3200 (NH2), 2925, 2855 (CHaliph.), 1591 (C=N or C=C), 1238 (C=S). 1H-NMR (400 MHz, DMSO-*d*6) δ (ppm): 8.51 (d, 1H, Ar-H, Ha, *J* = 8.2 Hz), 7.82 (t, 1H, Ar-H, Hc, *J* = 7.6 Hz, *J* = 7.8 Hz), 7.69 (d, 1H, Ar-H, Hd, *J* = 7.6 Hz), 7.57 (t, 1H, Ar-H, Hb, *J* = 7.6 Hz), 7.04 (s, 2H, NH2, exchangeable with D2O), 3.06 (t, 2H, N=CCH2, *J* = 7.2 Hz, *J* = 8.0 Hz), 1.81 (quintet, 2H, N=CCH2CH2, *J* = 7.6 Hz, *J* = 7.2 Hz), 1.42−1.32 (m, 4H, CH3CH2CH2), 0.88 (t, 3H, CH3, *J* = 6.8 Hz, *J* =7.2 Hz). 13C NMR (100 MHz, DMSO-*d*6) δ (ppm): 182.1, 155.5, 142.0, 134.5, 130.6, 128.1 (2), 127.4, 34.4, 31.3, 25.5, 22.4, 14.3. MS *m*/*z* (%): 247 (M<sup>+</sup>; 74.3). Anal. Calcd. for C13H17N3S (247.36): C, 63.12; H, 6.93; N, 16.99; S, 12.96. Found: C, 63.19; H, 6.96; N, 17.11; S, 12.82.

#### *3.2. Cytotoxicity and Antiproliferative Evaluation*

#### MTT Assay

The implement of MTT methodology for the antiproliferative screening of quinazolinone derivatives **5**–**14** along with compound **3** against two cell lines, namely, hepatocellular carcinoma (HepG2) and mammary gland (MCF-7) were obtained from ATCC through the Holding company for biological products and vaccines (VACSERA), Cairo, Egypt. The reference anticancer drug used was Doxorubicin. The MTT assay was carried out at the pharmacology department, Faculty of pharmacy, Mansoura University, Egypt according to the reported literatures [42,43,54]. The cells were cultured in a RPMI-1640 medium with 10% fetal bovine serum, followed by the addition of antibiotics (100 units/mL penicillin and 100 μg/mL streptomycin) at 37 ◦C in a 5% CO2 incubator. The cells were seeded in a 96-well plate at a density of (1.0 <sup>×</sup> 10<sup>4</sup> cells/well) at 37 ◦C for 48 h under 5% CO2. Treatment of cells with different concentrations of compounds such as 100, 50, 25, 12.5, 6.25, 3.125, and 1.56 μM was carried out and placed in the incubator for 24 h. Then, 20 μL of MTT solution at 5 mg/mL was added and incubated for 4 h. DMSO (100 μL) was added into each well to dissolve the purple formazan formed. At 570 nm absorbance the colorimetric assay was measured and recorded by using a plate reader (BioTek EL ×800 Microplate Reader, BioTek Instruments, Inc, Winooski, VT, USA).

Calculation of the relative cell viability (%) = (A of treated samples /A of untreated sample) × 100.

#### *3.3. Antioxidant Assay*

#### 3.3.1. Antioxidant Activity Screening Assay

#### ABTS Method

By the bleaching of ABTS derived radical cations, the detections of antioxidant activities were estimated. The radical cation was prepared by the reaction of ABTS [2,2 -azino-bis(3-ethyl benzothiazoline-6-sulfonic acid)] (60 μL) with MnO2 (3 mL, 25 mg/mL) in a phosphate buffer solution (10 μM, pH 7, 5 mL). The solution was shaken for 3 min, centrifuged, filtered, and recorded at λmax 734 nm the absorbance *A*(control) of the resulting ABTS radical solution (green-blue). Upon the addition of the tested sample solution (20 μl) with different concentrations of compounds such as 200, 100, 50, 25, and 12.5 μM in spectroscopic grade MeOH/buffer (1:1 *v*/*v*) to the ABTS solution, the absorbance *A*(test) was measured. The decreasing in the absorbance is expressed as % inhibition which was calculated according to the following equation [55]:

$$^{0\text{\textquotedblleft}I\text{\textquotedblright}I\text{\textquotedblright}} = [A\_{\text{(control)}} - A\_{\text{(test)}} / A\_{\text{(control)}}] \times 100\tag{1}$$

where; the reference and standard antioxidant compound in this test is the ascorbic acid solution (20 μL, 2 mM) and the blank sample was performed by the solvent without ABTS.

#### DPPH Method

According to the methodology described by Brand-Williams et al. [56], the measurement of the DPPH radical scavenging activity was implemented. The samples with different concentrations of compounds such as 200, 100, 50, 25, and 12.5 μM were allowed to react with the stable DPPH radical in ethanol solution. Whereas, the reaction mixture consisted of sample (0.5 mL), absolute ethanol (3 mL), and DPPH radical solution (0.3 mL) 0.5 mM in ethanol. DPPH is reduced when it reacts with an antioxidant compound, which can donate hydrogen. The changes in color (from deep violet to light yellow) were recorded [absorbance (*Abs*)] at λmax 517 nm after 100 min of reaction using a UV-Vis spectrophotometer (Schimadzu Co., Tokyo, Japan). The blank solution was prepared by mixing ethanol (3.3 mL) and the sample (0.5 mL). Meanwhile, the mixture of ethanol (3.5 mL) and DPPH radical solution (0.3 mL) serve as a positive control.

The scavenging activity percentage (*AA* %) was determined according to Mensor et al. [57]:

$$AA\ \%=100-\left[\left(Abs\_{\text{(sample)}}-Abs\_{\text{(blank)}}\right)A\text{bs}\_{\text{(control)}}\right]\times100\right] \tag{2}$$

#### *3.4. Computational Procedures*

All theoretical calculations and results of the studied compounds were implemented by utilizing Gaussian(R) 09 D.01 [58] (Semichem Inc., Shawnee Mission, KS, USA) by applying the DFT operation with the hybrid functional B3LYP level [59,60] in conjunction with the 6−31G(d,p) basis set. The visualization of these results was achieved using GaussView 6.0.16 software (Semichem Inc., Shawnee Mission, KS, USA).

#### **4. Conclusions**

In conclusion, this work focused on the study of the antiproliferative and antioxidant activities in vitro in addition to the theoretical calculation of the DFT theory of some novel quinazolinone(thione) derivatives **6**–**14**. Two main points were the principal targets; firstly, by comparing the activities of quinazolinone and quinazolinthione derivatives. Secondly, comparing the activities of four series of Schiff bases, that have quinazolinone moiety. The results of this study imply that the quinazolinthione derivatives **6** and **14** have promising potent antiproliferative activity comparable with quinazolinone derivatives **5** and **9**, respectively. According to the DFT study, compounds **6** and **14** have a smaller

energy gap and a higher chemical softness than that of compounds **5** and **9**, respectively. Additionally, screening of various aryl aldehyde hydrazone derivatives **(11a**–**d)** analogs exhibited that the potency increased with increasing the electron donating group in *p*-position due to increasing of the conjugated system, and that was supported by the DFT study.

On the other hand, compounds **6** and **11d** showed promising antioxidant activity using ABTS assay. While in the DPPH assay, compounds **6**, **11d,** and **14** have showed potent activities comparable to the ascorbic acid which was used as a reference drug. Noteworthy, the results of both antiproliferative and antioxidant activities for each compound individually are nearly the same.

**Author Contributions:** The listed authors contributed to this work as described in the following: A.A.E.-S., M.F.I., and A.E.-G.E.A. designed the research idea, implemented the synthesis and characterization of novel compounds, and contributed to the data interpretation. A.E.-G.E.A. and A.M.N. contributed to discuss the results, writing the original draft manuscript, and revisions. All authors read and approved the final manuscript.

**Funding:** The authors are grateful to the Deanship of Scientific Research, King Saud University for funding this work through research group project "RGP-172".

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


**Sample Availability:** Samples of the compounds are available from the authors.

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