human foreskin fibroblasts (HFF); human gastric cancer cells-803 (MGC-803); hepatocellular carcinoma bel-7404 (BEL-7404); large cell lung cancer cells (NCI-H460); and bladder cancer cells (T24); LO2 human normal liver cells (LO2).

#### *2.3. Antitumor Mechanism Studies*

2.3.1. Apoptosis and Cell-Cycle Analysis

Apoptosis and the cell-cycle play a central role in cancer, since their induction in cancer cells is critical to a successful therapy [17,18]. Therefore, the most active compounds, including **5d** and **6h** or **4h** and **6h** were selected to study their effect on apoptosis and cell cycle profiles in the MGC80-3 or T24 cell lines, respectively.

The apoptosis ratios of MGC80-3 or T24 cell lines induced by the selected compounds at the concentration of IC<sup>50</sup> and 0.5 IC<sup>50</sup> were quantitatively determined by flow cytometry. Four quadrant images (Q1, Q2, Q3 and Q4) were observed by flow cytometric analysis. The results of apoptosis ratios (including the early and late apoptosis ratios) after 12 h are presented in Figure 6 (MGC80-3) and Figure 7 (T24). Figure 6 revealed that compounds **5d** and **6h** could induce apoptosis in MGC80-3 cells in a concentration dependent manner. The apoptosis percentage of compound **5d** measured at different concentrations were found to be 6.616% (2.76 µM) and 17.51% (5.52 µM), while the value for control was 0.586%. Treatment was also accompanied by a decrease in the percentage of live cells, with values of 93.0% in control and 81.2% in treated cells. After treatment with compound **6h**, 5.62% (3.43 µM) and 14.25% (6.85 µM) of the cells were apoptotic. These were higher percentages than the one observed in the control (0.586%)**.** These results further demonstrate that apoptosis was induced by compounds **5d** and **6h** in addition to cell proliferation inhibition. From the results of Figure 7, compounds **4h** and **6h** led to an increase in the number of apoptotic cells in T24 with the increase of the concentration (from 0.5 IC<sup>50</sup> to IC50), and their apoptosis ratios at their IC<sup>50</sup> concentrations were increased to 12.377% and 10.749%, respectively, when compared with the control (1.18%). All compounds had little effect on late apoptosis of MGC80-3 or T24, and some normal cells were found to be necrotic in Q1 region. The results evidently illustrate that representative compounds **5d** and **6h** or **4h** and **6h** could suppress cell proliferation by inducing apoptosis in the early apoptotic period.

**Figure 6.** Apoptosis ratio detection of compounds **5d** and **6h** by Annexin V-FITC and PI. (**a**,**d**) The MGC80-3 cells not treated with compounds **5d** or **6h** were used as controls; (**b**,**c**) compound **5d** treated MGC80-3 cells for 24 h at concentrations of 2.76 and 5.52 μM, respectively; (**e**,**f**) compound **6h** treated MGC80-3 cells for 24 h at concentrations of 3.34 and 6.85 μM, respectively. **Figure 6.** Apoptosis ratio detection of compounds **5d** and **6h** by Annexin V-FITC and PI. (**a**,**d**) The MGC80-3 cells not treated with compounds **5d** or **6h** were used as controls; (**b**,**c**) compound **5d** treated MGC80-3 cells for 24 h at concentrations of 2.76 and 5.52 µM, respectively; (**e**,**f**) compound **6h** treated MGC80-3 cells for 24 h at concentrations of 3.34 and 6.85 µM, respectively.

**Figure 7.** Apoptosis ratio detection of compounds **4h** and **6h** by Annexin V-FITC and PI. (**a**,**d**) The T24 cells not treated with compounds **4h** and **6h** were used as controls; (**b**,**c**) compound **4h** treated T24 cells for 24 h at concentrations of 4.03 and 8.05 μM, respectively; (**e**,**f**) compound **6h** treated T24 cells for 24 h at concentrations of 4.50 and 8.93 μM, respectively. **Figure 7.** Apoptosis ratio detection of compounds **4h** and **6h** by Annexin V-FITC and PI. (**a**,**d**) The T24 cells not treated with compounds **4h** and **6h** were used as controls; (**b**,**c**) compound **4h** treated T24 cells for 24 h at concentrations of 4.03 and 8.05 µM, respectively; (**e**,**f**) compound **6h** treated T24 cells for 24 h at concentrations of 4.50 and 8.93 µM, respectively.

The cell cycle distributions of T24 and MGC80-3cells after 48 h of treatment with the most active compounds, **4h** and **5d,** at their IC50 concentrations are shown in Figure 8. Compared to control, both compounds **4h** and **5d** interfered with the cell cycles of T24 and MGC80-3 cells, respectively. As shown in Figure 8a,b, the S-phase population of T24 cells increased by 30.04% compared to the control cells (22.89%), indicating that compound **4h** might inhibit the growth of tumor cells by arresting the cells in S phase during the DNA synthesis period. However, compound **5d** could induce a significant cell cycle arrest in the G2 phase, resulting in a concomitant population increase (13.32%) compared with the control cells (8.91%) at a concentration of 5.52 μM (Figure 8c,d). These results suggest that compound **5d** may inhibit the growth of tumor cells by arresting cells in the G2 phase in the late stage of DNA synthesis. The cell cycle distributions of T24 and MGC80-3cells after 48 h of treatment with the most active compounds, **4h** and **5d,** at their IC<sup>50</sup> concentrations are shown in Figure 8. Compared to control, both compounds **4h** and **5d** interfered with the cell cycles of T24 and MGC80-3 cells, respectively. As shown in Figure 8a,b, the S-phase population of T24 cells increased by 30.04% compared to the control cells (22.89%), indicating that compound **4h** might inhibit the growth of tumor cells by arresting the cells in S phase during the DNA synthesis period. However, compound **5d** could induce a significant cell cycle arrest in the G2 phase, resulting in a concomitant population increase (13.32%) compared with the control cells (8.91%) at a concentration of 5.52 µM (Figure 8c,d). These results suggest that compound **5d** may inhibit the growth of tumor cells by arresting cells in the G2 phase in the late stage of DNA synthesis.

**Figure 8.** Cell cycle analysis of compound **4h** treated T24 cells (**b**) and compound **5d** treated MGC80- 3 cells (**d**) at their IC50 concentrations (8.05 μM and 5.52 μM) for 48 h. And the T24 and MGC80-3 cells not treated with compounds **4h** and **5d** were used as control, (**a**,**c**). (G1: Prophase of DNA synthesis; S: stage of dna synthesis; G2: Late stages of DNA synthesis). **Figure 8.** Cell cycle analysis of compound **4h** treated T24 cells (**b**) and compound **5d** treated MGC80-3 cells (**d**) at their IC<sup>50</sup> concentrations (8.05 µM and 5.52 µM) for 48 h. And the T24 and MGC80-3 cells not treated with compounds **4h** and **5d** were used as control, (**a**,**c**). (G1: Prophase of DNA synthesis; S: stage of dna synthesis; G2: Late stages of DNA synthesis).

#### 2.3.2. Evaluation of Topo I Inhibitory Activity 2.3.2. Evaluation of Topo I Inhibitory Activity

DNA topoisomerase I (topo I) has become the main molecular target in anticancer drugs on account of its significance in all living organisms, participating in replication, transcription, recombination, and repair in many cellular metabolic processes. The topo I inhibitory activity of the compounds with the known topo I inhibitor camptothecin (CPT) is depicted in Figure 9. Only compounds **4e**, **5c**, and **6h** exhibit potent topo I inhibitory activity at 100 µM. Compounds that have little to no inhibitory activity may have other mechanisms for their anticancer effects. Molecular docking studies of the selected compounds were carried out by the Surflex-Dock algorithm of Sybyl-X 2.0 (Tripos Inc., St. Louis, MI, USA). The molecular docking approach was verified by our previously published methods (RMSD (root-mean-square deviation) value was 0.4438 Å) [19]. The binding affinities of protein-ligand complexes were expressed as a total score and shown in Figure 10. compounds **4e**, **5c** and **6h** exhibited good binding affinities, with total scores of 9.79, 7.81 and 9.66, respectively. Potent Topo I inhibitory activity of these compounds DNA topoisomerase I (topo I) has become the main molecular target in anticancer drugs on account of its significance in all living organisms, participating in replication, transcription, recombination, and repair in many cellular metabolic processes. The topo I inhibitory activity of the compounds with the known topo I inhibitor camptothecin (CPT) is depicted in Figure 9. Only compounds **4e**, **5c**, and **6h** exhibit potent topo I inhibitory activity at 100 µM. Compounds that have little to no inhibitory activity may have other mechanisms for their anticancer effects. Molecular docking studies of the selected compounds were carried out by the Surflex-Dock algorithm of Sybyl-X 2.0 (Tripos Inc., St. Louis, MI, USA). The molecular docking approach was verified by our previously published methods (RMSD (root-mean-square deviation) value was 0.4438 Å) [19]. The binding affinities of proteinligand complexes were expressed as a total score and shown in Figure 10. compounds **4e**, **5c** and **6h** exhibited good binding affinities, with total scores of 9.79, 7.81 and 9.66, respectively. Potent Topo I inhibitory activity of these compounds may be attributed to the formation of hydrophobic residue, hydrogen bond, and π–π stacking with the same amino acid residue DA113, DC112, TGP11 as CPT.

may be attributed to the formation of hydrophobic residue, hydrogen bond, and π–π

stacking with the same amino acid residue DA113, DC112, TGP11 as CPT.

**Figure 9.** DNA topoisomerase I (Topo I) inhibitory activity of CPT and all target compounds (**4a**–**4j, 5a**–**5j, 6a**–**6j**) at 1 mM. **Figure 9.** DNA topoisomerase I (Topo I) inhibitory activity of CPT and all target compounds (**4a**–**4j, 5a**–**5j, 6a**–**6j**) at 1 mM. *Int. J. Mol. Sci.* **2022**, *23*, x FOR PEER REVIEW 11 of 21

**Figure 10.** The best pose of the binding mode of compounds (**4e**, **5c**, **6h** and **CPT**) with DNA Topo I complex (PDB:1T8I). **Figure 10.** The best pose of the binding mode of compounds (**4e**, **5c**, **6h** and **CPT**) with DNA Topo I complex (PDB:1T8I).

Many antitumor drugs inevitably have side effects on normal cells, such as bone mar-

to improve the possibility of clinical application of acridine-heterocyclic derivatives, the effective and low-toxicity antitumor drugs were screened using a zebrafish model. In this experiment, 2% DMSO was used to dissolve the target products, and the abnormal rate (MAR) and mortality rate (MOR) of zebrafish embryos (72 hpf) were used for statistics. At different concentrations (1–2 mg/mL) of the selected compounds, various deformities were observed, such as failure to hatch, embryo necrosis, severe angulation of the spine and severe pericardial edema (Figure 11). The mortality and malformation rates of embryos increased with a dose-effect relationship (Table 2). Almost all compounds in the compound **4** series were toxic. Particularly, compounds **4a**, **4b**, **4f** and **4h** had a total mortality and malformation rate of 100% at 2.0 mg.L−1, exhibiting the strongest embryonic toxicity. It is worth mentioning that compounds **5d** and **5h** displayed high levels of antitumor activities but were less toxic to zebrafish embryos. At the highest concentration of 2.0 mgL−1, the mortality rate of zebrafish embryos was close to 0% and the malformation rate was less than 15%. Moreover, there was hardly any toxicity observed in compound **6**  at lower concentrations (1.0 mgL−1). Compound **6i** exhibited very low toxicity at a high

concentration of 2.0 mg.L−1 with 0% mortality rate and 25% malformation rate.

2.3.3. Toxicity and Anti-angiogenesis in the Zebrafish Model

#### 2.3.3. Toxicity and Anti-angiogenesis in the Zebrafish Model 2.3.3. Toxicity and Anti-angiogenesis in the Zebrafish Model Many antitumor drugs inevitably have side effects on normal cells, such as bone

complex (PDB:1T8I).

*Int. J. Mol. Sci.* **2022**, *23*, x FOR PEER REVIEW 11 of 21

Many antitumor drugs inevitably have side effects on normal cells, such as bone marrow suppression, liver and kidney injury, and abnormal blood cells. Therefore, in order to improve the possibility of clinical application of acridine-heterocyclic derivatives, the effective and low-toxicity antitumor drugs were screened using a zebrafish model. In this experiment, 2% DMSO was used to dissolve the target products, and the abnormal rate (MAR) and mortality rate (MOR) of zebrafish embryos (72 hpf) were used for statistics. At different concentrations (1–2 mg/mL) of the selected compounds, various deformities were observed, such as failure to hatch, embryo necrosis, severe angulation of the spine and severe pericardial edema (Figure 11). The mortality and malformation rates of embryos increased with a dose-effect relationship (Table 2). Almost all compounds in the compound **4** series were toxic. Particularly, compounds **4a**, **4b**, **4f** and **4h** had a total mortality and malformation rate of 100% at 2.0 mg.L−<sup>1</sup> , exhibiting the strongest embryonic toxicity. It is worth mentioning that compounds **5d** and **5h** displayed high levels of antitumor activities but were less toxic to zebrafish embryos. At the highest concentration of 2.0 mg.L−<sup>1</sup> , the mortality rate of zebrafish embryos was close to 0% and the malformation rate was less than 15%. Moreover, there was hardly any toxicity observed in compound **6** at lower concentrations (1.0 mg.L−<sup>1</sup> ). Compound **6i** exhibited very low toxicity at a high concentration of 2.0 mg.L−<sup>1</sup> with 0% mortality rate and 25% malformation rate. marrow suppression, liver and kidney injury, and abnormal blood cells. Therefore, in order to improve the possibility of clinical application of acridine-heterocyclic derivatives, the effective and low-toxicity antitumor drugs were screened using a zebrafish model. In this experiment, 2% DMSO was used to dissolve the target products, and the abnormal rate (MAR) and mortality rate (MOR) of zebrafish embryos (72 hpf) were used for statistics. At different concentrations (1–2 mg/mL) of the selected compounds, various deformities were observed, such as failure to hatch, embryo necrosis, severe angulation of the spine and severe pericardial edema (Figure 11). The mortality and malformation rates of embryos increased with a dose-effect relationship (Table 2). Almost all compounds in the compound **4** series were toxic. Particularly, compounds **4a**, **4b**, **4f** and **4h** had a total mortality and malformation rate of 100% at 2.0 mg.L−1, exhibiting the strongest embryonic toxicity. It is worth mentioning that compounds **5d** and **5h** displayed high levels of antitumor activities but were less toxic to zebrafish embryos. At the highest concentration of 2.0 mg.L−1, the mortality rate of zebrafish embryos was close to 0% and the malformation rate was less than 15%. Moreover, there was hardly any toxicity observed in compound **6** at lower concentrations (1.0 mg.L−1). Compound **6i** exhibited very low toxicity at a high concentration of 2.0 mg.L−<sup>1</sup> with 0% mortality rate and 25% malformation rate.

**Figure 10.** The best pose of the binding mode of compounds (**4e**, **5c**, **6h** and **CPT**) with DNA Topo I

**Figure 11.** Normal and abnormal zebrafish embryos (**a**) Normal zebrafish embryos (**b**) Normal zebrafish (**c**) Normal pericardium of zebrafish (**d**) Embryo necrosis (**e**) Severe angulation of the spine (**f**) Severe pericardial edema. **Figure 11.** Normal and abnormal zebrafish embryos (**a**) Normal zebrafish embryos (**b**) Normal zebrafish (**c**) Normal pericardium of zebrafish (**d**) Embryo necrosis (**e**) Severe angulation of the spine(**f**) Severe pericardial edema.

Currently, the zebrafish has emerged as a valuable model organism to substitute traditional models for studying angiogenesis inhibitors [20]. The genes of zebrafish show 70–80% similarity to humans, and the vascular structure of zebrafish has high similarity to that of other vertebrates [21–23]. Therefore, the subintestinal veins (SIVs) in the zebrafish embryos are used as evaluation indicators for anti-angiogenesis inhibitors. In this study,

NBT/BCIP vascular staining was used to observe the angiogenesis effect of representative drugs (**4h**, **4f**, **5d**, **5h**, **6g** and **6h**) in a zebrafish model. As shown in Figure 12**,** SIVs grew well in the blank group, naturally extending into a network in the abdomen with many branches. The length of SIVs of zebrafish was measured by Image J software and is shown in Figure 10b. Compared with the blank control group, the area of the meshed pattern vessel and the number of vascular branches in the network decreased after the administration of compounds **4h**, **5d**, **5h** and **6h.** Among these compounds, compounds **5d** and **5h** exhibited the strongest antiangiogenic effects that led to a nearly 50% reduction in the vessel length compared to the mean vessel length for the controls. Compounds **4a** and **6g** could reduce the area of blood vessels, but at the same time, additional blood vessels were formed on the blood vessel edge. this study, NBT/BCIP vascular staining was used to observe the angiogenesis effect of representative drugs (**4h**, **4f**, **5d**, **5h**, **6g** and **6h**) in a zebrafish model. As shown in Figure 12**,** SIVs grew well in the blank group, naturally extending into a network in the abdomen with many branches. The length of SIVs of zebrafish was measured by Image J software and is shown in Figure 10b. Compared with the blank control group, the area of the meshed pattern vessel and the number of vascular branches in the network decreased after the administration of compounds **4h**, **5d**, **5h** and **6h.** Among these compounds, compounds **5d** and **5h** exhibited the strongest antiangiogenic effects that led to a nearly 50% reduction in the vessel length compared to the mean vessel length for the controls. Compounds **4a** and **6g** could reduce the area of blood vessels, but at the same time, additional blood vessels were formed on the blood vessel edge.

Currently, the zebrafish has emerged as a valuable model organism to substitute traditional models for studying angiogenesis inhibitors [20]. The genes of zebrafish show 70–80% similarity to humans, and the vascular structure of zebrafish has high similarity to that of other vertebrates [21–23]. Therefore, the subintestinal veins (SIVs) in the zebrafish embryos are used as evaluation indicators for anti-angiogenesis inhibitors. In

**Table 2.** The mortality rate (MOR) and malformation rate (MAR) of some of the selected compounds. **Table 2.** The mortality rate (MOR) and malformation rate (MAR) of some of the selected compounds.


*Int. J. Mol. Sci.* **2022**, *23*, x FOR PEER REVIEW 12 of 21

Note: <sup>a</sup> = *p* < 0.01; <sup>b</sup> = *p* < 0.001; <sup>c</sup> = *p* < 0.002, compared with the control group. **6i** 0 0 0 0 0 25% c 0 10% b 0 0

Note: a = *p* < 0.01; b = *p* < 0.001; c = *p* < 0.002, compared with the control group.

**Figure 12.** Effects of representative compounds (**4f, 4h, 5d, 5h**, **6g** and **6h**) and control on the subintestinal veins (SIVs) length of 72 hpf zebrafish embryos (x ± s, *n* = 12), *p* < 0.05.

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

All commercially available chemicals were reagent grade and bought from Aladdin Reagent Co., LTD (Shanghai, China); NBT/BCIP kit was bought from Tiangen Biochemical Technology Co., LTD (Shanghai, China); AnnexinV-FITC apoptosis detection kit was bought from Nanjing KGI Biotechnology Development Co., Ltd. (Nanjing, China); The spectra such as NMR, MS, and IR were all evaluated and recorded on a Bruker DRX-400 (1H: 400 MHz, <sup>13</sup>C: 100 MHz) (Rheinstetten, Germany), a Thermo Fisher LCQ Fleet (ESI) instrument (Waltham, MA, USA), and FT-IR Thermo Nicolet Avatar 360 using a KBr pellet (Waltham, MA, USA). And the melting points were measured by the XT-4 A melting point apparatus (Shanghai, China) without correction. Other instruments include BD FACSAria II Flow cytometer (Franklin Lakes, NJ, USA), MCO96 carbon dioxide incubator (Osaka, Japan) and Bio Tek EL × 800 microplate reader (Winooski, VT, USA), etc.

#### *3.1. Synthesis Methods*

3.1.1. Synthesis of N-phenyl-o-aminobenzoic acid (**1**) and 9-chlorine acridine (**2**)

The synthesis of N-phenyl-o-aminobenzoic acid (**1**) and 9-chlorine acridine (**2**) was carried out according to our previously published procedure, with slight modifications [24]. Compound **1** could proceed to the next step without further purification.

2-methoxyl-9-chlorine acridine (**2a**): Yellow-green needle crystal, yield 85.2%, m.p. 158–159 ◦C. ESI-MS m/z: 244 ([M + H]<sup>+</sup> ); <sup>1</sup>H NMR (CDCl3, 400 MHz) 8.00 (dd, 2H, *J* = 8.00, ArH), 7.93 (d, 2H, *J* = 8.20, ArH), 7.55–7.60 (m, 2H, ArH), 7.50 (d, 1H, *J* = 8.40, ArH), 7.30 (s, 1H, ArH), and 3.73 (s, 3H, -OCH3).

2-methyl-9-chlorine acridine (**2b**): Pale green needle crystal, yield 78.5%, m.p. 122– 123 ◦C. ESI-MS m/z: 228 ([M + H])+ 1H NMR (CDCl3, 400 MHz), δ: 8.05 (dd, 2H, *J* = 8.00, ArH), 8.00 (d, 2H, *J* = 9.20, ArH), 7.61–7.68 (m, 2H, ArH), 7.50 (d, 1H, *J* = 5.40, ArH), 7.43 (s, 1H, ArH), 2.35 (s, 3H, -CH3).

#### 3.1.2. Synthesis of 9-acridinyl Isothiocyanate (**3**)

To a solution of chlorine acridine **2** (5 mmol) in acetone (50 mL), NaSCN (0.81 g, 10 mmol) and tetrabutylammonium bromide (0.32 g, 1 mmol) were added, and the mixture was then refluxed at 60 ◦C for 1 h. After cooling to room temperature, crystals of **3a** were immediately precipitated in the reaction mixture, and crystals of **3b** were precipitated in an ice bath. At the end of the procedure, the crystals were filtered, washed with water, and dried under vacuum, and no further purification was carried out.

2-methoxyl-9-acridinyl isothiocyanate (**3a**): bright yellow crystal, yield 88.0%, m.p. 149–150 ◦C; ESI-MS m/z: 267 ([M + H]<sup>+</sup> ); <sup>1</sup>H NMR (CDCl3, 400 MHz), δ: 8.25 (d, 2H, *J* = 8.50, ArH), 8.15 (d, 1H, *J* = 9.20, ArH), 7.77–7.81 (q, 1H, ArH), 7.66–7.68 (t, 1H, ArH), 7.51 (d, 1H, *J* = 8.00, ArH), 7.40 (s, 1H, ArH), 4.08(s, 3H, -OCH3); <sup>13</sup>C NMR (CDCl3, 100 MHz) δ: 158.66, 130.48, 127.54, 127.04, 123.48, 122.62, 122.28, 98.50, 55.90; IR (KBr) ν: 2967, 2098 (-N=C=S), 1356–1557 cm−<sup>1</sup> .

2-methyl-9-acridinyl isothiocyanate (**3b**): faint yellow needle crystal, yield 94%, m.p. 128–129 ◦C; ESI-MS m/z: 351 ([M + H]<sup>+</sup> ); <sup>1</sup>H NMR (CDCl3, 400 MHz), δ: 8.26–8.28 (m, 2H, ArH), 8.15 (d, 1H, *J* = 8.40 Hz, ArH), 8.04 (s, 1H, ArH), 7.83 (t, 1H, ArH), 7.64–7.70 (m, 2H, ArH), 7.40 (s, 1H, ArH), 2.67 (s, 3H, -CH3); <sup>13</sup>C NMR (CDCl3, 100 M Hz) δ: 137.59, 130.46, 127.08, 125.21, 122.92, 122.21, 121.17, 22.12; IR (KBr) ν: 2903, 2143 (-N=C=S), 1411– 1630 cm−<sup>1</sup> .

#### 3.1.3. General Procedure for the Synthesis of Acridinyl Aroyl Thiourea Derivatives **4a**–**4f**

To a solution of 9-isothiocyanatoacridine **3** (2mmol) in absolute ethyl alcohol (60 mL), the appropriate substituted hydrazides (2 mmol) were added, and the reaction mixture was refluxed until the reactants had been consumed (monitored by TLC). The precipitate of **4a**–**4f** was prepared, filtered off, washed with 95% ethyl alcohol, and dried at room temperature.

1-20 -methoxyl acridinyl-3-40 -pyridinamide thiourea (**4a**): Yellow powder, Yield 91%, m.p. 200–206 ◦C; ESI-MS m/z: 404 ([M + H]<sup>+</sup> ); <sup>1</sup>H NMR (400 MHz, DMSO-*d 6* ), δ: 11.26 (br, s, 1H, -NH), 10.41 (br, s, 1H, -NH), 10.22 (br, s, 1H, -NH), 8.80 (s, 1H, ArH), 8.05–8.15 (m, 2H, ArH), 7.93 (s, 1H, ArH), 7.77 (t, 1H, ArH), 7.63 (t, 1H, ArH), 8.15 (d, 1H, *J* = 8.40 Hz, ArH), 8.04 (s, 1H, ArH), 7.83 (t, 1H, ArH), 7.53 (d, 1H, *J* = 9.20 Hz, ArH), 7.45 (s, 1H, ArH),

4.02 (s, 3H, -OCH3); <sup>13</sup>C NMR (DMSO-*d 6* , 100 MHz) δ: 183.19, 167.30, 165.41, 158.40, 150.74, 148.72, 141.00, 139.61, 131.03, 129.59, 125.82, 122.40, 113.46, 109.76, 56.09; IR (KBr) ν: 3108, 2948 (N–H), 1695 (-C=O), 1291 (-C=S) cm−<sup>1</sup> .

1-20 -methoxyl acridinyl-3-benzoyl thiosemicarbazides (**4b**): Yellow powder, Yield 76%, m.p. 190–192 ◦C; ESI-MS m/z: 403 ([M + H]<sup>+</sup> ); <sup>1</sup>H NMR (400 MHz, DMSO-*d 6* ), δ: 10.96 (br, s, 1H, -NH), 10.40 (br, s, 1H, -NH), 10.10 (br, s, 1H, -NH), 8.01–8.14 (m, 4H, ArH), 7.76 (s, 2H, ArH), 7.52–7.58 (m, 6H, ArH), 4.02 (s, 3H, -OCH3); <sup>13</sup>C NMR (101 MHz, DMSO-*d 6* ) δ: 183.14, 166.84, 164.80, 157.06, 156.21, 144.80, 141.24, 139.31, 132.72, 129.80, 128.80, 127.28, 125.95, 122.46, 122.11, 112.10, 111.51, 56.22; IR (KBr) ν: 3102, 2941 (-N—H), 1686 (-C=O), 1289 (-C=S) cm−<sup>1</sup> .

1-20 -methoxyl acridinyl-3-40 -methoxy benzoyl thiosemicarbazides (**4c**): Yellow powder, Yield 87%, m.p. 202–203 ◦C; ESI-MS m/z: 455 ([M + Na]<sup>+</sup> ); <sup>1</sup>H NMR (400 MHz, DMSO-*d 6* ), δ: 10.81 (br, s, 1H, -NH), 10.37 (br, s, 1H, -NH), 10.04 (br, s, 1H, -NH), 8.02–8.09 (m, 4H, ArH), 7.32–7.87 (m, 6H, ArH), 7.06 (s, 1H, ArH), 3.82 (s, 3H, -OCH3), 3.32 (s, 3H, -OCH3); <sup>13</sup>C NMR (101 MHz, DMSO-*d 6* ) δ: 182.97, 166.60, 162.03, 156.30, 153.82, 140.77, 136.15, 132.16, 129.58, 128.77, 127.25, 125.82, 121.52, 111.00, 108.32, 55.90; IR (KBr) ν: 3107, 2945 (-N—H), 1677 (-C=O), 1256 (-C=S) cm−<sup>1</sup> .

1-2'-methoxyl acridinyl-3-40 -nitro benzoyl thiosemicarbazides (**4d**): Yellow powder, Yield 94%, m.p. 223–227 ◦C; ESI-MS m/z: 448 ([M + H]<sup>+</sup> ); <sup>1</sup>H NMR (400 MHz, DMSO-*d 6* ), δ: 11.30 (br, s, 1H, -NH), 10.45 (br, s, 1H, -NH), 10.22 (br, s, 1H, -NH), 8.38 (s, 2H, ArH), 8.26 (s, 2H, ArH), 8.02–8.16 (m, 2H, ArH), 7.53-7.63 (m, 4H, ArH), 7.46 (s, 1H, ArH), 4.02 (s, 3H, -OCH3); <sup>13</sup>C NMR (101 MHz, DMSO-*d 6* ) δ: 183.22, 167.59, 162.87, 157.42, 149.87, 147.71, 146.73, 140.26, 138.66, 131.49, 131.11, 129.64, 125.67, 124.67, 124.15, 123.98, 117.74, 110.08, 100.87, 56.20; IR (KBr) ν: 3105, 2947 (-N—H), 1697 (-C=O), 1527 (-C=S) cm−<sup>1</sup> .

1-2'-methoxyl acridinyl-3-4'-nitro benzoyl thiosemicarbazides (**4d**): Yellow powder, Yield 94%, m.p. 223–227 ◦C; ESI-MS m/z: 448 ([M+H]<sup>+</sup> ); <sup>1</sup>H NMR (400 MHz, DMSO-*d 6* ), δ: 11.30 (br, s, 1H, -NH), 10.45 (br, s, 1H, -NH), 10.22 (br, s, 1H, -NH), 8.38 (s, 2H, ArH), 8.26 (s, 2H, ArH), 8.02-8.16 (m, 2H, ArH), 7.53–7.63 (m, 4H, ArH), 7.46 (s, 1H, ArH), 4.02 (s, 3H, -OCH3); <sup>13</sup>C NMR (101 MHz, DMSO-*d 6* ) δ: 183.22, 167.59, 162.87, 157.42, 149.87, 147.71, 146.73, 140.26, 138.66, 131.49, 131.11, 129.64, 125.67, 124.67, 124.15, 123.98, 117.74, 110.08, 100.87, 56.20; IR (KBr) ν: 3105, 2947 (-N—H), 1697 (-C=O), 1527 (-C=S) cm−<sup>1</sup> .

1-20 -methyl acridinyl-3-40 -pyridinamide thiourea (**4f**): Orange powder, Yield 82%, m.p. 176–180 ◦C; ESI-MS m/z: 410 ([M + Na]<sup>+</sup> ); <sup>1</sup>H NMR (400 MHz, DMSO-*d 6* ), δ: 11.59 (br, s, 1H, -NH), 10.99 (br, s, 1H, -NH), 10.21 (br, s, 1H, -NH), 8.81 (s, 2H, ArH), 7.89 (s, 3H, ArH), 7.42–7.89 (m, 6H, ArH), 2.33 (s, 3H, -CH3); <sup>13</sup>C NMR (101 MHz, DMSO-*d 6* ) δ: 183.47, 167.14, 155.61, 150.76, 140.45, 140.16, 138.18, 133.65, 131.24, 126.31, 124.88, 117.52, 111.80, 21.93; IR (KBr) ν: 3104, 2918 (-N—H), 1556 (-C=O), 1471 (-C=S) cm−<sup>1</sup> .

1-20 -methyl acridinyl-3-benzoyl thiosemicarbazides (**4g**): Orange powder, Yield 82%, m.p. 171–173 ◦C; ESI-MS m/z: 409 [M + Na]<sup>+</sup> ; <sup>1</sup>H NMR (400 MHz, DMSO-*d 6* ), δ: 10.68 (br, s, 1H, -NH), 10.41 (br, s, 1H, -NH), 10.13 (br, s, 1H, -NH), 8.58–8.75 (m, 4H, ArH), 7.42–7.57 (m, 5H, ArH), 7.39 (d, *J* = 8.5 Hz, 2H, ArH), 7.09 (s, 1H, ArH), 2.42 (s, 3H, -CH3); <sup>13</sup>C NMR (101 MHz, DMSO-*d 6* ,) δ: 181.29, 166.81, 153.65, 150.24, 148.96, 140.25, 138.24, 133.58, 130.84, 130.35, 126.22, 125.07, 122.13, 121.38, 117.44, 116.40, 111.44, 21.25; IR (KBr) ν: 3102, 2917 (-N—H), 1569 (-C=O), 1471 (-C=S) cm−<sup>1</sup> .

1-20 -methyl acridinyl-3-4'-methoxy benzoyl thiosemicarbazides (**4h**): Orange-yellow powder, Yield 93%, m.p. 210–212 ◦C; ESI-MS m/z: 439 ([M + Na]<sup>+</sup> ); <sup>1</sup>H NMR (400 MHz, DMSO-*d 6* ), δ: 10.53 (br, s, 1H, -NH), 10.38 (br, s, 1H, -NH), 10.08 (br, s, 1H, -NH), 7.97–8.18 (m, 4H, ArH), 7.34–7.55 (m, 3H, ArH), 7.34–7.55 (m, 3H, ArH), 7.07 (s, 1H, ArH), 3.44 (s, 3H, -OCH3), 2.42 (s, 3H, -CH3); <sup>13</sup>C NMR (101 MHz, DMSO-*d 6* ) δ: 181.09, 166.30, 153.39, 148.81, 140.15, 138.18, 130.79, 130.04, 126.17, 125.32, 121.21, 117.39, 114.12, 111.37, 55.89, 22.24; IR (KBr) ν: 3094, 2914 (-N—H), 1556 (-C=O), 1471 (-C=S) cm−<sup>1</sup> .

1-20 -methyl acridinyl-3-4'-nitro benzoyl thiosemicarbazides (**4i**): Orange-yellow powder, Yield 73%, m.p. 187–188 ◦C; ESI-MS m/z: 457 ([M + Na]<sup>+</sup> ); <sup>1</sup>H NMR (400 MHz, DMSO-*d 6* ), δ: 11.56 (br, s, 1H, -NH), 10.76 (br, s, 1H, -NH), 10.15 (br, s, 1H, -NH), 8.41 (d, *J*

= 8.5 Hz, 2H), 7.96–8.26 (m, 4H, ArH), 7.26–7.57 (m, 4H, ArH), 7.11 (s, 1H, ArH), 2.44 (s, 3H, -CH3); <sup>13</sup>C NMR (101 MHz, DMSO-*d 6* ) δ: 183.42, 166.78, 155.67, 149.20, 140.24, 140.14, 131.87, 130.89, 130.41, 124.68, 117.93, 111.56, 22.50; IR (KBr) ν: 3095, 2918 (-N—H), 1598 (-C=O), 1483 (-C=S) cm−<sup>1</sup> .

1-20 -methyl acridinyl-3-40 -chloro benzoyl thiosemicarbazides (**4j**): Orange-yellow powder, Yield 86%, m.p. 178–179 ◦C; ESI-MS m/z: 443 ([M + Na]<sup>+</sup> ); <sup>1</sup>H NMR (400 MHz, DMSO-*d 6* ), δ: 10.77 (br, s, 1H, -NH), 10.48 (br, s, 1H, -NH), 10.08 (br, s, 1H, -NH), 8.18–8.34 (m, 4H, ArH), 7.57–7.83 (m, 3H, ArH), 7.33–7.57 (m, 3H, ArH), 7.07 (s, 1H, ArH), 2.42 (s, 3H, -CH3); <sup>13</sup>C NMR (101 MHz, DMSO-*d 6* ) δ: 182.61, 166.39, 155.38, 149.09, 140.26, 139.92, 131.03, 130.08, 129.29, 124.20, 117.53, 116.31, 111.58, 104.34, 22.83; IR (KBr) ν: 3094, 2915 (-N—H), 1567 (-C=O), 1480 (-C=S) cm−<sup>1</sup> .

#### 3.1.4. General Procedure for the Synthesis of Acridinyl 1,2,4-triazole Derivatives **5a**–**5f**

The appropriate acyl thiosemicarbazides (**4a**–**4i**, 1 mmol) and 5% aqueous sodium carbonate (40 mL) were refluxed for 5 h. After cooling, the precipitate was filtered off and the filtrate was acidified by hydrochloric acid to a pH of 2. The precipitates were formed, filtered off and then crystallized from ethyl alcohol.

4-(2-methoxyacridin-9-yl)-5-(pyridin-4-yl)-2,4-dihydro-3H-1,2,4-triazole-3-thione (**5a**): Light yellow powder, Yield 75%, m.p. 272–273 ◦C; ESI-MS m/z: 384 ([M + H]<sup>+</sup> ); <sup>1</sup>H NMR (400 MHz, DMSO-*d 6* ), δ: 14.79 (br, s, 1H, -NH), 8.38 (dd, *J* = 4.6, 1.5 Hz, 2H, ArH), 8.33–8.11 (m, 2H, ArH), 7.94–7.76 (m, 1H, ArH), 7.08 (dd, *J* = 4.6, 1.6 Hz, 2H, ArH), 6.87 (d, *J* = 2.6 Hz, 1H), 3.86 (s, 3H, -OCH3); <sup>13</sup>C NMR (100 MHz, DMSO-*d 6* ) δ: 169.99, 159.21, 157.94, 150.89, 149.46, 134.90, 130.31, 129.15, 126.69, 125.10, 124.89, 120.97, 56.52; IR (KBr) ν: 3069, 2906, 2745 (-N—H), 1505–1633 (-C=N),1480 (-C=S) cm−<sup>1</sup> .

4-(2-methoxyacridin-9-yl)-5-phenyl-2,4-dihydro-3H-1,2,4-triazole-3-thione (**5b**): Light yellow powder, 60%, m.p. 259–261 ◦C; ESI-MS m/z: 385 ([M + H]<sup>+</sup> ); <sup>1</sup>H NMR (400 MHz, DMSO-*d 6* ), δ: 14.56 (s, 1H, -NH), 8.22 (dd, *J* = 12.2, 9.1 Hz, 2H, ArH), 7.89–7.75 (m, 1H, ArH), 7.70–7.58 (m, 2H, ArH), 7.56 (d, *J* = 8.6 Hz, 1H, ArH), 7.26 (t, *J* = 6.6 Hz, 1H, ArH), 7.21–7.07 (m, 4H, ArH), 6.82 (d, *J* = 2.6 Hz, 1H, ArH), 3.85 (s, 3H, -OCH3); <sup>13</sup>C NMR (100 MHz, DMSO-*d 6* ) δ: 169.50, 159.01, 151.69, 147.45, 146.68, 132.13, 131.27, 130.24, 130.13, 129.26, 128.94, 127.42, 126.58, 125.76, 124.95, 123.68, 122.58, 98.59, 56.44; IR (KBr) ν: 3056, 2912, 2749 (-N—H), 1500–1632 (-C=N), 1476 (-C=S) cm−<sup>1</sup> .

4-(2-methoxyacridin-9-yl)-5-(4-methoxyphenyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione (**5c**): Light yellow powder, Yield 58%, m.p. 262–267 ◦C; ESI-MS m/z: 415 ([M + H]<sup>+</sup> ); <sup>1</sup>H NMR (400 MHz, DMSO-*d 6* ), δ: 14.45 (s, 1H, -NH), 8.23 (dd, *J* = 11.8, 9.1 Hz, 2H, ArH), 7.93–7.73 (m, 1H, ArH), 7.64 (s, 2H, ArH), 7.54 (s, 1H, ArH), 7.08 (d, *J* = 8.9 Hz, 2H, ArH), 6.80 (s, 1H, ArH), 6.70 (d, *J* = 8.9 Hz, 2H, ArH), 3.85 (s, 3H, -OCH3), 3.58 (s, 3H, -OCH3); <sup>13</sup>C NMR (100 MHz, DMSO-*d 6* ) δ: 169.30, 161.33, 159.01, 151.58, 147.51, 146.73, 134.08, 132.17, 130.20, 128.95, 126.56, 125.02, 123.76, 122.59, 117.89, 114.76, 98.57, 56.41, 55.60; IR (KBr) ν: 3066, 2883, 2726 (N—H), 1500–1632 (C=N), 1476 (C=S) cm−<sup>1</sup> .

4-(2-methoxyacridin-9-yl)-5-(4-nitrophenyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione (**5d**): Light yellow powder, Yield 40%, m.p. 253–255 ◦C; ESI-MS m/z: 430 ([M + H]<sup>+</sup> ); <sup>1</sup>H NMR (400 MHz, DMSO-*d 6* ), δ: 14.79 (s, 1H, -NH), 8.24 (t, *J* = 9.3 Hz, 2H, ArH), 8.02 (d, *J* = 8.9 Hz, 2H, ArH), 7.93–7.75 (m, 1H, ArH), 7.63 (dd, *J* = 9.5, 2.7 Hz, 2H, ArH), 7.58 (s, 1H, ArH), 7.44 (d, *J* = 8.9 Hz, 2H, ArH), 6.91 (s, 1H, ArH), 3.87 (s, 3H, -OCH3); <sup>13</sup>C NMR (100 MHz, DMSO-*d 6* ) δ: 170.00, 159.22, 149.93, 148.90, 147.47, 146.78, 133.20, 132.22, 131.44, 130.25, 129.15, 128.69, 126.70, 124.93, 124.61, 123.51, 122.38, 98.64, 56.54; IR (KBr) ν: 3066, 2883, 2726 (-N—H), 1421–1633 (-C=N), 1345 (-C=S) cm−<sup>1</sup> .

4-(4-chlorophenyl)-4-(2-methoxyacridin-9-yl)-2,4-dihydro-3H-1,2,4-triazole-3-thione (**5e**): Light yellow powder, Yield 78%, m.p. 286–287 ◦C; ESI-MS m/z: 419 ([M + H]<sup>+</sup> ); <sup>1</sup>H NMR (400 MHz, DMSO-*d 6* ), δ: 14.61 (s, 1H, -NH), 8.23 (dd, *J* = 11.4, 9.2 Hz, 2H, ArH), 7.93–7.76 (m, 1H, ArH), 7.73–7.60 (m, 2H, ArH), 7.55 (d, *J* = 8.6 Hz, 1H, ArH), 7.25 (d, *J* = 8.6 Hz, 2H, ArH), 7.17 (d, *J* = 8.6 Hz, 2H, ArH), 6.85 (s, 1H, ArH), 3.86 (s, 3H, -OCH3); <sup>13</sup>C NMR (100 MHz, DMSO-*d 6* ) δ: 169.59, 159.10, 150.72, 147.44, 146.71, 136.12, 133.51, 132.17, 130.22, 129.54, 129.11, 126.64, 124.93, 124.62, 123.57, 122.49, 98.61, 56.49; IR (KBr) ν: 3056, 2909, 2748 (-N—H), 1344–1632 (-C=N), 1503 (-C=S) cm−<sup>1</sup> .

4-(2-methylacridin-9-yl)-5-(pyridin-4-yl)-2,4-dihydro-3H-1,2,4-triazole-3-thione (**5f**): Light yellow powder, Yield 48%, m.p. 264–268 ◦C; ESI-MS m/z: 370 ([M + H]<sup>+</sup> ); <sup>1</sup>H NMR (400 MHz, DMSO-*d 6* ) δ: 14.84 (s, 1H, -NH), 8.37 (d, *J* = 6.0 Hz, 2H, ArH), 8.29 (d, *J* = 8.7 Hz, 1H, ArH), 8.23 (d, *J* = 8.9 Hz, 1H, ArH), 7.96–7.84 (m, 1H, ArH), 7.84–7.74 (m, 1H, ArH), 7.72–7.59 (m, 2H, ArH), 7.50 (s, 1H, ArH), 7.04 (d, *J* = 6.1 Hz, 2H, ArH), 2.52 (s, 3H, -CH3); <sup>13</sup>C NMR (100 MHz, DMSO-*d 6* ) δ: 170.32, 150.92, 149.15, 148.75, 148.53, 139.37, 134.60, 134.56, 132.85, 131.15 130.21, 130.00, 129.04, 123.54, 123.40, 122.87, 120.84, 120.70, 56.49; IR (KBr) ν: 3066, 2917, 2757 (-N—H), 1279–1600 (-C=N), 1426 (-C=S) cm−<sup>1</sup> .

4-(2-methylacridin-9-yl)-5-phenyl-2,4-dihydro-3H-1,2,4-triazole-3-thione (**5g**): White powder, Yield 78%, m.p. 292–293 ◦C; ESI-MS m/z: 369 ([M + H]<sup>+</sup> ); <sup>1</sup>H NMR (400 MHz, DMSO-*d 6* ) δ: 14.60 (s, 1H, -NH), 8.26 (d, *J* = 8.8 Hz, 1H, ArH), 8.20 (d, *J* = 8.9 Hz, 1H, ArH), 7.90–7.82 (m, 1H, ArH), 7.77 (d, *J* = 9.0 Hz, 1H, ArH), 7.72–7.56 (m, 2H, ArH), 7.48 (s, 1H, ArH), 7.30–7.17 (m, 1H, ArH), 7.13 (d, *J* = 4.4 Hz, 4H, ArH), 2.51 (s, 3H, -CH3); <sup>13</sup>C NMR (100 MHz, DMSO-*d 6* ) δ: 169.73, 151.47, 148.71, 148.47, 139.08, 135.20, 134.51, 131.29, 131.07, 130.11, 129.91, 129.28, 128.83, 127.33, 125.66, 123.71, 123.58, 123.02, 120.86, 22.15; IR (KBr) ν: 3066, 2917, 2757 (-N—H), 1279–1600 (-C=N), 1426 (-C=S) cm−<sup>1</sup> .

4-(4-methoxyphenyl)-4-(2-methylacridin-9-yl)-2,4-dihydro-3H-1,2,4-triazole-3-thione (**5h**): White powder, Yield 83%, m.p. 268–269 ◦C; ESI-MS m/z: 399 ([M + H]<sup>+</sup> ); <sup>1</sup>H NMR (400 MHz, DMSO-*d 6* ) δ: 14.48 (s, 1H, -NH), 8.27 (d, *J* = 8.8 Hz, 1H), 8.21 (d, *J* = 8.9 Hz, 1H, ArH), 7.86 (t, 1H, ArH), 7.78 (dd, *J* = 9.0, 1.5 Hz, 1H, ArH), 7.65 (d, *J* = 6.5 Hz, 1H, ArH), 7.60 (d, *J* = 8.5 Hz, 1H, ArH), 7.46 (s, 1H, ArH), 7.05 (d, *J* = 8.9 Hz, 2H, ArH), 6.68 (d, *J* = 8.9 Hz, 2H, ArH), 3.57 (s, 3H, -OCH3), 2.52 (s, 3H, -CH3); <sup>13</sup>C NMR (100 MHz, DMSO-*d 6* ) δ: 169.51, 161.33, 151.35, 148.77, 148.53, 139.05, 135.42, 134.49, 131.06, 130.06, 128.85, 123.71 123.02, 120.86, 117.78, 114.78, 55.61, 22.17; IR (KBr) ν: 3095, 2925, 2750 (-N—H), 1360–1613 (-C=N), 1514 (-C=S) cm−<sup>1</sup> .

4-(2-methylacridin-9-yl)-5-(4-nitrophenyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione (**5i**): Light yellow powder, Yield 53%, m.p. 245–247 ◦C; ESI-MS m/z: 414 ([M + H]<sup>+</sup> ); <sup>1</sup>H NMR (400 MHz, DMSO-*d 6* ) δ: 14.79 (s, 1H, -NH), 8.24 (t, *J* = 9.3 Hz, 2H, ArH), 8.02 (d, *J* = 8.9 Hz, 2H, ArH), 7.83 (s, 1H, ArH), 7.71–7.59 (m, 2H, ArH), 7.57 (d, *J* = 8.7 Hz, 1H, ArH), 7.44 (d, *J* = 8.9 Hz, 2H, ArH), 6.91 (s, H, ArH), 2.51 (s, 3H, -CH3), <sup>13</sup>C NMR (100 MHz, DMSO*d 6* ) δ: 169.99, 150.53, 148.55, 139.72, 135.26, 131.39, 130.41, 129.19, 128.69, 126.71, 124.62, 123.78, 123.49, 120.98, 22.19; IR (KBr) ν: 3054, 2918, 2755 (-N—H), 1432-1633 (-C=N), 1376 (-C=S) cm−<sup>1</sup> .

4-(4-chlorophenyl)-4-(2-methylacridin-9-yl)-2,4-dihydro-3H-1,2,4-triazole-3-thione (**5j**): Yellow powder, Yield 80%, m.p. 259–263 ◦C; ESI-MS m/z: 403 ([M + H]<sup>+</sup> ); <sup>1</sup>H NMR (400 MHz, DMSO-*d 6* ) δ: 14.65 (s, 1H, -NH), 8.27 (d, *J* = 8.8 Hz, 1H, ArH), 8.20 (d, *J* = 8.9 Hz, 1H, ArH), 7.88 (dd, *J* = 10.5, 4.0 Hz, 1H, ArH), 7.77 (d, *J* = 8.8 Hz, 1H, ArH), 7.73–7.57 (m, 2H, ArH), 7.49 (s, 1H, ArH), 7.24 (d, *J* = 8.5 Hz, 2H, ArH), 7.14 (d, *J* = 8.5 Hz, 2H, ArH); <sup>13</sup>C NMR (100 MHz, DMSO-*d 6* ) δ: 169.85, 150.49, 148.74, 148.52, 139.19, 136.15, 134.86, 134.52, 131.08, 130.17, 129.97, 129.55, 129.00, 124.51, 123.56, 122.98, 120.81, 22.15; IR (KBr) ν: 3097, 2933 (-N—H), 1258-1600 (-C=N), 1497 (-C=S) cm−<sup>1</sup> .

#### 3.1.5. General Procedure for the Synthesis of Acridinyl 1,3,4-thiadiazol Derivatives (**6**)

About 3 mL of 98% concentrated sulfuric acid was added to a 50 mL round-bottom flask and stirred in an ice bath for 10 min at 0 ◦C. Then, intermediate **4** (0.5 mmol) was added into the solution in small portions over the course of 1 h. The reaction was continued at room temperature for 24–48 h, and 10 mL pure water was slowly added to reaction mixture in an ice bath. The final product **6** was precipitated, filtered off, washed with water, dried, and crystallized from ethyl alcohol.

7-methoxy-N-(5-(pyridin-4-yl)-1,3,4-thiadiazol-2-yl)-10,10a-dihydroacridin-9(8aH)-imine (**6a**): Orange solids, Yield 80%, m.p. 272–275 ◦C; ESI-MS m/z: 386 ([M + H]<sup>+</sup> ); <sup>1</sup>H NMR (400 MHz, DMSO-*d 6* ), δ: 8.78 (d, *J* = 5.7 Hz, 2H, ArH), 8.22 (d, *J* = 8.6 Hz, 1H, ArH), 8.00–7.94

(m, 3H, ArH), 7.90 (d, *J* = 6.2 Hz, 2H, ArH), 7.75 (dd, *J* = 9.3, 2.7 Hz, 1H, ArH), 7.58 (s, 1H, ArH), 7.53 (dt, *J* = 8.3, 4.0 Hz, 1H, ArH), 3.86 (s, 3H, -OCH3); <sup>13</sup>C NMR (DMSO-*d 6* , 100 MHz) δ: 156.40, 149.19, 140.03, 135.03, 130.65, 128.43, 127.65, 127.49, 127.29, 126.86, 125.02, 121.72, 119.38, 103.95, 56.20; IR (KBr) ν: 3055, 3011, 2837 (-C—H, -N—H), 1379-1631 (-C=N) cm−<sup>1</sup> .

7-methoxy-N-(5-phenyl-1,3,4-thiadiazol-2-yl)-10,10a-dihydroacridin-9(8aH)-imine (**6b**): Orange- yellow solid, Yield 81%, m.p. 263–265 ◦C; ESI-MS m/z: 385 ([M + H]<sup>+</sup> ); <sup>1</sup>H NMR (400 MHz, DMSO-*d 6* ), δ: 8.19 (d, *J* = 8.7 Hz, 1H, ArH), 7.92 (q, *J* = 9.3 Hz, 3H, ArH), 7.80 (dd, *J* = 6.6, 2.9 Hz, 2H, ArH), 7.68 (dd, *J* = 9.2, 2.7 Hz, 1H, ArH), 7.58 (s, 1H, ArH), 7.54–7.44 (m, 4H, ArH), 3.83 (s, 3H, -OCH3); <sup>13</sup>C NMR (101 MHz, DMSO-*d 6* ) δ: 156.06, 140.22, 136.58, 134.49, 131.24, 130.46, 129.83, 127.72, 126.95, 126.79, 124.37, 121.59, 120.02, 120.33, 119.26, 116.76, 104.39, 56.03; IR (KBr) ν: 2771 (-C—H, N—H), 1329-1632 (-C=N) cm−<sup>1</sup> .

7-methoxy-N-(5-(4-methoxyphenyl)-1,3,4-thiadiazol-2-yl)-10,10a-dihydroacridin-9(8aH)-imine (**6c**): Orange-red solid, Yield 54%; m.p. 230–232 ◦C; ESI-MS m/z: 415 ([M + H]<sup>+</sup> ); <sup>1</sup>HNMR (400 MHz, DMSO-*d 6* ) δ: 8.35 (d, *J* = 8.5 Hz, 1H, ArH), 8.10 (d, *J* = 8.1 Hz, 3H, ArH), 8.01 (d, *J* = 9.8 Hz, 1H, ArH), 7.96 (s, 1H, ArH), 7.91–7.79 (m, 1H, ArH), 7.76–7.55 (m, 3H, ArH), 7.06 (t, *J* = 9.1 Hz, 1H, ArH), 3.93 (s, 3H, -OCH3), 3.81 (s, 3H, -OCH3); <sup>13</sup>C NMR (101 MHz, DMSO-*d 6* ) δ: 156.91, 149.55, 144.54, 140.78, 136.88, 135.34, 129.01, 128.50, 126.77, 124.39, 121.00, 116.85, 112.99, 103.39, 103.21, 56.65, 56.17; IR (KBr) ν: 2771 (-C—H, -N—H), 1567 (-C=N) cm−<sup>1</sup> .

7-methoxy-N-(5-(4-nitrophenyl)-1,3,4-thiadiazol-2-yl)-10,10a-dihydroacridin-9(8aH) imine (**6d**): Orange-red solid, Yield 33%; m.p. 258–260 ◦C; ESI-MS m/z: 430 ([M + H]<sup>+</sup> ); <sup>1</sup>H NMR (DMSO-*d 6 ,* 400 MHz) δ: 8.32 (d, *J* = 8.9 Hz, 2H, ArH), 8.29 (d, *J* = 8.6 Hz, 1H, ArH), 8.04 (dd, *J* = 6.9, 4.8 Hz, 5H, ArH), 7.81 (dd, *J* = 9.3, 2.7 Hz, 1H, ArH), 7.61 (dd, *J* = 8.5, 2.5 Hz, 2H, ArH), 3.89 (s, 3H, -OCH3); <sup>13</sup>C NMR (101 MHz, DMSO-*d 6* ) δ: 156.85, 148.76, 145.74, 142.43, 139.80, 136.85, 135.92, 135.35, 129.32, 127.93, 126.55, 125.67, 125.01, 121.90, 120.38, 119.60, 117.03, 103.59, 56.32; IR (KBr) ν: 2829 (-C—H, -N—H), 1346-1633 (-C=N) cm−<sup>1</sup> .

7-methoxy-N-(5-(4-chlorophenyl)-1,3,4-thiadiazol-2-yl)-10,10a-dihydroacridin-9(8aH) imine (**6e**): Orange-yellow solid, Yield 51%; m.p. 203–205 ◦C; ESI-MS m/z: 419 ([M + H]<sup>+</sup> ); <sup>1</sup>H NMR (400 MHz, DMSO-*d 6* ) δ: 8.21 (d, *J* = 8.6 Hz, 1H, ArH), 7.94 (d, *J* = 8.1 Hz, 3H, ArH), 7.82 (s, 2H, ArH), 7.72 (dd, *J* = 9.2, 2.6 Hz, 1H, ArH), 7.58 (dd, *J* = 5.9, 2.6 Hz, 3H, ArH), 7.55–7.41 (m, 1H, ArH), 3.85 (s, 3H, -OCH3); <sup>13</sup>C NMR (101 MHz, DMSO-*d 6* ) δ: 156.36, 140.09, 135.90, 134.8, 129.89, 129.19, 128.58, 128.25, 126.65, 124.87, 121.70, 120.01, 119.45, 116.90, 104.12, 102.60, 56.21; IR (KBr) ν: 2781 (-C—H, -N—H), 1467 (-C=N) cm−<sup>1</sup> .

7-methyl-N-(5-(pyridin-4-yl)-1,3,4-thiadiazol-2-yl)-10,10a-dihydroacridin-9(8aH)-imine **(6f**): Orange-yellow solid, Yield 43%; m.p. 276–278 ◦C; ESI-MS m/z: 370 ([M + H]<sup>+</sup> ); <sup>1</sup>H NMR (400 MHz, DMSO-*d 6* ) δ: 8.34 (d, *J* = 8.7 Hz, 2H, ArH), 8.23 (d, *J* = 8.9 Hz, 1H, ArH), 8.14 (d, *J* = 8.3 Hz, 1H, ArH), 8.05 (t, *J* = 9.3 Hz, 3H, ArH), 8.01–7.91 (m, 2H, ArH), 7.88 (s, 1H, ArH), 7.51 (d, *J* = 8.2 Hz, 1H, ArH), 2.48 (s, 3H, -CH3); <sup>13</sup>C NMR (101 MHz, DMSO-*d 6* ) δ: 158.09, 155.78, 151.07, 140.54, 135.49, 133.66, 131.53, 127.77, 126.46, 123.78, 121.88, 118.38, 118.11, 116.14, 21.31; IR (KBr) ν: 2824 (-C—H, -N—H), 1347-1600 (-C=N) cm−<sup>1</sup> .

7-methyl-N-(5-phenyl-1,3,4-thiadiazol-2-yl)-10,10a-dihydroacridin-9(8aH)-imine (**6g**): Yellow solid, Yield 56%; m.p. 294–296 ◦C; ESI-MS m/z: 369 ([M + H]<sup>+</sup> ); <sup>1</sup>H NMR (400 MHz, DMSO-*d 6* ) δ: 8.17 (d, *J* = 8.5 Hz, 1H, ArH), 8.07 (s, 1H, ArH), 7.97–7.85 (m, 2H, ArH), 7.80 (d, *J* = 6.9 Hz, 4H, ArH), 7.59-7.49 (m, 3H, ArH), 7.48–7.34 (m, 1H, ArH), 2.45 (s, 3H, -CH3); <sup>13</sup>C NMR (101 MHz, DMSO-*d 6* ) δ: 160.12, 157.49, 140.60, 138.98, 137.32, 135.07, 133.97, 131.26, 130.45, 129.82, 127.17, 127.15, 125.56, 124.06, 119.50, 119.21, 118.10, 117.00, 21.44; IR (KBr) ν: 2792 (C—H, N—H), 1366-1627 (C=N) cm−<sup>1</sup> .

7-methyl-N-(5-(4-methoxyphenyl)-1,3,4-thiadiazol-2-yl)-10,10a-dihydroacridin-9(8aH) imine (**6h**): Orange-yellow solid, Yield 84%; m.p. >300 ◦C; ESI-MS m/z: 399 ([M + H]<sup>+</sup> ); <sup>1</sup>H NMR (400 MHz, DMSO-*d 6* ) δ: 8.33 (d, *J* = 8.7 Hz, 1H, ArH), 8.18 (s, 1H, ArH), 8.10–8.01 (m, 3H, ArH), 7.98 (d, *J* = 8.8 Hz, 2H, ArH), 7.91–7.77 (m, 1H, ArH), 7.68 (dd, *J* = 8.6, 2.3 Hz, 1H, ArH), 7.62 (dd, *J* = 14.4, 7.9 Hz, 1H, ArH), 7.08 (d, *J* = 8.7 Hz, 1H, ArH), 2.48 (s, 3H, -CH3); <sup>13</sup>C NMR (101 MHz, DMSO-*d 6* ) δ: 162.17, 159.02, 140.47, 139.22, 137.04, 136.13, 135.63,

128.91, 127.11, 127.06, 126.92, 126.85, 125.57, 125.06, 120.71, 120.03, 119.59, 118.29, 117.35, 113.02, 56.20, 21.52; IR (KBr) ν: 3090, 2920, 2830 (-C—H, -N—H), 1486-1628 (-C=N) cm−<sup>11</sup> .

7-methyl-N-(5-(4-nitrophenyl)-1,3,4-thiadiazol-2-yl)-10,10a-dihydroacridin-9(8aH) imine (**6i**): Purple-red solid, Yield 53%; m.p. 224–226 ◦C; ESI-MS m/z: 414 ([M + H]<sup>+</sup> ); <sup>1</sup>H NMR (400 MHz, DMSO-*d 6* ) δ: 12.17 (s, 1H, -NH), 8.35 (d, *J* = 8.9 Hz, 2H, ArH), 8.15 (d, *J* = 8.9 Hz, 2H, ArH), 7.96 (d, *J* = 5.1 Hz, 2H, ArH), 7.72 (t, *J* = 7.0 Hz, 1H, ArH), 7.65–7.56 (m, 2H, ArH), 7.54 (d, *J* = 8.5 Hz, 1H, ArH), 7.18 (t, *J* = 7.6 Hz, 1H, ArH), 2.35 (s, 3H, -CH3); <sup>13</sup>C NMR (100 MHz, DMSO-*d 6* ) δ: 165.41, 148.81, 143.72, 140.52, 139.40, 138.81, 136.22, 135.69, 135.63, 127.93, 126.99, 125.65, 125.02, 120.12, 119.72, 118.15, 117.34, 113.14, 21.54; IR (KBr) ν: 2918 (-C—H, -N—H), 1340-1627 (-C=N) cm−<sup>1</sup> .

7-methyl-N-(5-(4-chlorophenyl)-1,3,4-thiadiazol-2-yl)-10,10a-dihydroacridin-9(8aH) imine (**6j**): Orange-yellow solid, Yield 67%, m.p. 258–260 ◦C; ESI-MS m/z: 403 ([M + H]<sup>+</sup> ); <sup>1</sup>H NMR (400 MHz, DMSO-*d 6* ) δ: 8.23 (d, *J* = 8.6 Hz, 1H, ArH), 8.12 (s, 1H, ArH), 8.04–7.93 (m, 2H, ArH), 7.88 (s, 2H, ArH), 7.81 (d, *J* = 8.5 Hz, 2H, ArH), 7.58 (d, *J* = 8.5 Hz, 2H, ArH), 7.51 (t, *J* = 7.5 Hz, 1H, ArH), 2.49 (s, 3H, -CH3); <sup>13</sup>C NMR (101 MHz, DMSO-*d 6* ) δ: 160.71, 146.36, 140.56, 139.09, 137.87, 135.87, 135.50, 129.89, 129.14, 128.58, 127.04, 125.34, 124.71, 119.72, 119.37, 118.16, 117.13, 21.47; IR (KBr) ν: 2795 (-C—H, -N—H), 1366–1627 (-C=N) cm−<sup>1</sup> .

3.1.6. Preparation of Single Crystal Compounds **5b** and **6d** and Their X-ray Single Crystal Diffraction Method

A single crystal of **5b** and **6d** suitable for X-ray diffraction study was cultivated from 95% ethyl alcohol and N, N-dimethylformamide respectively, by a slow evaporation method at room temperature. All measurements were performed with Mo Kα radiation (λ = 0.7107 Å) on a Brucker SMART 1000 CCD X diffractometer (Billerica, MA, USA). The structure was solved by direct methods with SHELXS-97 [25] and refined by SHELXL-97 [26]. All non-hydrogen atoms were refined with anisotropic thermal parameters. The final full-matrix least-squares refinement of **5b** gave R = 0.0914, ω = 1/[s<sup>2</sup> (Fo<sup>2</sup> ) + (0.0431 *p*) <sup>2</sup> + 0.2721 *p*] where *p* = (Fo<sup>2</sup> + 2Fc<sup>2</sup> )/3, S = 1.043, (∆/*σ*)max = 0.237 and (∆/σ)min <sup>=</sup> <sup>−</sup>0.267 e/Å<sup>3</sup> . In addition, the final full-matrix least-squares refinement of **6d** gave R = 0.0914, ω = (1/[s<sup>2</sup> (Fo<sup>2</sup> ) + (0.0650 *p*) <sup>2</sup> + 0.0224 *p*] where *p* = (Fo<sup>2</sup> + 2Fc<sup>2</sup> )/3, S = 1.028, (∆/*σ*)max = 0.236 and (∆/*σ*)min <sup>=</sup> <sup>−</sup>0.197 e/Å<sup>3</sup> .

#### *3.2. In-Vivo Antitumor Activity*

#### 3.2.1. Antiproliferative Activity

Test samples, including compounds **4**–**6** and commercial classical anticancer drugs (5-FU and cis-platinum), were screened for their anti-cancer activity against HFF human foreskin fibroblast cells, MGC-803 human gastric cancer cells, BEL-7404 human hepatocellular carcinoma cells, NCI-H460 human large cell carcinoma cells, and T24 human bladder carcinoma cells using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) assay method cited in the literature [27]. The initial concentration of all the test samples was 100 µg/mL, which was serially diluted in complete medium with ten-fold dilutions to give six concentrations per compound. Their cytotoxicity was determined in 96-well flat bottomed microtiter plates. All the test samples were tested in triplicate. The results were expressed as the drug concentration that inhibited cell growth by 50% as compared to the controls (IC50). The IC<sup>50</sup> values were calculated from regression lines obtained from the percent cell growth inhibition plotted as a function of the logarithm of the dose.

#### 3.2.2. Apoptosis and Cell Cycle Analysis

The apoptosis assay and the cell cycle analysis were carried out by cytometry (FACS-Verse, BD, Piscataway, NJ, USA) at an excitation wavelength of 488 nm according to the method described in the literature with slight modifications [28]. The cells were seeded at

<sup>2</sup> <sup>×</sup> <sup>10</sup>6/well and washed by cold PBS. The buffer solutions were prepared using 0.1 M pH 7.4 Hepes/NaOH, 1.4 M NaCl, and 25 mM CaCl2.

#### 3.2.3. Topo I Inhibitory Activity

Topo I and pBR322 were obtained commercially from Takara Bio Inc. (Shiga, Japan). And the enzyme inhibitory activity was determined by our previous methods [19].

#### 3.2.4. Anti-Angiogenic Effect Using the Zebrafish Model

(a) Zebrafish toxicity assay

The zebrafish embryos were collected at 6 hpf and randomly divided into naive control (embryos maintained in distilled water), vehicle control (embryos treated with 2% DMSO), and drug groups. Stock solutions of all drugs were prepared in 2% DMSO as a solubilizing agent and diluted to three concentrations (0.5 mg/mL, 1 mg/mL, and 2 mg/mL). Each group had 20 embryos per test concentration. The zebrafish embryos were maintained in an incubator at 28 ◦C and read at 72 hpf for their mortality and teratogenicity (including non-hatching, egg condensation, spinal curvature, pericardial enlargement, etc.). Each compound was evaluated in three independent biological experiments.

(b) Angiogenesis assay

The 24 hpf zebrafish embryos were dechorionated with a 1 gL−<sup>1</sup> pronase treatment and maintained in distilled water in 12-well cell culture plates (each well contained 20 embryos). A negative control group containing zebrafish embryos in distilled water and a vehicle treatment group that was treated with 2% dimethyl sulfoxide (DMSO) were prepared. The anti-angiogenic compounds were diluted to 1 mg/mL. After incubating at 28 ◦C for 72 h, the embryos were immersed in 4% paraformaldehyde and dehydrated by gradient ethanol. Subsequently, the embryos were balanced in NTMT buffer (5 M NaCl + 1 M Tris [pH 9.0–9.5] + 1 M MgCl2+ 10% Tween), and nitrotetrazolium blue chloride (NBT) and p-toluidine salt (BCIP) staining were performed. To evaluate the effect of compounds on the angiogenesis of zebrafish embryos, the growth of embryonic sub-intestinal veins (SIVs) at 72 hpf was observed using an IX71 Olympus microscope (Hamburger, Germany). The length of the SIVs was calculated using the image J 1.8.0 software (Bethesda, MD, USA).

#### **4. Conclusions**

A new series of acridine-triazole and acridine-thiadiazole derivatives were synthesized and characterized by spectral studies. All the synthesized compounds were evaluated for their in vitro cytotoxic activities against HFF, MGC-803, BEL-7404, NCI-H460, and T24 by the MTT assay method. Most of the compounds were sensitive to MGC-803 and T24 cell lines. Compared to all the prepared compounds, **4a**, **5d** and **6h** exhibited the best anticancer activity against MGC-803 cell lines, and compounds **4h**, **5h** and **6h** showed the most excellent antitumor activity against T24. Preliminary studies of antitumor mechanisms revealed that the representative compounds (**5d** and **6h** or **4h** and **6h**) could suppress cell proliferation by inducing apoptosis in the Q3 period of MGC-803 or T24 cell lines. Compound **5d** might inhibit the growth of tumor cells by arresting cells in the G2 phase, while compound **4h** had a great effect on the S phase. In the zebrafish experiment, compound **5d** displayed a superior antiangiogenic effect and lower toxicity than other compounds. Therefore, compound **5d** has the potential to be an antitumor drug with high efficiency and low toxicity.

**Author Contributions:** Contributed to the synthesis of compounds, L.H., X.L. and H.X.; performed pharmacological activity testing and docking studies of the target products, R.C., Y.N., Z.J. and Y.L.; wrote the manuscript, L.H. and R.C.; supervision of the project and design of experiment, L.H., R.C. and R.L.; contributed to improvising the manuscript, provided intellectual inputs, and edited the language of the manuscript, Y.J. and L.W. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work acknowledges the support of the National Natural Science Foundation of China (22067001) and the Inheritance and innovation team of Guangxi Traditional Chinese Medicine (2022B005). We also thank the Guangxi Key Laboratory of Zhuang and Yao Ethnic Medicine [(2013) No. 20], the Collaborative Innovation Center of Zhuang and Yao Ethnic Medicine [(2014) No. 32], the Guangxi Key Discipline Zhuang Pharmacology ((2013) No. 16), the First-class Discipline in Guangxi of traditional Chinese Pharmacology (Direction of Ethnic Medicine) ((2018) No. 12) and Guangxi Joint Graduate education Base (Guangxi Degree (2021) No. 6).

**Institutional Review Board Statement:** There are no any ethical issues with animals.

**Informed Consent Statement:** These studies were not involving humans.

**Data Availability Statement:** We have presented all of our main data in the form of tables and figures. CCDC 2214949 contain supplementary crystallographic data for compound **5b** and CCDC 2214923 contain supplementary crystallographic data for compound **6d**. These datas can be obtained free of charge via http://www.ccdc.cam.ac.uk/conts/retrieving.html (accessed on 20 December 2022) or the Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; Fax: +44-1223-336-033; or e-mail: deposit@ccdc.cam.ac.uk.

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

#### **References**


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