*Article* **Design and Synthesis of Acridine-Triazole and Acridine-Thiadiazole Derivatives and Their Inhibitory Effect against Cancer Cells**

**Lini Huo 1,†, Xiaochen Liu 1,†, Yogini Jaiswal <sup>2</sup> , Hao Xu <sup>1</sup> , Rui Chen 1,\*, Rumei Lu 1,\*, Yaqin Nong <sup>1</sup> , Leonard Williams <sup>2</sup> , Yan Liang <sup>3</sup> and Zhiruo Jia <sup>1</sup>**


**Abstract:** We report herein the design and synthesis of a series of novel acridine-triazole and acridinethiadiazole derivatives. The newly synthesized compounds and the key intermediates were all evaluated for their antitumor activities against 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). Most of the compounds exhibited high levels of antitumor activity against MGC-803 and T24 but low toxicity against human normal liver cells (LO2), and their effect was even better than the commercial anticancer drugs, 5-fluorouracil (5-FU) and cis-platinum. Further, pharmacological mechanisms such as topo I, cell cycle, cell apoptosis, and neovascularization were all evaluated. Only a few compounds exhibited potent topo I inhibitory activity at 100 µM. In addition, the most active compounds with an IC<sup>50</sup> value of 5.52–8.93 µM were chosen, and they could induce cell apoptosis in the G2 stage of MGC-803 or mainly arrest T24 cells in the S stage. To our delight, most of the compounds exhibited lower zebrafish cytotoxicity but could strongly inhibit the formation of zebrafish sub-intestinal veins, indicating a potential for clinical application.

**Keywords:** acridine-triazole; acridine-thiadiazole; topoisomerase I; anti-angiogenesis; zebrafish

#### **1. Introduction**

Today, cancer is one of the major health problems in the world. With the development of molecular biology and molecular pharmacology, the pathogenesis of cancer is being explored at the gene level. Pharmacological mechanisms such as signal transduction, neovascularization, telomerase, topoisomerase, cell cycle and cell apoptosis have major impacts on cancerous cells and can be used as targets in cancer therapy [1].

Acridines are an important classe of nitrogen-containing heterocyclic compounds. Due to their structural characteristics as planar tricyclic aromatic molecules, acridines intercalate tightly but reversibly to the DNA helix [2,3]. These compounds reveal a wide variety of biological activities, including anticancer [4], antimicrobial [5,6], anti-acetylcholinesterase [7], etc. A number of acridine derivatives serve as chemotherapeutic agents, especially in the field of antitumor DNA-binding agents [8]. An example of one such compound is 9-amsacrine, which has been clinically used for the treatment of leukemia [9].

Due to their beneficial characteristics, triazole and thiadiazole derivatives can serve as potential antitumor agents and thus are of pharmaceutical interest. In drug development, the triazole ring is often used to replace the amino group to reduce the resistance of some anticancer drugs and enhance their anticancer activity [10]. Thiadiazole groups are

**Citation:** Huo, L.; Liu, X.; Jaiswal, Y.; Xu, H.; Chen, R.; Lu, R.; Nong, Y.; Williams, L.; Liang, Y.; Jia, Z. Design and Synthesis of Acridine-Triazole and Acridine-Thiadiazole Derivatives and Their Inhibitory Effect against Cancer Cells. *Int. J. Mol. Sci.* **2023**, *24*, 64. https://doi.org/ 10.3390/ijms24010064

Academic Editor: Laura Paleari

Received: 31 October 2022 Revised: 5 December 2022 Accepted: 6 December 2022 Published: 21 December 2022

**Copyright:** © 2022 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 (https:// creativecommons.org/licenses/by/ 4.0/).

commonly introduced in the design of anticancer drugs because of their high anticancer activity. Kumar et al. recently reported the synthesis and anticancer activity of a series of benzpyrole-thiadiazole derivatives and revealed the important role of the thiadiazole ring in cytotoxicity [11]. groups are commonly introduced in the design of anticancer drugs because of their high anticancer activity. Kumar et al. recently reported the synthesis and anticancer activity of a series of benzpyrole-thiadiazole derivatives and revealed the important role of the thiadiazole ring in cytotoxicity [11]. acridine-chlormethine hybrid (c) [15] (Figure 1).

such compound is 9-amsacrine, which has been clinically used for the treatment of

*Int. J. Mol. Sci.* **2022**, *23*, x FOR PEER REVIEW 2 of 21

such compound is 9-amsacrine, which has been clinically used for the treatment of

as potential antitumor agents and thus are of pharmaceutical interest. In drug development, the triazole ring is often used to replace the amino group to reduce the resistance of some anticancer drugs and enhance their anticancer activity [10]. Thiadiazole groups are commonly introduced in the design of anticancer drugs because of their high anticancer activity. Kumar et al. recently reported the synthesis and anticancer activity of a series of benzpyrole-thiadiazole derivatives and revealed the important role of the

Due to their beneficial characteristics, triazole and thiadiazole derivatives can serve

Designing hybrid drugs with multiple effects is a common strategy in the recent

search for new anticancer drugs [12]. In recent years, many structurally diverse hybrid molecules at the 9-position of the acridine skeleton have been reported for the enhancement of anti-cancer activity. Examples of such compounds include acridinemycophenolic acid hybrid (a) [13], acridine-thiazolidinedione hybrid [14] (b), and

Due to their beneficial characteristics, triazole and thiadiazole derivatives can serve as potential antitumor agents and thus are of pharmaceutical interest. In drug development, the triazole ring is often used to replace the amino group to reduce the resistance of some anticancer drugs and enhance their anticancer activity [10]. Thiadiazole

*Int. J. Mol. Sci.* **2022**, *23*, x FOR PEER REVIEW 2 of 21

thiadiazole ring in cytotoxicity [11].

leukemia [9].

leukemia [9].

Designing hybrid drugs with multiple effects is a common strategy in the recent search for new anticancer drugs [12]. In recent years, many structurally diverse hybrid molecules at the 9-position of the acridine skeleton have been reported for the enhancement of anticancer activity. Examples of such compounds include acridine-mycophenolic acid hybrid (a) [13], acridine-thiazolidinedione hybrid [14] (b), and acridine-chlormethine hybrid (c) [15] (Figure 1). Designing hybrid drugs with multiple effects is a common strategy in the recent search for new anticancer drugs [12]. In recent years, many structurally diverse hybrid molecules at the 9-position of the acridine skeleton have been reported for the enhancement of anti-cancer activity. Examples of such compounds include acridinemycophenolic acid hybrid (a) [13], acridine-thiazolidinedione hybrid [14] (b), and acridine-chlormethine hybrid (c) [15] (Figure 1).

**Figure 1.** Structures of some hybrid molecules (**a**–**c**) at the 9-position of the acridine skeleton. **Figure 1.** Structures of some hybrid molecules (**a**–**c**) at the 9-position of the acridine skeleton. thiadiazole nucleus to obtain a new class of compounds such as the acridine-triazole

Considering these facts, our strategy was to couple an acridine and a triazole or thiadiazole nucleus to obtain a new class of compounds such as the acridine-triazole hybrid or acridine-thiadiazole hybrid (Figure 2). The anticancer activities of the synthesized compounds were assessed based on various mechanisms of action and Considering these facts, our strategy was to couple an acridine and a triazole or thiadiazole nucleus to obtain a new class of compounds such as the acridine-triazole hybrid or acridine-thiadiazole hybrid (Figure 2). The anticancer activities of the synthesized compounds were assessed based on various mechanisms of action and molecular docking. hybrid or acridine-thiadiazole hybrid (Figure 2). The anticancer activities of the synthesized compounds were assessed based on various mechanisms of action and molecular docking.

**2. Results and Discussion Figure 2.** Strategy for the design of acridine-triazole or acridine-thiadiazole hybrids. **Figure 2.** Strategy for the design of acridine-triazole or acridine-thiadiazole hybrids.

#### *2.1. Chemistry* **2. Results and Discussion**

#### The general synthetic approach for aroyl thiourea derivatives (**4**), acridinyl 1,2,4- **2. Results and Discussion** *2.1. Chemistry*

Scheme 1.

molecular docking.

triazole derivatives (**5**) and acridinyl 1,2,4-thiadiazole derivatives (**6**) is illustrated in Scheme 1. *2.1. Chemistry* The general synthetic approach for aroyl thiourea derivatives (**4**), acridinyl 1,2,4- The general synthetic approach for aroyl thiourea derivatives (**4**), acridinyl 1,2,4 triazole derivatives (**5**) and acridinyl 1,2,4-thiadiazole derivatives (**6**) is illustrated in Scheme 1.

triazole derivatives (**5**) and acridinyl 1,2,4-thiadiazole derivatives (**6**) is illustrated in

**Scheme 1.** Syntheses of acridinyl derivatives. Reagents and conditions: (i) Cu, K2CO3, 140 °C; (ii) POCl3, 140 °C; (iii) NaSCN/ tetrabutylammonium bromide; (iv) ; (v) Na2CO3, reflux, or 98% H2SO4, 0 °C. R2 C O NHNH2 **Scheme 1.** Syntheses of acridinyl derivatives. Reagents and conditions: (i) Cu, K2CO3, 140 ◦C; (ii)POCl3, 140 ◦C; (iii) NaSCN/ tetrabutylammonium bromide; (iv) **Scheme 1.** Syntheses of acridinyl derivatives. Reagents and conditions: (i) Cu, K2CO3, 140 °C; (ii) POCl3, 140 °C; (iii) NaSCN/ tetrabutylammonium bromide; (iv) ; (v) Na2CO3, reflux, or 98% H2SO4, 0 °C. R2 C O NHNH2; (v) Na2CO3, reflux, or98% H2SO4, 0 ◦C.

The target compounds of 1,2,4-triazolethiones (**5**) and 1,2,4-thiadiazoles (**6**) were synthesized by means of a ring closure reaction using aroyl thiourea derivatives (**4**) in sodium carbonate or concentrated sulfuric acid conditions, respectively. The synthesis of aroyl thiourea derivatives (**4**) was carried out according to the known procedure of the addition of substituted hydrazides to acridin-9-yl isothiocyanate (**3**). It is important to note that the precipitate **3a** is formed at room temperature, while **3b** needs to be cooled in an ice bath. The key intermediates (**4**) were obtained in 95% EtOH without purification with a yield of 73–92% w/w. The target compounds of 1,2,4-triazolethiones (**5**) and 1,2,4-thiadiazoles (**6**) were synthesized by means of a ring closure reaction using aroyl thiourea derivatives (**4**) in sodium carbonate or concentrated sulfuric acid conditions, respectively. The synthesis of aroyl thiourea derivatives (**4**) was carried out according to the known procedure of the addition of substituted hydrazides to acridin-9-yl isothiocyanate (**3**). It is important to note that the precipitate **3a** is formed at room temperature, while **3b** needs to be cooled in an ice bath. The key intermediates (**4**) were obtained in 95% EtOH without purification with a yield of 73–92% w/w. The target compounds of 1,2,4-triazolethiones (**5**) and 1,2,4-thiadiazoles (**6**) were synthesized by means of a ring closure reaction using aroyl thiourea derivatives (**4**) in sodium carbonate or concentrated sulfuric acid conditions, respectively. The synthesis of aroyl thiourea derivatives (**4**) was carried out according to the known procedure of the addition of substituted hydrazides to acridin-9-yl isothiocyanate (**3**). It is important to note that the precipitate **3a** is formed at room temperature, while **3b** needs to be cooled in an ice bath. The key intermediates (**4**) were obtained in 95% EtOH without purification with a yield of 73–92% *<sup>w</sup>*/*w*.

As expected, auto-condensation cyclization proceeded effectively in the refluxing condition of 5% Na2CO3 or 98% concentrated sulfuric acid in an ice bath. It is reported that acridinyl 1,2,4-triazole derivatives (**5**) possibly exist in one of two tautomeric forms (Figure 3), thione (**a**) or thiol (**b**) [16]. And the thione form (**a**) was established by comparison of the HSQC and HMBC spectra and DFT calculations. To further confirm the structure of our synthesized products, a single crystal of compound **5b** was cultivated in absolute ethyl alcohol, and the molecular structure was confirmed as indicated in Figure 3c. The corresponding single crystal structural data for compound **5b** is provided in the supporting information (CCDC 2214949). As expected, auto-condensation cyclization proceeded effectively in the refluxing condition of 5% Na2CO3 or 98% concentrated sulfuric acid in an ice bath. It is reported that acridinyl 1,2,4-triazole derivatives (**5**) possibly exist in one of two tautomeric forms (Figure 3), thione (**a**) or thiol (**b**) [16]. And the thione form (**a**) was established by comparison of the HSQC and HMBC spectra and DFT calculations. To further confirm the structure of our synthesized products, a single crystal of compound **5b** was cultivated in absolute ethyl alcohol, and the molecular structure was confirmed as indicated in Figure 3c. The corresponding single crystal structural data for compound **5b** is provided in the supporting information (CCDC 2214949). As expected, auto-condensation cyclization proceeded effectively in the refluxing condition of 5% Na2CO<sup>3</sup> or 98% concentrated sulfuric acid in an ice bath. It is reported that acridinyl 1,2,4-triazole derivatives (**5**) possibly exist in one of two tautomeric forms (Figure 3), thione (**a**) or thiol (**b**) [16]. And the thione form (**a**) was established by comparison of the HSQC and HMBC spectra and DFT calculations. To further confirm the structure of our synthesized products, a single crystal of compound **5b** was cultivated in absolute ethyl alcohol, and the molecular structure was confirmed as indicated in Figure 3c. The corresponding single crystal structural data for compound **5b** is provided in the supporting information (CCDC 2214949).

R1

N

R2 S

NHN

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N

N

R2 SH

NN

**Figure 3.** The molecular structure of compound **5b. Figure 3.** The molecular structure of compound **5b**. temperature and reaction time. The reaction temperature had to be maintained below 0 10 atom of the acridinyl moiety captured a proton and thus resulted in the formation of a 9′,10′-dihydroacridine structure (b, Figure 4), which was verified through X-ray

The success of the cyclization of compound **6** mainly depended on reaction temperature and reaction time. The reaction temperature had to be maintained below 0 °C. When R2 was an electron-withdrawing group such as pyridyl and nitrophenyl, the reaction time had to be extended almost to 48 h. Interestingly, the final structure of compound **6** was not the desired acridine skeleton (a, Figure 4) for the compound. The N-10 atom of the acridinyl moiety captured a proton and thus resulted in the formation of a 9′,10′-dihydroacridine structure (b, Figure 4), which was verified through X-ray crystallographic analysis (c). The corresponding single crystal structural data of compound **6d** is provided in the supporting information (CCDC 2214923). The exchangeable NH protons of acridine thiosemicarbazides are reported in the literature The success of the cyclization of compound **6** mainly depended on reaction temperature and reaction time. The reaction temperature had to be maintained below 0 ◦C. When R<sup>2</sup> was an electron-withdrawing group such as pyridyl and nitrophenyl, the reaction time had to be extended almost to 48 h. Interestingly, the final structure of compound **6** was not the desired acridine skeleton (a, Figure 4) for the compound. The N-10 atom of the acridinyl moiety captured a proton and thus resulted in the formation of a 90 ,100 -dihydroacridine structure (b, Figure 4), which was verified through X-ray crystallographic analysis (c). The corresponding single crystal structural data of compound **6d** is provided in the supporting information (CCDC 2214923). The exchangeable NH protons of acridine thiosemicarbazides are reported in the literature (Figure 5) [16]. °C. When R2 was an electron-withdrawing group such as pyridyl and nitrophenyl, the reaction time had to be extended almost to 48 h. Interestingly, the final structure of compound **6** was not the desired acridine skeleton (a, Figure 4) for the compound. The N-10 atom of the acridinyl moiety captured a proton and thus resulted in the formation of a 9′,10′-dihydroacridine structure (b, Figure 4), which was verified through X-ray crystallographic analysis (c). The corresponding single crystal structural data of compound **6d** is provided in the supporting information (CCDC 2214923). The exchangeable NH protons of acridine thiosemicarbazides are reported in the literature (Figure 5) [16]. crystallographic analysis (c). The corresponding single crystal structural data of compound **6d** is provided in the supporting information (CCDC 2214923). The exchangeable NH protons of acridine thiosemicarbazides are reported in the literature (Figure 5) [16]. R'

The success of the cyclization of compound **6** mainly depended on reaction

R1

**5b**

R'

N

(Figure 5) [16].

**6d 6d** R S N

**6d**

**a acridine skeleton b 9',10'-dihydroacridine structure c single-crystal structure**

activities in comparison to the reference compounds, 5-FU and cis-platinum. Compounds

**a acridine skeleton b 9',10'-dihydroacridine structure c single-crystal structure Figure 4.** The molecular structure of compound **6d. Figure 4.** The molecular structure of compound **6d**. **Figure 4.** The molecular structure of compound **6d.** 

N H

**Figure 5.** Reported structure of acridine thiosemicarbazides [13]. *2.2. In-Vitro Anticancer Activity Assay and Structure-Activity Analysis* **Figure 5.** Reported structure of acridine thiosemicarbazides [13]. **Figure 5.** Reported structure of acridine thiosemicarbazides [13].

*2.2. In-Vitro Anticancer Activity Assay and Structure-Activity Analysis* All newly synthesized acridinyl derivatives (**4**–**6**) were screened for their anticancer *2.2. In-Vitro Anticancer Activity Assay and Structure-Activity Analysis*

All newly synthesized acridinyl derivatives (**4**–**6**) were screened for their anticancer activities in comparison to the reference compounds, 5-FU and cis-platinum. Compounds activities in comparison to the reference compounds, 5-FU and cis-platinum. Compounds *2.2. In-Vitro Anticancer Activity Assay and Structure-Activity Analysis* All newly synthesized acridinyl derivatives (**4**–**6**) were screened for their anticancer All newly synthesized acridinyl derivatives (**4**–**6**) were screened for their anticancer activities in comparison to the reference compounds, 5-FU and cis-platinum. Compounds

**4**–**6** were tested for their in vitro antitumor activities against HFF, MGC-803, BEL-7404, NCI-H460, and T24 tumor cell lines, and human normal liver cells (LO2), and the results are shown in Table 1. Most of the compounds had strong selective potency against MGC-803 and T24 cancer cells. In the MGC-803 cell line assay, almost all of the compounds displayed better cytotoxicity than the positive control 5-FU (IC<sup>50</sup> = 30.45 ± 2.87 µM), with an IC<sup>50</sup> of 5.52–34.99 µM. This indicates that the introduction of the triazole and thiadiazole groups on the acridine skeleton could improve the antitumor activity against MGC-803. In addition, except for compounds **4c**, **4d**, **4e**, **4i**, **4j**, **5a**, **5g**, **6b**, **6d**, and **6i**, almost all of the compounds demonstrated better cytotoxicity inhibition than cis-platinum (IC<sup>50</sup> = 15.97 ± 1.53 µM). Particularly, the IC<sup>50</sup> values of compounds **5d**, **5g**, **5i**, **6g**, **6e**, and **6h** were all below 10 µM, and the IC<sup>50</sup> of them were 5.52 ± 1.04 µM, 8.5 ± 1.85 µM, 8.92 ± 0.99 µM, 9.01 ± 1.32 µM, 9.95 ± 1.03 µM, and 6.85 ± 0.84 µM, respectively. In the T24 cell line assay, many compounds, especially the series of compound **4**, had significant activity against T24. This implies that there is a significant increase in potency after the introduction of the aroyl thiourea group. Among these compounds, R<sup>1</sup> = -CH<sup>3</sup> and R<sup>2</sup> = -OCH<sup>3</sup> might help to improve the antitumor activity of acridine nuclear, such as compounds **4h**, **5h** and **6h**, all of which exhibited the best inhibition compared with other analogues, with IC<sup>50</sup> values of 8.05 ± 1.06, 11.25 ± 1.16, and 8.93 ± 1.25 µM, respectively. In particular, compounds **4h** and **6h** had better antitumor activities than the two commercial anticancer drugs 5-FU (IC<sup>50</sup> = 32.04 ± 1.23) and cis-platinum (IC<sup>50</sup> = 9.13 ± 1.54 µM). To our delight, most 1,2,4-triazolethiones (**5**) and 1,2,4-thiadiazoles (**6**) have low toxicity to LO2 compared with the positive control. Compounds **5d** and **6h** were the most active but had lower toxicities than 5-FU and *cis*-platinum. Therefore, compounds **5d** and **6h** or **4h** and **6h** exhibited good cytotoxicity inhibition against MGC-803 or T24 cancer cells and were selected for further exploration to identify their mechanisms of cancer cell growth inhibition.




**Table 1.** *Cont*.
