Development of 1,2,3‐Triazolopyridazinone Derivatives as Potential Caspase 3 and Apoptosis Inducers: Design, Synthesis and Anticancer Activity Studies

ABSTRACT

Herein, the synthesis, anticancer activity and apoptotic pathways of 1,2,3‐triazolopyridazinones compounds, which are similar to DNA bases not previously found in the literature have been investigated. To achieve this goal, it is designed the hybrid molecules combining triazole and pyridazinone/pyridazithione structures, bearing a lipophilic group (benzyl/phenyl) at the one position and benzene with electron withdrawing or donating groups at five positions, with high pharmacophoric properties on the same scaffold structure. The representative compounds in this series 5a, 5c, 6a and 8c exhibited higher anticancer activity than other compounds and cisplatin control against breast (MCF‐7) and lung (A549) cell lines. These compounds were less toxic when tested against the noncancerous L929 cell line. In addition, the apoptotic effect mechanisms of these compounds were confirmed by AO/EB staining and caspase 3 activity results. These findings indicate that some derivatives of these compounds could be effective therapeutic agents for the treatment of cancer disease with an apoptosis‐promoting.

Herein, the synthesis, anticancer activity and apoptotic pathways of 1,2,3‐triazolopyridazinones compounds, which are similar to DNA bases not previously found in the literature have been investigated. The representative compounds in this series 5a, 5c, 6a and 8c exhibited higher anticancer activity than other compounds and cisplatin control against breast (MCF‐7) and lung (A549) cell lines. These compounds were less toxic when tested against the noncancerous L929 cell line.

1. Introduction

Apoptosis is a tightly regulated process that involves a cascade of molecular events. It plays important roles in the development and maintenance of homeostasis, as well as in the elimination of damaged or unnecessary cells. Identifying and developing pharmaceutical agents that selectively modulate apoptotic pathways may provide an effective strategy for preventing and treating many diseases. It has been suggested in various studies that signals leading to the activation of caspases, a family of intracellular cysteine proteases, may play a crucial role in the initiation and execution of apoptosis induced by different stimuli. Caspase 3, previously known as CPP32/Yama/Apopain, is among the most well‐characterized caspases. Caspase 3 is synthesized as an inactive proenzyme that requires proteolytic activation. Its activation may involve another aspartate‐specific protease. Furthermore, caspase 3 can act as a general mediator of apoptosis in various cells and tissues [1, 2]. In this context, scientific advancements in the fight against cancer focus intensively on understanding this complexity at the genetic and cellular levels [3, 4, 5, 6, 7]. The goal is to develop more effective strategies such as caspase 3 and apoptosis inducer for the treatment and prevention of this disease.

It is reported that the chemistry of pyridazinones has been a fascinating ring structure in nowadays. Pyridazinone rings have become target molecules in pharmaceutical chemistry due to their structural similarity to DNA bases. You can rather say previous studies indicated that many pyridazine‐based molecules have been extensively investigated for a broad array of biological activities, such as antiviral, antioxidant, anticancer, and so on [8, 9, 10, 11, 12, 13, 14, 15]. Especially, many studies put forward that pyridazin‐3‐ones and their derivatives can be considered as anticancer and anticardiovascular agents [16, 17, 18, 19]. Anticancer properties of pyridazinone structures and their derivatives have investigated in the literature. For example, anticancer properties of 30 different pyridazino[4,5‐b]indole derivatives containing alkyl‐, benzyl‐ and phenacyl‐derived 1,2,3‐triazolylmethyl units were investigated on MDA‐MB‐231, MCF7, U‐87 and IMR‐32 cell lines. Especially, the IC₅₀ values for the MCF7 cell were found to be between 0.5 and 38 µM, which are considerable results [20]. In another study, a series of benzimidazoles were synthesized and their cytotoxic and apoptotic effects were investigated using MCF7 and HEK‐293 cell lines. It has been revealed that HEK‐293 is not effective and has an effect on breast cancer [21]. Similar to our previous study, benzimidazole derivatives were studied in MCF7 human breast cancer and A549 human lung cancer cell lines. The IC50 values affected cell viability in the range of 18–120 µg/mL in MCF7 and in the range of 20–80 µg/mL in A549 [22]. Xiong at al. investigated anticancer properties of pridinamidtriazole derivatives on MCF7, HeLa and A549 cell lines. The IC₅₀ values were found at the range of 19–358 and 42–332 µM for A549 and MCF7, respectively [23]. These data show that 1,2,3‐triazolopyridazinone/pyridazithione derivatives have exhibited anticancer properties.

Sulfur structure is an important field with applications ranging from pharmaceuticals to materials science. As a result of the interaction of pyridazin‐3‐one molecules with sulfurizing reagents, pyridazine‐3‐thione structures are obtained. These molecules, like pyridazin‐3‐ones, are active compounds in pharmacological structures [24, 25, 26]. Their structural diversity and reactivity make them valuable candidates for further investigation in the quest for new therapeutic agents. These compounds may exhibit various biological effects, such as antimicrobial, antiviral, anticancer, or antioxidant properties. Additionally, pyridazine‐3‐thione derivatives provide the basis for designing novel drugs targeting specific biological pathways or disease conditions [27].

Here, we designed 1,2,3‐triazolopyridazinone/pyridazithione molecules, which are structures similar to DNA bases that were not previously available in the literature. We determined the molecules we designed as hybrid cores that combine triazole and pyridazinone/pyridazithone structures with high pharmacophoric properties on the same skeletal structure. We conducted extensive research and experiments on the designed skeletal structures, both synthetically and in terms of their biological activity. In these hybrid core structures, lipophilic groups (benzyl and phenyl) are present in the 1‐position, aromatic rings with electron‐withdrawing or releasing groups at position 5 were preferred. While lipophilic tips were preferred so that the cells used during anticancer screenings could easily pass through the fat tissue, triazolopyridazinone/pyridazithione hybrid structures with electron‐withdrawing and releasing groups were preferred to show effective pharmacophoric properties through intermolecular interactions within the cell. The main focus of the synthesis designs of these core structures was to create purine‐like structures that could show anticancer activity. The findings showed that the designed structures indeed exhibited promising anticancer activity.

2. Experimental Section

2.1. Material Method

All starting compounds were obtained from Sigma, Fluka and Merck. The reagent grade solvents used in the purification step were purified before use. For the synthesis of the precursor compounds 1‐aryl‐5‐formyl‐1H‐1,2,3‐triazole‐4‐carboxylate (1 and 2), Yagiz et al. and Erdemir et al. were followed [28, 29, 30, 31]. The reaction's progress was monitored using thin‐layer chromatography (TLC), which was performed on aluminum plates coated with silica gel 60 F254. The product was purified using Kieselgel 60 column chromatography. Mass spectrometry (using HRMS‐TOF or HRMS‐QTOF), infrared (IR) and nuclear magnetic spectroscopy (¹H and ¹³C‐APT NMR) were used to confirm the identity and purity of the produced compounds. The melting points of the derivatives were recorded by the STUART (SMP‐30) apparatus.

2.2. General Procedures for 1,2,3‐Triazolopyridazin‐4‐one (5a‐e and 6a‐e) Derivatives

Phenyl hydrazines that are p‐substituted were gradually added to the agitated solution of methyl 1‐aryl‐5‐formyl‐1H‐1,2,3‐triazole‐4‐carboxylate (1.0 equiv.) in tetrahydrofuran (THF) (1.1 equiv.). Following the addition process, the reaction mixture was heated for 3 h at 60°C. The reaction mixture was added to cold water after 3 h, and the solids from 3a–e and 4a–e were filtered out. Following the dissolution of these 3a–e and 4a–e compounds (1.0 equiv.) in toluene‐THF, para‐toluenesulfonic acid (p‐TsOH) in THF was added gradually and heated at 100°C for 2–7 days, depending on the substituent. Volatiles were eliminated using a rotary evaporator when the reaction was finished (monitored by TLC), and the crude residue was then applied using column chromatography to get the intended products (5a‐e and 6a‐e) [32, 33]. They were purified by column chromatography from the EtOAc:Hexane (40:60, v/v) solvent system using silica gel.

2.3. General Procedures for 1,2,3‐Triazolopyridazin‐4‐thione (7a‐d and 8a‐d) Derivatives

In inert atmosphere, 1,2,3‐triazolopyridazinone derivatives (5a‐d and 6a‐d) (1.0 mmol) were dissolved in 3 mL of toluene. Lawesson's reagent (LR) (2.1 mmol) was then added to this solution. The reaction mixture was heated under a reflux condenser for 24 h. After the reaction was complete, it was cooled and poured into a solution of ice‐saturated ammonium chloride. The resulting mixture was diluted with water and extracted twice with ethyl acetate. The organic phases were collected, and the solvent was removed. The obtained derivatives 7a‐d and 8a‐d were purified by column chromatography on silica gel using an ethyl acetate/hexane solvent system, starting from 20% and increasing to 60% [34, 35].

1‐Benzyl‐5‐phenyl‐1,5‐dihydro‐4 H ‐[1–3]triazolo[4,5‐ d ]pyridazin‐4‐one (5a): White solid. 71%, mp. 132°C–133°C. IR (ATR, cm ^‐1 ): 3063, 2922, 1699, 1596. ¹ H NMR (300 MHz, CDCl ₃ ) δ 7.92 (s, 1H, ‐N = C‐H), 7.55–7.26 (m, 10H, Ar‐H), 5.87 (s, 2H, ‐CH₂‐), ¹³ C‐APT NMR (75 MHz, CDCl ₃ ) δ 155.2, 140.9, 140.6, 133.0, 132.4, 129.5, 129.4, 128.9, 128.3, 128.2, 125.9, 124.0, 53.8. HRMS (ESI‐TOF) m/z: [M + H]⁺ calculated C₁₇H₁₄N₅O⁺: 304.1192, found: 304.1193.

1‐Benzyl‐5‐(4‐flourophenyl)‐1,5‐dihydro‐4 H ‐[1–3]triazolo[4,5‐ d ]pyridazin‐4‐one (5b): White solid. 68%, mp. 135°C–136°C. IR (ATR, cm ^‐1 ): 3048, 1697, 1603, 1556. ¹ H NMR (300 MHz, CDCl ₃ ) δ 7.93 (s, 1H, ‐N = C‐H), 7.52 (dd, J = 8.9, 4.8 Hz, 2H, Ar‐H), 7.45–7.36 (m, 5H, Ar‐H), 7.14 (t, J = 8.6 Hz, 2H, Ar‐H), 5.87 (s, 2H, ‐CH₂‐), ¹³ C‐APT NMR (75 MHz, CDCl ₃ ) δ 161.9 (d, J = 248.3 Hz), 155.1, 140.5, 136.8, 132.9, 132.4, 129.6, 129.5, 128.2, 127.7 (d, J = 8.5 Hz), 124.0, 115.7 (d, J = 23.1 Hz), 53.8. HRMS (ESI‐TOF) m/z: [M + H]⁺ calculated C₁₇H₁₃N₅OF⁺: 322.1099, found: 322.1117.

1‐Benzyl‐5‐(4‐methylphenyl)‐1,5‐dihydro‐4 H ‐[1–3]triazolo[4,5‐ d ]pyridazin‐4‐one (5c): Yellow solid. 54%, mp. 142°C–143°C. IR (ATR, cm ^‐1 ): 3035, 2920, 1691, 1549, 1511. ¹ H NMR (300 MHz, CDCl ₃ ) δ 7.91 (s, 1H, ‐N = C‐H), 7.44 (m, 7H, Ar‐H), 7.26 (d, J = 8.2 Hz, 2H, Ar‐H), 5.86 (s, 2H, ‐CH₂‐), 2.39 (s, 3H, ‐CH₃), ¹³ C‐APT NMR (75 MHz, CDCl ₃ ) δ 155.2, 144.7, 140.7, 138.4, 132.9, 132.4, 129.6 (2 C), 129.5, 128.2, 125.6, 123.5, 53.8, 21.2. HRMS (ESI‐TOF) m/z: [M + H]⁺ calculated C₁₈H₁₆N₅O⁺: 318.1349, found: 318.1357.

1‐Benzyl‐5‐(4‐methoxyphenyl)‐1,5‐dihydro‐4 H ‐[1–3]triazolo[4,5‐ d ]pyridazin‐4‐one (5d): White solid. 50%, mp. 148°C–49°C. IR (ATR, cm ^‐1 ): 3077, 2951, 1677, 1586. ¹ H NMR (300 MHz, CDCl ₃ ) δ 7.91 (s, 1H, ‐N = C‐H), 7.45–7.34 (m, 5H, Ar‐H), 7.44 (d, J = 8.9 Hz, 2H, Ar‐H), 6.97 (d, J = 9.0 Hz, 2H, Ar‐H), 5.86 (s, 2H, ‐CH₂‐), 3.84 (s, 3H, ‐OCH₃), ¹³ C‐APT NMR (75 MHz, CDCl ₃ ) δ 158.6, 154.6, 139.9, 133.2, 132.3, 131.7, 128.9, 128.8, 127.5, 126.4, 122.9, 113.4, 54.9, 53.1. HRMS (ESI‐TOF) m/z: [M + H]⁺ calculated C₁₈H₁₆N₅O₂ ⁺: 334.1299, found: 334.1307.

1,5‐Diphenyl‐1,5‐dihydro‐4 H ‐[1–3]triazolo[4,5‐ d ]pyridazin‐4‐one (6a): White solid. 48%, mp. 189°C–190°C. IR (ATR, cm ^‐1 ): 3056, 2956, 2852, 1701, 1596, 1556. ¹ H NMR (300 MHz, CDCl ₃ ) δ 8.47 (s, 1H, ‐N = C‐H), 7.79–7.44 (m, 10H, Ar‐H), ¹³ C‐APT NMR (75 MHz, CDCl ₃ ) δ 149.5, 135.3, 135.0, 129.8, 126.2, 124.9, 124.8, 123.4, 122.9, 120.3, 118.4, 117.5. HRMS (ESI‐TOF) m/z: [M + H]⁺ calculated C₁₆H₁₂N₅O⁺: 290.1036, found: 290.1047.

5‐(4‐Fluorophenyl)‐1‐phenyl‐1,5‐dihydro‐4 H ‐[1–3]triazolo[4,5‐ d ]pyridazin‐4‐one (6b): White solid. 55%, mp. 191°C–192°C. IR (ATR, cm ^‐1 ): 3066, 2923, 1708, 1604, 1556. ¹ H NMR (300 MHz, CDCl ₃ ) δ 8.46 (s, 1H), 7.75 (d, J = 8.3 Hz, 2H, Ar‐H), 7.70–7.58 (m, 5H, Ar‐H), 7.20 (d, J = 8.4 Hz, 2H, Ar‐H), ¹³ C‐APT NMR (75 MHz, CDCl ₃ ) δ 166.8 (d, J = 248.6 Hz), 159.8, 145.2, 141.5, 140.0, 136.6, 135.3, 135.2, 132.5 (d, J = 8.7 Hz), 128.9, 127.8, 120.6 (d, J = 23.0 Hz). HRMS (ESI‐TOF) m/z: [M + H]⁺ calculated C₁₆H₁₁N₅OF⁺: 308.0942, found: 308.0956.

5‐(4‐Methylphenyl)‐1‐phenyl‐1,5‐dihydro‐4 H ‐[1–3]triazolo[4,5‐ d ]pyridazin‐4‐one (6c): White solid. 40%, mp. 186°C–188°C. IR (ATR, cm ^‐1 ): 3065, 2923, 1699, 1591, 1507. ¹ H NMR (300 MHz, CDCl ₃ ) δ 8.45 (s, 1H, ‐N = C‐H), 7.78–7.63 (m, 5H, Ar‐H), 7.48 (d, J = 8.5 Hz, 2H, Ar‐H), 7.31 (d, J = 8.7 Hz, 2H, Ar‐H), 2.42 (s, 3H, ‐CH₃), ¹³ C‐APT NMR (75 MHz, CDCl ₃ ) δ 155.1, 140.5, 138.4, 138.4, 135.3, 131.7, 130.4, 130.4, 129.5, 125.6, 123.8, 123.0, 21.1. HRMS (ESI‐TOF) m/z: [M + H]⁺ calculated C₁₇H₁₄N₅O⁺: 304.1193, found: 304.1207.

5‐(4‐Methoxyphenyl)‐1‐phenyl‐1,5‐dihydro‐4 H ‐[1–3]triazolo[4,5‐ d ]pyridazin‐4‐one (6d): White solid. 45%, mp. > 300°C‐Decomposed. IR (ATR, cm ^‐1 ): 3067, 2967, 2835, 1683, 1605, 1590. ¹ H NMR (300 MHz, DMSO‐d ₆ ) δ 8.86 (s, 1H, ‐N = C‐H), 7.95–7.68 (m, 5H, Ar‐H), 7.48 (d, J = 8.9 Hz, 2H, Ar‐H), 7.08 (d, J = 9.0, 2H, Ar‐H), 3.83 (s, 3H, ‐OCH₃), ¹³ C‐APT NMR (75 MHz, DMSO‐d ₆ ) δ 162.4, 158.4, 143.3, 138.6, 137.5, 135.8, 133.8, 133.8, 131.1, 129.3, 127.0, 117.5, 59.0. HRMS (ESI‐TOF) m/z: [M + H]⁺ calculated C₁₇H₁₄N₅O₂ ⁺: 320.1142, found: 320.1156.

1‐Benzyl‐5‐phenyl‐1,5‐dihydro‐4 H ‐[1–3]triazolo[4,5‐ d ]pyridazine‐4‐thione (7a): Yellow solid. 54%, mp. 132°C–133°C. IR (ATR, cm ^‐1 ): 3035, 1582, 1492, 1187. ¹ H NMR (300 MHz, CDCl ₃ ) δ 8.05 (s, 1H, ‐N = C‐H), 7.53–7.35 (m, 10H, Ar‐H), 5.88 (s, 2H, ‐CH₂‐), ¹³ C‐APT NMR (75 MHz, CDCl ₃ ) δ 176.9, 149.6, 143.3, 131.9, 128.9, 128.9, 128.5, 128.3, 127.4, 126.1, 126.0, 124.6, 53.3. HRMS (ESI‐TOF) m/z: [M + H]⁺ calculated C₁₇H₁₄N₅S⁺: 320.0964, found: 320.0970.

1‐Benzyl‐5‐(4‐flourophenyl)‐1,5‐dihydro‐4 H ‐[1–3]triazolo[4,5‐ d ]pyridazine‐4‐thione (7b): Yellow solid. 56%, mp. 124°C–125°C. IR (ATR, cm ^‐1 ): 3035, 2918, 1601, 1582, 1192. ¹ H NMR (300 MHz, CDCl ₃ ) δ 7.99 (s, 1H), 7.38–7.27 (m, 7H, Ar‐H) 7.12 (m, 2H, Ar‐H), 5.79 (s, 2H, ‐CH₂‐), ¹³ C‐APT NMR (75 MHz, CDCl ₃ ) δ 176.9, 161.5 (d, J = 247.5 Hz) 149.5, 139.1, 131.7, 128.8, 128.8, 128.0, 127.3, 126.1, 124.4, 115.4 (d, J = 23.1 Hz), 53.3. HRMS (ESI‐TOF) m/z: [M + H]⁺ calculated C₁₇H₁₃N₅SF⁺: 338.0870, found: 338.0880.

1‐Benzyl‐5‐(4‐methylphenyl)‐1,5‐dihydro‐4 H ‐[1–3]triazolo[4,5‐ d ]pyridazine‐4‐thione (7c): Yellow solid. 66%, mp. 134°C–135°C. IR (ATR, cm ^‐1 ): 3030, 2918, 1579, 1508, 1180. ¹ H NMR (300 MHz, CDCl ₃ ) δ 8.05 (s, 1H, Ar‐H), 7.45–7.30 (m, 9H, Ar‐H), 5.87 (s, 2H, ‐CH₂‐), 2.41 (s, 3H, ‐CH₃), ¹³ C‐APT NMR (75 MHz, CDCl ₃ ) δ 177.6, 150.3, 141.7, 139.2, 132.8, 129.9, 129.6, 129.6, 128.2, 127.1, 126.5, 125.4, 54.0, 21.3. HRMS (ESI‐TOF) m/z: [M + H]⁺ calculated C₁₈H₁₆N₅S⁺: 334.1121, found: 334.1131.

1‐Benzyl‐5‐(4‐methoxyphenyl)‐1,5‐dihydro‐4 H ‐[1–3]triazolo[4,5‐ d ]pyridazine‐4‐thione (7d): Red solid. 36%, mp. 136°C–138°C. IR (ATR, cm ^‐1 ): 3042, 2919, 1595, 1505, 1190. ¹ H NMR (300 MHz, CDCl ₃ ) δ 8.09 (s, 1H), 7.43–7.36 (m, 5H), 7.33 (d, J = 8.0 Hz, 2H), 6.99 (d, J = 8.0 Hz, 2H), 5.87 (s, 2H), 3.84 (s, 3H), ¹³ C‐APT NMR (75 MHz, CDCl ₃ ) δ 177.1, 159.1, 149.6, 136.4, 132.1, 129.0, 127.5, 127.3, 126.3, 124.7, 113.7, 54.9, 53.4. HRMS (ESI‐TOF) m/z: [M + H]⁺ calculated C₁₈H₁₆N₅OS⁺: 350.1070, found: 350.1073.

1,5‐Diphenyl‐1,5‐dihydro‐4 H ‐[1–3]triazolo[4,5‐ d ]pyridazine‐4‐thione (8a): Yellow solid. 41%, mp. 162°C–163°C. IR (ATR, cm ^‐1 ): 3067, 2919, 1594, 1574, 1214. ¹ H NMR (300 MHz, CDCl ₃ ) δ 8.62 (s, 1H, N = C‐H), 7.77–7.50 (m, 10H, Ar‐H), ¹³ C‐APT NMR (75 MHz, CDCl ₃ ) δ 176.9, 149.7, 143.4, 134,4, 129,9, 129.8, 128.7, 128.5, 126.3, 126.1, 124.1, 122.3. HRMS (ESI‐TOF) m/z: [M + H]⁺ calculated C₁₆H₁₂N₅S⁺: 306.0808, found: 306.0823.

5‐(4‐Fluorophenyl)‐1‐phenyl‐1,5‐dihydro‐4 H ‐[1–3]triazolo[4,5‐ d ]pyridazine‐4‐thione (8b): Yellow solid. 56%, mp. 164°C–165°C. IR (ATR, cm ^‐1 ): 3068, 2922, 1574, 1499, 1213. ¹ H NMR (300 MHz, CDCl ₃ ) δ 8.61 (s, 1H, N = C‐H), 7.76–7.68 (m, 4H, Ar‐H), 7.49‐7.45 (m, 3H, Ar‐H), 7.23‐7.21 (m, 2H, Ar‐H), ¹³ C‐APT NMR (75 MHz, CDCl ₃ ) δ 177.0, 163.3‐160.0 (d, J = 249.7 Hz), 139.2, 134.3, 129.9, 129.8, 128.1, 127.9, 126.4, 124.0, 122.3, 115.7‐115.4 (d, J = 23.1 Hz). HRMS (ESI‐TOF) m/z: [M + H]⁺ calculated C₁₆H₁₁N₅SF⁺: 324.0714, found: 324.0729.

5‐(4‐Methylphenyl)‐1‐phenyl‐1,5‐dihydro‐4 H ‐[1–3]triazolo[4,5‐ d ]pyridazine‐4‐thione (8c): Yellow‐orange solid. 38%, mp. 164°C–165°C. IR (ATR, cm ^‐1 ): 3063, 2919, 1500, 1219. ¹ H NMR (300 MHz, CDCl ₃ ) δ 8.61 (s, 1H, ‐N = C‐H), 7.77–7.26 (m, 9H, Ar‐H), 3.84 (s, 3H, ‐CH₃), ¹³ C‐APT NMR (75 MHz, CDCl ₃ ) δ 177.1, 141.1, 138.8, 136.5, 134.5, 130.0, 129.9, 129.4, 126.4, 125.9, 124.2, 122.5, 20.8, HRMS (ESI‐TOF) m/z: [M + H]⁺ calculated C₁₇H₁₄N₅S⁺: 320.0964, found: 320.0983.

2.4. Anticancer and Cytotoxicity Activity

A549 (Human Lung Cancer) and MCF7 (Human Breast Cancer) cell lines and L929 (Mouse Fibroblast Cell Line) obtained from Merck were cultured in culture medium containing sterilized Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal calf serum (FBS) and 1% antibiotic‐antimycotic solution. After the cells reached a certain proliferation number, samples and cisplatin were added at different concentrations and the cells were treated for 24 h. These solutions contained titrations of different concentrations used in the treatments applied to the cells. MTT (3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide) assay method was used to determine the effects of the materials. This assay was performed by measuring formazan production, which reflects the metabolic activity of cells, to determine cell viability. Cells incubated with MTT solution induced a reaction that led to the production of formazan after a certain period of time. This formazan production was measured by absorbing light at a wavelength of 540 nanometers with a spectrophotometer. These data were also used by calculating IC₅₀ values.

2.5. Acridine Orange/Ethidium Bromide (AO/EB) Staining and Imaging

AO/EB staining method was applied to evaluate the apoptotic effects of 5a, 5c, 6a and 8c materials, which exhibit the highest anticancer activity on cells. Green Nucleus (Acridine Orange): AO stains cell nuclei with green fluorescence. This indicates that the cells are healthy, alive and have complete nuclei. This green color is usually seen in normal cells. Red Nucleus (Ethidium Bromide): EB stains cell nuclei with red fluorescence. This can indicate that the cells are damaged or dead. EB can only enter damaged or dead cells and interact with DNA. Based on the results of AO/EB staining, the following comments can be made, If the green color is dominant. The majority of cells are healthy and live. If the red color is dominant: Some of the cells may be damaged or dead. If a yellow or orange color appears: This is a mixture of AO and EB and indicates that the cells have entered the apoptosis pathway or are slightly damaged. 1% AO/EB mixture was prepared from stock solutions in PBS buffer. The medium of the cells exposed to the IC₅₀ concentration for 24 h in the well plate was removed and washed once with PBS buffer. After removal of the PBS buffer, a 1% dye mixture was added to each sample at a depth of 2–3 mm, depending on the surface area of the samples, and removed after 1 min. Fluorescence was visualized using an inverted microscope [36] and color intensity were analyzed using Image J according to the following formula.

Red Intensity Percentage (Cell Death Percentage): To calculate the percentage of cell death, it was used the red intensity values (R_measured) from the cellular images. The red intensity value was normalized against 255 (the maximum red intensity, representing the most dead cells), and this ratio was used to calculate the cell death percentage. The calculation is as follows:

$$\mathit{Cell\; Death\; Percentage}=\left(\frac{{\mathit{R}}_{\mathit{measured}}}{\mathbf{255}}\right)\times \mathbf{100}$$

Cell Survival Percentage: To determine the cell survival rate, it was subtracted the cell death percentage from 100. This calculation represents the percentage of cells that remained healthy:

$$\mathit{Cell\; Survival\; Percentage}=\mathbf{100}-\mathit{Cell\; Death\; Percentage}$$

Normalized Cell Survival Percentage: To ensure that the results were comparable across different experimental conditions, it was normalized all survival percentages using the positive control (C_positive) and negative control (C_negative) values. This normalization process adjusted all the values to fall within the 0%–100% range:

$$\mathit{Normalized\; Cell\; Survival\; Percentage}=\left(\frac{\mathit{Cell\; Survival\; Percentage}-{\mathit{C}}_{\mathit{negative}}}{{\mathit{C}}_{\mathit{positive}}-{\mathit{C}}_{\mathit{negative}}}\right)\times \mathbf{100}$$

2.6. Caspase 3 Activity Analysis

Caspase 3 activity was examined to determine the apoptosis process in cells exposed to 5a, 5c, 6a and 8c samples at the IC₅₀ concentrations in A549 cell line. Caspase 3 activities of these cells were determined using an assay kit (Wuhan USCN Business Co. Ltd.) with a fluorescence spectrophotometer. Using GraphPad Prism 8, a one‐way analysis of variance (ANOVA) was performed to determine statistical significance at a p value of less than 0.05. Three independent tests (n = 3) were used to calculate the mean and standard deviation.

3. Results and Discussion

3.1. Synthetic Part

The synthetic design of the study is depicted in Scheme 1. Using the condensation and cyclization technique, a series of studies were carried out to create 1,2,3‐triazolopyridazin‐4‐one and subsequently 1,2,3‐triazolopyridazin‐4‐thione by using Lawesson's reagent (LR). Initially, the synthesis of the 1,2,3‐triazole skeleton structure was carried out. 4‐Formyl‐5‐carboxylate 1,2,3‐triazole main skeleton structure was obtained in three steps with around 75% yields by the method developed by our group [32, 33, 34]. This step is important in terms of eliminating the cytotoxicity problem caused by undesirable metal catalyst residues, especially in pharmacophoric studies, which was introduced to the literature by our group, in which the 1,2,3‐triazole ring containing an asymmetric carbonyl group is obtained without a metal catalyst. Then, molecules 3a‐e and 4a‐e were interacted with p‐substituted phenyl hydrazines in DMF medium to obtain the designed purine‐like structures. This reaction is a two‐stage condensation reaction sequence: firstly, hydrazone derivatives are formed between the formyl group and hydrazine group, and then it consists of a cyclization step in which the condensation of the methanol molecule is achieved by boiling these hydrazones under acidic conditions in the same environment. As a result of such simple but effective organic interactions, 5a‐d and 6a‐d structures were synthesized with 40%–71% yields. In this synthesis step, the targeted derivatives were successfully synthesized in the presence of strong/weak electron emitting groups. However, in the presence of a strong electron‐withdrawing ‐nitro group, it was observed that the reaction ended in the first step, where only hydrazone products were formed and 5e and 6e derivatives were not formed. This effect was consistent with what we expected from basic organic theories. The strong electron withdrawal of the nitro group caused this synthesis to fail, as the electrons on the N atom remaining free in the hydrazone structure were delocalized to the aromatic ring instead of the nucleophilic attack. In the second step of the study, to examine the effect of the sulfur atom, which has an important place in biological applications, in this study, triazolopyridazithione structures in which the ‐C = O group was changed to ‐C = S were synthesized with LR, which is the sulfurization reagent of these purine‐like triazolopyridazinone structures. While 7a‐d and 8a‐c derivatives were synthesized with 35%–66% yield, 8d molecule could not be synthesized. The fact that the 6 d structure, which is the starting compound, has a very low solubility in organic solutions, caused problems in the formation of this reaction. Although this synthesis step was attempted to be carried out in more polar or non‐polar solvents/solvent mixtures, unfortunately this derivative could not be obtained.

SCHEME 1.

Synthesis approach of 1,2,3‐triazolopyridazin‐4‐one and 1,2,3‐triazolopyridazin‐4‐thione derivatives.

3.2. Characterization Part

The successful synthesis of triazolopyridazin‐4‐ones and triazolopyridazin‐4‐thiones was validated by the infrared spectra, as illustrated in Figure S61. Notably, the IR spectra of 5a revealed a band related to lactam type stretching (‐N‐C = O) at 1697 cm^‐1, whereas the IR spectra of 1 disappeared separate peaks belonging to ester stretching (‐O‐C = O) and formyl (‐C = O) stretching at roughly 1714 and 1690 cm^‐1, respectively. The ‐C = O stretching vibration at 1697 cm^‐1 was found to vanish in pyridazinthione compounds. Consequently, it was possible to successfully synthesis the purine‐like compounds pyridazinone and pyridazinthione, which made it suitable for later anticancer procedures. A thorough NMR investigation was carried out to clarify the chemical structure of the cyclization derivatives further, the corresponding spectra are shown in Figure S62 and SI. The synthesized triazolopyridazin‐4‐ones were confirmed by the appearance of an ‐N = C‐H resonance around 8 ppm in the ¹H NMR spectra, and the disappearance of methyl‐H's from the ‐OCH₃ group of the starting compound at around 4 ppm. The structural characterization of pyridazinthione molecules was conducted using FTIR, NMR, and mass spectroscopy. In the ¹³C‐APT NMR spectrum, the C peak associated with the ‐C = O group in the pyridazinone ring disappeared at around 160 ppm, while the C atom of the ‐C = S group in the pyridazinthione ring resonated at approximately 190 ppm (Figure S62). Furthermore, their characterization was completed using ¹³C‐APT NMR and MS analyses (see supporting information).

3.3. Anticancer Activity Results

The anticancer activity properties of triazolpyridazin‐4‐one/triazolepyridazin‐4‐thione compounds were applied on A549 and MCF7 cell lines and IC₅₀ results were given in the Figure 1.

FIGURE 1.

IC₅₀ values (µM) of triazolpyridazin‐4‐one/triazolepyridazin‐4‐thione compounds against A549 and MCF7 cell lines.

In the design of the synthesized derivatives, the anticancer activities of lipophilic cores at the 1‐position of the purine‐like structures and electron‐releasing or attracting groups in the aromatic ring at the 5‐position were screened. As known in the literature, substituents such as methyl, methoxy and fluorine have significant effects on the biological activity and pharmacokinetics of pyridazinone derivatives. Substituents such as methyl, methoxy and fluorine have been preferred in phenyl structures at the 5‐position of the pyridazinone ring. These groups may accelerate the passage in the fat tissue in their intracellular behaviors, increase the binding affinity in certain enzyme or receptor active sites, and contribute to metabolic stability. In line with all these effects, these substituents have also been preferred for the pyridazinone ring. Adding a methyl group to the structures increases lipophilicity (fat solubility) and thus improves membrane permeability, while fluorine is known in the literature to increase metabolic stability since it resists oxidative degradation by enzymes in the liver. Screenings were performed in A549 and MCF7 cell lines and the results were calculated in IC₅₀. It was noted that in pyridazinone derivatives (5a‐e, and 6a‐e), the electron‐withdrawing group rather than the electron‐releasing group at position 5, which is generally independent of the lipophilic end, and the unsubstituted derivative showed the highest activity in the A549 line. In the pyridazinthione skeleton structure (7a‐d) of the same cell line, derivatives with ‐F and ‐CH₃ groups at the 5‐position independent of the lipophilic group showed higher activity. In cell screens in MCF7 cell line, the results were quite different from A549 cell line. The lipophilic end of the pyridazinone derivatives (5a‐e and 6a‐e), the phenyl derivatives, showed less activity compared to the derivatives containing benzyls. This result can be explained by the direct pi‐pi flow of the phenyl ring to the piridazinone ring and the formation of the planarity of the structure and the reduction of the interaction surface with the cell line in this planarity, resulting in a decrease in activity. In MCF7 cell line, derivatives 8a‐c showed more effective results compared to A549 cell line. In all analysis results, it is predicted that the derivatives, which are purine‐like structures, are more effective structures compared to cisplatin and these structures will be possible structures that can be used in future novel drug designs.

3.4. AO/EB Staining and Caspase 3 Activity Results

Images of AO/EB staining performed to show the apoptotic effects of the synthesized compounds on A549, MCF7 and L929 cells were given in Figures 2, 3 and 4 respectively. In images using the AO staining method, a color tone between green and orange is observed in the early apoptosis phase of the cell. In case of late apoptosis, color tones between orange and red become evident. In healthy cells, this situation will express itself with a completely green color. These color changes indicate different stages of the cell in the apoptotic process. In images obtained with EB, another staining method, the genetic material interacts with more ethidium bromide as a result of the damage suffered by the cell nucleus. This is observed as a glow between yellow and white instead of red. Also, in the merged photos, the yellow tones, which are the combination of green and red lights, appear to be more prominent. The observation of nearly yellow cells with shining nuclei in the merged photographs indicates that there is damage to the genetic material in these cells and they are undergoing apoptosis. On the other hand, if cells turn red in AO staining and no nuclear brightness is observed in EB or merged photographs, this indicates that the cell has entered apoptosis as a result of a metabolic inhibition [22, 23, 37]. According to our results, it is clearly seen that compounds 5a, 5c, 6a and 8c cause apoptosis in A549 cell lines. In addition, green color intensity reduced with similar pattern at A549 and MCF7 exposed 5a, 5c, 6a and 8c. However, A549 and MCF7 cell lines are cell types that may respond differently to membrane damage. A549 lung cancer may be more sensitive to membrane damage [37], in this case, dyes such as ethidium bromide can quickly pass through the cell membrane and reach the nucleus, which is highlighted by the red‐orange color in dead cells. MCF7, as a breast cancer cell line, may have different characteristics in terms of membrane structure and apoptosis process. The membranes of these cells are more durable and may respond more slowly to the cell death process. In this case, because the cell membranes are impermeable to ethidium bromide, dead cells show less red color. In addition to membrane permeability, other cell death mechanisms, such as the slow progression of apoptosis, may cause the lack of red color observed in MCF7 cells.

FIGURE 2.

(A–E) Visible light microscope images. (F) IC₅₀ Value of samples. (G–K) AO/EB staining images of A549 cells. (L) Color intensity of A549 cells in AO/EB staining.

FIGURE 3.

(A–E) Visible light microscope images. (F) IC₅₀ Value of samples. (G–K) AO/EB staining images of MCF7 cells. (L) Color intensity of MCF7 cells in AO/EB staining.

FIGURE 4.

(A–E) Visible light microscope images. (F) IC₅₀ Value of samples. (G–K) AO/EB staining images of L929 cells. (L) Color intensity of L929 cells in AO/EB staining.

Caspases regulate various signaling pathways within the cell and respond to stimuli during apoptosis. Caspase 3 is often referred to as an effector caspase at this stage and plays an important role in executing apoptosis. This enzyme triggers the death of the cell by breaking down various target proteins within the cell. The importance of using caspase 3 in apoptosis is to ensure that cell death occurs in an orderly and controlled manner. This programmed death helps eliminate harmful or unnecessary cells in the developing organism. In our study, caspase 3 activity for A549 cell line was determined in the samples with results given in Figure 5. A significant increase (p < 0.05) in compound 5a was seen in the A549 cell line. These images are overall consistent with AO/EB staining.

FIGURE 5.

Caspase 3 activity in A549 cell line exposed 5a, 5c, 6a and 8c for 24 h. (*p < 0.05).

4. Conclusions

In summary, 15 compounds, 1‐aryl‐5‐(p‐substitutedphenyl)‐1,5‐dihydro‐4H‐[1–3]triazolo[4,5‐d]pyridazin‐4‐one (5a‐d, 6a‐d) and 1‐aryl‐5‐(p‐substitutedphenyl)‐1,5‐dihydro‐4H‐[1–3]triazolo[4,5‐d]pyridazine‐4‐thione (7a‐d, 8a‐c) derivatives, were designed, synthesized and evaluated for their biological activities. In vitro antiproliferative activity against A549, MCF7 and L929 cancer cell lines was investigated for the synthesized 5a‐d, 6a‐d, 7a‐d and 8a‐c based triazoles derivatives. When compared against the references caspase 3 the results showed that 5a, 5c, 6a and 8c were the most potent. In the screening of cytotoxicity, 5a, 5c, 6a and 8c showed higher activity than other compounds with IC50 range from 36.35 to 184.72 µM, 30.66 to 154.87 µM and 152.14 to 253.76 µM against A549, MCF7 and L929 cells, respectively. According to these results, the 5‐position of the phenyl ring with ‐H, ‐F, ‐CH₃, ‐OCH₃ groups was the more effective in terms of cytotoxicity. Meanwhile, AO/EB and caspase 3 activity assay results on A549 cell indicated that 5a, 5c, 6a, and 8c could induced apoptosis in A549 cells. Further studies on the biological activities of these compounds led to promising results in the development of anticancer drugs.

Author Contributions

Guler Yagiz Erdemir: investigation, validation, data curation, writing – original draft, visualization, formal analysis. Ali Kuruçay: investigation, validation, formal analysis, data curation. Burhan Ates: conceptualization, methodology, writing – review and editing, validation, supervision. Aliye Altundas: conceptualization, methodology, writing – original draft, writing – review and editing, project administration, visualization, supervision.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Supporting information.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.