Design, synthesis and evaluation of novel 1,2,4-triazole derivatives as promising anticancer agents

Herein, we reported the synthesis of nineteen novel 1,2,4-triazole derivatives including 1,3-diphenyl-2-(1H-1,2,4-triazol-1-yl) propan-1-ones (7a-e), 1-(1,3-diphenylpropan-2-yl)-1H-1,2,4-triazole (8a-c) and 1,4-diphenyl-2-(1H-1,2,4-triazol-1-yl) butane-1,4-diones (10a-k). The structures of these derivatives were confirmed by spectroscopic techniques like IR, 1H-NMR, Mass spectroscopy and Elemental analysis. The cytotoxic activities of the synthesized compounds were evaluated against three human cancer cell lines including MCF-7, Hela and A549 using MTT assay. Compounds 7d, 7e, 10a and 10d showed a promising cytotoxic activity lower than 12 μM against Hela cell line. The safety of these compounds was also, evaluated on MRC-5 as a normal cell line and relieved that most of the synthesized compounds have proper selectivity against normal and cytotoxic cancerous cell lines. Finally, molecular docking studies were also, done to understand the mechanism and binding modes of these derivatives in the binding pocket of aromatase enzyme as a possible target. Supplementary Information The online version contains supplementary material available at 10.1186/s13065-022-00887-x.


Introduction
Cancer is characterized by the uncontrolled growth and proliferation of abnormal cells and is the second leading cause of morbidity and mortality in the world [1,2]. According to the global cancer statistics, 9.6 million deaths and also, more than 18 million new cancer occurred in 2018 [3]. It is expected that the cancer mortality rate will rise dramatically in the future [3].
Various internal and external factors cause abnormal cell proliferation, leading to development of various cancers, including genetics, viruses, drugs, diet and smoking [4,5]. Currently, three strategies including chemotherapy, radiotherapy, and surgery are used for the treatment of cancer. Chemotherapy is the most common treatment for cancer disease, in which various chemotherapeutic agents are utilized to kill the cancer cells with minimum harmful effect on normal cells [4,6]. However, drug resistance, non-selectivity and toxicity of many anticancer drugs have limited their clinical uses [1,7]. Hence, the discovery and development of more effective and potent anticancer agents is one of the most clinical challenges in modern medicinal chemistry [8].

Design
The target 1,2,4-triazole derivatives were designed based on the chemical structures of Letrozole (a), Anastrozole (b) and 4-triazolylflavans (c) which act as aromatase inhibitors. Aromatase is a member of the cytochrome P450 superfamily that catalyzes the estrogen biosynthesis and can be considered as a therapeutic target due to its overexpression in breast cancer. Anastrozole and Letrozole are potent aromatase inhibitors that use in the treatment of ER-positive breast cancer. In addition, it has been previously reported that 4-triazolylflavans derivatives exhibited aromatase inhibitory effect [38,39]. In these aromatase inhibitors, nitrogen atoms of 1,2,4-triazole ring bind to the iron in the heme moiety of CYP-450 and phenyl moieties have a key interaction in the active site of enzyme. Furthermore, carbonyl group is incorporated in the designed structures due to its ability to form hydrogen bonds. Therefore, 1,2,4-triazole moiety, phenyl rings and carbonyl groups were incorporated in the designed scaffolds (Fig. 3).

Chemistry
The synthesis of the desired compounds (7a-e, 8a-c, 10a-k) was carried out according to the synthetic pathway illustrated in Fig. 4. The phenacyl chloride derivatives 3a-f were prepared in high yield through Fridel Crafts acylation of mono or di-substituted benzene (1a-f) with chloroacetyl chloride (2) using aluminum trichloride (AlCl 3 ) as a strong Lewis acid catalyst [40,41]. In the second step, intermediates 5a-f were synthesized from the reaction of intermediates 3a-f with 1,2,4-triazole (4) in the presence of sodium bicarbonate (NaHCO 3 ). Compounds 7a-e were synthesized by treating benzyl bromide (6a) or benzhydryl bromide (6b) with intermediates 5a-f in the presence of NaH as a strong base catalyst in acetonitrile. Subsequently, Huang Minlon reduction of compounds 7a and 7c-d in the presence of N 2 H 4 .H 2 O and KOH yielded compounds 8a-c [42]. Compounds 10a-k were prepared from the reaction  2-chloro-1,2-diphenylethanone (desyl chloride) or 2-chloro-1-phenylethanone derivatives with key intermediates 5a-f utilizing NaH as base and in acetonitrile as solvent. The structures of the synthesized compounds were confirmed through 1 H-NMR, Mass and IR techniques. In the IR spectra of compounds 7a-e, a signal for C = O group was observed at 1690-1723 cm −1 , while this signal was removed for compounds 8a-c. The IR absorption spectra of 10a-k were characterized the presence of two signals for C = O groups at 1650-1712 cm −1 . 1 H-NMR spectrum of compounds 7a-e showed two singlet peaks at 8.12-8.33 and 7.77-7.91 ppm assigned to the 1,2,4-triazole ring. In addition, in compounds 7a and 7b, the CH proton was observed at 6.17-6.24 ppm as a doublet of doublet peak and in compounds 7c-e the CH proton was observed at 6.73-6.79 ppm as a doublet peak. 1 H-NMR spectrum of compounds 8a-c showed two singlet peaks at 8.17-8.29 ppm and 7.78-7.85 ppm assigned to the 1,2,4-triazole ring and a multiplet peak at 4.59-4.67 ppm assigned to the CH proton. 1 H-NMR spectrum of compounds 10a-k showed two singlet peaks at 7.97-8.35 ppm and 7.79-8.03 ppm assigned to the 1,2,4-triazole ring. Furthermore, in compounds 10a-e with R = H, the CH proton appeared at 6.52-6.69 ppm as a triplet peak whereas in compounds 10f-k with R = Ph, the CH proton appeared at 6.45-6.81 ppm as a doublet peak. All

Evaluation of anticancer activity
All of the synthetic compounds (7a-e, 8a-c and 10a-k) were screened for their in vitro cytotoxic effects against three human cancerous cell lines (MCF-7, Hela and A549) using MTT assay [43][44][45]. The biological results were expressed in terms of IC 50 (Table 1). Generally, the best anticancer effect was seen on breast (MCF-7) and cervical (Hela) cell lines and less on lung cancer (A549) cell line. In this assay, cis platin was used as positive control [46][47][48]. As shown in Table 1, some of the compounds such as 7b, 7d-7e, 10a and 10c-d showed better antiproliferative activity compared to cis platin in all studied cancer cell lines. Assessments on propane-1-one derivatives (7a-e), compound 7e bearing X 1 :X 2 = Cl, R = Ph showed significant activity with IC 50 = 4.7, 2.9 and 9.4 μM against MCF-7, Hela and A549 cell lines, respectively, followed by compound 7d possessing X 1 = Br, X 2 = H, R = Ph with IC 50 = 9.8, 12.1 and 43.4 μM. The obtained results indicated that the absence of electronegative groups at X position and also, the absence of phenyl ring at R position in analogue 7a (unsubstituted one), dramatically decreased the cytotoxic effects against all studied cancer cell lines. On the other hand, propane-1-yl-derivatives (8a-c), showed less cytotoxicity effect compared to other studied compounds (7a-e and 10a-k), which can be attributed to the reduction of carbonyl group at position 1 of propane chain. Further, a comparison of the cytotoxicity of compounds 7a-e on Hela cell line showed that the anticancer activities of different substitutions on phenyl ring followed the order 2,4-di-Cl > 4-Br > 4-Cl > H, and interestingly, the relative order of the substitution effect on cytotoxicity for compounds 8a-c was 4-Br > 4-Cl > H which was in line with above-mentioned results. In the case of butane-1,4-dione derivatives (10a-k), compound 10a was found to have promising anticancer activity with IC 50 = 6.43, 5.6 and 21.1 μM against MCF-7, Hela and A549 cell lines, respectively. The substitution of Br and Cl at X 1 and X 3 position produced appropriate derivative (10d) with IC 50 values of 10.2, 9.8 and 16.5 μM. Besides, the cytotoxic activity of compounds 10a-e on Hela cell line showed that the anticancer activities of different substitutions on the phenyl rings followed the order of H > 4Br > 2,4-di-Cl > 4-Cl. The incorporation of phenyl group at R position decreased cytotoxic activity. Among these compounds, 10j bearing 2,4-diflouro group at the phenyl moiety demonstrated adequate cytotoxic effect. The safety of these compounds was also, evaluated on MRC-5 as normal cell line. The resulted indicated that most of the synthesized compounds have proper selectivity against cancer cell lines (Fig. 5).

Molecular docking
The docking information of four 1,2,4-triazole derivatives possessing the highest (10a and 10d) and lowest (7c and 8c) cytotoxic activity were shown in Fig. 6 and Fig. 7.
Redocking of 4-androstene-3-17-dione as co-crystal ligand, was done to assessment the docking results. The RMSD was obtained 0.40 Å in comparison to its coordination in the crystal structure. The all interaction and score binding of all studied compounds was shown in Table 2.
Compound 10a through its carbonyl group interacted with Ser 478 via hydrogen bond and also, formed π-cation and π-π interactions with HEM. The phenyl ring of 10a formed π-sigma bond with Leu 477 and also, some π-alkyl interactions with Val 373, Ile 133, Val 370 and Ala 306 residues were observed. In addition, hydrophobic interactions with Thr 310, Trp 224, Arg 115, Met 374, Phe 221, Leu 372, and Phe 134 residues were existence which led to desire affinity to aromatase enzyme. On the other hand, compound 10d had same similarity interactions with 10a as one of the best compound such as, π-π, π-sigma and π-alkyl with Leu 477, Val 370, Val 369, Phe 221, Leu 372, Met 374 and Ile 133 residues. Compound 10d also, formed halogen bond and π-cation interaction with Ala 306 amino acid residue and HEM group, respectively. These interactions confirmed that compounds 10a and 10d were successfully bound to the aromatase enzyme via most of the previously binding interactions of co-crystal ligand of aromatase enzyme (3EQM) (natural substrate androstenedione (AD)), such as Phe134, Trp224, Val370, Val373, Met374 and HEM [49,50].

Fig. 5
Cytotoxic effect of compounds 7a-e, 8a-c and 10a-k on MCF-7, Hela and A549 cell lines Taken to gather, regarding the cytotoxic evaluations on 7a-e and 8a-c derivatives, it can be realizing that 7e was the most potent derivative against all three tested cell lines. The structure activity relationship disclosed that electronegative substitution such as Cl and Br at para position of phenyl ring (X 1 ) and also, the presence of phenyl ring at R position could increase the inhibitory activity significantly in a 7a-e series. Also, the presence of one-carbonyl group showed necessary for pharmacological effect. In addition, propane-1-yl-derivatives (8a-c) had least effect on cytotoxic activity. In the case of 10a-k, replacement of H with Ph moiety at R position led to decreased cytotoxic activity (10f-k) and also, no substituted analogue (10a) had favorable pharmacological effect on MCF-7 and Hela cell lines. The cytotoxicity of all synthesized compounds were shown in Table 1.
In silico ADME modeling study ADME properties of the synthesized compounds were determined using SwissADME online software [51]. As depicted in Table 3, all of the compounds represented admissible molecular weight (MW < 500). They had desire lipophilicity (logP) values. In addition, the hydrogen bond properties including hydrogen bond donor (HBD), hydrogen bond acceptor (HBA) and rotatable bond (RB) are reasonable. According to rule of five, total polar surface area of all compounds are in accepted range ≤ 140 Å. Based on our results, all of the compounds indicated desire potential for oral bioavailability [52].

Chemistry
All reagents and solvents with analytical grade were purchased from commercial sources (Merck & Sigma Aldrich) and used without further purification. IR spectra (KBr, cm −1 ) were recorded using a Perkin Elmer IR instrument. 1 H-NMR spectra were recorded in CDCl 3 on Bruker 500 MHz spectrophotometer using tetramethylsilane (TMS) as an internal standard. Chemical shift values were expressed in ppm scale (δ) and coupling constants were reported in hertz (Hz). An Agilent spectrometer was used for Mass spectra recordation. All melting points were measured with an Electro-thermal (Van der waals: green, dark pink: π-π, light pink: π-alkyl, purple: π-sigma, orange: π-cation, blue: halogen bond) IA 9100 apparatus and were uncorrected. The progress of reactions was monitored using thin layer chromatography (TLC) sheets pre-coated with UV fluorescent silica gel Merck 60F 254 and the spots were visualized using UV lamp. The purification of the synthesized compounds was performed by column chromatography.

General procedure for the synthesis of intermediates 3a-f
The appropriate AlCl 3 (60 mmol) was added to a stirred solution of phenyl halides (50 mmol) in dichloromethane (30 mL). After stirring for 30 min at room temperature, the mixture was cooled to 0 ο C and a solution of chloroacetyl chloride (54 mmol) in dichloromethane (20 mL) was added dropwise to it. The resulting mixture was stirred at room temperature for 24 h. After this time, 50 mL of HCl solution (5%) was slowly added and the reaction mixture was extracted with dichloromethane (3 × 30 mL) and then washed with NaHCO 3 (20 mL), water (2 × 20 mL) and brine (20 mL), respectively. The organic layer was dried over anhydrous Na 2 SO 4 and concentrated under vacuum. Finally, the obtained precipitate was recrystallized from n-hexane to give intermediates 3a-f.

General procedure for the synthesis of intermediates 5a-f
1,2,4-triazole (48 mmol) and NaHCO 3 (48 mmol) were added to a stirred solution of intermediates 3af (40 mmol) in toluene. The reaction mixture was refluxed for 20 h. After this time, the reaction mixture was quenched with an ice bath and extracted with ethyl acetate (3 × 30 mL). Then, the organic phase was washed with water (2 × 20 mL) and brine (10 mL), dried over anhydrous Na 2 SO 4 and concentrated in vacuum. Finally, the residue was recrystallized from diethyl ether to afford the desired compounds 5a-f. (Van der waals: green, dark pink: π-π, light pink: π-alkyl, purple: π-sigma, orange: π-cation, blue: halogen bond)

General procedure for the synthesis of compounds 7a-e
A solution of intermediates 5a-f (6 mmol) in acetonitrile (10 mL) was added to a suspension of NaH (8 mmol) in acetonitrile (30 mL) and the resulting mixture was stirred at room temperature for 1 h. Then, a solution of benzhydryl bromide or benzyle bromide (6 mmol) in 10 mL acetonitrile was added dropwise and the mixture was heated under reflux for 24 h. After cooling to room temperature, the solvent was evaporated in vacuum, 50 mL water was added and the mixture was extracted with dichloromethane (3 × 30 mL). In the following, the organic layers were dried over Na 2 SO 4 and purified by column chromatography on silica gel eluting with ethyl acetate and petroleum ether (3:1) to afford desired products 7a-e.    7a-e, 8a-c and 10a-

General procedure for the synthesis of compounds 8a-c
A mixture of compound 7a or 7c-d (1.6 mmol), hydrazine monohydrate (8 mmol) and potassium hydroxide (8 mmol) in ethylene glycol (50 mL) was heated at 170 ˚C for 4 h. Then, the reaction mixture was cooled to room temperature, quenched with water (500 mL) and acidified to pH = 1 with concentrated hydrochloric acid and extracted with chloroform (3 × 30 mL). Afterwards, the organic layer was washed with brine and dried over Na 2 SO 4 . The crude product was purified by column chromatography on silica gel eluting with ethyl acetate and petroleum ether (1:1) to give pure compounds 8a-c.

General procedure for the synthesis compounds 10a-k
A solution of intermediates 5a-f (6 mmol) in acetonitrile (10 mL) was added to a suspension of NaH (8 mmol) in acetonitrile (30 mL) and the resulting mixture was stirred at room temperature for 1 h. Then, a solution of 2-chloro-2-phenyl acetophenone or halogenated phenacyl chloride (6 mmol) in 10 mL acetonitrile was added dropwise and the mixture was heated under reflux for 24 h. After cooling to room temperature, the solvent was evaporated in vacuum, 50 mL water was added and the mixture was extracted with dichloromethane (3 × 30 mL). In the following, the organic layers were dried over Na 2 SO 4 and purified by column chromatography on silica gel eluting with ethyl acetate and petroleum ether (3:1) to afford desired products 10a-k.

In vitro cytotoxic assay Chemicals
Fetal bovine serum (FBS) and RPMI-1640 medium were purchased from Gibco Invitrogen Co. (Scotland, UK) and sigma Aldrich, respectively. Dimethyl sulfoxide (DMSO), doxorubicin, penicillin, streptomycin was also purchased from Sigma Aldrich.

Cell cultures
Cell cultures were obtained from the human lung cancer cell line (A549), human cervical cancer cell line (Hela), human breast cancer cell line (MCF-7) and Normal cells isolated from human lung tissue (MRC-5) taken from the National Cell Bank of Iran (NCBI, Pasteur Institute, Tehran, Iran). All cells were cultured in RPMI-1640 medium supplemented with 10% FBS, antibiotics (penicillin 100 U/mL, streptomycin 100 lg/mL), at 37 ˚C in a humidified atmosphere containing 5% CO 2 .

MTT assay
Cytotoxic activities of all the synthesized compounds were assessed by standard 3-(4,5-dimethylthiazol-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay [53]. The assay was performed according to a known protocol [54][55][56]. The cells were seeded and plated in 96-well microplates at a density of 1 × 10 4 cells per well in 180 μL complete culture media. After 24 h incubation, the cells were treated with 100 mL of different concentrations of synthesized compounds ranging from 1 × 10 -4 to 1 × 10 -7 M at identical conditions in triplicates. After 72 h, media were replaced with 150 μL media containing 0.5 mg/mL of MTT solution [57]. The plates were incubated at 37 °C for additional four hours. Then, media containing MTT were discarded and 150 μL dimethylsulfoxide (DMSO, > 99%) was added to each well to dissolve the formazan crystals. The absorbance in each well was determined at 570 nm by a BioRad microplate reader (Model 680) and then the IC 50 values were calculated and demonstrated as mean ± SEM [58].

Molecular docking study
Molecular docking studies were performed by Auto-Dock 4.2 program and AutoDock Tools 1.5.4 to investigate the binding mode of derivative in the active site of receptor [59]. The X-ray crystallographic structures of aromatase (PDB: 3EQM) (39) were obtained from the protein data bank (http:// www. rcsb. org). Then, all water molecules and co-crystallized ligand were removed and hydrogen atoms were added to the protein and finally saved as pdbqt format. In the following, 3D structures of ligands were drawn and minimized under Molecular Mechanics MM + and semi-empirical AM1 methods, using HyperChem software and saved as pdbqt format. The box dimensions were set at 65 × 65 × 65 with 0.375 Å grid spacing. Docking validation was done by re-docking the original ligand into the active site of receptor. Finally, conformations with the lowest free energies of binding were selected for analysis.

Conclusion
In summary, we have designed and synthesized nineteen new 1,2,4-triazole-based derivatives starting from different phenyl halide analogues through three or four different steps. Their chemical structures were fully confirmed by IR, 1 H-NMR, Mass spectra and elemental analysis. In vitro cytotoxic activity of the synthesized compounds were evaluated against three human cancer cell lines including MCF-7, Hela and A549, using MTT assay. The obtained results indicated that the synthesized compounds possessed relatively high to moderate antiproliferative activities against MCF-7 and Hela cancer cell lines . Compounds 7d, 7e, 10a and 10d were the most potent ones against three tested cell lines. Based on structure activity relationship (SAR) studies, it was found that the presence of electronegative substituents on the phenyl ring, as well as the presence of onecarbonyl group, resulted in a relative increase in the cytotoxic activity of the synthesized compounds. The outcome results relived that these active derivatives can be considered as a lead compound for anticancer treatments.