Development of di-arylated 1,2,4-triazole-based derivatives as therapeutic agents against breast cancer: synthesis and biological evaluation

Abstract

The synthesis, anticancer activity, and metabolic stability of di-arylated 1,2,4-triazole molecules have been reported. Utilizing an efficient programmed arylation technique which starts from commercially available 3-bromo-1H-1,2,4-triazole, a series of therapeutic agents have been synthesized and screened against three human breast cancer cell lines, MDA-MB-231, MCF-7, and ZR-75-1, via an in vitro growth inhibition assay. At 10 μM concentration, 4k, 4m, 4q, and 4t have displayed good anticancer potency in the MCF-7 cell line, among which 4q has shown the best efficacy (IC₅₀ = 4.8 μM). Mechanistic investigations of 4q have indicated the elevation of the pro-apoptotic BAX protein in the malignant cells along with mitochondrial outer membrane permeabilization which are hallmarks of apoptosis. Further metabolic stability studies in diverse liver microsomes have provided insights into the favorable pharmacokinetic properties of 4q in humans, establishing it as a promising lead compound of this series that deserves further investigation.

The most potent derivative (4q, IC₅₀ = 4.8 μM) of the newly synthesized diarylated 1,2,4-triazole series displays several hallmarks of apoptosis in the MCF-7 breast cancer cells and favourable metabolic stability in diverse liver microsomes.

Introduction

The prevalence of triazoles in various biologically active molecules, natural products, FDA-approved marketed drugs, and active pharmaceutical agents (APIs) has made it one of the privileged nitrogen-containing heterocyclic scaffolds in the pharmaceutical industry.¹ Due to the unique structural and medicinal properties of the 1,2,4-triazole ring, it has become an integral part of many structure-guided drug discovery programs (SGDDs).² In recent years, a plethora of research work associated with various cancer-related drug targets has shown the ubiquity of 1,2,4-triazole as an active pharmacophore (Fig. 1). For example, 1,2,4-triazole-containing VEGFR-2 inhibitor (1a),³ TNKS2 inhibitor (1b),⁴ tubulin polymerization inhibitor (1c),⁵ CDK1 inhibitor (1d),⁶etc. have exhibited promising cytotoxicity against tumor cells. Moreover, it is proven to be an important key feature in several MYOF inhibitors, potent therapeutics for the pancreas (1e), and gastric cancer cell lines (1f).^7,8 Due to these significant applications, the facile formation of 1,2,4-triazole-containing molecules has captivated the interest of medicinal chemists over the past few years.

Fig. 1. Anticancer compounds containing 1,2,4-triazole pharmacophore.

While working on a medicinal chemistry program related to the development of novel small molecules having antiproliferative activity for various human cancer cells, we envisioned the need for direct functionalization of the 1,2,4-triazole pharmacophore in the lead molecules during the structure–activity relationship (SAR) studies. Despite several traditional methods, like condensation between arylhydrazine hydrochlorides with formamides,⁵ annulation between isocyanides and aryldiazonium salts,^9a or reaction between nitrilimines and Vilsmeier reagent^9b for constructing the 1,2,4-triazole ring, there is a definite scope for more straightforward synthetic strategies to functionalize the commercially available triazole molecules for the establishment of robust methodology required in a drug discovery and development process.

The ubiquity of N-aryl derivatives in innumerable molecules of medicinal interest has imparted great importance in the C–N bond formation techniques. For this, one of the emerging medicinal chemistry toolboxes is the boronic acid-mediated Cu-catalysed Chan–Evans–Lam (CEL)¹⁰ coupling reaction. Apart from the other two well-established N-arylation techniques, Ullmann–Goldberg¹¹ and Buchwald–Hartwig,¹² this CEL coupling technique has several advantages like mild reaction conditions, room temperature, open-air reaction, and good functional group tolerance which are important requirements during SAR studies and late-stage modifications in drug candidates. Till now, our group has surveyed various aspects of the possible applications of CEL coupling for different N-heterocyclic molecules like 7-azaindole, hydantoin, and azoles along with mechanistic investigations and potential biological applications.¹³ Lately, several developments regarding the N-arylation of different azoles have already been reported.¹⁴ However, the N-arylation techniques of halogen-containing 1,2,4-triazoles remain very limited with relatively few reports.^15,16 In Pd- or Cu-catalysed C–N cross-couplings, free halogen moiety sometimes hinders the desired reaction because the use of high temperature and strong base triggers the halogen tolerability with several possible side reactions.

Considering the challenges, we tried to develop a general and efficient synthetic toolbox for functionalizing the commercially available 3-bromo-1H-1,2,4-triazole. We utilized this technique to develop a small compound library of therapeutic agents targeting breast cancer since 1,2,4-triazole-containing FDA-approved marketed drugs letrozole and anastrozole are used for the same. Herein, we report a series of novel 1,3-diarylated triazole derivatives using programmed arylation (Scheme 1). After optimizing a CEL cross-coupling reaction condition for the same using a catalytic amount of Cu(OAc)₂, we have employed it along with standard conditions of the Pd-catalysed Suzuki–Miyaura cross-coupling reaction.¹⁷ The synthesized molecules are examined through the MTT assay to check their potency for inhibiting the growth in three human breast cancer cell lines MDA-MB-231, MCF-7, and ZR-75-1. After identifying the most active molecule that targets the MCF-7 cell line, more investigations are performed to gain mechanistic insights into the possible biological pathways. Subsequently, metabolic stability assay using liver microsomes from diverse species was performed to assess its pharmacokinetic activity.

Scheme 1. Sequential arylation of 3-bromo-1H-1,2,4-triazole.

The rationale of design

A thorough literature survey of various triazole-containing anticancer therapeutics and other marketed anticancer drugs has shown that FDA-approved medications for breast cancer like letrozole and anastrozole have the 1,2,4-triazole core.¹⁸ These therapeutics are mainly used against hormone receptor-positive (HR+) breast cancer.¹⁹ On the other hand, several natural products with potential antiproliferative activities, for example, combretastatin and colchicine, have a particular substitution pattern. In combretastatin, two aromatic rings are joined with a cis-olefinic bond, and the anticancer activity crucially depends on the 3,4,5-trimethoxy substitution in one phenyl ring.²⁰ Several anticancer agents have a similar type of substitution pattern.

In our present work, keeping these points in mind we have designed 1,3-diarylated triazole scaffolds where we followed a similar substitution pattern like combretastatin and replaced the cis-olefinic bond with a 1,2,4-triazole ring which is part of marketed drugs for breast cancer (Fig. 2). This type of pharmacophoric hybridization strategy has been used in several anticancer drug discovery programs where different heterocyclic/non-heterocyclic rings have been incorporated as a replacement for the cis-olefinic bond of combretastatin.²¹ In our designed derivatives, one phenyl ring (Ar₁) has been decorated with 3,4-dimethoxy, 3,4-methylenedioxy, and 3,4,5-trimethoxyphenyl substitution. On the other side, the phenyl ring (Ar₂) was decorated using several electron-withdrawing, electron-donating, halogen-containing groups to form a range of therapeutics that have been studied for potential anticancer effects.

Fig. 2. Rationale of designing the diarylated 1,2,4-triazole scaffolds.

Results and discussion

Chemistry

To establish optimized reaction conditions for the N-arylation of halo-substituted triazole molecule, we commenced our investigation by using 3-bromo-1H-1,2,4-triazole 1 (0.676 mmol, 1 equiv.) and phenylboronic acid 2a (0.811 mmol, 1.2 equiv.) as the substrates for Cu-catalyzed Chan–Evans–Lam (CEL) coupling reaction (Table 1).

Optimization of the copper-catalyzed N-arylation reaction conditions of 3-bromo-1H-1,2,4-triazole^a.

Entry	Catalyst	Base	Solvent	Yieldb (%)
1	Cu(OAc)2	DBU	DCM	57
2	Cu(OAc)2	DBU	DCE	49
3	Cu(OAc)2	DBU	MeOH	34
4	Cu(OAc)2	DBU	Toluene	n. r.
5	Cu(OAc)2	DBU	THF	41
6	Cu(OAc)2	Pyridine	DCM	5
7	Cu(OAc)2	Et3N	DCM	n. r.
8	Cu(OAc)2	DMAP	DCM	n. r.
9	Cu(OAc)2	K3PO4	DCM	n. r.
10	CuCl2	DBU	DCM	33
11	CuBr2	DBU	DCM	37
12	CuF2	DBU	DCM	33
13	Cu(OTf)2	DBU	DCM	31
14	CuBr	DBU	DCM	41
15	CuI	DBU	DCM	44
16c	Cu(OAc)2	DBU	DCM	52
17 d	Cu(OAc) 2	DBU	DCM	77
18e	Cu(OAc)2	DBU	DCM	57
19f	Cu(OAc)2	DBU	DCM	55
20g	Cu(OAc)2	DBU	DCM	58

^aReaction conditions: 1 (0.676 mmol), 2a (0.811 mmol), catalyst (50 mol%), base (2.028 mmol), solvent (3–4 mL), 4 Å molecular sieves, rt, air, 10 h.

^bIsolated yield.

^c1,10-Phenanthroline was used as a ligand.

^dThe reaction was carried out in the presence of an O₂ balloon.

^e0.676 mmol of 2a was used.

^f25 mol% catalyst was used.

^g1.014 mmol base was used. n. r. = no reaction.

By using Cu(OAc)₂ (50 mol%) as the catalyst and DBU (2.028 mmol, 3 equiv.) as a base in DCM solvent, we acquired our desired product 3a with 57% yield (Table 1, entry 1). The reaction was performed for about 10 hours at room temperature and in open-air conditions. By changing the solvent from DCM to DCE, MeOH, and THF, the yield of the reaction was decreased to 49%, 34%, and 41%, respectively (Table 1, entries 2, 3, and 5). Performing the reaction in toluene furnished no product formation (Table 1, entry 4). After optimizing the solvent, various bases were investigated for maximum efficiency of the reaction. While pyridine as a base showed inferior results in product formation (Table 1, entry 6), in the presence of other bases like Et₃N, DMAP, and K₃PO₄, the reaction did not proceed (Table 1, entries 7–9). After that, we examined various copper catalysts, copper(ii) salts like CuCl₂, CuBr₂, CuF₂, Cu(OTf)₂, and copper(i) salts like CuBr and CuI in our optimization studies. The yield of the desired product was found to be diminished further (Table 1, entries 10–15). Despite using 1,10-phenanthroline as an external ligand, the reaction yield did not improve (Table 1, entry 16). Next, the reaction was carried out in an O₂ atmosphere. The yield of the desired product formation was enhanced up to 77% (Table 1, entry 17).

The use of a lesser amount (1 equiv.) of 2a and lower catalyst loading (25 mol%) again decreased the product formation to 57% and 55% (Table 1, entries 18 and 19), respectively. Finally, the reaction yield dropped to 58% upon decreasing the amount of DBU from 3 equiv. to 1.5 equiv. (Table 1, entry 20).

Identifying the optimized condition, we examined its scope and limitations by using 3-bromo-1H-1,2,4-triazole 1 along with a range of boronic acids with diverse aryl- and heteroaryl substitution (Scheme 2). The use of phenylboronic acid 2a as the coupling partner produced the desired N-arylated product 3a with an isolated yield of 77%. Monosubstituted phenylboronic acids containing electron-donating methoxy groups in para and meta positions furnished a good yield of the desired products 3b (62%) and 3c (61%), respectively. In the case of phenylboronic acid with the amino group as meta-substitution, the isolated yield of the product 3d (57%) decreased slightly. For electron-withdrawing groups in para (fluoro, chloro, bromo, cyano) and meta (chloro and trifluoromethyl) positions, the N-arylated products 3e (66%), 3f (69%), 3g (71%), 3h (69%), 3i (65%), and 3j (69%), respectively, are formed in good yield. The yield marginally dropped to 51% for the N-arylated product 3k when (3-formylphenyl)boronic acid was used as a coupling partner. After monosubstitution, we incorporated disubstituted phenylboronic acids as the coupling partner. (3,4-Difluorophenyl)boronic acid and (3-fluoro-4-methoxyphenyl)boronic acid gave good yields of N-arylated products 3l (60%) and 3m (69%), respectively, but in the case of (3,4-dimethoxyphenyl)boronic acid, the yield of desired product 3n (59%) somewhat decreased. Exploring the trisubstituted boronic acid, (3,4,5-trimethoxyphenyl)boronic acid produced a moderate yield of the N-arylated product 3o (55%).

Scheme 2. The substrate scope of the Chan–Evans–Lam cross-coupling reaction of 3-bromo-1H-1,2,4-triazole under optimized conditions. Reaction conditions: 1 (0.676 mmol), 2 (0.811 mmol), Cu(OAc)₂ (50 mol%), DBU (2.028 mmol), DCM (3–4 mL), 4 Å molecular sieves, rt, O₂ balloon, 10 h.

Extending our investigation toward boronic acid containing bicyclic rings, it was found that 1-napthylboronic acid and benzo[d][1,3]dioxol-5-ylboronic acid formed the desired products 3p and 3q with moderate yield (59%). Our developed synthetic protocol was found to be also efficient for heteroaryl boronic acids; for example, pyridine-4-ylboronic acid, pyridine-3-yl boronic acid, and (6-methoxypyridin-3-yl)boronic acid delivered the desired N-arylated products 3r (54%), 3s (60%), and 3t (55%), respectively, in moderate to good yields.

However, with o-tolylboronic acid, the N-arylation was unsuccessful with no desired product 3u formation.

Next, we focused on the sequential functionalization of the N-arylated derivatives at the C3 position of the 1,2,4-triazole ring utilizing the palladium-catalyzed Suzuki–Miyaura cross-coupling reaction. Using N-arylated derivatives 3 (0.446 mmol, 1.0 equiv.) and aryl-/heteroaryl boronic acids 2 (0.535 mmol, 1.2 equiv.) as coupling partners, Pd(PPh₃)₄ as catalyst (5 mol%), and K₂CO₃ as base (1.338 mmol, 3.0 equiv.), the C3-arylation reaction was carried out in 1,4-dioxane/H₂O (2 : 1) solvent for about 15 hours under reflux conditions in an atmosphere of nitrogen.¹⁷ The reaction condition was excellent, constructing several 1,3-diarylated 1,2,4-triazole derivatives (4a–4v) with good to excellent yields and functional group tolerance (Scheme 3). We investigated the efficiency of this sequential arylation technique to develop the desired molecules necessary for the SAR studies in our medicinal chemistry program focused on anticancer drug discovery. We initiated our investigation by the C-3 arylation of the N-arylated derivative 3a with (4-methoxyphenyl)boronic acid, which resulted in the desired product 4a formation in an excellent yield of 95%. Next, utilizing N-arylated triazole derivative 3n, we developed several 1,3-disubstituted derivatives (4b–4f) in good to excellent yields while varying the boronic acid coupling partner in the Suzuki cross-coupling reaction. The use of (4-chlorophenyl), (4-fluorophenyl), (4-trifluoromethylphenyl), (3-trifluoromethylphenyl), and (3,4-difluorophenyl)boronic acid resulted in the formation of 4b (89%), 4c (67%), 4d (75%), 4e (78%), and 4f (80%), respectively. Again, changing the N-arylated derivative to 3q, the desired product 4g (80%) with benzo[d][1,3]dioxol-5-ylboronic acid and 4h (77%) with 1-napthylboronic acid were observed in excellent yields.

Scheme 3. The substrate scope for the C3-arylation of the N-arylated triazole derivatives. Reaction conditions: 3 (0.446 mmol), 2 (0.535 mmol), Pd(PPh₃)₄ (5 mol%), K₂CO₃ (1.338 mmol), 1,4-dioxane : H₂O (2 : 1, 3 mL), reflux, nitrogen atmosphere, 15 h. ^bBoronic acid pinacol ester (0.535 mmol) was used.

The 3,4,5-trimethoxyphenyl group has been a part of various therapeutics having anticancer effects. Thus we inquired about the scope of our synthetic strategy choosing the N-arylated derivative with this group as N-aryl substitution (3o) along with a range of boronic acids. Boronic acid having an electron-withdrawing group (fluoro) in the para position resulted in an excellent yield of desired product 4i (91%). Electron-donating groups (hydroxy, amino, methoxy) in the meta position produced the subsequent products 4j (85%), 4k (86%), and 4l (54%) with moderate to excellent yields. Successful desired N-arylated products 4m (89%) and 4n (86%) were observed with phenylboronic acids having electron-withdrawing groups (trifluoromethyl and formyl) in the meta position with notable yields. Next disubstituted boronic acids (3,4-dimethoxyphenyl)boronic acid, (3,4-difluorophenyl)boronic acid, (3-fluoro-4-methoxyphenyl)boronic acid, and (3,5-dichlorophenyl)boronic acid delivered the respective diarylated products 4o (85%), 4p (89%), 4q (88%), and 4r (93%) successfully.

The use of heteroaryl boronic acid (6-methoxypyridin-3-yl)boronic acid gave the desired product 4s in an excellent yield of 96%. The 1H-benzo[d]imidazole-6-ylboronic acid pinacol ester as coupling partner resulted in a good yield of 70% for the desired product 4t. Again, boronic acids containing bicyclic rings produced the respective diarylated products with remarkable yields; for example, 1-napthylboronic acid gave 4u (87%), and benzo[d][1,3]dioxol-5-ylboronic acid produced 4v (88%) successfully.

Biology

Growth inhibition studies

The di-arylated 1,2,4-triazole derivatives 4 were investigated for potential antiproliferative effects on tumor cells. For this study, three human breast cancer cell lines MDA-MB-231, MCF-7, and ZR-75-1 were chosen. MDA-MB-231 is a triple-negative breast cancer cell line. MCF-7 is an estrogen receptor (ER) and progesterone receptor (PR) positive but a human epithelial receptor (HER2) negative cancer cell line. ZR-75-1 is ER+, PR+/−, and HER2-negative. The rationale behind choosing these three cell lines was that from the literature survey, it has been observed that the 1,2,4-triazole-containing marketed drugs (letrozole and anastrozole) are active for HR-positive breast cancers. Thus, MCF-7 and ZR-75-1 were selected for study along with MDA-MB-231 which is HR-negative.²²

The in vitro cytotoxicity was examined using a 10 μM concentration of compounds 4a–4vvia MTT assay (Table 2). The assay showed that the compounds 4k, 4m, 4q, and 4t had legitimate cytotoxicity for the MCF-7 breast cancer cell line, where the percentages of cell growth inhibition were more than 48%. Therefore, these four analogs were taken up for IC₅₀ value calculation in all three breast cancer cell lines (Table 3). Here it was found that 4q and 4t were the potent derivatives of the series, having IC₅₀ values of 4.8 μM and 5.2 μM, respectively. Doxorubicin, one of the standard cytotoxic agents in drug development studies related to breast cancer, was used as a positive control to ensure that the experimental setup was functioning correctly and provided the standard cytotoxic activity in the chosen cell lines.

Percentage of growth inhibition by the compounds 4a–4v measured via MTT assay^a.

Entry	Compound	MDA-MB-231	MCF-7	ZR-75-1
1	4a	00	0.85325	00
2	4b	00	9.20334	00
3	4c	16.79	17.62	—
4	4d	15.81	21.27	—
5	4e	12.78	18.35	—
6	4f	7.98	16.10	—
7	4g	17.09	8.99	—
8	4h	15.27	5.714	—
9	4i	23.06	20.18	—
10	4j	10.4327	32.79543	8.544045
11	4k	22.74293	57.59924	12.09783
12	4l	16.41	16.41	—
13	4m	5.766997	48.63073	2.392276
14	4n	22.62	13.37	—
15	4o	23.50	28.87	—
16	4p	23.35	23.34	—
17	4q	32.11751	51.2877	6.413226
18	4r	17.43	00	—
19	4s	5.251687	35.85265	4.94602
20	4t	31.61213	58.51736	21.21026
21	4u	22.62	13.37	—
22	4v	17.92	21.21	—
23	Positive controlb	37.90	45.47	45.49

^aThe experiment was performed with a 10 μM concentration of each compound for 48 h.

^bDoxorubicin (1 μM) was used as a positive control.

IC₅₀ values (μM)^a.

Entry	Compound	MDA-MB-231	MCF-7	ZR-75-1
1	4k	21 ± 1.99	5.7 ± 0.99	71 ± 6.88
2	4m	94 ± 7.14	11 ± 0.98	98 ± 7.18
3	4q	17 ± 1.12	4.8 ± 0.87	34 ± 2.1
4	4t	15 ± 1.01	5.2 ± 0.21	93 ± 7.21
5	Positive controlb	1.7 ± 0.16	1.2 ± 0.88	1.2 ± 0.19

^a In vitro IC₅₀ value in human breast cancer cell lines. The experiment was conducted for 48 h.

^bDoxorubicin was used as a positive control.

To ensure the selectivity of these derivatives for breast cancer cells, we decided to conduct a cytotoxicity study with the MCF 10A breast cell line which is a non-tumorigenic mammalian epithelial cell line. The IC₅₀ values for both 4q and 4t were found to be more than 100 μM (Fig. 3). This cytotoxicity study exhibited more selectivity of our developed analogs toward breast cancer cells rather than normal healthy breast cells.

Fig. 3. In vitro IC₅₀ value determination in the MCF 10A cell line.

Since the compound 4q (4.8 μM) was the most active analog of the developed series, having an IC₅₀ value less than 5 μM in the MCF-7 breast cancer cell line (Fig. 4A), we decided to choose this derivative for further investigation to identify the biological pathways involved through which the compound might show an antiproliferative effect.

Fig. 4. Biological activity evaluation of 4q. (A) Growth inhibition curve for 4q. (B) Microscopic images of MCF-7 cells treated with different concentrations of 4q using various stainings and assays. (C) SDS-PAGE electrophoresis showing pro-apoptotic protein levels in the MCF-7 cells treated with 4q.

The MCF-7 cells were treated with three different concentrations (5 μM, 10 μM, and 20 μM) of 4q and examined through DAPI staining to check nuclear defragmentation and rhodamine 123 staining to check mitochondrial outer membrane permeabilization. The DCFH-DA assay was performed to investigate ROS generation and acridine orange staining for the generation of autophagy. The microscopic images of the cells undergoing these experiments are shown in Fig. 4B.

Taking account of all the microscopic images, it can be noticed that the compound 4q was involved in the disruption of the mitochondrial outer membrane. Mitochondrial membrane permeabilization has been established as the convergence of various biochemical pathways leading to the induction of programmed cell death or apoptosis.²³

Autophagy-mediated cell death is again another significant mechanism that closely regulates various aspects of apoptosis.²⁴4q had increased the production of autophagosomes which can be seen in acridine orange staining.

Moreover, in the cells treated with higher concentrations of 4q, there were indications of reactive oxygen species (ROS) which are associated with noxious effects on various essential cellular components.²⁵

Upregulation of pro-apoptotic protein levels is directly associated with mitochondrial outer membrane potential permeabilization (MOMP) mediated cell death mechanism.²⁶ Considering that, we further inspected the 4q-treated MCF-7 cells for pro-apoptotic protein levels through SDS-PAGE electrophoresis (Fig. 4C). It was found that 4q had elevated the levels of BAX and cleaved PARP in the cancer cells, which are the hallmarks of the programmed cell death machinery.

Metabolic stability studies

The liver is the primary organ responsible for xenobiotic metabolism, with approximately 90% of clinically administered medicines being metabolized in the liver.²⁷ Thus, assessment of metabolic stability is a crucial step in the early phases of the drug development process as it can provide hints on the clearance of test candidates by the liver so that plasma exposure time can be predicted before plunging into expensive preclinical investigations. Therefore, estimation of the metabolic stability of 4q was performed in commercially available liver microsomes of various species using a substrate depletion approach (Fig. 5). Along with that, the in vivo behavior of 4q was predicted based on the in vitro data (Table 4). For the metabolic stability studies, human liver microsomes (HLMs), monkey liver microsomes (MkLMs), dog liver microsomes (DLMs), rat liver microsomes (RLMs), and mouse liver microsomes (MLMs) were chosen. The study was carried out using verapamil as a positive control in HLM and the observed results closely aligned with the reported literature findings (Fig. 5A).^28,29 Control incubation was performed in the absence of NADPH as a negative control in all the species-specific microsomes to assess any chemical instability or non-NADPH-dependent enzymatic degradation, but 4q was found to be stable under these conditions. Based on the % compound remaining data, results displayed the following order of metabolic stability for 4q in the experimental time frame: HLM > MkLM > MLM > RLM > DLM (Fig. 5B–E).

Fig. 5. Metabolic stability studies of 4q using substrate depletion method in HLM, MkLM, DLM, RLM, and MLM. Data are represented as mean ± SEM (n = 3).

Prediction of in vivo behavior of 4q from in vitro data in liver microsomes of different species. Data are represented as mean ± SEM (n = 3).

Parameters	HLM	MkLM	DLM	RLM	MLM
Half-life in liver microsomes (min)	106 ± 5.5	60 ± 2.3	7 ± 0.5	14 ± 1.1	34 ± 2.0
Intrinsic clearance (μL min−1 mg−1 protein)	13 ± 0.7	23 ± 0.9	188 ± 11.7	103 ± 8.3	41 ± 2.6
Hepatic intrinsic clearance (mL min−1 kg−1)	15 ± 0.8	32 ± 1.2	271 ± 16.9	185 ± 14.9	164 ± 10.2
Hepatic clearance (mL min−1 kg−1)	9 ± 0.3	18 ± 0.4	28 ± 0.2	42 ± 0.8	58 ± 1.2
Hepatic extraction ratio	0.42 ± 0.012	0.42 ± 0.009	0.90 ± 0.006	0.77 ± 0.014	0.64 ± 0.014

The test candidate 4q exhibited moderate intrinsic clearance in HLM (between 8.6 and 47.0 μL min⁻¹ mg⁻¹ protein), MkLM (between 13.8 and 75.4 μL min⁻¹ mg⁻¹ protein), and MLM (between 13.1 and 71.1 μL min⁻¹ mg⁻¹ protein). However, high intrinsic clearance was observed in DLM (>53.0 μL min⁻¹ mg⁻¹ protein) and RLM (>86.1 μL min⁻¹ mg⁻¹ protein). The in vitro half-life of 4q was determined to be less than 30 min in DLM/RLM, between 30 and 60 min in MLM/MkLM, and greater than 60 min in the case of HLM. Based on the calculated results of the hepatic extraction ratio, 4q belongs to the moderate to high (<0.3: low and >0.7: high) hepatic extraction ratio category of molecule depending on the particular species.^30,31 Amitriptyline, a widely used antidepressant drug, is reported to have a similar line of species-specific metabolic stability.³² Thus, overall data of 4q, especially from HLM, suggest that the molecule has adequate metabolic stability, and further in vivo investigations using the preclinical model can be performed to ascertain the resemblance of in vitro data-based in vivo prediction behavior and subsequent efficacy studies. Hence, all the biological investigations have deduced that the developed molecule 4q can serve as a potent candidate in lead-oriented anticancer drug discovery programs.

Conclusions

To conclude, a series of di-arylated 1,2,4-triazole molecules have been generated, some of which successfully suppressed the growth of malignant cells in the MCF-7 breast cancer cell line in vitro. The developed synthetic toolbox is competent and straightforward, utilizing commercially available starting materials and common cross-coupling reactions. The most potent derivative 4q generates the hallmarks of apoptosis in malignant cells by inducing mitochondrial outer membrane permeabilization along with autophagosome and ROS elevation. Moreover, it raises the pro-apoptotic BAX and cleaved PARP protein levels that cause antiproliferative activity. Metabolic stability studies in liver microsomes along with the in vitro data-based in vivo prediction indicated that 4q can have promising pharmacokinetic behavior in humans. Thus the encouraging biological efficacy of 4q validates the developed 1,3-diarylated triazole series as promising anticancer agents targeting breast cancer. This series can be subjected to further SAR studies to identify lead anticancer drug candidates and the developments are currently underway in our laboratory.

Experimental

Chemistry

General information

All reactions were carried out under an air atmosphere of oven-/flame-dried glassware and standard syringe/septa techniques.

All heating reactions were performed in an oil bath. Unless otherwise noted, all commercial reagents and solvents were obtained from a commercial provider and used without further purification. All the catalysts and boronic acids were purchased from Sigma-Aldrich and Tokyo Chemical Industry Co., Ltd. (TCI). Thin-layer chromatography (TLC) was performed using precoated silica gel 60 F254 (Merck). TLC plates were visualized by exposing to UV light, iodine vapors, or potassium permanganate stain. Organic solutions were concentrated by rotary evaporation on an R-300 rotary evaporator and vacuum pump V-300 (Buchi, Switzerland). Flash column chromatography was performed on Merck flash silica gel 100–200 size. The melting points of solid compounds were determined on a TempStar KPM-207 A melting point apparatus and are uncorrected. ¹H (400 MHz), ¹³C{¹H} (101 MHz), and ¹⁹F (377 MHz) NMR spectra were recorded with Bruker 400 MHz NMR instruments. Chemical shifts (δ) were reported in parts per million (ppm) with respect to tetramethyl silane (TMS) as an internal standard. Coupling constants (J) are quoted in hertz (Hz). Proton and carbon magnetic resonance spectra (¹H NMR and ¹³C{¹H} NMR) were recorded using TMS in CDCl₃ solvent as the internal standard (¹H NMR: TMS at 0.00 ppm, CDCl₃ at 7.26 ppm; ¹³C{¹H} NMR: CDCl₃ at 77.0 ppm) and DMSO-d₆ solvent as the internal standard (¹H NMR: TMS at 0.00 ppm, DMSO-d₆ at 2.50 ppm; ¹³C{¹H} NMR: DMSO-d₆ at 39.92 ppm). All the NMR spectra were processed using MestReNova software. HRMS spectra were recorded with Waters Xevo G2-XS QToF instrument. Diffraction intensities were collected on a single crystal with a SuperNova, single source at offset/far, EosS2 diffractometer, with mirror-monochromated Mo-Kα (λ = 0.71073 Å) radiation at 293(2) K.

General synthetic procedure A for the preparation of compounds 3a–3t

A 50 mL oven-dried round-bottom flask with a magnetic bar was charged with 3-bromo-1H-1,2,4-triazole (100 mg, 0.676 mmol, 1.0 equiv.), arylboronic acid (0.811 mmol, 1.2 equiv.), Cu(OAc)₂ (0.338 mmol, 0.5 equiv.), DBU (2.028 mmol, 3.0 equiv.), and 4 Å molecular sieves (700 mg). After adding DCM (3–4 mL), the reaction mixture was stirred at room temperature under an atmosphere of oxygen for 10 hours. Completion of the reaction was monitored by TLC. After that, DCM (50 mL) was added to the reaction mixture. The reaction mixture was washed with (2 × 25 mL) saturated ammonium chloride solution. Then, the combined organic layer was dried over anhydrous sodium sulfate (Na₂SO₄) and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel to obtain the desired product.

3-Bromo-1-phenyl-1H-1,2,4-triazole (3a)^15a

The title compound 3a was synthesized according to procedure A. Compound 3a was obtained after column chromatography (EtOAc/petroleum ether = 17 : 83) as a white solid (116 mg, 77%). mp: 65–67 °C; ¹H NMR (400 MHz, CDCl₃) δ 8.42 (s, 1H), 7.63–7.60 (m, 2H), 7.49 (ddd, J = 8.1, 4.6, 1.2 Hz, 2H), 7.42–7.38 (m, 1H); ¹³C{¹H} NMR (101 MHz, CDCl₃) δ 142.14, 141.26, 136.36, 129.88, 128.77, 119.87.

3-Bromo-1-(4-methoxyphenyl)-1H-1,2,4-triazole (3b)¹⁶

The title compound 3b was synthesized according to procedure A. Compound 3b was obtained after column chromatography (EtOAc/petroleum ether = 20 : 80) as a beige solid (107 mg, 62%). mp: 51–53 °C; ¹H NMR (400 MHz, CDCl₃) δ 8.30 (s, 1H), 7.47 (d, J = 9.0 Hz, 2H), 6.94 (d, J = 9.0 Hz, 2H), 3.80 (s, 3H); ¹³C{¹H} NMR (101 MHz, CDCl₃) δ 159.76, 142.11, 140.71, 129.72, 121.70, 114.83, 55.65.

3-Bromo-1-(3-methoxyphenyl)-1H-1,2,4-triazole (3c)

The title compound 3c was synthesized according to procedure A. Compound 3c was obtained after column chromatography (EtOAc/petroleum ether = 20 : 80) as a beige solid (105 mg, 61%). mp: 102–104 °C; ¹H NMR (400 MHz, CDCl₃) δ 8.41 (s, 1H), 7.38 (t, J = 8.2 Hz, 1H), 7.20 (t, J = 2.2 Hz, 1H), 7.17 (ddd, J = 7.9, 2.0, 0.8 Hz, 1H), 6.94 (ddd, J = 8.4, 2.5, 0.8 Hz, 1H), 3.86 (s, 3H); ¹³C{¹H} NMR (101 MHz, CDCl₃) δ 160.74, 142.20, 141.21, 137.39, 130.66, 114.60, 111.54, 105.80, 55.68; HRMS (ESI) m/z calcd for C₉H₉BrN₃O [M + H]⁺: 253.9924, found 253.9922.

3-(3-Bromo-1H-1,2,4-triazol-1-yl)aniline (3d)

The title compound 3d was synthesized according to procedure A. Compound 3d was obtained after column chromatography (EtOAc/petroleum ether = 36 : 64) as a brown solid (92 mg, 57%). mp: 123–125 °C; ¹H NMR (400 MHz, CDCl₃) δ 8.37 (s, 1H), 7.23 (t, J = 8.0 Hz, 1H), 6.96 (t, J = 2.1 Hz, 1H), 6.92 (dd, J = 8.0, 1.4 Hz, 1H), 6.68 (dd, J = 8.1, 1.7 Hz, 1H), 3.94 (s, 2H); ¹³C{¹H} NMR (101 MHz, CDCl₃) δ 148.00, 142.10, 140.97, 137.30, 130.63, 115.05, 109.02, 106.23; HRMS (ESI) m/z calcd for C₈H₈BrN₄ [M + H]⁺: 238.9927, found 238.9939.

3-Bromo-1-(4-fluorophenyl)-1H-1,2,4-triazole (3e)¹⁶

The title compound 3e was synthesized according to procedure A. Compound 3e was obtained after column chromatography (EtOAc/petroleum ether = 15 : 85) as a white solid (108 mg, 66%). mp: 115–117 °C; ¹H NMR (400 MHz, CDCl₃) δ 8.38 (s, 1H), 7.63–7.59 (m, 2H), 7.19 (dd, J = 8.9, 8.1 Hz, 2H); ¹³C{¹H} NMR (101 MHz, CDCl₃) δ 162.32 (d, ¹J_C–F = 249.5 Hz), 142.20, 141.35, 132.63 (d, ⁴J_C–F = 3.1 Hz), 122.02 (d, ³J_C–F = 8.6 Hz), 116.87 (d, ²J_C–F = 23.4 Hz); ¹⁹F NMR (377 MHz, CDCl₃) δ −111.77 (s, 1F).

3-Bromo-1-(4-chlorophenyl)-1H-1,2,4-triazole (3f)

The title compound 3f was synthesized according to procedure A. Compound 3f was obtained after column chromatography (EtOAc/petroleum ether = 15 : 85) as a white solid (120 mg, 69%). mp: 113–115 °C; ¹H NMR (400 MHz, CDCl₃) δ 8.41 (s, 1H), 7.61–7.58 (m, 2H), 7.50–7.47 (m, 2H); ¹³C{¹H} NMR (101 MHz, CDCl₃) δ 142.05, 141.58, 134.85, 134.55, 130.08, 121.06; HRMS (ESI) m/z calcd for C₈H₆BrClN₃ [M + H]⁺: 257.9428, found 257.9431.

3-Bromo-1-(4-bromophenyl)-1H-1,2,4-triazole (3g)

The title compound 3g was synthesized according to procedure A. Compound 3g was obtained after column chromatography (EtOAc/petroleum ether = 20 : 80) as a white solid (145 mg, 71%). mp: 128–130 °C; ¹H NMR (400 MHz, CDCl₃) δ 8.42 (s, 1H), 7.65–7.62 (m, 2H), 7.55–7.52 (m, 2H); ¹³C{¹H} NMR (101 MHz, CDCl₃) δ 142.01, 141.61, 135.34, 133.05, 122.37, 121.27; HRMS (ESI) m/z calcd for C₈H₆Br₂N₃ [M + H]⁺: 301.8923, found 301.8925.

4-(3-Bromo-1H-1,2,4-triazol-1-yl)benzonitrile (3h)¹⁶

The title compound 3h was synthesized according to procedure A. Compound 3h was obtained after column chromatography (EtOAc/petroleum ether = 30 : 70) as a white solid (116 mg, 69%). mp: 154–156 °C; ¹H NMR (400 MHz, CDCl₃) δ 8.54 (s, 1H), 7.83 (s, 4H); ¹³C{¹H} NMR (101 MHz, CDCl₃) δ 142.50, 142.26, 139.10, 134.08, 119.81, 117.56, 112.40.

3-Bromo-1-(3-chlorophenyl)-1H-1,2,4-triazole (3i)

The title compound 3i was synthesized according to procedure A. Compound 3i was obtained after column chromatography (EtOAc/petroleum ether = 14 : 86) as a white solid (113 mg, 65%). mp: 80–82 °C; ¹H NMR (400 MHz, CDCl₃) δ 8.45 (s, 1H), 7.70 (t, J = 1.9 Hz, 1H), 7.55–7.51 (m, 1H), 7.44 (t, J = 8.0 Hz, 1H), 7.41–7.37 (m, 1H); ¹³C{¹H} NMR (101 MHz, CDCl₃) δ 142.16, 141.71, 137.15, 135.81, 130.97, 128.84, 120.18, 117.61; HRMS (ESI) m/z calcd for C₈H₆BrClN₃ [M + H]⁺: 257.9428, found 257.9436.

3-Bromo-1-(3-(trifluoromethyl)phenyl)-1H-1,2,4-triazole (3j)¹⁶

The title compound 3j was synthesized according to procedure A. Compound 3j was obtained after column chromatography (EtOAc/petroleum ether = 20 : 80) as a colorless liquid (135 mg, 69%). mp: NA; ¹H NMR (400 MHz, CDCl₃) δ 8.52 (s, 1H), 7.94 (s, 1H), 7.85 (dd, J = 5.3, 3.4 Hz, 1H), 7.67 (d, J = 7.4 Hz, 2H); ¹³C NMR (101 MHz, CDCl₃) δ 142.23, 141.92, 136.68, 132.63 (q, ²J_C–F = 33.3 Hz), 130.69, 130.10, 125.35 (q, ³J_C–F = 3.6 Hz), 123.22 (q, ¹J_C–F = 272.8 Hz), 122.69, 118.95, 116.87 (q, ³J_C–F = 3.9 Hz); ¹⁹F NMR (377 MHz, CDCl₃) δ −62.90 (s, 3F).

3-(3-Bromo-1H-1,2,4-triazol-1-yl)benzaldehyde (3k)

The title compound 3k was synthesized according to procedure A. Compound 3k was obtained after column chromatography (EtOAc/petroleum ether = 25 : 75) as an off-white solid (86 mg, 51%). mp: 128–130 °C; ¹H NMR (400 MHz, CDCl₃) δ 10.09 (s, 1H), 8.55 (s, 1H), 8.18–8.16 (m, 1H), 7.98–7.93 (m, 2H), 7.73 (t, J = 7.9 Hz, 1H); ¹³C{¹H} NMR (101 MHz, CDCl₃) δ 190.62, 142.21, 141.91, 137.74, 137.12, 130.89, 129.98, 125.15, 119.51; HRMS (ESI) m/z calcd for C₉H₇BrN₃O [M + H]⁺: 251.9767, found 251.9763.

3-Bromo-1-(3,4-difluorophenyl)-1H-1,2,4-triazole (3l)^15b

The title compound 3l was synthesized according to procedure A. Compound 3l was obtained after column chromatography (EtOAc/petroleum ether = 14 : 86) as a beige solid (105 mg, 60%). mp: 85–87 °C; ¹H NMR (400 MHz, CDCl₃) δ 8.40 (s, 1H), 7.55 (ddd, J = 10.2, 6.7, 2.6 Hz, 1H), 7.41–7.37 (m, 1H), 7.31 (dt, J = 17.1, 8.6 Hz, 1H); ¹³C{¹H} NMR (101 MHz, CDCl₃) δ 151.68 (dd, J = 52.6, 13.0 Hz), 149.18 (dd, J = 52.1, 13.1 Hz), 142.20, 141.71, 132.63 (dd, J = 7.9, 3.5 Hz), 118.54 (d, J = 19.0 Hz), 115.70 (dd, J = 6.6, 4.0 Hz), 110.20 (d, J = 21.8 Hz); ¹⁹F NMR (377 MHz, CDCl₃) δ −132.87 (d, J = 21.2 Hz, 1F), −136.00 (d, J = 21.2 Hz, 1F).

3-Bromo-1-(3-fluoro-4-methoxyphenyl)-1H-1,2,4-triazole (3m)

The title compound 3m was synthesized according to procedure A. Compound 3m was obtained after column chromatography (EtOAc/petroleum ether = 28 : 72) as a brown solid (127 mg, 69%). mp: 73–75 °C; ¹H NMR (400 MHz, CDCl₃) δ 8.33 (s, 1H), 7.41 (dd, J = 11.2, 2.4 Hz, 1H), 7.34 (dd, J = 8.8, 1.0 Hz, 1H), 7.04 (t, J = 8.7 Hz, 1H), 3.93 (s, 3H); ¹³C{¹H} NMR (101 MHz, CDCl₃) δ 152.32 (d, ¹J_C–F = 249.6 Hz), 148.10 (d, ³J_C–F = 10.5 Hz), 142.08, 141.15, 129.44 (d, ³J_C–F = 9.0 Hz), 115.79 (d, ⁴J_C–F = 3.8 Hz), 113.79 (d, ⁴J_C–F = 2.2 Hz), 109.25 (d, ²J_C–F = 22.9 Hz), 56.54; ¹⁹F NMR (377 MHz, CDCl₃) δ −130.92 (s, 1F); HRMS (ESI) m/z calcd for C₉H₈BrFN₃O [M + H]⁺: 271.9829, found 271.9834.

3-Bromo-1-(3,4-dimethoxyphenyl)-1H-1,2,4-triazole (3n)

The title compound 3n was synthesized according to procedure A. Compound 3n was obtained after column chromatography (EtOAc/petroleum ether = 28 : 72) as an off-white solid (113 mg, 59%). mp: 95–97 °C; ¹H NMR (400 MHz, CDCl₃) δ 8.33 (s, 1H), 7.19 (d, J = 2.5 Hz, 1H), 7.09 (dd, J = 8.6, 2.5 Hz, 1H), 6.92 (d, J = 8.6 Hz, 1H), 3.94 (s, 3H), 3.92 (s, 3H); ¹³C{¹H} NMR (101 MHz, CDCl₃) δ 149.95, 149.46, 142.15, 140.85, 129.98, 111.93, 111.21, 104.73, 56.28, 56.22; HRMS (ESI) m/z calcd for C₁₀H₁₁BrN₃O₂ [M + H]⁺: 284.0029, found 284.0017.

3-Bromo-1-(3,4,5-trimethoxyphenyl)-1H-1,2,4-triazole (3o)

The title compound 3o was synthesized according to procedure A. Compound 3o was obtained after column chromatography (EtOAc/petroleum ether = 35 : 65) as a beige solid (117 mg, 55%). mp: 120–122 °C; ¹H NMR (400 MHz, CDCl₃) δ 8.37 (s, 1H), 6.81 (s, 2H), 3.90 (s, 6H), 3.85 (s, 3H); ¹³C{¹H} NMR (101 MHz, CDCl₃) δ 154.01, 142.26, 141.01, 138.29, 132.22, 97.89, 61.04, 56.47; HRMS (ESI) m/z calcd for C₁₁H₁₃BrN₃O₃ [M + H]⁺: 314.0135, found 314.0126.

3-Bromo-1-(naphthalen-1-yl)-1H-1,2,4-triazole (3p)

The title compound 3p was synthesized according to procedure A. Compound 3p was obtained after column chromatography (EtOAc/petroleum ether = 17 : 83) as a white solid (109 mg, 59%). mp: 127–129 °C; ¹H NMR (400 MHz, CDCl₃) δ 8.31 (s, 1H), 8.01 (d, J = 7.8 Hz, 1H), 7.97–7.93 (m, 1H), 7.73–7.69 (m, 1H), 7.60–7.52 (m, 4H); ¹³C{¹H} NMR (101 MHz, CDCl₃) δ 146.12, 141.23, 134.21, 132.83, 130.81, 128.42, 128.40, 128.14, 127.27, 124.93, 123.68, 122.13; HRMS (ESI) m/z calcd for C₁₂H₉BrN₃ [M + H]⁺: 273.9974, found 273.9970.

1-(Benzo[d][1,3]dioxol-5-yl)-3-bromo-1H-1,2,4-triazole (3q)

The title compound 3q was synthesized according to procedure A. Compound 3q was obtained after column chromatography (EtOAc/petroleum ether = 27 : 73) as a white solid (107 mg, 59%). mp: 115–117 °C; ¹H NMR (400 MHz, CDCl₃) δ 8.30 (s, 1H), 7.11 (d, J = 2.2 Hz, 1H), 7.07–7.04 (m, 1H), 6.87 (d, J = 8.3 Hz, 1H), 6.06 (s, 2H); ¹³C{¹H} NMR (101 MHz, CDCl₃) δ 148.76, 148.06, 142.16, 140.91, 130.85, 113.73, 108.56, 102.35, 102.22; HRMS (ESI) m/z calcd for C₉H₇BrN₃O₂ [M + H]⁺: 267.9716, found 267.9723.

4-(3-Bromo-1H-1,2,4-triazol-1-yl)pyridine (3r)

The title compound 3r was synthesized according to procedure A. Compound 3r was obtained after column chromatography (EtOAc/petroleum ether = 70 : 30) as a beige solid (82 mg, 54%). mp: 138–140 °C; ¹H NMR (400 MHz, CDCl₃) δ 8.76 (d, J = 5.9 Hz, 2H), 8.61 (s, 1H), 7.62 (dd, J = 4.7, 1.5 Hz, 2H); ¹³C{¹H} NMR (101 MHz, CDCl₃) δ 151.82, 142.59, 142.37, 142.27, 112.88; HRMS (ESI) m/z calcd for C₇H₆BrN₄ [M + H]⁺: 224.9770, found 224.9761.

3-(3-Bromo-1H-1,2,4-triazol-1-yl)pyridine (3s)

The title compound 3s was synthesized according to procedure A. Compound 3s was obtained after column chromatography (EtOAc/petroleum ether = 65 : 35) as a beige solid (91 mg, 60%). mp: 97–99 °C; ¹H NMR (400 MHz, CDCl₃) δ 8.95 (d, J = 2.5 Hz, 1H), 8.68 (dd, J = 4.7, 1.1 Hz, 1H), 8.50 (s, 1H), 8.01 (ddd, J = 8.3, 2.6, 1.5 Hz, 1H), 7.48 (dd, J = 8.3, 4.8 Hz, 1H); ¹³C{¹H} NMR (101 MHz, CDCl₃) δ 149.95, 142.36, 142.18, 140.99, 133.08, 127.63, 124.35; HRMS (ESI) m/z calcd for C₇H₆BrN₄ [M + H]⁺: 224.9770, found 224.9759.

5-(3-Bromo-1H-1,2,4-triazol-1-yl)-2-methoxypyridine (3t)

The title compound 3t was synthesized according to procedure A. Compound 3t was obtained after column chromatography (EtOAc/petroleum ether = 60 : 40) as a brown solid (94 mg, 55%). mp: 120–122 °C; ¹H NMR (400 MHz, CDCl₃) δ 8.41–8.38 (m, 1H), 8.34 (s, 1H), 7.82 (dd, J = 8.9, 2.8 Hz, 1H), 6.86 (dd, J = 8.9, 0.5 Hz, 1H), 3.97 (s, 3H); ¹³C{¹H} NMR (101 MHz, CDCl₃) δ 164.10, 142.46, 141.47, 138.92, 131.88, 127.73, 111.96, 54.15; HRMS (ESI) m/z calcd for C₈H₈BrN₄O [M + H]⁺: 254.9876, found 254.9862.

General synthetic procedure B for the preparation of compounds 4a–4v

A solution of K₂CO₃ (1.338 mmol, 3.0 equiv.) in 1,4-dioxane and water (2 : 1, 3 mL) was taken in a round-bottom flask and was purged with a nitrogen balloon for 5 min at room temperature. A mixture of 3a (100 mg, 0.446 mmol, 1.0 equiv.) and arylboronic acid (0.535 mmol, 1.2 equiv.) was added to this reaction mixture, and it was again purged with nitrogen for 5 min. After adding Pd(PPh₃)₄ (0.022 mmol, 0.05 equiv.) and purging with nitrogen, the reaction mixture was refluxed for 15 hours. After completion of the reaction, the crude mixture was filtered through Celite and extracted with ethyl acetate (2 × 25 mL). The combined organic layer was dried over anhydrous sodium sulfate (Na₂SO₄) and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel to obtain the desired product.

3-(4-Methoxyphenyl)-1-phenyl-1H-1,2,4-triazole (4a)^9b

The title compound 4a was synthesized according to procedure B. Compound 4a was obtained after column chromatography (EtOAc/petroleum ether = 27 : 73) as a white solid (106 mg, 95%). mp: 115–117 °C; ¹H NMR (400 MHz, CDCl₃) δ 8.52 (s, 1H), 8.16–8.12 (m, 2H), 7.72 (dd, J = 8.5, 1.0 Hz, 2H), 7.49 (t, J = 7.9 Hz, 2H), 7.36 (t, J = 7.4 Hz, 1H), 7.00–6.97 (m, 2H), 3.84 (s, 3H); ¹³C{¹H} NMR (101 MHz, CDCl₃) δ 163.07, 160.78, 141.35, 137.19, 129.72, 128.01, 127.87, 123.36, 119.78, 114.05, 55.33.

3-(4-Chlorophenyl)-1-(3,4-dimethoxyphenyl)-1H-1,2,4-triazole (4b)

The title compound 4b was synthesized according to procedure B from 3n (126.7 mg, 0.446 mmol). Compound 4b was obtained after column chromatography (EtOAc/petroleum ether = 35 : 65) as a white solid (125 mg, 89%). mp: 137–139 °C; ¹H NMR (400 MHz, CDCl₃) δ 8.46 (s, 1H), 8.13–8.10 (m, 2H), 7.44–7.40 (m, 2H), 7.27 (d, J = 2.5 Hz, 1H), 7.16 (dd, J = 8.6, 2.5 Hz, 1H), 6.92 (d, J = 8.6 Hz, 1H), 3.96 (s, 3H), 3.92 (s, 3H); ¹³C{¹H} NMR (101 MHz, CDCl₃) δ 162.05, 149.88, 149.04, 141.70, 135.37, 130.64, 129.21, 128.86, 127.81, 112.02, 111.30, 104.74, 56.25, 56.22; HRMS (ESI) m/z calcd for C₁₆H₁₅ClN₃O₂ [M + H]⁺: 316.0847, found 316.0841.

1-(3,4-Dimethoxyphenyl)-3-(4-fluorophenyl)-1H-1,2,4-triazole (4c)

The title compound 4c was synthesized according to procedure B from 3n (126.7 mg, 0.446 mmol). Compound 4c was obtained after column chromatography (EtOAc/petroleum ether = 30 : 70) as a white solid (89 mg, 67%). mp: 144–146 °C; ¹H NMR (400 MHz, CDCl₃) δ 8.46 (s, 1H), 8.18 (dd, J = 8.8, 5.5 Hz, 2H), 7.29 (d, J = 2.4 Hz, 1H), 7.20–7.13 (m, 3H), 6.95 (d, J = 8.6 Hz, 1H), 3.98 (s, 3H), 3.94 (s, 3H); ¹³C{¹H} NMR (101 MHz, CDCl₃) δ 163.68 (d, ¹J_C–F = 248.6 Hz), 162.23, 149.90, 149.03, 141.65, 130.73, 128.44 (d, ³J_C–F = 8.3 Hz), 126.95 (d, ¹J_C–F = 3.1 Hz), 115.65 (d, ²J_C–F = 21.9 Hz), 112.03, 111.33, 104.79, 56.26, 56.24; ¹⁹F NMR (377 MHz, CDCl₃) δ −111.75 (s, 1F); HRMS (ESI) m/z calcd for C₁₆H₁₅FN₃O₂ [M + H]⁺: 300.1143, found 300.1150.

1-(3,4-Dimethoxyphenyl)-3-(4-(trifluoromethyl)phenyl)-1H-1,2,4-triazole (4d)

The title compound 4d was synthesized according to procedure B from 3n (126.7 mg, 0.446 mmol). Compound 4d was obtained after column chromatography (EtOAc/petroleum ether = 30 : 70) as a white solid (117 mg, 75%). mp: 86–88 °C; ¹H NMR (400 MHz, CDCl₃) δ 8.49 (s, 1H), 8.29 (d, J = 8.1 Hz, 2H), 7.70 (d, J = 8.2 Hz, 2H), 7.28 (d, J = 2.4 Hz, 1H), 7.18 (dd, J = 8.6, 2.4 Hz, 1H), 6.93 (d, J = 8.6 Hz, 1H), 3.97 (s, 3H), 3.92 (s, 3H); ¹³C{¹H} NMR (101 MHz, CDCl₃) δ 161.69, 149.89, 149.15, 141.88, 134.03, 131.12 (q, ²J_C–F = 32.4 Hz), 130.53, 126.71, 125.60 (q, ³J_C–F = 3.6 Hz), 124.13 (q, ¹J_C–H = 272.2 Hz), 112.08, 111.28, 104.72, 56.23, 56.19; ¹⁹F NMR (377 MHz, CDCl₃) δ −62.65 (s, 3F); HRMS (ESI) m/z calcd for C₁₇H₁₅F₃N₃O₂ [M + H]⁺: 350.1111, found 350.1115.

1-(3,4-Dimethoxyphenyl)-3-(3-(trifluoromethyl)phenyl)-1H-1,2,4-triazole (4e)

The title compound 4e was synthesized according to procedure B from 3n (126.7 mg, 0.446 mmol). Compound 4e was obtained after column chromatography (EtOAc/petroleum ether = 28 : 72) as a white solid (121 mg, 78%). mp: 118–120 °C; ¹H NMR (400 MHz, CDCl₃) δ 8.50 (s, 1H), 8.47 (s, 1H), 8.37 (d, J = 7.8 Hz, 1H), 7.67 (d, J = 7.8 Hz, 1H), 7.58 (t, J = 7.8 Hz, 1H), 7.30 (d, J = 2.4 Hz, 1H), 7.20 (dd, J = 8.6, 2.5 Hz, 1H), 6.95 (d, J = 8.6 Hz, 1H), 3.99 (s, 3H), 3.94 (s, 3H); ¹³C{¹H} NMR (101 MHz, CDCl₃) δ 161.75, 149.95, 149.17, 141.85, 131.52, 131.14 (q, ²J_C–F = 32.5 Hz), 130.58, 129.65, 129.15, 125.98 (q, ³J_C–F = 3.6 Hz), 124.08 (q, ¹J_C–F = 272.3 Hz), 123.41 (q, ³J_C–F = 3.7 Hz), 112.10, 111.33, 104.80, 56.29, 56.23; ¹⁹F NMR (377 MHz, CDCl₃) δ −62.67 (s, 3F); HRMS (ESI) m/z calcd for C₁₇H₁₅F₃N₃O₂ [M + H]⁺: 350.1111, found 350.1112.

3-(3,4-Difluorophenyl)-1-(3,4-dimethoxyphenyl)-1H-1,2,4-triazole (4f)

The title compound 4f was synthesized according to procedure B from 3n (126.7 mg, 0.446 mmol). Compound 4f was obtained after column chromatography (EtOAc/petroleum ether = 30 : 70) as a white solid (113 mg, 80%). mp: 137–139 °C; ¹H NMR (400 MHz, CDCl₃) δ 8.46 (s, 1H), 8.00 (ddd, J = 11.2, 7.7, 2.0 Hz, 1H), 7.93 (ddd, J = 8.6, 4.1, 1.7 Hz, 1H), 7.28–7.16 (m, 3H), 6.94 (d, J = 8.6 Hz, 1H), 3.98 (s, 3H), 3.93 (s, 3H); ¹³C{¹H} NMR (101 MHz, CDCl₃) δ 161.28, 152.12 (dd, J = 73.0, 12.6 Hz), 149.92, 149.65 (dd, J = 70.3, 12.6 Hz), 149.12, 141.78, 130.56, 127.85 (dd, J = 6.6, 3.7 Hz), 122.77 (dd, J = 6.6, 3.7 Hz), 117.55 (d, J = 17.6 Hz), 115.67 (d, J = 19.0 Hz), 112.02, 111.31, 104.72, 56.26, 56.23; ¹⁹F NMR (377 MHz, CDCl₃) δ −136.50 (d, J = 21.1 Hz, 1F), −137.43 (d, J = 21.2 Hz, 1F); HRMS (ESI) m/z calcd for C₁₆H₁₄F₂N₃O₂ [M + H]⁺: 318.1049, found 318.1041.

1,3-Bis(benzo[d][1,3]dioxol-5-yl)-1H-1,2,4-triazole (4g)

The title compound 4g was synthesized according to procedure B from 3q (119.6 mg, 0.446 mmol). Compound 4g was obtained after column chromatography (EtOAc/petroleum ether = 28 : 72) as a white solid (110 mg, 80%). mp: 195–197 °C; ¹H NMR (400 MHz, DMSO-d₆) δ 9.13 (s, 1H), 7.61 (dd, J = 8.1, 1.5 Hz, 1H), 7.50 (dd, J = 9.1, 1.8 Hz, 2H), 7.35 (dd, J = 8.4, 2.2 Hz, 1H), 7.03 (dd, J = 19.2, 8.2 Hz, 2H), 6.11 (s, 2H), 6.07 (s, 2H); ¹³C{¹H} NMR (101 MHz, DMSO-d₆) δ 161.77, 148.83, 148.59, 148.17, 147.12, 143.49, 131.75, 125.00, 120.74, 113.32, 109.09, 109.05, 106.51, 102.46, 101.86, 101.72; HRMS (ESI) m/z calcd for C₁₆H₁₂N₃O₄ [M + H]⁺: 310.0822, found 310.0824.

1-(Benzo[d][1,3]dioxol-5-yl)-3-(naphthalen-1-yl)-1H-1,2,4-triazole (4h)

The title compound 4h was synthesized according to procedure B from 3q (119.6 mg, 0.446 mmol). Compound 4h was obtained after column chromatography (EtOAc/petroleum ether = 28 : 72) as an off-white solid (108 mg, 77%). mp: 146–148 °C; ¹H NMR (400 MHz, CDCl₃) δ 9.13 (d, J = 8.6 Hz, 1H), 8.56 (s, 1H), 8.28 (dd, J = 7.2, 1.0 Hz, 1H), 7.93 (dd, J = 12.0, 8.2 Hz, 2H), 7.63–7.52 (m, 3H), 7.31 (d, J = 2.1 Hz, 1H), 7.20 (dd, J = 8.3, 2.2 Hz, 1H), 6.90 (d, J = 8.3 Hz, 1H), 6.05 (s, 2H); ¹³C{¹H} NMR (101 MHz, CDCl₃) δ 163.30, 148.69, 147.47, 140.97, 134.05, 131.74, 130.88, 130.18, 128.49, 128.16, 127.58, 126.93, 126.43, 125.95, 125.26, 113.44, 108.56, 102.36, 102.06; HRMS (ESI) m/z calcd for C₁₉H₁₄N₃O₂ [M + H]⁺: 316.1081, found 316.1077.

3-(4-Fluorophenyl)-1-(3,4,5-trimethoxyphenyl)-1H-1,2,4-triazole (4i)

The title compound 4i was synthesized according to procedure B from 3o (140.1 mg, 0.446 mmol). Compound 4i was obtained after column chromatography (EtOAc/petroleum ether = 40 : 60) as a white solid (133 mg, 91%). mp: 150–152 °C; ¹H NMR (400 MHz, CDCl₃) δ 8.48 (s, 1H), 8.17 (dd, J = 8.8, 5.5 Hz, 2H), 7.14 (t, J = 8.7 Hz, 2H), 6.91 (s, 2H), 3.93 (s, 6H), 3.87 (s, 3H); ¹³C{¹H} NMR (101 MHz, CDCl₃) δ 163.71 (d, ¹J_C–F = 248.9 Hz), 162.30, 154.00, 141.73, 137.91, 132.98, 128.49 (d, ³J_C–F = 8.4 Hz), 126.80 (d, ⁴J_C–F = 3.1 Hz), 115.66 (d, ²J_C–F = 21.8 Hz), 98.01, 61.05, 56.44; ¹⁹F NMR (377 MHz, CDCl₃) δ −111.54 (s, 1F); HRMS (ESI) m/z calcd for C₁₇H₁₇FN₃O₃ [M + H]⁺: 330.1248, found 330.1241.

3-(1-(3,4,5-Trimethoxyphenyl)-1H-1,2,4-triazol-3-yl)phenol (4j)

The title compound 4j was synthesized according to procedure B from 3o (140.1 mg, 0.446 mmol). Compound 4j was obtained after column chromatography (EtOAc/petroleum ether = 60 : 40) as a white solid (124 mg, 85%). mp: 134–136 °C; ¹H NMR (400 MHz, CDCl₃) δ 8.56 (s, 1H), 7.75 (d, J = 1.9 Hz, 1H), 7.70 (d, J = 7.7 Hz, 1H), 7.29 (t, J = 7.9 Hz, 1H), 6.93–6.89 (m, 3H), 3.89 (s, 6H), 3.87 (s, 3H); ¹³C{¹H} NMR (101 MHz, CDCl₃) δ 162.49, 156.75, 153.93, 141.70, 137.87, 132.78, 131.30, 130.13, 118.53, 117.19, 113.65, 97.95, 61.06, 56.41; HRMS (ESI) m/z calcd for C₁₇H₁₈N₃O₄ [M + H]⁺: 328.1292, found 328.1295.

3-(1-(3,4,5-Trimethoxyphenyl)-1H-1,2,4-triazol-3-yl)aniline (4k)

The title compound 4k was synthesized according to procedure B from 3o (140.1 mg, 0.446 mmol). Compound 4k was obtained after column chromatography (acetone/petroleum ether = 50 : 50) as a white solid (125 mg, 86%). mp: 111–113 °C; ¹H NMR (400 MHz, CDCl₃) δ 8.48 (s, 1H), 7.61–7.58 (m, 1H), 7.54–7.53 (m, 1H), 7.25 (t, J = 7.8 Hz, 1H), 6.92 (s, 2H), 6.77–6.74 (m, 1H), 3.95 (s, 8H), 3.88 (s, 3H); ¹³C{¹H} NMR (101 MHz, CDCl₃) δ 163.21, 153.99, 146.70, 141.58, 137.88, 133.10, 131.44, 129.67, 116.95, 116.37, 113.06, 98.10, 61.07, 56.47; HRMS (ESI) m/z calcd for C₁₇H₁₉N₄O₃ [M + H]⁺: 327.1452, found 327.1446.

3-(3-Methoxyphenyl)-1-(3,4,5-trimethoxyphenyl)-1H-1,2,4-triazole (4l)

The title compound 4l was synthesized according to procedure B from 3o (140.1 mg, 0.446 mmol). Compound 4l was obtained after column chromatography (EtOAc/petroleum ether = 40 : 60) as an orange solid (82 mg, 54%). mp: 150–152 °C; ¹H NMR (400 MHz, CDCl₃) δ 8.52 (s, 1H), 7.85–7.81 (m, 1H), 7.76 (dd, J = 2.4, 1.4 Hz, 1H), 7.41 (t, J = 8.0 Hz, 1H), 7.01 (ddd, J = 8.2, 2.6, 0.9 Hz, 1H), 6.96 (s, 2H), 3.98 (s, 6H), 3.93 (s, 3H), 3.91 (s, 3H); ¹³C{¹H} NMR (101 MHz, CDCl₃) δ 163.02, 159.90, 154.01, 141.69, 137.90, 133.06, 131.82, 129.77, 119.06, 116.04, 111.24, 98.11, 61.09, 56.48, 55.45; HRMS (ESI) m/z calcd for C₁₈H₂₀N₃O₄ [M + H]⁺: 342.1448, found 342.1453.

3-(3-(Trifluoromethyl)phenyl)-1-(3,4,5-trimethoxyphenyl)-1H-1,2,4-triazole (4m)

The title compound 4m was synthesized according to procedure B from 3o (140.1 mg, 0.446 mmol). Compound 4m was obtained after column chromatography (EtOAc/petroleum ether = 40 : 60) as an orange solid (150 mg, 89%). mp: 92–94 °C; ¹H NMR (400 MHz, CDCl₃) δ 8.55 (s, 1H), 8.49 (s, 1H), 8.39 (d, J = 7.8 Hz, 1H), 7.69 (d, J = 7.8 Hz, 1H), 7.60 (t, J = 7.8 Hz, 1H), 6.95 (s, 2H), 3.97 (s, 6H), 3.90 (s, 3H); ¹³C{¹H} NMR (101 MHz, CDCl₃) δ 161.85, 154.03, 141.93, 138.01, 132.86, 131.38, 131.12 (q, ²J_C–F = 32.5 Hz), 129.69, 129.18, 126.07 (q, ³J_C–F = 3.6 Hz), 124.06 (q, ¹J_C–F = 272.5 Hz), 123.42 (q, ³J_C–F = 3.8 Hz), 98.01, 61.06, 56.46; ¹⁹F NMR (377 MHz, CDCl₃) δ −62.66 (s, 3F); HRMS (ESI) m/z calcd for C₁₈H₁₇F₃N₃O₃ [M + H]⁺: 380.1217, found 380.1215.

3-(1-(3,4,5-Trimethoxyphenyl)-1H-1,2,4-triazol-3-yl)benzaldehyde (4n)

The title compound 4n was synthesized according to procedure B from 3o (140.1 mg, 0.446 mmol). Compound 4n was obtained after column chromatography (EtOAc/petroleum ether = 50 : 50) as a white solid (130 mg, 86%). mp: 162–164 °C; ¹H NMR (400 MHz, CDCl₃) δ 10.09 (s, 1H), 8.68 (s, 1H), 8.54 (s, 1H), 8.45–8.42 (m, 1H), 7.94–7.91 (m, 1H), 7.61 (t, J = 7.7 Hz, 1H), 6.94 (s, 2H), 3.94 (s, 6H), 3.87 (s, 3H); ¹³C{¹H} NMR (101 MHz, CDCl₃) δ 192.04, 161.88, 154.05, 141.93, 138.06, 136.84, 132.87, 132.22, 131.66, 129.94, 129.41, 128.31, 98.01, 61.05, 56.49; HRMS (ESI) m/z calcd for C₁₈H₁₈N₃O₄ [M + H]⁺: 340.1292, found 340.1288.

3-(3,4-Dimethoxyphenyl)-1-(3,4,5-trimethoxyphenyl)-1H-1,2,4-triazole (4o)

The title compound 4o was synthesized according to procedure B from 3o (140.1 mg, 0.446 mmol). Compound 4o was obtained after column chromatography (acetone/petroleum ether = 30 : 70) as a brown solid (141 mg, 85%). mp: 140–142 °C; ¹H NMR (400 MHz, CDCl₃) δ 8.46 (s, 1H), 7.78 (dd, J = 8.3, 1.8 Hz, 1H), 7.70 (d, J = 1.7 Hz, 1H), 6.95 (d, J = 8.4 Hz, 1H), 6.91 (s, 2H), 3.98 (s, 3H), 3.94 (s, 6H), 3.93 (s, 3H), 3.87 (s, 3H); ¹³C{¹H} NMR (101 MHz, CDCl₃) δ 163.03, 154.00, 150.33, 149.10, 141.67, 137.92, 133.07, 123.43, 119.52, 111.13, 109.47, 98.24, 61.05, 56.48, 56.06, 55.95; HRMS (ESI) m/z calcd for C₁₉H₂₂N₃O₅ [M + H]⁺: 372.1554, found 372.1549.

3-(3,4-Difluorophenyl)-1-(3,4,5-trimethoxyphenyl)-1H-1,2,4-triazole (4p)

The title compound 4p was synthesized according to procedure B from 3o (140.1 mg, 0.446 mmol). Compound 4p was obtained after column chromatography (EtOAc/petroleum ether = 35 : 65) as a white solid (138 mg, 89%). mp: 122–124 °C; ¹H NMR (400 MHz, CDCl₃) δ 8.48 (s, 1H), 7.99 (ddd, J = 11.2, 7.7, 2.0 Hz, 1H), 7.94–7.90 (m, 1H), 7.24–7.19 (m, 1H), 6.90 (s, 2H), 3.94 (s, 6H), 3.87 (s, 3H); ¹³C{¹H} NMR (101 MHz, CDCl₃) δ 161.37, 154.03, 152.13 (dd, J = 78.4, 12.6 Hz), 149.66 (dd, J = 75.5, 12.6 Hz), 141.85, 138.04, 132.84, 127.73 (dd, J = 6.6, 3.8 Hz), 122.82 (dd, J = 6.6, 3.7 Hz), 117.55 (d, J = 17.6 Hz), 115.70 (d, J = 19.1 Hz), 98.00, 61.05, 56.45; ¹⁹F NMR (377 MHz, CDCl₃) δ −136.33 (d, J = 21.2 Hz, 1F), −137.39 (d, J = 21.1 Hz, 1F); HRMS (ESI) m/z calcd for C₁₇H₁₆F₂N₃O₃ [M + H]⁺: 348.1154, found 348.1165.

3-(3-Fluoro-4-methoxyphenyl)-1-(3,4,5-trimethoxyphenyl)-1H-1,2,4-triazole (4q)

The title compound 4q was synthesized according to procedure B from 3o (140.1 mg, 0.446 mmol). Compound 4q was obtained after column chromatography (EtOAc/petroleum ether = 50 : 50) as an off-white solid (141 mg, 88%). mp: 120–122 °C; ¹H NMR (400 MHz, CDCl₃) δ 8.47 (s, 1H), 7.93–7.89 (m, 2H), 7.01 (d, J = 8.4 Hz, 1H), 6.91 (s, 2H), 3.94 (s, 6H), 3.93 (s, 3H), 3.87 (s, 3H); ¹³C{¹H} NMR (101 MHz, CDCl₃) δ 162.06 (d, ⁴J_C–F = 2.6 Hz), 154.01, 152.39 (d, ¹J_C–F = 245.3 Hz), 150.73, 148.86 (d, ³J_C–F = 10.8 Hz), 141.68, 137.87, 132.99, 123.77 (d, ³J_C–F = 7.4 Hz), 122.71 (d, ⁴J_C–F = 3.4 Hz), 114.43 (d, ²J_C–F = 20.2 Hz), 113.20, 105.18, 97.97, 97.19, 61.09, 56.47, 56.27; ¹⁹F NMR (377 MHz, CDCl₃) δ −135.03 (s, 1F); HRMS (ESI) m/z calcd for C₁₈H₁₉FN₃O₄ [M + H]⁺: 360.1354, found 360.1360.

3-(3,5-Dichlorophenyl)-1-(3,4,5-trimethoxyphenyl)-1H-1,2,4-triazole (4r)

The title compound 4r was synthesized according to procedure B from 3o (140.1 mg, 0.446 mmol). Compound 4r was obtained after column chromatography (EtOAc/petroleum ether = 30 : 70) as a white solid (157 mg, 93%). mp: 136–138 °C; ¹H NMR (400 MHz, CDCl₃) δ 8.49 (s, 1H), 8.04 (d, J = 1.9 Hz, 2H), 7.35 (s, 1H), 6.89 (s, 2H), 3.93 (s, 6H), 3.87 (s, 3H); ¹³C{¹H} NMR (101 MHz, CDCl₃) δ 160.72, 154.01, 141.88, 138.02, 135.33, 133.37, 132.71, 129.26, 125.68, 124.87, 123.22, 97.82, 61.05, 56.45; HRMS (ESI) m/z calcd for C₁₇H₁₆Cl₂N₃O₃ [M + H]⁺: 380.0563, found 380.0559.

2-Methoxy-5-(1-(3,4,5-trimethoxyphenyl)-1H-1,2,4-triazol-3-yl)pyridine (4s)

The title compound 4s was synthesized according to procedure B from 3o (140.1 mg, 0.446 mmol). Compound 4s was obtained after column chromatography (EtOAc/petroleum ether = 55 : 45) as a white solid (146 mg, 96%). mp: 178–180 °C; ¹H NMR (400 MHz, CDCl₃) δ 8.97 (dd, J = 2.3, 0.6 Hz, 1H), 8.48 (s, 1H), 8.32 (dd, J = 8.6, 2.4 Hz, 1H), 6.91 (s, 2H), 6.82 (dd, J = 8.6, 0.6 Hz, 1H), 3.99 (s, 3H), 3.94 (s, 6H), 3.87 (s, 3H); ¹³C{¹H} NMR (101 MHz, CDCl₃) δ 164.91, 161.10, 154.01, 145.66, 141.69, 137.93, 136.82, 132.95, 120.23, 110.79, 98.02, 61.07, 56.46, 53.72; HRMS (ESI) m/z calcd for C₁₇H₁₉N₄O₄ [M + H]⁺: 343.1401, found 343.1407.

6-(1-(3,4,5-Trimethoxyphenyl)-1H-1,2,4-triazol-3-yl)-1H-benzo[d]imidazole (4t)

The title compound 4t was synthesized according to procedure B from 3o (140.1 mg, 0.446 mmol). Compound 4t was obtained after column chromatography (MeOH/CH₂Cl₂ = 6 : 94) as a white solid (110 mg, 70%). mp: >220 °C; ¹H NMR (400 MHz, DMSO-d₆) δ 12.62 (s, 1H), 9.32 (s, 1H), 8.30 (s, 2H), 8.00 (d, J = 8.4 Hz, 1H), 7.70 (s, 1H), 7.24 (s, 2H), 3.89 (s, 6H), 3.69 (s, 3H); ¹³C{¹H} NMR (101 MHz, DMSO-d₆) δ 162.95, 154.02, 153.53, 151.51, 143.84, 143.77, 137.06, 133.28, 124.88, 120.84, 104.36, 97.65, 79.63, 60.68, 56.73; HRMS (ESI) m/z calcd for C₁₈H₁₈N₅O₃ [M + H]⁺: 352.1404, found 352.1402.

3-(Naphthalen-1-yl)-1-(3,4,5-trimethoxyphenyl)-1H-1,2,4-triazole (4u)

The title compound 4u was synthesized according to procedure B from 3o (140.1 mg, 0.446 mmol). Compound 4u was obtained after column chromatography (EtOAc/petroleum ether = 37 : 63) as an off-white solid (140 mg, 87%). mp: 118–120 °C; ¹H NMR (400 MHz, CDCl₃) δ 9.05 (d, J = 8.5 Hz, 1H), 8.63 (s, 1H), 8.24 (dd, J = 7.2, 0.9 Hz, 1H), 7.93 (dd, J = 14.0, 8.2 Hz, 2H), 7.61–7.51 (m, 3H), 6.99 (s, 2H), 3.94 (s, 6H), 3.90 (s, 3H); ¹³C{¹H} NMR (101 MHz, CDCl₃) δ 163.48, 154.04, 141.23, 137.92, 134.04, 133.12, 130.94, 130.22, 128.49, 128.20, 127.60, 126.90, 126.31, 125.97, 125.24, 98.14, 61.09, 56.49; HRMS (ESI) m/z calcd for C₂₁H₂₀N₃O₃ [M + H]⁺: 362.1499, found 362.1492.

3-(Benzo[d][1,3]dioxol-5-yl)-1-(3,4,5-trimethoxyphenyl)-1H-1,2,4-triazole (4v)

The title compound 4v was synthesized according to procedure B from 3o (140.1 mg, 0.446 mmol). Compound 4v was obtained after column chromatography (EtOAc/petroleum ether = 40 : 60) as a beige solid (139 mg, 88%). mp: 208–210 °C; ¹H NMR (400 MHz, CDCl₃) δ 8.45 (s, 1H), 7.73 (dd, J = 8.1, 1.6 Hz, 1H), 7.65 (d, J = 1.6 Hz, 1H), 6.91 (s, 2H), 6.89 (d, J = 8.1 Hz, 1H), 6.01 (s, 2H), 3.94 (s, 6H), 3.87 (s, 3H); ¹³C{¹H} NMR (101 MHz, CDCl₃) δ 162.84, 153.98, 148.82, 147.98, 141.51, 137.79, 133.06, 124.74, 120.83, 108.50, 106.99, 101.31, 97.93, 61.07, 56.45; HRMS (ESI) m/z calcd for C₁₈H₁₈N₃O₅ [M + H]⁺: 356.1241, found 356.1238.

Biology

Cell line and cell culture

All the cell lines were procured from ATCC, USA, and cultured as per the protocol provided. Cells were grown in a CO₂ incubator (Thermo) at 37 °C with 98% humidity and 5% CO₂ gas environment.³³

Cell viability assay

Inhibition of cell proliferation by different compounds was measured with the 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide or MTT assay. The compounds were tested for cell viability against different human breast carcinoma lines using an MTT assay. 4 × 10⁴ cells were seeded into each well of a 96-well cell culture plate and allowed to adhere overnight. Additionally, the cells were incubated for 48 hours with 10 μM of each compound. Following the removal of the compound-containing medium, 20 μL of fresh MTT (2.5 mg mL⁻¹) was added to each well. The MTT-containing medium was removed after 4 hours of incubation. After that, 150 μL of DMSO was added to each well to dissolve the formazan crystals and produce a blue color. The optical density of the treated and untreated cells was measured using Cytation 5 (Agilent, BioTek, USA) at 570 nm and percentage inhibition was calculated.³³

IC₅₀ determination on cancer and normal breast cell lines

IC₅₀ values of 4q and 4t were determined on breast cancer cells (MCF-7) and normal breast cells (MCF-10A). Cells were seeded in tissue culture 96-well plates and incubated in a humidified chamber with 5% CO₂. The normal cell line was exposed to 10, 50, and 100 μM concentration of test article for 24 h, while MCF-7 was exposed to 0.625, 1.25, 2.5, 5, 10, 20, and 40 μM concentrations of test articles for 48 hours. After that, 20 μL of MTT (2.5 mg mL⁻¹) was added to each well and incubated for 4 hours followed by medium removal and formazan solubilization using 150 μL of DMSO. The optical density of the treated and untreated cells was measured using Cytation 5 (Agilent, BioTek, USA) at 570 nm and percentage growth inhibition was calculated.³³

Nuclear staining for apoptosis

Nuclear staining and fluorescence microscopy. The MCF-7 cells were treated with 5, 10, and 20 μM concentrations of 4q for 48 hours. After treatment, cells were collected, washed twice with PBS, and fixed in 400 μL of cold acetic acid : methanol (1 : 3, v/v) overnight at 4 °C. The next day, cells were washed and dispensed in 50 μL of fixing solution. After that, the cells were spread out on a clean slide and allowed to dry at room temperature overnight. The cells were stained with DAPI (5 μg mL⁻¹) for 30 min at room temperature, then the slides were rinsed with distilled water followed by washing with PBS. About 30–40 μL of mounting fluid (PBS : glycerol, 1 : 1) was poured over it and then sealed with a glass coverslip. Cells were observed under a microscope for any nuclear morphological changes that occur during apoptosis. For phase-contrast microscopy, cells were simply photographed using a microscope after treatment.³³

Rhodamine 123 staining

To examine MMP-mediated apoptosis triggered by compound 4q in MCF-7 cells, rhodamine staining was utilized. Rhodamine dyes, under normal physiological conditions, tend to gather in mitochondria that are functional and exhibit polarization, giving rise to strong fluorescence. In contrast, a disturbed mitochondrial membrane potential prevents the dye from entering the mitochondria, causing a noticeable drop in the fluorescence intensity of rhodamine. The staining procedure conducted with rhodamine was as follows: MCF-7 cells underwent treatment with concentrations of 5, 10, and 20 μM of 4q and just 30 min before concluding the experiment, RH-123 (1 μg) (Catalogue Number R8004) was introduced. Cells were then cleansed with PBS before being evaluated using a fluorescence microscope.³³

DCFDA staining for ROS generation

This dye measures ROS generation after chemical treatment or genetic modifications. This staining is useful for determining cellular oxidative stress upon environmental stress, providing clues to cell death in mechanistic studies. After treatment with compound 4q, the cells were stained with 20 μM DCFDA and incubated for 30 min at 37 °C. Once the incubation was completed, images were captured in a fluorescence microscope (Zeiss Axiovert A1).³³

Acridine orange staining for autophagy

Vital stain acridine orange will intercalate with nucleic acid, altering the dye's optical properties and causing it to flash brilliant orange when exposed to ultraviolet light. Every cell that has nucleic acids will glow orange. After treatment with compound 4q cells were stained with 200 nM acridine orange dye and incubated for 10 min at 37 °C. Once the incubation was completed, images were captured in a fluorescence microscope (Zeiss Axiovert A1).³³

Western blot analysis

Cells (MCF-7) were seeded in 100 mm dishes at a density of 1 × 10⁶ cells per dish in 8 mL RPMI 1640 medium containing 10% FBS and 1× pen–strep in a CO₂ incubator. Seeded cells were treated with 4q at different concentrations (5, 10, 15, and 20 μM) in an incubator with 5% CO₂ at 37 °C for 48 hours. After treatment, the cells were washed thrice with PBS then trypsinized and pelleted down at 1100 RPM for 5 min at 4 °C. Cell pellets were washed again with PBS and centrifuged for 5 min at 1100 RPM. The supernatant was discarded and the cell pellets were lysed with RIPA buffer containing protease inhibitor. The cell lysate was centrifuged at 12 000g for 15 min and the supernatant was collected for further use. The concentrations of protein in supernatants were assayed using the Bio-Rad Protein Assay Dye (Bio-Rad Laboratories, Inc., Hercules, CA, USA) with bovine serum albumin (Bio-Rad Laboratories, Inc.) as standard. The aforementioned supernatant from cell lysate was mixed with an equal volume of 2× loading dye and the solution was heated for 5 min at 100 °C. A total of 50 μg proteins were loaded to each lane and were resolved using SDS-PAGE electrophoresis. After electrophoresis, the gel was transferred onto PVDF membranes for immunoblotting. The membranes were blocked with 5% BSA in TBST for 1 hour at room temperature. Primary and secondary antibodies for cell signalling-related proteins including blot were scanned on ChemiDoc and Bio-Rad systems. The bands were quantified using Image Lab software (Bio-Rad).³³

Antibodies

Antibodies for Western blot were procured from Cell Signaling Technology (CST): cleaved PARP (#5625 1 : 1000), Bax (#sc-7480 1 : 400).

Metabolic stability assessment

Verapamil hydrochloride (purity >99%), NADPH tetrasodium salt (purity ≥98%), diazepam (purity ≥98%), and phosphate-buffered saline (PBS) were purchased from Sigma-Aldrich. HLM (Lot No #PL050H-A), MkLM (Lot No #MK065-B), DLM (Lot No #DG035-C), RLM (Lot No #RT064-B), and MLM, (Lot No #2513990-A) were procured from Gibco. MgCl₂ hexahydrate and DMSO were obtained from Rankem and Loba Chemie, respectively. MS-grade methanol, acetonitrile, formic acid, and water were received from Thermo Fisher Scientific. All other reagents/solvents were of analytical grade or above.

The metabolic stability study of 4q was performed in HLM, MkLM, DLM, RLM, and MLM using our earlier reported protocol.^28,34 The stock solution of the test compound was prepared in DMSO, and further dilutions were prepared in PBS. Briefly, the reaction mixture (100 μL) contained PBS (100 mM, pH 7.4), MgCl₂ (3.3 mM), microsomal protein (0.5 mg mL⁻¹), and NADPH (1.2 mM). The reaction mixture was pre-incubated (5 min) in a pre-heated shaking water bath (37 °C). Then test candidate (5 μM) was spiked into it, followed by further incubation in a pre-heated shaking water bath (37 °C) for 0 min, 15 min, and 30 min. Following incubation, the reaction was quenched by adding chilled acetonitrile (100 μL) containing diazepam (250 ng mL⁻¹) as the internal standard (IS). The samples were then vortex-mixed (2 min), centrifuged (14 000 rpm, 10 min), transferred to vials, and analyzed by LC-MS/MS. The reaction was carried out in triplicate. Verapamil (5 μM) was used as a positive control in HLM. The reaction without NADPH served as the negative control. The percent substrate remaining in the samples was calculated by comparing the data obtained at 0 min and considering it as 100% of the substrate. Then, the concentration data obtained at each time point was utilized for log-linear plotting to determine various parameters for predicting the in vivo behavior of the test compound.^35–38

Abbreviations used

DAPI

4′,6-Diamidino-2-phenylindole

DCHF-DA

2′,7′-Dichlorodihydrofluorescein diacetate

DLM

Dog liver microsome

HLM

Human liver microsome

IC₅₀

Half-maximal inhibitory concentration

MkLM

Monkey liver microsomes

MLM

Mouse liver microsomes

MOMP

Mitochondrial outer membrane permeabilization

MTT

3-[4,5-Dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide

ROS

Reactive oxygen species

PARP

Poly(ADP-ribose) polymerase

RLM

Rat liver microsome

SAR

Structure–activity relationship

SDS-PAGE

Sodium dodecyl sulfate polyacrylamide gel electrophoresis

Data availability

The data underlying this study are available in the published article and its ESI.†

Conflicts of interest

There are no conflicts to declare.