Novel hybrid motifs of 4-nitroimidazole-piperazinyl tagged 1,2,3-triazoles: Synthesis, crystal structure, anticancer evaluations, and molecular docking study

Abstract

4-((4-(1-benzyl-2-methyl-4-nitro-1H-imidazole-5-yl)piperazine-1-yl)methyl)-1-substituted-1H-1,2,3-triazole motifs are designed and synthesized via click chemistry. The reaction of 1-(N¹-benzyl- 2-methyl-4-nitro-1H-imidazole- 5-yl)-4-(prop-2-yn-1-yl) piperazine 5 as new scaffold with diverse primary azides to selectively produce 1,4-disubstituted-1,2,3-triazoles 9a–k, 10a–c and 11a–q. Physicochemical methods: when ¹H NMR, ¹³C NMR, and HRMS are utilized to fully characterize all synthesized compounds. X-ray structural determination and analysis for compound 9a is also performed. The newly designed chromophores are assessed for their anti-proliferative potency against three selected human cancer cell lines (MCF-7, HepG2, and PC3), and one normal cell line (Dermal/Fibroblast). Compounds 9g and 9k have shown potent activities against the MCF-7 cell line with IC₅₀ values of (2.00 ± 0.03 μM) and (5.00 ± 0.01 μM) respectively. ADMET studies and Molecular docking investigations are performed on the most active hybrid nitroimidazole derivatives 9g and 9k with 4-hydroxytamoxifen (4-OHT) at the human estrogen receptor alpha (hER) during binding active sites to study the ligand–protein interactions and free binding energies at atomic levels. The triazole ring in the 9g derivative forms a hydrogen bond with Asp58 with distance 3.2 Å. And it is found that polar contact with His231 amino acid residue.

In silico assessment of the compounds showed very good pharmacokinetic properties based on their physicochemical values, also the ADMET criteria of the most active hybrid systems are within the acceptable range.

Graphical abstract

A diverse library of 1,4-disubstituted-1,2,3-triazolo-tethered piperazine-nitroimidazole conjugates were designed, synthesized, and evaluated for anti-proliferative activity against three of human cancer cell lines. Particularly conjugates 9g and 9k exhibited promising cytotoxicity against MCF-7 cell line, with IC₅₀ values of 2 μM and 5 μM, respectively, compared to the standard Doxorubicin (0.64 μM) and Cisplatin (14 μM). On the top of that, docking at the human estrogen receptor alpha (hER) binding active site to study the ligand–protein interactions and free binding energies for selected compounds were investigated.

1. Introduction

Heterocyclic compound [1], as well as heterocyclic scaffolds, are among the most important organic compounds which are frequently present in molecules of interest in medicinal chemistry [2]. Furthermore, nitrogen containing heterocycles are of great importance to life science, where some representative alkaloids and other nitrogen showing diverse biological activities. Several of them are even prescribed drugs such as: serotonin [3], thiamine, which is also called vitamin B1 [4], atropine [5], notorious morphine [6], and most of the vitamins, nucleic acid, enzymes, co-enzymes, hormones, and alkaloids contain N-based heterocycles as scaffolds [7]. Due to exhibiting diverse biological activities, nitrogen heterocyclic compounds have always been attractive targets to synthetic organic chemists, these facts disclose and emphasize the vital role of heterocycles in modern drug design and drug discovery [8,9].

Molecular hybridization of heterocyclic motifs is a well-known concept in drug design and development that rely on the combination of pharmacophoric moieties of different bioactive substances. They produce a new hybrid system with improved activities when compared to the parent drugs [10]. Nitroimidazole-containing compounds are one of the most well-studied compounds in nature as well as in the synthetic world [[11], [12], [13]]. Imidazole's have attracted intense interest in recent years due to their diverse biological and pharmacological properties [14,15]. Besides clinically proven anticoagulant and antithrombotic actions, various triazoles have exhibited anticancer [16,17] and antimicrobial [18,19] activities. Additionally, this strategy can result in compounds presenting modified selectivity profile, different and/or dual modes of action and reduced undesired side effects [[20], [21], [22]]. The rational planning of new synthetic prototypes has been using a series of methods of structural modification that aim, a priori, at the generation of new compounds presenting optimized pharmacodynamic and pharmacokinetic properties, exploring bioactive substances' fragments (Fragment-Based Drug Design) [23], active metabolites of drugs [24], bioisosterism [25], selective optimization of side effects of drugs [26] and drug latentiation [27]. 1,2,3-Triazoles occupy a prominent place in drug discovery due to their facile synthesis through click chemistry [28,29] and wide biological profiles, which include antibacterial [30,31] and anticancer activities [32,33]. Potential structural features of bioactive triazoles include their stability to metabolic degradation, high selectivity and capability of hydrogen bonding that could be favorable in the binding of biomolecular targets [34]. It is demonstrated that different types of chemical bridges at C-4 of the 1,2,3-triazole core eliminate planarity, thereby they could ameliorate the drug-ability and facilitate the binding of compounds to their possible receptor through induced fit [35].

Over the last fifteen years, our laboratory has been synthesizing several derivatives of new 5-substituted piperazinyl-4-nitroimidazole derivatives and their evaluation for anti-HIV activity [36,37]. The nitroimidazoles are a common class of compounds that have been extensively studied as molecular probes because they diffuse freely in the body and are irreversibly trapped by nucleophilic covalent binding to proteins in low oxygen environments [38]. 4-Nitroimidazoles derivatives have been studied as radiopharmaceuticals for probing tumor hypoxia and in therapeutic treatment, antifertility potential, and anti-tubercular profile [39,40]. Other efforts have focused on the synthesis of very successful hybridization system reported by our group [41,42].

Estrogen hormone is a steroid compound that plays vital modulatory roles in physiological process specially in females, even though the adrenal cortex of males can produce estrogen [43,44]. Estrogen is produced mainly by the ovaries and placenta cells as in mediated by its interaction and activation of estrogen receptors ERα and ERβ, which are members of the nuclear receptor superfamily of transcription factors and characterized by highly conserved DNA- and ligand-binding domains [[45], [46], [47], [48]]. Estrogen receptors are located in the tissues of the female reproductive tract, breast, and diverse tissues such as the liver, bone, brain, skin, colon, and salivary gland [[49], [50], [51]]. Therefore, ERα regulates multiple complex physiological processes in humans [44]. However, ER is involved in generating tumors through estrogen signaling ERα protein, which is located on the surface of tumors, can bind to estrogen (17b-estradiol, E2) and then be involved in generating tumors through estrogen signaling. Thus, activating the hormone-responsive genes that promote DNA synthesis and cell reproduction [44]. This enhances the growth and spreading of cancer cells in various tumors, including reproductive system (endometriosis, and breast, ovarian, and prostate cancer), bone, lung cancer, cardiovascular disease, gastrointestinal disease, urogenital tract disease, neurodegenerative disorders, and cutaneous melanoma [49].

ERα is overextended in breast cancer, which is around 50–80% in breast cancer tissues compared to 10% in healthy tissues [52]. Therefore, estrogen has been identified as a key stimulant in the development of ERα positive breast cancer, which constitutes around 70–80% of all breast cancers [[53], [54], [55]]. The human breast cancer cell line MCF-7 contains estrogen receptors and responds to estrogens with an increase in growth rate and to antiestrogens with a decrease in growth rate as a selective estrogen receptor modulator (SERM), 4-Hydroxytamoxifen (4-OHT) [56] (Table 1). shows the role of ERs in different kinds of cancer cells used in this study. On the one hand, it can be noted that Erα shows promising roles in breast cancer development, whereas the role of ERβ remains arguable [49,[57], [58], [59], [60], [61]]. On the other hand, ERα and ERβ have significant effects on prostate cancer, where ERβ employs tumor growth-suppressive effects, and ERα exerts tumor growth-promoting effects [49,62,63]. ERβ shows an inhibiting role in the progress of liver cancer by reducing the fibrosis and immune response, and the role of ERα remains arguable and promotes the proliferation of hepatocellular carcinoma cells in the liver when ERα works together with ERβ [49,[64], [65], [66], [67], [68]].

Table 1.

The role of estrogen receptors in various cancers [49].

No.	Cancer type	ERα	ERβ
1	Breast cancer	Promotes tumor [57].	Inhibits tumor [58,59]. Promote tumor [60,61].
2	Prostate cancer	Promote cell proliferation [62,63].	Inhibits tumor [63].
3	Liver cancer	Promote liver cancer [64] Inhibits the liver cancer [65,66].	Inhibits tumor [67,68].

1,2,3-triazole offers a wide range of biological profiles for an extensive SAR analysis against a variety of molecular targets (Fig. 1). shows the successful hybridization strategy to produce new compounds with broad-spectrum activity towards cancer cell lines: MCF-7, MGC-803, and PC-3 [69] triazole compounds [70].

Fig. 1.

1,2,3-Triazole-based derivatives as binary-effective inhibitors against different kinds of cancer cell lines, a) Dithiocarbamate hybrids, b) Nitroimidazole-Isatin Conjugates, and c) Coumarin scaffold.

The Coumarin-tagged triazole hybrids, (Fig. 1c), was identified as dual-effective inhibitors against hER and the results showed cytotoxic activity against MCF-7 cancer cell lines [71,72]. Another successful hybridization can be shown in the 1,2,3-Triazole tethered Nitroimidazole−Isatin conjugates, which inhibited the development of breast cancer as shown in (Fig. 1b) [73].

Herein, in this work, a library of 4-((4-(1-benzyl-2-methyl-4-nitro-1H-imidazole-5-yl)piperazine-1-yl)methyl)-1-substituted-1H-1,2,3-triazole motifs has been designed and synthesized, on the new hybrid compounds were designed based on the click chemistry approach. Assessment of the synthesized compounds against three selected human cancer cell lines (MCF-7, HepG2, and PC3), and one normal cell line (Dermal/Fibroblast) were performed.

2. Experimental

2.1. Materials and methods

The chemicals are purchased from Aldrich (Riedstraβe, Germany), Fluka (Buchs, Switzerland), and Scharlau, are used without further purification, unless otherwise stated. The NMR spectra are recorded on a Bruker Avance III-(500 MHz) spectrometer with TMS as an internal standard. 1H NMR (500 MHz), 13C NMR (125 MHz) and 2D are recorded in deuterated Chloroform (CDCl3) or Dimethylsulfoxide (DMSO‑d₆). Chemical shifts are expressed in (δ) units and are reported in ppm and J-coupling values for 1H–1H coupling constants are given in Hertz (Hz). High-resolution mass spectra (HRMS) are measured (in positive/or negative ion mode) using the electrospray ion trap (ESI pos low mass) technique by collision-induced dissociation on a Bruker APEX-IV (7 T) instrument. Single crystal X-ray diffraction data is measured using Oxford diffraction Xcalibure: Eos diffractometer. The crystal is kept at 293(2) K, (λ = 0.71073 Å) during data collection. Using Olex2 (Dolomanov et al., 2009), the structure is solved with the ShelXT (Sheldrick, 2015) structure solution program using Intrinsic Phasing and refined with the ShelXL (Sheldrick, 2015), refinement package for Empirical absorption corrections is applied using Least Squares minimization. Melting points (m.p, uncorrected in °C) are determined on the electrothermal melting point apparatus and Stuart (SMP-10) melting point apparatus.

2.2. Cytotoxicity assay

Cell lines are seeded in 96-well flat-bottomed microplates in 100 μL culture medium at the following densities: MCF-7 and PC3 cells (3 × 10³ cells per well); HepG2 and Dermal fibroblast (4.0 × 10³ cells per well). Cells are allowed to adhere for 24 h. After work the medium is freshly replaced with a medium with the solvent or with the tested compounds at increasing concentrations from 0 to 250 μM on the cancerous cell lines and concentrations of 0–500 μM for the normal dermal fibroblast cells. The reference drugs Doxorubicin (0–100 μM) and Cisplatin (0–10 μM) are included as positive controls for growth inhibition. After 72 h, cell viability is assayed using an (MTT) assay. All of the experimental conditions are performed and repeated independently at least three times. Half maximal inhibitory concentrations (IC₅₀, the concentration required for 50% in vitro inhibition of growth) are calculated for each experiment using GRAPHPAD PRISM software (Version 8, San Diego, CA, USA). IC₅₀ values are reported as mean ± SD.

2.3. Dose response study

Traditionally, dose response studies of anti-cancer agents require testing a range of concentrations of the compounds of interests and determining the area under curve in animal models. However, recent validated software(s) that create a transformed “dose-response” curve with new metrics that measure growth rate (GR) effects due to drug treatment as well as other perturbations, for example genetic manipulation or changes in seeding density of cells have been widely used in clinical research.

In this study, we used GR calculator [74] software to calculate “GR curves” and summary metrics after determining the percent viability curves and traditional metrics in the conventional MTT assays described above. The following metrics were calculated:

E_inf: drug efficacy extrapolated to an infinitely high drug concentration as determined from the asymptote of a traditional dose–response curve. Which is similar to E_max in conventional studies.

GEC₅₀: analogous to EC₅₀, the concentration of drug at half-maximal effect. GEC₅₀ is relevant for drugs having poor efficacy for which the response does not reach GR values below 0.5. The normalized growth rate inhibition values and the associated dose–response curve was then extrapolated.

For partially cytostatic treatments (where growth is slowed, but not completely halted) GR values fall between 1 and 0. A GR value of zero represents cytostasis, or completely halted population growth. Cytotoxic treatments (where cell population declines) produce GR values between 0 and -1. Finally, a GR value of greater than one signifies a treatment that promotes growth.

2.4. Preparation of ligand and protein structures

Human estrogen receptor alpha (hER) was identified from the crystal structure (pdb ID: 3ert). First, hydrogen atoms were added to the proteins. The missing residues of the protein were built using the SWISS-MODEL server [75]. The grid box utility in AutoDock 4.2.6 was used to select the active site of the co-crystal ligand within the protein structure [76]. The 3-D structure of the compounds was built using the Gaussview program [77], the semiempirical PM3 method available in Gaussian 2003 was used for the ligands optimization to achieve the low energy level [78,79].

2.5. Molecular docking protocol

Docking calculations were performed using a Lamarckian Genetic Algorithm available in Autodock 4.2.6 [80]. The receptor and the ligands were prepared using the Autodock Tools program. Gasteiger partial charges were computed for each molecule and rotatable bonds were defined as free for the ligands and rigid for the receptor. The grid spacing was set at 0.375 Å. In this study, the docking runs were set to terminate after a maximum of 25 × 10⁶ energy evaluations, a maximum number of 27,000 generations, a mutation rate of 0.02, and the population size was set to use 300 randomly placed individuals. A total of 100 independent docking runs were carried out for the docking system. Finally, the best three conformations with the lowest score were selected for analysis using Chimera and Discovery Studio 4.0 programs [81,82].

2.6. ADMET analysis

Protocols of Swiss ADME was used to assess the drug-like properties of most potential compounds to predict the ‘Drug-likeness’ by analyzing the Lipinski's rule of 5 and the Veber rule [83].

2.7. Chemistry

2.7.1. Synthesis of 1-(N1-benzyl-2-methyl-4-nitro-imidazole-5-yl)-4-(prop-2-yn-1-yl)piperazine (5)

To a suspension of sodium hydride NaH (60 w% dispersion in mineral oil) (0.35 g, 1.42 × 10-2 mol) in THF (25 mL) under N2 gas, compound 4 [84](1.0 g, 3.32 × 10-3 mol) in 3 mL of THF is added dropwise at 0 °C. The solution is stirred for 30 min, after which (2.6–3.0 equiv) propargyl bromide (0.64 mL, 8.63 × 10-3 mol) is added dropwise.

The resulting solution is stirred for 4 h at 0 °C. The reaction is monitored by TLC. After completion of the reaction, ammonium chloride NH4Cl (0.50 g, 7.47 × 10-3 mol) that is dissolved in H2O (20 mL) is added to the reaction mixture with stirring to quench the reaction. The product is extracted with CH2Cl2. The organic phase is dried over Na2SO4, filtered, and evaporated to dryness.

The crude crystalized to give product 5 as yellow flakes, 1.12 g (82%); m.p. 122–124 °C; 1H NMR (500 MHz, DMSO‑d₆) δ: 2.25 (s, 3H, -CH3), 2.46 (br.s, 4H, H-3ʹʹ, H-5ʹʹ), 2.99 (br.s, 4H, H-2ʹʹ, H-6ʹʹ), 3.17 (t, J = 2.2, 1H, H-1ʹʹʹ), 3.28 (d, J = 2.2, 2H, -CH2 C CH), 5.17 (s, 2H, NCH2-Ph), 7.12 (d, J = 7.4 Hz, 2H, H-2ʹ, H-6ʹ), 7.32 (t, J = 7.3 Hz, 1H, H-4ʹ), 7.39 (pseudo t, 2H, H-3ʹ, H-5ʹ); 13C NMR (125 MHz, DMSO‑d₆) δ: 14.1 (-CH3), 46.2 (NCH2-Ph), 46.4 (CH2C CH), 48.5 (C-2ʹʹ, C-6ʹʹ), 51.4 (C-3ʹʹ, C-5ʹʹ), 76.3 (C-1ʹʹʹ), 79.6 (C-2ʹʹʹ), 126.9 (C-2ʹ, C-6ʹ), 128.1 (C-4ʹ), 129.4 (C-3ʹ, C-5ʹ), 136.6 (C-1ʹ), 138.8 (C-4), 140.2 (C-2), 140.9 (C-5); HRMS (ESI, m/z): Calculated for C18H21N5O2Na (362.15875), found (362.15719) [M+Na] +.

2.7.2. General procedure for synthesis of 1-((N1-substituted benzyl-1H-1,2,3-triazol-4-yl)methyl)-4- (N1-benzyl-2-methyl-4-nitro-imidazole-5-yl) piperazine 9a-k

1-(N1-benzyl-2-methyl-4-nitro-1H-imidazole-5-yl)-4-(prop-2-yn-1-yl) piperazine 5 (0.5 g, 1 equiv) was charged into a 50 mL one-necked round bottom flask, with CuI (1/10 = 0.1 equiv) in toluene (10–15 mL), the mixture was stirred for 15–30 min Et3N (3.7 equiv) was added with stirring for 15 min. The corresponding substituted-(azidomethyl) benzene [85](1.3 equiv) were added. The reaction mixture was stirred overnight and monitored with TLC. Chloroform (20 mL) was added to the reaction mixture. The reaction was filtered, and the filtrate was washed with water (10–15 mL). The organic phase was extracted and evaporated under a vacuum until dry. The product was purified using column chromatography (eluent: 5% EtOAc: n-hexane) to furnish the pure compounds.

2.7.2.1. 1-((N1-benzyl-1H-1,2,3-triazol-4-yl)methyl)-4-(N1-benzyl-2-methyl-4-nitro-imidazole-5- yl)piperazine 9a

Pale-yellow solid, 0.50 g (83%); m.p. 167–169 °C; 1H NMR (500 MHz, CDCl3) δ: 2.27 (s, 3H, -CH3), 2.46 (br.s, 4H, H-3ʹʹ, H-5ʹʹ), 3.02 (br.s, 4H, H-2ʹʹ, H-6ʹʹ), 3.62 (s, 2H, -CH2 CCH), 5.05 (s, 2H, NCH2-Ph), 5.48 (s, 2H, NCH2–C), 6.95 (d, J = 6.7, 2H, H-2ʹ, H-6ʹ), 6.95 (d, J = 6.7, 2H, H-2ʹʹʹʹ, H-6ʹʹ’ʹ), 7.27 (t, J = 6.9, 1H, H-4ʹ), 7.27 (t, J = 6.9, 1H, H-4ʹʹʹʹ), 7.29 (m, 2H, H-3ʹʹʹʹ, H-5ʹʹʹʹ), 7.29 (m, 2H, H-3ʹ, H-5ʹ), 7.41 (S, 1H, H-5ʹʹʹ); 13C NMR (125 MHz,CDCl3) δ: 14.1(-CH3), 46.3 (NCH2-Ph), 49.1 (C-2ʹʹ, C-6ʹʹ), 53.3 (C-3ʹʹ, C-5ʹʹ), 53.6 (CH2CCH), 54.2 (NCH2–C), 122.6 (C-5ʹʹʹ), 125.9 (C-2ʹ, C-6ʹ), 128.1 (C-2ʹʹʹʹ, C-6ʹʹʹʹ), 128.2 (C-4ʹ), 128.7 (C-4ʹʹʹʹ), 129.1 (C-3ʹʹʹʹ, C-5ʹʹʹʹ), 129.2 (C-3ʹ, C-5ʹ), 134.6 (C-5), 135.2 (C-1ʹʹʹʹ), 135.2 (C-1ʹ), 139.2 (C-4), 140.4 (C-2), 145.0 (C-4ʹʹʹ); HRMS (ESI) m/z: Calculated for C25H28N8O2Na (495.22274), found (495.22140) [M+Na] +, Calculated for C25H29N8O2 (473.24080), found (473.23973) [M+H] +.

2.7.2.2. 1-(N1-benzyl-2-methyl-4-nitro-imidazole-5-yl)-4-((N1-(4-bromobenzyl)-1H-1,2,3-triazol-4- yl)methyl)piperazine 9b

Off-white solid, 0.33 g (82%); m.p. 146–148 °C; 1H NMR (500 MHz, CDCl3) δ: 2.25 (s, 3H, - CH3), 2.48 (br.s, 4H, H-3ʹʹ, H-5ʹʹ), 3.05 (br.s, 4H, H-2ʹʹ, H-6ʹʹ), 3.65 (s, 2H, -CH2 CCH), 5.04 (s, 2H, NCH2-Ph), 5.42 (s, 2H, NCH2–C), 6.94 (d, J = 6.7, 2H, H-2ʹ, H-6ʹ), 7.10 (d, J = 8.1, 2H, H-2ʹʹʹʹ, H-6ʹʹ’ʹ), 7.27 (t, J = 6.9, 1H, H-4ʹ), 7.29 (m, 2H, H-3ʹ, H-5ʹ), 7.42 (d, J = 8.1, 2H, H-3ʹʹʹʹ, H-5ʹʹʹʹ), 7.50 (S, 1H, H-5ʹʹʹ); 13C NMR (125 MHz,CDCl3) δ: 14.1 (-CH3), 46.4 (NCH2-Ph), 48.8 (C-2ʹʹ, C-6ʹʹ), 53.1 (C-3ʹʹ, C-5ʹʹ), 53.3 (CH2CCH), 53.5 (NCH2–C), 122.9 (C-4ʹʹʹʹ), 123.1 (C-5ʹʹʹ), 125.9 (C-2ʹ, C-6ʹ), 128.2 (C-4ʹ), 129.2 (C-3ʹ, C-5ʹ), 129.7 (C-2ʹʹʹʹ, C-6ʹʹʹʹ), 132.3 (C-3ʹʹʹʹ, C-5ʹʹʹʹ), 133.7 (C-5), 135.2 (C-1ʹ), 139.1 (C-1ʹʹʹʹ), 139.4 (C-4), 140.5 (C-2), 144.5 (C-4ʹʹʹ); HRMS (ESI) m/z: Calculated for C25H27BrN8O2Na (573.13326 : 575.13157), found (573.13341 : 575.13163) [M+Na] +.

2.7.2.3. 1-(N1-benzyl-2-methyl-4-nitro-imidazole-5-yl)-4-((N1-(4-fluorobenzyl)-1H-1,2,3-triazol-4- yl)methyl)piperazine 9c

Pale -yellow solid, 0.30 g (92%); m.p. 165–167 °C; 1H NMR (500 MHz, CDCl3) δ: 2.26 (s, 3H, -CH3), 2.97 (br.s, 4H, H-2ʹʹ, H-6ʹʹ), 2.47 (br.s, 4H, H-3ʹʹ, H-5ʹʹ), 3.64 (s, 2H, -CH2 CCH), 5.04 (s, 2H, NCH2-Ph), 7.45 (S, 1H, H-5ʹʹʹ), 5.44 (s, 2H, NCH2–C), 6.95 (d, J = 6.7, 2H, H-2ʹ, H-6ʹ), 7.27 (t, J = 6.9, 1H, H-4ʹ), 7.28 (m, 2H, H-3ʹ, H-5ʹ), 7.29 (Pseudo t, 2H, H-2ʹʹʹʹ, H-6ʹʹ’ʹ), 7.01 (Pseudo t, 2H, H-3ʹʹʹʹ, H-5ʹʹʹʹ); 13C NMR (125 MHz,CDCl3) δ: 14.1 (-CH3), 46.4 (NCH2- Ph), 48.9 (C-2ʹʹ, C-6ʹʹ), 53.2 (C-3ʹʹ, C-5ʹʹ), 53.4 (CH2CCH), 53.4 (NCH2–C), 116.2 (d, 2JC-F = 21.8, C-3ʹʹʹʹ, C-5ʹʹʹʹ), 122.7 (C-5ʹʹʹ), 125.9 (C-2ʹ, C-6ʹ), 128.2 (C-4ʹ), 129.2 (C-3ʹ, C-5ʹ), 130.0 (d, 3JC-F = 8.3, C-2ʹʹʹʹ, C-6ʹʹʹʹ), 130.5 (C-5), 135.2 (C-1ʹ), 139.1 (C-1ʹʹʹʹ), 139.4 (C-4), 140.5 (C-2), 144.9 (C-4ʹʹʹ), 162.8 (d, 1JC-F = 248.5, C-4ʹʹʹʹ); HRMS (ESI) m/z: Calculated for C₂₅H₂₈FN₈O₂ (491.23138), found (491.23036) [M+H] +, and Calculated for C₂₅H₂₇FN₈O₂Na (513.21332), found (513.21209) [M+Na] +.

2.7.2.4. 1-(N1-benzyl-2-methyl-4-nitro-imidazole-5-yl)-4-((N1-(3-nitrobenzyl)-1H-1,2,3-triazol-4- yl)methyl)piperazine 9d

Yellow solid, 0.15 g (72%); m.p. 85–87 °C; 1H NMR (500 MHz, CDCl3) δ: 2.26 (s, 3H, -CH3), 2.52 (br.s, 4H, H-3ʹʹ, H-5ʹʹ), 2.98 (br.s, 4H, H-2ʹʹ, H-6ʹʹ), 3.69 (s, 2H, -CH2 CCH), 5.05 (s, 2H, NCH2-Ph), 5.61 (s, 2H, NCH2–C), 6.95 (d, J = 6.6, 2H, H-2ʹ, H-6ʹ), 7.28–7.31 (m, 3H, H-3ʹ, H-5ʹ, H-4ʹ), 7.53 (d, J = 7.8, 1H, H-2ʹʹʹʹ), 7.57 (Pseudo t, 1H, H-3ʹʹʹʹ), 7.62 (S, 1H, H-5ʹʹʹ), 8.07 (s, H-6ʹ’ʹʹ), 8.16 (d, J = 7.2, 1H, H-4ʹʹʹʹ); 13C NMR (125 MHz,CDCl3) δ: 14.1 (-CH3), 46.4 (NCH2-Ph), 48.8 (C-2ʹʹ, C-6ʹʹ), 53.1 (C-3ʹʹ, C-5ʹʹ), 53.2 (CH2CCH), 53.3 (NCH2–C), 122.8 (C-4ʹʹʹʹ), 123.3 (C-5ʹʹʹ), 123.7 (C-6ʹʹʹʹ), 125.9 (C-2ʹ, C-6ʹ), 128.2 (C-4ʹ), 129.2 (C-3ʹ, C-5ʹ), 130.3 (C-3ʹʹʹʹ), 133.8 (C-5), 135.2 (C-1ʹ), 136.8 (C-2ʹʹʹ), 139.1 (C-1ʹʹʹʹ), 139.4 (C-4), 140.6 (C-2), 145.0 (C-4ʹʹʹ), 148.6 (C-5ʹʹʹʹ); HRMS (ESI) m/z: Calculated for C25H27N9O4Na (540.20782), found (540.20787) [M+Na] +, Calculated for C25H28N9O4 (518.22588), found (518.22577) [M+Na] +.

2.7.2.5. 1-(N1-benzyl-2-methyl-4-nitro-imidazole-5-yl)-4-((N1-(3-methylbenzyl)-1H-1,2,3-triazol-4- yl)methyl)piperazine 9e

Yellow solid, 0.50 g (96%); m.p. 70–72 °C; 1H NMR (500 MHz, CDCl3) δ: 2.26 (s, 3H, -CH3), 2.29 (s, 3H, -CH3), 2.47 (br.s, 4H, H-3ʹʹ, H-5ʹʹ), 3.02 (br.s, 4H, H-2ʹʹ, H-6ʹʹ), 3.63 (s, 2H, -CH2 CCH), 5.04 (s, 2H, NCH2-Ph), 5.42 (s, 2H, NCH2–C), 6.95 (d, J = 7.3, 2H, H-2ʹ, H-6ʹ), 7.04 (Pseudo t, 1H, H-3ʹʹʹʹ), 7.12 (d, J = 7.5, 1H, H-2ʹʹʹʹ), 7.20 (d, J = 7.5, 1H, H-4ʹʹʹʹ), 7.24 (s, H-6ʹʹ’ʹ), 7.28–7.31 (m, 3H, H-3ʹ, H-5ʹ, H-4ʹ), 7.44 (S, 1H, H-5ʹʹʹ); 13C NMR (125 MHz,CDCl3) δ: 14.1 (-CH3), 21.3 (-CH3), 46.4 (NCH2-Ph), 49.0 (C-2ʹʹ, C-6ʹʹ), 53.2 (C-3ʹʹ, C-5ʹʹ), 53.5 (CH2CCH), 54.2 (NCH2–C), 122.8 (C-5ʹʹʹ), 125.2 (C-2ʹʹʹ), 125.9 (C-2ʹ, C-6ʹ), 128.2 (C-4ʹ), 128.9 (C-4ʹʹʹʹ), 129.0 (C-3ʹʹʹʹ), 129.2 (C-3ʹ, C-5ʹ), 129.5 (C-6ʹʹʹʹ), 134.5 (C-5), 135.2 (C-1ʹ), 138.9 (C-5ʹʹʹʹ), 139.2 (C- 1ʹʹʹʹ), 139.4 (C-4), 140.5 (C-2), 145.1 (C-4ʹʹʹ); HRMS (ESI) m/z: Calculated for C26H30N8O2Na (509.23839), found (509.23922) [M+Na] +.

2.7.2.6. 1-(N1-benzyl-2-methyl-4-nitro-imidazole-5-yl)-4-((N1-(3-bromobenzyl)-1H-1,2,3-triazol-4- yl)methyl)piperazine 9f

Yellow solid, 0.70 g (90%); m.p. 54–56 °C; 1H NMR (500 MHz, CDCl3) δ: 2.26 (s, 3H, -CH3), 2.49 (br.s, 4H, H-3ʹʹ, H-5ʹʹ), 2.98 (br.s, 4H, H-2ʹʹ, H-6ʹʹ), 3.65 (s, 2H, -CH2 CCH), 5.05 (s, 2H, NCH2-Ph), 5.45 (s, 2H, NCH2–C), 6.95 (d, J = 7.3, 2H, H-2ʹ, H-6ʹ), 7.17 (d, J = 7.8, 1H, H-2ʹʹʹʹ), 7.24 (Pseudo t, 1H, H-3ʹʹʹʹ), 7.27–7.32 (m, 3H, H-3ʹ, H-5ʹ, H-4ʹ), 7.37 (s, H-6ʹʹ’ʹ), 7.45 (d, J = 7.2, 1H, H-4ʹʹʹʹ), 7.49 (S, 1H, H-5ʹʹʹ); 13C NMR (125 MHz,CDCl3) δ: 14.1 (-CH3), 46.4 (NCH2-Ph), 48.9 (C-2ʹʹ, C-6ʹʹ), 53.1 (C-3ʹʹ, C-5ʹʹ), 53.1 (CH2CCH), 53.4 (NCH2–C), 123.1 (C-5ʹʹʹʹ), 125.9 (C-2ʹ, C-6ʹ), 126.6 (C-5ʹʹʹ), 128.2 (C-4ʹ), 129.2 (C-3ʹ, C-5ʹ), 130.7 (C-2ʹʹ’ʹ), 130.7 (C-4ʹʹʹʹ), 131.0 (C-3ʹʹʹʹ), 131.9 (C-6ʹʹʹʹ), 135.2 (C-1ʹ), 136.8 (C-5), 139.1 (C-1ʹʹʹʹ), 139.4 (C-4), 140.5 (C-2), 144.8 (C-4ʹʹʹ); HRMS (ESI) m/z: Calculated for C25H27BrN8O2Na (573.13326 : 57513157), found (573.13381 : 575.13088) [M+Na] +.

2.7.2.7. 1-(N1-benzyl-2-methyl-4-nitro-imidazole-5-yl)-4-((N1-(3-fluorobenzyl)-1H-1,2,3-triazol-4- yl)methyl)piperazine 9g

Pale-yellow solid, 0.60 g (83%); m.p. 68–70 °C; 1H NMR (500 MHz, CDCl3) δ: 2.25 (s, 3H, - CH3), 2.46 (br.s, 4H, H-3ʹʹ, H-5ʹʹ), 2.97 (br.s, 4H, H-2ʹʹ, H-6ʹʹ), 3.63 (s, 2H, -CH2CCH), 5.04 (s, 2H, NCH2-Ph), 5.46 (s, 2H, NCH2–C), 6.90 (d, J = 9.3, 1H, H-2ʹʹʹʹ), 6.95 (d, J = 7.2, 2H, H-2ʹ, H-6ʹ), 7.01 (d, J = 7.3, 1H, H-4ʹʹʹʹ), 7.25 (Pseudo t, 1H, H-3ʹʹʹʹ), 7.27–7.31 (m, 3H, H-3ʹ, H-5ʹ, H-4ʹ), 7.46 (S, 1H, H-5ʹʹʹ), 7.96 (s, H-6ʹʹ’ʹ); 13C NMR (125 MHz,CDCl3) δ: 14.1 (-CH3), 46.3 (NCH2-Ph), 49.0 (C-2ʹʹ, C-6ʹʹ), 53.2 (C-3ʹʹ, C-5ʹʹ), 53.2 (CH2CCH), 53.5 (NCH2–C), 114.9 (d, 2JC-F = 22.4, C-6ʹʹʹʹ), 115.8 (d, 2JC-F = 21.1, C-4ʹʹʹʹ), 122.8 (C-5ʹʹʹ), 123.6 (C-2ʹʹ’ʹ), 125.9 (C-2ʹ, C-6ʹ), 128.2(C-4ʹ), 129.2 (C-3ʹ, C-5ʹ), 130.8 (d, 3JC-F = 8.5, C-3ʹʹʹʹ), 135.2 (C-1ʹ), 137.0 (C-5), 139.2 (C-1ʹʹʹʹ), 139.4 (C-4), 140.5 (C-2), 145.0 (C-4ʹʹʹ), 162.9 (d, 1JC-F = 247.9, C-5ʹʹʹʹ); HRMS (ESI) m/z: Calculated for C25H27FN8O2Na (513.21332), found (513.21178) [M+Na] +.

2.7.2.8. 1-(N1-benzyl-2-methyl-4-nitro-imidazole-5-yl)-4-((N1-(2-nitrobenzyl)-1H-1,2,3-triazol-4- yl)methyl)piperazine 9h

Pale-brown solid, 0.30 g (71%); m.p. = 64–66 °C; 1H NMR (500 MHz, CDCl3) δ: 2.25 (s, 3H, -CH3), 2.51 (br.s, 4H, H-3ʹʹ, H-5ʹʹ), 3.05 (br.s, 4H, H-2ʹʹ, H-6ʹʹ), 3.69 (s, 2H, -CH2 CCH), 5.03 (s, 2H, NCH2-Ph), 5.88 (s, 2H, NCH2–C), 6.96 (d, J = 7.3, 2H, H-2ʹ, H-6ʹ), 7.05 (d, J = 7.7, 1H, H-6ʹʹʹʹ), 7.26–7.31 (m, 3H, H-3ʹ, H-5ʹ, H-4ʹ), 7.48 (Pseudo t, 1H, H-4ʹʹʹʹ), 7.56 (t, J = 7.5, 1H, H-5ʹʹʹʹ), 7.68 (S, 1H, H-5ʹʹʹ), 8.07 (d, J = 8.1, 1H, H-3ʹʹʹʹ); 13C NMR (125 MHz, CDCl3) δ: 14.1 (-CH3), 46.4 (NCH2-Ph), 48.9 (C-2ʹʹ, C-6ʹʹ), 50.9 (C-3ʹʹ, C-5ʹʹ), 53.1 (CH2CCH), 53.3 (NCH2–C), 124.0 (C-5ʹʹʹ), 125.4 (C-3ʹʹʹʹ), 125.9 (C-2ʹ, C-6ʹ), 128.2 (C-4ʹ), 129.2 (C-3ʹ, C-5ʹ), 129.7 (C-4ʹʹʹʹ), 130.4 (C-6ʹʹʹʹ), 130.5 (C-5), 134.4 (C-5ʹʹʹʹ), 135.2 (C-1ʹ), 139.2 (C-1ʹʹʹʹ), 139.3 (C-4), 140.5 (C-2), 144.6 (C-4ʹʹʹ), 147.5 (C-2ʹʹʹ); HRMS (ESI) m/z: Calculated for C25H27N9O4Na (540.20782), found (540.21015) [M+Na] +.

2.7.2.9. 1-(N1-benzyl-2-methyl-4-nitro-imidazole-5-yl)-4-((N1-(2-bromobenzyl)-1H-1,2,3-triazol-4- yl)methyl)piperazine 9i

Brown solid, 0.50 g (78%); m.p. 167–169 °C; 1H NMR (500 MHz, CDCl3) δ: 2.22 (s, 3H, - CH3), 2.47 (br.s, 4H, H-3ʹʹ, H-5ʹʹ), 3.03 (br.s, 4H, H-2ʹʹ, H-6ʹʹ), 3.64 (s, 2H, -CH2 CCH), 5.04 (s, 2H, NCH2-Ph), 5.60 (s, 2H, NCH2–C), 6.95 (d, J = 7.3, 2H, H-2ʹ, H-6ʹ), 7.12 (d, J = 7.3, 1H, H-6ʹʹʹʹ), 7.18 (t, J = 7.6, 1H, H-4ʹ), 7.23–7.26 (m, 2H, H-4ʹʹʹʹ, H-5ʹʹʹʹ), 7.28–7.32 (m, 2H, H-3ʹ, H-5ʹ), 7.51 (S, 1H, H-5ʹʹʹ), 7.56 (d, J = 7.9, 1H, H-3ʹʹʹʹ); 13C NMR (125 MHz,CDCl3) δ: 14.1 (-CH3), 46.3 (NCH2-Ph), 49.1 (C-2ʹʹ, C-6ʹʹ), 53.2 (C-3ʹʹ, C-5ʹʹ), 53.5 (CH2CCH), 53.9 (NCH2–C), 123.1 (C-5ʹʹʹ), 123.6 (C-2ʹʹʹ), 125.9 (C-2ʹ, C-6ʹ), 128.2 (C-4ʹ), 129.2 (C-3ʹ, C-5ʹ), 130.4 (C-4ʹʹʹʹ),130.4 (C-5ʹʹʹʹ), 133.3 (C-3ʹʹʹʹ), 133.3 (C-6ʹʹʹʹ), 134.1 (C-5), 135.2 (C-1ʹ), 139.2 (C-1ʹʹʹʹ), 139.4 (C-4), 140.44 (C-2), 144.7 (C-4ʹʹʹ); HRMS (ESI) m/z: Calculated for C25H27BrN8O2Na (573.13326: 575.13157), found (573.13621: 575.13330) [M+Na] +.

2.7.2.10. 1-(N1-benzyl-2-methyl-4-nitro-imidazole-5-yl)-4-((N1-(2-fluorobenzyl)-1H-1,2,3-triazol-4- yl)methyl)piperazine 9j

Brown solid, 0.40 g (84%); m.p. 169–171 °C; 1H NMR (500 MHz, CDCl3) δ: 2.22 (s, 3H, - CH3), 5.04 (s, 2H, NCH2-Ph), 3.02 (br.s, 4H, H-2ʹʹ, H-6ʹʹ), 2.46 (br.s, 4H, H-3ʹʹ, H-5ʹʹ), 3.62 (s, 2H, -CH2 CCH), 7.51 (S, 1H, H-5ʹʹʹ), 5.52 (s, 2H, NCH2–C), 6.93 (d, J = 7.2, 2H, H-2ʹ, H-6ʹ), 7.28–7.32 (m, 3H, H-3ʹ, H-5ʹ, H-4ʹ), 7.21–7.26 (m, 2H, H-3ʹʹʹʹ, H-4ʹʹʹʹ), 7.08 (m, 1H, H-5ʹʹʹʹ), 7.06 (d, J = 6.1, 1H, H-6ʹʹʹʹ); 13C NMR (125 MHz, CDCl3) δ: 14.1 (-CH3), 46.3 (NCH2-Ph), 47.7 (C-2ʹʹ, C-6ʹʹ), 49.0 (C-3ʹʹ, C-5ʹʹ), 53.2 (CH2CCH), 53.4 (NCH2–C), 115.9 (d, 2JC-F = 21.2, C-3ʹʹʹʹ), 121.9 (d, 4JC-F = 14.7, C-5ʹʹʹʹ), 122.9 (C-5ʹʹʹ), 124.8 (C-4ʹ’ʹʹ), 125.9 (C-2ʹ, C-6ʹ), 128.2 (C-4ʹ), 129.2 (C-3ʹ, C-5ʹ), 130.8 (d-d, 3JC-F = 8.3, C-6ʹʹʹʹ), 135.2 (C-5), 135.2 (C-1ʹ), 139.2 (C-1ʹʹʹʹ), 139.4 (C-4), 140.5 (C-2), 144.7 (C-4ʹʹʹ), 160.6 (d, 1JC-F = 248.0, C-2ʹʹʹʹ); HRMS (ESI) m/z: Calculated for C25H27FN8O2Na (513.21332), found (513.21101) [M+Na] +.

2.7.2.11. 1-(N1-benzyl-2-methyl-4-nitro-imidazole-5-yl)-4-((N1-(4-methylbenzyl)-1H-1,2,3-triazol-4- yl)methyl)piperazine 9k

Off-white solid, 0.80 g (82%); m.p. 168–170 °C; 1H NMR (500 MHz, CDCl3) δ: 2.26 (s, 3H, - CH3), 2.31 (s, 3H, -CH3C-4ʹʹʹʹ), 2.43 (br.s, 4H, H-3ʹʹ, H-5ʹʹ), 3.01 (br.s, 4H, H-2ʹʹ, H-6ʹʹ), 3.61 (s, 2H, -CH2 CCH-5ʹʹʹ), 5.04 (s, 2H, NCH2-Ph), 5.42 (s, 2H, NCH2–C-1ʹʹʹʹ), 6.96 (d, J = 7.2, 2H, H-2ʹ, H-6ʹ), 7.13 (s, 4H, H-2ʹʹʹʹ, H-3ʹʹʹʹ, H-5ʹʹʹʹ, H-6ʹʹʹʹ), 7.23–7.31 (m, 3H, H-3ʹ, H-5ʹ, H-4ʹ), 7.39 (s, 1H, H-5ʹʹʹ); 13C NMR (125 MHz,CDCl3) δ: 14.1 (-CH3), 21.2 (-CH3C-4ʹʹʹʹ), 46.3 (NCH2-Ph), 49.1 (C-2ʹʹ, C-6ʹʹ), 53.2 (C-3ʹʹ, C-5ʹʹ), 53.6 (CH2CCH-5ʹʹʹʹ), 54.0 (NCH2–C-1ʹʹʹʹ), 122.6 (C-5ʹʹʹ), 125.9 (C-2ʹ, C-6ʹ), 128.2 (C-4ʹ), 129.2 (C-3ʹ, C-5ʹ), 129.8 (C-2ʹʹʹʹ, C-3ʹʹʹʹ, C-5ʹʹʹʹ, C-6ʹʹʹʹ), 131.5 (C-5), 135.2 (C-1ʹ), 138.7 (C-4ʹʹʹʹ), 139.2 (C-1ʹʹʹʹ), 139.4 (C-4), 140.5 (C-2), 144.9 (C-4ʹʹʹ); HRMS (ESI) m/z: Calculated for C26H30N8O2Na (509.23839), found (509.23711) [M+Na] +.

2.7.3. General procedure for synthesis of 2-(4-((4-(N1-benzyl-2-methyl-4-nitro-1H-imidazole-5-yl)piperazin-1-yl)methyl)-1H-1,2,3-triazol-1-yl)-1-substituted phenylethanone 10a-c

To a solution of copper sulfate (0.8 mmol) and sodium l-ascorbate (1.1 mmol) in a mixture of DMF: H2O (3:2, v/v, 5 mL) was added with stirring a mixture of compound 5 (0.2 g, 0.97 mmol) and a stoichiometric amount of corresponding acetophenone azides 7a-c [86] (1.0 mmol) in DMF: water (10 mL). Then, the reaction mixture was heated at 80 °C for 8 h. Upon the completion of the reaction, ice-water was added to quench the reaction. The solid formed was collected by filtration, washed with a saturated solution of ammonium chloride, and recrystallized from ethanol to give the desired 1,2,3-triazoles 7a-c.

2.7.3.1. 2-(4-((4-(1-benzyl-2-methyl-4-nitro-1H-imidazole-5-yl) piperazin-1-yl) methyl)-1H-1,2,3- triazol-1-yl)-1-phenylethanone 10a

Sanden solid, 0.15 g (70%); m.p. 124–126 °C (decom.); 1H NMR (500 MHz, CDCl3) δ: 2.28 (s, 3H, -CH3), 2.78 (br.s, 4H, H-3ʹʹ, H-5ʹʹ), 3.01 (br.s, 4H, H-2ʹʹ, H-6ʹʹ), 3.99 (s, 2H, -CH2 CCH5ʹʹʹ), 5.05 (s, 2H, NCH2-Ph), 5.86 (s, 2H, NCH2–CO), 6.95 (d, J = 7.2, 2H, H-2ʹ, H-6ʹ), 7.23–7.31 (m, 3H, H-3ʹ, H-5ʹ, H-4ʹ), 7.48 (ps.t, 2H, H-3ʹʹʹʹ, H-5ʹʹʹʹ), 7.61 (t, J = 7.3, 1H, H-4ʹʹʹʹ), 7.41 (s, 1H, H-5ʹʹʹ), 7.94 (d, J = 7.2, 2H, H-2ʹʹʹʹ, H-6ʹʹʹʹ); 13C NMR (125 MHz,CDCl3) δ: 14.1 (-CH3), 46.7 (NCH2-Ph), 47.8 (C-2ʹʹ, C-6ʹʹ, C-3ʹʹ, C-5ʹʹ), 52.4 (CH2CCH-5ʹʹʹʹ), 55.7 (NCH2–CO), 125.9 (C-2ʹ, C-6ʹ), 128.2 (C-2ʹʹʹʹ, C-6ʹʹʹʹ), 128.3 (C-5ʹʹʹ), 128.3 (C-4ʹ), 129.2 (C-3ʹʹʹʹ, C-5ʹʹʹʹ), 129.3 (C-3ʹ, C-5ʹ), 133.9 (C-1ʹʹʹʹ), 134.6 (C-4ʹʹʹʹ), 135.2 (C-1ʹ), 138.4 (C-4), 139.4 (C-4ʹʹʹ), 139.4 (C-5), 140.8 (C-2), 190.4 (-CO); HRMS (ESI) m/z: Calculated for C26H28N8O3Na (523.21766), found (523.21849) [M+Na] +.

2.7.3.2. 2-(4-((4-(1-benzyl-2-methyl-4-nitro-1H-imidazole-5-yl) piperazin-1-yl)methyl)-1H-1,2,3- triazol-1-yl)-1-(4-bromophenyl)ethanone 10b

Yellow solid, 0.15 g (39%); m.p. 163–166 °C (decom.); 1H NMR (500 MHz, CDCl3) δ: 2.28 (s, 3H, -CH3), 2.61 (br.s, 4H, H-3ʹʹ, H-5ʹʹ), 2.97 (br.s, 4H, H-2ʹʹ, H-6ʹʹ), 3.85 (s, 2H, CH2 CCH-5ʹʹʹ), 5.05 (s, 2H, NCH2-Ph), 5.78 (s, 2H, NCH2–CO), 6.95 (d, J = 7.2, 2H, H-2ʹ, H-6ʹ), 7.30 (m, 3H, H-3ʹ, H-5ʹ, H-4ʹ), 7.44 (s, 1H, H-5ʹʹʹ), 7.64 (d, J = 7.7, 2H, H-3ʹʹʹʹ, H-5ʹʹʹʹ), 7.78 (d, J = 7.8, 2H, H-2ʹʹʹʹ, H-6ʹʹʹʹ); 13C NMR (125 MHz,CDCl3) δ: 14.1 (-CH3), 46.5 (NCH2-Ph), 48.3 (C-2ʹʹ, C-6ʹʹ, C-3ʹʹ, C-5ʹʹ), 52.7 (CH2CCH-5ʹʹʹʹ), 55.4 (NCH2–CO), 125.9 (C-2ʹ, C-6ʹ), 128.3 (C-4ʹ), 128.8 (C-4ʹʹʹʹ), 129.3 (C-3ʹ, C-5ʹ), 129.5 (C-2ʹʹʹʹ, C-6ʹʹʹʹ), 131.5 (C-5ʹʹʹ), 131.9 (C-1ʹʹʹʹ), 132.6 (C-3ʹʹʹʹ, C-5ʹʹʹʹ), 135.1 (C-1ʹ), 135.1 (C-4), 138.7 (C-5), 139.5 (C-4ʹʹʹ), 140.7 (C-2), 189.5 (-CO); HRMS (ESI) m/z: Calculated for C26H28BrN8O3 (579.14623 : 581.1445), found (579.14636 : 581.14582) [M+H] +.

2.7.3.3. 2-(4-((4-(1-benzyl-2-methyl-4-nitro-1H-imidazole-5-yl) piperazin-1-yl) methyl)-1H-1,2,3- triazol-1-yl)-1-(3-methoxyphenyl)ethanone 10c

Light-yellow solid, 0.15 g (83%); m.p. 114–116 °C (decom.); 1H NMR (500 MHz, CDCl3) δ: 2.27 (s, 3H, -CH3), 2.91 (br.s, 4H, H-3ʹʹ, H-5ʹʹ), 3.64 (br.s, 4H, H-2ʹʹ, H-6ʹʹ), 3.73 (s, 2H, -CH2.

CCH-5ʹʹʹ), 3.79 (s, 3H, -OCH3), 5.05 (s, 2H, NCH2-Ph), 5.79 (s, 2H, NCH2–CO), 6.94 (br.s, 2H, H-2ʹ, H-6ʹ), 7.13 (br.s, 1H, H-4ʹʹʹʹ), 7.29–7.36 (m, 3H, H-3ʹ, H-5ʹ, H-4ʹ), 7.47 (m, 2H, H-2ʹʹʹʹ, H- 6ʹʹʹʹ), 7.75 (s, 1H, H-5ʹʹʹ), 7.97 (m, 1H, H-5ʹʹʹʹ); 13C NMR (125 MHz,CDCl3) δ: 14.1 (-CH3), 46.6 (NCH2-Ph), 47.8 (C-2ʹʹ, C-6ʹʹ, C-3ʹʹ, C-5ʹʹ), 52.4 (CH2CCH-5ʹʹʹʹ), 55.4 (NCH2–CO), 55.5 (-OCH3), 112.4 (C-4ʹʹʹʹ), 120.5 (C-6ʹʹʹʹ), 121.0 (C-5ʹʹʹ), 121.0 (C-5ʹʹʹʹ), 125.9 (C-2ʹ, C-6ʹ), 128.3 (C-4ʹ), 129.3 (C-3ʹ, C-5ʹ), 130.1 (C-2ʹʹʹʹ), 135.2 (C-1ʹʹʹʹ), 135.2 (C-1ʹ), 139.4 (C-4ʹʹʹ), 139.2 (C-5), 139.6 (C-4), 140.8 (C-2), 160.1 (C-3ʹʹʹʹ), 190.2 (-CO); HRMS (ESI) m/z: Calculated for C27H31N8O4 (531.24628), found (531.24398) [M+H] +.

2.7.4. General procedure for synthesis of 1-((N1-substituted aryl -1H-1,2,3-triazol-4-yl)methyl)-4- (N1-benzyl-2-methyl-4-nitro-imidazole-5-yl) piperazine 11a-q

1-(N1-benzyl-2-methyl-4-nitro-1H-imidazole-5-yl)-4-(prop-2-yn-1-yl) piperazine 5 (0.5 g, 1 equiv) was charged into a 50 mL one-necked round bottom flask, with CuI (1/10 = 0.1 equiv) in toluene (10–15 mL), the mixture was stirred for 15–30 min Et3N (3.7 equiv) was added with stirring for 15 min. The corresponding substituted azidobenzene [85] (1.3 equiv) were added. The reaction mixture was stirred overnight and monitored with TLC. Chloroform (20 mL) was added to the reaction mixture. The reaction was filtered, and the filtrate was washed with water (10–15 mL). The organic phase was extracted and evaporated under a vacuum to dry. The product was purified using column chromatography (eluent: 5% EtOAc: n-hexane) to furnish the pure compounds.

2.7.4.1. 1-(N1-benzyl-2-methyl-4-nitro-1H-imidazole-5-yl)-4-((N1-(4-bromophenyl)-1H-1,2,3-triazol-4- yl)methyl)piperazine 11a

Ivory solid, 0.40 g (86%); m.p. 176–178 °C; 1H NMR (500 MHz, CDCl3) δ: 2.22 (s, 3H, - CH3), 2.49 (br.s, 4H, H-3ʹʹ, H-5ʹʹ), 3.01 (br.s, 4H, H-2ʹʹ, H-6ʹʹ), 3.69 (s, 2H, -CH2 CCH), 5.04 (s, 2H, NCH2Ph), 6.93 (d, J = 6.3, 2H, H-2ʹ, H-6ʹ), 7.26 (m, 3H, H-3ʹ, H-5ʹ, H-4ʹ), 7.56 (br.s, 4H, H-2ʹʹʹʹ, H-3ʹʹʹʹ, H-5ʹʹʹʹ, H-6ʹʹʹʹ), 7.96 (s, 1H, H-5ʹʹʹ); 13C NMR (125 MHz,CDCl3) δ: 14.1(-CH3), 46.4 (NCH2Ph), 49.0 (C-2ʹʹ, C-6ʹʹ), 53.2 (C-3ʹʹ, C-5ʹʹ), 53.4 (-CH2CCH), 120.9 (C-5ʹʹʹ), 121.8 (C-2ʹʹʹʹ, C-6ʹʹʹʹ), 122.2 (-CBr), 125.9 (C-2ʹ, C-6ʹ), 128.2 (C-4ʹ), 129.2 (C-3ʹ, C-5ʹ), 132.9 (C-3ʹʹʹʹ, C-5ʹʹʹʹ), 135.2 (C-1ʹ), 135.2 (C-4), 136.0 (C-1ʹʹʹʹ), 139.3 (C-5), 140.6 (C-2), 145.6 (C-4ʹʹʹ); HRMS (ESI) m/z: Calculated for C24H26BrN8O2 (537.13566 : 539.13395), found (537.13684 : 539.13417) [M+H] +.

2.7.4.2. 1-(N1-benzyl-2-methyl-4-nitro-1H-imidazole-5-yl)-4-((N1-(2-(methylthio) phenyl)-1H-1,2,3- triazol-4-yl)methyl)piperazine 11b

Cappuccino solid, 0.35 g (91%); m.p. 149–151 °C; 1H NMR (500 MHz, CDCl3) δ: 2.28 (s, 3H, -CH3), 2.34 (s, 3H, -SCH3), 3.27 (br.s, 4H, H-3ʹʹ, H-5ʹʹ), 3.68 (br.s, 4H, H-2ʹʹ, H-6ʹʹ), 4.52 (s, 2H, -CH2 CCH), 5.11 (s, 2H, NCH2Ph), 6.98 (br.s, 2H, H-2ʹ, H-6ʹ), 7.23 (s, 2H, H-3ʹʹʹʹ, H-4ʹʹʹʹ), 7.35–7.26 (m, 4H, H-3ʹ, H-5ʹ, H-4ʹ, H-5ʹʹʹʹ), 7.44 (d, J = 7.0, 1H, H-2ʹʹʹʹ), 8.69 (s, 1H, H-5ʹʹʹ); 13C NMR (125 MHz,CDCl3) δ: 14.3(-CH3), 16.1 (-SCH3), 46.4 (NCH2Ph), 47.6 (C-2ʹʹ, C-6ʹʹ), 51.5 (C-3ʹʹ, C-5ʹʹ), 51.5 (-CH2CCH), 125.8 (C-2ʹ, C-6ʹ), 125.8 (C-5ʹʹʹ), 125.8 (C-6ʹʹʹʹ), 126.8 (C-2ʹʹʹʹ), 127.3 (C-4ʹʹʹʹ), 128.5 (C-4ʹ), 129.5 (C-3ʹ, C-5ʹ), 129.5 (C-3ʹʹʹʹ), 130.7 (C-5ʹʹʹʹ), 134.8 (C-5), 135.1 (C-4), 135.6 (C-1ʹ), 135.6 (C-1ʹʹʹʹ), 141.3 (C-4ʹʹʹ), 141.3 (C-2); HRMS (ESI) m/z: Calculated for C25H29N8O2S (505.21287), found (505.21114) [M+H] +.

2.7.4.3. 1-(N1-benzyl-2-methyl-4-nitro-1H-imidazole-5-yl)-4-((N1-(2-ethoxyphenyl)-1H-1,2,3-triazol-4- yl)methyl)piperazine 11c

Caramel crystal, 0.35 g (96%); m.p. 162–164 °C; 1H NMR (500 MHz, CDCl3) δ: 1.33 (t, J = 6.8, 3H, -OCH2CH3), 2.26 (s, 3H, -CH3), 2.53 (br.s, 4H, H-3ʹʹ, H-5ʹʹ), 3.06 (br.s, 4H, H-2ʹʹ, H-6ʹʹ), 3.76 (s, 2H, -CH2 CCH), 4.07 (q, J = 6.9, 2H, -OCH2CH3), 5.06 (s, 2H, NCH2Ph), 6.98 (d,J = 7.3, 2H, H-2ʹ, H-6ʹ), 7.03 (m, 1H, H-3ʹʹʹʹ), 7.04 (d, J = 7.3, 1H, H-5ʹʹʹʹ), 7.34–7.23 (m, 3H, H-3ʹ, H-5ʹ, H-4ʹ), 7.74 (d, J = 7.8, 1H, H-2ʹʹʹʹ), 8.09 (s, 1H, H-5ʹʹʹ); 13C NMR (125 MHz,CDCl3) δ:14.1 (-CH3), 14.7 (-OCH2CH3), 46.3 (NCH2Ph), 49.2 (C-2ʹʹ, C-6ʹʹ), 53.1 (C-3ʹʹ, C-5ʹʹ), 53.6 (-CH2CCH), 64.6 (-OCH2CH3), 113.2 (C-5ʹʹʹʹ), 121.1 (C-3ʹʹʹʹ), 125.0 (C-5ʹʹʹ), 125.3 (C-2ʹʹʹʹ), 125.9 (C-2ʹ, C-6ʹ), 126.5 (C-1ʹʹʹʹ), 128.2 (C-4ʹ), 129.2 (C-3ʹ, C-5ʹ), 129.9 (C-4ʹʹʹʹ), 135.2 (C-1ʹ), 139.3 (C-5), 139.4 (C-4), 140.5 (C-2), 143.7 (C-4ʹʹʹ), 150.4 (C-6ʹʹʹʹ); HRMS (ESI) m/z: Calculated for C26H30N8O3Na (525.23331), found (525.23142) [M+Na]+.

2.7.4.4. 1-(N1-benzyl-2-methyl-4-nitro-1H-imidazole-5-yl)-4-((N1-phenyl-1H-1,2,3-triazol-4- yl)methyl)piperazine 11d

Light-brown solid, 0.40 g (93%); m.p. 188–190 °C; 1H NMR (500 MHz, CDCl3) δ: 2.28 (s, 3H, -CH3), 2.59 (br.s, 4H, H-3ʹʹ, H-5ʹʹ), 3.10 (br.s, 4H, H-2ʹʹ, H-6ʹʹ), 3.79 (s, 2H, -CH2 CCH), 5.06 (s, 2H, NCH2Ph), 6.95 (d, J = 7.3, 2H, H-2ʹ, H-6ʹ), 7.32 7.23 (m, 3H, H-3ʹ, H-5ʹ, H-4ʹ), 7.40 (t, J = 7.4, 1H, H-4ʹʹʹʹ), 7.49 (ps.t, 2H, H-3ʹʹʹʹ, H 5ʹʹʹʹ), 7.70 (d, J = 7.9, 2H, H-2ʹʹʹʹ, H-6ʹʹʹʹ), 8.03 (s, 1H, H-5ʹʹʹ); 13C NMR (125 MHz,CDCl3) δ: 14.1(-CH3), 46.4 (NCH2Ph), 48.9 (C-2ʹʹ, C-6ʹʹ), 53.1 (C-3ʹʹ, C-5ʹʹ), 53.3 (-CH2CCH), 120.5 (C-2ʹʹʹʹ, C-6ʹʹʹʹ), 121.3 (C-5ʹʹʹ), 135.2 (C-1ʹ), 125.9 (C-2ʹ, C-6ʹ), 128.2 (C-4ʹ), 128.8 (C-4ʹʹʹʹ), 129.2 (C-3ʹ, C-5ʹ), 129.8 (C-3ʹʹʹʹ, C-5ʹʹʹʹ), 137.0(C-5), 139.0 (C-1ʹʹʹʹ), 139.4 (C-4), 140.5 (C-2), 144.8 (C-4ʹʹʹ); HRMS (ESI) m/z: Calculated for C24H26N8O2Na (481.20709), found (481.20592) [M+Na] +.

2.7.4.5. 1-(N1-benzyl-2-methyl-4-nitro-1H-imidazole-5-yl)-4-((N1-(p-tolyl)-1H-1,2,3-triazol-4- yl)methyl)piperazine 11e

Off-white solid, 0.25 g (92%); m.p. 160–162 °C; 1H NMR (500 MHz, CDCl3) δ: 2.28 (s, 3H, - CH3), 2.39 (s, 3H, -CCH3), 2.54 (br.s, 4H, H-3ʹʹ, H-5ʹʹ), 3.08 (br.s, 4H, H-2ʹʹ, H-6ʹʹ), 3.74 (s, 2H, -CH2 CCH), 5.06 (s, 2H, NCH2Ph), 6.97 (d, J = 7.4, 2H, H-2ʹ, H-6ʹ), 7.32–7.23 (m, 3H, H-3ʹ, H-5ʹ, H-4ʹ), 7.29–7.26 (m, 2H, H-3ʹʹʹʹ, H-5ʹʹʹʹ), 7.57 (d, J = 8.3, 2H, H-2ʹʹʹʹ, H-6ʹʹʹʹ), 7.92 (s, 1H, H-5ʹʹʹ); 13C NMR (125 MHz,CDCl3) δ: 14.1(-CH3), 21.1 (-CCH3), 46.4 (NCH2Ph), 49.1 (C-2ʹʹ, C-6ʹʹ), 53.2 (C-3ʹʹ, C-5ʹʹ), 53.5 (-CH2CCH), 120.3 (C-3ʹʹʹʹ, C-5ʹʹʹʹ), 120.9 (C-5ʹʹʹ), 125.9 (C-2ʹ, C-6ʹ), 128.2 (C-4ʹ), 129.2 (C-3ʹ, C-5ʹ), 130.2 (C-2ʹʹʹʹ, C-6ʹʹʹʹ), 134.8 (C-5), 135.2 (C-1ʹ), 138.8 (C-4ʹʹʹʹ), 139.2 (C-1ʹʹʹʹ), 139.4 (C-4), 140.5 (C-2), 145.1 (C-4ʹʹʹ); HRMS (ESI) m/z: Calculated for C25H28N8O2Na (495.22274), found (495.22240) [M+Na] +.

2.7.4.6. 1-(N1-benzyl-2-methyl-4-nitro-1H-imidazole-5-yl)-4-((N1-(4-methoxyphenyl)-1H-1,2,3-triazol- 4-yl)methyl)piperazine 11f

Off-white solid, 0.15 g (87%); m.p. 116–118 °C; 1H NMR (500 MHz, CDCl3) δ: 2.27 (s, 3H, - CH3), 2.55 (br.s, 4H, H-3ʹʹ, H-5ʹʹ), 3.07 (br.s, 4H, H-2ʹʹ, H-6ʹʹ), 3.74 (s, 2H, -CH2 CCH), 3.83 (s, 3H, -OCH3), 5.06 (s, 2H, NCH2Ph), 6.95 (d, J = 7.6, 2H, H-2ʹ, H-6ʹ), 6.99 (d, J = 8.5, 2H, H-3ʹʹʹʹ, H-5ʹʹʹʹ), 7.32–7.23 (m, 3H, H-3ʹ, H-5ʹ, H-4ʹ), 7.59 (d, J = 8.4, 2H, H-2ʹʹʹʹ, H-6ʹʹʹʹ), 7.89 (s, 1H, H-5ʹʹʹ); 13C NMR (125 MHz,CDCl3) δ: 14.1 (-CH3), 46.4 (NCH2Ph), 49.1 (C-2ʹʹ, C-6ʹʹ), 53.2 (C-3ʹʹ, C-5ʹʹ), 53.5 (-CH2CCH), 55.6 (-OCH3), 114.8 (C-3ʹʹʹʹ, C-5ʹʹʹʹ), 121.2 (C-5ʹʹʹ), 122.1 (C-2ʹʹʹʹ, C-6ʹʹʹʹ), 125.9 (C-2ʹ, C-6ʹ), 128.2 (C-4ʹ), 129.2 (C-3ʹ, C-5ʹ), 130.5 (C-1ʹʹʹʹ), 135.2 (C-1ʹ), 139.2 (C-5), 139.4 (C-4), 140.5 (C-2), 144.9 (C-4ʹʹʹ), 159.8 (C-4ʹʹʹʹ); HRMS (ESI) m/z: Calculated for C25H28N8O3Na (511.21766), found (511.21764) [M+Na] +, and Calculated for C25H29N8O3 (489.23571), found (489.23574) [M+H]+.

2.7.4.7. 1-(N1-benzyl-2-methyl-4-nitro-1H-imidazole-5-yl)-4-((N1-(o-tolyl)-1H-1,2,3-triazol-4- yl)methyl)piperazine 11g

Cummins solid, 0.40 g (86%); m.p. 177–179 °C; 1H NMR (500 MHz, CDCl3) δ: 2.17 (s, 3H, - CCH3), 2.28 (s, 3H, -CH3), 2.57 (br.s, 4H, H-3ʹʹ, H-5ʹʹ), 3.09 (br.s, 4H, H-2ʹʹ, H-6ʹʹ), 3.73 (s, 2H, -CH2 CCH), 5.07 (s, 2H, NCH2Ph), 6.96 (d, J = 7.3, 2H, H-2ʹ, H-6ʹ), 7.32–7.23 (m, 3H, H-3ʹ, H-5ʹ, H-4ʹ), 7.29 (d, J = 6.9, 1H, H-6ʹʹʹʹ), 7.31 (d, J = 7.2, 1H, H-3ʹʹʹʹ), 7.37–7.34 (m:ps.t, 2H, H-4ʹʹʹʹ, H-5ʹʹʹʹ), 7.73 (s, 1H, H-5ʹʹʹ); 13C NMR (125 MHz,CDCl3) δ: 14.1(-CH3), 17.9 (-CCH3), 46.4 (NCH2Ph), 49.0 (C-2ʹʹ, C-6ʹʹ), 53.2 (C-3ʹʹ, C-5ʹʹ), 53.4 (-CH2CCH), 124.6 (C-5ʹʹʹ), 125.9 (C-2ʹ, C-6ʹ), 125.9 (C-6ʹʹʹʹ), 126.9 (C-5ʹʹʹʹ), 128.2 (C-4ʹ), 129.2 (C-3ʹ, C-5ʹ), 129.9 (C-4ʹʹʹʹ), 131.5 (C-3ʹʹʹʹ), 133.6 (C-1ʹʹʹʹ), 135.2 (C-1ʹ), 136.5 (C-2ʹʹʹʹ), 139.2 (C-5), 139.4 (C-4), 140.5 (C-2), 144.2 (C-4ʹʹʹ); HRMS (ESI) m/z: Calculated for C25H28N8O2Na (495.22274), found (495.22311) [M+Na] +.

2.7.4.8. 1-(N1-benzyl-2-methyl-4-nitro-1H-imidazole-5-yl)-4-((N1-(2-ethylphenyl)-1H-1,2,3-triazol-4- yl)methyl)piperazine 11h

Brown crystal, 0.80 g (82%); m.p. 194–196 °C; 1H NMR (500 MHz, CDCl3) δ: 1.06 (t, J = 7.6, 3H, -CCH2CH3), 2.28 (s, 3H, -CH3), 2.47 (q, J = 7.5, 2H, -CCH2CH3), 2.58 (br.s, 4H, H-3ʹʹ, H- 5ʹʹ), 3.06 (br.s, 4H, H-2ʹʹ, H-6ʹʹ), 3.78 (s, 2H, -CH2 CCH), 5.07 (s, 2H, NCH2Ph), 6.96 (d, J = 7.2, 2H, H-2ʹ, H-6ʹ), 7.33–7.23 (m, 5H, H-3ʹ, H-5ʹ, H-4ʹ, H-3ʹʹʹʹ, H-6ʹʹʹʹ), 7.44–7.34 (m, 2H, H-4ʹʹʹʹ, H-5ʹʹʹʹ), 7.72 (s, 1H, H-5ʹʹʹ); 13C NMR (125 MHz,CDCl3) δ: 14.1 (-CH3), 14.9 (-CCH2CH3), 24.3 (-CCH2CH3), 46.4 (NCH2Ph), 49.0 (C-2ʹʹ, C-6ʹʹ), 53.2 (C-3ʹʹ, C-5ʹʹ), 53.4 (-CH2CCH), 124.8 (C-5ʹʹʹ), 125.9 (C-2ʹ, C-6ʹ), 126.3 (C-6ʹʹʹʹ), 126.8 (C-5ʹʹʹʹ), 128.2 (C-4ʹ), 129.2 (C-3ʹ, C-5ʹ), 129.9 (C-4ʹʹʹʹ), 130.2 (C-3ʹʹʹʹ), 135.2 (C-1ʹ), 135.9 (C-1ʹʹʹʹ), 139.2 (C-2ʹʹʹʹ), 139.4 (C-5), 139.8 (C-4), 140.5 (C-2), 144.7 (C-4ʹʹʹ); HRMS (ESI) m/z: Calculated for C26H30N8O2Na (509.23839), found (509.23686) [M+Na] +.

2.7.4.9. 1-(N1-benzyl-2-methyl-4-nitro-1H-imidazole-5-yl)-4-((N1-(2-nitrophenyl)-1H-1,2,3-triazol-4- yl)methyl)piperazine 11i

Yellow solid, 0.30 g (94%); m.p. 93–95 °C; 1H NMR (500 MHz, CDCl3) δ: 2.27 (s, 3H, -CH3), 5.06 (s, 2H, NCH2Ph), 3.06 (br.s, 4H, H-2ʹʹ, H-6ʹʹ), 2.54 (br.s, 4H, H-3ʹʹ, H-5ʹʹ), 3.78 (s, 2H, -CH2 CCH), 7.83 (s, 1H, H-5ʹʹʹ), 6.96 (d, J = 7.4, 2H, H-2ʹ, H-6ʹ), 7.32–7.23 (m, 3H, H-3ʹ, H-5ʹ, H-4ʹ), 8.06 (d, J = 8.0, 1H, H-3ʹʹʹʹ), 7.67 (t, J = 7.7, 1H, H-4ʹʹʹʹ), 7.77 (t, J = 7.6, 1H, H-5ʹʹʹʹ), 7.61 (d, J = 7.8, 1H, H-6ʹʹʹʹ); 13C NMR (125 MHz,CDCl3) δ: 14.1(-CH3), 46.4 (NCH2Ph), 49.1 (C-2ʹʹ, C-6ʹʹ), 53.0 (C-3ʹʹ, C-5ʹʹ), 53.2 (-CH2CCH), 124.6 (C-5ʹʹʹ), 125.6 (C-3ʹʹʹʹ), 125.9 (C-2ʹ, C-6ʹ), 127.9 (C-6ʹʹʹʹ), 128.2 (C-4ʹ), 129.2 (C-3ʹ, C-5ʹ), 130.4 (C-1ʹʹʹʹ), 130.8 (C-4ʹʹʹʹ), 133.9 (C-5ʹʹʹʹ), 135.2 (C-1ʹ), 139.2 (C-5), 139.4 (C-4), 140.5 (C-2), 144.5 (C-4ʹʹʹ), 144.9 (C-2ʹʹʹʹ); HRMS (ESI) m/z: Calculated for C24H25N9O4Na (526.19217), found (526.19014) [M+Na] +.

2.7.4.10. 1-(N1-benzyl-2-methyl-4-nitro-1H-imidazole-5-yl)-4-((N1-(4-chlorophenyl)-1H-1,2,3-triazol-4- yl)methyl)piperazine 11j

Beige solid, 0.80 g (79%); m.p. 166–168 °C; 1H NMR (500 MHz, CDCl3) δ: 2.27 (s, 3H, - CH3), 2.54 (br.s, 4H, H-3ʹʹ, H-5ʹʹ), 3.06 (br.s, 4H, H-2ʹʹ, H-6ʹʹ), 3.73 (s, 2H, -CH2 CCH), 5.06 (s, 2H, NCH2Ph), 6.95 (d, J = 7.2, 2H, H-2ʹ, H-6ʹ), 7.32–7.23 (m, 3H, H-3ʹ, H-5ʹ, H-4ʹ), 7.47 (d, J = 8.4, 2H, H-3ʹʹʹʹ, H-5ʹʹʹʹ), 7.66 (d, J = 8.4, 2H, H-2ʹʹʹʹ, H-6ʹʹʹʹ), 7.96 (s, 1H, H-5ʹʹʹ); 13C NMR (125 MHz,CDCl3) δ: 14.1 (-CH3), 46.4 (NCH2Ph), 49.1 (C-2ʹʹ, C-6ʹʹ), 53.3 (C-3ʹʹ, C-5ʹʹ), 53.5 (-CH2CCH), 120.9 (C-5ʹʹʹ), 121.6 (C-2ʹʹʹʹ, C-6ʹʹʹʹ), 125.9 (C-2ʹ, C-6ʹ), 128.2 (C-4ʹ), 129.2 (C-3ʹ, C-5ʹ), 129.9 (C-3ʹʹʹʹ, C-5ʹʹʹʹ), 134.5 (C-4ʹʹʹʹ), 135.2 (C-1ʹ), 135.5 (C-1ʹʹʹʹ), 139.2 (C-5), 139.4 (C-4), 140.5 (C-2), 145.6 (C-4ʹʹʹ); HRMS (ESI) m/z: Calculated for C24H25ClN8O2Na (515.16812), found (515.16634) [M+Na] +.

2.7.4.11. 1-(N1-benzyl-2-methyl-4-nitro-1H-imidazole-5-yl)-4-((N1-(4-nitrophenyl)-1H-1,2,3-triazol-4- yl)methyl)piperazine 11k

Yellow solid, 0.60 g (94%); m.p. 86–88 °C; 1H NMR (500 MHz, CDCl3) δ: 2.27 (s, 3H, -CH3), 2.55 (br.s, 4H, H-3ʹʹ, H-5ʹʹ), 3.05 (br.s, 4H, H-2ʹʹ, H-6ʹʹ), 3.76 (s, 2H, -CH2 CCH), 5.07 (s, H, NCH2Ph), 6.96 (d, J = 7.2, 2H, H-2ʹ, H-6ʹ), 7.32–7.23 (m, 3H, H-3ʹ, H-5ʹ, H-4ʹ), 7.96 (d, J = 8.7, 2H, H-2ʹʹʹʹ, H-6ʹʹʹʹ), 8.13 (s, 1H, H-5ʹʹʹ), 8.36 (d, J = 8.7, 2H, H-3ʹʹʹʹ, H-5ʹʹʹʹ); 13C NMR (125 MHz,CDCl3) δ: 14.1 (-CH3), 46.4 (NCH2Ph), 49.0 (C-2ʹʹ, C-6ʹʹ), 53.3 (C-3ʹʹ, C-5ʹʹ), 53.4 (-CH2CCH), 120.4 (C-2ʹʹʹʹ, C-6ʹʹʹʹ), 120.9 (C-5ʹʹʹ), 125.6 (C-3ʹʹʹʹ, C-5ʹʹʹʹ), 125.9 (C-2ʹ, C-6ʹ), 128.2 (C-4ʹ), 129.2 (C-3ʹ, C-5ʹ), 135.2 (C-1ʹ), 139.1 (C-5), 139.5 (C-4), 140.6 (C-2), 141.2 (C-1ʹʹʹʹ), 146.5 (C-4ʹʹʹ), 147.2 (C-4ʹʹʹʹ); HRMS (ESI) m/z: Calculated for C24H25N9O4Na (526.19217), found (526.19393) [M+Na] +.

2.7.4.12. 1-(N1-benzyl-2-methyl-4-nitro-1H-imidazole-5-yl)-4-((N1-(3-methoxyphenyl)-1H-1,2,3-triazol- 4-yl)methyl)piperazine 11l

Pale-brown solid, 0.65 g (90%); m.p. 158–160 °C; 1H NMR (500 MHz, CDCl3) δ: 2.27 (s, 3H, -CH3), 2.54 (br.s, 4H, H-3ʹʹ, H-5ʹʹ), 3.05 (br.s, 4H, H-2ʹʹ, H-6ʹʹ), 3.74 (s, 2H, -CH2 CCH), 3.84 (s, 3H, -OCH3), 5.06 (s, 2H, NCH2Ph), 6.93 (d, J = 6.3, 1H, H-4ʹʹʹʹ), 6.94 (s, 1H, H-2ʹʹʹʹ), 6.95 (d, J = 7.3, 2H, H-2ʹ, H-6ʹ), 7.32–7.23 (m, 3H, H-3ʹ, H-5ʹ, H-4ʹ), 7.37 (ps.t, 1H, H-5ʹʹʹʹ), 7.27 (d, J = 7.1, 1H, H-6ʹʹʹʹ), 7.96 (s, 1H, H-5ʹʹʹ); 13C NMR (125 MHz,CDCl3) δ: 14.1 (-CH3), 46.4 (NCH2Ph), 49.1 (C-2ʹʹ, C-6ʹʹ), 53.2 (C-3ʹʹ, C-5ʹʹ), 53.5 (-CH2CCH), 55.7 (-OCH3), 106.2 (C-2ʹʹʹʹ), 112.3 (C-4ʹʹʹʹ), 114.7 (C-6ʹʹʹʹ), 121.1 (C-5ʹʹʹ), 125.9 (C-2ʹ, C-6ʹ), 128.2 (C-4ʹ), 129.2 (C-3ʹ, C-5ʹ), 130.5 (C-5ʹʹʹʹ), 1353.2 (C-1ʹ), 138.1 (C-1ʹʹʹʹ), 139.2 (C-5), 139.4 (C-4), 140.5 (C-2), 145.1 (C-4ʹʹʹ), 160.6 (C-3ʹʹʹʹ); HRMS (ESI) m/z: Calculated for C25H28N8O3Na (511.21766), found (511.21628) [M+Na] +.

2.7.4.13. 1-(N1-benzyl-2-methyl-4-nitro-1H-imidazole-5-yl)-4-((N1-(m-tolyl)-1H-1,2,3-triazol-4- yl)methyl)piperazine 11m

Grey solid, 0.80 g (87%); m.p. 174–176 °C; 1H NMR (500 MHz, CDCl3) δ: 2.27 (s, 3H, -CH3), 2.41 (s, 3H, -CCH3), 2.53 (br.s, 4H, H-3ʹʹ, H-5ʹʹ), 3.05 (br.s, 4H, H-2ʹʹ, H-6ʹʹ), 3.73 (s, 2H, -CH2CCH), 5.06 (s, 2H, NCH2Ph), 6.95 (d, J = 7.3, 2H, H-2ʹ, H-6ʹ), 7.20 (d, J = 7.6, 1H, H-4ʹʹʹʹ), 7.32–7.23 (m, 3H, H-3ʹ, H-5ʹ, H-4ʹ), 7.35 (ps.t, 1H, H-5ʹʹʹʹ), 7.47 (d, J = 7.9, 1H, H-6ʹʹʹʹ), 7.54 (s, 1H, H-2ʹʹʹʹ), 7.93 (s, 1H, H-5ʹʹʹ); 13C NMR (125 MHz,CDCl3) δ: 14.1 (-CH3), 21.4 (-CCH3), 46.4 (NCH2Ph), 49.1 (C-2ʹʹ, C-6ʹʹ), 53.3 (C-3ʹʹ, C-5ʹʹ), 53.6 (-CH2CCH), 117.5 (C-6ʹʹʹʹ), 120.9 (C-5ʹʹʹ), 121.1 (C-5ʹʹʹʹ), 125.9 (C-2ʹ, C-6ʹ), 128.2 (C-4ʹ), 129.2 (C-3ʹ, C-5ʹ), 129.5 (C-4ʹʹʹʹ), 129.8 (C-2ʹʹʹʹ), 135.2 (C-1ʹ), 137.0 (C-1ʹʹʹʹ), 139.2 (C-5), 139.4 (C-4), 140.0 (C-3ʹʹʹʹ), 140.5 (C-2), 145.3 (C-4ʹʹʹ); HRMS (ESI) m/z: Calculated for C25H28N8O2Na (495.22274), found (495.22381) [M+Na]+.

2.7.4.14. 1-(N1-benzyl-2-methyl-4-nitro-1H-imidazole-5-yl)-4-((N1-(3-chlorophenyl)-1H-1,2,3-triazol-4- yl)methyl)piperazine 11n

Pale-Beige solid, 0.80 g (83%); m.p. 176–178 °C; 1H NMR (500 MHz, CDCl3) δ: 2.26 (s, 3H,-CH3), 2.54 (br.s, 4H, H-3ʹʹ, H-5ʹʹ), 3.04 (br.s, 4H, H-2ʹʹ, H-6ʹʹ), 3.73 (s, 2H, -CH2 CCH), 5.06 (s, 2H, NCH2Ph), 6.95 (d, J = 7.2, 2H, H-2ʹ, H-6ʹ), 7.32–7.23 (m, 3H, H-3ʹ, H-5ʹ, H-4ʹ), 7.36 (d, J = 7.9, 1H, H-4ʹʹʹʹ), 7.41 (ps.t, 1H, H-5ʹʹʹʹ), 7.61 (d, J = 6.9, 1H, H-6ʹʹʹʹ), 7.77 (s, 1H, H-2ʹʹʹʹ), 7.99 (s, 1H, H-5ʹʹʹ); 13C NMR (125 MHz,CDCl3) δ: 14.1 (-CH3), 46.5 (NCH2Ph), 49.1 (C-2ʹʹ, C-6ʹʹ), 53.2 (C-3ʹʹ, C-5ʹʹ), 53.3 (-CH2CCH), 118.4 (C-2ʹʹʹʹ), 120.7 (C-6ʹʹʹʹ), 125.1 (C-5ʹʹʹ), 125.9 (C-2ʹ, C-6ʹ), 128.2 (C-4ʹ), 128.8 (C-4ʹʹʹʹ), 129.2 (C-3ʹ, C-5ʹ), 130.9 (C-5ʹʹʹʹ), 135.2 (C-1ʹ), 135.2 (C-1ʹʹʹʹ), 135.6 (C-3ʹʹʹʹ), 138.0 (C-5), 139.5 (C-4), 140.6 (C-2), 145.5 (C-4ʹʹʹ); HRMS (ESI) m/z: Calculated for C24H25ClN8O2Na (515.16812), found (515.16677) [M+Na].

2.7.4.15. 1-(N1-benzyl-2-methyl-4-nitro-1H-imidazole-5-yl)-4-((N1-(5,6,7,8-tetrahydronaphthalen-1-yl)- 1H-1,2,3-triazol-4-yl)methyl)piperazine 11o

Deep purple solid, 0.35 g (87%); m.p. 106–108 °C; 1H NMR (500 MHz, CDCl3) δ: 1.21 (m:s, 2H, H-5ʹʹʹʹ), 1.74–1.67 (m:s, 4H, H-6ʹʹʹʹ, H-7ʹʹʹʹ), 2.29 (s, 3H, -CH3), 2.41 (br.s, 2H, H-8ʹʹʹʹ), 2.80 (br.s, 4H, H-3ʹʹ, H-5ʹʹ), 3.04 (br.s, 4H, H-2ʹʹ, H-6ʹʹ), 4.26 (s, 2H, -CH2 CCH), 5.09 (s, 2H, NCH2Ph), 6.98 (d, J = 6.6, 2H, H-2ʹ, H-6ʹ), 7.09 (br.s, 1H, H-3ʹʹʹʹ), 7.18 (br.s, 1H, H-4ʹʹʹʹ), 7.23 (br.s, 1H, H-2ʹʹʹʹ), 7.27 (t, J = 6.7, H-4ʹ), 7.32 (pseudo t, 2H, H-3ʹ, H-5ʹ), 8.32 (s, 1H, H-5ʹʹʹ); 13C NMR (125 MHz,CDCl3) δ: 14.1(-CH3), 22.4 (C-6ʹʹʹʹ, C-7ʹʹʹʹ), 24.9 (C-8ʹʹʹʹ), 29.5 (C-2ʹʹ, C-6ʹʹ), 29.7 (C-5ʹʹʹʹ), 47.0 (NCH2Ph), 52.0 (C-3ʹʹ, C-5ʹʹ), 52.0(-CH2CCH), 123.5 (C-5ʹʹʹ), 125.8 (C-2ʹ, C-6ʹ), 125.9 (C-2ʹʹʹʹ, C-4ʹʹʹʹ), 128.4 (C-4ʹ), 129.4 (C-3ʹ, C-5ʹ), 131.2 (C-3ʹʹʹʹ), 132.9 (C-1ʹʹʹʹ), 135.1 (C-1ʹ), 136.2 (C-4ʹʹʹʹa, C-8ʹʹʹʹa), 137.8 (C-5), 139.5 (C-4), 141.1 (C-2), 141.1 (C-4ʹʹʹ); HRMS (ESI) m/z: Calculated for C28H32N8O2Na (535.25404), found (535.25552) [M+Na] +, and Calculated for C28H33N8O2 (513.27210), found (513.27376) [M+H] +.

2.7.4.16. 1-((N1-(benzo[d] [1,3]dioxol-5-yl)-1H-1,2,3-triazol-4-yl)methyl)-4-(N1-benzyl-2-methyl-4- nitro-1H-imidazole-5-yl) piperazine 11p

Pale-grey solid, 0.50 g (88%); m.p. 159–161 °C; 1H NMR (500 MHz, CDCl3) δ: 2.32 (s, 3H,

-CH3), 2.92 (br.s, 4H, H-3ʹʹ, H-5ʹʹ), 3.23 (br.s, 4H, H-2ʹʹ, H-6ʹʹ), 4.44 (s, 2H, -CH2 CCH), 5.10 (s, 2H, NCH2Ph), 6.02 (s, 2H, H-5ʹʹʹʹ), 6.85 (d, J = 7.4, 1H, H-3ʹʹʹʹ), 6.97 (d, J = 6.0, 2H, H-2ʹ, H-6ʹ), 7.15 (d, J = 6.2, 1H, H-2ʹʹʹʹ), 7.23 (s, 1H, H-7ʹʹʹʹ), 7.28 (t, J = 6.8, H-4ʹ), 7.32 (pseudo t, 2H, H-3ʹ, H-5ʹ), 8.76 (s, 1H, H-5ʹʹʹ); 13C NMR (125 MHz, CDCl3) δ: 14.1(-CH3), 47.0 (NCH2Ph), 47.3 (C-2ʹʹ, C-6ʹʹ), 51.4 (C-3ʹʹ, C-5ʹʹ), 51.4 (-CH2CCH), 102.2 (C-5ʹʹʹʹ), 102.8 (C-7ʹʹʹʹ), 108.6 (C-2ʹʹʹʹ), 114.6 (C-3ʹʹʹʹ), 114.6 (C-5ʹʹʹ), 125.7 (C-2ʹ, C-6ʹ), 128.5 (C-4ʹ), 129.5 (C-3ʹ, C-5ʹ), 130.7 (C-1ʹʹʹʹ), 130.7 (C-1ʹ), 134.9 (C-5), 137.8 (C-4), 141.2 (C-2), 148.4 (C-3ʹʹʹʹa, C-6ʹʹʹʹa), 148.7 (C-4ʹʹʹ); HRMS (ESI) m/z: Calculated for C25H26N8O4Na (525.19692), found (525.19510) [M+Na]+, and Calculated for C25H27N8O4 (503.21498), found (503.21325) [M+H]+.

2.7.4.17. 1-(N1-benzyl-2-methyl-4-nitro-1H-imidazole-5-yl)-4-((N1-(naphthalen-1-yl)-1H-1,2,3-triazol- 4-yl)methyl)piperazine 11q

Yellow solid, 0.15 g (69%); m.p. 172–174 °C; 1H NMR (500 MHz, CDCl3) δ: 2.28 (s, 3H, - CH3), 2.62 (br.s, 4H, H-3ʹʹ, H-5ʹʹ), 3.10 (br.s, 4H, H-2ʹʹ, H-6ʹʹ), 3.84 (s, 2H, -CH2 CCH), 5.08 (s, 2H, NCH2Ph), 6.98 (d, J = 7.4, 2H, H-2ʹ, H-6ʹ), 7.23 (s, 1H, H-5ʹʹʹ), 7.28 (t, J = 7.1, H-4ʹ), 7.33 (pseudo t, 2H, H-3ʹ, H-5ʹ), 7.51 (d, J = 7.7, 1H, H-2ʹʹʹʹ), 7.55 (m, 3H, H-6ʹʹʹʹ, H-7ʹʹʹʹ,H-5ʹʹʹʹ), 7.92 (m, 2H, H-5ʹʹʹʹ, H-8ʹʹʹʹ), 7.98 (m, 1H, H-4ʹʹʹʹ); 13C NMR (125 MHz,CDCl3) δ: 14.1(-CH3), 46.4 (NCH2Ph), 49.1 (C-2ʹʹ, C-6ʹʹ), 53.3 (C-3ʹʹ, C-5ʹʹ), 53.5 (-CH2CCH), 122.3 (C-3ʹʹʹʹ), 123.5 (C-6ʹʹʹʹ), 124.9 (C-7ʹʹʹʹ), 125.6 (C-5ʹʹʹ), 125.9 (C-2ʹ, C-6ʹ), 127.1 (C-5ʹʹʹʹ), 127.9 (C-4ʹʹʹʹ), 128.3 (C-4ʹ), 128.3 (C-8ʹʹʹʹ), 128.5 (C-4ʹʹʹʹa), 129.3 (C-3ʹ, C-5ʹ), 130.4 (C-2ʹʹʹʹ), 133.7 (C-8ʹʹʹʹa), 134.2 (C-1ʹʹʹʹ), 135.2 (C-1ʹ), 139.2 (C-5), 139.4 (C-4), 140.5 (C-2), 144.3 (C-4ʹʹʹ); HRMS (ESI) m/z: Calculated for C28H28N8O2Na (531.22274), found (531.22195) [M+Na]+.

3. Results and discussion

Quite recently, we have synthesized the new compound 5 [1-(N1-benzyl-2-methyl-4-nitro-1H-imidazole-5-yl)-4-(prop-2-yn-1-yl) piperazine], which has been selected as a main scaffold with an active arm for building new potentially active hybrid systems. The benzylation of the commercially available 4-nitroimidazoles 1 is carried out with benzyl chloride to produce compound 2 [87]. The bromination of N1-benzyl-2- methyl-4-nitroimidazole 2, with liquid bromine in dimethylformamide (DMF) in the presence of potassium carbonate as a base has afforded the desired product 3 [88]. The reaction of 5-bromo-N1- benzyl-2-methyl-4- nitroimidazole 3 with piperazine using isopropanol as a solvent gives compound 4 [88]. The major scaffold 5 is prepared by the reaction of the piperazine derivative 4 with propargyl bromide using NaH [89] as yellow flakes with 82% yield (Scheme 1).

Scheme 1.

Reagents and conditions: i. Benzyl chloride, NaH, DMF; ii. Br₂, DMF, K₂CO₃, 60–70 °C, 4 h; iii. Piperazine, isopropanol, 90–100 °C, 5 h; iv. Propargyl bromide, NaH, THF, 0 °C, 30 min.

The successful propargylation for the newly synthesized compound 5 is confirmed by the absence of the –NH signal of the piperazine ring in compound 4 and the appearance of two new signals. The signals are assigned to the two protons of the propargyl resonate as a doublet at δ = 3.28 ppm, and the terminal alkyne proton as triplet at δ = 3.17 ppm. In the 13C NMR signals for the C-4 and -CH3 resonated at about δ = 138.8 and 14.1 ppm respectively. The three signals of methylene carbons on the N1-benzyl nitro imidazole ring and the piperazine carbons appear at δ = 46.2, 48.5 and 51.4 ppm, respectively. While the three new signals corresponding to the propargyl carbons resonate at δ = 46.4, 76.3 and 79.6 ppm.

Scheme 2 depicts the chemistry employed in our designed target compounds, where the alkyne moiety in compound 5 is decorated via click chemistry with three different types of azides namely (Azidomethyl) benzene derivatives 6a–k, 2-azido-1-phenyl ethanone derivatives 7a–c, and azidobenzene precursor's 8a–q. These precursors are utilized in preparing our library consisting of three different classes of hybrid systems: 9a–k, 10a–c, and 11a–q. Regio-selectively forming the 1,4- disubstituted-1,2,3-triazole ring via click chemistry in the presence of copper (I).

Scheme 2.

Reagents and conditions: i. 1 mol % CuI/TEA,Toluene, r.t., 12h; ii. CuSO₄.5H₂O, Na l-ascorbate, DMF:H₂O (3:2 v/v), r.t., 2 h.

The synthetic protocols for click reaction to generate the triazole moieties can be easily achieved by using toluene as a solvent (Scheme 2). Treatment of compound 5 with benzyl azides 6a–k, which are prepared directly by the reaction of substituted-benzyl chloride/bromide with an excess of sodium azide in DMF [90]. The target compounds 9a–k are isolated and purified with a 71–96% yield, while the structures of the compounds 9a–k are determined using NMR (1H and 13C) and high-resolution mass spectrometry (HRMS). 1H NMR spectra reveals the disappearance of the triple bond signal in compound 5. A new singlet signal for H-5ʹʹʹ appears at δ = 7.41 ppm, which is assigned to the newly formed triazole ring.

Such signals are the main characteristics for the new library of compounds. Besides that, two new methylene proton signals resonate at δ = 5.48 ppm are assigned to the benzyl group that is connected to the triazole ring. Another feature of the 1H NMR spectra is the down field shift of the methylene protons that are connected to the piperazine ring from δ = 3.28 ppm in compound 5 to δ = 3.64 ppm in compound 9a. This down field shift is a main characteristic feature of all the hybrid compounds which are synthesized in this class. All other protons are assigned unambiguously based on HMQC and HMBC analysis. The weak signal in HMQC for the piperazine carbons can be attributed to the dynamic equilibrium that restrict the rotation around the N1–C-5 bond at the nitroimidazole moiety (Fig. 2) [91].

Fig. 2.

Chair conformation of the piperazine ring, showed hindered, and chemical non-equivalence.

13C NMR spectrum is also in agreement with the indicated structure for compound 9a as well as all the series of compounds 9a–k. The triple bond signals in compound 5 that resonate at δ = 76.3 and 79.6 ppm are replaced with two characteristic signals for the triazole ring which resonate at δ = 145.0 and 122.6 ppm as quaternary and –CH carbons, respectively. These signals are considered to be the main features of the triazole ring construction. The down field shift for the methylene carbon which is connected to the piperazine ring from δ = 46.4 ppm in compound 5 to δ = 54.2 ppm in compound 9a is also another significant feature in this class. All other carbon signals resonate at their expected chemical shifts with small variations when compared with compound 5. Additional support for the proposed structures come from HRMS, which shows the correct molecular ions, [M+H] +, as suggested by their molecular formulas.

On the other hand, 2-Azido-1-phenyl ethanone precursors 7a–c, are prepared via the reaction of substituted 2-bromo-1-phenylethanone with sodium azide in acetonitrile to furnish the targeted azides 7a–c [92]. The formations of novel nitroimidazole-piperazine-1,2,3-triazole hybrids 10a–c are achieved by the reaction of compound 5 with 2-azido-1-phenyl ethanone precursors 7a–c in the presence of CuI/sodium l-ascorbate/CuSO4·5H2O in DMF: H2O (3:2 v/v) as the solvent afforded after chromatographic purification (eluent: 5% EtOAc: n-hexane) compounds 10a–c (39–83%) (Scheme 2).

The success of the click approaches to produce the 1,4-disubstituted-1,2,3-triazoles 10a–c has been evident by NMR (1H and 13C), HRMS analysis and DEPT–NMR. The 1H NMR spectra shows the appearance of one distinct singlet that resonated at δ = 7.48 ppm for compound 10a and is attributable to the 1,2,3-triazolyl proton. The eight methylene protons of the piperazine ring that resonate at δ = 2.78 and 3.01 ppm, appear as multiplets of the proton signals. While the new methylene protons, which are resonating at δ = 3.99 ppm, are assigned to the ethanone group which is linked to the triazole ring. 13C NMR spectra shows the absence of sp-carbon's signal of the terminal alkyne from compound 5 and the appearance of two new triazole ring signals of compound 10a resonate at δ = 128.3 and 139.4 ppm respectively. These signals are considered the main indication for the construction of the triazole ring. Moreover, the new carbonyl signal of the new substituents appears at δ = 190.4 ppm. The carbon atoms of the piperazine ring exhibit only one distinct peak at δ = 47.8 ppm. DEPT-NMR shows a complete contraction and shrinking signal of all of the carbon atoms which are related to the piperazine ring. This finding can be attributed to the restricted rotation regarding the N4–CH2 bond of the triazole ring moiety besides the restricted rotation around the N1–C-5 bond of the nitroimidazole ring moiety [93]. Hence positioning of the carbonyl group of the acetophenone in such a way is to impose a chemical equivalence upon the carbons C-3ʹʹ, C-5ʹʹ, C-2ʹʹ, and C-6ʹʹ, (Fig. 3).

Fig. 3.

Chair conformation of piperazine ring, showed hindered, and chemical non-equivalence.

Under the same optimized copper (I) catalyzed click synthesis, new nitroimidazole-piperazine-1,2,3-triazole hybrids 11a–n are tagged with different substituted aryl groups. They have been synthesized with good to excellent (68–95%) yield through the ligation of azidobenzene precursors 8a–n with the appropriate platform 5 in toluene (Scheme 2). Herein, substituted anilines are subjected to diazotization followed by reaction with sodium azide [94,95] to produce the azides 8a–q, which are used without further purifications.

The tagged nitroimidazole-piperazine-1,2,3-triazole hybrids 11o-q are fused with our synthesized library. It has been diversified further utilizing bicyclic azides namely 1,2,3,4-tetrahydronaphthalene 8o, benzo[d] [1,3]dioxole 8p, and naphthalene 8q. These azides produce the new compounds 11o-q with a 69–89% yield (Scheme 3).

Scheme 3.

Reagents and conditions: i. 1 mol % CuI/TEA,Toluene, r.t., 12h.

1H NMR confirms the building of our newly designed compounds 11a–q through the disappearance of the sp-proton signal from the starting compound 5, and the appearance of new signals in the aromatic region resonating exactly at δ = 7.0–8.0 ppm. The aromatic signals for compound 11a, which contain p-bromophenyl moiety, appear as one broad signal. The two protons of –NCH2Ph resonate at δ = 5.04 ppm as a singlet, while the other two protons of – CH2CCH appear at δ = 3.69 ppm. The 13C NMR spectra of compound 11a shows new signals in the aromatic region which resonate at δ = 120.9, 145.6 ppm to C-5ʹʹʹ and C-4ʹʹʹ respectively. All the carbon signals for the substituted aryl and benzyl groups appear as expected in the aromatic region. Two methylene carbons C-3ʹʹ, C-5ʹʹ in the piperazine ring resonate at δ = 53.2 ppm and appear in our compound as an overlapping signal that is assigned to –CH2CCH at δ = 53.4 ppm. DEPT spectrum show a shrunk signal of C-2ʹʹ and C-6ʹʹ, due to the dynamic equilibrium and the restricted rotation around N1–C-5 bond in the nitroimidazole moiety (Fig. 2). Considering the bulkiness of the nitroimidazole moiety, it may be possible that the inter conversion between the two chair conformations of the piperazine ring are hindered. Thus, it slows enough to impose the piperazine's non-equivalence carbons [91]. The molecular mass confirms the success of the construction of our new target compound by HRMS analysis of 11a which gives a molecular ion of [M+H]+ = 537.13684 corresponding to the molecular formula of C24H26BrN8O2.

3.1. The crystal structure of compound 9a

The undisputed proof for connecting building blocks of compound 9a is manifested via X-ray structural analysis. The compound is crystallized in the monoclinic system and p21/c space group (Fig. 4). shows the single structure for the newly synthesized hybrid compound.

Fig. 4.

Thermal Displacement ellipsoid plot of the asymmetric unit of the molecular structures in the target compound 9a.

The imidazole ring is closer to ideal planarity and the deviations from the least-squares plane by five ring atoms are smaller than the phenyl group. The twist between the rings is large, while the nitro groups are close to co-planarity with the imidazole ring. Geometrical parameters, bond lengths, and bond angles for 9a are shown in (Table 2).

Table 2.

Crystallographic data, data collection and structure refinement parameters for 9a.

Empirical formula	C25H28N8O2
Formula weight	472.55
Temperature/K	293(2)
Crystal system	monoclinic
Space group	P21/c
a/Å	11.585(4)
b/Å	10.620(7)
c/Å	20.415(8)
β/°	100.45(3)
Volume/Å3	2470(2)
Z	4
ρcalcg/cm3	1.271
μ/mm−1	0.085
F(000)	1000.0
Crystal size/mm3	0.2 × 0.1 × 0.06
Radiation	MoKα (λ = 0.71073)
2Θ range for data collection/°	6.222 to 58.654
Index ranges	−15 ≤ h ≤ 15, −12 ≤ k ≤ 13, −28 ≤ l ≤ 27
Reflections collected	13,315
Independent reflections	5743 [Rint = 0.0614, Rsigma = 0.1129]
Data/restraints/parameters	5743/0/318
Goodness-of-fit on F2	0.971
Final R indexes [I ≥ 2σ (I)]	R1 = 0.0603, wR2 = 0.1160
Final R indexes [all data]	R1 = 0.1729, wR2 = 0.1646
Largest diff. Peak/hole/e Å−3	0.16/-0.17

3.2. Biological evaluation

Al3l the newly synthesized hybrid compounds are tested against a panel of cancer cell lines, namely MCF-7 (human breast adenocarcinoma), HepG2 (hepatocellular carcinoma), PC3 (human prostate carcinoma), and one normal human dermal/fibroblasts (Table 3). The antitumor activity is compared with that of the known and FDA approved antitumor drugs. Doxorubicin [96] and Cisplatin [97]. The compounds 9g and 9k exhibit a promising activity against the (MCF-7) cell line with IC₅₀ = 2.0 and 5.0 μM, respectively. The Growth rate GR values of 9g and 9k were provided in supplementary files. The GR value relates naturally to a treatment's effects on cell population growth. According to our results, and after treatment of MCF-7 cell lines with 9g and 9k for 24 h, 9k showed cytotoxic effect on the concentration range from 75 to 300 μg/mL, and cytostatic effects on the concentration ranges from below 37.5 to 1 μg/mL. In contrast, 9g did not show cytotoxic effects on the range of tested concentrations 1–300 μg/mL but only was cytostatic (Fig. 5). The rest of the compounds have not shown notable activities against the tested cell lines. Furthermore, the selectivity toward MCF-7 cancer cell lines has encouraged us to investigate these two compounds and reveal their biological target at the molecular level.

Table 3.

Half-maximal inhibitory concentrations (IC₅₀) of tested compounds in human breast adenocarcinoma, hepatocellular carcinoma, human prostate carcinoma, and fibroblasts. Values mean ± SD expressed in (μM).

	IC50 ± SD (μM)
	Breast cancer cells		Liver cancer cells		Prostate cancer cells	Normal cells
Compound ID	MCF7	MDA-231	HEPG-2	PC-3	DU145	Fibro dental
Compound 2	0.068 ± 0.02	NT	0.58 ± 0.13	0.62 ± 0.24	NT	2.58 ± 0.45
Compound 3	4.2 ± 0.168	NT	>500	>500	NT	>500
Compound 4	0.085 ± 0.002	NT	3.96 ± 0.19	>500	NT	>500
Compound 5	0.084 ± 0.002	NT	2.85 ± 0.497	2.85 ± 0.498	NT	>500
Compound 9a	0.055 ± 0.04	NT	1.85 ± 0.50	1.65 ± 0.13	NT	>500
Compound 9b	0.079 ± 0.021	NT	2.65 ± 0.501	2.85 ± 0.502	NT	>500
Compound 9c	0.024 ± 0.0019	NT	>500	>500	NT	>500
Compound 9d	0.04 ± 0.015	NT	1.18 ± 0.54	1.85 ± 0.04	NT	3.85 ± 0.15
Compound 9e	0.079 ± 0.021	NT	>500	>500	NT	>500
Compound 9f	0.053 ± 0.06	NT	0.33 ± 0.16	1.73 ± 0.07	NT	2.73 ± 0.28
Compound 9g	0.002 ± 0.00003	NT	1.44 ± 0.22	1.84 ± 0.23	NT	>500
Compound 9h	0.048 ± 0.004	NT	1.3 ± 1.244	2.85 ± 0.508	NT	>500
Compound 9i	0.106 ± 0.0123	NT	>500	>500	NT	>500
Compound 9j	0.151 ± 0.02	NT	>500	>500	NT	>500
Compound 9k	0.005 ± 0.002	NT	4 ± 0.397	2.85 ± 0.511	NT	>500
Compound 10a	0.112 ± 0.03	1.153 ± 0.50	NT	0.262 ± 0.06	3.16 ± 0.03	NT
Compound 10b	0.372 ± 0.03	1.328 ± 0.9	NT	0.112 ± 0.06	8.95 ± 1.7	NT
Compound 10c	0.585 ± 0.037	1.38 ± 1.3	NT	0.149 ± 0.06	14.95 ± 1.6	>500
Compound 11a	2.12 ± 0.31	NT	>500	>500	NT	>500
Compound 11b	0.136 ± 0.06	NT	1.6 ± 0.079	1.2 ± 0.080	NT	NT
Compound 11c	0.399 ± 0.079	NT	2.96 ± 0.73	2.56 ± 0.17	NT	2.46 ± 0.38
Compound 11d	0.312 ± 0.065	NT	2.6 ± 0.179	2.2 ± 0.180	NT	2.9 ± 0.181
Compound 11e	0.219 ± 0.012	NT	2.3 ± 0.139	2.3 ± 0.140	NT	3.3 ± 0.141
Compound 11f	0.251 ± 0.071	NT	1.24 ± 0.46	1.44 ± 0.07	NT	>500
Compound 11g	0.183 ± 0.01	NT	2.56 ± 0.136	2.56 ± 0.137	NT	2.56 ± 0.138
Compound 11h	0.088 ± 0.037	NT	2.6 ± 0.037	2.6 ± 0.038	NT	3.6 ± 0.039
Compound 11i	0.185 ± 0.033	NT	1.6 ± 0.279	1.6 ± 0.280	NT	>500
Compound 11j	0.279 ± 0.02	NT	>500	>500	NT	>500
Compound 11k	0.671 ± 0.14	NT	1.6 ± 0.037	1.6 ± 0.03	NT	3.6 ± 0.39
Compound 11l	0.198 ± 0.01	NT	1.24 ± 0.46	1.24 ± 0.07	NT	3.64 ± 0.422
Compound 11 m	0.161 ± 0.016	NT	>500	>500	NT	>500
Compound 11n	0.155 ± 0.07	NT	1 ± 0.258	1 ± 0.38	NT	>500
Compound 11o	0.131 ± 0.03	NT	4 ± 0.198	>500	NT	>500
Compound 11p	0.052 ± 0.03	11.65 ± 1.6	NT	0.069 ± 0.03	0.803 ± 0.04	NT
Compound 11q	0.27 ± 0.04	NT	2.1 ± 0.037	2.8 ± 0.08	NT	2.9 ± 0.039
Doxorubicin	0.64 ± 0.04	2.3 ± 1.3	0.61 ± 0.05	0.62 ± 0.06	0.66 ± 0.04	>100
Cisplatin	14.6 ± 2.8	16.2 ± 1.6	9.64 ± 1.7	8.6 ± 1.5	8.5 ± 1.7	>100

a The IC_50s were determined by MTT method. Assays were performed duplicate in parallel. Data are shown as mean ± SD.

NT: Not Tested.

Fig. 5.

Logistic dose-response curve of 9g and 9k based on the calculated GR values. The (x-axis) is the range of treatment concentration. GR values greater than one are not allowed since the fitted curve to less than one represents the growth-inhibiting and cytotoxic treatments.

3.3. Molecular docking

Molecular docking of the synthesized compounds 9g and 9k are with human estrogen receptor alpha (hER) active site to study the ligand–protein interactions and free binding energies at atomic level. The Autodock scores show the same range of the binding free energies for the compounds hER within the value of (−10.22 ± 0.5 kcal mol⁻¹). The compound 9g (−9.32 kcal/mol⁻¹) exhibits best docking energy, whereas compound 9k (−9.24 kcal/mol⁻¹) has relatively lower binding affinity. Later, 1,4-disubstituted-1,2,3-triazoles 9g and 9k are considered for the further analysis of hER interactions. The chemical structure and binding energy are shown in (Table 4).

Table 4.

Docking binding energies and chemical structures of (9g, 9k) compounds and the 4-hydroxytamoxifen (4-OHT).

Compound	Chemical structure	Binding energy, ΔG
4-OHT_redocked		−10.22
9g		−9.32
9k		−9.24

The overlay structures docked pose of the compounds 9g and 9k with 4-hydroxytamoxifen (4-OHT) at the hER binding site is shown in (Fig. 6). It can be observed that both compounds form various hydrophobic interactions with Leu53, Met50, Thr54, Trp90, Met95, Met128, Leu232 amino acid residues and polar contact with His231 amino acid residue. While the triazole ring in the 9g derivative forms a hydrogen bond with Asp58 with distance 3.2 Å. Furthermore, the docking results showed that the derivatives have the same binding mode along with the 4-hydroxytamoxifen drug as shown in (Fig. 6). This study suggests that the modifications of these hybrid nitroimidazole skeleton may be worth exploring for the preparation of new anticancer agents.

Fig. 6.

The docking-poses of the human estrogen receptor hER pocket with (a) Compound 9g (purple), (b) Compound 9k (green), shows the important amino acid residues involved in the polar and non-polar interactions. (c) The docked poses for the 9g and 9k with the 4-OHT drug.

3.4. ADMET predictions

ADMET screening was applied for the potential compounds to predict the ‘Drug-likeness’ and ADMET properties. Physicochemical and ADMET properties have been evaluated by analysing the Lipinski's rule of 5 and the Veber rule [98,99]. In the ADMET studies the compounds were subjected to TPSA <100, log P < 5, rotatable bonds <25. The molecular weight <500 indicates good oral bioavailability, whereas the number hydrogen bond acceptors <10 and donors <5 indicates good intestinal availability. These predictions were calculated and used for analysing the physicochemical properties using the Swiss ADME tool [100]. And the results is summarized in (Table 5). We found that the hybrid 1,4-disubstituted-1,2,3-triazoles compounds were within the acceptable range of the ADMET criteria. In silico assessment of the compounds also showed that they have very good pharmacokinetic properties based on their physicochemical values. Detailed analysis is shown in (Table 5).

Table 5.

Physicochemical and drug likeness results of 9g and 9k compounds.

Compound	M.wt	Rotatable bonds	H-bond acceptors	H-bond donors	TPSA (Å2)	Lipinski violations	Ghose violations	Veber violations	Egan violations
9g	490.53	8	7	0	100.83	0	2	0	0
9k	486.57	8	6	0	100.83	0	2	0	0

4. Conclusions

In the search for new compounds with potent biological activities, many compounds with different moieties are synthesized via several steps. These substituted moieties include: 1-((N1-substituted benzyl-1H-1,2,3-triazol-4-yl)methyl)-4-(N1-benzyl-2-methyl-4-nitro- imidazole-5-yl) piperazine 9a-k, 2-(4-((4-(N1-benzyl-2-methyl-4-nitro-1H-imidazole-5-yl)piperazin-1-yl)methyl)-1H-1,2,3-triazol-1-yl)-1-substituted phenylethanone 10a-c, and 1- ((N1-substituted aryl -1H-1,2,3-triazol-4-yl)methyl)-4-(N1-benzyl-2-methyl-4-nitro-imidazole- 5-yl) piperazine 11a-q. Upon testing all of the newly synthesized compounds against different solid tumors derived cell lines, 9g and 9k are the most potent anticancer agents on only the MCF-7 cell line. The anticancer activity results suggest that these two compounds might act as lead promising anticancer agents for further structural modification. In addition, the docking results of the compounds 9g and 9k with 4-hydroxytamoxifen (4-OHT) at the hER binding site, show that form various hydrophobic interactions with Leu53, Met50, Thr54, Trp90, Met95, Met128, Leu232 amino acid residues and polar contact with His231 amino acid residue. The molecular docking results confirm that 9g and 9k targeted hER binding site in breast cancer cells, causing their apoptosis without affecting normal cells (fibroblasts) at the same conditions and examined concentrations. The ADMET screening of the most active compounds indicates good intestinal availability caused by the number of hydrogen bond acceptors <10 and donors <5, and indicates good oral bioavailability. Due to being subjected to TPSA <100, and rotatable bonds <25, these findings suggest that these two compounds could carry great potential activity against cancer cell lines such as: brain and lung cancers. However, these interesting findings need more investigation to confirm it.

Author contribution statement

Sadeekah Omar Saber: Conceived and designed the experiments; Performed the experiments; Analyzed and interpreted the data; Wrote the paper. Raed Al-Qawasmeh; Yaseen Al-Soud: Conceived and designed the experiments; Wrote the paper. Luay Abu-Qatouseh; Amneh Shtaiwi; Monther Khanfar: Analyzed and interpreted the data; Contributed reagents, materials, analysis tools or data.

Data availability statement

Data included in article/supplementary material/referenced in article.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A. Supplementary data

The following are the Supplementary data to this article: