Synthesis, anticancer, antioxidant activities and in silico studies of novel benzhydrylpiperazine bearing Δ 2 -1,2,3-triazoline hydrides

doi:10.21203/rs.3.rs-3156279/v1

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Synthesis, anticancer, antioxidant activities and in silico studies of novel benzhydrylpiperazine bearing Δ 2 -1,2,3-triazoline hydrides

https://doi.org/10.21203/rs.3.rs-3156279/v1

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To investigate the antiproliferative activity associated with the piperazine framework, a series of benzhydryl piperazine derivatives 8–18 were synthesized and characterized both spectroscopically and structurally. The antiproliferative activity of these compounds against eight human tumor cell lines was assessed. Among the tested compounds, compound 11 exhibited the highest potency, effectively inhibiting the proliferation of three selected human cancer cell lines, HL-60, Z138, and DND-41 with IC₅₀ values of 16.80, 18.50 and 19.20 µM, respectively. Compound 10 displayed IC₅₀ values of 19.90, 18.00 and 18.50 µM against the cell lines HL-60, Z138 and DND-41, whereas compound 13 showed IC₅₀ value of 19.90 µM against cell line DND-41. However, all compounds exhibited IC₅₀ values ranging from 22.95 and 58.45 µM against other tested cancer cell lines. These finding suggest that derivative 11 would be a promising potential lead compound for the development of novel antiproliferative agents. Further compounds 8–18 were evaluated for their antioxidant activity. Additionally, predictive docking studies were performed on the three-dimensional structures of acute myeloid leukemia (CDK2/cyclin A2, PDB: 7B7S, and protein kinase Akt1 PKB alpha, PDB: 4GV1).

Anticancer activity

Antioxidant activity

Benzhydrylpiperazine

Docking study analysis

3-Triazoline hybrids

Numerous derivatives of benzhydrylpiperazine have gained recognition as effective antihistaminic medications [1–6]. Additionally, these compounds exhibit a wide range of other pharmacological activities, such as antimicrobial and antifungal properties [7–12], calcium channel blocking effects [12–18], antiviral capabilities [19, 20], dopaminergic influences [21], selective dopamine D₃ receptor ligands [22], and enhance blood flow to the brain and alleviates vestibular-related syndroms [23]. Recent studies have revealed the diverse pharmacological activities attributed to the diphenylpiperazine component. The diphenylpiperazine fragment can be found in various compounds, which include antihistamines like cetirizine (Fig. 1, 1) and buclizine, calcium channel blockers drugs such as cinnarizine (Fig. 1, 2), the antimigrane agent lomerizine (Fig. 1, 3), the anticancer agent known as MS-209 (Fig. 1, 4), and the tranquillizing drug hydroxyzine (Fig. 1, 5). Furthermore, certain derivatives of benzhydrylpiperazine have shown promising potential as anticancer agents. Kumar et al. [24] reported the cytotoxic effects of various 1-benzhydrylpiperazine derivatives substituted with sulfonyl chlorides, acid chlorides and isothiocyanates. Gurdal et al. [25] examined the cytotoxicity of derivatives of 1-(substituted-benzoyl)-4-benzhydrylpiperazine and 1-[(substituted-phenyl) sulfonyl]-4-benzhydryl piperazine. Furthermore, Gan et al. [26] reported that benzhydryl derivatives incorporating imidazole and 1,2,3-triazole displayed significant activity against the human prostatic carcinoma (PC-3) cell line. Moreover, Yarim et al. [27] outlined the synthesis of 1-(4-substituted-benzoyl)-4-(4-chloro-benzhydryl)piperazine derivatives and their inhibitory impact on cancer cell lines derived from liver, breast, colon, gastric, and endometrial samples. A series of derivatives of 1-benzhydryl-sulfonyl-piperazine were synthesized and assessed for their effectiveness in inhibiting MDA-MB-231 breast cancer cells proliferation. Notably, compound 1-benzhydryl-4-(4-tert-butyl-benzenesulfonyl) piperazine demonstrated significant inhibitory activity [28]. In a recent study by Ruzic et al. [29], the synthesis of novel isoform-selective histone deacetylase (HDAC) inhibitors based on 1-benzhydrylpiperazine was demonstrated. One analogue, containing phenyl hydroxamic acid exhibited selective inhibition of HDAC6 inhibition with an IC₅₀ value of 30 nM. Valkova et al. [30] described the synthesis of novel 1-arylpiperazine derivatives and evaluated their affinity for 5-HT1A/5-HT2A receptors. One of these analogues exhibited the highest affinity value of 58.6 nM. However, a comprehensive review on the synthesis and biomedical application on benzhydryl amines in the pharmaceutical field from 2015–2019 as reported by Panda and colleagues [31].

In light of the biological significance of benzhydrylpiperazine derivatives, we present in this study the synthesis of innovative derivatives of benzhydrylpiperazine and the assessment of their anticancer and antioxidant activities, along with predictive molecular docking investigations.

Chemistry

1-Ally-4-((4-chlorophenyl)(phenyl)methyl)piperazine (7), derived from the reaction of 1-benzhydryl piperazine (6) and allyl bromide in the presence K₂CO₃/KI, has been selected as a crucial intermediate for synthesising novel 1-benzhydryl piperazine-based Δ²-1,2,3-triazoline hybrids. A yield of 79% was obtained for the formation of 7. Diez-Gonzales et al. [32] have documented the synthesis of Δ²-1,2,3- triazoline derivatives using deep eutectic solvent (DES) via the cycloaddition of aryl azides with olefines. In this study, our object is to investigate the synthesis of novel benzhydrylpiperazine analogues, inspired by the work of Diez-Gonales and his group. Additionally, we aim to assess the antiproliferative activity against various cancer cells. Thus, one-pot cycloaddition of 7 with various substituted 2-zido-N-aryl-acetamides (B): 2-azido-N-(o-tolyl)acetamide, 2-azido-N-(4-formylphenyl)acetamide, N-(4-acetylphenyl)-2-azidoacetamide, N-(3-acetylphenyl)-2-azidoacetamide, 2-(2-azidoacetamido)benzoic acid, 2-azido-N-(4-cyanophenyl) acetamide, 2-azido-N-(4-bromophenyl)acetamide, 2-azido-N-(4-hydroxyphenyl) acetamide, 2-azido-N-(4-nitrophenyl)acetamide, 2-azido-N-(pyridine-2-yl) acetamide, prepared in situe from the chloro analogue (A), in the presence of DES (choline chloride/urea 1:2, 0.5 M) at 80 ^oC gave the benzhydrazylpiperazine Δ²-1,2,3-triazoline analogues 8–16 in 45–60% yield. (Scheme 1).

Similary, treatment of 7 with 4-bromobenzyl azide, which was prepared in situ from 4-(bromomethyl)benzonitrile, under the same conditions resulted in the formation of Δ²-1,2,3-triazoline derivatives 17 and 18 in 59 and 52 yield, respectively (Scheme 2).

The structures of 8–18 were confirmed by their IR, ¹H, ¹³C and 2D NMR spectra. The Δ²-1,2,3-triazoline scaffold showed a silimar pattern. In the ¹H NMR spectra of 8–18, the singlet in the range d = 2.40–2.50 ppm were assigned to CH₂ protons, while multiplet in the range 2.68–2.95 and 2.73-3.00 ppm were attributed to the eight piperazine protons. H_triazoline-4’ appeared as broad singlet in the range d = 3.69–4.04 ppm, while H_triazoline-4’ resonated as multiplet or broad singlet in the range d = 2.59–2.73 ppm. H_bridge proton appeared as singlet in the range d = 5.12–5.22 ppm. The singlet in the range d = 3.41–3.57 ppm assigned to CH₂O protons of 8–16, whereas the two singlets at d = 6.57 ppm attributed to CH₂Ph-Br and CH₂Ph-CN of 17 and 18. The other aliphatic and aromatic protons were fully analyzed (c.f. Experimental section). In the ¹³C NMR spectra of 8–16, the carbonyl carbon atoms resonated in the ranges d = 165.9-172.7 ppm, while the resonances at d = 195.4, 197.8 and 176.6 ppm assigned to carbonyl carbon atoms of 2xCOMe and CO₂H, respectively. The resonances in the ranges d = 44.5–51.7 and 51.1–53.0 ppm were attributed to piperazine carbon atoms, while the signals in the range d = 52.6–56.5 ppm were assigned to CH₂ carbon atoms. Carbon-4’ of Δ²-1,2,3-triazoline ring

appeared in the range d = 60.3–61.2 ppm, whereas carbon-5’ of the same moiety resonated in the range d = 62.2–68.9 ppm. The resonances in the range d = 73.0-74.6 ppm were attributed to the bridge carbon atoms. CH₂O carbon atom of 8–16 appeared in the range d = 56.5–59.2 ppm. The other aliphatic and aromatic carbon atoms were fully analyzed (c.f. Experimental section).

Compound 10 was selected for more detailed NMR studies. In the gradient-selected HMBC spectrum [33] of 10, the carbonyl carbon atom at δ 172.5 ppm showed a ²J_C,H correlation with methylene protons (CH₂O) at δ_Η 3.41 ppm, while the methylene protons (CH₂) adjacent to piperazine ring at δ_Η 2.53 ppm showed a ³J_C,H correlation with carbon-5’ of Δ²-1,2,3-triazoline ring at δ_C 63.5 ppm. In addition, a ²J_C,H coupling between H_triazoline-4’ at δ_H 3.82 ppm and the CH₂ carbon atom adjacent to piperazine ring at δ_C 52.9 ppm was observed. Furthermore, methylene proton at carbon-5’ of Δ²-1,2,3-triazoline scaffold at δ_H 2.69 ppm showed a ²J_C,H coupling with carbon-4’ of Δ²-1,2,3-triazoline ring at δ_C 61.0 ppm (Fig. 2).

Biological evaluations

In vitro cytotoxic activity

The anticancer activity of the newly synthesized compounds 8–18 as evaluated on a diverse range of of human cancer cell lines including pancreatic adenocarcinoma (Capan)-1, colorectal carcinoma (HCT-116), glioblastoma (LN-229), acute lymphoblastic leukemia (DND-41), acute myeloid leukemia (HL-60), chronic myeloid leukemia (K-562), and non-Hodgkin lymphoma (Z-138) cancer cells (Table 1). The screening was conducted using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) viability assay [34] with etoposide and nocodazole used as reference drugs for comparison. Compound 11 exhibited the highest activity among the derivatives, inhibiting the proliferation of the selected tested human cancer cell lines HL-60, Z138, and DND-41 with IC₅₀ values of 16.80, 18.50 and 19.20 µM, respectively, while it demonstrated a higher micromolar IC₅₀ values towards the Capan-1, HTC-116, LN229 and K562 cell lines with IC₅₀ values of 40.90, 36.25, 43.90 and 36.10 µM, respectively. Compound 10 displayed IC₅₀ values of 19.90, 18.00 and 18.50 µM against the cell lines HL-60, Z138 and DND-41. It exhibited moderate activity against Capan-1, HTC-116, LN229 and K562 cell lines (ranging from 36.10 to 43.90 mM). In addition, compound 13 showed IC₅₀ value of 19.90 µM against DND-41 cell line, while compound 14 showed activity against HL-60 cell line with IC₅₀ value of 27.75 µM. Furthermore, compound 16 demonstrated selectivity towards the HL-60 and Z-138 cell lines with IC₅₀ values of 27.10 and 22.95 µM, respectively.

The SAR study indicated that the anticancer activity of compounds 7–18 depwnded on the substituents of the aryl amide ring. Compound 11, which featured an acetyl substituent at position meta of the phenyl-amide ring, displayed the most significant antitumoral activity among the tested compounds, especially against the HL-60 cancer cell line. Moreover, compound 11 displayed marginally lower antitumoral activity against DND-41 and Z138 cell lines compared to the HL-60 cell line. These finding provide encouragement to synthesize various substituted 1,2,3-triazoline derivatives, as they may enhance the antiproliferative effects on different cancer cell lines.

Mode of action

Acute myeloid leukemia (AML) is a malignant disease of the hematopoietic system characterized by uncontrolled cell proliferation, impaired cell differentiation, and infiltration of bone marrow, peripheral blood, or other tissues [35]. A crucial protein involved in various cellular processes, such as cell proliferation, apoptosis, and transcription, is Akt, also known as protein kinase B (PKB) or Rac. In AML, Akt1, a specific serine/threonine-specific protein kinase, plays a significant role by activating the PI3K/AKT pathways and serving as a key signaling protein [36]. As a result, Akt1 is commonly targeted in AML therapy, and multiple Akt1 inhibitors have been developed for this purpose. The mode of action of compound 11 can be attributed to its ability to inhibit the PI3K/AKT pathway, as assured by predictive docking studies.

Table 1

*In vitro* cytotoxicity^a of some benzhydrylpiperqazine analogues given as IC₅₀^b in µM
Comp.				IC₅₀ (µM)
				Cell Lines
	Capan-1	HCT-116	LN229	DND-41	HL-60	K562	Z138
8	44.30	51.25	58.45	38.85	38.45	44.15	36.85
9	46.00	38.90	52.05	30.15	31.30	37.75	28.40
10	46.35	37.45	35.40	18.50	19.90	39.40	18.00
11	40.90	36.25	43.90	19.20	16.80	36.10	18.50
12	45.15	45.95	44.45	39.45	42.10	44.65	41.45
13	41.95	40.00	37.70	19.90	43.6	40.55	40.65
14	45.25	29.00	34.05	33.9	27.75	41.95	37.90
15	47.40	51.25	41.55	36.70	38.75	46.00	39.90
16	45.25	42.75	40.70	31.70	27.10	36.9	22.95
17	44.35	51.25	46.95	37.10	42.55	49.75	39.90
18	45.00	43.70	47.45	53.60	50.0	47.3	32.9
ETP	0.10	3.50	1.65	2.00	0.30	2.15	0.35
NDZ	0.02	0.135	0.085	0.025	0.025	0.03	0.025

ETP: Etoposide, NDZ: Nocodazole. ^a Cytotoxicity as IC₅₀ for each cell line is the concentration of tested compound with reduced by 50% the optical density of treated cells with respect to untreated cells using the MTT assay. ^b Data represent mean values of at least two independent experiments.

Antioxidant activity

The evaluation of antioxidant activity was carried using DPPH (1,1-diphenyl-2-picryl hydrazyl) radical scavenging assay [37], with ascorbic acid serving as a positive control for comparison. This method is based on the reduction of DPPH radicals solution in the presence of hydrogen donating antioxidant, resulting in the formation of the non-radical form DPPH-H. The results of antioxidant activity of compounds 8–18 are presented in Table 2. The percentage inhibition of radical scavenging was measured at various concentrations (1000, 900, 800, 700, 600, 500, 250, 125, 62.5 and 31.25 µM), representing the concentration required to scavenge 50% of DPPH radicals. As shown in Table 2, most compounds exhibited low or negligible antioxidant activity in the DPPH assay, with inhibition percentages ranging from 0 to 33.7 µM at a solution concentration of 31.25 µM. Compound 13 exhibited the highest antioxidant activity, inhibiting 33.7% at a concentration of 31.25 and 73.2 µM at a concentration of 1000 µM, compared to the standard ascorbic acid (93.6 and 99.7%, respectively). Compounds 17 and 18, containing 4-bromobenzyl and 4-cyanobenzyl-1,2,3-triazoline groups, exhibited antioxidant activity of 25.4 and 30.9% at a concentration of 31.25 µM, respectively, and inhibition of 86.2 and 84.8% at a concentration of 1000 µM 54.4 and 54.5%, respectively. Specifically, among the series, only compound 13 displayed moderate DPPH scavenging activity. Therefore, 13 can be considered as a lead compound for further development in terms of DPPH scavenging activity.

Table 2

Antioxidant activity of new benzhydrylpiperazine analogues **8–18**
Compd. % Inhibition conc.
	1000	900	800	700	600	500	250	125	62.5	31.25
8	84.5	81.8	80.9	77.9	72.4	50.3	46.1	39.2	33.7	21.8
9	8.8	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0
10	68.8	58.6	34.0	25.4	1.4	0.0	0.0	0.0	0.0	0.0
11	39.2	22.1	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0
12	17.1	1.7	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0
13	73.2	71.0	69.9	68.2	62.7	57.5	54.1	49.4	47.2	33.7
14	76.0	75.7	72.7	68.5	63.8	58.3	53.9	45.3	32.3	20.7
15	44.8	32.9	24.6	17.4	13.3	6.1	0.0	0.0	0.0	0.0
16	85.1	77.6	75.4	69.1	64.1	58.6	56.6	44.8	30.9	8.8
17	86.2	79.3	74.9	72.1	69.1	61.3	55.8	47.0	29.3	25.4
18	84.8	79.8	75.4	72.7	69.1	64.1	58.6	47.8	45.3	30.9
A.A.	99.7	99.2	98.3	98.1	97.5	97.2	96.1	95.3	94.8	93.6

Docking study analysis

The objective of conducting molecular docking calculations is to anticipate the most propable binding between specific protein and a ligand. In this study, Autodock4 [38] was employed for the docking calculations, and the obtained docking results were visualized and analyzed using MGL Tools. The receptor molecule was prepared by elimination water molecules from their structures and then subjected to correction and 3D protonation refinment.

To explore a potential anticancer lead derivative, compound 11 was chosen for molecular docking investigations. The three-dimensional structures of acute myeloid leukemia (cell cycle-related protein CDK2/cyclin A2, PDB: 7B7S), and protein kinase Akt1 PKB alpha, PDB: 4GV1) were used for the docking procedure using the Autodock4 program. The ligand of interest was ranked based on the highest energy of the best conformations. The calculated binding energy scores for compound 11 were − 10.00 kcal mol-1 for both 4GV1 and 7B7S. These results suggest selective binding of this analogue to the active site of the protein receptor pockets in 7B7S, and 4GV1.

In Fig. 3 (A), the docking results depicted the appropriate positioning of compound 11 for its binding with the protein receptors of cell cycle-related protein CDK2/cyclin A2 (PDB: 7B7S). Two hydrogen bonds were formed between the N-3 and N-4 of the tetrazole moiety and Cys694. Furthermore, a π-π stacking interaction occurred between the phenyl ring of the benzyl group and the phenyl residue of Arg834. Additionally, a π-H interaction was observed between the phenyl group of the tetrazole ring and Leu616. Surrounding compound 11, various non-bonded amino acid residues, including Asp829, Asn816, Arg815, Glus831, Cys828, Val624, Gly697, Lys644, and 644, were observed in the receptor structure.

In Fig. 3 (B), the docking analysis revealed that compound 11 binds to the protein kinase Akt1 PKB alpha (PDB: 4GV1). A hydrogen bond (1.876 Å) formed between the oxygen of the carbonyl group attached to the phenyl residue and Ala230. Additionally, the phenyl group of the benzhydryl piperazine scaffold displayed π - cation and π - anion interactions with Glu191 and Lys179, along with a π - alkyl interaction with Leu181. Furthermore, the phenyl group, which was conjugated with

a 1,2,3-triazoline moiety, exhibited a π - σ interaction with Val164 and π - alkyl interactions with both Met281 and Ala177. The same phenyl group also showed a π - sulfur interaction with Met227.

The chloro residue demonstrated an alkyl interaction with Leu295. In the vicinity of compound 11, there were non-bonded amino acids present in the receptor structure, including Asp274, Lys276, Thr195, Gly157, Glys294, Tyr229, Thr291, Gly159, Asp292, and Phe161. The docked complex exhibited acceptable RMSD scores.

Experimental section

General information

Melting points were determined using a Mel-Temp device melting point apparatus and are uncorrected. The IR spectra were recorded, on an FT-IR Spectrophotometer (Thermo Nicolet Corp. USA), using KBr discs. ¹H and ¹³C NMR spectra were acquired on Bruker Avance (400 MHz) (¹H) and 75 MHz (¹³C) spectrometers, using DMSOd₆ solvent containing tetramethylsilane as an internal standard (chemical shifts expressed in δ in ppm). The progress of reactions were monitored by thin layer chromatography (eluent: hexane-EtOAc 4: 1), and the spots were visualized using iodine and U.V. Microwave supported reactions were performed in microwave reaction vials (2.5 mL) with Teflon septum and an aluminium crimp top, utilizing a microwave cavity operating at 2.4 GHz and temperature ranging from 0-300 ^oC .

1-Allyl-4-((4-chlorophenyl)(phenyl)methyl)piperazine (7)

To a solution of 1-benzhydryl piperazine (6) (820 mg, 2.86 mmol) in dry acetone (15 mL) was added allyl bromide (0.30 mL, mmol) and anhyd. K₂CO₃ (789 mg, 5.72 mmol) followed by the addition of KI (0.28 mg, 0.17 mmol) and the reaction mixture was heated under reflux 6 h. After the completion of the reaction (followed by TLC), the solvent was removed under reduced pressure; the residue was poured into ice cold water and extracted with CHCl₃ (3x20 mL). The combined organic layer was dried (MgSO₄), filtered and evaporated to dryness. The crude product was purified on short column of SiO₂ (5 g) using hexane-EtOAc (3:2) as eluent. To give 7 (738 mg, 79%) as yellow solid product, m.p.: 83–85 ^oC, R_f =0.34. FT-IR (KBr, cm^− 1): 2964, 2809 3059, 3025, 3100 (C-H), 1646 (C = C_allyl), 1599, 1486 (C = C_arom.). ¹H NMR (400 MHz, DMSO) δ 2.71, 2.99 (m, 8H, H_piperazine), 3.12 (s, 2H, CH₂-CH = CH₂), 5.14 (s, 1H, H_bridge), 5.16, 5.20 (d, J = 12.0 Hz, 2H, CH₂-CH = CH₂), 5.70 (dd, J = 5.0, 12.1 Hz, 1H, H-15), 7.17–7.77 (m, 9H, H_arom.). ¹³C NMR (100 MHz, DMSO) δ 44.6, 52.9 (C_piperazine), 61.0 (CH₂-CH = CH₂), 74.1 (C_bridge), 118.3 (CH₂CH = CH₂), 127.5, 128.0, 129.0, 129.1, 129.8, 131.8, 134.4, 141.6, 142.7 (C_arom.+CH₂-CH = CH₂). Anal. calc. for C₂₀H₂₃ClN₂ (326.87): C, 73.49; H, 7.09; N, 8.57. Found: C, 73.41; H, 7.01; N, 8.49

General procedure for the synthesis of 1,2,3-triazoline derivatives (8–15)

A mixture of 2-chloro-N-aryl-acetamide or 4-substituted benzyl bromide derivatives (1.0 mmol) and sodium azide (84.5 mg, 1.3 mmol) dissolved in water (5 mL) was subjected to microwave irradiation (70–100 W) at 120 ^oC for a duration of 30 min. After cooling, the mixture was extracted with CHCl₃ (3x15 mL), and the combined organic extracts was dried (MgSO₄), filtered and evaporated to dryness. The collected azide derivative was used directly for the next step. To a solution of 7 (287 mg, 0.92 mmol) in deep eutectic solvent (choline chloride / urea 1:2, 0.5 M) was added the appropriate aryl azide (1.84 mmol) and the rection mixture was stirred at 80 ^oC for 20–24 h. After colling to room temperature, water (10 mL) was added and extracted with EtOAc (3x10 mL). The combined organic extracted was dried (MgSO4), filtered an evaporated to dryness. The crude product was purified by SiO₂ column (20 g) using, in gradient, hexane (0–40%) and EtOAc as eluent to give the desired product.

2-(4-((4-((4-Chlorophenyl)(phenyl)methyl)piperazin-1-yl)methyl)-4,5-dihydro-1H-1,2,3-triazol-1-yl)-N-(o-tolyl)acetamide (8)

Yield: 290 mg (56%) as a dark brown semisolid. R_f =0.63. FT-IR (KBr, cm^− 1): 3390 (N-H), 3082, 3058, 3027, 2973, 2936, 2854 (C-H), 1645 (C = O), 1520, 1486 (C = C). ¹HNMR (400 MHz, DMSO) δ 2.52 (CH₂), 2.59 (br s., 2H, H_triazoline-5’), 2.95, 2.73 (m, 8H, H_piperazine), 3.52 (s, 2H, CH₂CO), 3.99 (br s., 1H, H_triazoline-4’), 5.22 (s, 1H, H_bridge), 7.18–7.58 (m, 13H, H_arom.), 9.91 (br., 1H, NH). ¹³C NMR (100 MHz, DMSO) δ 9.0 (Me), 45.8, 51.1 (C_piperazine), 52.6 (CH₂), 57.9 (CH₂-CO), 60.5 (CH_triazoline-4’), 62.2 (CH_2pyrazoline-5’), 73.0 (C_bridge), 125.1, 125.5, 127.5, 129.8, 131.8, 132.2, 133.5, 134.23, 141.9, 142.3 (C_arom.), 172.7 (C = O). Anal. calc. for C₂₉H₃₃ClN₆O (517.07): C, 67.36; H, 6.43; N, 16.25. Found: C, 67.18; H, 6.27; N, 16.08

2-(4-((4-((4-Chlorophenyl)(phenyl)methyl)piperazin-1-yl)methyl)-4,5-dihydro-1H-1,2,3-triazol-1-yl)-N-(4-formylphenyl)acetamide (9)

Yield: 260 mg (49), as a brown solid, m.p.: 63–65 ^oC, R_f =0.68. FT-IR (KBr, cm^− 1): 3059, 2973, 2935, 2854 (C-H), 1721, 1640 (C = O), 1596, 1523, 1486 (C = C). ¹H NMR (400 MHz, DMSO) δ 2.53 (CH₂), 2.62 (m, 2H, H_triazoline-5’), 2.71, 2.89 (m, 8H, H_piperazine), 3.57 (s, 2H, CH₂CO), 3.75 (br s., 1H, H_triazoline-4’), 5.20 (s, 1H, H_bridge), 7.18–7.58 (m, 13H, H_arom.), 9.58 (s, 1H, NH), 9.89 (s, 1H, CHO). ¹³C NMR (100 MHz, DMSO) δ 46.0, 52.5 (C_piperazine), 53.5 (CH₂), 59.2 (CH₂-CO), 60.3 (CH_triazoline-4’), 62.4 (CH_2pyrazoline-5’), 74.2 (C_bridge), 125.4, 127.6, 128.0, 129.0, 129.1, 129.8, 131.9, 132.3, 131.9, 133.5, 141.4, 142.1, 142.5 (C_arom.), 172.5 (C = O), 190.0 (CHO). Anal. calc. for C₂₉H₃₁ClN₆O₂ (531.06): C, 65.59; H, 6.68; N, 15.83. Found: C, 65.39; H, 6.47; N, 15.62.

N-(4-Acetylphenyl)-2-(4-((4-((4-chlorophenyl)(phenyl)methyl)piperazin-1-yl)methyl)-4,5-dihydro-1H-1,2,3-triazol-1-yl)acetamide (10)

Yield: 327 mg, (60%) as a brown semisolid, R_f =0.74. FT-IR (KBr, cm^− 1): 3396 (NH), 3057, 2974, 2936, 2854 (C-H), 1731, 1651 (C = O), 1597, 1556, 1486 (C = C). ¹H NMR (400 MHz, DMSO) δ 2.50 (s, 3H, COMe), 2.53 (s, 2H, CH₂), 2.69 (m, 2H, H_triazoline-5’), 2.71, 2.98 (m, 8H, H_piperazine), 3.41 (s, 2H, CH₂CO), 3.82 ((br s., 1H, H_triazoline-4’), 5.15 (s, 1H, H_bridge),7.21–7.67 (m, 13H, H_arom.), 10.73 (br s., 1H, NH). ¹³C NMR (100 MHz, DMSO) δ 26.3 (COMe), 45.8, 51.6 (C_piperazine), 52.9 (CH₂), 57.4 (CH₂-CO), 61.0 (CH_triazoline-4’), 63.5 (CH_triazoline-5’), 74.5 (C_bridge), 121.6, 127.5, 129.0, 129.3, 129.8, 131.0, 135.3, 141.9, 142.4, 142.8 (_Carom.) 172.5, 195.4 (2xC = O). Anal. calc. for C₃₀H₃₃ClN₆O₂ (545.08): C, 66.11; H, 6.10; N, 15.42. Found: C, 66.00; H, 5.99; N, 15.32

N-(3-Acetylphenyl)-2-(4-((4-((4-chlorophenyl)(phenyl)methyl)piperazin-1-yl)methyl)-4,5-dihydro-1H-1,2,3-triazol-1-yl)acetamide (11)

Yield: 311 mg (57%) as a semisolid, R_f = 0.74. FT-IR (KBr, cm^− 1): 3375 (NH), 3058, 2929, 2821 (C-H), 1720, 1678 (C = O), 1600 (C = C). ¹H NMR (400 MHz, DMSO) δ 2.51 (s, 3H, COMe), 2.56 (s, 2H, CH₂), 2.68 (m, 2H, H_triazoline-5’), 2.71, 2.95 (m, 8H, H_piperazine), 3.43 (s, 2H, CH₂CO), 3.89 ((br s., 1H, H_triazoline-4’), 5.13 ((s., 1H, H_bridge), 7.09–7.67 (m, 13H, H_arom.), 10.53 (br., 1H, NH). ¹³C NMR (100 MHz, DMSO) δ 27.2 (COMe), 45.9, 51.4 (C_piperazine), 52.8 (CH₂), 57.4 (CH₂-CO), 60.8 (CH_triazoline-4’), 68.9 (CH_triazoline-5’), 73.0 (C_bridge), 119.0, 125.4, 127.5,127.9, 129.0, 129.1, 129.3, 129.8, 131.8, 132.2, 138.1, 141.9, 142.3, 142.7 (C_arom.), 176.2, 197.8 (2xC = O). Anal. calc. for C₃₀H₃₃ClN₆O₂ (545.08): C, 66.11; H, 6.10; N, 15.42. Found: C, 65.98; H, 5.99; N, 15.31

2-(2-(4-((4-((4-Chlorophenyl)(phenyl)methyl)piperazin-1-yl)methyl)-4,5-dihydro-1H-1,2,3-triazol-1-yl)acetamido)benzoic acid (12)

Yield: 246 mg (45%), as a brown amorphous, R_f = 0.86. FT-IR (KBr, cm^− 1): 3059, 3028, 2974, 2937, 2824 (C-H), 1700, 1648 (C = O), 1612, 1582 (C = C). ¹H-NMR (400 MHz, DMSO) δ 2.40 (s, 2H, CH₂), 2.68 (br s., 2H, H_triazolin-5’), 2.70, 2.98 (m, 8H, H_piperazine), 3.42 (s, 2H, CH₂CO), 3.82 (br s., 1H, H_triazolin-4’), 5.14 (s., 1H, H_bridge), 7.20, 7.22, 7.24, 7.29, 7.31, 7.33, 7.35, 7.38, 7.40, 7.42, 7.45, 7.46, 7.48 (m, 13H, H_arom.), 10.61 (s, 1H, NH), 13.36 (s,1H, OH). ¹³C NMR (100 MHz, DMSO) δ 45.8, 51.7 (C_piperazine), 53.0 (CH₂), 57.4 (CH₂-CO), 61.1 (CH_triazoline-4’), 65.7. (CH_triazoline-5’), 74.6 (C_bridge), 115.0 (C_arom.-1’), 118.1 (C_arom.-3’), 125.4, 127.5, 128.0, 129.0, 129.1, 129.8, 131.7, 131.8, 135.7, 141.5, 142.4, 142.8 (C_arom.), 170.3 (C = O), 176.6 (CO₂H). Anal. calc. for C₂₉H₃₁ClN₆O₃ (547.06): C, 63.67; H, 5.71; N, 15.36. Found: C, 63.60; H, 5.64; N, 15.30

N-(4-bromophenyl)-2-(4-((4-((4-chlorophenyl)(phenyl)methyl)piperazin-1-yl)methyl)-4,5-dihydro-1H-1,2,3-triazol-1-yl)acetamide (13)

Yield: 291 mg (50%), as a brown amorphous, R_f = 0.58. FT-IR (KBr, cm^− 1) : 3374 (N-H), 3083, 3059, 3027, 2927, 2853, 2811 (C-H), 1646 (C = O), 1600, 1487 (C = C). ¹H-NMR (400 MHz, DMSO) δ 2.42 (CH₂), 2.60 (br s., 2H, H_triazolin-5’), 2.71, 2.95 (m, 8H, H_piperazine), 3.43 (s, 2H, CH₂CO), 3.69 ((br s., 1H, H_triazoline-4’), 5.12 (s., 1H, H_bridge), 7.16–7.77 (m, 13H, H_arom.), 10.32 (br s., 1H, NH). ¹³C NMR (100 MHz, DMSO) δ 45.8, 51.6 (C_piperazine), 53.0 (CH₂), 57.4 (CH₂-CO), 61.1 (CH_triazoline-4’), 66.9 (CH_triazoline-5’), 74.5 (C_bridge), 118.3, 121.6, 127.5, 127.9, 128.0, 129.0, 129.3, 129.4, 129.8, 131.8, 132.3, 135.4, 141.9, 142.4 (C_arom.), 172.5 (C = O). Anal. calc. for C₂₈H₃₀BrClN₆O (581.94): C, 57.79; H, 5.20; N, 14.44. Found: C, 57.67; H, 5.08; N, 14.21

2-(4-((4-((4-Chlorophenyl)(phenyl)methyl)piperazin-1-yl)methyl)-4,5-dihydro-1H-1,2,3-triazol-1-yl)-N-(4-hydroxyphenyl)acetamide (14)

Yield: 265 mg (51%), as a yellow-brown solid, m.p.: 99–101 ^oC, R_f = 0.91. FT-IR (KBr, cm^− 1): 3400 (OH), 3204 (NH), 3061, 2964, 2926, 2814 (C-H), 1676 (C = O), 1606 (C = C). ¹H-NMR (400 MHz, DMSO) δ 2.52 (CH₂), 2.64 (br s., 2H, H_triazolin-5’), 2.73, 2.98 (m, 8H, H_piperazine), 3.49 (s, 2H, CH₂CO), 3.72 ((br s., 1H, H_triazoline-4’), 5.16 (s., 1H, H_bridge), 6.74–7.53 (m, 13H, H_arom.), 9.47 (s, 1H, NH), 10.02 (s, 1H, OH). ¹³C NMR (100 MHz, DMSO) δ 48.7, 51.8 (C_piperazine), 53.1 (CH₂), 56.5 (CH₂-CO), 61.2 (CH_triazoline-4’), 67.8 (CH_triazoline-5’), 74.6 (C_bridge), 115.6, 121.6, 127.5, 128.0, 129.0, 129.1, 129.8, 130.5, 131.8, 142.8, 142.4 (C_arom.), 154.2 (C-OH), 165.9 (C = O). Anal. calc. for C₂₈H₃₁ClN₆O₂ (519.05): C, 64.79; H, 6.02; N, 16.19. Found: C, 64.70; H, 5.93; N, 16.11

2-(4-((4-((4-Chlorophenyl)(phenyl)methyl)piperazin-1-yl)methyl)-4,5-dihydro-1H-1,2,3-triazol-1-yl)-N-(4-nitrophenyl)acetamide (15)

Yield: 323 mg (59%), as a brown solid, m.p.: 91.94 ^oC, R_f = 0.77. FT-IR (KBr, cm^− 1): 3059, 3028, 2924, 2814 (C-H), 1634 (C = O), 1597, 1556 (C = C). ¹H-NMR (400 MHz, DMSO) δ 2.48 (CH₂), 2.73 (br s., 2H, H_triazolin-5’), 2.68, 2.93 (m, 8H, H_piperazine), 3.43 (s, 2H, CH₂CO), 3.84 ((br s., 1H, H_triazoline-4’), 5.14 ((s., 1H, H_bridge), 6.74–8.26 (m, 13H, H_arom.), 10.52 (br s., 1H, NH). ¹³C NMR (100 MHz, DMSO) δ 51.7, 53.0 (C_piperazine), 56.5 (CH₂), 57.4 (CH₂-CO), 61.1 (CH_triazoline-4’), 63.0 (CH_triazoline-5’), 74.6 (C_bridge), 118.2, 125.4, 127.5, 129.0, 129.1, 129.3, 129.8, 132.2, 141.9, 142.4, 142.8 (C_arom.), 172.6 (C = O). Anal. calc. for C₂₈H₃₀ClN₇O₃ (548.04): C, 61.37; H, 5.52; N, 17.89. Found: C, 61.18; H, 5.35; N, 17.70.

2-(4-((4-((4-Chlorophenyl)(phenyl)methyl)piperazin-1-yl)methyl)-4,5-dihydro-1H-1,2,3-triazol-1-yl)-N-(pyridin-2-yl)acetamide (16)

Yield: 242 mg (48%), as a light green amorphous, R_f = 0.63. FT-IR (KBr, cm^− 1): 3391 (NH), 3060, 3026, 2960, 2928, 2808 (C-H), 1645 (C = O), 1599 (C = C). ¹H-NMR (400 MHz, DMSO) δ 2.45 (CH₂), 2.70 (br s., 2H, H_triazolin-5’), 2.72, 3.00 (m, 8H, H_piperazine), 3.43 (s, 2H, CH₂CO), 4.04 ((br s., 1H, H_triazoline-4’), 5.20 ((s., 1H, H_bridge), 7.13–7.77 (m, 12H, H_arom.), 8.22 (m, 1H, H_pyridine-6’). ¹³C NMR (100 MHz, DMSO) δ 44.6, 51.5 (C_piperazine), 52.9 (CH₂), 57.4 (CH₂-CO), 60.9 (CH_triazoline-4’), 63.0 (CH_triazoline-5’), 74.5 (C_bridge), 118.5 (Cpyridin-3’), 125.5, 127.5, 128.0, 129.0, 129.8, 131.8, 138.1, 141.9, 142.4, 146.3 (C_arom.+C_pyridin.), 146.3 (C_pyridine-6’), 158.5 (C_pyridin.-1’), 171.3 (C = O). Anal. calc. for C₂₇H₃₀ClN₇O (504.04): C, 64.34; H, 6.00; N, 19.45 Found: C, 64.19; H, 5.87; N, 19.30

1-((1-(4-Bromobenzyl)-4,5-dihydro-1H-1,2,3-triazol-4-yl)methyl)-4-((4-chlorophenyl) (phenyl)methyl)piperazine (17)

Yield: 318 mg (59%), as a brown amorphous, R_f = 0.47. FT-IR (KBr, cm^− 1): 3050, 3020, 2960, 2933, 2812 (C-H), 1596 (C = C). ¹H-NMR (400 MHz, DMSO) δ 2.52 (CH₂), 2.71 (br s., 2H, H_triazolin-5’), 2.78, 2.91 (m, 8H, H_piperazine), 3.75 ((br s., 1H, H_triazoline-4’), 5.16 (s, 1H, H_bridge), 6.57 (s, 2H, CH₂-Ph-Br), 7.16–7.72 (m, 13H, H_arom.). ¹³C NMR (100 MHz, DMSO) δ 44.5, 51.8 (C_piperazine), 56.5 (CH₂ + CH₂-Ph-Br), 61.1 (C_triazoline-4’), 63.8 (C_triazoline-5’), 74.6 (C_bridge), 120.3, 127.5, 128.0, 131.7, 132.1, 135.9, 142.4, 142.8 (C_arom.). Anal. calc. for C₂₇H₂₉BrClN₅ (538.92): C, 60.18; H, 5.42; N, 13.00. Found: C, 60.02; H, 5.26; N, 12.83

4-((4-((4-((4-Chlorophenyl)(phenyl)methyl)piperazin-1-yl)methyl)-4,5-dihydro-1H-1,2,3-triazol-1-yl)methyl)benzonitrile (18)

Yield: 252 mg (52%), as a brown amorphous, R_f = 0.50. FT-IR (KBr, cm^− 1): 3095, 2931, 2813 (C-H), 2228 (CN), 1617, 1510, 1472 (C = C). ¹H-NMR (400 MHz, DMSO) δ 2.50 (CH₂), 2.69 (br s., 2H, H_triazolin-5’), 2.72, 2.92 (m, 8H, H_piperazine), 3.85 ((br s., 1H, H_triazoline-4’), 5.17 (s., 1H, H_bridge), 6.57 (s, 2H, CH₂-Ph-Br), 7.25–7.85 (m, 13H, H_arom.). ¹³C NMR (100 MHz, DMSO) δ 44.6, 51.8 (C_piperazine), 53.1 (CH₂ + CH₂-Ph-Br), 61.1 (CH_triazoline-4’), 64.6 (CH_triazoline-5’), 74.6 (C_bridge), 111.2 (C-CN), 117.9 (CN), 127.5, 128.0, 129.0, 129.5, 129.8, 131.7, 133.1, 135.9, 141.9, 142.5 (C_arom.). Anal. calc. for C₂₈H₂₉ClN₆ (485.03): C, 69.34; H, 6.03; N, 17.33. Found: C, 69.24; H, 5.93; N, 17.22.

Biological assays

Anticancer activity in vitro

Cancer Cell Lines

The human cancer cell lines, including Capan-1, HCT-116, LN-229, NCI-H460, HL-60, K-562, H and Z-138 cancer cells, were sourced from the American Type Culture Collection (ATCC, Manassas, VA, USA) for this manuscript, Additionally, the DND-41 cell line was obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ Leibniz-Institut, Germany). The culture media, except for those used for other cell lines which were purchased from Sigma, were acquired from Gibco Life Technologies, USA, and supplemented with 10% fetal bovine serum (HyClone, GE Healthcare Life Sciences, USA),

Proliferation Assays

For adherent cell lines (LN-229, HCT-116, NCI-H460, and Capan-1 cells), 384-well tissue culture plates (Greiner) were seeded at a density ranging from 500 to 1500 cells per well. Following overnight incubation, the cells were treated with seven different concentrations of the test compounds, ranging from 100 to 0.006 µM. On the other hand, suspension cell lines (HL-60, K-562, Z-138, and DND-41) were seeded in 384-well culture plates with the test compounds at the same concentration points, and cell densities ranging from 2500 to 5500 cells per well. The cells were then incubated with the compounds for 72 h. and subsequently analyzed using the CellTiter 96® AQueous One Solution Cell Proliferation Assay (MTS) reagent (Promega), following the manufacturer’s instructions. Absorbance measurments were measured at 490 nm using a SpectraMax Plus 384 (Molecular Devices), and the OD values were utilized to determine the 50% inhibitory concentration (IC₅₀). The compounds underwent testing in two independent experiments. For HeLa cells, seeding was performed in 96-well microtiter plates with a cell density raging from 1 × 10⁴ to 3 × 10⁴ cells/mL, depending on the doubling times specific to the cell line. Test agents were added at five 10-fold dilutions (10 − 8 to 10 − 4 M), with working dilutions freshly prepared on the day of testing. After 72 h. of incubation, the cell growth rate was assessed using the MTS assay as previously described [34]. Absorbance measurments was taken at 570 nm, and the OD values were used to calculate the 50% inhibitory concentration (IC₅₀). Each test was performed in duplicate, across at least two individual experiments.

Antioxidant assay by using DPPH radical scavenging method.

The antioxidant activity of the synthesized compounds 3–10 was assessed using DPPH (1,1-diphenyl-2-picrylhydrazyl) free radical scavenging assay. Initially, a 0.2 mM solution of DPPH in EtOH at various concentrations (1000, 900, 800, 700, 600, 500, 250, 125, 62.5, 31.25 µg/mL). This mixture was vigorously shaken and left at room temperature for 30 min. Absorbance then measured at 517 nm using a UV-VIS spectrophotometer (UV-VIS Shimadzu). To determine the antioxidant activity of the tested compounds, the percentage of DPPH neutralization by them was calculated and compared to the standard antioxidant ascorbic acid. The percent DPPH scavenging effect (%) or % inhibition was calculated using following equation:

DPPH scavenging effect (%) or % inhibition = Ac(0) – AA(t)1 / Ac(0) × 100.

where Ac(0) represents the absorbance of the control at t = 0, and AA(t) is the absorbance of the antioxidant at t = 30 min. All measurements were performed in triplicate

Docking and virtual screening

Preparations of ligands and proteins.

The 3D structures of ligand 11 were first generated using Avogadro software (v. 1.0.1) [39] and saved in PDB format. Subsequently, the ligand was prepared by selecting torsions, and the structures were converted from PDB format to PDBQT format. This conversion was done using the MGLTools software, which also performed united atom Kollman charges, fragmental volumes, and solvation parameter calculations for both the proteins and the ligand. To ensure an accurate docking process, the ligand's structure was subjected to energy minimization using the MMFF94 force field. The native ligands and crystallographic water molecules were then removed from the PDB structures, and polar hydrogens were added before commencing with the docking.

Molecular docking simulations

The Autodock program was utilized for molecular docking simulations. Prior to docking, the protein structures were prepared using UCSF Chimera 1.15 [40]. For pose sampling in Autodock, the Lamarkian Genetic Algorithm (LGA) was employed, and the number of energy simulations was set to 2,500,000. The default scoring function was utilized to calculate the docking scores. To generate the necessary maps, Autogrid was employed. The resulting docking outcomes were visualized using Biovia Discovery Studio 2020 software [41], followed by an analysis of the docking results. To validate the method, all docking simulations were performed using the ligands found in the crystal structures, and they were successful in reproducing the ligand-protein interaction geometries. Figure 3 illustrates the image of the native ligand for 7B7S and 4GV1 in comparison to the redocked native ligand with AutoDock. The root-mean-square deviation of atomic positions (RMSD) for these cases was 0.400 and 0.4008 Å, respectively.

In this study, a series of benzhydryl piperazine bearing substituted methyl 1,2,3-triazoline 8–16 were synthesized, where 1-allyl-4-((4-chlorophenyl)(phenyl)methyl)piperazine (7) served as a key intermediate. The key intermediate was further modified by coupling with the aryl azide compounds, leading to the synthesis of new analogues 17 and 18. The synthesized compounds were then evaluated for their anticancer activity against seven different human cancer cell lines (Capan-1, HCT-116, LN229, NCI-H460, DND-41, HL-60, K562, and Z138). Notably, derivatives 11 exhibited the most promising activity among the series. It displayed also low micromolar activity against DND-41 and Z-138 cell lines. Furthermore, the antioxidant activity of all compounds were evaluated. Out of the entires series, only compounds 13 exhibited moderate DPPH scavenging activity. A molecular docking study of compound 11 revealed intriguing binding interactions between its substituents and the amino acid Asp351. These findings provide valuable insights for the design of novel substituted-benzhydrylpiperazine analogues with potential as promising anticancer agents, as well as for further structural modifications

Acknowledgments

We acknowledge Leentje Persoons and Dirk Daelemans from the Laboratory of Virology and Chemotherapy, KU Leuven, Belgium for the antitumoral testing of the compounds.

Electronic Supplementary material

The online version of this article (http://doi.org..….) contains supplementary material, which is available to authorized users.

Compliance with ethical standards

Conflict of interest No potential conflict of interest was reported by the authors.

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Schemes are available in Supplementary Files section.

No competing interests reported.

GraphicalAbstract.png
Supplementary.Information.docx.docx
Scheme1.png
Scheme 1. Reagents and conditions: (i) allyl bromide, dry acetone, K₂CO₃, KI, reflux 6 h; (ii) NaN₃, DMF, 100 ^oC, 8 h; (iii) DES, 80 ^oC, 16-20 h.
Scheme2.png
Scheme 2. Synthesis of novel benzhydrazyl-piperazine-Δ²-1,2,3-triazoline analogues 17 and 18

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Synthesis, anticancer, antioxidant activities and in silico studies of novel benzhydrylpiperazine bearing Δ 2 -1,2,3-triazoline hydrides

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Abstract

Figures

Introduction

Results and Discussion

Chemistry

Biological evaluations

In vitro cytotoxic activity

Mode of action

Antioxidant activity

Docking study analysis

Experimental section

General information

1-Allyl-4-((4-chlorophenyl)(phenyl)methyl)piperazine (7)

General procedure for the synthesis of 1,2,3-triazoline derivatives (8–15)

2-(4-((4-((4-Chlorophenyl)(phenyl)methyl)piperazin-1-yl)methyl)-4,5-dihydro-1H-1,2,3-triazol-1-yl)-N-(o-tolyl)acetamide (8)

2-(4-((4-((4-Chlorophenyl)(phenyl)methyl)piperazin-1-yl)methyl)-4,5-dihydro-1H-1,2,3-triazol-1-yl)-N-(4-formylphenyl)acetamide (9)

N-(4-Acetylphenyl)-2-(4-((4-((4-chlorophenyl)(phenyl)methyl)piperazin-1-yl)methyl)-4,5-dihydro-1H-1,2,3-triazol-1-yl)acetamide (10)

N-(3-Acetylphenyl)-2-(4-((4-((4-chlorophenyl)(phenyl)methyl)piperazin-1-yl)methyl)-4,5-dihydro-1H-1,2,3-triazol-1-yl)acetamide (11)

2-(2-(4-((4-((4-Chlorophenyl)(phenyl)methyl)piperazin-1-yl)methyl)-4,5-dihydro-1H-1,2,3-triazol-1-yl)acetamido)benzoic acid (12)

N-(4-bromophenyl)-2-(4-((4-((4-chlorophenyl)(phenyl)methyl)piperazin-1-yl)methyl)-4,5-dihydro-1H-1,2,3-triazol-1-yl)acetamide (13)

2-(4-((4-((4-Chlorophenyl)(phenyl)methyl)piperazin-1-yl)methyl)-4,5-dihydro-1H-1,2,3-triazol-1-yl)-N-(4-hydroxyphenyl)acetamide (14)

2-(4-((4-((4-Chlorophenyl)(phenyl)methyl)piperazin-1-yl)methyl)-4,5-dihydro-1H-1,2,3-triazol-1-yl)-N-(4-nitrophenyl)acetamide (15)

2-(4-((4-((4-Chlorophenyl)(phenyl)methyl)piperazin-1-yl)methyl)-4,5-dihydro-1H-1,2,3-triazol-1-yl)-N-(pyridin-2-yl)acetamide (16)

1-((1-(4-Bromobenzyl)-4,5-dihydro-1H-1,2,3-triazol-4-yl)methyl)-4-((4-chlorophenyl) (phenyl)methyl)piperazine (17)

4-((4-((4-((4-Chlorophenyl)(phenyl)methyl)piperazin-1-yl)methyl)-4,5-dihydro-1H-1,2,3-triazol-1-yl)methyl)benzonitrile (18)

Biological assays

Anticancer activity in vitro

Cancer Cell Lines

Proliferation Assays

Docking and virtual screening

Molecular docking simulations

Conclusion

Declarations

References

Schemes

Additional Declarations

Supplementary Files

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