Synthesis, In Vitro Cytotoxicity Evaluation and GSK-3β Binding Study of Some Indole–Triazole-Linked Pyrazolone Derivatives

Abstract

Glycogen synthase kinase-3 beta (GSK-3β) is a multifunctional serine/threonine kinase mediating multiple cellular functions, such as differentiation, apoptosis, and cell proliferation. Because of their ability to alter carcinogenic pathways, GSK-3β inhibitors are being explored for the development of anticancer molecules. In the present study, we synthesized and evaluated the cytotoxic properties of a series of twenty indole–triazole-linked pyrazolone derivatives, 10Aa–Ed. All derivatives were characterized by FTIR, ¹H/¹³C NMR, and high-resolution mass spectrometry (HRMS) methods. All compounds and standards, sunitinib and 5-Fluorouracil (5-FU), were screened against four adherent cell lines, including pancreatic adenocarcinoma (Capan-1), colorectal carcinoma (HCT-116), glioblastoma(LN229), and lung carcinoma (NCI-4460), and four non-adherent cell lines, including acute myeloid leukemia (HL-60), chronic myeloid leukemia (K562), T lymphoblast (MOLT4), and non-Hodgkin lymphoma (Z138). Among the screened derivatives, molecule 10Aa showed cytotoxicity against MOLT 4, Z138, and HL60 with CC₅₀ values of 14.45 μM, 15.34 μM, and 17.56 μM, respectively. GSK-3β kinase inhibition was evaluated with the 10Aa, which is capable of inhibiting GSK-3β in a dose-dependent manner. Additionally, molecular docking was performed to estimate the correlation between invitro data and GSK-3β binding affinity. The outcomes of the invitro experiments demonstrated strong concordance with the insilico data. The discovery yielded compounds 10Aa and 10Cd, which can be modified to create effective anticancer agents that target GSK-3β.

1. Introduction

Cancer is a major global challenge, affecting individuals across all segments of society. It significantly contributes to global morbidity and mortality. The National Cancer Registry Programme (NCRP), India, has reported approximately 1.46 million cancer cases in 2022, with nearly 395,400 deaths [1]. Among different types of cancer, breast cancer leads the list with an estimated 98,300 deaths, while cervical, colorectal, and lung cancer accounted for approximately 79,900, 41,000, and 75,000 deaths, respectively, in 2022 across India [2]. These statistics are scary and need immediate attention. The existing anticancer regime is not only toxic but also costly to afford for the common human being. Hence, there is an unmet need for an effective lead that can be translated into a clinical drug to treat cancer.

In this context, the development of small nitrogen-containing heterocyclic scaffolds, such as indole and triazole hybrids, can be promising in cancer research. The nitrogen-containing heterocyclic compounds are an integral part of medicinal chemistry by virtue of low toxicity, efficient receptor binding in biological systems, and improved aqueous solubility [3]. The literature reports indole as a synthetically versatile scaffold with promising activities against cancer, human immunodeficiency virus (HIV), tuberculosis, hypertension, diabetes, and microbial infections [4]. Bajad et al. had reviewed indole as a promising scaffold for the discovery and development of potential anti-tubercular agents targeting cell-wall synthesis and membrane depolarization in mycobacterial cells [5]. Hasan et al. have reviewed indole derivatives as a potential anticancer lead acting via kinase inhibition, apoptosis, DNA intercalation, and disruption of cell signaling pathways [6]. The antidiabetic property of indole-linked thiazolidine-2,4-dione inhibiting α-glucosidase was reported by Hu et al. [7] and Mo et al. [8]. Recently, Madarakhandi et al. have reported some hydrazide-2-oxindole derivatives as potent GSK-3β kinase inhibitors against cancer [9]. Likewise, indole [10], pyrazole [11], and triazole [12] show remarkable binding capabilities with receptors and enzymes in biological systems and elicit promising pharmacological responses against microbes, free radicals, inflammatory responses, cancer, and viruses. Sucheta et al. [13] reviewed the multifaceted biological potential of pyrazole scaffolds, while Mallisetty et al., in a separate study, reported the pyrazole-linked 1,2,4-triazole hybrid as a topoisomerase-IIa inhibitor and evaluated its antibacterial and anticancer properties against Staphylococcus aureus and MCF7 cell lines, respectively [14]. Further, the study by Glomb et al. [15], Li et al. [16], and Matta et al. [17] underscores the biological significance of pyrazole- and triazole-based scaffolds. Figure 1 depicts some clinically used indole, pyrazole, and 1,2,3-triazole hybrids.

Glycogen synthase kinase-3 beta (GSK-3β) is a multifunctional serine/threonine kinase involved in several cellular functions, such as differentiation, apoptosis, and proliferation [18]. GSK-3β plays dual roles in cancer, depending on the tumor types and biochemical pathways involved. It not only suppresses tumors by blocking β-catenin in the Wnt pathway but also promotes the growth of new tumors by promoting survival signaling mediated by NF-κB and epithelial-to-mesenchymal transition [19]. GSK-3β is a possible therapeutic target because it has been shown to exhibit aberrant activation in malignancies such as glioblastoma, colorectal, and pancreatic tumors. Because of GSK-3β’s ability to alter several carcinogenic pathways, its inhibitors are being researched for anticancer treatment [20]. Considering the diverse pharmacological relevance of the indole, pyrazole, and triazole nuclei, along with the significance of GSK-3β inhibition in cancer research, we synthesized and evaluated the cytotoxicity of a series of twenty indole–triazole-linked pyrazolone derivatives in the present study. The indole-substituted triazoles were synthesized via a Cu(I)-catalyzed click reaction between indole-substituted alkynes and benzyl azides in the presence of CuSO₄·5H₂O and sodium ascorbate. Subsequently, substituted pyrazolones bearing an active methylene (-CH₂) group were condensed with the triazole-linked 3-formyl indole derivatives in methanol using piperidine as a base catalyst to get 10Aa–Ed [21,22,23,24,25]. Furthermore, the molecular docking of compound 10Aa was carried out against GSK-3β (PDB ID: 1Q41) to elucidate its receptor binding affinity. Figure 2 illustrates selected nitrogen-containing GSK-3β inhibitors that are clinically approved or currently under investigation.

2. Materials and Methods

2.1. Chemicals and Reagents

Spectrochem Pvt. Ltd., Bengaluru, India and BLD Pharma Hyderabad provided all chemicals and reagents. On pre-coated silica gel plates 60 F254 (Merck, Darmstadt, Germany), the progress of the reaction was monitored. A digital melting point instrument (5067/2, DBK, Mumbai, India) was used to measure melting points (m.p), which were then presented uncorrected. The ¹H and ¹³C nuclear magnetic resonance (NMR) spectra were acquired on 400 Mega Hertz (MHz) Avance-III NMR spectrometer (Bruker, MA, USA) in deuterated dimethyl sulfoxide (DMSO-d₆, 99.8%, Eurisotop, Saint-Aubin, France). The peaks’ positions (Chemical shift, J) were reported in hertz (Hz). A Fourier transform infra-red (FTIR, JASCO 460+, Tokyo, Japan) spectrophotometer was used to get FT-IR spectra. SynaptG2 QTOF high-resolution mass spectrometer (HRMS, Waters, MA, USA) was used to determine the molecular weight (M.W) of compounds 10Aa–Ed by the electrospray ionization (ESI) method. Intermediate 1-(prop-2-yn-1-yl)-1H-indole-3-carbaldehyde (3) [21], aryl azides (5a–e) [22], 1-((1-(4-substituted-benzyl)-1H-1,2,3-triazol-4-yl)methyl)-1H-indole-3-carbaldehyde (6a–e) [23] and 1-substituted-3-methyl-1H-pyrazol-5(4H)-one (9a–d) [24,25] were obtained as per literature. Compounds 1 (98%, BLD Pharm, Hyderabad, India), 2 (80%, Spectrochem, Mumbai, India), 4 (97%, BLD Pharm, Hyderabad, India), 7 (98%, SD Fine Chemical Limited, Mumbai, India), and 8a–d (95%, BLD Pharm, Hyderabad, India) were procured commercially.

2.2. General Procedure for Synthesis of 4-((1-((1-Benzyl-1H-1,2,3-triazol-4-yl)methyl)-1H-indol-3-yl)methylene)-3-methyl-1H-pyrazol-5(4H)-one (10Aa–Ed)

An equimolar quantity of different 1-((1-(4-substituted-benzyl)-1H-1,2,3-triazol-4-yl) methyl)-1H-indole-3-carbaldehyde (6a–e) and 1-substituted-3-methyl-1H-pyrazol-5 (4H)-one (9a–d) were refluxed using a catalytic amount of piperidine (98%, SD Fine Chemical Limited, Mumbai, India) in methanol (99.5%, Loba Chemie, Mumbai, India) to get respective4-((1-((1-benzyl-1H-1,2,3-triazol-4-yl)methyl)-1H-indol-3-yl)methylene)-3-met-hyl-1H-pyrazol-5 (4H)-one (10Aa–Ed). Recrystallized the precipitated product from appropriate solvents.

2.2.1. 4-((1-((1-Benzyl-1H-1,2,3-triazol-4-yl)methyl)-1H-indol-3-yl)methylene)-3-methyl-1H-pyrazol-5(4H)-one (10Aa)

Yellow crystals, m.p: 244.0–245.1 °C, % yield: 73, IR (KBr)ν: 3145, 3121, 1670, 1585 cm⁻¹. ¹H-NMR (DMSO-d₆, 400 MHZ): δ = 11.07 (1H, s, -NH), 9.78 (1H, s), 8.26 (1H, s, ar-H), 8.11–8.09 (1H, d J = 6.8Hz, ar-H), 7.90 (1H, s, ar-H), 7.74–7.72 (1H, d J = 7.6Hz, ar-H), 7.34–7.26 (7H, m, ar-H), 5.62 (2H, s, -N-CH₂), 5.55 (2H, s, -N-CH₂), 2.23 (3H, s, -CH₃). ¹³C-NMR (DMSO-d₆, 100 MHZ): δ = 13.43, 31.15, 42.39, 53.31, 111.88, 112.05, 119.21, 119.73, 122.65, 123.83, 124.55, 128.33, 128.62, 128.62, 129.24, 129.24, 134.90, 136.31, 136.57, 139.45, 142.81, 150.00, 166.70. M.F: C₂₃H₂₀N₆O, HRMS m/z (ESI): [M + H]⁺397.1753 (397.4445).

2.2.2. 4-((1-((1-Benzyl-1H-1,2,3-triazol-4-yl)methyl)-1H-indol-3-yl)methylene)-3-methyl-1-phenyl-1H-pyrazol-5(4H)-one, (10Ab)

Orange crystals, m.p: 205.7–206.5 °C, % yield: 68, IR (KBr)ν: 3126, 3080, 1671, 1600, 1469 cm⁻¹. ¹H-NMR (DMSO-d₆, 400 MHZ): δ = 9.97 (1H, s), 8.02–8.00 (2H, d J = 10.0 Hz, ar-H), 7.91–7.89 (1H, m, ar-H), 7.83 (1H, s, ar-H), 7.65–7.63 (1H, m, ar-H), 7.47–7.43 (2H, t J = 17.6 Hz, ar-H), 7.40–7.33 (6H, m, ar-H), 7.23–7.19 (3H, m, ar-H), 5.60 (2H, s, -N-CH₂), 5.47 (2H, s, -N-CH₂), 2.45 (3H, s, -CH₃). ¹³C-NMR (DMSO-d₆, 100 MHZ): δ = 13.48, 31.16, 42.39, 53.33, 112.12, 112.31, 118.60, 118.60, 119.17, 119.41, 123.10, 124.20, 124.59, 124.59, 128.36,128.36, 128.64, 129.26, 129.26, 129.26, 134.90, 136.30, 136.72, 137.08, 139.24, 140.69, 142.72, 151.53, 163.21. M.F: C₂₉H₂₄N₆O, HRMS m/z (ESI): [M + H]⁺ 473.2061 (473.5405).

2.2.3. 4-((1-((1-Benzyl-1H-1,2,3-triazol-4-yl)methyl)-1H-indol-3-yl)methylene)-3-methyl-1-(p-tolyl)-1H-pyrazol-5(4H)-one, (10Ac)

Yellow powder, m.p: 228.6–229.1 °C, % yield: 71, IR (KBr) ν: 3100, 3064, 1668, 1602, 1469 cm⁻¹. ¹H-NMR (DMSO-d₆, 400 MHZ): δ = 9.97 (1H, s), 7.91–7.85 (3H, m, ar-H), 7.82 (1H, s, ar-H), 7.64–7.62 (1H, m, ar-H), 7.54 (1H, s, ar-H), 7.40–7.34 (6H, m, ar-H), 7.26–7.21 (3H, m, ar-H), 5.60 (2H, s, -N-CH₂), 5.47 (2H, s, -N-CH₂), 2.44 (3H, s, -CH₃), 2.39 (3H, s, -CH₃). ¹³C-NMR (DMSO-d₆, 100 MHZ): δ = 13.45, 20.96, 31.15, 42.53, 53.33, 112.12, 112.31, 118.60, 118.60, 119.17, 119.41, 123.10, 124.20, 124.59, 124.59, 128.36,128.36, 128.64, 129.26, 129.26, 129.26, 134.90, 136.30, 136.72, 137.08, 139.24, 140.69, 142.72, 151.53, 163.21. M.F: C₃₀H₂₆N₆O, HRMS m/z (ESI): [M + H]⁺ 487.2153 (487.5670).

2.2.4. 4-((1-((1-Benzyl-1H-1,2,3-triazol-4-yl)methyl)-1H-indol-3-yl)methylene)-3-methyl-1-(4-nitrophenyl)-1H-pyrazol-5(4H)-one, (10Ad)

Green powder, m.p: 242.6–244.1 °C, % yield: 78, IR (KBr)ν: 3110, 3052, 1681, 1469, 1351 cm⁻¹. ¹H-NMR (DMSO-d₆, 400 MHZ): δ = 9.91 (1H, s), 8.33 (4H, s, ar-H), 7.91–7.87 (2H, m, ar-H), 7.67–7.65 (1H, m, ar-H), 7.41–7.35 (6H, m, ar-H), 7.25–7.23 (2H, m, ar-H), 5.63 (2H, s, -N-CH₂), 5.50 (2H, s, -N-CH₂), 2.47 (3H, s, -CH₃). ¹³C-NMR (DMSO-d₆, 100 MHZ): δ = 13.15, 31.15, 42.65, 53.35, 112.45, 112.45, 117.62, 117.62, 117.89, 123.39, 124.62, 124.62, 125.51, 125.51, 128.39, 128.39, 128.39, 128.66, 129.30, 129.30, 129.30, 129.30, 136.81, 138.34, 142.61, 153.75. M.F: C₂₉H₂₃N₇O₃, HRMS m/z (ESI): [M + H]⁺518.1940 (518.5380).

2.2.5. 4-((1-((1-(4-Chlorobenzyl)-1H-1,2,3-triazol-4-yl)methyl)-1H-indol-3-yl)methylene)-3-methyl-1H-pyrazol-5(4H)-one, (10Ba)

Orange powder, m.p: 260.1–261.5 °C, % yield: 63, IR (KBr)ν: 3118, 2980, 1670, 1578 cm⁻¹. ¹H-NMR (DMSO-d₆, 400 MHZ): δ = 11.06 (1H, s, -NH), 9.81 (1H, s), 8.29 (1H, s, ar-H), 8.13–8.11 (1H, d J = 8.8Hz, ar-H), 7.89 (1H, s, ar-H), 7.76–7.74 (1H, d J = 8.8Hz, ar-H), 7.43–7.41 (2H, d J = 8.4Hz, ar-H), 7.35–7.30 (4H, m, ar-H), 5.65 (2H, s, -N-CH₂), 5.58 (2H, s, -N-CH₂), 2.25 (3H, s, -CH₃). ¹³C-NMR (DMSO-d₆, 100 MHZ): δ = 13.43, 31.15, 42.36, 52.52, 111.89, 112.03, 119.22, 119.73, 122.66, 123.85, 124.65, 129.15, 129.15,129.15, 130.31, 130.31, 130.31,133.35, 134.91, 136.59, 139.41, 142.82, 150.01, 166.71.M.F: C₂₃H₁₉ClN₆O, HRMS m/z (ESI): [M + H]⁺431.1393 (431.8896).

2.2.6. 4-((1-((1-(4-Chlorobenzyl)-1H-1,2,3-triazol-4-yl)methyl)-1H-indol-3-yl)methylene)-3-methyl-1-phenyl-1H-pyrazol-5(4H)-one, (10Bb)

Orange crystals, m.p: 203.2–204.1 °C, % yield: 70, IR (KBr)ν: 3104, 3068, 1675, 1597, 1437 cm⁻¹. ¹H-NMR (DMSO-d₆, 400 MHZ): δ = 9.97 (1H, s), 8.01–7.99 (2H, d J = 8.8 Hz, ar-H), 7.91–7.89 (1H, m, ar-H), 7.83 (1H, s, ar-H), 7.63–7.61 (1H, m, ar-H), 7.48–7.44 (2H, t J = 16.0 Hz, ar-H), 7.39–7.37 (3H, m, ar-H), 7.33–7.30 (2H, m, ar-H), 7.23–7.20 (1H, t J = 15.8 Hz, ar-H), 7.17–7.15 (2H, d J = 8.4 Hz, ar-H), 5.60 (2H, s, -N-CH₂), 5.44 (2H, s, -N-CH₂), 2.45 (3H, s, -CH₃). ¹³C-NMR (DMSO-d₆, 100 MHZ): δ = 13.48, 31.15, 42.51, 52.53, 112.12, 112.30, 118.62, 118.62, 119.18, 119.41, 123.10, 124.21, 124.60, 124.66, 129.21, 129.21,129.21, 129.26, 130.32, 130.32,130.32, 133.35, 136.28, 136.73, 137.07, 139.23, 140.66, 142.76, 151.52, 163.21. M.F: C₂₉H₂₃ClN₆O, HRMS m/z (ESI): [M] 506.1718 (506.9855).

2.2.7. 4-((1-((1-(4-Chlorobenzyl)-1H-1,2,3-triazol-4-yl)methyl)-1H-indol-3-yl)methylene)-3-methyl-1-(p-tolyl)-1H-pyrazol-5(4H)-one, (10Bc)

Orange crystals, m.p: 216.3–217.1 °C, % yield: 74, IR (KBr)ν: 3108, 3055, 1668, 1600, 1469 cm⁻¹. ¹H-NMR (DMSO-d₆, 400 MHZ): δ = 9.97 (1H, s), 7.91–7.82 (4H, m, ar-H), 7.63–7.61 (1H, m, ar-H), 7.39–7.36 (3H, m, ar-H), 7.32–7.29 (2H, m, ar-H), 7.26–7.24 (2H, m, ar-H), 7.17–7.15 (2H, d J = 8.0 Hz, ar-H), 5.60 (2H, s, -N-CH₂), 5.44 (2H, s, -N-CH₂), 2.44 (3H, s, -CH₃), 2.39 (3H, s, -CH₃). ¹³C-NMR (DMSO-d₆, 100 MHZ): δ = 13.45, 20.96, 31.15, 42.48, 52.52, 112.08, 112.28, 118.66, 118.66, 119.32, 119.40, 123.07, 124.18, 124.68, 129.21, 129.21, 129.21, 129.28, 129.65, 129.65, 130.30, 130.30, 133.34, 133.65, 135.29, 136.72, 136.89, 140.58, 142.76, 151.24, 163.00. M.F: C₃₀H₂₅ClN₆O, HRMS m/z (ESI): [M] 521.1871 (521.0121).

2.2.8. 4-((1-((1-(4-Chlorobenzyl)-1H-1,2,3-triazol-4-yl)methyl)-1H-indol-3-yl)methylene)-3-methyl-1-(4-nitrophenyl)-1H-pyrazol-5(4H)-one, (10Bd)

Green powder, m.p: 251.3–252.9 °C, % yield: 68, IR (KBr)ν: 3130, 1672, 1590, 1321 cm⁻¹. ¹H-NMR (DMSO-d₆, 400 MHZ): δ = 9.91 (1H, s), 8.35 (4H, s, ar-H), 7.92–7.87 (1H, m, ar-H), 7.66–7.63 (1H, m, ar-H), 7.42–7.40 (3H, m, ar-H), 7.34–7.32 (3H, m, ar-H), 7.19–7.17 (2H, d J = 8.4 Hz, ar-H), 5.63 (2H, s, -N-CH₂), 5.47 (2H, s, -N-CH₂), 2.47 (3H, s, -CH₃). ¹³C-NMR (DMSO-d₆, 100 MHZ): δ = 13.62, 42.64, 52.52, 112.46, 112.46, 117.60, 117.95, 119.54, 123.29, 124.33, 124.70, 125.52, 129.21, 129.21, 129.21, 129.21, 129.21, 130.36, 130.36, 130.36, 130.36, 133.34, 135.28, 136.83, 141.20, 142.70, 144.36, 148.27, 159.33. M.F: C₂₉H₂₂ClN₇O₃, HRMS m/z (ESI): [M + H]⁺ 552.1574 (552.9831).

2.2.9. 3-Methyl-4-((1-((1-(4-methylbenzyl)-1H-1,2,3-triazol-4-yl)methyl)-1H-indol-3-yl)methylene)-1H-pyrazol-5(4H)-one, (10Ca)

Yellow crystals, m.p: 237.8–239.2 °C, % yield: 67, IR (KBr)ν: 3111, 3040, 1671, 1585, 1468 cm⁻¹. ¹H-NMR (DMSO-d₆, 400 MHZ): δ = 11.06 (1H, s, -NH), 9.82 (1H, s), 8.22 (1H, s, ar-H), 8.13–8.11 (1H, m, ar-H), 7.89 (1H, s, ar-H), 7.77–7.74 (1H, m, ar-H), 7.35–7.29 (2H, m, ar-H), 7.20–7.18 (4H, m, ar-H), 5.64 (2H, s, -N-CH₂), 5.51 (2H, s, -N-CH₂), 2.27 (3H, s, -CH₃), 2.25 (3H, s, -CH₃). ¹³C-NMR (DMSO-d₆, 100 MHZ): δ = 13.43, 21.13, 31.35, 53.13, 111.87, 119.26, 119.79, 122.65, 123.84, 124.35, 128.41, 128.41, 128.41, 129.15, 129.77, 129.77, 129.77, 133.28, 134.97, 136.64, 138.09, 139.46, 142.78, 150.01. M.F: C₂₄H₂₂N₆O, HRMS m/z (ESI): [M + H]⁺ 411.1936 (411.4711).

2.2.10. 3-Methyl-4-((1-((1-(4-methylbenzyl)-1H-1,2,3-triazol-4-yl)methyl)-1H-indol-3-yl)methylene)-1-phenyl-1H-pyrazol-5(4H)-one, (10Cb)

Yellow powder, m.p: 204.8–206.1 °C, % yield: 60, IR (KBr)ν: 3114, 3061, 1671, 1597, 1488 cm⁻¹. ¹H-NMR (DMSO-d₆, 400 MHZ): δ = 9.96 (1H, s), 8.02–7.99 (2H, d J = 9.6 Hz, ar-H), 7.91–7.88 (1H, m, ar-H), 7.83 (1H, s, ar-H), 7.65–7.63 (1H, m, ar-H), 7.47–7.43 (2H, t J = 16.0 Hz, ar-H), 7.39–7.36 (2H, m, ar-H), 7.34 (1H, s, ar-H), 7.23–7.19 (2H, t J = 15.4 Hz, ar-H), 7.16–7.11 (3H, m, ar-H), 5.59 (2H, s, -N-CH₂), 5.42 (2H, s, -N-CH₂), 2.45 (3H, s, -CH₃), 2.33 (3H, s, -CH₃). ¹³C-NMR (DMSO-d₆,100 MHZ): δ = 13.48, 21.11, 31.15, 42.51, 53.13, 112.11, 112.31, 118.61, 118.61, 119.17, 119.41, 123.10, 124.20, 124.43, 124.59, 128,40, 128.40, 129.26, 129.26,129.26, 129.76, 129.76,129.76, 133.27, 136.75, 137.09, 138.00, 139.24, 140.68, 142.68, 151.53. M.F: C₃₀H₂₆N₆O, HRMS m/z (ESI): [M + H]⁺ 487.2221 (487.5670).

2.2.11. 3-Methyl-4-((1-((1-(4-methylbenzyl)-1H-1,2,3-triazol-4-yl)methyl)-1H-indol-3-yl)methylene)-1-(p-tolyl)-1H-pyrazol-5(4H)-one, (10Cc)

Yellow powder, m.p: 228.9–230.1 °C, % yield: 62, IR (KBr)ν: 3108, 3061, 1669, 1600, 1469 cm⁻¹. ¹H-NMR (DMSO-d₆, 400 MHZ): δ = 9.96 (1H, s), 7.90–7.82 (4H, m, ar-H), 7.65–7.63 (1H, m, ar-H), 7.38–7.33 (4H, m, ar-H), 7.24 (3H, s, ar-H), 7.17–7.11 (4H, m, ar-H), 5.59 (2H, s, -N-CH₂), 5.42 (2H, s, -N-CH₂), 2.44 (3H, s, -CH₃), 2.39 (3H, s, -CH₃), 2.33 (3H, s, -CH₃). ¹³C-NMR (DMSO-d₆, 100 MHZ): δ = 13.44, 21.10, 21.10, 31.15, 53.13, 112.42, 118.04, 118.04, 118.65, 118.65, 119.31, 119.39, 123.04, 124.43, 128.39, 128.39, 128.39, 129.27, 129.65, 129.65, 129.76, 129.76, 129.76, 133.27, 133.62, 134.10, 136.88, 137.99, 140.60, 142.69, 151.25. M.F: C₃₁H₂₈N₆O, HRMS m/z (ESI): [M + H]⁺ 501.2451 (501.5936).

2.2.12. 3-Methyl-4-((1-((1-(4-methylbenzyl)-1H-1,2,3-triazol-4-yl)methyl)-1H-indol-3-yl)methylene)-1-(4-nitrophenyl)-1H-pyrazol-5(4H)-one, (10Cd)

Yellow powder, m.p: 266.4–268.1 °C, % yield: 69, IR (KBr)ν: 3119, 1670, 1590, 1468, 1324 cm⁻¹. ¹H-NMR (DMSO-d₆, 400 MHZ): δ = 9.90 (1H, s), 8.35 (4H, s, ar-H), 7.91–7.87 (2H, m, ar-H), 7.67–7.65 (1H, m, ar-H), 7.41–7.38 (3H, m, ar-H), 7.17–7.13 (4H, m, ar-H), 5.61 (2H, s, -N-CH₂), 5.45 (2H, s, -N-CH₂), 2.47 (3H, s, -CH₃), 2.34 (3H, s, -CH₃). ¹³C-NMR (DMSO-d₆, 100 MHZ): δ = 13.63, 21.13, 53.13, 112.51, 112.51, 117.62, 117.62, 117.90, 119.56, 123.30, 124.44, 124.44, 125.54, 125.54, 128.45, 128.45, 128.45, 129.75, 129.75, 129.75, 129.75, 133.31, 136.85, 138.40, 142.59, 143.09, 149.32, 149.32, 151.47, 153.75. M.F: C₃₀H₂₅N₇O₃, HRMS m/z (ESI): [M + H]⁺ 532.2106 (532.5646).

2.2.13. 4-((1-((1-(4-Methoxybenzyl)-1H-1,2,3-triazol-4-yl)methyl)-1H-indol-3-yl)methylene)-3-methyl-1H-pyrazol-5(4H)-one, (10Da)

Orange crystals, m.p: 223.8–225.1 °C, % yield: 64, IR (KBr)ν: 3132, 2947, 1678, 1590 cm⁻¹. ¹H-NMR (DMSO-d₆, 400 MHZ): δ = 9.87 (1H, s), 8.59 (1H, s, ar-H), 7.88–7.86 (1H, d J = 8.8Hz, ar-H), 7.89 (1H, s, ar-H), 7.63–7.61 (1H, d J = 8.8Hz, ar-H), 7.37–7.33 (3H, m, ar-H), 7.20–7.18 (2H, d J = 8.8Hz, ar-H), 5.57 (2H, s, -N-CH₂), 5.41 (2H, s, -N-CH₂), 3.81 (3H, s, -OCH₃), 2.34 (3H, s, -CH₃). ¹³C-NMR (DMSO-d₆, 100 MHZ): δ = 13.43, 31.15, 42.38, 52.88, 55.58, 111.87, 112.06, 114.59, 114.59, 119.21, 119.73, 122.64, 123.84, 124.16, 128.20, 129.14, 130.05, 130.05, 134.90, 136.57, 139.45, 142.77, 150.01, 159.59, 166.70. M.F: C₂₄H₂₂N₆O₂, HRMS m/z (ESI): [M + H]⁺ 427.1900 (427.4705).

2.2.14. 4-((1-((1-(4-Methoxybenzyl)-1H-1,2,3-triazol-4-yl)methyl)-1H-indol-3-yl)methylene)-3-methyl-1-phenyl-1H-pyrazol-5(4H)-one, (10Db)

Yellow powder, m.p: 201.1–202.3 °C, % yield: 66, IR (KBr)ν: 3110, 3064, 1670, 1595, 1438 cm⁻¹. ¹H-NMR (DMSO-d₆, 400 MHZ): δ = 9.95 (1H, s), 8.02–8.00 (2H, d J = 9.6 Hz, ar-H), 7.91–7.88 (1H, m, ar-H), 7.83 (1H, s, ar-H), 7.65–7.63 (1H, m, ar-H), 7.47–7.43 (2H, t J = 16.0 Hz, ar-H), 7.39–7.36 (2H, m, ar-H), 7.33 (1H, s, ar-H), 7.23–7.17 (3H, m, ar-H), 6.87–6.85 (2H, d J = 8.8 Hz, ar-H), 5.58 (2H, s, -N-CH₂), 5.40 (2H, s, -N-CH₂), 3.78 (3H, s, -OCH₃), 2.45 (3H, s, -CH₃). ¹³C-NMR (DMSO-d₆, 100 MHZ): δ = 13.47, 31.15, 42.52, 52.89, 55.54, 112.11, 112.31, 114.57, 114.57, 114.57, 118.57, 118.57, 119.17, 119.40, 123.09, 124.23, 123.57, 128.18, 129.26, 129.26, 130.06, 130.06, 130.06, 136.72, 137.06, 139.25, 140.68, 142.67, 151.52, 159.58, 163.21. M.F: C₃₀H₂₆N₆O₂, HRMS m/z (ESI): [M + H]⁺503.2213 (503.5664).

2.2.15. 4-((1-((1-(4-Methoxybenzyl)-1H-1,2,3-triazol-4-yl)methyl)-1H-indol-3-yl)methylene)-3-methyl-1-(p-tolyl)-1H-pyrazol-5(4H)-one, (10Dc)

Yellow powder, m.p: 224.6–226.1 °C, % yield: 65, IR (KBr)ν: 3107, 3055, 1670, 1602, 1469 cm⁻¹.¹H-NMR (DMSO-d₆, 400 MHZ): δ = 9.96 (1H, s), 7.89–7.86 (3H, m, ar-H), 7.82 (1H, s, ar-H), 7.65–7.63 (1H, m, ar-H), 7.38–7.36 (2H, s, ar-H), 7.32 (1H, s, ar-H), 7.26–7.24 (2H, m, ar-H), 7.19–7.17 (2H, d J = 8.4 Hz, ar-H), 6.87–6.85 (2H, d J = 8.8 Hz, ar-H), 5.58 (2H, s, -N-CH₂), 5.40 (2H, s, -N-CH₂), 3.79 (3H, s, -OCH₃), 2.44 (3H, s, -CH₃), 2.39 (3H, s, -CH₃). ¹³C-NMR (DMSO-d₆, 100 MHZ): δ = 13,45, 20.96, 31.15, 42.50, 52.89, 55.54, 112.07, 112.30, 114.57, 114.57, 118.59, 118.59, 119.31, 119.38, 123.05, 124.14, 124.22, 128.18, 129.27, 129.65, 129.65, 130.06, 130.06, 130.06, 133.61, 136.71, 136.92, 140.61, 142.69, 151.25, 159.58, 162.99. M.F: C₃₁H₂₈N₆O₂, HRMS m/z (ESI): [M + H]⁺ 517.2357 (517.5930).

2.2.16. 4-((1-((1-(4-Methoxybenzyl)-1H-1,2,3-triazol-4-yl)methyl)-1H-indol-3-yl)methylene)-3-methyl-1-(4-nitrophenyl)-1H-pyrazol-5(4H)-one, (10Dd)

Yellow powder, m.p: 223.8–225.2 °C, % yield: 67, IR (KBr)ν: 3145, 3105, 1685, 1591, 1320 cm⁻¹. ¹H-NMR (DMSO-d₆, 400 MHZ): δ = 9.90 (1H, s), 8.33 (4H, s, ar-H), 7.91–7.87 (2H, m, ar-H), 7.68–7.65 (1H, m, ar-H), 7.41–7.37 (3H, m, ar-H), 7.21–7.19 (2H, d J = 8.4 Hz, ar-H), 6.89–6.86 (2H, d J = 8.8 Hz, ar-H), 5.61 (2H, s, -N-CH₂), 5.43 (2H, s, -N-CH₂), 3.79 (3H, s, -OCH₃), 2.47 (3H, s, -CH₃). ¹³C-NMR (DMSO-d₆, 100 MHZ): δ = 13.58, 31.15, 42.61, 52.91, 55.54, 112.44, 114.56, 114.56, 117.58, 117.58, 117.86, 119.50, 123.37, 124.28, 124.46, 125.49, 128.16, 129.26, 130.09, 130.09, 130.09, 136.80, 138.28, 141.30, 142.55, 142.95, 144.35, 153.04, 153.73, 159.58, 163.97. M.F: C₃₀H₂₆N₇O₄, HRMS m/z (ESI): [M + H]⁺ 548.2011 (548.5640).

2.2.17. 3-Methyl-4-((1-((1-(4-nitrobenzyl)-1H-1,2,3-triazol-4-yl)methyl)-1H-indol-3-yl)methylene)-1H-pyrazol-5(4H)-one, (10Ea)

Yellow powder, m.p: 279.7–280.9 °C, % yield: 65, IR (KBr)ν: 3172, 3118, 1666, 1440, 1348 cm⁻¹. ¹H-NMR (DMSO-d₆, 400 MHZ): δ = 11.08 (1H, s, -NH), 9.79 (1H, s), 8.33 (1H, s, ar-H), 8.21–8.18 (2H, d J = 8.8Hz, ar-H), 8.12–8.10 (1H, d J = 8.8Hz, ar-H), 7.90 (1H, s, ar-H), 7.75–7.73 (1H, d J = 8.8Hz, ar-H), 7.51–7.49 (2H, d J = 8.8Hz, ar-H), 7.35–7.32 (2H, m, ar-H), 5.74 (2H, s, -N-CH₂), 5.65 (2H, s, -N-CH₂), 2.23 (3H, s, -CH₃). ¹³C-NMR (DMSO-d₆, 100 MHZ): δ =13.43, 31.15, 42.35, 52.44, 111.90, 112.03, 119.23, 119.75, 122.67, 123.87, 124.36, 124.36, 125.01, 129.15, 129.49, 129.49, 134.91, 136.59, 139.40, 142.96, 143.70, 147.71, 150.02, 166.71. M.F: C₂₃H₁₉N₇O₃, HRMS m/z (ESI): [M + H]⁺ 442.1632 (442.4421).

2.2.18. 3-Methyl-4-((1-((1-(4-nitrobenzyl)-1H-1,2,3-triazol-4-yl)methyl)-1H-indol-3-yl)methylene)-1-phenyl-1H-pyrazol-5(4H)-one, (10Eb)

Orange crystals, m.p: 232.6–234.7 °C, % yield: 65, IR (KBr)ν: 3107, 3073, 1664, 1595, 1334 cm⁻¹. ¹H-NMR (DMSO-d₆, 400 MHZ): δ = 9.97 (1H, s), 8.20–8.17 (2H, d J = 13.6 Hz, ar-H), 8.00–7.97 (2H, d J = 9.6 Hz, ar-H), 7.92–7.90 (1H, m, ar-H), 7.83 (1H, s, ar-H), 7.62–7.60 (1H, m, ar-H), 7.47–7.43 (3H, t J = 16.0 Hz, ar-H), 7.40–7.35 (4H, m, ar-H), 7.23–7.19 (1H, t J = 16.0 Hz, ar-H), 5.63 (2H, s, -N-CH₂), 5.58 (2H, s, -N-CH₂), 2.45 (3H, s, -CH₃). ¹³C-NMR (DMSO-d₆, 100 MHZ): δ =13.48, 31.15, 42.33, 52.44, 112.14, 112.30, 118.60, 118.60, 119.21, 119.43, 123.12, 124.24, 124.35, 124.35, 124.59, 125.05, 129.25, 129.25, 129.48, 129.48,136.74, 137.08, 139.21, 140.66, 140.66, 142.89, 143.69, 146.79, 147.68, 151.53, 163.22. M.F: C₂₉H₂₃N₇O₃, HRMS m/z (ESI): [M + H]⁺ 518.1981 (518.5380).

2.2.19. 3-Methyl-4-((1-((1-(4-nitrobenzyl)-1H-1,2,3-triazol-4-yl)methyl)-1H-indol-3-yl)methylene)-1-(p-tolyl)-1H-pyrazol-5(4H)-one, (10Ec)

Orange crystals, m.p: 208.3–209.5 °C, % yield: 60, IR (KBr)ν: 3146, 3110, 1671, 1467, 1351 cm⁻¹. ¹H-NMR (DMSO-d₆, 400 MHZ): δ = 9.98 (1H, s), 8.20–8.18 (2H, d J = 8.8 Hz, ar-H), 7.92–7.90 (1H, m, ar-H), 7.85–7.82 (3H, m, ar-H), 7.62–7.60 (1H, m, ar-H), 7.43 (1H, s, ar-H), 7.40–7.36 (4H, m, ar-H), 7.26–7.24 (2H, d J = 8.0 Hz, ar-H), 5.63 (2H, s, -N-CH₂), 5.58 (2H, s, -N-CH₂), 2.44 (3H, s, -CH₃), 2.39 (3H, s, -CH₃). ¹³C-NMR (DMSO-d₆, 100 MHZ): δ =13.44, 20.95, 31.15, 42.48, 52.44, 112.10, 112.27, 118.62, 118.62, 119.35, 119.40, 123.08, 124.20, 124.20, 124.35, 125.06, 129.28, 129.28, 129.46, 129.46, 129.46, 129.63, 133.64, 136.73, 136.88, 140.56, 142.89, 143.68, 147.68, 151.25, 163.00. M.F: C₃₀H₂₅N₇O₃, HRMS m/z (ESI): [M + H]⁺ 532.2106 (532.5646).

2.2.20. 3-Methyl-4-((1-((1-(4-nitrobenzyl)-1H-1,2,3-triazol-4-yl)methyl)-1H-indol-3-yl)methylene)-1-(4-nitrophenyl)-1H-pyrazol-5(4H)-one, (10Ed)

Orange powder, m.p: 279.8–281.2 °C, % yield: 60, IR (KBr)ν: 3136, 3112, 1682, 1589 cm⁻¹. ¹H-NMR (DMSO-d₆, 400 MHZ): δ = 9.83 (1H, s), 8.39 (1H, s, ar-H), 8.32–8.25 (4H, m, ar-H), 8.19–8.13 (4H, m, ar-H), 7.82–7.79 (1H, d J = 8.8 Hz, ar-H), 7.50–7.48 (2H, d J = 8.8 Hz, ar-H), 7.41–7.35 (2H, m, ar-H), 5.76 (2H, s, -N-CH₂), 5.74 (2H, s, -N-CH₂), 2.43 (3H, s, -CH₃). ¹³C-NMR (DMSO-d₆, 100 MHZ): δ = 13.61, 42.62, 52.43, 112.48, 117.54, 117.93, 119.55, 123.33, 124.36, 124.36, 124.36, 125.11, 125.48, 125.48, 129.30, 129.30, 129.30, 129.49, 129.49, 136.85, 138.29, 141.29, 142.83, 142.94, 143.75, 144.36, 147.67, 153.69, 163.96.

2.3. Biological Investigations

2.3.1. Cancer Cell Lines

The American Type Culture Collection (ATCC, Manassas, VA, USA) provided the human cancer cell lines Capan-1, HCT-116, NCI-H460, LN-229, HL-60, K-562, MOLT4, and Z-138. All cell lines were stored in accordance with the suppliers’ instructions. Gibco (Gibco Life Technologies, Merelbeke, Belgium) provided the culture media, which were supplemented with 10% fetal bovine serum (HyClone, Cytiva, Marlborough, MA, USA). The reference and test molecules were dissolved in DMSO (99.5%, Sigma, Hoeilaart, Belgium) to a concentration of 100 micromolar (µM).

2.3.2. Cytotoxicity Assays

Adherent cells were seeded at densities between 500 and 1500 cells/well in 384-well plates (Greiner Bio-One, Vilvoorde, Belgium). After 24 h of incubation, cells were exposed to seven varying concentrations of the test compounds (100–0.006 µM). The untreated cells served as negative controls. Suspension cell lines (non-adherent) were placed at densities between 2500 and 5000 cells/well in 384-well culture plates with the test compounds of the same concentration (100 to 0.006 µM). All cell lines were incubated for 72 h with test compounds and were subsequently assessed using the Cell Titer 96^® AQueous One Solution Cell Proliferation Assay (MTS) reagent (Promega, Leiden, Netherlands) following the supplier’s guidelines. The absorbance was recorded at 490 nanometers (nm) with a SpectraMaxPlus 384 (Molecular Devices, San Jose, CA, USA), and optical density (OD) was measured to determine the 50% cytotoxic concentration (CC50) of test compounds. All experiments were performed in triplicate [26], Table 2.

2.3.3. GSK-3β Inhibition Assay

This assay was performed using the GSK-3β Kinase Enzyme System (Promega V1991, Madison, WI, USA) and ADP-Glo Kinase Assay (Promega V6930, Madison, USA) kits. A reaction mix was prepared using 1X reaction buffer, 50 µM ATP, 50 µM DTT, 1 µg/µL GSK3β substrate, and 0.5 ng/µL GSK3β enzyme with increasing concentrations of 10Aa drug dissolved in DMSO. A reaction mix containing DMSO served as the vehicle control, and 12 nM Laduviglusib (MedChemExpress, New Jersey, USA), a GSK-3β inhibitor, was used as a positive control for the experiment. This reaction mix was incubated for 60 min at room temperature. Following incubation, 5 µL of ADP-Glo Reagent was added and incubated additionally for 40 min at room temperature. Following this, 10 µL of kinase detection reagent was added, and the mixture was incubated for 30 min at room temperature. Luminescence was measured using a microplate reader with an integration time of 0.5 s [9] (Figure 3).

2.3.4. InSilico Study [27,28,29]

Drug-Likeness and InSilico Absorption, Distribution, Metabolism, Excretion, and Toxicity (ADMET) Prediction

The physicochemical and pharmacokinetic parameters, such as molecular weight, hydrogen-bond donors (HBD), hydrogen-bond acceptors (HBA), lipophilicity (Log P), solubility, and compliance with Lipinski’s rule of five (RO5), along with bioavailability, gastrointestinal (GI) absorption, and toxicity profiles of the synthesized derivatives 10Aa-Ed, were predicted using Swiss-ADME (version-23, University of Lausanne and the SIB Swiss Institute of Bioinformatics, Switzerland) [27]. The blood–brain barrier (BBB) permeability and unbound fraction were also evaluated, together with CYP1A2 substrate binding affinity and half-lives. Toxicity indicators, including overall safety and skin sensitization potential, were predicted and summarized in Table 3 and in the Supplementary (S80–S99).

Protein Preparation

The crystal structure of the protein was retrieved from the PDB and prepared by removing co-crystallized ligands, buffer ions, and non-essential water molecules. The catalytic or structural waters known to mediate conserved H-bonding with the reference ligand were retained with the structures. Missing side-chain atoms were rebuilt and locally optimized. The polar hydrogens were added, and receptor protonation was set for pH 7.4. Gasteiger charges were assigned for docking compatibility [28,29].

Docking Protocol

The ligand molecules 10Aa–Ed and bioacetoxime were sketched using Chem Draw-Version 20.1 and subjected to energy minimization in Chem3D (v20.0, PerkinElmer Inc., Waltham, MA, USA). Molecular docking was performed utilizing PyRx v.0.8 software (Source Forge, San Diego, CA, USA; AutoDockVina engine). The binding orientations and interaction profiles were analyzed in BIOVIA Discovery Studio Visualizer 2021 (Dassault Systèmes Biovia Corp., San Diego, CA, USA). The docking protocol was validated by re-docking with native ligands into their specific binding sites, with an acceptance criterion of RMSD ≤ 2.0 Å. The MM-GBSA binding free-energy computations were carried out on snapshots taken from molecular dynamics trajectories utilizing the g_mmpbsa package (a GROMACS-compatible version of MM-PBSA/MM-GBSA) to obtain refined ΔG_bind estimates. The pharmacokinetic and drug-likeness evaluations were conducted through Swiss-ADME (Swiss Institute of Bioinformatics, Lausanne, Switzerland) and pkCSM (Bioinformatics & Computational Biology Biosig Lab, The University of Queensland, Brisbane, Australia), facilitating the prediction of physicochemical characteristics, ADMET profiles, and overall drug-likeness potential (Figures 4–7, Table 4).

MM-GBSA Binding Free Energy Calculations

Refined binding free energies (ΔG_bind) were calculated using the MM-GBSA method implemented in Schrödinger Prime (release 2023-1, Schrodinger, Mangaluru, India). Snapshots from the MD trajectories were used to derive ligand-protein binding free-energy estimates. Calculations were performed on 200 snapshots per replica (last 40 ns, stride = 0.2 ns) using the VSGB 2.1 solvation model, with ligand- and receptor-reparametrized partial charges consistent with CHARMM36 mapping. Results were reported as mean ± SD. MM-PBSA calculations were performed on snapshots extracted every 0.2 ns from the last 40 ns of each replica (≥200 frames/replica), using a Poisson-Boltzmann polar solvation term and a SASA-based non-polar term. For each complex, the per-frame ΔG_bind(t) traces and the time-averaged ΔG_bind ± SD across replicas convergence were checked via block-averaging (Table 5).

3. Results and Discussion

3.1. Chemistry

The compound 2-(3-formyl-1H-indol-1-yl)acetonitrile (3) was synthesized by reacting 3-formylindole (1) with propargyl bromide (2) in dimethylformamide (DMF, 99%, SD Fine Chemical, Bengaluru, India) using potassium carbonate (100%, SD Fine Chemical, Bengaluru, India) as a base. The respective intermediates 1-((1-aralkyl-1H-1,2,3-triazol-4-yl)methyl)-1H-indole-3-carbaldehyde (6a–d) were subsequently obtainedvia a click reaction between 2-(3-formyl-1H-indol-1-yl)acetonitrile (3) and various aralkylazides (5a–d). Separately, 5-methyl-2-substituted or unsubstituted-2,4-dihydro-3H-pyrazol-3-ones (9a–e) were prepared by condensing ethyl acetoacetate (7) with hydrazine or arylhydrazines (8a–d) in ethanol (100%, Bio Liqua Research, Bengaluru, India). Finally, the target molecles, 4-((1-((1-aralkyl-1H-1,2,3-triazol-4-yl)methyl)-1H-indol-3-yl)methylene)-5-methyl-2-aryl-2,4-dihydro-3H-pyrazol-3-ones (10Aa–Ed),were synthesized by coupling indole-3-carbaldehyde derivatives (6) with pyrazol-3-one derivatives (9) in methanol, using piperidine as a catalytic (Scheme 1,

Table 1. Details of the compounds 10Aa–Ed.

Code	R	R’	Code	R	R’	Code	R	R’
10Aa	H	H	10Bd	Cl	4-NO2-C6H4	10Dc	OCH3	4-CH3-C6H4
10Ab	H	C6H5	10Ca	CH3	H	10Dd	OCH3	4-NO2-C6H4
10Ac	H	4-CH3-C6H4	10Cb	CH3	C6H5	10Ea	NO2	H
10Ad	H	4-NO2-C6H4	10Cc	CH3	4-CH3-C6H4	10Eb	NO2	C6H5
10Ba	Cl	H	10Cd	CH3	4-NO2-C6H4	10Ec	NO2	4-CH3-C6H4
10Bb	Cl	C6H5	10Da	OCH3	H	10Ed	NO2	4-NO2-C6H4
10Bc	Cl	4-CH3-C6H4	10Db	OCH3	C6H5

).

All the compounds were confirmed for their structure using FTIR, ¹H/¹³C NMR, and HRMS data. The FT-IR spectra of compounds 10Aa–Ed displayed characteristic absorption bands. The indole -NH stretching vibrations were observed in the region of 3172–3100 cm⁻¹, while signals corresponding to aromatic and aliphatic -CH groups appeared between 3121 and 2947 cm⁻¹. A strong absorption due to carbonyl (>C=O) stretching was evident in the range of 1685 and 1664 cm⁻¹. In the ¹H-NMR spectra, the -NH proton resonances were detected at 11.08–11.06 δparts per million (ppm). Aromatic protons resonated between 8.35 and 6.85 δ ppm, whereas the -N-CH₂ protons appeared in the 5.76–5.40 δ ppm region. Signals for methoxy (-OCH₃) groups were found at 3.81–3.78 δ ppm, and methyl (-CH₃) protons resonated between 2.47 and 2.23 δ ppm. In the ¹³C-NMR spectra, the carbonyl carbons resonated between 166.71 and 163.00 ppm, while aromatic carbons were distributed across 159.59–111.87 δ ppm. The methoxy carbon appeared at 55.58–55.54 δ ppm, and the -N-CH₂ methylene carbons were observed between 53.33 and 31.15 δ ppm. Methyl carbons (-CH₃) resonated in the 21.00–13.15 δ ppm range. Finally, the mass spectrometric values (m/z) of all compounds closely matched their calculated molecular weights, confirming the synthesized structures.

3.2. Biology

3.2.1. Cytotoxicity Evaluation

The cytotoxic activities of compounds 10Aa–Ed were assessed across a panel of eight cancer cell lines (adherent and non-adherent types). The adherent cell lines included pancreatic adenocarcinoma (Capan-1), colorectal carcinoma (HCT-116), glioblastoma (LN229), and lung carcinoma (NCI-H460), while the non-adherent group consisted ofmyeloid leukemia (HL-60), chronic myeloid leukemia (K562), T-lymphoblasts (MOLT4), and non-Hodgkin lymphoma (Z138) cells. Sunitinib and 5-Fluorouracil (5-FU) were employed as standard reference drugs. Compound 10Cd exhibited notable cytotoxic activity against glioblastoma (LN226) cells (CC₅₀: 13 µM), which is comparable to Sunitinib (CC₅₀: 12 µM, BLD Pharm, Hyderabad, India) but less potent than 5-Fluorouracil (CC₅₀: 7 µM, Sun Pharmaceuticals, Baroda, India). Among the tested cell lines, LN226glioblastoma cells showed increased sensitivity to 10Bd, 10Cd, and 10Dd. In addition, compound 10Aa demonstrated cytotoxic effects across multiple cell lines, including Capan-1, NCI-H460, MOLT4, HL-60, and Z138 (CC₅₀: 14 to 34 µM). Based on these findings, molecule 10Aa was identified as moderately active against six different cell lines. All experiments were performed in triplicate (

Table 2. The cytotoxic activity of compounds 10Aa–Ed at a concentration of 50 µM against Capan-1, HCT-116, LN229, NCI-H460, MOLT-4, HL-60, K562, and Z138 cell lines.

Compound	Capan-1	HCT-116	LN229	NCI-H460	Molt-4	HL-60	K562	Z138
	Adherent Cells (CC50)				Suspension Cells (CC50)
10Aa	23.87 ± 6.98	>50	>50	34.06 ± 22.43	14.45 ± 1.46	17.56 ± 7.04	>50	15.34 ± 0.72
10Ab	>50	>50	>50	>50	>50	>50	>50	>50
10Ac	>50	>50	>50	>50	>50	>50	>50	>50
10Ad	>50	>50	>50	>50	>50	>50	>50	>50
10Ba	>50	>50	>50	>50	>50	>50	>50	>50
10Bb	>50	>50	>50	>50	>50	>50	>50	>50
10Bc	>50	>50	>50	>50	>50	>50	>50	>50
10Bd	>50	>50	35.78 ± 17.81	>50	>50	>50	>50	>50
10Ca	>50	>50	>50	>50	>50	>50	>50	>50
10Cb	>50	>50	>50	>50	>50	>50	>50	>50
10Cc	>50	>50	>50	>50	>50	>50	>50	>50
10Cd	>50	>50	13.41 ± 1.75	>50	>50	>50	>50	>50
10Da	>50	>50	>50	>50	>50	>50	>50	>50
10Db	>50	>50	>50	>50	>50	>50	>50	>50
10Dc	>50	>50	>50	>50	>50	>50	>50	>50
10Dd	>50	>50	41.22 ± 6.65	>50	>50	>50	>50	>50
10Ea	>50	>50	>50	>50	>50	>50	>50	>50
10Eb	>50	>50	>50	>50	>50	>50	>50	>50
10Ec	>50	>50	>50	>50	>50	>50	>50	>50
10Ed	>50	>50	>50	>50	>50	>50	>50	>50
Sunitinib (100 µM)	1.5 ± 0.01	4.25 ± 0.71	11.80 ± 1.41	8.85 ± 3.04	NT	5.00 ± 1.27	8.4 ± 2.12	8.15 ± 1.49
5-Fluorouracil (100 µM)	2.7 ± 0.01	2.3 ± 0.71	7.35 ± 0.35	5.0 ± 1.70	NT	5.90 ± 0.99	12.90 ± 0.42	7.85 ± 0.71

Note: All data are expressed as mean ± standard deviations (SD) from three independent experiments. CC₅₀: 50% cytotoxic concentration.

).

3.2.2. Assessment of GSK-3βinhibition by Compound 10Aa

GSK-3β kinase inhibition was evaluated using a luminescence-based kinase assay with the 10Aa. Increasing concentrations of 10Aa produced a dose-dependent reduction in GSK-3β kinase activity, indicating that 10Aa is capable of inhibiting GSK-3β, with 100 µM 10Aa showing a significant 25% inhibition (Figure 3). This dose-dependent inhibition further supports the binding affinity of 10Aa toward GSK-3β.

3.2.3. InSilico Study

Drug Likeness and ADMET Prediction

In silico ADMET evaluation indicated that the synthesized compounds possess acceptable drug-like physicochemical properties. Molecular weights ranged from 396.44 to 562.54 Da, with hydrogen bond acceptors (HBA, 4–8), hydrogen bond donors (HBD, 0–1), and rotatable bonds (5–8) largely within acceptable limits. The calculated LogP values (1.99–5.00) suggest moderate to high lipophilicity, supporting membrane permeability, while ESOL-predicted aqueous solubility (LogS −4.18 to −6.62) indicates low to moderate solubility, typical of hetero-aromatic scaffolds. Compounds from the 10A and 10C series showed blood–brain barrier (BBB) permeability. Skin permeability (log Kp −5.60 to −7.18) values indicated moderate transdermal penetration. Drug-likeness assessment using Lipinski, Ghose, Veber, Egan, and Muegge filters revealed minimal violations across the series, suggesting favorable oral bioavailability. All compounds were predicted to act as P-glycoprotein (P-gp) inhibitors and to exhibit high plasma protein binding. Compounds 10Ab, 10Ac, 10Ba, 10Ba, 10Bc, 10Cb, 10Cc, 10Db, and 10Dc were identified as CYP1A2 substrates, indicating potential phase-I metabolic clearance via this enzyme. The predicted short half-life (<3 h) suggests moderate systemic clearance. Overall, the ADMET analysis confirms that the majority of the compounds exhibit excellent drug-likeness, high oral absorption, acceptable metabolic behavior, and promising safety characteristics (Table 3).

Table 3. Predicted physicochemical, pharmacokinetic, and toxicity profile of 10Aa–Ed.

Compounds	MW	HBA	RB	HBD	LogP	LogS (ESOL)	LogKp	BBB	LV	GV	VV	EV	MV	BA
10Aa	396.44	4	5	1	2.82	−4.18	−6.78	Yes	0	0	0	0	0	0.55
10Ab	472.54	4	6	0	4.22	−5.72	−6.01	Yes	0	1	0	0	0	0.55
10Ac	486.57	4	6	0	4.44	−6.02	−5.84	Yes	0	2	0	0	0	0.55
10Ad	517.54	6	7	0	3.42	−5.78	−6.41	Yes	1	2	0	0	0	0.55
10Ba	430.89	4	5	1	3.3	−4.77	−6.55	No	0	0	0	0	0	0.55
10Bb	506.99	4	6	0	4.67	−6.31	−5.78	Yes	2	2	0	0	1	0.17
10Bc	521.01	4	6	0	5.0	−6.62	−5.60	No	2	2	0	0	1	0.17
10Bd	551.98	6	7	0	4.02	−6.38	−6.17	No	1	2	0	0	0	0.55
10Ca	410.47	4	5	1	3.09	−4.48	−6.61	Yes	0	0	0	0	0	0.55
10Cb	486.57	4	6	0	4.55	−6.09	−6.23	Yes	1	2	0	0	0	0.55
10Cc	500.59	4	6	0	4.80	−6.32	−5.67	Yes	2	2	0	0	1	0.17
10Cd	531.56	6	7	0	3.83	−6.09	−6.32	No	1	2	0	0	0	0.55
10Da	426.47	5	8	1	2.76	−4.25	−6.98	No	0	0	0	0	0	0.55
10Db	502.57	5	7	0	4.18	−5.80	−6.21	No	1	2	0	0	0	0.55
10Dc	516.59	5	7	0	4.56	−6.10	−6.04	No	1	2	0	0	0	0.55
10Dd	547.56	7	8	0	3.49	−5.87	−6.61	No	2	2	0	0	0	0.17
10Ea	441.44	6	6	1	1.99	−4.24	−7.18	No	0	1	0	0	0	0.55
10Eb	517.54	6	7	0	3.5	−5.78	−6.41	No	1	2	0	0	0	0.55
10Ec	531.56	6	7	0	3.71	−6.02	−5.84	No	0	2	0	0	0	0.55
10Ed	562.54	8	8	0	2.6	−5.85	−6.81	No	2	2	1	1	1	0.17

MW: molecular weight, HBA: hydrogen bond acceptor(s), RB: rotatable bonds, HBD: number of hydrogen bond donor(s). LogP: log of octanol/water partition coefficient, LogS: aqueous solubility, LogKp: skin permeability partition coefficient, BBB: blood–brain barrier, LV: Lipinski violation(s), GV: Ghose violation(s), VV: Veber violation(s), EV: Egan violation(s), MV: Muegge violation(s), BA: bioavailability.

Table 3. Predicted physicochemical, pharmacokinetic, and toxicity profile of 10Aa–Ed.

Compounds	MW	HBA	RB	HBD	LogP	LogS (ESOL)	LogKp	BBB	LV	GV	VV	EV	MV	BA
10Aa	396.44	4	5	1	2.82	−4.18	−6.78	Yes	0	0	0	0	0	0.55
10Ab	472.54	4	6	0	4.22	−5.72	−6.01	Yes	0	1	0	0	0	0.55
10Ac	486.57	4	6	0	4.44	−6.02	−5.84	Yes	0	2	0	0	0	0.55
10Ad	517.54	6	7	0	3.42	−5.78	−6.41	Yes	1	2	0	0	0	0.55
10Ba	430.89	4	5	1	3.3	−4.77	−6.55	No	0	0	0	0	0	0.55
10Bb	506.99	4	6	0	4.67	−6.31	−5.78	Yes	2	2	0	0	1	0.17
10Bc	521.01	4	6	0	5.0	−6.62	−5.60	No	2	2	0	0	1	0.17
10Bd	551.98	6	7	0	4.02	−6.38	−6.17	No	1	2	0	0	0	0.55
10Ca	410.47	4	5	1	3.09	−4.48	−6.61	Yes	0	0	0	0	0	0.55
10Cb	486.57	4	6	0	4.55	−6.09	−6.23	Yes	1	2	0	0	0	0.55
10Cc	500.59	4	6	0	4.80	−6.32	−5.67	Yes	2	2	0	0	1	0.17
10Cd	531.56	6	7	0	3.83	−6.09	−6.32	No	1	2	0	0	0	0.55
10Da	426.47	5	8	1	2.76	−4.25	−6.98	No	0	0	0	0	0	0.55
10Db	502.57	5	7	0	4.18	−5.80	−6.21	No	1	2	0	0	0	0.55
10Dc	516.59	5	7	0	4.56	−6.10	−6.04	No	1	2	0	0	0	0.55
10Dd	547.56	7	8	0	3.49	−5.87	−6.61	No	2	2	0	0	0	0.17
10Ea	441.44	6	6	1	1.99	−4.24	−7.18	No	0	1	0	0	0	0.55
10Eb	517.54	6	7	0	3.5	−5.78	−6.41	No	1	2	0	0	0	0.55
10Ec	531.56	6	7	0	3.71	−6.02	−5.84	No	0	2	0	0	0	0.55
10Ed	562.54	8	8	0	2.6	−5.85	−6.81	No	2	2	1	1	1	0.17

MW: molecular weight, HBA: hydrogen bond acceptor(s), RB: rotatable bonds, HBD: number of hydrogen bond donor(s). LogP: log of octanol/water partition coefficient, LogS: aqueous solubility, LogKp: skin permeability partition coefficient, BBB: blood–brain barrier, LV: Lipinski violation(s), GV: Ghose violation(s), VV: Veber violation(s), EV: Egan violation(s), MV: Muegge violation(s), BA: bioavailability.

Molecular Docking Analysis

The information derived from the molecular docking analysis, detailed in

Table 4. Docking scores of 10Aa–Ed against the target protein, highlighting top binding molecules.

Compounds	Binding Affinity	rmsd/ub	rmsd/lb	Compounds	Binding Affinity	rmsd/ub	rmsd/lb
10Aa	−10.6	6.437	3.808	10Cc	−11.7	8.518	1.639
10Ab	−10.4	6.673	2.496	10Cd	−09.9	9.642	2.876
10Ac	−10.7	6.349	2.258	10Da	−10.7	9.394	2.712
10Ad	−09.6	6.568	3.911	10Db	−09.6	2.042	1.370
10Ba	−10.3	6.817	3.421	10Dc	−10.7	9.394	2.712
10Bb	−10.6	6.482	2.682	10Dd	−11.2	4.604	2.928
10Bc	−10.0	7.911	3.339	10Ea	−10.4	3.729	2.422
10Bd	−11.1	8.926	4.480	10Eb	−10.3	5.205	2.570
10Ca	−10.6	5.214	2.575	10Ec	−09.7	9.843	3.199
10Cb	−10.4	6.590	1.641	10Ed	−10.4	8.226	3.539

rmsd/ub-root mean square deviation upper bound; rmsd/lb-root mean square deviation lower bound.

and graphically represented in Figure 4 and Figure 5, indicated that molecules spanning from 10Aa–10Ed exhibited substantial binding affinities towards GSK-3β (identified by PDB ID: 1Q41). Despite the observation of intermediate-level interactions with compounds 10Ac and 10Da, compounds 10Cc, 10Dd, and 10Bd displayed the most pronounced binding energies, quantified as −11.7 kcal/mol, −11.2 kcal/mol, and −11.1 kcal/mol, respectively. The binding affinity of the reference ligand bioacetoxime, as shown in Figure 6, is −8.6 kcal/mol, which is lower than the binding strength of Molecule 10Dc, which is −10.7 kcal/mol. In short, these findings lend credence to the notion that the chosen molecules improved interactions with the designated protein, implying that they ought to continue to be developed into molecular dynamics models and possibly confirmed through laboratory testing. Figure 7 illustrates the binding mode of 10Cc, demonstrating its extensive penetration into the ATP-binding site of GSK-3β and the generation of several contacts that enhance stability. A typical hydrogen bond in the hinge region secures the ligand, and numerous van der Waals interactions with amino acids such as VAL, ALA, and GLY reinforce the pocket fit. An additional π-cation interaction enhances electrostatic compatibility, while the aromatic components interact with hydrophobic residues like ILE, VAL, and LEU through π-σ and π-alkyl interactions. These combined interactions lead to a stable and energetically favorable binding arrangement. Figure 4 shows that compound 10Dd also takes on a very stable orientation when bound to ATP. A multitude of van der Waals interactions involving GLY, VAL, ALA, and ASN contribute to the close-knit hydrophobic arrangement, and the ligand forms essential hydrogen bonds, securing its location in proximity to the flexible joint area. The binding of ASP133 to π-anion, ILE, LEU, and VAL, coupled with π-alkyl and π-σ activities, fosters greater stabilization. Additionally, the π-sulfur interaction reinforces the binding at the functionally important site. These interactions reveal that 10Dd forms a robust connection with GSK-3β, suggesting its capacity for broad biological effects. Furthermore, the docking score for the compound 10Aa reached −10.6 kcal/mol. The indole fragment interacted with Val70, Ala83, Lys85, Leu132, and Cys199, whereas the pyrazole segment of 10Aa produced a hydrogen bond with Ile62. In addition, the triazolyl ring created hydrogen bonds with Asn186 and Val135, showing the positive agreement between the in vitro and in silico results of 10Aa (Figure 5), along with the stabilization of the ligand-protein complex by multiple van der Waals interactions involving Gly63, Asn64, Val119, Tyr134, Tyr140, Arg141, Val135, Val135, Pro136, Thr138, and Gln185 residues, making it an attractive candidate for future studies.

MM-GBSA Binding Free Energy Analysis

The synthetic compounds exhibit varied degrees of binding stability within the target protein, as indicated by the wide range of MM-GBSA binding free energy (ΔG_bind) values, which range from −25.05 to −41.98 kcal/mol. The most favorable binding energies were observed in 10Cc (−41.98 kcal/mol), 10Ec (−41.05 kcal/mol), 10Db (−37.14 kcal/mol), and 10Dd (−40.70 kcal/mol). These molecules were found to form strong and thermodynamically stable ligand-protein complexes, as shown in Table 2. The binding energies were mainly contributed to by van der Waals interactions (ΔG_vdw, −38 to −52 kcal/mol) and lipophilic interactions (ΔG_Lipo, −16 to −22 kcal/mol), indicating that hydrophobic contacts were crucial in stabilizing these ligands within the active site. Electrostatic (coulombic) contributions were also considerable for numerous high-affinity compounds, particularly 10Ab, 10Db, 10Bd, 10Ca, and 10Eb, indicating further stabilization through charged or polar contacts. On the other hand, the solvation energy (ΔG_solvGB) had a negative impact on the total binding in all compounds because of the anticipated desolvation penalties when the ligand entered the hydrophobic pocket. While hydrogen bonds and covalent bonds did play a role in binding, they were not the main factors and did vary across molecules. The docking results were supported by the MM-GBSA findings, which showed that compounds 10Cc, 10Db, 10Ec, and 10Dd were the most promising. These compounds exhibited strong binding affinity and stability within the protein active site, as well as highly favorable van der Waals, lipophilic, and electrostatic interactions (

Table 5. MM-GBSA binding free energy (ΔG_bind) and energy decomposition parameters for compounds 10Aa–Ed against the target protein.

Compounds	ΔG Bind	ΔG Bind Coulomb	ΔG Bind Covalent	ΔG Bind Hbond	ΔG Bind Lipo	ΔG Bind solvGB	∆G Bind vdw
10Aa	−30.92	−04.85	03.77	−2.94	−18.53	34.92	−40.52
10Ab	−35.98	−13.61	02.18	−2.28	−16.73	37.22	−46.72
10Ac	−37.20	−06.25	00.82	−2.08	−18.25	38.89	−46.27
10Ad	−30.87	−17.94	05.33	−3.15	−18.38	45.90	−38.69
10Ba	−27.08	−08.93	11.23	−1.01	−18.47	43.29	−49.60
10Bb	−25.38	−02.08	04.31	−2.27	−19.12	44.28	−47.18
10Bc	−29.31	−11.02	04.77	−2.44	−16.25	38.30	−40.12
10Bd	−41.55	−19.74	08.64	−2.24	−16.80	39.09	35.09
10Ca	−26.66	−12.57	03.83	−3.39	−20.27	58.92	−50.59
10Cb	−29.50	−10.26	02.59	−2.56	−21.13	46.78	−42.46
10Cc	−41.98	−11.40	03.92	−0.83	−20.31	46.78	−49.59
10Cd	−27.92	−13.95	03.13	−3.28	−20.84	52.96	−43.57
10Da	−32.18	−11.79	03.35	−2.56	−22.42	48.62	−44.91
10Db	−38.70	−13.22	06.34	−1.02	−19.72	40.35	−49.09
10Dc	−26.13	−08.56	07.66	−0.98	−19.19	46.62	−47.28
10Dd	−40.73	−09.63	04.25	−0.98	−19.29	43.92	−50.71
10Ea	−25.05	−06.66	10.39	−0.98	−16.96	44.93	−52.09
10Eb	−28.37	−11.16	06.46	−2.68	−20.25	46.23	−44.73
10Ec	−39.05	00.53	04.10	−1.21	−17.11	28.99	−51.73
10Ed	−40.23	−03.82	03.58	−2.66	−20.28	39.87	−47.93

ΔG Bind—binding free energy; ΔG Bind coulomb—change in Gibbs free energy of binding that is specially attributed to electrostatic interactions (coulomb interactions) between a ligand and a protein or receptor; ΔG Bind covalent—binding free energy of covalent bond; ΔG Bind Hbond—binding free energy of hydrogen bonds; ΔG Bind Lipo—lipophilic contribution to the overall binding free energy of a ligand–protein complex; ΔG Bind solvGB—binding free energy of solvation; ΔG Bind vdw—van der Waals force contribution to the overall binding free energy of a ligand–protein complexes.

). Therefore, these compounds were identified as promising leads for further biological evaluation.

Correlation Between Docking and MM-GBSA Results

The predicted ligand–protein interactions were found to be reliable, as evidenced by the strong agreement between the molecular docking scores and the MM-GBSA binding free energy values. In the MM-GBSA analysis, compounds with better docking scores, especially 10Cc, 10Bd, 10Bd,10Dd, 10Ed, and 10Ec, confirmed their great thermodynamic stability at the active site by showing very favorable ΔG_bind values. The fact that the structural properties that lead to high docking scores, like hydrophobic contact, van der Waals packing, and polar interactions, are faithfully reflected in the free energy decomposition is supported by this consistency. The association was further supported by the fact that molecules with moderate docking scores also showed lower MM-GBSA energies. Generally, the consistent patterns observed in the two computational approaches support the reliability of the binding predictions and further solidify the selection of these top-ranked compounds as potential leads for additional biological investigation.

4. Conclusions

In this study, twenty compounds were synthesized and characterized using various spectral techniques. Their cytotoxic effects were assessed against a variety of cancer cell lines, including pancreas, lung, blood, colon, and brain. The cytotoxicity data indicate that compound 10Aa exhibits greater sensitivity toward hematological cancer cell lines, including Molt-4, Z138, and HL-60, whereas the brain cancer cell line LN226 was more susceptible to compound 10Cd. Increasing concentrations of 10Aa produced a dose-dependent reduction in GSK-3β kinase activity, indicating that 10Aa was capable of inhibiting GSK-3β. Additionally, the higher docking score and significant stability in the MD simulation indicate that the synthesized compounds acted via GSK-3β inhibition. Further, mechanism-based investigation is required to determine the pharmacological potential of 10Aa as a therapeutic candidate.