Synthesis and Evaluation of 1,3,5-Triaryl-2-Pyrazoline Derivatives as Potent Dual Inhibitors of Urease and α-Glucosidase Together with Their Cytotoxic, Molecular Modeling and Drug-Likeness Studies

Abstract

In the present work, a concise library of 1,3,5-triaryl-2-pyrazolines (2a-2q) was designed and synthesized by employing a multistep strategy, and the newly synthesized compounds were screened for their urease and α-glucosidase inhibitory activities. The compounds (2a-2q) were characterized using a combination of several spectroscopic techniques including FT-IR, ¹H NMR, ¹³C NMR, and EI-MS. All the synthesized compounds, except compound 2i, were potent against urease as compared to the standard inhibitor thiourea (IC₅₀ = 21.37 ± 0.26 μM). These analogs disclosed varying degrees of urease inhibitory activities ranging from 9.13 ± 0.25 to 18.42 ± 0.42 μM. Compounds 2b, 2g, 2m, and 2q having IC₅₀ values of 9.36 ± 0.27, 9.13 ± 0.25, 9.18 ± 0.35, and 9.35 ± 0.35 μM, respectively, showed excellent inhibitory activity as compared to standard thiourea (IC₅₀ = 21.37 ± 0.26 μM). A kinetic study of compound 2g revealed that compound 2g inhibited urease in a competitive mode. Among the synthesized pyrazolines, the compounds 2c, 2k, 2m, and 2o exhibited excellent α-glucosidase inhibitory activity with the lowest IC₅₀ values of 212.52 ± 1.31, 237.26 ± 1.28, 138.35 ± 1.32, and 114.57 ± 1.35 μM, respectively, as compared to the standard acarbose (IC₅₀ = 375.82 ± 1.76 μM). The compounds (2a-2q) showed α-glucosidase IC₅₀ values in the range of 114.57 ± 1.35 to 462.94 ± 1.23 μM. Structure-activity relationship revealed that the size and electron-donating or -withdrawing effects of substituents influenced the activities, which led to the urease and α-glucosidase inhibiting properties. Compound 2m was a dual potent inhibitor against urease and α-glucosidase due to the presence of 2-CF₃ electron-withdrawing functionality on the phenyl ring. To the best of our knowledge, these synthetic compounds were found to be the most potent dual inhibitors of urease and α-glucosidase with minimum IC₅₀ values. The cytotoxicity of the compounds (2a-2q) was also investigated against human cell lines MCF-7 and HeLa. Compound 2l showed moderate cytotoxic activity against MCF-7 and HeLa cell lines. Moreover, in silico studies on most active compounds were also performed to understand the binding interaction of most active compounds with active sites of urease and α-glucosidase enzymes. Some compounds exhibited drug-like characteristics due to their lower cytotoxic and good ADME profiles.

Figure 1

Some identified representative motifs already reported in the literature.

Figure 2

Structural framework of a versatile heterocycle (1,3,5-triaryl-2-pyrazoline).

Scheme 1. Synthesis of Chalcones (1a–1q) and Pyrazolines (2a–2q)

Scheme 1. Synthesis of Chalcones (1a–1q) and Pyrazolines (2a–2q)

Figure 3

Graphical representation of IC₅₀ values of analogs 2a–2q based on the urease enzyme.

Figure 4

Graphical representation of IC₅₀ values of analogs 2a–2q based on α-glucosidase enzyme.

Figure 5

Enzyme kinetics of analog 2g, (A) Lineweaver Burk plot, (B) secondary replot (Lineweaver Burk), and (C) Dixon plot for the urease inhibition activity. Note: V_max is the maximum velocity of enzymatic activity at a 20 mM inhibitor concentration, K_m is the Michaelis–Menten constant at a 20 mM inhibitor concentration, and K_i (μM) is the value calculated from the Dixon plot.

Figure 6

Impact of the core nucleus and variable substituents on SAR of pyrazolines.

Figure 7

Cytotoxicity of 2l on MCF-7 and HeLa cells after 72 h.

Figure 8

Green stain (annexin-FITC) signifies induction of early apoptosis and red (PI) late apoptosis in MCF-7 cells treated with; (A,B) 2l for 6 and 9 h; (C,D) CP for 6 and 9 h; and (E,F) negative control for 6 and 9 h incubation.

Figure 9

(A–D) Presence of green stain (FITC), which indicates DNA fragmentation in MCF-7 cells treated with: (A) 2l; (B) CP; and (C) negative control (untreated) for 36 h incubation.

Figure 10

Graphical depiction of docking scores (Kcal/mol) of synthesized pyrazolines (2a–2q).

Figure 11

(a) Putative binding interactions of ligand 2c against urease. (b) Interactions of the ligand 2c in 3D space. Interactions with specific amino acid residues are shown in the box. The 3D ribbon represents the enzyme-stick model of the lowest energy conformers of the inhibitor 2c along with amino acids of urease interacting with it.

Figure 12

(a) Putative binding interactions of ligand 2c against α-Glucosidase. (b) Interactions of the ligand 2c in 3D space. Interactions with specific amino acid residues are shown in the box. The 3D ribbon represents the enzyme-stick model of the lowest energy conformers of the inhibitor 2c along with amino acids of α-glucosidase interacting with it.

Figure 13

(a) Putative binding interactions of ligand 2g against Urease. (b) Interactions of the ligand 2g in 3D space. Interactions with specific amino acid residues are shown in the box. The 3D ribbon represents the enzyme-stick model of the lowest energy conformers of the inhibitor 2g along with amino acids of urease interacting with it.

Figure 14

(a) Putative binding interactions of ligand 2o against α-glucosidase. (b) Interactions of the ligand 2o in 3D space. Interactions with specific amino acid residues are shown in the box. The 3D ribbon represents the enzyme-stick model of the lowest energy conformers of the inhibitor 2o along with amino acids of α-glucosidase interacting with it.

Figure 15

Drug-likeness score bar chart.