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.

1. Introduction

According to the contemporary research of drug discovery, enzyme inhibition plays an important role in this field. Urease and α-glucosidase are two imperative enzymes that are meticulously concomitant with various clinical fields.^1,2 Urease (urea amidohydrolase, EC 3.5.1.5, nickel-based) comes from the superfamily of phosphotriesterase and amidohydrolase that initiates the hydrolysis of urea, eventually leading to their conversion into ammonia and carbon dioxide. The urease enzyme is widely spread from prokaryotes to eukaryotes. The urease enzyme’s hyperactivity releases an excessive amount of ammonia causing alkalinity in the stomach, consequently, enhancing the permeability of the gastric mucosa.³⁻¹⁰ Although humans lack the urease enzymes, urea is synthesized as the end product of protein metabolism in them that is usually eliminated from the body in the form of urine.¹¹⁻¹³ Action of urease enzymes in cattle and many other animals also facilitates monitoring and regulating their nitrogen metabolism.^14,15 Different pathogenic conditions can arise due to the elevated levels of these enzymes, especially, they assist in the flourishing of various bacterial infections, leading to some grave adverse effects.¹⁶ The growth of Helicobacter pylori (HP) is eased via the assistance of low pH of the stomach in humans, eventually causing the peptic and gastric ulcers, finally leading to cancer.¹⁷⁻²¹ Moreover, various metabolic dysregulations can arise due to the elevated level of ammonia abolishing the gastrointestinal tract (GIT) epithelium.^22,23 Furthermore, kidney stone formation is also instigated via a high urease level.²⁴

Excessive urease activity is generated in urea fertilization leading to intense environmental pollution ultimately causing major economic losses.²⁵⁻³⁰ This also leads to plant mutilation by controlling the intake of necessary nutrients, enhancing the soil pH and secondary ammonia toxicity.^31,32 Thus, it is pivotal to distinguish new urease inhibitors with enhanced stability, prominent bioavailability, increased selectivity, extraordinary potency, and least toxicity.³³⁻³⁷

Diabetes mellitus (DM) is a prolonged metabolic disorder caused by hyperglycemia.³⁸ Diabetes mellitus metabolic disease gives rise to solemn health disorders, for instance cardiovascular disorders, nephropathy, retinopathy, amputations, neuropathy, cancer, and reduced ability of wound healing. The control of glycemia is an illustrious method to cure diabetes mellitus.³⁹⁻⁴³ α-Glucosidase is a significant enzyme present on the epithelial wall of the small intestine that is involved in carbohydrate metabolism, and the inhibition of this enzyme lowers the glucose level in the blood facilitating the treatment of DM.^44,45 Many detrimental effects such as gastrointestinal diseases, flatulence, and diarrhea are associated with typical available clinical drugs such as voglibose, miglitol, and acarbose.^46,47 The inhibition of α-glucosidase via these inhibitors lowers the absorption of glucose that is released from the pancreas and salivary glands for the conversion of oligosaccharides into simple sugar via hydrolysis, consequently, lowering the postprandial blood glucose level, which is a beneficial approach to cure diabetes.^48,49

Subsequently, because of the use of pyrazoline in the organic field due to its considerable bioavailability, it has always been interesting to design facile, effective, and eco-friendly methodologies for the synthesis of pyrazoline.^50,51

Generally, a combination therapy is recommended for patients suffering from diabetes mellitus and peptic ulcer simultaneously. As diabetic patients show slow healing ability, peptic ulcer can cause various health problems tallying the damage of diabetes mellitus itself. Therefore, the combined urease and α-glucosidase inhibition could be an ideal therapeutic approach for patients who are suffering from both DM and peptic ulcer simultaneously (Figure 1).⁵²⁻⁵⁴

Figure 1.

Some identified representative motifs already reported in the literature.

Despite the important efforts made in the research, the management of malignancies in humans is still a major challenge in contemporary medicinal chemistry. Unfortunately, the significant toxicity exerted by most of the anticancer agents and the development of cellular drug resistance remain the primary hurdle for effective chemotherapy.^55,56 Cancer is the second most fatal disease liable for ∼21% annual deaths globally.⁵⁷ The malignant neoplastic disease is mainly caused by uncontrolled cell growth, metastasis, and invasion. Cancer cells proliferate quickly and their inhibition is most vital within the treatment of cancer exploiting metastatic tumor compounds.⁵⁸ The difficulties in the diagnosis of the disease at the earliest stages, slender therapeutic indices of therapeutic agents, and, therefore, the development of multidrug resistance lead to a number of key obstacles, making cancer treatment complex and causing a high death rate worldwide.^59,60 On the other hand, pyrazole-containing compounds have received considerable attention owing to their diverse chemotherapeutic potential including versatile antineoplastic activities. In addition, it was found that the introduction of a pyrazole nucleus between two aryl rings of chalcones played an integral role in increasing the cytotoxic potential.⁶¹

Recently, pyrazoline has been conferred as one of the most valuable heterocyclic scaffolds in medicinal chemistry on account of its wide spectrum in clinical drugs.⁵⁴ It has been emerged as the most privileged source to design various synthons for drug synthesis (Figure 2). This class contains structure-targeting compounds in different fields such as synthetic and medicinal chemistry exhibiting a good affinity for various biotargets of heterocyclic compounds of this class.⁶²⁻⁶⁴ These scaffolds were utilized in the advancement of drug research and agricultural products.⁶⁵ The pyrazolinic scaffolds also show a variety of applications in pharmacology due to their exceptional antioxidant, anti-inflammatory, antifungal, analgesic, antipyretic, antibacterial, antiangiogenic, antiviral, and antitumoral activities.⁶⁶⁻⁶⁸

Figure 2.

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

Presently, our research group focusing on the designing of facile synthetic approaches of small compounds and exploring their potential biological activity.⁶⁹⁻⁷⁶ In recent years, pyrazolinic compounds have been drawing massive attention of our research group due to their remarkable inhibitory effects against urease and α-glucosidase enzymes.

Encouraged by the biological significance of pyrazoline structural motifs in the medicinal chemistry, herein, we report the synthesis, characterization, and biological evaluation of new pyrazoline derivatives as dual inhibitors of urease and α-glucosidase enzymes. The synthesized pyrazoline compounds with different substitution patterns were evaluated for their urease and α-glucosidase inhibitory activities, and these analogs were assessed, remarkably, to be the potent heterocyclic class among the already reported urease inhibitors. Furthermore, the synthesized compounds were investigated using cytotoxic and molecular modeling studies.

2. Results and Discussion

2.1. Chemistry

The title analogs (2a–2q) were synthesized from easily available and simple precursors as shown in Scheme 1. The first step involves a base-catalyzed condensation of 2′-hydroxyacetophenone with substituted aryl aldehydes in methanol to produce substituted 2′-hydroxychalcones (1a–1q). These intermediate compounds (1a–1q) were characterized by FTIR and NMR spectroscopy and subjected to the next step, which involves the cycloaddition of these intermediate 2′-hydroxychalcones (1a–1q) with phenylhydrazine hydrochloride in the presence of glacial acetic acid to furnish the target compounds (2a–2q) in good to excellent yields. The desired compounds (2a–2q) were purified through recrystallization by ethanol. The structures of novel pyrazoline analogs were elucidated using common spectral techniques such as FTIR, UV–Vis, NMR, and mass spectrometry. For example, the IR spectra of the pyrazoline derivatives depicted the absorption bands around 3357 for the OH group, 3090 for aromatic (C–H), 1592 for (C=N), and 1500 for (C=C). Similarly, the ¹H NMR spectra of these compounds revealed a characteristic singlet signal around δ 12.00 ppm for the OH group. The aromatic protons were observed as multiplets in the region of δ 6.64–7.92 ppm. The presence of other protons in the pyrazoline moiety was observed as a doublet of doublets in the region of δ 2.98–3.04, 3.79–3.86, and 5.37–5.42 ppm corresponding to three protons in HA, HB, and HX patterns, respectively. Moreover, the molecular masses of all the newly synthesized compounds were corroborated by EI-MS, thus all the spectral data unequivocally confirm the proposed structures of the target 1,3,5-triaryl-2-pyrazolines.

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

The spectral data of previously reported 2′-hydroxychalcones (1a–1q) are given in the following reference articles 1a, 1e, 1g, 1j,⁸²1b,⁸³1c,⁸⁴1d,⁸⁵1f,⁸⁶1h,⁸⁷1i,⁸⁸1k,⁸⁹1l,⁹⁰1m, 1n, 1o(91), 1p(92) and 1q.⁹³ The spectral data of all the synthesized pyrazolines (2a–2q) are given here:

2.1.1. 1-Phenyl-3(2-hydroxy-phenyl)-5-(4-flouro-phenyl)-2-pyrazoline (2a)

Colorless solid; mp 145–147 °C; yield: 80%; UV λ_max (MeOH) = 260 nm; FTIR (cm^–1): 3284 (OH), 3012 (C–H), 1548 (C=N), 1493 (C=C), 1245 (C–N), 840 (C–F); ¹H NMR (500 MHz, CDCl₃): δ 10.23 (br s, 1H, OH), 7.98 (d, J = 10.0 Hz, 2H, Ar–H), 7.72 (d, J = 10.0 Hz, 2H, Ar–H), 7.10–7.01 (m, 3H, Ar–H), 6.91–6.80 (m, 6H, Ar–H), 5.10 (dd, J = 15.0, 10.0 Hz, 1H, aliphatic), 3.91 (dd, J = 15.0, 10.0 Hz, 1H, aliphatic), 3.50 (dd, J = 15.0, 10.0 Hz, 1H, aliphatic); ¹³C NMR (126 MHz, CDCl₃): δ 168.4, 164.1, 163.6, 162.8, 146.7, 144.0, 138.0, 129.8, 129.0, 128.5, 127.7, 127.5, 127.1, 126.8, 122.3, 120.7, 118.0, 117.0, 116.7, 116.1, 115.8, 115.3, 66.0, 44.6; accurate mass (EI-MS) of [M]⁺: calcd for C₂₁H₁₇FN₂O, 332.1325; found, 332.1316.

2.1.2. 1-Phenyl-3(2-hydroxy-phenyl)-5-(4-tertbutyl-phenyl)-2-pyrazoline (2b)

Dirty-white crystalline solid; mp 134–136 °C; yield: 75%; UV λ_max (MeOH) = 345 nm; FTIR (cm^–1): 3287 (O–H), 3021 (C–H), 1554 (C=N), 1475 (C=C), 1375 (C–H tertbutyl), 1285 (C–N); ¹H NMR (500 MHz, CDCl₃): δ 13.08 (br s, 1H, OH), 7.42 (d, J = 10.0 Hz, 2H, Ar–H), 7.22 (d, J = 10.0 Hz, 2H, Ar–H), 7.10–7.01 (m, 4H, Ar–H), 6.85–6.76 (m, 5H, Ar–H), 5.22 (dd, J = 15.0, 10.0 Hz, 1H, aliphatic), 3.93 (dd, J = 15.0, 10.0 Hz, 1H, aliphatic), 3.55 (dd, J = 15.0, 10.0 Hz, 1H, aliphatic), 2.50 (d, J = 10.0 Hz, 2H, −CH₂CH(Me)₂), 1.87 (m, 1H, −CH₂CH(Me)₂), 0.93 (d, J = 10.0 Hz, 6H, −CH₂CH(Me)₂); ¹³C NMR (126 MHz, CDCl₃): δ 169.0, 166.8, 148.4, 144.4, 137.6, 133.8, 132.4, 129.6, 128.7, 128.2, 127.6, 126.3, 119.7, 115.9, 114.6, 112.2, 65.3, 45.2, 43.8, 30.1, 22.7; accurate mass (EI-MS) of [M]⁺: calcd for C₂₅H₂₆N₂O, 370.2045; found, 370.2035.

2.1.3. 1-Phenyl-3(2-hydroxy-phenyl)-5-(4-bromo-phenyl)-2-pyrazoline (2c)

Colorless crystalline solid; mp 149–151 °C; yield: 78%; UV λ_max (MeOH) = 322 nm; FTIR (cm^–1): 3295 (OH), 3015 (C–H), 1584 (C=N), 1480 (C=C), 1280 (C–N), 640 (C–F); ¹H NMR (500 MHz, CDCl₃): δ 13.02 (br s, 1H, OH), 7.98 (d, J = 10.0 Hz, 2H, Ar–H), 7.72 (d, J = 10.0 Hz, 2H, Ar–H), 7.10–7.01 (m, 3H, Ar–H), 6.91–6.80 (m, 6H, Ar–H), 5.10 (dd, J = 15.0, 10.0 Hz, 1H, aliphatic), 3.91 (dd, J = 15.0, 10.0 Hz, 1H, aliphatic), 3.52 (dd, J = 15.0, 10.0 Hz, 1H, aliphatic); ¹³C NMR (126 MHz, CDCl₃): δ 167.6, 162.0, 146.8, 144.5, 133.1, 132.4, 129.7, 128.7, 128.5, 127.4, 125.0, 119.7, 118.5, 115.7, 114.2, 67.3, 45.5; accurate mass (EI-MS) of [M]⁺: calcd for C₂₁H₁₇⁷⁹BrN₂O, 392.0524; found, 392.0510.

2.1.4. 1-Phenyl-3(2-hydroxy-phenyl)-5-(3, 4, 5-trimethoxyphenyl)-2-pyrazoline (2d)

Light yellow crystalline solid; mp 142–144 °C; yield: 50%; UV λ_max (MeOH) = 324 nm; FTIR (cm^–1): 3295 (OH), 3015 (C–H), 1584 (C=N), 1480 (C=C), 1295 (C–N), 833 (OCH₃); ¹H NMR (500 MHz, CDCl₃): δ 12.99 (br s, 1H, OH), 7.88 (d, J = 5.0 Hz, 2H, Ar–H), 7.70–7.64 (m, 2H, Ar–H), 7.27–7.28 (m, 3H, Ar–H), 7.00–6.90 (m, 4H, Ar–H), 5.09 (dd, J = 15.0, 10.0 Hz, 1H, aliphatic), 3.90 (dd, J = 15.0, 10.0 Hz, 1H, aliphatic), 3.50 (dd, J = 15.0, 10.0 Hz, 1H, aliphatic), 3.83 (s, 3H, −OCH₃), 3.81 (s, 6H, −OCH₃); ¹³C NMR (126 MHz, CDCl₃): δ 167.5, 164.8, 162.5, 147.7, 143.8, 136.6, 134.2, 129.1, 128.3, 127.8, 126.1, 123.8, 121.7, 118.4, 116.7, 114.3, 67.4, 56.7, 55.6, 44.1; accurate mass (EI-MS) of [M]⁺: calcd for C₂₄H₂₄N₂O₄, 404.1736; found, 404.1740.

2.1.5. 1-Phenyl-3(2-hydroxy-phenyl)-5-(4-methoxyphenyl)-2-pyrazoline (2e)

Light yellow solid; mp 157–159 °C; yield: 73%; UV λ_max (MeOH) = 278 nm; FTIR (cm^–1): 3485 (OH), 3040 (C–H), 1575 (C=N), 1487 (C=C), 1291 (C–N), 855 (OCH₃); ¹H NMR (500 MHz, CDCl₃): δ 7.62 (d, J = 10.0 Hz, 2H, Ar–H), 7.32 (d, J = 10.0 Hz, 2H, Ar–H), 7.05–6.99 (m, 4H, Ar–H), 6.95–6.72 (m, 5H, Ar–H), 5.11 (dd, J = 15.0, 10.0 Hz, 1H, aliphatic), 3.93 (dd, J = 15.0, 10.0 Hz, 1H, aliphatic), 3.53 (dd, J = 15.0, 10.0 Hz, 1H, aliphatic), 3.80 (s, 3H, OMe); ¹³C NMR (126 MHz, CDCl₃): δ 166.7, 162.5, 144.7, 137.5, 132.6, 129.2, 128.8, 127.8, 127.5, 126.5, 121.4, 117.6, 114.3, 65.0, 55.6, 43.8; accurate mass (EI-MS) of [M]⁺: calcd for C₂₂H₂₀N₂O₂, 344.1525; found, 344.1517.

2.1.6. 1-Phenyl-3(2-hydroxy-phenyl)-5-(4-N,N-dimethylphenyl)-2-pyrazoline (2f)

Light yellow crystalline solid; mp 149–151 °C; yield: 55%; UV λ_max (MeOH) = 352 nm; FTIR (cm^–1): 3431 (OH); 1527 (C=N), 1387 (C–N), 750 (N(CH₃)₂); ¹H NMR (500 MHz, CDCl₃): δ 11.98 (br s, 1H, OH), 7.82 (d, J = 10.0 Hz, 2H, Ar–H), 7.10 (d, J = 10.0 Hz, 2H, Ar–H), 7.05–6.98 (m, 3H, Ar–H), 7.05–6.98 (m, 3H, Ar–H), 6.87–6.80 (m, 3H, Ar–H), 5.14 (dd, J = 15.0, 10.0 Hz, 1H, aliphatic), 3.95 (dd, J = 15.0, 10.0 Hz, 1H, aliphatic), 3.80 (s, 6H, −N(CH₃)₂), 3.56 (dd, J = 15.0, 10.0 Hz, 1H, aliphatic); ¹³C NMR (126 MHz, CDCl₃): δ 166.7, 161.8, 147.7, 145.0, 139.0, 136.8, 132.8, 129.6, 128.5, 127.3, 126.1, 124.8, 121.7, 118.5, 116.8, 114.3, 64.1, 55.8, 45.1; accurate mass (EI-MS) of [M]⁺: calcd for C₂₃H₂₃N₃O, 357.1841; found, 357.1830.

2.1.7. 1-Phenyl-3(2-hydroxy-phenyl)-5-(3,4-dimethoxyphenyl)-2-pyrazoline (2g)

White solid; mp 125–127 °C; yield: 76%; UV λ_max (MeOH) = 259 nm; FTIR (cm^–1): 3340 (OH), 2980 (C–H), 1595 (C=N), 1580 (C=C), 876 (OCH₃); ¹H NMR (500 MHz, CDCl₃): δ 10.85 (br s, 1H, OH), 7.89 (dd, J = 10.0, 5.0 Hz, 1H, Ar–H), 7.78 (d, J = 5.0 Hz, 1H, Ar–H), 7.20 (dd, J = 10.0, 5.0 Hz, 1H, Ar–H), 6.95–6.90 (m, 4H, Ar–H), 6.80–6.68 (m, 5H, Ar–H), 5.10 (dd, J = 15.0, 10.0 Hz, 1H, aliphatic), 3.94 (dd, J = 15.0, 10.0 Hz, 1H, aliphatic), 3.86 (s, 3H, −OCH₃), 3.82 (s, 3H, −OCH₃), 3.55 (dd, J = 15.0, 10.0 Hz, 1H, aliphatic); ¹³C NMR (126 MHz, CDCl₃): δ 167.3, 162.6, 146.4, 143.2, 137.3 130.0, 128.8, 127.7, 126.6, 124.8, 122.0, 117.3, 115.1, 114.5, 113.3, 68.0, 55.6, 55.0, 45.5; accurate mass (EI-MS) of [M]⁺: calcd for C₂₃H₂₂N₂O₃, 374.1630; found, 374.1622.

2.1.8. 1-Phenyl-3(2-hydroxy-phenyl)-5-(4-carboxyphenyl)-2-pyrazoline (2h)

Light yellow crystalline solid; mp 129–131 °C; yield: 76%; UV λ_max (MeOH) = 374 nm; FTIR (cm^–1): 3500 (COOH), 3284 (OH), 3010 (C–H), 1548 (C=N), 1493 (C=C), 1245 (C–N); ¹H NMR (500 MHz, CDCl₃): δ 11.6 (br s, 1H, OH), 8.89 (s, 1H, −COOH), 8.15 (d, J = 10.0 Hz, 2H, Ar–H), 7.80 (d, J = 10.0 Hz, 2H, Ar–H), 7.16–7.07 (m, 3H, Ar–H), 6.98–6.90 (m, 4H, Ar–H), 6.78–6.70 (m, 2H, Ar–H) 5.16 (dd, J = 15.0, 10.0 Hz, 1H, aliphatic), 3.94 (dd, J = 15.0, 10.0 Hz, 1H, aliphatic), 3.53 (dd, J = 15.0, 10.0 Hz, 1H, aliphatic); ¹³C NMR (126 MHz, CDCl₃): δ 172.8, 168.1, 162.3, 159.5, 147.0, 142.5, 136.7, 129.2, 128.7, 127.4, 126.5, 124.0, 121.6, 117.3, 116.8, 114.3, 68.5, 47.6; accurate mass (EI-MS) of [M]⁺: calcd for C₂₂H₁₈N₂O₃, 358.1317; found, 358.1306.

2.1.9. 1-Phenyl-3(2-hydroxy-phenyl)-5-thiophene-2-pyrazoline (2i)

Light brown needles; mp 145–147 °C; yield: 81%; UV λ_max (MeOH) = 300 nm; FTIR (cm^–1): 3443.86 (OH); 3012 (CH), 1548 (C=N), 1493 (C=C), 1245 (C–N); ¹H NMR (500 MHz, CDCl₃): δ 12.88 (br s, 1H, OH), 7.98–7.90 (m, 2H, Ar–H), 7.62–7.55 (m, 2H, Ar–H), 7.21–7.13 (m, 2H, Ar–H), 7.11–6.99 (m, 6H, Ar–H), 5.11 (dd, J = 15.0, 10.0 Hz, 1H, aliphatic), 3.95 (dd, J = 15.0, 10.0 Hz, 1H, aliphatic), 3.50 (dd, J = 15.0, 10.0 Hz, 1H, aliphatic); ¹³C NMR (126 MHz, CDCl₃): δ 168.4, 163.8, 159.8, 148.7, 142.0, 136.0, 129.4, 128.6, 127.8, 122.5, 117.5, 116.8, 114.7, 113.2, 67.8, 42.6; accurate mass (EI-MS) of [M]⁺: calcd for C₁₉H₁₆N₂OS, 320.0983; found, 320.0970.

2.1.10. 1-Phenyl-3(2-hydroxy-phenyl)-5-(4-acetamidophenyl)-2-pyrazoline (2j)

Light brown crystals; mp 135 °C; yield: 45%; UV λ_max (MeOH) = 380 nm; FTIR (cm^–1): 3456 (OH), 3006 (CH), 1750 (C=O amide); 1535 (C=N), 1483 (C=C), 1230 (C–N); ¹H NMR (500 MHz, CDCl₃): δ 12.2 (br s, 1H, OH), 10.53 (br s, 1H, NHCOCH₃), 8.80 (d, J = 10.0 Hz, 2H, Ar–H), 8.00 (d, J = 10.0 Hz, 2H, Ar–H), 7.81–7.70 (m, 2H, Ar–H), 7.52–7.47 (m, 2H, Ar–H), 7.08–6.97 (m, 5H, Ar–H), 5.12 (dd, J = 15.0, 10.0 Hz, 1H, aliphatic), 3.94 (dd, J = 15.0, 10.0 Hz, 1H, aliphatic), 3.53 (dd, J = 15.0, 10.0 Hz, 1H, aliphatic), 2.18 (s, 3H, COCH₃); ¹³C NMR (126 MHz, CDCl₃): δ 171.3, 167.6, 162.6, 159.7, 147.5, 143.8, 133.4, 129.6, 128.7, 126.1, 121.7, 117.5, 116.3, 115.8, 114.3, 67.0, 47.6, 25.7; accurate mass (EI-MS) of [M]⁺: calcd for C₂₂H₁₉N₃O₂, 357.1477; found, 357.1480.

2.1.11. 1-Phenyl-3(2-hydroxy-phenyl)-5-(4-N,N diethyl phenyl)-2-pyrazoline (2k)

Pale yellow crystalline solid; mp 149–151 °C; yield: 65%; UV λ_max = 345 nm; FTIR (cm^–1): 3400 (OH), 3017 (C–H), 1530 (C=N), 1489 (C=C), 1235 (C–N); ¹H NMR (500 MHz, CDCl₃): δ 13.80 (s, 1H, OH), 8.10 (d, J = 10.0 Hz, 2H, Ar–H), 7.97 (d, J = 10.0 Hz, 2H, Ar–H), 7.74–7.63 (m, 3H, Ar–H), 7.46–7.40 (m, 2H, Ar–H), 7.01–6.95 (m, 4H, Ar–H), 5.15 (dd, J = 15.0, 10.0 Hz, 1H, aliphatic), 3.95 (dd, J = 15.0, 10.0 Hz, 1H, aliphatic), 3.52 (dd, J = 15.0, 10.0 Hz, 1H, aliphatic), 3.40 (m, 4H, −CH₂CH₃), 1.16, (t, J = 10.0 Hz, 6H, −CH₂CH₃); ¹³C NMR (126 MHz, CDCl₃): δ 166.8, 161.6, 147.8, 139.7, 138.6, 129.1, 128.5, 128.3, 127.2, 126.4, 123.8, 122.0, 118.6, 117.5, 116.8, 116.5, 113.5, 67.1, 48.1, 25.4, 20.6; accurate mass (EI-MS) of [M]⁺: calcd for C₂₅H₂₇N₃O, 385.2154; found, 385.2141.

2.1.12. 1-Phenyl-3(2-hydroxy-phenyl)-5-phenyl-2-pyrazoline (2l)

Yellow solid; mp 107–109 °C; yield: 78%; UV λ_max (MeOH) = 324 nm; FTIR (cm^–1): 3330 (OH), 3006 (C–H), 1535 (C=N), 1483 (C=C), 1230 (C–N); ¹H NMR (500 MHz, CDCl₃): δ 11.10 (br s, 1H, OH), 8.10–7.80 (m, 6H, Ar–H), 7.42–7.26 (m, 5H, Ar–H), 7.05–6.96 (m, 3H, Ar–H), 5.08 (dd, J = 15.0, 10.0 Hz, 1H, aliphatic), 3.90 (dd, J = 15.0, 10.0 Hz, 1H, aliphatic), 3.48 (dd, J = 15.0, 10.0 Hz, 1H, aliphatic); ¹³C NMR (126 MHz, CDCl₃): δ 166.8, 162.2, 146.8, 144.6, 137.9, 129.7, 128.6, 127.8, 127.2, 126.3, 117.4, 116.7, 115.4, 114.8, 113.3, 68.3, 49.0; accurate mass (EI-MS) of [M]⁺: calcd for C₂₁H₁₈N₂O, 314.1419; found, 314.1423.

2.1.13. 1-Phenyl-3(2-hydroxy-phenyl)-5-(2-triflouromethyl-phenyl)-2-pyrazoline (2m)

Colorless crystalline solid; mp 141–143 °C; yield: 63%; UV λ_max (MeOH) = 370 nm; FTIR (cm^–1): 3456.56 (OH), 3019 (C–H), 1550 (C=N), 1454 (C=C), 1276 (C–N), 750 (C–F); ¹H NMR (500 MHz, CDCl₃): δ 12.51 (br s, 1H, OH), 8.72–8.55 (m, 2H, Ar–H), 7.85–7.74 (m, 2H, Ar–H), 7.64–7.50 (m, 4H, Ar–H), 7.32–7.22 (m, 2H, Ar–H), 7.10–6.98 (m, 3H, Ar–H), 5.16 (dd, J = 15.0, 10.0 Hz, 1H, aliphatic), 3.96 (dd, J = 15.0, 10.0 Hz, 1H, aliphatic), 3.56 (dd, J = 15.0, 10.0 Hz, 1H, aliphatic); ¹³C NMR (126 MHz, CDCl₃): δ 168.0, 164.1, 163.7, 162.4, 146.5, 143.8, 139.6, 137.5, 129.3, 128.6, 127.4, 127.6, 127.3, 126.5, 122.6 (q, J = 270.8 Hz, CF₃), 120.6, 118.6, 117.8, 116.3, 115.4, 113.3, 68.8; accurate mass (EI-MS) of [M]⁺: calcd for C₂₂H₁₇F₃N₂O, 382.1293; found, 382.1285.

2.1.14. 1-Phenyl-3(2-hydroxy-phenyl)-5-(3-triflouromethyl phenyl)-2-pyrazoline (2n)

Colorless crystalline solid; mp 145–147 °C; yield: 70%; UV λ_max (MeOH) = 370 nm; FTIR (cm^–1): 3456 (OH), 3019 (C–H), 1550 (C=N), 1454 (C=C), 1276 (C–N), 780 (C–F); ¹H NMR (500 MHz, CDCl₃): δ 12.61 (br s, 1H, OH), 8.55 (s, 1H, Ar–H), 8.48 (dd, J = 10.0, 5.0 Hz, 1H, Ar–H), 8.30 (m, 1H, Ar–H), 8.16 (dd, J = 10.0, 5.0 Hz, 1H, Ar–H), 7.99–7.90 (m, 3H, Ar–H), 7.70–7.52 (m, 3H, Ar–H), 7.01–6.91 (m, 3H, Ar–H), 5.17 (dd, J = 15.0, 10.0 Hz, 1H, aliphatic), 3.96 (dd, J = 15.0, 10.0 Hz, 1H, aliphatic), 3.55 (dd, J = 15.0, 10.0 Hz, 1H, aliphatic); ¹³C NMR (126 MHz, CDCl₃): δ 169.1, 164.7, 163.5, 157.1, 145.2, 142.8, 138.6, 129.7, 128.4, 127.4, 127.7, 126.8, 127.2, 127.0, 122.8 (q, J = 270.8 Hz, CF₃), 118.7, 117.3, 116.2, 115.8, 114.3, 68.0; accurate mass (EI-MS) of [M]⁺: calcd for C₂₂H₁₇F₃N₂O, 382.1293; found, 382.1280.

2.1.15. 1-Phenyl-3(2-hydroxy-phenyl)-5-(4-triflouromethyl phenyl)-2-pyrazoline (2o)

Colorless crystalline solid; mp 139–141 °C; yield: 45%; UV λ_max (MeOH) = 370 nm; FTIR (cm^–1): 3456 (OH), 3019 (C–H), 1550 (C=N), 1454 (C=C), 1276 (C–N), 823 (C–F); ¹H NMR (500 MHz, CDCl₃): δ 13.10 (br s, 1H, OH), 8.51 (d, J = 10.0 Hz, 2H, Ar–H), 8.21 (d, J = 10.0 Hz, 2H, Ar–H), 8.01–7.85 (m, 5H, Ar–H), 7.31–6.92 (m, 4H, Ar–H), 5.16 (dd, J = 15.0, 10.0 Hz, 1H, aliphatic), 3.94 (dd, J = 15.0, 10.0 Hz, 1H, aliphatic), 3.53 (dd, J = 15.0, 10.0 Hz, 1H, aliphatic); ¹³C NMR (126 MHz, CDCl₃): δ 167.8, 164.6, 161.7, 155.6, 146.2, 143.5, 137.8, 129.6, 128.4, 127.7, 127.6, 127.3, 126.8, 122.7 (q, J = 270.8 Hz, CF₃), 119.8, 118.7, 116.6, 116.1, 115.0, 114.3, 68.0; accurate mass (EI-MS) of [M]⁺: calcd for C₂₂H₁₇F₃N₂O, 382.1293; found, 382.1285.

2.1.16. 1-Phenyl-3(2-hydroxy-phenyl)-5(2,4-dimethyl-phenyl)-2-pyrazoline (2p)

Light yellow crystalline solid; mp 153–155 °C; yield: 77%; UV λ_max (MeOH) = 335 nm; FTIR (cm^–1): 3378 (OH), 3022 (C–H), 1521 (C=N), 1453 (C=C), 1267 (C–N); ¹H NMR (500 MHz, CDCl₃): δ 12.54 (br s, 1H, OH), 8.35 (d, J = 5.0 Hz, 1H, Ar–H), 8.10 (dd, J = 10.0, 5.0 Hz, 1H, Ar–H), 7.80–7.72 (m, 4H, Ar–H), 7.52 (d, J = 10.0 Hz, 1H, Ar–H), 7.12–7.01 (m, 3H, Ar–H), 6.98–6.90 (m, 2H, Ar–H), 5.11 (dd, J = 15.0, 10.0 Hz, 1H, aliphatic), 3.89 (dd, J = 15.0, 10.0 Hz, 1H, aliphatic), 3.47 (dd, J = 15.0, 10.0 Hz, 1H, aliphatic), 2.40 (s, 3H, −CH₃), 2.35 (s, 3H, −CH₃); ¹³C NMR (126 MHz, CDCl₃): δ 166.8, 163.8, 158.7, 146.2, 143.6, 137.6, 129.5, 128.6, 127.6, 127.4, 125.2, 121.8, 117.7, 115.1, 112.3, 67.3, 44.8, 22.2, 21.5; accurate mass (EI-MS) of [M]⁺: calcd for C₂₃H₂₂N₂O, 342.1732; found, 342.1721.

2.1.17. 1-Phenyl-3(2-hydroxy-phenyl)-5(4-benzonitrile)-2-pyrazoline (2q)

Yellow crystalline solid; mp 154–156 °C; yield: 80%; UV λ_max (MeOH) = 346 nm; FTIR (cm^–1): 3406 (OH), 3087 (C–H), 2259 (C=N), 1550 (C=N), 1454 (C=C), 1276 (C–N); ¹H NMR (500 MHz, CDCl₃): δ 13.01 (br s, 1H, OH), 8.56 (d, J = 10.0 Hz, 2H, Ar–H), 8.28 (d, J = 10.0 Hz, 2H, Ar–H), 8.11–7.98 (m, 3H, Ar–H), 7.42–7.10 (m, 4H, Ar–H), 7.05–6.97 (m, 2H, Ar–H), 5.18 (dd, J = 15.0, 10.0 Hz, 1H, aliphatic), 3.97 (dd, J = 15.0, 10.0 Hz, 1H, aliphatic), 3.55 (dd, J = 15.0, 10.0 Hz, 1H, aliphatic); ¹³C NMR (126 MHz, CDCl₃): δ 169.0, 163.4, 161.8, 146.8, 142.5, 129.7, 128.4, 127.6, 127.6, 126.3, 123.7, 119.8, 117.3, 116.7, 115.6, 114.2, 68.8, 45.6; accurate mass (EI-MS) of [M]⁺: calcd for C₂₂H₁₇N₃O, 339.1372; found, 339.1375.

2.2. Biological Activities

2.2.1. Urease Inhibitory Activity

All synthesized pyrazoline analogs (2a–2q) were evaluated for their in vitro urease inhibitory activity. It is noteworthy that except compound 2i, the rest of the derivatives were found to be significant urease inhibitors with IC₅₀ values in the range of 9.13 ± 0.25 to 18.42 ± 0.42 μM as compared to the standard thiourea (IC₅₀ = 21.37 ± 0.26 μM) (Table 1). The varying activities of synthetic derivatives might be due to the varied substituents on aromatic rings. 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 activities due to the isobutyl, (−OCH₃)₂, −CF₃, and −CN groups. Excitingly, analog 2m bearing a −CF₃ group on the phenyl ring at the second position exhibited most potent activity having an IC₅₀ value of 9.18 ± 0.35 μM compared to compounds 2n and 2o having −a CF₃ group on the phenyl ring at the third and fourth positions having IC₅₀ values of 18.42 ± 0.42 and 12.63 ± 0.56 μM, respectively. All these analogs showed excellent activity as compared to the standard thiourea (IC₅₀ = 21.37 ± 0.26 μM). The substitution pattern of −F and −OCH₃ functionalities at the aryl ring in compounds 2a and 2e is the crucial factor of enzyme inhibition of these compounds. The derivatives with electron-donating groups (EDGs) attached to the phenyl ring have a better urease inhibitory activity than the analogs with electron-withdrawing groups (EWGs) attached to phenyl rings (Table 1) and (Figure 3).

Table 1. Urease and α-Glucosidase Inhibitory Activities of Pyrazolines (2a–2q).

compound no.	urease IC50 ± SEM (μM)	α-glucosidase IC50 ± SEM (μM)
2a	13.54 ± 0.34	445.83 ± 1.25
2b	9.36 ± 0.27	462.94 ± 1.23
2c	12.57 ± 0.43	212.52 ± 1.31
2d	13.28 ± 0.25	421.34 ± 1.27
2e	15.39 ± 0.38	384.73 ± 1.45
2f	15.57 ± 0.42	407.28 ± 1.24
2g	9.13 ± 0.25	419.45 ± 1.32
2h	11.26 ± 0.51	NA
2i	29.51 ± 0.35	NA
2j	13.67 ± 0.47	NA
2k	13.29 ± 0.25	237.26 ± 1.28
2l	NA*a	457.82 ± 1.25
2m	9.18 ± 0.35	138.35 ± 1.32
2n	18.42 ± 0.42	434.26 ± 1.29
2o	12.63 ± 0.56	114.57 ± 1.35
2p	12.24 ± 0.38	NA
2q	9.35 ± 0.35	NA
thiourea (standard)	21.37 ± 0.26
acarbose (standard)		375.82 ± 1.76

^aNA* = no activity.

Figure 3.

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

2.2.2. α-Glucosidase Inhibitory Activity

In continuation to our previous studies on α-glucosidase,^73c pyrazolines (2a–2q) were assessed for their antidiabetic activity as described in the Method section. All the tested compounds (2a–2q) showed moderate to good inhibition activity against the α-glucosidase enzyme. The table below illustrates the IC₅₀ values of the tested compounds and reference acarbose (IC₅₀ = 375.82 ± 1.76 μM). The compounds (2a–2q) showed IC₅₀ values in the range of 114.57 ± 1.35 to 462.94 ± 1.23 μM (Table 1 and Figure 4). 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. Therefore, compounds 2c, 2k, 2m, and 2o bearing −Br, −N(C₂H₅)₂, 2-CF₃, and 4-CF₃, respectively, could be recommended as potential drug candidates for the treatment of diabetes mellitus in the future.

Figure 4.

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

Overall, it is concluded that among the synthesized pyrazoline series, compound 2m is a dual potent inhibitor against urease and α-glucosidase due to the presence of 2-CF₃ electron-withdrawing functionality on the phenyl ring.

2.3. Kinetic Study

The mechanism of inhibition was further examined by a complete kinetic analysis of the most powerful derivative 2g (urease inhibitor) at various concentrations (0–20 μM) and substrates (0.5–4.0 mM). Enzymatic kinetics were used to establish the mode of inhibition and the inhibition constant (K_i). Lineweaver Burk plots used to determine the inhibition mode by measuring the kinetics of most active compound 2g are presented in Figure 5. To calculate K_i values, Lineweaver Burk secondary plots were drawn between the slope of each line and different inhibitor concentrations. To confirm the K_i value of the inhibitor, Dixon plots were constructed between the reciprocal of the rate of reaction and different concentrations of inhibitors. It was determined from kinetic study that 2g was a competitive inhibitor with a K_i value of 19.11 μM, a K_m value of 0.93 mM, and a V_max value of 5.76 μM/min at a 20 μM inhibitor concentration.

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.

2.4. Structure–Activity Relationship Based on Urease, α-Glucosidase Inhibition Assay

All the synthetic analogs (2a–2q) were investigated for their urease and α-glucosidase inhibitory activities (in vitro). Structure–activity relationship (SAR) studies were carried out purely based on the central core and substitution patterns on the pyrazoline scaffold. Based on the SAR, it can be asserted that the variations observed in urease and α-glucosidase inhibitory activities of 1,3,5-triaryl-2-pyrazoline analogs (2a–2q) were owing to different substituents. Limited SAR was established based on the substitution pattern on the pyrazoline scaffold and are accountable for influencing bioactivities (Figure 6).

Figure 6.

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

In this regard, it is noteworthy that compounds 2c (IC₅₀ = 12.57 ± 0.43 μM for urease and 212.52 ± 1.31 μM for α-glucosidase), 2k (IC₅₀ = 13.29 ± 0.25 μM for urease and 237.26 ± 1.28 μM for α-glucosidase), 2m (IC₅₀ = 9.18 ± 0.35 μM for urease and 138.35 ± 1.32 μM for α-glucosidase), and 2o (IC₅₀ = 12.63 ± 0.56 μM for urease and 114.57 ± 1.35 μM for α-glucosidase) were found to be the most potent dual inhibitors among the members of the series. All these synthetic analogs showed even higher activity than the standards thiourea (IC₅₀ = 21.37 ± 0.26 μM) and acarbose (IC₅₀ = 375.82 ± 1.76 μM). Compounds 2c, 2k, 2m, and 2o have various EDGs and EWGs at o- and p-positions of the aryl ring. The analogs 2m and 2o possess an inductively strong EWG, trifluoromethyl (−CF₃) group, at the o-position and the p-position of the phenyl ring. The highest inhibition by 2m and 2o may be attributed to their position and strong interactions with the active pocket of the enzyme. The analog 2c bears an EWG (−Br) at the p-position on ring B. It seems that this atom is also suitably positioned on the ring to foster favorable interaction with the active pocket of the enzyme. The analog 2k has a −N(C₂H₅)₂ group present on the pyrazoline framework and is accountable for enhanced inhibitory activity due to excellent interaction with the active pockets of the enzyme. Almost certainly, this functional group fits effectively into the large active pocket of the enzyme.

Interestingly, the next most potent inhibitor is compound 2g (IC₅₀ = 9.13 ± 0.25 μM for urease) among the remaining analogs of the series. It is also more active than the standards, thiourea and acarbose. This compound 2g has methoxy at position-3 and -4 on ring B of the pyrazoline carbon skeleton and thus was found to be a highly potent urease and glucosidase inhibitor as well. The high inhibition potential might be due to the electronegativity of one methoxy group at 3rd and 4th positions of the phenyl ring, which suitably interacts with the enzyme active site.

Subsequently, the next synthetic analog with a comparable IC₅₀ value is compound 2q (IC₅₀ = 9.35 ± 0.35 μM). This compound also showed a strong inhibitory activity against the urease enzyme than the reference standard inhibitor thiourea. This might be a result of the strong chelation of benzonitrile moiety derivative 2q with the active electrophilic center of the urease enzyme.

Furthermore, the replacement of the aromatic ring with other heterocyclic rings such as thiophene in compound 2i (IC₅₀ = 29.51 ± 0.35 μM) showed a decreased inhibition activity as compared to the standard (Table 1). The urease inhibition by compound 2i relied on the ability of a sulfur atom to coordinate within the active site of the urease enzyme. Afterward, the next compounds manifesting inhibitory activities are 2h (IC₅₀ = 11.26 ± 0.51 μM) and 2j (IC₅₀ = 13.67 ± 0.47 μM), where the aromatic ring B is substituted at position-4 with −COOH and acetamido (NHCOCH₃) groups, respectively. Interestingly, the highly hydrophilic p-substituted analogs 2h and 2j exhibited excellent activity against urease.

Additionally, compounds 2a (IC₅₀ = 13.54 ± 0.34 μM for urease and 445.83 ± 1.25 μM for α-glucosidase), 2b (IC₅₀ = 9.36 ± 0.27 μM for urease and 462.94 ± 1.23 μM for α-glucosidase), 2d (IC₅₀ = 13.28 ± 0.25 μM for urease and 421.34 ± 1.27 μM for α-glucosidase), 2e (IC₅₀ = 15.39 ± 0.38 μM for urease and 384.73 ± 1.45 μM for α-glucosidase), 2f (IC₅₀ = 15.57 ± 0.42 μM for urease and 407.28 ± 1.24 μM for α-glucosidase), 2g (IC₅₀ = 9.13 ± 0.25 μM for urease and 419.45 ± 1.32 μM for α-glucosidase), 2l (IC₅₀ = 457.82 ± 1.25 μM for α-glucosidase), 2n (IC₅₀ = 18.42 ± 0.42 μM for urease and 434.26 ± 1.29 μM for α-glucosidase), and 2p (IC₅₀ = 12.24 ± 0.38 μM for urease) were found to be more active against the envisioned enzyme urease and less active against α-glucosidase. These findings reflect that various EWGs and EDGs (i.e., −F, −isobutyl, −(OCH₃)₃, p-OCH₃, −N(CH₃)₂, −(OCH₃)₂, −(OCH₃)₂, phenyl, m-CF₃, and −(CH₃)₂) on the aryl ring are accountable for their excellent inhibitory activities because of increased interaction with the urease enzyme. Moreover, these findings also suggest that highly hydrophilic and hydrophobic (i.e., −F, −isobutyl, −(OCH₃)₃, p-OCH₃, −N(CH₃)₂, −(OCH₃)₂, −(OCH₃)₂, phenyl, m-CF₃, and −(CH₃)₂) groups on the aromatic ring are accountable for low inhibitory activities because of reduced favorable interactions with the active site of the target enzyme (α-glucosidase) due to conformational, steric, and electronic factors. Comparatively, the synthesized derivatives (2a–2q) are more active against urease as compared to the α-glucosidase enzyme.

Overall, it should be noted that these studies reveal that the nature, position, and number of substituents on the aryl ring greatly influence the activities of these analogs. In addition, it is evident from the results that bulky and polar functional groups present at the p-position on the phenyl ring play an essential role in controlling the inhibitory potential of the pyrazoline scaffold against the tested enzymes.

2.5. Cytotoxicity in Treated Cell Lines

The cytotoxic effects of 2a–2q on human breast carcinoma, MCF-7 and cervical cancer, HeLa cell lines were investigated. There was a dose-dependent increase in cytotoxicity of 2l on both cancer cell lines as shown in Figure 7. Interestingly, MCF-7 was shown to be more sensitive as compared to HeLa cells after treatment with 2l having EC₅₀ of 120 μM/mL and EC₅₀ of 510 μM/mL, respectively. Similarly, 2k exhibited a lower EC₅₀ (250 μM/mL) on MCF-7 as compared to HeLa cell (EC₅₀ 500 μM/mL). A similar cytotoxic effect was observed on MCF-7 and HeLa cells after treatment with 2a–2j and 2m–2q, where both cell lines possessed EC₅₀ > 500 as shown in Table 2. Furthermore, 2l is more sensitive toward MCF-7 cells as compared to the capecitabine (CP) drug with an EC₅₀ of 980 μM/mL. MCF-7 was more sensitive toward 2l as compared to HeLa cells in our studies. Therefore, 2l was selected for MCF cells to further investigate the mode of cell death (apoptosis).

Figure 7.

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

Table 2. EC₅₀ Values of 1–17 (μM/m).

	EC50
compound no.	MCF-7	HeLa
2a	>500	>500
2b	>500	>500
2c	>500	>500
2d	>500	>500
2e	>500	>500
2f	>500	>500
2g	>500	>500
2h	>500	>500
2i	>500	>500
2j	>500	>500
2k	250 ± 0.09	>500
2l	120 ± 0.10	510
2m	>500	>500
2n	>500	>500
2o	>500	>500
2p	>500	>500
2q	>500	>500

2.5.1. Apoptosis Study (Early Apoptosis)

In order to determine the mode of MCF-7 cell death, Annexin V-FITC was used. MCF-7 cells were treated with 2l at a concentration of EC₅₀ at 72 h for 6 and 9 h along with positive (CP) and negative control (without treatment). 2l was shown to induce early apoptosis by exposure of phosphoserine (PS) from inward to outward of the plasma membrane (PM) of cancer cell, which was detectable in cells by the green stain of Annexin-FITC after 6 h incubation treatment. Remarkably, both early (Annexin-FITC V⁺) and late apoptosis propidium iodide positive (PI⁺) cells were visible after 9 h. Besides, most of the cell death occurred due to apoptosis (AnnexinV⁺/PI⁺) with very few cells undergoing necrosis (Annexin V^–/PI⁺) as shown in Figure 8. Apoptosis (programmed cell death) is a highly regulated mechanism noticeable by various morphological features of cell such as cytoplasmic shrinkage and reduction of cell size, permeabilization of the PM, nuclear condensation, and DNA fragmentation.⁹⁴ Externalization of the PS protein to the outer leaflet of a cell membrane is among the early steps in the induction of apoptosis, which provides a signal to immune cells, probably phagocytes to further engulfment of apoptotic cells.⁹⁵ Excitingly, the results revealed the cytotoxic effects of 2l by apoptosis in the MCF-7 cell line.

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.

2.5.2. Apoptosis Study (Late Apoptosis)

In order to further validate the cytotoxicity by induction of apoptosis in the MCF-7 cell line, a fluorescence terminal nick end labeling of DNA (TUNEL) detection system was used. The in situ fluorometric determination of DNA fragmentation via TUNEL is a sensitive and accurate method of detection of apoptosis in cancer cells. Rows A and B in Figure 9 present the cells treated with 2l and CP alone, respectively, at a concentration of EC₅₀ of 72 h. The nuclei of MCF-7 cells treated with 2l were morphologically detectable (green) due to the polymerization reaction of labeled nucleotides by a transferase enzyme (TdT) at the site of fragmented DNA after 36 h incubation treatment. Accordingly, DNA fragmentation is considered as a hallmark of apoptosis, which also plays a major role in the detection of apoptosis in cancer cells.⁹⁶ Also, green-stained nuclei were observed in CP-treated cells. However, nuclei of negative control (no treatment) and the DNA of MCF-7 cells remain intact and the lack of green stain is noticeable under an Image Xpress Micro XLS Widefield High-Content Analysis System (HCS) (Sunnyvale, USA).

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.

2.6. In Silico Studies

2.6.1. Docking Analysis

ChemDraw was used to create 2D structures of compounds (2a-2q), which were then transformed into 3D using Chem Pro3D software. The ligands were docked to the protein using AutoDock 4.2.0 after being energetically minimized using Merck Molecular Force Field (MMFF). Table 3 and Figure 10 show the docking results, with just the best conformers and dock scores for each ligand. The ligand with the lowest docking score will produce the most stable drug–receptor combination. Hydrogen bonding, hydrophobic type interactions, and van der Waal’s interactions were investigated in the drug–receptor complexes of several powerful ligands.

Table 3. Binding Energies (Kcal mol^–1) of Synthesized Analogs (2a–2q) against Urease and α-Glucosidase.

	docking study against urease		docking study against α-glucosidase
compound no.	lowest binding energy (Kcal mol–1)	mean binding energy (Kcal mol–1)	lowest binding energy (Kcal mol–1)	mean binding energy (Kcal mol–1)
2a	–7.98	–7.63	–6.68	–6.14
2b	–8.74	–7.70	–7.20	–6.59
2c	–8.65	–8.59	–7.29	–6.64
2d	–8.24	–7.51	–6.31	–5.72
2e	–8.23	–7.87	–6.78	–6.16
2f	–8.47	–8.00	–6.78	–6.16
2g	–9.16	–8.31	–6.59	–5.95
2h	–8.40	–7.81	–6.25	–5.59
2i	–7.81	–7.49	–7.19	–6.35
2j	–8.68	–8.15	–7.58	–6.79
2k	–8.41	–7.86	–6.65	–6.05
2l	–7.85	–7.38	–7.67	–6.69
2m	–7.74	–7.14	–6.53	–6.04
2n	–7.74	–7.32	–6.51	–5.99
2o	–7.78	–7.33	–7.48	–5.91
2p	–7.65	–7.14	–6.57	–5.93
2q	–8.32	–7.96	–7.35	–7.35
standard	–8.31 (AHA)	–8.11 (AHA)	–7.98 (MIG)	–7.61 (MIG)

Figure 10.

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

Root mean square deviation (RMSD) is used to assess the validity of docking research. To calculate the RMSD value, the cocrystalline ligand was removed from the active pockets of the selected substrate and redocked. The docked poses of AHA and MIG overlapped with the poses in the X-ray crystal structure (PDBs: 4H9M and 5NN6) at RMSD values of 0.6 and 0.8 Å, respectively, which validates the docking process.

2c, an important ligand (lowest binding energy, −8.65 Kcal mol^-1), expressed promising inhibitory potential against the urease enzyme. This ligand 2c developed conventional hydrogen bonding with the His593 amino acid residue of biological-targeted moiety 4H9M. In addition to hydrogen bonding, this ligand 2c also built up several important interactions such as hydrophobic π-alkyl and electrostatic π-anion with Met637, Asp633, Ala636, Ala436, and Arg439 inside the active pockets of under study protein 4H9M as shown in Figure 11a,b.

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.

In the same fashion, this ligand 2c (lowest binding energy −7.29 Kcal mol^–1) is also a potential inhibitor of glucosidase enzyme. This ligand 2c performed its action by building conventional hydrogen bonds with Arg600 and Asp616 amino acid residues of the targeted biological specimen 5NN6. Trp481, Trp376, Phe649, and Met519 amino acid residues also developed hydrophobic π–π T-shaped, hydrophobic π–π stacked, and hydrophobic alkyl type associations inside the active pockets of under study molecule 5NN6. The inhibitory effect of this ligand also increases due to the electrostatic π anion interaction against Asp282 and Asp518 inside 5NN6 as shown in Figure 12a,b.

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.

Another potent ligand 2g (lowest binding energy −9.16 Kcal mol^–1) reveals its inhibitory potential against urease enzyme via various intermolecular attractions. This ligand 2g exhibits hydrogen bonding with Arg609, Arg439, and Gln635 amino acid residues of under study protein 4H9M. This ligand also undergoes electrostatic π anion type interactions with Asp633 of 4H9M. Ala636, Arg439, Ala440, and His492 amino acid residues of 4H9M protein associate with ligand 2g via hydrophobic alkyl, hydrophobic π alkyl, hydrophobic π–sigma, and hydrophobic π–π T-shaped type interactions as shown in Figure 13a,b.

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.

Similarly, ligand 2o (lowest binding energy −7.48 Kcal mol^–1) is a potent inhibitor against the glucosidase enzyme. The flouro group of the ligand 2o involves in hydrogen bonding with Ser523 of 5NN6. The hydroxyl group of the ligand 2o also undergoes hydrogen bonding with Arg600 and Asp616 amino acid residues of the targeted molecule 5NN6. Aromatic delocalized π electronic cloud of the ligand 2o develops electrostatic π cation, electrostatic π anion, and hydrophobic π–π stacked type associations with Arg600, Asp282, Trp376, and Asp518 amino acid residues of targeted biological specimen 5NN6. Met519 amino acid of the 5NN6 protein builds up hydrophobic alkyl and hydrophobic π alkyl type interactions with ligand 2o as shown in Figures 14a,b.

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.

2.6.2. Drug-Likeness Study

The expected chemoinformatics properties, for instance, solubility, polarizability, and polar surface area were evaluated computationally. Already reported data were used to establish a standard value for the number of atoms (20–70) and molar molecular weight (160–480).^73c,75 Inferences have shown that expected values of 2c, 2g, and 2o are better than those of all other synthesized compounds as well as standard values. Furthermore, the Lipinski’s rule of five (RO5) does not state anything about structural features or specific chemistry present in drugs or non-drugs. The computational results predicted that 2c, 2g, and 2o possess 2, 4, and 2 hydrogen bond acceptor (HBA) (≤10), 1, 1, and 1 hydrogen bond donor (HBD) (≤5), 0.42, 2.23, and 0.40 mg/L Log S, and PSA 30.47, 45.73, and 30.47 A² (≤120 A²) values, respectively, which considerably indicated their drug-like behavior with drug-like model scores 0.01, 0.27, and −0.32. Besides, their molecular weights (392.05, 374.16, and 382.13 g/mol, respectively) were also better than the standard value (Table 4 and Figure 15). These findings illustrate that many derivatives show drug-like effects without violating any of the rules regarding their medicinal potential.

Table 4. Physicochemical Properties of Synthesized Pyrazoline Analogs (2a–2q).

compound no.	mol. formula	mol. wt (g/mol)	no. HBAa	no. HBDb	mol. Log Pc	mol. Log Sd (mg/L)	mol. PSAe A2	drug-likeness model score
2a	C21H17FN2O	332.13	2	1	5.79	0.65	30.47	0.19
2b	C25H26N2O	370.20	2	1	7.49	0.45	30.47	0.96
2c	C21H17BrN2O	392.05	2	1	6.56	0.42	30.47	0.01
2d	C24H24N2O4	404.17	5	1	5.72	1.63	53.53	0.42
2e	C22H20N2O2	344.15	3	1	5.68	1.27	38.01	0.24
2f	C23H23N3O	357.18	2	1	5.79	1.02	33.27	0.08
2g	C23H22N2O3	374.16	4	1	5.40	2.23	45.73	0.27
2h	C22H18N2O3	358.13	4	2	5.53	2.13	58.88	0.41
2i	C19H16N2OS	320.10	3	1	5.25	8.50	31.49	–0.36
2j	C22H19N3O2	357.15	3	3	4.37	4.05	64.53	0.57
2k	C25H27N3O	385.22	2	1	6.53	0.51	33.20	0.23
2l	C21H18N2O	314.14	2	1	5.73	0.76	30.47	–0.45
2m	C22H17F3N2O	382.13	2	1	6.55	0.46	30.47	–0.69
2n	C22H17F3N2O	382.13	2	1	6.60	0.30	30.47	–0.44
2o	C22H17F3N2O	382.13	2	1	6.67	0.40	30.47	–0.32
2p	C23H22N2O	342.17	2	1	6.82	0.49	30.47	–0.06
2q	C22H17N3O	339.14	3	1	5.33	6.10	47.52	–0.29

^aThe table above depicts all of Lipinski’s RO5 components, that is, the number of HBAs.

^bNumber of HBDs.

^cOctanol–water partition coefficient.

^dMeasured solubility.

^eTotal polar surface area.

Figure 15.

Drug-likeness score bar chart.

3. Conclusions

In conclusion, we have synthesized a series of 1,3,5-triaryl-2-pyrazoline derivatives using precedent methodologies and evaluated their inhibitory potential against urease and α-glucosidase. Their cytotoxic activities were also investigated. The synthesized compounds showed varying degrees of urease inhibitory activity ranging from 9.13 ± 0.25 to 18.42 ± 0.42 μM. Among the series of compounds, the compound 2g exhibited the lowest IC₅₀ value (9.13 ± 0.25 μM) against urease, and other compounds in the series also showed excellent antiurease activity as compared to standard thiourea (IC₅₀ = 21.37 ± 0.26 μM). Compounds 2a, 2b, 2c, 2d, 2e, 2f, 2h, 2j, 2k, 2m, 2n, 2o, 2p, and 2q having IC₅₀ values of 13.54 ± 0.34, 9.36 ± 0.27, 12.57 ± 0.43, 13.28 ± 0.25, 15.39 ± 0.38, 15.57 ± 0.42, 11.26 ± 0.51, 13.67 ± 0.47, 13.29 ± 0.25, 9.18 ± 0.35, 18.42 ± 0.42, 12.63 ± 0.56, 12.24 ± 0.38, and 9.35 ± 0.35 μM, respectively, showed excellent inhibitory activity as compared to standard thiourea (IC₅₀ = 21.37 ± 0.26 μM). The inhibition mechanism of biologically assayed compound 2g (competitive inhibition) was investigated by Lineweaver Burk and Dixon plots to determine the V_max, K_m, and K_i values. The compounds (2a–2q) also exhibited α-glucosidase IC₅₀ values in the range of 114.57 ± 1.35 to 462.94 ± 1.23 μM. Furthermore, the compounds 2c, 2k, 2m, and 2o exhibited excellent α-glucosidase inhibitory activity with the lowest IC₅₀ = 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). Additionally, compound 2m acts as a potential dual inhibitor against urease and α-glucosidase. Exploring the urease and α-glucosidase inhibiting activities of the synthesized pyrazolines, we herein report that pyrazolines are potent dual urease and α-glucosidase inhibitors. Moreover, the cytotoxicity studies were performed and findings revealed that compound 2l displayed moderate and selective anticancer activity against MCF-7 and HeLa cell lines compared to CP. It is noteworthy to mention that the synthesized compounds are comparatively more active against urease than the α-glucosidase enzyme. In addition, compound 2l significantly induced apoptosis in both cell lines. The SAR study established that the nature, as well as the position of varying groups attached to the aryl group, played crucial roles in defining the urease and α-glucosidase inhibition activities. The molecular docking results agreed with the in vitro biological assay data. Pharmacological studies revealed that synthesized analogs 2a–2q obey Lipinski’s rule. Drug-likeness parameter assessment showed that these analogs show considerable lead-like characteristics with the least toxicity and can serve as templates in drug designing.

4. Experimental Section

4.1. Materials and Methods

All the chemicals and solvents were purchased from Merck and Sigma-Aldrich and were utilized as received. The melting points were determined on an electrothermal digital instrument, which are uncorrected. The IR spectra were recorded on a Bio-Rad spectrophotometer. The NMR spectra were obtained using a Bruker spectrometer (¹H, 500 MHz, ¹³C, 126 MHz). NMR chemical shift values were defined in δ (ppm) units. TLC was used to check/monitor reaction progress and completion and spots were visualized under a UV lamp (254 nm). The QUARTZ cell was used to record the absorption spectra in ethanol on the Jasco UV-VIS V-660 instrument. Accurate mass measurements were carried out with the Fisons VG sector-field instrument (EI) and a FT-ICR mass spectrometer.

4.2. General Procedures for the Syntheses of Chalcones (1a–1q) and 1,3,5-Triaryl-2-pyrazolines (2a–2q)

In the first step, chalcones were synthesized by Claisen–Schmidt condensation according to the literature procedure.^75,76 A mixture of 2′-hydroxyacetophenone (1.0 mmol) and aqueous NaOH solution (30%, 5.0 mL) was dissolved in distilled methanol (15 mL) and stirred for 30 min at ambient temperature, followed by the dropwise addition of aromatic aldehyde (1.0 mmol). Reactions were monitored by TLC using ethyl acetate/n-hexane (1:3) as a solvent system. After the reaction completion (checked by TLC), the reaction content was poured onto the ice-water and neutralized by HCl (10%). The product obtained was filtered, washed with cold water, and was recrystallized from ethanol to obtain the purified compounds (1a–1q) in good yields. In the second step, pyrazolines were synthesized according to the literature.⁷⁷ Appropriate chalcones (1 mmol) were dissolved in glacial acetic acid. To this mixture, phenylhydrazine HCl (1 mmol) was added and the reaction mixture was refluxed for 16–18 h. The reaction was monitored by TLC using ethyl acetate/n-hexane (1:3) as a solvent system. After the reaction completion, the reaction mixture was then poured onto crushed ice accompanied by vigorous stirring. It was left at room temperature to obtain a crystalline compound. The obtained solid was filtered and recrystallized from ethanol to obtain a pure product (2a–2q) in good yields.

4.3. Enzyme Inhibition Assay

4.3.1. Urease Inhibitory Assay

A reported method was used to perform the urease inhibitory potential of newly synthesized pyrazoline derivatives (2a–2q).^1,78 Reaction mixtures containing 55 μL buffer solution comprising of 100 mM urea and 25 μL enzyme solution were incubated with 5 μL newly synthesized compounds (1 mM concentration) at 30 °C in 96-well plates for 15 min. The production of ammonia was determined via utilizing the indophenol procedure to analyze the activity of urease. Momentarily, both 70 μL alkali reagent (0.5% w/v NaOH and 0.1% active chloride NaOCl) and 45 μL phenol reagent (1% w/v phenol and 0.005% w/v sodium nitroprusside) were added to each well. The enhanced absorbance was measured at 630 nm via utilizing a microplate reader (Molecular Device, USA) after 50 min. All steps were performed three times in a final volume of 200 μL. The final measurements (change in absorbance per min) were obtained via utilizing the SoftMax Pro software (Molecular Device, USA) for the abstraction of the results. Assays were performed at pH 8.2 (0.01 M K₂HPO₄·3H₂O, 1 mM EDTA, and 0.01 M LiCl). Acetohydroxamic acid was used as the standard inhibitor of urease.

Percentage of inhibition was calculated from the following formula

4.3.2. α-Glucosidase Inhibitory Assay

The α-glucosidase inhibitory activity of newly designed derivatives of pyrazolines (2a–2q) was determined by following the already reported procedure with minor amendments.^73c 250 mL of acarbose at different concentrations (250–1000 mg/mL) was incubated with 500 mL of 1.0 U/mL of α-glucosidase solution in 100 mM phosphate buffer (pH 6.8) for 20 min at 37 °C. 250 mL of 4-nitrophenyl-b-d-glucopyranoside solution and 250 mL of 1% starch⁴² dissolved in 100 mM phosphate buffer (pH 6.8), respectively, were then added to the reaction mixture and incubated for 1 h at 37 °C.²⁰ Then, 1.0 mL of the 3,5-dinitrosalicylic acid coloring reagent was added to the reaction mixture and it was boiled for 10 min. The absorbance of the final reaction mixture was determined for α-glucosidase at 405 nm. The preparation of blank was carried out for correcting absorbance. Acarbose solution was utilized as positive control. The inhibitory activity was stated as a percentage of a control sample without the inhibitors.⁴⁷

4.4. Kinetic Study

The kinetics of the binding mechanism of most dominant compound 2g (inhibitor), which has an IC₅₀ value comparable with thiourea (standard), was investigated. Five different inhibitor concentrations (0–20 μM) were reacted with different substrate concentrations (urea, 0.5–4.0 mM) to determine whether the inhibitor is competitive, noncompetitive (mixed), and uncompetitive after calculating/assessing the K_m (app) V_max (app) values from the Lineweaver Burk plot, and the K_i values (inhibition constant) were also determined by the Dixon plot using PRISM 7.0.⁷⁹

4.5. Cytotoxicity Study

Culture ware and other plastic consumables were purchased from Nunc, Denmark. Cancer cell lines were purchased from American Type Cell Culture, ATCC, USA. The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) reagent was obtained from thermofisher scientific, USA, ApoAlert Annexin V, Clontech, USA, DeadEnd Fluorometric TUNEL Promega, USA. All required chemicals and cell culture reagents were obtained from Sigma-Aldrich, USA.

4.5.1. Cell Culturing

The human cell lines MCF-7 (human breast adenocarcinoma) and HeLa (cervical cancer) were grown in RPMI 1640 medium (Gibco Life Technologies) containing 10% fetal calf serum (FCS; Gibco) and 1% antibiotics (100 IU/mL penicillin and 100 μg/mL streptomycin) in a humidified incubator at 37 °C in 5% CO₂ and 95% air.

4.6. Cytotoxicity Studies

4.6.1. Cell Viability in Treated Cell Lines (MTT Assay)

Cytotoxicity study was carried out using the MTT assay.⁸⁰ The cell viability was determined by reading the absorbance of the metabolized product (purple formazan) of the MTT reagent by living cells. The cells were plated in a 96-well plate at a density of 6000 cells/well and incubated at 37 °C in a CO₂ incubator for 24 h. Next, the old medium was replaced with fresh media containing treatments at given concentrations (0, 15.62, 31.25, 62.5, 125, 250, 500, and 1000 μM/mL). After 72 h of incubation, 20 μL of MTT (5 mg/mL) was added to each well and incubated for 4 h. Subsequently, after removal of media containing treatments, 100 μL of DMSO was added to each well to dissolve crystals of formazan. Using an Elisa reader, the absorbance was measured at 570 nm (Multiskan, Thermo fisher Scientific USA). The treatment was repeated three times, with the results calculated as percentage of growth inhibition values based on the mean of two independent values (SEM). The EC₅₀ (half effective inhibitory concentration) value was calculated by Graphpad Prism 7 using a non-linear regression model (curve fit) based on a sigmoidal dose–response curve (variable) (GraphPad, San Diego, California, USA).

4.6.2. Mode of Cell Death Study

4.6.2.1. Determination of Early Apoptosis in Treated Cells

The apoptotic effect of the cytotoxic crystal 2l (having lower EC₅₀ value) on the MCF-7 was evaluated according to the previously reported method.⁸¹ Briefly, MCF-7 cells were plated at a cell density of 10,000 cells/well and incubated at 37 °C for 24 h. After 24 h, the medium was removed and replaced with fresh media containing 2l at a concentration of EC₅₀ along with positive (cisplatin) and negative controls (without treatment). Next, the cells were incubated in 200 μL of binding buffer containing Annexin V-FITC (green) and PI (red) for 10 min at 37 °C. The cells were observed under an Image Xpress Micro XLS Widefield High-Content Analysis System (HCS) (Sunnyvale, USA).

4.6.2.2. Determination of Late Apoptosis in Treated Cells

A TUNEL assay was carried out using the DeadEnd Fluorometric TUNEL system (Promega, USA) for investigating in situ DNA fragmentation occurring in treated cells. The assay was performed according to the manufacturer’s protocol. Briefly, The MCF-7 cells were plated in Labtek Chamber Slides (Nunc, Denmark) with a density of 2 × 10⁴ and incubated at 37 °C for 24 h. Next, both treated and control (without treatment) cells were fixed by 4% paraformaldehyde solution in PBS (pH 7.4) for 25 min at 4 °C. Subsequently, the cells were washed with phosphate buffer saline (PBS) for 5 min. After this, fixed cells were permeabilized for 5 min using 0.2% triton X-100 in PBS. Then, the permeabilized cells were washed with PBS; each step of washing was done thrice for 5 min. The nick ends of DNA or fragmented ends were labeled by adding a 50 μL reaction mixture, containing equilibration Buffer, Nucleotide Mix, and enzyme rTdT in a ratio and incubated in the dark at 37 °C. 20× SSC (1:10) was diluted with deionized water and added enough to fill a standard chamber. Additionally, propidium iodide (PI) solution was freshly prepared (1 μg/mL in PBS) and added to stain the nuclear DNA for 15 min at room temperature in the dark. The Image Xpress Micro XLS Widefield High-Content Analysis System (HCS) (Sunnyvale, USA) was used to detect the green fluorescence of FITC-labeled apoptotic cells.

4.7. Molecular Modeling Assay

AutoDock 4.2.0 was applied in order to study the protein–ligand associations. The X-ray crystallographic structure of Jack bean urease (JBU) (in complex with acetohydroxamic acid, AHA) (PDB ID: 4H9M) and α-glucosidase (in complex with MIG ((2R,3R,4R,5S)-1-(2-hydroxyethyl)-2-(hydroxymethyl)piperidine-3,4,5-triol) (PDB ID: 5NN6, species Homo sapiens) (www.rcsb.org) was obtained, from RCSB protein data bank (PDB), as a model. 3D grids of AHA and MIG in the binding pocket of urease and α-glucosidase were estimated by using discovery studio 4.0. The protein structure was improved, and side-chain hydrogens were added.^73a,73b

4.8. Drug-Likeness Study

The Molsoft tool has been utilized to determine the drug-like characteristics of the predicted compounds. Lipinski’s RO5 was used to establish a framework for assessing the bioavailability and pharmacokinetics of a given molecule when administered orally, using a set of physiochemical parameters.^73c,75

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.1c06694.

• FTIR spectra of some of the synthesized compounds and ¹H NMR and ¹³C NMR spectra of the some of the synthesized compounds (PDF)

Author Contributions

R.M. contributed in experiments and first-draft preparation; A.S. contributed in main idea, supervision, and final writing of the manuscript; R.I.A. contributed in experiments and first-draft preparation; E.U.M. contributed in main idea, cosupervision, and final writing of the manuscript; M.A.A. contributed in first-draft preparation and data analysis and collection; N.N. contributed in first-draft preparation, data analysis and collection, and drug-likeness study; A.J. contributed in docking studies; M.M.A.-R. contributed in first-draft preparation and data analysis and collection; G.-S.C. performed enzyme inhibition and cytotoxicity studies; and S.A.A. contributed in main idea, cosupervision, and final writing of the manuscript.

The authors declare no competing financial interest.