Highly efficient, catalyst-free, one-pot sequential four-component synthesis of novel spiroindolinone-pyrazole scaffolds as anti-Alzheimer agents: in silico study and biological screening

Abstract

Alzheimer's disease is a neurodegenerative disorder that impacts memory, thinking, and behavior, and currently, there is no effective cure available for its treatment. This study explored a one-pot strategy for synthesizing spiroindolinone-pyrazole derivatives through a sequential four-component condensation reaction. These derivatives were further investigated for their potential as anti-Alzheimer's disease agents. The developed synthetic procedure provides remarkable advantages, including a clean reaction profile, abundant starting materials, operational simplicity, and easy purification without traditional methods with good to excellent yields (84–96%). Next, the biological potencies of the newly synthesized spiroindolinone-pyrazole derivatives against AChE and BChE as Alzheimer's disease-related targets were determined. Also, the kinetic study and cytotoxicity of the most potent derivative were investigated. Furthermore, molecular docking and molecular dynamics evaluations were performed employing in silico tools to investigate the interaction, orientation, and conformation of the potent analog over the active site of the enzyme.

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1. Introduction

Alzheimer's disease (AD) is the most common cause of dementia, especially in the elder population, which causes memory and cognitive impairments with behavioral abnormalities. AD is estimated to affect 115.4 million people worldwide by 2050, which requires urgent attention.^1,2

The pathogenesis of AD is still inconclusive and undefined, and different proposed mechanisms, including amyloid beta (Aβ), hyperphosphorylation of T-protein, protein deposits, oxidative stress, and metal dyshomeostasis, were defined. The consistent loss of cholinergic neurons and a considerable decline in choline acetyltransferase activity are the most prominent phenotypes of AD progression.^3,4 As a result, cholinergic system dysfunction happens with synaptic damage accompanied by neuronal loss in the brain, particularly in the hippocampus and cerebral cortex.⁵

Acetylcholine (ACh) is known as one of the main neurotransmitters in the central nervous system (CNS), synthesized from acetyl coenzyme A and choline by the enzyme choline acetyltransferase. It regulates many processes, including memory, learning, attention, and behavior.⁶ In detail, ACh exerts excitatory effects at the neuromuscular junction by binding to either nicotinic or muscarinic ACh receptors, thereby initiating its action. The termination of ACh's activity within the synaptic cleft is facilitated by two primary cholinesterases (ChEs), namely acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). These enzymes hydrolyze ACh into choline and acetate, halting synaptic transmission.^7,8

The loss of cholinergic neurons and the reduction of ACh in patients with AD make AChE and BChE key therapeutic targets to modify the condition of the patients. It was reported that as the disease progresses, AChE activity decreases and BChE activity increases, making it an ideal target for the late stage of the disease.⁷ Approved ChE inhibitors are tacrine (withdrawal), donepezil, rivastigmine, galantamine, and huperzine A (approved by CFDA). However, these drugs are associated with severe side effects, including nausea, vomiting, diarrhea, muscle weakness, and weight loss. As a result, excessive efforts have been made to design novel Che inhibitors.⁹

Multicomponent reactions (MCRs) are versatile protocols in which multiple reactions are combined in one synthetic operation and are widely used to synthesize a variety of complex heterocycles and natural products.¹⁰ These reactions are characterized by convergence, ease of execution, efficiency, reducing waste, high yields, and atom economy.^11,12 With the rising interest in generating heterocyclic compound libraries, exploring fresh multicomponent and synthetic reactions for academic and industrial research is an engaging research endeavor.

Pyrazolines are one of the important classes of nitrogen heterocyclic compounds, and their analogs are of great importance in medicinal chemistry.^13–15 They are one of the groups of studied compounds among the azole family. Indeed, various synthesis methods and synthetic analogs have been reported over the years. The presence of the pyrazole core in different structures leads to various applications in different fields such as technology, agriculture, and medicine. These kinds of compounds possess a broad spectrum of biological activity, such as anticancer,¹⁶ antimicrobial,¹⁷ anti-inflammatory,¹⁸ anticonvulsant¹⁹ antitumor,²⁰ and antiviral²¹ activities.

Isatin, also known as 1H-indole-2,3-dione, was initially synthesized by Erdmann and Laurent through the oxidation of indigo using nitric acid and/or chromic acid.^22,23 Isatin and its derivatives occur naturally in certain plants like the Isatis genus, Calanthe discolor LINDL,²⁴ and Couroupita guianensis Aubl.²⁵ It has also been studied in the parotid gland of Bufo frogs.²⁶ In humans, isatin is a metabolic byproduct of adrenaline.^27–29 Isatin and its derivatives are fascinating and versatile building blocks widely used to synthesize important and bioactive heterocyclic compounds.^30–35

The spirooxindole framework^36,37 is a significant structural arrangement found in various bioactive natural compounds, including horsfiline, coerulescine, spirotryprostatin A, welwitindolinone A, elacomine, and alstonisine. Spirotryprostatin A, a natural alkaloid obtained from the fermentation broth of Aspergillus fumigatus, has been recognized as a new inhibitor of microtubule assembly. Additionally, pteropodine and isopteropodine have demonstrated the ability to regulate the function of muscarinic serotonin receptors^38–40 (Fig. 1).

Fig. 1 Representative structure of spiroxindole derivatives.

The significance of the indole core in medicinal and organic chemistry has led to a heightened focus on synthesizing oxo-indole derivatives. Various methods for crafting spiroxindole derivatives have been documented.^41–44

Most of these synthetic routes can be categorized based on the following key reactions: (1) Mannich/Pictet–Spengler;^45,46 (2) 1,3-dipolar cycloaddition;^47,48 (3) Morita–Baylis–Hillman,^49,50 intermolecular alkylation;^51,52 (4) electrocyclization;⁵³ (5) sigmatropic rearrangement reactions.^54,55 For instance, Hongyu Guo and coworkers reported the synthesis of spirooxindole-pyrazolines and pyrazolones via P(NMe₂)₃-mediated substrate-controlled annulations of azoalkenes with α-dicarbonyl compounds.⁵⁶ Assem Barakat and coworkers reported a new series of functionalized spirooxindoles linked with the 3-acylindole scaffold in methanol solvent.⁵⁷ Alizadeh et al. have also reported the synthesis of these compounds using isatin derivatives, BMTN, ammonia, 1,3-dicarbonyl, and p-TSA as catalysts in water.¹⁹

In continuation of our research on multicomponent reaction and synthesis of spirooxindole derivatives and also considering the possible biological effects of pyrazoline and spiroxindole parts in an organic compound, synthesizing a series of new spiropyrazoline derivatives can be useful. In line with our ongoing interest in synthesizing biologically active heterocyclic compounds as anti-AD agents, we present a concise and efficient method for synthesizing novel spiro pyrazoline derivatives. This one-pot, sequential four-component synthesis involves the reaction of 1,1-bis(methylthio)-2-nitroethylene (BMTNE), arylamines, hydrazine hydrate, and isatine derivatives, all under catalyst-free conditions in EtOH at reflux. Next, all the derivatives were screened for their anti-AChE and BuChE activities. A kinetic study was also performed on the most potent compound against the respective enzyme. Molecular docking and molecular dynamics simulations were carried out on the active analogs to gain insights into the binding interactions. Furthermore, the neurotoxicity of these compounds was evaluated against the SH-SY5Y neuroblastoma cell line.

2. Experimental section

2.1 General

All chemicals were purchased from Merck or Fuluka chemical companies. ¹H-NMR 300 MHz and ¹³C-NMR (75 MHz) spectra were run on a Bruker Avance 300 MHz instrument in DMSO-d₆. A Unicom Galaxy Series FT-IR 4600 spectrophotometer was used to obtain infra-red spectra. Melting points were recorded on a Buchi B-545 apparatus in open capillary tubes. Reaction progress was screened by TLC using silica gel polygram SIL G/UV254 plates.

2.2 General procedure for the synthesis of compounds 5a–q

Initially, to prepare N-aryl-1-(methylthio)-2-nitroethenamine 3a, a mixture of BMTNE 1 (1 mmol) and arylamine 2 (1 mmol) was stirred in ethanol (5 mL) at reflux for 12 h. After that, isatin derivatives 4 (1 mmol) and NH₂NH₂ (80% aq.) (1.2 mmol) were added and were heated at reflux. After completion of the reaction (monitored by TLC analysis), the crude solid formed, was filtered and washed with ethanol (5 ml) to give the pure product 5.

4′-Nitro-5′-(o-tolylamino)-1′,2′-dihydrospiro[indoline-3,3′-pyrazol]-2-one (5a). Orange powder, yield: 89%, m.p: 240–242 °C; (TLC; hexane–EtOAc, 1 : 4, R_f = 0.272); IR (KBr): 3359, 3286, 3055, 2973, 1714, 1641, 1619, 1598, 1477, 1355, 1226, 1199, 1108, 1077, 871, 630; ¹H NMR (300 MHz, DMSO-d₆) δ: 10.60 (1H, s, NH), 9.90 (1H, s, NH), 7.64–7.52 (2H, m, Ar), 7.42–7.28 (3H, m, Ar), 7.22 (1H, td, J = 7.7, 1.2 Hz, Ar), 6.80 (1H, d, J = 7.8 Hz, Ar), 6.68 (1H, t, J = 7.6 Hz, Ar), 5.95 (2H, s, NH), 2.32 (3H, s, Me); ¹³C NMR (75 MHz, DMSO-d₆) δ: 165.7, 159.0, 146.1, 143.7, 136.6, 133.5, 132.0, 131.1, 127.4, 127.1, 126.7, 126.4, 121.8, 117.6, 110.4, 71.6, 18.2; anal. calcd for C₁₇H₁₅N₅O₃: C, 60.53; H, 4.48; N, 20.76. Found: C, 60.88; H, 4.64; N, 20.93.

4′-Nitro-5′-(p-tolylamino)-1′,2′-dihydrospiro[indoline-3,3′-pyrazol]-2-one (5b). Orange powder, yield: 91%, m.p: 178–180 °C; (TLC; hexane–EtOAc, 1 : 4, R_f = 0.269); IR (KBr): 3326, 3026, 3162, 1722, 1644, 1573, 1475, 1353, 1299, 1255, 1203, 1153, 1106, 877, 815, 626. ¹H NMR (300 MHz, DMSO-d₆) δ: 10.69 (1H, s, NH), 10.36 (1H, s, NH), 8.24 (1H, d, J = 7.5 Hz, Ar), 7.72 (2H, d, J = 8.2 Hz, Ar), 7.35–7.28 (3H, m, Ar), 6.97 (1H, t, J = 7.6 Hz, Ar), 6.88 (1H, d, J = 7.7 Hz, Ar), 5.89 (2H, s, NH), 2.35 (3H, s, Me); ¹³C NMR (75 MHz, DMSO-d₆) δ: 165.7, 157.4, 145.7, 144.0, 136.4, 134.4, 132.4, 129.9, 126.6, 122.2, 121.5, 117.4, 110.7, 72.8, 21.0.

5′-((4-Hydroxyphenyl)amino)-4′-nitro-1′,2′-dihydrospiro[indoline-3,3′-pyrazol]-2-one (5c). Orange powder, yield: 91%, m.p: 201–203 °C; (TLC; hexane–EtOAc, 1 : 4, R_f = 0.265); IR (KBr): 3289, 3261, 3170, 3077, 2952, 1716, 1635, 1590, 1514, 1494, 1427, 1259, 1106, 1018, 941, 854, 800, 713; ¹H NMR (300 MHz, DMSO-d₆) δ: 13.42 (1H, s, OH), 11.27 (1H, s, NH), 10.86 (1H, s, NH), 8.22 (1H, d, J = 7.6 Hz, Ar), 7.45–735 (3H, m, Ar), 7.10 (1H, t, J = 7.6 Hz, Ar), 6.93 (2H, dd, J = 15.7, 7.7 Hz, Ar), 6.98–6.75 (3H, m, Ar), 6.17 (2H, s, NH); ¹³C NMR (75 MHz, DMSO-d₆) δ: 163.0, 159.3, 151.7, 145.5, 142.9, 135.9, 134.3, 132.1, 129.6, 122.9, 122.5, 119.9, 117.1, 111.7, 74.9; anal. calcd for C₁₆H₁₃N₅O₄: C, 56.64; H, 3.86; N, 20.64. Found: C, 56.23; H, 3.65; N, 20.85.

5′-((4-Methoxyphenyl)amino)-4′-nitro-1′,2′-dihydrospiro[indoline-3,3′-pyrazol]-2-one (5d). Orange powder, yield: 93%, m.p: 215–217 °C; (TLC; hexane–EtOAc, 1 : 4, R_f = 0.265); IR (KBr): 3293, 3218, 3075, 2971, 1716, 1645, 1508, 1477, 1334, 1218, 1108, 1068, 900, 815, 732; ¹H NMR (300 MHz, DMSO-d₆) δ: 10.65 (1H, s, NH), 10.32 (1H, s, NH), 8.19 (1H, dd, J = 7.7, 1.3 Hz, Ar), 7.78–7.67 (2H, m, Ar), 7.31 (1H, td, J = 7.7, 1.3 Hz, Ar), 7.10–7.05 (2H, m, Ar), 6.97 (1H, td, J = 7.6, 1.0 Hz, Ar), 6.86 (1H, d, J = 7.7 Hz, Ar), 5.86 (2H, s, NH), 3.82 (3H, s, OMe); ¹³C NMR (76 MHz, DMSO-d₆) δ: 165.7, 157.5, 156.8, 145.3, 143.9, 132.2, 131.8, 126.7, 123.3, 122.2, 117.5, 114.6, 110.6, 72.7, 55.8; anal. calcd for C₁₇H₁₅N₅O₄: C, 57.79; H, 4.28; N, 19.82. Found: C, 57.85; H, 4.10; N, 19.88. Found: C, 57.53; H, 4.10; N, 19.66.

5′-((3,4-Dimethylphenyl)amino)-4′-nitro-1′,2′-dihydrospiro[indoline-3,3′-pyrazol]-2-one (5e). Orange powder, yield: 90%, m.p: 202–204 °C; (TLC; hexane–EtOAc, 1 : 4, R_f = 0.268); IR (KBr): 3322, 3265, 3162, 3027, 1725, 1644, 1299, 1260, 1205, 1106, 813, 688, 625; ¹H NMR (300 MHz, DMSO-d₆) δ: 10.68 (1H, s, NH), 10.32 (1H, s, NH), 8.32 (1H, dd, J = 7.7, 1.2 Hz, Ar), 7.83 (1H, d, J = 2.3 Hz, Ar), 7.43–7.27 (2H, m, Ar), 7.23 (1H, d, J = 8.2 Hz, Ar), 6.96 (1H, td, J = 7.6, 1.0 Hz, Ar), 6.88 (1H, d, J = 7.8 Hz, Ar), 5.88 (2H, s, NH), 2.31 (3H, s, Me), 2.27 (3H, s, Me); ¹³C NMR (75 MHz, DMSO-d₆) δ: 165.7, 157.6, 145.6, 144.0, 137.2, 136.6, 133.2, 132.4, 130.3, 126.5, 122.7, 122.0, 118.9, 117.4, 110.7, 72.8, 19.9, 19.4; anal. calcd for C₁₈H₁₇N₅O₃: C, 61.53; H, 4.88; N, 19.93. Found: C, 61.04; H, 4.56; N, 20.21.

5′-((3-Chlorophenyl)amino)-4′-nitro-1′,2′-dihydrospiro[indoline-3,3′-pyrazol]-2-one (5f). Yellow powder, yield: 89%, m.p: 202–204 °C; (TLC; hexane–EtOAc, 1 : 4, R_f = 0.272); IR (KBr): 3361, 3288, 3170, 3075, 2977, 1722, 1640, 1600, 1523, 1482, 1336, 1228, 1130, 1073, 918, 835, 634; ¹H NMR (300 MHz, DMSO-d₆) δ: 10.70 (1H, s, NH), 10.40 (1H, s, NH), 8.24 (1H, d, J = 7.6 Hz, Ar), 7.81 (1H, s, Ar), 7.50 (2H, t, J = 7.8 Hz, Ar), 7.32 (1H, td, J = 7.7, 1.3 Hz, Ar), 7.24 (1H, t, J = 7.4 Hz, Ar), 6.95 (1H, t, J = 7.6 Hz, Ar), 6.88 (1H, d, J = 7.8 Hz, Ar), 5.90 (2H, s, NH); ¹³C NMR (75 MHz, DMSO-d₆) δ: 165.6, 157.5, 146.1, 144.1, 138.9, 132.6, 129.5, 126.7, 125.1, 122.2, 121.6, 117.3, 110.8, 72.8; anal. calcd for C₁₆H₁₂ClN₅O₃: C, 53.72; H, 3.38; N, 19.58. Found: C, 53.49; H, 3.45; N, 19.25.

5′-((4-Chlorophenyl)amino)-4′-nitro-1′,2′-dihydrospiro[indoline-3,3′-pyrazol]-2-one (5g). Orange powder, yield: 92%, m.p: 220–222 °C; (TLC; hexane–EtOAc, 1 : 4, R_f = 0.269); IR (KBr): 3307, 3261, 3120, 3062, 2970, 1735, 1644, 1604, 1508, 1477,1388, 1245, 1216, 1184, 1066, 905, 845, 777, 628; ¹H NMR (300 MHz, DMSO-d₆) δ: 10.71 (1H, s, NH), 10.48 (1H, s, NH), 8.17 (1H, dd, J = 7.6, 1.2 Hz, Ar), 7.90–7.82 (2H, m, Ar), 7.60–7.54 (2H, m, Ar), 7.34 (1H, td, J = 7.7, 1.3 Hz, Ar), 7.01 (1H, td, J = 7.7, 1.1 Hz, Ar), 6.88 (1H, d, J = 7.7 Hz, Ar), 5.88 (2H, s, NH); ¹³C NMR (75 MHz, DMSO-d₆) δ: 165.5, 157.2, 146.5, 144.2, 137.9, 132.7, 129.4, 128.7, 126.7, 123.0, 122.4, 117.2, 110.8, 62.5.

5-Methoxy-5′-((4-methoxyphenyl)amino)-4′-nitro-1′,2′-dihydrospiro[indoline-3,3′-pyrazol]-2-one (5h). Brown powder, yield: 96%, m.p: 215–217 °C; (TLC; hexane–EtOAc, 1 : 4, R_f = 0.255); IR (KBr): 3313, 3253, 3080, 2992, 1722, 1633, 1513, 1426, 1344, 1261, 1072, 850, 808; ¹H NMR (300 MHz, DMSO-d₆) δ: 10.47 (1H, s, NH), 10.27 (1H, s, NH), 7.53 (2H, d, J = 8.1 Hz, Ar), 7.25–7.19 (2H, m, Ar), 6.98 (1H, d, J = 2.4 Hz, Ar), 6.81–6.66 (2H, m, Ar), 5.68 (2H, s, NH), 3.88 (3H, s, OMe), 3.74 (3H, s, OMe); ¹³C NMR (76 MHz, DMSO-d₆) δ: 177.7, 161.3, 155.6, 153.3, 150.6, 136.0, 135.0, 131.9, 124.7, 118.6, 116.7, 112.8, 110.7, 109.6, 60.6, 56.0, 55.9; anal. calcd for C₁₈H₁₇N₅O₅: C, 56.39; H, 4.47; N, 18.27. Found: C, 56.69; H, 4.41; N, 18.63.

5′-((3,4-Dimethylphenyl)amino)-5-methoxy-4′-nitro-1′,2′-dihydrospiro[indoline-3,3′-pyrazol]-2-one (5i). Brown powder, yield: 95%, m.p: 215–217 °C; (TLC; hexane–EtOAc, 1 : 4, R_f = 0.262); IR (KBr): 3345, 3288, 3166, 3096, 2992, 1722, 1644, 1608, 1504, 1357, 1292, 1236, 1201, 1139, 1020, 730, 568; ¹H NMR (300 MHz, DMSO-d₆) δ: 10.46 (1H, s, NH), 10.28 (1H, s, NH), 7.88 (1H, d, J = 2.6 Hz, Ar), 7.61 (1H, s, Ar), 7.48 (1H, dd, J = 8.1, 2.3 Hz, Ar), 7.20 (1H, d, J = 8.1 Hz, Ar), 6.91 (1H, dd, J = 8.5, 2.7 Hz, Ar), 6.77 (1H, d, J = 8.4 Hz, Ar), 5.87 (2H, s, NH), 3.59 (3H, s, OMe), 2.28 (3H, s, Me), 2.25 (3H, s, Me); ¹³C NMR (75 MHz, DMSO-d₆) δ: 165.8, 157.7, 155.0, 145.8, 137.6, 137.4, 136.5, 133.4, 130.4, 122.9, 119.3, 118.0, 117.8, 112.9, 111.0, 72.7, 55.9, 19.8, 19.3; anal. calcd for C₁₉H₁₉N₅O₄: C, 59.84; H, 5.02; N, 18.36. Found: C, 59.21; H, 4.97; N, 18.66.

4′,5-Dinitro-5′-(o-tolylamino)-1′,2′-dihydrospiro[indoline-3,3′-pyrazol]-2-one (5j). Brown powder, yield: 88%, m.p: 223–225 °C; (TLC; hexane–EtOAc, 1:4, R_f = 0.270); ¹H NMR (300 MHz, DMSO-d₆) δ: 11.30 (1H, s, NH), 10.25 (1H, s, NH), 8.48 (1H, s, Ar), 8.17 (1H, dd, J = 8.7, 2.5 Hz, Ar), 7.64–7.55 (1H, m, Ar), 7.51–7.25 (3H, m, Ar), 6.98 (1H, d, J = 8.6 Hz, Ar), 5.99 (2H, s, NH), 2.35 (3H, s, Me); ¹³C NMR (75 MHz, DMSO-d₆) δ: 165.9, 160.5, 148.9, 143.1, 142.4, 135.9, 132.9, 131.5, 128.1, 127.4, 127.1, 125.9, 122.1, 117.2, 110.6, 63.5, 18.2; anal. calcd for C₁₇H₁₄N₆O₅: C, 53.40; H, 3.69; N, 21.98. Found: C, 54.11; H, 3.77; N, 22.28.

5′-((2,5-Dimethoxyphenyl)amino)-4′,5-dinitro-1′,2′-dihydrospiro[indoline-3,3′-pyrazol]-2-one (5k). Brown powder, yield: 89%, m.p: 210–212 °C; (TLC; hexane–EtOAc, 1 : 4, R_f = 0.272); IR (KBr): 3396, 3360, 3268, 1741, 1646, 1506, 1479, 1335, 1191, 1106, 1066, 898, 644; ¹H NMR (300 MHz, DMSO-d₆) δ: 11.35 (1H, s, NH), 10.10 (1H, s, NH), 8.92 (1H, d, J = 2.5 Hz, Ar), 8.22 (1H, dd, J = 8.7, 2.5 Hz, Ar), 7.73 (1H, d, J = 3.0 Hz, Ar), 7.14 (1H, d, J = 9.1 Hz, Ar), 7.03 (1H, d, J = 8.6 Hz, Ar), 6.87 (1H, dd, J = 9.0, 3.1 Hz, Ar), 6.04 (2H, s, NH), 3.88 (3H, s, OMe), 3.62 (3H, s, OMe); ¹³C NMR (75 MHz, DMSO-d₆) δ: 165.8, 159.2, 153.6, 149.0, 145.3, 143.5, 142.4, 128.4, 127.0, 121.9, 117.2, 113.1, 111.4, 110.8, 110.6, 72.3, 56.1, 55.5; anal. calcd for C₁₈H₁₆N₆O₇: C, 50.47; H, 3.76; N, 19.62. Found: C, 50.68; H, 3.97; N, 19.21.

5′-((4-Chlorophenyl)amino)-4′,5-dinitro-1′,2′-dihydrospiro[indoline-3,3′-pyrazol]-2-one (5l). Brown powder, yield: 86%, m.p: 224–226 °C; (TLC; hexane–EtOAc, 1 : 4, R_f = 0.270); IR (KBr): 3343, 3282, 3143, 2923, 1716, 1644, 1504, 1349, 1191, 1016, 887, 757; ¹H NMR (300 MHz, DMSO) δ: 11.40 (1H, s, NH), 10.82 (1H, s, NH), 9.00 (1H, d, J = 2.4 Hz, Ar), 8.29–8.21 (1H, m, Ar), 7.91–7.80 (2H, m, Ar), 7.60–7.51 (2H, m, Ar), 7.04 (1H, d, J = 8.7 Hz, Ar), 5.92 (2H, s, NH); ¹³C NMR (75 MHz, DMSO) δ: 165.7, 158.9, 149.4, 143.9, 142.5, 137.3, 129.7, 129.3, 128.7, 123.4, 121.8, 116.8, 110.9, 72.9; anal. calcd for C₁₆H₁₁ClN₆O₅: C, 47.71; H, 2.75; N, 20.87. Found: 48.09; H, 2.62; N, 20.51.

4′-Nitro-5′-(o-tolylamino)-7-(trifluoromethyl)-1′,2′-dihydrospiro[indoline-3,3′-pyrazol]-2-one (5m). Yellow powder, yield: 84%, m.p: 214–216 °C; (TLC; hexane–EtOAc, 1 : 4, R_f = 0.270); IR (KBr): 3315, 3289, 3162, 3052, 2960, 1712, 1641, 1550, 1348, 1297, 1249, 1114, 1031, 890, 817; ¹H NMR (300 MHz, DMSO-d₆) δ: 11.09 (1H, s, NH), 10.21 (1H, s, NH), 7.84 (1H, d, J = 7.6 Hz, Ar), 7.54–7.46 (2H, m, Ar), 7.44–7.31 (3H, m, Ar), 6.83 (1H, t, J = 7.8 Hz, Ar), 5.98 (2H, s, NH), 2.31 (3H, s, Me); ¹³C NMR (75 MHz, DMSO-d₆) δ: 166.0, 160.1, 143.4, 140.5, 140.5, 136.4, 133.7, 131.2, 130.6, 127.5, 126.9, 126.6, 121.8, 119.0, 111.5, 111.0, 71.6, 18.1; anal. calcd for C₁₈H₁₄F₃N₅O₃: C, 53.34; H, 3.48; N, 17.28. Found: C, 52.89; H, 3.30; N, 17.13.

5′-((3-Chlorophenyl)amino)-4′-nitro-7-(trifluoromethyl)-1′,2′-dihydrospiro[indoline-3,3′-pyrazol]-2-one (5n). Yellow powder, yield: 85%, m.p: 191–193 °C; (TLC; hexane–EtOAc, 1 : 4, R_f = 0.262); IR (KBr): 3396, 3366, 3265, 1731, 1646, 1508, 1475, 1332, 1191, 1108, 1066, 890, 634; ¹H NMR (300 MHz, DMSO-d₆) δ: 11.23 (1H, s, NH), 10.77 (1H, s, NH), 8.53 (1H, d, J = 7.6 Hz, Ar), 8.24 (1H, t, J = 2.0 Hz, Ar), 7.64 (1H, d, J = 8.1 Hz, Ar), 7.58–7.46 (2H, m, Ar), 7.34 (1H, dt, J = 7.2, 1.9 Hz, Ar), 7.16 (1H, t, J = 7.8 Hz, Ar), 5.92 (2H, s, NH); ¹³C NMR (75 MHz, DMSO-d₆) δ: 165.8, 158.5, 152.3, 144.2, 141.3, 140.0, 138.1, 133.8, 131.3, 129.8, 127.1, 125.9, 125.0, 121.4, 120.1, 118.6, 72.7; anal. calcd for C₁₇H₁₁ClF₃N₅O₃: C, 47.96; H, 2.60; N, 16.45; O, 11.27. Found: C, 48.55; H, 2.78; N, 16.79.

5′-(Naphthalen-1-ylamino)-4′-nitro-7-(trifluoromethyl)-1′,2′-dihydrospiro[indoline-3,3′-pyrazol]-2-one (5o). Yellow powder, yield: 87%, m.p: 228–230 °C; (TLC; hexane–EtOAc, 1 : 4, R_f = 0.265); ¹H NMR (300 MHz, DMSO-d₆) δ: 11.05 (1H, s, NH), 10.75 (1H, s, NH), 8.20–8.06 (2H, m, Ar), 8.02 (1H, d, J = 8.1 Hz, Ar), 7.79 (1H, d, J = 7.3 Hz, Ar), 7.72–7.56 (3H, m, Ar), 7.40 (1H, d, J = 8.1 Hz, Ar), 7.31 (1H, d, J = 7.6 Hz, Ar), 6.56 (1H, t, J = 7.8 Hz, Ar), 6.10 (2H, s, NH); ¹³C NMR (75 MHz, DMSO-d₆) δ: 165.9, 160.6, 143.7, 140.5, 134.3, 133.9, 130.6, 128.7, 127.7, 127.0, 126.9, 126.0, 123.9, 123.5, 121.2, 118.7, 111.4, 110.9, 71.8; anal. calcd for C₂₁H₁₄ F₃N₅O₃: C, 57.15; H, 3.20; F, 12.91; N, 15.87. Found: C, 57.44; H, 3.63; N, 15.88.

5-Chloro-4′-nitro-5′-(o-tolylamino)-1′,2′-dihydrospiro[indoline-3,3′-pyrazol]-2-one (5p). Orange powder, yield: 90%, m.p: 185–187 °C; (TLC; hexane–EtOAc, 1 : 4, R_f = 0.282); IR (KBr): 3380, 3320, 3215, 3093, 2967, 1735, 1714, 1650, 1585, 1480, 1360, 1240, 1216, 1070, 934, 734, 588; ¹H NMR (300 MHz, DMSO-d₆) δ: 10.78 (1H, s, NH), 10.51 (1H, s, NH), 8.22 (1H, d, J = 2.2 Hz, Ar), 7.64 (2H, d, J = 8.2 Hz, Ar), 7.39–7.25 (3H, m, Ar), 6.85 (1H, d, J = 8.3 Hz, Ar), 5.90 (2H, s, NH), 2.35 (3H, s, Me); ¹³C NMR (75 MHz, DMSO-d₆) δ: 165.4, 158.4, 144.6, 142.4, 137.7, 135.9, 135.0, 131.5, 129.9, 126.3, 126.0, 122.3, 118.5, 114.2, 112.0, 72.5, 21.1; anal. calcd for C₁₇H₁₄ClN₅O₃: C, 54.92; H, 3.80; N, 18.84. Found: 54.49; H, 3.82; N, 19.11.

5-Bromo-4′-nitro-5′-(o-tolylamino)-1′,2′-dihydrospiro[indoline-3,3′-pyrazol]-2-one (5q). Orange powder, yield: 88%, m.p: 230–232 °C; (TLC; hexane–EtOAc, 1 : 4, R_f = 0.282); IR (KBr): 3378, 3338, 3261, 1741, 1716, 1650, 1481, 1334, 1194, 1097, 1068, 838, 694; ¹H NMR (300 MHz, DMSO-d₆) δ: 10.57 (1H, s, NH), 9.88 (1H, s, NH), 7.60 (1H, d, J = 7.6 Hz, Ar), 7.43–7.16 (4H, m, Ar), 6.80 (1H, d, J = 7.7 Hz, Ar), 6.68 (1H, t, J = 7.6 Hz, Ar), 5.95 (2H, s, NH), 2.32 (3H, s, Me); ¹³C NMR (75 MHz, DMSO-d₆) δ: 165.7, 159.0, 146.1, 143.7, 136.7, 133.5, 132.0, 131.1, 127.4, 127.1, 126.7, 126.5, 121.8, 117.7, 110.4, 71.6, 18.2; anal. calcd for: C₁₆H₁₃N₅O₄.

2.3 Screening of ChE inhibitory activity

The assay was performed according to previously reported procedures.^58,59

2.4 Enzyme kinetic studies

The mode of inhibition of the most active compound 5f, identified with the lowest IC50, was investigated against AChE activity with different concentrations of acetylthiocholine (0.1–1 mM) as the substrate in the absence and presence of 5f at different concentrations. A Lineweaver–Burk plot was generated to identify the type of inhibition, and the Michaelis–Menten constant (K_m) value was determined from the plot between the reciprocal of the substrate concentration (1/[S]) and reciprocal of the enzyme rate (1/V) over the various inhibitor concentrations. The experimental inhibitor constant (K_i) value was constructed by secondary plots of the 5f concentration versus K_m.⁶⁰

2.5 Molecular docking study

The SMILE format of 5f was converted to a three-dimensional structure within the Maestro software package. The X-ray structures of AChE (PDB code: 4EY7). and BChE (PDB code: 4BDS) were prepared with the Protein Preparation Wizard interface of Maestro via removing the ligand and water molecules, adding hydrogen atoms, and optimizing their position according to PROPKA prediction at pH 7.0. The molecular docking was performed using IFD mode with the ligand as flexible, the force field was set as OPLS-2005, and all other parameters were set to default. The binding site was used to generate the grid for IFD calculation. The maximum 5 poses with receptor and ligand van der Waals radii of 0.7 and 0.5, respectively were considered. Residues within 15 Å of the crystallographic ligands at the active site were refined, followed by side-chain optimization. Structures with prime energy of more than 30 kcal mol⁻¹ are eliminated. A re-docking experiment to validate the used docking protocol was done, and the RMSD value of 0.79 indicates that the docking experiment is reliable.

2.6 Molecular dynamics simulation

The molecular dynamics (MD) simulation of this study was performed by using the Desmond v5.3 module (https://www.schrodinger.com/products/desmond) implemented in the Maestro interface (from Schrödinger 2018-4 suite). The appropriate pose for the MD simulation procedure of the compounds was obtained by the IFD method. To build the system for MD simulation, the protein–ligand complexes were solvated with SPC explicit water molecules and placed in the center of an orthorhombic box of appropriate size in the periodic boundary condition. Sufficient counter-ions and a 0.15 M solution of NaCl were also utilized to neutralize the system and to simulate the real cellular ionic concentrations, respectively. The MD protocol involved minimization, pre-production, and production MD simulation steps. In the minimization procedure, the system was allowed to relax for 2500 steps by the steepest descent approach. Then the system's temperature was raised from 0 to 300 K with a small force constant on the enzyme to restrict any drastic changes. MD simulations were performed via the NPT (constant number of atoms, constant pressure i.e. 1.01325 bar, and constant temperature i.e. 300 K) ensemble. The Nose–Hoover chain method was used as the default thermostat with a 1.0 ps interval and Martyna–Tobias–Klein as the default barostat with a 2.0 ps interval by applying the isotropic coupling style. Long-range electrostatic forces were calculated using the particle-mesh-based Ewald approach with the cut-off radius for coulombic forces set to 9.0 Å. Finally, the system was subjected to produce MD simulations for 100 ns for the protein–ligand complex. During the simulation, every 1000 ps of the actual frame was stored. The systems' dynamic behavior and structural changes were analyzed by calculating the root mean square deviation (RMSD) and RMSF. Subsequently, the energy-minimized structure calculated from the equilibrated trajectory system was evaluated to investigate each ligand–protein complex interaction.

2.7 Toxicity assay on SH-SY5Y

SH-SY5Y cells were maintained in Dulbecco's modified Eagle medium with Ham's F12 medium (DMEM/F12) containing 15% fetal bovine serum, 100 units per ml penicillin and 100 μg ml⁻¹ streptomycin. Cells were seeded into flasks containing the supplemented medium and maintained at 37 °C in a humidified atmosphere of 5% CO₂ and 95% air. Cell viability, virtually the mitochondrial activity of living cells, was measured by quantitative colorimetric assay with MTT, as described previously. The MTT reagent, at a final concentration of 0.5 mg ml⁻¹, was added to each well at the end of the incubation period, and the plate was placed in a humidified incubator for an additional two h periods. Metabolically active cells convert the yellow MTT tetrazolium compound to a purple formazan product. Then, the insoluble formazan was dissolved with dimethylsulfoxide; colorimetric determination of MTT reduction was measured at 540 nm. Control cell treated media were taken as 100% viability.³

3. Results and discussion

3.1 Design

Structural features and key binding sites of AChE and BChE were clarified by X-ray crystallographic studies with known inhibitors. It was demonstrated that both enzymes consist of the peripheral anionic site (PAS), and catalytic active site (CAS) which contain anionic subsite (AS), oxyanion hole, and acyl binding pocket. The PAS is located at the gorge's entrance, and the CAS at the bottom of the gorge. It was confirmed that interactions with the CAS and PAS induce significant changes in the activity of the enzymes.⁶¹ Donepezil (Fig. 2) as a ChE inhibitor mimics the binding mode of the ACh neurotransmitter so that its inden-1-one moiety occupies the PAS pocket and benzyl piperidine occupies the CAS pocket. Regarding the structural future of donepezil, different ChE inhibitors were developed. For years, the indole and indolinone scaffolds have proven invaluable to medicinal chemistry researchers in developing various ChE inhibitors. These scaffolds have been particularly effective in targeting the PAS pocket, facilitating the design of potent inhibitors.

Fig. 2 The design strategy of spiroindolinone-pyrazole derivatives.

Indolinone-bearing pyridine derivatives were designed (Fig. 2, compound B) as dual-binding inhibitors of AChE, and the most potent derivative was 32-fold more potent than donepezil as a reference drug.⁶² A library of indolinone-based hydrazinecarbothioamide derivatives (Fig. 2, compound C) was synthesized and tested for their anti-ChE activities in the past year. The most active analog exhibited K_i = 0.52 ± 0.11 μM with a selectivity of SI = 37.69 for AChE over BuChE.⁶³ In another study, a series of benzyl-indolinones (Fig. 2, compound D) were designed using molecular modeling. In vitro evaluations exhibited significant inhibition of derivatives, in which the most potent analog exhibited more than 2-fold potency compared with donepezil.⁶⁴ The structure–activity relationship (SAR) also demonstrated that substitution at the different positions of the indolidinone ring significantly affects the potency.

It has been reported in the literature that various compounds carrying the pyrazole skeleton have ChE inhibitory effects.⁶⁵ Gutti et al. presented a linear pyrazole derivative as a ChE inhibitor (compound E). The most potent compound demonstrated significant inhibition of ChE activity and effectively reduced metal-induced aggregation of Aβ_1–42 at a concentration of 20 μM. Furthermore, the compound exhibited a high cell viability of nearly 90%.⁶⁶ A set of hybrid derivatives bearing a pyrazole and coumarin scaffold and the most potent entry, compound F, showed high potential AChE inhibitory activities with IC₅₀ values of 4.41 ± 0.53 μg ml⁻¹.⁶⁷ Another study in 2020 reported that compound G was the most potent mixed-type inhibitor acting as a dual site inhibitor by occupying the CAS and PAS, which was further supported by enzyme kinetic study. The compounds did not show any cytotoxicity against HEK-293 cells.⁶⁸ In a review, the limited SAR of pyrazole was developed, and it was exhibited that the smaller groups attached to the pyrazole ring promote ChE inhibition compared to bulky ones. Also, electron-withdrawing atoms increase activity.⁶⁹

In light of this information, we aimed to develop and synthesize an efficient and convenient one-pot reaction of novel spiroindolinone-pyrazole derivatives in the present study. To investigate the structure–activity relationships (SARs) of ChE inhibition, a substituted phenyl ring (small ring) was coupled with the pyrazole group. Additionally, the designed scaffold featured a nitro moiety known for its strong electron-withdrawing properties, which could enhance interactions with the enzyme's binding site. This incorporation provided an added value to the scaffold, potentially contributing to increased potency in ChE inhibition. All the synthesized compounds were evaluated for their ChE activities, and the kinetic study of the most potent derivative was executed. In addition, docking studies and molecular dynamic simulations of the most potent compound were performed. The present work aimed to identify agents that may lead to further development as potential anti-Alzheimer drugs.

3.2 Chemistry

During our laboratory efforts on developing multicomponent reactions for synthesizing biologically important heterocyclic compounds, new spiro pyrazoline derivatives were synthesized in the present work. Initially, BMTNE 1 (1 mmol) and arylamine 2 (1 mmol) were reacted in EtOH (5 mL) at reflux to form N-aryl-1-(methylthio)-2-nitroethenamine 3, after 12 h, NH₂NH₂ (80% aq.) (1.2 mmol) and isatin derivatives 4 (1 mmol) were added to obtain the desired product 5. To optimize the reaction conditions for the synthesis of 4′-nitro-5′-(o-tolylamino)-1′,2′-dihydrospiro[indoline-3,3′-pyrazol]-2-one 5a, initially intermediate 2-methyl-N-(1-(methylthio)-2-nitrovinyl)aniline 3a was prepared according to the reported procedure;⁷⁰ then, NH₂NH₂ (80% aq.) and isatin 4a were used as substrates in the model reaction to determine the optimal reaction conditions. Different solvents and reaction temperatures were carefully evaluated. The results are shown in Table 1. The product yield was low when the reaction was carried out in water (entry 1). Ethanol at reflux condition provided higher yields than other organic solvents (compare entry 5 with entries 2–4). It should be mentioned that the reaction was carried out in ethanol at different temperatures (room temperature, 40 °C, 60 °C Table 1 entries 6–8). These studies showed that refluxing ethanol provided the highest yield. Based on the optimal reaction conditions, we studied two-step one-pot sequential synthesis of 5a from BMTNE 1, 2-methylaniline 2a, NH₂NH₂ (80% aq.), and isatin 4a, and we were able to get the same result for the final product (93%). At first, we added BMTNE 1 (1 mmol) and 2-methylaniline 2a (1 mmol) in refluxing ethanol for 12 h then, the other components were added to form the final product 5a.

Table 1 Optimization of the reaction conditions for the synthesis of 5a^a

Entry	Solvent	T (°C)	Yield^b (%)
a Reaction conditions: 2-methyl-N-(1-(methylthio)-2-nitrovinyl)aniline (1 mmol), NH2NH2 (80% aq.) (1.2 mmol), isatin (1 mmol), solvent 5 mL. b Isolated yield.
1	H2O	80	25
2	CH3CN	Reflux	—
3	THF	Reflux	30
4	Toluene	80	45
5	EtOH	Reflux	93
6	EtOH	r.t	30
7	EtOH	40	55
8	EtOH	60	80

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The scope and efficiency of the reaction were discovered using a range of structurally diverse aryl amine and isatin derivatives to form the corresponding products 5a–q.

With the optimal reaction conditions, the substrate scope of N-aryl-1-(methylthio)-2-nitroethenamine 3 and isatin derivatives 4 was investigated. As shown in Table 1, the results showed that various substituent groups of N-aryl-1-(methylthio)-2-nitroethenamine 3 usually had a slight effect on the yields, and we could not ascertain the effect of any specific substituent. All the N-aryl-1-(methylthio)-2-nitroethenamine substrates can be used in the reaction, producing compound 5 in good to excellent yields. The effect of substituted groups (R₁) of isatins was also evaluated. The results revealed that different isatin substrates 4 could be transformed into the desired product 5 (Table 1). The substrates with electron-donating groups (R₁ = OMe) or electron-withdrawing groups (R¹ = Cl, Br, NO₂, CF₃) and isatin were well-tolerated during this transformation. The substrates with electron-donating groups usually provided the target compounds in higher yields than isatin or substrates with electron-withdrawing groups (Table 2).

Table 2 Synthesis of new spiroindolinone-pyrazoles (5a–q)^a,b

a Reaction conditions: BMTNE (1 mmol) and arylamine (1 mmol) were added to ethanol (5 mL) at reflux, and after 12 h, isatin derivatives (1 mmol) and NH₂NH₂ (80% aq.) (1.2 mmol) were added to obtain the desired product 5. b Isolated yield.

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According to a literature survey,⁷¹ a plausible mechanism for forming product 5 is shown in Scheme 1. The first synthetic pathway to produce 5 is initiated with intermediate 6 formed from the nucleophilic substitution of the NH₂ group of arylamine and the NH₂ group of the hydrazine molecule with two methylsulfanyl groups of BMTNE 1. Then, the one-component 1,1-enediamines 6 and the isatin derivatives 4 undergo an aza-ene reaction to give intermediate 7, resulting in the formation of product 5.

Scheme 1 Proposed mechanism for the formation of compound 5

3.3 AChE inhibitory activity of 5a–q derivatives

All the synthesized compounds were evaluated for their inhibitory activity against AChE. The results are summarized in Table 3.

Table 3 The anti-AChE and anti-BChE activity of 5a–q expressed in terms of mean ± S.E^a

Compound	R^1	R^2	AChE mean ± S.E		BChE
			% inhibition at 50 μM	IC50 (μM)	% inhibition at 50 μM	IC50 (μM)
a Data presented here are mean ± S.E. b Positive control.
5a	H	2-CH3	35.38 ± 4.61	—	60.07 ± 4.62	19.95 ± 2.23
5b	H	4-CH3	30.48 ± 5.82	—	56.49 ± 2.53	25.08 ± 1.59
5c	H	4-OH	40.96 ± 4.22	—	12.91 ± 1.79	—
5d	H	4-OCH3	47.77 ± 3.29	—	53.35 ± 8.21	34.67 ± 3.68
5e	H	3,4-diCH3	29.44 ± 3.89	—	31.08 ± 1.08	—
5f	H	3-Cl	75.47 ± 3.68	6.02 ± 2.59	13.94 ± 3.68	—
5g	H	4-Cl	13.95 ± 2.18	—	14.90 ± 5.73	—
5h	5-OCH3	4-OCH3	29.74 ± 1.19	—	Not active
5i	5-OCH3	3,4-diCH3	Not active	—	52.29 ± 5.76	47.30 ± 5.75
5j	5-NO2	2-CH3	71.57 ± 8.94	15.48 ± 2.69	38.94 ± 7.36	—
5k	5-NO2	2,5-diOCH3	55.15 ± 6.84	42.55 ± 4.11	25.13 ± 1.58	—
5l	5-NO2	4-Cl	65.89 ± 4.73	37.15 ± 3.52	21.84 ± 2.10	—
5m	7-CF3	2-CH3	63.13 ± 4.32	16.98 ± 2.43	37.53 ± 7.53
5n	7-CF3	3-Cl	20.63 ± 1.57	—	18.8 ± 4.58
5o	7-CF3	Naphtyl	39.07 ± 4.73	—	Not active	—
5p	5-Cl	2-CH3	21.92 ± 1.71	—	73.76 ± 1.95	9.13 ± 1.82
5q	5-Br	2-CH3	51.76 ± 2.21	41.68 ± 6.93	34.00 ± 6.37
Donepezil^b				0.079 ± 0.05		10.6 ± 2.1

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Among 5a–e bearing H at the R¹ position and an electron-donating group at R², it was understood that 5d (R² = 4-OCH₃) recorded 47.77% inhibition at 50 μM and bioestric replacement of methoxy with hydroxyl resulted in 5c with a slight reduction in the activity (inhibition at 50 μM = 40.96%). Ortho (5a) and para methyl (5b) substitution, as well as 3,4-diCH₃ (5e) substitution, did not significantly improve the activity compared with 5d. Contrarily, among analogs featuring halogen substitutions at the R² position, the compound bearing a 3-Cl substitution at R² (5f) demonstrated the highest potency, with an IC₅₀ value of 6.02 ± 2.59 μM. However, when the substitution was shifted from the meta to the para position (5g), the potency considerably decreased to 13.95 ± 2.18% inhibition at 50 μM, indicating a significant deterioration in activity.

As can be seen in 5h and 5i, the introduction of 5-OCH₃ as an electron-donating group at the R¹ position, regardless of the type of substitution at R², brought a reduction in the inhibitory activities compared to unsubstituted derivatives (5a–g; R¹ = H).

A notable enhancement in potency was observed in compounds 5j, 5k, and 5l upon replacing the OCH₃ group at the R¹ position with the strong electron-withdrawing group NO₂, possessing both resonance and inductive effects. Comparatively, these substitutions exhibited higher potencies than others at the same position. Additionally, the trend observed for R² substitutions was as follows: 2-CH₃ (5j, IC₅₀ = 15.48 ± 2.69 μM) > 4-Cl (5l, IC₅₀ = 37.15 ± 3.52 μM) > 2,5-diOCH₃ (5k, IC₅₀ = 42.55 ± 4.11 μM). This indicates that the presence of a methyl group at the 2-position (R²) resulted in the highest potency, followed by a chlorine substitution at the 4-position, while the presence of two methoxy groups at the 2 and 5 positions displayed slightly lower potency.

The trifluoromethyl (–CF₃) group, one of the most powerful electron-withdrawing groups, was substituted at the 7 position of the indolinone ring (5m–o). Among –CF₃ containing derivatives, 2-methyl substitution recorded better potency (5m; IC₅₀ = 16.98 ± 2.43 μM) in comparison with chlorine (5n) as electron-withdrawing and naphthyl (5o) as bulk moieties.

It was observed that the presence of a 2-methyl group at the R² position exhibited better potency when combined with electron-donating groups such as NO₂ and CF₃ at the R¹ position. Building upon this observation, the 2-methyl moiety was retained, and chlorine (5p) and bromine (5q) halogen groups were introduced at the R¹ position. Interestingly, the bulkier bromine moiety was more favorable than the chlorine group. This suggests that the size and steric properties of the substituents play a crucial role in the overall activity and interaction of the compound with the target site.

3.4 BChE inhibitory activity of 5a–q derivatives

Next, all derivatives 5a–q were screened against BChE, and results are presented in Table 3. Assessments of 5a–g (R¹ = H) showed that 5a bearing a 2-CH₃ moiety was the most potent derivative in R¹ = H derivatives. Replacement of the position from ortho to para lightly reduced the activity in 5b with IC₅₀ = 25.08 ± 1.59 μM. Also, multi-methyl substitutions (5e) deteriorated the potency to 31.08 ± 1.08% inhibition at 50 μM. Also, replacing 4-CH₃ with 4-OH (5c) and 4-OCH₃ (5d) did not improve the activity vs.5a. On the other hand, halogen substitutions significantly reduced the activity. In this group, it was understood that the electron-donating group at the R² position is more favorable.

The results obtained from the investigation of compound 5h (R²: 4-OCH₃) and compound 5i (R²: 3,4-diCH₃), both possessing a 5-OCH₃ substituent at R¹, revealed that the introduction of the 3,4-diCH₃ substitution resulted in a more potent BChE inhibitor with an IC₅₀ value of 47.30 ± 5.75 μM. These findings suggest that increased lipophilicity, conferred by the 3,4-diCH₃ substitution, improves BChE inhibition.

Relative to the activity of 5j–l with a 5-nitro substituent at the R¹ position, similar to R¹ = H derivatives, the introduction of 2-CH₃ substitution increased the activity in comparison with 2,5-diOCH₃ (5k) and 4-Cl (5l).

Among 7-CF₃ containing derivatives (5m–o), similar results were recorded in which the 2-CH₃ moiety was identified as a potent analog followed by 5n (R²: 3-Cl; 18.8% inhibition at 50 μM). Also, 5o was completely inactive.

Moreover, 5-Cl (5p) and 5-Br (5q) groups as halogen-substituted derivatives did function positively in the anti-BChE assay, and the best biological results were seen in 5p (R¹: 5-Cl, R²: 2-CH₃) with an IC₅₀ value of 9.13 ± 1.82 μM.

3.5 Summary of SARs

In the case of the electron-withdrawing group at R¹, introducing a 2-methyl substitution was found to be more favorable in most cases. However, when R¹ was not substituted (R¹ = H), the 3-Cl moiety was identified as the most potent analog among the tested compounds. These results suggest that the specific substitution pattern and electronic properties of the group attached to R¹ play a significant role in determining the inhibitory potency of the compounds. The anti-BChE result reflects the good impact of the 2-methyl substitution to get the optimum BChE activity. 5f showed a remarkable improvement in AChE inhibition (6.02 ± 2.59 μM) with no BChE inhibitory activity. 5p exhibited selective and significant BChE inhibitory potency (IC₅₀ = 9.13 ± 1.82 μM). Also, the presence of a nitro moiety increases the selectivity against AChE.

3.6 Enzyme kinetic studies of 5f against AChE

According to Fig. 3a, the Lineweaver–Burk plot showed that the K_m gradually increased and V_max changed with increasing inhibitor concentration, indicating a mix-type inhibition. The results showed that 5f might bind to the free enzyme or the enzyme that is already bound to the substrate. Furthermore, the slope plot versus different inhibitor concentrations gave an estimate of the inhibition constant, K_i of 5.59 μM (Fig. 3b).

Fig. 3 Kinetics of AChE inhibition by 5f. (a) The Lineweaver–Burk plot in the absence and presence of different concentrations of sample 5f; (b) the secondary plot between slope and various concentrations of 5f.

3.7 Molecular docking

AChE consists of two main pockets, a PAS near the entrance of the gorge and a CAS at the bottom of the gorge, approximately 20 Å deep. The PAS consists of amino acids of tryptophan 286, tyrosine 337, and phenylalanine 338, which guide molecules to the CAS pocket. CAS comprises the catalytic triad with glutamic acid 334, serine 203, and histidine 447 as the main residues and the anionic subsite of tryptophan 72. Also, there are two residues at the bottleneck, tyrosine 121 and phenylalanine 330, of the enzyme's binding site that guide molecules toward the active site.

First, the validity and reliability of molecular docking were assessed by redocking the AChE and BChE natural ligands inside the binding site of this relevant enzyme. RMSD less than 2 Å was recorded as the ideal value.

In the pursuit of identifying the most potent AChE inhibitor, compound 5f has emerged as the primary candidate, warranting further investigation. It is worth noting that 5f exists as an enantiomeric mixture, necessitating a comprehensive analysis of both its R and S isomers within the AChE active site. Molecular docking assessments were conducted on both the S and R enantiomers of compound 5f to elucidate their respective modes of action. This research aimed to uncover these enantiomers' binding orientations and poses within the AChE binding pocket. The results of the molecular docking calculations yielded significant insights. The S enantiomer exhibited a binding energy of −9.87 kcal mol⁻¹, while the R enantiomer displayed a binding energy of −7.63 kcal mol⁻¹. These findings were derived from a detailed examination of the binding interactions and their associated energetics within the AChE binding site.

The interactions of S and R isomers of 5f and in the AChE binding pocket are presented in Fig. 4 and 5, respectively.

Fig. 4 2D interaction diagram of the S isomer of 5f in the AChE binding site.

Fig. 5 2D interaction diagram of the R isomer of 5f in the BChE binding site.

The molecular docking study showed that the S enantiomer of 5f was appropriately oriented in the active site of AChE (Fig. 4). The indolineone moiety participated in H-bound interaction with Tyr124 (2.94 Å) and pi–pi stacking interaction with Trp86 (3.78 Å). Pyrazole recorded two H-bound interactions with Asp74 (2.60 Å) and Tyr337 (2.28 Å) and one pi–pi stacking interaction with Tyr337 (4.20 Å). The nitro moiety of 5f also participated in pi–cation interaction with Tyr341 (6.10 Å), and the 3-Cl substituted group displayed a halogen bond with Phe295 (3.51 Å).

On the other hand, the R isomer of 5f against the AChE binding site (Fig. 5) displayed notable interactions with key residues, including Phe295 (3.46 Å, halogen interaction), Ser203 (2.97 Å, hydrogen bond), Tyr341 (3.76 Å, pi–pi stacking), and His447 (4.10 Å, pi–cation) and His447 (4.36 Å, pi–pi stacking).

Furthermore, the molecular docking study results involving all derivatives against both AChE and BChE, along with details regarding the types of interactions and corresponding distances, were comprehensively presented in the ESI within Tables S1 and S2.†

3.8 Molecular dynamics simulation

Molecular dynamics simulations were conducted for both the R and S enantiomers of 5f to evaluate protein flexibility.

To study the stability of the system's dynamic behavior, the best IFD pose of R and S enantiomers of compound 5f and apo enzyme was implemented to predict the motion of complex systems at an atomistic level. Root mean square deviation (RMSD) values indicate the conformational stability and perturbations of the system, and RMSD value changes around 1–3 Å are optimum values for small globular protein and the complex has reached equilibrium.²¹

The RMSD plot for AChE (in blue) displayed a notable initial increase followed by fluctuations around 1.5 Å. Meanwhile, the AChE-R isomer of complex 5f (in green) exhibited a slightly lower RMSD value of 1.2 Å. It is worth noting that the R isomer displayed comparatively lower stability. In contrast, the AChE-S isomer of complex 5f (in orange) displayed remarkable stability throughout the simulation, with an RMSD value of 0.7 Å maintained over the entire 100 ns duration.

These observations suggest that the simulation time employed was sufficient to attain an equilibrium structure during the simulations. Additionally, it appears that the S isomer exhibited greater stability compared to the R isomer (Fig. 6).

Fig. 6 RMSD values of the AChE-S isomer of complex 5f (orange), the AChE-R isomer of complex 5f (green), and AChE (blue) throughout the simulation time.

RMSF values illustrate the protein backbone flexibility throughout the simulation time. Notably, the RMSF values indicate that the S isomer of compound 5f plays a substantial role in stabilizing crucial regions, including the PAS pocket (indicated by the blue dashed line), the catalytic triad (highlighted by the green dashed line), and the anionic subsite (represented by the red dashed line), as illustrated in Fig. 7.

Fig. 7 RMSF values of the AChE-S isomer of complex 5f (orange) and the AChE-R isomer of complex 5f (green) in angstroms throughout the simulation.

Furthermore, the 2D interaction diagram of the S isomer of compound 5f in complex with the enzyme is depicted in Fig. 8. These interactions happened for at least 30% of the duration of the MD simulation time. Fig. 8 shows that the carbonyl oxygen of the indolinone moiety participated in two H-bound interactions with Tyr72 (32% of MD simulation time) and Asn87 (63% of MD simulation time) of the anionic subsite. Also, NH of indolinone interacted with Ser125 through hydrogen bonds for about 82% of the MD simulation time. The pyrazole ring demonstrated two H-bound interactions with Tyr337 (95% of the simulation time) and Tyr 341 (74% of the MD simulation time) of the PAS. Also, the 3-chlorophenyl group stabilized through hydrophobic π–π stacking non-bonding interaction by the Tyr341 residue for about 34% of MD simulation time. All these results aligned with the kinetic study confirming mix-type inhibition of the 5f derivative.

Fig. 8 2D interaction diagram of the S isomer of compound 5f within the AChE binding site occurring over 30% of MD simulation time.

Furthermore, the 2D interaction diagram of the R isomer of compound 5f in complex with the enzyme is depicted in Fig. 9. Briefly, indolinone demonstrated pi–cation interaction for around 81% of MD run plus pi–pi stacking interaction (48% of MD simulation time) with His447. The C=O moiety of indolinone also recorded H-bound interaction with Ser203 mediated with water (35% of MD duration time). The nitro moiety also participates in another H-bound interaction with Tyr133 (35% of MD duration time). Another pi–pi stacking interaction was also seen between imidazole and Tyr337 (31% of MD duration time).

Fig. 9 2D interaction diagram of the R isomer of compound 5f within the AChE binding site occurring over 30% of MD simulation time.

3.9 Effect of 5f on SH-SY5Y cell viability

The SH-SY5Y neuroblastoma cell line is widely used as an in vitro neuronal model for evaluating neurodegenerative disorders. As a result, the toxicity of 5f as the most potent derivative was evaluated against the SH-SY5Y cell line, which was examined. As shown in Fig. 10, the compound exhibited 64.81 ± 0.11% viability at 50 μM with no significant toxicity at the lower dose.

Fig. 10 Cell viability of 5f on the SHSY-5Y cell line.

3.10 ADMET properties and in silico toxicity

ADMET encompasses the assessment of absorption, distribution, metabolism, excretion, and toxicity of pharmaceutical compounds within a biological system. These properties are crucial for drug developers in evaluating the safety and effectiveness of drugs during both the preclinical and clinical development stages. Table 4 demonstrates drug-likeness prediction for 5a–q, and Table 5 presents the ADMET predictions for all the compounds, calculated using the pkCSM and SwissADME online servers. The estimated human intestinal absorption (HIA) for the selected compounds indicates a high likelihood of efficient absorption within the gastrointestinal tract. Furthermore, all the compounds are projected to possess Caco-2 permeability levels suitable for oral administration. Regarding metabolism, it is noteworthy that none of the compounds are predicted to inhibit CYP2D6, which is a favorable characteristic. However, they are expected to act as inhibitors of CYP3A4. The derivatives exhibit acceptable toxicity profiles, making them suitable candidates for further drug design and refinement.

Table 4 Drug-likeness prediction for 5a–q

Compound	Molecular weight	Log P	Rotatable bonds	Acceptors	Donors	Surface area
5a	337.339	1.80812	3	6	4	142.596
5b	337.339	1.80812	3	6	4	142.596
5c	339.311	1.2053	3	7	5	141.025
5d	353.338	1.5083	4	7	4	147.710
5e	351.366	2.11654	3	6	4	148.961
5f	357.757	2.1531	3	6	4	146.534
5g	357.757	2.1531	3	6	4	146.534
5h	383.364	1.5169	5	8	4	159.188
5i	381.392	2.12514	4	7	4	160.439
5j	382.336	1.71632	4	8	4	157.249
5k	428.361	1.4251	6	10	4	173.841
5l	402.754	2.0613	4	8	4	161.187
5m	405.336	2.82692	3	6	4	161.458
5n	425.754	3.1719	3	6	4	165.396
5o	441.369	3.6717	3	6	4	177.774
5p	371.784	2.46152	3	6	4	152.899
5q	416.235	2.57062	3	6	4	156.464

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Table 5 ADMETa prediction of the synthesized 5a–q

Compd.	Absorption^a		Distribution^a		Metabolism^a				Excretion^a	Toxicity^a
	HIA%	Caco2 permeability	VDss (log L kg^−1)	BBB permeability	CYP3A4 inhibition	CYP2D6 inhibition	CYP2C9 inhibition	CYP2C19 inhibition	Total clearance	Oral rat acute toxicity (LD50)	Oral rat chronic toxicity (LOAEL)	hERG1 inhibitor
a HIA (human intestinal absorption): >80% is high and <30% is poor; VDss (steady-state volume of distribution): log L kg^−1: >0.45 is high and <−0.15 is low. The software could not predict parameters for these compounds (−).
5a	81.109	0.137	0.048	−0.492	No	No	No	No	0.509	2.127	1.877	No
5b	81.109	0.137	0.048	−0.492	No	No	No	No	0.509	2.127	1.877	No
5c	77.373	−0.253	0.238	−0.663	No	No	No	No	0.371	2.308	2.411	No
5d	81.98	−0.117	−0.065	−0.681	No	No	No	No	0.437	2.076	1.971	No
5e	81.578	0.2	0.074	−0.504	No	No	No	No	0.509	2.154	1.759	No
5f	81.817	0.024	−0.01	−0.646	No	No	No	No	−0.375	2.112	1.924	No
5g	81.817	0.024	−0.01	−0.646	No	No	No	No	−0.375	2.112	1.924	No
5h	78.877	−0.057	−0.235	−0.677	No	No	No	No	0.427	2.003	1.483	No
5i	78.475	0.112	−0.082	−0.5	No	No	No	No	0.458	2.082	1.241	No
5j	78.490	−0.05	−0.316	−0.594	No	No	No	No	0.453	2.704	2.187	No
5k	75.561	−0.175	−0.495	−0.981	No	No	No	No	0.461	2.795	2.058	No
5l	79.198	−0.07	−0.362	−0.747	No	No	No	No	−0.417	2.737	2.233	No
5m	83.776	0.164	−0.105	−0.681	Yes	No	Yes	Yes	0.124	2.036	1.403	No
5n	84.484	0.052	−0.161	−0.835	Yes	No	Yes	Yes	−0.256	2.023	1.45	No
5o	90.348	−0.081	−0.328	−0.676	Yes	No	Yes	Yes	0.142	2.294	1.329	No
5p	82.467	0.11	0.038	−0.542	No	No	No	Yes	−0.477	2.066	1.715	No
5q	82.203	0.115	0.048	−0.563	Yes	No	Yes	Yes	−0.499	2.074	1.698	No

---

4. Conclusions

In conclusion, we have successfully developed a one-pot sequential four-component reaction for synthesizing spiro pyrazoline derivatives under catalyst-free conditions. This reaction provides a novel, rapid, and efficient route for the preparation of a variety of spiropyrazoline derivatives in good to excellent yields from readily available building blocks of N-aryl-1-(methylthio)-2-nitroethenamine, hydrazine hydrate and isatin derivatives. Next, all the derivatives were tested as possible AChE and BChE inhibitors. Results exhibited the promising potency and selectivity of 5f bearing H at R¹ and 3-Cl at R² with an IC₅₀ value of 6.02 ± 2.59 μM with no significant activity against BChE. On the other hand 5p (R¹: 5-Cl, R²: 2-CH₃) recorded IC₅₀ = 9.13 ± 1.82 μM against BChE. SAR suggested that the specific substitution pattern and electronic properties of the group attached to R¹ and R² played a significant role in determining the inhibitory potency of the compounds. The kinetic study of 5f demonstrated mix-type inhibition with a K_i of 5.59 μM.

Furthermore, based on the MD studies, the S enantiomer of compound 5f depicted noticeable interactions with Tyr72 (H-bound) and Asn87 (H-bound) of the anionic subsite plus Tyr337 (H-bound) and Tyr 341 (H-bound & pi–pi stacking) of the PAS confirming its high potency. Also, 5f was tested against the SHSY-5Y cell line, exhibiting no significant toxicity at 25 μM. It can be understood that this set of compounds can serve as structural outlines to design and expand potential anti-AD agents.

Consents

There are no consents to declare.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

The authors wish to acknowledge the financial support of the Vice-Chancellor for Research of Shiraz University of Medical Sciences (grant number: IR.SUMS.REC.1401.728).

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Footnote

† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d3md00255a

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