Synthesis, biological evaluation and in silico study of 4-(benzo[d]thiazole-2-yl) phenols based on 4-hydroxy coumarin as acetylcholinesterase inhibitors

Bi Bi Fatemeh Mirjalili; Seyedeh Azita Fazeli Attar; Fereshteh Shiri

doi:10.1038/s41598-024-74001-7

. 2024 Nov 2;14:26459. doi: 10.1038/s41598-024-74001-7

Synthesis, biological evaluation and in silico study of 4-(benzo[d]thiazole-2-yl) phenols based on 4-hydroxy coumarin as acetylcholinesterase inhibitors

Bi Bi Fatemeh Mirjalili ^1,^✉, Seyedeh Azita Fazeli Attar ¹, Fereshteh Shiri ^2,^✉

PMCID: PMC11531508 PMID: 39488512

Abstract

Alzheimer’s disease, characterized by cognitive decline and memory loss, is associated with decreased acetylcholine levels due to acetylcholinesterase (AChE) activity. Compounds containing a coumarin heterocyclic core coupled with thiazole exhibit excellent acetylcholinesterase inhibitory activity. In this work, we designed and synthesized a series of 4-(benzo[d]thiazole-2-yl) phenols based on 4-hydroxycoumarin. The compounds were synthesized and their inhibitory activities were evaluated through in vitro biological assays. Of the compounds investigated, 3i exhibited the strongest inhibitory activity, with an IC50 value of 2.7 µM. Molecular docking and molecular dynamics simulations were employed to elucidate the binding interactions and stability of the synthesized compounds with AChE. The results demonstrated promising inhibitory activity, suggesting potential therapeutic applications for Alzheimer’s disease. This research contributes to the development of coumarin-based heterocyclic compounds as effective AChE inhibitors.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-024-74001-7.

Keywords: Coumarine heterocyclic, Acetylcholinesterase inhibitors, Alzheimer’s disease, Molecular docking, Molecular dynamic

Subject terms: Biochemistry, Chemistry

Introduction

Alzheimer’s disease is an age-related neurodegenerative disease that is associated with advanced cognitive impairment, memory loss, decreased language skills, psychological and behavioral disorders, and limitations in daily activities. Low levels of acetylcholine (ACh) play an important role in the pathophysiology of this disease^1–3. The enzyme acetylcholinesterase converts acetylcholine into inactive metabolites choline and acetate. The oldest and most common strategy for treating Alzheimer’s disease is to inhibit acetylcholinesterase, so that the cholinergic neurotransmitter in the brain increases as acetylcholine levels increase⁴. Figure 1shows the structures of a number of common therapeutic agents for Alzheimer’s disease, namely tacrine, donepezil, and rivastigmine and galantamine, all of which are acetylcholinesterase inhibitors⁵.

Fig. 1 — The structures of several common therapeutic agents for Alzheimer’s disease, along with coumarins coupled with tacrine, quinoline, thiazole, and lipoic acid, known for their acetylcholinesterase inhibitory activity.

Given the complexity of Alzheimer’s disease pathogenesis, a drug targeting only a single mechanism is insufficient to prevent the disease’s progression or provide effective treatment. As a result, developing multifunctional anti-Alzheimer’s strategies has emerged as a key focus in research.

Coumarin (2H-1-benzopyran-2-one) is a bioactive compound found in various traditional Chinese medicines, including Angelica, Cnidium, and Psoralea. It has garnered significant interest due to its wide range of pharmacological activities, particularly in addressing neurological disorders like Alzheimer’s disease. Coumarin derivatives inhibit the catalytic activity of cholinesterase enzymes, which helps maintain acetylcholine levels in the brain, thereby functioning as anti-Alzheimer’s agents. Research indicates that both natural and synthetic coumarin analogs possess acetylcholinesterase inhibitory effects, along with antioxidant properties. Consequently, in this study, coumarin was chosen as the core structure for developing anti-Alzheimer’s disease agents.

The literature survey shows that compounds containing a coumarin heterocyclic section have valuable pharmaceutical activity, and molecular hybridization, which combines different pharmacophores, has always been an effective tool to design new bioactive compounds and enhance the desired biological effects^6–9. Coupling of coumarine with tacrine¹⁰, quinoline¹¹, thiazole¹²and lipoic acid¹³ have excellent acetylcholine esterase inhibitor activity (Fig. 1). In this research, we aimed to design and synthesize a series of 4-(benzo[d]thiazole-2-yl) phenols based on 4-hydroxycoumarin, with the goal of developing new acetylcholinesterase inhibitors. Following positive theoretical studies, we have synthesized these compounds, designated as 3a-3o, and tested them for their in vitro activity. We then performed computational studies to investigate their binding interactions with the active site of acetylcholinesterase.

Materials and methods

General procedure for synthesis of 4-(n-bromoalkoxy)-2H-chromene-2-one

In a 25 mL flask, a mixture of 4-hydroxy coumarine (1 mmol), K₂CO₃ (1 mmol) and DMF (3 mL) was mixed for 30 min in room temperature. Then, 3 mmol of dibromo alkane (1,4-dibromobutane, 1,5-dibromo pentane or 1,6-dibromo hexane) was added to mixture and mixed until completion of reaction. The progress of reaction was monitored by TLC (solvent, n-Hexane: Ethylacetate, 7:3). After completion of reaction, water was added to reaction mixture to precipitate the product. The obtained precipitate was washed with n-Hexane and the recrystallized in hot ethanol.

Spectral data of 4-(n-bromoalkoxy)-2H-chromene-2-ones

4-(4-Bromobutoxy)-2H-chromen-2-one (1a).

White solid, m.p. 102–104 °C. FT-IR (ATR) ῡ (cm^-1): 3086, 2958, 2850, 1731, 1629, 1567, 1379, 1272, 1236, 1106, 1029, 905, 771, 751, 557. ¹H NMR (CDCl₃, 500 MHz)/δ (ppm): 7.81 (d, J = 7.5 Hz, 1H), 7.56 (t, J = 7.0 Hz, 1H), 7.32 (d, J = 8.5 Hz, 1H), 7.28 (t, J = 7.0 Hz, 1H), 5.67 (s, 1H), 4.19 (brs, 2 H), 3.52 (brs, 2 H), 2.12 (brs, 4H). ¹³C NMR (CDCl₃, 125 MHz)/δ (ppm): 165.4, 153.3, 132.4, 123.9, 122.9, 116.8, 90.6, 68.4, 32.8, 29.2, 27.2.

4-((5-Bromopentyl)oxy)-2H-chromen-2-one (1b).

White solid, m.p. 90–92 °C. FT-IR (ATR) ῡ (cm^-1): 2943, 2867, 1704, 1619, 1604, 1561, 1378, 1237, 1176, 1108, 923, 810, 784, 646. ¹H NMR (DMSO-d₆, 400 MHz)/δ (ppm): 7.79 (d, J = 7.6 Hz, 1H), 7.66 (t, J = 7.6 Hz, 1H), 7.32-7.41 (m, 2 H), 5.88 (s, 1H), 4.22 (t, J = 5.6 Hz, 2 H), 3.58 (t, J = 6.4 Hz, 2 H), 1.8-1.97 (m, 4 H), 1.49-1.69 (m, 2 H). ¹³C NMR (DMSO-d₆, 100 MHz)/δ (ppm): 165.4, 162.1, 153.2, 133.2, 124.6, 123.2, 116.9, 115.7, 90.9, 69.9, 69.7, 61.0, 35.4, 32.2, 27.5, 24.6.

4-((6-Bromohexyl)oxy)-2H-chromen-2-one (1c).

White solid, m.p. 104–106 °C. FT-IR (ATR) ῡ (cm^-1): 2943, 2862, 1709, 1609, 1559, 1373, 1271, 1235, 1104, 976 926, 782, 751, 643. ¹H NMR (CDCl₃, 400 MHz)/δ (ppm): 7.85 (d, J = 7.2 Hz, 1H), 7.58 (m, 1H), 7.28-7.37 (m, 2 H), 5.70 (s, 1H), 4.17 (m, 2 H), 3.47 (m, 2 H), 1.96 (brs, 4H), 1.59 (brs, 4H). ¹³C NMR (CDCl₃, 100 MHz)/δ (ppm): 165.7, 163.0, 153.4, 132.4, 123.9, 123.0, 116.8, 115.8, 90.5, 69.2, 33.7, 32.5, 28.4, 27.8, 25.7, 25.3.

General procedure for synthesis of 4-(benzo[d]thiazole-2-yl)-phenols.

In a 25 mL flask, a mixture of 2-aminothiophenol (1 mmol), 4-hydroxy benzaldehyde (1 mmol) and PEG-400 was mixed in room temperature. The progress of reaction was monitored by TLC (solvent, n-Hexane: Ethyl acetate, 7:3). After completion of reaction, the mixture was solved in hot ethanol and cooled. Water was added to cold mixture and crushed to precipitate the product. The solid product was crystalized with ethanol.

Spectral data of 4-(benzo[d]thiazole-2-yl)-phenols

4-(Benzo[d]thiazol-2-yl)phenol (2a).

Pale yellow solid, m.p. 223–225 °C (219–220 °C. FT-IR (ATR) ῡ (cm⁻¹): 2904, 2673, 2589, 1602, 1520, 1480, 1427, 1382, 1283, 1221, 1162, 974, 824, 753, 721.). ¹HNMR (CDCl₃, 500 MHz)/δ (ppm): 10.20 (s, 1H), 8.08 (d, J = 8.0 Hz, 1H), 7.98 (d, J = 8.0 Hz, 1H), 7.94 (d, J = 8.5 Hz, 2H), 7.50 (t, J = 7.5 Hz, 1H), 7.40 (t, J = 7.5 Hz, 1H), 6.94 (d, J = 8.0 Hz, 2H).

4-(Benzo[d]thiazol-2-yl)-2-methoxyphenol (2b).

Cream solid, m.p. 169–171 °C (171–173 °C). FT-IR (ATR) ῡ (cm^-1): 3098, 3005, 2934, 1604, 1583, 1525, 1476, 1425, 1275, 1193, 1124, 1010, 871, 753, 725, 651. ¹HNMR (CDCl₃, 400 MHz)/δ (ppm): 8.05 (d, J = 8.4 Hz, 1H), 7.88 (d, J = 8.0 Hz, 1H), 7.77 (s, 1H), 7.54 (d, J = 8.4 Hz, 1H), 7.48 (t, J = 7.6 Hz, 1H), 7.37 (t, J = 7.6 Hz, 1H), 7.01 (d, J = 8.0 Hz, 1H), 6.13 (s, 1H), 4.03 (s, 3H).

4-(Benzo[d]thiazol-2-yl)-2-ethoxyphenol (2c).

Pale yellow solid, m.p. 120–123 °C (125–126 °C) 1577, 1481, 1426, 1365, 1319, 1204, 1171, 1024, 977, 958, 833, 753, 727. ¹H NMR (DMSO-d₆, 400 MHz)/δ (ppm): 9.78 (brs, 1H), 8.06 (d, J = 7.6 Hz, 1H), 7.97 (d, J = 8.0 Hz, 1H), 7.59 (s, 1H), 7.46–7.49 (m, 2H), 7.39 (t, J = 7.6 Hz, 1H), 6.93 (d, J = 8.0 Hz, 1H), 4.13 (q, J = 6.8 Hz, 2H), 1.38 (t, J = 6.8 Hz, 3H).

4-(Benzo[d]thiazol-2-yl)-2,6-dimethoxyphenol (2d).

Pale yellow solid, m.p. 151–153 °C (153–156 °C). FT-IR (ATR) ῡ (cm⁻¹): 3113, 2992, 2942, 2839, 1608, 1529, 1482, 1450, 1422, 1333, 1279, 1196, 1172, 1108, 1078, 1050, 1024, 889, 858. ¹H NMR (DMSO-d₆, 400 MHz)/δ (ppm): 9.22 (s, 1H), 8.08 (d, J = 8.0 Hz, 1H), 8.00 (d, J = 8.4 Hz, 1H), 7.50 (t, J = 7.2 Hz, 1H), 7.40 (t, J = 7.2 Hz, 1H), 7.30 (s, 2H), 3.88 (s, 6H).

4-(Benzo[d]thiazol-2-yl)-3-methoxyphenol (2e).

Pale yellow solid, m.p. 234–236 °C. FT-IR (ATR) ῡ (cm^-1): 2939, 2834, 1613, 1577, 1481, 1426, 1365, 1319, 1277, 1203, 1129, 1023, 876, 833, 753. ¹H NMR (DMSO-d₆, 400 MHz)/δ (ppm): 10.30 (brs, 1H), 8.25 (dd, J = 8.8 Hz, J = 2.8 Hz, 1H), 8.04 (d, J = 7.6 Hz, 1H), 7.94 (d, J = 7.6 Hz, 1H), 7.47 (t, J = 7.2 Hz, 1H), 7.35 (t, J = 7.6 Hz, 1H), 6.61 (brs, 1H), 6.57 (dd, J = 8.4 Hz, J = 2.0 Hz, 1H), 3.98 (s, 3H).

General procedure for synthesis of 4-(Benzo[d]thiazol-2-yl)- phenols Based on 4-hydroxy coumarine.

In a 25 mL flask, a mixture of 4-(n-bromoalkoxy)-2H-chromen-2-one (1 mmol), 4-(Benzo[d]thiazol-2-yl)- phenols (1mmol), K₂CO₃ (1 mmol) and DMF (3 mL) was mixed at 75 °C. The progress of reaction was monitored by TLC (solvent, n-Hexane: Ethyl acetate, 7:3). After completion of the reaction, the product was extracted with CHCl₃, evaporate solvent, and recrystallized in hot ethanol.

Spectroscopic data of 4-(Benzo[d]thiazol-2-yl)- phenols Based on 4-hydroxy coumarine:

4-(4-(4-(Benzo[d]thiazol-2-yl)phenoxy)butoxy)-2H-chromen-2-one (3a).

White solid, m.p. 183–185 °C. FT-IR (ATR) ῡ (cm^-1): 3084, 2944, 2876, 1711, 1606, 1487, 1415, 1372, 1237, 1174, 1107, 1036, 959, 930, 812, 753, 728, 689. ¹H NMR (CDCl₃, 500 MHz)/δ (ppm): 8.03 (d, J = 8.0 Hz, 3H), 7.88 (d, J = 8.0 Hz, 1H), 7.78 (d, J = 8.0 Hz, 1H), 7.53 (t, J = 7.5 Hz, 1H), 7.47 (t, J = 7.5 Hz, 1H), 7.36 (t, J = 7.5 Hz, 1H), 7.31 (d, J = 8.5 Hz, 1H), 7.22–7.26 (m, 1H), 7.00 (d, J = 8.0 Hz, 2H), 5.69 (s, 1H), 4.23 (t, J = 6.0 Hz, 2H), 4.15 (t, J = 6.0 Hz, 2H), 2.15 (quintet, J = 6 Hz, 2H), 2.08 (quintet, J = 6 Hz, 2H).¹³C NMR (CDCl₃, 125 MHz)/δ (ppm): 167.8, 165.5, 162.8, 161.1, 153.9, 153.3, 134.7, 132.5, 132.4, 129.2, 126.4, 126.3, 124.8, 123.8, 122.9, 122.7, 121.5, 116.8, 115.6, 114.8, 90.5, 68.9, 67.3, 25.8, 25.4.

4-(4-(4-(Benzo[d]thiazol-2-yl)-2-methoxyphenoxy)butoxy)-2H-chromen-2-one (3b).

Cream solid, m.p. 152 °C. FT-IR (ATR) ῡ (cm^-1): 3068, 2938, 1723, 1622, 1565, 1489, 1416, 1265, 1236, 1139, 1106, 926, 864, 751. ¹H NMR (CDCl₃, 400 MHz)/δ (ppm): 8.19 (brs, 1H), 7.88 (d, J = 8.0 Hz, 2H), 7.78 (d, J = 7.2 Hz, 1H), 7.63 (d, J = 7.6 Hz, 1H), 7.52 (brs, 2H), 7.43 (t, J = 7.2Hz, 1H), 7.30 (d, J = 7.2 Hz, 1H), 7.23–7.26 (m, 1H), 6.95 (d, J = 8.0 Hz, 1H), 5.71 (s, 1H), 4.28 (brs, 2H), 4.22 (brs, 2H), 4.04 (s, 3H), 2.16 (brs, 4H). ¹³C NMR (CDCl₃, 100 MHz)/δ (ppm): 167.8, 165.6, 163.0, 154.1, 153.3, 150.7, 149.6, 134.9, 132.4, 126.8, 124.9, 123.8, 122.9, 122.8, 121.5, 121.0, 116.8, 116.7, 115.7, 115.2, 112.2, 110.1, 90.5, 69.1, 68.4, 56.1, 25.7, 25.6.

4-(4-(4-(Benzo[d]thiazol-2-yl)-2-ethoxyphenoxy)butoxy)-2H-chromen-2-one (3c).

Light brown solid, m.p. 150–152 °C. FT-IR (ATR) ῡ (cm⁻¹): 3076, 2928, 2874, 1723, 1626, 1489, 1393, 1267, 1243, 1188, 1143, 1050, 930, 749. ¹H NMR (DMSO-d₆, 400 MHz)/δ (ppm): 8.08 (d, J = 7.6 Hz, 1H), 8.00 (d, J = 8.0 Hz, 1H), 7.76 (d, J = 7.6 Hz, 1H), 7.55–7.62 (m, 3H), 7.50 (t, J = 7.6 Hz, 1H), 7.41 (t, J = 7.6 Hz, 1H), 7.36 (d, J = 8.0 Hz, 1H), 7.30 (t, J = 7.6 Hz, 1H), 7.13 (d, J = 8.0 Hz, 1H), 5.89 (s, 1H), 4.32 (brs, 2H), 4.17 (brs, 2H), 4.11 (q, J = 6.8 Hz, 2H), 1.99 (brs, 4H), 1.34 (t, J = 6.4 Hz, 3H). ¹³C NMR (DMSO-d₆, 100 MHz)/δ (ppm): 167.7, 165.4, 162.1, 154.1, 153.2, 151.5, 148.9, 134.7, 133.1, 127.0, 126.1, 125.6, 124.5, 123.2, 122.9, 122.6, 121.3, 116.9, 115.7, 113.7, 111.4, 91.0, 69.7, 68.5, 64.5, 25.5, 25.3.

4-(4-(4-(Benzo[d]thiazol-2-yl)-2,6-dimethoxyphenoxy)butoxy)-2H-chromen-2-one (3d).

Cream solid, m.p. 131–132 °C. FT-IR (ATR) ῡ (cm^-1): 3074, 2953, 1721, 1623, 1415, 1372, 1241, 1179, 1124, 1029, 925, 849, 755. ¹H NMR (CDCl₃, 400 MHz)/δ (ppm): 8.12 (d, J = 7.6 Hz, 1H), 8.05 (d, J = 8.0 Hz, 1H), 7.78 (d, J = 7.2 Hz, 1H), 7.62 (t, J = 6.4 Hz, 1H), 7.52 (t, J = 7.6 Hz, 1H), 7.44 (t, J = 7.2 Hz, 1H), 7.28–7.38 (m, 4H), 5.90 (s, 1H), 4.28-4.38 (m, 2H), 4.00–4.01 (m, 2H), 3.89 (s, 6 H), 1.98–2.08 (m, 2H), 1.80–1.90 (m, 2H). ¹³C NMR (CDCl₃, 100 MHz)/δ (ppm): 167.8, 165.7, 163.0, 153.7, 153.4, 139.5, 135.0, 132.5, 132.3, 129.2, 126.4, 125.2, 123.8, 123.1, 123.0, 121.6, 116.8, 115.8, 104.8, 90.5, 72.6, 69.2, 56.4, 26.6, 25.3.

4-(4-(4-(Benzo[d]thiazol-2-yl)-3-methoxyphenoxy)butoxy)-2H-chromen-2-one (3e).

Cream solid, m.p. 161–162 °C. FT-IR (ATR) ῡ (cm^-1): 3082, 2957, 2887, 1716, 1610, 1455, 1418, 1372, 1268, 1189, 1107, 1018, 932, 849, 751. ¹H NMR (CDCl₃, 400 MHz)/δ (ppm): 8.49 (d, J = 8.8 Hz, 1H), 8.07 (d, J = 8.0 Hz, 1H), 7.93 (d, J = 8.0 Hz, 1H), 7.81 (t, J = 8.0 Hz, 2H), 7.56 (t, J = 8.0 Hz, 1H), 7.50 (t, J = 7.6 Hz, 1H), 7.35–7.40 (m, 2H), 6.70 (d, J = 8.8 Hz, 1H), 6.61 (s, 1H), 5.74 (s, 1H), 4.26–4.32 (m, 2H), 4.20 (t, J = 5.6 Hz, 2H), 4.06 (s, 3H), 2.18–2.24 (m, 2H), 2.07–2.15 (m, 2H). ¹³C NMR (CDCl₃, 100 MHz)/δ (ppm): 165.6, 163.2, 162.9, 162.7, 158.6, 153.4, 152.2, 135.6, 132.4, 130.8, 125.8, 124.2, 123.9, 122.9, 122.3, 121.1, 116.8, 115.8, 115.5, 106.2, 99.0, 90.5, 68.9, 67.5, 55.7, 25.9, 25.4.

4-((5-(4-(Benzo[d]thiazol-2-yl)phenoxy)pentyl)oxy)-2H-chromen-2-one (3f).

Cream solid, m.p. 135–136 °C. FT-IR (ATR) ῡ (cm^-1): 3068, 2933, 1717, 1626, 1487, 1381, 1248, 1178, 1111, 1036, 914, 831, 762. ¹H NMR (DMSO-d₆, 400 MHz)/δ (ppm): 8.10 (d, J = 8.0 Hz, 1H), 8.02 (d, J = 8.8 Hz, 3H), 7.81 (d, J = 8.0 Hz, 1H), 7.66 (t, J = 7.6 Hz, 1H), 7.53 (t, J = 8.0 Hz, 1H), 7.35–7.46 (m, 3H), 7.11 (d, J = 8.0 Hz, 2H), 5.91 (s, 1H), 4.26 (t, J = 6.0 Hz, 2H), 4.12 (t, J = 6.4 Hz, 2H), 1.86 (quintet, J = 6.4 Hz, 2H), 1.92 (quintet, J= 6.4 Hz, 2H), 1.65 (quintet, J = 8 Hz, 2H). ¹³C NMR (DMSO-d₆, 100 MHz)/δ (ppm): 167.5, 165.4, 162.2, 161.6, 154.1, 153.2, 133.2, 129.3, 126.9, 125.8, 125.5, 124.6, 122.9, 122.6, 116.9, 115.7, 115.6, 90.0, 69.8, 68.1, 28.6, 28.1, 22.5.

4-((5-(4-(Benzo[d]thiazol-2-yl)-2-methoxyphenoxy)pentyl)oxy)-2H-chromen-2-one (3 g).

Light green solid, m.p. 129–132 °C. FT-IR (ATR) ῡ (cm^-1): 3070, 2943, 2867, 1707, 1619, 1470, 1415, 1370, 1238, 1138, 1008, 924, 867, 764. ¹H NMR (DMSO-d₆, 400 MHz)/δ (ppm): 8.06 (d, J = 7.6 Hz, 1H), 8.02 (d, J = 8.0 Hz, 1H), 7.75 (d, J = 7.2 Hz, 1H), 7.60–7.64 (m, 2H), 7.49–7.55 (m, 2H), 7.41 (t, J = 7.6 Hz, 1H), 7.30–7.37 (m, 2H), 7.06 (d, J = 8.0 Hz, 1H), 5.86 (s, 1H), 4.19 (brs, 2H), 4.05 (brs, 2H), 3.89 (s, 3H), 1.80–1.91 (m, 4H), 1.58–1.70 (m, 2H). ¹³C NMR (DMSO-d₆, 100 MHz)/δ (ppm): 167.7, 165.3, 162.1, 154.1, 151.4, 149.7, 134.7, 133.0, 126.9, 125.9, 125.5, 124.5, 123.2, 122.9, 122.5, 121.3, 116.8, 115.7, 113.2, 110.0, 90.9, 69.8, 68.6, 56.1, 28.7, 0.28.1, 22.5.

4-((5-(4-(Benzo[d]thiazol-2-yl)-2-ethoxyphenoxy)pentyl)oxy)-2H-chromen-2-one (3h).

Light brown solid, m.p. 128–131 °C. FT-IR (ATR) ῡ (cm^-1): 3079, 2931, 1726, 1623, 1488, 1425, 1375, 1243, 1183, 1036, 831, 751. ¹H NMR (DMSO-d₆, 400 MHz)/δ (ppm): 8.09 (d, J = 8.0 Hz, 1H), 8.02 (d, J = 8.0 Hz, 1H), 7.80 (d, J = 7.6 Hz, 1H), 7.63-7.68 (m, 2H), 7.58 (d, J = 8.4 Hz, 1H), 7.53 (t, J = 7.6 Hz, 1H), 7.33-7.45 (m, 3H), 7.13 (d, J = 8.4 Hz, 1H), 5.91 (s, 1H), 4.25 (t, J = 5.6 Hz, 2H), 4.09-4.16 (m, 4H), 1.93 (quintet, J = 6.0 Hz, 2H), 1.87 (quintet, J = 6.8 Hz, 2H), 1.66 (quintet, J = 7.6 Hz, 2H), 1.36 (t, J = 6.8 Hz, 3H). ¹³C NMR (DMSO-d₆, 100 MHz)/δ (ppm): 167.7, 165.4, 162.1, 154.1, 153.2, 151.7, 148.9, 134.7, 133.1, 126.9, 126.0, 125.6, 124.6, 123.2, 122.9, 122.6, 121.4, 116.9, 115.7, 113.7, 111.5, 90.9, 69.8, 68.7, 64.6, 28.6, 28.1, 22.6, 15.1.

4-((5-(4-(Benzo[d]thiazol-2-yl)-2,6-dimethoxyphenoxy)pentyl)oxy)-2H-chromen-2-one (3i).

Light brown solid, m.p. 139–141 °C. FT-IR (ATR) ῡ (cm^-1): 3090, 2937, 2879, 1710, 1618, 1514, 1483, 1413, 1371, 1333, 1244, 1183, 1127, 929, 768. ¹H NMR (DMSO-d₆, 400 MHz)/δ (ppm): 8.14 (d, J = 8.0 Hz, 1H), 8.08 (d, J = 8.0 Hz, 1H), 7.84 (d, J = 7.6 Hz, 1H), 7.67 (t, J = 8.0 Hz, 1H), 7.56 (t, J = 7.2 Hz, 1H), 7.47 (t, J = 8.0 Hz, 1H), 7.38–7.42 (m, 2H), 7.35 (s, 2H), 5.93 (s, 1H), 4.27 (t, J = 6.0 Hz, 2H), 4.01 (t, J = 6.0 Hz, 2H), 3.91 (s, 6 H), 1.92 (quintet, J = 6.4 Hz, 2H), 1.78 (quintet, J = 6.4 Hz, 2H), 1.67 (quintet, J = 7.2 Hz, 2H). ¹³C NMR (DMSO-d₆, 100 MHz)/δ (ppm): 167.6, 165.5, 162.2, 154.0, 153.9, 153.3, 133.2, 127.1, 124.7, 123.3, 123.2, 122.7, 116.9, 104.9, 90.9, 72.9, 70.0, 29.7, 28.2, 22.5.

4-((5-(4-(Benzo[d]thiazol-2-yl)-3-methoxyphenoxy)pentyl)oxy)-2H-chromen-2-one (3j).

Pale yellow solid, m.p. 180–181 °C. FT-IR (ATR) ῡ (cm^-1): 2945, 2868, 1708, 1605, 1457, 1270, 1240, 1205, 1109, 1022, 929, 818, 764. ¹H NMR (DMSO-d₆, 400 MHz)/δ (ppm): 8.37 (d, J = 8.8 Hz, 1H), 8.10 (d, J = 7.6 Hz, 1H), 8.00 (d, J = 8.4 Hz, 1H), 7.83 (t, J = 8.0 Hz, 1H), 7.68 (t, J = 7.6 Hz, 1H), 7.52 (t, J = 7.2 Hz, 1H), 7.39–7.43 (m, 3H), 6.83 (s, 1H), 6.78 (d, J = 8.8 Hz, 1H), 5.94 (s, 1H), 4.30 (t, J = 5.6 Hz, 2H), 4.18 (t, J = 6.0 Hz, 2H), 4.06 (s, 3H), 1.93–1.97 (m, 2H), 1.87-1.93 (m, 2H), 1.67–1.73 (m, 2H). ¹³C NMR (DMSO-d₆, 100 MHz)/δ (ppm): 165.5, 162.2, 158.9, 153.3, 152.1, 135.3, 133.2, 130.4, 126.6, 124.9, 124.7, 123.3, 122.4, 122.1, 116.9, 115.8, 107.8, 99.5, 91.0, 69.8, 68.3, 56.6, 28.7, 28.1, 22.5.

4-((6-(4-(Benzo[d]thiazol-2-yl)phenoxy)hexyl)oxy)-2H-chromen-2-one (3k).

White solid, m.p. 152–153 °C. FT-IR (ATR) ῡ (cm^-1): 2942, 2864, 1716, 1607, 1478, 1415, 1244, 1173, 1108, 930, 831, 757. ¹H NMR (CDCl₃, 400 MHz)/δ (ppm): 8.05 (d, J = 8.8 Hz, 2H), 7.91 (d, J = 8.0 Hz, 1H), 7.84 (t, J = 8.0 Hz, 1H), 7.57 (t, J = 7.6 Hz, 1H), 7.50 (t, J = 7.2 Hz, 1H), 7.32–7.41 (m, 2H), 7.26–7.29 (m, 2H), 7.17 (d, J = 8.8 Hz, 2H), 5.71 (s, 1H), 4.19 (t, J = 6.4 Hz, 2H), 4.10 (t, J = 6.4 Hz, 2H), 2.00 (m, 2H), 1.91 (m, 2H), 1.66 (m, 4H). ¹³C NMR (CDCl₃, 100 MHz)/δ (ppm): 167.9, 165.7, 163.0, 161.4, 154.2, 153.4, 134.8, 132.4, 132.3, 129.1, 126.4, 126.2, 124.8, 123.9, 122.9, 122.8, 121.5, 116.8, 115.8, 114.8, 90.5, 69.2, 69.0, 67.9, 29.1, 28.5, 28.4, 25.8, 25.6.

4-((6-(4-(Benzo[d]thiazol-2-yl)-2-methoxyphenoxy)hexyl)oxy)-2H-chromen-2-one (3 L).

Cream solid, m.p. 135–138 °C. FT-IR (ATR) ῡ (cm^-1): 2940, 2857, 1723, 1621, 1492, 1415, 1265, 1240, 1177, 1132, 1031, 916, 749, 648. ¹H NMR (CDCl₃, 400 MHz)/δ (ppm): 8.15 (d, J = 7.2 Hz, 1H), 7.80–7.89 (m, 3H), 7.62 (d, J = 7.6 Hz, 1H), 7.50–7.56 (m, 2H), 7.42 (t, J = 7.2 Hz, 1H), 7.31 (d, J = 8.0 Hz, 1H), 7.26-7.28 (m, 1H), 6.95 (d, J = 8.0 Hz, 1H), 5.68 (s, 1H), 4.08–4.15 (m, 4H), 4.03 (s, 3H), 1.96 (brs, 4H), 1.63 (brs, 4H).¹³C NMR (CDCl₃, 100 MHz)/δ (ppm): 165.7, 154.2, 151.1, 149.6, 134.9, 132.4, 126.6, 126.3, 124.9, 123.9, 123.0, 122.8, 121.5, 121.1, 116.8, 115.8, 112.3, 110.1, 90.4, 69.2, 68.7, 56.2, 28.9, 28.4, 25.8, 25.7.

4-((6-(4-(Benzo[d]thiazol-2-yl)-2-ethoxyphenoxy)hexyl)oxy)-2H-chromen-2-one (3 m).

Cream solid, m.p. 151–152 °C. FT-IR (ATR) ῡ (cm^-1): 3071, 2937, 1726, 1625, 1489, 1429, 1268, 1243, 1178, 1141, 998, 923, 762. ¹H NMR (DMSO-d₆, 400 MHz)/δ (ppm): 8.12 (d, J = 7.6 Hz, 1H), 8.03 (d, J = 8.0 Hz, 1H), 7.82 (d, J = 8.0 Hz, 1H), 7.60–7.68 (m, 3H), 7.54 (t, J = 7.2 Hz, 1H), 7.34-7.46 (m, 3H), 7.14 (d, J = 8.4 Hz, 1H), 5.91 (s, 1H), 4.26 (t, J = 5.2 Hz, 2H), 4.09–4.16 (m, 4H), 1.81–1.89 (m, 4H), 1.58 (brs, 4H), 1.37 (t, J = 6.0 Hz, 3H). ¹³C NMR (DMSO-d₆, 100 MHz)/δ (ppm): 167.7, 165.5, 162.2, 154.1, 153.2, 151.8, 149.0, 134.7, 133.2, 127.0, 126.0, 125.6, 123.2, 122.9, 122.6, 121.5, 116.9, 115.7, 113.8, 111.6, 90.9, 69.8, 68.8, 64.6, 28.9, 28.3, 25.5, 15.2.

4-((6-(4-(Benzo[d]thiazol-2-yl)-2,6-dimethoxyphenoxy)hexyl)oxy)-2H-chromen-2-one (3n).

Cream solid, m.p. 113–115 °C. FT-IR (ATR) ῡ (cm^-1): 2945, 1717, 1623, 1485, 1412, 1334, 1130, 1065, 984, 806, 766. ¹H NMR (CDCl₃, 400 MHz)/δ (ppm): 8.08 (d, J = 8.4 Hz, 1H), 7.92 (d, J = 8.0 Hz, 1H), 7.85 (d, J = 7.6 Hz, 1H), 7.50–7.57 (m, 2H), 7.41 (t, J = 7.2 Hz, 1H), 7.35 (s, 2H), 7.25-7.32 (m, 2H), 5.69 (s, 1H), 4.17 (t, J = 4.8 Hz, 2H), 4.11 (t, J = 6.4 Hz, 2H), 4.00 (s, 6 H), 1.98 (brs, 2H), 1.87 (brs, 2H), 1.65 (brs, 4H). ¹³C NMR (CDCl₃, 100 MHz)/δ (ppm): 167.8, 165.7, 163.0, 154.1, 153.8, 153.4, 139.9, 135.0, 132.3, 128.9, 126.4, 125.1, 123.8, 123.1, 128.0, 121.6, 116.8, 115.8, 104.9, 90.4, 73.3, 69.4, 56.4, 30.0, 28.5, 25.8, 25.6.

4-((6-(4-(Benzo[d]thiazol-2-yl)-3-methoxyphenoxy)hexyl)oxy)-2H-chromen-2-one (3o).

Cream solid, m.p. 156–157 °C. FT-IR (ATR) ῡ (cm^-1): 2943, 2863, 1710, 1609, 1568, 1463, 1415, 1269, 1244, 1185, 1027, 916, 826. ¹H NMR (CDCl₃, 400 MHz)/δ (ppm): 8.47 (d, J = 8.8 Hz, 1H), 8.06 (d, J = 8.0 Hz, 1H), 7.92 (d, J = 8.0 Hz, 1H), 7.82 (d, J = 7.6 Hz, 1H), 7.55 (t, J = 7.6 Hz, 1H), 7.48 (t, J = 7.2 Hz, 1H), 7.26–7.37 (m, 3H), 6.67 (dd, J = 8.4 Hz, J = 1.2 Hz, 1H), 6.58(s, 1H), 5.68 (s, 1H), 4.14 (t, J = 6.0 Hz, 2H), 4.08 (t, J = 6.0 Hz, 2H), 4.04 (s, 3H), 1.96 (brs, 2H), 1.89 (brs, 2H), 1.62 (brs, 4H). ¹³C NMR (CDCl₃, 100 MHz)/δ (ppm): 165.7, 163.3, 163.0, 162.4, 158.6, 153.4, 152.2, 135.6, 132.4, 130.7, 125.8, 124.1, 123.9, 122.9, 122.3, 121.1, 116.8, 115.8, 115.5, 106.4, 98.9, 90.4, 69.2, 67.9, 55.7, 41.0, 29.1, 28.4, 25.8.

In vitro acetylcholinesterase inhibition assay

The inhibition potential of the synthesized compounds toward AChE (Electrophorus electricus) was assessed using spectrophotometric method of Ellman. Donepezil was selected as reference drug. Five different concentrations of each compound were tested to obtain 20–80% enzyme inhibition. The test medium, consisted of 2 mL phosphate buffer (0.1 M, pH 8.0), 60 µL of 5,5′-dithio-bis(2-nitrobenzoic acid) (DTNB, 0.01 M), enzyme (20 µL of 2.5 unit/mL) and 30 µL of compound solution was incubated at 25 °C for 5 min and then, 20 µL of substrate (acetylthiocholine iodide) was added. As a reference, a solution containing all ingredients except the synthesized compounds was used. Changes in absorbance were measured at 412 nm for 2 min at 25 °C and inhibition percent of compounds was determined. The IC₅₀ values were obtained by the method of plotting log concentration vs. percentage of inhibition.

Molecular modeling

Molecular dynamics simulation

Molecular dynamics was employed to establish a stable protein structure prior to conducting molecular docking, considering specific physiological and environmental influences affecting proteins. Thorough examination and analysis of MD simulations were performed as outlined previously. In earlier research, MD simulations were conducted under the following parameters: The protein’s three-dimensional structure was acquired from the Protein Data Bank (PDB) using the PDB code 4EY7 for AChE with donepezil. The MD simulation was carried out utilizing the AMBER 12.0 simulation package¹⁴. MD simulations were conducted employing the Amber99sb-ildn force field¹⁵, maintaining a constant temperature of 300 K and physiological pH of 7. The topology, coordinate files, and partial charges for donepezil were generated using the Antechamber program, an included tool within the AmberTools suite^16,17. A solute-box distance of 1.0 nm was established, ensuring a minimum separation of 2 nm between every two periodic images of a protein complex and the boundary of the box. Two protein models were hydrated with the TIP3P water molecules, and seven Na⁺ counter-ions were added into the system by substituting water molecules, ensuring overall charge neutrality. The system underwent energy minimization employing the steepest descent algorithm over a 100 ps duration until reaching a tolerance level of 10 kJ mol⁻¹.Then, the protein was targeted to a restriction so that the atom positions of the macromolecule and ligand were restrained, and the solvent molecules and counter-ions were soaked into the macromolecule during a 100 ps NVT and then a 100 ps NPT MD runs. The adequately equilibrated structures were subsequently utilized for 100 ns production runs, during which atomic coordinates were stored in the trajectory file every 10 ps. The long-ranged electrostatic contributions were calculated utilizing the particle-mesh Ewald (PME) algorithm¹⁸ with a direct interaction cut-off of 10 nm. The LINCS algorithm (Linear Constraint Solver), known for its three to four times faster computation compared to the SHAKE algorithm, was employed to maintain the lengths of covalent bonds. Upon completion, the trajectory underwent corrections concerning periodic boundary conditions, followed by down sampling where only 1000 frames out of 10,000 were preserved from the simulation process. Subsequently, the root-mean-square deviation (RMSD) in Å for the protein backbone atoms of each frame was calculated throughout the entire duration of the MD simulation, referenced to the initial frame.

Molecular docking studies

A molecular docking investigation was conducted to suggest various potential protein binding sites and assess the binding affinity for each binding mode. The synthesized compounds were subjected to molecular docking into the X-ray 3D receptor structure using the Smina program¹⁹. The 3D structures of the compounds were built using ChemDraw Pro 12.0 software and energy minimized. The crystal structures of the protein complex encoded 4EY7 were taken from the RCSB Protein Data Bank (http://www.rcsb.org). All bound water molecules and ligands were removed from the protein and polar hydrogens were added to the proteins. In the docking studies conducted on protein active sites, the ligand was treated as flexible, and both the binding pose and internal torsions were sampled using the biased probability Monte Carlo (BPMC) minimization procedure. This procedure involved local energy minimization following each random move²⁰. In the docking scenarios, the binding mode was determined by selecting the lowest energy docked conformation of the compounds based on the Smina scoring function. The output generated by Smina was visualized and analyzed using the BIOVIA Discovery Studio client 2016 (http://www.accelrys.com).

In silico ADMET study

Predicting ADMET (absorption, distribution, metabolism, excretion, and toxicity) properties is widely recognized as a crucial and challenging aspect in identifying and refining lead compounds throughout the drug discovery process^21,22. The properties of synthesized compounds were forecasted using Qikprop (http://www.schrodinger.com/QikProp) and ORSIS Data Warrior software²³.

Results and discussion

Synthesis of 4-(benzo[d]thiazol-2-yl)- phenols based on 4-hydroxy coumarine

The synthesis of 4-(benzo[d]thiazole-2-yl) phenols based on 4-hydroxy coumarine was done in three steps according to Scheme 1. These compounds were characterized by spectroscopic methods and were investigated their pharmaceutical effects.

In the first step, three component of 4-(n-bromoalkoxy)-2H-chromene-2-one were synthesized from reaction of 4-hydroxy coumarine with 1, n-dibromoalkane (n = 4,5,6) in the presence of K₂CO₃ in DMF at room temperature (Table 1).

Table 1.

Synthesis of 4-(n-bromo alkoxy)-2H chromene-2-one.


Entry	n	product	Time (h)	Yield (%)*
1a	2		4.5	85
1b	3		5	86
1c	4		5.5	88

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^*Isolated yield.

In the second step, five derivatives of 4-(benzo[d]thiazole-2-yl)phenols were synthesized from reaction of 2-aminothiophenol with various 4-hydroxy benzaldehydes in PEG-400 at room temperature (Table 2).

Table 2.

Synthesis of 4-(benzo[d]thiazole-2-yl)phenols in PEG-400 at room temperature.


Entry	R¹	Product	Time (h)	Yield (%)*
2a	H		7	95
2b	3-OMe		8	93
2c	3-OEt		8.5	94
2d	3,5-(OMe)₂		9	90
2e	2-OMe		10	85

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*Isolated yield.

In the third step, for finding the best reaction condition for synthesis of 4-(benzo[d]thiazole-2-yl)phenols based on 4-hydroxy coumarine, the reaction of 4-(4-bromo butoxy)-2H-chromen-2-one with 4-(benzo[d]thiazole-2-yl)phenol was selected as model reaction and done under various condition (Table 3).

Table 3.

Synthesis of 4-(4-(4-(benzo[d]thiazole-2-yl)phenoxy)butoxy)-2H-chromenr-2-one under various condition.


Entry	Condition	Solvent	Base	Yield (%)*
1	75 °C	DMF	NaH	50
2	75 °C	DMF	CH₃COONa	30
3	75 °C	DMF	NaHSO₄	–
4	75 °C	DMF	Pyridine	–
5	75 °C	DMF	Pipyridine	98
6	75 °C	DMF	K₂CO₃	98
7	R.T.	DMF	K₂CO₃	10
8	Reflux	Ethylacetate	K₂CO₃	30
9	R.T.	Ethylacetate	K₂CO₃	–
10	Reflux	Acetone	K₂CO₃	10
11	R.T.	Acetone	K₂CO₃	–
12	Reflux	Acetonitril	K₂CO₃	30
13	R.T.	Acetonitril	K₂CO₃	–

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*Isolated yield.

According to the best condition, some compounds from coupling of 4-(n-bromo alkoxy)-2H-chromen-2-one with 4-(benzo[d]thiazole-2-yl)phenols were synthesized and characterized (Table 4).

Table 4.

Synthesis of 3a-o by coupling of 4-(n-bromo alkoxy)-2H-chromene-2-ones with 4-(benzo[d]thiazole-2-yl)phenols.


Entry	Product	Compound 1	Compound 2	Time (h)	Yield (%)*
1		1a	2a	4	92
2		1a	2b	4	94
3		1a	2c	4.5	95
4		1a	2d	5	91
5		1a	2e	5.5	85
6		1b	2a	4	93
7		1b	2b	4	94
8		1b	2c	4.5	96
9		1b	2d	5	93
10		1b	2e	5.5	87
11		1c	2a	4	94
12		1c	2b	4	93
13		1c	2c	4.5	96
14		1c	2d	5	92
15		1c	2e	5.5	88

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*Isolated yield.

According to obtained data in Table 4, all compounds 3a–3o were synthesized with good yields. The phenolic compounds with electron releasing group on ortho position of OH, have produced higher yield of products than the phenolic compounds with electron releasing group on meta position of OH.

Investigation of acetylcholinesterase inhibitor activity of 3a–3o

The acetylcholine esterase activity of 3a–3o was investigated and the obtained results were tabulated in Table 5. All of the synthesized compounds have good acetylcholine esterase activity in the range of 2.7–8.35 µM. The compounds with five carbon linker (n = 3, 3f–3j), have higher activity than four or six carbon linker (n = 2, 4). The compounds 3f–3j have good flexibility with optimum size for coupling with enzyme cavity. The best activity was shown from compound 3i with two methoxy groups in 2 and 6 position of phenolic ring of benzothiazole section.

Table 5.

In vitro investigation about acetylcholine esterase inhibitory of 3a–3o.


Compound	n	R¹	IC₅₀ (µM) AChE
3a	2	H	4.23 ± 0.11
3b	2	2-OMe	16.5 ± 0.23
3c	2	2-OEt	15.8 ± 0.18
3d	2	2,6-(OMe)₂	6.8 ± 0.10
3e	2	3-OMe	18.7 ± 0.28
3f	3	H	8.5 ± 0.18
3g	3	2-OMe	6.8 ± 0.13
3h	3	2-OEt	6.5 ± 0.12
3i	3	2,6-(OMe)₂	2.7 ± 0.10
3j	3	3-OMe	7.5 ± 0.16
3k	4	H	35.8 ± 0.30
3l	4	2-OMe	33.2 ± 0.28
3m	4	2-OEt	27.9 ± 0.20
3n	4	2,6-(OMe)₂	12.1 ± 0.19
3o	4	3-OMe	30.7 ± 0.23

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Structure-activity relationships (sars)

Based on the enzyme inhibition activity experiments, the structure-activity relationships (SARs) of the compounds 3a-3o can be summarized as follows. The compounds with a five-carbon linker (n = 3, 3f, IC₅₀ = 8.5 ± 0.18; 3 g, IC₅₀ = 6.8 ± 0.13; 3 h, IC₅₀ = 6.5 ± 0.12; 3i, IC₅₀ = 2.7 ± 0.10; and 3j, IC₅₀ = 7.5 ± 0.16 µM) exhibit greater activity compared to those with a four- or six-carbon linker (n = 2, 4). Among the compounds with a four-carbon linker, 3d (IC₅₀ = 6.8 ± 0.10), a five-carbon linker, 3i (IC50 = 2.7 ± 0.10), and a six-carbon linker, 3n (IC₅₀ = 12.1 ± 0.19), an increase in inhibitory activity is observed when the R¹ group is 2,6-(OMe)₂. In summary, the following recommendations are crucial for further modification of the structure of 4-(benzo[d]thiazole-2-yl) phenols based on 4-hydroxy coumarin derivatives. First, the length of the carbon linker and the inclusion of the 2,6-(OMe)₂ group as the R¹ group are important for enhancing enzyme inhibitory activity.

Molecular dynamics analysis

Molecular dynamics (MD) simulation has emerged as a contemporary tool for examining the physical movements of atoms and molecules, significantly impacting the understanding of interactions between proteins and drugs. Drawing from previous research on molecular dynamics, 100 ns MD simulations were conducted for both ligand-free and ligand-bound systems to capture multiple conformations of the protein structure. To assess the conformational stability of the enzyme and ensure accurate investigation, the trajectory stability and flexibility of both ligand-free and ligand-bound forms were confirmed using root-mean-square deviation (RMSD) over time (see Fig. 2). Both the enzyme-donepezil complex and ligand-free enzyme exhibited an initial sharp rise in RMSD for the initial frames, followed by a consistent profile. Hence, the trajectories of the MD simulation post-equilibrium were deemed reliable for subsequent analyses.

Fig. 2 — The root-mean-square deviation (RMSD) (Å) of AChE enzyme for the backbone atoms in 100 ns MD simulation for two states of apo (blue) and complex (red).

Analysis of molecular docking results

The goal of molecular docking is to predict the optimal binding pose of a ligand within a protein’s binding site and assess its binding affinity through a scoring function. The process involved in molecular docking was utilized to identify key residues and binding modes of synthesized compounds within the active site of the AChE protein. The results presented in Table 6 indicate that the interaction energy between compound 3i and AChE is the lowest among the compounds studied. Compound 3i exhibited the highest potency with an IC50 value of 2.7 µM in cytotoxicity assessment. Additionally, the binding modes of compounds 3i and AChE were visualized in both 3D and 2D formats, as shown in Fig. 3. The proposed binding mode of compound 3i demonstrated an affinity value of − 9.06 kcal/mol, indicating significant interaction with the AChE protein, primarily through multiple hydrophobic interactions and two hydrogen bonds. Specifically, TYR69 formed pi-sulfur and two pi-pi interactions with 3i, while two hydrogen bonds were formed between TRP277 and TYR332 and 3i. Furthermore, van der Waals interactions were observed between 3i and surrounding receptor amino acids such as LEU280, TYR121, and TYR328, as illustrated in Fig. 2.

Table 6.

The docking predicted minimized affinity of the synthesized compounds.

Comp.	IC₅₀ (µM) AChE	Minimized affinity (kcal/mol)
3a	4.23	− 9.04
3b	16.5	− 8.33
3c	15.8	− 8.34
3d	6.8	− 8.5
3e	18.7	− 8.21
3f	8.5	− 8.42
3g	6.8	− 8.63
3h	6.5	− 8.78
3i	2.7	− 9.06
3j	7.5	− 8.45
3k	35.8	− 8
3l	33.2	− 8.08
3m	27.9	− 8.18
3n	12.1	− 8.36
3o	30.7	− 8.14

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Fig. 3 — 3D and 2D binding mode of compound 3i with AChE, only interacting residues were labeled. the hydrogen and hydrophobic interactions are represented in green dashed and orange and pink lines, respectively.

In silico ADMET analysis

ADMET information aids chemists in optimizing the pharmacokinetic properties of compounds during rational drug design. These descriptors encompass various factors such as logBB (blood-brain barrier permeability), skin permeability coefficient (logKp), apparent Caco-2 and Madin-Darby canine kidney (MDCK) cell permeability (where higher MDCK values indicate greater cell permeability), aqueous solubility (log S), number of metabolic reactions, logKhsa for serum protein binding, human oral absorption in the gastrointestinal tract, logP for octanol/water partition coefficient governing the hydrophilicity or lipophilicity of molecules, and toxicity risk assessment parameters including mutagenicity, carcinogenicity, irritant effects, and reproductive effects. Prediction of tissue distribution is a crucial aspect of drug development. Molecular descriptors like logP, logBB (blood-brain barrier permeability), and logKhsa (serum protein binding) have demonstrated utility in modeling distribution patterns. As indicated in Table 7, the synthesized compounds demonstrate a very low ability to penetrate the blood-brain barrier (BBB). Consequently, it is anticipated that all synthesized compounds are safe for the central nervous system and exhibit favorable distribution characteristics. The process of drug absorption following oral administration is complex yet convenient for medication. The values of Caco-2 and MDCK for the synthesized compounds indicate favorable membrane permeability properties. Moreover, descriptors such as logP, aqueous solubility logarithm, and polar surface area (PSA) have been proposed to assess the absorption process. In summary, nearly all synthesized compounds fall within acceptable ranges for the properties analyzed, aligning with 95% of all known drugs (refer to Table 7).

Table 7.

Prediction of ADME properties of the retrieved hits compound using Qikprop toxicity risk assessment using data warrior.

Descriptors	3a	3b	3c	3d	3e	3f	3 g	Stand. range^[a]
Apparent Caco-2 permeability (nm/s)	1654	1651	2049	1648	1776	1706	1698	< 25 poor, > 500 great
Apparent MDCK permeability (nm/s)	1388	1380	1756	1377	1488	1428	1427	< 25 poor, > 500 great
logS (aqueous solubility)	− 5.382	− 5.4	− 5.7	− 5.418	− 5.4	− 5.652	− 5.67	− 6.5/0.5
% human oral absorption in GI (± 20%)	100	100	100	92.358	100	100	100	< 25% is poor
log BB for brain/blood	− 0.707	− 0.788	− 0.687	− 0.842	− 0.745	− 0.771	− 0.825	− 3.0 to 1.2
logKhsa (serum protein binding)	0.801	0.782	0.78	0.713	0.781	0.938	0.864	− 1.5 to 1.5
logP for octanol/water	5.696	5.784	5.952	5.764	5.806	6.106	6.072	− 2.0 to 6.5
Skin-permeability coefficient (logKp)	− 0.499	− 0.55	− 0.254	− 0.592	− 0.48	− 0.377	− 0.431	− 8.0 to − 1.0, Kp in cm/h
PSA (topological polar surface area)	66.01	69.788	72.219	72.965	71.172	66.131	69.648	≤ 140 is great
Mutagenicity	No risk	No risk	No risk	No risk	No risk	No risk	No risk	−
Tumorigenicity	No risk	No risk	No risk	No risk	No risk	No risk	No risk	−
Irritating effects	No risk	No risk	No risk	No risk	No risk	No risk	No risk	−
Reproductive effects	No risk	No risk	No risk	No risk	No risk	No risk	No risk	−

Descriptors	3h	3i	3j	3k	3l	3m	3n	3o
Apparent Caco− 2 permeability (nm/s)	1688	1776	1879	1673	1672	1687	1681	1799
Apparent MDCK permeability (nm/s)	1415	1521	1562	1405	1399	1418	1384	1506
logS(aqueous solubility)	− 5.97	− 5.688	− 5.67	− 5.922	− 5.94	− 6.24	− 5.958	− 5.94
% human oral absorption in GI (± 20%)	100	94.443	100	100	100	100	100	100
log BB for brain/blood	− 0.93	− 0.846	− 0.85	− 0.863	− 0.945	− 1.011	− 1.028	− 0.898
logKhsa (serum protein binding)	1.036	0.767	1.006	1.067	1.041	1.18	0.991	1.027
logP for octanol/water	6.556	6.021	6.407	6.486	6.559	6.972	6.56	6.547
Skin− permeability coefficient (logKp)	− 0.332	− 0.399	− 0.381	− 0.302	− 0.351	− 0.243	− 0.446	− 0.298
PSA (topological polar surface area)	69.097	74.987	71.096	65.63	69.568	69.028	77.593	71.118
Mutagenicity	No risk	No risk	No risk	No risk	No risk	No risk	No risk	–
Tumorigenicity	No risk	No risk	No risk	No risk	No risk	No risk	No risk	-
Irritating effects	No risk	No risk	No risk	No risk	No risk	No risk	No risk	–
Reproductive effects	No risk	No risk	No risk	No risk	No risk	No risk	No risk	–

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[a] For 95% of known drugs, based on -Qikprop v.3.2 (Schrçdinger, USA, 2009) software results.

Conclusion

Herein, we have synthesized and evaluated a series of 4-(benzo[d]thiazole-2-yl) phenols based on 4-hydroxy coumarin as highly potent acetylcholinesterase inhibitors. The compounds demonstrated anti-acetylcholinesterase activity with IC50 values ranging from 2.7 to 35.8 µM.

The molecular docking study of 3i into AChE revealed that this molecule was fully nestled within the AChE pocket, exhibiting two pi-pi stacked interactions and a pi-sulfur interaction with TYR69. Additionally, two hydrogen bonding interactions with TRP277 and TYR332 were also observed. Nearly all synthesized compounds demonstrated satisfactory predictions regarding pharmacokinetic characteristics and toxicity properties. The present findings suggest that the length of the carbon linker and the inclusion of the 2,6-(OMe)₂ group as the R¹ group are important for enhancing the enzyme inhibitory activity of 4-(benzo[d]thiazole-2-yl) phenols based on 4-hydroxy coumarin derivatives. Therefore, it can be concluded that the compounds have the potential to be developed into safe and effective acetylcholinesterase inhibitors. In summary, this study has identified a new class of 4-(benzo[d]thiazole-2-yl) phenols derived from 4-hydroxycoumarin, which could serve as lead structures for the development of novel acetylcholinesterase inhibitors.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary Material 1^{(5.4MB, docx)}

Acknowledgements

The authors thank the Research Council of Yazd University for the support of this research.

Author contributions

BFM, SF and AF designed and performed the research, analysed the data, interpreted the results, and prepared the manuscript. AF performed the assay and conducted the optimization, and purification of compounds. All authors read and approved the final manuscript.

Funding

This study was financially supported by Yazd University. The funding bodies played no role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript.

Data availability

All data generated or analyzed during this study are included in this published article.

Declarations

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Contributor Information

Bi Bi Fatemeh Mirjalili, Email: fmirjalili@gmail.com.

Fereshteh Shiri, Email: fereshteh.shiri@gmail.com.

References

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22.Nazarshodeh, E., Shiri, F. & Ghasemi, J. B. 3D-QSAR and virtual screening studies in identification of new rho kinase inhibitors with different scaffolds. J. Iran. Chem. Soc.12 (11), 1945–1959 (2015). [Google Scholar]
23.Sander, T., Freyss, J., Von Korff, M. & Rufener, C. DataWarrior: an open-source program for chemistry aware data visualization and analysis. J. Chem. Inf. Model.55 (2), 460–473 (2015). [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Material 1^{(5.4MB, docx)}

Data Availability Statement

All data generated or analyzed during this study are included in this published article.

[CR1] 1.Shiri, F., Pirhadi, S. & Ghasemi, J. B. Dynamic structure based pharmacophore modeling of the acetylcholinesterase reveals several potential inhibitors. J. Biomol. Struct. Dyn.37 (7), 1800–1812 (2019). [DOI] [PubMed] [Google Scholar]

[CR2] 2.Salehi, N. et al. Synthesis and biological evaluation of new N-benzylpyridinium-based benzoheterocycles as potential anti-alzheimer’s agents. Biorg Chem.83, 559–568 (2019). [DOI] [PubMed] [Google Scholar]

[CR3] 3.Akrami, H. et al. 9H-Carbazole derivatives containing the N-benzyl-1,2,3-triazole moiety as new acetylcholinesterase inhibitors. Arch. Pharm. Chem. Life Sci.348, 1–9 (2015). [DOI] [PubMed] [Google Scholar]

[CR4] 4.de Almeida, J. S. et al. Docking and molecular dynamics studies of peripheral site ligand–oximes as reactivators of sarin-inhibited human acetylcholinesterase. J. Biomol. Struct. Dyn.34 (12), 2632–2642 (2016). [DOI] [PubMed] [Google Scholar]

[CR5] 5.Marucci, G. et al. Efficacy of acetylcholinesterase inhibitors in Alzheimer’s disease. Neuropharmacology. 190, 108352 (2021). [DOI] [PubMed] [Google Scholar]

[CR6] 6.Xu, X-T. et al. Synthesis and biological evaluation of coumarin derivatives as α-glucosidase inhibitors. Eur. J. Med. Chem.189, 112013 (2020). [DOI] [PubMed] [Google Scholar]

[CR7] 7.Hussain, R. et al. Design, synthesis and in vitro biological evaluation of new coumarin containing oxazole hybrid derivatives as multitarget inhibitor of α-amylase and α-glucosidase for the treatment of diabetes. Res. Chem.9, 101638 (2024). [Google Scholar]

[CR8] 8.Lu, L. et al. Novel coumarin derivatives as potential tyrosinase inhibitors: synthesis, binding analysis and biological evaluation. Arab. J. Chem.16, 104724 (2023). [Google Scholar]

[CR9] 9.Luo, S. et al. Synthesis, biological evaluation, and docking study of new triazole-phenylacetamide derivatives as α-glucosidase inhibitors. Bioorg. Chem.141, 106844 (2023). [DOI] [PubMed] [Google Scholar]

[CR10] 10.Sun, Q. et al. Syntheses of coumarin–tacrine hybrids as dual-site acetylcholinesterase inhibitors and their activity against butylcholinesterase, Aβ aggregation, and β-secretase. Bioorg. Med. Chem.2 (17), 4784–4791 (2014). [DOI] [PubMed] [Google Scholar]

[CR11] 11.Hirbod, K. et al. Coumarin derivatives bearing benzoheterocycle moiety: synthesis, cholinesterase inhibitory, and docking simulation study. Iran. J. Basic. Med. Sci.20 (6), 631 (2017). [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR12] 12.Sonmez, F. et al. Design, synthesis and docking study of novel coumarin ligands as potential selective acetylcholinesterase inhibitors. J. Enzym Inhibit Med. Chem.32 (1), 285–297 (2017). [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR13] 13.Jalili-Baleh, L. et al. Novel 3-phenylcoumarin–lipoic acid conjugates as multi-functional agents for potential treatment of Alzheimer’s disease. Bioorg. Chem.79, 223–234 (2018). [DOI] [PubMed] [Google Scholar]

[CR14] 14.Van Der Spoel, D. et al. GROMACS: fast, flexible, and free. J. Comput. Chem.26 (16), 1701–1718 (2005). [DOI] [PubMed] [Google Scholar]

[CR15] 15.Lindorff-Larsen et al. Improved side‐chain torsion potentials for the Amber ff99SB protein force field. Proteins Struct. Funct. Bioinform.78 (8), 1950–1958 (2010). [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR16] 16.Case, D. A. et al. The Amber biomolecular simulation programs. J. Comput. Chem.26 (16), 1668–1688 (2005). [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR17] 17.Pearlman, D. A. et al. AMBER, a package of computer programs for applying molecular mechanics, normal mode analysis, molecular dynamics and free energy calculations to simulate the structural and energetic properties of molecules. Comput. Phys. Commun.91 (1–3), 1–41 (1995). [Google Scholar]

[CR18] 18.Darden, T., York, D. & Pedersen, L. Particle mesh Ewald: an N⋅ log (N) method for Ewald sums in large systems. J. Chem. Phys.98 (12), 10089–10092 (1993). [Google Scholar]

[CR19] 19.Koes, D. R., Baumgartner, M. P. & Camacho, C. J. Lessons learned in empirical scoring with smina from the CSAR 2011 benchmarking exercise. J. Chem. Inf. Model.53 (8), 1893–1904 (2013). [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR20] 20.Huey, R., Morris, G. M., Olson, A. J. & Goodsell, D. S. A semiempirical free energy force field with charge-based desolvation. J. Comput. Chem.28 (6), 1145–1152 (2007). [DOI] [PubMed] [Google Scholar]

[CR21] 21.Shiri, F., Shahraki, A. & Nejati-Yazdinejad, M. 3D-QSAR and molecular docking study on maleimide-based glycogen synthase kinase 3 (GSK-3) inhibitors as stimulators of steroidogenesis. Polycycl. Arom. Comp. 1–15 (2018).

[CR22] 22.Nazarshodeh, E., Shiri, F. & Ghasemi, J. B. 3D-QSAR and virtual screening studies in identification of new rho kinase inhibitors with different scaffolds. J. Iran. Chem. Soc.12 (11), 1945–1959 (2015). [Google Scholar]

[CR23] 23.Sander, T., Freyss, J., Von Korff, M. & Rufener, C. DataWarrior: an open-source program for chemistry aware data visualization and analysis. J. Chem. Inf. Model.55 (2), 460–473 (2015). [DOI] [PubMed] [Google Scholar]

PERMALINK

Synthesis, biological evaluation and in silico study of 4-(benzo[d]thiazole-2-yl) phenols based on 4-hydroxy coumarin as acetylcholinesterase inhibitors

Bi Bi Fatemeh Mirjalili

Seyedeh Azita Fazeli Attar

Fereshteh Shiri

Abstract

Supplementary Information

Introduction

Fig. 1.

Materials and methods

General procedure for synthesis of 4-(n-bromoalkoxy)-2H-chromene-2-one

Spectral data of 4-(n-bromoalkoxy)-2H-chromene-2-ones

Spectral data of 4-(benzo[d]thiazole-2-yl)-phenols

In vitro acetylcholinesterase inhibition assay

Molecular modeling

Molecular dynamics simulation

Molecular docking studies

In silico ADMET study

Results and discussion

Synthesis of 4-(benzo[d]thiazol-2-yl)- phenols based on 4-hydroxy coumarine

Scheme 1.

Table 1.

Table 2.

Table 3.

Table 4.

Investigation of acetylcholinesterase inhibitor activity of 3a–3o

Table 5.

Structure-activity relationships (sars)

Molecular dynamics analysis

Fig. 2.

Analysis of molecular docking results

Table 6.

Fig. 3.

In silico ADMET analysis

Table 7.

Conclusion

Electronic supplementary material

Acknowledgements

Author contributions

Funding

Data availability

Declarations

Competing interests

Footnotes

Contributor Information

References

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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