Cytotoxic effect, enzyme inhibition, and in silico studies of some novel N-substituted sulfonyl amides incorporating 1,3,4-oxadiazol structural motif

Abstract

Abstract

The acetylcholinesterase and carbonic anhydrase inhibitors (AChEIs and hCAIs) remain key therapeutic agents for many bioactivities such as anti-Alzheimer and antiobesity antiepileptic, anticancer, antiinfective, antiglaucoma, and diuretic effects. Here, it has been attempted to discover novel multi-target AChEIs and hCAIs that are highly potent, orally bioavailable, may be brain penetrant, and have higher effectiveness at lower doses than tacrine and acetazolamide. After detailed investigations both in vitro and in silico, novel N-substituted sulfonyl amide derivatives (6a–j) were determined to be highly potent inhibitors for AChE and hCAs (K_Is are in the range of 23.11–52.49 nM, 18.66–59.62 nM, and 9.33–120.80 nM for AChE, hCA I, and hCA II, respectively). Moreover, according to the cytotoxic effect studies, such as the ADME-Tox, cortex neuron cells, and neuroblastoma SH-SY5Y cell line, compounds 6a, 6d, and 6h, which are the most potent representative versus the target enzymes, were identified as orally bioavailable, highly selective, and brain preferentially distributed AChEIs and hCAIs. The docking studies revealed precise binding modes between 6a, 6d, and 6h and hCA II, hCA I, and AChE, respectively. The results presented here might provide a solid basis for further investigation into more potent AChEIs and hCAIs.

Graphical abstract

Supplementary Information

The online version contains supplementary material available at 10.1007/s11030-022-10422-8.

Introduction

Heterocyclic compounds play an essential role in medicinal chemistry. These agents are present in various drugs, most vitamins, many natural products, biomolecules, and biologically active compounds [1–7]. Oxadiazoles, an essential family of this class, have significant biological activities, such as analgesic, anti-inflammatory, anticonvulsive, antiemetic, fungicidal, diuretic, muscle relaxant, and antioxidant activity [8–10]. Oxadiazoles have different isomers. Among them, 1,3,4-oxadiazoles have become a significant construction motif for developing new drugs and have been determined to be more effective than other isomers in terms of biological activity. Compounds containing 1,3,4-oxadiazole cores, depending on the group of which it is a member, have a widespread biological activity spectrum, including antibacterial, antiviral, anticancer, antifungal, antihypertensive, analgesic, anticonvulsant, anti-inflammatory, and antidiabetic [11]. Due to this broad range of biological activities, the oxadiazole ring is used in various chemical reactions as a core part of a molecule. Moreover, many 1,3,4-oxadiazole motif-containing compounds are widely used in clinical medicine and drugs available in the market. For example, raltegravir, zibotentan, furamizole, and tiodazosin may be exhibited as antiretroviral [12], anticancer [13], antibacterial [14], and antihypertensive agents [15], respectively (Scheme S1).

Sulfonamides, which have strong pharmacological effects such as anticancer, antitumor, protease inhibitor, antibacterial, antifungal, antiprotozoal, anti-inflammatory, and anticonvulsants, have been used as medicines for nearly 100 years [16–21]. Since the molecular structure of sulfonyl amides is similar to the structure of the p-aminobenzoic acid being synthesized in our body, it interferes with the reactions controlled by this agent and affects the functioning of the metabolism [22, 23]. The more acidic character of the hydrogens on the nitrogen of the N-substituted sulfonyl amides in this class causes it to form stronger hydrogen bonds and thus increase its activity [24–29] (Scheme S2).

Alzheimer’s disease (AD) is an age-related, complex, and multifactorial, chronic neurodegenerative disease which accounts for most cases of dementia. AD usually leads to cognitive dysfunction accompanied by some behaviors such as difficulty performing familiar tasks, memory loss, problems with language, anxiety, and depression. Many factors have been associated with AD development, and different hypotheses have been proposed, such as the cholinergic hypothesis, tau hypothesis, amyloid hypothesis, oxidative stress, and neuroinflammation. Although many potential drugs targeting these hypotheses have been tested to treat AD, there is no definitive cure. Acetylcholinesterase (AChE; EC 3.1.1.7) is currently one of the only well-validated molecular targets for AD [30, 31]. The AChE inhibitors (AChEIs) used in this direction decrease the hydrolysis of acetylcholine (ACh) into acetate and choline (Ch) and, with this, raise the ACh levels at the synaptic cleft that may stimulate cholinergic receptors and further promote memory function [32–34]. For example, AChE inhibitors such as tacrine, galantamine, rivastigmine, and donepezil remain the leading choice for treating AD today [35].

Carbonic anhydrases (CAs; EC 4.2.1.1) [36] are zinc metalloenzymes [37] that activate the reversible reaction of bicarbonate ions and carbon dioxide in eukaryotes and prokaryotes [38]. CAs are contained in many crucial biosynthetic reactions, such as glucogenesis, lipogenesis, and ureagenesis. Moreover, they are also involved in numerous physiological processes like acid–base balance, electrolyte secretion, calcification, and transport of carbon dioxide and bicarbonate between tissues [39, 40]. hCA activators and/or inhibitors (hCAIs) display many bioactivities containing anti-Alzheimer, antiobesity, antiepileptic, anticancer, antiinfective, antiglaucoma, and diuretic effects [41]. Therefore, hCA I (expressed in erythrocytes) and hCA II (expressed in testis, bone osteoclasts, brain, gastrointestinal tract, kidney, erythrocytes, eye, gastrointestinal tract, lung) among the isoenzymes in this class are the most studied ones.

In light of all this literature information, our current efforts were to design novel multi-target AChEIs and hCAIs, mainly aiming to decrease administration dose within the safety limits of commercially available drugs. In this direction, a novel series N-substituted sulfonyl amide derivative (6a–j) were designed, synthesized, characterized, and investigated the biological activities of these compounds on mentioned above target enzymes by this strategy. Because AChEIs generally have high cytotoxicity, the possibility that they may counteract their application as central nervous system-targeting therapeutics also has been considered. Therefore, this study also was investigated cytotoxicity and neurotoxicity profiles of compounds (6a, 6d, and 6h) being the most potent representative versus the enzymes mentioned above on the cortex neuron cells and neuroblastoma SH-SY5Y cell line. Additionally, in silico studies were performed to assess those inhibitors’ inhibition mechanisms against AChE and hCAs.

Experimental

General procedure for the preparation of the compounds

Melting points were determined by a Yanagimoto micro-melting point apparatus and are uncorrected. IR spectra were acquired on a SHIMADZU Prestige-21 (200 VCE) spectrometer. ¹H and ¹³C NMR spectra were acquired at VARIAN Infinity Plus in 300 and 75 Hz, respectively. ¹H and ¹³C chemical shifts are referenced to the internal deuterated solvent. The elemental analysis was carried out with a Leco CHNS-932 instrument. All chemicals were purchased from Sigma-Aldrich.

Ethyl 4-(aminosulfonyl)benzoate (2)

4-Sulfamoylbenzoic acid (10 mmol) was refluxed for 24 h in 50 mL of ethanol and 1.0 mL of sulfuric acid was used as a catalyst. At the end of the reaction, the solvent was evaporated and the obtained product was washed with cold water and dried.

4-sulfonylamidebenzohydrazide (3)

Ethyl-4-(aminosulfonyl)benzoate (10 mmol) and hydrazine hydrate (25 mmol) in ethanol were refluxed for 24 h at 70 °C. The reaction mixture was cooled to room temperature and the solid was filtered then washed with water and dried.

4-(5-thioxo-4,5-dihydro-1,3,4-oxadiazol-2-yl)benzenesulfonamide (4)

Carbon sulfur (2.5 mmol) and 4-sulfonylamidebenzohydrazide (3) (1 mmol) were dissolved in DMF (6 mL) and K₂CO₃ (1 mmol) was added to the mixture. Then, it was stirred for 12 h at room temperature. After the reaction was completed, the mixture was cooled to room temperature and poured into ice cold water. It was then filtered, dried, and crystallized.

4-(5-(ethylthio)-1,3,4-oxadiazol-2-yl)benzenesulfonamide (5)

Iodoethane (1.2 mmol) and 4-(5-thioxo-4,5-dihydro-1,3,4-oxadiazol-2-yl)benzenesulfonamide (1 mmol) were dissolved in DMF (6 mL) and K₂CO₃ (1 mmol) was added to the mixture, then, stirred for 12 h at room temperature. After the reaction was completed. The mixture was cooled to room temperature and poured into ice cold water. It was then filtered, dried, and crystallized.

N-substituted ((ethylthio-1,3,4-oxadiazol-2-yl)phenyl)sulfonyl amide derivatives (6a–j)

Acylhalide derivatives (1.2 mmol) and 4-(5-thioxo-4,5-dihydro-1,3,4-oxadiazol-2-yl)benzenesulfonamide (1 mmol) were dissolved in pyridine (5 mL). Then, it was heated for 12 h at 60 °C. After the reaction was completed, the mixture was cooled to room temperature and poured into ice cold water. It was then filtered, dried, and crystallized from acetone. The prepared compounds shown in Scheme 1 were characterized by ¹H NMR, ¹³C NMR, IR, and elemental analysis.

Scheme 1.

Synthesis of the novel N-substituted sulfonyl amide derivatives (6a–j)

N-([4-(5-(ethylthio)-1,3,4-oxadiazol-2-yl)phenyl]sulfonyl)benzamide (6a)

Yield 75%, m.p. 178 °C. IR (ν_max, cm⁻¹): 3666 (N–H), 1683 (C=O), 1458 and 1386 (C–H), 1255 and 1168 (S=O). ¹H NMR (300 MHz, DMSO-d₆, ppm): 8.21 (2H, d,=CH), 8,16 (2H, d,=CH), 7,87 (2H, d,=CH), 7,64 (1H, m,=CH), 7,51 (2H, m,=CH), 3.40 (2H, m, –CH₂) 1.45 (3H, t, –CH₃). ¹³C NMR (75 MHz, DMSO-d₆, ppm): 170.1, 165.7, 164.2, 149.7, 147.3, 134.3, 131.8, 129.4, 129.1, 127.7, 27.4, 15.5. Anal. calcd. for C₁₇H₁₅N₃O₄S₂: C, 52.43; H, 3.88; N, 10.79; O, 16.43; S, 16.47; found: C, 52.47; H, 3.92; N, 10.83; O, 16.55; S, 16.54.

N-([4-(5-(ethylthio)-1,3,4-oxadiazol-2-yl)phenyl]sulfonyl)-4-methylbenzamide (6b)

Yield 80%, m.p. 191 °C. IR (ν_max, cm⁻¹): 3678 (N–H), 1732 (C=O), 1452 and 1388 (C–H), 1171 and 1068 (S=O). ¹H NMR (300 MHz, DMSO-d₆, ppm): 8.25 (2H, d,=CH), 8,21 (2H, d,=CH), 7,82 (2H, d,=CH), 7,31 (2H, d,=CH), 3.37 (2H, m, –CH₂), 2.35 (3H, s, –CH₃), 1.43 (3H, t, –CH₃). ¹³C NMR (75 MHz, DMSO-d₆, ppm): 171.2, 165.5, 164.6, 144.6, 142.5, 129.8, 129.4, 129.2, 129.0, 127.7, 27.4, 21.8, 15.5. Anal. calcd. for C₁₈H₁₇N₃O₄S₂: C, 53.58; H, 4.25; N, 10.41; O, 15.86; S, 15.89; found: C, 53.61; H, 4.33; N, 10.50; O, 15.99; S, 15.94.

N-([4-(5-(ethylthio)-1,3,4-oxadiazol-2-yl)phenyl]sulfonamido)-2-oxoethyl acetate (6c)

Yield 84%, m.p. 209 °C. IR (ν_max, cm⁻¹): 3672 (N–H), 1738 (C=O), 1451 and 1382 (C–H), 1230 and 1190 (S=O). ¹H NMR (300 MHz, DMSO-d₆, ppm): 8.23 (2H, d,=CH), 8,09 (2H, d,=CH), 4.55 (2H, s, –CH₃), 3.38 (2H, m, –CH₂), 2.00 (3H, s, –CH₃), 1.46 (3H, t, –CH₃). ¹³C NMR (75 MHz, DMSO-d₆, ppm): 171.4, 166.9, 164.5, 155.3, 129.6, 128.4, 127.7, 126.0, 64.7, 27.4, 21.0, 15.3. Anal. calcd. for C₁₄H₁₅N₃O₆S₂: C, 43.63; H, 3.92; N, 10.90; O, 24.91; S, 16.64; found: C, 43.74; H, 3.95; N, 10.96; O, 24.99; S, 16.70.

N-([4-(5-(ethylthio)-1,3,4-oxadiazol-2-yl)phenyl]sulfonyl)butyramide (6d)

Yield 72%, m.p. 142 °C. IR (ν_max, cm⁻¹): 3675 (N–H), 1708 (C=O), 1455 and 1385 (C–H), 1113 and 1106 (S=O). ¹H NMR (300 MHz, DMSO-d₆, ppm): 8.20 (2H, d,=CH), 8,08 (2H, d,=CH), 3.33 (2H, m, –CH₂), 2.51 (2H, m, –CH₂), 2.20 (2H, m, –CH₂), 1.44 (3H, t, –CH₃), 0.75 (3H, t, –CH₃). ¹³C NMR (75 MHz, DMSO-d₆, ppm): 177.1, 165.5, 160.5, 142.7, 129.2, 127.5, 119.2, 38.1, 27.6, 18.4, 15.5, 13.8. Anal. calcd. for C₁₄H₁₇N₃O₄S₂: C, 47.31; H, 4.82; N, 11.82; O, 18.01; S, 18.04; found: C, 47.35; H, 4.87; N, 11.86; O, 18.06; S, 18.10.

N-([4-(5-(ethylthio)-1,3,4-oxadiazol-2-yl)phenyl]sulfonyl)pentanamide (6e)

Yield 76%, m.p. 140 °C. IR (ν_max, cm⁻¹): 3662 (N–H), 1712 (C=O), 1452 and 1389 (C–H), 1168 and 1071 (S=O). ¹H NMR (300 MHz, DMSO-d₆, ppm): 8.23 (2H, d,=CH), 8,09 (2H, d,=CH), 3.36 (2H, m, –CH₂), 2.23 (2H, t, –CH₂), 1.44 (3H, t, –CH₃), 1.38 (2H, m, –CH₂), 1.16 (2H, m, –CH₂), 0.79 (3H, t, –CH₃). ¹³C NMR (75 MHz, DMSO-d₆, ppm): 173.0, 166.1, 164.8, 142.4, 129.6, 128.7, 128.0, 36.0, 27.1, 25.8, 22.2, 16.3, 14.6. Anal. calcd. for C₁₅H₁₉N₃O₄S₂: C, 48.76; H, 5.18; N, 11.37; O, 17.32; S, 17.36; found: C, 48.82; H, 5.20; N, 11.42; O, 17.38; S, 17.42.

N-([4-(5-(ethylthio)-1,3,4-oxadiazol-2-yl)phenyl]sulfonyl)hexanamide (6f)

Yield 85%, m.p. 122 °C. IR (ν_max, cm⁻¹): 3681 (N–H), 1695 (C=O), 1454 and 1385 (C–H), 1115 and 1071 (S=O). ¹H NMR (300 MHz, DMSO-d₆, ppm): 8.23 (2H, d,=CH), 8,11 (2H, d,=CH), 3.40 (2H, m, –CH₂), 2.20 (2H, t, –CH₂), 1.44 (3H, t, –CH₃), 1.38 (2H, m, –CH₂), 1.25 (2H, m, –CH₂), 1.11 (2H, m, –CH₂), 0.78 (3H, t, –CH₃). ¹³CNMR (75 MHz, DMSO-d₆, ppm): 171.8, 166.1, 163.6, 142.1, 129.6, 128.6, 127.4, 34.7, 31.4, 27.5, 24.9, 22.2, 15.6, 14.6. Anal. calcd. for C₁₆H₂₁N₃O₄S₂: C, 50.11; H, 5.52; N, 10.96; O, 16.69; S, 16.72; found: C, 50.18; H, 5.57; N, 10.99; O, 16.74; S, 16.75.

N-([4-(5-(ethylthio)-1,3,4-oxadiazol-2-yl)phenyl]sulfonyl)heptanamide (6g)

Yield 74%, m.p. 109 °C. IR (ν_max, cm⁻¹): 3669 (N–H), 1714 (C=O), 1453 and 1387 (C–H), 1160 and 1068 (S=O). ¹H NMR (300 MHz, DMSO-d₆, ppm): 8.21 (2H, d,=CH), 8,13 (2H, d,=CH), 3.42 (2H, m, –CH₂), 2.21 (2H, t, –CH₂), 1.43 (3H, t, –CH₃), 1.37 (2H, m, –CH₂), 1.23 (2H, m, –CH₂), 1.19 (2H, m, –CH₂), 1.10 (2H, m, –CH₂), 0.79 (3H, t, –CH₃). ¹³C NMR (75 MHz, DMSO-d₆, ppm): 171.8, 166.1, 163.9, 142.4, 129.2, 128.0, 127.7, 35.9, 31.4, 28.5, 27.4, 24.5, 22.5, 15.5, 14.5. Anal. calcd. for C₁₇H₂₃N₃O₄S₂: C, 51.36; H, 5.83; N, 10.57; O, 16.10; S, 16.13; found: C, 51.42; H, 5.86; N, 10.62; O, 16.14; S, 16.15.

N-([4-(5-(ethylthio)-1,3,4-oxadiazol-2-yl)phenyl]sulfonyl)octanamide (6h)

Yield 80%, m.p. 99 °C. IR (ν_max, cm⁻¹): 3678 (N–H), 1733 (C=O), 1455 and 1389 (C–H), 1168 and 1071 (S=O). ¹H NMR (300 MHz, DMSO-d₆, ppm): 8.22 (2H, d,=CH), 8,08 (2H, d,=CH), 3.36 (2H, m, –CH₂), 2.52 (2H, m, –CH₂), 2.19 (2H, t, –CH₂), 1.42 (3H, t, –CH₃), 1.42 (2H, m, –CH₂), 1.23 (2H, m, –CH₂), 1.13 (2H, m, –CH₂), 0.79 (2H, m, –CH₂), 0.77 (3H, t, –CH₃). ¹³C NMR (75 MHz, DMSO-d₆, ppm): 172.5, 165.6, 164.8, 142.4, 129.2, 128.0, 127.7, 36.0, 34.33, 31.8, 29.7, 28.9, 24.6, 22.7, 15.5, 14.5. Anal. calcd. for C₁₈H₂₅N₃O₄S₂: C, 52.53; H, 6.12; N, 10.21; O, 15.55; S, 15.58; found: C, 52.46; H, 6.18; N, 10.24; O, 15.67; S, 15.61.

N-([4-(5-(ethylthio)-1,3,4-oxadiazol-2-yl)phenyl]sulfonyl)nonanamide (6i)

Yield 60%, m.p. 110 °C. IR (ν_max, cm⁻¹): 3662 (N–H), 1739 (C=O), 1458 and 1385 (C–H), 1117 and 1071 (S=O). ¹H NMR (300 MHz, DMSO-d₆, ppm): 8.23 (2H, d,=CH), 8,12 (2H, d,=CH), 3.37 (2H, m, –CH₂), 2.21 (2H, m, –CH₂), 1.43 (3H, t, –CH₃), 1.37 (2H, m, –CH₂), 1.34–1.00 (10H, m, –CH₂), 0.85 (3H, t, –CH₃). ¹³C NMR (75 MHz, DMSO-d₆, ppm): 173.8, 165.9, 164.8, 142.4, 129.2, 128.0, 127.6, 35.7, 33.5, 31.9, 29.2, 28.8, 27.3, 25.1, 22.7, 15.5, 14.5. Anal. calcd. for C₁₉H₂₇N₃O₄S₂: C, 53.62; H, 6.39; N, 9.87; O, 15.04; S, 15.07; found: C, 53.69; H, 6.42; N, 9.99; O, 15.12; S, 15.20.

N-([4-(5-(ethylthio)-1,3,4-oxadiazol-2-yl)phenyl]sulfonyl)dodecanamide (6j)

Yield 76%, m.p. 88 °C. IR (ν_max, cm⁻¹): 3680 (N–H), 1695 (C=O), 1454 and 1384 (C=N), 1167 and 1068 (S=O). ¹H NMR (300 MHz, DMSO-d₆, ppm): 8.22 (2H, d,=CH), 8,09 (2H, d,=CH), 3.38 (2H, m, –CH₂), 2.20 (2H, m, –CH₂), 1.42 (3H, t, –CH₃), 1.37 (2H, m, –CH₂), 1.33–0.98 (16H, m, –CH₂), 0.85 (3H, t, –CH₃). ¹³C NMR (75 MHz, DMSO-d₆, ppm): 174.2, 166.6, 164.2, 142.7, 129.3, 127.6, 127.3, 36.1, 34.3, 33.5, 32.7, 30.5, 29.4, 28.5, 27.8, 26.4, 25.2, 23.8, 15.9, 14.3. Anal. calcd. for C₂₂H₃₃N₃O₄S₂: C, 56.50; H, 7.11; N, 8.99; O, 13.69; S, 13.71; found: C, 56.58; H, 7.16; N, 9.06; O, 13.72; S, 13.83.

Biological studies

AChE and hCAs activity assay

In the present work, AChE from Electrophorus electricus (Sigma C2888) was purchased from Sigma‐Aldrich Chemie GmbH. In vitro effects on AChE activity of the newly synthesized N-substituted sulfonyl amides (6a–j) incorporating 1,3,4-oxadiazol structural motif and reference compound, THA, were evaluated by the method of Ellman et al. [42, 43]. Analysis results were obtained spectrophotometrically at 412 nm using acetylthiocholine iodide (PubChem CID: 74629, Sigma 01480) as a substrate as in our previous assays [44, 45]. Also, hCAs (hCA I and II) were purified from human erythrocytes by Sepharose‐4B‐l‐tyrosine‐sulfanilamide affinity chromatography. The inhibition effects of these N-substituted sulfonyl amide derivatives (6a–j) and reference compound, AAZ versus the esterase activity of the hCAs were determined by following the change in absorbance at 348 nm according to the assay defined by Verporte et al. [46–48]. hCAs activities were measured using 4-nitrophenyl acetate (PubChem CID: 13,243, Sigma N8130). All the measurements were repeated thrice.

AChE and hCAs kinetic assay

To investigate the in vitro inhibitory mechanisms of the novel synthesized N-substituted sulfonyl amides (6a–j) incorporating 1,3,4-oxadiazol structural motif, kinetic studies were made with the variable compound and substrate concentrations, and IC₅₀ curves, Michaelis–Menten graphs [49–51], and Lineweaver–Burk curves [52–54] were generated as previously reported by Türkeş et al. [55–57]. Solutions of the novel synthesized all agents (6a–j), THA, and AAZ were prepared in dimethyl sulfoxide (PubChem CID: 679, Sigma D8418, DMSO) at an initial concentration of 1 mg/mL. The concentration of DMSO in the final reaction mixture was approx. 1%. IC₅₀ and K_I values for these derivatives were computed from the observed data, and the types of inhibition of AChE and hCAs were determined as in our previous studies [58–60]. Analysis of the data and drawing of graphs were realized using GraphPad Prism version 8 for Mac (GraphPad Software, La Jolla California USA). The inhibition constants were calculated by SigmaPlot version 12 for Windows (Systat Software, San Jose California USA). The fit of enzyme inhibition models was compared using the extra sum-of-squares F test and Akaike’s corrected information criterion approach. The results were exhibited as mean ± standard error of the mean (95% confidence intervals). Differences between data sets were considered statistically significant when the p value was less than 0.05.

Cell-based assay

The cortex neuron cells and neuroblastoma SH-SY5Y cell line was obtained from the Department of Medical Pharmacology, Faculty of Medicine, Atatürk University (Erzurum, Turkey). The cells were cultured in Dulbecco’s modified Eagle’s medium (Gibco, Grand Island, NY, USA) supplemented with 15% fetal bovine serum, and 1% antibiotic (penicillin, streptomycin, and amphotericin B), in a humidified atmosphere of 95% air and 5% CO₂ at 37 °C, and grown to 80% confluence [61, 62]. Before cell treatment, the complete medium was replaced with a reduced serum medium (i.e., with 2% fetal bovine serum). The novel synthesized N-substituted sulfonyl amide derivatives 6a, 6d, and 6h, which are the most potent representative versus the target enzymes, were prepared as stock solutions in DMSO at an initial concentration of 1 mg/mL. The cytotoxicity of these derivatives 6a, 6d, and 6h was compared with standard compound cisplatin. They were administered in four different doses (10, 100, 500, and 1000 µM) in a quadruplicate. After 24 h, on cortex neuron cells and SH-SY5Y cell line treated by seeding in 96-well plates with compounds 6a, 6d, and 6h, the administration was terminated, and the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) cytotoxicity test was performed. After the medium aspirating process of 10 μL, an MTT solution was added to each well. The plates containing the applications were kept for 4 h in an environment containing 5% CO₂ at 37 °C with MTT solution [63]. To dissolve the formazan crystals formed as a result of MTT, 100 μL DMSO was added to each well [64]. To determine the density of formazan crystals, data were obtained by reading plates with a Multiskan Go Microplate Spectrophotometer reader at a wavelength of 570 nm. Additionally, morphological changes occurring in the cortex neuron cells and SH-SY5Y cell line were visualized with an inverted microscope (Leica Microsystems, Wetzlar, Germany). The images obtained were recorded with × 200 magnification of the microscope [65]. GraphPad Prism version 8 for Mac (GraphPad Software, La Jolla California USA) was used for the statistical analyses to assess the results obtained in the study. The One-way ANOVA method was used to analyze the data, and the significance values were compared with the control group. Differences between data sets were considered statistically significant when the p value was less than 0.05.

Computational studies

ADME-Tox assay

In silico ADME-Tox assays were computed using Schrödinger Small-Molecule Drug Discovery Suite (Schrödinger Release 2021–1 panels for Mac: Maestro [66], LigPrep [67], QikProp [68, 69]; Schrödinger, LLC, NY, USA) and SwissADME platform for the novel synthesized N-substituted sulfonyl amides (6a–j) incorporating 1,3,4-oxadiazol structural motif. The ligands (compounds 6a–j and the reference compounds, THA and AAZ) were firstly sketched using ChemDraw [70] version 19.1 for Mac (PerkinElmer, Inc., Waltham, MA, USA), were prepared by LigPrep, and were lastly evaluated utilizing QikProp in normal processing mode. QikProp properties and prediction ranges for ADME-Tox include: MW (130.0–725.0), Dipole (1.0–12.5), Volume (500.0–2000.0), QPlogPoct (8.0–35.0), QPlogPw (4.0–45.0), QPlogPo/w (–2.0 to 6.5), QPlogS (–6.5 to 0.5), QPPCaco (< 25 poor, great > 500), QPlogBB (–3.0 to 1.2), QPPMDCK (< 25 poor, great > 500), QPlogKp (–8.0 to –1.0), QPlogKhsa (–1.5 to 1.5), HOA (< 25% poor, high > 80%), PSA (7.0–200.0), number of violations of Lipinski’s rule of five (max. 4) [71], number of violations of Jorgensen’s rule of three (max. 3) [72], and PAINS alert [73].

Molecular docking assay

The potential modes of the binding of for the novel synthesized N-substituted sulfonyl amides (6a–j) incorporating 1,3,4-oxadiazol structural motif to target proteins (AChE, hCA I, and hCA II) were investigated with Schrödinger Small-Molecule Drug Discovery Suite 2021–1 for Mac (Schrödinger, LLC, NY, USA), under the Maestro graphical user interface. Other associated panels included Protein Preparation Wizard [74], SiteMap [75], LigPrep [76], Receptor Grid Generation [77], Ligand Docking [78], and Prime MM-GBSA [79]. The crystal structures of AChE [80] (Species: Homo sapiens; PDB code 4EY7; Resolution: 2.35 Å; R-Values free, work, and observed: 0.211, 0.175, and 0.177, respectively), hCA I [81] (Species: Homo sapiens; PDB code 6I0L; Resolution: 1.40 Å; R-Values free, work, and observed: 0.237, 0.204, and 0.206, respectively), and hCA II [82] (Species: Homo sapiens; PDB code 5NY3; Resolution: 1.40 Å; R-values free, work, and observed: 0.172, 0.156, and 0.157, respectively) were obtained from Protein Data Bank (http://www.rcsb.org/) [83] were applied for in silico molecular docking. The 4EY7, 6I0L, and 5NY3 in the crystal structures were minimized using the Protein Preparation Wizard tool [84] has been used for preparing the protein structure where bond orders were assigned and hydrogen atoms were added as well as restrained minimization step has also been done using optimized potential liquid simulations 4 (OPLS4) force field [85] at pH 7.4 ± 0.5 [86]. The active sites of theirs were predicted utilizing the SiteMap panel [87]. The ligand-binding sites were calculated using the Receptor Grid Generation tool [88]. LigPrep module was used for preparing all the synthesized N-substituted sulfonyl amides (6a–j) where bond order and the bond angle were assigned as well as minimization was done using OPLS4 force field [89]. The prepared small molecules were docked into the binding sites of the target enzymes by the Ligand Docking module with Glide extra precision (XP) mode [90–92]. MM-GBSA relative binding free energy computations [93–95] of target proteins (4EY7, 6I0L, and 5NY3) with the agents were carried out using the Prime tool in the VSGB energy model and OPLS4 force field [96, 97].

Results and discussion

Drug design strategy and chemistry

Ethyl 4-(aminosulfonyl)benzoate was prepared from sulfamoylbenzoic acid in ethanol with catalytic amount of sulfuric acid by refluxing for 24 h. The ester group of the ethyl 4-(aminosulfonyl)benzoate was converted to 4-sulfonylamidebenzohydrazide with hydrazine hydrate in ethanol at 70 °C for 24 h. 4-(5-thioxo-4,5-dihydro-1,3,4-oxadiazol-2-yl)benzene sulfonamide was prepared by reacting Carbon disulfur and 4-sulfonylamide benzohydrazide in DMF in the presence of K₂CO₃. 4-(5-(ethylthio)-1,3,4-oxadiazol-2-yl) benzene sulfonamide was synthesized using Iodoethane and the compound 4 in DMF in basic condition. The final targeted compounds (6a–j) were prepared by acylhalide derivatives and the compound 5 in pyridine at 60 °C. The prepared compounds (6a–j) shown in Scheme 1 were characterized by ¹H NMR, ¹³C NMR, IR, and elemental analysis. From the ¹H NMR spectra of the compounds, sulfanilamide NH resonances at very low field due to electron delocalization and the =CH proton peaks on aromatic ring come between 8.05 and 8.25 ppm. The signals of carbonyl and oxadiazole ring are seen at around 170 ppm and 165 ppm, respectively, in the ¹³C NMR spectra. In the infrared spectra of compounds 6a–j, it was possible to observe the absorptions around 3660 cm⁻¹ and 1700 cm⁻¹ relating to N–H and C=O stretchings, respectively. As seen in the literature [98], there are two peaks assigned to S=O as asymmetric and symmetric stretching which are appeared around 1300 and 1100 cm⁻¹, respectively. All spectra and elemental analyses support the structure of the synthesized compounds.

Biological studies

Inhibition study and structure–activity relationship assay

The hCAs and AChE inhibitory impact for all herein prepared target N-substituted sulfonyl amides (6a–j) incorporating 1,3,4-oxadiazol structural motif, as well as the standards, hCAI acetazolamide (AAZ; K_Is for hCA I and hCA II 439.17 ± 9.30 and 98.28 ± 1.69 nM, respectively) and AChEI tacrine (THA; K_Is for AChE 155.29 ± 0.82 nM), were estimated versus the ubiquitous cytosolic hCA I, II isoforms, and AChE by the use of the Verpoorte’s and Ellman’s methods, respectively. Certain structure–activity relationships (SAR) could be drawn from the shown inhibition data in Table 1.

Table 1.

Inhibition data of AChE and hCA I, II isoforms with novel synthesized N-substituted sulfonyl amides (6a–j) incorporating 1,3,4-oxadiazol structural motif

Compound		AChE		hCA I		hCA II		Selectivity index a
ID	R	KI (nM)	Inhibition type	KI (nM)	Inhibition type	KI (nM)	Inhibition type	THAb/AChE	AAZc/hCA I	AAZc/hCA II	hCAI/hCA II	hCA II/hCA I
6a	Phenyl	52.49 ± 1.27	Competitive	21.64 ± 0.25	Competitive	9.33 ± 0.13	Competitive	2.96	20.29	10.53	2.32	0.43
6b	Tolyl	47.03 ± 1.71	Competitive	59.62 ± 0.53	Noncompetitive	17.84 ± 0.23	Competitive	3.30	7.37	5.51	3.34	0.30
6c	Acetoxymethyl	47.01 ± 1.95	Competitive	29.62 ± 0.28	Competitive	22.20 ± 0.22	Competitive	3.30	14.83	4.43	1.33	0.75
6d	Propyl	48.38 ± 1.75	Competitive	18.66 ± 0.21	Competitive	23.18 ± 0.26	Competitive	3.21	23.54	4.24	0.81	1.24
6e	Butyl	41.23 ± 0.95	Noncompetitive	47.15 ± 0.36	Competitive	37.24 ± 0.14	Competitive	3.77	9.31	2.64	1.27	0.79
6f	Pentyl	39.34 ± 1.13	Competitive	33.10 ± 0.47	Competitive	29.19 ± 0.10	Competitive	3.95	13.27	3.37	1.13	0.88
6g	Hexyl	49.70 ± 2.13	Competitive	23.01 ± 0.29	Competitive	30.71 ± 0.09	Competitive	3.12	19.09	3.20	0.75	1.33
6h	Heptyl	23.11 ± 0.77	Competitive	34.61 ± 0.51	Competitive	120.80 ± 0.34	Noncompetitive	6.72	12.69	0.81	0.29	3.49
6i	Octyl	47.56 ± 1.75	Competitive	38.40 ± 0.46	Competitive	112.70 ± 0.36	Competitive	3.27	11.44	0.87	0.34	2.93
6j	Undecanoyl	46.70 ± 1.64	Competitive	31.01 ± 0.41	Competitive	29.40 ± 0.13	Competitive	3.33	14.16	3.34	1.05	0.95
THAc	–	155.29 ± 0.82	Competitive	–	–	–	–	–	–	–	–	–
AAZd	–	–	–	439.17 ± 9.30	Noncompetitive	98.28 ± 1.69	Noncompetitive	–	–	–	–	–

^aThe K_I rates are indicative of selectivity index: A potent selective inhibitor is characterized by a high-value ratio

^bTacrine

^cAcetazolamide

Regarding the hCAs inhibitory activities of N-substituted sulfonyl amides, all derivatives (6a–j) displayed potent inhibitory action against the ubiquitous cytosolic hCA I isoform with K_I constants ranging from 18.66 ± 0.21 to 59.62 ± 0.53 nM (Table 1). In particular, N-substituted sulfonyl amide 6d (propyl substituted), 6a (phenyl substituted), and 6g (hexyl substituted) exhibited the best hCA I inhibitory activity with two-digit nanomolar activities with K_Is equal 18.66 ± 0.21, 21.64 ± 0.25, and 23.01 ± 0.29 nm, respectively. Interestingly, the kinetic value of the compound 6d, which is the lowest in the series, displayed a 23.5-fold lower K_I when compared to the reference AAZ (K_I = 439.17 ± 9.30 nM). It is worth stressing that replacement of a propyl group (derivative 6d; K_I = 18.66 ± 0.21 nM) with a tolyl group (derivative 6b; K_I = 59.62 ± 0.53 nM) resulted in a more than threefold inhibition increase for the cytosolic hCA I isoform. On the other hand, it is determined that elongation of the sulfonyl acetamide linker in the butyl tail-bearing compound 6e (K_I = 47.15 ± 0.36 nM) resulted in an increase in isoform hCA I inhibitory activity (derivatives 6f, 6g, 6h, 6i, and 6j; with K_Is of 33.10 ± 0.47, 23.01 ± 0.29, 34.61 ± 0.51, 38.40 ± 0.46, and 31.01 ± 0.41 nM, respectively).

Moreover, exploring the inhibitory activity of herein reported N-substituted sulfonyl amide derivatives (6a–j) versus the physiologically dominant hCA II isoform revealed that it was effectively inhibited by sulfonyl amides (6a–j) with K_Is spanning in the range 9.33 ± 0.13–120.80 ± 0.34 nM (Table 1). Superiorly, compound 6a (phenyl substituted) exerted single-digit nanomolar inhibitory activity versus hCA II isoform (K_Is = 9.33 ± 0.13 nM), thus resulting in 10.5-fold higher potency when compared to the reference AAZ (K_I = 98.28 ± 1.69 nM), besides, compounds 6b, 6c, and 6d displayed K_I constants close to each other 17.84 ± 0.23, 22.20 ± 0.22, and 23.18 ± 0.26 nM, respectively. Contrariwise, hCA II isoform was weakly inhibited by compounds 6h and 6i with K_Is of 120.80 ± 0.34 and 112.70 ± 0.36 nM, respectively. It is worth mentioning that replacing the tolyl tail of derivative 6b (K_I = 17.84 ± 0.23 nM) with a phenyl one, as in compound 6a, resulted in about twofold enhanced inhibitory potency against hCA II isoform (K_I = 9.33 ± 0.13 nM). Furthermore, branching of the sulfonyl acetamide linker and its elongation decreased the hCA II inhibitory action for the heptyl tail-bearing derivative 6h. Moreover, it is worth highlighting that hCAIs profiles presented in Table 1 hinted out that compound 6b demonstrated interesting selectivity versus the ubiquitous cytosolic hCA I isoform (selectivity index; S_I = 3.34), whereas compound 6h has shown selectivity against the dominant hCA II isoform (S_I = 3.49). Hence, further structural modifications are required to optimize the hCA II/I selectivity. This selectivity for compounds 6b and 6h makes understand them as exciting and promising candidates for further improvement as potential hCAIs.

Recently, an increasing number of different hCAIs than the sulfonamides and their bioisosteres have been identified. In this context, the study by Sharma et al. [99] reported that two novel series of 1,3,4-oxadiazole benzenesulfonamide hybrids 3 and 4, having twenty novel compounds, have been designed and synthesized to assess their inhibition potential as hCAIs against hCA I, II, IX, and XII. They found that potent inhibitory activity versus hCA I has been exhibited by derivatives 3g and 4j, 3.5-fold of order better than standard drug AAZ and derivative 4j effectively inhibited glaucoma-associated hCA II isoform as well as tumor-associated hCA IX isoform. Rutkauskas et al. [100] reported that a series of N-aryl-β-alanine derivatives and diazobenzenesulfonamides containing aliphatic rings were designed, characterization, synthesized, and their binding to hCA I, II, VI, VII, XII, and XIII isoenzymes was studied using the fluorescent thermal shift assay and isothermal titration calorimetry. They determined that 4-substituted diazobenzenesulfonamides were more potent hCA binders than N-aryl-β-alanine derivatives.

The obtained inhibition constants for the AD-related AChE (Table 1) showed that all sulfonyl amides (6a–j) were capable of inhibiting this enzyme in the low nanomolar range (K_Is of 23.11 ± 0.77–52.49 ± 1.27 nM). Derivatives 6f (pentyl substituted) and 6h (heptyl substituted) emerged as the most potent herein reported AChEI endowed with two-digit nanomolar K_I values equal 39.34 ± 1.13 and 23.11 ± 0.77 nM, respectively (Table 1). In addition, the derivative 6h showed a K_I value 6.7-fold higher when compared to the reference THA (K_I = 155.29 ± 0.82 nM). Similar to the SAR for inhibition of hCA I; replacement of a tolyl group (compound 6b; K_I = 47.03 ± 1.71 nM) with a heptyl group (compound 6h; K_I = 23.11 ± 0.77 nM) was advantageous for inhibitory activity versus AChE. Also, elongation of the sulfonyl acetamide linker incorporated in the propyl tail-bearing derivative 6d was more beneficial for AChE inhibition (compound 6d; K_I = 48.38 ± 1.75 nM), while the elongation of such linker resulted in about twofold increased activity versus AChE (compound 6h; K_I = 23.11 ± 0.77 nM).

Cell-based assay

Because the cortex neuron cells and neuroblastoma SH-SY5Y cell line express one or more neurofilament proteins, specific norepinephrine uptake, and neuronal marker enzyme activity, since the early 1980s, it is commonly employed in experimental neurological studies, including metabolism, function, and neuronal differentiation analysis, connected to neuroprotection, neurotoxicity, neuroadaptive, and neurodegenerative processes [101]. Thus, to gain further insight into the therapeutic potential for the treatment for AD of these selected novel synthesized N-substituted sulfonyl amide derivatives 6a, 6d, and 6h incorporating 1,3,4-oxadiazol structural motif, which are the most potent representative versus the target enzymes, their cell viability, and proliferation activities were determined using the cortex neuron cells and neuroblastoma SH-SY5Y cell line. The colorimetric MTT assay (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) was carried to examine the potential cytotoxic effects of these agents. Images of cortex neuron cells and SH-SY5Y cell line obtained by inverted microscope are given in Fig. 1, respectively. The derivatives 6a, 6d, and 6h evaluated in our study caused a decrease in cell viability after 24 h of incubation, depending on the dose (IC₅₀s = 0.95, 0.88, 0.82, and 0.64 µM for 6a, 0.99, 0.93, 0.87, and 0.74 µM for 6d, and 0.98, 0.92, 0.65, and 0.58 µM for 6h, in four different doses, 10, 100, 500, and 1000 µM, respectively). Moreover, they have been determined to display a propensity for high cell viability and neuroprotection at low concentrations (at 10 and 100 μM). However, these compounds showed cytotoxicities on the cortex neuron cells and neuroblastoma SH-SY5Y cells in a concentration-dependent manner, while N-substituted sulfonyl amide derivatives 6a, 6d, and 6h were found as non-toxic agents at their effective concentrations on target enzymes (AChE and hCAs) (Figs. 2 and 3). Finally, when examining the information obtained in this study, 6a, 6d, and 6h compounds can be considered promising precursors for new design and development of therapeutics against AD. As a result, these agents exhibit that they have AChE inhibitory activity, which supports their use to treat neurological disorders.

Fig. 1.

Inverted microscope images of compounds 6a, 6d, and 6h on cell viability in cortex neuron cells

Fig. 2.

Morphological changes of SH-SY5Y after 24 h of incubation with concentrations (10–1000 μM) of compounds 6a, 6d, and 6h the results presented are from that were carried out and photographed microscopically

Fig. 3.

Cytotoxicity study of compounds 6a, 6d, and 6h on SH-SY5Y cells. The treatment of SH-SY5Y cells was performed with these groups at concentrations varying between 10 and 1000 μM. Every bar represents the mean ± standard error of the mean (95% confidence intervals) of three separate tests. A Viability rates for cortex neuron cells and B viability rates for neuroblastoma SH-SY5Y cell line

Computational studies

ADME-Tox assay

All novel N-substituted sulfonyl amides (6a–j) incorporating 1,3,4-oxadiazol structural motif were evaluated in silico using the ADME-Tox prediction program QikProp, and SwissADME platform, and the findings are reported in Table 2. All properties computed were satisfactory pharmacodynamic and pharmacokinetic properties for the novel synthesized N-substituted sulfonyl amide derivatives (6a–j). Additionally, diagrams showing “drug-likeness” descriptors for 6a, 6d, and 6h agents, which are the most active derivatives in this series, are given in Fig. S1. Only one (compound 6j) of all N-substituted sulfonyl amide derivatives (6a–j) were displayed one Lipinski’s rule violation, and five agents (compounds 6b and 6g–j) showed only one Jorgensen’s rule violation. Namely, computed in silico ADME-Tox properties confirmed newly synthesized these sulfonyl amides (6a–j) as hit agents displaying suitable drug-like properties.

Table 2.

ADME-Tox-related parameters of novel synthesized N-substituted sulfonyl amides (6a–j) and standard inhibitors tacrine (THA), and acetazolamide (AAZ)

Principal descriptors	6a	6b	6c	6d	6e	6f	6g	6h	6i	6j	THA	AAZ	Standard range
MW	389.44	403.47	385.41	355.43	369.45	383.48	397.51	411.53	425.56	467.64	198.27	222.24	130.0–725.0
Dipole	10.28	8.29	7.53	10.66	8.49	7.85	11.66	10.28	10.77	10.60	4.22	10.75	1.0–12.5
Volume	1173.83	1231.90	1166.05	1121.54	1182.85	1237.73	1282.52	1352.00	1426.62	1603.47	710.28	634.45	500.0–2000.0
QPlogPoct	20.62	20.65	20.04	18.82	18.83	19.17	20.22	20.59	21.34	22.81	10.75	17.57	8.0–35.0
QPlogPw	12.63	12.31	13.56	11.08	10.95	10.76	10.47	10.33	10.42	9.97	6.38	15.15	4.0–45.0
QPlogPo/w	2.82	3.12	1.37	2.26	2.64	2.95	3.31	3.68	4.26	5.36	2.59	– 1.75	– 2.0–6.5
QPlogS	– 5.33	– 5.86	– 4.68	– 4.87	– 5.30	– 5.54	– 5.79	– 6.03	– 7.12	– 8.29	– 3.14	– 1.55	– 6.5–0.5
QPPCaco	208.55	212.48	71.86	204.55	203.17	191.94	243.56	208.35	271.45	263.90	2.931.42	36.88	< 25 poor, great > 500
QPlogBB	– 1.54	– 1.58	– 2.27	– 1.64	– 1.75	– 1.85	– 1.81	– 1.94	– 2.07	– 2.37	0.37	– 1.74	– 3.0–1.2
QPPMDCK	154.30	157.85	48.88	150.99	149.90	141.32	186.65	154.11	205.18	199.23	1.582.00	24.13	< 25 poor, great > 500
QPlogKp	– 3.11	– 3.30	– 4.53	– 3.70	– 3.61	– 3.58	– 3.31	– 3.36	– 2.92	– 2.67	– 1.78	– 5.90	– 8.0–1.0
QPlogKhsa	0.97	0.25	– 0.39	– 0.85	0.27	0.13	0.20	0.35	0.49	0.84	0.72	– 0.97	– 1.5–1.5
HOA	84.96	86.85	68.18	81.54	83.68	85.10	89.02	90.02	95.43	88.71	100.00	44.77	< 25% poor, high > 80%
PSA	110.34	110.37	151.43	111.88	111.91	111.58	109.39	110.49	111.60	111.67	34.21	134.97	7.0–200.0
Rule of Five	0	0	0	0	0	0	0	0	0	1	0	0	max. 4
Rule of Three	0	1	0	0	0	0	1	1	1	1	0	0	max. 3
PAINS	0	0	0	0	0	0	0	0	0	0	0	0	–

Various computational pharmacodynamic and pharmacokinetic parameters of synthesized compounds in this research were predicted such as molecular weight of the compound (MW), computed dipole moment of the compound (dipole), total solvent-accessible volume in cubic angstroms using a probe with a 1.4 Å radius (volume), octanol/gas partition coefficient (QPlogPoct), water/gas partition coefficient (QPlogPw), octanol/water partition coefficient (QPlogPo/w), aqueous solubility (QPlogS), apparent Caco-2 cell permeability in nm/sec (QPPCaco), brain/blood partition coefficient (QPlogBB), apparent MDCK cell permeability in nm/sec (QPPMDCK), skin permeability (QPlogKp), prediction of binding to human serum albumin (QPlogKhsa), human oral absorption (HOA), van der Waals surface area of polar nitrogen and oxygen atoms (PSA), number of violations of Lipinski’s rule of five, number of violations of Jorgensen’s rule of three, and pan-assay interference compounds (PAINS) alert

Molecular docking assay

To gain major insights into the causes of SARs researched for novel synthesized N-substituted sulfonyl amides (6a–j) incorporating 1,3,4-oxadiazol structural motif, molecular docking studies were performed. Firstly, for the redocking computes, the native ligands, E20 (1-Benzyl-4-[(5,6-dimethoxy-1-indanon-2-yl)methyl]piperidine), GZH (1-[4-chloranyl-3-(trifluoromethyl)phenyl]-3-[2-(4-sulfamoylphenyl)ethyl]urea), and 9E8 (1-(4-Chlorophenyl)-3-[2-(4-sulfamoylphenyl)ethyl]urea), in the receptors’ binding sites (AChE, PDB code 4EY7; hCA I, PDB code 6I0L; and hCA II, PDB code 5NY3, respectively) were used. The docked poses of E20, GZH, and 9E8 overlapped with the poses in the X-ray crystal structures of the AChE, hCA I, and hCA II at a root mean square deviation (RMSD) values of 0.62, 1.51, and 1.20 Å, respectively (Fig. 4). These redocking assays were instrumental for selecting the best model structures that could host all the newly synthesized AChE, hCA I, and hCA II inhibitors, namely, novel N-substituted sulfonyl amide derivatives (6a–j). After that, the constructed binding model was used to perform docking calculations of the most potent AChE, hCA I, and hCA II inhibitors, compounds 6h, 6d, and 6a, respectively, in this series (6a–j) employing the Ligand Docking panel.

Fig. 4.

3D interactions and 2D docking poses of the native ligands A E20 (C₂₄H₂₉NO₃: 1-benzyl-4-[(5,6-dimethoxy-1-indanon-2-yl)methyl]piperidine) with the key amino acids within the active site of AChE (PDB iD: 4EY7), B GZH (C₁₆H₁₅ClF₃N₃O₃S: 1-[4-chloro-3-(trifluoromethyl)phenyl]-3-[2-(4-sulfamoylphenyl)ethyl]urea) with the key amino acids within the active site of hCA I (PDB iD: 6I0L), and (C) 9E8 (C₁₅H₁₆ClN₃O₃S: 1-(4-chlorophenyl)-3-[2-(4-sulfamoylphenyl)ethyl]urea) with the key amino acids within the active site of hCA II (PDB iD: 5NY3)

The predicted binding mode (docking score of − 7.34 kcal/mol and MM-GBSA value of − 49.23 kcal/mol) between compound 6h and AChE showed that an H-bond interaction was generated between the oxygen atom of the sulfonamide moiety and Phe295 residue (distance of 2.25 Å). Also, Tyr124 (distance of 2.34 Å) made an H-bond with the carboxy group. However, both benzyl ring and oxadiazole-moiety stacked against Trp286 in the peripheral anionic site. Furthermore, hydrophobic interactions were monitored between derivative 6h and Tyr72, Ala204, Leu289, Val294, Phe297, Tyr337, Phe338, and Tyr341 residues (Fig. 5). A docking score of − 4.10 kcal/mol and MM-GBSA value of − 11.96 kcal/mol indicated that compound 6d is a tight binder for hCA I. A further look into the structural properties revealed agent 6d coordinated to the Zn(II) ion employing the deprotonated sulfonamide moiety, which in turn is involved in an H-bond interaction with the amide nitrogen of Thr200 (distance of 2.28 Å); further, the oxadiazole-moiety of derivative 6d displayed the strong H-bond involvement with Gln92 residue (distance of 2.60 Å). Moreover, it was exhibited that residues Phe91, Ala121, Leu131, Ala132, Ala135, Leu141, Val143, Leu198, Pro202, Val207, and Trp209 play significant roles in the binding of agent 6d with hCA I (Fig. 6). One oxygen atom of the sulfonamide moiety of compound 6a (docking score of − 4.58 kcal/mol and MM-GBSA value of − 26.13 kcal/mol) formed an H-bond with the Asn62 (distance of 2.76 Å), while the other oxygen atom made two H-bonds with the Asn67 and Gln92 (distances of 2.48 and 1.91 Å, respectively) from the binding site residues of hCA II. Also, the oxadiazole-moiety displayed π − π stacking interaction with Trp5 and His64. Additionally, compound 6a interacted with the hydrophobic pocket formed by Ala65, Val121, Leu141, Val143, Leu198, Pro201, Pro202, Val207, and Trp209, in the active site (Fig. 7).

Fig. 5.

A 3D interaction of the derivative 6h (C₁₈H₂₅N₃O₄S₂: N-([4-(5-(ethylthio)-1,3,4-oxadiazol-2-yl)phenyl]sulfonyl)octanamide) with the key amino acids within the active site of AChE (PDB iD: 4EY7). B 2D docking pose of the derivative 6h (C₁₈H₂₅N₃O₄S₂: N-([4-(5-(ethylthio)-1,3,4-oxadiazol-2-yl)phenyl]sulfonyl)octanamide) with the key amino acids within the binding site of AChE (PDB iD: 4EY7)

Fig. 6.

A 3D interaction of the derivative 6d (C₁₄H₁₇N₃O₄S₂: N-([4-(5-(ethylthio)-1,3,4-oxadiazol-2-yl)phenyl]sulfonyl)butyramide) with the key amino acids within the active site of hCA I (PDB iD: 6I0L). B 2D docking pose of the derivative 6d (C₁₄H₁₇N₃O₄S₂: N-([4-(5-(ethylthio)-1,3,4-oxadiazol-2-yl)phenyl]sulfonyl)butyramide) with the key amino acids within the binding site of hCA I (PDB iD: 6I0L)

Fig. 7.

A 3D interaction of the derivative 6a (C₁₇H₁₅N₃O₄S₂: N-([4-(5-(ethylthio)-1,3,4-oxadiazol-2-yl)phenyl]sulfonyl)benzamide) with the key amino acids within the active site of hCA II (PDB iD: 5NY3). B 2D docking pose of the derivative 6a (C₁₇H₁₅N₃O₄S₂: N-([4-(5-(ethylthio)-1,3,4-oxadiazol-2-yl)phenyl]sulfonyl)benzamide) with the key amino acids within the binding site of hCA II (PDB iD: 5NY3)

Conclusion

The small safety window of current AChEIs and hCAIs has limited their maximum therapeutic application in the therapy of some metabolic disorders, such as AD, epilepsy, cancer, and glaucoma. Here, we have attempted to design desirable novel multi-target AChEIs and hCAIs to overcome this deficiency. By this strategy, novel N-substituted sulfonyl amide derivatives (6a–j) were synthesized, and all of them displayed excellent inhibitory effects against AChE and hCAs. As expected, their effective doses were much lower than that of THA and AAZ, and in silico molecular docking studies on the X-ray co-crystal complexes for three highly potent derivatives (6a, 6d, and 6h) were provided precise binding modes between the individual agents and hCA II, hCA I, and AChE, respectively. Overall, according to both in silico ADME-Tox and cytotoxic effect studies on cortex neuron cells and neuroblastoma SH-SY5Y cell line were especially determined compounds 6a, 6d, and 6h be orally bioavailable, highly potent, and brain penetrant AChEIs and hCAIs. These favorable outcomes motivate us to detect further therapeutic values; more efforts for discovering novel multi-target AChEIs and hCAIs are currently underway.

Supplementary Information

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Declarations

Conflict of interest

The authors declare that there is no conflict of interest.