
==== Front
Molecules
Molecules
molecules
Molecules
1420-3049
MDPI

10.3390/molecules27072121
molecules-27-02121
Article
Design, Synthesis, and Evaluation of Novel 2H-Benzo[b][1,4]thiazin-3(4H)-one Derivatives as New Acetylcholinesterase Inhibitors
Haji Ali Sazan 12
https://orcid.org/0000-0002-0499-436X
Osmaniye Derya 23
https://orcid.org/0000-0002-0151-6266
Sağlık Begüm Nurpelin 23
https://orcid.org/0000-0003-3692-163X
Levent Serkan 23
https://orcid.org/0000-0001-8815-153X
Özkay Yusuf 23
https://orcid.org/0000-0003-2252-0923
Kaplancıklı Zafer Asım 2*
Geronikaki Athina Academic Editor
1 Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Hawler Medical University, Erbil 44000, Iraq; hwall@example.com
2 Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Anadolu University, Eskişehir 26470, Turkey; hwall@example.com (D.O.); hwall@example.com (B.N.S.); hwall@example.com (S.L.); hwall@example.com (Y.Ö.)
3 Central Research Laboratory, Faculty of Pharmacy, Anadolu University, Eskişehir 26470, Turkey
* Correspondence: hwall@example.com
25 3 2022
4 2022
27 7 212128 2 2022
22 3 2022
© 2022 by the authors.
2022
https://creativecommons.org/licenses/by/4.0/ Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
Alzheimer's disease (AD) is a slowly progressive neurodegenerative disease that causes dementia in people aged 65 and over. In the present study, a series of thiadiazole hybrid compounds with benzothiazine derivatives as acetylcholinesterase inhibitors were developed and evaluated for their biological activity. The AChE and BChE inhibition potentials of all compounds were evaluated by using the in vitro Ellman method. The biological evaluation showed that compounds 3i and 3j displayed significant inhibitory activity against AChE. Compounds 3i and 3j showed IC50 values of 0.027 µM and 0.025 µM against AChE, respectively. The reference drug donepezil (IC50 = 0.021 µM) also showed significant inhibition against AChE. Further docking simulation also revealed that these compounds (3i and 3j) interacted with the active site of the enzyme similarly to donepezil. The antioxidant study revealed that compounds 3i and 3j exhibited greater antioxidant effects. An in vitro blood–brain barrier permeability study showed that compounds 3i and 3j are promising compounds against AD. The cytotoxicity study of compounds 3i and 3j showed non-cytotoxic with an IC50 value of 98.29 ± 3.98 µM and 159.68 ± 5.53 µM against NIH/3T3 cells, respectively.

Alzheimer's disease
thiadiazole
benzothiazine
acetylcholinesterase
molecular docking
antioxidant activity
blood–brain barrier permeability
cytotoxicity
==== Body
pmc1. Introduction

A silent epidemic in Europe, and worldwide in general, this is one of the most common types of dementia in people over the age of 65, known as Alzheimer's disease (AD). According to the 2021 WHO global status report, the prevalence of AD and estimated cases with dementia will increase dramatically in the coming decades, from 55 million in 2019 to 139 million in 2050 [1]. AD is still an intricate health issue and remains an incurable disease in modern medicine. Unfortunately, this complication in treatment is due to many factors, such as erroneous differential diagnoses, unclear pathophysiology, and individual differences in symptoms; rarely do two people experience symptoms of dementia in the same way.

AD is a progressive, destructive neurodegenerative disorder. The main characteristics and clinical demonstrations of AD are cognitive impairment and memory loss, changes in mood and behavior, difficulty in performing familiar tasks, impairment in daily physical activities, in addition to a variety of neuropsychiatric symptoms [2]. The duration of the disease, during which the patient suffers from difficulty in performing daily activities to the final stage of memory loss and immobility, is protracted, about 8–10 years [3].

The etiology of AD, leading to the neurodegeneration and destruction of neurons, is explained by several factors. The aggregation of beta-amyloid proteins (Ab), the destruction of cholinergic neurons, neuroinflammation, mitochondrial damage, oxidative stress, and the degradation of tau proteins are the major predisposing factors for the progression of AD [4,5,6]. To date, the causes of neurodegeneration in AD patients are not well understood, so effective and curative drugs cannot yet be developed.

The prominent neuronal alteration in AD is a change in the level of cholinergic neurotransmitters such as acetylcholine ACh concentration in the cortex and hippocampus [7]. Studies on cholinergic neurons have shown that with disease progression, premature loss and severe damage to cholinergic neurons in the basal forebrain region can be observed. [8]. Consequently, the inhibition of the AChE enzyme to prevent the hydrolysis of ACh is the most important strategy in the treatment of AD. Currently, the clinically used AChEI drugs only have symptom-relieving effects and improve the quality of life of patients in mild to moderate stages of the disease. So far, there is no AChEI that can prevent the progression of the disease [9,10,11,12]. In addition, AChEIs do not show the same pharmacological effect in all AD patients, and to date, it remains unclear why some patients respond while others do not. [13,14]. According to some clinical studies regarding responders and non-responders to AChEIs, it was reported that the probability of response to donepezil in patients with temporal lobe atrophy is very low, while patients with a high level of an allele known as APOE-ε4 are more likely to show a response to donepezil [13,15,16,17]. On the other hand, oxidative stress, characterized by the production of free radical reactive oxygen species (ROS), is one of the predisposing factors for the progression of AD. The overproduction of ROS plays an important role in the age-related progression of neurodegeneration and cognitive impairment [6,18].

Compounds with dual binding affinity to both active binding sites of the AChE enzyme, such as donepezil, have an excellent AChE inhibition profile [19,20]. In continuation of our previous research [21], in this work, we designed and synthesized a series of donepezil-based molecules with an evaluation of their AChE inhibitor activity in comparison to donepezil, as shown in Figure 1.

In drug development, heterocyclic rings are usually used as the main constituent of the molecular structure. In medicinal chemistry, molecules with nitrogen and sulfur as functional groups have shown a variety of biological activities [22]. Five membered 1,3,4-thiadiazole rings have demonstrated various biological activities, which includes acetylcholinesterase inhibitors [23], antibacterial [24,25], antifungal [26,27], anti-cancer [28,29], antioxidant [30], analgesic, and anti-inflammatory activity [31].

In the present study, the hybridization of two bioactive molecules, thiadiazole with benzothiazine, as a single biologically active moiety was designed. The newly combined molecules were tested for their biological activity as an acetylcholinesterase inhibitor in the treatment of Alzheimer's disease.

2. Results and Discussion

2.1. Chemistry

Compounds 3a–3j were synthesized in several steps, as shown in Scheme 1. In addition, the formulas of the compounds obtained are presented in Table 1. Initially, the synthesis was started with the derivatives of isothiocyanate in reaction with hydrazine hydrate. The series of 1a–1j compounds were obtained and, in another step, reacted with CS2 to achieve compounds 2a–2j. Finally, the target compounds 3a–3j were obtained via a nucleophilic substitution reaction between thiadiazole ring derivatives 2a–2j and a 6-(2-chloroacetyl)-2H-benzo[b][1,4]thiazin-3(4H)-one molecule. The structures of the achieved compounds were verified using spectroscopic methods, namely IR, 1H-NMR, 13C-NMR, and HRMS (Supplementary Data).

2.2. Cholinesterase Enzymes Inhibition Assay

The inhibitory activities of all of the obtained chalcone derivatives (3a–3j) against cholinesterase enzymes were evaluated using the previously described in vitro modified Ellman's spectrophotometric method [32,33,34,35,36,37,38,39,40].

The assay was completed in two steps. The first step was achieved by means of all the chalcones (3a–3j) and reference agents, namely donepezil and tacrine, at concentrations of 1000 and 100 µM. The enzyme activity results of the first step are presented in Table 2. Next, the selected compounds (3i and 3j) that displayed more than 50% inhibitory activity at concentrations of 1000 and 100 µM were further tested, along with the reference agents, at concentrations of 10 to 0.001 µM. The IC50 values of the test compounds and reference agents are presented in Figure 2.

As a result of the enzyme inhibition test, it was observed that all compounds were more effective against AChE. None of the compounds exhibited greater than 50% inhibitory activity against BChE in the first step. However, especially compounds with an aromatic substituent showed inhibitory activity comparable to that of donepezil against AChE. Compounds 3i and 3j passed the second step of the enzyme activity assay, and their IC50 values were calculated by performing an enzyme inhibition study at concentrations of 10 to 0.001 µM. The IC50 values of compounds 3i and 3j were calculated as 0.027 and 0.025 µM, respectively.

When the building activity relations were examined, derivatives containing aromatic substitutions were found to be more active. Derivatives with an electron-withdrawing substituent in the fourth position of the phenyl ring were more active than the non-substituted phenyl ring. In this case, it can be said that the substituent in the fourth position was necessary for the activity. One of the derivatives containing substituents in this position carried methoxy and the other chlorine substituents. It was observed that the chlorine-bearing substituent was more active. Molecular docking studies were carried out to elucidate the reason for this.

2.3. DPPH Free Radical Scavenging Antioxidant Activity

This activity method is a method based on measuring the scavenging effects of the DPPH• (1,1-Diphenyl-2-picrylhydrazil) radical, which is a stable organic nitrogen radical of antioxidants. One of the few stable organic nitrogen radicals, the DPPH• radical is dark violet in color. This method, which is based on the scavenging of the DPPH radical via antioxidants, is a redox reaction. When this radical interacts with hydrogen donors, it is reduced to hydrazine. The dark purple color of the methanolic DPPH• solution turns yellow with the addition of antioxidants, and the change in absorbance is measured spectrophotometrically. More illumination is determined by a greater reduction in the absorbance of the reaction mixture and indicates higher radical scavenging capacity [41,42].

For the DPPH free radical scavenging activity test, test compounds were prepared at 10 µM, 1 µM, 0.1 µM, and 0.01 µM concentrations, as shown in Figure 3. By using the absorbance changes determined because of the spectroscopic measurement, the % DPPH free radical scavenging activities of the synthesized compounds and reference materials (ascorbic acid and citric acid) were determined. The % antioxidant activities of all tested compounds were calculated based on the control. Among the test compounds, all compounds at a concentration of 10 µM showed more than 50% antioxidant activity. Compounds 3i and 3j showed inhibition of 90.00% ± 2.40; 92.00% ± 1.80, respectively, at this concentration. They showed inhibition of 82.00% ± 1.20; 85.00% ± 2.00, respectively, at 1 µM concentration. Compounds 3i and 3j showed inhibition of 75.00% ± 1.80; 73.00% ± 1.40, respectively, at a concentration of 0.1 µM. They showed inhibition of 65.00% ± 1.10; 68.00% ± 1.50, respectively, at a concentration of 0.01 µM. Compounds 3i and 3j were observed to have a higher antioxidant activity compared to other obtained compounds. As a result of these findings, it was revealed that compounds 3i and 3j may have the potential effects on patients suffering from AD.

2.4. In Vitro BBB Permeability Assay

As it is known, the ability of drugs to be used in diseases related to the central nervous system to pass the blood–brain barrier is an indispensable feature. These properties of the synthesized compounds were estimated using physicochemical parameters. Additionally, according to these parameters, it is seen that they have physicochemical properties that can pass the BBB. To verify the validity of these estimated data, in vitro PAMPA tests were performed on the most active derivatives. Additionally, the results are presented in Table 3. Compounds 3i and 3j was found to have high BBB permeability. This valuable evidence once again proved that compounds 3i and 3j are promising compounds against AD.

2.5. Cytotoxicity Assay

Compounds 3i and 3j exhibited potent AChE inhibition profiles and were further tested for toxicity using the MTT assay in the NIH/3T3 cell line; the IC50 values of these compounds were calculated. Compound 3i and 3j showed non-cytotoxic with an IC50 value of 98.29 ± 3.98 µM and 159.68 ± 5.53 µM against NIH/3T3 cells, respectively. This result suggests that the compounds did not show cytotoxic activity.

2.6. Molecular Docking Studies

Molecular docking studies were performed to verify the compounds' inhibition capabilities in silico. For this purpose, the crystal structure of human acetylcholine esterase enzyme (PDB ID: 4EY7) [19] was retrieved from the Protein Data Bank server (www.pdb.org) (accessed on 1 January 2022). The resulting docking poses are presented in Figure 4, Figure 5 and Figure 6.

Figure 4 shows the localization of all active compounds (3i and 3j) and donepezil to the enzyme active site. All compounds were localized to the enzyme active site.

Figure 5 and Figure 6 show that the indanone ring of donepezil exhibited one π–π interaction with the indole ring of amino acid Trp286. The carbonyl group of the indanone formed a hydrogen bond with the amine group of Phe295, and this group formed an aromatic hydrogen bond with phenyl of Phe338. The nitrogen of piperidine ring exhibited three π–π interactions with the phenyl ring of Phe338, Tyr337, and the indole ring of Trp86. The phenyl ring of the benzyl group attached to the piperidine ring exhibited one π–π interaction and three aromatic hydrogen bonds. While the π–π interaction occurred with the indole ring of Trp86, the aromatic hydrogen bonds were formed between the carbonyl group of His447, the carbonyl group of Glu202, and the hydroxyl group of Glu202.

Figure 7 and Figure 8 show that the 2H-benzo[b][1,4]thiazin-3(4H)-one ring of compound 3i exhibited π–π interactions with the indole ring of amino acid Trp286. The carbonyl group and amino group of 2H-benzo[b][1,4]thiazin-3(4H)-one ring formed two hydrogen bonds with the hydroxy group of Ser293 and carbonyl group of Ser293. The carbonyl group of compound 3i formed a hydrogen bond with the hydroxy group of Phe295. The thiadiazol ring of compound 3i formed a π–π interaction with the phenyl ring of Tyr337. The phenyl ring of the compound 3i exhibited π–π interactions with the indole ring of Trp86.

Figure 9 and Figure 10 show that the 2H-benzo[b][1,4]thiazin-3(4H)-one ring of compound 3j exhibited a π–π interaction with the indole ring of amino acid Trp286. The carbonyl group of 2H-benzo[b][1,4]thiazin-3(4H)-one ring formed a hydrogen bond with the amine group of Ser293. The carbonyl group of compound 3j formed a hydrogen bond with the amino group of Phe295, and this group formed an aromatic hydrogen bond with phenyl rings of Phe338. The thiadiazol ring of compound 3j formed two π–π interactions with phenyl rings of Tyr341 and Tyr337. The phenyl ring of compound 3j formed a π–π interaction with the indole ring of Trp86. Additionally, this phenyl ring formed two aromatic hydrogen bonds with carbonyl groups of His447 and Glu202.

In the light of the above information, it is seen that the aromatic ring is required for the thiadiazole ring. According to the activity results, active derivatives carry aromatic substituents. This seems to be due to the interaction between aromatic substituents and Trp86 in the CAS region of the enzyme. In addition, if it carries halogen on the aromatic structure here, it also establishes aromatic hydrogen bonds with Glu202 and His447. These bonds are also made by donepezil, and their properties show selectivity against AChE.

3. Materials and Methods

3.1. Chemistry

3.1.1. General

All reagents were purchased from commercial suppliers and were used without further purification. Melting points (M.p.) were determined using the Mettler Toledo-MP90 Melting Point System and were uncorrected. A 1H-NMR (nuclear magnetic resonance) Bruker DPX 300 FT-NMR spectrometer and a 13C-NMR, Bruker DPX 75 MHz spectrometer (Bruker Bioscience, Billerica, MA, USA) were used. A total of 10 mg of compound was weighed and dissolved in 600 µL of DMSO-d6. In this way, the NMR procedure was carried out. Mass spectra were recorded on an LCMS-IT-TOF (Shimadzu, Kyoto, Japan) using ESI. In total, 10 mg of compound was weighed and dissolved in 1500 µL of MeOH. In this way, the HRMS procedure was carried out.

3.1.2. Synthesis of Hydrazine Carbosulfanylamide Derivatives (1a–1j)

Isothiocyanate derivatives (0.007 mol) and an excess of hydrazine hydrate were dissolved in ethanol (50 mL) and mixed at 0 °C. At the end of the reaction, ethanol was evaporated, and the precipitated product was purified via recrystallization.

3.1.3. Synthesis of 5-(R-amino)-1,3,4-thiadiazole-2-sulfanyl Derivatives (2a–2j)

Compounds 1a–1j (0.006 mol) were dissolved in absolute ethanol (20 mL). To the reaction mixture, 1 equivalent of NaOH and 1.2 equivalent of CS2 were added. The reaction mixture was refluxed for 8–10 h and followed up by TLC. The finished reaction mixture was acidified with 20% HCl, and the precipitates were filtered and purified through recrystallization in ethanol.

5-(Ethylamino)-1,3,4-thiadiazole-2-thiol (2a)

Yield: 90%, 1H-NMR (300 MHz, DMSO-d6): δ = 1.08 (3H, t, J = 7.1, -CH3), 3.1–3.36 (2H, q, J1 = 5.4, J2= 7.2, -CH2), 7.52 (1H, s, thiazin-NH-), 13.27 (1H, s, SH).

5-(Propylamino)-1,3,4-thiadiazole-2-thiol (2b)

Yield: 85%, 1H-NMR (300 MHz, DMSO-d6): δ = 0.84 (2H, m, -CH2), 1.4–1.52 (3H, t, J = 7.1, -CH3) 3.05–3.08 (2H, t, J1 = 7.1, -CH2), 7.56 (1H, s, thiazin-NH-), 13.26 (1H, s, SH).

5-(Isopropylamino)-1,3,4-thiadiazole-2-thiol (2c)

Yield: 92%, 1H-NMR (300 MHz, DMSO-d6): δ = 1.12 (6H, s, -2CH3) 3.61 (1H, m, -CH), 7.49 (1H, s, thiazin-NH-), 13.26 (1H, s, SH).

5-(Butyllamino)-1,3,4-thiadiazole-2-thiol (2d)

Yield: 89%, 1H-NMR (300 MHz, DMSO-d6): δ = 0.84 (3H, t, J = 7.15 -CH3), 1.26–1.33 (2H, m, -CH2), 1.45–1.49 (2H, m, -CH2), 3.05–3.43 (2H, s, -CH2), 7.57 (1H, s, thiazin-NH-), 13.26 (1H, s, SH).

5-(Isobutylamino)-1,3,4-thiadiazole-2-thiol (2e)

Yield: 92%, 1H-NMR (300 MHz, DMSO-d6): δ = 0.86 (6H, d, J = 6.6 -2CH3), 1.75–1.88 (1H, m, -CH), 2.95–2.99 (2H, d, J = 6.1 -CH2), 7.77 (1H, s, thiazin-NH-), 13.24 (1H, s, SH).

5-(Cyclohexylamino)-1,3,4-thiadiazole-2-thiol (2f)

Yield: 88%, 1H-NMR (300 MHz, DMSO-d6): δ = 1.17–1.29 (4H, m, cyclohexane), 1.51–1.66 (2H, m, cyclohexane), 1.86–1.88 (4H, m, cyclohexane), 1.9 (1H, m, cyclohexane), 7.49 (1H, s, thiazin-NH-), 13.24 (1H, s, SH).

5-(Phenylamino)-1,3,4-thiadiazole-2-thiol (2g)

Yield: 91%, 1H-NMR (300 MHz, DMSO-d6): δ = 6.97 (1H, t, J = 7.4, phenyl), 7.02 (2H, m, phenyl), 7.42 (2H, dd, J1 = 1.75, J2 = 8.1, phenyl), 10.23 (1H, s, thiazin-NH-), 13.67 (1H, s, SH).

5-(p-Tolylamino)-1,3,4-thiadiazole-2-thiol (2h)

Yield: 91%, 1H-NMR (300 MHz, DMSO-d6): δ = 2.5 (3H, s, -CH3), 7.1–7.3 (4H, dd, J1 = 1.7, J2 = 7.79, phenyl), 10.06 (1H, s, thiazin-NH-), 13.56 (1H, s, SH).

5-(4-Methoxyphenylamino)-1,3,4-thiadiazole-2-thiol (2i)

Yield: 87%, 1H-NMR (300 MHz, DMSO-d6): δ = 3.7 (3H, s, -CH3), 6.89–7.33 (4H, dd, J1 = 1.7, J2 = 7.79, phenyl), 9.98 (1H, s, thiazin-NH-), 13.39 (1H, s, SH).

5-((4-Chlorophenyl)amino)-1,3,4-thiadiazole-2-thiol (2j)

Yield: 84%, 1H-NMR (300 MHz, DMSO-d6): δ = 7.35–7.45 (4H, dd, J1 = 1.69, J2 = 7.71, phenyl), 10.4 (1H, s, thiazin-NH-), 13.72 (1H, s, SH).

3.1.4. Synthesis of Target Compounds (3a–3j)

The thiadiazole derivatives (2a–2j) (0.001 mol) and 6-(2-chloroacetyl)-2H-benzo[b] [1,4] thiazin-3(4H)-one (0.001 mol) were dissolved in acetone (20 mL) with the addition of 0.001 mol of K2CO3, and the mixture was refluxed overnight. After the completion of the reaction, the mixture was cooled, and precipitated product was filtered and purified using the recrystallization method in ethanol.

6-(2-((5-(Ethylamino)-1,3,4-thiadiazol-2-yl)sulfanyl)acetyl)-2H-benzo[b][1,4]thiazin-3(4H)-one (3a)

Yield: 90%, M.P.: 196.9–199 °C. IR (cm−1 bands): 3190 (N-H), 3093 (C-H), 1670 (C=O), 1394 (C-O); 1H-NMR (300 MHz, DMSO-d6): δ = 1.13 (3H, t, J = 7.18, -CH3), 3.21–3.25 (2H, q, J1 = 5.39, J2 = 7.15, -CH2), 3.56 (2H, s, 1,4-thiazine-3(4H)-one), 4.72 (2H, s, Sulfanylacetyl-H), 7.48–7.53 (2H, m, benzothiazine -H), 7.63–7.66 (1H, dd, J1 = 1.77, J2 = 8.13 benzothiazine, -H), 7.77–7.80 (1H, t, J1 = 5.26 Hz, propylamin-NH), 10.77 (1H, s, thiazin-NH-); 13C-NMR (75 MHz, DMSO-d6): δ = 11.81, 22.19, 28.87, 41.81, 46.83, 123.60, 124.64, 126.79, 128.98, 134.11, 138,09, 165.28, 192.77. HRMS (m/z): [M + H]+ calcd for C14 H14 N4 O2 S3: 367.0352; found: 367.0370.

6-(2-((5-(Propylamino)-1,3,4-thiadiazol-2-yl)sulfanyl)acetyl)-2H-benzo[b][1,4]thiazin-3(4H)-one (3b)

Yield: 89%, M.P.: 191.3–193.8 °C. IR (cm−1 bands): 3226 (N-H), 3095 (C-H), 1695 (C=O), 1369 (C-O); 1H-NMR (300 MHz, DMSO-d6): δ = 0.9 (3H, t, J = 7.93, -CH3), 1.50–1.57 (2H, m, -CH2), 3.1–3.2 (2H, q, -CH2), 3.56 (2H, s, 1,4-thiazine-3(4H)-one), 4.72 (2H, s, Sulfanylacetyl-H), 7.48–7.53 (2H, m, benzothiazine -H), 7.63–7.66 (1H, dd, J1 = 1.79, J2 = 8.1 benzothiazine, -H), 7.79–7.83 (1H, t, J1 = 5.42 Hz, propylamin-NH), 10.76(1H, s, thiazin-NH-); 13C-NMR (75 MHz, DMSO-d6): δ = 11.81, 22.19, 28.87, 41.81, 46.83, 123.60, 124.64, 126.79, 128.98, 134.11, 138,09, 165.28, 192.77. HRMS (m/z): [M + H]+ calcd for C15 H16 N4 O2 S3: 381.0508; found: 381.0513.

6-(2-((5-(Isopropylamino)-1,3,4-thiadiazol-2-yl)sulfanyl)acetyl)-2H-benzo[b][1,4]thiazin-3(4H)-one (3c)

Yield: 92%, M.P.: 212.5–214.4 °C. IR (cm−1 bands): 3219 (N-H), 2983 (C-H), 1699 (C=O), 1369 (C-O); 1H-NMR (300 MHz, DMSO-d6): δ = 1.14–1.16 (6H, d, J = 6.44, CH3- isopropyl), 3.68–3.79 (1H, m, -CH), 3.56 (2H, s, 1,4-thiazine-3(4H)-one), 4.72 (2H, s, Sulfanylacetyl-H), 7.48–7.53 (2H, m, benzothiazine -H), 7.63–7.66 (1H, dd, J1 = 1.81, J2 = 8.13 benzothiazine, -H), 7.70–7.72 (1H, d, J1 = 7.1 Hz, isopropylamin-NH), 10.76 (1H, s, thiazin-NH); 13C-NMR (75 MHz, DMSO-d6): δ = 22.57, 28.86, 41.78, 46.98,116.54, 123.59, 126.85, 127.88, 134.06, 138,09, 140.66, 148.77, 165.27, 169.19, 192.77. HRMS (m/z): [M + H]+ calcd for C15 H16 N4 O2 S3: 381.0508; found: 381.0529.

6-(2-((5-(Butylamino)-1,3,4-thiadiazol-2-yl)sulfanyl)acetyl)-2H-benzo[b][1,4]thiazin-3(4H)-one (3d)

Yield: 70%, M.P.: 177–180 °C. IR (cm−1 bands): 3240 (N-H), 3184 (C-H), 1666 (C=O), 1398 (C-O); 1H-NMR (300 MHz, DMSO-d6): δ = 0.8–0.9 (3H, t, J = 7.3 Hz -CH3), 1.27–1.35 (2H, m, methylene), 1.46–1.53 (2H, m, methylene), 1.02–1.07 (2H, t, J = 6.99, methylamine-CH2), 3.56 (2H, s, 1,4-thiazine-3(4H)-one), 4.99 (2H, s, Sulfanylacetyl-H), 7.5–7.54 (2H, m, benzothiazine -H), 7.65–7.66 (1H, dd, J1 = 1.8, J2 = 8.13 benzothiazine, -H), 7.94 (1H, s, butylamine-NH), 10.76 (1H, s, thiazin-NH-). HRMS (m/z): [M + H]+ calcd for C16 H18 N4 O2 S3:395.0665; found: 395.0659.

6-(2-((5-(Isobutylamino)-1,3,4-thiadiazol-2-yl)sulfanyl)acetyl)-2H-benzo[b][1,4]thiazin-3(4H)-one (3e)

Yield: 92%, M.P.: 194.1–195.8 °C. IR (cm−1 bands): 3242 (N-H), 3196 (C-H), 1668 (C=O), 1398 (C-O); 1H-NMR (300 MHz, DMSO-d6): δ = 0.87–0.89 (6H, d, J = 6.67 Hz, 2CH3- isobutyl), 1.79–1.88 (1H, m, CH-isobutyl), 3.02–3.06 (2H, dd, J1 = 1.01, J2 = 12.44, CH2-isobutyl), 3.56 (2H, s, 1,4-thiazine-3(4H)-one), 4.71 (2H, s, Sulfanylacetyl-H), 7.48–7.53 (2H, m, benzothiazine -H), 7.63–7.66 (1H, dd, J1 = 1.81, J2 = 8.13 benzothiazine, -H), 7.81–7.85 (1H, t, J1 = 5.59 Hz, isobutylamin-H), 10.76 (1H, s, thiazin-NH-); 13C-NMR (75 MHz, DMSO-d6): δ = 20.50, 27.95, 28.87, 41.79, 52.65, 116.56, 123.60, 126.86, 127.89, 134.07, 138,09, 165.28, 170.38, 192.78. HRMS (m/z): [M + H]+ calcd for C16 H18 N4 O2 S3: 395.0665; found: 395.0665.

6-(2-((5-(Cyclohexylamino)-1,3,4-thiadiazol-2-yl)sulfanyl)acetyl)-2H-benzo[b][1,4]thiazin-3(4H)-one (3f)

Yield: 88%, M.P.: 229–232 °C. IR (cm−1 bands): 3242 (N-H), 3221 (C-H), 1674 (C=O), 1388 (C-O); 1H-NMR (300 MHz, DMSO-d6): δ = 1.14–1.35 (4H, m, cyclohexane), 1.52–1.69 (2H, m, cyclohexane), 1.91–1.93 (4H, m, cyclohexane), 2.7 (1H, m, cyclohexane), 3.56 (2H, s, 1,4-thiazine-3(4H)-one), 4.70 (2H, s, Sulfanylacetyl-H), 7.48–7.53 (2H, m, benzothiazine -H), 7.62–7.66 (1H, dd, J1 = 1.83, J2 = 8.13 benzothiazine, -H), 7.73–7.75 (1H, t, J1 = 7.29 Hz, isobutylamin-H), 7.95 (1H, s, benzothiazine, -H), 10.76 (1H, s, thiazin-NH); 13C-NMR (75 MHz, DMSO-d6): δ = 24.65, 25.65, 28.86, 32.44, 41.76, 53.91, 116.55, 123.60, 126.85, 127.88, 134.08, 138,09, 165.28, 169.22, 192.80. HRMS (m/z): [M + H]+ calcd for C18 H20 N4 O2 S3: 421.0821; found: 421.0821.

6-(2-((5-(Phenylamino)-1,3,4-thiadiazol-2-yl)sulfanyl)acetyl)-2H-benzo[b][1,4]thiazin-3(4H)-one (3g)

Yield: 91%, M.P.: 243–245 °C. IR (cm−1 bands): 3197 (N-H), 3074 (C-H), 1662 (C=O), 1398 (C-O); 1H-NMR (300 MHz, DMSO-d6): δ = 3.56 (2H, s, 1,4-thiazine-3(4H)-one), 4.87 (2H, s, Sulfanylacetyl-H), 6.96–7.0 (1H, t, J = 7.34, phenyl), 7.29–7.35 (2H, t, J = 15.91, phenyl), 7.50–7.56 (2H, d, J = 6.83, benzothiazine -H), 7.67–7.70 (2H, dd, J1 = 1.79, J2 = 8.14, phenyl), 7.94 (1H, s, benzothiazine, -H)), 10.37 (1H, s, phenylamine-H), 10.78 (1H, s, thiazin-NH-); 13C-NMR (75 MHz, DMSO-d6): δ = 28.86, 41.61, 116.53, 117.81, 122.46, 123.59, 126.96, 127.94, 129.58, 134.07, 138,12, 140.79, 152.34, 165.29, 165.32, 192.57. HRMS (m/z): [M + H]+ calcd for C18 H14 N4 O2 S3: 415.0352; found: 415.0357.

6-(2-((5-(p-Tolylamino)-1,3,4-thiadiazol-2-yl)sulfanyl)acetyl)-2H-benzo[b][1,4]thiazin-3(4H)-one (3h)

Yield: 95%, M.P.: 245–247.7 °C. IR (cm−1 bands): 3244 (N-H), 3192 (C-H), 1662 (C=O), 1400 (C-O); 1H-NMR (300 MHz, DMSO-d6): δ = 2.24 (3H, s, methyl), 3.56 (2H, s, 1,4-thiazine-3(4H)-one), 4.85 (2H, s, Sulfanylacetyl-H), 7.11–7.14 (2H, d, J = 8.3, phenyl), 7.40–7.43 (2H, d, J = 8.48, phenyl), 7.66–7.69 (2H, dd, J1 = 1.79, J2 = 8.14 benzothiazine), 7.94 (1H, s, benzothiazine-H), 10.26 (1H, s, phenylamine-H), 10.77 (1H, s, thiazin-NH-); 13C-NMR (75 MHz, DMSO-d6): δ = 20.78, 28.86, 41.62, 116.52, 117.94, 123.58, 126.95, 127.93, 129.96, 131.46, 134.07, 138,11, 138.42, 151.83, 165.29, 165.56, 192.58. HRMS (m/z): [M + H]+ calcd for C19 H16 N4 O2 S3: 429.0508; found: 429.0509.

6-(2-((5-((4-Methoxyphenyl)amino)-1,3,4-thiadiazol-2-yl)sulfanyl)acetyl)-2H-benzo[b][1,4]thiazin-3(4H)-one (3i)

Yield: 96%, M.P.: 217–220 °C. IR (cm−1 bands): 3234 (N-H), 3197 (C-H), 1681 (C=O), 1244 (C-O); 1H-NMR (300 MHz, DMSO-d6): δ = 3.71 (3H, s, methyl), 3.56 (2H, s, 1,4-thiazine-3(4H)-one), 4.83 (2H, s, Sulfanylacetyl-H), 6.89–6.92 (2H, m, phenyl), 7.43–7.55 (2H, m, phenyl), 7.66 (1H, d, J1 = 1.79, benzothiazine-H), 7.69 (1H, d, J1 = 1.79, benzothiazine-H), 7.94 (1H, s, benzothiazine-H), 10.17 (1H, s, phenylamine-H), 10.77 (1H, s, thiazin-NH-); 13C-NMR (75 MHz, DMSO-d6): δ = 28.86, 41.67, 55.70, 114.76, 116.52, 119.70, 123.59, 126.94, 127.92, 134.06, 134.29, 138.11, 151.29, 155.07, 165.29, 166.05, 192.62. HRMS (m/z): [M+H]+ calcd for C19 H16 N4 O3 S3: 445.449; found: 445.457.

6-(2-((5-((4-chlorophenyl)amino)-1,3,4-thiadiazol-2-yl)sulfanyl)acetyl)-2H benzo[b][1,4]thiazin-3(4H)-one (3j)

Yield: 95%, M.P.: 241–244 °C. IR (cm−1 bands): 3234 (N-H), 3194 (C-H), 1660 (C=O), 1244 (C-O); 1H-NMR (300 MHz, DMSO-d6): δ = 3.56 (2H, s, 1,4-thiazine-3(4H)-one), 4.88 (2H, s, Sulfanylacetyl-H), 7.35–7.38 (2H, m, phenyl), 7.50–7.59 (2H, m, phenyl), 7.67–7.70 (2H, dd, J1 = 1.8, J2 = 8.13 benzothiazine), 7.94 (1H, s, benzothiazine-H), 10.50 (1H, s, phenylamine-H), 10.78 (1H, s, thiazin-NH-); 13C-NMR (75 MHz, DMSO-d6): δ = 28.86, 41.57, 116.52, 119.34, 123.59, 125.82, 126.97, 127.94, 129.39, 134.08, 138,12, 139.67, 153.02, 164.92, 165.29, 192.53. HRMS (m/z): [M + H]+ calcd for C19 H16 N4 O2 S3 Cl:448.9962; found: 448.9976.

3.2. Cholinesterase Enzymes Inhibition Assay

The in vitro AChE and BChE inhibition potencies of the synthesized compounds (3a–3j) were evaluated according to the modified Ellman's spectrophotometric method [33]. The reagents and materials used in the enzyme inhibition assay were supplied commercially by Sigma-Aldrich (St. Louis, MO, USA) and Fluka (Steinheim, Germany). The cholinesterase enzyme inhibition procedure was applied as reported in our previous research papers [32,34,35,36,37,38,39,40].

3.3. DPPH Free Radical Scavenging Antioxidant Activity

This is a method based on measuring the scavenging effects of the DPPH (1,1-Diphenyl-2- picrylhydrazyl) radical, a stable organic nitrogen-centered free radical. To prepare the DPHH solution, 9.86 mg of DPPH was measured and topped up to 25 mL with methanol. A total of 100 µL of DPPH solution and 100 µL of the test solutions were placed in the test wells. Only 200 µL of methanol was used for blank reading; 100 µL of methanol and 100 µL of DPPH solution were used for control. After incubation, spectrophotometric reading was performed at 517 nm [41,42].

3.4. In Vitro BBB Permeability Assay

To observe the BBB permeability of the active compounds 3i and 3j, the parallel artificial membrane permeability assay (PAMPA) was performed as previously described [21]. Briefly, the solutions of each compound were prepared in dimethyl sulfoxide (DMSO) at 10 mM and then diluted with PBS (Phosphate-Buffered Saline; pH = 7.4) to obtain the donor drug solution with the nominal final concentration of 100 μM (297 μL of buffer + 3 μL of DMSO drug solution). An amount of 100 μM is suitable for direct HPLC-DAD measurements. This solution was shaken for 1 h at room temperature in a 96-well polypropylene plate (Agilent, Waldbronn, Germany) and then filtered to avoid solid particles. The data were measured in 3 replicates on each plate. After the preparation of the solutions, the assay procedure was performed according to the kit method [43,44,45].

3.5. Cytotoxicity Assay

The NIH/3T3 mouse embryonic fibroblast cell line (ATCC® CRL-1658™, London, UK) was used for cytotoxicity assays. The incubation period of NIH/3T3 cells was based on the supplier's recommendation. NIH/3T3 cells were seeded at 1 × 104 cells into each well of the 96-well plates. The MTT assay was carried out in accordance with the standards previously described [32,33,34].

3.6. Molecular Docking

A structure-based in silico procedure was applied to discover the binding modes of compounds 3i and 3j to the hAChE enzyme active site. The crystal structure of hAChE (PDB ID: 4EY7), which was crystallized with donepezil, was retrieved from the Protein Data Bank server (www.pdb.org) (accessed on 1 January 2022).

The structures of the ligands were built using the Schrödinger Maestro [46] interface and then were submitted to the Protein Preparation Wizard protocol of the Schrödinger Suite 2020 Update 2 [47]. The ligands were prepared using LigPrep 3.8 [48] to assign the protonation states at pH 7.4 ± 1.0 and the atom types correctly. Bond orders were assigned, and hydrogen atoms were added to the structures. The grid was generated using Glide 7.1 [49]. Flexible docking runs were performed with single precision docking mode (SP).

4. Conclusions

The designed series of 6-(2-((5-(substituted-amino)-1,3,4-thiadiazol-2-yl) sulfanyl) acetyl)-2H-benzo[b][1,4]thiazin-3(4H)-one compounds were successfully synthesized and characterized for their biological activity as acetylcholine esterase inhibitor agents. All compounds were analyzed using 1HNMR, 13CNMR, and HRMS spectrometric techniques. The compounds 3a–3j showed no inhibitory activity toward BChE, while their enzyme inhibition activity was observed in vitro. As a result of the enzyme inhibition test, it was observed that all compounds were more effective against AChE. During the examination the biological activity of the compounds 3a–3j, it was observed that derivatives with an electron-withdrawing substituent at position 4 of the phenyl ring showed greater inhibitory activity than non-substituted phenyl and aliphatic derivatives. Therefore, it can be said that the substituent in the fourth position is necessary for this activity. One of the derivatives containing substituents in this position carries methoxy 3i and the other chlorine substituents 3j. It was observed that the chlorine-bearing substituent 3i was more active, with the IC50 value of 0.027 M, and 3j that bears a methoxy group at position 4 showed an IC50 value of 0.025 M. Molecular docking studies were carried out to elucidate the reason for this. According to the docking study of the designed compounds 3a–3j, it was demonstrated that the aromatic ring is required for the thiadiazole ring to exert its activity. This seems to be due to the interaction between aromatic substituents and Trp86 in the CAS region of the enzyme. In addition, if the aromatics moiety carries electronegative substitution at the para position, it also establishes aromatic hydrogen bonds with Glu202 and His447 and enhances its inhibitory activity. These bonds are also made by donepezil, and their properties show selectivity against AChE.

The antioxidant activities of compounds 3i and 3j showed great results in comparison to citric acid and ascorbic acid as antioxidative agents. As a result of these findings, it was revealed that compounds 3i and 3j may have potential effects on patients suffering from AD. In conclusion, based on this study, further molecular modification can be carried out on thiadiazole ring derivatives, and new molecules with higher affinity to AChE can be designed and may be subjected to future studies.

Acknowledgments

As the authors of this study, we thank the Anadolu University Faculty of Pharmacy Central Research Laboratory for their support and contributions.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/molecules27072121/s1, Figures S1–S10. HRMS of 3a–3j; Figures S11–S20. 1HNMR of 3a–3j; Figures S21–S29. 13CNMR of 3a–3c, 3e–3j; Figures S30–S39. IR spectrum of 3a–3j; Figures S40–S49. 1HNMR of 2a–2j.

Click here for additional data file.

Author Contributions

D.O., Y.Ö. and Z.A.K. conceived and designed the experiments; D.O. and S.H.A. performed the synthesis; B.N.S. and D.O. performed the activity tests and molecular docking studies; S.H.A. and S.L. performed analysis studies; D.O. and S.H.A. wrote the paper. All authors have read and agreed to the published version of the manuscript.

Funding

This study was financially supported by Anadolu University Scientific Projects Fund, Project No: 2108S151.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not available.

Conflicts of Interest

The authors declare no conflict of interest.

Sample Availability

Samples of the compounds 2a–2j and 3a–3j are available from the authors.

Figures, Scheme and Tables

Figure 1 Acetylcholine esterase enzyme-binding sites.

molecules-27-02121-sch001_Scheme 1 Scheme 1 Synthesis pathway of compounds (3a–3j).

Figure 2 IC50 graphs of compounds 3i and 3j and donepezil on AChE enzyme (the graphs were formed using GraphPad Prism Version 6 via regression analyses).

Figure 3 % DPPH antioxidant activity of the synthesized compounds 3a–3j and reference agents.

Figure 4 The three-dimensional superimposition poses of compounds 3i, 3j and donepezil in the active region of human AChE enzyme (PDB ID: 4EY7). The important residues in the active site and related compounds are presented by tube model and colored with turquoise, pink, black and yellow, respectively.

Figure 5 The two-dimensional interacting mode of donepezil in the active region of AChE (PDB ID: 4EY7).

Figure 6 The three-dimensional interacting mode of donepezil in the active region of AChE (PDB ID: 4EY7). The inhibitor and important residues in the active site of enzyme are presented by tube model colored with red and turquoise, respectively.

Figure 7 The two-dimensional interacting mode of compound 3i in the active region of AChE (PDB ID: 4EY7).

Figure 8 The three-dimensional interacting mode of compound 3i in the active region of AChE (PDB ID: 4EY7). The inhibitor and important residues in the active site of enzyme are presented by tube model colored with pink and turquoise, respectively.

Figure 9 The two-dimensional interacting mode of compound 3j in the active region of AChE (PDB ID: 4EY7).

Figure 10 The three-dimensional interacting mode of compound 3j in the active region of AChE (PDB ID: 4EY7). The inhibitor and important residues in the active site of enzyme are presented by tube model colored with purple and turquoise, respectively.

molecules-27-02121-t001_Table 1 Table 1 Synthesized compounds (3a–3j).

Compounds	R	
3a	-CH2-CH3	
3b	-CH2-CH2-CH3	
3c	-CH(CH3)2	
3d	-CH2(CH2)2-CH3	
3e	-CH2-CH(CH3)2	
3f	-Cyclohexyl	
3g	-Phenyl	
3h	-p-Tolyl	
3i	-4-Methoxyphenyl	
3j	-4-Chlorophenyl	

molecules-27-02121-t002_Table 2 Table 2 % Inhibition of the synthesized compounds, donepezil and tacrine against AChE and BChE.

Compound	AChE % Inhibition	BChE % Inhibition	
10−3 M	10−4 M	10−3 M	10−4 M	
3a	78.2 ± 1.2	48.8 ± 0.8	31.8 ± 0.8	25.1 ± 0.8	
3b	75.4 ± 1.4	45.6 ± 0.9	34.9 ± 0.8	26.9 ± 0.6	
3c	70.6 ± 1.0	40.1 ± 0.9	29.1 ± 0.8	20.4 ± 0.7	
3d	72.7 ± 1.5	47.3 ± 0.9	36.1 ± 0.9	21.4 ± 0.9	
3e	69.9 ± 1.2	46.2 ± 0.8	37.3 ± 0.8	24.6 ± 0.9	
3f	65.4 ± 1.3	45.0 ± 1.0	30.5 ± 0.8	26.2 ± 0.8	
3g	80.1 ± 1.2	44.5 ± 0.8	39.6 ± 0.9	24.8 ± 0.8	
3h	82.2 ± 1.8	42.7 ± 0.8	35.6 ± 0.7	23.5 ± 0.7	
3i	93.2 ± 1.4	90.5 ± 1.1	40.2 ± 0.9	28.0 ± 0.8	
3j	96.5 ± 1.2	92.3 ± 1.3	42.5 ± 0.9	27.9 ± 0.7	
Donepezil	99.3 ± 2.1	97.4 ± 1.9	-	-	
Tacrine	-	-	98.3 ± 1.9	95.5 ± 1.3	

molecules-27-02121-t003_Table 3 Table 3 Type of blood–brain barrier (BBB) penetration of compounds 3i and 3j.

Classification	Type of BBB Permeation	Compounds	Type of BBB Permeation	
CNS+	High BBB permeation Pe (10−6 cm s−1) > 4.0	3i	CNS+ High BBB permeation	
3j	CNS+ High BBB permeation	
CNS−	Low BBB permeation Pe (10−6 cm s−1) < 2.0			
CNS±	BBB permeation uncertain 2.0 < Pe (10−6 cm s−1) < 4.0			

Publisher's Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.
==== Refs
References

1. Gauthier S. Rosa-Neto P. Morais J.A. Webster C. World Alzheimer Report 2021: Journey through the Diagnosis of Dementia Alzheimer's Disease International London, UK 2021
2. Tarawneh R. Holtzman D.M. The Clinical Problem of Symptomatic Alzheimer Disease and Mild Cognitive Impairment Cold Spring Harb. Perspect. Med. 2012 2 a006541-51-4932/cshperspect.a006148 22553492
3. Huber A. Stuchbury G. Burkle A. Burnell J. Munch G. Neuroprotective Therapies for Alzheimers Disease Curr. Pharm. Des. 2006 12 705 541-51-4932/138161206775474251 16472161
4. Godyń J. Jończyk J. Panek D. Malawska B. Therapeutic Strategies for Alzheimer's Disease in Clinical Trials Pharmacol. Rep. PR 2016 68 127 541-51-4932/j.pharep.2015.07.006 26721364
5. Henstridge C.M. Pickett E. Spires-Jones T.L. Synaptic Pathology: A Shared Mechanism in Neurological Disease Ageing Res. Rev. 2016 28 72 84 10.1016/j.arr.2016.04.005 27108053
6. Madeo J. The Role of Oxidative Stress in Alzheimer' s Disease J. Alzheimer's Dis. Park. 2013 3 116 541-51-4932/2161-0460.1000116
7. Mesulam M. The Cholinergic Lesion of Alzheimer's Disease: Pivotal Factor or Side Show? Learn. Mem. 2004 11 43 49 10.1101/lm.69204 14747516
8. Bartus R.T. On Neurodegenerative Diseases, Models, and Treatment Strategies: Lessons Learned and Lessons Forgotten a Generation Following the Cholinergic Hypothesis Exp. Neurol. 2000 163 495 541-51-4932/exnr.2000.7397 10833325
9. Atri A. Frölich L. Ballard C. Tariot P.N. Molinuevo J.L. Boneva N. Windfeld K. Raket L.L. Cummings J.L. Effect of Idalopirdine as Adjunct to Cholinesterase Inhibitors on Change in Cognition in Patients With Alzheimer Disease: Three Randomized Clinical Trials JAMA 2018 319 130 541-51-4932/jama.2017.20373 29318278
10. Courtney C. Farrell D. Gray R. Hills R. Lynch L. Sellwood E. Edwards S. Hardyman W. Raftery J. Crome P. Long-Term Donepezil Treatment in 565 Patients with Alzheimer's Disease (AD2000): Randomised Double-Blind Trial Lancet Lond. Engl. 2004 363 2105 2541-51-4932/S0140-6736(04)16499-4
11. Doody R.S. Dunn J.K. Clark C.M. Farlow M. Foster N.L. Liao T. Gonzales N. Lai E. Massman P. Chronic Donepezil Treatment Is Associated with Slowed Cognitive Decline in Alzheimer's Disease Dement. Geriatr. Cogn. Disord. 2001 12 295 541-51-4932/000051272 11351141
12. Lahiri D.K. Farlow M.R. Greig N.H. Sambamurti K. Current Drug Targets for Alzheimer's Disease Treatment Drug Dev. Res. 2002 56 267 541-51-4932/ddr.10081
13. Connelly P.J. Prentice N.P. Fowler K.G. Predicting the Outcome of Cholinesterase Inhibitor Treatment in Alzheimer's Disease J. Neurol. Neurosurg. Psychiatry 2005 76 320 541-51-4932/jnnp.2004.043539 15716519
14. Lemstra A.W. Richard E. van Gool W.A. Cholinesterase Inhibitors in Dementia: Yes, No, or Maybe? Age Ageing 2007 36 625 541-51-4932/ageing/afm117 17881419
15. Bizzarro A. Marra C. Acciarri A. Valenza A. Tiziano F.D. Brahe C. Masullo C. Apolipoprotein E Epsilon4 Allele Differentiates the Clinical Response to Donepezil in Alzheimer's Disease Dement. Geriatr. Cogn. Disord. 2005 20 254 541-51-4932/000087371 16103669
16. Choi S.H. Kim S.Y. Na H.R. Kim B.-K. Yang D.W. Kwon J.C. Park M.Y. Effect of ApoE Genotype on Response to Donepezil in Patients with Alzheimer's Disease Dement. Geriatr. Cogn. Disord. 2008 25 445 541-51-4932/000124752 18401173
17. Craig L.A. Hong N.S. McDonald R.J. Revisiting the Cholinergic Hypothesis in the Development of Alzheimer's Disease Neurosci. Biobehav. Rev. 2541-51-4932 1541-51-4932/j.neubiorev.2011.03.001 21392524
18. Gella A. Durany N. Oxidative Stress in Alzheimer Disease Cell Adhes. Migr. 2009 3 88 93 10.4161/cam.3.1.7402
19. Cheung J. Rudolph M.J. Burshteyn F. Cassidy M.S. Gary E.N. Love J. Franklin M.C. Height J.J. Structures of Human Acetylcholinesterase in Complex with Pharmacologically Important Ligands J. Med. Chem. 2541-51-49322 10541-51-4932/jm300871x 23035744
20. Lan J.-S. Zhang T. Liu Y. Yang J. Xie S.-S. Liu J. Miao Z.-Y. Ding Y. Design, Synthesis and Biological Activity of Novel Donepezil Derivatives Bearing N-Benzyl Pyridinium Moiety as Potent and Dual Binding Site Acetylcholinesterase Inhibitors Eur. J. Med. Chem. 2017 133 184 541-51-4932/j.ejmech.2017.02.045 28388521
21. Sağlık B.N. Osmaniye D. Acar Çevik U. Levent S. Kaya Çavuşoğlu B. Özkay Y. Kaplancıklı Z.A. Design, Synthesis, and Structure–Activity Relationships of Thiazole Analogs as Anticholinesterase Agents for Alzheimer's Disease Molecules 2541-51-4932 10.3390/molecules25184312
22. Kerru N. Gummidi L. Maddila S. Gangu K.K. Jonnalagadda S.B. A Review on Recent Advances in Nitrogen-Containing Molecules and Their Biological Applications Molecules 2541-51-4932 10.3390/molecules25081909 32326131
23. Kulshreshtha A. Piplani P. Ameliorative Effects of Amide Derivatives of 1,3,4-Thiadiazoles on Scopolamine Induced Cognitive Dysfunction Eur. J. Med. Chem. 2016 122 557 541-51-4932/j.ejmech.2016.06.046 27448914
24. Karcz D. Matwijczuk A. Kamiński D. Creaven B. Ciszkowicz E. Lecka-Szlachta K. Starzak K. Structural Features of 1,3,4-Thiadiazole-Derived Ligands and Their Zn(II) and Cu(II) Complexes Which Demonstrate Synergistic Antibacterial Effects with Kanamycin Int. J. Mol. Sci. 2541-51-4932 10.3390/ijms21165735 32785125
25. Su S. Zhou X. Liao G. Qi P. Jin L. Synthesis and Antibacterial Evaluation of New Sulfone Derivatives Containing 2-Aroxymethyl-1,3,4-Oxadiazole/Thiadiazole Moiety Mol. J. Synth. Chem. Nat. Prod. Chem. +1-352-717-4705.3390/molecules22010064 28042864
26. Karaburun A.Ç. Acar Çevik U. Osmaniye D. Sağlık B.N. Kaya Çavuşoğlu B. Levent S. Özkay Y. Koparal A.S. Behçet M. Kaplancıklı Z.A. Synthesis and Evaluation of New 1,3,4-Thiadiazole Derivatives as Potent Antifungal Agents Molecules 2541-51-4932 10.3390/molecules23123129 30501053
27. Pal S. Singh V. Kumar R. Gogoi R. Design and Development of 1,3,4-Thiadiazole Based Potent New Nano-Fungicides J. Mol. Struct. 2020 1219 128541-51-4932/j.molstruc.2020.128507
28. Gür M. Yerlikaya S. Şener N. Özkınalı S. Baloglu M.C. Gökçe H. Altunoglu Y.C. Demir S. Şener İ. Antiproliferative-Antimicrobial Properties and Structural Analysis of Newly Synthesized Schiff Bases Derived from Some 1,3,4-Thiadiazole Compounds J. Mol. Struct. 2020 1219 128541-51-4932/j.molstruc.2020.128570
29. Szeliga M. Karpińska M. Rola R. Niewiadomy A. Design, Synthesis and Biological Evaluation of Novel 1,3,4-Thiadiazole Derivatives as Anti-Glioblastoma Agents Targeting the AKT Pathway Bioorg. Chem. 2020 105 104541-51-4932/j.bioorg.2020.104362 33074121
30. Zabiulla Nagesh Khadri M.J. Bushra Begum A. Sunil M.K. Khanum S.A. Synthesis, Docking and Biological Evaluation of Thiadiazole and Oxadiazole Derivatives as Antimicrobial and Antioxidant Agents Results Chem. 2020 2 100541-51-4932/j.rechem.2020.100045
31. El-Hazek R.M.M. El-Sabbagh W.A. El-Hazek R.M. El-Gazzar M.G. Anti-Inflammatory and Analgesic Effect of LD-RT and Some Novel Thiadiazole Derivatives through COX-2 Inhibition Arch. Pharm. 2020 353 e2000541-51-4932/ardp.202000094
32. Sağlık B.N. Ilgın S. Özkay Y. Synthesis of New Donepezil Analogues and Investigation of Their Effects on Cholinesterase Enzymes Eur. J. Med. Chem. 2016 124 1026 1541-51-4932/j.ejmech.2016.10.042 27783974
33. Ellman G.L. Courtney K.D. Andres V. Feather-Stone R.M. A New and Rapid Colorimetric Determination of Acetylcholinesterase Activity Biochem. Pharmacol. 1961 7 88 95 10.1016/0006-2952(61)90145-9 13726518
34. Demir Özkay Ü. Can Ö.D. Sağlık B.N. Acar Çevik U. Levent S. Özkay Y. Ilgın S. Atlı Ö. Design, Synthesis, and AChE Inhibitory Activity of New Benzothiazole-Piperazines Bioorg. Med. Chem. Lett. 2541-51-4932 5541-51-4932/j.bmcl.2016.10.041 27789142
35. Cevik U.A. Levent S. Saglık B.N. Ozkay Y. Kaplancıklı Z.A. Synthesis of Novel 4-(Dimethylaminoalkyl)Piperazine-1-Carbodithioa t e Derivatives as Cholinesterase Inhibitors Lett. Drug Des. Discov. 2017 14 528 541-51-4932/1570180813666160923105636
36. Levent S. Acar Çevik U. Sağlık B.N. Özkay Y. Can Ö.D. Özkay Ü.D. Uçucu Ü. Anticholinesterase Activity Screening of Some Novel Dithiocarbamate Derivatives Including Piperidine and Piperazine Moieties Phosphorus Sulfur Silicon Relat. Elem. 2017 192 469 541-51-4932/10426507.2016.1259228
37. Hussein W. Sağlık B.N. Levent S. Korkut B. Ilgın S. Özkay Y. Kaplancıklı Z.A. Synthesis and Biological Evaluation of New Cholinesterase Inhibitors for Alzheimer's Disease Molecules 2541-51-4932 10.3390/molecules23082033
38. Acar Cevik U. Saglik B.N. Levent S. Osmaniye D. Kaya Cavuşoglu B. Ozkay Y. Kaplancikli Z.A. Synthesis and AChE-Inhibitory Activity of New Benzimidazole Derivatives Molecules 2019 24 541-51-4932/molecules24050861
39. Osmaniye D. Sağlık B.N. Acar Çevik U. Levent S. Kaya Çavuşoğlu B. Özkay Y. Kaplancıklı Z.A. Turan G. Synthesis and AChE Inhibitory Activity of Novel Thiazolylhydrazone Derivatives Molecules 2541-51-4932 10.3390/molecules24132392
40. Tok F. Koçyiğit-Kaymakçıoğlu B. Sağlık B.N. Levent S. Özkay Y. Kaplancıklı Z.A. Synthesis and Biological Evaluation of New Pyrazolone Schiff Bases as Monoamine Oxidase and Cholinesterase Inhibitors Bioorg. Chem. 2019 84 41 50 10.1016/j.bioorg.2018.11.016 30481645
41. Kosar M. Dorman H.J.D. Bachmayer O. Baser K.H.C. Hiltunen R. An Improved On-Line HPLC-DPPH Method for the Screening of Free Radical Scavenging Compounds in Water Extracts of Lamiaceae Plants Chem. Nat. Compd. 2003 39 161 541-51-4932/A:1024853628326
42. Kumarasamy Y. Byres M. Cox P.J. Jaspars M. Nahar D.L. Sarker S.D. Screening Seeds of Some Scottish Plants for Free-Radical Scavenging Activity Phytother. Res. 2007 21 615 541-51-4932/ptr.2129 17357975
43. Könczöl Á. Müller J. Földes E. Béni Z. Végh K. Kéry Á. Balogh G.T. Applicability of a Blood–Brain Barrier Specific Artificial Membrane Permeability Assay at the Early Stage of Natural Product-Based CNS Drug Discovery J. Nat. Prod. 2013 76 655 541-51-4932/np300882f 23565574
44. Müller J. Esső K. Dargó G. Könczöl Á. Balogh G.T. Tuning the Predictive Capacity of the PAMPA-BBB Model Eur. J. Pharm. Sci. Off. J. Eur. Fed. Pharm. Sci. 2015 79 53 60 10.1016/j.ejps.2015.08.019 26344358
45. Parallel Artificial Membrane Permeability Assay-BBB Kit|BioAssay Systems Available online: https://www.bioassaysys.com/PAMPA-BBB-Kit.html (accessed on 17 March 2022)
46. Schrödinger Maestro 10.6 Schrödinger LLC New York, NY, USA 2020
47. Schrödinger Suite 2020 Update 2 Schrödinger LLC New York, NY, USA 2020
48. Schrödinger LigPrep, Version 3.8 Schrödinger LLC New York, NY, USA 2020
49. Schrödinger Glide, Version 7.1 Schrödinger LLC New York, NY, USA 2020


{'source': 'PubMedDataset'}