Acetylcholinesterase Inhibition Activity of Some ... - Springer Link

ISSN 10681620, Russian Journal of Bioorganic Chemistry, 2015, Vol. 41, No. 2, pp. 170–177. © Pleiades Publishing, Ltd., 2015.

Acetylcholinesterase Inhibition Activity of Some Quinolinyl Substituted Triazolothiadiazole Derivatives1 Muhammad Rafiqa, Muhammad Saleemb, Muhammad Hanifc, Qamar Abbasa, Ki Hwan Leeb, and SungYum Seoa, 2 a Department

of Biology, Kongju National University, Kongju, 314701 Korea Department of Chemistry, Kongju National University, Kongju, 314701 Korea c Department of Chemistry, QuaidiAzam University, Islamabad, 45320 Pakistan b

Received May 12, 2014; in final form, August 28, 2014

Abstract—A series of aralkanoic acids was converted into aralkanoic acid hydrazides through their esters for mation. The aralkanoic acid hydrazides upon treatment with carbon disulfide and methanolic potassium hydroxide yielded potassium dithiocarbazinate salts, which on refluxing with aqueous hydrazine hydrate yielded 5aralkyl4amino3mercapto1,2,4triazoles. The target compounds, 3aralkyl6(substitut edquinolinyl) [1,2,4]triazolo[3,4b][1,3,4]thiadiazoles, were synthesized by condensing various quinolinyl substituted carboxylic acids with 5aralkyl4amino3mercapto1,2,4triazoles in phosphorus oxychloride. The structures of the newly synthesized triazolothiadiazoles were characterized by IR, 1H NMR, 13C NMR, and elemental analysis studies. The structure of one of the 5aralkyl4amino3mercapto1,2,4triazoles was unambiguously deduced by single crystal Xray diffraction analysis. All the synthesized compounds were screened for their acetylcholinesterase inhibition activities. Four of the triazolothiadiazoles exhibited excel lent acetylcholinesterase inhibition activities as compared to the reference inhibitor. Keywords: quinolinyl substituted triazolothiadiazole, Xray diffraction analysis, acetylcholinesterase inhibition assay DOI: 10.1134/S1068162015020089 21

INTRODUCTION

Acetylcholinesterase is a serine hydrolase (AChE, acetylcholine hydrolase, EC 3.1.1.7) that plays an essential role in the cholinergic synapses. Hydrolysis of the neurotransmitter acetylcholine in the nervous system by acetylcholinesterase is known to be one of the most efficient enzyme catalytic reactions [1]. The basis of this high efficiency has been sought by means of ligandbinding studies using various substrates and has led to the suggestion that the active center is com posed of a cationic esteratic subsite containing the active serine, an anionic site, which accommodates the choline moiety of acetylcholine, and a peripheral anionic site (PAS) [2]. The primary physiologic that the active center is composed of a cationic esteratic subsite containing the active serine, an anionic site, which accommodates the choline moiety of acetyl choline, and a peripheral anionic site (PAS) [2]. The primary physiologic role of the acetylcholinesterase peripheral site is to accelerate the hydrolysis of acetyl choline at low substrate concentrations [3, 4]. 1 The article is published in the original. 2 Corresponding author: email: [email protected].

The role of cholinergic system has been an inten sive issue of interest in Alzheimer disease, which is a neurodegenerative disorder causing deterioration of memory and other cognitive functions [5, 6]. In Alzhe imer’s disease, a cholinergic deficiency in the brain has been reported [7]. Therefore, the synthesis and study of inhibitors of acetylcholinesterase may aid to the development of therapeutically useful compounds to treat such neurological disorders. Acetylcholinest erase inhibitors donepezil hydrochloride, galantamine hydrobromide, and rivastigmine tartrate are the cur rent approved drugs for the treatment of Alzheimer’s patients [8]. However, acetylcholinesterase inhibitors present some limitations, such as their short halflives and excessive side effects caused by activation of peripheral cholinergic systems, as well as hepatotoxic ity, which is the most frequent and important side effects of these drug therapies [9–11]. For this reason, alternative and complementary therapies need to be developed. The present work includes the synthesis of new series of biheterocycles containing triazole and thiadiazole ring coupled with substituted quinolinyl carboxylic acids that show good acetylcholinesterase inhibition activity.

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RESULTS AND DISCUSSION Synthetic pathway adopted for the synthesis of 5aralkyl4amino3mercapto1,2,4triazoles (Va–h) and 3aralkyl6(substitutedquinolinyl)[1,2,4]triazo lo[3,4b][1,3,4]thiadiazoles (VIa–v) is illustrated in Scheme 1. Briefly, substituted aralkanoic esters (IIa–h) were synthesized by the reaction of corresponding substituted aralkanoic acid (Ia–h) in the presence of catalytic amount of sulfuric acid. The synthesized es ters (IIa–h) were converted into corresponding aralkanoic acid hydrazides (IIIa–h) by refluxing with hydrazine hydrate (80%) in methanol; the reaction of hydrazides with carbondisulfide in methanolic potas sium hydroxide at low temperature yielded an inter mediate, potassium dithiocarbazinate (IVa–h), which on reflux with aqueous hydrazine gave 5aralkyl4 amino3mercapto1,2,4triazoles (Va–h). Structures of 5aralkyl4amino3mercapto1,2,4triazoles (Va–h) were confirmed by IR and NMR spectroscopy. The tautomeric structureof one of the triazoles (Vf) was determined using single crystal Xray diffraction

OH

O

i

n

n

O

O

X (Ia–h)

X (IIa–h)

I⎯Va, X = 4Br b, X = 3Br c, X = 4OCH3 d, X = 3OCH3 e, X = 2OCH3 f, X = 2F g, X = 3F h, X = 4OCH3 VIa, X = 4Br b, X = 3Br c, X = 4OCH3 d, X = 3OCH3 e, X = 2OCH3 f, X = 2F g, X = 3F h, X = 4OCH3 i, X = 4Br j, X = 2F k, X = 3F

analysis. Condensation of the 5aralkyl4amino3 mercapto1,2,4triazoles (Va–h) with quinolinyl sub stituted carboxylic acids in phosphorus oxychloride resulted in the synthesis of target compounds, 3aralkyl6(substitutedquinolinyl)[1,2,4]triazolo[3,4 b][1,3,4]thiadiazoles (VIa–v). Formation of triazo lothiadiazoles (VIa–v) was deduced from the IR spec tral data by the disappearance of signals typical to NH absorption for the primary and secondary NH group attached to triazole moiety. In the 1H NMR spectra, the disappearance of singlet peaks for NH and NH2 protons of 5aralkyl4amino3mercapto1,2,4tria zoles (Va–h) confirms the ring closure of thiadiazole. Condensation of quinolinyl carboxylic acids with 5aralkyl4amino3mercapto1,2,4triazoles (Va–h) was further confirmed by increased number of signals in the aromatic region in both 1H NMR and 13C NMR spectra of 3aralkyl6(substitutedquinoli nyl)[1,2,4]triazolo[3,4b][1,3,4]thiadiazoles (VIa–v). Experimental values of elemental analysis were in good aggrement with the calculated ones.

ii

H N

n

NH2

H N

n

iii

O

O

S N H

SK

X

X (IIIa–h)

(IVa–h) iv

n=1 n=1 n=1 n=1 n=1 n=1 n=1 n=2 Y = 2Quinolinyl Y = 2Quinolinyl Y = 2Quinolinyl Y = 2Quinolinyl Y = 2Quinolinyl Y = 2Quinolinyl Y = 2Quinolinyl Y = 3Quinolinyl Y = 3Quinolinyl Y = 3Quinolinyl Y = 3Quinolinyl

171

X

N N

n

v

n

N N

S

X

NH2 N S N NH

Y n=1 n=1 n=1 n=1 n=1 n=1 n=1 n=2 n=1 n=1 n=1

(VIa–v) l, X = 3OCH3 m, X = 2OCH3 n, X = 2F o, X = 4OCH3 p, X = 4Br q, X = 2F r, X = 4OCH3 s, X = 4Br t, X = 2F u, X = 4OCH3 v, X = 4Br

(Va–h) Y = 3Quinolinyl n = 1 Y = 3Quinolinyl n = 1 Y = 4Quinolinyl n = 1 Y = 4Quinolinyl n = 1 Y = 4Quinolinyl n = 1 Y = 5Quinolinyl n = 1 Y = 5Quinolinyl n = 1 Y = 5Quinolinyl n = 1 Y = 6Quinolinyl n = 1 Y = 6Quinolinyl n = 1 Y = 6Quinolinyl n = 1

Scheme 1. Synthesis of 3aralkyl6(substitutedquinolinyl)[1,2,4]triazolo[3,4b][1,3,4]thiadiazoles (VIa–v). Reagents and conditions: (i) H2SO4(conc.), methanol, reflux, 8–10 h; (ii) Hydrazine hydrate (80%), methanol, reflux, 10–12 h; (iii) CS2, KOH, methanol, stirring, 0°C, 1 h; (iv) Hydrazine hydrate (80%), methanol, reflux, 10–12 h; (v) POCl3,substituted quinolinyl carboxylic acid, reflux, 4–6 h. RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY

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MUHAMMAD RAFIQ et al.

cause the labile hydrogen can be attached to the nitrogen or sulfer atom, as shown in the thiol–thione tautomeric forms below. In order to investigate the major contributing structure, we have selected com pound (Vf) for single crystal XRDanalysis. The results indicated that triazole derivative predominantly existed in thione conformation as shown in (Figs. 1, 2).

N1 N2 C7 C8 F1

C5 C6

C9 C1

C4

C3 C2

N3 N4

S1

R Fig. 1. The molecular structure of one of the triazole mol ecules (Vf) with the atomnumbering scheme. Displace ment ellipsoids are drawn at the 50% probability level.

R N

R

R N S

SH

N N H (b)

N N (a) Scheme 2.

Crystal data. C9H9FN4S, Mw = 224.26, triclinic, space group P1, a = 6.54460(10), b = 6.74380(10), c = 11.0531(2) Å, α = 96.6600(10)°, β = 100.2400(10)°, γ = 96.1280(10)°, V = 472.758(13) Å3 at 100 K, Z = 2, Dx = 1.575 mg/m3, µ = 0.326 mm–1, F (000) = 232. Acetylcholinesterase Inhibition Activity

Fig. 2. Packing diagram of compound (Vf) showing inter molecular hydrogen bonding.

Crystal Structure Determination of Compound (Vf) For the disubstituted 1,2,4triazoles, two tauto meric forms (i.e., 4,5disubstituted4H1,2,4triaz ole3thiol (a) and 3,4disubstituted1H1,2,4triaz ole5(4H)thione (b)) are theoretically possible be Results of acetylcholinesterase inhibition assay of triazo lothiadiazole derivatives (VIa–v) Compounds

IC50, µM

Compounds

IC50, µM

(VIa) (VIb) (VIc) (VId) (VIe) (VIf) (VIg) (VIh) (VIi) (VIj) (VIk) Neostig mine methyl sulfate

80.31 ± 2.8 41.00 ± 0.32 71.30 ± 3.0 25 ± 0.81 38.29 ± 1.71 34.22 ± 0.82 43 ± 0.65 37.40 ± 1.76 0.89 ± 0.03 29.62 ± 1.8 1.13 ± 0.08 2.42 ± 0.01

(VIl) (VIm) (VIn) (VIo) (VIp) (VIq) (VIr) (VIs) (VIt) (VIu) (VIv) Neostig mine methyl sulfate

63.42 ± 0.71 75 ± 3.10 2.89 ± 0.06 52.91 ± 1.6 33.54 ± 0.78 67.40 ± 2.3 27.23 ± 0.04 2.08 ± 0.03 45.86 ± 0.76 86.67 ± 3.33 96.46 ± 4.12 2.42 ± 0.01

All the synthesized triazolothiadiazoles (VIa–v) were evaluated for acetylcholinesterase inhibition ac tivity (table). Neostigmine methyl sulfate with IC50 value of 2.42 ± 0.01 µM was used as a reference inhib itor. Almost all compounds exhibit moderate to excel lent activities as compared to the reference inhibitor. Compound (VIi) with IC50 value of 0.89 ± 0.03 µM showed maximum acetylcholinesterase inhibition ac tivity among the whole synthesized series. It has 4 bromobenzyl group as substituent X and 3quinolinyl group as substitent Y. Compounds (VIk) and (VIs) with IC50 values of 1.13 ± 0.08 and 2.08 ± 0.03 µM showed excellent inhibition exceeding that of the stan dard inhibitor. Compound (VIn) with IC50 values of 2.89 ± 0.06 µM showed comparable activity to the ref erence inhibitor. These compounds possesed 3fluo robenzyl, 2fluorobenzyl, and 4bromobenzyl groups as substituent X and 3quinolinyl, 5quinolinyl, and 4quinolinyl groups as substituent Y, respectively. Compound (VIv) with 4bromo group as substitent X and 6quinolinyl group as substitent Y with IC50 values of 96.46 ± 4.12 µM showed minimum acetylcholinest erase inhibition activity. The rest of the compounds showed moderate activities with IC50 values in the range of 25 ± 0.81 to 86.67 ± 3.33 µM. EXPERIMENTAL All the common solvents and chemicals were of an alytical grade or dry distilled. The qualitative analysis of the synthesized compounds was ascertained by thin layer chromatography and the Rf values were deter mined by employing precoated silica gel aluminium plates, Kieslgel 60 F254 from Merck (Germany), us ing chloroform–methanol, 9 : 1, as an eluent; TLC

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was visualized under UV lamp (VL4. LC, France) at 254 and 365 nm. Melting points were determined on a Stuart melting point apparatus (SMP3) and are uncor rected. The IR spectra (υ, cm–1) were recorded on a Bruker Optics Alpha FTIR spectrophotometer. NMR spectra were recorded in CDCl3 on a Bruker Avance 300 MHz spectrometer with TMS as an inter nal standard and reported in δ, ppm. The multiplici ties were expressed as s = singlet, d = doublet, t = trip let. Single crystal data was obtained by using Bruker SMART APEX II Xray Diffractometer CCD equipped with MO Xray tube and graphite mono chromator at the ambient temperature. Elemental analysis was performed on Leco CHNS932 Elemen tal Analyzer, Leco Corporation (USA). General Procedure for the Synthesis of Substituted Aromatic Esters (IIa–h) Aralkanoic acid (Ia–h) (0.2 mol) was dissolved in methanol (50 mL) in a round bottom flask equipped with a reflux condenser and a calcium chloride drying tube. Concentrated sulfuric acid (0.002 mol) was add ed and the reaction mixture subjected to reflux for 8– 10 hours while monitored by thin layer chromatogra phy using chloroform–methanol, 9 : 1, solvent system. After completion of the reaction, the excess alcohol was removed under reduced pressure and resulting oil was poured into water. The oily layer was separated and the aqueous portion extracted with diethyl ether (3 × 50 mL). The combined organic layer was washed with a dilute solution of sodium hydrogen carbonate (100 mL) to remove unreacted sulfuric acid. The or ganic layer was dried over anhydrous sodium sulphate. The solvent was removed on a rotary evaporator after filtration to afford corresponding esters (IIa–h) as oily liquids. General Procedure for the Synthesis of Aralkanoic Acid Hydrazides (IIIa–h) The respective substituted aromatic esters (IIa–h) (0.02 mol) were dissolved in methanol (100 mL) in a round bottom flask fitted with a reflux condenser and a calcium chloride drying tube. Hydrazine hydrate (80%, 0.04 mol) was added slowly, and the reaction was monitored by thin layer chromatography. After completion of the reaction, the mixture was concen trated under reduced pressure. The resulting crude solid was filtered, washed with water, and crystallized from aqueous ethanol to get aralkanoic acid hydrazides (IIIa–h). General Procedure for the Synthesis of 5Aralkyl4 Amino3Mercapto1,2,4Triazoles (Va–h) The aralkanoic acid hydrazides (IIIa–h) (0.01 mol) were treated with a solution of potassium hydroxide (0.125 mol) in methanol (50 mL) at 0°C under stir RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY

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ring. Carbon disulfide (0.125 mol) was added slowly dropwise and the reaction mixture was stirred for 1 h at 0°C. The solid product of potassium dithiocarbazi nates (IVa–h) formed was filtered, washed with chilled diethyl ether, and dried. It waws directly used for the next step without further purification. The potassium dithiocarbazinates (IVa–h) were taken in 100 mL round bottom flask with distilled water (20 mL), hydrazine hydrate (0.250 mol), and refluxed over night. The reaction mixture turned green with evolu tion of hydrogen sulphide and finally it became homo geneous. It was then poured in ice and neutralized with concentrated hydrochloric acid. The white pre cipitate was filtered, washed with cold water, and crys tallized from aquous methanol to afford 5aralkyl4 amino3mercapto1,2,4triazoles (Va–h). General Procedure for the Synthesis of 3Aralkyl6 (Substituted Quinolinyl)[1,2,4]triazolo[3,4 b][1,3,4]thiadiazoles (VIa–v) A mixture of 4amino5aralkyl [1,2,4]triazole3 thione (1 mmol) and substituted quinolinyl carboxylic acids (1.1 mmol) in POCl3 (8–10 mL) was refluxed for 4–6 hours. The reaction mixture was slowly poured in crushed ice with stirring and neutralized with sodium hydrogen carbonate. Solid material was fil tered, washed with cold water, and dried to furnish 3aralkyl6(substitutedquinolinyl)[1,2,4]triazolo[3,4 b][1,3,4]thiadiazoles (VIa–v) as colored powder [12]. 3(4Bromobenzyl)6(quinolin2yl)[1,2,4]tria zolo[3,4b][1,3,4]thiadiazole (VIa). Light brown sol id; yield 56%; mp 140–142°C; Rf 0.71 (chloroform– methanol, 9 : 1); IR: 3061 (sp2 CH), 2927, 2829 (sp3 CH), 1590 (C=N), 1502, 1478, 1471 (C=C), 1056 (C–S); 1H NMR (300 MHz): 8.36 (d, 1H, J = 8.4 Hz, ArH), 8.19 (d, 2H, J = 8.4 Hz, ArH), 7.96–7.90 (m, 1H, ArH), 7.88–7.81 (m, 1H, ArH), 7.74–7.66 (m, 1H, ArH), 7.54–7.47 (m, 2H, ArH), 7.38 (d, 2H, J = 8.4 Hz, ArH), 4.49 (s, 2H, CH2); 13C NMR (75 MHz): 163.21, 159.12, 147.14, 146.32, 143.21, 140.12, 143.21, 133.95, 131.95, 130.93, 129.75, 129.34, 128.82, 127.88, 121.37, 117.34, 30.82; Anal. calcd. for C19H12BrN5S: C, 54.04; H, 2.86; N, 16.58; S, 7.59; Found: C, 53.94; H, 2.80; N, 16.51; S, 7.45. 3(3Bromobenzyl)6(quinolin2yl)[1,2,4]tri azolo[3,4b][1,3,4]thiadiazole (VIb). Magenta color solid; yield 56%; mp 196–198°C; Rf 0.58 (chloro form–methanol, 9 : 1); IR: 3035 (sp2 CH), 2939, 2899 (sp3 CH), 1588 (C=N), 1563, 1502, 1485 (C=C), 1032 (C–S); 1H NMR (300 MHz): 8.63–8.59 (m, 1H, ArH), 8.44–8.40 (m, 1H, ArH), 8.39–8.36 (m, 1H, ArH), 8.34–8.29 (m, 1H, ArH), 8.23 (d, 1H, J = 8.7 Hz, ArH), 7.97–7.92 (m, 1H, ArH), 7.89– 7.81 (m, 1H, ArH), 7.52–7.43 (m, 1H, ArH), 4.03 (s, 2H, CH2); 13C NMR (75MHz): 170.18, 155.71, 147.65, 147.09, 145.20, 137.76, 133.32, 131.02, Vol. 41

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130.56, 129.73, 129.19, 128.94, 127.93, 127.50, 125.33, 124.83, 122.99, 117.41, 30.08. 3(4Methoxybenzyl)6(quinolin2yl)[1,2,4] triazolo[3,4b][1,3,4]thiadiazole (VIc). Brown solid; yield 65%; mp 197–199°C; Rf 0.67 (chloroform– methanol, 9 : 1); IR: 3028 (sp2 CH), 2910, 2832 (sp3 CH), 1578 (C=N), 1512, 1498, 1473 (C=C), 1039 (C–S); 1H NMR (300 MHz): 8.23–8.15 (m, 2H, Ar H), 7.92 (d, 1H, J = 8.1 Hz, ArH), 7.88–7.78 (m, 1H, ArH), 7.72–7.64 (m, 1H, ArH), 7.43 (d, 2H, J = 8.7 Hz, ArH), 7.34 (m, 1H, ArH), 6.90 (d, 2H, J = 8.7 Hz, ArH), 4.47 (s, 2H, CH2), 3.79 (s, 3H, OCH3); 13C NMR (75 MHz): 169. 35, 158.80, 154.49, 147.61, 147.44, 146.31, 137.55, 130.87, 130.11, 129.73, 129.30, 128.74, 127.86, 127.01, 117.39, 114.16, 55.30, 30.54; Anal. calcd. for C20H15N5OS: C, 64.33; H, 4.05; N, 18.75; S, 8.59; Found: C, 64.23; H, 4.01; N, 18.65; S, 8.49. 3(3Methoxybenzyl)6(quinolin2yl)[1,2,4] triazolo[3,4b][1,3,4]thiadiazole (VId). Brown solid; yield 76%; mp 214–215°C; Rf 0.66 (chloroform– methanol, 9 : 1); IR: 3032 (sp2 CH), 2923, 2852 (sp3 CH), 1584 (C=N), 1553, 1539, 1500, 1482 (C=C), 1070 (C–S); 1H NMR (300 MHz): 8.63–8.59 (m, 1H, ArH), 8.44–8.40 (m, 1H, ArH), 8.39–8.36 (m, 1H, ArH), 8.34–8.29 (m, 1H, ArH), 8.23 (d, 1H, J = 8.7 Hz, ArH), 7.97–7.92 (m, 1H, ArH), 7.89– 7.81 (m, 1H, ArH), 7.74–7.65 (m, 2H, ArH), 7.52– 7.43 (m, 1H, ArH), 4.25 (s, 2H, CH2), 3.21 (s, 3H, OCH3); 13C NMR (75MHz): 170.18, 155.71, 152.65, 147.09, 145.20, 137.76, 133.32, 131.02, 130.56, 129.73, 129.19, 128.94, 127.93, 127.50, 124.83, 123.07, 122.95, 117.41, 55.56, 25.60; Anal. calcd. for C20H15N5OS: C, 64.33; H, 4.05; N, 18.75; S, 8.59; Found: C, 64.24; H, 4.01; N, 18.65; S, 8.51. 3(2Methoxybenzyl)6(quinolin2yl)[1,2,4] triazolo[3,4b][1,3,4]thiadiazole (VIe). Light yellow solid; yield 65%; mp 203–205°C; Rf 0.61 (chloro form–methanol, 9 : 1); IR: 3006 (sp2 CH), 2969, 2827 (sp3 CH), 1589 (C=N), 1502, 1494, 1486 (C=C), 1067 (C–S): 1H NMR (300 MHz): 8.34 (d, 1H, J = 8.7 Hz, ArH), 8.22–8.14 (m, 2H, ArH), 7.91– 7.28 (m, 1H, ArH), 7.87–7.78 (m, 1H, ArH), 7.73– 7.64 (m, 1H, ArH), 7.47–7.40 (m, 1H, ArH), 7.34– 7.24 (m, 1H, ArH), 7.19–7.10 (m, 2H, ArH), 4.58 (s, 2H, CH2), 3.01 (s, 3H, OCH3); 13C NMR (75 MHz): 169.44, 159.23, 154.61, 150.60, 146.77, 146.00, 137.58, 135.74, 133.56, 131.03, 129.30, 128.74, 127.86, 124.42, 121.95, 120.56, 117.41, 115.74, 55.25, 24.25; Anal. calcd. for C20H15N5OS: C, 64.33; H, 4.05; N, 18.75; S, 8.59; Found: C, 64.28; H, 4.01; N, 18.63; S, 8.49. 3(2Fluorobenzyl)6(quinolin2yl)[1,2,4]tri azolo[3,4b][1,3,4]thiadiazole (VIf). Brown solid; yield 61%; mp 150–152°C; Rf 0.58 (chloroform– methanol, 9 : 1); IR: 3053 (sp2 CH), 2927, 2840 (sp3

CH), 1593 (C=N), 1502, 1478, 1473 (C=C), 996 (C– S); 1H NMR (300 MHz): 8.34 (d, 1H, J = 8.7 Hz, Ar H), 8.22–8.14 (m, 2H, ArH), 7.91 (d, 1H, J = 8.1 Hz, ArH), 7.87–7.78 (m, 1H, ArH), 7.73–7.64 (m, 1H, ArH), 7.47–7.40 (m, 1H, ArH), 7.34–7.24 (m, 1H, ArH), 7.19–7.10 (m, 2H, ArH), 4.58 (s, 2H, CH2); 13C NMR (75 MHz): 169.44, 162.51, 159.23, 154.61, 150.60, 146.00, 137.58, 135.31, 133.82, 131.03, 130.96, 129.30, 128.74, 127.86, 124.42, 121.95, 117.41, 115.74, 24.25; Anal. calcd. for C19H12FN5S: C, 63.15; H, 3.35; N, 19.38; S, 8.87; Found: C, 63.08; H, 3.25; N, 19.35; S, 8.77. 3(3Fluorobenzyl)6(quinolin2yl)[1,2,4]tria zolo[3,4b][1,3,4]thiadiazole (VIg). Off white solid; yield 67%; mp 199–201°C; Rf 0.62 (chloroform– methanol, 9 : 1); IR: 3022 (sp2 CH), 2900, 2819 (sp3 CH), 1588 (C=N), 1543, 1535, 1501, 1492 (C=C), 995 (C–S); 1H NMR (300 MHz): 8.63–8.59 (m, 1H, ArH), 8.44–8.40 (m, 1H, ArH), 8.39–8.36 (m, 1H, ArH), 8.34–8.23 (m, 2H, ArH), 8.23 (d, 1H, J = 8.7 Hz, ArH), 7.39–7.32 (m, 1H, ArH), 7.12– 7.06 (m, 3H, ArH), 4.25 (s, 2H, CH2); 13C NMR (75MHz): 170.18, 158.71, 152.65, 150.62, 147.09, 144.20, 139.76, 137.46, 134.02, 130.56, 131.73, 129.19, 128.94, 127.93, 127.50, 124.83, 123.07, 117.41, 25.60; Anal. calcd. for C19H12FN5S: C, 63.15; H, 3.35; N, 19.38; S, 8.87; Found: C, 63.09; H, 3.34; N, 19.32; S, 8.81. 3(4Methoxyphenethyl)6(quinolin3yl)[1,2,4] triazolo[3,4b][1,3,4]thiadiazole (VIh). Brown solid; yield 58%; mp 221–224°C; Rf 0.63 (chloroform– methanol, 9 : 1); IR: 3012 (sp2 CH), 2929, 2856 (sp3 CH), 1579 (C=N), 1541, 1512, 1479 (C=C), 1013 (C–S); 1H NMR (300 MHz): 8.55–8.49 (m, 1H, Ar H), 8.24–8.17 (m, 1H, ArH), 8.01–7.95 (m, 1H, Ar H), 7.94–7.85 (m, 1H, ArH), 7.75–7.67 (m, 1H, Ar H), 7.40–7.35 (m, 1H, ArH), 7.40 (d, 2H, J = 8.4 Hz, ArH), 6.92 (d, 2H, J = 8.4 Hz, ArH), 3.89 (s, 3H, OCH3), 3.01 (t, 2H, J = 8.4 Hz, CH2), 2.90 (t, 2H, J = 8.4 Hz, CH2); 13C NMR (75 MHz): 163.46, 157.61, 159.38, 154.46, 147.03, 146.31, 135.28, 132.11, 128.81, 128.61, 128.37, 126.88, 123.16, 122.29, 120.60, 117.58, 55.55, 30.38, 25.66; Anal. calcd. for C21H17N5OS: C, 65.10; H, 4.42; N, 18.08; S, 8.28; Found: C, 65.00; H, 4.35; N, 18.01; S, 8.21. 3(4Bromobenzyl)6(quinolin3yl)[1,2,4]tria zolo[3,4b][1,3,4]thiadiazole (VIi). Red solid; yield 66%; mp 170–173°C; Rf 0.68 (chloroform–metha nol, 9 : 1); IR: 3056 (sp2 CH), 2929, 2871 (sp3 CH), 1597 (C=N), 1541, 1488, 1477 (C=C), 1059 (C–S); 1H NMR (300 MHz): 8.60–8.39 (m, 1H, ArH), 8.34–8.27 (m, 1H, ArH), 8.11–7.99 (m, 1H, ArH), 7.94–7.84 (m, 1H, ArH), 7.75–7.67 (m, 1H, ArH), 7.40–7.35 (m, 1H, ArH), 7.4 (d, 2H, J = 8.4 Hz, ArH), 6.92 (d, 2H, J = 8.4 Hz, ArH), 4.56 (s, 2H, CH2); 13C NMR (75 MHz): 163.46, 159.38, 156.92,


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147.55, 148.03, 139.28, 135.11, 131.76, 129.81, 128.61, 128.37, 126.88, 123.16, 122.29, 120.60, 117.58, 25.66; Anal. calcd. for C19H12BrN5S: C, 54.04; H, 2.86; N, 16.58; S, 7.59; Found: C, 54.01; H, 2.80; N, 16.50; S, 7.40. 3(2Fluorobenzyl)6(quinolin3yl)[1,2,4]tria zolo[3,4b][1,3,4]thiadiazole (VIj). Yellow solid; yield 61%; mp 150–152°C; Rf 0.72 (chloroform–metha nol, 9 : 1); IR: 3064 (sp2 CH), 2983, 2852 (sp3 CH), 1583 (C=N), 1542, 1543, 1492 (C=C), 1009 (C–S); 1 H NMR (300 MHz): 8.55–8.49 (m, 1H, ArH), 8.24–8.17 (m, 1H, ArH), 8.01–7.95 (m, 1H, ArH), 7.94–7.85 (m, 1H, ArH), 7.75–7.67 (m, 1H, ArH), 7.40–7.35 (m, 1H, ArH), 7.34–7.25 (m, 2H, ArH), 7.00–6.90 (m, 2H, ArH), 4.56 (s, 2H, CH2); 13 C NMR (75 MHz): 163.46, 157.38, 156.35, 155.46, 147.03,145.35, 135.28, 132.11, 130.52, 128.81, 128.61, 128.37, 126.88, 123.16, 122.29, 120.60, 116.58, 55.55, 25.66; Anal. calcd. for C19H12FN5S: C, 63.15; H, 3.35; N, 19.38; S, 8.87; Found: C, 63.08; H, 3.28; N, 19.31; S, 8.76. 3(3Fluorobenzyl)6(quinolin3yl)[1,2,4]tria zolo[3,4b][1,3,4]thiadiazole (VIk). Brown powder; yield 58%; mp 206–208°C; Rf 0.59 (chloroform– methanol, 9 : 1); IR: 3045 (sp2 CH), 2953, 2896 (sp3 CH), 1587 (C=N), 1543, 1519, 1496 (C=C), 1034 (C–S); 1H NMR (300 MHz): 8.63–8.59 (m, 1H, Ar H), 8.44–8.40 (m, 1H, ArH), 8.39–8.36 (m, 1H, Ar H), 8.34–8.29 (m, 1H, ArH), 8.24–8.17 (m, 1H, J = 8.7 Hz, ArH), 7.97–7.92 (m, 1H, ArH), 7.39– 7.32 (m, 1H, ArH), 7.12–7.06 (m, 3H, ArH), 4.25 (s, 2H, CH2); 13C NMR (75MHz): 170.18, 155.71, 154.51, 153.65, 147.09, 145.20, 137.76, 133.32, 131.02, 130.56, 129.73, 129.19, 128.94, 127.93, 127.50, 124.83, 123.07, 117.41, 25.60; Anal. calcd. for C19H12FN5S: C, 63.15; H, 3.35; N, 19.38; S, 8.87; Found: C, 63.01; H, 3.18; N, 19.14; S, 8.64. 3(3Methoxybenzyl)6(quinolin3yl)[1,2,4]tri azolo[3,4b][1,3,4]thiadiazole (VIl). Saddle brown solid; yield 59%; mp 230–232°C; Rf 0.62 (chloro form–methanol, 9 : 1); IR: 3027 (sp2 CH), 2945, 2871 (sp3 CH), 1580 (C=N), 1533, 1509, 1480 (C=C), 1021 (C–S); 1H NMR (300 MHz): 8.63–8.59 (m, 1H, ArH), 8.44–8.40 (m, 1H, ArH), 8.39–8.36 (m, 1H, ArH), 8.34–8.29 (m, 1H, ArH), 8.24–8.17 (d, 1H, J = 8.7 Hz, ArH), 7.97–7.92 (m, 1H, ArH), 7.89– 7.81 (m, 1H, ArH), 7.74–7.65 (m, 2H, ArH), 7.52– 7.43 (m, 1H, ArH), 4.25 (s, 2H, CH2), 3.10 (s, 3H, OCH3); 13C NMR (75MHz): 170.18, 156.99, 155.71, 151.65, 147.09, 145.20, 137.76, 133.32, 131.02, 130.56, 129.73, 129.19, 128.94, 127.93, 127.50, 124.83, 123.07, 117.41, 55.25, 25.60; Anal. calcd. for C20H15N5OS: C, 64.33; H, 4.05; N, 18.75; S, 8.59; Found: C, 64.23; H, 4.01; N, 18.65; S, 8.49. 3(2Methoxybenzyl)6(quinolin3yl)[1,2,4] triazolo[3,4b][1,3,4]thiadiazole (VIm). Light yellow RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY

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solid; yield 66%; mp 203–205°C; Rf 0.61 (chloro form–methanol, 9 : 1); IR: 3006 (sp2 CH), 2969, 2851 (sp3 CH), 1589 (C=N), 1533, 1494, 1489, (C=C), 1011 (C–S): 1H NMR (300 MHz): 8.34–8.29 (m, 1H, ArH), 8.20–8.11 (m, 2H, ArH), 7.91–7.84 (m, 1H, ArH), 7.87–7.78 (m, 1H, ArH), 7.73–7.64 (m, 2H, ArH), 7.47–7.40 (m, 1H, ArH), 7.34–7.10 (m, 2H, ArH), 4.58 (s, 2H, CH2), 3.01 (s, 3H, OCH3); 13C NMR (75 MHz): 169.44, 159.23, 154.61, 152.60, 146.00, 145.36, 137.58, 135.36, 133.27, 131.03, 129.30, 128.74, 127.86, 124.42, 121.95, 120.98, 117.41, 115.74, 55.25, 24.25; Anal. calcd. for C20H15N5OS: C, 64.33; H, 4.05; N, 18.75; S, 8.59; Found: C, 64.28; H, 3.95; N, 18.66; S, 8.49. 3(2Fluorobenzyl)6(quinolin4yl)[1,2,4]tria zolo[3,4b][1,3,4]thiadiazole (VIn). White solid; yield 71%; mp 142–144°C; Rf 0.61 (chloroform–metha nol, 9 : 1); IR: 3033 (sp2 CH), 2922, 2843 (sp3 CH), 1581 (C=N), 1542, 1492 (C=C), 1019 (C–S); 1H NMR (300 MHz): 8.84–8.67 (m, 1H, ArH), 8.52 (d, 1H, J = 8.4 Hz, ArH), 7.91–7.83 (m, 1H, ArH), 7.75–7.68 (m, 1H, ArH), 7.67–7.64 (m, 2H, ArH), 7.37–7.27 (m, 1H, ArH), 7.20–7.16 (m, 2H, ArH), 6.90–6.84 (m, 1H, ArH), 4.61 (s, 2H, CH2): 13C NMR (75 MHz): 163.79, 162.58, 159.30, 154.93, 149.68, 149.15, 146.39, 133.65, 131.31, 131.26, 130.63, 130.51, 129.48, 129.00, 124.88, 123.62, 121.86, 115.87, 24.69; Anal. calcd. for C19H12FN5S: C, 63.15; H, 3.35; N, 19.38; S, 8.87; Found: C, 63.09; H, 3.29; N, 19.25; S, 8.71. 3(4Methoxybenzyl)6(quinolin4yl)[1,2,4] triazolo[3,4b][1,3,4]thiadiazole (VIo). Brown solid; yield 65%; mp 150–152°C; Rf 0.72 (chloroform– methanol, 9 : 1); IR: 3064 (sp2 CH), 2983, 2843 (sp3 CH), 1583 (C=N), 1542, 1543, 1492 (C=C), 1022 (C–S); 1H NMR (300 MHz): 8.64–8.57 (m, 1H, Ar H), 8.45 (d, 1H, J = 8.1 Hz, ArH), 8.25 (d, 1H, J = 8.4 Hz, ArH), 7.93–7.84 (m, 1H, ArH), 7.76– 7.64 (m, 2H, ArH), 7.40 (d, 2H, J = 8.4 Hz, ArH), 6.92 (d, 2H, J = 8.4 Hz, ArH), 4.50 (s, 2H, CH2), 3.81 (s, 3H, OCH3); 13C NMR (75MHz): 163.47, 158.91, 157.11, 152.97, 149.70, 149.08, 147.81, 133.80, 130.71, 130.49, 128.97, 126.69, 124.89, 123.67, 121.85, 114.21, 55.34, 30.93; Anal. calcd. for C20H15N5OS: C, 64.33; H, 4.05; N, 18.75; S, 8.59; Found: C, 64.25; H, 3.95; N, 18.69; S, 8.49. 3(4Bromobenzyl)6(quinolin4yl)[1,2,4]tri azolo[3,4b][1,3,4]thiadiazole (VIp). Red solid; yield 67%; mp 176–178°C; Rf 0.64 (chloroform–metha nol, 9 : 1); IR: 3054 (sp2 CH 8.40 (d 2869 (sp3 CH), 1577 (C=N), 1551, 1521, 1502, 1499 (C=C), 1034 (C–S); 1H NMR (300 MHz): 8.77–8.64 (m, 1H, ArH), 8.40 (d, 1H, J = 8.1 Hz, ArH), 8.19 (d, 1H, J = 8.4 Hz, ArH), 8.17–8.09 (m, 1H, ArH), 7.93–7.84 (m, 2H, ArH), 7.76–7.64 (m, 2H, ArH), 6.97–6.84 (d, 2H, J = 8.4 Hz, ArH), 4.50 (s, 2H, CH2); 13C NMR Vol. 41

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(75 MHz): 164.46, 159.33, 157.12, 147.63, 146.48, 140.11, 137.62, 133.95, 131.95, 130.93, 129.75, 129.34, 128.82, 127.88, 121.37, 117.34, 30.82; Anal. calcd. for C19H12BrN5S: C, 54.04; H, 2.86; N, 16.58; S, 7.59; Found: C, 54.01; H, 2.71; N, 16.49; S, 7.50. 3(2Fluorobenzyl)6(quinolin5yl)[1,2,4]triazo lo[3,4b][1,3,4]thiadiazole (VIq). Light brown solid; yield 65%; mp 188–190°C; Rf 0.64 (chloroform– methanol, 9 : 1); IR: 3043 (sp2 CH), 2938, 2871 (sp3 CH), 1587 (C=N), 1541, 1501, 1487 (C=C), 1042 (C–S); 1H NMR ( 300 MHz): 8.70–8.63 (m, 1H, ArH), 8.52–8.46 (m, 1H, ArH), 8.38–8.32 (m, 1H, ArH), 7.96–7.91 (m, 1H, ArH), 7.88–7.81 (m, 2H, ArH), 7.60–7.54 (m, 1H, ArH), 7.20–7.13 (m, 1H, ArH), 7.01–6.94 (m, 2H, ArH), 4.61 (s, 2H, CH2); 13 C NMR (75 MHz): 165.06, 162.58, 159.31, 156.37, 148.29, 146.21, 133.69, 131.32, 129.97, 129.36, 128.65, 126.23, 125.33, 124.47, 123.07, 122.02, 121.81, 115.85, 24.65; Anal. calcd. for C19H12FN5S: C, 63.15; H, 3.35; N, 19.38; S, 8.87; Found: C, 63.07; H, 3.28; N, 19.27; S, 8.74. 3(4Methoxybenzyl)6(quinolin5yl)[1,2,4] triazolo[3,4b][1,3,4]thiadiazole (VIr). Brown solid; yield 63%; mp 147–149°C; Rf 0.59 (chloroform– methanol, 9 : 1); IR: 3030 (sp2 CH), 2969, 2836 (sp3 CH), 1583 (C=N), 1511, 1501, 1499 (C=C), 1066 (C–S); 1H NMR (300 MHz): 9.09–9.03 (m, 1H, ArH), 8.84–8.77 (m, 1H, ArH), 8.39–8.32 (m, 1H, ArH), 7.94–7.88 (m, 1H, ArH), 7.87–7.79 (m, 1H, ArH), 7.55–7.47 (m, 1H, ArH), 7.39 (d, 2H, J = 8.7 Hz, ArH), 6.92 (d, 2H, J = 8.7 Hz, ArH), 4.21 (s, 2H, CH2), 3.74 (s, 3H, OCH3); 13C NMR (75 MHz): 164.68, 158.88, 156.55, 155.40, 148.27, 147.58, 134.43, 130.33, 129.87, 126.34, 125.36, 125.32, 124.12, 122.99, 119.87, 114.14, 55.35, 30.98; Anal. calcd. for C20H15N5OS: C, 64.33; H, 4.05; N, 18.75; S, 8.59; Found: C, 64.28; H, 4.01; N, 18.56; S, 8.49. 3(4Bromobenzyl)6(quinolin5yl)[1,2,4]tria zolo[3,4b][1,3,4]thiadiazole (VIs). Brown solid; yield 66%; mp 154–157°C; Rf 0.59 (chloroform–meth anol, 9 : 1); IR: 3068 (sp2 CH), 2933, 2826 (sp3 CH), 1589 (C=N), 1557, 1511, 1496 (C=C), 1054 (C–S); 1H NMR (300 MHz): 8.84–8.77 (m, 1H, ArH), 8.39–8.32 (m, 1H, ArH), 7.90–7.84 (m, 1H, ArH), 7.89–7.73 (m, 1H, ArH), 7.70–7.65 (m, 1H, ArH), 7.50–7.43 (m, 1H, ArH), 7.35 (d, 2H, J = 8.5 Hz, ArH), 6.82 (d, 2H, J = 8.5 Hz, ArH), 4.31 (s, 2H, CH2); 13C NMR (75 MHz): 166.04, 156.22, 155.91, 149.74, 146.77, 136.80, 133.84, 131.95, 131.23, 129.18, 127.94, 127.48, 126.59, 125.33, 121.43, 119.12, 30.88; Anal. calcd. for C19H12BrN5S: C, 54.04; H, 2.86; N, 16.58; S, 7.59; Found: C, 53.94; H, 2.81; N, 16.51; S, 7.50. 3(2Fluorobenzyl)6(quinolin6yl)[1,2,4]tria zolo[3,4b][1,3,4]thiadiazole (VIt). Light yellow sol id; yield 62%; mp 140–142°C; Rf 0.63 (chloroform– methanol, 9 : 1); IR: 3011 (sp2 CH), 2969, 2891 (sp3

CH), 1587 (C=N),1573, 1477, (C=C), 1069 (C–S); 1 H NMR (300 MHz): 9.08–9.03 (m, 1H, ArH), 8.35–8.21 (m, 1H, ArH), 7.56–7.47 (m, 1H, ArH), 7.48–7.40 (m, 1H, ArH), 7.35–7.30 (m, 1H, ArH), 7.30–7.24 (m, 1H, ArH), 7.24–7.15 (m, 4H, ArH), 4.58 (s, 2H, CH2); 13C NMR (75 MHz): 165.77, 162.51, 159.24, 152.73, 149.73, 146.28, 136.78, 131.19, 131.08, 129.36, 129.25, 127.59, 126.63, 124.42, 122.64, 122.05, 120.89, 115.50, 24.38; Anal. calcd. for C19H12FN5S: C, 63.15; H, 3.35; N, 19.38; S, 8.87; Found: C, 63.09; H, 3.25; N, 19.31; S, 8.7. 3(4Methoxybenzyl)6(quinolin6yl)[1,2,4] triazolo[3,4b][1,3,4]thiadiazole (VIu). Yellow solid; yield 61%; mp 170–172°C; Rf 0.69 (chloroform– methanol, 9 : 1); IR: 3031 (sp2 CH), 2912, 2843 (sp3 CH), 1593 (C=N), 1520, 1486, (C=C), 1052 (C–S); 1 H NMR (300 MHz): 9.00–8.94 (m, 1H, ArH), 7.56–7.47 (m, 1H, ArH), 7.48–7.40 (m, 1H, ArH), 7.36 (d, 2H, J = 8.7 Hz, ArH), 7.35–7.26 (m, 1H, ArH), 7.20–7.10 (m, 2H, ArH), 6.82 (d, 2H, J = 8.7 Hz, ArH), 4.58 (s, 2H, CH2), 3.82 (s, 3H, OCH3); 13C NMR (75 MHz): 165.77, 162.51, 159.24, 152.73, 149.73, 147.48, 138.78, 134.19, 129.36, 129.25, 127.59, 126.63, 124.42, 122.37, 122.05, 115.79, 55.35, 24.38; Anal. calcd. for C20H15N5OS: C, 64.33; H, 4.05; N, 18.75; S, 8.59; Found: C, 64.29; H, 3.95; N, 18.65; S, 8.51. 3(4Bromobenzyl)6(quinolin6yl)[1,2,4]tria zolo[3,4b][1,3,4]thiadiazole (VIv). Light green solid; yield 62%; mp 176–177°C; Rf 0.64 (chloroform– methanol, 9 : 1); IR: 3059 (sp2 CH), 2900, 2831 (sp3 CH), 1580 (C=N), 1561, 1509, 1497 (C=C), 1041 (C–S); 1H NMR (300 MHz): 8.83–8.77 (m, 1H, ArH), 7.65–7.57 (m, 1H, ArH), 7.48–7.40 (m, 1H, ArH), 7.39 (d, 2H, J = 8.7 Hz, ArH), 7.35–7.26 (m, 1H, ArH), 7.20–7.13 (m, 2H, ArH), 6.82 (d, 2H, J = 8.7 Hz, ArH), 4.58 (s, 2H, CH2); 13C NMR (75 MHz): 165.77, 162.51, 159.24, 152.73, 149.73, 147.48, 138.78, 134.19, 131.08, 130.65, 129.36, 129.25, 127.94, 127.59, 126.63, 124.42, 122.64, 119.15, 24.38; Anal. calcd. for C19H12BrN5S: C, 54.04; H, 2.86; N, 16.58; S, 7.59; Found: C, 53.91; H, 2.81; N, 16.48; S, 7.56. XRDAnalysis Data collection. A colorless crystal of 0.22 × 0.20 × 0.19 mm3 was mounted on a glass fiber in inert oil and transferred to the cold gas stream of the spectropho tometer. A total of 8741 intensities leading to 2358 in dependent reflections were collected from theta range of data collections 1.89° to 28.41°, using monochro matic Kα radiation. Structure solution and refinemen. The molecular structure was solved by routine direct methods and re fined on F2 using the program SHELXL97 (Sheld rick, 2008). The final wR2 was 0.1076 for all reflections


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and 136 parameters with a conventional R1 of 0.0395; S = 0.923, max. Δρ = –0.883 e Å–3. Acetylcholine Esterase Inhibition Assay Protocol The inhibitory activities of newly synthesized com pounds were determined spectrophotometrically us ing acetylthiocholine iodide as substrate from the method of Ellman [13]. The assay solution consisted of 180 µL of 50 mM TrisHCl buffer, pH 8.0, contain ing 0.1 M sodium chloride and 0.02 M magnesium chloride, and 20 µL of enzyme (AChE, acetylcholine hydrolase, EC 3.1.1.7, acetyl cholinesterase from hu man erythrocytes) solution (0.03 U/mL); increasing concentrations of test compounds (10 µL) were added to the assay solution and preincubated for 30 min at 4°C. 5,5'Dithiobis(2nitrobenzoic acid) (0.3 mM, 20 µL) and acetylthiocholine iodide (1.8 mM, 20 µL) were added to the reaction mixture and incubated at 37°C for 10 min, followed by the measurement of ab sorbance at 412 nm. For nonenzymatic reaction, the assays were carried out with a blank containing all components except acetyl cholinesterase. The assay measurements were carried out by using a micro plate reader (OPTIMax, Tunable Micro plate Reader; wave length range 340–850 nm; for 96well plates). The re action rates were compared and the percent inhibition due to the presence of tested inhibitors was calculated. Neostigmine methylsulfate was used as reference in hibitor. Each concentration was analyzed in three in dependent experiments run in triplicate. The IC50 val ues were determined by the data analysis and graphing software, Origin 8.6, 64bit. ACKNOWLEDGMENTS This work was supported by Business for Coopera tive R&D between Industry, Academy, and Research Institute funded Korea Small and Medium Business Administration in 2012 (grant no. C0036335) and by


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“Leades IndustryUniversity Cooperation” Project, supported by the Ministry of Education, Science & Technology (MEST) and the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF–2011–0015056). REFERENCES 1. Quinn, D.M., Chem. Rev., 1987, vol. 87, pp. 955–979. 2. Colletier, J.P., Fournier, D., Greenblatt, H.M., Stogan, J., Sussman, J.L., Zaccai, G., Silman, I., and Weik, M., EMBO J., 2006, vol. 25, pp. 2746–2756. 3. Mallender, W.D., Szeglets, T., and Rosenberry, T.L., J. Biol. Chem., 1999, vol. 274, pp. 8491–8499. 4. Szeglets, T., Mallender, W.D., Thomas, P.J., and Rosenberry, T.L., Biochemistry, 1999, vol. 38, pp. 122– 133. 5. Abramov, A.Y., Canvari, L., and Duchen, M.R., J. Neurosci., 2003, vol. 23, pp. 5088–5095. 6. Butterfield, D.A., Chem. Res. Toxicol., 1997, vol. 10, pp. 495–506. 7. Greenblatt, H.M., Dvir, H., Silman, I., and Sussman, J.L., J. Mol. Neurosci., 2003, vol. 20, pp. 369–383. 8. Ellis, J.M., J. Am. Osteopth. Assoc., 2005, vol. 105, pp. 145–158. 9. Farlow, M., Gracon, S.I., Hershey, L.A., Lewis, K.W., Sadowsky, C.H., and Ureno, J.D., J. Am. Med. Assoc., 1992, vol. 268, pp. 2523–2529. 10. Lahiri, D.K., Farlow, M.R., Grieg, N.H., and Sam bamurti, K., Drug. Dev. Res., 2002, vol. 56, pp. 267– 281. 11. Rogers, S.L., Farlow, M.R., Doody, R.S., Mohs, R., and Friedhoff, L.T., Neurology, 1998, vol. 50, pp. 136– 145. 12. Eugene, L., Piatnitski, C., Hassan, M.E., and John, B., Tetrahedron. Lett., 2008, vol. 49, pp. 6709–6711. 13. Ellman, G.L., Courtney, K.D., Andres, V., and Feath erstone, R.M., Biochem. Pharmacol., 1961, vol. 7, pp. 88–90.

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