Summary
Triazol ve altı üyeli halka sistemleri ile kondense olmuş triazoller; tıp, tarım ve sanayi alanında farklı kullanım alanlarına sahiptir. Bu çalışmada, 1,2,4-triazol ve 1,2,4-triazolo[3,4-b][1,3,4]tiyadiazin türevleri kolinesteraz inhibitörleri olarak sentezlendi. Tiyokarbohidrazid ile 1H-indol-3-asetik asit reaksiyona sokularak, 4-amino-3-merkapto-5- [(1H-indol-3-il)metil]-4H-1,2,4-triazol elde edildi. Arilaldehidler ile triazol etanol içinde reaksiyona sokularak 4-arilidenamino-3-merkapto-5-[(1H-indol-3-il)metil]-4H-1,2,4-triazoller elde edildi. 3-[(1H-indol-3-il)metil]-6-aril-7H- 1,2,4-triazolo[3,4-b][1,3,4]tiyadiazinler, triazoller ve fenasilbromürlerin absolu etanol içinde kondensasyonu ile elde edildi. Bileşiklerin kimyasal yapıları, IR, 1H-NMR ve FAB+-MS spektral verileri ve elementel analiz sonuçları yardımı ile aydınlatıldı. Modifiye edilmiş Ellman spektrofotometrik metodu kullanılarak tüm bileşiklerin asetilkolinesteraz (AChE) inhibisyonları incelendi. Bileşikler 1b ve 1c, sırasıyla gösterdikleri 96.45±8.14 ve 76.24±6.42 μM IC50 AChE inhibisyonu değerleri ile Donepezil (IC50 =0.056±0.001μM) ile kıyaslandıklarında ümit verici sonuçlar vermişlerdir.Introduction
In drug design; for a more efficient and beneficial therapy for the patients, it has become more important to discover novel and improved drugs, in means of selectivity and potency, through enzyme inhibition[1,2]. Cholinesterase inhibitors (ChEIs) have appealed a great deal of interest among researchers owing to their importance in the treatment of myasthenia gravis, glaucoma and Alzheimer's disease[1–3]. It becomes more important since the frequency of these diseases, especially Alzheimer's disease, have been increasing in the world. As known, in humans two cholinesterases are present: acetylcholinesterase (AChE), which selectively hydrolyses acetylcholine, and butyrylcholinesterase (BuChE), which is a non-specific cholinesterase. The main difference between two types of cholinesterase is the respective preferences for substrates: the former hydrolyses acetylcholine more quickly; the latter hydrolyses butyrylcholine more quickly. The main function of AChE is the termination of cholinergic neurotransmission, but the function of BuChE is not so clear[1,4]. Acetylcholinesterase inhibitors (AChEIs) exert their therapeutic action by inhibiting AChE, which results in the enhancement of cholinergic action. Especially, in medication of the most common age-related neurodegenerative disorder, Alzheimer's disease, AChEIs play a leading role in the first-line treatment against its symptoms[5–7].1,2,4-Triazole derivatives are well-known with their different biological activities, therefore various 1,2,4-triazole derivatives and their N-bridged heterocyclic analogs have been extensively studied. Also, triazole fused six-membered ring systems are found to possess diverse applications in the fields of medicine[8-12]. The commonly known systems are triazole fused with pyridines[8], pyridazines[9], pyrimidines[10], pyrazines[11] and triazines[12]. The literature survey reveals that there are not many examples of triazoles fused with thiadiazines. Triazolothiadiazines bear a nucleus incorporating the pharmacophoric N-C-S linkage as in the skeleton of 1,2,4-triazolo[ 3,4-b][1,3,4]thiadiazine, that plays a main role in cholinesterase inhibition[13-14].
On the other hand, cholinesterase inhibitor activity has also been reported to be associated with the indolic nucleus[15,16].
In the design of new drugs, the development of hybrid molecules through the combination of different pharmacophores in one frame may lead to compounds with interesting biological profiles.
In the present study, prompted by these observations, the synthesis and cholinesterase inhibitor activity of 1,2,4-triazoles and 1,2,4-triazolo[3,4-b][1,3,4]thiadiazines as hybrid molecules including different pharmacophores were aimed.
EXPERIMENTAL
Chemistry
All reagents were used as purchased from commercial suppliers
without further purification. Melting points were determined
by using a Gallenkamp apparatus and are uncorrected.
IR: Shimadzu IR-435 spectrophotometer; 1H NMR: Bruker 250
MHz spectrometer; MS: fast atom bombardment mass spectra
(FAB-MS) were obtained by VG Quattro mass spectrometer.
Microanalytical data were obtained by the Microanalytical
Section of Service Center (CNRS, Ecole Normale de Chimie de
Montpellier, France).
4-Amino-3-mercapto-5-[(1H-indol-3-yl)methyl]-1,2,4-triazole (A). Equimolar mixture of thiocarbohydrazide (0.1 mol) and 1H-indol-3-acetic acid was heated in an oil-bath at 160–170°C for 2 h. The fused mass thus obtained was dispersed with hot water to obtain the triazole. The product was recrystallized from methanol[17].
IR (KBr) υmax (cm–1): 3305-3181 (N-H), 1574-1423 (C=C and C=N), 1338 (C=S).
% Anal. Calc. for C11H11N5S: C, 53.86; H, 4.52; N, 28.55, Found: C, 53.82; H, 4.50; N, 28.52.
General procedure for the preparation of 5-[(1H-indol-3-yl) methyl]-4-arylideneamino-3-mercapto-1,2,4-triazoles (1ae). To a suspension of aryl aldehyde (0.005 mol) in ethanol (10 ml), was added an equimolar amount of triazole. The suspension was heated until a clear solution was obtained. A few drops of conc. sulfuric acid were added as a catalyst and the solution was refluxed for 3 h on a water bath. The precipitated solid was filtered off and recrystallized from ethanol[18].
General procedure for the preparation of 3-[(1H-indol-3-yl) methyl]-6-aryl-7H-1,2,4-triazolo[3,4-b]-1,3,4-thiadiazines (2ae). A solution of triazole (0.005 mol) and phenacyl bromide (0.005 mol) in absolute ethanol (30 ml) was heated under reflux for 1 h, cooled to room temperature and then neutralized with ammonium hydroxide. The product thus obtained was recrystallized from ethanol[18].
1a: R:-H, Yield: 60%, M.p.:185-7 °C, IR (KBr) υmax (cm–1): 3401- 3131 (N-H), 1595–1442 (C=C and C=N), 1372 (C=S). 1H-NMR (250 MHz) (DMSO-d6) δ (ppm): 4.45 (2H, s, CH2), 6.95-8.05 (10H, m, aromatic protons), 10.30 (1H, s, N=CH), 11.10 (1H, s, indole NH), 13.85 (1H, s, triazole NH). MS (FAB); m/z: 334 [M+1].
% Anal. Calc. C18H15N5S: C, 64.84; H, 4.53; N, 21.00. Found: C, 64.85; H, 4.56; N, 20.97.
1b: R:-Cl, Yield: 72%, M.p.:195-6 °C, IR (KBr) υmax (cm–1): 3389- 3105 (N-H), 1611–1435 (C=C and C=N), 1370 (C=S). 1H-NMR (250 MHz) (DMSO-d6) δ (ppm): 4.40 (2H, s, CH2), 7.10-7.50 (5H, m, indole protons), 8.05-8.45 (4H, m, phenyl protons), 10.30 (1H, s, N=CH), 11.20 (1H, s, indole NH), 13.75 (1H, s, triazole NH). MS (FAB); m/z: 368 [M+1].
% Anal. Calc. C18H14ClN5S: C, 58.77; H, 3.84; N, 19.04. Found: C, 58.73; H, 3.87; N, 19.02.
1c: R:-CH3, Yield: 70%, M.p.:205-8 °C, IR (KBr) υmax (cm–1): 3415-3150 (N-H), 1601–1435 (C=C and C=N), 1378 (C=S). 1H-NMR (250 MHz) (DMSO-d6) δ (ppm): 2.25 (3H, s, CH3), 4.35 (2H, s, CH2), 7.05-8.20 (9H, m, aromatic protons), 10.25 (1H, s, N=CH), 11.05 (1H, s, indole NH), 13.85 (1H, s, triazole NH).
MS (FAB); m/z: 348 [M+1].
% Anal. Calc. C19H17N5S: C, 65.68; H, 4.93; N, 20.16. Found: C, 65.71; H, 4.96; N, 20.20.
1d: R:-NO2, Yield: 81%, M.p.:237-9 °C, IR (KBr) υmax (cm–1): 3396-3120 (N-H), 1630–1430 (C=C and C=N), 1380 (C=S). 1H-NMR (250 MHz) (DMSO-d6) δ (ppm): 4.35 (2H, s, CH2), 6.95-7.50 (5H, m, indole protons), 8.05-8.45 (4H, dd J=8.75 Hz and 8.71 Hz, phenyl protons), 10.20 (1H, s, N=CH), 10.90 (1H, s, indole NH), 13.85 (1H, s, triazole NH). MS (FAB); m/z: 379 [M+1].
% Anal. Calc. C18H14N6O2S: C, 57.13; H, 3.73; N, 22.21. Found: C, 57.17; H, 3.76; N, 22.24.
1e: R:-N(CH3)2, Yield: 61%, M.p.:200-1 °C, IR (KBr) υmax (cm–1): 3412-3141 (N-H), 1571–1439 (C=C and C=N), 1365 (C=S). 1H-NMR (250 MHz) (DMSO-d6) δ (ppm): 3.10 (6H, s, N(CH3)2), 4.25 (2H, s, CH2), 6.85-7.85 (9H, m, aromatic protons), 10.25 (1H, s, N=CH), 11.00 (1H, s, indole NH), 13.75 (1H, s, triazole NH). MS (FAB); m/z: 377 [M+1].
% Anal. Calc. C20H20N6S: C, 63.81; H, 5.35; N, 22.32. Found: C, 63.80; H, 5.35; N, 22.29.
2a: R:-H, Yield: 50%, M.p.:162-3 °C, IR (KBr) υmax (cm–1): 3425-3301 (N-H), 1615–1444 (C=C and C=N). 1H-NMR (250 MHz) (DMSO-d6) δ (ppm): 4.35-4.60 (4H, m, CH2 and C7 protons of triazolothiadiazine), 6.85-8.05 (10H, m, aromatic protons), 10.90 (1H, s, indole NH). MS (FAB); m/z: 346 [M+1].
% Anal. Calc. C19H15N5S: C, 66.07; H, 4.38; N, 20.27. Found: C, 66.09; H, 4.34; N, 20.31.
2b: R:-Cl, Yield: 45%, M.p.:221-2 °C, IR (KBr) υmax (cm–1): 3431- 3291 (N-H), 1622–1431 (C=C and C=N). 1H-NMR (250 MHz) (DMSO-d6) δ (ppm): 4.30-4.45 (4H, m, CH2 and C7 protons of triazolothiadiazine), 6.90-8.25 (9H, m, aromatic protons), 11.15 (1H, s, indole NH). MS (FAB); m/z: 380 [M+1].
% Anal. Calc. C19H14ClN5S: C, 60.08; H, 3.71; N, 18.44. Found: C, 60.10; H, 3.72; N, 18.43.
2c: R:-CH3, Yield: 45%, M.p.:108-11 °C, IR (KBr) υmax (cm–1): 3405-3311 (N-H), 1601–1461 (C=C and C=N). 1H-NMR (250 MHz) (DMSO-d6) δ (ppm): 2.35 (3H, s, CH3), 4.25-4.55 (4H, m, CH2 and C7 protons of triazolothiadiazine), 6.75-8.00 (9H, m, aromatic protons), 11.25 (1H, s, indole NH). MS (FAB); m/z: 360 [M+1].
% Anal. Calc. C20H17N5S: C, 66.83; H, 4.77; N, 19.48. Found: C, 66.82; H, 4.77; N, 19.51.
2d: R:-NO2, Yield: 48%, M.p.:225-6 °C, IR (KBr) υmax (cm–1): 3440-3299 (N-H), 1622–1450 (C=C and C=N). 1H-NMR (250 MHz) (DMSO-d6) δ (ppm): 4.30-4.55 (4H, m, CH2 and C7 protons of triazolothiadiazine), 7.00-8.15 (9H, m, aromatic protons), 11.30 (1H, s, indole NH). MS (FAB); m/z: 391 [M+1].
% Anal. Calc. C19H14N6O2S: C, 58.45; H, 3.61; N, 21.53. Found: C, 58.46; H, 3.64; N, 21.54.
2e: R:-N(CH3)2, Yield: 39%, M.p.:216-7 °C, IR (KBr) υmax (cm–1): 3417-3320 (N-H),1631–1431 (C=C and C=N). 1H-NMR (250 MHz) (DMSO-d6) δ (ppm): 3.10 (6H, s, N(CH3)2), 4.25-4.40 (4H, m, CH2 and C7 protons of triazolothiadiazine), 7.05-8.15 (9H, m, aromatic protons), 11.25 (1H, s, indole NH). MS (FAB); m/z: 389 [M+1].
% Anal. Calc. C21H20N6S: C, 64.93; H, 5.19; N, 21.63. Found: C, 64.95; H, 5.22; N, 21.62.
Pharmacology
AChE Inhibition
All compounds were subjected to a slightly modified method
of Ellman's test[19] in order to evaluate their potency to inhibit
the AChE. The spectrophotometric method is based on
the reaction of released thiocholine to give a coloured product
with a chromogenic reagent 5,5-dithio-bis(2-nitrobenzoic)acid
(DTNB). AChE, (E.C.3.1.1.7 from Electric Eel, 500 units), and
Donepezil hydrochloride were purchased from Sigma–Aldrich
(Steinheim, Germany). Potassium dihydrogen phos-phate, DTNB, potassium hydroxide, sodium hydrogen carbonate,
gelatine, acetylthiocholine iodide (ATC) were obtained
from Fluka (Buchs, Switzerland). Spectrophotometric measurements
were performed on a 1700 Shimadzu UV-1700 UV–
Vis spectrophotometer. Cholinesterase activity of the compounds
(1a-e and 2a-e) was measured in 100 mM phosphate
buffer (pH 8.0) at 25 °C, using ATC as substrates, respectively.
DTNB (10 mM) was used in order to observe absorbance
changes at 412 nm. Donepezil hydrochloride was used as a
positive control (Table 1)[20].
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TABLE 1: % AChE inbition of the compounds and IC50 values.
Enzymatic assay
Enzyme solutions were prepared in gelatin solution (1%), at
a concentration of 2.5 units/mL. AChE and compound solution
(50 μL) which is prepared in 2% DMSO at a concentration
range of 10-1-10-6 mM were added to 3.0 mL phosphate
buffer (pH 8±0.1) and incubated at 25 °C for 5 min. The reaction
was started by adding DTNB) (50 μL) and ATC (10 μL)
to the enzyme-inhibitor mixture. The production of the yellow
anion was recorded for 10 min at 412 nm. As a control,
an identical solution of the enzyme without the inhibitor is
processed following the same protocol. The blank reading
contained 3.0 mL buffer, 50 μL 2% DMSO, 50 μL DTNB and
10 μL substrate. All processes were assayed in triplicate.
The inhibition rate (%) was calculated by the following
equation:
Inhibition % = (AC–AI) / AC x 100
Where AI is the absorbance in the presence of the inhibitor, AC is the absorbance of the control and AB is the absorbance of blank reading. Both of the values are corrected with blankreading value. SPSS for Windows 15.0 was used for statistical analysis. Data were expressed as Mean ± SD.
Results
In this present work, a series of ten compounds were synthesized (Scheme 1). The structure of the compounds was elucidated by IR, 1H-NMR, FAB+-MS spectral data and elemental analysis. IR spectra of all compounds C=N and C=C bands were observed at about 1630-1430 cm-1 region. According to the IR spectroscopic data of the compounds 1a-e which have triazoline-3-thione structure, the C=S stretching bands observed at about 1380–1365 cm–1. While the NH bands of compounds 1a-e observed at about 3415-3105 cm-1 regions, the NH bands of compounds 2a-e observed at about 3440-3291 cm-1 regions.){kind=link}
 Click Here to Zoom |
SCHEME 1: The synthetic protocol of the compounds. |
In the 1H-NMR spectra of compounds, NH proton of the indole ring was seen as singlet at about 10.90-11.30 ppm. The signal due to indol-CH2 methylene protons, presented in all compounds, appeared at 4.20–4.60 ppm, as singlets. Due to the electron withdrawing effects of phenyl and triazole ring, the N=CH proton of the compounds (1a-e) appeared at 10.20-10.30 ppm as singlet as a result of chemical shift. All the other aromatic and aliphatic protons were observed at the expected regions. Mass spectra (MS (FAB)) of compounds showed M+1 peaks, in agreement with their molecular formula.
The anticholinesterase effects of the compounds (1a-e and 2ae) were determined by modified Ellman's spectrophotometric method (Table 1). Among these compounds (1a-e and 2a-e), compounds 1b and 1c can be identified as promising anticholinesterase agents due to their inhibitory effect on AChE with IC50 value of 96.45±8.14 and 76.24±6.42 μM respectively when compared with Donepezil (IC50 =0.056±0.001μM). Although compounds 1b and 1c include triazole nuclei they showed different levels of anticholinesterase activity. The former bearing Cl atom and methyl group on phenyl ring exhibited the inhibitory effect on AChE with IC50 value of 96.45±8.14 and 76.24±6.42 μM, whereas the other substitutions exhibited the inhibitory effect on AChE with >100 μM. On the other hand the trizolothiadiazine derivatives (2a-e) did not show notable inhibitory effect on AChE.
As a result, we can say that the triazole derivatives are more active than triazolothiadiazines when their anticholinesterase activity is compared.
ACKNOWLEDGEMENTS
The author would like to thank the staff of Anadolu University
Faculty of Pharmacy, Department of Pharmaceutical
Chemistry for their valuable suggestions regarding the manuscript.
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